Cross-Reference to Related Applications

[001] This application claims the benefit of priority to U.S. Provisional Application No. 62/657,309, filed April 13, 2018, and U.S. Provisional Application No. 62/731,988, filed September 17, 2018, the disclosures of which are each expressly incorporated by reference herein in their entireties.

Background of the Invention

[002] There is an on-going need for new feed ingredients of improved feed qualities, including higher crude protein content and higher content of essential amino acids.

Summary of the Invention

[003] According to an embodiment, provided is a feed ingredient comprising at least 20% by weight Clostridium tyrobutyricum, wherein said Clostridium tyrobutyricum comprises at least 70% of dry weight crude protein and optionally at least 7% of dry weight lysine and/or at least 1% of dry weight methionine. According to an embodiment, said Clostridium tyrobutyricum is genetically modified. According to an embodiment, said Clostridium tyrobutyricum is adaptively evolved.

[004] According to an embodiment, said feed ingredient further comprises at least 20% by weight Clostridium pasteurianum. According to an embodiment, said feed ingredient further comprises at least 20% by weight an acetogenic bacteria. According to an embodiment, said feed ingredient further comprises at least 20% by weight Butyribacterium methylotrophicum. According to an embodiment, said feed ingredient further comprises at least 10% by weight Clostridium pasteurianum and at least 10% by weight Butyribacterium methylotrophicum.

[005] According to an embodiment, said feed ingredient is characterized by water solubility greater than 25% by weight. According to an embodiment, said Clostridium tyrobutyricum is sporulated. According to an embodiment, said Clostridium tyrobutyricum is in clostridial form.

[006] According to an embodiment, provided is an animal feed comprising said feed ingredient. According to an embodiment, said feed ingredient further comprises at least one of butyric acid and propionic acid. According to an embodiment, provided is an aqua feed comprising said feed ingredient.

[007] According to an embodiment, provided is a probiotic preparation comprising said feed ingredient. According to an embodiment, provided is a poultry probiotic preparation comprising said feed ingredient. According to an embodiment, provided is a swine probiotic preparation comprising said feed ingredient. According to an embodiment, provided is a companion animal probiotic preparation comprising said feed ingredient. According to an embodiment, provided is a human probiotic preparation comprising said feed ingredient. According to an embodiment, said probiotic preparation further comprising at least one of Enterococcus faecium, Bacillus subtilis, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus plantarum, Pediococcus acidilactici and Clostridium butyricum.

[008] According to an embodiment, provided is a method for producing said feed ingredient, comprising culturing in a fermentation medium Clostridium tyrobutyricum, whereby Clostridium tyrobutyricum cell mass is formed. According to an embodiment, said fermentation medium is kept at a pH greater than 5.25.

[009] According to an embodiment, said method further comprises co-culturing Clostridium pasteurianum, whereby said cell mass further comprises Clostridium pasteurianum. According to an embodiment, said method further comprises co-culturing an acetogenic bacteria, whereby said cell mass further comprises an acetogenic bacteria. According to an embodiment, said method further comprises co-culturing Butyribacterium methylotrophicum, whereby said cell mass further comprises Butyribacterium methylotrophicum. According to an embodiment, said method further comprises co-culturing Clostridium pasteurianum and Butyribacterium methylotrophicum, whereby said cell mass further comprises Clostridium pasteurianum and Butyribacterium methylotrophicum. According to an embodiment, said method further comprises mechanical separation of formed cell mass and at least one of washing, drying, pulverizing and pelletizing.

[OOIO] According to an embodiment, provided is a method for maintaining the good health of an animal comprising feeding said animal an animal feed comprising said feed ingredient. According to an embodiment, said animal is selected from the group consisting of larvae, fry, juvenile, and adult of crustacean, finfish and shellfish, poultry, swine, companion animals, other ruminants and humans.According to an embodiment, said maintaining good health comprises at least one of reduced susceptibility to enteric pathogens, reduction in bowel inflammation, reduced mortality, improved feed conversion and improved weight gain.

[ooil] According to an embodiment, provided is a method for maintaining the good health of an animal comprising feeding said animal a probiotic preparation comprising said feed ingredient. According to an embodiment, said animal is selected from the group consisting of larvae, fry, juvenile, and adult of crustacean, finfish and shellfish, poultry, swine, companion animals, other ruminants and humans. According to an embodiment, said maintaining good health comprises at least one of reduced susceptibility to enteric pathogens, reduction in bowel inflammation, reduced mortality, improved feed conversion and improved weight gain.

Detailed Description of the Invention

[0012] The particulars shown herein are by way of example and for purposes of illustrative discussion of the various embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0013] The present invention will now be described by reference to more detailed embodiments. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms“a,” “an,” and“the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. [0015] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term“about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0016] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0017] Additional advantages of the invention will be set forth in part in the description, which follows, and in part will be obvious from the description, or may be learned by practice of the invention. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0018] According to an embodiment, provided is a feed ingredient comprising at least 20% by weight (20%wt) Clostridium tyrobutyricum, wherein said Clostridium tyrobutyricum comprises at least 70% of dry weight crude protein and optionally at least 7% of dry weight lysine and/or at least 1% of dry weight methionine. According to an embodiment, provided is a feed ingredient comprises at least 30%wt, at least 40%wt, at least 50%wt, at least 60%wt, at least 70%wt, at least 80%wt, at least 90%wt or at least 95%wt Clostridium tyrobutyricum. According to an embodiment, said Clostridium tyrobutyricum comprises at least 75% of dry weight crude protein, at least 80%, at least 85% or at least 90%. According to an embodiment, said Clostridium tyrobutyricum comprises at least 7% of dry weight lysine, at least 7.5%, at least 8%, at least 8.5% or at least 9%. According to an embodiment, said Clostridium tyrobutyricum comprises at least 1 % of dry weight methionine, at least 1.5%, at least 2%, or at least 2.5%.

[0019] According to an embodiment, said feed ingredient further comprises at least 20%wt Clostridium pasteurianum, at least 30%wt, at least 40%wt, at least 50%wt, at least 60%wt or at least 70%wt. According to an embodiment, said feed ingredient comprises at least l%wt glycogen, at least 5%wt, at least l0%wt, at least l5%wt, at least 20%wt, at least 25%wt or at least 30%wt. According to an embodiment, said feed ingredient further comprises at least 20%wt Butyribacterium methylotrophicum, at least 30%wt, at least 40%wt or at least 50%wt.

[0020] According to an embodiment, said feed ingredient further comprises at least 20%wt of acetogenic bacteria, at least 30%wt, at least 40%wt, at least 50%wt, at least 60%wt or at least 70%wt.

[0021] According to an embodiment, said feed ingredient further comprises at least 20%wt Butyribacterium methylotrophicum at least 30%wt, at least 40%wt, at least 50%wt, at least 60%wt or at least 70%wt.

[ ] According to an embodiment, said feed ingredient further comprises at least l0%wt Clostridium pasteurianum, at least 20%wt or at least 30%wt and at least 10% by weight Butyribacterium methylotrophicum, at least 20%wt or at least 30%wt.

[0023] According to an embodiment, said animal feed is characterized by water solubility greater than 25%wt, greater than 40%wt, greater than 60%wt, greater than 80%wt, or greater than 80%wt.

[0024] According to an embodiment, said Clostridium tyrobutyricum is sporulated. According to an embodiment, said Clostridium tyrobutyricum is in clostridial form.

[0025] According to an embodiment, said Clostridium tyrobutyricum is genetically modified. According to an embodiment, said Clostridium tyrobutyricum is genetically modified by removing or inserting genetic material out of or into the chromosome. According to an embodiment, said genetic modification is not plasmid-based. According to an embodiment, said Clostridium tyrobutyricum is genetically modified to produce propionic acid. According to an embodiment, said Clostridium tyrobutyricum is genetically modified to reduce the production of graulose. According to an embodiment, said Clostridium tyrobutyricum is adaptively evolved. According to an embodiment, said Clostridium tyrobutyricum is adaptively evolved to have a higher growth rate. According to an embodiment, said Clostridium tyrobutyricum is adaptively evolved to have an improved substrate utilization rate. According to an embodiment, said Clostridium tyrobutyricum is adaptively evolved to have a higher crude protein content.

[0026] According to an embodiment, said clostridial form is characterized by the formation of granulose. According to an embodiment, said granulose comprises amylopectin and optionally also amylose.

[0027] According to an embodiment, provided is an animal feed comprising said feed ingredient. According to an embodiment, said feed ingredient forms at least 2%wt of said animal feed, at least 4%wt, at least 6%wt, at least 8%wt or at least l0%wt. According to an embodiment, said animal feed further comprises at least one of butyric acid and propionic acid. According to an embodiment, said animal feed further comprises amylopectin and amylose with amylopectin to amylose weight/weight ratio of about 1 to 4.

[0028] According to an embodiment, provided is an aqua feed comprising said feed ingredient. According to an embodiment, said feed ingredient forms at least 2%wt of said aqua feed, at least 4%wt, at least 6%wt, at least 8%wt or at least l0%wt.

[0029] According to an embodiment, provided is a probiotic preparation comprising said feed ingredient. According to an embodiment, said feed ingredient forms at least 20%wt of said probiotic preparation, at least 40%wt, at least 60%wt, at least 80%wt or at least 90%wt. According to an embodiment, said probiotic preparation further comprising at least one of Enterococcus faecium, Bacillus subtilis, Bacillus amyloliquefaciens, Lactobacillus acidophilus, Lactobacillus plantarum, Pediococcus acidilactici and Clostridium butyricum.

[0030] According to an embodiment, provided is a poultry probiotic preparation comprising said feed ingredient. According to an embodiment, said feed ingredient forms at least 20%wt of said poultry probiotic preparation, at least 40%wt, at least 60%wt, at least 80%wt or at least 90%wt. [0031] According to an embodiment, provided is a human probiotic preparation comprising said feed ingredient. According to an embodiment, said feed ingredient forms at least 20%wt of said human probiotic preparation, at least 40%wt, at least 60%wt, at least 80%wt or at least 90%wt.

[0032] According to an embodiment, provided is a method for producing said feed ingredient, comprising culturing in a fermentation medium Clostridium tyrobutyricum, whereby fermentation broth comprising Clostridium tyrobutyricum cell mass is formed. According to an embodiment, said fermentation broth further comprises at least one of acetic acid and butyric acid. According to an embodiment, said fermentation medium is kept at a pH greater than 5.25, greater than 5.30, greater than 5.35, greater than 5.40, greater than 5.45, or greater than 5.50.

[0033] According to an embodiment, said method comprises co-culturing Clostridium pasteurianum, whereby said cell mass further comprises Clostridium pasteurianum. According to an embodiment, said method comprises co-culturing an acetogenic bacteria, whereby said cell mass further comprises an acetogenic bacteria. According to an embodiment, said method comprises co-culturing Butyribacterium methylotrophicum, whereby said cell mass further comprises Butyribacterium methylotrophicum. According to an embodiment, said method comprises co-culturing Clostridium pasteurianum and Butyribacterium methylotrophicum, whereby said cell mass further comprises Clostridium pasteurianum and Butyribacterium methylotrophicum. According to an embodiment, said fermentation medium comprises a first feedstock comprising carbohydrates, glycerol, methanol, or combinations thereof. According to an embodiment, said fermentation medium comprises a second feedstock comprising CO, C0 ₂ , carbonate, bicarbonate, ¾, glycerol, methanol, formate, urea or mixtures thereof. According to an embodiment, said first feedstock, said second feedstock or both are metabolized during said culturing.

[0034] According to an embodiment, said method comprises mechanical separation of formed cell mass and at least one of washing, drying pulverizing, extrusion and pelletizing. According to an embodiment, said method comprises processing the cell mass to have a water solubility greater than 25%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. [0035] According to an embodiment, provided is a method for maintaining the good health of an animal comprising feeding said animal an animal feed comprising said feed ingredient. According to an embodiment, said animal is selected from the group consisting of larvae, fry, juvenile, and adult of crustacean, finfish and shellfish, poultry, swine, companion animals, such as dogs and cats, other ruminants and humans. According to an embodiment, said maintaining good health comprises at least one of reduced susceptibility to enteric pathogens, reduction in bowel inflammation, reduced mortality, improved feed conversion, and improved weight gain.

[0036] According to an embodiment, provided is a method for maintaining the good health of an animal comprising feeding said animal a probiotic preparation comprising said feed ingredient. According to an embodiment, said animal is selected from the group consisting of larvae, fry, juvenile, and adult of crustacean, finfish and shellfish, poultry, swine, companion animals, other ruminants and humans. According to an embodiment, said maintaining good health comprises at least one of reduced susceptibility to enteric pathogens, reduction in bowel inflammation, consumption of lactic acid, reduced mortality, improved feed conversion, and improved weight gain.

Examples

Example 1. Clostridium tyrubutyricum fermentation.

[0037] Clostridium tyrobutyricum was grown on a hydrolyzed corn starch medium with 10 mL/L trace elements solution (2.0 g/L nitrilotriacetic acid, 1.0 g/L MnS04 H20, 0.8 g/L Fe(S04)2(NH4)2· 6H20, 0.2 g/L CoCl2- 6H20, 0.2 mg/L ZnS04-7H20, 20.0 mg/L CuCl2-2H20, 20.0 mg/L NiCL2- 6H20, 20.0 mg/L Na2Mo04-2H20, 20.0 mg/L Na2Se04, and 20.0 mg/L Na2W04) and 10 mL/L Wolfe’s vitamins (2.0 mg/L biotin, 2.0 mg/L folic acid, 10.0 mg/L pyridoxine hydrochloride, 5.0 mg/L thiamine-HCl, 5.0 mg/L riboflavin, 5.0 mg/L nicotinic acid, 5.0 mg/L calcium D-(+)-pantothenate, 0.1 mg/L vitamin B12, 5.0 mg/L p-aminobenzoic acid, and 5.0 mg/L thioctic acid). The estimated glucose concentration was 65 g/L. The cells were grown under anaerobic conditions (low C02 sparge) at 37 °C. The pH was bottom controlled at 5.5 with 8M NH40H. Table 1 shows the increase in optical density (OD) over time along with butyric acid. Table 1. Clostridium tyrobutyricum fermentation.

Concentration (g/L)

Fermentation

OD* Lactic acid Acetic acid Butyric acid Ethanol time (h)

0 0.44 054 048 021 0.09

6 2.53 0.29 0.48 1.08 0.16

12 11.50 0.88 1.46 4.72 0.29

18 36.30 0.10 2.47 10.09 0.41

* OD measured at 600 nm

At 18 hours, the cells were harvested, washed three times in water, and dried with a dual drum dryer. The resulting dried cell mass had the characteristics listed in Table 2.

Table 2. Characteristics of Clostridium tyrobutyricum cell mass.

Characteristic _ As is % _ Dried %

Moisture 4.7

Crude protein 79.9 83.9

Ash 7.85 8.24

Lysine - 8.33

Methionine - 1.42

Example 2. Co-culture fermentation.

[0038] A co-culture of Clostridium tyrobutyricum and Butyribacterium

methylotrophicum was prepared and grown on hydrolyzed corn starch medium. The seed culture was generated by mixing equal volumes of a pure culture of C. tyrobutyricum and B. methylotrophicum. The estimated glucose concentration was 100 g/L. The cells were grown under anaerobic conditions (low CO2 sparge) at 37°C. The pH was bottom controlled at 5.5 with 8M NH ₄ OH. Table 3 shows the increase in optical density (OD) over time along with butyric acid.

Table 3. Co-culture fermentation.

Concentration (g/L)

Fermentation

OD* Lactic acid Acetic acid Butyric acid Ethanol time (h)

0 0.56 0.69 0.70 0.23 0.65

16 11.20 0.00 1.30 6.08 0.20

24 17.60 0.00 2.78 13.61 1.05

42 22.20 0.00 2.99 26.86 1.49 * OD measured at 600 nm

At 42 hours, the cells were harvested and washed three times in water. The resulting wet cell mass was analyzed and had the characteristics listed in Table 4.

Table 4. Characteristics of Clostridium tyrobutyricum cell mass.

Characteristic As is % Dried %

Moisture 69.1

Crude protein 24.5 79.3

Ash 1.8 5.8

Examples 3-28

[0039] Feed ingredient cell-mass compositions

to Example 29. Early survival of whiteleg shrimp (Litopenaeus vannamei)

[0040] Four 140-liter tanks with 100 whiteleg shrimp (Litopenaeus vannamei) at growing stage PL10 (2 to 3 gr.) growth in a recirculating aquaculture saltwater (25 ppt) system, water maintained at ~27°C and >80% saturation of dissolved oxygen. The shrimps were feed with a diet including Clostridium tyrobutyricum in accordance to claim 1. the tanks diets were: tank 1 - control, tank 2- 5% Clostridium tyrobutyricum, tank 3- 10% Clostridium tyrobutyricum, tank 4- 15% Clostridium tyrobutyricum.

the aim of the experiment was to check the influence of Clostridium tyrobutyricum feed on early mortality syndrome (EMS) in diets of whiteleg shrimp

Table 5. survival of whiteleg shrimp.

Tank 1 - Tank 2 Tank 3 Tank 4 day

control 5 % diet 10% diet 15% diet

0 100 100 100 100

7 44 66 100 97

14 41 61 96 96