CN103013871A - Method for screening bacteriophage insensitive mutants of streptococcus thermophilus - Google Patents
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Abstract
The invention discloses a method for screening bacteriophage insensitive mutants of streptococcus thermophilus, and relates to a streptococcus thermophilus genetic breeding technique in fermented dairy products industry. The method comprises the following steps: step (1), primarily culturing sensitive bacterial strains and concentrating thalli; step (2), culturing and taming under a high bacteriophage concentration, and gradient diluting and primarily screening; and step (3), secondarily screening and confirming the bacteriophage insensitive mutants. Compared with the prior art, the technical problems of large screening amount, low efficiency and poor production performance of mutant thalli are solved, and the possibility of obtaining the bacteriophage insensitive mutants is greatlty increased; the resistance separation ratio is increased; and the thalli production performance is good. The method is suitable for the streptococcus thermophilus genetic breeding in the fermented dairy product industry.
Description
Technical field
The present invention relates to the thermophilus streptococcus Biotechnology in Genetic Breeding in the cultured milk prod industry, relate in particular to a kind of thermophilus streptococcus phage resistance screening mutant method.
Background technology
Thermophilus streptococcus is one of main fermented bacterium of sour milk, cheese production, and it produces in the earlier fermentation quick fermentation, and acid suppresses living contaminants, the product exocellular polysaccharide improves the aspect important roles such as sour milk matter structure.But because thermophilus streptococcus mostly is virulent phage, compares with Bacterium lacticum and more easily be subjected to Phage Infection.After infecting, phage can cause thermophilus streptococcus lysis, cause ferment slow, sour-milk product taste flavor variation [Ma Chengjie etc., the thermophilus streptococcus phage is on the impact of sour milk quality, Food science, 2009,30(21) 7:328-331], become the worldwide technological puzzle of restricted fermentation Dairy Industry development, thereby the seed selection of milk-acid bacteria phage resistant strains and research enjoy domestic and international concern.
In China, sour milk is topmost cultured milk prod, and 2011 annual production have reached 2,000,000 tons.Pollution of Phage problem for thermophilus streptococcus, carried out the screening operation of some phage resistance bacterial strains both at home and abroad, mainly be under spontaneous mutation or physics, chemomorphosis condition, with double-deck agar plate method, secondary growth method screening thermophilus streptococcus phage resistance bacterial strain [Binetti A G
Et al. Spontaneous phage-resistant mutants of
Streptococcus thermophilus: isolation and technological characteristics. International Dairy Journal, 2007,17 (4): 343-349].Take yoghurt production strain streptococcus thermophilus S2 as starting strain, [cold one is non-etc., the seed selection of thermophilus streptococcus phage resistant strains to adopt the method for double-deck agar plate, natural seed selection to filter out the phage-resistant non-lysogeny mutant strain of 4 strains such as cold one non-grade, China brewages, 2010,7,118-120].The flat band method such as Li Yalei has been carried out phage-resistant thermophilus streptococcus screening [Li Yalei etc., phage-resistance thermophilus streptococcus mutant strain by spontaneous mutation, ultraviolet mutagenesis, ethyl sulfate mutagenesis
Streptococcus thermophilusThe seed selection of-21UD, foodstuffs industry science and technology, 2008,29(11), 118-121].But (generally only have 10 because microorganism spontaneous mutation probability is extremely low
-8～10
-9), the orthomutation probability that occurs in the phage resistance aspect is then lower.(bacterium is counted content 10 and traditional double-layer plate method, secondary growth method are got 0.1～1.0mL sensitive strain zymocyte liquid
7～10
8Cfu/mL) be coated with flat board, the sensitive strain cell quantity is less than 10
8, suppose that phage resistance orthomutation probability is 10
-10, then to be coated with at least in theory 100 flat boards and just can obtain 1 phage resistant mutant, the primary screening probability of success is very low.On the other hand, owing to only being in the thalline of competence (being generally the microorganism cells that is in logarithmic phase) just to phage-sensitive, and the zymocyte liquid cell that traditional method is got differs to establish a capital and is in competence, therefore the multiple sieve of a lot of warps of bacterium colony that grows with secondary growth method or double-layer plate method is not real resistant mutants, this has further strengthened our screening difficulty and workload.And the physical chemistry mutagenesis such as use ultraviolet, ethyl sulfate, nitrosoguanidine, though the mutation probability of microorganism is increased, but because these physical chemistry mutagenesis are not directed mutagenesis, may induce in extraneous factor and lower other sudden changes have also occured when the phage resistance sudden change occur, cause phage resistant mutant product acid, produce that the production performance such as sticking descends or even in cow's milk nonfermented produce acid.
Summary of the invention
The objective of the invention is for traditional thermophilus streptococcus phage resistance mutant (bacteriophage insensitive mutant, the technical barriers such as BIM) the screening method workload is large, efficient is low, the mutants which had production performance is not good provide a kind of thermophilus streptococcus phage resistance screening mutant method.
The object of the present invention is achieved like this:
By concentration, domestication primary dcreening operation and 3 steps of multiple sieve checking, realized efficient, the rapid screening of thermophilus streptococcus phage resistance mutant.
Concrete steps are as follows:
1. secondary cultivation and the concentration of sensitive strain
Access thermophilus streptococcus sensitive strain in the substratum of certain volume, be cultured to the logarithmic growth initial stage, press the phage splitting liquid of MOI 〉=1 access purifying, be cultured to cracking complete, continue to cultivate and to carry out secondary growth, carry out the concentrated of cracking thalline not and collect by the method such as centrifugal;
2. cultivation domestication and the gradient dilution primary dcreening operation under the high phage concentration
In the culture medium with the concentrated thalline access smaller size smaller of step in 1., and access simultaneously the high density phage splitting liquid of purifying, cultivate, domestication 1～20 time, be coated with flat board behind the gradient dilution, obtain single bacterium colony, get the primary dcreening operation of BIMs;
3. multiple sieve, the affirmation of BIMs
The BIMs that primary dcreening operation obtains carries out multiple sieve, phage-resistance confirming performance through turbidity test, the sour inhibition test of product;
Substratum confession under directions thermophilus streptococcus growth, the stock essential nutraceutical matrix of described step in 1. generally all contains carbon source, nitrogenous source, inorganic salt (comprising trace element) and VITAMIN and water etc., and it preferably is LM17 or LM17-Ca substratum;
Culture medium confession under directions thermophilus streptococcus growth and breeding, the fermentation and acid used nutraceutical matrix of described step in 2., it can be the substratum described in the step 1, also can make sour milk, the used various dairy milk starting materials of cheese, such as raw milk, skimmed milk powder and lactalbumin powder etc.;
It can be that the M17 solid medium is coated with flat board that the gradient dilution of described step in 2. is coated with dull and stereotyped primary dcreening operation, it also can be double-deck agar plate method, these are the ordinary method of microorganism operation, in order further to eliminate false phage-resistible mutant, raising phage separation rate, the phage that can add higher concentration in the dull and stereotyped primary dcreening operation process;
The turbidity test of described step in 3. for the checking bacterial strain whether to a kind of method of phage-sensitive, press MOI 〉=1, with supplying in the examination thermophilus streptococcus LM17-Ca liquid nutrient medium of phage splitting liquid access logarithmic growth, try thermophilus streptococcus LM17-Ca liquid nutrient medium as contrast take the confession that does not contain phage, 37～42 ℃ of cultivations, the opacity that compares both, be muddiness such as experimental group, control group, illustrate that strains tested is insensitive to phage, muddy such as control group, the experimental group clarification illustrates that strains tested is to phage-sensitive;
The product acid inhibition test of described step in 3. is whether the checking bacterial strain is to the another kind of method of phage-sensitive, whether it suppresses streptococcus thermophilus fermentation and produces acid and judge that bacterial strain is to phage sensitivity [Ma Chengjie etc. whether to add phage, Pollution of Phage in the yoghurt production, the Agriculture in Jiangxi journal, 2007,19 (7): 90～91];
Described MOI is infection multiplicity (multiplicity of infection, MOI), the ratio of virus and cell quantity when being infection;
Described resistance separation rate is phage resistance bacterial strain and the ratio of supposing the phage resistance bacterial strain.
When 2. step is coated with flat board, can contain the phage of higher concentration in the substratum.
Principle of work of the present invention:
Because microorganism spontaneous mutation probability is extremely low, the present invention is by carrying out concentration with uncracked thalline in the secondary growth, and carries out subsequent experimental, greatly increases so that obtain the probability of BIMs in the experiment material.Produce the probability of phage resistance as 10 take a certain sensitive strain spontaneous mutation
-10Meter, traditional double-layer plate method, secondary growth method are got the 0.5mL zymocyte liquid, and (content is with 10
8The cfu/mL meter) be coated with flat board, the probability that then obtains 1 BIM only has 0.5%.The present invention uses 200 mL level growth secondary fermentation bacterium liquid, is coated with flat board behind the centrifugal concentrating, can obtain 200 mL * 10 in theory
8Cfu/mL * 10
-10=2, the probability that namely obtains in theory 2 BIMs is 100%, is 400 times of traditional method.Therefore, the invention solves traditional method and obtain the extremely low technical barrier of BIMs probability.
Not real resistant mutants, sieve the problems such as workload is large again through multiple sieve for low, a lot of doubtful bacterium colony of secondary growth method or double-layer plate method resistance separation rate, step 2 of the present invention is by repeatedly cultivating target BIMs under high density phage condition, utilize the non-phage resistance mutant strain of phage natural selection, the resistance separation rate is improved greatly, effectively reduce the workload of screening breeding.
The problems such as the production performances such as phage-resistance unstable properties, the mutant strain product for BIMs is sticking, product acid are not good, step 2 of the present invention is tamed in the culture mediumes such as cow's milk for many times by BIMs, and observe the abilities such as its working up curd, and carry out the BIMs screening, can obtain the phage-resistance stable performance, produce the sour good phage resistance mutant strains of leavening property such as sticking that produce.
Compared with prior art, the present invention has the following advantages and positively effect:
1. obtaining the BIMs probability increases greatly
Because the present invention concentrates the thalline that secondary growth obtains, and is used for next step BIMs primary dcreening operation with the bacteria used thereby body, greatly increase therefore obtain the probability of BIMs, should be in theory the n of traditional method doubly, n=V
1/ V
2, V wherein
1The culture volume of cultivating for carrying out sensitive strain in the step 1, usually, V
1Be 100～1000mL, also can get as required larger numerical value, V
2For bacterium liquid in the traditional method is coated with dull and stereotyped sample volume, usually, V
2Be 0.1～1.0mL, thus n generally 100～10000, i.e. the present invention has increased about 1000 times with respect to the probability that traditional method obtains BIMs.
Because it is insensitive to phage during physiological stage that the sensitive strain thalline is in paracme etc., therefore it still can be survived when secondary grown cultures, cause being coated with dull and stereotyped primary dcreening operation and obtain bacterial strain and much be apparent resistance, the phage resistance separation rate is low, workload is large.Step 2 of the present invention is utilized the non-phage resistance mutant strain of phage splitting by target BIMs is repeatedly cultivated under high density phage condition, the BIMs growth and breeding improves the resistance separation rate greatly.
The major objective of screening BIMs is can phage-resistance to pollute, and keep or further improve its produce acid, produce sticking, produce the production performances such as flavour substances, can be used for better the cultured milk prod productions such as sour milk, cheese.Step 2 of the present invention is tamed in the culture mediumes such as cow's milk for many times by BIMs, and observe its working up curd, produce sticking, produce the abilities such as fragrant, and carry out the BIMs screening, can obtain the phage-resistance stable performance, produce the sour good phage resistance mutant strains of leavening property such as sticking that produce.
Be suitable for the thermophilus streptococcus genetic breeding in the cultured milk prod industry.
Embodiment
Describe in detail below in conjunction with embodiment:
Protection scope of the present invention is not subjected to the restriction of specific embodiment, and only as the single example of illustrating each side of the present invention, the scope of the invention also comprises component and the method for functional equivalent to described embodiment.
Among the following embodiment, raw material and source thereof:
1, skimming milk: raw milk centrifugal degreasing or skimmed milk powder make by the 12-15% reduction, and skimmed milk powder derives from New Zealand dairy company Fonterra;
2, raw milk: derive from Wuhan bright dairy products company limited;
3, M17 substratum: derive from the Merck company of Germany or the Oxoid company of Britain, LM17-Ca is for to add lactose (final concentration 5 ‰, mass percent), CaCl at the M17 medium base
2(final concentration 0.05mM), wherein lactose, CaCl
2Be commercially available analytical pure or chemically pure reagent;
4, strains of streptococcus thermophilus ST01: use the separation of M17 selective medium, purifying from MYE96 direct putting type ferment agent for sour milk [Ma Chengjie etc., the thermophilus streptococcus phage is on the impact of sour milk quality, Food science, 2009,30(21) 7:328-331], MYE96 derives from red Masco Corp., and strains of streptococcus thermophilus ST01 is hereinafter to be referred as ST01;
5, strains of streptococcus thermophilus ST03: from the Y903 direct putting type ferment agent for sour milk, Y903 derives from Denmark Ke Sen company with the separation of M17 selective medium, purifying, and strains of streptococcus thermophilus ST03 is hereinafter to be referred as ST03;
6, the thermophilus streptococcus phage phi 01: take strains of streptococcus thermophilus ST01 as indicator, separation is from sour-milk product commercially available, that the MYE96 starter is made, virulent phage for ST01, by the laboratory qualification of the bright dairy products in Wuhan company limited, preservation [Ma Chengjie etc., Pollution of Phage in the yoghurt production, the Agriculture in Jiangxi journal, 2007,19 (7): 90～91];
7, the thermophilus streptococcus phage phi 02: take strains of streptococcus thermophilus ST01 as indicator, separate from sour-milk product commercially available, that the MYE96 starter is made, be another virulent phage of ST01, by the laboratory qualification of the bright dairy products in Wuhan company limited, preservation;
8, the thermophilus streptococcus phage phi 903: take strains of streptococcus thermophilus ST03 as indicator, separate from sour-milk product commercially available, that the Y903 starter is made, be the virulent phage of ST03, by the laboratory qualification of the bright dairy products in Wuhan company limited, preservation.
Embodiment 1: the spontaneous mutation strain screening of thermophilus streptococcus ST01 phage-resistance φ 01
1. secondary cultivation and the concentration of sensitive strain: access thermophilus streptococcus sensitive strain ST01(inoculum size 2% in the LM17-Ca of 100mL substratum), 42 ℃ are cultured to the logarithmic growth initial stage, press phage phi 01 lysate of MOI=1 access purifying, be cultured to cracking complete, continue to cultivate 8hrs and carry out secondary growth, 8000rpm, 10min are centrifugal that uncracked ST01 concentrates thalline;
2. cultivation domestication and the gradient dilution primary dcreening operation under the high phage concentration: with the concentrated thalline of step in 1. in the φ 01 lysate system of the sterilization skimming milk of 3mL, MOI=10 cultured continuously 5 times (namely the 1st time take concentrated thalline as bacterial classification, the 2nd～5 time take fermented skim milk as bacterial classification, inoculum size 2%), incubation time is 24hrs, 42 ℃ of culture temperature, the fermented skim milk of getting the 5th carries out being coated with the LM17 flat board behind the gradient dilution, obtains single bacterium colony, gets the primary dcreening operation of BIMs;
3. multiple sieve, the affirmation of BIMs: selecting step 2. in the doubtful BIMs that obtains of primary dcreening operation, through turbidity test, produce whether sour inhibition test is checking, the affirmation of φ 01 resistant mutants.
The single bacterium colony of the doubtful BIMs that 20 primary dcreening operations choosing obtain through turbidity test, produce sour inhibition test, 19 are produced acid and are not subjected to φ 01 phage to suppress, add the LM17-Ca substratum that φ 01 phage can not make thalline to become clarification, namely these 19 bacterial strains are BIMs; Only have 1 to produce the LM17-Ca substratum change clarification that acid is subjected to the inhibition of φ 01 phage, adds φ 01 phage thalline, namely this bacterial strain is sensitive strain still, and the phage resistance sudden change does not occur, and the phage separation rate is 95%.Compare with starting strain ST01,19 BIMs are stable to φ 01 resistance, and sour-milk product viscosity, mouthfeel, local flavor and the ST01 of making are basically identical, and acid production speed is slightly slow than ST01, can be used for actual production.
Embodiment 2: the spontaneous mutation strain screening of thermophilus streptococcus ST01 phage-resistance φ 02
1. secondary cultivation and the concentration of sensitive strain: access thermophilus streptococcus sensitive strain ST01(inoculum size 2% in the LM17-Ca of 500mL substratum), 39 ℃ are cultured to the logarithmic growth initial stage, press phage phi 02 lysate of MOI=3 access purifying, be cultured to cracking complete, continue to cultivate 10hrs and carry out secondary growth, 8000rpm, 10min are centrifugal that uncracked ST01 concentrates thalline;
2. cultivation domestication and the gradient dilution primary dcreening operation under the high phage concentration: the concentrated thalline of step in 1. cultivated 48hrs in the φ 02 lysate system of the sterilization raw milk of 10mL, MOI=5, culture temperature is 42 ℃, cow's milk carries out being coated with the LM17 flat board behind the gradient dilution after getting fermentation, obtain single bacterium colony, get the primary dcreening operation of BIMs;
3. multiple sieve, the affirmation of BIMs: selecting step 2. in the doubtful BIMs that obtains of primary dcreening operation, through turbidity test, produce whether sour inhibition test is checking, the affirmation of φ 02 resistant mutants.
The single bacterium colony of the doubtful BIMs that 20 primary dcreening operations obtain through turbidity test, produce sour inhibition test, 16 are produced acid and are not subjected to φ 02 phage to suppress, add the LM17-Ca substratum that φ 02 phage can not make thalline to become clarification, namely these 16 bacterial strains are BIMs; 4 are produced the LM17-Ca substratum change clarification that acid is subjected to the inhibition of φ 02 phage, adds φ 02 phage thalline, and namely these 4 bacterial strains are sensitive strain still, and the phage resistance sudden change does not occur, and the phage separation rate is 80%.Compare with starting strain ST01,16 BIMs are stable to φ 02 resistance, and the sour-milk product viscosity of making, mouthfeel, local flavor, acidifying speed and ST01 are basically identical, can be used for actual production.
Embodiment 3: the spontaneous mutation strain screening (primary dcreening operation adds phage) of thermophilus streptococcus ST01 phage-resistance φ 02
1. secondary cultivation and the concentration of sensitive strain: access thermophilus streptococcus sensitive strain ST01(inoculum size 2% in the LM17-Ca of 300mL substratum), 39 ℃ are cultured to the logarithmic growth initial stage, press phage phi 02 lysate of MOI=2 access purifying, be cultured to cracking complete, continue to cultivate 12hrs and carry out secondary growth, 8000rpm, 10min are centrifugal that uncracked ST01 concentrates thalline;
2. cultivation domestication and the gradient dilution primary dcreening operation under the high phage concentration: the concentrated thalline in the step 1 is cultivated 48hrs in the φ 02 lysate system of the sterilization raw milk of 5mL, MOI=5, culture temperature is 42 ℃, getting after the fermentation cow's milk carries out being coated with the M17 flat board behind the gradient dilution (the M17 substratum has added φ 02 phage, and its final concentration is 10
8Pfu/mL), obtain single bacterium colony, get the primary dcreening operation of BIMs;
3. multiple sieve, the affirmation of BIMs: whether the doubtful BIMs that primary dcreening operation obtains in the selecting step 2 is checking, the affirmation of φ 02 resistant mutants through turbidity test, the sour inhibition test of product.
The single bacterium colony of the doubtful BIMs that 30 primary dcreening operations obtain through turbidity test, produce sour inhibition test, 28 are produced acid and are not subjected to φ 02 phage to suppress, add the LM17-Ca substratum that φ 02 phage can not make thalline to become clarification, namely these 28 bacterial strains are BIMs; 2 are produced acid and are subjected to φ 02 phage to suppress, add the LM17-Ca substratum change clarification of φ 02 phage thalline, be that these 2 bacterial strains still are sensitive strain, the phage resistance sudden change does not occur, the phage separation rate is 93%, the dull and stereotyped interpolation of primary dcreening operation phage has further been improved the phage separation rate.Compare with starting strain ST01,28 BIMs are stable to φ 02 resistance, and the sour-milk product viscosity of making, mouthfeel, local flavor, acidifying speed and ST01 are basically identical, can be used for actual production.
Embodiment 4: the spontaneous mutation strain screening (LM17 domestication) of thermophilus streptococcus ST03 phage-resistance φ 903
1. secondary cultivation and the concentration of sensitive strain: access thermophilus streptococcus sensitive strain ST03(inoculum size 2% in the LM17-Ca of 200mL substratum), 39 ℃ are cultured to the logarithmic growth initial stage, press phage phi 903 lysates of MOI=5 access purifying, be cultured to cracking complete, continue to cultivate 6hrs and carry out secondary growth, 8000rpm, 10min are centrifugal that uncracked ST03 concentrates thalline;
2. cultivation domestication and the gradient dilution primary dcreening operation under the high phage concentration: with the concentrated thalline of step in 1. in the φ 903 lysate systems of 3mL sterilization LM17-Ca substratum, MOI=5 cultured continuously 10 times (namely the 1st time to concentrate thalline as bacterial classification, the 2nd～10 the LM17-Ca zymocyte liquid take ST03 is as bacterial classification, inoculum size 1%), incubation time is 12hrs, 37 ℃ of culture temperature, the zymocyte liquid of getting the 10th time carries out being coated with flat board behind the gradient dilution, obtains single bacterium colony, gets the primary dcreening operation of BIMs;
3. multiple sieve, the affirmation of BIMs: selecting step 2. in the doubtful BIMs that obtains of primary dcreening operation, through turbidity test, produce whether sour inhibition test is checking, the affirmation of φ 903 resistant mutants.
The single bacterium colony of the doubtful BIMs that 50 primary dcreening operations obtain through turbidity test, produce sour inhibition test,, 48 are produced acid and are not subjected to φ 903 phages to suppress, add the LM17-Ca substratum that φ 903 phages can not make thalline to become clarification, namely these 48 bacterial strains are BIMs; 2 are produced the LM17-Ca substratum change clarification that acid is subjected to the inhibition of φ 903 phages, adds φ 903 phage thalline, and namely this bacterial strain is sensitive strain still, and the phage resistance sudden change does not occur, and the phage separation rate is 96%.Compare with starting strain ST03,48 BIMs are stable to φ 903 resistances, and the Yoghourt fermentation speed of making, product viscosity, mouthfeel, local flavor etc. are basically identical with ST03, can be used for actual production.
Embodiment 5: the spontaneous mutation strain screening (cow's milk domestication) of thermophilus streptococcus ST03 phage-resistance φ 903
1. secondary cultivation and the concentration of sensitive strain: access thermophilus streptococcus sensitive strain ST03(inoculum size 3% in the LM17-Ca of 1000mL substratum), 42 ℃ are cultured to the logarithmic growth initial stage, press phage phi 903 lysates of MOI=1 access purifying, be cultured to cracking complete, continue to cultivate 10hrs and carry out secondary growth, 8000rpm, 10min are centrifugal that uncracked ST03 concentrates thalline;
2. cultivation domestication and the gradient dilution primary dcreening operation under the high phage concentration: with the concentrated thalline of step in 1. in 10mL sterilizes the φ 903 lysate systems of skimming milk, MOI=3 cultured continuously 20 times (namely the 1st time take concentrated thalline as bacterial classification, the 2nd～20 fermented skim milk take ST03 is as bacterial classification, inoculum size 3%), incubation time is 16hrs, 42 ℃ of culture temperature, the zymocyte liquid of getting the 20th time carries out being coated with flat board behind the gradient dilution, obtains single bacterium colony, gets the primary dcreening operation of BIMs;
3. multiple sieve, the affirmation of BIMs: selecting step 2. in the doubtful BIMs that obtains of primary dcreening operation, through turbidity test, produce whether sour inhibition test is checking, the affirmation of φ 903 resistant mutants.
The single bacterium colony of the doubtful BIMs that 30 primary dcreening operations obtain through turbidity test, produce sour inhibition test, the bacterial strain that 30 primary dcreening operations obtain produces acid and all is not subjected to φ 903 phages to suppress, add the LM17-Ca substratum that φ 903 phages can not make thalline to become clarification, be that these 30 bacterial strains are BIMs, the phage separation rate is 100%.Compare with starting strain ST03,30 BIMs are stable to φ 903 resistances, and the Yoghourt fermentation speed of making, product viscosity, mouthfeel, local flavor etc. are all better, and part bacterial strain product is sticking, product glues ability and makes moderate progress than starting strain ST03, can be used for actual production.
Claims (2)
1. thermophilus streptococcus phage resistance mutants which had screening method is characterized in that:
1. secondary cultivation and the concentration of sensitive strain
Access thermophilus streptococcus sensitive strain in the substratum of certain volume, be cultured to the logarithmic growth initial stage, press the phage splitting liquid of MOI 〉=1 access purifying, be cultured to cracking complete, continue to cultivate and carry out secondary growth, carry out the not concentrated and collection of cracking thalline by centrifugal method;
2. cultivation domestication and the gradient dilution primary dcreening operation under the high phage concentration
In the culture medium with the concentrated thalline access smaller size smaller of step in 1., and access simultaneously the high density phage splitting liquid of purifying, cultivate, domestication 1～20 time, be coated with flat board behind the gradient dilution, obtain single bacterium colony, get the primary dcreening operation of BIMs;
3. multiple sieve, the affirmation of BIMs
The BIMs that primary dcreening operation obtains carries out multiple sieve, phage-resistance confirming performance through turbidity test, the sour inhibition test of product;
Substratum confession under directions thermophilus streptococcus growth, the stock essential nutraceutical matrix of described step in 1. generally all contains carbohydrate, nitrogenous substances, inorganic salt and VITAMIN and water, and it preferably is LM17 or LM17-Ca substratum;
Culture medium confession under directions thermophilus streptococcus growth and breeding, the fermentation and acid used nutraceutical matrix of described step in 2., it can be the substratum of step described in 1., also can make sour milk, the used various dairy milk starting materials of cheese, such as raw milk, skimmed milk powder and lactalbumin powder;
It is that the M17 solid medium is coated with flat board that the gradient dilution of described step in 2. is coated with dull and stereotyped primary dcreening operation, or double-deck agar plate method;
The turbidity test of described step in 3. for the checking bacterial strain whether to a kind of method of phage-sensitive, press MOI 〉=1, with supplying in the examination thermophilus streptococcus LM17-Ca liquid nutrient medium of phage splitting liquid access logarithmic growth, try thermophilus streptococcus LM17-Ca liquid nutrient medium as contrast take the confession that does not contain phage, 37～42 ℃ of cultivations, the opacity that compares both, be muddiness such as experimental group, control group, illustrate that strains tested is insensitive to phage, muddy such as control group, the experimental group clarification illustrates that strains tested is to phage-sensitive;
The product acid inhibition test of described step in 3. be the checking bacterial strain whether to the another kind of method of phage-sensitive, whether it suppresses streptococcus thermophilus fermentation and produces acid and judges that bacterial strain is to phage sensitivity whether to add phage;
Described MOI is infection multiplicity, the ratio of virus and cell quantity when being infection;
Described BIMs is the phage resistance mutant;
Described resistance separation rate is phage resistance bacterial strain and the ratio of supposing the phage resistance bacterial strain.
2. thermophilus streptococcus phage resistance mutants which had screening method according to claim 1 when it is characterized in that 2. step is coated with flat board, can contain the phage of higher concentration in the substratum.
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| CN111575209A (en) * | 2020-05-25 | 2020-08-25 | 浙江大学 | Method for screening bacteriophage-resistant lactobacillus plantarum strain |
| CN116814465A (en) * | 2023-03-17 | 2023-09-29 | 微康益生菌(苏州)股份有限公司 | Streptococcus thermophilus with phage resistance and application thereof |
| CN116814465B (en) * | 2023-03-17 | 2024-03-26 | 微康益生菌(苏州)股份有限公司 | Streptococcus thermophilus with phage resistance and application thereof |
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