Leuconostoc mesenteroides subsp. Mesenteroides MW135 and application thereof

Abstract

The invention belongs to the technical field of microbial fermentation, and discloses a leuconostoc mesenteroides subsp. Mesenteroides MW135 and application thereof. The strain is Leuconostoc mesenteroides subsp. Mesenteroides, is obtained by separation and purification from fresh wolfberry fruits, has the characteristics of tolerance to high alcohol, high acid and high temperature and the like, is preserved in China General Microbiological Culture Collection Center (CGMCC) on June 25, 2024, and has a preservation number of CGMCC No.31066. The invention further discloses a preparation method of the Leuconostoc mesenteroides subsp. Mesenteroides strain. The strain provided by the invention is suitable for preparation of high-acid, high-alcohol and other fermented foods. When the strain is used for fermenting sour beer, the acidity of the beer can be adjusted, the sour taste is softer, and the irritation caused by acetic acid is reduced, so that the sour beer with more pleasant sour taste is produced; when the strain is used for fermenting fruit and vegetable juice, not only can rich bubbles and aroma substances be brought, but also the content of non-volatile acid of the fruit and vegetable juice can be remarkably increased, so that the flavor and the taste of the fruit and vegetable juice are improved, and a beverage is more refreshing and rich in layering sense.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Leuconostoc mesenteroides subspecies mesenteroides MW135 and an application thereof. Background Art

[0002] Leuconostoc mesenteroides is an important species of the genus Leuconostoc, commonly used in fermented dairy products, silage, pickles and fruit wine, and is a safe strain that can be used in food. Studies have shown that Leuconostoc mesenteroides can ferment sugars to produce a variety of acids and alcohols, as well as substances with antioxidant and antagonistic abilities to pathogenic bacteria. At the same time, Leuconostoc mesenteroides can produce lactic acid and carbon dioxide during glucose metabolism through the pentose phosphate pathway, and generate a variety of flavor compounds (diacetyl, acetoin, acetate and 2,3-butanediol, etc.), thereby improving product texture and flavor. For example, invention patent application 202210583781.6 discloses a strain of Leuconostoc mesenteroides, which can promote epidermal cell proliferation and repair, inhibit the growth of skin pathogens, and have anti-inflammatory effects; invention patent application 202410229738.9 discloses a strain of Leuconostoc mesenteroides, which can tolerate high-sugar environments and is suitable for the fermentation of foods with high sugar content.

[0003] Although some strains of Leuconostoc mesenteroides can tolerate high-sugar environments, their growth and fermentation abilities may be inhibited under extreme conditions (such as high temperature, high acidity or high alcohol content), limiting their application in certain special fermented foods. Summary of the invention

[0004] The present invention aims at the technical bottleneck of Leuconostoc mesenteroides in the preparation of fermented food, and provides a strain of Leuconostoc mesenteroides subspecies mesenteroides MW135 and its application. The strain is a natural probiotic selected from fresh wolfberry fruit, and has a significant ability to withstand high alcohol, high acid and high temperature. It is not only suitable for traditional dairy product fermentation, but also can play an important role in beer, fruit wine, vinegar drink, pickles and other fermented foods that require higher tolerance.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In the first aspect, the present invention provides a strain of Leuconostoc mesenteroides subsp. mesenteroides MW135, whose classification name is Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ), deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on June 25, 2024, and its deposit number is CGMCC No.31066; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The Leuconostoc mesenteroides subspecies mesenteroides MW135 provided by the invention has a raised colony, a smooth and moist surface, neat edges, an opaque and creamy appearance, and shows a positive result after Gram staining.

[0007] Its 16S rDNA sequence (as shown in SEQ ID No.1) is as follows:

[0008] Compared with the prior art, the Leuconostoc mesenteroides subspecies mesenteroides MW135 provided by the present invention with a deposit number of CGMCC No. 31066 has the following advantages: (1) Tolerance to high alcohol pressure: can tolerate 12% ethanol; (2) Tolerance to low pH pressure: can tolerate pH 3.0; (3) Withstand high temperature pressure: can withstand high temperature of 57°C; (4) Tolerance to high acetic acid pressure: can tolerate 10% acetic acid.

[0009] The Leuconostoc mesenteroides subspecies mesenteroides MW135 of the present invention is not only suitable for single or mixed lactic acid bacteria fermentation, but also suitable for synergistic action with brewer's yeast in the alcohol fermentation stage, and suitable for fermentation in collaboration with other bacteria under high temperature and high acidity environment.

[0010] In a second aspect, the present invention also provides the use of the above-mentioned Leuconostoc mesenteroides subspecies mesenteroides MW135 in the preparation of food starter.

[0011] In a third aspect, the present invention further provides a bacterial agent comprising the above-mentioned Leuconostoc mesenteroides subspecies mesenteroides MW135.

[0012] The active microorganisms contained in the bacterial agent provided by the present invention can be Leuconostoc mesenteroides subspecies mesenteroides MW135 or a probiotic composition containing Leuconostoc mesenteroides subspecies mesenteroides MW135; and can also include other substances required for the preparation of the bacterial agent such as carriers, nutrients, protective agents, fillers, etc.

[0013] Preferably, the bacterial agent is a food fermentation agent.

[0014] In a fourth aspect, the present invention also provides an application of the above-mentioned Leuconostoc mesenteroides subspecies mesenteroides MW135 in the preparation of fermented food, wherein the Leuconostoc mesenteroides subspecies mesenteroides MW135 or a probiotic composition comprising the Leuconostoc mesenteroides subspecies mesenteroides MW135 is used to prepare fermented food.

[0015] The fermented food of the present invention includes but is not limited to dairy products, vegetable products and beverages.

[0016] Leuconostoc mesenteroides subsp. mesenteroides is included in the "List of Bacteria that Can Be Used in Foods" and is known for its excellent fermentation ability. It can ferment a variety of sugars, generate lactic acid and other metabolites, effectively regulate the balance of intestinal microecology, and improve the flavor and texture of food. Therefore, the Leuconostoc mesenteroides subsp. mesenteroides MW135 provided by the present invention meets the food production standards and is suitable for use in the preparation of various fermented foods.

[0017] Preferably, the fermented food is a fermented beverage; the bacterial cell concentration of the Leuconostoc mesenteroides subspecies mesenteroides MW135 inoculated in the fermentation substrate of the fermented beverage is 10 4 -10 6 CFU / mL.

[0018] Preferably, the fermented beverage is sour beer or carbonated fruit and vegetable juice beverage.

[0019] In a fifth aspect, the present invention further provides a sour beer, which is produced by fermentation with the above-mentioned Leuconostoc mesenteroides subspecies mesenteroides MW135.

[0020] Using this strain to ferment sour beer can enrich the sour substances in the sour beer, make the sour taste softer, reduce the irritation caused by acetic acid, and is also beneficial to the production of carbon dioxide in the beer, thereby enhancing the flavor and taste of the sour beer.

[0021] Preferably, the cell concentration of the Leuconostoc mesenteroides subspecies mesenteroides MW135 inoculated into the fermentation substrate of sour beer is 6×10 4 -3.2×10 6 CFU / mL; fermentation conditions: fermentation temperature 36-38℃, fermentation time 36-48 h.

[0022] The sour beer fermented by Leuconostoc mesenteroides subspecies MW135 of the present invention has an alcohol content of 5.0±0.5%vol, a total acid content of 1.8-2.35 mL / 100mL, and a diacetyl content of 0.04±0.01 mg / L. It has a straw color in appearance, fine foam, a typical aroma, a strong killing taste, a mild sour taste, and a refreshing taste.

[0023] Exemplarily, a method for brewing sour beer is provided, comprising the following steps: S1. The crushed malt raw material is mixed with water and then saccharified to obtain a saccharified mash; the saccharified mash is filtered to obtain a clarified wort; S2. The clarified saccharified mash is heated and boiled as described in S1, and hops are added to the clarified wort during the heating and boiling process, and the boiling is completed and cooled to obtain cooled wort; S3. The cooled wort obtained in S2 is connected to a fermentation tank, and then activated yeast is added to the fermentation tank for the first fermentation to obtain a fermentation liquid; S4. Siphon the supernatant of the fermentation liquid in step S5, put the supernatant into a pressure tank, adjust the sugar content and then add the above-mentioned Leuconostoc mesenteroides subsp. mesenteroides MW135 or a probiotic composition containing the above-mentioned Leuconostoc mesenteroides subsp. mesenteroides MW135 for fermentation, and obtain sour beer after the fermentation is completed.

[0024] Preferably, the mass ratio of the malt raw material to water in S1 is 1:5-7, and the pH of the water is 5.4-5.7; the saccharification treatment adopts two-step saccharification, the temperature of the first step saccharification is 50-55°C, and it is kept warm for 50 min; the temperature of the second step saccharification is 65-70°C, and it is kept warm until the saccharified liquid no longer shows blue when tested with iodine solution.

[0025] Preferably, the heating and boiling in S2 is divided into two stages, the boiling time of the first stage is 50-60 min, the boiling intensity is 4-6% / h, and hops are added in this stage, and the amount of hops added accounts for 0.0012%-0.0015% of the weight of the clarified wort; the boiling time of the second stage is 5-7 min, and the boiling intensity is 8-12% / h; the cooling is to 13-20°C.

[0026] Preferably, the yeast inoculated into the wort in S3 has a cell concentration of 6.2×10 6 -1.5×10 7 CFU / mL; the fermentation condition of the first fermentation is sealed and carried out in a constant temperature environment of (13-20) ± 0.5°C, and the fermentation is terminated when the residual sugar in the fermentation liquid drops to 4-5° Brix to obtain a fermentation liquid.

[0027] Preferably, in S4, the sugar content is adjusted by adding fruit and vegetable juice or white sugar to 15-20° Brix.

[0028] Preferably, the Leuconostoc mesenteroides subsp. mesenteroides MW135 described in S4 is introduced in the form of seed liquid.

[0029] More preferably, the culture medium of the seed liquid is fruit and vegetable juice puree.

[0030] More preferably, the culture medium of the seed solution is wolfberry puree, grape puree, citrus puree or carrot puree.

[0031] In a sixth aspect, the present invention further provides a fruit and vegetable juice beverage, characterized in that the fruit and vegetable juice beverage is produced by fermentation of the above-mentioned Leuconostoc mesenteroides subspecies mesenteroides MW135 or a probiotic combination including the above-mentioned Leuconostoc mesenteroides subspecies mesenteroides MW135.

[0032] The carbonated fruit and vegetable juice beverage is prepared by fermenting the Leuconostoc mesenteroides subspecies mesenteroides MW135 provided by the present invention. By utilizing its CO2 production characteristic during fermentation, the beverage can have a carbonation characteristic without the need for external addition of CO2, thereby giving the beverage a refreshing taste and a sense of bubbles. At the same time, the non-volatile acid content and the flavor substance content of the fruit and vegetable juice can be increased, making the fruit and vegetable juice sweet and sour and improving the flavor of the fruit and vegetable juice.

[0033] When preparing fruit and vegetable juice beverages, other lactic acid bacteria may be selected for use in combination with Leuconostoc mesenteroides subsp. mesenteroides MW135. Other lactic acid bacteria include but are not limited to Lactobacillus fermentum, Lactobacillus helveticus and Lactobacillus plantarum.

[0034] Preferably, the cell concentration of the Leuconostoc mesenteroides subspecies mesenteroides MW135 inoculated into the fermentation substrate of the carbonated fruit and vegetable juice beverage is 2×10 4 -4×10 6 CFU / mL; fermentation conditions: fermentation temperature 36-38℃, fermentation time 36-48h.

[0035] Exemplarily, a method for preparing a carbonated fruit and vegetable juice beverage is provided, comprising the following steps: S1. Wash the fruit and vegetable raw materials and beat them to obtain fruit and vegetable juice; S2. Pectinase is added to the fruit and vegetable juice for enzymolysis, and after the enzymolysis is completed, the solid-liquid separation is performed, and the pH and sugar content of the liquid phase are adjusted and sterilized to obtain sterilized fruit and vegetable juice; S3. After the sterilized fruit and vegetable juice in S2 is cooled, the above-mentioned Leuconostoc mesenteroides subsp. mesenteroides MW135 or a probiotic composition comprising the above-mentioned Leuconostoc mesenteroides subsp. mesenteroides MW135 is added for fermentation. After the fermentation is completed, a carbonated fruit and vegetable juice beverage is obtained.

[0036] Preferably, the amount of pectinase added in S2 is 0.15%-0.35%, the enzymatic hydrolysis temperature is 50°C for 2h; the pH is adjusted to 6.5-7.0, and the sugar content is adjusted to 15-20°Brix; and the sterilization method is pasteurization.

[0037] Preferably, the Leuconostoc mesenteroides subspecies mesenteroides MW135 described in S3 is introduced in the form of activated seed liquid.

[0038] Preferably, the culture medium of the seed liquid is the fruit and vegetable juice described in S1.

[0039] Preferably, the raw material of the fruit and vegetable juice is at least one of fruit and vegetables. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 This is the plate colony picture of Leuconostoc mesenteroides subsp. mesenteroides MW135; Figure 2This is an optical microscope examination of Leuconostoc mesenteroides subspecies mesenteroides MW135; Figure 3 The brewing process flow chart of the sour beer in Examples 4 and 5 is as follows; Figure 4 This is a process flow chart of the fermentation process of carbonated fruit and vegetable juice beverages in Examples 6-8. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0043] The strains used in the following examples of the present invention are derived from: Leuconostoc mesenteroides subsp. mesenteroides MW135 ( Leuconostoc mesenteroides subsp. mesenteroid es ), deposit number CGMCC No.31066; Lactobacillus mucilaginosus fermentation Limosilactobacillus fermentum )CGMCC 1.1880, provided by China Microorganism Culture Collection Center; Lactobacillus helveticus ( Lactobacillus helveticus )CGMCC No.12062, provided by China Microorganism Culture Collection Center; Lactobacillus plantarum ( Lactiplantibacillus plantarum )CGMCC 21791, provided by China Microorganism Culture Collection Center; The culture medium used in the following embodiments of the present invention is: MRS medium (g / L): glucose 20 g / L, peptone 10 g / L, beef extract powder 8 g / L, yeast extract powder 4 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1 g / L, pH 6.0, sterilized at 121℃ for 20 min. If preparing solid medium, add 15.0 g / L.

[0044] The malt and hops used in the examples are commercially available, wherein the hops are from the American bitter hop Magnum (with an α-acid content of 12%-14%) and the Czech aromatic hop Saaz (with an α-acid content of 2.8%-3.3%); the yeast is active dry yeast A-CN36 purchased from Angel Yeast Co., Ltd.

[0045] Table of sensory evaluation standards for finished fermented fruit and vegetable juice beverages in this embodiment: Table 1 Sensory evaluation criteria for fermented fruit and vegetable juice beverages

[0046] Unless otherwise specified, the fermentation conditions used in the examples of the present invention are conventional fermentation conditions in the art. Unless otherwise specified, the reagents used in the examples of the present invention are commercially available.

[0047] Example 1 Isolation, identification and sequencing of strains 1. Isolation and screening of Leuconostoc mesenteroides subsp. mesenteroides MW135 Take 25 g of fresh wolfberry fruit sample and put it into a conical flask filled with 225 mL of sterile physiological saline. Vortex and shake at 180 r / min for 30 min and ultrasonicate for 15 min to obtain wolfberry extract. Dilute the wolfberry extract 10 times with sterile water to form 10 -1 Up to 10 -7 Different concentration gradients, select 10 -4 -10 -7 100 μL of each of the four gradient dilutions was evenly spread on the MRS solid plate. The spread medium was incubated in a constant temperature incubator at 37°C for 48 h, and colonies with the same number of colonies were selected from the surface of the plate, streaked on the corresponding plate, and pure colonies were obtained. A strain was isolated and named MW135, which was stored in a refrigerator at 4°C.

[0048] 2. Identification of Leuconostoc mesenteroides subsp. mesenteroides MW135 2.1 Colony and bacterial morphology of MW135 strain Colony morphology of the strain: white, round, with protrusions, smooth and rounded surface, neat edges, opaque and creamy; see Figure 1 .

[0049] Morphological characteristics of bacteria: Gram-positive, spherical; see Figure 2 .

[0050] 2.2 Sequencing analysis of 16S rDNA of MW135 strain After the strains were enriched and cultured, the cells were collected by centrifugation and handed over to Suzhou Anshengda Biotechnology Co., Ltd. for 16S rDNA sequencing. After obtaining the 16S rDNA sequencing results of the strains, the NCBI database was logged in, and the 16S rDNA sequences of the strains were compared and searched with the database of the website by BLAST homology to obtain the identification results of the microorganisms. It can be seen that MW135 is similar to Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ) similarity reached 100%, it can be determined that strain MW135 is a strain of Leuconostoc mesenteroides subsp.

[0051] In summary, combined with 16S rDNA sequence analysis and physiological and biochemical identification results, MW135 was identified as Leuconostoc mesenteroides subsp. mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides ) strain and named it Leuconostoc mesenteroides subsp. mesenteroides MW135.

[0052] The MW135 strain of Leuconostoc mesenteroides subsp. mesenteroides was deposited in the General Microbiology Center of China Culture Collection on June 25, 2024, and its deposit number is CGMCC No.31066; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0053] 3. Tolerance of Leuconostoc mesenteroides subsp. mesenteroides MW135 strain Seed solution of Leuconostoc mesenteroides subsp. mesenteroides MW135 strain: Pick a single colony of the MW135 strain from the MRS plate medium and add it to 1 mL of sterile saline, gently mix it to obtain a bacterial suspension. Inoculate the MW135 bacterial suspension into 10 mL of MRS liquid medium at an inoculum rate of 1% (v / v), place it in a shaker at 150 rpm at 37°C and culture it for 24 h to obtain a seed solution.

[0054] 3.1 Determination of alcohol resistance Prepare MRS liquid culture medium, sterilize in autoclave at 121℃ for 20 min, adjust the alcohol concentration of MRS liquid culture medium to 2%, 4%, 6%, 8%, 10%, 12%, 14%, and 16% with anhydrous ethanol under sterile conditions, and dispense it into sterilized test tubes, 5 mL each. Under sterile conditions, inoculate MW135 strain seed liquid into the above-mentioned ethanol-containing MRS liquid culture medium at a 5% inoculation rate, and incubate in a 37℃ constant temperature incubator for 24 h after inoculation. Measure the OD value of the liquid in each test tube by using a spectrophotometer. 600 Value (the bacterial solution at 0 h of culture was used as the control, and its OD 600 =0) to determine the growth of MW135 strain. The results are shown in Table 2.

[0055] 3.2 Determination of acid resistance Prepare MRS liquid medium, sterilize in an autoclave at 121°C for 20 min, adjust the acetic acid concentration of the MRS liquid medium to 2%, 4%, 6%, 8%, 10%, 12%, 14%, and 16% with glacial acetic acid under sterile conditions, and dispense it into sterilized test tubes, 5 mL each. Under sterile conditions, inoculate the MW135 strain seed liquid into the above-mentioned MRS liquid medium containing acetic acid at a 5% inoculation rate, and incubate it in a 37°C constant temperature incubator for 24 h after inoculation. Measure the OD value of the liquid in each test tube by using a spectrophotometer. 600Value (the bacterial solution at 0 h of culture was used as the control, and its OD 600 =0) to determine the growth of MW135 strain. The results are shown in Table 2.

[0056] 3.3 Determination of temperature resistance The MW135 strain seed liquid was inoculated into normal MRS liquid medium at a 5% inoculum volume, mixed and cultured at 27°C, 32°C, 37°C, 42°C, 47°C, 52°C, 57°C, and 62°C for 24 h. The OD value of the liquid in each test tube was measured by a spectrophotometer. 600 Value (the bacterial solution at 0 h of culture was used as the control, and its OD 600 =0) to determine the growth of MW135 strain. The results are shown in Table 2.

[0057] Table 2 Tolerance of MW135 under different environmental conditions

[0058] “+” indicates OD 600 ≥0.2; “-” indicates OD 600 =0.

[0059] Table 2 shows the tolerance characteristics of the MW135 strain. The results show that the MW135 strain can still grow under the condition of 12% ethanol concentration, has good alcohol tolerance, and can be used in the fermentation production of beer, wine and other fruit wines. In addition, the MW135 strain tolerates more than 10% acetic acid. It has been determined that the pH of the MRS liquid culture medium with a 10% acetic acid concentration is 3.40; the temperature tolerance experiment shows that the MW135 strain can grow in an environment of 57°C, but its growth is restricted at a temperature of 62°C, so the temperature it can tolerate is 57°C.

[0060] Example 2 Application of Leuconostoc mesenteroides subspecies mesenteroides MW135 in the preparation of food starter This embodiment provides a food fermentation agent. White sugar is added to wolfberry pulp to adjust the sugar content to 15-20° Brix, and the mixture is pasteurized and then cooled; the cultured Leuconostoc mesenteroides subsp. mesenteroides MW135 bacterial liquid is centrifuged at 5000 r / min for 15 min, the supernatant is discarded, the mixture is washed twice with sterile saline, and the MW135 bacterial mud obtained by centrifugation is mixed with sterile saline to prepare a fermentation bacterial liquid; finally, 3% of the fermentation bacterial liquid is inoculated into the sterilized wolfberry pulp, and the food fermentation agent is obtained after fermentation for 24 hours.

[0061] Example 3 Application of Leuconostoc mesenteroides subsp. mesenteroides MW135 in the preparation of food starter The present embodiment provides a food fermentation agent. The glycerol-preserved (-80°C) strain of Leuconostoc mesenteroides subspecies MW135 was thawed from the storage state, 0.1 mL was transferred to 10 mL of MRS liquid culture medium for activation, and cultured at 37°C for 24 hours. After activation, 1 mL of the activation solution was inoculated into 100 mL of MRS liquid culture medium for expansion culture, and cultured in a shaker or fermenter at 37°C until the bacteria reached the end of the logarithmic growth phase or the stable phase. The cultured Leuconostoc mesenteroides subspecies MW135 bacterial solution was centrifuged at 5000 r / min for 15 min, the supernatant was poured out, and the culture was washed twice with sterile saline and resuspended to remove the residual culture medium components. 4% glycerol protective agent and 20 g skim milk powder were added to the bacterial suspension and mixed thoroughly to 100 mL, and then spray-dried to prepare bacterial powder after mixing.

[0062] The obtained food starter can be activated by using sterilized fruit and vegetable juice, and then added to the fermentation substrate after being mixed into seed liquid.

[0063] Example 4 Application of Leuconostoc mesenteroides subsp. mesenteroides MW135 in fermentation of sour beer This embodiment provides a method for preparing sour beer. The process flow is as follows: Figure 3 As shown, the following steps are included: (1) Grinding malt: Take barley malt and wheat malt as raw materials for grinding, and the mass ratio of barley malt to wheat malt is 6:4; the malt grinding requires that the bran is broken but not crushed, and is as complete as possible, and the white endosperm material is required to be crushed as completely as possible.

[0064] (2) Two-step saccharification: Mix the crushed malt with water in a saccharification pot at a weight ratio of 1:5, where the pH of the water is required to be 5.4, and then adjust the temperature to 50°C and keep it still for 40 minutes for the first step of saccharification; then slowly raise the temperature to 65°C and continue to keep it still for the second step of saccharification, and keep it still for 40-60 minutes until the saccharification liquid no longer shows blue when tested with iodine solution, and the saccharification is completed. Use a vacuum filtration device to filter the saccharified crude wort in the form of vacuum filtration to obtain clarified wort.

[0065] (3) Pour the filtered wort into a boiling pot and heat it in two stages. The first stage has a boiling time of 50-60 min and a boiling intensity of 4% / h. Hops are added at this stage. The hops are American bitter magnum hops, and the added amount accounts for 0.0012% of the weight of the clarified wort. The second stage has a boiling time of 5 min and a boiling intensity of 8% / h. The boiled wort is cooled to about 13°C.

[0066] (4) First fermentation: The cooled wort is sent to the fermentation tank, and activated yeast is added to the fermentation tank. The yeast inoculation amount is 6.2×10 6 CFU / mL, sealed, and the first fermentation was carried out at a constant temperature of 20±0.5℃. The first fermentation time was 9-12 days until the residual sugar in the first fermentation liquid dropped to 4°Brix, and the fermentation was terminated.

[0067] (5) Secondary fermentation: Siphon the supernatant of the fermentation liquid in step 4, put the supernatant into a pressure tank, add apple juice to the tank to adjust the sugar content to 4.7° Brix, and add the food starter provided in Example 2, with a MW135 cell concentration of 6×10 4 CFU / mL, fermented at 37℃ for 36 h, and then post-fermented at 4℃.

[0068] (6) After the post-fermentation is completed, fresh beer is obtained by bottling; or after the beer fermentation liquid is clarified, it is instant-killed and filled or filled for pasteurization to obtain cooked beer.

[0069] Example 5 Application of Leuconostoc mesenteroides subsp. mesenteroides MW135 in fermentation of sour beer This embodiment provides a method for preparing sour beer. The process flow is as follows: Figure 3 As shown, the following steps are included: (1) Grinding malt: Take barley malt and wheat malt as raw materials for grinding, and the mass ratio of barley malt to wheat malt is 6:4; the malt grinding requires that the bran is broken but not crushed, and is as complete as possible, and the white endosperm material is required to be crushed as completely as possible.

[0070] (2) Two-step saccharification: Mix the crushed malt with water in a saccharification pot at a weight ratio of 1:7, where the pH of the water is required to be 5.7, and then adjust the temperature to 55°C and keep it still for 60 minutes for the first step of saccharification; then slowly raise the temperature to 70°C and continue to keep it still for the second step of saccharification, and keep it still for 40-60 minutes until the saccharification liquid no longer shows blue when tested with iodine solution, and the saccharification is completed. Use a vacuum filtration device to filter the saccharified crude wort in the form of vacuum filtration to obtain clarified wort.

[0071] (3) Pour the filtered wort into a boiling pot and heat and boil it in two stages. The first stage has a boiling time of 60 minutes and a boiling intensity of 6% / h. When the wort has been boiling for 10 minutes, add American bitter hops Magnum hops at a ratio of 0.0005% of the weight of the clarified wort. When the wort has been boiling for 30 minutes, add Czech aromatic hops Saaz hops at a ratio of 0.001% of the weight of the clarified wort. The second stage has a boiling time of 7 minutes and a boiling intensity of 12% / h. After the boiling is completed, cool to 20°C.

[0072] (4) First fermentation: The cooled wort is sent to the fermentation tank, and activated yeast is added to the fermentation tank. The yeast inoculation amount is a bacterial concentration of 1.5×10 7 CFU / mL, sealed, and the first fermentation was carried out at a constant temperature of 20±0.5℃. The first fermentation time was 9-12 days until the residual sugar in the first fermentation liquid dropped to 5°Brix, and the fermentation was terminated.

[0073] (5) Secondary fermentation: Siphon the supernatant of the fermentation liquid in step 4, put the supernatant into a pressure tank, add white sugar to the tank to adjust the sugar content to 5.0° Brix, and add the food starter provided in Example 3, with a MW135 cell concentration of 3.2×10 6 CFU / mL, fermented at 37℃ for 48 h, and then post-fermented at 4℃.

[0074] (6) After the post-fermentation is completed, fresh beer is obtained by bottling; or after the beer fermentation liquid is clarified, it is instant-killed and filled or filled for pasteurization to obtain cooked beer.

[0075] The sour beer prepared in Examples 4 and 5 was tested, and its alcohol content was 5.0±0.5%vol, total acid was 1.8-2.35 mL / 100 mL, diacetyl was 0.04±0.01 mg / L, appearance was straw color, foam was fine, there was a typical aroma, strong taste, mild sourness, and a refreshing taste.

[0076] Example 6 Application of Leuconostoc mesenteroides subsp. mesenteroides MW135 in the preparation of gas-containing fermented fruit and vegetable juice beverages This embodiment provides a method for preparing a gas-containing fermented fruit and vegetable juice beverage. The process flow is as follows: Figure 4 As shown, the following steps are included: (1) Preparation of fruit and vegetable juice: Wash wolfberry, mango, carrot, and broccoli, and beat them in a ratio of 1:1:1:1 to obtain fruit and vegetable juice. Add 0.15% pectinase to the fruit and vegetable juice and perform enzymolysis at 50°C for 2 h. After the enzymolysis, add baking soda to the filtered juice to adjust the pH to 6.5, use white sugar to adjust the sugar content to 15°Brix, and then pasteurize the filtered juice.

[0077] (2) After the filtered juice obtained in step 1 is cooled, the food starter prepared in Example 3 is added to the fruit and vegetable juice. The concentration of Leuconostoc mesenteroides subspecies mesenteroides MW135 in the fruit and vegetable juice is 10 5 -10 6 CFU / mL, and fermented in a sealed container at 37°C for 36 h to obtain a carbonated fermented fruit and vegetable juice beverage.

[0078] The carbonated fruit and vegetable juice beverage prepared by the method has a good flavor, with a total acid content of 0.95-1.25 g / 100 mL, a mild and non-irritating sour taste, a strong mouthfeel, a pleasant and comfortable fruity aroma, and a sensory score of 80.12 points.

[0079] GC-MS was used to detect the volatile aroma substances in the prepared carbonated fermented fruit and vegetable juice beverages. There were 69 volatile aroma substances in the beverages, including 6 acids, 11 alcohols, 10 esters, 10 ketones, 9 aldehydes, and 23 other substances. See Table 3 for details.

[0080] Table 3 Volatile flavor substances in carbonated fermented fruit and vegetable juice beverages

[0081] Example 7 Application of Leuconostoc mesenteroides subsp. mesenteroides MW135 in the preparation of fermented fruit and vegetable juice beverages This embodiment provides a method for preparing a carbonated fermented apple juice beverage.

[0082] (1) Select ripe, non-rotten and insect-free apples for juicing, and remove the core and skin before juicing.

[0083] (2) Add 0.35% pectinase and perform enzymolysis at 50°C for 4 h. Filter the juice through four layers of gauze. Determine the sugar content of the apple juice and adjust the sugar content to 20°Brix with white sugar.

[0084] (3) The method for preparing the seed culture medium is as follows: inoculate Leuconostoc mesenteroides subsp. mesenteroides MW135 into MRS liquid medium for culture at 37°C for 36 h. The cultured bacterial solution is centrifuged at 5000 r / min for 15 min, the supernatant is discarded, and the culture is washed twice with sterile saline. The lactic acid bacteria obtained by centrifugation are then mixed with sterile saline (the amount of saline used is the same as the volume of the supernatant) to prepare a suspension of bacteria; 3% of the suspension of bacteria is added to sterilized fruit and vegetable juice, and the seed culture medium is obtained after fermentation for 24 h.

[0085] (4) Lactic acid bacteria fermentation: Using clarified apple juice with adjusted sugar content as raw material, seed culture solution of Leuconostoc mesenteroides subsp. mesenteroides MW135 was added. The concentration of MW135 bacteria in the apple juice was 10 5 -10 6 CFU / mL, sealed and fermented at 37℃ for 48 h to obtain a carbonated apple juice fermented beverage.

[0086] The fermented apple juice prepared by this method has a good flavor, with a total acid content of 0.89-1.19 g / 100 mL, a mild and non-irritating sour taste, a sweet and sour taste, a delicate taste, a pleasant and comfortable aroma, and a sensory score of 81.36 points.

[0087] Example 8 Application of Leuconostoc mesenteroides subsp. mesenteroides MW135 in the preparation of fermented fruit and vegetable juice beverages This embodiment provides a method for preparing a carbonated fermented fruit and vegetable juice beverage.

[0088] (1) Preparation of fruit and vegetable juice: Wash wolfberry, apple, citrus, carrot, broccoli and other fruit and vegetable raw materials, and beat them in a ratio of 1:1:1:1:1 to obtain fruit and vegetable juice. Add 0.25% pectinase to the fruit and vegetable juice and perform enzymolysis at 50°C for 3 h. After the enzymolysis is completed, filter it, add baking soda to adjust the pH to 7.0, adjust the sugar content to 18°Brix, and then pasteurize the filtered juice.

[0089] (2) Preparation of lactic acid bacteria liquid: Leuconostoc mesenteroides subsp. mesenteroides MW135, Lactobacillus fermentum, Lactobacillus helveticus and Lactobacillus plantarum were inoculated into MRS liquid medium and cultured at 37°C for 36 h. The cultured liquid was centrifuged at 5000 r / min for 15 min, the supernatant was discarded, and the culture was washed twice with sterile saline. The lactic acid bacteria obtained by centrifugation were then mixed with sterile saline (the amount of saline was the same as the volume of the supernatant) to prepare a suspension liquid; 3% of the suspension liquid was inoculated into sterilized fruit and vegetable juice, and after fermentation for 24 h, the four lactic acid bacteria seed liquids were obtained, and the seed liquids were mixed according to the volume ratio of 2:2:1:1 (Leuconostoc mesenteroides subsp. mesenteroides MW135: Lactobacillus fermentum: Lactobacillus helveticus: Lactobacillus plantarum = 2:2:1:1) to obtain lactic acid bacteria liquid.

[0090] (3) Fruit and vegetable juice fermentation: After the fruit and vegetable juice obtained in step 1 is cooled, the prepared lactic acid bacteria liquid is inoculated into the fruit and vegetable juice. The concentration of Leuconostoc mesenteroides subsp. mesenteroides in the fruit and vegetable juice is 10 5 -10 6 CFU / mL, and fermented at 37°C for 42 hours to obtain a carbonated fermented fruit and vegetable juice beverage.

[0091] The fruit and vegetable juice drink prepared by this method has good flavor, with a total acid content of 1.08-1.30 g / 100 mL, a mild and non-irritating sour taste, a strong floral and fruity aroma, and a harmonious sour-sweet taste. The sensory score is 82.15 points.

[0092] In summary, the Leuconostoc mesenteroides subspecies mesenteroides MW135 provided by the present invention can tolerate high alcohol, high acid and high temperature, and is suitable for various fermented foods. It has the ability to produce acid and alcohol resistance, and is suitable for the preparation of sour beer; it is applied to fruit and vegetable juice, utilizes fructose metabolism to produce acetic acid and CO2, and enriches the aroma substances of fruit and vegetable juice, increases the non-volatile acid content of fruit and vegetable juice, and thus improves the flavor and taste of fruit and vegetable juice.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Leuconostoc mesenteroides subspecies mesenteroides MW135, characterized in that: Its classification name is Leuconostoc mesenteroides subsp. mesenteroides. It was deposited in the General Microbiology Center of China Culture Collection on June 25, 2024. Its deposit number is CGMCC No.31066. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of Leuconostoc mesenteroides subspecies mesenteroides MW135 according to claim 1 in preparing food starter.

3. A bacterial agent, characterized in that: It comprises the Leuconostoc mesenteroides subspecies mesenteroides MW135 described in claim 1; preferably, the bacterial agent is a food fermentation agent.

4. The use of Leuconostoc mesenteroides subspecies mesenteroides MW135 according to claim 1 in preparing fermented food, characterized in that: The Leuconostoc mesenteroides subspecies mesenteroides MW135 or a probiotic composition comprising the Leuconostoc mesenteroides subspecies mesenteroides MW135 is used for preparing fermented food.

5. The use according to claim 4, characterized in that: The fermented food is a fermented drink; the Leuconostoc mesenteroides subspecies MW135 is inoculated in the fermentation substrate of the fermented drink at a concentration of 10 4 -10 6 CFU / mL; preferably, the fermented beverage is sour beer or carbonated fruit and vegetable juice beverage.

6. A sour beer, characterized in that The sour beer is prepared by fermentation with the Leuconostoc mesenteroides subspecies mesenteroides MW135 described in claim 1.

7. The sour beer according to claim 6, characterized in that The cell concentration of the Leuconostoc mesenteroides subspecies MW135 inoculated in the fermentation substrate of sour beer is 6×10 5 -3.2×10 6 CFU / mL; fermentation conditions: fermentation temperature 36-38℃, fermentation time 36-48h.

8. A carbonated fruit and vegetable juice beverage, characterized in that: The carbonated fruit and vegetable juice beverage is prepared by fermenting the Leuconostoc mesenteroides subspecies mesenteroides MW135 described in claim 1 or a probiotic combination including the Leuconostoc mesenteroides subspecies mesenteroides MW135 described in claim 1.

9. The carbonated fruit and vegetable juice beverage according to claim 8, characterized in that: The cell concentration of the Leuconostoc mesenteroides subspecies MW135 inoculated in the fermentation substrate of the carbonated fruit and vegetable juice beverage is 2×10 4 -4×10 6 CFU / mL; fermentation conditions: fermentation temperature 36-38℃, fermentation time 36-48h.

10. The carbonated fruit and vegetable juice beverage according to claim 8, characterized in that: The raw material of the fruit and vegetable juice is at least one of fruits and vegetables.

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