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Title:
INTEGRATED PROCESS EXTRACTION OF PINEAPPLE BIOMASS INTO FIBERS AND NATURAL PRODUCTS
Document Type and Number:
WIPO Patent Application WO/2015/138584
Kind Code:
A1
Abstract:
The invention relates to a method for disassembling and extracting process for the sustainable production of natural fibers and several natural bio-based products obtained from pineapple plant biomass. The embodiment of the present invention, comprises a simultaneous mechanical hydrodynamic disruption and homogenization processing, a combination of extracting disruptive fractionating action that has the capacity to crack and breakdown thoroughly plant cells and plant tissues from different parts of the pineapple plant to unlock biofibers and multiple natural components at cytologic level and plant tissues.

Inventors:
PUTILIN BETTY (US)
BAEZ-VASQUEZ MARCO A (US)
Application Number:
PCT/US2015/019918
Publication Date:
September 17, 2015
Filing Date:
March 11, 2015
Export Citation:
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Assignee:
ECOTECH DEV LLC (US)
International Classes:
A61K36/00; C12N9/50; D01C1/00
Foreign References:
US3002891A1961-10-03
US4925693A1990-05-15
TH2145U
Other References:
BANIK ET AL.: "Utilization of pineapple leaf agro-waste for extraction of fibre and the residual biomass for vermicroposting", INDIAN JOURNAL OF FIBRE & TEXTILE RESAERCH, vol. 36, June 2011 (2011-06-01), pages 172 - 177
MOHAMED ET AL.: "Characterization of pineapple leaf fibers from selected Malaysian cultivars.", JOURNAL OF FOOD, AGRICULTURE & ENVIRONMENT., vol. 7, no. 1, 2009, pages 235 - 240
Attorney, Agent or Firm:
LAMPEL, Justin (P.C.555 Skokie Blvd., Suite 50, Northbrook IL, US)
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Claims:
What is claimed is:

Claim 1. A method for isolating and extracting a plurality of constituents from, a pineapple plant comprising

obtaining a pineapple plant

separating said pineapple plant into at least two portions, one first portion containing pineapple plant stems and one second portion containing pineapple plant leaves t wherein each of said portions are processed to produce constituents from said pineapple plant;

isolating a plurality of constituents frora each of said first portion contain pineapple plant stems and said second portion containing pineapple plant leafs.

Claim 2. The method fo isolating and extracting a plurality of cons ituen s from a pineapple plant according to Claim 1 further comprising the stop of subjecting said pineapple plant to hydro-mechanical procedures to remove any dirt or foreig debris positioned on said plant.

Claim 3. The method for isolating and extracting a plurality of constituents from a pineapple plant according to

Claim 3 wherein said pineapple plant Smootii Cayenne, Red Spanish, Queen and Abaca:■: i , Hilo, Sugar Loaf, White Sugar Loaf, Kona Sugar Loaf, Natal Queen, Fernambuco, Queen, Green Spanish, Manzaria, DelMonte Gold, P&rolera, Maipuri , Singapore Spanish, Singapore Canning, Hawaiian Gold, Super Sweet, Ultra Sweet, MD2, or combinations thereof.

Claim 4, The method for isolating and extracting a plurality of constituents from a pineapple plant according to Claim 1 wherein said stems are subjected to one or more processes to form a plurality of first stem, separation fractions and said leaves are -.subjected to one or more processes to form a plurality of first leaf separation fractions.

Claim 5. The method for isolating and extracting a plurality of constituents from a pineapple plant according to Claim 1 wherein said step of preparing said plant for isolation of one or more various plant based constituents includes subjecting said plant to hydxomeehanic i procedures, thermomeeftanicai procedures, extractive chemical pracedares, or combinations thereof. Claim 6. The method for isolating and extracting a plurality of constituents from & pineapple plant according to Claim I further including the step of subjecting said leaf portions to a delaminatiori process*

Claim- 7. The method for isolating and extracting a plurality of constituents from a pineapple plant according to Claim 5 wherein said delamination. process separates said leaf portions Into at least three components,

Claim 81 The method isolating and extracting a plurality of constituents a pine pple plant to Claim 1 wherein said pineapple plant been removed from the ground no longer' than 24 hours,.

Claim 9. The method for isolating and extracting a plurality of constituent from a pineapple plant according to Claim 1 -whe ein, said stem portion and said leaf portions are further processed to isolate additional, products .

Claim 10. The method for isolating and extracting a pluralit of const ituents from a pineapple plant to Claim 1 further including the steps of fractionation. Claim 11. The method for isolating and extracting a plurality of constituents from a pineapple plant to Claim .1 further including the steps of ce.ntrifugation.

Claim 12. The method for isolating and extracting a pluralit of constituents from a pineapple plant to Claim 1 wherein said pineapple plant stems are processed to provide bromelain -

Claim .13. The method for isolating and extracting a plurality of constituen s from a pineapple plant to Claim 1 wherein said pineapple plant stems" are processed to provide one or more types of fibers.

Claim 14. The method for isolating and expecting a plurality of constituents fro.ni a pineapple plant to Ciaiiu 1 wherein said pineapple plant stems are processed to provide sug rs ,

Claim 15, The ethod for isolating and extracting plurality of constituents from a pineapple plant, to l im wherein said pineapple plant steins are processed to provid flour. Claim 16. The method for isolating and extracting a plurality of constituents from a pineapple plant to Claim 1 wherein said pineapple plant leaves are processed to provide fiber .

Claim 17. The method. or isolating and extracting a plurality of .constituents from a pineapple plant to Claim 1 wherein said pineapple plant leaves are processed to provide lumen .

Claim 18. The method for isolating and extracting a plurality of const.itusnts f rtiai a pineapple plant to: Claim 1 wherein said pineapple plant leaves are processed to provide paper ,

Claim 19. A method for isolating and extracting a plurality of constituents from the stems of a pineapple plant comprising obtaining stems from a pineapple plant;

subjecting said stems from a pineapple plant to a first set of procedures to form at least two first leaf .separation f actions

isolating a plurality of constituents f om each said first leaf separation fractions. Claim 20. A method for isolating and extracting a plurality of constituents from the leaves of a pineapple plant comprising obtaining leaves from a pineapple plant ;

subjecting said leaves from a pineapple plant to one or; sore pro.ces-s.es to form a plurality of first leaf separation fr c ions-;

isolating; a plurality of constituents rom each said first leaf separation fractions.

Description:
INTEGRATED PROCESS EXTRACTION OF PINEAPPLE BIOMASS

INTO FIBERS AND NATURAL PRODUCTS

FIELD OF THE INVENTION

[00013 The present invention relates to methods and products obtained from plants; and more particularly to a method of extracting multiple components, including fibers, active bio- catalytic enzymes and several natural bio-organic compounds from eap le piants .

BACKGROUND OF THE INVENTION

[0002] Rising global populations- have lead to a increasing demand for energy, food.,. shelter and natural., green, and renewable raw materials. As petroleum and petrole derivatives maybe reaching saturation and the ever-growing envi ron-nental pressure caused by the -widespread consump ion of petroleum based fuels, or e roc emicals , a- .shift into the development of biodegradable and environmentally acceptable mats-rials from natural resources is increasing, vfit this comes an increasing demand to develop substitutes that replace petroleum based materials with renewable natural materials that reduce pollution and minimize envi onmental footprint.

[0003] One such focus is on the use of sust inable and renewable materials. Pineapple (Anan s o osus] belongs to the Bromeliaaeae family. it is a very popular plant worldwide because of the palatable pleasant fruit that it produces. The first native plant varieties originated from South America and are now widely cultivated in the great majority of the tropical and subtropical countries . Given the widespread growth, pineapple plants have gained attention as a renewable source for the production of chemical , natural products, food additives and foiofaels. A variety of natural products can foe extracted from pineapple residual plants. For ex mple, the pineapple plant is a very important source of natural virgin fibers that exhibit- high specific strength, stiffness and hygroscopic properties. Th superior mechanical properties of pineapple leaf fibers are associated with high cellulos content, up to 5% to 90%, and comparatively low microfibrillar angle. Due to the unique properties- exhibited by pineapple ' leaf fiber, they can he used: as excellent potential reinforcement in biocomposite matrices and. functional bioxnateriais.,

[0004} I general, the processing of pineapple begins with the harvesting of the pineapple fruit and/or collecting of the fruit for the subsequent packaging process:. Once the fruit collection i complete,, the re aining pineapple plan is typically left on the ground for subsequent disposal processes. In order to clear and prepare the ground fo the next cultivation cycle, pineapple growers often use herbicides such as $ί' -dlmethyl~ , .f ' -bipyridinrum d.ichloride, to aid in the process. Such herbicides are applied onto the surface of the remaining pineapple plant- Microbial cell degradation digests the remaining plant tissue, followed by the pineapple plant field burning and final mechanical grinding up for complete elimination .

[00053 While an effective means for eliminating the waste, field burning enhances the release of multiple fine particles and/or air pollutants such as carbon compounds, nitrogen oxides { θχί , Sulfur Oxides or Dioxides (Sox) , Carbon Monoxide {CO) and methane {€¾},- formate iKCQO) , acetate ions (Cf¾C00) , oxalyate ions (C 2 0. 2 -4) , sulfur dioxide (SQ Z ) r nitrates (Nt¾) and so forth. In addition to the use of chemic ls, the field burning process involves production of green house gases due to the herbicide treated plant burning process. In .addition, slow organic decomposition of the plants on the ground causes the production of insects which affect dairy farms and livestock of adjacent farmland. The present invention addresses some of the issues involved in the processing of pineapples by providing for methods that exhibit a significant capacity to utilize the pineapple plant biomass waste left at the cultivation ground after the fruit has been harvested. The remaining plant biorriass represents an agricultural residue and a renewable biomass feedstock generated immediately after the fruit has been collected < Γ0006Ι The present invention helps to introduce sustainable practices for hio ass waste managemen and pollution prevention, by means of eliminating the massive use of herbicides. The present invention is adapted to process most of the pineapple plant biqmass, thereby eliminating the eq i ement of landfill space, reducing the use of expensive and contaminant trad.itio.nai disposal methods which use the combination of chemical., biological, combustion, and .mechanical processes to decompose the pineapple plant bioraass after fruit harvesting. I« addition to waste management and pollut on ' prevention, a system and method designed to create natural product from the pineapple plant can address othe issues, such hs the econom c and/or environmental impacts relating to the Stable Fly { Sto oxys c.<3J ci ' trans) .

[0007] I su s such as outbreaks .and/or parasite infection tend to increase due to pineapple waste accumulation.. For example, during 2009 and 2011 in Central Amer can, countries, a

68% increase by about in outbreaks and attacks by Stable Fly was observed, particularly near pineapple fields. Stabl Fly exhibit strong reproduction cycles on plant pineapple waste and the problem becomes raore intense during high humidity and heavy rain periods. As a result of the high frequency fly outbreaks, livestock such as cows exhibit a dramatic reduction in milk yields (up to 50% growth and weigh gain. These effects on the animal can be translated into economic losses for livestock farmers. · It has been estimated that an animal may foe prevented fro gaining as much as 600 grains per day if continuously attacked by the Stabl Fly, i.e. for 2 hours. Therefore, the elimination of this parasite benefits cattle growers by securing higher production yields for meat and dairy.

100083 Accordingly, the present invention could provide multiple ' economical and sustainable environmental benefits by preventing the use of herbicide-cheniical-burning-mechanical disposal process and providing pineapple growers a mechanism to save millions of dollars through fast and economical means to prepare the ground for the ' next pineapple cultiva ion cycle. In addition. to the above, the present invention will bring economical and sustainable environmental benefits for the cattle and dairy producers by disrupting the Fly Stable cycle and eliminating attacks o dairy and livestock farms.

SUMMARY OF THE I VE TION

[0009] The present invention describes an innovative process for the extraction of pineapple biomass- components, including fibers, active biocatalytic enzymes and several natural bio- organic compounds, Such materials are extracted using a process which includes the disassembling extraction of Pineapple plant agricultural waste, defined a any portion of the plant but the fruit of the plant as well as any waste rom processing the plant. The process further includes downstream steps that exhibit a direct interdependence;, i.e. each subsequent step in the processing relies on previous steps to form a processing cascade, during the extraction of natural substances, biocatalysts and commercial biofiber products from Pineapple p1ant biomas .

[0010] In a preferred embodiment , innovative method includes provides a single and integrated extraction of pineapple plant biofibers, multiple natural products and functional derivatives from the tissues of the pineapple plant biomass components. The present invention, therefore, enables a fast, integrated and cost-effective extraction of for example, dietary fiber for human consumption, sulfydril endopeptidases Bromelain enzyme complex, and other hydrolases including earbohydrases, fiber for paper production, functional starchy flour used in rood and industrial applications, xylose from the hyaroiytic pretreatment of fiber, xylitol for low caloric natural sweeteners and lumen for organic fertilizer, compos or anim l feed; in addition fibers can be further extracted to produce microfibers and nanofiber for multiple industrial applications .

[0011] Preferably, the process is adapted to provide a minimal steps resulting in disassembling of the pineapple plant biomass to extract various components or constituents using a combination of mechanical, the ' rnso-mechani al and chemical procedures in order to obtain biofibers, natural products and derivatives of commercial relevance. An embodiment of the present invention comprises a simultaneous mechanical hydrodynamic disruption and omogenization processing, a combination of .extracting disruptive fractionating action th t has the capacity to crack, and breakdown plant cells and plant tissues from, many different parts of th pineapple plant, thereby .unlocking biofibers and multiple natural components at cytologic level and plant tissues. In the disassembling process it is possible to: fractionate to homogeneity and obtain consistent uniform fractions. In the embodiment of the present invention the disassembling extracting process comprises hydrodynamic abrasion processes. Ά series of extractive products are obtained from, the disassembling extraction of the : pineapple plant foiomass. The pineapple biomass was subjected to. an extraction process and the treated material is subjecte processes to separate the water soluble fiber material, including water soluble sugars, proteins and complex carbohydrate .

[0012] The pineapple ioaiass disassembling extraction process in accordance with the present, invention can be used to as a single process to isolate various components from the pineapple plant using two or more parts of the plant. Alternatively,, the pineapple biomass disassembling extraction process can be adapted to provide processes for isolating constituents from only one part of the pineapple plant. Accordingly, in one embodiment of the invention, a method for isolating and extracting a plurality of constituents front a pineapple plan comprises: obtaining a pineapple plant; separating said pineapple plant into at least two portions, one first portion containing pineapple plant -stems and .one second portion containing pineapple plant leaves, wherein each of said portions .are processed to: produce constituents from said pineapple plant; isolating a plurality of constituents from each of said first portion, -.contain pineapple plant :stems and said, .second portion containing pineapple plant leafs. The initial steps provide for a first stem separation fraction and a first leaf separation fraction. The process further includes: steps to further process the .first separation fractions, thereby isolating additional components from the first ctem : or leaf.

[00131 In an alternative embodiment , a method for isolating and extracting a plurality of constituents f om: the stems of a pineapple plant comprises: obtaining ste s from a pineapple plant; subjecting said stems from a pineapple plant to a first set of procedures to form at least two first leaf separation fractions; isolating a plurality or " constituents from each said first leaf separation fractions. The process further includes steps to further process the first separation fractions, thereby isolating additional components from the first stem or f actions ,

[0014] In an alternative eMbodiroent , a method for isolating and extracting a plurality of constituentis. frorn the leaves of a pineapple plant comprises: obtaining leaves from a pineapple plant; subjecting said leaves from a pineapple plant to one or tttore processes to form a plurality of first leaf separation fractions;; ' isolating .a plurality o. constituents from each said first leaf separation fractions. The process further includes steps to- farther process the first separation fractions, thereby isolating additional components frorn the first leaf fractions. £00153 Accordingly, it is a objective of the present invention to teach an Integrated process tor extraction ' of ■pineapple hio ass into fibers and natural products.

[00163 It is a further objective of the present Invention to teach fibers and natural products obtained f om pineapple.

[0017] It is yet another objective of the present invention to teach fibers and natural product obtained from a process of extraction ffora a pineapple.

[0018] It is a still further objective of the invention to teach the extraction of pineapple bloraass fibers., active biocatalytic enayotes and natural bio-organic compounds. t0019] .It is a further objective of the present invention to teach the extraction of pineapple biomass fibers, active biocatalytic enzymes and natural bio-organic compounds using a one step process which includes the disassembling extraction of. Pineapple plant agricultural residues,

(0020! It is yet another objective of the invention to teach the extraction of pineapple biomass fibers, active biocatalytic enzymes and natural bio-organic compounds using the pineapple stem,,

(0021! is> yet another objective of the invention, to teach the extraction of pineapple biomass fibers, active biocataiytic enzymes and natural bio-organic compounds using the pineapple fruit pulp and core..

[0022! It is yet another objective of the invent ion to teach the extraction of pi.neapp.ie .biomass fibers f active biocatalytic enzym s and natural bio-erganic compounds using the pineapple crow leaves >

[00231 It is yet another objective of the inventio to teach the extraction of pineapple biomass fibers, active .bipcatalytlc enzymes and natural bio-organic compounds using the pineapple leaves ,

[0024] It is yet another objective of the invention teach the extraction of pinea ple : biomass fibers, active: biocatalytic enzymes and natural bio-organic compounds using the pineapple fruit peel,

£.00253 It is a still further objective of the invention to teach the extraction of pineapple bioraass fibers, active bxocatalytie enzymes and natural bio-organic compounds using the pineapple st rn fruit pulp and core, fruit peel, crown leaves and leaves.

£0026 J Other objectives and advantages of this invention will become apparent froro the following description, taken in conjunction with any accompanying drawings wherein, are set forth, by way of illustration and example, certain embo imen s of this invention. Any drawings contained herein constitute a part of this specification and include exemplary embodiments of the present inventio and illustrate various objects and features thereof!

BRIEF DESCRIPTIOH O THE FIGURES

[00273 Figure 1ft is a flow chart illustrating various end products or substitu.ent , suc as bi©fibers, natural products and derivatives, isolated and obtained frora the extraction process in accordance wi h the present invention-?

[0028] Figure IB is a flow chart illustrating the general steps to obtaining end products or plant const! Lu.snts, such as biofibers, natural products and derivatives, isolated and

1! obtained from the extraction process in accordance with the present invention;

10029] Figure 2 illustrates a pi.0eapp.le plant, showing several of the major structural components of a typical, plant,'

[00303 Figure 3 is a bar graph illustrating the bromelain activity in different pineapple varieties, including Cayenne Lisa MD2 f Ma.-nza.na, and Perolera }

0031J Figure 4 illustrates the effects of pH variation on residual bromelain activity;

[00323 Figure -5. is a bar graph representing the total monosaccharide, production during in vitro digestibility test of lumen, from pineapple natural, fibers.

DETAILED DESCRIPTION OF THE INVENTION

[00333 While the present invention is susceptible of embodiment in various forms, there is shown in t e drawings ana will hereinafter be described a presently preferred, albeit not limiting, embodiment with the understanding that the present disclosure is to be considered an exemplification of the present invention and is not intended to limit the invention to the specific embodiments illustr ted.

[0034] The present invention, is d rected towards a process for extracting various products from a pineapple plant,, and is reiereed to generally as a pineapple bioraass disassembling extraction process 10. The pineapple biomass disassembling extractio process 10 involves disassembling and extracting processes that utilises the agricultural waste or residues of the pineapple plant biomass and fruit as a raw material to extract various components. The pineapple biomass disassembling extraction process 10 may include using stems only, leaves only, or combinations of stems and leaves as a starting point.

Accordingly, the processing cascade ma include stems for isolating, components from the stems, leaves, or combina ions, of stems and. leaves. The extraction process is completed using, in combination one or more . , hydro-mechanical, thermo-mechanical and extractive: chemical procedures to obtain long, short, micro, nano-foiofiberS and natural products of commercial relevance.

The extractantS: mainly contain cellulose, hemicellulose, xylose, pectin, functional starches, chlorophyll,,, com lex carbohydrates, natural fibers., natural enzymes ' , dietary fiber and other bioorgahio derivatives and nutraceutical compounds. As will be described in greater detail, the pineapple plant may be separated into stems and leaves. The- leaves may undergo a de-lamination process in which the stems ere processed so that portions of the plants inay be stripped away to form individual components, forming soluble components, semi-solid components, and solid components. As part of the same process or cascade, or as a separate individual cascade, the stems may undergo a maceration process to form a solid component and a liquid component. Each of these initial round processing components can further b processed to form additional components.

[O03S3 Referring to Figure lA, a flow chart illustrating various end products such as faiofibers, natural products and derivatives, isolated and obtained from the extraction process in accordance with th presen invention is shown. The pineapple: biomass disassembling extraction process ID utilizes various steps involving one or more hydro-mechanical, therrao- mechanical and extractive chemical procedures to process all parts of the pineapple plant. Referring to Figure 2, a standard pineappl plant 12 is illustrated. The pineapple plant 12 contains a mother stump 14, a first rat©or. stump 16> a butt 1-, and a sucker 19. The pineapple fruit 20 rests at of near the highest point of the plant 12, The pineapple fruit 20 contains a crown 22 with crown leaves 24. A slip 26. acts as a rudimentary fruit with an exaggerated crown. The slip 26 develops from buds i the axils of the leaves ·2¾ extending from the fruit stalk or stem 18. Roots 30 help provid the plant 12 needed ntitrients and water. The present invention provides for a mechanism to utilize most, in not all of the pineapple plant 12.

[0036] Referring ' back to Figure 1A, the pineapple biomass disassembling extraction process 1G is designed to process stem homogenate 32 , fruit pulp or core 34, fruit peel 36, crown leaves 38, leave homogenate 40, individually or in combinatio . Each of the components of the plant is further processed to provide additional sub-components. For example, the stem 32 is initially processed to provide an aqueous phase 42 and a solid fiber residue phase 44. Each of the two phases can be further processed to provide for additional isolated components. The processed liquids 42 can be further processed to provide one o more enzymes 46, such, as but not limited to SH-esterases I, flour 48, such as functional starch flour containing at least one enzyme, suc as bromelain, sugar 50 such as glucose and glucose derivatives, and stem homogenate residuals 52. The solid fiber residue 44 can be processed to provide dietary fiber -lation fiber 52. Such fibers can form the basis of nutraceutlcals 54. The solid fiber residue phase 44 can be further processed to form sugars 56 such as Xylose, Arihinose and other C5 and C6 monomeric sugars, biosurfactants 58, and food additives 60 and cosmoceutioias €1. The sugars 56 can foe processed and/or used for sugar alcohols 56ft or low caloric natural sweeteners 568.

[0037] The fruit pulp and core can be processed to provide for fiber juice 62, enzymes such as but not limited to SH- esterases I and other enz me extracts 6 . The enzymes 64 can be used to provide biotherapeutics 66. The fruit peel 36 is further processed to plant hydro! yzate-s 68, syrups 70, organic acids 72 and biobased chemicals 74.

The fract onated crown leaves 38 can be processed to provide lumen 76, short fibers 80, and micro and nanofibers 80. In addition, to the same components processed from the crown leaves, the other leaves of the pineapple plant can be processed to isolate fibers 84 for paper , natural pigments 86, iignl and derivatives 8-8, and other residuals 90 which m y be useful for advanced biofueis,

[00383 Figure IS provides a generalized overview of the disassembling. extraction process 10. The disassembling extraction process 10 starts with the obtaining or provided of plants, such as one or more varieties of pineapples, see step

100. The plants are inspected to insure the plants meet organoleptic and biochemical criteria. The pineapples are washed, step 102, In the washing phase, waste such as dirt and dust is washed away. ft second inspection is performed to remove any unwanted raw materials. The plant m y be dissected to separate- various components, step 1.04, into for example leaves

106, stems 108, and other components 110, such as fruit body, peels. The leaves 106, stems 108, and other components 110 may be further processed separately. The stems 108 are then fractioned by cutting into predetermined sizes, ste 112... The fragmented material can be subjec to macerat on in water in the blending stage, 114. After maceration, the slurry formed is separated' into solids and liquids, The solids are processed to form for example fibers and k-latiori. The liquid portion is processed to form for example, flour and bromelain. Various components are separated (i.e. fiber from flour), step 116. Additional separation and centrifugation steps, 120 and 122, are performed. Additional processes are undertaken to extract va ious plant constituents or products, step 124, such as bromelain / . 126, fiber 128, flour 130, or sugars 132.

10039] ' To the separated leaves IG ' 6, several steps are applied thereto to provid additional plant consti uents o products, the leaves are preferably processed using a deiaminatation step . 1.3.3 i which layers of plant tissue are removed.: For example, the delaminatation process strips away various plant layers, including, plant waxes,- dermis, epidermis and/or pericarp. The delaminating process forms various components, I } solubles, which include chlorophyll and nan . o~£ibers, 2} semi solids, which include: some na o--fibers, and lumen, and 3} solids, which include long fibers. These leaf first building block can be further processed ' to form additional isolated product . The leaves 10-6 undergo fraction steps 134, separations steps, 136, and isolation of products 138, such as fiber 138, lumen 140. A. paper extraction process " 142 occurs via additional steps, such

1? as pre-treatment. 144, washing s eps 146, bleaching 148, arid separation techniques 150.

[00 03 The pineappie biomass disassembling extraction process

10 includes a minimal step d sassembly extraction that efficiently enhances the molecular, plant cellular and tissues. separation of the biomass pineappie components. This innovative procedure facilitates the fast separation of the main pineapple plant fibers bi-opolymerie constituents and other natural compounds towards their further isolation, purification, refinement and deriratization. The final products can be alternatively separated by specific -molecular weights, according to their physieoehemicai, hydrodyna ic, biochemical, mechanical, thermo-mechanica.I and Theological properties, and/or by sorting them into homogenous materials. By separating the various components of agricultural materials, the pineapple biomass disassembling extraction process 10 offers significant functional ' extractab iity, and cost-effective production of fibers and natural compounds, as well as back-end economic and environmental advantages to minimize the land filing of residual agricultural biomass, preventing the production of GRG and other toxic substances, and the elimination of traditional contaminant procedures such as the open field burning. The pineapple biomass disassembling extraction process 10 has many possible applications for both organic end non-organic products. [0041] The pineapple hioraass disassembling extraction process 10 is preferably designed as a one step disassembly and extracting method for preparing multiple extractants containing fractions as described above and illustrated in Figure 1A. The pineapple plant or feedstock comprises natural plant biopolyraers listed in . Table 1. As shown in the Table l f the typical pineapple plant includes hemicelluloses, cellulose, lig i as well as starches and multiple active enzymatic systems, constitutive proteins, dietary fiber as well as sugar monomers {glucose, fructose, arabinose, galactose, mannose, xylose) and oligosaccharides sugars (arabinoxyians, glucoraarmas, arahinogalatahs, xyioglucans) .

[00421 Table 1: Analysis of the Main Constituents in the Pineapple Plant

[0043] The pineapple blomass disassembling extraction process 10 results in the production of fibers and several natural products obtained from the pineapple plant such as fibers, enzymes functional biopolyrn rs and multiple natural compounds. In particular the pineapple blomass disassembling extraction process 10 provides a method of preparing, comprising fractionation, extraction and concentrating the naturai products from pineapple plant biomass that comprises the extraction of biopoiymeric fibers dietary fiber, functional starches, proteolytic enzymes as phytotherapeut.ic agents, a and β-.D- Mannopyranosidases, natural pigments, monome ic sugars and low caloric natural sweeteners, comprising; ther o-meehanical maceration and blending of the leaves and stems of the residual pineapple plant. The pineapple biomass utilized includes ail- components of the pineapple plant and most preferable, the leaves, stexas as well as the pineapple peel, crown and center- heart of the fruit.

[0044] Various pineapple plant varieties can be used, and include species such as Smooth Cayenne, Red Spanish, Q een and

Ahacaxi and most preferable pineapple cultlvar varieties such as: Hilo ' r Sugar Loaf, White Sugar Loaf, Kona Sugar Loaf, Natal

Queen, Perna huco, ' Queen, Green S nis / Manzana, DelMonte Gold,

Perolera , M&ipuri , Singapore Spanish, Singapore Canning,

Hawaiian Gold, Super S eet / ' Ultra Sweet and MD2. Liquid streams, semisolid and solid residues are produced and separated after the pineapple biomass has been subjected to the mechanical disassembling extracting process. Further downstream stages are designed to achieve higher yields and efficiency whil maintaining process -efficiency using minima.! number o stages.

Kediletion. in -stages reduces energy, residence time, lowers manufacturing and capital costs. In. rder to isolate specific fractions according to particle size and consistency various techniques ' are used, individuall or combination, including separation techniques, chromatographic techniques, decanting

(decanter centrifuge, hydrOcyelon and elarifier, hig speed and ultracentrifugej and filtration systetns (ultraf ltrat on f nanoftitration, reverse osmosis, belt filters, disc and drum filters:}, including drying support ifreere drier, spray drier, wind tunnel drier } . Such system allows for higher levels of separation and purity of the final compounds and products.

[0045] The extraction Of multiple types of fibers and natural compounds was carried out using several pineapple eultivars such as Smooth Cayenne Lisa, Ma.na;za.na-, D2 and Perolera, widely cultivated in the Central ¾meripa. and South America Regions,

Table 2 illustrates several pineapple varieties tested using the process in accordance with the present invention. The table also indicates the presence of several components found in the stems and leaves of each of the varieties. £00463 Table 2, Composition of Stems and leaves in varies ineapple varieties:

[0047} Samples were collected from pineapple growe s located within 50 to 70 miles distanc from the< processing facility, The pineapple laio ass was collected from pineapple growers that com ly with the characteristics of the biomass required for our process mainly to: keep the freshness of the plants to avoid deterioration and reduction on the concent, ration of main components that -could: -diminish the extraction yield of the final p oducts -

[004-8] .As part. of. the process, collected plact. material from t ' field was cleaned i a aqueous media, sach as water, to remove soil, fertilizer and other then-deal impurities.

Preferably, th.e : plant material is ollected within 24 hours from the time the plant, is picked or harceeted for i.ts fruit.. The plant biomass was mechanically fract ion ted, (i.fe. via. deiamination process, abrasion processes , use of water to mix within the tissues and/or starches of the plant, or hoffiogenization process) in order to disassembly the foliage and the rhizome plant components■ The pineapple plane varieties utilized for the extractive method were selected in terms of the characteristics of the main parts of the remaining plant such as the stem and leaves to be subjected to the disassembling extractive process. These components were sub-f actionated into small pieces, preferably having a length of 10 mm to 100 iron, preferable 20 mm to 80 asm, and mos preferably 25 mm to 50 mm. In the pineapple plant processing, the pineapple biomass was subjected to a disassembly disruptive combination of thermo™ rnec.hanicai and hydro-mechanical processing that comprises a simultaneous mechanical disrupt on, plant tissue attrition and homogenisation processing, with an attrition abrasion speed between 708 rpm to 5, 000 rpm, preferably 8S0 rpm to 4350 rpm, and most preferable 975 rpm. to 3200 rpm for about between 2 to 35. Preferably, the abrasion was carried out for between 7 to 28 min, and moat preferably, between 12 to 24 rain. The mechanically treated fractionated pineapple biomass feedstock was further subjected into a series of extractive- arid downst eam stages as part of the integrated bio-refining process towards the production of various types of biofibers, several natural products and derivatives.

[00493 Stem Processing

£0050-] Production of Bro eiain/Sngymes ; The rh zome stem fraction of the plant fa.ioma.ss was further extracted in. order to enhance the solab lity of the target bromelain protein system (£.0.3.4 33.4) and pineapple thiol-endopeptidases . The processing of the stems as well as pineapple core and peel required an equilibration into a solution of 1.5% to 3.7% of NaCI, .more preferable 2...5% to 3.0%., most preferable 2.01 to. 2.7% for about 120 .minutes to about 210 minutes, most preferably 150 minutes. The material was .subjected to extractive homogenisation in the presence of ' sodium acetat buffer 0.Ό1 at a working pH 5. to 7.2, more^ preferably pH 5.2 to 7.0, and most preferably pl-S 5.5 to 6.87 The extractive homogenisation proceeded at 5, 000 to 45, 000 rpm, more preferably 8,00:0 rpm to 35,000 rpm and most preferably 12, 000 rpm to 28,000 rpm in a temperature range irotrs. S ' C Up to 1,5 * G. The ho ogeoization process took place for a period of at least 70 seconds , and up to about . 5 minutes.

[00513 Following the mechanical homogen lea c ion process, a filtration separation stage was performed to further .isolate the bromelain (cystein-endopeptidases] crude extract. The filtratio process resulted in ob aining an aqueous filtrate and a solid residue. The aqueous filtrate was further decanted at a temperature of 5 Q C to 9°C, more preferably at 4°C to 7°G, most preferable at 3°C to 5*C„ A clear liquid phase was separated from the soft semisolid residue and the liquid phase was further ceritrifuged at 16G0g for 25 minutes, more preferably at 2700g for 10 minutes, and most preferable at 2100g per 20 minutes. The clarified liquid, fraction was further characterised by determining the. protein content using techniques known to one of skill in the art, such as the Bradford method and enzyme activity * After the centxifujatidn process, the semisolid precipitant was concentrated and comprised, a bio oiyraeric mixture of 2% pectins, 5% hemicellutoses and 97% starchy type polymers. Usually the. starch frac ion is stored in the stem of the plant instead of th fruit. Prio to fruit ripening, the starch reserves are enzymat ically converted to racno eric sugars and further accumulated in the fruit . , thereby increasing the sweetness .

[00523 The aqueous phase, that contains th bromelain enzyme system was subjected to a f actional precipitation stage comparing both ammonium sulfate and ethanol as- complementary step, Prior to that, the pH of crude enzyme aqueous solution was adjusted to a pH 5.3 to pH 8.2, more preferably between H

5,7 to pH 7.5, most preferably between pit 6.2 to pH 7.0. The initial fractional precipi ation was carried out fay combining the addition of ammonium sulfate to achieve different ercentages of saturation, ranging from, for example, 20% to 95%, more preferably 35% to 50% and most preferably 41% to 47%, The f actional precipitation was carried out at a temperature between 2°C to 7 e C.

[00531 ftmm.e?niuav- sulfate was added to achieve saturation under constant stirring using a magnetic stirrer. after adding the: ammonium sulfate, stirring was continued for 15 more minutes to allow attainment of equilibrium between:, the dissolved and aggregated protein. The salt enriched solution: was then subjected to high speed centrifugation at IS.OOg .for 25 minates at 4°C, The precipitate pellet was collected and resuspended in

0.0Βί Phosphate, buffer pH 7.2. The clear supern tant was, recollected and the volume was utilised for further saturation:

Ki h aroisorium sulfate. Anothe fraction of the crude soluble extract was precipitated stepwise by mixing with differen concentrations of cold ethahoi iv/ ) until a percentage of 20% to 85%, more preferable 30% to: 65% ana most preferable 40% to

75%, was achieved, The final ethahoi enzy e crude extract solution was centrifuged at " ISOOg per 5.mi.ri at 4°C, The protein pellet was. resuspended in 0.01M Phosphate buffer pH 7.2.

[005 J As depicted in Table 3, the fractional precipitation with Ammonium sulfate or ethanoi was both .efficacious alternatives to precipitate the bromelain. enzyiRe system:. it is possible to achieve up to 27% yield with the above precipitation tools. Alternative to, or in addition to the selective centrifugation process, mierofi.i ra ion r ultrafiltration and lyophilization or othe drying processes may be utilized. The use of fractional precipitation with Ammonium :sul£ate and ethanoi appears to concentrate the target enzyme products, thereby minimising the crude enzyme working volume to facilitate further downstream operations,. Such action optimizes time and cost savings in order to reduc the purification and downstream stages of the bromelain enzyme- system process.

C00551 Table 3, Fractional Precipitation of Bromelain with Ammonium sulfate and Ethanoi:

0056] The activity content of bromelain among d fferent varieties of pineapple was indicated at Figure 3:. As indicated the MD2 exhibited the highest bromelain c i ity 3256 GDti/mg, followed by tbe varieties anzana and cayenne lisa and peroiera .

2? Figure 4 further indicates the Η variation effect on the residual Bromelain activity, illustrating the range of enzymatic activity at various pH ranges,

[00573 STEMS: FLOOR STARCH/FIBER: After mechanical pretreatment of the pineapple biomass, the liquid fraction and the solid fiber residue were separated for further extraction. The liquid fraction was further decanted at a temperature range of about 0°€ to about 17° c, preferably about 0 to about 12 °C, more preferably from about 2°C to about 9°iZ. f to obtain a precipitant. The precipitant contained mixture of insoluble biopolymers. After the decanting process was concluded, the supernatant was removed from the precipitan material * The precipitant fraction was constituted mainly of natural materials such as hemicelluloses, pectin and complex starch.

[00.58.3 Table 3, Composition and /or Characteristics of the isolated Flou .

[005S] these natural materials can be isolated and processed further according to specific industrial applications. The crude residual fraction after -homogenization of the stems was mixed with water in a proportion of 2:1. Mtsrn.ati.vely, the proportion can be 2.5:1 or 3; 1.5. The homogeneous aqueous phase was isolated after the de.cantation stage. The pH on this working solution was adjusted at a pH of between 4.5 and 7.5, preferably 4.7 to. 7,2, Most preferably from 5.5 to 6.8.· Once adjusted, fractional precipitation was performed by adding solid ammonium sulfate to saturation between 45% to 79% at cold temperature ^ After standing for a couple of hours, the precipitate obtained contained active crude protein fraction, mainly sulfhydryi cysteine proteinases (EC 3.4.22.32) comprising a mixture, of More than five isoforrtia. The five isoforros exhibited multiple p st- ' t rah la ionai modifica ions, The extract also contained other accessory enzymes having activitie such as peroxidases, oeliulases, vryianases, phosphatases, acetyi.xyi nesterases, D-galaetas leases » β-D-M-

Aeetyiglucosaminidase pectinases^ o---.D-'Manncsida.ses and β-Β- Hannopyranosidase . The presence- of cystein protease and glycosidase activities in the bromelain mix; indicated that a cleavage effect on. the carbohydrate giycosyllc fraction of multiple proteins besides ' the prgteoilfic activity, thereby expanding the radius of industrial, medical and pharmaceutical applications of the bromelain extract.

[0060J Dietary Fiber Applications: The delignifiied solid residue was a fibrous material with a high content of cellulose. The material was further dried and milled to produce nanofibers that can be incorporated into dietary fiber. Such dietary fiber can be used for food consumption incorporated into multiple food ingredient applications. In the embodiment of the present invention, the solid plant tnaterial of the pineapple biomass was subjected to mechanical disassembling disruption and homogenization process, exerting processing attrition conditions for mechanical disruption and homogenization effects. Such processes are performed in combination with abrasion and maceration during this edge-to-surface stage. A mechanical treatment and frictio between fibers provided fiber-to-fiber treatment and vortexed effect at higher rotation speed between 700 rpm to 5,000 x-p , more preferable 850 rpm to 4350 rpm and most preferable 975 rpm to 3200 rpm. for about 2 to 35 minutes, more preferable 7 to 28 minutes and more preferable 12 to 24 minutes, in order to detach pericar tissue.

[0061] The process enables concentrating significant higher levels of biofiber of different lengths "Long fibers" and "Short

Fibers" ' as well as "nvicrc fibers" and "nano fibers" respectively, as well as several cors eunds distributed, within the water soluble and insoluble fractions. Separation of the* soluble from the solid residual fractions was performed through the filtrating stage which involves a temperature in the range of from 3 e C to 10° G, for a period of 2 hours and preferable from 2 hours to 24 hours, preferable at a temperature range of 2°Q to 4 a G. The present method may i clude .further separation, nanofi.ltration, microftitration and ' ultrafiltratio steps, including additional chromatographic techniques such as gel filtration,, hydrophobic: interaction, anion exchange, cation exchange, affinity chromatography .steps for the isolation and polishing to homogeneity multiple natural .proteins, peptides and enzymes, s well as monome ic sugars, oligosaccharides, biopolymers, pigments and complex carbohydrates and. fibers from each of the produced fraction

[00621 LU S i During the disassembly of the pineapple plant biomass, another fibrous fraction was produced. A. fraction with more short length fibers and pericarp tissue fragments was isolated. This product is the material once the: long fibers are removed. The lumen portion may therefore' contain pieces of short fiber, leaves, epidermal tissu and chlorophyll. This wet fraction was highly regarded for its cellulose content and other plant components. The reformulation of this lumen fraction can be amenable for the production of cattle feed,- aquaeulfure, pet food and other food arid nutritional rel at ed applications. Further extractive operations on this lumen fraction such as delignlficat ion. and bleaching can foe done to incorporate the refined fibers into paper manufacturing or higher value applica ions >

10-0633 CHLOROPHYLL : An additional component isolated from the mechanical extraction process undertaken by the pineapple biomass plant was natural pigment Chlorophyll. This organic compound can be obtained in riough levels as a by-product and further concentrated into either liquid o powder form. R primary use fox The Chlorophyll includes industrial applications as natural pigment ' s in substitution of petroleum derived ■pigments- Chlorophyll may also: be used In food applications, pharmaceutical .applications:, as well as the medical and diagnostics applications .

[00643 Extraction Process Fiber K-Lation: fiber K-Latlon can be obtained from isolating the cruder residue after the extractive homogenization of the pineapple biomass rhizome stems. The product is sub j ect to a steam pretreatmerit, then milled and dried under mild air conditions. The utilisation of steam regulated conditions helps- to strip certain sugars, complex gums and pectin derivatives to improve ine concentration of active and functional ingredients in the product. The product contains active and. functional components: a mixture of endogenous constitutive natural plant eocytnes such as cystein- proteinases, cellulases and xyianases in -combirxation with soluble and insoluble dietary fiber. The plant enzymes and the soluble and insoluble dietary fiber may be mixed with another fiber source from the plan Trichantera gigantean. The proximal composition of the K-Lation product is indicated in able 5, where ratios are given based on 100g of product and amino acid content, Table 6. The K-Lation product, has use for multiple applications, including as a dietary supplement to trap cholesterol, accelerate fat degradation and improve function of the GI tract.

[0065 ] Table 5 Nutrient Composition of the K-Lation Product

[00661 Table 6 . Content of Essential and Non-Essen ial Amino Acids in K-Lation Product :

[00.67] As part of the p eparation ox the K-Lation mixture, the solid residue .or fiber was washed with hot water three (3) times t eliminate the sugar content. Immediately following the washing, a second product, TricJxantera i3.iga.ntea, was added to enable, fat burning action. After mixing " of the. ingredients, a frac ionating process was carried out. The fractionating process was carried simultaneously during a grinding process. Bulk fiber was obt ined with the particle: diameter selected in accordance to the eharactericalion of .a dietary supplement, regulated by TNVTMA (FDA).... The product was subjected to direct heating to eliminate water content until reaching 8-10% humidity. The selected fiber may be encapsulated in capsules or further processed for oral use.

10068] Xylose Extraction; The crude residual fraction after the extractive bomogenization stage was mixed with water in a proportion. 2:1 (v/w) and diluted with sulfuric acid iO.7%). The mix was heated under recirculation for about 30 minutes to 200 minutes. The heated mixture was filtrated to obtain a liquid phase and a solid residue. As a result of the acid hydrol sis process upon the hemicelluloaes fiber component o the crude residual fraction, the liquid fraction contained a higher concentration of Xylose sugar as well as other Xylose derivatives. The sugar was further concentrated and refined, using for example additional processes such as evaporation, dewatering, or centrifugation, to obtain a product ready for several food, nutritional and preparation of low caloric sweeteners. ' At present the regiospeoific hydrolysis of a a i osiylans in pineapple biomass becomes an important requirement to enhance the utilisation of pineapple heriiicelluioses and heterosubstitnted arahinoxylans, giyoomannas, arabinogalatans, xyloqiocans in the advance liquid biofueis sector, food, pharmaceutical, low caloric nutritional products and so for h , [ Q06d] An alternative mechanism to produce loonomeric sugars included utilization of specific hydro!ytic enzyme systems that have the capacity to produce xylose and complex heterosufostituted xylose derivatives that in mild reaction conditions and high speed reaction rates, becomes a moderate and less aggressive way to produce higher y elds of xylose and xylose: derivatives than the use of sulfuric acid. Therefore in the embodiment of the present invention the enzymatic hydrolysis of hemicelluioses pineapple biopolymers allowed for. -production of significant yields, of ' xylose in a more sustainable way than, the use of sulfuric acid. For the isolation of xylose and xylose derivatives, it i necessar to carry out starch and. protein removal .

[0070] Pineapple Stem Fiber suspension lOOg: Stein fiber was treated with wheat bran (2 uL/q wheat bran! at a temperature range of 80° C to 90° C, more preferable 83° C to 85°C and most preferable at the range B2 0: C to about 87' s C per about 60 minutes. Th mixture was filtered through bolting cloth , , re-suspended and repeatedly treated with Thermozyme for 90 minutes, more preferable 80 minutes, most preferable 120 mirt. The suspension was filtered, washed with water and Tris-HCl buffer iO.1 H, pH 7.8} . The obtained destarched PSP was resaspended in Tris-HCl buffer and treated with Protol ichether protease {20 mg/g destarched stem fiber, ^5 ' ¾, 20 hours} followed by filtration. Pretreated PSF solids .were resuspended in water arid boiled for 20-30 minutes, at a temperature of between 85° C and 100° C for enzyme Insctiva ion. The slurry was washed and filtered 5 to 7 times with deionized (DI) water and air-dried at 65°C for 10 hours to 18 hours. As a. result, 23.8 g of. destarched and deproteinized stem fiber was obtained.

[0071] Preparation of xylose and xylose derivatives from pineapple stems fiber as follows: destarched and deproteinized stem fiber was suspended in NaOAc buffer {0,1 M) at a pH 5.0, more preferable pH 5,3, most preferable pH 5,7 to give 100 g/L suspension. Xyianolytic, Celluloiytic, Pectinol tic Enzymes were added on the following dosage ranges 0.4 U/g to 15 U/g DD-stern fiber and incubated at 50° C to 60° C. Samples were subtracted after 24, 65, and 110 hours of hydrolysis, centrifuged, and subjected to HPLC analysis. A chromatographic method that uses HPASC-PAD High Pressure Anxo Exchange Chromatography with Pulse Araperometrie Detection was developed as follows. The DD-PSF samples after hydrolysis were cent ifuged at ll,Q00g 16,000a, more preferable 1 Q00g, most preferable 16, 0 ' OOg per 10 to 15 minutes, heated at 85-100°C f for 10 minutes. After dilution in deionized water in the presence of 0.053% to 0.75 % NaN 3 , samples were subjected to HPLC analysis. The analysis was performed with an Agilent 1100 HPLC system with Esa Couiochem. III electrochemical detector and Dionex CarboPac PA-100 column (HFAEC-PAD) .

[00721 Xylose carbohydrates and xylose derivatives were separated by anion exchange chromatography HPAEC-PAD at high pH and detected by pulsed electrochemical detection. Electrochemical detection was used to measure the current resulting from oxidation or reduction of analyte molecules at the surface of. a working gold electrode. The chromatographic running conditions used were: Ceduron.: .CarboPac. P&10 Q (4 * 200 mm} and Guard: ( χ 50 mm), Eluent; Ά; 50 ταΜ, preferable 100 ΐίΐ , most preferable 175ru NaOH; B: 250 ιΜ, preferable 370 mM, most preferable 40OmM of ISSaOAc in 65 f more preferable 7¾rr¾3 » more preferable lOOmM of MaOH Gradient 0-62, SI B in 30 minutes, 62/5- 100% in 15 min, followed by 75¾κΜ, mor preferable S ' OrftH, more preferable lOOmM KaOH for reconditioning for 12 minutes to regene te the coIximn , Flow a e .: 0.75mL/min , mxe ρretera.bie 1.00 mL ffiin, :ni:ost preferable 1.5 mL/min. Temperature; ambient Detection: Pulsed electrochemica r TAu electrode. Wave ormr Quadruple potential, Arabiaose, xylose, xylp.biose, xylotriose, and xyiotetraoo were used as standards.

[0073] Enzymatic combinations were diluted in ¾0m a.O¾c buffer pB 5..7, more preferable pH 6.3 and most preferable pM 7 O: screeh multiple enzyme dosaqes ranges such as 0, 0/g to 0.7 y./.g; more prefer ble 2 0/g to 4 XJ/gy most preferable 10 b/g to preferable 45 minutes to 180 minutes, most preferable 60 minutes to 120 minutes. At the end of the heating step, the mixture was filtrated, the fibrous solid residue was separated with the liquid fraction containing mainly polymers of lignin. Furthe concentration of this material can be utilized fo the production of flavor {vanillin} and other food ingredients «

10076] Leaves: The embodiment of the present technology invention involves an innovative method of one step: disassembly single and integrated extraction of pineapple biofibers in which the pineapple biomass was subjected to mechanical disassembl ng disruption and hornogenixation process,. exerting processing attrition conditions to: facilitate the mechanical disruption of the plant tissue. In the embodiment of the present invention mult le combined effects were exerted to efficiently increase the disassembly disrupting effects through and mainly external and internal d fib illa on, del ami.naticn and multipl micrcco pressions on the pineapple biomass plant cell walls. In the embodiment of the present invention the simultaneous disassembling disruptive effects,, most preferabl in combination with abrasion, delamination and maceration effects performed during this edge-to-surface stage providing hydro-mechanreal effect and maintaining the extraction environment at pH range of

4.9 to 7.5, more preferable pH S,S to 6.2, and most preferable at. a pH range of 5,7 to 7.3 and a fiber ex raction tempera ure 15 U/g DD-PSF in the hydrolysis mixture and incubated at BQ°C f more preferable at 54°C and most preferable at 57°C, Samples were subtracted after 24, 65 ? and 110 hours of hydrolysis. Calibration of anion-exchange .column was performed .using xylose and xylooligosaccharides with DP 2- . The coefficients for calculation of peak area to concentration were approximated using extrapolation of the calibration plot.

[0Q7 J . Xyiitol: The Xylose obtained in the me hods as described above was subjected to alternative chemleal, hydrogenation under the following working conditions using hydrogen gas under the presence of a. chemical catalyst. As an illustrative exaisple, Nickel was used as the chemical catalyst under the wo ding conditions of 1Q atmosphere of pressure and 55 <! C to 95°e, more preferable 6-5 * € t 9D C, m&t preferable 70*C bo. S5°e during a process im - of around :3£) minutes to 200 minutes,. .more, preferable 4.5 minutes to ISO minutes., most preferable 60 minutes to 120 minutes.

[00751 LIGNIN: The first Solid residue obtained after the ext active roaeeratian of the pineapple biomass was subjected to a treatment with. 1% to 3.5% F¾OH f .more preferable 1 * 5% to 3.0%, and most preferabl 1.8 to 2151 HaOH, in a propor ion 2:1

{v lume to weight) . The mixture was heated between 55°C to

95¾, more preferable. 65 U C to 90°C f, most preferable 70 °c to 85°C during a process time of around 0 imPuis s to 200 minutes, more

39 of .15*C to 25 * C, more preferable at the range of 1?°C to 27"C and most preferable at a temperature range of 12 °C to 21 e C, in the embodiment of the present invention the process induces natural surface active friction and abrasion between fibers gives enabling fiber~to-fiber treatment. In the embodiment of the present, invention our unique integrated process helps to enhance favorable biostructurai fiber arrangements and molecular redistribution from the inner areas of the fiber bundles to. the exterior, including the release and exposure of multiple natural biopolymers and com lex carbohydrates within the plant leaves, .matrix., such as.; pectin, hemi eiiuioses, cellulose, some iignin species, protein-sugar and protein~0li,go.sa:ccharid. s colloidal materials, the overall process improve the interfiber surface area interactio which .en ance3· the reac ivity of the fibers fox further industrial processing and applictat ions .

[0077] The pineapple foiomass disassembling extraction process

10 enhances the. molecular, plant cellular and efficient .plant tissues separation of the biomass pineapple leaf components.

The plant biomass was mechanically frantdonated in combination with the del amination of the foliage the pineapple leaves and further subjected to a disassembly disruptive combination of ther o-inechanical and hydro-mechanical processing that comprises a simultaneous mechanical disruption, plant tissue attrition and maceration processing, with an attrition abrasion: speed .between 700 rpm to 5,000 p , more preferable 850 rpm to 4350 rpm. and most preferable 975 rpm to 3200 rpm for about 2 to 35 min, more preferable 7 to 28 min and more preferable 12 to 24 min, to efficiently detach components of the pericarp tissue. At this stage the fibers aggregates and ate not free to move independently. Instead t-hey form floes of 2mm to 7m, and under the continuous shear, de-laminating and turbulence hydrodynamic forces, some bundles of fibers, may be further defiforiliated . I the embodiment of the present invention the. mechanicall treated fractionated pineapple ornass was further subjected into a series of extractive and downstream stages as part of the integrated biorefining process towards the production of various types of biofibers, several natural products and derivatives.

In the -embodiment of th present invention- from the fibrous material which was subjected to the disassembling fractionat on process, three streams were produced that corresponded mainly to the long fibers, Lumen (short fibers} with fines and water soluble residuals. The process enabled concentrating significant higher- levels of biofiber of different lengths long, short, micro nano fibers respectively, as ie.ll as several compounds distributed within the water soluble and insoluble fractions. The present fret hod may include further separation, rianofiitration, raicrofi It rat ion and ultrafiltration steps, including additional chromauo-gra hi-c techniques such as gel filtration, hydrophobic interaction, anion exchange, cation exchange, affinity chromatography steps for the isolation and polishing to homogeneity multiple natural proteins, peptides and enzymes, as well as monomeric sugars, oligosaccharides,, bi©polymers, pigments and complex carbohydrates and fibers from each of the produced fractions.

{0.0783 During the i a ' semfoling of the plant biomass it was possible to develop a positive refining effect to the pineapple natural fibers. In some regions of the fibers this refining effect brought several biostrueturai fiber enhancement effects. The fiber walls were to some extent removed through external, fibrillation, or rooting o t" and del ir-ination effects which creates fines and debris in suspension in the form of micro (rpra to 3,μτη> sizes, such. as : elementary fiber and nano fibers {3nm to n¾nj . Internal biost ueturai change ' s occurre in the ceil walls such, s swelling and internal fibr illation .

[0079] In an embodiment of the present invention, it was possible to enhance fiber mechanical characte istics as indicated in TABLE 7. Tabic 7 depicts; the mechanic l characteristics of pineapple leaf fibers from vari us pineapple cultivars. Some- of the differences on the rerchanical character i sties among the fibers free:; e ch pineapple variety were attributable to the differences in fiber cent orrnation, orientation, , internal and external bio-structural rear r riu nientt; occurring during the extracting disassembling process. In addition the possible reactivity among hydroxy! groups along the fiber may enhance the interfacial fiber-fiber interaction to form strong bundles and micro iber to nano iber aggregates. Exogenous forces, such as stressing, deforming,- elongating, tensile, impact strength and fiexurai, may induc morphological and biostructur&l changes . The strength and nature composition of the pineapple fibers, with more than 82% cellulose, results in a relatively stable polymer, with the fiber-t - ' fiber and interfacial adhesion in the fiber bundles attributing to the density on the hydrogen bond network. As a result, the pineapple cellulose fibers have a good flexibility and elasticity, allowing for maintaining high processing characteristics. These improved mechanical properties may enhance the properties- of the .materials where pineapple fiber are incorporated, for instance in. the papers making process, foioco posi es-, biopoiymers of multi le fiber industrial applications.

[00803 Table No. 7, Mechanical Properties Natural Fibers fror Multiple Pineapple Varieties.

[00813 The mechanical properties displayed in Table ¥io. 7, help t . o better apply pineapple fibers according to the intrinsic mechanical properties to bette maximize industrial and commercial value- of the pineapple libera. Every pineapple eultivar from which fibers were extracted displayed diverse mechanical properties:. These mechanical properties, may increase during fiber processing to increase the internal bonding strength to maximis , drainage resistance, the stiffness, burst and tensile s ength. The parameters indicated the tensile strength was higher for the Cayenne Lisa variety followed by the Peroiera, ΜΏ2 and. ¾an¾ana . Cayenne; Lis exhibited higher Young's Modulus and density. The mechanical properties that, pineappl fibers exhibited helped to further develop structural combi ations with other biopoiymers and resins which bring new mecha ical properties for further industrial application ' s. [00821 After the process disassembling and extractive stage, the main fiber streams such as long fibers and short fibers or lumen were obtained. Table Mo.2 indicates the chemical composition of pineapple fibers from various- cultivars showing the percentage of cellulose, heraicellnloses, iignin and moisture content . Preferably, the fibers obtained as result of the extractive disassembling process were enhanced. For example, the long fibers exhibited fiber content from 83,11% to 87.16%.

[0083] Table No. 8, Chemical Composition of Pineapple Fibers Before and After Bleaching

[0084] Bleaching treatment was necessary to remove Iignin, ligni.n derivatives and residual hemice11u 1oses - The bleaching process further enhanced the mechanical properties of the fibers. The pineapple foiomass di assembl i q ex raction process 10 further results in increase str ngth of the pineapple fibers.. These can he further processed through oleachi^g or hiobleachirr process conditions with the aiteraaL ve use of a m x of hydrogen peroxide 0,1% to 0.42%,· more preferable 0.15% to 0.35%., and roost preferable 0.37% to 0.5% in combination with sodium hypochlorite in a concentration of 3% to and 6%, more preferable 2.8% to 4.5%, most preferable 3.8% to 5.2% respectively. After the extraction of the long ibers, additional lignin and heroieelluloses, were removed. As indicated in the levels of cellulose content after the bleaching stage were from 95.74% in Ferolera bleached fibers to a maximum of 98.24% obtained with MD2 bleached fibers respectively.

[008.52 Pineapple Natural Fibers in Pulp and Paper

Utilization. The pineapple bioraass disassembling' extraction process 10 was used to prepare long fibers of pineapple plant,. having fibers that exhibit unique mechanical properties, higher cellulose composition, and -orientation. The pineapple biomass disassembling extraction process 10 confers excellent surface fiber surface treatment to enhance the. mechanical properties of the fibers that are also transferred to t e materials where these fibers are incorporated. The pineapple biomass disassembling extraction process 10 may also foe adapted to produce both long and short fibers source multiple types of materials and mixture co posi es, to increase the weightless, mechanical resistance and enhanced optical properties of multiple types of paper and packaging products, light and resistant materials and so fo t , u . most, important the key function of the pineapple fibers is to bring strength, weightless to. the final products. More natural fibers are required to substitute chemically recycled fibers which have already been processed and carrying higher concentration of chemicals. In the embodiment of the present invention the process becomes a source of natural fibers to substitute chemically recycle fibers for paper, food packaging, textiles and multiple ' ether applications. The combinatorial extracting disassembling process helps to increase fiber width that amplifies the surface contact areas to increase the fiber .bond.

This affects favorably on paper properties. Using the pineapple biomass disassembling extraction process 10, the fiber coarseness decreases after the extracting processing. The decrease of coarseness increases -the fiber surface and increases the bonding ability of fibers., This property supports an increase in paper strength, and paper smoothness. Fiber width increase means the contact surface of fiber is wider and larger then it increases the bonding between fibers. The higher the fiber width, approached, the higher the tearing resistance.

Fiber width increase their width may because of flattening of fiber coarseness, and collapse ability. The incorpora ion of pineapple fiber in the paper mix brings superior mechanical characteristics that, also determine the quality and industrial appl cations of the final produc . £00863 The pineapple fibers generated by pineapple foicroass disassembling extraction process 10 can be utilized individually or in combination with recycled fibers. The pineapple fibers created have a natural white color that allows production of higher quality papers. Paper formed could be for example, art paper and other fine paper applications that, exhibit strong and smooth paper sheet with good printing properties » The long pineapple fibers (LPE'} obtained, have ah average of 1.5 cm to 3,5 cm ' length. Traditional fibers have smaller lengths.* averaging lengths o 0.9cm to 1.3 cm, The mechanical properties of the L? .obtained bring strength., flexibility and. great appearance to paper, such as brightness, opacity, resistance, and long shell life. Samples .of paper were made using fibers obtained form the pineapple .b omaSs disassembl ng extraction process 10.

[0087} In addition,, the use of combined long and short pineappl fibers, fibers obtained from, the pineapple biomass disassembling extraction process 10 can be Incorporated to mak a good sheet formation or to develop other pulp properties such as absorbency, porosity, brightness or optical properties specifically a r a given paper grade. In as embodiment of tire present invention, paper samples we e prepared according to the

TAcPI standards in which fiber concentrations were va ed £¾¾ to

35%, second casa 4.5% to 50%, third case 65% to 75% and fourth case S % to 97%, as i dicated in Table 9. cable 1 indicates various optical properties of the paper samples with increasing fibers being ' incorporated in the pulp mix.

[00883 Table No. 9, Characteriza on of Paper made with

Naty.rr.al Pinea le Fibers

brightness levels reached up to 63.54, which after additional washes, but without bleaching reached, was increased to 80. In some other cases, if the concentration of fibers exceed optimal concentration,, then the brightness, opacity, whiteness of hand sheet paper may decreased because of increase the density of fiber, fiber contact areas, fiber width, coarseness, levels of hemicelluloses and lignin being released. The paper made with pineapple fibers can be recycled between 3 to 7 times.

ί00901 Long Fiber and Lumen, in vitro diges iveiy results

{illustrating the breakdown o fiber into monomerίσ sugar components}. In an embodiment of the present invention, the lumen mainly arid long fibers being valorised for its potential for preparing animal feeding and also with potential as organic fertilizer In both oases the possibility to supply high quality sugars and energy for growt improvement in both animal and plant systems. In a range of 15g to 25g, more preferable 17g to

2,1.g, most, preferable 17g to 20g dry weight of lumen or long fiber material are placed in 250 ral flasks. A buffer solution of Phosphate buffer range 5.7 to ? .0 , made 1 , 4 of dibasic

R;dii¾ in 200ml of .0,1 water where the pH is B.75, also 0.78 monobasic Κ¾Ρί¾ in 300ml 01 w&ter, pH .5, add Sodium aside

SOOmM to get 0.03%, more preferable 0.025%, most preferable

0.02%, keep the buffer at cool temperature between 3 to l^ ' C, more preferable 2 t® 5 Q C t most preferable- 4 to 8*G, The buffer solution is added to the flasks containing the lumen and long fibers respectively, also add the appropriate amount of enzyme dosages to each flask the reactions are incubated at a controlled temperature range 45°C to 4$"C, more preferable 53 e C to 55 e C f most preferable 50°C to 60°C with vigorous shaking range 200 rpm to 250 rpm, more preferable 275 rpm to 285 rpm f most preferable 290 rpm to 325 r m, for the specified time 24 hours, 48 hours and 72 hours. As depicted in Figure 5, the lumen samples were enzymatically digested under specific hydrolytic reaction conditions, to show that the combination of enzymes produces from30 g/L to 35 g/L which outperform over the untreated control samples. At 24 hours it is possible to obtain up to 30 g/L of combined sugars which is excellent for initiating the energy intake within the first 24 hours. The above results display the efficacious enzymatic digestibility of the lumen fraction and the long fibers prepared as value added products by the present invention.

[0091] Ail patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publ cation, was specifically and. individually indicated to be incorporated by reference. [0092] It is to foe understood that while a certain form of the invention is illustrated, it is not to be limited to the specific form or arrangement herein described and show < It will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown and described in the speci cation and any drawings/figures included herein *

[0093] One skilled: in the art will readily -appreciate that the present invention is well adapted to carry out the objectives and: obtain the ends and advantages mentioned, as well as those inherent therein. h : embodiments r methods, procedures and techniques described herein are presently representative of the: preferred embodiments, are intended to foe exem lar and are not intended as limi tati.ons oh the scope. Changes therein and other uses: will ©cctir to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the appended claims. Although the invention has been described in connection with specific preferred embodiments, it should foe understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the nvent ion which are obvious to those skilled in the art are intended to be within the scope of the following claims ,