CN103923835A - Method for preparing lactobacillus bulgaricus freeze-dried powder - Google Patents
Method for preparing lactobacillus bulgaricus freeze-dried powder Download PDFInfo
- Publication number
- CN103923835A CN103923835A CN201410171964.2A CN201410171964A CN103923835A CN 103923835 A CN103923835 A CN 103923835A CN 201410171964 A CN201410171964 A CN 201410171964A CN 103923835 A CN103923835 A CN 103923835A
- Authority
- CN
- China
- Prior art keywords
- freeze
- lactobacillus bulgaricus
- polysaccharide
- white hyacinth
- hyacinth bean
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention relates to a method for preparing lactobacillus bulgaricus freeze-dried powder. The method mainly comprises the following steps: a) strain activation and multiplication culture; b) thallus collection; and c) prefreezing and vacuum freeze drying. By using white hyacinth bean polysaccharide and the like as anti-freezing factors, lactobacillus bulgaricus has better anti-freezing capability; meanwhile, coix seed and yam mixed polysaccharide extract and the like are used as freeze-drying protective agents, so that the bacterial powder has higher survival rate, and medicament resources in 'homology of medicine and food' in China are better developed and utilized.
Description
Technical field
The invention belongs to lactobacillus bulgaricus production of articles technical field, relate to a kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof.
Background technology
Lactobacillus bulgaricus (Lactobacillus bulgaricus) is the important milk-acid bacteria of fermentative production milk-product, it can give tart flavour and fragrance that yogurt product is soft, can improve quality and the nutrition of product, also have the critical functions such as the micro-ecological environment of maintaining, anti-mutation, antitumor, reducing blood-fat and raising immunizing power, be therefore the focus that the fields such as foodstuffs industry, medicine and microecology are paid close attention to always.
Bacterial classification is in the time of vacuum lyophilization, and the loss of activity of enzyme is few, and can keep bacterial classification form, color and luster, and the rehydration of lyophilize bacterium powder is good, and thoroughly, long preservative period, stores transport and use all very convenient in dehydration.But in the time of actual use, in freezing dry process, the situation such as the dehydration of the crystallization of water, cell makes thalline occur dead or damage, very unfavorable for preservation, the production and application of bacterial classification.Report, thalline can make its survival rate very low when freeze-drying under without any sfgd., is even low to moderate 0.1%.
At present, oneself has a lot of reports to utilize freeze drying technology preparation and storage lactobacillus bulgaricus bacterium powder.In actual use, need to obtain the bacterium powder of high viable count and reduce the damage of cell in freeze-drying process.Current topmost method is before lyophilize, to add lyophilized vaccine to prepare freeze-dried vaccine powder; as disclosed a kind of lactobacillus bulgaricus freeze-drying composite protectant in CN102952755A, its formula is as follows: 20～30 weight part lactose, 20～30 weight part skimmed milk powders, 3～5 weight part sodium ascorbates, 0.2～0.6 weight part Macrogol 4000,0.2～0.8 weight account polyethylene pyrrolidone, 0.5～2.0 weight part bovine serum albumin and 100 parts of distilled water.Also there is in addition the report about lactobacillus bulgaricus freeze-dried vaccine powder in a large number.For the increasing bacterium of lactobacillus bulgaricus, patent CN102952722A discloses a kind of enrichment medium and preparation method of lactobacillus bulgaricus, and its enrichment medium formula is as follows: every 100mL distilled water comprises the oxyproline that 0.4g lactose, 0.4mL new-born calf serum, 0.8g casein hydrolysate, 0.090mL mass concentration are 1%, L-glutamic acid and the 0.59g K that 0.050mL mass concentration is 1%
2hPO
4.Patent CN1844354A also discloses a kind of lactobacillus bulgaricus enrichment medium, and its main component is: jerusalem artichoke juice basic medium 100mL, 5-15mL tomato juice, 0.5-2g peptone, 0.1-1.0g lactose.Also have many investigators also to report lactobacillus bulgaricus enrichment medium.Patent CN103387939A has reported a kind of preparation method of the Bulgarian freeze-dried vaccine powder that is suitable for 25 DEG C of fermented yogurts.
The use of lyophilized vaccine has greatly improved the survival rate of thalline under vacuum lyophilization and the stability of freeze-dried vaccine powder; can also in substratum, add some molecular substances; these materials can be in the time that lactobacillus bulgaricus be cultivated in cylinder accumulation or change into other material, and then suppress the generation of cell injury in freeze-drying process.The interpolation of these materials also can change lactobacillus bulgaricus cell membrane fat acid composition; even inducing cell produces a large amount of antifreeze proteins; thereby effectively improve the freezing tolerance of lactobacillus bulgaricus thalline, can reach minimizing even avoid thalline Freeze-drying Damage, improve viable bacteria survival rate and bacterium powder viable count, reduce compound lyophilized vaccine addition, extend the object of freeze-drying lactobacillus bulgaricus bacterium powder normal temperature preservation period.Also do not retrieve at present about increasing with lactobacillus bulgaricus report and the patent of invention that the freeze proof substratum of bacterium is prepared freeze-dried vaccine powder.Meanwhile, use active substance in " integration of drinking and medicinal herbs " medicine stimulate the growing microorganism of lactobacillus bulgaricus, as the freeze proof factor and also less as the report of lyophilized vaccine.
Summary of the invention
The object of this invention is to provide a kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof of China's natural resources of Chinese medicinal materials as lyophilized vaccine, raising thalline freezing tolerance that develop better.
Technical scheme of the present invention comprises the following steps:
A) actication of culture and multiplication culture: lactobacillus bulgaricus is inoculated in MRS liquid nutrient medium and is activated, the bacterial classification having activated is inoculated in to multiplication culture in the freeze proof substratum of propagation, obtain the bacterium liquid of propagation;
B) collect thalline: by the bacterium liquid centrifugal treating of above-mentioned propagation, abandoning supernatant, obtains bacterium mud;
C) dry: the phosphoric acid buffer, 0.1-0.3g Semen Coicis and the Chinese yam mixing polysaccharide extract that in prepared bacterium mud, add 0.2-0.24g skim-milk, 0.06-0.1g sodium ascorbate, 0.8-1.4mL by every gram of bacterium mud, mix final vacuum lyophilize, obtain lactobacillus bulgaricus freeze-dried vaccine powder;
The freeze proof culture medium prescription of described propagation consists of: lactose 10-20g, trehalose 2-6g, White Hyacinth Bean polysaccharide 0.2-0.6g, xylo-oligosaccharide 2-4g, yeast extract 4-10g, soy peptone 8-10g, Sodium Glutamate 0.6-1.0g, diammonium hydrogen citrate 1.0-2.0g, sodium acetate 1.0-2.0g, dipotassium hydrogen phosphate 1.0-2.0g, MnSO
40.01-0.06g, tween 80 0.5-1.0mL, distilled water 1000mL.
Described step a) in activation refer to lactobacillus bulgaricus be inoculated in MRS liquid nutrient medium in 35-38 DEG C and cultivate 22-26h, again in the ratio of every 100mL inoculation of medium 3-5mL bacterial classification, be inoculated in the substratum identical with above-mentioned MRS liquid nutrient medium, and be activated under the same conditions the third generation.
The described step a) inoculative proportion of middle multiplication culture is that every 100mL breeds the bacterial classification that freeze proof inoculation of medium 3-5mL has activated.
Described step a) middle multiplication culture is to cultivate 16-18h at 35-38 DEG C.
The configuration step of the freeze proof substratum of described propagation comprises:
First take according to quantity lactose, White Hyacinth Bean polysaccharide, yeast extract, soy peptone, diammonium hydrogen citrate, sodium acetate, dipotassium hydrogen phosphate, MnSO
4be dissolved in distilled water a with tween 80, heating for dissolving, stir, then adjust pH is to 6.4-6.6, is contained in anaerobism bottle according to loading amount 70%-90%, in 118-121 DEG C of sterilizing 15min, is cooled to room temperature, obtains sterilized substratum;
Taking trehalose, xylo-oligosaccharide and Sodium Glutamate is dissolved in distilled water b again, the total amount of described distilled water a and distilled water b is 1000mL, mix rear filtration sterilization, filtrate is required to mix according to aseptic technique with above-mentioned sterilized substratum, obtain lactobacillus bulgaricus and breed freeze proof substratum.
The preparation process of described White Hyacinth Bean polysaccharide comprises:
First after 5 portions of dry White Hyacinth Beans being pulverized, be soaked in 100-150 part distilled water, regulate pH value to 5.0-6.8;
Secondly under the bath temperature of 40-55 DEG C, add the polygalacturonase that cellulase that White Hyacinth Bean quality 0.5-2%, vigor are 8000-10000U/g and White Hyacinth Bean quality 1-2%, vigor are 20000-30000U/g, the extremely boiling of insulation 1.5-2.5h post-heating, then be incubated 15-20min;
Then suction filtration filtrate is concentrated adds the long-pending dehydrated alcohol of triploid in concentrated solution, and suction filtration after standing 10-14h, in 45-55 DEG C of oven dry, obtains White Hyacinth Bean Crude polysaccharides by gained filter residue;
Finally getting dry White Hyacinth Bean Crude polysaccharides is dissolved in the distilled water that water-bath is heated to 50-70 DEG C, the ratio that is dissolved in 15-20mL distilled water in every 0.5g White Hyacinth Bean Crude polysaccharides is calculated, obtain polysaccharide soln, in polysaccharide soln, add the papoid of White Hyacinth Bean Crude polysaccharides quality 1-4%, after enzymolysis 0.5-1h, obtain enzymolysis solution, enzymolysis solution uses the Sevag reagent of polysaccharide soln 15-25% volume except protein 20-30min; After the centrifugal 15-20min of 2000-4000rpm, get supernatant liquor, centrifugal treating three times repeatedly, the supernatant liquor obtaining obtains White Hyacinth Bean polysaccharide after vacuum lyophilization.
Described step b) middle centrifugal treating is 0-4 DEG C, the centrifugal 15-20min of 8000-10000rpm.
The preparation process of described Semen Coicis and Chinese yam mixing polysaccharide extract comprises:
First after the Chinese yam mixture of 5 parts of dry Semen Coiciss and peeling being pulverized, be soaked in 150-200 part distilled water, wherein the Chinese yam of Semen Coicis and peeling is pressed 1:1 mixing, regulate pH value to 6.0-7.0, under 80-100 DEG C of bath temperature, extract 1-2h, be cooled to 40-55 DEG C;
Then under 40-55 DEG C of water-bath, add the polygalacturonase of the Chinese yam mixture quality 1-2.5% that accounts for above-mentioned Semen Coicis and peeling and the α-amylase of 0.8-1%, and be incubated 1.5-2.0h post-heating to boiling, then be incubated 10-15min;
Then suction filtration by concentrated filtrate, to 95% ethanol that adds 3-4 times of volume in concentrated solution, leaves standstill suction filtration after 10-14h, and gained filter residue is thick mixing polysaccharide;
Finally wash thick mixing polysaccharide with 80% ethanolic soln, after filtration, gained is deposited in to 45-55 DEG C of oven dry, obtain Semen Coicis and Chinese yam mixing polysaccharide extract.
Described step c) middle vacuum lyophilization is in Freeze Drying Equipment, to carry out 18-24h.
Described step c) in before vacuum lyophilization at-40 DEG C of pre-freeze 6-14h.
Compared with prior art, lactobacillus bulgaricus freeze-dried vaccine powder of the present invention is to breed in the freeze proof substratum of propagation containing materials such as White Hyacinth Bean polysaccharide, the bacterium liquid of propagation is through centrifugal treating, add again the material such as Semen Coicis and Chinese yam mixing polysaccharide extract, after dry, obtain freeze-dried vaccine powder, in the present invention, adopt White Hyacinth Bean polysaccharide etc. as the freeze proof factor, make lactobacillus bulgaricus there is better freezing tolerance, use Semen Coicis and Chinese yam mixing polysaccharide extract etc. is lyophilized vaccine simultaneously, make bacterium powder have higher survival rate, lactobacillus bulgaricus freeze-dried vaccine powder prepared in accordance with the present invention can make its freeze-drying survival rate reach as high as 75.21%, bacterium powder viable count is up to 1.96 × 10
11cFU/g, and cultivate, use lactobacillus bulgaricus bacterium powder freeze-drying survival rate that phosphoric acid buffer prepared as lyophilized vaccine only up to 19.72% taking the business MRS broth culture commonly used, viable count is only up to 7.8 × 10
9cfu/g.In the present invention, in White Hyacinth Bean polysaccharide, contained various carbohydrates have hormesis, are also the freeze proof factors of perviousness the growth of lactobacillus bulgaricus, in addition, its have anti-oxidant, strengthen the effects such as immunity, human body is had to larger benefit; Many oligose in Semen Coicis and Chinese yam polysaccharide have good cryoprotection and cultivation effect to lactobacillus bulgaricus; be conducive to equally HUMAN HEALTH; the present invention has developed the drug resource in China's " integration of drinking and medicinal herbs " better; improve Chinese medicine use value, can increase the use value of lactobacillus bulgaricus.
Embodiment
Below in conjunction with embodiment, the present invention will be further described.
Embodiment 1
A) actication of culture and multiplication culture:
Lactobacillus bulgaricus is inoculated in sterilized MRS liquid nutrient medium, cultivate 22-26h in 37 DEG C, the ratio that (v/v) is 3% is more by volume inoculated in substratum same as described above, be every 100mL inoculation of medium 3mL bacterial classification, and be activated under the same conditions the third generation.
In the freeze proof substratum of propagation, access the lactobacillus bulgaricus that 4% (v/v) activated, 36 DEG C of constant temperature culture 16h.
B) collect thalline:
Then centrifugal 20min under 4 DEG C, 8000rpm, abandoning supernatant, obtains bacterium mud.
C) pre-freeze and vacuum lyophilization:
In every gram of bacterium mud, add 0.24g skim-milk, 0.06g sodium ascorbate, 1.0mL phosphoric acid buffer, 0.1g Semen Coicis and Chinese yam mixing polysaccharide extract, mix, in-40 DEG C of pre-freeze 10h, then put into Freeze Drying Equipment and carry out vacuum lyophilization 24h under-56 DEG C, 5Pa, obtain lactobacillus bulgaricus freeze-dried vaccine powder.
Its freeze-drying survival rate can be 72.31% (control medium is 19.72%), and bacterium powder viable count reaches 1.34 × 10
11(control medium is 7.1 × 10 to CFU/g
9cFU/g).
Wherein, the configuration step that lactobacillus bulgaricus is bred freeze proof substratum comprises:
First accurately take lactose 20g, White Hyacinth Bean polysaccharide 0.4g, yeast extract 4g, soy peptone 8g, diammonium hydrogen citrate 1.5g, sodium acetate 1.2g, dipotassium hydrogen phosphate 1.5g, MnSO
40.05g and tween 80 0.8mL are dissolved in 950mL distilled water, heating for dissolving, stir, then adjust pH are to 6.4-6.6, be contained in anaerobism bottle according to loading amount 70%, and on the rubber plug of bottle cap to interpolation 1mL syringe needle, after 121 DEG C of sterilizing 15min, pull up syringe needle, be cooled to room temperature;
Then accurately taking 3g trehalose, 3g xylo-oligosaccharide and 1.0g Sodium Glutamate is dissolved in 50mL distilled water, mix, carry out filtration sterilization with the aseptic filter membrane of 0.22 μ m again, filtrate is required to mix according to aseptic technique in Bechtop with above-mentioned sterilized substratum, obtain lactobacillus bulgaricus and breed freeze proof substratum.
The preparation method of White Hyacinth Bean polysaccharide comprises the following steps: after 5 portions of dry White Hyacinth Beans are pulverized, be soaked in 150 parts of distilled water, regulate pH value to 5.0-6.8, under the bath temperature of 45 DEG C, add White Hyacinth Bean quality 1.5%, vigor is cellulase and the White Hyacinth Bean quality 1% of 8000U/g, vigor is the polygalacturonase of 30000U/g, after insulation 2h, on electromagnetic oven, be heated to boiling, thereafter regulating electromagnetism rate power is 1000W insulation 20min, then use Büchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, in concentrated solution, add the long-pending dehydrated alcohol of triploid, suction filtration after standing 10h in refrigerator, gained filter residue is thick White Hyacinth Bean polysaccharide, by gained filter residue in 50 DEG C of oven dry, obtain White Hyacinth Bean Crude polysaccharides.Be dissolved in by the dry White Hyacinth Bean Crude polysaccharides of every 0.5g in the distilled water of 20mL, heating in water bath to 55 DEG C and calculate, obtain polysaccharide soln, in polysaccharide soln, add the papoid of White Hyacinth Bean Crude polysaccharides quality 1%, after enzymolysis 0.9h, obtain enzymolysis solution, enzymolysis solution uses the Sevag reagent of polysaccharide soln 15% volume except protein 20 min, wherein chloroform: propyl carbinol=4:1 in Sevag reagent; After the centrifugal 15min of 2000rpm, get supernatant liquor, centrifugal treating three times repeatedly, the supernatant liquor obtaining obtains White Hyacinth Bean polysaccharide after vacuum lyophilization.
The preparation of Semen Coicis and Chinese yam mixing polysaccharide extract: be soaked in 150 parts of distilled water after the Chinese yam mixture of 5 parts of dry Semen Coiciss and peeling is pulverized, wherein the Chinese yam of Semen Coicis and peeling is pressed 1:1 mixing, regulate pH value to 6.0-7.0, under 80 DEG C of bath temperatures, extract 2h, be cooled to after 40 DEG C, under 40 DEG C of water-baths, add the polygalacturonase of the Chinese yam mixture quality 1% that accounts for above-mentioned Semen Coicis and peeling and 0.8% α-amylase, and be incubated 2.0h, in electromagnetic oven on be heated to boiling thereafter, insulation 10min, then use Büchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, to 95% ethanol that adds 3 times of volumes in concentrated solution, suction filtration after standing 10h in refrigerator, gained filter residue is thick mixing polysaccharide, with 80% ethanolic soln washing Crude polysaccharides, after filtration, gained is deposited in to 45 DEG C of oven dry, obtain Semen Coicis and Chinese yam mixing polysaccharide extract.
Embodiment 2
A) actication of culture and multiplication culture:
Lactobacillus bulgaricus is inoculated in sterilized MRS liquid nutrient medium, in 35 DEG C of cultivation 22-26h, then is inoculated in substratum same as described above in 4% (v/v) ratio, and is activated under the same conditions the third generation.
In the freeze proof substratum of propagation, access the lactobacillus bulgaricus that 3% (v/v) activated, 37 DEG C of constant temperature culture 16h.
B) collect thalline:
Then centrifugal 15min under 4 DEG C, 10000rpm, abandoning supernatant, obtains bacterium mud.
C) pre-freeze and vacuum lyophilization:
In every gram of bacterium mud, add 0.24g skim-milk, 0.06g sodium ascorbate, 0.8mL phosphoric acid buffer, 0.2g Semen Coicis and Chinese yam mixing polysaccharide extract, mix, in-40 DEG C of pre-freeze 10h, then put into Freeze Drying Equipment and carry out vacuum lyophilization 20h under-56 DEG C, 5Pa, obtain lactobacillus bulgaricus freeze-dried vaccine powder.
Its freeze-drying survival rate can be 74.82% (control medium is 10.2%), and bacterium powder viable count reaches 1.96 × 10
11(control medium is 6.4 × 10 to CFU/g
9cFU/g).
Wherein, the configuration step that lactobacillus bulgaricus is bred freeze proof substratum comprises:
First accurately take lactose 16g, White Hyacinth Bean polysaccharide 0.5g, yeast extract 6g, soy peptone 10g, diammonium hydrogen citrate 1.8g, sodium acetate 1.0g, dipotassium hydrogen phosphate 1.2g, MnSO
40.04g and tween 80 1.0mL are dissolved in 950mL distilled water, heating for dissolving, stir, then adjust pH are to 6.4-6.6, be contained in anaerobism bottle according to loading amount 80%, and on the rubber plug of bottle cap to interpolation 1mL syringe needle, after 121 DEG C of sterilizing 15min, pull up syringe needle, be cooled to room temperature;
Then accurately taking 5g trehalose, 2g xylo-oligosaccharide and 0.6g Sodium Glutamate is dissolved in 50mL distilled water, mix, carry out filtration sterilization with the aseptic filter membrane of 0.22 μ m again, filtrate is required to mix according to aseptic technique in Bechtop with above-mentioned sterilized substratum, obtain lactobacillus bulgaricus and breed freeze proof substratum.
The preparation method of White Hyacinth Bean polysaccharide comprises the following steps: after 5 portions of dry White Hyacinth Beans are pulverized, be soaked in 125 parts of distilled water, regulate pH value to 5.0-6.8, under the bath temperature of 40 DEG C, add White Hyacinth Bean quality 1%, vigor is cellulase and the White Hyacinth Bean quality 1.5% of 9000U/g, vigor is the polygalacturonase of 20000U/g, after insulation 2.5h, on electromagnetic oven, be heated to boiling, thereafter regulating electromagnetism rate power is 1000W insulation 18min, then use Büchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, in concentrated solution, add the long-pending dehydrated alcohol of triploid, suction filtration after standing 10h in refrigerator, gained filter residue is thick White Hyacinth Bean polysaccharide, by gained filter residue in 55 DEG C of oven dry, obtain White Hyacinth Bean Crude polysaccharides.Be dissolved in by the dry White Hyacinth Bean Crude polysaccharides of every 0.5g in the distilled water of 15mL, heating in water bath to 50 DEG C and calculate, obtain polysaccharide soln, in polysaccharide soln, add the papoid of White Hyacinth Bean Crude polysaccharides quality 3%, after enzymolysis 0.6h, obtain enzymolysis solution, enzymolysis solution uses the Sevag reagent (chloroform: propyl carbinol=4:1) of polysaccharide soln 18% volume except protein 25 min, after the centrifugal 20min of 3000rpm, get supernatant liquor, centrifugal treating three times repeatedly, the supernatant liquor obtaining obtains White Hyacinth Bean polysaccharide after vacuum lyophilization.
The preparation of Semen Coicis and Chinese yam mixing polysaccharide extract: be soaked in 200 parts of distilled water after the Chinese yam of 5 parts of dry Semen Coiciss and peeling (1:1 mixing) is pulverized, regulate pH value to 6.0-7.0, under 100 DEG C of bath temperatures, extract 1h, be cooled to after 45 DEG C, under 45 DEG C of water-baths, add the polygalacturonase of the Chinese yam mixture quality 2% that accounts for above-mentioned Semen Coicis and peeling and 1% α-amylase, and be incubated 1.5h, in electromagnetic oven on be heated to boiling thereafter, insulation 15min, then use Büchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, to 95% ethanol that adds 3 times of volumes in concentrated solution, suction filtration after standing 12h in refrigerator, gained filter residue is thick mixing polysaccharide, with 80% ethanolic soln washing Crude polysaccharides, after filtration, gained is deposited in to 50 DEG C of oven dry, obtain Semen Coicis and Chinese yam mixing polysaccharide extract.
Embodiment 3
A) actication of culture and multiplication culture:
Lactobacillus bulgaricus is inoculated in sterilized MRS liquid nutrient medium, in 38 DEG C of cultivation 22-26h, then is inoculated in substratum same as described above in 5% (v/v) ratio, and is activated under the same conditions the third generation.
In the freeze proof substratum of propagation, access the lactobacillus bulgaricus that 4% (v/v) activated, 35 DEG C of constant temperature culture 16h.
B) collect thalline:
Then centrifugal 15min under 0 DEG C, 10000rpm, abandoning supernatant, obtains bacterium mud.
C) pre-freeze and vacuum lyophilization:
In every gram of bacterium mud, add 0.2g skim-milk, 0.08g sodium ascorbate, 1.2mL phosphoric acid buffer, 0.3g Semen Coicis and Chinese yam mixing polysaccharide extract, mix, in-40 DEG C of pre-freeze 14h, then put into Freeze Drying Equipment and carry out vacuum lyophilization 22h under-56 DEG C, 5Pa, obtain lactobacillus bulgaricus freeze-dried vaccine powder.
Its freeze-drying survival rate can be 75.20% (control medium is 15.4%), and bacterium powder viable count reaches 1.80 × 10
11(control medium is 7.8 × 10 to CFU/g
9cFU/g).
Wherein, the configuration step that lactobacillus bulgaricus is bred freeze proof substratum comprises:
First accurately take lactose 18g, White Hyacinth Bean polysaccharide 0.6g, yeast extract 5g, soy peptone 10g, diammonium hydrogen citrate 1.2g, sodium acetate 1.0g, dipotassium hydrogen phosphate 1.8g, MnSO
40.03g and tween 80 1.0mL are dissolved in 950mL distilled water, heating for dissolving, stir, then adjust pH are to 6.4-6.6, be contained in anaerobism bottle according to loading amount 90%, and on the rubber plug of bottle cap to interpolation 1mL syringe needle, after 120 DEG C of sterilizing 15min, pull up syringe needle, be cooled to room temperature;
Then accurately taking 4g trehalose, 4g xylo-oligosaccharide and 0.8g Sodium Glutamate is dissolved in 50mL distilled water, mix, carry out filtration sterilization with the aseptic filter membrane of 0.22 μ m again, filtrate is required to mix according to aseptic technique in Bechtop with above-mentioned sterilized substratum, obtain lactobacillus bulgaricus and breed freeze proof substratum.
The preparation method of White Hyacinth Bean polysaccharide comprises the following steps: after 5 portions of dry White Hyacinth Beans are pulverized, be soaked in 100 parts of distilled water, regulate pH value to 5.0-6.8, under the bath temperature of 55 DEG C, add White Hyacinth Bean quality 2%, vigor is cellulase and the White Hyacinth Bean quality 2% of 10000U/g, vigor is the polygalacturonase of 20000U/g, after insulation 1.5h, on electromagnetic oven, be heated to boiling, thereafter regulating electromagnetism rate power is 1000W insulation 20min, then use Büchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, in concentrated solution, add the long-pending dehydrated alcohol of triploid, suction filtration after standing 14h in refrigerator, gained filter residue is thick White Hyacinth Bean polysaccharide, by gained filter residue in 50 DEG C of oven dry, obtain White Hyacinth Bean Crude polysaccharides.Be dissolved in by the dry White Hyacinth Bean Crude polysaccharides of every 0.5g in the distilled water of 20mL, heating in water bath to 70 DEG C and calculate, obtain polysaccharide soln, in polysaccharide soln, add the papoid of White Hyacinth Bean Crude polysaccharides quality 4%, after enzymolysis 1h, obtain enzymolysis solution, enzymolysis solution uses the Sevag reagent (chloroform: propyl carbinol=4:1) of polysaccharide soln 25% volume except albumen 30min, after the centrifugal 20min of 4000rpm, get supernatant liquor, centrifugal treating three times repeatedly, the supernatant liquor obtaining obtains White Hyacinth Bean polysaccharide after vacuum lyophilization.
The preparation of Semen Coicis and Chinese yam mixing polysaccharide extract: be soaked in 200 parts of distilled water after the Chinese yam of 5 parts of dry Semen Coiciss and peeling (1:1 mixing) is pulverized, regulate pH value to 6.0-7.0, under 90 DEG C of bath temperatures, extract 1.5h, be cooled to after 50 DEG C, under 50 DEG C of water-baths, add the polygalacturonase of the Chinese yam mixture quality 1.5% that accounts for above-mentioned Semen Coicis and peeling and 1% α-amylase, and be incubated 2.0h, in electromagnetic oven on be heated to boiling thereafter, insulation 15min, then use Büchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, to 95% ethanol that adds 4 times of volumes in concentrated solution, suction filtration after standing 14h in refrigerator, gained filter residue is thick mixing polysaccharide, with 80% ethanolic soln washing Crude polysaccharides, after filtration, gained is deposited in to 55 DEG C of oven dry, obtain Semen Coicis and Chinese yam mixing polysaccharide extract.
Embodiment 4
A) actication of culture and multiplication culture:
Lactobacillus bulgaricus is inoculated in sterilized MRS liquid nutrient medium, in 36 DEG C of cultivation 22-26h, then is inoculated in substratum same as described above in 3% (v/v) ratio, and is activated under the same conditions the third generation.
In the freeze proof substratum of propagation, access the lactobacillus bulgaricus that 5% (v/v) activated, 38 DEG C of constant temperature culture 17h.
B) collect thalline:
Then centrifugal 16min under 1 DEG C, 9000rpm, abandoning supernatant, obtains bacterium mud.
C) pre-freeze and vacuum lyophilization:
In every gram of bacterium mud, add 0.22g skim-milk, 0.1g sodium ascorbate, 1.4mL phosphoric acid buffer, 0.3g Semen Coicis and Chinese yam mixing polysaccharide extract, mix, in-40 DEG C of pre-freeze 8h, then put into Freeze Drying Equipment and carry out vacuum lyophilization 21h under-56 DEG C, 5Pa, obtain lactobacillus bulgaricus freeze-dried vaccine powder.
Its freeze-drying survival rate can be 75.21% (control medium is 18.63%), and bacterium powder viable count reaches 1.85 × 10
11(control medium is 7.6 × 10 to CFU/g
9cFU/g).
Wherein, the configuration step that lactobacillus bulgaricus is bred freeze proof substratum comprises:
First accurately take lactose 13g, White Hyacinth Bean polysaccharide 0.3g, yeast extract 8g, soy peptone 9g, diammonium hydrogen citrate 2.0g, sodium acetate 1.5g, dipotassium hydrogen phosphate 1.0g, MnSO
40.06g and tween 80 0.6mL are dissolved in 950mL distilled water, heating for dissolving, stir, then adjust pH are to 6.4-6.6, be contained in anaerobism bottle according to loading amount 70%, and on the rubber plug of bottle cap to interpolation 1mL syringe needle, after 119 DEG C of sterilizing 15min, pull up syringe needle, be cooled to room temperature;
Then accurately taking 2g trehalose, 4g xylo-oligosaccharide and 0.7g Sodium Glutamate is dissolved in 50mL distilled water, mix, carry out filtration sterilization with the aseptic filter membrane of 0.22 μ m again, filtrate is required to mix according to aseptic technique in Bechtop with above-mentioned sterilized substratum, obtain lactobacillus bulgaricus and breed freeze proof substratum.
The preparation method of White Hyacinth Bean polysaccharide comprises the following steps: after 5 portions of dry White Hyacinth Beans are pulverized, be soaked in 120 parts of distilled water, regulate pH value to 5.0-6.8, under the bath temperature of 48 DEG C, add White Hyacinth Bean quality 1%, vigor is cellulase and the White Hyacinth Bean quality 1% of 8000U/g, vigor is the polygalacturonase of 20000U/g, after insulation 1.8h, on electromagnetic oven, be heated to boiling, thereafter regulating electromagnetism rate power is 1000W insulation 17min, then use Büchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, in concentrated solution, add the long-pending dehydrated alcohol of triploid, suction filtration after standing 13h in refrigerator, gained filter residue is thick White Hyacinth Bean polysaccharide, by gained filter residue in 53 DEG C of oven dry, obtain White Hyacinth Bean Crude polysaccharides.Be dissolved in by the dry White Hyacinth Bean Crude polysaccharides of every 0.5g in the distilled water of 16mL, heating in water bath to 60 DEG C and calculate, obtain polysaccharide soln, in polysaccharide soln, add the papoid of White Hyacinth Bean Crude polysaccharides quality 2%, after enzymolysis 0.8h, obtain enzymolysis solution, enzymolysis solution uses the Sevag reagent (chloroform: propyl carbinol=4:1) of polysaccharide soln 20% volume except protein 23 min, after the centrifugal 16min of 3000rpm, get supernatant liquor, centrifugal treating three times repeatedly, the supernatant liquor obtaining obtains White Hyacinth Bean polysaccharide after vacuum lyophilization.
The preparation of Semen Coicis and Chinese yam mixing polysaccharide extract: be soaked in 170 parts of distilled water after the Chinese yam of 5 parts of dry Semen Coiciss and peeling (1:1 mixing) is pulverized, regulate pH value to 6.0-7.0, under 100 DEG C of bath temperatures, extract 1.5h, be cooled to after 45 DEG C, under 45 DEG C of water-baths, add the polygalacturonase of the Chinese yam mixture quality 2% that accounts for above-mentioned Semen Coicis and peeling and 0.8% α-amylase, and be incubated 1.9h, in electromagnetic oven on be heated to boiling thereafter, insulation 13min, then use Büchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, to 95% ethanol that adds 3 times of volumes in concentrated solution, suction filtration after standing 11h in refrigerator, gained filter residue is thick mixing polysaccharide, with 80% ethanolic soln washing Crude polysaccharides, after filtration, gained is deposited in to 48 DEG C of oven dry, obtain Semen Coicis and Chinese yam mixing polysaccharide extract.
Embodiment 5
A) actication of culture and multiplication culture:
Lactobacillus bulgaricus is inoculated in sterilized MRS liquid nutrient medium, in 37 DEG C of cultivation 22-26h, then is inoculated in substratum same as described above in 3% (v/v) ratio, and is activated under the same conditions the third generation.
In the freeze proof substratum of propagation, access the lactobacillus bulgaricus that 5% (v/v) activated, 38 DEG C of constant temperature culture 18h.
B) collect thalline:
Then centrifugal 17min under 2 DEG C, 8000rpm, abandoning supernatant, obtains bacterium mud.
C) pre-freeze and vacuum lyophilization:
In every gram of bacterium mud, add 0.21g skim-milk, 0.07g sodium ascorbate, 1.0mL phosphoric acid buffer, 0.2g Semen Coicis and Chinese yam mixing polysaccharide extract, mix, in-40 DEG C of pre-freeze 6h, then put into Freeze Drying Equipment and carry out vacuum lyophilization 18h under-56 DEG C, 5Pa, obtain lactobacillus bulgaricus freeze-dried vaccine powder.
Its freeze-drying survival rate can be 73.46% (control medium is 15.4%), and bacterium powder viable count reaches 1.65 × 10
11(control medium is 6.9 × 10 to CFU/g
9cFU/g).
Wherein, the configuration step that lactobacillus bulgaricus is bred freeze proof substratum comprises:
First accurately take lactose 10g, White Hyacinth Bean polysaccharide 0.2g, yeast extract 10g, soy peptone 9g, diammonium hydrogen citrate 1.0g, sodium acetate 2.0g, dipotassium hydrogen phosphate 2.0g, MnSO
40.01g and tween 80 0.5mL are dissolved in 950mL distilled water, heating for dissolving, stir, then adjust pH are to 6.4-6.6, be contained in anaerobism bottle according to loading amount 80%, and on the rubber plug of bottle cap to interpolation 1mL syringe needle, after 118 DEG C of sterilizing 15min, pull up syringe needle, be cooled to room temperature;
Then accurately taking 6g trehalose, 3g xylo-oligosaccharide and 0.9g Sodium Glutamate is dissolved in 50mL distilled water, mix, carry out filtration sterilization with the aseptic filter membrane of 0.22 μ m again, filtrate is required to mix according to aseptic technique in Bechtop with above-mentioned sterilized substratum, obtain lactobacillus bulgaricus and breed freeze proof substratum.
The preparation method of White Hyacinth Bean polysaccharide comprises the following steps: after 5 portions of dry White Hyacinth Beans are pulverized, be soaked in 140 parts of distilled water, regulate pH value to 5.0-6.8, under the bath temperature of 50 DEG C, add White Hyacinth Bean quality 0.5%, vigor is cellulase and the White Hyacinth Bean quality 2% of 10000U/g, vigor is the polygalacturonase of 30000U/g, after insulation 2.5h, on electromagnetic oven, be heated to boiling, thereafter regulating electromagnetism rate power is 1000W insulation 15min, then use Büchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, in concentrated solution, add the long-pending dehydrated alcohol of triploid, suction filtration after standing 12h in refrigerator, gained filter residue is thick White Hyacinth Bean polysaccharide, by gained filter residue in 45 DEG C of oven dry, obtain White Hyacinth Bean Crude polysaccharides.Be dissolved in by the dry White Hyacinth Bean Crude polysaccharides of every 0.5g in the distilled water of 18mL, heating in water bath to 65 DEG C and calculate, obtain polysaccharide soln, in polysaccharide soln, add the papoid of White Hyacinth Bean Crude polysaccharides quality 2.5%, after enzymolysis 0.5h, obtain enzymolysis solution, enzymolysis solution uses the Sevag reagent (chloroform: propyl carbinol=4:1) of polysaccharide soln 23% volume except protein 28 min, after the centrifugal 18min of 2000rpm, get supernatant liquor, centrifugal treating three times repeatedly, the supernatant liquor obtaining obtains White Hyacinth Bean polysaccharide after vacuum lyophilization.
The preparation of Semen Coicis and Chinese yam mixing polysaccharide extract: be soaked in 180 parts of distilled water after the Chinese yam of 5 parts of dry Semen Coiciss and peeling (1:1 mixing) is pulverized, regulate pH value to 6.0-7.0, under 90 DEG C of bath temperatures, extract 1h, be cooled to after 55 DEG C, under 55 DEG C of water-baths, add the polygalacturonase of the Chinese yam mixture quality 2.5% that accounts for above-mentioned Semen Coicis and peeling and 0.9% α-amylase, and be incubated 1.8h, in electromagnetic oven on be heated to boiling thereafter, insulation 12min, then use Büchner funnel suction filtration, filtrate is concentrated on Rotary Evaporators, to 95% ethanol that adds 4 times of volumes in concentrated solution, suction filtration after standing 13h in refrigerator, gained filter residue is thick mixing polysaccharide, with 80% ethanolic soln washing Crude polysaccharides, after filtration, gained is deposited in to 52 DEG C of oven dry, obtain Semen Coicis and Chinese yam mixing polysaccharide extract.
Claims (10)
1. a lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof, is characterized in that: comprise the following steps:
A) actication of culture and multiplication culture: lactobacillus bulgaricus is inoculated in MRS liquid nutrient medium and is activated, the bacterial classification having activated is inoculated in to multiplication culture in the freeze proof substratum of propagation, obtain the bacterium liquid of propagation;
B) collect thalline: by the bacterium liquid centrifugal treating of above-mentioned propagation, abandoning supernatant, obtains bacterium mud;
C) dry: the phosphoric acid buffer, 0.1-0.3g Semen Coicis and the Chinese yam mixing polysaccharide extract that in prepared bacterium mud, add 0.2-0.24g skim-milk, 0.06-0.1g sodium ascorbate, 0.8-1.4mL by every gram of bacterium mud, mix final vacuum lyophilize, obtain lactobacillus bulgaricus freeze-dried vaccine powder;
The freeze proof culture medium prescription of described propagation consists of: lactose 10-20g, trehalose 2-6g, White Hyacinth Bean polysaccharide 0.2-0.6g, xylo-oligosaccharide 2-4g, yeast extract 4-10g, soy peptone 8-10g, Sodium Glutamate 0.6-1.0g, diammonium hydrogen citrate 1.0-2.0g, sodium acetate 1.0-2.0g, dipotassium hydrogen phosphate 1.0-2.0g, MnSO
40.01-0.06g, tween 80 0.5-1.0mL, distilled water 1000mL.
2. a kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof according to claim 1, it is characterized in that: described step a) in activation refer to lactobacillus bulgaricus be inoculated in MRS liquid nutrient medium in 35-38 DEG C and cultivate 22-26h, again in the ratio of every 100mL inoculation of medium 3-5mL bacterial classification, be inoculated in the substratum identical with above-mentioned MRS liquid nutrient medium, and be activated under the same conditions the third generation.
3. a kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof according to claim 2, is characterized in that: the described step a) inoculative proportion of middle multiplication culture is that every 100mL breeds the bacterial classification that freeze proof inoculation of medium 3-5mL has activated.
4. a kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof according to claim 3, is characterized in that: described step a) middle multiplication culture is to cultivate 16-18h at 35-38 DEG C.
5. according to a kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof described in claim 1 to 4 any one, it is characterized in that: the configuration step of the freeze proof substratum of described propagation comprises:
First take according to quantity lactose, White Hyacinth Bean polysaccharide, yeast extract, soy peptone, diammonium hydrogen citrate, sodium acetate, dipotassium hydrogen phosphate, MnSO
4be dissolved in distilled water a with tween 80, heating for dissolving, stir, then adjust pH is to 6.4-6.6, is contained in anaerobism bottle according to loading amount 70%-90%, in 118-121 DEG C of sterilizing 15min, is cooled to room temperature, obtains sterilized substratum;
Taking trehalose, xylo-oligosaccharide and Sodium Glutamate is dissolved in distilled water b again, the total amount of described distilled water a and distilled water b is 1000mL, mix rear filtration sterilization, filtrate is required to mix according to aseptic technique with above-mentioned sterilized substratum, obtain lactobacillus bulgaricus and breed freeze proof substratum.
6. a kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof according to claim 1, is characterized in that: the preparation process of described White Hyacinth Bean polysaccharide comprises:
First after 5 portions of dry White Hyacinth Beans being pulverized, be soaked in 100-150 part distilled water, regulate pH value to 5.0-6.8;
Secondly under the bath temperature of 40-55 DEG C, add the polygalacturonase that cellulase that White Hyacinth Bean quality 0.5-2%, vigor are 8000-10000U/g and White Hyacinth Bean quality 1-2%, vigor are 20000-30000U/g, the extremely boiling of insulation 1.5-2.5h post-heating, then be incubated 15-20min;
Then suction filtration filtrate is concentrated adds the long-pending dehydrated alcohol of triploid in concentrated solution, and suction filtration after standing 10-14h, in 45-55 DEG C of oven dry, obtains White Hyacinth Bean Crude polysaccharides by gained filter residue;
Finally getting dry White Hyacinth Bean Crude polysaccharides is dissolved in the distilled water that water-bath is heated to 50-70 DEG C, the ratio that is dissolved in 15-20mL distilled water in every 0.5g White Hyacinth Bean Crude polysaccharides is calculated, obtain polysaccharide soln, in polysaccharide soln, add the papoid of White Hyacinth Bean Crude polysaccharides quality 1-4%, after enzymolysis 0.5-1h, obtain enzymolysis solution, enzymolysis solution uses the Sevag reagent of polysaccharide soln 15-25% volume except protein 20-30min; After the centrifugal 15-20min of 2000-4000rpm, get supernatant liquor, centrifugal treating three times repeatedly, the supernatant liquor obtaining obtains White Hyacinth Bean polysaccharide after vacuum lyophilization.
7. a kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof according to claim 1, is characterized in that: described step b) middle centrifugal treating is 0-4 DEG C, the centrifugal 15-20min of 8000-10000rpm.
8. a kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof according to claim 1, is characterized in that: the preparation process of described Semen Coicis and Chinese yam mixing polysaccharide extract comprises:
First after the Chinese yam mixture of 5 parts of dry Semen Coiciss and peeling being pulverized, be soaked in 150-200 part distilled water, wherein the Chinese yam of Semen Coicis and peeling is pressed 1:1 mixing, regulate pH value to 6.0-7.0, under 80-100 DEG C of bath temperature, extract 1-2h, be cooled to 40-55 DEG C;
Then under 40-55 DEG C of water-bath, add the polygalacturonase of the Chinese yam mixture quality 1-2.5% that accounts for above-mentioned Semen Coicis and peeling and the α-amylase of 0.8-1%, and be incubated 1.5-2.0h post-heating to boiling, then be incubated 10-15min;
Then suction filtration by concentrated filtrate, to 95% ethanol that adds 3-4 times of volume in concentrated solution, leaves standstill suction filtration after 10-14h, and gained filter residue is thick mixing polysaccharide;
Finally wash thick mixing polysaccharide with 80% ethanolic soln, after filtration, gained is deposited in to 45-55 DEG C of oven dry, obtain Semen Coicis and Chinese yam mixing polysaccharide extract.
9. a kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof according to claim 1, is characterized in that: described step c) middle vacuum lyophilization is in Freeze Drying Equipment, to carry out 18-24h.
10. according to a kind of thermophilus streptococcus freeze-dried vaccine powder, preparation method thereof described in claim 1 or 9, it is characterized in that: described step c) in before vacuum lyophilization at-40 DEG C of pre-freeze 6-14h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410171964.2A CN103923835B (en) | 2014-04-25 | 2014-04-25 | A kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410171964.2A CN103923835B (en) | 2014-04-25 | 2014-04-25 | A kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103923835A true CN103923835A (en) | 2014-07-16 |
| CN103923835B CN103923835B (en) | 2016-02-10 |
Family
ID=51142234
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410171964.2A Active CN103923835B (en) | 2014-04-25 | 2014-04-25 | A kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103923835B (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611256A (en) * | 2014-12-17 | 2015-05-13 | 光明乳业股份有限公司 | Microbial freeze-dried protective agent and preparation method and application thereof |
| CN105685223A (en) * | 2016-02-01 | 2016-06-22 | 陕西科技大学 | Preparation method of antioxidant lactobacillus acidophilus fermented goat milk |
| CN106234589A (en) * | 2016-09-23 | 2016-12-21 | 陕西科技大学 | A kind of preparation method of antioxidation probiotics fermention breast |
| CN106417601A (en) * | 2016-09-23 | 2017-02-22 | 陕西科技大学 | Preparation method of antioxidant peptide-containing probiotics milk beverage |
| CN106962478A (en) * | 2017-05-05 | 2017-07-21 | 广州赛莱拉干细胞科技股份有限公司 | A kind of store method of beneficial bacteria of intestinal tract |
| CN107858388A (en) * | 2017-10-17 | 2018-03-30 | 陕西科技大学 | A kind of method that sheep breast Antihypertensive Peptides are prepared using probiotic lactobacilli protease |
| CN108018211A (en) * | 2018-02-06 | 2018-05-11 | 内蒙古双奇药业股份有限公司 | A kind of culture medium and its application process for improving lactobacillus bulgaricus freexing tolerance |
| CN108013261A (en) * | 2017-12-30 | 2018-05-11 | 陕西石羊农业科技股份有限公司 | One breeder probiotics feed and preparation method thereof |
| CN108244337A (en) * | 2017-12-30 | 2018-07-06 | 陕西石羊农业科技股份有限公司 | One boar probiotics feed and preparation method thereof |
| CN109989284A (en) * | 2019-02-25 | 2019-07-09 | 黄彬彬 | A kind of environmental protection is grease proofing to use paper |
| CN114451485A (en) * | 2022-01-26 | 2022-05-10 | 江苏省淡水水产研究所 | Traditional Chinese medicine polysaccharide extract freeze-drying protective agent, direct-vat-set starter and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2172223A1 (en) * | 2007-06-22 | 2010-04-07 | Kaneka Corporation | Composition containing physiologically active substance |
| CN102952755A (en) * | 2012-11-19 | 2013-03-06 | 陕西科技大学 | Lactobacillus bulgaricus freeze-drying protecting agent and preparation method thereof |
| CN102952772A (en) * | 2012-11-19 | 2013-03-06 | 陕西科技大学 | Lactobacillus bulgaricus enrichment culture medium and preparation method thereof |
| CN102977221A (en) * | 2012-12-04 | 2013-03-20 | 天津科技大学 | Preparation method of coix seed polysaccharide and application of coix seed polysaccharide |
-
2014
- 2014-04-25 CN CN201410171964.2A patent/CN103923835B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2172223A1 (en) * | 2007-06-22 | 2010-04-07 | Kaneka Corporation | Composition containing physiologically active substance |
| CN102952755A (en) * | 2012-11-19 | 2013-03-06 | 陕西科技大学 | Lactobacillus bulgaricus freeze-drying protecting agent and preparation method thereof |
| CN102952772A (en) * | 2012-11-19 | 2013-03-06 | 陕西科技大学 | Lactobacillus bulgaricus enrichment culture medium and preparation method thereof |
| CN102977221A (en) * | 2012-12-04 | 2013-03-20 | 天津科技大学 | Preparation method of coix seed polysaccharide and application of coix seed polysaccharide |
Non-Patent Citations (3)
| Title |
|---|
| XIANGYI LU ET AL: "A polysaccharide fraction of adlay seed (Coix lachryma-jobi L.) induces apoptosis in human non-small cell lung cancer A549 cells", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》, vol. 430, 31 December 2013 (2013-12-31), pages 846 - 851 * |
| 史娟: "白扁豆多糖的超声波辅助提取及其稳定性研究", 《中国食品添加剂》, 31 December 2012 (2012-12-31), pages 155 - 160 * |
| 肖小年: "薏苡仁多糖的提取及分离纯化", 《食品科学》, vol. 31, no. 22, 31 December 2010 (2010-12-31), pages 1 - 5 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611256A (en) * | 2014-12-17 | 2015-05-13 | 光明乳业股份有限公司 | Microbial freeze-dried protective agent and preparation method and application thereof |
| CN104611256B (en) * | 2014-12-17 | 2017-09-15 | 光明乳业股份有限公司 | A kind of microorganism lyophilized formulations and preparation method thereof |
| CN105685223A (en) * | 2016-02-01 | 2016-06-22 | 陕西科技大学 | Preparation method of antioxidant lactobacillus acidophilus fermented goat milk |
| CN106234589A (en) * | 2016-09-23 | 2016-12-21 | 陕西科技大学 | A kind of preparation method of antioxidation probiotics fermention breast |
| CN106417601A (en) * | 2016-09-23 | 2017-02-22 | 陕西科技大学 | Preparation method of antioxidant peptide-containing probiotics milk beverage |
| CN106962478A (en) * | 2017-05-05 | 2017-07-21 | 广州赛莱拉干细胞科技股份有限公司 | A kind of store method of beneficial bacteria of intestinal tract |
| CN107858388A (en) * | 2017-10-17 | 2018-03-30 | 陕西科技大学 | A kind of method that sheep breast Antihypertensive Peptides are prepared using probiotic lactobacilli protease |
| CN108013261A (en) * | 2017-12-30 | 2018-05-11 | 陕西石羊农业科技股份有限公司 | One breeder probiotics feed and preparation method thereof |
| CN108244337A (en) * | 2017-12-30 | 2018-07-06 | 陕西石羊农业科技股份有限公司 | One boar probiotics feed and preparation method thereof |
| CN108018211A (en) * | 2018-02-06 | 2018-05-11 | 内蒙古双奇药业股份有限公司 | A kind of culture medium and its application process for improving lactobacillus bulgaricus freexing tolerance |
| CN109989284A (en) * | 2019-02-25 | 2019-07-09 | 黄彬彬 | A kind of environmental protection is grease proofing to use paper |
| CN114451485A (en) * | 2022-01-26 | 2022-05-10 | 江苏省淡水水产研究所 | Traditional Chinese medicine polysaccharide extract freeze-drying protective agent, direct-vat-set starter and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103923835B (en) | 2016-02-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103923835B (en) | A kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof | |
| CN102366023B (en) | Method for preparing cow feed additive containing traditional Chinese medicine active probiotics | |
| CN102952755B (en) | Lactobacillus bulgaricus freeze-drying protecting agent and preparation method thereof | |
| CN101731511B (en) | Probiotic active product and preparation method thereof | |
| CN110141585A (en) | A kind of composite probiotics ferment microbial inoculum and preparation method thereof for adjusting intestinal flora | |
| CN105685475A (en) | Method for preparing feed antibiotic substitute from subtilin producing bacillus subtilis fermented Chinese herbal medicine | |
| CN107897368A (en) | A kind of probiotics fermention breast and preparation method thereof | |
| CN1911118B (en) | Kefir mushroom freeze-dried powder, production method and use thereof | |
| CN108112987A (en) | A kind of functional preparation for adjusting gastrointestinal health and its preparation method and application | |
| CN105420111A (en) | Composite protective agent for increasing freeze-drying survival rate of lactobacillus plantarum and preparation method thereof | |
| CN108244432A (en) | A kind of fermentation Cordyceps militaris probiotic beverage and preparation method thereof | |
| CN106509100A (en) | Production method of solid yogurt powder containing high active bacteria | |
| CN108559717A (en) | A kind of lactobacillus acidophilus high density fermentation culture medium and its bacterium powder preparation method | |
| CN105746881A (en) | Method for preparing substituent of antibiotics for feed by fermenting Chinese herbal medicines through lipopeptide-producing bacillus subtilis | |
| CN102986869A (en) | Solid Kefir dairy product and preparation method thereof | |
| CN102453681B (en) | Protective agent for vacuum freeze drying of lactobacillus johnsonii, and application thereof | |
| CN102329759B (en) | Method for improving freeze-drying survival rate of Lactobacillus acidophilus | |
| CN102367416B (en) | Traditional Chinese medicine probiotic solid culture medium for cow feed, culture method and feed additive | |
| CN103571767B (en) | Utilize double application lactobacillus powder of edible agar and preparation method thereof | |
| CN109364114A (en) | A kind of fermented tcm and preparation method thereof improving cattle and sheep growth and production performance | |
| CN103937725B (en) | A kind of streptococcus thermophilus freeze-dried vaccine powder, preparation method thereof | |
| CN105230782A (en) | Preparation method of hair weed yoghourt | |
| CN103875922B (en) | A kind of composite probiotics micro-ecological additive agent for feeding and preparation method thereof | |
| CN102334684A (en) | Preparation method of microbial product with specific function | |
| CN102408992B (en) | Lactobacillus acidophilus anti-freeze culture medium and application method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| CB03 | Change of inventor or designer information |
Inventor after: Chen He Inventor after: Chen Shiwei Inventor after: Chen Hongli Inventor after: Wu Yanyan Inventor after: Shu Guowei Inventor after: Wang Chunling Inventor after: He Yulan Inventor before: Chen He Inventor before: Chen Shiwei Inventor before: Shu Guowei |
|
| COR | Change of bibliographic data | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20160105 Address after: 710021 Shaanxi province Xi'an Weiyang University Park No. 1 Applicant after: Shaanxi University of Science and Technology Applicant after: XINJIANG SHIHEZI HUAYUAN DAIRY INDUSTRY CO., LTD. Address before: 710021 Shaanxi province Xi'an Weiyang University Park No. 1 Applicant before: Shaanxi University of Science and Technology |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |