A method for increasing flavonoid content in citrus peel

Abstract

The present invention discloses a method for increasing the content of flavonoids in citrus peels. Flavonoids are important functional components of citrus fruits. They not only have physiological effects such as affecting fruit flavor and color and improving plant stress resistance, but also have multiple physiological activities such as anticancer, antiviral, anti-inflammatory, and antibacterial. Using citrus fruits as raw materials, the present invention discovered that treating the fruits with an Al solution before harvest or soaking them after harvest can increase the content of flavonoids such as naringin. Consequently, a simple and easy method has been developed to rapidly increase the content of flavonoids in citrus peels.

Description

Technical Field

[0001] The invention relates to a citrus fruit processing method, in particular to a processing method capable of rapidly increasing the flavonoid content of citrus peel. Background Art

[0002] Citrus is the world's largest fruit, and its planting area and output rank first among all kinds of fruits. my country is the origin of citrus and has rich variety resources. In 2022, my country's annual citrus output was 60.0389 million tons, ranking first in the world. In recent years, with the continuous improvement of people's living standards and the continuous improvement of health awareness, consumers' requirements for the quality characteristics and health attributes of citrus fruits have become increasingly diversified. As one of the main sources of polyphenols in the human diet, citrus is favored by the food and pharmaceutical industries because it is rich in various flavonoids. Tangerine peel, green peel, bitter orange, Buddha's hand and tangerine peel processed from citrus are all well-known traditional Chinese medicines. They are all rich in flavonoids, and their medicinal value is also closely related to the flavonoid content. For example, naringin and wild lacquercoside are the main medicinal ingredients in tangerine peel. [1-3] Modern medical clinical experimental studies have shown that citrus flavonoid compounds such as flavanones and polymethoxyflavones have significant inhibitory effects on some bacteria, fungi and cancer cells. [4] .

[0003] However, the current production of citrus flavonoids is very limited, unable to meet the growing demand for healthcare. To increase production, citrus varieties are constantly being improved through genetic modification, but this also has a series of impacts on citrus yield, quality, and disease resistance, and the prospects for industrial application are poor. Alternatively, improved extraction processes can be used to isolate more flavonoids, but this method has the disadvantage of being costly. Summary of the Invention

[0004] The purpose of the present invention is to provide a method that is simple, easy and can quickly increase the flavonoid content in citrus peels.

[0005] To achieve the above objectives, the applicant used two citrus fruits, Huazhou pomelo (Citrus grandis, the source plant of Huajuhong) and 'Flame' grapefruit (Citrus paradisi), as materials, and used Al solution to treat them before harvest and soak them after harvest. The changes in flavonoid content in citrus fruits under Al stress were analyzed. The results showed that both treatments could significantly increase the flavonoid content in citrus peels. It is speculated that the expression of flavonoid synthesis genes in citrus fruits may be promoted under Al stress.

[0006] Specifically, the method comprises spraying an Al solution on the citrus fruits before picking, or soaking the citrus fruits in the Al solution and then placing them aside after picking.

[0007] Furthermore, the spraying time is 8-12 days before picking, and the placement time after soaking is 2-3 weeks.

[0008] Wherein, the flavonoids include naringin.

[0009] Preferably, the concentration of the Al solution is 10-50 mM.

[0010] The beneficial effects of the present invention are:

[0011] The present invention significantly increases the flavonoid content in citrus peels by simply spraying or soaking citrus fruits with a low-cost aluminum-containing solution. The entire process can be controlled within 10-15 days, offering advantages such as a short cycle and low cost. This method has no special requirements for citrus germplasm and does not affect citrus yield and quality. It has great potential for promotion and application in the fields of citrus deep processing and flavonoid production. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : Postharvest treatment of 'Flame' grapefruit fruits with aluminum solution.

[0013] Figure 2 : HPLC detection spectrum of flavonoids in pre-harvest fruiting treatment (1 is naringin, 2 is rhubarb glycoside).

[0014] Figure 3 : Total contents of naringin and naringin in fruits treated before harvest.

[0015] Figure 4 : HPLC detection spectrum of flavonoids treated with post-harvest soaking (1 is naringin, 2 is naringin).

[0016] Figure 5 : Total contents of naringin and naringin in postharvest soaking treatment. DETAILED DESCRIPTION

[0017] The present invention will be described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the various experimental procedures involved in the embodiments are all routine techniques in the art. For any part not specifically described herein, those skilled in the art can refer to various commonly used reference books, scientific literature, or relevant specifications, manuals, etc. before the filing date of the present invention for implementation.

[0018] Adverse stress is one of the important factors affecting the biosynthesis of plant secondary metabolites. Flavonoids, as an important type of plant secondary metabolites, play an important role in the interaction between plants and adverse stress. They constitute part of the plant defense system. When subjected to abiotic stresses such as ultraviolet radiation, low temperature, drought, and salt stress, plants can respond to adverse stress by increasing the content of flavonoid metabolites. [5-7] After blueberry fruit was treated with UV-B radiation, the anthocyanin content in the body increased significantly. [8] Low temperature conditions are conducive to the accumulation of flavonoid metabolites in Ginkgo biloba [9] Corn roots exposed to aluminum poisoning release high concentrations of the flavonoid compound quercetin, whose ability to chelate metal ions becomes an important regulatory mechanism to improve aluminum poisoning.

[10] In Huazhou pomelo young fruits, the naringin content was significantly positively correlated with the Mn content in the leaves, and also with the available Cu and available S contents in the soil.

[11] Under adverse stress, increasing the content of flavonoids can effectively enhance the plant's ability to respond to adversity. Therefore, we can promote plant flavonoid metabolism through adverse stress treatment, thereby promoting the enrichment of flavonoid compounds in natural plants.

[0019] The peels of common cultivated citrus fruits, such as pomelo and grapefruit, are a major source of flavonoid compounds. We treated Huazhou pomelo and 'Flame' grapefruit fruits with aluminum-containing solutions. Analysis revealed that treatment significantly increased the content of flavonoids such as naringin, naringrin, and naringrin in the peels. Therefore, external application of aluminum solutions at appropriate concentrations can be an effective method for increasing the flavonoid content of citrus fruits. This provides a promising approach for the large-scale production and extraction of natural flavonoids directly from citrus peels.

[0020] 1. Materials

[0021] Al solution preparation: This experiment used the commonly used Al ion supply reagent AlCl3·6H2O. Considering the treatment effect on citrus fruit (yellow peel) in a short period of time, we prepared a 30mM AlCl3·6H2O solution and added 0.4% Tween 80 as an extender.

[0022] Citrus fruit material preparation: Huazhou pomelo (Citrus grandis, the source plant of Huajuhong) and 'Flame' grapefruit (Citrus paradisi), 70 days after anthesis (or 6 cm in diameter), were selected for pre-harvest hanging on the tree and post-harvest soaking. This study used Huazhou pomelo (Citrus grandis, the source plant of Huajuhong) and 'Flame' grapefruit (Citrus paradisi) as the citrus materials.

[0023] 2. Methods

[0024] 2.1 Treatment methods

[0025] 2.1.1 Pre-harvest Fruiting Treatment: Six Huazhou pomelo trees with relatively uniform growth were selected and divided into an AlCl₃·6H₂O treatment group and a water control group, with three trees in each group. Fruit in the treatment group was marked with pink tags, while fruit in the control group was marked with pure white tags. The fruit surface was sprayed with AlCl₃·6H₂O solution once in the morning and evening, and the fruit surface was promptly covered with absorbent paper to prevent rapid loss and evaporation of the solution. Fruit samples were collected and tested 5 days after treatment, with three fruits from each treatment and control group tested each time.

[0026] 2.1.2 Postharvest Soaking Treatment: Eighteen mature 'Flame' grapefruits of uniform size and shape were divided into two groups. The treatment group was soaked in AlCl₃·6H₂O solution, while the control group was soaked in water. Nine fruits were included in each group. The fruits were soaked for 1 hour in the morning and evening, then placed on trays for undisturbed soaking. The treatment lasted for a total of 3 weeks, with three fruits from each group tested weekly.

[0027] 2.2 Detection method

[0028] After the fruits of the treatment and control groups were treated accordingly, the peels were freeze-dried and ground into fine powder using a mortar. The flavonoid content was detected according to the method of Peng et al. (2021)

[12] The specific steps were as follows: 0.1 g of dry sample powder was weighed and added to 5 mL of 80% methanol. The cells were disrupted by ultrasonication for 1 hour. During this period, the sample was removed and flipped several times every 20 minutes to ensure sufficient contact between the sample and the extract. After the ultrasonication, the sample was centrifuged in a high-speed refrigerated centrifuge at 10,000 rpm for 10 minutes. Subsequently, 1 mL of the extract was aspirated with a syringe and filtered through a 0.22 μm microporous membrane into a sample injection bottle. Finally, the sample was placed in an HPLC for flavonoid detection.

[0029] 3. Results and Analysis

[0030] 3.1 Results and analysis of pre-harvest fruiting treatment: The liquid chromatography results of pre-harvest fruiting treatment (10 days) are as follows Figure 2 As shown in the figure, the peak areas of two components in the Al treatment group increased significantly, and these two components were qualitatively identified as naringin and succinoside using standard substances. Subsequently, the fruits of the fruiting treatment were quantitatively tested for 5 days, 10 days, and 15 days. The results are shown in Table 1. From the test results, it can be seen that after 5 days of fruiting on the tree, the content of naringin and succinoside in the treatment group was significantly reduced compared with the control group (P<0.05); after 10 days of fruiting, the content of naringin in the treatment group was significantly increased compared with the control group (P<0.05), and the content of succinoside increased but was not much different from that of the control group; after 15 days of fruiting, the content of both components in the treatment group was not much different from that of the control group (P>0.05).

[0031] Table 1 Flavonoid content of pre-harvest fruiting treatment

[0032]

[0033] The total contents of naringin and scutellarin at different stages were calculated, and the results were as follows: Figure 3 As shown in the figure, after 10 days of fruiting, the total content of naringin and naringin in the treatment group was significantly higher than that in the control group. In addition, the analysis of the test results of the same treatment method with different treatment times showed that after the control group was treated with water, the flavonoids showed a significant decrease, which is basically consistent with the trend of flavonoids gradually decreasing during the growth and development period of the fruit.

[12] However, the flavonoid content in the Al-treated group showed an overall increasing trend. Al treatment promoted the increase in flavonoid content, thereby offsetting the decrease in fruit flavonoid content.

[0034] 3.2 Results and analysis of post-harvest fruit soaking treatment: The liquid chromatography results of post-harvest soaking treatment (14 days) are as follows Figure 4 As shown in the figure, the peak areas of two components in the Al treatment group also increased significantly, which were qualitatively determined to be naringin and naringin using standard substances. Subsequently, the fruits soaked for 3 weeks were quantitatively tested, and the results are shown in Table 2. The test results show that after 7 days of soaking, the content of naringin and naringin in the treatment group was significantly reduced compared with the control group (P<0.05); after 14 days of soaking, the content of naringin and naringin in the treatment group was significantly increased compared with the control group (P<0.05); after 21 days of soaking, the content of naringin in the treatment group was significantly increased compared with the control group (P<0.05), while the content of naringin in the treatment group was not much different (P>0.05).

[0035] Table 2 Flavonoid content of post-harvest soaking treatment

[0036]

[0037] The total contents of naringin and naringin in different periods were calculated, and the results were as follows: Figure 5 After 7 days, the total content of naringin and naringin in the treatment group was significantly lower than that in the control group. However, after 14 and 21 days of immersion, the content in the treatment group was significantly higher than that in the control group. There was a significant upward trend from the 2nd week, and then a slight decline in the 3rd week. The content reached the highest value after 14 days of immersion in Al solution. This may be because the flavonoid content of the fruit is related to the time of response to mineral element stress.

[0038] In summary, this experiment used aluminum solution to treat citrus fruit before and after harvest, and found that the content of the main flavonoids in the yellow peel of the fruit increased significantly after 10 or 14 days of treatment, respectively. The effects of the two treatments differed slightly, possibly due to the different responses of aluminum treatment to genes in the fruit flavonoid biosynthesis pathway in vitro and in vivo. In short, both methods significantly increased the flavonoid content of citrus fruit. The present invention uses a low-cost aluminum solution to promote the increase of naringin and flavonoids in citrus peel through simple treatment, which is a simple and effective method.

[0039] References:

[0040] [1]LIP L,LIU MH,HU JH,et al.Systematic chemical profiling of'Tomentosa'by ultra-fast liquid chromatography / diode-array detector / quadrupoletime-of-flight tandem mass spectrometry[J].Journal of Pharmaceutical andBiomedical Analysis,2014,90(167-79).

[0041] [2]CHEN

[0042] [3]WEI L,YU XX,LI H,et al.Optimization of solvent-free microwaveextraction of essential oil from the fresh peel of Citrus medicaL.var.arcodactylis Swingle by response surface methodology,chemical composition and activity characterization[J].Sci Hortic-Amsterdam,2023,309

[0043] [4]GAO Z,GAO W,ZENG SL,et al.Chemical structures,bioactivities andmolecular mechanisms of citrus polymethoxyflavones[J].J Funct Foods,2018,40(498-509).

[0044] [5]ZORATTI L,SARALA M,CARVALHO E,et al.Monochromatic light increasessanthocyanin content during fruit development in bilberry[J].Bmc PlantBiology,2014,14

[0045] [6]AZUMA A,YAKUSHIJI H,KOSHITA Y,et al.Flavonoid biosynthesis-related genes in grape skin are differentially regulated by temperature and lightconditions[J].Plant,2012,236(4):1067-80.

[0046] [7]NAKABAYASHI R,YONEKURA-SAKAKIBARA K,URANO K,et al.Enhancement ofoxidative and drought tolerance in Arabidopsis by overaccumulation ofantioxidant flavonoids[J].Plant J,2014,77(3):367-79.

[0047] [8]NGUYEN CTT,LIM S,LEE JG,et al.VcBBX,VcMYB21,and VcR2R3MYBTranscription Factors Are Involved in UV B-Induced Anthocyanin Biosynthesisin the Peel of Harvested Blueberry Fruit[J].Journal of Agricultural and FoodChemistry,2017,65(10):2066-73.

[0048] [9]GUO J, ZHOU

[0049]

[10] KIDD PS, LLUGANY M, POSCHENRIEDER C, et al. The role of rootexudates in aluminum resistance and silicon-induced amelioration ofaluminium toxicity in three varieties of maize(Zea mays L.)[J]. Journal of Experimental Botany, 2001, 52(359):1339-52.

[0050]

[11] Lin Lanwen, Zhong Jihong, Luo Bosheng, et al. Distribution of trace elements in soils of Citrus aurantium production areas and their correlation with the medicinal active ingredients of Citrus aurantium [J]. Ecological Environment, 2008(03):1179-1183. DOI:10.16258 / j.cnki.1674-5906.2008.03.071.

[0051]

[12] PENG Z X,ZHANG H P,LI W Y,et al.Comparative profiling and naturalvariation of polymethoxylated flavones in various citrus germplasms[J].FoodChemistry,2021,354。

Claims

1. A method for increasing the flavonoid content of citrus peel, characterized by: Using Al solution to treat citrus fruits can promote the expression of flavonoid synthesis genes under adverse stress, thereby rapidly increasing the content of flavonoids in citrus peels. The treatment involves spraying an Al solution on Huazhou pomelo (Citrus grandis) citrus fruits 8-12 days before harvest to increase the content of naringin and naringin, or soaking grapefruit (Citrus paradisi) citrus fruits in the Al solution after harvest and then leaving them for 2-3 weeks to increase the content of naringin and naringin. The concentration of the Al solution is 10-50 mM.

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