Lactobacillus paracasei from shrimp intestine and its application in shrimp farming

Abstract

The invention discloses a Lactobacillus paracasei derived from the intestinal tract of a shrimp and application thereof in shrimp farming. The invention is characterized in that the strain is an M23 strain and is classified and named as Lactobacillus paracasei. Lacticaseibacillus paracasei , the deposit number is CGMCC No.26155. The strain is used in the preparation of Vibrio parahaemolyticus inhibitors, New Caledonian Vibrio inhibitors and / or Pseudomonas aeruginosa inhibitors, in biological fermentation feeds, and in the preparation of shrimp probiotic additives. The advantages are that it has a good inhibitory effect on Vibrio parahaemolyticus, New Caledonian Vibrio and / or Pseudomonas aeruginosa, can promote the growth of shrimp fry and improve the survival rate of young shrimp, while reducing the feed coefficient and improving the yield and disease resistance of shrimp.

Description

Technical Field

[0001] The invention relates to a strain of Lactobacillus paracasei, in particular to a strain of Lactobacillus paracasei originating from the intestinal tract of a shrimp and application of the strain in shrimp farming. Background Art

[0002] Penaeus vannamei (Latin name Penaeus vannamei ), also known as Penaeus vannamei, is the main species of aquaculture in my country. In recent years, the outbreak of diseases has become increasingly serious, causing huge losses to the industry and has become the main bottleneck restricting the sustainable development of the industry. Although microbial agents such as lactic acid bacteria are widely used for disease prevention and control in the breeding process, the strains of lactic acid bacteria preparations commonly used for shrimp are mostly isolated from terrestrial animals such as humans or livestock and poultry. When used in aquatic environments, especially in marine aquaculture environments, they often have shortcomings such as slow growth and weak colonization ability, and their probiotic disease prevention effects are not ideal. In addition, human-derived lactobacilli used in aquaculture will induce zebrafish intestinal epithelial cell pyroptosis and intestinal flora disorder, leading to intestinal damage in fish and even causing host death, revealing that the application of non-host-derived probiotics has great safety risks. Aquatic animal breeding environments or intestines contain a large number of lactic acid bacteria resources. Screening excellent strains with significant probiotic disease prevention effects, evaluating their probiotic effects, and applying them in aquatic animal breeding is a new way to replace traditional lactic acid bacteria preparations and effectively promote the health of aquatic animals. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a Lactobacillus paracasei derived from the intestinal tract of shrimp, which can promote the growth of shrimp fry and improve the survival rate of young shrimp, reduce the feed conversion ratio, and improve the yield and disease resistance of shrimp, and the application of the Lactobacillus paracasei in shrimp farming.

[0004] The technical solution adopted by the present invention to solve the above technical problems is: a Lactobacillus paracasei strain from the intestinal tract of shrimp, the strain is the M23 strain, and is classified and named as Lactobacillus paracasei ( Lacticaseibacillus paracasei ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 15, 2022, with the deposit number CGMCC No.26155.

[0005] The colonies of this strain are milky white, translucent protrusions with neat and smooth edges. Gram staining microscopy shows that it is Gram-positive, rod-shaped, and has a very strong sour taste. This strain can grow rapidly at a culture temperature between 25℃ and 45℃, with the optimal growth temperature being 30℃. It is in the exponential growth phase within 12 hours of culture in MRS broth medium, and enters the stable phase after 24 hours of culture, with the pH value dropping from 5.7 to 3.7. This strain has good stability in subculture, and its growth and acid production abilities remain unchanged after 10 consecutive subcultures.

[0006] The present invention also provides the use of the Lactobacillus paracasei derived from the shrimp intestine in the preparation of Vibrio parahaemolyticus inhibitor, Vibrio New Caledoniensis inhibitor and / or Pseudomonas aeruginosa inhibitor.

[0007] The invention also provides the use of the Lactobacillus paracasei derived from the shrimp intestine in the preparation of biological fermentation feed.

[0008] The invention also provides the use of the Lactobacillus paracasei derived from the shrimp intestine in the preparation of a shrimp probiotic additive.

[0009] Compared with the prior art, the invention has the advantages that: the invention discloses a Lactobacillus paracasei derived from the intestinal tract of a shrimp and its application in shrimp farming; the strain is isolated from the intestinal tract of a healthy shrimp; the strain has good growth at 25°C to 45°C, good passage stability, and the OD value of the fermentation liquid after culturing for 12 hours is 600 The value is above 1.5; the strain has good acid production ability and can withstand acid. After 12 hours of cultivation, the pH value of the fermentation liquid is below 4.0, and it has a distinct sour taste; the strain also has antibacterial activity, especially for common bacterial pathogens in shrimp farming, such as Vibrio; using this strain for feed fermentation can significantly increase the activity of feed protease, cellulase and amylase, and increase the content of acid-soluble protein and lactic acid in feed; using this strain in shrimp seedling cultivation can significantly promote the growth of young shrimp, improve the vitality and survival rate of shrimp seedlings; using this strain as a probiotic additive for shrimp farming can improve the growth and disease resistance of shrimp; therefore, Lactobacillus paracasei M23 has great economic value in aquatic animal farming, especially in the research of new green feed additives and probiotics for shrimp.

[0010] The above-mentioned Lactobacillus paracasei from the shrimp intestine is a strain M23, which is classified and named Lactobacillus paracasei ( Lacticaseibacillus paracasei ), deposited in the General Microbiology Center of China Microorganism Culture Collection on November 15, 2022, with the deposit number CGMCC No. 26155, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 pH and OD of fermentation broth of Lactobacillus paracasei M23 at different culture times (A) and temperatures (B) 600 Value changes;

[0012] Figure 2 pH tolerance (A) and passage stability (B) of Lactobacillus paracasei M23;

[0013] Figure 3The cluster tree was constructed based on the 16S rRNA gene sequence of Lactobacillus paracasei M23;

[0014] Figure 4 The changes in pH value (A), cellulase activity (B), amylase activity (C) and trypsin activity (D), as well as acid-soluble protein content (E) and lactic acid content (F) of the control feed and the fermented feed supplemented with Lactobacillus paracasei M23 at different times;

[0015] Figure 5 The weight, length (A) and survival rate (B) of juvenile shrimps supplemented with Lactobacillus paracasei M23 during the juvenile shrimp standardization period;

[0016] Figure 6 The culture yield (A), feed conversion ratio (B) and infection survival rate (C) of shrimp supplemented with Lactobacillus paracasei M23. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below with reference to the accompanying drawings.

[0018] Example 1: Isolation and identification of target strain Lactobacillus paracasei M23.

[0019] 1. Isolation and purification of target strains: Healthy white shrimp with full intestines, vigorous vitality and no diseases were randomly collected from Ningbo Xiangshan Farm, and quickly placed in an ice box and brought back to the laboratory. After the surface of the shrimp was disinfected with 75% ethanol, the shrimp intestine was picked up with a sterile toothpick and placed in a sterile 2 mL centrifuge tube on a sterile clean bench. 1.5 mL of sterile PBS buffer with pH = 7.0 was added, and the mixture was ground with a sterile pestle. The mixture was placed in a shaking incubator at 30°C and 180 rpm for 30 min. The culture solution was evenly spread on MRS solid culture medium using the gradient dilution method and inverted for 1 to 3 days at 30°C. A single colony was picked up with a sterile inoculation loop, and after purification and culture on MRS solid culture medium for two more times, 20% of the volume of the bacterial solution was added for seed preservation and stored in an ultra-low temperature refrigerator at -80°C.

[0020] 2. Biological characteristics of the target strain: After the target strain M23 isolated in step 1 was activated on MRS solid medium, a single colony was picked and placed in MRS liquid medium and cultured overnight at 30°C as seed liquid for strain growth, acid production and antibacterial assay. The seed liquid was inoculated into MRS liquid medium at a volume percentage of 2.0%, cultured at 30°C and 200 rpm, and its OD was measured every 12 h. 600 The changes in pH and pH were measured for 3 consecutive days. Figure 1 As shown in A, the OD of the fermentation liquid after 12 h of cultivation of the strain was 600The value is above 1.5, the pH value of the fermentation liquid is below 4.0, and it has a distinct sour taste, indicating that the strain has a good acid production ability. Figure 1 As shown in B, after the seed solution was inoculated into the MRS liquid medium and cultured at different temperatures, the strain could grow well at 25℃~45℃. Figure 2 As shown in Figure A, the strain can grow in MRS liquid medium with a pH of 2 to 4, and Figure 2 As shown in B, after 10 generations of continuous culture in MRS, the strain M23 can still maintain its rapid growth and acid production ability, indicating that the strain M23 has good acid resistance and stability in succession.

[0021] The formula of the above-mentioned MRS liquid medium is: peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, diammonium citrate 2.0 g, glucose 20.0 g, Tween 80 1.0 mL, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, distilled water 1000 mL, pH 4.5-6.0. MRS solid medium is agar added to MRS liquid medium at 1.5%-2.0% of the mass of MRS liquid medium.

[0022] After strain M23 was cultured overnight, the OD of the fermentation broth was measured. 600 value and adjust it to 0.6-0.8 for later use. Various pathogenic indicator bacteria include Vibrio parahaemolyticus ( Vibrio parahemolyticus ), Vibrio caledoniensis ( Vibrio neocaledonicus )、Vibrio brasiliensis( Vibrio brasiliensis ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Staphylococcus aureus ( Staphylococcus aureus )、Aeromonas hydrophila( Aeromonas hydrophila ) and Listeria monocytogenes ( Listeria monocytogenes ) were cultured in 2216E medium (or LB medium) at 30°C overnight, and then mixed with 2216E medium (or LB medium) containing 1.0 wt% agar to make the OD 600 The value was 0.1. After mixing evenly, pour it into a disposable culture dish to prepare a culture medium plate containing pathogens. After air-drying on an ultra-clean bench for 30 min, take 5 μL of the M23 strain fermentation liquid and drop it vertically in the center of the plate. After the bacterial liquid was air-dried, it was cultured at 30 °C for 24 h and the size of the inhibition zone was measured. It was found that the strain M23 had a good inhibitory effect on the following three pathogens, as shown in Table 1.

[0023] Table 1 Inhibitory ability of Lactobacillus paracasei M23 against common pathogens

[0024]

[0025] Note: + / - indicates the presence / absence of inhibitory effect, respectively.

[0026] The target strain screened in this example was cultured on MRS agar medium at 30°C for 24 h, and the colonies were milky white, translucent, and convex with neat and smooth edges. After being cultured overnight in MRS liquid medium, the fermentation liquid had a sour taste.

[0027] The preparation method of the 2216E medium used in this example is as follows: 37.4 g of commercial 2216E medium (Difco™ Marine Broth 2216) was weighed and dissolved in 1000 mL of distilled water, and the pH was adjusted to 7.6; the preparation method of the LB medium used was as follows: 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl were weighed and dissolved in 1000 mL of distilled water, and the pH was adjusted to 7.0.

[0028] 3. Identification of target strain: The strain was identified by 16S rRNA gene sequencing. The PCR amplification process of the 16S rRNA gene was as follows: DNA of the screened strain M23 was extracted by boiling method, and the 16S rRNA gene of the strain genome was amplified by commercial Taq Master Mix premix. The nucleotide sequence of the primer 27F used was shown in SEQ ID NO.1: 5'-AGAGTTTGATCCTGGCTCAG-3'; the nucleotide sequence of the primer 1492R was shown in SEQ ID NO.2: 5'-GGTTACCTTGTTACGACTT-3'.

[0029] The PCR reaction system was as follows: template 2 μL, 2×PCR Master Mix (Cat. No: K0171; manufacturer: Thermo Scientific™) 10 μL, primer 27F 0.5 μL, primer 1492R 0.5 μL, ddH2O 7 μL, and a total volume of 20 μL.

[0030]

[0031] After submitting the sequence to the EZBioCloud database (a microbial model strain database, mainly used for strain identification and microbial classification), it was found that the strain was Lacticaseibacillus paracasei The similarity with strain JCM1171(T) is 100%. The 16S rRNA gene clustering tree of this strain is attached. Figure 3 Therefore, the strain was named Lactobacillus paracasei Lacticaseibacillus paracasei M23 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 15, 2022, with the deposit number CGMCC No. 26155.

[0032] Example 2: The Lactobacillus paracasei M23 screened in Example 1 was used in a feed fermentation test.

[0033] The commercial feed required for the feed fermentation experiment was purchased from Zhejiang Qiangpu Biotechnology Co., Ltd. The experiment was divided into a control group and a treatment group, with 5 replicates in each group. After overnight fermentation of Lactobacillus paracasei M23, the fermentation liquid OD was adjusted with sterile growth medium (Sterilized Growth Medium) 600 The value is 1.0, 10 mL of fermentation liquid is added to 190 mL of sterile water, mixed and evenly mixed with 500 g of feed, put into a self-sealing bag, compacted, and placed in a 30°C constant temperature incubator for culture. The control group was replaced with 200 mL of sterile water. The fermentation time was 72 h, and samples were taken every 12 h to measure the pH value of the fermented feed and the changes in the activities of cellulase, amylase and trypsin. After the fermentation was completed, the acid-soluble protein and lactic acid content of the feed were measured. Figure 4 As shown in Figure A, after adding Lactobacillus paracasei M23, the pH value of the feed gradually decreased with the fermentation time, from 6.4 at 0 h to 5.3, while the pH value of the control group feed remained basically unchanged; Figure 4 As shown in Figures B, C, and D, the addition of Lactobacillus paracasei M23 also significantly increased the activities of cellulase, amylase, and trypsin in the feed, which all showed a trend of first increasing and then decreasing over time. Among them, amylase and trypsin reached their highest values ​​at 36 h of fermentation, which were 5.2 and 13290.0 U / mgprot, respectively, while cellulase reached its highest value of 504.7 U / mgprot at 48 h. After the fermentation was completed, the fermented feed in the group with the addition of Lactobacillus paracasei M23 had a distinct sour aroma, such as Figure 4 As shown in E, the acid-soluble protein content was 4.5 times that of the control group; Figure 4 As shown in F, the lactic acid content is 2.0 times that of the control group. This example shows that the addition of Lactobacillus paracasei M23 can significantly improve the nutritional level and palatability of feed.

[0034] Example 3: The Lactobacillus paracasei M23 screened in Example 1 was used in a crude shrimp fry culture experiment.

[0035] This experiment was conducted in 10 500 L round canvas culture barrels with consistent specifications. The culture barrels were equipped with consistent oxygenation, drainage and water exchange devices. Each culture barrel was filled with 400 L of sand-filtered clean seawater and 8,000 shrimp fry of P5 specification (the common name for shrimp on the 5th day, which is used for subsequent culture after 15-25 days of roughing). A control group and an M23 bacterial agent addition group were set up respectively. The M23 bacterial powder was obtained by centrifugation and removal of the supernatant after overnight fermentation in MRS liquid medium and freeze-drying. After the bacterial powder was expanded, it was added by spraying. The expansion method was as follows: 50 g brown sugar and 0.1 g of the above-mentioned M23 bacterial powder were added to each liter of tap water, stirred and mixed, and then sealed. Fermented bacterial liquid was obtained by anaerobic fermentation at room temperature and used after 2-3 days. The M23 bacterial agent addition group was sprayed with 100 mL of fermented bacterial liquid every day, and the control group was not sprayed with bacterial liquid. Each group was set up with 5 replicates. Feeding was done 5 times a day during the culture period, and the initial daily feeding amount accounted for 10% of the shrimp weight. After one month of culture, the weight, length and survival rate of the shrimp were measured. Figure 5 As shown in Figure A, the average body length and weight of shrimp in the group with M23 bacterial agent increased by more than 40% compared with the control group; Figure 5 As shown in B, the survival rate of shrimp culture was 35% higher than that of the control group, indicating that the addition of M23 bacterial agent can improve the growth and survival rate of shrimp seedlings in the standard coarse stage.

[0036] Example 4: The Lactobacillus paracasei M23 screened in Example 1 was used in a shrimp farming experiment.

[0037] This experiment was carried out in a 1.5-ton round plastic aquaculture barrel with consistent specifications. The aquaculture pond was equipped with a consistent aeration system, drainage and water exchange device, etc. Each aquaculture barrel was filled with 1000 L of clean sand-filtered seawater and 1000 shrimp seedlings with a size of 3-4 cm. A control group and an M23 bacterial agent addition group were set up respectively. The bacterial agent expansion and splashing addition methods were the same as in Example 3. 200 mL of fermented bacterial liquid was splashed per barrel every day, and the control group was not splashed with bacterial liquid. Each group was set up with 5 repetitions. During the breeding period, the water was changed every two days, and the water change amount was 50% each time; feeding was 3 times a day (7:00, 12:00, 17:00), and the initial daily feeding amount accounted for 5% of the shrimp weight. After 80 days of breeding, the shrimp was harvested, the shrimp yield was counted, and the feed coefficient was calculated.

[0038] 60 shrimps were randomly selected from each bucket and divided equally into two culture boxes filled with 50 L of clean seawater, one of which was used for Vibrio infection and the other for control. The Vibrio infection method was as follows: Vibrio parahaemolyticus was cultured in 2216E broth medium to an OD of 0. 600 The value is about 1.0 (the bacterial concentration is about 1.0×10 9cfu / mL), 50 mL of bacterial solution was added to the culture tank to make the concentration of Vibrio parahaemolyticus in the water body reach 1.0×10 6 cfu / mL. Figure 6 As shown in Figure A, compared with the control group, the unit yield of shrimp in the group with M23 bacterial agent increased by 30%; Figure 6 As shown in B, the bait coefficient is reduced by more than 20%; Figure 6 As shown in C, the survival rate after Vibrio infection increased by more than 35%. This example shows that adding M23 bacterial agent to culture can promote the growth of shrimp, improve feed digestion efficiency and enhance the disease resistance of shrimp, thereby improving the yield and quality of shrimp culture.

[0039] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A Lactobacillus paracasei from the intestinal tract of a shrimp, characterized in that: The strain is M23, which is classified as Lactobacillus paracasei ( Lacticaseibacillus paracasei ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 15, 2022, with the deposit number CGMCC No. 26155.

2. Use of the Lactobacillus paracasei derived from the shrimp intestine according to claim 1 in the preparation of a Vibrio parahaemolyticus inhibitor, a Vibrio New Caledonianus inhibitor and / or a Pseudomonas aeruginosa inhibitor.

3. Use of the Lactobacillus paracasei derived from the shrimp intestine according to claim 1 in preparing biological fermentation feed.

4. Use of the Lactobacillus paracasei derived from the shrimp intestine according to claim 1 in preparing a shrimp probiotic additive.

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