CN115960775B - Lactobacillus salivarius capable of improving productivity of gosling and resisting gout - Google Patents
Lactobacillus salivarius capable of improving productivity of gosling and resisting gout Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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Abstract
The invention provides a lactobacillus salivarius which increases the productivity of gosling and resists gout, and is preserved in the common microorganism management center of China Committee for culture Collection of microorganisms, the preservation address is No.1 and No. 3 of North Chen West Lu 1 of Beijing, china, named lactobacillus salivarius @, which is 9 months and 16 days of 2022Lactobacillus salivarius) r12 with the preservation number of CGMCC NO.25732; the invention can increase the production performance of gosling, antagonize hyperuricemia and liver and kidney injury caused by high protein ration, and has the potential of developing new antibacterial medicines. The feed additive can increase the weight gain rate of gosling for 5 days, reduce the feed-meat ratio of the gosling, protect the liver function and the kidney function of the gosling without harmful effects, remarkably improve the growth performance of the gosling, and can be prepared into feed for feeding the gosling, thereby having the potential of improving the economic benefit of farmers and having great application prospect.
Description
Technical Field
The invention relates to a novel lactobacillus salivarius, and further provides a lactobacillus salivarius capable of increasing the production performance of gosling and resisting gout, belonging to the technical field of microorganisms.
Background
Lactobacillus salivarius belongs to the genus Lactobacillus of the order Thick-walled phylum, and is a catalase-negative, gram-positive, non-motile, non-sporulation, obligate heterotypic fermentation bacterium. The study shows that the lactobacillus salivarius has better tolerance to gastrointestinal tract environment and has certain tolerance to artificial gastric juice, intestinal juice and bile salt. These properties make lactobacillus salivarius have the potential to be a probiotic added to animal diets and resistant to stress and can be effectively utilized.
Lactobacillus salivarius widely exists in intestinal tracts of human beings and animals, has strong intestinal adhesion capability, and has important effects of inhibiting reproduction of pathogenic bacteria in gastrointestinal tracts, regulating animal immunity, reducing animal stress, reducing animal morbidity, regulating intestinal flora, preventing or relieving diarrhea, improving feed utilization efficiency, promoting animal growth, reducing feed-to-weight ratio and the like. Based on the advantages, the lactobacillus salivarius is a microecological preparation with higher development value.
Disclosure of Invention
The invention discloses lactobacillus salivarius capable of improving the productivity of gosling and resisting gout, which is a novel strain separated from the cecum faeces of healthy gosling, can improve the productivity of gosling and reduce uric acid, and has the potential of developing novel fermented feed.
The invention provides a lactobacillus salivarius capable of improving the productivity of gosling and resisting gout, which is preserved in the common microorganism management center of China Committee for culture Collection of microorganisms, wherein the preservation address is North Chen Silu No.1, 3 of Beijing, china, the preservation date is 2022, 9 months and 16 days, the preservation number is CGMCC No.25732, and the invention is named as: lactobacillus salivarius @Lactobacillus salivarius）r12。
The lactobacillus salivarius of the inventionLactobacillus salivarius) Application of r12 in preparing gosling feed additive with viable count not less than 1×10 9 CFU/mL or 1X 10 9 CFU/g。
The lactobacillus salivarius of the inventionLactobacillus salivarius) Application of r12 in antagonizing gout caused by high protein feed for gosling.
The lactobacillus salivarius of the inventionLactobacillus salivarius) Application of r12 in preparing gosling blood uric acid reducing preparation.
The screening and culturing method of the lactobacillus salivarius capable of increasing the production performance of gosling and resisting gout of gosling comprises the following specific steps:
lactobacillus selective media (LBS agar): 10.0g/L peptone, 10.0g/L beef extract powder, 2.0g/L yeast extract powder, 2.0g/L dipotassium hydrogen phosphate, 5.0g/L sodium acetate, 2.0g/L triammonium citrate, 0.2g/L magnesium sulfate, 0.05g/L manganese sulfate, 801.0g/L tween, 20.0g/L glucose, 5.0g/L calcium carbonate, 25.0g/L agar and pH value of 6.0-6.5; collecting a healthy gosling cecum fecal sample for separating and culturing the strain: purifying to obtain the lactobacillus salivarius of the inventionLactobacillus reuteriGram staining positive; streaking lactobacillus salivarius on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain single colony; selecting single bacterial colony to inoculate in MRS liquid culture medium, and standing for 6h at 37 ℃ to obtain bacterial liquid; adding glycerol with 20-30% of the total volume into the bacterial liquid, and storing at-80deg.C.
The sequence obtained by sequencing the screened strain of the invention is subjected to nucleic acid sequence comparison in NCBI, and the result shows that the strain is lactobacillus salivarius and is named as lactobacillus salivariusLactobacillus salivarius r12。
The lactobacillus salivariusLactobacillus salivariusThallus characteristics of r 12: a milky white colony with a round shape and a complete edge is formed on the culture medium.
The lactobacillus salivariusLactobacillus salivariusGrowth characteristics of r 12: the strain is a gram-positive facultative anaerobe, and grows optimally at 37 ℃.
The application of the lactobacillus salivarius in preparing the feed for increasing the weight of gosling is that the viable count of the lactobacillus salivarius in the feed is not less than 1 multiplied by 10 9 CFU/mL or 1X 10 9 CFU/g。
The invention has the positive effects that:
the lactobacillus salivarius r12 which is a new strain is screened, so that the blood uric acid of gosling can be reduced, the average weight of the gosling can be increased, the liver function and the kidney function of the gosling are not affected, the growth performance of the gosling can be remarkably improved, the feed can be prepared for feeding the gosling, the feed has the potential of improving the economic benefit of farmers, and the application prospect is huge.
Drawings
FIG. 1 is a graph showing the average 5-day weight gain versus the feed conversion ratio for two groups of gosling according to the present invention;
FIG. 2 shows the blood uric acid of two groups of gosling; liver function: serum glutamic pyruvic transaminase and serum glutamic oxaloacetic transaminase; renal function: serum urea nitrogen, serum creatinine; a comparison of liver and kidney tissue sections;
FIG. 3 shows the transcript levels of liver xanthine oxidase, adenylate deaminase of two groups of gosling according to the present invention; comparison of transcript levels of renal ATP-binding superfamily G member 2 and glucose transporter 9.
Detailed Description
The present invention is further illustrated by the following specific embodiments, which are some, but not all embodiments of the invention, and which are not intended to limit the invention to the embodiments described. The experimental procedures, which are not specified in the following examples, were carried out according to conventional methods or according to the commercial specifications.
Example 1
MRS broth: 10.0g/L of peptone, 8.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 2.0g/L of dipotassium hydrogen phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 801.0g/L of tween and pH value of 5.7+/-0.2;
MRS solid medium: 10.0g/L of peptone, 8.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 2.0g/L of dipotassium hydrogen phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 14.0g/L of agar, 801.0g/L of tween and pH value of 6.5+/-0.2;
the preparation method of the lactobacillus salivarius r12 bacterial suspension comprises the following steps:
streaking lactobacillus salivarius on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain single colony; selecting single bacterial colony to inoculate in MRS liquid culture medium, and standing for 6h at 37 ℃ to obtain bacterial liquid; adding glycerol accounting for 20-30% of the total volume into the bacterial liquid, and storing at-80 ℃;
isolation of Lactobacillus salivarius r12
1. Sample collection
Collecting a cecum fecal sample of a healthy gosling in a goose farm in Liaoyang city, placing the sample in a centrifuge tube filled with glycerol with the total volume of 20-30%, storing the sample in a heat preservation box filled with an ice bag, taking the sample back to a laboratory, and rapidly placing the sample in a refrigerator with the temperature of-80 ℃ for separation and screening.
2. Isolation culture of strains
(1) And (3) dilution coating: about 0.5g of the content stored in 20-30% glycerol is taken and added into a 10mL centrifuge tube filled with 4.5mL of physiological saline under aseptic environment to obtain 10 -1 And (3) diluting the solution, repeating the diluting steps to sequentially obtain 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 A dilution liquid;
(2) And (3) streaking culture: dipping 10 in the step (1) by using an inoculating loop -1 、10 -2 、10 -3 、10 -4 Streaking four gradient dilutions on a lactobacillus selective medium, and culturing for 48 hours at 37 ℃ to obtain a streaking plate;
(3) Coating and culturing: respectively sucking 50 mu L of 10 in the step (1) -5 、10 -6 、10 -7 、10 -8 Coating four gradient dilutions on a lactobacillus selective culture medium uniformly by using a coating rod, and culturing for 48 hours at 37 ℃ to obtain a diluted coating plate;
(4) Culturing bacterial liquid: picking the streak plate in the step (2) and the single colony of the dilution coating plate in the step (3) to be respectively inoculated in MRS liquid culture medium, standing for 6 hours in a 37 ℃ incubator until the colony number is 1 multiplied by 10 9 Adding glycerol with 20% -30% of the total volume after CFU/mL, and storing at-80 ℃;
3. identification of species
Delivering the bacterial liquid obtained in the step 2 (4) to gene sequencing of Jilin Kumei biotechnology Co., ltd; BLAST (http:// www.ncbi.nlm.nih.g) uploading sequenced spliced sequences to NCBIov/BLAST) for species validation; the comparison result shows that the lactobacillus salivarius isLactobacillus salivariusThe 16S rDNA amplification sequence is shown as SEQ ID NO. 1:
4. preservation of bacterial species
The strain of the invention is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 9 months and 16 days in 2022, and has a preservation address of North Chen Silu No.1, 3 of Beijing, china, and is named as: lactobacillus salivarius @Lactobacillus salivarius）r12 with the preservation number of CGMCC NO.25732.
The lactobacillus salivarius r12 has the following properties:
1) The weight gain rate and the feed conversion ratio of gosling can be increased;
2) Antagonizing the harmful effect of high protein feed on liver function and kidney function of gosling;
3) Improving the growth performance of gosling and having the potential of improving the economic benefit of farmers.
From the above examples, it can be seen that the present invention provides a new strain of lactobacillus and illustrates the basic characteristics, cultivation method and application of the new strain, but it can also be seen that some modifications can be made thereto based on the present invention, and therefore, the modifications or improvements made thereto without departing from the spirit and principles of the present invention fall within the scope of the present invention as claimed.
Experimental example 1
Influence of Lactobacillus salivarius r12 on growth performance of gosling
1. Preparation of lactobacillus salivarius r12 bacterial liquid
Streaking the lactobacillus salivarius r12 obtained in the example 1 in MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain single colony; picking single colony on MRS solid culture medium, inoculating into MRS liquid culture medium, standing in 37 deg.C incubator for 6 hr to obtain bacterial liquid; adding glycerol with 20-30% of the total volume into the bacterial liquid, and storing at-80deg.C. Taking lactobacillus salivarius r12 suspension for centrifugation before experiment, pouring out supernatant, washing with sterile physiological saline with twice volume for resuspension to obtain lactobacillus salivarius r12 bacterial liquid (1X 109 CFU/mL);
2. experimental animal
Keeping the temperature of the young goose of 1 day old at 37 ℃ by a warm lamp in the initial feeding period, keeping the room temperature for one week, and keeping the animal room clean and ventilated all the time;
3. experimental grouping
40 healthy gosling of 1 day old are randomly divided into 4 groups of 10 gosling each, and the 4 groups are respectively: normal proteomes, normal protein lactobacillus salivarius r12 intervention group, high proteomes and high protein lactobacillus salivarius r12 intervention group;
4. experimental details
(1) Normal proteome: feeding normal protein daily ration;
(2) Normal protein lactobacillus salivarius r12 intervention group: feeding normal protein ration, centrifuging lactobacillus salivarius r12, pouring out supernatant, adding two times of sterile physiological saline, mixing, and feeding to gosling;
(3) High protein group: feeding high protein daily ration;
(4) High protein lactobacillus salivarius r12 intervention group: feeding high protein ration, centrifuging lactobacillus salivarius r12, pouring out supernatant, adding two times of sterile physiological saline, mixing, and feeding to gosling;
(5) Weighing and recording the weight and the grain consumption of 4 groups of gosling in the morning;
(6) The experimental period was 25 days;
5. experimental results
As shown in fig. 1, lactobacillus salivarius r12 was able to increase the rate of weight gain and feed conversion of gosling compared to normal proteomes.
Experimental example 2
The influence of the lactobacillus salivarius r12 on the blood uric acid of gosling and the liver and kidney is as follows:
1. preparation of lactobacillus salivarius r12 bacterial liquid is the same as that of experimental example 1;
2. experimental animals are the same as in experimental example 1;
3. experimental grouping is the same as experimental example 1;
4. experimental details
(1) Normal proteome: feeding normal protein daily ration;
(2) Normal protein lactobacillus salivarius r12 intervention group: feeding normal protein ration, centrifuging lactobacillus salivarius r12, pouring out supernatant, adding two times of sterile physiological saline, mixing, and feeding to gosling;
(3) High protein group: feeding high protein daily ration;
(4) High protein lactobacillus salivarius r12 intervention group: feeding high protein ration, centrifuging lactobacillus salivarius r12, pouring out supernatant, adding two times of sterile physiological saline, mixing, and feeding to gosling;
(5) The experimental period was 25 days;
(6) The gosling was sacrificed on day 26 and 4 groups of blood, liver and kidney tissues were collected, a portion of the liver, kidney tissues were sectioned, and liver-related indices were examined: serum uric acid (Serum UA), serum glutamic pyruvic transaminase (Serum ALT), serum glutamic pyruvic transaminase (Serum AST); kidney related index: serum urea nitrogen (Serum BUN), serum creatinine (Serum CREA);
5. experimental results
As shown in fig. 2, the high protein group and the normal protein group and the high protein lactobacillus salivarius r12 intervene in serum uric acid, serum glutamic pyruvic transaminase, serum urea nitrogen and serum creatinine of group gosling, and the difference of the results is remarkable; the high protein group and the normal protein group and the high protein lactobacillus salivarius r12 interfere with the liver and kidney tissues of the gosling, and the fact that the high protein feed has damage to liver and kidney functions and the lactobacillus salivarius r12 can antagonize the damage and has no harmful effect on liver functions and kidney functions of the gosling is shown.
Experimental example 3
The lactobacillus salivarius r12 affects the metabolism ability of gosling livers and kidneys, and comprises the following specific steps:
1. preparation of lactobacillus salivarius r12 bacterial liquid is the same as that of experimental example 1;
2. experimental animals are the same as in experimental example 1;
3. experimental grouping is the same as experimental example 1;
4. experimental details
(1) Normal proteome: feeding normal protein daily ration;
(2) Normal protein lactobacillus salivarius r12 intervention group: feeding normal protein ration, centrifuging lactobacillus salivarius r12, pouring out supernatant, adding two times of sterile physiological saline, mixing, and feeding to gosling;
(3) High protein group: feeding high protein daily ration;
(4) High protein lactobacillus salivarius r12 intervention group: feeding high protein ration, centrifuging lactobacillus salivarius r12, pouring out supernatant, adding two times of sterile physiological saline, mixing, and feeding to gosling;
(5) The experimental period was 25 days;
(6) The gosling was sacrificed on day 26 and 4 groups of gosling liver and kidney tissues were collected, the liver and kidney tissues were ground in liquid nitrogen to extract RNA, the RNA was reverse transcribed into cDNA, and fluorescence quantification pcr detection of relevant indicators was performed: adenosine Deaminase (ADA), xanthine Oxidase (XOD), ATP-binding nuclear superfamily G member 2 (ABCG 2), glucose transporter 9 (GLUT 9);
5. experimental results
In the liver, the changes in the levels of transcription of enzymes involved in purine production are more chaotic, but in the kidney, the transcription of proteins involved in uric acid transport is significantly reduced in the high protein group, whereas the high protein lactobacillus salivarius r12 intervention group antagonizes this change, enhancing the transcription of the relevant transport proteins.
Claims (4)
1. Lactobacillus salivarius @Lactobacillus salivarius) r12 is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is 1 # 3, and the preservation date is 2022, 9 and 16, and the preservation number is CGMCC NO.25732.
2. The lactobacillus salivarius according to claim 1Lactobacillus salivarius) Application of r12 in preparing anti-gout gosling feed additive, and viable count is not less than 1×10 9 CFU/mL or 1X 10 9 CFU/g。
3. The lactobacillus salivarius according to claim 1Lactobacillus salivarius) The application of r12 in preparing antagonistic high protein feed for gosling.
4. The lactobacillus salivarius according to claim 1Lactobacillus salivarius) Application of r12 in preparing gosling blood uric acid reducing preparation.
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