CN103421712B - For animal bifidobacteria and the subtilis of fodder additives probiotic solid fermentation - Google Patents
For animal bifidobacteria and the subtilis of fodder additives probiotic solid fermentation Download PDFInfo
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- CN103421712B CN103421712B CN201310321979.8A CN201310321979A CN103421712B CN 103421712 B CN103421712 B CN 103421712B CN 201310321979 A CN201310321979 A CN 201310321979A CN 103421712 B CN103421712 B CN 103421712B
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Abstract
The present invention relates to and a kind ofly produce the fermented type dregs of beans with special sour fragrance and containing the probiotic solid fermentation that can be used for food and feed additive field of high viable count and high protein degradation rate.For the subtilis (Bacillus of solid fermentation? subtilis) HM-66 and animal bifidobacteria (Bifidobacterium? animalis) V9 is probiotic bacterium, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, does is deposit number respectively CGMCC? No.6733 and CGMCC? No.5470, preservation date is respectively on October 29th, 2012 and on November 18th, 2011, and preservation address is: Datun Road, Chaoyang District, Beijing City.The invention also discloses its application method.
Description
Technical field
The invention belongs to the probiotic solid fermentation field of fodder additives, relate to and a kind ofly can produce the fermented type dregs of beans with special sour fragrance and high (Bacillussubtilis) HM-66 of the subtilis for fodder additives probiotic solid fermentation of the viable count of bean pulp fermentation product degradation rate that is high and dregs of beans protein and animal bifidobacteria (Bifidobacteriumanimalis) V9, this technology can be widely used in the aspects such as animal feedstuff additive.
Background technology
At present, probiotic composition many employings probiotic bacterium directly with liquid cow's milk and soya-bean milk fermentation, obtain functional yogurt milk or soybean milk yoghurt, all the other then adopt liquid state fermentation, and directly collection probiotic bacterium thalline, is made into probiotic bacterium pulvis, tablet or capsule.Adopt liquid state fermentation can produce a large amount of waste water, environmental pollution is comparatively large,
Aftertreatment is more difficult, machinery equipment complex operation.
Summary of the invention
The object of the invention is to adopt subtilis HM-66 and animal bifidobacteria V9 to carry out mixed solid fermentation dregs of beans, provides a kind of and can produce the fermented type dregs of beans with special sour fragrance and subtilis (Bacillussubtilis) HM-66 of the high probiotic solid fermentation for fodder additives of the viable count of bean pulp fermentation product degradation rate that is high and dregs of beans protein and animal bifidobacteria (Bifidobacteriumanimalis) V9.
The present invention is achieved through the following technical solutions.
For animal bifidobacteria and the subtilis of fodder additives probiotic solid fermentation, it is characterized in that: described subtilis (Bacillussubtilis) HM-66 and animal bifidobacteria (Bifidobacteriumanimalis) V9 is probiotic bacterium, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is respectively CGMCCNo.6733 and CGMCCNo.5470, preservation date is respectively on October 29th, 2012 and on November 18th, 2011, and preservation address is: Datun Road, Chaoyang District, Beijing City.
For the animal bifidobacteria of fodder additives probiotic solid fermentation and the application of subtilis, it is characterized in that: adopt subtilis (Bacillussubtilis) HM-66 and animal bifidobacteria (Bifidobacteriumanimalis) V9 to carry out mixed culture solid state fermentation dregs of beans, prepare the nutritious bean pulp fermentation goods also containing a large amount of probiotic bacterium.
For the animal bifidobacteria of fodder additives probiotic solid fermentation and the using method of subtilis, it is characterized in that comprising the following steps: that the cultivation of subtilis (Bacillussubtilis) HM-66 and animal bifidobacteria (Bifidobacteriumanimalis) V9 and solid state fermentation thereof are cultivated, obtain the solid state fermentation soybean products of probiotic bacterium, described subtilis HM-66 and animal bifidobacteria V9 seed culture medium are: raffinose 2%, dregs of beans 1%, add water to 100%, pH7.0-7.2.Solid-state fermentation culture medium: 58% dregs of beans, the sugar of 2%, moisture content 40%.
Described subtilis HM-66 seed culture condition is: be positioned on shaking table, rotating speed is 150r/min, and temperature is 37 DEG C, cultivates 20h.The seed culture condition of described animal bifidobacteria V9, is placed in anaerobic box quiescent culture, culture temperature 37 DEG C, and incubation time is 20h.
Described solid state fermentation culture condition is: subtilis HM-66 and animal bifidobacteria V9 inoculum size are respectively 0.6% and 6.0%.
At the fermentation initial stage, subtilis HM-66 and animal bifidobacteria V9 mixed fermentation, pH is all on a declining curve, and pH minimum value is 5.78, and in the later stage of fermentation, its pH is all in rising trend, and after fermentation 48h, its pH is 6.71.
After subtilis HM-66 and animal bifidobacteria V9 mixed fermentation 48h, its free ammonical nitrogen content is: 301.9umol/g.
Subtilis HM-66 and animal bifidobacteria V9 mixed fermentation, after fermentation 48h, little peptide (<1000Da) content adds 30.7%, and large peptide (>10000Da) decreases 34.3%.
Advantage of the present invention and positively effect:
The application adopts subtilis HM-66 and animal bifidobacteria V9 mixed solid fermentation dregs of beans, and adopt solid state fermentation, its production cost is low, and the fund of investment is less, and convenient in downstream processing, pollute little, device structure is simple, easy to operate.
Animal bifidobacteria V9 is adopted to ferment, the tart flavour such as lactic acid and acetic acid small-molecule substance can be produced, these small-molecule substances give improvement and the variation of the organoleptic features such as the distinctive sour fragrance of product, color and luster and mouthfeel, make bean pulp fermentation product more easily by human consumer is accepted; Adopt subtilis HM-66 fermented bean dregs, the protein in dregs of beans can be made to be broken down into little peptide and total free aminoacids, be beneficial to digesting and assimilating of stomach.
Accompanying drawing explanation
Fig. 1 is that animal bifidobacteria V9 inoculum size affects schematic diagram to viable count;
Fig. 2 is that subtilis HM-66 inoculum size affects schematic diagram to animal bifidobacteria V9 viable count;
Fig. 3 is the present invention affects schematic diagram in the solid state fermentation time to animal bifidobacteria V9 viable count;
Fig. 4 is subtilis HM-66 and animal bifidobacteria V9 when carrying out mixed culture solid state fermentation, and pH is schematic diagram over time;
Fig. 5 is subtilis HM-66 and animal bifidobacteria V9 when carrying out mixed culture solid state fermentation, and amino-acid nitrogen content is schematic diagram over time;
Fig. 6 is after subtilis HM-66 and animal bifidobacteria V9 carry out mixed culture solid state fermentation 36h, the percentage composition schematic diagram of different polypeptide molecular weight.
Embodiment
Below in conjunction with example, the present invention is further described, and following example is illustrative, is not determinate, can not limit protection scope of the present invention with following example.
For animal bifidobacteria and the subtilis of fodder additives probiotic solid fermentation, it is characterized in that: described subtilis (Bacillussubtilis) HM-66 and animal bifidobacteria (Bifidobacteriumanimalis) V9 is probiotic bacterium, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is respectively CGMCCNo.6733 and CGMCCNo.5470, preservation date is respectively on October 29th, 2012 and on November 18th, 2011, and preservation address is: Datun Road, Chaoyang District, Beijing City.
For the animal bifidobacteria of fodder additives probiotic solid fermentation and the application of subtilis, it is characterized in that: adopt subtilis (Bacillussubtilis) HM-66 and animal bifidobacteria (Bifidobacteriumanimalis) V9 to carry out mixed culture solid state fermentation dregs of beans, prepare the nutritious bean pulp fermentation goods also containing a large amount of probiotic bacterium.
For the animal bifidobacteria of fodder additives probiotic solid fermentation and the using method of subtilis, it is characterized in that comprising the following steps: that the cultivation of subtilis (Bacillussubtilis) HM-66 and animal bifidobacteria (Bifidobacteriumanimalis) V9 and solid state fermentation thereof are cultivated, obtain the solid state fermentation soybean products of probiotic bacterium, described subtilis HM-66 and animal bifidobacteria V9 seed culture medium are: raffinose 2%, dregs of beans 1%, add water to 100%, pH7.0-7.2.Solid-state fermentation culture medium: 58% dregs of beans, the sugar of 2%, moisture content 40%.
At the fermentation initial stage, subtilis HM-66 and animal bifidobacteria V9 mixed fermentation, pH is all on a declining curve, and pH minimum value is 5.78, and in the later stage of fermentation, its pH is all in rising trend, and after fermentation 48h, its pH is 6.71.
After subtilis HM-66 and animal bifidobacteria V9 mixed fermentation 48h, its free ammonical nitrogen content is: 301.9umol/g.
Subtilis HM-66 and animal bifidobacteria V9 mixed fermentation, after fermentation 48h, little peptide (<1000Da) content adds 30.7%, and large peptide (>10000Da) decreases 34.3%.
1. the seed liquor preparation of thalline
Being received from the glycerine pipe of cold storage by subtilis HM-66 is equipped with in the substratum of liquid seeds, and be positioned on shaking table, rotating speed is 150r/min, and temperature is 37 DEG C, cultivates 20h.Animal bifidobacteria V9 seed culture condition, is placed in constant-temperatureanaerobic anaerobic incubator quiescent culture, culture temperature 37 DEG C, and incubation time is 20h.Wherein liquid seed culture medium (g/L) is: raffinose 20, dregs of beans 10, pH7.0-7.2.
2. solid state fermentation
Subtilis HM-66 and animal bifidobacteria V9 is connect bacterium to the wet dregs of beans substratum gone out with 0.6% and 6.0% inoculum size respectively, sealing be placed on constant incubator quiescent culture.
Solid-state fermentation culture medium is: the dregs of beans of water content 60%, 115 DEG C of autoclaving 25min.
3. measure viable count
General employing colony counting method, operation steps is: take the bean pulp fermentation goods 10g prepared, be placed in the stroke-physiological saline solution of 90ml, be placed on shaking table and shake 30min, shaking up the bacterium liquid that rear liquid-transfering gun gets 1ml adds in the stroke-physiological saline solution of another 9ml, dilute, according to national standard method, take turns doing ten times of gradient dilutions.After having diluted, get suitable extent of dilution 1ml respectively with liquid-transfering gun and throw flat board into, pour into substratum mixing, take turns doing three parallel, be placed in incubator at 37 DEG C of quiescent culture 48h.
The mensuration of 4.pH value
Take the soybean products after 10g fermentation, put into triangular flask, add the distilled water of 90ml.Use magnetic stirrer 30min, after leaving standstill 10min, determine its pH value (see Fig. 4) with Accurate pH measurement.
5. the mensuration of amino-acid nitrogen content
Get the dregs of beans after 10g fermentation, broken with homogenizer, mix with the distilled water of 90ml afterwards, place 1 day at 4 DEG C of refrigerators.The centrifugal 7000r/min of sample, centrifugal 15min.Supernatant liquor 0.8ml is placed in test tube, then adds 3.2ml distilled water, 2.0ml developer, and mixing, puts into boiling water bath and heat 15min, make blank simultaneously.Then cold water cooling, adds the mixing of 5.0ml40% ethanolic soln, uses 1cm cuvette after placing 15min, and returning to zero in 570nm place with blank tube measures A value (see Fig. 5).
6. dregs of beans peptide molecule flow measurement
Get the dregs of beans after 10g fermentation, after homogenizer fragmentation, by gained fermented product by 5 times of stroke-physiological saline solution at 4 DEG C of lixiviate 4h, centrifuging and taking supernatant liquor under 4500r/min condition, powder is got in lyophilize, does not separately inoculate solid state fermentation thing as blank (see Fig. 6).
Chromatographic condition
Chromatographic column: GESuperdex
tMpeptide10/300GL(10 × 300nm);
Moving phase: 50mM phosphate buffer soln (pH is 7.2, and containing 0.15MNacl);
Post pressure: be less than 18bar;
Flow velocity: 0.5ml/min;
Determined wavelength: 214nm
Column temperature: room temperature
Sample size: 10ul.
7. the packaging of fermented product
There is a large amount of viable bacterias in bean pulp fermentation goods, therefore bean pulp fermentation product must carry out sealing packaging, and in cryopreservation, the quality guaranteed period is approximately 6 months.
Below the determination of some parameters of the production method to the probiotics fermention product taking dregs of beans as matrix, and the optimization of some culture condition.
One. inoculum size is on the impact of viable count
In Fig. 1, solid-state fermentation culture medium is: 58% dregs of beans, the sugar of 2%, moisture content 40%, 115 DEG C of autoclaving 25min.
When the bacterium amount that connects of animal bifidobacteria V9 is 1%, culture temperature 37 ± 1 DEG C, incubation time 28h, recording plant lactobacillus HM-20 viable count is 3.8 × 10
7cfu/g.
When the bacterium amount that connects of animal bifidobacteria V9 is 4%, culture temperature 37 ± 1 DEG C, incubation time 28h, recording plant lactobacillus HM-20 viable count is 3.4 × 10
7cfu/g.
When the bacterium amount that connects of animal bifidobacteria V9 is 6%, culture temperature 37 ± 1 DEG C, incubation time 28h, recording plant lactobacillus HM-20 viable count is 4.1 × 10
7cfu/g.
Result: the suitableeest inoculum size of animal bifidobacteria V9 is respectively 6%.
Two. soak water to the impact of viable count
When the pH soaking water is 6.0, when the inoculum size of animal bifidobacteria V9 is respectively 4%, culture temperature 37 ± 1 DEG C, incubation time 32h, recording animal bifidobacteria V9 viable count is 7.8 × 10
7cfu/g.
When the pH soaking water is 6.5, when the kind amount of animal bifidobacteria V9 is respectively 4%, culture temperature 37 ± 1 DEG C, incubation time 32h, recording animal bifidobacteria V9 viable count is 8.9 × 10
7cfu/g.
When the pH soaking water is 7.0, when the kind amount of animal bifidobacteria V9 is respectively 4%, culture temperature 37 ± 1 DEG C, incubation time 32h, recording animal bifidobacteria V9 viable count is 7.9 × 10
7cfu/g.
Result: when the pH soaking water is 6.5, the viable count of animal bifidobacteria V9 is the highest.
Three. subtilis HM-66 inoculum size is on the impact of viable count
In Fig. 2, when the inoculum size of subtilis HM-66 is 0.6%, when animal bifidobacteria V9 is inoculum size 4%, soaking water pH is 6.5, and culture temperature 37 ± 1 DEG C, incubation time 32h, recording animal bifidobacteria V9 viable count is 7.6 × 10
7cfu/g.
When the inoculum size of subtilis HM-66 is 1.0%, when the kind amount of animal bifidobacteria V9 is 4%,
Soaking water pH is 6.5, and culture temperature 37 ± 1 DEG C, incubation time 32h, recording animal bifidobacteria V9 viable count is 8.5 × 10
7cfu/g.
When the inoculum size of subtilis HM-66 is 2.0%, when the kind amount of animal bifidobacteria V9 is 4%, soaking water pH is 6.5, and culture temperature 37 ± 1 DEG C, incubation time 32h, recording animal bifidobacteria V9 viable count is 6.3 × 10
7cfu/g.
Result: when subtilis HM-66 and animal bifidobacteria V9 mixed fungus fermentation, its suitableeest inoculum size is 0.6%.
Four. the solid state fermentation time is on the impact of viable count
In Fig. 3, bacterial growth experienced by lag phase, logarithmic phase, balance period and paracme respectively, and at different times, growing state is different, carries out live bacterial count respectively to its sampling.
Solid-state fermentation culture medium is: 58% dregs of beans, the sugar of 2%, moisture content 40%, 115 DEG C of autoclaving 25min.
Inoculum size and culture condition: subtilis HM-66 and animal bifidobacteria V9 inoculum size are respectively 0.6% and 6.0%, soak water pH6.5, culture temperature 37 ± 1 DEG C, incubation time 28h, recording animal bifidobacteria V9 viable count is 3.2 × 10
7cfu/g.
Subtilis HM-66 and animal bifidobacteria V9 inoculum size are respectively 0.6% and 6.0%, and soak water pH6.5, culture temperature 37 ± 1 DEG C, incubation time 36h, recording animal bifidobacteria V9 viable count is 1.1 × 10
8cfu/g.
Subtilis HM-66 and animal bifidobacteria V9 inoculum size are respectively 1.0% and 4.0%, and soak water pH6.5, culture temperature 37 ± 1 DEG C, incubation time 48h, animal bifidobacteria V9 viable count is 1.4 × 10
8cfu/g.
Result: when considering production cost and production cycle, as subtilis HM-66 and animal bifidobacteria V9 mixed fungus fermentation, the suitableeest fermentation time is 36h.
Conclusion
When subtilis HM-66 and animal bifidobacteria V9 inoculum size are respectively 0.6% and 6.0%, the pH soaking water is 6.5, and culture temperature 37 ± 1 DEG C, after incubation time 36h, its viable count is higher, and the viable count recording animal bifidobacteria V9 is 1.1 × 10
8cfu/g.
Claims (2)
1. for the mixing thalline of fodder additives probiotic solid fermentation, comprise subtilis (
bacillussubtilis) HM-66 and animal bifidobacteria (
bifidobacteriumanimalis) V9, it is characterized in that: described subtilis and described animal bifidobacteria are probiotic bacteriums, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center; Deposit number is respectively CGMCCNo.6733 and CGMCCNo.5470, and preservation date is respectively on October 29th, 2012 and on November 18th, 2011.
2. the mixing thalline for fodder additives probiotic solid fermentation according to claim 1 is applied, and it is characterized in that: described subtilis (
bacillussubtilis) HM-66 and animal bifidobacteria (
bifidobacteriumanimalis) V9 be used for fermented bean dregs.
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| CN105200000B (en) * | 2015-11-17 | 2018-09-28 | 山东西王糖业有限公司 | A kind of method of Bifidobacterium and bacillus subtilis symbiosis culture |
| CN116196353A (en) * | 2023-02-27 | 2023-06-02 | 广东生和堂健康食品股份有限公司 | Lung-moistening and cough-relieving composition, and preparation method and application thereof |
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