CSOSMIbEEERENCB TO RELATED APPLICATIONS

This is a P€T application of Ser. No. 16 680,294 of U.S patent application, filed No ember 11, 2QI¾ which Is a continuation-in-part application of U.S. patent application Ser. o. 16/333,179, filed March 14, 2Q19 which is a emifionation of U.S. patent application Ser No. 14/969,519, filed December 15, 20I5 hich claims priority to Bulgarian Utility Model No. 2935 fifed on January 16, 2015 lie content of each of th aforementioned applications is incorporated by reference herein in their entireties.

FIELD QF THE EMBODIMENTS

This Invention characterises beverages comprising hydrolyzed collagen, deri e from animal raw materials. Thos raw materials ma vary according to their origin, including mammals, fish, bi s, spalls; or tap various parts of carcass.

BACKGROUND OF THE EMBODIMENTS

Many of the beneficial properties of collagen are applicable both to the medical field and cosmetics. Collagen is a; «tr or protein of the human connective tissue including, for example, cartilage, cornea, arteries, an skin. Robust and inmrtensible, collagen possesses great tensile strength. It is the principal component of cartilage, tendons, articular joints, hone as well as teeth. Collagen can also he found i the walls of capillaries, veins and arteries l imparts to tbe sPPngtb and elasticity ¹ for the effective transport of blood throughout the body. Collagen is o vital importance for muscle Amotion The: collagen molecules also provide _: muscle fibers _: with strength and structure necessary for their functioning over long periods of time. Collagen not only supports skeletal muscle fibers, but also smooth muscles present in cardiac, bladder and genital muscles.

Collagen is permanently being replaced in the human body. However, after the age of 25 this proces decelerates and progressively slackens with advancing age. Disorders of collagen buildup can also be provoked in si tuations of permanent overloading (for example In power sports, or in cas

INCORPORATED BY REFERENCE (RULE 20.6) of intensive physical activity). With aging, the amoun ai de novo synthesized collagen is reduced as result of a slowdown in metabolism which results in a gradual decline in the resilience _: of both soft tissues and bones (osteoporosis). The c llagen deficit due to advancing age, leads to visibl effects including shin drying and wrinkling, limite mobility due to stif&ess and joint pain, bone fractures caused by osteoporotic changes, the appearance of pointed and visible capillaries o th shin surface as well as the appearance of varicose veins

Specific references t relevant prior art ate herein described as follows:

IJ S. Patent Publication Mo. 2biO/IBIb?b8 and International Patent Publication No. W02904O23§99 disclose a shelf-stable, ready to use, water-like composition for humansfan mais; m m a innct to fiber-water, and/or sale drinking water, consumed directly, tube feedings, or in the preparafion/reeonstitution of kiod(s)/hevcrage(s). These disclosures teach a fortified fiber-water with _: adde delivery systems: Eucapsulsticns/partieles, of different rizefs), shapefs), mat d aifs). colors, non-visibie, serving one or more functions: improved taste, odor-masking; controlled release: applications, bio-availabiiit of actives, avoid hygroscop ty; minimized interactions, improved thermal, end dative, and shelf-life; decorative.

U S. Patent Publication No. 2014/02125¾S and International Patent Publication No. 02013040540 teach a clear high protein beverage comprising water; between about 4% and. about S% by weight protein; and a flavoring. W02OO?O9§§93 teaches aa alkaline soluble fiber compositions nd methods f¾j ^· preparing dm same. Soluble fiber for use in the compositions of the invention i contributed from one or more sources and is preferably Inui , FOB and/or soFOS. In some cases, th compositions have a pH of between 8,0 and 95,

The cthod aird solutions known in the ait fell to remedy all the problems lauglt by the present disclosure. There Is an ongoing unntet nee In the art lor improved thods of restoring collagen lost as a result of aging.

SUMMARY OF THE EMBODIMENTS

The prefeoted embodiments of the presentinvention will nowbe described, Reference will sow he made in detail to ©geh embodiment of dm present invention Such embtxlisbents are provided by way of explanation of the present Invention, which is not intended to be limited thereto. In feet, those of ordinary skill In the art may appreciate upon reading the present specification that various modifications and variations ca be a e _: hereto.

INCORPORATED BY REFERENCE (RULE 20.6) The disclosure described a hydrolyzed collages everage that facilitates trie absorption of the hydrolyzed collagen in trie: digestive tract.

In a fimt aspect, a beverage is disclosed coasted eg of anon-protein portion comprising water, gad a protein portion consisting of collagen la certain emhodimmits f thefirst speel, the collagen is fiom oomhuomn ammafs.

In certaia embodiments of tl e first aspect, the collagen is isolated from a plant expressing recombinant collagen.

In certain embodiments of tbs first aspect, the collagen is hydrol zed in eertain _: embodiments of trie first aspect the hydcolyzed eollagen comprises peptides having a molecular weight of no more t haft shoot 2060 dal tons. in certain embodiments of th first aspect, the collagen concentration range is between 1 milfigram/liter to about 8440 milfigram/liter, la certain embodiments of the first aspect, the collagen concentration range is between about 8440 mliilgranVtiter and dOg/llter

¾ certain embodiments of the first aspect, the water is carbonated.

In certain embodiments of the first aspect the son-protein port on of the beverage does not comprise sodium acid sulphate, in certa n embodiments of the first aspect, the non-protein portion of the beverage does not comprise ny one of citric acid, phosphoric acid, mafic acid, tartaric acid and firmarie acid.

In certain embodiments of the first aspect the non-protein portion of the beverage fiuther comprises a vitamin, in certain embodiments of the first aspect, the vitamin is in an amount equal to die US: “Recommended Daily Allowance (RD )/

In certain embodiments of the first aspect, the vitamin comprises at Jeast one of Vitamin A, Vitamin 1% Vitamin E, Vitami K, Vitamin C, Thiami (Vitami B 1), Riboflavin (Vitamin B2), Niacin (Vitamin 03), Vitamin 86, Folic acid (Vitamin 89), Vitamin 812, Biotin (Vitamin 87), Pantothenic acid (Vitamin 85),

In certain embodiments of the first aspect, the vitamin comprises any one of Vitamin 86, Biotin (Vitamin 87), Vitamin € and Vitamin 812

INCORPORATED BY REFERENCE (RULE 20.6) Its certain embodiments of the first as ect, the vitamin comprises any two of ita n Bb, Biotin (Vitamin B7), Vitamin G aud Vitamin B12.

In certain embodiments of the first aspect, the vitamin comprises any three of Vitamin BS, Biotin (Vitamin 87), Vitamin C an Vitamin B12

In certain embodiments of the first aspect, the vitamin consistsof Vitamin Id, Biotin (Vitamin B?5, Vitamin C and Vitamin BI2.

In certai embodiments of the first aspect, the non-protein portion of the beverage further comprises a mineral.

In certai n embodiments of the first aspect, the mineral is chosen from at least one of Potassiu (fo) Clhoride (Cl), Caie m Cn), Phospbo s (P), Magnesium (Mg). Iron (t¾), Zinc (Zn), Coppe (€¾), Manganese (Mn), Fluoride (F), Selenium (Sc), Chromium ( r), Molybdenum: (Mo) and Iodine (0

I certain embodiments of the first aspect, the mineral consists of manganese and or magnesium.

In certain embodiments of the first aspect, the non-protein portion of the beverage comprises water, a vitamin and a mineral.

In certain embodiments of the first aspect th mineral is manganese ndMr magnesium. in certain embodiments of the first aspect, the vitamin Is Vitamin Bb, Biotin (Vitamin B7), Vitamin C and Vitamin B 12.

In a second aspect, a beverage is disclosed comprising a non-protein portion comprising water, and a protein portion, wherein the protein portion comprises bydrolyae collages and no whey protein.

Is a third aspect, a beverage is disclosed consisting of water and hydrolyzed collagen.

In certai n embodiments of the third aspect, the beverage has a pH of about 6

In a fourth aspect, ametbod of Increasing the absorption of hy rolyaed collagen in the iiestrve tract of a sab) eel is disclosed comprising any one of the aforementioned beverages. fn certain embodiments, & beverage Is disclosed having water and a concentration range of about .001 grams per liter to 8.44 grams per liter of collagen, wherein the collagen is: hydrolyred. Th collagen concentration range may he between I milligra per liter to about 8440 illigram per liter or I part per million to about 8440 parts per million, The hydrolyaed collagen can have collagen

INCORPORATED BY REFERENCE (RULE 20.6) peptides roduced through hy rolysis of a plurality collagen I s® a variet of sources, ineindtng, for exam le, animal raw materials, animals raised in non-orgaine or organic ferms, animals fro various animal species and various parts of the animal carcass. In certain embodiments, collagen, as used herein, e&n be recombinant collagen that is expresse for exampl in plants, yeast, tissue culture cells or bacteria. Is certain embodiments, the beverage contains water Including, for example, tap water, spring or mineral wafer. In certain embodiments, the beverage may be carbonated or nbn- earbotiated.

Is c rtain embodiments, a beverage is disclos having water, : concentration range of about .001 grams per liter to 8,44 grams per lifer of collagen; and at least one a ditive In certain embod ents, the additive may comprise a stabilizing preservative. The stabilizing _: preservative may have chemical substances which dissolve in the beverage and are only parts of its content or hav chemically active substances: used in the production of the bevemgebui subsequently dissolvet otlrer ingredients. I certain embodiments, the additive mag comprise a preservative Ih certain embodiments, the beverage may be pasteurized In certain embodiments, the additive ma comprise a packaging gas and/or a coloratd:. The coloraiti may be synthetic or organic and derived from vegetable, Iry.it or anbnaJ origin The coioraul may have a cmtcentrafiOP range of at least one of ,0001 fi¾ to aboyt %, .001 % to about ,1 and 003 % to about .2 . In certain embodiments, the additive may comprise a plurality of sweeteners. The sweeteners may be in liquid form, hard form, natural sugars, refined sugars, low caloric, and ®on~reiined sugars, In certain embodiments, the additive may cotnprise a plurality offruit juices or fruit jmee concentrates In certain enthodi meats, the additive may comprise a plurality of acidity regulating; or E-nymbe additives In certain embodiments, the additive may comprise a thickener, flavor enhancers, vitamins, minerals, electrolytes and/or a mo: acids The additive may be at least one other protein with a concentration range: sum total of ,0001 grams per liter to S.44 graft!»- per liter. In certain embodiments, the additive pay comprise a dairy component probiotic ingredients and/or extracts. In certain embodiments, dm beverage ma be carbonated or non- carbonated.

In certain embodiments, absorption of the collagen hydrolysate in the intestinal tract can be modulated depending on the molecular weight of the collagen peptides and the presence of certain addi tives, The optimal concentration of 10,000 dal ton collagen hydrolysate for intestinal absorption is _: about 8,4g/l which does not appreciably increase even ifthe collagen concentration Is increased to 20, 30 or even 40g/I in the presence or absence of whey protein. In contrast, the absorption of a 2,Q00 dalton collage hydrolysate composition, that does not contain whey protein, is shown to be enhanced

INCORPORATED BY REFERENCE (RULE 20.6) b the additiou of supplements including vita ins andfor CIOs and/or pro-hyp hyp-gly. The absorption is shown to increase: with collagen concentration up to an optimal concentration of about 20g/l. At collagen concentrations: greater than about SOg/h the absorpti on of the, coll agen pepti des is maintained or decreases,

BRIEF DESCMIFflOK OF THE DRAWINGS FKI LA ki exemplary schematic sfte Aag ¾Qw peptides are absorbed across the Intestinal wall;

FIS !B shows an exemplary embodiment of a Iraoswell assay testing lor the transport of collagen peptides across a Caco 2, cell monol yer;

: FIS, I C shows an exemplary embodiment of peptide transport across a Caeo 2 cell monolayer as a function of collagen peptide concentration;

FIG 2 shows ah exemplary embodiment of the optimal DV (dally v&lnef dose of Vitamin Package elements for absorption via Flydrol red Collagen _: Beverage (500 nil heverage is 1 dose);

FIS. 3: shows an exemplary emhodlMenfof collagen hydrolysate beverage on absorption;

FI©. 4 show exemplary comparisons of the effect Of different concentrations of various collagen: hydrolysate co positions ear absorption

DETAILED DESC PT 1QN OF THE INVBGTIOM

Unless explained otherwi e, all technical and seiemifie terms used herein have the same meaning as commonly understood to, one of ordinary skill in the art to which this di closure belongs. Although methods and materials similar or eputvaleni to those described herein can he used in the practice or testing of the present disclosure:, the materials, methods:, and examples are illustrative only and no! Intended to be limiting, Other features of the disclosure are apparent from the following detailed description and the claims.

Definitions

As used herein, the singula forms “a; “any and “they are intended to inohsde the: plural forms as well unless the context clearl indicates otherwise.

The phrase ¾nd/ory as used herein in the specification and in the claims, should he understood to mean “either or hot¾” of foe elements so conjoined,, he., elements that are eonjuncdvel present in

INCORPORATED BY REFERENCE (RULE 20.6) some eases md disjunctive^ present in other eases, Thus, as a son-limiting exa le, a reference to “ A and/or BA when used i n conjunction with open-ended language such as “com risingr can refer, in one embodiment, ΐo _: A only (optionally including elements other than If); in another embodiment, to B only (optionall including elements other than A); In et another embodiment, to both A and B (optionally including other elements) etc.

As use herein in the specification and is the claims, the phrase “at least one," is reference to a list of one or moreefements, should be understood to mean at least one element selected from any one or more of the elements in th list of elements, buf not necessaril including at least one of eaeh and every element specifically listed within the list of elements and not excluding any combinations of elements is the list of elements. This definition also allows that elements may optionally fee present Other than the elements specifically identified within the list of elements to which the phrase ¾ feast OK" refers, whether related os- unrelate to those elements specifically identified, Thus, as a non- ll itisg examp!eAfe feast one of A and B” (dr, equivalently, “at least one of A ©rB," or, equivalently ⁱ at least one of A an d/or B ^>s ) _: esn refer, is on embodiment, to at least one, optionally mefebing more than one, A, with no B present fend optionally ineludisg dements other than B); In anothe embodi e t, t© at least one, optionally including more than one, B, with no A present (and optionall including elements other than A); in yet another embodiment, to si l east one. ptionally in i siding more t an one, A, and all east pee, optionally mciad g more than one, B (and optionally including other elements); etc.

When the term “about" is used in conjunction wMr a numerical range, it modifies that range b extending the boundaries above d below those numerical values. In general, the term ^gfeoat’is used beret h to modify a numerical value above and belo the staled value by a variance of 205% 1014 5%, or 1%. In certain embodiments, the term “ahouf ^' is: used t© modify a numerical value above and below the stated value by a variance of 10%. In certain embodiments, the term “about” is used to modify a mnnerlc value above and fie!ew the stated value by a vari nce of 514 I certain embod nts, the term “about” is used to modify a numerical value above and below the stated valu by a variance of T%

When a range ©f values; is _: listed herein, it is intended to encompass each value and sub-range within that range. For example, “1~5 ng" or a range “between 1 ng and 5$rg" is intended to encompass I ng,2 ng, 3 ug, 4 ng, 5 ng, 1-2 ng, I -3 ng, 1-4 ng, 1-5 ng, 2-3 ng, 2~4 ng, 2~S ng, 3-4 ng, 3-5 ng, mi 4-5: ng.

INCORPORATED BY REFERENCE (RULE 20.6) It will e further nderstood that “including^ when use herein, specify the presence of stated features, integers, steps, operations, ele ents, and/or components, hut do not preclude the presence or a ition of one or mote other features, integers, steps, operations, elements, components, and or groups thereo

As use _: heroin. Hep refers tohydroxyprol e.

As used herein, the terur Isolated” refers to a sample in a nonmamraily occurring state outside an organ! sot (e.g., isolated from organism or a biological sa ple fknn an mgaoism).

A'nRΐrheT ⁵ is a verteh me, preferably a mammal e g., a non-hnntgn antmai), om preferably a primate an still more preferably a hitman. Mammals include, hut am nqt Itmiied to, primates, humans, fan» animals, rodents, sport animals, and pets.

As used herein, a A Vitamin Package” refers to the core vitanrin/minerai additive package (see FIO:. 2) which aids in absorption of higher percentages of collagen hydrolysate in a hydrolyzed collagen beverage and will he set at the concentrations specified in FIG. 2. The specific dose set out delivers the optimal dose of viismitm to absorption of coiky p hydroly sate Hydrolyzed Collagen Beverage (one SOd ml, beverage is 1 dose) to allow maxi um absorption while avoiding formation of sediment ansi without significant effect on beverag taste. These core vitamins will always part of the Vitamin package. Other vitamins and minerals can he added without negatively affecting collagen absorption. Additional vitainins/ lnerals o increased concentration may, however, may have a significant effect on product Taste and may cause the formation of sediment in the beverage.

As used herein, RDA is the acronym for “Reco mmende Pally Allowance ”

As used herein, the maxi um RDA refers to the estimated Maximum percentage of RDA of elements of th Vitamin Package which could _: be effectively absorbed via a hydrolyzed collagen beverage in its original state, the digestive system is incapable of enc aiically degrading collagen in part because to its great length and tire etcsslinking between collagen fibers. Thug administration of collagen to an individual Is best achi eved usi ng: ''hydrolyzed” Collagen that facilitates its absorption in the digestive tract Collagen can be administered is the fornt of tablets, capsules, or I the form of & drink.

Recent scientific studies show, that drinking collagen in licfuid form results In more than 20% absorption of collagen in: the digestive tract, which is absolutely unachievable when collagen is either

INCORPORATED BY REFERENCE (RULE 20.6) In tablet « t psole lbrtn The iquM collagen form has demonstrated better results, higher efficiency of active ingredients and unrivalled absorption. Moreover, all dietary supplement forms tablets, capsules, saebets ete- deinitelv butden ore on tbe d^sfive system. In contrast to the liquid icon for drinking, which is easily assimilated by the stomach. The accurate dosage is of particular importance for tie con sinners of cell agen drin ks.

Known collagen drinks have a different percentage of hydrolyae collagen and loci a o many additive like stabilizers, preservatives, aromalizers, artificial colors, flavor enhancers etc. These components are put in products with more concentrated amount of added hydrolyzed collagen. Because of the absence of any additional components, except drinking water and hydrolyzed collagen, the disclosed beverage represents m Important benefit winch ensures Its adi mstratioo without; limitations regarding age and health Mate of the expected consumers.

The risk of forming intolerance, or exacerbation of hyperesthesia of the organism, which normally Is provoked by additionall added unnatural chemical additives, is completel avoided.

In certain embodiments of the present Invention, the optimal concentration of hydrolyze collagen in the drink is Completel clear in taste and almost wholly corresponding to the nsutml taste of the clean drinking water. Furthermore, the drink is maximal simplified in functionally and tbat is why it doesn’t burden the consumer with additional calories. A a whole, the consumption of the created non~carbo«aied aIeohd!~§se drink leads to the improvement of the skin structure, the ski ensity. Skin turgor, m usds ne as well as to the Improvement of the condi tion and the flexibility of a 8o!¾eei s joints:.

In certain embodiments, the hydrolyzed collagen drink can he the sole source of a subject daily Intake of water.

The market offers a wide variety of beverages containing rious health ingredients. Contemporary society strives to improve its nutrition by selecting food and beverages with high added value, provide by the various forms of Ingredients, beneficial far the body , include in their contents. This way, the need of consumers to reach bette shape and tonus, to li ve a fell lift, feel good and preventively enhance their health can be satisfied. Beverages, which nclude in their content hydrolyzed collagen, may be offered in convenient and easy for consumption form. Th collagen In these drinks may fee a source of valuable, proteins In the body tha can characterize with high and fid! assimilation. At the same time, when collagen is added in beverages, it combines the advantage, the daily intake of fluids to be stimulated, which usually Is what underlies most recommended healthy

INCORPORATED BY REFERENCE (RULE 20.6) diets, te sufficient Intake of fluids i& extremely imp rt®† for nxany groups of eople having specific and increased needs of Holds, such as, active sbbetes m people tose work and li fe cum prise high physicsl actlvity or mental ressure The additional intake of h r lyzed collagen Is a valuahlo soume of one s dally nutrition . Aim, sol I agbn offers one a ltd I set of all aisino acids needed for the synthesis of collagen matrix in the human body. This is patficolarty i portant for active people, the elderly, pregnant and laetatlng women, people undergoing aesthetic surgery and others

In certain enibodi ments of the presen inventi n beverages are disclosed comprising functional ingredient hydrolyzed collagen in concentration; fro® 0,001 g/L to 8,44 g L, which may also be expressed as: from I ppm to 8440 ppm, This concentration of hydrolyaed collagen in the beverages shall be reviewe regarding ready for consumption beverages or regarding beverages obtained after dilution by adding additional water in the prescribed proportion to produced eoncentrates In the form of powder or liquids; The functional ingredient hydrolyesd collagen, also known as collage peptides, is produced through hydrolysis in controlled conditions of are materials such as collagen sources from various animal origin.

In certain embodiments, th disclosed beverage comprises hydrolyzed collagen at a concentration of abend 0,01- lg/1, about 0 J}f -2g i _s , about 0.01 ~3g/L about 0,01-4g/i, _: about 0,0! -Sgrt,, about 0,01~6g/I, abend 0.0J~?g i, about 0,01 ~fe/I, about 001 -Og l, about 0.0!~10g/l, about 0,01-f!g/I, about 0.01~!2grt, about (I.Oi-Og/l, about Q Ql-14g l, about 0.01-lSg/l, about 0,01~lig/l, about 0.01~ I 7g/l , about 0.01 1 g/!, ab out 0.01 -1 %/l or about 0.01 -20g/i , about 0.01 ~21 g/1, about0.0 ! -22g 1 , about 0.01 ~E3g/l, about ø ø l 24g/l, about O,0i-25g/f, , about O,01~26g/I ₅ abotd 001-27g/i, about 0,01 ~28g I, about 0,0 !-29g/l, about O.OT-iOg/l, about 0:01-3 lg/1, about 001~32g/l, about 0,01-33 /l, about 0/01- 34g/i, abO _: Ut0.01~35g/I, about 0.Ol~36g/1, about 0.0I~3?g/i, about 0,01 ~38g/k abou 0.0 I~39g i or about 0,01- 40gd or more.

In certain embodiments, the disclosed beverage comprises hydrolyzed collagen having a molecular weight of Ί-10QO0, 1-9000, 1-8060, 127000, 1-6000, 1-5000, 1-4000, 1-3000, 1-2000, 1- 1600 or 1-500. in certain embodiments, the disclosed beverage composes hydrolyzed ollagen havin a olecular weigh! between !OOOO-SOO, 10000-1000, 10000-2000, 10000-3000, 10000-4000, 10000- 5000, 10000-6000, 10000-7000, 10000-8000, 10000-0000 or 10000-9500.

INCORPORATED BY REFERENCE (RULE 20.6) Iis certain embodi ents, the disclosed beverage comprises hydrolyzed: collagen haying a molecular weight of about 50Q, about 1900, about 2000, about 3000, about 4000, about 5000, about 00, about 7000, about b D, about 9000 or about 10000.

.¾ certain embodiments, the disclosed beverage comprises hydrolyzed collagen at a concentration of at least Igd, at feast 2g/l, at feast ¾/f, at least 4gd, at least Sg/I, at least 6g/l, at least 7g/l, at least ¾, at feast 9gd, at least lOg/l, a least 1 lg/1, at least 12g/l, at least 13^1, at least 14g/i, at least 15gd, at least lbg/1, at least !Tgd, at least iSg/l, at leas 19g/l or at least 2itg/l

In certain embodiments, the disclose beverage comprises hydrolyzed collagen at a concentration of no more than about 20g/f.

Is certain embodiments, the disclosed beverage comprises 2000 Dalton hydrolyzed collagen at a concentratio of no more titan about 20g/l

The beverages containing hydrolyzed collagen characterize with great diversity, depending on f e other combined ingredients that they may include. Beverages may contain only hydrolyzed collagen and water.

In certain embodiments, the water for producing the beverages may be: tap water ®r spring or mineral water or water, gained by icebergs (glaciers). When tap water is used, th tap water shall mandatorilf be supplied: b central water supply system or extracted underground water: or extracte Irora natural or artificially constructed ponds. Also, preventively or in case of heed, the water can be additionally purified, or processed approved by th appropriate healthcare authority's methods of additional: purification or processing:

Examples of such methods are:

- mechanical filtration; through filtration systems,

- filtration through chemically active litem to separate the: unwanted Saits of iron (Fe) and manganese (Ma), decrease of total mineralization through ultrafiltration - using ullrafi!tration (reverse osmosis systems;

~ decrease of total mineralization through filirauon in loo exchange filter systems,

- decrease of total mineralization through distillation of water (evaporation and farther condensation),

- deconiaminadon by OWfilter (DV-ia ps),

INCORPORATED BY REFERENCE (RULE 20.6) - decontamluMion fey using chemical processing agents s$c¾ ay chlorine (CD), chlorine dioxide (CIO¾), ozone ({¾);, active oxygen (<¾} and others.

Also, water, used for the beverages ma be additionally carbonated through ad ing Carbon Dioxide - CCfc. The quantity of the Carbon Dioxide added may reach ?,Q volume units (7 _? 0v/v) in the most co mon beverages combination, it may usually ysry between 0,5 and 5,0 v/y in certain embodiments, beverages containing hydrolysed collagen May be produced by siuhiliaing preservatives. The preservative used shall be permitted for use ibr the category of beverages. Respectively, the preservatives may fee chemical substances (as Potassium Sorfeat f E2S2:}, Sodium benaoate (ESI 1] and others), which dissolute in the product and are nly parts of its content or may be chemically active substances, used In the production of the product but subsequentl dissolnte to other ingredients (such as Dimethyl loarbooate [B242])

In certain embodiments, beverages eontatning feydrolyeed. collagen may be produced without any preservatives as well as, their stabilization in those cases is ensured through applying appropriate iechuologicaJ production Methods.

Such methods are for example:

- pasteurization of tire product in combination wit a "clean Till" and subsequent storage at reifigerstioo conditions,

- pasteurisatio of the product by UffT-steri!ioation Ibliosved by aseptic filling (filling in sterile environment) of the product;

- pasi irixa i on of tire product followed by hot felling of ire product, filling of the product and further tunnel pasteririaafeon ensuring reaching commercial sterility of the product;

- pasteurization of the product and using special nanomaferiais when packaging the product in eoMfematlon with further storage at: refrigerator conditions.

In certain embodiments, I tire process of felling lbs beverages, regardless of whether the beverage is produeed by adding preservatives or with no added preservatives, packaging gases may ho used and added in the package prior to filling, doling the filling or after it. Packaging ga&ev are gases, diflhrent than air, and are Inert gases such as: Mitogen [E941 j, Carbon dioxide [E2fi0j, !de!iu o [E939] and others. These gases are added in the fellin process with the purpose of protecting the produe from adverse processes such as oxidation, change in taste, aroma and appearance. These gases

INCORPORATED BY REFERENCE (RULE 20.6) contribute to eimering the shelf life of the product w ile li ping to p eser e ihe quallty of the added ftutr ests in wtein embodiments,, beverages containing Ipdrolyzed collagen y fe colourless or e oered, clear or cloudy and may contain, a floe precipitate of larger particles m _. the botom anrhor to have unifor ly dispersed particl es in thelupi They may be calm flui s or carbonate! in certain embodiments, beverages containing hydrolyzed collagen that may fee colored, various eolopursMey fee added such as synthetic or organic ones from vegetable or animal orsghi or mm fee from various fruit and/or vegetable concentrates, vegetable extracts. Added colourants, classified as additives with an E-number may be permited for use In the beverage. The exac amount of coloring agent used will vary, depending on the agents used and the intensity desired in the finishe product. Generally, if utiMae , the coloring agent should be pesent at a level of fro about 0,1X101% to about 0,5%, from about 0.00154 to about . i%, or ihp about 0.003% to about 02%, by weight or volume of the composition, Certain formulations will have no added colorant.

In certain embodiments, beverages containing hydrolyzed collagen may fee produced i sweetened or sweetened through adding various sweeteners or mixtures of seve l ones.

In certain embodiments, sweetening the present beverage may fee achieved by adding sweeteners, most of will eh : are I ow-calori e ones, as well as bard: sweeteners ma be used such as crystals or granules. Examples lor sweeteners include:

* in a liquid form are the polyols; Sorbitol (E420), Xyiitol fE9fe?], Mannitol [B42I ], Maltltols fEtfeSJ and others,

• in a bas form are; Aspartame [BbSlf, Oyelapatss [11962], Neotame |Efeil|, Aeesu!ianm K JE9S0J and others.

In certain emfeodiments, sweetening beverages may b achieved by adding various types of sugars derived from natural sources. These sugars pay be respectively pon-reisned or refined to various extend. Examples of such sugars am: refined white sugar, unrefined or partly refined sugar,honey, fipctpe, glucose, giecosetrnetose syrups, syrups derived from fi'uits, plant extracts,

In certain embodiments, tip desired degree of sweetness of the present Inyeftion’s beverage may vary from , Ox-16,0 (bri units) or in cases when using artificial or namm! low calorie sweeteners, the desired sweetness intensity shall matc equivalences of "Sx^l:,O%&0 for ready 19

INCORPORATED BY REFERENCE (RULE 20.6) drink beverages, Clue krk unit ( ^¾ Bx) coHm mds to !!¾ water a ueous sugar so! ulon, i.e, 1,0 g sugar per 100 ml water.

In certain embodiments, for regulating tile aci ity and/or giving sour taste of beverages containing hydrolyzed collagen, fruit juices or fhdt juice concentrates m ay he added These ihmjuiees may ha e a sour taste, acidity regulating additives, or Bmimfemmdditives such as, Citric acid [B330], Phosphoric acid [£331], Phosphates m 1340; E343, E343], Citrates [E331; E33¾ E333] and others . The titratahle acidity of a preferred embodiments of the present i ayentiorf $: beverage containing hydrolyzed: collagen may he a value wilt» the range of front about 0,01: f$w/V to: about 0 5 %wf about OT ¾w/v to about 045 %w/v, 0,12 ¾w/v to about 0 35 %w/v or any ranges r single valueswithin these ranges.

In certain embodiments, for beverages containing: hydrolyzed collagen, other ingredients teay be adde such as: thickeners an / or stabilizers. Thickeners are substances which increase the viscosity of a foodstuff Stabilisers are substances which ale It possible to maintain the phy ico-ebe ical state of a foodstuff. tabil sers include substances which enable the maintenance of a homogenous dispersion of two or more imnuseible substances in a fbodsmtfi Substances: which stabilise retai or intensify an existing colour of a foodstuff an substances which increase the binding capacity of fee feed by the fetnstion of cross-inks between proteins. Exa pl for ingred ents used as thickeners ari stabilizers are the substances: Gum arable [E414], Guar gum [E412f, Carrageenan fE407}, _: Pectins _: |E440] and others.

In certain embodiments, for enhancing and modifying _: fee intensity of taste of fee ptesem invention's beverage containing hydrolyzed collagen, Ingredients such as flavour enhancers may he added. These are substaneea hich enhance the existing taste and/or odour ofa foodsiuff Such a?¾ l r example: Glutamic acid [E620], Guanylc acid [E¾:26} _; luosinie add E630] an others. For the purpose of adding flavour for taste, organic substances may fee added to the present Invention’s beverage. Such organic substances include, for example: table salt, extracts and iiuei Ps of plants, bulk seeds, yeast extract and others.

In certain embodiments, fruit an vegetable juices, fitfe and vegetable puree, concentrates from fruit and vegetables: as well as extracts or tinctures of fruit, part of final or plants may all be added to the present inventlojfs hydr y&ed collagen beverage or it may contain such substances. The diversity of this category of additives is high as it is conditioned on the natural diversity of plants. The juices, purees, concentrates or extracts that ma he added to the present Invention’s beverage may be dear of cloudy, contain particles of fruit, vegetables or plants aa seeds, parts of the peel, pulp, cells and, so on

INCORPORATED BY REFERENCE (RULE 20.6) exanipie of clear juices that « he a ed fa the present Inventlon i beverage mac be Juices made from: apples, cherries, raspberries· _» strawberries, lemon, orange, grapefruit etc. Cloudy juices and popes can h© produced from frails; apple, gpu cherry, raspberry, le ony orange, plu , pear, apricot, peach , etc. In certain embo iments, the beverage containing hydr yaed collagen disclosed here may optionally include one or more juices (e g,, one or more of sioglewtrength fruit, berry, or vegetable juice, well as extracts, concentrates, purees, milks, and other forms) prasent at a level foom about 0 00354 to about § ,10%, about 0001% to about 20%, about 0905% to about 15%, about 0.01 % to about 10 , about 0:95% to about 5%, about 0.91 % to about 10%, abou 0.05 to about S%, _: or about 0,1% to about 2.5% by weight of the beverage, or at a level of about 0001%, 0.002S%, 0 005%, 00075%, 0,01%, 002%, 0.03%, 0.04%, 0 05%, 0.00%, 007%, §.08%, _: 0.99%, 0 1%, 0,2%, 0.3%, 0,4%, 0 5%, 0 6%, 0.7%, 0.8%, 0,9%, 1.0%, 2.9%, 3 054, 49%, 505%, 0.0%, 7.0%, 8.0%, 9.0%, 10 11.0%, 12.0%, 13.0%, 149%, !S9%, 100%, 179¾¾, 1805%, 199%, 29.0%, 21.0%, 22.0%, 23.0%, 24.0%, 25.05%, 26.0%, 279%, 28.0%, 299%, 30.0% _: , 31.0%, 32.0%, 33,954, 349%, 359%, 36.0%, 37.0%, 38.0%, 39.0%, 40.0%, 41.0%, 429%, 439%, 44.0%, 45.0%, 469%, 47.0%, 48.0%, 499%,

50 0%, 51.0%, 529%, 53 %, 549%, 55.0%, 569%, 57.0%, SS 0%, 59.0%, 609%, 61 0%, 62 0%,

63.0%, 64.0%, 65.0%, 66.0%, 07:0%, 689%, 699%, 70.0%, 7LD%, 72.0%, 739%, 74,0%, 759%,

76.9%, 77,054, 789%, 79 ø%, 89.0%, 81.0%, 92.0%, 83.054, 84.0%, 85.954, 869%, 8705 88.056,

899%, 90.0%, 91.0%, 92.0%, 939%, 94 94 95.0%, 96.0%, 979%, 98.054, 999 or about 99.16%, about by wei ght of the beverage, or any ranges or single val ues within these ranges. Typically, juie can be used, if at a! I , I a an amount _: of front about 0.001 % to about 45% fey wei ght . Fruit and vegetab! e juices, felt and vegetable puree, concentrates fro iislt gsd vegetables as well as extracts or tinctures of fruit, pari of fruit or plants ma be mixed in all possible ©oafomabeos with each other and then added to the present invention's hydrolyaed collagen beverage

In certain embodiments, for flavouring the present inveniiorfs beverage, aromas as well as ingredients with flavouring properties may be added. The beverage containin !wdr yced cel!agen disclosed here may optionally contain one or more flavourings pr Savour compositions. For example, natural and/or sy nthetic fruit flavours, botanical flavours, other flavours, and mixtures thereof Added: concentrated aromas may fee extracted _: front natural sources as: fruit, food, parts of plants, smoke and so on but may also consist of synthetic flavourings, approved for use with foodstuff For flavouring foe present invention/ beverage, ingredients with flavouring properties such as herbs and spices extracts, fruit extracts and fruit concentrates with Intense aroma may be used.

INCORPORATED BY REFERENCE (RULE 20.6) In certain embodiments, the present i wntionV hydrolysed collagen beverag disclose here may optionally include doe or more flavourings present at a level from about Q.0005% to d it 5%, about 0,001% to admit 4%, about 0,005% to about 3%, ad it 0,01% to admit about 0.05% to about 1%,, dr about 0,1% to about O.Sbf by weight of the beverage, or at a level of about 0.001%, 0.0025%, 0.005%. .0075%, 0.0!%. 0.02%, 0,03%, 0,04%, 0.05%, 0.06%, 0,07%, 0.08%, 0.09%, 0.1%, 0,2%, 0.3%, 0.4%, 0.5%, 0.6%, 0,7%, 0J%, 0.0% or about !.ø% by weight of the beverage, or any ranges or single values within these ranges.

In certaiioembodiments, yitoniBS and minerals wife the purpose of enriehing their nutritional value may be added to the present invention’s beverage. Vitamins include, but are not limited to. Vitamin A, Vitamin D, Vitamin E, Vitamin K, Vitamin C, Thiamin (Vitamin Br) _> RiboEgvin (Vitamin By), Niacin (Vitamin By), Vitamin B« _* Foflc/seid (Vitamin By), Vitamin BJJ, Biotin (Vitamin B% Fautoibe ne acid (Vitamin Ids). Minera!sindude, hid are not limite to, Potassium (!£}, Chloride (Cl), Cal eium(€&), Phosphorus (P¾ Magnesium (Mg), Iron (Fe), Zinc a), Copper (Cu), Manganese (Mo), Fluoride ;( ), Selenium (Set Chromium (Cr). Molybdenum (Mo) iodine (I).

In certain e hoiiitnents, supplements areiygleally present amounts generally accepte unde good m& sfeotu ing practices and are typically present in amounts between about 7,5% to about 100% RDM, where such MW are established, But may present at a level from about 101% to about 150% BDV,,abouf 120*12 to shout 200% RE>¥, about 150% to about 300% RDV, about 200% fo about 500% RDV.

In certain embodiments, vitamins and minerals are used to enrich the beverage nutritional profile. For exa pte, according to legislative food sources, a goo source of Vitamin A can be found In substances such as; retinol, refety! acetate, retinyi palmitate, beta~earoteoe, As Magnesium (Mg sourees used may he; magnesium carbonate, magnesium chloride, magnesium gluconate and etc, may he added to the present invention ^' s beverage.

In certain embodiments, emfehffieni of beverages with vitamins and minerals may be one with organic sources of vitamins and mineral s. For example, such vl tatnlns are; acero!a extracts, rose hips extracts and others, as orgartic sources of minerals use may he: organic concsotrales by seawater, seaweed, eggshell extracts and others The use of vitamins and minerals may vary to 100% and mor than 100%, according to established Recommended Daily Value (MW) for vitamins _: and minerals.

INCORPORATED BY REFERENCE (RULE 20.6) In certain embodiments, the beverag containing hydrolyzed collegers mag he produced by adding appropriate electrolytes, often categorized s specialize beverage category, seed as: kafeafrc sport beve ages, hypertonic sport beverages, s» hypotonic sped beve ages, in certain embodiments;, additional aoiino adds ay be added ter the preseat invention’ s beverage. Additional enrichment wi h amino acids y he done by udcJtog amino adds is their L- for . For example, L-giotamine, Lfrsoleucine, Lfreoeioe, L~vaii¾e, Lrtaur e and others: may b added to the present invention’s beverage. Additionally, amino acids ma be added as separate amino adds as well as in eoeth atton to other amino adds.

In Pertain embodiments, other types of proteins, different than collagen may be a ed to the present invention^ hydrolyzed collagen containing beverage. These proteins may he hydrolyzed proteins with higher or lower molecular wdglrt: For additional enrichment oilier proteins may be added to toe present invention, such as proteins from vegetable as well as animal origin proteins. For example, proteins from soy, peas, milk, egg and others may be added to the present in entions beverage

In certain embodiments, the beverage of hydrolyzed collagen frees not contain whey protei n.

In certain embodiments, the beverage of hydrolyzed collagen does not contain sodium aci sulphate.

I certain embodiments, tbe beverage of hydrolyzed collagen does not contain citric acid, phosphoric add, malic acid, tartaric acid or torn an c add.

In certain embodiments, where other types of proteins and / or protein hydroly sates, other than collagen are added, the total concentration of binding _: proteins in tbe beverage should be varied, from 0.001 g/1 g/L to 844 g/ ^' L, which may also he expresse as: I ppm to 8440 ppm. In these embodiments, the total protein ooneentrattoa in the beverage is th sum of all ptotein sources conta ned in th beverages, inctodirsg the added hydrolyzed collagen.

Alternative embodiments of the present invention’s beverage may be produced which do not contain lactose but may also include lactos as an indi vidual ingredient or have added compositions which arc sources of 1 actose.

In certain embodiments, the present invention’s beverage may include additional added ingre ients, such as rimy con wtomb which can be different fractions derived front milk For example: whey proteins, casein, caseinates, etc.) or fractions derived from the colostrum (for example:

INCORPORATED BY REFERENCE (RULE 20.6) antibodies, immunoglobulin, growth factors), Staled eity components include, but ere not limited to: itpid proteins (casein, lastogiobaiin}, antibodies, minerals and salts (phosphates, calcium, magnesium, sodium, potassium, etc,), Vitamins (A _* ¾ _.· fi _,i¾5 D, K, B, thi&mlne, nladn, biotin, pautothenk acid, etc,), c Tboby f les (lactose), enzymes and others.

In eertai n em bo l m ents, the present i nvemion s beverage may be produced with other additives snob ax; fresh milk, reconstitutedmilk or milk fomented ith lactic ad cultures so accordingly each of the following types of milk can be skimmed or whol to various extend, Fermented milk is usually derived from using the following starters; Strefnoeoccus fherroophilus « Lactobacillus detbrueckil suhsp, feulgarieiis, as well as various starters mostly from the Lactobacillus, Lactoeoceus, Bill dobacteri am and Sir; Thermophil us kinds. During fermentation probiotic cultures may be added. Pre-pregared probiotic: conoeowafes (probiotics) ma also o added as individual ingredients to the present invention’s beverage. bs present invention’s beverage containing hydrolyzed collagen ma also contain added prehiorie ingredients which are fibers which can be soluble as wei! as insoluble.

In certain embodiments, variou other Ingredients with beneficial properties: may be added to tbe present invention’ s beverage containing hydrolyze collagen inch Ingredients may add value to the present inventions beverage containing hydrolyzed collagen, All these additional health ingredients may vary in different combinations as may be Individually added to beverages containi g: hydrolyzed collagen or be combined. Examples for such healthy ingredients are:

- L-carniline,

- Coenzyme QK),

Omega 3 and Omega 6 essential fatty acids,

- Bee products, such as natural honey, bee pollen, royal jelly, propolis,

Vegetable extracts from plant with healthy effects that may: Include various pails of plants as; fruit, blossoms, peels, roots, stables. For example, plants with health effects are; Fanax Ginseng, C’amo iia, Lavender, Bleitsria cardamomum, Glnfcgp biloba and others,

- Tea extracts (black, green, white etc.)

- Soy isoftsvoaes, - Hyaluronic acid and/or its salts,

Alpha lipoi c Acid,

- Extracts of seaweed as wel I as purified finely ground seaweed,

- Mushrooms extracts, edible and special types such as: Letriimiia edodes, Orifbla Iro oss, Cordyceps Sinensis, Mericium etinaceus, Coprinus contains and others.

INCORPORATED BY REFERENCE (RULE 20.6) - Extraets of polyphenols . resveratr , astaxanthiu,

- Carotenoids, iododing lutein and xeaxuniMn,

- Green coffee extracts,

- Gai!e e.

As shown in FI€¾, 3 and 4, the optimal concentration for the absorption of a 10,000 Dalton collagen hydrolysate as 8.44 g/1 with or without whey protein in contrast, the optimal concentration for the absorption of ,000 Dalton collagen hydrolysate was 20g/I if the whey: protein was removed and replaced with additives, including a vitamin package as defined herein and/or CCfe end/or pn hyp/hyp¾% In all instances tested, the % absorption of the 2000 Dalton collagen hydrolysate decreased at condentrafions greater than 20g/l .

EXA FLEI QF FEEFEMED EMBODIMENTS:

EXAMPLE I

EXAMPLE 1 (as shown in TABLE f) eotnprises a oloudy, eo!oafed boveia e contsinisg liy rol aesl eollagem The ingredients expressed i n weight percentage sre listed belo in Table I. Used Ingredients are as follows: water psriEed y «ItraEJifatios teehsslogy. Hydrolyzed bovine collagen Feptan B 5O0OHD (femsseiQt lac ), crystalline fructose, peach aroma, citric acid jE330}, antioxidant: ascorbic add, acidity regulator: sodium citrates [E33I j, _: apowder ix of wtamlos and minerals: D-peutotbeoate calcium; Pyrldpxine hydrochloride; Bydtoxoeobalsmin, D--biotin; Calciu gluconate; Magnesium gluconate; Zinc chloride, RDM levels: Pantothenic acid - 15%; Vitamin B _& ~ 15%; Vitamin B« - 15%: Biotin ~ !S¾; €a - 1S%; g ~ 1S%; ¾i - 13%;), clear concen rated peach juice, carrageenan |E407], guar gunt |B l¾ colour: car tenes _: The beverage Is aseptically filled into plastic disposable bottles 37Sm! (12.680 S, ox). it is sterilized in lIUT-sterilizer st: 13?* € for 4.2 seconds. It Iras a shelf life of 12 onths when stored at 2S ^W €, after opening shall be kept in the refrigerator up to 48 boors.

TABLE !

INCORPORATED BY REFERENCE (RULE 20.6)

EXAMPLE 2

Exa ple: 2 (as sho n ip Tabl 2) comprises a e oar, colourless beverage containing hydrolyzed collage». The ingredients, expressed in weight percentages, are listed below- I» Table 2, Used ingredients are as follows: water purified % ultrafiltration technology. Hydrolyzed bovine collagen _: Feptan B 5000HU (Rousse!ot nc.). The beverage: is aseptically filled Into plastic disposable botties _: 3?SmI (12.680 II. oz). fi ls sterilized in UHT-sferifizer at 13T C for 4.2 seconds. It has a shelf life of ! 2 months when store at 2S° C, after opening shall be kept in the: refrigerator up to 48 boors.

TABLE 2:

EXAM PLE 3

EXAMPLE 3 I as shown m Table 3 comprises ¾n opaque, coloured beverage, eomaiplng hydrolyzed collagen. Tbe compeslfeha, expressed in weight percentages, are listed belo In Table , Used Inpudle s are as fellows: water purified by e¾r¾f traao» technology, Hydrolysed fish collagen PeptanT) F 2C)O01-ID (Kemaselof Inc ), crystalline fructose, white crystal sugar, natural peach aroma, natural mango aroma, natural guava aro a _¾ amidaled pectin |E44 ], citric sold [E330], &ntiosddaBi

INCORPORATED BY REFERENCE (RULE 20.6) ascorbic eld [E3O0], acidity regulator: calcium lactate [B327], peach puree sin le strength, concentrate mango puree, colour: Ribofkvina [BlOl]. The beverage Is asept aliy filled info disposable piastre bottles 37SiBi _: (i2 80 ll oe) It is botaegea e at 150 bar sa sterilised in OHT- s!eriliaer at 137 C for 4.2 seconds, It has a shelf life: of 12 months when stored at 23° €„ alter opening shall be kept la the refrigerator up to 48 hours.

TABLE 3

EXAMPLE 4

EXAMPLE 4 as shown in Table 4} comprises a transparent coloured beverage coats) pi ng ¾yfro!y;aed e lagem Ingredients expressed is we) gilt percentage, _: are listed below la Table 4. Use Ingredients ate as follows: water purified by uitraSibailoa technology, Bydrof eed fish collagen Pepfa«€ F 2600BD (Roasseiot Ihe crystalline irueiose, brown sugar, natural strawberry aroma, natural lime aroma, natural dry green tea extract, eliri:e ackt|E330j, acidity regulator: sodium citrates, clear strawberry juice concentrate, colour chlorophyll ins (EMI], The beverage is aseghealiy filled into plastic disposable bottles 375ml (12.680 fi end It is sterilized in: lIMT-sterlliacf at 137° € for 42

INCORPORATED BY REFERENCE (RULE 20.6) seconds. 1 has a shelf life of 12 months when stored at 25 C, after opening shall be kepi in the refrigerator up to 48 b ors.

TABLE 4

I

EXAM LE 5

EXAMPLE 5 (as sho n in Table 5) comprises an opaefue, col cured beverage containing hydrolyzed collagen ¾ ingredients, expressed in weight percentages, are listed below In Table 5: Used ingredients are as follows: water poo Had b aiftafiltrstioo technology, Hydrolyzed bovine collagen Pepiafei B SOfifiHD fEousselot Inc.), so protein isolat SIIBEO® FLU 2640 DS (DAN!SCP), natural mango aro a, natural ripe apple aroma, amidated pectin (E440Ii J, brown cane sugar, citric acid [H 30J, anflr tdaof; ascorbic acid fEBOO], acidity regulator: calcium lactate fB322], apple puree single strength, saffron extract. The beverage is aseptieally filled into disposable plastic bottles 3T5«d (I2T80 fl, ox). if is homogenized at I50 bar and stertl!aed in XJBT-storil er at !37 ^¾ for 4.2 seconds. I has a shelf life of 12 months when stored at 25* C, after opening shall be kept in the refrigerator up io 41 hours.

TABLE S

INCORPORATED BY REFERENCE (RULE 20.6)

EXAMPLE 6

A preferred alternative etab¾ ifM»t of the pr sent invention's beverage comprises a dear, colourless beverage containing hydrolyaed collagen, with vitamins and annemls as shown in Tafele 6.

TABLE 6

INCORPORATED BY REFERENCE (RULE 20.6) * ~ Vitamin Premia Matenal-N IJF40105368; produced by I3S Xumiilona! Products Europe Lid lie Vitamin Prsmin to covers 15t¾ EDA / 100ml of: Vitatoio B 12, Mg and 2n

EXAMPLE 7

Au alternative preferred embodiment of the beverage comprises a of sg colourless beverage ^: eet¾si««\g riydroi eed collagen, with vitamins anti minerals as sbown id Table ?:

TABLE ?

* - Vitamin EmmixMaierisI-Mo. 0740105368:; produced by DSM Nutritional Products Europe lid. Trie VitarnM Premia to covers 15% EDA oT Vitamin B 12, Mg and Zm

EXAMPLE 8

An alternative preferred e riodlmerd of trie everage comprises a dear, colourless beverage containing hydrolysed collagen, with vitamins and minerals as shown ia Table 8,

TABLE S

INCORPORATED BY REFERENCE (RULE 20.6)

* ~ Wimm Pffcntx Msierial-NG. 1JF40107368; prodoeed by SM Nutritional ProduGs Europe Ltd. The Elfegrin Pre ia to covers 15% M)A of; Vitamin B12, Vita in B? and Zn

E AMPLE 9

Aft alternative preferred embodiment of ids beverage comprises a ©leap colourless beverage coMafekg!iydrolyaed and mineral wafer as show© in Table 9.

~ Bieafeor te ®€0:r5rmg/i: 270;0;

- Chloride (O mg1; 460;

- Sulphate ISOs · ). g/i : 65.80;

- dry residue at ISO ^:5 € _s mg/h 340.3

INCORPORATED BY REFERENCE (RULE 20.6) EXAMPLE 10

RXBMPLARY METHOD OF PREPARATION:

Collagen powder is ra ed with heated water until dlssciutiou. After eosi l eti n of a filtration step, the concentrated solution is transferred to the second mixing container cohere other ingredient such m flavorings, vitamins, minerals etc ate added followed by ultra hig fcmper tnre (OUT) sterilisation, quality control an packaging is sterilised bottles

EXAMPLE I I

A hydrolysed collagen beverage with hydrol zed collages as the sole protein (no other proteins present in the beverage) presents superior human gastrointestinal absorption of collagen when compared to similarly formulated collagen beverages which include other sources of protein due to the absence of receptor competition By contrast, the inclusion of one or more additional sources of protein in an otherwise Identical hydrolyzed collagen beverage will have reduced efficacy of collagen absorption pe; to receptor competition among the different protein types.

As shown in Fig. 1 A, In the human gastrointestinal traet (GIT), food proteins can be digeste Into enormous numbers of free amino acid (A As) and peptides by various digestive enzymes or microbial fermentation. Some of these peptides containing 2---20 AA8 have various biological activities and are named htoaeilve peptides (BAPs). (1) Small peptides can: be transported vi peptide transporter I (FcpTl)(2)

Foo protein in the lumen Is hydrolysed to peptides by proteases In the brush-border membrane of the small Intestine. The peptides may b absorbed (i) via transport across the intestinal epithelial cell monolayers throug carrier-medtated routes; (is), via the paracellular route: via tight junctions; (ill) by iraoseytosis via vesicles; an (tv) via passive ftanseelhriar di fusion. For PepTl- mediated: trans ort _s peptides are coupled wi h protons (BY), which are then transporte out of the enteroeyies by ITBffiaf exchangers, Inside the ceils and bloodstream, the peptides ma be -partly hydrolyzed to a ipo acids by peptidases (Adapted from previous reports 0, 4, 5)

Thousands of di- and tripeptides are taken ¹ up into enteroeytes via FepTl, which is mainly distributed in the intestinal brash -border membrane. FepTl deficiency in mammals impairs nutrient absorption and causes ma! absorption that becomes visible on high protein and high fat feeding (iff As mentioned above, FepTl can transport various sraaifRAPs in udiug Pro-Gyp (FO). Collagen-derived

INCORPORATED BY REFERENCE (RULE 20.6) G!y-Ihw-Fiyp (CrPO) is hydrolyeed by imes uiil ucosal a ical proteases and lire resulting i e ii e PCI is transported across the intestinal niembrarse via PepTl {?), Shimizu e? al (1) demonstrated t at collagen peptides (GAXGLXGF) are transported by energy-independent passive difluslon.

As shown in- Figs. 1B _: and 1C, at low BAP, concentrations (AErn) PepTl is the major eontri fetor to the total transport rate. However, as: the BAP eoncentraboR increases, tbe contribution of passive transport becomes more apparent. Because Peg! 1 -mediated transport is saturated at Vws* all further increases in tbe total transport rate are attributable to passive transport.

Accordingly, it may be concluded that tile presence of other (non-collagen) low molecular weight peptides in a hydrolyzed collagen: beverage would competi tively and adversely reduce the rate and efficiency of absorption of the collagen peptides that are present

Cited references _:

:1 , Xu, Q„ Hong, !i, Wu, I., $ Yen, X. (2019). Bioavall ability of h Inactive peptides derived §wm food proteins across the intestinal epithelial membrane; A review. Trends in food science technology.

2. Daniel, Fi- (2004). Molecular and. integrative physiolog of intestinal peptide transport. Anna. Rev Physiol., 66, 361-3:84.

3. Gilbert, E. R., Wong, E. A,, & Webb 3r, K. E (2008) Board-invited review: pepti e absorption and utilization: implications for animal nutrition and health Journal of animal science, 8:6(9), 2135-2155.

4. Wada, Y„ A: Loennerdal,: B. (2014). Bloeetlve peptides derived Bom huma milk proteins— mechanisms of action The journal of nu tri tional biochemistry, 25 51, 503-514.

5 Xu, Q., Fan, H., ¾ ff, Fiqsg, H. _s & Wu, 3. (201:7). Transport study of egg-derived and hyperten ive peptides (Ikp and iqw) using caeo-2 and b|29 coeulture mon ayers. Journal of agricultural and oo chemistry. 65(34), 7406-7414

6 Daniel, FI., & Eietek, T. (2015) Taste and move: glucose and peptide transporters in the gastrointestinal tract. Experimental physiology, 100(12), 1441-1450,

7, Alio, inone, M. lamkcyrirm, B, Fan, M. Z., Sato, R., & Mine, Y. (200?). Transport of a iripeptlde, Gly-FroAIyp, across the porcine iotesdn i brush-borde membrane. Journal of peptide science: an official publication of the: European Peptide: Soc ety , 13(7), 468-474

INCORPORATED BY REFERENCE (RULE 20.6) 8, Shimizu, K., Safo, M. _s Zhang, ¥„ Kouguchi, T,, lakahaia, ¥„ Morlmatsu, F,, 22 Shimizu, Ml (2010). 1¾e biosvallahle octapepdde Oly-iylaHi pTli -Leu-H p-Giy-Pro stimulates nitric oxide: synthesis in vascular endothelial cells. Journal of agdoultuml and food chemistry , 58(1 !),

6960-4905,

EXAMPLE 12

A peptide weig t of 2,000 Bal o s the optimal level for c llagen peptides in /hydrolyzed collagen beverage. Cienera!ly smaller peptides are superior for absorption of hydrolyzed collagen because less energy required !¾' thi eeefiy-slepeaderil process. However, 2,000 Ha Is at or very near the optimal weight because the energy abso ption sa ings achieved by fedher reducing peptide weight significantly below 2,000 Da would be minimal

Hou et at, demonstrated that collagen polypeptides had good moisture absorption and retention propert es, and collage polypeptide fractions €1 2 (Mr < 2 kDa| was better than collagen polypeptide fractions CP I (2 IcDa < Mr < 6 kPa), Ultraviolet radiation could cause skin plmtodamage and collagen polypeptide couk! aileviate the damage induced by U ^' V radabori. The action mechanisms of collagen polypeptide mainly Involved enhancing immunity, reducing: the loss of moi sture and lipid, promotin anii-oxidative: properties, inhibiting th increase of glyeosa inoglyeans, repairin endogenous: collagen and elastic protein fibres, and maintaining the ratio of type III to type I collagen (I)

The use of collagen hydrolysates (€%) as a aniraeeutlo&f agent i skin aging has gained: increasing attention. Here, the eilems of various doses and moie nlar weights of €H from Silver casp skin on photoaging in mice were investigated:. The ingestion of CH at SO, 100 and 200 g per !feg b y weight led to a dose-dependent inmease in the bydmwypfoSine, hyaluronic aci and moistur contents ofthe shin, hut it had no significant effect on dm mice body weight, or on the spleen or thymus index. Furthermore, ingesting €11 wi h lower (LMCH, 200-1000 Da, 6S¾¾ and higher molecular weight (HMCM, > 1000 Da, 2286) significastiy increased the skin components and improved the amioyidatl ve enzyme activities in both serum and skin (p < 0.05); IJVIGH performe better than HMCB. By contrast, gelatin (>120 hf>&) ingestion did not bring a significant change compared to model mice. These results indicated tha LMQT exerted a stronger beneficial effect on the skis than did either HMCH and gelatin, which supported the feasibility of using LMCH as a dietary supplement from sliver carp skin to combat photoaging. (2), The UPs f2O0 Da), used i the H Song et al, study , ha been demonstrated to have a stronger beneficial effect oil the aging skin than other collagen; hydrolysates (including gelatin an CPs wi th higher molecular weigh >1 00 Dal (2,3),

INCORPORATED BY REFERENCE (RULE 20.6) E Proksc at at. conducted a «lonoeenirk, doabliddinded, randmnixed, ptaceho-oontroiled supplementation study on the effects of a specific CH w th molecular weigh! of 2.0 kBa. The results: clearly revealed that oral intake of i e specific €H ha a besefiel l effect on skin physio ogy, as indicated by Increased skid elasticity af er weeks of daily consumption, The observed effect was statistically significant: (p< 0,05) afterd d S weeksta the tFeat!scotgroyps compared to placebo. (4)

In a placebo controlled clinical trial I Asserin et al found that oral supplementation with Ush eeflagen peptides (Pep¾n®f molecular weight of 2000 Da.) in women Increased the collagen density in the dermis by 9%, as titeasisre by high frequenc ultrasound; highly effi aciously increased the amount of water-binding glyeosamipogfyeans, the collagen content of human skin improved skin hydration as well as the density and stiueture of the collagen network of the derails if)

Some n vitro studies have demonstrated that Pro-Hyp an Hyp-Gly exert che otaxis on dermal fibroblasts and enhance ceil prohfe tloo Additionally, Pro-Hyp enhances the production of hyaluronic acid! by dermal fibroblasts These Endings suggest that the amounts of Pro-Hyp and Hyp- Gly in blood are important factors to show the efficacy of collagen hydrolysates on: skin health, Som study demonstrated that the; use o the collagen hydrolysate with ahigher content of Pro-Hyp and _: Hyp- Gly led to more improvement in facial skin conditions. Including banal skin moisture, elasticity, wrinkles an roughness fh)

Studies hy N okl et al suggest the possibility for the use of collagen peptide and ornithine to improve skin conditions by increasing plasma IGF-! levels - the used weight of the collagen peptide was 2001) Pa, (?)

All studies with mice showed no significant difference I final bod weight between the compari son groups, There are some studies on the effeetivehess of lower molecular weight collage peptides, hut the most studied an confirmed effect on skin relates to collagen is weighing 2000 Da Accordingly, It is logical to conclude that 2,060 Da is at or very near the optimal peptide weight hv respect of a beverage of simple hydrolyze collagen and water without additives or supplements because tits energy absorption savings achieved by further reducing peptide weight significantly below 2,600 Da wou be minimal

In addition, it is logical to consider the possibility to concentrating collagen with a higher content of Pro-Hyp an Hyp-Gly. A higher content of Pro-Hy and Hyp-Gly could be expected to result in ayueasurahie inerease in the optimal peptide weight.

INCORPORATED BY REFERENCE (RULE 20.6) Cited References

1, Bern, H , , B., Zhang, X., Xm, Yu, Ό., Wang,!, ... & Su, S (2012), Moisture absorption and retention properties, and acti vity in alleviating s n pboto amage of collagen polypeptide from: mar ne f h sk in Fix ckmiMrv M3bc 1432-4439.

2, Song H, eng M, Cheag X, elat The effect of collagen by droty sales from silver carp

^ypophiMmickihys skin on UV-inclneed photoagiag in mice: molecular weight affects skin rep ir, Food Funci. 2017; 8; 1538-46.

3, Song, FI., Zhang, L., L·»©, ¥., Zhang, S., 14, B, (2018). Effects of collagen peptides intake on sldn ageing and platelet release in chronologl cal l aged mice revealed by eytokine array analysis ,/, C A Med, 22(1% 277-288.

4, Pfdkscfe, :£., Segger, D., Deg ert, I., Sehnnek, M., Zsgug V,, &. Oesser, S. (20:14). Ora!

Suppl era eolation of Specific Collagen Pepli es Fins Beneficial Effects on Human Ski Physiology; A Double-Blind, Placebo-Controlled Study. Skm Pharm M Plt ioi 27, 47-55.

5, Assents, |„ Lath E,, Shioya, T., & Prawitt, 1. (2015), The eSee! of oral collagen peptide supplementation on shift moisture and the dernml collages network: evidence from an ev vivo model an ran o ize . plueehoosontrolled clinical trials. J rmd af ima ucBam olagi?,

14(4), m -301. - Inmuy K, _? Suglhara, P. k Wang, X, (2016) Ingestion of Moaeiive collagen hydrolysates enhance facial skin mo sture _: an elasticity and reduce fecial ageing signs In a randornke doubicfeSlnd piaeebo-Oontrolied clinical study . Jmmai Mike S&imm >ff Oo mttl Agrimilme, 96(12), 4077-4011.

7, lie, N., Se d, S. _s & Ueda, F. 12018), Effects of Cmnposite Supplement Containing Collagen Peptide and Ornithine on Skin Conditions and Plasma. IGF-I Levels — A Ran o ized, Double-Blind, Placebo-Controlled Trial Marine rJrgp, (!2), 482.

EXAMPLE 13

CARBOMATIOM IMPROVES ABSORPTION Of A FIVDROLVZEO COLLAGEN BEVERAGE.

Introducing €02 to hydrolyzed collagen beverage: can materially increase eoMa ca abortion by slowing the natural increase the _: pH level in the gastroi testinal s ste during the digestive; process.

INCORPORATED BY REFERENCE (RULE 20.6) Typical carbonated beverages s ch as ordinary soda water and Coeaffioia eraualn approximately 6.2 grams/liter of €02 on average.

GaHrotnicsfloal (01) digestion of a dietar protein is inflnenced by the physicochemical characteristics of the protein A complex; combination of mechanical, physicochemical, and physiological processes is involved in the CH digestion of proteins in humans, Digestion of dietary protein In the gastrointestinal system involves stomach and intestinal proteolytic eneymes. li protein digestion begins In the stomach, where pepsin breaks down the protein into smaller peptides Pepsin exhibits maximum activity at acidic pH 20 and is inactivated at pH 6,S in the duodenum, The pH in the duodenum is gradually increased to 5 -7.5, due to the secretio of bicarbonate and pancreatic juices. The Increased pH inacti vates the gastri c enevmes and gives: the optima! activi ty of the duodenal euxymes (trypsio hym rypsin). When the semidigested peptide mix roaches the dtrobenom _:> the Intcstina] enaymes continue to break d own the pepti des. The final stage of digestion of proteins occurs on the surface of intestinal enterocyies by brush border enaymes, wbefe peptides are bydrolyced to amino acids as well as di- an tripeptidcs. The nutrients are then: absorbed by the enteroeytes of the; jejunum and ileum and can be further degraded by intracellular proteases before entering th blood stream (1 )

At standard te peralnre an pressures, about 1ml (2mg) of carbon: dioxide dissolves In I ml of neotral solution (gH ^:::: 7}. In alkaline solutions, the gas is converted Into carbonates and Mcarbonaies, aud it Is in this form that any ingested carbon diexldels likely to be present in the intestinal tract. In general, one can assume that the daily average i make of carbon dioxide in food is _{: g} cfulvaieui to approximately ig daily of sodium b carbonate or carbonate and therefore represents only a very small fraction of the amounts of these compounds in normal diets (2), I the past, several studies demonstrated that _: carbon dioxide plays a major role in the process of hydrochloric acid formation an secretion in the parietal cells of the gastric mucos , after drinking carbonate beverages some of the carbon dioxide may be absorbe through the gastric wall, partly contributing, together with the carbon dioxide from the interstitial fluid and plasma, to the formation of hydrochloric acid. (3)

Ho evidence has been found that the addition of carbon dioxide to the GI while collagen peptides arc present af!ccis the peptides molecular weight However, a hydreiyced collagen beverage to which earfednatlon has been included to a level of 6:2 g/1 can be expected to materially slow the norma! increase in pH in the GI during the absorption process. Accordingly, the introduction; of €(¾ at 62 g/1 will slgnlricaMly extend the ti e period during which the pH level In the GC remains below 65 pH, thereby increasing the overall efficacy and rate of absorbtion of collage peptides in the Gl

INCORPORATED BY REFERENCE (RULE 20.6) Cited References

1 Tapal, A-, Tiku, P !L (2019). Nubitlor l a Nutraceutleal I provement fey Emwn lie

Modification of Food Proteins la in Pood Biotechnology (pp. 471-4S1). Academic

Press

2 Select committee on GRAS Substances Evaluation of the health aspects of carbonates and biearbonates as food ingredients (8CQ6S-26 Life Belences Research Office, Federation of A erican Societies for Experimental Biology, Bcthesda (1925)

3 Cuonio, R., Samel!!, G., Savarese, M F„ & Buyebx, M. (20t ⁾ 9). Carbonated beverages and gastreinre inal s atenr: between myth an realit Nutrition,. Metabolism arid Cardiovascular Di seases, 19(10), 6S3-4B9:

EXAMPLE 19

USING PLANT-BASED COLLAGEN

Osage of a plan tffiased collagen source can fee exp cte to provide certain health benefits However, there is cot evidence that plant-based collagen course (m opposed to anisnai -based) improves th : absorption of collagen is the gastroiniesh a al system:

Gorsssen et al observed that plant-based proteins have relatively low essential amino acid arid leucine contents when compared with anl af-based proteins and Ir nan skeletal mu scle pwkem. is addition, some bet not all plant-based protein isolates are low in lysine and/of methionine contents. As there is a large variability In amino acid composition among the various plant-based protein sources, & balanced combination of different plant-based proteins may provide s lilghercjuailty protein blend. (I ) Plant-based diets are known to (!) improved plasms lipid concentrations. {¾ reduce blood pressure, and (i it ) as part of a lifestyle intervention, the regression of atherosclerotic lesions. (2, 3} At the: same time, Vliet et al provi ed evidence that some plant proteins: produce a lower muscle protein synthetic response compared to ani al -based proteins (4) in conclusion, therels evidence of heal th benefi ts front consumption of plant-based proteins including collagen, However, there is no evidence that utilisation of plant-based collagen in a hydrolyae collagen beverage lusmoG qf animal -base collagen improves the rate or efficacy of collagen absorption in the hum an gastrointesti ual sy s e _: .

INCORPORATED BY REFERENCE (RULE 20.6) Cited References

1. Cterissea, S IT, €remhag, _{: ,} 1 T., Eenden, 3. Ni, a ered, W. IT, Bientu, I, Verdijiu L E , & van Loon, L. I (2018) Protein content an amino acid cmrpQsifidn of commercial ly available plant-based proteinisolates. Amino acids, 50(12), 1685-1095

2. Najjar, E. S„ Moore, Cl E., & Montgomery, 1. D. (2018) A defined, pianteb&sed diet util tee in an outpatient cardiovascular ellnie effectively treats hyperoholesteroterak and hypertension and reduces medications. Clinical cardiology, 41(¾ 30T-313

3. Najjar, P, S., Moore, C K, & Montgomery, B, I> (2018). Consumption of a ^n^ plsiib based diet reduces lipoprotein {a), inflammation,, and other atherogenic lipoproteins: and particles within 4 weeks. Clinical cardiology, 41(8¾ 10624062.

4. van Yliet, Bnrd, N. A., _: & van Loon, L. I (2015) The skeletal muscle anabolic response to plant- versos anint al-hased protei a consumption The Journal of nutrition, 145(0), 1981 991.

EN AMPLE 15

AOBITIO OF CERTAIN VITAMINS ANB MINERALS (VITAMIN FA€KAQE ^¾ )

Tho inclusion of certain targeted vitamins and minerals in Iwdrolyaed collagen beverage can iMpr ve absotption of collagen peptides In the gastrointestinal system and can improve efficacy of collagen that ban been absorbed. It can he concluded that following vitamins and minerals provide significant improvements

« Afagmsrmi * M ig wse ® wiiA m b ¾«¾a M$)

* t akttitih (Fiiamm BP)

* m rb mid/Fti&mm Q

MBUitm Mammsmm:

Magnesiu stimulates collagen synthesis expressed by fibroblasts in culture. Magnesiu inhlhitsproly! and lysyl hydroxylases and couhihe considered as antifibrodC _: Magnesium is associated with el ash s and plays a protective role in maintaining the extensibility of elastic. Magnesium

⁵ Vitamin Package: ngneslunnmanganese, pyrldotene/86, eotoi«is/8i2, and ascorbic acid

INCORPORATED BY REFERENCE (RULE 20.6) associated proteoglycans in cartilage prevent the swelling degradation of this tissue. Magnesium regulates the fonetional activity of Integrins (I, 2, 3)

Studies suggest the presence of overall skin I prove em in the us of Magn siu _: independently. I s Improve nt was net demonstrate to he due to its eifost on the absorption of eel lagers peptides. However, as Magnesium has been shown to stimtdaie collagen synthesis expressed by fibroblasts in culture, consumption , of magnesium concurrently with hydrolyzed oo!lageti can improv the net efficacy of orally ingested hydrolyzed collagen.

Manganese (Mn) is an essential metal that Is critical for human health. It is the hffir nrost abundant metal and twelfth most abundant dement overall on earth, Mn is involved in several significant hysi og!ca! processes, including development, reproduction, Immune function, energy metabolism, and antioxidant detenses. Mn is, n essential micronutrient that is required for the activity of a diverse set of enaymaitc proteins (1) Mult humans absorb approvi steiy 3~S% of ingested Mu, ingested Mn readily absorbed in ie Intestine (2) Studies using the Caeo-2 intestinal cell line describe a triphasic uptake process in which Mn transport achieves a steady-state condition alter a btlef period of egoillhratiou between intracellular an extracellular components p). Frolldase is metalloprotease, requiring manganese for catalytic activity, an enzyme involved in collagen metabolism, cleaves Imi cslipeptides containing G-termioal proline, providing large amount of gfoime for collagen synthesis, 4).

Among the multilayered and Interdependent antioxidant system, which consists of non- enzymatic as well as enzymatic components, the mitochondria! superoxide dismotase 2 (8CID2, MnSOD is, a xubfeef of pSitienkr interest, as it is located in the itochondrial matrix where it represents the first line, of antioxidant defense against superoxide anions produced as byproducts of oxidative phosphorylation MnSOD ovefexpressing Sbrobiast populated collagen lattices revealed significantly enhanced contramion compare to collage lattices populated with vector control cells (5), Manganese is neede for producing the amino acid proiine, whic is essential for collagen formation and wound hsaling In huma skin cell s, Manganese pi ays an important role in the formation of collagen in skin cells (6),

L _: Homing Cktlio S , Tijp KG, Bowias AB Aschner M. Manganese Is Essential for Meuronal Healt A»»nifeF.A¾/r. 20t5;3S;7I49!k dpi: 10J l46/apnare¥~s»str O?I7I4~O344JE Egub 2015 Ma 13. PMIO: 2S9:?4&¾ PMQD; E CA52S78S,

INCORPORATED BY REFERENCE (RULE 20.6) 2, finley JW, Johnses» PE, Johnson L . 1994. Sex affects manganese afosorptien and retention y humans from a diet a equate In mangaaes Am. I. Clin, Nutr 00:949-55 Lebfondei _s AIM» 1 99, Manganese transport by €aco~2 eel!s. Blok Trace Mefeehi let 97; 13-28

4. ., 19S2 Optimal conditions for prolidase assay by prdline eolodtoesie delernunaifon; application to iraidodipepddnria. €¾ia ChiM. Acta 125, 193-205,

5. Week 1, Srenoeisen l¾ Wlaschek M, Fos ig A, llriviba K, O drjef 1i¾ et al. Stable ovefexpresston of manganese supciOxide <tsmmase ip mitochondria Idepitles by rogea peroxide as a ajor oxidan la ike A F-h· mediated iadnchon: of matrlxfoegrabirsg me!alioprotease-L I Bi l Cham 1999; 274:: 25:369 76.

6. Par j¾. &ofe of mierauUtrieats n akin health and ilmeiion. Blomol Tber tSeoni), 2015 M y ;23(3t:307-tf. doi: i0.4062/biiimo0ker.2015093 Tpub 2011 May I. RMΪ ; 259958 iff PMC IBr 1 ³ MC8428712 positive influence os human ksratinoeytes mi fibroblasts. In particular, pyri oxine promotes fibroblast Migration, Further, a statisticallySignific nt induction of keratinoeyte proliferation i observed with In conjunction wife human i ngestion of pyridoxine. (4, 5).

Cobalamit (Bl ) is a naturally occurring, organometallic compound containing cobalt that serves as an important water-soluble vitamin for human health. The recommended aily intake for cobalsmin Is 2,4 gg (6) This vitamin functions as a cofadtor for two classes of human enxyross, na ely, Isomerascs and mefeyliraasfc ses. Consequently, sobaiamin eficienc can cause disturbances in cell division, leading to neuropathy, nervous system disease and pernicious anemia. Vi tamin B:1 deficiency i s common m people of all ages who con sum e a low intake of animal ~sooree foods, including populations ip developing countr es. It is aiso prevalent among the elderly, even in wealthier countries, due to their mal absorption of B12 from food, (7),

Altered eobalamtn levels ca lead to dermatological manifestations, which may indieate deficiency or excess of this vitamin. The biochemistry and metaholism of cobaiamln is complex, and

INCORPORATED BY REFERENCE (RULE 20.6) resses can he associated with alterations of t is metabolic pathway, The cutaneous manifestations of eohafemm deficiency include LygerpigmeBtation (m st comraooly}; h&hr an si ail changes- sa oral changes, pli*4iitg glossitis;. ditioaally, several dermatologic cooditionS including vitiligo _* aphthous stomatitis, atopic ermatitis, and acne am related to eobalsniie excess or deficiency, (¾}

The addition of cohalamm can improve the skin condition, however, was not carried out Fesear i stodyiag the impact of cehaia io on the adsor tion of peptides, on the contrary discovered that whey protein can improve the absorption o cobalsniin. (9, !0)

Several studies hav shown that the presence of ascorbic acid (Vitamin C) can increase the natural collagen production of the hody 0¾ 13, :4}

Qptimat Concentrations of Targeted Vitamins and Minerals

Dietary reference intakes are set of reference values used to plan an assess nutrient intakes of healthy people. The Baity Value (PV) is a valve published hy the US Food and Bm Administration (FDA) containing the recommended Intake of specified vitamins and minerals and has been determined sufficient to meet tire nutrient rerfui remeufs of nearly all ($174·?/») of healthy people withottf taking I nto account age an sex.

In general, the concentration of each of the targeted vitamins and minerals iohekm me kw <m t should not exceed 10% of the FDA recommended D¥ per standard 500 Ml collagen beverage, or 20% of the FDA recommended D¥ per liter. Highe concentrations of the targeted vitamins are possible. Higher concentration of any of the targete vitamins and min als risks formation of sediment and material changes In the taste of tie beverage,

TABLE 10

EXAMPLE 16

IDCIBASEO EATIO PRO-HYP AND HYP-GLY INCREASES EFFICACY OF COLL CEH

INCORPORATED BY REFERENCE (RULE 20.6) two: different types o eoilagen dipeptides, prolyl dwdroxyproline (Fro-Pyp) and hydroxyprolyl-glyeme (Hyp-Giv} form a portion of peptides in hydrolyned collagen. Both Pro-Hyp an Hyp-Gly are available at high ooneeoteaftons for several hours in the uman bloodstream following ora! administration Some m viim studies lave: demonstrate that :Pto-By and I-lyfi-Gly exert ehemotaxis on dermal fibroblasts and enhance cell proliferation. Additionally, Pro-Hy enhances the pOdftctios of hyaluronic acid b dermal fibroblasts. These findings suggest that the amounts of Pro- Hyp and Hyp-Ci!y in bloo are important festers to show the efficac of collage hydrolysates on skin health Some study ds emstrsted that the use of the eoilagen hydrol sate ith a highe content of Pn Hyp and Hyp-Gly led to more Improvement; iu fecial skin conditions, including facial skin moisture, elasticity, wrinkles and toughness.

Pro-Hyp and Byp-Oiy may a en low ratio of dipep ide-te-pOduet content, about ø,! g kg ^~ l of product (LCP), or It may also have a high ratio of dipeptide-to-product content, greater than 2 g, kg l of product HCP), A recent clinical provides evidcftee tha both LCP and HCP are effective supplements for increasing skin elasticity, reducing wrmldes. improving skin moisture and reducing the feeling of skin dryness and roughness. This study furthe indicate that HCP provides greater ifttprovemeni in skin elasticity, increased moisture and reducing wrinkles on fecial skin than LCP (16).

EXAMPLE 17

MODIFIG AXIOMS ALLOW INCREASED CONCENTRATION Of COLLAGEN.

Fro S g to 40giL Simple hydrolyzed collagen will a peptide weight of 10,00 Daltons (common supplements and commercial flood p- ducts cooiai ng coilsgen) and no firrther additives can fee expected to hsveaa optimal concentration radge of approximately 844 g/l. in studies oft the absorption, collagen peptides were shown to have a dose-depeftdeot effect and increased concentrations of hydrolyzed collagen Increases absorption of col agen peptides path corresponding increase effect on the skin: In studies on the absorption, collagen peptides were shown to have a dose-dependent effect. (17). However', there is significant evidence of a limit to the saturation of peptides, _: to a point where a rise in concentration increasingl loses effectiveness (18) The optimal range for absorption and heoeildsl effect car skin in respect of simple hydrolyzed collagen with peptide weight of 10,060 Daltons (common in supplements and commercial foo products containing collagen) and containing no further additives can he expected to he approximatel than 8 4 g/l.

INCORPORATED BY REFERENCE (RULE 20.6) Ho ever, the optimal range can be expecte to fcetttaie wit the introduction of addi tional factors and modifications.

Theoretical oaladatlon of optimal concentration of collagen in hydrolyved collagen bev rage;

TABLE ] 1: Theoretical Esti ate Absorption ates of Collagen Hydtelvaate % Absorbed

As shown TABLE l i, the optima! concentration of collagen is shoot *0 g/i withat least (1 } no whey , (¾ a molecular weight of about 2,000 da, (3) a Vitamin PaekageA and addition of €02 and pro-hyp / hyp-gly improving absorption feslher, hut not changing the optimal concentration

3. Sasaki, Y Sathi, Q. A., & Yamamoto, O, (2017). Wound healing effect of bioactive ion released from Mg-snieetite Maier isSeiwm mdB gifi ring: C, ff 52-57.

4. Fn)li, K, Kafiwara, T, & Knrose, B, (fOff , Effect of vitamin B| deficiency on the crosslink fensaioa of collagen. PEBSi&k s, 77(1), 103-195.

5, Rernbe, 1. D., Fromm-Domieden, C., & Sfoeoner, E K, (20!¾), Effcts of vitamin B comple and vitamin C o human skin cells: is the perceived effect measurable'? JAmwmi m skm wci ml , 31(5}, 225-233.

INCORPORATED BY REFERENCE (RULE 20.6) 6. B.R, Rucker, J.W.; Settle, S.B, Mc anii k, LJ Machilin Handbook of vitamin t Marcel DeFfeer Inc, Mew York 2001 )

8. Allen, L. H (2010). Bk vailabilit of vitamin E12 International Journal ¾r Vi tamin and Nutrition Research, 80(4)2330

9. Brescdll, 3 , it Baveiuy, S, (2615). A review of Wiamin 12 in dermatology. American journal of clinical dermatology, ½()), 27-33.

10. Wang, !-l, Stag ¥., Ehu, X., Xu. Y.. Sin. L., Xiang, 8,, ... & Man, X (2010), Stability of vitamin B12 with tfee protection of whey prolei us and their effects on the gut mierohlotne. Food ehenirstry, 276, 208-366.

11. Do, G., Yang, ί,, Wang, Y., Liu, _r tk €hert _s 1, f2Bi9), _: Prote -Iipid composite nanoparticles for the oral delivery of vitamiri BT2: Impact of protein saeoinyiation os naaopartMe physicochemical and biological properties. Food Bydrecolloids, 92, 189-197.

12. Paxton, Jennifer Z., Liam M. Grover, and Keith Baar. "Engineering an in vitro model of a functional li ament from hone to hone ” Tissue engineering part A 16.11 (2616): 3615-3525

13. Shaw, G., Lee-Barthel, A., Ross,M. 3L, Wang, B:., & Baar, K (2016), Vitamin C-enriched gelatin suppleroentatiors hefdre infenuideaf activity augments collagen synthesis. The American Journal ofclinical nutrition, T05(I), 136443

14. Lis, D, M., & Baar, K. (2019). Effects of Different Vitamin C-Tmriehed Collagen Derivatives on Gol!agen Synthesi s. International journal of sport nutriti on mi exercise meiafeol ism ,(øø}, 1-6.

I S. (IS. Department of H eal th and Human Services, Food and Drug Administration; available htips;Awws¥ accessdai8 Ha.goW¾C5ipts/interactiveHntritioHFaotsLah _: el/ fteisheetVYitam i m and _ Mi ne i, C ad pdf

16. Iheue, N,, Sogihara, F„ & Wang, X (2Q16). Ingestion of bldacdve collagen hykOly sales enhance facial skin moisture and el astie!ty and reduce facial ageing si s in a randomised double-blind plaeebo-cotttralfed clinical study.. m c fifths Science fFekxdwitf dg w&fc, 96(12}, 4077-4081.

17. Shigemura, Y , Kuho ura, D,, Sato, ¥., & Sato, K. (2014) Dose-dependent changes in the levels of free and peptide forms of hydrosyproline in human plasma after collage hydrolysate ingestion. Food chemistry , 159, 328-33

INCORPORATED BY REFERENCE (RULE 20.6) I S. Ohara, SI, Mikawa, S- Maisonioto, II Akiyama, M , Fu)tmoip, hi , Kohayashi, T., &

Taping, S. (2018) CoUagen-deftved ipepbde, ptoIihe :rexypj¾i s, sti ulate cell pjstiiS jt p and hyaluronic acid synthesis in cultured hu an; denia ihrohlnstS t¾e !ournai of dermatology, 3?<¾ 330420.

Wlisle this diroldsuro refers to exe lary embodiments, it will be understood by those skilled in the art that canons changes may be matte* and: equivalents may be substituted fer elements thereof without departing from the scop of the disclosure in addition, many modification will beappreciated by those skilled in the ait to adapt a particular instrument, situation or material to the teachings of the disclosure without departing from the spirit thereof.

References _: and citations to other documents, such as patents, patent applications, patent pu ieatiorss, journals, hooks, papers, web soofcms, have been made In this disclosure. All such documents are hereby incorporated herein by referenoe in their entirety for all purposes. Any material, or portion thereof, that is sai to be incorporated by reference herein, but which coofiieis with existing definitions, statements, or other disclosure materia! explicitly set ferth herein Is only incorporated to the extent that no conf!kt arises between that hieorpo tedroateria! and the present disci osure materia! In the event of a conflict, the conflict is to be resolved in favor of flic present disclosure: as the preferred disclosure.

INCORPORATED BY REFERENCE (RULE 20.6) BEVERAGE WITH COLLAGEN AND ADDITIONAL ADDITIVES

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a PCT application of Ser. No. 16/680,294 of U.S. patent application, filed November 11, 2019, which is a continuation-in-part of Ser. No. 16/353,179, filed March 14, 2019, which is a continuation of U.S. patent application Ser. No. 14/969,519, filed December 15, 2015, which claims priority to Bulgarian Utility Model No. 2935 filed on January 16, 2015. The content of each of the aforementioned applications is incorporated by reference herein in their entireties.

FIELD OF THE EMBODIMENTS

This invention characterizes beverages comprising hydrolysed collagen, derived from animal raw materials. Those raw materials may vary according to their origin, including mammals, fish, birds, snails; or from various parts of a carcass.

BACKGROUND OF THE EMBODIMENTS

Many of the beneficial properties of collagen are applicable both to the medical field and cosmetics. Collagen is a major protein of the human connective tissue including, for example, cartilage, cornea, arteries, and skin. Robust and inextensible, collagen possesses great tensile strength. It is the principal component of cartilage, tendons, articular joints, bone as well as teeth. Collagen can also be found in the walls of capillaries, veins and arteries. It imparts to them strength and elasticity for the effective transport of blood throughout the body. Collagen is of vital importance for muscle function. The collagen molecules also provide muscle fibers with strength and structure necessary for their functioning over long periods of time. Collagen not only supports skeletal muscle fibers, but also smooth muscles present in cardiac, bladder and genital muscles.

Collagen is permanently being replaced in the human body. However, after the age of 25 this process decelerates and progressively slackens with advancing age. Disorders of collagen buildup can also be provoked in situations of permanent overloading (for example in power sports, or in case of intensive physical activity). With aging, the amount of de novo synthesized collagen

ERRONEOUSLY FILED (RULE 20.5bis) is reduced as result of a slowdown in metabolism which results in a gradual decline in the resilience of both soft tissues and bones (osteoporosis). The collagen deficit due to advancing age, leads to visible effects including skin drying and wrinkling, limited mobility due to stiffness and joint pain, bone fractures caused by osteoporotic changes, the appearance of pointed and visible capillaries on the skin surface, as well as the appearance of varicose veins.

Specific references to relevant prior art are herein described as follows:

U.S. Patent Publication No. 2010/0316768 and International Patent Publication No. W02004023899 disclose a shelf-stable, ready to use, water-like composition for humans/animals; as an adjunct to fiber-water, and/or safe drinking water, consumed directly, tube feedings, or in the preparation/reconstitution of food(s)/beverage(s). These disclosures teach a fortified fiber- water with added delivery systems: Encapsulations/particles, of different size(s), shape(s), material(s), colors, non-visible, serving one or more functions: improved taste, odor-masking; controlled release applications; bio-availability of actives, avoid hygroscopicity; minimized interactions, improved thermal, oxidative, and shelf-life; decorative.

U.S. Patent Publication No. 2014/0212565 and International Patent Publication No. WO2013049540 teach a clear high protein beverage comprising water; between about 4% and about 8% by weight protein; and a flavoring. W02007098593 teaches an alkaline soluble fiber compositions and methods for preparing the same. Soluble fiber for use in the compositions of the invention is contributed from one or more sources and is preferably inulin, FOS and/or scFOS. In some cases, the compositions have a pH of between 8.0 and 9.5.

The method and solutions known in the art fail to remedy all the problems taught by the present disclosure. There is an ongoing unmet need in the art for improved methods of restoring collagen lost as a result of aging.

SUMMARY OF THE EMBODIMENTS

The preferred embodiments of the present invention will now be described. Reference will now be made in detail to each embodiment of the present invention. Such embodiments are provided by way of explanation of the present invention, which is not intended to be limited thereto. In fact, those of ordinary skill in the art may appreciate upon reading the present specification that various modifications and variations can be made thereto.

ERRONEOUSLY FILED (RULE 20.5bis) The disclosure described a hydrolyzed collagen beverage that facilitates the absorption of the hydrolyzed collagen in the digestive tract.

In a first aspect, a beverage is disclosed consisting of a non-protein portion comprising water, and a protein portion consisting of collagen.

In certain embodiments of the first aspect, the collagen is from non-human animals.

In certain embodiments of the first aspect, the collagen is isolated from a plant expressing recombinant collagen.

In certain embodiments of the first aspect, the collagen is hydrolyzed.

In certain embodiments of the first aspect, the hydrolyzed collagen comprises peptides having a molecular weight of no more than about 2000 daltons.

In certain embodiments of the first aspect, the collagen concentration range is between 1 milligram/liter to about 8440 milligram/liter.

In certain embodiments of the first aspect, the collagen concentration range is between about 8440 milligram/liter and 40g/liter

In certain embodiments of the first aspect, the water is carbonated.

In certain embodiments of the first aspect, the non-protein portion of the beverage does not comprise sodium acid sulphate.

In certain embodiments of the first aspect, the non-protein portion of the beverage does not comprise any one of citric acid, phosphoric acid, malic acid, tartaric acid and fumaric acid.

In certain embodiments of the first aspect, the non-protein portion of the beverage further comprises a vitamin.

In certain embodiments of the first aspect, the vitamin is in an amount equal to the US “Recommended Daily Allowance (RDA).”

In certain embodiments of the first aspect, the vitamin comprises at least one of Vitamin A, Vitamin D, Vitamin E, Vitamin K, Vitamin C, Thiamin (Vitamin Bl), Riboflavin (Vitamin B2), Niacin (Vitamin B3), Vitamin B6, Folic acid (Vitamin B9), Vitamin B12, Biotin (Vitamin B7), Pantothenic acid (Vitamin B5).

ERRONEOUSLY FILED (RULE 20.5bis) In certain embodiments of the first aspect, the vitamin comprises any one of Vitamin B6, Biotin (Vitamin B7), Vitamin C and Vitamin B 12.

In certain embodiments of the first aspect, the vitamin comprises any two of Vitamin B6, Biotin (Vitamin B7), Vitamin C and Vitamin B 12.

In certain embodiments of the first aspect, the vitamin comprises any three of Vitamin B6, Biotin (Vitamin B7), Vitamin C and Vitamin B 12.

In certain embodiments of the first aspect, the vitamin consists of Vitamin B6, Biotin (Vitamin B7), Vitamin C and Vitamin B 12.

In certain embodiments of the first aspect, the non-protein portion of the beverage further comprises a mineral.

In certain embodiments of the first aspect, the mineral is chosen from at least one of Potassium (K), Chloride (Cl), Calcium (Ca), Phosphorus (P), Magnesium (Mg), Iron (Fe), Zinc (Zn), Copper (Cu), Manganese (Mn), Fluoride (F), Selenium (Se), Chromium (Cr), Molybdenum (Mo) and Iodine (I).

In certain embodiments of the first aspect, the mineral consists of manganese and/or magnesium.

In certain embodiments of the first aspect, the non-protein portion of the beverage comprises water, a vitamin and a mineral.

In certain embodiments of the first aspect, the mineral is manganese and/or magnesium.

In certain embodiments of the first aspect, the vitamin is Vitamin B6, Biotin (Vitamin B7), Vitamin C and Vitamin B 12.

In a second aspect, a beverage is disclosed comprising a non-protein portion comprising water, and a protein portion, wherein the protein portion comprises hydrolyzed collagen and no whey protein.

In a third aspect, a beverage is disclosed consisting of water and hydrolyzed collagen.

In certain embodiments of the third aspect, the beverage has a pH of about 6.8.

In a fourth aspect, a method of increasing the absorption of hydrolyzed collagen in the digestive tract of a subject is disclosed comprising any one of the aforementioned beverages.

ERRONEOUSLY FILED (RULE 20.5bis) In certain embodiments, a beverage is disclosed having water and a concentration range of about .001 grams per liter to 8.44 grams per liter of collagen, wherein the collagen is hydrolyzed. The collagen concentration range may be between 1 milligram per liter to about 8440 milligram per liter or 1 part per million to about 8440 parts per million. The hydrolyzed collagen can have collagen peptides produced through hydrolysis of a plurality of collagen from a variety of sources, including, for example, animal raw materials, animals raised in non-organic or organic farms, animals from various animal species and various parts of the animal carcass. In certain embodiments, collagen, as used herein, can be recombinant collagen that is expressed for example in plants, yeast, tissue culture cells or bacteria. In certain embodiments, the beverage contains water including, for example, tap water, spring or mineral water. In certain embodiments, the beverage may be carbonated or non-carbonated.

In certain embodiments, a beverage is disclosed having water, a concentration range of about .001 grams per liter to 8.44 grams per liter of collagen; and at least one additive. In certain embodiments, the additive may comprise a stabilizing preservative. The stabilizing preservative may have chemical substances which dissolve in the beverage and are only parts of its content or have chemically active substances used in the production of the beverage but subsequently dissolve to other ingredients. In certain embodiments, the additive may comprise a preservative. In certain embodiments, the beverage may be pasteurized. In certain embodiments, the additive may comprise a packaging gas and/or a colorant. The colorant may be synthetic or organic and derived from vegetable, fruit or animal origin. The colorant may have a concentration range of at least one of .0001 % to about .05 %, .001 % to about .1 %, and .003 % to about .2 %. In certain embodiments, the additive may comprise a plurality of sweeteners. The sweeteners may be in liquid form, hard form, natural sugars, refined sugars, low caloric, and non-refined sugars. In certain embodiments, the additive may comprise a plurality of fruit juices or fruit juice concentrates. In certain embodiments, the additive may comprise a plurality of acidity regulating or E-number additives. In certain embodiments, the additive may comprise a thickener, flavor enhancers, vitamins, minerals, electrolytes and/or amino acids. The additive may be at least one other protein with a concentration range sum total of .0001 grams per liter to 8.44 grams per liter. In certain embodiments, the additive may comprise a dairy component, probiotic ingredients and/or extracts. In certain embodiments, the beverage may be carbonated or non-carbonated.

ERRONEOUSLY FILED (RULE 20.5bis) BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an exemplary schematic showing how peptides are absorbed across the intestinal wall;

FIG. IB shows an exemplary embodiment of a transwell assay testing for the transport of collagen peptides across a Caco 2 cell monolayer;

FIG. 1C shows an exemplary embodiment of peptide transport across a Caco 2 cell monolayer as a function of collagen peptide concentration;

FIG. 2 shows an exemplary embodiment of the absorption of collagen peptides in the intestine as a function of peptide concentration;

FIG. 3 shows an exemplary embodiment of the effect of carbonated water on the absorption of collagen peptides in the intestine;

FIG. 4 shows an exemplary embodiment of the effect of peptide length on the absorption of collagen peptides in the intestine;

FIG. 5 shows an exemplary embodiment of the effect of vitamins on the absorption of collagen peptides in the intestine;

FIG. 6 shows an exemplary embodiment of the maximum “Recommended Daily Allowance” for magnesium, manganese, vitamin B12, vitamin B6, vitamin B7, zinc and vitamin C.

FIG. 7 shows an exemplary embodiment of the optimal DV (daily value) dose of Vitamin Package elements for absorption via Hydrolyzed Collagen Beverage (500 ml. beverage is 1 dose);

FIG. 8 shows an exemplary embodiment of collagen hydrolysate beverage on absorption;

FIG. 9 shows an exemplary embodiment of the benefit of selected collagen hydrolysate beverages on absorption;

FIGs. 10A-10E show exemplary comparisons of the effect of different concentrations of various collagen hydrolysate compositions on absorption.

DETAIFED DESCRIPTION OF THE INVENTION

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure

ERRONEOUSLY FILED (RULE 20.5bis) belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

Definitions

As used herein, the singular forms “a,” “an,” and “the,” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Thus, as a non limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below those numerical values. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20%,

ERRONEOUSLY FILED (RULE 20.5bis) 10%, 5%, or 1%. In certain embodiments, the term “about” is used to modify a numerical value above and below the stated value by a variance of 10%. In certain embodiments, the term “about” is used to modify a numerical value above and below the stated value by a variance of 5%. In certain embodiments, the term “about” is used to modify a numerical value above and below the stated value by a variance of 1%.

When a range of values is listed herein, it is intended to encompass each value and sub range within that range. For example, “1-5 ng” or a range “between 1 ng and 5ng” is intended to encompass 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 1-2 ng, 1-3 ng, 1-4 ng, 1-5 ng, 2-3 ng, 2-4 ng, 2-5 ng, 3-4 ng, 3-5 ng, and 4-5 ng.

It will be further understood that the terms “comprises,” “comprising,” “includes,” and/or “including,” when used herein, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

As used herein, the term “isolated” refers to a sample in a non-naturally occurring state outside an organism (e.g., isolated from organism or a biological sample from an organism).

A “subject” is a vertebrate, preferably a mammal (e.g., a non- human mammal), more preferably a primate and still more preferably a human. Mammals include, but are not limited to, primates, humans, farm animals, rodents, sport animals, and pets.

As used herein, a “Vitamin Package” refers to the core vitamin/mineral additive package (see FIG. 6) which aids in absorption of higher percentages of collagen hydrolysate in a hydrolyzed collagen beverage. These core vitamins will always be part of the Vitamin package. Addition of other vitamins and minerals can be added without negatively affecting collagen absorption. Additional vitamins/minerals may, however, may have a significant effect on product taste.

As used herein, RDA is the acronym for “Recommended Daily Allowance.”

As used herein, the maximum RDA refers to the estimated Maximum percentage of RDA of elements of the Vitamin Package which could be effectively absorbed via a hydrolyzed collagen beverage.

In its original state, the digestive system is incapable of enzymatically degrading collagen in part because to its great length and the crosslinking between collagen fibers. Thus, administration of collagen to an individual is best achieved using “hydrolyzed” collagen that

ERRONEOUSLY FILED (RULE 20.5bis) facilitates its absorption in the digestive tract. Collagen can be administered in the form of tablets, capsules, or in the form of a drink.

Recent scientific studies show, that drinking collagen in liquid form results in more than 90% absorption of collagen in the digestive tract, which is absolutely unachievable when collagen is either in tablet or capsule form. The liquid collagen form has demonstrated better results, higher efficiency of active ingredients and unrivalled absorption. Moreover, all dietary supplement forms - tablets, capsules, sachets etc. definitely burden more on the digestive system, in contrast to the liquid form for drinking, which is easily assimilated by the stomach. The accurate dosage is of particular importance for the consumers of collagen drinks.

Known collagen drinks have a different percentage of hydrolyzed collagen and include many additives like stabilizers, preservatives, aromatizers, artificial colors, flavor enhancers etc. These components are put in products with more concentrated amount of added hydrolyzed collagen. Because of the absence of any additional components, except drinking water and hydrolyzed collagen, the disclosed beverage represents an important benefit, which ensures its administration without limitations regarding age and health state of the expected consumers.

The risk of forming intolerance, or exacerbation of hyperesthesia of the organism, which normally is provoked by additionally added unnatural chemical additives, is completely avoided. In the present invention, the optimal concentration of hydrolyzed collagen in the drink is completely clear in taste and almost wholly corresponding to the neutral taste of the clean drinking water. Furthermore, the drink is maximal simplified in functionally and that is why it doesn’t burden the consumer with additional calories. As a whole, the consumption of the created non- carbonated alcohol-free drink leads to the improvement of the skin structure, the skin density, skin turgor, muscle tone as well as to the improvement of the condition and the flexibility of a subject’s joints.

In certain embodiments, the hydrolyzed collagen drink can be the sole source of a subject daily intake of water.

The market offers a wide variety of beverages containing various healthy ingredients. Contemporary society strives to improve its nutrition by selecting food and beverages with high added value, provided by the various forms of ingredients, beneficial for the body, included in their contents. This way, the need of consumers to reach better shape and tonus, to live a full life, feel good and preventively enhance their health can be satisfied. Beverages, which include in their

ERRONEOUSLY FILED (RULE 20.5bis) content hydrolysed collagen, may be offered in convenient and easy for consumption form. The collagen in these drinks may be a source of valuable proteins in the body that can characterize with high and full assimilation. At the same time, when collagen is added in beverages, it combines the advantage, the daily intake of fluids to be stimulated, which usually is what underlies most recommended healthy diets. The sufficient intake of fluids is extremely important for many groups of people having specific and increased needs of fluids, such as, active athletes or people whose work and life comprise high physical activity or mental pressure. The additional intake of hydrolysed collagen is a valuable source of one’s daily nutrition. Also, collagen offers one a full set of all amino acids needed for the synthesis of collagen matrix in the human body. This is particularly important for active people, the elderly, pregnant and lactating women, people undergoing aesthetic surgery and others

This invention regards to beverages comprising functional ingredient hydrolysed collagen in concentration: from 0.001 g/L g/L to 8.44 g/L, which may also be expressed as: from 1 ppm to 8440 ppm. This concentration of hydrolysed collagen in the beverages shall be reviewed regarding ready for consumption beverages or regarding beverages obtained after dilution by adding additional water in the prescribed proportion to produced concentrates in the form of powder or liquids. The functional ingredient hydrolysed collagen, also known as collagen peptides, is produced through hydrolysis in controlled conditions of raw materials such as collagen sources from various animal origin.

The beverages containing hydrolysed collagen characterize with great diversity, depending on the other combined ingredients that they may include. Beverages may contain only hydrolysed collagen and water.

The water for producing the beverages may be tap water or spring or mineral water or water, gained by icebergs (glaciers). When tap water is used, the tap water shall mandatorily be supplied by central water supply system or extracted underground water or extracted from natural or artificially constructed ponds. Also, preventively or in case of need, the water can be additionally purified, or processed approved by the appropriate healthcare authority’s methods of additional purification or processing:

Examples of such methods are: mechanical filtration through filtration systems,

ERRONEOUSLY FILED (RULE 20.5bis) filtration through chemically active filters to separate the unwanted salts of iron (Fe) and manganese (Mn), decrease of total mineralization through ultrafiltration - using ultrafiltration (reverse osmosis systems; decrease of total mineralization through filtration in ion exchange filter systems, decrease of total mineralization through distillation of water (evaporation and further condensation), decontamination by UV-filter (UV-lamps), decontamination by using chemical processing agents such as chlorine (Ch), chlorine dioxide (CIO2), ozone (O3), active oxygen (O2) and others.

Also, water, used for the beverages may be additionally carbonated through adding Carbon Dioxide - CO2. The quantity of the Carbon Dioxide added may reach 7,0 volume units (7,0 v/v). In the most common beverages combination, it may usually vary between 0,5 and 5,0 v/v.

Beverages containing hydrolysed collagen may be produced by stabilizing preservatives. The preservatives used shall be permitted for use for the category of beverages. Respectively, the preservatives may be chemical substances (as Potassium Sorbate [E202], Sodium benzoate [E211] and others), which dissolute in the product and are only parts of its content or may be chemically active substances, used in the production of the product but subsequently dissolute to other ingredients (such as Dimethyl dicarbonate [E242]).

Beverages containing hydrolysed collagen may be produced without any preservatives as well as their stabilization in those cases is ensured through applying appropriate technological production methods.

Such methods are for example: pasteurization of the product in combination with a "clean fill" and subsequent storage at refrigeration conditions, pasteurization of the product by UHT-sterilization followed by aseptic filling (filling in sterile environment) of the product; pasteurization of the product followed by hot filling of the product, filling of the product and further tunnel pasteurization ensuring reaching commercial sterility of the product;

ERRONEOUSLY FILED (RULE 20.5bis) pasteurization of the product and using special nanomaterials when packaging the product in combination with further storage at refrigerator conditions.

In the process of filling the beverages, regardless of whether the beverage is produced by adding preservatives or with no added preservatives, packaging gases may be used and added in the package prior to filling, during the filling or after it. Packaging gases are gases, different than air, and are inert gases such as: Nitrogen [E941], Carbon dioxide [E290], Helium [E939] and others. These gases are added in the filling process with the purpose of protecting the product from adverse processes such as oxidation, change in taste, aroma and appearance. These gases contribute to ensuring the shelf life of the product while helping to preserve the quality of the added nutrients.

Regarding the appearance of the ready product, beverages containing hydrolysed collagen may be colourless or coloured, clear or cloudy and may contain a fine precipitate of larger particles on the bottom and/or to have uniformly dispersed particles in the liquid. They may be calm fluids or carbonated.

In other beverages containing hydrolysed collagen that may be coloured, various colourants may be added such as synthetic or organic ones from vegetable or animal origin or may be from various fruit and/or vegetable concentrates, vegetable extracts. Added colourants, classified as additives with an E-number may be permitted for use in the beverage. The exact amount of coloring agent used will vary, depending on the agents used and the intensity desired in the finished product. Generally, if utilized, the coloring agent should be present at a level of from about 0.0001% to about 0.5%, from about 0.001% to about 0.1%, or from about 0.003% to about 0.2%, by weight or volume of the composition. Certain formulations will have no added colorant.

Beverages containing hydrolysed collagen may be produced unsweetened or sweetened through adding various sweeteners or mixtures of several ones.

Sweetening the present beverage may be done by adding sweeteners, most of which are low-calorie ones. Liquid as well as hard sweeteners may be used such as crystals or granules. Examples for sweeteners include: in a liquid form are the polyols: Sorbitol (E420), Xylitol [E967], Mannitol [E421], Maltitols [E965] and others,

ERRONEOUSLY FILED (RULE 20.5bis) in a hard form are: Aspartame [E951], Cyclamates [E952], Neotame [E961], Acesulfame K [E950] and others,

Sweetening beverages may be done by adding various types of sugars derived from natural sources. These sugars may be respectively non-refined or refined to a various extend. Examples of such sugars are: refined white sugar, unrefined or partly refined sugar, honey, fructose, glucose, glucose-fructose syrups, syrups derived from fruits, plant extracts.

The desired degree of sweetness of the present invention’s beverage may vary from °Bx=l,0÷16,0 (brix units) or in cases when using artificial or natural low caloric sweeteners, the desired sweetness intensity shall match equivalences of °Bx=l,0÷16,0 for ready to drink beverages. One brix unit (°Bx) corresponds to 1% water aqueous sugar solution, i.e. 1,0 g sugar per 100 ml water.

For regulating the acidity and/or giving a sour taste of beverages containing hydrolysed collagen, fruit juices or fruit juice concentrates may be added. These fruit juices may have a sour taste, acidity regulating additives, or E-number additives such as, Citric acid [E330], Phosphoric acid [E338], Phosphates [E339; E340; E341, E343], Citrates [E331; E332; E333] and others. The titratable acidity of a preferred embodiments of the present invention’s beverage containing hydrolysed collagen may be a value within the range of from about 0.01 %w/v to about 0.85 %w/v, about 0.1 %w/v to about 0.45 %w/v, 0.12 %w/v to about 0.35 %w/v or any ranges or single values within these ranges.

For beverages containing hydrolysed collagen, other ingredients may be added such as: thickeners and / or stabilizers. Thickeners are substances which increase the viscosity of a foodstuff. Stabilisers are substances which make it possible to maintain the physico-chemical state of a foodstuff. Stabilisers include substances which enable the maintenance of a homogenous dispersion of two or more immiscible substances in a foodstuff. Substances which stabilise retain or intensify an existing colour of a foodstuff and substances which increase the binding capacity of the food by the formation of cross-links between proteins. Example for ingredients used as thickeners and stabilizers are the substances: Gum arabic [E414], Guar gum [E412], Carrageenan [E407], Pectins [E440] and others.

For enhancing and modifying the intensity of taste of the present invention’s beverage containing hydrolysed collagen, ingredients such as flavour enhancers may be added. These are substances which enhance the existing taste and/or odour of a foodstuff. Such are for example:

ERRONEOUSLY FILED (RULE 20.5bis) Glutamic acid [E620], Guanylic acid [E626], Inosinic acid [E630] and others. For the purpose of adding flavour for taste, organic substances may be added to the present invention’s beverage. Such organic substances include, for example: table salt, extracts and tinctures of plants, fruit, seeds, yeast extract and others.

Fruit and vegetable juices, fruit and vegetable puree, concentrates from fruit and vegetables as well as extracts or tinctures of fruit, part of fruit or plants may all be added to the present invention’s hydrolysed collagen beverage or it may contain such substances. The diversity of this category of additives is high as it is conditioned on the natural diversity of plants. The juices, purees, concentrates or extracts that may be added to the present invention’s beverage may be clear or cloudy, contain particles of fruit, vegetables or plants as seeds, parts of the peel, pulp, cells and so on. An example of clear juices that can be added to the present invention’s beverage may be juices made from: apples, cherries, raspberries, strawberries, lemon, orange, grapefruit etc. Cloudy juices and purees can be produced from fruits: apple, sour cherry, raspberry, lemon, orange, plum, pear, apricot, peach, etc. In certain embodiments, the beverage containing hydrolysed collagen disclosed here may optionally include one or more juices (e.g., one or more of single-strength fruit, berry, or vegetable juice, as well as extracts, concentrates, purees, milks, and other forms) present at a level from about 0.0005% to about 99,16%, about 0.001% to about 20%, about 0.005% to about 15%, about 0.01 % to about 10%, about 0.05% to about 5%, about 0.01 % to about 10%, about 0.05% to about 5%, or about 0.1% to about 2.5% by weight of the beverage, or at a level of about 0.001%, 0.0025%, 0.005%, 0.0075%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 21.0%, 22.0%, 23.0%, 24.0%, 25.0%, 26.0%, 27.0%, 28.0%, 29.0%, 30.0%,

31.0%, 32.0%, 33.0%, 34.0%, 35.0%, 36.0%, 37.0%, 38.0%, 39.0%, 40.0%, 41.0%, 42.0%,

43.0%, 44.0%, 45.0%, 46.0%, 47.0%, 48.0%, 49.0%, 50.0%, 51.0%, 52.0%, 53.0%, 54.0%,

55.0%, 56.0%, 57.0%, 58.0%, 59.0%, 60.0%, 61.0%, 62.0%, 63.0%, 64.0%, 65.0%, 66.0%,

67.0%, 68.0%, 69.0%, 70.0%, 71.0%, 72.0%, 73.0%, 74.0%, 75.0%, 76.0%, 77.0%, 78.0%,

79.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%,

91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0%, 99.0 or about 99.16%, about by weight of the beverage, or any ranges or single values within these ranges. Typically, juice can be used, if at all, in an amount of from about 0.001 % to about 45% by weight. Fruit and vegetable juices, fruit and vegetable puree, concentrates from fruit and vegetables as well as extracts or

ERRONEOUSLY FILED (RULE 20.5bis) tinctures of fruit, part of fruit or plants may be mixed in all possible combinations with each other and then added to the present invention’s hydrolysed collagen beverage

For flavouring the present invention’s beverage, aromas as well as ingredients with flavouring properties may be added. The beverage containing hydrolysed collagen disclosed here may optionally contain one or more flavourings or flavour compositions. For example, natural and/or synthetic fruit flavours, botanical flavours, other flavours, and mixtures thereof. Added concentrated aromas may be extracted from natural sources as: fruit, food, parts of plants, smoke and so on but may also consist of synthetic flavourings, approved for use with foodstuff. For flavouring the present invention’s beverage, ingredients with flavouring properties such as herbs and spices extracts, fruit extracts and fruit concentrates with intense aroma may be used.

In certain embodiments, the present invention’s hydrolysed collagen beverage disclosed here may optionally include one or more flavourings present at a level from about 0.0005% to about 5%, about 0.001% to about 4%, about 0.005% to about 3%, about 0.01% to about 2%, about 0.05% to about 1%, or about 0.1% to about 0.5% by weight of the beverage, or at a level of about 0.001%, 0.0025%, 0.005%, 0.0075%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or about 1.0% by weight of the beverage, or any ranges or single values within these ranges.

Vitamins and minerals with the purpose of enriching their nutritional value may be added to the present invention’s beverage. Vitamins include, but are not limited to, Vitamin A, Vitamin D, Vitamin E, Vitamin K, Vitamin C, Thiamin (Vitamin Bi), Riboflavin (Vitamin B2), Niacin (Vitamin B3), Vitamin Be, Folic acid (Vitamin B9), Vitamin B12, Biotin (Vitamin B7), Pantothenic acid (Vitamin Be). Minerals include, but are not limited to, Potassium (K), Chloride (Cl), Calcium (Ca), Phosphorus (P), Magnesium (Mg), Iron (Fe), Zinc (Zn), Copper (Cu), Manganese (Mn), Fluoride (F), Selenium (Se), Chromium (Cr), Molybdenum (Mo), Iodine (I).

Supplements are typically present in amounts generally accepted under good manufacturing practices and are typically present in amounts between about 7.5% to about 100% RDV, where such RDV are established. But may present at a level from about 101% to about 150% RDV, about 120% to about 200% RDV, about 150% to about 300% RDV, about 200% to about 500% RDV.

Vitamins and minerals are used to enrich the beverage nutritional profile. For example, according to legislative food sources, a good source of Vitamin A can be found in substances such

ERRONEOUSLY FILED (RULE 20.5bis) as: retinol, retinyl acetate, retinyl palmitate, beta-carotene. As Magnesium (Mg) sources used may be: magnesium carbonate, magnesium chloride, magnesium gluconate and etc. may be added to the present invention’s beverage.

Enrichment of beverages with vitamins and minerals may be done with organic sources of vitamins and minerals. For example, such vitamins are: acerola extracts, rose hips extracts and others, as organic sources of minerals used may be: organic concentrates by seawater, seaweed, eggshell extracts and others. The use of vitamins and minerals may vary to 100% and more than 100%, according to established Recommended Daily Value (RDV) for vitamins and minerals.

The present invention beverage containing hydrolysed collagen may be produced by adding appropriate electrolytes, often categorized as specialized beverage category, such as: isotonic sport beverages, hypertonic sport beverages, and hypotonic sport beverages.

Additional amino acids may be added to the present invention’s beverage. Additional enrichment with amino acids may be done by adding amino acids in their L-forms. For example, F-glutamine, F-isoleucine, F-leucine, F-valine, F-taurine and others may be added to the present invention’s beverage. Additionally, amino acids may be added as separate amino acids as well as in combination to other amino acids.

In certain embodiments, other types of proteins, different than collagen may be added to the present invention’s hydrolysed collagen containing beverage. These proteins may be hydrolysed proteins with higher or lower molecular weight. For additional enrichment, other proteins may be added to the present invention, such as proteins from vegetable as well as animal origin proteins. For example, proteins from soy, peas, milk, egg and others may be added to the present invention’s beverage.

In certain embodiments, the beverage of hydrolysed collagen does not contain whey protein.

In certain embodiments, the beverage of hydrolysed collagen does not contain sodium acid sulphate.

In certain embodiments, the beverage of hydrolysed collagen does not contain citric acid, phosphoric acid, malic acid, tartaric acid or fumaric acid.

Where other types of proteins and / or protein hydrolysates, other than collagen are added, the total concentration of binding proteins in the beverage should be varied, from 0.001 g/1 g/L to

ERRONEOUSLY FILED (RULE 20.5bis) 8.44 g/L, which may also be expressed as: 1 ppm to 8440 ppm. In these embodiments, the total protein concentration in the beverage is the sum of all protein sources contained in the beverages, including the added hydrolysed collagen.

Alternative embodiments of the present invention’s beverage may be produced which do not contain lactose but may also include lactose as an individual ingredient or have added compositions which are sources of lactose.

The present invention’s beverage may include additional added ingredients, such as dairy components which can be different fractions derived from milk. For example: whey proteins, casein, caseinates, etc.) or fractions derived from the colostrum (for example: antibodies, immunoglobulin, growth factors). Stated dairy components include, but are not limited to: lipids, proteins (casein, lactoglobulin), antibodies, minerals and salts (phosphates, calcium, magnesium, sodium, potassium, etc.), Vitamins (A, Be, B12, D, K, E, thiamine, niacin, biotin, pantothenic acid, etc.), carbohydrates (lactose), enzymes and others.

The present invention’s beverage may be produced with other additives such as; fresh milk, reconstituted milk or milk fermented with lactic acid cultures and accordingly each of the following types of milk can be skimmed or whole to various extend. Fermented milk is usually derived from using the following starters: Streptococcus thermophilus n Lactobacillus delbrueckii subsp. bulgaricus, as well as various starters mostly from the Lactobacillus, Lactococcus, Bifidobacterium and Str. Thermophilus kinds. During fermentation probiotic cultures may be added. Pre-prepared probiotic concentrates (probiotics) may also be added as individual ingredients to the present invention’s beverage. The present invention’s beverage containing hydrolysed collagen may also contain added prebiotic ingredients which are fibers which can be soluble as well as insoluble.

Various other ingredients with beneficial properties may be added to the present invention’s beverage containing hydrolysed collagen. Such ingredients may add value to the present invention’s beverage containing hydrolysed collagen. All these additional healthy ingredients may vary in different combinations as may be individually added to beverages containing hydrolysed collagen or be combined. Examples for such healthy ingredients are:

L- carnitine,

Coenzyme Q 10,

Omega 3 and Omega 6 essential fatty acids,

ERRONEOUSLY FILED (RULE 20.5bis) Bee products, such as natural honey, bee pollen, royal jelly, propolis,

Vegetable extracts from plants with healthy effects that may include various parts of plants as: fruit, blossoms, peels, roots, stables. For example, plants with healthy effects are: Panax Ginseng, Camomila, Lavender, Elettaria cardamomum, Ginkgo biloba and others,

Tea extracts (black, green, white etc.)

Soy isoflavones,

Hyaluronic acid and/or its salts,

Alpha Lipoic Acid,

Extracts of seaweed as well as purified finely ground seaweed,

Mushrooms extracts, edible and special types such as: Lentinula edodes, Grifola frondosa, Cordyceps Sinensis, Hericium erinaceus, Coprinus comatus and others,

Extracts of polyphenols - resveratrol, astaxanthin,

Carotenoids, including lutein and zeaxanthin,

Green coffee extracts,

Caffeine.

EXAMPLES OF PREFERRED EMBODIMENTS EXAMPLE 1

EXAMPLE 1 (as shown in Table 1) comprises a cloudy, coloured beverage containing hydrolysed collagen. The ingredients expressed in weight percentage are listed below in Table 1. Used ingredients are as follows: water purified by ultrafiltration technology, Hydrolysed bovine collagen Peptan® B 5000HD (Rousselot Inc.), crystalline fructose, peach aroma, citric acid [E330], antioxidant: ascorbic acid, acidity regulator: sodium citrates [E331], a powder mix of vitamins and minerals: D-pantothenate calcium; Pyridoxine hydrochloride; Hydroxocobalamin, D-biotin; Calcium gluconate; Magnesium gluconate; Zinc chloride, RDV levels: Pantothenic acid - 15%; Vitamin Bd - 15%; Vitamin B12 - 15%; Biotin - 15%; Ca - 15%; Mg - 15%; Zn - 15%;), clear concentrated peach juice, carrageenan [E407], guar gum [E412], colour: carotenes. The beverage is aseptically filled into plastic disposable bottles 375ml (12.680 fl. oz). It is sterilized in UHT-sterilizer at 137° C for 4.2 seconds. It has a shelf life of 12 months when stored at 25° C, after opening shall be kept in the refrigerator up to 48 hours.

TABLE 1

ERRONEOUSLY FILED (RULE 20.5bis)

EXAMPLE 2

Example 2 (as shown in Table 2) comprises a clear, colourless beverage containing hydrolysed collagen. The ingredients, expressed in weight percentages, are listed below in Table 2. Used ingredients are as follows: water purified by ultrafiltration technology, Hydrolysed bovine collagen Peptan® B 5000HD (Rousselot Inc.). The beverage is aseptically filled into plastic disposable bottles 375ml (12.680 fl. oz). It is sterilized in UHT-steribzer at 137° C for 4.2 seconds. It has a shelf life of 12 months when stored at 25° C, after opening shall be kept in the refrigerator up to 48 hours.

ERRONEOUSLY FILED (RULE 20.5bis) TABLE 2

EXAMPLE 3

EXAMPLE 3 (as shown in Table 3) comprises an opaque, coloured beverage, containing hydrolysed collagen. The compositions, expressed in weight percentages, are listed below in Table 3. Used ingredients are as follows: water purified by ultrafiltration technology, Hydrolysed fish collagen Peptan® F 2000HD (Rousselot Inc.), crystalline fructose, white crystal sugar, natural peach aroma, natural mango aroma, natural guava aroma, amidated pectin [E440ii], citric acid [E330], antioxidant ascorbic acid [E300], acidity regulator: calcium lactate [E327], peach puree single strength, concentrated mango puree, colour: Riboflavins [ElOl] The beverage is aseptically filled into disposable plastic bottles 375ml (12.680 fl. oz). It is homogenized at 150 bar and sterilized in UHT-sterilizer at 137° C for 4.2 seconds. It has a shelf life of 12 months when stored at 25° C, after opening shall be kept in the refrigerator up to 48 hours.

TABLE 3

ERRONEOUSLY FILED (RULE 20.5bis)

EXAMPLE 4

EXAMPLE 4 (as shown in Table 4) comprises a transparent, coloured beverage containing hydrolysed collagen. Ingredients expressed in weight percentage, are listed below in Table 4. Used ingredients are as follows: water purified by ultrafiltration technology, Hydrolysed fish collagen Peptan® F 2000HD (Rousselotlnc.), crystalline fructose, brown sugar, natural strawberry aroma, natural lime aroma, natural dry green tea extract, citric acid [E330], acidity regulator: sodium citrates, clear strawberry juice concentrate, colour: chlorophylbns [E 141]. The beverage is aseptically filled into plastic disposable bottles 375ml (12.680 fl. oz). It is sterilized in UHT- sterilizer at 137° C for 4.2 seconds. It has a shelf life of 12 months when stored at 25° C, after opening shall be kept in the refrigerator up to 48 hours.

TABLE 4

ERRONEOUSLY FILED (RULE 20.5bis)

EXAMPLE 5

EXAMPLE 5 (as shown in Table 5) comprises an opaque, coloured beverage containing hydrolysed collagen. The ingredients, expressed in weight percentages, are listed below in Table 5. Used ingredients are as follows: water purified by ultrafiltration technology, Hydrolysed bovine collagen Peptan® B 5000HD (Rousselot Inc.), soy protein isolate SUPRO® PLUS 2640 DS (DANISCO), natural mango aroma, natural ripe apple aroma, amidated pectin [E440ii], brown cane sugar, citric acid [E330], antioxidant: ascorbic acid [E300], acidity regulator: calcium lactate [E327], apple puree single strength, saffron extract. The beverage is aseptically filled into disposable plastic bottles 375ml (12.680 fl. oz). It is homogenized at 150 bar and sterilized in UHT-steribzer at 137° C for 4.2 seconds. It has a shelf life of 12 months when stored at 25° C, after opening shall be kept in the refrigerator up to 48 hours.

TABLE 5

ERRONEOUSLY FILED (RULE 20.5bis) EXAMPLE 6

A preferred alternative embodiment of the present invention’s beverage comprises a clear, colourless beverage containing hydrolysed collagen, with vitamins and minerals as shown in Table 6

TABLE 6

* - Vitamin Premix Material-No. UF40105368; produced by DSM Nutritional Products Europe Ltd. The Vitamin Premix to covers 15% RDA / 100ml of: Vitamin B 12, Mg and Zn.

EXAMPLE 7

An alternative preferred embodiment of the beverage comprises a clear, colourless beverage containing hydrolysed collagen, with vitamins and minerals as shown in Table 7. TABLE 7

ERRONEOUSLY FILED (RULE 20.5bis)

* - Vitamin Premix Material-No. UF40105368; produced by DSM Nutritional Products Europe Ltd. The Vitamin Premix to covers 15% RDA of: Vitamin B12, Mg and Zn.

EXAMPLE 8

An alternative preferred embodiment of the beverage comprises a clear, colourless beverage containing hydrolysed collagen, with vitamins and minerals as shown in Table 8. TABLE 8

* - Vitamin Premix Material-No. UF40107368; produced by DSM Nutritional Products Europe Ltd. The Vitamin Premix to covers 15% RDA of: Vitamin B12, Vitamin B7 and Zn.

EXAMPLE 9

An alternative preferred embodiment of the beverage comprises a clear, colourless beverage containing hydrolysed and mineral water as shown in Table 9.

TABLE 9

* The mineral water has the following characteristics:

ERRONEOUSLY FILED (RULE 20.5bis) - Electrical conductivity at 20°C(pS/cm): 493.5;

- Potassium (K ⁺ ), mg/1: 0.60,

- Magnesium (Mg ^2- ), mg/1: 28.0;

- Calcium (Ca ²⁺ ), mg/1: 77.30;

- Ammonium (Mi-), mg/1: < 0.05;

- Bicarbonate (liCOs-), mg/1: 270.0;

- Chloride (Cl-), mg/1: 4.60;

- Sulphate (SO4 ^2- ), mg/1: 65.80;

- dry residue at 180 °C, mg/1: 340.3

EXAMPLE 10

EXEMPLARY METHOD OF PREPARATION:

The drink is obtained in the following method. The preliminary hydrolyzed collagen dissolution takes place in a container, made of stainless steel, with a maximal capacity 2500 1 or others), equipped with a stirrer and a flow heater on the way of the supplied water. The water in the container warms to a temperature: (min. 60 °C ÷ max. 90) ± 5 °C through the flow heater. The container stirrer starts working. The preliminary weighed amount of collagen in powder form is fed to the mixing chamber of a mixing unit, which automatically receives the fed powdery collagen, wets it uniformly with water, which circulates between the mixing unit and the mixing container, sucks it by means of vacuum effect and forwards it to the container for preliminary dissolution. This process takes at least 5 minutes. Thereupon the mixing unit is stopped, i.e. the circulation between the mixing container and the mixing unit is discontinued. The obtained preliminary solution of hydrolyzed collagen in a definite ratio with water is stirred additionally by the stirrer of the container for preliminary dissolution. The stirring takes at least 10 minutes.

The obtained preliminary concentrated dissolution of hydrolyzed collagen is subject to filtration through filter sleeves with permeability at least 20 pm. The concentrated solution is supplied through a pump to a filter unit, after which it is forwarded to a second mixing container, made of stainless steel, with a maximal capacity 105001 for preparation of the drink itself.

After completion of the filtration, the concentrated solution is transferred to the second mixing container. The second container is equipped with two stirrers - lower stirrer for a low level and an upper stirrer, as well as with an automatic level gauge, which reads off the actual liquid

ERRONEOUSLY FILED (RULE 20.5bis) volume, while the level gauge gives a signal to the stop valve, dosing drink water to the container. The lower stirrer starts working automatically after the liquid level in the second mixing container reaches up to 2000 1. Drinking water is added to the available quantity of 2000 1 solution upon already working lower stirrer, and when the liquid level in the second mixing container reaches up to 45001, the upper stirrer also starts working automatically.

For other stock keeping units (or SKU' s) where the beverage contains except collagen other ingredients such as: flavorings, vitamins, minerals etc... All additional ingredients, previously precisely measured, dissolved similarly to the collagen solution in a 10001 water for example, but with the temperature (6 ÷ 25 °C). This reconstituted solution is transferred in the same way as already transferred collagen solution. Adding of drinking water with the temperature (6 ÷ 25 °C) continues until the level gauge reads off 100001. Subsequently, the level gauge automatically gives a signal to the stop valve and the water feeding is discontinued. The stirring of the solution upon simultaneous operation both of the upper and the lower stirrer continues minimum 10 minutes. Thereafter the upper stirrer is stopped, and the ready drink is let to be stirred only by the lower stirrer.

The ready drink represents a solution of hydrolyzed collagen with concentration of 10 kg per 10000 1 water, i.e. 1 g/1 (1000 mg/1). This stirring continues and the drink is forwarded for sterilization and filling. When the liquid level in the second mixing container subsides under 2000 1 due to the continuous taking out of the product to the sterilizer of the filling line, this stirrer stops automatically. Before forwarding for sterilization and filling the ready drink has to be analyzed in order the quality property of the drink to be confirmed. For that purpose, a sample of the ready drink is taken through a faucet, which the second mixing container is equipped with. This sample is tested at a production laboratory about the conformity of the standardized indices regarding appearance, clarity, smell and flavor. In case of confirming evaluation, the ready drink can be forwarded to the filling block.

The tested drink is fed to a sterilizing block, which is located before the filling and closure block. The drink is sterilized, which ensures a microbiological stability against spoilage and worsening of its characteristics. Since the used raw material of drinking water and hydrolyzed collagen is not sterile and the process of preliminary dissolution and final dissolution to a standard drink itself is performed in a non-sterile environment, it is supposed, that the ready drink is polluted with microorganisms. A sterilization, which is known under the trade name UHT - sterilization,

ERRONEOUSLY FILED (RULE 20.5bis) is applied at ultra-high temperature for a short time (some seconds). This sterilization process guarantees the termination of the active cell’s microorganisms, ensuring the commercial sterility of the ready product. Furthermore, the stabilized by such kind of sterilization ready drink can be transported, exposed at commercial premises and sold at room temperature, i.e. it is not necessary to support a cold chain, offering uninterrupted transport and distribution of the products under refrigeration conditions. The so-called UHT sterilization allows maximally to preserve the usefulness of the active ingredients of the drink, while the freshness of the natural and original taste and flavor are kept, and the appearance of taste defects is avoided.

The drink comes into the first section of a pasteurizer - sterilizer, which comprises three sections: for heating, for maintaining the temperature and for cooling. Each of the three sections is equipped with fully insulated double-rooms, which indirectly contact each other through a tight separating heat-exchange surface. The drink, coming into the first preheating section, meets in an indirect reverse flow the already treated drink, which leaves the maintaining section. In this way an energy savings is ensured, while the incoming drink absorbs a part of the heat of the leaving drink. The preheating of the incoming drink facilitates and guarantees the easily and effective prompt achievement of the desired temperature of maintenance - the actual sterilization, which is performed in the maintaining section. In the maintaining section the drink meets in an indirect reverse flow (through the heat-exchange surface) the heat medium - a hot steam. The inputted sterilizing temperature and the maintenance time (stay in the maintaining section) are controlled automatically by means of computer system. After the time of maintenance at the inputted sterilizing temperature is over, the drink automatically is pushed into the preheating section, in order to meet again in a reverse flow, the incoming drink and to give a part of its heat. Subsequently, the already semi-cooled sterilized drink comes into the cooling section, where its final refrigeration takes place by means of ice-cold water in a reverse flow, until the desired given temperature of filling the drink is reached.

The recommendable mode of UHT sterilization of the drink is: sterilization at temperature 137 °C and maintenance for 4,2 s under 13000 1/h flow rate;

(it's possible these parameters to fluctuate according to the production/filling line characteristics ) the sterilized cooled product leaves the sterilizer at temperature 20 °C.

The filling of the ready sterilized drink is performed by means of an aseptic filling and closure mono-block. The filling takes place in a closed sterile system in order to prevent the

ERRONEOUSLY FILED (RULE 20.5bis) eventual pollution. The filling is performed under controlled conditions by means of sterile compartments, while each of them is cleaned through chemical detergents and is sterilized in advance.

A definite pressure of sterile air is maintained, and each filling block section has anti bacterial filters, which preserve the machine from microbiological pollution during the filling process. The maintained pressure difference (the sterile air pressure is bigger) ensures a barrier, which doesn’t allow the entering of non-sterile air. The insulation from the environment is also guaranteed by means of a liquid seal (disinfectant). The disinfectant represents a liquid barrier between the inside of the sterilizing, the filling and the closing carousel and the environment, and protect the machine against microbiological contamination.

The bottles where the product is filled in, are prepared after blowing from blanks - PET performs. The bottles are forwarded into the aseptic filling and closure block by means of a pneumatic belt conveyor. The blowing process of bottles by prompt ultra-heating at high temperature about 140 °C is performed just before their feeding to the filling and closure block, which guarantees the clean packaging. The packs are sterilized finally before filling by spraying with a hydrogen peroxide solution and a subsequent prompt drying by means of sterile, dry and warm air. The caps, which are used for the pack closure, are also sterilized in a compartment for a preliminary sterilization of caps. The sterilization of bottles and caps (also called “dry sterilization”) is performed at temperatures: 140 °C (for bottles) and 150 °C accordingly (for caps) in two steps:

VHP - injection (of hydrogen peroxide in evaporated phase); activation and drying with dry, sterile air.

The cooled drink for filling is forwarded at temperature 20 °C and is non-contact filled (there is no physical contact between the bottle and the filling valve during the dosage of the product). The temperature and the flow of the drink, fed for filling, are controlled automatically during the filling process. The filling of the drink is performed in nitrogen environment, ensuring an inert atmosphere, which additionally preserves the product from oxidation during the storage period.

The closed and labeled bottles are packed in stacks by means of a foil machine. The stacks are palletized automatically, and the ready pallets are forwarded to the storage area.

ERRONEOUSLY FILED (RULE 20.5bis) EXAMPLE 11

A hydrolyzed collagen beverage with hydrolyzed collagen as the sole protein (no other proteins present in the beverage) presents superior human gastrointestinal absorption of collagen when compared to similarly formulated collagen beverages which include other sources of protein due to the absence of receptor competition. By contrast, the inclusion of one or more additional sources of protein in an otherwise identical hydrolyzed collagen beverage will have reduced efficacy of collagen absorption due to receptor competition among the different protein types.

As shown in Fig. 1A, in the human gastrointestinal tract (GIT), food proteins can be digested into enormous numbers of free amino acids (AAs) and peptides by various digestive enzymes or microbial fermentation. Some of these peptides containing 2-20 AAs have various biological activities and are named bioactive peptides (BAPs). (1) Small peptides can be transported via peptide transporter 1 (PepTl) (2).

Food protein in the lumen is hydrolyzed to peptides by proteases in the brush-border membrane of the small intestine. The peptides may be absorbed (i) via transport across the intestinal epithelial cell monolayers through carrier-mediated routes; (ii), via the paracellular route via tight junctions; (iii) by transcytosis via vesicles; and (iv) via passive transcellular diffusion. For PepTl -mediated transport, peptides are coupled with protons (H+), which are then transported out of the enterocytes by H+/Na+ exchangers. Inside the cells and bloodstream, the peptides may be partly hydrolyzed to amino acids by peptidases. (Adapted from previous reports (3, 4, 5).

Thousands of di- and tripeptides are taken up into enterocytes via PepTl, which is mainly distributed in the intestinal brush-border membrane. PepTl deficiency in mammals impairs nutrient absorption and causes malabsorption that becomes visible on high protein and high fat feeding (6). As mentioned above, PepTl can transport various small BAPs including Pro-Hyp (PO). Collagen-derived Gly-Pro-Hyp (GPO) is hydrolyzed by intestinal mucosal apical proteases and the resulting dipeptide PO is transported across the intestinal membrane via PepTl (7). Shimizu et al. (8) demonstrated that collagen peptides (GAXGLXGP) are transported by energy- independent passive diffusion.

As shown in Figs. IB and 1C, at low BAP, concentrations (<Km), PepTl is the major contributor to the total transport rate. However, as the BAP concentration increases, the contribution of passive transport becomes more apparent. Because PepTl -mediated transport is saturated at Vmax, all further increases in the total transport rate are attributable to passive transport.

ERRONEOUSLY FILED (RULE 20.5bis) Accordingly, it may be concluded that the presence of other (non-collagen) low molecular weight peptides in a hydrolyzed collagen beverage would competitively and adversely reduce the rate and efficiency of absorption of the collagen peptides that are present.

Cited references

1. Xu, Q., Hong, H., Wu, J., & Yan, X. (2019). Bioavailability of bioactive peptides derived from food proteins across the intestinal epithelial membrane: A review. Trends in food science & technology.

2. Daniel, H. (2004). Molecular and integrative physiology of intestinal peptide transport. Annu. Rev. Physiol., 66, 361-384.

3. Gilbert, E. R, Wong, E. A., & Webb Jr, K. E. (2008). Board- invited review: peptide absorption and utilization: implications for animal nutrition and health. Journal of animal science, 86(9), 2135-2155.

4. Wada, Y., & Loennerdal, B. (2014). Bioactive peptides derived from human milk proteins — mechanisms of action. The Journal of nutritional biochemistry, 25(5), 503-514.

5. Xu, Q., Fan, H., Yu, W., Hong, H., & Wu, J. (2017). Transport study of egg-derived antihypertensive peptides (lkp and iqw) using caco-2 and ht29 coculture monolayers. Journal of agricultural and food chemistry, 65(34), 7406-7414.

6. Daniel, H., & Zietek, T. (2015). Taste and move: glucose and peptide transporters in the gastrointestinal tract. Experimental physiology, 100(12), 1441-1450.

7. Aito-Inoue, M., Lackeyram, D., Fan, M. Z., Sato, K., & Mine, Y. (2007). Transport of a tripeptide, Gly-Pro-Hyp, across the porcine intestinal brush-border membrane. Journal of peptide science: an official publication of the European Peptide Society, 13(7), 468-474.

8. Shimizu, K., Sato, M., Zhang, Y., Kouguchi, T., Takahata, Y., Morimatsu, F., & Shimizu, M. (2010). The bioavailable octapeptide Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro stimulates nitric oxide synthesis in vascular endothelial cells. Journal of agricultural and food chemistry, 58(11), 6960-6965.

EXAMPLE 12

A peptide weight of 2,000 Daltons is the optimal level for collagen peptides in hydrolyzed collagen beverage. Generally smaller peptides are superior for absorption of hydrolyzed collagen

ERRONEOUSLY FILED (RULE 20.5bis) because less energy is required for this energy-dependent process. However, 2,000 Da. is at or very near the optimal weight because the energy absorption savings achieved by further reducing peptide weight significantly below 2,000 Da. would be minimal.

Hou et al. demonstrated that collagen polypeptides had good moisture absorption and retention properties, and collagen polypeptide fractions CP2 (Mr < 2 kDa) was better than collagen polypeptide fractions CPI (2 kDa < Mr < 6 kDa). Ultraviolet radiation could cause skin photodamage and collagen polypeptide could alleviate the damage induced by UV radation. The action mechanisms of collagen polypeptide mainly involved enhancing immunity, reducing the loss of moisture and lipid, promoting anti-oxidative properties, inhibiting the increase of glycosaminoglycans, repairing endogenous collagen and elastin protein fibres, and maintaining the ratio of type III to type I collagen (1).

The use of collagen hydrolysates (CHs) as a nutraceutical agent in skin aging has gained increasing attention. Here, the effects of various doses and molecular weights of CH from silver carp skin on photoaging in mice were investigated. The ingestion of CH at 50, 100 and 200 mg per kg body weight led to a dose-dependent increase in the hydroxyproline, hyaluronic acid and moisture contents of the skin, but it had no significant effect on the mice body weight, or on the spleen or thymus index. Furthermore, ingesting CH with lower (LMCH, 200-1000 Da, 65%) and higher molecular weight (HMCH, >1000 Da, 72%) significantly increased the skin components and improved the antioxidative enzyme activities in both serum and skin (p < 0.05); LMCH performed better than HMCH. By contrast, gelatin (>120 kDa) ingestion did not bring a significant change compared to model mice. These results indicated that LMCH exerted a stronger beneficial effect on the skin than did either HMCH and gelatin, which supported the feasibility of using LMCH as a dietary supplement from silver carp skin to combat photoaging. (2). The CPs (200 Da), used in the H Song et al. study, has been demonstrated to have a stronger beneficial effect on the aging skin than other collagen hydrolysates (including gelatin and CPs with higher molecular weight >1000 Da) (2,3).

E. Proksch et al. conducted a monocentric, double-blinded, randomized, placebo- controlled supplementation study on the effects of a specific CH with molecular weight of 2.0 kDa. The results clearly revealed that oral intake of the specific CH had a beneficial effect on skin physiology, as indicated by increased skin elasticity after 4 weeks of daily consumption. The

ERRONEOUSLY FILED (RULE 20.5bis) observed effect was statistically significant (p < 0.05) after 4 and 8 weeks in the treatment groups compared to placebo. (4)

In a placebo-controlled clinical trial J Asserin et al. found that oral supplementation with fish collagen peptides (Peptan®F molecular weight of 2000 Da.) in women increased the collagen density in the dermis by 9%, as measured by high-frequency ultrasound; highly efficaciously increased the amount of water-binding glycosaminoglycans; the collagen content of human skin improved skin hydration as well as the density and structure of the collagen network of the dermis.

(5)

Some in vitro studies have demonstrated that Pro-Hyp and Hyp-Gly exert chemotaxis on dermal fibroblasts and enhance cell proliferation. Additionally, Pro-Hyp enhances the production of hyaluronic acid by dermal fibroblasts. These findings suggest that the amounts of Pro-Hyp and Hyp-Gly in blood are important factors to show the efficacy of collagen hydrolysates on skin health. Some study demonstrated that the use of the collagen hydrolysate with a higher content of Pro-Hyp and Hyp-Gly led to more improvement in facial skin conditions, including facial skin moisture, elasticity, wrinkles and roughness. (6)

Studies by Naoki Ito et. al. suggest the possibility for the use of collagen peptide and ornithine to improve skin conditions by increasing plasma IGF-1 levels - the used weight of the collagen peptide was 2000 Da. (7)

All studies with mice showed no significant differences in final body weight between the comparison groups. There are some studies on the effectiveness of lower molecular weight collagen peptides, but the most studied and confirmed effect on skin relates to collagen is weighing 2000 Da. Accordingly, it is logical to conclude that 2,000 Da. is at or very near the optimal peptide weight in respect of a beverage of simple hydrolyzed collagen and water without additives or supplements because the energy absorption savings achieved by further reducing peptide weight significantly below 2,000 Da. would be minimal.

In addition, it is logical to consider the possibility to concentrating collagen with a higher content of Pro-Hyp and Hyp-Gly. A higher content of Pro-Hyp and Hyp-Gly could be expected to result in a measurable increase in the optimal peptide weight.

ERRONEOUSLY FILED (RULE 20.5bis) Cited References Hou, H., Li, B., Zhang, Z., Xue, C, Yu, G., Wang, J., ... & Su, S. (2012). Moisture absorption and retention properties, and activity in alleviating skin photodamage of collagen polypeptide from marine fish skin. Food chemistry, 135(3), 1432-1439. Song H, Meng M, Cheng X, et al The effect of collagen hydrolysates from silver carp

( hypophthalmichthys molitrix) skin on UV-induced photoaging in mice: molecular weight affects skin repair. Food Funct. 2017; 8: 1538-46. Song, H., Zhang, L., Luo, Y., Zhang, S., & Li, B. (2018). Effects of collagen peptides intake on skin ageing and platelet release in chronologically aged mice revealed by cytokine array analysis. /. Cell. Mol. Med, 22(1), 277-288. Proksch, E., Segger, D., Degwert, J., Schunck, M., Zague, V., & Oesser, S. (2014). Oral Supplementation of Specific Collagen Peptides Has Beneficial Effects on Human Skin Physiology: A Double-Blind, Placebo-Controlled Study. Skin Pharmacol Physiol, 27, 47- 55. Asserin, J., Lati, E., Shioya, T., & Prawitt, J. (2015). The effect of oral collagen peptide supplementation on skin moisture and the dermal collagen network: evidence from an ex vivo model and randomized, placebo-controlled clinical trials. Journal of cosmetic dermatology, 14(4), 291-301. Inoue, N., Sugihara, F., & Wang, X. (2016). Ingestion of bioactive collagen hydrolysates enhance facial skin moisture and elasticity and reduce facial ageing signs in a randomised double-blind placebo-controlled clinical study. Journal of the Science of Food and Agriculture, 96(12), 4077-4081. Ito, N., Seki, S., & Ueda, F. (2018). Effects of Composite Supplement Containing Collagen Peptide and Ornithine on Skin Conditions and Plasma IGF-1 Levels — A Randomized, Double-Blind, Placebo-Controlled Trial. Marine drugs, 76(12), 482.

ERRONEOUSLY FILED (RULE 20.5bis) EXAMPLE 13

CARBONATION IMPROVES ABSORPTION OF A HYDROLYZED COLLAGEN BEVERAGE.

Introducing C02 to hydrolyzed collagen beverage can materially increase collagen abortion by slowing the natural increase the pH level in the gastrointestinal system during the digestive process. Typical carbonated beverages such as ordinary soda water and Coca-Cola contain approximately 6.2 grams/liter of C02 on average.

Gastrointestinal (GI) digestion of a dietary protein is influenced by the physicochemical characteristics of the protein. A complex combination of mechanical, physicochemical, and physiological processes is involved in the GI digestion of proteins in humans. Digestion of dietary proteins in the gastrointestinal system involves stomach and intestinal proteolytic enzymes. The protein digestion begins in the stomach, where pepsin breaks down the protein into smaller peptides. Pepsin exhibits maximum activity at acidic pH 2.0 and is inactivated at pH 6.5 in the duodenum. The pH in the duodenum is gradually increased to 5-7.5, due to the secretion of bicarbonate and pancreatic juices. The increased pH inactivates the gastric enzymes and gives the optimal activity of the duodenal enzymes (trypsin/chymotrypsin). When the semidigested peptide mix reaches the duodenum, the intestinal enzymes continue to break down the peptides. The final stage of digestion of proteins occurs on the surface of intestinal enterocytes by brush border enzymes, where peptides are hydrolyzed to amino acids as well as di- and tripeptides. The nutrients are then absorbed by the enterocytes of the jejunum and ileum and can be further degraded by intracellular proteases before entering the blood stream. (1)

At standard temperature and pressures, about 1ml (2mg) of carbon dioxide dissolves in 1ml of neutral solution (pH=7). In alkaline solutions, the gas is converted into carbonates and bicarbonates, and it is in this form that any ingested carbon dioxide is likely to be present in the intestinal tract. In general, one can assume that the daily average intake of carbon dioxide in food is equivalent to approximately lg daily of sodium bicarbonate or carbonate and therefore represents only a very small fraction of the amounts of these compounds in normal diets (2). In the past, several studies demonstrated that carbon dioxide plays a major role in the process of hydrochloric acid formation and secretion in the parietal cells of the gastric mucosa, after drinking carbonated beverages some of the carbon dioxide may be absorbed through the gastric wall, partly

ERRONEOUSLY FILED (RULE 20.5bis) contributing, together with the carbon dioxide from the interstitial fluid and plasma, to the formation of hydrochloric acid. (3)

No evidence has been found that the addition of carbon dioxide to the GI while collagen peptides are present affects the peptides molecular weight. However, a hydrolyzed collagen beverage to which carbonation has been included to a level of 6.2 g/1 can be expected to materially slow the normal increase in pH in the GI during the absorption process. Accordingly, the introduction of C02 at 6.2 g/1 will significantly extend the time period during which the pH level in the GI remains below 6.5 pH, thereby increasing the overall efficacy and rate of absorbtion of collagen peptides in the GI.

Cited References

1. Tapal, A., & Tiku, P. K. (2019). Nutritional and Nutraceutical Improvement by Enzymatic Modification of Food Proteins. In Enzymes in Food Biotechnology (pp. 471- 481). Academic Press.

2. Select committee on GRAS SubstancesEvaluation of the health aspects of carbonates and bicarbonates as food ingredients (SCOGS-26) Life Sciences Research Office, Federation of America Societes for Experimental Biology, Bethesda( 1975)

3. Cuomo, R., Sarnelli, G, Savarese, M. F., & Buyckx, M. (2009). Carbonated beverages and gastrointestinal system: between myth and reality. Nutrition, Metabolism and Cardiovascular Diseases, 19(10), 683-689.

EXAMPLE 14

USING PLANT-BASED COLLAGEN CAN BE EXPECTED TO PROVIDE CERTAIN HEALTH BENEFITS. HOWEVER, IT DOES NOT IMPROVE ABSORPTION OF COLLAGEN IN THE GASTROINTESTINAL SYSTEM.

Gorissen et al observed that plant-based proteins have relatively low essential amino acid and leucine contents when compared with animal-based proteins and human skeletal muscle protein. In addition, some but not all plant-based protein isolates are low in lysine and/or methionine contents. As there is a large variability in amino acid composition among the various plant-based protein sources, a balanced combination of different plant-based proteins may provide a higher quality protein blend. (1) Plant-based diets are known to (i) improved plasma

ERRONEOUSLY FILED (RULE 20.5bis) lipid concentrations, (ii) reduce blood pressure, and (iii) as part of a lifestyle intervention, the regression of atherosclerotic lesions. (2, 3) At the same time, Vliet et al. provided evidence that some plant proteins produce a lower muscle protein synthetic response compared to animal- based proteins (4).

In conclusion, there is evidence of health benefits from consumption of plant- based proteins including collagen. However, there is no evidence that utilization of plant-based collagen in a hydrolyzed collagen beverage instead of animal-based collagen improves the rate or efficacy of collagen absorption in the human gastrointestinal system.

Cited References

1. Gorissen, S. H., Crombag, J. J., Senden, J. M, Waterval, W. H., Bierau, J., Verdijk, L.

B., & van Loon, L. J. (2018). Protein content and amino acid composition of commercially available plant-based protein isolates. Amino acids, 50(12), 1685-1695.

2. Najjar, R. S., Moore, C. E., & Montgomery, B. D. (2018). A defined, plant-based diet utilized in an outpatient cardiovascular clinic effectively treats hypercholesterolemia and hypertension and reduces medications. Clinical cardiology, 41(3), 307-313.

3. Najjar, R. S., Moore, C. E., & Montgomery, B. D. (2018). Consumption of a defined, plant-based diet reduces lipoprotein (a), inflammation, and other atherogenic lipoproteins and particles within 4 weeks. Clinical cardiology, 41(8), 1062-1068.

4. van Vliet, S., Burd, N. A., & van Loon, L. J. (2015). The skeletal muscle anabolic response to plant-versus animal-based protein consumption. The Journal of nutrition, 145(9), 1981-1991.

EXAMPLE 15

ADDITION OF CERTAIN VITAMINS AND MINERALS (VITAMIN PACKAGE ¹ ).

The inclusion of certain targeted vitamins and minerals in hydrolyzed collagen beverage can improve absorption of collagen peptides in the gastrointestinal system, and can improve efficacy of collagen that has been absorbed. It can be concluded that following vitamins and minerals provide significant improvements:

• Magnesium

¹ Vitamin Package: magnesium, maganese, pyridoxine/B6, cobalamin/B12, and ascorbic acid

ERRONEOUSLY FILED (RULE 20.5bis) • Manganese

• pyridoxine ( Vitamin B6)

• cobalamin (Vitamin B12)

• ascorbic acid (Vitamin C)

Addition of Magnesium

Magnesium stimulates collagen synthesis expressed by fibroblasts in culture. Magnesium inhibits prolyl and lysyl hydroxylases and could be considered as antifibrotic. Magnesium is associated with elastin and plays a protective role in maintaining the extensibility of elastin. Magnesium associated proteoglycans in cartilage prevent the swelling and degradation of this tissue. Magnesium regulates the functional activity of integrins (1, 2, 3)

Studies suggest the presence of overall skin improvement in the use of Magnesium independently. This improvement was not demonstrated to be due to its effect on the absorption of collagen peptides. However, as Magnesium has been shown to stimulate collagen synthesis expressed by fibroblasts in culture, consumption of magnesium concurrently with hydrolyzed collagen can improve the net efficacy of orally ingested hydrolyzed collagen.

Addition of Manganese

PLEASE COMPLETE]

Addition of pyridoxine (Vitamin B6)

Studies demonstrate that pyridoxine (B6) induces a positive influence on human keratinocytes and fibroblasts. In particular, pyridoxine promotes fibroblast migration. Further, a statistically significant induction of keratinocyte proliferation is observed with in conjunction with human ingestion of pyridoxine. (4, 5).

Addition of Cobalamin (Vitamin B12)

Cobalamin (B12) is a naturally occurring organometallic compound containing cobalt that serves as an important water-soluble vitamin for human health. The recommended daily intake for cobalamin is 2.4 pg (6). This vitamin functions as a cofactor for two classes of human enzymes, namely, isomerases and methyltransferases. Consequently, cobalamin deficiency can cause disturbances in cell division, leading to neuropathy, nervous system disease and pernicious anemia. Vitamin B12 deficiency is common in people of all ages who consume a low intake of animal-

ERRONEOUSLY FILED (RULE 20.5bis) source foods, including populations in developing countries. It is also prevalent among the elderly, even in wealthier countries, due to their malabsorption of B12 from food. (7).

Altered cobalamin levels can lead to dermatological manifestations, which may indicate a deficiency or excess of this vitamin. The biochemistry and metabolism of cobalamin is complex, and diseases can be associated with alterations of this metabolic pathway. The cutaneous manifestations of cobalamin deficiency include hyperpigmentation (most commonly); hair and nail changes; and oral changes, including glossitis. Additionally, several dermatologic conditions, including vitiligo, aphthous stomatitis, atopic dermatitis, and acne are related to cobalamin excess or deficiency. (8)

The addition of cobalamin can improve the skin condition, however, was not carried out research studying the impact of cobalamin on the absorption of peptides, on the contrary discovered that whey protein can improve the absorption of cobalamin. (9, 10)

Addition of Vitamin C (Ascorbic Acid)

Several studies have shown that the presence of ascorbic acid (Vitamin C) can increase the natural collagen production of the body (12, 13, 14)

Optimal Concentrations of Targeted Vitamins and Minerals

Dietary reference intakes are set of reference values used to plan and assess nutrient intakes of healthy people. The Daily Value (DV) is a value published by the US Food and Drug Administration (FDA) containing the recommended intake of specified vitamins and minerals and has been determined sufficient to meet the nutrient requirements of nearly all (97+%) of healthy people without taking into account age and sex.

In general, the concentration of each of the targeted vitamins and minerals ( magnesium , pyridoxine, cobalamin, ascorbic acid) should not exceed 5% of the FDA) recommended DV per standard 500 ml. collagen beverage, or 10% per liter. Higher concentration of any of the targeted vitamins and minerals can be expected to significantly reduce the efficiency of absorption and can increase the risk of overconsumption.

ERRONEOUSLY FILED (RULE 20.5bis) TABLE 10

EXAMPLE 16

INCREASED RATIO PRO-HYP AND HYP-GLY INCREASES EFFICACY OF COLLAGEN

Two different types of collagen dipeptides, prolyl-hydroxyproline (Pro-Hyp) and hydroxyprolyl-glycine (Hyp-Gly) form a portion of peptides in hydrolyzed collagen. Both Pro- Hyp and Hyp-Gly are available at high concentrations for several hours in the human bloodstream following oral administration. Some in vitro studies have demonstrated that Pro-Hyp and Hyp- Gly exert chemotaxis on dermal fibroblasts and enhance cell proliferation. Additionally, Pro-Hyp enhances the production of hyaluronic acid by dermal fibroblasts. These findings suggest that the amounts of Pro-Hyp and Hyp-Gly in blood are important factors to show the efficacy of collagen hydrolysates on skin health. Some study demonstrated that the use of the collagen hydrolysate with a higher content of Pro-Hyp and Hyp-Gly led to more improvement in facial skin conditions, including facial skin moisture, elasticity, wrinkles and roughness.

Pro-Hyp and Hyp-Gly may have a low ratio of dipeptide-to-product content, about 0.1 g kg-1 of product (LCP), or it may also have a high ratio of dipeptide-to-product content, greater than 2 g. kg-1 of product (HCP). A recent clinical provides evidence that both LCP and HCP are effective supplements for increasing skin elasticity, reducing wrinkles, improving skin moisture and reducing the feeling of skin dryiness and roughness. This study further indicates that HCP provides greater improvement in skin elasticity, increased moisture and reducing wrinkles on facial skin than LCP (16).

EXAMPLE 17

MODIFICATIONS ALLOW INCREASED CONCENTRATION OF COLLAGEN.

Increased Range:

From 8.44g./l to 40g./l. Is the lack of whey in our product sufficient to overcome obviousness (or other) objections? It seems that this may be best for the collagen and water-only product. If we use it for flavored products, we increase the risk of an obviousness rejection. At

ERRONEOUSLY FILED (RULE 20.5bis) the same time, we must remain mindful that Prof. Pavlov stated that the optimal levels for absorption is 8.44 and under. We can go as high as we want as long as the high level is and can be supported as being critical. That’s the whole argument - ie. that whatever range or exact rate we use must be supported by the fact that (a) it hasn’t been previously disclosed by a prior patent or (b) even if it has been disclosed by a prior patent that our number is critical and causes untaught and unexpected results that the prior patent that may have been cited against us doesn’t disclose or teach.

Simple hydrolyzed collagen with a peptide weight of 10,000 Daltons (common in supplements and commercial food products containing collagen) and no further additives can be expected to have an optimal concentration range of approximately than 8.44 g/1.

In studies on the absorption, collagen peptides were shown to have a dose-dependent effect and increased concentrations of hydrolyzed collagen increases absorption of collagen peptides with a corresponding increased effect on the skin. In studies on the absorption, collagen peptides were shown to have a dose-dependent effect. (17). However, there is significant evidence of a limit to the saturation of peptides, to a point where a rise in concentration increasingly loses effectiveness. (18) The optimal range for absorption and beneficial effect on skin in respect of simple hydrolyzed collagen with a peptide weight of 10,000 Daltons (common in supplements and commercial food products containing collagen) and containing no further additives can be expected to be approximately than 8.44 g/1. However, the optimal range can be expected to fluctuate with the introduction of additional factors and modifications. Theoretical calculation of optimal concentration of collagen in hydrolyzed collagen beverage:

TABLE 11 . Theoretical Estimated Absorption Rates of Collagen Hydrolysate - % Absorbed

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Cited References

1. Galland, L. D., Baker, S. M, & McLellan, R. K. (1986). Magnesium deficiency in the pathogenesis of mitral valve prolapse. Magnesium, 5(3-4), 165-174.

2. Senni, K., Foucault-Bertaud, A., & Godeau, G. (2003). Magnesium and connective tissue. Magnesium research, 76(1), 70-74.

3. Sasaki, Y., Sathi, G. A., & Yamamoto, O. (2017). Wound healing effect of bioactive ion released from Mg-smectite. Materials Science and Engineering: C, 77, 52-57.

4. Fujii, K., Kajiwara, T., & Kurosu, H. (1979). Effect of vitamin B6 deficiency on the crosslink formation of collagen. FEBS letters, 97(1), 193-195.

5. Rembe, J. D., Fromm-Dornieden, C., & Stuermer, E. K. (2018). Effects of vitamin B complex and vitamin C on human skin cells: is the perceived effect measurable? Advances in skin & wound care, 31(5), 225-233.

6. B.R Rucker, J.W. Suttie, B.D. McCormick, L. J. Machilin Handbook of vitamin

7. Marcel Dekker Inc, New York (2001)

8. Allen, L. H. (2010). Bioavailability of vitamin B12. International Journal for Vitamin and Nutrition Research, 80(4), 330.

9. Brescoll, J., & Daveluy, S. (2015). A review of vitamin B12 in dermatology. American journal of clinical dermatology, 16(1), 27-33.

10. Wang, H, Shou, Y., Zhu, X., Xu, Y., Shi, L., Xiang, S., ... & Han, J. (2019). Stability of vitamin B 12 with the protection of whey proteins and their effects on the gut microbiome. Food chemistry, 276, 298-306.

11. Liu, G, Yang, J., Wang, Y., Liu, X., & Chen, L. (2019). Protein-lipid composite nanoparticles for the oral delivery of vitamin B12: Impact of protein succinylation on

ERRONEOUSLY FILED (RULE 20.5bis) nanoparticle physicochemical and biological properties. Food Hydrocolloids, 92, 189- 197.

12. Paxton, Jennifer Z., Liam M. Grover, and Keith Baar. "Engineering an in vitro model of a functional ligament from bone to bone." Tissue engineering part A 16.11 (2010): 3515- 3525

13. Shaw, G., Lee-Barthel, A., Ross, M. L., Wang, B., & Baar, K. (2016). Vitamin C- enriched gelatin supplementation before intermittent activity augments collagen synthesis. The American journal of clinical nutrition, 105(1), 136-143.

14. Lis, D. M., & Baar, K. (2019). Effects of Different Vitamin C-Enriched Collagen Derivatives on Collagen Synthesis. International journal of sport nutrition and exercise metabolism, (00), 1-6.

15. U.S. Department of Health and Human Services, Food and Drug Administration; available factsheets/V itamin_and_Mineral_Chart. pdf.

16. Inoue, N., Sugihara, F., & Wang, X. (2016). Ingestion of bioactive collagen hydrolysates enhance facial skin moisture and elasticity and reduce facial ageing signs in a randomised double-blind placebo-controlled clinical study. Journal of the Science of Food and Agriculture, 96(12), 4077-4081.

17. Shigemura, Y., Kubomura, D., Sato, Y., & Sato, K. (2014). Dose-dependent changes in the levels of free and peptide forms of hydroxyproline in human plasma after collagen hydrolysate ingestion. Food chemistry, 159, 328-332.

18. Ohara, H, Ichikawa, S., Matsumoto, H, Akiyama, M., Fujimoto, N., Kobayashi, T., & Tajima, S. (2010). Collagen-derived dipeptide, proline-hydroxyproline, stimulates cell proliferation and hyaluronic acid synthesis in cultured human dermal fibroblasts. The Journal of dermatology, 37(4), 330-338.

While this disclosure refers to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted for elements thereof without departing from the scope of the disclosure. In addition, many modifications will be appreciated by those skilled in the art to adapt a particular instrument, situation or material to the teachings of the disclosure without departing from the spirit thereof.

ERRONEOUSLY FILED (RULE 20.5bis) References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure.

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