The invention provides a composition for use in providing slow-feeding aquatic animals, with defined amino acids, said composition comprising the polymer cyanophycin (also abbreviated CGP, Cyanophycin Granule Peptide) that is essentially comprised of a poly(L-aspartic acid) backbone covalently bound to other amino acid residues, and has a molecular weight between 15 and 100 kDa. While these are mostly arginine residues, one or more structurally similar amino acids such as lysine, ornithine, glutamicacid, citrul line and canavanine, may partially replace the arginine residues of CGP depending on the bacterial strain and/or environmental/cultivation conditions.

Background of the Invention

Global amino acids market demand was around 6.5 million tons in 2014 and is expected to exceed 10 million tons by 2022 (Global Amino Acids Market. July 2015. Online summary, ISBN : 978-1-68038-453-6. Grand View Research, CA, USA). The forecasted market growth is due to increasing application in various end-use segments such as dietary supplements, health food and sports nutrition, cosmetics, artificial sweeteners, but is mainly driven by the increasing demand in the area of animal feed.

Numerous studies have investigated the physiological roles of amino acids in humans and animals, and thereby laid the basis for many scientifically proven applications in nutrition and therapy. However, the importance of each amino acid varies among living organisms, for example : L-arginine is considered essential in most poultry and fish species, and varies among mammals from being semi- essential as for human adults, over being regarded as nutritionally critical as in the case of swine, to being essential for human infants and cats. Since natural sources of protein such as soy, corn, and wheat are usually deficient in certain essential amino acids, targeted supplementation with these amino acids, such as lysine, arginine, or methionine, contributes to balancing and/or improving the nutritional value of food and animal feed. On the other hand, and after being familiar with factors limiting the application of the free form of amino acids, an increasing demand for related but superior forms of amino acids emerged. Among these limiting factors, the following two are considered to be major:

A) Physiological limitation : absorption capacity, which is - in the case of several amino acids - largely influenced by the competition for shared transporters, and

B) Physical limitation : through the properties of the free amino acid itself where, for example, the pH value or the solubility of the free amino acid can be limiting in a certain formulation/application or advantageous in another.

Both kinds of limitations are relevant for human and animal nutrition. Besides numerous studies; however, their impact is most clear in the feed area due to the decisive role amino acids play in means of productivity. While often only the physiological limitation is relevant, for example in the field of poultry farming, where the amino acids L-lysine, L-arginine and L-methionine are known to strongly interact (Chamruspollert et al., Br. J. Nutr. 88(6) :655-60(2002)), both kinds of limitation coexist for example in the area of aquaculture, where a high solubility of the amino acid leads to disadvantageous leaching in the aquatic milieu.

The present invention provides a novel approach in the supplementation of certain amino acids in the form of the polypeptide cyanophycin, which can overcome the above mentioned limitations in the areas of food and feed. In order to best demonstrate the effectiveness of this approach in overcoming both limitations at the same time, the field of aquaculture was chosen for the experimental part of the present invention. Shrimp were chosen as model organism due to the sensitivity to unbalanced amino acid content in their nutrition (limitation A) and due to their slow- feeding behavior, resulting in high levels of leaching of amino acids from feed (limitation B).

Intensive efforts have been made in the past to avoid or overcome known problems of supplementing amino acids in the free form, including the above mentioned two limitations. For example, the effective feeding of fish and crustaceans in aquaculture often requires a suitable chemical or physical protection to render them sufficiently stable during feeding in the aqueous environment and prevent them from leaching out of the feed pellet. On the other hand, the formulation must allow the amino acid product to be taken up by the animal . Thus, many attempts have been conducted to develop suitable or optimized forms of amino acids, e.g. :

US20030099689 describes the use of synthetic peptides as growth-promoting feed additives for aquatic animals with a peptide proportion of 6-50% (wt/wt) of the total feed formulation . The enantioselective synthesis of these synthetic oligo- and polypeptides is, however, very complicated, expensive, and difficult to scale up . Moreover, the efficacy of homopolypeptides consisting of one individual amino acid is disputed due to their very slow or even absent conversion to free amino acids under physiological conditions (Boebel and Baker, J . Nutr. 112 : 1130-1132 ( 1982)) . WO2002088667 describes the enantioselective synthesis and use of oligomers of Methionine Hydroxy Analog (M HA) and amino acids, e .g . methionine, as feed additives. It is claimed that faster growth can be achieved as a result. The oligomers described are synthesized by an enzyme-catalyzed reaction and have a very wide distribution of chain lengths of the individual oligomers. As a consequence, the method, and thereby the product, is unselective, expensive, and complicated in execution and purification .

US20110295006 describes various known problems of the use of crystalline essential amino acids with certain species of fishes and crustaceans. It cites several previously published trials for the synthesis of chemically protected oligopeptides in the form of diketopiperazines (cyclo-dipeptides, dehydrodipeptides) as a solution, and also the disadvantages thereof. The invention relates to feed additives containing synthetic chemically-protected diketopiperazines of essential limiting amino acids. The dipeptide cyclo-DL-Met-DL-Met was preferably used .

In addition to trials to develop and/or use chemical derivatives of amino acids for aquaculture, various possibilities for physical protection were also investigated, e.g. coatings. For example, Alam et al ., Aquaculture 248 : 13-19 (2005) showed that coated methionine and lysine, in comparison to their uncoated form, have a very positive influence on the growth of young kuruma shrimp. Although the use of a special coating prevented leaching of methionine and lysine from the feed pellet, there are some serious disadvantages. Coating of amino acids is generally a technically complicated process and is therefore expensive, and the protective layer can easily be damaged by mechanical stresses during feed processing. Furthermore, coating or related techniques reduce the content of amino acid and are therefore often uneconomical.

In general, a realistic practical use of a functional peptidic molecule depends largely on the complexity of its synthesis method and whether it can be successfully scaled up. These main factors determine the economic feasibility of the product itself and thereby the industrial applicability of the functional peptidic molecule. For the chemical synthesis, the overview article of Pattabiraman and Bode, Nature 480 :471-479 (2011) describes the development in this field, which largely did not change until today: "Improved methods for the synthesis of amide functionality, whether catalytic and waste-free or chemoselective and suitable for fragment coupling, are in great demand." The author also refers to the vote of the American Chemical Society Green Chemistry Institute (comprising members from major pharmaceutical industries worldwide) in 2007 on the "amide formation avoiding poor atom economy reagents" as a "top challenge for the organic chemistry". Furthermore, the chemical peptide synthesis as well as promising biotechnological approaches such as L-amino acid ligase remains particularly challenging when it comes to basic amino acids like arginine and lysine, due to their properties.

On the other hand, WO2009150252 discloses the preparation of unprotected amino acids in the form of β-dipeptides, such as L-arginine-p-L-aspartic acid and L-lysine- β-L-aspartic acid by hydrolysis of CGP (see also Sallam and Steinbuchel, J. Appl. Microbiol 107:474-484 (2009); Sallam and Steinbuchel, Appl . Microbiol. Biotechnol . 87 :815-828 (2010)) .

In applicant's copending PCT/EP2016/075432 it has been furthermore shown that these β-dipeptides are a suitable source for essential amino acids for several omnivorous, herbivorous, and carnivorous fish and crustacean species that live in salt water or freshwater. Although the β-dipeptide form showed superiority to the free form of amino acids even for the slow-feeding kuruma shrimp, its potential is thought to be not fully reached due to its high leaching rate in the aqueous milieu at feeding.

The present invention provides a further solution towards better food and feed formulations. The bioavailability of CGP can be explained either by the presence of CGP-degrading bacteria in gut flora which was previously shown during an in-vitro screening study on several mammalian, avian, and fish gut flora (Sallam and Steinbuchel, J . Appl. Microbiol. 107 :474-484 (2009)), or by the degradation of CGP via endogenous digestive enzymes. In both cases, the positive overall nutritional effect is most probably due to the absorption of the dipeptides resulting from CGP- degradation . Although the absorbed ratio of dipeptides might be - due to the necessary degradation step of the biopolymer - less than that in case if dipeptides were supplemented directly, the overall nutritional benefit appears to be better in such application due to bypassing the physiological and/or physical limitations known for free amino acids.

Thus, said composition can serve as a substitute for the widely applied free amino acids as a component/additive for food and animal feed formulations with very high biological value, and has good handling, storage, and stability properties in the usual conditions of mixed feed processing. Furthermore, it renders efforts and costs for protecting the respective free amino acids unnecessary. In this way, additional efficient sources of the essential amino acids constituting the biopolymer could be made available. The use of the said composition in the present invention could solve or effectively participate in solving known problems of amino acids in the areas of food and feed.

Short Description of the Invention

The invention thus provides

(1) a composition for use in providing human beings and animals with defined amino acids, said composition comprising the polymer CGP, wherein CGP comprises essentially a poly(L-aspartic acid) backbone covalently bound to other amino acid residues, and has a molecular weight between 15 and 100 kDa;

(2) a preferred embodiment of (1) above, wherein the composition is for use in culturing aquatic animals, notably for culturing slow-feeding aquatic animals;

(3) the use of the composition as described in (1) above as food or (animal) feed or as food/feed additive; and

(4) a method for providing human beings and animals with defined amino acids, said method comprising supplementing the human beings and animals with the composition as described in ( 1) above. Detailed Description of the Invention

In nature, and in addition to several heterotrophic bacteria, most cyanobacterial species (blue-green algae) accumulate the polymer CGP of the composition of aspect (1) of the present invention . CGP is a reserve material for carbon and nitrogen which is mostly composed of the two amino acids aspartic acid and arginine and exhibits low solubility under physiological conditions. One or more amino acids that are structurally similar to arginine, such as lysine, ornithine, glutamicacid, citrulline, and canavanine, may replace the arginine content of CGP depending on the bacterial strain and/or the environmental/cultivation conditions. The latter factors also determine the length, and thereby the molecular weight, of the polydisperse polymer which ranges from 15 to 100 kDa.

Thus, "CGP" according to the present invention is a polypeptidic structure essentially comprised of a poly(L-aspartic acid) backbone and covalently bound amino acid residues, preferably said residues are composed of the amino acids arginine, lysine, glutamic acid, citrulline, ornithine, canavanine, and the like.

CGP is prepared by culturing a prokaryotic or eukaryotic cell line. The producing cell line may be any cell line capable of producing CGP. It is preferred that the producing cell line is selected from Escherichia coli, Ralstonia eutropha, Acinetobacter bayiyi, Corynebacterium giutamicum, Pseudomonas putida, yeast strains, and plant biomass. Particularly preferred producing cell lines are Ralstonia eutropha H 16-PHB ^" 4-Aec/a (pBBRlMCS-2 : cphA ₆₃ w/ edaH 16) and E. coli DH 1 (pMa/c5-914: : c Mpcc6803) .

The above process may further comprise the steps of isolating, purifying and/or chemically or enzymatically modifying the CGP product obtained by cultivating the producing cell line. Such isolation, purification, modification and separation may be applied by methods well established in the art.

Compared to chemically-synthesized peptides, the compositions issued in the present invention are natural and stereospecific substances that are produced from biomass in a biotechnological and environmentally-friendly way. Their production furthermore requires much less technological expense and effort, very little time, and significantly less financial effort. In a further embodiment, one or more of said polymeric structures comprise a reduced content of amino acid residues or are furtherly modified . Such modification includes phosphorylation, farnesylation, ubiquitination, glycosylation, acetylation, formylation, amidation, sumoylation, biotinylation, N-acylation, esterification, and cyclization . In a preferred embodiment, the composition of aspect ( 1) comprises from 0.01 to 50 wt.% of the said polymeric structures, most preferably from 0.01 to 5 wt.% . The composition of aspect ( 1) may further comprise one or more free amino acids or salts thereof. These free amino acids are preferably selected from arginine, lysine, and methionine. The content of free amino acids in the composition is preferably from 0.01 to 10 wt.%.

The polymeric products described above are highly stable under several conditions, and are suitable for being admixed with conventional food or feed components, e.g . cereals, vegetables, legumes, fishmeal, but also in combination with supplemented free amino acids, proteins, peptides, dipeptides, carbohydrates, vitamins, minerals, fats and/or oils. The composition may also include active ingredients such as plant extracts, prebiotic compounds, probiotics, yeast extracts, short chain fatty acids, medium chain fatty acids, unsaturated long chain fatty acids, vitamins, and toxin absorbing compounds.

In particular, the compositions of aspect (1) are basal diets that are supplemented with the said polymeric products to a concentration of 0.01 to 50 wt.% of said polymeric products, preferably from 0.01 to 5 wt.%. These basal diets can be formulated from individual components in common use.

Furthermore, the composition of aspect (1) displays good pressing, pelletizing, and extrusion stability.

The composition of aspect (1) is preferred for use as food or feed or food/feed additive where it may serve as a growth promoter and/or enhancer for vitality, survival rates, and immune response for animals and human beings. Animals that use said composition comprise omnivorous, carnivorous, and herbivorous mammals, birds and aquatic animals (including fish and crustaceans (shellfish)) . The fish and crustaceans according to the invention include fry, juvenile fish and adult fish and larvae, postlarvae, juvenile and adult shellfish, respectively. Particularly preferred in the culture of slow-feeding aquatic animals such as fry and juvenile fish, and larvae, post larvae, juvenile and adult shellfish and fish and shellfish with feed uptake from the bottom (bottom feeders) . Aspects (3) and (4) of the invention pertain to the use of the composition (1) in supplementing the above mentioned animals with defined amino acids, namely those constituting the CGP, in the bound (polymeric) form including aspartic acid, arginine, lysine, ornithine, glutamic acid, citrulline, canavanine and the like.

The invention will be further described in the following Examples, which are not to be construed as limiting the invention .

Experimental examples

Materials and Methods

Production of CGP: A recombinant strain of Ralstonia eutropha H 16-PHB ^" 4-Aec/a (pBBRlMCS-2 : :cphA ₆₃ o ₈ /eda 16) was used for the production of CGP in a 500L fermentation. CGP was then extracted from the produced biomass, purified, and analyzed for purity via HPLC, and finally dried to a powder (WO2009150252 and Sallam et al., AEM 75 :29-38(2009)). The pure CGP powder was added to the feed formulations for the supplementation experiments.

Aquaculture experiments: In order to best demonstrate the effectiveness of the novel approach of the present invention in overcoming the addressed limitations known for using free amino acids in food and feed, kuruma shrimps (Marsupenaeus japonicas) were chosen as an ideal test-model organism due to 1) the sensitivity of balancing the amino acid content in their ration, and 2) their slow-feeding behavior resulting in maximum leaching of amino acids from feed.

Two experiments (examples 1 and 2) were conducted using CGP consisting of arginine and aspartic acid and different basal diets. Basal diets were formulated from individual components in common use in the aquaculture feed industry. For this, the published amino acid requirements of the species were considered to ensure arginine limitation in the diet for Example 2. Each basal diet was supplemented with the CGP to a concentration of 2.5 % (wt/wt), or with corresponding isonitrogenous concentrations of an amino acid mixture to determine the effect of free amino acids alone.

Shrimp were maintained in seawater and the experiments started with postlarvae. Water quality (oxygen levels, nitrite, and nitrate) was monitored weekly. Partial water replacements were performed if the desired levels were exceeded. (< 3 mg 0 ₂ /l, detectable nitrite, > 25 mg/l nitrate). At the end of the experiments, the individual weights were recorded and the data analyzed statistically.

Example 1 :

In this experiment, the used commercial diet contained wheat & Wheat bran, Soybean meal from husked seed (toasted), hemoglobin, fishmeal, fish oil, wheat gluten, sun flower meal, soybean oil, and had the following analysis values: crude protein 41%, crude fat 12 %, carbohydrates 29 %, crude ash 8 %, crude fiber 2.5 %, Lysine 2.5 %, phosphorus 1.4 %, energy 17.6 MJ/kg, Vitamin A 9.000 I.E./kg, vitamin E 150 mg/kg.

Results:

After two months of feeding. Start/test group: 15 postlarvae (120 days old) . Arg/Asp: free amino acids mixture; CGP: cyanophycin.

Kuruma shrimps responded well to the addition of the CGP product to the diet which increased the average final weight to around 157 % of the average weight showed by the test group fed on free amino acid.

Example 2 :

The arginine-limited basal diets used in this experiment contained a calculated arginine content of about 1.5% (of diet), which is also lower than the minimum requirements of Kuruma shrimps : Corn 40 %, soybean meal 21 %, rice polishings 14 %, gluten 11 %, wheat bran 5 %, herring meal 5 %, monocalcium phosphate 0.8 %, limestone 0.7 %, L-lysine HCI 0.7 %, fish oil 0.5 %, vitamin mineral premix 0.3 %, sodium bicarbonate 0.15 %, salt 0.15 %, pellet binder 0.05 %.

Results:

After six weeks of feeding. Start/test group: 48 postlarvae (75 days old) . Arg/Asp: free amino acids mixture; CGP: cyanophycin. Shrimps responded well to the addition of CGP to the basal diet in this experiment as well . The supplementation increased the average final weight to around 153 % of the average weight showed by the control test group fed on the basal diet alone. The supplementation with free amino acid mixture increased the average final weight to around 129 % of the average weight showed by the control test group fed on the basal diet alone.

Results of both experiments (1 and 2) provide clear evidence on the superiority of the polymer CGP issued in the present invention when compared to the free form of amino acids. Thus, the present invention provides a novel possibility for supplementing amino acids in a form which can overcome known limitations of using their free form.