Application of lactobacillus plantarum
By using Lactobacillus plantarum WQ-01 to synthesize the precursor substance of phenethyl acetate in the alfalfa silage fermentation process, the problem of insufficient fermentation quality and flavor in the prior art was solved, and the effect of improving the feed intake of ruminants and the quality of livestock products was achieved.
Patent Information
- Application Number
- CN202510183378.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2025-04-18
AI Technical Summary
The prior art has failed to effectively improve its quality and flavor in regulating the fermentation of alfalfa silage, affecting the feed intake of ruminants and the quality of livestock products.
During the fermentation process of Lactobacillus plantarum WQ-01, the fermentation quality and flavor of alfalfa silage were improved by synthesizing the precursor substance phenethyl acetate, phenylethyl acetate.
It significantly improves the fermentation quality of alfalfa silage, promotes the formation of high-quality flavor substances, increases the feed intake of ruminants, and improves the quality of livestock products.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology and relates to an application of Lactobacillus plantarum, in particular to the application of Lactobacillus plantarum in alfalfa silage fermentation. Background Art
[0002] Alfalfa is rich in protein, digestible fiber, minerals and vitamins, and is known as the "king of forage grass". Alfalfa silage is one of the important types of alfalfa products. It has high nutritional value and good palatability. It occupies an important position in the diet of ruminants such as dairy cows and sheep, especially its contribution to protein and total fiber content. Silage quality and flavor not only affect the feed intake of ruminants, but also affect the flavor and quality of livestock products. At present, there are many studies on microbial regulation of fermentation processes in the field of fermented foods, such as Daqu, rice wine, sauerkraut, sour bamboo shoots, etc. There is less research information on how microorganisms regulate high-quality flavor compounds in alfalfa silage to increase animal feed intake. Summary of the invention
[0003] In order to further improve the fermentation quality of alfalfa silage and optimize its flavor, thereby increasing the feed intake of ruminants, the present invention is committed to determining the key microorganisms that dominate alfalfa fermentation, and studying the regulatory mechanism of these core microorganisms on the formation of key flavor substances in high-quality alfalfa silage. By deeply analyzing its regulatory mechanism, the purpose of the present invention is to provide a method for effectively regulating the flavor of alfalfa silage, so as to significantly improve the quality of alfalfa silage, enhance its attractiveness to ruminants, and achieve an increase in breeding benefits.
[0004] Application of Lactobacillus plantarum provided by the present invention in preparing a bacterial agent for fermenting alfalfa silage;
[0005] The fermented alfalfa silage is characterized by:
[0006] 1) Improve the fermentation quality of alfalfa silage;
[0007] 2) Promoting the formation of flavor compounds in alfalfa silage, wherein the flavor compound is phenylethyl acetate.
[0008] In the present invention, the Lactobacillus plantarum WQ-01 can use phenylacetaldehyde in alfalfa silage to synthesize phenylethanol, a precursor of phenylethyl acetate.
[0009] In the above application, the plant lactobacillus is Lactobacillus plantarum, whose strain number is WQ-01, and its preservation number in the General Microbiological Center of China National Microbiological Culture Collection is CGMCC No.13318.
[0010] In the above application, the improvement of the fermentation quality of alfalfa silage is manifested in at least one of the following:
[0011] a) Reduce the pH value of alfalfa silage;
[0012] b) reducing the acetic acid content in alfalfa silage;
[0013] c) Reduce the ammonia nitrogen content in alfalfa silage;
[0014] d) Increase the lactic acid content in alfalfa silage.
[0015] The present invention also provides the use of Lactobacillus plantarum and / or a bacterial agent containing Lactobacillus plantarum in alfalfa silage fermentation in the following 1) and / or 2):
[0016] 1) Improve the fermentation quality of alfalfa silage;
[0017] 2) Promoting the formation of flavor compounds in alfalfa silage, wherein the flavor compound is phenylethyl acetate.
[0018] In the above application, the improvement of the fermentation quality of alfalfa silage is manifested in at least one of the following:
[0019] a) Reduce the pH value of alfalfa silage;
[0020] b) reducing the acetic acid content in alfalfa silage;
[0021] c) Reduce the ammonia nitrogen content in alfalfa silage;
[0022] d) Increase the lactic acid content in alfalfa silage.
[0023] The present invention also provides the use of Lactobacillus plantarum in the preparation of feed and / or an attractant for increasing animal feed intake by fermenting alfalfa silage as a raw material.
[0024] In the above applications, the animals include ruminants.
[0025] In the above application, the ruminant includes sheep.
[0026] The present invention further provides a method for preparing a feed or an attractant for increasing animal feed intake, comprising the following steps: using alfalfa silage as a raw material, adding the above-mentioned Lactobacillus plantarum for fermentation, and obtaining the feed or the attractant for increasing animal feed intake.
[0027] In the above preparation method, the amount of the plant lactobacillus added to the alfalfa silage can be 5×10 5 CFU / g~5×10 6 CFU / gCFU / g, specifically 1×10 6 CFU / g.
[0028] In the above preparation method, the fermentation conditions are as follows: vacuum, light-proof, storage for 25 to 180 days (specifically 25 days), and temperature of 20 to 25°C.
[0029] The present invention has the following beneficial effects:
[0030] The plant lactobacillus of the present invention improves the fermentation quality of alfalfa silage, promotes the formation of flavor compounds (phenethyl acetate) in alfalfa silage, improves the high-quality flavor of alfalfa silage, and thus increases the feed intake of ruminants.
[0031] Collection Instructions
[0032] Classification and nomenclature of biological materials: Lactobacillus plantarum.
[0033] Latin name of the biomaterial: Lactobacillus plantarum.
[0034] Reference biological material (strain): WQ-01.
[0035] Full name of the depository unit: General Microbiology Center of China Microbiological Culture Collection Administration.
[0036] Abbreviation of the depository: CGMCC.
[0037] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
[0038] Date of preservation: 2016.11.18.
[0039] Deposit number: CGMCC No.13318. BRIEF DESCRIPTION OF THE DRAWINGS
[0040] Figure 1 The fermentation quality of alfalfa silage fermented for 70 days under different treatments.
[0041] Figure 2 The flavor compounds in the CK group changed significantly after fermentation.
[0042] Figure 3 These are the flavor compounds that changed significantly after fermentation in the LP group.
[0043] Figure 4 is the relative content of phenylacetaldehyde and phenylethanol under different treatments. DETAILED DESCRIPTION
[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0045] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0046] Embodiment 1,
[0047] The alfalfa silage fermentation method provided by the present invention comprises the following steps:
[0048] 1) The dry matter content of alfalfa is adjusted to 35% for later use.
[0049] 2) Activating bacterial agent: Lactobacillus plantarum WQ-01 (preservation number CGMCC No. 13318) was cultured in MRS liquid medium (Beijing Solebaugh Technology Co., Ltd., catalog number M8540-250g) at 37°C for 24 hours. The amount of strain added to alfalfa silage was 1×10 6 CFU / gFM (wet weight).
[0050] 3) Spray the bacterial agent obtained in step 2 evenly onto the alfalfa silage obtained in step 1, stir evenly, take 300 g and put it into a polyethylene bag, vacuumize and store it in a dark place for 70 days, with the average indoor temperature between 20 and 25° C., to obtain a silage sample. The control group is alfalfa silage without adding bacterial agent, recorded as CK; the experimental group is alfalfa silage with adding bacterial agent, recorded as LP; the fresh sample is recorded as FS; 6 replicates are set for each group.
[0051] The nutritional quality, fermentation quality, flavor compounds and microbial community of alfalfa silage after 70 days of fermentation were tested.
[0052] The functional verification test of Lactobacillus plantarum WQ-01 includes the following steps:
[0053] The blank liquid MRS medium was used as the control group (CK), and the liquid MRS medium supplemented with phenylacetaldehyde was used as the experimental group (AP). The AP group was inoculated with Lactobacillus plantarum WQ-01 and cultured at 37°C for 48 hours. The phenylacetaldehyde and phenylethanol in the supernatant of the bacterial liquid were extracted with ethyl acetate, and the standard products of phenylacetaldehyde and phenylethanol were used to make mixed standards. The relative contents of phenylacetaldehyde and phenylethanol were determined using liquid chromatography-mass spectrometry (LC-MS) technology.
[0054] The animal feeding experiment included the following steps:
[0055] 1) Alfalfa silage preparation
[0056] a. Adjust the dry matter content of alfalfa to 35% for later use.
[0057] b. Activating bacterial agent: Lactobacillus plantarum WQ-01 (CGMCC No. 13318) was cultured in MRS liquid medium (Beijing Solebaugh Technology Co., Ltd., item number M8540-250g) at 37°C for 2 hours. The strain was added to alfalfa silage at a viable count of 1×10 6 CFU / gFM (wet weight);
[0058] Addition of phenylethyl acetate: the addition amount was 50 mg / kg FM (wet weight).
[0059] c. Spray the bacterial agent and phenylethyl acetate obtained in step b evenly onto the alfalfa silage obtained in step a, stir evenly, ferment at room temperature for 25 days with an average indoor temperature of 20-25° C. to obtain alfalfa silage. The control group is alfalfa silage added with Lactobacillus plantarum WQ-01, denoted as LP; the experimental group is alfalfa silage added with Lactobacillus plantarum WQ-01 and phenylethyl acetate, denoted as LPP.
[0060] 2) Animal feeding and management
[0061] a. Experimental design: 8 healthy mutton sheep (snowflake sheep and ewes) aged 8 months with no significant difference in body weight were selected and raised in single cages. The feeding trough was designed with two troughs. One trough was placed with alfalfa silage supplemented with Lactobacillus plantarum WQ-01, which was recorded as the LP group; the other trough was placed with alfalfa silage supplemented with Lactobacillus plantarum WQ-01 and phenylethyl acetate, which was recorded as the LPP group. 500g of alfalfa silage was placed in each trough each time.
[0062] b. Feeding method: Alfalfa silage was fed twice a day at 6:00 and 16:00, each feeding for 30 minutes. After 30 minutes, the remaining silage was collected and the LP and LPP intake of each mutton sheep was weighed. The total intake of the two feedings was taken as the daily LP and LPP intake of each mutton sheep, and the feed preference was calculated based on the daily intake. After 30 minutes of feeding alfalfa silage, the animals were fed a complete mixed diet consisting of corn silage and a mixture of cereals to meet the nutritional needs of the animals. The adaptation period was 7 days, the transition period was 6 days, and the formal trial was 8 days.
[0063] c. Animal Management: During the experiment, all animals had equal access to feed and water to ensure that all animals received the same nutritional level and management conditions.
[0064] Table 1 Nutritional quality of alfalfa silage fermented for 70 days under different treatments
[0065]
[0066] In Table 1, FS: fresh alfalfa sample; CK: no bacterial agent added; LP: addition of Lactobacillus plantarum WQ-01; different lowercase letters in the same row indicate significant differences among the treatments (P<0.05).
[0067] Results and analysis:
[0068] 1. Effects of Lactobacillus plantarum WQ-01 on nutritional quality and fermentation quality of alfalfa silage
[0069] The nutritional qualities of alfalfa silage under different treatments are shown in Table 1, including the contents of dry matter, crude protein, neutral detergent fiber, acid detergent fiber and water-soluble carbohydrates. There was no significant difference in the nutritional qualities between the LP group and the CK group. Figure 1 As shown, the pH of the LP group (4.36) was significantly (P<0.0001) lower than that of the CK group (4.85); the lactic acid content (5.26% DM) was significantly (P<0.05) higher than that of the CK group (3.72% DM); the acetic acid content (0.68% DM) was significantly (P<0.001) lower than that of the CK group (1.21% DM); the ammonia nitrogen content (0.23% DM) was significantly (P<0.01) lower than that of the CK group (0.42% DM). The results showed that inoculation with Lactobacillus plantarum WQ-01 had little effect on the nutritional quality of alfalfa silage, but significantly improved the fermentation quality of alfalfa silage.
[0070] 2. Effect of Lactobacillus plantarum WQ-01 on flavor substances in alfalfa silage
[0071] Flavor substances are key quality indicators that affect fermented products and are closely related to their overall sensory quality. Figure 2 , 3 As shown in the results, the number and types of key flavor substances in different treatment groups were different, among which phenylethyl acetate with fruity and sweet aroma was significantly upregulated in the CK group and the LP group. This indicates that fermentation changed the composition of the flavor structure of alfalfa silage and promoted the production of high-quality ester compounds. The addition of Lactobacillus plantarum WQ-01 may promote the production of flavor compounds in alfalfa silage.
[0072] 3. Functional verification test of Lactobacillus plantarum WQ-01
[0073] In order to verify whether Lactobacillus plantarum WQ-01 plays an important role in the production of phenylethyl acetate, specifically whether phenylacetaldehyde can be used to produce phenylethanol, the precursor of phenylethyl acetate. Figure 4As shown, the contents of phenylacetaldehyde and phenylethanol in the AP group were significantly higher than those in the CK group (P<0.0001). And phenylethanol was only detected in the AP group, indicating that Lactobacillus plantarum WQ-01 can produce phenylethanol from phenylacetaldehyde. Studies have reported that an acidic environment is conducive to the esterification reaction of phenylethanol with acetic acid to form phenylethyl acetate. Therefore, phenylethyl acetate can be used as a target metabolite to enhance the flavor of alfalfa silage, which may increase the feed intake of animals and may become an attractant for ruminants in animal husbandry production. Lactobacillus plantarum WQ-01 has the potential to become a functional strain that promotes the formation of attractants for ruminants.
[0074] 4. Effect of alfalfa silage supplemented with Lactobacillus plantarum WQ-01 and phenylethyl acetate on the feed intake of mutton sheep
[0075] Table 2 Effects of fermented alfalfa silage with phenylethyl acetate on feed preference and feed intake of mutton sheep
[0076]
[0077] In Table 2, LP a : Add Lactobacillus plantarum WQ-01 as the control group; LPP b : Add Lactobacillus plantarum WQ-01 and phenethyl acetate as the experimental group.
[0078] As shown in Table 2, the feed intake of the LPP group was significantly higher (P<0.01) than that of the LP group, and the dry matter intake, crude protein intake, and neutral detergent fiber intake were significantly higher (P<0.01) than those of the LP group. The feed preference of the LPP group was significantly higher (P<0.001) than that of the LP group, indicating that phenylethyl acetate is a high-quality flavor compound that can significantly increase the intake of alfalfa silage.
[0079] In summary, the feed obtained by using Lactobacillus plantarum WQ-01 as an exogenous additive for alfalfa silage has a higher fermentation quality. At the same time, it can promote the production of high-quality flavor substance phenylethyl acetate in alfalfa silage, thereby increasing the feed intake of mutton sheep and improving the utilization rate of high-quality alfalfa silage.
[0080] More importantly, Lactobacillus plantarum WQ-01 can use phenylacetaldehyde in alfalfa silage to synthesize phenylethanol, the precursor of phenylethyl acetate. This discovery highlights the potential of Lactobacillus plantarum to improve the flavor quality of alfalfa silage and opens up a new path for the development of high-quality silage additives and ruminant attractants.
Claims
1. Application of Lactobacillus plantarum in the preparation of bacterial agents for fermenting alfalfa silage; The fermented alfalfa silage is characterized by: 1) Improve the fermentation quality of alfalfa silage; 2) Promoting the formation of flavor compounds in alfalfa silage, wherein the flavor compound is phenylethyl acetate.
2. The use according to claim 1, characterized in that: The plant lactobacillus is Lactobacillus plantarum, the strain number of which is WQ-01, and the preservation number of which in the General Microbiological Center of China National Microbiological Culture Collection is CGMCC No.13318.
3. The use according to claim 1, characterized in that: The improvement of the fermentation quality of alfalfa silage is manifested in at least one of the following: a) Reduce the pH value of alfalfa silage; b) reducing the acetic acid content in alfalfa silage; c) Reduce the content of ammonia nitrogen in alfalfa silage; d) Increase the lactic acid content in alfalfa silage.
4. Application of Lactobacillus plantarum and / or a bacterial agent containing Lactobacillus plantarum in alfalfa silage fermentation as follows 1) and / or 2): 1) Improve the fermentation quality of alfalfa silage; 2) Promoting the formation of flavor compounds in alfalfa silage, wherein the flavor compound is phenylethyl acetate.
5. The use according to claim 4, characterized in that: The improvement of the fermentation quality of alfalfa silage is manifested in at least one of the following: a) Reduce the pH value of alfalfa silage; b) reducing the acetic acid content in alfalfa silage; c) Reduce the content of ammonia nitrogen in alfalfa silage; d) Increase the lactic acid content in alfalfa silage.
6. Application of Lactobacillus plantarum in the preparation of feed and / or attractant for increasing animal feed intake by fermenting alfalfa silage as raw material.
7. The use according to claim 6, characterized in that: The animals include ruminants.
8. The use according to claim 7, characterized in that: The ruminants include sheep.
9. A method for preparing a feed or an attractant for increasing animal feed intake, comprising the following steps: using alfalfa silage as a raw material, adding the plant lactobacillus described in any one of claims 1 to 8 for fermentation, and obtaining the feed or the attractant for increasing animal feed intake.
10. The preparation method according to claim 9, characterized in that: The amount of Lactobacillus plantarum added to the alfalfa silage is 5×10 5 CFU / g~5×10 6 CFU / g; The fermentation conditions are as follows: vacuum, light-proof, storage for 25 to 180 days, and temperature of 20 to 25°C.