Application of phytobacterium plantarum FMBL L23251 FJX in removal of beany flavor and preparation of fermented bean products

Abstract

The invention relates to the technical field of biology, in particular to application of plant lactobacillus FMBL L23251FJX to removal of beany flavor and preparation of fermented bean products, the plant lactobacillus FMBL L23251FJX is preserved in the China Center for Type Culture Collection on June 26, 2023, the preservation number is CCTCC NO: M20231100, the plant lactobacillus FMBL L23251FJX fermented chickpeas has DPPH free radical scavenging capacity, and the plant lactobacillus FMBL L23251FJX can be used for removing the beany flavor and preparing fermented bean products. The chickpea yoghurt prepared by compounding the chickpea powder has the advantages that the antioxidant capacity of the yoghurt is further improved on the basis of ensuring the storage viable count, the release of volatile flavor substances is stimulated, and the health care effect of the chickpea yoghurt is improved, so that the health care effect of the chickpea yoghurt is improved, and the health care effect of the chickpea yoghurt is improved. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of Lactiplantibacillus plantarum FMBL L23251 FJX in removing the beany flavor and preparing fermented soy products. Background Art

[0002] The beany flavor of beans is a familiar taste to many people. Beans contain lipoxygenase. After beans are broken (such as processed into soy milk, etc.), the cell structure is damaged, and lipoxygenase comes into contact with polyunsaturated fatty acids (such as linoleic acid, linolenic acid, etc.) in the beans, catalyzing the oxidation reaction of these fatty acids to generate hydroperoxides. These hydroperoxides are unstable and will further decompose into small molecule aldehydes, ketones, alcohols and other volatile compounds with special odors. Some of these substances are the main components causing the beany flavor. At the same time, the proteins in beans may degrade under certain conditions (such as microbial contamination, improper storage, etc.) to produce some nitrogen-containing compounds with bad odors, such as amines, ammonia, etc. These substances also affect the formation of the beany flavor. In addition, beans also contain some substances with odors themselves, such as phospholipids, sulfur-containing compounds, etc., which contribute to the flavor characteristics of the beany flavor to a certain extent.

[0003] The beany flavor will affect the taste and flavor of bean products, making them unacceptable to some consumers. For example, for common soy products such as soy milk and tofu, if the beany flavor is too strong, it will cover up their original bean aroma and mellow taste, reducing the quality and market acceptance of the products. Methods for removing the beany flavor include heat treatment, soaking treatment, adding additives, etc. Among them, through heat treatment methods such as boiling, steaming, frying, etc., lipoxygenase can be inactivated, thereby inhibiting the oxidation reaction it catalyzes and reducing the generation of beany flavor substances, which is a commonly used method. However, it will cause the loss of nutrients. Soaking treatment can remove part of the beany flavor, but if the soaking time is not well controlled, it will lead to the growth of microorganisms and affect the quality; adding cyclic oligosaccharides such as β-cyclodextrin can encapsulate the beany flavor substances through molecular inclusion, thereby reducing their volatility and alleviating the beany flavor; some spices, sweeteners, etc. can also be added to adjust the flavor of the product and cover up the beany flavor. However, the restricted use of additives limits their application. How to remove the beany flavor without affecting the quality of bean products and without losing their nutritional value has become a technical problem that needs to be solved urgently by those skilled in the art.

[0004] Chickpeas are rich in bioactive substances such as galactooligosaccharides, unsaturated fatty acids, vitamins, isoflavones, etc., as well as trace elements such as iron, zinc, magnesium, calcium, and selenium. They also contain volatile aldehyde substances such as hexanal and benzaldehyde, alcohol substances such as n-hexanol, ketone substances such as 2-heptanone, and acetone, etc. When chickpeas are processed, they produce a strong beany flavor and pungent smell, increasing the complexity of the beany flavor and reducing consumers' desire to purchase chickpeas.

[0005] The research group where the inventors are located has been long-term committed to the research of probiotics. In the previous research, several strains of probiotics that can ferment chickpeas were discovered. For example, the invention patent CN117535185B discloses a Lactobacillus paracasei FMBL L23249 FJX suitable for chickpea fermentation, which can efficiently utilize chickpea milk to achieve rapid proliferation and is used for preparing chickpea yogurt; the invention patent CN117511794B discloses a Lactiplantibacillus plantarum that can efficiently utilize chickpea milk, which can efficiently utilize chickpea milk to achieve rapid proliferation and can be used for preparing chickpea yogurt.

[0006] During the continuous research process, the inventors unexpectedly found that the newly screened Lactiplantibacillus plantarum FMBLL23251 FJX can also efficiently utilize chickpeas for proliferation, and the viable cell rate is higher than that of Lactiplantibacillus plantarum FMBLL23252FJX. On this basis, Lactiplantibacillus plantarum FMBL L23251 FJX can also reduce the beany flavor of bean products and prepare yogurt with a better taste, having broad application prospects. Summary of the Invention

[0007] The primary object of the present invention is to provide the application of Lactiplantibacillus plantarum FMBL L23251 FJX in reducing the beany flavor in beans or bean products. The Lactiplantibacillus plantarum FMBLL23251 FJX was deposited at the China Center for Type Culture Collection on June 26, 2023, with the deposit number CCTCC NO: M20231100.

[0008] Preferably, the beans include one or more of chickpeas, soybeans, broad beans, peas, mung beans, miscellaneous beans, pigeon peas, and sword beans.

[0009] Preferably, the bean products are prepared by processing beans.

[0010] Preferably, the bean products are prepared by non-high-temperature processing of beans.

[0011] The second object of the present invention is to provide a method for reducing the beany flavor in bean products. Using the bean products as a substrate, adding Lactiplantibacillus plantarum FMBL L23251 FJX for fermentation can reduce the beany flavor of the obtained products. The Lactiplantibacillus plantarum FMBL L23251 FJX was deposited at the China Center for Type Culture Collection on June 26, 2023, with the deposit number CCTCCNO: M20231100.

[0012] The third object of the present invention is to provide the application of Lactiiplantibacillus plantarum FMBL L23251 FJX in fermenting beans to prepare soy products. The Lactiiplantibacillus plantarum FMBL L23251 FJX was deposited at the China Center for Type Culture Collection on June 26, 2023, with the deposit number CCTCC NO: M20231100.

[0013] The fourth object of the present invention is a fermented soy milk with reduced beany flavor, which contains Lactiiplantibacillus plantarum FMBL L23251 FJX. The Lactiiplantibacillus plantarum FMBL L23251 FJX was deposited at the China Center for Type Culture Collection on June 26, 2023, with the deposit number CCTCC NO: M20231100.

[0014] The fifth object of the present invention is to provide the application of Lactiiplantibacillus plantarum FMBL L23251 FJX in preparing fermented chickpea yogurt. The Lactiiplantibacillus plantarum FMBL L23251 FJX was deposited at the China Center for Type Culture Collection on June 26, 2023, with the deposit number CCTCC NO: M20231100.

[0015] The sixth object of the present invention is to provide the application of Lactiiplantibacillus plantarum FMBL L23251 FJX in producing short-chain fatty acids by fermenting chickpea milk. The Lactiiplantibacillus plantarum FMBL L23251 FJX was deposited at the China Center for Type Culture Collection on June 26, 2023, with the deposit number CCTCC NO: M20231100.

[0016] The seventh object of the present invention is to provide the application of Lactiiplantibacillus plantarum FMBL L23251 FJX in producing B vitamins by fermenting chickpea milk. The Lactiiplantibacillus plantarum FMBL L23251 FJX was deposited at the China Center for Type Culture Collection on June 26, 2023, with the deposit number CCTCC NO: M20231100.

[0017] The eighth object of the present invention is to provide the application of Lactiiplantibacillus plantarum FMBL L23251 FJX in fermenting chickpeas for inhibiting the activity of dipeptidyl peptidase IV or preparing a dipeptidyl peptidase IV inhibitor. The Lactiiplantibacillus plantarum FMBL L23251 FJX was deposited at the China Center for Type Culture Collection on June 26, 2023, with the deposit number CCTCC NO: M20231100.

[0018] The ninth object of the present invention is to provide the application of Lactiiplantibacillus plantarum FMBL L23251 FJX in fermenting chickpeas to prepare hypoglycemic products or enhancing the hypoglycemic effect of chickpea products. The Lactiiplantibacillus plantarum FMBL L23251 FJX was deposited at the China Center for Type Culture Collection on June 26, 2023, with the deposit number CCTCC NO: M20231100.

[0019] The beneficial effects of the present invention are as follows: The present invention provides the application of Lactiiplantibacillus plantarum FMBL L23251 FJX in removing the beany flavor and preparing fermented soy products. The Lactiiplantibacillus plantarum FMBL L23251 FJX was deposited at the China Center for Type Culture Collection on June 26, 2023, with the deposit number CCTCC NO: M20231100. The fermented chickpeas by Lactiiplantibacillus plantarum FMBLL23251 FJX have DPPH free radical scavenging ability, ABTS free radical scavenging ability and inhibitory activity against dipeptidyl peptidase IV, can produce short-chain fatty acids and B vitamins, and can also significantly reduce the beany flavor in soy products. When compounded with chickpea powder to prepare chickpea yogurt, on the basis of ensuring the viable bacteria count during storage, the antioxidant capacity of the yogurt is further improved, and the release of volatile flavor substances is also stimulated, having broad application prospects. Description of the Drawings

[0020] Figure 1 Phylogenetic tree of Lactiiplantibacillus plantarum FMBL L23251 FJX

[0021] Figure 2 Antioxidant activity of fermented chickpea milk

[0022] Figure 3 pH value, acidity and sensory scores of fermented milk with different chickpea concentrations

[0023] Figure 4 Antioxidant capacity of chickpea yogurt

[0024] Figure 5 Volatile flavor substances of chickpea yogurt

[0025] Figure 6 Changes in viable cell counts of Lactobacillus plantarum FMBL L23251 FJX during the storage period of plants Specific implementation manners

[0026] The protection scope of the present invention will be described in detail below in conjunction with specific implementation manners. It should be noted that the protection scope of the present invention is not limited by the following embodiments.

[0027] Chickpea (Cicer arietinum L.), also known as nohut, is a kind of legume with high nutritional value, a good source of carbohydrates and proteins (accounting for 80% of the dry weight), and can be an important part of a vegetarian diet. Chickpeas are rich in bioactive substances such as galactooligosaccharides, unsaturated fatty acids, vitamins, isoflavones, etc., as well as trace elements such as iron, zinc, magnesium, calcium, and selenium. Due to the rich active substances in chickpeas, it has certain medicinal value. As early as more than 2,500 years ago, the Uyghur residents in China used chickpeas as herbs to treat hypertension and diabetes. Modern research has found that chickpeas can improve cardiovascular diseases, reduce cholesterol levels, improve type 2 diabetes, reduce hypertension, promote the balance of intestinal flora, and improve constipation, etc. Therefore, chickpeas can be considered a good "functional food". According to historical records, the Xinjiang region has a cultivation history of chickpeas of more than 2,000 years. Mulei County is famous for its unique geographical and natural environment and is known as the hometown of chickpeas, with rich resources. How to effectively utilize these resources has become an urgent technical problem to be solved.

[0028] It should be noted that the following embodiments are experiments based on chickpeas. However, other legumes with a beany smell can all be fermented using the Lactobacillus plantarum FMBL L23251 FJX described in the present invention to obtain legume products with significantly reduced beany smell, and their action mechanisms are the same.

[0029] In the following embodiments, unless otherwise specified, the methods described are all conventional methods; the reagents used can all be purchased on the market.

[0030] Formulation of MRS medium: Beef extract 8 g / L; Magnesium sulfate 0.2 g / L; Peptone 10 g / L; Sodium acetate anhydrous 5 g / L; Glucose 20 g / L; Manganese sulfate 0.05 g / L; Dipotassium hydrogen phosphate 2 g / L; Tween-80 1 mL / L; Agar 20 g / L; Yeast extract powder 5 g / L; Ammonium citrate dibasic 2 g / L;

[0031] LAMVAB solid medium (1 L): Peptone 10 g; Beef extract 8 g; Yeast extract 4 g; Glucose 20 g, Tween 80 1 mL; K 2 HPO 42 g; 5 g of sodium acetate, 2 g of diammonium hydrogen citrate; MgSO 4 ·7H 2 O 0.2 g; MnSO 4 ·4H 2 O 0.05 g; 20 g of agar; 0.5 g of L-cysteine hydrochloride; 20 mg of vancomycin hydrochloride, sterilized at 115 °C for 20 min.

[0032] In the following examples, Lactiplantibacillus plantarum WCFS1 (CGMCC 1.6971) was purchased from the China General Microbiological Culture Collection Center.

[0033] Example 1: Screening and Identification of Strains

[0034] 1. Isolation and Purification of Strains

[0035] Strains were isolated from fecal samples of the Han population in Wuwei area according to the conventional method. The collected fresh children's feces were placed in a sampling tube. Take 1 g of fecal sample and inoculate it into 9 mL of MRS liquid medium, mix well, and dilute the fecal sample to 10 -3 、10 -4 、10 -5 , and spread them on LAMVAB agar medium, and culture them at 37 °C for 24 - 48 hours under anaerobic conditions. Pick the suspected lactic acid bacteria colonies and purify them 3 times, then store the isolates in MRS liquid medium supplemented with 25% glycerol, and store them in a -80 °C ultra-low temperature freezer.

[0036] 2. Strain Identification

[0037] Extract the genomic DNA of the strain using a bacterial genomic DNA rapid extraction kit. Use species-level specific primers to perform PCR amplification on the groEL gene of the strain, as shown in Table 1. After the reaction, detect the PCR product by agarose gel electrophoresis. After passing the test, sequence it by Suzhou Genewiz Biotechnology Co., Ltd. Submit the obtained strain sequence to the NCBI database for BLAST alignment, download the homologous species sequences of the strain, and construct a phylogenetic tree based on the neighbor-joining method using the software MEGA 11.0.

[0038] Table 1 PCR Amplification of groEL Gene

[0039]

[0040] The phylogenetic tree of the strain FMBL L23251 FJX is as Figure 1As shown. After identification, the obtained strain was Lactiplantibacillus plantarum, with the Latin name: Lactiplantibacillus plantarum. It was named Lactiplantibacillus plantarum FMBL L23251 FJX and was deposited in the China Center for Type Culture Collection on June 26, 2023, with the deposit number CCTCC NO: M 20231100. The deposit address is: Wuhan University, Wuhan, China, and the phone number is 027 - 68754052.

[0041] In the following examples, Lactiplantibacillus plantarum FMBL L23251 FJX will be abbreviated as Lactiplantibacillus plantarum FMBL L23251 FJX.

[0042] Example 2. Comparison of the characteristics of chickpea milk fermented by different strains

[0043] 1. Activation of strains and preparation of seed liquid

[0044] Using Lactiplantibacillus plantarum FMBL L23252 FJX and the model strain WCFS1 as controls (Table 2). All strains were streaked and activated twice on MRS agar plates, and single colonies were picked and subcultured continuously in MRS liquid medium at 37°C for 2 times, each time for 24 h, with an inoculation amount of 2%. The fermentation broth was centrifuged at 10000 r / min for 10 min, and the cells were washed twice with sterile physiological saline and then resuspended. The concentration of the cell suspension was adjusted to 1.0×10 9 CFU / mL (OD600 to about 1.0) to obtain the seed liquid of the strain.

[0045] Table 2 Strain information

[0046]

[0047] 2. Preparation and fermentation of chickpea milk

[0048] Weigh 100 g of chickpeas without pests, diseases and mechanical damage. Soak them at a ratio of bean:water of 1:2 for 12 h, then filter and drain the water. Then, according to the ratio of bean:water of 1:6, add 60°C hot water and 0.45% α - medium - temperature amylase, grind in a blender for 8 min, and enzymatically hydrolyze at 60°C for 24 min. Then, boil the chickpea homogenate for 5 min to inactivate the enzyme activity. After cooling, filter through a 200 - mesh sterilized filter cloth to obtain chickpea milk. Inoculate the above - prepared seed liquid into the chickpea milk at an inoculation amount of 2% and ferment at 37°C for 12 h to obtain chickpea milk fermented by Lactiplantibacillus plantarum FMBL L23251 FJX.

[0049] 3. Antioxidant capacity

[0050] (1) DPPH radical scavenging ability

[0051] Weigh 39.4 mg of DPPH, dissolve it with 75% methanol and make up the volume to 100 mL to obtain a 1 mmol / L DPPH solution, which is stored in the dark at 0 - 4 °C for later use. Take 4 mL of chickpea milk sample, centrifuge it at 10000 r / min for 10 min, take the supernatant and filter it through a 0.45 μm filter membrane to obtain the sample solution. Take 1 mL of the sample solution, dissolve it in 0.25 mL of DPPH mixture, place it at room temperature in the dark for 30 min, measure the absorbance at 517 nm, and record it as Ai; take 1 mL of the sample solution, dissolve it in 0.25 mL of 75% methanol solution, place it at room temperature in the dark for 30 min, measure the absorbance at 517 nm, and record it as Aj; take 1 mL of 75% methanol solution, dissolve it in 0.25 mL of DPPH mixture, place it at room temperature in the dark for 30 min, measure the absorbance at 517 nm, and record it as A0;

[0052]

[0053] (2) ABTS radical scavenging ability

[0054] Weigh 6 mg of ABTS, add 1.47 mL of distilled water to prepare a 7.4 mmol / L ABTS stock solution; weigh 2 mg of K 2 S 2 O 8 , add 2.86 mL of distilled water to prepare a 2.6 mmol / L ABTS stock solution. Mix the above two solutions in a 1:1 ratio, place them in the dark at room temperature for 12 h, and then dilute them 40 - 50 times with PBS buffer to make the absorbance at OD 734 nm within the range of 0.7 ± 0.02 to obtain the ABTS mixed solution. The sample treatment is the same as that for DPPH. Take 0.8 mL of the ABTS mixed solution and mix it with 0.2 mL of PBS buffer for 10 s, let it stand for 6 min, measure the absorbance at 734 nm, and record it as A0. Take 0.8 mL of the ABTS mixed solution and mix it with 0.2 mL of the sample solution for 10 s, let it stand for 6 min, measure the absorbance at 734 nm, and record it as A.

[0055]

[0056] Antioxidant activity is an important indicator for evaluating functionality. The antioxidant components produced by fermentation can help the human body scavenge free radicals, reduce oxidative stress, and prevent human self-metabolic diseases such as cardiovascular diseases and diabetes. The antioxidant activity results of fermented chickpea milk are as Figure 2As shown. Compared with before fermentation, the ABTS free radical scavenging rate of Lactiplantibacillus plantarum FMBL23251 FJX fermented chickpea milk increased significantly. Among them, the ABTS scavenging rate increased by 22.92% (59.18% before fermentation and 82.10% after fermentation), and the ABTS scavenging rate of Lactiplantibacillus plantarum FMBL L23252 FJX increased by 13.33%. The DPPH scavenging rates of the three strains of bacteria fermented chickpea milk were all higher than those before fermentation. Among them, Lactiplantibacillus plantarum FMBL L23251 FJX had the highest DPPH scavenging rate, which increased by 26.73% compared with before fermentation (46.99% before fermentation and 73.72% after fermentation). In summary, Lactiplantibacillus plantarum FMBLL23251 FJX had the strongest antioxidant capacity in fermented chickpea milk.

[0057] 4. Determination of short-chain fatty acid content

[0058] Take 1 mL of chickpea milk, centrifuge at 4 °C and 10,000 r / min for 10 min, take the supernatant, and filter it through a 0.22 μm aqueous filter membrane to obtain the sample to be measured.

[0059] Preparation of standard solution: Accurately weigh 10 mg of each standard of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid, add distilled water to a 10 mL brown volumetric flask to a constant volume, and the mass concentration is 1 mg / mL.

[0060] Temperature programming: Keep at 50 °C for 3 min, rise to 114 °C at 6 °C / min, keep for 1 min, then rise to 160 °C at 6 °C / min, and finally rise to 220 °C at 30 °C / min and keep for 10 min. Carrier gas: N 2 Flow rate 3 mL / min, H 2 Flow rate 47 mL / min, air flow rate 400 mL / min. Injection volume: 1 μl; split ratio 3:1; injection port temperature 220 °C; detector temperature 240 °C. Chromatographic column: DB-Wax.

[0061] Higher short-chain fatty acid content can promote the antioxidant activity of chickpea milk and its functionality; at the same time, these short-chain fatty acids can bring a unique fermentation flavor to the extract, which helps to improve the flavor of chickpea milk. The short-chain fatty acid contents of the three strains of bacteria fermented chickpea milk are shown in Table 3. Among them, Lactiplantibacillus plantarum FMBL L23251 FJX had the best short-chain fatty acid production ability among all strains. The contents of 7 short-chain fatty acids all increased significantly after fermentation. Among them, the acetic acid content increased by 1.31 times compared with before fermentation; the propionic acid content increased by 1.98 times; the isobutyric acid content increased by 1.97 times; the butyric acid content increased by 1.81 times; the isovaleric acid content increased by 6.71 times; the valeric acid content increased by 3.77 times; the caproic acid content increased by 1.87 times.

[0062] Table 3 Short-chain fatty acid content in fermented chickpea milk

[0063]

[0064] Note: Different letters indicate significant differences between groups

[0065] 5. Determination of B vitamin content

[0066] Weigh 5.00 g of the sample (accurate to 0.01 g) into a 100 mL conical flask, dissolve it with 25.0 mL of 0.1% formic acid solution, ultrasonically extract for 20 min, then add 2 mL of 20% zinc acetate solution, shake well, transfer it to a 50 mL volumetric flask, make up the volume with ultrapure water, centrifuge at 10000 r / min for 10 min, take the supernatant and filter it through a 0.22 μm water-based filter membrane for use on the machine. Standard solutions are prepared with VB1, VB3, and VB6 as standards for the quantitative analysis of B vitamins in the sample

[0067] Chromatographic conditions: Waters ACQUITY PREMIER HSS T3 chromatographic column (100 mm × 2.1 mm, 1.7 μm); flow rate: 0.3 mL / min; sample volume: 1 μL; column oven: 30 °C. The mobile phase is 0.1% formic acid - water - acetonitrile, and the gradient elution program is shown in Table 4 below

[0068] Table 4 Gradient elution program

[0069]

[0070]

[0071] B vitamins have the effects of maintaining skin health, maintaining the normal function of the nervous system, and participating in the body's metabolism, etc., and are important indicators for evaluating the nutritional value of foods. The results are shown in Table 5. Lactobacillus plantarum WCFS1 significantly increased the content of VB1, while Lactobacillus plantarum FMBL L23251 FJX and FMBL L23252 FJX significantly decreased the content of VB1 in fermented chickpea milk. However, compared with each other, the decrease in the VB1 content in fermented chickpea milk by Lactobacillus plantarum FMBL L23251 FJX was significantly lower than that by FMBL L23252 FJX. The contents of VB3 and VB6 in fermented chickpea milk both increased significantly. The contents of VB3 and VB6 in fermented chickpea milk by Lactobacillus plantarum FMBL L23251 FJX were 1.90 times and 1.58 times the pre-fermentation contents respectively, and were significantly higher than those by Lactobacillus plantarum FMBL L23252 FJX and WCFS1

[0072] Table 5 Determination of vitamin content

[0073]

[0074] Note: Different letters indicate significant differences between groups.

[0075] 6. Determination of the ability of Lactiplantibacillus plantarum FMBL L23251 to degrade beany flavor

[0076] Take 5 g of soy milk, add 1 g of NaCl and 2 μl of 2-methyl-3-heptanone at 0.1 mg / mL as the internal standard, place it in a 20 mL headspace vial, tighten the cap, and after equilibrating at 50 °C for 20 min, insert a DVB / CAR / PDMS extraction head to extract and adsorb for 30 min.

[0077] Chromatographic conditions: Agilent HP-innowax chromatographic column (30 m × 0.25 μm); carrier gas: He; flow rate 1.5 mL / min; split ratio 10:1. Temperature programming: hold at 35 °C for 3 min, increase to 210 °C at 6 °C / min, and hold for 10 min. Then increase to 240 °C at 30 °C / min and hold for 5 min. Mass spectrometry conditions: electron impact ionization source, electron energy 70 eV, ion source temperature 230 °C; mass spectrometry range: m / z, 33 - 350 amu.

[0078] The beany flavor substances in chickpea milk are the most critical factors that make it unacceptable to consumers. The beany flavor is mainly composed of small molecule substances such as aldehydes (such as hexanal), alcohols (such as hexanol), ketones (such as 2-butanone), furans (such as 2-pentylfuran), etc. When these substances are mixed together, they form the characteristic unpleasant odor of soybeans, which usually manifests as stench, fishy smell, bitterness, grassy smell, astringency, etc. By measuring the changes in flavor substances in chickpea milk before and after fermentation, the results are shown in Table 6. A total of 32 flavor substances were identified in the unfermented group and fermented chickpea milk, including 6 organic acids, 5 alcohols, 12 aldehydes, 5 ketones, and 3 furans, and there were significant differences in the contents of flavor substances between different groups. Before fermentation, the main volatile substances in chickpea milk were mainly aldehydes, alcohols, and furan compounds, which were the main sources of unfavorable flavors such as astringency and beany flavor in chickpea milk. After fermentation by Lactiplantibacillus plantarum FMBL L23251 FJX and Lactiplantibacillus plantarum FMBL L23252 FJX, the contents of these substances in chickpea milk were significantly reduced. However, the degradation rate of Lactiplantibacillus plantarum FMBL L23251 FJX for compounds related to beany flavor was significantly higher than that of Lactiplantibacillus plantarum FMBL L23252 FJX, while the contents of compounds related to beany flavor in chickpea milk fermented by Lactiplantibacillus plantarum WCFS1 mostly increased significantly. Through fermentation, Lactiplantibacillus plantarum FMBL L23251 FJX reduced the total amount of furan substances in chickpea milk by more than 98%, reduced the total amount of aldehyde substances by more than 83%, and reduced the total amount of ketone substances by nearly 70%; among them, the degradation rates of 2-n-pentylfuran, nonenal, heptenal, nonadienal, benzaldehyde, n-pentanol, acetone and other substances were more than 50% higher than those of Lactiplantibacillus plantarum FMBL L23252 FJX.

[0079] Table 6 Flavor substances of fermented chickpea milk

[0080]

[0081] 4. Determination of DPP-IV inhibitory activity

[0082] In a 96-well microplate, add 25 μl of glycine-p-nitroaniline (0.2 mM) and 25 μl of fermented soy milk, and pre-incubate at 37 °C for 10 minutes. Then, add 50 μl of DPP-IV (0.01 U / mL) and incubate at 37 °C for 60 minutes; then add 100 μl of sodium acetate buffer (1 M, pH 4.0) to terminate the reaction, and measure the absorbance of the sample at 405 nm. In the reaction system, a PBS solution with pH 6.8 and 0.1 mol / L was used as the blank control for the DPP-IV solution and the sample to be measured.

[0083] DPP-IV inhibition rate = [1 - (A - B) / (C - D)] * 100%

[0084] Wherein: A is the measured absorbance value containing DPP-IV solution and the sample to be tested; B is the measured absorbance value containing the sample to be tested but without DPP-IV solution; C is the measured absorbance value containing DPP-IV solution but without the sample; D is the measured absorbance value without DPP-IV solution and the sample to be tested.

[0085] Table 7 DPP-IV inhibition rate of chickpea milk fermented by strains

[0086]

[0087] abc represents significant difference, P < 0.05.

[0088] The results are shown in Table 7. The inhibition rate of DPP-IV by Lactiplantibacillus plantarum FMBL L23251 FJX fermented chickpea milk is 63.81%, which is 1.85 times that of unfermented chickpea milk; while the inhibition rate of DPP-IV by Lactiplantibacillus plantarum WCFS1 fermented chickpea milk reaches 40.42%. This indicates that Lactiplantibacillus plantarum FMBL L23251 FJX has great application potential in reducing blood sugar and improving diabetes, and can be used for the preparation of blood sugar-lowering and diabetes products.

[0089] Example 3: Process optimization of Lactiplantibacillus plantarum FMBL L23251 FJX fermented chickpea yogurt

[0090] 1. Activation of strains

[0091] Lactiplantibacillus plantarum FMBL L23251 FJX was deposited at the China Center for Type Culture Collection on June 26, 2023, with the deposit number CCTCC NO: M 20231100.

[0092] Basic starter culture strains: Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus subsp. were purchased from Chr. Hansen A / S, Denmark. After the strains were activated on MRS agar plates, single colonies were picked and inoculated into MRS liquid medium, and subcultured continuously at 37 °C for 2 times, 24 h each time, with an inoculation amount of 2%. The obtained fermentation broth was centrifuged at 10000 r / min for 10 min, and the cells were washed twice with sterile physiological saline and then resuspended, and the concentration of the cell suspension was adjusted to 1.0×10 8 CFU / mL for standby.

[0093] 2. Preparation of chickpea yogurt

[0094] (1) Take chickpeas without pests, diseases and mechanical damage, soak them in a ratio of bean:water of 1:2 for 12 h, then filter and drain the water, and dry them at 40 - 60 °C or freeze-dry them to constant weight. The dried chickpeas were pulverized with an ultrafine pulverizer and passed through a 200-mesh sieve to obtain chickpea powder;

[0095] (2) Mix the chickpea powder obtained in step (1) with fresh milk in proportion and evenly, and compound 8% sucrose and 0.20% - 0.30% of compound esterified pectin, sodium carboxymethylcellulose, and xanthan gum. Stir well until completely dissolved and homogenized, then sterilize at 95°C for 10 min, and cool to room temperature for standby;

[0096] (3) Inoculate the bacterial suspensions of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus subsp. salivarius, and Lactiplantibacillus plantarum FMBL L23251 FJX in proportion and stir evenly. Ferment at a constant temperature of 40°C until solidified, and then ripen at 4°C for 16 h to obtain fermented chickpea yogurt.

[0097] 3. Determination of pH value and acidity of chickpea yogurt

[0098] Take 10 mL of chickpea yogurt and measure the pH value of the fermented milk with a pH meter. Refer to the national standard GB 5009.239-2016 "Determination of Acidity in Foods" to determine the acidity of chickpea yogurt.

[0099] 4. Sensory evaluation of chickpea yogurt

[0100] Take 30 mL of the fermented product and evaluate it according to the "RHB 104-2020 Sensory Evaluation Rules for Fermented Milks" promulgated by the China Dairy Industry Association.

[0101] 5. qPCR colony counting of Lactiplantibacillus plantarum FMBL L23251 FJX

[0102] Use a kit to extract the genomic DNA of microorganisms in the fermented milk, and use the qPCR method to determine the viable count of Lactiplantibacillus plantarum FMBL L23251 FJX in the fermented milk. Perform absolute quantification according to the reaction system and reaction conditions of the standard curve (Table 8). Substitute the obtained Ct value into the corresponding standard curve, and calculate the viable count of Lactiplantibacillus plantarum FMBL L23251 FJX in the yogurt sample in combination with the sampling volume and the dilution factor of the fermented milk sample DNA. The result is expressed as lgcfu / mL.

[0103] Preparation of the standard curve: Test the concentration of the extracted DNA on a micro nucleic acid quantifier to obtain the known concentration. Then perform 10-fold serial dilutions until diluted to 10 -6 times. Using the nucleic acid dilutions of each concentration gradient as templates, draw a standard amplification curve according to the reaction system in Table 6.

[0104] Reaction conditions: 95°C, pre-denaturation for 30 s, 95°C, denaturation for 10 s, 55°C, annealing for 30 s, 72°C, extension for 25 s, a total of 40 cycles.

[0105] Table 8 qPCR primers and reaction system

[0106]

[0107] 6. Effect of chickpea addition on yogurt quality

[0108] According to the preparation process of chickpea yogurt, chickpea flour is mixed with fresh milk at a ratio of 1%-5%, and 8% sucrose and 0.20%-0.30% of composite esterified pectin, sodium carboxymethyl cellulose, and xanthan gum are compounded, fully stirred until completely dissolved and homogenized, sterilized at 95℃ for 10min, inoculated with bacterial suspensions of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus salivarius thermophilus subsp. and Lactobacillus plantarum FMBL L23251 FJX at a ratio of 1:1:1 and stirred evenly, fermented at a constant temperature of 40℃ until coagulation, and post-ripened at 4℃ for 16h. The effect of chickpea addition on the quality of fermented milk was analyzed by measuring the acidity, pH, and viable count of Lactobacillus plantarum FMBL L23251FJX in fermented milk and sensory analysis.

[0109] Adding chickpeas can provide Lactobacillus plantarum with plant-derived proteins, polysaccharides and other nutrients, thereby promoting the growth and metabolism of Lactobacillus plantarum FMBL L23251 FJX in fermented milk. Figure 3 As shown in Table 9. The pH of fermented milk decreased rapidly with the increase of chickpea concentration, and too low pH would have an adverse effect on the taste of yogurt. Within the range of 2%, with the increase of chickpea concentration, the biomass and acidity of Lactobacillus plantarum FMBL L23251 FJX showed an upward trend. When the chickpea concentration was ≥2%, the biomass and acidity of Lactobacillus plantarum FMBL L23251 FJX basically tended to balance, without a significant increase trend.

[0110] Table 9 The number of viable lactic acid bacteria at different chickpea concentrations

[0111]

[0112] Sensory evaluation found that when the chickpea concentration was ≤2%, the sensory score of fermented milk was positively correlated with the chickpea concentration, which was related to the fact that the addition of chickpeas could increase the viscosity of fermented milk and give the fermented milk a plant-flavored taste; however, when the chickpea concentration was greater than 2%, as the chickpea concentration increased, the chickpea yogurt gradually became too sour, had a beany flavor, and had an astringent taste. Therefore, the next experiment will select the 2% chickpea addition amount with the best taste for subsequent experiments.

[0113] 7. Effect of the addition ratio of Lactobacillus plantarum FMBL L23251 FJX on yogurt quality

[0114] According to the preparation process of chickpea yogurt, mix chickpea powder with fresh milk evenly at a ratio of 2%, and compound 8% sucrose and 0.20% - 0.30% of compound esterified pectin, sodium carboxymethyl cellulose, and xanthan gum. After stirring thoroughly until completely dissolved and homogenized, sterilize at 95°C for 10 min. Inoculate the suspensions of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus subsp. salivarius, and Lactiplantibacillus plantarum FMBL L23251 FJX at ratios of 1:1:1, 1:1:2, 1:1:3, 1:1:4, and 1:1:5 respectively, stir evenly, and ferment at a constant temperature of 40°C until solidified, then ripen at 4°C for 16 h. Measure the acidity, pH of the fermented milk, and analyze the effect of the inoculation ratio of Lactiplantibacillus plantarum FMBL L23251 FJX on the quality of the fermented milk by sensory evaluation.

[0115] During the production of yogurt, the types and addition amounts of starter cultures and adjunct cultures are important factors determining the final product's flavor, texture, and sensory quality. The effects of different inoculation ratios of the adjunct culture Lactiplantibacillus plantarum FMBL L23251 FJX on the quality of the fermented milk are shown in Table 10. As the addition ratio of Lactiplantibacillus plantarum FMBL L23251 FJX increases, the acidity of the fermented milk rises rapidly, the pH value gradually decreases, and the coagulation time of the acid milk fermentation accelerates continuously. When the inoculation ratio is ≤1:1:2, the changes in the acidity and pH of the acid milk are small, and the sensory score of the acid milk increases with the increase in the inoculation ratio. When the inoculation ratio ≥1:1:3, the change range of the pH of the acid milk also becomes larger, and the sensory score of the acid milk gradually decreases significantly with the increase in the inoculation ratio. This is because too high acidity and too low pH will have an adverse effect on the sensory properties of the acid milk. When the inoculation ratio is greater than 1:1:4, a large amount of whey is separated out after coagulation, and the too-fast coagulation is not conducive to the formation of flavor substances.

[0116] Considering the pH, acidity, and sensory score of the fermented acid milk comprehensively, the acid milk fermented by Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus subsp. salivarius, and Lactiplantibacillus plantarum FMBL L23251 FJX at a ratio of 1:1:2 is the best. Based on this condition, the chickpea yogurt prepared has the best taste and meets the requirements of the "GB 19302-2010 National Food Safety Standard Fermented Milk".

[0117] Table 10 pH values, acidities, and sensory scores of fermented milk with different inoculation ratios

[0118]

[0119] Example 4. Stability and flavor of Lactiplantibacillus plantarum FMBL L23251 FJX-fermented chickpea yogurt

[0120] 1. Experimental grouping

[0121] The preparation process of the fermented milk was the same as that in Example 4.

[0122] The experiment was divided into 4 groups, including 3 control groups (M-2, M-3, D-2) and 1 experimental group (D-3). The details are shown in Table 11.

[0123] Table 11 Design of experimental groups

[0124]

[0125] 2. Antioxidant capacity of chickpea yogurt

[0126] The operation method was the same as that in Example 2 above.

[0127] The antioxidant capacities of the four groups of fermented milk were as Figure 4 shown. Among them, the antioxidant capacity of M-2 was the lowest, and the DPPH and ABTS scavenging rates were 42.79% and 47.95% respectively; the antioxidant capacity of D-3 was the highest, and the DPPH and ABTS scavenging rates were 67.86% and 91.27% respectively; by comparing M-2 and M-3, and D-2 and D-3, it can be obtained that adding Lactiplantibacillus plantarum FMBL L23251 FJX as an adjunct starter can significantly improve the antioxidant capacity of yogurt. By comparing M-2 and D-2, and M-3 and D-3, it can be obtained that the combination of Lactiplantibacillus plantarum FMBL L23251 FJX and chickpeas can further significantly improve the antioxidant capacity of yogurt.

[0128] 3 Physicochemical characteristics of chickpea yogurt

[0129] 3.1 Water holding capacity of chickpea yogurt

[0130] Select a 50 mL centrifuge tube, weigh the centrifuge tube, and record the mass as m 1 ; take about 10 g of yogurt into the centrifuge tube, and record the mass of the centrifuge tube and yogurt as m 2 ; centrifuge the centrifuge tube at a speed of 5000 r / min at room temperature for 30 minutes, pour out the supernatant, invert the centrifuge tube for 10 minutes and then weigh it, and record the mass as m 3 , and the calculation formula for the water holding capacity (WHC) of yogurt is as follows:

[0131] WHC = (m 3 - m 1 ) / (m 2 - m 1 )

[0132] The water-holding capacity of different groups is shown in Table 12. Generally speaking, the water-holding capacity of components D-2 and D-3 with chickpea milk added is higher than that of components M-2 and M-3 without chickpea. This may be because the soluble proteins in chickpea milk can fill into the three-dimensional gel-like network structure, enhancing the stability of the three-dimensional gel-like structure. The water-holding capacity of M-3 and D-3 with Lactiplantibacillus plantarum added as an adjunct starter is higher than that of the group without Lactiplantibacillus plantarum.

[0133] Table 12 Water-holding capacity of fermented milk

[0134]

[0135] 3.2 Texture of chickpea yogurt

[0136] Take the yogurt samples after 12 h of ripening for texture testing. Use an A / BE probe with a diameter of 30 mm and test in a 250 mL glass container (diameter 86 mm, height 80 mm). The speeds before, during, and after testing are 1.0, 1.0, and 5.0 mm / s respectively, the penetration distance is 20 mm, and the surface trigger force is 10 g.

[0137] The texture characteristics of fermented milk of different groups are shown in Table 13. The hardness, viscosity, chewiness, and resilience of the four groups of fermented milk show an increasing trend. This result is consistent with the determination result of the water-holding capacity, proving that the three-dimensional network gel structure of casein in D-3 is the densest and that in M-2 is the lowest. This result is due to the combined action of chickpea milk and Lactiplantibacillus plantarum FMBL L23351 FJX. The soluble proteins in chickpea milk can fill into the three-dimensional gel-like network structure, enhancing the stability of the three-dimensional gel-like structure.

[0138] Table 13 Texture characteristics of fermented milk

[0139]

[0140] 4. Determination of volatile flavor substances

[0141] The operation method is the same as that in Example 2 above.

[0142] For fermented milk, its popularity is often determined by its flavor. The detection results of volatile flavor substances of the four fermented milks are as Figure 5As shown in the figure. A total of 32 flavor substances were identified, including 7 alcohols, 6 aldehydes, 6 ketones, 8 acids, 2 esters, 2 alkanes, and 1 furan. Generally speaking, there were significant differences in volatile flavor substances among the four groups. The main flavor substances in chickpea yogurt D-3 fermented with Lactiplantibacillus plantarum FMBL L23351 FJX were formic acid, acetic acid, oxalic acid, malonic acid, butyric acid, hexanoic acid, ethyl acetate, hexanol, n-octanol, 2,3-butanediol, 2,3-butanedione, 2-heptanone. These flavor substances could bring unique fermented fragrance, buttery flavor and creamy flavor to chickpea milk, and the contents of these substances in D-3, such as hexanoic acid, butyric acid, ethyl acetate, acetic acid, n-octanol, etc., were mostly significantly higher than those in the control group. While the contents of substances related to the beany flavor in chickpea yogurt D-2 without the addition of the auxiliary starter Lactiplantibacillus plantarum FMBL L23351 FJX, such as 2-pentylfuran, acetone, pentanol, heptanal and other compounds, were significantly higher than those in D3 and M3, and most of these substances in D3 were degraded by Lactiplantibacillus plantarum FMBL L23351 FJX.

[0143] 5. Changes in the viable count of Lactiplantibacillus plantarum FMBL L23251 FJX during the storage period of chickpea yogurt

[0144] DNA was extracted from the yogurt stored at 4°C for 0, 7, 14, 21 and 28 days respectively, and the qPCR method was used to detect the changes in the viable count of Lactiplantibacillus plantarum FMBL L23251 FJX in the prepared yogurt during the shelf life. The operation method was the same as that in Example 3 above.

[0145] The changes in the viable count of Lactiplantibacillus plantarum FMBL L23251 FJX during the storage period of fermented yogurt are as Figure 6 shown. The viable count of Lactiplantibacillus plantarum FMBL L23251 FJX in the prepared chickpea yogurt was significantly about 1 order of magnitude higher than that of the yogurt without chickpeas after fermentation, reaching 1.2×10 9 cfu / mL. In the first seven days of the storage period, the viable count of Lactiplantibacillus plantarum FMBL L23251 FJX in the yogurt showed a slow growth trend; after seven days, the viable count of Lactiplantibacillus plantarum FMBL L23251 FJX in the fermented milk began to gradually decline; after 21 days of storage, although the viable count of Lactiplantibacillus plantarum FMBL L23251 FJX in the fermented chickpea yogurt decreased slightly, it still reached 7.9×10 8 cfu / mL, far higher than the requirements specified in the national standard "GB 19302-2010 National Food Safety Standard Fermented Milk".

[0146] In summary, the present invention provides the application of Lactiplantibacillus plantarum FMBL L23251 FJX in removing the beany flavor and preparing fermented soy products. The Lactiplantibacillus plantarum FMBL L23251 FJX was deposited at the China Center for Type Culture Collection on June 26, 2023, with the deposit number CCTCC NO: M20231100. The Lactiplantibacillus plantarum FMBL L23251 FJX-fermented chickpeas have DPPH free radical scavenging ability, ABTS free radical scavenging ability and inhibitory activity against dipeptidyl peptidase IV, can produce short-chain fatty acids and B vitamins, and can also significantly reduce the beany flavor in soy products. When compounded with chickpea powder to prepare chickpea yogurt, on the basis of ensuring the viable bacteria count during storage, it further enhances the antioxidant capacity of the yogurt and also stimulates the release of volatile flavor substances, having broad application prospects.

Claims

1. Application of Lactiiplantibacillus plantarum FMBL L23251 FJX in reducing bean smell in beans or bean products, characterized in that: The plant lactobacillus FMBL L23251 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M20231100.

2. The use according to claim 1, characterized in that The beans include one or more of chickpeas, soybeans, broad beans, peas, mung beans, miscellaneous beans, pigeon peas and sword beans.

3. The use according to claim 1, characterized in that The bean product is prepared by processing beans.

4. A method for reducing beany smell in bean products, characterized in that: The bean product is taken as a substrate, and the bean smell of the product obtained by adding plant lactobacillus FMBL L23251 FJX for fermentation is reduced. The plant lactobacillus FMBL L23251 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, and the deposit number is CCTCC NO: M20231100.

5. Application of Lactibacillus plantarum FMBL L23251 FJX in the preparation of bean products by fermenting beans, characterized in that: The plant lactobacillus FMBL L23251 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M20231100.

6. A fermented soymilk with reduced beany smell, characterized in that: The fermented soy milk contains plant lactobacillus FMBL L23251 FJX, which was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCCNO: M20231100.

7. Application of Lactobacillus plantarum FMBL L23251 FJX in the preparation of fermented chickpea yogurt, characterized in that: The plant lactobacillus FMBL L23251 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M20231100.

8. Application of Lactiiplantibacillus plantarum FMBL L23251 FJX in fermenting chickpea milk to produce short-chain fatty acids, characterized in that: The plant lactobacillus FMBL L23251 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M20231100.

9. Use of chickpea fermented with Lactobacillus plantarum FMBL L23251 FJX in inhibiting dipeptidyl peptidase IV activity or preparing dipeptidyl peptidase IV inhibitor, characterized in that: The plant lactobacillus FMBL L23251 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCCNO: M20231100.

10. Use of Lactiiplantibacillus plantarum FMBL L23251 FJX in fermenting chickpeas to prepare hypoglycemic products or to enhance the hypoglycemic effect of chickpea products, characterized in that: The plant lactobacillus FMBL L23251 FJX was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCCNO: M20231100.

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