Streptococcus paris W3B and application of fermentation product of streptococcus paris W3B in fresh keeping of fish meat

Abstract

The invention relates to application of Streptococcus paris W3B and a fermentation product of the Streptococcus paris W3B in fresh keeping of fish meat. The preservation number of the Streptococcus paris W3B is CGMCC (China General Microbiological Culture Collection Center) No.32831. The invention further relates to a preparation method of the Streptococcus paris W3B and the fermentation product of the Streptococcus paris W3B. The streptococcus paris W3B and / or the fermentation product of the streptococcus paris W3B disclosed by the invention have / has an antibacterial effect and / or a sterilization effect on dominant putrefying bacteria of fish meat, and the streptococcus paris W3B and / or the fermentation product of the streptococcus paris W3B have / has an antibacterial effect and / or a sterilization effect; the fish preservative can delay oxidation of fish protein and fat, can be applied to fresh keeping of fish as a fresh keeping agent, and can improve the fresh keeping effect of the fish and prolong the shelf life of the fish.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial application, and particularly relates to the application of Streptococcus Parisi W3B and its fermentation products in fish preservation. Background Art

[0002] Fish storage (or preservation) has become a challenge hindering the industry's development. A report by the Food and Agriculture Organization of the United Nations (FAO) indicates that approximately 35% of fish and aquatic products are lost and wasted annually in the global supply chain. Microbial contamination accounts for 10-12 million tons of fish losses, representing approximately 15-25% of total losses. Therefore, inhibiting the growth and reproduction of microorganisms in post-slaughter fish, preventing spoilage, extending its shelf life, and reducing fish losses and waste are pressing challenges for my country's fish storage, preservation, and processing industries.

[0003] With the advancement of technology, the mainstream methods of fish storage and preservation are low temperatures, vacuum packaging, and the addition of preservatives. However, fish meat, with its high moisture content and rich protein and unsaturated fatty acids, is susceptible to spoilage even when refrigerated and vacuum-packed. Therefore, the addition of preservatives is undoubtedly an effective way to preserve fish meat. However, reports of chemical synthetic preservatives causing cancer, teratogenicity, and chronic diseases have led consumers to place higher demands on the safety of preservatives. Biological preservatives, such as probiotics, have gained consumer recognition for their nutritional, health, and safety attributes.

[0004] Lactic acid bacteria, a general term for Gram-positive bacteria that ferment sugars into lactic acid, are widely found in nature as probiotics. As a normal member of the human intestinal flora, lactic acid bacteria offer unique advantages in safety and effectiveness. They are also a key component of various fermented foods, not only harmless to human health but also beneficial to human health. They are excellent alternatives to synthetic preservatives. Therefore, screening for lactic acid bacteria strains suitable for fish preservation will contribute significantly to the development of fish storage and preservation technologies in my country and around the world. Summary of the Invention

[0005] The purpose of the present invention is to overcome the defects in the prior art and provide the use of Streptococcus Parisi W3B and its fermentation products in fish preservation. The fermentation products have good antibacterial effects on the dominant spoilage bacteria in fish, can delay the oxidation of proteins and lipids in fish during storage, improve the preservation effect of fish, and extend the shelf life of fish.

[0006] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0007] Application of Streptococcus lutetiensis W3B and / or its fermentation products in fish preservation.

[0008] As a further technical solution, the Streptococcus lutetiensis W3B is deposited in the China General Microorganism Culture Collection Center with the deposit number CGMCC No.32831.

[0009] As a further technical solution, the nucleotide sequence of the 16S rDNA of Streptococcus lutetiensis W3B is shown in SEQ ID NO.1.

[0010] As a further technical solution,

[0011] The Streptococcus lutetiensis W3B and / or its fermentation product is used as a preservative.

[0012] As a further technical solution, the Streptococcus lutetiensis W3B and / or its fermentation products have broad-spectrum antibacterial properties and have antibacterial effects on both Gram-negative bacteria and Gram-positive bacteria.

[0013] As a further technical solution, the Streptococcus lutetiensis W3B and / or its fermentation products have an antibacterial and / or sterilizing effect on dominant spoilage bacteria in fish.

[0014] As a further technical solution, the Streptococcus Parisi W3B and / or its fermentation products can delay the oxidation of fish protein and fat.

[0015] As a further technical solution, the Streptococcus lutetiensis W3B is a Streptococcus lutetiensis W3B that produces high amounts of exopolysaccharides.

[0016] As a further technical solution, a bacterial suspension of Streptococcus lutetiensis W3B, a bacterial fermentation liquid of Streptococcus lutetiensis W3B, or a sterile fermentation liquid of Streptococcus lutetiensis W3B is applied to the surface of the fish meat, and then the fish meat is placed in a sterile packaging bag and refrigerated for storage.

[0017] The method for preserving fish meat by using Streptococcus lutetiensis W3B and / or its fermentation products comprises the following steps: applying a bacterial suspension of Streptococcus lutetiensis W3B, a bacterial fermentation liquid of Streptococcus lutetiensis W3B, or a sterile fermentation liquid of Streptococcus lutetiensis W3B to the surface of the fish meat, and then placing the fish meat in a sterile packaging bag for refrigerated storage.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The Streptococcus Parisi W3B and its fermentation products obtained by screening in the present invention have good organic acid and exopolysaccharide production capabilities, good safety, antibiotic sensitivity, antioxidant and adhesion capabilities. They can not only inhibit common pathogens but also have good antibacterial ability against dominant spoilage bacteria in fish, inhibit the oxidation of fish protein and fat during fish storage, and achieve fish preservation during storage. They can be used as preservatives in fish preservation.

[0020] The Streptococcus Parisi W3B of the present invention is isolated and obtained from Yunnan Shilin milk cake, is a probiotic, and is safe, environmentally friendly and has no side effects compared to traditional preservatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Plate inhibition graphs of strains W3B, W3C, W3D, and W3E;

[0022] Figure 2 The images are of hemolysis circles of strains W3B, W3C, W3D, and W3E;

[0023] exist Figure 2 Middle, A: positive control; B: W3B; C: W3C; D: W3D; E: W3E;

[0024] Figure 3 Colony morphology of Streptococcus lutetiensis W3B;

[0025] Figure 4 This is the Gram staining image of Streptococcus lutetiensis W3B;

[0026] Figure 5 This is the phylogenetic tree of Streptococcus lutetiensis W3B; DETAILED DESCRIPTION

[0027] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0028] In the present invention,

[0029] MRS medium: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L sucrose, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium citrate, 5 g / L sodium acetate, 0.4 g / L magnesium sulfate heptahydrate, 0.1 g / L manganese sulfate tetrahydrate, 20 g / L agar powder added to solid medium, pH = 6.1; LB medium: 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract;

[0030] MSgg medium: 5 mmol / L potassium phosphate, 100 mmol / L 3-(N-morpholino)propanesulfonic acid sodium salt, 2 mmol / L magnesium chloride, 0.7 mmol / L calcium chloride, 0.05 mmol / L ferric chloride, 0.05 mmol / L manganese chloride, 1 μmol / L zinc chloride, 1 μmol / L thiamine, 0.5% glycerol, 0.5% glutamic acid, 50 mg / L tryptophan, 50 mg / L phenylalanine;

[0031] Sporulation medium: 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast powder, 0.1 g / L calcium chloride, 0.04 g / L manganese sulfate tetrahydrate, 20 g / L agar powder.

[0032] Unless otherwise specified, the raw materials used in the present invention are all commercially available raw materials.

[0033] Example 1: Isolation of strains

[0034] 1. Isolation of strains: Weigh 10 g of homemade milk cake from farmers in Shilin, Yunnan, add 90 mL of sterilized water, shake on a shaker at 180 rpm for 20 min to prepare a bacterial suspension, draw 1 mL of the bacterial suspension for gradient dilution, and take 10 -3 , 10 -4 , 10 -5 The three gradients were evenly spread on MRS solid plates, cultured at 37°C for 1-3 days, single colonies were picked, purified to pure culture, and named W3A, W3B, W3C... in sequence. The inhibition zone of Aeromonas hydrophila was used as an indicator to screen lactic acid bacteria strains that could inhibit Aeromonas hydrophila.

[0035] 2. Results: A total of 12 single colonies were selected and named W3A-W3K. Among them, strains W3B, W3C, W3D, and W3E had antibacterial effects, and W3B had the largest inhibition zone ( Figure 1 ).

[0036] Example 2: Antibacterial activity study (strain screening)

[0037] 1. Preparation of pathogen suspension: Take out the frozen pathogen suspension (Escherichia coli ATCC35218, Salmonella Enteritidis BNCC184946, Salmonella Paratyphi CMCC 50094, Pseudomonas aeruginosa ATCC 27853, Vibrio parahaemolyticus ATCC 17802, Aeromonas hydrophila 3CA, Aeromonas welchii 5SB, Streptococcus pyogenes ATCC 19615, Listeria monocytogenes ATCC 19115, Bacillus cereus ATCC 14579, Staphylococcus aureus ATCC 29213) from the -80℃ freezer, streak it onto LB solid plate, culture it in a 37℃ incubator overnight, then take a single colony and transfer it to 3 mL LB liquid medium, culture it at 37℃, 180 rpm overnight, dilute the cultured bacterial suspension with physiological saline to prepare a bacterial suspension (1×10 8 CFU / mL).

[0038] 2. Preparation of sterile lactic acid bacteria fermentation broth: Remove frozen W3B, W3C, W3D, and W3E bacterial cultures from a -80°C freezer, streak onto MRS solid plates, and incubate in a 37°C incubator overnight. Then, transfer a single colony from the culture to 50 mL of MRS liquid medium and incubate at 37°C, 180 rpm, and shake for 36 hours to prepare the seed culture. Transfer the seed culture to 250 mL of MRS liquid medium at a 2% inoculum volume and incubate at 37°C, 180 rpm, and shake for 36 hours. Centrifuge at 4°C, 10,000 rpm, and collect the supernatant. Filter through a 0.22 μm filter to prepare the sterile fermentation broth.

[0039] 3. Determination of antibacterial activity (inhibitory spectrum):

[0040] Take 50 μL of pathogen suspension and evenly spread it on an LB solid plate. After drying, punch a hole on the plate and add 50 μL of Streptococcus lutetiensis W3B sterile fermentation broth to each well. Use 50 μL of sterile MRS liquid medium as a negative control. After incubation at 37°C for 24 h, observe and measure the diameter of the inhibition zone. The results are shown in Table 2.

[0041] 4. Results

[0042] Table 1 Antibacterial spectrum of each strain

[0043]

[0044] Note: The value indicates the diameter of the inhibition zone (mm), and - indicates no inhibition zone.

[0045] The data in Table 1 show that strain W3B has a broad-spectrum antibacterial activity against Gram-negative bacteria such as Escherichia coli ATCC 35218, Salmonella Enteritidis BNCC184946, Salmonella Paratyphi CMCC 50094, Pseudomonas aeruginosa ATCC 27853, Vibrio parahaemolyticus ATCC17802, Aeromonas hydrophila 3CA, and Aeromonas vermiformis 5SB, and Gram-positive bacteria such as Streptococcus pyogenes ATCC 19615, Listeria monocytogenes ATCC 19115, Bacillus cereus ATCC 14579, and Staphylococcus aureus ATCC 29213. The antibacterial effect is superior to that of strains W3C, W3D, and W3E.

[0046] Example 3: Antioxidant Study (Strain Screening)

[0047] 1. Preparation of sterile fermentation broth: same as Example 2;

[0048] 2. Determination of antioxidant properties:

[0049] 0.5 ml of sterile fermentation broth of W3B, W3C, W3D and W3F was taken respectively, and 1.5 ml of 2 mmol / L FeSO4, 1.5 ml of 6 mmol / L salicylic acid and 1.5 ml of 1 mmol / L H2O2 solution were added to a 10 ml centrifuge tube in sequence. After mixing, the mixture was placed in the dark at 37°C for 30 minutes and the absorbance at 562 nm was measured. Hydroxyl radical (·OH) scavenging rate (%) = [1-(A s -A c ) / A b ]×100, where A s is the absorbance value of the reaction system with fermentation broth added, A c is the absorbance value of the reaction system when deionized water is substituted, A b The absorbance values ​​of the reaction system in which deionized water was used instead of the fermentation liquid are shown in Table 2.

[0050] 3. Results

[0051] Table 2: Antioxidant properties of various strains

[0052] strain W3B W3C W3D W3E OH (%) 95.26±0.38 87.56±0.22 86.06±0.09 93.94±0.19

[0053] The antioxidant capacity of a strain can generally be measured by evaluating its ability to scavenge hydroxyl radicals. As shown in Table 2, strains W3B, W3C, W3D, and W3E are capable of scavenging hydroxyl radicals, with W3B having the highest hydroxyl radical scavenging rate, reaching 95.26%, and therefore possessing the highest antioxidant activity.

[0054] Example 4: Safety Performance Study of Lactic Acid Bacteria (Strain Screening)

[0055] 1. Hemolytic assay

[0056] Hemolysis is an important indicator for evaluating the safety of strains. Strains W3B, W3C, W3D, and W3E were spotted onto Columbia blood agar plates and cultured at 37°C for 24 hours to observe the hemolysis zone. Bacillus cereus ATCC 14579 was used as a positive control. The results are shown in Table 1. Figure 2 .

[0057] 2. Measurement results: Figure 2 It can be seen that strains W3B, W3C, W3D, and W3E did not form a hemolytic zone, so there was no hemolysis phenomenon and they can be used safely.

[0058] Example 5: Antibiotic sensitivity study (strain screening)

[0059] 1. Test method: Use commercial drug sensitivity paper to determine antibiotic sensitivity. Strain W3B, W3C, W3D, and W3E bacterial suspensions were diluted with physiological saline to a concentration of 1×10 8 CFU / mL, and then 50 μL of the diluted bacterial solution was evenly spread on MRS solid culture medium. After drying, 13 antibiotic sensitivity papers were placed on the plate respectively. After incubation at 37°C for 24 h, the antibacterial effect was observed and the diameter of the inhibition zone was measured. The results are shown in Table 3.

[0060] 2. Test results

[0061] Table 3 Antibiotic sensitivity of each strain

[0062] antibiotic W3B W3C W3D W3E penicillin 20 32 16 21 Ampicillin 24 28 18 20 Oxypiperazine penicillin 18 17 17 14 Cefazolin 18 24 14 16 Cefoperazone 16 24 8 18 Butamican 18 15 14 26 Gentamycin 16 31 0 18 Streptomycin 18 32 15 20 tetracycline 20 31 34 16 Minocycline 14 33 16 17 Vancomycin 20 0 25 0 doxycycline 15 28 18 24

[0063] Note: Diameter of inhibition zone (mm)

[0064] Antibiotic susceptibility is also an important indicator for evaluating strain safety. In this test, 12 common antibiotics, including penicillin (10 μg), ampicillin (10 μg), piperacillin (100 μg), cefazolin (30 μg), cefoperazone (75 μg), amikacin (30 μg), gentamicin (10 μg), streptomycin (10 μg), tetracycline (30 μg), minocycline (30 μg), vancomycin (30 μg), and doxycycline (30 μg), all formed inhibition zones on the W3B plate. Therefore, strain W3B is sensitive to all 12 antibiotics and can be safely used in food. Strains W3C and W3E are resistant to vancomycin, and W3D is resistant to gentamicin. The spread of resistant strains within the food chain should be considered when using strains W3C, W3D, and W3E in food preservation.

[0065] In summary, strain W3B has the best antibacterial, antioxidant and safety properties. Therefore, the present invention selected strain W3B for further research.

[0066] Example 6: Identification of strain W3B

[0067] 1. Morphological and Gram staining identification: Figure 3 As shown, after strain W3B was cultured on MRS plates at 37°C for 2 days, the colonies were small, milky white, round and convex, with smooth edges and a moist surface. Figure 4 As shown, Gram staining is blue, the bacterial cells are spherical, and they are Gram-positive bacteria.

[0068] 2. 16S rRNA molecular identification and phylogenetic tree

[0069] Strain W3B was identified by 16S rRNA sequencing. Universal primers 27F (5′-GTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) were used for PCR amplification. The PCR products were sequenced and analyzed. The nucleotide sequence of 16S rDNA of strain W3B is shown in SEQ ID NO. 1. Phylogenetic tree was constructed using MEGA 5.1 software. The results are shown in Figure 5 .

[0070] SEQ ID NO.1:

[0071]

[0072] The 16S rDNA gene sequence of strain W3B was 1451 bp in length.

[0073] After identification, it was determined that strain W3B belongs to Streptococcus lutetiensis, named Streptococcus lutetiensis W3B, and deposited in the China General Microbiological Culture Collection, the deposit address is: No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit date is November 28, 2024; the deposit number is CGMCC No.32831.

[0074] Example 7: Study on the inhibition of pathogen biofilm and spore germination by Streptococcus Parisi W3B

[0075] 1. Determination of inhibition of biofilm formation of Bacillus cereus and Pseudomonas aeruginosa

[0076] 50 μL of Bacillus cereus suspension was added to 1 mL of MSgg medium and mixed thoroughly. Simultaneously, 50 μL of Pseudomonas aeruginosa suspension was added to 1 mL of LB medium and mixed thoroughly. These served as seed solutions for Bacillus cereus and Pseudomonas aeruginosa, respectively. 100 μL of the pathogen seed solution was incubated at 37°C for 12 h. Then, 50 μL of W3B sterile fermentation broth was added to each of the two cultures. A control group was treated with 50 μL of sterile culture medium, shaken thoroughly, and incubated at 37°C for another 24 h. The relative biofilm formation was determined using crystal violet staining. The results are shown in Table 4.

[0077] Biofilm formation inhibition rate (%) = (A 对照 -A 处理 ) / A 对照 ×100;

[0078] Where: A 处理 is the absorbance of the sterile fermentation broth group at 595 nm, A 对照 is the absorbance at 595 nm of the control group;

[0079] For the crystal violet staining method, the 96-well cell culture plate was incubated at 37°C for 24 hours. The liquid culture medium was gently removed, leaving the surface biofilm. Each well was washed twice with 200 μL of sterile saline solution, then fixed with 200 μL of methanol for 20 minutes. After removing the methanol, the plate was dried at 60°C for 20 minutes. Then, 250 μL of crystal violet was added for staining for 10 minutes. The plate was rinsed with sterile saline to remove excess dye. After drying at 60°C for 20 minutes, anhydrous ethanol was added and the plate was incubated at 37°C for 30 minutes to dissolve the crystal violet. The absorbance at 595 nm in each well was measured, indicating the biofilm content.

[0080] 3) Determination of inhibition of Bacillus cereus spore germination

[0081] Take 50 μL of Bacillus cereus suspension and spread it on the spore-forming medium. After culturing at 30°C for 7 days, scrape the colonies on the plate, resuspend and wash with 10 mL of sterile saline, and centrifuge at 10,000 r / min for 10 minutes until milky white. Resuspend the precipitate with 10 mL of 50% ethanol solution and let it stand at 4°C for 12 hours. Then wash it three times with sterile saline and resuspend the precipitate with 10 mL of saline. This is the Bacillus cereus spore. The spore concentration after counting is 5×10 8 100 μL of Bacillus cereus spores were added to 900 μL of LB medium, followed by 500 μL of W3B sterile fermentation broth. 500 μL of sterile culture medium was added to the control group, and the mixture was shaken and allowed to stand at 37°C for 12 h. 100 μL of the mixture was diluted to an appropriate multiple, and the colony counts were performed on the plates. The spore germination inhibition rate was calculated. The results are shown in Table 4.

[0082] Bacillus germination inhibition rate (%) = (N 对照 -N 处理 ) / N 对照 ×100

[0083] Where: N 处理 is the number of spore colonies in the sterile fermentation broth group, N 对照 is the number of spore colonies in the control group.

[0084] Table 4. Properties of Streptococcus Parisi W3B in inhibiting pathogen biofilm and spore germination

[0085]

[0086] Biofilm formation has long been a serious problem in the food processing industry. Pathogen biofilm formation can enhance bacterial adaptability to harsh conditions such as environmental stress, nutrient deprivation, and sterilization, making it difficult to remove during food storage and processing. Furthermore, spores formed by Bacillus cereus are also highly resistant to common physical sterilization methods and various preservatives. Consequently, the formation of pathogen biofilms and Bacillus cereus spores often causes food spoilage, resulting in significant economic losses for producers. As shown in Table 4, Streptococcus lutetiensis W3B can inhibit the formation of Pseudomonas aeruginosa and Bacillus cereus biofilms, with inhibition rates of 58.40 ± 1.32% and 64.40 ± 1.39%, respectively. Streptococcus lutetiensis W3B can also inhibit the germination of Bacillus cereus spores, with an inhibition rate of 76.00 ± 4.54%.

[0087] Example 8: Study on the Surface Characteristics (i.e. Adhesion Ability) of Streptococcus Parisi W3B Cells

[0088] 1. Test method

[0089] 1) Preparation of bacterial suspension: Overnight cultures of Streptococcus lutetiensis W3B, Escherichia coli, and Staphylococcus aureus were centrifuged at 10,000 rpm for 10 min. The pellet was collected, washed 2-3 times with PBS, and resuspended. The OD was adjusted. 600 When the concentration of HCl is 0.7, the bacterial suspension is obtained for use.

[0090] 2) Hydrophobicity determination: 3 mL of Streptococcus lutetiensis W3B suspension was added with 1 mL of xylene, n-hexane, dichloromethane, ethyl acetate, and n-hexadecane, respectively. The mixture was vortexed and allowed to stand at 37°C for 3 h to allow separation. 1 mL of the lower aqueous phase was aspirated and the OD was measured. 600 The absorbance value of PBS buffer was used as blank control. Hydrophobicity (%) = (1-A t / A0)×100, where A0 and A t are the absorbance values ​​measured after mixing with the solvent for 0 h and 3 h, respectively.

[0091] 3) Self-aggregation ability: Take 3 mL of W3B bacterial suspension and let it stand at 37°C for 3 h, then take 1 mL of the upper bacterial suspension to measure the OD 600 The absorbance value of the self-aggregation rate (%) = (1-A t / A0)×100, where A0 and A t These are the absorbance values ​​measured at 0 h and 3 h, respectively.

[0092] 4) Coaggregation ability: Take 2 mL of W3B bacterial suspension, add 2 mL of Escherichia coli or Staphylococcus aureus suspension, vortex mix, and let stand at 37°C for 3 hours. Then take 1 mL of the upper bacterial suspension to measure the OD 600 Absorbance value. Co-aggregation rate (%) = (1-A t / A0)×100, where A0 and A t Respectively represent the absorbance values ​​at 0 h and 3 h after W3B was mixed with pathogenic bacteria.

[0093] 2. Test results

[0094] Table 5 Hydrophobicity and aggregation ability of Streptococcus lutetiensis W3B

[0095]

[0096]

[0097] Bacterial surface properties are one of the most important driving forces that determine the nonspecific adhesion of bacteria to various biological and non-biological surfaces and interfaces. The hydrophobicity of the bacterial surface has very important ecological significance for the interaction between bacteria and organisms and other bacteria. It can compete with spoilage bacteria for adhesion and colonization on the surface of fish meat, thereby inhibiting harmful bacteria in fish meat. Studies have shown that the adhesion ability of lactic acid bacteria is related to the hydrophobicity of the strain surface, self-aggregation and co-aggregation ability, etc. Strains with higher adhesion ability also have high surface hydrophobicity and coagulation ability.

[0098] The results of this study showed that Streptococcus lutetiensis W3B exhibited strong hydrophobicity, self-aggregation, and co-aggregation abilities. The hydrophobicity of strain W3B in xylene, n-hexane, dichloromethane, ethyl acetate, and n-hexadecane was 12.10±0.59%, 27.71±0.99%, 28.90±1.08%, 39.73±1.08%, and 33.97±0.80%, respectively. Its self-aggregation rate was 44.87±0.36%, its co-aggregation rate with Escherichia coli was 10.65±0.30%, and its co-aggregation rate with Staphylococcus aureus was 29.06±0.20% (Table 5). In this study, the microbial adhesion to hydrocarbons (MATH) method was used to evaluate the surface hydrophobicity, self-aggregation ability, and co-aggregation ability of the strain towards two pathogenic bacteria, Escherichia coli and Staphylococcus aureus, providing a basis for using the surface hydrophobicity of the strain to control pathogens in food matrices.

[0099] Example 9: Study on the ability of Streptococcus Parisi W3B to produce exopolysaccharides

[0100] 1. Preparation of sterile fermentation broth: Streptococcus lutetiensis W3B was inoculated into 3 mL of liquid MRS medium and cultured at 37°C for 24 h. Then, 2 mL was transferred to 100 mL of liquid MRS medium and cultured at 37°C for 36 h. The supernatant was collected and filtered through a 0.22 μm filter to prepare the sterile fermentation broth.

[0101] 2. Extraction of extracellular polysaccharides: trichloroacetic acid was added to the sterile fermentation broth to a final concentration of 4%. After standing at 4°C for 12 h, the solution was centrifuged at 10,000 r / min for 10 min. Two volumes of anhydrous ethanol were added to the supernatant. After standing at 4°C for 12 h, the solution was centrifuged at 10,000 r / min for 10 min. The precipitate was re-dissolved in sterile deionized water and dialyzed for 24 h, during which the water was changed every 6 h to obtain a crude polysaccharide solution. The crude polysaccharide was then freeze-dried in vacuo to obtain the crude polysaccharide.

[0102] 3. Determination of extracellular polysaccharide content: The phenol-sulfuric acid method was used to determine the extracellular polysaccharide production, and a standard curve was drawn using glucose as the standard. 100 μL of 50, 100, 200, 400, 600, 800, and 1000 mg / L glucose standard solutions were taken, 300 μL of deionized water was added, and then 200 μL of 6% phenol was added. Mix well, and quickly add 1 mL of concentrated sulfuric acid. Boil in a boiling water bath for 15 minutes, and measure the optical density at a wavelength of 490 nm. The standard curve was drawn with glucose mass concentration as the horizontal axis and optical density as the vertical axis, and the regression equation y=0.0039x+0.0033(R 2 =0.9994). Under the same conditions, the optical density of the exopolysaccharide solution at 490 nm was measured, and the exopolysaccharide yield was calculated using the regression equation.

[0103] 4. Result analysis:

[0104] The exopolysaccharides produced by lactic acid bacteria have antibacterial, anti-inflammatory, antioxidant, anti-tumor, antiviral, and immunomodulatory properties, and have broad application prospects in the fields of food, medicine, and biotechnology. Therefore, determining the exopolysaccharide production capacity of Streptococcus lutetiensis W3B can, to a certain extent, reflect its effect on fish preservation. The results of this experiment showed that the exopolysaccharide yield of Streptococcus lutetiensis W3B was relatively high, at 461.48±8.62 mg / L.

[0105] Example 10: Study on the ability of Streptococcus Parisi W3B to produce organic acids

[0106] 1. Preparation of sterile fermentation broth: same as Example 9;

[0107] 2. Determination of organic acid production: Dilute 1 mL of sterile fermentation broth 10-fold with CO2-free distilled water, mix thoroughly, add 2-3 drops of 1% phenolphthalein indicator, and titrate with a standardized 0.1 mol / L NaOH solution. The titration endpoint is when a slightly reddish color does not fade within 30 seconds. Record the volume of NaOH. Use MRS medium as a blank control. The formula for calculating acid production is:

[0108] Acid production (g / L) = [(V1-V2)×C×F×0.09] / V0×1000;

[0109] Where: C refers to the molar concentration of the NaOH standard solution (0.1 mol / L); V1 refers to the volume (mL) of the NaOH standard solution used for sample titration; V2 refers to the volume (mL) of the NaOH standard solution used for blank control titration; F is the dilution factor; V0 is the volume (mL) of the sample or fermentation broth; 0.09 is the organic acid to lactic acid conversion coefficient.

[0110] 3. Results and Analysis

[0111] During their metabolism, lactic acid bacteria produce antimicrobial substances such as organic acids (primarily lactic acid), thereby inhibiting the growth and reproduction of pathogens. Therefore, evaluating the acid-producing capacity of Streptococcus lutetiensis W3B can, to a certain extent, reflect its ability to inhibit spoilage bacteria on the surface of fish. In this study, the organic acid content of Streptococcus lutetiensis W3B was 73.4 ± 3.4 g lactic acid / L.

[0112] Example 11: Study on the Inhibition of Streptococcus Parisi W3B, a Dominant Spoilage Bacteria in Fish

[0113] 1. Test method:

[0114] 1) Preparation of a suspension of dominant spoilage bacteria: Pseudomonas aeruginosa, Aeromonas hydrophila, and Bacillus cereus were selected as the dominant spoilage bacteria, and the preparation method of the bacterial suspension was the same as that in Example 2;

[0115] 2) Preparation of sterile fermentation broth of strain W3B: same as in Example 2;

[0116] 3) Fish antibacterial test: Grass carp (1.5 kg) was stunned, the scales, skin and internal organs were removed, the fish meat was removed, rinsed with sterile water, and minced in a meat grinder. Subsequently, Bacillus cereus, Pseudomonas aeruginosa and Aeromonas hydrophila were inoculated into the minced fish meat respectively, so that the final density of each pathogen reached 3 log CFU / g. Then, 2.0 mL of the sterile fermentation broth of strain W3B was mixed with 20.0 g of fish meat, and commercial nisin (0.5 g / kg, commercially available) was used as a positive control, and sterile distilled water was used as a negative control. The fish meat was placed in a sterile packaging bag and placed in a refrigerator at 4°C. The number of pathogens in the fish meat was determined using differential culture medium on the 2nd and 4th days. The results are shown in Table 6.

[0117] 2. Test results and analysis

[0118] Table 6 Inhibitory and killing effects of Streptococcus lutetiensis W3B on dominant spoilage bacteria

[0119]

[0120] Pseudomonas aeruginosa and Aeromonas hydrophila can also grow and reproduce in low-temperature environments and are the dominant spoilage bacteria in refrigerated fish. Bacillus cereus can form spores and biofilms and has strong stress resistance, making it a key bacterial species causing fish food poisoning. These three bacteria are important bacterial groups that cause spoilage during fish storage. As shown in Table 6, the number of pathogens in the sterile water group increased with the number of days of refrigerated storage: Pseudomonas aeruginosa increased from 3.63 Log10 CFU / g at the initial storage to 3.95 Log10 CFU / g on the fourth day, Aeromonas hydrophila increased from 3.63 Log10 CFU / g at the initial storage to 4.73 Log10 CFU / g on the fourth day, and Bacillus cereus increased from 3.45 Log10 CFU / g at the initial storage to 3.83 Log10 CFU / g on the fourth day. In the Streptococcus lutetiensis W3B and nisin-treated groups, the number of Bacillus cereus and Pseudomonas aeruginosa decreased with extended storage time, while the number of Aeromonas hydrophila initially decreased and then increased, but remained below the number of pathogens in the sterile water group. Furthermore, Streptococcus lutetiensis W3B treatment was more effective against Aeromonas hydrophila than commercial nisin, while its inhibitory effect against Bacillus cereus and Pseudomonas aeruginosa was not significantly different from that of commercial nisin.

[0121] The results of this experiment show that Streptococcus lutetiensis W3B has a good effect on inhibiting and killing dominant spoilage bacteria in fish meat, laying the foundation for solving the problem of fish meat deterioration caused by the growth of spoilage microorganisms during fish storage.

[0122] Example 12: Study on the preservation of fish meat with Streptococcus Parisi W3B

[0123] 1. Preparation of sterile fermentation broth of strain W3B: same as in Example 2;

[0124] 2. Fish Preservation Test: Grass carp (1.5 kg) were stunned, and the scales, skin, and internal organs removed. The fish meat was removed, rinsed with sterile water, and minced in a meat grinder. 2.0 mL of the sterile fermentation broth of strain W3B was mixed with 20.0 g of fish meat. Nisin (0.5 g / kg) was used as a positive control, and sterile distilled water was used as a negative control. The fish meat was placed in sterile packaging and refrigerated at 4°C. Samples were taken on the second and fourth days to determine the total bacterial count, volatile basic nitrogen, and malondialdehyde content. The results are shown in Table 7.

[0125] 1) The determination of total colony count was carried out in accordance with the national standard GB / T 4789.2-2022 "National Food Safety Standard Food Microbiology Examination: Determination of Total Colony Count", and the amount of fish sample was modified to 1.0g.

[0126] 2) Determination of volatile basic nitrogen

[0127] The volatile basic nitrogen value was determined by referring to the microdiffusion method in the national standard GB 5009.228-2016 "National Food Safety Standard Determination of Volatile Basic Nitrogen in Food", and the amount of fish sample was modified to 1.0g.

[0128] 3) Determination of malondialdehyde (TBA)

[0129] Take 1.0g of minced fish sample, add 5mL of 7.5% trichloroacetic acid solution (containing 0.1% EDTA), homogenize for 1 minute, place on a constant temperature oscillator and shake for 30 minutes, filter with double-layer filter paper, take 1mL of the filtrate and add 1mL of 0.02mol / L thiobarbituric acid solution, bathe at 95℃ for 30 minutes, cool to room temperature, add 2mL of chloroform, shake well and let stand to separate, then take the supernatant and measure the absorbance at 532nm and 600nm. The TBA value is expressed as the mass (mg) of malondialdehyde contained in 1kg of fish sample. The TBA value is calculated as follows: TBA value (mg / kg) = (A 532 -A 600 )

[0130] ×4.64 / w, where: A 532 、A 600 is the absorbance of the sample at 532nm and 600nm; w is the mass of the fish sample (g); 4.64 is the extinction constant.

[0131] 3. Test results and analysis

[0132] Table 7 Effect of Streptococcus lutetiensis W3B on the preservation of fish

[0133]

[0134] The total colony count is closely related to the spoilage of fish meat, and volatile basic nitrogen and malondialdehyde are important indicators for evaluating the quality of fish meat.

[0135] From Table 7 we can see that:

[0136] 1) The total colony count in the fish meat treated with sterile water increased with the increase in refrigerated storage days, from an initial 4.29Log10 CFU / g to 6.66Log10 CFU / g on the fourth day. The total colony counts in the Streptococcus lutetiensis W3B and nisin-treated groups decreased with the extension of storage time, reaching 2.97 and 3.01Log10 CFU / g on the fourth day, respectively.

[0137] 2) The volatile basic nitrogen and malondialdehyde values ​​of fish meat showed an upward trend during refrigeration; however, after treatment with strain W3B or commercial nisin, the increasing rate was significantly inhibited, and the inhibitory effect of strain W3B on malondialdehyde production was better than that of commercial nisin.

[0138] In conclusion, Streptococcus lutetiensis W3B has a significant effect on the preservation of refrigerated fish and can be used as a preservative in the preservation of fish, thereby achieving the purpose of extending the shelf life of fish.

[0139] The above-described embodiments are only preferred embodiments of the present invention and are not exhaustive of all feasible implementations of the present invention. For those skilled in the art, any obvious modifications made thereto without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. Application of Streptococcus lutetiensis W3B and / or its fermentation products in fish preservation.

2. The use according to claim 1, characterized in that The Streptococcus lutetiensis W3B is deposited in the China General Microorganism Culture Collection Center with the deposit number of CGMCC No.32831.

3. The use according to claim 1, characterized in that The nucleotide sequence of the 16S rDNA of Streptococcus lutetiensis W3B is shown in SEQ ID NO.

1.

4. The use according to claim 1, characterized in that The Streptococcus lutetiensis W3B and / or its fermentation product is used as a preservative.

5. The use according to claim 1, characterized in that The Streptococcus lutetiensis W3B and / or its fermentation product has broad-spectrum antibacterial properties.

6. The use according to claim 1, characterized in that The Streptococcus lutetiensis W3B and / or its fermentation products have an antibacterial and / or sterilizing effect on dominant spoilage bacteria in fish.

7. The use according to claim 1, characterized in that The Streptococcus lutetiensis W3B and / or its fermentation product can delay the oxidation of fish protein and fat.

8. The use according to claim 1, characterized in that The Streptococcus lutetiensis W3B is a Streptococcus lutetiensis W3B that produces high amounts of exopolysaccharides.

9. The use according to claim 1, characterized in that Streptococcus Parisi lutetiensis W3B, a bacterial suspension of Streptococcus lutetiensis W3B, or a sterile fermentation liquid of Streptococcus lutetiensis W3B is applied to the surface of the fish meat, and then the fish meat is placed in a sterile packaging bag and refrigerated for storage.

10. A method for preserving fish meat using Streptococcus lutetiensis W3B and / or its fermentation product as claimed in claim 1, characterized in that: The method comprises the following steps: applying a bacterial suspension of Streptococcus lutetiensis W3B, a bacterial fermentation liquid of Streptococcus lutetiensis W3B or a sterile fermentation liquid of Streptococcus lutetiensis W3B to the surface of fish meat, and then placing the fish meat in a sterile packaging bag for refrigerated storage.

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