CN102382874B - Biochemistry method for researching white spirit omagari microbial community structure - Google Patents
Biochemistry method for researching white spirit omagari microbial community structure Download PDFInfo
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Abstract
The invention relates to a biochemistry method for researching white spirit omagari microbial community structure, namely phospholipid fatty acid spectrogram technology. The biochemistry method comprises the following main steps: firstly, weighing certain mass of omagari; secondly, adding extracting solution and organic solvent, thus obtaining organic phase containing lipid; thirdly, utilizing the organic solvent for eluting the organic phase by a silicon pillar; fourthly, collecting eluent containing polar lipid; fifthly, conducting methyl esterification reaction on the obtained eluent under the weak base condition, thus obtaining fatty acid methyl ester; sixthly, utilizing a gas chromatograph-mass spectrometer for nature determining and quantifying on the fatty acid methyl ester; and seventhly, calculating the biomass live weight of living microorganism of characterization of the phospholipid fatty acid. The biochemistry method provides an accurate and fast technical measure for the white spirit omagari microbial community structure and related researching, controls the production process quality of the omagari, and has important and practical meaning of promoting and stabilizing the quality level of the white spirit omagari.
Description
Technical field:
Lipoid fatty acid (PLFA) spectrum analysis belongs to the biochemical research field.This method can the quantitative analysis microorganism, be mainly used in studying the diversity of microorganism in the complicated group, this method mainly is applied to the research of the aspects such as microbes biomass, structure of community, nutritional status and metabolic activity of testing environment sample in microbial ecology research.
Background technology:
Daqu is the carrier of net for catching fish or birds, enrichment, cultivation beneficial microorganism and meta-bolites thereof, as saccharifying agent, starter and living pastil in the brewed spirit process, aspects such as the yield of liquor of liquor, quality, quality there is great influence, it is the important substance guarantee of the fermented distilled yeast wine of conventional solid-state, the Daqu biological community structure has determined its metabolic function, biological community structure changes and will cause the change of group's metabolic function, finally influences the quality of liquor.
The vital role of Daqu in brewed spirit mainly is that microorganism by wherein and the relevant enzymes of generation thereof realize, Given this, domestic relevant enterprise and scientific research institution have carried out big quantity research to the Daqu microorganism all the time, Daqu microflora composition there has been certain understanding, but research in the past focuses mostly on traditional plate method, and there is bigger limitation in traditional microbial technique based on substratum in microbial ecology research.Under present condition, the most of microbe that nature exists can not or be difficult in the laboratory and carries out artificial culture and obtain its pure growth and carry out characteristic research such as physiology, form and heredity, there are some researches show that cultural method only can isolation identification go out to account for 0.1%～10% of environmental microorganism sum, seldom a part of information of microbial community structures can only be provided, and because the cultivation of microorganism process orthoselection has changed original structure in the sample, can't reflect the original appearance of biological community structure in the sample.
PLFA is the important component that constitutes the active somatic cell film, has structure diversity and high biological specificity, is effective especially biomarker.The microorganism of different monoids can form different PLFA by different biochemical route, has different PLFA kind and quantity in the different microorganisms monoid, the PLFA feature spectrogram difference of different floras, have diversity on height specificity basis, can be used as the marker of different groups in the microflora.It is relatively significantly constant that phospholipids content in the biological cell is considered to usually, the change of PLFA spectrogram mainly since in the sample microbe species and quantity change and cause.Because PLFA will very fast degraded to a few hours inner cell enzymic hydrolysis and release phosphatide in several minutes after the necrocytosis.Therefore the different microorganisms monoid microorganism biological total amount of potential fecundity can quantitative response can be bred or have to the content by measuring PLFA, and this is that PLFA is as the basis of distinguishing living microorganism group mark.
PLFA spectrogram technology is a kind of method of quick, reliable, reproducible analysis biological community structure, can be used for being characterized in dominant microflora quantitatively, and comprising can not culturing micro-organisms.Sample is not had particular requirement, and the information of obtaining is provided by the microorganism of sample basically, and operation is easy relatively in addition, and analysis time is shorter.
Utilize Phospholipid Fatty Acid Profile diagram technology research Daqu biological community structure can overcome the limitation of traditional method to a great extent, this method need not be through cultivating, do not change the original appearance of microflora in the sample, the biomass information of obtaining is comprised and can not cultured microorganism be provided by the living microorganism of sample basically.
The Phospholipid Fatty Acid Profile diagram technology is studied more aspect farmland, artificial swamp, soil microbial community diversity, and do not appearing in the newspapers as yet aspect the research of Daqu microflora, PLFA spectrogram technology is introduced in the Daqu research on microbial community structure, the useful information of spirit quality stability and good and bad degree can be provided, also provide certain thinking and technical support for setting up spirit quality evaluation technique system simultaneously.
Summary of the invention:
The objective of the invention is do not carrying out under the prerequisite of microorganism culturing, a kind of biochemical method of studying liquor Daqu biological community structure is provided, solves and to reflect in the research of Daqu microflora that at present living microorganism (comprise can not culturing micro-organisms) forms the problem of overall picture.
The concrete scheme of the present invention is as follows:
A) get 5g Daqu sample and in the 50mL centrifuge tube of tinfoil parcel, add the 15mL chloroform: methyl alcohol: citrate buffer solution=1: 2: 0.8, V: V: V fully shakes up, the low-temperature dark standing over night;
B) continue to add the 7.5mL chloroform in the centrifuge tube in the step a), 7.5mL phosphoric acid buffer, low-temperature dark standing over night again after shaking up;
C) the centrifugal 10min of 6000r/min room temperature carefully sops up supernatant liquor with disposable sterilized suction pipe, and to the little triangular flask of tinfoil parcel, gained liquid dries up with high pure nitrogen with filter paper filtering in lower floor;
D) add 300 μ L chloroforms dissolvings in the triangular flask in step c) after, with suction pipe with whole sample transfer to silicagel column, carry out wash-out with 6mL chloroform, 10mL acetone, 6mL anhydrous methanol respectively, collect the anhydrous methanol elutriant with the tool plug scale test tube of tinfoil parcel at last, gained liquid dries up with high pure nitrogen;
E) add 1mL methyl alcohol in the test tube in step d) respectively: toluene mixture liquid 1: 1, V: V, 1mL0.2mol/L potassium hydroxide methanol solution, behind 37 ℃ of water-bath 15min, add 0.3mL1mol/L acetic acid again, 2mL normal hexane chloroform mixed solution=4: 1, V: V and 2mL aseptic deionized water, fully concussion, after leaving standstill the 30min layering, the upper strata carefully is transferred in another new clean test tube, high pure nitrogen dries up, add and contain the interior target normal hexane of a certain amount of nondecylic acid methyl ester (nonadecanoic acid methyl ester): chloroform=4: 1, V: V solution, to be measured;
F) gas chromatograph-mass spectrometer is measured the liquid to be measured of step e).Chromatographic condition is as follows: keep 1min at 50 ℃ earlier behind the sample introduction, rise to 180 ℃ and keep 2min with the speed of 12 ℃/min, speed with 6 ℃/min rises to 220 ℃ again, keep 2min, speed with 15 ℃/min rises to 240 ℃ afterwards, keep 1min, rise 260 ℃ with 15 ℃/min at last, and keep 15min.Injector temperature is 230 ℃, and gas phase is 280 ℃ with the temperature that is connected between the mass spectrum, and adopts the split stream sampling pattern, splitting ratio 10: 1, and carrier gas is high-purity helium.Mass spectrograph adopts electron ionization (EI) mode, and electron energy is 70eV;
G) according to mass spectrum and java standard library detected lipoid fatty acid is identified, adopted marker method to calculate the relative content of every kind of lipoid fatty acid;
H) corresponding with the microorganism of PLFA sign, and the calculation sample microbes biomass.
Advantage of the present invention is the limitation of traditional microbial technique in the research of Daqu microflora that has overcome based on substratum, the microorganism that can reflect in the sample survival or have a potential fecundity comprises can not cultured microorganism biomass information, has simply, characteristics fast and accurately.
The invention has the beneficial effects as follows improving the Daqu production technique, stablizing the spirit quality level and be of great practical significance, because the change of PLFA spectrogram mainly since in the sample microbe species and quantity change and cause, be that change has taken place biological community structure, can influence significant process procedure to microflora for investigation and definite Daqu production process, propose control and innovative approach on this basis.
Embodiment:
For a better understanding of the present invention, describe in conjunction with example.
Be experimental subjects with a certain place of production different batches and dissimilar Daqu, under aseptic condition, with the knee-piece fragmentation, and cross 20 mesh sieves, standby; Get 5g Daqu powder and in the 50mL centrifuge tube of tinfoil parcel, add the 15mL chloroform: methyl alcohol: citrate buffer solution=1: 2: 0.8, V: V:V fully shakes up, and stand at low temperature is spent the night.In centrifuge tube, continue to add the 7.5mL chloroform, the 7.5mL phosphoric acid buffer, after shaking up again stand at low temperature spend the night; The centrifugal 10min of 6000r/min room temperature carefully sops up supernatant liquor with disposable sterilized suction pipe, and to the little triangular flask of tinfoil parcel, gained liquid dries up with high pure nitrogen with filter paper filtering in lower floor; After in triangular flask, adding the dissolving of 300 μ L chloroforms, with suction pipe with whole sample transfer to silicagel column, carry out wash-out with 6mL chloroform, 10mL acetone, 6mL anhydrous methanol respectively, collect the anhydrous methanol elutriant with the tool plug scale test tube of tinfoil parcel at last, gained liquid dries up with high pure nitrogen; In above-mentioned test tube, add 1mL methyl alcohol respectively: toluene mixture liquid=1: 1, V: V, 1mL0.2mol/L potassium hydroxide methanol solution, behind 37 ℃ of water-bath 15min, add 0.3mL1mol/L acetic acid, 2mL normal hexane chloroform mixed solution=4: 1 again, V: V and 2mL aseptic deionized water, fully concussion, after leaving standstill the 30min layering, the upper strata carefully is transferred in another new clean test tube, high pure nitrogen dries up, add and contain target normal hexane in a certain amount of nondecylic acid methyl ester: chloroform=4: 1, V: V solution, to be measured.
Adopt gas chromatograph-mass spectrometer that liquid to be measured is measured, chromatographic condition is as follows: keep 1min at 50 ℃ earlier behind the sample introduction, rise to 180 ℃ and keep 2min with the speed of 12 ℃/min, speed with 6 ℃/min rises to 220 ℃ again, keep 2min, the speed with 15 ℃/min rises to 240 ℃ afterwards, keeps 1min, rise 260 ℃ with 15 ℃/min at last, and keep 15min.Injector temperature is 230 ℃, and gas phase is 280 ℃ with the temperature that is connected between the mass spectrum, and adopts the split stream sampling pattern, splitting ratio 10: 1, and carrier gas is high-purity helium.Mass spectrograph adopts electron ionization (EI) mode, and electron energy is 70eV.
According to mass spectrum and java standard library detected lipoid fatty acid is identified, and calculated the relative content of every kind of lipoid fatty acid, corresponding with the microorganism that PLFA characterizes according to table 1, and calculation sample living microorganism biomass.
Table l characterizes the PLFA of specified microorganisms
Table 2 is that different batches and the dissimilar Daqu biological community structure in a certain place of production formed and biomass, and wherein PB represents dull and stereotyped song, BB representative bag Bao Qu, the digitized representation production batch behind the letter.
Table 2 is that certain place of production of basic calculation biological community structure dissimilar and the different batches Daqu is formed and biomass with PLFA
Claims (2)
1. biochemical method of studying liquor Daqu biological community structure is characterized in that may further comprise the steps:
A) select for the Daqu sample of analyzing;
B) under the aseptic condition after the fragmentation, take by weighing 5g Daqu sample, add the 15mL chloroform: methyl alcohol: citrate buffer solution=1: 2: 0.8, V: V: V after the lucifuge standing over night, adds 7.5mL chloroform and 7.5mL citrate buffer solution respectively again, lucifuge standing over night again obtains to contain the organic phase of lipid;
C) with the organic phase that obtains in the step b) through silicagel column, use different organic solvents that dissimilar lipids are carried out wash-out respectively, and collect the elutriant that contains polar lipid;
D) with the elutriant that contains polar lipid that obtains in the step c), under certain temperature and weak basic condition, carry out esterification reaction of organic acid, obtain fatty acid methyl ester, wherein the solvent that adds in this step is the mixed solution of 1: 1 volume ratio of 1mL methanol toluene, 1mL0.2mol/L potassium hydroxide methanol solution, and at 37 ℃ of water-bath 15min;
E) with the fatty acid methyl ester that obtains in the step d), add 0.3mL1mol/L acetic acid, 2mL normal hexane: chloroform=4: 1, the mixed solution of V: V and 2mL deionized water, vibration separates, and standing demix extracts organic phase;
F) organic phase that obtains in the step e) is dried up with high pure nitrogen, add and contain in a certain amount of interior target organic solvent;
G) use gas chromatograph-mass spectrometer that the solution that obtains in the step f) is measured, obtain kind and the quantity information of lipoid fatty acid in the sample;
H) by the biomass of the resulting data computation sign of step g) living microorganism, know information of microbial community structures in the Daqu sample.
2. the method for claim 1 is characterized in that, the laboratory sample in the step a) can be the different places of production, different batches and dissimilar Daqu samples.
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| CN109752510B (en) * | 2019-03-11 | 2021-06-29 | 中国科学院亚热带农业生态研究所 | Phospholipid fatty acid biomarker of rhizobium meliloti and biomass measurement and calculation method |
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Non-Patent Citations (4)
| Title |
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| 徐泽江等.芝麻香型白酒堆积过程微生物群落结构特征的PLFA分析.《食品与发酵工业》.2012,第38卷(第4期),20-24. |
| 磷脂脂肪酸谱图分析方法及其在微生物生态学领域的应用;齐鸿雁等;《生态学报》;20030831;第23卷(第8期);1576-1582 * |
| 芝麻香型白酒堆积过程微生物群落结构特征的PLFA分析;徐泽江等;《食品与发酵工业》;20120430;第38卷(第4期);20-24 * |
| 齐鸿雁等.磷脂脂肪酸谱图分析方法及其在微生物生态学领域的应用.《生态学报》.2003,第23卷(第8期),1576-1582. |
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