Lactobacillus plantarum SQ03 and its fermentation preparation and application

Abstract

The present invention discloses Lactobacillus plantarum SQ03, its fermentation preparation and application, belonging to the technical field of microbial preparations. Lactobacillus plantarum SQ03 is isolated and purified from the visceral coelomic fluid of sea cucumbers, and is preserved in the China Center for Type Culture Collection. The preservation date is May 14, 2024, and the preservation number is CCTCC NO: M 2024921. The advantages of the present invention are as follows: Lactobacillus plantarum SQ03 can grow rapidly even under low-temperature refrigeration conditions, can effectively inhibit the browning of fresh fruits and improve the antibacterial and fresh-keeping effect of seafood under low-temperature refrigeration conditions, and at the same time has the function of fermenting fruit and vegetable enzymes, greatly broadening the application scope of Lactobacillus plantarum.

Description

Technical Field

[0001] The present invention relates to a bacterium, its fermentation preparation and application, specifically to Lactobacillus plantarum SQ03, its fermentation preparation and application, belonging to the technical field of microbial preparations. Background Art

[0002] At present, Lactobacillus plantarum is mostly isolated from dairy products and fermented products. These Lactobacillus plantarum generally reproduce under relatively high temperature conditions (35°C - 40°C) and grow slowly under low-temperature refrigeration conditions (0°C - 4°C), which greatly limits the application scope of the bacterial strains. Summary of the Invention

[0003] To solve the deficiencies of the prior art, the purpose of the present invention is to provide a strain of Lactobacillus plantarum that can reproduce under relatively high temperature conditions (35°C - 40°C) and can also grow rapidly under low-temperature refrigeration conditions (0°C - 4°C).

[0004] To achieve the above goal, the present invention adopts the following technical solutions:

[0005] Lactobacillus plantarum SQ03, isolated and purified from the visceral coelomic fluid of sea cucumbers, with the Latin name Lactobacillus plantarum SQ03, is preserved in the China Center for Type Culture Collection. The preservation date is May 14, 2024, and the preservation number is CCTCC M2024921. The location of the preservation unit is Wuhan, China.

[0006] A microbial preparation prepared by fermenting the aforementioned Lactobacillus plantarum SQ03, and the microbial preparation is prepared by the following method:

[0007] (1) Inoculate the Lactobacillus plantarum SQ03 described in claim 1 onto an MRS solid medium and incubate it in an inverted position at 37°C;

[0008] (2) Pick the bacterial colonies from the above MRS solid medium and transfer them to an MRS liquid medium, and incubate them with shaking at 37°C to obtain a seed solution;

[0009] (3) Take the above seed solution and inoculate it into fermentation medium A, and incubate it statically at 37°C to obtain fermentation broth A. Among them, the formula of fermentation medium A is: glucose 21.0 g / L, peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 8.0 g / L, dipotassium hydrogen phosphate 3.5 g / L, sodium acetate 6.5 g / L, diammonium citrate 2 g / L, magnesium sulfate 0.2 g / L, adenine 0.13 g / L, phenylalanine 0.051 g / L, Tween 80 1 mL / L;

[0010] (4) Transfer the above fermentation broth A to fermentation medium B and culture it with shaking at 37°C to obtain fermentation broth B. The formula of fermentation medium B is as follows: glucose 16.0 g / L, peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 6.0 g / L, dipotassium hydrogen phosphate 3.5 g / L, sodium acetate 6.5 g / L, diammonium hydrogen citrate 2.0 g / L, magnesium sulfate 0.5 g / L, calcium chloride 1.0 g / L, L-glutamic acid 5.0 g / L, manganese sulfate 0.1 g / L, vitamin C 0.01 g / L, adenine 0.15 g / L, phenylalanine 0.05 g / L, Tween 80 1 mL / L;

[0011] (5) Transfer the above fermentation broth B to fermentation medium A and culture it with shaking at 37°C until the final maximum viable cell count of the fermentation broth reaches 9.4×10 9 CFU / mL to obtain fermentation broth C;

[0012] (6) Perform solid-liquid separation on the above fermentation broth C, and collect the fermentation supernatant and the bacterial sludge separately;

[0013] (7) Perform ultrafiltration concentration, add the cryoprotectant trehalose, homogenize, and perform low-temperature spray drying on the above fermentation supernatant in sequence to obtain microbial preparation 1;

[0014] (8) Add wall material, homogenize, and perform vacuum freeze-drying on the above bacterial sludge in sequence to obtain microbial preparation 2.

[0015] Preferably, in steps (3) and (5), fermentation medium A is prepared with purified seawater and the pH is adjusted to 6.2 ± 0.1.

[0016] Preferably, in step (4), fermentation medium B is prepared with purified seawater.

[0017] Preferably, in step (5), sterile aeration is performed once every 12 h during fermentation.

[0018] Preferably, in step (7), the cryoprotectant trehalose is added to the concentrated solution at a weight ratio of 1:10.

[0019] Preferably, in step (8), the wall material is selected from any one or more of gum arabic, maltodextrin, whey protein, and alginate; more preferably, the wall material is obtained by mixing gum arabic, maltodextrin, whey protein, and alginate at a weight ratio of 5:4:1:5, and the weight ratio of the composite wall material to the bacterial sludge is 1:1.

[0020] The application of the aforementioned microbial preparation 1 in the following aspects: (1) antioxidant of fruits, vegetables, and fruit / vegetable-related solid / liquid beverages and skin care products; (2) antibacterial preservation of seafood.

[0021] Use of the aforementioned microbial agent 2 in the following aspects: (1) antibacterial preservation of seafood; (2) fermentation of fruit and vegetable enzymes.

[0022] The beneficial effects of the present invention are as follows:

[0023] (1) Lactobacillus plantarum SQ03 was isolated and purified from the visceral coelomic fluid of the marine animal sea cucumber. Different from the existing Lactobacillus plantarum, Lactobacillus plantarum SQ03 can grow rapidly even under low-temperature refrigeration conditions, can effectively inhibit the browning of fresh fruits and improve the antibacterial preservation effect of seafood under low-temperature refrigeration conditions. At the same time, it also has the function of fermenting fruit and vegetable enzymes, greatly expanding the application scope of Lactobacillus plantarum;

[0024] (2) The Lactobacillus plantarum microbial agent developed in the present invention is safe, non-toxic and has no drug resistance, and can be used with confidence for a long time. Description of the Drawings

[0025] Figure 1 It is the colony morphology diagram of Lactobacillus plantarum SQ03;

[0026] Figure 2 It is the cell shape diagram of Lactobacillus plantarum SQ03;

[0027] Figure 3 It is the growth curve diagram of Lactobacillus plantarum SQ03 under the condition of 4°C;

[0028] Figure 4 It is the detection result diagram of the scavenging ability of microbial agent 1 made from the fermentation supernatant of Lactobacillus plantarum SQ03 against various free radicals;

[0029] Figure 5 It is the diagram of the effect of microbial agent 1 made from the fermentation supernatant of Lactobacillus plantarum SQ03 on the browning rate of fresh-cut apples;

[0030] Figure 6 It is the diagram of the effect of microbial agent 1 made from the fermentation supernatant of Lactobacillus plantarum SQ03 and microbial agent 2 made from the bacterial sludge of Lactobacillus plantarum SQ03 on the sensory score of sea bass meat;

[0031] Figure 7 It is the diagram of the effect of microbial agent 1 made from the fermentation supernatant of Lactobacillus plantarum SQ03 and microbial agent 2 made from the bacterial sludge of Lactobacillus plantarum SQ03 on the total volatile basic nitrogen (TVBN) of sea bass;

[0032] Figure 8 It is the diagram of the effect of microbial agent 1 made from the fermentation supernatant of Lactobacillus plantarum SQ03 and microbial agent 2 made from the bacterial sludge of Lactobacillus plantarum SQ03 on the thiobarbituric acid (TBA) of sea bass;

[0033] Figure 9 Effect diagram of microbial preparation 1 made from the fermentation supernatant of Lactobacillus plantarum SQ03 and microbial preparation 2 made from the bacterial sludge of Lactobacillus plantarum SQ03 on the total number of colonies of sea bass;

[0034] Figure 10 Effect diagram of microbial preparation 1 made from the fermentation supernatant of Lactobacillus plantarum SQ03 and microbial preparation 2 made from the bacterial sludge of Lactobacillus plantarum SQ03 on the hardness of sea bass meat;

[0035] Figure 11 Effect diagram of microbial preparation 1 made from the fermentation supernatant of Lactobacillus plantarum SQ03 and microbial preparation 2 made from the bacterial sludge of Lactobacillus plantarum SQ03 on the elasticity of sea bass meat. Detailed implementation mode

[0036] The present invention will be specifically introduced below in conjunction with the accompanying drawings and specific embodiments.

[0037] I. Culture medium

[0038] 1. MRS liquid medium

[0039] The formula of MRS liquid medium is as follows: peptone 10.0 g / L, beef extract powder 5.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, sodium acetate 5.0 g / L, ammonium citrate 2.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, prepared with purified seawater.

[0040] Autoclave at 121 °C for 20 min, and after sterilization, cool to room temperature for standby.

[0041] 2. MRS solid medium

[0042] The formula of MRS solid medium is as follows: peptone 10.0 g / L, beef extract powder 5.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, sodium acetate 5.0 g / L, ammonium citrate 2.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, agar 15.0 g / L, prepared with purified seawater.

[0043] Autoclave at 121 °C for 20 min, and after sterilization, cool to 45 °C, and pour into a sterile petri dish in a laminar flow cabinet and let it solidify for standby.

[0044] 3. Fermentation medium A

[0045] The formulation of fermentation medium A is as follows: glucose 21.0 g / L, peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 8.0 g / L, dipotassium hydrogen phosphate 3.5 g / L, sodium acetate 6.5 g / L, diammonium hydrogen citrate 2 g / L, magnesium sulfate 0.2 g / L, adenine 0.13 g / L, phenylalanine 0.051 g / L, Tween 80 1 mL / L. It is prepared with purified seawater and the pH is adjusted to 6.2 ± 0.1.

[0046] Autoclave at 121 °C for 20 min. After sterilization, cool to room temperature and set aside.

[0047] 4. Fermentation medium B

[0048] The formulation of fermentation medium B (optimized fermentation medium) is as follows: glucose 16.0 g / L, peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 6.0 g / L, dipotassium hydrogen phosphate 3.5 g / L, sodium acetate 6.5 g / L, diammonium hydrogen citrate 2.0 g / L, magnesium sulfate 0.5 g / L, calcium chloride 1.0 g / L, L-glutamic acid 5.0 g / L, manganese sulfate 0.1 g / L, vitamin C 0.01 g / L, adenine 0.15 g / L, phenylalanine 0.05 g / L, Tween 80 1 mL / L. It is prepared with purified seawater.

[0049] Autoclave at 121 °C for 20 min. After sterilization, cool to room temperature and set aside.

[0050] II. Isolation, purification and species identification of Lactobacillus plantarum SQ03

[0051] 1. Isolation and purification of Lactobacillus plantarum SQ03

[0052] Lactobacillus plantarum SQ03 involved in the present invention is isolated and purified from the visceral coelomic fluid of Apostichopus japonicus in Changdao, Penglai, Yantai. The isolation and purification process is as follows:

[0053] (1) Take 10 mL of the visceral coelomic fluid of Apostichopus japonicus and add it to 100 mL of a sterile NaCl solution with a concentration of 0.85% (w / v) to make a turbid solution.

[0054] (2) Gradient dilute the above turbid solution. Take 100 μL of the turbid solution diluted 10,000 times and spread it onto MRS solid medium by the plate coating method. A total of 3 plates are coated. Then place the 3 plates coated with the turbid solution in an incubator at 37 °C and incubate them upside down for 36 h. After incubation, about 30 - 60 single colonies grew on each plate.

[0055] (3) Randomly pick 3 single colonies from each plate onto a new MRS solid medium, and purify the single colonies by the method of streaking on the plate. After streaking, place the 9 plates in an incubator at 37°C and incubate them upside down for 24 h. After the incubation, about 20 single colonies grew on each plate. Pick the white colonies with a calcium dissolution zone onto a new MRS solid medium for further isolation and purification. Finally, preserve a single colony that is round, medium-sized, convex in the middle, neat at the edge, slightly yellowish white, moist on the surface, and opaque. This single colony is the final single colony screened out by the present invention. Correspondingly, the strain in this single colony is the final strain screened out by the present invention.

[0056] 2. Species identification of Lactobacillus plantarum SQ03

[0057] (1) Observe the colony morphology and cell shape

[0058] Spread the strain finally screened out by the present invention onto an MRS solid medium by the method of spreading on the plate, and incubate it upside down in an incubator at 37°C for 24 h. After the incubation, multiple single colonies were isolated on the plate, as Figure 1 shown. These single colonies are round, medium-sized, convex in the middle, neat at the edge, slightly yellowish white, moist on the surface, and opaque.

[0059] Take a loopful of the strain from the above plate and disperse it into an MRS liquid medium. Gram stain the strain according to "Bergey's Manual of Determinative Bacteriology" and observe the cell morphology under a microscope. As Figure 2 shown, this strain is a Gram-positive bacterium, showing short and thick rod-shaped, with blunt ends, arranged singly or in pairs, and without endospores.

[0060] (2) Gel electrophoresis detection

[0061] Inoculate the strain finally screened out by the present invention into an MRS liquid medium, and culture it at 37°C and 150 rpm for 24 h to obtain a bacterial solution.

[0062] Extract the DNA of the bacteria in the above bacterial solution with a bacterial genomic DNA extraction kit, and then use the extracted DNA as a template, use 27F (sequence: AGA GTT TGATCC TGG CTCAG) as the upstream primer, and use ITS1 (sequence: TCCGTAGGTGAACCTGCGG) as the downstream primer for sequence amplification.

[0063] The PCR reaction program is: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 1 min, 31 cycles; extension at 72°C for 5 min, and the amplification is completed.

[0064] The amplified products were detected by 1% (w / v) agarose gel electrophoresis. The detection results showed that the PCR amplification with universal primers was successful.

[0065] (3) 16S rRNA identification

[0066] The strain finally screened out in the present invention was sent to Beijing Aoke Dingsheng Biotechnology Co., Ltd. for 16S rRNA sequence determination, and the 16S rRNA sequence of the strain was retrieved by homologous sequence alignment in GenBank. The results of the alignment and retrieval showed that the similarity between the strain finally screened out in the present invention and Lactobacillus plantarum reached 100%.

[0067] Finally, the strain finally screened out in the present invention was identified as Lactobacillus plantarum, denoted as Lactobacillus plantarum SQ03.

[0068] III. Growth of Lactobacillus plantarum SQ03 under low temperature conditions

[0069] Lactobacillus plantarum SQ03 was continuously cultured at a constant temperature on MRS liquid medium at 4°C and 120 rpm for 72 h, and samples were taken every 6 h to measure the OD 600 value of Lactobacillus plantarum SQ03 to judge the growth of the strain under low temperature conditions.

[0070] The growth curve of Lactobacillus plantarum SQ03 is as Figure 3 shown. The results showed that the strain grew rapidly under the low temperature condition of 4°C.

[0071] IV. Physiological and biochemical identification of Lactobacillus plantarum SQ03

[0072] The physiological and biochemical identification items of Lactobacillus plantarum SQ03 include: cellobiose, esculin, maltose, sucrose, inulin, lactose, salicin, sorbitol, melezitose, palatinose, stevioside. The identification results are shown in Table 1.

[0073] Table 1 Physiological and biochemical identification results of Lactobacillus plantarum SQ03

[0074] Identification item Identification result Cellobiose + Esculin + Maltose + Sucrose + Inulin + Lactose + Salicin + Sorbitol + Melezitose - Isomaltulose - Stevioside -

[0075] Note: + indicates that the strain is positive, - indicates that the strain is negative.

[0076] The results showed that Lactobacillus plantarum SQ03 could utilize cellobiose, esculin, maltose, sucrose, inulin, lactose, salicin and sorbitol, and could not utilize melezitose, palatinose and stevioside.

[0077] V. Cultivation, fermentation of Lactobacillus plantarum SQ03 and preparation of microbial agents

[0078] 1. Cultivation of Lactobacillus plantarum SQ03

[0079] (1) Plate cultivation of Lactobacillus plantarum SQ03

[0080] Inoculate the purified Lactobacillus plantarum SQ03 onto MRS solid medium and incubate it upside down at 37°C for 48 h.

[0081] (2) Seed liquid cultivation of Lactobacillus plantarum SQ03

[0082] Pick 1 loop of independent bacterial colonies from the MRS solid medium obtained after 48 h of the above cultivation and transfer them to 200 mL of MRS liquid medium. Seal it with plastic wrap and continuously culture it at 37°C and 120 rpm for 24 h to obtain the seed liquid.

[0083] 2. Fermentation of Lactobacillus plantarum SQ03

[0084] (1) Fermentation of Lactobacillus plantarum SQ03

[0085] Take the seed liquid obtained after 24 h of the above cultivation and inoculate it into 3000 mL of fermentation medium A at an inoculation amount of 5%. Seal it with plastic wrap and statically culture it at 37°C for 36 h to obtain fermentation broth A.

[0086] (2) Optimized fermentation of Lactobacillus plantarum SQ03

[0087] Transfer all of the fermentation broth A obtained after 36 h of the above cultivation to 20 L of fermentation medium B (contained in a 30 L fermenter) and continuously culture it at 37°C and 120 rpm for 36 h to obtain fermentation broth B. Subsequently, transfer all of the 20 L of fermentation broth B to 200 L of fermentation medium A (contained in a 300 L fermenter) and continuously ferment it at 37°C and 120 rpm for 48 h until the final maximum viable cell count of the fermentation broth can reach 9.4×10 9 CFU / mL. Sterile aeration is carried out once every 12 h during fermentation to obtain fermentation broth C.

[0088] 3. Preparation of Lactobacillus plantarum SQ03 microbial preparation

[0089] After the optimized fermentation of Lactobacillus plantarum SQ03, the obtained fermentation broth C is subjected to solid-liquid separation using a disc centrifuge, centrifuged at 5000 rpm for 200 s, and the fermentation supernatant and bacterial sludge are collected respectively. Among them: (1) The above fermentation supernatant is ultrafiltered and concentrated using a ceramic membrane, and the ultrafiltration conditions are: 0.5 Mpa, 0.02 μm. Then, add the cryoprotectant trehalose to the concentrated solution at a weight ratio of 1:10 (m 浓缩液 : m 海藻糖= 1:10), and shear and stir with a homogenizer until evenly mixed. The homogenization conditions are: 60 °C, 20 MPa, 6000 rpm. Then, perform low-temperature spray drying with an inlet air temperature of 110 °C and an outlet air temperature of 60 °C to obtain microbial preparation 1; (2) Add wall material (any one or any combination of gum arabic, maltodextrin, whey protein, and alginate) to the above-mentioned bacterial sludge at a weight ratio of 1-10:1. The types and proportions of the wall material added to the bacterial sludge are different, and the biological activities of the prepared microbial preparations are slightly different, but the differences are not very significant. In this specific embodiment, a composite wall material obtained by mixing gum arabic, maltodextrin, whey protein, and alginate in a weight ratio of 5:4:1:5 is mixed with the bacterial sludge, and the weight ratio of the composite wall material to the bacterial sludge is 1:1. Then, shear and stir with a homogenizer until evenly mixed. The homogenization conditions are: 60 °C, 20 MPa, 6000 rpm. This process is also a process of crosslinked microencapsulation modification. Then, perform vacuum freeze-drying. The vacuum freeze-drying conditions are: (i) Pre-freezing stage: shelf temperature -50 °C, cold trap temperature -50 °C, vacuum degree 200 mTorr; (ii) Main drying stage: dry at -40 °C for 200 min, -30 °C for 360 min, -20 °C for 480 min, 0 °C for 240 min, 20 °C for 240 min, vacuum degree 100 mTorr; (iii) Secondary drying stage: shelf temperature 25 °C, vacuum degree 650 mTorr, maintain for 5 min to obtain microbial preparation 2.

[0090] After the seed liquid culture of Lactobacillus plantarum SQ03, the cultivated seed liquid is directly inoculated into fermentation medium A and continuously cultured at 37 °C and 120 rpm. Sterile aeration is carried out once every 12 h until the maximum viable count of the fermentation broth can reach 9.4×10 9 CFU / mL without optimized fermentation. After fermentation, collect the fermentation supernatant and bacterial sludge using the same method as before. Ultrafiltration concentration, addition of trehalose, homogenization, and low-temperature spray drying are successively carried out on the fermentation supernatant to obtain microbial preparation 3. Wall material addition, homogenization, and vacuum freeze-drying are successively carried out on the bacterial sludge to obtain microbial preparation 4.

[0091] After the optimized fermentation of Lactobacillus plantarum SQ03, the obtained fermentation broth C is not subjected to solid-liquid separation. The fermentation broth C (containing bacterial sludge) is directly subjected to 2-fold vacuum concentration. The concentration conditions are: vacuum degree ≤0.095 MPa, steam pressure: 0.3 MPa, heating area: 2 m 2 , condensation area: 3.5 m 2 . Collect the concentrated fermentation broth (containing bacterial sludge). The concentrated fermentation broth (containing bacterial sludge) is successively subjected to addition of trehalose, homogenization, and low-temperature spray drying using the same method as before to obtain microbial preparation 5.

[0092] After the optimized fermentation of Lactobacillus plantarum SQ03, the bacterial sludge was collected by the same method as before. The bacterial sludge was not subjected to cross-linked microencapsulation modification and was directly vacuum freeze-dried by the same method as before to obtain the microbial preparation 6.

[0093] VI. Verification of the antibacterial activity of Lactobacillus plantarum SQ03

[0094] Indicator bacteria: Shewanella, Pseudomonas, Vibrio alginolyticus, Staphylococcus aureus, and Aeromonas hydrophila were used as indicator bacteria.

[0095] Detection method: The antibacterial effect of the microbial preparation of Lactobacillus plantarum SQ03 was determined by the Oxford cup plate diffusion method.

[0096] Antibacterial plate: Shewanella, Pseudomonas, Vibrio alginolyticus, Staphylococcus aureus, Aeromonas hydrophila, and Escherichia coli were added to the molten MRS solid medium at a concentration of 1% (v / v), and then poured into different petri dishes respectively. After cooling, the corresponding antibacterial plates were obtained.

[0097] 1. Antibacterial activity of microbial preparation 1

[0098] The microbial preparation 1 was prepared into a preparation solution with a concentration of 1 mg / mL with ultrapure water for later use.

[0099] Four Oxford cups were placed on each antibacterial plate. One Oxford cup was used as a control group, and the other three Oxford cups were used as parallel experimental groups. 100 μL of sterile water was added to the Oxford cup in the control group, and 100 μL of the above-mentioned preparation solution was added to each Oxford cup in the parallel experimental groups. The plates were incubated at 35 °C for 24 h in an upright position, and then the diameters (mm) of the antibacterial zones on each antibacterial plate were measured and recorded.

[0100] The detection results of the antibacterial activity of the microbial preparation 1 are shown in Table 2.

[0101] Table 2 Antibacterial effect of the microbial preparation 1

[0102]

[0103] As can be seen from Table 2, the microbial preparation 1 prepared from the fermentation supernatant of Lactobacillus plantarum SQ03 has obvious inhibitory effects on Shewanella, Pseudomonas, Vibrio alginolyticus, Staphylococcus aureus, and Aeromonas hydrophila. Among them, the inhibitory effects on the pathogenic bacteria Shewanella, Aeromonas hydrophila, Pseudomonas, and Vibrio alginolyticus are the most obvious.

[0104] 2. Antibacterial activity of microbial preparation 2

[0105] The microbial preparation 2 was prepared into a preparation solution with a concentration of 1 mg / mL with ultrapure water for later use.

[0106] Place 4 Oxford cups on each antibacterial plate, with 1 Oxford cup as the control group and the remaining 3 Oxford cups as parallel experimental groups. Add 100 μL of sterile water to the Oxford cup in the control group, and add 100 μL of the above-mentioned preparation solution to each Oxford cup in the parallel experimental groups. Incubate at 35 °C upright for 48 h, and then measure and record the diameter (mm) of the antibacterial zone on each antibacterial plate.

[0107] The detection results of the antibacterial activity of microbial preparation 2 are shown in Table 3.

[0108] Table 3 Antibacterial effect of microbial preparation 2

[0109]

[0110]

[0111] As can be seen from Table 3, microbial preparation 2 prepared by cross-linked microencapsulation of the bacterial sludge of Lactobacillus plantarum SQ03 has a very significant inhibitory effect on Shewanella, Pseudomonas, Vibrio alginolyticus, Staphylococcus aureus and Aeromonas hydrophila, and has a stronger inhibitory effect than microbial preparation 1. This may be because after cross-linked microencapsulation modification and freeze-drying of Lactobacillus plantarum SQ03, the retention rate of the strain's own activity is higher. Further, the activity and concentration of antibacterial substances (bacteriocins, antibacterial peptides, acids, etc.) accumulated by direct fermentation are also higher. Furthermore, wall materials such as arabic gum, maltodextrin, whey protein, and alginate have an obvious inhibitory effect on a variety of common food spoilage bacteria, and synergistically enhance the antibacterial effect of the antibacterial substances produced by Lactobacillus plantarum SQ03, resulting in a significant improvement in the antibacterial effect of the entire fermentation system.

[0112] 3. Antibacterial activity of microbial preparations 3, 4, 5 and 6

[0113] Prepare preparation solutions of microbial preparations 3, 4, 5 and 6 with ultrapure water at a concentration of 1 mg / mL respectively. Correspondingly, obtain preparation solutions 3, 4, 5 and 6 for use.

[0114] Use the same method as before to detect the antibacterial effect. Among them, preparation solutions 3 and 5 are incubated at 35 °C upright for 24 h, and preparation solutions 4 and 6 are incubated at 35 °C upright for 48 h.

[0115] The detection results of the antibacterial activity of microbial preparations 3, 4, 5 and 6 are shown in Table 4.

[0116] Table 4 Antibacterial effects of microbial preparations 3, 4, 5 and 6 (average value of antibacterial zone diameter, mm)

[0117] Indicator bacteria Microbial agent 3 Microbial agent 4 Microbial agent 5 Microbial agent 6 Shewanella 9.7 13.6 14.2 10.7 Pseudomonas 10.5 14.7 17.8 9.3 Vibrio alginolyticus 9.4 14.3 17.4 8.6 Staphylococcus aureus 9.2 11.5 12.7 9.2 Aeromonas hydrophila 8.3 12.7 13.3 8.7

[0118] As can be seen from Table 4, microbial agents 3, 4, 5, and 6 all have certain inhibitory effects on Shewanella, Pseudomonas, Vibrio alginolyticus, Staphylococcus aureus, and Aeromonas hydrophila, but their inhibitory abilities are relatively weaker than those of microbial agents 1 and 2.

[0119] Compared with microbial agent 1, there are obvious differences in the antibacterial effects of the two, which may be due to the fact that microbial agent 3 was not fermented by fermentation medium B (optimized fermentation medium) during the preparation process, resulting in a low accumulation of its antibacterial substances (bacteriocins, antimicrobial peptides, acids, etc.).

[0120] Compared with microbial agent 2, the antibacterial effect of the former is weaker, which may be because microbial agent 4 was not fermented by fermentation medium B (optimized fermentation medium), and the strain itself was not domesticated by fermentation medium B, so its growth and metabolic activity characteristics are not as significant as those of the strains in microbial agent 2.

[0121] Microbial agent 5 is characterized by the coexistence of fermentation broth and bacterial sludge during preparation. Through the average size of the antibacterial zones of each indicator bacterium, it is found that its antibacterial ability has little difference compared with microbial agents 1 and 2, and its antibacterial effects on Pseudomonas and Vibrio alginolyticus are higher than those of microbial agents 1 and 2. This shows that preparing antibacterial agents by combining fermentation broth and bacterial sludge is also a feasible solution, but in actual application, factors such as preparation efficiency, energy consumption, and application scenarios and methods need to be comprehensively considered. The separate preparation mode of microbial agents 1 and 2 may be a better choice.

[0122] Microbial agent 6 is characterized by directly freeze-drying the obtained bacterial sludge without crosslinked microencapsulation modification. From the diameter of the antibacterial zone, it is found that its antibacterial ability is quite different from that of microbial agent 2. The reason may be that the Lactobacillus plantarum SQ03 strain was not encapsulated by crosslinked microencapsulation modification, and its growth and metabolic activities were restricted by external environmental factors, resulting in a low efficiency of producing antibacterial substances, and thus a relatively weak antibacterial effect. Therefore, crosslinked microencapsulation modification of Lactobacillus plantarum SQ03 helps to synergistically enhance its antibacterial ability.

[0123] VII. Verification of the antioxidant activity of Lactobacillus plantarum SQ03

[0124] Because microbial agent 1 contains substances such as polysaccharides, oligosaccharides, and oxidases produced by the fermentation of Lactobacillus plantarum SQ03, while microbial agent 2 contains encapsulated strains (not yet fermented), it is predicted that microbial agent 1 has antioxidant activity.

[0125] 1. Determination of the scavenging ability of Lactobacillus plantarum SQ03 preparation on free radicals

[0126] Using a vitamin E solution with a concentration of 1 mg / mL as a comparative experiment and pure water as a blank experiment, the scavenging ability of microbial preparation 1 on DPPH free radicals, ABTS+ free radicals and superoxide anion free radicals was detected.

[0127] The microbial preparation 1 was prepared into a preparation solution with a concentration of 1 mg / mL using ultrapure water for later use.

[0128] (1) DPPH free radical scavenging ability

[0129] Take 100 μL of the preparation solution and 100 μL of a 0.1 mmol / L DPPH solution, mix them, and let them react in the dark for 30 minutes. Use an enzyme reader to measure the absorbance A1 at 517 nm.

[0130] 100 μL of the preparation solution and 100 μL of 95% (v / v) ethanol solution were mixed and allowed to react in the dark for 30 min. The absorbance A2 was measured at a wavelength of 517 nm using an ELISA reader.

[0131] Take 100 μL of ultrapure water and 100 μL of 0.1 mmol / L DPPH solution, mix them, and let them react in the dark for 30 minutes. Use an enzyme reader to measure the absorbance A2 at 517 nm.

[0132] The DPPH free radical scavenging rate (H, %) was calculated according to the following formula:

[0133] H = [1 - (A1 - A2) / A0] x 100%.

[0134] (2) ABTS+ free radical scavenging ability

[0135] Take 180 μL of ABTS working solution with a concentration of 7 mmol / L, add 20 μL of preparation solution, react in the dark for 6 min, and use an enzyme reader to measure the absorbance A1 at a wavelength of 734 nm.

[0136] Take 180 μL of ultrapure water, add 20 μL of the preparation solution, react in the dark for 6 minutes, and use an enzyme reader to measure the absorbance A2 at a wavelength of 734 nm.

[0137] Take 180 μL of 7 mmol / L ABTS working solution, add 20 μL of ultrapure water, react in the dark for 6 min, and use an enzyme reader to measure the absorbance A0 at a wavelength of 734 nm.

[0138] The ABTS+ free radical scavenging rate (B, %) was calculated according to the following formula:

[0139] B = [1 - (A1 - A2) / A0] x 100%.

[0140] (3) Superoxide anion radical scavenging ability

[0141] Take 225 μL of Tris-HCl solution with a concentration of 0.05 mol / mL and preheat it at 25 °C for 20 min. Add 50 μL of the preparation solution and 20 μL of pyrogallol solution with a concentration of 25 mmol / mL. React at 25 °C for 5 min, and then quickly add 20 μL of concentrated hydrochloric acid with a concentration of 12 mol / L to terminate the reaction. Measure the absorbance A1 at a wavelength of 320 nm using an enzyme-labeled instrument.

[0142] Take 225 μL of Tris-HCl solution with a concentration of 0.05 mol / mL and preheat it at 25 °C for 20 min. Add 50 μL of the preparation solution and 20 μL of ultrapure water. React at 25 °C for 5 min, and then quickly add 20 μL of concentrated hydrochloric acid with a concentration of 12 mol / L to terminate the reaction. Measure the absorbance A2 at a wavelength of 320 nm using an enzyme-labeled instrument.

[0143] Take 225 μL of Tris-HCl solution with a concentration of 0.05 mol / mL and preheat it at 25 °C for 20 min. Add 50 μL of ultrapure water and 20 μL of pyrogallol solution with a concentration of 25 mmol / mL. React at 25 °C for 5 min, and then quickly add 20 μL of concentrated hydrochloric acid with a concentration of 12 mol / L to terminate the reaction. Measure the absorbance A0 at a wavelength of 320 nm using an enzyme-labeled instrument.

[0144] Calculate the superoxide anion radical scavenging rate (O, %) according to the following formula:

[0145] O = [1 - (A1 - A2) / A0] × 100%.

[0146] The detection results of the scavenging abilities of microbial preparation 1 against DPPH radicals, ABTS+ radicals, and superoxide anion radicals are shown in Figure 4 .

[0147] From Figure 4 it can be seen that the fermentation metabolites of Lactobacillus plantarum SQ03 have strong antioxidant activity, and the antioxidant activity of microbial preparation 1 is significantly higher than that of vitamin E at the same concentration.

[0148] The above results indicate that Lactobacillus plantarum SQ03 and its fermentation preparation can be applied to the antioxidant of fruits, vegetables, and related solid / liquid beverages.

[0149] 2. Determine the anti-browning (oxidation) of Lactobacillus plantarum SQ03 preparation on fresh-cut apples

[0150] Wash fresh apples with a 0.1% (w / v) sodium hypochlorite solution for 2 min, then rinse thoroughly with sterile distilled water and dry the surface moisture of the apples with absorbent paper. After peeling and coring the apples on a sterile workbench, cut them evenly into 6 slices, and every 6 slices of apple chunks are taken as one treatment.

[0151] Prepare a preparation solution with a concentration of 1 mg / mL of microbial preparation 1 using ultrapure water for later use.

[0152] Experimental group: Immerse the fresh-cut apple chunks in the preparation solution for 2 min.

[0153] Control group: Immerse the fresh-cut apple chunks in pure water for 2 min.

[0154] After the immersion, dry all the fresh-cut apple chunks, then pack them in plastic boxes, seal them with breathable PE plastic wrap, and store them refrigerated at 4°C for 5 days.

[0155] Use the 0 - 4 scoring method to evaluate the browning of fresh-cut apples, where:

[0156] "0" score indicates no browning on the surface of the fresh-cut apples;

[0157] "1" score indicates slight browning on the surface of the fresh-cut apples (the browning area on the surface of the fresh-cut apples reaches about 5%);

[0158] "2" score indicates moderate browning on the surface of the fresh-cut apples (the browning area on the surface of the fresh-cut apples reaches 5% - 20%);

[0159] "3" score indicates medium-severe browning on the surface of the fresh-cut apples (the browning area on the surface of the fresh-cut apples reaches 20% - 50%);

[0160] "4" score indicates complete browning on the surface of the fresh-cut apples (the browning area on the surface of the fresh-cut apples reaches more than 50%).

[0161] Calculate the browning rate (%) according to the following formula:

[0162] Browning rate (%) = [Σ(k × f) / N × D] × 100%.

[0163] In the formula, k represents the assigned score for the degree of browning, f represents the number of apple chunks corresponding to the assigned score, N represents the number of apple chunks in each group, and D represents the assigned score when complete browning occurs.

[0164] The calculation results of the browning rates of the fresh-cut apples in the experimental group and the control group are shown in Figure 5 .

[0165] By Figure 5It can be seen that with the extension of storage time, the browning rate of fresh-cut apples continuously increases and reaches the maximum value at the end of the storage period. The browning rate of the experimental group is much lower than that of the control group, indicating that the microbial preparation of Lactobacillus plantarum SQ03 has a significant antioxidant effect, and this antioxidant characteristic can be further applied to functional beverages and skin care products.

[0166] VIII. Bacteriostatic and fresh-keeping effects of Lactobacillus plantarum SQ03 on seafood

[0167] Because microbial preparation 1 contains substances such as organic acids, bacteriocins, and lipopeptides fermented by Lactobacillus plantarum SQ03, the strains in microbial preparation 2 will continue to ferment to produce bacteriostatic substances, and the strain embedding material also has certain bacteriostatic characteristics, so it is predicted that both microbial preparation 1 and microbial preparation 2 have bacteriostatic and fresh-keeping effects.

[0168] Take appropriate amounts of microbial preparation 1 and microbial preparation 2, dissolve them evenly with sterile water respectively, and prepare preparation solution 1 and preparation solution 2 with a concentration of 25 mg / 100 mL.

[0169] The shelf life of sea bass is very short and it is extremely perishable. Although the storage period can be appropriately extended at 4°C, it will deteriorate if not carefully handled. Taking the fresh-keeping of sea bass as an example, the present invention verifies the fresh-keeping effect of Lactobacillus plantarum SQ03.

[0170] Process the fresh sea bass into fish slices weighing 20 g and 0.5 mm thick. Randomly divide the samples into 3 groups, with each group being 20.0 ± 0.2 kg, namely experimental group 1, experimental group 2, and the control group. Among them, experimental group 1 uses the above-mentioned preparation solution 1, experimental group 2 uses the above-mentioned preparation solution 2, and the control group uses sterile water.

[0171] Fresh-keeping treatment method: Spray 15 mL of preparation solution 1 onto experimental group 1; spray the same dose of preparation solution 2 onto experimental group 2; spray the same dose of sterile water onto the control group. All samples are independently packaged in vacuum packaging bags and stored in a low-temperature refrigerator at 4 ± 1°C for 15 d. Random samples are taken every 3 d for determination of relevant indicators.

[0172] 1. Determination of sensory evaluation of refrigerated sea bass meat

[0173] Use a 10-point scale to score the color, odor, and tissue state of refrigerated sea bass meat. The lowest quality score is 0 points, the highest quality score is 10 points, and the lowest acceptable score is 5 points. According to the sensitivity of the evaluation team members to the indicators, determine the weights of each item: odor: 0.4; tissue state: 0.3; color: 0.3. The specific evaluation criteria are shown in Table 5.

[0174] Table 5 Sensory evaluation criteria for refrigerated sea bass meat

[0175]

[0176] Calculate the sensory score of fish meat according to the following formula:

[0177] Sensory score of fish meat = odor × 0.4 + texture × 0.3 + color × 0.3.

[0178] After treating sea bass with microbial agent 1 and microbial agent 2 for preservation, the sensory evaluation results of the fish meat are shown in Figure 6 .

[0179] As Figure 6 can be seen, with the extension of storage time, the sensory score of sea bass fish meat shows a downward trend. Under the storage condition of 4°C, the sensory scores of experimental group 1 and experimental group 2 are significantly higher than those of the control group, and the sensory scores of experimental group 1 and experimental group 2 are not lower than the lowest acceptable range (5 points). The sensory score of the control group was 4.5 points on the 9th day, which was already lower than the acceptable range, while the sensory scores of experimental group 1 and experimental group 2 were 6.2 points and 5.7 points respectively on the 12th day, both of which were not lower than the lowest acceptable range.

[0180] The above results indicate that both microbial agent 1 made from the fermentation supernatant of Lactobacillus plantarum SQ03 and microbial agent 2 made from the bacterial sludge of Lactobacillus plantarum SQ03 can effectively delay the deterioration of the quality of sea bass during storage.

[0181] 2. Determination of total volatile basic nitrogen (TVBN) in refrigerated sea bass fish meat

[0182] After treating sea bass with microbial agent 1 and microbial agent 2 for preservation, the determination results of TVBN in the fish meat are shown in Figure 7 .

[0183] As Figure 7 can be seen, during storage, TVBN in the sea bass fish meat of experimental group 1, experimental group 2 and the control group all showed an upward trend, but the growth rates of experimental group 1 and experimental group 2 were slower. After 6 days of storage, TVBN in the fish meat of the control group reached 20.2 mg / 100 g, which had reached the inedible state. During the same period, TVBN in the fish meat of experimental group 1 and experimental group 2 were 12.2 mg / 100 g and 13.8 mg / 100 g respectively, and had not reached the inedible standard yet. With the extension of storage time, the difference in TVBN between experimental group 1 and experimental group 2 and the fish meat of the control group gradually increased, and experimental group 1 and experimental group 2 were significantly lower than the control group.

[0184] The above results indicate that both microbial agent 1 made from the fermentation supernatant of Lactobacillus plantarum SQ03 and microbial agent 2 made from the bacterial sludge of Lactobacillus plantarum SQ03 can significantly inhibit the growth of microorganisms in fish meat (most of the production of TVBN is caused by spoilage microorganisms in fish meat), thereby reducing the production of alkaline substances caused by microbial action.

[0185] 3. Determination of Thiobarbituric Acid (TBA) in Refrigerated Sea Bass Fillets

[0186] After treating sea bass with microbial agent 1 and microbial agent 2 for preservation, the determination results of TBA in the fish fillets are shown in Figure 8 .

[0187] As can be seen from Figure 8 , as the storage time prolongs, the TBA in sea bass fillets first increases and then decreases. After 6 days of storage, the TBA in experimental group 1 and experimental group 2 is significantly lower than that in the control group. The TBA in experimental group 1, experimental group 2 and the control group did not exceed 2 mg / kg during storage, that is, they did not exceed the national safety standard. Generally speaking, compared with the control group, the TBA in experimental group 1 and experimental group 2 is significantly reduced, and the quality of sea bass fillets during storage can be better maintained.

[0188] The above results indicate that microbial agent 1 made from the fermentation supernatant of Lactobacillus plantarum SQ03 and microbial agent 2 made from the bacterial sludge of Lactobacillus plantarum SQ03 can both effectively slow down the lipid oxidation and rancidity of sea bass fillets.

[0189] 4. Determination of Total Colony Count

[0190] Accurately weigh 3 g of fresh sea bass fillets in a super clean and sterile workbench and cut them into pieces. Then add them into test tubes containing 9 mL of sterile distilled water (control group), test tubes containing 9 mL of preparation solution 1 (experimental group 1), and test tubes containing 9 mL of preparation solution 2 (experimental group 2). Each test tube contains 1 g. Vortex thoroughly to make the fish fillets fully mixed. Take 1 mL of dilution solution with different dilution gradients, place it in a sterilized petri dish, mix it with plate count agar medium, and perform pour plate culture. Continuously culture at 30 ± 1 °C for 5 days, record the colony number every day, and calculate the total colony count according to the dilution factor. The results are expressed as the logarithm of the total colony count.

[0191] After treating sea bass with microbial agent 1 or microbial agent 2, the determination results of the total colony count of the fish fillets are shown in Figure 9 .

[0192] As can be seen from Figure 9 , as the refrigeration time prolongs, the total colony count of sea bass fillets in experimental group 1, experimental group 2 and the control group all increases rapidly, indicating that microorganisms can still grow and reproduce in large numbers during refrigeration at 4 °C. The total number of bacteria in the control group reached the maximum limit of secondary freshness (total food bacteria (CFU / g) ≤ 5×10 5 is secondary freshness) on the 3rd day, while the total number of bacteria in experimental group 1 and experimental group 2 did not reach the maximum limit of primary freshness (total food bacteria (CFU / g) ≤ 10 5When it comes to the first-class freshness, the total number of bacteria in experimental group 1 and experimental group 2 has always been much smaller than that in the control group.

[0193] The above results show that both microbial agent 1 and microbial agent 2 can effectively inhibit the growth of spoilage bacteria and have significant antibacterial effects.

[0194] 5. Determination of the hardness and elasticity of refrigerated sea bass meat

[0195] After using microbial agent 1 and microbial agent 2 to preserve sea bass, the measurement results of the hardness of sea bass meat are shown in Figure 10 , and the measurement results of elasticity are shown in Figure 11 .

[0196] From Figure 10 and Figure 11 , it can be seen that after culturing for a period of time, the hardness and elasticity of the meat quality of sea bass in experimental group 1, experimental group 2 and the control group all showed a downward trend, but the decrease in elasticity and hardness in experimental group 1 and experimental group 2 was slower.

[0197] The above results show that microbial agent 1 and microbial agent 2 can maximize the storage quality of seafood.

[0198] IX. Research on the fermentation of fruit and vegetable enzyme by Lactobacillus plantarum SQ03

[0199] Since microbial agent 1 does not contain live bacteria, while microbial agent 2 contains cross-linked and embedded live bacteria and can continue to ferment in an acidic environment, it is predicted that microbial agent 2 has the potential to ferment fruit and vegetable enzymes.

[0200] Using mulberry as the fermentation raw material and Lactobacillus plantarum SQ03 as the fermentation strain, the preparation of mulberry enzyme was studied.

[0201] 1. Preparation method of mulberry enzyme

[0202] Take fresh mulberry, first wash it with pure water, then wash it with a 0.1% (w / v) sodium hypochlorite solution for 2 min, and finally rinse it with sterile distilled water. Use a juicer to juice the washed mulberry, and then filter to remove the mulberry residue to obtain the mulberry stock solution. Place the mulberry stock solution in a glass jar, pasteurize it and cool it to room temperature. Inoculate microbial agent 2 into the mulberry stock solution at an inoculation amount of 10 g / L, and continuously ferment at 30 °C for 72 h to obtain mulberry enzyme.

[0203] 2. Quality evaluation of mulberry enzyme

[0204] Using the mulberry stock solution without inoculating microbial agent 2 as the control group and the mulberry enzyme obtained above as the experimental group, the total phenol content, soluble solid content and pH value were measured, and a sensory evaluation was carried out. The quality evaluation results are shown in Table 6.

[0205] Table 6 Comparison results of physical and chemical indexes between mulberry juice stock solution and mulberry enzyme

[0206]

[0207] As can be seen from Table 6, the total phenol content of mulberry enzyme is 2.6 times that of mulberry juice stock solution, and the former has strong antioxidant ability; the pH value of mulberry enzyme decreases compared with that of mulberry juice stock solution, which will effectively improve the flavor and physical quality of the drink; the soluble solid content of mulberry enzyme is 5.64%, meeting the standard that the soluble solid content of fermented fruit and vegetable juice should be greater than 5.0% specified in GB / T31121-2014 "Fruit and Vegetable Juices and Their Beverages"; the sensory evaluation of mulberry enzyme is significantly better than that of mulberry juice stock solution.

[0208] The above results show that Lactobacillus plantarum SQ03 has good application potential in the field of fermentation preparation of fruit and vegetable enzymes.

[0209] X. Preservation of Lactobacillus plantarum SQ03

[0210] From the use effects of microbial preparation 1 and microbial preparation 2 prepared by fermenting with Lactobacillus plantarum SQ03 provided above, it can be seen that Lactobacillus plantarum SQ03 screened in the present invention has very high practical application value in the fields of antioxidant, antibacterial preservation, and fermentation of fruit and vegetable enzymes. Therefore, the strain of Lactobacillus plantarum SQ03 was preserved, and the preservation information is as follows:

[0211] The preservation name is Lactobacillus plantarum SQ03, the Latin name is Lactobacillus plantarum SQ03, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation date is May 14, 2024, the preservation number is CCTCC M 2024921, and the location of the preservation unit is Wuhan, China.

[0212] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention and are not limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. Lactobacillus plantarum ( Lactobacillus plantarum ) SQ03, characterized in that, It was isolated and purified from the visceral coelomic fluid of sea cucumber and deposited in China Center for Type Culture Collection on May 14, 2024 with the deposit number CCTCC M 2024921.

2. A microbial preparation prepared by fermentation of Lactobacillus plantarum SQ03 according to claim 1, characterized in that: Prepared by the following method: (1) inoculating the Lactobacillus plantarum SQ03 described in claim 1 onto MRS solid culture medium and inverting and culturing at 37°C; (2) Pick up the bacterial moss from the above-mentioned MRS solid culture medium, transfer it to the MRS liquid culture medium, and culture it at 37°C with shaking to obtain the seed liquid; (3) The seed solution was inoculated into fermentation medium A, and the mixture was statically cultured at 37° C. to obtain fermentation liquid A, wherein the formula of fermentation medium A was as follows: 21.0 g / L glucose, 10.0 g / L peptone, 8.0 g / L beef extract powder, 8.0 g / L yeast extract powder, 3.5 g / L dipotassium hydrogen phosphate, 6.5 g / L sodium acetate, 2 g / L diammonium hydrogen citrate, 0.2 g / L magnesium sulfate, 0.13 g / L adenine, 0.051 g / L phenylalanine, and 1 mL / L Tween 80; (4) The fermentation liquid A was transferred to fermentation medium B, and the culture was shaken at 37°C to obtain fermentation liquid B, wherein the formula of fermentation medium B is as follows: glucose 16.0 g / L, peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 6.0 g / L, dipotassium hydrogen phosphate 3.5 g / L, sodium acetate 6.5 g / L, diammonium hydrogen citrate 2.0 g / L, magnesium sulfate 0.5 g / L, calcium chloride 1.0 g / L, L-glutamic acid 5.0 g / L, manganese sulfate 0.1 g / L, vitamin C 0.01 g / L, adenine 0.15 g / L, phenylalanine 0.05 g / L, Tween 80 1 mL / L; (5) Transfer the fermentation broth B to fermentation medium A and culture at 37°C with shaking until the maximum viable count of the fermentation broth reaches 9.4×10 9 CFU / mL, and fermentation broth C was obtained; (6) performing solid-liquid separation on the fermentation liquid C, and collecting the fermentation supernatant and the bacterial sludge respectively; (7) The fermentation supernatant is sequentially subjected to ultrafiltration concentration, addition of protective agent trehalose, homogenization, and low-temperature spray drying to obtain microbial preparation 1; (8) The above-mentioned bacterial sludge is sequentially added with wall materials, homogenized, and vacuum-freeze-dried to obtain microbial preparation 2.

3. The microbial preparation according to claim 2, characterized in that In step (3) and step (5), the fermentation medium A is prepared with purified seawater, and the pH is adjusted to 6.2±0.

1.

4. The microbial preparation according to claim 2, characterized in that In step (4), the fermentation medium B is prepared using purified seawater.

5. The microbial preparation according to claim 2, characterized in that: In step (5), sterile ventilation is performed once every 12 hours during the fermentation period.

6. The microbial preparation according to claim 2, characterized in that In step (7), the protective agent trehalose is added to the concentrated solution at a weight ratio of 1:

10.

7. The microbial preparation according to claim 2, characterized in that In step (8), the wall material is selected from any one or any combination of gum arabic, maltodextrin, whey protein and alginate.

8. The microbial preparation according to claim 7, characterized in that The wall material is obtained by mixing gum arabic, maltodextrin, whey protein and alginate in a weight ratio of 5:4:1:5, and the weight ratio of the composite wall material to the bacterial mud is 1:

1.

9. The use of the microbial preparation 1 described in claim 2 in the following aspects: (1) Antioxidant effects of fruits and vegetables, fruit and vegetable-related solid / liquid beverages, and skin care products; (2) Antibacterial preservation of seafood for non-disease treatment purposes, where the pathogenic bacteria are Shewanella, Pseudomonas, Vibrio alginolyticus, Staphylococcus aureus or Aeromonas hydrophila.

10. Use of the microbial preparation 2 described in claim 2 in the following aspects: (1) Antibacterial preservation of seafood for non-disease treatment purposes, where the pathogenic bacteria are Shewanella, Pseudomonas, Vibrio alginolyticus, Staphylococcus aureus or Aeromonas hydrophila; (2) Fruit and vegetable enzyme fermentation.

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