CN102093967B - A strain of Enterococcus faecium derived from mink and its application - Google Patents

Abstract

The invention relates to an Enterococcus faecium strain and application thereof. Specifically related to Enterococcus faecium MDF1104, patent preservation number CGMCC No.4237. It also relates to the application of Enterococcus faecium MDF1104 in carbohydrate fermentation and cholesterol degradation; especially related to the application of Enterococcus faecium MDF1104 alone or in combination with conventional carriers can increase mink feed intake, daily weight gain, enhance the immune function of the body, and can prevent or Treating seasonal diarrhea in minks.

Description

A strain of Enterococcus faecium derived from mink and its application

technical field

The invention relates to a strain of Enterococcus faecium isolated from mink, and particularly relates to its application in the ration of fur animals.

technical background

Microecological preparations are live probiotic preparations that play a beneficial role by improving the balance of the host's intestinal flora. With their advantages of green safety, no toxic side effects, and no residues, they have gradually become the main force to replace antibiotic additives. Bacteria of the genus Enterococcus are widely distributed in nature and are common in some vegetables and plants. They are also a type of microorganisms that normally exist in the digestive tract of humans and animals, and are mainly colonized in the ileum and colon. The application of Enterococcus as a probiotic mainly focuses on Enterococcus faecalis and Enterococcus faecium. Enterococcus faecium belongs to lactic acid bacteria. Because it grows fast, has good adhesion ability, and can produce lactic acid and some antibacterial substances, it is widely used as Probiotic preparations. Studies have shown that the probiotics composed of Enterococcus faecium can increase the body weight of young livestock and poultry, improve the digestibility of nutrients, improve feed remuneration, reduce the rate of diarrhea, relieve stress, and reduce mortality. In addition to the above functions, Enterococcus can also directly participate in the metabolism of cholesterol, and adjust the abnormal metabolism to make it return to normal. As a microecological agent, Enterococcus is widely used in the production of broiler chickens, fattening pigs, fattening cattle, piglets and dairy cows. In addition, probiotics have obvious immune-promoting effects on the body, can promote the development of the neonatal intestinal immune system, and can also promote the local and systemic immune function of the intestinal mucosa, and can regulate both specific and non-specific immune responses. Maintain the normal level of immune function when the body is in a healthy state. Although probiotics have many of the above-mentioned immunomodulatory effects, for bacterial species, not only are there differences in immune effects between species, but also differences between strains. In addition, the intake time, supply method and addition amount also have an impact on the immune regulation effect. The host also produces different effects due to differences in species, age, physiological conditions, and living environments.

Enterococcus faecium exists widely in the environment and belongs to the intestinal symbiotic bacteria. It is dominant in many animals within 2 to 3 days after birth, and is a facultative anaerobic lactic acid bacteria, which is very suitable for production and application. At present, there are many products on the market that contain Enterococcus faecium strains, such as South Korea's "Mami Love", probiotic products Ventrux, Bioflorin and Fiormil produced in Germany and Sweden, etc., the number of live bacteria is about 10 ^8-9 / g products, currently products containing Enterococcus faecium have also been widely used in animal production, such as the feed additive "Ubita" produced in South Korea. Feeding direct-fed microorganisms containing Enterococcus faecium to dairy cows during the transition period can promote forage digestion in the rumen and increase milk production. Feeding beef cattle direct feed microorganisms containing Enterococcus faecium can promote the adaptation of beef cattle to high-concentrate diets and prevent acidosis. Properly applied Enterococcus faecium can not only promote the digestion and absorption of nutrients, reduce serum cholesterol levels, maintain the balance of intestinal flora, but also regulate the immune function of the body, stimulate the development of immune organs such as the thymus, spleen and bursa of Fabricius, and enhance macrophages. Phagocytic activity of cells, activation of intestinal-associated lymphoid tissue, promotion of immunoglobulin A production, regulation of immune function of the body.

Regarding the lactic acid bacteria additive, the research and application in the production of fur animals is less researched at home and abroad. Based on the probiotic effect of the probiotic additive, the present invention proposes a safe, stable and highly active lactic acid bacteria derived from fur animals. and test its advantages.

Contents of the invention

Isolate lactic acid bacteria from the intestinal tract of healthy adult mink, take 20g of small intestinal content, add 100ml of sterile water, and shake on a shaker for 30min to fully dissolve the particulate matter, gradient dilution, isolation and culture by pouring culture method, 37 ° C for 72 hours to select those with soluble bacteria The colony of the calcium circle was purified by streaking on a plate, purified for 3-4 generations, and examined under a microscope. The purified lactic acid bacteria were inserted into the MRS liquid medium to detect their microbiological characteristics, such as morphological and physiological characteristics (Table 1 ) and the ability of sugar catabolism (Table 2), and the degradation function of cholesterol in vitro culture (Table 3). Carry out mouse safety (Table 4) detection and the impact on mink production performance (Table 5).

The Enterococcus faecium MDF1104 was preserved on October 21, 2010 in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences) No. CGMCC No.4237. Its classification is named: Enterococcus faecium, Latin name: Enterococcusfaecium.

Description of drawings

Fig. 1 Degradation of cholesterol by Enterococcus faecium MDF1104 under in vitro culture conditions, the abscissa is the culture time (unit: hour), and the ordinate is the degradation rate (%).

Below in conjunction with embodiment and figure further illustrate the present invention:

Example I

Morphological observation, microscopic examination and detection of physiological and biochemical indicators of Enterococcus faecium MDF1104

Under sterile conditions, use an inoculation loop to pick up Enterococcus faecium MDF1104 cultured for 18-24 hours, smear it on a clean glass slide, make slides, and observe the morphology and staining results with Gram staining. Enterococcus faecium was inoculated in the corresponding medium or culture conditions under aseptic conditions, and the physiological and biochemical indicators were detected and observed. The test results are shown in Table 1

Table 1 Morphological and physicochemical test results of Enterococcus faecium MDF1104

Detection Indicator Features Morphology Spherical, two chained or scattered a Positive Sporulation no Catalase -

Oxidase - grow in air + Grow at 65°C + Grow at 45°C + Grow at 15°C + pH value (cultivation 24h) 4.29 6.5% NaCl growth + pH9.6 growth + pH4.5 growth +

Example II

Detection of different carbohydrate fermentation activities of Enterococcus faecium MDF1104

Under sterile conditions, use an inoculation loop to pick a small amount of Enterococcus faecium MDF1104 that grows vigorously, and culture it for 18-24 hours, inoculate it into different sugar fermentation tubes produced by Beijing Land Bridge Technology Co., Ltd., and culture it at a constant temperature of 37°C for 18-24 hours, and observe the fermentation The color of the liquid changes to judge the fermentation of different carbohydrates.

Table 2 Fermentation activity of different carbohydrates of Enterococcus faecium MDF1104

Detection Indicator Features Detection Indicator Features Glucose + Raffinose + Mannose + Sorbitol + Melezitose - Melibiose + Fructose + Galactose + Salicin + Mannitol + Laetrile + Arabic candy + Ribose + Sodium Gluconate - Trehalose + Sucrose + xylose + Cellobiose + D + Aesculin + Maltose + Fermenting glucose to produce gas - Lactose +

It can be seen from Tables 1 and 2 that Enterococcus faecium MDF1104 is Gram-positive, can grow in a wide temperature range, has strong acid production ability after 24-hour culture, and can widely use carbohydrates to generate energy. It is a lactic acid bacterium with excellent characteristics.

Example III

Cholesterol degradation by Enterococcus faecium MDF1104 in vitro

Inoculate the purified and cultivated Enterococcus faecium MDF1104 into the newly prepared and sterilized MRS liquid medium, culture at 37°C for 18-24 hours, take samples every four hours, centrifuge to get the supernatant, and use the o-phthalaldehyde method to determine The ability of lactic acid bacteria to degrade cholesterol within 24 hours.

Table 3 Degradation rate of Enterococcus faecium MDF1104 to cholesterol

sampling time Cholesterol degradation rate% 4 38.45 8 32.05 12 32.05 16 32.55 twenty four 31.07

It can be seen from Table 3 and Figure 1 that Enterococcus faecium MDF1104 can degrade cholesterol under in vitro culture conditions, and can be degraded efficiently throughout the culture period, with an average degradation rate of 33.23%, and a maximum of 38.45% in the logarithmic division phase , which shows that the mink-derived Enterococcus faecium MDF1104 has a good degradability to cholesterol, and its degradation efficiency does not decrease much during the in vitro culture period, which also shows that MDF1104 has its unique advantages in the degradation of high-cholesterol food or feed.

Example IV

Effects of Enterococcus faecium MDF1104 on the safety and production performance indexes of mice

Inoculate Enterococcus faecium MDF1104 into 10ml of liquid medium with an inoculum size of 1%, culture at 37°C for 18 hours, centrifuge at 5000rmp/min for 10 minutes, and dilute to 10 ⁷ cfu/ml and 10 ⁹ cfu/ml with physiological saline. Select healthy adult Kunming mice, 15 mice in each group, each mouse in the experimental group was orally perfused with 0.3ml of MDF1104 dilution solution of different concentrations every day, and the control group was perfused with the same amount of normal saline every day, continuous perfusion for 20 days, and observation for 10 days , observe every day, record the situation of food intake and body weight. Regular weighing, daily monitoring of feed intake, slaughter experiment at 30 days, weighing of thymus, spleen weight, calculation of immune organ index.

Table 4. Effect of Enterococcus faecium MDF1104 on the safety and production performance indicators of mice

group Only number of deaths Average daily weight gain g Average daily feed intake g Immune Organ Index control group 2 9.90 12.98 0.57% MDF1104-10 ⁷ 0 10.80 18.19 0.56% MDF1104-10 ⁹ 0 11.63 18.27 0.59%

It can be seen from Table 4 that the mice were perfused with different doses of Enterococcus faecium MDF1104 from mink, and no mice died, and the daily feed intake and average daily weight gain of the mice in the MDF1104 group were significantly higher than those in the control group. The organ index of the low-dose group was lower than that of the control group, and that of the high-dose group was slightly higher than that of the control group. It can be known that Enterococcus faecium MDF1104 is not lethal to mice. And it can promote the daily weight gain and average daily feed intake of mice, and the high-dose group can promote the immune function of mice.

Example V

Effect of Enterococcus faecium MDF1104 on the performance of mink

Inoculate Enterococcus faecium into liquid culture medium at 1% inoculum amount, ferment at 37°C for 24 hours, select healthy minks in the breeding period, 20 for each group, and feed each mink with feed 10 ⁹ cfu/g dry per day in the test group. The substance Enterococcus faecium MDF1104 was not added to the control group, and the control group was weighed regularly. The feeding situation was monitored daily, and the diarrhea situation of each mink was tracked. They were fed continuously for 30 days, slaughtered, and the weight of the immune organs was weighed to calculate the immune organ index.

Table 5 Effect of Enterococcus faecium MDF1104 on mink performance

group Only a few diarrhea Only number of deaths Average daily weight gain g Average daily feed intake g Immune organ index g control group 11 4 22.28 368.81 0.36 MDF1104-10 ⁹ cfu 4 1 23.33 384.38 0.40

It can be seen from Table 5 that adding Enterococcus faecium MDF1104 to the mink diet during the breeding period can reduce the incidence of seasonal diarrhea in mink during the period from the end of August to the beginning of September. Compared with the control group, the diarrhea rate decreased by 40%, and the mortality rate decreased. 10%, can significantly increase the mink daily feed intake by more than 4%, the average daily weight gain rate increases by about 5%, and the immune organ index increases slightly.

in conclusion

The above results show that Enterococcus faecium MDF1104 involved in the present invention can increase the daily feed intake and daily gain of mice and minks, and enhance the immune function of animals. Enterococcus faecium MDF1104 can be used alone or combined with conventional carriers to prevent Occurrence of seasonal diarrhea in mink, improved animal performance.

Claims (3)

1. a faecium (Enterococcus faecium) MDF1104 preserves numbering CGMCC No.4237.

2. faecium MDF1104 claimed in claim 1 is improving the mink daily ingestion amount, the application of day weight gain aspect.

3. feed pre-mixing material, feed pre-mixing material contains the described faecium MDF1104 of claim 1.

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