CN101548716A - A porcine lactobacillu plantarurn freeze-dry preparation and its preparation method - Google Patents

Abstract

The invention relates to "a freeze-dried preparation of pig-derived lactobacillus plantarum and a preparation method thereof", belonging to the field of microorganism application. A freeze-dried preparation of Lactobacillus plantarum derived from pigs, characterized in that the weight percent of the freeze-dried protective agent in the freeze-dried preparation is: 16% of skimmed milk powder, 1.5% of sodium glutamate, 0.5% of L-cysteine, and 2% of lactose , 10% dextrin, and the rest is water. This freeze-drying protective agent not only makes the survival rate of bacteria high, but also has good storage stability, making it very suitable for adding to feed; adding the freeze-dried preparation of the present invention to nursery pigs In the feed, the result proves that it has the function of reducing pig diarrhea rate, improving pig daily gain and feed utilization rate, significantly exceeding the effect of feeding antibiotics (chlortetracycline); Bacteria culture medium, the culture medium is better than ordinary MRS culture medium, and the number of viable bacteria is increased by 2 orders of magnitude.

Description

A kind of freeze-dried preparation of pig-derived Lactobacillus plantarum and preparation method thereof

technical field

The present invention relates to the field of microorganisms and applications thereof, in particular to a freeze-dried preparation of lactobacillus derived from porcine and a preparation method thereof

Background technique

There are hundreds of kinds of bacteria living in animals, the number of which exceeds one billion billion. Among them, probiotics are beneficial to animal health, represented by lactic acid bacteria and bifidobacteria, and harmful bacteria are harmful to humans and animals. When the probiotics are dominant (accounting for more than 80% of the total), the animals maintain a healthy state, otherwise they are in a sub-healthy or non-healthy state. For a long time, farmers have often limited themselves to immediate economic benefits and blindly added antibiotics to feed, which not only killed harmful bacteria, but also inhibited the killing of beneficial bacteria, resulting in an imbalanced state of intestinal micro-ecological system. The results of scientific research in recent years have proved that adding probiotics to feed can have the opposite effect to antibiotics. It can inhibit the number of harmful bacteria, increase the number of beneficial bacteria, and maintain the balance of the intestinal micro-ecological system. . Among them, probiotic preparations represented by lactic acid bacteria are the most widely used and most widely used. It is well known that lactic acid bacteria are beneficial bacteria that are essential to animals and have important physiological functions. Their number directly affects the health of animals. . At present, a large number of studies have reported that this kind of probiotic preparation has good biological functions, and it has been proved that it can treat and prevent certain intestinal diseases of animals or play a role in health care clinically, and has been widely used in the feed industry.

Lactobacillus plantarum (Lactobacillus plantarum) is a beneficial lactic acid bacillus commonly found in the intestinal tract of humans and animals. Studies have shown that Lactobacillus plantarum can be colonized in the intestinal tract of animals, metabolize to produce lactic acid, acetic acid and other substances, which can inhibit the growth and reproduction of harmful intestinal bacteria, and can ferment a variety of carbohydrates, such as simple sugars (glucose, mannose, semi Lactose, etc.), disaccharides (sucrose, lactose, trehalose, etc.), oligosaccharides (raffinose, melezitose, etc.), are one of the feed-grade microbial strains allowed by the Ministry of Agriculture of my country. At present, a large number of such probiotic products have emerged on the market, but due to the imperfect standards of the new lactic acid bacteria products, the products have problems such as unstable quality, low number of viable bacteria, and poor stability. An effective method of preservation, because lactic acid bacteria can easily cause damage to some cells, death and passivation of some enzyme protein molecules during the freeze-drying process, many studies have found that adding appropriate freeze-drying protective agents to lactic acid bacteria before freeze-drying can Reduce or prevent the damage to the cells caused by the two processes of freezing and drying, thereby protecting the bacteria. Since lactic acid bacteria are species-specific, even under the same external conditions, the same bacteria from different sources will be different during the freeze-drying process. It may also show different characteristics. At present, there is no freeze-dried preparation of Lactobacillus plantarum with good effect and suitable for adding to animal feed. Although some freeze-dried formulations have a high survival rate of bacteria after freeze-drying, they have poor storage stability. It is easy to be inactivated during the packaging process, making it difficult to exert its excellent characteristics after being added to the feed. Some research reports on lyoprotectants such as the lyophilization disclosed by Li Jiapeng et al. in 2008 ("response surface optimization research on the formula of Lactobacillus plantarum lyoprotectant", Food Science, 2008, Vol.29, No.06) The formulation of the protective agent can obtain a higher bacterial survival rate, but its effect on the storage stability of the bacteria has not been reported, but the storage stability of the freeze-dried preparation is very important. From the preparation of the freeze-dried preparation to before feeding to animals, The number of viable bacteria in the lyophilized preparation depends on the storage stability provided by the lyoprotectant, and the number of viable bacteria is the key to determine whether the lyophilized preparation of the bacteria can exert a beneficial effect. Moreover, the characteristics of its formula are not suitable for actual production. In actual production, such as the production above the pilot test level, the formula adopted should not only ensure high bacterial survival rate and good stability, but also take into account the adaptability of production equipment, operability, and product quality. Yield and other factors, the optimal formula provided by this document uses trehalose, the cost is too high, so it is not suitable for production into a freeze-dried preparation added to feed. Many of the above problems affect the application and development of probiotic preparations in animal feed.

Contents of the invention

Aiming at the deficiencies in the above-mentioned fields, the present invention provides a freeze-dried preparation of pig-derived Lactobacillus plantarum and a preparation method thereof. The freeze-dried preparation has a high number of live bacteria and good storage stability, and can be fully utilized in direct feeding or adding to feed. Ecological balance function of Lactobacillus in animals.

A freeze-dried preparation of Lactobacillus plantarum derived from pigs, characterized in that the percentage by weight of the freeze-drying protective agent used when preparing the freeze-dried preparation is: 16% of skimmed milk powder, 1.5% of sodium glutamate, 0.5% of L-cysteine, 2% lactose, 10% dextrin, and the rest is water.

The number of viable bacteria in the freeze-dried preparation of the porcine-derived Lactobacillus plantarum is more than or equal to ^{10 12} cfu/g.

The pig-derived Lactobacillus plantarum is derived from the cecal mucosa of nursery pigs.

The preparation method of the above-mentioned freeze-dried preparation of Lactobacillus plantarum of pig origin comprises the steps of: carrying out enrichment culture of Lactobacillus plantarum liquid of pig origin, centrifuging the enrichment culture solution at 6000rpm for 10min, taking the sludge, adding a freeze-drying protective agent, and freezing Dry time is 20-28 hours.

The freeze-drying time is 26 hours.

The weight percent of the medium for the enrichment culture is: tryptone 0.5%, lactose 1%, yeast extract powder 0.5%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2%, diammonium hydrogen citrate 0.2%, sulfuric acid tetrahydrate Manganese 0.025%, magnesium sulfate heptahydrate 0.0058%, Tween-80 0.1%, and the rest is water.

The parameters of the enrichment culture are as follows: the pH of the culture medium is 6.4-6.5, the inoculation amount of Lactobacillus plantarum is 1%, cultured at 37° C. for 24 hours, and the rotation speed is 100 rpm.

Application of the above freeze-dried preparation in animal feed additives. The animal is a pig.

In order to make Lactobacillus plantarum well applied in animal feed and give full play to the effect of its probiotics, the present invention provides a formula of freeze-drying protectant, adding the freeze-dried preparation made of Lactobacillus plantarum derived from pigs to the conservation In the pig feed, carry out 33 days feeding test, prove that 0.3% Lactobacillus plantarum+basic diet group shows the best effect, see table 13, significantly exceed the effect of feeding antibiotics such as aureomycin, illustrate in the present invention provides Among the freeze-drying protective agents available, Lactobacillus plantarum has a good survival rate and storage stability, and can fully play the function of reducing pig diarrhea rate, improving pig daily gain and feed utilization rate. Through a large number of experimental analysis, the present invention has obtained a group of freeze-drying protectant formulas with high bacterial survival rate and high storage stability from the aspects of bacterial survival rate and bacterial storage stability. The weight percentage is: skimmed milk powder 16%, sodium glutamate 1.5%, L-cysteine 0.5%, lactose 2%, dextrin 10%, and the rest is water. Choose skimmed milk powder as the suspended substance, skimmed milk powder can form a protein film outside the bacteria to protect the cells; lactose is a reducing sugar, it can inhibit the phase transition of the plasma membrane, and has a significant protective effect on the freeze-drying and dehydration of lactic acid bacteria , and the cost is low, suitable for actual production and use; studies have shown that L-cysteine has a better protective effect on the bacteria, which can cause the cells to undergo cytoplasmic separation before freezing, and accumulate between the cell wall and the cell membrane to form a barrier The buffer zone formed by ice crystals provides mechanical protection for the cell membrane; dextrin, which is easy to dissolve in water, is selected and does not need to be dissolved by heating, which simplifies the pilot production procedure. The number of viable bacteria in the freeze-dried preparation prepared by the lyoprotectant in the pilot production was 10 ¹² cfu/g, and the number of viable bacteria in the freeze-dried preparation could still maintain 3.3×10 ⁹ cfu/g when stored at room temperature for 12 months. g, good storage stability and suitable production cost make it suitable to be added to conventionally produced feed.

In the present invention, the culture medium used for the enrichment culture adopts a special culture medium, and the percentage by weight is preferably: tryptone 0.5%, lactose 1%, yeast extract powder 0.5%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2%, citric acid Diammonium hydrogen diammonium 0.2%, manganese sulfate tetrahydrate 0.025%, magnesium sulfate heptahydrate 0.058%, Tween-80 0.1%, and the rest is water, and its bacterial enrichment effect is 2 orders of magnitude higher than that of ordinary MRS viable bacteria, see Table 8 .

Since the Lactobacillus plantarum of the freeze-dried preparation of pig-derived Lactobacillus plantarum obtained in the present invention is a common probiotic in the intestinal tract of common animals, the freeze-dried preparation can be added to animal feed.

Since the source of Lactobacillus plantarum of the present invention is pigs, the freeze-dried preparation is preferably used in pig feed.

In summary, the freeze-drying protectant of the freeze-dried preparation of pig-derived Lactobacillus plantarum provided by the present invention can provide good freeze-drying protection for Lactobacillus plantarum, can obtain higher bacterial survival rate and storage stability, and can produce The process is simple and the cost is reasonable, so that the freeze-dried preparation of the invention is very suitable for application in animal feed.

Detailed ways

Example 1 Preparation of pig-derived Lactobacillus plantarum freeze-dried preparation

Step 1 Isolation and identification of pig-derived Lactobacillus plantarum

Aseptically scrape off the intestinal contents of healthy nursery pigs, rinse the intestinal tract with sterilized physiological saline, scrape a certain amount of intestinal mucosa with a glass slide, weigh 1g of the sample, and place it in a triangular flask filled with 99ml sterile physiological saline disperse it evenly, draw 1ml of the bacterial solution into a test tube filled with 9ml of sterile saline, the dilution is 10 ^-1 , repeat the above process, and make 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 bacterial liquid. Select three dilutions of 10 ^-4 , 10 ^-5 , and 10 ^-6 , draw 0.1ml of the bacterial solution and drop it on the MRS modified medium plate, and culture it at 37°C for 24 to 48 hours by the candle jar method, and pick the colonies with different shapes. Streak inoculation on the selection medium for pure culture, and then store in a 4°C refrigerator for later use.

Then pick the pure culture for staining and microscopic examination; select non-spore-forming Gram-positive bacilli for biochemical experiments such as contact enzymes, nitrate reduction, gelatin liquefaction, and sugar alcohol fermentation tests. The results are consistent with the "Common Bacteria System Identification Manual", "Classification and Experimental Methods of Lactic Acid Bacteria" was compared to determine the strains.

Then sent to China Industrial Microorganism Culture Collection Center for identification test. The results of molecular biology identification show that the similarity of this bacterial strain with Lactobacillus pentosus and Lactobacillus plantarum is 99.7%. The gene sequence based on it is shown in Table 1. Further biochemical identification is carried out, and its biochemical characteristics are shown in Table 1. It is the closest to Lactobacillus plantarum. The bacterium is Lactobacillus plantarum.

The Lactobacillus plantarum used in the present invention is a common bacterium on the pig cecal mucosa, which is stored in the laboratory of the Institute of Animal Husbandry, Beijing Academy of Agriculture and Forestry Sciences, and can be distributed to the public; it is isolated by conventional methods or the method described in step 1 and referred to its physiological and biochemical Characterization for identification is also available.

Table 1 Molecular biology identification report of Lactobacillus plantarum

Gene sequence (Sequences) 1   GATACATGCA AGTCGAACGA ACTCTGGTAT TGATTGGTGCTTGCATCATG51  ATTCTACTAT TTGAGTGAGT GGCGAACTGG TGAGTAACACGTGGGAAACC101 TGCCCAGAAG CGGGGGATAA CACCTGGAAA CAGATGCTAATACCGCATAA151 CAACTTGGAC CGCATGGTCC GAGCTTGAAA GATGGCTTCGGCTATCACTT201 TTGGATGGTC CCGCGGCGTA TTAGCTAGAT GGTGGGGTAACGGCTCACCA251 TGGCAATGAT ACGTAGCCGA CCTGAGAGGG TAATCGGCCACATTGGGACT301 GAGACACGGC CCAAACTCCT ACGGGAGGCA GCAGTAGGGAATCTTCCACA351 ATGGACGAAA GTCTGATGGA GCAACGCCGC GTGAGTGAACAAGGGTTTCG401 GCTCGTAAAA CTCTGTTGTT AAAGAAGAAC ATATCTGAGAGTAACTGTTC451 AGGTATTGAC GGTATTTAAC CAGAAAGCCA CGGCTAACTACGTGCCAGCA501 GCCGCGGTAA TACGTAGGTG GCAAGCGTTG TCCGGATTTATTGGGCGTAA551 AGCGAGCGCA GGCGGTTTTT TAAGTCTGAT GTGAAAGCCTTCGGCTCAAC601 CGAAGAAGTG CATCGGAAAC TGGGAAACTT GAGTGCAGAAGAGGACAGTG651 GAACTCCATG TGTAGCGGTG AAATGCGTAG ATATATGGAAGAACACCAGT701 GGCGAAGGCG GCTGTCTGGT CTGTAACTGA CGCTGAGGCTCGAAAGTATG

  751  GGTAGCAAAC AGGATTAGAT ACCCTGGTAG TCCATACCGTAAACGATGAA801  TGCTAAGTGT TGGAGGGTTT CCGCCCTTCA GTGCTGCAGCTAACGCATTA851  AGCATTCCGC CTGGGGAGTA CGGCCGCAAG GCTGAAACTCAAAGGAATTG901  ACGGGGGCCC GCACAAGCGG TGGAGCATGT GGTTTAATTCGAAGCTACGC951  GAAAAACCTT ACCAGGTCTT GACATACTAT GCAAATCTAAAAGATTAGAC1001 GTTCCCTTCG GGGACATGGA TACAGGTGGT GCATGGTTGTCGTCAGCTCG1051 TGTCGTGAGA TGTTGGGTTA AGTCCCGCAA CGAGCGCAACCCTTATTATC1101 GGAGGAAGGT GGGGATGACG TCAAATCATC ATGCCCCTTATGACCTGGGC1151 GGAGGAAGGT GGGGATGACG TCAAATCATC ATGCCCCTTATGACCTGGGC1201 TACACACGTG CTACAATGGA TGGTACAACG AGTTGCGAA

Table 2 Test report on physiological and biochemical characteristics of pig-derived Lactobacillus plantarum

Note: "+" in the table means ≥90% of the strains are positive, "-" means ≥90% of the strains are negative, and "ND" means not determined.

Step 2: Acid-resistant and bile-resistant characteristics of pig-derived Lactobacillus plantarum

Inoculate pig-derived Lactobacillus plantarum into MRS liquid medium, culture for 24 hours, dilute the bacterial solution gradiently, take 10 ^-5 diluted solution 1ml and inoculate on the MRS plate containing 0.3% bile salt and the MRS plate without bile salt. Plates were incubated at 37°C for 48 hours, colonies were counted, and the survival rate was calculated.

Survival rate (%)=(the number of viable bacteria in the MRS solid medium containing 0.3% bile salt/the number of viable bacteria in the control MRS solid medium)×100. The above test process was repeated twice, and the results were averaged.

Inoculate pig-derived Lactobacillus plantarum into MRS liquid medium, cultivate for 24 hours, add 1% of the inoculum into the MRS medium with a pH of 3.0, make two parts, one part is gradiently diluted with sterilized physiological saline, and take 10 ^- Inoculate 1ml of the diluted solution of ⁵ on the MRS plate and incubate at 37°C for 48 hours, and count the colonies, that is, the number of viable bacteria in 0 hours. After the other part was treated at room temperature for 1 hour, it was further diluted with sterilized physiological saline, and 1ml of the 10 ^-5 dilution was inoculated on the MRS plate, and incubated at 37°C for 48 hours, and counted colonies, that is, the number of viable bacteria in 1 hour, and Calculate the survival rate of lactic acid bacteria.

Survival rate (%)=(the number of viable bacteria in 1 h/the number of viable bacteria in 0 h)×100. The above test process was repeated twice, and the results were averaged.

Table 3 results of the bile-resistant and acid-resistant tests of pig-derived Lactobacillus plantarum

Step 3: Enrichment and cultivation of pig-derived Lactobacillus plantarum

Enrichment medium:

Nitrogen source: fish peptone, tryptone, soybean peptone; all are biochemical grade reagents.

Carbon sources: lactose, sucrose, glucose; all are analytically pure reagents.

Growth Factors: Vitamin C, Yeast Extract Powder, Beef Extract.

Buffer salts: K ₂ HPO ₄ , diammonium hydrogen citrate, sodium acetate, MgSO ₄ ·7H ₂ O, MnSO ₄ ·4H ₂ O; all are analytical reagents.

3.1 Preparation of enrichment medium

The alternative materials for the enrichment medium are based on the nutrient composition of the MRS medium, and are determined by preliminary tests with reference to the food composition table. Select nitrogen source (A), carbon source (B), growth factor (C) and (D) salts (salts include inorganic salts and buffer salts) as experimental factors, and design an orthogonal experiment to obtain the optimal composition of the medium. There may be a synergistic effect among the nitrogen source, carbon source and other nutrient components of the medium, but the difference between factor D and the other three factors is large, so the interaction between it and other factors can be ignored. The ratio of each nutrient component is: 0.5% of nitrogen source, 1% of carbon source, 0.5% of growth factor, the composition of inorganic salt and buffer salt remains unchanged, and the rest is water. The specific factor level project design and the formulation of lactic acid bacteria enrichment medium are shown in Table 4 and Table 5.

Table 4 Orthogonal test factor level table of medium

Table 5 Enrichment medium formula table

Preparation of the enrichment medium: the culture medium is heated and boiled, and then filtered through filter paper to make the enrichment solution completely clear and transparent for easy color comparison. The enrichment solution is sterilized at 105°C for 15 minutes.

3.2 Determination of the absorbance value (OD ₆₀₀ ) of the bacterial solution

After preparing various culture media, they were inoculated at 1% respectively, cultured at 37°C for 24 hours, and the absorbance value of the bacterial solution was measured colorimetrically at 600 nm with a UV4802 ultraviolet spectrophotometer. The pH value of the bacterial solution was measured at room temperature with a pH213 acidity meter. See Table 6 for the results of the orthogonal test on the component screening of the Lactobacillus plantarum enrichment medium, and Table 7 for the range analysis results of the indicators in the Lactobacillus plantarum enrichment medium.

Table 6 Orthogonal test results of screening of components of pig-derived Lactobacillus plantarum enrichment medium

Table 7 The results of the range analysis of the indicators in the pig source Lactobacillus plantarum enrichment medium

Note: The order of the influence of various factors on the enrichment effect of Lactobacillus plantarum from pigs is: B, C, A.

Orthogonal test analysis draws the optimal level of enrichment medium weight percent as: tryptone 0.5%, lactose 1%, yeast extract powder 0.5%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2%, diammonium hydrogen citrate 0.2%, manganese sulfate tetrahydrate 0.025%, magnesium sulfate heptahydrate 0.058%, Tween-80 0.1%, and the rest is water.

3.3 Comparison of the number of viable bacteria between the enrichment medium and the MRS medium

The enrichment solution prepared according to the optimal level, inoculate the activated second generation of bacteria into the enrichment medium and MRS medium with 1% inoculation amount for culture, and after 24 hours of culture, dilute it with sterile normal saline To 10 ^-9 , 10 ^-7 , 10 ^-8 , and 10 ^-9 , 10ul were inoculated in the MRS counting medium. The results are shown in Table 8. The enrichment effect of the enrichment medium was significantly better than that of the MRS medium.

Table 8 compares the number of viable bacteria between the enrichment medium and the MRS medium

The optimization of step 4 lyoprotectant formula

Material

Lactose, Sucrose, Soluble Starch, Glycerin, Ascorbic Acid, L-Cysteine, Skimmed Milk Powder, Monosodium Glutamate, L-Glutamic Acid, Dextrin.

4.1 Preparation of lyoprotectant

Eight kinds of protective agents were selected and based on skim milk powder, 8 kinds of protective agents were randomly combined according to different concentrations to form 10 groups of protective agent formulas as shown in Table 9. Culture Lactobacillus plantarum in the best enrichment medium for 24 hours, centrifuge at 6000r/min for 10 minutes, discard the supernatant, remove the sludge, add a protective agent equal to the volume of the sludge to make a bacterial suspension, and take 0.5ml of the bacterial suspension Dilute to 10 ^-7 with physiological saline, take 10ul to inoculate MRS medium, and count. The remaining bacterial suspension was subpackaged and pre-frozen at ultra-low temperature for 2 hours, then quickly put into a small freeze dryer, and vacuum freeze-dried.

The impact of different protective agents of table 9 on the survival rate of plantaractobacillus

group Protectant Formula Survival rate (%) 1 12% Skim Milk Powder + 4% Lactose + 2% Soluble Starch + 2% Ascorbic Acid + 2% Monosodium Glutamate 67.50 2 8% Skim Milk Powder + 2% Soluble Starch + 2% Lactose + 2% Glycerin + 2% Ascorbic Acid 83.08 3 10% skimmed milk powder + 4% lactose + 2% soluble starch + 0.5% L-cysteine + 1.5% sodium glutamate 13.08 4 10% skimmed milk powder + 4% lactose + 2% ascorbic acid + 1% L-cysteine + 2% sodium glutamate 27.66 5 16% skim milk powder + 2% lactose + 10% dextrin + 0.5% L-cysteine + 1.5% sodium glutamate 88.89 6 10% Skim Milk Powder + 4% Lactose + 10% Dextrin + 2% Ascorbic Acid + 1% L-Glutamic Acid 8.00 7 10% skim milk powder + 8% dextrin + 2% ascorbic acid + 1% L-glutamic acid + 0.4% L-cysteine 24.34 8 6% Skim Milk Powder + 2% Sucrose + 4% Glycerin + 3% Ascorbic Acid + 1% L-Glutamic Acid 0 9 10% Skim Milk Powder + 5% Sucrose + 4% Glycerin + 2% Ascorbic Acid + 1% L-Glutamic Acid 7.23 10 16% skimmed milk powder + 1% sucrose + 3% glycerin + 1% L-glutamic acid 15.50

Counting medium is MRS medium: glucose 20.0g, peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, sodium acetate 5.0g, diammonium citrate 2.0g, Tween 80 1.0mL, K ₂ HPO ₄ 2.0g, MgSO ₄ ·7H ₂ O 0.58g, MnSO ₄ ·4H ₂ O 0.25g, agar 20g, distilled water 1000mL, pH 6.5, sterilized at 121°C for 15min.

The results showed that when the protective agent combination was 16% skimmed milk powder + 2% lactose + 10% dextrin + 0.5% L-cysteine + 1.5% sodium glutamate, the survival rate of the freeze-dried preparation of Lactobacillus plantarum derived from pig The highest, up to 88.89%, therefore, identified as the best lyoprotectant.

4.2 Pilot production of Lactobacillus plantarum

After the Lactobacillus plantarum is expanded and cultivated with the enrichment medium, centrifuge at 6000r/min for 10min, discard the supernatant, take the bacteria sludge, and use the best protective agent to make a bacteria suspension. Take 0.5ml of the bacteria suspension and dilute it with normal saline to 10 ^-7 , take 10ul to inoculate MRS medium, and count. The remaining bacterial suspensions were subpackaged and placed in a medium-sized freeze dryer for freeze-drying. The results are shown in Table 10.

Table 10 Lactobacillus plantarum pilot production viable count

Protective ingredients The total amount of bacterial suspension before freeze-drying (L) Freeze-drying time (h) The total amount of bacteria powder after freeze-drying (kg) The number of live bacteria in bacterial powder (CFU/g) 16% skimmed milk powder + 0.5% sodium glutamate + 10% dextrin + 2% lactose + 1.5% cysteine 25 26 6.7 1.6×10 ¹²

4.3 Determination of storage stability of Lactobacillus plantarum freeze-dried powder

The freeze-dried Lactobacillus plantarum powder was packaged in an aluminum foil bag, and stored at room temperature for 6 months, 9 months, and 12 months. After the bacterial powder was taken, it was serially diluted with sterile saline, and the viable bacteria were determined by MRS medium. number. The results are shown in Table 11. It can be seen from Table 11 that after 6 months of storage at room temperature for freeze-dried Lactobacillus plantarum powder, the number of viable bacteria can still reach 10 ¹¹ cfu/g, and the survival rate reaches 68.75%. After 12 months of storage at room temperature, the number of viable bacteria can still reach 10 ⁹ cfu/g.

Table 11 Determination of storage stability of Lactobacillus plantarum

strain name The number of live bacteria (CFU/g) Save time (month) The number of viable bacteria (CFU/g) Save time (month) The number of viable bacteria (CFU/g) Save time (month) The number of viable bacteria (CFU/g) Lactobacillus plantarum 1.6×10 ¹² 6 1.1×10 ¹² 9 1.1×10 ¹¹ 12 3.3×10 ⁹

Step 5 Preparation of porcine source Lactobacillus plantarum freeze-dried preparation

After the enrichment of Lactobacillus plantarum, collect the culture medium, centrifuge at 6000r/min for 10 minutes to get the bacteria sludge, add the bacteria sludge to the freeze-dried protective agent configured with the optimal ratio, and make the concentration of the culture medium before centrifugation the same bacterial suspension. Pour the bacterial suspension into the material drying tray of a medium-sized freeze dryer, and vacuumize to start freeze-drying. After 26 hours of freeze-drying, the freeze-dried product of Lactobacillus plantarum was obtained, and the number of viable bacteria after determination was 1.6×10 ¹² cfu/g.

Step 6 Determination of the safety performance of the freeze-dried preparation of pig-derived Lactobacillus plantarum

The lyophilized preparation of Lactobacillus plantarum derived from pigs was sent to the Veterinary Drug Safety Supervision, Inspection and Testing Center of the Ministry of Agriculture to carry out the acute toxicity LD ₅₀ experiment in rats and mice. ₅₀ are greater than 5000mg/kg body weight, which proves that the freeze-dried preparation belongs to a safe feed additive.

Antibacterial test of embodiment 2 pig source lactobacillus plantarum freeze-dried preparation

Table 12 is the bacteriostasis test result of Lactobacillus plantarum. It can be seen that the strain has a strong inhibitory effect on pathogenic Escherichia coli, Salmonella, and Staphylococcus aureus, and they are all judged as moderately sensitive. Among them, the diameter of the inhibition zone against Salmonella is the largest, and the antibacterial effect is the best; Escherichia coli was next; Staphylococcus aureus was the worst. It has been indirectly proved that Lactobacillus plantarum has a preventive effect on Escherichia coli, Salmonella and other diseases that easily cause intestinal infection and diarrhea in pigs.

Table 12 Lactobacillus plantarum in vitro antibacterial test results

Application effect of embodiment 3 pig source Lactobacillus plantarum freeze-dried preparation in the production of nursery pigs

The freeze-dried preparations were added to the feed of 28-day-old nursery pigs according to different addition ratios. The experiment was randomly divided into 4 groups, with 16 pigs in each group, and the test period was 33 days. The test was divided into an antibiotic group, a 0.3% freeze-dried preparation group of Lactobacillus plantarum derived from pigs, a freeze-dried preparation group of 0.5% Lactobacillus plantarum derived from pigs, and a freeze-dried preparation group of 0.7% Lactobacillus plantarum derived from pigs. Nursery pigs are raised on net beds, the house temperature is kept at 25-27°C, they are fed with powder, and they have free access to food and water. The test results are shown in Table 12.

Table 13 pig-derived Lactobacillus plantarum freeze-dried preparation feeding piglet test

group Test period (days) Number of heads (heads) Oral weight gain during the test period (g/d) Ratio of feed to meat during the test period Mortality during the trial period (%) OD ₆₃₀ value of swine fever antibody (33 days) Control (chlortetracycline+basal diet) 33 16 393 ^{a 1.98a} 0 ^0.41a 0.3% freeze-dried preparation of Lactobacillus plantarum + basal diet 33 16 ^{405a 1.95a} 0 ^0.78b 0.5% freeze-dried preparation of Lactobacillus plantarum + basal diet 33 16 ^{374b 2.03b} 6.25 ^0.74b 0.7% Freeze-dried preparation of Lactobacillus plantarum + basal diet 33 16 ^{364b 2.10b} 12.50 ^0.72b

Note: In the longitudinal comparison, the data with different superscript letters have significant difference (P<0.05), but the data with the same superscript letter have no significant difference (P>0.05).

It can be seen from Table 13 that when feeding the freeze-dried preparation of Lactobacillus plantarum derived from pigs for 33 days, the 0.3% Lactobacillus plantarum freeze-dried preparation + basal diet group had the best effect, and the daily gain, feed-to-meat ratio, mortality rate and pig The indicators such as plague titer are the best, and exceed the similar effect of feeding antibiotics (chlortetracycline).

appendix:

sequence listing

<110> Beijing Academy of Agriculture and Forestry Sciences

<120> A freeze-dried preparation of pig-derived Lactobacillus plantarum and its preparation method

<130>P09140/NLK

<160>1

<170>PatentIn version 3.3

<210>1

<211>1239

<212>DNA

<213> Gene sequence for the identification of Lactobacillus plantarum

<400>1

gatacatgca agtcgaacga actctggtat tgattggtgc ttgcatcatg attctactat 60

ttgagtgagt ggcgaactgg tgagtaacac gtgggaaacc tgcccagaag cgggggataa 120

cacctggaaa cagatgctaa taccgcataa caacttggac cgcatggtcc gagcttgaaa 180

gatggcttcg gctatcactt ttggatggtc ccgcggcgta ttagctagat ggtggggtaa 240

cggctcacca tggcaatgat acgtagccga cctgagaggg taatcggcca cattgggact 300

gagacacggc ccaaactcct acgggaggca gcagtaggga atcttccaca atggacgaaa 360

gtctgatgga gcaacgccgc gtgagtgaac aagggtttcg gctcgtaaaa ctctgttgtt 420

aaagaagaac atatctgaga gtaactgttc aggtattgac ggtatttaac cagaaagcca 480

cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggattta 540

ttgggcgtaa agcgagcgca ggcggttttt taagtctgat gtgaaagcct tcggctcaac 600

cgaagaagtg catcggaaac tgggaaactt gagtgcagaa gaggacagtg gaactccatg 660

tgtagcggtg aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctgtctggt 720

ctgtaactga cgctgaggct cgaaagtatg ggtagcaaac aggattagat accctggtag 780

tccataccgt aaacgatgaa tgctaagtgt tggagggttt ccgcccttca gtgctgcagc 840

taacgcatta agcattccgc ctggggagta cggccgcaag gctgaaactc aaaggaattg 900

acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagctacgc gaaaaacctt 960

accaggtctt gacatactat gcaaatctaa aagattagac gttcccttcg gggacatgga 1020

tacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080

cgagcgcaac ccttattatc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta 1140

tgacctgggc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc 1200

tacacacgtg ctacaatgga tggtacaacg agttgcgaa 1239

Claims (9)

1. A freeze-dried preparation of pig-derived Lactobacillus plantarum, characterized in that the percentage by weight of the freeze-drying protectant used when preparing the freeze-dried preparation is: skimmed milk powder 16%, sodium glutamate 1.5%, L-cysteine 0.5% %, lactose 2%, dextrin 10%, and the rest is water. 2. The freeze-dried preparation of Lactobacillus plantarum derived from porcine according to claim 1, wherein the number of viable bacteria in the freeze-dried formulation of Lactobacillus plantarum derived from porcine is ≥ 1012 cfu/g. 3. The freeze-dried preparation of porcine-derived Lactobacillus plantarum according to claim 1, wherein said porcine-derived Lactobacillus plantarum is derived from the cecal mucosa of nursery pigs. 4. the preparation method of the arbitrary described porcine source lactobacillus plantarum freeze-dried preparation of claim 1 to 3, the step is: the pig source lactobacillus plantarum bacterium liquid is carried out enrichment culture with enrichment culture medium, enrichment culture solution is in Centrifuge at 6000rpm for 10min, take the sludge, add freeze-drying protective agent, and freeze-dry for 20-28 hours. 5. The preparation method according to claim 4, the freeze-drying time is 26 hours. 6. The preparation method according to claim 4, the percentage by weight of the enrichment medium is: tryptone 0.5%, lactose 1%, yeast extract powder 0.5%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2%, lemon Diammonium hydrogen acid 0.2%, manganese sulfate tetrahydrate 0.025%, magnesium sulfate heptahydrate 0.058%, Tween-80 0.1%, and the rest is water. 7. The preparation method according to claim 6, the parameters of the enrichment culture are: the pH of the enrichment medium is 6.4 to 6.5, the inoculum size of Lactobacillus plantarum is 1%, cultivated at 37° C. for 24 hours, and the rotating speed is 100 rpm . 8. The application of the freeze-dried preparation of porcine-derived Lactobacillus plantarum described in any one of claims 1 to 3 in animal feed additives. 9. The use according to claim 8, wherein the animal is a pig.