CN115197885A - Lactococcus garvieae LGM15, microbial inoculum and application - Google Patents

Lactococcus garvieae LGM15, microbial inoculum and application Download PDF

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CN115197885A
CN115197885A CN202211111216.6A CN202211111216A CN115197885A CN 115197885 A CN115197885 A CN 115197885A CN 202211111216 A CN202211111216 A CN 202211111216A CN 115197885 A CN115197885 A CN 115197885A
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赵勇
陈亮
钟儒清
张宏福
姜岳
张鹏飞
周业勋
韩慧
郝亚男
卢东欣
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Abstract

The invention relates to the field of microorganisms, and in particular relates to lactococcus garvieae LGM15, a microbial inoculum and application. Lactococcus garvieae of the present invention: (Lactococcus garvieae) M15 can improve metabolism of animal body, increase beneficial metabolites, and increase beneficial metabolites in testis such as melatoninThe sperm and the unsaturated fatty acid which are beneficial to spermatogenesis, thereby improving the sperm quality and the fertility of male animals; can be fixedly planted in the animal body, and has strong tolerance to artificial bile salt, artificial gastric acid and artificial intestinal juice.

Description

Lactococcus garvieae LGM15, microbial inoculum and application
Technical Field
The invention relates to the field of microorganisms, and in particular relates to lactococcus garvieae LGM15, a microbial inoculum and application.
Background
At present, the male sterility rate of male animals is greatly improved, mainly because the male reproductive system is more easily influenced by environmental factors. However, there is currently no good way to improve semen quality to treat male infertility. In recent years, researches show that intestinal flora is closely related to the health of human and other animals, however, the relationship between the intestinal flora and the male reproductive axis is not confirmed, and although researches show that the intestinal flora mixture can change the semen quality of male animals, beneficial intestinal beneficial flora can improve the semen quality of the animals, no report is made on which beneficial bacteria can improve the semen quality of the animals and improve the reproductive capacity of the male animals, and no document discloses which specific intestinal bacteria can improve the reproductive capacity of the male.
Disclosure of Invention
The invention aims to provide lactococcus garvieae (lactococcus garvieae)Lactococcus garvieae) Strain M15.
Another object of the present invention is to provide a pharmaceutical composition comprising the aforementioned lactococcus garvieae (lactococcus garvieae) (S)Lactococcus garvieae) Bacterial agent of the strain M15.
Another object of the present invention is to provide the aforementioned lactococcus garvieae (Lactococcus garvieae) Application of the strain M15.
Lactococcus garvieae of the present invention: (Lactococcus garvieae) The strain M15 was deposited in China general microbiological culture Collection center (CGMCC, accession No. 3 of Xilu No. 1 of Beijing, the national institute of sciences, the postal code 100101) at 7 months and 18 days in 2022, and was classified and named as lactococcus garvieaeLactococcus garvieaeAnd the preservation number is CGMCC No.25328.
The present invention provides a pharmaceutical composition comprising lactococcus garvieae (lactococcus garvieae)Lactococcus garvieae) The microbial inoculum of the strain M15 can be a liquid microbial inoculum or a solid microbial inoculum, and is prepared by adding auxiliary materials allowed in the field of microbial preparations by adopting conventional technical means.
The present invention provides lactococcus garvieae (Lactococcus garvieae) Use of the strain M15 for the preparation of a formulation for the use,
(1) Improving intestinal health;
(2) Improving blood and testicular metabolome;
(3) Increasing male sperm concentration;
(4) Increasing sperm motility in males;
(5) The mating success rate of male animals and the pregnancy rate of mated female animals are improved; and/or
(6) Increasing the number of live litter produced by a female mated with a male.
Lactococcus garvieae of the present invention: (Lactococcus garvieae) The strain M15 can improve the metabolism of animal organism, increase beneficial metabolites, thereby increasing testis beneficial metabolites such as melatonin and unsaturated fatty acid beneficial to spermatogenesis, and further improving the sperm quality and fertility of male animals; and lactococcus garvieae: (A), (B)Lactococcus garvieae) The strain M15 can be fixedly planted in an animal body and has strong tolerance to artificial bile salt, artificial gastric acid and artificial intestinal juice.
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FIG. 1 shows lactococcus garvieae (lactococcus garvieae) of the present inventionLactococcus garvieae) Growth curve of strain M15 (MRS medium);
FIG. 2 shows the weight change of the test mice at 35 days of the test period in example 2 of the present invention;
FIG. 3 shows sperm concentration of the test mice in example 2 of the present invention;
FIG. 4 shows sperm motility of mice tested in example 2 of the present invention;
FIG. 5 shows sperm acrosome integrity of mice tested in example 2 of the present invention;
FIG. 6 shows the acrosome teration rates of the sperm of the test mice in example 2 of the present invention;
FIG. 7 shows pregnancy rates of mice tested in example 2 of the present invention;
FIG. 8 shows the number born alive of the test mice in example 2 of the present invention;
FIG. 9 shows the amount of the test boars harvested in example 3 of the present invention;
FIG. 10 shows the concentration of sperm of the test boar in example 3 of the present invention;
FIG. 11 shows sperm motility of test boars in example 3 of the present invention;
FIG. 12 shows acrosome integrity of boar sperm tested in example 3 of the present invention;
FIG. 13 shows the teratogenicity of the sperm of the test boar in example 3 of the present invention;
FIG. 14 shows the concentrations of sperm of the test goat in example 4 of the present invention;
FIG. 15 shows the motility of sperm of the test goat in example 4 of the present invention;
FIG. 16 shows acrosome integrity of a goat sperm tested in example 4 of the present invention;
FIG. 17 shows the teratogenicity of the sperm of the goat tested in example 4 of the present invention.
Lactococcus garvieae of the present invention: (Lactococcus garvieae) Strain M15, classified and named lactococcus garvieaeLactococcus garvieaeThe preservation number is CGMCC No.25328, and the strain is preserved in China general microbiological culture Collection center (abbreviated as China Committee for culture Collection of microorganisms) at 7 month and 18 days 2022CGMCC, address No. 3 of Xilu No. 1 of Beijing, chaoyang, institute of microbiology, china academy of sciences, postal code 100101).
Detailed Description
The present invention provides lactococcus garvieae (Lactococcus garvieae) Strain M15, abbreviated LGM15, further validated lactococcus garvieae (R) ((R))Lactococcus garvieae) The strain M15 has a beneficial effect on the fertility of male animals, and a mouse asthenospermia and oligospermia model is established by Busulfan (Busulfan), so that the strain can improve the concentration, the activity and the acrosome integrity of sperms, reduce the teratospermia rate, improve the mating success rate of male mice and normal female mice, and improve the pregnancy rate and the survival number of the female mice after mating. In summer, the high temperature and high humidity can cause the quality of the semen of the boars and the rams to be reduced, thereby causing the pregnancy rate and the number of born alive piglets of the sows and the ewes to be reduced, and lactococcus garvieae: (Lactococcus garvieae) The strain M15 can improve the sperm concentration and activity and acrosome integrity of boars and rams in high temperature and high humidity state in summer and reduce the sperm teratogenesis rate.
Example 1 lactococcus garvieae: (Lactococcus garvieae) Isolation of Strain M15
1. Strain separation: grignard reagent lactococcus (C)Lactococcus garvieae) Strain M15 was isolated from intestinal contents. Taking intestinal contents, carrying out 10-fold gradient dilution by using sterile physiological saline, sucking 0.1mL of diluent with proper dilution, uniformly coating on MRS agar, culturing at 37 ℃ for 48h, and inoculating grown colonies to a new MRS solid culture medium by virtue of streaking for purification and culturing for 48h.
2. Strain enrichment: the purified and cultured strains are inoculated into a sterile MRS broth culture medium one by one on a sterile operation table by using inoculating loops, placed in a shaking table, cultured for 48h at 37 ℃, subjected to 16S rRNA identification, and the rest part is mixed with physiological saline containing 25 percent of glycerol and stored in a refrigerator at-80 ℃.
3. 16S rRNA identification: carrying out colony PCR amplification on the enriched bacterial liquid by using a bacterial universal primer, carrying out 16S rDNA sequencing identification, comparing the 16S rDNA sequence of each strain with the 16S rDNA sequences of all tested bacteria in a database on NCBI (national center of Biotechnology information), wherein 4 strains and the 16S rDNA sequences of all tested bacteria in the database are foundOf the genus lactococcusLactococcus garvieaeThe 16S rDNA sequence of def2 has the highest homology similarity, the similarity is more than or equal to 99.8 percent, and the 4 strains are determined to be lactococcus garvieae (Lactococcus garvieae)。
Example 2 lactococcus garvieae: (Lactococcus garvieae) Characterization of Strain M15
1. Grignard reagent lactococcus (C)Lactococcus garvieae) The microbiological properties of strain M15 are as follows:
(1) Colony morphology: the bacterial colony is a circular single bacterial colony with the diameter of about 1 to 2mm, is milky and opaque, and has smooth and slightly-protruded surface and regular edge.
(2) After staining, the bacteria are in a short rod shape, have no flagella and do not move.
(3) Growth characteristics: culturing in MRS liquid culture medium at 37 deg.C by shaking table for 5 hr, starting logarithmic growth phase, and 16 hr for plateau phase with maximum viable count of 2.98 × 10 8 CFU/mL (FIG. 1).
2. Bile salt resistance assay
Activated bacteria liquid concentration is 1 × 10 8 1mL of CFU/mL bacterial liquid is respectively inoculated into 9mL of sterile physiological saline containing pig bile salt with the concentration of 0.3g/100mL, and the viable count is determined by adopting a plate coating method after 1 h.
The results show (Table 1) that lactococcus garvieae: (lactococcus garvieae: (lactococcus garvieae) at a bile salt concentration of 0.3g/100mL, as compared with other 3 strains of lactococcus garvieaeLactococcus garvieae) The strain M15 has the best bile salt resistance, and the survival rate is about 20.15%.
TABLE 1 survival rate of lactococcus garvieae at a concentration of 0.3% strong bile salt%
Figure 942166DEST_PATH_IMAGE001
3. Artificial gastric juice resistance assay
Activated bacterial liquid concentration is 1X 10 8 1mL of each CFU/mL bacterial solution was inoculated into 9mL of artificial gastric juice with pH =1.5, and the viable cell count was measured by the plate coating method after 1 hour.
Results show (Table 2)Lactococcus garvieae after 1h of artificial gastric juice at pH1.5, in comparison with other 3 strains of lactococcus garvieae (Lactococcus garvieae) The strain M15 has the best artificial gastric juice resistance, and the survival rate is about 22.33%.
TABLE 2 survival Rate of lactococcus garvieae in artificial gastric juice at pH 1.5%
Figure 859306DEST_PATH_IMAGE002
4. Detection of resistant artificial intestinal juice
Activated bacteria liquid concentration is 1 × 10 8 1mL of each CFU/mL bacterial solution was inoculated into 9mL of pH =6.8 artificial gastric juice, and viable cell count was measured by a plate coating method after 4 hours.
The results show (Table 3) that after 4h of artificial intestinal juice, lactococcus garvieae: (lactococcus garvieae) was compared with other 3 strains of lactococcus garvieaeLactococcus garvieae) The strain M15 has the best capability of resisting the artificial intestinal juice, and the survival rate is about 21.54 percent.
TABLE 3 survival rate of lactococcus garvieae in artificial intestinal juice%
Figure 311147DEST_PATH_IMAGE003
Example 3 Effect of Lactobacillus gasseri M15 on the Productivity of male mice in the Busulfan model
1. Experimental methods
1. 100 21 day old Balb/c male mice were selected. During the test, the temperature of the rat room was controlled at 22 + -2 deg.C, the light/dark time was 12 hours, and the rat room was freely fed with water and drunk.
2. All mice were randomized into 5 groups (20 mice per group): control group (Con group), busulfan group (B group), B + LGM15 (10 ^ 6) group, B + LGM15 (10 ^ 8) group, B + LGM15 (10 ^ 10) group. Con group: in the test process, the stomach is irrigated with 0.1ml of physiological saline once a day for 14 days; group B: in the test process, the stomach is irrigated with 0.1ml of physiological saline once a day for 14 days; b + LGM15 (10 ^ 6) group: during the test, the gavage was performed once a day at a concentration of 0.1ml of 1X 10 6 CFU/mL bacterial liquid for 14 days; b + LGM15 (10 ^ 8) group: during the test, the gavage was performed once a day at a concentration of 0.1ml of 1X 10 8 CFU/mL bacterial liquid for 14 days; b + LGM15 (10 ^ 10) group: during the test, the gavage was performed once a day at a concentration of 0.1ml of 1X 10 10 CFU/mL of bacterial suspension, for 14 days. Then, all mice were normally kept for 21 days (3 weeks) without any treatment, and during the test, the body weights of the mice were measured every day, and the states, survival conditions, presence or absence of clinical abnormal symptoms, and the like of the mice were observed and recorded. After a total of 35 days (14 +21 days), at day 36, 10 mice per group were randomly selected and samples were collected for analysis. The remaining 10 mice of each group were mated with normal female mice, and each male mouse was mated with 2 female mice for 4 days. 4 days after mating, the female mice are raised in a single cage, and the male mice are collected, detected and analyzed. After 21 days of mating, the pregnancy rate of the female mouse is counted, and the number born alive is counted.
2. Detecting the index
1. During the test period, the weight of the mice is counted once every 2 days;
2. measuring the sperm concentration, the motility, the acrosome integrity and the teratogenesis rate of the male mouse;
3. counting the pregnancy rate and the number born alive of the female mouse 21 days after mating;
4. and blood indicators and testicular metabolites of the mice were tested.
3. Results of the experiment
1. Growth conditions were as follows: growth changes in mice throughout the experiment are shown in figure 2, with the weight gain of busulfan mice becoming slower, starting on day 1 of treatment and continuing through the end of the experiment, with the busulfan group weighing significantly less than the control group, and the 3 groups fed LGM15 after busulfan treatment weighing significantly more than the busulfan group.
2. Sperm concentration: as shown in FIG. 3, the concentration of sperm in the mice in the busulfan model group was much lower than that in the control group, and the concentration of sperm in the 3 groups fed with LGM15 after busulfan treatment was much higher than that in the busulfan group, almost reached the level of the control group, and the concentration dependence increased.
3. Sperm motility: as shown in fig. 4, the sperm motility of the mice in the busulfan model group was much lower than that of the control group, and the sperm motility of the 3 groups fed with LGM15 after the busulfan treatment was much higher than that of the busulfan group, almost reached the level of the control group, and the concentration dependence was increased.
4. Sperm acrosome integrity: as shown in fig. 5, the integrity of the sperm acrosome of mice in busulfan model group was deteriorated and was much lower than that of the control group, and the integrity of the sperm acrosome of 3 groups fed LGM15 after busulfan treatment was much higher than that of the busulfan group, almost reached the level of the control group, and the concentration dependency was increased.
5. Sperm teratogenesis rate: as shown in FIG. 6, the sperm aberration rate of the mice in the Busulfan model group was much higher than that of the control group, and the sperm aberration rate of the 3 groups fed with LGM15 after Busulfan treatment was much lower than that of the Busulfan group, which was equivalent to that of the control group, and the concentration dependency decreased.
6. Pregnancy rate: as shown in fig. 7, the pregnancy rate of mice in busulfan model group after mating with normal mother mice was much lower than that of the control group, and the pregnancy rate of mice in 3 groups fed with LGM15 after busulfan treatment after mating with normal mother mice was much higher than that of busulfan group, almost reaching the level of the control group.
7. Number of born alive offspring: as shown in fig. 8, after the mice in the busulfan model group were mated with the normal mice, the number of live animals born by the mice was much smaller than that of the control group, and after the mice in the 3 groups fed with LGM15 after the busulfan treatment were mated with the normal mice, the number of live animals born by the mice was much larger than that of the busulfan group, and almost reached the level of the control group.
8. In addition, analysis of blood and testis metabolites of treated mice shows that Busulfan increases harmful metabolites in blood and testis and reduces beneficial metabolites, while feeding LGM15 after Busulfan treatment increases beneficial metabolites in blood and testis, such as melatonin, unsaturated fatty acids, etc.
Example 4 Effect of Lactobacillus gasseri strain M15 on boar semen quality
1. Experimental method
A healthy boar in a boar station with 40 heads (2-3 years old) and the weight of about 300 kilograms (kg) is selected. All boars were housed individually in closed piggeries and provided with commercial feed and free drinking water. The pigsty environment is kept clean, ventilated and disinfected regularly. The experiment was conducted during the summer months of 7,8.
All boars were randomly divided into 4 groups: control group (Con); LGM15 (10 ^ 9) group; LGM15 (10 ^ 12) group; LGM15 (10 ^ 15) group; each group had 10 boars. Con group: feeding commercial feed; LGM15 (10 ^ 9) group: feeding commercial feed +10 9 CFU/head/lactococcus lactis LGM15; LGM15 (10 ^ 12) group: feeding commercial feed +10 12 CFU/head/day LGM15; LGM15 (10 ^ 15) group: feeding commercial feed +10 15 CFU/head/day LGM15; LGM15 was fed for two weeks (14 days) and then all groups were fed commercial feed for 56 days (total experimental period 70 days), 2 times daily, dosed. During the test, the boar status was observed and recorded. Boar semen was harvested 70 days later and tested for analysis.
2. Detecting the index
1. During the test period, counting the physical signs of the boars every day;
2. boars were examined for semen quality starting on day 71.
3. Results of the experiment
1. And (3) precision extraction: as shown in fig. 9, the 3 groups fed LGM15 had more harvest than the control group and increased concentration dependence;
2. sperm concentration: as shown in fig. 10, the sperm concentration of the 3 groups fed LGM15 was higher than that of the control group, and the concentration-dependently increased;
3. sperm motility: as shown in fig. 11, the sperm motility of 3 groups fed LGM15 was higher than that of the control group, and the concentration dependence increased;
4. sperm acrosome integrity: as shown in fig. 12, the sperm acrosome integrity of the 3 groups fed LGM15 was higher than the control group and increased in concentration dependence;
5. sperm teratogenesis rate: as shown in fig. 13, the sperm teratogenicity was lower in the 3 groups fed LGM15 than in the control group, and the concentration dependence was reduced.
Example 5 Effect of Lactobacillus gasseri strain M15 on the quality of sperm of a goat
1. Experimental method
40 healthy goats (2-3 years old) are selected, and the weight is about 100 kilograms (kg). All goats were housed individually in closed sheeppens and provided with commercial feed (concentrate and forage) and free drinking water. The sheepfold environment is kept clean, ventilated and periodically disinfected. The experiment was performed in summer months 7,8.
All the goats were randomized into 4 groups: control group (Con); LGM15 (3 \9674; 10^ 8) group; LGM15 group; LGM15 (3 \9674; 10^ 14) group; each group contained 10 goats. Con group: feeding commercial feed; LGM15 (3 \9674; 10^ 8) group: feeding commercial feed +3 \967410 8 CFU/head/day LGM15; LGM15 (3 \9674; 10^ 11) group: feeding commercial feed +3 \967410 11 CFU/head/day LGM15; LGM15 (3 \9674; 10^ 14) group: feeding commercial feed +3 \967410 14 CFU/head/day LGM15; LGM15 was fed for two weeks (14 days) and then all groups were fed commercial feed for 56 days (total experimental period 70 days), dosed. During the test, the state of the goats was observed and recorded. Ram semen was harvested 70 days later and tested for analysis.
2. Detecting the index
1. During the test period, the ram was counted daily for signs.
2. The sperm quality of the rams was measured starting on day 71.
3. Results of the experiment
1. Sperm concentration: as shown in fig. 14, the sperm concentration of the 3 groups fed LGM15 was higher than that of the control group, and the concentration dependence was increased.
2. Sperm motility: as shown in FIG. 15, the sperm motility of the 3 groups fed with LGM15 was higher than that of the control group, and the concentration dependence was increased.
3. Sperm acrosome integrity: as shown in fig. 16, the sperm acrosome integrity of the 3 groups fed LGM15 was higher than the control group and increased in concentration dependence.
4. Sperm teratogenesis rate: as shown in fig. 17, the sperm teratogenicity was lower in the 3 groups fed LGM15 than in the control group, and the concentration dependence was reduced.
Therefore, compared with lactococcus garvieae of the same genus, LGM15 has the greatest advantages of improving animal reproductive performance and having strong tolerance to artificial bile salts, artificial gastric acid and artificial intestinal juice.
The above embodiments are only used to explain the technical solutions of the present application, and do not limit the scope of protection of the present application.

Claims (4)

1. Grignard reagent lactococcus (C)Lactococcus garvieae) M15, characterized in that said lactococcus garvieae (S.) (Lactococcus garvieae) The preservation number of M15 is CGMCC No.25328.
2. Comprising the lactococcus garvieae (lactococcus garvieae) of claim 1 (c.) (Lactococcus garvieae) M15 bacteria agent.
3. The microbial inoculum according to claim 2, which is a liquid microbial inoculum or a solid microbial inoculum.
4. Lactococcus garvieae (lactococcus garvieae) according to claim 1Lactococcus garvieae) The use of M15 for the preparation of a formulation for the use,
(1) Formulations for improving intestinal health;
(2) Preparations for improving blood and testicular metabolome;
(3) An agent that increases sperm concentration in males;
(4) Agents that increase sperm motility in males;
(5) An agent for increasing the mating success rate of male animals and the pregnancy rate of mated female animals; and/or
(6) A preparation for increasing the number of live calves of a female animal mated with a male animal.
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CN116083307B (en) * 2023-01-10 2024-05-07 四川轻化工大学 Lactococcus garvieae for producing esterase and ester compounds and application thereof

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