CN102031235B - Enterococcus faecium ANSE228 and application thereof - Google Patents

Abstract

The invention provides an Enterococcus faecium ANSE228 of which the collection number is CGMCC No.4082. The invention also provides application of the Enterococcus faecium ANSE228 to inhibition of salmonella pullorum and/or Escherichia coli and/or Staphylococcus aureus. The Enterococcus faecium ANSE228 is obtained by processes of repeated separation, purification, rejuvenation and the like, and has high biological activity, obvious probiotic property, high adversity resistance and the like. The invention also provides a microecological agent which contains the Enterococcus faecium ANSE228. When the microecological agent is added into drinking water and/or feeds for breeding animals, the Enterococcus faecium ANSE228 can be quickly activated and reproduced and a dominant beneficial flora can be formed after the Enterococcus faecium ANSE228 is fed into intestinal canals of the animals, and the Enterococcus faecium ANSE228 has the effects of reducing a harmful flora in the intestinal canals, adjusting microecological balance of the intestinal canals, substituting for medicaments such as antibiotic and the like, and improving weight increment of the animals and the utilization rate of the feeds.

Description

A kind of Enterococcus faecalis ANSE228 and its application

technical field

The invention relates to an enterococcus faecalis and its application. the

Background technique

With the development of society and science, people pay more and more attention to food safety, because unsafe food not only endangers human health and life, but may even have adverse effects on future generations. The society calls for green and safe food, and the safety of feed is the safety of food. the

Antibiotics, as feed additives, play an important role in promoting animal growth and development, and improving disease resistance. However, its long-term and large-scale use has led to some negative effects, such as destroying the micro-ecological balance of animals, increasing the incidence of digestive tract diseases in livestock and poultry, and causing pathogenic bacteria to mutate into drug-resistant strains. More importantly, the residues of antibiotics in animals are transmitted to humans through the food chain, bringing hidden dangers to human health. In view of this, many countries have prohibited and restricted the use of antibiotics as feed additives in feed. Therefore, the development of alternative products to antibiotics has become the focus of research in countries all over the world. Among them, due to their unique advantages, such as broad-spectrum non-specific antibacterial and bactericidal effects, green environmental protection, and no residue, etc., more and more attention is being paid to the field of antibiotic substitution. the

This study takes Enterococcus faecium as the research object, integrates the existing theoretical and practical understanding of probiotic Enterococcus faecium, screens out the probiotic Enterococcus faecium with antibacterial activity, stress resistance and safety, and optimizes the probiotic Enterococcus faecium. The fermentation process of bacteria and its effect on animal production performance as a microecological agent are studied, laying the foundation for the application and practice of probiotic Enterococcus faecalis. the

Contents of the invention

The object of the present invention is to provide a kind of enterococcus faecalis. the

Another object of the present invention is to provide the application of the above Enterococcus faecalis. the

The object of the present invention is also to provide a probiotics. the

Another object of the present invention is to provide the application of the above-mentioned probiotics. the

In order to realize the purpose of the present invention, the present invention provides a kind of Enterococcus faecium (Enterococcusfaecium) ANSE228, and its preservation number is CGMCC No.4082, depository unit: China Microorganism Culture Preservation Management Committee Common Microbiology Center (CGMCC), address: Beijing Municipality No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, date of preservation: August 16, 2010. The Enterococcus faecalis ANSE228 is obtained through repeated isolation, purification, rejuvenation and other processes, and has the advantages of strong biological activity, remarkable probiotics and good stress resistance. the

The cultivation method of Enterococcus faecalis ANSE228 described in the present invention is as follows: get Enterococcus faecalis ANSE228 seed liquid 1-5ml (seed liquid is that the single bacterium colony of picking is cultivated in seed liquid culture medium, and its live bacteria concentration is 10 ⁹ CFU /ml meter; the medium and culture conditions of the seed liquid are the same as the culture medium and culture conditions of fermentation), inoculated into 50-100ml culture medium to carry out shake flask fermentation culture. The culture medium is composed of the following components: corn flour 10-25g/L, soybean meal 10-30g/L, glucose 0-15g/L, dipotassium hydrogen phosphate 0.1-0.2g, potassium dihydrogen phosphate 1.0-2.0g, Manganese sulfate monohydrate 0.5-2.0g, magnesium sulfate heptahydrate 0.5-2.0g, distilled water 800-1200mL, pH value 6.5-7.5.

Among them, the preferred ones are: 15g corn flour, 20g soybean meal, 5g glucose, 0.15g dipotassium hydrogen phosphate, 1.5g potassium dihydrogen phosphate, 1.0g manganese sulfate monohydrate, 1.5g magnesium sulfate heptahydrate, 1000mL distilled water, pH 7.0 . the

The conditions for shake flask fermentation in the present invention are: fermentation temperature 25-45°C, fermentation time 18-36h, pH value 6.5-7.5, static culture; preferably: fermentation temperature 37°C, fermentation time 24h, pH value 7.0 . the

The 10L fermenter pilot test medium is the same as the shake flask fermentation medium. The pilot test fermentation conditions of 10L fermenter are: liquid volume is 4-7L medium, inoculum volume is 30-100ml shake flask seed liquid, fermentation temperature is 25-45℃, fermentation time is 18-36h, pH value is 6.5-7.5, The stirring speed is 200-400r/min. Among them, it is preferred that the liquid volume is 5L of culture medium, the inoculum volume is 50ml, the shake flask seed liquid fermentation temperature is 37°C, the fermentation time is 24h, the pH value is 7.0, and the stirring speed is 300r/min. the

The present invention also provides the application of Enterococcus faecalis ANSE228 in inhibiting Salmonella pullorum, Escherichia coli and/or Staphylococcus aureus. The bacterial strain has significant probiotic properties and can significantly inhibit the growth and reproduction of pathogenic bacteria such as Salmonella pullorum, Escherichia coli and Staphylococcus aureus. the

The present invention also provides the application of the Enterococcus faecalis ANSE228 in simulated gastric juice and/or simulated bile salt and/or high temperature environment of 75-90°C. The bacterial strain has strong stress resistance, can tolerate simulated gastric acid, simulated bile salt and high temperature environment, and can maintain a high survival rate of more than 80% of live bacteria. the

The probiotic testing method of Enterococcus faecalis ANSE228 of the present invention is as follows:

Prepare an MRS plate with a thickness of about 4 mm on a sterile operating table, and spread 100-200 μL of a bacterial suspension of pathogenic bacteria (E. coli, Staphylococcus aureus, and Salmonella) at a concentration of 10 ⁶ on the plate, and Place a sterilized stainless steel small tube (circular small tube with an inner diameter of 6 mm, an outer diameter of 8 mm, and a height of 10 mm, with both ends smooth) on the culture medium, pressurize gently to make it contact with the culture medium without gaps, and wait for several minutes Finally, drop a certain amount of Enterococcus faecalis ANSE228 activated strain bacteria liquid into each small tube respectively to prevent it from overflowing, incubate at 35°C-40°C for 18-36h, and then measure the diameter of the inhibition zone. Each experiment was repeated three times, and the average value was taken.

Wherein, the MRS plate is the MRS culture medium sterilized at 121° C., poured into the sterilized plate, and cooled to form a 4 mm thick MRS plate with a smooth surface. The formula of the MRS medium is: peptone 10g, beef extract 10g, yeast extract 5g, K ₂ HPO ₄ 2g, diammonium hydrogen citrate 2g, glucose 20g, sodium acetate 5g, Tween-801mL, MgSO ₄ 7H ₂ O0.58g, MnSO ₄ ·4H ₂ O 0.25g, agar 16g, distilled water 1000ml, pH=7.0.

The test method of Enterococcus faecalis resistance of the present invention is as follows:

1. High temperature tolerance test: Take 5mL Enterococcus faecalis ANSE228 live bacteria liquid and inject it into the centrifuge tube 1, dilute it step by step with sterile normal saline ten times, choose the appropriate diluent, and spread it on the MRS plate 1 . Another 5mL live Enterococcus faecalis ANSE228 bacteria solution was injected into centrifuge tube 2, placed in a 75-90°C water bath and heated for 10-20min, and the heated Enterococcus faecalis ANSE228 live bacteria solution was treated with sterile saline Ten-fold serial dilutions were made, and the dilutions with the same dilution as above were selected and coated on MRS plate 2. Plate 1 and plate 2 were cultured at 30-45° C. for 18-36 hours, and the number of Enterococcus faecalis before and after heating was calculated. the

2. Tolerance test of simulated gastric juice: Dissolve pepsin in 0.5%-0.85% normal saline to make the final concentration 3g/L, and adjust the pH value to 2.0-4.0 with concentrated hydrochloric acid or 10% NaOH. Take 0.5mL of Enterococcus faecalis ANSE228 suspension and add it to 4.5mL of simulated gastric juice (i.e., ten-fold serial dilution), and quickly mix it fully on a shaker, and then place it at 30-45°C for static culture for 2-4h. The culture solution was taken out at 2h and 4h respectively, and the number of residual viable bacteria was counted immediately, and compared with the original number of viable bacteria. the

3. Tolerance test of simulated bile salts:

Use pancreatin to make a solution of 1g/L-1.5g/L, and add 0.3% porcine bile salt to the solution, adjust the pH to 7.0-9.0 with 10% NaOH, then filter with a 0.45μm microfiltration membrane and remove bacteria. Inoculate 0.5ml of Enterococcus faecalis ANSE228 live bacteria solution into 4.5ml of simulated bile salt, cultivate for 24 hours to obtain a culture solution, and count the number of viable bacteria of Enterococcus faecalis ANSE228 remaining. Dilute the bacterial solution tenfold in sterilized normal saline, spread it on an MRS plate, and then place it at 30-45°C for static culture for 18-36 hours (Enterococcus faecalis generally needs to be cultured for more than 18 hours before it can grow into an obvious single colony ). the

Counting methods in simulated gastric juice and simulated bile salt tolerance test:

Samples were serially diluted ten-fold with sterile normal saline, and an appropriate dilution was selected for counting. Using MRS agar medium, 0.5mL of the diluted solution was spread on the plate, and cultured at 37°C for 24h. the

The present invention also provides a microecological preparation, which contains the above-mentioned Enterococcus faecalis ANSE228. The micro-ecological preparation is made of Enterococcus faecalis ANSE228 obtained through repeated separation, purification, rejuvenation and other processes, which has strong biological activity, significant probiotics and good stress resistance, and then undergoes production processes such as liquid submerged fermentation to obtain a fermented liquid, and then dried made later. the

The present invention also provides the application of the above-mentioned probiotics in breeding, which is to add the probiotics as an additive to drinking water or feed of farmed animals. Wherein, the addition amount of Enterococcus faecalis ANSE228 in drinking water is 10 ⁸ -10 ¹¹ CFU/L drinking water; the addition amount of Enterococcus faecalis ANSE228 in feed is 10 ⁷ -10 ¹⁰ CFU/kg feed. The farm animals mentioned in the present invention include broiler hens, laying hens and pigs, and also include fish, ducks or ruminants such as cattle or sheep.

The Enterococcus faecalis ANSE228 of the present invention can significantly improve the production performance of animals when the amount of Enterococcus faecalis ANSE228 is only 10 ⁷ CFU/kg in the feed, reduce the feed-to-meat ratio of broilers by 33.14%, and reduce the death rate of piglets to 0 %, the feed-to-egg ratio of laying hens is reduced by 19.8%, which significantly improves the production performance of animals and reduces production costs.

The beneficial effects of the present invention are:

1. Enterococcus faecalis ANSE228 of the present invention has significant probiotics, can significantly inhibit the growth and reproduction of pathogenic bacteria such as Salmonella pullorum, Escherichia coli and Staphylococcus aureus. the

2. The Enterococcus faecalis ANSE228 of the present invention has strong stress resistance, can tolerate simulated gastric acid, simulated bile salt and high temperature environment, and can maintain a high survival rate of viable bacteria, and the survival rate of viable bacteria can reach 80% above. Due to the high temperature in the process of feed processing and pelleting, reaching 75-90°C, the strain needs to tolerate this temperature range to ensure that the animals eat live bacteria, and live bacteria have probiotic properties. Tolerance to gastric acid and bile salts is required to allow Enterococcus faecalis to perform its role in the animal's digestive tract. the

3. The present invention discovers new uses of the known Enterococcus faecalis and opens up a new application field. the

4. After the microecological preparation made of Enterococcus faecalis ANSE228 enters the intestinal tract of animals, the powerful Enterococcus faecalis ANSE228 rapidly activates and proliferates, and at the same time forms beneficial dominant flora in the intestinal microecological environment. the

5. Enterococcus faecalis ANSE228 of the present invention has significant probiotic and stress resistance, not only can improve the feed utilization efficiency of animals, save costs, but also improve the stability of the internal environment of the digestive tract in animals, and promote the absorption and utilization of nutrients. The effects of promoting animal growth and improving economic benefits indicate a good application prospect. the

Description of drawings

Fig. 1 is the inhibition zone of Enterococcus faecalis ANSE228 to Salmonella pullorum. the

Figure 2 is the inhibition zone of Enterococcus faecalis ANSE228 on Escherichia coli. the

Figure 3 is the inhibition zone of Enterococcus faecalis ANSE228 to Salmonella aureus. the

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. the

The preparation of embodiment 1 enterococcus faecalis ANSE228 fermented liquid

Take Enterococcus faecalis ANSE228 (preservation number is CGMCC No.4082, the concentration of viable bacteria is 10 ⁹ CFU/ml), inoculate it in 50ml culture medium for shake flask fermentation culture, the fermentation temperature is 37 ℃, pH value 7.0, static culture , fermentation time 24h.

Among them, the shake flask fermentation medium is composed of the following components: corn flour 15g, soybean meal 20g, glucose 5g, dipotassium hydrogen phosphate 0.15g, potassium dihydrogen phosphate 1.5g, manganese sulfate monohydrate 1.0g, sulfuric acid heptahydrate Magnesium 1.5g, distilled water 1000mL, pH value 7.0. the

After the shake flask fermentation, carry out the fermenter pilot test, take 50ml of shake flask fermentation seed liquid and inoculate it into a 10L fermenter, the liquid volume is 5L, the fermentation temperature is 37°C, the pH value is 7.0, the stirring speed is 300r/min, the fermentation time 36h. The composition of the medium in the pilot test of the fermenter was the same as that of the shake flask fermentation. the

After the fermentation, the fermentation broth was stored in a refrigerator at 4°C for later use. the

Example 2 Probiotic verification of Enterococcus faecalis ANSE228

Prepare an MRS plate with a thickness of about 4 mm, spread 100 μL of bacterial suspension with a concentration of 10 ⁶ of pathogenic bacteria (E. (a small round tube with an inner diameter of 6 mm, an outer diameter of 8 mm, and a height of 10 mm, the two ends of the tube should be smooth), placed on the medium, and gently pressurized to make it contact with the medium without gaps. Add 200-300 microliters of the fermented liquid (activated strain bacterial liquid) prepared in Example 1 dropwise in each small tube, do not allow it to overflow, cultivate at a temperature of 37° C. for 24 hours, and then measure the diameter of the inhibition zone. Each experiment was repeated three times, and the average value was taken. The results are shown in Figure 1-3.

Wherein, the MRS plate is the MRS culture medium sterilized at 121° C., poured into the sterilized plate, and cooled to form a 4 mm thick MRS plate with a smooth surface. The formula of the MRS medium is: peptone 10g, beef extract 10g, yeast extract 5g, K ₂ HPO ₄ 2g, diammonium hydrogen citrate 2g, glucose 20g, sodium acetate 5g, Tween-801mL, MgSO ₄ 7H ₂ O 0.58g, MnSO ₄ ·4H ₂ O 0.25g, agar 16g, distilled water 1000ml, pH=7.0.

The diameter of the inhibition zone of Enterococcus faecalis ANSE228 against Salmonella pullorum was 1.20 cm, and the diameter of the inhibition zone against Escherichia coli and Staphylococcus aureus was 1.16 cm and 1.12 cm, respectively. the

Example 3 Stress resistance verification of Enterococcus faecalis ANSE228

1. Take the preserved 5mL fermented liquid prepared in Example 1 and pour it into the centrifuge tube 1, use ten-fold serial dilution, and spread it on the MRS plate 1. Then put the centrifuge tube 2 containing 5mL of the fermented liquid prepared in Example 1 in a water bath at 80°C and heat it for 15 minutes, take the heated Enterococcus faecalis bacterial liquid and carry out ten-fold stepwise dilution, and spread it on the MRS plate 2 . Finally, the plates before and after heating were cultured at 37° C. for 24 hours, and the number of Enterococcus faecalis ANSE228 before and after heating was calculated. the

The results showed that the survival rate of live bacteria reached 85%. the

2. Tolerance test of simulated gastric juice: Dissolve pepsin in 0.5% normal saline to make the final concentration 3g/L, and adjust the pH value to 2.0 with concentrated hydrochloric acid or 10% NaOH. Take 0.5mL Enterococcus faecalis ANSE228 bacterial suspension and add it into 4.5mL simulated gastric juice (i.e. ten-fold dilution), and quickly mix it fully on a shaker, and then place it at 37°C for static incubation for 2h. After 2 hours of treatment, the culture solution was taken out and the number of residual viable bacteria was counted immediately, and compared with the original number of viable bacteria. The results showed that the survival rate of live bacteria was 96%. the

3. Tolerance test of simulated bile salts: Use pancreatin to make a 1g/L solution, add 0.3% porcine bile salts to the solution, adjust the pH to 8.0 with 10% NaOH, and then use a 0.45 μm microfiltration membrane Filter and sterilize. Inoculate 0.5ml of Enterococcus faecalis ANSE228 live bacteria solution into 4.5ml of simulated bile salt, cultivate for 24 hours to obtain a culture solution, and count the number of viable bacteria of Enterococcus faecalis ANSE228 remaining. The bacterial solution was diluted tenfold in sterilized physiological saline, spread on the MRS plate, and then cultured at 37°C for 24 hours. The results showed that the survival rate of live bacteria was 96%. the

Embodiment 4 broiler growth performance test

120 30-day-old healthy broilers were selected, weighed, and randomly divided into 4 treatment groups. Each treatment had 5 replicates, and each replicate had 6 chickens, 1/2 of which were male and female. The test drinking water is: (1) ordinary drinking water (control group); (2) the concentration of Enterococcus faecalis ANSE228 in drinking water is 10 ¹⁰ CFU/L; (3) the concentration of Enterococcus faecalis ANSE228 in drinking water is 10 ⁹ CFU /L; (4) The concentration of Enterococcus faecalis ANSE228 in drinking water was 10 ⁸ CFU/L. The experimental chickens had free access to food and water, and the feeding management and immunization procedures were in accordance with the broiler feeding management manual.

Table 1 Effect of Enterococcus faecalis ANSE228 on growth performance of broilers

Note: The shoulders of peer data without the same letters indicate significant differences (P<0.05) (the same below)

The results of this experiment showed that Enterococcus faecalis ANSE228 significantly increased the average daily gain (P=0.0123) and feed conversion efficiency (P=0.0006) of broilers. At the same time, when the concentration of Enterococcus faecalis ANSE228 in drinking water was 10 ¹⁰ CFU/L, the feed conversion rate of broilers was the highest.

Embodiment 5 laying hen production performance test:

The experiment was a single-factor design, and 576 commercial laying hens were randomly divided into 3 treatments, each treatment had 6 replicates, each replicate had 8 cages, and each cage had 4 chickens. Treatment 1 was a negative control, fed a basal ration without any drug feed additives; treatment 2 was a positive control, and bacitracin zinc 20 mg/kg was added to the basal ration; Add 3×10 ⁹ CFU/kg Enterococcus faecalis ANSE228 probiotics to the basal diet. The experimental chickens had free access to food and water, and the feeding management and immunization procedures were referred to the laying hen feeding management manual.

Table 2 Effect of Enterococcus faecalis ANSE228 on the performance of laying hens

The results of this test show that Enterococcus faecalis ANSE228 can significantly improve the feed-to-egg ratio of laying hens and increase the egg production of laying hens (p<0.05), and can reduce production costs, improve the production performance of laying hens, increase economic benefits, and effectively It can replace antibiotic additives and is suitable for large-scale promotion and use. the

Embodiment 6 piglet growth performance test

In this experiment, 90 weaned piglets of 35±1 days old and with an average body weight of 7.86±0.06kg were selected and randomly assigned to three treatments. Each treatment had 5 replicates, and each replicate had 6 piglets. For piglets (half male and half female), there was no significant difference in initial body weight among the repeated groups (p>0.05). the

The three treatments were: blank control group, antibiotic control group (75 mg/kg aureomycin) and test group (adding 4×10 ⁹ CFU/kg Enterococcus faecalis ANSE228 probiotics). The experimental pigs had free access to food and water, and the feeding management and immunization procedures were referred to the piglet feeding management manual.

Table 3 Effect of Enterococcus faecalis ANSE228 on growth performance of piglets

The results of this experiment show that Enterococcus faecalis ANSE228 can significantly increase the average daily gain of piglets (p<0.05), improve the feed-to-meat ratio, and significantly reduce the diarrhea rate and death rate of piglets (p<0.05). The production performance and economic benefits of piglets are improved, the antibiotic additives can be effectively replaced, and the method is suitable for large-scale popularization and use. the

Although, the present invention has been described in detail with general description, specific embodiments and experiments above, but on the basis of the present invention, some modifications or improvements can be made to it, which will be obvious to those skilled in the art . Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention. the

Claims (7)

1. An Enterococcus faecium (Enterococcus faecium) ANSE228, the preservation number of which is CGMCC No.4082. 2. a method for cultivating Enterococcus faecalis ANSE228 described in claim 1, is characterized in that, gets Enterococcus faecalis ANSE228 seed liquid 1-5ml, inoculates in the 50-100ml medium and carries out shake flask fermentation culture. 3. The cultivation method according to claim 2, wherein the culture medium is composed of the following components: corn flour 10-25g/L, soybean meal 10-30g/L, glucose 0-15g/L, hydrogen phosphate Dipotassium 0.1-0.2g, potassium dihydrogen phosphate 1.0-2.0g, manganese sulfate monohydrate 0.5-2.0g, magnesium sulfate heptahydrate 0.5-2.0g, distilled water 800-1200mL, pH value 6.5-7.5. 4 . The cultivation method according to claim 2 , wherein the shake flask fermentation conditions are: fermentation temperature 25-45° C., fermentation time 18-36 h, pH value 6.5-7.5, static cultivation. 5. A probiotics, characterized in that it contains Enterococcus faecalis ANSE228 according to claim 1. 6. the application of the probiotics described in claim 5 in breeding is characterized in that, the probiotics is added as an additive to the drinking water or feed of farmed animals, and the farmed animals are broilers, laying hens, pigs, Duck, cow or sheep. 7. The application according to claim 6, characterized in that the addition amount of Enterococcus faecalis ANSE228 in drinking water is 10 ⁸ -10 ¹¹ CFU/L drinking water; the addition amount of Enterococcus faecalis ANSE228 in feed It is 10 ⁷ -10 ¹⁰ CFU/kg feed.

Images