Probiotic composition, compound fermented feed and application

Abstract

The invention discloses a probiotic composition, a compound fermented feed and application, and belongs to the technical field of livestock and poultry breeding. Comprising leuconostoc mesenteroides and lactococcus, the leuconostoc mesenteroides is named as a leuconostoc mesenteroides LM01 strain and is preserved in China General Microbiological Culture Collection Center on November 21, 2025, the preservation number is CGMCC: 36737, and the lactococcus is named as a lactococcus LC01 strain and is preserved in China General Microbiological Culture Collection Center on November 21, 2025. The strain is preserved in the China General Microbiological Culture Collection Center on November 21, 2025, and the preservation number is CGMCC: 36736. The compound fermented feed disclosed by the invention is fermented by specific compound probiotics, so that the physicochemical properties and the nutritional value of the feed are effectively improved, and the compound fermented feed shows the effects of increasing the laying rate, the daily egg weight and the feed intake in laying hen breeding, and has extremely high popularization and application values.

Description

Technical Field

[0001] This invention belongs to the field of livestock and poultry breeding technology, specifically relating to a probiotic composition, compound fermented feed, and its application. Background Technology

[0002] Currently, the egg-laying hen farming industry faces the dual challenges of improving production efficiency and ensuring product safety. In traditional farming models, antibiotics are often used to maintain the health and production performance of laying hens. However, this leads to food safety and public health risks such as antibiotic residues and bacterial resistance, prompting the industry to seek alternative antibiotic solutions.

[0003] Meanwhile, improving the egg production rate and average daily egg weight of laying hens is key to enhancing the economic benefits of poultry farming. The utilization rate of macromolecular nutrients in ordinary feed is relatively low, making it difficult to maximize the egg production needs of laying hens; therefore, feed utilization efficiency needs to be improved.

[0004] Probiotic fermented feed, as a green and efficient feed processing technology, can improve the palatability of feed and increase the bioavailability of nutrients such as protein and fat through microbial metabolic activities. At the same time, it can optimize the intestinal microecology of animals and help reduce dependence on drugs.

[0005] However, existing technologies lack specific compound probiotic fermentation systems that can significantly improve the production performance of laying hens. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention aims to provide a probiotic composition and compound fermented feed, which has excellent fermentation quality, high nutritional value, and can improve the egg production rate, average daily egg weight and feed intake of laying hens.

[0007] The objective of this invention is achieved through the following technical solutions: A probiotic composition that significantly improves the nutritional quality of feed and greatly enhances the laying hen production performance, wherein the fermented feed includes Leuconostoc mesenteroides (Leuconostoc mesenteroides). Leuconostoc mesenteroides ) and lactococci ( Lactococcus cremoris Among them, Leuconostoc mesenteroides was named Leuconostoc mesenteroides LM01 strain, which was deposited at the China General Microbiological Culture Collection Center on November 21, 2025, with accession number CGMCC:36737. Lactococcus was named Lactococcus LC01 strain, which was deposited at the China General Microbiological Culture Collection Center on November 21, 2025, with accession number CGMCC:36736.

[0008] The present invention also provides a compound fermented feed obtained using the above-mentioned probiotic composition, the preparation method of which includes the following steps: (1) Preparation of compound bacterial solution: After activating Leuconostoc mesenteroides LM01 and Lactococcus lactis LC01, they were inoculated into MRS liquid medium and cultured for 48 hours. Then, the Leuconostoc mesenteroides LM01 and Lactococcus lactis LC01 were mixed to prepare a compound bacterial solution. (2) Substrate preparation: Select corn, soybean meal and wheat bran as fermentation substrates and mix them evenly; (3) Inoculation and mixing: Inoculate the compound fermentation liquid into the substrate and adjust the moisture content to 40%~50% to obtain a mixture; (4) Fermentation: The mixture is placed in a fermentation container and fermented in a sealed container at 30℃~40℃ for 3~7 days to obtain compound fermented feed.

[0009] Preferably, the ratio of Leuconostoc mesenteroides LM01 to Lactococcus lactis LC01 is 1:1.

[0010] Preferably, the ratio of corn, soybean meal and wheat bran in step (2) is 3:1:1.

[0011] Preferably, in step (3), the ratio of fermentation liquid to substrate is 2% (v / w) (i.e., 2L of fermentation liquid is added for every 100kg of substrate).

[0012] The present invention also provides an application of the above-mentioned compound fermented feed, which can be used to improve the production performance of laying hens.

[0013] This invention employs traditional bacterial isolation methods to isolate two dominant bacterial strains from fresh raw milk of healthy dairy cows: *Leuconostoc mesenteroides* strain LM01 and *Lactococcus cremoris* strain LC01. 16S rRNA gene sequencing of both strains revealed that strain LM01 showed a 99% similarity to *Leuconostoc mesenteroides*, and strain LC01 showed a 99% similarity to *Lactococcus cremoris*. A phylogenetic tree was constructed using MEGA 6.0 software. Strain LM01 is listed below. Figure 5 LC01 strain seen Figure 6 .

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The compound fermented feed of this invention, through fermentation with specific compound probiotics, effectively improves the physicochemical properties and nutritional value of the feed, and shows the effect of increasing egg production rate, daily egg weight and feed intake in laying hen farming, and has extremely high promotion and application value.

[0015] Improve feed quality: Excellent sensory characteristics: The fermented feed is yellowish-brown, soft and non-sticky, with a slightly sour and alcoholic taste, and good palatability. Improved nutritional value: After fermentation with probiotics, the crude protein content in the feed increased from 8.90% to 9.91%; the crude fat content increased from 1.4% to 2.6%. The significant increase in crude protein and crude fat content indicates that the fermentation process effectively degraded some indigestible substances, improving nutrient content and bioavailability. Reduced moisture and ash content: The moisture content decreased from 47.4% to 45.5%, and the ash content decreased from 5.6% to 5.3%, which is beneficial for feed storage and quality stability.

[0016] Significantly improves laying hen production performance: Increased egg production rate: Laying hens fed the compound fermented feed of this invention showed a 4.7% increase in egg production rate compared to those fed the ordinary feed group. Increased average daily egg weight: Laying hens fed the compound fermented feed of this invention showed a 6.99g increase in average daily egg weight compared to those fed the ordinary feed group. Increased feed intake: Feeding the fermented compound feed of this invention significantly increased the feed intake of laying hens. Attached Figure Description

[0017] Figure 1 A comparison of the appearance characteristics of fermented feed and unfermented feed; Figure 2 The graph shows the production performance results. Figure 3 Figure showing the effect of fermented feed on the cecal microbial composition of laying hens at the phylum level; Figure 4 Figure showing the effect of fermented feed on the cecal microbial composition of laying hens at the genus level; Figure 5 Phylogenetic tree of strain LM01; Figure 6 This is the phylogenetic tree of strain LC01. Detailed Implementation

[0018] The method of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0019] Example 1 A compound fermented feed, the preparation method includes the following steps: (1) Preparation of compound bacterial solution: After activating Leuconostoc mesenteroides LM01 and Lactococcus lactis LC01, they were inoculated into MRS liquid medium and cultured for 48 hours. Then, Leuconostoc mesenteroides LM01 and Lactococcus lactis LC01 were mixed 1:1 to prepare compound bacterial solution. (2) Substrate preparation: Select corn, soybean meal and wheat bran as fermentation substrates according to a mass ratio of 3:1:1, and mix them evenly; (3) Inoculation and mixing: Inoculate the compound fermentation liquid into the substrate at a ratio of 2% (v / w) of the substrate mass (i.e., add 2L of bacterial solution per 100kg substrate) and adjust the moisture content to 40%~50% to obtain a mixture; (4) Fermentation: The mixture is placed in a fermentation container and fermented in a sealed container at 37°C for 5 days to obtain compound fermented feed.

[0020] Product characteristics: The fermented feed is yellowish-brown in color, soft and non-sticky in texture, can be squeezed into shape but does not form water droplets, and will crumble easily when gently kneaded. It has a slightly sour and slightly alcoholic aroma. Figure 1 The sensory characteristics and conventional nutritional components of the fermented feed are shown in Table 1.

[0021] Table 1. Changes in nutrient composition of fermented feed for future use

[0022] Example 2 Application Trial of Compound Fermented Feed in Laying Hen Farming 1. Experimental Objective The effect of feeding the compound fermented feed of the present invention (prepared according to Example 1) on the laying performance of laying hens was verified.

[0023] 2. Experimental Design Experiment location: Shuangyin Breeding Farm, Pinggu District, Beijing.

[0024] Experimental animals: 120 healthy laying hens of similar age and weight were randomly divided into two groups, with 5 replicates in each group and 12 laying hens in each replicate. Control group (normal feed group): fed with basic normal feed.

[0025] Experimental group (fermented feed group): The feed used to replace 10% of the basal diet with the compound fermented feed of this invention.

[0026] Feeding and management: Two groups of laying hens were raised under the same environmental and management conditions for a period of 6 weeks.

[0027] 3. Experimental Results After the trial period, the production performance and egg quality indicators of the two groups of laying hens were statistically analyzed. The production performance results are shown in the table below. Figure 2 Egg quality indicators are shown in Table 2.

[0028] Table 2 Egg Quality Indicators

[0029] Conclusion: Feeding hens with the compound fermented feed of this invention significantly improves their laying performance. Increased egg production rate: The egg production rate of laying hens in the compound fermented feed group was 4.7% higher than that in the ordinary feed group, indicating that the fermented feed of the present invention can effectively improve the production efficiency of laying hens.

[0030] Increased egg weight: The average daily egg weight produced in the compound fermented feed group was 6.99g higher than that in the ordinary feed group, which directly increased the economic benefits of farming.

[0031] Improved feed intake and production performance: The compound fermented feed group can significantly increase the feed intake of laying hens, which is consistent with the rich sour aroma and excellent palatability of the fermented feed in Example 1, thereby effectively improving the overall production performance of laying hens.

[0032] Egg quality improvement: Compared with the control group, the albumen height and Haugh units of the FF group were significantly improved (P<0.05), indicating that the present invention can effectively improve albumen structure and egg quality. There were no significant differences in average egg weight, yolk color, and shell strength between the two groups (P>0.05).

[0033] Example 3 Analysis of the effects of fermented feed on the cecal microbial community of laying hens This embodiment aims to analyze the changes in the microbial community structure and diversity of the cecal contents of laying hens after feeding them the fermented feed of the present invention using high-throughput sequencing technology, so as to elucidate the mechanism of action of the compound strain of the present invention.

[0034] 1. Sample collection and sequencing methods (1) Sample collection: After the trial period of Example 2, five laying hens were randomly selected from the basal diet group (CK) and the experimental fermented feed group (FF) for slaughter. The cecal contents of each hen were aseptically collected, immediately frozen in liquid nitrogen, and then transferred to a -80°C freezer for storage.

[0035] (2) DNA extraction and sequencing: Total DNA was extracted from the cecal contents using a kit. PCR amplification was performed using universal primers for the V3-V4 variable region of the bacterial 16S rRNA gene. After purification, the amplified products were sequenced using the Illumina NovaSeq platform.

[0036] 2. Community Alpha Diversity Analysis: The Alpha diversity index of each group of samples was calculated by clustering the effective sequences. The results are shown in Table 3.

[0037] Table 3. Alpha diversity index of each sample group

[0038] Conclusions: The ACE and Chao indices of the FF group were slightly higher than those of the CK group, indicating that fermented feed helps improve the richness of the cecal bacterial community. The Shannon index also showed an increasing trend, while the Simpson index decreased, indicating that the evenness and diversity of the bacterial community structure in the FF group were improved. The Coverage values ​​of both groups were close to 1, indicating sufficient sequencing depth and reliable data. The overall results show that the fermented feed described in this invention can optimize the cecal bacterial community structure of laying hens, improve the level of bacterial community diversity, and thus further promote intestinal health.

[0039] 3. The effect of fermented feed on the cecal microbial composition of phylum-level laying hens is shown in [reference needed]. Figure 3 .

[0040] The analysis results showed that the relative abundance of Firmicutes (p_Bacillota) significantly increased from 30.60% to 37.23% in the fermented feed group. Furthermore, the relative abundance of potentially pathogenic phyla significantly decreased: Fusobacteriota (p_Fusobacteriota) and Campylobacterota (p_Campylobacterota) decreased sharply in the fermented feed group (from 0.79% to 0.02% and from 0.60% to 0.08%, respectively). These phyla contain many potentially pathogenic bacteria, and their reduction reflects the antagonistic and inhibitory effects of the compound microbial fermented feed of this invention. Simultaneously, the abundance of Bacteroidetes (p_Bacteroidota) decreased, and the Firmicutes / Bacteroidetes (F / B) ratio increased, which is generally considered a marker of improved digestive function and immune regulation.

[0041] This indicates that feeding the fermented feed of this invention can significantly optimize the composition and structure of the cecal microbiota in laying hens, mainly manifested in a significant increase in the abundance of beneficial bacteria such as Firmicutes and a significant decrease in the abundance of potentially harmful bacteria such as Fusobacterium and Campylobacter. This regulatory effect is an important mechanistic basis for the improved production performance and egg quality observed in Example 5.

[0042] 4. The results of the effect of fermented feed on the cecal microbial composition of laying hens are shown in [the table below]. Figure 4 The analysis results showed that the relative abundance of known short-chain fatty acid (SCFA) producing bacteria, including *Phascolarctobacterium* and *Megasphaera* (belonging to the phylum Firmicutes) and unclassified genera of the family Ruminaceae, all increased in the fermented feed group. SCFA is a major energy source for intestinal epithelial cells, and its increase is beneficial for maintaining intestinal barrier function and nutrient absorption. The abundance of major Bacteroidetes (such as *Bacteroides* and *Rikenellaceae*) decreased, and the potentially pathogenic phyla *Fusobacterium* and *Campylobacterium* were significantly suppressed at the phylum level. This indicates that *Leuconostoc mesenteroides* and *Lactococcus* in the fermented feed successfully altered the intestinal environment and inhibited the growth of harmful bacteria through competitive exclusion and metabolic acid production.

[0043] In summary, the fermented feed of this invention can significantly optimize the cecal microbial community structure of laying hens by increasing the abundance of core beneficial bacteria (such as SCFA-producing bacteria) and inhibiting potentially harmful bacteria, thereby improving the intestinal microecological balance. This regulatory effect is an important mechanism for ensuring the health of laying hens, improving feed utilization efficiency, and enhancing egg quality.

Claims

1. A probiotic composition, characterized in that, It can significantly improve the nutritional quality of feed and greatly enhance the production performance of laying hens, including Leuconostoc mesenteroides (Leuconostoc mesenteroides). Leuconostoc mesenteroides ) and lactococci ( Lactococcus cremoris Among them, Leuconostoc mesenteroides was named Leuconostoc mesenteroides LM01 strain, which was deposited at the China General Microbiological Culture Collection Center on November 21, 2025, with accession number CGMCC:36737. Lactococcus was named Lactococcus LC01 strain, which was deposited at the China General Microbiological Culture Collection Center on November 21, 2025, with accession number CGMCC:36736.

2. The compound fermented feed obtained using the probiotic composition according to claim 1, characterized in that, The preparation method includes the following steps: (1) Preparation of compound bacterial solution: After activating Leuconostoc mesenteroides LM01 and Lactococcus lactis LC01, they were inoculated into MRS liquid medium and cultured for 48 hours. Then, the Leuconostoc mesenteroides LM01 and Lactococcus lactis LC01 were mixed to prepare a compound bacterial solution. (2) Substrate preparation: Select corn, soybean meal and wheat bran as fermentation substrates and mix them evenly; (3) Inoculation and mixing: Inoculate the compound fermentation liquid into the substrate and adjust the moisture content to 40%~50% to obtain a mixture; (4) Fermentation: The mixture is placed in a fermentation container and fermented in a sealed container at 30℃~40℃ for 3~7 days to obtain compound fermented feed.

3. The compound fermented feed according to claim 2, characterized in that, The ratio of Leuconostoc mesenteroides LM01 bacterial solution to Lactococcus LC01 bacterial solution is 1:

1.

4. The compound fermented feed according to claim 2, characterized in that, In step (2), the ratio of corn, soybean meal and wheat bran is 3:1:

1.

5. The compound fermented feed according to claim 2, characterized in that, The volume-to-mass ratio of fermentation broth to substrate is 2%.

6. An application of a compound fermented feed, characterized in that, The compound fermented feed according to any one of claims 2 to 5 can be used to improve the production performance of laying hens.

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