CN102115777B - Screening method of lactic acid bacteria strains of high-yield DL-3-phenyllactic acid - Google Patents
Screening method of lactic acid bacteria strains of high-yield DL-3-phenyllactic acid Download PDFInfo
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 100
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Abstract
The invention belongs to the field of analytical chemistry and applied microbiology, and relates to a screening method of lactic acid bacteria strains of high-yield DL-3-phenyllactic acid, which applies HPLC (high-performance liquid chromatography) to analyze the content of DL-3-phenyllactic acid in MRS (DeMan-Rogosa-Sharpe broth) fermentation supernatant fluid qualitatively and quantitatively, so as to rapidly screen the lactic acid bacteria strains of high-yield DL-3-phenyllactic acid. Through reversed-phase ion suppression technology, 0.5 percent (V/V) of phosphoric acid is added to a mobile phase, so as to suppress the dissociation of DL-3-phenyllactic acid; an Agilent Zorbax SB-C18 (4.6mm multiplied by 150mm, 5mu.m) chromatographic column is used to separate DL-3-phenyllactic acid from impurities in the lactic acid bacteria MRS fermentation supernatant fluid; and DL-3-phenyllactic acid is detected qualitatively through the comparison between the retention time of a standard sample and the retention time of a phenyllactic acid sample in the lactic acid bacteria MRS fermentation supernatant fluid and a spectrogram, and is detected quantitatively through an external standard method. The screening method has the advantages of rapidity, accuracy and the like, and the sample is easy to pre-treat, thereby proving a technical means for rapid screening and qualitative and quantitative detection of the lactic acid bacteria strains of the high-yield phenyllactic acid.
Description
Technical field:
The invention belongs to analytical chemistry and applied microbiology field, be specifically related to measure phenyllactic acid content (phenyllactic acid, PLA) in MRS fermentation supernatant by using HPLC, thus the method for screening high yield phenyllactic acid lactic bacterium strains.
Background technology:
In recent years, Chemical Preservative and residue of veterinary drug be the health of harm humans and animal day by day, and the exploitation of wide spectrum, efficient, stable, safe novel antibacterial material has become the task of top priority with research.Milk-acid bacteria is the normal microflora in human body and animal intestinal, is also the food-grade microorganisms (Food-grade Microorganisms) of generally recognized as safe (GRAS, Generally Recognized As Safe).milk-acid bacteria not only gives leavened food distinctive local flavor in the food fermentation process, quality and structure, and can also produce the multiple bacteriocin (bacteriocin) that can effectively suppress the growth and reproduction of food-borne pathogenic microorganism, ring dipeptides (cyclic dipeptides), organic acid (organic acid), hydrogen peroxide (hydrogen peroxid), the antibacterial factors such as two second phthaleins (diacetyl), based on the security of milk-acid bacteria and the critical role in foodstuffs industry, research emphasis and the focus in biological preservation (biopreservation) field have been become with milk-acid bacteria and meta-bolites development of new biological preservative thereof.
DL-3 phenyllactic acid (DL-3-phenyllactic acid, PLA), namely PLA is a kind of New Biological Preservatives that can be produced by part milk-acid bacteria secretion of discovered in recent years, and two kinds of corresponding isomer of D-phenyllactic acid and L-phenyllactic acid be arranged.It can suppress the multiple fungi that causes gram-positive microorganism, Gram-negative bacteria and the generation mycotoxin of food spoilage preferably.It is safe, nontoxic that phenyllactic acid has, and do not affect the local flavor of food and mouthfeel under lower concentration, have the advantages such as the livestock immunity of raising and meat as novel fodder additive.In addition, one of derivative of DL-3 phenyllactic acid or Salvianic acidA (β-3,4-DLA sodium) has and the similar pharmacological action of Salvianic acidA, can anticoagulant, suppress thrombosis and improve microcirculation, ischemic heart disease is had certain pharmaceutical use.Along with the pay attention to day by day of China to food safety, the development and utilization of DL-3 phenyllactic acid is with a wide range of applications.
The at present known lactic bacterium strains output that can secrete the generation phenyllactic acid is lower, take MRS (De Man-Rogosa-Sharpe broth) as substratum, in the situation that be not 99mg/L through any condition optimizing production peak, seriously limited biosynthesizing and the application of DL-3 phenyllactic acid, so the screening of this organic acid bacterial strain of high yield is imperative.Yet screening method must be based upon a kind of easy, fast and on detection method basis that simultaneously can be qualitative and quantitative.The detection method of phenyllactic acid mainly contains thin-layer chromatography, high resolution gas chromatography mass spectrometry, LC-MS and high performance liquid chromatography at present.Thin-layer chromatography can only detect phenyllactic acid by quantitative and semi-quantitative, and high resolution gas chromatography mass spectrometry and LC-MS have high-throughput, level of automation and resolving power advantages of higher, but price is high, is not suitable for Routine Test Lab and carries out general sieve; And the HPLC detection method of known phenyllactic acid need to be such as the complicated sample pre-treatments such as liquid-liquid extraction, liquid-solid extraction and pH regulator, and completing a sample detection needs 2-3 hour at least, can not adapt to the needs that real-time analysis detects.MRS substratum (DeMan-Rogosa-Sharpe broth) is the selective medium of milk-acid bacteria, is also the first-selected substratum that milk-acid bacteria is carried out related science research.Therefore, set up the fast and convenient method that detects benzene DL-3 phenyllactic acid from the MRS fermented liquid, significant for rapid screening and the qualitative and quantitative detection of high yield DL-3 phenyllactic acid lactic bacterium strains.
Summary of the invention:
The objective of the invention is to use HPLC DL-3-phenyllactic acid in milk-acid bacteria MRS fermentation supernatant is carried out after qualitative and quantitative measures, set up a kind of screening system of quick and easy high yield phenyllactic acid lactic bacterium strains.
Above-mentioned purpose of the present invention is to be achieved by following technical scheme:
The screening method of DL-3-phenyllactic acid lactic bacterium strains comprises that the configuration of substratum and sample pre-treatments, HPLC carry out qualitative and quantitative analysis to DL-3-phenyllactic acid content in MRS fermentation supernatant.
4 ℃ of milk-acid bacteria MRS fermented liquids are adopted in the preparation of said culture supernatant, the centrifugal 10min of 6000rpm, and getting supernatant liquor, to cross 0.45 μ m filter membrane be milk-acid bacteria MRS fermentation supernatant.
Said sample pre-treatments is configuration DL-3-phenyllactic acid standard stock solution and working fluid: accurately take (100 ± 0.01) mg DL-3-phenyllactic acid standard substance, be settled to 100mL with dissolve with methanol, be 1g/LDL-3-phenyllactic acid standard stock solution, in-4 ℃ of preservations; Draw DL-3-phenyllactic acid standard stock solution, take methyl alcohol as the solvent constant volume, compound concentration is 0,60,100,200,400,600, the DL-3-phenyllactic acid standard operation liquid of 800mg/L.
Said is to adopt reversed phase ion inhibition technology with PLC to DL-3-phenyllactic acid content qualitative detection in MRS fermentation supernatant, add people 0.5% phosphoric acid V/V in moving phase, suppressing the DL-3-phenyllactic acid dissociates, utilize Agilent Zorbax SB-C18 (4.6mm * 150mm, 5 μ m) chromatographic column, the DL-3-phenyllactic acid is separated with impurity in milk-acid bacteria MRS fermented supernatant fluid, carry out DL-3-phenyllactic acid qualitative detection by retention time and the spectrogram that contrasts DL-3-phenyllactic acid in standard specimen and milk-acid bacteria MRS fermentation supernatant.
It is DL-3-phenyllactic acid standard operation liquid sample introduction analysis with a series of concentration known that said HPLC detects DL-3-phenyllactic acid content quantitative in MRS fermentation supernatant, take DL-3-phenyllactic acid sample concentration as X-coordinate, peak area is ordinate zou Criterion curve, and calculates regression equation.
4 ℃ of milk-acid bacteria MRS fermented liquids are adopted in the configuration of said substratum, the centrifugal 10min of 6000rpm, and getting supernatant liquor, to cross 0.45 μ m filter membrane be milk-acid bacteria MRS fermentation supernatant; Said sample pre-treatments is configuration DL-3-phenyllactic acid standard stock solution and working fluid: accurately take (100 ± 0.01) mg DL-3-phenyllactic acid standard substance, be settled to 100mL with dissolve with methanol, be 1g/L DL-3-phenyllactic acid standard stock solution, in-4 ℃ of preservations; Draw DL-3-phenyllactic acid standard stock solution, take methyl alcohol as the solvent constant volume, compound concentration is 0,60,100,200,400,600, the DL-3-phenyllactic acid standard operation liquid of 800mg/L; Said HPLC is to adopt reversed phase ion inhibition technology to DL-3-phenyllactic acid content qualitative detection in MRS fermentation supernatant, add people 0.5% phosphoric acid V/V in moving phase, suppressing the DL-3-phenyllactic acid dissociates, utilize AgilentZorbax SB-C18 (4.6mm * 150mm, 5 μ m) chromatographic column, the DL-3-phenyllactic acid is separated with impurity in milk-acid bacteria MRS fermented supernatant fluid, carry out DL-3-phenyllactic acid qualitative detection by retention time and the spectrogram that contrasts DL-3-phenyllactic acid in standard specimen and milk-acid bacteria MRS fermentation supernatant; It is DL-3-phenyllactic acid standard operation liquid sample introduction analysis with a series of concentration known that said HPLC detects DL-3-phenyllactic acid content quantitative in MRS fermentation supernatant, take DL-3-phenyllactic acid sample concentration as X-coordinate, peak area is ordinate zou Criterion curve, and calculates regression equation.
Further this screening method being carried out precision, repeatability, stability and recovery of standard addition detects, it is to get that concentration is respectively 100,200, the DL-3-phenyllactic acid standard operation liquid of 400mg/L repeats respectively sample introduction and analyzes for 5 times, and calculates 3 peak area relative standard deviations that concentration is corresponding that said precision detects; Said repeatability and Detection of Stability be within a working days duplicate detection concentration be respectively 100,200, the DL-3-phenyllactic acid standard operation liquid 5 times (withinday precision) of 400mg/L, and every 24h, sample solution is detected, repetitive operation 5 times (day to day precision), and the peak area relative standard deviation of calculating; It is that DL-3-phenyllactic acid standard substance with 3 concentration gradients add in the known milk-acid bacteria MRS fermentation supernatant culture supernatant of concentration that recovery of standard addition detects, and it is carried out replication 5 times, and the peak area relative standard deviation of calculating.
Technical scheme of the present invention can be summarized as: the rapid screening system of the lactic bacterium strains of high yield DL-3-phenyllactic acid.the DL-3-phenyllactic acid is acidulous material (pKa=3.46), easily occur from solution in neutral moving phase, adopt reversed phase ion to suppress technology, add people's 0.5% phosphoric acid (V/V) in moving phase, suppressing the DL-3-phenyllactic acid dissociates, utilize Agilent Zorbax SB-C18 (4.6mm * 150mm, 5 μ m) chromatographic column, the DL-3-phenyllactic acid is separated with impurity in milk-acid bacteria MRS fermented supernatant fluid, retention time and spectrogram by phenyllactic acid sample in contrast standard specimen and milk-acid bacteria MRS fermentation supernatant carry out DL-3-phenyllactic acid qualitative detection, external standard method is carried out detection by quantitative.
The HPLC chromatographic condition: the high performance liquid chromatograph model is Agilent 1200: chromatographic column: Agilent Zorbax SB-C18 (4.6mm * 150mm, 5 μ m) chromatographic column; Moving phase: A:0.5% phosphoric acid (V/V), B:0.5% phosphoric acid acetonitrile solution (V/V); Flow velocity 1mL/min; Column temperature: 30 ℃; Sample size: 5 μ L; Detect wavelength 210nm.
Description of drawings:
Fig. 1 is D-phenyllactic acid and two kinds of corresponding isomer of L-phenyllactic acid;
Fig. 2 is DL-3-phenyllactic acid standard substance ultraviolet spectrograms;
Fig. 3 is DL-3-phenyllactic acid ultraviolet spectrogram in milk-acid bacteria MRS fermentation supernatant;
Fig. 4 is DL-3-phenyllactic acid standard substance color atlass;
Fig. 5 is milk-acid bacteria MRS fermentation supernatant color atlas;
Fig. 6 is DL-3-phenyllactic acid typical curve;
Fig. 7 is for producing the rapid screening result of DL-3 phenyllactic acid lactic bacterium strains.
Specific embodiments:
Configuration and the sample pre-treatments of embodiment 1:1. substratum:
1.1MRS the preparation of substratum:
MRS (De Man-Rogosa-Sharpe broth) substratum: be purchased from Britain Oxoid company, substratum forms: peptone 10.0g/L, Lab-lemco Powder8.0g/L, Yeast extract4.0g/L, Glucose20.0g/L, Tween-80 1.0ml, dipotassium hydrogen phosphate 2.0g/L, sodium-acetate 5.0g/L, anhydrous magnesium sulfate 0.2g/L, four water manganous sulfate 0.05g/L, Triammonium citrate 2.0g/L.Take MRS powder 52g, fully use ddH after dissolving
2O is settled to 1L, test tube packing, every 5mL, the wrapping of jumping a queue, mark, 121 ℃ of autoclaving 15min, cooling rear 4 ℃ of preservations.
1.2 milk-acid bacteria MRS fermented liquid preparation:
The milk-acid bacteria pure strain that-80 ℃ of refrigerator and cooled is frozen preservation takes out, and is placed in Biohazard Safety Equipment, and after it melted liquefaction, mixing was got 10 μ L with micropipet and is inoculated in 5mL sterilization MRS broth culture, is placed in (36 ± 1) ℃ shaking table and cultivates 24h.
1.3 the preparation of milk-acid bacteria MRS fermentation supernatant:
4 ℃ of milk-acid bacteria MRS fermented liquids, the centrifugal 10min of 6000rpm gets and is milk-acid bacteria MRS fermentation supernatant after supernatant liquor is crossed 0.45 μ m filter membrane.
2. the high performance liquid chromatography real-time analysis of DL-3-phenyllactic acid in milk-acid bacteria MRS fermentation supernatant
2.1DL-3-phenyllactic acid standard stock solution preparation:
Accurately take (100 ± 0.01) mg DL-3-phenyllactic acid standard substance, be settled to 100mL with methyl alcohol (chromatographically pure) dissolving, be 1g/LDL-3-phenyllactic acid standard stock solution ,-4 ℃ of preservations.
2.2DL-3-the configuration of phenyllactic acid standard operation liquid:
Draw DL-3-phenyllactic acid standard stock solution, take methyl alcohol as the solvent constant volume, compound concentration is 0,60,100,200,400,600, the DL-3-phenyllactic acid standard operation liquid of 800mg/L.
2.3 the qualitative analysis of DL-3-phenyllactic acid in milk-acid bacteria MRS fermentation supernatant:
HPLC chromatographic condition: chromatographic column: Agilent Zorbax SB-C18 (4.6mm * 150mm, 5 μ m) chromatographic column; Moving phase: A:0.5% phosphoric acid (V/V), B:0.5% phosphoric acid acetonitrile solution (V/V); Flow velocity 1mL/min; Column temperature: 30 ℃; Sample size: 5 μ L; Detect wavelength 210nm; Elution program sees Table 1.Adopt the retention time of phenyllactic acid sample in standard specimen and milk-acid bacteria MRS fermentation supernatant and the method for spectrogram contrast to carry out the phenyllactic acid qualitative analysis.
Table 1 HPLC gradient elution program
The DL-3-phenyllactic acid is acidic substance, easily occurs from solution in neutral moving phase, and during as moving phase, the DL-3-phenyllactic acid is shorter in the retention time of stationary phase, can't separate with other components in the MRS culture supernatant, and the chromatographic peak hangover is serious with water and acetonitrile.Therefore this patent has adopted reversed phase ion inhibition technology, adopt 0.5% phosphate aqueous solution (V/V) and: 0.5% phosphoric acid acetonitrile solution (V/V) is during as moving phase, in sample the chromatographic peak of phenyllactic acid with other not the principal component chromatographic peak can access good separating (R＞1.5), peak shape is improved simultaneously, has reduced the chromatographic peak conditions of streaking.Adopt the retention time of standard specimen and sample and the method for spectrogram contrast to carry out qualitative analysis.Carry out contained DL-3-phenyllactic acid full wavelength scanner in DL-3-phenyllactic acid standard model and sample between 190～400nm, both absorbing state basically identical (Fig. 2-3).Under 2.3 chromatographic conditions, in milk-acid bacteria MRS fermentation supernatant, DL-3-phenyllactic acid and other compositions can reach good separation, resolution＞1.5.The retention time of DL-3-phenyllactic acid is 8.879min, and 15min can complete the real-time analysis (Fig. 4-5) of a sample.
2.4 the foundation of DL-3-phenyllactic acid quantitative analysis typical curve in milk-acid bacteria MRS fermentation supernatant:
Under 2.3 chromatographic conditions, to 60,100,200,400,600, the DL-3-phenyllactic acid standard operation liquid sample introduction of 800mg/L analyzes, each concentration repeats sample introduction three times.Take DL-3-phenyllactic acid sample concentration as X-coordinate, peak area is ordinate zou Criterion curve, and calculates regression equation (Fig. 6).Result shows, DL-3-phenyllactic acid chromatographic peak area and concentration are linear dependence, and its equation of linear regression is: y=14.723x+0.1325, r=0.9999 (x is sample concentration, and y is peak area).
The methodological study of the HPLC rapid assay methods of DL-3-phenyllactic acid in 3 milk-acid bacteria MRS fermentation supernatants:
3.1 precision:
Get that concentration is respectively 100,200, the DL-3-phenyllactic acid standard operation liquid of 400mg/L repeats respectively sample introduction and analyzes for 5 times.Peak area relative standard deviation corresponding to above-mentioned 3 concentration is respectively 0.84%, 0.55%, 0.27%, shows that present method precision is good.
3.2 repeatability and stable:
Under 2.3 chromatographic conditions, within a working days duplicate detection concentration be respectively 100,200, the DL-3-phenyllactic acid standard operation liquid 5 times (withinday precision) of 400mg/L, and every 24h, sample solution is detected, repetitive operation 5 times (day to day precision), the results are shown in Table 2, show that this detection method has good reproducibility and measurement result is stable in 72h.
3.3 recovery of standard addition experiment:
The DL-3-phenyllactic acid standard substance of 3 concentration gradients are added in the known milk-acid bacteria MRS fermentation supernatant of concentration, prepare liquid to be measured according to the method for cleanup standard product, utilize the method for determining under 2.3 chromatographic conditions to carry out replication 5 times to it, detected result sees Table 3.Result shows, present method rate of recovery is high.
Table 2 DL-3-phenyllactic acid recovery of standard addition measurement result
Table 3 DL-3-phenyllactic acid in a few days with the day to day precision measurement result
4. produce the rapid screening of DL-3 phenyllactic acid lactic bacterium strains:
Use above-mentioned definite HPLC detection system, DL-3-phenyllactic acid in 7 strains of lactic acid bacteria MRS fermentation supernatants is carried out Fast Measurement, detected result is seen (Fig. 7).
Claims (5)
1.DL-3-the screening method of phenyllactic acid lactic bacterium strains comprises that the configuration of substratum and sample pre-treatments, HPLC carry out qualitative and quantitative analysis to DL-3-phenyllactic acid content in MRS fermentation supernatant;
4 ℃ of milk-acid bacteria MRS fermented liquids are adopted in the configuration of said substratum, the centrifugal 10min of 6000rpm, and getting supernatant liquor, to cross 0.45 μ m filter membrane be milk-acid bacteria MRS fermentation supernatant;
Said sample pre-treatments is configuration DL-3-phenyllactic acid standard stock solution and working fluid: accurately take (100 ± 0.01) mg DL-3-phenyllactic acid standard substance, be settled to 100mL with dissolve with methanol, be 1g/L DL-3-phenyllactic acid standard stock solution, in-4 ℃ of preservations; Draw DL-3-phenyllactic acid standard stock solution, take methyl alcohol as the solvent constant volume, compound concentration is 0,60,100,200,400,600, the DL-3-phenyllactic acid standard operation liquid of 800mg/L;
Said HPLC is to adopt reversed phase ion inhibition technology to DL-3-phenyllactic acid content qualitative detection in MRS fermentation supernatant, add people 0.5% phosphoric acid V/V in moving phase, suppressing the DL-3-phenyllactic acid dissociates, utilize AgilentZorbax SB-C18 (4.6mm * 150mm, 5 μ m) chromatographic column, the DL-3-phenyllactic acid is separated with impurity in milk-acid bacteria MRS fermented supernatant fluid, carry out DL-3-phenyllactic acid qualitative detection by retention time and the spectrogram that contrasts DL-3-phenyllactic acid in standard specimen and milk-acid bacteria MRS fermentation supernatant;
It is DL-3-phenyllactic acid standard operation liquid sample introduction analysis with a series of concentration known that said HPLC detects DL-3-phenyllactic acid content quantitative in MRS fermentation supernatant, take DL-3-phenyllactic acid sample concentration as X-coordinate, peak area is ordinate zou Criterion curve, and calculates regression equation.
2. screening method as claimed in claim 1 is characterized in that 4 ℃ of milk-acid bacteria MRS fermented liquids are adopted in the configuration of said substratum, the centrifugal 10min of 6000rpm, and getting supernatant liquor, to cross 0.45 μ m filter membrane be milk-acid bacteria MRS fermentation supernatant.
3. screening method as claimed in claim 1, it is characterized in that said sample pre-treatments is configuration DL-3-phenyllactic acid standard stock solution and working fluid: accurately take (100 ± 0.01) mgDL-3-phenyllactic acid standard substance, be settled to 100mL with dissolve with methanol, be 1g/L DL-3-phenyllactic acid standard stock solution, in-4 ℃ of preservations; Draw DL-3-phenyllactic acid standard stock solution, take methyl alcohol as the solvent constant volume, compound concentration is 0,60,100,200,400,600, the DL-3-phenyllactic acid standard operation liquid of 800mg/L.
4. screening method as claimed in claim 1, it is characterized in that said HPLC is to adopt reversed phase ion inhibition technology to DL-3-phenyllactic acid content qualitative detection in MRS fermentation supernatant, add people 0.5% phosphoric acid V/V in moving phase, suppressing the DL-3-phenyllactic acid dissociates, utilize Agilent Zorbax SB-C18 (4.6mm * 150mm, 5 μ m) chromatographic column, the DL-3-phenyllactic acid is separated with impurity in milk-acid bacteria MRS fermented supernatant fluid, carry out DL-3-phenyllactic acid qualitative detection by retention time and the spectrogram that contrasts DL-3-phenyllactic acid in standard specimen and milk-acid bacteria MRS fermentation supernatant.
5. screening method as claimed in claim 1, it is characterized in that it is DL-3-phenyllactic acid standard operation liquid sample introduction analysis with a series of concentration known that said HPLC detects DL-3-phenyllactic acid content quantitative in MRS fermentation supernatant, take DL-3-phenyllactic acid sample concentration as X-coordinate, peak area is ordinate zou Criterion curve, and calculates regression equation.
As claim 1～
Described screening method, it is characterized in that further this screening method being carried out precision, repeatability, stability and recovery of standard addition detects, it is to get that concentration is respectively 100,200, the DL-3-phenyllactic acid standard operation liquid of 400mg/L repeats respectively sample introduction and analyzes for 5 times, and calculates 3 peak area relative standard deviations that concentration is corresponding that said precision detects; Said repeatability and Detection of Stability be within a working days duplicate detection concentration be respectively 100,200, the DL-3-phenyllactic acid standard operation liquid of 400mg/L 5 times, and every 24h, sample solution is detected, repetitive operation 5 times, and the peak area relative standard deviation of calculating; It is that DL-3-phenyllactic acid standard substance with 3 concentration gradients add in the known milk-acid bacteria MRS fermentation supernatant culture supernatant of concentration that recovery of standard addition detects, and it is carried out replication 5 times, and the peak area relative standard deviation of calculating.
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