CN101606552A - Freezing direct vat inoculation of a kind of bifidobacterium deep and composite frozen protective agent thereof - Google Patents

Freezing direct vat inoculation of a kind of bifidobacterium deep and composite frozen protective agent thereof Download PDF

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CN101606552A
CN101606552A CNA2009101365153A CN200910136515A CN101606552A CN 101606552 A CN101606552 A CN 101606552A CN A2009101365153 A CNA2009101365153 A CN A2009101365153A CN 200910136515 A CN200910136515 A CN 200910136515A CN 101606552 A CN101606552 A CN 101606552A
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bifidobacterium
deep
liquid
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CN101606552B (en
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刘松玲
任发政
蒋菁莉
刘爱萍
赵亮
张明
杨海莺
田寒友
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Zhongyuan Food Laboratory
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Abstract

The invention discloses agent of a kind of bifidobacterium deep frozen fermented and cryoprotector thereof.Obtain the Bifidobacterium thalline by High Density Cultivation, centrifugal collection bacterium mud, and utilize the resuspended thalline of preferred composite protective agent, in-80 ℃ of deep layer freezings, effectively kept bacterium number and the functional characteristic of Bifidobacterium.Bifidobacterium deep frozen fermented of the present invention agent is as a kind of auxiliary leavening, and effectively being applied to the dairy products fermentation for probio has remarkable result.

Description

Freezing direct vat inoculation of a kind of bifidobacterium deep and composite frozen protective agent thereof
Technical field
The present invention relates to freezing direct vat inoculation of a kind of bifidobacterium deep and composite frozen protective agent thereof.
Background technology
As a kind of probio of classics, the benefit of Bifidobacterium is given birth to function and has been obtained general understanding, and the application in functional food has also obtained paying close attention to widely, and wherein the application in dairy products is the most representative.But Bifidobacterium has all drawbacks on fermenting property and production performance.At first, Bifidobacterium is extremely responsive for external environment, experiences low temperature in the preparation process of leavening, and drying stimulates survival rate lower etc. adverse circumstance; Secondly, Bifidobacterium is an obligate anaerobe, and fermentation condition is had relatively high expectations, and the aerobic fermentation acid production speed is low, and local flavor is relatively poor; The difficult control of bacterial classification ratio in the preparation of mixed bacteria direct vat inoculation, the viable count instability in the final products.Bifidobacterium is prepared into direct vat inoculation, mix use as auxiliary leavening with traditional lactic acid bacteria direct vat inoculation, can guarantee the quantity of probio in the product, can guarantee the quality and the taste of sour milk again, the control curdled milk time, in the function dairy products, be with a wide range of applications.
Bifidobacterium effectively is applied to preparation and the preservation technology that the dairy products fermentation mainly depends on leavening.The exploitation of bifidobacterium fermentation agent at present mainly concentrates on freeze drying and spray-drying field, but these technology can cause a series of side effects: at first survival rate only is about 30%-40% in the dry run, because Bifidobacterium is had relatively high expectations to nutrition and growth conditions, the thalline production cost is higher, and the survival rate that improves in the leavening preparation process is significant for production application; Secondly the low moisture activity that continues in leavening preparation and the preserving process can cause cell membrane, cell membrane, and the damage of DNA etc., the cell of a lot of survivals can lose its physiological function, thereby loses the value of probio; The 3rd, since cellular damage, oxygen in the ability of thalline tolerance adverse circumstance and tolerance product and the enteron aisle, acid, the ability of cholate etc. also can descend, thereby makes the bacterium number that reaches enteron aisle and grow surely in enteron aisle can not guarantee that this also is the bottleneck in the Bifidobacterium application process.
The deep layer Refrigeration Technique is generally used for tissue, cell, and the preservation of bacterial classification etc. has good effect for bioactive maintenance, and the preservation time, long preservation was effective.Utilize effective protective agent, the preparation survival rate that the deep layer Refrigeration Technique is used for leavening can have important function for the cultivation cost that reduces Bifidobacterium near 100%.Deep cooling protective agent commonly used comprises dimethyl sulfoxide (DMSO), polyethylene glycol, and price costlinesses such as serum, and be not suitable for being used for food, limited its application in leavening is produced.The present invention relates to a kind of bifidobacterium deep frozen fermented agent of eating source property, be used for the preparation of Bifidobacterium direct vat inoculation, to guarantee Bifidobacterium bacterium number and functional characteristic in product.
According to the difference of the protective agent mechanism of action, protective agent can be divided into permeability protective agent, semipermeability protective agent and impermeability protective agent.Permeability protective agent porous is to cell interior, in cell fully water and, prevent the cell transition dehydration, can reduce the toxicity of high salt pair cell, also can prevent the formation of ice crystal in the cell.The semipermeability protective agent can prevent freezing precellular plasmolysis, can concentrate on the growth that prevents ice crystal between cell membrane and the cytoplasm as a cushion, and cell membrane plays the chemoproection effect.The surface that the impermeability protective agent is adsorbed on microbial cell forms the higher protective layer of viscosity and stops the growth of ice crystal by the viscosity that increases solution, forms impalpable structure near cell.The cryoprotector of various mechanism is united the maintain significant effect of use for viable count and bacterial strain function, is the key of the efficient deep cooling bifidobacterium fermentation of preparation agent.
Skimmed milk is a kind of good buffer system; itself containing amounts of protein, carbohydrate etc. is a kind of desirable protective medium; can stop cellular damage by the protection cell membrane stability; protein wherein also can be used as tunicle and pair cell plays a protective role, and adds its protection effect of enhancing that different protective agents also can be in various degree in skimmed milk.
Carbohydrate its role is to as semi-permeable protective agent, by forming full-bodied glass state material protein is played a protective role in refrigerating process; Prevent the sex change of protein as the substitute of water with protein combination.Except the 26S Proteasome Structure and Function of protected protein matter, trehalose and other carbohydrate can also reduce the phase transition temperature of cell membrane by the hydrophilic head that replaces lipid, thereby can prevent the phase transformation of cell membrane and the cell membrane that causes thus run-down.
Thereby the cryoprotection effect of glutamic acid is the carboxyl combination of protectant amino and microprotein plays the stabilization to protein, and glutamic acid also may improve the fixedly moisture in the cell.
Bifidobacterium is the strictly anaerobic bacterium leavening agent, and coercing of oxygen understood the survival and the vigor that influence thalline in the preparation process, and the interpolation antioxidant keeps having remarkable result to bacterium number and the vigor of Bifidobacterium in the composition of leavening.
Summary of the invention
The objective of the invention is to prepare the agent of a kind of bifidobacterium deep frozen fermented and effective cryoprotector thereof.It is low to solve in the conventional bifidobacterium fermentation agent preparation process survival rate, the problem that the bacterial strain vigor descends.
The protective agent weight fraction that the present invention is used for the freezing direct vat inoculation of bifidobacterium deep consists of: skimmed milk 6%-16%; Trehalose 2.5%-7.5%; Fructose 2.5%-7.5%; Glycerine 1%-4%; Vc0.01%-0.1%; L-Glu0.01%-0.1%.
The freezing direct vat inoculation of described bifidobacterium deep preferably is grouped into by following weight: skimmed milk 8%; Trehalose 5%; Fructose 5%; Glycerine 3%; Vc0.05%; L-Glu0.05%.
The freezing direct vat inoculation of bifidobacterium deep of the present invention's preparation is after the Bifidobacterium thalline adds above-mentioned protective agent, obtains after-80 ℃ of deep layers are freezing.
Above-mentioned protective agent fully mixes freezing in-80 ℃ rapidly according to the 20/1-1/5 adding of bifid bar original bacteria liquid volume.
Described Bifidobacterium thalline uses Dried Corn Steep Liquor Powder culture medium or NPNL culture medium or MRS to cultivate based on 37 ℃, and the permanent pH anaerobism of pH6.5-pH7.0 is cultivated the 15-24h cultivation and obtained.
Described thalline is by 4000rpm-8000rpm, and 8-20min is centrifugal to be obtained.
Described Dried Corn Steep Liquor Powder culture medium is made by following component: Dried Corn Steep Liquor Powder 55g/L, glucose 10g/L, L cysteine hydrochloride 0.05g/L, A liquid 10mL/L, B liquid 5ml/L.
Described A liquid is made up of following component: KH 2PO 4100g/L, K 2HPO 4100g/L.
Described B liquid is made up of following component: MgSO 4.7H 2O 2g/L,, FeSO 4.7H 2O 2g/L, Nacl 2g/L, MnSO 41.3g/L.
Described NPNL culture medium is made up of following component: ox meat extract powder 3g/L, peptone 10g/L, tryptone 5g/L plants peptone 3g/L, Yeast diffusion juice 5g/L, beef liver soaks powder 10g/L, glucose 10g/L, soluble starch 0.5g/L, solution A 10ml/L, solution B 5ml/L, Tween 80 1g/L, CYSTEAMINE HCL hydrochlorate 0.5g/L.
Described A liquid is made up of following component: KH 2PO 4100g/L, K 2HPO 4100g/L.
Described B liquid is made up of following component: MgSO 4.7H 2O 2g/L,, FeSO 4.7H 2O 2g/L,, NaCl 2g/L, MnSO 41.3g/L.
Described MRS culture medium is made up of following component: peptone 10g/L, beef extract 10g/L, dusty yeast 5g/L, glucose 20g/L sodium acetate 5g/L, dibasic ammonium citrate 2g/L, KH 2PO 42g/, Tween 80 1g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.28g/L, CYSTEAMINE HCL hydrochlorate 0.5g/L.
Experiment shows that Bifidobacterium prepares the deep layer cryoprotector under protective agent effect of the present invention, and freezing back bacterium number keeps stable, and titratable acidity is measured the bacterial strain vigor and kept good, and duration of storage has good stability, and is used for Yoghourt fermentation product viable count and reaches 10 7More than the cfu/mL, reach the direct vat inoculation requirement.
Description of drawings
Fig. 1: Bifidobacterium BBMN68 viable bacteria logarithm value changes in Yoghourt fermentation and the storage.
Fig. 2: Bifidobacterium BBMN23 viable bacteria logarithm value changes in Yoghourt fermentation and the storage.
Fig. 3: Bifidobacterium BBMN01 viable bacteria logarithm value changes in Yoghourt fermentation and the storage.
The specific embodiment
Embodiment 1-24
(1) strain culturing: the bifidobacterium longum BBMN68 (being preserved in China Agricultural University) of preservation gone down to posterity to rejuvenate for twice, and microscopy nutrient solution strain morphology will activate bacterial strain with 10 7Cfu/mL is inoculated in the Dried Corn Steep Liquor Powder culture medium, and permanent pH7.0 anaerobism is cultivated 24h.
The Dried Corn Steep Liquor Powder culture medium is made by following component: Dried Corn Steep Liquor Powder 55g/L, glucose 10g/L, L cysteine hydrochloride 0.05g/L, A liquid 10mL/L, B liquid 5ml/L.
Described A liquid is made up of following component: KH 2PO 4100g/L, K 2HPO 4100g/L.
Described B liquid is made up of following component: MgSO 4.7H 2O 2g/L, FeSO 4.7H 2O 2g/L, Nacl 2g/L, MnSO 41.3g/L.
(2) collect thalline: (1) is obtained bacterium liquid 4000rpm, and the centrifugal thalline that obtains of 10min is collected bacterium mud.
(3) add protective agent:
The bifidobacterium deep cryoprotector consists of:
Table 1
With the thalline of collecting, add the described protective agent of stating of table 1 of 1/10 original bacteria liquid volume, stirring and evenly mixing obtains concentrating thallus suspension liquid, rapidly with-80 ℃ of freezings.
(4) bacterial strain vitality test
Frozen concentrate is inoculated in 12% skimmed milk, and inoculum concentration 1% (V/V) is cultivated 7h for 37 ℃, measures titratable acidity (° T).
Described titratable acidity assay method is: get the above-mentioned acidified milk of 10mL, use the 20mL distilled water diluting, add 2 1% phenolphthalein, use the 0.1mol/LNaOH titration, the NaOH milliliter number that consumes multiply by 10, and with the required 0.1mol/LNaOH milliliter number of 100mL acidified milk, consuming 1mL is 1 ° of T in being.The vigor that can reflect bacterial strain.
Above-mentioned table 1 is listed result show, freezing back bacterial strain vigor keeps good under the protective agent effect, and optimal result is that mass fraction is 8% skimmed milk; Mass fraction is 5% trehalose; Mass fraction is 5% fructose; Volume fraction is 3% glycerine; Mass fraction is 0.05% Vc; Mass fraction is 0.05% L-Glu.
The preparation and the application of embodiment 25 bifidobacterium deep frozen fermented agent
(1) strain culturing: the bifidobacterium longum BBMN68 (being preserved in China Agricultural University) of preservation gone down to posterity to rejuvenate for twice, and microscopy nutrient solution strain morphology will activate bacterial strain with 10 7Cfu/mL is inoculated in the Dried Corn Steep Liquor Powder culture medium, and permanent pH7.0 anaerobism is cultivated 24h.
The Dried Corn Steep Liquor Powder culture medium is made by following component: Dried Corn Steep Liquor Powder 55g/L, glucose 10g/L, L cysteine hydrochloride 0.05g/L, A liquid 10mL/L, B liquid 5ml/L.
Described A liquid is made up of following component: KH 2PO 4100g/L, K 2HPO 4100g/L.
Described B liquid is made up of following component: MgSO 4.7H 2O 2g/L,, FeSO 4.7H 2O 2g/L,, Nacl 2g/L, MnSO 41.3g/L.
(2) collect thalline: (1) is obtained bacterium liquid 4000rpm, and the centrifugal thalline that obtains of 10min is collected bacterium mud.
(3) add protective agent:
It is described that the bifidobacterium deep cryoprotector consists of embodiment 24.
With the thalline of collecting, add the above-mentioned protective agent of 1/10 original bacteria liquid volume, stirring and evenly mixing obtains concentrating thallus suspension liquid, rapidly with-80 ℃ of freezings.
(4) leavening is used for Yoghourt fermentation
12% skimmed milk is inoculated in above-mentioned leavening that makes and traditional zymotic agent simultaneously, and 42 ℃ ferment to sour milk and solidify, and Bifidobacterium bacterium number between preservation term is measured in 4 ℃ of preservations.Fig. 1 is that sour milk duration of storage viable count changes.
The bifidobacteria viable bacteria counting uses Bifidobacterium selectivity counting culture medium in the above-mentioned sour milk.
Bifidobacterium selectivity counting culture medium consists of: ox meat extract powder 3g/L, peptone 10g/L, tryptone 5g/L, plant peptone 3g/L, Yeast diffusion juice 5g/L, beef liver soak powder 10g/L, glucose 10g/L, soluble starch 0.5g/L, solution A 10ml/L, solution B 5ml/L, C liquid 40ml/L, Tween 80 1g/L, CYSTEAMINE HCL hydrochlorate 0.5/L, agar 15g/L.
Described A liquid is made up of following component: KH 2PO 4100g/L, K 2HPO 4100g/L.
Described B liquid is made up of following component: MgSO 4.7H 2O 2g/L,, FeSO 4.7H 2O 2g/L,, Nacl 2g/L, MnSO 41.3g/L.
Described solution C consists of: paromomycin sulfate 100mg, and neomycinsulphate 400mg, sodium propionate 15g, lithium chloride 3g places the 40ml sterilized water, and 0.45 μ m filtering with microporous membrane is now with the current.
The preparation and the application of embodiment 26 bifidobacterium deep frozen fermented agent
(1) strain culturing: the bifidobacterium adolescentis BBMN23 (being preserved in China Agricultural University) of preservation gone down to posterity to rejuvenate for twice, and microscopy nutrient solution strain morphology will activate bacterial strain with 10 7Cfu/mL is inoculated in the MRS culture medium, and permanent pH6.5 anaerobism cultivates 18.
The MRS culture medium is made up of following component: peptone 10g/L, beef extract 10g/L, dusty yeast 5g/L, glucose 20g/L sodium acetate 5g/L, dibasic ammonium citrate 2g/L, KH 2PO 42g/, Tween 80 1g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.28g/L, CYSTEAMINE HCL hydrochlorate 0.5g/L.
(2) collect thalline: (1) is obtained bacterium liquid 4500rpm, and the centrifugal thalline that obtains of 8min is collected bacterium mud.
(3) add protective agent:
It is described that the bifidobacterium deep cryoprotector consists of embodiment 10.
With the thalline of collecting, add the above-mentioned protective agent of 1/20 original bacteria liquid volume, stirring and evenly mixing obtains concentrating thallus suspension liquid, rapidly with-80 ℃ of freezings.
(4) leavening is used for Yoghourt fermentation
12% skimmed milk is inoculated in above-mentioned leavening that makes and traditional zymotic agent simultaneously, and 42 ℃ ferment to sour milk and solidify, and Bifidobacterium bacterium number between preservation term is measured in 4 ℃ of preservations.Fig. 2 is that sour milk duration of storage viable count changes.
The bifidobacteria viable bacteria counting uses Bifidobacterium selectivity counting culture medium in the above-mentioned sour milk.
Bifidobacterium selectivity counting culture medium consists of: ox meat extract powder 3g/L, peptone 10g/L, tryptone 5g/L, plant peptone 3g/L, Yeast diffusion juice 5g/L, beef liver soak powder 10g/L, glucose 10g/L, soluble starch 0.5g/L, solution A 10ml/L, solution B 5ml/L, C liquid 40ml/L, Tween 80 1g/L, CYSTEAMINE HCL hydrochlorate 0.5/L, agar 15g/L.
Described A liquid is made up of following component: KH 2PO 4100g/L, K 2HPO 4100g/L.
Described B liquid is made up of following component: MgSO 4.7H 2O 2g/L,, FeSO 4.7H 2O 2g/L,, Nacl 2g/L, MnSO 41.3g/L.
Described solution C consists of: paromomycin sulfate 100mg, and neomycinsulphate 400mg, sodium propionate 15g, lithium chloride 3g places the 40ml sterilized water, and 0.45 μ m filtering with microporous membrane is now with the current.
The preparation and the application of embodiment 27 bifidobacterium deep frozen fermented agent
(1) strain culturing: the animal bifidobacteria BBMN01 (being preserved in China Agricultural University) of preservation gone down to posterity to rejuvenate for twice, and microscopy nutrient solution strain morphology will activate bacterial strain with 10 7Cfu/mL is inoculated in the NPNL culture medium, and permanent pH6.8 anaerobism cultivates 12.
Described NPNL culture medium is made up of following component: ox meat extract powder 3g/L, peptone 10g/L, tryptone 5g/L plants peptone 3g/L, Yeast diffusion juice 5g/L, beef liver soaks powder 10g/L, glucose 10g/L, soluble starch 0.5g/L, solution A 10ml/L, solution B 5ml/L, Tween 80 1g/L, CYSTEAMINE HCL hydrochlorate 0.5g/L.
Described A liquid is made up of following component: KH 2PO 4100g/L, K 2HPO 4100g/L.
Described B liquid is made up of following component: MgSO 4.7H 2O 2g/L, FeSO 4.7H 2O 2g/L, Nacl 2g/L MnSO 41.3g/L.
(2) collect thalline: (1) is obtained bacterium liquid 5000rpm, and the centrifugal thalline that obtains of 15min is collected bacterium mud.
(3) add protective agent:
It is described that the bifidobacterium deep cryoprotector consists of embodiment 18.
With the thalline of collecting, add the above-mentioned protective agent of 1/5 original bacteria liquid volume, stirring and evenly mixing obtains concentrating thallus suspension liquid, rapidly with-80 ℃ of freezings.
(4) leavening is used for Yoghourt fermentation
12% skimmed milk is inoculated in above-mentioned leavening that makes and traditional zymotic agent simultaneously, and 42 ℃ ferment to sour milk and solidify, and Bifidobacterium bacterium number between preservation term is measured in 4 ℃ of preservations.Fig. 3 is that sour milk duration of storage viable count changes.
The bifidobacteria viable bacteria counting uses Bifidobacterium selectivity counting culture medium in the above-mentioned sour milk.
Bifidobacterium selectivity counting culture medium consists of: ox meat extract powder 3g/L, peptone 10g/L, tryptone 5g/L, plant peptone 3g/L, Yeast diffusion juice 5g/L, beef liver soak powder 10g/L, glucose 10g/L, soluble starch 0.5g/L, solution A 10ml/L, solution B 5ml/L, C liquid 40ml/L, Tween 80 1g/L, CYSTEAMINE HCL hydrochlorate 0.5/L, agar 15g/L.
Described A liquid is made up of following component: KH 2PO 4100g/L, K 2HPO 4100g/L.
Described B liquid is made up of following component: MgSO 4.7H 2O 2g/L,, FeSO 4.7H 2O 2g/L,, Nacl 2g/L, MnSO 41.3g/L.
Described solution C consists of: paromomycin sulfate 100mg, and neomycinsulphate 400mg, sodium propionate 15g, lithium chloride 3g places the 40ml sterilized water, and 0.45 μ m filtering with microporous membrane is now with the current.

Claims (8)

1, a kind of composite protectant that is used to prepare the agent of bifidobacterium deep frozen fermented is characterized in that this protective agent is made up of following components by part by weight: skimmed milk 6%-16%; Trehalose 2.5%-7.5%; Fructose 2.5%-7.5%; Glycerine 1%-4%; Vc0.01%-0.1%; L-Glu0.01%-0.1%; All the other are water.
2, the composite protectant that is used to prepare the agent of bifidobacterium deep frozen fermented according to claim 1 is characterized in that this protective agent is made up of following components by part by weight: skimmed milk 8%; Trehalose 5%; Fructose 5%; Glycerine 3%; Vc0.05%; L-Glu0.05%; All the other are water.
3, the freezing direct vat inoculation of a kind of bifidobacterium deep is characterized in that in the Bifidobacterium thalline adding as behind arbitrary described composite protectant among the claim 1-2, obtains through deep layer is freezing.
4, the freezing direct vat inoculation of bifidobacterium deep according to claim 3 is characterized in that, its preparation method is:
(1) Bifidobacterium is inoculated in Dried Corn Steep Liquor Powder culture medium or NPNL culture medium or the MRS culture medium, in 37 ℃, anaerobism is cultivated 15-24h under the pH6.5-pH7.0, and is centrifugal, 4000rpm-8000rpm, and 8-20min collects thalline;
(2) thalline of collecting is added the above-mentioned composite protectant of 1/20-1/5 original bacteria liquid volume, stirring and evenly mixing obtains concentrating thallus suspension liquid, rapidly in-80 ℃ of following freezings.
5, the freezing direct vat inoculation of bifidobacterium deep according to claim 4, it is characterized in that: described Dried Corn Steep Liquor Powder culture medium is made by following component: Dried Corn Steep Liquor Powder 55g/L, glucose 10g/L, L cysteine hydrochloride 0.05g/L, A liquid 10mL/L, B liquid 5ml/L;
Described A liquid is made up of following component: KH 2PO 4100g/L, K 2HPO 4100g/L;
Described B liquid is made up of following component: MgSO 4.7H 2O 2g/L,, FeSO 4.7H 2O 2g/L,, Nacl 2g/L,, MnSO 41.3g/L.
6, the freezing direct vat inoculation of bifidobacterium deep according to claim 4 is characterized in that: described NPNL culture medium is made up of following component: ox meat extract powder 3g/L, peptone 10g/L, tryptone 5g/L, plant peptone 3g/L, Yeast diffusion juice 5g/L, beef liver soak powder 10g/L, glucose 10g/L, soluble starch 0.5g/L, solution A 10ml/L, solution B 5ml/L, Tween 80 1g/L, CYSTEAMINE HCL hydrochlorate 0.5g/L.
Described A liquid is made up of following component: KH 2PO 4100g/L, K 2HPO 4100g/L;
Described B liquid is made up of following component: MgSO 4.7H 2O 2g/L, FeSO 4.7H 2O 2g/L, Nacl 2g/L, MnSO 41.3g/L.
7, the freezing direct vat inoculation of bifidobacterium deep according to claim 4 is characterized in that: described MRS culture medium is made up of following component: peptone 10g/L, beef extract 10g/L, dusty yeast 5g/L, glucose 20g/L, sodium acetate 5g/L, dibasic ammonium citrate 2g/L, KH 2PO 42g/, Tween 80 1g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.28g/L, CYSTEAMINE HCL hydrochlorate 0.5g/L.
8, as the application of the claim 3-7 freezing direct vat inoculation of each described bifidobacterium deep in fermented yoghourt.
CN 200910136515 2009-05-06 2009-05-06 Bifidobacterium deep freezing direct vat starter culture and composite cryoprotectant thereof Active CN101606552B (en)

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CN102358884A (en) * 2011-10-13 2012-02-22 陕西农产品加工技术研究院 Bifidobacterium bifidum lyophilization composite protection agent and application method thereof
CN102408993A (en) * 2011-11-23 2012-04-11 陕西农产品加工技术研究院 Bifidobacterium bifidum anti-freeze culture medium and application method thereof
CN102429016A (en) * 2011-12-28 2012-05-02 石家庄市兄弟伊兰食品配料有限公司 Deep-frozen yoghurt starter and preparation method thereof
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CN102334605A (en) * 2011-07-15 2012-02-01 天津科技大学 Direct vat set clostridium butyricum starter and preparation method thereof
CN102358884A (en) * 2011-10-13 2012-02-22 陕西农产品加工技术研究院 Bifidobacterium bifidum lyophilization composite protection agent and application method thereof
CN102408993A (en) * 2011-11-23 2012-04-11 陕西农产品加工技术研究院 Bifidobacterium bifidum anti-freeze culture medium and application method thereof
CN102408993B (en) * 2011-11-23 2013-06-19 陕西农产品加工技术研究院 Bifidobacterium bifidum anti-freeze culture medium and application method thereof
CN102429016A (en) * 2011-12-28 2012-05-02 石家庄市兄弟伊兰食品配料有限公司 Deep-frozen yoghurt starter and preparation method thereof
CN105961587A (en) * 2016-04-28 2016-09-28 陈鹤 Preparation method and application of selenium-enriched yoghourt
CN107772477A (en) * 2017-11-27 2018-03-09 内蒙古普泽生物制品有限责任公司 A kind of compound probiotic powder preparation rich in selenium and preparation method thereof

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