Lactobacillus salivarius expanding culture combined microbial agent and expanding culture method and application of lactobacillus salivarius expanding culture combined microbial agent
By combining Lactobacillus salivary with Lactobacillus plantarum and Lactobacillus acidophilus for fermentation, and equipped with a special culture medium, the problem of expanding and application of Lactobacillus salivary in livestock and poultry breeding was solved, and the production and meat quality of livestock and poultry were significantly improved.
Patent Information
- Application Number
- CN202411980504.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-18
AI Technical Summary
The disadvantages of Lactobacillus salivary have poor thermal stability, poor storage stability and high usage cost, which limit their promotion and use in the livestock and poultry breeding field. Moreover, it germinates slowly during the expansion and cultivation process and is prone to infection with mixed bacteria, has special nutritional requirements, strict culture environment requirements, and is susceptible to metabolites.
By selecting Lactobacillus plantarum and Lactobacillus acidophilus with fast germination speed and strong pH reduction ability, and combining Lactobacillus salivary for complex fermentation, and scientifically comparing special culture medium, the shortcomings of Lactobacillus salivary alone were overcome and Lactobacillus salivary expansion liquid was prepared.
It improves the production performance, immune performance, antioxidant ability, intestinal digestion and absorption capacity and meat quality of livestock and poultry, solves the technical challenges of Lactobacillus salivary in the promotion and use of livestock and poultry breeding, and achieves efficient expansion and application.
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Figure CN119842529A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of livestock and poultry breeding, and specifically relates to a Lactobacillus salivarius expansion culture combined bacterial agent and an expansion culture method and application thereof. Background Art
[0002] Lactobacillus salivarius is a common Gram-positive bacterium in the intestine of animals. It has a strong ability to adhere to the intestinal mucosa, can compete for nutrients and adhesion sites in the intestine, inhibit the growth of various pathogens, and produce a variety of beneficial enzymes and vitamins to promote the healthy growth of animals. In addition, it also plays an important role in maintaining intestinal health, improving the body's immunity, and improving digestive function. However, the poor thermal stability, poor storage stability, and high cost of use of Lactobacillus salivarius limit its use. Therefore, on-site fermentation has brought new opportunities for the use of Lactobacillus salivarius. The expansion of Lactobacillus salivarius has special nutritional requirements (high requirements for growth factors, specific requirements for the types and concentrations of carbon and nitrogen sources), strict requirements for the culture environment (sensitive to temperature, pH value, and oxygen conditions), and is easily inhibited by metabolites. Therefore, the development of a method for the expansion of Lactobacillus salivarius is of great significance for the promotion and use of Lactobacillus salivarius in the field of livestock and poultry breeding. Summary of the invention
[0003] The invention provides a combined bacterial agent for the expansion of Lactobacillus salivarius, a method for the expansion of Lactobacillus salivarius and an application thereof. The Lactobacillus salivarius has a good expansion and fermentation effect, can well improve the production performance, immune performance, antioxidant capacity, intestinal digestion and absorption capacity and meat quality of livestock and poultry, and has good water solubility and is not easy to clog water lines.
[0004] In order to achieve the above-mentioned object of the invention, the present invention is implemented by the following technical solutions: The invention provides a combined bacterial agent for the expansion of Lactobacillus salivarius, which comprises Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus acidophilus and yeast.
[0005] Furthermore, the bacterial content of Lactobacillus salivarius in the combined bacterial agent is 4 billion / L-8 billion / L, the bacterial content of Lactobacillus plantarum is 2 billion / L-6 billion / L, the bacterial content of Lactobacillus acidophilus is 3 billion / L-9 billion / L, and the bacterial content of yeast is 60 million / L-120 million / L.
[0006] Furthermore, the combined bacterial agent also includes 5g / L-10g / L lactose, 45g / L-50g / L brown sugar, 10g / L-20g / L fermented corn syrup powder and 0.8g / L-1.5g / L trisodium phosphate.
[0007] Furthermore, the Lactobacillus salivarius is Lactobacillus salivarius XJP2 with a preservation number of CGMCC No.11386, the Lactobacillus plantarum is Lactobacillus plantarum GBW-LP001 with a preservation number of CGMCC No.22078, and the Lactobacillus acidophilus is Lactobacillus acidophilus L100 with a preservation number of CGMCC No.10701.
[0008] The invention also provides a method for expanding and culturing Lactobacillus salivarius, which comprises the following steps: mixing the combined bacterial agent for expanding and culturing Lactobacillus salivarius in tap water at a mass ratio of 2-5:100, stirring, dissolving and fermenting to obtain a Lactobacillus salivarius expansion liquid.
[0009] Furthermore, the fermentation temperature is 25°C-35°C, and the fermentation time is 24h-48h.
[0010] The present invention also provides application of the Lactobacillus salivarius expansion culture combination bacterial agent or the Lactobacillus salivarius expansion culture liquid in livestock and poultry breeding.
[0011] Furthermore, the livestock and poultry include broiler chickens, laying hens, broiler ducks, laying ducks, sows, piglets, and medium and large pigs.
[0012] Furthermore, under normal breeding conditions, the dosage of the Lactobacillus salivarius expansion solution is 1 L / 5000 birds / day to 2L / 5000 birds / day for poultry, 30 ml / head / day to 50 ml / head / day for sows, 10 ml / head / day to 20 ml / head / day for piglets, and 20 ml / head / day to 30 ml / head / day for medium and large pigs.
[0013] Furthermore, in an emergency, the dosage of the Lactobacillus salivarius expansion solution is 3 L / 5000 birds / day to 5L / 5000 birds / day for poultry, 100 ml / head / day to 200 ml / head / day for sows, 30 ml / head / day to 50 ml / head / day for piglets, and 100 ml / head / day to 200 ml / head / day for medium and large pigs.
[0014] Compared with the prior art, the advantages and technical effects of the method of the present invention are: 1. The present invention selects plant lactobacillus and acidophilus with fast germination speed and strong pH lowering ability to carry out compound fermentation with Lactobacillus salivarius, which effectively solves the problem that Lactobacillus salivarius fermented alone has slow germination speed and is easily infected by miscellaneous bacteria.
[0015] 2. The special culture medium of the present invention is scientifically formulated to overcome the problems of special nutritional requirements, strict culture environment requirements, and susceptibility to inhibition by metabolites in on-site fermentation of Lactobacillus salivarius. In addition, the culture medium is entirely made of water-soluble materials to prevent water line blockage during application.
[0016] 3. The Lactobacillus salivarius culture solution prepared by the present invention can greatly improve the production performance, immune performance, antioxidant capacity, intestinal digestion and absorption capacity and meat quality of livestock and poultry, which is of great significance for the promotion and use of Lactobacillus salivarius in the field of livestock and poultry breeding. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 To evaluate the single-bacterium fermentation of Lactobacillus salivarius and its complex bacteria.
[0018] Figure 2 Comparison of fermentation appearance for Lactobacillus salivarius culture medium optimization experiment.
[0019] Figure 3 Comparison of water solubility for Lactobacillus salivarius culture medium optimization experiment.
[0020] Figure 4 This is a picture of broiler muscle fiber tissue section. DETAILED DESCRIPTION
[0021] The technical solution of the present invention is further described in detail with reference to the following specific examples.
[0022] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.
[0023] The Lactobacillus salivarius used in the present invention is Lactobacillus salivarius XJP2, which is deposited at the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; deposit date: September 16, 2015; Lactobacillus salivarius Lactobacillus salivarius The deposit number is CGMCC No.11386.
[0024] The plant lactobacillus used is plant lactobacillus GBW-LP001, which is deposited at the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: March 26, 2021; plant lactobacillus Lactobacillus plantarum The deposit number is CGMCC No.22078.
[0025] The Lactobacillus acidophilus used was Lactobacillus acidophilus L100, which was deposited at the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; deposit date: April 9, 2015; Lactobacillus acidophilus Lactobacillus acidophilus The deposit number is CGMCC No.10701.
[0026] Example 1: On-site culture of Lactobacillus salivarius 1. Evaluation of single bacterial proliferation ability During the expansion process, Lactobacillus salivarius germinates slowly and is easily infected by foreign bacteria, resulting in expansion failure. Therefore, it is combined with strains that have fast germination speed and strong pH-lowering ability for fermentation. In order to evaluate the fermentation ability of Lactobacillus salivarius and candidate combined strains, the culture medium in Table 1 was designed, and single-bacteria fermentation tests of different strains (Lactobacillus plantarum, Lactobacillus acidophilus, Bacillus coagulans, Lactobacillus casei and Lactobacillus salivarius) were carried out using it. The test results show (see Tables 2 and Figure 1 ): The bacterial counts of the groups adding Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus salivarius all showed significant growth, and the fermentation effect was good; the bacterial counts of the groups adding Lactobacillus casei and Bacillus coagulans were low, and the fermentation effect of the group adding Bacillus coagulans was not good, the pH was high, and there were no bubbles on the surface of the fermentation liquid. Therefore, from the perspective of fermentation, Lactobacillus salivarius has good fermentation ability, and Lactobacillus plantarum and Lactobacillus acidophilus have good potential for compound addition.
[0027] Table 1 Design of culture medium for Lactobacillus salivarius expansion
[0028] Table 2 Evaluation of single bacterial proliferation ability
[0029] 2. Evaluation of bacterial strain combinations The inoculation amount of the strain directly affects the fermentation level. Different strains may affect each other. Therefore, in order to determine the optimal combination and dosage of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus salivarius and yeast, a 4-factor 3-level orthogonal experiment was designed and implemented with fermentation bacteria and pH as evaluation indicators (Table 3). The results of the orthogonal experiment (Table 3) show that the inoculation amount of Lactobacillus plantarum has the largest range among the four factors, so it has the greatest impact on the fermentation bacteria amount; with the increase of the inoculation amount of the strain, the fermentation bacteria amount under the influence of Lactobacillus acidophilus, Lactobacillus plantarum and yeast factors all tend to increase first and then decrease. The inoculation amounts of the three strains are 60×10 8 CFU / L, 40×10 8 CFU / L and 9000×10 6 CFU / L, the fermentation bacteria amount was the highest; the higher the inoculation amount of Lactobacillus salivarius, the higher the fermentation bacteria amount. 8 CFU / L, the increase in the inoculation amount of Lactobacillus salivarius slowed down the trend of fermentation bacteria. Considering the production cost, the best inoculation amount of saliva was 8 billion / L. Based on the above results, the best bacterial combination was obtained (see Table 4).
[0030] Table 3 Orthogonal test of bacterial strain combination
[0031] Table 4 Optimal orthogonal bacterial quantity combination
[0032] 3. Culture medium optimization test This experiment optimized the nitrogen source in the culture medium for two main reasons: 1. Lactobacillus salivarius has substrate specificity in the use of nitrogen sources and tends to use small-molecule amino acids and peptides that are easy to digest and absorb; 2. Lactobacillus salivarius has high requirements for growth factors. In addition to the nitrogen element provided by the nitrogen source, it also requires other nutrients such as vitamins, growth hormones, and trace elements; 3. Fermented soybean meal is a non-water-soluble nitrogen source, which may cause poor appearance or inconvenience in application when added to drinking water on site. Fermented corn pulp powder is rich in nutrients, and most of the nitrogen-containing substances in it have been decomposed into small-molecule amino acids, peptides, etc. after fermentation. It is a high-quality soluble organic nitrogen source. Therefore, this experiment selected two different brands of fermented corn pulp powder to replace the fermented soybean meal powder in the original culture medium. The test results show (see Table 5, Figure 2 and Figure 3 ): The bacterial count, fermentation appearance and water solubility of the two groups with added fermented corn slurry powder were improved compared with the fermented soybean meal group. Among them, the bacterial count of the fermented corn slurry powder 2 group was the highest, the fermented bacterial film was thicker, and the bubbles were more intense. Based on this, it was confirmed that the fermented corn slurry powder 2 replaced the original fermented soybean meal powder.
[0033] Table 5 Culture medium optimization experiment
[0034] 4. Formula combination optimization Through the above experiments, the components of each material in the formula, including bacterial strains, carbon sources, nitrogen sources and buffer salts, as well as their approximate dosages, were basically determined. In order to explore and determine the optimal dosage combination of each material at present, a 4-factor 3-level orthogonal experiment was designed and implemented for bacterial strains, carbon sources, nitrogen sources and inorganic salts (Table 6). The results of the orthogonal experiment (Table 6) showed that: with the increase of bacterial strain inoculation amount and carbon source amount, the fermentation bacterial amount showed an upward trend. Considering the production cost and product form, it was decided to keep the bacterial amount and carbon source amount unchanged; with the increase of nitrogen source amount, the fermentation bacterial amount showed a trend of first increasing and then decreasing. The fermentation bacterial amount was the best when the original nitrogen source amount was used; with the increase of buffer salt amount, the fermentation bacterial amount showed a downward trend. Considering PO 4 3- It can buffer pH, so a small amount of buffer salt should be added. Based on the above results, several batches of repeated verification tests were carried out to determine the best formula combination (see Table 7).
[0035] Table 6 Formula combination optimization
[0036] Table 7 Optimal formula combination
[0037] 5. Exploration of the best fermentation time As shown in Table 8, the pH of the fermentation liquid of Lactobacillus salivarius at room temperature for 0-24h showed a decreasing trend, and the pH showed an increasing trend at 24-120h. The pH reached the lowest value at 24h, and the pH was most suitable at 48h. The bacterial count showed a decreasing trend at 24-120h, and reached the highest value at 24h. Comprehensive evaluation showed that room temperature fermentation for 24-48h was most suitable.
[0038] Table 8 Changes in pH and bacterial count during fermentation
[0039] 6. On-site culture steps of Lactobacillus salivarius After cleaning the fermentation barrel (tank), add drinking water (you can add 2 / 3 of water first, and then adjust the volume after the bacteria bag and culture medium are all added). The formula of Lactobacillus salivarius expansion liquid: tap water weight = 3:100. Add the culture medium bag, stir and dissolve, continue to add the fermentation bacteria bag, stir evenly, control the temperature at 25-35℃, ferment for 24-48h, and test the fermentation liquid with pH test paper. If the pH value is lower than 4.2, and you can smell the sour and fragrant fermentation smell without any odor, then the fermentation is considered successful.
[0040] Example 2: Application of Lactobacillus salivarius culture solution in broiler farming The experiment selected 180 7-day-old white-feathered broilers and randomly divided them into 3 treatment groups: a control group, a group with Lactobacillus salivarius removed from the best formula combination in Table 7 of Example 1 (experimental group A), and a group with Lactobacillus salivarius expansion culture solution (the best formula combination in Table 7 of Example 1, experimental group B), with 6 replicates in each group, 10 birds in each replicate, and the addition amount of fermented liquid in experimental group A and experimental group B was 5 ml / 20 birds / day, and the drinking water was added centrally. During the feeding period, the chickens were free to eat and drink water, and routine immunization was performed. During the feeding period, the feed consumption and weight gain were counted every 7 days in units of replicates, and the number of deaths and eliminations was recorded. When the chickens were fed to the 42nd day, 2 broilers were randomly selected from each replicate, 10 ml of venous blood was collected from the neck, placed in a centrifuge at 4°C 3000 r / min for 10 min, and the separated serum was temporarily stored at -20°C for the detection of immune and antioxidant indicators. The abdominal cavity was opened after dissection, and the pectoral muscles on both sides were carefully removed. The left pectoral muscle was stored at 4°C for the determination of physicochemical and texture parameters, and the right pectoral muscle was placed in a 4% paraformaldehyde solution and stored at room temperature for the determination of muscle fiber characteristics. The jejunum was carefully removed, the intestinal segment was rinsed with precooled saline, and then the intestinal mucosa was scraped with a clean slide, and then quickly frozen in liquid nitrogen and stored at -80°C. The cecal chyme was collected and temporarily stored at -20°C for the determination of volatile fatty acids.
[0041] The results showed that: in terms of production performance (see Table 9), the average daily weight gain of broilers in the experimental group B at 42 days was significantly higher than that of the control group and the experimental group A, the average daily feed intake was 2% and 1.3% higher than that of the control group and the experimental group A, and the feed-to-meat ratio was reduced by 0.04 and 0.02; in terms of immunity (see Table 10), compared with the control group, the levels of serum immunoglobulin IgG and jejunal mucosal secretory immunoglobulin sIgA in broilers in the experimental groups A and B were significantly increased ( P <0.05, and the improvement effect of test group B was better than that of test group A. In terms of inflammation control (see Table 10), the level of IL-10 in the serum of broiler chickens in test groups A and B was significantly higher than that in the control group ( P <0.05), the level of IL-6 was significantly lower than that of the control group ( P <0.05); in terms of antioxidant (see Table 11), the MDA content in the serum of broilers in the experimental group B was reduced by 14% and 3.8% compared with the control group and the experimental group A, respectively, the SOD content was increased by 27.5% and 32.9%, respectively, and the T-AOC was increased by 14.7% and 2.6%, respectively; in terms of intestinal digestion and absorption (see Table 12), the total VFA content in the cecal chyme of broilers in the experimental group B was increased by 8.9% and 6.4% compared with the control group and the experimental group A, respectively, and the total branched-chain VFA was reduced by 9.8% and 14.2%, respectively; in terms of meat quality (see Table 13), the drip loss of the breast muscle of broilers in the experimental group B was reduced by 2.1% and 1.4%, the cooking loss was reduced by 5.1% and 3.1%, respectively, and the shear force was reduced by 1.3% and 0.8%, respectively, compared with the control group and the experimental group A, respectively. The HE results showed (see Figure 4 ), the muscle fibers of test group B were thinner than those of control group and test group A, and the muscle fiber diameter decreased by 7.3% and 5.9% respectively, the muscle fibers were more densely arranged, and the muscle fiber density increased by 2.9% and 2.5% respectively, indicating that Lactobacillus salivarius culture fluid greatly improved the tenderness of the meat.
[0042] Table 9 Effects of Lactobacillus salivarius culture medium on broiler production performance
[0043] Table 10 Effects of Lactobacillus salivarius culture fluid on immunity and inflammation levels in broilers
[0044] Table 11 Effects of Lactobacillus salivarius culture medium on antioxidant capacity of broilers
[0045] Table 12 Effects of Lactobacillus salivarius culture medium on intestinal digestion and absorption capacity of broiler chickens
[0046] Table 13 Effects of Lactobacillus salivarius culture medium on broiler breast muscle quality
[0047] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.
Claims
1. A Lactobacillus salivarius expansion culture combined bacterial agent, characterized in that: The combined bacterial agent comprises Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus acidophilus and yeast.
2. The Lactobacillus salivarius expansion culture combined bacterial agent according to claim 1, characterized in that: The bacterial content of Lactobacillus salivarius in the combined bacterial agent is 4 billion / L-8 billion / L, the bacterial content of Lactobacillus plantarum is 2 billion / L-6 billion / L, the bacterial content of Lactobacillus acidophilus is 3 billion / L-9 billion / L, and the bacterial content of yeast is 60 million / L-120 million / L.
3. The Lactobacillus salivarius expansion culture combined bacterial agent according to claim 1, characterized in that: The combined bacterial agent also includes 5g / L-10g / L lactose, 45g / L-50g / L brown sugar, 10g / L-20g / L fermented corn steep liquor powder and 0.8g / L-1.5g / L trisodium phosphate.
4. The Lactobacillus salivarius expansion culture combined bacterial agent according to claim 1, characterized in that: The Lactobacillus salivarius is Lactobacillus salivarius XJP2 with a preservation number of CGMCC No.11386, the Lactobacillus plantarum is Lactobacillus plantarum GBW-LP001 with a preservation number of CGMCC No.22078, and the Lactobacillus acidophilus is Lactobacillus acidophilus L100 with a preservation number of CGMCC No.10701.
5. A method for culturing Lactobacillus salivarius, characterized in that: The steps of the expansion method are: mixing the Lactobacillus salivarius expansion combination bacterial agent according to claim 1 in water at a mass ratio of 2-5:100, stirring, dissolving and fermenting to obtain the Lactobacillus salivarius expansion solution.
6. The method for expanding the culture according to claim 5, characterized in that: The fermentation temperature is 25°C-35°C, and the fermentation time is 24h-48h.
7. Use of the Lactobacillus salivarius expansion culture combination bacterial agent according to claim 1 or the Lactobacillus salivarius expansion culture solution according to claim 5 in livestock and poultry breeding.
8. The use according to claim 7, characterized in that: The livestock and poultry animals include broiler chickens, laying hens, meat ducks, laying ducks, sows, piglets, and medium and large pigs.
9. The use according to claim 8, characterized in that: Under normal breeding conditions, the dosage of the Lactobacillus salivarius expansion solution is 1 L / 5000 birds / day to 2L / 5000 birds / day for poultry, 30 ml / head / day to 50 ml / head / day for sows, 10 ml / head / day to 20 ml / head / day for piglets, and 20 ml / head / day to 30 ml / head / day for medium and large pigs.
10. The use according to claim 8, characterized in that: In emergency, the dosage of the Lactobacillus salivarius expansion solution is 3 L / 5000 birds / day to 5 L / 5000 birds / day for poultry, 100 ml / head / day to 200 ml / head / day for sows, 30 ml / head / day to 50 ml / head / day for piglets, and 100 ml / head / day to 200 ml / head / day for medium and large pigs.