Compound fermentation inoculant capable of prolonging shelf life of prefabricated vegetables and preparation method of compound fermentation inoculant

Abstract

The invention discloses a compound fermentation inoculant for prolonging the shelf life of prefabricated vegetables and a preparation method of the compound fermentation inoculant, and relates to the technical field of fermentation, the compound fermentation inoculant is prepared from lactobacillus fermentum CZ22, lactobacillus buchneri-like CZ23 and digestion associated lactobacillus CZ24 according to the viable count ratio of 1: 1: 1 and the inoculum size of 3%, the preservation number of the lactobacillus fermentum CZ22 is CGMCC NO.31233, the preservation number of the lactobacillus buchneri-like CZ23 is CGMCC NO.31233, and the preservation number of the digestion associated lactobacillus CZ24 is CGMCC NO.31233. The preservation number of the lactobacillus buchneri CZ23 is CGMCC (China General Microbiological Culture Collection Center) NO.31986; the preservation number of the digestion associated lactobacillus CZ24 is CGMCC (China General Microbiological Culture Collection Center) NO.31987; according to the compound fermentation inoculant disclosed by the invention, lactobacillus fermentum CZ22, lactobacillus buchneri-like CZ23 and digestion associated lactobacillus CZ24 are adopted, a plurality of harmful microorganisms can be effectively inhibited in the pretreatment of the cooked prefabricated vegetables, the antibacterial effect is good, and the shelf life of food is prolonged.

Description

Technical Field

[0001] The present invention relates to the technical field of fermentation, and particularly relates to a compound fermentation bacterium agent for extending the shelf life of prefabricated dishes and a preparation method thereof. Background Art

[0002] Lactic acid bacteria are a class of Gram-positive bacteria without spores that can ferment carbohydrates to produce lactic acid. They are a recognized probiotic with food safety, and have various effects such as reducing the intestinal pH value, regulating the intestinal flora, inhibiting the growth of harmful bacteria, and preventing lactose intolerance. They are also important strains for making common fermented foods such as yogurt and pickles in life. Like most bacteria, lactic acid bacteria can also produce biofilms in some environments, enabling lactic acid bacteria to inhibit a variety of pathogenic bacteria that have an antagonistic effect on them.

[0003] In the food field, fermented lactic acid bacteria agents are mostly used as leavening agents in dairy products, baked products, and traditional fermented foods to promote the fermentation process of foods and improve the flavor and texture of foods; they can also be used as substitutes for preservatives in raw food products to inhibit the growth of microorganisms and extend the shelf life of foods. However, there are currently few similar products of fermented lactic acid bacteria agents, and their effects in inhibiting the growth of microorganisms and extending the shelf life of foods are not ideal. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a compound fermentation bacterium agent for extending the shelf life of prefabricated dishes and a preparation method thereof to overcome the defect that the existing fermented lactic acid bacteria agents are not ideal in inhibiting the growth of microorganisms and extending the shelf life of foods; and to achieve the purpose of being able to be applied in the pretreatment of cooked prefabricated dishes and having good antibacterial effects.

[0005] To solve the above technical problem, the technical solution of the present invention is: A compound fermentation bacterium agent for extending the shelf life of prefabricated dishes, which is prepared from Lactobacillus fermentum CZ22, Lentilactobacillus parabuchner i CZ23, and Lactobacillus parafarraginis CZ24 in a ratio of viable bacteria numbers of 1:1:1 and an inoculation amount of 3%; Companilactobacillus alimentarius Among them, the Lactobacillus fermentum CZ22 was deposited on July 9, 2024, at the China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and its deposit number is: CGMCC NO. 31233; Lactobacillus fermentum The Lentilactobacillus parabuchner CZ23, Lentilactobacillus parabuchneri) CZ23, deposited on September 18, 2024 at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, China General Microbiological Culture Collection Center, with the deposit number: CGMCC NO. 31986; The Lactobacillus parafarraginis ( Companilactobacillus alimentarius ) CZ24, deposited on September 18, 2024 at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, China General Microbiological Culture Collection Center, with the deposit number: CGMCC NO. 31987.

[0006] Further, the acid tolerance pH of the composite fermentation agent is 3.23 - 3.3.

[0007] Further, the preparation method of the composite fermentation agent for extending the shelf life of prefabricated dishes includes: Step a, pour Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22, Lactobacillus brantae ( Lentilactobacillus parabuchneri ) CZ23, Lactobacillus parafarraginis ( Companilactobacillus alimentarius ) CZ24 into MRS liquid medium respectively on the sterile operating table, activate at 37 °C for 24 h, and the OD600 value in the above three MRS liquid media is 2.1 - 2.3 to obtain the starter seed liquid; Step b, sterilize the fermentation broth at 121 °C for 15 min, immediately cool to 42 °C - 43 °C after sterilization, inoculate the starter seed liquid, inoculate it into the fermentation broth at a ratio of viable bacteria 1:1:1 and an inoculation amount of 3%, stir evenly and then immediately culture at a constant temperature of 37 °C. When the acidity reaches 1.5% - 2.0%, the fermentation can be ended to obtain the composite fermentation agent, and after ripening at 5 °C - 10 °C for 24 h, the finished product is obtained.

[0008] Further, in the step a, the MRS liquid medium: peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, glucose 20.0 g, diammonium citrate 2.0 g, sodium acetate 5.0 g, MgSO 4 2.0 g, MnSO 4 0.05 g, K 2 HPO 4 2.0 g, Tween 80 1.0 mL; distilled water 1000 mL; adjust the pH to 6.2 - 6.4, culture temperature 37 °C, culture time 16 h.

[0009] Further, in the step b, the components of the fermentation broth are glucose 30.00 g / L, lactose 30.00 g / L, soy peptone 30.00 g / L, K 2 HPO 42.29 g / L, MgSO 4 ·7H 2 O 0.80 g / L, tomato juice addition 50 ml / L, peanut sprout juice addition 50 ml / L, and histidine 0.15 g / L, culture temperature 37 °C, culture time 16 h.

[0010] Adopting the above technical solutions, compared with the prior art, the present invention has the following advantages: The compound fermentation bacterium agent of the present invention uses Lactobacillus fermentum CZ22, Lactobacillus brantae CZ23, and Lactobacillus parafarraginis CZ24, which can effectively inhibit a variety of harmful microorganisms in the pretreatment of cooked prefabricated dishes, has good antibacterial effect, and prolongs the shelf life of food. Specific embodiments

[0011] The embodiments of the present invention are described in detail below. The examples of the embodiments are intended to explain the present invention and should not be construed as limiting the present invention. For the specific technologies or conditions not specified in the embodiments, they are carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0012] Example 1. A preparation method of a compound fermentation bacterium agent for extending the shelf life of prefabricated dishes The strains used include Lactobacillus fermentum ( Lactobacillus fermentum ), CZ22, Lactobacillus brantae ( Lentilactobacillus parabuchneri ), CZ23, and Lactobacillus parafarraginis ( Companilactobacillus alimentarius ), CZ24.

[0013] Lactobacillus fermentum ( Lactobacillus fermentum ), CZ22, was isolated from pickled vegetable mother liquor. The collection time was May 7, 2024, and the collection location was Chengdu, Sichuan Province, China. Lactobacillus fermentum ( Lactobacillus fermentum ), CZ22, was deposited on July 9, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, at the China General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms. Its deposit number is: CGMCC NO. 31233. Lactobacillus fermentum CZ22 has the DNA sequence of SEQ ID NO. 1 in the sequence listing.

[0014] Lactobacillus brantae ( Lentilactobacillus parabuchneri ), CZ23, was isolated from pickled vegetable mother liquor. The collection time was May 28, 2024, and the collection location was Zunyi, Guizhou Province, China. Lactobacillus brantae ( Lentilactobacillus parabuchneri)CZ23 was deposited on September 18, 2024 at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, China General Microbiological Culture Collection Center, with the deposit number: CGMCC NO. 31986. Lactobacillus delbrueckii subsp. bulgaricus CZ23 has the DNA sequence of SEQ ID NO. 2 in the sequence listing.

[0015] Lactobacillus parafarraginis ( Companilactobacillus alimentarius )CZ24, isolated from pickled vegetable mother liquor, collected on May 16, 2024, at the collection site of Chongqing, China. Lactobacillus parafarraginis ( Companilactobacillus alimentarius )CZ24 was deposited on September 18, 2024 at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, China General Microbiological Culture Collection Center, with the deposit number: CGMCC NO. 31987. Lactobacillus parafarraginis CZ24 has the DNA sequence of SEQ ID NO. 3 in the sequence listing.

[0016] The medium used was MRS liquid medium: peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, glucose 20.0 g, diammonium citrate 2.0 g, sodium acetate 5.0 g, MgSO 4 2.0 g, MnSO 4 0.05 g, K 2 HPO 4 2.0 g, Tween 80 1.0 mL; distilled water 1000 mL; adjust the pH to 6.2 - 6.4, culture temperature 37 °C, culture time 16 h.

[0017] A preparation method of a composite fermentation agent for extending the shelf life of prefabricated dishes, comprising the following steps: (1)Pour Lactobacillus fermentum ( Lactobacillus fermentum )CZ22, Lactobacillus delbrueckii subsp. bulgaricus ( Lentilactobacillus parabuchneri )CZ23, Lactobacillus parafarraginis ( Companilactobacillus alimentarius )CZ24 into MRS liquid medium respectively on a sterile operating table, and culture at 37 °C for 24 h. The OD600 values of the above three MRS liquid media are 2.1 - 2.3 to obtain the fermentation agent seed liquid.

[0018] MRS liquid medium: peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, glucose 20.0 g, diammonium citrate 2.0 g, sodium acetate 5.0 g, MgSO 4 2.0 g, MnSO 4 0.05 g, K 2 HPO 42.0 g, Tween 80 1.0 mL; distilled water 1000 mL; adjust the pH to 6.2 - 6.4, culture temperature 37°C, culture time 16 h.

[0019] (2) Sterilize the MRS liquid medium at 121°C for 15 min, and immediately cool it to 42°C - 43°C after sterilization. Inoculate the starter culture seed liquid, and inoculate it into the fermentation broth at an inoculation amount of 3% according to the ratio of viable bacteria count of 1:1:1. After stirring evenly, immediately incubate it at a constant temperature of 37°C. When the acidity reaches 1.5% - 2.0%, the fermentation can be ended to obtain the compound fermentation agent, and it is refrigerated at 5°C - 10°C for 24 h for after-ripening to obtain the finished product.

[0020] Components of the fermentation broth: glucose 30.00 g / L, lactose 30.00 g / L, soy peptone 30.00 g / L, K 2 HPO 4 2.29 g / L, MgSO 4 ·7H 2 O 0.80 g / L, tomato juice addition amount 50 ml / L, peanut sprout juice addition amount 50 ml / L, and histidine 0.15 g / L, culture temperature 37°C, culture time 16 h.

[0021] Example 2. Determination of the pH value of the compound fermentation agent during fermentation After activating Lactobacillus fermentum CZ22, Lactobacillus brantae subsp. CZ23, and Lactobacillus alimentarius CZ24 for 2 generations in the MRS liquid medium, inoculate them into the MRS liquid medium at an inoculation amount of 5% according to the ratio of viable bacteria count of 1:1:1, and incubate them at a constant temperature of 37°C. Measure the pH value of the mixed bacterial liquid every 2 h. The pH change of the compound fermentation agent is shown in the following table.

[0022] Time (h) pH 0 6.19 2 6.14 4 5.89 6 5.33 8 4.21 10 4.04 12 3.85 14 3.65 16 3.3 18 3.25 20 3.25 22 3.24 24 3.23

[0023] It can be seen from this experiment that Lactobacillus fermentum CZ22, Lactobacillus brantae subsp. CZ23, and Lactobacillus alimentarius CZ24 can survive and reproduce in an environment with a relatively low pH value, reflecting the acid resistance of Lactobacillus fermentum CZ22, Lactobacillus brantae subsp. CZ23, and Lactobacillus alimentarius CZ24. After 16 h of culture time, the pH value no longer drops significantly and basically reaches stability. The fermentation of the fermentation broth is carried out at 37°C for 16 h.

[0024] Example 3. Determination of the viable bacteria count of the compound fermentation agent After activating Lactobacillus fermentum CZ22, Lactobacillus brantae - like CZ23, and Lactobacillus parafarraginis CZ24 for 2 generations in MRS liquid medium, inoculate them into the fermentation broth at an inoculation amount of 5% according to the ratio of viable cell counts of 1:1:1, and incubate them at a constant temperature of 37°C. The viable cell counts are determined by the plate colony counting method every 4 hours. Prepare MRS agar medium, autoclave it at 121°C for 15 minutes. After sterilization, wait for the medium to cool to 48°C and then pour the plates. Pour about 15 - 20 ml of MRS agar medium into each petri dish. Take samples for gradient dilution. Use a pipette to aspirate 1 ml of the bacterial suspension into a test tube containing 9 ml of sterile normal saline, and dilute the sample to 10 -1 , and repeat the operation to dilute it to 10 -7 . Use a pipette to aspirate 200 μl of 10 -5 , 10 -6 , and 10 -7 diluted bacterial suspensions onto the solidified MRS agar medium, spread them evenly with a sterile spreading rod, and incubate them upside down in a constant temperature incubator at 37°C for 16 hours and then count.

[0025] The changes in the viable cell counts of the complex fermentation bacterium agent during the fermentation process are shown in the following table: Cultivation time Total number of colonies 4 <![CDATA[4.34×10 4 > 8 <![CDATA[7.62×10 6 > 12 <![CDATA[3.77×10 8 > 16 <![CDATA[1.12×10 9 > When Lactobacillus fermentum CZ22, Lactobacillus brantae - like CZ23, and Lactobacillus parafarraginis CZ24 are co - fermented, the maximum viable cell count in the complex fermentation bacterium agent is 1.12×10 9 CFU / ml.

[0026] Example 4: Experiment on the antibacterial, bactericidal, anti - spoilage and preservation effects of the complex fermentation bacterium agent in beef, pork and fish Sample treatment: Completely immerse the raw materials (beef, pork, fish) in the preservative and seal them, and soak them for 8 - 10 hours under refrigeration conditions at 0 - 4°C.

[0027] Detection: Detect Staphylococcus aureus (GB 4789.10 - 2016), Listeria monocytogenes (GB4789.30 - 2016), total colony count (GB 4789.2 - 2022), coliforms (GB 4789.3 - 2016), Salmonella (GB4789.4 - 2024), Escherichia coli (GB 4789.38 - 2012), and molds (GB 4789.15 - 2016).

[0028] Experimental groups: Conventional preservative group (adding nisin, the mass ratio of nisin to raw materials is 1:2000), experimental group (adding the complex fermentation bacterium agent, the mass ratio of the complex fermentation bacterium agent to raw materials is 1:2000).

[0029] The conventional preservative group and the test group were respectively set with three kinds of samples: beef, pork, and fish. Five parallel tests were conducted for each sample, and Staphylococcus aureus, Listeria monocytogenes, total colony count, coliform bacteria, Salmonella, Escherichia coli, and molds were detected. The results are shown in the following table:

[0030] When the test sample was beef, the total colony count detected in the test group was 3.52×10 5 CFU / g, and the total colony count detected in the conventional preservative group was 2.6×10 4 CFU / g; the coliform bacteria detected in the test group was <10 CFU / g, and the coliform bacteria detected in the conventional preservative group was 1.21×10 2 CFU / g; the molds detected in the test group was <10 CFU / g, and the molds detected in the conventional preservative group was 2.0×10 2 CFU / g.

[0031] When the test sample was pork, the total colony count detected in the test group was 6.58×10 5 CFU / g, and the total colony count detected in the conventional preservative group was 4.04×10 2 CFU / g.

[0032] When the test sample was fish, the total colony count detected in the test group was 3.30×10 6 CFU / g, and the total colony count detected in the conventional preservative group was 5.02×10 3 CFU / g; the coliform bacteria detected in the test group was <10 CFU / g, and the coliform bacteria detected in the conventional preservative group was 13 CFU / g; the molds detected in the test group was 5 CFU / g, and the molds detected in the conventional preservative group was 35 CFU / g.

[0033] Experimental conclusion: The test groups of beef, pork, and fish added with the compound fermentation agent had significantly better effects in inhibiting coliform bacteria and molds than the conventional preservative group, proving that the compound fermentation agent can effectively inhibit a variety of harmful microorganisms in the pretreatment of beef, pork, and fish ready-to-eat dishes, with good antibacterial effects and extended food shelf life.

[0034] Example 5. Effect of adding compound fermentation agent on the shelf life of pre-packaged crucian carp Experimental conditions: From October 15, 2024 to January 17, 2025 in Weifang, Shandong, with the temperature controlled at 18℃ - 38℃.

[0035] Sample treatment: Completely immerse the crucian carp in the preservative and seal it, and soak it for 8 - 10 hours under refrigeration conditions of 0 - 4℃.

[0036] Experimental group: Conventional preservative group (adding nisin, the mass ratio of nisin to crucian carp is 1:2000), experimental group (adding compound fermentation agent, the mass ratio of compound fermentation agent to crucian carp is 1:2000).

[0037] The conventional preservative group and the experimental group were each conducted with 5 parallel tests. The appearance of pre-packaged crucian carp was detected every 10 days. Staphylococcus aureus was detected according to GB 4789.10, Salmonella was detected according to GB 4789.4, and Escherichia coli was detected according to GB 4789.3. The two samples were compared as shown in the following table.

[0038]

[0039] In summary, it shows that the experimental group adding the compound bacteria agent has significantly better effect in inhibiting Staphylococcus aureus than the conventional preservative group, and significantly extends the shelf life of pre-packaged crucian carp, up to 6 times.

[0040] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A composite fermentation agent for extending the shelf life of prepared dishes, characterized in that: It is made from Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22, Lactobacillus lentus ( Lentilactobacillus parabuchneri ) CZ23, digestive associated lactobacillus ( Companilactobacillus alimentarius ) CZ24 was prepared at a ratio of 1:1:1 of viable count and 3% inoculum size; wherein the fermented Lactobacillus ( Lactobacillus fermentum )CZ22, deposited on July 9, 2024 at the General Microbiology Center of China Microorganism Culture Collection Committee, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with the deposit number: CGMCC NO.31233; The Lactobacillus lentus ( Lentilactobacillus parabuchneri )CZ23, deposited on September 18, 2024 at the General Microbiology Center of China Microorganism Culture Collection Committee, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with the deposit number: CGMCC NO.31986; The digestive associated lactobacillus ( Companilactobacillus alimentarius ) CZ24, deposited on September 18, 2024 at the General Microbiology Center of China Microorganism Culture Collection, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and its deposit number is: CGMCC NO.31987.

2. The composite fermentation agent for extending the shelf life of prepared dishes according to claim 1, characterized in that: The composite fermentation bacteria agent has an acid-resistant pH of 3.23-3.

3.

3. A method for preparing a composite fermentation agent for extending the shelf life of pre-prepared dishes as claimed in claim 1 or 2, characterized in that: The preparation method comprises: Step a, fermentation of Lactobacillus ( Lactobacillus fermentum ) CZ22, Lactobacillus lentus ( Lentilactobacillus parabuchneri ) CZ23, digestive associated lactobacillus ( Companilactobacillus alimentarius ) CZ24 were poured into MRS liquid culture medium on a sterile operating table and activated at 37°C for 24 h. The OD600 values ​​of the three MRS liquid culture media were 2.1-2.3, and the starter seed liquid was obtained; Step b, the fermentation liquid is sterilized at 121° C. for 15 minutes, and immediately cooled to 42° C. to 43° C. after sterilization, and the starter seed liquid is inoculated into the fermentation liquid at a ratio of 1:1:1 of the number of viable bacteria, and 3% of the inoculum is inoculated into the fermentation liquid, and immediately cultured at a constant temperature of 37° C. after stirring evenly, and when the acidity reaches 1.5% to 2.0%, the fermentation is terminated to obtain the composite fermentation agent, which is refrigerated at 5° C. to 10° C. for 24 hours and then ripened to obtain the finished product.

4. The preparation method according to claim 3, characterized in that: In the step a, the MRS liquid culture medium includes: 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 20.0 g of glucose, 2.0 g of diammonium citrate, 5.0 g of sodium acetate, 2.0 g of MgSO4, 0.05 g of MnSO4, 2.0 g of K2HPO4, 1.0 mL of Tween 80; 1000 mL of distilled water; the pH is adjusted to 6.2-6.4, the culture temperature is 37° C., and the culture time is 16 hours.

5. The preparation method according to claim 3, characterized in that: In the step b, the fermentation liquid components are 30.00 g / L glucose, 30.00 g / L lactose, 30.00 g / L soy peptone, 2.29 g / L K2HPO4, 0.80 g / L MgSO4·7H2O, 50 ml / L tomato juice, 50 ml / L peanut sprout juice and 0.15 g / L histidine, the culture temperature is 37°C, and the culture time is 16 hours.

Images