CN106497842A - The preparation method and application of a kind of lactic acid bacteria and aspergillus niger mixed solid fermentation preparation - Google Patents
The preparation method and application of a kind of lactic acid bacteria and aspergillus niger mixed solid fermentation preparation Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Abstract
The present invention provides the preparation method of a kind of lactic acid bacteria and aspergillus niger mixed solid fermentation preparation, after lactobacillus casei (Lactobacillus casei) zhang with aspergillus niger (Aspergillus niger) KT An8 mixed solid fermentations, both the content of the metabolites such as lactic acid bacterium number and lactic acid in fermentation preparation had been improved, the acid protease content that the metabolism of aspergillus niger institute is produced had been improve again.Wherein, the deposit number of the deposit number CGMCC No.5469 of the lactobacillus casei zhang, the aspergillus niger KT An8 is CGMCC No.12866.Can be applied in animal feed directly as probiotics preparation by fermentation preparation product obtained in above-mentioned preparation method of the invention, this mixed fermentation method can greatly reduce fermentation costs, improve production efficiency has greatly help to production.
Description
Technical field
The present invention relates to a kind of lactic acid bacteria and preparation method and the application of aspergillus niger mixed solid fermentation preparation, belong to micro- life
State preparation technique field.
Background technology
With the continuous understanding to microbiologic population's interaction mechanism, people are using mixed fermentation technology with culture of the same race
Base is fermented, and the mixed fermentation product for obtaining high viable count is also increasingly taken seriously.Mixed fermentation can utilize the mutual of them
Sharp relation, makes mixed bacteria learn from other's strong points to offset one's weaknesses, displays one's respective advantages, and produces the unique products of low cost, Functionality, quality and appealing design.
In cud animal microecological formulation, lactic acid bacteria and aspergillus niger all have unique effect to animal intestinal, wherein, black
Aspergillosis can produce the protease material such as substantial amounts of acid protease, accelerate the digestion rate of nutrient substance in cud, while black
Aspergillosis have good microbe adherence trait, heavy metal ion that can be in active adsorption feedstuff, reduce heavy metal ion to animal
Toxic.Adding aspergillus niger in cattle diet can make its increased weight 7.5%；Adding aspergillus niger in milch cow produces can which
Milk amount increases by 20%.For lactic acid bacteria, wherein as intestinal in common flora, its can by secreting bacteria element, hydrogen peroxide,
The materials such as organic acid (including lactic acid, acetic acid, propanoic acid, butanoic acid etc.), decline can intestinal environment pH value, suppress the micro- life of harmful cause of disease
Thing grows.
But numerous studies personnel are studied to the inhibition of mycete just for lactic acid bacteria, be seldom related to lactic acid bacteria and
Research and development in terms of the mixed fermentation of mycete.In lactic acid bacteria and aspergillus niger mixed cultivation process, it is former that aspergillus niger can consume solid fermentation
Remaining air in material, the growth for lactic acid bacteria provide preferable anaerobic environment；It is a large amount of that aspergillus niger can produce a large amount of protease
Decomposition of protein class material generates a large amount of peptides, which solves the underdeveloped problem of lactobacillus protein enzyme system, is that the growth of lactic acid bacteria is carried
Abundant nitrogen source is supplied, the breeding and metabolism of lactic acid bacteria has been promoted, is reduced the pH of fermentation raw material；At the same time, relatively low acid
Property environmental benefits in the synthesis of Aspergillus niger acidic protease, so as to form a kind of benign cycle.Product obtained by after this kind of fermentation
Product, the sour fragrance not only having after lactic acid bacteria fermentation and abundant lactic acid bacteria and substantial amounts of lactic acid, and there is aspergillus niger generation
Thank to the enzyme material enriched in product.The aspects such as flavor taste therefore, in product, nutritive value and physiological function are better than any
The product of single bacterium fermentation, has good using value in the cultivation of animal.
Content of the invention
Present invention is primarily intended to providing the preparation method of a kind of lactic acid bacteria and aspergillus niger mixed solid fermentation preparation, pass through
Lactobacillus casei (Lactobacillus casei) zhang and aspergillus niger (Aspergillus niger) KT-An8 hybrid solid-states
The content of the metabolites such as lactic acid bacterium number and lactic acid in fermentation preparation after fermentation, had both been improved, aspergillus niger institute had been improve again
The acid protease content that metabolism is produced.
The purpose of the present invention is achieved by the following technical solution：
A kind of lactic acid bacteria and the preparation method of aspergillus niger mixed solid fermentation preparation, including：Lactobacillus casei is prepared respectively
Zhang lyophilizing mycopowder and aspergillus niger KT-An8 seed liquor, then carry out following mixed solid fermentation：The quality of solid-state fermentation culture medium
Fraction is consisted of：Bean cake 15%, feed grade whitewashing skin of Semen Maydis 10%, bran of Fagopyrum esculentum Moench 30%, glucose 5%, water 40% need not go out
Bacterium, initial pH are 6.5, and the inoculum concentration of lactobacillus casei zhang is 1 × 106CFU/g solid-state fermentation culture mediums, aspergillus niger KT-
The inoculum concentration of An8 be 10g seed liquor/1Kg solid-state fermentation culture mediums, 30 DEG C of fermentation temperature, fermentation time 6 days.
Further, the preparation method of described aspergillus niger KT-An8 seed liquor is as follows：
(1) first order seed is prepared in Cha Shi solid slant culture bases：NaNO32g/L, K2HPO41g/L, KCl 0.5g/L,
MgSO40.5g/L, FeSO40.01g/L, sucrose 30g/L, agar 15g/L, distilled water are prepared, and pH natures are dispensed in test tube,
121 DEG C of sterilizing 20min rear-inclineds put natural cooling solidification, are rule on inclined-plane with inoculating loop picking original strain, are placed in 30 DEG C
Culture 2 days；
(2) prepared by secondary seed：Culture matrix is pressed mass fraction and is constituted, bean cake 20%, bran of Fagopyrum esculentum Moench 40%, water 40%,
121 DEG C of sterilizing 15min, picking first order seed sterilized water 10mL dilutions shake up makes bacteria suspension, and inoculum concentration is trained for 10mL/10kg
Foster substrate, 30 DEG C culture 4 days after be obtained be aspergillus niger KT-An8 seed liquor.
Further, the preparation method of described lactobacillus casei zhang lyophilizing mycopowder is as follows：
(1) high density fermentation of lactobacillus casei zhang：Soybean protein powder 60g/L, glucose 40g/L, anhydrous sodium acetate
10g/L, K2HPO47g/L, sodium citrate 9g/L, yeast powder 7g/L, tween 80 0.1g/L, distilled water are prepared, initial pH7.0,
Inoculation lactobacillus casei zhang 6% (v/v), 37 DEG C of fermentation 5h logical nitrogen holding pressure 0.03MPa, rotating speed 60r/min, permanent
pH 5.9；Lactobacillus casei zhang high density fermentation liquid is prepared, lactobacillus casei zhang viable counts reach 1010CFU/mL
More than；
(2) above-specified high density fermentation liquid directly adds protective agent dry powder blend uniformly into bacteria suspension, freeze-dried obtains
Lyophilizing mycopowder, in lyophilizing mycopowder, lactobacillus casei zhang viable counts reach 2 × 1011More than CFU/g；
Further, the protective agent is made up of skimmed milk, trehalose, soybean protein powder and vitamin C；
Further, the protective agent is as follows with the mass ratio of lactobacillus casei zhang high density fermentation liquid composition：De-
Fat breast 3%, trehalose 1.5%, soybean protein powder 15%, vitamin C 0.4%, balance of lactobacillus casei zhang high density
Fermentation liquid.
Lactobacillus casei zhang (patent CN102813004A is disclosed) of the present invention is from the Inner Mongol in calendar year 2001
The lactic acid bacteria of the one plant of performance brilliance separated in natural fermentation koumiss sample on Xilinguole League's Zhenglan Banner grassland, its
There is good pipe intestinal digesting liquid tolerance, can survive in human body intestinal canal, be colonized and breed, make intestinal microflora year
Lightization；There is blood fat reducing, improve the effect of antioxidant ability of organism, the liver protecting and prevention type Ⅱdiabetes mellitus.The bacterial strain in
On November 18th, 2011 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC), ground
Location is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica (postcode 100101), and deposit number is
CGMCC No.5469, Classification And Nomenclature are lactobacillus casei (Lactobacillus casei).
Aspergillus niger KT-An8 of the present invention is isolated from the Feed Sample of natural fermentation, from 30 plants of Aspergillus niger strains
In gone out by fermented bean cake and wheat bran Screening matrix, the ability with the various metabolism enzyme activity of high yield.The bacterial strain is in September, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC) is preserved within 8th, address is court of Beijing
No. 3 Institute of Microorganism, Academia Sinica of institute of positive area's North Star West Road 1 (postcode 100101), deposit number is CGMCC
No.12866 Classification And Nomenclatures are aspergillus niger (Aspergillus niger).
Aspergillus niger KT-An8 bacterial strains of the present invention cultivate the various enzyme activities of 3d such as on aforementioned base fermentation medium
Under：Saccharifying enzyme 3000U/mL culture fluid, xylanase activity 3000U/mL culture fluid, acid protease 2012.52U/mL are cultivated
Liquid, pectase 5000.16U/mL culture fluid, cellulase 4361.37U/mL culture fluid, beta-glucosidase 2012.52U/mL
Culture fluid.The abundant enzyme system that aspergillus niger KT-An8 fermented bean cake and wheat bran are produced, can preferably act on bean cake and wheat bran, drop
Polysaccharide, protein and cellulose in solution bean cake etc..
By lactic acid yield in fermentation preparation product obtained in above-mentioned preparation method of the invention compared with lactobacillus casei zhang
Individually fermentation improves 10%；In gained fermented product, the viable count of lactic acid bacteria is 1.02 × 1010CFU/g, compared with lactobacillus casei
Zhang individually ferment viable count improve 10 times；In gained fermented product, pH value is 4.22, individually sends out compared with lactobacillus casei zhang
Ferment pH value 4.67 is significantly reduced；In gained fermented product, acid protease yield individually ferments raising compared with aspergillus niger KT-An8
15.6%.
Can be applied to move directly as probiotics preparation by fermentation preparation product obtained in above-mentioned preparation method of the invention
In thing feedstuff, the mass ratio being preferably added in feedstuff is 10-80%.This mixed fermentation method can greatly reduce fermentation
Cost, improve production efficiency have greatly help to production.
Specific embodiment
Below by the specific embodiment narration present invention.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment be interpreted as illustrative, and unrestricted the present invention
Scope, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
On the premise of invention spirit and scope, various changes that the material component and consumption in these embodiments is carried out are changed
Belong to protection scope of the present invention.
The percentage sign " % " being related in embodiment, if not specified, refers to that mass percent, the percentage ratio of solution refer to
Grams containing solute in 100mL, the percentage ratio between liquid refer to the volume ratio of the solution at 25 DEG C.
Embodiment 1：
The preparation of 1.1 aspergillus niger KT-An8 seed liquor：
(1) first order seed is prepared in Cha Shi solid slant culture bases：NaNO32g, K2HPO41g, KCl 0.5g, MgSO4
0.5g, FeSO40.01g, sucrose 30g, agar 15g, distilled water 1L, pH natures are dispensed in 30mL test tubes, 121 DEG C of sterilizings
20min rear-inclineds put natural cooling solidification, are rule on inclined-plane with inoculating loop picking original strain, are placed in 30 DEG C and cultivate 2 days；
(2) prepared by secondary seed：Mass fraction is constituted, bean cake 20%, bran of Fagopyrum esculentum Moench 40%, water 40%, 121 DEG C of sterilizings
15min, picking first order seed sterilized water 10mL dilution shakes up and makes bacteria suspension, and inoculum concentration is 10mL/10kg culture matrixes, and 30
DEG C culture 4 days after be obtained be aspergillus niger KT-An8 seed liquor.
The preparation of 1.2 lactobacillus casei zhang lyophilizing mycopowder：
(1) high density fermentation of lactobacillus casei zhang：Soybean protein powder 60g, glucose 40g, anhydrous sodium acetate 10g,
K2HPO47g, sodium citrate 9g, yeast powder 7g, tween 80 0.1g, distilled water 1L, initial pH7.0, are inoculated with lactobacillus casei
Zhang 6% (v/v), 37 DEG C of fermentation 5h logical nitrogen holding pressure 0.03MPa, rotating speed 60r/min, permanent pH 5.9；It is prepared into
Lactobacillus casei zhang fermentation liquids are arrived, lactobacillus casei zhang viable counts reach 1010More than CFU/mL；
(2) above-specified high density fermentation liquid directly adds protective agent dry powder blend uniformly into bacteria suspension, freeze-dried obtains
Lyophilizing mycopowder, in lyophilizing mycopowder, lactobacillus casei zhang viable counts reach 2 × 1011More than CFU/g；
The protective agent is as follows with the mass ratio of lactobacillus casei zhang high density fermentation liquid composition：Skimmed milk 3%, sea
Algae sugar 1.5%, soybean protein powder 15%, vitamin C 0.4%, balance of lactobacillus casei zhang high density fermentation liquid.
1.3 test assay methods
Viable lactic acid bacteria is counted：Typically using pour plate counting method, operating procedure is：According to national standard method, shake up and send out
Zymotic fluid, draws 1mL fermentation liquids and is placed in the physiological saline solution of 9mL, carries out 10 times of dilutions, is subsequently diluted to required ladder successively
Degree.By last dilution gradient tube dilution shake even after, draw during 1mL diluents are poured into flat board, to culture medium is entered, shake up, put
Solidification, in 37 DEG C of incubator culture 48h.Flat board does two gradients respectively, and each gradient two is parallel.
Determine viable count of lactobacillus culture medium：MRS+ cycloheximide solid mediums.
PH is determined：Fermentation liquid pH value is directly determined using accurate pH meter.
Lactic acid content is determined：Using high performance liquid chromatography detection.
Acid protease is detected：Using acidic protein enzyme detection kit, detected by microplate reader.
1.4 lactobacillus casei zhang and the preparation method of aspergillus niger KT-An8 mixed solid fermentation preparations：
Solid-state fermentation culture medium mass fraction is constituted：Bean cake 15%, feed grade whitewashing skin of Semen Maydis 10%, bran of Fagopyrum esculentum Moench
30%, glucose 5%, water 40%, without the need for sterilizing, initial pH is 6.5, the inoculum concentration of lactobacillus casei zhang is 1 ×
106CFU/g solid mediums, the inoculum concentration of aspergillus niger KT-An8 is 10g seed liquor/1Kg solid-state fermentation culture mediums, fermentation temperature
30 DEG C, fermentation time 6 days.
Matched group 1：Bean cake 15% (m/m), whitewashing skin of Semen Maydis 10% (m/m), bran of Fagopyrum esculentum Moench 30% (m/m), glucose 5%
(m/m), water 40% (m/m), without the need for sterilizing, Preliminary fermentation pH is 6.5, and lactobacillus inoculum amount is 1 × 106CFU/g, fermentation temperature
30 DEG C of degree, fermentation time 6 days.
Matched group 2：Bean cake 15% (m/m), whitewashing skin of Semen Maydis 10% (m/m), bran of Fagopyrum esculentum Moench 30% (m/m), glucose 5%
(m/m), water 40% (m/m), without the need for sterilizing, Preliminary fermentation pH is 6.5, and aspergillus niger inoculum concentration is 10g secondary seeds/1Kg solid-states
Fermentation medium, 30 DEG C of fermentation temperature, fermentation time 6 days.
1.5 result of the test
(1) situation of change of viable count of lactobacillus
Table 1：The situation of change of viable count of lactobacillus
After lactobacillus casei zhang and aspergillus niger KT-An8 mixed solid fermentations, lactic acid bacteria in gained fermentation preparation product
Viable count be 1.02 × 1010CFU/g, compared with lactobacillus casei zhang individually ferment viable count improve 5.58 times, viable lactic acid bacteria
Number is significantly improved.
(2) situation of change of pH value after fermenting
Table 2：The situation of change of pH value after fermentation
After lactobacillus casei zhang and aspergillus niger KT-An8 mixed solid fermentations, in gained fermentation preparation product, pH value is
4.22, significantly reduce compared with the lactobacillus casei zhang pH 4.67 that individually ferment, illustrate that mixed fermentation can significantly reduce the acid of feedstuff
Degree, increased product acid amount.
(3) situation of change of lactic acid after fermenting
Table 3：The situation of change of lactic acid after fermentation
After lactobacillus casei zhang and aspergillus niger KT-An8 mixed solid fermentations, in gained fermentation preparation product, lactic acid is produced
Raw amount is individually fermented increase by 30% compared with lactobacillus casei zhang, dramatically increases lactic acid content.
(4) situation of change of acid protease after fermenting
Table 4：The situation of change of acid protease after fermentation
Independent compared with aspergillus niger KT-An8 according to acid protease yield in the fermentation process gained fermentation preparation product
Fermentation improves 15.6%, illustrates that the sour environment that lactic acid bacteria lactobacillus casei zhang is produced is conducive to aspergillus niger KT-An8 acid
The generation of protease.
The fermentation preparation product being prepared by the method for the present invention, not only have lactic acid bacteria fermentation after sour fragrance and enrich
Lactic acid bacteria and substantial amounts of lactic acid, and with the enzyme material enriched in aspergillus niger metabolite.Therefore, in the local flavor of product
The aspects such as mouthfeel, nutritive value and physiological function have in the cultivation of animal good better than the product of any single bacterium fermentation
Using value.
Claims (8)
1. the preparation method of a kind of lactic acid bacteria and aspergillus niger mixed solid fermentation preparation, including：Lactobacillus casei is prepared respectively
Zhang lyophilizing mycopowder and aspergillus niger KT-An8 seed liquor, then carry out following mixed solid fermentation：The quality of solid-state fermentation culture medium
Fraction is consisted of：Bean cake 15%, feed grade whitewashing skin of Semen Maydis 10%, bran of Fagopyrum esculentum Moench 30%, glucose 5%, water 40% need not go out
Bacterium, initial pH are 6.5, and the inoculum concentration of lactobacillus casei zhang is 1 × 106CFU/g solid-state fermentation culture mediums, aspergillus niger KT-
The inoculum concentration of An8 be 10g seed liquor/1Kg solid-state fermentation culture mediums, 30 DEG C of fermentation temperature, fermentation time 6 days；Wherein, described dry
The deposit number of the deposit number CGMCC No.5469 of Lactobacillus paracasei zhang, the aspergillus niger KT-An8 is CGMCC
No.12866.
2. the preparation method of a kind of lactic acid bacteria and aspergillus niger mixed solid fermentation preparation as claimed in claim 1, its feature exist
In the preparation method of described aspergillus niger KT-An8 seed liquor is as follows：
(1) first order seed is prepared in Cha Shi solid slant culture bases：NaNO32g/L, K2HPO41g/L, KCl 0.5g/L, MgSO4
0.5g/L, FeSO40.01g/L, sucrose 30g/L, agar 15g/L, distilled water are prepared, and pH natures are dispensed in test tube, 121 DEG C
Sterilizing 20min rear-inclineds put natural cooling solidification, are rule on inclined-plane with inoculating loop picking original strain, are placed in 30 DEG C of cultures 2
My god；
(2) prepared by secondary seed：Culture matrix is pressed mass fraction and is constituted, bean cake 20%, bran of Fagopyrum esculentum Moench 40%, water 40%, 121 DEG C
Sterilizing 15min, picking first order seed sterilized water 10mL dilution shakes up makes bacteria suspension, and inoculum concentration is 10mL/10kg culture medium
Matter, 30 DEG C culture 4 days after be obtained be aspergillus niger KT-An8 seed liquor.
3. the preparation method of a kind of lactic acid bacteria and aspergillus niger mixed solid fermentation preparation as claimed in claim 1, its feature exist
In the preparation method of described lactobacillus casei zhang lyophilizing mycopowder is as follows：
(1) high density fermentation of lactobacillus casei zhang：Soybean protein powder 60g/L, glucose 40g/L, anhydrous sodium acetate 10g/
L, K2HPO47g/L, sodium citrate 9g/L, yeast powder 7g/L, tween 80 0.1g/L, distilled water are prepared, initial pH7.0, inoculation
Lactobacillus casei zhang 6% (v/v), 37 DEG C of fermentation 5h logical nitrogen holding pressure 0.03MPa, rotating speed 60r/min, permanent pH
5.9；Lactobacillus casei zhang high density fermentation liquid is prepared, lactobacillus casei zhang viable counts reach 1010CFU/mL with
On；
(2) above-specified high density fermentation liquid directly adds protective agent dry powder blend uniformly into bacteria suspension, freeze-dried obtains lyophilizing
Mycopowder, in lyophilizing mycopowder, lactobacillus casei zhang viable counts reach 2 × 1011More than CFU/g.
4. the preparation method of a kind of lactic acid bacteria and aspergillus niger mixed solid fermentation preparation as claimed in claim 3, its feature exist
It is made up of skimmed milk, trehalose, soybean protein powder and vitamin C in the protective agent.
5. the preparation method of a kind of lactic acid bacteria and aspergillus niger mixed solid fermentation preparation as claimed in claim 3, its feature exist
As follows with the mass ratio of lactobacillus casei zhang high density fermentation liquid composition in the protective agent：Skimmed milk 3%, trehalose
1.5%, soybean protein powder 15%, vitamin C 0.4%, balance of lactobacillus casei zhang high density fermentation liquid.
6. lactic acid bacteria and aspergillus niger mixed solid fermentation formulation products obtained in the arbitrary preparation method of claim 1-5.
7. the lactic acid bacteria described in claim 6 and application of the aspergillus niger mixed solid fermentation formulation products in animal feed.
8. lactic acid bacteria as claimed in claim 7 and application of the aspergillus niger mixed solid fermentation formulation products in animal feed,
Characterized in that, the lactic acid bacteria is added on the mass ratio in feedstuff for 10- with aspergillus niger mixed solid fermentation formulation products
80%.
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| CN111424003A (en) * | 2020-05-19 | 2020-07-17 | 北京科拓恒通生物技术股份有限公司 | Lactobacillus casei and application thereof in preventing and treating chronic gastroenteritis |
| CN113440465A (en) * | 2021-08-23 | 2021-09-28 | 云南英格生物技术有限公司 | Rice fermentation product, rice fermentation extract, preparation method and application |
| CN113440465B (en) * | 2021-08-23 | 2022-08-26 | 云南英格生物技术有限公司 | Rice fermentation product, rice fermentation extract, preparation method and application |
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