Phytobacterium plantarum and application thereof in processing of small yellow croakers

Abstract

The invention discloses plant lactobacillus and application thereof in processing of small yellow croakers, the plant lactobacillus is classified and named as plant lactobacillus, the strain name is WZ-33, and the plant lactobacillus is preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 28, 2024, and the preservation number is CGMCC NO. 30800. The invention further discloses a preparation method of the plant lactobacillus and application of the plant lactobacillus in processing of the small yellow croakers. When the plant lactobacillus provided by the embodiment of the invention is applied to a small yellow croaker product, the quality and the nutritional function of the product can be effectively improved, and industrial production is easy to realize.

Description

Technical Field

[0001] The invention relates to the fields of microorganisms and food processing, and in particular to a plant lactobacillus and its application in processing small yellow croaker. Background Art

[0002] Lactic acid bacteria (LAB) are a type of facultative anaerobic bacteria that use carbohydrate fermentation to produce lactic acid as the main metabolite. They are commonly found in the fermentation process of traditional foods (yogurt, kimchi, pickled meat, pickled fish, etc.). Lactic acid bacteria are internationally recognized intestinal probiotics and have multiple physiological functions. As normal flora in the intestines of humans and animals, lactic acid bacteria play an important role in maintaining the microecological balance of the intestine and regulating its metabolism; moreover, lactic acid bacteria can ferment in the intestine to produce lactic acid, hydrogen peroxide and antibiotics, which have obvious effects on the prevention and treatment of gastrointestinal diseases; some lactic acid bacteria strains can accelerate the decomposition of cholesterol by producing bile salt hydrolase, effectively reducing the amount of cholesterol ingested by the human body; in addition, lactic acid bacteria can interact with the host through their own secreted metabolites and cell wall surface components, activate related immune cell signal transduction, regulate the release of inflammatory cytokines and the secretion of antibodies, and help the body form a natural immune barrier (Tao Hongyan, Xu Xinwei, Wang Mingjin, Li Shanglin, Wei Jia. Research progress on the probiotic properties of lactic acid bacteria and their applications. Anhui Agricultural Science Bulletin, 2021, 27(21):45-48).

[0003] Small yellow croaker (Larimichthys polyacti), also known as small yellow flower, yellow croaker, small yellow croaker, etc., belongs to the order Perciformes, family Sciaenidae, and is similar in shape to large yellow croaker, with a golden belly and a gray-brown back. The body length is generally 15 to 25 cm. Small yellow croaker is a warm-temperate near-bottom fish and one of my country's main marine economic fishes. It is widely distributed in the East China Sea, the Yellow Sea and the Bohai Sea. It is rich in production and is known as one of China's three major marine fishes along with large yellow croaker and hairtail. Small yellow croaker is rich in protein, minerals, vitamins and other nutrients needed by the human body. It also has tender meat with less bones and is one of the high-quality edible fish in life. As a low-value fish, it is cheap, has abundant production and tastes delicious, and is deeply loved by consumers.

[0004] The water content of yellow croaker is 70%-80%. Its rich nutrition is also a good matrix for microorganisms. Therefore, after the death of yellow croaker, it carries a large number of bacteria and multiplies. At the same time, the enzyme system in the body causes autolysis, which makes the yellow croaker quickly corrupt after being caught and difficult to store for a long time. At present, in addition to some fresh food, it is mainly processed into dried yellow croaker, canned yellow croaker, yellow croaker crisps, etc. through drying and frying.

[0005] In order to improve the quality and nutritional function of small yellow croaker processed products, the present invention isolates a plant lactobacillus WZ-33 with beneficial activity from pickled small yellow croaker, and integrates it into small yellow croaker processing after culturing to produce small yellow croaker products rich in lactic acid bacteria. Summary of the invention

[0006] The invention aims to provide a plant lactobacillus and application thereof in processing of small yellow croaker, so as to make the small yellow croaker more nutritious and beneficial to the stomach and intestines.

[0007] To achieve the above object, the technical solution of the present invention is to screen and obtain plant lactobacillus with beneficial activity, and its preservation number is: CGMCC NO.30800.

[0008] The plant lactobacillus of the present invention has been deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration Committee on May 28, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101. The strain name of the strain is: WZ-33; the classification name is plant lactobacillus (Lactiplantibacillus plantarum); the deposit number is: CGMCC NO.30800. The nucleotide sequence of the plant lactobacillus (Lactiplantibacillus plantarum) is shown in SEQUENCE LISTING WZ-33.

[0009] The present invention also provides an application of the plant lactobacillus in the processing of small yellow croaker, comprising the following steps: (1) Slant culture: inoculating the plant lactobacillus onto a slant culture medium and culturing at 30-34° C. for 22-26 hours; (2) Seed culture: Inoculate the bacteria grown on the slant medium into fresh seed culture medium and culture at 30-34°C for 22-26 hours to prepare seed solution; (3) Fermentation: 100 g skim milk powder, 200 g yellow croaker meat, 20 g glucose, and 1000 g water are placed in a fermentation container, sterilized at 85° C. for 30 minutes, and after cooling, 1 to 5 wt % of the plant lactobacillus seed solution is added, and the mixture is incubated at 30 to 34° C. for 22 to 26 hours to obtain plant lactobacillus fermentation liquid; (4) mixing: mixing the eviscerated, headless, seasoned and dried small yellow croaker with the Lactobacillus plantarum fermentation liquid, wherein the ratio of the small yellow croaker to the Lactobacillus plantarum fermentation liquid is 1:1 to 3:1 (by weight); (5) Drying: The mixed small yellow croaker and Lactobacillus plantarum fermentation liquid are dried at low temperature.

[0010] Preferably, the slant culture medium and seed culture medium are MRS culture medium, wherein the slant culture medium composition is (g / L): casein peptone 10, beef extract 10, yeast extract 5, glucose 5, dipotassium hydrogen phosphate 2, sodium acetate 5, ammonium citrate 2, Tween 80 1, magnesium sulfate 0.2, manganese sulfate 0.05, calcium carbonate 10, agar 18, pH 6.8; the seed culture medium composition is (g / L): casein peptone 10, beef extract 10, yeast extract 5, glucose 5, dipotassium hydrogen phosphate 2, sodium acetate 5, ammonium citrate 2, Tween 80 1, magnesium sulfate 0.2, manganese sulfate 0.05, calcium carbonate 10, pH 6.8.

[0011] Preferably, the ratio of small yellow croaker to Lactobacillus plantarum fermentation broth is 2:1 (by weight).

[0012] Preferably, the low-temperature drying is vacuum drying or vacuum freeze drying.

[0013] The invention produces the small yellow croaker product with lactic acid bacteria, can effectively improve the quality and nutritional function of the product, and is easy to realize industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0015] Figure 1 is a colony of Lactobacillus plantarum WZ-33; Figure 2 The MEGA7.0 software was used to display the phylogenetic tree of 16S rDNA sequences of strain CGMCC No. and related species in the GeneBank database by the proximity joining method. DETAILED DESCRIPTION

[0016] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0017] The experimental methods in the following examples without specifying specific conditions were carried out according to conventional methods and conditions, or selected according to the product instructions.

[0018] The bacterial culture medium used in the following examples is as follows: Lactic acid bacteria screening medium (g / L): casein peptone 10, beef extract 10, yeast extract 5, glucose 5, dipotassium hydrogen phosphate 2, sodium acetate 5, triammonium citrate 2, Tween 80 1, magnesium sulfate 0.2, manganese sulfate 0.05, calcium carbonate 10, agar 15, pH 6.8, 0.6% bromocresol green 1.

[0019] Slant culture medium (g / L): casein peptone 10, beef extract 10, yeast extract 5, glucose 5, dipotassium hydrogen phosphate 2, sodium acetate 5, triammonium citrate 2, Tween 80 1, magnesium sulfate 0.2, manganese sulfate 0.05, calcium carbonate 10, agar 15, pH 6.8.

[0020] Seed culture medium (g / L): casein peptone 10, beef extract 10, yeast extract 5, glucose 5, dipotassium hydrogen phosphate 2, sodium acetate 5, triammonium citrate 2, Tween 80 1, magnesium sulfate 0.2, manganese sulfate 0.05, calcium carbonate 10, pH 6.8.

[0021] Fermentation medium (g / L): skimmed milk powder 100, small yellow croaker meat 200, glucose 20.

[0022] Example 1 Screening and Identification of Lactiplantibacillus plantarum WZ-33 1. Lactiplantibacillus plantarum was isolated from pickled yellow croaker. 10 mL of pickling solution was taken into 3 15 mL centrifuge tubes and 10 -3 , 10 -4 , 10 -5 , 10 -6 The gradient concentrations were diluted and 100 μl was spread on the lactic acid bacteria screening medium plate. Each concentration was repeated three times and cultured at 32°C for 48 hours. The round, slightly flat white colonies and the surrounding medium that turned yellow were acid-producing bacteria. The acid-producing bacteria were selected and purified several times to obtain 5 strains. The purified strains were inoculated on the slant culture medium, cultured at 32°C for 24 hours, and then stored in a 4°C refrigerator for use.

[0023] 2. Analysis of beneficial life The strain stored in a refrigerator at 4°C was inoculated into the seed culture medium and cultured at 32°C for 24 hours to obtain the seed solution.

[0024] (1) The inoculum was 3% (v / v) and inoculated into MRS liquid culture medium with pH values ​​of 2.0, 3.0, and 6.5, respectively. The culture was carried out at 32°C for 3 h. The number of viable bacteria was determined by plate count method. The pH 6.5 group was used as the control. The survival rate was calculated and the acid tolerance of the test strain was tested. The results are shown in Table 1.

[0025] Table 1 Survival rate of isolated strains under different pH conditions (2) The strains were inoculated with 3% (v / v) in MRS liquid culture medium containing 0, 0.1%, and 0.2% ox bile salts, respectively, and cultured anaerobically at 32°C for 3 h. The number of viable bacteria was determined by plate count method. The survival rate was calculated using the group without bile salts as the control. The bile salt tolerance of the test strains was tested. The results are shown in Table 2.

[0026] Table 2 Survival rate of isolated strains under different bile salt concentrations (3) Add Escherichia coli solution (10 9 cfu / mL) was mixed into MRS medium at about 50°C, shaken, and poured into a sterilized plate. After cooling, a sterilized Oxford cup was placed vertically on the plate, and 200 μL of the fermentation seed supernatant of the isolated strain was added. The culture was carried out at a constant temperature of 32°C for 24 hours, and the diameter of the inhibition zone was observed and measured. The results are shown in Table 3.

[0027] Table 3 Antibacterial activity of isolated strains against Escherichia coli strains Diameter of inhibition zone (mm) WZ-31 - WZ-32 6.8 WZ-33 11.2 WZ-34 3.5 WZ-35 4.1 According to the test results, strain WZ-33 has good acid tolerance, bile salt tolerance and ability to inhibit Escherichia coli, and has a strong probiotic effect. Figure 1 .

[0028] 2. Molecular biological identification of Lactiplantibacillus plantarum WZ-33 The target strain was cultured on a slant medium for 24 hours, genomic DNA was extracted, and 16S rDNA of the strain was amplified by PCR technology. The primers used were forward primer (5'-CAGAGTTTGATCCTGGCT-3') and reverse primer (5'-AGGAGGTGATCCAGCCGCA-3'); The composition of the PCR reaction system is as follows: Reagents Volume (μl) Template (genomic DNA 20-50ng / μl) 0.5 <![CDATA[10×Buffer (containing Mg 2+ )]]> 2.5 dNTP(2.5mM) 1 Forward primer (10 μM) 0.5 Reverse primer (10 μM) 0.5 Taq enzyme 0.2 Add double distilled water to 25 The PCR conditions are shown in the following table: The PCR products were sequenced and compared with the 16S rDNA in the GeneBank database. Figure 2, and found that it had the highest homology with Lactiplantibacillus plantarum strain HPM2 (GenBank accessionNo.PP355672.1), up to 100%. The strain was determined to be Lactiplantibacillus plantarum, which was deposited in the General Microbiology Center of China Microbiological Culture Collection (CGMCC) on May 28, 2024, with the deposit number: CGMCC NO.30800.

[0029] Example 2 Preparation of small yellow croaker product containing Lactobacillus plantarum The small yellow croaker is gutted and headed, cleaned, and soaked in sauce for 35-40 minutes (the ratio of fish to sauce is 2:1; the sauce composition is: 5% salt, 4% sugar, 3% monosodium glutamate, 5% spices), and then vacuum fried and dried at a vacuum degree of -90 kpa, a temperature of 95°C, and a time of 90 minutes; after vacuum frying, centrifugal deoiling is performed to obtain dried small yellow croaker.

[0030] Inoculate plant lactobacillus CGMCC No.30800 on a slant culture medium, culture at 30-34°C, preferably at 32°C for 22-26 hours, preferably for 24 hours, then inoculate the grown strains on the slant culture medium into a fresh seed culture medium, culture at 30-34°C, preferably at 32°C for 24 hours to obtain a seed solution. Then, take 100 grams of skimmed milk powder, 200 grams of small yellow croaker meat, 20 grams of glucose, and 1000 grams of water, place them in a fermentation container, sterilize at 85°C for 30 minutes, add 2% (wt) of the plant lactobacillus seed solution after cooling, culture at 30-34°C, preferably at 32°C for 24 hours, and obtain plant lactobacillus fermentation liquid; The mixture was mixed with Lactobacillus plantarum fermentation liquid, and the ratio of small yellow croaker to Lactobacillus plantarum fermentation liquid was 1:1; the mixed small yellow croaker and Lactobacillus plantarum fermentation liquid were vacuum dried, with a vacuum degree of -90 kPa, a temperature of 40 °C, and a drying time of 6 hours to obtain a small yellow croaker product containing Lactobacillus plantarum. The content of Lactobacillus plantarum was 6×10 7 cfu / g.

[0031] Example 3 Preparation of small yellow croaker product containing Lactobacillus plantarum The preparation of dried small yellow croaker and Lactobacillus plantarum fermentation liquid is the same as in Example 2; The mixture was mixed with Lactobacillus plantarum fermentation liquid, the ratio of small yellow croaker to Lactobacillus plantarum fermentation liquid was 2:1; the mixed small yellow croaker and Lactobacillus plantarum fermentation liquid were vacuum dried, the vacuum degree was -90 kpa, the temperature was 40 °C, and the drying time was 6 hours to obtain a small yellow croaker product containing Lactobacillus plantarum. The content of Lactobacillus plantarum was 2×10 7 cfu / g.

[0032] Example 4 Preparation of small yellow croaker product containing Lactobacillus plantarum The preparation of dried small yellow croaker and Lactobacillus plantarum fermentation liquid is the same as in Example 2; The mixture was mixed with Lactobacillus plantarum fermentation liquid, the ratio of small yellow croaker to Lactobacillus plantarum fermentation liquid was 2:1; the mixed small yellow croaker and Lactobacillus plantarum fermentation liquid were vacuum freeze-dried, the vacuum degree was -0.05 bar, the cold trap temperature was -80°C, the drying temperature was 30°C, and the drying time was 18 hours to obtain a small yellow croaker product containing Lactobacillus plantarum. The content of Lactobacillus plantarum was 3.5×10 7 cfu / g.

[0033] Example 5 Preparation of small yellow croaker product containing Lactobacillus plantarum The preparation of dried small yellow croaker and Lactobacillus plantarum fermentation liquid is the same as in Example 2; The mixture was mixed with Lactobacillus plantarum fermentation liquid, and the ratio of small yellow croaker to Lactobacillus plantarum fermentation liquid was 3:1. The mixed small yellow croaker and Lactobacillus plantarum fermentation liquid were vacuum freeze-dried, with a vacuum degree of -0.05 bar, a cold trap temperature of -80°C, a drying temperature of 30°C, and a drying time of 18 hours to obtain a small yellow croaker product containing Lactobacillus plantarum. The content of Lactobacillus plantarum was 1.8×10 7 cfu / g.

[0034] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A plant lactobacillus, characterized in that: The plant lactobacillus is classified and named Lactiplantibacillus plantarum, the strain name is WZ-33, and it was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on May 28, 2024, with the deposit number CGMCC NO. 30800.

2. Application of plant lactobacillus according to claim 1 in processing of small yellow croaker.

3. The use according to claim 2, characterized in that: The steps include: (1) Slant culture: inoculating the plant lactobacillus onto a slant culture medium and culturing at 30-34°C for 22-26°C for 24 hours; (2) Seed culture: inoculate the bacteria grown on the slant culture medium into fresh seed culture medium and culture at 30-34°C for 24 hours to obtain Lactobacillus plantarum seed solution; (3) Fermentation: 100 g skim milk powder, 200 g yellow croaker meat, 20 g glucose, and 1000 g water were placed in a fermentation container, sterilized at 85° C. for 30 minutes, and after cooling, 1-5 wt % of the plant lactobacillus seed solution was added, and the mixture was incubated at 30-34° C. for 22-26 hours to obtain plant lactobacillus fermentation liquid; (4) Mixing: Mixing the eviscerated, headless, seasoned and dried small yellow croaker with the Lactobacillus plantarum fermentation liquid, wherein the ratio of the small yellow croaker to the Lactobacillus plantarum fermentation liquid is 1:1 to 3:1 (by weight); (5) Drying: The mixed small yellow croaker and Lactobacillus plantarum fermentation liquid are dried at low temperature.

4. The use according to claim 3, characterized in that: The slant culture medium and seed culture medium are MRS culture medium.

5. The use according to claim 3, characterized in that: The low-temperature drying is vacuum drying or vacuum freeze drying.

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