Bifidobacterium-containing complex microbial inoculant and application thereof

Abstract

The invention discloses a complex microbial inoculant containing bifidobacterium and application of the complex microbial inoculant. Comprising two or more of bifidobacterium lactis BL9, bifidobacterium longum subsp. Longum KS1, clostridium pratensis GZSY3214, Leuconostoc mesenteroides PM10, and Gluconacetobacter xylinus Q1, and further comprises a preparation method of the bifidobacterium lactis BL9, a preparation method of the bifidobacterium longum subsp. Longum KS1, a preparation method of the bifidobacterium longum subsp. Longum GZSY3214, a preparation method of the bifidobacterium longum, a preparation method of the bifidobacterium longum, a preparation method of the bifidobacterium longum and a preparation method of the bifidobacterium longum, and a preparation method of the bifidobacterium longum. The complex microbial inoculant can be used for preparing milk powder, weight-losing food and fruit and vegetable beverages, and is wide in application range.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a composite bacterial agent containing bifidobacteria and application thereof. Background Art

[0002] Bifidobacterium is a type of anaerobic probiotic bacteria beneficial to human health, primarily found in the intestines, mouths, and vaginas of humans and animals. As a core member of the intestinal flora, it plays an important role in maintaining microecological balance, promoting digestion, and enhancing immunity. Bifidobacteria ferment dietary fiber to produce short-chain fatty acids (such as acetic acid and lactic acid), which lower intestinal pH and inhibit harmful bacteria (such as Escherichia coli and Salmonella); relieve constipation, diarrhea, and irritable bowel syndrome (IBS); stimulate the intestinal immune system, and regulate inflammatory responses.

[0003] Infants and children need milk and milk powder during their development. Bifidobacteria are often added to milk and milk powder. Since the human intestine contains a variety of bifidobacteria and other beneficial bacteria, supplementing with a single type of bifidobacterium is not very beneficial for infants and children. It is necessary to find a compound strain of bacteria to improve its effectiveness.

[0004] Obesity is a chronic metabolic disease caused by multiple factors, which is mainly manifested by excessive accumulation of fat in the body, leading to weight gain and potentially causing various health problems. The World Health Organization (WHO) defines obesity as a body mass index (BMI) ≥ 30 kg / m 2 (Asian population may have BMI ≥ 27.5 kg / m 2 As a standard). Obesity is a preventable and controllable chronic disease that requires comprehensive management with diet, exercise, psychological and medical means. If BMI is ≥30 or accompanied by complications (such as diabetes, hypertension), it is recommended to seek medical attention as soon as possible to develop a personalized plan. Obesity is closely related to intestinal flora imbalance, and probiotics (such as bifidobacteria, lactic acid bacteria, etc.) may assist in weight management by regulating intestinal microecology, improving metabolism and reducing inflammation. Currently, there are few beneficial bacteria and beneficial bacteria combinations for the treatment of obesity, and more research and development efforts are needed.

[0005] The preparation of fermented fruit and vegetable beverages is currently a focus of research. Beverages made from fruits or vegetables fermented with natural microorganisms (yeast, lactic acid bacteria, etc.) are rich in probiotics, enzymes, and organic acids, and have unique flavors and potential health benefits. Free amino acids (FAAs) are small molecules produced by microbial enzymatic hydrolysis of proteins during the fermentation process. They are easily absorbed by the human body and affect the flavor, nutritional, and functional properties of beverages. They are the most important nutrients and flavor substances in fermented beverages. How to increase the content of free amino acids in fermented fruits and vegetables is a problem that people in this field need to solve. Summary of the Invention

[0006] The purpose of the present invention is to provide a composite bacterial agent containing bifidobacteria and application thereof.

[0007] A composite bacterial agent containing bifidobacteria, comprising two or more of Bifidobacterium lactis BL9, Bifidobacterium longum subsp. longum KS1, Faecalibacterium prausnitzii GZSY3214, Leuconostoc mesenteroides PM10, and Gluconacetobacter xylinus Q1.

[0008] The Bifidobacterium lactis BL9 was purchased from the China General Microbial Culture Collection Center under the China National Center for Type Culture Collection with a collection number of CGMCC No. 14536; the Bifidobacterium longum subsp. longum KS1 was purchased from the China Center for Type Culture Collection with a collection number of CCTCC NO: M 20221509; the Faecalibacterium prausnitzii GZSY3214 was purchased from the Guangdong Provincial Center for Type Culture Collection with a collection number of GDMCC No: 64992; the Leuconostoc mesenteroides PM10 was purchased from the China Center for Type Culture Collection with a collection number of CC TCC NO: M 20232146; and the Gluconacetobacter xylosus xylinus)Q1, purchased from China Center for Type Culture Collection, with the collection number CCTCC No: M2014353.

[0009] Preferably, the composite bacterial agent comprises Bifidobacterium lactis BL9 and Bifidobacterium longum subsp. longum KS1 in a mass ratio of (2-4):1; the number of viable bacteria of Bifidobacterium lactis BL9 in the bacterial agent is 1-4x10 8 cfu / mL, the viable count of Bifidobacterium longum subsp. longum KS1 is 2-5x10 8 cfu / mL.

[0010] Preferably, the composite bacterial agent comprises Bifidobacterium lactis BL9, Faecalibacterium prausnitzii GZSY3214 and Leuconostoc mesenteroides PM10 in a mass ratio of (1-5): (1-3): 1; the number of viable bacteria of Bifidobacterium lactis BL9 in the bacterial agent is 1-3x10 8 cfu / mL, and the viable count of Faecalibacterium prausnitzii GZSY3214 was 1-3x10 8 cfu / mL, the number of viable bacteria of Leuconostoc mesenteroides PM10 is 1-4x10 8 cfu / mL.

[0011] Preferably, the composite bacterial agent comprises Leuconostoc mesenteroides PM10 and Gluconacetobacter xylinus Q1; the number of viable bacteria of Leuconostoc mesenteroides PM10 in the bacterial agent is 2-5x10 8 cfu / mL, the viable count of Gluconacetobacter xylinus Q1 is 1-4x10 8 cfu / mL.

[0012] The invention relates to an application of the composite bacterial agent containing bifidobacteria in the preparation of milk powder for promoting the improvement of memory ability.

[0013] The invention relates to an application of the composite bacterial agent containing bifidobacteria in the preparation of fermented fruit beverages.

[0014] The invention relates to an application of the composite bacterial agent containing bifidobacteria in the preparation of fermented vegetable beverages.

[0015] Application of the bifidobacterium-containing composite bacterial agent in the preparation of food additives.

[0016] The invention relates to an application of the composite bacterial agent containing bifidobacteria in the preparation of weight-loss food.

[0017] The beneficial effects of the present invention are as follows: the composite bacterial agent Bifidobacterium lactis BL9 and Bifidobacterium longum subsp. longum KS1 of the present invention can promote neural development and improve memory, and is suitable for preparing milk powder for children; the composite bacterial agent Bifidobacterium lactis BL9, Faecalibacterium prausnitzii GZSY3214 and Leuconostoc mesenteroides PM10 can promote weight loss, and is suitable for preparing weight loss food or food additives; the composite bacterial agent Leuconostoc mesenteroides PM10 and Gluconacetobacter xylinus Q1 can increase the content of amino acid nitrogen (free amino acids) in fermented fruits and vegetables, and improve the flavor. DETAILED DESCRIPTION

[0018] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0019] Example 1

[0020] The purchased Bifidobacterium lactis BL9 and Bifidobacterium longum subsp. longum KS1 were activated and cultured using conventional methods, and the number of viable bacteria in the culture medium of Bifidobacterium lactis BL9 was adjusted to 3×10 8 cfu / mL, the number of viable bacteria in the KS1 culture medium of Bifidobacterium longum subsp. longum was 3x10 8 cfu / mL.

[0021] Morris Water Maze (MWM) is a classic behavioral experiment widely used in neuroscience and psychology to study the spatial learning and memory ability of animals. Newly weaned rats were selected and first fed with basal feed for 1 week to adapt to the environment. They were then randomly divided into control group, BL9 group, KS1 group, and BL9 and KS1 group according to the random number table method, with 10 rats in each group. The BL9 group was gavaged with 1 mL of Bifidobacterium lactis BL9 culture medium every day, the KS1 group was gavaged with 1 mL of Bifidobacterium longum subsp. longum KS1 culture medium every day, the BL9 and KS1 groups were gavaged with 0.75 mL of Bifidobacterium lactis BL9 culture medium and 0.25 mL of Bifidobacterium longum subsp. longum KS1 culture medium every day, and the control group was gavaged with 1 mL of water once a day. The mice had free access to food and water for 28 days. A five-day water maze experiment was performed five days before the end of the feeding period.

[0022] The water maze apparatus consisted of a circular water tank, an automatic video recorder, and a computer analysis and processing system. The water tank had a diameter of 1.5 meters and a height of 0.5 meters, and the water temperature was controlled at 23 ± 2°C. The circular water tank was divided into four quadrants, with a marked entry point in the middle of each quadrant for the rat to enter. The platform was located underwater in the second quadrant. The water maze experiment was conducted according to the method described by Guo Yanfen et al. (Guo Yanfen et al., 2011).

[0023] The experimental results were statistically analyzed using SPSS 24.0 software, and the measurement data were analyzed using The data were expressed as mean ± standard deviation. The Kolmogorov-Smirnov test was used to test the normality of the data. For data that met the normal distribution, the t-test was used to compare the mean differences between the two groups. The difference was considered statistically significant when P < 0.05. The results are shown in Table 1:

[0024] Table 1

[0025]

[0026] Note: * represents P < 0.05 compared with BL9 and KS1 groups, ** represents P < 0.01.

[0027] This experiment demonstrated that both Bifidobacterium lactis BL9 and Bifidobacterium longum subsp. longum KS1 have the ability to improve mammalian memory and promote neural development. The combined use of the two bacteria has a synergistic effect and is suitable for the development of milk powder and functional foods that promote children's neural development and memory ability.

[0028] Example 2

[0029] The purchased Bifidobacterium lactis BL9, Faecalibacterium prausnitzii GZSY3214 and Leuconostoc mesenteroides PM10 were activated and cultured using conventional methods, and the number of viable bacteria in the culture medium of Bifidobacterium lactis BL9 was adjusted to 2×10 8 cfu / mL, the number of viable bacteria in the culture medium of Faecalibacterium prausnitzii GZSY3214 is 2x10 8 cfu / mL, the number of viable bacteria in PM10 culture medium of Leuconostoc mesenteroides is 2x10 8 cfu / mL.

[0030] SPF male C57BL / 6J mice (8 weeks old, 18-22 g) were randomly divided into 5 groups, with 10 mice in each group, namely blank group, model group, group 1, group 2 and group 3. The mice were first adaptively fed for 7 days. Starting from the 8th day, the model group mice and groups 1-3 mice were fed with high-fat diet. The blank group still used ordinary diet. Group 1 was gavaged with 0.4 mL of Bifidobacterium lactis BL9 culture medium and 0.6 mL of Faecalibacterium prausnitzii GZSY3214 culture medium every day; Group 1 was gavaged with 0.4 mL of Bifidobacterium lactis BL9 culture medium and 0.6 mL of Leuconostoc mesenteroides PM10 culture medium every day; Group 3 was gavaged with 0.4 mL of Bifidobacterium lactis BL9 culture medium and 0.4 mL of Faecalibacterium prausnitzii GZSY3214 culture medium every day. prausnitzii) GZSY3214 culture medium and 0.2mL Leuconostoc mesenteroides PM10 culture medium; the blank group and model group were gavaged with equal volumes of PBS solution. After the experiment, blood was collected and the mice were killed. The abdominal fat of the mice was removed and weighed. The experimental results were statistically analyzed using SPSS 24.0 software. The quantitative data results were analyzed using The data were expressed as mean ± standard deviation. The Kolmogorov-Smirnov test was used to test the normality of the data. For data that met the normal distribution, the t-test was used to compare the mean differences between the two groups. The difference was considered statistically significant when P < 0.05. The results are shown in Table 2:

[0031] Table 2

[0032]

[0033] Note: * represents P < 0.05 compared with group 3, ** represents P < 0.01.

[0034] As can be seen from Table 2, Bifidobacterium lactis BL9, Faecalibacterium prausnitzii GZSY3214, and Leuconostoc mesenteroides PM10 can significantly reduce the abdominal fat weight of mice and can be developed into food additives and foods with weight loss effects.

[0035] Example 3

[0036] The purchased Leuconostoc mesenteroides PM10 and Gluconacetobacter xylinus Q1 were activated and cultured using conventional methods to adjust the number of viable bacteria in the culture medium of Leuconostoc mesenteroides PM10 to 3×10 8 cfu / mL, the number of viable bacteria in the Q1 culture medium of Gluconacetobacter xylinus was 3x10 8 cfu / mL.

[0037] The fruit fermented beverage is prepared according to the following steps: washing snow pear and Bali pineapple, peeling and cored, mixing the peeled and cored snow pear and Bali pineapple in a mass ratio of 1:1, adding 6 times the weight of water, beating until no obvious particulate matter is present to obtain a slurry, adding 5% sucrose by weight of the slurry, mixing evenly, sterilizing at high temperature at 115°C for 20 minutes, cooling to room temperature (25°C) to obtain a mixed solution, adding a fermentation agent with a total amount of 1% of the volume of the mixed solution, fermenting at 33°C for 96 hours, filtering, sterilizing at high temperature at 115°C for 20 minutes, cooling to room temperature (25°C), and canning to obtain the fruit fermented beverage. Among them, the access scheme of the fermentation bacteria agent is as follows: Group 1: accessed with 1% Leuconostoc mesenteroides PM10 culture solution; Group 2: accessed with 1% Gluconacetobacter xylinus Q1 culture solution; Group 3: accessed with 0.5% Leuconostoc mesenteroides PM10 and 0.5% Gluconacetobacter xylinus Q1, and the control group did not access any bacteria agent.

[0038] The amino acid nitrogen content in the fruit fermented beverages prepared by groups 1-3 and the control group was determined according to the method specified in "GB 5009.235-2016 National Food Safety Standard Determination of Amino Acid Nitrogen in Foods"; SPSS 24.0 software was used for statistical analysis, and the quantitative data results were used to analyze the results. The data were expressed as mean ± standard deviation. The Kolmogorov-Smirnov test was used for data normality test. For data that met the normal distribution, the t-test was used to compare the mean differences between the two groups. The difference was considered statistically significant when P < 0.05. The results are shown in Table 3:

[0039] Table 3

[0040]

[0041] Note: * represents P < 0.05 compared with group 3, ** represents P < 0.05.

[0042] Example 4

[0043] The purchased Leuconostoc mesenteroides PM10 and Gluconacetobacter xylinus Q1 were activated and cultured using conventional methods to adjust the number of viable bacteria in the culture medium of Leuconostoc mesenteroides PM10 to 3×10 8 cfu / mL, the number of viable bacteria in the Q1 culture medium of Gluconacetobacter xylinus was 3x10 8 cfu / mL.

[0044] The vegetable fermented beverage is prepared according to the following steps: high-calcium vegetables are washed, 5 times the weight of water is added, and pulped until no obvious particles are present to obtain a slurry, 6% sucrose by weight of the slurry is added, and mixed evenly, sterilized at high temperature at 115°C for 20 minutes, cooled to room temperature (25°C) to obtain a mixed liquid, and fermentation agents are added with a total amount of 1% of the volume of the mixed liquid, fermented at 33°C for 96 hours, filtered, sterilized at high temperature at 115°C for 20 minutes, cooled to room temperature (25°C), and canned to obtain a fruit fermented beverage. Among them, the access scheme of the fermentation bacteria agent is as follows: Group 1: accessed with 1% Leuconostoc mesenteroides PM10 culture solution; Group 2: accessed with 1% Gluconacetobacter xylinus Q1 culture solution; Group 3: accessed with 0.5% Leuconostoc mesenteroides PM10 and 0.5% Gluconacetobacter xylinus Q1, and the control group did not access any bacteria agent.

[0045] The amino acid nitrogen content in the fruit fermented beverages prepared by groups 1-3 and the control group was determined according to the method specified in "GB 5009.235-2016 National Food Safety Standard for Determination of Amino Acid Nitrogen in Foods"; SPSS24.0 software was used for statistical analysis, and the quantitative data results were used to analyze the results. The data were expressed as mean ± standard deviation. The Kolmogorov-Smirnov test was used for data normality test. For data that met the normal distribution, the t-test was used to compare the mean differences between the two groups. The difference was considered statistically significant when P < 0.05. The results are shown in Table 4:

[0046] Table 4

[0047]

[0048]

[0049] Note: * represents P < 0.05 compared with group 3, ** represents P < 0.05.

[0050] Example 5

[0051] Bifidobacterium lactis BL9 and Bifidobacterium longum subsp. longum KS1 were cultured according to conventional methods to obtain culture fluids; the culture fluids were centrifuged to obtain bacterial cells; the bacterial cells were washed three times with a phosphate buffer having a pH of 7.2 and then resuspended with a trehalose freeze-drying protective agent having a trehalose concentration of 100 g / L (the mass ratio of the freeze-drying protective agent to the bacterial cells was 2:1) to obtain bacterial cell concentrations of 2×10 8 cfu / mL, 3×10 8 cfu / mL of the resuspension; and freeze-drying the resuspension using a vacuum freezing method to obtain Bifidobacterium lactis BL9 bacterial powder and Bifidobacterium longum subsp. longum KS1 bacterial powder.

[0052] Bifidobacterium lactis BL9 powder, Bifidobacterium longum subsp. longum KS1 powder and maltodextrin were mixed at a ratio of 1:1:1 to obtain a solid beverage.

[0053] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A composite bacterial agent containing bifidobacteria, characterized in that Including two or more of Bifidobacterium lactis BL9, Bifidobacterium longum subsp. longum KS1, Faecalibacterium prausnitzii GZSY3214, Leuconostoc mesenteroides PM10, and Gluconacetobacter xylinus Q1.

2. The composite bacterial agent containing bifidobacteria according to claim 1, characterized in that The Bifidobacterium lactis BL9 has a preservation number of CGMCC No. 14536; the Bifidobacterium longum subsp. longum KS1 has a preservation number of CCTCC NO: M 20221509; the Faecalibacterium prausnitzii GZSY3214 has a preservation number of GDMCC No: 64992; the Leuconostoc mesenteroides PM10 has a preservation number of CCTCC NO: M20232146; and the Gluconacetobacter xylinus Q1 has a preservation number of CCTCC No: M2014353.

3. The composite bacterial agent containing bifidobacteria according to claim 1, characterized in that The composite bacterial agent comprises Bifidobacterium lactis BL9 and Bifidobacterium longum subsp. longum KS1 in a mass ratio of (2-4):1; the number of viable bacteria of Bifidobacterium lactis BL9 in the bacterial agent is 1-4×10 8 cfu / mL, the viable count of Bifidobacterium longum subsp. longum KS1 is 2-5x10 8 cfu / mL.

4. The composite bacterial agent containing bifidobacteria according to claim 1, characterized in that The composite bacterial agent comprises Bifidobacterium lactis BL9, Faecalibacterium prausnitzii GZSY3214, and Leuconostoc mesenteroides PM10 in a mass ratio of (1-5):(1-3):1; the number of viable bacteria of Bifidobacterium lactis BL9 in the bacterial agent is 1-3×10 8 cfu / mL, and the viable count of Faecalibacterium prausnitzii GZSY3214 was 1-3x10 8 cfu / mL, the number of viable bacteria of Leuconostoc mesenteroides PM10 is 1-4x10 8 cfu / mL.

5. The composite bacterial agent containing bifidobacteria according to claim 1, characterized in that The composite bacterial agent comprises Leuconostoc mesenteroides PM10 and Gluconacetobacter xylinus Q1; the number of viable bacteria of Leuconostoc mesenteroides PM10 in the bacterial agent is 2-5×10 8 cfu / mL, the viable count of Gluconacetobacter xylinus Q1 is 1-4x10 8 cfu / mL.

6. Use of the bifidobacterium-containing composite bacterial agent according to claim 1 in the preparation of milk powder for promoting memory improvement.

7. Use of the bifidobacterium-containing composite bacterial agent according to claim 1 in the preparation of fermented fruit beverages.

8. Use of the bifidobacterium-containing composite bacterial agent according to claim 1 in the preparation of fermented vegetable beverages.

9. Use of the composite bacterial agent containing bifidobacteria according to claim 1 in the preparation of food additives.

10. Use of the composite bacterial agent containing bifidobacteria according to claim 1 in the preparation of weight-loss food.

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