CN103202386B - Application of Lactobacillus casei fermentation liquor to feeds - Google Patents
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Abstract
The invention relates to application of Lactobacillus casei fermentation liquor to feeds, wherein the collection name of the Lactobacillus casei is Lactobacillus casei lyT-7, the collection unit is China Center for Type Culture Collection (CCTCC), and the collection number is CCTCC M2010197. 10 tons of Lactobacillus casei fermentation liquor obtained through fermentation is dried via spray and then added into detoxified rapeseed meal to prepare the feed additive with microbial content of the Lactobacillus casei being 109 living bacteria/g. After candida and the Lactobacillus casei for fermentation are utilized, not only is the role of detoxification played, but also the content of rapeseed meal protein and the nutritional ingredient of the rapeseed meal are improved; and when the Lactobacillus casei is applied to conventional feeds, the piglet feeding experiments prove that the survival rate, the disease resistance and the weight increment effect are effectively improved.
Description
Technical field
The invention belongs to biofermentation technique and field of fodder, be specifically related to the application of a kind of Lactobacillus casei in feed.
Background technology
Antibiotic is widely used as feed addictive, but the problem such as bacterial drug resistance and medicament residue that antibiotic produces as feed addictive can work the mischief to human health.Therefore, studying effective antibiotic feed additive substitute products comes into one's own day by day.
Antibacterial peptide molecular weight is little, heat endurance is strong.On the one hand, high temperature when antibacterial peptide can tolerate feed granulating; On the other hand, in the time of scale fermenting and producing, through high temperature enrichment process, can fully kill miscellaneous bacteria body and not cause antibacterial peptide inactivation, and product there will not be the Environmental and ecological problems causing because of the diffusion of engineering bacteria after applying.Meanwhile, because of antibacterial peptide molecular weight, be difficult for thering is immunogenicity.In view of these advantages of antibacterial peptide, its research and development are become to the frontier nature problem of studying in the world Substitutes For Antibiotic at present.
At present, domestic is mainly Cecropin AD---yeast preparation as Additive Production application.Most livestock and poultry test is also carried out around this antibacterial peptide.Pertinent literature report has: 1, " antimicrobial peptide preparation replaces the effect of antibiotic in Weaned Growing Pig Diets " (" Chinese feed " calendar year 2001 the 18th phase 13-14 page), by replace antibiotic with antibacterial peptide in the feed of weanling pig, diarrhoea, the production performance etc. of piglet are contrasted.Result shows, antibacterial peptide Antidiarrheic effect during stress of baby pigs caused ablaction is poor unlike antibiotic.Appropriate antibacterial peptide is better than the somatotrophic effect of antibiotic, but the antibacterial peptide of high dose has reduced the growth of piglet.2, " the effect comparison of antibacterial peptide and antibiotic feeding dorking " (" Guangdong feed " the 02nd phase in 2004), find that antibacterial peptide has the growth of promotion and improves immunity broiler chicken.Compared with Chinese herbal medicine antibiotic, difference that there are no significant at aspects such as the rate of animals delivered to the slaughter-house, average weight, feed efficiencies, and within 4 days before delivering for sale, stop to feed sampling observation noresidue.3, " impact of Cecropin AD-yeast preparation on the yellow chicken enteron aisle digestive ferment in Guangdong and feed quality " (" Chinese poultry resource " the 7th phase in 2004) the feeding experiment result of adding antibacterial peptide to the yellow chicken in Guangdong by water way shows, antibacterial peptide can promote chicken growth and reduce excreta nitrogen content.In addition, abroad report, antibacterial peptide also has certain effect to feedstuff mildew.
Up to the present, only have minority antibacterial peptide to carry out small-scale experimental study as feed addictive, the result of experiment is all positive-effect; The antibacterial peptide on a large scale application in feed industry is also very few.
In addition, domestic Lactobacillus casei industry development is still in the starting stage, research to bacterial classification and the research of relevant leavening are less, the Lactobacillus casei bacterial classification of domestic use in enormous quantities and leavening are mostly from external well-known manufacturer, domestic Lactobacillus casei industry production business or distributors's sales volume are smaller, and therefore Lactobacillus casei industry also has larger development space.And at present the production technology of domestic scholar to Lactobacillus casei bacteriocinogeny and as the production technology aspect of feed addictive also in the starting stage, commercially produced product is also very limited, therefore urgently develops on a large scale.
At present about the patent application of Lactobacillus casei aspect is as follows: application number: 201210277454.4, denomination of invention " a strain Lactobacillus casei and the application in feed thereof ", this patent relates to a strain Lactobacillus casei and the application in feed thereof, and specifically disclose this bacterial strain can ferment produce phospholipase A2, yield of enzyme is high, this bacterial strain is applied to field of fodder, taking containing the oil crops of plant phosphatide or its processed side product as fermentation substrate, with ammonium sulfate, sodium chloride, cornstarch is as energy donor, using this bacterial strain as fermentation strain, prepare feed addictive by solid state fermentation.But this patent does not relate to the utilization of antibacterial peptide, do not relate to the large-scale production process of fermentation manufacturing technique and Lactobacillus casei feed addictive yet.
Application number: 201010277557.1, denomination of invention " a kind of lactobacillus casei bacteriocin and the application in feed thereof ", discloses a kind of lactobacillus casei bacteriocin and the application in feed thereof.Specifically disclose a strain Lactobacillus casei (Lactobacillus casei), preservation name is called Lactobacillus casei lyT-7, depositary institution: Chinese Typical Representative culture collection center, preserving number is: CCTCC M2010197, and the method for producing bacteriocin, and the application of bacteriocin crude extract in feed.The bacteriocin producing obtains through ammonium sulfate precipitation, Sephadex G-100 gel filtration and RP-HPLC chromatography.This bacteriocin is stable to heat, acid, can be by proteasome degradation, and antimicrobial spectrum is wide.Bacteriocin is added in feed, and growth that can anti-bacteria, has the good prospect as feed addictive application.But for concrete application process, the large-scale production process that relates in particular to fermentation manufacturing technique, rape cake detoxification process and the Lactobacillus casei feed addictive of this bacterial strain does not have further disclosure, therefore more needs to provide a kind of industrialized manufacturing technique of Lactobacillus casei feed addictive.
Summary of the invention
The object of the present invention is to provide a kind of industrialized manufacturing technique of Lactobacillus casei feed addictive, this Lactobacillus casei (Lactobacillus casei) preservation name is called Lactobacillus casei lyT-7, depositary institution: Chinese Typical Representative culture collection center, preserving number is: CCTCC M2010197.The present invention utilizes this bacterial strain to produce to have the bacteriocin (having another name called antibacterial peptide) of broad spectrum antibacterial, to all inhibited characteristics of gram-positive bacteria, Gram-negative bacteria and aspergillus flavus, using this bacterial strain as fermentation strain, by the foundation of the fermentation manufacturing technique to this bacterial strain, large-scale production process to rape cake detoxification process, Lactobacillus casei feed addictive, thereby the industrialization that has realized Lactobacillus casei feed addictive is produced.
Model of the present invention good fermentation manufacturing technique; Secondly, utilizing after saccharomycete and lactobacillus-fermented, can not only play the effect of detoxification, and can also improve the content of rape cake albumen and the utilization rate of rape cake nutritional labeling; Again, set up the large-scale production process of good Lactobacillus casei feed addictive, by the feed that has added Lactobacillus casei bacterium powder, to the pig experiment of feeding, between feed period, the survival rate of interpolation group, resistance against diseases, gaining effect etc. are all better than control group.
Realize the technical scheme that the object of the invention adopts as follows:
The application of a kind of Lactobacillus casei zymotic fluid in feed, described Lactobacillus casei (Lactobacillus casei) preservation name is called Lactobacillus casei lyT-7, depositary institution: Chinese Typical Representative culture collection center, preserving number is: CCTCC M2010197, it is characterized in that: the Lactobacillus casei zymotic fluid that 10 tons of scale fermentations are obtained, by spraying, rape cake dry and after adding detoxification is made containing Lactobacillus casei bacterium number 10
9viable bacteria/gram feed addictive.
The Lactobacillus casei zymotic fluid technological process of production of described 10 tons of scales comprises:
The inclined plane inoculating of A, Lactobacillus casei
B, 500ml triangular flask are cultivated
C, the seeding tank fermentation of 0.5 ton
D, 10 tons of bulk fermentations 48 hours
Culture medium in described zymotic fluid production technology is: glucose 2.21%, peptone 2.41%, yeast extract 0.64%.
The suitableeest cultivation temperature that described triangular flask is cultivated is 30 DEG C, and the suitableeest initial pH is 6.0, and optimum inoculation amount is 2%, and the suitableeest shaking flask loading amount is 75ml/100ml.
Fermentation condition in described fermentation tank is: optimum inoculation amount 5%, before fermentation, in 12h, give intermittent stirring every 1h, and revolution 100rpm, the 30min that holds time, carries out a feed supplement in fermentation 30 hours time, adds 0.01% ammoniacal liquor.
Described inoculum concentration is the percent by volume in zymotic fluid.
The addition of described Lactobacillus casei zymotic fluid in feed is 7%(g/g) time, fungistatic effect is best.
The technological process of production of described Lactobacillus casei feed addictive is:
Lactobacillus casei zymotic fluid is dried to water content below 8% in 60 DEG C of sprayings, obtains dry bacterium powder, then add rape cake after detoxification, making containing Lactobacillus casei bacterium number is 10
9viable bacteria/gram feed addictive.
Every gram of living preparation of lactobacillus number containing of described dry bacterium powder is 10
11.
Described 10
9viable bacteria/gram the bacteriocin content of Lactobacillus casei be equivalent to 2.4 × 10
5the terramycin of individual unit/gram.
The addition of described Lactobacillus casei feed addictive in feed is that feed per ton adds 2 kilograms.
After described detoxification, the preparation technology of rape cake is as follows:
Rape cake adds water, condition: rape cake: wheat bran=9:1(g/g), ratio of water to material is 1.0:0.7(g/g), Initial pH 6.0, by mixer batching, at 1kg/cm
2, under 110 DEG C of vapour pressures, sterilize 20～30 minutes, be cooled to 35 DEG C, in inoculation pavier, access bacterial classification, described bacterial classification is Candida: Lactobacillus casei=2:1(g/g), total inoculum concentration is 4.50 × 107 cfu/g, windrow, material thickness is controlled at 3 centimeters, fermenting cellar temperature is controlled at 32 DEG C, and relative humidity is 95%, fermentation time 3 days, dry with pneumatic drier, then product is ground into the powder below 100 orders and get final product with pulverizer.
Useful technique effect of the present invention:
1, adopt preservation name to be called Lactobacillus casei (the Lactobacillus casei bacterial strain of Lactobacillus casei lyT-7, the bacteriocin that energy generation has broad spectrum antibacterial has another name called (antibacterial peptide), all inhibited to gram-positive bacteria, Gram-negative bacteria and aspergillus flavus, acid and alkali resistance, high temperature resistant, in pH2 to pH11 scope, still keep good bacteriostatic activity, under 121 DEG C of conditions, process 20min and still can keep 88.42% activity, good antimicrobial effect.
The Lactobacillus casei zymotic fluid production technology of 2, carrying out under the fermentation condition after optimization, fermentation yield has improved 225% before optimizing, simultaneously carry out in 10 tons of fermentation tanks 10 batches continuously ferment in middle trial production, the Lactobacillus casei bacterium number in zymotic fluid reaches 1.4 × 10
9viable bacteria/ml, using staphylococcus aureus as indicator bacteria, terramycin is product in contrast, and the bacteriocin of measuring in zymotic fluid is tired, and measurement result shows, and the bacteriocin content in zymotic fluid reaches and is equivalent to 3.4 × 10
5the terramycin of the terramycin/ml unit of individual unit, ferment effect is good.
3, zymotic fluid spray-dried at 60 DEG C after, as in the Lactobacillus casei bacterium powder finished product of feed addictive containing Lactobacillus casei bacterium number be 10
9viable bacteria/gram, bacteriocin content reaches and is equivalent to 2.4 × 10
5the terramycin of individual unit/gram, bacterial population content is high, good as feed addictive effect.
4, adopt saccharomycete and Lactobacillus casei mixed culture fermentation detoxification, be specially Candida: Lactobacillus casei=2:1(g/g), total inoculum concentration is 4.50 × 107 cfu/g, detoxification efficiency is obvious, detoxification efficiency can, up to more than 60%, can be reduced to 0.15% by glucosinolate content, has reached below national glucosinolate content control criterion, the forage protein source that can be used as livestock and poultry completely, security is good; Meanwhile, utilizing after saccharomycete and Lactobacillus casei fermentation, not only played the effect of detoxification, but also improved the content of rape cake albumen and the utilization rate of rape cake nutritional labeling.
5, produce and prove by continuously fermenting, bacterial strain production performance is stable, fermentation manufacturing technique maturation, and whole production process environmentally safe, for further suitability for industrialized production provides the foundation.Lactobacillus casei feed addictive of the present invention is applied to conventional feed, through feeding, the piglets of 25-30 kilogram proves, compare with control group (same do not add any antibiotic), daily gain improves more than 4% than control group, its resistance against diseases effectively improves, and between trimestral feed period, its incidence of disease is zero, and the incidence of disease of control group is 8.3%, fully show the resistant effect of Lactobacillus casei feed addictive uniqueness.
Brief description of the drawings
The growth curve of Fig. 1 Lactobacillus casei and product love song line
The impact of the inoculum concentration of Fig. 2 Lactobacillus casei on fungistatic effect
The confirmatory experiment design sketch of Fig. 3 fermentation medium
Wherein, No. 1-6 experiment for being undertaken by caluclate table, No. 7 is control experiment, and fungistatic effect is 100% relatively, and experiment condition is: glucose 2%, peptone 2%, yeast extract 0.63%
The fungistatic effect of Fig. 4 feed addictive under normal condition
Wherein, Tc1 addition is by adding respectively 2.5mg bacteriocin crude extract in every 1000g feed; C addition is by adding respectively 5mg bacteriocin crude extract in every 1000g feed; Tc3 addition is by adding respectively 10mg bacteriocin crude extract in every 1000g feed; CKc is blank.
The fungistatic effect of Fig. 5 feed addictive under intensified condition,
Wherein, Tq1 addition is by adding respectively 2.5mg bacteriocin crude extract in every 1000g feed; Tq2 addition is by adding respectively 5mg bacteriocin crude extract in every 1000g feed; Tq3 addition is by adding respectively 10mg bacteriocin crude extract in every 1000g feed; CK is blank.
Detailed description of the invention
embodiment 1the fermentation condition of Lactobacillus casei and Study on Fermentation
1.1 laboratory condition bottom fermentation condition optimizings
1.1.1 the mensuration of growth curve
First the growth curve to this Lactobacillus casei (T1) and product love song line are measured, and result is as Fig. 1.
Lactobacillus casei T1 approximately enters exponential phase since 4 h as can be seen from Figure 1, enters stationary phase to 36 h, starts decline after 84 h.Can find out from producing love song line, in the scope of 1-4 h, there is obvious decline in pH, and this explanation T1 had started growth, its lag phase < 4 h before 4 h.Can find out from producing love song line, the acid producing ability of T1 is more intense, and in the time of 4 h, pH has reduced to 4.35, and along with the Fast Growth of thalline, pH declines gradually, finishes to exponential phase, and pH touches the bottom.
1.1.2 experiment of single factor
1.1.2.1 the impact of carbon source
Taking fermentation medium as basis, add 2% different carbon source to replace glucose, leave standstill at 30 DEG C and cultivate 48h, the centrifugal removal thalline of zymotic fluid, is adjusted to 5.0 survey fungistatic effects by the pH of CFS, the results are shown in Table 1.As can be seen from Table 1, during taking glucose as carbon source, Lactobacillus casei element is tired the highlyest, and cornstarch takes second place, and the fungistatic effect of cornstarch has declined 12.3% compared with the fungistatic effect of glucose.So employing glucose is carbon source.
Note: card punch diameter is 7mm
1.1.2.2 the impact of nitrogenous source
Taking fermentation medium as basis, taking 2% glucose as carbon source, add 2% different nitrogen sources.At 30 DEG C, leave standstill and cultivate 48h, the pH of CFS is adjusted to 5.0 mensuration fungistatic effects, the results are shown in Table 2.
Note: card punch diameter is 7mm
As can be seen from Table 2, during taking 2% peptone as nitrogenous source, fungistatic effect is best, the peptone taking 1% and 1% corn steep liquor during as mixed nitrogen fungistatic effect take second place.Peptone taking 1% and 1% corn steep liquor be during as mixed nitrogen, and the fungistatic effect of its fungistatic effect during taking peptone as only nitrogen source declines 6.7%.
1.1.2.3 the impact of initial pH
Fermentation medium is adjusted to respectively to pH4.0-9.0 with the NaOH of 1mol/L or the HCl of 1mol/L, leaves standstill and cultivate 48h at 30 DEG C, the pH of CFS is adjusted to 5.0 mensuration fungistatic effects, when result shows initial pH6.0, fungistatic effect is best.
1.1.2.4 the impact of shaking flask loading amount
In the triangular flask of 100ml, be respectively charged into the culture medium of 10ml, 25ml, 50ml, 75ml and 100ml, adjusting initial pH is 6.0, leaves standstill and cultivates 48h at 30 DEG C, and the pH of CFS is adjusted to 5.0 mensuration fungistatic effects.The suitableeest loading amount is 75ml.When shaking flask loading amount is 50ml, 86.8% when antibacterial efficiency is 75ml, when shaking flask loading amount is 100ml, 97.2% when antibacterial efficiency is 75ml, illustrates relatively oxytolerant of Lactobacillus casei T1.
1.1.2.5 the impact of inoculum concentration
In the triangular flask of 50mL, pack 35mL culture medium into, adjust pH6.0, with different vaccination amount inoculation, leave standstill at 30 DEG C and cultivate 48 h, the pH of CFS is adjusted to 5.0 mensuration fungistatic effects, the results are shown in Figure 2.As can be seen from the figure the obvious effect of inoculum concentration to fungistatic effect: inoculum concentration fungistatic effect in the scope of 1%-2% is better, after this, along with the increase gradually of inoculum concentration, fungistatic effect obviously weakens.The suitableeest inoculum concentration is 2% as seen from the figure.
1.1.2.6 the impact of cultivation temperature
In the triangular flask of 50mL, pack 35mL culture medium into, adjust pH6.0, with 2% inoculum concentration inoculation, under different cultivation temperature, leave standstill and cultivate 48 h, the pH of CFS is adjusted to 5.0 mensuration fungistatic effects.Result demonstration, the suitableeest cultivation temperature is 30 DEG C, between 30 DEG C-37 DEG C, fungistatic effect changes not obvious, in the time of 37 DEG C, 97% when fungistatic effect is 30 DEG C.
1.1.3 response surface analysis
1.1.3.1 experimental design
From experiment of single factor, find, Carbon and nitrogen sources is larger to the bacteriocin yield effect of T1, therefore selects Carbon and nitrogen sources, and the yeast extract of nitrogenous source and growth factor does Box-Behnken experiment as a supplement, taking average diameter of inhibition zone as response, experimental design table and result are as follows simultaneously:
A, glucose; B, peptone; C, yeast extract;
Utilize Design Expert software to analyze the above results, obtain the secondary multinomial regression equation of antibacterial circle diameter (Y) to concentration of glucose (A), peptone concentration (B), yeast extract concentration (C):
Y=+16.82+0.090A+1.06B-0.28C-0.020AB-0.11AC-0.27BC-1.06A
2-2.50B
2-0.91C
2
From the analysis of variance table 4 of equation, testing selected model P value is 0.001, extremely significantly (
p﹤ 0.05).Coefficient of determination R
2=0.9825, illustrate that this model can predict its response, this experiment R
2be 0.9601, illustrate that the variation of response has 96.01% to derive from selected variable, this equation model situation is better.
R
2=0.9825; Proofread and correct R
2=0.9601
1.1.3.2 response surface analysis map analysis
Can make the response surface analysis figure of the different factors according to regression equation, from response surface analysis figure, can find out, these three factors of concentration of glucose, peptone concentration and yeast extract concentration are in the impact of fungistatic effect, peptone concentration is the most obvious on the impact of fungistatic effect, and the impact of concentration of glucose is taken second place.
1.1.3.3 confirmatory experiment
Assistant software Design Expert optimizes and cultivates formula and obtain theoretical the best and cultivate formula and see the following form by experiment:
According to the experimental design of caluclate table, carry out confirmatory experiment, the optimum condition obtaining taking experiment of single factor is contrast, experiment knot is shown in accompanying drawing 3.
From experimental result, best fermentative medium formula is: concentration of glucose is 2.21%, and peptone concentration is 2.41%, and yeast extract concentration is 0.64%, do bacteriostatic experiment with the zymotic fluid obtaining after this medium culture T1 48h, average diameter of inhibition zone reaches 17.14mm.Find to adopt the experiment condition that dopes of response surface analysis and the condition of confirmatory experiment confirmation to have good fitness by confirmatory experiment, illustrate with the analysis that the method is carried out condition of culture be feasible.
1.1.4 effect comparison before and after optimizing
Drawing ancient cooking vessel to the antibacterial standard of staphylococcus aureus the curve of tiring according to grace, can determine T1 bacteriocinogeny tiring as 1171IU/mL before fermentation condition optimization, is 3812IU/mL after optimizing.Before and after optimizing, bacteriocin output has improved 225%.Illustrate that this fermentation condition optimization has obtained good effect, the fungistatic effect after optimization obviously improves.
1.2 industrial fermentation condition researchs
1.2.1 lab scale condition
Select the fermentation tank of 2 liters to carry out lab scale experiment, feed 1.7 liters.Glucose taking 2.2% is carbon source, peptone taking 2.4% is nitrogenous source, 0.6% yeast extract is growth factor fermentation, 30 DEG C of fermentation temperatures, initial pH6.0, incubation time 48h, inoculum concentration 2%, the lab scale that ferments, lab scale fermentation results shows, ferment after 48 hours, the Lactobacillus casei bacterium number in zymotic fluid reaches 4.4 × 10
9g/L, using staphylococcus aureus as indicator bacteria, terramycin is product in contrast, record in zymotic fluid bacteriocin and tire to reach and be equivalent to 6.8 × 10
5terramycin/the mL of individual unit.
1.2.2 pilot plant conditions
Select the fermentation tank of 10 liters to carry out pilot experiment, feed 9 liters.Glucose taking 2.2% is carbon source, nitrogen concentration 2%, and wherein the ratio of peptone and dregs of beans is 1:0.5,0.4% yeast extract is that growth factor is carried out fermenting experiment.30 DEG C of fermentation temperatures, initial pH6.0, incubation time 45h, inoculum concentration 5%, before fermentation, in 12h, each 1h gives intermittent stirring, revolution is 100rpm, hold time as 30min, carry out a feed supplement in fermentation 30 hours time, add 0.01% ammoniacal liquor, ferment 48 hours, the Lactobacillus casei cell concentration of zymotic fluid is 4 × 10
9individual/ml, then using staphylococcus aureus as indicator bacteria, terramycin is product in contrast, and the bacteriocin of measuring in zymotic fluid is tired, and result shows that the tiring of bacteriocin in zymotic fluid is equivalent to 5.2 × 10
5terramycin/the mL of individual unit.
embodiment 2the effect research that bacteriocin superior strain adds in feed
2.1 bacteriocin crude extracts add the antibacterial effect research in feed to
2.1.1 the fungistatic effect under normal condition
By adding respectively 2.5mg in every 1000g feed, 5 mg, the amount of 10 mg adds bacteriocin crude extract to be determined at the fungistatic effect of normal condition, found that fungistatic effect the best when addition is 10mg.The results are shown in accompanying drawing 4.
2.1.2 the fungistatic effect under intensified condition
By adding respectively 2.5mg in every 1000g feed, 5mg, the amount of 10mg adds bacteriocin crude extract to be determined at the fungistatic effect of intensified condition, found that fungistatic effect the best when addition is 10mg.The results are shown in accompanying drawing 5.
2.1.3 the effect comparison of bacteriocin crude extract and positive control
Under normal condition and intensified condition, be determined at respectively under different additive amount condition, the fungistatic effect of bacteriocin crude extract and positive control, the fungistatic effect and the grace that found that T1 crude extract in lowest dose level group draw ancient cooking vessel suitable, and the fungistatic effect of T1 crude extract is slightly better than grace and draws ancient cooking vessel in high dose group.
2.2 zymotic fluids add the antibacterial effect research in feed to
Measure initial total number of bacteria, within later every 7 days, measure total number of bacteria once.Experimental result shows that T1 zymotic fluid has obvious inhibitory action to bacterium, still has stronger bacteriostasis while being saved to 3 months, and to 20 weeks, rear total number of bacteria increased to some extent.Fungistatic effect best (close with positive control fungistatic effect) when zymotic fluid addition is 7%, its reason is that wherein active ingredient bacteriocin amount is few at least for zymotic fluid addition, bacteriostasis is not obvious, zymotic fluid adds too much,, because other nitrogenous sources that contain in fermentation medium, carbon source nutriment provide condition for bacterial growth, total number of bacteria can increase on the contrary.Passing in time of the fungistatic effect of zymotic fluid and reducing, reaches best at the 10th week left and right fungistatic effect, and fungistatic effect progressively declines afterwards.
The survival effect of 2.3 probioticses in feed
The Lactobacillus casei bacterium liquid of fermenting and producing, the living preparation of lactobacillus number in fermenation raw liquid is 1.4 × 10
10, through the vacuum spray drying of 60 DEG C, the lactobacillus dry bacterium powder (water content is below 8%) of acquisition, contains 10
11living preparation of lactobacillus/gram dry bacterium powder, by the amount of 1.5-2 kilogram of feed interpolation per ton, adds in feed, in every gram of feed, contains 10
5living preparation of lactobacillus (i.e. 200,000 living preparation of lactobacillus/gram feeds).Preserve at normal temperatures after 3 six months, measure Lactobacillus casei viable bacteria in feed and the content of bacteriocin, measurement result shows, the Lactobacillus casei viable count in feed is 1.6 × 10
5/ gram feed, bacteriocin content does not change, and still remains on the terramycin/gram feed that is equivalent to 46.8 units.
embodiment 3the large-scale production process research of Lactobacillus casei feed addictive
Lower batch of fermentation test of 3.1 pilot plant conditions
The trial production of continuously fermenting in the fermentation tank of 10 tons, continuously ferment and produce 10 batches, produce nearly 10 tons of Lactobacillus casei bacterium powder, produce by continuously fermenting and prove that bacterial strain production performance is stable, fermentation manufacturing technique maturation, ferment 48 hours, the Lactobacillus casei cell concentration in zymotic fluid is 1.4 × 10
9viable bacteria/ml, then using staphylococcus aureus as indicator bacteria, terramycin is product in contrast, and the bacteriocin of measuring in zymotic fluid is tired, and measurement result shows, and the bacteriocin in zymotic fluid is tired as being equivalent to 3.4 × 10
5terramycin/the mL of individual unit.Zymotic fluid is spray-dried, then the Lactobacillus casei microbial inoculum that adds corresponding inserts (the rape cake after detoxification) to prepare, containing Lactobacillus casei 10
9viable bacteria/gram, then using staphylococcus aureus as indicator bacteria, terramycin is product in contrast, and the bacteriocin of measuring in Lactobacillus casei microbial inoculum is tired, and measurement result shows, and the bacteriocin in Lactobacillus casei microbial inoculum is tired and is equivalent to 2.4 × 10
5the terramycin of individual unit/gram.
3.2 Lactobacillus casei bacterium powder process studies
Lactobacillus casei is carried out to scale fermenting and producing, obtain Lactobacillus casei zymotic fluid, then by Lactobacillus casei zymotic fluid by spray-dired method, the dry bacterium powder (water content is below 8%) of making of spraying at 60 DEG C, and adding corresponding inserts (the rape cake after detoxification), the viable count of measuring in Lactobacillus casei microbial inoculum is 10
9viable bacteria/gram; Bacteriocin content reaches and is equivalent to 2.4 × 10
5the terramycin of individual unit/gram.Add again the amount of 2 kilograms by feed per ton, add in conventional feed, in every gram of conventional feed, contain 2 × 10
6lactobacillus casei viable bacteria (being 150-200 ten thousand living preparation of lactobacillus/gram conventional feed).Using staphylococcus aureus as indicator bacteria, terramycin is product in contrast, measure and are added with the feed of Lactobacillus casei feed addictive, and measurement result shows, in feed, contain be equivalent to the terramycin of 480 units/gram.
The experiment of feeding of 3.3 Lactobacillus casei feed addictives
3.3.1 add the effect research that Lactobacillus casei bacterium powder is fed pig
210 of the weanling pigs of 25-30 kilogram of left and right of selection, as feeding experiment, piglet is divided into 2 groups at random, and A organizes the test group (150) of having added 2 kilograms of dry bacterium feed addictives of cheese milk for feed per ton, the control group (60) that B group is conventional feed (not adding any antibiotic).The cycle of feeding is 3 months, and feed 5 first and second month every day, and feed 4 a 3rd month every day.Between feed period, observe and take food situation, palatability, life habit, growing state, Gain weight, incidence etc., result show between feed period two groups of A, B taking food, the aspect such as palatability, life habit is without significant difference, aspect daily gain, A group has improved 4.2%. than B group daily gain, aspect the disease incidence of pig, during feeding experiment, the disease incidence of A group is zero (being none example morbidity), and the disease incidence of B group to be 8.3%(have 5 pigs morbidities).Therefore, feeding experiment result shows, is added with the feed of Lactobacillus casei feed addictive, not adding in any antibiotic situation, after the pig of feeding, can resist the infection of the various diseases of pig completely.
embodiment 4
The cultivation of 4.1 detoxification microorganisms
4.1.1 saccharomycetic cultivation
Use malt juice liquid medium: by 4～6 layers of filtered through gauze for malt amylase liquid (being purchased from new capital brewery), filtrate is as muddiness, and available Egg-white clarification is about to an Egg-white about 20ml that adds water, till while mixing well to raw foam, be then poured on stirring in saccharified liquid and refilter after boiling.Filtrate is diluted to 5～6 Baume degrees, pH approximately 6.4.121 DEG C of sterilizings 20 minutes.Candida is inoculated in to malt juice liquid medium, sterilizing, inoculation, 28 DEG C of cultivations of shaking table (150r/min) 3 days.
4.1.2 the cultivation of Lactobacillus casei: use MRS fluid nutrient medium:
Peptone 10g; Beef extract 10g; Yeast extract 5g; K
2hPO
43H
2o 2g; Sodium acetate 5g; Glucose 20g; Tween 80 1g; Dibasic ammonium citrate 2g; MgSO
47H
2o 0.58g; MnSO
44H
2o 0.25g; Adding distil water 1000mL; PH6.2-6.4,121 DEG C, 15min sterilizing is for subsequent use.
Lactobacillus casei is inoculated in to MRS fluid nutrient medium, and sterilizing, inoculation, leave standstill and cultivate, and 30 DEG C, 2～3 days.
4.2 rape cake microbial detoxification
4.2.1 single culture fermentation
By the liquid fermentation and culture Candida of 3 days and Lactobacillus casei, be inoculated in respectively fermentation medium (rape cake: wheat bran=9:1, ratio of water to material: 1.0:0.7, Initial pH 6.0) and carry out solid fermentation, be divided into four experimental group by the difference of inoculum concentration, inoculum concentration is respectively 2.25 × 10
7cfu/g, 4.50 × 10
7cfu/g, 6.75 × 10
7cfu/g, 9.00 × 10
7cfu/g.Fermentation condition: 32 DEG C, moisture content: 30%, cultivate respectively 7d.
4.2.2 saccharomycete and Lactobacillus casei mixed culture fermentation
By Candida and Lactobacillus casei (Candida: Lactobacillus casei=1:2 by a certain percentage; Saccharomycete: Lactobacillus casei=2:1; Candida: Lactobacillus casei=1:1) be inoculated in fermentation medium after mixing and carry out solid fermentation (total inoculum concentration is about 4.50 × 10
7cfu/g).Fermentation condition is the same.
Within 3 days, occur obvious detoxification efficiency, but detoxification efficiency afterwards do not have clear improvement, therefore the reduce toxicity time was advisable with 3-5 days later.Inoculum concentration is too low is unfavorable for detoxification.Mixed bacteria is better than single culture detoxification efficiency.Candida hybrid mode virus elimination rate different from Lactobacillus casei comparative result shows, Candida: Lactobacillus casei=2:1/1:1 > yeast: the fermentation of lactic acid=1:2 > single culture.What detoxification efficiency was best can, up to 60%, can be reduced to 0.15% by glucosinolate content.
Can find out from result of study, can not only play the effect of detoxification after culture propagation, can also improve the content of rape cake albumen, the rape cake protein content after 5 days that ferments can increase more than 5%.And the fermentation of lactic acid can improve the utilization rate of rape cake nutritional labeling.In addition, microorganism fermentation can also improve the mouthfeel of rape cake widely, and microorganism utilizes self metabolism, can produce the precursor substance of fragrance matter or fragrance, improves the fragrance after the detoxification of rape cake.
The foundation of 4.3 industrial fermentation production technologies
By tests such as inoculum concentration, mixed culture fermentation bacterial classification inoculative proportion, medium component, amount of water, fermentation pH value, fermentation periods, set up the industrialized producing technology of rape cake microbial detoxification: use culture medium (the rape cake: wheat bran=9:1 that adds water, ratio of water to material: 1.0:0.7, Initial pH 6.0), by mixer batching, at 1kg/cm
2, under 110 DEG C of vapour pressures, sterilize 20～30 minutes, be cooled to 35 DEG C, (Candida: Lactobacillus casei=2:1, total inoculum concentration is about 4.50 × 10 in inoculation pavier, to access bacterial classification
7cfu/g), windrow, material thickness is controlled at 3 centimeters, and fermenting cellar temperature is controlled at 32 DEG C, and relative humidity is 95%, and fermentation time 3 days is dry with pneumatic drier, then product is ground into the powder below 100 orders with pulverizer.
embodiment 5the feeding effect of Lactobacillus casei feed addictive
Lactobacillus casei feed addictive is added in conventional feed, feed per ton adds by the addition of 2 kilograms, do not add in addition any antibiotic, add after Lactobacillus casei feed addictive, after measured, containing 105 Lactobacillus casei viable bacteria/gram feeds, the bacteriocin containing is equivalent to the terramycin of 74 units.Prove through the piglets of 25-30 kilogram of feeding, compare with control group (same do not add any antibiotic), daily gain provides more than 4% than control group, resistance against diseases improves greatly, between trimestral feed period, its incidence of disease is zero, and the incidence of disease of control group is 8.3%.Fully show the resistant effect of Lactobacillus casei feed addictive uniqueness.
Claims (5)
1. the Lactobacillus casei zymotic fluid application in feed, described Lactobacillus casei (Lactobacillus casei) preservation name is called Lactobacillus casei lyT-7, depositary institution: Chinese Typical Representative culture collection center, preserving number is: CCTCC M2010197, it is characterized in that: the Lactobacillus casei zymotic fluid that 10 tons of scale fermentations are obtained, by spraying, rape cake dry and after adding detoxification is made containing Lactobacillus casei bacterium number 10
9viable bacteria/gram feed addictive;
The Lactobacillus casei zymotic fluid technological process of production of described 10 tons of scales comprises:
The inclined plane inoculating of A, Lactobacillus casei
B, 500ml triangular flask are cultivated
C, the seeding tank fermentation of 0.5 ton
D, 10 tons of bulk fermentations 48 hours
Culture medium in described zymotic fluid production technology is: glucose 2.21%, peptone 2.41%, yeast extract 0.64%;
The cultivation temperature that described triangular flask is cultivated is 30 DEG C, and initial pH is 6.0, and inoculum concentration is 2%, and shaking flask loading amount is 75ml/100ml;
Fermentation condition in described fermentation tank is: inoculum concentration 5%, before fermentation, in 12h, give intermittent stirring every 1h, and revolution 100rpm, the 30min that holds time, carries out a feed supplement in fermentation 30 hours time, adds 0.01% ammoniacal liquor.
2. the application of Lactobacillus casei zymotic fluid in feed according to claim 1, is characterized in that: the technological process of production of described Lactobacillus casei feed addictive is:
Lactobacillus casei zymotic fluid is dried to water content below 8% in 60 DEG C of sprayings, obtains dry bacterium powder, then add rape cake after detoxification, making containing Lactobacillus casei bacterium number is 10
9viable bacteria/gram feed addictive.
3. the application of Lactobacillus casei zymotic fluid in feed according to claim 2, is characterized in that: every gram of Lactobacillus casei viable count containing of described dry bacterium powder is 10
11.
4. the application of Lactobacillus casei zymotic fluid in feed according to claim 2, is characterized in that: the addition of described Lactobacillus casei feed addictive in feed is that feed per ton adds 2 kilograms.
5. the application of Lactobacillus casei zymotic fluid in feed according to claim 2, is characterized in that: after described detoxification, the preparation technology of rape cake is as follows:
Rape cake adds water, condition: rape cake: wheat bran=9:1g/g, and ratio of water to material is 1.0:0.7g/g, Initial pH 6.0, by mixer batching, at 1kg/cm
2under 110 DEG C of vapour pressures, sterilize 20～30 minutes, be cooled to 35 DEG C, in inoculation pavier, access bacterial classification, windrow, material thickness is controlled at 3 centimeters, fermenting cellar temperature is controlled at 32 DEG C, and relative humidity is 95%, fermentation time 3 days, dry with pneumatic drier, then product is ground into the powder below 100 orders and get final product with pulverizer; Described bacterial classification is Candida: Lactobacillus casei=2:1g/g, and total inoculum concentration is 4.50 × 10
7cfu/g.
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