Pediococcus acidilactici, microbial inoculum containing pediococcus acidilactici, application of microbial inoculum, sorghum fermented food and preparation method of sorghum fermented food

Abstract

The invention relates to the technical field of food engineering, and discloses pediococcus acidilactici, a microbial agent containing the pediococcus acidilactici, application of the microbial agent, sorghum fermented food and a preparation method of the sorghum fermented food. The preservation number of the pediococcus acidilactici is GDMCC (China General Microbiological Culture Collection Center) No.65676, and the pediococcus acidilactici can be used for preparing the pediococcus acidilactici. The microbial agent contains pediococcus acidilactici and auxiliary bacteria. According to the sorghum fermented food obtained through fermentation treatment of the fungicide, antinutritional factors such as tannin in sorghum can be converted into urolithin, hydrolysis of protein, fat and starch can be promoted, the utilization rate of the sorghum is increased, the quality and flavor of fermented products are improved, the fermentation period is shortened, the shelf life is prolonged, and high economic benefits are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of food engineering, and particularly to a Pediococcus acidilactici, a bacterial agent containing the Pediococcus acidilactici and its application, a sorghum fermented food and a preparation method thereof. Background Art

[0002] As an ancient food processing technology, sorghum fermentation can not only improve the nutritional value of sorghum, increase the digestibility and bioavailability of proteins, but also produce a series of health-beneficial active substances, such as organic acids, vitamins and antioxidants. In addition, the fermentation process can also improve the flavor and texture of sorghum foods, extend their shelf life, and at the same time promote intestinal health and enhance immunity. Therefore, sorghum fermentation is not only an effective means to improve food quality, but also an important way to achieve a healthy diet.

[0003] However, there are still some deficiencies in sorghum fermentation. Antinutrients in sorghum, such as phenolic compounds like tannins, form complexes with proteins through multiple hydrogen bonds, hydrophobic binding, etc., leading to protein precipitation and limiting the utilization rate of nutrients. And urolithin, as the ultimate material basis of tannins, exerts physiological activities such as antioxidant, anti-inflammatory and anti-cancer with the advantage of high bioavailability. Therefore, in actual operation, it is of great significance to deeply process it to improve the selectivity of converting antinutritional factors in sorghum, such as tannins, into natural metabolite urolithin, improve the utilization rate of sorghum, and increase the conversion rate of starch, protein and fat in sorghum into easily absorbable nutrients such as sugars, amino acids and fatty acids.

[0004] However, when the existing microorganisms capable of synthesizing urolithin are used for fermentation with sorghum as the raw material, the fermentation performance is poor, it is difficult to efficiently convert sorghum raw materials, the utilization rate of sorghum is low, and the ability to synthesize urolithin in sorghum is significantly reduced. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problem in current sorghum fermentation that antinutrients such as phenolic compounds like tannins form complexes with proteins through multiple hydrogen bonds, hydrophobic binding, etc., resulting in protein precipitation, being unable to effectively convert tannins into urolithin, and having a low utilization rate of sorghum. The present invention provides a Pediococcus acidilactici, a bacterial agent containing the Pediococcus acidilactici and its application, a sorghum fermented food and a preparation method thereof. This Pediococcus acidilactici can not only increase the content of urolithin in sorghum fermented foods, but also effectively improve the bioavailability of sorghum (such as promoting the conversion of starch, protein and fat in sorghum into sugars, amino acids and fatty acids), thereby improving the nutritional value of fermented foods, improving the quality and flavor of sorghum fermented foods, shortening the fermentation cycle, extending the shelf life, and increasing economic benefits.

[0006] To achieve the above object, a first aspect of the present invention provides a Pediococcus acidilactici with a preservation number of GDMCC No. 65676.

[0007] A second aspect of the present invention provides a bacterial agent containing the Pediococcus acidilactici as described above.

[0008] Preferably, the bacterial agent further contains an auxiliary bacterium selected from any one of Streptococcus thermophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp., Bifidobacterium bulgaricum, Bifidobacterium longum, Bifidobacterium bifidum, and Bifidobacterium breve.

[0009] More preferably, the auxiliary bacterium is Streptococcus thermophilus.

[0010] Preferably, the bacterial agent is selected from at least one of a liquid bacterial agent, a semi-liquid bacterial agent, and a solid bacterial agent.

[0011] Preferably, the bacterial agent is a liquid bacterial agent, and the concentration of the Pediococcus acidilactici is 1×10 7 -1×10 8 CFU / mL, and the concentration of the auxiliary bacterium is 1×10 7 -1×10 11 CFU / mL.

[0012] A third aspect of the present invention provides an application of the Pediococcus acidilactici as described above and / or the bacterial agent as described above in the preparation of sorghum fermented foods.

[0013] Preferably, the sorghum fermented foods are selected from at least one of pasta, seasonings, and liquors.

[0014] More preferably, the sorghum fermented food is an alcoholic beverage.

[0015] A fourth aspect of the present invention provides a preparation method of a sorghum fermented food, which includes: contacting the bacterial agent as described above with a sorghum substrate for fermentation culture.

[0016] Preferably, relative to 100 g of the sorghum substrate, the dosage of the bacterial agent is 2 - 4.5 g.

[0017] Preferably, the sorghum substrate is sorghum, and more preferably, sorghum is used as the sorghum substrate after being crushed, washed, and steamed.

[0018] Preferably, the conditions for steaming include: pressure of 0.1 - 0.2 MPa and time of 20 - 40 min.

[0019] Preferably, the particle size of the sorghum substrate is less than 1 mm.

[0020] Preferably, the conditions for the fermentation culture include: the temperature is 42 - 45°C, the time is 29 - 33 days, and the mass ratio of the sorghum substrate to the microbial agent is 30:0.8 - 1.3.

[0021] The fifth aspect of the present invention provides a sorghum fermented food prepared by the method described above.

[0022] Through the above technical solutions, the beneficial effects of the present invention are as follows:

[0023] The microbial agent provided by the present invention can effectively improve the selectivity of converting anti-nutritional factors such as tannins in sorghum into the natural metabolite urolithin; Pediococcus acidilactici and the auxiliary bacteria cooperate with each other, which can not only promote the fermentation of sorghum fermented food, improve the ability to degrade anti-nutritional factors (such as tannins) in sorghum and convert them into urolithin, improve the antioxidant, anti-inflammatory and anti-cancer and other health functions of sorghum fermented food, but also further decompose starch, protein and fat into sugars, amino acids and fatty acids, improve the bioavailability of sorghum, enhance the nutritional value of sorghum fermented food, improve the quality and flavor of sorghum fermented food, and improve economic benefits.

[0024] Biological preservation

[0025] Pediococcus acidilactici is preserved in the Guangdong Provincial Microbial Culture Collection Center (Address: 5th Floor, Building 59, No. 100 Yard, Middle Xianlie Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, Postcode 510070, the abbreviation of the preservation unit is GDMCC), with the preservation number of GDMCC No. 65676 and the preservation date of December 25, 2024. Description of the drawings

[0026] Figure 1 It is the morphological diagram of the single colony strain isolated in Example 1 on the MRS solid medium;

[0027] Figure 2 It is the cell morphological diagram of the single colony strain isolated in Example 1 under the microscope. Detailed implementation manners

[0028] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0029] The present invention provides a Pediococcus acidilactici, and the preservation number of the Pediococcus acidilactici is GDMCC No. 65676.

[0030] The above-mentioned Pediococcus acidilactici is preserved in the Guangdong Provincial Culture Collection of Microorganisms (address: 5th Floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, postal code 510070, the abbreviation of the preservation unit is GDMCC), the preservation number is GDMCC No. 65676, and the preservation date is December 25, 2024.

[0031] Exemplarily, the screening process of the above-mentioned Pediococcus acidilactici is as follows:

[0032] S1. Sample collection and culture: Select volunteers who have not used antibiotics and have no history of gastrointestinal diseases within 3-4 months, let them eat 10-15 g of peanuts every day for two consecutive weeks, collect their fecal samples to prepare a bacterial suspension, and inoculate the prepared bacterial suspension into a liquid medium containing tannin for enrichment culture.

[0033] S2. Preliminary determination and screening: Use high-performance liquid chromatography to screen out the bacterial suspension that can convert tannin into urolithin A and urolithin B, and spread the bacterial suspension on a solid medium;

[0034] S3. Re-screening: Pick single colonies with different shapes and sizes and inoculate them into a liquid medium added with tannin for culture, and use high-performance liquid chromatography to screen out the single bacteria that can convert tannin into urolithin A and urolithin B.

[0035] Pick the isolated single bacterial colonies onto a solid medium for morphological identification. After culturing at room temperature for 20-26 h, take a small amount of the bacterial liquid for Gram staining and microscopic observation. The observation results are as Figure 1 shown. The morphological of the colony strains on the medium is round or oval, arranged singly, in pairs or in short chains; Figure 2 It can be seen that the cell morphology under the microscope is round or oval.

[0036] The 16S rDNA sequence of the isolated strain was cloned and sequenced. Its nucleotide sequence is shown in SEQ ID NO.1. After alignment with the sequence of Pediococcus acidilactici in NCBI, the 16S rDNA sequence of the strain of the present invention has a homology of 99.32% with the species sequence of Pediococcus acidilactici. Therefore, this strain was identified as Pediococcus acidilactici and was deposited in the Guangdong Microbial Culture Collection Center on December 25, 2024, with the deposit number GDMCC No.65676.

[0037] The inventors' research found that the Pediococcus acidilactici provided by the present invention can effectively improve the selectivity of converting anti-nutritional factors such as tannins in sorghum into the natural metabolite urolithin, significantly increase the content of urolithin in sorghum fermented foods, and improve the antioxidant, anti-inflammatory and anti-cancer functions of sorghum fermented foods; Pediococcus acidilactici and auxiliary bacteria cooperate with each other, can promote the fermentation of sorghum fermented foods, improve the ability to degrade anti-nutritional factors (such as tannins) in sorghum and convert them into urolithin, improve the health care functions such as antioxidant, anti-inflammatory and anti-cancer of sorghum fermented foods, can further decompose starch, protein and fat into sugars, amino acids and fatty acids, improve the bioavailability of sorghum, enhance the nutritional value of sorghum fermented foods, improve the quality and flavor of sorghum fermented foods, and improve economic benefits.

[0038] The Pediococcus acidilactici provided by the present invention can produce a large number of viable cells of Pediococcus acidilactici after cultivation. The present invention has no particular limitation on the cultivation method, as long as the viable cells of the Pediococcus acidilactici can be proliferated in large quantities by this cultivation method. For example, Pediococcus acidilactici can be inoculated into a seed medium for seed cultivation to obtain a seed liquid, and then the seed liquid can be inoculated into a fermentation medium for fermentation cultivation.

[0039] In the present invention, there is no particular limitation on the seed medium and fermentation medium used, as long as they can provide the nutrients required for the growth and proliferation of Pediococcus acidilactici. Preferably, the composition of the liquid medium includes: peptone 5 - 15 g / L, meat extract (or beef powder) 8 - 10 g / L, yeast extract powder (or yeast paste powder) 4 - 5 g / L, glucose 15 - 25 g / L, dipotassium hydrogen phosphate 1.5 - 2.5 g / L; the composition of the solid medium includes: peptone 5 - 15 g / L, meat extract (or beef powder) 8 - 10 g / L, yeast extract powder (or yeast paste powder) 4 - 5 g / L, agar 18.0 - 22.0 g / L, glucose 15 - 25 g / L, dipotassium hydrogen phosphate 1.5 - 2.5 g / L.

[0040] In the second aspect of the present invention, a bacterial agent is provided, which contains Pediococcus acidilactici as described above.

[0041] According to the present invention, preferably, the bacterial agent further contains an auxiliary bacterium, and the auxiliary bacterium is selected from any one of Streptococcus thermophilus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp., Bifidobacterium bulgaricum, Bifidobacterium longum, Bifidobacterium spp. and Bifidobacterium breve. More preferably, the auxiliary bacterium is Streptococcus thermophilus.

[0042] In the present invention, there is no particular limitation on the method for obtaining the bacterial agent, and it can be obtained by mixing Pediococcus acidilactici and an auxiliary bacterium. For example, the viable cells of Pediococcus acidilactici and the auxiliary bacterium can be respectively inoculated into a liquid medium at an inoculation amount of not less than 0.5% by volume, and after culturing at a temperature of 35 - 45 °C for 12 - 14 h, a Pediococcus acidilactici bacterial solution and an auxiliary bacterium bacterial solution are respectively obtained, and then the two bacterial solutions are mixed, or the two bacterial solutions are separated to obtain the bacterial cells and then mixed. There is no particular limitation on the separation method, as long as the bacterial cells can be enriched from the culture solution, for example, it can be achieved by centrifugation and / or filtration, and the conditions of the centrifugation and the filtration can be conventional conditions in the art, which are well known to those skilled in the art and will not be elaborated herein.

[0043] According to the present invention, there is no particular limitation on the dosage form of the bacterial agent. Depending on different intended uses, it can be prepared into different dosage forms and corresponding excipients and other components are added. For example, the bacterial agent can be a liquid bacterial agent and / or a solid bacterial agent and / or a semi - liquid bacterial agent. Among them, which excipients are added to which dosage form of the bacterial agent is well known to those skilled in the art and will not be elaborated in detail herein.

[0044] The bacterial agent of the present invention is preferably a liquid bacterial agent, and the concentration of Pediococcus acidilactici is 1×10 7 -1×10 8 CFU / mL, specifically it can be 1×10 7 CFU / mL, 3×10 7 CFU / mL, 8×10 7 CFU / mL, 1×10 8 CFU / mL, or any value within the range formed by any two of the above numerical values; more preferably 2.5×10 7 -7.5×10 7 CFU / mL. Preferably, the concentration of the auxiliary bacterium is 1×10 7 -1×10 11 CFU / mL, specifically it can be 1×10 7 CFU / mL, 1×108 CFU / mL, 1×10 9 CFU / mL, 1×10 11 CFU / mL, or any value within the range formed by any two of the above numerical values; more preferably 1×10 8 -1×10 10 CFU / mL. The inventors have found through research that the bacterial agent obtained by the above preferred method has a higher ability to convert tannins in sorghum into urolithin, and can promote the conversion of starch, protein, and fat in sorghum into sugars, amino acids, and fatty acids, improving the nutritional value of fermented foods and the quality and flavor of sorghum fermented foods.

[0045] Exemplarily, the method for preparing the bacterial agent of the present invention includes:

[0046] S1. Regularly monitor the growth of the strains in the Pediococcus acidilactici seed liquid and Streptococcus thermophilus seed liquid as described above, adjust the concentration of the seed liquid, and ensure that the concentration of the Pediococcus acidilactici seed liquid is 2×10 7 -2×10 8 CFU / mL, and the concentration of the auxiliary bacteria seed liquid is 2×10 7 -2×10 11 CFU / mL;

[0047] S2. Mix the adjusted Pediococcus acidilactici and auxiliary bacteria in equal proportions under sterile conditions, monitor the concentration of Pediococcus acidilactici and auxiliary bacteria. If the concentration of the mixed strains is too high, an appropriate diluent (such as sterile water, physiological saline, or the seed culture medium as described above) can be used for dilution to reach the required concentration range;

[0048] S3. Fill the prepared mixed bacterial agent into a sealed container and store it in the dark at 2 - 8°C. This mixed bacterial agent is the bacterial agent prepared by the present invention;

[0049] The third aspect of the present invention provides an application of the Pediococcus acidilactici as described above and / or the bacterial agent as described above in the preparation of sorghum fermented foods.

[0050] According to the present invention, preferably, the sorghum fermented food is selected from at least one of pasta, seasonings, and alcoholic beverages. More preferably, the sorghum fermented food is an alcoholic beverage. The inventors have found through research that the application obtained by the above preferred method can further increase the conversion rate of urolithin, further improve the utilization rate of sorghum, and make the content of urolithin and nutritional components in sorghum fermented foods higher.

[0051] The fourth aspect of the present invention provides a method for preparing a sorghum fermented food, which includes: contacting the bacterial agent as described above with a sorghum substrate for fermentation culture.

[0052] The inventors of the present invention have found that the isolated Pediococcus acidilactici has a high selectivity for sorghum substrates and can efficiently ferment and utilize sorghum. Preferably, the sorghum substrate is sorghum. The inventors have studied and found that by adopting the above preferred embodiment, the fermentation of the microbial agent can be more sufficient, greatly improving the utilization rate of sorghum, significantly enhancing the conversion rates of starch, protein, and fat in sorghum into sugars, amino acids, and fatty acids, and making the ability of tannins in sorghum to be converted into urolithin higher.

[0053] According to the present invention, preferably, the sorghum substrate is obtained by cleaning, cooking, and pulverizing sorghum. There are no particular limitations on the pulverization, and it can be selected from any one of the methods such as pulverization by a mill, pulverization by an electric pulverizer, pulverization by a hammer mill, and pulverization by a double-roll flattening machine, as long as it can expose sorghum starch, increase the surface area of the raw material, and is conducive to sorghum particles absorbing moisture and cooking and gelatinization. The pulverization method used in the present invention is pulverization by a mill, which can break sorghum into the required particle size in a short time, significantly improving the processing efficiency of sorghum. Moreover, the mill can make full use of sorghum residues, reduce waste, and improve the resource utilization rate. The pulverized sorghum particles increase the contact area with water and the microbial agent, which is conducive to the hydrolysis during the fermentation process and significantly improves the utilization rate of sorghum.

[0054] According to the present invention, preferably, relative to 100 g of the sorghum substrate, the dosage of the microbial agent is 2 - 4.5 g, specifically it can be 2 g, 3 g, 3.5 g, 4 g, 4.5 g, or any value between the above two values; the dosage of the microbial agent is further preferably 2.5 - 4 g. Preferably, the sorghum substrate is sorghum, and more preferably, sorghum after pulverization, cleaning, and cooking is used as the sorghum substrate. According to the present invention, preferably, the conditions for cooking include: the pressure is 0.1 - 0.2 MPa, specifically it can be 0.1 MPa, 0.12 MPa, 0.15 MPa, 0.18 MPa, 0.20 MPa, or any value between the above two values; the time for the cooking treatment is 20 - 40 min, specifically it can be 20 min, 25 min, 30 min, 35 min, or any value between the above two values; the particle size of the sorghum substrate is less than 1 mm.

[0055] According to the present invention, preferably, the conditions for fermentation culture include: the temperature is 42 - 45 °C; the time is 29 - 33 d. The inventors have studied and found that by adopting the above preferred method, sorghum particles can better absorb moisture, increase the humidity inside, thereby promoting the fermentation process, contributing to the gelatinization of starch, and improving the saccharification efficiency.

[0056] Taking sorghum-fermented food as an alcoholic beverage as an example, its specific preparation process includes:

[0057] S1. Clean and remove impurities from Northeast red sorghum and crush it into powder of less than 1 mm. Weigh the sorghum powder and add it into a fermentation barrel. Add 70℃-80℃ water to moisten the flour for the first time, and then pile it up and air-dry it for 18-22 hours to cool it down. Steam the air-dried and cooled sorghum powder with water at a pressure of 0.1-0.2MPa for 20-40min, then add 70℃-80℃ water to moisten the flour for the second time, and stir and crush the sorghum at the same time.

[0058] S2. Add Luzhou-flavor medium-temperature Daqu powder (crushed particle size of about 1.2 mm, and the mass ratio of Daqu powder to sorghum powder of 8-12:100) and the aforementioned bacterial agent (the mass ratio of sorghum powder to bacterial agent of 100:2-4.5) into a fermentation barrel, set the fermentation temperature of the fermentation barrel to 42-45°C, fully stir with the sorghum powder, and then put into a tank for fermentation for 29-33 days. After the fermentation is completed, further distill it using the retort distillation method to obtain the white wine base wine (the middle wine and the tail wine are taken from the base wine, and the head wine is discarded).

[0059] The inventors have found that the liquor prepared by the above method has higher alcohol content, urolithin content and nutritional value.

[0060] A fifth aspect of the present invention provides a fermented sorghum food prepared by the method described above.

[0061] The present invention will be described in detail below through examples.

[0062] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0063] Streptococcus thermophilus, numbered GDMCC NO.1.2800, was purchased from Guangdong Microbiological Culture Collection Center; sorghum was purchased from Yixing Yihong Rice Industry; koji was purchased from Luzhou Laojiao; MRS broth medium was purchased from Nantong Kaiheng Biotechnology Development Co., Ltd.; MRS solid culture medium was purchased from Hangzhou Microbiological Reagent Co., Ltd.

[0064] The following culture medium was used in the following examples:

[0065] MRS broth medium: peptone 10g / L, meat extract (or beef powder) 10g / L, yeast extract powder 5g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, triammonium citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.1g / L and Tween 80 0.1g / L.

[0066] MRS solid medium: peptone 10 g / L, meat extract (or beef powder) 10 g / L, yeast extract powder 5 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.28 g / L, agar 20 g / L, and Tween 80 0.1 g / L.

[0067] The high performance liquid chromatography analysis parameters of urolithin are as follows:

[0068] Mobile phase: Phase A: 0.1% formic acid aqueous solution (v / v); Phase B: 100% acetonitrile. Mix Phase A and Phase B evenly and sonicate in an ultrasonic cleaner for 30 min.

[0069] The elution gradient is: 0 - 6 minutes, 10 - 100% acetonitrile; 6 - 7 minutes, 100 - 100% acetonitrile; 7 - 9 minutes, 100 - 10% acetonitrile; 9 - 10 minutes, 10 - 10% acetonitrile.

[0070] Chromatographic column model: MP C18 (250×4.6 mm / 5 μm) column; mobile phase flow rate: 0.5 mL / min; detection wavelength: 306 nm; detection temperature: 40 °C.

[0071] Example 1

[0072] This example illustrates the screening and identification of Pediococus acidilactici

[0073] S1. Sample collection: Seven volunteers without a history of gastrointestinal diseases (age: 22 - 27 years) were selected, and it was ensured that they had not used antibiotics in the 3 months before the study. After they ate 10 - 15 g of peanuts every day for two consecutive weeks, fecal samples were collected. A small amount of fresh human feces was quickly picked with a sterilized bamboo stick and placed into a sterilized test tube containing 4.5 mL of physiological saline (10 -1 ), shaken well to prepare a bacterial suspension;

[0074] S2. Enrichment culture: The bacterial suspension prepared in step S1 was inoculated into MRS broth medium supplemented with tannin for enrichment culture for 36 h;

[0075] S3. Preliminary HPLC determination: The bacterial suspension that could convert tannin into urolithin A and urolithin B was screened by high performance liquid chromatography;

[0076] S4. Dilution and plating: Take the bacterial suspension detected in step S3 that could convert tannin into urolithin A, and take 100 μL to coat it on the corresponding MRS solid medium;

[0077] S5, rescreening: pick single colonies of different shapes and sizes and inoculate them into MRS broth medium supplemented with tannin and culture for 36 hours;

[0078] S6. HPLC determination: Use high performance liquid chromatography to screen out single bacterial strains that can convert tannins to produce urolithin A and urolithin B.

[0079] Morphological identification: Pick the isolated single colony strain (prepared in step S6 above) and place it in MRS solid culture medium. After culturing at room temperature for 24 hours, take a small amount of bacterial liquid for Gram staining microscopy observation. Figure 1 and Figure 2 As shown by Figure 1 It can be seen that the strains on the culture medium are round or oval in shape, arranged singly, in pairs or in short chains; Figure 2 It can be seen that the cell morphology under the microscope is round or oval.

[0080] Molecular identification: The 16S rDNA sequence of the single bacterium obtained in the above step S6 was cloned and sequenced, and its nucleotide sequence was shown in SEQ ID NO.1. After comparison with the sequence of Pediococcus acidilactici in NCBI, the 16S rDNA sequence of the strain of the present invention had a homology of 99.32% with the sequence of Pediococcus acidilactici. Therefore, the strain should be Pediococcus acidilactici and was used for the study of subsequent examples.

[0081] The Pediococus acidilactici is deposited in the Guangdong Microbial Culture Collection Center (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, Postal Code 510070, the abbreviation of the depository is GDMCC), with the deposit number GDMCC No.65676 and the deposit date of December 25, 2024.

[0082] Example 2

[0083] S1. Preparation of bacterial agent (the whole process is carried out in a clean bench): the lactic acid Pediococcus prepared in Example 1 is inoculated in an MRS broth medium at an inoculum amount of not less than 0.5% by volume to prepare a lactic acid Pediococcus seed solution, and Streptococcus thermophilus is inoculated in the MRS broth medium to prepare a thermophilus Streptococcus seed solution. The lactic acid Pediococcus seed solution and the thermophilus Streptococcus seed solution are cultured at 35° C. for 12 h, and the thermophilus Streptococcus seed solution and the lactic acid Pediococcus seed solution are mixed, diluted with physiological saline, and the concentration of the lactic acid Pediococcus is adjusted to 5×10 7 CFU / mL, adjust the concentration of thermophilic Streptococcus to 1×10 9 CFU / mL, the concentration was observed under a microscope, the pH was adjusted to 6.8, and the mixture was sealed and stored away from light at 2-8°C.

[0084] S2. Clean and remove impurities from Northeast red sorghum and grind it into powder of less than 1 mm. Under barrel fermentation conditions, weigh 6 kg of sorghum powder and add it into a fermentation barrel, add 3.6 kg of 80°C water for the first moistening, and then pile it up and spread it out for 20 hours to cool; use 100°C water to steam the cooled sorghum powder at normal pressure for 35 minutes, then add 3.6 kg of 80°C water for the second moistening, and stir and crush the sorghum at the same time.

[0085] S3. Add 0.6 kg of Luzhou-flavor medium-temperature Daqu powder (crushed particle size of about 1.2 mm) and 0.2 kg of the bacterial agent prepared in step S1 into the fermentation barrel, fully stir with the sorghum powder, and then put into a tank for fermentation for 31 days. Set the fermentation temperature to 43°C. After the fermentation is completed, further distill it using the retort distillation method to obtain the white wine base wine (the middle wine and the tail wine are taken from the base wine, and the head wine is discarded).

[0086] The obtained residual mash is recorded as C1, and the obtained base wine is recorded as J1.

[0087] Example 3

[0088] In this embodiment, sorghum is used as a sorghum substrate to ferment and obtain the raw material (white wine base wine) of the alcoholic beverage containing urolithin.

[0089] S1. Preparation of bacterial agent (the whole process is carried out in a clean bench): the lactic acid Pediococcus prepared in Example 1 is inoculated in an MRS broth medium at an inoculum amount of not less than 0.5% by volume to prepare a lactic acid Pediococcus seed solution, and Streptococcus thermophilus is inoculated in the MRS broth medium to prepare a thermophilus Streptococcus seed solution. The lactic acid Pediococcus seed solution and the thermophilus Streptococcus seed solution are cultured at 35° C. for 12 h, and the thermophilus Streptococcus seed solution and the lactic acid Pediococcus seed solution are mixed, and diluted with physiological saline to adjust the concentration of the lactic acid Pediococcus to 2.5×10 7 CFU / mL, adjust the concentration of thermophilic Streptococcus to 1×10 8 CFU / mL, the concentration was observed under a microscope, the pH was adjusted to 6.8, and the mixture was sealed and stored away from light at 2-8°C.

[0090] S2. Clean and remove impurities from Northeast red sorghum and grind it into powder of less than 1 mm. Under barrel fermentation conditions, weigh 6 kg of sorghum powder and add it into a fermentation barrel, add 3.6 kg of 80°C water for the first moistening, and then pile it up and spread it out for 20 hours to cool; steam the air-dried and cooled sorghum powder with 100°C water for 20 minutes, then add 3.6 kg of 80°C water for the second moistening, and stir and crush the sorghum at the same time.

[0091] S3. Add 0.6 kg of Luzhou-flavor medium-temperature Daqu powder (crushed particle size of about 1.2 mm) and 0.15 kg of the bacterial agent prepared in step S1 into the fermentation barrel, fully stir with the sorghum powder, and then put into a tank for fermentation for 29 days. Set the fermentation temperature to 42°C. After the fermentation is completed, further distill it using the retort distillation method to obtain the white wine base wine (the middle wine and the tail wine are taken from the base wine, and the head wine is discarded).

[0092] The obtained residual mash is recorded as C2, and the obtained base wine is recorded as J2.

[0093] Example 4

[0094] In this embodiment, sorghum is used as a sorghum substrate to ferment and obtain the raw material (white wine base wine) of the alcoholic beverage containing urolithin.

[0095] The preparation method comprises the following steps:

[0096] S1. Preparation of bacterial agent (the whole process is carried out in a clean bench): the lactic acid Pediococcus prepared in Example 1 is inoculated in an MRS broth medium at an inoculum amount of not less than 0.5% by volume to prepare a lactic acid Pediococcus seed solution, and Streptococcus thermophilus is inoculated in the MRS broth medium to prepare a thermophilus Streptococcus seed solution. The lactic acid Pediococcus seed solution and the thermophilus Streptococcus seed solution are cultured at 35° C. for 12 h, and the thermophilus Streptococcus seed solution and the lactic acid Pediococcus seed solution are mixed, and diluted with physiological saline to adjust the concentration of the lactic acid Pediococcus to 7.5×10 7 CFU / mL, adjust the concentration of thermophilic Streptococcus to 1×10 10 CFU / mL, the concentration was observed under a microscope, the pH was adjusted to 6.8, and the mixture was sealed and stored away from light at 2-8°C.

[0097] S2. Clean and remove impurities from Northeast red sorghum and grind it into powder of less than 1 mm. Under barrel fermentation conditions, weigh 6 kg of sorghum powder and add it into a fermentation barrel, add 3.6 kg of 80°C water for the first moistening, and then pile it up and spread it out for 20 hours to cool; steam the air-dried and cooled sorghum powder with 100°C water for 40 minutes, then add 3.6 kg of 80°C water for the second moistening, and stir and crush the sorghum at the same time.

[0098] S3. Add 0.6 kg of Luzhou-flavor medium-temperature Daqu powder (crushed particle size of about 1.2 mm) and 0.24 kg of the bacterial agent prepared in step S1 into the fermentation barrel, fully stir with the sorghum powder, and then put into a tank for fermentation for 33 days. Set the fermentation temperature to 45°C. After the fermentation is completed, further distill it using the retort distillation method to obtain the white wine base wine (the middle wine and the tail wine are taken from the base wine, and the head wine is discarded).

[0099] The obtained residual mash is recorded as C3, and the obtained base wine is recorded as J3.

[0100] Example 5

[0101] In this embodiment, sorghum is used as a sorghum substrate to ferment and obtain the raw material (white wine base wine) of the alcoholic beverage containing urolithin.

[0102] The preparation method comprises the following steps:

[0103] S1. Preparation of bacterial agent (the whole process is carried out in a clean bench): the lactic acid Pediococcus prepared in Example 1 is inoculated in an MRS broth medium at an inoculum amount of not less than 0.5% by volume to prepare a lactic acid Pediococcus seed solution, and Streptococcus thermophilus is inoculated in the MRS broth medium to prepare a thermophilus Streptococcus seed solution. The lactic acid Pediococcus seed solution and the thermophilus Streptococcus seed solution are cultured at 35° C. for 12 h, and the thermophilus Streptococcus seed solution and the lactic acid Pediococcus seed solution are mixed, diluted with physiological saline, and the concentration of the lactic acid Pediococcus is adjusted to 1×10 7 CFU / mL, adjust the concentration of thermophilic Streptococcus to 1×10 7 CFU / mL, the concentration was observed under a microscope, the pH was adjusted to 6.8, and the mixture was sealed and stored away from light at 2-8°C.

[0104] S2. Clean and remove impurities from Northeast red sorghum and grind it into powder of less than 1 mm. Under barrel fermentation conditions, weigh 6 kg of sorghum powder and add it into a fermentation barrel, add 3.6 kg of 80°C water for the first moistening, and then pile it up and spread it out for 20 hours to cool; steam the air-dried and cooled sorghum powder with 100°C water for 20 minutes, then add 3.6 kg of 80°C water for the second moistening, and stir and crush the sorghum at the same time.

[0105] S3. Add 0.6 kg of Luzhou-flavor medium-temperature Daqu powder (crushed particle size of about 1.2 mm) and 0.12 kg of the bacterial agent prepared in step S1 into the fermentation barrel, fully stir with the sorghum powder, and then put into a tank for fermentation for 29 days. Set the fermentation temperature to 42°C. After the fermentation is completed, further distill it using the retort distillation method to obtain the white wine base wine (the middle wine and the tail wine are taken from the base wine, and the head wine is discarded).

[0106] The obtained residual mash is recorded as C4, and the obtained base wine is recorded as J4.

[0107] Example 6

[0108] In this embodiment, sorghum is used as a sorghum substrate to ferment and obtain the raw material (white wine base wine) of the alcoholic beverage containing urolithin.

[0109] The preparation method comprises the following steps:

[0110] S1. Preparation of bacterial agent (the whole process is carried out in a clean bench): the lactic acid Pediococcus prepared in Example 1 is inoculated in an MRS broth medium at an inoculum amount of not less than 0.5% by volume to prepare a lactic acid Pediococcus seed solution, and Streptococcus thermophilus is inoculated in the MRS broth medium to prepare a thermophilus Streptococcus seed solution. The lactic acid Pediococcus seed solution and the thermophilus Streptococcus seed solution are cultured at 35° C. for 12 h, and the thermophilus Streptococcus seed solution and the lactic acid Pediococcus seed solution are mixed, diluted with physiological saline, and the concentration of the lactic acid Pediococcus is adjusted to 1×10 9 CFU / mL, adjust the concentration of thermophilic Streptococcus to 1×10 11 CFU / mL, the concentration was observed under a microscope, the pH was adjusted to 6.8, and the mixture was sealed and stored away from light at 2-8°C.

[0111] S2. Clean and remove impurities from Northeast red sorghum and grind it into powder of less than 1 mm. Under barrel fermentation conditions, weigh 6 kg of sorghum powder and add it into a fermentation barrel, add 3.6 kg of 80°C water for the first moistening, and then pile it up and spread it out for 20 hours to cool; steam the air-dried and cooled sorghum powder with 100°C water for 40 minutes, then add 3.6 kg of 80°C water for the second moistening, and stir and crush the sorghum at the same time.

[0112] S3. Add 0.6 kg of Luzhou-flavor medium-temperature Daqu powder (crushed particle size of about 1.2 mm) and 0.27 kg of the bacterial agent prepared in step S1 into the fermentation barrel, fully stir with the sorghum powder, and then put into a tank for fermentation for 33 days. Set the fermentation temperature to 45°C. After the fermentation is completed, further distill it using the retort distillation method to obtain the white wine base wine (the middle wine and the tail wine are taken from the base wine, and the head wine is discarded).

[0113] The obtained residual mash is recorded as C5, and the obtained base wine is recorded as J5.

[0114] Example 7

[0115] The liquor base was prepared according to the method of Example 2, except that the concentration of Pediococcus acidilactici in step S1 of Example 2 was adjusted to 0.5×10 7 CFU / mL, the concentration of thermophilic Streptococcus was adjusted to 0.5×10 7 CFU / mL.

[0116] The obtained residual mash is recorded as C6, and the obtained base wine is recorded as J6.

[0117] Example 8

[0118] The liquor base was prepared according to the method of Example 2, except that the concentration of Pediococcus acidilactici in step S1 of Example 2 was adjusted to 5×10 8 CFU / mL, the concentration of thermophilic Streptococcus was adjusted to 5×1011 CFU / mL.

[0119] The obtained residual fermented grains are denoted as C7, and the obtained base liquor is denoted as J7.

[0120] Example 9

[0121] The preparation of the base liquor for Baijiu was carried out according to the method of Example 2, except that the process of crushing sorghum in step S2 of Example 2 was replaced with: cleaning, removing impurities from Northeast Red Sorghum and crushing it into powder with a particle size of less than 2 mm.

[0122] The obtained residual fermented grains are denoted as C8, and the obtained base liquor is denoted as J8.

[0123] Example 10

[0124] The preparation of the base liquor for Baijiu was carried out according to the method of Example 2, except that the fermentation time in step S3 of Example 2 was adjusted to 20 d.

[0125] The obtained residual fermented grains are denoted as C9, and the obtained base liquor is denoted as J9.

[0126] Example 11

[0127] The preparation of the base liquor for Baijiu was carried out according to the method of Example 2, except that the mass of the microbial agent in step S3 of Example 2 was adjusted to 0.1 kg.

[0128] The obtained residual fermented grains are denoted as C10, and the obtained base liquor is denoted as J10.

[0129] Example 12

[0130] The preparation of the base liquor for Baijiu was carried out according to the method of Example 2, except that the steaming time in step S2 of Example 2 was adjusted to 15 min.

[0131] The obtained residual fermented grains are denoted as C11, and the obtained base liquor is denoted as J11.

[0132] Example 13

[0133] The preparation of the base liquor for Baijiu was carried out according to the method of Example 2, except that Streptococcus thermophilus in Example 2 was replaced with Lactobacillus casei (strain number: GDMCC NO.1.2885, purchased from Guangdong Provincial Microbial Culture Collection Center).

[0134] The obtained residual fermented grains are denoted as C12, and the obtained base liquor is denoted as J12.

[0135] Comparative Example 1

[0136] The preparation of the base liquor for Baijiu was carried out according to the method of Example 2, except that sorghum in Example 2 was replaced with rice.

[0137] The resulting residual fermented grains are denoted as CA, and the resulting base liquor is denoted as JA.

[0138] Comparative Example 2

[0139] The preparation of the base liquor for Baijiu was carried out according to the method of Example 2, except that Pediococcus acidilactici in Example 2 was adjusted to Clostridium butyricum (purchased from Guangdong Provincial Microbial Culture Collection Center, strain number GDMCC NO. 1.676).

[0140] The resulting residual fermented grains are denoted as CB, and the resulting base liquor is denoted as JB.

[0141] Test Example

[0142] This example illustrates the detection of the physical and chemical properties of the base liquor.

[0143] 1. The content of urolithin in the base liquor was detected by high performance liquid chromatography, and the steps were as follows:

[0144] S1. Parameter settings for high performance liquid chromatography: Mobile phase: Phase A: 0.1% formic acid aqueous solution (v / v); Phase B: 100% acetonitrile. Mix Phase A and Phase B evenly and ultrasonicate in an ultrasonic cleaner for 30 min.

[0145] The elution gradient of this program was: 0 - 6 minutes, 10 - 100% acetonitrile; 6 - 7 minutes, 100 - 100% acetonitrile; 7 - 9 minutes, 100 - 10% acetonitrile; 9 - 10 minutes, 10 - 10% acetonitrile.

[0146] Chromatographic column model: MP C18 (250×4.6 mm / 5 μm) column; Mobile phase flow rate: 0.5 mL / min; Detection wavelength: 306 nm; Detection temperature: 40 °C.

[0147] S2. Respectively pipette 15 groups of base liquor (J1, J2, J3, J4, J5, J6, J7, J8, J9, J10, J11, J12, JA, and JB), 5 ml of each group of base liquor, and dilute it 10 times with deionized water.

[0148] S3. Use an autosampler to inject the base liquor diluted in step S2 into the high performance liquid chromatograph.

[0149] The detection results are shown in Table 1.

[0150] 2. Calculate the alcohol content of the base liquor using alcohol, and the steps are as follows:

[0151] Absorb 15 groups of base wines (J1, J2, J3, J4, J5, J6, J7, J8, J9, J10, J11, J12, JA, and JB) separately, with 50 ml for each group, and inject them into a 100-ml beaker. Place the alcohol meter vertically into the wine, ensure it is level with the liquid surface, wait for 3 minutes for the alcohol meter to stabilize, and ensure accurate readings. The test results are shown in Table 1.

[0152] 3. Refer to GB5009.5—2016, use the Kjeldahl method to measure the protein content in the sorghum powder before fermentation and the residual fermented grains substrates (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, CA, and CB) after the second-round fermentation, and convert the conversion rate. The test results are shown in Table 1.

[0153] 4. Refer to GB5009.9—2023, use the acid hydrolysis method to measure the total starch content in the sorghum powder before fermentation and the residual fermented grains substrates (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, CA, and CB) after the second-round fermentation, and convert the conversion rate. The test results are shown in Table 1.

[0154] 5. Refer to GB5009.6—2016, use the Soxhlet extraction method to measure the fat content in the sorghum powder before fermentation and the residual fermented grains substrates (C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, CA, and CB) after the second-round fermentation, and convert the conversion rate. The test results are shown in Table 1.

[0155] Table 1

[0156]

[0157] The results in Table 1 show that the urolithin content of the base wine prepared in the examples and the utilization rate of the sorghum substrates are significantly higher than those in the comparative examples. Therefore, the urolithin content, the utilization rate of sorghum, and the nutritional value of the sorghum fermented food prepared by the method provided by the present invention are better.

[0158] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A Pediococus acidilactici, characterized in that: The deposit number of the Pediococcus acidilactici is GDMCC No.65676.

2. A bacterial agent, characterized in that The bacterial agent contains the Pediococcus acidilactici according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent further contains auxiliary bacteria, and the auxiliary bacteria are selected from any one of thermophilic Streptococcus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii subspecies, Bifidobacterium bulgaricus, Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium breve; Preferably, the auxiliary bacteria is Streptococcus thermophilus.

4. The bacterial agent according to claim 3, characterized in that The bacterial agent is selected from at least one of a liquid bacterial agent, a semi-liquid bacterial agent and a solid bacterial agent; Preferably, the bacterial agent is a liquid bacterial agent, and the concentration of the lactic acid Pediococcus is 1×10 7 -1×10 8 CFU / mL, the concentration of the auxiliary bacteria is 1×10 7 -1×10 11 CFU / mL.

5. Use of the Pediococcus acidilactici according to claim 1 and / or the bacterial agent according to any one of claims 2 to 4 in the preparation of sorghum fermented food.

6. The use according to claim 5, characterized in that: The fermented sorghum food is selected from at least one of pasta, condiments and alcoholic beverages; Preferably, the fermented sorghum food is an alcoholic beverage.

7. A method for preparing fermented sorghum food, characterized in that: The method comprises: contacting the Pediococcus acidilactici according to claim 1 and / or the bacterial agent according to any one of claims 2 to 4 with a sorghum substrate for fermentation culture.

8. The method according to claim 7, characterized in that The method comprises: contacting the bacterial agent with a sorghum substrate for fermentation culture; Preferably, relative to 100g of the sorghum substrate, the dosage of the bacterial agent is 2-4.5g; Preferably, the sorghum substrate is obtained by crushing, cleaning and steaming sorghum; Preferably, the steaming conditions include: pressure of 0.1-0.2 MPa, time of 20-40 min; Preferably, the particle size of the sorghum substrate is less than 1 mm.

9. The method according to claim 7 or 8, characterized in that: The fermentation culture conditions include: temperature of 42-45°C and time of 29-33 days.

10. The fermented sorghum food prepared according to the method according to any one of claims 7 to 9.

Images