CN110229770A - A kind of bacillus subtilis formulation and preparation method - Google Patents
A kind of bacillus subtilis formulation and preparation method Download PDFInfo
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Abstract
The invention discloses a kind of bacillus subtilis formulation and preparation methods.Fermentation liquid, the in mass ratio skimmed milk power of 10wt% are prepared by culture medium fermentation of bean curd yellow pulp water, the bacillus subtilis SB13 fermentation liquid of 10wt% wheat bran and 80wt% is obtained by freeze-drying.The survival rate of bacterium SB13 is up to 97.71% in the bacteria preparation.The bacteria preparation is in artificial simulation gastric juices and pancreatic juice environment after 37 DEG C of processing 4h, and bacterium survival rate retains 15.61% and 43.80%, and endo-glucanase enzyme activity storage rate is respectively 34.17% and 42.00%, and the clump count detected is respectively 2.2 and 5.65*107(cfu/ml).The bacteria preparation has significant saccharification to pannage, chicken feed and ox feed, is added in the animal and fowl fodders such as pig, chicken and ox, is expected to improve livestock and poultry to the absorption and use efficiency of feed.
Description
Technical field
The invention belongs to bacteria preparation technical fields, and in particular to a kind of bacillus subtilis bacteria preparation and preparation method.
Background technique
Bacillus subtilis (Bacillus subtilis) because have resistance is strong, growth cycle is short, to nutritional requirement not
It is high, easily the unique biological characteristics such as storage and be widely used.The bacterium because of pollution-free, noresidue, the spies such as do not develop drug resistance
Point is given the ratification in China can be used as a kind of probiotic bacteria of feed addictive.Due to there is efficient extracellular protein secretory
Energy, extremely low nutritional requirement, stores the unique biological characteristics such as simple at good gene expression basis, it is considered to be has very much
The industrial potential strain of biorefinery ability.The bacterium, which enters animal intestinal tract, can sprout into rapidly the nutrition with metabolism
Type bacterium adjusts intestinal flora balance, enhances animal body immunity, some bacillus subtilises can secrete higher inscribe Portugal
Dextranase, hydrolysis or cellulose substances or derivative in dilation feed improve absorption of the animal to high-fiber feeding stuff
Utilization rate reduces feed cost.
Bean curd yellow pulp water, is the bean curd draining that bean curd is formed in process of production, and yield is the 3-5 of soybean dry weight
Times.Bean curd yellow pulp water solid content accounts for about 1%, mainly by soluble protein, oligosaccharide, vitamin, lipid, microelement
Deng composition.Bean curd yellow pulp water is the chief component of tofu wastewater, is developed and utilized to bean curd yellow pulp water, then can reduce
Its pollution to environment, help enterprise save processing sewage generate it is high-cost simultaneously, produce additional valuable production
Product.
The present invention produces bacillus subtilis fermentation liquor using bean curd yellow pulp water as fermentation raw material, passes through best gemma shape
At the screening of rate fermentation parameter, protective agent and carrier, additional amount and drying process screening, bacillus subtilis bacterium system is obtained
Agent and preparation process are preferably applied to livestock and poultry cultivation for bacillus subtilis, improve the saccharifying of cellulose in feed, mention
High livestock and poultry provide help to the absorption and use efficiency of feed.
Summary of the invention
The purpose of the present invention is to provide a kind of bacillus subtilis formulation and preparation methods.Bacillus subtilis of the invention
Bacterium is processed into powder, and the raising of bacterium survival rate therein is preferably applied to animal and fowl fodder for bacteria preparation, improves fiber in feed
It is helpful to the absorption and use efficiency of feed to improve livestock and poultry for the saccharification of element.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of bacillus subtilis formulation, raw material include bacillus subtilis SB13 fermentation liquid, skimmed milk power, wheat bran；By weight
Percentage meter includes: bacillus subtilis SB13 fermentation liquid 80%, skimmed milk power 10%, wheat bran 10%.
A kind of preparation method of bacillus subtilis formulation, comprising the following steps:
(1) preparation of bacillus subtilis SB13 mother liquor: bacillus subtilis SB13 the connecing by 1wt % that 20% glycerol is saved
Kind of amount is inoculated in basal medium that (10 g/L of peptone, 5 g/L of beef extract, sodium chloride 5 g/L, pH7.0,121 DEG C of high pressures are gone out
Bacterium 25min), 37 DEG C of 160r/min constant temperature incubations for 24 hours, obtain bacterium SB13 mother liquor；
(2) preparation of bacillus subtilis SB13 fermentation liquid: with bean curd yellow pulp water, the NaOH solution that concentration is 10wt% is added to adjust
The pH value of bean curd yellow pulp water is inoculated with bacillus subtilis SB13 by the inoculum concentration of 1wt % to 7.0,121 DEG C of high pressure sterilization 30min
Mother liquor, 160r/min, 35 DEG C of constant temperature incubation 72h obtain the bacillus subtilis SB13 fermentation liquid that spore content is greater than 90 %；
It (3) is by mass percentage fermentation liquid 80% made from step (1), skimmed milk power 10%, wheat bran 10% mix three kinds of raw materials
It closes uniformly, using -80 DEG C of precoolings, -50 DEG C of temperature, the dry 48h of 23 pa freeze drying process of vacuum degree obtains bacillus subtilis
Preparation.
Wheat bran is the 60 mesh fine powder of mistake by grinding in above-mentioned steps (3).
A kind of application of bacillus subtilis formulation in animal and fowl fodder.
The beneficial effects of the present invention are:
It is screened by fermentation time and fermentation temperature, obtains the gemma of bacillus subtilis SB13 after 35 DEG C of constant temperature incubation 72h
Formation rate is best, by the screening to different protective agents and carrier, obtains the skimmed milk power and 10wt% wheat that additional amount is 10wt%
Bran has good protective effect to bacillus subtilis SB13, is screened by different drying process, obtains freeze drying process
Best to bacillus subtilis survival rate protecting effect, the bacteria preparation is after artificial simulation gastric juices and pancreatic juice, the every milli of clump count
It rises and still remains with up to 107Viable count.The bacteria preparation can significantly promote pannage, in ox feed and chicken feed cellulose to more
The conversion of sugar is conducive to improve livestock and poultry to the absorption and use efficiency of feed, can be applied to livestock and poultry cultivation field, have fabulous answer
With value.
Specific embodiment
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention
Technical solution is described further, but the present invention is not limited only to this.
Skimmed milk power and wheat bran used are that purchase obtains in the market, and yellow serofluid used is Tofu processing factory in Tofu processing
Discarded bean curd draining in the process, the bacillus subtilis SB13 in the present invention is that strain is had in laboratory by oneself, and technology is embodied
It is as follows.
The acquisition of the best spore forming rate of embodiment 1
1.1 fermentation times are on the sporiferous influence of bacterium
The preparation of bacillus subtilis SB13 mother liquor: the bacillus subtilis SB13 that 20% glycerol is saved presses the inoculum concentration of 1wt %
(10 g/L of peptone, 5 g/L of beef extract, sodium chloride 5 g/L, pH7.0,121 DEG C of high pressure sterilizations are inoculated in basal medium
25min), 37 DEG C of 160r/min constant temperature incubations for 24 hours, obtain bacterium SB13 mother liquor.
Bacillus subtilis SB13 fermentation mother liquor is placed in bean curd yellow pulp water culture medium according to the inoculum concentration of 1wt%,
160r/min, 37 DEG C of constant temperature incubations sample when cultivating 12,24,36,48,72,96h, using Malachite green stain method to production
Raw gemma is counted, and calculates gemma generation rate, calculation formula are as follows: gemma according to gemma (green) and vegetative cell (red)
Generation rate=gemma number/(gemma number+vegetative cell number) * 100%, is as a result shown in 1.Known to bacterium SB13 when fermenting to 72h, gemma generates
Rate highest, reaches 78.8%.
The different incubation time spore forming rates (%) of table 1
The spore forming rate of bacillus subtilis SB13 under 1.2 condition of different temperatures
Bacterium SB13 mother liquor is prepared, is inoculated in bean curd yellow pulp water culture medium by the inoculum concentration of 1wt%, is respectively placed in 20,25,30,35
With 40(DEG C) it cultivates under the conditions of temperature, 160r/min cultivation and fermentation 72h, sample detection gemma number the results are shown in Table 2.It is found that bacterium
SB13 spore forming rate when fermentation temperature is 35 DEG C is best, is 92.48%.
Spore forming rate (%) under the conditions of the different cultivation temperatures of table 2
The screening of 2. protective agent of embodiment
The screening of 2.1 best protection agent types
It ferments preparation bacterium SB13 fermentation liquid using yellow serofluid as culture medium, is separately added into 10 % glycerol in the fermentation liquid of 100ml, 10
% skimmed milk power, 10wt% trehalose, 10 wt% maltodextrins, 10 wt% sucrose, 0.5wt % sodium alginate, the room temperature at 25 DEG C
Drying, is settled to 100ml for each sample after drying again, takes 1ml that 9mL physiological saline is added, is diluted to different multiples, plate
Coating measures clump count, for control, to calculate survival rate, survival rate=(dry the viable bacteria of 1ml sample liquid after sample constant volume before drying
The viable count of 1ml sample liquid before protective agent is added in number/fermentation liquid) * 100%.As a result table is shown in 3: as seen from the table, skimmed milk power is to withered
The protecting effect of careless bacillus SB13 is best, and survival rate is up to 94.2 ± 2.1%, is significantly higher than other protective agents.
Survival rate of the different protective agents of table 3. to bacterium
Concentration screening is added in 2.2 best protection agent
Using skimmed milk power as protective agent, the skimmed milk power of the amount of 2wt%-12wt% is added in the bacterium SB13 zymocyte liquid of 100mL,
After room temperature drying, the normal saline dilution of sample 100mL will be dried, take 100ul to be diluted to -6, -8 powers, 100ul is taken to be coated with
It counts, clump count is with log10(cfu/ml) it counts, the results are shown in Table 4.As seen from the table, skimmed milk power with additional amount be 10wt% when, bacterium
The clump count that SB13 drying obtains is maximum.
The screening of 4. best protection agent additional amount of table
3. vector selection of embodiment
3.1 most suitable carrier screenings
Using soya-bean milk yellow serofluid as culture medium, the fermentation preparation SB13 fermentation liquid under the optimum condition of embodiment 1, in the hair of 100ml
Be separately added into zymotic fluid 10wt% Mai ?, 10wt% rice bran, 10wt% rice husk, 10wt% dregs of beans, 10wt% starch, the room temperature at 25 DEG C
Drying, is settled to 100ml for each sample after drying again, takes 1ml that 9mL physiological saline is added, is diluted to different multiples, plate
Coating measures clump count, preceding for control to dry, and calculates survival rate.As known from Table 5, wheat bran, dregs of beans and rice husk are to withered grass gemma
The protecting effect of bacillus SB13 is preferable, and there were significant differences between three, but the survival rate of wheat bran is up to 90.2 ± 1.1%, therefore
Select wheat bran for the optimum carrier of bacterium SB13.
Influence of 5. different carriers of table to bacterium survival rate
3.2 most suitable carrier additional amount screenings
Using wheat bran as carrier, the wheat bran of the amount of 2wt %-10wt %, room temperature drying are added in the bacterium SB13 zymocyte liquid of 100mL
Afterwards, drying sample is settled to 100mL with physiological saline, 1ml is taken to be diluted to -6, -8 powers, take 100ul coating to count, bacterium colony
Number is by log10(cfu/ml) in terms of, it the results are shown in Table 6.As seen from the table, wheat bran with additional amount be 10wt% when, bacterium SB13 drying obtain
Clump count it is maximum.
The screening of 6. optimum carrier additional amount of table
4. finished product preparation process of embodiment
It is fermented using bean curd yellow pulp water as culture medium and prepares bacterium SB13, under 1 optimum condition of embodiment in 100ml bacterium SB13 respectively
Protective agent concentration is added: 10wt% skimmed milk power and 10wt% wheat bran are dried using following drying process after mixing
Dry, wherein room temperature is dried: 25 degree, being dried 72 hours.Spray drying: 85 degree of inlet temperature, 60 degree of outlet temperature.Freeze-drying :-
80 degree of precoolings, temperature -50 are spent, 23 pa of vacuum degree, dry 48h.60 degree of drying: it 60 degree, dries 24 hours.Drying the results are shown in Table
7, as known from Table 7, the preferable technique of drying condition is air drying and freeze-drying, since air drying is easy sample occur
The problems such as agglomeration, uneven drying weighs, Gu using freeze-drying preferred aspect drying process.
Influence of the different drying conditions of table 7. to bacterium protecting effect
5. bacteria preparation finished product survival rate of embodiment calculates
Respectively plus with optimal addn zymocyte liquid is added in the mixture of best protection agent, carrier and best protection agent and carrier
In, freeze-drying, the fermenation raw liquid with unprotected dose and carrier is control.It is de- that 10wt% is individually added into 100ml fermentation liquid
Rouge milk powder and 10wt% wheat bran, freeze-drying, are settled to 100ml for each sample after drying again, take 1ml that 9mL physiology salt is added
Water carries out bacterium colony counting after diluting different multiples.It the results are shown in Table 8, compared with stoste, protective agent be added and carrier survival rate is significant
Raising has no that there were significant differences between three processing that statistics display is added, bacterium SB13 after addition skimmed milk power+wheat bran mixture
Survival rate is up to 97.71%.The bacteria preparation clump count of acquisition reaches 6.4*108(cfu/ml)
Several protective agents of table 8. bacterium survival rate under the conditions of freeze-drying
The external stomach and intestine simulated environment survival condition analysis of 6. enzyme bacteria preparation of embodiment
Gut simulation experiment: respectively to the simulated gastric fluid of 450mL (KCl 6.9mmol/L, KH2PO4 0.9 mmol/L、NaHCO3
25 mmol/L、NaCl 47.2 mmol/L、MgCl2 0.1 mmol/L、(NH4)2CO3 0.5mmol/L、CaCl2 0.075
Mmol/L adjusts pH value to 3.0 with HCl, pepsin is added before use to 2000U/mL) and simulated intestinal fluid (KCl
6.8mmol/L、KH2PO4 0.8 mmol/L、NaHCO3 85 mmol/L、NaCl 38.4 mmol/L、MgCl20.33 mmol/
L、CaCl20.6 mmol/L adjusts pH value to 7.0 with HCl, trypsase is added before use to 100U/mL.) in be added 50g
SB13 bacteria preparation, ZRS-8GD intelligently dissolution 37 DEG C of laboratory apparatus set temperature, 120 turns/min of mixing speed, respectively at
5mL sample is taken out when 0h, 2h, 4h, it is to be measured.It takes in 1mL sample and 9mL physiological saline, is diluted to 10-5, then 100uL is taken to be coated with.
Bacterium colony, which counts, calculates survival rate, and DNS method tests and analyzes endo-glucanase enzyme activity and keeps situation.It the results are shown in Table 9, it can from table
Know, after artificial simulation gastric juices are handled, there is apparent decline in the survival rate of bacterium SB13, after handling 2h, survival rate difference
For 18.96 % and 22.29 %, clump count is respectively 2.55*107And 3.8*107Cfu/ml, after treatment endoglucanase
Also there is apparent decline in the storage rate of enzyme activity, but fall is lower than clump count.
The analysis of the survival condition of the in-vitro simulated gastric juice of 9. bacteria preparation of table and pancreatic juice
7. enzyme bacteria preparation of embodiment detects the saccharifying power of high microsteping substance
Choose pannage (feed corporation,Ltd, Hua Long group, Fujian Province), chicken feed (feed corporation,Ltd, Hua Long group, Fujian Province)
It is saccharification target with ox feed (Hebei Ji Tai feed companies), adds water to mix in each feed and the water ratio of 1:1 in mass ratio equal
It is even, the SB13 bacillus subtilis formulation of all kinds of processing feed inoculation 5wt% of 100g is weighed respectively, is placed in 37 DEG C of ferment at constant temperature,
0,2,9,12,24(h) sample detection total sugar content, three repetitions of each processing the results are shown in Table 8.As seen from the table: with do not ferment
It compares, after 2h ferments, significant rising all occurs in the total sugar content of all feeds, illustrates bacterium SB13 in each feed
Fiber is known as saccharification result, illustrates to add the absorption and use efficiency that bacteria preparation is beneficial to livestock and poultry to feed.Generally, bacteria preparation
Significant difference is occurred as soon as when cultivating 2h to the carbonization power of pannage, total sugar content is not shown substantially in culture later
Difference is write, this may be further metabolized with total reducing sugar by bacterium related；After bacteria preparation fermentation, chicken feed contains in fermentation 2-12h total reducing sugar
Amount successively reaches peak, after fermentation for 24 hours, significant decline occurs, thus it is speculated that total reducing sugar is further metabolized by bacterium to be caused；Ox
After feed fermentation, until fermenting for 24 hours, the total reducing sugar of ox feed is in rising trend always, this may be with content of cellulose in ox feed
It is high related.
The saccharifying power (mg/mL) to feed of 10 enzyme bacteria preparation of table
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with repair
Decorations, are all covered by the present invention.
Claims (4)
1. a kind of bacillus subtilis formulation, it is characterised in that: raw material includes bacillus subtilis SB13 fermentation liquid, defatted milk
Powder, wheat bran；Percentage includes: bacillus subtilis SB13 fermentation liquid 80%, skimmed milk power 10%, wheat bran 10%.
2. a kind of preparation method of bacillus subtilis formulation as described in claim 1, which comprises the following steps:
(1) preparation of bacillus subtilis SB13 mother liquor: the bacillus subtilis SB13 that 20% glycerol is saved, by connecing for 1wt%
Kind amount is inoculated in basal medium 37 DEG C, and 160r/min constant temperature incubation for 24 hours, obtains bacterium SB13 mother liquor；
(2) preparation of bacillus subtilis SB13 fermentation liquid: using bean curd yellow pulp water as culture medium, adding concentration is the NaOH of 10wt%
Solution adjusts the pH value of bean curd yellow pulp water to 7.0,121 DEG C of high pressure sterilization 30min, is inoculated with bacillus subtilis by the inoculum concentration of 1wt%
Bacterium SB13 mother liquor, 160r/min, 35 DEG C of constant temperature incubation 72h obtain bacillus subtilis SB13 of the spore content greater than 90% and ferment
Liquid；
It (3) is by weight percentage fermentation liquid 80% made from step (1), milk powder 10%, wheat bran 10% mix three kinds of raw materials
Uniformly, using -80 DEG C of precoolings, -50 DEG C of temperature, the dry 48h of 23 pa freeze drying process of vacuum degree obtains bacillus subtilis system
Agent.
3. a kind of preparation method of bacillus subtilis formulation according to claim 2, it is characterised in that: in step (3)
Wheat bran is the 60 mesh fine powder of mistake by grinding.
4. a kind of application of bacillus subtilis formulation as described in claim 1 in animal and fowl fodder.
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Cited By (3)
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| CN110819579A (en) * | 2019-12-24 | 2020-02-21 | 中国海洋大学 | Preparation method of solid bacillus subtilis microbial inoculum |
| CN113773996A (en) * | 2021-09-24 | 2021-12-10 | 南京工业大学 | Preparation method and application of feeding bacillus subtilis |
| CN115025231A (en) * | 2022-06-15 | 2022-09-09 | 江西省药品检验检测研究院 | Dry inoculation method of traditional Chinese medicinal materials |
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| CN110819579A (en) * | 2019-12-24 | 2020-02-21 | 中国海洋大学 | Preparation method of solid bacillus subtilis microbial inoculum |
| CN113773996A (en) * | 2021-09-24 | 2021-12-10 | 南京工业大学 | Preparation method and application of feeding bacillus subtilis |
| CN115025231A (en) * | 2022-06-15 | 2022-09-09 | 江西省药品检验检测研究院 | Dry inoculation method of traditional Chinese medicinal materials |
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