Probiotic composition, bean-based fermented food and application of bean-based fermented food

Abstract

The invention discloses a probiotic composition, a bean-based fermented food and application of the bean-based fermented food. The probiotic composition is prepared from bacillus subtilis SP-8-5, lactobacillus plantarum YM-4-3, lactobacillus plantarum AY01 and pediococcus acidilactici LH01. The fermented soya bean has the advantages that the prepared fermented soya bean can regulate the composition of intestinal microbial populations, reduce the abundance of harmful bacteria, improve the abundance of beneficial bacteria, enhance the immunity of intestinal tracts and maintain the integrity of intestinal barriers of people with exercise requirements through a specific compounding method. Meanwhile, plasma serine can be reduced, cell damage can be reduced, sleep quality can be improved, and the health-care product can be used as an effective exercise supplement to be applied to people with exercise requirements.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a probiotic composition, a fermented soy-based food and application thereof. BACKGROUND

[0002] Intestinal microbes regulate the functions of the body's nervous system and immune system through various metabolic pathways. When the diversity of the intestinal microbiota decreases, it can cause changes in various systems of the body through the microbiome-gut-brain axis, leading to psychological distress and other problems. Microbiota depletion is one of the potential causes of impaired athletic performance. Sleep quality is influenced by various factors such as mental health, depression, anxiety, and stress, and improving sleep quality can improve mental health.

[0003] Currently, dietary supplements for improving sleep quality and regulating intestinal flora mainly focus on the supplementation of amino acids, melatonin, vitamins, and various inorganic ions, with relatively few probiotic supplements. Taking appropriate probiotic supplements can improve the structure of the human intestinal flora, allowing the host to spontaneously or rely on the intestinal flora to continuously produce beneficial substances and regulate the body's metabolism. People with exercise needs have higher requirements for physical fitness. Currently, there are relatively few probiotic supplements suitable for people with exercise needs, so it is necessary to develop a probiotic supplement suitable for people with exercise needs. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a probiotic composition and fermented douchi suitable for people with exercise needs.

[0005] The present application provides a probiotic composition comprising Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, and Pediococcus acidilactici LH01. Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, and Pediococcus acidilactici LH01 are all selected by the inventors and have been preserved.

[0006] Bacillus subtilis SP-8-5 Bacillus subtilis SP-8-5) was preserved in the China Center for Type Culture Collection on March 06, 2023, with the preservation number CCTCC NO: M2023254, and the address of preservation is Luo Jia Mountain, Bao Yi Road, Wuchang District, Wuhan City, Hubei Province, China, and the contact number is 027-68754052.

[0007] Lactobacillus plantarum YM-4-3 Lactobacillus plantarumYM-4-3 was deposited at the China Center for Type Culture Collection on March 6, 2023, with accession number CCTCCNO:M2023253. Address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. Tel: 027-68754052.

[0008] Lactobacillus plantarum AY01 ( Lactobacillus plantarum AY01 was deposited at the China Center for Type Culture Collection on September 27, 2022, with accession number CCTCC M 20221517. Address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. Tel: 027-68754052.

[0009] Pediococcus acidilactici LH01( Pediococcusacidilactici LH01 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 26, 2023, with accession number GDMCCNo: 63840; the deposit address is: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province; contact number: 020-87137633.

[0010] Furthermore, the ratio of Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, and Pediococcus lactis LH01 is 2:1:1:1.

[0011] The present invention also provides a fermented soybean paste, which is made by fermenting soybeans using a probiotic composition.

[0012] Furthermore, the preparation method of fermented soybeans is as follows: Weigh 10-20 kg of soybeans, soak them in purified water for 12 hours, then steam them at 100℃ for 20 minutes. After cooling to room temperature, inoculate with Bacillus subtilis SP-8-5 to a final concentration of 1.0 × 10⁻⁶. 7 ~1.0×10 8 After fermentation at 37℃ for 1 day at CFU / mL, a mixed fermentation broth containing three strains of *Lactobacillus plantarum* YM-4-3, *Lactobacillus plantarum* AY01, and *Pediococcus lactis* LH01 was inoculated to achieve a final concentration of 1.0 × 10⁻⁶ CFU / mL. 8 ~1.0×10 9 CFU / mL; after mixing, ferment at 37℃ for 4 days to obtain fermented soybean paste.

[0013] This invention also relates to the application of probiotic compositions in the preparation of products that regulate intestinal microbial population composition, reduce plasma serine, reduce cell damage, and improve sleep quality. The probiotic compositions include Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, and Pediococcus lactis LH01.

[0014] Further, the product is a sports supplement, which is fermented soy sauce prepared by using the probiotic composition as a substrate.

[0015] In the preparation of the fermented soy sauce, in addition to the bacterial liquid, a bacterial agent dosage form such as a freeze-dried composite bacterial powder can also be selected according to actual needs. Common adjuvants in the art can also be added.

[0016] The sports supplement can also select other fermentation substrates and use the probiotic composition of the present application to prepare other fermented foods.

[0017] The present application also relates to the application of fermented soy sauce in regulating the composition of intestinal microbial population, reducing plasma serine, reducing cell damage, and improving sleep quality. The probiotic composition includes Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, and Pediococcus acidilactici LH01.

[0018] The present application has the following advantages: The fermented soy sauce prepared by the present application can regulate the composition of intestinal microbial population, reduce the abundance of harmful bacteria, increase the abundance of beneficial bacteria, enhance intestinal immunity, and maintain the intestinal barrier integrity of people with exercise needs through a specific compounding method. At the same time, it can reduce plasma serine, reduce cell damage, and improve sleep quality, and can be used as an effective sports supplement for people with exercise needs. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Figure 3 is a diagram of the change in the composition ratio of human intestinal microorganisms by fermented soy sauce of 3.1 in Example 3; Figure 2 Figure 4 is a metabolite heat map of targeted neurotransmitter metabolomics of 3.2 in Example 3; Figure 3 Figure 5 is a sleep quality column chart of 3.3 in Example 3. DETAILED DESCRIPTION

[0020] The specific embodiments of the present application will be further described below in conjunction with the accompanying drawings. It should be noted that the description of these embodiments is used to help understand the present application, but does not constitute a limitation on the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0021] Example 1 A probiotic composition includes Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, and Pediococcus acidilactici LH01.

[0022] Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, Pediococcus acidilactici LH01 have been preserved.

[0023] Bacillus subtilis SP-8-5 Bacillus subtilis SP-8-5) has been preserved in the China Center for Type Culture Collection on March 6, 2023, with a preservation number of CCTCC NO: M2023254, and an address of Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a contact number of 027-68754052.

[0024] Lactobacillus plantarum YM-4-3 Lactobacillus plantarum YM-4-3) has been preserved in the China Center for Type Culture Collection on March 6, 2023, with a preservation number of CCTCC NO: M2023253, and an address of Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a contact number of 027-68754052.

[0025] Lactobacillus plantarum AY01 Lactobacillus plantarum AY01) has been preserved in the China Center for Type Culture Collection on September 27, 2022, with a preservation number of CCTCC M 20221517, and an address of Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a contact number of 027-68754052.

[0026] Pediococcus acidilactici LH01 Pediococcusacidilactici LH01) has been preserved in the Guangdong Microbial Culture Collection Center on September 26, 2023, with a preservation number of GDMCC No: 63840, and an address of Experimental Building 5th Floor, No. 100, Martyrs Road, Yuexiu District, Guangzhou City, Guangdong Province, with a contact number of 020-87137633.

[0027] Specifically, the ratio of Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, and Pediococcus acidilactici LH01 is 2:1:1:1.

[0028] Example 2 A fermented douchi is prepared as follows: After 10-20 kg of soybeans are soaked in pure water for 12 h, they are cooked at 100℃ for 20 min and then cooled to room temperature. Bacillus subtilis SP-8-5 is inoculated to make the final concentration 1.0×10 7 ~1.0×10 8 CFU / mL, and then fermented at 37℃ for 1 d. Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01, and Pediococcus acidilactici LH01 are inoculated to make the final concentration 1.0×10 8 ~1.0×109 CFU / mL; after mixing, fermentation at 37℃ for 4 d, fermented douchi can be obtained.

[0029] The method for preparing the bacterial solution is as follows: Preparation of Bacillus subtilis SP-8-5 bacterial solution: the Bacillus subtilis SP-8-5 stored at low temperature was taken out, inoculated into NB liquid medium at an inoculation amount of 4‰, and incubated at 37℃ in a constant temperature incubator for 24 h, then subcultured once more. The bacterial solution activated twice and in logarithmic growth phase was inoculated into 50 mL NB liquid medium for expansion culture. The concentration of the cultured bacterial solution was calculated by viable bacterial counting method, and the concentration was adjusted to 1×10 7 ~1×10 9 CFU / mL, for subsequent experiments.

[0030] Preparation of Lactobacillus plantarum YM-4-3 bacterial solution: the Lactobacillus plantarum YM-4-3 stored at low temperature was taken out, inoculated into MRS liquid medium at an inoculation amount of 4‰, and incubated at 37℃ in a constant temperature incubator for 24 h, then subcultured once more. The bacterial solution activated twice and in logarithmic growth phase was inoculated into 50 mL MRS liquid medium for expansion culture. The concentration of the cultured bacterial solution was calculated by viable bacterial counting method, and the concentration was adjusted to 1×10 9 ~1×10 11 CFU / mL, for subsequent experiments.

[0031] Preparation of Lactobacillus plantarum AY01 bacterial solution: the method was the same as that for preparing the YM-4-3 bacterial solution, except that the bacterial strain was replaced by Lactobacillus plantarum AY01.

[0032] Preparation of Pediococcus acidilactici LH01 bacterial solution: the method was the same as that for preparing the YM-4-3 bacterial solution, except that the bacterial strain was replaced by Pediococcus acidilactici LH01.

[0033] Preparation of mixed fermentation bacterial solution: the Lactobacillus plantarum YM-4-3 bacterial solution, Lactobacillus plantarum AY01 bacterial solution, and Pediococcus acidilactici LH01 bacterial solution prepared above were inoculated into 500 mL MRS liquid medium at an inoculation amount of 1% and cultured for 18-24 h. The concentration of the cultured mixed bacterial solution was calculated by viable bacterial counting method, and the concentration was adjusted to 1×10 9 ~1×10 11 CFU / mL, for subsequent experiments.

[0034] MRS liquid medium: 10 g of proteose peptone, 8 g of beef powder, 4 g of yeast powder, 2 g of potassium phosphate dibasic, 5 g of sodium acetate, 2 g of triammonium citrate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 20 g of glucose, 1 mL of Tween-80, and distilled water were added to make up to 1000 mL, mixed, and adjusted to pH 6.2±0.2.

[0035] NB liquid medium: 10 g of proteose peptone, 3 g of beef extract, 5 g of sodium chloride, 1 g of glucose, add distilled water to 1000 mL, mix, adjust pH to 7.4±0.1.

[0036] The application also relates to application of the probiotic composition in regulating intestinal microbial population composition, reducing plasma serine, reducing cell damage and improving sleep quality, and the probiotic composition comprises Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01 and Pediococcus acidilactici LH01.

[0037] Specifically, the product is a sports supplement, which is fermented soy sauce prepared by using the probiotic composition and taking soybeans as a substrate.

[0038] During preparation of the fermented soy sauce, in addition to the bacterial liquid, a bacterial agent dosage form such as a freeze-dried composite bacterial powder can also be selected according to actual needs. Common auxiliary materials in the field can also be added.

[0039] The sports supplement can also select other fermentation substrates, and other fermented foods can be prepared by using the probiotic composition.

[0040] The application also relates to application of the fermented soy sauce in regulating intestinal microbial population composition, reducing plasma serine, reducing cell damage and improving sleep quality, and the probiotic composition comprises Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01 and Pediococcus acidilactici LH01.

[0041] In order to verify the application effect of the fermented soy sauce, the following test is carried out: Example 3 The subjects in the study include 10 people, including 6 males and 4 females. The basic information of the subjects is shown in Table 1. All the subjects are randomly divided into a probiotic group (group A, n=5) and a control group (group B, n=5). The experimental period is 2 weeks. From day 1 to day 7, all the subjects are given unified diet and training programs. From day 8 to day 14, the subjects in group A are supplemented with the fermented soy sauce prepared by the application before dinner every day, and the subjects in group B are supplemented with a placebo, once a day, 50 g each time. During the whole experimental period, all the subjects execute unified diet and training programs.

[0042] The placebo is soybeans which are high-temperature cooked, cooled and then packaged. The fermented soy sauce prepared by the application and the placebo have no difference in specification, model and appearance, and the taste is basically the same after seasoning.

[0043] Table 1 Basic information of the probiotic group (group A) and the control group (group B)

[0044] Collection of plasma and fecal samples Plasma: 5 mL of venous blood was collected in a heparin sodium blood collection tube under fasting condition, and plasma was collected by centrifugation at 3000 rpm / min for 10 min using a low-temperature centrifuge for subsequent plasma neurotransmitter-targeted metabolomics.

[0045] Feces: Fecal samples were collected before and after the experiment and stored frozen throughout the experiment.

[0046] 3.1 Fecal microbiome sequencing Genomic DNA of the feces collected during the experiment was extracted (DNA extraction used a fecal genomic extraction kit), and the V3-V4 region of the 16S rRNA gene was amplified using primers 515F and 909R; the reaction system was as follows: 100 ng of DNA template, 2 μL of each of the upstream and downstream primers (10 μmol / L), and 25 μL of 2x Taq Master Mix (Novozyme). The reaction program was as follows: 95 °C pre-denaturation for 3 min, 95 °C denaturation for 15 s, 55 °C annealing for 15 s, 72 °C extension for 30 s, a total of 30 cycles, and finally 72 °C final extension for 10 min. The PCR product was used as a template for a second PCR, and the number of cycles was set to 5 to reduce the influence of non-specific amplification products. The PCR product after the second amplification was sent to Shanghai Yuanxu Biotechnology Co., Ltd. for Illumina second-generation sequencing.

[0047] The results of the effect of fermented douchi on human intestinal microorganisms are shown in Figure 1 The abundance of beneficial bacteria such as Bifidobacterium, Butyricicoccus, and Ruminococcus increased, and the abundance of harmful bacteria such as uncultured Pasteurellaceae and Enterobacteriaceae decreased. The results show that the fermented douchi prepared in the present application has an important influence on the reduction of harmful bacteria and the increase of beneficial bacteria in the human intestinal flora, can improve intestinal flora imbalance, and maintain intestinal flora health balance. These changes in intestinal microbiota can improve exercise performance through material metabolism, improve sleep quality, reduce inflammation levels, promote energy supplementation and exercise recovery.

[0048] 3.2 Plasma neurotransmitter-targeted metabolomics detection Take 50 μl sample with 200 μL pre-cooled 1.25% formic acid acetonitrile / methanol (1:1 by volume) solution vortex mixing 30 s, then ultrasonic in ice bath for 30 min. Incubate at -20℃ for 1 h to precipitate the protein. After centrifugation at 4℃, 14000 rcf / min for 20 min, the supernatant was taken, 37 μL Na2CO3 (200 mg / mL) was added and vortexed for 60 min. 37 μL Na2CO3 (200 mg / mL) was added to the supernatant and vortexed, then 50 μL DSCl solution (10 mg / mL) was added, and the reaction was carried out at 30℃ constant temperature shaker for 60 min, then 1 μL 50% formic acid aqueous solution was added to adjust the pH of the solution to 5. Centrifuge at 4℃ for 5 min, and take the supernatant for detection. Agilent 1290 Infinity LC ultra-high performance liquid chromatography system was used for separation, column temperature 40℃, mobile phase A: 50 mM ammonium formate aqueous solution + 0.425% formic acid, mobile phase B: methanol solution, flow rate 400 μL / min, injection volume 2 μL.

[0049] The target neurotransmitter metabolomics metabolite heat map is shown in Figure 2 As shown in the figure, a total of 18 neurotransmitters were detected in the plasma. After intervention, the content of D-serine in the plasma of the subjects in group A was significantly lower than that in group B (p<0.05). D-serine and glutamate are both activators of N-methyl-D-aspartate receptor (NMDAR), and the increase of D-serine may directly affect glutamate neurotransmitters, causing excitotoxicity and causing nerve cell damage in patients with amyotrophic lateral sclerosis. In this study, the plasma serine content of group A was significantly lower than that of group B (p<0.05), which was beneficial to reduce cell damage.

[0050] 3.3 Sleep quality monitoring On the 7th day (before), the 11th day (middle), and the 14th day (after) of the experiment, the sleep quality of the five subjects in the probiotic group was monitored. The Firstbeat monitoring system was used to determine the sleep quality, and the stress and recovery report was generated. The average heart rate (Average heart beat), root mean square of successive R-R interval differences (RMSSD), sleep duration, sleep index, and recovery index in the report were used.

[0051] The average heart rate, sleep RMSSD, sleep duration, sleep index, and recovery index of the five people in the probiotic group are shown in Table 2: Table 2 Sleep quality detection results of probiotic group

[0052] Sleep quality histogram as shown in Figure 3 The monitoring results show that after the probiotic intervention, the average heart rate does not change, the sleep RMSSD and recovery index show an increasing trend, and the sleep duration and sleep index show a decreasing trend (sleep quality enhancement and sleep duration shortening are normal phenomena); RMSSD is one of the core indicators for measuring heart rate variability (HRV) and reflects the activity of the parasympathetic nervous system (vagus nerve) in the autonomic nervous system. The higher the value, the more sensitive the heart is to parasympathetic nervous regulation, which is usually related to better cardiovascular adaptability and stress recovery ability.

[0053] The above describes the embodiments of the present application in detail in combination with the drawings, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of the embodiments can be made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application.

Claims

1. Use of a probiotic composition for the preparation of a product for modulating the composition of the intestinal microbial population, reducing plasma serine, reducing cell damage and improving sleep quality, characterized in that: The probiotic composition comprises Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01 and Pediococcus acidilactici LH01.

2. Use according to claim 1, characterized in that: The product is a sports supplement.

3. Use according to claim 3, characterized in that: The sports supplement is fermented soy sauce fermented by the probiotic composition.

4. A probiotic composition, characterized in that: The probiotic composition comprises Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01 and Pediococcus acidilactici LH01.

5. The probiotic composition according to claim 4, characterized in that: The ratio of the Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01 and Pediococcus acidilactici LH01 is 2:1:1:

1.

6. A fermented tempeh, characterized by, The fermented soy sauce is fermented by the probiotic composition of claim 4 or 5.

7. The fermented soybean paste according to claim 6, characterized in that: The preparation method is as follows: The soybeans are soaked, cooked and cooled to room temperature, then the Bacillus subtilis SP-8-5 is inoculated for primary fermentation, then the mixed fermentation inoculum of Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01 and Pediococcus acidilactici LH01 is inoculated in sequence for secondary fermentation, and the fermented soy sauce is obtained.

8. The use of the fermented tempeh according to claim 7 or 8 for modulating the composition of the intestinal microbial population, reducing plasma serine, reducing cell damage, and improving sleep quality, characterized in that: The probiotic composition comprises Bacillus subtilis SP-8-5, Lactobacillus plantarum YM-4-3, Lactobacillus plantarum AY01 and Pediococcus acidilactici LH01.

Images