A method for identifying the postharvest cold storage time of rose-scented grapes based on aroma substances

Abstract

The invention discloses a method for identifying the post-harvest cold storage time of rose-scented grape fruits based on aroma substances, and belongs to the field of biological diagnosis research and development. The method comprises the following steps: using the mature fruits of the main varieties of rose-scented grapes as test materials, setting low-temperature storage treatments for different times, and then respectively carrying out aroma substance detection on the fruits of different cold storage times, and then screening out aroma compounds whose compound content is significantly correlated with the length of cold storage time. The identification method provided by the invention is based on detecting the aroma substances of grape fruits, and has a fast detection speed; the identification accuracy rate is high, and the instrument can operate continuously, which is more suitable for continuous and real-time detection, and is convenient for large-scale promotion and application, and provides an objective, accurate and new method for identifying the cold storage time of grapes.

Description

Technical Field

[0001] The invention relates to a method for identifying the post-harvest cold storage time of rose-scented grape fruits based on aroma substances, and belongs to the field of biological diagnosis research and development. Background Art

[0002] The information disclosed in this background technology section is only intended to increase some understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to ordinary technicians in this field.

[0003] The ultimate goal of fruit cultivation is to obtain delicious fruits. Aroma is one of the elements that constitute the quality of fruits. Rich aroma is loved by consumers. Fruit aroma is mainly contributed by some volatile compounds, and the types and contents of these compounds are controlled by the corresponding synthetic genes. It is worth mentioning that the synthetic genes of fruit aroma substances have obvious variety specificity, resulting in different types and contents of aroma substances among different fruits, and the effects are also different under the influence of environmental factors. For example, in low-temperature refrigeration, the limonene content in longan juice decreased, but there was no significant change in blood orange. Similarly, up to 113 aroma substances were detected in citrus fruits, while 100 were detected in grape fruits. According to the aroma type, grapes are divided into three types: rose aroma, strawberry aroma and Foxy. Compared with the other two types of grapes, rose-scented grapes have obvious differences in aroma characteristics and aroma substances, and are deeply loved by consumers because of their rich rose aroma. Grapes are berries and are not resistant to storage and transportation at room temperature. In order to extend the market supply period of fruits, low-temperature refrigeration (4℃ storage) is the main storage method for grapes at present. During low-temperature storage, the quality of the fruit usually declines to varying degrees, especially the content of some aroma compounds changes significantly. In the market, the longer the refrigeration time, the less fresh the fruit is, and the less consumers like it. In particular, fruits that have been refrigerated for two months or more have a significant effect on the taste. In order to promote sales, some merchants will falsely mark the refrigeration time, and often use fruits that have been refrigerated for more than two months to sell as fruits that have been refrigerated for less than two months. Therefore, it is of great significance to study and develop methods that can identify the refrigeration time of rose-scented grape fruits under low-temperature storage. Although some fruits have reported the evaluation of the refrigeration time of fruits by detecting changes in flavor substances, these flavor substances cannot be applied to the grape industry due to differences in fruit aroma characteristics. At present, most studies focus on the changing characteristics of the quality of refrigerated grape fruits, and the development of technologies to delay the decline in fruit quality, but lack methods that can effectively identify the refrigeration time. Summary of the invention

[0004] In view of the existing technology, in order to develop a method for identifying the refrigeration time, the present invention screens out key aroma compounds that can identify the refrigeration time that have not been reported in the literature by detecting the changes in the content of aroma substances in fruits under different refrigeration times. The content of these substances is significantly negatively correlated with the refrigeration time, and the refrigeration time can be identified by detecting the content of these substances.

[0005] The technical solution adopted by the present invention is as follows:

[0006] In a first aspect of the present invention, a method for identifying the post-harvest cold storage time of rose-scented grape fruits based on aroma substances is provided, the method comprising the following steps:

[0007] The mature fruits of the main rose-scented varieties were used as experimental materials. Low-temperature storage treatments of different durations were set up, and then the fruits of different refrigeration times were tested for aroma substances. Finally, the aroma compounds whose compound content was significantly correlated with the length of refrigeration time were screened out.

[0008] In one or some embodiments of the present invention, mature fruits of three typical rose-scented main varieties (Sunshine Rose, Giant Rose, 87-1) are used as test materials. Rose-scented fresh-eating grape fruits contain some common aroma substances and a small amount of variety-specific aroma substances (substances not present in other varieties). Sunshine Rose, Giant Rose and 87-1 are confirmed typical rose-scented fresh-eating grape varieties. Selecting three varieties to detect and screen common aroma substances at the same time can effectively remove the specific substances of each variety. Therefore, the screened substances are common substances of rose-scented grapes, and are also applicable to rose-scented grapes other than these three varieties.

[0009] In one or some embodiments of the present invention, the temperature of the low-temperature refrigeration treatment is 4-8°C; preferably 4°C.

[0010] In one or some embodiments of the present invention, the method for detecting aroma substances comprises the following steps:

[0011] (1) Extraction of fruit aroma substances.

[0012] Free aroma substances: Separate the pulp and peel of the fruit, put the peel into the extract, and shake it in the dark; filter and centrifuge, and use the supernatant for the detection of peel aroma compounds; add ascorbic acid to the pulp, crush it, centrifuge and filter the pulp, and use the supernatant for the detection of pulp aroma compounds.

[0013] Extraction of bound aroma substances: Peel extract or juice supernatant is loaded through a solid phase column and rinsed with ultrapure water and dichloromethane respectively; eluted with methanol, the eluate is evaporated to dryness, and citric acid / phosphate buffer and glucosidase are added to dissolve.

[0014] (2) Extraction of aroma substances. The extraction of aroma substances uses headspace solid phase microextraction (SPME) technology. Take NaCl, internal standard (2-octanol) and the extract in step (1) and add them to the headspace bottle. The headspace bottle is balanced, and the extraction head is inserted into the headspace bottle for extraction; after the extraction is completed, the extraction head is inserted into the GC port for thermal analysis.

[0015] (3) Quantitative and qualitative detection of aroma substances. Use gas chromatography-mass spectrometry (GC-MS) to qualitatively and quantitatively identify aroma compounds. Qualitative analysis of aroma substances: Conduct qualitative analysis of compounds based on full ion scanning mass spectra, and use the chromatographic retention time and mass spectrum information of standard compounds to qualitatively analyze grape compounds. Quantitative analysis of aroma substances: Prepare various standard compounds into mother liquors with continuous gradient dilutions of different concentrations, establish standard curves for aroma substances, and use the corresponding standard curves of each compound for quantitative analysis.

[0016] In one or some embodiments of the present invention, the contents of free and bound aroma substances in the peel and pulp are respectively correlated with the refrigeration time to obtain compounds with significant negative correlation in each variety; then, the significant negative correlation compounds that exist in rose-scented grape varieties are screened out as compounds for identifying the refrigeration time; by detecting and comparing the contents of these substances in fresh fruits and test samples, substances that are easy to detect (high content, >5μg / kg) and whose content changes with the refrigeration time are determined. Finally, by checking the content of any one substance in the sample and the mature fresh fruit, the extent of the decrease in content is calculated to achieve the identification of the refrigeration time. Specific method: Compared with the mature fruit, the content of these substances in the sample decreases by less than 50% for fruits that have been refrigerated for 2-6 weeks, and the decrease is more than 50% for fruits that have been refrigerated for 8 weeks or more. For example, if the screened substances include α-terpineol in free form in the pulp, the content of which is 66 μg / kg in fresh ripe fruit. The content of which is greater than 33 μg / kg is in fruit that has been refrigerated for 2-6 weeks, while the content of which is less than 33 μg / kg is in fruit that has been refrigerated for 8 weeks or more.

[0017] In the second aspect of the present invention, a compound for identifying the cold storage time of rose-scented grape fruits after harvest is provided, and the compound includes nerol and geraniol in the free state of the peel and cis-linalool oxide, nerolidol, α-terpineol and nerol in the free state of the pulp. Firstly, by carrying out the correlation analysis between the content of fruit aroma substances and the cold storage time, 9 substances with significant negative correlation and 1 substance with positive correlation with the cold storage time are preliminarily obtained; secondly, by observing the content and changes of various substances during the cold storage process of three kinds of grapes, 4 substances with low content (less than 5μg / kg) and no obvious changes are eliminated, including β-ionone in the free state of the peel and 2-methylbutenol, (E,E)-2,4-heptadienal and 2-octanone in the free state of the pulp. Finally, six substances were obtained that were easy to detect quantitatively and qualitatively and whose content changes could characterize the refrigeration time, including nerol and geraniol in the free state of the peel or cis-linalool oxide, nerolidol, α-terpineol, and nerol in the free state of the pulp.

[0018] Compared with the related art known to the inventor, one of the technical solutions of the present invention has the following beneficial effects:

[0019] The present invention can realize and fill the gap in the identification of rose-scented grape fruit cold storage time. The identification method provided by the present invention is based on the detection of grape fruit aroma substances, with a fast detection speed and high identification accuracy, and the instrument can operate continuously, which is more suitable for continuous and real-time detection, and is convenient for large-scale promotion and application, providing an objective, accurate and new method for the identification of grape cold storage time. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 . Venn diagram: The number and names of compounds with significant positive correlation between the changes in the content of aroma substances in different grape varieties under low temperature refrigeration and the refrigeration time. Note: FS: free skin, FP: free pulp, BS: bound to skin, BP: bound to pulp. The values ​​in the figure represent the number of aroma substances.

[0022] Figure 2 . Venn diagram: The number of compounds with significant negative correlation between the content of aroma substances in different grape varieties under low temperature cold storage and the cold storage time. Note: FS: free skin, FP: free pulp, BS: bound to skin, BP: bound to pulp. The values ​​in the figure represent the number of aroma substances.

[0023] Figure 3 .The average contents of four aroma substances in three varieties that were significantly correlated with changes in refrigeration time during refrigeration.

[0024] Figure 4 .The average contents of six aroma compounds in three varieties that were significantly correlated with changes in refrigeration time during refrigeration. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0026] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.

[0027] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0028] The mature fruits of three typical rose-scented main varieties (Sunshine Rose, Giant Rose, 87-1) were used as test materials. Different storage times (0, 2, 4, 6 and 8 weeks) at 4°C were set up, and then the fruits of different cold storage times were tested for aroma substances. Then, the aroma compounds with significant correlation between the compound content and the cold storage time were screened out. The specific method is as follows:

[0029] 1. Extraction of fruit aroma substances. Free aroma substances: Separate the pulp and peel of the fruit, put the peel into the extract (pH 3.2, 50 mg / L ascorbic acid and 3 g / L tartaric acid), and shake at 20℃ for 24h in the dark. Filter, centrifuge (4℃, 12000 rpm) for 20min, and the supernatant is used for the detection of peel aroma compounds. Add ascorbic acid (50 mg / L) to the pulp, grind it in a grinder for 1min, centrifuge the pulp (4℃, 10,000 rpm) for 10min and filter, and the supernatant is used for the detection of pulp aroma compounds. Extraction of bound aroma substances: 25mL of peel extract or juice supernatant is loaded through a solid phase column (SPE, activated with 5mL methanol and 5mL ultrapure water in advance), and rinsed with 15mL ultrapure water and 15mL dichloromethane respectively. Eluted with 25 mL of methanol, evaporated the eluate to dryness (rotary evaporator, 40°C), added 25 mL of citric acid / phosphate buffer (pH = 5) and 2 mL of AR2000 enzyme to dissolve, and maintained at 40°C for 16 h.

[0030] 2. Extraction of aroma substances. The extraction of aroma substances uses headspace solid phase microextraction (SPME) technology. Take 1.5g NaCl, 3μL internal standard (2-octanol) and 6mL extract in 1 and add them to the headspace bottle (20mL). The headspace bottle is balanced at 40℃ for 15 min (program: speed 250rpm, shaking 3s, rest 4s), and the 50 / 30-μm DVB / CAR / PDMS extraction head is inserted into the headspace bottle for extraction for 40 min. After the extraction is completed, the extraction head is inserted into the GC port, and thermal desorption is performed for 3 min, which is repeated three times.

[0031] 3. Quantitative and qualitative detection of aroma substances. GC-MS was used to qualitatively and quantitatively identify aroma compounds. The capillary column was HP-INNOWAX 30 mm × 0.25 mm × 0.25 µm. The carrier gas was high-purity helium at 1 mL / min; SPME was automatically injected without split flow. The column oven program was: 40°C for 5 min, then increased to 240°C at a rate of 5°C / min; then increased to 260°C at a rate of 20°C / min and maintained for 5 min. The ionization mode was EI, the mass spectrometry interface temperature was 280°C, the ion energy was 70 eV, the ion source temperature was 230°C, and the mass scan was 20-400 m / z. Qualitative analysis of aroma substances: Qualitative analysis of compounds was carried out based on the full ion scanning mass spectrum, and qualitative analysis of grape compounds was performed using the chromatographic retention time and mass spectrometry information of the standard compounds. Quantification of aroma substances: Prepare various standard compounds into mother solutions and continuously dilute them into 12 different concentrations to establish a standard curve for aroma substances. Quantification is performed using the corresponding standard curves of each compound.

[0032] 4. Screening of compounds related to cold storage time. The free and bound aroma substances in the peel and pulp obtained in the 3 methods were used for correlation analysis with the cold storage time (weeks), and significant negatively correlated compounds of each variety were obtained (P<0.05, |r|>0.6). Then, significant correlated compounds that existed in the three rose-scented grape varieties were screened out, including one positively correlated ( Figure 1 ) and 9 negatively correlated compounds ( Figure 2 ). By observing the average content of these substances in various varieties and their changing trends during cold storage, it was found that the free β-ionone in the peel and the free 2-methylbutenol, (E,E)-2,4-heptadienal, and 2-octanone in the pulp were all below 5μg / kg, making them difficult to detect ( Figure 3 ); The remaining 6 negatively correlated substances not only have high contents, but also have significantly decreased contents after refrigeration compared with ripe fresh fruits ( Figure 4 Among them, the 6 negatively correlated substances all showed the same trend of change. The decrease in content after refrigeration for 2-6 weeks was similar, all less than 50%, while the decrease in fruit after refrigeration for 8 weeks was greater than 50% ( Figure 4). These six substances are nerol and geraniol in the free state of the peel and cis-linalool oxide, nerolidol, α-terpineol and nerolidol in the free state of the pulp. In the market, the refrigeration time is identified by detecting and comparing the content of any one of the six substances in fresh fruits and test samples. Since the content of other substances does not change regularly after refrigeration, they cannot be used to characterize the refrigeration time. Although the aroma quality is more affected by environmental factors than the quality of sugar and acid, the aroma substances are stable under the influence of external factors (refrigeration). Therefore, the screened characterization substances can effectively characterize the refrigeration time.

[0033] The present invention uses Sunshine Rose, Giant Rose, and 87-1 rose-scented grape varieties from different producing areas to carry out verification tests. The test results show that the contents of the six substances, free nerol and geraniol in the peel and free cis-linalool oxide, nerolidol, α-terpineol, and nerolidol in the pulp, have the following changes during refrigeration for 2-8 weeks: Figure 4 The changing pattern of can effectively characterize the refrigeration time.

[0034] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for identifying the post-harvest cold storage time of rose-scented grape fruits based on aroma substances, characterized in that: The method comprises the following steps: The fruits of the main rose-scented varieties at the mature stage were used as test materials. Different low-temperature storage treatments were set up, and then the fruits of different cold storage times were tested for aroma substances. The contents of free and bound aroma substances in the peel and pulp were analyzed for correlation with the cold storage time, and the significantly negatively correlated compounds of each variety were obtained. Then, the significantly negatively correlated compounds that also existed in rose-scented grape varieties were screened out as compounds for identifying the cold storage time. The cold storage time was identified by detecting and comparing the contents of these substances in fresh fruits and test samples. Among them, the mature fruits of three typical rose-scented main varieties were used as test materials, including Sunshine Rose, Giant Rose, and 87-1; The aroma substance detection method comprises the following steps: (1) Extraction of fruit aroma substances; (2) Extraction of aroma substances; (3) Quantitative and qualitative detection of aroma substances; The specific method of step (1) is: Free aroma substances: the pulp and peel of the fruit are separated, the peel is placed in the extract, and shaken in the dark; filtered, centrifuged, and the supernatant is used for the detection of peel aroma compounds; ascorbic acid is added to the pulp, crushed, the pulp is centrifuged and filtered, and the supernatant is used for the detection of pulp aroma compounds; Extraction of bound aroma substances: Peel extract or juice supernatant is loaded onto a solid phase column and washed with ultrapure water and dichloromethane respectively; eluted with methanol, the eluate is evaporated to dryness, and citric acid / phosphate buffer and glucosidase are added to dissolve; The specific method of step (2) is as follows: the aroma substances are extracted by headspace solid phase microextraction technology; NaCl, internal standard and the extract in step (1) are added to a headspace bottle, the headspace bottle is balanced, and the extraction head is inserted into the headspace bottle for extraction; after the extraction is completed, the extraction head is inserted into the GC port for thermal analysis; The extract comprises: 50 mg / L ascorbic acid and 3 g / L tartaric acid, pH 3.2; The compounds include cis-linalool oxide, nerolidol and α-terpineol in free state in the pulp.

2. The method for identifying the post-harvest cold storage time of rose-scented grape fruits based on aroma substances as claimed in claim 1, characterized in that: The temperature of low temperature refrigeration treatment is 4~8℃.

3. The method for identifying the post-harvest cold storage time of rose-scented grape fruits based on aroma substances as claimed in claim 1, characterized in that: The specific method of step (3) is as follows: qualitative and quantitative analysis of aroma compounds is performed using a gas chromatography-mass spectrometer; qualitative analysis of aroma substances is performed using a total ion scanning mass spectrum, and qualitative analysis of grape compounds is performed using the chromatographic retention time and mass spectrum information of the standard compounds; quantitative analysis of aroma substances is performed using a mother liquor of various standard compounds that are continuously diluted to different concentrations to establish a standard curve for aroma substances, and quantitative analysis is performed using the corresponding standard curves of each compound.

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