CN116286556B - Lactococcus garvieae ABML2022003, microbial starter and preparation method thereof - Google Patents

Lactococcus garvieae ABML2022003, microbial starter and preparation method thereof Download PDF

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CN116286556B
CN116286556B CN202310501538.XA CN202310501538A CN116286556B CN 116286556 B CN116286556 B CN 116286556B CN 202310501538 A CN202310501538 A CN 202310501538A CN 116286556 B CN116286556 B CN 116286556B
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lactococcus garvieae
culture medium
microbial starter
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王凯
王路英
沈娟娟
杨华
黎萍
严晶
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Jiangmen Aobao Biological Technology Co ltd
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Abstract

The application is applicable to the technical field of microorganisms, and provides a lactococcus garvieae ABML2022003, a microbial starter and a preparation method thereof. The application is separated from healthy penaeus vannamei boone intestinal tracts, is applied to aquatic animals, is more beneficial to bacterial strain colonization, has reasonable bacterial strain adopted by a bacterial agent and strong bacterial strain activity, can generate various digestive enzymes (protease, lipase, amylase and the like), organic acids, vitamins and various growth promoting factors in the metabolic process, and can promote the digestion, absorption, nutrition and metabolism of the cultivated animals and improve the feed availability. The method can promote the proliferation of lactobacillus in the intestinal tracts of aquatic animals, form dominant lactobacillus flora, maintain the microecology balance of the intestinal tracts, effectively inhibit the reproduction of pathogenic bacteria, promote the growth of aquatic animals and reduce the consumption of feed; the stability is good, no toxic or side effect is caused, no residue exists, and the safety and pollution are avoided; in addition, the production is carried out by adopting a modern fermentation tank, so that the production cost and the use cost are reduced, the application is wide, and the effect is remarkable.

Description

Lactococcus garvieae ABML2022003, microbial starter and preparation method thereof
Technical Field
The application belongs to the technical field of microorganisms, and particularly relates to a lactococcus garvieae ABML2022003, a microbial starter and a preparation method thereof.
Background
A large number of researches show that the lactobacillus microecological live bacteria preparation can regulate the normal flora balance of the gastrointestinal tract of the organism, improve the nutritive value of the feed, improve the flavor of the feed, improve the digestibility, inhibit the growth and reproduction of pathogenic bacteria in the intestinal tract and in water environment, degrade harmful substances in the water body and stabilize the pH value of the water body, and is a microbial preparation which is expected to replace antibiotics.
At present, most of probiotics preparations are mainly prepared from lactobacillus, including lactobacillus casei, lactobacillus acidophilus, lactobacillus plantarum and the like, which are derived from intestinal tracts of human beings and livestock and poultry animals, but the problems of unreasonable strain, weak strain activity and unobvious improvement on feed availability are generally solved.
Disclosure of Invention
The embodiment of the application aims to provide the lactococcus garvieae ABML2022003, and aims to solve the problems that the existing probiotic preparation is unreasonable in strain, weak in strain activity and not obvious in improving the feed utilization effect.
The embodiment of the application is realized in such a way that the preservation number of the lactococcus garvieae ABML2022003 is CCTCC NO: m2022223.
Another object of an embodiment of the present application is a microbial starter culture, characterized in that the microbial starter culture comprises the above-mentioned lactococcus garvieae ABML2022003.
Another object of the embodiment of the present application is to provide a method for preparing a microbial starter, wherein the microbial starter is obtained by culturing the lactobacillus gasseri ABML2022003 serving as a starting strain in a culture medium of the lactobacillus gasseri ABML 2022003; the culture medium of the lactococcus garvieae ABML2022003 comprises a liquid culture medium and a solid culture medium.
The lactococcus garvieae ABML2022003 provided by the embodiment of the application is separated from the intestinal tracts of healthy penaeus vannamei, is more beneficial to bacterial strain colonization when being applied to aquatic animals, has reasonable bacterial strain adopted by the microbial inoculum and strong bacterial strain activity, can generate various digestive enzymes (protease, lipase, amylase and the like), organic acid, vitamins and various growth promoting factors in the metabolic process, and can promote the digestion, absorption and nutrition metabolism of the cultivated animals and improve the feed availability. The method can promote the proliferation of lactobacillus in the intestinal tracts of aquatic animals, form dominant lactobacillus flora, maintain the microecology balance of the intestinal tracts, effectively inhibit the reproduction of pathogenic bacteria, promote the growth of aquatic animals and reduce the consumption of feed; the stability is good, no toxic or side effect is caused, no residue exists, and the safety and pollution are avoided; in addition, the production is carried out by adopting a modern fermentation tank, so that the production cost and the use cost are reduced, the application is wide, and the effect is remarkable.
Drawings
Fig. 1 is a flowchart of a preparation process of a microbial starter according to an embodiment of the present application.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present application more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In order to solve the problems that the existing probiotics preparation is unreasonable in strain, weak in strain activity and unobvious in improvement of feed utilization effect, the embodiment of the application provides the lactococcus garvieae ABML2022003 which is separated from the intestinal tracts of healthy penaeus vannamei, is more beneficial to strain field planting when being applied to aquatic animals, is reasonable in strain adopted by the microbial inoculum, strong in strain activity, and can produce various digestive enzymes (protease, lipase, amylase and the like), organic acids, vitamins and various growth promoting factors in the metabolic process, and the substances can promote digestion, absorption, nutrition metabolism and feed utilization rate of the cultured animals. The method can promote the proliferation of lactobacillus in the intestinal tracts of aquatic animals, form dominant lactobacillus flora, maintain the microecology balance of the intestinal tracts, effectively inhibit the reproduction of pathogenic bacteria, promote the growth of aquatic animals and reduce the consumption of feed; the stability is good, no toxic or side effect is caused, no residue exists, and the safety and pollution are avoided; in addition, the production is carried out by adopting a modern fermentation tank, so that the production cost and the use cost are reduced, the application is wide, and the effect is remarkable.
In the embodiment of the present application, the preservation number of the lactococcus garvieae ABML2022003 is CCTCC NO: m2022223, classified and namedLactococcus garvieae ABML2022003, with a deposit address of university of Wuhan, china, a deposit unit of China center for type culture Collection, and a deposit date of 2022, 3 months and 9 days.
In the embodiment of the application, the gene sequence of the 16S rDNA of the lactococcus garvieae ABML2022003 is shown as SEQ ID NO. 1.
SEQ ID NO:1
TGCAAGTCGAGCGATGATTAAAGATAGCTTGCTATTTTTATGAAGAGCGGCGAACGGGTGAGTAACGCGTGGGAAATCTGCCGAGTAGCGGGGGACAACGTTTGGAAACGAACGCTAATACCGCATAACAATGAGAATCGCATGATTCTTATTTAAAAGAAGCAATTGCTTCACTACTTGATGATCCCGCGTTGTATTAGCTAGTTGGTAGTGTAAAGGACTACCAAGGCGATGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACGTTAAGTAGAGTGGAAAATTACTTAAGTGACGGTATCTAACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGTGGTTTCTTAAGTCTGATGTAAAAGGCAGTGGCTCAACCATTGTGTGCATTGGAAACTGGGAGACTTGAGTGCAGGAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGAGGCGAAAGCGGCTCTCTGGCCTGTAACTGACACTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGCTGTAGGGAGCTATAAGTTCTCTGTAGCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATACTCGTGCTATCCTTAGAGATAAGGAGTTCCTTCGGGACACGGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCATCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCCAACCCGCGAGGGTGCGCTAATCTCTTAAAACCATTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGAAGTTGGGAGTACCCAAAGTAGGTTGCCTAACCGCAAGGAGGGCGCT
The embodiment of the application also provides a microbial starter, which comprises the lactococcus garvieae ABML2022003.
In the examples of the present application, the number of bacteria of the lactococcus garvieae-containing ABML2022003 per 1mL of the microbial fermentation agent was 1×l0 8 cfu / mL~5×10 9 cfu / mL。
The embodiment of the application also provides a preparation method of the microbial starter, wherein the microbial starter is obtained by taking the lactococcus garvieae ABML2022003 as a starting strain and culturing the starting strain in a culture medium of the lactococcus garvieae ABML 2022003; the culture medium of the lactococcus garvieae ABML2022003 comprises a liquid culture medium and a solid culture medium.
When the culture medium is a liquid culture medium, the culture medium comprises the following raw materials in percentage by weight in 1L of water: yeast extract powder 0.1-1%, peptone 0.1-1%, glucose 2-5%, sodium acetate 0.2-1.4%, dipotassium hydrogen phosphate 0.1-0.4%, diamine hydrogen citrate 0.1-0.3%, magnesium sulfate 0.01-0.03% and manganese sulfate 0.005-0.01%;
when the culture medium is a solid culture medium, the culture medium comprises the following raw materials in percentage by weight in each 1L of water and 15g of agar: yeast extract powder 0.1-1%, peptone 0.1-1%, glucose 2-5%, sodium acetate 0.2-1.4%, dipotassium hydrogen phosphate 0.1-0.4%, diamine hydrogen citrate 0.1-0.3%, magnesium sulfate 0.01-0.03% and manganese sulfate 0.005-0.01%.
In the examples herein, the pH of the medium is between 6.0 and 6.5.
According to the embodiment of the application, through optimizing each component of the culture medium, the influence of the change of the components of the culture medium on fermentation indexes such as lactic acid, pH, lactic acid bacteria activity and the like is obvious.
Optionally, the preparation method of the microbial starter comprises the following steps:
placing the lactococcus garvieae ABML2022003 in the solid culture medium, and culturing at 30-35 ℃ for 48h to obtain a solid culture;
inoculating the solid culture into the liquid culture medium, and culturing for 16-20h at 30-35 ℃ to obtain primary seed liquid;
transferring the first-stage seed liquid into a liquid culture medium for expansion culture, wherein the transfer amount is 1-10%, and standing culture is carried out at 30-35 ℃ for 12-20h to obtain a second-stage seed liquid;
inoculating the secondary seed liquid into a fermentation tank according to the concentration of 1-10%, intermittently stirring and culturing for 16-48h at the temperature of 30-35 ℃, and reducing the pH value to 3.0-4.5 to obtain the microbial starter.
In the embodiment of the application, purified water, yeast extract, peptone, glucose, sodium acetate, dipotassium hydrogen phosphate, diammonium hydrogen citrate, magnesium sulfate and manganese sulfate are mixed in the fermentation tank in advance, and after high-temperature heating, the temperature and pressure are maintained at 100-115 ℃ for 15-30min, and then the temperature is reduced to 30-35 ℃.
More specifically, the preparation process of the microbial starter can be performed with reference to the process flow shown in fig. 1.
Examples of certain embodiments of the present application are given below, which are not intended to limit the scope of the present invention.
Example 1
The embodiment provides a method for culturing lactococcus garvieae ABML2022003, wherein the culture medium comprises the following components:
yeast soaking powder: 0.25%; peptone: 0.25%; glucose: 2%; sodium acetate: 0.4%; dipotassium hydrogen phosphate: 0.2%; hydrogen diamine citrate: 0.2%; magnesium sulfate: 0.01%; manganese sulfate: 0.005%, agar: 15g, distilled water: 1L, pH6.0-6.5.
Inoculating bacteria on the solid culture medium, and culturing at 30-35deg.C for 48 hr to obtain culture; inoculating the cultured fresh solid culture into the liquid culture medium without agar, and culturing for 16h at 35 ℃ to obtain primary seed liquid; transferring the primary seed liquid into a liquid culture medium for expansion culture, wherein the transfer amount is 1%, standing and culturing for 16h at 35 ℃ to obtain a secondary seed liquid, and then inoculating the secondary seed liquid cultured for 16h into a fermentation tank according to the concentration of 1%.
Adding yeast extract powder into a fermentation tank: 0.1%; peptone: 0.1%; glucose: 2%; sodium acetate: 0.2%;dipotassium hydrogen phosphate: 0.1%; hydrogen diamine citrate: 0.1%; magnesium sulfate: 0.01%; manganese sulfate: 0.005% of pure water and the balance of pure water, maintaining 115 ℃ after heating at high temperature, preserving heat and pressure for 15 minutes, then cooling to 35 ℃, inoculating according to 1% concentration, intermittently stirring and culturing at 35 ℃ for 36 hours, and reducing the pH to 4.32 to obtain the microbial starter containing 1.1X10% of viable lactococcus garvieae 9 CFU/mL, lactic acid content of 8.5. 8.5 mg/mL, and aseptically canning after taking out of the pot at room temperature.
Example 2
The embodiment provides a method for culturing lactococcus garvieae ABML2022003, wherein the culture medium comprises the following components:
yeast soaking powder: 0.25%; peptone: 0.25%; glucose: 2%; sodium acetate: 0.4%; dipotassium hydrogen phosphate: 0.2%; hydrogen diamine citrate: 0.2%; magnesium sulfate: 0.01%; manganese sulfate: 0.005%, agar: 15g, distilled water: 1L, pH6.0-6.5.
Inoculating bacteria on the solid culture medium, and culturing at 30-35deg.C for 48 hr to obtain culture; inoculating the cultured fresh solid culture into the liquid culture medium without agar, and culturing for 16h at 35 ℃ to obtain primary seed liquid; transferring the primary seed liquid into a liquid culture medium for expansion culture, wherein the transfer amount is 1%, standing and culturing for 16h at 35 ℃ to obtain a secondary seed liquid, and then inoculating the secondary seed liquid cultured for 16h into a fermentation tank according to the concentration of 1%.
Adding yeast extract powder into a fermentation tank: 0.5%; peptone: 0.5%; glucose: 3%; sodium acetate: 1%; dipotassium hydrogen phosphate: 0.2%; hydrogen diamine citrate: 0.2%; magnesium sulfate: 0.02%; manganese sulfate: 0.008 percent of pure water and the balance of pure water, maintaining 115 ℃ after heating at high temperature, preserving heat and pressure for 15 minutes, then cooling to 35 ℃, inoculating according to 1 percent concentration, intermittently stirring and culturing for 36 hours at 35 ℃, and reducing the pH to 4.17, wherein the obtained microbial starter contains 2.5x10 viable lactococcus garvieae 9 CFU/mL, lactic acid content 11.2mg/mL, and aseptically canning after taking out of the pot at room temperature.
Example 3
The embodiment provides a method for culturing lactococcus garvieae ABML2022003, wherein the culture medium comprises the following components:
yeast soaking powder: 0.25%; peptone: 0.25%; glucose: 2%; sodium acetate: 0.4%; dipotassium hydrogen phosphate: 0.2%; hydrogen diamine citrate: 0.2%; magnesium sulfate: 0.01%; manganese sulfate: 0.005%, agar: 15g, distilled water: 1L, pH6.0-6.5.
Inoculating bacteria on the solid culture medium, and culturing at 30-35deg.C for 48 hr to obtain culture; inoculating the cultured fresh solid culture into the liquid culture medium without agar, and culturing for 16h at 35 ℃ to obtain primary seed liquid; transferring the primary seed liquid into a liquid culture medium for expansion culture, wherein the transfer amount is 1%, standing and culturing for 16h at 35 ℃ to obtain a secondary seed liquid, and then inoculating the secondary seed liquid cultured for 16h into a fermentation tank according to the concentration of 1%.
Adding yeast extract powder into a fermentation tank: 1%; peptone: 1%; glucose: 5%; sodium acetate: 1.4%; dipotassium hydrogen phosphate: 0.4%; hydrogen diamine citrate: 0.3%; magnesium sulfate: 0.03%; manganese sulfate: 0.01 percent of pure water and the balance of pure water, maintaining 115 ℃ after heating at high temperature, preserving heat and pressure for 15 minutes, then cooling to 35 ℃, inoculating according to 1 percent concentration, intermittently stirring and culturing for 36 hours at 35 ℃, and reducing the pH to 4.24, wherein the obtained microbial starter contains 3.2x10 percent of viable lactococcus garvieae 9 CFU/mL, lactic acid content 12.0mg/mL, and aseptically canning after taking out of the pot at room temperature.
Test case
The experimental method comprises the following steps:
the test is carried out in a circulating water culture system, fish fries are temporarily cultured in a glass jar, basic feed is fed every day, after the test fish adapt to the environment of the glass jar, after the feed intake is normal, the initial weight of the tilapia juvenile fish with strong physique and good vitality is selected and randomly divided into 12 glass fish tanks with the specification of 70cm multiplied by 40cm, 30 fish are subjected to each test, the formal test time is 60d, the feed is fed twice a day, the feed is fed 9:00 a day earlier, 17:00 a day later, the residual feed and excrement are sucked out by a siphon pipe after the feed feeding is finished for 2 hours until the fish swarm is basically not robbed, and water is changed 1/3 a day. Keeping the water temperature at (25+/-1) ℃, and keeping the pH value of 7.7-8.1, wherein the dissolved oxygen concentration is more than 6mg/L, and continuously oxygenating for 24 hours.
The test is provided with 7 treatment groups, each treatment group is provided with 3 replicates, wherein 1 control group A is fed with basic feed,3 test groups B, C, D and 3 control groups E, F, G, the test group feed was added by spraying 5X 10 on the base feed 6 CFU / mL、5×10 7 CFU / mL、5×10 8 The CFU/mL of the fermentation liquid (microbial starter) of the lactococcus garvieae is made into feed, and the feed is naturally air-dried, so that sunlight irradiation is avoided, lactobacillus is killed by ultraviolet rays, and the feed is stored at 4 ℃ and in a dark place after being air-dried. A comparative test group feed was prepared in the same manner using Lactobacillus acidophilus.
And (3) collecting samples 60d after formal culture, fasting for 24 hours before collecting the samples, taking out the live fish, wiping the body surface with gauze, weighing and measuring the length. And randomly taking 6 fish, opening abdominal cavity of fish body under aseptic operation, taking out intestinal tracts, respectively placing into corresponding aseptic centrifuge tubes, and storing in a refrigerator at-80 ℃ for standby.
Measurement of growth index:
weight Gain Ratio (WGR) = (end body weight-initial body weight)/initial body weight×100%
Feed Coefficient (FCR) =bait weight/body weight gain
Specific Growth Rate (SGR) = (Ln final body weight/Ln initial body weight)/days of trial x 100%
The experimental results of the effect of lactococcus garvieae on the growth performance of tilapia are shown in table 1.
TABLE 1
Group of Initial average weight (g) Last average weight (g) Weight gain Rate (%) Specific growth rate (%/d) Feed coefficient (%)
A 2.47±0.14 35.22±0.43 1329.45±25.07 4.43±0.02 1.17±0.14
B 2.45±0.12 37.65±0.32 1440.34±17.66 4.56±0.03 1.15±0.18
C 2.44±0.20 39.87±0.34 1521.45±27.96 4.65±0.03 1.08±0.10
D 2.51±0.13 38.78±0.24 1455.32±14.09 4.57±0.02 1.09±0.12
E 2.45±0.15 36.43±0.21 1437.42±16.04 4.49±0.02 1.10±0.11
F 2.46±0.22 38.57±0.23 1512.22±16.13 4.58±0.02 1.09±0.10
G 2.50±0.10 38.28±0.31 1458.32±16.45 4.56±0.02 1.08±0.13
Compared with the control group A, the weight gain rates of the test group B, C, D and the comparative example E, F, G are obviously improved, wherein the concentration of the C group, namely the added lactococcus garvieae, is 5 multiplied by 10 7 When CFU/mL is carried out, the weight gain rate of the tilapia is highest and reaches 1521.45 +/-27.96, which is obviously higher than that of the control group A and the comparative group; the specific growth rates of both test group B, C, D and comparative example E, F, G were significantly improved compared to control group a; compared with the control group A, the feed coefficients of the test group B, C, D and the control group E, F, G are reduced to different degrees, and the feed coefficient of the group C is the lowest; research shows that various digestive enzymes produced by lactic acid bacteria are helpful for digestion and absorption of fish and promote the growth of fish. The experimental result shows that the addition of a proper amount of lactococcus garvieae in the feed can increase the weight gain rate and the specific growth rate of tilapia in different degrees.
The experimental results of the effect of lactococcus garvieae on the intestinal flora of tilapia are shown in table 2.
TABLE 2
Group of Bacterial count (lg CFU/g) Vibrio count (lg CFU/g) Lactic acid bacteria count (lg CFU/g)
A 6.43±0.02 2.53±0.07 4.09±0.03
B 6.26±0.06 1.94±0.03 4.35±0.04
C 6.23±0.05 1.66±0.11 4.57±0.05
D 6.36±0.01 1.78±0.06 4.60±0.04
E 6.33±0.02 1.88±0.10 4.25±0.03
F 6.27±0.04 1.76±0.05 4.33±0.05
G 6.34±0.05 1.73±0.06 4.46±0.06
The total bacteria count was reduced to a different extent in test group B, C, D compared to comparative example E, F, G compared to control group a; the addition of lactobacillus in the feed can reduce the vibrio number in the intestinal tract of tilapia, the difference of the vibrio number between the test group B, C, D and the comparison example E, F, G and the comparison group A is obvious, and the group C has the best effect of reducing the vibrio; lactic acid bacteria are added into the feed to increase the number of lactic acid bacteria in the intestinal tracts of tilapia mossambica, the number of lactic acid bacteria in the test group B, C, D is increased to different degrees compared with that in the comparison group A of the comparison example E, F, G, lactic acid and other organic acids generated by the lactic acid bacteria can reduce the pH value of the intestinal tracts, inhibit the growth and reproduction of pathogenic bacteria, regulate the composition of intestinal flora and maintain the microecological balance of the intestinal tracts. The test result shows that the addition of a proper amount of lactococcus garvieae in the feed can reduce the bacterial count and the vibrio count in the intestinal tract of tilapia and increase the lactic acid bacteria count.
The experimental results of the effect of lactococcus garvieae on intestinal digestive enzymes of tilapia are shown in table 3.
TABLE 3 Table 3
Group of Lipase (U/gprot) Amylase (U/mgprot) Trypsin (U/mgprot)
A 82.41±1.43 0.72±0.01 779.56±10.65
B 90.25±1.37 0.85±0.02 818.11±9.54
C 96.33±1.83 0.95±0.02 866.12±12.32
D 94.37±1.65 0.89±0.01 846.22±16.15
E 90.32±1.32 0.81±0.01 856.36±13.33
F 92.51±1.53 0.85±0.01 865.42±11.36
G 94.79±1.47 0.84±0.01 847.72±16.73
The activity of the digestive enzyme of the fish can reflect basic digestive physiological characteristics, and can also be used as an important index for measuring the digestion, absorption and utilization of the nutrient components of the feed by the fish, and the activity of the digestive enzyme determines the digestion and absorption capacity of the nutrient components of the fish. The activity of lipase, amylase and trypsin is improved, so that the decomposition of nutrients and the digestion and absorption of baits by fish can be promoted, and the growth of fish is promoted. The test result shows that the lipase, amylase and trypsin activities in the intestinal tracts of the tilapia can be obviously improved by adding the lactococcus garvieae to feed the tilapia, the lipase, amylase and trypsin activities of the test group B, C, D and the comparative example E, F, G are improved to different degrees compared with those of the control group A, and the three enzyme activities of the test group C are higher than those of other test groups. The test result proves that the lactococcus garvieae plays a role in the tilapia body to a certain extent, enhances the digestive enzyme activity of the body, enhances the decomposition and utilization of the bait by the tilapia, and promotes the growth of the tilapia.
In addition, the application also carries out related researches on the influence of main components of a culture medium of the lactococcus garvieae ABML2022003 on fermentation indexes in the early development process, specifically, 1% of the lactococcus garvieae ABML2022003 is inoculated on the basis of components of the culture medium shown in the table 4, and fermentation is carried out for 48 hours at the temperature of 34 ℃, the fermentation indexes are detected, and the detection results are shown in the table 5.
TABLE 4 addition amount of glucose in the Medium
Experimental group Peptone Yeast extract powder Glucose Diammonium hydrogen citrate Acetic acid sodium salt Dipotassium hydrogen phosphate Manganese sulfate Magnesium sulfate
1 0.25% 0.25% 1% 0.18% 0.45% 0.36% 0.01% 0.018%
2 0.25% 0.25% 3% 0.18% 0.45% 0.36% 0.01% 0.018%
3 0.25% 0.25% 5% 0.18% 0.45% 0.36% 0.01% 0.018%
4 0.25% 0.25% 7% 0.18% 0.45% 0.36% 0.01% 0.018%
TABLE 5 influence of the glucose addition on the lactococcus garvieae
Experimental group Residual sugar (mg/mL) Lactic acid (mg/mL) pH Lactic acid bacteria (cfu/mL)
1 0 10.5 4.16 2.3×10 9
2 0 10.7 4.17 3.7×10 9
3 10 12.0 4.24 3.6×10 9
4 50 12.0 4.24 2.6×10 9
From tables 4 to 5, it is clear that the amount of glucose added to the medium increases, the number of lactic acid bacteria increases gradually after fermentation for 24 hours, the amount of glucose added reaches 3% of the total number of viable bacteria, no significant synergy is achieved in the accumulation of viable bacteria and the metabolic product lactic acid, and the amount of glucose added reaches 7% of the total number of viable bacteria, which inhibits the number of lactic acid bacteria, and more residual glucose in the fermentation broth is not available, so that the optimal amount of glucose added is between 1% and 5%.
Table 6 addition amount of peptone in the Medium
Experimental group Peptone Yeast extract powder Glucose Diammonium hydrogen citrate Acetic acid sodium salt Dipotassium hydrogen phosphate Manganese sulfate Magnesium sulfate
1 0.25% 0.25% 2% 0.18% 0.45% 0.36% 0.01% 0.018%
2 0.5% 0.25% 2% 0.18% 0.45% 0.36% 0.01% 0.018%
3 1% 0.25% 2% 0.18% 0.45% 0.36% 0.01% 0.018%
4 2% 0.25% 2% 0.18% 0.45% 0.36% 0.01% 0.018%
5 3% 0.25% 2% 0.18% 0.45% 0.36% 0.01% 0.018%
TABLE 7 influence of peptone addition on lactococcus garvieae
Experimental group Residual sugar (mg/mL) Lactic acid (mg/mL) pH Lactic acid bacteria (cfu/mL)
1 0 10.4 4.15 1.8×10 9
2 0 11.6 4.16 2.2×10 9
3 0 12.4 4.18 3.8×10 9
4 0 12.6 4.15 3.6×10 9
5 0 12.4 4.15 3.7×10 9
From tables 6 to 7, it is understood that the number of viable bacteria of lactococcus garvieae is continuously increased with the increase of the addition amount of peptone, and when the addition amount reaches 1% of the number of viable bacteria to the maximum, no remarkable synergy is caused to the accumulation of the number of viable bacteria and the lactic acid of the metabolite by increasing the use amount of peptone, so that the optimal addition amount of peptone is between 0.1 and 1%.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims (9)

1. The utility model provides a lactococcus garvieae ABML2022003 which is characterized in that the preservation number of the lactococcus garvieae ABML2022003 is cctccc NO: m2022223.
2. The lactococcus garvieae ABML2022003 of claim 1, wherein the gene sequence of the 16S rDNA of the lactococcus garvieae ABML2022003 is shown in SEQ ID No. 1.
3. A microbial starter culture, characterized in that it comprises the lactococcus garvieae ABML2022003 of claim 1 or 2.
4. A microbial starter according to claim 3, wherein the number of bacteria of the lactococcus garvieae-containing ABML2022003 per 1mL of the microbial starter is 1 x l0 8 cfu / mL~5×10 9 cfu / mL。
5. A method for preparing a microbial starter, which is characterized in that the microbial starter is obtained by taking the lactococcus garvieae ABML2022003 as a starting strain and culturing the starting strain in a culture medium of the lactococcus garvieae ABML 2022003; the culture medium of the lactococcus garvieae ABML2022003 is one or two selected from a liquid culture medium and a solid culture medium.
6. The method for producing a microbial starter according to claim 5, wherein the liquid medium comprises the following raw materials in weight percent per 1L of water: yeast extract powder 0.1-1%, peptone 0.1-1%, glucose 2-5%, sodium acetate 0.2-1.4%, dipotassium hydrogen phosphate 0.1-0.4%, diamine hydrogen citrate 0.1-0.3%, magnesium sulfate 0.01-0.03% and manganese sulfate 0.005-0.01%;
the solid culture medium comprises the following raw materials in percentage by weight in every 1L of water and 15g of agar: yeast extract powder 0.1-1%, peptone 0.1-1%, glucose 2-5%, sodium acetate 0.2-1.4%, dipotassium hydrogen phosphate 0.1-0.4%, diamine hydrogen citrate 0.1-0.3%, magnesium sulfate 0.01-0.03% and manganese sulfate 0.005-0.01%.
7. The method for producing a microbial starter according to claim 5, wherein the pH of the medium is 6.0 to 6.5.
8. The method for producing a microbial starter according to claim 5, comprising:
placing the lactococcus garvieae ABML2022003 of claim 1 or 2 in the solid culture medium, and culturing for 48 hours at 30-35 ℃ to obtain a solid culture;
inoculating the solid culture into the liquid culture medium, and culturing for 16-20h at 30-35 ℃ to obtain primary seed liquid;
transferring the first-stage seed liquid into the liquid culture medium for expansion culture, wherein the transfer amount is 1-10%, and standing culture is carried out for 12-20h at 30-35 ℃ to obtain a second-stage seed liquid;
inoculating the secondary seed liquid into a fermentation tank according to the concentration of 1-10%, intermittently stirring and culturing for 16-48h at the temperature of 30-35 ℃, and reducing the pH value to 3.0-4.5 to obtain the microbial starter.
9. The method for preparing a microbial starter according to claim 8, wherein purified water, yeast extract, peptone, glucose, sodium acetate, dipotassium hydrogen phosphate, diammonium hydrogen citrate, magnesium sulfate and manganese sulfate are mixed in advance in the fermenter, heated at a high temperature, maintained at 100-115 ℃ for 15-30min, and cooled to 30-35 ℃.
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