Lactobacillus fermentum CZ22, microbial inoculum, microbial inoculum preparation method and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses lactobacillus fermentum CZ22, a fungicide, a fungicide preparation method and application, the lactobacillus fermentum CZ22 is preserved in China General Microbiological Culture Collection Center on July 9, 2024, and the preservation number of the lactobacillus fermentum CZ22 is CGMCC NO.31233. The lactobacillus fermentum CZ22 has a DNA sequence of SEQ ID NO.1 in a sequence table; lactobacillus fermentum CZ22 fermentation liquor has the characteristic of inhibiting growth of harmful microorganisms and can regulate and control fence factors, the pH value of a lactic acid solution is 3.28-3.32, and lactobacillus fermentum CZ22 can survive and breed in an environment with a relatively low pH value and is good in acid resistance and high in survival amount.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a Lactobacillus fermentum CZ22, a bacterial agent, a method for preparing the bacterial agent, and applications thereof. Background Art

[0002] In the processes of food processing and preservation, sterilization and bacteriostasis are key steps to ensure food safety and extend the shelf life of food. Currently, the commonly used sterilization and bacteriostasis methods in the food industry mainly include high-temperature sterilization, pasteurization, room-temperature sterilization, adding preservatives, etc. High-temperature sterilization usually refers to the sterilization treatment at a temperature of 121°C, which is sufficient to kill most bacteria, molds, and yeasts, including spores. To further enhance the preservation effect of food, a small amount of preservatives, antioxidants, and other additives are usually added during or after the sterilization process; the sterilization conditions for pasteurization are 61 - 63°C for 30 min, or 72 - 75°C for 10 - 15 min, such conditions can kill some pathogenic bacteria but cannot completely eliminate them, and preservatives (such as potassium sorbate, sodium benzoate, etc.) are usually added; room-temperature sterilization usually refers to the sterilization treatment at room temperature conditions, mainly relying on physical methods (such as ultraviolet rays, filtration) or chemical methods (such as preservatives).

[0003] However, high-temperature sterilization can lead to a decline in the taste, color, and nutritional value of food, especially for heat-sensitive vitamins and proteins. Pasteurization and room-temperature sterilization cannot kill most bacteria, molds, and yeasts, there are certain food safety risks, and a large amount of preservatives need to be used, and long-term intake may have an adverse impact on health.

[0004] Therefore, how to achieve food safety and food quality while performing sterilization and bacteriostasis is an important problem commonly faced by the food industry. At present, no perfect solution to the above problems has been found. Summary of the Invention

[0005] The problem to be solved by the present invention is to provide a Lactobacillus fermentum CZ22, a bacterial agent, a method for preparing the bacterial agent, and applications thereof, and to verify the application and effect of this strain in food production.

[0006] To achieve the above object, the present invention provides the following technical solution: A Lactobacillus fermentum CZ22, the Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22 was deposited in the China General Microbiological Culture Collection Center on July 9, 2024, and the deposit number is: CGMCC NO. 31233; The Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22 has the DNA sequence of SEQ ID NO.1 in the sequence listing.

[0007] A bacterial agent, the bacterial agent contains the Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22.

[0008] Furthermore, the viable count of Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22 contained in the bacterial agent is (1.52 ± 0.45) × 10 9 CFU / mL.

[0009] Furthermore, the acid tolerance pH is 3.28 - 3.32.

[0010] The preparation method of the above bacterial agent is as follows: The Lactobacillus fermentum CZ22 bacterial liquid is cultured in MRS medium at 37°C for 24 h, and the bacterial liquid is inoculated into the fermentation medium at an inoculation amount of 3%, and then cultured in an incubator at 37°C to obtain the Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22 bacterial agent; Fermentation medium: Glucose 15.00 g / L, lactose 15.00 g / L, soy peptone 30.00 g / L, K2HPO4 2.29 g / L, MgSO4·7H2O 0.80 g / L, addition amount of barley green juice 5% and histidine 0.15 g / L.

[0011] Furthermore, the application of the Lactobacillus fermentum CZ22 or the bacterial agent in food production.

[0012] Compared with the prior art, the beneficial effects of the present invention are: The fermentation broth of Lactobacillus fermentum CZ22 has the characteristic of inhibiting the growth of harmful microorganisms, can regulate the hurdle factors, and the pH value of the lactic acid solution is 3.28 - 3.32. Lactobacillus fermentum CZ22 can survive and reproduce in an environment with a relatively low pH value, has good acid tolerance and a high survival amount, can significantly extend the shelf life of food, and the sensory score of the food is good. Description of the drawings

[0013] Figure 1 It is the plate colony morphology diagram of Lactobacillus fermentum CZ22; Figure 2 It is the Gram staining microscope diagram of Lactobacillus fermentum CZ22; Figure 3 It is the phylogenetic tree constructed from the 16S rDNA sequence of Lactobacillus fermentum CZ22; Figure 4 It is the antibacterial circle effect diagram of Lactobacillus fermentum CZ22 on Bacillus subtilis; Figure 5 It is the antibacterial circle effect diagram of Lactobacillus fermentum CZ22 on Staphylococcus aureus; Figure 6 It is the antibacterial circle effect diagram of Lactobacillus fermentum CZ22 on Escherichia coli. Detailed Implementation Modes

[0014] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0015] Please refer to Figures 1 - 6 , the present invention provides a Lactobacillus fermentum CZ22, which is isolated from pickled vegetables. The collection time is May 2024, and the collection place is Chengdu, Sichuan Province, China. Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 9, 2024. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is: CGMCC NO. 31233.

[0016] Example 1: Isolation, purification and identification of Lactobacillus fermentum CZ22 1. Isolation and purification of strains Lactobacillus fermentum CZ22 is isolated from pickled vegetables. The isolation and purification method is as follows: Select pickled vegetables as the raw material, take a small amount of pickled vegetable juice and add it to the MRS liquid medium containing antibiotics, and culture for 24 h. After the culture is completed, add sterilized calcium carbonate to the MRS liquid medium at a ratio of 2%, and cool the medium to about 46 °C in a constant temperature water bath. Shake it while cooling to mix the calcium carbonate evenly, and pay attention not to generate bubbles. After mixing, mix the MRS liquid medium containing 2% calcium carbonate with an appropriate amount of agar, heat it until the agar is completely dissolved, and then immediately pour it into a petri dish and let it stand and solidify to obtain the MRS solid medium. Under sterile conditions, pipette 200 μL of the sample and spread it on the surface of the solidified MRS solid medium plate, and culture it in an inverted position at a constant temperature of 37 °C. Observe the colony characteristics after culturing for 24-48 h, pick the single colony with a large calcium dissolution circle, streak-culture it on the MRS solid medium to obtain a single colony strain, and streak-separate it on the plate and purify it repeatedly.

[0017] 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 the pH to 6.2-6.4, culture temperature 37 °C, culture time 16 h.

[0018] 2. Preliminary identification of colonies Hydrogen peroxide catalase test: Use an inoculation loop to pick up one loop of the colony after isolation and purification onto a clean glass slide, and add one drop of 30% hydrogen peroxide. If there are bubbles, it is catalase positive; otherwise, it is negative.

[0019] Gram staining: Pick the colony after isolation and purification for Gram staining and observe it under a microscope. The results of the hydrogen peroxide catalase test and Gram staining of the lactic acid bacteria after isolation and purification are shown in the following table: Number Catalase (±) Gram stain (G±) Microscopic examination results CZ22 - G+ Rod-shaped, occurring singly or in pairs Conclusion: As can be seen from the above table, the strain is negative for hydrogen peroxide catalase, positive for Gram staining, and rod-shaped. This strain is labeled as CZ22.

[0020] 3. Physiological and biochemical tests Select different kinds of sugars to replace glucose as the carbon source to prepare MRS liquid medium. Absorb the liquid medium into a 96-well plate and measure its OD600. Inoculate CZ22 into the MRS liquid medium with different carbon sources at an inoculation amount of 1%, and culture it at 37°C for 24 hours. Absorb the bacterial liquid into a 96-well plate and measure its OD600. Based on "Bergey's Manual of Systematic Bacteriology" and "Classification and Identification and Experimental Methods of Lactic Acid Bacteria", conduct physiological and biochemical identification of the strain. A positive reaction result is indicated by (+), and a negative result is indicated by (-), so as to make a preliminary identification of the genus and species of the target strain. The results are shown in the following table: Test items CZ22 Sorbitol - Mannitol + Arabinose - Lactose + Cellobiose - Raffinose - Trehalose + Ribose - Melezitose - Conclusion: Based on "Bergey's Manual of Systematic Bacteriology" and "Classification and Identification and Experimental Methods of Lactic Acid Bacteria", the target strain CZ22 is preliminarily identified as the genus Lactobacillus fermentum.

[0021] 4. 16S rDNA sequence analysis Use a bacterial genome extraction kit to extract the genomic DNA of the target strain. Using the genomic DNA of the target strain as a template, select 27F and 1492R as universal primers for PCR amplification of 16S rDNA. PCR reaction system (25 μL): 1 μL each of upstream and downstream primers and genomic template, 12.5 μL of Pre-mix Ex Taq, and 9.5 μL of sterilized ddH2O. PCR amplification conditions: pre-denaturation at 95°C for 4 min, denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 2 min, a total of 30 cycles, and finally extension at 72°C for 7 min to end the reaction. The PCR product is sent to BGI Tech Solutions Co., Ltd. for purification and sequencing. The obtained PCR product sequence and the 16S rDNA sequences of lactic acid bacteria with relatively close genetic relationships are used to construct a phylogenetic tree by MEGA 5.05 software. The phylogenetic tree is as shown in the appendix Figure 3 shown.

[0022] The sequencing result is: CTGACGCTGAGGCTCGAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTAACGCCCAAAGTCGGTGGCCTAACCTTTATGGAGGGAGCCGCCTAAGGCGGGACAGATGACTGGGGGAGCAAAAAAAAAAC Based on the sequencing results, the target strain CZ22 was identified as Lactobacillus fermentum ( Lactobacillus fermentum ).

[0023] Example 2. Changes in pH value during the fermentation of Lactobacillus fermentum CZ22 The bacterial solution of Lactobacillus fermentum CZ22 was cultured in MRS medium at 37 °C for 24 h, and then the bacterial solution was inoculated into the fermentation medium at an inoculation amount of 3% and cultured in an incubator at 37 °C.

[0024] MRS medium: 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g ammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 20 g calcium carbonate, 1000 mL distilled water.

[0025] Fermentation medium: 15.00 g / L glucose, 15.00 g / L lactose, 30.00 g / L soy peptone, 2.29 g / L K2HPO4, 0.80 g / L MgSO4·7H2O, 5% addition of barley seedling green juice, and 0.15 g / L histidine.

[0026] Preparation methods of MRS medium and fermentation medium: Add the above components to distilled water, heat to dissolve, and autoclave at 121 °C for 15 min.

[0027] The effects of Lactobacillus fermentum CZ22 fermentation on pH value are shown in the following table: Fermentation culture time (h) pH value 0 6.24 12 4.31 18 3.68 24 3.32 30 3.28 Conclusion: The pH value of the fermentation broth will decrease with the fermentation and growth of Lactobacillus fermentum CZ22. The pH value drops rapidly at the beginning and then slowly decreases and levels off after 18 h. It can be seen from this experiment that Lactobacillus fermentum CZ22 can survive and reproduce in an environment with a relatively low pH value, reflecting the acid tolerance of Lactobacillus fermentum CZ22. The pH values at 24 h and 30 h of fermentation are basically unchanged, and 24 h is selected as the fermentation time of Lactobacillus fermentum CZ22.

[0028] Example 3. Determination of viable cell count in the lactic acid solution added with Lactobacillus fermentum CZ22 Take three batches of the above fermentation broth and use the plate coating method to determine the viable cell count. Prepare MRS agar medium, autoclave at 121 °C for 15 min. After sterilization, wait for the medium to cool to 48 °C and then pour the plates. Pour about 15 - 20 mL / plate of MRS medium into the petri dishes. Take samples with tomato juice addition amounts of 50 mL / L and 100 mL / L respectively for gradient dilution. Use a pipette to suck 1 mL of the bacterial solution into a test tube containing 9 mL of sterile normal saline, and dilute the sample to 10 -1 , and repeat the operation to dilute to 10 -7 . Use a pipette to suck 200 μl of 10 -5 , 10 -6 , 10 -7 diluted bacterial suspensions onto the solidified MRS agar medium, spread evenly with a sterile spreading rod, and incubate in an inverted position in a constant temperature incubator at 37 °C for 24 h and then count. The results show that the viable cell count can reach (1.52 ± 0.45) × 10 9 CFU / mL.

[0029] Example 4: Bacteriostatic Characteristics of Lactobacillus fermentum The bacteriocin was determined by the Oxford cup double-layer agar diffusion method. Take the sample to be tested, centrifuge it at 4000 r / min for 3 min, and take the supernatant. Using Escherichia coli, Bacillus subtilis, and Staphylococcus aureus as indicator bacteria, take 200 μL of the supernatant and place it in the Oxford cup. The diameter of the Oxford cup is 8.0 mm, and culture it in an incubator at 37 °C for 24 - 48 h, observe the inhibition zone, measure and record the diameter of the inhibition zone. The results are shown in the following table and appendix Figures 4 - 6 as follows: Indicator bacteria Inhibition zone diameter (mm) Escherichia coli 10.8±0.05 Bacillus subtilis 11.10±0.10 Staphylococcus aureus 11.6±0.10 Conclusion: Lactobacillus fermentum CZ22 has an inhibitory effect on the growth of Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. Among them, the inhibitory effect on Staphylococcus aureus is the best, and the diameter of the inhibition zone can reach 11.6 mm; the inhibitory effect on Bacillus subtilis is the second best, and the average diameter of the inhibition zone is 11.1 mm; when Escherichia coli is used as the indicator bacteria, the average diameter of the inhibition zone is 10.8 mm. Lactobacillus fermentum CZ22 has different degrees of inhibitory effects on common pathogenic bacteria.

[0030] Example 5: Effects of Adding Different Amounts of Lactobacillus fermentum CZ22 on the Quality of Soft Pre-packaged Braised Beef Inoculate the activated Lactobacillus fermentum CZ22 at an inoculation amount of 3% by mass ratio, and ferment it at 37 °C for 24 h to obtain the fermentation broth. The sensory quality comprehensive evaluation was used to test the effects of the fermentation broth with different amounts of Lactobacillus fermentum CZ22 added on the quality of soft pre-packaged braised beef. The groups were divided into: blank group (without adding any preservatives or preservatives), test group 1 (the fermentation broth and the sample were added in a ratio of 1:100), test group 2 (the fermentation broth and the sample were added in a ratio of 2:100), test group 3 (the fermentation broth and the sample were added in a ratio of 4:100), test group 4 (the fermentation broth and the sample were added in a ratio of 8:100), and the above ratios were all mass ratios. Among them, the sensory quality comprehensive evaluation included 4 evaluation indicators: taste, flavor, aroma, and color. Each indicator was evaluated on a scale of "1 - 10", and the higher the score, the better the quality. The specific evaluation results are shown in the following table.

[0031] Group Taste (score) Flavor (score) Aroma (score) Color (score) Average score (score) Blank group 9 9 9 9 9.00±0.00 Test group 1 8 8 8 9 8.00±0.00 Test group 2 8 7 7 8 7.50±0.58 Test group 3 8 7 7 7 7.25±0.50 Test group 4 4 6 5 4 4.75±0.96 Conclusion: As can be seen from the above table, with the increase in the addition amount of the fermentation broth of Lactobacillus fermentum CZ22, the sensory quality of the soft pre-packaged braised beef decreases. In order to ensure the quality of the soft pre-packaged braised beef to the greatest extent, the addition ratio of its fermentation broth and the sample does not exceed 4:100.

[0032] Example 6: Effects of Adding the Fermentation Broth of Lactobacillus fermentum CZ22 on the Shelf Life of Soft Pre-packaged Braised Beef Inoculate the activated fermented lactic acid bacteria CZ22 at an inoculation amount of 3% by mass ratio, and ferment at 37 °C for 24 h to obtain a fermentation broth. Test the antibacterial, bactericidal, anti-corrosion and freshness preservation effects of the soft fermentation broth of fermented lactic acid bacteria CZ22 added to soft pre-packaged marinated beef.

[0033] Experimental conditions: From August 15, 2024 to October 17, 2024 in Weifang, Shandong, at a normal temperature of 18 °C - 38 °C throughout the day.

[0034] Experimental groups: Conventional preservative group (adding nisin, with an addition amount of 0.5 g / kg), Experimental group 1 (adding the fermentation broth and the sample in a ratio of 4:100), Experimental group 2 (adding the fermentation supernatant and the sample in a ratio of 4:100).

[0035] The conventional preservative group, Experimental group 1 and Experimental group 2 were each subjected to 5 parallel tests. The appearance of the soft pre-packaged marinated beef was detected every 7 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 three samples were compared, as shown in the following table.

[0036] Group Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Conventional preservative group Normal Only Staphylococcus aureus detected Only Staphylococcus aureus detected, with browning and swelling Only Staphylococcus aureus detected, with browning and juice leakage Only Staphylococcus aureus and Escherichia coli detected, with browning and juice leakage Only Staphylococcus aureus and Escherichia coli detected, with browning and black water leakage Test group 1 Normal Normal Normal Normal Normal Staphylococcus aureus detected Test group 2 Normal Normal Normal Normal Staphylococcus aureus detected Staphylococcus aureus detected, with browning and swelling

[0037] In summary, the effects of Experimental group 1 and Experimental group 2 in inhibiting Staphylococcus aureus and Escherichia coli were significantly better than those of the conventional preservative group. The antibacterial effect of the fermentation broth of fermented lactic acid bacteria CZ22 was better than that of the fermentation supernatant, and the fermentation broth of fermented lactic acid bacteria CZ22 significantly extended the shelf life of the soft pre-packaged marinated beef, up to 5 times.

[0038] The above description is only a preferred embodiment of the present invention, and it is not intended to limit the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification made to the above examples based on the technical essence of the present invention without departing from the technical solution content of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims

1. A Lactobacillus fermentum CZ22, characterized in that: The Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22 was deposited at the General Microbiological Center of the China National Culture Collection Center on July 9, 2024, with the deposit number: CGMCC NO. 31233; The Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22 has the DNA sequence of Sequence Listing SEQ ID NO.

1.

2. A bacterial agent, characterized in that: The bacterial agent contains Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22.

3. The microbial agent according to claim 2, characterized in that: The bacterial agent contains Lactobacillus fermentum ( Lactobacillus fermentum ) and the viable count of CZ22 is (1.52 ± 0.45) × 10 9 CFU / mL.

4. The microbial agent according to claim 2, wherein: The acid tolerance pH is 3.28 - 3.

32.

5. The microbial agent according to claim 2, characterized in that: The bacterial agent is prepared according to the following method: The Lactobacillus fermentum CZ22 bacterial liquid was cultured in MRS medium at 37 °C for 24 h. The bacterial liquid was inoculated into the fermentation medium at an inoculation amount of 3% and placed in an incubator at 37 °C for cultivation to obtain the Lactobacillus fermentum ( Lactobacillus fermentum ) CZ22 bacterial agent; Fermentation medium: glucose 15.00 g / L, lactose 15.00 g / L, soy peptone 30.00 g / L, K2HPO4 2.29 g / L, MgSO4·7H2O 0.80 g / L, addition amount of barley green juice 5% and histidine 0.15 g / L.

6. Use of the Lactobacillus fermentum CZ22 according to claim 1 or the bacterial agent according to claim 2 in food production.

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