CN114814022B - Application of ganoderma lucidum glycol and glycerol-3-phosphorylcholine as biomarkers in detecting effectiveness of heat treatment of papaya - Google Patents
Application of ganoderma lucidum glycol and glycerol-3-phosphorylcholine as biomarkers in detecting effectiveness of heat treatment of papaya Download PDFInfo
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Abstract
The invention provides an application of ganoderma lucidum glycol and glycerol-3-phosphorylcholine serving as biomarkers in detecting the effectiveness of heat treatment of papaya, belonging to the technical field of metabonomics detection. The invention uses ganoderma lucidum glycol and glycerol-3-phosphorylcholine as biological markers through metabonomics detectionAnd (3) carrying out data analysis to accurately and intuitively determine whether the papaya fruits are subjected to effective heat treatment. Judging whether the heat treatment is carried out or not and whether the heat treatment is effective or not, wherein only whether the pawpaw fruit red glossy ganoderma diol and the glycerol-3-phosphorylcholine are obviously different or not before and after the heat treatment is needed, and the Log of the substance concentration of the two substances 2 Whether the absolute value of the fold factor of (2) is greater than 1.
Description
Technical Field
The invention belongs to the technical field of metabonomics detection, and particularly relates to application of ganoderma lucidum glycol and glycerol-3-phosphorylcholine serving as biomarkers in detecting heat treatment effectiveness of papaya.
Background
Papaya is a evergreen cork tree of papaya, which is transferred into China in 17 th century and is a fruit tree widely planted in tropical and subtropical areas, and in recent years, the planting area in the south of China is larger and larger. Papaya is a whole body treasured, which is a famous fruit and a special raw crop. The fruit contains sugar, protein, calcium and multiple vitamins, especially vitamin A, vitamin B, vitamin C, etc., and has effects of regulating qi, promoting digestion, and treating gastropathy. Papain contained in olive has been widely used in medical, cosmetic and food industries. Papaya can be processed into various products, and Chinese olive can be used as vegetable food and also is good poultry feed.
The heat treatment means that after fruits and vegetables are picked, the fruits and vegetables are treated at a proper temperature, pathogenic bacteria are inhibited, the enzyme activity in the fruits and vegetables is regulated, the stress resistance of the fruits and vegetables is improved, and finally, the fruits and vegetables maintain good fresh-keeping quality in the store process. The heat treatment has the advantages of low energy consumption, low investment, simplicity, effectiveness, no toxicity, no pesticide residue and the like, and has become a physical fresh-keeping technology widely studied at home and abroad. The fresh-keeping effect is effectively achieved on various fruits such as papaya, bananas, peaches, mangoes, strawberries, apples and the like. Although the heat treatment has beneficial effects on the quality, softening and disease resistance of the fruits, it is noted that in practical application, the effect of prolonging the preservation period of the fruits can be achieved only by selecting proper heat treatment conditions due to different fruit varieties, maturity and even storage conditions. However, there is no study in the current technology on how to effectively, accurately and rapidly detect whether fruits and vegetables, especially papaya fruits, are subjected to effective heat treatment.
Disclosure of Invention
In view of the above, the invention aims to provide an application and a detection method of using ganoderma lucidum glycol and glycerol-3-phosphorylcholine as biomarkers in detecting the effectiveness of heat treatment of papaya, wherein ganoderma lucidum glycol and glycerol-3-phosphorylcholine are used as biomarkers to detect the quality of fruits before and after heat treatment of papaya, and accurately judge the effectiveness of heat treatment of papaya.
In order to achieve the aim of the invention, the invention provides application of the ganoderma lucidum glycol and the glycerol-3-phosphorylcholine as biomarkers in testing the effectiveness of heat treatment of papaya.
The invention also provides a method for detecting the effectiveness of the heat treatment of the papaya, which comprises the following technical scheme: metabolic group detection and OPLS-DA analysis are carried out on papaya fruit materials before and after heat treatment, t-test detection is adopted, when the ganoderma lucidum glycol and the glycerol-3-phosphorylcholine have obvious difference, and the Log of the substance concentration of the two substances 2 The absolute value of fold factor of >1, indicates that the heat treatment of papaya is effective.
Preferably, the metabonomic assay is a GC-MS analysis.
More preferably, the GC-MS analysis conditions are:
chromatographic conditions: waters UPLC liquid chromatographic column, liquid chromatography phase A is aqueous solution containing 0.1% formic acid, phase B is acetonitrile; the temperature of the column temperature box is 40 ℃, the temperature of the automatic sampler is 4 ℃, and the sampling volume is 2 mu L;
mass spectrometry conditions: ESI ion sources, mass spectrometry in multi-reaction monitoring MRM mode.
The invention also provides a method for screening the biomarker for the effectiveness of the heat treatment of the papaya, which comprises the following steps: preparing papaya fruits of a group to be heat-treated and a control group, carrying out heat treatment on the papaya fruits of the group to be heat-treated, respectively carrying out metabonomics detection on the papaya fruits of the group to be heat-treated and the papaya fruits of the control group, carrying out OPLS-DA analysis, and screening differential metabolites; the t-test detection is adopted, the metabolite content between the screening control group and the heat treatment group is obviously different, and the Log is used 2 And (3) taking the papaya as a bottom, wherein the differential metabolite with fold multiple larger than 1 is the biomarker for the heat treatment effectiveness of the papaya.
Preferably, the papaya fruits of the group to be heat-treated and the control group have uniform sizes and consistent maturity.
Preferably, the heat treatment adopts an alternating damp-heat heating program, and the treatment is continued for 60min after the temperature of the fruit cores reaches 47.2 ℃.
Preferably, the metabonomic assay is a GC-MS analysis.
More preferably, the GC-MS analysis conditions are:
chromatographic conditions: waters UPLC liquid chromatographic column, liquid chromatography phase A is aqueous solution containing 0.1% formic acid, phase B is acetonitrile; the temperature of the column temperature box is 40 ℃, the temperature of the automatic sampler is 4 ℃, and the sampling volume is 2 mu L;
mass spectrometry conditions: ESI ion sources, mass spectrometry in multi-reaction monitoring MRM mode.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides application of ganoderma lucidum glycol and glycerol-3-phosphorylcholine as biomarkers in detecting the effectiveness of heat treatment of papaya and a corresponding method for detecting the effectiveness of heat treatment of papaya.
Therefore, whether the heat treatment is carried out or not and whether the heat treatment is effective or not is judged, and whether the pawpaw fruit ganoderma lucidum diol and the glycerol-3-phosphorylcholine are obviously different or not is only needed, and the Log of the substance concentration of the two substances 2 Whether the absolute value of the fold factor of (2) is greater than 1.
Drawings
Fig. 1: and (5) carrying out OPLS-DA score graphs before and after heat treatment of papaya.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Papaya heat treatment
The fruit of the seven-ripe Hainan papaya with uniform size and quality (smooth surface, uniform color and no scratch, disease spot or gummosis) is selected and has the quality of 700-900 g. The papaya is subjected to forced hot air treatment by using a fruit heat treatment box, an alternating damp-heat heating program (see table 1) is adopted, and meanwhile, the temperature of the core of the papaya is monitored and recorded by using a temperature recorder.
TABLE 1 alternating damp-heat warming procedure
Opening the fruit heat treatment box, putting papaya into the box, and continuously treating for 60min after the temperature of the fruit core reaches 47.2 ℃. The control group papaya was placed under room temperature (25 ℃ C.).
After the treatment, the papaya of the treatment group and the control group are put into a water tank (1L) with the temperature of 25 ℃ for water cooling treatment, after the temperature of the fruit core is reduced to the room temperature (about 30 min), the fruit core is put into a sodium hypochlorite solution (with the concentration of 50 ppm) for disinfection for 2-5min, taken out and dried for sampling. Samples of both the heat treatment group and the control group were flash frozen with liquid nitrogen and stored in a-80 ℃ refrigerator. Each treatment was run in 5 replicates.
2. Metabolome pretreatment of samples
Freeze-drying the sample, and grinding (50 Hz,60 s); weighing 20mg of sample into a centrifuge tube, adding two small steel balls, adding 500 μl of extracting solution (methanol water, volume ratio of 3:1, precooling at-40deg.C, and containing internal standard L-2-phenylalanine); vortex 30s, homogenizing for 4min at 35Hz, and ultrasonic treating for 5min in ice water bath; repeating homogenization and ultrasonic treatment for 3 times; overnight at 4 ℃ on a mixing instrument; the sample was centrifuged at 12000rpm (centrifugal force 13800 (. Times.g), radius 8.6 cm) at 4℃for 15min; carefully taking the supernatant and filtering the supernatant through a 0.22 mu m microporous filter membrane; 30 mu L of each sample is mixed into QC samples; storing at-80 ℃ until on-machine detection.
3. Papaya sample metabolome GC/MS analysis
Chromatograph condition parameters: the target compound was chromatographed on a Waters UPLC liquid chromatography column using an existing LC System (SCIEX) ultra-high performance liquid chromatograph. The phase A of liquid chromatography is aqueous solution containing 0.1% formic acid, and the phase B is acetonitrile. The column temperature was 40℃and the autosampler temperature was 4℃with a sample volume of 2. Mu.L.
Sciex QTrap 6500 Mass Spectrometry Instrument parameter set up: the present project uses a SCIEX 6500qtrap+ triple quadrupole mass spectrometer equipped with a IonDrive Turbo VESI ion source for mass spectrometry in multi-reaction monitoring (MRM) mode. The ion source parameters are as follows: ionSprayVoltage: +5500/-4500V,Curtain Gas:35psi,Temperature:400 ℃, ion Source Gas 1:60psi,Ion Source Gas 2:60psi,DP: + -100V.
4. Data processing
In this project, all mass spectrum data acquisition and target compound quantitative analysis were completed by SCIEX Analyst Work Station Software (Version 1.6.3). The mass spectrum was originally converted to TXT format using msconcnter software. And then, the self-writing R program package is combined with a self-built database to finish the work of peak lifting, annotation and the like.
5. Orthogonal partial least squares discriminant analysis (OPLS-DA)
And filtering signals irrelevant to model classification, namely orthogonal signals, by adopting orthogonal partial least squares discriminant analysis (OPLS-DA) to establish a reliable OPLS-DA model. The model quality parameters are as follows: r is R 2 Y(cum)＝1，Q 2 (cum) =0.472, model quality was good. The OPLS-DA score graph is shown in FIG. 1. After filtering out noise signals that are not related to classification, two sets of samples are sampled at PC1 (i.e., t 1]P) has good metabolic profile separation, i.e.two groups of samples are each in the principal component (PC 1, t 1]P) positive and negative sides. Variability in the control group was significantly greater than in the heat treated group, as evidenced by greater variability between samples in the control group.
6. Differential metabolite and structural identification thereof
The obtained differential metabolites are more reliable, since uncorrelated orthogonal signals are filtered out. The VIP (Variable Importance in the Projection) value (threshold > 1) of the first principal component of the OPLS-DA model was used in conjunction with the p value (threshold 0.05) of the t-test to find differentially expressed metabolites. The qualitative method of the differential metabolites comprises the following steps: the NIST commercial database was searched (comparison of mass spectra and chromatographic retention times RT or retention index RI) and determined using standard data alignment.
Therefore, the method can separate the difference between the heat treatment group and the control group, has high sensitivity and specificity, has 84 metabolites, 56 metabolites are reduced, 28 metabolites are increased (see table 2), confirms the metabolic difference between the heat treatment group and the control group, and can be used for detecting the heat treatment effectiveness of papaya. In the subsequent assays, the above metabonomics assays and OPLS-DA analysis were only required for these 84 metabolites in table 2.
TABLE 2 differential metabolites between heat treated and control groups
* The ratio of the mean of the heat treated group to the control group (base 2) is shown as a positive sign for the heat treated group rising relative to the control group and a negative sign for the heat treated group falling.
On the basis of the scheme, biomarkers which can represent effective heat treatment are further screened from the 84 differential metabolites, so that the detection procedure is further simplified on the basis of ensuring the detection accuracy.
7. ROC curve analysis and biomarker substance identification of substance peak identification heat treatment after metabonomics GC-MS analysis
According to the metabonomics detection result, t-test detection is adopted for diagnosis of heat treatment, and significant difference (P < 0.05) exists between screening control and heat treatment group, so that Log is used 2 For the bottom, the Fold ratio (Fold change) is greater than 1. Meanwhile, an ROC curve is drawn with (1-specificity) as the abscissa and sensitivity as the ordinate. ROC curve analysis relates the sensitivity and specificity of a test, and is a comprehensive and scientific method for evaluating detection items. The larger the area under the curve (AUC), the greater the diagnostic value, and when the AUC is close to 0.5, the diagnostic significance is not realized; AUC < 0.7, which indicates lower diagnostic accuracy; AUC is 0.7-0.9, which indicates moderate diagnostic accuracy; AUC > 0.9 indicates a higher accuracy of diagnosis.
As shown in Table 3, in the heat-treated group, the metabolites of Ganoderma lucidum glycol (Lucidadiol) and glycerol-3-phosphate choline (Glycerol) had AUC of 0.96 and 1.00, respectively, after ROC analysis, and t-test P value < 0.5, log 2 Fold multiples of-3.63 and 2.92, respectively. The two substances are heat treated biomarkers. Therefore, whether the heat treatment is performed or not and whether the heat treatment is effective or not is judged, and the group to be detected and the control group are required to be comparedWhether the sesame glycol and the glycerin-3-phosphorylcholine have obvious difference or not, and the Log of the substance concentration of the two substances 2 Whether fold multiple (absolute value) of (a) is > 1.
TABLE 3 Heat treatment biomarker identified by ROC Curve analysis
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (1)
1. The application of ganoderma lucidum glycol and glycerol-3-phosphorylcholine as biomarkers in the detection of the effectiveness of heat treatment of papaya comprises the following steps: the method comprises the steps of performing metabonomics detection on papaya fruit materials before and after heat treatment, adopting t-test detection, wherein when the contents of the ganoderma lucidum glycol and the glycerol-3-phosphorylcholine are obviously different, and the Log of the substance concentrations of the two substances 2 The absolute value of fold multiple of (2) is more than 1, which indicates that the papaya heat treatment is effective;
the heat treatment is as follows: the papaya is subjected to forced hot air treatment by using a fruit heat treatment box, an alternating damp-heat heating program is adopted, and meanwhile, the temperature of the core of the papaya is monitored and recorded by using a temperature recorder; opening a fruit heat treatment box, putting papaya into the fruit heat treatment box, and continuously treating the fruit for 60 minutes after the temperature of the fruit core reaches 47.2 ℃;
the alternating damp-heat temperature rise program is as follows:
the metabonomic assay is an LC-MS assay under the following conditions:
chromatographic conditions: watersUPLC liquid chromatographic column, liquid chromatography phase A is aqueous solution containing 0.1% formic acid, phase B is acetonitrile;
the temperature of the column temperature box is 40 ℃, the temperature of the automatic sampler is 4 ℃, and the sampling volume is 2 mu L;
mass spectrometry conditions: ESI ion sources, mass spectrometry in multi-reaction monitoring MRM mode.
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