Composite fermentation agent for extending shelf life of prepared dishes and preparation method thereof

Abstract

The invention discloses a composite fermentation agent for extending the shelf life of pre-prepared dishes and a preparation method thereof, relating to the technical field of fermentation. The composite fermentation agent is prepared by using Lactobacillus fermentum CZ22, Lactobacillus lentus-like buchneri CZ23, and Lactobacillus associated with digestion CZ24 in a ratio of 1:1:1 in terms of viable bacteria count and an inoculation amount of 3%. The preservation number of Lactobacillus fermentum CZ22 is CGMCC No. 31233; the preservation number of Lactobacillus lentus-like buchneri CZ23 is CGMCC No. 31986; and the preservation number of Lactobacillus associated with digestion CZ24 is CGMCC No. 31987. The composite fermentation agent adopts Lactobacillus fermentum CZ22, Lactobacillus lentus-like buchneri CZ23, and Lactobacillus associated with digestion CZ24, can effectively inhibit multiple harmful microorganisms in the pre-treatment of cooked pre-prepared dishes, has good antibacterial effect, and extends the shelf life of food.

Description

Technical Field

[0001] The present invention relates to the technical field of fermentation, and in particular to a composite fermentation agent for extending the shelf life of prepared dishes and a preparation method thereof. Background Art

[0002] Lactic acid bacteria are non-spore-forming, Gram-positive bacteria that ferment carbohydrates to produce lactic acid. They are recognized as safe probiotics for consumption, with numerous benefits, including lowering intestinal pH, regulating intestinal flora, inhibiting the growth of harmful bacteria, and preventing and treating lactose intolerance. They are also important strains for making common fermented foods like yogurt and kimchi. Like most bacteria, lactic acid bacteria can form biofilms in certain environments, enabling them to inhibit a variety of pathogens that antagonize them.

[0003] In the food industry, lactic acid bacteria fermentation agents are primarily used as starter cultures in dairy products, baked goods, and traditional fermented foods, promoting the fermentation process and improving flavor and texture. They can also replace preservatives in raw food products, inhibiting microbial growth and extending shelf life. However, currently, there are relatively few similar lactic acid bacteria fermentation agent products, and their effectiveness in inhibiting microbial growth and extending shelf life is unsatisfactory. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the above shortcomings and provide a composite fermentation agent for extending the shelf life of pre-prepared dishes and a preparation method thereof, thereby overcoming the unsatisfactory defects of existing fermentation lactic acid bacteria agents in inhibiting the growth of microorganisms and extending the shelf life of food; and achieving the purpose of being able to be applied in the pre-treatment of cooked pre-prepared dishes and having a good antibacterial effect.

[0005] In order to solve the above technical problems, the technical solution of the present invention is: a composite fermentation agent for extending the shelf life of pre-prepared dishes, which is composed of Lactobacillus fermentum CZ22, Lentilactobacillus parabuchner i ) CZ23, digestive associated lactobacillus ( Companilactobacillus alimentarius ) CZ24 was prepared at a ratio of 1:1:1 of viable bacteria count and a 3% inoculum size;

[0006] wherein the fermented Lactobacillus ( Lactobacillus fermentum ) CZ22, deposited on July 9, 2024, at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with the deposit number: CGMCC NO.31233;

[0007] The Lactobacillus lentus-like buchneri ( Lentilactobacillus parabuchneri) CZ23, deposited on September 18, 2024 at the General Microbiology Center of China Culture Collection, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with the deposit number: CGMCC NO.31986;

[0008] The digestive associated lactobacillus ( Companilactobacillus alimentarius ) CZ24, deposited on September 18, 2024 at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and its deposit number is: CGMCC NO.31987.

[0009] Furthermore, the composite fermentation bacteria agent has an acid-resistant pH of 3.23-3.3.

[0010] Furthermore, the preparation method of the composite fermentation agent for extending the shelf life of pre-prepared dishes comprises:

[0011] Step a, fermentation of Lactobacillus ( Lactobacillus fermentum ) CZ22, Lactobacillus lentus ( Lentilactobacillus parabuchneri ) CZ23, digestive associated lactobacillus ( Companilactobacillus alimentarius ) CZ24 was 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.

[0012] 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 viable bacteria count, and 3% of the inoculum amount is inoculated into the fermentation liquid. After stirring evenly, the fermentation is immediately cultured at a constant temperature of 37° C. 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 matured to obtain the finished product.

[0013] Furthermore, in step a, MRS liquid culture 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, MgSO4 2.0 g, MnSO4 0.05 g, K2HPO4 2.0 g, Tween 80 1.0 mL; distilled water 1000 mL; adjust pH 6.2-6.4, culture temperature 37 ° C, culture time 16 h.

[0014] Furthermore, in step b, the fermentation broth 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 h.

[0015] The present invention adopts the above technical scheme and has the following advantages compared with the existing technology: the composite fermentation bacteria agent of the present invention adopts fermentation Lactobacillus CZ22, Brucella-like Lactobacillus CZ23, and digestive-associated Lactobacillus CZ24, which can effectively inhibit a variety of harmful microorganisms in the pre-treatment of cooked pre-cooked dishes, has a good antibacterial effect, and extends the shelf life of food. DETAILED DESCRIPTION

[0016] The embodiments of the present invention are described in detail below. The examples of the embodiments are intended to be used to explain the present invention and are not to be construed as limiting the present invention. Specific techniques or conditions are not indicated in the embodiments, but are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used are not indicated by manufacturer and are all conventional products that can be obtained commercially.

[0017] Example 1: A method for preparing a composite fermentation agent for extending the shelf life of prepared dishes

[0018] The bacteria used include Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22, Lactobacillus lentus ( Lentilactobacillus parabuchneri ) CZ23, digestive associated lactobacillus ( Companilactobacillus alimentarius )CZ24.

[0019] Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22, isolated from kimchi mother water, collected on May 7, 2024, in Chengdu, Sichuan Province, China, Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22 was deposited on July 9, 2024 at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and its deposit number is: CGMCC NO.31233. Lactobacillus fermentum CZ22 has the DNA sequence of SEQ ID NO.1 in the sequence list.

[0020] Lactobacillus lentus ( Lentilactobacillus parabuchneri ) CZ23, isolated from kimchi mother water, collected on May 28, 2024, in Zunyi City, Guizhou Province, China, Lactobacillus tarda ( Lentilactobacillus parabuchneri) CZ23 was deposited on September 18, 2024 at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and its deposit number is: CGMCCNO.31986. The bacterium CZ23 has the DNA sequence of SEQ ID NO.2 in the sequence list.

[0021] Digestive Lactobacillus ( Companilactobacillus alimentarius ) CZ24, isolated from kimchi mother water, collected on May 16, 2024, in Chongqing, China, and digestive associated lactobacillus ( Companilactobacillus alimentarius ) CZ24 was deposited on September 18, 2024 at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and its deposit number is: CGMCC NO.31987. The digestive-associated lactobacillus CZ24 has the DNA sequence of SEQ ID NO.3 in the sequence list.

[0022] The culture medium used was MRS liquid medium: 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 was adjusted to 6.2-6.4, the culture temperature was 37°C, and the culture time was 16 h.

[0023] A method for preparing a composite fermentation agent for extending the shelf life of prepared dishes comprises the following steps:

[0024] (1) Lactobacillus fermentum ( 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 cultured 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.

[0025] 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, MgSO4 2.0 g, MnSO4 0.05 g, K2HPO4 2.0 g, Tween 80 1.0 mL; distilled water 1000 mL; adjust pH 6.2-6.4, culture temperature 37°C, culture time 16 h.

[0026] (2) Sterilize the MRS liquid culture medium at 121°C for 15 minutes, then immediately cool it to 42°C to 43°C. Inoculate the starter seed liquid with a 3% inoculum volume in a ratio of 1:1:1 viable bacteria count into the fermentation liquid. Stir evenly and then incubate at 37°C. When the acidity reaches 1.5% to 2.0%, the fermentation is complete, and the composite fermentation agent is obtained. Refrigerate at 5°C to 10°C for 24 hours to mature and obtain the finished product.

[0027] Fermentation broth ingredients: glucose 30.00 g / L, lactose 30.00 g / L, soy peptone 30.00 g / L, K2HPO4 2.29 g / L, MgSO4·7H2O 0.80 g / L, tomato juice 50 ml / L, peanut sprout juice 50 ml / L and histidine 0.15 g / L, culture temperature 37℃, culture time 16 h.

[0028] Example 2: Determination of pH value of composite fermentation agent during fermentation

[0029] After Lactobacillus fermentum CZ22, Lactobacillus lentus-like CZ23, and Lactobacillus pepticus-associated CZ24 were activated for two generations in MRS liquid culture medium, 5% inoculum size was inoculated into MRS liquid culture medium at a ratio of 1:1:1 of viable bacteria, and cultured at a constant temperature of 37°C. The pH value of the mixed culture solution was measured every 2 hours. The pH changes of the composite fermentation agent are shown in the following table.

[0030] 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

[0031] This experiment shows that Lactobacillus fermentum CZ22, Lactobacillus lentus-like Bruckeri CZ23, and Lactobacillus associated with digestion CZ24 can survive and reproduce in an environment with a relatively low pH value, reflecting the acid resistance of Lactobacillus fermentum CZ22, Lactobacillus lentus-like Bruckeri CZ23, and Lactobacillus associated with digestion CZ24. After 16 hours of culture, the pH value no longer drops significantly and basically reaches stability. The fermentation broth is fermented at 37°C for 16 hours.

[0032] Example 3: Determination of viable bacterial count of composite fermentation agent

[0033] After activating Lactobacillus fermentum CZ22, Lactobacillus lentus CZ23, and Lactobacillus associating with digestion CZ24 for 2 generations in MRS liquid culture medium, 5% of the inoculum was inoculated into the fermentation liquid at a ratio of 1:1:1 in terms of the number of viable bacteria. The culture was kept at a constant temperature of 37°C, and the number of viable bacteria was determined every 4 hours by plate colony counting method. MRS agar culture medium was prepared and sterilized at 121°C for 15 minutes. After sterilization, the culture medium was cooled to 48°C before pouring the plate. About 15-20 ml of MRS agar culture medium was poured into the plate. The sample was taken for gradient dilution. 1 ml of bacterial solution was pipetted into a test tube containing 9 ml of sterile saline solution with a pipette. The sample was gradient diluted to 10 -1 Repeat the gradient dilution to 10 -7 . Use a pipette to draw 200μl 10 -5 、 10 -6 , 10 -7 Dilute the bacterial suspension onto the solidified MRS agar medium, spread it evenly with a sterile spreader stick, and incubate it upside down in a constant temperature incubator at 37°C for 16 hours before counting.

[0034] The changes in the number of viable bacteria in the composite fermentation agent during the fermentation process are shown in the following table:

[0035] 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 ]]>

[0036] The maximum number of viable bacteria in the composite fermentation agent was 1.12×10 9 CFU / ml.

[0037] Example 4: Experimental study on the antibacterial, sterilizing, antiseptic and fresh-keeping effects of the composite fermentation agent on beef, pork and fish

[0038] Sample processing: Soak the raw materials (beef, pork, fish) completely in preservatives and seal them, then soak them at 0-4℃ for 8-10 hours.

[0039] Testing: Testing for Staphylococcus aureus (GB 4789.10-2016), Listeria monocytogenes (GB4789.30-2016), total colony count (GB 4789.2-2022), coliform bacteria (GB 4789.3-2016), Salmonella (GB4789.4-2024), Escherichia coli (GB 4789.38-2012), and mold (GB 4789.15-2016).

[0040] Experimental groups: conventional preservative group (nisin was added, the mass ratio of nisin to raw materials was 1:2000) and experimental group (composite fermentation bacteria was added, the mass ratio of composite fermentation bacteria to raw materials was 1:2000).

[0041] The conventional preservative group and the experimental group were set up with three kinds of samples, namely beef, pork and fish. Each sample was tested in parallel 5 times. Staphylococcus aureus, Listeria monocytogenes, total colony count, coliform group, Salmonella, Escherichia coli and mold were tested, as shown in the following table:

[0042]

[0043] When the test sample was beef, the total number of colonies detected in the test group was 3.52×10 5 CFU / g, and the total number of colonies detected in the conventional preservative group was 2.6×10 4 CFU / g; the coliform bacteria detected in the experimental group was <10 CFU / g, and the coliform bacteria detected in the conventional preservative group was 1.21×10 2 CFU / g; the mold detected in the experimental group was <10 CFU / g, and the mold detected in the conventional preservative group was 2.0×10 2 CFU / g.

[0044] When the sample was pork, the total number of colonies detected in the test group was 6.58×10 5 CFU / g, and the total number of colonies detected in the conventional preservative group was 4.04×10 2 CFU / g.

[0045] When the sample was fish, the total number of colonies detected in the test group was 3.30×10 6 CFU / g, and the total number of colonies detected in the conventional preservative group was 5.02×10 3 CFU / g; the coliform bacteria detected in the experimental group was <10CFU / g, and the coliform bacteria detected in the conventional preservative group was 13CFU / g; the mold was detected in the experimental group at 5CFU / g, and the mold was detected in the conventional preservative group at 35CFU / g.

[0046] Experimental conclusion: The beef, pork and fish test groups with added composite fermentation bacteria were significantly better than those with conventional preservatives in inhibiting coliform bacteria and mold, proving that composite fermentation bacteria can effectively inhibit a variety of harmful microorganisms in the pretreatment of beef, pork and fish pre-cooked dishes, have good antibacterial effects, and extend the shelf life of food.

[0047] Example 5: Effect of adding a composite fermentation agent on the shelf life of prepackaged crucian carp

[0048] Experimental conditions: Weifang, Shandong, October 15, 2024 - January 17, 2025, temperature controlled at 18°C-38°C.

[0049] Sample processing: completely immerse the crucian carp in preservatives and seal them, then refrigerate them at 0-4℃ for 8-10 hours.

[0050] Experimental groups: conventional preservative group (added nisin, the mass ratio of nisin to crucian carp was 1:2000) and experimental group (added composite fermentation agent, the mass ratio of composite fermentation agent to crucian carp was 1:2000).

[0051] Five parallel tests were conducted on the conventional preservative group and the experimental group. The appearance of the pre-packaged crucian carp was tested every 10 days. Staphylococcus aureus was tested according to GB 4789.10, Salmonella was tested according to GB 4789.4, and Escherichia coli was tested according to GB 4789.3. The two samples were compared, as shown in the table below.

[0052]

[0053] In summary, the experimental group with the addition of the composite bacterial agent was significantly better than the conventional preservative group in inhibiting Staphylococcus aureus, and significantly extended the shelf life of pre-packaged crucian carp by up to 6 times.

[0054] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A composite fermentation agent for extending the shelf life of prepared dishes, characterized in that: It is composed of 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 bacteria count and a 3% inoculum size; wherein the fermented Lactobacillus ( Lactobacillus fermentum ) CZ22, deposited on July 9, 2024, at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with the deposit number: CGMCC NO.31233; The Lactobacillus lentus-like buchneri ( Lentilactobacillus parabuchneri ) CZ23, deposited on September 18, 2024 at the General Microbiology Center of China Culture Collection, 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 Culture Collection Administration, 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 was 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 viable bacteria count, and 3% of the inoculum amount is inoculated into the fermentation liquid. After stirring evenly, the fermentation is immediately cultured at a constant temperature of 37° C. 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 matured to obtain the finished product.

4. The preparation method according to claim 3, wherein In 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, and 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 h.

5. The preparation method according to claim 3, wherein In the step b, the fermentation liquid composition is 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.

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