CN102041314A - Method for testing content of biotins - Google Patents
Method for testing content of biotins Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, particularly discloses a method for testing the content of biotins. The method comprises the following steps of: 1, cultivating AS1.299 in a seed culture medium, then removing the seed culture medium, and diluting AS1.299 into an AS1.299 bacteria solution; 2, respectively adding biotins with gradient contents and the AS1.299 bacteria solution with the same volume to testing culture media with the same volume, cultivating after settling, and drawing a standard curve according to an OD620 value to obtain a linear equation; 3, adding raw materials to be tested and the AS1.299 bacteria solution to the testing culture media in the step 2, cultivating after settling, then testing the OD620 value, and calculating the content of the biotins in the raw materials to be tested by using the linear equation. The method can be used for effectively eliminating effects caused by culture media, inoculum size and raw material components and increasing the testing accuracy of biotins with a microbiological method, and can be widely applied in actual production.
Description
Technical field
The present invention relates to a kind of detection method of biological technical field, be specifically related to a kind of method of detection of biological cellulose content.
Background technology
Vitamin H claims vitamin H, vitamin H again, and molecular formula is C
6H
16N
2O
3S is a kind of human body natural's of keeping growth and the necessary water-soluble vitamins of normal human's function.Vitamin H also is the microbial growth factor except as human body normal growth, growth and the healthy necessary nutritive element, is vital material in the glutamic acid fermentation.In glutamic acid fermentation, vitamin H content for control thalline transition, produce acid phase transformation efficiency and whole output all has remarkably influenced.
At present, the main vitamin H suboptimal dose method zymotechnique that adopts carries out glutamic acid fermentation in the monosodium glutamate industry, promptly adjusts the fermentation raw material consumption, makes that vitamin H content is in suboptimal dose in the fermented liquid, thereby allows thalline secrete accumulation L-glutamic acid in a large number.In actual production, determine that the fermentation raw material consumption often will show vitamin H content from analysis fermentation raw material directly perceived according to experience, jar feelings earlier, and then grope and to determine through what multiple tank was criticized.But this intuitive analysis has a lot of uncertain factors, can't guarantee that vitamin H content is in the suboptimal dose scope.In addition, every batch of fermentation raw material vitamin H content is all different, all needs to grope again consumption behind the replacing raw material.Even same batch fermentation raw material, because fluctuation appears in the improper vitamin H content that also can cause of condition of storage, this has brought difficulty to a great extent to glutamic acid fermentation.
Therefore, before fermentation, need fermentation raw material vitamin H content is accurately detected.Main measuring method has microbial method, high performance liquid chromatography and marked by fluorescein isothiocyanate avidin method etc. at present.Wherein, high performance liquid chromatography and marked by fluorescein isothiocyanate avidin method precision are better, but need expensive precision instrument and reagent, and the sample preparation process is complicated, are difficult to apply on producing.Microbial method is used widely on producing owing to highly sensitive, easy and simple to handle.
Microbial method is the biological detecting method that utilizes the vitamin H deficient strain that the dependency of vitamin H is set up, and can detect the vitamin H of biologically active delicately, and this is the maximum characteristics that microbial method is different from other method.The microorganism ratio juris is under certain condition, the height of biotin concentration with identify linear with the bacteria growing amount, by the drawing standard curve and then try to achieve vitamin H content.But, traditional microbial method has very mistake in actual production, even occurs the error on the order of magnitude sometimes, and reason mainly is: amino nitrogen in (1) substratum, inorganic salt, micro-growth factor equal size are higher, growth to thalline has considerable influence, directly detection is impacted; (2) measure process at every turn can't guarantee the inoculum size unanimity; (3) in the testing process, the amino nitrogen in the raw material, Vb
1Bigger to detecting influence, cause error bigger, thereby can't effectively instruct production.These reasons make microbial method can not effectively instruct production.
Summary of the invention
In view of this, the invention provides a kind of method of detection of biological cellulose content, this method is eliminated the influence that factors such as substratum, inoculum size and raw material cause effectively, reduces error, has improved the tolerance range of utilizing microorganism detection vitamin H content.
The method of a kind of detection of biological cellulose content of the present invention specifically comprises:
Step 1, AS1.299 bacterial strain are cultivated the back in seed culture medium and are removed seed culture medium, make AS1.299 bacterium liquid after the dilution, and described seed culture medium comprises 5% glucose, 0.8% urea, 3% corn steep liquor, 0.14%K
2HPO
4, 0.06%MgSO
4, 2 μ g/mL MnSO
4, 2 μ g/mL FeSO
4
Step 2, respectively to volume identical respectively detect in the substratum AS1.299 bacterium liquid that adds gradient content biotin solution and equal volume, cultivate behind the constant volume, then according to OD
620Value drawing standard curve, draw linear equation, described detection substratum comprises 5% glucose, 0.8% urea, the aspartic acid of 16mg/100mL, the Serine of 8mg/100mL, the glycine of 16mg/100mL, the Threonine of 10mg/100mL, the L-Ala of 10mg/100mL, the arginine of 14mg/100mL, the tyrosine of 4mg/100mL, the Xie Ansuan of 7mg/100mL, the methionine(Met) of 3mg/100mL, the phenylalanine of 10mg/100mL, the Isoleucine of 7mg/100mL, the leucine of 16mg/100mL, the Methionin of 19mg/100mL, the proline(Pro) of 2mg/100mL, the Vb of 20 μ g/L
1, 0.14%K
2HPO
4, 0.06%MgSO
4, 2 μ g/mLMnSO
4With 2 μ g/mL FeSO
4
Step 3, get raw material to be measured and AS1.299 bacterium liquid and be added in the described detection substratum of step 2 and cultivate behind the constant volume, detect OD then
620Value, calculate the vitamin H content of raw material to be measured by described linear equation, the volume of the volume of the volume of the volume of described AS1.299 bacterium liquid, the volume that detects substratum, constant volume and the time of cultivation and the described AS1.299 bacterium of step 2 liquid, the volume of detection substratum, constant volume and the time of cultivation are consistent.
Wherein, described AS1.299 bacterial strain is available from China Committee for Culture Collection of Microorganisms common micro-organisms center, and described typical curve linearity range is 0.2-1 μ g/L, and the described constant volume of step 2 is for being settled to 100mL, and the time of the described cultivation of step 2 is 12-14h.
Because glucose is the main source of the required carbon source of thalli growth, urea can be thalli growth and provides inorganic nitrogen-sourced and have the effect of regulating the pH value, the two influence to thalli growth is greater than the influence of other compositions, so the present invention is to the glucose of seed culture medium and detection substratum, the urea initial content is optimized, make bacterial strain in cultivation and testing process, can not be subjected to the restriction of carbon source and nitrogenous source substrate concn substantially, guarantee that simultaneously initial content can not produce obvious restraining effect to the growth of bacterial strain, thereby improved bacterial strain maximum growth amount (corresponding to maximum OD value) in the vitamin H linearity range, the testing process error is dwindled relatively, improved the tolerance range that detects.
Because amino nitrogen and Vb
1Strain growth is had remarkably influenced, and the present invention's raw material to be measured is corn steep liquor or molasses, wherein all contains a certain amount of amino nitrogen and Vb
1So the present invention additionally adds amino acid and Vb in 14 in detecting substratum
1, make amino nitrogen and Vb
1Amount surpass scope influence strain growth, thereby these two factors are to the interference in the testing process in the elimination raw material.
In addition, the present invention carried out centrifuge washing three times to the bacterial strain after the seed culture medium cultivation before detecting, can effectively remove the seed culture based component, clearance reaches more than 99.9%, remaining amino nitrogen and micro-growth factor can be ignored the influence of testing process, avoid the seed culture medium nutritive ingredient to be brought into thus and detect in the substratum, detection is caused error.
The present invention also breaks up thalline by granulated glass sphere and makes it mixing before inoculation, guaranteed the consistence of inoculation, and the present invention carries out sample determination at every turn and all will repaint typical curve, makes each typical curve corresponding with the actual thalline quantity of inoculating.
The present invention finds that through test in the logarithmic growth stage, the linear dependence of typical curve does not have direct relation with the minute of choosing point, and then different time points mensuration OD value gained vitamin H content results has collimation.Therefore, the present invention takes a sample by many time points (more than 5) are set at logarithmic phase, to reduce the error that culturing process and sampling and measuring process produce.
By above technical scheme as can be known, the present invention can eliminate the influence that factors such as substratum, inoculum size and raw material cause effectively, has reduced error, has improved the tolerance range of utilizing microorganism detection vitamin H content, this method is highly sensitive, easy and simple to handle, can be widely used in actual production.
Embodiment
The invention discloses a kind of method of detection of biological cellulose content, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Method of the present invention is described by preferred embodiment, and the related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
According to the present invention, the method for described a kind of detection of biological cellulose content comprises:
Step 1, AS1.299 bacterial strain are cultivated the back in seed culture medium and are removed seed culture medium, make AS1.299 bacterium liquid after the dilution, and described seed culture medium comprises 5% glucose, 0.8% urea, 3% corn steep liquor, 0.14%K
2HPO
4, 0.06%MgSO
4, 2 μ g/mL MnSO
4, 2 μ g/mL FeSO
4
Step 2, respectively to volume identical respectively detect in the substratum AS1.299 bacterium liquid that adds gradient content biotin solution and equal volume, cultivate behind the constant volume, then according to OD
620Value drawing standard curve, draw linear equation, described detection substratum comprises 5% glucose, 0.8% urea, the aspartic acid of 16mg/100mL, the Serine of 8mg/100mL, the glycine of 16mg/100mL, the Threonine of 10mg/100mL, the L-Ala of 10mg/100mL, the arginine of 14mg/100mL, the tyrosine of 4mg/100mL, the Xie Ansuan of 7mg/100mL, the methionine(Met) of 3mg/100mL, the phenylalanine of 10mg/100mL, the Isoleucine of 7mg/100mL, the leucine of 16mg/100mL, the Methionin of 19mg/100mL, the proline(Pro) of 2mg/100mL, the Vb of 20 μ g/L
1, 0.14%K
2HPO
4, 0.06%MgSO
4, 2 μ g/mLMnSO
4With 2 μ g/mL FeSO
4
Step 3, get raw material to be measured and AS1.299 bacterium liquid and be added in the described detection substratum of step 2 and cultivate behind the constant volume, detect OD then
620Value, calculate the vitamin H content of raw material to be measured by described linear equation, the volume of the volume of the volume of the volume of described AS1.299 bacterium liquid, the volume that detects substratum, constant volume and the time of cultivation and the described AS1.299 bacterium of step 2 liquid, the volume of detection substratum, constant volume and the time of cultivation are consistent.
Wherein, described typical curve linearity range is 0.2-1 μ g/L, and described raw material to be measured is corn steep liquor or molasses, and the described constant volume of step 2 is for being settled to 100mL, and the time of the described cultivation of step 2 is 12-14h.
Adopt the method for the invention to detect, the linear dependence degree is more than 0.99, relative error is less than 5%, and same materials detects gained vitamin H content and present method through marked by fluorescein isothiocyanate avidin method, and to survey vitamin H content approaching, no significant difference, show that the method for the invention tolerance range is higher, to survey the vitamin H content data credible.
In addition, the present invention has carried out vitamin H standard substance content rate of recovery detection test respectively to corn steep liquor and molasses, corn steep liquor vitamin H standard substance average recovery rate is 110%, molasses vitamin H standard substance average recovery rate is 95%, both are all near 100%, and the result shows that further the method for the invention accuracy is higher.
Below in conjunction with embodiment, further set forth the present invention.
Embodiment 1: glucose and urea initial content are analyzed in the method for the invention
The present invention is by first sugar, just urinate the single factor experiment of consumption and AS1.299 strain growth, and seed culture medium and the glucose, the urea initial content that detect substratum are optimized, and test-results is referring to table 1.
Table 1 just sugar, just urinate the single factor experiment of consumption and AS1.299 strain growth
The result shows that glucose was at 5% o'clock, and the AS1.299 bacterial strain reaches the maximum growth amount, and enters stationary stage at 12-14h; Urea was at 0.8% o'clock, and the AS1.299 bacterial strain reaches the maximum growth amount, and entered stationary stage at 12-14h.Therefore, just sugar, just urinate consumption and be respectively 5% and 0.8%.
Embodiment 2: the method for the invention detects vitamin H content in the corn steep liquor
1, test reagent and preparation
Urea soln: 115 ℃ of sterilizations of the urea 3min for preparing 40% content is used to prepare substratum;
Physiological saline: 15min is standby for 121 ℃ of sterilizations of preparation 0.85%NaCl solution;
Corn steep liquor solution: the corn steep liquor raw material is provided by Club Biological Technology Group Co., Ltd's production plant, will get 10g behind the abundant mixing of corn steep liquor raw material, and it is standby that thin up is settled to 100mL sterilization back;
The vitamin H mother liquor: the biotin solution sterilization back of preparation 5.000mg/L is standby.
Seed culture medium: 5% glucose, 0.8% urea, 3% corn steep liquor, 0.14%K
2HPO
4, 0.06%MgSO
4, 2 μ g/mL MnSO
4, 2 μ g/mL FeSO
4
Detect substratum: 5% glucose, 0.8% urea, 4% amino acid, 0.14%K
2HPO
4, 0.06%MgSO
4, 2 μ g/mL MnSO
4, 2 μ g/mL FeSO
4, 20 μ g/L Vb
1
The methionine(Met) of the tyrosine of the L-Ala of the glycine of the aspartic acid of 4% amino acid: 16mg/100mL, the Serine of 8mg/100mL, 16mg/100mL, the Threonine of 10mg/100mL, 10mg/100mL, the arginine of 14mg/100mL, 4mg/100mL, the Xie Ansuan of 7mg/100mL, 3mg/100mL, the phenylalanine of 10mg/100mL, the Isoleucine of 7mg/100mL, the leucine of 16mg/100mL, the Methionin of 19mg/100mL and the proline(Pro) of 2mg/100mL.
2, detection method
Encircle thalline in seed culture medium by aseptic technique picking 2 from AS1.299 activated inclined plane substratum, eight layers of gauze seal, in shaking table 32 ℃, 220r/min cultivates 10h and obtains seed liquor, according to being sub-packed in the 10mL centrifuge tube (every pipe dress 7mL) with measuring required seed liquor, 4000rpm, after 10min is centrifugal, in centrifuge tube, add the 5mL stroke-physiological saline solution after the supernatant discarded, the centrifugal again supernatant of abandoning after concussion suspends, add the 5mL stroke-physiological saline solution again, the centrifugal again supernatant of abandoning after concussion suspends, after adding the suspension of 7mL stroke-physiological saline solution bacterium liquid in each centrifuge tube is merged in the aseptic triangular flask with granulated glass sphere of packing into, place revolution shaking table 220rpm, 32 ℃ obtain bacterium liquid after breaing up 15min.
To detect every bottle of constant volume packing of substratum 96mL, accurately getting vitamin H mother liquor 0,4,8,12,16,20 μ L joins respectively in the detection substratum, detect substratum by aseptic technique to every bottle and accurately add 2000 μ L bacterium liquid, be settled to 100mL, eight layers of gauze seal, 32 ℃, 220r/min are cultivated 12h in shaking table, detect every bottle and detect OD in the substratum
620, drawing X-coordinate is biological cellulose content, ordinate zou is Δ OD
620Typical curve, Δ OD
620=add the OD of vitamin H
620-do not add the OD of vitamin H
620, and adopt the LINEST function calculation among the EXCEL to obtain slope according to typical curve, and adopt the INTERRUPT function calculation to obtain intercept, obtain linear equation at last.
To detect every bottle of constant volume packing of substratum 96mL, accurately getting corn steep liquor solution 0,500 μ L joins respectively in the detection substratum, detect substratum by aseptic technique to every bottle and accurately add 2000 μ L bacterium liquid, be settled to 100mL, eight layers of gauze seal, 32 ℃, 220r/min are cultivated 12h equally in shaking table, detect every bottle and detect OD in the substratum
620, try to achieve Δ OD
620=add the OD of corn steep liquor
620-the OD of corn steep liquor not
620, try to achieve the vitamin H content in the corn steep liquor of surveying according to linear equation.Test repeats 4 times, and sampling point detection time is 14,16,18,20h, repeats to prepare bacterium liquid, drawing standard curve again at every turn.
3, test-results
Detect vitamin H content in the corn steep liquor by aforesaid method, test-results is specifically referring to table 2 and table 3.
The Δ OD of each point in time sampling of table 2
620
| Vitamin H content | 0.2μg/L | 0.4μg/L | 0.6μg/L | 0.8μg/L | 1.0μg/L | The corn steep liquor sample |
| 12hΔOD 620 | 0.127 | 0.215 | 0.310 | 0.396 | 0.491 | 0.237 |
| 14hΔOD 620 | 0.132 | 0.234 | 0.331 | 0.436 | 0.509 | 0.251 |
| 16hΔOD 620 | 0.133 | 0.244 | 0.338 | 0.445 | 0.528 | 0.265 |
| 18hΔOD 620 | 0.138 | 0.257 | 0.360 | 0.464 | 0.574 | 0.271 |
| 20hΔOD 620 | 0.136 | 0.258 | 0.364 | 0.470 | 0.592 | 0.276 |
The dependency of table 3 typical curve and vitamin H cubage result
The result shows that vitamin H content is 887 ± 11 μ g/kg in the corn steep liquor, and the dependency of each typical curve is all more than 0.99, and relative error is 1.2%, shows that the method for the invention has good linearity and less relative error, has than high degree of accuracy.
Embodiment 3: the method for the invention detects vitamin H content in the molasses
1, test reagent and preparation
Urea soln: 115 ℃ of sterilizations of the urea 3min for preparing 40% content is used to prepare substratum;
Physiological saline: 15min is standby for 121 ℃ of sterilizations of preparation 0.85%NaCl solution;
Molasses solution: molasses raw material is provided by Club Biological Technology Group Co., Ltd's production plant, will get 10g behind the abundant mixing of molasses raw material, and it is standby that thin up is settled to 100mL sterilization back;
The vitamin H mother liquor: the biotin solution sterilization back of preparation 5.000mg/L is standby.
Seed culture medium: 5% glucose, 0.8% urea, 3% corn steep liquor, 0.14%K
2HPO
4, 0.06%MgSO
4, 2 μ g/mL MnSO
4, 2 μ g/mL FeSO
4
Detect substratum: 5% glucose, 0.8% urea, 4% amino acid, 0.14%K
2HPO
4, 0.06%MgSO
4, 2 μ g/mL MnSO
4, 2 μ g/mL FeSO
4, 20 μ g/L Vb
1
The methionine(Met) of the tyrosine of the L-Ala of the glycine of the aspartic acid of 4% amino acid: 16mg/100mL, the Serine of 8mg/100mL, 16mg/100mL, the Threonine of 10mg/100mL, 10mg/100mL, the arginine of 14mg/100mL, 4mg/100mL, the Xie Ansuan of 7mg/100mL, 3mg/100mL, the phenylalanine of 10mg/100mL, the Isoleucine of 7mg/100mL, the leucine of 16mg/100mL, the Methionin of 19mg/100mL and the proline(Pro) of 2mg/100mL.
2, detection method
Encircle thalline in seed culture medium by aseptic technique picking 2 from AS1.299 activated inclined plane substratum, eight layers of gauze seal, in shaking table 32 ℃, 220r/min cultivates 10h and obtains seed liquor, according to being sub-packed in the 10mL centrifuge tube (every pipe dress 7mL) with measuring required seed liquor, 4000rpm, after 10min is centrifugal, in centrifuge tube, add the 5mL stroke-physiological saline solution after the supernatant discarded, the centrifugal again supernatant of abandoning after concussion suspends, add the 5mL stroke-physiological saline solution again, the centrifugal again supernatant of abandoning after concussion suspends, after adding the suspension of 7mL stroke-physiological saline solution bacterium liquid in each centrifuge tube is merged in the aseptic triangular flask with granulated glass sphere of packing into, place revolution shaking table 220rpm, 32 ℃ obtain bacterium liquid after breaing up 15min.
To detect every bottle of constant volume packing of substratum 96mL, accurately getting vitamin H mother liquor 0,4,8,12,16,20 μ L joins respectively in the detection substratum, detect substratum by aseptic technique to every bottle and accurately add 2000 μ L bacterium liquid, be settled to 100mL, eight layers of gauze seal, 32 ℃, 220r/min are cultivated 8h in shaking table, detect every bottle and detect OD in the substratum
620, drawing X-coordinate is biological cellulose content, ordinate zou is Δ OD
620Typical curve, Δ OD
620=add the OD of vitamin H
620-do not add the OD of vitamin H
620, and adopt the LINEST function calculation among the EXCEL to obtain slope according to typical curve, and adopt the INTERRUPT function calculation to obtain intercept, obtain linear equation at last.
To detect every bottle of constant volume packing of substratum 96mL, accurately getting molasses solution 0,250 μ L joins respectively in the detection substratum, detect substratum by aseptic technique to every bottle and accurately add 2000 μ L bacterium liquid, be settled to 100mL, eight layers of gauze seal, 32 ℃, 220r/min are cultivated 8h equally in shaking table, detect every bottle and detect OD in the substratum
620, try to achieve Δ OD
620=add the OD of molasses
620-the OD of molasses not
620, try to achieve the vitamin H content in the molasses of surveying according to linear equation.Test repeats 5 times, and sampling point detection time is 10,12,14,16,18h, repeats to prepare bacterium liquid, drawing standard curve again at every turn.
3, test-results
Detect vitamin H content in the molasses by aforesaid method, test-results is specifically referring to table 4 and table 5.
The Δ OD of each point in time sampling of table 4
620
| Vitamin H content | 0.2μg/L | 0.4μg/L | 0.6μg/L | 0.8μg/L | 1.0μg/L | The molasses sample |
| 8hΔOD 620 | 0.083 | 0.153 | 0.210 | 0.259 | 0.328 | 0.246 |
| 10hΔOD 620 | 0.104 | 0.175 | 0.237 | 0.296 | 0.377 | 0.302 |
| 12hΔOD 620 | 0.090 | 0.170 | 0.258 | 0.342 | 0.403 | 0.308 |
| 14hΔOD 620 | 0.105 | 0.193 | 0.302 | 0.373 | 0.457 | 0.330 |
| 16hΔOD 620 | 0.095 | 0.208 | 0.330 | 0.400 | 0.501 | 0.393 |
| 18hΔOD 620 | 0.090 | 0.222 | 0.349 | 0.432 | 0.526 | 0.425 |
The dependency of table 5 typical curve and vitamin H cubage result
The result shows that vitamin H content is 1507 ± 72 μ g/kg in the molasses, and the dependency of each typical curve is all more than 0.99, and relative error is 5%, shows that the method for the invention has good linearity and less relative error, has higher precision.
Embodiment 4: the method for the invention accuracy analysis
Corn steep liquor among the embodiment 2,3 and molasses are measured with marked by fluorescein isothiocyanate avidin method respectively, and detected result compares with the result among the embodiment 2,3, specifically referring to table 6.
Table 6 the method for the invention accuracy analysis
| Corn steep liquor vitamin H content (μ g/kg) | Molasses vitamin H content (μ g/kg) | |
| Microbial method | 887 | 1507 |
| Marked by fluorescein isothiocyanate avidin method | 895 | 1540 |
The result shows that the result who adopts marked by fluorescein isothiocyanate avidin method to measure is respectively 895 μ g/kg and 1540 μ g/kg, and approaching with embodiment 2,3 measured results, no significant difference shows that the method for the invention accuracy is higher.
Embodiment 5: the method for the invention accuracy analysis
For further embodying the accuracy of the method for the invention, the present invention carries out the detection of the vitamin H standard substance content rate of recovery to corn steep liquor and the molasses in embodiment 2,3 each tests.
Rate of recovery detection method: add a certain amount of vitamin H standard substance in the sample by corn steep liquor in embodiment 2,3 each tests and molasses, the content that deducts sample with detected content behind the mark-on is the background amount, can obtain adding the detected value of scalar, this detected value is exactly the rate of recovery with the ratio that adds scalar, if there is the factor that makes detected result produce deviation in detection method, the detected value that adds scalar will be subjected to the influence of this factor and be higher or on the low side, thereby embodies with the form of the rate of recovery.Concrete outcome is referring to table 7 and table 8.
Table 7 corn steep liquor vitamin H standard substance rate of recovery detected result
| Parallels | Background amount (μ g/kg) | Add scalar (μ g/kg) | Vitamin H standard substance content detection value (μ g/kg) | The rate of recovery |
| 1 | 887 | 800 | 1703 | 102％ |
| 2 | 878 | 800 | 1782 | 113％ |
| 3 | 907 | 800 | 1771 | 108％ |
| 4 | 877 | 800 | 1765 | 111％ |
| 5 | 886 | 800 | 1814 | 116％ |
| Mean value | 887 | 800 | 1767 | 110％ |
Table 8 molasses vitamin H standard substance rate of recovery detected result
| Parallels | Background amount (μ g/kg) | Add scalar (μ g/kg) | Vitamin H standard substance content detection value (μ g/kg) | The rate of recovery |
| 1 | 1464 | 1500 | 2724 | 84％ |
| 2 | 1585 | 1500 | 3130 | 103％ |
| 3 | 1478 | 1500 | 3008 | 102％ |
| 4 | 1399 | 1500 | 2764 | 91％ |
| 5 | 1543 | 1500 | 2953 | 94％ |
| 6 | 1574 | 1500 | 3014 | 96％ |
| Mean value | 1507 | 1500 | 2932 | 95％ |
The result shows that corn steep liquor vitamin H standard substance average recovery rate is 110%, and molasses vitamin H standard substance average recovery rate is 95%, and both show further that all near 100% the method for the invention accuracy is higher.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (5)
1. the method for a detection of biological cellulose content is characterized in that, comprising:
Step 1, AS1.299 bacterial strain are cultivated the back in seed culture medium and are removed seed culture medium, make AS1.299 bacterium liquid after the dilution, and described seed culture medium comprises 5% glucose, 0.8% urea, 3% corn steep liquor, 0.14%K
2HPO
4, 0.06%MgSO
4, 2 μ g/mL MnSO
4, 2 μ g/mL FeSO
4
Step 2, respectively to volume identical respectively detect in the substratum AS1.299 bacterium liquid that adds gradient content biotin solution and equal volume, cultivate behind the constant volume, then according to OD
620Value drawing standard curve, draw linear equation, described detection substratum comprises 5% glucose, 0.8% urea, the aspartic acid of 16mg/100mL, the Serine of 8mg/100mL, the glycine of 16mg/100mL, the Threonine of 10mg/100mL, the L-Ala of 10mg/100mL, the arginine of 14mg/100mL, the tyrosine of 4mg/100mL, the Xie Ansuan of 7mg/100mL, the methionine(Met) of 3mg/100mL, the phenylalanine of 10mg/100mL, the Isoleucine of 7mg/100mL, the leucine of 16mg/100mL, the Methionin of 19mg/100mL, the proline(Pro) of 2mg/100mL, the Vb of 20 μ g/L
1, 0.14%K
2HPO
4, 0.06%MgSO
4, 2 μ g/mLMnSO
4With 2 μ g/mL FeSO
4
Step 3, get raw material to be measured and AS1.299 bacterium liquid and be added in the described detection substratum of step 2 and cultivate behind the constant volume, detect OD then
620Value, calculate the vitamin H content of raw material to be measured by described linear equation, the volume of the volume of the volume of the volume of described AS1.299 bacterium liquid, the volume that detects substratum, constant volume and the time of cultivation and the described AS1.299 bacterium of step 2 liquid, the volume of detection substratum, constant volume and the time of cultivation are consistent.
2. according to the described method of claim 1, it is characterized in that described typical curve linearity range is 0.2-1 μ g/L.
3. according to the described method of claim 1, it is characterized in that described raw material to be measured is corn steep liquor or molasses.
4. according to the described method of claim 1, it is characterized in that the described constant volume of step 2 is for being settled to 100mL.
5. according to the described method of claim 1, it is characterized in that the time of the described cultivation of step 2 is 12-14h.
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| CN2010105514821A CN102041314B (en) | 2010-11-19 | 2010-11-19 | Method for testing content of biotins |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102680420A (en) * | 2012-05-30 | 2012-09-19 | 国药集团威奇达药业有限公司 | Method for rapidly determining biotins in miniaturized manner |
| CN106222233A (en) * | 2016-08-03 | 2016-12-14 | 四川剑南春（集团）有限责任公司 | A kind of daqu fermentation power detection culture medium, the method for its detection daqu fermentation power of preparation method and application |
| CN110618281A (en) * | 2019-09-10 | 2019-12-27 | 沈阳农业大学 | Method for measuring biotin content in insect body |
| CN110796389A (en) * | 2019-11-08 | 2020-02-14 | 高培（广州）乳业有限公司 | Milk powder production quality control analysis system and method |
| CN112683625A (en) * | 2020-12-31 | 2021-04-20 | 上海微谱检测技术有限公司 | Method for detecting content of free biotin in infant formula food |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680420A (en) * | 2012-05-30 | 2012-09-19 | 国药集团威奇达药业有限公司 | Method for rapidly determining biotins in miniaturized manner |
| CN106222233A (en) * | 2016-08-03 | 2016-12-14 | 四川剑南春（集团）有限责任公司 | A kind of daqu fermentation power detection culture medium, the method for its detection daqu fermentation power of preparation method and application |
| CN110618281A (en) * | 2019-09-10 | 2019-12-27 | 沈阳农业大学 | Method for measuring biotin content in insect body |
| CN110618281B (en) * | 2019-09-10 | 2022-09-27 | 沈阳农业大学 | Method for measuring content of biotin in insect body |
| CN110796389A (en) * | 2019-11-08 | 2020-02-14 | 高培（广州）乳业有限公司 | Milk powder production quality control analysis system and method |
| CN112683625A (en) * | 2020-12-31 | 2021-04-20 | 上海微谱检测技术有限公司 | Method for detecting content of free biotin in infant formula food |
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| CN102041314B (en) | 2012-11-21 |
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