CN103627637A - Preparation method for pediococcus acidilactici strain freeze-drying preparation - Google Patents
Preparation method for pediococcus acidilactici strain freeze-drying preparation Download PDFInfo
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Abstract
The invention discloses a preparation method for a pediococcus acidilactici strain freeze-drying preparation. The preparation method comprises the following steps: firstly, activating a pediococcus acidilactici strain PA003 strain in a liquid MRS culture medium and utilizing a growth culture medium for cultivation so as to optimize the activity and survivability; then utilizing the centrifuging method of a refrigerated centrifuge to collect the thallus, suspending the thallus in a sterilized freeze-drying preservative culture medium after washing, freezing the solution by the progressive freezing method, and preparing for freeze drying after the solution is frozen; finally, placing the frozen sample in a freeze dryer for final freeze drying; placing the finally obtained freeze-drying preparation at the room temperature in a sealed manner and in the absence of light for storage. The pediococcus acidilactici strain prepared by the preparation method has the survival rate up to 81.96%, has the fermentation property of lactic acid bacteria after renaturation while keeping the bacteriostatic activity to mononucleosis Listeriosis, is suitable for large-scale industrial production after development, and has great potential when serving as a biological preservative or a feed additive in the industries of foods and feed.
Description
Technical field
The present invention relates to a kind of preparation method of freeze-dried preparation, particularly a kind of preparation method of Pedicoccus acidilacticii strain freeze-dried preparation and using method.
Background technology
Since using milk-acid bacteria to produce leavened food, the mankind at least had the history of more than 4000 year, mainly for the production of cultured milk prod, fermented vegetables and fermented meat prods etc.Lactic acid bacteria fermented food unique flavor, has prebiotic effect to human and animal, and even some bacterial classification has preservative activity, and these milk-acid bacterias are worldwide liked deeply.At present, pediococcus acidilactici can be given tart flavour and the fragrance that food is soft, improves quality and the nutrition of food, also have antibacterial, reduce cholesterol, maintain micro-ecological environment, antitumor, resistance is different and the important biomolecule such as strengthening immunity is learned function.It uses in multinational food as fermented bacterium is a large amount of, is the profitable strain that enjoys people to like and accept.In addition, its significant fungistatic effect, makes pediococcus acidilactici hide huge using value and wide application prospect.
At present for the research of pediococcus acidilactici bacteriostatic action, be mostly all devoted in the research of pediocin both at home and abroad, mainly by the whole bag of tricks, to obtain the pediocin of efficient high yield, as: optimize and cultivate and various condition, the methods such as genetic engineering technique obtain the bacteriocin of efficient high yield.But as the genus lactubacillus pediococcus acidilactici of human body probiotics, the research of the use of its viable bacteria and viable bacteria bacteriostatic test report is but rarely seen at home and abroad.The present invention's Pedicoccus acidilacticii strain used (pediococcus acidilatici) PA003 is the peculiar bacterial classification of the Zhou Zhijiang of chemical engineering institute of University Of Tianjin professor research department, (open ，Ke Cong University Of Tianjin's chemical engineering institute's department of food science food biotechnology research department obtains in patent CN102115720B) that separation and purification obtains from a kind of Fermented Cabbage (northeast acid Chinese cabbage) at first.This pediococcus acidilactici PA003 can generate pediocin by fermenting carbohydrate---and be the natural biological antiseptic agent of a peptide species, belong to thermally-stabilised polypeptide bacteriocin, can suppress the gram-positive microorganism in very wide scope, there is broad spectrum antibacterial.And this sheet coccus is a kind for lactic-acid-bacterium, some food can ferment, possess again anti-corrosion function, people can also be directly edible, and when it uses as crude substance, the current controversial transgenosis material of ratio and synthetic material or compound more can obtain popular recognition and acceptance.
There is not yet at present the report of pediococcus acidilactici freeze-dried preparation research both at home and abroad, therefore, for the stable Long-term Storage of freeze-dried preparation energy that guarantees to produce, and after renaturation, there is higher survival rate and more stable bacteriostatic activity, and in operation, storage, sells, convenient feasible in use, just must conscientiously study for the various influence factors of preparing Pedicoccus acidilacticii strain freeze-dried preparation.And the angle combining from technology and business, the growth of bacterial classification and carry out lyophilize, renaturation, lyophilize after the impact that is subject in Long-term Storage process be all the important research core that this freeze-dried preparation has high reactivity and failure-survival capability after renaturation.And main influence factor in above-mentioned research process is divided into: intrinsic internal factor, comprising: the composition of the size of cell, gene, cell walls and cytolemma; Growth factor, comprising: can compatible Solute accumulation thing, the generation of exocellular polysaccharide is, the variation of cytolemma; Semilethal is processed; Dehydrated medium, comprising: skimmed milk powder, perviousness compound, semipermeability compound, impervious compound; Storage and renaturation, storage comprises: oxygen, moisture, temperature, renaturation comprises: the pH value of renaturation solution, temperature, capacity, nutritive substance composition and content, energy matter composition and content.Therefore, for different affecting factors, for kinds different in Pseudomonas, there is the different results that affects, the problem that optimum condition between not of the same race also may not occur mutually on an equal basis, add pediococcus acidilactici PA003 bacterial strain after being made as freeze-dried preparation, in later stage use and standing storage process, must possess bacteriostatic activity, must prepare the three large influence factors that affect pediococcus acidilactici PA003 freeze-dried preparation survival rate and bacteriostatic activity in freeze-dried preparation and renaturation process (that is: growth medium to freeze-drying so, freezing drying protective agent, renaturation condition) study one by one.Develop the best approach of preparing Pedicoccus acidilacticii strain freeze-dried preparation.Finally can allow Pedicoccus acidilacticii strain freeze-dried preparation effectively, reasonably, as natural biological antiseptic agent, starter, fodder additives, apply in various food and related industries easily.Thereby it is natural, harmless, pollution-free to show pediococcus acidilactici PA003 freeze-dried preparation, Fermentation Function and the biological antiseptic effect of safety.Above all research inventions are all researching value and the meanings that tool is very high.
Summary of the invention
The preparation method who the object of this invention is to provide a kind of Pedicoccus acidilacticii strain freeze-dried preparation, fills up the blank of domestic and international pediococcus acidilactici freeze-dried preparation, solves the problem that Pedicoccus acidilacticii strain is applied to large-scale industrial production difficulty.
The present invention is achieved through the following technical solutions:
A preparation method for Pedicoccus acidilacticii strain freeze-dried preparation, comprises the steps:
(1) activation pediococcus acidilactici PA003 bacterial classification, in liquid MRS substratum, utilizes growth medium to cultivate, and makes its activity and viability best;
(2) utilize refrigerated centrifuge centrifuging to collect thalline, after washing, thalline is suspended in sterilized freezing drying protective agent substratum, by progressive freezing, solution is freezing, after freezing, it prepares to carry out lyophilize;
(3) sample that will freeze is positioned over and in freeze drier, carries out lyophilize; The freeze-dried preparation sealing lucifuge finally obtaining is positioned under room temperature preserves.
In described step (1), growth medium is that 10% (w/v) skimmed milk is as the MRS-glucose-fructose-sucrose medium of solution matrix.Described MRS-glucose-fructose-sucrose medium formula refers to glucose 0.66%, fructose 0.66%, and sucrose 0.66% replaces the glucose 2% of MRS culture medium prescription.
In described step (1), inoculum size is 1%, 37 ℃ of culture temperature, incubation time 14h.
Described step (2) freezing drying protective agent substratum is 10% (w/v) skimmed milk solution of 5% sucrose.
Described step (2) refrigerated centrifuge rotating speed 10000rpm, centrifugal 10min at 4 ℃.
Described step (3) lyophilize temperature-53 ℃, sublimation drying 24h.
A using method for Pedicoccus acidilacticii strain freeze-dried preparation, utilizes physiological saline as renaturation solution, at 22 ℃, to carry out renaturation 30min during use.
Beneficial effect of the present invention is: the present invention utilizes the MRS substratum after improvement, and it is best that the growth of this pediococcus acidilactici can reach.The pediococcus acidilactici survival rate of preparing with the present invention is up to 81.96%, when thering is the fermentation character of genus lactubacillus after renaturation, still keep the bacteriostatic activity to monokaryon increase disease listeria spp, be suitable for developing rear large-scale industrial production, in grocery trade and feed industry, as biological preservative or fodder additives, use and there are very large potentiality.
Accompanying drawing explanation
The different carbohydrates of Fig. 1 (matrix is distilled water) survival rate of pediococcus acidilactici PA003 freeze-dried preparation during as freezing drying protective agent under different concns separately;
Different carbohydrate (matrix the is 10%(w/v) skimmed milks of Fig. 2) survival rate of pediococcus acidilactici PA003 freeze-dried preparation during as freezing drying protective agent under different concns separately;
The different high polymers of Fig. 3 (matrix is distilled water) survival rate of pediococcus acidilactici PA003 freeze-dried preparation during as freezing drying protective agent under different concns separately;
Different high polymer (matrix the is 10%(w/v) skimmed milks of Fig. 4) survival rate of pediococcus acidilactici PA003 freeze-dried preparation during as freezing drying protective agent under different concns separately;
The different polyvalent alcohols of Fig. 5 (matrix is distilled water) survival rate of pediococcus acidilactici PA003 freeze-dried preparation during as freezing drying protective agent under different concns separately;
Different polyvalent alcohol (matrix the is 10%(w/v) skimmed milks of Fig. 6) survival rate of pediococcus acidilactici PA003 freeze-dried preparation during as freezing drying protective agent under different concns separately;
Other compounds of Fig. 7 (matrix is distilled water) survival rate of pediococcus acidilactici PA003 freeze-dried preparation during as freezing drying protective agent under different concns separately;
Other compounds of Fig. 8 (matrix is 10%(w/v) skimmed milk) survival rate of pediococcus acidilactici PA003 freeze-dried preparation during as freezing drying protective agent under different concns separately;
The impact of Fig. 9 a renaturation solution on pediococcus acidilactici PA003 freeze-dried preparation survival rate;
The impact of Fig. 9 b renaturation solution on pediococcus acidilactici PA003 freeze-dried preparation survival rate;
The impact of Figure 10 renaturation temperature on pediococcus acidilactici PA003 freeze-dried preparation survival rate;
The impact of Figure 11 renaturation time on pediococcus acidilactici PA003 freeze-dried preparation survival rate;
Figure 12 Pedicoccus acidilacticii strain freeze-dried preparation suppresses the bacteriostatic activity of monokaryon increase disease listeria spp CVCC1595.
Embodiment
Below in conjunction with accompanying drawing, the present invention is elaborated.
Prepare Pedicoccus acidilacticii strain freeze-dried preparation
(1) by being stored in the inoculum size by 1% of the pediococcus acidilactici PA003 bacterial classification on MRS inclined-plane at 4 ℃, be inoculated in liquid MRS substratum, after inoculation, at 37 ℃, cultivate 14h, liquid MRS bacteria suspension is inoculated in liquid growth medium by 1% inoculum size again, is positioned at 37 ℃ and cultivates 14h.
(2) utilize refrigerated centrifuge centrifugal (10000rpm, 4 ℃, 10min), abandon supernatant, collect solid precipitation (thalline); By the sterilized Ringer ' s of same volume solution washed twice, abandon equally supernatant, collect solid precipitation (thalline); After this thalline is suspended in sterilized freezing drying protective agent substratum, in 25 ℃ of standing 1h, standing 1h again at 4 ℃ afterwards, be finally positioned over-20 ℃ standing, after it freezes, prepare to carry out lyophilize.
(3) sample that will freeze is positioned over (53 ℃) in freeze drier and carries out lyophilize 24h; The freeze-dried preparation sealing lucifuge obtaining is positioned under room temperature preserves.
Embodiment 2
The survival rate of Pedicoccus acidilacticii strain freeze-dried preparation
(1) live bacterial count of Pedicoccus acidilacticii strain before lyophilize: after above-mentioned 25 ℃ of standing 1h; from freezing drying protective agent substratum, respectively taking out this protectant bacteria suspension of 1mL joins in the sterilized physiological saline of 9mL; it,, after suitable dilution, is reached to 10
-6, 10
-7time, the bacteria suspension under this dilution gradient respectively to be got to 100 μ L and be added on MRS solid medium plate and carry out bacterium colony coating (adjacent two gradients are respectively coated with 3 plates), coated plate is positioned at 37 ℃ and cultivates 48h, carries out afterwards plate count.The colony number of survival represents with cfu/mL.
(2) after lyophilize, with the renaturation solution of same volume, freeze-dried preparation is carried out to renaturation at once, make it under renaturation condition, form renaturation bacteria suspension.From this renaturation bacteria suspension, respectively take out 1mL bacteria suspension and join in the sterilized physiological saline of 9mL, it is reached to 10 after suitable dilution
-6, 10
-7, the bacteria suspension under this dilution gradient to be got to 100 μ L and be added on MRS solid medium plate and carry out bacterium colony coating (adjacent two gradients are respectively coated with 3 plates), coated plate is positioned at 37 ℃, carries out enumeration after cultivating 48h.The colony number of survival represents with cfu/mL.
(3) survival rate is by (No) before lyophilize, on the MRS solid culture ware that (Nf) is corresponding afterwards, the ratio of colony number represents: the numerical value of twice parallel test of survival rate (V) %=(Nf/No) * 100 utilizes Software SPSS 18.0 statistics softwares to process, general linear model for variance analysis (GLM) variance analysis; Multiple comparisons utilizes duncan's new multiple range method analysis.
Embodiment 3
The bacteriostatic activity of Pedicoccus acidilacticii strain freeze-dried preparation
Pediococcus acidilactici PA003 after lyophilize is inoculated in MRS solid culture ware from renaturation solution, is positioned at 37 ℃ and cultivates 24h, treat that it grows the visible bacterium colony of naked eyes; In the semi-solid TSYEB of the 5mL of 50 ℃ of left and right, access 1 μ L monokaryon increase disease listeria spp CVCC1595, after being mixed, pour in the above-mentioned MRS solid culture ware that has grown the visible bacterium colony of naked eyes, this layer of semisolid medium is laid on solid MRS substratum, afterwards this flat board is positioned at 37 ℃ and cultivates 12h, observe whether in pediococcus acidilactici periphery of bacterial colonies, occur inhibition zone.(Figure 12)
By Figure 12, found out, the freeze-dried preparation of producing carries out after renaturation, and the pediococcus acidilactici PA003 of survival is carried out to bacteriostatic test, finds that this periphery of bacterial colonies can see inhibition zone clearly, shows that this freeze-dried preparation still has stable bacteriostatic activity.
Embodiment 4
Optimize and prepare each influence factor in Pedicoccus acidilacticii strain freeze-dried preparation preparation process
(1) in preparing freeze-dried preparation process by utilize two factors to test comprehensively, growth medium to Pedicoccus acidilacticii strain is optimized, the growth medium of finally selecting best preparation lyophilize bacteria preparation, 10% (w/v) skimmed milk is as the MRS-glucose-fructose-sucrose medium of solution matrix.(table 1,2,3,4)
The different growth mediums of table 1
The impact of the different growth mediums of table 2 on pediococcus acidilactici PA003 freeze-dried preparation survival rate
Numerical value is mean value.
*p<0.05,
*p<0.01, NS is not remarkable.
Letter representation after numerical value utilizes duncan's new multiple range method to carry out the result (p<0.05) of multiple comparisons.
Table 3 affects the grown cultures based substrate of pediococcus acidilactici PA003 freeze-dried preparation survival rate, different grown cultures based formulas and interactive variance analysis between them
*p<0.05,
*p<0.01, NS is not remarkable.
Table 4 affects the multiple comparisons (employing duncan's new multiple range method) between the different grown cultures based formulas of growth medium of pediococcus acidilactici PA003 freeze-dried preparation survival rate
Letter representation utilizes duncan's new multiple range method to carry out the result of multiple comparisons.
Each culture medium prescription is:
Pediococcus acidilactici MRS culture medium prescription (w/v): dipotassium hydrogen phosphate 0.2%, sodium acetate (anhydrous) 0.5% magnesium sulfate 0.058%, ammonium citrate 0.2%, manganous sulfate 0.025%, glucose 2%, Tryptones 1%, extractum carnis 0.8%, yeast extractive substance 0.5%, tween 80 0.1%, pH6.2-6.5.
MRS-sucrose medium formula (w/v): sucrose 2% replaces the glucose 2% of MRS culture medium prescription
MRS-fructose culture medium prescription (w/v): fructose 2% replaces the glucose 2% of MRS culture medium prescription
MRS-glucose-sucrose medium formula (w/v): glucose 1%, sucrose 1%, the glucose 2% of replacement MRS culture medium prescription.
MRS-glucose-fructose culture medium prescription (w/v): glucose 1%, fructose 1%, the glucose 2% of replacement MRS culture medium prescription.
MRS-sucrose-fructose culture medium prescription (w/v): sucrose 1%, fructose 1% replaces the glucose 2% of MRS culture medium prescription.
MRS-glucose-fructose-sucrose medium formula (w/v): glucose 0.66%, fructose 0.66%, sucrose 0.66%, the glucose 2% of replacement MRS culture medium prescription.
(2) in preparing freeze-dried preparation process; to prepare freezing drying protective agent in Pedicoccus acidilacticii strain freeze-dried preparation process according to carbohydrate; high polymer; polyvalent alcohol; other materials are divided into 4 major parts; by utilizing three factor equilibrium/unbalanced comprehensive experimental designs, after optimization, select best freezing drying protective agent, contain 10% (w/v) skimmed milk solution of 5% sucrose.(Fig. 1-8, table 5-12)
As seen from Figure 1: when the matrix of freezing drying protective agent is distilled water, with 5.00% sucrose and 2.00% lactose, as freezing drying protective agent formula, can make the survival rate of this freeze-dried preparation be greater than 60%; When freezing drying protective agent formula is 5.00% sucrose, the survival rate of this freeze-dried preparation can be up to 78.12%.
As seen from Figure 2: when the matrix of freezing drying protective agent is 10%(w/v) during skimmed milk, with 1.25%, 2.00%, 5.00%, 10.00% sucrose, 5.00% sorbose and 1.25%, 2.00% rhamnosyl, as freezing drying protective agent formula, can make the survival rate of this freeze-dried preparation be greater than 60%; Freezing drying protective agent formula is that 5.00% sucrose can make the survival rate of this freeze-dried preparation up to 81.96%.
As seen from Figure 3: when the matrix of freezing drying protective agent is distilled water, with 10.00% Star Dri 5 and 25.00%, 20.00% skimmed milk, make freezing drying protective agent formula, can make the survival rate of this freeze-dried preparation be greater than 80.00%; When freezing drying protective agent formula is 25.00% skimmed milk, make the survival rate of this freeze-dried preparation up to 83.82%.
As seen from Figure 4: when the matrix of freezing drying protective agent is 10%(w/v) during skimmed milk, only have with 1.00% Zulkovsky starch as freezing drying protective agent formula, just can make the survival rate of this freeze-dried preparation be greater than 50.00%, its value is 51.12%.
As seen from Figure 5: when the matrix of freezing drying protective agent is distilled water, with 1.25% and 5.00%D-sorbyl alcohol as freezing drying protective agent formula, can make the survival rate of pediococcus acidilactici PA003 freeze-dried preparation be greater than 50.00%; When freezing drying protective agent formula is 1.25%D-sorbyl alcohol, can make the survival rate of this freeze-dried preparation up to 86.88%.
As seen from Figure 6: when the matrix of freezing drying protective agent is 10%(w/v) during skimmed milk, only have with 5.00% N.F,USP MANNITOL as freezing drying protective agent formula, just can make the survival rate of this freeze-dried preparation be greater than 50.00%, its value is 59.26%.
As seen from Figure 7: when the matrix of freezing drying protective agent is distilled water, only have with 5.00% Sodium Glutamate (MSG) as freezing drying protective agent formula, just can make the survival rate of this freeze-dried preparation be greater than 50.00%, its value is 54.02%.
As seen from Figure 8: when the matrix of freezing drying protective agent is 10%(w/v) during skimmed milk; Sodium Glutamate with 1.00% (MSG) and 1.00% vitamins C can make the survival rate of this freeze-dried preparation be greater than 50.00% as freezing drying protective agent formula; when wherein freezing drying protective agent formula is 1.00% Sodium Glutamate (MSG), can make the survival rate of this freeze-dried preparation up to 59.91%.
Table 5 affects the freezing drying protective agent matrix of pediococcus acidilactici PA003 freeze-dried preparation survival rate, carbohydrate, the concentration of carbohydrate and interactive variance analysis between them
*p<0.05,
*p<0.01, NS is not remarkable.
Table 6 affects multiple comparisons and the multiple comparisons between the concentration of carbohydrate (employing duncan's new multiple range method) between the freezing drying protective agent carbohydrate of pediococcus acidilactici PA003 freeze-dried preparation survival rate
Letter representation utilizes duncan's new multiple range method to carry out the result of multiple comparisons.
Table 7 affects the freezing drying protective agent matrix of pediococcus acidilactici PA003 freeze-dried preparation survival rate, high polymer, the concentration of high polymer and interactive variance analysis between them
*p<0.05,
*p<0.01, NS is not remarkable.
Table 8 affects multiple comparisons and the multiple comparisons between the concentration of high polymer (employing duncan's new multiple range method) between the freezing drying protective agent high polymer of pediococcus acidilactici PA003 freeze-dried preparation survival rate
Letter representation utilizes duncan's new multiple range method to carry out the result of multiple comparisons.
Table 9 affects the freezing drying protective agent matrix of pediococcus acidilactici PA003 freeze-dried preparation survival rate, polyvalent alcohol, the concentration of polyvalent alcohol and interactive variance analysis between them
*p<0.05,
*p<0.01, NS is not remarkable.
Table 10 affects multiple comparisons and the multiple comparisons between the concentration of polyvalent alcohol (employing duncan's new multiple range method) between the freezing drying protective agent polyvalent alcohol of pediococcus acidilactici PA003 freeze-dried preparation survival rate
Letter representation utilizes duncan's new multiple range method to carry out the result of multiple comparisons.
Table 11 affects the freezing drying protective agent matrix of pediococcus acidilactici PA003 freeze-dried preparation survival rate, different compounds, the concentration of different compounds and interactive variance analysis between them
*p<0.05,
*p<0.01, NS is not remarkable.
Table 12 affects multiple comparisons and the multiple comparisons between the concentration of different compounds (employing duncan's new multiple range method) between the different compounds of freezing drying protective agent of pediococcus acidilactici PA003 freeze-dried preparation survival rate
Letter representation utilizes duncan's new multiple range method to carry out the result of multiple comparisons.
(3) when using Pedicoccus acidilacticii strain freeze-dried preparation, to its renaturation condition: renaturation solution, renaturation time, renaturation temperature are carried out the optimization of three factor three water product orthogonal tests and variance analysis, after optimizing, best renaturation condition is:: renaturation solution is physiological saline, renaturation temperature is 22 ℃, and the renaturation time is 30min.(Fig. 9-11, table 13-20)
By Fig. 9 a Fig. 9 b, can be found out, the descending order of this freeze-dried preparation survival rate is: pH6.5 ultrapure water >1% magnesium sulfate (MgSO
47H
2o) > physiological saline >pH7.0 ultrapure water >pH8.0 ultrapure water >pH6.0 ultrapure water >pH7.5 ultrapure water >2%(w/v) sucrose > meat soup >1% potassium primary phosphate (KH
2pO
4) >Ringer ' solution>10% (w/v) skimmed milk >1% manganous sulfate (MnSO
44H
2o) >MRS+0.1% calcium carbonate (CaCO
3) >pH5.0 ultrapure water.When pH6.5 ultrapure water is during as renaturation solution, can make the survival rate of this freeze-dried preparation up to 70.87%, and 1% magnesium sulfate (MgSO
47H
2o) make the survival rate of this freeze-dried preparation reach 70.65%.
As seen from Figure 10, when renaturation temperature is 25 ℃, make the survival rate of this freeze-dried preparation up to 42.41%, when renaturation temperature is 4 ℃, make the survival rate of this freeze-dried preparation be low to moderate 19.59%.In the time of can significantly observing renaturation temperature rise to 25 ℃ from 4 ℃ from scheming, the survival rate of this freeze-dried preparation rises rapidly, when renaturation temperature is 25 ℃, reach maximum value, while progressively rising to 50 ℃ along with renaturation temperature afterwards, the survival rate of this freeze-dried preparation declines to some extent.
As seen from Figure 11, the renaturation time makes the survival rate of this freeze-dried preparation up to 72.93% while being 30min, and the renaturation time makes the survival rate of this freeze-dried preparation be low to moderate 20.43% while being 120min.From scheming, can significantly observe the renaturation time while extending to 30min by 0min, the survival rate of this freeze-dried preparation changes little, when the renaturation time is 30min, reach maximum value, while extending to 120min gradually along with the renaturation time afterwards, the survival rate of this freeze-dried preparation declines rapidly.
Table 13 renaturation solution is to the variance analysis of pediococcus acidilactici PA003 freeze-dried preparation survival rate and the multiple comparisons between various renaturation solution (employing duncan's new multiple range method)
Numerical value is mean value.
*p<0.05,
*p<0.01, NS is not remarkable.
Letter representation utilizes duncan's new multiple range method to carry out the result of multiple comparisons.
Table 14 renaturation temperature is to the multiple comparisons (employing duncan's new multiple range method) between the variance analysis of pediococcus acidilactici PA003 freeze-dried preparation survival rate and various renaturation temperature
Numerical value is mean value.
*p<0.05,
*p<0.01, NS is not remarkable.
Letter representation utilizes duncan's new multiple range method to carry out the result of multiple comparisons.
The table 15 renaturation time is the multiple comparisons (employing duncan's new multiple range method) between the time to the variance analysis of pediococcus acidilactici PA003 freeze-dried preparation survival rate and various renaturation
Numerical value is mean value.
*p<0.05,
*p<0.01, NS is not remarkable.
Letter representation utilizes duncan's new multiple range method to carry out the result of multiple comparisons.
Table 16 renaturation condition orthogonal test factor table
The two-element list of table 17 renaturation solution (S) and renaturation temperature (T) collocation
The two-element list of table 18 renaturation solution (S) and renaturation time (t) collocation
The two-element list of table 19 renaturation temperature (T) and renaturation time (t) collocation
Table 20 orthogonal test analysis of variance table
*p<0.05,
*p<0.01, NS is not remarkable.
With reference to accompanying drawing, the present invention is schematically described above, this description does not have restricted.The common construction technical staff of this area all can be understood, and in actual applications, in the present invention, some change all may occur the set-up mode of each parts, and other staff also may make similar Design under its enlightenment.Only it is pointed out that otherwise depart from design aim of the present invention, all apparent changes and similar Design thereof, within being all included in protection scope of the present invention.
Claims (8)
1. a preparation method for Pedicoccus acidilacticii strain freeze-dried preparation, is characterized in that, comprises the steps:
(1) activation pediococcus acidilactici PA003 bacterial classification, in liquid MRS substratum, utilizes growth medium to cultivate, and makes its activity and viability best;
(2) utilize refrigerated centrifuge centrifuging to collect thalline, after washing, thalline is suspended in sterilized freezing drying protective agent substratum, by progressive freezing, solution is freezing, after freezing, it prepares to carry out lyophilize;
(3) sample that will freeze is positioned over and in freeze drier, carries out lyophilize; The freeze-dried preparation sealing lucifuge finally obtaining is positioned under room temperature preserves.
2. the preparation method of Pedicoccus acidilacticii strain freeze-dried preparation according to claim 1, is characterized in that, in described step (1), growth medium is that 10% (w/v) skimmed milk is as the MRS-glucose-fructose-sucrose medium of solution matrix.
3. the preparation method of Pedicoccus acidilacticii strain freeze-dried preparation according to claim 2, it is characterized in that, described MRS-glucose-fructose-sucrose medium formula refers to glucose 0.66%, fructose 0.66%, and sucrose 0.66% replaces the glucose 2% of MRS culture medium prescription.
4. the preparation method of Pedicoccus acidilacticii strain freeze-dried preparation according to claim 1, is characterized in that, in described step (1), inoculum size is 1%, 37 ℃ of culture temperature, incubation time 14h.
5. the preparation method of Pedicoccus acidilacticii strain freeze-dried preparation according to claim 1, is characterized in that, described step (2) freezing drying protective agent substratum is 10% (w/v) skimmed milk solution of 5% sucrose.
6. the preparation method of Pedicoccus acidilacticii strain freeze-dried preparation according to claim 1, is characterized in that described step (2) refrigerated centrifuge rotating speed 10000rpm, centrifugal 10min at 4 ℃.
7. the preparation method of Pedicoccus acidilacticii strain freeze-dried preparation according to claim 1, is characterized in that described step (3) lyophilize temperature-53 ℃, sublimation drying 24h.
8. a using method for Pedicoccus acidilacticii strain freeze-dried preparation, is characterized in that, utilizes physiological saline as renaturation solution, at 22 ℃, to carry out renaturation 30min during use.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388345A (en) * | 2014-11-05 | 2015-03-04 | 中国农业科学院哈尔滨兽医研究所 | New strain of pediococcus acidilactici and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104388345A (en) * | 2014-11-05 | 2015-03-04 | 中国农业科学院哈尔滨兽医研究所 | New strain of pediococcus acidilactici and application thereof |
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| CN110279117A (en) * | 2018-03-19 | 2019-09-27 | 医迈霖科技公司 | A kind of probiotics frozen dried food |
| CN110804553A (en) * | 2019-11-21 | 2020-02-18 | 华南农业大学 | Culture medium for improving preservation survival rate of lactic acid bacteria and application thereof |
| CN110804553B (en) * | 2019-11-21 | 2022-08-12 | 华南农业大学 | Culture medium for improving preservation survival rate of lactic acid bacteria and application thereof |
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