Compound lactic acid bacteria and application thereof in silage

Abstract

The invention relates to the technical field of microorganisms, in particular to compound lactic acid bacteria and application of the compound lactic acid bacteria in silage, the compound lactic acid bacteria are obtained by compounding a strain XH146 and a strain XH352, the two strains are obtained by self-screening of an applicant, and compared with same lactic acid bacteria, the compound lactic acid bacteria have the advantages that the compound lactic acid bacteria are more stable in performance; the compound lactic acid bacteria can well survive in the pennisetum purpureum-sugarcane tail leaf mixed silage to form flora advantages, object grass-sugarcane tail leaves are subjected to mixed silage, detection shows that the strain plays a good role in fermentation of the pennisetum purpureum-sugarcane tail leaf mixed silage, the fermentation quality of the pennisetum purpureum-sugarcane tail leaf mixed silage is effectively improved, and the yield of the pennisetum purpureum-sugarcane tail leaf mixed silage is increased. Through breeding verification, the feed has a very good effect on the milk buffalo, and can achieve the effects of improving the milk yield of the milk buffalo and reducing the diarrhea rate of the milk buffalo; the strain has the effects of improving the milk protein content, the non-milk fat solid content and the lactose content of buffalo milk, and is a compound strain with an excellent effect, and silage fermented by using the strain also has good quality.

Description

[Technical field]

[0001] The invention relates to the technical field of microorganisms, in particular to composite lactic acid bacteria and application thereof in silage. [Background technology]

[0002] Elephant grass is a high-yield and high-quality forage grass cultivated in Guangxi. It is soft and juicy, has good palatability, and has high nutritional value. It is an important high-quality green feed for ruminants. At present, in order to better utilize elephant grass in the feeding process, it is usually prepared into silage. After ensiling, it can effectively solve the problems of feed storage, transportation and seasonal roughage shortage. However, in the ensiling process of elephant grass, due to the relatively low content of dry matter and water-soluble carbohydrates in elephant grass, it is difficult to prepare high-quality silage by ensiling alone. Therefore, in the prior art, we usually add sugarcane tail leaves with a high sugar content to adjust the sugar content of the substrate. Sugarcane tail leaves contain high sugar, water and crude fiber, which can effectively improve the problem of low dry matter and water-soluble carbohydrate content of elephant grass.

[0003] However, during the fermentation process of the elephant grass-sugarcane tail leaf substrate, we found that the silage effect of the silage microorganisms on the market was not good, and in order to adapt to the silage of more raw materials, some microorganisms often use multiple genera for compounding, such as the combined use of Bacillus subtilis and lactic acid bacteria. This leads to the fact that lactic acid bacteria that generally have intestinal regulatory functions may not necessarily form a dominant flora, and their colonization and intestinal regulatory abilities will be greatly reduced. At the same time, we also found that not all lactic acid bacteria are suitable for silage of elephant grass-sugarcane tail leaves. Therefore, in order to improve the silage quality of elephant grass-sugarcane tail leaves and improve the quality of elephant grass-sugarcane tail leaf feed, it is necessary for us to continuously screen lactic acid bacteria suitable for silage fermentation. Only when the strains are enriched can we better utilize the elephant grass-sugarcane tail leaves to prepare composite silage. [Summary of the invention]

[0004] In view of the above, it is necessary to provide a lactic acid bacteria that can ensilage grass-sugarcane tail leaves, and use the lactic acid bacteria to prepare a composite bacterial agent, which can well utilize grass-sugarcane tail leaves to prepare silage.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention comprises composite lactic acid bacteria, which is composed of Lactobacillus paracasei strain XH146 and Lactobacillus plantarum strain XH352;

[0007] The composite lactic acid bacteria is Lactobacillus paracasei strain XH146, the deposit number of which is CCTCC NO: M20232336; the strain is deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: M20232336 and a deposit date of November 27, 2023.

[0008] The Lactobacillus plantarum strain XH352 has a deposit number of CCTCC NO: M20232337. The strain is deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China. The deposit number is CCTCC NO: M20232337, and the deposit date is November 27, 2023.

[0009] Furthermore, the mass ratio of the Lactobacillus paracasei strain XH146 to the Lactobacillus plantarum strain XH352 is 3-6:3-6.

[0010] The present invention also includes the use of the composite lactic acid bacteria in preparing elephant grass-sugarcane tail leaf silage.

[0011] The present invention also includes a method for preparing the elephant grass-sugarcane tail leaf silage, which comprises: crushing the elephant grass and the sugarcane tail leaf, inoculating the composite lactic acid bacteria for silage fermentation; the mass ratio of the elephant grass, the sugarcane tail leaf, the strain XH146 and the strain XH352 is 100-200:100-150:3-6:3-6.

[0012] The present invention also includes the use of the composite lactic acid bacteria in preparing medicines for preventing diarrhea.

[0013] The present invention also includes the application of the elephant grass-sugarcane tail leaf silage in improving the yield of dairy buffalo.

[0014] The present invention also includes the use of the elephant grass-sugarcane tail leaf silage in increasing the milk protein content of buffalo milk, increasing the non-milk fat solid content of buffalo milk and / or increasing the lactose content of buffalo milk.

[0015] The present invention has the following beneficial effects:

[0016] 1. The composite lactic acid bacteria of the present invention are obtained by compounding strain XH146 and strain XH352. Both of these lactic acid bacteria strains are obtained by self-screening by the applicant. It has been verified that compared with the same type of lactic acid bacteria, the composite lactic acid bacteria can survive well in elephant grass-sugarcane tail leaves, form a bacterial community advantage, and ensilage the elephant grass-sugarcane tail leaves. After testing, the strain has played a good effect in the fermentation of elephant grass-sugarcane silage, and effectively improved the silage efficiency of elephant grass-sugarcane tail leaves. After feeding verification, the feed has a good effect on dairy buffaloes, and can play the role of: increasing the buffalo milk production of dairy buffaloes and reducing the diarrhea rate; increasing the milk protein, non-fat solids content and lactose content of buffalo milk. It is a composite strain with excellent effect, and the fermented elephant grass-sugarcane tail leaf silage has good quality.

Brief Description of the Drawings

[0017] Figure 1 This is a developmental tree diagram of the XH352 strain of the present application. [Specific implementation method]

[0018] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0019] Any feature disclosed in this specification (including any appended claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features.

[0020] Example 1

[0021] This embodiment is a verification method for Lactobacillus paracasei strain XH146 and Lactobacillus plantarum strain XH352, which is as follows:

[0022] The 16S rDNA amplification products of the selected strains: X122, X14, X184, X368, XH1, XH124, X335, H356, XH15, X348, X360, X372, X378, X380, XH209, XH341, XH343, XH345, XH352 and XH49 were sequenced and the phylogenetic tree was obtained by BLAST similarity comparison analysis in the NCBI database. Figure 1 As shown, the specific comparison results are shown in Table 1:

[0023] Table 1 Blast alignment results of 16S rDNA sequences of lactic acid bacteria strains

[0024]

[0025] from Figure 1 As can be seen in Table 1, the XH146 strain and Lactobacillus paracasei are clustered together, so the XH146 strain is identified as Lactobacillus paracasei; the XH352 strain and Lactobacillus plantarum are clustered together, so the XH352 strain is identified as Lactobacillus plantarum.

[0026] The specific deposit information of the above strain is as follows:

[0027] Lactobacillus paracasei strain XH146, whose deposit number is CCTCC NO: M20232336, is deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: M20232336 and a deposit date of November 27, 2023;

[0028] Lactobacillus plantarum strain XH352, whose preservation number is CCTCC NO: M20232337, is deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, with a preservation number of CCTCC NO: M20232337 and a preservation date of November 27, 2023.

[0029] Example 2

[0030] This example is the screening of lactic acid bacteria strains using elephant grass-sugarcane tail leaf fermentation substrate:

[0031] The same batch of strains X122, XH146, X184, X368, XH1, XH124, X335, XH356, XH15, X348, X360, X372, X378, X380, XH209, XH341, XH343, XH345, XH352 and XH49 were tested for their activity on fermentation substrates containing sugarcane tail leaves and elephant grass, as follows:

[0032] 1. Preparation of culture medium containing elephant grass-sugarcane tail leaves:

[0033] ① After mixing elephant grass and water in a solid-liquid mass ratio of 2:1, filtering the filtrate to obtain elephant grass filtrate;

[0034] ② After mixing the sugarcane tail leaves and water at a solid-liquid mass ratio of 2:1, filtering the filtrate to obtain the sugarcane tail leaf filtrate;

[0035] ③ The elephant grass filtrate and the sugarcane tail leaf filtrate were mixed in a volume ratio of 1:1 to obtain the elephant grass-sugarcane tail leaf filtrate, and then the filtrate was mixed with the liquid MRS medium, the amount of the filtrate added was 10% of the volume percentage of the liquid MRS medium, after sufficient mixing, the above strains were inoculated at an inoculation rate of 5%, and after culturing for 24 days, the liquid MRS medium without the addition of the elephant grass-sugarcane tail leaf filtrate was used as the control group (CK), and then the pH value of the above strains and the OD of each strain were measured. 600 , the specific results are shown in Table 1:

[0036] Table 1 Effects of sugarcane tail leaf substrate culture medium on different lactic acid bacteria

[0037]

[0038]

[0039] As shown in Table 1, the above strains showed different growth activities on the substrate with the addition of elephant grass-sugarcane tail leaf filtrate. The pH value of the experimental group of lactic acid bacteria XH1, XH209, and XH341 was greater than that of the control group and OD 600 The experimental group with a pH value of 0.04 was lower than the control group, indicating that the filtrate of elephant grass-sugarcane tail leaves had an inhibitory effect on these lactic acid bacteria; the pH value of the experimental group of lactic acid bacteria XH146 and XH352 was lower than that of the control group and OD 600 The experimental group with a pH value of 0.04 was greater than the control group, indicating that the filtrate of elephant grass-sugarcane tail leaves had a promoting effect on these lactic acid bacteria; while the pH value and OD of other lactic acid bacteria 600 The values ​​are not much different, indicating that the filtrate of elephant grass-sugarcane tail leaves has no obvious promoting or inhibiting effect on other strains. Therefore, based on the above experimental results, we selected strain XH146 and strain XH352 as composite strains for the next elephant grass-sugarcane tail leaf silage fermentation experiment.

[0040] Example 3

[0041] This example is a fermentation experiment of elephant grass-sugarcane tail leaf mixed feed, and the specific scheme is as follows:

[0042] The strain XH146, strain XH352, elephant grass and sugarcane tail leaves were mixed according to the test groups in Table 2 and Table 3 to prepare silage. The silage method was as follows: the fresh sugarcane tail leaves and elephant grass were fully crushed, and then inoculated with strain XH146 and strain XH352 respectively, with 3 replicates for each treatment. A small-scale fermentation method was used, and the samples were weighed and mixed evenly according to the mixed storage design ratio, and then packed into polyethylene film bags, 500g per bag, and vacuum-sealed with a vacuum packaging machine, and fermented at room temperature for 60 days away from light. Samples were taken for determination of relevant indicators, and the total colony count of lactic acid bacteria, in vitro dry matter disappearance rate and total VFA were used as evaluation indicators, as shown in Table 2 and Table 3.

[0043] Table 2 Orthogonal experimental factor levels of composite bacteria on grass-sugarcane tail leaf silage

[0044]

[0045] Table 3 Orthogonal experiment and results

[0046]

[0047] As shown in Table 3, in Experiment 1, only elephant grass and sugarcane were used for silage, and the colony count of lactic acid bacteria could reach 5.46; from the perspective of other experimental groups, the colony counts of Experiments 3, 5, 6, 8 and 9 were lower than those of Experiment 1, which showed that the proportion of this experimental group did not promote the growth of lactic acid bacteria. This may be because the proportion of the pure plant base material did not significantly promote the growth of lactic acid bacteria; from the extreme value, sugarcane tail leaves> strain XH352> strain XH146> elephant grass; for the total colony count of lactic acid bacteria, the greatest influence was sugarcane tail leaves, followed by strain XH352, then strain XH146 and elephant grass.

[0048] From the perspective of in vitro dry matter disappearance rate, the in vitro dry matter disappearance rates of Experiments 1 and 3 were similar, both of which were the lowest, and the other experimental groups were higher than Experiment 1, indicating that the addition of strain XH146 and / or strain XH352 can effectively promote the fermentation of elephant grass-sugarcane tail leaves; the in vitro dry matter disappearance rates of Experiments 2, 4 and 7 can reach more than 60%; from the extreme point of view, strain XH146> strain XH352> sugarcane tail leaves> elephant grass; for the in vitro dry matter disappearance rate, strain XH146 had the greatest impact, followed by strain XH352, and then sugarcane tail leaves and elephant grass.

[0049] From the perspective of total VFA content, the total VFA of Experiment 1 and Experiment 5 were similar, both of which were the lowest, and the other experimental groups were higher than Experiment 1, indicating that the addition of strain XH146 and / or strain XH352 can effectively promote the fermentation of elephant grass-sugarcane tail leaves; the total VFA of Experiment 2, Experiment 4 and Experiment 7 can reach more than 23mmol / l; from the perspective of the extreme difference, strain XH146> strain XH352> sugarcane tail leaves> elephant grass; for the in vitro dry matter disappearance rate, the strain XH146 had the greatest impact, followed by strain XH352, and then sugarcane tail leaves and elephant grass.

[0050] Based on the above experimental results, we believe that the fermentation effects of Experiments 2, 4 and 7 are better and significantly better than those of Experiment 1. Therefore, when strain XH146 and strain XH352 are used to prepare a composite strain, the optimal silage quality ratio is: elephant grass: sugarcane tail leaves: strain XH146: strain XH352 = 100-200:100-150:3-6:3-6.

[0051] Example 4

[0052] Based on the experimental results of Example 3, we selected fermented feed to conduct animal experiments on dairy buffaloes. The specific scheme is as follows:

[0053] Experimental design: Based on the principle of random pairing, 28 experimental buffaloes (Neri-Lafite buffaloes) were randomly divided into an experimental group and a control group, with 7 buffaloes in each group. The experimental period was 2 months, and the feeding experiment was carried out according to the following feeding formula:

[0054] Experimental group 1: silage prepared by the method of Experiment 2 in Table 3 was directly fed;

[0055] Experimental group 2: silage prepared by the method of Experiment 4 in Table 3 was directly fed;

[0056] Experimental group 3: silage prepared by the method of Experiment 7 in Table 3 was directly fed;

[0057] Control group: silage prepared by the method of Experiment 1 in Table 3 was directly fed.

[0058] During the experiment, the dry matter intake and milk production of each group of dairy buffaloes were calculated every day. The milk production of dairy cows was recorded by milkers every day, and the milk composition was tested once a week. The number of cows with diarrhea during the experimental period was observed and recorded, and the incidence of diarrhea was calculated; the dry matter intake and milk production were calculated as follows:

[0059] The dry matter intake calculation formula is: dry matter intake = amount of feed fed - amount of remaining feed; after calculating the dry matter intake every day, take the average value for the entire experimental period.

[0060] Milk production calculation method: average daily milk production = total milk production during the test period / number of test days;

[0061] The diarrhea incidence rate was calculated by counting the number of diarrhea occurrences in dairy buffaloes during the experimental period: one diarrhea occurrence was recorded as 1, and two diarrhea occurrences were recorded as 2. After the experimental period, the number of diarrhea occurrences was divided by the sample base: 60 (days) × 7 (heads) to calculate the diarrhea incidence rate;

[0062] The method for determining milk composition is as follows: 200 mL is collected at 8:00 and 16:00 every Wednesday, filtered and shaken, and then the milk fat rate, milk protein, total solids, non-milk fat solids and lactose are determined using a FOSS milk composition analyzer.

[0063] The specific experimental results are shown in Table 4:

[0064] Table 4 Culture experiment results

[0065] Group Dry matter intake (kg / d) Average daily milk production (kg / d) Diarrhea incidence Experimental Group 1 16.10 8.06 No diarrhea was found during the experimental period Experimental Group 2 15.06 7.98 No diarrhea was found during the experimental period Experimental Group 3 16.31 8.01 0.24％ Control group 14.65 7.31 5.71％

[0066] As can be seen from Table 4, the dry matter intake of experimental groups 1 to 3 is not much different, but they are all significantly higher than the control group, indicating that in the experiment of elephant grass-sugarcane tail leaf silage, the addition of composite bacterial species silage can effectively improve the palatability of feed and increase feed intake compared with the control group; the milk production of experimental groups 1 to 3 is not much different, and is significantly higher than that of the control group; indicating that in the experiment of elephant grass-sugarcane tail leaf silage, the addition of composite bacterial species silage can effectively increase the milk production of dairy buffaloes compared with the control group; the incidence of diarrhea in experimental groups 1 to 3 is significantly lower than that in the control group, and even no diarrhea occurs, indicating that in the experiment of elephant grass-sugarcane tail leaf silage, the addition of composite bacterial species silage has a good effect on preventing dairy cow diarrhea compared with the control group. Therefore, on the whole, the composite bacterial species of the present application have a good intestinal regulating effect.

[0067] Table 5 Milk composition test results

[0068] Group Milk fat percentage (%) Milk protein (%) Total solids (%) Non-milk solids (%) lactose(%) Experimental Group 1 7.36 6.03 18.63 11.03 5.53 Experimental Group 2 7.32 5.98 18.67 11.03 5.47 Experimental Group 3 7.34 5.93 18.57 11.07 5.50 Control group 7.34 4.87 18.36 10.36 4.92

[0069] As can be seen from Table 5, the milk fat rate and total solid content of experimental groups 1 to 3 are not much different from those of the control group, indicating that the silage with the addition of composite bacteria has no effect on the milk fat rate and total solid content of dairy buffaloes; the milk protein content of experimental groups 1 to 3 is higher than that of the control group, indicating that the silage with the addition of composite bacteria has an improving effect on the milk protein of dairy buffaloes; the non-milk fat solid content of experimental groups 1 to 3 is higher than that of the control group, indicating that the silage with the addition of composite bacteria has an improving effect on the non-milk fat solids of dairy buffaloes; the lactose content of experimental groups 1 to 3 is higher than that of the control group, indicating that the silage with the addition of composite bacteria has an improving effect on the lactose content of dairy buffaloes.

[0070] In summary, the composite strain of the present application is used to ferment elephant grass-sugarcane tail leaves, which can be used to ensilage the elephant grass-sugarcane tail leaves, with high fermentation efficiency and good feed quality. Feeding the silage to dairy buffaloes can effectively increase the milk production of dairy buffaloes and prevent diarrhea in dairy buffaloes. In addition, it also has an effect of increasing the milk protein and non-milk fat solids of buffalo milk, and has an effect of increasing lactose. It is a feed probiotic that can effectively improve the quality of buffalo milk and the growth of dairy buffaloes.

[0071] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A composite lactic acid bacteria, characterized in that The composite lactic acid bacteria consists of Lactobacillus paracasei strain XH146 and Lactobacillus plantarum strain XH352; The composite lactic acid bacteria is Lactobacillus paracasei strain XH146, the deposit number of which is CCTCC NO: M20232336; The deposit number of the Lactobacillus plantarum strain XH352 is CCTCC NO: M20232337.

2. The composite lactic acid bacteria according to claim 1, characterized in that The mass ratio of the Lactobacillus paracasei strain XH146 to the Lactobacillus plantarum strain XH352 is 3-6:3-6.

3. Use of the composite lactic acid bacteria as claimed in claim 1 in preparing elephant grass-sugarcane tail leaf silage.

4. A method for preparing the elephant grass-sugarcane tail leaf silage as claimed in claim 3, characterized in that: The method comprises the following steps: crushing elephant grass and sugarcane tail leaves, inoculating composite lactic acid bacteria for silage fermentation, and obtaining the product; the mass ratio of the elephant grass, sugarcane tail leaves, strain XH146 and strain XH352 is 100-200:100-150:3-6:3-6.

5. Use of the composite lactic acid bacteria as claimed in claim 1 in the preparation of drugs for preventing diarrhea.

6. Use of the elephant grass-sugarcane tail leaf silage as claimed in claim 3 in improving the yield of dairy buffalo.

7. Use of the elephant grass-sugarcane tail leaf silage as claimed in claim 3 in increasing the milk protein content of buffalo milk, increasing the non-milk fat solids content of buffalo milk and / or increasing the lactose content of buffalo milk.

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