Application of saponin in inhibition of penicillium

Abstract

The invention relates to application of saponin in inhibition of penicillium. The saponin is at least one of oleanolic acid type saponin, hedera helix type saponin and phytolaccae acid type saponin. On the basis, the citrus preservative can be used for citrus preservation to improve the quality and prolong the shelf life of citrus so as to guarantee the commodity value of fruits.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bacteriostatic agents, in particular to the application of bacteriostatic agents in citrus preservation. BACKGROUND

[0002] Citrus penicillium disease can cause fruit rot, and is one of the most important postharvest diseases of citrus. It has a significant negative impact on the whole chain of storage, transportation and sales of citrus, seriously affecting the commodity quality and market value of the fruit, and causing direct economic losses to fruit farmers and related industries. The pathogen of this disease is Penicillium italicum Wehmer, which has strong infectivity and can multiply rapidly even at 10-20℃, making it easy to spread the disease. Penicillium italicum

[0003] In view of the above, it is urgent to find an efficient, environmentally friendly and effective preservation method for citrus penicillium disease. SUMMARY

[0004] One of the present application provides the application of saponins in inhibiting Penicillium italicum Wehmer or preserving citrus by inhibiting Penicillium italicum Wehmer, wherein the saponins are at least one of oleanolic acid type saponins (e.g., which can be an extract of Ligustrum lucidum), hedera type saponins (e.g., which can be an extract of Hedera helix) and dianthric acid type saponins (e.g., which can be an extract of Phytolacca acinosa). Penicillium Penicillium In one embodiment, the saponins are at least one of total saponins of Chenopodium quinoa, an extract of Ligustrum lucidum, an extract of Hedera helix and an extract of Phytolacca acinosa.

[0005] In one embodiment, the saponins are at least one of total saponins of Chenopodium quinoa, an extract of Ligustrum lucidum, an extract of Hedera helix and an extract of Phytolacca acinosa.

[0006] In one embodiment, the Penicillium italicum Wehmer is Penicillium italicum Wehmer. Penicillium italicum

[0007] In one embodiment, the citrus is at least one of orange, mandarin, grapefruit and orange.

[0008] In one embodiment, the saponins are a combination of oleanolic acid type saponins, hedera type saponins and dianthric acid type saponins.

[0009] In one embodiment, the saponins are total saponins of Chenopodium quinoa, wherein the total saponins of Chenopodium quinoa are a combination of oleanolic acid type saponins, hedera type saponins and dianthric acid type saponins in a mass ratio of 6:3:1.

[0010] The second aspect of the present application provides a method for preserving citrus, comprising the following steps: 1) preparing saponins into a total saponin solution of Chenopodium quinoa, wherein the saponins are at least one of oleanolic acid type saponins, hedera type saponins and dianthric acid type saponins; 2) soaking the citrus in the saponin solution and then taking it out to dry.​​​

[0011] In one specific embodiment, the saponin is at least one of quinoa total saponins, privet extract, ivy extract, and pokeweed extract.

[0012] In one specific embodiment, the citrus fruit is at least one of mandarin orange, tangerine, grapefruit, and orange.

[0013] In one specific embodiment, the saponin is a composition of oleanolic acid type saponin, ivy type saponin and phytolacca acid type saponin.

[0014] In one specific embodiment, the saponin is total saponin from quinoa.

[0015] In one specific embodiment, the concentration of the saponin in the saponin solution is greater than or equal to 20 mg / mL.

[0016] In one specific embodiment, the concentration of the saponin in the saponin solution is from 20 mg / mL to 40 mg / mL.

[0017] In one specific embodiment, the citrus fruit inhibits Penicillium (… Penicillium Preservation.

[0018] In one specific embodiment, the Penicillium is Penicillium italicum (… Penicillium italicum ).

[0019] In one specific embodiment, the saponin solution is an aqueous solution of saponin.

[0020] In one specific embodiment, the method further includes step 3): packaging the citrus fruits after drying.

[0021] The beneficial effects of this invention are as follows: This invention discovers that oleanolic acid saponins, ivy-type saponins, and phytolacca acid saponins have inhibitory effects on Penicillium, especially their combinations, such as total saponins from quinoa, which have a superior inhibitory effect on Penicillium. Based on this, they can be used for the preservation of citrus fruits to improve their quality and shelf life, thereby ensuring the commercial value of the fruit.

[0022] Citrus fruits have a relatively thick peel, a natural barrier that effectively blocks the bitterness of quinoa saponins, preventing them from penetrating the pulp and affecting the taste. Therefore, overall, saponin-based citrus preservation methods possess multiple advantages, including high efficiency, safety, and environmental friendliness, and have broad application prospects and high promotional value, potentially providing strong support for the sustainable development of the citrus industry.

[0023] In addition, a large amount of bran is produced in the processing of quinoa, which is usually discarded, undoubtedly causing waste of resources. Efficiently extracting total saponins from the bran can promote the full use of this resource, which has important resource recycling value. DETAILED DESCRIPTION

[0024] The above content of the present application is further described in detail in the form of preferred embodiments, but it does not constitute a limitation on the present application.

[0025] Unless otherwise specified, the reagents and other materials in the embodiments of the present application can be purchased through commercial channels.

[0026] The total saponin crude powder of quinoa was purchased from Ningxia Xiangcao Biotechnology Co., Ltd. The total saponin content of quinoa was 97wt%.

[0027] The components in the total saponin crude powder of quinoa were determined by high performance liquid chromatography, and it was found that the total saponin of quinoa was mainly composed of oleanolic acid type saponin, ivy type saponin and phytolaccagenic acid type saponin, and the mass ratio of the three was 6:3:1.

[0028] The extract of ligustrum lucidum was purchased from Xi'an Tianguangyuan Biotechnology Co., Ltd. Among them, 98wt% of the extract of ligustrum lucidum was oleanolic acid type saponin.

[0029] The extract of ivy was purchased from Shaanxi Snoot Biotechnology Co., Ltd. Among them, 96wt% of the extract of ivy was ivy type saponin.

[0030] The extract of phytolacca was purchased from Shanghai Linkamei Biotechnology Co., Ltd. Among them, 98wt% of the extract of phytolacca was phytolaccagenic acid type saponin.

[0031] Potato glucose agar medium (PDA): wash the potato, peel and cut into small pieces, weigh 200g, add 1000mL distilled water, boil for 30min, then filter with four layers of gauze, add 20g glucose to the filtrate, continue to heat and stir to mix, after cooling, supplement distilled water to 1000mL, add 15g agar powder, heat continuously with stirring until the agar is completely dissolved, sterilize at 121 degrees Celsius for 20min.

[0032] Penicillium italicum (P. Penicillium italicum ) CGMCC 3.4040 was purchased from China General Microbiological Culture Collection Center. Example 1: Bacteriostatic test of Penicillium italicum

[0033] The PDA culture medium was heated and melted, and different masses of quinoa total saponins, female privet fruit extract, ivy extract and pokeweed extract which had been dry heat sterilized (165 degrees Celsius for 2.5 hours) were added respectively under sterile operating conditions, and shaken well, so that the mass concentration of the quinoa total saponins, female privet fruit extract, ivy extract and pokeweed extract in the PDA was 2.5, 5, 10, 20 and 40 mg / mL respectively. The PDA culture medium containing each saponin was prepared by pouring into a sterile culture dish before the medium solidified.

[0034] The Italian Penicillium was inoculated on a PAD plate and cultured at 26 degrees Celsius for 2 days to activate, so as to obtain activated Italian Penicillium.

[0035] The activated Italian Penicillium with a diameter of 6 mm was inoculated in the central part of the PAD culture medium (blank control), PDA culture medium containing quinoa total saponins, PDA culture medium containing female privet fruit extract, PDA culture medium containing ivy extract and PDA culture medium containing pokeweed extract, and cultured at 26 degrees Celsius for 6 days. The concentration of each saponin in the culture medium was 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL and 40 mg / mL respectively. One plate was one repetition, and there were a total of 3 repetitions. The colony diameter was measured by the cross method, and then the relative inhibition rate of each saponin at different concentrations on the Italian Penicillium was calculated, and then the virulence regression equation of the same saponin at different concentrations on the Italian Penicillium was obtained, and the half-inhibitory concentration EC 50 was calculated based on the virulence regression equation. The relative inhibition rates of different concentrations of each saponin in the culture medium are shown in Table 1; the virulence regression equation, correlation coefficient and EC 50 of the antibacterial ability of each saponin on the Italian Penicillium are shown in Table 2. The calculation of the colony expansion diameter is shown in formula (1), and the relative inhibition rate is shown in formula (2).

[0036] Colony expansion diameter (mm) = colony diameter average value (mm) - (6 mm) Formula (1).

[0037] Relative inhibition rate = (blank control colony expansion diameter - treatment colony expansion diameter) x 100% / blank control colony expansion diameter Formula (2).

[0038] Table 1

[0039] Note: Lowercase letters represent significant differences at the 5% level.

[0040] According to the results of Table 1, the total saponins of quinoa, extract of ligustrum lucidum, extract of Hedera helix and extract of pokeweed all have inhibitory effect on the pathogenic bacteria of citrus penicillium; at the same concentration, the inhibitory effect of the total saponins of quinoa is significantly better than that of the extract of ligustrum lucidum, the extract of Hedera helix and the extract of pokeweed.

[0041] Table 2

[0042] Note: X is the logarithm with base 10 of the concentration of each saponin in the PAD medium, and Y is the probability unit (Probit value) of the relative inhibitory rate.

[0043] According to the results of Table 2, the EC 50 of the total saponins of quinoa is 2.34 times the EC 50 of the extract of ligustrum lucidum, 4.98 times the EC 50 of the extract of Hedera helix and 4.03 times the EC 50 of the extract of pokeweed, which is significantly smaller than the EC 50 of the extract of ligustrum lucidum, the extract of Hedera helix and the extract of pokeweed. Example 2: Preservative effect of total saponins of quinoa on navel oranges

[0044] The total saponins of quinoa subjected to dry heat sterilization (165 degrees Celsius for 2.5 hours) were dissolved in sterilized water to prepare total saponins of quinoa aqueous solutions with concentrations of 10 mg / mL, 20 mg / mL and 40 mg / mL, respectively.

[0045] 25% prochloraz emulsifiable concentrate was purchased from Andomax Hui Feng (Jiangsu) Co., Ltd., and was diluted with sterile water to 0.5 mg / mL prochloraz solution for use.

[0046] Uniform-sized, disease-free, mature, mechanically unharmed navel orange fruits were selected, washed with clean water, soaked in 1.0% sodium hypochlorite solution for 2 min, and then rinsed with flowing clean water for 3 times to ensure no sterilization water residue, and then dried at room temperature. A total of 10 mg / mL total saponins of quinoa aqueous solution, 20 mg / mL total saponins of quinoa aqueous solution, 40 mg / mL total saponins of quinoa aqueous solution, 0.5 mg / mL prochloraz solution (positive control) and sterilized water (negative control) were set up, with 4 repeats for each treatment, and 30 fruits in each repeat.

[0047] The navel orange fruits were soaked in the solutions of the 5 treatments for 2 min, and then dried in a sterilized clean bench. A 3 mm diameter and 3 mm deep wound was made on the equator of the navel orange fruit with a sterilized needle, and 15 microliters of 1×10 6The spore water suspension of Penicillium italicum CFU / mL was loaded into a sterile box after the wound holes were completely dried, 10 per box, and then packaged with a polyethylene plastic bag, and stored in an incubator at a temperature of 27±1 degrees Celsius and a relative humidity of 90% to 95%. The incidence of disease was observed at 3 days, 6 days, and 9 days, respectively. The incidence of disease was calculated based on formula (3), and the results are shown in Table 3.

[0048] Incidence of disease = (number of diseased fruits / total number of fruits) x 100% Formula (3).

[0049] Table 3

[0050] Note: Lowercase letters represent significant differences at the 5% level.

[0051] As can be seen from Table 3, the incidence of disease in the negative control group reached 56.7% at 3 days of storage, while the positive control group and different concentrations of total saponins of chenopodium did not develop disease. At 6 days of storage, the incidence of disease in the low concentration of total saponins of chenopodium was significantly higher than that in the medium and high concentrations of total saponins of chenopodium and the positive control group, and was significantly lower than that in the negative control group. At 9 days of storage, the incidence of disease increased, but the significant difference was the same as that at 6 days. The incidence of disease in the medium and high concentrations of total saponins of chenopodium was not significantly different from that in the positive control group. This shows that soaking navel oranges in 20 mg / mL total saponin solution of chenopodium can improve the disease resistance of the fruit after harvest and control the penicillium disease of navel oranges. Example 3: Effect of saponins on sensory evaluation of navel orange fruits

[0052] The total saponin powder of chenopodium, which had been subjected to dry heat sterilization (165 degrees Celsius for 2.5 hours), was dissolved in sterilized water to prepare a 40 mg / mL total saponin solution of chenopodium.

[0053] The extract of ligustrum lucidum, which had been subjected to dry heat sterilization (165 degrees Celsius for 2.5 hours), was dissolved in sterilized water to prepare a 40 mg / mL extract solution of ligustrum lucidum.

[0054] The extract of ivy, which had been subjected to dry heat sterilization (165 degrees Celsius for 2.5 hours), was dissolved in sterilized water to prepare a 40 mg / mL extract solution of ivy.

[0055] The extract of pokeweed, which had been subjected to dry heat sterilization (165 degrees Celsius for 2.5 hours), was dissolved in sterilized water to prepare a 40 mg / mL extract solution of pokeweed.

[0056] The uniform size, no disease and insect pests, maturity, no mechanical injury, and the navel orange fruits were washed with clean water, soaked in 1.0% sodium hypochlorite solution for 2 min, and then washed with flowing clean water for 3 times to ensure no disinfectant water residue, and then placed in a ventilated room at room temperature. A total of 40 mg / mL total saponins of Chenopodium quinoa water solution, 40 mg / mL extract of Ligustrum lucidum water solution, 40 mg / mL extract of Hedera helix water solution, 40 mg / mL extract of Daturae radix water solution, and sterilized water (negative control) were set up, 50 fruits were randomly selected for each treatment.

[0057] The navel orange fruits were soaked in the solution of the five treatments for 2 min, and then completely dried at room temperature. The fruits were placed in a fresh-keeping box with air holes, 10 fruits per box, and stored in the refrigerator (temperature 4±1 degrees Celsius, relative humidity 90% to 95%). The sensory evaluation of the fruits was carried out at 10 days.

[0058] The sensory evaluation of the fruits was carried out in the laboratory of Inner Mongolia Qingyuan Bioscience Technology Co., Ltd. Based on the requirements of 6.1 manual sensory evaluation in GH / T 1471-2024, 20 healthy, non-sensory defect, and citrus-consuming consumer representatives were recruited as sensory evaluators. The evaluators were trained for 10 min in advance, and the purpose, process, and question options of the test were explained.

[0059] During the sensory evaluation, the fruit peduncle and fruit bottom were cut off along the parallel direction, and only the equatorial part of the fruit with a thickness of 1.5 to 2 cm was reserved. The fruit was then divided into four equal parts along the meridian direction to form uniform petal samples. To reduce the interference of sample numbers on the test, all sample numbers were encoded with three random numbers, and the sample presentation was random. Each evaluator was presented with four petal samples in each round, a total of four rounds. The evaluators scored the smell and taste of the samples based on the 6.1 manual sensory evaluation in GH / T 1471-2024 (each index scored 100 points). In the manual sensory evaluation, the bitterness was added (no bitterness: 70 to 100 points; slightly bitter: 30 to 70 points; obvious bitterness: 0 to 30 points). The results are shown in Table 4.

[0060] Table 4

[0061] Note: Lowercase letters represent 5% level of significant difference.

[0062] From the results in Table 4, it can be seen that after treatment with total saponins of Chenopodium quinoa, extract of Ligustrum lucidum, extract of Hedera helix, and extract of Daturae radix, the cold storage for 10 days had no significant difference in smell and taste compared with the negative control. Example 4: Detection of saponin residues

[0063] The quinoa total saponins raw powder which was subjected to dry heat sterilization (165 degrees Celsius for 2.5 hours) was dissolved with sterilized water to prepare a quinoa total saponins water solution with a concentration of 40 mg / mL.

[0064] The lonicera extract which was subjected to dry heat sterilization (165 degrees Celsius for 2.5 hours) was dissolved with sterilized water to prepare a lonicera extract water solution with a concentration of 40 mg / mL.

[0065] The ivy extract which was subjected to dry heat sterilization (165 degrees Celsius for 2.5 hours) was dissolved with sterilized water to prepare an ivy extract water solution with a concentration of 40 mg / mL.

[0066] The pokeweed extract which was subjected to dry heat sterilization (165 degrees Celsius for 2.5 hours) was dissolved with sterilized water to prepare a pokeweed extract water solution with a concentration of 40 mg / mL.

[0067] Uniform size, no disease and insect pests, maturity, no mechanical injury, and no sterile water residue, the navel orange fruits were selected, washed with water, soaked in 1.0% sodium hypochlorite solution for 2 min, and then washed with flowing water for 3 times. After being dried at room temperature, 40 mg / mL quinoa total saponins water solution, 40 mg / mL lonicera extract water solution, 40 mg / mL ivy extract water solution, 40 mg / mL pokeweed extract water solution, and sterilized water (negative control) were set up, with 30 fruits in each treatment.

[0068] The navel orange fruits were soaked in the solutions of the five treatments for 2 min, and then dried at room temperature. After being dried, the fruits were put into fresh-keeping boxes with air holes, 10 fruits in each box, and stored in the refrigerator (temperature 4±1 degrees Celsius, relative humidity 90% to 95%) for 21 days.

[0069] Residual detection was performed at the 0th day, 14th day and 21st day of storage. The residual detection was performed as follows: 5 navel oranges were randomly selected from each treatment, respectively, the peel and pulp of each treatment were separated, and were cut into uniform small pieces, and were put into a pulverizer, and the peel or pulp was beaten into a paste to obtain the peel paste and pulp paste of each treatment; 5 g of the peel paste or pulp paste of each treatment was placed in a 150 mL centrifuge tube, 10 mL of acetonitrile was added, and after being mixed thoroughly, 10 mg of sodium chloride powder and 40 mg of anhydrous magnesium sulfate were added, and ultrasonic oscillation was performed for 2 min, and centrifugation was performed at 4500 rpm for 5 min to obtain the first peel supernatant or first pulp supernatant of each treatment; 6 mL of the first peel supernatant or first pulp supernatant of each treatment was taken in a 10 mL centrifuge tube, 10 mg of anhydrous magnesium sulfate and 20 mg of Quattro filler purification adsorbent were added, ultrasonic oscillation was performed for 2 min to mix thoroughly, and centrifugation was performed at 4500 rpm for 5 min to obtain the second peel supernatant or second pulp supernatant of each treatment; 2 mL of the second peel supernatant or second pulp supernatant of each treatment was filtered through a 0.22 micron filter membrane to obtain the peel purified liquid or pulp purified liquid of each treatment for chromatographic detection; oleanolic acid, hederagenin and pokosanton were used as standard products, and dilute solutions of the standard products were prepared using methanol; the chromatographic detection conditions were as follows: a chromatographic column, a mobile phase of acetonitrile + 0.02% phosphoric acid solution (volume ratio of 32:68); detection wavelength of 203 nm; flow rate of 1.0 mL / min; column temperature of 30 degrees Celsius; injection volume of 5 microliters. The mass concentration of saponin in each purified liquid was calculated based on formula (4), and the content (residual amount) of saponin in the peel or pulp was calculated based on formula (5), and the results are shown in Table 5. 18 a chromatographic column, a mobile phase of acetonitrile + 0.02% phosphoric acid solution (volume ratio of 32:68); detection wavelength of 203 nm; flow rate of 1.0 mL / min; column temperature of 30 degrees Celsius; injection volume of 5 microliters. The mass concentration of saponin in each purified liquid was calculated based on formula (4), and the content (residual amount) of saponin in the peel or pulp was calculated based on formula (5), and the results are shown in Table 5.

[0070] C1=A1C2 / A2 (4).

[0071] wherein C1 is the mass concentration of saponin in each purified liquid, expressed in mg / L; C2 is the mass concentration of saponin in the saponin standard solution, expressed in mg / L; A1 is the average of the peak areas of saponin in the two purified liquids; and A2 is the average of the peak areas of saponin in the two saponin standard solutions.

[0072] C3=C1xVxn / m (5).

[0073] wherein C3 is the residual amount of saponin in the peel or pulp, expressed in mk / kg; C1 is the mass concentration of saponin in each purified liquid, expressed in mg / L; V is the volume of the extraction solution, V=10 mL; and n is the dilution factor, n=1.

[0074] Table 5

[0075] The National Food Safety Standard GB2763-2021, "Maximum Residue Limits for Pesticides in Food," stipulates that the maximum residue limits for prochloraz and imazalil, widely used for preserving citrus fruits, in citrus fruits are 5 mg / kg. Based on this standard, the residues of saponins used in this invention in the peel or pulp of navel oranges comply with the National Food Safety Standard GB2763-2021.

[0076] Quinoa originated in the Andes Mountains of South America and was once a staple food of the indigenous people. Modern scientific research shows that quinoa is suitable for a wide range of people due to its rich nutritional content. Therefore, quinoa is considered a relatively safe food. Furthermore, the article "Research Progress on Quinoa Saponins" by Huo Junqi et al. points out that quinoa saponins are mainly distributed in the quinoa pericarp and seeds, primarily including oleanolic acid, ivy-type, and phytolacca-type, with sweet quinoa containing 200 to 400 mg / kg and bitter quinoa containing 1130 mg / kg. The residual amount of saponins used in this invention in the peel or pulp of navel oranges is far lower than the original content in the quinoa pericarp and seeds, therefore it is safe for humans.

Claims

1. Saponins inhibit Penicillium (… Penicillium ) or by inhibiting Penicillium ( Penicillium The application of saponins in the preservation of citrus fruits, wherein the saponins are at least one of oleanolic acid type saponins, ivy type saponins and phytolacca acid type saponins.

2. The application according to claim 1, characterized in that, The saponins are at least one of the following: total saponins from quinoa, privet fruit extract, ivy extract, and pokeweed extract; and / or The Penicillium species mentioned is Penicillium italicum ( ) Penicillium italicum ); and / or The citrus fruit is at least one of mandarin orange, tangerine, grapefruit, and orange.

3. The application according to claim 1, characterized in that, The saponins are a combination of oleanolic acid type saponins, ivy type saponins and phytolacca acid type saponins.

4. The application according to claim 3, characterized in that, The saponins are total saponins from quinoa.

5. A method for preserving citrus fruits, comprising the following steps: 1) Prepare a saponin solution by preparing the saponin, wherein the saponin is at least one of oleanolic acid type saponin, ivy type saponin and phytolacca acid type saponin; 2) Soak the citrus fruits in a saponin solution, then remove and air dry.

6. The method according to claim 5, characterized in that, The saponins are at least one of the following: total saponins from quinoa, privet fruit extract, ivy extract, and pokeweed extract; and / or The citrus fruit is at least one of mandarin orange, tangerine, grapefruit, and orange.

7. The method according to claim 5, characterized in that, The saponins are a combination of oleanolic acid type saponins, ivy type saponins and phytolacca acid type saponins.

8. The method according to claim 5, characterized in that, The saponins are total saponins from quinoa.

9. The method according to claim 5, characterized in that, The concentration of the saponin in the saponin solution is greater than or equal to 20 mg / mL.

10. The method according to claim 5, characterized in that, The concentration of the saponin in the saponin solution is from 20 mg / mL to 40 mg / mL.

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