CN103082092A - Optimal selection method of preparation parameters of bacillus licheniformis-xylo-oligosaccharide synbiotics, preparation process and application - Google Patents
Optimal selection method of preparation parameters of bacillus licheniformis-xylo-oligosaccharide synbiotics, preparation process and application Download PDFInfo
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Abstract
The invention relates to an optimal selection method of preparation parameters of bacillus licheniformis-xylo-oligosaccharide synbiotics, a preparation process and an application. The method comprises the following steps of: inoculating bacterial liquid of activated bacillus licheniformis CICC20446 into a liquid culture medium containing 2.5-4 mg.mL<1> of xylo-oligosaccharide according to the capacity ratio of 1%, and placing the mixture in an incubator for shake cultivation for 3.5-4.5h under the conditions of 40 DEG C and 120 r.min<1>. The bacillus licheniformis-xylo-oligosaccharide synbiotics provided by the invention can be capable of obviously improving the daily gain of animals and reducing material-to-weight ratio and diarrhea rate, and has an obvious improvement effect on the growth of the animals.
Description
Technical field
The invention belongs to a kind of animal feeding microecologic regulator, specifically method for optimizing, preparation technology and the application of the first preparation parameter of a kind of bacillus licheniformis-wood oligose symphysis.
Background technology
Enteron aisle is the digestive organs of body maximum, is also maximum immune organ.Stable intestinal microecology group can promote the perfect of function of intestinal canal, can improve production performance and the immune performance of animal.Body suffer external or interior under various factors (as disease, wean etc.) spread effect, enteric microorganism flora structure changes, pernicious bacteria (as Escherichia coli) increases, profitable strain (as Bacillus acidi lactici, bacillus) quantity can reduce, will cause the dysfunction of enteron aisle, occur diarrhoea, poor appetite, to food hypersenstivity, resistance descend, the problem such as sick repeatedly.All the time, the control of function of intestinal canal disorder is take antibiotic as main, but along with the going deep into of research, it is found that, antibiotic effect to microorganism in enteron aisle is logical killing: not only harmful microorganism is had killing action, simultaneously beneficial microbe is also had killing action.In addition, not killed part microorganism to treatment-resistant, it is as a whole no longer responsive to some antibiotic that its result causes certain bacterial classification to do, and finally forms drug resistance by natural evolution modes such as gene mutations.Therefore seek nontoxic, noresidue in natural resources, become the focus of animal and veterinary industry research without the effectively alternative preparation of drug-fast antibiotic.
Probio is to bring into play beneficial effect to improving host's gut flora ecological balance, reaches the active bacteria formulation and the metabolite thereof that improve host health level and healthy good attitude.Have the normal function of keeping enteron aisle, have to promote nutrient absorption, regulate immunity of organism, improve organism metabolism, purify many-sided effect such as intestinal environment.Bacillus licheniformis bacillus licheniformis is common probio in animal body, and it has good benefit to give birth to function.Bacillus licheniformis survives in low pH and bile and can implant intestinal mucosa, and it is good to have a security, and the characteristics that are easy to store are considered to optimal microbe additive.In recent years, along with the raising of China's animal feeding level, the effect of probio descends to some extent.The active bacteria formulation that it is found that probio have poor stability, product category single, be of limited application, the shortcoming such as effect is unstable, have larger difficulty in the product application facet.
A kind of dietary supplements of prebiotics, thus the growth that it can be by optionally stimulating the bacterium in a kind of or minority kind bacterium colony with active and to the host produce wholesome effect improve host health can not be digested food composition.Successful prebiotics should be when the UGI, and major part is digested and can be by gut flora fermented.The most important thing is that it just stimulates the growth of profitable strain, rather than the harmful bacteria of potential pathogenic or corrupt activity is arranged.Prebiotics provides " food " to probio, can be decomposed by beneficial bacteria in enteron aisle to absorb, and promotes the beneficial bacteria growth and breeding.Prebiotics mainly comprises various oligosaccharide kind materials.Wood oligose claims again xylo-oligosaccharide, is described as " superpower Oilgosaccharkdes ", is considered to have the effect of regulating gastrointestinal microflora, is the probiotic that promotes the growth of beneficial bacterium and suppress the harmful bacteria growth.The researcher combines both according to the characteristics of prebiotics and probio, becomes symphysis unit.
Symphysis unit merges use with probio and prebiotics.Symphysis unit has probio and prebiotics double action effect: the physiological bacterial activity that both can bring into play probio, the alternative quantity that increases this bacterium makes prebiotic effect more lasting again, and symphysis unit is to adjusting the intestinal microecology balance and be better than probio and prebiotics using separately.It is a kind of good antibiotic substitute, is also extraordinary probiotics.On the market the first product of bacillus licheniformis-wood oligose symphysis is not reported at present, therefore produced and use significant symphysis is first for bacillus licheniformis-wood oligose symphysis unit's preparation parameter and technical study.
Summary of the invention
The purpose of this invention is to provide method for optimizing, preparation technology and the application of the first preparation parameter of a kind of bacillus licheniformis-wood oligose symphysis, test by in vitro culture, science is chosen the first preparation parameter of bacillus licheniformis-wood oligose symphysis, realize optimum preparation condition, guarantee the result of use of the first product of symphysis.
Principle of the present invention: according to the growth curve of bacillus licheniformis, determine cultivation optimum temperature, the rotating speed of bacillus licheniformis.Utilize bacterium liquid OD value, determine suitable wood oligose addition.Again according to bacillus licheniformis growth for the time G, determine the best incubation time of symphysis unit, finally determine the best preparation technology of bacillus licheniformis-wood oligose symphysis unit.
The method for optimizing of the first preparation parameter of bacillus licheniformis provided by the invention-wood oligose symphysis is as follows:
1, determining of the drafting of bacillus licheniformis growth curve and cultivation temperature, rotating speed: the bacillus licheniformis CICC20446 bacterium liquid after activating is inoculated in the LB fluid nutrient medium of determining pH value (PH=7.0), respectively at 34 ℃-120rmin with the inoculum concentration of Capacity Ratio 1%
-1, 37 ℃-120rmin
-1, 40 ℃-120rmin
-1With 34 ℃-150rmin
-1, 37 ℃-150rmin
-1, 40 ℃-150rmin
-1And 34 ℃-180rmin
-1, 37 ℃-180rmin
-1, 40 ℃-180rmin
-1Condition under shaken cultivation, every 0.5h sampling 1 time, Continuous Cultivation 12h measures its absorbance under the 600nm wavelength, take the time as abscissa, absorbance is the ordinate curve plotting, determines to cultivate optimum temperature, rotating speed according to growth curve;
2, wood oligose determining of the suitableeest addition: add the wood oligose of different proportion in the LB fluid nutrient medium, the concentration of wood oligose is respectively: 0.5mgmL
-1, 1.5mgmL
-1, 2.5mgmL
-1, 3mgmL
-1, 3.5mgmL
-1, 4mgmL
-1, 4.5mgmL
-1, 5mgmL
-1, 6mgmL
-1, 7mgmL
-1, in the conical flask of packing into the culture medium equivalent of each concentration.
Bacillus licheniformis liquid after activation is added respectively in the above-mentioned wood oligose fluid nutrient medium that contains variable concentrations with the inoculum concentration of Capacity Ratio 1%, put shaken cultivation in 40 ℃-120r/min incubator, sample when 2.5～7.5h respectively, according to measuring absorbance under the 600nm wavelength, to determine the wood oligose broiler diets;
3, bacillus licheniformis-wood oligose symphysis unit determining of the suitableeest incubation time: after the suitableeest addition of wood oligose is determined, OD value according to the suitableeest interpolation concentration group of measuring, utilize formula calculate for the time G, thereby according to bacillus licheniformis each time period for the time to determine the suitableeest incubation time of symphysis unit;
G=(t2-t1)/3.322(lgx2-lgx1)
In formula: t1, t2 represent respectively incubation time, and x1, x2 represent respectively the OD value of different time symphysis unit;
4, bacillus licheniformis-wood oligose bacterium liquid viable count detects: utilize the method for plate culture count that bacillus licheniformis-wood oligose bacterium liquid viable count is detected.
The preparation technology of unit is as follows in bacillus licheniformis provided by the invention-wood oligose symphysis:
1, according to the growth curve of bacillus licheniformis, determine that the cultivation optimum temperature of bacillus licheniformis and rotating speed are 40 ℃-120r/min.
2, with the LB fluid nutrient medium, add wood oligose, the optimum content that makes wood oligose is 3.5 mgmL
-1
3, the Bacillus licheniformis liquid after activating is inoculated in content as 3.5mgmL take the inoculum concentration of Capacity Ratio 1%
-1The wood oligose fluid nutrient medium in, put shaken cultivation in 40 ℃-120r/min incubator, the suitableeest incubation time is 4h, namely gets the bacillus licheniformis of buff-wood oligose symphysis unit product after taking-up.
Good effect of the present invention:
1, science is chosen the first preparation parameter of bacillus licheniformis-wood oligose symphysis, realizes optimum preparation condition, guarantees the result of use of the first product of symphysis.
2, consumption is little, and the best interpolation of wood oligose concentration is 3.5mgmL
-1, the Bacillus licheniformis amount is 1.9 * 10
8mL
-1, the raw material consumption of the probiotics of the present invention's preparation can not produce toxic and side effect to animal itself, can not increase production cost simultaneously yet;
3, in the first bacterium liquid of bacillus licheniformis of the present invention-wood oligose symphysis, viable count is 10
8CFUmL
-1Above, viable count is higher.
Description of drawings
Fig. 1 is growth curve under bacillus licheniformis (CICC20446) 120R different temperatures.
Fig. 2 is growth curve under bacillus licheniformis (CICC20446) 150R different temperatures.
Fig. 3 is growth curve under bacillus licheniformis (CICC20446) 180R different temperatures.
Fig. 4 be in different time sections symphysis unit bacterium liquid bacillus licheniformis for the time.
The specific embodiment
(1) drafting of bacillus licheniformis growth curve and condition of culture determines
In the aseptic experiment chamber, bacillus licheniformis liquid after activating in the bacterium amount that connects of 1% capacity ratio is inoculated in (composition of LB culture medium and the ratio: peptone 10.0g in the 100mL conical flask of LB fluid nutrient medium that 50mL determined pH value (PH=7.0) that be equipped with, dusty yeast 5.0g, NaCl10.0g, agar 15.0g, distilled water 1.0L, PH7.0), respectively at 34 ℃-120rmin
-1, 37 ℃-120rmin
-1, 40 ℃-120rmin
-1With 34 ℃-150rmin
-1, 37 ℃-150rmin
-1, 40 ℃-150rmin
-1And 34 ℃-180rmin
-1, 37 ℃-180rmin
-1, 40 ℃-180rmin
-1Condition under shaken cultivation, every 0.5h sampling 1 time, Continuous Cultivation 12h, measure its absorbance under the 600nm wavelength, take the time (h) as abscissa, absorbance (OD value) is ordinate curve plotting (seeing Fig. 1,2,3), determines that according to growth curve bacillus licheniformis cultivates optimum temperature, rotating speed.As seen from Figure 1, the growth that this bacterial strain can be good under three temperature, rotating speed, but 40 ℃, 120rmin
-1The growing state of bacillus licheniformis under condition is better than under other temperature, speed conditions.The optimum condition of therefore cultivating this bacterial strain is 40 ℃, 120rmin
-1
Calculate bacterial population with the method for plate culture count after bacillus licheniformis activation of the present invention, determine that bacteria containing amount is 1.9 * 10
8CFUmL
-1
(2) wood oligose determining of the suitableeest addition
To join in sterilized LB fluid nutrient medium through the wood oligose (xylo-oligosaccharide, Shandong Longli Biology Science and Technology Co., Ltd produces and sells) of 0.22 μ m miillpore filter degerming, the concentration of wood oligose is respectively: 0.5mgmL
-1, 1.5mgmL
-1, 2.5mgmL
-1, 3mgmL
-1, 3.5mgmL
-1, 4mgmL
-1, 4.5mgmL
-1, 5mgmL
-1, 6mgmL
-1, 7mgmL
-1The culture medium of each concentration is packed in the 100mL conical flask, every bottle of 50mL.
Bacillus licheniformis liquid after activation is added respectively in the above-mentioned wood oligose fluid nutrient medium that contains variable concentrations with the inoculum concentration of capacity ratio 1%, put 40 ℃-120rmin
-1Shaken cultivation in incubator respectively in 2.5～7.5h sampling, every 0.5h sampling 1 time, is measured its absorbance (take LB fluid nutrient medium group as the blank group, seeing Table 1) under the 600nm wavelength.
By as seen from Table 1, along with the prolongation of incubation time, bacterium liquid OD value is in rising trend, and namely bacillus licheniformis CICC20446 is in continuous growth.Interpolation concentration is 3.5mgmL
-1In the culture medium of wood oligose, bacillus licheniformis CICC20446 bacterial concentration reaches the highest.The test explanation, appropriate wood oligose can promote the growth of bacillus licheniformis, excessive wood oligose can suppress its growth.The suitableeest addition of wood oligose is 3.5mgmL
-1
Table 1 variable concentrations wood oligose is to bacillus licheniformis CICC20446 affects on the growth (OD value)
(3) bacillus licheniformis-wood oligose symphysis unit determining of the suitableeest incubation time
After the suitableeest addition of wood oligose is determined, according to the OD value of the suitableeest interpolation concentration group of measuring, utilize formula calculate for the time G, thereby according to bacillus licheniformis each time period for the time (see Table 2, Fig. 2) with definite their the suitableeest incubation time;
G=(t2-t1)/3.322(lgx2-lgx1)
In formula: t1, t2 represent respectively incubation time, and x1, x2 represent respectively the OD value of different time symphysis unit.
By table 2 and Fig. 4 as seen, add 3.5mgmL
-1The bacillus licheniformis of wood oligose group reproduction speed when 4h is the fastest, namely 4h required for the time the shortest.As seen, add 3.5mgmL
-1Wood oligose can promote bacillus licheniformis CICC20446 growth, and the suitableeest incubation time is 4h.
The different incubation times of table 2 are to bacillus licheniformis affects on the growth in the first bacterium liquid of symphysis
| Time (h) | The OD value | For the time G(h) |
| 2.5 | 0.134 | - |
| 3 | 0.225 | 0.669 |
| 3.5 | 0.325 | 0.943 |
| 4 | 0.486 | 0.374 |
| 4.5 | 0.576 | 2.04 |
| 5 | 0.701 | 1.765 |
| 5.5 | 0.843 | 1.879 |
| 6 | 1.019 | 1.827 |
| 6.5 | 1.178 | 2.39 |
| 7 | 1.312 | 3.218 |
| 7.5 | 1.438 | 3.779 |
(4) the first viable count of bacillus licheniformis-wood oligose symphysis detects
Utilize known the method for plate culture count that bacillus licheniformis-wood oligose bacterium liquid viable count is detected, in the first bacterium liquid of bacillus licheniformis-wood oligose symphysis, viable count is 10
9CFUmL
-1
The preparation technology of unit is as follows in bacillus licheniformis-wood oligose symphysis:
1, with the LB fluid nutrient medium, add wood oligose, the content that makes wood oligose is 3.5mgmL
-1
2, the Bacillus licheniformis liquid after activating is inoculated in content as 3.5mgmL take the inoculum concentration of Capacity Ratio 1%
-1The wood oligose fluid nutrient medium in, put 40 ℃-120rmin
-1Shaken cultivation in incubator, the suitableeest incubation time are 4h, namely get the bacillus licheniformis of buff-wood oligose symphysis unit product after taking-up.
The present invention carries out following feeding experiment:
One, materials and methods
(1) experimental animal and design
Adopt the design of completely random group experiment, select the tri-crossbreeding (duroc * Landrace * Large White) of 36 21 age in days wean; Average weight is 4.25kg ± 0.71kg, is divided at random 4 groups, every group of 3 repetitions, and each repeats 3 pigs.Each group examination pig is filled with respectively and feeds LB fluid nutrient medium (control group), LB fluid nutrient medium+3.5mgmL
-1Wood oligose+1.9 * 10
8CFUmL
-1Bacillus licheniformis (symphysis tuple), LB fluid nutrient medium+1.9 * 10
8CFUmL
-1Bacillus licheniformis (bacillus licheniformis group), LB fluid nutrient medium+3.5mgmL
-1Wood oligose (wood oligose group), each organizes to fill with and feeds once every day, fills with the amount of feeding and is 15mLd
-1Experimental period is 14 days.
(2) test diet
The experimental basis daily ration is corn-soybean meal diet, and nutritional requirements meets NRC(1998) the feeding piglet standard.
(3) growth performance is measured
Duration of test is observed piglet mental status, diarrhoea situation and duration every day.Reached the test 20:00 of last 1 day before on-test and stop feeding, as usual supply water, morning next day, the examination pig is pursued and only weigh; After off-test, to repeat (hurdle) as unit claims surplus material amount, record feed consumption rate, calculate the average daily gain (ADG) of experimental period, average daily ingestion amount (ADFI), material anharmonic ratio (F/G) and diarrhea rate.
Diarrhea rate (%)=(a diarrhoea number * diarrhoea number of days every day)/(test pig's head number * test number of days) * 100.
(2) experimental result and analysis
Table 3 bacillus licheniformis-wood oligose symphysis unit affects Growth Performance of Weaning Piglets
The different letter representation significant differences (P＜0.05) of colleague's numerical value shoulder motes are without letter representation difference not significantly (P〉0.05).
As shown in Table 3, symphysis tuple, Di clothing Ya Bao Rod bacterium group, wood oligose group are compared all with control group and can be improved the weanling pig daily gain, but the ratio that the symphysis tuple improves is higher than other groups, can improve 50.67%; Reduce simultaneously material anharmonic ratio and the diarrhea rate of weanling pig, the ratio that the symphysis tuple reduces is wanted height than bacillus licheniformis group and wood oligose group, improves respectively 18.73% and 3.35%, illustrates that symphysis unit is improved effect to piglet growth performance.
Claims (4)
1. a bacillus licheniformis is closed-method for optimizing of wood oligose symphysis unit preparation parameter, it is characterized in that:
Determining of a, growth curve and cultivation temperature: the bacillus licheniformis CICC20446 bacterium liquid after activating is inoculated in definite pH value in 7.0 LB fluid nutrient medium take the inoculum concentration of Capacity Ratio 1%, cultivate under different temperatures and different revolution, every 0.5 h sampling 1 time, Continuous Cultivation 12h, measure its absorbance under 600 nm, take the time as abscissa, absorbance draws growth curve as ordinate, determines optimum growth temperature, rotating speed;
B, wood oligose be determining of the suitableeest addition: the wood oligose after 0.22 μ m millipore filter degerming is added the LB fluid nutrient medium of sterilization, the concentration of wood oligose is respectively: 0.5 mgmL
-1, 1.5 mgmL
-1, 2.5 mgmL
-1, 3 mgmL
-1, 3.5 mgmL
-1, 4 mgmL
-1, 4.5 mgmL
-1, 5 mgmL
-1, 6 mgmL
-1, 7 mgmL
-1, the culture medium of each concentration to be packed in container bottle, the liquid in each container bottle equates;
Bacillus licheniformis liquid after activation is added respectively in the above-mentioned wood oligose fluid nutrient medium that contains variable concentrations with the inoculum concentration of Capacity Ratio 1%, put 40 ℃ of-120 rmin
-1Shaken cultivation in incubator is sampled 1 time every 0.5 h in 2.5 ~ 7.5 h, according to measuring absorbance under 600 nm wavelength, to determine the wood oligose broiler diets;
C, bacillus licheniformis close-wood oligose symphysis unit determining of the suitableeest incubation time: after the suitableeest addition of wood oligose is determined, OD value according to the suitableeest interpolation concentration group of measuring, utilize formula calculate for the time G, thereby according to bacillus licheniformis each time period for the time to determine their the suitableeest incubation time;
G?=(t2-t1)/3.322(lgx2-lgx1)
In formula: t1, t2 represent respectively incubation time, and x1, x2 represent respectively the OD value of different time symphysis unit;
D, bacillus licheniformis close-wood oligose symphysis unit bacterium liquid viable count detects: utilize the method for plate culture count to bacillus licheniformis close-wood oligose bacterium liquid viable count detects.
One kind as claimed in claim 1 bacillus licheniformis close-preparation technology of the method for optimizing of wood oligose symphysis unit preparation parameter is as follows:
A, with the LB fluid nutrient medium, add wood oligose, the content that makes wood oligose is 2.5 ~ 4mgmL
-1
B, the Bacillus licheniformis liquid after activating are inoculated in content as 3.5 ~ 4.5 mgmL take the inoculum concentration of Capacity Ratio 1%
-1The wood oligose fluid nutrient medium in, put 40 ℃ of-120 rmin
-1Shaken cultivation in incubator is taken out after 2.5 ~ 7.5 h, namely gets the bacillus licheniformis of buff-wood oligose symphysis unit product.
3. the preparation technology of the method for optimizing of bacillus licheniformis according to claim 2-wood oligose symphysis unit preparation parameter, it is characterized in that: the optimum content of the wood oligose in the LB fluid nutrient medium is 3.5 mgmL
-1Bacillus licheniformis is inoculated in the wood oligose fluid nutrient medium at 40 ℃ of-120 rmin
-1Shaken cultivation in incubator, the suitableeest incubation time are 4 h.
4. application that yellow bacillus licheniformis-the wood oligose symphysis preparation technology of unit produces product as claimed in claim 2: fill with every day and feed once, fill with and feed amount 15 ~ 20 mL.
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| CN106689798A (en) * | 2016-11-17 | 2017-05-24 | 大连理工大学 | Marine synbiotic additive, preparation method thereof, as well as application of marine synbiotic additive in breeding of stichopus japonicus |
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Application publication date: 20130508 |