Process for improving the nutritional quality of silkworm pupae, silkworm pupae meal and silkworm waste (SPM) by solid-state fermention using sequential co-culture method

Abstract

The present invention relates to compositions and methods for improving the nutritional quality of a silkworm-based substrate, such as silkworm pupae, silkworm pupae meal or silkworm waste (SPM). The method utilizes a sequential solid-state fermentation process that synergistically increases both the crude protein content and the digestibility of the substrate. The process also improves the amino acid profile by increasing the concentration of lysine and methionine, enhances the fatty acid profile by reducing saturated fats, and decreases undesirable components like carbohydrates and non-protein nitrogen. The resulting fermented product is a highly digestible, protein-rich animal feed component particularly suitable for use in aquaculture feeds, but may also be useful for humans.

Description

[0001] PROCESS FOR IMPROVING THE NUTRITIONAL QUALITY OF SILKWORM PUPAE, SILKWORM PUPAE MEAL AND SILKWORM WASTE (SPM) BY SOLID-STATE FERMENTION USING SEQUENTIAL CO-CULTURE METHOD

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application 63 / 764,709, PROCESS FOR IMPROVING THE NUTRITIONAL QUALITY OF SILKWORM PUPAE, SILK WORM PUPAE MEAL AND SILKWORM WASTE (SPM) BY SOLID-STATE FERMENTION USING SEQUENTIAL CO-CULTURE METHOD.

[0004] BACKGROUND OF THE INVENTION

[0005] The present invention relates generally to compositions and methods of making silkworm pupae meal more digestible using solid state fermentation.

[0006] Protein is the most important nutrient in fish and shrimp feed, with fishmeal and soybean meal being major sources. However, unsustainable production, inconsistent supplies and fluctuating prices have the feed industry looking for suitable alternatives. Proteins from microbes (fungi and bacteria), insects and algae are being developed as the next generation protein sources, with high protein content and other beneficial properties. Their advantage is that they are not seasonal and can be produced with low carbon footprint.

[0007] Silkworm pupae is a byproduct of silk reeling process (1kg silk produces about 2kg of dry pupae) which is either discarded or dried and ground to silkworm pupae meal and used in animal feed / pet food. China and India are the largest silk producers (88,913 MT of silk annually, 2023), this material is identified as one of the sustainable sources for protein production available at a low price. SPM contains 50-55% crude protein with a balanced amino acid profile but with low digestibility (<70%) compared to fishmeal (minimum 90%).Solid state fermentation (SSF) is a fermentative process in which microorganisms grow on a solid substrate in the limited presence of free water whereby the moisture level required to guarantee the biological activity is absorbed or complexed in the solid matrix. The past decade has witnessed an unprecedented interest in SSF for the development of bioprocesses such as bioremediation and biodegradation of hazardous compounds, biological detoxification of agro-industrial residues, bio pulping and production of value-added products, such as biologically active secondary metabolites, including antibiotics, alkaloids, plant growth factors, enzymes, organic acids, biosurfactants, and aroma compounds.

[0008] By conducting SSF on a solid substrate with low moisture content, a high product concentration may be achieved with significantly less energy being used. There are a number of advantages to microorganism growth and oxygen transport in SSF, which is achieved when the substrate absorbs the required water content inside a solid matrix, including effective gaseous conveyance, decreased water consumption, use of cellulosic waste, management of pH, and the need for smaller fermenters, leading to less strain for further processing. As a medium for seed growth, SSF commonly makes use of solid materials like agricultural byproducts, such as rice straw, wheat straw, sugarcane bagasse, rice hulls, and corn cobs. The substrate typically contains low moisture, typically between about 20% to 70%, in contrast to liquid fermentation which typically has a moisture content of from 95-100%. SSF has several advantages over liquid fermentation in being cost-effective when using inexpensive agricultural residues, having a higher product concentration, and producing products that are sensitive to high water content.SSF can break down complex organic molecules into simpler ones, making nutrients more accessible. In animal feed production, SSF can improve digestibility by breaking down antinutritional components and increasing the concentration of digestible minerals. SSF is particularly useful for manufacturing industrial enzymes such cellulases, proteases, and amylases. These enzymes are essential for a variety of sectors, including biofuels, food, and textiles. SSF can stimulate the creation of bioactive substances such as antibiotics, vitamins, and organic acids. These molecules have potential uses in medicines, food preservation, and nutritional supplements. Traditional fermented foods produced by SSF, such as tempeh, koji, and miso, benefit from increased tastes, textures, and nutritional profiles as a result of the fermentation process.

[0009] In SSF, an aerobic bioreactor is generally filled with a solid substrate and then inoculated with the specific strain of interest in order to generate the intended bioproduct. Following production, this bioproduct can be retrieved, however in certain instances the resulting fermented solid can serve as the ultimate product. Various reactor types have been employed in the generation of solid-state fuel: packed bed reactors, mechanically stirred reactors, tray reactors, and plug flow topologies.

[0010] There is therefore a need to make SPM a more digestible, value-added but economical protein for animal feed and / or pet food.

[0011] For these and other reasons, there is a need for the present invention.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention relates to the use of silkworm material, such as silkworm pupae, waste and silkworm pupae meal (collectively referred to herein as " SPM”] as asubstrate for single cell protein production and use of the resulting microbes to improve the digestibility of silkworm protein. The invention comprises a microbially-based system that uses solid state fermentation ( " SSF”) to turn insect protein plant matter into a highly digestible, concentrated protein source that can be used as animal feed or consumed by humans.

[0014] The substrate of the invention may include SPM either alone or in combination with other carbon sources, nitrogen and / or other nutrients. The invention preferably includes a sequential co-culturing process using Saccharomyces cerevisiae and Yarrowia lipolytica to prepare a combination of fermented silkworm protein and single cell protein. The fermented silkworm pupae meal includes the presence of microbial peptides beneficial for the growth of shrimps and fishes. The fermented SPM may further be used as a digestible protein source in animal feed.

[0015] DETAILED DESCRIPTION OF THE INVENTION

[0016] Before the present composition, methods, and methodology are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0017] As used in this specification and the appended claims, the singular forms "a”, "an”, and "the” include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, as it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure.

[0018] As used herein, "about,” "substantially” and "significantly" will be understood by a person of ordinary skill in the art and will vary in some extent depending on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about” and "approximately” will mean plus or minus ≤10% of particular term and "substantially" and "significantly” will mean plus or minus >10% of the particular term.

[0019] As used herein, "consisting essentially of” means, the particular component and may include other components, which other components do not change the novel properties or aspects of the particular component.

[0020] As used herein, the term "animal” includes, without limitation, humans, birds (e.g. poultry), mammals (e.g. cattle, swine, goat, sheep, cat, dog, mouse and horse) as well as aquaculture organisms such as fish (e.g. trout, salmon, perch), mollusks (e.g. clams) and crustaceans (e.g. lobster and shrimp). The term "fish” includes all vertebrate fishes.

[0021] As already noted, the present invention is directed to a method of improving the digestibility of silkworm pupae, silkworm pupae meal, and silkworm waste (SPM) using fermentation. Protein is the most important nutrient in fish and shrimp feed, with fishmeal and soybean meal being major sources. Unsustainable production, inconsistent supplies and fluctuating prices have the feed industry looking for suitable alternatives. SPM is a byproductof silk reeling process (1kg silk produces 2kg of dry pupae] that is either discarded or dried to use in animal feed / pet food. It was identified as one of the sustainable and cost-effective protein sources containing 50-55% crude protein with a balanced amino acid profile but with low digestibility of <70% (not defatted] compared to fishmeal.

[0022] A main objective of the present invention is to utilize the SPM as a substrate (i.e. a "silkworm-based substrate”] for single cell protein production and in turn utilize the growing microbes to improve the digestibility of silkworm protein. In this regard, a series of preliminary experiments were performed, both submerged and solid-state fermentation (SSF], Initial studies showed that supplementing carbon, nitrogen, and some key minerals in basal amount to SPM is required for the microbial growth and substantially increase the digestibility.

[0023] Solid state fermentation has been practiced for centuries, most often in connection with food production, and can be defined as a technique for growing microorganisms, such as fungi, yeast and bacteria, on moist solid substrates. Solid state fermentation devices provide several advantages over the commonly used process of submerged fermentation in terms of product yield, costand ease of use.

[0024] Solid state fermentation (SSF) devices fall within two categories, those categories being static systems and agitated systems. In static systems, the solid media is stationary throughout the fermentation process. Examples of static systems used for solid state fermentation include flasks, petri dishes, trays, fixed bed columns, and ovens. Agitatedsystems provide a means for mixing the solid media during the fermentation process. Examples of agitated systems include rotating drums and stirred vessel bioreactors.

[0025] Solid-state fermentation (SSF] involves the growth of microorganisms on moist particles of solid materials in beds in which the spaces between the particles are filled with a continuous gas phase. As used herein, "fermentation” refers to any controlled microbial process without implying that the microorganism is using fermentative metabolic pathways. A typical bioreactor for SSF involves three primary components: (1) the body of the bioreactor itself; [2] a bulk gas phase, which, if it is above the bed, is typically referred to as the headspace; and [3] the substrate bed. The substrate bed itself may be thought of as consisting of two subphases, namely the particles of solid material, to which the growing microorganism is attached, and the interparticle gas phase.

[0026] According to the methods described herein, the solid growth medium or substrate of the invention comprises or primarily comprises silkworm pupae meal (SPM]. SPM has carbohydrate and nitrogen that is complex for microbes to utilize and provide secondary metabolites. The SPM may be used as the sole substrate or nutrient source for the SSF or may be supplemented with other nutrient sources such as, but not limited to, carbon, nitrogen and / or minerals to augment the nutritional capacity and shorten the lag phase. These may include carbon sources such as carbohydrates (sugars, starch], proteins or fats, nitrogen sources in organic form (proteins, amino acids] or inorganic nitrogen salts (ammonium and nitrate salts, urea], trace elements or other growth factors (vitamins, pH regulators, inorganic salts]. The solid growth medium may contain aids for structural composition, suchas super absorbents, for example polyacrylamides. The substrate may further comprise various organic or inorganic carriers. The inorganic carriers may include, but are not limited to, vermiculite, perlite, amorphous silica or granular clay. These types of materials are commonly used because they form loose, airy granular structure having preferably a particle size of 0.5-50 mm and a high surface area. The organic carriers are preferably cereal grains, bran, sawdust, peat or wood chips.

[0027] According to one embodiment of the invention, the SPM is supplemented with from about 0.1-10% by weight glucose, from about 0.1-10% by weight nitrogen source, and from about 0.05-2% by weight key minerals. In an embodiment of the invention, the SPM is supplemented with about 1-3% by weight glucose, from about 1-2% by weight ammonium sulfate, and from about 0.001-0.1% by weight potassium dihydrogen phosphate, magnesium sulfate, and / or sodium chloride.

[0028] The SPM and / or other supplemental ingredients are generally prepared by grinding and / or milling, steaming, and / or treating with chemicals to break down the ingredients and increase surface area. The initial moisture level in the substrate should be sufficient to dissolve the other media ingredients and allow for microbial proliferation and will typically range from about 40-90% by weight. In one embodiment, the moisture level is about 50% by weight. While not required, the substrate may be sterilized prior to treatment to eliminate unwanted microbes prior to inoculation using conventional methods, such as autoclaving, steam, etc.The microorganism to be cultivated and inoculated on the solid growth medium may comprise fungi, including yeasts, for example such as Saccharomyces cerevisiae, Phlebiopsis gigantea, Gliocladium sp,, Nectria pityrodes, Chondrostereum purpureum, Pseudozyma flocculosa, Coniothyrium minitans, Trichoderia sp., Metarrhizium sp., Verticillium sp., Yarrowia lipolytica or Beauveria bassiana. In one embodiment, the microorganism is Saccharomyces cerevisiae. The fungi may additionally include edible mushrooms such as Agaricus bisporus, Lentinus edodes or Pleurotus ostreatus. The microorganism according to the invention can also be bacteria such as, but not limited to, Acetobacter pasteurianus, Bacillus subtilis, Bacillus pumilis, Bacillus licheniformis, Clostridium kluyveri, and / or Ralstonia eutropha.

[0029] In one embodiment, a sequential fermentation method is used whereby the silkworm-based substrate is first fermented with a first strain(s) to increase digestibility of the substrate followed by fermentation with a second strain(s). The use of sequential fermentation has been surprisingly found to improve digestibility of the SPM as well as enhanced utilization of nutrients. In one embodiment, the silkworm-based substrate is first fermented with Saccharomyces cerevisiae for a time period of about 1-3 days, followed by fermentation with either Yarrowia lipolytica or Bacillus BFI9 and R eutropha for about 2-4 days to enhance utilization of nutrients in the meal, resulting in increased protein (i.e. by 7% or more).

[0030] In another embodiment, the silkworm-based substrate is first fermented with S. cerevisiae followed by fermentation with Y. lipolytica, which has been found to result inimproved amino acid and fatty acid profiles. While it is acceptable to ferment the silkwormbased substrate for longer periods of time, such extended fermentation (i.e. > 3 days) has not been found to significantly improve bacterial growth.

[0031] In monoculture fermentation in accordance with the invention, S. cerevisiae showed the highest improvement in digestibility of SPM reaching to 90.83%. In addition, an increase in protein content of the fermented product was observed with some of the strains, highest among them being Y. lipolytica, where the protein content increased from 43.67% to 50.51%. This increase in protein content may be correlated to a reduction in fat and carbohydrate content indicating the utilization of both.

[0032] In a preferred embodiment, the method maybe summarized as the following. SPM is ground into fine particles, after which the ground SPM is enriched with nutrients, water, and strain-1. The mixture with strain-1 ferments for 48 hours. After 48 hours, the second strain is incorporated into the mixture. After the addition of strain-2, the mixture ferments for another 72 hours. The fermented material is then dried and milled to form the final product. In general, the fermentation conditions include a pH range of from about 5.0 to about 7.0, for instance from about 5.5 to about 6.8, which is a suitable range for the described microorganisms. The incubation temperature will generally be in the range of about 37°C for Bacillus strains and R. eutropha and around 30°C for S. cerevisiae and Y. lipolytica.

[0033] The resulting fermented SPM in accordance with the invention displays improved protein content (an increase of ~8 percentage points) with improved digestibility, comparable to that of fish meal, as well as a reduction in total carbohydrate and crude fiber.As noted, once the fermentation step(s) are completed, the resulting fermented product is dried, milled if needed, then packaged.

[0034] The methods of the present invention provide numerous advantages, including providing effective digestion of SPM to provide a sustainable and cost-effective source of protein for humans and animals. SPM provides a low-cost substrate for single cell protein production, and the highly digestible fermented SPM produced in accordance with these methods is high in protein content and can serve as a sustainable and economical protein source for aqua feed, as well as in other types of animal feed. It may further serve as a high protein supplement for humans. The produced silkworm protein feed product has good quality without odor of silkworms, has sour and delicious taste without organic solvent residues, contains protein easy to digest, has low production costand simple and convenient production process, and is suitable for large-scale production. The SPM product further has improved pepsin digestibility, improved amino acid profile, fatty acid profile and other nutritional parameters.

[0035] An object of the invention is to improve the nutritional quality of SPM. In some embodiments, an improvement in nutritional quality may include increased digestibility, increased crude protein content, increased concentration of essential amino acids critical for animal growth, reduction of non-protein nitrogen, reduction of fats or lipids, a reduction of total carbohydrates, and / or a reduction in crude fiber.

[0036] In an embodiment, the protein content may show a 10% increase, an 11% increase, a 12% increase, a 13% increase, a 14% increase, a 15% increase, a 16% increase, a 17%increase, an 18% increase, a 19% increase, or a 20% increase when compared to an unfermented SPM.

[0037] In an embodiment, the pepsin digestibility of the fermented product is increased. In certain embodiments, the final pepsin digestibility may be at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94%.

[0038] In an embodiment, the concentration of key essential amino acids in the fermented product is increased. In certain embodiments, the concentration of an essential amino acid such as lysine or methionine may be increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% when compared to an unfermented SPM. The essential amino acids may include one or more selected from the group consisting of lysine, methionine, histidine, and threonine.

[0039] In an embodiment, the non-protein nitrogen (NPN) content of the fermented product is reduced due to its utilization by the microbes. In certain embodiments, the NPN content may be reduced by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% when compared to an unfermented SPM.

[0040] In an embodiment, the overall crude fat content of the fermented product may be reduced. In certain embodiments, the crude fat content may be reduced by at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, or at least 8% when compared to an unfermented SPM.In an embodiment, the fatty acid profile of the fermented product is improved, wherein the improvement comprises a reduction in saturated fatty acids and a simultaneous increase in polyunsaturated fatty acids. For example, in certain embodiments, the concentration of saturated fatty acids such as palmitic acid or stearic acid may be reduced by at least 10%, at least 20%, at least 30%, or at least 40%, while the concentration of beneficial polyunsaturated fatty acids such as linoleic acid may be increased by at least 100%, 200%, 300%, 400%, or 500% when compared to an unfermented SPM.

[0041] In an embodiment, the total carbohydrate content of the fermented product is reduced. In certain embodiments, the total carbohydrate content may be reduced by at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% when compared to an unfermented SPM. In certain embodiments, the crude fiber content may be reduced by at least 5%, at least 8%, at least 10%, at least 12%, at least 15%, or at least 18% when compared to an unfermented SPM.

[0042] SSF fermentation provides a cost-effective method for the cultivation of microbes due to the solid-state approach and minimal downstream processes. These solid-state conditions also limit bacterial contamination due to the reduced water activity. In addition, the preferred sequential co-culture method is more robust and does not require strictly sterile conditions, leading to high product yield and lower operational costs. Moreover, as some bioactive substances may be produced during the fermentation process, the functionalities of the product can be improved. Furthermore, the fermented SPM can be dried withoutexpensive downstream processes, resulting in a mixture of hydrolyzed, highly digestible protein substrate together with microbial protein.

[0043] Another embodiment of the invention involves the use of the fermented SPM protein concentrate product produced by the method above which is included as an animal feed component in an amount of between about 0.1% to about 50% on a dry matter basis. In another embodiment, the protein concentrate is included in a composition, wherein the composition is a complete replacement for animal-based fishmeal in a fish / shrimp feed.

[0044] The following examples are intended to describe preferred embodiments of the invention in more detail. They are not intended to limit or restrict the invention in any manner.

[0045] EXAMPLES

[0046] Example 1 - Conversion of Silkworm Pupae Meal to a Sustainable Single Cell Protein Experiment 1 - Fermentation experiment to optimize the substrate. SPM contains carbohydrates and nitrogen, but these compounds that are difficult for the microbes to utilize, particularly for the production of secondary metabolites. Hence, in this experiment, additional supplementation of carbon, nitrogen and minerals were evaluated. Glucose or saccharification residue (SR) from corn syrup processing and ammonium sulphate ((NH4)2SO4) were used as carbon and nitrogen sources, respectively. Addition of minerals like magnesium sulphate (MgSO₄), potassium dihydrogen phosphate (KH2PO4) and sodium chloride (NaCl) to maintain the osmoregulation of the substrate was also evaluated in thisstudy. Different fermentation media were prepared as shown (Table 1). Bacillus subtilis BFI9 was used as inoculum. Different groups- SPM alone, SPM supplemented with and without glucose, ammonium sulphate, minerals, and SR (saccharification residue) replacing glucose as carbon source were evaluated.2% of glucose and 1.5% of ammonium sulphate was added to induce the growth of the microbes. SR (30% carbohydrate) was added at an inclusion rate of 6% to match 2% glucose. For fermentation, SPM substrate were distributed in 250ml Erlenmeyer’s flask to which all the other ingredients and water were added and sterilized. It was inoculated with 1% of culture of Bacillus subtilis BFI9, mixed carefully under sterile conditions and incubated at 37°C for 3 days. Separate controls were maintained for each of the groups with same media composition but without inoculum. Samples were drawn from the flasks (after mixing the contents well) at 0, 24, 48 and 72 hours to measure total plate counts. After the fermentation, dried at 60°C overnight and evaluated for moisture content, pH value of 5% solution, crude protein, total carbohydrates and pepsin digestibility.

[0047] Table 1. Different fermentation media based on the carbon and nitrogen inclusion B C D E

[0048] (SPM with (SPM with (SPM with (SPM with Media

[0049] ammonium glucose, no glucose and SR and composition

[0050]

[0051]

[0052] sulphate, no ammonium ammonium ammonium glucose) sulphate) sulphate) sulphate)

[0053] SPM 50 50 50 50 50

[0054] Water 50 50 50 50 50 (NH4)2SO4 1.5 1.5 1.5

[0055] Glucose 2 2

[0056] SR 6

[0057] MgSO40.025 0.025 0.025 0.025 KH2PO4 0.05 0.05 0.05 0.05

[0058] NaCl

[0059] 0.5 0.5 0.5 0.5Results (Table 2) showed that the crude protein content in all the unfermented control groups remained same. Among the fermented samples, increased protein content was observed in groups C, D and E, while A & B showed minimal change from controls. Highest reduction was observed in group E, could be due to presence of easily digestible carbohydrates in SR which correlates with the microbial growth in the respective group. Increased pepsin digestibility was observed in groups C, D & E fermented samples - 69.95%, 79.13% and 83.59% respectively, compared to control group average 66.3%. No change in digestibility was observed in groups A & B.

[0060] Table 2. Compositional analysis of the fermented samples from Experiment 1 - Optimization of the substrate

[0061] „, Total Pepsin

[0062] CrudeK

[0063] NI_

[0064] PKNT

[0065] Groups. carbohydrate digestibility

[0066] protein (%)f0 / ^5fo / o](%)

[0067] IT r. i 48.03 ±o„. 65.33 ± 1.21 ±

[0068] . Unfermented _ _ 8.24 ± 0.06a„ „ „

[0069] A 0.05a0.14a0.00a

[0070] (

[0071] 1only

[0072] JSPM)

[0073] Jr_ 4

[0074] Fermentedn7.84 ±e65.23 ± 1.09 ±

[0075] 5.25 ± 0.44bn n

[0076] 0.00a0.26a0.00a

[0077] B (SPM with „c, 48.45 ±o n nr. 66.27 ± 1.38 ±

[0078] 1Unfermentedn8.23 ± 0.05an nn. ammonium 0.07a0.12a0.00bsulphate & „t, 48.42 ±r.. 66.35 ± 1.20 ±

[0079] ,,. Fermented 5.47 ± 0.14

[0080] w / o glucose) 0.10a bn

[0081] 0.65a0.05b

[0082] C (SPM with 48.41 ±

[0083]

[0084] 1.21 ±

[0085] Unfermented 9.04 ± 0.05

[0086] glucose & 0.13a b

[0087] 0.43a 4

[0088] 0.00aw / o

[0089] 50.71 ±, 69.95 ± 1.16 ± ammonium Fermented 5.06± 0.01*066„

[0090] 0.66ab0.07asulphate

[0091] D (SPM with 48.76 ±

[0092]

[0093] 1.31 ±

[0094] Unfermented 9.05 ± 0.07 H

[0095] glucose and 0.35a

[0096]

[0097] 0.00bammonium 51.09 ± 7913 + 1.04 ±

[0098] Fermented

[0099] sulphate) 0.07b5-10 ± 0'47 ab0.50 e" 0.00a48.85 ± 1.34 ± E Unfermented 91 + 001b 66‘59 ±

[0100] 0.10a?.± _ u.u± o. O3a0.00b

[0101]

[0102] (

[0103] 1SPM withr J50.21 ± „ 83.59 ± 1.04 ±

[0104] SR) Fermented 4.64 ± 0.20a50.21 ± 0.23b83.59 ± 0.88d1.04 ± 0.02aValues are expressed as Mean ± SD; n=2. Columns with different superscripts show statistically significant difference between the groups, p<0.05.

[0105] Table 3. Microbial count of the fermented samples from Experiment 1 - Optimization of the substrate

[0106] No of colony forming units (CFU) / g

[0107] Groups Initial 24 hrs 48 hrs 72 hrs A (only SPM) 83×10591×10642×106B (SPM with

[0108] ^mmon'um97×105106×106210×106sulphate & w / o

[0109] glucose)

[0110] C (SPM with

[0111] glucose & w / o 84×106350×10549×10717×107ammonium

[0112] sulphate)

[0113] D (SPM with

[0114] glucose and 366×10540×10715×108ammonium

[0115] sulphate)

[0116] E (SPM with SR) 103×105101×10823×106Values are expressed as Mean; n=2

[0117] Microbial growth data is shown in Table 3. The inoculum suspension had a total microbial count of 3×107CFU / ml. Initial count in the substrate (after inoculation of 1%) was 84×106CFU / g. After 24 hours, there was 1-log difference observed in all the groups. After 48 hours, there was a 2-log increase observed in groups C & D, while 3-log increase was observed in E and no log increase was observed in A & B until day 3. After 72 hours, D showed a highest microbial count compared to other groups. This indicates fermentation medium D, with the supplementation of glucose and nitrogen at the concentrations of basal medium promotes the growth of the microbes, which was selected for further studies. Medium withSR supplementation performed equally well, indicating the possibility of using it as a substitute for glucose.

[0118] Experiment 2 - Fermentation experiment to select the strains. This study was conducted in two sets, in the first set, growth ofS. cerevisiae and Bacillus strains on SPM were compared while in the second set, the selected strains from set 1 were compared with Y. lipolytica and R. eutropha. Y. lipolytica and R. eutropha were evaluated in set 2 for their ability to utilize lipids present in SPM as energy source for their growth and to observe for improved protein content and digestibility. In both the sets of fermentation, SPM media with glucose and ammonium sulphate supplementation were distributed in 250ml Erlenmeyer's flask to which all the ingredients and water were added, mixed and sterilized. The cooled substrates were inoculated with 2% of prepared cultures in set 1 and 5% of prepared cultures in set 2. The contents were mixed carefully under sterile conditions and incubated for 5 days. SPM with high fat content was used as substrate in set 1 and SPM with less fat content in set 2. Control was maintained with the same media composition but without inoculum. One gram of sample was drawn from the flasks (after mixing the contents well) at 0, 24, 48 and 72 hours to measure total plate count. After the fermentation, the biomass dried and evaluated for moisture content, pH value, crude protein, total carbohydrates, crude fat, pepsin digestibility and microbial growth.

[0119] The compositional analysis of fermented samples is as shown in Table 4. Increased protein content compared to control was observed in B. subtilis BFI9 and S. cerevisiae while B. subtilis BFI1 and BFI2 did not show an increase. Reduced carbohydrate in fermented samples show that carbohydrates were used up for microbial growth. Highest reduction wasobserved in B. subtilis BFI9, and S. cerevisiae fermented samples. Increased pepsin digestibility was observed in groups B. subtilis BFI9 (similar to the previous study i.e.,79.13%), S. cerevisiae, and B. subtilis BFI29 fermented samples compared to control. No change in the digestibility observed in groups B. subtilis BFU & BFI2. Results showed that F19 Bacillus strain and S. cerevisiae could be used for further optimization. On analyzing the microbial count, higher microbial counts were observed in BFI9, and S. cerevisiae as shown in Table 5. A 2-log to 3-log decrease was observed in all the groups on day 1. On day 3, a 2-log increase was observed in group B. subtilis BFI9 and a 3-log increase in group S. cerevisiae fermented samples while no log increase was observed B. subtilis BFI29 and 1 log decrease in groups B. subtilis BFI1 & BFI2. Decline in the count was observed on day 5, indicating fermentation duration was 3 days.

[0120] Table 4. Compositional analysis of the fermented samples from Experiment 2 – Selection of strains (Set 1)

[0121] Total

[0122] Crude Pepsin Groups Crude fat % carbohydrate

[0123] protein % digestibility %

[0124] %

[0125] B. subtilis 47.39 ±

[0126] 13.73 ± 0.02 8.84 ± 0.54c66.37 ± 0.01a

[0127] BFI1 0.24a

[0128] B. subtilis 47.15 ±

[0129] 13.78 ± 0.06 7.54 ± 0.82bc67.88 ± 1.58a

[0130] BFI2 0.19a

[0131] B. subtilis 51.57 ±

[0132] 13.93 ± 0.03 6.55 ± 0.49ab80.58 ± 0.40c

[0133] BFI9 0.01c

[0134] B. subtilis 49.79 ±

[0135] 13.75 ± 0.04 9.03 ± 1.31c76.53 ± 0.60b

[0136] BFI29 0.19b

[0137] 51.53 ±

[0138] S. cerevisiae 13.88 ± 0.04 5.47 ± 0.02a83.77 ± 0.37d

[0139] 0.03c

[0140] 47.91 ±

[0141] Control 13.94 ± 0.02 9.11 ± 0.47c67.56 ± 0.02

[0142] 0.02a

[0143] Values are expressed as Mean ± SD; n=3. Columns with different superscripts show statistically significant difference between the groups, p<0.05.Table 5. Microbial count of the fermented samples from Experiment 2 – Selection of strains (Set 1)

[0144] No of colony forming units (CFU) / g

[0145] Groups Inoculum Initial Day 1 Day 3 Day 5

[0146] B. subtilis 1×10839×10546×1051×1029×102

[0147] Bill

[0148] B. subtilis 2×10719×10556×1049×10216×102

[0149] BF12

[0150] B. subtilis BFI9 3×10769×10561×10345×10615×105

[0151] BFI9 B. subtilis BFI29 5×10716×10520×10375×10510×103

[0152] S

[0153]

[0154] . cerevisiae 6×1062×10581×10319×10626×105

[0155] Values are expressed as Mean; n=2.

[0156] The first set of fermentation studies showed that fermentation of SPM with B. subtilis FI9 and S. cerevisiae can result in a meal with higher digestibility and a crude protein increase of 2-3%. In the second set of fermentation, two other microbial strains Y. lipolytica and R. eutropha were considered. Increased protein content was observed in all the treatment groups. Highest protein content was observed in S. cerevisiae and Y. lipolytica groups comparatively. Pepsin digestibility increased in all the treatment groups, compared to control. Highest pepsin digestibility was observed in groups S. cerevisiae (90.83 %). A slight reduction in the fat content was observed in Y. lipolytica group comparatively, showing its utilization of fat. Reduction in the total carbohydrate content was observed in all the treatment groups as shown in Table 6. After 72 hours, S. cerevisiae, B. subtilis BFI9 and Y. lipolytica groups showed the maximum log increase (3-log) in microbial counts (as shown in Table 7). This study showed that S. cerevisiae can improve the digestibility (78.71% to 90.8%) and Y. lipolytica can increase the protein content (43.7% to 50.51%).Table 6. Compositional analysis of the fermented samples from Experiment 2 - Selection of strains [Set 21

[0157] Total Pepsin

[0158] Crude protein Crude fat

[0159] Groups carbohydrate digestibility

[0160] % %

[0161] % %

[0162] B. subtilis 31.65 ± 80.40 ± 1.32

[0163] 47.57 ± 1.70b9.36 ± 1.29d

[0164] BFI9 2.12ab

[0165] S. 30.93 ±

[0166] 46.59 ± 1.90c7.09 ± 0.78ab90.83 ± 4.20ccerevisiae 0.48a

[0167] R. 30.16 ± 79.66 ± 0.40

[0168] 46.65 ± 1.33b5.71 ± 1.83a

[0169] eutropha 4.83ab Y.

[0170]

[0171] 82.82 ± 1.59

[0172] 50.51 ± 1.43c9.08 ± 0.58bc

[0173] lipolytica 1.08ab

[0174] 31.85 ±

[0175] Control 43.67 ± 2.20a10.34 ± 0.51c78.71 ± 0.31a

[0176] 0.30a

[0177] Values are expressed as Mean; n=2 Columns with different superscripts show statistically significant difference between the groups, p<0.05.

[0178] Table 7. Microbial count of the fermented samples from Experiment 2 - Selection of strains [Set 2]

[0179] No of colony forming units (CFU) / g

[0180] Groups Inoculum Day 1 Day 3

[0181] B. subtilis

[0182] BFI9 2×1077×10414×107

[0183] S. cerevisiae 19×10777×10551×108

[0184] R. eutropha 6×10815×10618×107

[0185] Y. lipolytica 8×10627×10420×107

[0186] Values are expressed as Mean; n=2.

[0187] Experiment 3 - Fermentation experiment to evaluate sequential co-culture method. In this study, two step fermentation was conducted with high fat SPM. S. cerevisiae, which resulted in highest improvement in digestibility, was selected as strain-1 and then combined with other strains and evaluated for increase in protein content and digestibility. SPM with other ingredients and water were added to 250ml Erlenmeyer’s flasks then mixed and sterilized. All flasks [except control) were inoculated with of S. cerevisiae under sterile conditions and incubated for 2 days. After the first phase of fermentation, the substrates were inoculated with strain 2 to the respective flasks and incubated at respective optimumtemperature for 3 days. Total plate count was measured at 0, 24, 48 and 72 hours. After the fermentation, the biomass was dried and evaluated for moisture content, pH value, crude protein, total carbohydrates, crude fat, pepsin digestibility and microbial growth.

[0188] The compositional analysis is shown in Table 8. Highest protein content (57.8%) was observed in S. cerevisiae + Y. lipofytica group. The pepsin digestibility remained approximately the same (above 90%) in all the groups, observed to be improved by just fermenting with S. cerevisiae. On evaluating the combinations, Groups S. cerevisiae + Y. lipofytica and S. cerevisiae + R. eutropha showed a decrease in carbohydrate content compared to other groups, indicating its utilization by the microbes for their growth. The crude fat content remained approximately the same in Control, S. cerevisiae + R. eutropha, S. cerevisiae + B. subtilis BFI9 groups, while a slight reduction was observed in S. cerevisiae + Y. lipofytica group. pH did not show a significant variation between all the groups. On evaluating the total plate count on each step of the inoculum addition and the final fermented sample, highest microbial growth was observed in S. cerevisiae + Y. lipofytica as shown in Table 10, which correlates with the improved protein content.

[0189] Table 8. Compositional analysis of the fermented samples from Experiment 3 - Sequential co-culture method

[0190] Total

[0191] Crude Crude fat Pepsin Groups carbohydrate

[0192] protein % % digestibility %

[0193] %

[0194] 49.55 ± 30.52 ±

[0195] Control 8.21 ± 0.08c92.46 ± 0.01b0.09a0.23a

[0196] S. cerevisiae + R. 51.03 ± 30.85 ±

[0197] 6.47 ± 0.01a92.71 ± 0.21 eutropha 0.58a0.02abb

[0198] S. cerevisiae + Y. 57.80 ± 29.07 ±

[0199] 7.40 ± 0.01b92.75 ± 0.03 lipofytica 0.05b0.11b b

[0200] S. cerevisiae + B. 52.52 ± 30.85 ±

[0201] 8.62 ± 0.01d91.77 ± 0.12asubtilis BFI9 0.61a0.27abValues are expressed as Mean ± SD; n=2. Columns with different superscripts show statistically significant difference between the groups, p<0.05.

[0202] Table 9. Microbial count of the fermented samples from Experiment 3 - Sequential co-culture method

[0203] No of colony forming units (CFU) / g

[0204] ,.,. Dayl- After Day 3- After

[0205] „ Inoculum Inoculum,.... Groups 2 adding adding Final

[0206]

[0207] inoculum 1 inoculum 2 Control 2×107- 26×10410×10611×107S. cerevisiae + R.

[0208] eutropha 2×10719×10727×10414×10756×107S. cerevisiae + Y. lipolytica 2×1076×10821×10419×10719×109S. cerevisiae + B. subtilis BFI9 2×1078×10622×10415×10750×107

[0209]

[0210] subtilis BFI9 Values are expressed as Mean; n=2.

[0211] Experiment 4 - Fermentation experiment to evaluate the reverse sequence of 2-step fermentation process. In this study, two-step fermentation of the strains that was observed with improved protein and digestibility (from Experiment 3) was evaluated by interchanging the strains between the 2 phases. SPM-containing media was sterilized and inoculated with Y. lipolytica, except in control flask. The contents were mixed carefully under sterile conditions and incubated for 2 days. After the first phase of fermentation, the flasks were inoculated with of S. cerevisiae (except control flask) and incubated for 3 days. After the fermentation, the biomass was dried at 60°C overnight and evaluated for moisture content, crude protein and pepsin digestibility.

[0212] The analyses of reverse sequence of 2-step fermentation process (Y. lipolytica + S. cerevisiae) is shown in Table 10. The protein content of Y. lipolytica + S. cerevisiae sequential addition was higher than the control by 5%, but was much lower than the original combination (seen in Experiment 3). Pepsin digestibility to 89.1% was observed in Y.lipolytica + S. cerevisiae. The study notably showed lower improvement than the original combination observed in Experiment 3.

[0213] Table 10. Compositional analysis of the fermented samples from Experiment 4 - Reverse combination process

[0214] „ Moisture Crude Pepsin Groups

[0215] protein % digestibility % Control (unfermented) 3 ± 0.08a49.4 ± 0.21a80.1 ± 0.09aReverse Combination (K lipolytica 3.1 ±

[0216] 52 ± 0.28b89.1 ± 0.34b

[0217] + S. cerevisiae') 0.02a

[0218] Values are expressed as Mean ± SD; n=2. Columns with different superscripts show statistically significant difference between the groups, p<0.05.

[0219] Experiment 5 - Fermentation experiment to reconfirm the selected sequential coculture method process. 150g of SPM substrate, water and nutrients were mixed in a 500ml Erlenmeyer’s flask. After sterilization, S. cerevisiae was added and incubated for 2 days. After the first phase of fermentation, the substrates were inoculated with Y. lipolytica, under sterile conditions and incubated for 3 days. Growth of the culture was evaluated by collecting 1 g of fermented sample on day 1, 2 and 3, and the inoculum was used for measuring total plate count. After the fermentation, the biomass was dried at 60°C overnight and evaluated for moisture content, pH value, crude protein, total carbohydrates, non-protein nitrogen (NPN), pepsin digestibility and microbial growth.

[0220] Fermented SPM showed an increase of 13 percentage in protein content compared to the unfermented control. The total carbohydrate was reduced by 26% and the NPN was reduced by 57% in the fermented group compared to control group, indicating its utilization for the growth of the inoculum. A slight reduction in the fat content and ash content was observed in the external lab analysis report as well. Pepsin digestibility increased to 93.14% in the fermented group from 82.53% in control group as shown in Table 11. Amino acid andfatty acid profiles of the unfermented and fermented samples are shown in Table 12 and Table 13. Amino acid profile of fermented SPM showed a relative increase in glycine, glutamic acid, histidine, lysine, methionine, and threonine. Fatty acid profile also improved with a reduction in saturated fatty acids and an increase in polyunsaturated fatty acid.

[0221] Table 11. Compositional analysis of the fermented samples from Experiment 5 - Confirmation of sequential co-culture method

[0222] In- house analysis* External lab analysis*

[0223] Fermented

[0224] ..tFermented

[0225] Parameters _t„, Silkworm Control Silkworm Control

[0226] pupae pupae meal

[0227] meal Moisture % 2.37 ± 0.07a2.33 ± 0.10a3.75 3.66 ± 0.08

[0228]

[0229] Protein % 60.99 ± 0.06a68.78 ± 0.86b60.88

[0230]

[0231] Total

[0232] carbohydrate 8.88 ± 0.13b6.55 ± 0.07a8.59 5.94 ± 0.03

[0233] %

[0234] 16.46 + Crude fat % 17.23 ± 0.12b16.59 ± 0.22a

[0235] 17 890.23 " Total ash % 6.11 ± 0.11b5.55 ± 0.10a6.56 5.19 ± 0.04 Crude Fiber % 3.93 ± 0.19b3.27 ± 0.35a4.12 3.25 ± 0.06

[0236] Not

[0237] Not measured.PepSin, n / 82.53 ± 0.18a93.14 ± 0.15

[0238] digestibility %b

[0239] measured Not

[0240] NPN% 0.79 ± 0.02b0.34± 0.01aNot measured,

[0241] measured

[0242] -I _, Not

[0243] pH 5.43 ± 0.03 5.74 ± 0.02 Not measured

[0244]

[0245] measured *Values are expressed as Mean ± SD; n=3 Columns with different superscripts show statistically significant difference between the groups, p<0.05.#Values are expressed as Mean ± SD; n=2 for treatment groups, n=l for control group Table 12. Amino acid profile of the fermented samples from Experiment 5 - Confirmation of sequential co-culture method

[0246] Amino acid (g / 100g) Control Fermented Silkworm pupae meal Alanine 4.4 5.05 ± 0.24

[0247] Arginine 5.8 5.61 ± 0.00

[0248] Aspartic acid 7.4 7.01 ± 0.00

[0249] Cystine 0.8 0.92 ± 0.07

[0250] Glutamic acid 8.3 9.47 ± 0.20

[0251] Glutamine BLQ BLQGlycine 3.7 4.44 ± 0.24

[0252] Histidine 1.74 2.25 ± 0.00

[0253] Leucine 4.76 4.51 ± 0.00

[0254] Lysine 5.1 7.52 ± 0.00

[0255] Methionine 2.1 3.08 ± 0.00 Phenylalanine 1.4 1.36 ± 0.00

[0256] Ornithine BLQ BLQ

[0257] Cysteine BLQ BLQ

[0258] Isoleucine 2.9 2.73 ± 0.00 Hydroxyproline BLQ BLQ

[0259] Aspargine BLQ BLQ

[0260] Threonine 3.8 4.51 ± 0.00

[0261] Serine 3.5 3.82 ± 0.85

[0262] Valine 3.9 4.2 ± 0.00

[0263] Sum of amino acids 59.72 66.48 ± 0.11

[0264] Values are expressed as Mean ± SD; n=2 for treatment groups, n=l for control group, BLQ- Below the limit of quantification

[0265] Table 13. Fatty acid profile of the fermented samples from Experiment 5 - Confirmation of sequential co-culture method

[0266] Fatty acid composition Fermented Silkworm pupae Control

[0267] (g / lOOg] meal

[0268] C 4:0 (Butyric acid] BLQ(LOQ:0.05] BLQ(LOQ:0.05]

[0269] C 6:0 (Caproic acid] BLQ(LOQ:0.05] BLQ(LOQ:0.05)

[0270] C 8:0 (Caprylic acid] 0.19 0.14 ± 0.06

[0271] C 10:0 (Capric acid] BLQ(LOQ:0.05] BLQ(LOQ:0.05]

[0272] C 11:0 (Undecanoic acid] BLQ(LOQ:0.05] 0.05 ± 0.00

[0273] C 12:0 (Lauric acid] 0.1 0.08 ± 0.00

[0274] C 13:0 (Tridecanoic acid] BLQ(LOQ:0.05] BLQ(LOQ:0.05]

[0275] C 14:0 (Myristic acid] 0.18 0.072 ± 0.00

[0276] C 14:1 (Myristoleic acid] 0.5 0.45 ± 0.07

[0277] C 15:0 (Pentadecanic acid] BLQ(LOQ:0.05] BLQ(LOQ:0.05]

[0278] C 15:1 (Pentadecanoic

[0279] BLQ(LOQ:0.05] BLQ(LOQ:0.05]

[0280] acid] + Isomers

[0281] C 16:0 (Palmitic acid] 8.4 6.84 ± 0.32

[0282] C 16:1 (Palmitoleic acid] 1.05 0.05 ± 0.00

[0283] C 17:0 (Heptadecanoic

[0284] 0.053 0.05 ± 0.00

[0285] acid]

[0286] C18:3n-6 (Gamma linolenic

[0287] 0.05 0.06 ± 0.01

[0288] acid ME]

[0289] C 18:0 (Stearic acid] 3.46 1.95 ± 0.11

[0290] C 18:1 (Oleic acid] 0.69 1.71 ± 0.11

[0291] C 18: ln9t (Elaidic acid] 0.2 0.08 ± 0.01C 18:2 (Linoleic acid] 0.31 1.91 ± 0.08

[0292] C 18:2t (Linolelaidic acid] 0.05 0.06 ± 0.00

[0293] C 18:3 n3 (Alpha- Linolenic acid) 1.02 0.06 ± 0.01

[0294] acid]

[0295] C 18:3 n6 (Gamma- Linolenic acid) 0.81 0.25 ± 0.07

[0296] Linolenic acid]

[0297] C 20:0 (Arachidic acid] BLQ(LOQ:0.05] BLQ(LOQ:0.05]

[0298] C 20:1 (Eicosenoic acid] 0.112 0.08 ± 0.00

[0299] C 20:2 (Eicosadienoic acid] BLQ(LOQ:0.05] BLQ(LOQ:0.05]

[0300] C 20:3 (Eicosatrienoic acid] BLQ(LOQ:0.05] BLQ(LOQ:0.05] Linolenic]('h°m°'gamma’ BLQ(LOQ:0.05] BLQ(LOQ:0.05] acid]2°4n6 ('AraChld°niCBLQ(LOQ:0.05] BLQ(LOQ:0.05]

[0301] C 20:5 (Eicosapentaenoic acid) BLQ(LOQ:0.05) BLQ(LOQ:0.05) C 21:0 (Heneicosanoic acid) BLQ(LOQ:0.05) BLQ(LOQ:0.05)

[0302] C 22:0 (Behenic acid] 0.05 1 ± 0.04

[0303] C 22:1 (Docosenoic acid) + Isomers BLQ(LOQ:0.05) 0.06 ± 0.01

[0304] Isomersv J

[0305] C22:ln-9 Erucic BLQ(LOQ:0.05] 0.94 ± 0.03

[0306] C 22:2 (Docosadienoic acid) BLQ(LOQ:0.05) 0.05 ± 0.00

[0307] acid]v J

[0308] C 22:6 (Docosahexaenoic acid) BLQ(LOQ:0.05) 0.08 ± 0.01

[0309] C 23:0 (Tricosanoic acid] BLQ(LOQ:0.05] BLQ(LOQ:0.05]

[0310] C 24:0 (Lignoceric acid] BLQ(LOQ:0.05] BLQ(LOQ:0.05)

[0311] C 24:1 (Nervonic acid] BLQ(LOQ:0.05] BLQ(LOQ:0.05]

[0312] Medium Chain Triglyceride (C6-C12) BLQ(LOQ:0.05) BLQ(LOQ:0.05)

[0313] (C6-C12] BLQ(LOQ:0.05] BLQ(LOQ:0.05]

[0314] Sum of fatty acids 17.225 15.99 ± 0.16

[0315] Values are expressed as Mean ± SD; n=2 for treatment groups, n=l for control group Table 14. Microbial count of the fermented samples from Experiment 5 - Confirmation of sequential co-culture method

[0316] S. No Sample details CFU / g

[0317] 1 Day 1 -After adding inoculum 1 14×105

[0318] inoculum 1

[0319] 2 Day 3-Before adding inoculum 2 12×107

[0320] inoculum 2

[0321] 3* Day 3- After adding inoculum 2 12×107

[0322] inoculum 24* Day 6- Before drying 15×108

[0323] 5* Final dried product 14×102

[0324] Values are expressed as Mean; n=2. *Includes the colony count of both the inoculum Microbial counts in the samples are shown in Table 14. On day 1, inoculum 1 was added with the concentration of 4×107CFU / ml, after adding it to the substrate there was a 2-log reduction in the count. On day 3, it was observed to increase by 1-log. Inoculum 2 with the concentration of 13×107was added on day 3 and allowed to ferment for 3 days. Totally 3-log increase in the count was observed during fermentation. In the final product, after drying, it was observed to have 14×102CFU / g.

[0325] Example 2 - Evaluation of fermented silkworm pupae in Atlantic salmon

[0326] The fermented silkworm pupae was incorporated into feed as a partial substitute for fishmeal and was used to conduct a 4-week growth performance trial in Atlantic salmon (Salmo salar). The composition of the fermented SPM used in the trial is shown below.

[0327] Table 15. Nutritional composition of fermented SPM Parameter Measured value

[0328] Moisture 2.3%

[0329] Crude protein 68.8%

[0330] Crude fat 16.6%

[0331] Ash 5.5%

[0332] Carbohydrates 6.5%

[0333] Pepsin 93.1%

[0334]

[0335] digestibility

[0336] The trial included a control group and a treatment group (Table 16), with each group having three replicate tanks.

[0337] Table 16. Experimental groups in the trial

[0338]

[0339] Group Diet Test product & inclusionControl 20% fishmeal diet -

[0340]

[0341] Treatment 15% fishmeal diet Silkworm protein [5%)

[0342] Experimental diets were manufactured for juvenile Atlantic salmon. The composition of experimental diets is provided in Table 17. A control diet containing 20% fishmeal and no test ingredient was formulated to exceed the species’ known nutrient requirements [NRC, 2011). In the test diet, 5% of the fishmeal was replaced with 5% silkworm protein. Other ingredients were adjusted as needed to maintain a similar overall nutritional composition.

[0343] Table 17. Composition of experimental diets

[0344] Ingredient Control Treatment

[0345] Fishmeal [herring) 20 15

[0346] Poultry meal 20 20

[0347] Corn gluten meal 7.00 7.00

[0348] Soy protein concentrate 2.91 2.85

[0349] Blood meal [spray 6.45 8.00

[0350] dried)

[0351] Wheat flour 18.66 19.16

[0352] Wheat gluten 6.00 5.35

[0353] Fish oil [sardine) 6.84 7.28

[0354] Canola oil 6.77 5.75

[0355] Soy lecithin 1.00 1.00

[0356] Monosodium phosphate 2.00 1.61

[0357] [23% P)

[0358] L-Lysine HC1 1.02 1.00

[0359] DL-Methionine 0.24 0.10

[0360] Choline chloride [60% 0.20 0.20

[0361] choline)

[0362] Vitamin C [Stay C-35) 0.30 0.30

[0363] Vitamin-mineral premix 0.40 0.40

[0364] Silkworm protein - 5.00

[0365] Nutrient composition (measured)

[0366] Moisture 7.90 6.90

[0367] Crude protein 52.50 53.30

[0368] Crude lipid 20.60 18.80

[0369]

[0370] Ash 9.76 9.05One hundred ninety-four fish (initial body weight 27.4g) were randomly distributed to fiberglass tanks, with each tank having 97 fish. Fish were acclimated for 12 days and given a commercial salmon feed. Animals in each tank were bulk weighed prior to feeding experimental diets. Fish were hand-fed three times a day to satiation until the end of the experiment. After four weeks of feeding the experimental diets, fish were bulk weighed again. Body weight gain (BWG), Feed intake (FI), economic feed conversion ratio (eFCR), specific growth rate (SGR) and survival rate were measured.

[0371] At the end of 4 weeks of feeding, the body weight increased by 48.14-49.13%. No statistically significant differences (p>0.05) were observed between the high-fishmeal control group and the low-fishmeal group containing silkworm protein in any of the growth indices (Table 18).

[0372] Table 18. Performance indices measured in the trial

[0373] Parameters Control Treatment Initial Body weight (g / fish) 27.46+0.27 27.40+0.14 Final Body weight (g / fish) 40.95+1.21 40.59+0.19 Weight gain (g / fish) 13.49+0.94 13.18+0.28 Daily Feed intake (g / fish)* 0.32 + 0.01 0.32 + 0.01 Specific Growth rate (% / day) 1.38 + 0.07 1.35 + 0.03 eFCR 0.69 + 0.03 0.71 + 0.02

[0374]

[0375] Survival 100% 100%

[0376] Values are expressed as Mean ± SD, n=3. No statistically significant difference observed;

[0377] p>0.05

[0378] The researchers observed that partial substitution of fishmeal with silkworm protein can deliver similar animal performance as that of a regular fishmeal diet.It should be appreciated that minor dosage and formulation modifications of the composition and the ranges expressed herein may be made and still come within the scope and spirit of the present invention.

[0379] Having described the invention with reference to particular compositions, theories of effectiveness, and the like, it will be apparent to those of skill in the art that it is not intended that the invention be limited by such illustrative embodiments or mechanisms, and that modifications can be made without departing from the scope or spirit of the invention, as defined by the appended claims. It is intended that all such obvious modifications and variations be included within the scope of the present invention as defined in the appended claims. The claims are meant to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates to the contrary.

[0380] The foregoing description has been presented for the purposes of illustration and description. It is not intended to be an exhaustive list or limit the invention to the precise forms disclosed. It is contemplated that other alternative processes and methods obvious to those skilled in the art are considered included in the invention. The description is merely examples of embodiments. It is understood that any other modifications, substitutions, and / or additions may be made, which are within the intended spirit and scope of the disclosure. From the foregoing, it can be seen that the exemplary aspects of the disclosure accomplish at least all of the intended objectives.

Claims

CLAIMS1. An animal feed component comprising:a fermented silkworm-based composition, wherein the fermented silkworm-based composition has a protein content above about 65% and a pepsin digestibility above about 90%.

2. The animal feed component of claim 1, wherein the pepsin digestibility of the composition is above about 90%.

3. The animal feed component of claim 1, wherein the protein content of the composition is above about 65%.

4. The animal feed component of claim 1, wherein the composition has a non-protein nitrogen level below 0.5%.

5. The animal feed component of claim 1, wherein the composition is further characterized by an increased concentration of one or more amino acids selected from the group consisting of lysine, methionine, histidine, and threonine, as compared to an unfermented silkworm-based substrate.

6. The animal feed component of claim 1, wherein the composition has a reduced crude fat and fiber content as compared to an unfermented silkworm-based substrate.

7. The animal feed component of claim 1, wherein the fermented silkworm-based composition is produced by a process comprising fermenting with Saccharomyces cerevisiae and / or sequentially fermenting an unfermented silkworm-based composition first with Saccharomyces cerevisiae and subsequently with Yarrowia lipolytica.

8. The component of claim 1, wherein the component is fed to fish and shrimp.

9. A method of solid-state fermenting a silkworm-based substrate comprising:fermenting the silkworm-based substrate with Saccharornyces cerevisiae in a first fermentation step; andsubsequently fermenting the silkworm-based substrate with Yarrowia lipolytica in a second fermentation step,wherein the method results in a fermented product having at least 10% more protein than the silkworm-based substrate prior to fermentation.

10. The method of claim 9, wherein the pepsin digestibility of the fermented product is above about 90%.

11. The method of claim 9, wherein the protein content of the fermented product is above about 65%.

12. The method of claim 9, wherein the fermented product has a non-protein nitrogen level below 0.5%.

13. The method of claim 9, wherein the first fermentation step lasts for approximately 48 hours and the second fermentation step lasts for approximately 72 hours.

14. The method of claim 9, wherein the silkworm-based substrate is supplemented with a carbon source and a nitrogen source prior to the first fermentation step.

15. A method of increasing the protein content and digestibility of a silkworm-based composition, comprising:fermenting the silkworm-based composition with Saccharornyces cerevisiae in a first fermentation step to produce an intermediate fermented product; andsubsequently fermenting the intermediate fermented product with Yarrowia lipolytica in a subsequent fermentation step to produce a final fermented product, wherein the final fermented product has a protein content at least 10% greater than the silkworm-based composition prior to the first fermentation and a pepsin digestibility of at least about 90%.

16. The method of claim 15, wherein the protein content of the final fermented product is at least about 65%.

17. The method of claim 15, wherein the final fermented product has a non-protein nitrogen level below 0.5%.

18. The method of claim 15, wherein the final fermented product has a reduction in saturated fatty acids and an increase in polyunsaturated fatty acids compared to an unfermented silkworm-based substrate.

19. The method of claim 15, wherein the final fermented product is further characterized by an increased concentration of one or more amino acids selected from the group consisting of lysine, methionine, histidine, and threonine, as compared to an unfermented silkworm-based substrate.

20. The method of claim 15, wherein the first fermentation step lasts for approximately 48 hours and the subsequent fermentation step lasts for approximately 72 hours.

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