CN103048403B - Method for detecting lactic acid in fermented soybean meal - Google Patents
Method for detecting lactic acid in fermented soybean meal Download PDFInfo
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- CN103048403B CN103048403B CN201210538871.XA CN201210538871A CN103048403B CN 103048403 B CN103048403 B CN 103048403B CN 201210538871 A CN201210538871 A CN 201210538871A CN 103048403 B CN103048403 B CN 103048403B
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- lactic acid
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- bean dregs
- fermented bean
- soybean meal
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Abstract
The invention provides a method for detecting lactic acid in fermented soybean meal. The method for detecting lactic acid in fermented soybean meal, comprises the following steps of: (1) pre-treating a sample, (2) determining chromatogram conditions, (3) preparing a calibration curve, (4) determining the sample, and (5) calculating to obtain a result. The proteins in the fermented soybean meal are settled by 80% ethanol, macro-molecule substances in a supernatant is removed by filtrating with a 0.45mum filter membrane, the lactic acid in the fermented soybean meal is separated by a mixture mobile phase of methanol and hydrochloric acid, and the content of lactic acid is calculated by an external standard method. The sample is pretreated by a specific manner, the impurity interference can be avoided by selecting a proper mobile phase, and then the content of lactic acid in the fermented soybean meal can be accurately measured.
Description
Technical field
The present invention relates to the assay method of lactic acid content in fermented bean dregs.
Background technology
At present, the multiplex lactic acid bacteria of dregs of beans is fermented, and thus contains a large amount of lactic acid in fermented bean dregs, simultaneously containing a small amount of propionic acid, butyric acid etc.
The assay method of conventional lactic acid mainly contains two kinds: neutralization titration and high performance liquid chromatography.Research finds, neutralization titration measured result error is comparatively large, the result reflection of titration be the content of total acid, inaccurate; Although high-efficient liquid phase technique can quantitative and qualitative analysis, but fermented bean dregs complicated component, containing protein, oligosaccharides, little peptide, organic acid, isoflavones etc., when causing utilizing efficient liquid phase to measure lactic acid, the interference of assorted peak is large, main peak separating effect is not ideal enough, and in fermented bean dregs, lactic acid content is difficult to Accurate Measurement.Not yet there is the assay method of lactic acid content in report fermented bean dregs at present.
Summary of the invention
In order to solve above-mentioned technical matters, the object of this invention is to provide the assay method of lactic acid in a kind of fermented bean dregs, the method is carried out pre-service by ad hoc fashion to sample, is adopted suitable mobile phase to avoid impurity to disturb, and is accurately measured by lactic acid content in fermented bean dregs.
In order to realize above-mentioned object, present invention employs following technical scheme:
An assay method for lactic acid in fermented bean dregs, is characterized in that the method comprises the following steps:
1) sample pretreatment
Accurately take fermented bean dregs 5.0000 g pulverized and sieved, be placed in 100 mL volumetric flasks, the ethanolic solution adding 80% concentration of volume percent is settled to 100ml, ultrasound wave 50 DEG C extracts 30 min, and be cooled to room temperature, sample liquid high speed freezing centrifuge is centrifugal, rotating speed 12000rpm, 4 DEG C of centrifugal 10min, 0.45um filter membrane crossed by supernatant, and filtrate is for subsequent use as sample solution;
2) chromatographic condition
Chromatograph: Agilent1100 high performance liquid chromatograph, UV213nm detecting device;
Chromatographic column: Walts sunfire C18,4.6 × 250 nm, 5 μm;
Column temperature: 35 DEG C;
Mobile phase: methyl alcohol: the hydrochloric acid solution=5:95 of 0.01% concentration of volume percent, above-mentioned ratio is volume ratio;
Flow velocity: 1.0 mL/min;
Sample size: 20 μ L;
3) preparation of calibration curve
Extracting lactic acid titer 5ul, 10ul, 15ul, 20ul, 25ul respectively, be equivalent to each lactic acid content 25ug, 50ug, 75ug, 100ug, 125ug injection liquid chromatography, carry out efficient liquid phase chromatographic analysis, measure chromatographic peak area or peak height, with standard lactic acid quality, typical curve is done to corresponding peak area or peak height, or ask regression equation by least square method;
4) sample determination
Under identical chromatographiccondition, get sample solution injection high performance liquid chromatograph analysis prepared by 20ul step 1), measure chromatographic peak area or peak height, compare with typical curve and determine lactic acid quality in sample introduction liquid, draw lactic acid chromatogram;
5) result calculates
In fermented bean dregs, the content of lactic acid presses formulae discovery below:
In formula:
X---the content of lactic acid in fermented bean dregs, %;
M
1---the quality of lactic acid, unit is g;
M---fermented bean dregs quality, unit is g;
V---sample solution volume, unit ul;
V
1---sampling volume, unit is ul.
The present invention utilizes the ethanol of 80% to be settled down by protein substance in fermented bean dregs, and supernatant is removed macromolecular substances with 0.45um membrane filtration again and removed, and adopts lactic acid in methyl alcohol and mixed in hydrochloric acid mobile phase separation and fermentation dregs of beans, and external standard method calculates lactic acid content.By ad hoc fashion, pre-service carried out to sample, adopt suitable mobile phase to avoid impurity to disturb, lactic acid content in fermented bean dregs is accurately measured.
Accompanying drawing explanation
Fig. 1 is lactic acid chromatogram schematic diagram in fermented bean dregs.
Embodiment
, reagent and material
Except as otherwise noted, it is pure that agents useful for same of the present invention is analysis, and water is ultrapure water.
1.1 methyl alcohol: chromatographically pure.
1.2 hydrochloric acid: chromatographically pure.
1.3 0.01% hydrochloric acid solutions: accurately draw 0.1ml hydrochloric acid (1.2), are settled to 1000ml by water-soluble solution.
1.4 lactate standard product: purity >=99.99%.
1.5 80% ethanolic solutions: measure 800ml absolute ethyl alcohol and be diluted with water to 1000ml.
1.6 lactate standard solution: take lactate standard product 0.5000g and be placed in 100ml volumetric flask, are settled to scale with 80% dissolve with ethanol solution, shake up.Every ml soln is containing lactic acid 5mg.Through 0.45um membrane filtration, filtrate is analyzed for HPLC.
1.7 fermented bean dregs: dregs of beans is connected lactic acid bacteria, inoculum concentration 5%, solid-liquid ratio is 2:1, sealed fermenting 4d in 37 DEG C of incubators, pulverizes 60 mesh sieves after 80 DEG C of oven dry, for subsequent use.
, instrument
2.1 high performance liquid chromatographs: attached differential refraction detector.
2.2 ultrasonic cleaner.
2.3 microbiological incubator.
2.4 analytical balances: sensibility reciprocal 0.0001g.
2.5 miillpore filters: aperture 0.45um.
2.6 high speed freezing centrifuge.
, operation steps
3.1 sample pretreatment
Accurately take fermented bean dregs 5.0000 g pulverized and sieved, be placed in 100 mL volumetric flasks, the ethanolic solution adding 80% is settled to 100ml, ultrasound wave 50 DEG C extracts 30 min, and be cooled to room temperature, sample liquid high speed freezing centrifuge is centrifugal, rotating speed 12000rpm, 4 DEG C of centrifugal 10min, 0.45um filter membrane crossed by supernatant, and filtrate is for subsequent use.
chromatographic condition
Chromatograph: Agilent1100 high performance liquid chromatograph, UV213nm detecting device;
Chromatographic column: Walts sunfire C18(4.6 × 250 nm, 5 μm);
Column temperature: 35 DEG C;
Mobile phase: V(methyl alcohol): V(0.01% hydrochloric acid solution)=5:95;
Flow velocity: 1.0 mL/min;
Sample size: 20 μ L.
the preparation of calibration curve
Extracting lactic acid titer 5ul, 10ul, 15ul, 20ul, 25ul (being equivalent to each lactic acid content 25ug, 50ug, 75ug, 100ug, 125ug) injection liquid chromatography respectively, carry out efficient liquid phase chromatographic analysis, measure chromatographic peak area (or peak height), with standard lactic acid quality, typical curve is done to corresponding peak area (or peak height), or ask regression equation by least square method.
sample determination
Under identical chromatographiccondition, get 20ul sample solution (3.1) and inject high performance liquid chromatograph analysis, measure chromatographic peak area (or peak height), compare with typical curve and determine lactic acid quality in sample introduction liquid, Fig. 1 is shown in by lactic acid chromatogram schematic diagram.
5, result calculates
In fermented bean dregs, the content of lactic acid presses formulae discovery below:
In formula:
x---the content (%) of lactic acid in fermented bean dregs;
M
1---the quality of lactic acid, unit is g;
M---fermented bean dregs quality, unit is g;
V---sample solution volume, unit ul;
V
1---sampling volume, unit is ul.
Claims (1)
1. the assay method of lactic acid in fermented bean dregs, is characterized in that the method comprises the following steps:
1) sample pretreatment
Accurately take fermented bean dregs 5.0000 g pulverized and sieved, be placed in 100 mL volumetric flasks, the ethanolic solution adding 80% concentration of volume percent is settled to 100ml, ultrasound wave 50 DEG C extracts 30 min, and be cooled to room temperature, sample liquid high speed freezing centrifuge is centrifugal, rotating speed 12000rpm, 4 DEG C of centrifugal 10min, 0.45 μm of filter membrane crossed by supernatant, and filtrate is for subsequent use as sample solution;
2) chromatographic condition
Chromatograph: Agilent1100 high performance liquid chromatograph, UV213nm detecting device;
Chromatographic column: Walts sunfire C18,4.6 × 250 nm, 5 μm;
Column temperature: 35 DEG C;
Mobile phase: methyl alcohol: the hydrochloric acid solution=5:95 of 0.01% concentration of volume percent, ratio is volume ratio;
Flow velocity: 1.0 mL/min;
Sample size: 20 μ L;
3) preparation of calibration curve
Extracting lactic acid titer 5 μ l, 10 μ l, 15 μ l, 20 μ l, 25 μ l respectively, be equivalent to each lactic acid content 25 μ g, 50 μ g, 75 μ g, 100 μ g, 125 μ g injection liquid chromatographies, carry out efficient liquid phase chromatographic analysis, measure chromatographic peak area or peak height, with standard lactic acid quality, typical curve is done to corresponding peak area or peak height, or ask regression equation by least square method;
4) sample determination
Under identical chromatographiccondition, get sample solution injection high performance liquid chromatograph analysis prepared by 20 μ l step 1), measure chromatographic peak area or peak height, compare with typical curve and determine lactic acid quality in sample introduction liquid, draw lactic acid chromatogram;
Result calculates
In fermented bean dregs, the content of lactic acid presses formulae discovery below:
In formula:
X---the content of lactic acid in fermented bean dregs, %;
M
1---the quality of lactic acid, unit is g;
M---fermented bean dregs quality, unit is g;
V---sample solution volume, unit μ l;
V
1---sampling volume, unit is μ l.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210538871.XA CN103048403B (en) | 2012-12-11 | 2012-12-11 | Method for detecting lactic acid in fermented soybean meal |
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| CN201210538871.XA CN103048403B (en) | 2012-12-11 | 2012-12-11 | Method for detecting lactic acid in fermented soybean meal |
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| CN103048403B true CN103048403B (en) | 2015-04-15 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108982718A (en) * | 2018-07-30 | 2018-12-11 | 湖北邦之德牧业科技有限公司 | A kind of fermented bean dregs fingerprint map construction method and its application in quality control |
| CN110426481A (en) * | 2019-09-16 | 2019-11-08 | 亚太星原农牧科技海安有限公司 | The method of lactic acid content and the measurement extracting method of prepare liquid in a kind of measurement fermented bean dregs |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1769888A (en) * | 2004-10-29 | 2006-05-10 | 上海唯依农业科技发展有限公司 | Soybean saponins detection method |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1769888A (en) * | 2004-10-29 | 2006-05-10 | 上海唯依农业科技发展有限公司 | Soybean saponins detection method |
Non-Patent Citations (4)
| Title |
|---|
| L-Lactic acid production by Lactobacillus casei fermentation with corn steep liquor-supplemented acid-hydrolysate of soybean meal;Zheng Li et al;《Biotechnology Journal》;20061231;第1卷(第12期);第1453-1458页 * |
| 反相高效液相色谱法测定发酵液中乳酸的含量;郑志等;《食品科学》;20031231;第24卷(第12期);第89-91页 * |
| 吉林省部分大豆品种(系)低聚糖含量分析;宋志峰等;《大豆科学》;20090630;第28卷(第3期);第517页第1.5.2节 * |
| 高效液相色谱法同时测定复合有机酸化剂中5种成分的含量;吴剑平等;《中国饲料》;20080731(第14期);第2.1,2.2,2.5,3.1节 * |
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