Strain composition for increasing content of short-chain fatty acid and application thereof

Abstract

The invention discloses a strain composition for increasing the content of short-chain fatty acid and application of the strain composition. The strain composition comprises pichia membranaefaciens, saccharomyces cerevisiae, acetobacter pasteurianus, bifidobacterium bifidum, lactobacillus acidophilus, lactobacillus bulgaricus and clostridium butyricum. The fermentation method comprises the following steps: inoculating pichia membranaefaciens and saccharomyces cerevisiae for fermentation; inoculating bifidobacterium bifidum, lactobacillus acidophilus and lactobacillus bulgaricus, and then fermenting; inoculating acetobacter pasteurianus and clostridium butyricum, and then fermenting. The strain composition provided by the invention is easy for large-scale industrial production, low in culture cost, short in period, stable in performance and high in yield; on the premise of ensuring food safety, the yield of the short-chain fatty acid in the plant-based organic acid fermentation puree is increased.

Description

Technical Field

[0001] The present invention relates to the field of microbial fermentation, and in particular to a bacterial composition for increasing the content of short-chain fatty acids and an application thereof. Background Art

[0002] Short-chain fatty acids (SCFA), also known as volatile fatty acids (VFA), are organic fatty acids with 1 to 6 carbon atoms, according to the number of carbon atoms in the carbon number. SCFAs in the human body are mainly produced by the fermentation of indigestible carbohydrates such as dietary fiber, resistant starch, and oligosaccharides by beneficial bacteria such as lactic acid bacteria and bifidobacteria in the colon. Acetic acid, propionic acid, and butyric acid are the main three, and the proportion of the three in the colon is about 60:20:20. SCFA has the functions of regulating the balance of intestinal flora, improving intestinal function, promoting colon epithelial cell proliferation and mucosal growth, regulating intestinal immune function, maintaining water and electrolyte balance, anti-inflammatory, anti-tumor and regulating gene expression. In recent years, a large number of academic journals and clinical medicine journals have described the various health benefits of SCFA and its use in the treatment of various diseases. For example, the 13th Annual Meeting of Danone Nutrition Center, "Research Progress on the Efficacy and Mechanism of Short-Chain Fatty Acids", explained the mechanism and application prospects of SCFA in regulating the balance of intestinal flora, improving intestinal function, and regulating gene expression in the ileum and colon. The article "Re-understanding of Short-Chain Fatty Acids" in Practical Clinical Medicine describes in detail the relationship between SCFA and leukocyte adhesion molecules and cancer cell differentiation and apoptosis, and explains the research and application prospects of SCFA in many aspects such as ulcerative colitis, intestinal inflammatory diseases, colon cancer, liver cancer, and glioma.

[0003] Pitaya, a plant of the Cactaceae family and the genus Hylocereus, is rich in nutrients and has unique functions. It contains plant albumin and anthocyanins that are rare in general plants, rich in vitamins and water-soluble dietary fiber. Albumin is a viscous, gelatinous substance that has a detoxifying effect on heavy metal poisoning. At present, pitaya is mainly eaten fresh, and there are few deep-processed products. Using pitaya as the raw material for enzyme fermentation without adding any preservatives, preservatives and other chemical inputs can not only retain the nutrients of the pitaya itself, but also produce a large amount of short-chain fatty acids during the fermentation process, which are more easily absorbed by the human body.

[0004] In the fermentation industry, the microorganisms used for fermentation play a vital role in the quality of the enzyme. Different fermentation strains with the same raw materials will have great differences in the types and contents of the fermentation end products. At present, there are no corresponding special fermentation bacteria for the production and fermentation of plant-based organic acids in my country. Most of them use natural fermentation, but there are often problems such as slow and incomplete fermentation, and large differences in taste and ingredients between different batches of products, which affect the flavor of the enzyme. Therefore, it is particularly important to screen out strains or strain combinations that can produce rich short-chain fatty acids suitable for plant-based organic acid fermentation. Summary of the invention

[0005] In view of the above-mentioned deficiencies of the current strains rich in short-chain fatty acids, the present invention provides a strain composition for improving the content of short-chain fatty acids and its application, which increases the yield of short-chain fatty acids in plant-based organic acid fermentation puree while ensuring food safety.

[0006] To achieve the above object, the embodiments of the present invention adopt the following technical solutions: On the one hand, a bacterial composition for increasing the content of short-chain fatty acids, the bacterial composition includes Pichia membranaceus ( Pichia membranaefaciens )、Saccharomyces cerevisiae( Saccharomyces cerevisiae )、Acetobacter pasteurianus( Acetobacter pasteurianus )、Bifidobacterium bifidum( Bifidobacterium bifidum )、Acid-tolerant Lactobacillus( Lactobacillus acetotolerans bulgaricus ( Lactobacillus delbrueckii subsp. Bulgarian ) and Clostridium butyricum ( Clostridium butyricum ).

[0007] Preferably, the ratio of the number of viable bacteria of Pichia membranaceus, Saccharomyces cerevisiae, Acetobacter pasteurianus, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus bulgaricus, and Clostridium butyricum is (1-10):(1-10):(1-5):(3-10):(3-10):(3-10):(1-5).

[0008] Preferably, the total viable count of the bacterial composition is 1.0*10 10 / g or above.

[0009] Preferably, the bacterial composition ensures that the short-chain fatty acid content of pitaya pulp after fermentation by the bacterial composition is ≥9%, and the short-chain fatty acids contained are ≥10 types.

[0010] On the other hand, a method for fermentation using the above bacterial composition comprises the following steps: Fermentation after inoculation with Pichia pastoris and Saccharomyces cerevisiae; Fermentation after inoculation with Bifidobacterium bifidum, Lactobacillus acidophilus and Lactobacillus bulgaricus; Fermentation was carried out after inoculation with Acetobacter pasteurianus and Clostridium butyricum.

[0011] Preferably, the fermentation time after inoculation of Pichia pastoris and Saccharomyces cerevisiae is 10 to 15 days.

[0012] Preferably, the fermentation time after inoculation with Bifidobacterium bifidum, acid-fast Lactobacillus and Lactobacillus bulgaricus is 10 to 15 days.

[0013] Preferably, after inoculation with Acetobacter pasteurianus and Clostridium butyricum, the fermentation is performed until the ethanol content is ≤0.2% and the total colony count is ≤100 CFU / g.

[0014] Preferably, the fermentation temperature after inoculation of Pichia membranaceus and Saccharomyces cerevisiae is 25-30°C, the fermentation temperature after inoculation of Bifidobacterium bifidum, Lactobacillus acid-fast and Lactobacillus bulgaricus is 25-37°C, and the fermentation temperature after inoculation of Acetobacter pasteurianus and Clostridium butyricum is 25-37°C.

[0015] In a third aspect, a use of the above-mentioned bacterial composition in the production of short-chain fatty acids by fermentation of edible plant-based organic acids.

[0016] Advantages of the implementation of the present invention: the bacterial composition provided by the present invention is easy to be industrially produced on a large scale, with low culture cost, short cycle, stable performance and large yield; the fermentation method of the bacterial composition provided by the present invention can increase the yield of short-chain fatty acids in plant-based organic acid fermentation raw materials, especially short-chain fatty acids such as acetic acid, butyric acid, lactic acid, malic acid, succinic acid, citric acid, etc., under the premise of ensuring food safety. It is tested that the yield of butyric acid, lactic acid, malic acid, succinic acid, etc. is more than 2 times higher than that of traditional fermentation methods. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] The Pichia pastoris strain mentioned in this article is from Lianzu Biotechnology, with the product number LZ-J63823.

[0019] The brewer's yeast strain mentioned in this article is of Mingzhou Biotechnology brand and its product number is B98028.

[0020] The Acetobacter pasteurianus strain mentioned in this article is CMCC brand and product number GIM1.67.

[0021] The Bifidobacterium bifidum strain mentioned in this article is from Mingzhou Biotechnology and its product number is B86053.

[0022] The acid-resistant Lactobacillus strain mentioned in this article is of the brand Mingzhou Bio, with the product number BMZ139546.

[0023] The Lactobacillus bulgaricus strain mentioned in this article is of the brand Mingzhou Bio, with the product number BMZ124642.

[0024] The Clostridium butyricum strain mentioned in this article is of Mingzhou Biotechnology brand and has the product number B90320.

[0025] The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market.

[0026] In some embodiments and Examples 1-3, the natural culture medium is pitaya pulp. The bacterial composition obtained by the above inoculation culture can be targeted for the three major fermentation stages of pitaya organic acid fermentation liquid fermented by the three-step directional fermentation method as mentioned in the "Red Heart Pitaya Enzyme, Preparation Method and Application" with patent number CN201810157407.3, and has a more ideal adaptability to the pitaya pulp fermentation environment, and can quickly increase the growth rate of the fermentation bacteria in the initial inoculation period, which is conducive to the rapid formation of dominant bacteria to occupy the dominant position in fermentation. And it can effectively increase the yield of various short-chain fatty acids in the fermentation process.

[0027] Embodiment 1

[0028] The bacterial composition of the method of the present invention is inoculated into the pitaya pulp in stages, and the number of live bacteria per single bacterium is 1*10 12 / g and above: The first step is aerobic and anaerobic fermentation. In this stage, the double-cultured yeast is inoculated, and 10.0g / L of Pichia pastoris and Saccharomyces cerevisiae are inoculated at 25-30℃ for about 10-15 days. The second step is complete anaerobic fermentation. In this stage, the lactic acid bacteria are inoculated with 3.0g / L of Bifidobacterium bifidum, Lactobacillus acidophilus, and Lactobacillus bulgaricus, respectively, at 30-35℃ for about 5-10 days. The third step is complete aerobic fermentation. In this stage, 5.0 g / L of composite acetic acid bacteria, butyric acid bacteria, Acetobacter pasteurianus and Clostridium butyricum are inoculated. Ferment at 25-30℃ until the ethanol content is ≤0.2% and the total number of colonies is ≤100 CFU / g, and then the fermentation is terminated.

[0029] Embodiment 2

[0030] The bacterial composition of the method of the present invention is inoculated into the pitaya pulp in stages, and the number of live bacteria per single bacterium is 1*10 12 / g and above: The first step is aerobic and anaerobic fermentation. In this stage, the compound yeast is inoculated, and 3.0g / L of Pichia pastoris and Saccharomyces cerevisiae are inoculated at 25-30℃ for about 10-15 days. The second step is complete anaerobic fermentation. In this stage, the compound lactic acid bacteria are inoculated, and Bifidobacterium bifidum, Lactobacillus acidophilus, and Lactobacillus bulgaricus are inoculated at 5.0g / L each, at 30-35℃, and fermented for about 5-10 days. The third step is complete aerobic fermentation. In this stage, 1.0 g / L of composite acetic acid bacteria, butyric acid bacteria, Acetobacter pasteurianus and Clostridium butyricum are inoculated. Ferment at 25-35℃ until the ethanol content is ≤0.2% and the total number of colonies is ≤100 CFU / g, and then the fermentation is terminated.

[0031] Embodiment 3

[0032] The bacterial composition of the method of the present invention is inoculated into the pitaya pulp in stages, and the number of live bacteria per single bacterium is 1*10 12 / g and above: The first step is aerobic and anaerobic fermentation. In this stage, the compound yeast is inoculated, and 1.0g / L of Pichia pastoris and Saccharomyces cerevisiae are inoculated at 25-30℃ for about 10-15 days. The second step is complete anaerobic fermentation. In this stage, the compound lactic acid bacteria are inoculated, and Bifidobacterium bifidum, Lactobacillus acidophilus, and Lactobacillus bulgaricus are inoculated at 10.0g / L each, at 30-37℃, and fermented for about 5-10 days. The third step is complete aerobic fermentation. In this stage, 5.0 g / L of composite acetic acid bacteria, butyric acid bacteria, Acetobacter pasteurianus and Clostridium butyricum are inoculated. Ferment at 25-30℃ until the ethanol content is ≤0.2% and the total number of colonies is ≤100 CFU / g, and then the fermentation is terminated.

[0033] Comparative Example 1 The pitaya pulp is naturally fermented at 25-30°C until the ethanol content is ≤0.2% and the total colony count is ≤100 CFU / g.

[0034] Comparative Example 2 The bacterial composition of the method of the present invention is inoculated into the pitaya pulp in stages, and the number of live bacteria per single bacterium is 1*10 12 / g and above: The first step is aerobic and anaerobic fermentation. In this stage, the double culture yeast is inoculated, and the membrane-fed Pichia pastoris is inoculated at 3g / L, at 25~30℃, and the fermentation lasts about 10~15 days; The second step is complete anaerobic fermentation. In this stage, the lactic acid bacteria are inoculated with 3g / L of Lactobacillus bulgaricus at 30-35℃ for about 5-10 days. The third step is complete aerobic fermentation. In this stage, 2 g / L of Acetobacter and Butyricum are inoculated. Ferment at 25-28℃ until the ethanol content is ≤0.2% and the total number of colonies is ≤100 CFU / g, and then the fermentation is terminated.

[0035] Comparative Example 3 The bacterial composition of the method of the present invention is inoculated into the pitaya pulp in stages, and the number of live bacteria per single bacterium is 1*10 12 / g and above: The first step is aerobic and anaerobic fermentation. In this stage, the double-cultured yeast is inoculated, and the brewer's yeast is inoculated at 3g / L, at 25~30℃, and the fermentation lasts about 10~15 days; The second step is complete anaerobic fermentation. In this stage, lactic acid bacteria are inoculated, and Bifidobacterium bifidum is inoculated at 3g / L, at 30-35℃, and fermented for about 5-10 days. The third step is complete aerobic fermentation. In this stage, 2 g / L of Acetobacter and Butyricum are inoculated. Ferment at 25-28℃ until the ethanol content is ≤0.2% and the total number of colonies is ≤100 CFU / g, and then the fermentation is terminated.

[0036] The performance data of the products obtained in each embodiment and comparative example are shown in Table 1.

[0037] Table 1: Performance data of the products obtained in Examples 1-3 and Comparative Examples 1-3 The experimental data show that the content of various short-chain fatty acids is significantly higher than that of single-strain fermentation by using a combination of multiple composite strains, and more than doubled compared to natural fermentation. The fermentation cycle of enzymes using a combination of multiple composite strains is shorter than that of single-strain fermentation, and is half the time of natural fermentation. Among the specific organic acids, the content of butyric acid and lactic acid has been greatly increased.

[0038] Advantages of the implementation of the present invention: the bacterial composition provided by the present invention is easy to be industrially produced on a large scale, with low culture cost, short cycle, stable performance and large yield; the fermentation method of the bacterial composition provided by the present invention can increase the yield of short-chain fatty acids in plant-based organic acid fermentation raw materials, especially short-chain fatty acids such as acetic acid, butyric acid, lactic acid, malic acid, succinic acid, citric acid, etc., under the premise of ensuring food safety. It is tested that the yield of butyric acid, lactic acid, malic acid, succinic acid, etc. is more than 2 times higher than that of traditional fermentation methods.

[0039] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A bacterial composition for increasing the content of short-chain fatty acids, characterized in that: The bacterial composition includes Pichia pastoris ( Pichia membranaefaciens )、Saccharomyces cerevisiae( Saccharomyces cerevisiae )、Acetobacter pasteurianus( Acetobacter pasteurianus )、Bifidobacterium bifidum( Bifidobacterium bifidum )、Acid-tolerant Lactobacillus( Lactobacillus acetotolerans bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ) and Clostridium butyricum ( Clostridium butyricum ).

2. The bacterial composition according to claim 1, characterized in that The ratio of the number of live bacteria of Pichia membranaceus, Saccharomyces cerevisiae, Acetobacter pasteurianus, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus bulgaricus and Clostridium butyricum is (1-10):(1-10):(1-5):(3-10):(3-10):(3-10):(1-5).

3. The bacterial composition according to claim 1, characterized in that The total number of viable bacteria in the bacterial composition is 1.0*10 10 / g or above.

4. The bacterial composition according to claim 1, characterized in that The bacterial composition ensures that the short-chain fatty acid content of pitaya pulp after fermentation by the bacterial composition is ≥9%, and the short-chain fatty acids contained are ≥10 types.

5. A method for fermentation using the bacterial composition according to any one of claims 1 to 4, characterized in that: The method uses fruit or plant puree as a fermentation substrate, and comprises the following steps: Fermentation after inoculation with Pichia pastoris and Saccharomyces cerevisiae; Fermentation after inoculation with Bifidobacterium bifidum, Lactobacillus acidophilus and Lactobacillus bulgaricus; Fermentation was carried out after inoculation with Acetobacter pasteurianus and Clostridium butyricum.

6. The method according to claim 5, characterized in that The fermentation time after inoculating Pichia pastoris and Saccharomyces cerevisiae is 10 to 15 days.

7. The method according to claim 5, characterized in that The fermentation time after inoculation of Bifidobacterium bifidum, acid-fast Lactobacillus and Lactobacillus bulgaricus is 10 to 15 days.

8. The method according to claim 5, characterized in that The method comprises inoculating Acetobacter pasteurianus and Clostridium butyricum and fermenting the mixture until the ethanol content is less than or equal to 0.2% and the total colony count is less than or equal to 100 CFU / g.

9. The method according to claim 5, characterized in that The fermentation temperature after inoculation of Pichia membranaceus and Saccharomyces cerevisiae is 25-30°C, the fermentation temperature after inoculation of Bifidobacterium bifidum, Lactobacillus acidotolerant and Lactobacillus bulgaricus is 25-37°C, and the fermentation temperature after inoculation of Acetobacter pasteurianus and Clostridium butyricum is 25-37°C.

10. Use of the bacterial composition according to any one of claims 1 to 3 in the production of short-chain fatty acids by fermentation of edible plant-based organic acids.

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