CN104946724B - For detecting the culture medium and detection method of lactobacillus in extract oral liquid preparation - Google Patents
For detecting the culture medium and detection method of lactobacillus in extract oral liquid preparation Download PDFInfo
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Abstract
It is used to detect in extract oral liquid preparation the present invention relates to one kind, if pollute the detection method of lactobacillus, and for detecting the culture medium of lactobacillus in extract oral liquid preparation, it includes（1）One kind is lactobacillus enriched medium：Peptone 5.0g, yeast extract powder 4.0g, glucose 10.0g, sucrose, 10.0g, dipotassium hydrogen phosphate 1.0g, magnesium sulfate 0.3g, polyoxyethylene sorbitan monoleate 5.0g, potassium sorbate 3g, water 1000mL；（2）Another kind is lactobacillus agar medium：Peptone 10.0g, glucose 5.0g, sucrose 5.0g, sodium chloride 3.0g, yeast extract powder 5.0g, L cystine 1.0g, dipotassium hydrogen phosphate 1.0g, agar 17g, potassium sorbate 3g, water 1000mL.The present invention is continuously monitored to Chinese medicine oral liquid production line key area, can find pollution hidden trouble existing for production line in time, is excluded in time, is prevented the generation of massive pollution event.
Description
Technical field
The present invention relates to medicine microorganism detection field, and in particular to and one kind is used to detect in extract oral liquid preparation,
Whether the detection method of lactobacillus is polluted, and for detecting the culture medium of lactobacillus in extract oral liquid preparation
Background technology
Extract oral liquid preparation be based on traditional Chinese herbal decoction, extract crude drug in active ingredient, add flavouring,
Bacteriostatic agent etc., then through standing, filtering, the technique such as sterilize is made, have that dosage is small, convenient to take, the rapid, taste that works is easy to connect
By the advantages that, clinical practice is extensive.However, traditional Chinese medicine oral liquid bulk drug is more based on natural animal and plant composition, protein content
Height, and preparation adds sucrose, honey etc. more and is used to adjust taste, rich in nutrition content；It is again in its raw material to carry largely more
Microorganism, wherein being no lack of heat-resisting bacillus, resistance to irradiated bacteria, spore and gemma etc., there is stronger resistance to poor environment.Therefore,
Product, easily by microorganism pollution, causes medicine to go bad during production, storage and use, does not have treatment not only and makees
With when serious, metabolic toxicities, drug ingedient change formed sensibiligen etc., or even entail dangers to patient caused by microorganism
Life and health.
Lactobacillus (Lactobacillaceae) is Grain-positive bacillus, and amphimicrobian is acidproof, is deposited extensively in nature
In the oral cavity, enteron aisle and the vagina that also parasitize warm-blooded animal.The tolerable 70 DEG C temperature of general lactobacillus, also has been reported that card
Real, certain plants lactobacillus can still grow in 80 DEG C of high temperature.As beneficial flora, lactobacillus should in modern food industry
With extensive, consulting literatures, the nearly no report on its harmfulness.But for extract oral liquid preparation, lactobacillus is but
Can cause serious Product quality and safety hidden danger, main cause have it is following some：1. carried in traditional Chinese medicine oral liquid bulk drug big
Lactobacillus is measured, the multipair poor environment of wild strain has stronger resistance.2. to be effectively retained the active component of medicine, production technology
Avoid high temperature to extract or sterilize as far as possible, advantage is created for the survival of lactobacillus.3. it is rich in sucrose, honey etc. in medicine
Flavor enhancement, it is the carbon source that lactobacillus is excelled at leveraging.Although 4. with the addition of the bacteriostatic agents such as potassium sorbate in product, the composition is only right
Mould, saccharomycete and aerobic bacteria have inhibitory action, nearly unavailable to genus lactubacillus.5. product is strictly sealed more, bottle
Interior remaining space is less, and living environment is close to anaerobism or micro- aerobic conditions, the growth and breeding suitable for lactobacillus.6. patient buys
Afterwards, it is placed in storage at normal temperature more, meets summer and autumn, temperature is suitable to the growth and breeding of lactobacillus especially.After product is polluted by lactobacillus, i.e.,
Just the pollution of extremely low level, under appropriate conditions, lactobacillus can quickly be bred, and product characteristics is changed in the short time.
Phenomena such as flatulence, precipitation, pH value decline and active ingredient reduces is mainly shown as, threatens patient health.If enterprise's production line
Polluted by the bacterium, can often cause the successive batches of massive pollutions of product, huge economic loss is brought to enterprise.In recent years
Come, the recall event of domestic several oral preparation of Chinese traditional medicinal is the pollution for being derived from the bacterium.
At present, Chinese medicine detection concerned countries standard, such as Chinese Pharmacopoeia, only by staphylococcus aureus, large intestine angstrom
The invasive organisms such as uncommon bacterium, salmonella, pseudomonas aeruginosa, Candida albicans list correlation as control bacterium
Detection method and result judgement standard.Lactobacillus in itself can't direct do harm to huamn body, therefore, medicine examination criteria is not
It is controlled as harmful bacteria.With the implementation of China's drug's GMP management system and perfect, provided in existing national standards
Several eliminations of the threat to drug quality safety of the pathogenic bacteria of detection, the non-pathogenic bacteria of some strong resistance or conditioned pathogen,
Such as lactobacillus of the present invention, rise to the main threatening factors of Chinese medicine preparation quality safety.
In viewpoint of Chinese food safety national standard, detection method containing lactic acid bacteria in dairy products has been recorded and containing biodiasmin
Food in lactic acid bacteria detection method, detected as probiotics.With in extract oral liquid preparation, by lactobacillus
Testing goal as harmful bacteria is different, and therefore, culture medium, detection method and result judgement standard are inapplicable.
Domestic and international pertinent literature is consulted, is showed no the detection method on traditional Chinese medicine oral liquid pollution lactobacillus.Therefore, establish
A set of liquid preparation suitable for extract oral, the detection method and standard of lactobacillus are polluted, specificity culture medium is developed, to true
Protect the drug safety of patient, promote the sound development of Chinese Medicine Industry that there is very positive meaning.
The content of the invention
It is an object of the invention to provide the culture medium and traditional Chinese medicine mouth for detecting lactobacillus in extract oral liquid preparation
Take the detection method of lactobacillus in liquid preparation, it is possible to prevente effectively from test sample all kinds of miscellaneous bacterias interference, test limit is less than
5cfu/100mL。
The invention discloses a kind of culture medium for being used to detect lactobacillus in extract oral liquid preparation, include
(1) one kind is lactobacillus enriched medium, is fluid nutrient medium, the preceding Zengjing Granule for lactobacillus；Its composition
For：Peptone 5.0g, yeast extract powder 4.0g, glucose 10.0g, sucrose, 10.0g, dipotassium hydrogen phosphate 1.0g, magnesium sulfate
0.3g, polyoxyethylene sorbitan monoleate 5.0g, potassium sorbate 3g, water 1000mL；
It is made of following methods：In mentioned component in addition to dextrose and saccharose, boil dissolving, then add glucose and
After sucrose dissolving, shake up, adjust pH value 5.5~6.0, packing, 115 DEG C, 30min sterilizes；
(2) another kind is lactobacillus agar medium, is solid agar medium, for the line culture of enrichment liquid, its
Composition is：Peptone 10.0g, glucose 5.0g, sucrose 5.0g, sodium chloride 3.0g, yeast extract powder 5.0g, CYSTINE
1.0g, dipotassium hydrogen phosphate 1.0g, agar 17g, potassium sorbate 3g, water 1000mL；
It is made of following methods：In mentioned component in addition to dextrose and saccharose, boil dissolving, then add glucose and
After sucrose dissolving, shake up, adjust pH value 5.5~6.0, packing, 115 DEG C, 30min sterilizes, pour plate.
The invention also discloses a kind of detection side for being used for lactobacillus in extract oral liquid preparation using above-mentioned culture medium
Method, it is characterised in that comprise the following steps：
(1) test sample is taken, is seeded in lactobacillus enriched medium, 37 DEG C ± 1 DEG C, anaerobism, shaken cultivation 18~24 is small
When, 48 hours can be extended to if necessary, 100~150r/min of shake speed；
(2) above-mentioned culture is taken, streak inoculation is into lactobacillus agar medium, 37 DEG C ± 1 DEG C, anaerobism, culture 24~
48 hours；
(3) if occurring bacterium colony on plate has following characteristics：Canescence, bacterium colony center color is deeper, circular protrusions, light
It is sliding, neat in edge, 1.0~1.5mm of colony diameter, and culture dish gives out lactic taste, then should select 2~3 bacterium colonies, respectively
Lactobacillus agar medium is inoculated in, 37 DEG C ± 1 DEG C, anaerobism, cultivates 24~48 hours, takes culture to carry out Grain stain microscopy
With suitable qualification test, judged whether to detect lactobacillus according to result；
(4) if grown on flat board without colony growth or without colonies typical, sentence product and do not detect lactobacillus.
The detection method of the invention described above, preferably：In the step (1), the dosage of lactobacillus enriched medium is to connect
5-10 times of kind test sample volume.
The detection method of the invention described above, preferably：The minimum amount of inspection of test sample is following (table 1),
Beneficial effects of the present invention are as follows：
The present invention establishes the detection method of extract oral liquid preparation infected milk bacillus, and develops two kinds of selective bacterium
Culture medium, one kind are lactobacillus enriched medium, and another kind is lactobacillus agar medium.Extract oral is carried out using the present invention
The test experience of liquid preparation lactobacillus, it is possible to prevente effectively from all kinds of aerobic bacterias, Gram-negative bacteria, mould and ferment in test sample
The interference of the miscellaneous bacterias such as female bacterium, test limit are less than 5cfu/100mL.Quality control is carried out to Chinese medicine oral liquid using the present invention
System, it can be detected to criticizing the product that dispatches from the factory, effectively prevent contaminated launch from selling；Using the present invention to traditional Chinese medicine oral liquid
Body preparation production line key area is continuously monitored, and can find pollution hidden trouble existing for production line in time, is excluded in time, is prevented
The only generation of massive pollution event.The present invention is for ensuring the drug safety of patient, promoting the sound development of Chinese Medicine Industry
With very positive meaning.
Embodiment：
It is the further explanation bright to this law below, rather than limitation of the present invention.
Embodiment 1：
Using bacillus subtilis (Bacillus subtilis) [CMCC (B) 63501], staphylococcus aureus
(Staphylococcus aureus) [CMCC (B) 26003], EHEC (Escherichia coli) [CMCC (B)
44102], clostridium sporogenes (Clostridium sporogenes) [CMCC (B) 64941], Candida albicans (Candida
Albicans) [CMCC (F) 98001] and aspergillus niger (Aspergillus niger) [CMCC (F) 98003] use bacterium as checking
Strain, represents bacillus common in environment, gram-positive cocci, gram-Negative bacillus, yeast and mold etc. respectively,
For verifying rejection ability of the heretofore described two kinds of lactobacillus culture mediums to miscellaneous bacteria；Using Lactobacillus plantarum plant subspecies
(Lactobacillus plantarum) [CICC20718] and Bu Shi lactobacillus (Lactobacillus buchneri) [CICC
20293] checking bacterial strain is used as, verifies the validity of extract oral liquid preparation lactobacillus detection method, checks two kinds of cultures
Growth promotion ability and instruction ability of base etc..
1st, bacterium solution prepares the fresh cultured thing extremely battalion of inoculation bacillus subtilis, staphylococcus aureus and EHEC
Support in broth bouillon, be inoculated with the fresh cultured thing of clostridium sporogenes into THIOGLYCOLLIC ACID salt broth, 30~35 DEG C of cultures
18~24 hours；The fresh cultured thing of Candida albicans is inoculated with to improveing in Martin's culture medium, 23~28 DEG C of cultures 24~48 are small
When, every 1ml is made containing bacteria suspension of the bacterium number less than 100cfu with 0.9% aseptic sodium chloride solution in above-mentioned culture.It is inoculated with black
To improveing on Martin's agar slant culture-medium, 23~28 DEG C are cultivated 5~7 days the fresh cultured thing of aspergillus, are added 3~5ml and are contained
0.9% aseptic sodium chloride solution of 0.05% polyoxyethylene sorbitan monoleate, spore is eluted, and the spore that spore count is less than 100cfu is then made
Sub- suspension.
2nd, culture medium growth promotion efficiency test takes above-mentioned Lactobacillus plantarum plant subspecies and Bu Shi lactobacillus bacteria suspensions each
1ml, it is inoculated in respectively in 100ml lactobacillus enriched medium, 37 DEG C, anaerobism, shaken cultivation 24 hours, shake speed 100~
150r/min；The ring of enrichment culture medium one is taken, streak inoculation 37 DEG C, anaerobism, is cultivated 48 hours in lactobacillus agar medium.Together
When, control medium operation repetitive is used as using culture medium specified in strain passage specification.
3rd, the inspection of culture medium rejection ability takes bacillus subtilis, staphylococcus aureus, EHEC and raw spore shuttle
Bacterium bacteria suspension 1ml, it is inoculated in 100ml lactobacillus enriched medium, 37 DEG C, anaerobism, shaken cultivation 24 hours, shakes respectively
100~150r/min of speed；The ring of enrichment culture medium one is taken, streak inoculation 37 DEG C, anaerobism, is cultivated in lactobacillus agar medium
48 hours.
4th, result observation and judgement
Lactobacillus plantarum plant subspecies and the culture experiment of Bu Shi lactobacillus, culture are carried out using culture medium of the present invention
Bacterium colony size, the morphological feature grown on base should be consistent with control medium；Withered grass is carried out using culture medium of the present invention
Bacillus, staphylococcus aureus, EHEC, clostridium sporogenes, the culture experiment of Candida albicans and aspergillus niger, are being advised
Cultivated under fixed cultivation temperature and most long incubation time, test organisms must not grow.It the results are shown in Table 2.
Growth promotion ability, rejection ability and the instruction efficiency test of the culture medium of table 2
Test result indicates that the checkout facility of lactobacillus in extract oral liquid preparation, Ke Yiyou are carried out using the present invention
Effect avoids the interference of the miscellaneous bacteria such as all kinds of aerobic bacterias, Gram-negative bacteria, yeast and mold in test sample, beneficial to the inspection of lactobacillus
Go out.
Embodiment 2：
The invention belongs to carry out qualitative detection to the lactobacillus in product, refer under the test condition of setting, in product
The minimum quantity for the microorganism that can be detected.It is sample microbe quantity contained before dilution or culture corresponding to test limit
Amount, rather than the micro organism quantity in detection process contained by the test liquid of a certain step.Detection method of the present invention, to breast
The test limit of bacillus is less than 5cfu/100mL.
For verify the method for the invention test limit, should be respectively adopted standard method and the method for the invention to containing
The positive of lactobacillus is tested, and compares the difference of two methods to detect result.At present, for extract oral liquid
The no standard method of the detection of lactobacillus in preparation, intend using colony counting method as a control group.It is sub- with Lactobacillus plantarum plant
Kind is used as test strain, 0~5cfu/mL bacteria suspension is made, then by randomly selecting the bacterial population in 1mL liquid in the bacteria suspension
Poission distributions are obeyed, accordingly, there exist in sampling 1mL bacteria suspensions, lactobacillus content is 0CFU/mL sample.In control group,
Count results are 0cfu/mL plate, are set as negative findings；Count results are more than 1cfu/mL plate, are set as positive knot
Fruit.Experiment bacteria suspension 1mL is taken immediately, is distinguished inoculation test group and illumination, intersection is carried out, 50 parts of samples of operation repetitive, according to
The difference that two groups of results contrast.
1st, prepared by bacterium solution takes the fresh cultured thing of Lactobacillus plantarum plant subspecies to be seeded to liquid as defined in strain specification
In culture, 37 DEG C are cultivated 18~24 hours, and the bacterium that every 1mL 0~5cfu containing bacterium number are made of 0.9% aseptic sodium chloride solution is hanged
Liquid.
2nd, control group takes Lactobacillus plantarum plant subspecies bacteria suspension 1mL to add in plate, then pours into temperature no more than 45
DEG C lactobacillus agar medium, and test group operation repetitive, intersect and carry out, altogether 50 parts of number, it is small to put 37 DEG C of cultures 18~24
When, result count is carried out, the results are shown in Table 3.
The control group bacterium colony count results of table 3
| Plate is numbered | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Count results (cfu/mL) | 3 | 1 | 6 | 0 | 2 | 1 | 4 | 3 | 3 | 2 |
| Plate is numbered | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| Count results (cfu/mL) | 0 | 3 | 1 | 4 | 4 | 5 | 0 | 2 | 3 | 7 |
| Plate is numbered | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| Count results (cfu/mL) | 0 | 1 | 7 | 4 | 3 | 5 | 3 | 5 | 2 | 3 |
| Plate is numbered | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| Count results (cfu/mL) | 2 | 5 | 1 | 0 | 3 | 4 | 3 | 5 | 3 | 7 |
| Plate is numbered | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 |
| Count results (cfu/mL) | 6 | 5 | 0 | 3 | 4 | 3 | 0 | 2 | 3 | 3 |
3rd, test group takes commercially available 50 bottles of certain traditional Chinese medical oral liquid for children, specification：50mL/ bottles, every bottle of inoculation 1mL plant breast bar
Bacterium plant subspecies bacteria suspension, with control group operation repetitive, intersect and carry out.After the completion of inoculation, test sample is shaken, makes bacteria suspension in sample
It is uniformly dispersed in product, sterile working, every bottle of test sample takes full dose, is seeded to respectively in 400mL lactobacillus enriched medium, 37
DEG C, anaerobism, shaken cultivation 24~48 hours, 100~150r/min of shake speed；The ring of enrichment culture medium one is taken, line respectively connects
Kind 37 DEG C, anaerobism, is cultivated 48 hours in lactobacillus agar medium.It is positive test sample to detect lactobacillus person, and non-detection person is
Negative test sample.It the results are shown in Table 4.
The test group testing result of table 4
| Test sample is numbered | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| As a result | Detection | Detection | Detection | Detection | Do not detect | Detection | Detection | Detection | Do not detect | Do not detect |
| Test sample is numbered | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| As a result | Detection | Detection | Do not detect | Detection | Detection | Detection | Detection | Do not detect | Detection | Do not detect |
| Test sample is numbered | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| As a result | Detection | Detection | Detection | Detection | Detection | Do not detect | Detection | Detection | Do not detect | Detection |
| Test sample is numbered | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| As a result | Do not detect | Detection | Detection | Detection | Do not detect | Detection | Detection | Detection | Detection | Detection |
| Test sample is numbered | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 |
| As a result | Detection | Do not detect | Detection | Detection | Detection | Do not detect | Detection | Detection | Detection | Detection |
4th, interpretation of result
In 50 parts of test samples of test group, positive findings is 38 parts, positive rate 76%；It is positive in 50 parts of samples of control group
As a result it is 42 parts, positive rate is 84% (being shown in Table 5).X is carried out to the positive rate of two groups of samples2Examine, P=0.2 ＞ 0.1, experiment
The positive rate of group and control group does not have significant difference.With reference to the specificity analysis to microorganism, the results showed that inspection of the present invention
Survey method, Chinese medicinal liquor oral liquid product of the lactobacillus content in 0~5CFU/mL can be gone out with effective detection, i.e., this method is to breast
The test limit of bacillus is less than 5cfu/100mL.
The comparison of 5 two groups of positive rates of table
| Processing | Positive findings | Negative findings | It is total | Positive rate (%) |
| Test group | 38 | 12 | 50 | 76 |
| Control group | 42 | 8 | 50 | 84 |
| It is total | 80 | 20 | 100 | 80 |
Embodiment 3：
To ensure assay accurately and reliably, when establishing the inspection method of certain microorganism, inspection method should be carried out
Checking, with confirmation used by method be suitable for the inspection of the product.If the constituent of product or original test condition occur
Change, when may have influence on the accuracy of assay, inspection method should be verified again.To heretofore described method
When carrying out method validation, made using Lactobacillus plantarum plant subspecies (Lactobacillus plantarum) [CICC 20718]
To verify bacterial strain.Embodiment 2 is by taking certain commercially available heat clearing Chinese medicine oral liquid as an example, specification：100mL/ bottles, introduce the operation side of the present invention
Method.
1st, prepared by bacterium solution takes the fresh cultured thing of Lactobacillus plantarum plant subspecies to be seeded to liquid as defined in strain specification
In culture, 37 DEG C are cultivated 18~24 hours, are made every 1mL 10~100cfu's containing bacterium number with 0.9% aseptic sodium chloride solution
Bacteria suspension.
2nd, direct inoculation takes the test sample of ormal weight to be seeded to respectively in lactobacillus enriched medium, is trained in each container
The test sample volume that the dosage of foster base should meet inoculation cannot be greater than the 20% of culture volume.Take 10 bottles of test sample, sterile behaviour
To make, every bottle of test sample takes 50mL, is seeded to respectively in 400mL lactobacillus enriched medium, 37 DEG C, anaerobism, shaken cultivation 24~
48 hours, 100~150r/min of shake speed；Taking the ring of enrichment culture medium one, streak inoculation is in lactobacillus agar medium respectively,
37 DEG C, anaerobism, cultivate 48 hours.
3rd, verification method takes 50mL test liquids and 10~100cfu bacteria suspension, adds 400mL lactobacillus enriched medium
In, the operation sequence according to direct inoculation is checked.If experiment detection test organisms, shows that this method can be used for the product
Lactobacillus detection；If experiment does not detect test organisms, show that this method is unfavorable for the recovery and breeding of lactobacillus, training should be increased
Support the usage amount of base or using membrane-filter procedure etc., eliminate unfavorable factor.
Whether the 4th, cultivate and should observe culture medium during observing culture day by day and record has bacteria growing, fills in inspection record
Table.Children's inducing diaphoresis oral liquid lactobacillus inspection result is shown in Table 6.
The result inspection record table of table 6
The 5th, result judges that if bacterium colony occur on any plate has following characteristics：Canescence, bacterium colony center color is deeper,
Circular protrusions, smooth, neat in edge, 1.0~1.5mm of colony diameter, and culture dish gives out lactic taste, then should select 2~3
Individual bacterium colony, lactobacillus agar medium is inoculated in respectively, 37 DEG C ± 1 DEG C, anaerobism, is cultivated 24~48 hours.Culture is taken to carry out
Grain stain microscopy and suitable qualification test, judged whether to detect lactobacillus according to result.It is if sterile on all flat boards
It is born long or is grown without colonies typical, sentences product and do not detect lactobacillus.When negative control has bacteria growing or positive control not to grow
Or growth bacterium get blamed Lactobacillus plantarum plant subspecies when, it is impossible to make survey report.Whole experiment process is looked back, discovery can
The factor of the failure of an experiment can be caused, and investigated.If experiment is confirmed invalid, should be retried.Again same amount test sample is taken,
Check in accordance with the law, if not detecting lactobacillus, sentence test sample and meet regulation；If detecting lactobacillus, it is against regulation to sentence test sample.
Claims (3)
1. a kind of detection method for being used to detect lactobacillus in extract oral liquid preparation, it is characterised in that comprise the following steps：
（1）Lactobacillus enriched medium is prepared, is fluid nutrient medium, the preceding Zengjing Granule for lactobacillus；Its composition is：Albumen
Peptone 5.0g, yeast extract powder 4.0g, glucose 10.0g, sucrose, 10.0g, dipotassium hydrogen phosphate 1.0g, magnesium sulfate 0.3g, poly- sorb
The 5.0g of ester 80, potassium sorbate 3g, water 1000mL；
It is made of following methods：In mentioned component in addition to dextrose and saccharose, dissolving is boiled, then adds dextrose and saccharose
After dissolving, shake up, adjust pH value 5.5 ~ 6.0, packing, 115 DEG C, 30min sterilizes；
（2）Lactobacillus agar medium is prepared, is solid agar medium, for the line culture of enrichment liquid, its composition is：Egg
White peptone 10.0g, glucose 5.0g, sucrose 5.0g, sodium chloride 3.0g, yeast extract powder 5.0g, CYSTINE 1.0g, phosphoric acid hydrogen two
Potassium 1.0g, agar 17g, potassium sorbate 3g, water 1000mL；
It is made of following methods：In mentioned component in addition to dextrose and saccharose, dissolving is boiled, then adds dextrose and saccharose
After dissolving, shake up, adjust pH value 5.5 ~ 6.0, packing, 115 DEG C, 30min sterilizes, pour plate；
（3）Test sample is taken, is seeded in lactobacillus enriched medium, 37 DEG C ± 1 DEG C, anaerobism, shaken cultivation 18~24 hours, is shaken
Shake 100~150r/min of speed；
（4）Above-mentioned culture is taken, streak inoculation is into lactobacillus agar medium, 37 DEG C ± 1 DEG C, anaerobism, and culture 24~48 is small
When；
（5）If occurring bacterium colony on plate has following characteristics：Canescence, bacterium colony center color is deeper, and circular protrusions are smooth,
Neat in edge, 1.0~1.5mm of colony diameter, and culture dish gives out lactic taste, then should select 2~3 bacterium colonies, connect respectively
Kind in lactobacillus agar medium, 37 DEG C ± 1 DEG C, anaerobism, cultivate 24~48 hours, take culture carry out Grain stain microscopy and
Suitable qualification test, judged whether to detect lactobacillus according to result；
（6）If grown on flat board without colony growth or without colonies typical, sentence product and do not detect lactobacillus.
2. detection method according to claim 1, it is characterised in that：The step（3）In, lactobacillus enriched medium
Dosage is be inoculated with test sample volume 5-10 times.
3. detection method according to claim 1, it is characterised in that：The minimum amount of inspection of test sample is as follows,
。
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| CN103031361A (en) * | 2011-09-30 | 2013-04-10 | 江苏春晖乳业有限公司 | Improved yoghourt lactic acid bacteria counting culture medium |
| JP5219058B2 (en) * | 2005-07-25 | 2013-06-26 | 株式会社明治 | Selection medium for lactic acid bacteria |
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| JP5219058B2 (en) * | 2005-07-25 | 2013-06-26 | 株式会社明治 | Selection medium for lactic acid bacteria |
| CN103031361A (en) * | 2011-09-30 | 2013-04-10 | 江苏春晖乳业有限公司 | Improved yoghourt lactic acid bacteria counting culture medium |
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