Streptococcus non-lactolyticus GX-7 and application of complex microbial inoculant thereof in sugarcane leaf silage

Abstract

The invention relates to the technical field of microorganisms, in particular to application of streptococcus non-lactolyticus GX-7 and a complex microbial inoculant thereof in sugarcane leaf silage, the strain GX-7 is separated from healthy pig manure by a research group, and through detection, the strain has good cholate resistance and is suitable for surviving in intestinal tracts, and the strain GX-7 has good cholate resistance and is suitable for being used as a feed additive. The antibacterial effect on pseudomonas fluorescens, listeria monocytogenes, salmonella choleraesuis and shigella flexneri is achieved; the strain has a good fermentation capability on sugarcane leaves, and the sugarcane leaves are inoculated with a complex microbial inoculant prepared from streptococcus non-lactolyticus GX-7 and streptococcus non-lactolyticus GX-2, so that a synergistic fermentation effect can be achieved; after the silage prepared from the complex microbial inoculant is fed to female buffalo in the gestation period, it is found that the silage can effectively increase the content of milk protein, milk total solids and milk non-fat solids in colostrum of the female buffalo and relieve diarrhea of the female buffalo, and the silage is safe and good.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to application of non-galactolytic Streptococcus GX-7 and a composite bacterial agent thereof in sugarcane leaf silage. Background Art

[0002] Guangxi is one of my country's major sugarcane producing areas. Sugarcane leaves, consisting of the two to three tender nodes at the top of the sugarcane plant and the intact leaves attached to them, account for approximately one-fifth of the weight of the entire sugarcane plant. They are rich in crude protein, neutral detergent fiber, and acid detergent fiber, making them suitable for use as roughage for livestock and poultry. However, due to the large amount of sugarcane leaves harvested in a short period of time, they cannot be fully digested and consumed by livestock and poultry, leading to much waste and discard. Improper storage can also lead to mold. Sugarcane leaves are highly nutritious. Fresh sugarcane leaves are rich in water, comprising approximately 70% of the plant's water content. After air-drying, the crude fiber content reaches approximately 30%. Sugarcane leaves also have a high nutritional value, with a crude protein content of 7%, a neutral detergent fiber content of 40%, and an acid detergent fiber content of up to 70%. This richness in nutrients makes sugarcane leaves highly palatable and beneficial for animal husbandry. However, sugarcane leaves are difficult to store and their nutrients are easily lost. In order to preserve sugarcane leaves well and enhance the utilization rate of sugarcane leaves in the prior art, we usually ensilage the sugarcane leaves and prepare silage to achieve effective utilization of the sugarcane leaves.

[0003] In the field of sugarcane leaf silage, some lactic acid bacteria, Bacillus and even yeast are often used for fermentation. However, in the actual fermentation process, we found that different strains of the same genus and species have very different fermentation abilities for sugarcane leaves. We generally prefer strains that can effectively maintain dry matter content, increase protein content, and reduce fiber content as indicators for evaluating fermentation ability. Of course, in addition to fermentation ability, resistance to pathogens is also essential. Improving the strain's resistance to pathogens will greatly reduce the incidence of livestock and have a good disease prevention effect.

[0004] In order to effectively develop higher-quality silage, we need to conduct extensive screening and verification of microorganisms, develop probiotics suitable for fermentation of sugarcane leaf substrates, produce sugarcane leaf silage, and verify the feasibility of this feed in feeding animals. Summary of the Invention

[0005] In view of the above, it is necessary to screen suitable microorganisms for sugarcane substrates and develop microorganisms that can effectively ferment sugarcane leaves and have a certain antibacterial ability against pathogens, provide theoretical support for the production of sugarcane leaf silage, and preliminarily verify the feasibility of sugarcane leaf silage in feeding animals.

[0006] To achieve the above objectives, the present invention screened out a new strain: Streptococcus alactolyticus strain GX-7, which is classified as Streptococcus alactolyticus GX-7, and its Chinese classification name is Streptococcus alactolyticus GX-7, with a preservation number of GDMCC NO: 65509; the strain is deposited in the Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation date is November 19, 2024.

[0007] The present invention also includes a composite bacterial agent comprising the non-lactolytic Streptococcus alactolyticus strain GX-7.

[0008] Furthermore, the composite bacterial agent also includes non-lactose-lytic Streptococcus alactolyticus strain GX-2; the non-lactose-lytic Streptococcus alactolyticus strain GX-2, its classification name is: Streptococcus alactolyticus GX-2, its Chinese classification name is: non-lactose-lytic Streptococcus GX-2, and its preservation number is GDMCC NO: 65508; the strain is deposited in the Guangdong Provincial Microbiological Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the preservation date is November 19, 2024.

[0009] Furthermore, the composite bacterial agent is prepared by mixing non-lactose-lytic Streptococcus alactolyticus strain GX-7 and non-lactose-lytic Streptococcus alactolyticus strain GX-2 in a volume ratio of 2-1:1.

[0010] Furthermore, the composite bacterial agent is prepared by mixing non-lactose-lytic Streptococcus alactolyticus strain GX-7 and non-lactose-lytic Streptococcus alactolyticus strain GX-2 in a volume ratio of 2:1.

[0011] The present invention also includes the use of the non-lactolytic Streptococcus alactolyticus strain GX-7 or the composite bacterial agent in preparing sugarcane leaf silage.

[0012] The present invention also includes the use of the non-lactolytic Streptococcus alactolyticus strain GX-7 in producing bile salt-resistant and / or diarrhea-relieving drugs for female buffaloes.

[0013] The present invention also includes the use of the non-lactolytic Streptococcus alactolyticus strain GX-7 in an antibacterial agent, wherein the antibacterial agent inhibits pathogenic bacteria such as Pseudomonas fluorescens, Listeria monocytogenes, Salmonella Choleraesuis, and Shigella flexneri.

[0014] The present invention also includes silage containing the non-galactolytic Streptococcus alactolyticus strain GX-7 or the composite bacterial agent according to claim 2, characterized in that the silage is prepared by inoculating the non-galactolytic Streptococcus alactolyticus strain GX-7 or the composite bacterial agent into sugarcane leaves at an inoculum rate of 5% by mass and silaging for 35 days.

[0015] The present invention also includes the use of the silage in increasing the content of milk protein, total milk solids and / or milk non-fat solids in the colostrum of female buffaloes.

[0016] The present invention also includes a method for feeding pregnant female buffaloes with the silage, which comprises: mixing 20%-30% by mass of the silage with 70%-80% by mass of the basic diet and then feeding the mixture to the pregnant female buffaloes.

[0017] The basic diet is whole-plant corn silage-based feed.

[0018] The present invention has the following beneficial effects: the strain GX-7 of the present invention was isolated from healthy pig feces by a research team. Testing showed that the strain has good bile salt tolerance and is suitable for survival in the intestine, and has an antibacterial effect on Pseudomonas fluorescens, Listeria monocytogenes, Salmonella choleraesuis, and Shigella flexneri. The strain has good fermentation ability on sugarcane leaves. Non-lactolytic Streptococcus GX-7 and non-lactolytic Streptococcus GX-2 are prepared into a composite bacterial agent and then inoculated into sugarcane leaves to achieve a synergistic fermentation effect. After feeding silage prepared from the composite bacterial agent to pregnant buffaloes, it was found that the feed can effectively increase the milk protein, total milk solids, and milk non-fat solids content in the colostrum of the buffaloes, alleviate diarrhea in the buffaloes, and is a safe and good silage.

[0019] Biomaterial deposit information

[0020] The strain information deposited in this application is: Streptococcus alactolyticus strain GX-7, its classification name is: Streptococcus alactolyticus GX-7, its Chinese classification name is: Streptococcus alactolyticus GX-7, and its preservation number is GDMCC NO: 65509; the strain is deposited in Guangdong Provincial Microbiological Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the preservation date is November 19, 2024.

[0021] The strain information deposited in this application is: Streptococcus alactolyticus strain GX-2, its classification name is: Streptococcus alactolyticus GX-2, its Chinese classification name is: Streptococcus alactolyticus GX-2, and the preservation number is GDMCC NO: 65508; the strain is deposited in Guangdong Provincial Microbiological Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation date is November 19, 2024. DETAILED DESCRIPTION

[0022] All features disclosed in this specification, or all steps in the disclosed methods or processes, except mutually exclusive features and / or steps, can be combined in any manner.

[0023] Any feature disclosed in this specification (including any accompanying claims and abstract), unless otherwise stated, is merely one example of a series of equivalent or similar features.

[0024] Example 1

[0025] This example is about the isolation and identification of strains.

[0026] 1. Strain Screening and Isolation: 10g of fresh feces was collected from healthy pigs. Place 100μL of the fecal sample in 90mL of sterile saline and shaken in a 250mL Erlenmeyer flask for 20 minutes. 100μL of each appropriately diluted bacterial solution was spread onto Ca-MRS (CaCO3: 2g / L) plates and incubated in a 37°C incubator for 24 hours. Morphological characteristics of the bacteria were recorded. Colonies with distinct morphology and color were streaked and purified three times. A single colony was selected and placed on a clean glass slide. 1-2 drops of 3% hydrogen peroxide solution were added and allowed to stand for observation. A positive result was identified if a large number of bubbles were produced within half a minute; a negative result was identified if no bubbles were produced. The isolated and purified single colony was Gram-stained and its color and morphology were observed under a microscope. Lactic acid bacteria are Gram-positive and should appear purple after Gram staining. The single colony screened was inoculated into MRS liquid medium and incubated at 37°C for 18-2 hours. Add the bacteria and glycerol into an EP tube, mix well (the final concentration of glycerol is 15%), and store in a -20°C refrigerator for later use.

[0027] 2. Morphological and molecular identification of the screened GX-7 and GX-2 strains:

[0028] ① The GX-7 strain was inoculated on MRS plate culture medium and cultured for 24 hours. The colonies were round and 0.9 mm in size. The colonies were white, opaque, smooth, round, convex, and had neat edges. Microscopic examination showed that the bacteria were spherical, single or in pairs, and not curved. After molecular identification, the strain was finally classified and named Streptococcus alactolyticus.

[0029] ② The GX-2 strain was inoculated on MRS plate culture medium and cultured for 24 hours. The colonies were round and 0.4 mm in size. The colonies were flesh-colored, transparent and matte. The colonies were round and convex, with a smooth surface and neat edges. Microscopic examination showed that the bacteria were oblate and clustered. After molecular identification, the strain was finally classified and named Streptococcus alactolyticus.

[0030] Example 2

[0031] This example is a test on the acid resistance, bile salt resistance and artificial gastrointestinal fluid resistance of strains GX-7 and GX-2.

[0032] 1. Acid and bile salt resistance test:

[0033] MRS liquid culture medium was prepared and adjusted to pH 2.0, 3.0, and 4.0 with 1 mol / L hydrochloric acid. A 2% inoculum was inoculated into each of the liquid culture media at different pH values. A medium without hydrochloric acid was used as a control. After incubation at 37°C for 3 hours, viable bacteria were counted. The survival rate was calculated as (viable bacteria count at 3 hours / viable bacteria count at 0 hours) × 100%. Simultaneously, a 2% inoculum was inoculated into liquid culture media containing different bile salt concentrations (0.3% and 0.5%). After incubation at 37°C for 4 hours, viable bacteria were counted. The survival rate was calculated as (viable bacteria count at 4 hours / viable bacteria count at 0 hours) × 100%. The results are shown in Table 1.

[0034] 2. Artificial gastrointestinal fluid experiment:

[0035] Artificial gastric fluid formula: 3.1g / L NaCl, 1.1g / L KCl, 0.15g / L CaCl2·2H2O, 0.6g / L NaHCO3, adjusted to pH 3.0, sterilized at 121°C for 20 minutes, and pepsin (10g / L) added in a clean hood. Artificial intestinal fluid formula: Weigh 6.8g KH2PO4 and dissolve it in 500mL of water. Adjust the pH of the mixture to 6.8 with 0.1M NaOH. Dissolve 1.0g of trypsin in water. Mix the two solutions and dilute to 1000mL with water to obtain artificial intestinal fluid. Inoculate the artificial gastric fluid at a 2% inoculum level. MRS medium without other ingredients is used as a control. After incubation at 37°C for 3 hours, count the viable bacteria. Survival rate = (number of viable cells at 3 hours / number of viable cells at 0 hours) × 100%. Similarly, when 2% of the inoculum was inoculated into artificial intestinal fluid and cultured for 4 hours, the survival rate = (number of viable cells at 4 hours / number of viable cells at 0 hours) × 100%. The results are shown in Table 1.

[0036] Table 1 Acid and bile salt resistance test results of strains GX-7 and GX-2

[0037]

[0038] As shown in Table 1, under the conditions of pH 2-4, the lower the pH, the lower the survival rate of the strains. When the pH reached 2, the survival rate of the two strains was only 8%. This shows that under the condition of pH 2, the two strains basically cannot survive. From the perspective of significance, there is no significant difference in the survival rate of the two strains (p>0.05); in the bile salt test, as the bile salt concentration increases, the survival rate of the two strains will decrease. Under the condition of 0.3% bile salt concentration, the survival rate of strain GX-7 is significantly higher than that of strain GX-2 (p<0.05). Under the condition of 0.5% bile salt concentration, the survival rate of strain GX-7 is extremely significantly higher than that of strain GX-2 (p<0.001). From the perspective of survival rate, the survival rate of GX-7 was higher than that of GX-2, indicating that strain GX-7 has better bile salt tolerance than strain GX-2. From the results of artificial gastric fluid treatment, the survival rate of GX-7 was significantly lower than that of GX-2 (p<0.05), indicating that strain GX-2 is more suitable for survival in the stomach than strain GX-7. From the results of artificial intestinal fluid treatment, the survival rate of GX-7 was extremely significantly higher than that of GX-2 (p<0.001), indicating that strain GX-7 is more suitable for survival in the intestine than strain GX-2.

[0039] Example 3

[0040] This example is a bacteriostasis experiment of strain GX-7.

[0041] Six indicator bacteria (Staphylococcus aureus, Escherichia coli, Pseudomonas fluorescens, Listeria monocytogenes, Salmonella Choleraesuis, and Shigella flexneri) were activated and cultured for later use. The antibacterial activity of strain GX-7 against these six indicator bacteria was determined using the punch agar diffusion method. The diameters of the clearing zones (inhibition zone diameter = measured diameter minus well diameter) were measured. The results are shown in Table 2.

[0042] Table 2 Antibacterial effect of strain GX-7 on common pathogens

[0043]

[0044] Note: “—” in the table indicates no inhibitory effect, different lowercase letters indicate significant differences in the same column of data (p<0.05), and the same letters indicate no significant differences in the same column (p>0.05). The same applies to the following tables.

[0045] As shown in Table 2, strain GX-7 has an antibacterial effect on Pseudomonas fluorescens, Listeria monocytogenes, Salmonella Choleraesuis and Shigella flexneri, but has no antibacterial effect on Staphylococcus aureus and Escherichia coli. From the inhibition zone, the inhibition zone of Pseudomonas fluorescens is significantly higher than that of other pathogens (p < 0.05), and the inhibition zone of Listeria monocytogenes and Salmonella choleraesuis is significantly higher than that of other pathogens (p < 0.05). Choleraesuis) had no significant difference in inhibition zone (p>0.05), and the inhibition zone of Shigella flexneri was significantly lower than that of other pathogens (p<0.05); this indicated that strain GX-7 had the best inhibitory effect against Pseudomonas fluorescens, followed by Listeria monocytogenes and Salmonella Choleraesuis, and then Shigella flexneri.

[0046] Example 4

[0047] This example shows the fermentation effects of different screened bacterial agents on sugarcane leaf silage.

[0048] 1. Experimental design: This study adopted a completely randomized experimental design, with three groups and three replicates in each group: a control group without any bacterial agent (CK group), a group with non-galactolytic Streptococcus GX-7 (GX-7), and a group with non-galactolytic Streptococcus GX-2 (GX-2).

[0049] Experimental method: After the sterilized sugarcane leaves were crushed, the water content was adjusted to about 40% with distilled water, and the inoculation amount was 5% by mass (the effective viable bacteria count was 10 7 -10 8cfu / g) were inoculated into sterilized sugarcane leaves with the corresponding inoculum, mixed well, and placed in 250 mL silage bottles for ensiling. Each bottle contained 200 g of sample, which was sealed with sealing film and fermented in the dark for 35 days. The nutritional components and fermentation parameters after fermentation were tested. The indicators of nutrient component determination included dry matter (DM), crude protein (CP), crude fiber (CF), neutral detergent fiber (NDF), and acid detergent fiber (ADF) content, and the results are shown in Table 3. The indicators of fermentation parameter determination included lactic acid, acetic acid, and ammonia nitrogen, and the results are shown in Table 4.

[0050] Table 3 Effects of different microbial agents on the nutritional components of sugarcane leaf silage

[0051]

[0052] As shown in Table 3, after the strain GX-7 fermented sugarcane leaves, the contents of crude fiber (CF), neutral detergent fiber (NDF), and acid detergent fiber (ADF) were significantly lower than those in the control group CK (p<0.05); the crude protein (CP) content was significantly increased compared with the control group CK (p<0.05), and the dry matter (DM) content was not significantly different from that in the control group CK (p>0.05).

[0053] After the strain GX-2 fermented sugarcane leaves, the crude fiber (CF), neutral detergent fiber (NDF), dry matter (DM) and crude protein (CP) contents were not significantly different from those of the control group CK (p>0.05); the acid detergent fiber (ADF) content was significantly lower than that of the control group CK (p<0.05); this shows that for sugarcane leaf substrates, the crude fiber degradation effect and protein enhancement effect of the strain GX-7 are the most significant, indicating that the strain GX-7 has the best fermentation effect on sugarcane leaf substrates.

[0054] Table 4 Results of fermentation parameters of sugarcane leaf silage with different inoculants

[0055]

[0056] As shown in Table 4, after the strain GX-7 fermented sugarcane leaves, the lactic acid content was significantly increased compared with the control CK (p<0.05), the ammonia nitrogen content was significantly decreased compared with the control CK (p<0.05), and the acetic acid content was not significantly different from that of the CK (p>0.05).

[0057] After strain GX-2 fermented sugarcane leaves, the lactic acid content was significantly increased compared with the control CK (p<0.05), while the ammonia nitrogen content and acetic acid content were not significantly different from those of CK (p>0.05).

[0058] Based on the nutritional composition and parameters of silage, the fermentation of sugarcane leaves using strain GX-7 is better than that using strain GX-2. Strain GX-2 has a certain fermentation effect on sugarcane leaves, and the lactic acid content will increase, but the overall fermentation effect is not as good as that of strain GX-7.

[0059] Example 5

[0060] This example shows the fermentation effect of the composite bacterial agent on sugarcane leaves.

[0061] According to the experimental results of Example 4, we found that: the fermentation of strain GX-7 can reduce the dry matter loss of sugarcane leaves, reduce the crude fiber, neutral detergent fiber, and acid detergent fiber levels of silage sugarcane leaves, increase the crude protein content, increase the lactic acid content, and reduce the NH3 / TN; the fermentation of strain GX-2 can reduce the dry matter loss of sugarcane leaves and increase the lactic acid content. In order to increase the diversity of strains and study the effect of the composite inoculum on the quality of sugarcane leaf silage, the research team considered preparing a composite inoculum with strains GX-7 and GX-2 according to the mass ratio in Table 5, and then inoculating the composite inoculum with sugarcane leaves at a mass percentage of 5% (the effective viable cell count was 10 7 -10 8 cfu / g) sugarcane leaf silage was prepared according to the method of Example 4. The optimal experimental group was selected based on the crude protein (CP), crude fiber (CF), lactic acid (LA), and ammonia nitrogen (NH3 / TN) contents as optimization indicators, as shown in Tables 5 and 6.

[0062] Table 5 Mass ratio of each strain of composite bacterial agent

[0063]

[0064] Table 6 Experimental results of composite microbial agents

[0065]

[0066] As can be seen from Table 6, from the perspective of crude protein (CP), compared with Table 3 of Example 4, the crude protein content of the T2 and T3 experimental groups was higher than that of the GX-7 fermentation group (5.60%), and the crude protein content of T1, T4 and T5 was lower than that of the GX-7 fermentation group (5.60%); from the perspective of the groups, the crude protein (CP) content of the T2 and T3 experimental groups was the highest, but the difference between the two groups was not significant (p>0.05), and the crude protein (CP) of the T2 and T3 experimental groups was significantly higher than that of the T1, T4 and T5 experimental groups (p<0.05).

[0067] From the perspective of crude fiber (CF), compared with Table 3 of Example 4, the crude fiber (CF) content of the experimental groups of T2, T3, T4 and T5 was lower than that of the GX-7 fermentation group (29.96%), and the crude fiber (CF) content of T1 was higher than that of the GX-7 fermentation group (29.96%); from the perspective of between groups, the crude fiber (CF) of the T2 experimental group was significantly lower than that of the T1, T3, T4 and T5 groups (p<0.05), and the difference in crude fiber (CF) content between the T4 group and the T5 group was not significant (p>0.05).

[0068] From the perspective of lactic acid (LA) content, compared with Table 3 of Example 4, the lactic acid content of the T2 and T3 experimental groups was higher than that of the GX-7 fermentation group (774.38 mmol·kg -1 ), the lactic acid contents of T1, T4 and T5 were significantly different from those of GX-7 fermentation group (774.38 mmol·kg -1 ) were basically consistent; from the perspective of the groups, the lactic acid content of the T2 experimental group was significantly higher than that of the other experimental groups: T1, T4 and T5 groups (p<0.05).

[0069] From the perspective of ammonia nitrogen (NH3 / TN) content, compared with Table 3 of Example 4, the NH3 / TN content of the T2 and T3 experimental groups was lower than that of the GX-7 fermentation group (95.65 mmol·kg -1 ), the NH3 / TN contents of the T1, T4 and T5 experimental groups were higher than those of the GX-7 fermentation group (95.65mmol·kg -1 ); From the perspective of the groups, the NH3 / TN content of the T2 and T3 experimental groups had no significant difference (p>0.05), and was significantly lower than that of the T1, T4 and T5 experimental groups.

[0070] The above results show that the ratio of T2 and T3 composite strains can significantly synergistically improve the fermentation effect of sugarcane leaves, while the ratio of T1, T4 and T4 composite strains cannot synergistically increase the fermentation effect of sugarcane leaves. Therefore, the mass ratio of the composite microbial agent is selected as: strain GX-7: strain GX-2 = 2-1:1. Compared with the T3 experimental group, the fermentation effect of the T2 experimental group is slightly better. The optimal mass ratio of the composite microbial agent is selected as: strain GX-7: strain GX-2 = 2:1.

[0071] Example 6

[0072] This example is an experiment on feeding sugarcane leaf silage to female buffaloes.

[0073] Fifty pregnant buffaloes of similar gestational age and good body condition were selected and divided into five groups for feeding. Sugarcane silage was fed 60 days before the expected birth date. The specific feeding method was: 50% of the daily feed intake was fed at 9:00 and 16:00 every day. The feed fed to each group was as follows:

[0074] CK: The basic diet is whole-plant corn silage-based feed.

[0075] Control 1: 20% by mass of sugarcane leaf silage without fungicide + 80% by mass of whole-plant corn silage basic feed.

[0076] Control 2: 30% by mass of sugarcane leaf silage without fungicide + 70% by mass of whole-plant corn silage basic feed.

[0077] Experiment 1: 20% by mass of sugarcane leaf silage containing bacterial agent (the bacterial agent was added according to the T2 experimental group of Example 5) + 80% by mass of whole plant corn silage basic feed.

[0078] Experiment 2: 30% by weight of sugarcane leaf silage containing the bacterial agent (the bacterial agent was added according to the T2 experimental group of Example 5) + 70% by weight of whole corn silage as a basic feed; the number of diarrhea cases in female buffaloes during the experimental period was recorded: 1 diarrhea event was recorded as 1, and 2 diarrhea events were recorded as 2.

[0079] Colostrum sample collection: Within 24 hours after the buffalo gave birth, 30 mL of mixed milk samples were collected from the front, middle, and back teats by hand expression and stored in a -20°C refrigerator for testing. The colostrum was tested for milk fat, milk protein, lactose, milk non-fat solids, and total milk solids content, as shown in Table 7.

[0080] Table 7 Effects of sugarcane leaf silage on colostrum composition of buffaloes

[0081]

[0082] As shown in Table 7, there was no significant difference in the milk fat and lactose content in colostrum among the experimental groups (p>0.05), indicating that sugarcane leaf silage had little effect on the milk fat and lactose content in colostrum.

[0083] In terms of milk protein content, the milk protein content of Experiment 1 and Experiment 2 was significantly higher than that of the control CK group (p<0.05), and the milk protein content increased with the increase of the addition amount of sugarcane leaf silage containing bacterial agents, but the difference in milk protein content between Experiment 1 and Experiment 2 was not significant (p>0.05), while the milk protein content of Control 1 and Control 2 was lower than that of the control CK group, and the difference between Control 2 and CK was significant (p<0.05), and the difference between Control 1 and CK was not significant (p>0.05). As the addition amount of sugarcane leaf silage without bacterial agents increased, the milk protein content decreased, but the difference between the two groups was not significant (p>0.05).

[0084] In terms of milk non-fat solids, the milk non-fat solids content of Experiment 1 and Experiment 2 was significantly higher than that of the control CK group (p<0.05), and the milk non-fat solids content increased with the increase of the addition of sugarcane leaf silage containing bacterial agents, but the difference in milk non-fat solids content between Experiment 1 and Experiment 2 was not significant (p>0.05), while the milk non-fat solids content of Control 1 and Control 2 was significantly lower than that of the control CK group (p<0.05), and the milk non-fat solids content decreased with the increase of the addition of sugarcane leaf silage without bacterial agents, but the difference between the two groups was not significant (p>0.05).

[0085] In terms of total milk solids, the total milk solids content of Experiment 1 and Experiment 2 was significantly higher than that of the control CK group (p<0.05), and the total milk solids content increased with the increase of the addition of sugarcane leaf silage containing bacterial agents, but the difference in milk non-fat solids content between Experiment 1 and Experiment 2 was not significant (p>0.05), while the total milk solids content of Control 1 and Control 2 was significantly lower than that of the control CK group (p<0.05), and the total milk solids content decreased with the increase of the addition of sugarcane leaf silage without bacterial agents, and the difference between the two groups was significant (p<0.05).

[0086] In terms of the number of diarrhea, the number of diarrhea in Experiment 2 was 0, and the number of diarrhea in Experiment 1 was 1, which were significantly lower than those in the CK group and Control 1 and Control 2.

[0087] The above experimental results show that the sugarcane leaf silage prepared by the composite bacterial agent of the present application can effectively increase the milk protein, total milk solids and milk non-fat solids content in the colostrum of female buffaloes, and relieve diarrhea in female buffaloes.

[0088] In summary, the non-lactose-solubilizing Streptococcus GX-7 screened by the applicant of the present invention has good bile salt tolerance, is suitable for survival in the intestine, and has an antibacterial effect on Pseudomonas fluorescens, Listeria monocytogenes, Salmonella choleraesuis, and Shigella flexneri. Non-lactose-solubilizing Streptococcus GX-7 has good fermentation ability on sugarcane leaves. A composite bacterial agent prepared by non-lactose-solubilizing Streptococcus GX-7 and non-lactose-solubilizing Streptococcus GX-2 is inoculated into sugarcane leaves for ensiling to prepare sugarcane leaf silage. The composite bacterial agent can effectively increase the milk protein, total milk solids, and milk non-fat solids content in the colostrum of female buffaloes, thereby alleviating diarrhea in female buffaloes.

[0089] The above-described embodiments merely illustrate several embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Streptococcus alactolyticus strain GX-7, characterized in that The deposit number of the Streptococcus alactolyticus strain GX-7 is GDMCC NO:65509.

2. A composite bacterial agent comprising the Streptococcus alactolyticus strain GX-7 according to claim 1.

3. The composite bacterial agent according to claim 2, characterized in that The composite bacterial agent further comprises a non-lactose-lytic Streptococcus alactolyticus strain GX-2; the deposit number of the non-lactose-lytic Streptococcus alactolyticus strain GX-2 is GDMCC NO:65508.

4. The composite bacterial agent according to claim 3, characterized in that The composite bacterial agent is prepared by mixing non-lactose-lytic Streptococcus alactolyticus strain GX-7 and non-lactose-lytic Streptococcus alactolyticus strain GX-2 in a mass ratio of 2-1:

1.

5. The composite bacterial agent according to claim 4, characterized in that The composite bacterial agent is prepared by mixing non-lactose-lytic Streptococcus alactolyticus strain GX-7 and non-lactose-lytic Streptococcus alactolyticus strain GX-2 in a mass ratio of 2:

1.

6. Use of the Streptococcus alactolyticus strain GX-7 according to claim 1 or the composite bacterial agent according to claim 2 in preparing sugarcane leaf silage.

7. Use of the Streptococcus alactolyticus strain GX-7 according to claim 1 in producing bile salt-tolerant and / or diarrhea-relieving drugs for female buffaloes.

8. Use of the Streptococcus alactolyticus strain GX-7 according to claim 1 in preparing an antibacterial agent, characterized in that: The pathogenic bacteria inhibited by the bacteriostatic agent are Pseudomonas fluorescens, Listeria monocytogenes, Salmonella Choleraesuis and Shigella flexneri.

9. Silage comprising the Streptococcus alactolyticus strain GX-7 according to claim 1 or the composite bacterial agent according to claim 2, characterized in that: The silage preparation method comprises the following steps: inoculating a non-lactolytic Streptococcus alactolyticus strain GX-7 or a composite bacterial agent into sugarcane leaves at an inoculation rate of 5% by mass and silaging for 35 days to obtain the silage.

10. Use of the silage according to claim 9 in increasing the content of milk protein, total milk solids and / or milk non-fat solids in colostrum of female buffaloes.

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