Method for producing butyrate products

Abstract

A method for producing a butyrate product is provided by fermenting a carbon source-containing fermentation feedstock with a Clostridium bacterium that naturally produces butyric acid while maintaining a pH of about 5 to 7, where ammonia is used to maintain the pH, thereby forming a fermentation broth containing ammonium butyrate, biomass, and optionally fermentation by-products; separating the biomass from the fermentation broth to form a separated biomass and a clarified fermentation broth, where the clarified fermentation broth contains the ammonium butyrate and optionally fermentation by-products; and adding an additive selected from the group consisting of a mineral acid, a mineral base, a soluble calcium salt, and a combination thereof to the clarified broth, whereby the butyrate product is formed in the clarified broth. Further provided are feed ingredients, animal feeds, and deicers containing the composition, and uses thereof.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority from U.S. Provisional Application No. 63 / 039,566, filed on Jun. 16, 2020, which is incorporated herein by reference in its entirety as if fully set forth herein.

[0002] Field of the Invention The present invention relates to a method for producing a butyrate product, more specifically, to the fermentation of a carbon - source - containing raw material using Clostridia bacteria that naturally produce butyric acid, and to further processing of the fermentation product to produce the butyrate product.

Background Art

[0003] Background There is a continuing need for improved butyrate products, such as feed components that better achieve improvements such as increased feed intake, improved feed conversion rate, faster daily weight gain, larger ileal surface area, and consistent results between genders when feeding animals.

Summary of the Invention

[0004] According to one aspect of some embodiments of the present invention, a method for producing a butyrate product, the method comprising: (i) providing a fermentation raw material containing a carbon source; (ii) fermenting the raw material using Clostridia bacteria that naturally produce butyric acid while maintaining a pH of about 5 to 7, wherein ammonia is used to maintain the pH, thereby forming a fermentation broth containing ammonium butyrate, biomass, and optionally fermentation by - products; (iii) separating the biomass from the fermentation broth to form separated biomass and clarified fermentation broth, wherein the clarified fermentation broth contains the ammonium butyrate and optionally the fermentation by - products; and (iv) Adding an additive selected from the group consisting of mineral acids, mineral bases, soluble calcium salts, and combinations thereof to the clarified broth, thereby forming the butyrate product in the clarified broth. A method including this is provided.

[0005] According to further embodiments of some of the present invention, an animal feed component comprising the butyrate product disclosed herein is provided.

[0006] According to further embodiments of some of the present invention, an anti-icing agent comprising a butyrate product disclosed herein is provided.

[0007] According to further embodiments of some of the present invention, a method for treating an animal is provided, comprising providing the animal with a feed containing the feed components disclosed herein. [Brief explanation of the drawing]

[0008] Several embodiments of the present invention are described herein with reference to the accompanying drawings. This specification, together with the drawings, will make it clear to those skilled in the art how several embodiments of the present invention can be carried out. The drawings are for illustrative purposes only, and no attempt has been made to show the structural details of the embodiments in any more detail necessary for a basic understanding of the present invention. For clarity, some of the objects depicted in the drawings are not to scale.

[0009] [Figure 1] Figure 1 is a line graph showing metabolites and cellularly produced and consumed glucose as a function of fermentation time for Clostridium tyrobchilicum batch fermentation using glucose as the raw material. [Modes for carrying out the invention]

[0010] Detailed description of the invention The present invention relates to a method for producing a butyrate product, comprising fermentation of a carbon source-containing raw material using Clostridium bacteria that naturally produce butyric acid, and further processing of the fermentation product for producing a butyrate product.

[0011] Further processing yields improved butyrate products compared to those obtained through the fermentation process alone.

[0012] Improved butyrate products may be purified butyrate products and / or solid-form butyrate products (e.g., butyrates, which may be calcium or sodium salts) that are advantageous for use in feed ingredients. Calcium and sodium butyrates have been found to be preferable to ammonium butyrate as feed ingredients.

[0013] The improved butyrate product may have a low phosphorus content, where the method involves removing phosphorus from a clarified fermentation broth. Such low-phosphorus products are advantageous for use as de-icing agents. Furthermore, it has been found that potassium salts of acetate or butyrate have a better de-icing effect than those obtained by the corresponding ammonium salts.

[0014] Improved butyrate products may be obtained by processes that are less costly than those known in the art for obtaining such products. The reduced cost may be due to the reuse of ammonia, which is released during the conversion of ammonium butyrate to butyrate products and / or from the use of less refined carbon sources containing impurities such as phosphate anions, as well as for the use of processing by-products such as calcium phosphate as feed components and fertilizers.

[0015] Further processing steps may include separating the butyrate product from the fermentation by-products, such as by distilling the butyrate product from the fermentation broth, by distilling or evaporating the fermentation by-products from the fermentation broth, or by forming insoluble salts of phosphate anions, such as phytates or phosphates, which are found as impurities in conventional fermentation methods, and removing the insoluble salts from the fermentation broth.

[0016] The details provided herein are illustrative and are solely for the purpose of illustrating various embodiments of the invention, and are presented to provide what is considered to be the most useful and readily understandable explanation of the principles and conceptual aspects of the invention. In this regard, no attempt has been made to provide details of the invention in any way necessary for a basic understanding of the invention, and this explanation will make it clear to those skilled in the art how some forms of the invention can be actually embodied.

[0017] The present invention will now be described with reference to more detailed embodiments. However, the present invention may be embodied in different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure is thorough and complete and fully conveys the scope of the invention to those skilled in the art.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the invention pertains. Technical terms used herein in describing the invention are solely for the purpose of describing specific embodiments and are not intended to limit the invention.

[0019] As used herein, the term "butyrate product" refers to compounds that contain or are derived from butyrate, such as butyrates, butyric acid, and butyrate esters.

[0020] As used herein, the term "feed ingredient" refers to an ingredient, part, component, or any combination / mixture for animal food.

[0021] As used herein, the term "fermentation raw material" refers to one or more components added to fermentation.

[0022] As used herein, the term "animal feed" refers to products for consumption by non-human animals such as livestock, poultry, and companion animals.

[0023] As used herein, the term "deicing agent" refers to a material that can remove ice from a surface and / or prevent or reduce the formation of ice on a surface, including anti-freezing agents.

[0024] As used herein with respect to butyrate, the term "protected" refers to the molecule provided in a bound or reversibly reacted form to allow the butyrate molecule to pass through the upper gastrointestinal tract.

[0025] As used herein, the term "treating" includes improving, alleviating, and reducing the reality of a disease or condition, or the symptoms of a disease or condition.

[0026] As used herein, both the terms "evaporating" and "distilling" mean the transfer from the liquid phase to the gas phase and are used interchangeably.

[0027] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0028] Unless otherwise indicated, all figures used in this specification and the claims, representing quantities of components, reaction conditions, etc., should be understood in all cases as being modified by the term “approximately.” Therefore, unless otherwise indicated, the numerical parameters described in the subsequent specification and the appended claims are approximations that may vary depending on the desired properties to be obtained by the invention. At a minimum, 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 interpreted in terms of a significant number of decimal places and common rounding techniques.

[0029] Although the numerical ranges and parameters describing the broad scope of the present invention are approximations, the numerical values ​​described in the specific examples are reported as accurately as possible. Each numerical value, however, contains a certain degree of error that inevitably arises from the standard deviation found in each of those test measurements. Any numerical range given throughout this specification will include any narrower numerical range that falls within such a broader range, as if all narrower numerical ranges were explicitly described herein. Additional advantages of the present invention are described in part in the subsequent specification, some of which are evident from the specification or can be learned through the practice of the invention. It should be understood that both the general description above and the subsequent detailed description are, as claimed, illustrative and for illustrative purposes only, and do not limit the invention. Where numerical values ​​are preceded by the term “about,” the term “about” as used herein is intended to indicate a range of ±10% of that value.

[0030] The terms “comprising,” “including,” “having,” and their grammatical variations as used herein should be understood as specifying a feature, integer, process, or component being described, but not precluding the addition of one or more additional features, integers, processes, components, or groups thereof. These terms encompass the terms “consisting of” and “substantially consisting of.”

[0031] According to one aspect of several embodiments of the present invention, a method for producing a butyrate product is provided, the method comprising: (i) providing a fermentation raw material comprising a carbon source; (ii) fermenting the raw material using Clostridium bacteria that naturally produce butyric acid, while maintaining a pH of about 5 to 7, wherein ammonia or ammonium hydroxide is used to maintain the pH, thereby forming a fermentation broth comprising ammonium butyrate, biomass, and optionally fermentation byproducts; (iii) separating the biomass from the fermentation broth to form a separated biomass and a clarified fermentation broth, wherein the clarified fermentation broth comprises ammonium butyrate and optionally fermentation byproducts; and (iv) adding an additive selected from the group consisting of mineral acids, mineral bases, soluble calcium salts and combinations thereof to the clarified broth, thereby forming the butyrate product in the clarified broth.

[0032] According to one embodiment, separating the biomass includes at least one of filtering and centrifuging.

[0033] According to one embodiment, the carbon source is selected from the group consisting of starch, dextrose, maltodextrin, glucose, liquefied corn mash, fructose, xylose, glycerol, sucrose, hemicellulose, cellulose, and combinations thereof.

[0034] According to one embodiment, the additive comprises a mineral acid, and the butyrate product formed in the clarified broth is butyric acid in free acid form. The method further includes separating the butyric acid in free acid form from the clarified broth by a method selected from the group consisting of distillation and liquid-liquid extraction of the butyric acid.

[0035] According to one embodiment, the additive comprises a mineral acid, and butyric acid in free acid form and an ammonium salt of the mineral acid are formed in the clarified broth. The method further comprises providing an alkanol, reacting the butyric acid in free acid form with the alkanol to form a butyric acid ester, and separating the butyric acid ester from the clarified broth by a method selected from the group consisting of distillation and liquid-liquid extraction of the butyric acid ester.

[0036] According to one embodiment, the alkanol is selected from the group consisting of methanol, ethanol, propanol, butanol, and combinations thereof.

[0037] According to one embodiment, the pH of the clarified broth is adjusted to 2-5 before or simultaneously with the reaction.

[0038] According to one embodiment, the mineral acid is selected from the group consisting of sulfuric acid, phosphoric acid, nitric acid, and combinations thereof.

[0039] According to one embodiment, the additive comprises a mineral base, ammonia is liberated in the clarified broth, and the butyrate product formed in the clarified broth is the butyrate of the mineral base, and the method further comprises separating the butyrate product from the liberated ammonia by evaporating the liberated ammonia. According to one embodiment, the evaporation is carried out at least partially at atmospheric pressure or a pressure lower than atmospheric pressure (e.g., 0.2 to 1 atmosphere) at a temperature lower than the boiling point of water at that pressure. According to one embodiment, the evaporation includes steam distillation.

[0040] According to one embodiment, the mineral base is selected from the group consisting of oxides, hydroxides, carbonates, and bicarbonates of sodium, potassium, calcium, and magnesium, as well as combinations thereof.

[0041] According to one embodiment, at least one selected from the group consisting of the clarified fermentation broth, the carbon source, and the carbon source-containing fermentation raw materials further comprises a phosphate anion selected from the group consisting of phosphates, phytes, and combinations thereof, wherein the additive comprises a soluble calcium salt, where a water-insoluble calcium salt of the phosphate anion is formed, and the method further comprises separating the butyrate product from the insoluble salt. According to some such embodiments, separating the butyrate product from the insoluble salt comprises removing the insoluble salt, for example, by filtration or centrifugation of the insoluble salt. According to one embodiment, the pH of the clarified broth is adjusted to 7.5 to 9.5 before or at the same time as the addition of the additive, but not before the removal.

[0042] According to one embodiment, the carbon source and / or the carbon source-containing fermentation material comprises a phytate, and the method further comprises contacting the carbon source and / or the carbon source-containing fermentation material with a phytase enzyme, thereby hydrolyzing the phytase to form a phosphate, wherein the contact with the phytase enzyme is performed before or simultaneously with the fermentation.

[0043] According to one embodiment, the soluble calcium salt has water solubility greater than 1% by weight, such as at least 1% by weight, at least 2% by weight, at least 5% by weight, at least 10% by weight, at least 20% by weight, at least 30% by weight, at least 40% by weight, at least 50% by weight, at least 60% by weight, or even at least 70% by weight.

[0044] According to one embodiment, the soluble calcium salt is selected from the group consisting of calcium acetate, calcium propionate, calcium butyrate, calcium chloride, and combinations thereof.

[0045] According to one embodiment, the fermentation broth further contains ammonium acetate.

[0046] According to one embodiment, the butyrate product is a feed component.

[0047] According to one embodiment, the butyrate product is an anti-icing agent.

[0048] According to one embodiment, the phosphorus content of the de-icing agent is less than 0.1% by weight, less than 0.08% by weight, less than 0.06% by weight, less than 0.04% by weight, less than 0.02% by weight, or less than 0.01% by weight.

[0049] According to one embodiment, the additive comprises a mineral base, the butyrate of the mineral base is formed in the clarified broth, and the method further comprises separating the butyrate of the mineral base from the clarified broth by at least one method selected from the group consisting of salt crystallization and electrodialysis.

[0050] According to one embodiment, the additive comprises a mineral acid, the ammonium salt of the mineral acid is formed in the clarified broth, and the method further comprises crystallizing the ammonium salt of the mineral acid from the clarified broth.

[0051] According to one embodiment, the ammonium salt of the mineral acid is selected from the group consisting of ammonium sulfate, ammonium phosphate, ammonium nitrate, and combinations thereof. According to one embodiment, a fertilizer containing the ammonium salt disclosed herein is provided.

[0052] According to one embodiment, the method further comprises reacting the ammonium salt of the mineral acid with a calcium base, thereby liberating ammonia and forming a precipitate, wherein the precipitate includes calcium sulfate, calcium phosphate, or a combination thereof. According to several such embodiments, calcium phosphate is formed. According to one embodiment, an animal feed containing calcium phosphate disclosed herein is provided.

[0053] According to another embodiment, a fertilizer containing calcium phosphate disclosed herein is provided.

[0054] According to one embodiment in which ammonia is liberated, the method further includes reusing the liberated ammonia and / or the evaporated ammonia for pH control during the fermentation.

[0055] According to one embodiment, the additive contains a calcium base, and potassium butyrate is the butyrate product.

[0056] According to one embodiment, the method further includes protecting at least one fraction of the butyrate in the clarified fermentation broth or its product, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or even at least 90% of the butyrate.

[0057] According to one embodiment, the method further includes removing water from the fermentation broth and / or from the clarified fermentation broth, at least partially.

[0058] According to some such embodiments, biomass is first separated from the fermentation broth, and the clarified fermentation broth is concentrated to produce a concentrated, clarified fermentation broth containing up to about 60% by weight of solute. According to some such embodiments, additives are added to the concentrated, clarified fermentation broth. According to an alternative embodiment, additives are added to the clarified fermentation broth before concentration. According to a further alternative embodiment, the clarified broth is partially concentrated, then additives are added, and the clarified broth is further concentrated.

[0059] According to one aspect of several embodiments of the present invention, an animal feed is provided comprising selected nutrients, a butyrate feed component or its products as disclosed herein, and optionally biomass.

[0060] According to one embodiment, the animal feed further comprises at least one mineral micronutrient. According to some such embodiments, the mineral micronutrient is a metal ion selected from the group consisting of iron ions, zinc ions, and combinations thereof. While we do not wish to be bound by any one theory, we hypothesize that such metal ions form complexes with butyrates (and / or acetates, if present) that increase their bioavailability.

[0061] According to one aspect of several embodiments of the present invention, a method for treating an animal is provided, comprising providing the animal with a feed disclosed herein.

[0062] According to one aspect of several embodiments of the present invention, a feed disclosed herein is provided for use in treating animals.

[0063] According to one embodiment, the feed contains butyrate at concentrations between 10 ppm and 20,000 ppm, such as at least 10 ppm, at least 50 ppm, at least 100 ppm, at least 500 ppm, at least 1,000 ppm, at least 5,000 ppm, at least 10,000 ppm, less than 20,000 ppm, less than 10,000 ppm, less than 5,000 ppm, less than 1,000 ppm, less than 500 ppm, less than 100 ppm, or less than 50 ppm.

[0064] According to one embodiment, at least 50% by weight of the butyrate in the feed, such as at least 50%, at least 60%, at least 70%, or at least 80%, comes from the feed components.

[0065] According to one embodiment, the animal is selected from a group consisting of birds, pigs, cattle, horses, sheep, fish, goats, dogs, cats, and rapines.

[0066] According to one embodiment, the method provides improved results compared to providing the same selected nutrients without the feed component.

[0067] According to one embodiment, the improved results include at least one of increased feed intake, improved feed conversion rate, faster daily weight gain, larger ileal surface area, and sex-matched results.

[0068] According to one embodiment, the improved results include at least one, at least two, at least three, or at least four selected from the group consisting of increased feed intake, improved feed conversion rate, faster daily weight gain, larger ileal surface area, sex-matched results, and combinations thereof.

[0069] According to one embodiment, the method provides improved results compared to providing the same selected nutrients without the feed component. According to one embodiment, the improved results include increased feed intake. According to one embodiment, feed intake increases by at least 0.5%, at least 1%, at least 2%, at least 3%, or at least 4%. According to one embodiment, the improved results include an improved feed conversion rate (FCR). According to one embodiment, the FCR improves by at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 1%, at least 2.5%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10%.

[0070] According to one embodiment, the improved result includes a faster daily weight gain. According to one embodiment, the daily weight gain increases by at least 0.1%, at least 0.2%, at least 0.3%, or at least 0.4%. [Examples]

[0071] Example 1: Glucose fermentation Glucose-based fermentation media containing 60–160 g / L of glucose were fermented in batch fermentation using Clostridium tyrobthyricum. Additional media components included those listed in Tables 1, 2, and 3. The temperature was maintained at 35°C, and the pH was controlled to a minimum of 5–7 using 8 M NH4OH. The results of glucose consumption, cell production, and metabolite production during 30 hours of fermentation are shown in Figure 1. Butyrate and cell concentrations reached approximately 60 g / L and 20 g / L, respectively.

[0072] [Table 1]

[0073] [Table 2]

[0074] [Table 3]

[0075] Example 2: Preparation of concentrated, clarified broth Fermentation was carried out in the same manner as in Example 1. The biomass was filtered off. The clarified fermentation broth was concentrated by water evaporation to a total solids concentration of 52.6% by weight to form a clarified broth. Its density was 1.076 kg / L. The formed clarified broth was analyzed. The results are summarized in Table 4.

[0076] [Table 4]

[0077] Example 3: Corn mash fermentation Fermentation was carried out in the same manner as in Example 1, except that corn mash was used as the raw material instead of glucose. The corn mash was diluted to a concentration of 60-160 g / L (assuming glucose concentration) and filtered, and amylase enzyme was added during fermentation. Only trace elements (Table 2) and vitamins (Table 3) were added to the fermentation. Other operating conditions were the same, and a similar production profile was achieved, along with a product similar to that of Example 2.

[0078] Example 4: Removal of phosphate ions A concentrated, clarified broth was prepared in the same manner as in Example 3. Its main components are shown in Table 5. Note that the total phosphorus content was approximately 1800 mg / kg, with half of it in phosphate form. The remainder was apparently in phytate form.

[0079] [Table 5]

[0080] A concentrated, clarified broth sample was treated with pH adjustment using NaOH solution to reach pH 8 or 9. A strong ammonia odor was detected at pH 9. Calcium acetate solution was added to a portion of the sample. The sample was then centrifuged, and the supernatant was analyzed. The amount of clarified broth sample was 50 grams. The concentration of calcium acetate in the added solution was 20% by weight. The results are summarized in Table 5.

[0081] [Table 6]

[0082] These results demonstrate that phosphate phosphorus is reduced very efficiently when processing such concentrated, clarified broth. Such a reduction is required for the use of the product in road de-icing agents.

Claims

1. A method for producing butyrate products, (i) To provide fermentation raw materials containing a carbon source; (ii) Fermenting the raw material using Clostridium bacteria that naturally produce butyric acid, while maintaining a pH of 5 to 7, wherein ammonia is used to maintain the pH, thereby forming a fermentation broth containing ammonium butyrate and biomass; (iii) Separating the biomass from the fermentation broth in order to form separated biomass and clarified fermentation broth, wherein the separation is carried out by a method selected from the group consisting of filtration and centrifugation, wherein the clarified fermentation broth contains the ammonium butyrate; (iv) Adding a mineral acid to the clarified broth, wherein butyric acid in free acid form and an ammonium salt of the mineral acid are formed in the clarified broth; (v) (a) Separating the butyric acid in its free acid form from the clarified broth by a method selected from the group consisting of distillation of the butyric acid, liquid-liquid extraction, or a combination thereof, (b) providing an alkanol, reacting the butyric acid in its free acid form with the alkanol to form a butyric acid ester, and separating the butyric acid ester from the clarified broth by a method selected from the group consisting of distillation of the butyric acid, liquid-liquid extraction, or a combination thereof; (vi) The ammonium salt is reacted with a calcium base, thereby liberating ammonia and forming a precipitate, wherein the precipitate contains calcium sulfate, calcium phosphate, or a combination thereof; and (vii) Reusing the liberated ammonia for pH control during fermentation. Methods including,

2. The method of claim 1, wherein at least one selected from the group consisting of the clarified fermentation broth, the carbon source, and the fermentation raw materials containing the carbon source further comprises a phosphate anion selected from the group consisting of phosphates, phytes, and combinations thereof, the additive comprises a soluble calcium salt, a water-insoluble calcium salt of the phosphate anion is formed, and the method further comprises separating the butyrate product from the water-insoluble calcium salt.

3. The method of claim 2, wherein the carbon source and / or fermentation material containing the carbon source contains a phytate, and the method further comprises contacting the carbon source and / or fermentation material containing the carbon source with a phytase enzyme, thereby hydrolyzing the phytase to form a phosphate, and the contact with the phytase enzyme is performed before or concurrently with the fermentation.

4. The method according to claim 1, wherein the mineral acid is selected from the group consisting of sulfuric acid, phosphoric acid, and combinations thereof.

5. The method of claim 2, wherein the soluble calcium salt has a water solubility greater than 1% by weight.

6. The method of claim 1, wherein the fermentation broth further comprises ammonium acetate.

7. The method of claim 1, wherein the butyrate product is a feed component.

8. The method according to claim 1, wherein the butyrate product is an anti-icing agent.

9. The method of claim 8, wherein the phosphorus content of the anti-icing agent is less than 0.1% by weight.

10. The method of claim 1, further comprising crystallizing the ammonium salt from the clarified broth.

11. The method according to claim 1, wherein the ammonium salt is selected from the group consisting of ammonium sulfate, ammonium phosphate, and combinations thereof.

12. The method according to claim 1, wherein calcium phosphate is formed.

13. The method of claim 7, further comprising protecting at least one fraction of the butyrate feed component in the clarified fermented broth.

14. The method of claim 1, further comprising removing water at least partially from the fermentation broth and / or the clarified fermentation broth.

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