CN103698292A

CN103698292A - Dual-wavelength ultraviolet spectrophotometry method for measuring content of natamycin in fermentation liquid

Info

Publication number: CN103698292A
Application number: CN201310745222.1A
Authority: CN
Inventors: 詹志春; 王冠; 徐丽
Original assignee: WUHAN SUNHY BIOLOGY CO Ltd
Current assignee: WUHAN SUNHY BIOLOGY CO Ltd
Priority date: 2013-12-30
Filing date: 2013-12-30
Publication date: 2014-04-02

Abstract

The invention relates to the field of analytical chemistry, in particular to a dual-wavelength ultraviolet spectrophotometry method for measuring the content of natamycin in fermentation liquid. The method comprises the following steps: (1) measuring light absorption values of a processed sample to be measured at the wavelengths of 303 nm and 290 nm respectively by taking a 70% methanol-water solution as a blank; (2) calculating the content of the natamycin according to the following formula: C sample=(k*delta A+b)*N. The precision degree, the accuracy inspection, the detection limit and the quantitation limit of the method are effectively evaluated, so that the method is guaranteed to be scientific and accurate.

Description

A kind of dual-wavelength ultraviolet spectrophotometry of measuring natamycin content in fermentation liquor

Technical field

The present invention relates to analytical chemistry field, be specifically related to a kind of dual-wavelength ultraviolet spectrophotometry of measuring natamycin content in fermentation liquor.

Background technology

Natamycin is that a kind of two sixteen-ring polyene macrocyclic lactones are antifungal agents based, and inhibition saccharomycete and the mould of obligate, be widely used as natural biological food preservative and antibacterial additives.Natamycin (Natamycin) has another name called pimaricin (Pimaricin), and the main generation bacterium such as Natal streptomycete (Streptomyces natalensis), streptomyces chatanoogensis (Streptomyces chattanovgensis) and brown yellow spore streptomycete (Streptomyces gilvosporeus) in streptomysin Pseudomonas by fermentation refining refinement form.

In biological fermentation process, in fermentation liquor, can produce a large amount of organic substances, as protein and some metabolic products, bring interference can to the mensuration of sample.HPLC method can be opened some relevant separating substances, but because HPLC systematic comparison is complicated, operating cost and personnel have relatively high expectations, especially comparatively strict to the pre-service requirement of sample, is not suitable for the detection of large number of biological fermented sample.

In American Pharmacopeia, the assay of natamycin finished product is to adopt high performance liquid chromatography, the domestic report that also has this method of employing to measure natamycin, but this method has distinct disadvantage for the extensive seed selection of natamycin Producing Strain and the online and quick offline inspection of sweat, as longer in determination period, sample preparation and instrument are had relatively high expectations, and are not suitable for batch samples etc.

The domestic report that also has natamycin in employing Fat by UV Spectrophotomotry fermentation liquor, but because natamycin fermentation liquor is obtained by biofermentation, many organic substances in fermentation medium, have been used, in these materials, contain some other material that has uv absorption, when with determined by ultraviolet spectrophotometry, can produce certain interference to measurement result, single length ultraviolet spectrophotometric method is difficult to get rid of the interference of various impurity to measurement result in fermentation liquor.

Summary of the invention

Therefore, the object of this invention is to provide a kind of dual-wavelength ultraviolet spectrophotometry of measuring natamycin content in fermentation liquor, can guarantee finding speed, thus again effectively the interference of despumation guarantee accuracy of measurement.

Dual-wavelength ultraviolet spectrophotometry according to natamycin content in mensuration fermentation liquor of the present invention, comprises step:

(1) get the testing sample of handling well, using 70% methanol-water solution as blank, measure respectively it at the light absorption value at 303nm, 290nm place;

(2) calculate natamycin content

C _sample=(k * Δ A+b) * N

C _sample: the content of natamycin in sample (μ g/ml); Δ A: sample solution is in the difference of 303nm and 290nm place light absorption value; K: the slope of typical curve; B: the intercept of typical curve; N: the extension rate of testing sample.

According to the application's method, natamycin standard items, natamycin fermentation liquor sample are carried out to full wavelength scanner, the absorption maximum of natamycin is respectively 290nm, 303nm, 319nm, and interfering material in the fermentation liquor absorption value at natamycin maximum absorption wavelength 290nm, 303nm place is respectively 0.051 and 0.047, basic not variation, and differ larger with the absorption value of natamycin, therefore can select 290nm, 303nm as natamycin assay wavelength, herein the assorted Δ A absorbing _{λ 2-λ 1}≈ 0, therefore can eliminate to greatest extent the assorted interference absorbing.

According to the dual-wavelength ultraviolet spectrophotometry of natamycin content in the application's mensuration fermentation liquor, the precision of the method, accuracy investigation, detectability and quantitative limit have all obtained Efficient Evaluation, thereby have guaranteed that the science of method is accurate.To the fermentation unit in sweat, adopt respectively dual-wavelength ultraviolet spectrophotometry and high performance liquid chromatography (HPLC assay method adopts 29 editions methods of American Pharmacopeia) to carry out tracking and measuring, the result of the two is analyzed relatively, and to determine two kinds of methods, whether there were significant differences.

Embodiment

Embodiment 1

1. material and equipment

Electronic balance (ten thousand/), uv-spectrophotometric instrument, ultrasonic cleaning machine, natamycin reference substance (the American Pharmacopeia council), natamycin fermentation liquor sample.

2. fermentation condition

Slant medium (%): peptone 0.05, Fructus Hordei Germinatus extract 0.3, glucose 1.0, agar 2.0, pH7.0;

Seed culture medium (%): peptone 0.6, corn steep liquor 0.6, glucose 2.0, sodium chloride 1.0, pH7.0;

Fermentation medium (%): cornstarch 2.0, yeast extract powder 0.5, peptone 0.5, glucose 4.0, pH7.0.

Slant culture in 28 ℃ of cultivations is dug to piece to be inoculated in the seed bottle that fills 25ml seed culture medium in right amount, 28 ℃, 200rpm cultivates 24～36h, according to 10% inoculum concentration, be inoculated in the automatic fermenter of 30L, 28 ℃, throughput 20L/min cultivates 144h, from 72h, every 8h, samples and obtain natamycin fermentation liquor sample.

3. the pre-service of sample

Get 2ml fermentation liquor in tool plug triangular flask, add 25ml methyl alcohol, ultrasonic processing 30min, with Filter paper filtering, removes bacterium slag.Get filtrate appropriate, with 70% methanol-water, suitably obtain testing sample (after dilution in sample natamycin content should at 0.5～6.0 μ g/ml) after dilution.

4. typical curve

Standard solution: accurately take appropriate natamycin standard items, use methyl alcohol ultrasonic dissolution, then it is standby to be diluted to suitable concentration with 70% methanol-water.

With 70% methanol-water solution, standard solution is diluted, concentration is respectively 0.5 μ g/ml, 1.0 μ g/ml, 2.0 μ g/ml, 3.0 μ g/ml, 4.0 μ g/ml, 5.0 μ g/ml, 6.0 μ g/ml, using 70% methanol-water solution as blank, measure respectively them at the light absorption value at 303nm, 290nm place.The standard dilute solution concentration of take is ordinate, and the difference DELTA A of wavelength 303nm, 290nm place light absorption value is as horizontal ordinate, drawing standard curve, and regression equation is y=19.33x+0.022, coefficient R ²=0.999, good in 0.5～6.0 μ g/ml scope internal linear.

5. the mensuration of sample

Get the testing sample of handling well, using 70% methanol-water solution as blank, measure respectively it at the light absorption value at 303nm, 290nm place.

6. the calculating of natamycin content

C _sample=(k * Δ A+b) * N

For example:

The Δ A of certain fermentation broth sample is 0.156, and extension rate is 1000 times, and according to typical curve y=19.33x+0.022, the content that can calculate Natamycin in this fermentation broth sample is that 3037 μ g/ml are 3.04g/L;

The Δ A of certain fermentation broth sample is 0.195, and extension rate is 5000 times, and according to typical curve y=19.33x+0.022, the content that can calculate Natamycin in this fermentation broth sample is that 18955 μ g/ml are 18.96g/L;

The Δ A of certain fermentation broth sample is 0.233, and extension rate is 10000 times, and according to typical curve y=19.33x+0.022, the content that can calculate Natamycin in this fermentation broth sample is that 45260 μ g/ml are 45.26g/L.

7. precision

Each sample should be got three parts of parallel samples and analyze mensuration, and relative error should not surpass 5.0%, and three's mean value is final natamycin assay value.

By standard solution formulation content, be about respectively the standard solution of 2.0 μ g/ml, 3.0 μ g/ml, 4.0 μ g/ml, with methanol-water (70%), as blank solution, by ultraviolet spectrophotometry, measure respectively 3 times, experimental result sees the following form.

The mensuration precision of ultraviolet spectrophotometry

Relative standard deviation is that coefficient of variation RSD (n=3) is respectively 0.57%, 0.38% and 0.49%, and mean value is 0.48%.This result shows, the method reappearance is better, and precision meets the requirements completely.

8. accuracy

Get fermentation broth sample and process according to sample treatment, with the content C of spectrophotometry natamycin _sample, then in this testing sample solution, add respectively the natamycin standard solution of 2.02 μ g/ml, 3.03 μ g/ml, 4.04 μ g/ml, and with the content C of spectrophotometry natamycin _mark-on, with (C _mark-on-C _sample)/C _{standard items}calculate its recovery.As shown in the table, the recovery, 98.51%～102.97%, illustrates that this method recovery is qualified, and accuracy meets the requirements completely.

The accuracy of measurement of ultraviolet spectrophotometry

9. detectability and quantitative limit

According to 3.3 times and 10 times of the residue standard deviation S D in linear equation, respectively the quantitative limit of ultraviolet spectrophotometry and detectability are estimated, the concentration of sample represents when measuring, detectability and quantitative limit are respectively 0.14 μ g/ml and 0.41 μ g/ml.

10. with the result comparison of HPLC determination method

The comparison of ultraviolet spectrophotometry and HPLC method measurement result

The testing result of ultraviolet spectrophotometry and HPLC method as shown above, obviously finds out that the testing result of the two is basically identical, according to there is no significant difference between two kinds of methods of t test and judge.

Claims

1. a dual-wavelength ultraviolet spectrophotometry of measuring natamycin content in fermentation liquor, is characterized in that, described method comprises step:

(2) calculate natamycin content

C _sample=(k * Δ A+b) * N

C _sample: the content μ g/ml of natamycin in sample; Δ A: sample solution is in the difference of 303nm and 290nm place light absorption value; K: the slope of typical curve; B: the intercept of typical curve; N: the extension rate of testing sample.