CN117229984B - A pig-derived animal combined lactobacillus and its application - Google Patents

Abstract

The invention provides a swine animal-derived combined lactobacillus and application thereof. The collection number of the pig source animal combined lactobacillus ZLA031 is CGMCC No.26855; the bacterium has good acid resistance, cholate resistance, bacteriostasis and lactic acid production characteristics. The invention also discloses a fermentation preparation containing the animal combined lactobacillus ZLA031, and the preparation is used as a feed additive for feeding growing pigs, can improve the intestinal health of the pigs, reduce the nitrogen emission and harmful bacteria emission in the feces, has the effects of enhancing the immunity of organisms, improving the production performance and reducing the environmental pollution, promotes the emission reduction and the efficiency improvement of the pig industry, and has better economic value.

Description

A pig-derived animal combined lactobacillus and its application

Technical field

The invention belongs to the technical field of microorganisms, and specifically relates to a pig-derived animal-associated Lactobacillus and its application.

Background technique

At present, pig breeding is mainly based on a relatively single feed structure of corn-soybean meal. Corn is the main energy raw material in the feed, and soybean meal is the main protein raw material. The protein content in the feed is usually used to evaluate its nutritional level. In recent years, with the rapid growth of large-scale pig breeding, its breeding volume accounts for about half of the world's total stock, requiring a large amount of feed grain every year. In response to the current problems of protein feed shortage and serious nitrogen emission pollution in the pig industry, the Ministry of Agriculture and Rural Affairs has proposed vigorously promoting the reduction and substitution of corn and soybean meal. Technologies such as low-protein diets and lactic acid bacteria fermented feed have received widespread attention. How to make low-protein diets maintain animal production performance without degrading while improving the utilization rate of nitrogen nutrients, thereby reducing production costs and nitrogen emissions, and further promoting emission reduction and efficiency improvement in the pig industry, is a technology that needs to be solved question.

Lactobacillus animalis ( Ligilactobacillus animalis ), formerly known as Lactobacillus animalis ( Lactobacillus animalis ), is a kind of lactic acid bacteria that exists in fermented foods and animal intestines or feces. It has antibacterial, anti-allergic, improved obesity and lowered cholesterol, etc. effect. The current patents related to Lactobacillus conjunctiva are: Application Announcement No. CN 115024382 A, titled "An anti-animal diarrhea Lactobacillus conjunctiva ZJUIDS-R2 and its application". This bacterium was isolated from the feces of healthy pet rabbits and has relatively high Strong bile salt hydrolase activity can be used to prevent pet diarrhea; and the patent application publication number CN 115887507 A, titled "Application of Lactobacillus animalis in the preparation of anti-tumor drugs", involves a strain of Lactobacillus animalis ATCC35046 in Application in the preparation of anti-melanoma drugs. In addition, the application announcement number CN112753875 A, titled "A low-protein and antibiotic-free fattening pig feed", discloses a feed containing Bacillus subtilis fermented paper mulberry, compound enzyme preparation, organic iron compound and antibiotic replacement core combination material, etc. Low protein antibiotic-free fattening pig feed formula. There are currently no reports involving the preparation of pig-derived animal combined Lactobacillus preparations and their application in pig production with low-protein diets.

Contents of the invention

The object of the present invention is to provide a pig-derived animal-associated Lactobacillus and its application. The animal-associated Lactobacillus is a strain with excellent performance selected from healthy growing pigs fed with low-protein feed, and has strong acid resistance and bile salt resistance. , strong adhesion, high lactic acid production and other characteristics, it has a certain inhibitory effect on common pig pathogenic bacteria and is sensitive to a variety of antibiotics.

In order to achieve the above object, the present invention adopts the following technical solutions:

A kind of pig-derived animal combined Lactobacillus, the Lactobacillus strain was collected in the General Microbiology Center of China Committee for the Collection of Microbial Cultures (No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on March 22, 2023) Postcode: 100101) for preservation, the preservation name is Ligilactobacillus animalis (ZLA031), and the preservation number is: CGMCC No. 26855.

The porcine-derived Lactobacillus animalis (CGMCC No. 26855) is a Gram-positive bacillus, arranged singly or in pairs; the colonies on the MRS plate culture medium are white, round, with a moist, opaque surface and neat edges.

Furthermore, the pig-derived animal-associated Lactobacillus (CGMCC No. 26855) also has biological properties such as acid resistance, bile salt resistance, strong adhesion, good bacteriostasis, and high lactic acid production:

1) The survival rate of this animal combined with Lactobacillus in an acidic environment of pH=3 is 70.34% for 2 hours and 72.03% for 4 hours; in an acidic environment of pH=4, the survival rate for 2 hours is 70.34%. The rate was 90.68%, and the survival rate after 4 hours of action was 107.63%.

2) The survival rate of this animal combined with Lactobacillus in a 0.05% bile salt environment was 145.46% for 2 hours and 177.62% for 4 hours; in a 0.1% bile salt environment, the survival rate for 2 hours The rate was 123.08%, and the survival rate after 4 hours of action was 111.89%.

3) The animal combined Lactobacillus has strong adhesion to caco-2 cells; the porcine animal combined Lactobacillus and caco-2 cells were co-cultured for 2 hours, and the adhesion index was 7.5 ± 0.3 bacteria/cell.

4) This animal combined lactobacillus has a strong inhibitory effect on three common pig pathogenic bacteria (pig-derived Escherichia coli, Salmonella, and Staphylococcus aureus), and can effectively control the occurrence of animal diseases.

5) The animal combined with Lactobacillus has a strong ability to produce lactic acid, and the lactic acid production reached 168.53mmol/L after being cultured in MRS liquid medium for 32 hours.

Application of the above-mentioned pig-derived animal combined lactobacilli in the preparation of fermented feed additives.

A fermentation preparation. Preferably, the fermentation preparation contains the pig-derived animal-associated Lactobacillus as described above.

The preparation method of the fermentation preparation as described above, which includes inoculating pig-derived animals with Lactobacillus and performing fermentation culture in a liquid medium, wherein the carbon source in the liquid medium is lactose, the nitrogen source is beef peptone, and the carbon-nitrogen ratio is 1 :1. The other components are the same as the MRS medium, the culture temperature is 33°C, and the culture time is 16 to 28 hours, preferably 24 hours.

Further, the formula of the liquid culture medium is lactose 20g/L, beef peptone 20g/L, sodium acetate 5g/L, K ₂ HPO ₄ •3H ₂ O 2g/L, diammonium hydrogen citrate 2g/L, MgSO ₄ •7H ₂ O 0.58g/L, MnSO ₄ 0.19g/L, Tween-80 1ml/L, pH 6.5.

A feed additive. Preferably, the feed additive is an animal-associated Lactobacillus preparation containing the pig-derived animal-associated Lactobacillus as described above.

The preparation method of the feed additive as described above includes inoculating the pig-derived animal combined Lactobacillus and fermenting it in a complete feed.

Further, the complete feed is a complete feed for growing pigs, and the complete feed is mixed with water at a weight ratio of 1:1. The inoculation amount of pig-derived animal combined Lactobacillus is 5.5×10 ⁸ CFU per kg of growing pig complete feed. The fermentation temperature is 34.5°C, the culture time is 24 hours, and the fermentation is sealed.

As mentioned above the application of feed additives in the pig industry.

As mentioned above, it is preferable to feed pigs with low protein diet and feed additives.

Further, the feed additive is 0.1% of the low-protein diet;

The low-protein diet has a crude protein content of 12.9%, and the feed additive contains a total effective viable bacterial count of more than 2.5×10 ⁹ CFU/g.

The beneficial effects of the present invention are:

The pig-derived animal-associated Lactobacillus ( Ligilactobacillus animalis ) ZLA031 (CGMCC No. 26855) provided by the invention is a strain with acid resistance, bile salt resistance, strong adhesion and good antibacterial properties. The fermented feed made from the animal combined with Lactobacillus was used as an additive (the final concentration of live bacteria was 2.5×10 ⁹ CFU/g) and was added to the low-protein feed of growing pigs at an amount of 0.1%. Compared with conventional protein content feed, The daily weight gain of growing pigs increased by 12.13%, the feed-to-weight ratio decreased by 9.99%; fecal nitrogen decreased by 31.19%, urinary nitrogen decreased by 14.70%, total nitrogen emissions decreased by 22.49%, and apparent nitrogen digestibility increased by 6.81%; The number of viable lactic acid bacteria in the feces increased by 171.15%, the number of Escherichia coli decreased by 74.14%, and the number of Staphylococcus aureus decreased by 71.02%; the serum total protein, globulin and IgG contents were significantly increased, and the serum aspartate aminotransferase, glutamic acid transaminase, and glutamic acid levels were significantly reduced. Alanine aminotransferase activity and urea nitrogen content. It can be seen that the animal combined Lactobacillus ZLA031 is fed as a feed additive to growing pigs, which can inhibit the reproduction of harmful bacteria, regulate the balance of intestinal microecology, enhance the body's immunity, improve production performance and reduce fecal nitrogen emissions.

On the basis of low-protein diet, the use of pig-derived animal combined Lactobacillus ZLA031 preparation can help improve the nutritional value of feed and the digestion and absorption of nutrients, improve the growth performance of animals, optimize the breeding environment, and promote the reduction of pig industry. It improves efficiency and has good economic value.

Description of the drawings

Figure 1 shows the colony morphology of ZLA031.

Figure 2 shows the cell morphology of Lactobacillus animalis ZLA031.

Figure 3 shows the results of lactic acid production by Animal Lactobacillus ZLA031.

Figure 4 shows the screening results of carbon sources in the culture conditions of Lactobacillus animalis ZLA031.

Figure 5 shows the screening results of nitrogen sources in the culture conditions of Lactobacillus animalis ZLA031.

Figure 6 shows the screening results of the carbon-nitrogen ratio in the culture conditions of Lactobacillus animalis ZLA031.

Figure 7 shows the screening results of culture temperature in the culture conditions of Lactobacillus animalis ZLA031.

Figure 8 shows the screening results of the culture time in the culture conditions of Lactobacillus animalis ZLA031.

Detailed ways

The probiotic function of lactic acid bacteria is closely related to the biological characteristics of the strain. Screening strains with excellent biological properties and exploring their intrinsic probiotic functions are currently hot topics in the research and development of lactic acid bacteria. Studies have found that lactic acid bacteria have potential nitrogen fixation effects and can reduce dietary nitrogen losses. Therefore, isolating, identifying and screening porcine lactobacillus strains from the intestinal tract of healthy pigs and preparing pig lactobacillus preparations with nitrogen-fixing functions are of great application value in promoting the digestion and absorption of nutrients and reducing protein feed consumption and nitrogen emissions. and market prospects. After a large number of experiments, the inventor screened and obtained a strain of Lactobacillus animalis ( Ligilactobacillus animalis ) ZLA031 (CGMCC No. 26855), which is a Gram-positive bacillus, arranged singly or in pairs, and its colonies on the MRS plate culture medium are white. , round, with a moist surface, opaque, and neat edges. It also has biological properties such as acid resistance, bile salt resistance, strong adhesion, and good antibacterial properties. The fermented feed using this lactic acid bacteria preparation in growing pigs can inhibit the proliferation of harmful bacteria, improve intestinal health, reduce nitrogen emissions and harmful bacteria emissions in feces, and can enhance the body's immunity, improve production performance, and reduce environmental pollution.

The following examples are used to further illustrate the present invention, but should not be understood as limiting the present invention. Without departing from the spirit and essence of the present invention, any modification or substitution made to the present invention shall fall within the scope of the present invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents used in the examples are all of analytical grade or above.

Example 1 Isolation, screening and identification of Lactobacillus combined with animals

From the fresh feces of growing pigs fed low-protein feed, perform aseptic operation, weigh an appropriate amount and add it to sterile saline, shake evenly and make ten-fold gradient dilution, spread on MRS culture medium plate, anaerobic tank method Incubate at 37°C for 24 to 48 hours. Select milky white colonies for preliminary screening. All strains that are Gram stain positive, H ₂ O ₂ enzyme test negative, and contact enzyme negative can be preliminarily determined to be lactic acid bacteria. After the 18 strains of bacteria obtained from the preliminary screening were activated respectively, they were separated by volume ratio. Inoculate 0.1% of the inoculum into liquid MRS medium containing 0.1% bile salts and pH=3. After incubation at 37°C for 6 hours, viable bacteria were counted to observe the acid and bile salt resistance of the strains. The results are as follows in Table 1.

Table 1 Screening results of acid and bile salt resistance of lactic acid bacteria

strain number Viable bacterial count (CFU/mL) strain number Viable bacterial count (CFU/mL) 1 5.2×10 ⁷ 10 6.55×10 ⁶ 2 4.5×10 ⁷ 11 2.33×10 ⁸ 3 6.75×10 ⁷ 12 7.85×10 ⁷ 4 1.32×10 ⁶ 13 2.1×10 ⁷ 5 5.3×10 ⁸ 14 2.03×10 ⁷ 6 9.45×10 ⁷ 15 9.5×10 ⁶ 7 2.47×10 ⁸ 16 8.35×10 ⁵ 8 9.5×10 ⁵ 17 2.6×10 ⁷ 9 3.5×10 ⁸ 18 1.05×10 ⁸

It can be seen from the results that these 18 strains of lactic acid bacteria have different tolerances to 0.1% bile salt and pH=3, indicating that there are large differences in characteristics between different strains. According to the screening results, the strain numbered 5 has the highest number of viable bacteria, indicating that it has excellent acid and bile salt resistance. It was named ZLA031 and sent to the China Industrial Microbial Culture Collection Center (CICC) for cell morphological characteristics, Identification of physiological and biochemical characteristics and 16S rRNA sequence. The primers for molecular biology PCR amplification (27F, 1492R) are: 27F (SEQ ID NO.1: 5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (SEQ ID NO.2: 5’-TACGACTTAACCCCAATCGC-3’).

After identification, ZLA031 bacteria were Gram-positive bacilli, arranged singly or in pairs. The colonies on the MRS plate medium are white, round, with a moist, opaque surface and neat edges. The colony characteristics are shown in Figure 1, the cell morphology is shown in Figure 2, the physiological and biochemical characteristics are shown in Table 2, and the 16S rRNA sequence is shown in SEQ ID NO.3. The bacterium was finally identified as Ligilactobacillus animalis .

Table 2 Physiological and biochemical characteristics of Lactobacillus combined animalis ZLA031

Note: “+” means positive; “–” means negative.

SEQ ID NO.3:

AACGAAACTT CTTTATCACC GAGTGCTTGC ACTCACCGAT AAAGAGTTGA

GTGGCGAACG GGTGAGTAAC ACGTGGGCAA CCTGCCCAAA AGAGGGGGAT

AACACTTGGA AACAGGTGCT AATACCGCAT AACCATAGTT ACCGCATGGC

AACTATGTAAAAGGTGGCTATGCTACCGCTTTTGGATGGG CCCGCGGCGC

ATTAGCTAGT TGGTGAGGTA AAGGCTTACC AAGGCAATGA TGCGTAGCCG

AACTGAGAGG TTGATCGGCC ACATTGGGAC TGAGACACGG CCCAAACTCC

TACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGG

AGCAACGCCG CGTGGGTGAA GAAGGTCTTC GGATCGTAAA ACCCTGTTGT

TAGAGAAGAA AGTGCGTGAG AGTAACTGTT CACGTTTCGA CGGTATCTAA

CCAGAAAGCC ACGGCTAACT ACGTGCCAGC AGCCGCGGTA ATACGTAGGT

GGCAAGCGTT ATCCGGATTT ATTGGGCGTA AAGGGAACGC AGGCGGTCTT

TTAAGTCTGA TGTGAAAGCC TTCGGCTTAA CCGGAGTAGT GCATTGGAAA

CTGGGAGACT TGAGTGCAGA AGAGGAGAGT GGAACTCCAT GTGTAGCGGT

GAAATGCGTA GATATATGGA AGAACACCAG TGGCGAAAGC GGCTCTCTGG

TCTGTAACTG ACGCTGAGGT TCGAAAGCGT GGGTAGCAAA CAGGATTAGA

TACCCTGGTA GTCCACGCCG TAAACGATGA ATGCTAAGTG TTGGAGGGTT

TCCGCCCTTC AGTGCTGCAG CTAACGCAAT AAGCATTCCG CCTGGGGAGT

ACGACCGCAA GGTTGAAACT CAAAGGAATT GACGGGGGCC CGCACAAGCG

GTGGAGCATG TGGTTTAATT CGAAGCAACG CGAAGAACCT TACCAGGTCT

TGACATCTTC TGACAATCCT AGAGATAGGA CTTTCCCTTC GGGGACAGAA

TGACAGGTGG TGCATGGTTG TCGTCAGCTC GTGTCGTGAG ATGTTGGGTT

AAGTCCCGCA ACGAGCGCAA CCCTTATTGT TAGTTGCCAG CATTAAGTTG

GGCACTCTAG CAAGACTGCC GGTGACAAAC CGGAGGAAGGTGGGGATGAC

GTCAAATCAT CATGCCCCTT ATGACCTGGG CTACACACGT GCTACAATGG

ACGGTACAAC GAGTCGCAAG ACGCGAGGT TTAGCAAATC TCTTAAAGCC

GTTCTCAGTT CGGATTGTAG GCTGCAACTC GCCTACATGA AGTCGGAATC

GCTAGTAATC GCGGATCAGC ATGCCGCGGT GAATACGTTC CCGGGCCTTG

TACACACCGC CCGTCACACC ATGAGAGTTT GTAACACCCA AAGCCGGTGG

GGTAACCTTT T

This lactobacillus strain was collected on March 22, 2023 at the General Microbiology Center of the China Microbial Culture Collection Committee (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) Patent deposited, the deposited strain is ZLA031, the classification name is Ligilactobacillus animalis , and the deposit number is CGMCC No. 26855.

Among them, MRS solid medium formula: 1g peptone, 1g beef extract, 0.5g yeast extract, 0.2g hydrogen citrate diamine, 2g glucose, KH ₂ P0 ₄ 0.2g, 0.2g sodium acetate, Tween-80 0.1ml, 0.058g magnesium sulfate, 0.02g manganese sulfate, 2g agar, 100m1 water, pH 6-6.5, autoclave at 121°C for 15 minutes.

Example 2 Study on the characteristics of Lactobacillus combined with animals

1. Determination of acid resistance

The Lactobacillus animalis ( Ligilactobacillus animalis ) ZLA031 obtained in Example 1 after activation twice was inserted into the liquid MRS culture medium with a pH of 3 and 4 at an inoculum volume ratio of 1%, 37 °C for 2 and 4 hours respectively, with 0 hour as the control, determine the number of viable bacteria, and calculate the survival rate according to the formula: survival rate (%) = number of viable bacteria after different hours of action/number of viable bacteria at 0 hour × 100, The results are shown in Table 3. It can be seen from the test results that the survival rate of this animal combined with Lactobacillus in an acidic environment of pH=3 for 2 hours is 70.34%, and the survival rate of 4 hours is 72.03%; The survival rate was 90.68%, and the 4-hour survival rate was 107.63%. The above results show that this animal combined Lactobacillus can tolerate acidic environments with pH of 3 and 4, and has strong tolerance to the gastric acid of animals.

Table 3 Survival rate (%) of Lactobacillus lactobacillus combined with gastric acid

2. Determination of bile salt resistance performance

The Lactobacillus lactobacillus ZLA031 after activation twice was inserted into the liquid MRS medium containing 0.05%, 0.1% and 0.2% porcine bile salts at a volume ratio of 1% to simulate the animal intestinal environment at 37°C. After 2 hours and 4 hours of action, with 0 hour as the control, the survival rate was calculated according to the formula: survival rate (%) = number of viable bacteria after different hours of action/number of viable bacteria at 0 hours × 100. The test results are shown in Table 4. It can be seen from the results that this animal combined with Lactobacillus ZLA031 can tolerate 0.05% and 0.1% bile salts, and the survival rate exceeds 100% at 2 hours and 4 hours. However, the survival rate drops sharply at a bile salt concentration of 0.2%. decline. The above results show that this animal combined with Lactobacillus can tolerate a 0.1% bile salt environment and has strong tolerance to the animal's intestinal bile salts.

Table 4 Survival rate of Lactobacillus combined with bile salts (%)

project 0 hours 2 hours 4 hours 0.05% 100 145.46±3.45 177.62±4.61 0.10% 100 123.08±2.43 111.89±4.02 0.20% 100 2.80±0.04 1.40±0.03

3. Antibacterial test

The Oxford cup method was used to detect the antibacterial effect of Lactobacillus animalis ZLA031 culture solution on three common swine pathogenic bacteria (E. coli, Salmonella cholera, and Staphylococcus aureus). It can be seen from the results in Table 5 that the diameters of the inhibition zones of the animal combined Lactobacillus ZLA031 against swine-derived Escherichia coli, Salmonella cholera, and Staphylococcus aureus are 31, 25, and 26.1 mm respectively, indicating that it has a strong antibacterial effect. .

Table 5 Animal combined Lactobacillus antibacterial test

Pathogens Inhibition zone diameter (mm) Sensitivity determination E. coli (C838901) 31.0±0.3 sensitive Salmonella cholera (C78-4) 25.0±0.2 sensitive Staphylococcus aureus (C56023) 26.1±0.2 sensitive

4. Adhesion test

The adhesion ability of lactic acid bacteria is one of the important indicators for evaluating the probiotic properties of lactic acid bacteria. Centrifuge the ZLA031 culture medium that has been activated twice (4000 rpm, 10 min), collect the cells, wash them three times with sterile PBS buffer (pH=7.4) and resuspend them to make a concentration of 1×10 ⁸ CFU/mL The bacterial suspension is ready for use. An appropriate amount of human colon adenocarcinoma cell line Caco-2 cells that had been passaged 3 times were put into a 24-well culture plate and cultured. When the cells reached 80% confluence, an adhesion test was performed. Aspirate the culture fluid in the well, add the same volume of Lactobacillus animalis ZLA031 bacterial suspension, incubate together for 2 hours at 37°C in a 5% CO ₂ incubator, then wash 3 times with PBS buffer to remove unadherent cells. Determine the number of viable bacteria on the MRS plate and calculate the adhesion index according to the formula: adhesion index/(CFU/Caco-2 cells) = number of adherent bacteria/(CFU/well)/number of Caco-2 cells/(cells/well). The results showed that the adhesion index of Lactobacillus sp. ZLA031 was 7.5 ± 0.3 CFU/Caco-2 cells, showing strong adhesion ability.

5. Drug sensitivity test

Antimicrobial microbial susceptibility testing using the agar disk diffusion method. The drug susceptibility test of 21 antibiotics (OXOID susceptibility disks) in 11 categories was conducted on Lactobacillus animalis ZLA031. The results are shown in Table 6. As can be seen from Table 6, the combined Lactobacillus species of this animal is sensitive to antibiotics such as ofloxacin, cefprozil, cephalosporin, and amoxicillin, moderately sensitive to antibiotics such as ampicillin, ceftizoxime, and amoxicillin, and Streptomycin, amikacin, gentamicin, ciprofloxacin, clindamycin, lincomycin, chloramphenicol, florfenicol, erythromycin, azithromycin, colistin sulfate, new Resistance to antibiotics such as oxazole and tetracycline.

Table 6 Result of drug susceptibility test of combined Lactobacillus in animals

Note: S means sensitive; I means moderately sensitive; R means resistant.

6. Lactic acid production test

The twice-activated Lactobacillus animalis ZLA031 was inserted into the liquid MRS medium at an inoculum volume of 1% at a volume ratio, and a lactic acid detection kit was used to detect the lactic acid content in the fermentation broth. The results are shown in Figure 3. As can be seen from Figure 3, as the culture time prolongs, Lactobacillus animalis grows rapidly in the MRS medium, and the lactic acid content it produces also increases rapidly, reaching the highest at 32 hours, which is 168.53mmol/L, and then Acid production begins to slowly decrease.

Example 3 Preparation of animal combined Lactobacillus fermentation feed additives

1. Optimization of animal combined Lactobacillus culture conditions

The twice-activated Lactobacillus lactobacilli ZLA031 was inoculated into different liquid culture media at an inoculation amount of 1%, and a single factor test method was used to test the carbon and nitrogen sources, carbon-nitrogen ratio, culture temperature and culture time of the culture medium. The culture condition parameters were studied, and the optimal culture conditions were determined by detecting the number of viable bacteria of Lactobacillus animalis in the fermentation broth. Specific operation methods: (1) Based on MRS liquid culture medium, select 7 different carbon sources (glucose, lactose, maltose, sucrose, fructose, xylose and raffinose) and 7 different nitrogen sources (peptone , yeast powder, beef extract, beef peptone, casein peptone, soy peptone and ammonium sulfate) to replace the carbon source and nitrogen source in the MRS medium, and on this basis, 5 different carbon to nitrogen ratios (2:1, 1.5: 1, 1:1, 1:1.5 and 1:2) to screen out the best carbon and nitrogen sources and carbon to nitrogen ratio; (2) Select 5 different culture temperatures (31, 33, 35, 37 and 39°C ) to screen out the best culture temperature; (3) Select different culture time points (8, 16, 20, 24, 28 and 32 hours) to screen out the best culture time. The results are shown in Figure 4-Fig. 8.

From the single-factor test results in Figures 4 to 8, it can be seen that the different culture condition parameters when Animal Combined Lactobacillus has a higher viable bacterial count are: the carbon source is lactose and maltose, the nitrogen source is beef peptone, and the carbon-nitrogen ratio is 2: 1. 1.5:1 and 1:1, the culture temperature is 33℃ and 35℃, and the culture time is 16, 20, 24 and 28 hours. Considering the product usage, culture cost and operation convenience, the optimal culture conditions of Lactobacillus animalis ZLA031 are set as follows: the carbon source is lactose, the nitrogen source is beef peptone, the carbon to nitrogen ratio is 1:1, and other ingredients Same as MRS medium; the optimal culture temperature is 33°C and the culture time is 24 hours. Under these conditions, the viable bacterial count of Lactobacillus lactobacillus ZLA031 can reach 2.2×10 ⁹ CFU/mL, which is more than 2.7 times higher than before optimization (8.1×10 ⁸ CFU/mL).

The optimized liquid culture medium composition is: lactose 20g/L, beef peptone 20g/L, sodium acetate 5g/L, K ₂ HPO ₄ •3H ₂ O 2g/L, diammonium hydrogen citrate 2g/L, MgSO ₄ •7H ₂ O 0.58g/L, MnSO ₄ 0.19g/L, Tween-80 1ml/L, pH 6.5, sterilized at 121°C for 15min and set aside.

2. Parameter optimization of animal combined Lactobacillus fermented feed

The above animal combined Lactobacillus fermentation liquid and the complete feed for growing pigs are used to prepare an animal combined Lactobacillus fermented feed additive. Response surface methodology was used to optimize the parameters of fermented feed. According to the central combination design principle of Box-Behnken, temperature, feed-to-water ratio and bacterial inoculum amount are used as investigation factors. Each factor takes three levels, and the number of viable bacteria in the fermented feed at 24 hours is used as the response value. A comprehensive experimental design was used to determine the optimal fermentation parameters of animal combined Lactobacillus fermented feed. The experimental design is shown in Table 7.

Table 7 Box-Behnken Experimental Design

level Temperature (℃) Material to water ratio (Kg/Kg) Inoculum volume (mL) -1 33 0.8:1 0.25 0 35 0.9:1 0.3 1 37 1:1 0.35

Table 8 Fermentation test results

The specific operations are as follows:

(1) Preparation of strain fermentation broth: Inoculate Lactobacillus animalis ZLA031 strain into MRS liquid medium at an inoculum volume ratio of 1%, and culture it at 33°C for 24 hours. After two times, an activated seed culture is obtained. ;

The seed culture was inoculated into the optimized liquid medium at an inoculation amount of 1%, and the temperature was set to 33°C and the culture time was 24 hours to obtain the bacterial fermentation liquid. According to the viable bacterial count test, the effective viable bacterial count of the fermentation broth is 2.2×10 ⁹ CFU/mL;

(2) Select 16 clean fermentation barrels, add different volumes of bacterial fermentation broth, water with different feed-to-water ratios and growing pig complete feed according to the factor combinations in different test groups in Table 8, and stir evenly. Compact, cover and seal;

(3) Put the fermentation barrel into a closed and clean culture device, and culture it at different temperatures for 24 hours according to the combination of factors in the different test groups in Table 8 to obtain the animal combined Lactobacillus preparation.

(4) Count the number of viable lactic acid bacteria in the 16 groups of preparations. The results are shown in Table 8.

Using the number of viable bacteria in Table 8 as the response value, Minitab 18.0 software was used to calculate the data, and the optimal process parameters for animal combined lactobacillus fermentation feed were obtained: temperature 34.5°C, feed-water ratio 1:1, and bacterial inoculation amount is 0.25mL (the effective viable bacterial count of the strain is 2.2×10 ⁹ CFU/mL, that is, the inoculation amount of pig-derived animal combined Lactobacillus is 5.5×10 ⁸ CFU per kg of full-price growing pig feed). At this time, the fermented feed The viable count of lactic acid bacteria can reach 2.5×10 ⁹ CFU/g, and the pH can reach below 4.30.

Example 4 The application effect of animal combined lactobacilli preparations in the production of growing pigs

1. Experimental animals and management

90 healthy growing pigs (length × size) with similar initial weight were selected and randomly divided into 3 groups: control group, test group 1 and test group 2. Each group had 3 repetitions, with 10 pigs in each repetition. The control group was fed a basic diet (crude protein content 15.6%), the experimental group 1 was fed a low-protein diet (crude protein content 12.9%), and the experimental group 2 was fed a low-protein diet (crude protein content 12.9%) + low 0.1% of the animal combined Lactobacillus preparation prepared in the above Example 3 of the protein diet (the final effective viable bacterial count in the complete feed is 2.5×10 ⁶ CFU/g). The basic diet is composed of corn, soybean meal, wheat bran, calcium hydrogen phosphate, stone powder, amino acids, vitamins, trace elements, etc. The nutritional level is prepared according to the "Pig Feeding Standards" (NY/T 65-2004) and the US NRC (2012) standards. . According to the experimental design, each experimental group added crystalline amino acids to the experimental basal diet to make the amino acid content consistent with that of the control group. The measured values of the nutritional components of the basal diet are shown in Table 9. During the test, the pigs had free access to food, water, and routine immunization. The test period was 30 days.

Table 9 Measured values of nutritional components of basal diet and low-protein diet (%)

At the beginning and end of the test, individuals were weighed on an empty stomach; during the test, the growth of the pigs was observed daily, and the feed consumption was recorded; after the test, 9 fresh fecal samples were taken from each group to determine the lactic acid bacteria, Escherichia coli and golden grapes in the feces. The number of viable bacteria such as cocci; 9 pigs from each group were randomly selected to collect blood, and biochemical and immune indicators in the serum were measured; the nitrogen content and emissions in feed and feces were detected and recorded, and the impact on the fecal nitrogen balance of growing pigs was calculated. Data statistics were performed using SPSS 25.0 statistical software for one-way analysis of variance, and the results were expressed as mean ± standard deviation.

2. Test results

2.1 Effect of animal combined lactobacillus preparations on the growth performance of growing pigs

The results of average daily weight gain, average daily feed intake, and feed-to-weight ratio are shown in Table 10.

Table 10 Effects of animal combined lactobacillus preparations on the growth performance of growing pigs

project control group Test 1 group Test 2 groups Average daily weight gain (Kg/d) 0.57± ^0.05b 0.59± ^0.03b 0.64± ^0.04a Average daily feed intake (Kg/d) 2.13±0.16 2.16±0.11 2.15±0.09 Material to weight ratio 3.76±0.03 ^a 3.67±0.04 ^a 3.39± ^0.08b

Note: Different labels on the same shoulder represent significant differences, and different lowercase letters represent significant differences ( P <0.05).

It can be seen from the results that compared with the control group and test group 1, the average daily weight gain of test group 2 increased by 12.13% ( P <0.05) and 6.67% ( P <0.05) respectively, and the feed-to-weight ratio decreased by 9.99% ( P <0.05) and 7.63% ( P <0.05). It can be seen that compared with conventional protein content feeds and low-protein feeds, adding a certain dose of Lactobacillus animalis to low-protein diets for growing pigs can better promote the growth rate and feed efficiency of growing pigs, thereby improving the efficiency of pig farming.

2.2 Effect of animal combined with Lactobacillus preparations on fecal nitrogen balance of growing pigs

The results of nitrogen intake, fecal nitrogen, urinary nitrogen, total nitrogen emissions, deposited nitrogen and apparent nitrogen digestibility are shown in Table 11.

Table 11 Effects of animal combined lactobacilli preparations on fecal nitrogen balance of growing pigs

project control group Test 1 group Test 2 groups Nitrogen intake (g/d) 60.51±4.64 53.53±2.30 51.73±2.10 Fecal nitrogen (g/d) 13.86± ^0.14b 9.97±0.44 ^a 9.26±0.15 ^a Urine nitrogen (g/d) 19.05± ^0.08b 17.00±0.20 ^a 16.25±0.23 ^a Total nitrogen emissions (g/d) 32.91± ^0.14b 26.97±0.64 ^a 25.51±0.10 ^a Deposited nitrogen (g/d) 27.60±4.75 26.56±1.84 26.21±2.06 Nitrogen apparent digestibility (%) 76.80±1.89 ^a 81.36± ^0.52b 82.03± ^0.76b Apparent biological value of nitrogen (%) 58.37±3.88 60.85±1.50 61.54±1.99

Note: Different labels on the same shoulder represent significant differences, and different lowercase letters represent significant differences ( P <0.05).

It can be seen from the results that compared with the control group, the fecal nitrogen of the test group 1 and the test group 2 were reduced by 28.07% ( P <0.05) and 33.19% ( P <0.05), and the urinary nitrogen was reduced by 10.76% ( P <0.05). ) and 14.70% ( P <0.05), total nitrogen emissions decreased by 18.05% ( P <0.05) and 22.49% ( P <0.05), and apparent nitrogen digestibility increased by 5.94% ( P <0.05) and 6.81%, respectively. ( P <0.05). Compared with the experimental group 1, the experimental group 2 had more fecal nitrogen, urinary nitrogen and total nitrogen emissions, and the apparent nitrogen digestibility was higher. It can be seen that adding a certain dose of Lactobacillus animalis to the low-protein diet of growing pigs is more conducive to promoting the absorption and utilization of nitrogen by growing pigs, reducing nitrogen emissions to the environment, and is conducive to environmental emission reduction.

2.3 Effect of animal combined Lactobacillus preparations on the number of microorganisms in growing pig manure

The results for lactic acid bacteria, Escherichia coli, and Staphylococcus aureus are shown in Table 12.

Table 12 Effects of animal combined lactobacilli preparations on the number of fecal microorganisms in growing pigs

project control group Test 1 group Test 2 groups Lactic acid bacteria (10 ⁹ CFU/g) 1.56±0.30 ^a 1.83±0.28 ^a 4.23± ^0.40b E. coli (10 ⁷ CFU/g) 2.32±0.58 ^b 2.04± ^0.21b 0.60±0.25 ^a Staphylococcus aureus (10 ⁶ CFU/g) 4.21± ^0.25b 3.89± ^0.22b 1.22±0.35 ^a

Note: Different labels on the same shoulder represent significant differences, and different lowercase letters represent significant differences ( P <0.05).

It can be seen from the results that compared with the control group and test group 1, the number of lactic acid bacteria in pig feces of test group 2 increased by 171.15% ( P <0.05) and 131.15% ( P <0.05) respectively, and the number of E. coli decreased by 74.14% respectively. % ( P <0.05) and 70.59% ( P <0.05), and the number of Staphylococcus aureus decreased by 71.02% ( P <0.05) and 68..64% ( P <0.05), respectively. It can be seen that compared with conventional protein content feeds and low-protein feeds, adding animal combined Lactobacillus preparations to low-protein feeds can increase the content of beneficial bacteria in the intestines of growing pigs and reduce the content of harmful bacteria, thereby maintaining the intestinal microecology of pigs. Environmental balance.

2.4 Effects of animal combined lactobacillus preparations on serum indicators of growing pigs

The results of total protein, albumin, globulin, aspartate aminotransferase, alanine aminotransferase, glucose, urea nitrogen, IgM, IgG, and IgA are shown in Table 13.

Table 13 Effects of animal combined lactobacillus preparations on serum indicators of growing pigs

project control group Test 1 group Test 2 groups Total protein (g/L) 58.63±2.61 ^a 58.85±3.02 ^a 63.67± ^1.35b Albumin(g/L) 20.71±2.32 21.24±177 22.78±1.62 Globulin (g/L) 37.93± ^0.97a 37.60±1.03 ^a 40.89± ^2.41b Aspartate aminotransferase (U/L) 55.21± ^4.09b 52.57± ^4.29b 43.58±6.58 ^a Alanine aminotransferase (U/L) 43.55±3.53 ^b 41.88± ^2.68b 34.75±4.16 ^a Glucose (mmol/L) 3.72±0.41 3.74±0.25 3.64±0.11 Urea nitrogen (mmol/L) 12.00± ^0.99b 11.08± ^0.67ab 10.57±1.40 ^a IgM (g/L) 1.07±0.02 1.19±0.05 1.22±0.08 IgG (g/L) 19.00±0.90 ^a 19.88± ^1.03ab 20.33± ^1.42b IgA (g/L) 2.14±0.04 2.22±0.04 2.37±0.05

Note: Different labels on the same shoulder represent significant differences, and different lowercase letters represent significant differences ( P <0.05).

It can be seen from Table 13 that compared with the control group and test group 1, test group 2 can significantly increase the serum total protein and globulin content ( P <0.05), and significantly reduce the serum aspartate aminotransferase and alanine aminotransferase activities ( P <0.05) ; Compared with the control group, test group 2 could significantly increase serum IgG content ( P <0.05) and significantly reduce urea nitrogen content ( P <0.05). Serum total protein can reflect the liver's ability to synthesize protein, while GLB is secreted by plasma cells and can reflect the body's immune status and resistance. Immunoglobulins (mainly including IgG, IgM and IgA) are the main antibodies of humoral immunity, and their content can reflect the body's immunity level to a certain extent. Alanine aminotransferase and aspartate aminotransferase are two indicators that reflect liver function. Increased activity indicates that liver cells may be damaged. It can be seen from the results that compared with conventional protein content feeds and low-protein feeds, adding a certain dose of animal combined Lactobacillus preparations to low-protein diets for growing pigs can protect the liver, improve the body's resistance, and promote animal health.

Claims (9)

1. Pig-derived animal combined lactobacillusLigilactobacillus animalis)ZLA031, which is deposited in China general microbiological culture Collection center, with accession number: CGMCC No.26855.

2. Use of a swine animal in combination with lactobacillus for the preparation of a fermented feed additive according to claim 1.

3. A fermented feed additive comprising the swine-derived animal-associated lactobacillus of claim 1.

4. The method for preparing the fermented feed additive according to claim 3, which is characterized by comprising the steps of inoculating the pig-derived animal combined lactobacillus into a liquid culture medium for fermentation culture, wherein the carbon source in the liquid culture medium is lactose, the nitrogen source is beef peptone, the carbon-nitrogen ratio is 1:1, other components are the same as the MRS culture medium, the culture temperature is 33 ℃, and the culture time is 16-28 hours; the liquid culture medium comprises lactose 20g/L, beef peptone 20g/L, sodium acetate 5g/L, K ₂ HPO ₄ •3H ₂ O2 g/L, diammonium hydrogen citrate 2g/L, mgSO ₄ •7H ₂ O 0.58g/L、MnSO ₄ 0.19g/L and Tween-80 1ml/L, pH 6.5.

5. A feed additive, characterized in that the feed additive is an animal-derived lactobacillus preparation comprising the swine-derived animal-derived lactobacillus preparation of claim 1.

6. The method for preparing the feed additive according to claim 5, wherein the method comprises inoculating the swine animal-derived lactobacillus in a complete feed and water for mixed fermentation.

7. The preparation method of claim 6, wherein the whole price is a growing pig whole price, and the weight ratio of the whole price to water is 1:1, and the inoculation amount of the pig source animal combined lactobacillus is 5.5X10 ⁸ The CFU is carried out per kg of whole pig feed, the fermentation temperature is 34.5 ℃, the fermentation time is 24 hours, and the sealed fermentation is carried out.

8. Use of the feed additive according to claim 5 in the pig industry.

9. The use according to claim 8, wherein the pigs are fed with a low protein ration and with a feed additive having a mass of 0.1%.