Application of rice leaf extract in inhibiting postharvest fungi of fruits and vegetables

Abstract

The present invention discloses the application of rice leaf extract in inhibiting postharvest fungi of fruits and vegetables. The present invention provides the application of rice leaf extract in any one of the following: inhibiting postharvest fungi of fruits and vegetables; preparing a postharvest fungi inhibitor of fruits and vegetables; storing fruits and vegetables; preparing a product for storing fruits and vegetables. The rice leaf extract provided by the present invention can inhibit postharvest fungi of fruits and vegetables and has an obvious inhibitory effect on Alternaria alternata, Botrytis cinerea, Penicillium expansum and Penicillium digitatum. The present invention is of great significance for the postharvest storage of fruits and vegetables.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and to the application of rice leaf extract in inhibiting postharvest fungi of fruits and vegetables. Background Art

[0002] The presence of pathogenic fungi such as Botrytis, Aspergillus, Penicillium, Rhizopus, Alternaria, etc. after fruits and vegetables are harvested can cause fruit and vegetable rot, resulting in significant economic losses (Ramos et al., 2013; Ersus et al., 2013). Currently, the use of chemical fungicides and biological preservation technologies are the main antibacterial means (Jennifer et al., 2001), but these methods have problems such as poor effect stability; easy to cause drug residues and endanger human health.

[0003] Flavonoids are a class of natural metabolites widely distributed in plants, containing the basic skeleton C6-C3-C6. According to the oxidation degree of the central three-carbon chain, whether it forms a ring, and the position of the B-ring connection (2- or 3-position), etc., the main natural flavonoids can be divided into flavonoids, flavonols, dihydroflavonoids, dihydroflavonols, anthocyanins, isoflavonoids, etc. Most flavonoids exist in the form of glycosides or carbon glycosides bound to sugars in plants, and some exist in free form. Flavonoids have strong anti-inflammatory and anti-cancer effects and have great application prospects in the treatment of diseases such as cardiovascular diseases and tumors.

[0004] The leaves of rice contain a large number of secondary metabolites, including flavonoids, and these compounds play important roles in aspects such as rice's resistance to ultraviolet rays and diseases (Peng et al., 2017; Zhan et al., 2020). However, currently, it is not very clear whether the rice leaf metabolites can inhibit postharvest fungi of fruits and vegetables. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of rice leaf extract in inhibiting postharvest fungi of fruits and vegetables.

[0006] In the first aspect, the present invention claims to protect the application of rice leaf extract in any one of the following:

[0007] P1, inhibiting postharvest fungi of fruits and vegetables;

[0008] P2, preparing an inhibitor for postharvest fungi of fruits and vegetables;

[0009] P3, storing fruits and vegetables;

[0010] P4, preparing a product for storing fruits and vegetables.

[0011] Among them, the rice leaf extract is an ethanol extract of rice leaves.

[0012] Furthermore, the rice leaf extract is an extract obtained by extracting rice leaves with 75% (volume percentage) ethanol.

[0013] Among them, the rice leaves can be fresh rice leaves.

[0014] Even further, the rice leaf extract can be prepared by a method including the following steps: adding rice leaves to 75% (volume percentage) ethanol at a ratio of 1 Kg (fresh weight) to 1 L, soaking for 30 min, extracting twice continuously, and obtaining the rice leaf extract from the soaked liquid.

[0015] Furthermore, after soaking, there is also a step of rotary evaporation and concentration.

[0016] Furthermore, the mass ratio of tricin-5-O-glucoside to isoorientin in the rice leaf extract can be (1 - 1.5):1, such as 1.35:1.

[0017] In a specific embodiment of the present invention, the total concentration of the target substances (tricin-5-O-glucoside and isoorientin) in the stock solution of the rice leaf extract obtained after rotary evaporation and concentration reaches about 2000 mg / L, such as 2040 mg / L (the concentration of tricin-5-O-glucoside is 1173 mg / L, and the concentration of isoorientin is 867 mg / L).

[0018] In a second aspect, the present invention claims to protect a method for inhibiting post-harvest bacteria of fruits and vegetables.

[0019] The method for inhibiting post-harvest bacteria of fruits and vegetables claimed by the present invention may include the following steps: applying the rice leaf extract described in the second aspect above to post-harvest fruits and vegetables (such as smearing or spraying on the surface of fruits and vegetables); wherein the total working concentration of the target substances (tricin-5-O-glucoside and isoorientin) in the rice leaf extract is 50 mg / L or more (such as 100 or more or 500 mg / L or more; further such as 50 - 1000 mg / L or 100 - 1000 mg / L or 500 - 1000 mg / L).

[0020] In the first aspect to the second aspect, the rice is indica rice.

[0021] Furthermore, the indica rice can be any variety of indica rice such as Montakcl, J.P.5, PD 46, PATNAI 6, Shui Ya Jien, and / or YOU-I B, etc.

[0022] In a specific embodiment of the present invention, the rice leaves are a quality mixture of Montakcl, J.P.5, PD 46, PATNAI6, Shui Ya Jien, and YOU-I B leaves.

[0023] In the first to second aspects, the post-harvest fungi of fruits and vegetables are selected from: Alternaria alternata, Botrytis cinerea, Penicillium expansum, and / or Penicillium digitatum.

[0024] When the post-harvest fungus of fruits and vegetables is Botrytis cinerea, the total working concentration of the target substances (tricin-5-O-glucoside and isoorientin) in the rice leaf extract described in the second aspect above is 500 mg / L or more, such as 500 - 1000 mg / L. When the post-harvest fungus of fruits and vegetables is Alternaria alternata, Penicillium expansum, and / or Penicillium digitatum, the total working concentration of the target substances (tricin-5-O-glucoside and isoorientin) in the rice leaf extract described in the second aspect above is 50 mg / L or more, such as 100 mg / L or more or 500 mg / L or more; further such as 50 - 1000 mg / L or 100 - 1000 mg / L or 500 - 1000 mg / L.

[0025] In the third aspect, the present invention claims the application of a mixture composed of tricin-5-O-glucoside and isoorientin in any one of the following:

[0026] P1, inhibiting post-harvest fungi of fruits and vegetables;

[0027] P2, preparing an inhibitor for post-harvest fungi of fruits and vegetables;

[0028] P3, storing fruits and vegetables;

[0029] P4, preparing a product for storing fruits and vegetables.

[0030] Wherein, in the mixture, the mass ratio of tricin-5-O-glucoside to isoorientin can be (1 - 1.5):1. Such as 1.35:1.

[0031] In the said application, dissolve the mixture composed of tricin-5-O-glucoside and isoorientin in ethanol (such as 75% ethanol), so that the total working concentration of tricin-5-O-glucoside and isoorientin is above 50 mg / L (such as above 100 or above 500 mg / L; or 50 - 1000 mg / L or 100 - 1000 mg / L or 500 - 1000 mg / L).

[0032] In the specific embodiments of the present invention, the postharvest fungi of fruits and vegetables are selected from: Alternaria alternata, Botrytis cinerea, Penicillium expansum, and / or Penicillium digitatum.

[0033] Experiments have proved that the rice leaf extract provided by the present invention can inhibit the postharvest fungi of fruits and vegetables, and has obvious inhibitory effects on Alternaria alternata, Botrytis cinerea, Penicillium expansum, and Penicillium digitatum. The present invention is of great significance for the postharvest storage of fruits and vegetables. Description of the Drawings

[0034] Figure 1 It is the inhibitory effect of low-concentration rice leaf extract on postharvest pathogenic bacteria.

[0035] Figure 2 It is the inhibitory effect of high-concentration rice leaf extract on postharvest pathogenic bacteria.

[0036] Figure 1 and Figure 2 Both are the results after 3 days of inoculation with postharvest pathogenic bacteria. Specific Embodiments

[0037] The following further describes the present invention in detail in combination with specific embodiments. The given embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0038] The experimental methods in the following embodiments, unless otherwise specified, are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0039] Example 1. Preparation of Rice Leaf Extract

[0040] (1) Collect fresh leaves of rice (indica rice) about 60 days after sowing (fresh leaves of indica rice varieties Montakcl, J.P.5, PD 46, PATNAI 6, Shui Ya Jien, and YOU-I B are mixed in equal mass. These indica rice varieties are all recorded in "Supplemental Table 1" of "Wang et al., The power of inbreeding: NGS based GWAS of rice reveals convergent evolution during rice domestication. 2016 Molecular Plant 9:975", which is publicly available from the applicant and can only be used for repeating the experiments of the present invention and not for other purposes), and then soak the rice leaves in 75% ethanol (volume percentage content, with the other 25% being water) at a ratio of 1 Kg of fresh leaves to 1 L of 75% ethanol for 30 min; after 30 min, collect the rice leaf extract obtained from the soaking solution, and add an appropriate amount of 75% ethanol to the leaves again and repeat the extraction once;

[0041] (2) Collect all the leaf extracts (the liquid after soaking) obtained and concentrate them using a rotary evaporator. During this process, continuously detect the concentration of the concentrate until the total concentration of the target substances (tricin-5-O-glucopyranoside and isoorientin) in the rice leaf extract reaches approximately 2000 mg / L. The detection method is as follows: Pipette 200 μL of the concentrate of the rice leaf extract into a 1.5 mL Eppendorf centrifuge tube, centrifuge at 4 °C and 12,000 rpm for 15 min. Pipette 100 μL of the supernatant into an Agilent injection vial containing a liner with a volume of 200 μL. Then detect it using UPLC-MS. Mobile phase A: 0.1% formic acid aqueous solution (% represents volume percentage); Mobile phase B: acetonitrile. Elution gradient: 0 - 2 min: 5% B - 10% B, 2 - 12 min: 10% B - 25% B, 12 - 18 min: 25% B - 70% B, 18 - 23 min: 70% B - 90% B, 23 - 25 min: 90% B - 100% B, 25 - 30 min: 100% B, post-run for 5 min, % all represent volume percentage. Flow rate 0.3 mL / min, column temperature: 40 °C, injection volume: 5 μL. Use an electrospray ionization source (ESI), detect in positive ion mode, the carrier gas is high-purity nitrogen, pressure 40 psi, temperature 325 °C. Finally, using the standard curve of the target substances (tricin-5-O-glucopyranoside and isoorientin) drawn, convert the peak area of the target substances in the obtained mass spectrum into the absolute content of the target substances in the concentrate. After detection, the total concentration of the target substances tricin-5-O-glucopyranoside and isoorientin in the concentrate is 2040 mg / L (the concentration of tricin-5-O-glucopyranoside is 1173 mg / L, and the concentration of isoorientin is 867 mg / L).

[0042] The structural formula of tricin-5-O-glucopyranoside is as follows:

[0043]

[0044] The structural formula of isoorientin is as follows:

[0045]

[0046] Example 2. Cultivation of postharvest fungi of experimental fruits and vegetables

[0047] The postharvest fungi used in this example are Alternaria alternata, Botrytis cinerea, Penicillium expansum, and Penicillium digitatum. The cultivation methods for each strain are as follows:

[0048] Spores of Alternaria alternata, Botrytis cinerea, Penicillium expansum, and Penicillium digitatum collected after culturing at 25°C for 7 - 10 d were formulated into a spore suspension with a concentration of 10 5 spores / mL using sterile water.

[0049] Example 3. Inhibitory effect of rice leaf extract on post - harvest fungi of fruits and vegetables

[0050] (1) Medium preparation: Appropriate amounts of the concentrated stock solution of rice leaf extract obtained in Example 1 (total concentration of vitexin - 5 - O - glucoside and isoorientin is 2040 mg / L) were respectively added to 200 mL of molten PDA medium heated, so that the final concentrations of the target substances (vitexin - 5 - O - glucoside and isoorientin) in the leaf extract were 50 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, and 1,000 mg / L respectively. After thorough mixing, the medium was poured into 90 - mm petri dishes and solidified in a laminar flow hood. The medium without adding rice leaf extract was used as a control. The PDA medium formula is as follows: 200 g of potato, boiled with ddH2O for 30 min. Then 10 g of D - glucose and 15 g of agar were added, made up to 1 L, and autoclaved (121°C, 20 min).

[0051] (2) Inoculation of post - harvest pathogenic bacteria: 3 μL of the spore suspension of each bacterium in Example 2 was respectively dropped onto the PDA plates containing different concentrations of rice leaf extract in (1), and cultured statically at 25°C for 7 d. Colony growth morphology was recorded every day starting from 3 d, and the colony diameter was measured by the cross - method. Each treatment had 5 replicates. Finally, the inhibition rate of each treatment was calculated using the following formula: (average colony diameter of the control group - colony diameter of each colony in the treatment group) / average colony diameter of the control group * 100%.

[0052] (3) Phenotypic observation: As Figure 1As shown, when the final concentration of the externally applied rice leaf extract was 50 mg / L, 100 mg / L, and 200 mg / L, with the increase of each treatment concentration, the inhibition rates of the rice leaf extract against Alternaria alternata, Penicillium expansum, and Penicillium digitatum increased significantly. However, each treatment concentration had no inhibitory effect on the growth of Botrytis cinerea. When the final concentration of the externally applied rice leaf extract was increased to 500 mg / L, the rice leaf extract could completely inhibit the growth of Alternaria alternata, Botrytis cinerea, and Penicillium digitatum (inhibition rate 100%), and the inhibition rate against Penicillium expansum was about 50%. When the final concentration of the externally applied rice leaf extract was increased to 1,000 mg / L, the rice leaf extract could completely inhibit the growth of each postharvest pathogen ( Figure 2 ), indicating that the rice leaf extract has a significant antibacterial effect.

[0053] The above has detailed the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use, or improvement of the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. Use of rice leaf extract in any of the following: P1, inhibiting post - harvest fungi of fruits and vegetables; P2, preparing a post - harvest fungi inhibitor of fruits and vegetables; The rice leaf extract is prepared by a method comprising the following steps: adding rice leaves in a ratio of 1 Kg fresh weight to 1 L into 75% (v / v) ethanol, soaking for 30 min, extracting twice continuously, and obtaining the rice leaf extract from the soaked liquid; after soaking, there is also a step of rotary evaporation and concentration; The rice is indica rice; the indica rice is a mass mixture of Montakcl, J.P. 5, PD 46, PATNAI 6, Shui Ya Jien, and YOU - I B, etc.; The post - harvest fungi of fruits and vegetables are selected from: Alternaria alternata ( Alternaria alternata ), Botrytis cinerea ( Botrytis cinerea ), Penicillium expansum ( Penicillium expansum ), and / or Penicillium digitatum ( Penicillium digitatum ).

2. The use according to claim 1, characterized in that: The mass ratio of tricin-5-O-glucoside to isoorientin in the rice leaf extract is (1 - 1.5):

1.

3. A method for inhibiting post - harvest fungi of fruits and vegetables, comprising the following steps: applying the rice leaf extract described in claim 1 or 2 to post - harvested fruits and vegetables; wherein the total working concentration of the target substances vitexin - 5 - O - glucoside and isoorientin in the rice leaf extract is above 50 mg / L; The post - harvest fungi of fruits and vegetables are selected from: Alternaria alternata ( Alternaria alternata ), Botrytis cinerea ( Botrytis cinerea ), Penicillium expansum ( Penicillium expansum ), and / or Penicillium digitatum ( Penicillium digitatum ).

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