CN105671184A

CN105671184A - Method for measuring concentration of thalli in culture media

Info

Publication number: CN105671184A
Application number: CN201610185344.3A
Authority: CN
Inventors: 李大攀; 李书至; 常坤; 郭双; 李俊; 陈浩然; 李冰
Original assignee: ZHUMADIAN HUAZHONG CHIA TAI CO Ltd
Current assignee: ZHUMADIAN HUAZHONG CHIA TAI CO Ltd
Priority date: 2016-03-29
Filing date: 2016-03-29
Publication date: 2016-06-15
Anticipated expiration: 2036-03-29
Also published as: CN105671184B

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to an improved method for measuring the concentration of thalli in compound culture media. The method includes steps of preserving heat for fermentation liquor; carrying out enzymatic hydrolysis; carrying out TCA (cellulose triacetate) extraction; measuring light absorbance; computing the concentration and the like. The improved method has the main advantages that the fermentation liquor is subjected to appropriate enzymatic hydrolysis treatment, accordingly, disturbance due to nucleic acid or analogs in materials such as soybean powder and peanut powder in solid components when the nucleic acid of the thalli is extracted from the culture media can be effectively prevented, the nucleic acid of the thalli in the fermentation liquor can be accurately and sufficiently extracted, the quantities of the thalli in the fermentation liquor can be truly reflected, reference and guidance can be effectively provided for fermentation production, and the improved method has excellent practical value and an excellent popularization and application significance.

Description

The assay method of cell concentration in a kind of culture medium

Technical field

The invention belongs to technical field of microbial fermentation, be specifically related to a kind of measure the method for the improvement of cell concentration in complex medium.

Background technology

The mensuration of cell concentration is an important indicator of reflection growth of microorganism metabolic condition, it is necessary to accurately data could better control every production procedure. In existing industrialization fermentation process, due to the commonly used fermentation medium containing a large amount of stop ingredients admittedly, such as soybean cake powder, peanut cake powder etc., and these solid constituents and thalline mix, the mensuration of cell concentration in fermentation liquid is made to become the problem of a complexity and difficulty, always undecided for a long time.

When current most antibiotics factory measures fermentation liquid cell concentration, generally individually is adopt loading amount volumetric method (cell accumulation volumetric measurement method, centrifugal compressed cyton area method, ThePacked-CellvolumeMethod). But in actually used process, this method is bigger by the impact of solid material composition in fermentation liquid, and when thalli growth stable phase uses the method, the result increasing mainly some lipids and secondary metabolite synthesis of surveyed cell concentration value, can not the change of authentic representative cell concentration. Thus the numerical value that loading amount volumetric method records is cell concentration and the result of two mutual adductions of variable of solid culture based component, real cell concentration can not be reacted. Further, the data that use the method measures without evident regularity, also cannot reflect the growth rhythm that thalline is real in whole sweat.

During existing cell concentration measures, another comparatively accurate and conventional assay method is to measure the concentration of nucleic acid contained by thalline in culture medium, and using the amount of nucleic acid as the index weighing cell concentration, and the extraction of thalline amplifying nucleic acid obtains mainly through TCA extraction. But in actually used process, the method is affected bigger by medium component. Such as, if containing insoluble composition such as analysis for soybean powder, peanut powder etc. in fermentation medium, it is likely to containing a small amount of nucleic acid or nucleic acid analog, and these materials are extracted in TCA together with the nucleic acid in thalline, thus bringing a degree of interference to mensuration; Especially, when adopting germ flour class material as nutritional labeling in culture medium, it contains the nucleic acid of high-load, and measured value often causes serious interference.Thus the method is still needed and improves further, to obtain cell concentration data more accurately.

Summary of the invention

It is an object of the invention to provide the TCA algoscopy of a kind of follow-on mensuration fermentation liquid amplifying nucleic acid concentration, thus reflecting cell concentration data more accurately.

Details are as follows for the technical scheme that the present invention takes.

The assay method of cell concentration in a kind of culture medium, specifically includes following steps:

(1) take fermentation liquid to be measured, fermentation liquid is incubated 20 ~ 40min at 45 DEG C ~ 50 DEG C, it is preferable that 50 DEG C of insulation 30min; The insulation of described fermentation liquid terminates after fermentation fluid viscosity and declines, and mobility is better;

Then adding cellulase and pectase in the fermentation liquid after insulation terminates, mix homogeneously is to carry out enzymolysis to fermentation liquid, and period can every 5min with reverse mixing;

Described fermentation liquid can be such as Aureomycin fermentation liquor, specifically such as normal fermentation 0 ~ 120h Aureomycin fermentation liquor or cultivate 0 ~ 36h chlortetracycline seed liquor;

Total addition is fermentation liquid quality the 2% ~ 4% of described cellulase and pectase; The mass ratio of its cellulase (enzyme activity >=250,000 U/g) and pectase (enzyme activity >=100,000 U/g) is 1:1.5 ~ 2.0, during enzymolysis, temperature controls at 35 DEG C ~ 50 DEG C, enzymolysis time >=1h, but no more than 5h, long-time thus affecting fermentation liquid quality to avoid enzymolysis to cross;

(2) fermentation liquid after enzymolysis in step (1) being completed, 0 DEG C, the centrifugal 10min of 3000 × g, abandon supernatant, retain precipitate;

(3) in the precipitate of step (2), add the TCA(trichloroacetic acid of 5% (V/V) of 2 DEG C ~ 4 DEG C) solution, it is sufficiently stirred for washing precipitate, 0 DEG C, centrifugal 10 minutes of 3000 × g, abandon supernatant; Repeat this step once;

The volume ratio of TCA solution proportion and sample solution (namely being taken fermentation liquid to be measured in step (1)) is 1:1 ~ 1.5; It is preferably in a proportion of 1:1.5, but owing to now TCA consumption is bigger than normal, thus can adjust in right amount according to practical situation;

(4) (V/V) TCA that adds 5% in the precipitation of step (3), is sufficiently stirred for, and then at the stirred in water bath extracting 10 ~ 15min of 100 DEG C, is subsequently placed in frozen water and cools down, 0 DEG C, centrifugal 10 minutes of 3000 × g, takes supernatant, abandons precipitate;

(5) step (4) supernatant 5% (V/V) TCA is suitably diluted, OD₂₆₀Measure absorbance, calculate nucleic acid concentration according to below equation:

In formula: OD_260nmFor the light absorption value that TCA extract is measured at 260nm place; N is the multiple of TCA extract dilution; 0.022 represents that nucleic acid concentration is light absorption value during 1 mcg/ml, and this numerical value is a constant.

The major technique thinking of the present invention is: the interpolation of cellulase and pectase, destroy the cell wall (microbial cell is not affected by enzyme) of part plant material in complex medium (analysis for soybean powder, Semen Maydis powder, peanut powder etc.), so that the nucleic acid in plant born of the same parents is discharged into extracellular, and the plant content discharged is by follow-up being centrifuged, going the steps such as supernatant to be removed along with other impurity, thus avoiding the interference that measurement result is caused by nucleic acid in culture medium raw material and the like.

In general, the invention belongs to a kind of follow-on TCA method measuring fermentation liquid amplifying nucleic acid concentration, its improvement is mainly reflected in: the method is by the suitable enzymolysis processing to fermentation liquid, better avoid from culture medium extract thalline nucleic acid time solid constituent in such as analysis for soybean powder, the interference of contained nucleic acid or the like in the materials such as peanut powder, can comparatively accurately and fully extract the nucleic acid of thalline in fermentation liquid, thus reflecting biomass in fermentation liquid more truly, thus provide reference and guidance for more preferably fermenting and producing, show practical value and popularization and application meaning preferably.

Accompanying drawing explanation

Fig. 1 is the present invention with existing assay method to the measurement result comparison diagram of the nucleic acid concentration of different fermentations time in certain batch of Aureomycin fermentation liquor.

Detailed description of the invention

Below in conjunction with embodiment, the present invention is further explained explanation. Before introducing specific embodiment, part test equipment in the present invention and material situation are briefly discussed below.

In following embodiment, Aureomycin fermentation liquor is provided by Huazhong Zhengda Co., Ltd., Zhumadian City, adopts streptomyces aureus to be prepared from, and the preparation flow of Aureomycin fermentation liquor is approximately as described:

(1) prepared by inclined-plane mycelia, and the streptomyces aureus spore preserved by sandy soil pipe adopts slant culture mode, cultivates 3 ~ 5 days preparation female bottle inclined-plane mycelia for 32 DEG C ± 1 DEG C;

(2) prepared by seed liquor, and by the inclined-plane mycelium inoculation culture bottle in bottle female in step (1), 26 DEG C ~ 32 DEG C, 230 revs/min shake-flask culture 18 ~ 24h prepare seed liquor;

(3) prepared by Aureomycin fermentation liquor, is inoculated in fermentation tank by seed liquor in step (2), 26 DEG C ~ 30 DEG C, cultivate under the condition of pH5.4 ~ 6.5, is Aureomycin fermentation liquor;

In fermentation tank, the concrete composition of chlortetracycline fermentation medium is (1L): peanut cake powder 20g, soybean cake powder 18g, corn starch 80g, yeast powder 7.0g, Semen Maydis pulp 12g, Oleum Glycines 3g, amylase 0.1g, sodium chloride 2.0g, ammonium sulfate 6g, calcium carbonate 7.0g, magnesium sulfate 0.2g, potassium dihydrogen phosphate 0.3g, pH nature.

Embodiment 1

The assay method of cell concentration in the culture medium that the present embodiment provides, specifically includes following steps:

(1) take the Aureomycin fermentation liquor 5mL of fermentation 0h (before namely not yet inoculating after the sterilization of chlortetracycline fermentation medium in fermentation tank), fermentation liquid is incubated 30min at 50 DEG C; Insulation terminates after fermentation fluid viscosity and declines, and mobility is better, without a large amount of deposited phenomenons in 3min.

It is to be understood that the viscosity differences of the fermentation liquid of different fermentations time is relatively big, when not inoculating, viscosity is within 1s, and the viscosity of the fermentation liquid in normal fermentation process is at about 1 ~ 15s, and fermentation broth viscosity adopts capillary viscosimeter to detect.

Being subsequently adding cellulase and pectase, mix homogeneously is to carry out enzymolysis to fermentation liquid, and period can every 5min with reverse mixing;

Total addition is fermentation liquid quality the 2.6% of cellulase and pectase, its cellulase (enzyme activity=250,000 U/g) and pectase (enzyme activity=100,000 U/g) consumption respectively 0.05,0.08g, hydrolysis temperature controls at 40 DEG C, enzymolysis time 3h;

(2) enzymolysis in step (1) is completed after fermentation liquid, 0 DEG C, the centrifugal 10min of 3000 × g, abandon supernatant;

(3) in the precipitate of step (2), add the 5%(V/V of 4 DEG C) TCA solution 5mL, be sufficiently stirred for washing precipitate, 0 DEG C, centrifugal 10 minutes of 3000 × g, abandon supernatant; Repeat this step once;

(4) in the precipitation of step (3), add 5%TCA5mL, be sufficiently stirred for, then at the stirred in water bath extracting 15min of 100 DEG C, be subsequently placed in frozen water and cool down, 0 DEG C, centrifugal 10 minutes of 3000 × g, take supernatant, abandon precipitate;

(5) step (4) supernatant 5%TCA is suitably diluted, OD₂₆₀Measure absorbance, calculate nucleic acid concentration according to below equation:

。

Calculating shows, now Aureomycin fermentation liquor amplifying nucleic acid concentration is 0.17mg/mL. Owing to now fermentation liquid not yet being inoculated Ferment of DM bacterial strain, thus it is believed that containing trace dna material in the solid materials such as peanut cake powder in culture medium, soybean cake powder, thus easily subsequent fermentations center measuring acid concentration being caused certain impact.

Comparative example

Under same operational approach, under omitting insulation and enzymolysis operational hypothesis (namely omitting above-mentioned steps (1)), same batch of Ferment of DM culture medium amplifying nucleic acid concentration is measured, it is shown that nucleic acid concentration is 2.73mg/mL.

From the testing result of the present embodiment and comparative example it can be seen that by, after enzymolysis processing, the impact of original fermentation liquid amplifying nucleic acid being greatly lowered, can provide for real cell concentration detection in production process and better ensure.

Embodiment 2

Identical with embodiment 1, the Aureomycin fermentation liquor amplifying nucleic acid concentration of same batch is measured, only in set-up procedure (1), fermentation liquid holding temperature is 45 DEG C, temperature retention time 30min. Last result of calculation shows, mycelium nucleic acid concentration is 0.29mg/mL.

Embodiment 3

Identical with embodiment 1, the Aureomycin fermentation liquor amplifying nucleic acid concentration of same batch is measured, only adjusts the ratio of enzyme during enzymolysis, the concrete consumption of enzyme is: cellulase 0.07g, pectase 0.13g, and finally calculating nucleic acid concentration is 0.06mg/mL. From this result it can be seen that now fermentation liquid original nucleic acid amount for subsequent measurements almost without impact.

According to this operational approach, cell concentration in Aureomycin fermentation liquor different fermentations time after fermentation liquid being measured, be measured according to existing (i.e. operational approach in comparative example) operational approach simultaneously, related assays result is as shown in Figure 1. It will be seen from figure 1 that the TCA algoscopy after improvement provided by the present invention, can better solve the fermentation culture early stage inaccurate drawback of bacterial strain measurement of concetration, more can reflect the real growth rhythm of thalline, thus can better instruct fermenting and producing.

Claims

1. the assay method of cell concentration in a culture medium, it is characterised in that the method specifically includes following steps:

(1) take fermentation liquid to be measured, fermentation liquid is incubated at 45 DEG C ~ 50 DEG C 20 ~ 40min; It is subsequently adding cellulase and pectase, so that fermentation liquid is carried out enzymolysis;

Total addition is fermentation liquid quality the 2% ~ 4% of described cellulase and pectase;

By quality ratio, cellulase pectase=1:1.5 ~ 2.0, wherein cellulose enzyme activity >=250,000 U/g, pectase enzyme >=10 ten thousand U/g alive;

Hydrolysis temperature controls at 35 DEG C ~ 50 DEG C, enzymolysis time >=1h, but less than 5h;

(2) fermentation liquid after being completed by enzymolysis in step (1) is centrifuged, and abandons supernatant, retains precipitate;

(3) in the precipitate of step (2), add the TCA solution of 5% volume fraction of 2 DEG C ~ 4 DEG C, washing precipitate, be centrifuged, abandon supernatant; Repeat this step once;

TCA solution is 1:1 ~ 1.5 with the middle volume ratio being taken fermentation liquid to be measured of step (1);

(4) in the precipitation of step (3), add the TCA solution of 5% volume fraction, stirring, then extracting 10 ~ 15min in the water-bath of 100 DEG C, then be placed in frozen water and cool down, centrifugal, take supernatant, abandon precipitate;

(5) by step (4) gained supernatant 5%TCA solution dilution, OD₂₆₀Measure absorbance, calculate nucleic acid concentration according to below equation:

In formula: OD_260nmFor the light absorption value that TCA extract is measured at 260nm place; N is the multiple of TCA extract dilution.

2. the assay method of cell concentration in culture medium as described in the appended claim 1, it is characterised in that described fermentation liquid is Aureomycin fermentation liquor.