Weissella fusiformis and application thereof

Abstract

The invention relates to a Weissella confusa strain, which is classified and named as Weissella confusa SY628, is preserved in China General Microbiological Culture Collection Center (CGMCC) on September 19, 2024, and has a preservation number of CGMCC No.32006. The Weissella confusa strain is named as Weissella confusa SY628, and the Weissella confusa strain is named as Weissella confusa SY628. The invention further discloses a microbial agent, a biological agent, a purification screening method and application of the fused Weissella. The fused Weissella SY628 disclosed by the invention is a lactic acid bacterium capable of producing exopolysaccharides at high yield, has better acid resistance and cholate resistance, has a good escherichia coli inhibition effect, is safe to eat, and can be used for remarkably improving the solidification effect of sour soybean milk.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a fusion Weissella strain and application thereof. Background Art

[0002] The growing global population and protein demand challenge traditional animal protein sources, plant protein emerges as a viable, environmentally friendly alternative, and plant-based dairy products are becoming increasingly popular.

[0003] After fermentation, plant-based dairy products not only aggregate proteins to form a unique texture, but also eliminate allergens, reduce the content of oligosaccharides that easily cause flatulence, and produce organic acids to inhibit the growth of intestinal saprophytes. They have anti-cancer effects, improve human gastrointestinal function, enhance immune function, and lower serum cholesterol. However, there are still few types of fermented plant-based dairy products.

[0004] Leguminous plants play an important role as one of the raw materials for the production of plant-based dairy products. They are rich in protein, complex carbohydrates, vitamins and dietary fiber, making beans an ideal substitute for dairy products. At present, there is no special fermentation agent for fermenting sour soy milk on the market. Public literature shows that the current sour soy milk fermentation often uses commercial fermentation agents used for fermenting yogurt. However, due to the large difference in the composition of soy milk / soy milk and animal-based dairy products such as milk, the sour soy milk obtained by fermentation has poor stability, whey is easy to precipitate, and has a strong beany smell. There is no successful sour soy milk product on the market. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art, provide a fused Weissella suitable for plant-based dairy product fermentation, and further provide a microbial agent, biological preparation, purification screening method and application of the fused Weissella.

[0006] The technical solution adopted by the present invention to solve its technical problems is as follows: a strain of Weissella confusa, classified and named Weissella confusa SY628, was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 19, 2024, with a deposit number of CGMCC No.32006.

[0007] As a general technical concept, the present invention also provides a microbial agent containing the fused Weissella.

[0008] As a general technical concept, the present invention also provides a biological preparation containing the fermentation product of the fused Weissella.

[0009] As a general technical concept, the present invention also provides an application of the fused Weissella: the fused Weissella or a microbial agent containing the fused Weissella is applied to the fermentation of food containing dietary fiber.

[0010] Preferably, the method is applied to the fermentation of beverages containing dietary fiber.

[0011] Preferably, the method is applied to the fermentation of dairy products containing dietary fiber.

[0012] Preferably, the method is applied to the fermentation of plant-based dairy products, or to the fermentation of products containing plant-based dairy products, or to the fermentation of animal-based dairy products containing dietary fiber.

[0013] Preferably, the plant-based dairy product is a seed-based plant-based dairy product.

[0014] Preferably, the plant-based dairy product is one or more of nut-based dairy products, cereal-based dairy products, and bean-based dairy products, and more preferably bean-based dairy products.

[0015] Preferably, the beans are one or more of soybeans, black beans, green beans, mung beans, red beans, chickpeas, kidney beans, peas, broad beans, and lentils. More preferably, soybeans are used.

[0016] Preferably, the fused Weissella or a microbial agent containing the fused Weissella is inoculated into a fermentation substrate and fermented at a constant temperature.

[0017] As a general technical concept, the present invention also provides an application of fused Weissella: the fused Weissella or the microbial agent is used to produce polysaccharides.

[0018] As a general technical concept, the present invention also provides an application of fused Weissella: the fused Weissella or the microbial agent is used to decompose dietary fiber.

[0019] As a general technical concept, the present invention also provides an application of fused Weissella: the fused Weissella or the microbial agent is used for acid production.

[0020] As a general technical concept, the present invention also provides an application of fused Weissella: the fused Weissella, or the microbial agent, or the biological agent is used as a stabilizer.

[0021] As a general technical concept, the present invention also provides the use of the fused Weissella as a probiotic.

[0022] As a general technical concept, the present invention also provides a purification and screening method for the fused Weissella, wherein the screening medium is an MRS agar medium supplemented with 1-3 wt % CaCO3.

[0023] Preferably, the culture temperature during screening is 28-45°C.

[0024] Preferably, the screening is carried out from the microbial agent or fermentation product fused with Weissella.

[0025] The present invention has the following beneficial effects:

[0026] (1) Weissella fusion SY628 is a lactic acid bacterium with high exopolysaccharide production, with a polysaccharide production of 19.8±0.14 g / L, which is better than many existing reports on Weissella fusion;

[0027] (2) The fusion strain Weissella SY628 has good acid and bile resistance, indicating that the strain is relatively active in the intestine (strong probiotics); SY628 has significantly different antibiotic sensitivities to different antibiotics and has a good inhibitory effect on Escherichia coli. Through drug resistance tests and hemolytic tests, it is determined that SY628 is safe for human consumption and is a promising candidate probiotic strain for the development of new functional foods;

[0028] (3) The fusion of Weissella SY628 can significantly improve the coagulation effect of yogurt, and the fermentation effect is significantly better than that of commercial strains. Compared with other strains, the fermentation can save the expensive additive costs (stabilizers, etc.), making its fermented products have obvious price advantages.

[0029] (4) The fused Weissella SY628 has the ability to decompose dietary fiber and can be used to decompose dietary fiber; the decomposition of dietary fiber during fermentation by the fused Weissella SY628 can also make the fermented product more nutritious.

[0030] In addition to the above-described purposes, features and advantages, the present invention has other purposes, features and advantages. The present invention will be further described in detail below with reference to the accompanying drawings.

[0031] Description of microbial preservation:

[0032] The fused Weissella SY628 (Weissella confusa SY628) of the present invention was deposited on September 19, 2024 in the General Microbiology Center of China Microorganism Culture Collection Administration (referred to as CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, China), and the culture deposit number is CGMCC No. 32006. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0034] Figure 1 The morphological characteristics of the fused Weissella SY628 of the present invention are observed in the figure: the left figure shows the cell morphology of the fused Weissella SY628; the middle figure shows the colony morphology of the fused Weissella SY628 on the MRS solid culture medium; the right figure shows the colony morphology and colony wire drawing of the fused Weissella SY628 on the mMRS solid culture medium;

[0035] Figure 2 It is the species distribution map of Weissella SY628 fused with the present invention according to its gene annotation;

[0036] Figure 3 It is a growth curve diagram of the fusion Weissella SY628 of the present invention in MRS liquid culture medium;

[0037] Figure 4 It is a growth curve diagram of the fusion Weissella SY628 of the present invention in MRS liquid culture medium at 31-41°C;

[0038] Figure 5 It is a curve diagram of the production of extracellular polysaccharide (EPS) by the fusion Weissella SY628 of the present invention in MRS liquid culture medium;

[0039] Figure 6 is a curve diagram showing the survival of the fused Weissella SY628 of the present invention under acidic conditions;

[0040] Figure 7 is a curve diagram of the survival of the fused Weissella SY628 of the present invention under bile salt-containing conditions;

[0041] Figure 8 This is a diagram showing the antibacterial effect of the present invention fused with Weissella SY628 on Staphylococcus aureus;

[0042] Fig. 9 This is a diagram showing the antibacterial effect of the present invention fused with Weissella SY628 on Escherichia coli;

[0043] Fig.10 This is a graph showing the growth of the fused Weissella SY628 of the present invention on a blood agar plate;

[0044] Fig.11 The whey precipitation after demulsification of the sour soy milk prepared in Example 5 of the present invention is as follows: (A) fermented with Weissella SY628; (B) fermented with commercial yogurt bacteria; (C) fermented with Lactobacillus plantarum;

[0045] Fig.12 This is a photo of the plant cheese prepared in Example 6 of the present invention. DETAILED DESCRIPTION

[0046] In order to make the purpose, scheme and beneficial technology of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments and drawings. It should be pointed out that the embodiments described in this specification are only for explaining the present invention, not for limiting the present invention.

[0047] For simplicity, only some numerical ranges are explicitly disclosed herein. However, any lower limit can be combined with any upper limit to form an unambiguous range; and any lower limit can be combined with other lower limits to form an unambiguous range, and any upper limit can be combined with any other upper limit to form an unambiguous range. In addition, although not explicitly stated, each point or single value between the range endpoints is included in the range. Thus, each point or single value can be combined with any other point or single value as its own lower limit or upper limit or with other lower limits or upper limits to form an unambiguous range.

[0048] In the description of this article, it should be noted that, unless otherwise specified, “above” and “below” are inclusive of the number, and the “multiple” in “one or more” means two or more, and the “multiple” in “one or more” means two or more.

[0049] An embodiment of the present invention provides a strain of Weissella confusa, which is classified and named Weissella confusa SY628, and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 19, 2024, with a deposit number of CGMCC No.32006.

[0050] The fused Weissella provided by the embodiment of the present invention has the following advantages:

[0051] (1) Weissella fusion SY628 is a lactic acid bacterium with high exopolysaccharide production, with a polysaccharide production of 19.8±0.14 g / L, which is better than many existing reports on Weissella fusion;

[0052] (2) The fusion strain Weissella SY628 has good acid and bile resistance, indicating that the strain is relatively active in the intestine (strong probiotics); SY628 has significantly different antibiotic sensitivities to different antibiotics and has a good inhibitory effect on Escherichia coli. Through drug resistance tests and hemolytic tests, it is determined that SY628 is safe for human consumption and is a promising candidate probiotic strain for the development of new functional foods;

[0053] (3) The fusion of Weissella SY628 can significantly improve the coagulation effect of yogurt, and the fermentation effect is significantly better than that of commercial strains; it can be used to make a variety of fermented products such as coagulated yogurt that does not require stabilizers and plant cheese that does not require stabilizers, filling the market gap; compared with other strains, the fermentation can save expensive additive costs (stabilizers, etc.), making its fermented products have obvious price advantages;

[0054] (4) The fused Weissella SY628 has the ability to decompose dietary fiber and can be used to decompose dietary fiber; the decomposition of dietary fiber during fermentation by the fused Weissella SY628 can also make the fermented product more nutritious.

[0055] The embodiment of the present invention also provides a microbial agent containing the fused Weissella, which can be in various forms such as liquid, granules, and powder.

[0056] The embodiment of the present invention also provides a biological preparation containing the fermentation product of the fused Weissella.

[0057] The embodiment of the present invention also provides an application of fused Weissella: the fused Weissella or a microbial agent containing fused Weissella is applied to the fermentation of food containing dietary fiber. The fused Weissella of the present invention has the ability to decompose dietary fiber, can be used for the fermentation of food containing dietary fiber, and can make the taste of the fermented product more delicate and smooth.

[0058] In some embodiments of the present invention, the present invention is applied to the fermentation of beverages containing dietary fiber. The Weissella fusion of the present invention has the ability to decompose dietary fiber and can be used for the fermentation of beverages containing dietary fiber.

[0059] In some embodiments of the present invention, the present invention is applied to the fermentation of dairy products containing dietary fiber. The Weissella fusion of the present invention has the ability to decompose dietary fiber and can be used for the fermentation of dairy products containing dietary fiber.

[0060] In some embodiments of the present invention, the present invention is applied to the fermentation of plant-based dairy products, or to the fermentation of products containing plant-based dairy products, or to the fermentation of animal-based dairy products containing dietary fiber.

[0061] In some embodiments of the present invention, the plant-based dairy product is a seed-based plant-based dairy product.

[0062] In some embodiments of the present invention, the plant-based dairy product is one or more of nut-based plant-based dairy products, cereal-based plant-based dairy products, and bean-based plant-based dairy products.

[0063] In some embodiments of the present invention, the plant-based dairy product is a soy-based plant-based dairy product.

[0064] In some embodiments of the present invention, the beans are one or more of soybeans, black beans, green beans, mung beans, red beans, chickpeas, kidney beans, peas, broad beans, and lentils.

[0065] In some embodiments of the present invention, the beans are soybeans.

[0066] In some embodiments of the present invention, the fused Weissella or the microbial agent containing the fused Weissella is inoculated into the fermentation substrate and the fermentation is carried out under heat preservation. In some embodiments, the fermentation temperature is 28-45°C.

[0067] The embodiment of the present invention also provides an application of the fused Weissella: the fused Weissella or the microbial agent is used to produce polysaccharides. The fused Weissella of the present invention is a lactic acid bacterium with high production of extracellular polysaccharides and can be used to produce polysaccharides.

[0068] The embodiment of the present invention also provides an application of the fused Weissella: the fused Weissella or the microbial agent is used to decompose dietary fiber. The fused Weissella of the present invention has the ability to decompose dietary fiber.

[0069] The embodiment of the present invention further provides an application of the fused Weissella: the fused Weissella or the microbial agent is used for acid production. The fused Weissella of the present invention has the ability to produce acid.

[0070] The embodiment of the present invention also provides an application of the fused Weissella: the fused Weissella, or the microbial agent, or the biological agent is used as a stabilizer. The fused Weissella of the present invention has a high yield of viscous polysaccharides and can be used as a stabilizer.

[0071] The embodiment of the present invention also provides a use of the fused Weissella as a probiotic.

[0072] The embodiment of the present invention also provides a purification and screening method for the fused Weissella, wherein the screening medium is an MRS agar medium supplemented with 1-3 wt% CaCO3. The MRS agar medium can be prepared by oneself or purchased directly from the market, and conventional formulas are feasible.

[0073] In some embodiments of the present invention, a typical screening medium is composed of: peptone 10.0 g·L -1 , beef powder 8.0g·L -1 , Yeast powder 4.0g·L -1 , Glucose 20.0 g·L -1 , Potassium hydrogen phosphate 2.0 g·L -1 , diammonium hydrogen citrate 2.0 g·L -1, sodium acetate 5.0g·L -1 , magnesium sulfate 0.2 g·L -1 , Manganese sulfate 0.04g·L -1 , Tween 80 1.0g·L -1 , agar 15.0 g·L -1 , pH6.2±0.225℃g·L -1 , calcium carbonate 2%.

[0074] In some embodiments of the present invention, the culture temperature during screening is 28-45°C.

[0075] In some embodiments of the present invention, screening is performed from the microbial agent or fermentation product fused with Weissella.

[0076] In some embodiments of the present invention, the purification screening method comprises the following steps:

[0077] (1) taking a microbial agent or fermentation product containing the Weissella fusion, adding a diluent and mixing well to obtain a sample solution;

[0078] (2) diluting the sample solution to obtain a diluted solution;

[0079] (3) applying the diluted solution on a solid culture medium (screening culture medium) and culturing until colonies grow;

[0080] (4) Select typical colonies with transparent circles on the solid culture medium and transfer them to liquid culture medium for culture. After the obtained bacterial liquid is diluted, it is streaked on another solid culture medium (screening medium) to obtain pure colonies.

[0081] In some embodiments of the present invention, the purification screening method comprises the following steps:

[0082] (1) taking a microbial agent or fermentation product containing the Weissella fusion, adding a diluent and mixing well to obtain a sample solution;

[0083] (2) diluting the sample solution to obtain a diluted solution;

[0084] (3) applying the diluted solution on a solid culture medium (screening culture medium) and culturing until colonies grow;

[0085] (4) Pick typical colonies with transparent circles on the solid culture medium, transfer them to liquid culture medium for culture, dilute the resulting bacterial solution, and then streak culture on another solid culture medium (screening medium). Further pick typical colonies with transparent circles from the solid culture medium and repeat the above steps of picking typical colonies - culturing in liquid culture medium - diluting - streaking until pure colonies are obtained after streak culture.

[0086] In some embodiments of the present invention, the liquid culture medium is an MRS liquid culture medium. The MRS liquid culture medium can be prepared by itself or purchased directly from the market, and conventional formulas are feasible.

[0087] In some embodiments of the present invention, a typical liquid culture medium is composed of: peptone 10.0 g·L -1 , beef powder 8.0g·L -1 , Yeast powder 4.0g·L -1 , Glucose 20.0 g·L -1 , Potassium hydrogen phosphate 2.0 g·L -1 , diammonium hydrogen citrate 2.0 g·L -1 , sodium acetate 5.0g·L -1 , magnesium sulfate 0.2 g·L -1 , Manganese sulfate 0.04g·L -1 , Tween 80 1.0g·L -1 , pH6.2±0.225℃g·L -1 .

[0088] In some embodiments of the present invention, the diluent in step (1) is sterile saline.

[0089] In some embodiments of the present invention, in step (1), the amount of the sterile physiological saline is 8 to 10 times that of the microbial inoculum or fermentation product containing the Weissella fusion.

[0090] In some embodiments of the present invention, in step (2) or (4), 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 Multiple dilutions were prepared and appropriate gradients were selected for the next step of inoculation.

[0091] In some embodiments of the present invention, the fused Weissella is added to a sterilized 40% glycerol protective agent and stored at -80°C.

[0092] Weissella fusion belongs to the order Lactobacillales, family Leuconostocaceae, genus Weissella, and is a beneficial lactic acid bacterium, a Gram-positive bacterium with a rod-shaped or spherical shape. This microorganism has been included in the list of the International Dairy Federation (IDF) and verified by the Food Safety Committee (SKLM) of the German Science Foundation (DFG). It is widely used in traditional food fermentation and exists in fermented foods such as yogurt and kimchi. It can also colonize the intestines of humans and animals, with benefits such as preventing infection, promoting digestion, lowering cholesterol and reducing the risk of cancer.

[0093] Currently, lactic acid bacteria are widely used in yogurt fermentation. Although there are a few reports on the research of Weissella in yogurt, there are no reports on pure fermented yogurt.

[0094] Among plant-based yogurts, there is currently no special fermentation agent for fermenting soy milk on the market. Public literature shows that the current sour soy milk fermentation often uses commercial fermentation agents used for fermenting yogurt, but because the ingredients of soy milk / soy milk are quite different from those of animal-based dairy products such as milk, the sour soy milk fermented currently has poor stability, whey is easy to precipitate, and has a strong beany smell. There is no successful sour soy milk product on the market yet.

[0095] The Weissella strain of the invention is screened out from a bottle of naturally fermented coagulated sour soybean milk obtained accidentally. After the pure strain is inoculated back into soybean milk / soy milk, the fermentation effect is very good and the sour soybean milk obtained after fermentation has good quality.

[0096] Example

[0097] The following examples describe the disclosure of the present invention in more detail, and these examples are intended for illustrative purposes only, as it will be apparent to those skilled in the art that various modifications and variations will be made within the scope of the disclosure of the present invention. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are by weight. Unless otherwise stated, all reagents used in the examples are available through conventional commercial sources or are synthesized according to conventional methods and can be used directly without further processing. Unless otherwise stated, the instruments used in the examples are available through conventional commercial sources.

[0098] The MRS liquid culture medium mentioned in the following examples is composed of: peptone 10.0 g·L -1 , beef powder 8.0g·L -1 , Yeast powder 4.0g·L -1 , Glucose 20.0 g·L -1 , Potassium hydrogen phosphate 2.0 g·L -1 , diammonium hydrogen citrate 2.0 g·L -1, sodium acetate 5.0g·L -1 , magnesium sulfate 0.2 g·L -1 , Manganese sulfate 0.04g·L -1 , Tween 80 1.0g·L -1 , pH6.2±0.225℃g·L -1 .

[0099] The MRS solid medium mentioned in the following examples is composed of: peptone 10.0 g·L -1 , beef powder 8.0g·L -1 , Yeast powder 4.0g·L -1 , Glucose 20.0 g·L -1 , Potassium hydrogen phosphate 2.0 g·L -1 , diammonium hydrogen citrate 2.0 g·L -1 , sodium acetate 5.0g·L -1 , magnesium sulfate 0.2 g·L -1 , Manganese sulfate 0.04g·L -1 , Tween 80 1.0g·L -1 , agar 15.0 g·L -1 , pH6.2±0.225℃g·L -1 , calcium carbonate 2%.

[0100] The mMRS medium mentioned in the following examples is composed of: 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 1.0 g of Tween 80, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of anhydrous sodium acetate, 5.0 g of sodium citrate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 50.0 g of sucrose, and deionized water is added to 1 L, sterilized at 115° C. for 20 min, and 20 g / L of agar is added to the solid medium. The medium can be used for the screening of exopolysaccharide (EPS)-producing lactic acid bacteria, and the colony morphology of exopolysaccharide-producing lactic acid bacteria on the medium is mucous.

[0101] Example 1

[0102] SY628 was selected by the inventor from sour soy milk, which was accidentally obtained by the inventor in the Hunan Provincial Key Laboratory of Soy Products Processing and Safety Control of Shaoyang University. It is a solidified sour soy milk made by natural fermentation of soy milk. After fermentation test verification, it was confirmed that SY628 is the key strain that transforms soy milk into solidified sour soy milk in the natural fermentation.

[0103] Purification and screening method of SY628:

[0104] (1) Collect naturally fermented soy milk from the Hunan Provincial Key Laboratory of Soy Products Processing and Safety Control of Shaoyang University;

[0105] (2) Weigh 25 g of the yogurt sample and place it in 225 mL of sterile physiological saline, and shake it thoroughly in a shaking incubator at 36±1° C. and 200 rpm;

[0106] (3) Use a 1 mL sterile pipette to draw 1 mL of the sample solution and slowly inject it along the tube wall into a test tube containing 9 mL of sterile saline to prepare a 10-fold serial dilution of the sample solution. Each time the dilution is increased, a 1 mL sterile pipette is used; 10 -2 ~10 -9 Dilute the sample homogenate by 10 times;

[0107] (4) Select 1 to 3 appropriate sample homogenates, take 100 μL and evenly apply it onto MRS solid medium supplemented with 2% calcium carbonate using a spreader, and culture at 36±1°C. After the colonies grow, select single bacteria with obvious transparent circles and transfer them to MRS liquid medium for constant temperature culture at 36±1°C. Use the diluted solution of the obtained bacterial solution to streak and obtain pure strains.

[0108] The purified colonies were subjected to Gram staining, and Gram staining was positive, and the strain was preliminarily identified as lactic acid bacteria. The final bacteria were collected after three generations, added to a sterilized 40% glycerol protective agent, and stored at -80°C.

[0109] Morphological observation:

[0110] See also Figure 1 SY628 cell morphology Figure 1 As shown in the left figure, the bacteria are short rod-shaped, arranged singly or in pairs, and Gram-positive; the colony morphology of SY628 on MRS solid medium supplemented with 2% calcium carbonate is as follows Figure 1 As shown in the middle figure, the colonies are milky white, opaque, round, with irregular edges, flat and not raised; the colony morphology of SY628 on mMRS solid medium is as follows Figure 1 As shown in the right picture, the colony morphology is transparent and mucus-like, and appears to be stringy when picked up, indicating that the strain produces a high amount of viscous polysaccharides.

[0111] Genome Sequencing:

[0112] NR (Non-Redundant Protein Database) is a non-redundant protein database that contains protein sequences translated from the entire DNA sequence stored in NCBI GenBank. Figure 2The number of species genes annotated to SY628 is as follows: 1807 genes were annotated as Weissella confusa, indicating that SY628 is a member of Weissellaconfusa, and it was named Weissella confusa SY628.

[0113] The fusion Weissella SY628 was deposited on September 19, 2024 in the General Microbiology Center of the China Culture Collection Administration (CGMCC for short, address: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, China), and the culture collection number is CGMCC No.32006.

[0114] Example 2

[0115] This example tests the biological characteristics of Weissella SY628.

[0116] (1) Growth curve of strains

[0117] The fused Weissella SY628 was cultured in MRS liquid medium at 37°C for 24 h.

[0118] The OD value (at 600nm) and pH changes during the period are as follows Figure 3 As shown. The strain grew rapidly in MRS liquid medium, entered the logarithmic phase after 2 hours, entered the stable phase after 4 hours, reached the highest viable count at 8 hours, and began to decline after 10 hours. The pH value of the bacterial solution decreased rapidly between 0 and 12 hours, tended to decrease slowly after 12 hours, and its pH at 24 hours was above 4.5. This also shows the acid production ability of Weissella SY628.

[0119] (2) Optimal growth temperature of strains

[0120] The fused Weissella SY628 was cultured in MRS liquid medium at 31-41°C for 12 h, and the OD value (at 600 nm) of the fermentation broth was measured. Figure 4 As shown in the growth curve, the fusion Weissella SY628 can grow in all temperature ranges, and the optimal growth temperature is around 37°C.

[0121] (3) Sugar production of strains

[0122] The fusion strain Weissella SY628 was cultured in MRS liquid medium at 37°C. The polysaccharide production curve within 48 hours is as follows Figure 5 As shown, the results showed that the sugar production of the strain increased rapidly with the extension of the culture time. When the culture time was 12-18h, the sugar production was the highest, with a content of 19.8±0.14g / L.

[0123] During the fermentation process, with the increase of mucus, bacterial proliferation and enzyme secretion are inhibited, and the sugar production rate gradually decreases. After a certain period of fermentation, the synthesis of EPS is disturbed, and the glycosyltransferase at this time is more inclined to synthesize oligosaccharides. At the same time, bacteria will produce enzymes that can degrade polysaccharides in the later stage of fermentation, so long-term fermentation will reduce EPS production. Due to the high production of extracellular polysaccharides of this strain, the speculation that the extracellular polysaccharides produced by the fusion Weissella SY628 cross-link with plant proteins to form a more stable network structure is further verified, which explains the phenomenon that the fermented sour soy milk can well improve the water retention and texture stability, thereby maintaining the coagulation state.

[0124] Example 3

[0125] This example tests the probiotic properties of Weissella SY628.

[0126] (1) Gastrointestinal tolerance of strains

[0127] Preparation of artificial gastric juice: Prepare 100 g / L HCl, dilute with distilled water, adjust the pH to 2.5, 3.5, and 4.5, respectively, add 1 g / 100 ml of pepsin, mix well, and filter with a 0.22 μm microporous filter membrane for sterilization.

[0128] Preparation of artificial intestinal fluid: 1.36g K2HPO4 was weighed and dissolved in 100ml distilled water, and the pH value was adjusted to 6.8 with 4g / L NaOH, and then diluted to 200ml. 1g / 100ml trypsin was added, and after thorough mixing, the solution was sterilized by filtering with a 0.22μm microporous membrane.

[0129] Activation and preparation of strains: Take out the frozen tubes of strains stored in a -80℃ refrigerator and thaw them. Pick up the strains on a sterile operating table and put them into the sterilized MRS liquid culture medium. Culture them in a 37℃ incubator for 24 hours. This is the first generation of activation solution. Use a pipette to take the first generation of activated culture solution and add 2% to the MRS liquid culture medium for expansion culture. This is the second generation of activation solution. Repeat this operation until the third generation of activation solution is obtained. When the number of viable bacteria is 10 8 CFU / mL, it can be used as the strain fermentation liquid for inoculation and stored in a 4℃ refrigerator.

[0130] The fermentation liquid of the strain was added in an amount of 3 wt % and inoculated into artificial gastric juices of pH 2.5, 3.5 and 4.5 respectively. The culture conditions were 37° C. and 100 r / min shaking culture. After 3 hours, samples were taken for determination of total colony count. The results are shown in Table 1.

[0131] The fermentation liquid of the strain was added in an amount of 3 wt % and inoculated into artificial intestinal fluid. The culture condition was 37° C. The total number of colonies was detected by taking samples at 0 h, 2 h and 4 h. The results are shown in Table 2.

[0132] Table 1 Results of the artificial gastric juice experiment on bacterial strains

[0133]

[0134]

[0135] Table 2 Results of the artificial intestinal fluid experiment on strains

[0136]

[0137] From the experimental results in Table 1, it can be seen that strain SY628 has strong tolerance to gastric juice. When the pH is 2.5, the number of viable bacteria is 7.8*10 7 CFU / mL, when pH is 4.5, the number of viable bacteria is 1.04*10 8 CFU / mL, indicating that SY628 has good tolerance to gastric juice. From the experimental results in Table 2, it can be seen that, in general, with the extension of time, the number of live bacteria and the survival rate of this strain in artificial intestinal juice showed a downward trend; however, SY628 still had a relatively high number of live bacteria in artificial intestinal juice at 2h and 4h, indicating that this strain has good tolerance to artificial intestinal juice.

[0138] (2) Acid resistance of strains

[0139] The fusion Weissella SY628 was cultured in MRS liquid medium with pH values ​​of 1.5, 2.5, 3.5, 4.5, and 5.5 at 37°C for 24 hours, and the growth status of the number of viable bacteria of the strain at different pH values ​​was analyzed. Figure 6 As shown in the figure, the number of viable bacteria of SY628 increases with the increase of pH. When the pH is 1.5, the number of viable bacteria of SY628 is 0 cfu / mL, and when the pH is 2.5, the number of viable bacteria of SY628 is 2.43×10 9 ±0.20 cfu / mL, when pH was 5.5, the survival rate of SY628 was as high as 33.67×10 9 ±1.32 cfu / mL, indicating that the strain showed good tolerance in low pH environmental solutions.

[0140] (3) Bile tolerance of strains

[0141] The fusion Weissella SY628 was cultured in MRS liquid medium supplemented with bile salts (concentrations of 0.05%, 0.1%, 0.5%, 1.0%, and 1.5%) at 37°C for 24 hours, and the number of viable bacteria of the strain was determined. The results are as follows: Figure 7As shown in the figure, with the increase of bile salt concentration, the number of viable bacteria showed a downward trend, which may be related to the high osmotic pressure generated by high bile salt concentration outside the cells, which affected the bacterial cells and caused the strain tolerance to decrease. When the bile salt concentration was 0.05%, the number of viable bacteria of SY628 was 3.78×10 9 cfu / mL, bile salt concentration was 1.5%, the viable count of SY628 was still 3.1×10 9 cfu / mL, compared with the bile salt tolerance range of 0.05% to 0.3% reported by Zou Fanghong, the strain SY628 of the invention has a stronger high bile salt tolerance. This shows that the strain has a good bile salt tolerance, good probiotic function, and broad application prospects.

[0142] Example 4

[0143] This example tests the safety of fusion of Weissella SY628.

[0144] (1) Drug resistance of strains

[0145] The drug sensitivity paper agar diffusion method (K-B method) was used to test the drug resistance sensitivity of the isolated lactic acid bacteria to 8 antibiotics, including amikacin, erythromycin, penicillin, chloramphenicol, cefazolin, ampicillin, cotrimoxazole, and amoxicillin. The strains to be tested were activated for 2-3 generations, and the number of live bacteria reached 10 8 cfu / mL, centrifuge at 8000r / min, 4°C for 5min, and adjust the bacterial concentration to OD 600 =0.5, take 100 μL of bacterial solution for plate coating, then take out the drug-sensitive paper with sterile tweezers and place it in the plate, and set a blank drug-sensitive paper as a control. Place it in a 37°C constant temperature incubator for culture, and measure and record the diameter of the inhibition zone after 24 hours. According to the "Antimicrobial Susceptibility Test Implementation Standard" formulated by the American Clinical and Laboratory Standards Institute, the resistance of Weissella SY628 to antibiotics was evaluated. The test results are summarized in Table 3.

[0146] Table 3 Results of drug sensitivity test of fusion Weissella SY628

[0147]

[0148] Note: Resistant R; Intermediate I; Sensitive S

[0149] As shown in Table 3, SY628 was sensitive to amikacin, erythromycin, chloramphenicol, cefazolin, ampicillin, and amoxicillin, moderately sensitive to penicillin, and resistant to cotrimoxazole. As a potential probiotic lactic acid bacteria strain, SY628 showed significantly different antibiotic sensitivities to different antibiotics belonging to the same class of drugs, indicating that the SY628 strain may be a sensitive isolate, and its growth can be controlled by antibiotics when necessary, with good safety.

[0150] (2) Antibacterial activity of strains

[0151] The antibacterial activity of SY628 was determined by the Oxford cup agar diffusion method. Escherichia coli and Staphylococcus aureus were used as indicator bacteria. After incubation at 37°C for 48 hours, the supernatant was centrifuged at 8000rpm for 10 minutes, and the supernatant was filtered with a 0.22μm filter under a sterile environment. 100μL of Escherichia coli and Staphylococcus aureus were added to each well (h=7.8mm) of LAB solid culture medium, placed for 5 hours to diffuse, and then placed at 37°C for 24 hours to measure the size of the inhibition zone, with the diameter in millimeters (mm).

[0152] The bacteria used in the test are Escherichia coli and Staphylococcus aureus; the Escherichia coli is ATCC25922, purchased through commercial channels (purchased from the Institute of Microbiology, Guangdong Academy of Sciences); the Staphylococcus aureus is the bacteria used in GB4789.28 standard; antibiotics are tested, bacteria used for sterilization effect detection, positive control strains, and quality control strains of the 2015 edition of the Chinese Pharmacopoeia. (Purchased from the Institute of Microbiology, Guangdong Academy of Sciences).

[0153] The antibacterial effect of SY628 on Staphylococcus aureus is as follows Figure 8 As shown in Fig. 9 shown.

[0154] SY628 did not produce an inhibition zone against Staphylococcus aureus, which may be due to the difference in sensitivity between the selected Staphylococcus aureus PJ and other reported sensitive strains, which just fits the characteristic of targeted antibacterial effect of bacteriocins; SY628 had a good antibacterial effect on Escherichia coli, with an inhibition zone diameter of 27±1.20mm. Compared with the antibacterial effect of Weissella confusa F3c isolated and screened from horse intestine lactic acid bacteria by Yin Fangfang on Escherichia coli (inhibition zone of 10.535mm), SY628 showed a strong inhibitory effect on Escherichia coli.

[0155] (3) Hemolytic test

[0156] Streak the fused Weissella SY628 on Columbia blood agar medium and incubate at 37°C for 48 hours. Determine whether the strain is hemolytic based on whether a hemolytic zone is formed around the colony. Fig.10 As shown, no hemolytic zone appeared around the colonies of the fused Weissella SY628, indicating that the fused Weissella SY628 was not hemolytic and was a safe strain.

[0157] Example 5

[0158] This example tests the effect of actually fermenting sour soybean milk with Weissella SY628: soybeans are used as raw materials to make soy milk, fermentation bacteria are inoculated and cultured for a period of time, and the fermentation products are further analyzed.

[0159] The specific fermentation method is as follows:

[0160] (1) Soaking soybeans: Weigh 2 kg of soybeans and soak them in cold water at a mass ratio of 1:4 for 10 h;

[0161] (2) Grinding: The beans are mixed with water at a ratio of 1:8 (w / w, based on dry beans) and then put into a grinder for grinding;

[0162] (3) Cooking: Steam heating and cooking at 102°C for 15 minutes;

[0163] (4) Filtration: 200 mesh vibrating screen;

[0164] (5) Preparation: adding 6% sucrose by weight of soy milk;

[0165] (6) Pasteurization: 85°C, 20 min;

[0166] (7) Inoculation: Inoculate 5-60 mL of bacteria per liter of soy milk at a concentration of 10 6 -10 10 cfu / mL of fermentation broth;

[0167] (8) Fermentation: The inoculated liquid was placed at 37° C. for fermentation for 6 h;

[0168] (9) Post-ripening and refrigeration: Post-ripen at 4°C for 12 hours and store at this temperature.

[0169] This embodiment also conducted a comparative experiment, using Lactobacillus plantarum (purchased from the Institute of Microbiology, Guangdong Academy of Sciences) and commercial bacteria for producing yogurt (a 1:1 mixed bacterial agent of Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus, purchased online) to conduct experiments using the same fermentation method.

[0170] The yogurt fermented with the SY628 strain, commercial bacteria and Lactobacillus plantarum obtained above was tested after being post-ripened and refrigerated. The results of the measured physical and chemical indicators are summarized in Table 4.

[0171] Table 4 Physical and chemical indexes of sour soybean milk

[0172]

[0173] As shown in Table 4, there are no significant differences in protein content, pH value, and titrated acidity, but there are extremely significant differences in water holding capacity (P < 0.01). This shows that the acid production capacity of SY628 strain during the fermentation of yogurt is not significantly different from that of commercial bacteria and Lactobacillus plantarum, but the water holding capacity of yogurt obtained by SY628 strain is much higher than that of yogurt fermented by other bacteria, and it has a strong advantage in improving the easy precipitation of whey in yogurt. The whey precipitation of the fermented product after demulsification is shown in Figure 4. Fig.11 As shown: (A) SY628, no whey precipitation after demulsification, smooth and fine texture, good coagulation property; (B) commercial bacteria, some whey precipitation after demulsification, relatively smooth texture, general coagulation property; (C) plantarum Lactobacillus, a large amount of whey precipitation after demulsification, relatively brittle texture, poor coagulation property.

[0174] Example 6

[0175] In this embodiment, Weissella SY628 is used as a fermentation bacterium to prepare plant cheese, including the following steps:

[0176] 1) Grinding: Mix the beans with water at a ratio of 1:1 (w / w, based on dry beans) and put them into a two-stage series high-concentration fine grinder for grinding;

[0177] 2) Filtration: The soy paste obtained above is preliminarily filtered by a centrifuge with a 60-mesh screen to obtain raw soy milk;

[0178] 3) Cooking: Cook the raw soy milk obtained above in a micro-pressure sandwich pot, raise the temperature to 102°C, and keep it for 10 minutes to obtain cooked soy milk;

[0179] 4) Secondary filtration: The cooked soybean milk obtained above is filtered again with a vibrating screen having a mesh size of 60, and filtered soybean milk is obtained after filtration; the slurry is diluted with water to a protein concentration of 3.0 g / 100 g in the soybean milk;

[0180] 5) Adding sucrose: the weight of sucrose used is 8% of the weight of soy milk;

[0181] 6) Pasteurization: pasteurize the soy milk obtained above at 85° C. for 20 min;

[0182] 7) Fermentation: Inoculate the above soy milk with Weissella confusa SY628, add 20 mL of viable bacteria per liter of soy milk, and the number of live bacteria is 10 6 ~10 10 cfu / mL of bacterial solution for fermentation; the fermentation conditions are 35°C, 6 hours, and curd is formed after the fermentation is completed.

[0183] 8) Compression molding: The curd is placed in a mold and compression molded for about 24 hours until the moisture content is 50-60 wt%.

[0184] 9) Ripening: The dehydrated curd is cut into small pieces and vacuum packed, and then matured in an incubator at 4°C and 90% humidity for 40 days. During the fermentation and ripening process, the cheese will react and produce flavor substances.

[0185] The appearance of the resulting cheese is Fig.12 As shown, the obtained plant cheese product is light yellow, smooth and delicate, has a soft but hard texture, a smooth cut surface, a slight bean aroma, and a sweet but sour taste.

[0186] In this embodiment, only 60 meshes were sieved, and the bean dregs rich in dietary fiber were not completely filtered out, but the cheese product was still successfully obtained, and the obtained product had a smooth, uniform and compact texture, indicating that the bean dregs had been decomposed into smaller molecules (e.g., insoluble dietary fiber was decomposed into soluble dietary fiber), reflecting that Weissella fusion SY628 has the ability to decompose dietary fiber.

[0187] From the above examples, it can be seen that the fusion Weissella SY628 has good acid resistance and bile salt resistance, indicating that the strain is relatively active in the intestine (strong probiotics), and SY628 has significantly different antibiotic sensitivities to different antibiotics and has a good inhibitory effect on Escherichia coli. Through hemolytic analysis, it is determined that SY628 is safe for human consumption and is a candidate probiotic strain with the potential to develop new functional foods. In the application of yogurt, SY628 can significantly improve the coagulation effect of yogurt, which is significantly better than the fermentation effect of commercial strains.

[0188] Weissella fusion SY628 is a lactic acid bacterium with high extracellular polysaccharide production, with a polysaccharide production of 19.8±0.14 g / L, which is better than many existing reports on Weissella fusion.

[0189] The fusion strain Weissella SY628 was able to grow in MRS liquid medium with a pH of 2.5, and the viable count reached 2.43±0.20*10 9 CFU / mL, with good acid resistance; SY628 grew in an environment with a bile salt concentration of 1.5%, and the number of viable bacteria was 3.1±0.11×10 9 cfu / mL, and has good bile salt resistance; it is superior to many existing reports on Weissella fusion.

[0190] The Oxford agar diffusion method was used to measure the metabolites of Weissella SY628, which had a good inhibitory effect on Escherichia coli ATCC25922, with an inhibition zone of 27±1.20 mm, which was better than many existing reports on Weissella SY628.

[0191] Through drug resistance test and hemolytic test, it was determined that Weissella SY628 is safe for human consumption and is a candidate probiotic strain with the potential to develop new functional foods. Using it as a fermentation agent in the preparation of yogurt can significantly improve the coagulation effect of yogurt.

[0192] Although lactic acid bacteria generally have the ability to produce extracellular polysaccharides, they cannot achieve the fermentation effect of fused Weissella SY628. The inventors believe that on the one hand, it may be due to the higher level of extracellular polysaccharide production by fused Weissella SY628, and more importantly, it is due to the modification of plant protein by fused Weissella SY628, which causes gelation of plant protein during fermentation. The extracellular polysaccharides produced by fused Weissella SY628 are cross-linked with plant protein to form a more stable network structure, which greatly improves water retention and texture stability. The water retention is as high as 95.44%, which is a qualitative improvement compared to other fermentation bacteria products such as commercial bacteria, which have a water retention of less than 60%.

Claims

1. A fusion Weissella, characterized in that Its classification name is Weissella confusa SY628, and it was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 19, 2024, with the deposit number CGMCC No.32006.

2. A microbial agent containing the fused Weissella according to claim 1.

3. A biological preparation containing the fermentation product of the fused Weissella according to claim 1.

4. An application of fusion Weissella, characterized in that: The fused Weissella described in claim 1 or the microbial agent containing fused Weissella described in claim 2 is applied to the fermentation of food containing dietary fiber.

5. The use of the fused Weissella according to claim 4, characterized in that: Applied to the fermentation of beverages containing dietary fiber.

6. The use of the fused Weissella according to claim 4, characterized in that: Applied to the fermentation of dairy products containing dietary fiber.

7. The use of the fused Weissella according to claim 4, characterized in that: Applied to the fermentation of plant-based dairy products, or to the fermentation of products containing plant-based dairy products, or to the fermentation of animal-based dairy products containing dietary fiber.

8. The use of the fused Weissella according to claim 7, characterized in that: The plant-based dairy product is a seed-based plant-based dairy product.

9. The use of the fused Weissella according to claim 7, characterized in that: The plant-based dairy product is one or more of nut-based plant-based dairy products, cereal-based plant-based dairy products, and bean-based plant-based dairy products.

10. The use of the fused Weissella according to claim 9, characterized in that: The beans are one or more of soybeans, black beans, green beans, mung beans, red beans, chickpeas, kidney beans, peas, broad beans and lentils.

11. The use of the fused Weissella according to any one of claims 4 to 10, characterized in that: The fused Weissella or the microbial agent containing the fused Weissella is inoculated into the fermentation substrate and the fermentation is carried out under heat preservation.

12. An application of fusion Weissella, characterized in that: The fused Weissella described in claim 1 or the microbial agent described in claim 2 is used to produce polysaccharides.

13. An application of fusion Weissella, characterized in that: The fused Weissella described in claim 1 or the microbial agent described in claim 2 is used to decompose dietary fiber.

14. An application of fusion Weissella, characterized in that: The fused Weissella described in claim 1 or the microbial agent described in claim 2 is used for acid production.

15. An application of fusion Weissella, characterized in that: The fused Weissella described in claim 1, the microbial agent described in claim 2, or the biological agent described in claim 3 is used as a stabilizer.

16. Use of the fused Weissella according to claim 1 as a probiotic.

17. The purification and screening method of fused Weissella according to claim 1, characterized in that: The screening medium is MRS agar medium supplemented with 1-3 wt% CaCO3.

18. The method for purifying and screening fused Weissella according to claim 17, characterized in that: The culture temperature during screening was 28-45°C.

19. The method for purifying and screening fused Weissella according to claim 17 or 18, characterized in that: Screening is performed from the microbial agents or fermentation products fused with Weissella.

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