CN103981116A - Culture method and application of enterococcus faecium - Google Patents
Culture method and application of enterococcus faecium Download PDFInfo
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Abstract
The invention relates to a method for cultivating an enterococcus faecium strain and its application. The enterococcus faecium WEI-9 is inoculated to a special liquid fermentation medium by the inoculation amount of 1% and fermented under the conditions of a 50 L fermentation tank, 37 DEG C, 50 rpm and stuffiness for 10-12 h; and the yield of number of live bacteria reaches up to 2*10<9>-3*10<9>CFU / mL. The fermentation broth has thalli collected by a high speed centrifuge tubular-type centrifuge, is mixed with a special protective agent in the volume ratio of 1:5, and subjected to pre-freezing at -70 DEG C for 2-3 h, and drying freezing for 44h in a vacuum freezer dryer; and the obtained dry bacterium powder is crushed by a pulverizer and mixed with bran in the same size according to a certain proportion, so as to obtain probiotics products. The product can be used as a microbial feed additive and added to animal feed, can adjust intestinal microecological balance of animals, improve the production performance of livestock and poultry and disease resistant ability, and is expected to become the alternative products of antibiotics.
Description
Technical field
The present invention relates to cultural method and the application thereof of an Enterococcus faecalis, belong to microbial technology field.
Background introduction
The growth promotion health-care agents such as abuse of antibiotics in current feed and livestock and poultry cultivation, chemical synthetic drug, hormone, beta-stimulants, heavy metal, tranquilizer, cause series of problems and consequence, comprising: animal products drug residue is serious; Bacterial drug resistance strengthens, Resistant strain increases; Various resistance germs can be passed to people by food, produce the disease that is difficult to healing.These problems and serious consequence thereof are greatly threatening food safety, affect the health of animals and humans.
In view of the problems that exist in above-mentioned feed and livestock and poultry cultivation, China starts " feed safety engineering ", and has forbidden a collection of toxicity and residual larger medicine.< < fodder industry " 12 " the development program > > of country explicitly points out the production of the fodder additives of exploitation and production, the especially substitute antibiotics that will accelerate novel fodder additive.Therefore, probiotics becomes the focus of industrialization development as the fodder additives of a kind of green, safety, is one of potential product of substitute antibiotics.
As autochthonous flora in digestive tube, milk-acid bacteria has regulating intestinal canal colony balance, suppresses the effects such as harmful bacteria growth, strengthening immunity.Current commercially available probiotics product has raising livestock and poultry production performance in actual use, the effect of control disease of alimentary tract of livestock husbandry and birds, but also there are some defects in a lot of product: viable count does not reach the amount of product annotation, causes the effect of product to be had a greatly reduced quality; Post-treatment condition too fierceness causes dried bacterium powder product to contain mortality thalline, and microorganism active is lost in a large number.
Therefore, develop a kind of low cost, high yield and can keep for a long time active lactobacillus micro-ecological preparation, for development green feed, guaranteeing that feed safety has great importance.
Summary of the invention
The present invention is directed to the deficiencies in the prior art provides large scale fermentation cultural method and the application in producing probiotics thereof of an Enterococcus faecalis (Enterococcus faecium) WEI-9CGMCC No.7745.Faecium (Enterococcus faecium) WEI-9, and in June, 2013 19Song China Committee for Culture Collection of Microorganisms common micro-organisms center preservation, be numbered CGMCC No.7745, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of Chinese Academy of Sciences postcode: 100101.
The large scale fermentation cultural method of faecium provided by the present invention (Enterococcus faecium) WEI-9CGMCC No.7745, be that faecium (Enterococcus faecium) WEI-9CGMCC No.7745 is inoculated in the dedicated liquid substratum of this bacterium, standing or stuffiness is cultivated at 30-40 ℃.The formula of described dedicated liquid substratum is: in every 1000mL water, contain: whey powder 10-30g, peptone 10-30g, ammonium citrate 1-3g, sodium acetate trihydrate 2-8g, bitter salt 0.1-0.5g, Manganous sulfate monohydrate 0.02-0.08g, three hypophosphite monohydrate hydrogen dipotassium 1-3g, tween 80 0.5-1.5g, pH value is 6.5-7.5.
In above-mentioned cultural method, inoculative proportion is 1%; Culture temperature is 37-40 ℃; Standing or stuffiness is cultivated, and incubation time is 10-12h.
The formula of preferred dedicated liquid substratum is: in every 1000mL water, contain: whey powder 21.34g, peptone 21.94g, ammonium citrate 2g, sodium acetate trihydrate 5g, bitter salt 0.2g, Manganous sulfate monohydrate 0.05g, three hypophosphite monohydrate hydrogen dipotassium 2g, tween 80 1g, pH value is 7.
Under 50L fermentor tank condition, utilize above-mentioned substratum at 37 ℃, 50rpm, faecium (Enterococcus faecium) the WEI-9CGMCC No.7745 fermented liquid viable count that stuffiness condition bottom fermentation obtains reaches 2 * 10
9-3 * 10
9cFU/mL, and under similarity condition, the viable count of faecium (Enterococcus faecium) WEI-9CGMCC No.7745 in MRS substratum is 6.5 * 10
8cFU/mL, the substratum after optimization makes viable count improve 3-4 doubly.
The application of faecium provided by the present invention (Enterococcus faecium) WEI-9CGMCC No.7745 in probiotics preparation; that the fermented liquid that faecium (Enterococcus faecium) WEI-9CGMCC No.7745 is obtained according to above-mentioned substratum and culture condition fermentation is collected after thalline by high speed tubular-bowl centrifuge; mix with protective material; prior to-70 ℃ of pre-freezes; then be placed in the dry rear active microbe powder that obtains of vacuum freeze drier; bacterium powder, with mixing with the wheat bran of same granularity after pulverizer fragmentation, is obtained to probiotics product.
The rotating speed that described high speed tubular-bowl centrifuge is collected thalline is 15000-20000rpm.
The described thalline protective material formula of processing for vacuum lyophilization is: every 1000mL water contains: skim-milk 100g, maltodextrin 10g, trehalose 15g, glycerine 5g, sorbyl alcohol 20g.Protective material with centrifugal after bacterium mud according to volume mass, than 5: 1, mix; in-70 ℃ of pre-freeze 2-3h; then after being placed in vacuum freeze drier vacuum-freeze-dry 44h, finish; freeze-drying parameter is as follows: vacuum tightness is less than 30Pa; condenser temperature is-40 ℃ to-50 ℃; the dry bacterium powder obtaining, with mixing according to a certain percentage with the wheat bran of same granularity after pulverizer fragmentation, obtains probiotics product.Former bacterium powder viable count after freeze-drying is 2 * 10
11-3 * 10
11cFU/g, the wheat bran of same granularity of take can dilute the probiotics bacterium powder product as containing different viable counts as carrier.
In a word, according to above-mentioned cultural method and fermented liquid post-treating method, can prepare low cost, highly active probiotics bacterium powder product, as livestock and poultry microorganism feed addictive, can improve Production of Livestock and Poultry ability, resistance against diseases, finally reduce the use of feeding antibiotic.
Accompanying drawing explanation
Fig. 1 is the optimization of faecium (Enterococcus faecium) the suitableeest fermentation initial pH value of WEI-9CGMCC No.7745.
Fig. 2 is faecium (Enterococcus faecium) the WEI-9CGMCC No.7745 optimization of suitable leavening temperature.
Fig. 3 is the optimization of faecium (Enterococcus faecium) the suitableeest technical grade compounded carbons of WEI-9CGMCC No.7745.
Fig. 4 is faecium (Enterococcus faecium) the WEI-9CGMCC No.7745 optimization of suitable nitrogenous source.
Fig. 5 is the growth curve of faecium (Enterococcus faecium) WEI-9CGMCC No.7745 under 50L fermentor tank condition.
Embodiment
Embodiment mono-: the optimization of suitable fermentation culture conditions of faecium (Enterococcus faecium) WEI-9CGMCC No.7745
One, the optimization of the most suitable growth envrionment conditions of faecium (Enterococcus faecium) WEI-9CGMCC No.7745
1, the optimization of suitable initial pH value
Investigate the impact of different initial pH value on faecium (Enterococcus faecium) WEI-9CGMCC No.7745 growth.From the preservation inclined-plane (MRS substratum) of faecium (Enterococcus faecium) WEI-9CGMCC No.7745, choosing a ring lawn is connected to the 250mL triangular flask that 100mL liquid MRS substratum is housed, in 37 ℃, 180rpm shaking culture 11h, 1% the inoculum size of take is connected to respectively and 300mL pH is housed as 5.5,6.0,6.5,7.0, in the 500mL triangular flask of 7.5 liquid MRS substratum, in 37 ℃, standing cultivation 16h, after starting to cultivate respectively at 2h, 4h, 6h, 10h, 12h, 14h, 16h sampling and measuring OD
600, and when cultivate finishing, measure the viable count of each sample.Optimum result shows, faecium (Enterococcus faecium) WEI-9CGMCC No.7745 the most suitable growth initial pH value scope is 6.5-7.5.
2, the optimization of suitable leavening temperature
Investigate the impact of differing temps on faecium (Enterococcus faecium) WEI-9CGMCC No.7745 growth.From the preservation inclined-plane (MRS substratum) of faecium (Enterococcus faecium) WEI-9CGMCC No.7745, choosing a ring lawn is connected to the 250mL triangular flask that 100mL liquid MRS substratum is housed, in 37 ℃, 180rpm shaking culture 11h, 1% the inoculum size of take is connected in the 500mL triangular flask that the liquid MRS substratum that 300mL pH is 7.0 is housed, respectively at 25 ℃, 30 ℃, 37 ℃, 40 ℃, 42 ℃, 45 ℃, 48 ℃, standing cultivation 10h, after starting to cultivate respectively at 2h, 4h, 6h, 8h, 10h sampling and measuring OD
600, and when cultivate finishing, measure the viable count of each sample.Optimum result shows, faecium (Enterococcus faecium) WEI-9CGMCC No.7745 optimum growth temperature scope is 37-40 ℃.
3, the optimization of suitable rotating speed
Investigate the impact of different rotating speeds on faecium (Enterococcus faecium) WEI-9CGMCC No.7745 growth.From the preservation inclined-plane (MRS substratum) of faecium (Enterococcus faecium) WEI-9CGMCC No.7745, choosing a ring lawn is connected to the 250mL triangular flask that 100mL liquid MRS substratum is housed, in 37 ℃, 180rpm shaking culture 11h, 1% the inoculum size of take is connected in the 500mL triangular flask that the liquid MRS substratum that 300mL pH is 7.0 is housed, in 37 ℃, rotating speed is adjusted to respectively 50rpm, 100rpm, 200rpm, and take standing cultivation as contrast, cultivate 10h, after starting to cultivate respectively at 2h, 4h, 6h, 8h, 10h sampling and measuring OD
600, and when cultivate finishing, measure the viable count of each sample.Optimum result shows, different rotating speeds does not have significant difference to the impact of faecium (Enterococcus faecium) WEI-9CGMCC No.7745 growth, the growth that shows faecium (Enterococcus faecium) WEI-9CGMCC No.7745 is not remarkable to the demand of oxygen, in real attenuation process in order to save the energy, can without ventilation condition bottom fermentation.
Two, the optimization of the suitableeest fermention medium component of faecium (Enterococcus faecium) WEI-9CGMCC No.7745
1, the optimization of suitable carbon source
Adopt above-mentioned optimum growing condition, investigated faecium (Enterococcus faecium) WEI-9CGMCC No.7745 to single carbon source, comprise glucose, lactose, sucrose, maltose, glycerine and industrial conventional compounded carbons, comprise the situation of utilizing of molasses, whey powder, glucose syrup, oligomeric isomaltose and dextrin.Adopt and above-mentioned same inoculation method, fixedly nitrogenous source is peptone 10g/L, beef powder 5g/L, yeast powder 5g/L, the pH value of the substratum that contains different carbon sources (content is 20g/L) is 7.0, culture temperature is 37 ℃, standing cultivation 10h, after starting to cultivate respectively at 2h, 4h, 6h, 8h, 10h sampling and measuring OD
600, and when cultivate finishing, measure the viable count of each sample.Consider raw materials cost and thalli growth situation, finally determine that the suitableeest carbon source of faecium (Enterococcus faecium) WEI-9CGMCC No.7745 growth is whey powder.
2, the optimization of suitable nitrogenous source
Adopt above-mentioned optimum growing condition, investigated the utilize situation of faecium (Enterococcus faecium) WEI-9CGMCC No.7745 to several nitrogenous sources such as peptone, extractum carnis, corn steep liquor, corn starch, yeast extract paste, urea, SODIUMNITRATE and ammonium sulfate.Adopt and above-mentioned same inoculation method, fixedly carbon source is lactose 20g/L, the pH value of the substratum that contains different nitrogen sources (content converts according to the nitrogen content of the every kind of nitrogenous source principle consistent with nitrogen content in MRS substratum) is 7.0, culture temperature is 37 ℃, standing cultivation 10h, after starting to cultivate respectively at 2h, 4h, 6h, 8h, 10h sampling and measuring OD
600, and when cultivate finishing, measure the viable count of each sample.Consider raw materials cost and thalli growth situation, finally determine that the suitableeest nitrogenous source of faecium (Enterococcus faecium) WEI-9CGMCC No.7745 growth is peptone.
3, Plackett-Burman test
The fermention medium that is used for cultivating faecium (Enterococcus faecium) WEI-9CGMCC No.7745 contains 8 kinds of components, utilizes Plackett-Burman design to filter out the factor that viable bacteria output is had to remarkably influenced from 8 kinds of raw materials.Each factor and level thereof are in Table 1, Plackett-Burman test design and the results are shown in Table 2, and the regression analysis of data and significance analysis are in Table 3.The analytical results of table 3 shows, peptone is the most remarkable factor that affects faecium (Enterococcus faecium) WEI-9CGMCC No.7745 fermentation viable bacteria output, and the influence of all the other factors is followed successively by whey powder, sodium acetate trihydrate, tween 80, bitter salt, three hypophosphite monohydrate hydrogen dipotassiums, ammonium citrate, Manganous sulfate monohydrate.Wherein peptone, whey powder, Manganous sulfate monohydrate and three hypophosphite monohydrate hydrogen dipotassiums are positive correlation on the impact of viable count, and ammonium citrate, sodium acetate trihydrate, bitter salt, tween 80 are negative correlation on the impact of viable count.Choose whey powder, peptone and sodium acetate trihydrate and do further optimization Test.
Table 1Plackett-Burman test design factor level
Table 2Plackett-Burman test design and result
The significance analysis of table 3 regression coefficient and factor of influence
3, steepest hill climbing test
Response surface fit equation is ability sufficient approximation truth in the close region of investigating only, therefore set up effective fit equation after should first approaching maximum producing region again.3 important factors that affect faecium (Enterococcus faecium) WEI-9CGMCC No.7745 viable count selecting according to Plaekett-Burman testing sieve, use steepest hill climbing test to find out the region of viable bacteria output maximum.Steepest hill climbing test the results are shown in Table 4.As shown in Table 4, when whey powder concentration is that 20g/L, peptone concentration are 20g/L, sodium acetate trihydrate concentration while being 5g/L, viable count output is maximum, when condition in its both sides or when farther viable count output decline gradually.Can determine that thus concentration that viable count reaches three kinds of major influence factors in peaked fermention medium should be near region whey powder 20g/L, peptone 20g/L, sodium acetate trihydrate 5g/L.
Table 4 steepest climbing experimental result
4, Box-Behnken optimization design experimental result
According to steepest hill climbing test result, using whey powder concentration, be that 20g/L, peptone concentration are that 20g/L, sodium acetate trihydrate concentration are that 5g/L adopts Box-Behnken method further to optimize as center condition, each level of factor is in Table 5, and Optimum Design Results is in Table 6.Adopt the viable count data analysis in Design-Expert software his-and-hers watches 6, obtain simulation curve, corresponding simulation equation is:
Y＝16.24+0.91X1+1.45X2-0.11X4+0.5X1X2-0.025X1X4+0.2X2X4-2.06X1
2-2.03X2
2-2.11X4
2。
Simulation equation is carried out to variance analysis, the results are shown in Table 7.As shown in Table 7, the P < 0.05 of simulation equation, illustrates that simulation equation is significant on the impact of viable count.The cross term of simulation equation is all not remarkable on the impact of viable count, and the once item of whey powder and peptone and the quadratic term of three factors are on viable count impact significantly, wherein the quadratic term of three factors is utmost point remarkably influenced, this illustrates that the relation of each factor and viable count is not simple linear relationship, but a complicated secondary relation.
By utilizing the response surface tracing analysis of Design-Expert Software on Drawing, response surface curve exists the maximum value of viable count, using software prediction to go out viable count reaches peaked condition and is: whey powder 21.34g/L, peptone 21.94g/L, sodium acetate trihydrate 5g/L, viable count is 16.64 * 10 to the maximum
8cFU/mL.
In order to verify the reliability of above-mentioned optimum result, according to the Optimal compositions of fermentation medium filtering out and fermentation condition, carried out twice parallel laboratory test, viable count is respectively 15.89 * 10
8cFU/mL and 16.52 * 10
8cFU/mL.Experimental value and theoretical value are basically identical, illustrate that above-mentioned optimization method has good practicality and reliability.
Table 5Box-Behnken designs each level of factor
Table 6Box-Behnken design and result
The regression coefficient of each parameter in table 7 simulation equation
Embodiment bis-: the fermentation culture of faecium (Enterococcus faecium) WEI-9CGMCC No.7745
From the preservation inclined-plane (MRS substratum) of faecium (Enterococcus faecium) WEI-9CGMCC No.7745, choosing a ring lawn is connected to the 500mL triangular flask that the fermenting substratum that the above-mentioned optimization of 300mL is good is housed, in 37 ℃, standing cultivation 10-12h, 1% the inoculum size of take is connected in the 50L fermentor tank that the dedicated liquid fermention medium that 30L pH is 7.0 is housed, in 37 ℃, carry out fermentation culture, in fermenting process, rotating speed is controlled at 50rpm, 10-12h is cultivated in stuffiness, pH is down to 4.2 left and right, fermentation ends, fermented liquid viable count is greater than 2 * 10
9cFU/mL, and under similarity condition, the viable count in the MRS substratum of faecium (Enterococcus faecium) WEI-9CGMCC No.7745 before optimization is 6.5 * 10
8cFU/mL, the substratum after optimization makes viable count improve 3-4 doubly.Described dedicated liquid fermention medium is in embodiment mono-, to optimize the optimum fermention medium obtaining, formula is: in every 1000mL water, contain: whey powder 21.34g, peptone 21.94g, ammonium citrate 2g, sodium acetate trihydrate 5g, bitter salt 0.2g, Manganous sulfate monohydrate 0.05g, three hypophosphite monohydrate hydrogen dipotassium 2g, tween 80 lg, pH value is 7.
Embodiment tri-: the vacuum lyophilization of the fermented liquid of faecium (Enterococcus faecium) WEI-9CGMCC No.7745 is processed
The fermented liquid of faecium (Enterococcus faecium) the WEI-9CGMCC No.7745 making according to embodiment bis-is through high speed tubular-bowl centrifuge, centrifugal collection thalline under the condition of 15000-20000rpm.In order to reduce vacuum lyophilization processing cost, after raising freeze-drying, the survival rate of bacterium powder product, has selected 4 kinds of protective material formulas (in Table 8) to be compared.By 4 kinds of protective materials through sterilizing, according to volume mass, than 5: 1, the thalline after centrifugal evenly mixed; be placed in-70 ℃ of pre-freeze 2-3h, put into temperature and be down to-40 ℃--the cold-trap of the vacuum freeze drier of 50 ℃, open vacuum pump; suction is to 10Pa, about freeze-drying 44h.Take out bacterium powder, measure viable count, calculate survival rate; result shows; protective material 3 is suitable for the vacuum lyophilization of faecium (Enterococcus faecium) WEI-9CGMCC No.7745 fermented liquid and processes, and survival rate can reach 90%, and bacterium powder viable count is greater than 2 * 10
11cFU/g.
Table 8 protective material numbering and formula
Claims (8)
1. the cultural method of an Enterococcus faecalis and application, is characterized in that, described faecium (Enterococcus faecium) is WEI-9, and deposit number is CGMCC No.7745.
2. cultural method claimed in claim 1, is characterized in that: faecium (Enterococcus faecium) WEI-9CGMCC No.7745 is inoculated in the dedicated liquid substratum of this bacterium, and standing or stuffiness is cultivated at 30-40 ℃.The formula of described dedicated liquid substratum is: in every 1000mL water, contain: whey powder 10-30g, peptone 10-30g, ammonium citrate 1-3g, sodium acetate trihydrate 2-8g, bitter salt 0.1-0.5g, Manganous sulfate monohydrate 0.02-0.08g, three hypophosphite monohydrate hydrogen dipotassium 1-3g, tween 80 0.5-1.5g, pH value is 6.5-7.5.
3. cultural method according to claim 2, it is characterized in that: the screening formulation of described dedicated liquid substratum is: in every 1000mL water, contain: whey powder 21.34g, peptone 21.94g, ammonium citrate 2g, sodium acetate trihydrate 5g, bitter salt 0.2g, Manganous sulfate monohydrate 0.05g, three hypophosphite monohydrate hydrogen dipotassium 2g, tween 80 1g, pH value is 7.
4. according to cultural method described in claim 2-3, it is characterized in that: inoculative proportion is 1%; Culture temperature is 37-40 ℃; Standing or stuffiness is cultivated, and incubation time is 10-12h.
5. the cultural method of faecium claimed in claim 1 and application, is characterized in that: application is to prepare probiotics take faecium (Enterococcus faecium) WEI-9CGMCC No.7745 as main raw material.
6. application according to claim 5; it is characterized in that: the preparation method of described probiotics is: the fermented liquid of faecium (Enterococcus faecium) WEI-9CGMCC No.7745 after first order seed shaking flask and the enrichment of 50L fermentor tank collected by high speed tubular-bowl centrifuge; mix with protective material; prior to-70 ℃ of pre-freezes; then be placed in the dry rear active microbe powder that obtains of vacuum freeze drier; bacterium powder, with mixing with the wheat bran of same granularity after pulverizer fragmentation, is obtained to probiotics product.
7. application according to claim 6, is characterized in that: the rotating speed that described high speed tubular-bowl centrifuge is collected thalline is 15000-20000rpm; Protective material formula is: in every 1000mL water, contain: skim-milk 100g, maltodextrin 10g, trehalose 15g, glycerine 5g, sorbyl alcohol 20g.
8. according to the application described in claim 6-8; it is characterized in that: by protective material with centrifugal after bacterium mud according to volume mass, than 5: 1, mix; in-70 ℃ of pre-freeze 2-3h; then after being placed in vacuum freeze drier vacuum-freeze-dry 44h, finish; freeze-drying parameter is as follows: vacuum tightness is less than 30Pa, and condenser temperature is-40 ℃ to-50 ℃.
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| CN105002091A (en) * | 2015-08-25 | 2015-10-28 | 中农颖泰林州生物科园有限公司 | Enterococcus faecium spray drying protective agent and spray drying method |
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| CN104560717A (en) * | 2014-12-15 | 2015-04-29 | 北京伟嘉人生物技术有限公司 | Protection agent applied to feeding enterococcus faecium freeze-drying preparation and preparation method of protection agent |
| CN105087420A (en) * | 2015-03-30 | 2015-11-25 | 北京伟嘉人生物技术有限公司 | High-density fermentation medium and fermentation technology for forage-use enterococcus faecium |
| CN105002091A (en) * | 2015-08-25 | 2015-10-28 | 中农颖泰林州生物科园有限公司 | Enterococcus faecium spray drying protective agent and spray drying method |
| CN107177522A (en) * | 2016-03-09 | 2017-09-19 | 北京大伟嘉生物技术股份有限公司 | One plant height activity forage plant lactobacillus and its cultural method and application |
| CN106190926A (en) * | 2016-08-30 | 2016-12-07 | 林州中农颖泰生物肽有限公司 | A kind of enterococcus faecalis fermentation medium |
| CN108707569A (en) * | 2018-06-13 | 2018-10-26 | 青岛蔚蓝生物股份有限公司 | Enterococcus faecium high-efficiency fermenting culture medium and its fermentation culture method |
| CN108707569B (en) * | 2018-06-13 | 2022-03-29 | 青岛蔚蓝生物股份有限公司 | Enterococcus faecium efficient fermentation culture medium and fermentation culture method thereof |
| CN112522165A (en) * | 2020-12-29 | 2021-03-19 | 湖北华扬科技发展有限公司 | Liquid lactic acid bacteria for reducing disease rate of weever and application thereof |
| CN113717899A (en) * | 2021-10-09 | 2021-11-30 | 广州金水动物保健品有限公司 | Preparation method of feeding enterococcus faecium raw powder |
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