Lactococcus lactis subsp. Lactis strain and preparation and application of fermentation product of lactococcus lactis subsp. Lactis strain

Abstract

The invention relates to a Lactococcus lactis subsp. Lactis strain (Lactococcus lactis subsp. Lactis) CGMCC (China General Microbiological Culture Collection Center) No.28205 as well as a preparation method and an application of a fermented product of the Lactococcus lactis subsp. Lactis strain, and belongs to the technical field of industrial microorganisms. The lactococcus lactis subsp. Lactis strain CGMCC 28205 can utilize sucrose, peptone, yeast extract or natural fruit and vegetable juice as raw materials, and sucrose fermentation mash or fermented fruit and vegetable juice can be prepared through fermentation; performing ceramic membrane filtration, centrifugal separation, spray drying and the like on the sucrose fermentation mash to prepare a solid fermentation product; the solid fermentation product is used for agaricus bisporus coating preservation, and the shelf life of agaricus bisporus can be prolonged. The preparation method provided by the invention is simple and convenient for industrial production, a specific strain is selected, and the produced fermentation product is unique in flavor, has preservative and fresh-keeping activity, and can be used for seasoning and fresh-keeping of foods such as fruits and vegetables.

Description

Technical Field

[0001] The invention belongs to the technical field of industrial microorganisms and relates to a Lactococcus lactis subsp. lactis strain and the preparation and application of a fermentation product thereof. Background Art

[0002] Lactococcus lactis subsp. lactis is a common probiotic, which is widely used in food industry, agriculture, feed industry, etc. In the food industry, Lactococcus lactis subsp. lactis can be used as a fermentation agent to make yogurt, sour cheese, fermented vegetables and other dairy products and pickled products, and can also be used to ferment sucrose fermentation products with antibacterial and fresh-keeping functions.

[0003] Sucrose fermentation is a seasoning with antibacterial and preservative activity. It is a powdered fermentation product made from white sugar and yeast products through fermentation by Lactococcus lactis. Clean label fermentation products represented by whey fermentation and sucrose fermentation have good antibacterial activity and belong to clean label products, which reduces consumers' safety concerns about products with added preservatives and has broad application prospects in food production and processing.

[0004] Edible fungi are rich in nutrients and high in water content, and their quality is very likely to deteriorate after harvesting. Therefore, it is crucial for edible fungi production and management companies to use appropriate post-harvest preservation technology to extend the shelf life and reduce losses and increase efficiency. The biological coating preservation method is often based on macromolecular substances such as polysaccharides and proteins, and is made into a composite biological coating agent with antibacterial and film-forming materials. It can inhibit the respiration of fresh fruits and vegetables such as edible fungi and tomatoes, prevent the growth of spoilage bacteria, and thus delay deterioration.

[0005] Through searching, the following public documents related to the patent application of the present invention are found:

[0006] 1. A sucrose fermentation product and a preparation method thereof (CN 116725173 A), discloses a method for producing solid seasoning by mixed culture and fermentation of Lactococcus lactis subsp. lactis (CGMCC 1.1936), Lactococcus lactis subsp. Cremoris (CGMCC 1.3920), Lactobacillus plantarum and Weizmannella coagulans. The fermentation cycle of the method is 120 hours, the fermentation time is relatively long, the plate and frame filter press method is adopted in the bacterial liquid separation process, and a single maltodextrin auxiliary material is added in the spray drying process, but no in-depth study is conducted on the activity of the strain, the product yield and the application effect.

[0007] 2. A method for preparing a solid seasoning with antibacterial and fresh-keeping functions (CN 117481326A), which discloses a method for producing solid seasoning by fermenting Lactococcus lactis subsp. lactis (CICC 6242), wherein the auxiliary materials in the spray drying process are selected from one or more of sodium chloride, maltodextrin, starch, glucose, etc., but no in-depth research is conducted on the yield of active products, product application methods and effects, etc.

[0008] Based on the above situation, the present invention provides a new strain of Lactococcus lactis subsp. lactis CGMCC 28205 with unique flavor. After the strain is fermented with sucrose or fruit and vegetable juice, the fermented mash has antibacterial activity. Ceramic membrane filtration and centrifugation are adopted in the further separation process of the fermented mash, and two protective agents are added during the spray drying process to improve the yield of active ingredients in the fermented powder. The prepared fermented powder is used in the film coating preservation of Agaricus bisporus, which effectively prolongs the shelf life of Agaricus bisporus. By comparison, the patent application of the present invention is essentially different from the above patent disclosure. Summary of the invention

[0009] To solve the above technical problems, the object of the present invention is to provide a strain of Lactococcus lactis, which is a mutant strain obtained by ARTP mutagenesis screening, and its classification is named Lactococcus lactis subsp. lactis 1109, and its preservation number is CGMCC No.28205. It was deposited in the General Microbiology Center of China National Committee for the Preservation of Microorganisms on August 21, 2023, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the 16SrRNA gene sequence of the strain is shown in SEQ ID No.1.

[0010] SEQ ID No.1:

[0011]

[0012]

[0013] A preparation and application of a lactococcus lactis strain and its fermented product, comprising the following steps: (1) fermentation, using sucrose, peptone, yeast extract or natural fruit and vegetable juice as raw materials, and fermenting with Lactococcus lactis CGMCC 28205 to obtain sucrose fermented mash or fermented fruit and vegetable juice; (2) bacterial liquid separation, separating the bacterial bodies and insoluble matter in the sucrose fermented mash to obtain a clear and transparent separated liquid, wherein the bacterial liquid separation method is ceramic membrane filtration or centrifugal separation; (3) concentration, using rotary evaporation to obtain a concentrated liquid; (4) adding auxiliary materials, adding auxiliary materials to the concentrated liquid and stirring evenly; (5) drying, spray drying the mixed material to obtain a sucrose fermented product powder with antibacterial and fresh-keeping functions. (6) Application of the fermented fruit and vegetable juice and the sucrose fermented product powder.

[0014] In the above-mentioned method for preparing a fermentation product of a Lactococcus lactis subsp. lactis strain and its application, the bacterial liquid is separated in step (2) by ceramic membrane filtration, and the pore size of the ceramic membrane is 100-200 nm.

[0015] In the above-mentioned method for preparing a fermentation product of a Lactococcus lactis subsp. lactis strain and its application, the bacterial liquid is separated by centrifugation in the step (2) with the parameters of 7500 rpm and 15 min.

[0016] In the above-mentioned method for preparing a fermentation product of a Lactococcus lactis subsp. lactis strain and its application, the auxiliary materials in step (4) are selected from one or more of maltodextrin, water-soluble starch, trehalose, calcium carbonate, sodium chloride, xanthan gum, sodium glutamate, skimmed milk powder, pectin, etc.

[0017] In the above-mentioned method for preparing a solid seasoning with antibacterial and fresh-keeping functions, the auxiliary materials in step (4) are preferably maltodextrin and trehalose, with a ratio of 2:1-3:2 and an addition amount of 12.5-17.5%.

[0018] In the above-mentioned method for preparing a fermentation product of a Lactococcus lactis subsp. lactis strain and its application, the process parameters of spray drying in step (5) are: the inlet air temperature is set to 130-170° C., the outlet air temperature is set to 70-90° C., and the peristaltic pump efficiency is 15-25%.

[0019] The method for preparing a fermentation product of the Lactococcus lactis subsp. lactis strain and its application are characterized in that the step (6) is to prepare a biological coating agent by combining 0.05-0.25% of the fermentation product solids, 0.5-2.5% of pullulan and 1%-5% of calcium ascorbate and apply it on the surface of Agaricus bisporus, thereby extending the shelf life of Agaricus bisporus.

[0020] The beneficial effects of the present invention over the existing technology are as follows:

[0021] 1. The present invention provides a method for preparing a fermentation product of a Lactococcus lactis subsp. lactis strain and its application. Sucrose, peptone, yeast extract or fruit and vegetable juice can be used as raw materials to obtain sucrose fermentation liquid or fermented fruit and vegetable juice through fermentation by Lactococcus lactis CGMCC 28205; the sucrose fermentation liquid can also be further made into a solid fermentation product, which can be used for coating and preserving Agaricus bisporus, inhibiting the growth of pathogenic bacteria and microorganisms to a certain extent, and extending the shelf life of food.

[0022] 2. The present invention improves the spray drying process, and the composite ratio of the protective agent can significantly increase the total yield of the active ingredient. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Pear juice fermented by Lactococcus lactis CGMCC 28205.

[0024] Figure 2 The inhibitory effect of the supernatant of pear juice fermented by Lactococcus lactis CGMCC 28205 on Micrococcus luteus.

[0025] Figure 3 The effects of different spray drying conditions on the yield of active ingredients in sucrose fermentation products, including A: the effect of inlet air temperature on the yield; B: the effect of blowing efficiency on the yield; C: the effect of peristaltic speed on the yield.

[0026] Figure 4 It is a solid powder of sucrose fermentation product of Lactococcus lactis CGMCC 28205.

[0027] Figure 5 This is the inhibitory effect of the solid powder of sucrose fermentation product of Lactococcus lactis CGMCC 28205 on Micrococcus luteus.

[0028] Figure 6 The effect of solid powder of sucrose fermentation product of Lactococcus lactis CGMCC 28205 on the apparent quality of Agaricus bisporus during storage.

[0029] Figure 7 The effects of sucrose fermentation product composite coating on physiological and biochemical indicators of Agaricus bisporus during storage, including A: Effect of different coating treatments on PPO activity of Agaricus bisporus during storage; B: Effect of different coating treatments on POD activity of Agaricus bisporus during storage; C: Effect of different coating treatments on MDA of Agaricus bisporus during storage; D: Effect of different coating treatments on soluble protein of Agaricus bisporus during storage. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0031] The culture medium involved in the embodiments of the present invention has the following specific components:

[0032] Lactococcus lactis subsp. lactis CGMCC 28205 seed liquid culture medium and sucrose fermentation liquid culture medium: sucrose 20%, potassium dihydrogen phosphate 10%, peptone 10%, yeast extract 10%, sodium chloride 2%, water, the culture medium sterilization conditions are 115°C, 30min.

[0033] Solid culture medium for sucrose fermentation of Lactococcus lactis subsp. lactis CGMCC 28205: sucrose 20%, potassium dihydrogen phosphate 10%, peptone 10%, yeast extract 10%, sodium chloride 2%, agar powder 2.5%. The culture medium was sterilized at 115°C for 30 min.

[0034] Micrococcus luteus S1 medium: 8% tryptone, 5% glucose, 2% disodium hydrogen phosphate, 5% yeast extract, 5% sodium chloride, water. The medium is sterilized at 115°C for 30 minutes. 2.5% agar is added to the solid medium.

[0035] Example 1 Preparation of sucrose fermentation mash

[0036] The Lactococcus lactis subsp. lactis CGMCC 28205 strain was streaked on a sucrose fermentation solid culture medium plate and cultured at 30°C for 36 hours. After observing the colony morphology on the plate and confirming that it was correct under a microscope, single colonies on the plate were picked up and inoculated into a seed liquid culture medium of Lactococcus lactis subsp. lactis CGMCC 28205 and cultured at 30°C for 10 hours to obtain a seed solution. The seed solution was then inoculated into 500 mL of a sucrose fermentation liquid culture medium at an inoculum size of 3%, and the culture was carried out at 30°C for 8 hours to obtain a sucrose fermentation mash.

[0037] Example 2 Comparison of flavor and antibacterial properties of different fermentation strains:

[0038] 1. Strain Types The inventors purchased and isolated different strains of Lactococcus lactis subsp. lactis. The specific strains are shown in Table 1:

[0039] Table 1 Lactococcus lactis strains

[0040] CGMCC Fungus name Latin name 1.2030 Lactococcus lactis subsp. lactis Lactococcuslactissubsp.lactis 1.2281 Lactococcus lactis subsp. lactis Lactococcuslactissubsp.lactis 1.2829 Lactococcus lactis subsp. lactis Lactococcuslactissubsp.lactis 28205 Lactococcus lactis subsp. lactis Lactococcuslactissubsp.lactis

[0041] 2. Fermentation screening of Lactococcus lactis strains

[0042] The purchased Lactococcus lactis 1.2030, 1.2281, 1.2829 and the 28205 strain screened in this laboratory were fermented according to the fermentation method of Example 1 to obtain different Lactococcus lactis sucrose fermentation mashes, and the sucrose fermentation supernatant was obtained by centrifugation at 8000 rpm for 5 min.

[0043] In order to find a suitable strain, the inventors used sensory evaluation and antibacterial effect and two methods to conduct the selection. The antibacterial effect was evaluated based on the size of the antibacterial circle of solid Micrococcus luteus by the sucrose fermentation supernatant. The evaluation team was composed of 10 experimenters. The evaluation was conducted by random experiments. Each evaluation was performed with a random number corresponding to the fermentation sample of the strain. The evaluation score was based on a 100-point system. Each R&D staff member scored individually, and then the average score of all people was used as the evaluation result. Since the products of Lactococcus lactis are mainly flavor substances and antibacterial peptides, the selected scoring items and scoring criteria are shown in Table 2:

[0044] Table 2 Evaluation criteria for Lactococcus lactis strains

[0045]

[0046] The sucrose fermentation supernatants of different strains were used as evaluation samples, and the evaluation criteria in Table 2 were used to score and evaluate each strain. The following results were obtained (for the convenience of calculation, each person's score is an integer):

[0047] Table 3 Evaluation results of various strains of Lactococcus lactis

[0048]

[0049]

[0050] According to the comprehensive scoring effect of the strains, the total score of Lactococcus lactis 28205 was higher than that of other strains, so it was selected as the subsequent fermentation strain.

[0051] Example 3 Preparation and antibacterial effect of fermented fruit and vegetable juice

[0052] 1. Select crown pears with intact skin and ripeness, and wash the crown pears. After washing, take out the core, put the processed crown pears into a juicer, and filter with filter cloth to obtain crown pear juice;

[0053] 2. Prepare the fermentation medium of crown pear juice: mix crown pear juice and distilled water in a ratio of 5:3 (mass ratio) and sterilize at 100°C for 20 min.

[0054] 3. The fermented Lactococcus lactis 28205 strain seed solution in Example 1 was inoculated into the crown pear juice culture medium at a rate of 5%, and the fermented pear juice was obtained after static culture at 30° C. for 12 h. The fermented pear juice was centrifuged at 8000 rpm for 5 min to obtain the fermented pear juice supernatant.

[0055] 4. According to the above embodiment, the sensory evaluation of the fermented pear supernatant juice was conducted. The sensory evaluation team consisted of 10 experimenters, and the color, aroma and taste of the fermented pear juice were comprehensively scored. The evaluation score was based on a 60-point system. Each R&D staff member scored individually, and the average score was selected as the final sensory score.

[0056] Table 4 Sensory evaluation scoring criteria

[0057]

[0058] 5. Take 100 μl of the fermented Crown Pear juice supernatant and add it to the prepared S1 solid plate containing Micrococcus luteus for 36 hours to determine its antibacterial effect. The results are shown in Figure 2 ,The results showed that the fermented pear juice supernatant had a good antibacterial activity against Micrococcus luteus.

[0059] 6. According to the comprehensive evaluation of the experimenters, the color of the pear juice fermented by Lactococcus lactis subspecies CGMCC 28205 is light yellow ( Figure 1 ), the taste is more fragrant, sweet and sour than traditional pear juice, and it has a better antibacterial effect, which not only improves the flavor of the product but also inhibits the growth of bacteria.

[0060] Example 4 Optimization of Fermentation Product Powder Spray Drying Conditions

[0061] 1. Prepare sucrose fermentation liquid seeds using Lactococcus lactis subspecies lactis CGMCC 28205 strain according to the method of Example 1.

[0062] Fermentation in a 2.5 L fermenter: The prepared seeds were inoculated at an inoculation rate of 6% (v / v) into a 5 L fermenter filled with 3.5 L sucrose fermentation liquid culture medium and fermented at 30° C. for 22-26 h to obtain sucrose fermentation mash.

[0063] 3. Ceramic membrane sterilization or centrifugal sterilization: After fermentation, adjust the pH of the sucrose fermentation mash to 2-3, keep it at 80℃ for 25-30 minutes to inactivate the enzyme, cool it to below 50℃, and filter it through a ceramic membrane with a pore size of 200nm or centrifuge it at 7500rpm for 15 minutes to obtain the sucrose fermentation filtrate or supernatant.

[0064] 4. Rotary evaporation concentration: The sucrose fermentation filtrate or supernatant is concentrated by rotary evaporation at 60-70°C to 4 times to obtain sucrose fermentation concentrate.

[0065] 5. Add auxiliary materials: add 20% maltodextrin to the sucrose fermentation concentrate.

[0066] 6. Optimization of spray drying conditions: The drying conditions were set to an air inlet temperature of 130°C-170°C, a peristaltic speed of 15-35%, and an air blowing efficiency of 80-100%. Different powdered sucrose ferments were obtained by drying. The total yield of active ingredients in the ferments was Figure 3 The results show that with the increase of inlet air temperature, the total activity of the sample first increases and then decreases, and 150℃ is the optimal temperature ( Figure 3 A); The higher the fan efficiency, the higher the total recovery rate ( Figure 3 B); the peristaltic speed is the injection speed. When the peristaltic speed is too slow, the sample will be affected by the continuous high temperature, resulting in a decrease in the total yield ( Figure 3 C), when the creep speed is too fast, the liquid is not dried sufficiently and will stick to the wall; the final optimal spray drying conditions are an inlet air temperature of 150°C, a fan efficiency of 100%, and a creep speed of 25%.

[0067] Example 5 Optimization of spray drying auxiliary material types and addition amounts

[0068] 1. Prepare sucrose fermentation concentrate according to the method of Example 4.

[0069] 2. Adding auxiliary materials: Adding auxiliary materials as protective agents to the sucrose fermentation concentrate can greatly reduce the inactivation of active ingredients during the spray drying process. Different types of substances (sugars, inorganic salts, composites, etc.) were selected as protective agents, including different concentrations of resistant dextrin, maltodextrin, calcium carbonate, sodium chloride, xanthan gum, trehalose, sodium glutamate, skim milk powder, pectin, water-soluble starch, etc. The specific content ratio and total yield are shown in Table 5.

[0070] 3. Spray drying: The fermentation powder was prepared by spray drying according to the optimal spray drying conditions determined in Example 4, i.e., an inlet air temperature of 150°C, a peristaltic velocity of 25%, and an air blowing efficiency of 100%.

[0071] Table 5 Effect of different protective agents on total yield

[0072] Type of protective agent Addition amount (%) Average total yield (%) Maltodextrin + Trehalose Maltodextrin 10+Trehalose 7.5 69.94 Resistant dextrin 15 61.32 Maltodextrin 15 63.31 Calcium carbonate 10 62.8 Sodium chloride 15 46.93 Xanthan gum 10 43.21 Trehalose 15 62.79 Monosodium Glutamate 10 58.62 Skim milk powder 15 58.14 Pectin 10 56.98 Water-soluble starch 15 61.71

[0073] The calculation formula for the total yield is:

[0074] Total yield = spray-dried solid weight * total potency / (total potency of concentrate * single-step yield of concentrate) / volume of concentrate

[0075] The results showed that 10% maltodextrin + 7.5% trehalose was the best excipient, with a total yield of 69.94% of active ingredients, 6.63% higher than that of single maltodextrin and 8.62% higher than that of resistant dextrin. Figure 4 , is a milky white powder with a certain sour aroma. The antibacterial effect of the fermentation powder is shown in Figure 5 , 0.1 g of fermentation powder was added into 15 mL of 0.02 mol / L hydrochloric acid to fully dissolve, 100 μL was drawn and added onto the prepared solid plate of Micrococcus luteus with a sample addition aperture of 7 mm, and the average diameter of the inhibition zone was detected to be 15.87 mm.

[0076] Example 6 Preservative Screening

[0077] 1. Preparation of preservative solution: Using the 12 common biological preservatives in Table 6 as screening objects, ascorbic acid calcium (3.00 g / 100 mL), ergothioneine (0.1 g / mL), vitamin E (3.00 g / 100 mL), rosemary extract (2.00 g / 100 mL), natamycin (0.20 g / 100 mL), ε-polylysine (0.20 g / 100 mL), Nisin (0.20 g / 100 mL), sucrose fermentation powder (0.2 g / 100 mL), water-soluble chitosan (0.15 g / 100 mL) and pullulan (0.15 g / 100 mL) aqueous solutions were prepared with sterile distilled water. Preparation of xanthan gum preservative: 0.15 g xanthan gum powder was mixed with 100 mL 70°C sterile distilled water, ultrasonically mixed, and allowed to stand at room temperature for use. Preparation method of pectin preservative: 0.15g pectin powder is mixed with 100mL 95℃ sterile distilled water, ultrasonically mixed evenly, then kept at 95℃ for 10min until the sol is fully formed, and allowed to stand at room temperature for use.

[0078] Table 6 Classification of 12 common biological preservatives

[0079]

[0080] 2. Preservative treatment: Soak the prepared Agaricus bisporus in different preservative solutions for 90 seconds. After the timer is up, move it to a clean table. After the water evaporates completely, gently move it into a PET non-porous fresh-keeping box, mark it and store it in a refrigerator at 4°C. Soak it in sterile distilled water for 90 seconds as the control group (CK).

[0081] 3. Index determination method: In this embodiment, the whiteness value, weight loss rate, hardness and smell of the treated Agaricus bisporus were scored. The scoring standard is shown in Table 7. The measured objects are Agaricus bisporus on the 0th, 2nd, 4th and 6th days of storage period. Each treatment is repeated three times, and 5 fruiting bodies are randomly measured in each repeat.

[0082] Table 7 Scoring criteria for characteristic changes of Agaricus bisporus

[0083]

[0084] 4. Preservation results: In this embodiment, the changes in the total scores of Agaricus bisporus in each group on the 0th, 2nd, 4th and 6th days are shown in Table 8. The results show that the total score of the blank group Agaricus bisporus on the 6th day was 75, the total score of the calcium ascorbate treatment group in the antioxidant group was 80, which was higher than that of other treatments, the total scores of nisin and sucrose fermentation in the antibacterial group were 81 and 82, respectively, which were not much different, but sucrose fermentation was safer, and the total score of pullulan in the film-forming group was 79, which was higher than that of other control groups. Therefore, the highest-scoring preservative was selected from the above three types as the raw material for the subsequent preparation of the composite coating agent.

[0085] Table 8 Effects of different biological preservatives on storage of Agaricus bisporus

[0086]

[0087]

[0088] Example 7 Coating agent optimization

[0089] 1. This Example Based on the screening results in Example 6, a three-factor three-level orthogonal analysis experiment was conducted on three preservatives, namely, sucrose fermentation product, calcium ascorbate and pullulan. The experimental design is shown in Table 9. The prepared Agaricus bisporus was soaked in the corresponding preservative solutions of different concentrations for 90 seconds. After the timing was over, it was moved to a clean desktop. After the water was completely evaporated, it was gently moved into a PET non-porous fresh-keeping box, marked and stored in a refrigerator at 4°C. Soaking in sterile distilled water for 90 seconds was used as a negative control (CK).

[0090] Table 9 Orthogonal level table of coating agents

[0091]

[0092] 2. The treated Agaricus bisporus were grouped and scored, and the scoring criteria were the same as in Example 6. The test objects were Agaricus bisporus on the 0th, 2nd, 4th, 6th, and 8th days of storage. Each treatment was repeated three times, and 10 fruiting bodies were randomly measured in each repeat. The scoring results in Table 10 show the total scores of each treatment group on the 0th, 4th, and 8th days of storage.

[0093] Table 10 Effects of composite biological coating preservative treatment on storage of Agaricus bisporus

[0094]

[0095]

[0096] The results in Table 10 show that the group A2B2C3, i.e. 0.2% sucrose fermentation powder + 2% pullulan + 3% calcium ascorbate, has the best preservation effect on Agaricus bisporus.

[0097] Example 8 Application and Effect of Sucrose Fermentation Powder

[0098] 1. Select intact, disease-free, and mechanically undamaged Agaricus bisporus and randomly divide them into 3 groups, with 10 in each group. The control group (CK) was immersed in distilled water for 90 seconds; the antioxidant coating group (No. 1) was immersed in 2% pullulan + 3% calcium ascorbate for 90 seconds; the sucrose fermentation product composite coating group (No. 2) was immersed in 0.2% fermentation product powder + 2% pullulan + 3% calcium ascorbate for 90 seconds. Each treatment was repeated 3 times. All treated mushrooms were placed on drying paper to dry and then stored in a refrigerator at a temperature of (4±1)℃. The apparent changes of Agaricus bisporus in each group are shown in Figure 6 The results showed that after 8 days of storage, both treatment groups 1 and 2 were better than the control group, and treatment group 2 was better than treatment group 1.

[0099] 2. The enzyme activity determination method in this example refers to the instructions of the corresponding enzyme activity determination kit, and the Coomassie Brilliant Blue G-250 method is used to determine the soluble protein concentration, and the thiobarbituric acid method is used to determine the MDA. The polyphenol oxidase (PPO) activity, peroxidase (POD) activity, soluble protein content, and malondialdehyde (MDA) content of each group of Agaricus bisporus after different treatments on days 0, 2, 4, 6, and 8 of the storage period are determined. Each treatment is repeated three times, and the results are shown in Figure 7 .

[0100] PPO is activated when the body tissue is aged or damaged, and is directly related to browning. Therefore, the lower the PPO activity, the better. POD is an enzyme that can reduce hydrogen peroxide. Its main function is to protect plant cells from damage by hydrogen peroxide. The higher the POD activity, the greater the damage to the plant cells. Soluble protein is a key nutrient and osmotic regulating substance, as well as a component of metabolic enzymes for Agaricus bisporus. It can maintain and participate in the regulation of various metabolic processes. Its content is related to the degree of tissue damage. MDA is the main decomposition product of membrane lipid peroxidation. Its content reflects the severity of damage to cells in the plant body. The higher the MDA, the more serious the damage to Agaricus bisporus cells.

[0101] The results show that ( Figure 7 A) The PPO activity of the three treatment groups increased rapidly at first and then slowly with the extension of storage time. Among them, the CK group increased significantly on the second day, while the No. 2 preservative group was significantly lower than the CK group and the No. 1 group throughout the storage period (P < 0.05), which effectively controlled the increase of PPO activity. The POD activity of the No. 2 preservative group was significantly lower than that of the CK group during the entire storage period, indicating that this treatment reduced the intensity of plant damage ( Figure 7 B). The MDA content of the three groups of samples showed an overall increasing trend ( Figure 7C), while the MDA content of the sucrose fermentation product composite coating group (No. 2) was lower than that of the control group and the antioxidant coating group (No. 1) as a whole. The MDA content of the antibacterial agent composite coating group on the 8th day was significantly different from that of the other two groups (P<0.05). The sucrose fermentation product composite coating group and the antibacterial agent composite coating group inhibited the decline of soluble protein content throughout the storage process, among which the sucrose composite coating group had a more obvious inhibitory effect. In short, the various indicators of the sucrose fermentation product coating group are more advantageous than those of the blank and antioxidant composite preservative groups, which can effectively delay the shelf life of Agaricus bisporus and maintain its nutritional value.

[0102] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A strain of Lactococcus lactis subsp. lactis, deposited on September 6, 2023 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC No. 28205, and the 16S rRNA gene sequence of the strain is shown in SEQ ID No.

1.

2. A preparation and application of a Lactococcus lactis subsp. lactis strain and its fermentation product, comprising the following steps: (1) Fermentation: using sucrose, peptone, yeast extract or fruit and vegetable juice as raw materials, and fermenting with Lactococcus lactis to obtain sucrose fermentation mash or fermented fruit and vegetable juice; (2) Bacterial liquid separation: separating the bacteria and insoluble matter in the sucrose fermentation mash to obtain a clear and transparent separated liquid, wherein the bacterial liquid separation method is ceramic membrane filtration or centrifugal separation; (3) Concentration: using rotary evaporation to obtain a concentrated liquid; (4) Adding auxiliary materials: adding auxiliary materials to the concentrated liquid and stirring evenly; (5) Drying: spray drying the mixed material to obtain a sucrose fermentation powder with antibacterial and fresh-keeping functions. (6) Application of fermented fruit and vegetable juice and sucrose fermentation powder.

3. A preparation and application of a Lactococcus lactis subsp. lactis strain and its fermentation product, characterized in that: According to claim 2, the method for separating the bacterial liquid in step (2) is ceramic membrane filtration, and the pore size of the ceramic membrane is 200 nm.

4. A preparation and application of a Lactococcus lactis subsp. lactis strain and its fermentation product, characterized in that: According to claim 2, in step (2), the bacterial liquid is separated by centrifugation at a speed of 7500 rpm for 15 min.

5. A preparation and application of a Lactococcus lactis subsp. lactis strain and its fermentation product, characterized in that: According to claim 2, the auxiliary materials in step (4) are selected from one or more of maltodextrin, water-soluble starch, trehalose, calcium carbonate, sodium chloride, xanthan gum, sodium glutamate, skimmed milk powder, pectin, etc.

6. A preparation and application of a Lactococcus lactis subsp. lactis strain and its fermentation product, characterized in that: The auxiliary materials in step (4) according to claim 2 are preferably maltodextrin and trehalose, with a ratio of 2:1-3:2 and an addition amount of 12.5-17.5%.

7. A preparation and application of a Lactococcus lactis subsp. lactis strain and its fermentation product, characterized in that: The process parameters of the spray drying in step (5) according to claim 2 are: the air inlet temperature is set to 130-170°C, the air outlet temperature is set to 70-90°C, and the peristaltic pump efficiency is 15-25%.

8. A preparation and application of a Lactococcus lactis subsp. lactis strain and its fermentation product, characterized in that: According to the application of step (6) of claim 2, 0.05-0.25% of fermentation solids, 0.5-2.5% of pullulan and 1%-5% of calcium ascorbate are combined to prepare a biological coating agent and coated on the surface of Agaricus bisporus, which can extend the shelf life of Agaricus bisporus.

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