Lactobacillus strain for improving diarrhea in laying hens and application thereof

Abstract

The application discloses a lactic acid bacteria strain for improving diarrhea of laying hens and application of the lactic acid bacteria strain, and belongs to the field of lactic acid bacteria. The Lactobacillus fermentum is screened by the applicant. Compared with common lactic acid bacteria, the strain can well colonize in the intestinal tract of the laying hens, form a bacterial population advantage, and inhibit pathogenic bacteria infection. The strain plays a good role in fermentation of a protein feed composition, effectively improves the bacteriostatic activity of the feed, and has the effects of reducing the diarrhea incidence of the laying hens and improving the egg production rate of the laying hens. The protein feed composition is a lactic acid bacteria strain with excellent effects, and the protein feed composition prepared by using the strain also has good application value.

Description

Technical Field

[0001] This invention relates to the field of lactic acid bacteria, and in particular to a lactic acid bacteria strain that improves diarrhea in laying hens and its application. Background Technology

[0002] In egg-laying hen farming, frequent diarrhea is a key factor affecting profitability. This problem not only directly leads to a significant decrease in egg production but also reduces feed conversion efficiency and significantly increases flock mortality, thus severely impacting overall farming profits. Therefore, effective diarrhea control is crucial for ensuring the health of laying hens and improving production performance and economic efficiency.

[0003] Currently, egg-laying hen farming mainly relies on antibiotics to control pathogenic bacterial infections. However, long-term use can easily lead to drug resistance and drug residues, threatening food safety and public health. Existing research shows that intestinal pathogens in livestock and poultry, such as Salmonella pullorum and pathogenic Escherichia coli, are the main causes of diarrhea. Traditional probiotic preparations have shortcomings such as weak colonization ability and narrow antibacterial spectrum. Therefore, developing new lactic acid bacteria strains with highly efficient antibacterial activity that can improve the intestinal health of egg-laying hens is of great significance for promoting green farming and reducing antibiotic use. Summary of the Invention

[0004] In view of the above, it is necessary to provide a strain of lactic acid bacteria that can improve diarrhea in laying hens, and to use this lactic acid bacteria to prepare protein feed compositions.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention includes lactic acid bacteria, specifically Lactobacillus fermentum, with accession number CGMCC No. 36375. This strain is deposited at the China General Microbiological Culture Collection Center, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on October 28, 2025.

[0006] The present invention also includes a protein feed composition, wherein the mass percentages of each component in the composition are as follows: 28%-32% lactic acid bacteria strain liquid, 43%-47% soybean meal, 13%-17% corn gluten meal, and 8%-12% Bacillus subtilis liquid.

[0007] Furthermore, the concentration of viable bacteria in the lactic acid bacteria solution of the protein feed composition is not less than [amount missing]. CFU / g.

[0008] The present invention also includes the use of the protein feed composition in the preparation of a feed that reduces the incidence of diarrhea in laying hens.

[0009] Compared with the prior art, the advantages of this invention are: The *Lactobacillus fermentum* CGMCC No. 36375 selected in this program was obtained by the applicant through screening. Compared with common lactic acid bacteria, this strain has been verified to colonize well in the intestines of laying hens, forming a dominant flora and inhibiting pathogenic bacterial infection. This strain plays a good role in the fermentation of protein feed composition, effectively improving the antibacterial activity of the feed. Feeding verification shows that this protein feed composition can reduce the incidence of diarrhea in laying hens and increase the egg production rate. It is a high-performance lactic acid bacteria strain, and the prepared protein feed composition has great application value. Attached Figure Description

[0010] Figure 1 This is a developmental tree diagram of the present invention; Figure 2 This is a colony morphology diagram of the strain of the present invention; Figure 3 The results of a comparative test of *Lactobacillus fermentum* of this invention against six common pathogenic bacteria in livestock and poultry farming. Figure 1 ; Figure 4 The results of the comparative test of the fermenting lactobacillus of the present invention against six other common pathogenic bacteria in livestock and poultry farming. Figure 2 . Detailed Implementation

[0011] The technical solutions will now be clearly and completely described with reference to the accompanying drawings in the embodiments of the present invention.

[0012] Unless otherwise specified, when describing the content of a certain substance in this article, it refers to the percentage of the mass of that substance to the total content of the substance.

[0013] This invention, through extensive screening, yielded a lactic acid bacteria strain CGMCC No. 36375 that can improve diarrhea in laying hens. Experiments have demonstrated that this lactic acid bacteria has a significant inhibitory effect on 12 common pathogenic bacteria in livestock and poultry farming (experimental results are available in [link to results]). Figure 3 and Figure 4 Furthermore, this lactic acid bacteria strain CGMCC No. 36375 also has a certain effect in improving diarrhea in laying hens, thereby increasing egg production. For example, the lactic acid bacteria strain of this invention can be used in the preparation of protein feed for laying hens. Therefore, the lactic acid bacteria CGMCC No. 36375 of this invention has broad application prospects.

[0014] In at least some embodiments of the present invention, a protein feed composition is provided, the protein feed composition comprising the above-mentioned lactic acid bacteria strain. In some embodiments, the mass percentage of each component in the above-mentioned protein feed composition includes: 28%-32% lactic acid bacteria bacterial solution, 43%-47% soybean meal, 13%-17% corn gluten meal, and 8%-12% Bacillus subtilis solution; in some embodiments, the preferred composition of the above-mentioned protein feed composition is: 30% lactic acid bacteria bacterial solution, 45% soybean meal, 15% corn gluten meal, and 10% Bacillus subtilis solution; in some embodiments, the viable bacteria concentration in the lactic acid bacteria bacterial solution of the prepared protein feed composition is not less than CFU / g.

[0015] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0016] Example 1: Isolation and screening of Lactobacillus fermentum (CGMCC No. 36375) (1) Sample collection and preprocessing Ten fresh chicken feces samples (1 g each) were collected from an egg-laying hen farm in Tianjin and placed in 15 mL sterile centrifuge tubes. 9 mL of distilled water was added to dilute and suspend the samples. After vortexing for 20 min, the samples were allowed to settle until the solid matter had fully precipitated. The supernatant was then collected for bacterial isolation.

[0017] (2) Isolation and purification of lactic acid bacteria Transfer 1 mL of the supernatant to a 50 mL sterile centrifuge tube, add 9 mL of MRS liquid culture medium, and mix thoroughly. Incubate under anaerobic conditions at 36°C for 16-18 hours. Take 500 μL of the culture medium and... After serial dilution, 100 μL was evenly spread onto the surface of MRS solid medium. Incubate under anaerobic conditions at 36°C (inverted) for 16-18 hours. Select single, milky-white colonies with neat edges (see colony morphology section). Figure 2 Purification was carried out until a pure culture was obtained. After confirmation by Gram staining and microscopic examination, the culture was stored at -80°C in 20% glycerol protectant.

[0018] (3) Preparation of Salmonella Pullorum suspension Freshly cultured Salmonella pullorum was transferred to LB liquid medium at a 2% inoculum (v / v) and incubated at 37°C for 16-18 hours. The final concentration of the bacterial suspension was then adjusted to... CFU / mL, for later use.

[0019] (4) Screening of lactic acid bacteria antagonizing Salmonella pullorum Lactic acid bacteria exhibiting inhibitory activity against *Salmonella pullorum* were screened using the plate-punching method. Activated *Salmonella pullorum* was prepared into a bacterial suspension with a turbidity of 0.5 McFarland units, and 100 μL was evenly spread onto the surface of LB agar. After the agar surface dried, wells were prepared using a sterile 8 mm punch, and 100 μL of purified lactic acid bacteria culture was added to each well. After incubation at 36°C for 24 hours, inhibition zone formation was observed. Lactic acid bacteria strains producing clear inhibition zones were preserved and sent to a sequencing institution for 16S rRNA gene sequence analysis. The lactic acid bacteria rRNA sequencing results are as follows: The sequence obtained from sequencing was entered into the NCBI website and compared with the 16S rRNA sequence in GenBank using BLAST. The lactic acid bacteria obtained was identified as Lactobacillus fermentum. The strain was deposited with the accession number CGMCC No. 36375 at the China General Microbiological Culture Collection Center, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0020] Example 2: Effect test of Lactobacillus fermentum (CGMCC No. 36375). The above-mentioned Lactobacillus fermentum will be referred to as: Lactobacillus fermentum LF18. I. Drug sensitivity test Step 1: Antibiotic susceptibility testing using the paper disc method to determine the inhibition zone. After incubating the test strain overnight at 37°C under anaerobic conditions, the concentration of the bacterial suspension was adjusted to... CFU / mL was evenly spread on the surface of MRS solid medium and allowed to stand at room temperature for 30 min to allow the bacterial culture to be fully absorbed. Then, antibiotic susceptibility testing discs were placed on the surface of the medium and incubated at 37°C under anaerobic conditions for 24 h. The diameter of the inhibition zone was then measured.

[0021] Step 2: In vitro inhibition test of Salmonella pullorum growth (plate punch method) The antibacterial activity of postbiotics was evaluated using the plate-punching method. The indicator bacterium *Salmonella pullorum* was activated and prepared into a bacterial suspension with a turbidity of 0.5 McFarland. 100 μL of the suspension was evenly spread on the surface of LB agar plates. After the plates dried, 8 mm holes were punched in each hole. 100 μL of *Lactobacillus fermentum* broth was added to each hole, and the plates were allowed to stand for 2 h in a clean bench to promote diffusion. The plates were then transferred to a 36℃ incubator for 24 h. The formation of inhibition zones was observed using the cross-hatching method. The diameter of the inhibition zone around each agar well was measured with a ruler, and the average value was taken as the result.

[0022] Step 3, Cell Culture Chicken small intestinal epithelial mucosal cells were cultured in 25 mL cell culture flasks using a nutrient medium containing 10% fetal bovine serum. When the cells reached 80% adhesion, they were digested with 0.25% trypsin. The passaged cells were then seeded into 6-well cell culture plates at a density of [missing information - likely a specific density]. Cells; culture medium without any antibiotics, incubated at 37°C, 5%... 95% humidity Incubate overnight in an incubator.

[0023] Step 4: Analysis of the inhibitory effect of metabiotics on the adhesion ability of Salmonella pullorum in chickens. Overnight cultured cells were washed three times with PBS buffer (pH 7.4). 1 mL of culture medium containing 0.1 mg / mL streptomycin and 200 μL of post-biotic solution were added to each well of the cell culture plate; four groups were set up with post-biotic concentrations of 10, 20, and 40 mg / mL (a control group without post-biotic was also included); 2 h later, 200 μL of Salmonella pullorum suspension was added to each well. CFU / mL). The control group received only culture medium containing streptomycin and Salmonella pullorum suspension. After incubation at 37°C for 6 h, cells were washed three times with PBS buffer (pH 7.4); 200 μL of 0.25% trypsin digestion solution was added to each well, and digestion was carried out for 5 min followed by thorough mixing. The cell suspension was then subjected to... to The colonies of Salmonella pullorum adhering to the bacteria were counted using LB nutrient agar plates after serial dilution.

[0024] Step 5: Analysis of the inhibitory effect of metabiotics on the invasive ability of Salmonella pullorum in chickens. Overnight cultured cells were washed three times with PBS buffer (pH 7.4). 1 mL of culture medium containing 0.1 mg / mL streptomycin and 200 μL of post-biotic solution were added to each well of the cell culture plate. Four groups were set up with post-biotic concentrations of 10, 20, and 40 mg / mL (a control group without post-biotic was also included). After 2 h, 200 μL of Salmonella pullorum suspension was added to each well. (CFU / mL); the control group only added culture medium containing streptomycin and Salmonella pullorum suspension; after culturing at 37℃ for 6 h, the cells were washed 3 times with PBS buffer (pH 7.4); 1 mL of amikacin solution with a concentration of 200 μg / mL was added to each well and co-cultured with the cells for 20 min to kill extracellular bacteria; then, the Salmonella bacteria that invaded the cells were counted according to the method described in "Step 2, In vitro inhibition of Salmonella pullorum growth test (plate punching method)".

[0025] II. Test Results (1) Screening of lactic acid bacteria that inhibit Salmonella pullorum by Lactobacillus fermentum strain LF18 The antibacterial activity of suspected lactic acid bacteria was detected by the perforation diffusion method to screen for potential superior strains that inhibit Salmonella pullorum, providing a basis for further research. The results of the antibacterial experiment are shown in Table 1. After screening, strain P15 was found to have a significant inhibitory effect on Salmonella pullorum, with an inhibition zone diameter of (22.5±0.8) mm. Therefore, this strain was selected as the research object for subsequent experiments.

[0026] Table 1. Results of in vitro antibacterial activity of lactic acid bacteria against Salmonella pullorum.

[0027] Note: "-" indicates no antibacterial effect. (2) Results of drug sensitivity test The antibiotic susceptibility test results of *Lactobacillus fermentum* LF18 are shown in Table 2. The results indicated that this strain exhibited resistance to four antibiotics: ciprofloxacin, vancomycin, levofloxacin, and polymyxin B; susceptibility to nine antibiotics, including ampicillin, gentamicin, and streptomycin; and intermediate resistance to tetracycline. These results suggest that *Lactobacillus fermentum* LF18 possesses good safety characteristics.

[0028] Table 2. Antibiotic susceptibility results of Lactobacillus fermentum LF18

[0029] Note: R: drug resistance; I: intermediate; S: sensitive.

[0030] (3) Results of the test on external inhibition of Salmonella pullorum growth in chickens As shown in Table 3, the inhibition zone at the round hole numbered 1 represents the antibacterial activity of the fermentation supernatant of Lactobacillus fermentum LF18, and its diameter is (16.3±0.4) mm; the inhibition zone at the round hole numbered 2 represents the antibacterial effect of the fermentation supernatant of Lactobacillus fermentum LF18 after being inactivated by heat treatment at 80℃, and its diameter is (15.5±0.7) mm.

[0031] Table 3. Results of in vitro antibacterial activity against Salmonella pullorum LF18 postbiotic in chickens.

[0032] (4) Lactobacillus fermentum strain LF18 inhibits the adhesion and invasion of Salmonella pullorum in chicken small intestinal mucosal epithelial cells. As shown in Table 4, compared with the control group, samples treated with different concentrations of fermentation broth all showed significant inhibitory effects on the ability of Salmonella pullorum to adhere to the small intestinal mucosal epithelial cells of chickens (P<0.05). With decreasing fermentation broth concentration, its inhibitory effect on bacterial adhesion gradually weakened, leading to a corresponding increase in the number of adhered bacteria; among them, the postbiotic treatment group at a concentration of 40 mg / mL showed the most significant inhibitory effect, reducing the number of adhered bacteria from 2.0 × 10⁻⁶ to 1.5%. CFU / pore decreased to approximately 0.6× CFU / well.

[0033] Table 4. Effects of different concentrations of Lactobacillus fermentation LF18 broth on inhibiting the adhesion of Salmonella pullorum to chicken small intestinal epithelial cells.

[0034] Note: Different letters indicate significant differences between data points, P < 0.05 As shown in Table 5, Salmonella pullorum possesses the ability to invade and colonize the mucosal epithelial cells of chicken small intestine. Treatment with different concentrations of fermentation broth significantly reduced the number of Salmonella pullorum in the cells (P<0.05); the 40 mg / mL fermentation broth treatment group showed the most significant antibacterial effect, with the bacterial count decreasing from 2.0 × 10^7 CFU / well to approximately 4.7 × 10^4 CFU / well.

[0035] Table 5. Effects of different concentrations of Lactobacillus fermentation LF18 broth on Salmonella pullorum invasion of chicken small intestinal epithelial cells.

[0036] (5) Adhesion of Lactobacillus fermentum strain LF18 to chicken small intestinal epithelial mucosal cells The data in Table 6 show that, within a suitable range, the higher the content of LF18 strain, the stronger its adhesion to the epithelial mucosal cells of the chicken small intestine, and the greater the number of adherent bacteria.

[0037] Table 6. Adhesion of different amounts of Lactobacillus fermentum to chicken small intestinal mucosal epithelial cells.

[0038] Note: Different letters indicate significant differences (P < 0.01), the same applies below.

[0039] (6) Competitive effect of Lactobacillus fermentum strain LF18 on the adhesion of pathogenic bacteria to chicken small intestinal epithelial mucosal cells As shown in Tables 7 and 8, within a certain concentration range, the higher the content of Lactobacillus fermentum LF18 strain, the more significant its competitive inhibitory effect on pathogens; in particular, the competitive inhibitory effect on pathogenic Escherichia coli is significantly stronger than that on Salmonella; when pathogenic Escherichia coli or Salmonella are co-cultured with LF18 strain and chicken small intestinal mucosal epithelial cells, the adhesion ability of both pathogens is significantly reduced compared with the control group (P<0.01); in addition, it was found that co-culturing with the above pathogens also inhibits the adhesion ability of LF18 strain itself.

[0040] Table 7. Number and competition rate of Salmonella adhering to chicken small intestinal mucosal epithelial cells under different contents of Lactobacillus fermentum LF18 strain.

[0041] Table 8. Number and competition rate of pathogenic Escherichia coli adhering to chicken small intestinal mucosal epithelial cells under different contents of Lactobacillus fermentum LF18 strain.

[0042] (7) The repulsive effect of Lactobacillus fermentum strain LF18 on pathogenic bacteria adhering to chicken small intestinal epithelial mucosal cells According to the results shown in Tables 9 and 10, the rejection rates of pathogenic Escherichia coli and Salmonella by higher concentrations of Lactobacillus fermentum LF18 reached 89.28% and 88.71%, respectively, with an average rejection rate of approximately 43%. As the concentration of the strain decreased, its adhesion and rejection effect on the cells of the above-mentioned pathogenic bacteria significantly weakened. In addition, the adhesion ability of Lactobacillus fermentum LF18 itself to the epithelial mucosal cells of chicken small intestine also decreased accordingly.

[0043] Table 9. Number and rejection rate of Salmonella adhering to chicken small intestinal mucosal epithelial cells under different contents of Lactobacillus fermentum LF18 strain.

[0044] Table 10. Number and rejection rate of pathogenic Escherichia coli adhering to chicken small intestinal mucosal epithelial cells under different contents of Lactobacillus fermentum LF18 strain.

[0045] (8) The displacement effect of Lactobacillus fermentum strain LF18 on the adhesion of pathogenic bacteria to chicken small intestinal epithelial mucosal cells As shown in Tables 11 and 12, high concentrations of *Lactobacillus fermentum* LF18 strain exhibited a strong displacement effect on Salmonella cell adhesion, with a displacement rate of up to 70.34%. Its displacement effect on pathogenic *Escherichia coli* cells was even more significant, reaching a displacement rate of 87.21%. However, under low concentrations of *Lactobacillus fermentum* LF18, the adhesion rate of Salmonella cells was not inhibited by displacement; instead, it increased. These results indicate that the inhibitory effect of *Lactobacillus fermentum* LF18 on the adhesion of pathogenic *Escherichia coli* and Salmonella is concentration-dependent, and the strain's own adhesion ability also weakens as the concentration decreases.

[0046] Table 11. Number and replacement rate of Salmonella adhering to chicken small intestinal mucosal epithelial cells under different concentrations of Lactobacillus fermentum LF18 strain.

[0047] Table 12. Number and replacement rate of pathogenic Escherichia coli adhering to chicken small intestinal mucosal epithelial cells under different contents of Lactobacillus fermentum LF18 strain.

[0048] like Figure 3 and Figure 4 As shown, the left well is the control group, and the right well is the experimental group of fermentation broth of Lactobacillus fermentum strain LF18. The experimental results show that Lactobacillus fermentum strain LF18 exhibits significant antibacterial activity against 12 common pathogenic bacteria in livestock and poultry farming.

[0049] Example 3: Application of Lactic Acid Bacteria (CGMCC No. 36375) in the Preparation of Protein Feed Compositions This embodiment provides a protein feed composition, which is obtained by mixing different compositions in a certain proportion. Specifically, the composition consists of: 30% fermentation broth of lactic acid bacteria CGMCC No. 36375, 45% soybean meal, 15% corn gluten meal, and 10% Bacillus subtilis broth, wherein the viable bacteria concentration in the lactic acid bacteria broth is not less than 5.0 × 10⁻⁶. CFU / g.

[0050] The specific preparation method is as follows: after mixing the components evenly according to the proportion, ferment at a constant temperature of 37℃ for 24 hours, during which the aeration rate is maintained at 0.5 vvm and the pH is maintained at 5.8±0.2; after fermentation, dry at a low temperature of 45℃ until the moisture content is ≤10%, and crush through a 40-mesh sieve to obtain the finished protein feed composition.

[0051] In the formulated diet of laying hens, this protein feed composition can replace 10-20% of the recommended basic soybean meal in equal proportions.

[0052] (1) Feeding experiments were conducted in a laying hen farm using the above-mentioned protein feed.

[0053] Experimental grouping: 300 healthy Hy-Line Brown laying hens aged 28 weeks were randomly divided into an experimental group and a control group, with 150 birds in each group. The control group was fed a basal diet, while the experimental group was fed a basal diet supplemented with 400 g / t of the protein feed composition for 28 days. All groups were caged in the same environment (temperature 22±2℃, humidity 60%), with free access to feed and water, and other management conditions were the same.

[0054] Production performance: Daily feed intake (kg / d) and egg production (eggs / day) for each group were recorded, and the average daily feed intake and egg production rate were calculated.

[0055] Diarrhea rate: Observe the feces of laying hens daily and record the number of diarrhea episodes (diarrhea criteria: feces are watery or loose). Diarrhea incidence rate (%) = (total number of diarrhea episodes / (number of days in the experiment × number of chickens)) × 100%.

[0056] (2) Experimental results After 28 days of feeding, the experimental group was significantly better than the control group in terms of diarrhea rate, egg production and egg quality (P<0.05), and the specific data are shown in Table 13.

[0057] Table 13 Effects of Lactobacillus fermentum LF18 protein diet on laying hen production performance

[0058] *Note: Indicates a significant difference compared to the control group (P<0.05); data are mean ± standard deviation.

[0059] As shown in Table 13, the average daily feed intake of the experimental group increased by 4.3% (P<0.05) and the egg production rate increased by 5.0% (P<0.05) compared with the control group, indicating that the protein feed prepared by Lactobacillus fermentum LF18 can improve the appetite and egg production efficiency of laying hens.

[0060] The incidence of diarrhea in the experimental group was only 0.8%, significantly lower than the 5.2% in the control group (P<0.05), which is related to the antibacterial mechanism of *Lactobacillus fermentum* LF18. *Lactobacillus fermentum* LF18 can effectively colonize the intestines of laying hens, inhibiting pathogenic bacterial infection through competitive exclusion and antibacterial effects; its antibacterial effect has been confirmed in the in vitro experiments of Example 2. Simultaneously, the protein composition substrate (soybean meal and corn flour) provides a suitable growth environment for *Lactobacillus fermentum* LF18, and this protein feed composition has an effect on improving diarrhea.

[0061] In conclusion, through a 28-day feeding experiment, the protein feed composition prepared by Lactobacillus fermentum (CGMCC No. 36375) can effectively reduce the incidence of diarrhea in laying hens, improve egg production rate and egg quality, thus proving its practical value in laying hen farming.

[0062] The above description is merely a preferred embodiment of the present invention; it encompasses all the protection scope of the present invention. Any equivalent substitutions or modifications made by those skilled in the art within the technical scope disclosed in the present invention, based on the technical solutions and improved concepts of the present invention, should be covered within the protection scope of the present invention.

Claims

1. A lactic acid bacteria strain that improves diarrhea in laying hens, characterized by: The lactic acid bacteria is classified as Lactobacillus fermentum and is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 36375.

2. A protein feed composition, characterized in that: Includes the bacterial solution of the lactic acid bacteria as described in claim 1.

3. The protein feed composition according to claim 2, characterized in that: The mass percentages of each component in the composition include: 28%-32% lactic acid bacteria bacterial solution, 43%-47% soybean meal, 13%-17% corn gluten meal, and 8%-12% Bacillus subtilis solution.

4. The protein feed composition according to claim 3, characterized in that: The concentration of viable bacteria in the bacterial solution of the lactic acid bacteria is not less than CFU / g.

5. The use of the protein feed composition as described in claim 2 in the preparation of products that reduce the incidence of diarrhea in laying hens.

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