CN103937725B - A kind of streptococcus thermophilus freeze-dried vaccine powder, preparation method thereof - Google Patents
A kind of streptococcus thermophilus freeze-dried vaccine powder, preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of streptococcus thermophilus freeze-dried vaccine powder, preparation method thereof, mainly comprise the steps that a) actication of culture and Multiplying culture；B) thalline is collected；C) pre-freeze and vacuum freeze drying.The present invention breeds by being inoculated in by streptococcus thermophilus strain in the freeze proof culture medium of propagation containing materials such as Semen Lablab Album polysaccharide; the bacterium solution of propagation processes by centrifugation; add the freeze drying protectant containing coix seed and Chinese yam mixing polysaccharide extract etc.; pre-freeze and vacuum freeze drying process; obtain freeze-dried vaccine powder; its lyophilized survival rate and bacterium powder viable count are high, have preferably developed the drug resource in China's " integration of drinking and medicinal herbs " simultaneously.
Description
Technical field
The invention belongs to streptococcus thermophilus production of articles technical field, relate to a kind of streptococcus thermophilus freeze-dried vaccine powder
Preparation method.
Background technology
Streptococcus thermophilus (Streptococcus thermophilus) be fermenting and producing dairy products important lactic acid bacteria it
One, the distinctive local flavors of Yoghourt such as it in addition to carrying out Acid production metabolism, also produces 2 during the fermentation, 3-diacetyl
Material, moreover it is possible to promote the growth of lactobacillus bulgaricus, is also one of important probio simultaneously, and it has
The abilities such as regulation human gastrointestinal tract colony balance, anti-sudden change, enhancing immunity.
In leavening preparation process, conventional is vacuum freeze-drying method, and bacterial classification is after vacuum freeze drying:
The loss of activity of enzyme is few, lyophilized dry bacterium powder rehydration is good, dehydration thoroughly, long shelf-life, storage transport and make
With all very convenient, provide good basis for throw type leaven.But, when actual use, freezing
The situations such as the crystallization of water in dry run, the dehydration of cell, protein inactivation make thalline dead or damage occur,
Preservation, production and application for bacterial classification are the most unfavorable.It is thus desirable to bacterium powder, preparation method thereof is improved.
The improved method prepared for streptococcus thermophilus freeze-dried vaccine powder has a variety of, as carried out Multiplying culture, resisting
Freeze the preparation etc. that the factor is added, protective agent is filled a prescription.As patent CN102952771B discloses a kind of thermophilic chain
Coccus freeze drying protectant and preparation method, its frozen-dried protective agent prescription is as follows: skimmed milk powder 10-20 part, cottonseed
Sugar 3-10 part, soyabean oligosaccharides 4-10 part, dextran D401.5-5.0 part, sodium tripolyphosphate 0.3-0.8 part,
Propylgallate 0.005-0.02 part, distilled water 100 parts.Patent CN100429980C discloses a kind of straight
The production method of throwing formula ferment agent for sour milk, its culture medium prescription is (mass percent): skimmed milk powder 0.1-15%,
Whey powder 0.1-10%, yeast extract 0.1-5%, tomato juice 1-10%, remaining is water；Its protective agent formula is (matter
Amount percentage): sodium glutamate 0.1-5.0%, lactose 0.1-8.0%, glycerine 0.1-5.0%, polyvinylpyrrolidine
Ketone 0.1-3.0%, Vc0.1-5%.Patent CN101502287B also discloses that a kind of direct putting type ferment agent for sour milk
Preparation method, its culture medium used is (mass percent): 1-2% glucose, 0.5-1% peptone,
0.1-10% whey powder, 1-5% yeast extract, 0.1-0.5% dibasic ammonium citrate, 0.01-0.2% dipotassium hydrogen phosphate,
0.1-0.5% anhydrous sodium acetate；Its protective agent used is: the milk powder of percentage by weight 1-30% and 0.1-2%
Antioxidant (ISOASCORBIC ACID or glutamic acid or glycine).CN103404703A discloses a kind of stable
Streptococcus thermophilus active bacteria formulation of safety and preparation method thereof, its culture medium prescription is: yeast extract 7.5g, egg
White peptone 7.5g, glucose 10g, KH2PO42g, Tomato juice 100mL, Tween800.5mL, distillation
Water 900mL.Patent CN102766590B discloses a kind of streptococcus thermophilus enriched medium and preparation method,
Its formula is as follows: glucose 0.8-1.2g, yeast extract 0.5-1.0g, soy peptone 0.5-1.0g, tomato juice
8-12mL, xylo-oligosaccharide 0.2-0.4g, water 88-92mL, KH2PO40.1-0.2g and Tween 80 0.01-0.05mL.
CN102978150A discloses a kind of streptococcus thermophilus and is combined the freeze proof factor, and its formula is following (with mass fraction
Meter): glycine betaine 0.1-0.4 part, sodium chloride 3-5 part, mannose 0.5-3 part and calcium carbonate 0.1-0.4 part.Separately
Many Research Literatures are had outward to also disclose that the bacterium powder, preparation method thereof of streptococcus thermophilus, such as " the screening of streptococcus thermophilus
And prepared by high-active bacteria powder " in the proliferation culture medium formula that obtains of research be: dusty yeast 0.5%, tomato juice
7.5%, brewer's wort 7.5%, whey powder 7.5%；Protective agent formula is: trehalose 20%, lactose 2.5%,
Sodium glutamate 5%, glycerine 0.5%.
The use of freeze drying protectant drastically increases thalline survival rate under vacuum freeze drying and freeze-dried vaccine
The stability of powder, and add some in the medium and can promote that cell produces the molecular substance of cryoprotective effects also
Be conducive to the preparation of its freeze-dried vaccine powder.The interpolation of these materials also can change the acid of streptococcus thermophilus cell membrane fat
Composition, even inducing cell produce substantial amounts of antifreeze protein, thus are effectively improved the anti-of streptococcus thermophilus thalline
Refrigerating capacity, can reach to reduce and even avoid thalline Freeze-drying Damage, raising Viable detection and bacterium powder viable bacteria
Number, the mesh reducing the addition being combined freeze drying protectant, extending lyophilized streptococcus thermophilus bacterium powder normal temperature storage life
's.
" integration of drinking and medicinal herbs " medicine that Semen Lablab Album, coix seed and Chinese yam are promulgated as the Ministry of Public Health, its contained polysaccharide pair
Human body has the effects such as anti-oxidant, antitumor, reducing blood lipid, enhancing immunity, additionally, these polysaccharide also may be used
Growing microorganism with effective stimulus lactic acid bacteria, it is also possible to as the freeze proof factor, and in terms of frozen-dried protective also
There is good result.At present, this type polysaccharide is used to prepare the report of aspect at freeze-dried vaccine powder and invent specially
Profit is few.
Summary of the invention
It is an object of the invention to provide one preferably develop China's natural resources of Chinese medicinal materials as freeze drying protectant,
Improve the streptococcus thermophilus freeze-dried vaccine powder, preparation method thereof of thalline freezing tolerance.
Technical scheme comprises the following steps:
A) actication of culture and Multiplying culture: streptococcus thermophilus strain is inoculated in M17 fluid nutrient medium
Activation, is inoculated in the bacterial classification activated Multiplying culture in the freeze proof culture medium of propagation, obtains the bacterium solution of propagation；
B) thalline is collected: by the bacterium solution centrifugal treating of above-mentioned propagation, abandoning supernatant, obtain bacterium mud；
C) it is dried: in prepared bacterium mud, add 0.14-0.2g sucrose, 0.12-0.18g by every gram of bacterium mud
Soluble starch, the phosphate buffer of 0.002-0.004g Vc, 0.5-1.0mL, 0.1-0.3g coix seed and
Chinese yam mixing polysaccharide extract, mixes final vacuum freeze-drying, obtains streptococcus thermophilus freeze-dried vaccine powder；
Described grow freeze proof culture medium prescription and consist of: 10-20g glucose, 10-25g soy peptone,
5-10g casein hydrolysate, 0.005-0.010mg glutamic acid, 2-4g KH2PO4, 0.5-1mL tween
80, Semen Lablab Album polysaccharide 0.2-0.5g, distilled water 1000mL.
In described step a) activation refer to be inoculated into by streptococcus thermophilus strain in M17 fluid nutrient medium in
Cultivate 22-26h, then the ratio in every 100mL inoculation of medium 2-5mL bacterial classification, inoculation for 35-38 DEG C
In the culture medium identical with above-mentioned M17 fluid nutrient medium, and activate under the same conditions to the third generation.
In described step a), the inoculative proportion of Multiplying culture is that every 100mL breeds freeze proof inoculation of medium
The bacterial classification that 2-5mL has activated.
In described step a), Multiplying culture is cultivation 16-18h at 35-38 DEG C.
The sterilizing the most of described culture medium, and the freeze proof culture medium of described propagation is through 118-121 DEG C, 15min
Sterilization treatment room temperature cooling.
The preparation process of described Semen Lablab Album polysaccharide includes:
First being soaked in 100-150 part distilled water after being pulverized by 5 portions of dry Semen Lablab Albums, regulation pH value is extremely
5.0-6.8；
Secondly add Semen Lablab Album quality 0.5-2% under the bath temperature of 40-55 DEG C, vigor is
The cellulase of 8000-10000U/g and Semen Lablab Album quality 1-2%, vigor are the pectin of 20000-30000U/g
Enzyme, is heated to boiling after insulation 1.5-2.5h, then is incubated 15-20min；
Then suction filtration filtrate being concentrated, adds the absolute ethyl alcohol that triploid is long-pending in concentrate, stands 10-14h
Rear suction filtration, gained filter residue, in 45-55 DEG C of drying, obtains Semen Lablab Album Thick many candies；
Finally take dry Semen Lablab Album Thick many candies and be dissolved in heating water bath in the distilled water of 50-70 DEG C, by every 0.5g
Semen Lablab Album Thick many candies is dissolved in the ratio of 15-20mL distilled water and calculates, and obtains polysaccharide solution, in polysaccharide solution
Adding the papain of Semen Lablab Album Thick many candies quality 1-4%, obtain enzymolysis liquid after enzymolysis 0.5-1h, enzymolysis liquid makes
With the Sevag reagent removing protein 20-30min of polysaccharide solution 15-25% volume；2000-4000rpm is centrifuged
Taking supernatant after 15-20min, be centrifuged repeatedly process three times, the supernatant obtained is after vacuum freeze drying
Obtain Semen Lablab Album polysaccharide.
In described step b) centrifugal treating be 0-4 DEG C, 8000-10000rpm be centrifuged 15-20min.
The preparation process of described coix seed and Chinese yam mixing polysaccharide extract includes:
First it is soaked in 150-200 part after being pulverized by the Chinese yam mixture of 5 parts of dry coix seeds and peeling to steam
In distilled water, wherein the Chinese yam of coix seed and peeling presses 1:1 mixing, and regulation pH value is to 6.0-7.0, at 80-100 DEG C
Extract 1-2h under bath temperature, be cooled to 40-55 DEG C；
Then under 40-55 DEG C of water-bath, add the Chinese yam mixture quality 1-2.5% accounting for above-mentioned coix seed and peeling
Pectase and the AMS of 0.8-1%, and be heated to boiling after being incubated 1.5-2.0h, then be incubated 10-15min；
Then suction filtration and by filtrate concentrate, in concentrate add 3-4 times of volume 95% ethanol, stand
Suction filtration after 10-14h, gained filter residue is thick mixing polysaccharide；
Finally wash thick mixing polysaccharide with 80% ethanol solution, after filtration, gained be deposited in 45-55 DEG C of drying,
Obtain coix seed and Chinese yam mixing polysaccharide extract.
In described step c), vacuum freeze drying is to carry out 18-24h in freeze dryer.
At-40 DEG C of pre-freeze 6-14h before vacuum freeze drying in described step c).
Compared with prior art, in the present invention streptococcus thermophilus strain containing materials such as Semen Lablab Album polysaccharide
Breeding and carry out in freeze proof culture medium breeding, the bacterium solution of propagation processes by centrifugation, add containing coix seed and
The freeze drying protectant of Chinese yam mixing polysaccharide extract etc., pre-freeze and vacuum freeze drying process, and are lyophilized
Bacterium powder, in the present invention, employing Semen Lablab Album polysaccharide etc. are as the freeze proof factor, make streptococcus thermophilus have higher
Viable count and more preferable freezing tolerance, it is lyophilized for using coix seed and Chinese yam mixing polysaccharide extract etc. simultaneously
Protective agent, makes bacterium powder have higher survival rate, and streptococcus thermophilus freeze-dried vaccine powder prepared in accordance with the present invention can
Making its lyophilized survival rate reach as high as 80.19%, bacterium powder viable count is 2.32 × 1011cfu/g；And with conventional
Business M17 medium culture, use phosphate buffer be streptococcus thermophilus prepared by freeze drying protectant
Bacterium powder is lyophilized survival rate and is only up to 22.72%, and viable count is only up to 8.1 × 109cfu/g.And in above-mentioned
The health-care efficacy of medicine polysaccharide also substantially increases the use value of this bacterium powder, and the present invention preferably develops
Drug resource, developing lactic acid bacteria health care in China " integration of drinking and medicinal herbs ", improve natural resources of Chinese medicinal materials and lactic acid bacteria
Comprehensive use value.
Detailed description of the invention
Below in conjunction with embodiment, the present invention will be further described.
Embodiment 1
A) actication of culture and Multiplying culture:
Streptococcus thermophilus strain is inoculated in sterilized M17 fluid nutrient medium, in 37 DEG C of cultivations
22-26h, more by volume (v/v) be 3% ratio be inoculated in and above-mentioned M17 fluid nutrient medium phase
In same culture medium, i.e. every 100mL inoculation of medium 3mL bacterial classification, and activate under the same conditions
To the third generation.
To breeding, freeze proof culture medium accesses the streptococcus thermophilus that 3% (v/v) has activated, 37 DEG C of constant temperature trainings
Support 16h.
B) thalline is collected:
Then 0 DEG C, centrifugal 15min, abandoning supernatant, it is thus achieved that bacterium mud under 10000rpm.
C) pre-freeze and vacuum freeze drying:
0.14g sucrose, 0.14g soluble starch, 0.002gVc, 0.5mL is added to every gram of bacterium mud
Phosphate buffer, 0.1g coix seed and Chinese yam mixing polysaccharide extract, mix, in-40 DEG C of pre-freezes
8h, be then placed in freeze dryer in-56 DEG C, carry out vacuum freeze drying 24h under 5Pa, obtain thermophilus
Bacterium freeze-dried vaccine powder.
Its lyophilized survival rate can be 78.25% (control medium is 21.21%), and bacterium powder viable count reaches
1.62×1011(control medium is 7.2 × 10 to CFU/g9CFU/g)。
Wherein, the configuration of the freeze proof culture medium of streptococcus thermophilus propagation includes: accurately weigh 20g glucose, 10g
Soy peptone, 5g casein hydrolysate, 0.005mg glutamic acid, 3g KH2PO4, 1mL Tween 80,
0.4g Semen Lablab Album polysaccharide, and being dissolved in 1000mL distilled water, heating for dissolving, stirs, then adjusts pH
Value, to 6.4-6.6, is contained in anaerobism bottle according to loading amount 80%, and to interpolation 1mL on the rubber stopper of bottle cap
Syringe needle, pulls up syringe needle after 118 DEG C of sterilizing 15min, is cooled to room temperature, obtains streptococcus thermophilus and increases
Grow freeze proof culture medium.
The preparation method of Semen Lablab Album polysaccharide comprises the following steps: be soaked in after being pulverized by 5 portions of dry Semen Lablab Albums
In 150 parts of distilled water, regulation pH value, to 5.0-6.8, adds Semen Lablab Album quality under the bath temperature of 45 DEG C
1.5%, vigor is the cellulase of 8000U/g and Semen Lablab Album quality 1%, vigor are the pectin of 30000U/g
Enzyme, is heated to boiling on electromagnetic oven after insulation 2h, regulation electromagnetism rate power is 1000W insulation thereafter
20min, then uses Buchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, add in concentrate
The absolute ethyl alcohol that triploid is long-pending, stands suction filtration after 10h in refrigerator, and gained filter residue, in 50 DEG C of drying, to obtain final product
Semen Lablab Album Thick many candies.The Semen Lablab Album Thick many candies being dried by every 0.5g is dissolved in 20mL, the distillation of water-bath to 55 DEG C
Water calculates, obtains polysaccharide solution, polysaccharide solution adds the Papain of Semen Lablab Album Thick many candies quality 1%
Enzyme, obtains enzymolysis liquid after enzymolysis 0.6h, enzymolysis liquid uses the Sevag reagent removing protein of polysaccharide solution 15% volume
20min, wherein chloroform in Sevag reagent: n-butanol=4:1,2000rpm take supernatant after being centrifuged 15min
Liquid, is centrifuged repeatedly process three times, and the supernatant obtained i.e. obtains Semen Lablab Album polysaccharide after vacuum freeze drying.
Coix seed and the preparation of Chinese yam mixing polysaccharide extract: by 5 parts of dry coix seeds and the Chinese yam of peeling
Mixing after mixture pulverizing and be soaked in 150 parts of distilled water, wherein the Chinese yam of coix seed and peeling presses 1:1 mixing,
Regulation pH value, to 6.0-7.0, extracts 2h, after being cooled to 40 DEG C, under 40 DEG C of water-baths under 80 DEG C of bath temperatures
Add pectase and the AMS of 0.8% of the Chinese yam mixture quality 1% accounting for above-mentioned coix seed and peeling, and
Insulation 2.0h, is heated to thereafter boiling on electromagnetic oven, is incubated 10min, then uses Buchner funnel suction filtration,
Filtrate is concentrated on Rotary Evaporators, in concentrate, adds 95% ethanol of 3 times of volumes, quiet in refrigerator
Putting suction filtration after 10h, gained filter residue is thick mixing polysaccharide, washs Thick many candies with 80% ethanol solution, after filtration
Gained is deposited in 45 DEG C of drying, obtains coix seed and Dioscorea opposite Thunb polysaccharide mixed extract.
Embodiment 2
A) actication of culture and Multiplying culture:
Streptococcus thermophilus strain is inoculated in sterilized M17 fluid nutrient medium, in 35 DEG C of cultivations
22-26h, then it is inoculated in the culture medium identical with above-mentioned M17 fluid nutrient medium in 4% (v/v) ratio
In, and activate under the same conditions to the third generation.
To breeding, freeze proof culture medium accesses the streptococcus thermophilus that 4% (v/v) has activated, 36 DEG C of constant temperature trainings
Support 16h.
B) thalline is collected:
Then 0 DEG C, centrifugal 18min, abandoning supernatant, it is thus achieved that bacterium mud under 9000rpm.
C) pre-freeze and vacuum freeze drying:
0.18g sucrose, 0.17g soluble starch, 0.003gVc, 0.8mL is added to every gram of bacterium mud
Phosphate buffer, 0.2g coix seed and Chinese yam mixing polysaccharide extract, mix, in-40 DEG C of pre-freezes
10h, be then placed in freeze dryer in-56 DEG C, carry out vacuum freeze drying 20h under 5Pa, obtain thermophilic chain
Coccus freeze-dried vaccine powder.
Its lyophilized survival rate can be 77.82% (control medium is 22.72%), and bacterium powder viable count reaches
1.21×1011(control medium is 8.1 × 10 to CFU/g9CFU/g)。
Wherein, the configuration of the freeze proof culture medium of streptococcus thermophilus propagation includes: accurately weigh 18g glucose, 24g
Soy peptone, 8g casein hydrolysate, 0.008mg glutamic acid, 2gKH2PO4, 0.5mL tween
80,0.3g Semen Lablab Album polysaccharide, and being dissolved in 1000mL distilled water, heating for dissolving, stirs, then
Adjust pH value to 6.4-6.6, be contained in anaerobism bottle according to loading amount 80%, and to interpolation on the rubber stopper of bottle cap
1mL syringe needle, pulls up syringe needle after 118 DEG C of sterilizing 15min, is cooled to room temperature.Obtain thermophilus
Bacterium breeds freeze proof culture medium.
The preparation method of Semen Lablab Album polysaccharide comprises the following steps: be soaked in after being pulverized by 5 portions of dry Semen Lablab Albums
In 125 parts of distilled water, regulation pH value, to 5.0-6.8, adds Semen Lablab Album quality under the bath temperature of 40 DEG C
1%, vigor is the cellulase of 9000U/g and Semen Lablab Album quality 1.5%, vigor are the pectin of 20000U/g
Enzyme, is heated to boiling on electromagnetic oven after insulation 2.5h, regulation electromagnetism rate power is 1000W insulation thereafter
18min, then uses Buchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, add in concentrate
The absolute ethyl alcohol that triploid is long-pending, stands suction filtration after 10h in refrigerator, and gained filter residue, in 55 DEG C of drying, to obtain final product
Semen Lablab Album Thick many candies.The Semen Lablab Album Thick many candies being dried by every 0.5g is dissolved in 15mL, the distillation of water-bath to 50 DEG C
Water calculates, obtains polysaccharide solution, polysaccharide solution adds the Papain of Semen Lablab Album Thick many candies quality 3%
Enzyme, after enzymolysis 0.5h enzymolysis liquid, enzymolysis liquid use polysaccharide solution 18% volume Sevag reagent (chloroform:
N-butanol=4:1) removing protein 25min, 3000rpm take supernatant after being centrifuged 20min, is centrifuged repeatedly process three
Secondary, the supernatant obtained i.e. obtains Semen Lablab Album polysaccharide after vacuum freeze drying.
Coix seed and the preparation of Chinese yam mixing polysaccharide extract: by 5 parts of dry coix seeds and the Chinese yam of peeling
Mixing after (1:1 mixing) pulverizing and be soaked in 200 parts of distilled water, regulation pH value is to 6.0-7.0, at 100 DEG C of water
Extract 1h under bath temperature, after being cooled to 45 DEG C, under 45 DEG C of water-baths, add the mountain accounting for above-mentioned coix seed and peeling
The pectase of medicine mixture quality 2% and the AMS of 1%, and it is incubated 1.5h, heat on electromagnetic oven thereafter
To boiling, it is incubated 13min, then uses Buchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, to
Adding 95% ethanol of 3 times of volumes in concentrate, stand suction filtration after 12h in refrigerator, gained filter residue is thick mixed
Close polysaccharide, with 80% ethanol solution washing Thick many candies, after filtration, gained is deposited in 50 DEG C of drying, obtains the heart of a lotus seed
The seed of jog's tears and Dioscorea opposite Thunb polysaccharide mixed extract.
Embodiment 3
A) actication of culture and Multiplying culture:
Streptococcus thermophilus strain is inoculated in sterilized M17 fluid nutrient medium, in 38 DEG C of cultivations
22-26h, then it is inoculated in the culture medium identical with above-mentioned M17 fluid nutrient medium in 2% (v/v) ratio
In, and activate under the same conditions to the third generation.
To breeding, freeze proof culture medium accesses the streptococcus thermophilus that 5% (v/v) has activated, 35 DEG C of constant temperature trainings
Support 17h.
B) thalline is collected:
Then 4 DEG C, centrifugal 15min, abandoning supernatant, it is thus achieved that bacterium mud under 10000rpm.
C) pre-freeze and vacuum freeze drying:
0.2g sucrose, 0.12g soluble starch, 0.004gVc, 1.0mL is added in every gram of bacterium mud
Phosphate buffer, 0.3g coix seed and Chinese yam mixing polysaccharide extract, mix, in-40 DEG C of pre-freezes
14h, be then placed in freeze dryer in-56 DEG C, carry out vacuum freeze drying 22h under 5Pa, obtain thermophilic chain
Coccus freeze-dried vaccine powder.
Its lyophilized survival rate can be 79.60% (control medium is 20.42%), and bacterium powder viable count reaches
2.02×1011(control medium is 7.8 × 10 to CFU/g9CFU/g)。
Wherein, the configuration of the freeze proof culture medium of streptococcus thermophilus propagation includes: accurately weigh 15g glucose, 18g
Soy peptone, 10g casein hydrolysate, 0.010mg glutamic acid, 4gKH2PO4, 0.8mL tween
80,0.5g Semen Lablab Album polysaccharide, and being dissolved in 1000mL distilled water, heating for dissolving, stirs, then adjusts
PH value, to 6.4-6.6, is contained in anaerobism bottle according to loading amount 80%, and to interpolation 1mL on the rubber stopper of bottle cap
Syringe needle, pulls up syringe needle after 119 DEG C of sterilizing 15min, is cooled to room temperature, obtains streptococcus thermophilus and increases
Grow freeze proof culture medium.
The preparation method of Semen Lablab Album polysaccharide comprises the following steps: be soaked in after being pulverized by 5 portions of dry Semen Lablab Albums
In 100 parts of distilled water, regulation pH value, to 5.0-6.8, adds Semen Lablab Album quality under the bath temperature of 55 DEG C
2%, vigor is the cellulase of 10000U/g and Semen Lablab Album quality 2%, vigor are the pectase of 20000U/g,
Being heated to boiling after insulation 1.5h on electromagnetic oven, regulation electromagnetism rate power is that 1000W is incubated 20min thereafter,
Then use Buchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, in concentrate, add triploid
Long-pending absolute ethyl alcohol, stands suction filtration after 14h in refrigerator, and gained filter residue, in 50 DEG C of drying, obtains Semen Lablab Album
Thick many candies.The Semen Lablab Album Thick many candies being dried by every 0.5g is dissolved in 20mL, the distilled water of water-bath to 70 DEG C is fallen into a trap
Calculate, obtain polysaccharide solution, polysaccharide solution adds the papain of Semen Lablab Album Thick many candies quality 4%, enzyme
Obtaining enzymolysis liquid after solving 1h, enzymolysis liquid uses the Sevag reagent (chloroform: n-butanol of polysaccharide solution 25% volume
=4:1) removing protein 30min, 4000rpm take supernatant after being centrifuged 20min, is centrifuged repeatedly process three times,
To supernatant after vacuum freeze drying, i.e. obtain Semen Lablab Album polysaccharide.
Coix seed and the preparation of Chinese yam mixing polysaccharide extract: by 5 parts of dry coix seeds and the Chinese yam of peeling
Mixing after (1:1 mixing) pulverizing and be soaked in 200 parts of distilled water, regulation pH value is to 6.0-7.0,90 DEG C of water-baths
At a temperature of extract 1.5h, after being cooled to 50 DEG C, add under 50 DEG C of water-baths and account for the Chinese yam of above-mentioned coix seed and peeling
The pectase of mixture quality 1.5% and the AMS of 1%, and it is incubated 2.0h, heat on electromagnetic oven thereafter
To boiling, it is incubated 15min, then uses Buchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, to
Adding 95% ethanol of 4 times of volumes in concentrate, stand suction filtration after 14h in refrigerator, gained filter residue is thick mixed
Close polysaccharide, with 80% ethanol solution washing Thick many candies, after filtration, gained is deposited in 55 DEG C of drying, obtains the heart of a lotus seed
The seed of jog's tears and Dioscorea opposite Thunb polysaccharide mixed extract.
Embodiment 4
A) actication of culture and Multiplying culture:
Streptococcus thermophilus strain is inoculated in sterilized M17 fluid nutrient medium, in 36 DEG C of cultivations
22-26h, then it is inoculated in the culture medium identical with above-mentioned M17 fluid nutrient medium in 5% (v/v) ratio
In, and activate under the same conditions to the third generation.
To breeding, freeze proof culture medium accesses the streptococcus thermophilus that 2% (v/v) has activated, 38 DEG C of constant temperature trainings
Support 18h.
B) thalline is collected:
Then 2 DEG C, centrifugal 20min, abandoning supernatant, it is thus achieved that bacterium mud under 8000rpm.
C) pre-freeze and vacuum freeze drying:
0.16g sucrose, 0.18g soluble starch, 0.003gVc, 0.9mL is added in every gram of bacterium mud
Phosphate buffer, 0.2g coix seed and Chinese yam mixing polysaccharide extract, mix, pre-in-40 DEG C
Freeze 6h, be then placed in freeze dryer in-56 DEG C, carry out vacuum freeze drying 18h under 5Pa, obtain thermophilic
Streptococcus freeze-dried vaccine powder.
Its lyophilized survival rate can be 80.19% (control medium is 21.65%), and bacterium powder viable count reaches
2.32×1011(control medium is 7.9 × 10 to CFU/g9CFU/g)。
Wherein, the configuration of the freeze proof culture medium of streptococcus thermophilus propagation includes: accurately weigh 10g glucose, 25g
Soy peptone, 7g casein hydrolysate, 0.0007mg glutamic acid, 3gKH2PO4, 0.7mL tween
80,0.2g Semen Lablab Album polysaccharide, and being dissolved in 1000mL distilled water, heating for dissolving, stirs, then adjusts
PH value, to 6.4-6.6, is contained in anaerobism bottle according to loading amount 80%, and to interpolation 1mL on the rubber stopper of bottle cap
Syringe needle, pulls up syringe needle after 121 DEG C of sterilizing 15min, is cooled to room temperature, obtains streptococcus thermophilus and increases
Grow freeze proof culture medium.
The preparation method of Semen Lablab Album polysaccharide comprises the following steps: be soaked in after being pulverized by 5 portions of dry Semen Lablab Albums
In 140 parts of distilled water, regulation pH value, to 5.0-6.8, adds Semen Lablab Album quality under the bath temperature of 50 DEG C
0.5%, vigor is the cellulase of 10000U/g and Semen Lablab Album quality 2%, vigor are the pectin of 30000U/g
Enzyme, is heated to boiling on electromagnetic oven after insulation 2.5h, regulation electromagnetism rate power is 1000W insulation thereafter
15min, then uses Buchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, add in concentrate
The absolute ethyl alcohol that triploid is long-pending, stands suction filtration after 12h in refrigerator, and gained filter residue, in 45 DEG C of drying, to obtain final product
Semen Lablab Album Thick many candies.The Semen Lablab Album Thick many candies being dried by every 0.5g is dissolved in 18mL, the distillation of water-bath to 60 DEG C
Water calculates, obtains polysaccharide solution, polysaccharide solution adds the Papain of Semen Lablab Album Thick many candies quality 2%
Enzyme, after enzymolysis 0.8h enzymolysis liquid, enzymolysis liquid use polysaccharide solution 20% volume Sevag reagent (chloroform:
N-butanol=4:1) removing protein 28min, 3000rpm take supernatant after being centrifuged 18min, is centrifuged repeatedly process three
Secondary, the supernatant obtained i.e. obtains Semen Lablab Album polysaccharide after vacuum freeze drying.
Coix seed and the preparation of Chinese yam mixing polysaccharide extract: by 5 parts of dry coix seeds and the Chinese yam of peeling
Mixing after (1:1 mixing) pulverizing and be soaked in 180 parts of distilled water, regulation pH value is to 6.0-7.0,90 DEG C of water-baths
At a temperature of extract 1h, after being cooled to 55 DEG C, add under 55 DEG C of water-baths and account for the Chinese yam of above-mentioned coix seed and peeling
The pectase of mixture quality 2.5% and the AMS of 0.9%, and it is incubated 1.8h, heat on electromagnetic oven thereafter
To boiling, it is incubated 12min, then uses Buchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, to
Adding 95% ethanol of 4 times of volumes in concentrate, stand suction filtration after 13h in refrigerator, gained filter residue is thick mixed
Close polysaccharide, with 80% ethanol solution washing Thick many candies, after filtration, gained is deposited in 52 DEG C of drying, obtains the heart of a lotus seed
The seed of jog's tears and Dioscorea opposite Thunb polysaccharide mixed extract.
Claims (8)
1. a streptococcus thermophilus freeze-dried vaccine powder, preparation method thereof, it is characterised in that: comprise the following steps:
A) actication of culture and Multiplying culture: streptococcus thermophilus strain is inoculated in M17 fluid nutrient medium
Activation, is inoculated in the bacterial classification activated Multiplying culture in the freeze proof culture medium of propagation, obtains the bacterium solution of propagation；
B) thalline is collected: by the bacterium solution centrifugal treating of above-mentioned propagation, abandoning supernatant, obtain bacterium mud；
C) it is dried: in prepared bacterium mud, add 0.14-0.2g sucrose, 0.12-0.18g by every gram of bacterium mud
Soluble starch, the phosphate buffer of 0.002-0.004g Vc, 0.5-1.0mL, 0.1-0.3g coix seed and
Chinese yam mixing polysaccharide extract, mixes final vacuum freeze-drying, obtains streptococcus thermophilus freeze-dried vaccine powder；
Described grow freeze proof culture medium prescription and consist of: 10-20g glucose, 10-25g soy peptone,
5-10g casein hydrolysate, 0.005-0.010mg glutamic acid, 2-4g KH2PO4, 0.5-1mL tween
80, Semen Lablab Album polysaccharide 0.2-0.5g, distilled water 1000mL；
The preparation process of described Semen Lablab Album polysaccharide includes:
First being soaked in 100-150 part distilled water after being pulverized by 5 portions of dry Semen Lablab Albums, regulation pH value is extremely
5.0-6.8；
Secondly add Semen Lablab Album quality 0.5-2% under the bath temperature of 40-55 DEG C, vigor is
The cellulase of 8000-10000U/g and Semen Lablab Album quality 1-2%, vigor are the pectin of 20000-30000U/g
Enzyme, is heated to boiling after insulation 1.5-2.5h, then is incubated 15-20min；
Then suction filtration filtrate being concentrated, adds the absolute ethyl alcohol that triploid is long-pending in concentrate, stands 10-14h
Rear suction filtration, gained filter residue, in 45-55 DEG C of drying, obtains Semen Lablab Album Thick many candies；
Finally take dry Semen Lablab Album Thick many candies and be dissolved in heating water bath in the distilled water of 50-70 DEG C, by every 0.5g
Semen Lablab Album Thick many candies is dissolved in the ratio of 15-20mL distilled water and calculates, and obtains polysaccharide solution, in polysaccharide solution
Adding the papain of Semen Lablab Album Thick many candies quality 1-4%, obtain enzymolysis liquid after enzymolysis 0.5-1h, enzymolysis liquid makes
With the Sevag reagent removing protein 20-30min of polysaccharide solution 15-25% volume；2000-4000rpm is centrifuged
Taking supernatant after 15-20min, be centrifuged repeatedly process three times, the supernatant obtained is after vacuum freeze drying
Obtain Semen Lablab Album polysaccharide；
The preparation process of described coix seed and Chinese yam mixing polysaccharide extract includes:
First it is soaked in 150-200 part after being pulverized by the Chinese yam mixture of 5 parts of dry coix seeds and peeling to steam
In distilled water, wherein the Chinese yam of coix seed and peeling presses 1:1 mixing, and regulation pH value is to 6.0-7.0, at 80-100 DEG C
Extract 1-2h under bath temperature, be cooled to 40-55 DEG C；
Then under 40-55 DEG C of water-bath, add the Chinese yam mixture quality 1-2.5% accounting for above-mentioned coix seed and peeling
Pectase and the AMS of 0.8-1%, and be heated to boiling after being incubated 1.5-2.0h, then be incubated 10-15min；
Then suction filtration and by filtrate concentrate, in concentrate add 3-4 times of volume 95% ethanol, stand
Suction filtration after 10-14h, gained filter residue is thick mixing polysaccharide；
Finally wash thick mixing polysaccharide with 80% ethanol solution, after filtration, gained be deposited in 45-55 DEG C of drying,
Obtain coix seed and Chinese yam mixing polysaccharide extract.
A kind of streptococcus thermophilus freeze-dried vaccine powder, preparation method thereof the most according to claim 1, it is characterised in that:
In described step a) activation refer to be inoculated into by streptococcus thermophilus strain in M17 fluid nutrient medium in
Cultivate 22-26h, then the ratio in every 100mL inoculation of medium 2-5mL bacterial classification, inoculation for 35-38 DEG C
In the culture medium identical with above-mentioned M17 fluid nutrient medium, and activate under the same conditions to the third generation.
A kind of streptococcus thermophilus freeze-dried vaccine powder, preparation method thereof the most according to claim 2, it is characterised in that:
In described step a), the inoculative proportion of Multiplying culture is that every 100mL breeds freeze proof inoculation of medium 2-5mL
The bacterial classification activated.
A kind of streptococcus thermophilus freeze-dried vaccine powder, preparation method thereof the most according to claim 3, it is characterised in that:
In described step a), Multiplying culture is cultivation 16-18h at 35-38 DEG C.
5. according to a kind of streptococcus thermophilus freeze-dried vaccine powder, preparation method thereof described in Claims 1-4 any one,
It is characterized in that: the sterilizing the most of described culture medium, and the freeze proof culture medium of described propagation through 118-121 DEG C,
15min sterilization treatment room temperature cooling.
A kind of streptococcus thermophilus freeze-dried vaccine powder, preparation method thereof the most according to claim 1, it is characterised in that:
In described step b) centrifugal treating be 0-4 DEG C, 8000-10000rpm be centrifuged 15-20min.
A kind of streptococcus thermophilus freeze-dried vaccine powder, preparation method thereof the most according to claim 1, it is characterised in that:
In described step c), vacuum freeze drying is to carry out 18-24h in freeze dryer.
8. according to a kind of streptococcus thermophilus freeze-dried vaccine powder, preparation method thereof described in claim 1 or 7, its feature
It is: at-40 DEG C of pre-freeze 6-14h before vacuum freeze drying in described step c).
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| RU2822476C1 (en) * | 2023-11-20 | 2024-07-05 | Федеральное государственное бюджетное учреждение "Центр стратегического планирования и управления медико-биологическими рисками здоровью" Федерального медико-биологического агентства | Method for long-term preservation of obligate anaerobic bacteria by drying from frozen state |
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| CN105685223A (en) * | 2016-02-01 | 2016-06-22 | 陕西科技大学 | Preparation method of antioxidant lactobacillus acidophilus fermented goat milk |
| CN113143972B (en) * | 2021-05-19 | 2023-11-24 | 吉林省命之元生物科技有限公司 | Application of streptococcus thermophilus TIMR0705-6 in preparation of anti-hyperuricemia and anti-gout drugs |
| CN115558620B (en) * | 2022-09-30 | 2023-10-20 | 微康益生菌(苏州)股份有限公司 | Lactic acid bacteria freeze-drying protective agent and preparation method and application thereof |
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| EP2241615A1 (en) * | 2008-02-07 | 2010-10-20 | Snow Brand Milk Products Co., Ltd. | Microorganism-protecting agent, and method for production of frozen or lyophilized microbial cell |
| CN102952771A (en) * | 2012-11-19 | 2013-03-06 | 陕西科技大学 | Streptococcus thermophilus freeze-drying protective agent and preparation method thereof |
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| CN102952771A (en) * | 2012-11-19 | 2013-03-06 | 陕西科技大学 | Streptococcus thermophilus freeze-drying protective agent and preparation method thereof |
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| RU2822476C1 (en) * | 2023-11-20 | 2024-07-05 | Федеральное государственное бюджетное учреждение "Центр стратегического планирования и управления медико-биологическими рисками здоровью" Федерального медико-биологического агентства | Method for long-term preservation of obligate anaerobic bacteria by drying from frozen state |
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