CN107090485A - A kind of method and its special culture media and complex bacterial agent special for preparing active polysaccharide - Google Patents
A kind of method and its special culture media and complex bacterial agent special for preparing active polysaccharide Download PDFInfo
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- C12P19/00—Preparation of compounds containing saccharide radicals
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Abstract
The invention discloses a kind of method and its special culture media and complex bacterial agent special for preparing active polysaccharide.The method provided by the present invention for preparing active polysaccharide, in turn includes the following steps：(1) composite bacteria agent, fermentation raw material and water are mixed, obtains fermentation system；Fermentation, obtains tunning；(2) water extraction is carried out；(3) alcohol precipitation is carried out, active polysaccharide is obtained；The composite bacteria agent contains bacillus subtilis and saccharomyces cerevisiae；The fermentation raw material includes the urea of wheat bran, the bean cake powder of 8 10 mass parts, the corn flour of 9 12 mass parts and 0.15 0.25 mass parts of 78 82 mass parts.It is demonstrated experimentally that the method provided using the present invention can prepare active polysaccharide, with important application value.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of method and its special culture media for preparing active polysaccharide and
Complex bacterial agent special.
Background technology
China is large agricultural country, and wheat is one of staple food crop of China.The annual production of wheat bran is about 2000
Ten thousand tons, it is the processed side product of flour mill, is directly used in feed industry more；But due to containing substantial amounts of crude fibre, egg in wheat bran
White matter content is not high and second-rate, and amino acid content can not meet the nutritional need of pig, chicken, therefore often substitutes portion of energy and raise
Expect or as the carrier of additive premix, its economic value is simultaneously underused, and causes the significant wastage of resource.
Aquaculture development is swift and violent, but extensive and highdensity cultivation makes environment go from bad to worse, and causes cultivated animals itself
Immunity degradation, disease takes place frequently.People with prevention disease, promote to give birth to by adding the antibiotic less than therapeutic dose in feed
Production efficiency that is long or improving animal, but going deep into research, it has been recognized that the problem of antibiotic triggers is used for a long time
And consequence, therefore, seek the substitute of antibiotic turns into a key factor of aquaculture sustainable development.From 20th century 60
Since age, active polysaccharide is widely used to clinical medicine as a kind of immunopotentiator, is successfully applied to again in recent years
In the disease control of the aquatic livestocks such as fishes and shrimps, and show significant immunoenhancement result.In livestock, active polysaccharide is also
A kind of feed addictive with development prospect and the immunopotentiating agent with antibiotic and probiotic double action, increasingly by
To the attention of academia and aquaculture.
The content of the invention
The technical problems to be solved by the invention are how to prepare active polysaccharide using wheat bran etc..
In order to solve the above technical problems, present invention firstly provides a kind of method for preparing active polysaccharide.
The method provided by the present invention for preparing active polysaccharide, may include following steps successively：
(1) composite bacteria agent, fermentation raw material and water are mixed, obtains fermentation system；Fermentation, obtains tunning；
(2) water extraction is carried out；
(3) alcohol precipitation is carried out, active polysaccharide is obtained；
The composite bacteria agent contains bacillus subtilis and saccharomyces cerevisiae；
The fermentation raw material includes wheat bran, the bean cake powder of 8-10 mass parts, the corn of 9-12 mass parts of 78-82 mass parts
The urea of powder and 0.15-0.25 mass parts.
In the above method, the preparation method of the wheat bran is concretely：Take without the wheat bran gone mouldy, cross 20 mesh sieves, leave and take
Wheat bran on sieve.
In the above method, the preparation method of the bean cake powder is concretely：Dregs of beans is taken, is crushed, 40 mesh sieves, the sieve left and taken are crossed
Lower bean cake powder.
In the above method, the preparation method of the corn flour is concretely：By dry crush maize, 40 mesh sieves are crossed, sieve is left and taken
Lower corn flour.
In the above method, the composite bacteria agent can be specifically made up of bacillus subtilis and saccharomyces cerevisiae.
In the above method, the fermentation raw material can by 78-82 mass parts wheat bran, the bean cake powder of 8-10 mass parts, 9-12
The corn flour of mass parts and the urea composition of 0.15-0.25 mass parts.
In the above method, the fermentation raw material specifically can by 78 mass parts wheat bran, the bean cake powder of 10 mass parts, 12 mass
The corn flour of part and the urea composition of 0.15 mass parts.
In the above method, the fermentation raw material specifically can by 80 mass parts wheat bran, the bean cake powder of 10 mass parts, 10 mass
The corn flour of part and the urea composition of 0.15 mass parts.
In the above method, the fermentation raw material specifically can by 82 mass parts wheat bran, the bean cake powder of 8 mass parts, 9 mass parts
Corn flour and 0.25 mass parts urea composition.
In the step (1), in the fermentation system, the active component content of composite bacteria agent is 2.00 × 107cfu/g-
6.00×107Cfu/g (such as 4.00 × 107cfu/g-6.00×107cfu/g、2.00×107cfu/g-4.00×107cfu/g、
2.00×107cfu/g、4.00×107Cfu/g or 6.00 × 107cfu/g).The bacillus subtilis and the saccharomyces cerevisiae
CFU ratio be 1：(2-2.2) (such as 1：2、1：2.1 or 1：2.2).The mass ratio of the fermentation raw material and the water
For 1：(1-1.15) (such as 1：1 or 1：1.15).The temperature of the fermentation is 35 DEG C -36 DEG C (such as 35 DEG C or 36 DEG C), fermentation time
For 45h-48h (such as 45h-46h, 47h-48h, 45h, 46h, 47h or 48h).
In the fermentation system, the active component content of the composite bacteria agent concretely 2.00 × 107Cfu/g, wherein
The content of the bacillus subtilis is 0.67 × 107Cfu/g, the content of the saccharomyces cerevisiae is 1.34 × 107Cfu/g, now
The CFU ratio of the bacillus subtilis and the saccharomyces cerevisiae is 1：2.
In the fermentation system, the active component content of the composite bacteria agent concretely 6.00 × 107Cfu/g, wherein
The content of the bacillus subtilis is 1.94 × 107Cfu/g, the content of the saccharomyces cerevisiae 2.1190 is 4.06 × 107cfu/
G, now the CFU ratio of the bacillus subtilis and the saccharomyces cerevisiae is 1：2.1.
In the fermentation system, the active component content of the composite bacteria agent concretely 4.00 × 107Cfu/g, wherein
The content of the bacillus subtilis is 1.25 × 107Cfu/g, the content of the saccharomyces cerevisiae is 2.75 × 107Cfu/g, now
The CFU ratio of the bacillus subtilis and the saccharomyces cerevisiae is 1：2.2.
It is as follows the step of " the carry out water extraction " in the step (2)：By the tunning aeration-drying, crush, obtain
To tunning powder；20-25 mass parts (such as 20 mass parts or 25 mass are added into tunning powder described in 1 mass parts
Part) water extracted, centrifugation, obtains Aqueous extracts.The temperature of the aeration-drying can be 30 DEG C -40 DEG C (such as 30 DEG C or 40 DEG C).
The sieve mesh number of the crushing concretely 60 mesh.The extraction can extract to stand.The Extracting temperature is 80 DEG C -90 DEG C
(such as 80 DEG C -85 DEG C, 85 DEG C -90 DEG C, 80 DEG C, 85 DEG C or 90 DEG C).The extraction time is 30min-60min (such as 30min-
40min, 40min-60min, 30min, 60min or 40min).The parameter of the centrifugation can be 6000rpm-7000rpm centrifugations
5min-20min.Concretely 6000rpm centrifuges 10min to the parameter of the centrifugation.The parameter of the centrifugation is concretely
7000rpm centrifuges 10min.
It is as follows the step of " the carry out alcohol precipitation " in the step (3)：3-4 bodies are added into Aqueous extracts described in 1 parts by volume
Product part (such as 3 parts by volume or 4 volume integrals) 90-95% (v/v) ethanol water (90% (v/v) ethanol water or 95% (v/v)
Ethanol water) alcohol precipitation is carried out, precipitation is collected in centrifugation；By the precipitation aeration-drying, active polysaccharide is obtained.During the alcohol precipitation
Between be 24h-48h (such as 24h-36h, 36h-48h, 24h, 36h or 48h).The parameter of the centrifugation can be 6000rpm-7000rpm
Centrifuge 5min-20min.Concretely 6000rpm centrifuges 10min to the parameter of the centrifugation.The parameter of the centrifugation is concretely
7000rpm centrifuges 10min.The temperature of the aeration-drying can be 30 DEG C -35 DEG C (such as 30 DEG C or 35 DEG C).
The active polysaccharide prepared using any of the above-described methods described falls within protection scope of the present invention.
Any of the above-described composite bacteria agent, or, application of any of the above-described fermentation raw material in active polysaccharide is prepared
Belong to protection scope of the present invention.
The present invention also protects any of the above-described composite bacteria agent or any of the above-described fermentation raw material；It is any of the above-described described multiple
Closing microbial inoculum or any of the above-described fermentation raw material is used to prepare active polysaccharide.
Above, the bacillus subtilis concretely bacillus subtilis (Bacillus subtilis) CGMCC
1.0892.The saccharomyces cerevisiae concretely saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC 2.1190.It is withered
Careless bacillus (Bacillus subtilis) CGMCC 1.0892 and saccharomyces cerevisiae (Saccharomyces cerevisiae)
CGMCC 2.1190 is the strain of China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC).
Above, the monosaccharide component of the active polysaccharide have glucose, it is fucose, arabinose, xylose, galactolipin, sweet
Dew sugar and rhamnose.The mass ratio of each monose is followed successively by：176.39：54.63：22.13：13.39：7.79：6.34：1.18、
Or, 182.01：56.14：18.56：11.71：7.81：4.87：1.02 or, 183.21：59.01：20.66：12.00：6.58：
5.66：2.01.
It is demonstrated experimentally that the method provided using the present invention can prepare active polysaccharide, with important application value.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.
Experimental method in following embodiments, is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Active polysaccharide is determined in following embodiments (to be recorded in the Scavenging activity of DPPH free radicals using DPPH free radical methods
In following document：The preparation of Ge Li flower forulic acid oligosaccharide and its inoxidizability Quality Research [D] Harbin：Northeast forestry is big
Learn, 2007), comprise the following steps that：
(1) active polysaccharide is diluted respectively with distilled water, obtaining polysaccharide solution 1, concentration that concentration is 0.01mg/mL is
It is polysaccharide solution 4 that polysaccharide solution 3 that 0.10mg/mL polysaccharide solution 2, concentration are 0.50mg/mL, concentration are 1.00mg/mL, dense
The polysaccharide solution 6 that the polysaccharide solution 5 and concentration that degree is 5.00mg/mL are 10.00mg/mL.
(2) take 2mL polysaccharide solutions (polysaccharide solution 1, polysaccharide solution 2, polysaccharide solution 3, polysaccharide solution 4, polysaccharide solution 5 or
Polysaccharide solution 6), the DPPH ethanol solutions that 2mL concentration is 2mmol/L are added, is mixed, is stored at room temperature 30min, then determines
Absorbance A at 517nmi。
(3) 1.58mgDPPH and 2mL absolute ethyl alcohols are mixed, then determines the absorbance A at 517nmc。
(4) 2mL polysaccharide solutions are taken, 2mL absolute ethyl alcohols are added, is mixed, is stored at room temperature 30min, then determines at 517nm
Absorbance Aj。
(5) clearance rate of the polysaccharide solution to DPPH free radicals is calculated according to the following equation；Clearance rate is higher, then polysaccharide solution
It is stronger to the Scavenging activity of DPPH free radicals；
Clearance rate={ 1- (Ai-Aj)/Ac} × 100%.
Active polysaccharide is determined in following embodiments (following article is recorded in using salicylic acid method to the Scavenging activity of hydroxy radical
In offering：The extraction of Wang Xi barley polysaccharide and its bioactivity research [D] Nanjing：Jiangsu University, 2008), comprise the following steps that：
(1) active polysaccharide is diluted respectively with distilled water, obtaining polysaccharide solution 1, concentration that concentration is 0.01mg/mL is
It is polysaccharide solution 4 that polysaccharide solution 3 that 0.10mg/mL polysaccharide solution 2, concentration are 0.50mg/mL, concentration are 1.00mg/mL, dense
The polysaccharide solution 6 that the polysaccharide solution 5 and concentration that degree is 5.00mg/mL are 10.00mg/mL.
(2) take 2mL polysaccharide solutions (polysaccharide solution 1, polysaccharide solution 2, polysaccharide solution 3, polysaccharide solution 4, polysaccharide solution 5 or
Polysaccharide solution 6), add the FeSO that 0.5mL concentration is 9mmol/L4The aqueous solution and the H that 2mL concentration is 8.8mmol/L2O2It is water-soluble
Liquid, mixes, is stored at room temperature 10min；Then the salicylic acid ethanol solution that 2mL concentration is 9mmol/L is added, mixes, is stored at room temperature
30min；Finally determine the absorbance A at 510nmi。
(3) according to the method for above-mentioned steps (2), concentration is replaced with into distilled water for 9mmol/L salicylic acid ethanol solution,
Other steps are constant, obtain absorbance Aj。
(4) according to the method for above-mentioned steps (2), polysaccharide solution is replaced with into distilled water, other steps are constant, inhaled
Luminosity Ao。
(5) clearance rate of the polysaccharide solution to hydroxy radical is calculated according to the following equation；Clearance rate is higher, then polysaccharide solution pair
The Scavenging activity of hydroxy radical is stronger；
Clearance rate={ 1- (Ai-Aj)}/Ao× 100%.
Bacillus subtilis (Bacillus subtilis) CGMCC 1.0892 and saccharomyces cerevisiae (Saccharomyces
Cerevisiae) CGMCC 2.1190 is China Committee for Culture Collection of Microorganisms's common micro-organisms center's (abbreviation
CGMCC strain).Bacillus subtilis (Bacillus subtilis) CGMCC 1.0892 hereinafter abbreviation withered grass gemma
Bacillus 1.0892.Saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC 2.1190 hereinafter abbreviation saccharomyces cerevisiaes
2.1190。
Nutrient broth fluid nutrient medium and malt extract medium are the product of Huankai Microbes Tech Co., Ltd., Guangdong.
Embodiment 1, active polysaccharide prepared using culture medium first and composite bacteria agent first
First, composite bacteria agent first is prepared
The preparation method of composite bacteria agent first is as follows：
1st, the preparation of the seed liquor first of bacillus subtilis 1.0892
Bacillus subtilis 1.0892 is inoculated in nutrient broth fluid nutrient medium, then 34-35 DEG C of culture 18-24h, is obtained
To the seed liquor first of bacillus subtilis 1.0892.In the seed liquor first of bacillus subtilis 1.0892, bacillus subtilis 1.0892
Concentration be 2.0 × 108cfu/mL。
2nd, the preparation of the seed liquor first of saccharomyces cerevisiae 2.1190
Saccharomyces cerevisiae 2.1190 is inoculated in malt extract medium, then 31-33 DEG C of culture 18-24h, obtains saccharomyces cerevisiae
2.1190 seed liquor first.In the seed liquor first of saccharomyces cerevisiae 2.1190, the concentration of saccharomyces cerevisiae 2.1190 is 2.0 × 108cfu/mL。
3rd, the preparation of composite bacteria agent first
By the seed liquor first of bacillus subtilis 1.0892 of 1 parts by volume and the seed liquor of saccharomyces cerevisiae 2.1190 of 2 parts by volume
First is mixed, and obtains composite bacteria agent first；In composite bacteria agent first, the bacterium colony shape of bacillus subtilis 1.0892 and saccharomyces cerevisiae 2.1190
Into unit (cfu) than being 1:2.
The active component content of composite bacteria agent first is bacillus subtilis 1.0892 and wine brewing in every milliliter of composite bacteria agent first
The content sum of yeast 2.1190, is 2.0 × 108cfu/mL.Bacillus subtilis 1.0892 in every milliliter of composite bacteria agent first
Content is 0.67 × 108cfu/mL.The content of saccharomyces cerevisiae 2.1190 is 1.34 × 10 in every milliliter of composite bacteria agent first8cfu/mL。
2nd, culture medium first is prepared
1st, pretreatment of raw material
(1) take without the wheat bran gone mouldy, cross 20 mesh sieves, leave and take the upper wheat bran of sieve.Hereinafter, wheat bran is named as on the sieve left and taken
Wheat bran after sieve.
(2) dregs of beans is crushed, crosses 40 mesh sieves, leave and take the lower bean cake powder of sieve.Hereinafter, bean cake powder is referred to as under the sieve left and taken
Bean cake powder.
(3) by dry crush maize, 40 mesh sieves are crossed, the lower corn flour of sieve is left and taken.Hereinafter, corn flour abbreviation under the sieve left and taken
For corn flour.
2nd, culture medium first is prepared
(1) by wheat bran, the bean cake powder of 10 mass parts, the corn flour of 12 mass parts and 0.15 mass parts after the sieve of 78 mass parts
Urea mixing (pH value natural), obtain fermentation raw material first.
(2) by fermentation raw material first and etc. quality distilled water (i.e. m/m=1：1) mix, 121 DEG C of sterilizing 20min, cooling
Afterwards, culture medium first is obtained.
3rd, active polysaccharide is prepared
1st, by culture medium first and composite bacteria agent first according to mass volume ratio 10:1 (10g/1mL) is mixed, and obtains fermentation system
First.In fermentation system first, the content of bacillus subtilis 1.0892 is 0.67 × 107Cfu/g, the content of saccharomyces cerevisiae 2.1190
For 1.34 × 107cfu/g。
2nd, complete after step 1, fermentation system first is placed under conditions of 35 DEG C, ferment 45h, obtains tunning first.
3rd, complete after step 2, tunning first is placed in 30 DEG C of aeration-dryings, then crush (sieve mesh number：60 mesh), obtain
It is less than 0.3cm tunning powder first to granularity.
4th, complete after step 3, take 1 mass parts tunning powder first, add 20 mass parts distilled water, stand and extract
30min (Extracting temperature is 80 DEG C), then 6000rpm centrifuges 10min, and the supernatant of collection is Aqueous extracts first.
5th, complete after step 4, take 1 parts by volume Aqueous extracts first, add the ethanol water of 3 parts by volume 90% (v/v), alcohol precipitation
24h, then 6000rpm centrifugations 10min, collects precipitation first.The precipitation first is placed in 30 DEG C of forced air dryings, material first is obtained.
The monosaccharide component of material first is analyzed using HPLC derivatization methods.As a result show, the monosaccharide component in material first has Portugal
Grape sugar, fucose, arabinose, xylose, galactolipin, mannose and rhamnose, mass ratio are followed successively by：176.39：54.63：
22.13：13.39：7.79：6.34：1.18.It can be seen that, material first is a kind of active polysaccharide.
Active polysaccharide prepared by determination step three is to DPPH free radicals and the Scavenging activity of hydroxy radical, and experimental result is shown in Table
1.As a result show, active polysaccharide prepared by step 3 has good scavenging action to DPPH free radicals and hydroxy radical.
Table 1
Embodiment 2, active polysaccharide prepared using culture medium second and composite bacteria agent second
First, composite bacteria agent second is prepared
The preparation method of composite bacteria agent second is as follows：
1st, the preparation of the seed liquor second of bacillus subtilis 1.0892
Bacillus subtilis 1.0892 is inoculated in nutrient broth fluid nutrient medium, then 34-35 DEG C of culture 18-24h, is obtained
To the seed liquor second of bacillus subtilis 1.0892.In the seed liquor second of bacillus subtilis 1.0892, bacillus subtilis 1.0892
Concentration be 6.0 × 108cfu/mL。
2nd, the preparation of the seed liquor second of saccharomyces cerevisiae 2.1190
Saccharomyces cerevisiae 2.1190 is inoculated in malt extract medium, then 31-33 DEG C of culture 18-24h, obtains saccharomyces cerevisiae
2.1190 seed liquor second.In the seed liquor second of saccharomyces cerevisiae 2.1190, the concentration of saccharomyces cerevisiae 2.1190 is 6.0 × 108cfu/mL。
3rd, the preparation of composite bacteria agent second
By the seed of saccharomyces cerevisiae 2.1190 of the seed liquor second of bacillus subtilis 1.0892 of 1 parts by volume and 2.1 parts by volume
Liquid second is mixed, and obtains composite bacteria agent second；In composite bacteria agent second, the bacterium colony of bacillus subtilis 1.0892 and saccharomyces cerevisiae 2.1190
Unit (cfu) is formed than being 1:2.1.
The active component content of composite bacteria agent second is bacillus subtilis 1.0892 and wine brewing in every milliliter of composite bacteria agent second
The content sum of yeast 2.1190, is 6.0 × 108cfu/mL.Bacillus subtilis 1.0892 in every milliliter of composite bacteria agent second
Content is 1.94 × 108cfu/mL.The content of saccharomyces cerevisiae 2.1190 is 4.06 × 10 in every milliliter of composite bacteria agent second8cfu/mL。
2nd, culture medium second is prepared
1st, pretreatment of raw material
(1) take without the wheat bran gone mouldy, cross 20 mesh sieves, leave and take the upper wheat bran of sieve.Hereinafter, wheat bran is named as on the sieve left and taken
Wheat bran after sieve.
(2) dregs of beans is crushed, crosses 40 mesh sieves, leave and take the lower bean cake powder of sieve.Hereinafter, bean cake powder is referred to as under the sieve left and taken
Bean cake powder.
(3) by dry crush maize, 40 mesh sieves are crossed, the lower corn flour of sieve is left and taken.Hereinafter, corn flour abbreviation under the sieve left and taken
For corn flour.
2nd, culture medium second is prepared
(1) by wheat bran, the bean cake powder of 10 mass parts, the corn flour of 10 mass parts and 0.15 mass parts after the sieve of 80 mass parts
Urea mixing (pH value natural), obtain fermentation raw material second.
(2) by fermentation raw material second and etc. quality distilled water (i.e. m/m=1：1) mix, 121 DEG C of sterilizing 20min, cooling
Afterwards, culture medium second is obtained.
3rd, active polysaccharide is prepared
1st, by culture medium second and composite bacteria agent second according to mass volume ratio 9:1 (9g/1mL) is mixed, and obtains fermentation system second.
In fermentation system second, the content of bacillus subtilis 1.0892 is 1.94 × 107Cfu/g, the content of saccharomyces cerevisiae 2.1190 is
4.06×107cfu/g。
2nd, complete after step 1, fermentation system second is placed under conditions of 35 DEG C, ferment 45h, obtains tunning second.
3rd, complete after step 2, tunning second is placed in 30 DEG C of aeration-dryings, then crush (sieve mesh number：60 mesh), obtain
It is less than 0.3cm tunning powder second to granularity.
4th, complete after step 3, take 1 mass parts tunning powder second, add 20 mass parts sterilized waters, stand and extract
30min (Extracting temperature is 85 DEG C), then 6000rpm centrifuges 10min, and the supernatant of collection is Aqueous extracts second.
5th, complete after step 4, take 1 parts by volume Aqueous extracts second, add the ethanol water of 3 parts by volume 90% (v/v), alcohol precipitation
24h, then 6000rpm centrifugations 10min, collects precipitation second.The precipitation second is placed in 30 DEG C of forced air dryings, material second is obtained.
The monosaccharide component of material second is analyzed using HPLC derivatization methods.As a result show, the monosaccharide component in material second has Portugal
Grape sugar, fucose, arabinose, xylose, galactolipin, mannose and rhamnose, mass ratio are followed successively by：182.01：56.14：
18.56：11.71：7.81：4.87：1.02.It can be seen that, material second is a kind of active polysaccharide.
Active polysaccharide prepared by determination step three is to DPPH free radicals and the Scavenging activity of hydroxy radical, and experimental result is shown in Table
2.As a result show, active polysaccharide prepared by step 3 has good scavenging action to DPPH free radicals and hydroxy radical.
Table 2
Embodiment 3, active polysaccharide prepared using culture medium third and composite bacteria agent third
First, composite bacteria agent third is prepared
The preparation method of composite bacteria agent third is as follows：
1st, the preparation of the seed liquor third of bacillus subtilis 1.0892
Bacillus subtilis 1.0892 is inoculated in nutrient broth fluid nutrient medium, then 34-35 DEG C of culture 18-24h, is obtained
To the seed liquor third of bacillus subtilis 1.0892.In the seed liquor third of bacillus subtilis 1.0892, bacillus subtilis 1.0892
Concentration be 4.0 × 108cfu/mL。
2nd, the preparation of the seed liquor third of saccharomyces cerevisiae 2.1190
Saccharomyces cerevisiae 2.1190 is inoculated in malt extract medium, then 31-33 DEG C of culture 18-24h, obtains saccharomyces cerevisiae
2.1190 seed liquor third.In the seed liquor third of saccharomyces cerevisiae 2.1190, the concentration of saccharomyces cerevisiae 2.1190 is 4.0 × 108cfu/mL。
3rd, the preparation of composite bacteria agent third
By the seed of saccharomyces cerevisiae 2.1190 of the seed liquor third of bacillus subtilis 1.0892 of 1 parts by volume and 2.2 parts by volume
Liquid third is mixed, and obtains composite bacteria agent third；In composite bacteria agent third, the bacterium colony of bacillus subtilis 1.0892 and saccharomyces cerevisiae 2.1190
Unit (cfu) is formed than being 1:2.2.
The active component content of composite bacteria agent third is bacillus subtilis 1.0892 and wine brewing in every milliliter of composite bacteria agent third
The content sum of yeast 2.1190, is 4.0 × 108cfu/mL.Bacillus subtilis 1.0892 in every milliliter of composite bacteria agent third
Content is 1.25 × 108cfu/mL.The content of saccharomyces cerevisiae 2.1190 is 2.75 × 10 in every milliliter of composite bacteria agent third8cfu/mL。
2nd, culture medium third is prepared
1st, pretreatment of raw material
(1) take without the wheat bran gone mouldy, cross 20 mesh sieves, leave and take the upper wheat bran of sieve.Hereinafter, wheat bran is named as on the sieve left and taken
Wheat bran after sieve.
(2) dregs of beans is crushed, crosses 40 mesh sieves, leave and take the lower bean cake powder of sieve.Hereinafter, bean cake powder is referred to as under the sieve left and taken
Bean cake powder.
(3) by dry crush maize, 40 mesh sieves are crossed, the lower corn flour of sieve is left and taken.Hereinafter, corn flour abbreviation under the sieve left and taken
For corn flour.
2nd, culture medium third is prepared
(1) by wheat bran after the sieve of 82 mass parts, the bean cake powder of 8 mass parts, the corn flour of 9 mass parts and 0.25 mass parts
Urea mixing (pH value is natural), obtains fermentation raw material third.
(2) by 1 mass parts fermentation raw material third and distilled water (the i.e. m/m=1 of 1.15 mass parts：1.15) mix, 121 DEG C go out
Bacterium 20min, after cooling, obtains culture medium third.
3rd, active polysaccharide is prepared
1st, by culture medium third and composite bacteria agent third according to mass volume ratio 10:1 (10g/1mL) is mixed, and obtains fermentation system
Third.In fermentation system third, the content of bacillus subtilis 1.0892 is 1.25 × 107Cfu/g, the content of saccharomyces cerevisiae 2.1190
For 2.75 × 107cfu/g。
2nd, complete after step 1, fermentation system third is placed under conditions of 36 DEG C, ferment 48h, obtains tunning third.
3rd, complete after step 2, tunning third is placed in 40 DEG C of aeration-dryings, then crush (sieve mesh number：60 mesh), obtain
It is less than 0.3cm tunning powder third to granularity.
4th, complete after step 3, take 1 mass parts tunning powder third, add 25 mass parts sterilized waters, stand and extract
60min (Extracting temperature is 90 DEG C), then 7000rpm centrifuges 10min, and the supernatant of collection is Aqueous extracts third.
5th, complete after step 4, take 1 parts by volume Aqueous extracts third, add the ethanol water of 4 parts by volume 95% (v/v), alcohol precipitation
48h, then 7000rpm centrifugations 10min, collects precipitation third.The precipitation third is placed in 35 DEG C of forced air dryings, material third is obtained.
The monosaccharide component of material third is analyzed using HPLC derivatization methods.As a result show, the monosaccharide component in material third has Portugal
Grape sugar, fucose, arabinose, xylose, galactolipin, mannose and rhamnose, mass ratio are followed successively by：183.21：59.01：
20.66：12.00：6.58：5.66：2.01.It can be seen that, material third is a kind of active polysaccharide.
Active polysaccharide prepared by determination step three is to DPPH free radicals and the Scavenging activity of hydroxy radical, and experimental result is shown in Table
3.As a result show, active polysaccharide prepared by step 3 has good scavenging action to DPPH free radicals and hydroxy radical.
Table 3
Claims (10)
1. a kind of method for preparing active polysaccharide, in turn includes the following steps：
(1) composite bacteria agent, fermentation raw material and water are mixed, obtains fermentation system；Fermentation, obtains tunning；
(2) water extraction is carried out；
(3) alcohol precipitation is carried out, active polysaccharide is obtained；
The composite bacteria agent contains bacillus subtilis and saccharomyces cerevisiae；
The wheat bran of the fermentation raw material including 78-82 mass parts, the bean cake powder of 8-10 mass parts, the corn flour of 9-12 mass parts and
The urea of 0.15-0.25 mass parts.
2. the method as described in claim 1, it is characterised in that：In the step (1), in the fermentation system, composite bacteria agent
Active component content be 2.00 × 107cfu/g-6.00×107cfu/g；The bacillus subtilis and the saccharomyces cerevisiae
CFU ratio is 1：(2-2.2)；The mass ratio of the fermentation raw material and the water is 1：(1-1.15).
3. the method as described in claim 1, it is characterised in that：In the step (1), the temperature of the fermentation is 35 DEG C -36
DEG C, fermentation time is 45h-48h.
4. the method as described in claim 1, it is characterised in that：In the step (2), the step of " the carry out water extraction " such as
Under：By the tunning aeration-drying, crush, obtain tunning powder；Add into tunning powder described in 1 mass parts
Enter 20-25 mass parts water to be extracted, centrifuge, obtain Aqueous extracts.
5. method as claimed in claim 4, it is characterised in that：The temperature of the aeration-drying is 30 DEG C -40 DEG C；It is described to extract
Temperature is 80 DEG C -90 DEG C；The extraction time is 30min-60min；The parameter of the centrifugation centrifuges for 6000rpm-7000rpm
5min-20min。
6. the method as described in claim 1, it is characterised in that：In the step (3), the step of " the carry out alcohol precipitation " such as
Under：3-4 parts by volume 90-95% (v/v) ethanol water is added into Aqueous extracts described in 1 parts by volume and carries out alcohol precipitation, centrifuges, collects
Precipitation；By the precipitation aeration-drying, active polysaccharide is obtained.
7. method as claimed in claim 6, it is characterised in that：The alcohol precipitation time is 24h-48h；The parameter of the centrifugation is
6000rpm-7000rpm centrifuges 5min-20min；The temperature of the aeration-drying is 30 DEG C -35 DEG C.
8. the active polysaccharide prepared using any methods described of claim 1 to 7.
9. composite bacteria agent described in claim 1 or 2, or, fermentation raw material described in claim 1 or 2 are preparing active polysaccharide
In application.
10. composite bacteria agent described in claim 1 or 2 or the fermentation raw material；The composite bacteria agent or the fermentation raw material are used
In preparing active polysaccharide.
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