PRIORITY DOCUMENT

[0001] The present application claims priority from US Provisional Patent Application No62531894 titled "COMPOSITIONS OF AND METHODS FOR PRODUCING FERMENTED AGRICULTURAL BIOMASS AS FEED FOR AQUACULTURE" and filed on 13 July 2017, the content of which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure is directed to compositions comprising fermented agricultural biomass as feed for animals, and to methods for producing the compositions.

BACKGROUND

[0003] Current methods of food production will be unable to feed the world population, which is expected to be 9.7 billion by 2050 from the current population of 7.5 billion. Two major nutritional challenges to feed this burgeoning population are (1) the amount of food and (2) the quality of nutritional diets. Although alternative sources of proteins are being developed mainly from botanical or microbial sources to address the production problem, these sources are deficient in omega 3 fatty acids, an important essential fatty acid required for brain, eye and joint health from infants to the elderly. At the same time, the Western diet has a higher omega 6:3 ratio (14-20) of the inflammatory omega 6 fatty acids to anti-inflammatory omega 3 fatty acids. This high omega 6:3 ratio has been attributed to the cause of a wide range of chronic diseases such as cancer, diabetes and autoimmune diseases. Unfortunately, the Western diet is also permeating into the rest of the world's diet with the popularity of fast food chains. With the decreasing sources of omega 3 in the diet, the outlook for proper nutrition in the future appears to be bleak.

[0004] Fish are a known source of omega 3 fatty acids in human and animal diets. The main source of the more relevant omega 3 fatty acids, EPA and DHA, is other fish, which is incorporated in aqua and livestock feeds. The supply of wild fish just to feed farmed fish, however, is limited and there is an immediate need to fill the 30 million tons of wild fish currently used just to feed farmed fish. By the end of the century, 300 million tons a year of extra fish feed is required to meet demand. At that rate, it is unlikely that this will be met if the source comes from wild fish. Other sources of omega 3 are micro- and macroalgae, which are known to synthesize de novo their own omega 3s. On the other hand, soybean meal, a common source of plant-based protein incorporated in animal feeds, is high in omega 6 fatty acids such that animals fed with soybean meal tend to accumulate more omega 6 fatty acids than omega 3 fatty acids.

[0005] Furthermore, with the detrimental effects from the excessive use of antibiotics in livestock production and aquaculture, there is a growing trend of excluding antibiotics in feeds. Japan and Europe has been on the vanguard and have banned the incorporation of antibiotics in all livestock and aquaculture feeds.

[0006] There is thus a need to modify existing feed formulations to address the foregoing issues.

Microbes have been explored as a protein source. Single cell proteins, which are mainly derived from fermenting biomass with bacteria or unicellular eukaryotes like yeast, have shown potential as a source of protein and can provide some health benefits to livestock and aquatic animals. These single cell proteins have also shown some antibiotic effects, which can reduce or eliminate the need for using antibiotics. However, most of these single cell proteins do not produce the omega 3 fatty acids EPA and DHA.

Alternative protein sources such as plant proteins are being actively investigated but most if not all of these plant proteins are devoid of the relevant omega 3 fatty acids - DHA and EPA.

[0007] Most single cell proteins are directed as alternative protein sources and in some instances such as Novacq, the cocktail of organisms includes some cyanobacteria which will include some omega 3 (EPA and DHA) fatty acids in the final formulation. In some instances, these microbial sources have shown some antibiotic -like effects.

[0008] International Publication No. WO 2014/165936 Al relates to a feed product or feed ingredient comprising a microbial biomass, wherein the feed product or feed ingredient is substantially free of an aquatic animal-derived protein source and/or an aquatic animal derived-lipid source. The feed products or feed ingredients, however, described in this publication contain microorganisms that are predominantly bacteria, and do not increase omega-3 levels.

SUMMARY

[0009] According to a first aspect of the present disclosure, there is provided a composition comprising a fermented agricultural biomass that can be used as a viable feed product for promoting growth of an organism, wherein the biomass is fermented by a microorganism originating from an inoculant with a specifically defined microorganism profile. [0010] The composition of the first aspect may be used as a feed product on its own or, alternatively, it may be used as a component of a feed formulation. Thus, according to a second aspect of the present disclosure, there is provided a feed formulation comprising from about 10% to about 50% by weight the composition of the first aspect.

[0011] In certain embodiments, the agricultural biomass is fermented in a solid-state fermentation process in which the agricultural biomass is used as a substrate to support growth of the microorganism.

[0012] In certain embodiments the agricultural biomass is selected from one or more of the group consisting of: sweet potato, cassava, taro, wheat, brewers grain, rice bran, and corn bran. In certain specific embodiments the agricultural biomass is sweet potato, cassava, taro or any combination of two or more of the aforementioned.

[0013] In certain embodiments, the microorganism is a fungus. The fungus may belong to the genus selected from one or more of the group consisting of: Rhizopus, Aspergillus, and Trichoderma, such as one or more of the group consisting of: Aspergillus niger, Aspergillus oryzae, Aspergillius fumigatus, Pleurotus florida, and Pleurotus sajur cajo.

[0014] Thus, in certain embodiments, the organism is an aquatic animal. The aquatic animal may be selected from one or more of the group consisting of: finfish, flatfish, shellfish, eels and aquatic reptiles.

[0015] In certain embodiments, the aquatic animal is a finfish. The finfish may be selected from any one or more of the group comprising, but not limited to, tilapia, milkfish, kingfish, cobia, carp (including grass carp, silver carp, common carp, bighead carp, crucian carp, black carp), asari, pollock, catla, mackerel, rohu, cod, pilchard, sardine, anchovy, haddock, pompano, grouper, tuna, snapper, bream, sweetfish (Ayu), perch, catfish, bluegill, golden shiner, salmon, sturgeon, trout, walleye, herring, whiting, garfish, mullet, and seabass (baramundi).

[0016] In certain other embodiments, the aquatic animal is a shellfish. The shellfish may be a crustacean selected from the group consisting of shrimp, prawns, crabs, lobster, and crayfish. In certain other embodiments, the shellfish may be a mollusc, such as abalone.

[0017] In certain other embodiments, the aquatic animal is an aquatic reptile, such as a reptile.

[0018] According to a third aspect of the present disclosure, there is provided a feed formulation comprising the composition of the first aspect, wherein the feed formulation, when fed to an organism, increases the omega 3 levels of the organism. [0019] According to a fourth aspect of the present disclosure, there is provided a feed formulation comprising the composition of the first aspect, wherein the feed formulation has at least one antibiotic property. In certain embodiments, the antibiotic property is Vibrio-inhibitory activity.

[0020] According to a fifth aspect of the present disclosure, there is provided a feed formulation for tilapia comprising the composition of the first aspect. In certain embodiments, the feed formulation for tilapia comprises (1) 12.5-50% the composition of the first aspect; (2) 11-17% fish meal; (3)

12.5.0-23.5% soybean meal; (4) 4.5-24.5% rice bran; (5) 5-16% wheat flour; (6) 1.5-10% acetes meal; (7) 1% soybean oil; (8) 1% soy lecithin; (9) 1% vitamin premix; and (10) 1 % mineral premix, wherein the percentages are by weight of the total composition.

[0021] According to a sixth aspect of the present disclosure, there is provided a feed formulation for milkfish comprising the composition of the first aspect. In certain embodiments, the feed formulation for milkfish comprises (1) 12.5-50% the composition of the first aspect; (2) 7-19% fish meal; (3)

22.0-28% soybean meal; (4) 27-20.5% rice bran; (5) 5-.15% wheat flour; (6) 1.5-9% acetes meal; (7) 1- 3% fish oil; (8) 1% soy lecithin; (9) 1% vitamin premix; and (10) 2 % mineral premix,

wherein the percentages are by weight of the total composition.

[0022] According to a seventh aspect of the present disclosure, there is provided a feed formulation for shrimp comprising the composition of the first aspect. In certain embodiments, the feed formulation for shrimp comprises (1) 12.5-50% the composition of the first aspect; (2) 29.5-33% fish meal; (3) 5.5-9.0% soybean meal; (4) 18-24% rice bran; (5) 5-16% wheat flour; (6) 2-3% acetes meal; (7) 1-4% fish oil; (8) 1% soy lecithin; (9) 1% vitamin premix; and (10) 2 % mineral premix, wherein the percentages are by weight of the total composition.

[0023] According to an eighth aspect of the present disclosure, there is provided

a method for producing an animal feed from an agricultural biomass, the method comprising:

inoculating the agricultural biomass with a microorganism originating from an inoculant with a specifically defined microorganism profile;

allowing the microorganism to ferment the agricultural biomass, whereby protein content of the agricultural biomass increases.

[0024] In certain embodiments, the fermentation process is a solid-state fermentation with the agricultural biomass as a substrate to support growth of the fungus.

[0025] In certain embodiments, the agricultural biomass is selected from one or more of the group consisting of: sweet potato, cassava, taro, wheat, brewers grain, rice bran, and corn bran. In certain specific embodiments, the agricultural biomass is sweet potato, cassava, taro or any combination of two or more of the aforementioned.

[0026] In certain embodiments, the microorganism is a fungus. The fungus may belong to the genus selected from one or more of the group consisting of: Rhizopus, Aspergillus, and Trichoderma, such as one or more of the group consisting of: Aspergillus niger, Aspergillus oryzae, Aspergillius fumigatus, Pleurotus florida, and Pleurotus sajur cajo.

[0027] In certain embodiments, the protein content of the agricultural biomass increases from about 3- 6% to about 18-40% of the dry biomass weight.

[0028] According to a ninth aspect of the present disclosure, there is provided a method for increasing the ratio of omega 3 to omega 6 fatty acid content of an organism comprising feeding the organism with a feed formulation comprising the composition of the first aspect.

BRIEF DESCRIPTION OF DRAWINGS

[0029] Embodiments of the present disclosure will be discussed with reference to the accompanying figures wherein:

[0030] Figure 1 is a plot of weight gain of milkfish fed diets containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05);

[0031] Figure 2 is a plot of the survival rate of milkfish fed diets containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05);

[0032] Figure 3 is a plot of the specific growth rate of milkfish fed diets containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05); [0033] Figure 4 is a plot of the protein efficiency ratio rate of milkfish fed diets containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05);

[0034] Figure 5 is a plot of the feed conversion ratio (FCR) of milkfish fed diets containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05);

[0035] Figure 6 is a plot of the survival rate of tilapia fed diets containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05);

[0036] Figure 7 is a plot of weight gain of tilapia fed diets containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05);

[0037] Figure 8 is a plot of the specific growth rate of tilapia fed diets containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05);

[0038] Figure 9 is a plot of the feed conversion ratio of tilapia fed diets containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05);

[0039] Figure 10 is a plot of the protein efficiency ratio rate of tilapia fed diets containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05);

[0040] Figure 11 is a plot of the weight gain of P. vannamei fed containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05);

[0041] Figure 12 is a plot of the survival rate of P. vannamei fed diets containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05);

[0042] Figure 13 is a plot of the specific growth rate of P. vannamei fed diets containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05);

[0043] Figure 14 is a plot of the feed conversion ratio of P. vannamei fed diets containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05); and

[0044] Figure 15 is a plot of the protein efficiency ratio rate of P. vannamei fed diets containing graded levels (0, 12.5, 25, 50 g/lOOg of diet weight) of a feed formulation comprising a composition of the present disclosure (as feed ingredient) for 60 days. Bars represent mean values ± S.E. (standard error of the mean) of triplicate groups of experimental animals. Values having similar superscripts are not significantly different (a=0.05).

DESCRIPTION OF EMBODIMENTS

[0045] Compositions and methods of the present disclosure represent a solution to the need to develop alternative protein sources for feed for aquatic and terrestrial animals. The present disclosure is directed to a new type of animal feed in terms of providing a novel source of protein that can supplement or potentially replace fishmeal and the ability to modify the fatty acid profiles of livestock.

[0046] In certain embodiments, solid state fermentation of certain root crops such as sweet potato, cassava, and taro or certain grain matter such as wheat, brewers grain, rice bran, and corn bran using multi-cellular organisms results in an enhanced protein feed supplement with antibiotic -like properties and the ability to modify the fatty acid profile of some aquatic animals' flesh (higher omega 3 and lower omega 6) without the incorporation of extra source of these fatty acids.

[0047] To date, the applicant is not aware of any product devoid of omega 3 fatty acids that has the ability to confer a favorable omega 3 profile, presumably stimulating an animal fed with the product to accumulate more omega 3 and less omega 6 fatty acids.

[0048] The composition comprising a fermented agricultural biomass described herein can be used as a viable feed product for promoting growth of an organism. The biomass is fermented by a microorganism originating from an inoculant with a specifically defined microorganism profile under conditions to produce the fermented agricultural biomass.

[0049] Advantageously, the applicant has found that the fermented agricultural biomass of the present disclosure has antibiotic-like effects when used in a feed composition. Advantageously, the applicant has also found that the fermented agricultural biomass of the present disclosure has the ability to confer a favorable fatty acid profile to aquatic animals. The applicants work has shown that this property is applicable to other terrestrial livestock and their by-products (e.g. milk, eggs, and offal). Furthermore, the fermented agricultural biomass has been shown to replace at least 50% of fishmeal in feeding trials with no adverse event and in some instances increased the growth rate of prawns. Another protein enhanced product derived from solid state fermentation of copra meal has been shown to replace only up to 25% of fishmeal and did not show any improvement in the favorable accumulation of omega 3 fatty acids.

[0050] The fermented agricultural biomass of the present disclosure may replace fishmeal and soybean meal as sources of protein for animal feeds, providing a cheaper source of proteins and omega 3 fatty acids. Wild fish as a source for fishmeal/fish oil as well as DHA and EPA omega 3 fatty acids, is not sustainable and efforts to find alternative sources such as algae are in progress. Soybean meal having a higher omega 6 content is not favorable for a healthy diet.

[0051] By "fermented agricultural biomass" is meant plants or plant parts that have been exposed to the fermenting activity of microorganisms. [0052] By "viable feed product" is meant a feed ingredient or feed product whose performance to promote growth of an organism is good enough to reduce the need for a prior art feed ingredient or feed product generally acceptable to a person of ordinary skill in the art. Examples of a prior art feed ingredient include fish meal and soybean meal.

[0053] By "inoculant with a specifically defined microorganism profile" is meant an inoculant whose microorganism content is well characterized in terms of concentration and identity of each microorganism present.

[0054] By "about," which is followed by a number, is meant to include values higher and lower the indicated number that a person of ordinary skill in the art considers equivalent to the indicated number. For example, "about" is meant to include values that reflect variations inherent in methods employed in determining a measurement. As such, "about" may be ±20%, ±19%, ±18%, ±17%, ±16%, ±15%, ±14%, ±13%, ±12%, ±11%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, or ±0.1%, depending on the methods employed in determining the measurement.

[0055] The composition of the present disclosure comprises a fermented agricultural biomass that is fermented by a microorganism originating from an inoculant with a specifically defined microorganism profile. The composition may be used as a feed product on its own. For example, the composition may provide an adequate feed for herbivorous fish, such as tilapia. Alternatively, the composition may be used as a component of a feed formulation,. Thus, the present disclosure also provides an animal feed formulation comprising the fermented agricultural biomass to about 10 - 50% by weight. The animal feed formulation may be used to feed aquatic animals or terrestrial animals.

[0056] In certain embodiments, the present disclosure provides an animal feed produced by fermentation of the agricultural biomass with filamentous fungi to increase its protein content from 3-6% to 18-40% of the dry biomass.

[0057] In certain embodiments, the present disclosure provides an animal feed formulation containing fermented agricultural biomass that can increase the omega 3 to omega 6 fatty acid profile of the animals.

[0058] In certain embodiments, the present disclosure provides a feed formulation with Vibrio inhibitory activity and produced by fermentation of the agricultural biomass with filamentous fungi to increase its antibacterial activity. [0059] In certain embodiments, the present disclosure provides a composition comprising a fermented agricultural biomass that can be used as a viable feed product for promoting growth of an organism, wherein the biomass is fermented by a specific microorganism originating from an inoculant with a specifically defined microorganism profile. This composition may comprise by weight 10%, 12.5%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of a feed formulation.

[0060] The agricultural biomass may be a root crop such as sweet potato (Ipomoea batata), cassava (Manihot esculenta) or taro {Colocasia esculenta) and/or it may be a grain matter, such as wheat, brewers grain, rice bran or corn bran. The agricultural biomass could also be any combination of the

aforementioned materials. The agricultural biomass may comprise the flesh, the skin, the husk, the hull or any other part of the root or grain of the particular agricultural biomass used. The agicultural biomass may be in the form of whole root, grain, bran, hull, husk, etc. For root crops, the agicultural biomass is preferably in the form of chips, powder, granules, etc.

[0061] The microorganism fermenting the agricultural biomass may be a fungus or a combination of at least two fungi. The fungus may be a filamentous fungus, microfungus or a macrofungus. The fungus may belong to the genus of Rhizopus, Aspergillus, or Trichoderma. More specifically, the fungus may be Aspergillus niger, Aspergillus oryzae, Aspergillius fumigatus, Pleurotus florida, or Pleurotus sajur cajo.

[0062] Organisms whose growth is promoted by feeding with the feed formulations and fermented agricultural biomass of the present disclosure include, but are not limited to, aquatic animals and terrestrial animals.

[0063] Without being limited to a specific application, the compositions and feed formulations of the present disclosure are particularly suitable for feeding farmed aquatic animals. The aquatic animal may be selected from one or more of the group consisting of: finfish, flatfish, shellfish, eels and aquatic reptiles. The aquatic animal may be a sea water (i.e. marine) animal, a fresh water animal or a brackish water animal.

[0064] In certain embodiments, the aquatic animal is a finfish. The finfish may be selected from the group consisting of tilapia, milkfish, kingfish, cobia, carp (including grass carp, silver carp, common carp, bighead carp, crucian carp, black carp), asari, pollock, catla, mackerel, rohu, cod, pilchard, sardine, anchovy, haddock, pompano, groupers, tuna, snapper, bream, sweetfish (Ayu), perch, catfish, bluegill, golden shiner, salmon, sturgeon, trout, walleye, herring, whiting, garfish, mullet, and seabass

(baramundi).

[0065] In certain embodiments, the aquatic animal is a shellfish. The shellfish may be a crustacean or a mollusc. The crustacean may be selected from the group consisting of shrimp, prawns, crabs, lobster, and crayfish. The shrimp may be of the genera Penaeus including P. vannamei, P. stylirostris, P. monodon, P. chinensis. The prawns may be of the genus Macrobrachium. The crabs may be of the genus Scylla, such as Scylla serrata, and Scylla paramamosai.

[0066] In certain other embodiments, the aquatic animal is an aquatic reptile, such as a reptile.

[0067] In certain embodiments, the organism is an terrestrial animal. The terrestrial animal may be selected from one or more of the group consisting of: pigs, chicken, goats, cattle, sheep, horses, ducks, etc.

[0068] The feed formulations and fermented agricultural biomass of the present disclosure, when fed to an organism, increase the omega 3 levels of the organism. These feed formulations and fermented agricultural biomass also have at least one antibiotic property, including, but not limited to, Vibrio- inhibitory activity.

[0069] The present disclosure is also directed to a method for producing an animal feed from an agricultural biomass, comprising: (1) inoculating the agricultural biomass with a microorganism originating from an inoculant with a specifically defined microorganism profile; and (2) allowing the microorganism to ferment the agricultural biomass, whereby protein content of the agricultural biomass increases. The protein content of the agricultural biomass may increase from about 3-6% to about 18-40% of the dry biomass weight. The fermentation process may be a solid-state fermentation with the agricultural biomass as a substrate to support growth of the fungus.

[0070] The agricultural biomass used in the method may be in the form of chips, powder, granules, grains, etc.

[0071] The agricultural biomass may processed prior to inoculation. For example, the moisture content of the agricultural biomass may be adjusted prior to inoculation.

[0072] The microorganism may be allowed to ferment the agricultural biomass under room conditions for a short a period of time as possible, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days. The substrate may be mixed to loosen lumps regularly during the fermentation period.

[0073] After fermentation, the fermented agricultural biomass may be dried. The fermented agricultural biomass may be dried to about 10% moisture. [0074] The present disclosure also concerns an aquaculture feed formulation comprising fermented agricultural biomass to about 12.5 - 50% by weight. The feed formulation may be for tilapia, milkfish, and shrimp, comprising a fermented agricultural biomass in accordance with the present disclosure disclosure.

[0075] The feed formulation for tilapia may comprise (1) 12.5-50% the fermented agricultural biomass; (2) 11-17% fish meal; (3) 12.5.0-23.5% soybean meal; (4) 4.5-24.5% rice bran; (5) 5-16% wheat flour; (6) 1.5-10% acetes meal; (7) 1% soybean oil; (8) 1% soy lecithin; (9) 1% vitamin premix; and (10) 1 % mineral premix, wherein the percentages are by weight of the total composition.

[0076] The feed formulation for milkfish may comprise (1) 12.5-50% the fermented agricultural biomass; (2) 7-19% fish meal; (3) 22.0-28% soybean meal; (4) 27-20.5% rice bran; (5) 5-.15% wheat flour; (6) 1.5-9% acetes meal; (7) 1-3% fish oil; (8) 1% soy lecithin; (9) 1% vitamin premix; and (10) 2 % mineral premix, wherein the percentages are by weight of the total composition.

[0077] The feed formulation for shrimp may comprise (1) 12.5-50% the fermented agricultural biomass; (2) 29.5-33% fish meal; (3) 5.5-9.0% soybean meal; (4) 18-24% rice bran; (5) _. 5-.16% wheat flour; (6) 2- 3% acetes meal; (7) 1-4% fish oil; (8) 1% soy lecithin; (9) 1% vitamin premix; and (10) 2 % mineral premix, wherein the percentages are by weight of the total composition.

[0078] The present disclosure is also directed to methods for increasing the ratio of Omega3: Omega 6 fatty acid content of an organism comprising feeding the organism with a feed formulation comprising the composition of claim 1.

EXAMPLES

[0079] Below are examples that illustrate various embodiments of the disclosure. These examples should not be construed as restricting the scope of the disclosure as defined in the claims.

[0080] Throughout the following examples, reference is made to ProEnK. ProEnK is a trade name for the composition produced according to Example 1.

[0081] Example 1 - Preparation of Fermented Agricultural Biomass

[0082] Sweet potato chips (at least 90% dry matter) and nutrient solution were mixed at a ratio of 35-40 : 60-65. The nutrient solution (adopted from Pham, C.B., Lat, M.R.L.Y., Ramirez, T.J., Quinlat, MJ. and Pham, LJ. 1992. Enriching cassava protein using solid state fermentation. BIOTECH, UPLB) contained, per litre of water, the following: 35 g urea or CO(NH ₂ ) ₂

45.35 g ammonium phosphate or (NH ₄ ) ₃ P04

15 g ammonium sulfate or (NH ₄ ) ₂ S04

10 g sucrose (sugar)

3 tbsp cane vinegar

[0083] Manual mixing of sweet potato chips and nutrient solution was performed until a uniform mixture (substrate) was obtained. Substrate was packed in polypropylene bags and secured with plugs (PVC, cotton plug, paper cover, rubber band).

[0084] Bagged substrate was sterilized at 20 psi for 30 minutes. Sterilized substrate was allowed to cool. Under aseptic conditions, the substrate was inoculated with any of the following microbial cultures (following specified protocol to avoid contamination):

a. Aspergillus niger

b. Aspergillus oryzae

c. Aspergillius fumigatus

d. Pleurotus florida

e. Pleurotus sajur cajo

[0085] For a 2kg substrate, the size of the inoculum (grown in PDA) was 9 cm ² . The inoculated substrate was left to ferment for 21 days under room conditions. As soon as mycelial

growth/multiplication was visible, the substrate was mixed to loosen lumps and to ensure fast and proliferous microbial growth throughout the substrate. This was done regularly during the entire fermentation period.

[0086] After three weeks of fermentation, the fermented substrate (protein biomass) was subjected to sun drying to partially dry, ground to pellet-like form, and sundried again to about 10% moisture. The dried biomass was packed in 5-kg polypropylene bags, or 25-kg or 50-kg sacks and marked ProEnK.

[0087] Biochemical analysis of the generated ProEnK was conducted. Analysis of protein was conducted following the methods described by AOAC. Total lipid was measured by Bligh and Dyer, moisture by subjecting to oven drying and gravimetric measurement as described by AOAC. Ash was measured by combusting in an oven furnace. Fatty acid was measured by extracting the lipid, subjecting the lipid to esterification and the esters were subjected to a gas chromatography analysis to assess the profile of the fatty acid composition of the ProEnK. Further contaminating polyaromatic hydrocarbons (PAH) and aflatoxin that could bioaccumulate in fish and could cause harm to humans were analyzed using gas chromatography mass spectrometer and enzyme linked immunosorbent based assay. [0088] The results of the analyses are shown in Table 1. The analyses revealed that the fermentation increased the protein content of the sweet potato material. The final fermented meal had a protein content of around 18% with minimal lipid and ash content indicating that the material contained less metals and fats that could affect the overall growth performance of the test animal.

[0089] Table 1 - Biochemical composition of ProEnK (g/lOOg dry matter; Mean + S.E.)

[0090] The fermented material was further identified to contain a significant amount of starch measured as NFE that is about 60% of the dry biomass. This material could be a good source of energy when used as feed for growing animals. The high starch content may reflect the starch content of the raw material used that is the sweet potato tuber or pulp that is known to be mostly composed of starch.

[0091] The aflatoxin level found in the fermented material was low and was considered as a safe ingredient to be used as animal feed. Ten ppb and above of aflatoxin level in feed material is the level that is not allowable. The aflatoxin level found in ProEnK analysis is below lOppb making this ingredient safe for use as feed for aquatic animals.

[0092] Although the lipid content of the fermented material (ProEnK) was found minimal, it is worthy to know the fatty acid composition of the lipid present in this feed material. Analysis of the fatty acid profile of this fermented feed material showed that almost all of the fatty acids are saturated and no poly unsaturated fatty acids were found, Table 2.

[0093] Table 2 - Fatty acid profile of feed material

Percent of total

fatty acid

Fatty Acids

methyl esters

(%)

Benzeneacetic acid, methyl ester 3.05

Benzenepropanoic acid, methyl ester 1.03

Decanoic acid, methyl ester 2.4

3-Phenyl-2-propenoic acid, methyl ester 1.0 Percent of total

fatty acid

Fatty Acids

methyl esters

(%)

Laurie acid 7.4

Methyl tetradecanoate 1.2

Pentadecanoic acid 2.8

Myristic acid 3.3

Palmitic acid 18.65

14-Methylhexadecanoic acid, methyl ester 3.0

9,12-Octadecadienoic acid (Z,Z)-, methyl

2.9

ester

9-Octadecenoic acid (Z)-, methyl ester 10.45

11 -Hydroxylauric acid 35.61

10-Methyloctadecanoic acid, methyl ester 1.08

10-Nonadecenoic acid, methyl ester 1.08

[0094] The fatty acid profile indicates that the fatty acids are mostly of the medium chain type and no fatty acid with 20 or more carbon backbone was detected. Hydroxylauric acid was found to be dominant with 35% followed by palmitic acid at 18% and oleic acid at 10%. These fatty acids as component of this feed material are ideal as a metabolic energy source for aquatic animals since these are medium chain fatty acids. Medium chain fatty acids with less than 20 carbons are directly absorbed and utilized by tissues without undergoing complex processing in the liver.

[0095] The presence of phenyl acetic acid or benzyl acetic acid in this material could also improve the feed value of this material. Phenyl acetic acid is usually a metabolic by-product of Aspergillus species used in fermentation. This compound has been documented to be potent antibacterial agent and the presence of this in this ingredient may prevent gut infection in aquatic animals and may promote growth and better survival of the animals fed by this material.

[0096] In summary, the fermented agricultural biomass was found to be high in protein, contained medium chain fatty acids and some antibacterial metabolites produced by the fermenting agents. The material was also free of polyaromatic hydrocarbons.

[0097] The presence of carcinogenic polyaromatic hydrocarbons (PAH) in ProEnK was also evaluated since some of these compounds are produced by microbes and since ProEnK is a product of fungal fermentation there is a possibility of the presence of these compounds in this feed material. [0098] Analysis using gas chromatography mass spectrometry revealed that PAH is not present in this fermented feed material. No PAH was detected in the solvent extract of ProEnK, Table 3. This indicates that this feed ingredient is safe as feed material for aquatic animals.

[0099] Table 3 - Results of PAH analysis of ProEnK feed material

[00100] Collectively the data indicate that ProEnK is a feed ingredient containing a good amount of protein and significant amount of carbohydrates as energy source. The fatty acid profile also indicates that this material is a good source of medium chain fatty acids that are good sources of cellular energy to fuel tissue growth and for efficient metabolism. The aflatoxin level of this material is low and no polyaromatic hydrocarbon was detected in the solvent extract of this feed ingredient.

[00101] Example 2 - Characterization of Vibrio inhibitory activity of ProEnK

[00102] A fermented agricultural biomass, labelled ProEnK, was produced from fermentation of sweet potato with selected microbes. ProEnK has low molecular weight compounds and short chain fatty acid components. These molecules could act as an antibacterial. ProEnK was extracted using ethyl acetate and the low molecular weight residues were collected and assayed against a shrimp pathogenic bacterium Vibrio vulnificus. The assay consisted of a disc diffusion system wherein a dose of ProEnK extract was added to a disc that was laid on a lawn of V. vulnificus in a Luria Bertani media. The results obtained indicated that fermentation metabolites including low molecular weight compounds exhibit antibacterial activity. This makes ProEnK an interesting feed material for it contains low antibacterial activity against the pathogenic Vibrio vulnificus. [00103] Example 3 - Preparation of feed formulations comprising ProEnK

[00104] ProEnK was used as a dietary ingredient for the three major species commonly used for aquaculture, namely, milkfish (Chanos chanos), white shrimp (Penaeus vannamei) and the saline strain of Tilapia nilotica developed at the University of the Philippines Visayas that can be reared in full strength seawater conditions.

[00105] Milkfish Diets

[00106] The first diet to be formulated was a diet for juvenile milkfish, Chanos chanos. Four diets were formulated and theoretical nutritive composition derived based on the published data on the composition of the dietary ingredients. The value of the proximate composition analysis of ProEnK was used in the calculation to attain the target protein and lipid levels of the diets. The diets were formulated to be isonitrogenous and isolipidic meaning the overall protein and lipid contents of the diets were designed to be almost similar. The goal of this formulation was to maximize the dietary inclusion of ProEnK in the diet of milkfish and to assess at what particular amount this material could be added to milkfish diets without affecting the growth performance of this fish.

[00107] The diets were soybean and fish meal based formulations and primarily designed for the optimum requirement of milkfish. The diets were formulated to contain 30% protein and 8% fat that is known to promote optimum growth of this cultured species. The inclusion levels of ProEnK in these diets ranged from 0 to 12.5, 25, and 50% of actual ingredient weight in the diet. Since fishmeal is the costly ingredient in fish feed, the present formulation was also designed to decrease the amount of fishmeal in the diets as the levels of ProEnK inclusion is increased. To balance the levels of other nutrients, the other ingredients were also minimally varied as to attain balanced dietary protein and lipid contents in the diet. The composition of the formulation of the diets containing ProEnK for milkfish is presented in Table 4. The theoretical calculated composition of the diets are also presented and indicate that the lipid and protein contents (considered the major nutrients with significant influence on growth) of these formulations are almost similar.

[00108] Table 4 - Composition of diets containing different levels of ProEnK for milkfish

M0

M12.5 M25 M50

(Basal diet)

Ingredients

(g/lOOg Dry Matter)

Fish Meal 25 19 13 7

ProEnK 0 12.5 25 50

Soybean Meal 12 22 28 22 Rice Bran 31 27 20.5 0

Acetes Meal 1 1.5 3.5 9

Wheat Flour 27 13 5 5

Fish Oil 0 1 1 3

Lecithin 1 1 1 1

Vitamin Mix 1 1 1 1

Mineral Mix 2 2 2 2

Calculated Biochemical Composition

(g/lOOg Dry Matter)

Protein 30.90 30.80 30.63 30.14

Lipid 8.80 8.69 7.32 6.16

Ohers 60.30 60.51 62.05 63.7

[00109] Tilapia Diets

[00110] Similar to milkfish, four diets were also formulated to assess the maximum dietary inclusion levels of ProEnK in juvenile tilapia. The formulated diets were soybean and fish meal based formulations that were formulated to satisfy the requirement of tilapia to attain maximum growth. These diets were formulated to contain almost similar lipid and protein contents (30% protein and 8% lipid) to ensure that the effects observable in the trial could only be attributed to the inclusion levels of dietary ProEnK and not to the major nutrient variability. In contrast to the formulation used for milkfish, the dietary oil in the tilapia diet formulation was sourced from soybean oil that can provide essential fatty acids required by tilapia since this fish has the ability to chain elongate and desaturate fatty acids originating from plant oils. Milkfish like other marine fish species, on the other hand poorly utilize fatty acids with 18 carbons that are commonly found in soybean oil.

[00111] The four formulated diets contained varying inclusion levels of ProEnK that is 0, 12.5,

25, and 50% by weight of the formulated diet. Similar to the formulation made for milkfish, the fishmeal content of the diets was decreased as the amount of dietary ProEnK inclusion was increased. The levels of nutrients in the formulated diets were balanced by varying the amounts of other ingredients as not to affect the levels of other major dietary nutrients. The composition of the formulation of the diets containing ProEnK for Tilapia is presented in Table 5. The theoretical calculated composition of the diets are also presented and indicate that the lipid and protein contents (considered the major nutrients with significant influence on growth) of these formulations are almost similar. [00112] Table 5 - Composition of diets containing different levels ofProEnKfor tilapia

[00113] Shrimp Diets

[00114] In order to assess the viability of ProEnK as a dietary ingredient for shrimp culture, diets were formulated to contain varying levels of this feed ingredient. Four diets were designed containing graded levels of ProEnK that is 0 (the basal diet), 12.5%, 25% and 50% inclusion of ProEnK by weight. The diets were formulated to contain a good amount of fish meal since shrimp requires marine proteins to achieve maximum growth. The diets were formulated to be similar in lipid and protein contents and were formulated to contain 35% protein and 9% lipid. In contrast to tilapia and milkfish, shrimp requires a higher content of dietary protein and a good supply of dietary marine oil is considered essential for growth. Though the formulation for shrimp diets contains a higher amount of protein as compared to milkfish and tilapia, in this formulation the amount of dietary protein was also decreased as the inclusion levels of dietary ProEnK were increased.

[00115] The ingredient list and composition of the formulation of the diets containing ProEnK for shrimp is shown in Table 6. All the formulations were formulated to ensure that the levels of protein and lipid among the different formulations were almost similar. [00116] Table 6 - Composition of diets containing different levels ofProEnKfor shrimp

[00117] Process Flow in Formulation of the Diets

[00118] The diets were formulated following systematic processes involving a series of steps that includes:

1. Mathematical Formulations: Paper calculations and mathematical calculations were conducted to balance the nutrient levels of the dietary ingredients to be used in the diet formulation and to attain the optimum nutrient requirement essential for the need of the target organisms.

2. Sieving: The dietary ingredients were sieved to appropriate size (100 micrometer) and weighed based on the amounts needed for each dietary formulation. The wet and dry dietary ingredients were prepared and weighed separately.

3. Mixing: The dry Ingredients were mixed in a mechanical mixer to ensure complete homogenization of the individual dietary ingredients. Following the dry ingredient mixing wet ingredients were added and the resulting moist mash was further mixed mechanically.

4. Pelletizing: The moist mash resulting from the mixing was then subjected to pelletization by passing the mash through a cold mechanical extruder that produces moist pellets.

5. Drying: Following the extrusion the moist pellet was then subjected to drying in a hot air oven at 80°C to attain a moisture content of 8-10 percent. This drying would extend the shelf life of the feed and at this temperature the heat labile nutrients including vitamins and other micronutrients will not be degraded or oxidized. After the drying step the feeds were then stored in a refrigerator.

6. Analysis: Subsamples were taken from the finished formulated feeds and subjected to biochemical analyses to quantify the level of nutrients including protein, lipid, ash and NFE. Analyzed data were used in the computation of the feed value and the biological value of the feed.

[00119] Biochemical Analysis of the Feed Formulations

[00120] Biochemical or nutritive component analysis of the aforementioned prepared feed formulations was conducted.

[00121] A total of twelve diets were formulated with four diets containing graded levels of dietary

ProEnK for the three commonly cultured species including milkfish, tilapia and shrimp. The biochemical composition analyses were conducted following the AO AC method for analysis of feed nutrient including, protein, lipid, ash, NFE and fiber. Similar methods were also used in the analysis of the raw ProEnK as ingredient.

[00122] The analyses of the proximate nutrient composition of the formulated diets are presented in Tables 7, 8, and 9. Analysis revealed that the formulations satisfy the required nutrients needed for the optimum growth of tilapia, milkfish and shrimp. The analyzed protein and lipid contents of the different diets for a particular species were found not to be highly variable. Specifically, the analyzed protein content of the formulated diets intended for milkfish and tilapia are close to the theoretical value that is 30% of the total diet. The protein and lipid content of the formulated diets for shrimp is also close to the calculated value that is 35%. The analyzed nutrient variations among these diets were found not significant.

[00123] Table 7 - Biochemical composition (actual analysis) of diets containing different levels of ProEnK for milkfish

ProEnK inclusion levels g/lOOg diet

DIET TYPES

M0 M12.5 M25 M50

(0 level) (12.5%) (25%) (50%)

Biochemical

Composition

(g/lOOg diet)

Total Protein 30.43+0.20 32.3+0.34 32.26+0.20 31.80+0.47

Total Lipid 4.5+0.10 4.7+0.09 4.8+0.05 5.1+0.30

Total Carbohydrate

36.5+1.40 35.4+1.3 34.4+1.20 37.7+1.5

(NFE)

[00124] Table 8 - Biochemical composition (actual analysis) of diets containing different levels ofProEnKfor tilapia

[00125] Table 9 - Biochemical composition (actual analysis) of diets containing different levels of ProEnK for shrimp

Example 4 - Tilapia, milkfish, and shrimp feeding trials using feed formulations [00127] To assess the feed value of ProEnK for shrimp, pathogen free P. vannamei juveniles, were obtained from a commercial hatchery in Cebu Philippines and transported to Institute of

Aquaculture Multi-Species Hatchery (University of the Philippines Visayas, Miagao, Iloilo). Shrimps were stocked and acclimated in 2-ton fiberglass tank indoors for 7 days and were maintained with commercial feed given three times daily. Following the acclimation, three hundred juvenile shrimp were randomly collected and distributed in 12 tanks (60-L volume) to constitute the four experimental treatments in triplicate following a completely randomized design (CRD). Tanks were supplied with constant aeration maintaining oxygen near at saturation levels. Water temperature was at the range of 28°C to 30°C, at a salinity of 32 to 35 psu and the animals were exposed to normal photoperiod.

[00128] For the fish trials, Nile tilapia (Tilapia nilotica) and milkfish (Chanos chanos) juveniles were produced and were acclimatized to the laboratory conditions for two weeks. The animals were maintained fed the control diet at 10% body weight, four times daily (0800, 1100, 1400, and 1700 h). Feeding trials with both fish species were conducted in a recirculating culture system with 12 (95L) tanks equipped with a biological filter, sedimentation tank, and sediment filter. Continuous aeration was provided and water quality parameters were monitored regularly. Water temperature and pH were measured twice daily (0800 and 1400 h) using a laboratory mercury thermometer and hand-held digital pH meter, respectively. Normal photoperiod was used and levels of dissolved oxygen, total ammonia nitrogen, and nitrite were measured using test kits (Advance Pharma Co., Ltd., Bangkok, Thailand). No critical conditions for water temperature (27.11 ± 0.15°C), pH (8.87 ± 0.06), dissolved oxygen (8.50 ± 0.27 ppm), total ammonia nitrogen (0.11 ± 0.03 ppm), and nitrite (0.18 ± 0.05 ppm) were observed during the entire duration of the experimental period.

[00129] Following the feeding trial, all fish samples from each tank were pooled, weighed, and subjected to proximate analysis. Growth performance and nutrient utilization were measured by calculation of the following response parameters:

Survival (SR, %) = (final number of fish/ initial number of fish) x 100

Relative weight gain (WG, %) = (final weight - initial weight / initial weight) x 100

Specific growth rate (SGR, % day-1) = [(In final weight - In initial weight) / number of culture days] x 100

Feed conversion ratio (FCR) = total dry feed intake (g)/wet weight gain (g)

Nutrient retention (NR, % of intake) = 100 x [(% final carcass nutrient x final ABW) -

(% initial carcass nutrient x initial ABW)] / total nutrient intake.

[00130] All data were subjected to a statistical analysis with Shapiro-Wilks W test used to asses normality of data and Levene's test was performed to check homogeneity of variance. Apparent dry matter digestibility of diets and data from the growth trial (i.e. survival, weight gain, SGR, FCR, carcass composition, nutrient retention) were analyzed by one-way analysis of variance (ANOVA). Where significant difference was present, treatment means were compared using Tukey's HSD test at /?<0.05. Values are expressed as means ± S.E.M. All statistical calculations were performed using SPSS for windows (version 16).

[00131] Results of Feeding Trial on Milkfish

[00132] The results of a feeding trial of milkfish with increasing dietary inclusion of ProEnK indicate that this material could be included in the diet to 50% with no inhibitory activity on growth and overall physiological performance of the fish. The control with full fish meal based diet is not significantly different with that of the 50% inclusion levels of ProEnK in the diet (Figures 1 to 5).

[00133] Measured growth indices including specific growth rate (SGR), protein efficiency ratio

(PER), and the ability to convert eaten feeds to biomass, feed conversion ratio (FCR), were observed to be similar in treatments maintained with the control and those that received the highest inclusion levels of dietary ProEnK. No negative trend was found in the performance of milkfish as an effect of the increasing levels of ProEnK in diets. This indicates that overall ProEnK could be included in milkfish diets up to 50 % of the dietary ingredients (Figures 1 to 5).

[00134] Results of Feeding Trial on Tilapia

[00135] During the growth trial, fish survival was found high (above 80%) and was not significantly different among treatments. Fish given the control exhibited similar trends with that of the treatments receiving diets with ProEnK. No significant differences in biological indices including weight gain, SGR and survival were observed among the treatments including those receiving the highest dose of ProEnK inclusion in the diets. Moreover, ability of the animals to convert feeds to biomass is also not affected with ProEnK inclusion in the diet. In terms of feed protein utilization, ProEnK inclusion even at 50% of the diets does not affect the overall protein efficiency ratio of the experimental animals. Overall growth indices, survival and feed efficiency index have been shown in these data not to be affected by dietary ProEnK inclusion and the data suggest that ProEnK could be included in the diet up to 50% of the total diet biomass (Figures 6 to 10).

[00136] Results of Feeding Trial on Shrimp Penaeus vannamei

[00137] In trials with P.vannamei, growth indices including specific growth rate (SGR), protein efficiency ratio (PER), and the ability to convert eaten feeds to biomass, feed conversion efficiency, were observed to be similar in all treatments maintained with the diets containing ProEnK including the highest level and the control treatment that received the full fish meal based diet. In this trial, it was generally observed that higher inclusion levels beyond the 25% elicited improvement trend in shrimp growth performance in terms of SGR, FCE, and PER. Though the trend in growth parameters increases with increasing level of dietary ProEnK inclusion, the values are not statistically different among the treatments. Collectively the data indicate that similar to fish, P. vannamei could tolerate high level of ProEnK inclusion in the diet without jeopardizing overall growth performance. ProEnK could be included in the diet up to 50 % of the total diet.

[00138] Biochemical composition analyses ofmilkfish (C. chanos) fed with ProEnK

[00139] Dietary inclusion levels of ProEnK in milkfish were found not to affect the biochemical composition of the fish carcass. No significant differences in terms of total lipid, protein and ash were found among treatments (Table 10). There was no observable trend also on the pattern of major nutrient profile of the fish maintained with the control diet and those that received diets containing increasing inclusion levels of dietary ProEnK. The carcass composition of the treatment receiving the highest inclusion level is similar to that of the group receiving the full soybean meal diets.

[00140] Table 10 - Final carcass biochemical composition of milkfish fed increasing levels of dietary ProEnK

[00141 ] Fatty acid profile of milkfish fed with ProEnK

[00142] The fatty acid profile of milkfish fed ProEnK based diets is shown in Table 11. In terms of fatty acids the control group was compared with that of the group that received the highest dietary inclusion level of ProEnK and results indicate that this treatment did not alter the major fatty acid composition of the fish receiving the feed ingredient (ProEnK). In terms of omega 3 fatty acid, the treated group with ProEnK has higher ratio of omega 3 to omega 6 fatty acids suggesting accumulation of omega 3 fatty acids in the tissues of this fish. The DHA/ EPA ratio was found comparable between the two experimental groups. These results suggest that even at high inclusion levels of ProEnK the fatty acid profile of this fish is maintained and not significantly adversely altered. Table 11 - Fatty acid profile of milkfish fed increasing levels of dietary ProEnK

Fatty Acid Methyl Esters Milkfish Fed Milkfish Fed ProEnK based (FAME) Control diet Diet

(g/lOOg FAME) (g/lOOg FAME)

Dodecanoic acid, methyl ester 0.11+0.01 0.16+0.02

Tetradecanoic acid, methyl ester 0.98+0.21 1.69+0.03

Pentadecanoic acid, methyl ester 0.19+0.00 0.43+0.01

9-Hexadecenoic acid, methyl ester 2.70+0.29 4.25+0.12

Hexadecanoic acid, methyl ester 21.78+02.45 15.40+0.52

Heptadecanoic acid, methyl ester 0.30+0.01 7.10+0.29

9,12,15-Octadecatrienoic acid,

0.30+0.03

methyl ester (Linolenic, Omega 3) 0.00

6,9-Octadecadienoic acid, methyl 7.89+0.08

0.00

ester

9,12-Octadecadienoic acid, methyl

11.12+1.65

ester (linoleic, Omega 6) 00.00

9-Octadecenoic acid, methyl ester 22.77+0.44

21.47+1.81

(cis)

Octadecanoic acid, methyl ester 8.24+0.20

11.30+1.21

5,8,11,14-Eicosatetraenoic acid,

methyl ester (Omega 6, 5.53+0.15 5.82+0.13

Arachidonic)

5,8,11,14, 17-Eicosapentaenoic acid,

4.44+0.01

methyl ester (Omega 3, EPA) 4.95+0.26

7,10,13-Eicosatrienoic acid, methyl 1.64+0.06

2.16+0.14

ester (trans)

11-Eicosenoic acid, methyl ester 2.16+0.01

1.98+0.04

(cis)

Eicosanoic acid, methyl ester

0.16+0.01

0.34+0.02

4,7,10,13,16, 19-Docosahexaenoic

acid, methyl ester (DHA, Omega 3) 15.47+0.07 14.96+0.51

13-Docosenoic acid, methyl ester 0.00+0.0 0.78+0.06 Docosanoic acid, methyl ester 0.00+0.0 0.21+0.00

(n-3) / n-6) ratio 1.24 3.42

DHA / EPA ratio 3.48 3.02

[00144] Biochemical composition analyses oftilapiafed with ProEnK

[00145] Similar to milkfish, increasing dietary inclusion of ProEnK to tilapia diet did not significantly adversely affect the overall performance of the experimental fish and the corresponding flesh nutrient composition. No significant changes in body composition were observed in terms of protein, lipid and ash contents of tilapia fed the ProEnK based diets even at the highest dietary inclusion dose of 50% diet. This indicates that the ingredient satisfies the basic nutrient requirement of the fish to attain optimum growth and normal deposition of nutrient materials in the body. Even the highest inclusion level of ProEnK resulted in a body composition that is similar with that of the control receiving the fish meal based diets (Table 12).

[00146] Table 12 - Final carcass biochemical composition oftilapiafed increasing levels of dietary ProEnK

[00147] Fatty acid Profile oftilapiafed ProEnK at optimum inclusion level

[00148] Influence of ProEnK dietary inclusion on the fatty acid profile of tilapia is shown in

Table 13. The fatty acid profile indicates better accumulation of omega 3 fatty acid in the tissues of ProEnK fed tilapia as indicated by a higher omega 3/omega 6 ratios (n3:n6 ratio) as compared to the control fish meal based group. This indicates better biosynthesis and accumulation of omega 3 fatty acid by fish fed the ProEnK-based diets. The ratio of DHA/EPA in the treatment groups indicate a value of almost 1, suggesting optimum levels of these omega 3 fatty acids in the two dietary treatments. The similar values obtained in both treatments indicate that these fatty acids are not limiting for the requirement of the species and this explains the similar growth pattern observed in the ProEnK based diet and the fish meal based dietary regimen. [00149] Table 13 - Fatty acid profile oftilapiafed increasing levels of dietary ProEnK

[00150] Biochemical composition analyses of white shrimp (P.vannameiJ/eif with ProEnK [00151] Results of the trial on P. vannamei indicate a similar type of response as observed in both tilapia and milkfish treatment groups. Carcass biochemical composition of the group receiving the control fish meal based diet exhibited similar composition to those that received the experimental diets containing different levels of dietary ProEnK. There is no clear influence of dietary ProEnK on the carcass composition of the experimental shrimp. Nutrient contents including lipid, protein and ash contents were not found to be influenced by dietary levels of ProEnK (Table 14).

Table 14 - Carcass biochemical composition of P. vannamei fed increasing levels of

[00153] Fatty acid Profile of white shrimp (P. vannamei) fed with ProEnK

[00154] The fatty acid profile of shrimp (P. vannamei) fed ProEnK based diet is shown in Table

15. The fatty acid profile of the group receiving the ProEnK diets indicates that similar to fish, ProEnK as ingredient for shrimp diets has promoted the accretion of omega 3 fatty acids in the tissues as indicated by a higher N3:N6 fatty acid ratio in the group receiving the ProEnK diet. This suggests better accumulation of omega 3 fatty acids in shrimp tissues. The ratio of DHA:EPA in both treatments indicates that these fatty acids are not limiting in these experimental animals.

[00155] Table 15 - Fatty acid profile of P. vannamei/<?(f increasing levels of dietary ProEnK

Tilapia Fed Control diet Tilapia Fed ProEnK

Fatty Acid Methyl Esters (g/lOOg FAME) based Diet

(g/lOOg FAME )

2-Decenoic acid, methyl ester 0.29+0.03 0.00+0.00

Dodecanoic acid, methyl ester 0.10+0.00 0.07+0.00

Tetradecanoic acid, methyl ester 0.53+0.03 0.72+0.06

Pentadecanoic acid, methyl ester 0.33+0.02 0.24+0.01

9-Hexadecenoic acid, methyl 1.49+0.17

1.55+0.32

ester

Hexadecanoic acid, methyl ester 12.00+0.41 18.57+4.35 Heptadecanoic acid, methyl 1.90+0.29

0.90+0.09

ester

9,12-Octadecadienoic acid,

4.97+0.02

methyl ester (linoleic, Omega 6) 10.80+0.20

9-Octadecenoic acid, methyl

20.52+4.64

ester 25.93+2.50

Octadecanoic acid, methyl ester

8.01+0.12 9.50+2.45

5,8,11,14-Eicosatetraenoic acid,

methyl ester (arachidonic, 2.71+0.04 1.66+0.04

Omega 6)

5,8,11,14, 17-Eicosapentaenoic

acid, methyl ester (EPA, Omega 12.20+0.41 11.47+0.64

3)

11,13-Eicosadienoic acid,

1.00+0.12

methyl ester 2.73+0.42

11-Eicosenoic acid, methyl ester 3.50+0.12 3.53+0.08

Eicosanoic acid, methyl ester 0.51+0.02 0.25+0.00

4,7,10,13,16,19- Docosahexaenoic acid, methyl 15.21+1.47 10.74+2.03

ester (DHA, omega 3)

13-Docosenoic acid, methyl 1.37+0.08 14.14+1.04

ester

Docosanoic acid, methyl ester 0.40+0.02 0.19+0.02

(n-3) / n-6) ratio 2.02 3.35

DHA / EPA ratio 1.2 0.99

[00156] General Discussion

[00157] Results of the feeding trials on P. vannamei, milkfish and tilapia indicate that ProEnK can be included as ingredient in the diet up to 50% by weight without significantly affecting the overall growth and biological performance of these aquatic animals. The feed material was observed to be acceptable for both the fish and shrimp species used in the present study and increasing levels of dietary ProEnK did not affect the overall survival of the test aquatic animal species indicating absence of toxic substances or contaminants that has the potential to cause acute toxicological effects. Moreover the detected levels of heavy metals that include lead, cadmium, chromium and copper in the ProEnK are at the allowable level for a feed material, further supporting the high potential of this material as a feed ingredient.

[00158] Results of the feeding trials both in tilapia and milkfish indicate that growth of the fish meal based diet maintained groups is comparable with the group receiving the highest inclusion level of ProEnK (50% diet) and no significant decline in performance has been noted. Results in trials with tilapia and milkfish indicate that these two species could tolerate high levels of ProEnK in their diet. The normal growth responses of these species even at the highest replacement level that is 50% indicate that the nutrients in the formulated diets can satisfy their optimum requirement to attain normal growth. Previous studies with plant protein replacement indicate that most marine fish could not tolerate high level of dietary inclusion with plant protein. However studies regarding plant protein inclusion in the diets of omnivorous freshwater fish suggest that these species are able to tolerate and attain normal growth even at 100% replacement of fish meal with plant protein meals. Khan et al. (2003) reported successful replacement (100%) of fishmeal with soybean meal in the diets of Rohu, Labeo rohita. Also, Kasper et al. (2007) has shown that up to 47.6% of the fish meal could be replaced with SBM in the diets of yellow perch (Perca flavescens) without affecting the feed consumption, weight gain, feed efficiency or survival of the fish. These earlier findings support the current data obtained both for milkfish and tilapia, wherein 50% of the diet allotted to ProEnK does not adversely affect overall growth performance and overall body and tissue composition of the test animals. The optimum biological performance of these species at high dietary inclusion level of ProEnK inclusion could also be due to the feeding habits of these species.

Tilapia is considered an omnivore that can utilize plant material as energy source and milkfish is considered an herbivore that is adapted to plant material as a dietary source of energy for growth and survival.

[00159] The better performance of these animals at higher inclusion levels of ProEnK could also be attributed to the high digestibility of ProEnK as a dietary ingredient. Moreover growth in P. vannamei was found higher at the higher ProEnK dietary inclusion levels of 25-50% as compared to the other treatments. Higher growth was observed as compared to the control group that is a fish meal based diet. Specific Growth rate (SGR) was also found better in these treatment groups but feed conversion efficiency was found slightly higher than the control group. All other biological parameters showed no significant differences among the treatment groups.

[00160] The better growth performance of shrimp fed ProEnK as compared to the control may be attributed to the high carbohydrate content of this fed ingredient. Unlike for fish that cannot utilize carbohydrate for metabolic energy production, crustaceans on the other hand are able to utilize this nutrient for energy production and in the synthesis of their shell. Crustacean carapace or shell requires a lot of dietary protein and carbohydrate for its synthesis and it may appear that with the presence of high amount carbohydrate in ProEnK. This has led to the sparing of protein degradation for shell synthesis thus protein is used for body accretion. The better growth in shrimp as an effect of dietary ProEnK inclusion also resulted to a better protein content in the group receiving the highest inclusion level. This again confirms the protein sparing effects of carbohydrate in ProEnK. Lipid content of shrimp fed the ProEnK diet at 50 % inclusion level has a lower body lipid content. Ash was not found significant among the dietary treatments.

[00161] In recent years fatty acid composition of the flesh of farmed aquatic animals used as food has elevated concerns among consumers. It has been suggested dietary manipulation specifically the composition and quality of feed proteins may influence the profile of fatty acid composition of cultured fish. In the present study we examined the influence of dietary ProEnK on the fatty acid profile of tilapia, shrimp and milkfish. Results suggest that both in milkfish and tilapia highest inclusion level of ProEnK in the diet appears to influence the Omega 3/ Omega 6 ratio. Those that received the ProEnK diet have a better ratio of these fatty acids indicating less omega 6 fatty acid synthesis. The profile indicates that ProEnK diet treatments are better in terms of this Omega 3/ Omega 6 ratio as compared to those that received the fish meal based diets. Similar trend could also be observed in P. vannamei that is known to lack the capacity to synthesize EPA and DHA from 18 carbon precursors' fatty acids. The Ratio of DHA/ EPA appears to be similar both in the control and the treated groups in the fish and shrimp experiments. The high levels of Omega 3 fatty acids in the treatment groups receiving the ProEnK diets indicate that this diet ingredient may have promoted these animals to favor the biosynthesis of Omega 3 fatty acids rather than the omega 6 -series fatty acids. Both tilapia and milkfish are known to biosynthesize EPA and DHA from 18 carbon fatty acids precursors. Currently the mechanism and conditions that could trigger the biosynthesis of EPA and DHA in tilapia and milkfish are still unknown but the present results indicate that dietary inclusion of 50% ProEnK in the diet could result to high EPA and DHA fatty acids in tilapia and milkfish flesh. It could also be speculated that the antibacterial property of ProEnK may limit the degree of bacterial infection and inflammation of the gut. Natural infection and inflammation are known factors to activate biosynthesis of arachidonic fatty acid an omega 6 fatty acid. The low level of omega 6 fatty acid in the experimental animals in the presents study could be attributed to this antibacterial properties of ProEnK.

[00162] Collectively the present work indicates that ProEnK could be included in the diets of tilapia, shrimp, and milkfish to about 50% by weight without affecting the overall biological performance, feed efficiency and fatty acid profiles of these aquatic animals. ProEnK can be an economical feed ingredient for aquatic animals that could lower the usage of using fish meal in aquatic animal diets.

[00163] Example 5 - Broiler chicken feeding trials using feed formulations comprising ProEnK [00164] Fermented sweet potato (Ipomoea batata), taro (Colocasia esculenta) and cassava

(Manihot esculenta) were evaluated as broiler feeds. The growth performance and feed conversion efficiency of broilers fed with 10 per cent fermented root crop was assessed and the cost efficiency of using these high-protein feeds was evaluated.

[00165] Dried sweet potato and cassava tubers and taro corms were separately ground and mixed with a nutrient solution composed of urea, ammonium phosphate, ammonium sulfate, sucrose and vinegar to obtain the fermentable substrate following the method described in Example 1. This substrate was sterilized in an autoclave, allowed to cool and inoculated with either of two strains of fungi namely Aspergillus niger and Rhizopus oligosporus. Inoculated substrates were allowed to ferment for two weeks, after which they were harvested and sundried to eliminate the odorous gaseous metabolites.

Samples were taken and subjected to proximate analysis at the Animal Nutrition Analytical Laboratory of U.P. Los Banos. The fermented or protein-enriched root crops were incorporated as 10 per cent replacement of the ration and test-fed using straight- run broiler chicks. Broiler performance was evaluated in terms of weight gain and feed conversion efficiency. The feed cost per kilogram of broiler produced was also calculated.

[00166] The results showed that the poor feeding values of sweet potato, cassava and taro were significantly improved by fermentation using either A. niger or R. oligosporus. Increases in crude protein contents were as much as twelve-fold. Likewise, improvement in crude fat or ether extract decreased after fermentation. The suitability of utilizing the high-protein feeds in broiler rations was evidenced by the fact that broilers on test diets performed comparably well as those in the control lot. They exhibited similar weight responses and efficiency of feed utilization. Utilization of the high-protein fermented feeds proved more cost-efficient resulting in some amount of savings on feed costs (Tables 16 and 17).

[00167] Table 16 - Nutrient composition of sweet potato, taro and cassava before and after fermentation

% % % % % %

Root crops

DM CP NFE EE CF Ash

Fresh

Sweet potato 34.5 1.1 31.8 0.4 0.7 1.2

Taro 29.0 2.3 25.7 0.2 0.7 0.8

Cassava 35.0 0.7 31.4 0.1 0.9 1.1

Dehydrated Sweet potato 92.0 2.6 81.9 1.3 3.3 2.8

Taro 90.0 7.1 79.8 0.6 2.2 2.5

Cassava 90.0 2.8 79.6 1.0 1.8 3.0

Fermented

Sweet potato 80.0 30.8 38.8 2.6 2.4 5.5

Taro 81.6 30.7 39.4 1.3 2.0 8.2

Cassava 80.6 29.4 40.4 1.9 1.3 7.6

[00168] Table 17 - Nutrient composition of protein-enriched sweet potato, taro and cassava fermented by two strains of fungi

Example 6 - Preparation of Fermented Agricultural Biomass from Cassava

[00170] Table 18 shows the nutrient composition of cassava before and after it was fermented by three sub-culture species of microfungi namely Aspergillus niger, Aspergillus fumigatus, and Aspergillus oryzae following the method set out in Example 1. The technical analysis shows that the amount of crude protein and ash generally increased, while the amount of crude fat, crude fiber and nitrogen-free extract decreased after fermentation for 21 days. The highest increases in crude protein and ash were obtained when cassava was fermented by Aspergillus niger. On the other hand, the highest decrease in crude fat was obtained when cassava was fermented by Aspergillus oryzae. The decrease in nitrogen-free extract was highest when Aspergillus niger was used to ferment cassava. A similar study conducted by Oboh et al. (G. Oboh, M.K. Oladunmoye (2007) Nutr HeaZi/z.18(4):355-67) showed that solid substrate fermentation of cassava mash using Rhizopus oryzae and Saccharomyces cerevisae caused a significant (P < 0.05) increase in the protein and fat content. The experiment of Akindahunsi, et al. (Akindahunsi AA and Oboh G. (2003) Nutr Health. 17(2): 131-8) indicated that fungi fermentation of cassava mash significantly increased (P < 0.05) the in vitro multi-enzyme protein digestibility of the cassava products. In view of this, fungi fermentation could be used to improve the protein digestibility of cassava products without any significant (P > 0.05) effect on the energy-giving role of cassava products. Moreover, Oboh analyzed the dried fermented cassava peels and found that there was a significant (P < 0.05) increase in the protein content of the cassava peels fermented with squeezed liquid from the inoculated cassava pulp (21.5%) when compared with the unfermented cassava peel (8.2%) (Oboh G (2006) Electronic Journal of Biotechnology 9(1): 1-4).

[00171] Table 18 - Nutrient composition of cassava before and after fermentation

[00172] Example 7 - Biological response ofPekin ducks (Anas platyrhyncos) to diets containing fermented agricultural biomass [00173] Research was conducted to evaluate rations with protein biomass (from sweet potato) for

Pekin ducks in terms of growth, carcass yield, and sensory qualities. It also aimed to identify the most appropriate inclusion rates that could result in higher return on investment. The percentage by weight substitution of commercial ration with protein biomass served as the variable of the study and these were 0, 5, 10, 15, and 20%.

[00174] The results showed that using different levels of protein biomass (5% to 20% of the ration) elicited comparable effect on the growth, carcass yield and sensory qualities of duck as the control ration (commercial). It was noted, however, that at 20% inclusion rate there was a slight reduction (although non-significant).

[00175] The different inclusion levels of protein biomass in the commercial duck ration yielded no significant differences (P >.05) among experimental treatments in terms of dressing percentage, carcass yield, cut-up yield and cooking loss. The differences in scores on the appearance and color of steamed meat samples were statistically not significant, however, the scores for odor of meat sample had highly significant differences with duck that fed on ration with 10% protein biomass having more appealing odor. Percentage return on investment ranged from 25.41% (20% inclusion) to 30.31% (5% inclusion).

[00176] Example 8 - Utilization of ProEN-K and Probiotics in Growing Pigs

[00177] ProEn-K was used at 10% inclusion rate in growing swine ration which was

supplemented with probiotics (at a dose of 0.3, 0.6, and 0.9%). After a three-month feeding trial, pigs on diet without ProEn-K and those with 10% ProEn-K, regardless of probiotic supplementation had comparable gain in weight and feed conversion efficiency.

[00178] Throughout the specification and the claims that follow, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprising" and "including" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[00179] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.

[00180] It will be appreciated by those skilled in the art that the disclosure is not restricted in its use to the particular application described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.