Lactobacillus plantarum XH352 and application thereof
By screening and applying Lactobacillus plantarum XH352 strain to ferment the sugarcane tail leaves, the problem of poor microbial effect of sugarcane tail leaves was solved, and the effect of improving the utilization efficiency of sugarcane tail leaves and the growth performance of milk buffalo was achieved.
Patent Information
- Application Number
- CN202411480839.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-23
- Publication Date
- 2024-12-13
AI Technical Summary
In the prior art, the silage microorganisms of the tail leaves of sugar cane are not effective. The combined use of some microorganisms leads to the colonization and intestinal regulation of lactic acid bacteria. Not all lactic acid bacteria are suitable for silage of sugar cane tail leaves, resulting in the lack of sugar cane tail leaves resources. Can be used effectively.
A specific Lactobacillus plant strain XH352 was screened and used, and the sugarcane tail leaves were pulverized and fermented, and the sugarcane tail leaves were prepared to improve its viability and fermentation effect in the sugarcane tail leaves.
The XH352 strain can effectively improve the silage efficiency of sugarcane tail leaves, improve the dry matter intake and daily weight gain of milk buffaloes, reduce diarrhea rate, and show good probiotic properties and intestinal colonization ability.
Smart Images
Figure CN119120311A_ABST
Abstract
Description
[Technical field]
[0001] The invention relates to the technical field of microorganisms, in particular to Lactobacillus plantarum XH352 and applications thereof. [Background technology]
[0002] Sugarcane is an important sugar crop in my country. Sugarcane tail leaves contain high sugar, water and crude fiber. A large amount of residue in the sugarcane field will have an adverse effect on the agricultural management in the coming year. Therefore, in the existing technology, we usually use sugarcane tail leaves to develop feed and other products to provide a new way for ecological cattle breeding. In the processing of sugarcane tail leaves, it is usually considered to process the feed into silage to improve the efficiency of ecological breeding.
[0003] In the prior art, we found that due to the high sugar, water and crude fiber content in sugarcane tail leaves, the silage effect of silage microorganisms on the market is not good in the silage process using pure sugarcane tail leaves as raw materials. In addition, in order to adapt to the silage of more raw materials, some microorganisms often use multiple genera of microorganisms for compounding, such as the combined use of Bacillus subtilis and lactic acid bacteria. This results in lactic acid bacteria that generally have intestinal regulatory functions not necessarily forming a dominant flora, and their colonization and intestinal regulatory abilities will be greatly reduced. Guangxi is the largest sugarcane producing area in my country, with rich resources of sugarcane tail leaf by-products. It is imperative to strengthen the application of sugarcane tail leaves. During the screening process, we also found that not all lactic acid bacteria are suitable for silage of sugarcane tail leaves. Therefore, it is essential to screen probiotics targeting sugarcane tail leaves and suitable for silage of this raw material. Only when the strains are enriched can the sugarcane tail leaves be better utilized to prepare sugarcane tail leaf silage. [Summary of the invention]
[0004] In view of the above, it is necessary to provide a lactic acid bacteria capable of ensiling sugarcane tail leaves, and to utilize the lactic acid bacteria to better utilize the sugarcane tail leaves and prepare sugarcane tail leaf silage.
[0005] In order to achieve the above object, the technical solution adopted by the present invention is:
[0006] The present invention includes a Lactobacillus plantarum strain XH352, whose preservation number is CCTCC NO: M20232337. The strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, address: Wuhan University, Wuhan, China, with a preservation number of CCTCC NO: M20232337 and a preservation date of November 27, 2023.
[0007] The present invention also includes a bacterial agent comprising the Lactobacillus plantarum strain XH352.
[0008] The present invention also includes the use of the Lactobacillus plantarum strain XH352 and / or the bacterial agent in sugarcane tail leaf silage.
[0009] The present invention also includes a method for preparing sugarcane tail leaf silage using the Lactobacillus plantarum strain XH352 and / or the bacterial agent, which comprises crushing the sugarcane tail leaf, inoculating the strain XH352, and fermenting the resulting mixture.
[0010] The present invention also includes the application of the Lactobacillus plantarum strain XH352 and / or the bacterial agent in increasing dry matter intake and / or increasing daily weight gain of buffaloes.
[0011] The present invention also includes the use of the Lactobacillus plantarum strain XH352 and / or the bacterial agent in the preparation of a drug for preventing diarrhea.
[0012] The present invention has the following beneficial effects:
[0013] 1. The XH352 strain of the present invention is obtained by screening by the applicant. It has been verified that, compared with the same type of lactic acid bacteria, the strain can survive well in sugarcane tail leaves, can form a flora advantage, and can ensilage sugarcane tail leaves. After testing, the strain has played a good effect in the fermentation of sugarcane silage, and effectively improved the silage efficiency of sugarcane tail leaves. After feeding verification, the feed has a good effect on dairy buffaloes, and can play the role of: increasing the dry matter intake of dairy buffaloes, increasing the daily weight gain of buffaloes, reducing the diarrhea rate, and colonizing the intestines. It is a good probiotic that can ferment sugarcane tail leaf feed well.
Brief Description of the Drawings
[0014] Figure 1 This is a developmental tree diagram of the XH352 strain of the present application. [Specific implementation method]
[0015] 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.
[0016] 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.
[0017] Example 1
[0018] This example is a verification method for Lactobacillus plantarum strain XH352, which is as follows:
[0019] 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:
[0020] Table 1 Blast alignment results of 16S rDNA sequences of lactic acid bacteria strains
[0021]
[0022] from Figure 1 As can be seen from Table 1, strain XH352 and Lactobacillus plantarum were clustered together, so strain XH352 was identified as Lactobacillus plantarum.
[0023] The strain is preserved, and the preservation method is: the strain is preserved in the General Microbiology Center of China Culture Collection Administration, address: Wuhan University, Wuhan, China, the preservation number is CCTCC NO: M20232337, and the preservation date is November 27, 2023.
[0024] Example 2
[0025] The effects of the same batch of strains screened and identified as Lactobacillus paracasei: X122, X184, X378, Lactobacillus plantarum: XH1, XH352, X345 and Lactobacillus harbinensis strain X380 on the fermentation of sugarcane tail leaf substrate in this example are as follows:
[0026] 1. Preparation of culture medium containing sugarcane tail leaves: After mixing sugarcane tail leaves with water at a solid-liquid mass ratio of 2:1, filter the filtrate and mix it with liquid MRS culture medium. The amount of filtrate added is 10% of the volume percentage of the liquid MRS culture medium. After sufficient mixing, inoculate the above strains at an inoculum amount of 5%. After culturing for 24 days, use the liquid MRS culture medium without adding sugarcane tail leaf filtrate as the control group (CK) and then measure the pH value of the above strains and the OD of each strain 600 , the specific results are shown in Table 1:
[0027] Table 1 Effects of sugarcane tail leaf substrate culture medium on different lactic acid bacteria
[0028]
[0029] As shown in Table 1, the pH value, OD value and other parameters of the experimental groups of lactic acid bacteria X122, X378, XH1 and X380 in the substrate supplemented with sugarcane tail leaf filtrate were significantly different from those of the experimental groups of lactic acid bacteria X122, X378, XH1 and X380. 600 The values were not much different from those of the control group. The pH value of lactic acid bacteria X354 in the experimental group was greater than that in the control group. 600 The experimental group with a pH value was lower than the control group, indicating that the filtrate from sugarcane tail leaves had an inhibitory effect on the growth of strains and X354; the pH values of X184 and XH352 in the experimental group were lower than those in the control group, and OD 600 The experimental group had a greater value than the control group, indicating that the sugarcane tail leaf filtrate promoted the growth of the strain and X380; therefore, we selected these two strains for the next step of the sugarcane tail leaf silage fermentation experiment.
[0030] Example 3
[0031] In the experiment, fresh sugarcane tail leaves were fully crushed and inoculated with the above strains XH352 and X360, respectively. Sugarcane tail leaves without any lactic acid bacteria were used as the control group, and each treatment was repeated 3 times. A small-scale fermentation method was used, and the leaves were packed into polyethylene film bags, 500g per bag, and vacuum-sealed with a vacuum packaging machine. After fermentation at room temperature for 60 days away from light, samples were taken for determination of relevant indicators.
[0032] 1. Fermentation quality: After opening the bag, accurately weigh 20g of sample from each bag, put it into a 250mL wide-mouth bottle, add 180mL of distilled water, immerse the sample completely in water, seal the wide-mouth bottle cap with a sealing film, put it in a 4℃ refrigerator for extraction for 24h, and then filter it with 2 layers of gauze. The filtrate is used for the determination of pH, organic acid (lactic acid, acetic acid, propionic acid and butyric acid) and ammonia nitrogen content. The pH value is determined by a portable pH meter, the lactic acid, acetic acid, propionic acid and butyric acid content is determined by high performance liquid chromatography, and the ammonia nitrogen content is determined by colorimetry.
[0033] 2. Nutritional ingredients: Take the opened sample and dry it in an electric constant temperature air drying oven at 65°C to constant weight, then grind it to pass through a 1mm sieve. This sample is the air-dried sample. Determine the dry matter content of the sample. The crude protein is determined by the Kjeldahl method, and the neutral detergent fiber and acid detergent fiber contents are determined by the Van der Waals method.
[0034] The results obtained by fermentation are shown in Table 2 and Table 3:
[0035] Table 2 Effects of different strains on the fermentation quality of sugarcane tail leaf fermented feed
[0036] Group pH Lactic acid (g / kg) Acetic acid(g / kg) Propionic acid(g / kg) Butyric acid (g / kg) <![CDATA[NH3-N(g / kg)]]> XH352 4.17±0.08b 33.35±0.14a 30.35±1.86c 0.32±0.04b ND 8.69±0.09c X184 4.55±0.07a 20.38±0.86b 38.55±0.82b 2.28±0.20a ND 10.95±0.25b Comparison 4.65±0.07a 15.34±0.13c 43.08±1.03a 2.34±0.05a 1.66±0.04 16.31±0.21a
[0037] Note: Different lowercase letters in the same column indicate significant differences (P<0.05), the same below.
[0038] As shown in Table 2, the pH value of sugarcane tail leaves fermented by strain XH352 was significantly lower than that of strain X184 and the control group (P<0.05). At the same time, the lactic acid content was significantly higher than that of strain X184 and the control group (P<0.05), and the acetic acid content was significantly lower than that of strain X184 and the control group (P<0.05). No butyrate was produced, and the NH3-N content was significantly lower than that of strain X184 and the control group (P<0.05). This shows that strain XH352 is more suitable for sugarcane tail leaf fermentation than strain X184. After fermentation, the lactic acid production of sugarcane tail leaves is higher and the NH3-N production is lower.
[0039] Table 3 Effects of different strains on the nutritional components of fermented feed from sugarcane tail leaves
[0040] Group Dry matter (%) Crude protein (%) Organic matter (%) Neutral detergent fiber (%) Acid detergent fiber (%) XH352 28.24±1.03a 8.57±0.11a 83.61±0.63a 63.02±0.83b 33.01±0.88c X184 26.28±0.99ab 7.38±0.10b 81.68±1.24b 62.71±1.20b 38.29±0.78b Comparison 25.18±1.11b 7.23±0.09b 80.83±0.55b 65.69±0.57a 42.24±0.73a
[0041] As shown in Table 3, after the fermentation of strain XH352, the dry matter, crude protein and organic matter content of the fermented feed of sugarcane tail leaves were significantly higher than those of the sugarcane tail leaves fermented by strain X184 and the control group (P<0.05), the neutral detergent fiber content was significantly lower than that of the sugarcane tail leaves fermented by the control group (P<0.05), and the acid detergent fiber content was significantly lower than that of the sugarcane tail leaves fermented by strain X184 and the control group (P<0.05); it shows that strain XH352 is more suitable for sugarcane tail leaf fermentation than strain X184, and has higher dry matter and crude protein content. From this embodiment, it can be seen that although the filtrate of sugarcane tail leaves has a promoting effect on strain X184, after inoculation into the sugarcane tail leaf base material, the growth-promoting effect of strain X184 is not obvious. This is probably because the filtrate of sugarcane tail leaves has filtered out most of the fiber and other substances, and after inoculation of sugarcane tail leaves, the fiber content increases, and strain X184 cannot play a good fermentation role.
[0042] Example 4
[0043] From the experimental results of Example 3, we selected the effect of fermented feed on the growth of dairy buffalo. We conducted animal experiments on the fermented feed prepared in Example 3. The specific scheme is as follows:
[0044] Guangxi Buffalo Research Institute Breeding Farm, Experimental Design: The experimental period is 2 months. This experiment adopts the principle of random pairing. The experimental cattle are three-grade hybrid dairy buffaloes (local buffalo × Nili-Lafite buffalo × Mora buffalo). 30 dairy buffaloes (350±10kg) were randomly divided into an experimental group and a control group, with 10 dairy buffaloes in each group. The feeding experiment was carried out according to the following feeding formula:
[0045] Experimental group 1: strain XH352 was used to prepare silage according to the method of Example 3 and fed directly;
[0046] Experimental group 2: strain X184 was used to prepare silage according to the method of Example 3 and fed directly;
[0047] Control group: directly fed with the silage prepared by the control group of Example 3.
[0048] During the feeding period, the dry matter intake of each group of dairy buffaloes was calculated every day; after 60 days of feeding, the average daily weight gain of each group was calculated; the number of cattle with diarrhea during the experimental period was observed and recorded, and the incidence of diarrhea was calculated; the calculation formulas for the increase in dry matter intake and the average daily weight gain were as follows:
[0049] Dry matter intake = amount of feed fed - amount of remaining feed; after calculating the dry matter intake every day, the average value was taken during the entire experimental period.
[0050] The formula for calculating average daily weight gain is:
[0051] 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) × 20 (heads) to calculate the diarrhea incidence rate. The specific experimental results are shown in Table 4:
[0052] Table 4 Culture experiment results
[0053] Group Dry matter intake (kg / d) Average daily weight gain (g) Diarrhea incidence Experimental Group 1 12.82 931.11 No diarrhea was found during the experimental period Experimental Group 2 12.36 882.13 1.67％ Control group 11.62 820.14 5.83％
[0054] As can be seen from Table 4, the dry matter intake of experimental group 1 was significantly higher than that of experimental group 2 and the control group, indicating that the strain XH352 of the present application has better palatability than the strain X184 and the control group, and can effectively improve the appetite of dairy buffaloes; the average daily weight gain of experimental group 1 was significantly higher than that of experimental group 2 and the control group, indicating that the strain XH352 of the present application can effectively increase the weight of dairy buffaloes compared with the strain X184 and the control group; the incidence of diarrhea in experimental group 1 was significantly lower than that in experimental group 2 and the control group, and no diarrhea occurred, indicating that the strain XH352 of the present application has a good effect in preventing dairy cow diarrhea. Therefore, on the whole, the strain XH352 of the present application has a better intestinal regulation effect than other lactic acid bacteria.
[0055] In summary, the use of Lactobacillus plantarum XH352 of the present application can effectively ferment sugarcane tail leaves, and the prepared fermented sugarcane tail leaf feed has a good effect on the growth of dairy buffaloes and the prevention and treatment of diarrhea, and is a probiotic with good prebiotic effects.
[0056] 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 Lactobacillus plantarum strain XH352, whose deposit number is CCTCC NO: M20232337.
2. A bacterial agent comprising the Lactobacillus plantarum strain XH352 as claimed in claim 1.
3. Use of the Lactobacillus plantarum strain XH352 according to claim 1 and / or the bacterial agent according to claim 2 in sugarcane tail leaf silage.
4. A method for preparing sugarcane tail leaf silage using the Lactobacillus plantarum strain XH352 according to claim 1 and / or the bacterial agent according to claim 2, characterized in that: The method comprises the following steps: crushing the sugarcane tail leaves, inoculating the strain XH352 and fermenting the resulting mixture.
5. Use of the plant lactobacillus (Lactobacillus plantarum) strain XH352 as claimed in claim 1 and / or the bacterial agent as claimed in claim 2 in increasing the dry matter intake of dairy buffaloes and / or increasing the daily weight gain of buffaloes.
6. Use of the Lactobacillus plantarum strain XH352 according to claim 1 and / or the bacterial agent according to claim 2 in the preparation of a drug for preventing diarrhea.