Animal feed composition and method for producing the same

Abstract

Provided herein are animal feed compositions comprising fermented organic material, black soldier fly biomass, fermented black soldier fly production residues, and microbial biomass. Methods for producing the compositions are also provided. The compositions can be produced from food waste and provide an environmentally friendly alternative to existing protein sources for farmed animals, particularly fishmeal.

Description

[Technical Field]

[0001] Field The present invention relates to animal feed compositions based on fermented organic material and to methods for decomposing organic material that can be used to produce animal feed compositions. [Background technology]

[0002] background The global animal feed business is worth around $400 billion per year and generates one-twelfth of the world's greenhouse gas emissions. The primary protein source in many animal feeds is fishmeal, which is made by cooking, pressing, drying, and grinding whole fish into fishmeal powder. Fishmeal is used to feed a wide range of farmed animals, including pigs, poultry, and fish. Summary of the Invention [Problem to be solved by the invention]

[0003] However, the use of fishmeal in animal feed is controversial because high global demand for fishmeal has led to overfishing (especially around developing countries), which could lead to the collapse of fishing industries and damage to ecosystems. The combination of increased demand for animal feed and overfishing-induced fishing losses is expected to nearly double fishmeal prices by 2030, spurring a demand for alternative protein sources.

[0004] One alternative protein source is soybeans, which are already widely used in animal feed, but global soybean production is insufficient to replace fishmeal, and soybean production is also associated with environmental impacts, particularly deforestation to make way for land for soybean cultivation.

[0005] In recent years, the animal feed industry has shifted its attention to insects as ingredients in animal feed. Insects have high nutritional value, are easy and inexpensive to raise, and their use is environmentally and ethically sound. One insect of particular interest in the animal feed industry is the black soldier fly (BSF). BSF larvae have a protein content of over 40%, and therefore have great potential to replace fish meal in animal feed. Because the larvae have simple requirements for survival and growth, they can be grown on organic waste, such as food waste or even animal manure. As a result, BSF larvae are publicly used as fish food in small fish farms, where they are raised and fed directly to fish (Astuti & Wiryawan, Animal Bioscience 35(2): 356-363, 2022). BSF larvae meal has also been used in livestock feed; for example, on goat farms, BSF larvae milk replacer has been used successfully to feed pre-weaned kids, and BSF larvae meal-based diets have also been used successfully to raise weaned kids (Astuti & Wiryawan, supra).

[0006] BSF production residues (frass) have also attracted attention as an ingredient in animal feed, particularly in aquaculture. Frass is a by-product of larval development and includes larval waste, shed exoskeletons, and dead larvae, which contain approximately 20% protein in addition to other essential nutrients. The inclusion of BSF production residues in fish diets has been found to have a positive effect on the growth of herbivorous and detritivorous fish species, but it is not suitable as a feed for carnivorous fish (Banavar et al., Animals 12: 2407, 2022).

[0007] Another environmental challenge facing the world today is that of food waste. The Food and Agriculture Organization of the United Nations estimates that approximately one-third of products intended for human consumption are discarded or lost each year. In developing countries, losses occur primarily at the first step in the food supply chain, during harvesting and processing. In developing countries, food is also discarded by retailers and consumers. Many challenges are associated with reducing food waste, and some waste is unavoidable, especially in food processing. Therefore, attention is turning to the valorization of food waste. Fermentation and anaerobic digestion of food waste to produce energy in the form of biogas and biofuels are becoming popular. The use of microorganisms to produce biomaterials from food waste, such as bioplastics, is also emerging.

[0008] More recently, food waste has also been recognized as a potential source of high-value chemicals suitable for use, for example, as flavorings or fragrances. For example, agricultural waste, such as sugar beet pulp and grain bran, has been recognized as a source of ferulic acid, which can be bioconverted by natural or engineered microorganisms into vanillin, the component of vanilla responsible for its flavor / aroma (Saadoun et al., Foods 10: 707, 2021). [Means for solving the problem]

[0009] The present invention provides novel compositions suitable for use as animal feed, along with methods by which the animal feed can be made. The feed compositions are obtained by processing organic material, such as food waste, with BSF larvae and a microbial cocktail, which are also included in the final product. The present invention thus addresses two pressing environmental challenges (food waste and the unsustainable use of animal feed components) and produces nutritious and affordable animal feed with minimal environmental impact. The combination of both microbial and insect processing of organic material to produce the animal feed compositions of the present invention is unique and highly advantageous.

[0010] In a first aspect, the present invention provides an animal feed composition comprising fermented organic material, black soldier fly biomass, fermented black soldier fly production residue, and microbial biomass. In some embodiments, the composition comprises about 5-25% w / w black soldier fly biomass and about 0.05-5% w / w microbial biomass.

[0011] In a second aspect, the present invention provides a method for decomposing organic material, the method comprising the steps of contacting the organic material with a microbial composition comprising black soldier fly larvae and at least one microorganism capable of fermenting the organic material, and incubating the organic material with the black soldier fly larvae and the microbial composition so that the organic material is processed by the black soldier fly larvae and the at least one microorganism; The method produces a product composition comprising fermented organic material, black soldier fly larval biomass, and microbial biomass.

[0012] In an embodiment, the microbial composition ferments organic material. (i) at least one digestive microorganism, wherein the digestive microorganism produces digestive enzymes; (ii) at least one protein-enhancing microorganism, wherein the protein-enhancing microorganism increases the protein content of the product composition; and / or (iii) at least one microorganism that increases the nutritional and / or health value of the resulting composition and / or reduces the environmental impact of the method; Includes.

[0013] In a third aspect, the present invention provides the use of black soldier fly larvae and a microbial composition as defined in the second aspect to decompose organic material, thereby producing a product composition.

[0014] In a fourth aspect, the present invention provides a product composition obtainable from the method defined in the second aspect.

[0015] In a fifth aspect, the present invention provides a composition for decomposing organic material, the composition comprising black soldier fly larvae and a microbial composition as defined in the second aspect.

[0016] In a sixth aspect, the present invention provides a kit for decomposing organic material, the kit comprising black soldier fly larvae and a microbial composition as defined in the second aspect. DETAILED DESCRIPTION OF THE INVENTION

[0017] Description of the Invention The present invention relates to animal feed compositions (ie compositions suitable for use as animal feed or for feeding animals) and methods by which such compositions may be made.

[0018] composition An animal feed composition may alternatively and interchangeably be referred to herein as an animal feed composition. The composition may be obtained by a method comprising contacting organic material with a BSF larvae and microbial composition (or cocktail), and incubating the organic material with the BSF larvae and microbial composition, as described in detail below, so that the organic material is processed by the BSF larvae and the microorganisms contained in the microbial composition. Processing of the organic material includes both consumption and digestion of the organic material by the BSF larvae and fermentation of the organic material by the microorganisms of the microbial cocktail. Further processing steps may also be performed to obtain the animal feed composition of the present invention.

[0019] The animal feed composition thus comprises fermented organic material, black soldier fly biomass, fermentation production residues (usually fermented black soldier fly production residues), and microbial biomass. The animal feed composition according to the present invention can be used as or in any animal feed. That is, the animal feed composition can be used as a stand-alone animal feed, as a component of an animal feed that also includes other components, or as a supplement to an existing animal feed. Any existing animal feed known in the art may be supplemented with the animal feed composition of the present invention, and standard animal feed components that can be mixed with the animal feed composition of the present invention to thereby produce an animal feed are also known in the art.

[0020] The animal feed composition is particularly suitable for consumption by farmed animals, including mammals, birds, fish, and crustaceans. For example, the animal feed composition can be used to feed livestock, such as cattle, sheep, goats, or horses. Similarly, the animal feed composition can be used to feed poultry, such as chickens, turkeys, geese, or ducks. Most preferably, the animal feed composition is used in aquaculture, i.e., the farming of marine food species. The animal feed composition can be fed to farmed fish and crustaceans, and indeed to any type of farmed aquatic species (either saltwater or freshwater). The animal feed compositions of the present invention can contain high levels of protein and thus be particularly suitable as feed for carnivorous and omnivorous aquatic species, such as salmon (most commonly Atlantic salmon (Salmo salar) farmed in and around the North and South Atlantic Oceans), whiteleg shrimp (Litopenaeus vannamei, also known as king shrimp), farmed worldwide, particularly in Latin America, India, and Southeast Asia (e.g., Thailand and Indonesia), and shrimp, including tiger prawns (Penaeus monodon), farmed worldwide. The animal feed compositions of the present invention are also suitable as feed for other species (including herbivorous aquatic species), including similarly important farmed species, such as carp, tilapia, catfish, crayfish, and crab.

[0021] The animal feed compositions of the present invention comprise fermented organic material. While any organic material can be fermented and used in the compositions of the present invention, the most common organic material is food waste. Food waste refers to any organic waste product or by-product of the food industry, including agricultural or aquaculture waste, waste from food processing (e.g., from factories, mills, etc.), commercial food waste (e.g., from restaurants, hotels, supermarkets / stores, etc.), and household food waste. The food waste can be plant-based food waste, animal-based food waste, or a mixture thereof. The food waste can be from a single or mixed food species. The food waste can be from cooked or uncooked food.

[0022] In a preferred embodiment, the food waste is non-fibrous, since black soldier fly larvae prefer non-fibrous food to fibrous food. As used herein, "fibrous" food refers to food containing a large amount of cellulose fiber, such as vegetables. Examples of suitable food waste products that can be used as a base for the animal feed composition of the present invention include slaughterhouse waste (e.g., from chicken slaughter), flour mill waste (e.g., cassava pulp from cassava flour production), nut waste from nut milk production, palm oil decanter cake from palm oil production, and spent grain (i.e., brewers spent grain (BSG) or dregs), as well as other brewery waste streams. Typically, those skilled in the art can use types of food waste that are readily available in their own area. Also, while the animal feed compositions of the present invention are suitable for feeding to any animal, as described above, one skilled in the art can readily optimize the composition for a particular species of interest by selecting a base material (e.g., type of food waste) with a nutrient content appropriate to the nutritional requirements of the animal; for example, a composition intended for an animal with high dietary protein requirements can be made using a high-protein starting material (e.g., food waste).

[0023] While food waste is most commonly used as a base material for the compositions of the present invention, for example, due to its ready availability and low cost, any organic material may be used. Herein, the term "organic material" is used to refer to plant and animal products. In addition to food waste, other suitable sources of organic material include horticultural waste, forestry waste, gardening waste, and waste from other industries that process organic products for non-food manufacturing purposes, such as waste from papermaking, cosmetics, etc. The organic material processed to produce the compositions of the present invention is referred to as a base material, organic base material, base organic material, substrate, organic substrate, etc., and all of these terms may be used interchangeably herein.

[0024] In some embodiments, the organic material comprises feathers. Feathers of any type or size from any species of bird may be used. In such embodiments, the compositions of the present invention may comprise fermented feathers or feather fragments.

[0025] To produce the compositions of the present invention, the organic substrate is contacted with and incubated with the microbial composition and BSF larvae, allowing the organic substrate to be processed by the BSF larvae and microbial composition. As described above, this processing step involves both consumption and digestion of the substrate by the BSF larvae and fermentation of the substrate by the microbial composition (i.e., by the microorganisms in the microbial composition). The incubation step is described in more detail below. Fermentation, as defined herein, can be carried out under anaerobic or aerobic conditions. Fermentation is defined herein simply as the processing of an organic substrate by microbial processing (i.e., by the microorganisms of the microbial composition). A strict biochemical definition of the term (anaerobic metabolism of organic molecules) is not intended.

[0026] Thus, in the animal feed compositions of the present invention, the organic material is fermented (i.e., fermented). Fermented organic material is defined herein as material that has undergone fermentation by one or more microorganisms (the terms "microorganism," "microbial species," and "microbe" are used interchangeably herein). That is, fermented organic material is defined as material that has undergone processing by one or more microorganisms such that the structure or chemical composition of the material is at least partially changed relative to that prior to microbial processing. For example, the fermented organic material may have been at least partially decomposed, digested, or metabolized by one or more microorganisms. Fermentation of organic material may also result in the breakdown of carbohydrates (especially sugars) and the production of chemicals, such as lactic acid, lysine, and other products of fermentation, which may be present in the fermented organic material in the animal feed composition.

[0027] As described above, the organic material is also contacted with BSF larvae, which feed on (or "process") the organic material simultaneously with its fermentation by the microbial species applied in the microbial composition. Thus, the fermented organic material of the compositions of the present invention is material that is undergoing biological processing by both the microorganisms and the BSF larvae. As mentioned above, the BSF larvae consume and digest the organic substrate, which means that at least a portion of the organic substrate is consumed and digested during the processing of the organic substrate, and this is reflected in the animal feed compositions of the present invention.

[0028] The black soldier fly (Hermetia illucens) is an insect species found publicly worldwide. As a globally endemic species, it is not considered an invasive species and can therefore be used in essentially any country without concern. BSF larvae hatch from BSF eggs. BSF eggs are laid by adult female black soldier flies and hatch after approximately four days. BSF larvae are approximately 1 mm long when they hatch but can grow to approximately 25 mm long during their larval stage (which lasts approximately 3-5 weeks, depending on available food). The larvae then pupate and subsequently hatch into adult flies.

[0029] The animal feed composition of the present invention comprises black soldier fly (BSF) biomass. "BSF biomass" essentially means black soldier fly larvae in the context of the present invention. Thus, BSF biomass is composed of BSF larvae and may further comprise BSF eggs and / or BSF pupae. Typically, BSF biomass comprises dead BSF larvae; if it comprises BSF eggs and / or pupae, these are preferably non-viable eggs and / or pupae (i.e., eggs and / or pupae that cannot hatch). It is not excluded that BSF biomass may comprise adult BSF (especially dead adult BSF), but this is not typically the case. If BSF biomass comprises dead BSF larvae, they may be killed by any suitable method, with the understanding that the BSF biomass is part of the composition to be fed to animals (i.e., toxic chemicals cannot be used).

[0030] While the animal feed compositions of the present invention can contain any amount of BSF biomass, in certain embodiments, the composition contains about 5-25% w / w BSF biomass, e.g., 5-22, 5-20, 5-18, or 5-15%, 8-25, 8-22, 8-20, 8-18, or 8-15%, 10-25, 10-22, 10-20, 10-18, or 10-15%, 12-25, 12-22, 12-20, 12-18, or 12-15% w / w BSF biomass. In preferred embodiments, the composition contains 10-20% or 12-18% w / w BSF biomass. In certain embodiments, the composition contains about 5, 10, 15, 20, or 25% w / w BSF biomass. BSF biomass can be recognized, for example, as containing BSF DNA or RNA, or BSF protein.

[0031] The animal feed compositions of the present invention further comprise fermented BSF production residues. As described above, production residues are by-products of larval development and include larval waste (e.g., larval excrement), shed exoskeletons, and dead larvae. Processing of the base organic material by BSF larvae results in the production and deposition of production residues in and on the base material. Thus, the production residues are fermented by the microorganisms applied to the base material at the same time the base material is fermented.

[0032] The animal feed composition also includes microbial biomass. Microbial biomass is biomass (i.e., biological material) composed of microorganisms. Microbial biomass can include any microorganism. The microorganisms can be living or dead, but are preferably dead. Microbial biomass can include bacteria, archaea, fungi, and / or protists. In certain embodiments, the microbial biomass includes bacteria. In certain embodiments, the microbial biomass includes fungi. Preferably, the microbial biomass includes bacteria and fungi (i.e., bacterial biomass and fungal biomass).

[0033] When the microbial biomass includes bacterial biomass, depending on the species present, the bacterial biomass includes vegetative cells and may further include endospores.

[0034] In certain embodiments, the microbial biomass does not include any pathogenic microbial species. In other embodiments, the microbial biomass includes pathogenic microbial species. Preferably, the microbial biomass does not include any viable pathogenic microorganisms, and preferably, the microbial biomass does not include any pathogenic spore-forming bacteria. In certain embodiments, the microbial biomass does not include antibiotic-resistant strains of bacterial species (i.e., strains of bacterial species that have genes that make the bacterial species resistant to antibiotics to which the species is / was originally sensitive). For example, vancomycin-resistant Enterococci are antibiotic-resistant strains of Enterococcus species, such as E. faecalis and E. faecium. Also, in certain embodiments, the microbial biomass does not include fungal strains that are resistant to antifungal agents. Microbial biomass includes the microorganisms that are applied to the base organic material for fermentation purposes and all other microorganisms present in the composition (including the gut microbial community of BSF larvae and the microorganisms present in the base organic material prior to application of the BSF larvae and microbial composition).

[0035] The animal feed compositions of the present invention can contain any amount of microbial biomass, but in certain embodiments contain about 0.05-5% w / w microbial biomass. For example, the composition may contain about 0.05 to 4, 0.05 to 3, 0.05 to 2, 0.05 to 1, 0.05 to 0.9, 0.05 to 0.8, 0.05 to 0.7, 0.05 to 0.6, 0.05 to 0.5, 0.05 to 0.4, 0.05 to 0.3, 0.05 to 0.2, or 0.05 to 0.1%, 0.06 to 5, 0.06 to 4, 0.06 to 3, 0.06 to 2, 0.06 to 1, 0.06 to 0.9, 0.06 to 0.8, 0.06 to 0.7, 0.06 to 0.6, 0.06 to 0.5, 0.06 to 0.4, 0.06 to 0.3, 0.06 to 0.2, or 0.05 to 0.1%. 0.06-0.1%, 0.07-5, 0.07-4, 0.07-3, 0.07-2, 0.07-1, 0.07-0.9, 0.07-0.8, 0.07-0.7, 0.07-0.6, 0.07-0.5, 0.07-0.4, 0.07-0.3, 0.07-0.2, or 0.07-0.1%, 0.08-5, 0.08-4, 0.08-3, 0.08-2, 0.08-1, 0.08-0.9, 0.08-0.8, 0.08-0.7, 0.08-0.6, 0.08-0.5, 0.08-0.4, 0.08-0.3, 0.08-0. 2, or 0.08-0.1%, 0.09-5, 0.09-4, 0.09-3, 0.09-2, 0.09-1, 0.09-0.9, 0.09-0.8, 0.09-0.7, 0.09-0.6, 0.09-0.5, 0.09-0.4, 0.09-0.3, 0.09-0.2, or 0.09-0.1%, 0.1-5, 0.1-4, 0.1-3, 0.1-2, 0.1-1, 0.1-0.9, 0.1-0.8, 0.1-0.7, 0.1-0.6, or 0.1-0.5%, 0.2-5, 0.2-4, 0.2-3, 0.2-2, 0. 2-1, 0.2-0.9, 0.2-0.8, 0.2-0.7, 0.2-0.6, or 0.2-0.5%, 0.3-5, 0.3-4, 0.3-3, 0.3-2, 0.3-1, 0.3-0.9, 0.3-0.8, 0.3-0.7, 0.3-0.6, or 0.3-0.5%, 0.4-5, 0.4-4, 0.4-3, 0.4-2, 0.4-1, 0.4-0.9, 0.4-0.8, 0.4-0.7, 0.4-0.6, or 0.4-0.5%, 0.5-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1, 0.5-0.9, 0.5-0.8, 0.5-0.7, or 0.5-0.6%, 0.6-5, 0.6-4, 0.6-3, 0.6-2, 0.6-1, 0.6-0.9, 0.6-0.8, or 0.6-0.7%, 0.7-5, 0.7-4, 0.7-3, 0.7-2, 0.7-1, 0.7-0.9, or 0.7-0.8%, 0.8-5, 0.8-4, 0.8-3, 0.8-2, 0.8-1, or 0.8-0.9%, 0.9-5, 0.9-4, 0.9-3, 0.9-2, or 0.9-1%, or 1-5, 1-4, 1-3, or 1-2% w / w of microbial biomass.

[0036] Typically, the microbial biomass comprises at least two microbial species, and preferably comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 or more microbial species.

[0037] In certain embodiments, the microbial biomass comprises lactic acid-producing microorganisms; in certain embodiments, the microbial biomass comprises nitrogen-fixing microorganisms; in certain embodiments, the microbial biomass comprises methanotrophic microorganisms; in certain embodiments, the microbial biomass comprises probiotic microorganisms; in certain embodiments, the microbial biomass comprises microorganisms capable of secreting antimicrobial compounds; in certain embodiments, the microbial biomass comprises microorganisms capable of producing antioxidants and / or vitamins; in certain embodiments, the microbial biomass comprises microorganisms capable of degrading toxins; in certain embodiments, the microbial biomass comprises microorganisms that secrete amino acids, preferably lysine and / or glutamate. These capabilities are further described below.

[0038] The animal feed compositions of the present invention may therefore include lactic acid, one or more antimicrobial compounds, one or more antioxidants, and / or one or more vitamins.

[0039] In certain embodiments, the microbial biomass comprises: (i) at least one microorganism selected from Bacillus licheniformis, Enterococcus faecium, Eubacterium ruminantium, Bacillus megaterium (e.g., Bacillus megaterium var. phosphaticum), Bacillus polymyxa, and Streptomyces thermophilus; (ii) at least one microorganism selected from Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, and Streptomyces thermophilus; and / or (iii) at least one microorganism selected from Bacillus subtilis, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus, Aspergillus oryzae, Azotobacter vinelandii, Azospirillum species, Bacillus megaterium (e.g., Bacillus megaterium var. phosphaticum), and Bacillus polymyxa. In certain embodiments, the microbial biomass comprises at least one microbial species from each of (i), (ii), and (iii).

[0040] As described further below, broadly speaking, the three lists of species represent groups of bacterial species that perform different functions during fermentation of the base material. Note that certain species are listed in more than one list (e.g., B. subtilis is listed under point (ii) and point (iii)). Thus, inclusion of such a species covers both lists; for example, if the microbial biomass includes B. subtilis, in these embodiments, the microbial biomass can be considered to include microbial species from each of lists (ii) and (iii).

[0041] The microbial biomass of the compositions of the present invention may alternatively or additionally comprise at least one species selected from Corynebacterium glutamicum, Aneurinbacillus danicus, Methylococcus capsulatus, and Bacillus firmus.

[0042] Thus, in certain embodiments, the microbial biomass comprises: (i) at least one microorganism selected from Bacillus licheniformis, Enterococcus faecium, Eubacterium ruminantium, Bacillus megaterium var. phosphaticum, Bacillus polymyxa, and Streptomyces thermophilus; (ii) at least one microorganism selected from Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, and Streptomyces thermophilus; (iii) at least one microorganism selected from Bacillus subtilis, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus, Aspergillus oryzae, Azotobacter vinelandii, Azospirillum species, Bacillus megaterium var. phosphaticum, and Bacillus polymyxa; and (iv) at least one microorganism selected from Corynebacterium glitamicum, Aneurinbacillus danicus, Methylococcus capsulatus, and Bacillus firmus.

[0043] In other embodiments, the microbial biomass comprises at least one microbial species selected from Bacillus licheniformis, Enterococcus faecium, Eubacterium ruminantium, Bacillus megaterium var. phosphaticum, Bacillus polymyxa, Streptomyces thermophilus, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus, Azotobacter vinelandii, Azospirillum species, Corynebacterium gritamicum, Aneurinbacillus danicus, Methylococcus capsulatus, and Bacillus firmus. The microbial biomass may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of these species, or may include all of these species.

[0044] In other embodiments, the microbial biomass comprises at least four microbial species selected from Bacillus licheniformis, Enterococcus faecium, Eubacterium ruminantium, Bacillus megaterium var. phosphaticum, Bacillus polymyxa, Streptomyces thermophilus, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus, Azotobacter vinelandii, and Azospirillum species, preferably at least five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of these species, or may comprise all of these species, which may include at least one species from list (i) above, at least one species from list (ii) above, and at least one species from list (iii) above.

[0045] In other embodiments, the microbial biomass is selected from the group consisting of Bacillus licheniformis, Enterococcus faecium, Eubacterium ruminantium, Bacillus megaterium var. phosphaticum, Bacillus polymyxa, Streptomyces thermophilus, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus pediococcus, Bacillus spp. The microbial composition includes at least five microbial species selected from S. pentosaceus, Azotobacter vinelandii, Azospirillum species, Corynebacterium gritamicum, Aneurinbacillus danicus, Methylococcus capsulatus, and Bacillus firmus, and includes at least one species from list (i) above, at least one species from list (ii) above, at least one species from list (iii) above, and at least one species from list (iv) above.

[0046] In a preferred embodiment, the microbial biomass comprises B. subtilis.

[0047] Species contained in microbial biomass can be most easily identified by their DNA or RNA sequences. Routinely, microbial species can be identified by rRNA sequencing (in particular, bacterial species can be identified by their 16S rRNA sequences and fungal species by their 18S rRNA sequences). Those skilled in the art can easily determine the microbial species present in a particular composition of interest.

[0048] The above species can be obtained from cell banks or repositories, such as the ATCC. For example, strains of B. licheniformis are available from the ATCC under accession numbers 14580 and 9789, strains of E. faecium, among others, are available from the ATCC under accession numbers BAA-2317 and BAA-2127, and strains of E. ruminantium, among others, are available from the ATCC under accession number 17233, Bacillus megaterium var. phosphaticum is available from the DSM under accession number DSM-3228, and Bacillus polymyxa (Paenibacillus Bacillus subtilis is available from ATCC under accession numbers 6051 and 21332, among others; Saccharomyces cerevisiae is available from ATCC under accession numbers 9763 and 834, Aspergillus oryzae is available from ATCC under accession numbers 1011 and 42149, among others; and Lactobacillus plantarum is available from ATCC under accession numbers 8014 and 14917, among others. Among others, Lactobacillus acidophilus is available from ATCC under accession numbers 4356 and 314; Pediococcus pentosaceus is available from ATCC under accession numbers 33316 and 25745; among others, Azotobacter vinelandii is available from ATCC under accession numbers 9046 and 478; among others, Azospirillum species is available from ATCC under accession numbers 29145 and 29708; among others, Corynebacterium gritamicum is available from ATCC under accession numbers 13032 and 14067; among others, Aneurinbacillus danicus is available from DSMZ under accession number DSM 21566; Methylococcus capsulatus is available from ATCC under accession numbers 33009 and 19069; and Bacillus firmus is available from ATCC under accession numbers 14575 and 8247.

[0049] The animal feed composition of the present invention may be dry, i.e., it may have a low moisture or liquid content. In certain embodiments, the animal feed composition has a moisture content of about 15% (v / v) or less, for example, about 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5% (v / v) or less. Preferably, the moisture content is about 10% (v / v) or less. As described further below, a low moisture content may be achieved by heating the composition.

[0050] The animal feed composition may be crushed, ground, or blended to break down and fragment the fermented organic material. The crushing, grinding, or blending steps may also kill and fragment the BSF larvae in the composition. These processes make the composition more readily edible, especially for small animals.

[0051] If the animal feed composition is dry, it may be powdered (i.e., the animal feed composition may be a powder). Powdered compositions have advantages in terms of shelf life, ease of transportation, and ease of combining with other ingredients to make an animal feed. For the same reasons, the composition may further comprise a preservative. Suitable preservatives are known in the art and include, for example, propionic acid, formic acid, lactic acid, citric acid, sorbic acid, acetic acid (and salts of these acids, e.g., their sodium, potassium, and calcium salts), antioxidants such as ascorbic acid (vitamin C) and its salts, tocopherol (a form of vitamin E), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and propyl gallate, and antifungal agents such as natamycin. Similarly, the compositions of the present invention may be pelleted, i.e., provided in the form of pellets suitable for feeding to animals, as discussed further below.

[0052] In certain embodiments, the compositions of the present invention are free of viable organisms. That is, the compositions are both free of viable BSF and free of viable microorganisms. In this case, the BSF larvae (and any BSF adults) in the composition are dead, any BSF pupae are dead as well, and any BSF eggs are not viable (i.e., unable to hatch into BSF larvae). Vegetative bacterial cells are also dead, as are other types of microbial cells (e.g., fungal cells), although viable endospores may be present. The compositions may preferably be sterile (i.e., in this case, in addition to being free of viable organisms, the compositions also contain no viable endospores).

[0053] In another preferred embodiment, the composition of the present invention comprises endospores of spore-forming bacteria (or bacteria), in particular probiotic bacteria (or bacteria), such as B. subtilis, B. licheniformis, B. megaterium, or B. polymyxa. Preferably, the composition comprises endospores of B. subtilis.

[0054] The animal feed compositions of the present invention are nutritious compositions and typically contain high levels of protein, in addition to carbohydrates and fat, to support animal growth. The amount of each type of macronutrient can depend on the base material used for fermentation and the desired composition for the animal being fed. For example, on a dry matter basis, the animal feed composition can contain at least 15, 20, 25, 30, 35, or 40% (w / w) protein. In some embodiments, the composition preferably contains at least 40% protein, e.g., at least 45 or 50% protein. The animal feed composition can contain, on a dry matter basis, at least 15, 20, 25, 30, 35, or 40% (w / w) carbohydrate and / or at least 3, 5, 10, 15, 20, 25, or 30% (w / w) fat. In some embodiments, the animal feed composition is defatted and therefore contains a low amount of fat, e.g., about 3-5% fat.

[0055] In some embodiments, the animal feed composition comprises at least 20% protein, at least 20% carbohydrate, and at least 20% fat (all w / w on a dry matter basis). In some embodiments, the animal feed composition comprises at least 25% protein, at least 25%, 30%, or 35% carbohydrate, and optionally at least 20% fat (all w / w on a dry matter basis). In some embodiments, the composition comprises at least 50% protein and at least 30% fat (all w / w on a dry matter basis).

[0056] When the composition is defatted, it may contain at least 30% protein and at least 30%, 35%, or 40% carbohydrates, hi other embodiments, it may contain at least 50, 55, 60, 65, 70, or 75% protein (all w / w on a dry matter basis).

[0057] In some embodiments, the animal feed composition comprises a starch. For example, the animal feed composition may comprise a resistant starch, i.e., a starch that resists digestion into sugars (e.g., maltose and / or glucose) in the small intestine of an animal. The resistant starch may be fermented by the animal's gut microbial community, resulting in the production of short-chain fatty acids, such as butyrate.

[0058] In certain embodiments, the animal feed composition comprises resistant starch type 3 (RS3 starch), which may be obtained, for example, by heating and cooling resistant starch type 2 (RS2 starch) during production of the composition.

[0059] The animal feed compositions of the present invention may contain postbiotics, i.e., metabolic products of microbial biomass that provide physiological benefits to animals consuming the compositions. Postbiotics that may be included in the compositions include lactic acid (e.g., produced by lactic acid bacteria, e.g., Lactobacilli), short-chain fatty acids, and surfactin. Short-chain fatty acids (SCFAs) are defined herein as saturated aliphatic organic acids containing 1 to 6 carbon atoms. SCFAs that may be included in the compositions include acetate, propionate, and butyrate. SCFAs are commonly produced by Firmicutes, e.g., lactic acid bacteria. Surfactin is a cyclic lipopeptide antibiotic produced by B. subtilis.

[0060] Methods for decomposing organic materials In a second aspect, the present invention provides a method for decomposing organic material. As described above, the method can be used to produce the animal feed composition of the present invention, but the method can also be used for any other suitable purpose as desired by the skilled artisan. For example, the method can be used to produce fertilizer from organic waste, or simply for waste management purposes.

[0061] The organic material decomposed according to the present method can be any organic material, as defined above with respect to the base material for the animal feed composition. "Decomposition" means that the organic material is broken down or processed. As described in more detail below, the present method includes a step of fermenting the organic material, and therefore decomposition includes fermentation. However, decomposition also includes consumption and digestion of the organic material by the BSF larvae. Consumption of the organic material by the BSF larvae can reduce the total mass of the material.

[0062] In some embodiments, the organic material may be supplemented with one or more prebiotics before or during contact with the BSF larvae and microbial composition. "Prebiotic" is defined herein as a compound that promotes the growth of species in the microbial composition.

[0063] Suitable prebiotics for adding to organic materials include resistant starch, partial RS2 starch. RS2 starch is a type of starch that has a structure or composition that makes it inaccessible to starch-degrading enzymes, such as amylase or isoamylase. RS2 starch is usually granular, i.e., added to organic materials in the form of granules. RS2 starch is rapidly and efficiently fermented by the microbial composition, thereby improving the speed of the fermentation process.

[0064] Any suitable amount of RS2 starch can be added to the organic material, depending, for example, on the origin and content of the organic material and the metabolic requirements of the microbial composition. RS2 starch can be added to the organic material at a rate of about 5-15% w / w, e.g., 5-12%, 5-10%, 6-9%, or 7-8% w / w, on a dry weight basis. That is, RS2 starch can be added to the organic material so that it constitutes, for example, 5-10%, 6-9%, or 7-8% w / w of the resulting product on a dry weight basis.

[0065] The RS2 starch may be included in the context of refined starch granules, or it may be included in the context of a starchy raw material, such as uncooked potatoes or unripe bananas.

[0066] The method includes contacting the organic material with BSF larvae and a microbial composition. Contacting the organic material with BSF larvae can be accomplished by either applying BSF larvae directly to the organic material or by applying BSF eggs to the organic material and allowing the eggs to hatch into BSF larvae.

[0067] Larvae can be applied to organic material at any age or size. However, toward the end of the larval stage, BSF larvae enter a post-feeding phase in preparation for pupation. It is usually desirable for the BSF larvae to continue feeding on the organic material throughout the decomposition process, which should therefore be completed before the larvae enter the post-feeding phase. Because the larval stage usually lasts up to about 18 days before the post-feeding (or pre-pupal) stage begins, the larval instars used should be selected based on the length of the desired process, with the goal of completing the decomposition process before the post-feeding stage begins.

[0068] The degradation (i.e., incubation) step can be any suitable length required for sufficient degradation of the substrate. For example, the degradation step can last about 5 to 14 days, e.g., 5 to 12, 5 to 10, 5 to 8, 5 to 7, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 7 to 14, 7 to 12, 7 to 10, 8 to 14, 8 to 12, 8 to 10, 9 to 14, 9 to 12, 9 to 10, 10 to 14, 10 to 12, or 12 to 14 days, preferably about 6 to 12, 7 to 12, or 8 to 12 days. In preferred embodiments, the degradation step lasts about 6, 7, 8, 9, 10, 11, or 12 days. The length of the degradation step can be defined as the length of time the organic material is incubated with the BSF larvae and microbial composition. If the BSF larvae and microbial compositions are added at separate times, the length of the degradation process can be measured from either the time the first of the larvae and microbial compositions is applied to the organic material or the time the second of the larvae and microbial compositions is applied. That is, the length of the degradation process can be defined as the length of time the organic material is incubated with both the BSF larvae and microbial compositions, or the length of time the organic material is incubated with at least one of the BSF larvae and microbial compositions. Preferably, the length of the degradation process refers to the length of time the substrate is incubated with both the BSF larvae and microbial compositions, as described above.

[0069] Thus, the BSF larvae can be applied to the organic material at any age, taking into account the length of the decomposition process. In certain embodiments, the larvae are applied to the organic material at an age of up to about 12 days, e.g., up to 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. In embodiments, the larvae are applied to the organic material at an age of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days. In embodiments, the larvae are applied to the organic material when they are 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 6 to 10, 6 to 9, 6 to 8, 7 to 10, 7 to 9, or 8 to 10 days old. In preferred embodiments, the larvae are applied to the organic material when they are 1 to 5 or 4 to 6 days old, particularly about 5 days old.

[0070] When the larvae are applied directly to the organic material, the larvae may be applied to the material at a weight ratio of at least 1:250 (i.e., 1 g of larvae per 250 g of organic material), 1:225, 1:200, 1:175, 1:150, 1:125, 1:100, 1:75, or 1:50. Preferably, the larvae are applied to the organic material at a weight ratio of at least 1:150 (i.e., preferably at least 1 g of larvae is applied per 150 g of organic material), for example, at least 1:140, 1:130, 1:120, 1:110, 1:100, 1:90, 1:80, 1:70, 1:60, or 1:50. In preferred embodiments, the larvae are applied to the organic material at a weight ratio of about 1:250, 1:225, 1:200, 1:175, 1:150, 1:140, 1:130, 1:120, 1:110, 1:100, 1:90, 1:80, 1:70, 1:60, or 1:50. Higher weight ratios can be used because there is no technical maximum (within reason) for the number of larvae that can be used to decompose organic material of a given size, but above a certain number, competition for food can result in suboptimal performance of individual larvae, and using excessive numbers of larvae can be uneconomical.

[0071] As described above, organic material can be contacted with BSF larvae by applying BSF eggs to the organic material and allowing the eggs to hatch on or within the organic material. BSF eggs take approximately four days to hatch after being laid, so if BSF eggs are applied to the material, the overall length of the process may need to be extended to allow for hatching time. If BSF eggs are applied, they can be applied one day after being laid or any time thereafter, for example, one, two, three, or four days after the BSF eggs are laid.

[0072] One gram of BSF eggs produces approximately 30,000 larvae (by comparison, five-day-old larvae weigh on average about 0.02 g, or about 600 times the weight of an egg, so one gram of five-day-old larvae corresponds to about 50 larvae). Thus, a very small weight of BSF eggs is required to produce enough larvae to process the organic material. For example, suitable weight ratios of eggs to organic material are 1:5,000 to 1:100,000, e.g., 1:5,000 to 1:90,000, 1:5,000 to 1:80,000, 1:5,000 to 1:70,000, 1:5,000 to 1:60,000, 1:5,000 to 1:50,000, 1:5,000 to 1:40,000, 1:10,000, etc. ~1:100,000, 1:10,000~1:90,000, 1:10,000~1:80,000, 1:10,000~1:70,000, 1:10,000~1:60,000, 1:10,000~1:50,000, 1:10,000~1:40,000, 1:20,000~1:100,000, 1:20,000~1:90 ,000, 1:20,000~1:80,000, 1:20,000~1:70,000, 1:20,000~1:60,000, 1:20,000~1:50,000, 1:20,000~1:40,000, 1:30,000~1:100,000, 1:30,000~1:90,000, 1:30,000~1:80,000, 1 :30,000 to 1:70,000, 1:30,000 to 1:60,000, 1:30,000 to 1:50,000, 1:40,000 to 1:90,000, 1:40,000 to 1:80,000, 1:40,000 to 1:70,000, 1:40,000 to 1:60,000, or 1:50,000 to 1:100,000. In a preferred embodiment, a weight ratio of eggs to organic material of 1:20,000 to 1:40,000, e.g., 1:25,000 to 1:40,000, 1:30,000 to 1:40,000, 1:35,000 to 1:40,000, 1:20,000 to 1:35,000, 1:25,000 to 1:35,000, 1:25,000 to 1:30,000, 1:30,000 to 1:40,000, or 1:35,000 to 1:40,000, is used.In preferred embodiments, a weight ratio of eggs to organic material of about 1:20,000, 1:25,000, 1:30,000, 1:35,000, or 1:40,000 is used.

[0073] The BSF larvae or eggs may be applied to the organic material, for example, by pouring or placing the larvae / eggs onto the material, or alternatively, the organic material may be poured or placed onto the larvae / eggs. The larvae or eggs may be mixed with the organic material or deposited on the surface of the material and left to process the material (in the case of BSF eggs, allowed to hatch before processing the material).

[0074] The microbial composition with which the organic material is contacted is simply a composition comprising at least one microorganism that serves to decompose the organic material and, optionally, improve the properties of the resulting product composition with respect to its use, for example, as an animal feed composition. The contents of the composition are discussed further below.

[0075] The microbial composition can be applied to the organic material in the form of a liquid suspension or culture (e.g., in a growth medium or buffer). A liquid microbial composition can be applied to the organic material, for example, by spraying or pouring. Alternatively, the microbial composition can be applied to the organic material as a dry powder (e.g., a lyophilized or dehydrated powder), which can also be sprayed or poured onto the organic material. After application to the organic material, the microbial composition can be mixed with the organic material.

[0076] The microbial composition and BSF larvae (or eggs) can be applied to the organic material simultaneously or substantially simultaneously, e.g., consecutively, or within, e.g., 1, 2, 3, 4, 5, or 6 hours of each other. Alternatively, the microbial composition and BSF larvae / eggs can be applied to the organic material at separate times, e.g., at least 6, 12, 18, or 24 hours apart, or at least 1, 2, or 3 days apart. In this case, the microbial composition can be applied before the BSF larvae or eggs, or the BSF larvae or eggs can be applied before the microbial composition. Typically, however, the microbial composition and BSF larvae or eggs are applied to the organic material substantially simultaneously.

[0077] Prior to application of the BSF larvae or eggs, and typically also prior to application of the microbial composition, the organic material may be processed to make it easier for the BSF larvae and microorganisms to digest and decompose. In particular, the organic material may be crushed, ground, and / or blended prior to application of the BSF larvae or eggs and microbial composition. This processing may be carried out, for example, using a blender, food processor, or shredder.

[0078] A microbial composition comprises at least one microorganism capable of fermenting a substrate, i.e., at least one microbial species capable of growing and processing (e.g., digesting, decomposing, or metabolizing) the substrate, or a portion thereof, or a component thereof. Typically, a microbial composition comprises multiple microbial species.

[0079] In embodiments, the microbial composition comprises at least one digestive microorganism, at least one protein-enhancing microorganism, and / or at least one microorganism that increases the nutritional and / or health value of the product composition and / or reduces the environmental impact of the process. Preferably, the microbial composition comprises at least one microbial species of each of these three functional types. A single microorganism may fulfill two or even all three of these roles, depending on its characteristics.

[0080] "Digestive microorganisms," as defined herein, are microorganisms that secrete digestive enzymes. A digestive enzyme is any enzyme that breaks down macronutrients or components of organic materials into smaller parts; for example, it may break down polymers, multimers, or dimers into smaller molecules, such as their monomeric constituents. Examples of digestive enzymes that may be produced by at least one digestive microorganism include proteases (which break down proteins), lipases (which break down lipids, and in particular triglycerides into fatty acids and glycerol), and carbohydrases (which break down carbohydrates). Proteases that may be secreted by one or more digestive microorganisms also include endopeptidases and exopeptidases, as well as proteases defined by their mechanism of action, including serine proteases, cysteine ​​proteases, threonine proteases, aspartic acid proteases, glutamic acid proteases, and metalloproteases. Carbohydrases (also called glycosidases) that may be secreted by one or more digestive microorganisms include amylases (which break down starch), cellulases (which break down cellulose), hemicellulases including xylanases (which break down hemicellulose), chitinases (which break down chitin), ligninases (which break down lignin), lactase, maltase, isomaltase, sucrase, invertase, trehalase, and the like.

[0081] Digestive microorganisms for use in the microbial compositions of the present invention preferably secrete a plurality of different digestive enzymes (e.g., at least 2, 3, 4, 5, or 6 digestive enzymes) and / or secrete high levels of digestive enzymes and / or secrete particularly active digestive enzymes, so that the digestive microorganisms are particularly effective in breaking down organic material.

[0082] Preferably, the digestive microorganisms for use in the microbial compositions of the present invention secrete at least one (and preferably multiple) digestive enzymes, such as proteases, amylases, cellulases, etc., that break down polymer molecules into smaller components, e.g., monomers, as described above. Digestive enzymes that break down substrates classified as human dietary fiber (e.g., cellulose, hemicellulose, chitin, lignin) are particularly useful because such polymers are not readily digestible by BSF larvae.

[0083] In a preferred embodiment, at least one digestive microorganism secretes a protease and a carbohydrase. That is, at least one protease and at least one carbohydrase are secreted among the digestive microorganisms in the microbial composition. The protease and carbohydrase may be secreted by the same microbial species or by different species (e.g., one digestive microorganism may secrete a protease and a second digestive microorganism may secrete a carbohydrase). At least one digestive microorganism may further secrete a lipase (such that at least one digestive microorganism secretes a protease, a lipase, and a carbohydrase).

[0084] In certain embodiments, at least one digestive microorganism secretes a carbohydrase selected from cellulases, hemicellulases (especially xylanases), and amylases. Preferably, at least one digestive microorganism secretes at least two of these enzymes (e.g., cellulases and hemicellulases, e.g., xylanases). At least one digestive microorganism may secrete cellulases, hemicellulases (e.g., xylanases), and amylases. These one or more carbohydrases may be secreted by the one or more digestive microorganisms in addition to a protease and, optionally, a lipase.

[0085] Examples of species suitable for use as digestive microorganisms in the microbial composition include Bacillus licheniformis, Enterococcus faecium, Eubacterium ruminantium, Bacillus megaterium var. phosphaticum, Bacillus polymyxa, and Streptomyces thermophilus. In embodiments, the at least one digestive microorganism is selected from or comprises any one or more of these species.

[0086] Other suitable species for use as digestive microorganisms can be identified by examining the ability of the species / strain of interest to degrade the substrate of interest, by genetic analysis (by identifying microbial species that encode digestive enzymes with signal peptides in their genomes), or by routine microbiology / enzymology. Digestive microorganisms can also be engineered, for example, by modifying known microbial strains to express desired enzymes (e.g., by introducing the gene of interest into the microorganism on a plasmid or by inserting the gene of interest into the genome of the microorganism by standard techniques, such as CRISPR or homologous recombination), or by increasing expression of naturally encoded enzymes, for example, by replacing the native promoter with a more active promoter.

[0087] Alternatively or in addition to including one or more digestive microorganisms in the microbial composition, one or more digestive enzymes (described above) can be added directly to the organic substrate, where the digestive enzymes can be provided in purified or partially purified form, or in the context of crude culture supernatant or cell extract.

[0088] A "protein-enhanced" microorganism, as defined herein, is a microorganism that increases the protein content of a product composition. That is, a protein-enhanced microorganism is a microorganism that produces high levels of protein (particularly, produces more protein than it degrades or induces degradation by secreting proteases), and whose presence in a microbial composition causes the product composition to contain more protein than it would in the absence of the protein-enhanced microorganism. Such microorganisms can be routinely identified empirically by testing the methods of the present invention side-by-side with a control method (which does not include the microorganism of interest in the microbial composition but is otherwise identical to the test method). If the test method (including the microorganism of interest) produces a higher protein content in the product composition than the control method, the microorganism of interest is a protein-enhanced microorganism.

[0089] Examples of species suitable for use as protein-enhanced microorganisms in the microbial composition include Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, and Streptomyces thermophilus, and in embodiments, the at least one protein-enhanced microorganism is selected from or comprises any one or more of these species.

[0090] Protein-enhanced microorganisms can also be engineered by modifying known microbial species to produce more protein.

[0091] The microbial composition may include at least one microorganism that increases the nutritional and / or health value of the product composition and / or reduces the environmental impact of the method.

[0092] As described above, the method yields a product composition, which is an animal feed composition according to the first aspect of the invention (i.e., the product composition is officially an animal feed or a component of an animal feed, as described in the first aspect of the invention). Because the product composition is typically suitable for feeding animals, it is advantageous if the nutritional value (i.e., amount of nutrients) in the composition is increased, for example, by virtue of the production of vitamins or other micronutrients in the product composition by the microorganisms in the microbial composition.

[0093] For animal feed, it would also be advantageous if the health value of the product composition was increased. By "health value" is meant the value of the product composition to the health of the animal consuming the product composition, beyond its mere nutritional value. Thus, increasing the health value of a product composition means increasing the positive contribution of the product composition to the general health of the animal. As discussed further below, this can be achieved, for example, by including in the microbial composition microorganisms that secrete antimicrobial compounds (which may protect the consuming animal from infection), or by any other health-enhancing mechanism.

[0094] As described above, the method of the present invention has a significantly reduced environmental impact compared to existing methods for producing animal feed. Nevertheless, the method has an environmental impact in terms of the production of greenhouse gases, etc. The negative aspects of the environmental impact of the method can be eliminated by including microorganisms in the microbial composition that reduce the environmental impact of the method. Microorganisms that reduce the environmental impact of the method are microorganisms that reduce the environmental impact of the method, for example, that reduce greenhouse gas emissions associated with the method.

[0095] In embodiments, the microbial composition comprises at least one microorganism that increases the nutritional value and health value of the product composition, or at least one microorganism that increases the nutritional value of the product composition and reduces the environmental impact of the process, or at least one microorganism that increases the health value of the product composition and reduces the environmental impact of the process, or at least one microorganism that increases the nutritional value of the product, increases the health value of the product, and reduces the environmental impact of the process. In these embodiments, the two or three functions can be performed by a single microorganism that performs all three functions, or by multiple microorganisms that each perform one function.

[0096] In some embodiments, the microorganisms that increase the nutritional value of the product composition are nitrogen-fixing microorganisms, particularly nitrogen-fixing bacteria. Nitrogen-fixing microorganisms are microorganisms that convert atmospheric nitrogen (N) into organic nitrogen, such as ammonia. Nitrogen-fixing microorganisms may also be known as diazotrophs. Many diazotrophs that can be used in the present invention are known in the art and include bacteria of the Azotobacter and Azospirillum genera, such as Azotobacter vinelandii, Azospirillum brasilense, Azospirillum halopraeferens, and Azospirillum lipoferum, as well as Bacillus polymyxa (now also known as Paenibacillus polymyxa). Non-diazotrophic microorganisms can be modified to have nitrogen fixation activity, for example, by recombinant DNA technology, by inducing expression of nitrogenase enzymes and related proteins required for cofactor synthesis (as described in Tatemichi et al. (Bioscience, Biotechnology and Biochemistry 85(10): 2209-2216, 2021)). Nitrogen fixation increases the amount of organic nitrogen in and associated with substrate organic material, which can be converted to amino acids and other biologically important molecules, promoting the growth of microorganisms and BSF larvae that process the organic material and increasing the overall nutritional value of the resulting product composition.

[0097] In some embodiments, the microorganisms that increase the nutritional value of the resulting composition produce (preferably secrete) amino acids, preferably at high levels. While any amino acid may be secreted by such microorganisms, in certain embodiments, the microorganisms produce or secrete lysine and / or glutamate. Other amino acids that may be produced by microorganisms include all proteinogenic amino acids. Bacillus megaterium (also known as Priestia megatarium), including B. megaterium var. phosphaticum, is known to produce high levels of glutamate (in the form of poly-γ-glutamic acid) and may therefore be used in the present method for this purpose. Another known group of bacteria that may be used for this purpose is Corynebacterium glutamicum, which produces high levels of several amino acids, particularly lysine, but also glutamate, threonine, isoleucine, valine, serine, arginine, methionine, and cysteine. Other microbial species capable of producing high levels of amino acids and that can be used for this purpose in the present method include the Lactobacillus plantarum (now also known as Lactiplantibacillus plantarum) bacterial group and the Aspergillus oryzae fungal group. Microorganisms that produce high levels of amino acids increase the amount of amino acids available to other organisms that process organic materials, thus increasing total protein production. Other microorganisms that produce high levels of amino acids are known to those skilled in the art. Microorganisms can also be genetically modified to increase amino acid production by introducing or increasing expression in the microorganism of the gene responsible for the synthesis of the amino acid of interest.

[0098] In some embodiments, the microorganisms that increase the nutritional and / or health value of the resulting composition produce one or more vitamins and / or antioxidants. Vitamins are essential micronutrients required for metabolic function in animals. Vitamins that can be produced by microorganisms for use in the microbial composition include vitamin A (e.g., retinol), vitamin B (B1 (thiamine), B2 (flavin / riboflavin), B3 (e.g., niacin), B5 (pantothenic acid), B6 ​​(e.g., pyridoxine), B7 (biotin), B9 (folic acid), and B 12 Vitamins include vitamin C (including cobalamin), vitamin D (including D1 to D5), vitamin E, and vitamin K (including K1 and K2).

[0099] Antioxidants are compounds that inhibit oxidation and free radical production. Antioxidants prevent food spoilage (thus increasing the shelf life of the animal feed compositions produced according to the methods of the present invention), as well as have beneficial health effects on animals that consume them. Some vitamins are antioxidants (vitamins A, C, and E). Other chemicals with antioxidant activity are known as well.

[0100] Many microbial species that produce vitamins and antioxidants are known in the art and may be used in the methods of the present invention for this purpose, for example, Azospirillum species, which are known to produce B vitamins, including thiamine and riboflavin, vitamin B 12Examples of suitable bacterial species include Bacillus megaterium, known to produce vitamins such as niacin, pantothenic acid, riboflavin, and biotin, and Azotobacter vinelandii, known to produce B vitamins including niacin, pantothenic acid, riboflavin, and biotin. B. subtilis is known to produce vitamin K2. Other bacterial species can also be engineered to produce vitamins by expressing genes required for vitamin biosynthesis therein; for example, B. subtilis has been successfully engineered to produce B vitamins, including vitamins B1, B2, B5, B6, and B7 (Su et al., Microbial Cell Factories 19: 173, 2020). Thus, microorganisms that produce (and preferably secrete) vitamins and / or antioxidants can produce one or more vitamins and / or antioxidants. Such microorganisms can produce non-antioxidant vitamins, antioxidant vitamins, and / or non-vitamin antioxidants.

[0101] In some embodiments, the microorganisms that increase the health value of the product composition are microorganisms that produce (preferably secrete) antimicrobial compounds. An antimicrobial compound is defined as any compound that has a negative impact on the growth of one or more microbial species. Antimicrobial compounds include antibacterial, antifungal, antiprotozoan, and antiprotozoan agents. Microbial species may produce one or more antimicrobial compounds from one or more different classes (e.g., antibacterial and antifungal compounds). Antibacterial agents produced by microorganisms for use in the microbial compositions may be referred to as antibiotics. The terms "antibacterial" and "antibiotic" are used interchangeably herein. Any antibiotic may be produced by a microorganism for use in the microbial compositions, and may include known classes of antibiotics used in medicine, such as β-lactam antibiotics (e.g., penicillins, cephalosporins, and carbapenems), polymyxins, rifamycins, quinolones, sulfonamides, macrolides, lincosamides, tetracyclines, aminoglycosides, lipopeptides, and the like. Antibiotics, as defined herein, also include bacteriocins and lantibiotics. Antibiotics can be bacteriostatic or bacteriocidal.

[0102] Many microbial species are known to produce antimicrobial agents (indeed, other microorganisms are the primary source of antimicrobial agents used medically / industrially today). By including such microorganisms in a microbial composition, the antimicrobial agent can be included in the resulting composition to provide protection against microbial infection to animals consuming the resulting composition. Preferably, the microbial species used in the antimicrobial-producing microbial composition do not produce antimicrobial agents used in human medicine, thereby avoiding the animal feed composition from promoting microbial resistance to the antimicrobial agents contained therein. For example, B. subtilis can be used for this purpose because this species is known to produce over 20 antibiotic compounds, major lantibiotics, in addition to other antibiotics, such as surfactin. Other suitable microbial species that can be used for this purpose are known in the art or can be generated by expression of genes responsible for antimicrobial synthesis, e.g., by recombinant technology.

[0103] In some embodiments, the microorganisms that increase the health value of the product composition are those that reduce the amount of toxins in the product composition, e.g., degrade toxins. Toxins can be produced by microorganisms (particularly bacteria and fungi) present in the organic substrate material (including microorganisms applied to the material in the microbial composition and microorganisms present in the organic material prior to application of the microbial composition). In certain embodiments, such microorganisms can reduce the amount of mycotoxins in the product composition, and preferably remove mycotoxins from the product composition. Mycotoxins are toxic metabolites produced by fungi, including aflatoxins, ochratoxins, zearalenone, and trichothecenes. Certain bacterial species are known to have mycotoxin-degrading activity and may be used for this purpose in the methods of the invention, including Lactobacillus acidophilus, which has been shown to be able to degrade zearalenone and trichothecenes (and to a lesser extent aflatoxins and ochratoxins; see Ragoubi et al., Toxins 13(3): 185 (2021)).

[0104] In some embodiments, the microorganisms that increase the nutritional and / or health value of the product composition are lactic acid-producing microorganisms. Although many microorganisms can produce lactic acid (for example, during anaerobic respiration), preferably, the lactic acid-producing microorganisms of the microbial composition are lactic acid bacteria, i.e., bacteria that produce lactic acid as their main end product of carbohydrate metabolism. Lactic acid bacteria are known in the art and include, in particular, bacteria of the genera Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus. Any such lactic acid bacteria can be used. Preferred lactic acid bacteria for use in the microbial composition include Lactobacillus plantarum, Lactobacillus acidophilus, and Pediococcus pentosaceus.

[0105] The inclusion of lactic acid bacteria (or other lactic acid-producing microorganisms) in the microbial composition is advantageous because it results in the presence of lactic acid in the product composition. Lactic acid is a preservative and therefore acts to reduce spoilage or contamination of the product composition, which reduces the likelihood of food poisoning resulting from the composition. Lactic acid also acts as an antioxidant, improves gastrointestinal health, and may enhance absorption of other nutrients in the composition.

[0106] In some embodiments, the microorganisms that reduce the environmental impact of the methods of the present invention are methanotrophic microorganisms. Fermentation of organic material in the methods of the present invention induces the production of methane. Methane is a potential greenhouse gas with a stronger warming effect on the planet than CO2. Methanotrophic microorganisms are microorganisms (usually bacteria or archaea) that can metabolize methane (usually to formaldehyde and / or CO2), thereby reducing the environmental impact of the methods. Any methanotrophic microorganism may be used in the microbial composition, including, for example, Methylococcus capsulatus, Aneurinbacillus danicus, and Bacillus firmus. Other suitable microbial species that can be used for this purpose are known in the art or can be generated by expression of genes responsible for methane metabolism, for example, by recombinant technology.

[0107] Thus, in some embodiments, the microbial composition comprises: (a) Microorganisms that secrete antimicrobial compounds; (b) microorganisms that degrade toxins; (c) lactic acid-producing microorganisms, (d) antioxidant and / or vitamin producing microorganisms; (e) a microorganism secreting an amino acid, preferably lysine and / or glutamate; (f) nitrogen-fixing microorganisms, and / or (g) Methanotrophic microorganisms.

[0108] In an embodiment, the microbial composition comprises at least one microorganism that increases the nutritional and / or health value of the resulting composition and / or reduces the environmental impact of the method, and is selected from Bacillus subtilis, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus, Aspergillus oryzae, Azotobacter vinelandii, Azospirillum species, Bacillus megaterium (e.g., Bacillus megaterium var. phosphaticum), Bacillus polymyxa, Corynebacterium gritamicum, Aneurinbacillus danius, Methylococcus capsulatus, and Bacillus firmus.

[0109] As indicated above, the microbial composition may therefore contain any type of desired microorganism. The microbial composition may contain prokaryotes and / or eukaryotes. Prokaryotes that may be contained in the microbial composition include both bacteria and archaea. Both gram-negative and / or gram-positive bacteria may be contained in the microbial composition. Eukaryotes that may be contained in the microbial composition include, in particular, fungi. In a preferred embodiment, the microbial composition contains bacteria and fungi. That is, the microbial composition preferably contains at least one type of bacteria and at least one type of fungus.

[0110] The microorganisms in the microbial composition can be obligate aerobes, facultative anaerobes, or obligate anaerobes. Because aerobic conditions are required for the survival of BSF larvae, obligate aerobes and facultative anaerobes are preferred, although obligate anaerobes can also be used if the microbial composition is mixed into organic material prior to the incubation step, as the interior of the organic material may be anaerobic or have a low oxygen content.

[0111] In some embodiments, the microbial composition does not contain any pathogenic species. In other embodiments, however, the microbial composition does contain pathogenic species. As described below, the method may include a sterilization step (e.g., using heat), so even if the method is for the purpose of producing animal feed, the inclusion of pathogenic species in the composition may be acceptable as long as the pathogenic species are killed during the sterilization process. However, if pathogenic species are included in the microbial composition, it is preferable that the species do not produce exotoxins that survive the sterilization process and are therefore present and active in the product composition. For example, if a heat sterilization process is used, it is preferable that the microbial composition does not include microbial species that produce heat-stable exotoxins.

[0112] Similarly, endospores produced by bacteria in the microbial composition may also survive any sterilization process. Thus, in some embodiments, it may be preferable for the microbial composition not to contain any spore-forming (i.e., endospore-forming) bacteria. In other embodiments, however, the inclusion of spore-forming bacterial species (particularly probiotic bacteria, such as B. subtilis) is preferred. However, it is particularly preferred that pathogenic spore-forming bacteria not be included in the microbial composition.

[0113] The microbial species used in the microbial composition may be naturally occurring strains (i.e., wild-type) of the species (e.g., the species mentioned above) or genetically modified strains thereof, as indicated. The composition may contain a single strain of each species, multiple strains of each species, or a single strain of some species and multiple strains of other species. The microbial species included in the microbial composition may be obtained from any source, for example, a cell bank, such as the ATCC, as described above.

[0114] In some embodiments, the microbial composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 different microbial species. For example, the microbial composition can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following species: Bacillus licheniformis, Enterococcus faecium, Eubacterium ruminantium, Bacillus megaterium (e.g., Bacillus megaterium var. phosphaticum), Bacillus polymyxa, Streptomyces thermophilus, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus, Azotobacter vinelandii, and Azospirillum species. Preferably, the microbial composition includes at least 7, 10, or 12 of these species. In some embodiments, the microbial composition includes all 14 of these species, i.e., the microbial composition can include Bacillus licheniformis, Enterococcus faecium, Eubacterium ruminantium, Bacillus megaterium var. phosphaticum, Bacillus polymyxa, Streptomyces thermophilus, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus, Azotobacter vinelandii, and Azospirillum species.

[0115] The microbial composition can include at least one, two, or three species selected from Corynebacterium glitamicum, Aneurinbacillus danicus, Methylococcus capsulatus, and Bacillus firmus, hi some embodiments, the microbial composition includes all four of these species.

[0116] In some embodiments, the microbial composition comprises: (a) at least one species selected from Bacillus licheniformis, Enterococcus faecium, Eubacterium ruminantium, Bacillus megaterium var. phosphaticum, Bacillus polymyxa, Streptomyces thermophilus, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus, Azotobacter vinelandii, and Azospirillum species; and (b) at least one species selected from Corynebacterium glitamicum, Aneurinbacillus danicus, Methylococcus capsulatus, and Bacillus firmus;

[0117] In some embodiments, the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, or all, of the species listed in (a) and at least 2 or 3, or all, of the species listed in (b). In some embodiments, the composition comprises the following species: Bacillus licheniformis, Enterococcus faecium, Eubacterium ruminantium, Bacillus megaterium var. phosphaticum, Bacillus polymyxa, Streptomyces thermophilus, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus, Azotobacter vinelandii, Azospirillum species, Corynebacterium gritamicum, Aneurinbacillus danius, Methylococcus capsulatus, and Bacillus firmus.

[0118] Any suitable amount of the microbial composition can be applied to the organic material as determined by one of skill in the art. For example, the microbial composition can be applied in a total amount ranging from 1 to 10 g microbial composition / kg organic material, e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10 g microbial composition / kg organic material. In a preferred embodiment, 3 to 7 or 4 to 6 g microbial composition / kg organic material is used, for example, about 3, 3.5, 4, 4.5, 5, 5.5 or 6 g microbial composition / kg organic material.

[0119] The total amount of the microbial composition was approximately 5 × 10 9 ~5×10 11 cfu / kg of organic material, e.g., about 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 10 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , or 10 11 cfu / kg, ~5 × 10 11 , 4×10 11 , 3×10 11 , 2 × 10 11 , 10 11 , 9×10 10 , 8×10 10 , 7×10 10 , 6×10 10 , 5×10 10 , 4×10 10 , 3×10 10 , 2 × 1010 or 10 10 For example, the microbial composition may contain approximately 5×10 cfu / kg of total 9 ~10 10 cfu / kg, 10 10 ~5×10 10 cfu / kg, 5 × 10 10 ~10 11 cfu / kg, 10 11 ~5×10 11 cfu / kg or 10 10 ~10 11 It can be applied to organic materials in the range of cfu / kg.

[0120] Each microbial species present in the microbial composition may be present in any suitable amount so that a sufficient amount of the microorganisms is applied to the organic material to be effective in its processing. Each species in the microbial composition may be present in the same amount, or each may be present in different amounts, or different groups of species may be present in different amounts.

[0121] For example, the microbial species may be present in the microbial composition at a concentration of about 10 8 ~10 10 cfu / kg organic material, e.g., about 10 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 10 9 , 2 × 10 9 , 3×10 9 4×10 9 , or 5 x 10 9 cfu / kg organic material~, approx. 10 10 , 9×10 9 , 8×10 9 , 7×10 9 , 6×10 9 , 5×10 9 , 4×10 9 , 3×10 9 , 2 × 10 9 , 10 9 , 9×10 8, 8×10 8 , 7×10 8 , 6×10 8 , or 5 x 10 8 For example, the microbial species may be present in the microbial composition in an amount of about 10 cfu / kg of organic material. 8 ~10 9 cfu / kg, 5 × 10 8 ~10 9 cfu / kg, 5 × 10 8 ~5×10 9 cfu / kg, 10 9 ~5×10 9 cfu / kg, 10 9 ~10 10 cfu / kg, or 5 x 10 9 ~10 10 For example, the microbial species may be present in the microbial composition in an amount of about 10 cfu / kg. 8 , 5×10 8 , 10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 or 10 10 cfu / kg organic waste. The optimal amount of each microbial species in the microbial composition can be readily determined by one of skill in the art based, for example, on the composition to which the microbial composition is applied and the combination of species used in the organic material.

[0122] Cfu, as is standard in the art, means "colony forming unit."

[0123] Once the organic material has been contacted with the BSF larvae and microbial composition, it is incubated, i.e., the organic substrate is incubated with the BSF larvae and microbial composition.

[0124] During the incubation step, the organic substrate is processed by both the BSF larvae and the microbial composition. Processing of the organic substrate by the microbial composition is referred to herein as fermentation of the organic substrate.

[0125] The incubation step can be carried out under atmospheric conditions (e.g., at ambient temperature and humidity) or under controlled conditions. The organic material can be incubated at a temperature of about 20-35°C, e.g., 25-35°C, 26-34°C, 27-33°C, 28-32°C, 29-31°C, 25-30°C, 26-30°C, 27-30°C, 28-30°C, 29-30°C, 25-29°C, 25-28°C, 25-27°C, 26-29°C, 26-28°C, 26-27°C, 27-29°C, 27-28°C, or 28-29°C. Preferably, the organic material is incubated at 28-30°C, e.g., 28-29°C, 29-30°C, about 28°C, about 29°C, or about 30°C.

[0126] The relative humidity for incubation is preferably in the range of 60 to 100%, for example, 70 to 100, 80 to 100, 90 to 100, 60 to 90, 60 to 80, 60 to 70, 70 to 100, 70 to 90, 70 to 80, 75 to 85, 80 to 100, 80 to 90, or 90 to 100%. A preferred range of relative humidity is 75 to 85%, for example, about 80%.

[0127] Preferably, the organic material is incubated at about 28-30° C. and a relative humidity of about 75-80%.

[0128] Incubation is carried out under aerobic conditions because BSF larvae are aerobic organisms. Incubation can be carried out in ambient air. Incubation can be carried out under airflow, for example, at a rate of about 2,000 to 12,000 cfm, e.g., 3,000 to 11,000, 4,000 to 10,000, 5,000 to 9,000, or 6,000 to 8,000 cfm.

[0129] Advantageously, by fermenting the organic material while it is being processed by the BSF larvae, aerobic conditions can be provided without the use of any mechanical aeration devices, such as agitators or the like. Instead, the BSF larvae function as the aerators. Thus, in one embodiment, the method includes fermenting the organic material under aerobic conditions without the use of mechanical aerators. Alternatively, the method can be considered to include fermenting the organic material under aerobic conditions without the use of inanimate (i.e., non-living) aerators.

[0130] The incubation step can be carried out for any suitable length of time, as determined by one of skill in the art. Suitable lengths of time for the incubation step are described above (see discussion regarding length of degradation step).

[0131] In some embodiments, the incubation step involves parallel fermentation of two layers of organic material. In such embodiments, the two layers of organic material can be prepared separately for processing / fermentation by the addition of BSF larvae and / or a microbial composition, as described above. The second layer of material can then be placed on the first layer, such that the second layer is directly above the first layer, after which the incubation step is carried out. Alternatively, the first layer of organic material can be prepared for processing / fermentation, and the second layer of organic material can be placed on top of the first layer, and then the second layer can be prepared for processing / fermentation in situ. In this case, the upper second layer is not mixed with the lower first layer.

[0132] In some embodiments, both the first and second layers contain BSF larvae. In preferred embodiments, only the first (lower) layer contains BSF larvae. Typically, both the first and second layers contain a microbial composition for fermentation.

[0133] When a two-layer fermentation step is performed, the organic material forming the first layer can be the same as or different from the organic material forming the second layer. Preferably, the first and second layers are formed of different organic materials. In this case, the first and second layers can be of approximately the same density, or the second (top) layer can be less dense than the first layer.

[0134] Similarly, the same microbial composition can be used to ferment the organic material in both layers, or different microbial compositions can be used in each layer. In certain embodiments, the first layer is fermented with any microbial composition, as discussed above, and the second (top) layer is fermented with a microbial composition comprising Aspergillus oryzae. A. oryzae has been found to grow particularly well on soybean pulp (okara) and can be used as the top layer in some embodiments.

[0135] The use of a two-phase fermentation process has been found to improve the performance of BSF larvae. Processing of the two layers by BSF larvae during the incubation step usually results in homogenization of the two layers, resulting in the production of a single, homogenous product.

[0136] In some embodiments, a cooling pad is used during the incubation step. The cooling pad consists of or comprises a heat-absorbing material. The cooling pad can be placed near (e.g., opposite or near) the fermentation material (e.g., in the same room or building where fermentation is carried out) to prevent the fermentation material from overheating or to cool the fermentation site. In certain embodiments, the heat-absorbing material is water.

[0137] The cooling pad may be an evaporative cooling pad, particularly one that uses one or more sheets of cellulose-based material, such as cardboard or paper (e.g., kraft paper). Such cooling pads may commonly include corrugated sheets that may be adhered to one another using, for example, glue. The sheets may be treated with resins or other agents to provide water resistance, prevent corrosion, and / or improve the durability or lifespan of the cardboard. Such cooling pads function by flowing water through the cellulose-based sheet. The water extracts heat from the air and evaporates. Evaporative cooling sheets of this type are readily available commercially.

[0138] The cooling pad may include a water-repellent container (e.g., a metal container) that encases a sheet of cellulose-based material. A window in the container provides a passageway from the cellulose-based sheet to the environment. The cooling pad may include a bottom sump to collect water that runs down the cellulose-based sheet, and a pump to pump the water back to the top of the pad for reuse. The water may be replaced and / or replenished periodically.

[0139] Fermentation can result in the production of foul-smelling or toxic gases, especially ammonia, which can be dissolved in the water of the cooling pad and removed from the environment. This avoids the buildup of gases that could otherwise have a negative effect on the BSF larvae and microorganisms used in the fermentation, and can also improve the odour of the fermentation process.

[0140] Thus, the fermentation step may be carried out using an evaporative cooling pad to remove heat and / or gaseous by-products of the fermentation process from the environment.

[0141] Surprisingly, the use of cooling pads in this manner can make the step of processing organic materials a carbon-neutral or even carbon-negative process. Without being bound by theory, alkalinization of the water in the cooling pad by dissolving ammonia therein increases the solubility of CO2 in the water, so that excess CO2 can be withdrawn from the environment into the water.

[0142] The processing step of the organic material includes fermentation by the microorganisms and consumption by the BSF larvae to produce a product composition. The product composition includes the fermented organic material, the BSF larvae biomass, and the microbial biomass, as discussed above. The product composition may be an animal feed composition, as discussed above. The product composition typically further includes production residues (from the BSF larvae), particularly fermentation production residues (i.e., fermented BSF production residues), also as discussed above. The product composition may also include nutrients, postbiotics, etc., such as amino acids, vitamins, antioxidants, and / or antimicrobial compounds, produced by the microbial composition, as discussed above.

[0143] At the end of the incubation step, the product composition may be further processed to improve its suitability for use as an animal feed, for example to improve its transportability or storability. The product composition may be blended (as discussed above with respect to the optional blending step prior to the fermentation step). Thus, the method may include the further step of blending the product composition. The blending step may also kill BSF larvae.

[0144] In the methods of the present invention, typically only a single fermentation step is performed. Thus, once the incubation step is complete, no additional microbial composition is added. If a blending step is performed after the incubation step, no subsequent incubation or fermentation steps are typically performed.

[0145] Preservatives can be added to the product composition to increase its shelf life and prevent spoilage. Suitable preservatives are known in the art and are discussed above. If the product composition is also blended, the preservative can be added before or after the compositions are blended.

[0146] The product composition may be subjected to a sterilization step (i.e., the method may further comprise a sterilization step). In the sterilization step, the product composition is treated to kill any living organisms therein, particularly BSF larvae (if the blending step is not performed or if some larvae survive the process), and microorganisms from the microbial composition. The sterilization step kills all or essentially all living organisms in the product composition, although viable bacterial endospores may remain. The sterilization step may kill all living organisms in the product composition and destroy (i.e., render non-viable) any bacterial endospores in the composition.

[0147] The sterilization step can be carried out by any effective method known to those skilled in the art, but is preferably carried out by heat treatment of the product composition. If the product composition is also blended, the blending step can be carried out before, after, or simultaneously with the sterilization step. Similarly, if a preservative is added, it can be added before or after the sterilization step.

[0148] When the product composition is heat-treated, a sufficiently high temperature is applied to the composition for a sufficient length of time to sterilize the composition. For example, a temperature of at least about 50, 60, 70, 80, 90, or 100°C may be applied for at least 0.5, 1, 1.5, 2, 2.5, or 3 hours, or more. In some embodiments, a temperature of about 75-85°C (e.g., about 80°C) is applied to the product composition for 1.5-2.5 hours (e.g., about 2 hours). In other embodiments, temperatures of 80-100°C may be used, e.g., 80-90°C, 80-95°C, 85-100°C, 85-95°C, or 90-100°C.

[0149] If heat sterilization is performed, the heating step can also function as a drying step, drying and removing moisture from the product composition, which can have a low moisture content, as described above with respect to the animal feed compositions of the present invention.

[0150] In some embodiments, a two-step heating process is performed. In such embodiments, the first step is a preheating step, and the second step is a sterilization / drying step as described above. The preheating step is performed at a lower temperature than the drying step; for example, the preheating step can be performed at a temperature of about 50-70°C, e.g., 50-60°C, 50-65°C, 55-65°C, or 60-70°C. The preheating step can be performed for any suitable period of time, e.g., at least 0.5, 1, 1.5, 2, 2.5, or 3 hours, or more. The drying step performed after the preheating step can be performed at a temperature of, for example, 80-100°C, e.g., 80-90°C, 80-95°C, 85-100°C, 85-95°C, or 90-100°C, as described above.

[0151] The purpose of the preheating step is to stimulate sporulation of spore-forming probiotic bacteria (e.g., B. subtilis, B. licheniformis, B. megaterium, or B. polymyxa) to form endospores that can survive the higher temperatures used in the drying step. Thus, the preheating step is carried out at a temperature high enough to induce heat stress in such species to promote sporulation, but not so high that the bacteria are killed before sporulation is complete.

[0152] If a heat treatment step is performed, it can be at the end of the incubation step or immediately after the blending step (if a blending step is performed). By "immediately" we mean that no separate step is performed between the blending step and the heating step, and in particular no additional fermentation step is performed between the blending step and the heating step. Any interval between the blending step and the heating step is also typically short, allowing for progress between the steps, e.g., less than 24, 12, 10, 8, 6, 4, or 2 hours.

[0153] If RS2 starch is included in the organic material that is fermented by the microbial composition, the final heat treatment step of fermentation advantageously converts any remaining RS2 starch (which is particularly suitable for microbial fermentation as described above) into RS3 starch (which is beneficial to animals as described above) that is included in the product composition.

[0154] The resulting composition may be pelleted or pelletized (the terms are used interchangeably herein), i.e., formed into pellets. Pelleted animal feed is often preferred in aquaculture because pelleted food is associated with higher quality, lower pathogen content, lower dust levels, and easier handling than powdered feed. Feed pelleting processes are known in the art and typically involve moistening dry feed with the aid of steam injection and compressing it into pellets.

[0155] The product composition may have the nutritional composition (in terms of protein, carbohydrate, and fat content) described above for the animal feed composition. The product composition may optionally be defatted. The defatting step may be carried out by any method known in the art. For example, the defatting step may be accomplished by a screw press.

[0156] As demonstrated in the Examples, the methods of the present invention have high product yields in terms of their yield in terms of dry weight of the product composition relative to the starting dry weight (i.e., the combined dry weight of the substrate organic material and the BSF larvae / eggs and microbial composition applied thereto). For example, the methods of the present invention preferably produce a product composition having a dry weight that is at least 75, 80, 85, 90, or 95% of the starting dry weight.

[0157] The method of the present invention may include the further step of formulating the product composition into an animal feed. This formulating step may include mixing the product composition with one or more other ingredients. For example, if the product composition is used as a supplement to another (e.g., existing) animal feed, this step includes mixing the product composition with the other animal feed. The animal feed may be for (i.e., suitable for, and potentially designed for) any animal of interest. Typically, the animal may be a farmed animal, such as a fish or crustacean, as discussed above.

[0158] In a third aspect, the present invention provides the use of BSF larvae and a microbial composition for decomposing organic material, said use being as defined above in the method of the invention, thereby producing a product composition as defined above.

[0159] In a fourth aspect, the present invention provides a product composition obtainable (i.e., obtainable) from the method or use of the invention. Preferably, the product composition of the invention is obtained by the method or use of the invention as described above.

[0160] In a fifth aspect, the present invention provides a composition for decomposing organic material, the composition comprising the microbial composition defined above and black soldier fly larvae or black soldier fly eggs. The composition can be used to decompose organic material as described above in the methods of the present invention. Thus, the composition preferably comprises black soldier fly larvae or eggs and the microbial composition in ratios suitable for use in the methods of the present invention.

[0161] In a sixth aspect, the present invention provides a kit for decomposing organic material comprising black soldier fly larvae or black soldier fly eggs and a microbial composition, as defined above. In the kit, the BSF larvae / eggs may be provided separately from the microbial composition (e.g., in separate pouches or compartments) or combined with the microbial composition in a single mixture.

[0162] The invention may be better understood by reference to the following non-limiting examples and drawings. [Brief explanation of the drawings]

[0163] [Figure 1] FIG. 1 depicts the survival rate of farmed shrimp fed either a diet of standard shrimp food (control) or an experimental diet comprising standard shrimp food supplemented with 20% w / w of the animal feed composition of the present invention. [Figure 2] 2 depicts the relative survival rates of shrimp fed either a diet of standard shrimp food (control) or an experimental diet comprising standard shrimp food supplemented with 2% w / w of an animal feed composition of the present invention ("Full Circle Product"). For each day, the left bar represents the survival rate of the control and the right bar represents the survival rate of the experimental. Error bars indicate standard deviation. [Figure 3] 3 shows the average weight gain of Pacific white shrimp after 45 days of being fed either a control diet of standard shrimp food or a standard shrimp food supplemented with 2% w / w of an animal feed composition of the present invention ("complete rotation product"). Error bars indicate standard deviation. [Figure 4] FIG. 4 depicts representative Pacific white shrimp after 45 days of being fed either a control standard shrimp diet or standard shrimp diet supplemented with 2% w / w of an animal feed composition of the present invention (the "complete rotation product"). [Figure 5] 5 shows the feed conversion ratio of Pacific white shrimp after 45 days of being fed either a diet of control standard shrimp food or standard shrimp food supplemented with 2% w / w of an animal feed composition of the present invention ("complete rotation product"). Error bars indicate standard deviation. [Example]

[0164] (Example) (Example 1) Yield test The following organic samples (listed in Table 1) were used to determine the yield of dry product composition relative to the starting dry weight.

[0165] [Table 1] Table 1: Samples used in the yield experiment

[0166] BSF larvae were applied to the substrate along with a microbial composition containing a mixture of digestive microorganisms, protein-enhancing microorganisms, lactic acid bacteria, and nitrogen-fixing bacteria. The substrate was then incubated for 6 to 12 days (as shown in Table 2 below). The results are listed in Table 2 below.

[0167] [Table 2] Table 2: Experimental organic waste decomposition yields 1 Dry mass yield as a percentage of the original dry mass The average dry mass yield of the experiment was 86.52%, which was considered an excellent yield.

[0168] (Example 2) Nutrient content test As in Example 1 above, degradation experiments were conducted to investigate the nutrient content of animal feed compositions obtained by processing Amygdalus, Salmonid, Elaeis (oil palm), and a mixture of 75% Colocasia esculenta (taro) and 25% pig blood. The results are presented in Table 3.

[0169] [Table 3] Table 3: Nutrient content of animal feed compositions obtained by processing different substrates. PB = porcine blood.

[0170] As expected, the different substrates had substantially different nutrient compositions, and the starting nutrient composition was reflected in the product nutrient composition: in no case was there a significant decrease in protein content, and in some cases there was a significant increase in protein content.

[0171] The products obtained from the Amygdalus, Elaeis, and Salmonide substrates were defatted by screw press, leaving a total fat content of only 5% for each product. For the Amygdalus product, this gave a protein content of 32% and a carbohydrate content of 40.2% on a dry matter basis, for the Elaeis product, this gave a protein content of 16.9% and a carbohydrate content of 55% on a dry matter basis, and for the Salmonide product, this gave a protein content of 75.9% and a carbohydrate content of 11.3% on a dry matter basis.

[0172] (Example 3) Feeding white shrimp with the animal feed composition of the present invention An animal feed composition was produced by processing palm decanter cake with a BSF larvae and microbial composition, as used in Examples 1 and 2 above. The process was carried out in Thailand, and after application of the BSF larvae and microbial composition to the palm decanter cake, the substrate was incubated under ambient conditions for 10 days. At the end of the process, the mixture was blended, dried, and sterilized by heating at 80°C for 2 hours.

[0173] The resulting animal feed composition was added to a CP basal shrimp diet (CP Prima, Indonesia) at a level of 20% w / w. Shrimp were fed either the CP basal shrimp diet alone or a diet supplemented with 20% of the composition of the present invention, as described below, and their growth was compared.

[0174] Shrimp sample Fifty-eight juvenile white shrimp with an initial weight of approximately 3 g were obtained from a white shrimp farm in Chachoengsao Province, Thailand. Before the feeding experiment began, the shrimp were acclimated for 2 days in rearing water with a salinity of 10 ppt and a pH of 8.

[0175] Feeding test Shrimp (total initial weight of approximately 190 g) were divided into two tanks (0.6 m x 0.45 m x 0.8 m), each containing 29 shrimp, and reared in 10 ppt artificial seawater (Marinium®, Thailand) with continuous aeration at 29-30°C. Water quality parameters, such as pH, alkalinity, and ammonia, were measured every other day. Animals were fed 6% of their body weight of their respective diets using an automatic feeder set to release four meals every 24 hours. The study was conducted over a 24-day period, during which mortality was recorded in both groups.

[0176] Results and Discussion During the test, the number of shrimp mortality was recorded. A better survival rate was observed in the group fed with 20% of the animal feed composition of the present invention in the regular diet (Figure 1 and Table 4), but this was not statistically significant. To ensure proper culture conditions, water quality parameters such as pH, alkalinity, and ammonia (NH3) were recorded using commercially available kits, and the results showed that the water quality parameters were within normal values.

[0177] The results demonstrate, at a minimum, that the animal feed composition of the present invention is suitable for feeding to white shrimp (i.e., the animal feed composition of the present invention is not toxic to white shrimp). The animal feed composition may enhance the survival of white shrimp, but larger feeding trials are needed to determine this.

[0178] [Table 4] Table 4: Survival of white shrimp. "Control survival" indicates the survival of shrimp in the control group fed with CP basal shrimp diet, and "experimental survival" indicates the survival of shrimp in the experimental group fed with CP basal shrimp diet supplemented with the animal feed composition of the present invention.

[0179] (Example 4) Large-scale shrimp growth test Growth trials were conducted at the Aquaculture Business Research Center (ABRC), Faculty of Fisheries, Kasetsart University, 50 Phaholyothin Rd., Chatuchak, Bangkok, 10900, Thailand.

[0180] method This study used specific pathogen-free white shrimp, 2-4g in size. A total of 400 shrimp were transported from the farm to the laboratory at the Aquaculture Research Center, Faculty of Fisheries, Kasetsart University. The shrimp were acclimated in fiberglass tanks for 14 days.

[0181] In the experiment, a total of 8,500 liters of tanks were used to grow shrimp in seawater with a salinity of 25-30 ppt. Shrimp were stocked at a density of 40 shrimp per tank. Two experimental groups with two replicates were studied as follows: Diet 1: Control. Diet 2: Supplemented with 2% of the feed composition of the present invention as a feed additive. The compositions of the control and supplemented media are listed in Table 5 below.

[0182] Shrimp were fed a specific shrimp diet at a satiety rate four times daily. The feeding rate was adjusted according to shrimp weight throughout the 45-day experimental period. Water quality parameters, such as pH, dissolved oxygen (DO), alkalinity, ammonia, and nitrite, were maintained at optimal levels for shrimp growth and analyzed weekly throughout the experiment. Survival rates were recorded every two weeks. Shrimp were weighed at the end of the experiment. Feed conversion ratio (FCR), protein efficiency (PER), and feed efficiency (FER) were determined at the end of the experiment. At the end of the experiment, all detailed data from all experimental groups were statistically compared using t-tests.

[0183] [Table 5] Table 5: Composition of control shrimp feed and shrimp feed supplemented with 2% of the product of the present invention ("complete rotation product")

[0184] result There was no difference in survival rate of shrimp fed different diets throughout the test period (Table 6 and Figure 2). Shrimp fed diets supplemented with 2% of the feed additive of the present invention gained the most weight (Table 7 and Figures 3-4). This group gained significantly more shrimp weight than the control group. Regarding feed conversion ratio (FCR), shrimp fed diets supplemented with 2% of the feed additive of the present invention had a significantly lower FCR than the control group, indicating a greater efficiency in converting supplemented feed into shrimp weight (Table 7 and Figure 5). There was no difference in FER and PER between the experimental and control groups (Table 7).

[0185] [Table 6] Table 6: Mean survival rates of Pacific white shrimp at 14, 28, and 45 days after feeding different diets. Data are expressed as mean ± standard deviation. Mean values ​​in the same column with different superscripts are significantly different from each other (p<0.05).

[0186] [Table 7] Table 7: Mean weight gain, feed conversion ratio (FCR), feed efficiency (FER), and protein efficiency (PER) of Pacific white shrimp after 45 days of feeding different diets. Data are expressed as mean ± standard deviation. Means in the same column with different superscripts are significantly different from each other (p<0.05).

Claims

1. An animal feed composition comprising fermented organic material, black soldier fly biomass, fermented black soldier fly production residues, and microbial biomass.

2. 10. The composition of claim 1, wherein the composition comprises about 5-25% w / w black soldier fly biomass and about 0.05-5% w / w microbial biomass.

3. 3. The composition of claim 1 or 2, wherein the composition is crushed, ground or blended, optionally wherein the composition is powdered.

4. The composition according to any one of claims 1 to 3, wherein the composition has a water content of 10% (v / v) or less.

5. The composition of any one of claims 1 to 4, wherein the black soldier fly biomass comprises dead black soldier fly larvae.

6. The composition of any one of claims 1 to 5, wherein the composition is free of living organisms, and optionally the composition is sterile.

7. The composition of any one of claims 1 to 6, wherein the microbial biomass comprises bacterial biomass and fungal biomass.

8. The microbial biomass comprises: (i) at least one microorganism selected from Bacillus licheniformis, Enterococcus faecium, Eubacterium ruminantium, Bacillus megaterium var. phosphaticum, Bacillus polymyxa, and Streptomyces thermophilus; (ii) at least one microorganism selected from Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, and Streptomyces thermophilus; and (iii) at least one microorganism selected from Bacillus subtilis, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus, Aspergillus oryzae, Azotobacter vinelandii, Azospirillum species, Bacillus megaterium var. phosphaticum, and Bacillus polymyxa; The composition of any one of claims 1 to 7, comprising:

9. 9. The composition of claim 8, further comprising at least one of Corynebacterium glitamicum, Aneurinbacillus danicus, Methylococcus capsulatus, and Bacillus firmus.

10. 10. The composition of claim 8 or 9, comprising Bacillus subtilis.

11. the microbial biomass comprises at least four microbial species selected from Bacillus licheniformis, Enterococcus faecium, Eubacterium ruminantium, Bacillus megaterium var. phosphaticum, Bacillus polymyxa, Streptomyces thermophilus, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus, Azotobacter vinelandii, and Azospirillum species; Preferably, the microbial biomass comprises at least 7, at least 10, or at least 12 of the microbial species, or all of the microbial species. The composition according to any one of claims 8 to 10.

12. 12. The composition of claim 11, further comprising at least two, at least three, or preferably all of Corynebacterium glitamicum, Aneurinbacillus danicus, Methylococcus capsulatus, and Bacillus firmus.

13. A composition according to any one of claims 1 to 12, wherein the composition comprises endospores of one or more spore-forming species of bacteria, preferably spores of B. subtilis.

14. The composition according to any one of claims 1 to 13, wherein the composition comprises resistant starch type 3 (RS3 starch).

15. 15. The composition of any one of claims 1 to 14, wherein the fermented organic material is the product of fermenting food waste.

16. The composition of any one of claims 1 to 13, wherein the composition further comprises a preservative and / or is pelletized.

17. 1. A method for decomposing organic material, the method comprising the steps of contacting the organic material with black soldier fly larvae and a microbial composition comprising at least one microorganism capable of fermenting the organic material, and incubating the organic material with the black soldier fly larvae and the microbial composition; the method producing a product composition comprising fermented organic material, black soldier fly larval biomass, and microbial biomass; The method.

18. The microbial composition comprises: (i) at least one digestive microorganism, wherein said digestive microorganism secretes digestive enzymes; (ii) at least one protein-enhancing microorganism, wherein the protein-enhancing microorganism increases the protein content of the product composition; and (iii) at least one microorganism that increases the nutritional and / or health value of the product composition and / or reduces the environmental impact of the method; 18. The method of claim 17, comprising:

19. 19. The method of claim 17 or 18, wherein the organic material is food waste.

20. 20. The method of any one of claims 17 to 19, comprising the step of crushing, grinding and / or blending the organic material prior to applying the black soldier fly larvae and microbial composition.

21. A method according to any one of claims 17 to 20, wherein the product composition is an animal feed or a component of an animal feed.

22. The method of any one of claims 17 to 21, wherein the microbial composition comprises at least one bacterial species and at least one fungal species.

23. 23. The method according to any one of claims 18 to 22, wherein the proteases and carbohydrases are secreted by at least one digestive microorganism.

24. 24. The method of claim 23, wherein the cellulase, xylanase, and / or amylase is secreted by at least one digestive microorganism.

25. (iii) at least one microorganism (a) Decompose toxins, (b) secrete antimicrobial compounds; (c) a lactic acid-producing microorganism; (d) producing antioxidants and / or vitamins; (e) secrete amino acids, preferably lysine and / or glutamate; (f) is a nitrogen-fixing microorganism; and / or (g) is a methanotroph; The method according to any one of claims 18 to 24.

26. 26. The method of any one of claims 18 to 25, wherein the at least one digestive microorganism is selected from Bacillus licheniformis, Enterococcus faecium, Eubacterium ruminantium, Bacillus megaterium var. phosphaticum, Bacillus polymyxa, and Streptomyces thermophilus.

27. 27. The method of any one of claims 18 to 26, wherein the at least one protein-enhanced microorganism is selected from Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, and Streptomyces thermophilus.

28. 28. The method of any one of claims 18 to 27, wherein the at least one microorganism in (iii) is selected from Bacillus subtilis, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus, Aspergillus oryzae, Azotobacter vinelandii, Azospirillum species, Bacillus megaterium var. phosphaticum, Bacillus polymyxa, Corynebacterium gritamicum, Aneurinbacillus danicus, Methylococcus capsulatus, and Bacillus firmus.

29. 29. The method according to any one of claims 17 to 28, wherein the microbial composition comprises at least 4 microbial species, preferably at least 7, at least 10, at least 14, or at least 18 microbial species.

30. 30. The method of claim 29, wherein the microbial composition comprises Bacillus licheniformis, Enterococcus faecium, Eubacterium ruminantium, Bacillus megaterium var. phosphaticum, Bacillus polymyxa, Streptomyces thermophilus, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus, Azotobacter vinelandii, and Azospirillum species.

31. 31. The method of claim 30, wherein the microbial composition further comprises Corynebacterium glythamicum, Aneurinbacillus danicus, Methylococcus capsulatus, and Bacillus firmus.

32. The method of any one of claims 17 to 31, wherein the product composition further comprises a production residue, preferably a fermentation production residue.

33. 33. The method of any one of claims 17 to 32, wherein the organic material is incubated with the black soldier fly larvae and microbial composition for about 5 to 14 days.

34. (i) contacting a first layer of organic material with black soldier fly larvae and a first microbial composition comprising at least one microorganism capable of fermenting the organic material; (ii) contacting the second layer of organic material with a second microbial composition comprising at least one microorganism capable of fermenting the organic material; and (iii) incubating the organic material with black soldier fly larvae and the microbial composition; the second layer of organic material overlies the first layer of organic material; The method according to any one of claims 17 to 33.

35. 35. The method of claim 34, wherein the first and second layers of organic material are different.

36. 36. The method of claim 34 or 35, wherein the second microbial composition comprises Aspergillus oryzae.

37. The method of any one of claims 17 to 36, wherein a cooling pad containing water is used during incubation.

38. 38. The method of claim 37, wherein the cooling pad is an evaporative cooling pad comprising at least one sheet of a cellulose-based material.

39. (i) grinding, crushing, and / or blending the product composition; (ii) adding a preservative to the product composition; and / or (iii) pelletizing the product composition The method of any one of claims 17 to 38, further comprising:

40. The method of any one of claims 17 to 39, further comprising a sterilization step.

41. The sterilization step is a heating step, and the heating step comprises: (i) preheating the product composition at a first temperature, wherein the first temperature stimulates sporulation of spore-forming bacteria in the microbial composition; and (ii) heating the product composition at a second temperature, the second temperature being greater than the first temperature and rendering the product composition free of viable organisms; Including, after the heating step, the product composition comprises viable bacterial endospores; 41. The method of claim 40.

42. 42. The method of any one of claims 17 to 41, wherein the dry mass yield in the product composition relative to the starting dry mass is at least 75%, preferably at least 80%, 85% or 90%.

43. further comprising the step of incorporating said product composition into an animal feed, optionally including the step of mixing said product composition with one or more other ingredients; Optionally, the animal feed is for fish or crustaceans.

43. The method according to any one of claims 17 to 42.

44. 42. Use of black soldier fly larvae and a microbial composition as defined in any one of claims 17 to 31 for the decomposition of organic material, thereby producing a product composition, optionally wherein the use, organic material, and / or product composition is as defined in any one of claims 19 to 21 or claims 32 to 42.

45. A product composition obtainable from the method defined in any one of claims 17 to 44.

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