CN108020575B - Method for quantitatively determining oil in culture solution - Google Patents
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Abstract
The invention relates to a method for quantitatively determining oil in a culture solution, which comprises the following steps: and (3) measuring the sample liquid of the water system by using a low-field nuclear magnetic resonance spectrometer, and calculating the oil content in the fermentation liquid according to the area-mass ratio. Compared with the traditional detection method, the method provided by the invention is more convenient and accurate in result, and is a new and effective method.
Description
Technical Field
The invention belongs to the technical field of fermentation, and particularly relates to a method for quantitatively detecting grease in fermentation liquor by using a low-field nuclear magnetic resonance technology in fermentation detection of downstream microorganisms in fermentation engineering.
Background
In the industrial production of antibiotic fermentation, when oil is used as a carbon source, the catabolite repression effect is not easy to occur, so that the catabolite synthesis is facilitated, and the utilization rate of a fermentation tank is improved because the surface tension is low and bubbles are not easy to generate; meanwhile, the method also has the functions of strengthening oxygen transfer, a product extracting agent, a substrate solubilizer, a metabolism activator and the like. At present, the role of oil in the antibiotic fermentation industry is irreplaceable. In order to improve the fermentation efficiency, the oil needs to be quantitatively detected, and the fermentation process needs to be effectively controlled. However, the existing quantitative detection method for oil has many defects in the process of guiding fermentation regulation. Although traditional extraction technologies such as a Soxhlet extraction method are classical methods, the traditional extraction technologies have the defects of complex operation, complex process, time and labor waste and the like; the method for detecting grease by using a spectrophotometer in the dyeing of dyes such as Nile red (Nile red) has the defects of complex operation calculation, insecurity, large error and the like due to the characteristics and the limitations of the dyes; although the chromatographic techniques represented by a chromatography-mass spectrometry (GC-MS) method and the like have higher accuracy, the requirements on sample treatment are high, and the cost of money and time is higher when the sample is detected; the near infrared spectroscopy plays an important role in the conventional analysis development of the oil and fat, but the limitations are also various, and although the sensitivity is poor, the scanning time is long, and the samples need special treatment and the like to be improved to a certain extent, the method has certain defects in industrial production.
Low-field nuclear magnetic resonance (LF-NMR) generally refers to the phenomenon of nuclear magnetic resonance at constant magnetic field strengths below 0.5T.1The basic principle of H LF-NMR is toThe sample in the constant magnetic field is applied with radio frequency pulse to make hydrogen proton generate resonance, and the proton returns to the ground state after releasing the absorbed radio frequency wave energy in a non-radiative mode, and the process is called relaxation process. At present, the LF-NMR technology has certain application potential in the detection field of food science due to the characteristics of rapidness, no damage, less sample requirement, simple treatment and the like. Plum tide and the like obtain an oil content matched with a Soxhlet extraction method based on the ratio of the peak areas of fat relaxation spectra of dry peanuts and pure peanut oil in unit mass, Han Zhu and the like detect the Water Holding Capacity (WHC) of the longissimus of pigs by using a low-field nuclear magnetic resonance technology, Song Ping and the like research the influence of the water state and the change process and the seed soaking method on the water absorption capacity of rice seeds by nuclear magnetic resonance, and Wangle and the like identify edible vegetable oil adulterated with waste oil in catering industry and Geir H by using a nuclear magnetic resonance method.
And the like, using low-field nuclear magnetic resonance to detect fat and water content and the like in the meat. However, these studies are limited to short relaxation systems of low water solutions, while high water systems, and no technology for detection by using low-field nuclear magnetic resonance technology is available at home and abroad at present. In addition, in the prior art, many studies on low-field nuclear magnetic resonance technology are limited to qualitative studies, and quantitative studies are few and few.
In conclusion, the low-field nuclear magnetic resonance technology can accurately detect the content of the oil in a low-water system, but the fermentation liquid belongs to the water system (mostly the high-water system), and a quantitative detection method of the fermentation liquid is not reported in the literature at present. Therefore, there is a need in the art to find accurate methods for determining the amount of oil and fat in aqueous systems.
Disclosure of Invention
The invention aims to provide a method for quantitatively measuring oil in a culture solution.
In a first aspect of the invention, there is provided a method of quantitatively determining the oil content in an aqueous system, the method comprising: measuring the sample liquid of a water system by using a low-field nuclear magnetic resonance spectrometer, and calculating the oil content in the fermentation liquid according to the area-mass ratio; the area mass ratio is the peak area corresponding to each gram of oil.
In a preferred embodiment, in the method for quantitatively determining the content of oil in an aqueous system, the oil is vegetable oil; preferably said oils include (but are not limited to): soybean oil, rapeseed oil or palm oil.
In another preferred embodiment, in the method for quantitatively determining the oil content in the aqueous system, the aqueous system is a fermentation broth.
In another preferred embodiment, in the method for quantitatively determining the oil content in the water system, the fermentation broth is a fermentation broth for producing salinomycin by fermenting streptomyces albus.
In another preferred embodiment, in the method for quantitatively determining the oil content in the water system, the oil quality is obtained by performing linear fitting on the oil quality and the peak area.
In another preferred embodiment, in the method for quantitatively determining the oil content in an aqueous system, the mass of soybean oil is calculated according to formula (I):
Y＝12956.08927X-71.77
(I)；
in the formula, Y represents a peak area, and X represents an oil mass.
In another preferred embodiment, in the method for quantitatively determining the oil content in the water system, the supernatant in the fermentation liquid is determined by using a low-field nuclear magnetic resonance apparatus; preferably, the supernatant is obtained by removing solids from the fermentation broth by centrifugation.
In another preferred embodiment, in the method for quantitatively determining the oil content in an aqueous system, the solids comprise: bacteria and insoluble solids; preferably said insoluble solids comprise: soybean flour, germ flour.
In another preferred embodiment, the method for quantitatively determining the oil content in the water system, the centrifuging the fermentation liquid to remove solids and then determining by using a low-field nuclear magnetic resonance spectrometer, comprises: centrifuging the fermentation liquid, directly sucking the supernatant with a pipette, adding the supernatant into a glass test tube, and measuring with a low-field nuclear magnetic resonance apparatus.
In another preferred embodiment, in the method for quantitatively determining the oil content in the water system, the minimum detected oil content is 1% (w/v).
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
FIG. 1 inversion fitting of different masses of components.
A: an inverse fit atlas of water;
b: fitting an inversion map of the oil;
c: an inversion fitting map of the soybean meal;
d: and (4) carrying out inversion fitting on the germ powder.
FIG. 2 is a correlation fit with peak area and CPMG response values (abbreviated as "response values") (A: water, B: oil, C: medium). The peak area refers to the area obtained by inversion fitting under the relaxation time of T2.
FIG. 3 Linear fit of oil mass to peak area.
FIG. 4 correlation of n-hexane extraction with NMR method.
Detailed Description
The present inventors have made extensive studies in the field of biological fermentation, where the fermentation broth generally belongs to a water system and oil is added to the fermentation broth in case of fermentation needs. The existing technology for quantitatively determining the oil content has the disadvantages of complicated steps and low accuracy. Therefore, the present inventors have conducted extensive studies and have revealed that the method for detecting oil in a culture solution of the present invention.
As used herein, the term "aqueous system" refers to a solution system in which water is used as a solvent; preferably, the "water system" is a "high water system". The aqueous system contains oil, typically in an amount greater than 0.1% (w/v), more preferably from 1% to 70% (w/v).
The research process of the method comprises the following steps:
(1) injecting a certain amount of culture medium into a conical flask or a fermentation tank, and keeping the components of the culture medium dissolved uniformly;
(2) independently detecting main components of water, oil, germ powder and soybean meal in a culture medium, and determining the relaxation time of the culture medium under a low-field nuclear magnetic resonance spectrometer;
(3) sterilizing a conical flask or a fermentation tank added with a culture medium, and inoculating strains according to a certain inoculation amount;
(4) determining the amount of characterizing oil by comparing the correlation of water, oil quality with response values and corresponding peak areas;
(5) determining the influence of each component in the culture medium on oil detection through a single-factor variable, wherein each component takes the content in the culture medium as an upper limit, then sequentially decreasing to zero, and adding the culture medium in five gradients;
(6) the components of the single-factor experiment culture medium are respectively germ powder, soybean meal, potassium chloride, sodium chloride, ammonium tartrate, urea, magnesium sulfate heptahydrate, dipotassium phosphate trihydrate and calcium carbonate;
(7) taking 0.1-1 g of a small amount of oil to perform a sensitivity experiment of a low-field nuclear magnetic resonance spectrometer, and establishing an area-to-mass ratio;
(8) gradient experiments are carried out according to the oil contents of 0%, 0.1%, 0.2%, 0.5%, 0.8%, 1%, 2%, 5% and 8% respectively, and the stability and the accuracy of the method are verified;
(9) preparing oil-water mixtures with different oil contents for low-field nuclear magnetic resonance detection, eliminating the interference of other culture medium components, and verifying the accuracy and precision of the method again;
(10) comparing different sample processing modes;
(11) compared with the conventional oil detection method, the method is used for extracting n-hexane.
Through the research of the process, the inventor optimizes and obtains a method for detecting the oil content in the water system by analyzing various possible interference factors, wherein the method comprises the following steps: measuring by using a low-field nuclear magnetic resonance instrument, and calculating the oil content in the fermentation liquor according to the area-mass ratio; the area mass ratio is the peak area corresponding to each gram of oil.
In a preferred embodiment of the present invention, the water system is a fermentation broth system, and the oil is soybean oil. More preferably, the fermentation broth is a fermentation broth for producing salinomycin by fermenting streptomyces albus.
As a preferred mode of the present invention, the oil quality is obtained by linear fitting the oil quality and the peak area. More preferably, the mass of soybean oil is calculated according to formula (I):
Y＝12956.08927X-71.77 (I)；
in the formula, Y represents a peak area, and X represents an oil mass.
As a preferred mode of the invention, when the oil content in the water system is quantitatively determined, after sampling, the fermentation liquor is centrifuged and then determined by a low-field nuclear magnetic resonance spectrometer, so that the interference factors existing in the culture medium and the interference caused by bacteria are eliminated. Preferably, the fermentation broth is a fermentation broth for producing salinomycin by fermenting streptomyces albus; the removing solids comprises: fermented thallus, soybean powder and germ powder.
The inventor also researches the influence of several solid removing methods on the detection accuracy, and finds that the following methods are more accurate: the fermentation broth (2ml) was centrifuged, the centrifuged supernatant was directly aspirated by a pipette, and the supernatant was added to a glass test tube and measured using a low-field nuclear magnetic resonance spectrometer.
The invention establishes a convenient, rapid and nondestructive method for detecting grease in fermentation liquor by using a low-field nuclear magnetic resonance technology, which comprises the following steps: the relaxation time of the grease is determined by detecting each component in the culture medium; the single-factor variable experiment proves that except the soybean meal and the germ meal, the uneven sampling has certain influence on the oil peak area, and other components have no influence on the oil peak area; gradient experiments with different oil contents show that the low-field nuclear magnetic resonance technology has good stability and accuracy in oil detection. According to the results of the measurement, the present inventors excluded interference of factors affecting the detection by the centrifugation step.
The traditional n-hexane extraction determination oil content and the low-field nuclear magnetic resonance technology determination oil are subjected to linear correlation analysis, and the correlation is good. Moreover, compared with the traditional detection method, the result obtained by applying the low-field nuclear magnetic resonance technology to detect the fermentation liquor is more convenient and accurate, and the method is a new effective method.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
The strain is as follows: streptomyces albus (Streptomyces albus) A30, sublimation Baike Biotech, Inc. in Zhejiang.
The specific instruments and reagents used in the assay are shown in Table 1.
TABLE 1 reagents used in the culture media
| Name of reagent | Purity grade | Manufacturer of the product |
| Soybean flour | From Zhejiang sublimation Baike Biotech Co., Ltd | |
| Germ powder | From Zhejiang sublimation Baike Biotech Co., Ltd | |
| Soybean oil | From Zhejiang sublimation Baike Biotech Co., Ltd | |
| Ammonium tartrate | Molecular level | SANGON BIOTECH (SHANGHAI) Co.,Ltd. |
| Urea | Molecular level | SANGON BIOTECH (SHANGHAI) Co.,Ltd. |
| Sodium chloride | Analytical purity | Shanghai Lingfeng Chemicals Co., Ltd |
| Potassium chloride | Analytical purity | Shanghai Lingfeng Chemicals Co., Ltd |
| Magnesium sulfate heptahydrate | Analytical purity | Shanghai Lingfeng Chemicals Co., Ltd |
| Dipotassium hydrogen phosphate trihydrate | Analytical purity | Shanghai Lingfeng Chemicals Co., Ltd |
| Anhydrous calcium carbonate | Analytical purity | Shanghai Lingfeng Chemicals Co., Ltd |
| N-hexane | Analytical purity | Shanghai Lingfeng Chemicals Co., Ltd |
Preparation of culture medium (g/L):
germ powder 8, soybean powder 5, potassium chloride 2.2, urea 1.6, sodium chloride 1, ammonium tartrate 2, magnesium sulfate 0.1, dipotassium hydrogen phosphate 0.2, calcium carbonate 5 and soybean oil 150, adjusting pH to 7.0 with concentrated hydrochloric acid, and sterilizing at 121 deg.C for 30 min.
TABLE 2 instruments and devices
| Low field nuclear magnetic resonance apparatus | SHANGHAI NIUMAG Corp.,Ltd. |
| Oscillator | Shanghai Hualian medical instruments Co Ltd |
| Water bath pot | Shanghai Hualian medical instruments Co Ltd |
| Magnetic rotary stirrer | Hangzhou Instrument Motor Co.,Ltd. |
| Spectrophotometer | Shanghai cyanine instruments science and technology Limited |
The fermentation liquid is from shake flask fermentation culture, the components of the culture medium are the same as above, the inoculation amount of the strain is 10%, the culture condition is 32 ℃, 250r/min, and the shake flask level does not control ventilation, dissolved oxygen and the like. Samples were taken every other day ( days 2, 4, 6, 8, 10).
The fermentation method of the 50L fermentation tank is as follows: the culture solution is filled in 30L, and the inoculation amount is 5-10%. The control conditions were as follows: the air flow is 0.5-2.0VVM at 32 ℃, the DO is maintained at 10% or above, and the tank pressure is 0.03-0.08 MPa.
Example 1 relaxation time analysis of several Components
In this example, several component relaxation time analyses were performed.
The components in the culture medium are complex, and the main components of the culture medium, namely water, oil (soybean oil), soybean meal and germ powder, are separately detected firstly, so that the characteristics of the culture medium are researched, and a basis is provided for effectively analyzing the culture medium. The process is as follows: respectively measuring water, oil, soybean meal and germ meal with different masses into a detection test tube, wherein the height in the test tube is not higher than 2cm, and then searching for center frequency and parameter setting by using soybean oil. See the legend of figure 1 for the mass of each component.
The detection results are shown in FIG. 1. The relaxation time of water is about 2500ms, the relaxation time of oil is about 100ms, and the relaxation time of soybean meal and germ meal is about 0.1-1 ms, 2-15 ms, 20-400 ms and 2000-4000 ms.
Example 2 determination of the amount of oil characterized in the detection by Low-field NMR technique
In this example, the amount of the characterizing oil in the low-field nmr detection is determined as follows:
selecting pure water, pure oil and a culture medium as substances for determining which of a response value and an inversion peak area is a quantity representing the oil; respectively taking 0.5ml, 1.0ml, 1.5ml, 2.0ml and 2.5ml of pure water to a detection test tube, taking 0.6g, 0.9g, 1.4g, 1.9g and 2.6g of soybean oil to the detection test tube, taking 2ml of culture medium (without centrifugal operation) with the oil content of 0%, 1%, 2%, 5%, 8% and 10% to the detection test tube, and then carrying out low-field nuclear magnetic resonance detection; performing correlation fitting on the water volume, the response value and the corresponding oil peak area, wherein the result is shown in FIG. 2A, and the correlation between the peak area and the volume is greater than that between the response value and the water volume, so that the peak area represents the water volume better; the oil quality is linearly fitted with the peak area and the response value, and the result is shown in fig. 2B, wherein the correlation between the peak area and the oil quality is greater than the correlation between the response value and the oil quality, so the peak area represents the oil quality better; and (3) performing low-field nuclear magnetic resonance detection on culture media with the same volume and different oil contents, and performing correlation fitting on the oil peak area and the response value and the oil quality, wherein the result is shown in fig. 2C, the correlation between the oil peak area and the oil quality is far greater than that between the response value and the oil quality, and the oil peak area is used for representing that the quality of the oil in the culture media is better. In conclusion, the oil quality in the culture medium is more suitable to be quantitatively characterized by using peak area.
When pure oil is used as a test substance, the peak start time is about 24ms, the peak top time is about 100ms, and the peak end time is 305 ms. However, the composition of the medium is complicated, and each substance may affect the peak appearance time, peak top time, peak end time, peak area size, and the like of the oil peak.
Example 3 analysis of the Effect of Components on the assay
The entire process from the preparation of the medium to the detection of low-field nmr can cause errors in many operations or components. The present inventors conducted intensive analytical studies on each substance in order to determine the influence of each substance on the oil peak area. The study data were statistically analyzed and some of the results are shown in Table 3.
TABLE 3 oil Peak area under influence of Components
With each component as a variable, six gradients were set, with 3 replicates per gradient. The medium with each component as a variable was subjected to low-field nuclear magnetic resonance detection.
The inventors found that the oil peak area does not follow the regular change under the influence of each component as the content of the components of the culture medium increases. By calculating the variation coefficient of the average value of the peak area of each gradient oil, the variation coefficients of calcium carbonate, dipotassium hydrogen phosphate, magnesium sulfate, potassium chloride, sodium chloride, ammonium tartrate and urea are all less than 7%, the influence on the peak area of the oil is small, and the variation coefficients are within an acceptable range; the variation coefficients of the soybean meal and the germ meal are respectively 32.23 percent and 19.52 percent, and are relatively large, so that the area of the oil peak in the culture medium is greatly influenced.
Therefore, it is necessary to analyze such influencing factors and consider avoiding the influence of the soybean flour and the germ flour on the measurement results during the measurement.
Example 4 determination of NMR sensitivity to Small amounts of oil
A small amount of pure oil is measured and subjected to low-field nuclear magnetic resonance detection to determine the sensitivity of NMR on the small amount of oil, and therefore an area-mass ratio is established to express, and the area-mass ratio is the peak area corresponding to each gram of oil. As the oil mass increased, the area to mass ratio remained stable, as in table 4.
TABLE 4 NMR results of a small amount of oil
The average value is calculated to be 12687.61, the standard deviation is 252.6915, and the coefficient of variation CV is 1.9916%, which shows that the sensitivity of the low-field nuclear magnetic resonance apparatus to oil is not related to the quality of the oil. Meanwhile, linear fitting is performed on the oil mass and the peak area, as shown in fig. 3, a curve is fitted:
Y＝12956.08927X-71.77 (I)；
in the formula, Y represents the peak area, X represents the oil quality, the correlation is good, and this formula is used as the calculation of the oil content in the medium.
Media with different oil contents (not centrifuged) were prepared for low field nmr examination with five replicates per gradient and 2ml samples were taken for each examination, as in table 5. It is known that the change of the area of the oil peak in the medium containing less than 0.8% oil is small, and may be the influence of the soybean meal, germ meal, and the like in the medium, and the average value thereof is calculated to be 563.11. The average, standard deviation and relative standard deviation of the oil peak area of the five replicates of each gradient were calculated. The relative standard deviation of each gradient is in a reasonable variation range, the average value of the variation coefficient of each gradient is 4.42%, and the stability of the low-field nuclear magnetic instrument detection is good. Then, the area mass ratio of the oil peak in the culture medium with the oil content of more than 0.8 percent is calculated, the values are all consistent with the area mass ratio of the pure oil, the average value is 12160.01, and the accuracy is good.
TABLE 5 NMR detection of media with different oil contents
The LF-NMR was performed on an oil-water mixture prepared to have an oil content of 0%, 0.1%, 0.2%, 0.5%, 0.8%, 1%, and the results are shown in Table 6. It can be seen that the presence of oil can be detected only when the oil content reaches above 0.8%, the calculated mass ratio of the oil peak area in the oil-water mixture is substantially consistent with that of pure oil, and the influence of other components in the culture medium on the oil peak area is also proved to be extremely small.
TABLE 6 NMR detection of oil-water mixtures
Example 5 improvement of detection accuracy
As previously mentioned, soy flour and germ flour affect the accuracy of the assay. In addition, the inventor finds that the mycelium in the fermentation liquor seriously influences the detection of the oil in the fermentation liquor during the detection of the fermentation liquor. The mycelium in the fermentation broth is gradually increased as the fermentation time progresses, thereby resulting in a gradually increased area of the oil peak which should be gradually decreased.
Therefore, through repeated research, the inventor carries out different modes of processing on the sample, finds out a reasonable processing mode, and then accurately detects the oil in the fermentation liquor. And (3) determining that thalli in the fermentation liquor and the soybean meal and the germ meal which are determined to have influence on the measurement in the prior art are removed in a centrifugal mode. The inventor selects several centrifugal processing modes respectively, and the centrifugal processing modes sequentially comprise the following steps:
a: centrifuging 2ml of fermentation liquor (12000r/min for 10min), directly sucking the supernatant by a pipette, transferring into a glass test tube, and measuring by a low-field nuclear magnetic resonance spectrometer;
b: centrifuging 3ml of fermentation liquor (12000r/min for 10min), directly sucking the supernatant by a pipette, transferring into a glass test tube, and measuring by a low-field nuclear magnetic resonance spectrometer;
c: taking 3ml of fermentation liquor, centrifuging (12000r/min for 10min), directly pouring into a test tube, transferring into a glass test tube without a pipette, and measuring by using a low-field nuclear magnetic resonance spectrometer;
d: centrifuging a filter element centrifuge tube, filling 1ml of liquid, transferring into a glass test tube, and measuring by using a low-field nuclear magnetic resonance instrument;
g: n-hexane extraction: adding 20mL of normal hexane into 20mL of fermentation liquor, uniformly oscillating, and centrifuging at 5000r/min for 10 min; taking the supernatant and pouring the supernatant into a clean plate (the mass is called W)0) Extracting the rest fermentation liquor with 10mL of n-hexane again, oscillating uniformly, and centrifuging at 5000r/min for 10 min; the upper n-hexane layer was poured into the previous dish. Placing the culture dish in a 60 ℃ oven, taking out the dried culture dish after n-hexane volatilizes, and weighing the culture dish as W1And then calculating the content of residual oil in the fermentation liquor. Oil content in the fermentation broth:
along with the progress of the fermentation time, the response value shows irregular change, the peak area is gradually decreased, and the results are shown in tables 7 and 8, so that the LF-NMR measurement is directly carried out on the fermentation liquid without treatment, the response value and the peak area show an increasing trend along with the progress of the fermentation time due to the influence of the mycelium, and meanwhile, the fact that the peak area represents that the oil content in the fermentation liquid is more appropriate than the response value is verified.
TABLE 7 CPMG response values for different fermentation broth treatment modes
TABLE 8 Peak area for fermentation broths treated differently
TABLE 9 oil content per ml of fermentation broth obtained by different treatment modes of the fermentation broth
The oil content per ml of the fermentation broth and the oil content per ml of the fermentation broth extracted with n-hexane were calculated from equation (I) and the results are shown in Table 9. The linear fitting between the oil obtained by n-hexane extraction and the oil obtained by low-field nuclear magnetic resonance is good in correlation, and the result is shown in figure 4, which indicates that the low-field nuclear magnetic resonance detection is feasible. As can be seen from Table 9, the oil content per ml, B, A, G, and D, shows that the accuracy of the low-field nuclear magnetic resonance detection of the oil content is higher than that of the traditional oil detection method.
The results of the oil content measurements using n-hexane and the method of the invention (characterized by peak area using low field nuclear magnetic resonance, 2ml of the fermentation broth was centrifuged before measurement and the centrifuged supernatant was directly aspirated by a pipette) were determined by examining a medium (same composition as above) with known oil contents (1%, 3%, 5%, 8%, 10%).
TABLE 10 comparison of LF-NMR method with n-hexane extraction method
As can be seen from table 9 (for the fermentation broth during fermentation) and table 10 (for the fermentation broth given the oil content), both methods detected less oil than the true value, but the LF-NMR method of the invention was closer to the true value.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (5)
1. A method for quantitatively determining the oil content in an aqueous system, the method comprising: measuring the sample liquid of a water system by using a low-field nuclear magnetic resonance spectrometer, and calculating the oil content in the fermentation liquid according to the area-mass ratio; the area mass ratio is the corresponding peak area of each gram of oil, the oil is soybean oil, and the oil content in the fermentation liquor is more than 0.8 percent (w/v); the water system is fermentation liquor for producing salinomycin by fermenting streptomyces albus, insoluble solid content including thalli, soybean meal and germ meal in the fermentation liquor is removed, supernatant in the fermentation liquor is measured by using a low-field nuclear magnetic resonance spectrometer, and the oil quality is obtained by linearly fitting the oil quality and the peak area; and calculating the mass of soybean oil according to formula (I):
Y＝12956.08927X-71.77 (I)；
in the formula, Y represents a peak area, and X represents an oil mass.
2. The method of quantitatively determining the oil content in an aqueous system as claimed in claim 1, characterized in that the supernatant is obtained by removing solids from the fermentation broth by centrifugation.
3. The method of claim 2, wherein centrifuging the fermentation broth to remove solids and then measuring with a low field nuclear magnetic resonance spectrometer comprises: and (4) centrifuging the fermentation liquor, adding the supernatant into a glass test tube, and measuring by using a low-field nuclear magnetic resonance spectrometer.
4. The method of claim 1 wherein the minimum oil content detected by the method is 1% (w/v).
5. The method of claim 1, wherein the method detects oil in an aqueous system in a range of 1.0% to 70% (w/v).
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| 利用低场核磁共振技术研究山楂酒发酵过程中水分的变化规律;张文叶 等;《中国酿造》;20151231;第34卷(第12期);137-140 * |
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