Method for increasing content of esterified carotenoid in Chinese wolfberry

Abstract

The invention discloses a method for increasing the esterified carotenoid content of Chinese wolfberry. The method comprises the following steps: S1, picking Chinese wolfberry fruits; s2, on the day when the Chinese wolfberry fruits are picked, the Chinese wolfberry fruits are soaked in a melatonin aqueous solution for 15 min to 30 min, and the concentration of melatonin in the aqueous solution is 40 [mu] M to 60 [mu] M; s3, draining water after soaking, and performing breathable packaging and storage. The melatonin solution with proper concentration is adopted to soak the medlar fruits and then the medlar fruits are refrigerated and stored, the expression quantity of carotenoid esterifying enzyme genes in the medlar fruits is obviously higher, and the content of esterified carotenoids is also obviously higher, so that the loss of main bioactive components in the low-temperature storage period of the medlar fruits is avoided; the nutritional quality of the Chinese wolfberry fruits is guaranteed, the bright color of the Chinese wolfberry fruits can be maintained, and the commodity value of the Chinese wolfberry fruits is guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of post-harvest fruit storage and fresh-keeping, and in particular to a method for increasing the content of esterified carotenoids in Ningxia wolfberry fruit. Background Art

[0002] Ningxia wolfberry is a deciduous shrub whose fruit has many important biological functions, including antioxidant properties, immunomodulation, anti-tumor effects, neuroprotection, liver protection and anti-fatigue.

[0003] Esterified carotenoids are the main components of Ningxia wolfberry that exert pharmacological activity and can enhance the antioxidant activity of the fruit itself; esterified carotenoids are also the main pigment of mature Ningxia wolfberry fruit and the main material basis for the bright red color of Ningxia wolfberry fruit. Among them, zeaxanthin dipalmitate (ZDP) is the most important esterified carotenoid in wolfberry, accounting for more than 75% of the total esterified carotenoids.

[0004] However, as wolfberry fruit leaves the plant, the content of esterified carotenoids in the fruit will decrease during low-temperature storage, thus affecting the quality and color of the wolfberry fruit. Summary of the invention

[0005] Based on this, the purpose of the present invention is to increase the content of esterified carotenoids in wolfberry fruit, so as to ensure the nutritional quality of wolfberry fruit during low-temperature storage and maintain the color of the fruit.

[0006] The specific technical solutions for achieving the above-mentioned invention objectives include the following.

[0007] A first aspect of the present invention provides a method for increasing the content of esterified carotenoids in wolfberry, comprising the following steps:

[0008] S1. Picking wolfberry fruits;

[0009] S2. Within no more than 20 hours after picking wolfberry fruits, soaking wolfberry fruits in a melatonin aqueous solution for 15 min to 30 min, wherein the concentration of melatonin in the aqueous solution is 40 μM to 60 μM;

[0010] S3. Drain the water after soaking, pack in airtight packaging and store.

[0011] The second aspect of the present invention provides a method for increasing the expression level of wolfberry carotenoid esterase gene, comprising the following steps:

[0012] S1. Picking wolfberry fruits;

[0013] S2. Within no more than 20 hours after picking wolfberry fruits, soaking wolfberry fruits in a melatonin aqueous solution for 15 min to 30 min, wherein the concentration of melatonin in the aqueous solution is 40 μM to 60 μM;

[0014] S3. Drain the water after soaking, pack in airtight packaging and store.

[0015] The inventors of the present invention found that within no more than 20 hours after the wolfberry fruits were picked, the wolfberry fruits were immersed in a melatonin aqueous solution of appropriate concentration for a certain period of time. After 9 days of low-temperature storage, compared with the wolfberry fruits immersed in clear water, the expression level of the carotenoid esterase gene LbaZAT1 was significantly higher, and the content of zeaxanthin dipalmitate was also significantly higher. Therefore, the method of the present invention can increase the esterified carotenoid content of wolfberry, thereby avoiding the loss of the main bioactive components of wolfberry fruits during low-temperature storage, ensuring the nutritional quality of wolfberry fruits, maintaining the bright color of wolfberry fruits, and ensuring the commercial value of wolfberry fruits.

[0016] The exogenous melatonin used in the present invention is a novel, natural and safe ingredient, which provides a healthy and environmentally friendly solution for maintaining the quality of Ningxia wolfberry fruit after harvest. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are some wolfberry fruits that were treated with different concentrations of melatonin and stored for 9 days in Example 1 of the present invention.

[0018] Figure 2 The results are as follows: the zeaxanthin dipalmitic acid content in the Ningxia wolfberry fruit after the fruit was treated with melatonin at different concentrations and stored for 9 days in Example 1 of the present invention.

[0019] Figure 3 The expression level of the carotenoid esterase gene LbaZAT1 in the fruit after the Ningxia wolfberry fruit was treated with different concentrations of melatonin and stored for 9 days in Example 2 of the present invention. DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0021] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0022] Unless otherwise specified, the embodiments are carried out according to conventional experimental conditions or the conditions recommended in the manufacturer's instructions. The instruments, raw materials and reagents used in the present invention without indicating the manufacturer are all conventional products that can be purchased commercially.

[0023] In some embodiments of the present invention, a method for increasing the content of esterified carotenoids in wolfberry is disclosed, comprising the following steps:

[0024] S1. Picking wolfberry fruits;

[0025] S2. Within no more than 20 hours after picking wolfberry fruits, soaking wolfberry fruits in a melatonin aqueous solution for 15 min to 30 min, wherein the concentration of melatonin in the aqueous solution is 40 μM to 60 μM;

[0026] S3. After soaking, drain the water, package in a breathable bag (use a polyethylene plastic bag or plastic box with air holes), and store.

[0027] In some embodiments, the concentration of melatonin in the aqueous solution is 45 μM to 55 μM.

[0028] In some embodiments, the concentration of melatonin in the aqueous solution is 48 μM to 52 μM.

[0029] In some embodiments, the mixture is stored at 3°C ​​to 5°C for no more than 10 days.

[0030] In some embodiments, the mixture is stored at 4° C. for 8 to 9 days.

[0031] In some embodiments, the soaking time is 18 min to 22 min.

[0032] In some embodiments, the soaking time is 19 min to 21 min.

[0033] In some embodiments, the soaking is performed once or twice.

[0034] In some embodiments, the soaking is performed once.

[0035] In some embodiments, the esterified carotenoid is zeaxanthin dipalmitate.

[0036] In some embodiments, the wolfberry is Ningxia wolfberry.

[0037] In other embodiments of the present invention, a method for increasing the expression of wolfberry carotenoid esterase gene is disclosed, comprising the following steps:

[0038] S1. Picking wolfberry fruits;

[0039] S2. Within no more than 20 hours after picking wolfberry fruits, soaking wolfberry fruits in a melatonin aqueous solution for 15 min to 30 min, wherein the concentration of melatonin in the aqueous solution is 40 μM to 60 μM;

[0040] S3. After soaking, drain the water, package in a breathable bag (use a polyethylene plastic bag or plastic box with air holes), and store.

[0041] In some embodiments, the carotenoid esterase gene is the LbaZAT1 gene.

[0042] In some embodiments, the concentration of melatonin in the aqueous solution is 45 μM to 55 μM.

[0043] In some embodiments, the concentration of melatonin in the aqueous solution is 48 μM to 52 μM.

[0044] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] Example 1

[0046] Ningxia wolfberry fruits (same plant, same part) that were mature (32±2 days after flowering) and free of mechanical damage and pests were harvested on a sunny day. After the Ningxia wolfberry was picked, the Ningxia wolfberry fruits were randomly divided into 4 groups, with 30 fruits in each group, and soaked in 50μM, 100μM, and 150μM melatonin aqueous solutions (prepared with pure water as solvent) for 20 minutes; soaked in pure water for 20 minutes as CK. The Ningxia wolfberry fruits after soaking were taken out and the water on the surface of the Ningxia wolfberry fruits was drained at room temperature; the drained Ningxia wolfberry fruits were packaged in polyethylene plastic boxes with air permeable holes; the packaged Ningxia wolfberry fruits were stored at 4℃ (storage day 0). On the 9th day of storage, the esterified carotenoids in each group of Ningxia wolfberry fruits were extracted, and the content of zeaxanthin dipalmitate was determined. The determination was repeated 3 times and the average value was taken.

[0047] 1. Extraction of esterified carotenoids

[0048] The fruits of each group of Ningxia wolfberry were quickly frozen with liquid nitrogen and ground into powder (30 Hz, 1.5 min, stored at -80 °C). 50 mg of the powder was extracted with 0.5 mL of a mixed solution of n-hexane: acetone: ethanol (1:1:1, v / v / v). After vortexing at room temperature for 20 minutes, the supernatant was obtained by centrifugation (12000 rpm, 5 min, 4 °C). The residue was extracted a second time under the same conditions. The obtained supernatant was dried and dissolved with 100 μL of dichloromethane to obtain the esterified carotenoid extract.

[0049] 2. Determination of the content of zeaxanthin dipalmitate

[0050] The esterified carotenoid extract from step 1 was filtered through a 0.22 μm membrane and subjected to LC-MS / MS analysis.

[0051] Chromatographic column: YMC C30;

[0052] Mobile phase A: A methanol-acetonitrile mixture containing 0.01% 2,6-di-tert-butyl-p-cresol (BHT) and 0.1% formic acid (methanol:acetonitrile = 1:3, v / v)

[0053] Mobile phase B: methyl tert-butyl ether containing 0.01% 2,6-di-tert-butyl-p-cresol (BHT)

[0054] Gradient program: 0→3 min, 0% B; 3 min→5 min, 0% B→70% B; 5 min→9 min, 70% B→95% B, 10 min→11 min, 95% B→0% B;

[0055] Flow rate: 0.8 mL / min;

[0056] Temperature: 28℃.

[0057] The standard curve was prepared using the zeaxanthin dipalmitate standard. The esterified carotenoid extracts in each group of samples were compared with the zeaxanthin dipalmitate standard at the 450nm channel, and then the zeaxanthin dipalmitate content in each group of samples was calculated.

[0058] 3. Results Analysis

[0059] On the 9th day of storage, the wolfberry fruits in each treatment group remained red, and no significant difference could be seen with the naked eye (part of the fruits were Figure 1 ).

[0060] The results of the content of zeaxanthin dipalmitate in the samples of each treatment group are as follows: Figure 2 shown.

[0061] Figure 2The results showed that compared with the water immersion treatment, the content of zeaxanthin dipalmitate in the fruit of Ningxia wolfberry was higher after being treated with different concentrations of melatonin and stored for 9 days after harvest. In addition, the effect of the 50μM melatonin treatment group was significantly better than that of the 100μM and 150μM melatonin treatment groups.

[0062] Example 2

[0063] The Ningxia wolfberry fruits stored for 9 days in Example 1 (quickly frozen with liquid nitrogen, ground into powder, and stored at -80°C) were taken to measure the expression level of the carotenoid esterase gene LbaZAT1 therein.

[0064] Determination of carotenoid esterase gene expression: Total RNA from each group of powders was extracted, and after reverse transcription to obtain cDNA, the expression of carotenoid esterase gene was determined by real-time fluorescence quantitative PCR. Actin I was used as the internal reference gene, and MonampTM ChemoHS qPCR Mix was used as the PCR amplification reagent (the reaction system was prepared according to the instructions). The amplification program was as follows: 95°C, 10min; 95°C 10s; 60°C 30s, 40 cycles. The 2-ΔΔCt method was used for data analysis.

[0065] The results are as follows Figure 3 As shown, from Figure 3 It can be seen that when the Ningxia wolfberry was treated with different concentrations of melatonin after harvest and stored for 9 days, the expression level of the carotenoid esterase gene LbaZAT1 after melatonin treatment was significantly increased compared with CK, the 50μM melatonin treatment group was 5.09 times that of CK, the 100μM melatonin treatment group was 1.98 times that of CK, and the 150μM melatonin treatment group was 1.79 times that of CK; and the expression level of the carotenoid esterase gene LbaZAT1 in the 50μM melatonin treatment group was also significantly different from the expression levels of the carotenoid esterase gene LbaZAT1 in the 100μM melatonin treatment group and the 150μM melatonin treatment group.

[0066] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0067] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for increasing the content of esterified carotenoids in wolfberry, characterized in that: The following steps are involved: S1. Picking wolfberry fruits; S2. Within no more than 20 hours after picking wolfberry fruits, soaking wolfberry fruits in a melatonin aqueous solution for 15 min to 30 min, wherein the concentration of melatonin in the aqueous solution is 40 μM to 60 μM; S3. Drain the water after soaking, pack in airtight packaging and store.

2. The method for increasing the content of esterified carotenoids in wolfberry according to claim 1, characterized in that: The concentration of melatonin in the aqueous solution is 45 μM to 55 μM, preferably 48 μM to 52 μM.

3. The method for increasing the content of esterified carotenoids in wolfberry according to claim 1, characterized in that: The mixture is stored at 3°C ​​to 5°C for no more than 10 days; preferably, the mixture is stored at 4°C for 8 to 9 days.

4. The method for increasing the content of esterified carotenoids in wolfberry according to claim 1, characterized in that: The soaking time is 18 min to 22 min, more preferably 19 min to 21 min.

5. The method for increasing the content of esterified carotenoids in wolfberry according to claim 1, characterized in that: The soaking is performed once to twice, preferably once.

6. The method for increasing the content of esterified carotenoids in wolfberry according to any one of claims 1 to 5, characterized in that: The esterified carotenoid is zeaxanthin dipalmitate.

7. The method for increasing the content of esterified carotenoids in wolfberry according to any one of claims 1 to 5, characterized in that: The wolfberry is Ningxia wolfberry.

8. A method for increasing the expression of wolfberry carotenoid esterase gene, characterized in that: The following steps are involved: S1. Picking wolfberry fruits; S2. No more than 20 hours after picking wolfberry fruits, soaking wolfberry fruits in a melatonin aqueous solution for 15 min to 30 min, wherein the concentration of melatonin in the aqueous solution is 40 μM to 60 μM; S3. Drain the water after soaking, pack in airtight packaging and store.

9. The method for increasing the expression level of wolfberry carotenoid esterase gene according to claim 8, characterized in that: The carotenoid esterase gene is the LbaZAT1 gene.

10. The method for increasing the expression level of wolfberry carotenoid esterase gene according to claim 8, characterized in that: The concentration of melatonin in the aqueous solution is 45 μM to 55 μM, preferably 48 μM to 52 μM.

Images