CN101445822A - Method for detecting diversity of prokaryotic microbe in fermented vegetable - Google Patents
Method for detecting diversity of prokaryotic microbe in fermented vegetable Download PDFInfo
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- CN101445822A CN101445822A CNA2008101027608A CN200810102760A CN101445822A CN 101445822 A CN101445822 A CN 101445822A CN A2008101027608 A CNA2008101027608 A CN A2008101027608A CN 200810102760 A CN200810102760 A CN 200810102760A CN 101445822 A CN101445822 A CN 101445822A
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Abstract
The invention provides a method for detecting the diversity of prokaryotic microbe in fermented vegetable. In the method, total DNA of contained microbe is extracted from a sample directly, and the sequence of 16S rRNA gene is analyzed, so as to determine the species of the microbe in the sample. In the method, DNA is extracted from the sample directly without any screening process, so that the obtained DNA can exactly reflect the species of the microbe in the fermented vegetable, avoiding the disadvantage that by adopting a culture isolation method, the actual micro ecological condition of a fermented vegetable system can not be reflected truly, and by adopting the method of the invention, the relative content of certain microbe in the fermented vegetable in the fermented vegetable system can be evaluated. The method does not require separating strains, thereby being capable of shortening detection time greatly.
Description
Technical field
The present invention relates to the microorganism detection technology, specifically a kind of method of using protokaryon microbial diversity in the molecule means detection fermented vegetables.
Background technology
The vegetable fermentation system is a kind of micro-ecological environment of uniqueness.The quality and the local flavor of microorganism wherein (prokaryotic micro-organisms) and fermented vegetables products have direct relation, to the diversity of microorganism in the fermented vegetables and institute's figure thereof investigate for improve traditional vegetable fermentation technology, probe into fermentation dish local flavor form aspect such as mechanism provide can be for reference data.According to document announcement, forefathers use microbial culture method
[1](analyze the method for lipid acid with non-cultural method
[2-3]With the analysis of cells utilization of carbon source
[4-5]) milk-acid bacteria in the fermented vegetables done the research of phylogenetic systematics method.The method for culturing and separating of microorganism is to insert the fermented vegetables water sample on the substratum of artificial preparation, with method of microorganism in the cultivation and fermentation vegetables water under the certain culture condition, this method is owing to nutrition in the substratum that uses and culture condition and the fermented vegetables system, envrionment conditions are incomplete same, can cause the enrichment and the decline of bacterial strain inevitably, this just the people for a change in the original fermented vegetables system the little ecological of flora constitute, can cause bigger deviation to result of study.May be in the fermented vegetables system in the highest flight or the very high flora of content in substratum, can't cultivate or grow very poor.But find that with the bacterial classification of culture of isolated method isolated advantage from fermented vegetables these bacteriums only are owing to can be cultivated out in a large number on the substratum after after deliberation on the contrary, thereby by the actual content and the effect of having over-evaluated them.In addition, occurring in nature has suitable bacterial classification (90%-99% is arranged approximately) can't cultivate out with substratum.Therefore there is very large deviation with microbe species and in esse microorganism in the method reflection fermented vegetables system of culture of isolated.And need the time long with microbial culture method, complex steps.Form with lipid acid and to identify that distinguishing relation microorganism far away has certain difficulty when non-cultural method is analyzed in the fermented vegetables system microbial diversity, its application is restricted; The utilization of carbon source analytical procedure is mainly based on the evaluation of Gram-negative bacteria, can not actual response fermented vegetables system in a large amount of microorganisms.And can reflect microbial diversity in the fermented vegetables fast, accurately with molecular biology method of the present invention.
Summary of the invention
The objective of the invention is to provides a kind of novel method that detects microbial diversity in the fermented vegetables at above-mentioned deficiency, this method has been avoided microorganism culturing and has been separated link, taking directly to extract contained microorganism total DNA from sample analyzes, understand the kind of the microbial DNA that is wherein comprised, and can estimate the relative content of various microorganisms.
The inventive method comprises the steps: to extract contained microorganism total DNA from sample, with total DNA is template, with 16SrRNA gene universal primer is primer amplification 16S rRNA gene, reclaim amplified production and set up the clone bacterium storehouse of 16S rRNA gene, measure the sequence of positive colony 16S rRNA gene, relatively determine its identity in the back with gene database.
Sample of the present invention can be a fermented liquid.Employed universal primer can be a section on the intestinal bacteria 16S rRNA.And can according to the product types that will specifically increase further determine primer sequence.Conventional method can be adopted in foundation clone bacterium storehouse, amplified production is reclaimed, and be connected in the back importing competence intestinal bacteria with the T carrier, cultivates the clone bacterium storehouse that has obtained 16S rRNA gene through selectivity.Before order-checking, can further confirm with the PCR method, and then deliver order-checking company and check order and to reduce cost the positive colony bacterium.
And can determine the relative content of this bacterium in the fermented vegetables system by calculating the ratio that a certain positive colony bacterium accounts for total positives clone bacterium.
The inventive method is owing to directly extract DNA from sample, middle process without any screening property, therefore, gained DNA can reflect microbe species in the fermented vegetables exactly, avoided method for culturing and separating can not truly reflect the shortcoming of the little ecological practical situation of fermented vegetables system, and can make assessment the content of a certain microorganism in the fermented vegetables system in the fermented vegetables.Because the inventive method does not need bacterial classification is separated, thereby can shorten detection time greatly.
Description of drawings
Fig. 1 is the qualification program that adopts ordinary method.
Embodiment
Following embodiment is used for further specifying of the present invention, but is not used for limiting the scope of the invention.
Kind and the content of milk-acid bacteria in the embodiment 1 usefulness 16S rRNA gene test pickles water
Get 10 milliliters in Pickles, Sichuan Style water, filter paper filtering with sterilization, with bacterial genomes DNA extraction test kit (day root [TIANGEN BIOTECH (BEIJING) CO. of Time Inc., LTD]) extract microorganism total DNA in the fermented vegetables filtrate, with this DNA is template, with bacterial 16 S rRNA gene universal primer is that primer carries out pcr amplification, and the upstream and downstream primer is respectively:
fD1：5′-AGAGTTTGACCTCCTCCTGGCTCAG-3′
rD1：5′-AAGGAGGTGATCCAGCC-3′
Amplification condition: reaction system and reaction conditions see Table 1 and table 2 respectively.
Table 1 PCR reaction system
Tab.1 Reaction system of PCR
Table 2 PCR reaction conditions
Tab.2 Reaction conditions of PCR
Reclaim amplified production and be connected with the T carrier, to connect product and import competence intestinal bacteria ToP10, with selecting culture medium culturing, screening positive clone (containing the bacterium colony that the purpose segment connects product), further confirm positive colony with PCR method, with the positive colony order-checking, sequencing result is compared in the GenBank storehouse respectively, found that to have 8 kinds of milk-acid bacterias in this example.See Table 3.
The 16S rDNA The sequencing results of milk-acid bacteria in the table 3 fermentation dish halogen
*Clone bacterium 16S rRNA gene order is with the homology degree after comparing in the GenBank storehouse
Detect the multifarious method steps of milk-acid bacteria in the fermented vegetables system with ordinary method:
1. the separation of milk-acid bacteria in the fermentation dish halogen
The separating lactic acid bacterium is with MRS and M17 substratum.MRS agar is used for the separating lactic acid bacillus, the milk-acid bacteria of leukonid and Pediococcus.M17 is used to separate lactococcus and faecalis.The dish halogen that will ferment dilution (10
5, 10
6, 10
7) the back MRS that inoculates, the M17 flat board was cultivated 48 o'clock 30 ℃ of anaerobism.Picking colony is transferred in the 10ml test tube that aseptic MRS and M17 broth culture are housed at random from different high density flat boards, carry out kind identify before with isolating bacterium colony on suitable nutrient agar with continuous scribble method purifying.The bacterium of totally 103 strain purifying.
Qualification program and the experimental project of milk-acid bacteria in the 2 fermentation dish halogen
Qualification program can carry out with reference to Fig. 1, and experimental project comprises:
1) Gram-reaction test
2) catalase reaction test
3) glucose aerogenesis experiment
4) temperature and salt concn are to the influence of microorganism
By top property experiment milk-acid bacteria is sorted out, choosing several strains from same type is that representative is carried out the utilization of carbon source experiment with API50CH reagent strip and API50CHL substratum, and makes qualification result with software.
3. the level that identifies genus of isolating milk-acid bacteria
Use API50CH reagent strip and API50CHL substratum according to the (APIsystem of manufacturer, Bio-Merieux, France) indication explanation is carried out the sugar-fermenting reaction to isolating 30 bacterial strains (every class is got 5 strains), test-results is by APILABPLUS (Version3.3.3, Bio-Merieux, France) program and criteria classification method are described.
API50CH reagent strip composition sees the following form
Table 4 API50CH reagent strip composition
Table 4 Reagent component of AP150CH
Experimental result:
The phenotypic evaluation test-results of milk-acid bacteria in the table 5 fermentation dish halogen
| Genus lactubacillus | The milk-acid bacteria number | Separating lactic acid bacterium sum | Percentage composition |
| Lactobacillus Pentosus | 27 | 90 | 3000 |
| Lactobacillus plantarum | 31 | 90 | 3444 |
| Lactobacillus fermentum | 2 | 90 | 222 |
| Lactobacillus brevis | 25 | 90 | 2778 |
| Leuconostoc mesenterotdes | 2 | 90 | 222 |
| Lactobactlluslactics | 3 | 90 | 333 |
The analysis of two kinds of methods is compared:
1. the experimental period of ordinary method is about three months, only needs can finish once week with the inventive method. shortened about 13 times detection time.
2. the milk-acid bacteria of identifying with ordinary method has six kinds, is respectively LactobacillusPentosus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus brevis, Leuconostoc mesenteroides, Lactobacilluslactics.Wherein the content of Lactobacillus plantarum is maximum, for separating 34.44% of total milk-acid bacteria, next is Lactobacillus Pentosus and Lactobacillus brevis, be respectively 30% and 27.78%, the content of Lactobacillus fermentum, Leuconostocmesenteroides, Lactobacillus lactics is few.
3. the milk-acid bacteria of identifying with the inventive method has eight kinds, is respectively Lactobacillusacidifarinae (25.46%), Lactobacillus plantarum (36.36%), Pediococcussp. (5.46%), Weissella confusa (7.27%) Lactobacillus pentosus (9.01%), Lactobacillus curvatus (10.91%), Leuconostoc mesenteroides (3.64%), Lactobacillus hammesii (1.81%).Wherein the content of Lactobacillus plantarum is maximum.This result with the conventional sense method is consistent, secondly be Lactobacillusacidifarinae (25.46%), this bacterium and Pediococcus sp. (5.46%), Weissellaconfusa (7.27%), Lactobacillus curvatus (10.91%), Lactobacillushammesii (1.81%) are with the undetected milk-acid bacteria of ordinary method.And Lactobacillus fermentum, Lactobacillus brevis, Lactobacilluslactics that ordinary method detects do not detect with present method, may adapt to the growth of these bacterium and relevant to the difference of the identification of bacterium with the composition of substratum.The present invention does not relate to other experiments of culture medium preparation and artificial identification bacterium, and therefore lactic acid bacteria diversity is real in the reaction fermentation dish, and can make correct estimation to the content of a certain bacterium.
Reference:
1, Qiu Zhiguang. the variation of microorganism and flavour substances in the pickled cabbage salting process: [doctorate paper]. Taiwan: food scientific research institute of National Chung Hsing University, 1993
2、Lee J.S.，Jung M.C.，Lee K.C.，et al..Identification of lactic acid bacteriafrom kimchi by cellular FAMEs analysis.Korean Journal of Applied Microbiologyand Biotechnology，1996a，24：234～241
3、Lee J.S.，Chun C.O.，Kim，H.J.，et al..Analysis of cellular fatty acid methylesters FAMEs.For the identification of Leuconostoc strains isolated from kimchi.Journal of Microbiology，1996b，34：225～228
4、Lee J.S.，Chun C.O.，Jung M.C.，et al..Classification of isolatesoriginationg from kimchi using carbon-source utilization patterns.Journal ofMicrobiology and Biotechnology，1997a，7：68～74
5、Lee J.S.，Chun C.O.，Hector M.，et al..Identification of Leuconostocstrains isolated from kimchi using carbon-source utilization patterns.Journal ofMicrobiology，1997b，35：10～14
Claims (6)
1, a kind of method that detects microbial diversity in the fermented vegetables, comprise step: the total DNA that from sample, extracts contained microorganism, with total DNA is template, with 16S rRNA gene universal primer is primer amplification 16S rRNA gene, reclaim amplified production and set up the clone bacterium storehouse of 16SrRNA gene, measure the sequence of positive colony 16S rRNA gene, relatively determine its identity in the back with gene database.
2, the method for claim 1 is characterized in that, this method also comprises by calculating the ratio that a certain positive colony bacterium accounts for total positives clone bacterium determines the relative content of this bacterium in the fermented vegetables system.
3, method as claimed in claim 1 or 2 is characterized in that, described universal primer is a bacterium 16sRNA gene universal primer.
4, method as claimed in claim 3 is characterized in that, described universal primer is: fD1:5 '-AGAGTTTGACCTCCTCCTGGCTCAG-3 '
rD1：5′-AAGGAGGTGATCCAGCC-3′。
5, as right 1 or 2 described methods, it is characterized in that, after amplified production is reclaimed, be connected, and import in the competence intestinal bacteria, cultivate the clone bacterium storehouse that obtains the 16SrRNA gene through selectivity with the T carrier.
6, method as claimed in claim 1 or 2 is characterized in that, also comprises before order-checking the positive colony bacterium is further confirmed with the PCR method.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103589789A (en) * | 2013-10-23 | 2014-02-19 | 上海美吉生物医药科技有限公司 | Composition and method for detecting and checking microbial diversity |
| CN107937581A (en) * | 2017-12-28 | 2018-04-20 | 内蒙古农业大学 | Amplimer for lactic acid bacteria sequencing is to, lactic acid bacteria kind identification method and application |
-
2008
- 2008-03-26 CN CNA2008101027608A patent/CN101445822A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103589789A (en) * | 2013-10-23 | 2014-02-19 | 上海美吉生物医药科技有限公司 | Composition and method for detecting and checking microbial diversity |
| CN103589789B (en) * | 2013-10-23 | 2016-03-16 | 上海美吉生物医药科技有限公司 | Composition and the method for check and correction is detected for microbial diversity |
| CN107937581A (en) * | 2017-12-28 | 2018-04-20 | 内蒙古农业大学 | Amplimer for lactic acid bacteria sequencing is to, lactic acid bacteria kind identification method and application |
| CN107937581B (en) * | 2017-12-28 | 2021-09-17 | 内蒙古农业大学 | Amplification primer pair for lactobacillus sequencing, lactobacillus species identification method and application |
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Open date: 20090603 |