CN102505038A - Method for measuring content of nicotinic acid and nicotinamide in feed by using microorganisms - Google Patents
Method for measuring content of nicotinic acid and nicotinamide in feed by using microorganisms Download PDFInfo
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- CN102505038A CN102505038A CN2011103533860A CN201110353386A CN102505038A CN 102505038 A CN102505038 A CN 102505038A CN 2011103533860 A CN2011103533860 A CN 2011103533860A CN 201110353386 A CN201110353386 A CN 201110353386A CN 102505038 A CN102505038 A CN 102505038A
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Abstract
The invention provides a method for measuring the content of nicotinic acid and nicotinamide in a feed by using microorganisms. According to the method, when a culture medium contains nutrient substances required by the growth of all thalli except the nicotinic acid and the nicotinamide, the growth amount of lactobacillus plantarum in a certain range corresponds to the content of the nicotinic acid in a standard solution in a standard pipe and the effective content of the nicotinic acid and the nicotinamide in a solution which is to be measured and has unknown concentration, so that the content of the nicotinic acid and the nicotinamide in a feed sample can be quantitatively calculated. The microorganism method has the advantages of high repeatability, sensitivity and accuracy and the like, and the effective content of the nicotinic acid and the nicotinamide in the feed can be accurately reflected.
Description
Technical field
The present invention relates to a kind of method of measuring nicotinic acid and niacinamide content in the feed, specifically, relate to a kind of method of utilizing nicotinic acid and niacinamide content in the microbioassay feed.
Background technology
The measuring method of nicotinic acid and vitamin PP is mainly HPLC in the vitamin premix at present, although this method has fast and convenient advantage, this method is only applicable to the purity height and is mostly the sample of unbound state, such as vitamin premix.But the mensuration to animal complete diet pellet and natural feed raw material nicotinic acid and vitamin PP does not still have suitable method; This is because nicotinic acid content is extremely low in complete diet pellet and the natural feed raw material; Go out peak area little or sample peak and the adhesion of assorted peak when adopting high effective liquid chromatography for measuring, error is very big.
Mikrobe plant lactobacillus (Lactobacillus plantarum) has high specificity and susceptibility to nicotinic acid and vitamin PP; Its growth is suppressed when lacking nicotinic acid and vitamin PP, thus utilize this characteristic of plant lactobacillus can working sample in the effective content of nicotinic acid and vitamin PP.
Summary of the invention
The purpose of this invention is to provide a kind of method of utilizing nicotinic acid and niacinamide content in the microbioassay feed, adopt nicotinic acid and the existing deficiency of niacinamide content in high effective liquid chromatography for measuring animal complete diet pellet and the natural feed raw material to overcome.
In order to realize the object of the invention; A kind of method of utilizing nicotinic acid and niacinamide content in the microbioassay feed of the present invention; It is that plant lactobacillus is inoculated in the used substratum of the plant lactobacillus growth that contains except that nicotinic acid and vitamin PP; Then the increment of plant lactobacillus is corresponding with the content of nicotinic acid that is added and vitamin PP standard substance, and the drawing standard curve; Then to the processing that is hydrolyzed of feed sample; The preparation testing sample solution; The plant lactobacillus of equivalent is inoculated in the substratum that contains testing sample solution, thereby goes out the content of nicotinic acid and vitamin PP in the feed sample according to the increment quantitative Analysis of plant lactobacillus.
Particularly; Preceding method may further comprise the steps: the 1) preparation of nicotinic acid and vitamin PP standardized solution: accurately take by weighing the nicotinic acid standard substance; Be mixed with the solution of concentration known with ethanol; The ethanolic soln that in containing the used substratum of plant lactobacillus growth except that nicotinic acid and vitamin PP, adds this nicotinic acid is mixed with the nicotinic acid final concentration and is 0,2.5,5,10,15,20,25,30,35,40,45 and the standardized solution of 50ng/mL; 2) drafting of nicotinic acid and vitamin PP typical curve: quantitative plant lactobacillus is inoculated in respectively in the standardized solution of above-mentioned different nicotinic acid concentration, according to the increment of plant lactobacillus and the content drawing standard curve of nicotinic acid standard substance; 3) hydrolysis treatment of feed sample: in the 1mol/L sulphuric acid soln, the sterilization of ultrasonic back is regulated pH value 6.0-6.5 with NaOH, uses salt acid for adjusting pH value 4.5 then, and hydrolyzed solution is transferred to constant volume in the volumetric flask, obtains testing sample solution with the feed sample dissolution; 4) mensuration of nicotinic acid and niacinamide content in the feed sample: the plant lactobacillus of equivalent is inoculated in the substratum that contains testing sample solution, goes out the content of nicotinic acid and vitamin PP in the feed sample according to the increment quantitative Analysis of plant lactobacillus.
Used mikrobe is plant lactobacillus (Lactobacillus plantarum) ATCC 8014 in the microbiological method of the present invention; Inoculating used plant lactobacillus is bacterium liquid form; Its preparation process is following: the 1) activation of bacterial classification: the adding of plant lactobacillus ATCC 8014 lyophilized powders is filled in the anaerobism pipe of MRS broth culture, place 37 ± 1 ℃ of incubators to cultivate, cultivate 48h first; Later every 24h goes down to posterity once; Go down to posterity and transfer in the MRS nutrient agar with puncture method after 10 times ,-20 ℃ of preservations, switching is once weekly; 2) preparation of bacterium liquid: the bacterium liquid that will transfer in the MRS broth culture is poured in the 10mL centrifuge tube, the centrifugal 10min of 2000rpm, abandoning supernatant; Add 10mL 0.9% saline water, concussion mixing such as preceding centrifugal, twice repeatedly; Add 10mL saline water again and process bacteria suspension; With saline water is contrast, measures down its transmittance with spectrophotometer in the 550nm wavelength, and transmittance is 60～80% a bacterium liquid operable bacterium liquid during for inoculation.
The MRS broth culture that uses in the preceding method is: glucose 30g, Tryptones 10g, Carnis Bovis seu Bubali cream 10g, yeast soak powder 5g, ammonium citrate 2g, sodium-acetate 5g, potassium hydrogenphosphate 2g, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.3g, four water manganous sulfate 0.12g, tween 80 1mL and water 1000mL, pH6.7 ± 0.2; The MRS nutrient agar is: the weight ratio by 1.5%～1.8% in above-mentioned MRS broth culture is added agar.
Adopt the mentioned microorganism assay method to measure the content of nicotinic acid and vitamin PP in natural feed and the animal complete diet pellet, said animal complete diet pellet is chicken, duck, pig, ox, sheep complete diet pellet.
The principle of microbiological method is when containing the required nutritive substance of all thalli growths except that nicotinic acid and vitamin PP in the substratum; Then within the specific limits the increment of plant lactobacillus just with standard pipe in the solution to be measured of nicotinic acid content and unknown concentration of standardized solution the effective content of nicotinic acid and vitamin PP corresponding, thereby but quantitative Analysis goes out the content of nicotinic acid and vitamin PP in the sample.
The present invention provides microbial method that the content of nicotinic acid and vitamin PP in natural feed raw material and the animal complete diet pellet is measured; And its precision and accuracy analyzed; Result's proof utilizes this method to measure nicotinic acid and niacinamide content in the feed, favorable reproducibility, and susceptibility is high; Accuracy is high, for the mensuration of nicotinic acid in the feed and niacinamide content provides reference.
Description of drawings
Fig. 1 is the increment of plant lactobacillus of the present invention and the typical curve of nicotinic acid content.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment, the raw materials used commercial goods that is.
The mensuration of nicotinic acid and niacinamide content in 1 10 kinds of natural feeds of embodiment and the 8 kinds of complete diet pellets
Used mikrobe is plant lactobacillus (Lactobacillus plantarum) ATCC 8014 in the microbiological method of present embodiment.
The used substratum of plant lactobacillus growth is the MRS broth culture: glucose 30g, Tryptones 10g, Carnis Bovis seu Bubali cream 10g; Yeast soaks powder 5g, ammonium citrate 2g, sodium-acetate 5g; Potassium hydrogenphosphate 2g, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.3g, four water manganous sulfate 0.12g; Tween 80 1mL adds water 1000mL, regulates pH6.7 ± 0.2; MRS nutrient agar: with adding agar according to 1.5%～1.8% ratio in the above-mentioned MRS broth culture.
1, the resurrection of bacterial classification and preservation: plant lactobacillus ATCC 8014 lyophilized powders are changed in the anaerobism pipe that the MRS broth culture is housed; Cultivate in 37 ± 1 ℃ of incubators; Cultivate 48h first, later every 24h goes down to posterity once, goes down to posterity to transfer in the MRS nutrient agar with puncture method after 10 times;-20 ℃ of preservations, switching once weekly.
2, the preparation of bacterium liquid: the bacterium liquid that will transfer in the MRS broth culture falls in the 10mL centrifuge tube 2000 rev/mins of centrifugal 10min, abandoning supernatant; Add 10mL 0.9% saline water, concussion mixing such as preceding centrifugal, twice repeatedly; Add 10mL saline water again, mixing.Drawing an amount of this bacteria suspension in 10mL saline water, is contrast with saline water, measures its transmittance with spectrophotometer down in the 550nm wavelength, as between 60～80%, then using.
3, the treatment process of feed sample: take by weighing 0.5g sample (choosing 10 kinds of natural feeds and 8 kinds of complete diet pellets) in the 250mL triangular flask, add 1mol/L sulphuric acid soln 50mL dissolved samples, thorough mixing; Ultrasonic 10min in UW places high-pressure sterilizing pot to preserve 30min for 121 ℃ then, takes out and is cooled to room temperature; Regulate pH to 6.5 with NaOH, concuss is regulated pH to 4.5 with hydrochloric acid again; Hydrolyzed solution is transferred in the brown volumetric flask of 100mL; Constant volume filters, and this hydrolyzed solution can be stored in 4 ℃ of refrigerators and store.Draw an amount of this hydrolyzed solution, add 20mL zero(ppm) water, regulate pH to 6.8, be settled to suitable scale.
4, the drafting of nicotinic acid typical curve: nicotinic acid standard reserving solution (100 μ g/mL): take by weighing 20mg (exact value 0.1mg) nicotinic acid standard substance (sigma), with 25% dissolve with ethanol solution and be settled to 200mL, be stored in 4 ℃ of refrigerators, preservation period is 4 months.Nicotinic acid standard intermediate solution (10 μ g/mL): from the nicotinic acid standard reserving solution, draw in the brown volumetric flask of 10mL to 100mL, with 25% ethanolic soln constant volume, be stored in 4 ℃ of refrigerators, preservation period is 1 month.Nicotinic acid standard operation liquid (100ng/mL): from the standard intermediate liquid, draw 1.0mL to brown volumetric flask, water is settled to 100mL, faces and uses preceding preparation.
The preparation of typical curve pipe: nicotinic acid content is respectively (ng) in the standard pipe: 0,0,25,50,100,150,200,250,300,350,400,450,500; With nicotinic acid content in the standard pipe is X-coordinate, and the OD value that records is an ordinate zou drawing standard curve.The preparation such as the table 1 of typical curve pipe.
The preparation of table 1 typical curve pipe (unit, mL)
The preparation of sample hose: add zero(ppm) water, sample solution and MRS by table 2 order and cultivate based in the test tube, duplicate.
The preparation of table 2 sample hose (unit, mL)
The mensuration of nicotinic acid and vitamin PP: whole test tubes are put into 121 ℃ of sterilizations of high-pressure sterilizing pot 5min, take out back cooling rapidly, in super clean bench, in above-mentioned test tube, respectively add 50 μ L bacterium liquid (except the S0), add a cover, fully shake each test tube with whirlpool concussion appearance.Put into 37 ℃ ± 1 ℃ incubator and cultivate 16h, S0 pipe should be clarification, is contrast with the S1 pipe, under the 550nm wavelength, measures S12 pipe absorbancy, measures once more after 2 hours, and twice measured value≤2% item can take out whole pipes mensuration.
Typical curve fits to cubic equation:
y＝2.225×10
-8x
3-2.431×10
-5x
2+0.009x+0.024，R
2＝0.999
(Fig. 1) can find out from typical curve; Rising along with nicotinic acid content; The OD value rises thereupon, and this has also met the characteristic of the essential nicotinic acid of plant lactobacillus ATCC 8014 growths, but both are not exclusively linear; The OD value rises slowly when being enough to satisfy bacterial growth when nicotinic acid content is elevated to, and typical curve is smooth-out.
5, the mensuration of nicotinic acid and niacinamide content in 10 kinds of natural feed raw materials and the 8 kinds of complete feeds:
The calculation formula of nicotinic acid and niacinamide content is:
In the formula: nicotinic acid in X-sample (vitamin PP) content, unit is mg/kg; Nicotinic acid in Cx-sample hose (vitamin PP) content, unit is ng; F-extension rate; The quality of m-sample or volume, unit are g.
10 kinds of natural feed raw materials and 8 kinds of complete feeds of recording are seen table 3 at the content of nicotinic acid and vitamin PP.
Nicotinic acid in table 3 testing sample (vitamin PP) content (unit, mg/kg)
*Sd: standard deviation
6, method circulation ratio checking
Respectively dregs of beans, fish meal, fryer material 1 and fryer material 2 are carried out 6 times again with microbial method of the present invention and measure result such as table 4.
Table 4 method circulation ratio checking result
RSD＜10% shows that present method is stable.
7, method accuracy checking
Take by weighing the sample of a certain amount of known nicotinic acid and niacinamide content, add a certain amount of 100 μ g/mL nicotinic acid standardized solution, carry out sample preparation and mensuration, calculate recovery rate like preceding method.The result is as shown in table 5.
Table 5 microbial method determination of recovery rates of the present invention result
Can find out that from table 5 nicotinic acid that microbial method of the present invention the records recovery as a result is high, average recovery rate is 98.75%.
The mensuration of nicotinic acid and niacinamide content in 2 10 kinds of natural feed raw materials of embodiment and the 8 kinds of complete diet pellets
Except that the treatment process of sample slightly the difference, all the other determination steps are with embodiment 1.
The treatment process of sample: take by weighing the 1.0g sample in the 250mL triangular flask, add 1mol/l sulphuric acid soln 80mL dissolved samples, thorough mixing, ultrasonic 15min in UW; Place high-pressure sterilizing pot to preserve 20min for 121 ℃, take out and be cooled to room temperature, regulate pH to 6.0 with NaOH; Concuss is regulated pH to 4.8 with hydrochloric acid again, and hydrolyzed solution is transferred in the brown volumetric flask of 100mL; Constant volume filters, and this hydrolyzed solution can be stored in 4 ℃ of refrigerators and store.Draw an amount of this hydrolyzed solution, add 20mL zero(ppm) water, regulate pH to 6.6, be settled to suitable scale.
10 kinds of natural feeds and 8 kinds of complete feeds of recording are seen table 3 at the content of nicotinic acid and vitamin PP.
The mensuration of nicotinic acid and niacinamide content in 3 10 kinds of natural feeds of embodiment and the 8 kinds of complete diet pellets
Except that the treatment process of sample slightly the difference, all the other determination steps are with embodiment 1.
The treatment process of sample: take by weighing the 1.5g sample in the 250mL triangular flask, add 1mol/l sulphuric acid soln 100mL dissolved samples, thorough mixing, ultrasonic 20min in UW; Place high-pressure sterilizing pot to preserve 25min for 121 ℃, take out and be cooled to room temperature, regulate pH to 6.3 with NaOH; Concuss is regulated pH to 4.5 with hydrochloric acid again, and hydrolyzed solution is transferred in the brown volumetric flask of 100mL; Constant volume filters, and this hydrolyzed solution can be stored in 4 ℃ of refrigerators and store.Draw an amount of this hydrolyzed solution, add 20mL zero(ppm) water, regulate pH to 6.9, be settled to suitable scale.
10 kinds of natural feeds and 8 kinds of complete feeds of recording are seen table 3 at the content of nicotinic acid and vitamin PP.
Can find out from embodiment 1～3; Microbial method provided by the invention is measured nicotinic acid and niacinamide content in natural feed raw material and the animal complete diet pellet; Have favorable reproducibility, susceptibility is high, the accuracy advantages of higher; The effective content that can accurately reflect nicotinic acid and vitamin PP in the feed is for the mensuration of vitamin contents in the feed provides reference.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (7)
1. method of utilizing nicotinic acid and niacinamide content in the microbioassay feed; It is characterized in that; Plant lactobacillus is inoculated in the used substratum of the plant lactobacillus growth that contains except that nicotinic acid and vitamin PP; Then the increment of plant lactobacillus is corresponding with the content of nicotinic acid that is added and vitamin PP standard substance, and the drawing standard curve; Then to the processing that is hydrolyzed of feed sample; The preparation testing sample solution; The plant lactobacillus of equivalent is inoculated in the substratum that contains testing sample solution, thereby goes out the content of nicotinic acid and vitamin PP in the feed sample according to the increment quantitative Analysis of plant lactobacillus.
2. method according to claim 1 is characterized in that, may further comprise the steps:
1) preparation of nicotinic acid and vitamin PP standardized solution: accurately take by weighing the nicotinic acid standard substance; Be mixed with the solution of concentration known with ethanol; The ethanolic soln that in containing the used substratum of plant lactobacillus growth except that nicotinic acid and vitamin PP, adds this nicotinic acid is mixed with the nicotinic acid final concentration and is 0,2.5,5,10,15,20,25,30,35,40,45 and the standardized solution of 50ng/mL;
2) drafting of nicotinic acid and vitamin PP typical curve: quantitative plant lactobacillus is inoculated in respectively in the standardized solution of above-mentioned different nicotinic acid concentration, according to the increment of plant lactobacillus and the content drawing standard curve of nicotinic acid standard substance;
3) hydrolysis treatment of feed sample: in the 1mol/L sulphuric acid soln, the sterilization of ultrasonic back is regulated pH value 6.0-6.5 with NaOH, uses salt acid for adjusting pH value 4.5 then, and hydrolyzed solution is transferred to constant volume in the volumetric flask, obtains testing sample solution with the feed sample dissolution;
4) mensuration of nicotinic acid and niacinamide content in the feed sample: the plant lactobacillus of equivalent is inoculated in the substratum that contains testing sample solution, goes out the content of nicotinic acid and vitamin PP in the feed sample according to the increment quantitative Analysis of plant lactobacillus.
3. method according to claim 1 is characterized in that, said plant lactobacillus is plant lactobacillus (Lactobacillus plantarum) ATCC 8014.
4. method according to claim 3 is characterized in that, inoculating used plant lactobacillus is bacterium liquid form, and its preparation process is following:
1) activation of bacterial classification: the adding of plant lactobacillus ATCC 8014 lyophilized powders is filled in the anaerobism pipe of MRS broth culture; Place 37 ± 1 ℃ of incubators to cultivate; Cultivate 48h first, later every 24h goes down to posterity once, goes down to posterity to transfer in the MRS nutrient agar with puncture method after 10 times;-20 ℃ of preservations, switching is once weekly;
2) preparation of bacterium liquid: the bacterium liquid that will transfer in the MRS broth culture is poured in the 10mL centrifuge tube, 2000 rev/mins of centrifugal 10min, abandoning supernatant; Add 10ml 0.9% saline water, concussion mixing such as preceding centrifugal, twice repeatedly; Add 10ml saline water again and process bacteria suspension; With saline water is contrast, measures down its transmittance with spectrophotometer in the 550nm wavelength, and transmittance is the bacterium liquid that 60～80% bacterium liquid uses during for inoculation.
5. method according to claim 4; It is characterized in that; Said MRS broth culture is: glucose 30g, Tryptones 10g, Carnis Bovis seu Bubali cream 10g, yeast soak powder 5g, ammonium citrate 2g, sodium-acetate 5g, potassium hydrogenphosphate 2g, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.3g, four water manganous sulfate 0.12g, tween 80 1mL and water 1000mL, pH6.7 ± 0.2; Said MRS nutrient agar is: the weight ratio by 1.5%～1.8% in above-mentioned MRS broth culture is added agar.
6. according to each described method of claim 1-5, it is characterized in that said feed is natural feed or animal complete diet pellet.
7. method according to claim 6 is characterized in that, said animal complete diet pellet is chicken, duck, pig, ox, sheep complete diet pellet.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043030A (en) * | 2009-10-22 | 2011-05-04 | 北京万全阳光医学技术有限公司 | Method for measuring materials associated with niacin simvastatin sustained-release tablets by high performance liquid chromatography |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043030A (en) * | 2009-10-22 | 2011-05-04 | 北京万全阳光医学技术有限公司 | Method for measuring materials associated with niacin simvastatin sustained-release tablets by high performance liquid chromatography |
Non-Patent Citations (3)
| Title |
|---|
| 中华人民共和国卫生部: "食品安全国家标准 婴幼儿食品和乳品中烟酸和烟酰胺的测定", 《中华人民共和国国家标准GB 5413.15-2010》, 26 March 2010 (2010-03-26), pages 1 - 7 * |
| 殷晓红等: "微生物法测定食品中的烟酸和烟酰胺", 《中国乳品工业》, vol. 31, no. 2, 31 December 2003 (2003-12-31), pages 32 - 35 * |
| 董爽等: "微生物法测定饲料中烟酸含量", 《饲料加工与检测》, vol. 48, no. 15, 31 December 2012 (2012-12-31), pages 66 - 69 * |
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