CN107338198A - A kind of Lactobacillus plantarum and its application - Google Patents

Abstract

The present invention provides a kind of Lactobacillus plantarum and its application, named as Lactobacillus plantarum GRLP-25, preserved in China Typical Culture Collection Center, address Wuhan University, the preservation date is April 18, 2016, and the preservation number is CCTCC M NO : 2016197, Lactobacillus plantarum GRLP‑25 can have sensitive antibacterial activity on pathogenic bacteria that appear in aquatic animal breeding, and can improve the immune function of aquatic animals, improve animal health, and can also reduce ammonia nitrogen and nitrite in aquaculture water. content, improve the ecological environment of aquaculture, enable the healthy development of aquaculture production, increase the productivity of aquaculture, and achieve better economic and ecological benefits.

Description

A kind of lactobacillus plantarum and application thereof

technical field

The invention provides a plant lactobacillus and its application in aquaculture to obtain a functional bacterium capable of reducing ammonia nitrogen, nitrite and antibacterial, and belongs to the technical fields of aquaculture technology and ecological environment protection.

Background technique

Macrobrachium rosenbergii aquaculture is an important industry in the village and town economy in the Yangtze River Delta region of my country. But at present, the feed protein content is high, the breeding mode is rough, the utilization rate of feed protein is low, and the breeding wastewater has the characteristics of high nitrogen and phosphorus content. In a high-density breeding environment, the pollutants in the breeding wastewater include residual bait, manure and nutrients contained in excreta, namely N, P, etc. According to reports, 10-20% of the high-protein feed is not absorbed and utilized by fish and shrimp during the feeding process, and directly enters the aquaculture water body. At the same time, it is utilized by 80-90% of the ingested body, and only 20-25% of the nutrients are used as Synthesize the whole body's own protein, and 60-65% of the nutrients are excreted with the feces and enter the water body. During the breeding process, about 70-85% of the nutrients are discharged into the water body through direct and indirect means, and the excrement and residual bait are deposited in the water, which are quickly decomposed at a suitable temperature, making the ammonia nitrogen and nitrite in the water body The increase of the content will further deteriorate the ecological environment of the aquaculture pond, and easily lead to the aggravation of its diseases, which will easily cause a series of problems such as an increase in the use of aquatic animals, drug residues, and food safety. At the same time, the disorderly discharge of aquaculture wastewater has become an important cause of eutrophication in surrounding water bodies, which has seriously affected the quality and safety of water for life and production of residents in villages and towns, and has become a limiting factor restricting the healthy and sustainable development of aquaculture. Therefore, it is an urgent task to research and develop products that improve feed utilization and reduce ammonia nitrogen and nitrite in water.

To solve the most common diseases and water quality problems in intensive farming, common solutions include injection of immunization vaccines, addition of immune enhancers and probiotics to feed. However, due to its complicated operation, the development of vaccines is hindered to a certain extent, and the immune booster is only limited to the immune enhancement of animal organisms, and has little effect on water body changes.

Contents of the invention

The present invention provides a kind of Lactobacillus plantarum isolated from the intestinal tract of Macrobrachium rosenbergii, named Lactobacillus plantarum GRLP-25, preserved in China Typical Culture Collection Center, address Wuhan University, preservation date is April 18, 2016, The deposit number is CCTCC NO: M2016197.

Lactobacillus plantarum GRLP-25 can purify the water quality of aquaculture, improve and restore the ecological environment of aquaculture water; it can effectively inhibit intestinal bacteria, improve the immune function of aquatic animals, improve the disease resistance of animals, and improve the digestion and absorption of feed by aquatic animals and promote growth.

The specific steps for obtaining Lactobacillus plantarum GRLP-25 are as follows:

1. Isolation and identification of Lactobacillus plantarum

1.1 Isolation and purification of strains

After the intestinal tissue homogenate of Macrobrachium rosenbergii was diluted with sterile physiological saline by 10-fold dilution method, the dilution in the dilution range of 10 ^-5 to 10 ^-8 was spread on the MRS solid medium plate. Incubate at a constant temperature of 30°C for 48 hours, pick a typical single colony, and repeatedly streak and isolate until a pure strain is obtained.

1.2 Physiological and biochemical identification

The colony morphology of the strains was observed, Gram staining, and microscope observation. And refer to the method provided in the literature to carry out catalase test, nitrate reduction test, gelatin liquefaction test, methyl red test (MR test), indole test, hydrogen sulfide test, sugar fermentation test (micro-fermentation tube method) on the strains .

1.3 16S rRNA sequence analysis

DNA was extracted using a bacterial genomic DNA extraction kit and operated according to the instructions. Primers 27F (AGAGTTTGATCCTGGCTCAG) and 1492R (TACCTTGTTA

CTT) amplifies the 16S rDNA gene of bacteria, and the amplification system is carried out with reference to the literature. Amplification program: pre-denaturation at 95°C for 5 min, followed by PCR cycle, denaturation at 95°C for 30 s, annealing at 50°C for 60 s, extension at 72°C for 30 s, 40 cycles, and extension at 72°C for 5 min. The amplified products were electrophoresed on 1.0% agarose gel and recovered and purified. The primer synthesis and sequence determination were entrusted to Shanghai Yingjun Bioengineering Technology Co., Ltd. Submit the sequence to the GenBank database, use BLASTN to search for homologous sequences, download the homologous sequences, use software Meg 4.0 for phylogenetic analysis, and BioEdit software to analyze identity and identify strains.

2 Results and Analysis

2.1 Isolation of strains

A strain of lactic acid bacteria was isolated from the intestinal tract of Macrobrachium rosenbergii by using MRS medium. The colony was milky white translucent protrusions with neat and smooth edges; Gram staining microscopic examination showed Gram-positive, rod-shaped, and sour taste. It was numbered as GRLP-25.

2.2 Physiological and biochemical characteristics

The physiological and biochemical identification results of the strain GRLP-25 are shown in Table 1. Referring to "Bergey's Bacterial Identification Manual" (8th edition), it was preliminarily identified as Lactobacillus plantarum .

Table 1 Physiological and biochemical characteristics of strain GRLP-25 and standard strain

Note: + means positive reaction, - means negative reaction, (+) means slow reaction.

2.3 PCR amplification results and phylogenetic analysis of 16S rRNA gene

The 16S rRNA fragment was amplified by PCR, which was sequenced to 1448bp, and the GenBank accession number was JQ319663. Using BLASTN alignment, Clustal X constructs a Neighbor-Joining tree, the results are shown in Figure 1.

It can be seen from Figure 1 that the 16S rRNA of the strain GRLP -25 and the known Lactobacillus plantarum (L. plantarum) clustered on one branch, and the similarity was 100%. Based on the observation of morphological characteristics, physiological and biochemical tests, and molecular biology sequencing results, the strain isolated from the intestine of Macrobrachium rosenbergii was Lactobacillus plantarum, named Lactobacillus plantarum GRLP-25 ( Lactobacillus plantarum GRLP-25).

The isolated Lactobacillus plantarum GRLP-25 mainly has the following applications:

1. The antibacterial effect on pathogenic bacteria in aquatic animals, especially the inhibition of Macrobrachium rosenbergii pathogenic bacteria in the cultivation process of rosenbergii;

Preferred pathogenic bacteria that can inhibit aquatic animals are Enterobacter cloacae GY52, Aeromonas hydrophila SHB1, Citrobacter fischeri GYB8 and Proteus vulgaris HZL2;

2. Improve the immune function of Macrobrachium rosenbergii;

Preferably, the fermented liquid of Lactobacillus plantarum GRLP-25 is used to improve the immune function of Macrobrachium rosenbergii, and the fermented liquid is added to bait to make medicinal bait to feed Macrobrachium rosenbergii.

3. Reduce ammonia nitrogen and nitrite in aquaculture water

The Lactobacillus plantarum GRLP-25 provided by the present invention can have sensitive antibacterial activity to pathogenic bacteria occurring in aquatic animal breeding, and can improve the immune function of aquatic animals, improve the health level of animals, and can also reduce ammonia nitrogen and nitrous acid in aquaculture water The content of salt can improve the ecological environment of aquaculture, make the benign development of aquaculture production, increase the productivity of aquaculture, and achieve better economic and ecological benefits.

Description of drawings

Accompanying drawing 1 is the phylogenetic tree constructed based on the 16S rRNA gene sequence of strain GRLP-25.

detailed description

The present invention will be described in further detail below through specific examples.

Example 1:

Antibacterial effect of Lactobacillus plantarum GRLP-25 on pathogenic bacteria in aquatic animals

1. Method

1.1 Preparation of cell-free fermentation supernatant:

After activated culture of Lactobacillus plantarum GRLP-25, inoculate in MRS liquid medium with 4% inoculum amount, culture at 30°C until the stationary phase (24h~36h), centrifuge at 12000*g for 15min at 4°C, and collect the supernatant The supernatant was filtered with a 0.22 μm filter membrane to remove bacteria and other impurities to obtain a sterile fermentation supernatant. Store in a 4°C refrigerator for later use.

1.2 Bacteriostatic activity test on aquatic pathogens:

In this study, the aquatic pathogenic strains of Aeromonas hydrophila SHB1, BSK-10, Enterobacter cloacae GY52, Citrobacter fischeri GYB8, Proteus vulgaris HZL2, Vibrio parahaemolyticus DHY0708 , Vibrio harveyi GYC1108-1, and Streptococcus agalactiae L4 are test bacterial strains, adopt the agar plate punching method: pour 10ml heating and melting agar medium (suitable for test bacterium growth) into a sterile plate, wait for After it is fully cooled and solidified, put it into several sterilized Oxford cups. Take 30ml of TSA medium and heat it to melt, wait for it to cool down to about 50°C, add 0.15μl of the overnight cultured test bacteria solution, mix quickly and evenly, and pour it into a plate. After cooling, remove the Oxford cup with sterile forceps. Under aseptic operation, pipette 200 μl of the supernatant sample to be tested into the well of the Oxford cup, incubate at 30°C for 24 hours, and measure the diameter of the inhibition zone (mm).

2. Results

As shown in Table 2, the bacteriocins produced by Lactobacillus plantarum have a significant effect on the pathogens of Macrobrachium rosenbergii, Enterobacter cloacae GY52, Citrobacter fischeri GYB8H, Proteus vulgaris HZL2, Aeromonas hydrophila SHB1 and the aquatic pathogen Aeromonas All bacteria BSK-10 had sensitive antibacterial activity, and had stronger antibacterial activity against Vibrio parahaemolyticus and Vibrio harveyi, but weaker antibacterial activity against Streptococcus agalactiae L4.

Table 2 Antibacterial spectrum of bacteriocin produced by Lactobacillus plantarum GRLP-25

Example 2:

Effects of Lactobacillus plantarum GRLP-25 fermentation broth on the immune function of Macrobrachium rosenbergii

1. Method

The fermentation broth of Lactobacillus plantarum GRLP-25 was added into the bait at the mass fraction of 1%, 2%, 4%, and 8% to make medicinal bait, and 5 groups of three replicates with an average initial weight of (21.05 ± 3.18) g were fed respectively. Macrobrachium rosenbergii, one group of Macrobrachium rosenbergii was fed with no added Lactobacillus plantarum bait as a control, with 10-12 tails in each group. The experiment was carried out in 15 recirculating aquariums with a volume of 80L. The experimental water temperature is (25±1) ℃. The blood superoxide dismutase activity (SOD), acid phosphatase activity (ACP) and alkaline phosphatase activity (AKP) of Macrobrachium rosenbergii were detected after feeding for 8 weeks at intervals of 4 weeks and 8 weeks to study the bacterial cell Effects of components and metabolites on the immune function of Macrobrachium rosenbergii. SOD, ACP, and AKP enzyme activities were measured with reference to kits purchased from Nanjing Jiancheng Technology Co., Ltd.

2. Results

After feeding for 8 weeks, the effects of each Lactobacillus plantarum GRLP-25 fermentation broth addition group on SOD, ACP, AKP immune enzyme indicators in the blood of Macrobrachium rosenbergii are shown in Table 3, and the data were processed by variance analysis and p test methods. The results showed that the blood SOD enzyme activity was (44.5±3.4) U/ml in the control group without supplementation, (54.6±3.5) U/ml and (56.6±6.1) U/ml in the 2% and 4% dose groups, respectively, and compared with the control group There is a significant difference (p<0.05) between the two groups, and the 8% dose group is (65.9±3.7) U/ml, the difference is extremely significant (p<0.01); there is no significant difference between the ACP enzyme activity of each dose group and the control group; AKP enzyme activity without adding control group is (2.7±0.5) King’s unit/100ml, 1%～4% dosage group is (4.1±0.2, 4.4±0.3, 4.5±0.4) King’s unit/100ml, and the control group There was a significant difference (p<0.05) compared to the 8% dosage group (4.8±0.3) King’s units/100ml, and the difference was extremely significant (p<0.01) by T test. It can be seen that Lactobacillus plantarum GRLP-25 has a certain effect on improving the immune function of Macrobrachium rosenbergii.

Table 3 Effects of Lactobacillus plantarum GRLP-25 on the activities of various immune enzymes in the blood of Macrobrachium rosenbergii

Note: * indicates that there is a significant difference compared with the control group (p<0.05), ** indicates that the difference is extremely significant (p<0.01)

Example 3:

Effects of Lactobacillus plantarum GRLP-25 on ammonia nitrogen and nitrite

1 Materials and methods 1.1 Lactobacillus plantarum was isolated from the intestinal tract of Macrobrachium rosenbergii in our laboratory and cultured statically. Before use, the number of bacteria in the preparation was determined by plate counting.

1.2 The effect of oral Lactobacillus plantarum on ammonia nitrogen and nitrite in water

The Macrobrachium rosenbergii feed was pulverized by a pulverizer, and 0.2×10 ⁷ , 2×10 ⁸ , 2×10 ⁹ and 5×10 ⁹ cfu/g of Lactobacillus plantarum were added respectively. After 1 week of adaptive cultivation of Macrobrachium rosenbergii, they were randomly divided into 5 groups and placed in aquaculture tanks, 5 Macrobrachium rosenbergii were placed in each tank, and 5 L of aerated groundwater was placed in each tank, and the experiment was set up with 3 horizontal repetitions. Each group was gavaged once a day (9:0-10:00 a.m.), with 0.1 ml of feed containing Lactobacillus plantarum of different concentrations being gavaged each time, and the control group was gavaged with 0.1 ml of normal saline. %Feed 2 feeds. Continuous oral irrigation for 10 days. The gavage time was 9:00-9:30 am every day, and the water body in each tank was collected at 16:00 pm every day, and the contents of ammonia nitrogen and nitrite in the water body were measured. The results are shown in Table 4.

1.3 Mathematical statistics methods

Experimental data are expressed as mean ± standard error (Mean ± SE). SPSS11.5 software was used to conduct one-way ANOVA to test whether there were significant differences in the mean values of each index among different experimental groups. If there was a significant difference ( P <0.05), Duncan's multiple comparison analysis was performed.

2. Results

Table 4 Content of nitrite in water body (mg/L)

It can be seen from Table 4 that compared with the blank group, the content of nitrite in each concentration group decreased significantly. On the 7th day, the 2.5×10 ⁹ cfu/g concentration group decreased significantly (P<0.05), and 1×10 ⁹ The content of nitrite in the cfu/g concentration group decreased by 23.5%. On the 10th day, except the 1×10 ⁷ cfu/g concentration group, the other groups were significantly lower than the control group (P<0.05). It can be seen that although the content of nitrite in each concentration group has been on the rise, its content can be significantly reduced compared with the control, indicating that oral administration of Lactobacillus plantarum can reduce the content of nitrite in the cultured Macrobrachium rosenbergii water.

Table 5 Ammonia nitrogen content in water body (mg/L)

It can be seen from Table 5 that the content of each added concentration group has little change compared with the control group. On the 7th day, the 2.5×10 ⁹ cfu/g concentration group significantly decreased (P<0.05), and the 10 ⁹ cfu/g concentration group nitrous acid The salt content was reduced by 42.6%. On the 10th day, except the 1×10 ⁷ and 1×10 ⁸ cfu/g concentration groups, the other groups were significantly lower than the control group (P<0.05). At the same time, it can be seen from Table 4 that although the content of ammonia nitrogen in each concentration group has been on the rise, its content can be significantly reduced compared with the control, indicating that oral administration of Lactobacillus plantarum can reduce the content of ammonia nitrogen in the cultured Macrobrachium rosenbergii water. This may be due to the addition of Lactobacillus plantarum to the feed to feed Macrobrachium rosenbergii, its colonization in the intestinal tract may produce enzymes that accelerate the decomposition of feed and improve the utilization rate of feed, or it may be that some Lactobacillus plantarum is excreted through the feces of aquatic animals. Discharge into the water body, accelerate the decomposition of feces, reduce the pollution of the feces to the water body, or it itself has the function of degrading ammonia nitrogen or nitrite. Therefore, the Lactobacillus plantarum in this patent can improve the water quality to a certain extent, and the oral administration not only reduces the work intensity of the farmers, saves the cost, but also improves the utilization rate of the feed, which has a great prospect of popularization and application.

Claims (9)

1. A kind of Lactobacillus plantarum named Lactobacillus plantarum GRLP-25, preserved in China Center for Type Culture Collection, date of preservation is April 18, 2016, preservation number is CCTCC NO: M 2016197. 2. A kind of lactobacillus plantarum according to claim 1, characterized in that, said lactobacillus plantarum is isolated from the intestinal tract of Macrobrachium rosenbergii. 3. the application of a kind of plantarum lactobacillus as claimed in claim 1, is characterized in that, is used for the bacteriostasis of aquatic animal pathogenic bacteria. 4. the application of a kind of plantarum lactobacillus according to claim 3, is characterized in that, for suppressing Macrobrachium rosenbergii pathogenic bacteria. 5 . The application of a plantarum lactobacillus according to claim 3 , wherein the aquatic animal pathogens include Vibrio, Aeromonas hydrophila TPS-30 and Aeromonas hydrophila BSK-10. 6. the application of a kind of lactobacillus plantarum as claimed in claim 1, is characterized in that, is used for improving the immune function of Macrobrachium rosenbergii. 7. The application of a kind of Lactobacillus plantarum according to claim 1, characterized in that the fermented liquid of said Lactobacillus plantarum is used to improve the immune function of Macrobrachium rosenbergii. 8. the application of a kind of plantarum lactobacillus as claimed in claim 1, is characterized in that, is used for reducing the content of nitrite in the water body of cultured aquatic animals. 9. the application of a kind of plantarum lactobacillus as claimed in claim 1, is characterized in that, is used for reducing the content of ammonia nitrogen in the water body of cultured aquatic animals.