Psychrophilic Bacillus maritima PN-Ta3 from the intestine of turbot and its application

Abstract

The present invention discloses a strain of Psychrobacterium marinum PN‑Ta3 derived from the intestine of turbot and its application, belonging to the field of microbial technology. The Psychrobacterium marinum PN‑Ta3 is isolated from the intestine of healthy farmed turbot and is preserved in the China Center for Type Culture Collection, with the preservation address in Wuhan, China, the preservation date on January 25, 2024, and the preservation number as CCTCC NO: M2024201. The benefit of the present invention is that Psychrobacter marinum PN‑Ta3 is non-pathogenic to healthy turbot and has good colonization performance. After being made into freeze-dried bacterial powder and mixed with and added to turbot for feeding, it can not only promote the growth and intestinal development of farmed turbot and optimize the intestinal microbial structure, but also delay the morbidity and death of turbot caused by infection with Aeromonas salmonicida BHAS‑1.

Description

Technical Field

[0001] The invention relates to a bacterium and application thereof, in particular to Psychrophilic Bacillus maritimus PN-Ta3 derived from the intestine of turbot and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] turbot Scophthalmus maximus , commonly known as turbot, is a cold-water bottom-dwelling marine fish. With the continuous expansion of aquaculture, diseases are frequent, and improper use of drugs leads to excessive drug residues, which seriously affects the quality and safety of aquatic products. How to reduce the use of antibiotics and fishery drugs and seek green and healthy disease prevention methods is the best way to solve the current disease problem.

[0003] Biological disease prevention is green and pollution-free, and is widely used in the prevention and control of diseases in the fields of animal husbandry and planting. Probiotics, or beneficial microorganisms, are the first choice for biological disease prevention and have become one of the effective strategies for improving the health of farmed organisms in the aquaculture industry. The use of probiotics has played a positive role in reducing the invasion of pathogens, improving digestion performance, and enhancing the body's resistance to stress. It can achieve disease source prevention and control, and has gradually been promoted and applied in actual production. Summary of the invention

[0004] The present invention aims to provide a strain of Psychrophilus nautilus PN-Ta3 which is derived from the intestine of turbot and can promote the growth and intestinal development of turbot, improve the intestinal flora structure of turbot, and enhance the ability of turbot to resist infection by Aeromonas salmonicida BHAS-1, and its application as endogenous probiotics for turbot.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] Psychrophilic Bacterium PN-Ta3 was isolated from the intestine of healthy farmed turbot and named Psychrophilic Bacterium PN-Ta3 Psychrobacter nivimaris PN-Ta3 is deposited in China Center for Type Culture Collection, Wuhan, China. The deposit date is January 25, 2024, and the deposit number is CCTCC NO: M2024201.

[0007] The application of the aforementioned Psychrobacterium sibiricum PN-Ta3 includes:

[0008] (1) Use of the microbial agent in the preparation of a microbial agent for improving the growth performance of turbot, wherein the growth performance includes body length and body weight;

[0009] (2) Application in the preparation of bacterial agents for promoting intestinal development of turbot;

[0010] (3) Application in the preparation of bacterial agents for improving the intestinal flora structure of turbot;

[0011] (4) Application in the preparation of bacterial agents for the prevention of Aeromonas salmonicida BHAS-1 infection.

[0012] Preferably, the preparation method of the bacterial agent is as follows:

[0013] (1) Activating the strain: inoculating Psychrophilus maritimus PN-Ta3 into MRS liquid culture medium to obtain Psychrophilus maritimus PN-Ta3 bacterial liquid;

[0014] (2) Collecting the cells and preparing a bacterial suspension: Centrifuge the PN-Ta3 bacterial suspension at 4°C, wash the precipitate with sterile PBS buffer, collect the cells and prepare a suspension with a concentration of 1.0 × 10 10 CFU / mL of Psychrobacterium marinum PN-Ta3 bacterial suspension;

[0015] (3) Adding a lyophilized protective agent: uniformly mixing the lyophilized protective agent with the PN-Ta3 bacterial suspension of Psychrobacterium marissima to obtain a PN-Ta3 bacterial suspension of Psychrobacter marissima to which the lyophilized protective agent is added;

[0016] (4) Prefreezing: placing the suspension of Psychrophilus maritimus PN-Ta3 to which a lyophilization protective agent is added in a freezer for prefreezing to obtain prefrozen Psychrophilus maritimus PN-Ta3 bacterial cells;

[0017] (5) Freeze-drying: The pre-frozen PN-Ta3 bacterial cells are placed in a vacuum freeze dryer for freeze-drying to obtain freeze-dried bacterial powder of PN-Ta3.

[0018] More preferably, the lyoprotectant is prepared by mixing skim milk powder, trehalose, sodium glutamate and glycerol in a mass ratio of 3:2:1:1.

[0019] The present invention is beneficial in that:

[0020] (1) The Psychrophilic Bacillus PN-Ta3 disclosed in the present invention is screened from the intestines of healthy farmed turbot. Experiments have confirmed that the Psychrophilic Bacillus PN-Ta3 is a Gram-negative bacillus and is non-pathogenic to healthy turbot.

[0021] (2) PN-Ta3 was made into freeze-dried bacterial powder and then mixed with feed. The feeding results confirmed that exogenous supplementation of PN-Ta3 can promote the growth and intestinal development of farmed turbot and optimize the intestinal microbial structure. PN-Ta3 had a positive effect on the growth and intestinal development of turbot throughout the experiment, promoting growth, increasing the height, width and muscle thickness of intestinal villi, and increasing the relative abundance of several potential beneficial bacteria in the intestine and the level of intestinal microbial diversity.

[0022] (3) The challenge experiment confirmed that exogenous supplementation of Psychrophilic Bacillus marineus PN-Ta3 could delay the morbidity and mortality of turbot caused by infection with Aeromonas salmonicidae BHAS-1, effectively reduce the mortality rate of farmed turbot, and enhance the disease resistance of farmed turbot. This proves that Psychrophilic Bacillus marineus PN-Ta3 has good colonization performance and can continuously play a probiotic role in the body of farmed turbot. DETAILED DESCRIPTION

[0023] The present invention is described in detail below with reference to specific embodiments.

[0024] 1. Strain Screening

[0025] Potential probiotics were isolated and screened from the intestines of healthy turbot, which were sourced from a breeding farm of turbot, with individual weight ranging from 521g to 637g. The specific performance of healthy turbot is: the individual is vigorous, there are no obvious lesions on the body surface, and there is no parasitic infection on the body surface and gills under microscopic observation.

[0026] Take a number of the above healthy turbots, and take out the intestines of each healthy turbot aseptically, and keep the intestines of individuals without bacterial pathogen infection for later use.

[0027] The contents were taken out from the intestines of the individuals without bacterial pathogen infection, streaked on nutrient agar plates supplemented with 2 wt% NaCl, and cultured inverted at 28°C for 48 h. Then, dominant colonies with consistent morphology were picked for purification and culture until pure culture was obtained. Finally, the seeds were preserved in 40% glycerol (strain number: PN-Ta3) and stored in a -80°C refrigerator for future use.

[0028] 2. Strain Identification

[0029] The 16S rRNA of the obtained pure culture (strain number: PN-Ta3) was amplified by PCR using bacterial 16S rRNA universal primers (27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID No: 2), 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No: 3)) to obtain the 16S rRNA nucleotide sequence of strain PN-Ta3 (SEQ ID No: 1).

[0030] After NCBI online BLAST homology comparison, strain PN-Ta3 was finally identified as Psychrobacter maritima and recorded as Psychrobacter maritima PN-Ta3.

[0031] 3. Strain genome information

[0032] In order to better understand the genome information of Psychrobacter marissima PN-Ta3, the whole genome DNA of Psychrobacter marissima PN-Ta3 was extracted using a kit, and the whole genome sequencing was completed on the Illumina HISsq platform. FastQC was used to perform statistics on the sequencing data of the samples, and the statistical results showed that:

[0033] (1) After assembly and correction of the whole genome sequencing fragments, the total length of the whole genome sequence was 1228060963 bp, the GC content was 43.51%, and a total of 2629 coding genes were predicted.

[0034] (2) A total of 1,880 genes were annotated in the prokaryotic COG database, of which 193 were involved in amino acid transport and metabolism, 158 were involved in translation, ribosome structure and biogenesis, 150 were involved in energy production and conversion, 144 were involved in cell membrane biogenesis, 122 were involved in replication, recombination and repair, and 1,113 were genes of unknown function.

[0035] (3) In the GO database of gene function classification system, 841 genes were annotated, which were involved in 7 types of cell composition activities in different locations, and 1143 genes were involved in 11 types of molecular function realization.

[0036] (4) A total of 848 genes were annotated in the KEGG biological system database, which are involved in five metabolic pathways, including cellular processes, environmental information processing, genetic information processing, metabolism and organic systems.

[0037] (5) The protein sequences of the gene set were compared with the CAZy database, and 40 carbohydrate-active enzyme genes were annotated, including 4 oxidoreductases, 6 carbohydrate lipolytic enzymes, 3 glycoside hydrolases, and 7 glycosyltransferases; 604 transmembrane proteins, 241 signal peptide proteins, and 1986 lipoproteins were predicted among the 2629 proteins.

[0038] IV. Strain Safety Test

[0039] The PN-Ta3 strain of Psychrobacterium marinum stored in a -80°C refrigerator was inoculated into a nutrient agar plate supplemented with 2 wt% NaCl for activation. Then, a single colony in the exponential growth phase was selected and placed in nutrient broth. The culture was shaken and cultured at 28°C for 24 h. The culture fluid was collected and finally prepared into a 10% PBS buffer solution. 10 CFU / mL of bacterial suspension is reserved.

[0040] Healthy turbot fry with an average body weight of 0.21g±0.07g were purchased, kept for one week, and then randomly divided into a low-concentration injection group, a medium-concentration injection group, a high-concentration injection group, an injection control group and a blank control group, with 10 turbot fry in each group.

[0041] The low concentration injection group, medium concentration injection group and high concentration injection group were injected with 10 7 CFU / mL, 10 8 CFU / mL, 10 9 The control group was injected with 100 μL of PN-Ta3 suspension of CFU / mL of Psychrobacterium marinum, and the blank control group was injected with 100 μL of sterile PBS buffer, while the death of turbot fry in each group was closely observed.

[0042] After observation, 14 days later, 1, 1, and 0 turbot fry died in the low-concentration injection group, medium-concentration injection group, and high-concentration injection group, respectively; 1 turbot fry died in the injection control group; 2 turbot fry died in the blank control group; and the rest of the turbot fry were normal.

[0043] It can be determined that: injection within the above concentration range (10 7 CFU / mL~10 9 CFU / mL) of Psychrobacterium maritima PN-Ta3 is relatively safe and non-pathogenic to healthy turbot.

[0044] 5. Preparation of freeze-dried bacterial powder

[0045] For ease of application, Psychrobacterium sibiricum PN-Ta3 was prepared into freeze-dried bacterial powder, and the preparation method is as follows:

[0046] (1) Activating the strain: inoculate the PN-Ta3 strain of Pseudomonas aeruginosa stored in a -80°C refrigerator into MRS liquid culture medium and culture at 28°C with shaking for 24 h to obtain a bacterial liquid of the PN-Ta3 strain of Pseudomonas aeruginosa in the exponential growth phase;

[0047] (2) Collecting bacteria and preparing bacterial suspension: Place the PN-Ta3 bacterial suspension of Pseudomonas aeruginosa in the exponential growth phase in a 4°C centrifuge and centrifuge at 6000 rpm for 15 min. Discard the supernatant and wash the precipitate twice with sterile PBS buffer. Collect the bacteria and prepare a suspension with a concentration of 1.0 × 10 10 CFU / mL of Psychrobacterium marinum PN-Ta3 bacterial suspension;

[0048] (3) Adding a lyophilization protective agent: The lyophilization protective agent is prepared by mixing skim milk powder, trehalose, sodium glutamate and glycerol in a mass ratio of 3:2:1:1. The lyophilization protective agent is mixed evenly with the Psychrobacterium marissinensis PN-Ta3 bacterial suspension in a ratio of 35 g:100 mL to obtain the Psychrobacterium marissinensis PN-Ta3 bacterial suspension added with the lyophilization protective agent;

[0049] (4) Prefreezing: placing the suspension of Psychrophilus maritimus PN-Ta3 to which a lyophilization protective agent is added in a freezer and prefreezing it at -80°C for 4 hours to obtain prefrozen Psychrophilus maritimus PN-Ta3 cells;

[0050] (5) Freeze-drying: The pre-frozen PN-Ta3 bacterial cells were placed in a vacuum freeze dryer and freeze-dried for 24 hours to obtain freeze-dried bacterial powder of PN-Ta3.

[0051] 6. Probiotics Addition Experiment

[0052] Healthy turbot fry with an average body weight of 0.19 g ± 0.08 g were selected, kept for one week, and then randomly divided into a supplemented group and a control group, with 100 turbot fry in each group.

[0053] The supplemented group was fed with a compound feed supplemented with 35wt% freeze-dried bacterial powder of Psychrobacterium marinum PN-Ta3 every day, and the control group was fed with a compound feed without freeze-dried bacterial powder of Psychrobacter marinum PN-Ta3 every day. The daily feeding amount of the feed was 5% of the body weight of the turbot fry.

[0054] On the 30th and 60th days of feeding, 20 turbot fry were taken from each group to measure their body length and weight, and then the liver and intestine were removed by aseptic operation. The activities of three immune-related enzymes, catalase, alkaline phosphatase and superoxide dismutase, in the liver were tested, the intestine was bio-sectioned and HE-stained, and the changes in intestinal structure were observed by microscopy. In addition, intestinal (content-free) DNA was extracted and a small fragment library was constructed by double-end sequencing based on the Illumina NovaSeq platform. After sequencing, diversity analysis was performed to identify the differences in intestinal microorganisms in samples from different groups.

[0055] 1. Effect of adding Psychrobacterium maritima PN-Ta3 to feed on the growth performance of turbot larvae

[0056] The statistical results of body length and weight of turbot fry in each group on the 30th and 60th day of feeding are shown in Table 1.

[0057] Table 1 Changes in growth indicators of turbot larvae after adding Psychrobacterium maritima PN-Ta3 to feed

[0058]

[0059] Note: The same superscript letters in the same column indicate no significant difference between groups ( p >0.05), different superscript letters indicate significant differences among the groups ( p <0.05).

[0060] As shown in Table 1, compared with the control group without PN-Ta3, the exogenous addition of PN-Ta3 improved the comprehensive growth performance of turbot fry, and with the extension of feeding time, the increase in body length and weight became more and more significant. Among them, after feeding for 30 days, compared with the control group, the body length of the supplemented group increased by 3.94%, and the weight increased by 23.44%, and the difference in body length growth was not significant ( p >0.05), the difference in weight gain was significant ( p< 0.05); After 60 days of feeding, the body length of the supplemented group increased by 10.12% and the body weight increased by 29.32% compared with the control group, and the differences in body length and weight growth were significant ( p <0.05).

[0061] 2. Effect of adding Psychrophilus maritima PN-Ta3 to feed on the immunity of turbot larvae

[0062] The results of activity detection of immune-related enzymes (catalase, superoxide dismutase) in the liver of turbot fry in each group on the 30th and 60th days of feeding are shown in Table 2.

[0063] Table 2 Changes in the activities of immune-related enzymes in the liver of turbot larvae after adding Psychrobacterium maritima PN-Ta3 to feed

[0064]

[0065] Note: The same superscript letters in the same column indicate no significant difference between groups ( p >0.05), different superscript letters indicate significant differences among the groups ( p <0.05).

[0066] From Table 2, we can see that:

[0067] Compared with the control group without PN-Ta3, the exogenous addition of PN-Ta3 increased the activity of catalase in the liver of turbot larvae, especially after 60 days of addition, the activity of catalase increased significantly ( p< 0.01), exogenous addition of PN-Ta3 of Pseudomonas aeruginosa for 60 days can increase the activity of superoxide dismutase in the liver of turbot larvae, but the difference is not significant ( p >0.05).

[0068] In summary, the addition of Psychrobacterium marinum PN-Ta3 can improve the activity of immune-related enzymes to a certain extent, thereby promoting the improvement of the immune ability of turbot fry.

[0069] 3. Effect of adding Psychrobacterium marinum PN-Ta3 to feed on the intestinal structure of turbot larvae

[0070] The test results of the intestinal structure of turbot larvae in each group on the 30th and 60th days of feeding are shown in Table 3.

[0071] Table 3 Changes in intestinal structure of turbot larvae after adding Psychrobacterium maritima PN-Ta3 to feed

[0072]

[0073] As shown in Table 3, compared with the control group without the addition of PN-Ta3, the exogenous addition of PN-Ta3 for 30 days can increase the height of the intestinal villi of turbot larvae by 12.68%, the width of the intestinal villi by 7.69%, and the thickness of the muscular layer by 3.97%. The exogenous addition of PN-Ta3 for 60 days increased the height of the intestinal villi, the width of the intestinal villi, and the thickness of the muscular layer by 13.40%, 11.22%, and 9.85%, respectively. It can be seen that with the extension of feeding time, the increase in the height of the intestinal villi, the width of the intestinal villi, and the thickness of the muscular layer became more and more significant.

[0074] In summary, the addition of Psychrobacterium marinum PN-Ta3 can significantly promote the intestinal development of turbot larvae and improve the intestinal structure of turbot larvae.

[0075] 4. Effect of adding Psychrobacterium marinum PN-Ta3 to feed on intestinal flora of turbot larvae

[0076] The results of intestinal flora diversity detection of turbot larvae in each group on the 30th and 60th days of feeding are shown in Table 4.

[0077] Table 4 Differences in intestinal flora diversity of turbot larvae after adding Psychrobacterium maritimus PN-Ta3 to feed

[0078]

[0079] It can be seen from Table 4 that among the five indicators for evaluating the diversity of the microbiota, namely the Shannon index, Simpson index, ACE index, Chao index and PD index, the values ​​of the supplemented group were higher than those of the control group, regardless of whether they were fed for 30 days or 60 days. The abundance of intestinal microbiota of turbot fry in the supplemented group was higher, indicating that the addition of Psychrophilic Bacillus PN-Ta3 enriched the types of intestinal microorganisms of turbot fry to a certain extent and increased the diversity of intestinal microorganisms of turbot fry.

[0080] Specifically, after 30 days of feeding, the supplementation group increased the abundance of Proteobacteria, Firmicutes and Actinobacteriota, the core intestinal flora, at the phylum level, increased the abundance of γ-Proteobacteria, α-Proteobacteria and Clostridium at the class level, and increased the abundance of Enterobacter, Burkholderia, Lactobacillus and other bacteria at the order level; after 60 days of feeding, the supplementation group increased the abundance of Proteobacteria and Actinobacteriota, the core intestinal flora, at the phylum level, increased the abundance of γ-Proteobacteria, α-Proteobacteria, Clostridium, Verrucomicrobia and Acidmicrobacteria at the class level, and increased the abundance of Enterobacter, Rhodobacter, Burkholderia, Pseudomonas and other bacteria at the order level.

[0081] In summary, the addition of Psychrobacterium marinum PN-Ta3 can not only increase the number of core flora in the intestine of turbot larvae, but also increase the abundance and diversity of intestinal microorganisms in turbot larvae, which has a positive effect on turbot larvae's resistance to adverse external environment.

[0082] VII. Effect of Adding Psychrophilus maritima PN-Ta3 to Feed on Disease Resistance of Turbot Larvae

[0083] 1. Preparation of challenge strains

[0084] Aeromonas salmonicida BHAS-1 was selected for artificial infection.

[0085] After activation of Aeromonas salmonicida BHAS-1, it was eluted with sterile PBS and the concentration was adjusted to 10 8 CFU / mL and placed in a 4°C refrigerator for subsequent artificial infection.

[0086] 2. Artificial infection

[0087] After the probiotics addition experiment, the turbot fry in the addition group and the control group were temporarily reared for one week. During the period, they were fed with a basic diet and the culture conditions remained unchanged. Artificial infection was carried out one week later.

[0088] Thirty turbot fry were randomly selected from the supplementation group and the control group, and three parallels were set up in each group, with 10 fry in each parallel. Each group was intraperitoneally injected with 100 μL of Aeromonas salmonicida BHAS-1.

[0089] After infection, the number of deaths of turbot fry in each group was observed and recorded every day. When no deaths occurred for three consecutive days, the experiment was stopped and the survival rate was calculated.

[0090] It was observed that after artificial infection with Aeromonas salmonicida BHAS-1:

[0091] (1) Deaths began to occur in the control group on the second day after injection (the number of deaths in the three parallel groups was 0, 2, and 1, respectively), and the death peak occurred on the third day (the number of deaths in the three parallel groups was 2, 3, and 2, respectively) and the fourth day (the number of deaths in the three parallel groups was 4, 3, and 3, respectively). The mortality rates of the three parallel groups in the control group in the first four days were 60%, 80%, and 60%, respectively. The number of deaths decreased on the fifth day (the number of deaths in the three parallel groups was 1, 0, and 1, respectively). No deaths occurred from the sixth day onwards. The survival rates of the three parallel groups in the control group were 30%, 20%, and 30%, respectively, with an average survival rate of 26.67%;

[0092] (2) In the group supplemented with PN-Ta3 of Pseudomonas aeruginosa, only one fish died on the 7th day after injection. The survival rates of the three parallel experiments were 100%, 100%, and 90%, respectively, with an average survival rate of 96.67%.

[0093] Comparing the mortality of the added group and the control group, it can be seen that the exogenous addition of Psychrophilic Bacillus marine snow PN-Ta3 can effectively resist the morbidity and mortality caused by artificial infection with Aeromonas salmonicida BHAS-1, and significantly reduce the mortality rate.

[0094] 8. Strain Preservation

[0095] From the above studies, it can be seen that when Psychrobacterium cerevisiae PN-Ta3 is mixed with feed and fed to farmed turbot, it can promote the growth and intestinal development of farmed turbot and improve the intestinal flora structure, thus comprehensively improving the intestinal health. After 60 days of feeding, the results of the artificial toxin challenge (Aeromonas salmonicida BHAS-1) test showed that the exogenous addition of Psychrobacterium cerevisiae PN-Ta3 can effectively improve the ability of turbot to resist infection by Aeromonas salmonicida BHAS-1. At 60 days of feeding, the growth and intestinal development were more obvious than at 30 days, indicating that Psychrobacterium cerevisiae PN-Ta3 has good intestinal colonization ability and can continuously play a probiotic role.

[0096] In summary, the Psychrobacterium marinum PN-Ta3 screened by the present invention can be added as an exogenous probiotic to cultured turbot, which can improve growth performance, promote intestinal development, improve intestinal flora structure, and enhance the ability to resist infection by Aeromonas salmonicida BHAS-1.

[0097] The marine snow psychrophilic bacillus PN-Ta3 screened by the present invention has been deposited in the China Center for Type Culture Collection on January 25, 2024, with the deposit address in Wuhan, China, and the deposit number is CCTCC NO: M2024201.

[0098] It should be noted that the above embodiments do not limit the present invention in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation shall fall within the protection scope of the present invention.

Claims

1. Psychrobacterium marinum ( Psychrobacter nivimaris )PN-Ta3, isolated from the intestine of healthy farmed turbot, with the deposit number being CCTCC NO: M2024201.

2. The use of Psychrobacterium marinum PN-Ta3 according to claim 1, characterized in that: The applications include: (1) Use of the bacterial agent in the preparation of a microbial agent for improving the growth performance of turbot, wherein the growth performance is body length and body weight; (2) Application in the preparation of bacterial agents for promoting intestinal development of turbot; (3) Application in the preparation of bacterial agents for improving the intestinal flora structure of turbot; (4) Application in the preparation of bacterial agents for the prevention of Aeromonas salmonicida BHAS-1 infection.

3. The use according to claim 2, characterized in that: The preparation method of the bacterial agent is as follows: (1) Activating the strain: inoculating Psychrophilus maritimus PN-Ta3 into MRS liquid culture medium to obtain Psychrophilus maritimus PN-Ta3 bacterial liquid; (2) Collecting the cells and preparing a bacterial suspension: Centrifuge the PN-Ta3 bacterial suspension at 4°C, wash the precipitate with sterile PBS buffer, collect the cells and prepare a suspension with a concentration of 1.0 × 10 10 CFU / mL of Psychrobacterium marinum PN-Ta3 bacterial suspension; (3) Adding a lyophilized protective agent: uniformly mixing the lyophilized protective agent with the PN-Ta3 bacterial suspension of Psychrobacterium marissima to obtain a PN-Ta3 bacterial suspension of Psychrobacter marissima to which the lyophilized protective agent is added; (4) Prefreezing: placing the suspension of Psychrophilus maritimus PN-Ta3 to which a lyophilization protective agent is added in a freezer for prefreezing to obtain prefrozen Psychrophilus maritimus PN-Ta3 bacterial cells; (5) Freeze-drying: The pre-frozen PN-Ta3 bacterial cells are placed in a vacuum freeze dryer for freeze-drying to obtain freeze-dried bacterial powder of PN-Ta3.

4. The use according to claim 3, characterized in that: The freeze-drying protective agent is prepared by mixing skim milk powder, trehalose, sodium glutamate and glycerol in a mass ratio of 3:2:1:1.

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