Slurry water source functional strain extraction method and application thereof in food

Abstract

The invention discloses an extraction method of a slurry water source functional strain and application thereof in food, and belongs to the fields of microbial screening and food biology. The extraction method comprises the following steps: (1) sample pretreatment: obtaining a microbial suspension; (2) primarily screening to obtain primarily screened strains; (3) secondary screening: respectively carrying out uric acid reducing function detection, intestinal regulation function detection and tolerance detection on the primarily screened strains, and screening the strains meeting preset indexes; (4) strain purification: purifying the qualified strains subjected to secondary screening by adopting a streak separation method to obtain pure strains; (5) strain identification: carrying out variety identification on the pure strain through 16SrRNA gene sequencing; and (6) strain preservation: freezing and preserving the qualified pure strain by using a glycerol tube to obtain the slurry water source functional strain. According to the invention, efficient extraction and safe application of the pulp water source functional strain are realized, and a key technical support is provided for development of functional foods for reducing uric acid and regulating intestinal functions.

Description

Technical Field

[0001] This invention relates to a method for extracting functional bacterial strains from slurry and their application in food, and relates to the fields of microbial screening and food biology. Specifically, it relates to a method for selectively extracting specific bacterial strains with uric acid-lowering and intestinal function-regulating properties from traditional slurry, and the application of these strains in functional foods. Background Technology

[0002] Fermented vegetable juice (jiangshui) is an important fermentation substrate for traditional Chinese fermented foods. It is typically made from vegetables such as celery, cabbage, and radish, through natural fermentation by microorganisms such as lactic acid bacteria. It is widely consumed in northern and southwestern my country. During fermentation, the microbial community (mainly including lactic acid bacteria and yeast) metabolizes carbohydrates and organic acids in the vegetables, producing functional components such as lactic acid, acetic acid, vitamins, amino acids, and various bioactive peptides. This not only gives the fermented vegetable juice its unique flavor and preservation properties but also endows it with traditional health benefits such as promoting digestion and regulating intestinal microecology. With the deepening of research on functional foods, screening specific strains of microorganisms with specific physiological functions (such as lowering uric acid and targeted regulation of intestinal function) from traditional fermented vegetable juice has become a research hotspot in the field of food biotechnology. On the one hand, the microorganisms in fermented vegetable juice have long adapted to the vegetable fermentation environment, exhibiting strong environmental tolerance and metabolic stability; on the other hand, as food-derived microorganisms, their safety has been verified through long-term consumption history, laying the foundation for subsequent food applications.

[0003] The existing technology has the following problems: 1. Lack of targeted screening: Existing technologies mostly aim to obtain strains with fermentation capabilities, without designing screening systems for specific physiological functions such as lowering uric acid and regulating intestinal function, resulting in low screening efficiency and low acquisition rate of target strains; 2. Existing technologies are insufficient in detecting safety indicators such as drug resistance and the risk of generating harmful metabolites in screened strains, posing potential food safety hazards. Summary of the Invention

[0004] This invention provides a method for extracting functional bacterial strains from slurry water sources and its application in food, in order to solve the problems mentioned in the background art.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is to provide a method for extracting functional strains from slurry water and its application in food.

[0006] Firstly, a method for extracting functional bacterial strains from slurry water sources is provided, comprising the following steps: (1) Sample pretreatment: Collect slurry samples, centrifuge and filter membrane to remove impurities and bacteria, and obtain microbial suspension.

[0007] (2) Initial screening: The microbial suspension was spread on a selective medium containing uric acid, and after anaerobic culture, single colonies were selected to obtain the initial screening strains.

[0008] (3) Secondary screening: The strains screened in the primary screening were tested for uric acid lowering function, intestinal regulation function and tolerance, and strains that met the preset indicators were selected.

[0009] (4) Strain purification: The qualified strains were purified by streak stripping to obtain pure strains.

[0010] (5) Strain identification: The species of pure strains were identified by 16S rRNA gene sequencing.

[0011] (6) Preservation of strains: The qualified pure strains were frozen in glycerol tubes to obtain functional strains of the pulp water source.

[0012] A further improvement of the technical solution of the present invention is that: in step (1), the centrifugation conditions are 8000 r / min for 15 min and the filter membrane pore size is 0.22 μm.

[0013] A further improvement of the technical solution of the present invention is that: in step (2), the selective culture medium is composed of: 10 g / L tryptone, 5 g / L yeast extract, 2 g / L uric acid, 5 g / L glucose, 2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 20 g / L agar, pH 6.5; the anaerobic culture conditions are 37℃ for 48 h.

[0014] A further improvement of the technical solution of the present invention is that: in step (3), the uric acid-lowering function detection includes: inoculating the initial screening strain into a secondary screening liquid culture medium containing uric acid, anaerobic culture at 37°C for 72 hours, detecting the uric acid degradation rate and xanthine oxidase inhibition rate, and screening strains with a uric acid degradation rate ≥40% and a xanthine oxidase inhibition rate ≥30%.

[0015] A further improvement of the technical solution of the present invention is that: in step (3), the detection of intestinal regulatory function includes: inoculating the primary screening strain into the secondary screening liquid culture medium, anaerobic culture at 37°C for 72 hours, detecting the production of short-chain fatty acids and the effect of intestinal flora regulation, and screening strains with a total production of short-chain fatty acids ≥15mmol / L and a bifidobacteria count increased by more than 2 times.

[0016] A further improvement of the technical solution of the present invention is that: in step (3), the tolerance test includes the survival rate test of the simulated food processing environment (30-60℃ treatment for 30 min) and the digestive tract environment (pH2.0-3.0 acidic conditions treatment for 2 h, 0.3% bile salt treatment for 4 h), and the strains with a survival rate ≥60% under each condition are screened.

[0017] The further improvement of the technical solution of the present invention is that: in step (4), the streak separation method uses a double-screening solid culture medium (double-screening liquid culture medium with 20g / L agar added), anaerobic culture at 37℃ for 48h, and the streak is repeated 3 times.

[0018] Secondly, according to the first aspect above, an application of a functional strain of slurry source in food is also provided. The application involves adding the functional strain to food to prepare food with uric acid-lowering function or intestinal function regulation. The food is selected from fermented food, health food, or functional beverage.

[0019] A further improvement of the technical solution of the present invention is that: the fermented food includes fermented yogurt and fermented milk beverages, and the functional beverage includes plant-based fermented beverages; the amount of functional strain added to the food is 10. 6 -10 7 CFU / g (or CFU / mL).

[0020] A further improvement of the technical solution of the present invention is that the survival rate of functional strains in the food must meet the following requirement: ≥10 in fermented foods. 8 CFU / g, ≥10 in functional beverages 7 CFU / mL.

[0021] Due to the adoption of the above technical solution, the technical progress achieved by this invention compared to the prior art is as follows: 1. This invention systematically verifies the uric acid degradation capacity (in vitro uric acid degradation rate ≥40%), xanthine oxidase inhibitory activity (inhibition rate ≥30%), short-chain fatty acid production capacity (total production of acetic acid, propionic acid, and butyric acid ≥15mmol / L), and intestinal flora regulation effect (can increase the number of Bifidobacteria and Lactobacillus) of the strains, ensuring that the selected strains simultaneously have the ability to lower uric acid and regulate intestinal function, and that the function is stable.

[0022] 2. By introducing tolerance tests of simulated food processing environment (30-60℃ treatment for 30 min) and digestive tract environment (pH 2.0-3.0 acidic conditions treatment for 2 h, 0.3% bile salt treatment for 4 h) during the screening process, the survival rate of the screened strains is ≥60%, which significantly improves their activity stability in practical applications.

[0023] 3. The strains screened in this invention can simultaneously achieve the synergistic effect of lowering uric acid and regulating intestinal function. Improved intestinal function can promote the excretion of uric acid from the intestine, while lower uric acid levels can reduce the damage of uric acid to the intestinal flora. The two form a virtuous cycle, and compared with single-function strains, their effect on improving human health is more significant.

[0024] 4. This invention uses a two-step screening method of primary screening (selective culture medium enrichment) and secondary screening (precise detection of functional indicators) to directly target strains with the target function. Compared with traditional blind screening, the screening efficiency is improved and the acquisition rate of target strains is also improved.

[0025] 5. In the screening process, this invention introduces tolerance tests to simulated food processing environment (30-60℃ treatment for 30 min) and digestive tract environment (pH 2.0-3.0 acidic conditions treatment for 2 h, 0.3% bile salt treatment for 4 h). The survival rate of the screened strains is ≥60%, which significantly improves their activity stability or stability in practical applications.

[0026] 6. This invention screens strains with good compatibility with various food matrices by testing their growth rate and flavor contribution in different food matrices (milk-based, plant-based, and fruit and vegetable juice-based), achieving a growth rate ≥0.5h in food. -1 It does not produce off-flavor substances (such as hydrogen sulfide and amines) and can be widely used in various foods. Detailed Implementation

[0027] This invention provides a method for extracting functional bacterial strains from slurry water and its application in food.

[0028] Firstly, a method for extracting functional bacterial strains from slurry water sources is provided, comprising the following steps: (1) Sample pretreatment: Collect slurry samples, centrifuge and filter membrane to remove impurities and bacteria, and obtain microbial suspension. The centrifugation conditions are 8000 r / min for 15 min and the filter membrane pore size is 0.22 μm. (2) Initial screening: The microbial suspension was spread on a selective medium containing uric acid, and after anaerobic culture, single colonies were selected to obtain the initial screening strains. The selective medium consisted of: 10 g / L tryptone, 5 g / L yeast extract, 2 g / L uric acid, 5 g / L glucose, 2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 20 g / L agar, and pH 6.5. The anaerobic culture conditions were 37℃ for 48 h. (3) Secondary screening: The strains screened in the primary screening were subjected to tests for uric acid-lowering function, intestinal regulation function, and tolerance. Strains that met the preset indicators were selected, including: a. The uric acid-lowering function test includes: inoculating the primary screening strains into a secondary screening liquid culture medium containing uric acid, anaerobic culture at 37°C for 72 hours, and then detecting the uric acid degradation rate and xanthine oxidase inhibition rate. Strains with a uric acid degradation rate ≥40% and a xanthine oxidase inhibition rate ≥30% are selected. b. Intestinal regulatory function test includes: inoculating the primary screening strains into the secondary screening liquid culture medium, anaerobic culture at 37℃ for 72h, and then detecting the production of short-chain fatty acids and the effect of intestinal flora regulation. Strains with a total production of short-chain fatty acids ≥15mmol / L and a bifidobacteria count increased by more than 2 times were screened. c. Tolerance testing includes survival rate testing in simulated food processing environment (30-60℃ treatment for 30 min) and digestive tract environment (pH 2.0-3.0 acidic conditions treatment for 2 h, 0.3% bile salt treatment for 4 h), and screening for strains with a survival rate ≥60% under each condition; (4) Strains purification: The qualified strains were purified by streak separation method to obtain pure strains. The streak separation method used solid culture medium (solid culture medium with 20 g / L agar added) for screening, and anaerobic culture at 37℃ for 48 h. The streak was repeated 3 times. (5) Strain identification: The species of the pure strain was identified by 16S rRNA gene sequencing; (6) Preservation of strains: The qualified pure strains were frozen in glycerol tubes to obtain functional strains of the pulp water source.

[0029] Secondly, in conjunction with the first aspect above, an application of a functional strain of slurry source in food is also provided. The application involves adding the functional strain to food to prepare food with uric acid-lowering function or intestinal function regulation. The food is selected from fermented food, health food, or functional beverage.

[0030] Fermented foods include fermented yogurt and fermented milk beverages; functional beverages include plant-based fermented beverages. The recommended addition level of functional bacterial strains to food is 10%. 6 -10 7 CFU / g (or CFU / mL).

[0031] The viable number of functional bacterial strains in food must meet the following requirements: ≥10 in fermented foods. 8 CFU / g, ≥10 in functional beverages 7 CFU / mL.

[0032] The present invention will be further described in detail below with reference to the implementation scheme and embodiments.

[0033] Implementation Plan

[0034] (1) Material preparation

[0035] Fermented vegetable juice samples: 20 samples of traditional homemade fermented vegetable juice were collected, each with a volume of 500 mL. After collection, the samples were transported to the laboratory at 4℃ and processed within 24 hours. Culture medium: a. Primary screening medium (containing uric acid): tryptone 10g / L, yeast extract 5g / L, uric acid 2g / L, glucose 5g / L, potassium dihydrogen phosphate 2g / L, magnesium sulfate heptahydrate 0.5g / L, agar 20g / L, pH 6.5; b. Re-screening liquid culture medium: tryptone 10g / L, yeast extract 5g / L, glucose 10g / L, potassium dihydrogen phosphate 2g / L, magnesium sulfate heptahydrate 0.5g / L, pH 6.5; c. Food matrix: milk-based matrix (10% whole milk powder, 5% sucrose, pH 6.8 after sterilization), plant-based matrix (5% soy protein, 3% malt syrup, pH 6.2 after sterilization), fruit and vegetable juice matrix (80% carrot juice, 20% apple juice, pH 4.0 after sterilization). Reagents: Uric acid standard (purity ≥99%), xanthine oxidase kit, short chain fatty acid detection kit, 16S rRNA gene sequencing primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3'), glycerol (analytical grade).

[0036] (2) Extraction method steps

[0037] Sample pretreatment: Take 10 mL of slurry sample, place it in a centrifuge tube, centrifuge at 8000 r / min for 15 min, and take the supernatant; filter the supernatant through a 0.22 μm microporous membrane to remove bacteria and solid impurities, and obtain the sample filtrate; Primary screening: Take 100 μL of sample filtrate and spread it on a primary screening medium plate. Incubate anaerobically at 37°C for 48 h. After incubation, select single colonies with different morphologies (such as round, milky white, and neatly edged colonies). Select 5-8 colonies from each plate, for a total of 100 single colonies. Inoculate them onto the slant of the primary screening medium and incubate at 37°C for 24 h to obtain the primary screening strains.

[0038] Secondary screening: a. Uric acid-lowering function test: The primary screening strains were inoculated into secondary screening liquid culture medium containing uric acid (2 g / L) and anaerobically cultured at 37℃ for 72 h; after the culture was completed, the culture medium was centrifuged at 8000 r / min for 10 min, and the supernatant was collected. The uric acid concentration was detected by high performance liquid chromatography (HPLC), and the uric acid degradation rate was calculated (uric acid degradation rate = (initial uric acid concentration - uric acid concentration after culture) / initial uric acid concentration × 100%); at the same time, the inhibition rate of the culture medium on xanthine oxidase was detected by a xanthine oxidase kit; strains with a uric acid degradation rate ≥ 40% and a xanthine oxidase inhibition rate ≥ 30% were screened.

[0039] b. Intestinal regulatory function test: The strains that passed the uric acid-lowering function test were inoculated into the rescreening liquid culture medium and anaerobic cultured at 37℃ for 72 h. After the culture was completed, the culture medium was centrifuged at 8000 r / min for 10 min, and the supernatant was collected. The production of short-chain fatty acids (acetic acid, propionic acid, butyric acid) was detected by gas chromatography (GC). At the same time, the strains were co-cultured with human intestinal flora simulation solution (a mixed bacterial solution containing Bifidobacterium, Escherichia coli, and Enterococcus faecalis) for 48 h. The changes in the number of Bifidobacterium and Lactobacillus in the intestinal flora were detected by real-time quantitative PCR (qPCR). Strains with a total short-chain fatty acid production ≥15 mmol / L and a Bifidobacterium count increased by more than 2 times were screened. c. Tolerance test: The strains that meet the requirements for intestinal regulation function are inoculated into the secondary screening liquid culture medium and cultured until the logarithmic growth phase (OD600=1.0). ① Simulate food processing tolerance: Treat bacterial solutions at 30℃, 45℃, and 60℃ for 30 min respectively, and use plate counting method to detect the number of colonies before and after treatment, and calculate the survival rate; ② Simulating digestive tract tolerance: The bacterial culture was treated in acidic buffer solutions of pH 2.0 and pH 3.0 for 2 hours, and in 0.3% bile salt solution for 4 hours. The number of colonies before and after treatment was detected by plate counting method, and the survival rate was calculated. Strains with a survival rate of ≥60% under each condition were screened.

[0040] Strain purification: The qualified strains were inoculated into the rescreening solid medium (with 20 g / L agar added) using the streak separation method, and anaerobically cultured at 37℃ for 48 h. The streak was repeated 3 times to obtain pure strains.

[0041] Strain identification: Single colonies of pure strains were selected, genomic DNA was extracted, and PCR amplification was performed using 16S rRNA gene sequencing primers. The amplification products were sent to a sequencing company for sequencing. The sequencing results were compared with the GenBank database for homology to determine the strain species (such as Lactobacillus plantarum, Lactobacillus acidophilus, etc.). Strains were preserved by inoculating the qualified pure strains into a secondary screening liquid medium and culturing them to the logarithmic growth phase. The bacterial solution was then mixed with 50% glycerol at a volume ratio of 1:1 and stored at -80℃ to obtain functional strains of the slurry source.

[0042] (3) Functional verification

[0043] In vivo validation of uric acid-lowering function: The preserved strain was prepared into a bacterial agent (concentration 10). 10 CFU / g was administered orally to mice with hyperuricemia (uric acid level ≥400 μmol / L) at a dose of 10 μmol / g. 9CFU / mouse / day, administered by gavage for 21 consecutive days. Serum uric acid levels in mice were measured on days 7, 14, and 21 after gavage. The results showed that after 21 days of gavage, the serum uric acid level in mice dropped to below 250 μmol / L, demonstrating a significant uric acid-lowering effect. In vivo validation of intestinal regulatory function: In mice with gut microbiota dysbiosis (treated with antibiotics, with ≤10 Bifidobacteria), the gut microbiota was regulated. 6 The above-mentioned bacterial agent (CFU / g feces) was administered orally at a dose of 10 g / g. 9 CFU / mouse / day was administered via gavage for 14 consecutive days. The content of short-chain fatty acids in mouse feces and the composition of intestinal flora were then measured. Results showed that after 14 days of gavage, the total content of short-chain fatty acids in mouse feces increased to over 20 mmol / L, and the number of Bifidobacteria increased to 10. 8 When CFU / g in feces exceeds a certain level, the intestinal flora is restored to balance.

[0044] (4) Application verification

[0045] Application in fermented yogurt: The preserved strain (Lactobacillus plantarum) was prepared at a ratio of 10... 6 Inoculate the milk-based matrix with an addition of CFU / g, ferment at 37℃ for 12 hours to prepare functional yogurt, and test the number of viable bacteria in the yogurt (≥10). 8 The yogurt was tested for CFU / g, acidity (70-80°T), and flavor compounds (no off-odor). Volunteers were given 100g / person / day of this yogurt for 30 consecutive days. The volunteers' serum uric acid levels (average decrease of 15%) and fecal intestinal flora (average increase of 1.8 times in the number of Bifidobacteria) were measured to demonstrate its effectiveness.

[0046] Application in plant-based fermented beverages: The preserved strain (Lactobacillus acidophilus) was prepared at a ratio of 10... 6 Inoculate the plant-based substrate with CFU / mL, ferment at 30℃ for 24 hours, prepare a functional plant beverage, and test the number of viable strains in the beverage (≥10). 7 The effects of the beverage were demonstrated by measuring CFU / mL, pH (3.8-4.2), and sensory scores (≥85 points). Volunteers were given 200mL / person / day for 30 consecutive days. Serum uric acid levels (average decrease of 12%) and fecal short-chain fatty acid content (average increase of 25%) were measured to prove its efficacy.

[0047] The following embodiments are provided in conjunction with the first aspect, the second aspect, and the implementation scheme described above.

[0048] Example 1: Functional bacterial strains with uric acid-lowering function were extracted from traditional northern fermented rice water.

[0049] Sample collection: Five samples of traditional homemade celery juice from northern my country were collected, each 500 mL, and transported under refrigeration at 4℃.

[0050] Sample pretreatment: Take 10 mL of slurry sample, centrifuge at 8000 r / min for 15 min, take the supernatant and filter it through a 0.22 μm filter membrane.

[0051] Primary screening: Spread 100 μL of filtrate onto a primary screening medium plate and anaerobic culture at 37°C for 48 h. Select 12 single colonies with different morphologies and inoculate them onto slant culture.

[0052] Secondary screening: a. Uric acid-lowering function test: 12 primary screening strains were inoculated into secondary screening liquid culture medium containing uric acid and anaerobically cultured at 37℃ for 72 h; HPLC analysis showed that the uric acid degradation rates of 3 strains were 45%, 48%, and 52%, respectively, and the xanthine oxidase inhibition rates were 32%, 35%, and 38%, respectively. b. Intestinal regulatory function test: The total short-chain fatty acid production of the above three strains was 16 mmol / L, 18 mmol / L and 20 mmol / L, respectively, and the number of Bifidobacteria increased by 2.1 times, 2.3 times and 2.5 times, respectively. c. Tolerance test: The survival rates of the three strains after treatment at 60℃ for 30 min were 62%, 65%, and 70%, respectively; after treatment at pH 2.0 for 2 h, the survival rates were 61%, 63%, and 68%, respectively; and after treatment with 0.3% bile salts for 4 h, the survival rates were 63%, 66%, and 72%, respectively.

[0053] Strain purification and identification: The strain with the highest uric acid degradation rate (52%) was selected and streak purified three times to obtain a pure strain. 16S rRNA gene sequencing showed that the strain had 99.8% homology with Lactobacillus plantarum and was named Lactobacillus plantarum JS-01. Preservation: Prepare glycerol tubes from Lactobacillus plantarum JS-01 and store at -80℃.

[0054] Functional validation: Mice with hyperuricemia were administered Lactobacillus plantarum JS-01 bacterial agent (10 mg / L) via gavage. 9 (CFU / mouse / day), after 21 days, the serum uric acid level of mice decreased from 420 μmol / L to 230 μmol / L, showing a significant effect in lowering uric acid.

[0055] Application: Add Lactobacillus plantarum JS-01 at a ratio of 10... 6 CFU / g was added to the milk-based matrix, and yogurt was produced by fermentation at 37°C for 12 hours. The number of viable bacteria in the yogurt was 1.2 × 10⁻⁶. 8 CFU / g, after 30 days of consumption, the average serum uric acid level of volunteers decreased by 18%.

[0056] Example 2: Functional bacterial strains with intestinal regulation as the main function were extracted from traditional fermented rice water in Southwest China.

[0057] Sample collection: Five samples of traditional homemade radish juice from southwestern my country were collected, each 500 mL, and transported under refrigeration at 4℃.

[0058] Sample pretreatment: Take 10 mL of slurry sample, centrifuge at 8000 r / min for 15 min, take the supernatant, and filter through a 0.22 μm filter membrane; Primary screening: Spread 100 μL of filtrate onto a primary screening medium plate and anaerobic culture at 37°C for 48 h. Select 10 single colonies with different morphologies and inoculate them onto slant culture.

[0059] Secondary screening: a. Uric acid-lowering function test: Ten primary screening strains were inoculated into secondary screening liquid culture medium containing uric acid and anaerobic cultured at 37℃ for 72 h. HPLC analysis showed that the uric acid degradation rates of two strains were 41% and 43%, respectively, and the xanthine oxidase inhibition rates were 31% and 33%, respectively. b. Intestinal regulatory function test: The total short-chain fatty acid production of the two strains was 22 mmol / L and 25 mmol / L, respectively, and the number of Bifidobacteria increased by 2.8 times and 3.2 times, respectively. c. Tolerance test: The survival rates of the two strains after treatment at 60℃ for 30 min were 64% and 68%, respectively; after treatment at pH 2.0 for 2 h, the survival rates were 62% and 66%, respectively; and after treatment with 0.3% bile salts for 4 h, the survival rates were 65% and 71%, respectively.

[0060] Strain purification and identification: The strain with the highest short-chain fatty acid production (25 mmol / L) was selected and streak purified three times to obtain a pure strain; 16S rRNA gene sequencing showed that the strain had 99.7% homology with Lactobacillus acidophilus, and it was named Lactobacillus acidophilus SC-01. Preservation: Prepare glycerol tubes from Lactobacillus acidophilus SC-01 and store at -80°C.

[0061] Functional validation: Lactobacillus acidophilus SC-01 bacterial agent (10 mg / L) was administered orally to mice with intestinal flora imbalance model. 9 (CFU / mouse / day), after 14 days, the total short-chain fatty acid content in the mouse feces increased from 8 mmol / L to 23 mmol / L. The number of Bifidobacteria increased from 10. 5 CFU / g feces increased to 1.5×10 8 CFU / g feces, the intestinal flora is restored to balance.

[0062] Application: Add Lactobacillus acidophilus SC-01 at a ratio of 10...6 CFU / mL was added to the plant-based matrix, and fermentation was carried out at 30℃ for 24 hours to produce a plant-based beverage. The number of viable strains in the beverage was 1.5 × 10⁻⁶. 7 After 30 days of drinking the product, volunteers experienced an average 30% increase in fecal short-chain fatty acids (CFU / mL) and a significant increase in gut microbiota diversity.

[0063] The present invention has been described in detail above. However, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, any modifications or improvements that do not depart from the spirit of the present invention are within the scope of protection of the present invention.

Claims

1. A method for extracting functional bacterial strains from pulp water sources, characterized in that, Includes the following steps: (1) Sample pretreatment: Collect slurry samples, centrifuge and filter membrane to remove impurities and bacteria, and obtain microbial suspension; (2) Initial screening: The microbial suspension was spread on a selective medium containing uric acid, and after anaerobic culture, single colonies were selected to obtain the initial screening strains; (3) Secondary screening: The uric acid-lowering function, intestinal regulation function and tolerance of the initially screened strains were tested to screen strains that met the preset indicators; (4) Strains purification: The qualified strains were purified by streak plating to obtain pure strains; (5) Strain identification: The species of the pure strain was identified by 16S rRNA gene sequencing; (6) Preservation of strains: The qualified pure strains were frozen in glycerol tubes to obtain functional strains of the pulp water source.

2. The method for extracting functional bacterial strains from slurry water source according to claim 1, characterized in that, In step (1), the centrifugation conditions are 8000 r / min for 15 min and the filter membrane pore size is 0.22 μm.

3. The method for extracting functional bacterial strains from slurry water source according to claim 1, characterized in that, In step (2), the selective culture medium consists of: 10 g / L tryptone, 5 g / L yeast extract, 2 g / L uric acid, 5 g / L glucose, 2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 20 g / L agar, and pH 6.5; the anaerobic culture conditions are 37℃ for 48 h.

4. The method for extracting functional bacterial strains from pulp water source according to claim 1, characterized in that, In step (3), the uric acid-lowering function test includes: inoculating the initial screening strain into a secondary screening liquid culture medium containing uric acid, anaerobic culture at 37°C for 72 hours, detecting the uric acid degradation rate and xanthine oxidase inhibition rate, and screening strains with a uric acid degradation rate ≥40% and a xanthine oxidase inhibition rate ≥30%.

5. The method for extracting functional bacterial strains from slurry water source according to claim 1, characterized in that, In step (3), the intestinal regulatory function test includes: inoculating the primary screening strains into the secondary screening liquid culture medium, anaerobic culture at 37°C for 72 h, detecting the production of short-chain fatty acids and the intestinal flora regulation effect, and screening strains with a total short-chain fatty acid production ≥15 mmol / L and a bifidobacteria count that is more than 2 times higher.

6. The method for extracting functional bacterial strains from slurry water source according to claim 1, characterized in that, In step (3), the tolerance test includes the survival rate test of the simulated food processing environment (30-60℃ treatment for 30 min) and the digestive tract environment (pH 2.0-3.0 acidic conditions treatment for 2 h, 0.3% bile salt treatment for 4 h), and screening strains with a survival rate ≥60% under each condition.

7. The method for extracting functional bacterial strains from pulp water source according to claim 1, characterized in that, In step (4), the streak separation method uses a double-screening solid culture medium (double-screening liquid culture medium with 20 g / L agar added), anaerobic culture at 37°C for 48 h, and the streak is repeated 3 times.

8. The application of a functional strain of sap-derived water source in food, wherein the functional strain of sap-derived water source extracted by the extraction method according to any one of claims 1-7 is used in food, characterized in that... The application involves adding the functional strains to food to prepare food with uric acid-lowering and / or intestinal function-regulating functions, wherein the food is selected from fermented foods, health foods, or functional beverages.

9. The application according to claim 8, characterized in that, The fermented foods include fermented yogurt and fermented milk beverages, and the functional beverages include plant-based fermented beverages; the amount of functional bacterial strains added to the food is 10. 6 -10 7 CFU / g (or CFU / mL).

10. The application according to claim 8, characterized in that, The viable number of functional bacterial strains in the food must meet the following requirement: ≥10 in fermented foods. 8 CFU / g, ≥10 in functional beverages 7 CFU / mL.