CN103202386A - 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, yet problems such as the bacterial drug resistance that antibiotic produces as feed addictive and medicament residue can work the mischief to human health.Therefore, studying effective antibiotic feed additive substitute products comes into one's own day by day.
The antibacterial peptide molecular weight is little, heat endurance is strong.High temperature when on the one hand, antibacterial peptide can tolerate feed granulating; On the other hand, when the scale fermenting and producing, through the high temperature enrichment process, can fully kill assorted thalline and do not cause the antibacterial peptide inactivation, and the environmental ecology problem that the diffusion because of engineering bacteria causes can not appear in product after applying.Simultaneously, less because of the antibacterial peptide molecular weight, be difficult for having immunogenicity.In view of these advantages of antibacterial peptide, its research and development have been become the frontier nature problem of studying the antibiotic substitute at present in the world.
It is at present, domestic that what use as Additive Production mainly is silkworm antibacterial peptide AD---yeast preparation.Most livestock and poultry test is also carried out around this antibacterial peptide.The pertinent literature report has: 1, " antimicrobial peptide preparation replaces the effect of antibiotic in the weanling pig diet " (" Chinese feed " the 18th phase of calendar year 2001 13-14 page or leaf), by in the feed of weanling pig, replacing antibiotic with antibacterial peptide, the diarrhoea of piglet, production performance etc. are contrasted.The result shows that antibacterial peptide anti diar rhea effect during stress of baby pigs caused ablaction is poor unlike antibiotic.An amount of antibacterial peptide is better than the somatotrophic effect of antibiotic, but the antibacterial peptide of high dose has then reduced growth in piglets.2, " effect of antibacterial peptide and antibiotic feeding dorking relatively " (" Guangdong feed " the 02nd phase in 2004), find that antibacterial peptide has the growth of promotion and improves immunity broiler chicken.Compare with the 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 stopped to feed the sampling observation noresidue in preceding 4 days delivering for sale.3, " silkworm antibacterial peptide AD-yeast preparation is to the influence of the yellow chicken enteron aisle digestive ferment in Guangdong and feed quality " (" Chinese poultry " the 7th phase in 2004) show for the Guangdong feeding experiment result that yellow chicken is added antibacterial peptide by water way, antibacterial peptide can promote the chicken growth and reduce the excreta nitrogen content.In addition, report abroad that antibacterial peptide also has certain effect to feedstuff mildew.
Up to the present, only have the minority antibacterial peptide to carry out small-scale experimental study as feed addictive, result of experiment all is positive-effect; The antibacterial peptide application in the feed industry on a large scale is also very few.
In addition, domestic Lactobacillus casei industry development still is in the starting stage, research to the research of bacterial classification and the leavening of being correlated with is 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 merchant or distributors's sales volume are smaller, so the Lactobacillus casei industry also has bigger development space.And at present domestic scholar to the production technology of 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 demands large-scale development and use urgently.
The patent application of at present relevant 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 and can ferment and produce phospholipase A2, the yield of enzyme height, this bacterial strain is applied to field of fodder, be fermentation substrate with the oil crops or its processed side product that contain plant phosphatide, with ammonium sulfate, sodium chloride, cornstarch is as energy donor, as fermentation strain, prepare feed addictive by solid state fermentation with this bacterial strain.But this patent does not relate to the utilization of antibacterial peptide, does 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.One strain Lactobacillus casei (Lactobacillus casei) is specifically disclosed, the preservation name is called Lactobacillus casei lyT-7, depositary institution: Chinese typical culture collection center, preserving number is: CCTCC M2010197, and method and the application of bacteriocin crude extract in feed of producing bacteriocin.The bacteriocin that produces 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 the feed, can suppress the growth of bacterium, have the good prospect of using as feed addictive.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, and therefore a kind of industrialized manufacturing technique of Lactobacillus casei feed addictive more need be provided.
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 culture collection center, preserving number is: CCTCC M2010197.The bacteriocin (having another name called antibacterial peptide) that the present invention utilizes this bacterial strain to produce to have broad spectrum antibacterial, to the equal inhibited characteristic of gram-positive bacteria, Gram-negative bacteria and aspergillus flavus, with this bacterial strain as fermentation strain, by to the fermentation manufacturing technique of this bacterial strain, to the foundation of the large-scale production process of rape cake detoxification process, Lactobacillus casei feed addictive, thereby realized the industrialization production of Lactobacillus casei feed addictive.
The present invention has at first set up fermentation manufacturing technique preferably; Secondly, after utilizing 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 Lactobacillus casei feed addictive preferably, to the pig experiment of feeding, the survival rate of interpolation group, resistance against diseases, gaining effect etc. all are better than control group between feed period with the feed that added Lactobacillus casei bacterium powder.
The technical scheme that realization the object of the invention adopts is 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 culture collection center, preserving number is: CCTCC M2010197, it is characterized in that: the Lactobacillus casei zymotic fluid that the fermentation of 10 tons of scales is obtained, make by spray-drying and the rape cake after adding detoxification and to contain Lactobacillus casei bacterium several 10
9The feed addictive of viable bacteria/gram.
The Lactobacillus casei zymotic fluid technological process of production of described 10 tons of scales comprises:
The inclined-plane inoculation of A, Lactobacillus casei
B, 500ml triangular flask are cultivated
C, 0.5 ton seeding tank fermentation
D, 10 tons of bulk fermentations 48 hours
Culture medium in the 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 ℃, and the suitableeest initial pH is 6.0, and optimum inoculation amount is 2%, and the suitableeest to shake bottled amount be 75ml/100ml.
Fermentation condition in the described fermentation tank is: optimum inoculation amount 5%, before fermentation, give intermittently to stir every 1h in the 12h, and revolution 100rpm, the 30min that holds time carries out a feed supplement in fermentation in the time of 30 hours, 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:
With the Lactobacillus casei zymotic fluid in 60 ℃ of spray-dryings to water content below 8%, obtain dry bacterium powder, add rape cake after the detoxification then, make that to contain Lactobacillus casei bacterium number be 10
9The feed addictive of viable bacteria/gram.
The living preparation of lactobacillus number that the every gram of described dry bacterium powder contains is 10
11
Described 10
9The bacteriocin content of the Lactobacillus casei of viable bacteria/gram is equivalent to 2.4 * 10
5Terramycin/the gram of individual unit.
The addition of described Lactobacillus casei feed addictive in feed is that feed per ton adds 2 kilograms.
The preparation technology of rape cake is as follows after the described detoxification:
The rape cake adds water, condition: the rape cake: wheat bran=9:1(g/g), ratio of water to material is 1.0:0.7(g/g), initial pH value 6.0 is with the mixer batching, at 1kg/cm
2, sterilized 20～30 minutes under 110 ℃ of vapour pressures, be cooled to 35 ℃, in the inoculation pavier, insert 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 control is at 3 centimeters, the control of fermenting cellar temperature is at 32 ℃, and relative humidity is 95%, fermentation time 3 days, use the pneumatic drier drying, again with pulverizer product be ground into below 100 orders powder namely.
Useful technique effect of the present invention:
1, adopt the preservation name to be called Lactobacillus casei (the Lactobacillus casei bacterial strain of Lactobacillus casei lyT-7, can produce the bacteriocin with broad spectrum antibacterial and have 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 ℃ of conditions, handle 20min and still can keep 88.42% activity, good antimicrobial effect.
The Lactobacillus casei zymotic fluid production technology of 2, under the fermentation condition after the optimization, carrying out, fermentation yield has improved 225% before optimizing, in 10 batches the middle trial production of carrying out in 10 tons of fermentation tanks simultaneously of continuously fermenting, the Lactobacillus casei bacterium number in the zymotic fluid reaches 1.4 * 10
9Viable bacteria/ml, as indicator bacteria, terramycin is product in contrast with staphylococcus aureus, and the bacteriocin of measuring in the zymotic fluid is tired, and measurement result shows that the bacteriocin content in the 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 60 ℃ down spray-dried after, containing Lactobacillus casei bacterium number in the Lactobacillus casei bacterium powder finished product as feed addictive is 10
9Viable bacteria/gram, bacteriocin content reaches and is equivalent to 2.4 * 10
5Terramycin/the gram of individual unit, bacterial population content height is good as the 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 be reduced to 0.15% with glucosinolate content up to more than 60%, has reached below the national glucosinolate content control criterion, can be used as the livestock and poultry feed protein sources fully, security is good; Simultaneously, after utilizing the fermentation of saccharomycete and Lactobacillus casei, 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 proof by continuously fermenting, the bacterial strain production performance is stable, the fermentation manufacturing technique maturation, and the whole process of production environmentally safe is for further suitability for industrialized production provides the foundation.Lactobacillus casei feed addictive of the present invention is applied to conventional feed, piglet evidence through the 25-30 kilogram of feeding, compare with control group (not adding any antibiotic equally), 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%, has fully shown the resistant effect of Lactobacillus casei feed addictive uniqueness.
Description of drawings
The growth curve of Fig. 1 Lactobacillus casei and product love song line
The inoculum concentration of Fig. 2 Lactobacillus casei is to the influence of fungistatic effect
The confirmatory experiment design sketch of Fig. 3 fermentation medium
Wherein, the 1-6 number 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, the Tc1 addition is by adding 2.5mg bacteriocin crude extract in every 1000g feed respectively; The C addition is by adding 5mg bacteriocin crude extract in every 1000g feed respectively; The Tc3 addition is by adding 10mg bacteriocin crude extract in every 1000g feed respectively; CKc is blank.
The fungistatic effect of Fig. 5 feed addictive under intensified condition,
Wherein, the Tq1 addition is by adding 2.5mg bacteriocin crude extract in every 1000g feed respectively; The Tq2 addition is by adding 5mg bacteriocin crude extract in every 1000g feed respectively; The Tq3 addition is by adding 10mg bacteriocin crude extract in every 1000g feed respectively; CK is blank.
The specific embodiment
Embodiment 1The fermentation condition of Lactobacillus casei and Study on Fermentation
1.1 laboratory condition bottom fermentation condition optimizing
1.1.1 the mensuration of growth curve
At first growth curve and the product love song line of this Lactobacillus casei (T1) are measured result such as Fig. 1.
Lactobacillus casei T1 enters exponential phase since 4 h approximately as can be seen from Figure 1, enters stationary phase to 36 h, begins decline behind 84 h.From producing the love song line as can be seen, in the scope of 1-4 h, tangible decline has appearred in pH, and this explanation T1 had begun growth, its lag phase＜4 h in the past at 4 h.From producing the love song line as can be seen, the acid producing ability of T1 is more intense, and pH has reduced to 4.35 when 4 h, and along with the quick growth of thalline, pH descends gradually, finishes to exponential phase, and pH touches the bottom.
1.1.2 experiment of single factor
1.1.2.1 the influence of carbon source
Based on fermentation medium, add 2% different carbon source and replace glucose, leave standstill under 30 ℃ and cultivate 48h, the centrifugal removal thalline of zymotic fluid transfers to 5.0 with the pH of CFS and surveys fungistatic effects, the results are shown in Table 1.As can be seen from Table 1, the Lactobacillus casei element is tired the highlyest when being carbon source with glucose, and cornstarch takes second place, and the fungistatic effect of cornstarch has descended 12.3% than the fungistatic effect of glucose.So employing glucose is carbon source.
Annotate: the card punch diameter is 7mm
1.1.2.2 the influence of nitrogenous source
Based on fermentation medium, be carbon source with 2% glucose, add 2% different nitrogen sources.Leave standstill under 30 ℃ and cultivate 48h, the pH of CFS is transferred to 5.0 measure fungistatic effects, the results are shown in Table 2.
Annotate: the card punch diameter is 7mm
As can be seen from Table 2, fungistatic effect is best when being nitrogenous source with 2% peptone, and fungistatic effect took second place when the peptone with 1% and 1% corn steep liquor were mixed nitrogen.When the peptone with 1% and 1% corn steep liquor were mixed nitrogen, the fungistatic effect when its fungistatic effect is only nitrogen source with the peptone descended 6.7%.
1.1.2.3 the influence of initial pH
Fermentation medium is transferred to pH4.0-9.0 respectively with the NaOH of 1mol/L or the HCl of 1mol/L, leave standstill at 30 ℃ and cultivate 48h, the pH of CFS is transferred to 5.0 measure fungistatic effects, fungistatic effect was best when the result showed initial pH6.0.
1.1.2.4 shake the influence of bottled amount
Be respectively charged into the culture medium of 10ml, 25ml, 50ml, 75ml and 100ml in the triangular flask of 100ml, transferring initial pH is 6.0, leaves standstill at 30 ℃ and cultivates 48h, the pH of CFS is transferred to 5.0 measure fungistatic effects.The suitableeest loading amount is 75ml.Shake bottled amount when being 50ml, 86.8% when antibacterial efficient is 75ml shaken bottled amount when being 100ml, and 97.2% when antibacterial efficient is 75ml illustrates relatively oxytolerant of Lactobacillus casei T1.
1.1.2.5 the influence of inoculum concentration
The 35mL culture medium of packing in the triangular flask of 50mL is transferred pH6.0, with the inoculation of different vaccination amount, leaves standstill under 30 ℃ and cultivates 48 h, the pH of CFS is transferred to 5.0 measure fungistatic effects, the results are shown in Figure 2.As can be seen from the figure inoculum concentration is to the obvious effect of fungistatic effect: inoculum concentration fungistatic effect in the scope of 1%-2% is better, and 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 influence of cultivation temperature
The 35mL culture medium of packing in the triangular flask of 50mL is transferred pH6.0, and the inoculum concentration inoculation with 2% is left standstill under different cultivation temperature and cultivated 48 h, the pH of CFS is transferred to 5.0 measure fungistatic effects.The result shows that the suitableeest cultivation temperature is 30 ℃, and between 30 ℃-37 ℃, fungistatic effect changes not obvious, in the time of 37 ℃, and 97% when fungistatic effect is 30 ℃.
1.1.3 response surface analysis
1.1.3.1 experimental design
From experiment of single factor, find, therefore carbon source and nitrogenous source are bigger to the bacteriocin yield effect of T1, select carbon source and nitrogenous source, and the yeast extract of nitrogenous source and growth factor is done the Box-Behnken experiment as a supplement, be response with the average diameter of inhibition zone simultaneously, experimental design table and result are as follows:
A, glucose; B, peptone; C, yeast extract;
Utilize Design Expert software that The above results is analyzed, obtain antibacterial circle diameter (Y) to the multinomial regression equation of secondary of 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 as can be known, 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 the response surface analysis figure as can be seen, in these three the factor influences to fungistatic effect of concentration of glucose, peptone concentration and yeast extract concentration, peptone concentration is the most obvious to the influence of fungistatic effect, and the influence of concentration of glucose is taken second place.
1.1.3.3 confirmatory experiment
Assistant software Design Expert optimize to cultivate prescription and obtains theoretical the best and cultivate prescription and see the following form by experiment:
According to the experimental design of caluclate table, carry out confirmatory experiment, the optimum condition that obtains with experiment of single factor is contrast, the experiment knot is seen accompanying drawing 3.
By experimental result as can be known, 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 that obtains behind this medium culture T1 48h, average diameter of inhibition zone reaches 17.14mm.Find to adopt experiment condition that response surface analysis dopes and the condition of confirmatory experiment confirmation that fitness is preferably arranged by confirmatory experiment, illustrate that the analysis of carrying out condition of culture with the method is feasible.
1.1.4 effect relatively before and after optimizing
Draw ancient cooking vessel to the antibacterial standard of staphylococcus aureus the curve of tiring according to grace, can determine that T1 bacteriocinogeny tiring before fermentation condition optimization is 1171IU/mL, optimizing the back is 3812IU/mL.Bacteriocin output has improved 225% before and after optimizing.Illustrate that this fermentation condition optimization has obtained effect preferably, the fungistatic effect after the optimization obviously improves.
1.2 industrial fermentation condition research
1.2.1 lab scale condition
Select 2 liters fermentation tank to carry out the lab scale experiment, feed 1.7 liters.Glucose with 2.2% is carbon source, peptone with 2.4% is nitrogenous source, 0.6% yeast extract is the growth factor fermentation, 30 ℃ of fermentation temperatures, initial pH6.0, incubation time 48h, inoculum concentration 2%, the lab scale that ferments, the lab scale fermentation is the result show, ferment after 48 hours, the Lactobacillus casei bacterium number in the zymotic fluid reaches 4.4 * 10
9G/L, as indicator bacteria, terramycin is product in contrast with staphylococcus aureus, record in the 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 10 liters fermentation tank to carry out pilot experiment, feed 9 liters.Glucose with 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 ℃ of fermentation temperatures, initial pH6.0, incubation time 45h, inoculum concentration 5%, each 1h gives intermittently to stir in the 12h before fermentation, revolution is 100rpm, holding time is 30min, carries out a feed supplement in fermentation in the time of 30 hours, adds 0.01% ammoniacal liquor, fermented 48 hours, the Lactobacillus casei cell concentration of zymotic fluid is 4 * 10
9Individual/ml, then with staphylococcus aureus as indicator bacteria, terramycin is product in contrast, the bacteriocin of measuring in the zymotic fluid is tired, the result shows that the tiring of bacteriocin in the zymotic fluid is equivalent to 5.2 * 10
5Terramycin/the mL of individual unit.
Embodiment 2The effect research that the bacteriocin superior strain adds in feed
2.1 the bacteriocin crude extract adds the antibacterial effect research in the feed to
2.1.1 the fungistatic effect under the normal condition
By adding 2.5mg respectively in every 1000g feed, 5 mg, the amount of 10 mg adds the fungistatic effect that the bacteriocin crude extract is determined at 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 the intensified condition
By adding 2.5mg respectively in every 1000g feed, 5mg, the amount of 10mg adds the fungistatic effect that the bacteriocin crude extract is determined at 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 of bacteriocin crude extract and positive control relatively
Under normal condition and intensified condition, be determined at respectively under the 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 the lowest dose level group draw ancient cooking vessel suitable, and the fungistatic effect of T1 crude extract slightly is better than grace and draws ancient cooking vessel in high dose group.
2.2 zymotic fluid adds the antibacterial effect research in the feed to
Measure initial total number of bacteria, measured total number of bacteria once in later per 7 days.Experimental result shows that the T1 zymotic fluid has the obvious suppression effect to bacterium, and strong bacteriostasis is still arranged when being saved to 3 months, to 20 week the back totals number of bacteria increase to some extent.The zymotic fluid addition is 7% o'clock fungistatic effect best (close with the positive control fungistatic effect), its reason is that wherein active ingredient bacteriocin amount is few at least for the zymotic fluid addition, bacteriostasis is not obvious, zymotic fluid adds too much, then because other nitrogenous sources, the carbon source nutriment that contain in the fermentation medium provide condition for bacterial growth, total number of bacteria can increase on the contrary.The fungistatic effect of zymotic fluid in time passing and reduce, reach best at the 10th all left and right sides fungistatic effect, fungistatic effect progressively descends afterwards.
2.3 the survival effect of probiotics in feed
The Lactobacillus casei bacterium liquid of fermenting and producing, the living preparation of lactobacillus number in the fermenation raw liquid is 1.4 * 10
10, through 60 ℃ vacuum spray drying, the lactobacillus dry bacterium powder of acquisition (water content is below 8%) contains 10
11Living preparation of lactobacillus/gram dry bacterium powder by the amount that feed per ton adds the 1.5-2 kilogram, adds in the feed, in every gram feed, contains 10
5Living preparation of lactobacillus (i.e. 200,000 living preparation of lactobacillus/gram feeds).After preserving 3 six months at normal temperatures, the Lactobacillus casei viable bacteria in the mensuration feed and the content of bacteriocin, measurement result shows that the Lactobacillus casei viable count in the 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
3.1 following batch of fermentation test of pilot plant conditions
The trial production of in 10 tons fermentation tank, continuously fermenting, continuously ferment and produce 10 batches, produce nearly 10 tons of Lactobacillus casei bacterium powder, produce proof bacterial strain production performance by continuously fermenting stable, the fermentation manufacturing technique maturation, fermented 48 hours, the Lactobacillus casei cell concentration in the zymotic fluid is 1.4 * 10
9Viable bacteria/ml, then with staphylococcus aureus as indicator bacteria, terramycin is product in contrast, the bacteriocin of measuring in the zymotic fluid is tired, measurement result shows that the bacteriocin in the zymotic fluid is tired to being equivalent to 3.4 * 10
5Terramycin/the mL of individual unit.Zymotic fluid is spray-dried, adds the Lactobacillus casei microbial inoculum of corresponding inserts (the rape cake after the detoxification) preparation again, contains Lactobacillus casei 10
9Viable bacteria/gram, then with staphylococcus aureus as indicator bacteria, terramycin is product in contrast, the bacteriocin of measuring in the Lactobacillus casei microbial inoculum is tired, measurement result shows that the bacteriocin in the Lactobacillus casei microbial inoculum is tired and is equivalent to 2.4 * 10
5Terramycin/the gram of individual unit.
3.2 Lactobacillus casei bacterium powder process study
Lactobacillus casei is carried out the scale fermenting and producing, obtain the Lactobacillus casei zymotic fluid, then with the Lactobacillus casei zymotic fluid by spray-dired method, make bacterium powder (water content is below 8%) 60 ℃ of following spray-dryings, and adding corresponding inserts (the rape cake after the detoxification), the viable count of measuring in the Lactobacillus casei microbial inoculum is 10
9Viable bacteria/gram; Bacteriocin content reaches and is equivalent to 2.4 * 10
5Terramycin/the gram of individual unit.Add 2 kilograms amount again by feed per ton, add in the conventional feed, contain 2 * 10 in every gram conventional feed
6Lactobacillus casei viable bacteria (being ten thousand living preparation of lactobacillus of 150-200/gram conventional feed).As indicator bacteria, terramycin is product in contrast with staphylococcus aureus, measure the feed that is added with the Lactobacillus casei feed addictive, and measurement result shows, contains the terramycin/gram that is equivalent to 480 units in the feed.
The experiment 3.3 the Lactobacillus casei feed addictive is fed
3.3.1 add the effect research that Lactobacillus casei bacterium powder is fed pig
210 of weanling pigs about selection 25-30 kilogram, as feeding experiment, piglet is divided into 2 groups at random, the test group (150) that the A group has been added 2 kilograms of dried bacterium feed addictives of cheese milk for feed per ton, and the B group is the control group (60) of conventional feed (not adding any antibiotic).The cycle of feeding is 3 months, and feed 5 every day first and second month, and feed 4 the 3rd a month every day.Observe food situation, palatability, life habit, growing state, the weightening finish situation of getting between feed period, incidence etc., the result shows that between feed period two groups of A, B do not have significant difference at aspects such as getting food, palatability, life habit, aspect daily gain, the A group has improved 4.2%. than B group daily gain, aspect the disease incidence of pig, during the 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 is the result show, is added with the feed of Lactobacillus casei feed addictive, do not adding under any antibiotic situation, after the pig of feeding, can resist the infection of the various diseases of pig fully.
4.1 the cultivation of detoxification microorganism
4.1.1 saccharomycetic cultivation
Use malt juice liquid medium: with malt amylase liquid (purchasing in new capital brewery) with 4～6 layers of filtered through gauze, filtrate such as muddiness, available egg is clarified in vain, is about to an egg and adds the about 20ml of water in vain, till mixing well when giving birth to foam, be poured on then to stir in the saccharification liquid and refilter after boiling.Filtrate is diluted to 5～6 Baume degrees, and pH about 6.4.Sterilized 20 minutes for 121 ℃.Candida is inoculated in malt juice liquid medium, sterilization, inoculation, shaking table (150r/min) was cultivated 3 days for 28 ℃.
4.1.2 the cultivation of Lactobacillus casei: use the 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 ℃, the 15min sterilization is standby.
Lactobacillus casei is inoculated in the MRS fluid nutrient medium, and cultivation is left standstill in sterilization, inoculation, and 30 ℃, 2～3 days.
4.2 rape cake microbial detoxification
4.2.1 single culture fermentation
With 3 days Candida of liquid fermentation and culture and Lactobacillus casei, be inoculated in fermentation medium (rape cake: wheat bran=9:1, ratio of water to material: 1.0:0.7, initial pH value 6.0) respectively and carry out solid fermentation, difference by inoculum concentration is divided into four experimental group, and namely 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 ℃, moisture content: 30%, cultivate 7d respectively.
4.2.2 saccharomycete and Lactobacillus casei mixed culture fermentation
With Candida and Lactobacillus casei (Candida: Lactobacillus casei=1:2 by a certain percentage; Saccharomycete: Lactobacillus casei=2:1; Candida: be inoculated in fermentation medium after Lactobacillus casei=1:1) mixes and carry out solid fermentation (total inoculum concentration is about 4.50 * 10
7Cfu/g).Fermentation condition is the same.
Namely occurred tangible detoxification efficiency later in 3 days, but detoxification efficiency afterwards there is not obviously improvement, so the fermentation detoxification time was advisable with 3-5 days.Inoculum concentration is too low to be unfavorable for detoxification.Mixed bacteria is better than single culture detoxification efficiency.The different hybrid mode virus elimination rate with Lactobacillus casei of Candida comparative result shows, Candida: Lactobacillus casei=2:1/1:1＞yeast: lactic acid=1:2＞single culture fermentation.What detoxification efficiency was best can be reduced to 0.15% with glucosinolate content up to 60%.
From result of study as can be seen, can not only play the effect of detoxification behind the culture propagation, can also improve the content of rape cake albumen, fermenting, rape cake protein content can increase more than 5% after 5 days.And the fermentation of lactic acid can improve the utilization rate of rape cake nutritional labeling.In addition, microbial 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, the fragrance after the detoxification of raising rape cake.
4.3 the foundation of industrial fermentation production technology
By tests such as inoculum concentration, mixed culture fermentation bacterial classification inoculation ratio, medium component, amount of water, fermentation pH value, fermentation periods, set up the industrialized producing technology of rape cake microbial detoxification: use culture medium to add water (rape cake: wheat bran=9:1, ratio of water to material: 1.0:0.7, initial pH value 6.0), with the mixer batching, at 1kg/cm
2, sterilized 20～30 minutes under 110 ℃ of vapour pressures, be cooled to 35 ℃, (Candida: Lactobacillus casei=2:1, total inoculum concentration is about 4.50 * 10 to insert bacterial classification in the inoculation pavier
7Cfu/g), windrow, material thickness is controlled at 3 centimeters, and the fermenting cellar temperature is controlled at 32 ℃, and relative humidity is 95%, and fermentation time 3 days is used the pneumatic drier drying, with pulverizer product is ground into powder below 100 orders again.
Embodiment 5The feeding effect of Lactobacillus casei feed addictive
The Lactobacillus casei feed addictive is added in the conventional feed, feed per ton adds by 2 kilograms addition, do not add any antibiotic in addition, after adding the Lactobacillus casei feed addictive, after measured, contain 105 Lactobacillus casei viable bacteria/gram feeds, the bacteriocin that contains is equivalent to the terramycin of 74 units.Through the piglet evidence of the 25-30 kilogram of feeding, to compare with control group (not adding any antibiotic equally), 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%.The resistant effect that has fully shown Lactobacillus casei feed addictive uniqueness.
Claims (10)
1. the application of Lactobacillus casei zymotic fluid in feed, described Lactobacillus casei (Lactobacillus casei) preservation name is called Lactobacillus casei lyT-7, depositary institution: Chinese typical culture collection center, preserving number is: CCTCC M2010197, it is characterized in that: the Lactobacillus casei zymotic fluid that the fermentation of 10 tons of scales is obtained, make by spray-drying and the rape cake after adding detoxification and to contain Lactobacillus casei bacterium several 10
9The feed addictive of viable bacteria/gram.
2. according to the application of the described Lactobacillus casei zymotic fluid of claim 1 in feed, it is characterized in that: the Lactobacillus casei zymotic fluid technological process of production of described 10 tons of scales comprises:
The inclined-plane inoculation of A, Lactobacillus casei
B, 500ml triangular flask are cultivated
C, 0.5 ton seeding tank fermentation
D, 10 tons of bulk fermentations 48 hours
Culture medium in the 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 ℃, and initial pH is 6.0, and inoculum concentration is 2%, and shaking bottled amount is 75ml/100ml;
Fermentation condition in the described fermentation tank is: inoculum concentration 5%, before fermentation, give intermittently to stir every 1h in the 12h, and revolution 100rpm, the 30min that holds time carries out a feed supplement in fermentation in the time of 30 hours, adds 0.01% ammoniacal liquor.
3. according to the application of the described Lactobacillus casei zymotic fluid of claim 2 in feed, it is characterized in that: the addition of described Lactobacillus casei zymotic fluid in feed is 7%.
4. according to the application of the described Lactobacillus casei zymotic fluid of claim 1 in feed, it is characterized in that: the technological process of production of described Lactobacillus casei feed addictive is:
With the Lactobacillus casei zymotic fluid in 60 ℃ of spray-dryings to water content below 8%, obtain dry bacterium powder, add rape cake after the detoxification then, make that to contain Lactobacillus casei bacterium number be 10
9The feed addictive of viable bacteria/gram.
5. according to the application of the described Lactobacillus casei zymotic fluid of claim 4 in feed, it is characterized in that: the living preparation of lactobacillus number that the every gram of described dry bacterium powder contains is 10
11
6. according to the application of the described Lactobacillus casei zymotic fluid of claim 4 in feed, it is characterized in that: the addition of described Lactobacillus casei feed addictive in feed is that feed per ton adds 2 kilograms.
7. according to the application of the described Lactobacillus casei zymotic fluid of claim 4 in feed, it is characterized in that: the preparation technology of rape cake is as follows after the described detoxification:
The rape cake adds water, condition: rape cake: wheat bran=9:1g/g, ratio of water to material are 1.0:0.7g/g, and initial pH value 6.0 is with the mixer batching, at 1kg/cm
2Sterilized 20～30 minutes under 110 ℃ of vapour pressures, be cooled to 35 ℃, in the inoculation pavier, insert bacterial classification, windrow, material thickness control is at 3 centimeters, the control of fermenting cellar temperature is at 32 ℃, and relative humidity is 95%, fermentation time 3 days, use the pneumatic drier drying, again with pulverizer product be ground into below 100 orders powder namely; Described bacterial classification is Candida: Lactobacillus casei=2:1g/g, and total inoculum concentration is 4.50 * 10
7Cfu/g.
8. according to the application of the described Lactobacillus casei zymotic fluid of claim 7 in feed, it is characterized in that: described Candida is to be inoculated in malt juice liquid medium, sterilization, inoculation, and the 150r/min shaking table, 28 ℃ of cultivations obtained in 3 days.
9. the application of described Lactobacillus casei zymotic fluid in feed according to Claim 8, it is characterized in that: the preparation method of described malt juice liquid medium is: earlier with malt amylase liquid with 4～6 layers of filtered through gauze, to mix well with water then and empty saccharification liquid after the filtration to the egg that produces foam, stirring refilters after boiling, and filtrate is diluted to 5～6 Baume degrees, pH6.4,121 ℃ of sterilizations got final product in 20 minutes.
10. according to the application of the described Lactobacillus casei zymotic fluid of claim 7 in feed, it is characterized in that: described Lactobacillus casei adopts the 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 ℃, the 15min sterilization is standby, then Lactobacillus casei is inoculated in the MRS fluid nutrient medium, and cultivation is left standstill in sterilization, inoculation, and 30 ℃, 2～3 days are namely.
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| CN108641991A (en) * | 2018-07-11 | 2018-10-12 | 江苏恒丰强生物技术有限公司 | A kind of composite viable bacteria preparation and preparation method and its application |
| CN110317758A (en) * | 2019-07-19 | 2019-10-11 | 河南广安生物科技股份有限公司 | Double toxin antidotes of a kind of broiler chicken and preparation method thereof |
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