Lactococcus lactis and application thereof

Abstract

This invention discloses a strain of Lactococcus lactis, named Lactococcus lactis ( Lactococcus lactis ZY2, with accession number GDMCC No. 66499, is a *Lactococcus lactis* strain provided by this invention. It exhibits no antibiotic resistance, good gastrointestinal tolerance, and in vitro α-glucosidase inhibitory activity, making it suitable for preparing drugs with hypoglycemic functions and / or functional foods that help maintain healthy blood sugar levels. Fermenting chickpea milk using *Lactococcus lactis* ZY2 provided by this invention significantly improves the flavor, increases the total phenolic content, and enhances the inhibitory activity against α-glucosidase, thereby further improving the effect of chickpea milk in maintaining healthy blood sugar levels.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and mainly relates to a strain of lactococcus lactis and its applications. Background Technology

[0002] Traditional fermented milk mostly uses cow's milk as its raw material. However, due to the naturally occurring lactose and cholesterol in cow's milk, people with lactose intolerance experience metabolic disorders when consuming traditional dairy products, leading to discomfort. Fermented dairy products based on plant proteins, on the other hand, show great promise in the health food industry due to their unique nutritional composition and functional properties. Plant-based ingredients are rich in fermentable carbohydrates, amino acids, B vitamins, and minerals, providing ideal fermentation substrate conditions for lactic acid bacteria. Currently, common plant-based milk ingredients on the market mainly include legumes, grains, nuts, and seeds.

[0003] Lactic acid bacteria ( Lactic acid bacteria Lactic acid bacteria (LAB), as a potential probiotic, have attracted widespread attention due to their outstanding effectiveness in maintaining intestinal health and preventing and treating diarrhea. In the food industry, the use of lactic acid bacteria in the preparation of fermented foods is very common. Through the fermentation process, lactic acid bacteria can improve the flavor of plant-based milk, and the live bacteria themselves can also promote human health through various mechanisms such as regulating the intestinal microecological balance, promoting digestion and absorption, and enhancing the body's immunity. However, most existing commercial strains are derived from human feces or animal dairy products, making them more suitable for animal dairy substrates and less suitable for growth in plant-based substrates.

[0004] Chickpeas ( Cicer arietinum L. Chickpeas contain high-quality plant-based protein, dietary fiber, and various bioactive components, making them an excellent source of plant-based milk. However, the natural beany flavor of chickpeas limits their application in plant-based milk. Summary of the Invention

[0005] The main objective of this invention is to provide a strain of Lactococcus lactis ( Lactococcus lactis ZY2, to solve at least one of the above-mentioned technical problems.

[0006] According to a first aspect of the invention, a strain of Lactococcus lactis is provided.

[0007] The lactococcus lactis provided in this invention is derived from commercially available naturally fermented mustard greens from Guangdong, and has been identified as lactococcus lactis (Lactococcus lactis). Lactococcus lactis It was named Lactococcus lactis ZY2 ( Lactococcus lactis ZY2 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66499 and deposit date of June 12, 2025.

[0008] The Lactococcus lactis ZY2 provided by this invention is a lactic acid bacterium that is free from antibiotic resistance, has good gastrointestinal tolerance, and exhibits in vitro α-glucosidase inhibitory activity. It can be used to prepare drugs with hypoglycemic function and / or to prepare functional foods that help maintain healthy blood sugar levels.

[0009] According to a second aspect of the present invention, a microbial agent is provided containing *Lactococcus lactis* ZY2 provided by the present invention. This microbial agent can be used in the preparation of pharmaceuticals with hypoglycemic functions and / or in the preparation of functional foods that help maintain healthy blood sugar levels.

[0010] According to a third aspect of the invention, the application of Lactococcus lactis ZY2 in chickpea milk fermentation is provided.

[0011] According to a fourth aspect of the present invention, a method for preparing fermented chickpea milk using *Lactococcus lactis* ZY2 of the present invention is provided, comprising the following steps:

[0012] The product is obtained by inoculating a suspension of Lactococcus lactis ZY2 into sterilized chickpea milk and fermenting it.

[0013] In some embodiments, the method may further include the step of dissolving a sweetener in chickpea milk and sterilizing it to obtain sterilized chickpea milk. This increases the sweetness and improves the sourness of fermented chickpea milk.

[0014] In some embodiments, the sweetener may be selected from at least one of granulated sugar, glucose, fructose, lactose, and cellobiose.

[0015] In some implementations, the sweetener can be white sugar. This not only increases the sweetness of the product but also provides an additional carbon source for the lactic acid bacteria, promoting their fermentation.

[0016] In some embodiments, the method for preparing fermented chickpea milk using Lactococcus lactis ZY2 may include the following steps:

[0017] Dissolve 4-8% (w / v) of sweetener in chickpea milk, sterilize it, inoculate with a bacterial suspension of Lactococcus lactis ZY2 at 1-2% (v / v), ferment at 30-40℃ for 18-30 hours, and then ripen at 2-8℃ for 18-30 hours to obtain the product.

[0018] In some embodiments, sterilization can be performed by autoclaving at 100°C for 15 minutes.

[0019] In some embodiments, the concentration of the Lactococcus lactis ZY2 suspension can be 1×10⁻⁶. 7 ~1×10 9CFU / mL.

[0020] In some embodiments, the concentration of the Lactococcus lactis ZY2 suspension can be 1×10⁻⁶. 8 CFU / mL.

[0021] The preparation of fermented chickpea milk using the Lactococcus lactis ZY2 strain provided by this invention has the following advantages:

[0022] (1) After being fermented by Lactococcus lactis ZY2, chickpea milk can significantly reduce the content of beany substances and increase the content of aromatic substances, thereby significantly improving the flavor of chickpea milk.

[0023] (2) Fermented chickpea milk prepared by fermentation with Lactococcus lactis ZY2 has good water-holding capacity and pH, and the viable count is higher than 1×10⁻⁶. 8 CFU / mL.

[0024] (3) The application of Lactococcus lactis ZY2 to ferment chickpea milk can significantly increase the total phenol content and the inhibitory activity against α-glucosidase in chickpea milk, which is more conducive to improving the effect of chickpea milk in maintaining healthy blood sugar levels.

[0025] (4) The results of the simulated in vitro digestion experiment of mouth, stomach and intestine showed that the survival rate of live bacteria in the fermented chickpea milk prepared by fermentation with Lactococcus lactis ZY2 after digestion in the mouth, stomach and intestine was as high as 98%, indicating that chickpea milk has a certain protective effect on Lactococcus lactis ZY2, enabling it to survive effectively and play a role in maintaining healthy blood sugar levels in the intestine.

[0026] (5) The results of the simulated in vitro digestion experiment of mouth, stomach and intestine showed that after fermented chickpea milk prepared by fermentation with Lactococcus lactis ZY2 was digested by mouth, stomach and intestine, the total phenol content and α-glucosidase inhibitory activity of the fermented chickpea milk were significantly increased compared with those before digestion. This indicates that the bioactive components of fermented chickpea milk can be effectively released after digestion, and its effect of maintaining healthy blood sugar levels is also enhanced.

[0027] The fermented chickpea milk prepared by this invention helps maintain healthy blood sugar levels and can be used directly as a functional food to help maintain healthy blood sugar levels or applied to the preparation of functional foods to help maintain healthy blood sugar levels. Attached Figure Description

[0028] Figure 1 For the present invention Lactococcus lactis Colony morphology diagram of strain ZY2;

[0029] Figure 2 For the present invention Lactococcus lactis Cell morphology diagram of strain ZY2. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to the embodiments. The embodiments are for illustrative purposes only and do not limit the invention in any way. Unless otherwise specified, the raw materials and reagents used in the embodiments are conventional products that can be obtained commercially; experimental methods that do not specify specific conditions in the embodiments are generally performed under conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0031] In the embodiments of the present invention:

[0032] (1) Gram staining, morphological observation and commonly used physiological and biochemical identification culture media refer to Ling Daiwen's "Classification, Identification and Test Methods of Lactic Acid Bacteria", China Light Industry Press, 1999.

[0033] (2) Preparation of MRS liquid culture medium (g / L) (for the cultivation of lactic acid bacteria)

[0034] 10.0 g of casein digest, 10.0 g of beef extract powder, 4.0 g of yeast extract powder, 2.0 g of triammonium citrate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate (MgSO4·7H2O), 0.05 g of manganese sulfate (MnSO4·4H2O), 2.0 g of dipotassium hydrogen phosphate, 20.0 g of glucose, and 1.0 g of Tween-80 were added to distilled water to a final volume of 1 L. The pH was adjusted to 5.7 ± 0.2 (MRS solid medium is prepared by adding 1.5% agar to the liquid medium). The mixture was then sterilized at 121°C for 15 min.

[0035] (3) Assay of α-glucosidase inhibitory activity

[0036] Add 50 mg (or μL) of the sample to be tested, 500 μL of p-nitrophenyl-α-D-glucopyranoside (PNPG, 2.5 mmol / L), and 500 μL of PBS solution (0.1 mol / L, pH 6.8) to a centrifuge tube, and incubate (37℃, 10 min). Then add 100 μL of α-glucosidase (0.2 U / mL) and react at 37℃ for 20 min. Finally, add 800 μL of Na2CO3 solution (1 mol / L) to terminate the reaction. Centrifuge (10000×) g (10 min), take the supernatant and measure the OD value at a wavelength of 405 nm.

[0037] Physiological saline and PBS buffer solution were used instead of the test samples and α-glucosidase, while the amounts and order of other reagents remained unchanged. The specific experimental groups are shown in Table 1 below:

[0038] Table 1. Experimental Groups for Assay of α-glucosidase Inhibitory Activity

[0039]

[0040] The formula for calculating the α-glucosidase inhibition rate is:

[0041]

[0042] (4) The method for determining the number of viable lactic acid bacteria is in accordance with GB4789.35-2016 "National Food Safety Standard for Microbiological Examination of Food: Lactic Acid Bacteria Examination".

[0043] Example 1: Strain Screening and Identification

[0044] 1. Sampling and plate separation

[0045] Commercially available, naturally fermented mustard greens from Guangdong were collected using sterile sampling bottles and immediately diluted with sterile water to a concentration of 10. -3 10 -4 10 -5 The culture was spread onto MRS solid medium and incubated at 37°C for 48 hours. Suspected colonies were picked and streaked onto plates for isolation. This process was repeated 4-5 times until pure single colonies were obtained. The strain of the purified single colony was named ZY2. The purified single colony was then stab-inoculated into MRS solid medium and stored at 4°C.

[0046] 2. Morphological observation and physiological and biochemical experiments

[0047] like Figures 1 - 2 As shown, on MRS solid medium plates, the colonies of strain ZY2 are raised, round, generally 1-2 mm in diameter, white or grayish-white, opaque, moist and smooth. Figure 1 The bacteria are Gram-positive, non-spore-forming bacilli, arranged in spherical, paired, or clumped forms. Figure 2 ).

[0048] The results of routine physiological and biochemical experiments are shown in Table 2. Table 2 shows that strain ZY2 is Gram-positive, catalase-negative, hemolytic-negative, biogenic amine-negative, and non-motile.

[0049] Table 2 Physiological and biochemical characteristics of strain ZY2

[0050]

[0051] 3. Molecular identification

[0052] The ZY2 strain was activated and cultured, then sent to a professional testing institution for sequencing to obtain the 16S rDNA sequence (the specific sequence is shown in SEQ NO: 1). The results were compared with the NCBI gene database to find the standard strain MT545051.1, which is closely related to the ZY2 strain. Lactococcus lactis strain 4536), KX713999.1 ( Lactococcus lactis strain 14B4) and OR083513.1 ( Lactococcus lactis The 16S rDNA of strain ZY2 (strain QJQJ-7) was analyzed for similarity with the standard strain. The results are shown in Table 3. Strain ZY2 is similar to Lactococcus lactis. Lactococcus lactis The sequence homology of strain 4536 is 99.64%, indicating that they are the same strain.

[0053] Table 3. BLAST sequence alignment results of strain ZY2 based on its 16S rDNA sequence.

[0054]

[0055] Molecular identification results, combined with colony and cell morphology and physiological and biochemical characteristics, can identify strain ZY2 as *Lactococcus lactis* (Lactococcus lactis). Lactococcus lactis ), named it Lactococcus lactis ( Lactococcus lactis ZY2.

[0056] This strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66499 and deposit date of June 12, 2025.

[0057] The following uses Lactobacillus casei strain Shirota ( Lacticaseibacillus paracasei Shirota was used as a positive control to further measure the results provided in this application. Lactococcus lactis Relevant properties of ZY2.

[0058] Experimental Example 1 Lactococcus lactis Antibiotic susceptibility analysis of ZY2

[0059] (1) Preparation of bacterial suspension

[0060] Take a 10 mL test tube, add 10 mL of MRS liquid culture medium, sterilize at 121℃ for 15 min, and pick out portions of each. Lactococcus lactis ZY2 and Lacticaseibacillus paracasei A single Shirota colony was inoculated into MRS liquid medium and incubated at 36°C for 24 h; then, a 1% (v / v) inoculum was added to 50 mL of MRS liquid medium and incubated at 36°C for another 24 h. After incubation, the colonies were cultured at 4000 × 10⁻⁶ mcg.g The bacterial cells were obtained by centrifugation for 10 min, resuspended in sterile physiological saline, and the bacterial concentration was adjusted to 1×10⁻⁶. 9 CFU / mL, Lactococcus lactis ZY2 and Lacticaseibacillus paracasei Shirota bacterial suspension.

[0061] (2) Preparation of resistant plates

[0062] According to the European Food Safety Authority's criteria for determining bacterial resistance (2012 edition), an antimicrobial stock solution with a concentration of 5120 μg / mL was prepared using the antibiotics ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol. Before use, the solution was diluted to the required concentration using the dilution method to obtain an antimicrobial dilution, which was then added to the corresponding culture medium in proportion.

[0063] Dispense 9.0 mL of MRS agar medium into 10 mL test tubes, sterilize at 121°C for 15 min, and then keep warm in a water bath (50°C) for later use.

[0064] Pipette 1.0 mL of the diluted antibacterial drug solution into a large test tube containing 9.0 mL of MRS agar medium, vortex immediately to mix, pour into a sterile Petri dish, and allow to solidify to obtain an inhibition plate with a specific drug concentration (μg / mL). Simultaneously, prepare a control plate without the drug.

[0065] (3) Antibiotic susceptibility analysis

[0066] Take 1 μL of bacterial suspension (1×10 9 Inoculate the agar plate with CFU / mL and incubate at 37°C for 16–20 h. Use uninoculated blank plates as a control. Observe and record the critical concentration (MIC) at which the strain does not grow on the agar plate. The experiment was conducted in triplicate.

[0067] The criteria for judging the results of lactic acid bacteria antimicrobial susceptibility testing and the MIC values ​​are shown in Table 4 below:

[0068] Table 4. Criteria for judging the results of lactic acid bacteria drug susceptibility (MIC value)

[0069]

[0070] Table 4 shows the results compared to the positive control strain. Lacticaseibacillus paracasei Like Shirota, the present invention provides Lactococcus lactis ZY2's MIC values ​​for all nine antibiotics were below the resistance inflection point, meeting EFSA safety standards.

[0071] Experimental Example 2 Lactococcus lactisGastrointestinal tolerance of ZY2

[0072] The preparation process of the bacterial suspension is the same as in Experiment 1.

[0073] Preparation of simulated gastric juice: 0.35 g pepsin was diluted in 100 mL of sterile 0.3% (w / v) NaCl solution, the pH was adjusted to 3 with hydrochloric acid, and the solution was filtered through a 0.22 μm disposable syringe filter for sterilization.

[0074] Preparation of simulated intestinal fluid: 0.1 g trypsin was diluted in 100 mL of sterile 1% (w / v) phosphate buffer, the pH was adjusted to 8.0 with sodium hydroxide, and the solution was filtered through a 0.22 μm disposable syringe filter for sterilization.

[0075] Take 1 mL of bacterial suspension (approximately 1×10⁻⁶) 9 The CFU / mL solution was inoculated into 9 mL of simulated gastric fluid (pH=3) and incubated in a constant temperature incubator at 37°C. After 4 hours, 1 mL of the digestive fluid was transferred to 9 mL of simulated intestinal fluid (pH=8) and incubated in a constant temperature incubator at 37°C for 4 hours. After the incubation, samples were taken and the viable count was calculated using the dilution spread method.

[0076] The results showed that after digestion by simulated gastric and intestinal juices, Lactococcus lactis ZY2 had a remaining viable bacterial count of 8.87 log CFU / mL, with a survival rate of 90%; while the positive control bacteria... Lacticaseibacillus paracasei The remaining viable bacteria count of Shirota was 7.97 log CFU / mL, with a survival rate of 82%. This invention provides... Lactococcus lactis The remaining viable bacteria count and survival rate of ZY2 were significantly higher than those of the positive control bacteria. Lacticaseibacillus paracasei Shirota.

[0077] Experiment Example 3 Lactococcus lactis ZY2 in vitro α-glucosidase inhibitory activity

[0078] Take a 10 mL test tube, add 10 mL of MRS liquid culture medium, sterilize at 121℃ for 15 min, and pick out portions of each. Lactococcus lactis ZY2 and Lacticaseibacillus paracasei A single Shirota colony was inoculated into MRS liquid medium and incubated at 36°C for 24 h; then, a 1% (v / v) inoculum was added to 50 mL of MRS liquid medium and incubated at 36°C for another 24 h. After incubation, the colonies were cultured at 4000 × 10⁻⁶ mcg. g Centrifuge for 10 min and collect the supernatant for later use.

[0079] Take 50 μL of the bacterial culture supernatant for in vitro α-glucosidase inhibitory activity assay.

[0080] The results showed that Lactococcus lactisZY2 inhibited α-glucosidase by 86%, which was higher than that of the positive control bacteria. Lacticaseibacillus paracasei Shirota (83%).

[0081] Example 2, Application Lactococcus lactis ZY2 Fermented Chickpea Milk Preparation Fermented Chickpea Milk

[0082] Includes the following steps:

[0083] (1) Preparation of sterilized chickpea milk

[0084] Take 100 g of chickpeas, add 400 mL of 0.5% (w / v) NaHCO3 solution, soak overnight (14~16 h) at 4℃, discard the supernatant, and wash the chickpeas with distilled water; add 800 mL of distilled water to the chickpeas, grind them in a high-speed blender for 20 min, filter through a 180-mesh sieve to obtain chickpea milk, boil at 100℃, add 7% (w / v) white sugar, shake well to dissolve, sterilize by high-pressure steam at 100℃ for 15 min, and cool to room temperature before use.

[0085] (2) Lactococcus lactis Preparation of ZY2 bacterial suspension

[0086] Pick Lactococcus lactis A single colony of ZY2 was inoculated into sterile MRS liquid medium and incubated at 36°C for 24 h. After incubation, the culture was repeated at 4000 × 10⁻⁶. g The bacterial cells were obtained by centrifugation for 10 min, resuspended in sterile physiological saline, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL, Lactococcus lactis ZY2 bacterial suspension.

[0087] (3) Application Lactococcus lactis ZY2 Fermented Chickpea Milk

[0088] The inoculation was prepared in step (2) at a dose of 1% (v / v). Lactococcus lactis The ZY2 bacterial suspension was cultured in the sterilized chickpea milk obtained in step (1) at 37°C for 24 h and then ripened at 4°C for 24 h to obtain fermented chickpea milk.

[0089] In addition, Lactobacillus casei strain Shirota ( Lacticaseibacillus paracasei Shirota was used as the fermentation starter, and the above method was followed. Lacticaseibacillus paracasei Shirota fermented chickpea milk is produced by fermentation.

[0090] To apply Lactococcus lactis ZY2 Lacticaseibacillus paracasei Fermented chickpea milk prepared by Shirota fermentation was used as a sample to determine its relevant properties.

[0091] Experiment Example 4: Basic Characteristics and Detection of Volatile Flavor Compounds in Fermented Chickpea Milk

[0092] (1) Basic characteristics

[0093] The viable count, pH value, and water-holding capacity of fermented chickpea milk were determined.

[0094] The method for determining the viable count of lactic acid bacteria refers to GB4789.35-2016 "National Food Safety Standard - Microbiological Examination of Food - Lactic Acid Bacteria Examination".

[0095] pH values ​​are measured using a pH meter.

[0096] The method for determining water-holding capacity includes the following steps: Weigh the empty centrifuge tube and record the mass as . m 0; Place 12 mL of fermented chickpea milk sample into a 15 mL centrifuge tube and record the mass as 0. m 1. Centrifuge at 4000 rpm / min for 10 min, discard the supernatant, and record the mass. m 2. Calculate the water-holding capacity of the fermented chickpea milk sample using the following formula. W :

[0097]

[0098] The results are shown in Table 5 below. Application of this invention Lactococcus lactis The live bacteria count in the fermented chickpea milk produced by ZY2 fermentation meets the national standard GB / T 30885-2014 requirement for the live lactic acid bacteria count in fermented soy milk (1×10⁻⁶). 6 (CFU / mL), and compared with the positive control bacteria Lacticaseibacillus paracasei Shirota fermented chickpea milk is quite similar.

[0099] Table 5. Basic characteristics of fermented chickpea milk

[0100]

[0101] (2) Analysis of volatile flavor compounds

[0102] Volatile aromatic compounds in unfermented and fermented chickpea milk were analyzed using solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC / MS).

[0103] A method for analyzing volatile aromatic compounds using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC / MS): A 50 / 30 μm DVB / CAR / PDMS extraction fiber (SPME) was aged at the injection port (270 °C) for 30 min. 2.0 mL of the sample was weighed and added to a 25 mL headspace vial. The vial was heated to 40 °C and equilibrated for 10 min. The SPME needle was then inserted, and after adsorption for 30 min, it was inserted into the GC / MS injection port for analysis.

[0104] Gas chromatography conditions: Injector temperature 300℃; Temperature program: 35℃ for 2 min, then ramp at 5℃ / min to 110℃ and hold for 8 min; ramp at 15℃ / min to 240℃ and hold for 5 min. Split ratio 30:1.

[0105] Mass spectrometry conditions: mass spectrometer interface temperature 250℃; ion source temperature 230℃; quadrupole temperature 150℃; ionization mode: EI; scan mode: full scan; mass number range: 33~400 m / z; solvent delay: 0.1 min.

[0106] The results are shown in Table 6. The results demonstrate that the application of this invention... Lactococcus lactis Fermented chickpea milk prepared by strain ZY2 significantly reduced the content of volatile components responsible for the beany odor, such as hexanal (reduced by 99%), nonanal (reduced by 100%), and pentylfuran (reduced by 46%). In contrast, Lacticaseibacillus paracasei Shirota has limited ability to improve the beany odor; for example, it only reduces hexanal by 89% and pentylfuran by only 39%, and even increases the content of some volatile beany odor compounds such as 2-ethylfuran. Meanwhile, Lactococcus lactis The ZY2 strain can also enhance the composition and content of aroma components in soy milk, such as 2,3-butanedione, acetic acid, and hexanoic acid. Among these, 2,3-butanedione is a characteristic flavor compound of milk, indicating that the application of this invention... Lactococcus lactis Fermented chickpea milk made from ZY2 strain has a unique milky aroma.

[0107] Table 6. Results of Volatile Flavor Compound Analysis

[0108]

[0109] Experimental Example 5: Analysis of viable bacterial count, bioactive substances, and α-glucosidase inhibitory activity in fermented chickpea milk after simulated digestion.

[0110] (1) In vitro simulated digestion of fermented chickpea milk

[0111] Take 30 g of fermented chickpea milk, dilute it with 30 mL of NaCl (0.9%) solution, adjust the pH and simulate oral (1 min), gastric (2 h) and intestinal (2 h) digestion.

[0112] Simulated oral digestion: The pH of each sample was adjusted to 6.0 using 1 mol / L NaHCO3 solution. 0.3 mL of simulated saliva (prepared by dissolving α-amylase in 1 mmol / L CaCl2 solution, with an enzyme activity of 100 U / mL) was added. The mixture was subjected to simulated oral digestion at 37℃ and 200 rpm / min for 1 min. The reaction was then stopped by cooling in an ice-water bath.

[0113] Simulated gastric digestion: The pH of the sample after simulated oral digestion was adjusted to 2.0 with 1 mol / L HCl solution, and then simulated gastric juice (prepared by dissolving 250 mg of pepsin in 10 mL of 0.1 mol / L HCl) was added at a ratio of 0.05 mL / g of digested sample. The simulated gastric digestion was carried out at 37℃ and 100 rpm / min for 2 h, and the reaction was terminated by cooling in an ice-water bath.

[0114] Simulated intestinal digestion: The pH of the sample after simulated oral and gastric digestion was adjusted to 6.0 with 1 mol / L NaHCO3 solution, and then simulated intestinal fluid (prepared by dissolving 40 mg trypsin and 250 mg bovine bile salt in 10 mL 0.1 mol / L NaHCO3) was added at a ratio of 0.125 mL / g of digested sample. Simulated intestinal digestion was carried out at 37℃ and 100 rpm / min for 2 h, and the reaction was terminated by cooling in an ice-water bath.

[0115] Samples were collected after each digestion stage and subjected to 4℃ and 10000× g After centrifugation for 10 min, collect the supernatant, filter it through a 0.22 μm filter membrane, and set it aside for later use.

[0116] (2) Viable bacterial count analysis

[0117] Samples were collected after the simulated intestinal digestion was completed, and the viable bacterial count was calculated using the dilution plating method.

[0118] The results show that the application Lactococcus lactis The fermented chickpea milk produced by ZY2 fermentation, after oral, gastric, and intestinal digestion, showed a decrease in viable bacterial count from 8.36 log CFU / mL to 8.21 log CFU / mL, with a survival rate of 98%, compared to the positive control bacteria. Lacticaseibacillus paracasei Shirota levels were comparable (decreased from 8.51 log CFU / mL to 8.39 log CFU / mL, with a survival rate of 98.6%). This indicates that chickpea milk is effective against... Lactococcus lactis ZY2 has a certain protective effect, enabling it to survive effectively and play a role in maintaining healthy blood sugar levels in the intestines.

[0119] (3) Analysis of total phenol content

[0120] Samples were collected from unfermented, fermented, oral, gastric, and intestinal digested states, and subjected to a process at 4°C and 10000× g After centrifugation for 10 min, the supernatant was collected, filtered through a 0.22 μm filter membrane, and the total phenol content in the supernatant was determined.

[0121] Total phenol content determination method: Take 0, 0.4, 0.8, 1.2, 1.6, and 2.0 mL of gallic acid standard solution (50 mg / L) respectively, place them in test tubes, add distilled water to adjust the final volume to 2.0 mL, add 1 mL of Folin-phenol reagent and react at room temperature. After 10 min, add 3 mL of sodium carbonate solution (7.5%), shake slowly on a shaker, and incubate in a water bath (45℃, 1.5 h). Then measure the absorbance at 765 nm and plot a standard curve. Repeat the above steps for the sample to be tested, measure the absorbance at 765 nm, and calculate its content according to the standard curve.

[0122] The results showed that the total phenol content of unfermented chickpea milk was 10.99 mg / L. Lactococcus lactis After fermentation, the concentration of ZY2 increased to 25.94 mg / L, which was significantly higher than that obtained by other methods. Lacticaseibacillus paracasei The concentration of fermented chickpea milk produced by Shirota fermentation is 15.64 mg / L.

[0123] After being digested sequentially through the mouth, stomach, and intestines, Lactococcus lactis The total phenolic content of fermented chickpea milk obtained by ZY2 fermentation increased to 405.86 mg / L, which is 15.65 times that before digestion, and significantly higher than that of the positive control bacteria. Lacticaseibacillus paracasei Fermented chickpea milk (248.61 mg / L) obtained from Shirota fermentation. This indicates that after digestion, the product provided by this invention... Lactococcus lactis The bioactive components of fermented chickpea milk produced by ZY2 fermentation can be effectively released.

[0124] (4) α-glucosidase inhibitory activity

[0125] Samples were collected from unfermented, fermented, oral, gastric, and intestinal digested states, and subjected to a process at 4°C and 10000× g After centrifugation for 10 min, the supernatant was collected, filtered through a 0.22 μm filter membrane, and the α-glucosidase inhibitory activity of the supernatant was measured.

[0126] The results showed that unfermented chickpea milk had a 23% inhibitory activity against α-glucosidase. Lactococcus lactis ZY2 increased to 81% after fermentation, and after... Lacticaseibacillus paracasei The inhibitory activity of Shirota after fermentation was only 74%.

[0127] After being digested sequentially through the mouth, stomach, and intestines, Lactococcus lactis The fermented chickpea milk fermented by ZY2 showed 1.7 times higher inhibitory activity against α-glucosidase than the undigested product, which was higher than that of the positive control bacteria. Lacticaseibacillus paracasei 1.4 times that of Shirota.

[0128] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A strain of Lactococcus lactis, characterized in that, designated Lactococcus lactis (L. lactis) Lactococcus lactis ) ZY2, with the accession number GDMCC No. 66499.

2. An inoculant characterized in that, The bacterial agent contains the lactococcus lactis as described in claim 1.

3. The use of the Lactococcus lactis according to claim 1 or the microbial agent according to claim 2 in the preparation of drugs with hypoglycemic function and / or in the preparation of functional foods that help maintain healthy blood sugar levels.

4. The application of the Lactococcus lactis according to claim 1 or the inoculum according to claim 2 in chickpea milk fermentation.

5. A method of preparing a fermented chickpea milk that helps maintain a healthy level of blood sugar using the Lactococcus lactis of claim 1, characterized by, Includes the following steps: The bacterial suspension of Lactococcus lactis as described in claim 1 is inoculated into sterilized chickpea milk for fermentation to obtain the product.

6. The method of claim 5, wherein, It also includes the following steps: dissolving the sweetener in chickpea milk, sterilizing it, and obtaining sterilized chickpea milk.

7. The method of claim 6, wherein, The sweetener is selected from at least one of white sugar, glucose, fructose, lactose and cellobiose.

8. The method according to claim 7, characterized in that, Includes the following steps: Dissolve 4-8% (w / v) of sweetener in chickpea milk, sterilize it, and then inoculate it with 1-2% (v / v) of the lactococcus lactis suspension described in claim 1. Ferment at 30-40°C for 18-30 hours, and then ripen at 2-8°C for 18-30 hours to obtain the product.

9. The method according to any one of claims 5 to 8, characterized in that, The concentration of the bacterial suspension was 1 x 10 7 ~1 x 10 9 CFU / mL.

10. Fermented chickpea milk prepared by the method according to any one of claims 5 to 9, which helps maintain healthy blood sugar levels.

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