Compositions for modulating growth of microorganisms

Abstract

The present invention provides a composition for modulating the growth of microorganisms comprising benzoic acid and / or at least one salt thereof, and at least one additional organic acid and / or at least one salt thereof, and uses of the composition.

Description

Technical Field

[0001] The present invention relates to a composition for regulating the growth of microorganisms and uses of said composition. Background Art

[0002] In the field of microbiology, it is well known that the growth behavior of microorganisms depends on environmental conditions. Certain conditions may favor the growth of microorganisms, while other conditions may reduce growth. Notably, certain environmental conditions may cause a reduction or even a halt in the growth of some microorganisms, while other microorganisms may not be affected at all, or may even exhibit increased growth when exposed to the same conditions.

[0003] In practice, humans purposefully adjust environmental conditions to favor or disfavor the growth of microorganisms. For example, in the feed industry (such as the use of silage agents) or in the dairy processing industry, for example, for the production of yogurt or cheese, effective growth of selected suitable microorganisms (such as bacteria or yeast or fungi) is required. Additional examples of a controlled increase in favor of microorganism growth include fermentation processes, such as beer brewing or winemaking.

[0004] For example, in the feed industry (such as the use of silage inoculants) or in the milk processing industry (such as the production of yogurt or cheese), it is desirable to select suitable microorganisms (such as bacteria, yeast or fungi) for effective growth. Other examples of facilitating the control of microorganism growth also include fermentation processes, such as beer brewing or wine making.

[0005] On the other hand, attempts are made to avoid or at least delay the spoilage of nutritious products (such as food, raw materials or feed) due to the uncontrolled growth of unwanted microorganisms. In this regard, temperature conditions can be selected to disfavor the growth of microorganisms. Generally, products at risk of microbial spoilage can be kept frozen or at least stored at low temperatures, such as below 10 °C. Additional options for regulating or controlling the growth of microorganisms include, for example, removing water by lyophilization, smoking, salting, pickling, sugaring or souring. In some preservation processes, an acidic pH environment is established to disfavor the growth of adverse and potentially pathogenic microorganisms.

[0006] In this regard, acidifier products including organic acids can be used to maintain food and feed hygiene and prevent spoilage caused by microorganisms (such as bacteria or molds). Organic acids (such as formic acid, propionic acid or acetic acid) as described in EP 2642874 can be used to control the growth of microorganisms in food, foodstuffs or feed, with the aim of minimizing the risk of foodborne diseases. For this purpose, acidifier products can be added before and / or after the production of finished feed. In addition, acidifier products can be used to achieve a reduced growth of potentially pathogenic microorganisms in the gastrointestinal tract and can also improve feed conversion rate and weight gain after ingestion.

[0007] Notwithstanding the above, there remains a continuing need to improve the acidulant products, for example by improving the efficiency of the acids used. It has surprisingly been found that benzoic acid and / or at least one of its salts in combination with at least one further organic acid and / or at least one of its salts provides an enhanced antibacterial effect. Summary of the Invention

[0009] The present invention provides a composition for regulating microbial growth, the composition comprising a) benzoic acid and / or at least one of its salts, and b) at least one additional organic acid and / or at least one of its salts.

[0010] The present invention also provides the use of the above composition for regulating microbial growth, and a method for regulating microbial growth using the composition.

[0011] The present invention further provides the use of at least one additional organic acid and / or at least one of its salts in the composition as defined herein for enhancing the ability of benzoic acid and / or at least one of its salts to regulate microbial growth. Detailed Description of the Invention

[0013] In a first aspect, the present invention provides a composition for regulating microbial growth, wherein the composition comprises a) benzoic acid and / or at least one of its salts, and b) at least one additional organic acid and / or at least one of its salts.

[0014] In the present invention, at least one salt of benzoic acid may be selected from the group consisting of sodium benzoate, magnesium benzoate, manganese benzoate, potassium hypophosphite, aluminium benzoate, calcium benzoate and iron benzoate, preferably selected from sodium benzoate, magnesium benzoate, manganese benzoate and potassium benzoate, more preferably selected from sodium benzoate.

[0015] In the present invention, at least one additional organic acid is any organic acid other than benzoic acid and may be selected from the group consisting of short-chain monocarboxylic acids having 1 to 6 carbon atoms, saturated dicarboxylic acids, unsaturated dicarboxylic acids, unsaturated carboxylic acids, saturated carboxylic acids, hydroxycarboxylic acids, aromatic carboxylic acids and / or keto-carboxylic acids. By way of illustration only, examples of short-chain monocarboxylic acids having 1 to 6 carbon atoms are formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, 3-methylbutyric acid, 2-methylbutyric acid, 2-ethylbutyric acid, valeric acid and hexanoic acid. Examples of saturated dicarboxylic acids are adipic acid and succinic acid. Examples of unsaturated dicarboxylic acids are fumaric acid. Examples of unsaturated carboxylic acids are sorbic acid and oleic acid. Examples of saturated carboxylic acids are stearic acid, caprylic acid (also known as octanoic acid), capric acid (also known as decanoic acid) and lauric acid (also known as dodecanoic acid). Examples of hydroxycarboxylic acids are lactic acid, malic acid (D-malic acid or L-malic acid or D / L-malic acid), citric acid and tartaric acid. Examples of aromatic carboxylic acids are cinnamic acid. Examples of keto-carboxylic acids are pyruvic acid.

[0016] Preferably, the at least one additional organic acid is selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, 3-methylbutyric acid, 2-methylbutyric acid, 2-ethylbutyric acid, valeric acid, caproic acid, adipic acid, succinic acid, fumaric acid, sorbic acid, oleic acid, stearic acid, caprylic acid, capric acid, lauric acid, lactic acid, malic acid, citric acid, tartaric acid, cinnamic acid, pyruvic acid, gluconic acid, suberic acid, malonic acid, tannic acid, caffeic acid, ellagic acid, perillic acid, and gallic acid.

[0017] More preferably, the at least one additional organic acid is selected from formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, 3-methylbutyric acid, 2-methylbutyric acid, 2-ethylbutyric acid, valeric acid, caproic acid, adipic acid, succinic acid, fumaric acid, sorbic acid, oleic acid, stearic acid, caprylic acid, capric acid, lauric acid, lactic acid, malic acid, citric acid, tartaric acid, cinnamic acid, pyruvic acid, or at least one salt thereof.

[0018] Even more preferably, the at least one additional organic acid is selected from formic acid, acetic acid, propionic acid, fumaric acid, and succinic acid. Most preferably, the at least one additional organic acid is selected from fumaric acid and succinic acid.

[0019] In the present invention, at least one salt of the additional organic acid can be any one of metal salts such as potassium, sodium, or calcium salts, and ammonium salts. Examples of organic acid salts include, but are not limited to, ammonium formate, potassium diformate, sodium diacetate, calcium acetate, ammonium propionate, sodium propionate, calcium propionate, calcium lactate, potassium sorbate, sodium formate, calcium formate, sodium butyrate, sodium sorbate, potassium citrate, sodium citrate, and calcium citrate.

[0020] In some embodiments, the composition according to the present invention may comprise benzoic acid and / or at least one of its salts, in combination with more than one additional organic acid and / or at least one of its salts. For example, such a composition may comprise a combination of benzoic acid and / or at least one of its salts with two, three, four, five or even more additional organic acids. In a preferred embodiment, the composition comprises benzoic acid and / or a salt of benzoic acid and at least three additional organic acids, in particular formic acid, acetic acid and propionic acid, wherein the salt of benzoic acid is selected from the group consisting of sodium benzoate, manganese benzoate, magnesium benzoate and potassium benzoate, preferably sodium benzoate or manganese benzoate, more preferably sodium benzoate. In a more preferred embodiment, the composition according to the present invention comprises benzoic acid and / or benzoate and at least four additional organic acids, in particular formic acid, acetic acid, propionic acid and fumaric acid, wherein the benzoate is selected from the group consisting of sodium benzoate, manganese benzoate, magnesium benzoate and potassium benzoate, preferably sodium benzoate or manganese benzoate, more preferably sodium benzoate. In a more preferred embodiment, the composition according to the present invention comprises benzoic acid and / or benzoate and at least five additional organic acids, in particular formic acid, acetic acid, propionic acid, fumaric acid and succinic acid, wherein the benzoate is selected from the group consisting of sodium benzoate, manganese benzoate, magnesium benzoate and potassium benzoate, preferably sodium benzoate or manganese benzoate, more preferably sodium benzoate.

[0021] The composition of the present application can be formulated in the form of a feed additive (premix) or food additive for administration to animals or in the form of feed or food. Thus, such a composition may further comprise one or more of the following ingredients: fat-soluble or water-soluble vitamins, trace and / or macro minerals, (proteinogenic) amino acids, organic and / or inorganic adsorbents, polyunsaturated fatty acids, antimicrobial polypeptides, prebiotics and / or probiotics, flavoring agents and / or coloring agents, microorganisms and / or enzymes to promote better nutrient utilization and / or mitigate the adverse effects of feed / food contaminants.

[0022] According to the present invention, such a composition may be provided in a form in which one or more or all of the components are provided in solid form (such as salts, powders, granules, pellets, etc.) or in liquid form (such as aqueous solutions, gels, viscous substances). It is also contemplated that the composition may be provided in such a way that one component (such as one or more organic acids) is provided in liquid form and a second component is provided in solid form (such as benzoic acid), and the composition is formed by combining these two components either in a mixture or separately.

[0023] According to the present invention, the composition can be provided in a specific manner, wherein the molar ratio of at least one additional organic acid and / or at least one of its salts to benzoic acid and / or at least one of its salts is from 0.001:1 to 500:1, preferably from 0.005:1 to 300:1, more preferably from 0.01:1 to 200:1, such as 0.01:1 to 0.15:1, 0.02:1 to 0.1:1, 0.04:1 to 100:1, 0.05:1 to 100:1, 0.1:1 to 100:1, 1:1 to 100:1, 10:1 to 80:1 and 20:1 to 40:1. For example, for every 1 kg of the composition in solid form or every 1 liter of the composition in liquid form, at least one additional organic acid and / or at least one of its salts can be provided at any concentration from 0.2 mmol to 100 mmol, such as 0.2 mmol, 0.4 mmol, 0.8 mmol, 1.5 mmol, 3.1 mmol, 6.25 mmol, 12.5 mmol, 25.0 mmol, 50.0 mmol and 100 mmol, in combination with benzoic acid and / or at least one of its salts at any concentration from 0.2 mmol to 20 mmol (such as 0.3 mmol, 0.6 mmol, 1.3 mmol, 2.5 mmol, 5.0 mmol, 10 mmol and 20 mmol). Preferably, in the composition of the present invention, the molar ratio of at least one additional organic acid and / or at least one of its salts to benzoic acid and / or at least one of its salts is in the range of 0.001:1 to 0.15:1.

[0024] The composition of the present invention can be used to regulate the growth of microorganisms, i.e., inhibit the growth of pathogenic microorganisms and / or enhance the growth of beneficial (i.e., probiotic) microorganisms.

[0025] In the present invention, the microorganism can be any one or more of the following genera: Buttiauxella, Citrobacter, Cronobacter, Enterobacter, Escherichia, Edwardsiella, Klebsiella, Phytobacter, Plesiomonas, Pseudoescherichia, Raoultella, Salmonella, Shigella, Proteus, Yersinia, Vibrio, Aeromonas, Clostridium, Pseudomonas, Staphylococcus, Pasteurella, Brachyspira, Campylobacter, Listeria, Streptococcus, Haemophilus, Brucella, Moritella, Tenacibaculum, Lactobacillus, Bifidobacterium, Saccharomyces, Bacillus, Pediococcus, Enterococcus, Streptococcus, Propionibacterium, Pseudomonas, and Citrobacter. In particular, the microorganism in the present invention can be any one or more of the following: Escherichia coli, Salmonella enterica, Shigella sonnei, Clostridium perfringens, Lactobacillus reuteri, Enterococcus faecium, and Bacillus subtilis.

[0026] In particular, the pathogenic microorganisms in the present invention may be at least one of the following genera: Buchnera, Citrobacter, Cronobacter, Enterobacter, Escherichia, Edwardsiella, Klebsiella, Phytoplasma, Plesiomonas, Pseudescherichia, Raoultella, Salmonella, Shigella, Proteus, Yersinia, Vibrio, Aeromonas, Clostridium, Pseudomonas, Staphylococcus, Pasteurella, Brachyspira, Campylobacter, Listeria, Streptococcus, Haemophilus, Brucella, Moellerella, and Jejuia, and in particular, may be selected from the group consisting of Escherichia coli, Salmonella enterica, Shigella sonnei, and Clostridium perfringens.

[0027] In particular, the beneficial microorganisms in the present invention may be at least one of the following genera: Lactobacillus, Bifidobacterium, Saccharomyces, Bacillus, Pediococcus, Enterococcus, Streptococcus, Propionibacterium, Pseudomonas, and Citrobacter, and in particular, may be selected from the group consisting of Lactobacillus reuteri, Enterococcus faecium, and Bacillus subtilis.

[0028] Unexpectedly, it was found that the composition of the present invention can regulate the growth of microorganisms in a synergistic mode. In the present invention, the term "in a synergistic mode" means that in the test for regulating the growth of microorganisms, for example, according to Example 1 of the present invention, the observed value ("O") provided by the composition of the present invention exceeds the expected value ("E") produced by the individual components of the composition, namely benzoic acid and / or its salts and additional organic acids and / or their salts. In the present invention, the expected value ("E") can be calculated according to the formula developed by Colby et al. (1967. Weeds 15(1):20 - 22), which is incorporated herein by reference. In the present invention, the expression "the observed value ("O") exceeds the expected value ("E")" means that the result of subtracting the expected value from the observed value, i.e., E - O is greater than zero (0), preferably not less than 1, 2, 3, 5, 8, 10, and more preferably not less than 10.

[0029] Therefore, the present invention also provides a composition as described above, wherein in the test for regulating the growth of microorganisms, the observed value ("O") provided by the composition of the present invention exceeds the expected value ("E") produced by the individual components of the composition, namely benzoic acid and / or its salts and additional organic acids and / or their salts.

[0030] In the present invention, the composition may contain benzoic acid and / or at least one of its salts and at least one additional organic acid and / or at least one of its salts as the sole active ingredient for regulating the growth of microorganisms. However, in addition to benzoic acid and / or at least one of its salts and at least one additional organic acid and / or at least one of its salts, the composition of the present invention may also include other active ingredients, such as enzymes for regulating the growth of microorganisms.

[0031] In a second aspect, the present invention provides the use of the compositions described herein for modulating microbial growth, namely inhibiting the growth of pathogenic microorganisms and / or enhancing the growth of beneficial (i.e., probiotic) microorganisms.

[0032] As is known to those skilled in the art, microorganisms can be present in food and / or feed, and / or in the gastrointestinal tract of animals. In some embodiments, the present invention provides the use of the compositions described herein for modulating microbial growth in food and / or feed, particularly for inhibiting the growth of pathogenic microorganisms. In some embodiments, the present invention provides the use of the compositions described herein for modulating microbial growth in the gastrointestinal tract of animals, particularly in the intestine (such as the stomach, duodenum, jejunum, ileum, cecum, and / or large intestine). In some preferred embodiments, the present invention provides the use of the compositions described herein for improving the intestinal health of animals.

[0033] In some specific embodiments, the present invention provides the use of the compositions described herein for treating, improving, and / or preventing post-weaning diarrhea (PWD) in animals (such as pigs). PWD is typically caused by Escherichia coli strains in the gastrointestinal tract of animals (e.g., Fairbrother et al., 2005. Anim Health Res Rev. 6(1):17 - 39), causing significant economic losses and animal diseases. Notably, by administering the compositions described herein to animals (such as pigs), the growth of such strains in the gastrointestinal tract of animals (such as pigs) can be reduced, thereby treating, improving, and / or preventing PWD. As will be understood by any person skilled in the art, the characteristics of treating, improving, and / or preventing PWD can be, for example, an increase in fecal score. Preferably, in the present invention, the fecal score is increased by at least 1%, 2%, 3%, 5%, 10% or more.

[0034] In other specific embodiments, the present invention provides the use of the compositions described herein for improving the growth performance of animals. As would be expected by those skilled in the art, due to the role of the compositions described herein in modulating microbial growth in the gastrointestinal tract of animals, one or more growth performance characteristics such as weight gain, daily weight gain, feed intake, daily feed intake, feed conversion ratio, and / or mortality of animals can be improved. Preferably, due to the compositions described herein, any characteristic of growth performance is improved by at least 1%, 2%, 3%, 5%, 10%, 15%, 20% or more.

[0035] In the present invention, the terms "food" and "feed" refer to any component, ingredient, preparation, mixture or additive suitable or intended for oral administration to animals. Examples of food and / or feed include, but are not limited to, foodstuffs; fodder; feeds; silage; food additives; fodder additives; silage additives; feed or food components such as crops, grains such as wet distillers grains and dry distillers grains, hay, straw, protein sources such as soy protein, starches and carbohydrates, etc.; nutritional supplements; prebiotics; probiotics; their intermediates; and / or their mixtures.

[0036] In the present invention, the term "animal" (animal or animals) refers to any animal including humans. Examples of animals include ruminants such as sheep, goats, cattle, deer, antelopes, camels, llamas and kangaroos; and non-ruminants such as pigs (including but not limited to piglets, growing pigs and sows), poultry (including but not limited to turkeys, ducks, quails, guinea fowls, geese, pigeons and chickens), horses, crustaceans (such as freshwater shrimps and marine shrimps) and fish. Preferably, the animal is selected from the group consisting of sheep, goats, cattle, pigs (including but not limited to piglets, growing pigs and sows), poultry (including but not limited to turkeys, ducks, quails, guinea fowls, geese, pigeons and chickens) and horses.

[0037] Preferably, in the use of the present invention, the composition regulates the growth of microorganisms in a synergistic mode, i.e., the observed value ("O") it provides exceeds the expected value ("E") produced by the individual components in the composition (i.e., benzoic acid and / or its salts and additional organic acids and / or their salts) in regulating the growth of microorganisms.

[0038] In a third aspect, the present invention provides a method for regulating the growth of microorganisms, i.e., inhibiting the growth of pathogenic microorganisms and / or enhancing the growth of beneficial (i.e., probiotic) microorganisms, wherein the method comprises the following steps: a) providing the composition described herein; and b) contacting the composition of a) with the microorganisms.

[0039] In some embodiments, the present invention provides a method for regulating the growth of microorganisms in food and / or feed, particularly for inhibiting the growth of pathogenic microorganisms, wherein the method comprises the following steps: a) providing the composition described herein; b) contacting the composition of a) with the microorganisms in the food and / or feed.

[0040] In some other embodiments, the present invention provides a method for regulating the growth of microorganisms in the gastrointestinal tract of animals, particularly in the intestine (such as the stomach, duodenum, jejunum, ileum, cecum and / or large intestine) and / or improving the intestinal health of animals, wherein the method comprises the following steps: a) providing the composition described herein; and b) administering the composition of a) to the animals.

[0041] In some specific embodiments, the present invention provides a method for treating, improving, and / or preventing post-weaning diarrhea (PWD) in animals (such as pigs), wherein the method comprises the following steps: a) providing the composition described herein; and b) administering the composition of a) to the animal.

[0042] In other specific embodiments, the present invention provides a method for improving the growth performance of animals, wherein the method comprises the following steps: a) providing the composition described herein; b) administering the composition of a) to the animal.

[0043] Preferably, in the method of the present invention, the composition regulates the growth of microorganisms in a synergistic mode, and / or treats, improves, and / or prevents PWD in animals, and / or improves the growth performance of animals, that is, in terms of regulating the growth of microorganisms, and / or treating, improving, and / or preventing PWD in animals, and / or improving the growth performance of animals, the observed value ("O") it provides exceeds the expected value ("E") produced by the individual components of the composition, namely benzoic acid and / or its salts and additional organic acids and / or their salts.

[0044] In a fourth aspect, the present invention provides the use of at least one additional organic acid and / or at least one of its salts in the composition as defined above for enhancing the regulation of microbial growth by benzoic acid and / or at least one of its salts, that is, inhibiting the growth of pathogenic microorganisms and / or enhancing the growth of beneficial microorganisms.

[0045] In particular, the present invention provides a method for enhancing the effect of benzoic acid and / or at least one of its salts in regulating microbial growth, that is, inhibiting the growth of pathogenic microorganisms and / or enhancing the growth of beneficial microorganisms, wherein the method comprises: a) providing the composition described herein; and b) contacting the composition of a) with the microorganisms.

[0046] In particular, the present invention provides a method for enhancing the effect of benzoic acid and / or at least one of its salts in regulating the growth of microorganisms in the gastrointestinal tract, especially the growth of microorganisms in the intestine (such as the stomach, duodenum, jejunum, ileum, cecum, and / or large intestine), and / or improving the intestinal health of animals, and / or treating, improving, and / or preventing post-weaning diarrhea (PWD) in animals (such as pigs), wherein the method comprises the following steps: a) providing the composition described herein; and b) administering the composition of a) to the animal.

[0047] In the present invention, the term "boost" (or "boosting") means that benzoic acid and / or at least one of its salts and at least one additional organic acid and / or at least one of its salts in the composition according to the present invention regulate the growth of microorganisms in a synergistic mode, that is, in terms of regulating the growth of microorganisms, the observed value ("O") it provides exceeds the expected value ("E") produced by the individual components of the composition (i.e., benzoic acid and / or its salts and additional organic acids and / or their salts).

[0048] The following examples further illustrate the present invention. Example

[0049] Example 1: Regulation of microbial growth

[0050] In this example, the potential of the composition of the present invention in regulating microbial growth, i.e., inhibiting the growth of pathogenic microorganisms and promoting the growth of beneficial microorganisms, was studied. In a systematic method, the growth of the following microorganisms was recorded: pathogenic microorganisms: Escherichia coli (Escherichia coli U5 / 41 (DSMZ 30083)), Salmonella enterica (Salmonella enterica subsp. enterica serovar Typhimurium SL1344 (DSMZ 24522)), Shigella sonnei (Shigella sonnei ATCC 29930, type I virulent, WDCM 00127, CECT 4887 (DSMZ 5570)), and Clostridium perfringens (Clostridium perfringens (CCUG 47895; type A, as described in Johansson et al. 2004. Vet Microbiol. 99(3-4):251-257)); and beneficial microorganisms: Lactobacillus reuteri (Lactobacillus reuteri F 275 (DSMZ 20016)), Enterococcus faecalis (Enterococcus faecalis D, serotype 11 (DSMZ20477)), and Bacillus subtilis (Bacillus subtilis strain Marburg (DSMZ 10)), and the synergistic effect was evaluated.

[0051] Specifically, the composition of the present invention containing 0.2, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, or 100 mM formic acid, acetic acid, propionic acid, fumaric acid, or succinic acid; and / or 0.3, 0.6, 1.3, 2.5, 5, 10, or 20 mM benzoic acid was prepared in Mueller-Hinton medium and microfiltered and sterilized using a 0.22 μm filter. Any one of the above microorganisms was cultured in Mueller-Hinton medium (Mueller et al., 1941. Experimental Biology and Medicine 48(1):330-333) until the late exponential phase. These liquid cultures were used to inoculate the composition prepared as described above into 96-well microtiter plates to achieve a final density of 5*10 5 cfu / mL. As a sterile control, one well containing Mueller-Hinton culture broth was not inoculated with microorganisms. As a growth control, at least 4 wells of a 96-well microtiter plate were prepared, in which only Mueller-Hinton culture broth was inoculated with any one of the microorganisms being studied. In other words, the growth control was not treated with the composition containing any organic acids and / or benzoic acid mentioned herein.

[0052] The microtiter plates so inoculated were incubated overnight at 37 °C under anaerobic conditions provided by a saturated nitrogen atmosphere in an anaerobic jar to simulate the conditions in the gastrointestinal tract. To evaluate the growth of the microorganisms, at about 20 - 26 h after the start of incubation, usually after about 24 h, the optical density (OD600) at 600 nm of each well of the incubated microtiter plates was measured using a multimode microplate reader. The initial optical density value before the overnight incubation was subtracted from the final value obtained after the overnight incubation period to calculate the growth value. These growth values were used to calculate the amount of inhibition or enhancement compared to the growth value of the growth control wells. For example, if the growth value of the growth control was 0.8308 after about 10 h, the growth value of an E. coli culture containing 12.5 mM formic acid was 0.6624, the growth value of an E. coli culture containing 12.5 mM benzoic acid was 0.7128, and the growth value of an E. coli culture containing 12.5 mM formic acid and 12.5 mM benzoic acid was 0.3692, then the growth of the culture containing 12.5 mM formic acid compared to the growth of the growth control would be calculated as 0.6624 * 100 / 0.8308 = 79.72%, the growth of the culture containing 12.5 mM benzoic acid compared to the growth of the growth control would be calculated as 0.7128 * 100 / 0.8308 = 85.80%, and the growth of the culture containing 12.5 mM formic acid and 12.5 mM benzoic acid compared to the growth of the growth control would be calculated as 0.3692 * 100 / 0.8308 = 44.44%. In other words, the composition containing 12.5 mM formic acid inhibited microbial growth by up to 100 - 79.92 = 20.28%, the composition containing 12.5 mM benzoic acid inhibited microbial growth by up to 100 - 85.80 = 14.20%, and the composition containing 12.5 mM formic acid and 12.5 mM benzoic acid inhibited microbial growth by up to 100 - 44.44 = 55.56%.

[0053] Synergistic inhibition or enhancement was evaluated according to the following formula published by Colby et al. (1967. Weeds 15(1):20 - 22):

[0054] E = X + (Y / 100) * (100 - X)

[0055] Wherein, "E" represents the expected value; "X" represents the degree of inhibition or enhancement observed when treated with Substance 1 (in this article: organic acid and / or its salt); and "Y" represents the degree of inhibition or enhancement observed when treated with Substance 2 (in this article: benzoic acid and / or its salt). The expected value indicates the theoretical amount of inhibition or enhancement that would be observed if the combination of Substance 1 and Substance 2 only additively (i.e., non-synergistically) inhibits. Both "X" and "Y" are calculated as percentages of inhibition or enhancement. The growth control untreated with Substance 1 and / or Substance 2 is used as the base value without inhibition / enhancement, i.e., 0% inhibition / enhancement.

[0056] In the next step, the actually observed amount of inhibition or enhancement of the combination of Substance 1 and Substance 2 is compared with the expected value and used to quantify the synergy according to the following formula:

[0057] S = O - E

[0058] Wherein, "S" represents the difference between the calculated additive amount of inhibition or enhancement ("E") and the actually observed amount of inhibition or enhancement ("O"); "E" represents the expected value as described above; and "O" represents the observed amount of inhibition or enhancement. When "S" is a positive number, i.e., greater than 0, it is confirmed that the combination of Substance 1 and Substance 2 synergistically inhibits or enhances the growth of the microorganism under study.

[0059] For example, in the above embodiment, the composition containing 12.5 mM formic acid inhibits the growth of the microorganism by 20.28%, and the composition containing 12.5 mM benzoic acid inhibits the growth of the microorganism by 14.20%. Then the expected value "E" will be calculated as 20.28+(14.20 / 100)*(100 - 20.28)=31.60%. In other words, in the case of additive or non-synergistic inhibition, the composition containing both 12.5 mM formic acid and 12.5 mM benzoic acid is expected to inhibit the growth of the microorganism by 31.60%. However, contrary to this expectation, the experimental determination shows that the growth of the microorganism is inhibited by 55.56%. Therefore, "S" will be calculated as 55.56 - 31.60 = 23.96%, indicating synergistic inhibition.

[0060] It has been found that when treating with a composition comprising i) one or more organic acids and ii) benzoic acid, there is a synergistic regulation effect on microbial growth, especially synergistically inhibiting or enhancing the growth of pathogenic microorganisms. Tables 1 - 7 below show examples of the compositions according to the present invention synergistically inhibiting or enhancing microbial growth. Among them, the column marked "BA (mM)" represents the concentration of benzoic acid contained in the above composition. The column marked "OA (mM)" represents the concentration of the organic acid contained in the above composition. The column marked "BA:OA" represents the molar ratio of benzoic acid to the additional organic acid contained in the above composition. The column marked "Details" represents which organic acid is contained in the composition. In addition, the synergistic inhibition effect (average value "S" of two parallel experiments) is given in parentheses after the organic acid that shows a synergistic inhibition effect when used in combination with benzoic acid.

[0061] Table 1: Synergistic inhibition of the growth of Escherichia coli U5 / 41 (DSMZ 30083) after treatment with combinations of different concentrations of benzoic acid (BA) and different concentrations of organic acids.

[0062]

[0063]

[0064] Table 2: Synergistic inhibition of the growth of Salmonella enterica subsp. enterica serovar Typhimurium SL1344 (DSMZ 24522) after treatment with combinations of different concentrations of benzoic acid (BA) and different concentrations of organic acids.

[0065]

[0066]

[0067]

[0068]

[0069] Table 3: Synergistic inhibition of the growth of Shigella sonnei (ATCC 29930, I virulent, WDCM 00127, CECT 4887 (DSMZ 5570)) after treatment with combinations of different concentrations of benzoic acid (BA) and different concentrations of organic acids.

[0070]

[0071] Table 4: Synergistic inhibition of the growth of Clostridium perfringens (CCUG 47895; type A, as described by Johansson et al. 2004. Vet Microbiol. 99(3 - 4):251 - 257) after treatment with combinations of different concentrations of benzoic acid (BA) and different concentrations of organic acids.

[0072]

[0073]

[0074]

[0075] Table 5: Synergistic enhancement of the growth of Lactobacillus reuteri F 275 (DSMZ 20016) after treatment with combinations of different concentrations of benzoic acid (BA) and different concentrations of organic acids.

[0076]

[0077]

[0078] Table 6: Synergistic enhancement of the growth of Enterococcus faecium D (serotype 11 (DSMZ 20477)) after treatment with combinations of different concentrations of benzoic acid (BA) and different concentrations of organic acids.

[0079]

[0080]

[0081] Table 7: Synergistic enhancement of the growth of Bacillus subtilis strain Marburg (DSMZ 10) after treatment with combinations of different concentrations of benzoic acid (BA) and different concentrations of organic acids.

[0082]

[0083]

Claims

1. A composition for regulating the growth of microorganisms, wherein the composition comprises a) benzoic acid and / or at least one of its salts, and b) at least one additional organic acid and / or at least one of its salts, and wherein the composition regulates the growth of microorganisms in a synergistic mode.

2. The composition according to claim 1, wherein the at least one additional organic acid is selected from the group consisting of short monocarboxylic acids having 1 to 6 carbon atoms, saturated dicarboxylic acids, unsaturated dicarboxylic acids, unsaturated carboxylic acids, saturated carboxylic acids, hydroxycarboxylic acids, aromatic carboxylic acids, and / or keto-carboxylic acids.

3. The composition according to claim 1, wherein the at least one additional organic acid is selected from the group consisting of: formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, 3-methylbutyric acid, 2-methylbutyric acid, 2-ethylbutyric acid, valeric acid, caproic acid, adipic acid, succinic acid, fumaric acid, sorbic acid, oleic acid, stearic acid, caprylic acid, capric acid, lauric acid, lactic acid, malic acid, citric acid, tartaric acid, cinnamic acid, pyruvic acid, gluconic acid, suberic acid, malonic acid, tannic acid, caffeic acid, ellagic acid, perillic acid, and gallic acid.

4. The composition according to claim 1, wherein the at least one additional organic acid is selected from the group consisting of formic acid, acetic acid, propionic acid, fumaric acid, and succinic acid, preferably selected from fumaric acid and succinic acid.

5. The composition according to any one of claims 1-4, wherein at least one of the salts of the organic acid is any one of metal salts such as potassium, sodium, and calcium salts, and ammonium salts.

6. The composition according to claim 1, wherein at least one of the salts of benzoic acid is selected from the group consisting of sodium benzoate, magnesium benzoate, manganese benzoate, potassium hypophosphite, aluminum benzoate, calcium benzoate, and iron benzoate.

7. The composition according to any one of claims 1-6, wherein the molar ratio of the at least one additional organic acid and / or at least one of its salts to benzoic acid and / or at least one of its salts is from 0.001:1 to 500:1, preferably from 0.005:1 to 300:1, more preferably from 0.01:1 to 200:1, such as from 0.01:1 to 0.15:1, from 0.02:1 to 0.1:1, from 0.04:1 to 100:1, from 1:1 to 100:1, from 10:1 to 80:1, and from 20:1 to 40:

1.

8. The composition according to any one of claims 1-6, wherein, For every 1 kg of the composition in solid form or every 1 liter of the composition in liquid form, the at least one additional organic acid and / or at least one of its salts can be provided at any concentration from 0.2 mmol to 100 mmol, in combination with benzoic acid and / or at least one of its salts at any concentration from 0.2 mmol to 20 mmol.

9. The composition according to any one of claims 1-8, wherein the composition comprises benzoic acid and / or at least one of its salts and at least one additional organic acid and / or at least one of its salts as the sole active ingredients for regulating the growth of microorganisms.

10. The composition according to any one of claims 1-9, wherein the microorganism is any one or more of the following genera: Buttiauxella, Citrobacter, Cronobacter, Enterobacter, Escherichia, Edwardsiella, Klebsiella, Phytobacter, Plesiomonas, Pseudoescherichia, Raoultella, Salmonella, Shigella, Proteus, Yersinia, Vibrio, Aeromonas, Clostridium, Pseudomonas, Staphylococcus, Pasteurella, Brachyspira, Campylobacter, Listeria, Streptococcus, Haemophilus, Brucella, Moritella, Tenacibaculum, Lactobacillus, Bifidobacterium, Saccharomyces, Bacillus, Pediococcus, Enterococcus, Streptococcus, Propionibacterium, Pseudomonas and Citrobacter, preferably selected from the group consisting of Escherichia coli, Salmonella enterica, Shigella sonnei, Clostridium perfringens, Lactobacillus reuteri, Enterococcus faecium and Bacillus subtilis.

11. Use of the composition according to any one of claims 1-10 for regulating the growth of microorganisms.

12. Use of the composition according to any one of claims 1-10 for regulating the growth of microorganisms in the gastrointestinal tract of animals, and / or for improving the intestinal health of animals, and / or for treating, improving and / or preventing post-weaning diarrhea (PWD), and / or for improving the growth performance of animals.

13. Use according to claim 11 or 12, wherein the animal is selected from the group consisting of sheep, goats, cattle, pigs (including but not limited to piglets, growing pigs and sows), poultry (including but not limited to turkeys, ducks, quails, guinea fowls, geese, pigeons and chickens) and horses.

14. Use of at least one additional organic acid and / or at least one of its salts in the composition according to any one of claims 1-10 for enhancing the effect of benzoic acid and / or at least one of its salts in regulating the growth of microorganisms.

15. A method for regulating the growth of microorganisms, wherein the method comprises the following steps: a) Providing a composition according to any one of claims 1-10; and b) Contacting the composition of a) with the microorganism.

16. A method for regulating the growth of microorganisms in the gastrointestinal tract of animals, and / or improving the intestinal health of animals, and / or treating, improving and / or preventing post-weaning diarrhea (PWD), and improving the growth performance of animals, wherein the method comprises the following steps: a) Providing a composition according to any one of claims 1-10; and b) Administering the composition of a) to the animal.

17. A method for enhancing the effect of benzoic acid and / or at least one of its salts in regulating the growth of microorganisms, wherein the method comprises: a) Providing a composition according to any one of claims 1-10; and b) Contacting the composition of a) with the microorganism.

18. A method for enhancing the effect of benzoic acid and / or at least one of its salts in regulating the growth of microorganisms in the gastrointestinal tract of animals, and / or in improving the intestinal health of animals, and / or in treating, ameliorating and / or preventing post-weaning diarrhea (PWD) in animals, wherein the method comprises the following steps: a) Providing a composition according to any one of claims 1-10; and b) Administering the composition of a) to the animal.

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