Application of compound salimusk in bacteriostasis and preservation of strawberries

Abstract

The invention discloses an application of a compound salimusk in bacteriostasis and preservation of strawberries, starting from botryis cinerea which is a main pathogenic bacterium after strawberry fruits are picked, and the bacteriostasis effect of the salimusk on the botryis cinerea is studied. The saxellik belongs to important natural musk, contains terpene and alcohol as main components, and has biological activities such as antibacterial activity, anti-inflammatory activity and anti-oxidation activity. Experiments show that the salimusk can effectively inhibit mycelial growth and spore germination. And the destroying effect of the salimusk on fungal spores is deeply explored by observing the microstructure of the tested strain. The strawberry fresh-keeping agent can effectively delay reduction of weight and hardness and nutrient loss in the strawberry storage process, and is good in fresh-keeping effect. Results show that the salimusk has the potential to replace existing chemical bactericides, and a theoretical basis is provided for development of novel antibacterial preservatives.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial and fresh-keeping of fruits and vegetables, and relates to application of a compound salvinia in antibacterial and fresh-keeping of strawberries and an application method thereof. Background Art

[0002] Botrytis cinerea is a filamentous ascomycete in the family Sclerotiniaceae and the genus Sporodiscus. Botrytis cinerea has the characteristics of rapid reproduction, strong adaptability to the environment and a certain incubation period. Its conidia can enter the host fruits and vegetables through various pathways and complete the initial infection process. With the extension of transportation and storage time, a large number of gray molds will appear on the surface and inside of the fruit, growing rapidly in a short period of time and causing the fruit to rot. The surface of its sclerotium is coated with a blackened glucan hard film, which can resist ultraviolet radiation, dryness and microbial infection in the external environment, so that it can remain active under extreme conditions. Botrytis cinerea can cause gray mold in fruits and vegetables, which is the most common disease in fruits and vegetables. Compared with other postharvest pathogenic fungi, Botrytis cinerea has the characteristics of high reproduction rate, short life cycle and large genetic variation. Chemical control is the most common method for the preservation of fruits and vegetables against Botrytis cinerea. Commonly used chemical control drugs include dimethylimide, cyclohexylamine, benzimidazole and pyrimethanil, among which pyrimethanil has been verified and successfully used in fruit and vegetable preservation. The use of traditional chemical preservatives is highly efficient and has good antibacterial effects, but long-term and large-scale use of traditional fungicides can cause pathogens to develop drug resistance and reduce the control effect, and the residues of fungicides will have adverse effects on human health and the environment. Therefore, it is urgent to develop new natural, green, and pollution-free post-harvest fruit and vegetable disease control and anti-fungal preservation technologies.

[0003] Salix musk is an important natural musk, originally extracted from the outer protection of the Asian musk deer. The main components of Salix musk are terpenes and alcohols, which are non-toxic and will not enter the human body and be absorbed by the internal organs, making it safe to use. Salix musk has biological activities such as antibacterial, anti-inflammatory and antioxidant, and is also widely used as a fragrance-preserving and fixative in modern daily chemical products. Salix musk is used in some traditional prescriptions, and has the effects of calming the liver and regulating qi, promoting blood circulation and removing blood stasis, and has great market potential. Salix musk is generally considered to be a fairly safe compound and is widely used in many products.

[0004] Strawberry is a perennial herbaceous plant of the genus Fragaria in the Rosaceae family. Strawberry has high nutritional value and is rich in amino acids, vitamins, minerals and other nutrients, which are beneficial to human health. Strawberries are mainly eaten fresh, and some are used for processing, and are deeply loved by people. However, strawberries have high respiration intensity and are susceptible to mechanical damage. During storage, they are susceptible to erosion and decay by Botrytis cinerea, Anthocyaninus gloeosporioides and other fungi. Resisting the invasion of microorganisms and delaying their own metabolism are the main methods for preserving strawberries. Therefore, finding and developing a safe and efficient preservative is of great significance for the storage and preservation of strawberries. The application of saliforn in the process of strawberry preservation significantly prolongs the storage period of strawberries, proving that saliforn can be used as a potential fruit and vegetable preservative for subsequent application development. Summary of the invention

[0005] In view of the above shortcomings, the present invention provides an application of a natural compound, salvinia, in inhibiting fruit and vegetable fungi. Starting from the main pathogenic bacteria of post-harvest strawberry fruits, the antibacterial effect of salvinia on them is studied.

[0006] In order to achieve the above object, the technical solution of the present invention is to use a compound, salvinia, in inhibiting fungi in fruits and vegetables.

[0007] Furthermore, the compound salvinia of the present invention is used in inhibiting Botrytis cinerea.

[0008] Furthermore, the concentrations of the compound Salix salix that inhibit Botrytis cinerea are 80 ug / mL, 96 ug / mL, 112 ug / mL, and 128 ug / mL, respectively.

[0009] The natural compound Salix sargentii of the present invention has a significant inhibitory effect on Botrytis cinerea (IC50 is 64.303ug / mL). Salix sargentii is an important natural musk, the main components of which are terpenes and alcohols, and has biological activities such as antibacterial, anti-inflammatory and antioxidant. In view of the residual harm and resistance disadvantages of current chemical fungicides in the prevention and control of fruit and vegetable diseases, the present invention conducted an antibacterial test on Botrytis cinerea with Salix sargentii, observed the mycelial morphology, spore germination and the microstructure of the tested strains, and deeply explored the destructive effect of Salix sargentii on fungal spores. In addition, the present invention can effectively reduce the weight loss rate, hardness loss and nutritional loss during the storage of strawberries, and has a good preservation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The mycelial disc growth diagram of Botrytis cinerea after the compound Salix sargentii was used

[0011] Figure 2 This is the inhibition rate of mycelial growth of Botrytis cinerea after the compound Salisburymus acted on it

[0012] Figure 3 This is the mycelial growth state of Botrytis cinerea after the compound Salix sargentii acts on it

[0013] Figure 4 The graph shows the inhibition rate of spore germination of Botrytis cinerea after the compound Salix sargentii acts on it

[0014] Figure 5 The spore germination state of Botrytis cinerea after the compound Salix sarmentosum acts on it

[0015] Figure 6 Ultrastructure of spores of Botrytis cinerea after the compound Salisbury's musk was applied

[0016] Figure 7 Comparison of strawberry fruit status under different treatment methods

[0017] Figure 8 Comparison of the weight loss rate of strawberry fruit under different treatment methods

[0018] Fig. 9 Comparison of changes in strawberry fruit hardness under different treatment methods

[0019] Fig.10 Comparison of the changes in vitamin C in strawberry fruit under different treatment methods DETAILED DESCRIPTION

[0020] In order to further explain the technical methods and effects adopted by the present invention to achieve the predetermined purpose, the specific operation mode and effect results of the present invention are described in detail below in combination with the accompanying drawings and embodiments.

[0021] Embodiment 1:

[0022] Step 1: Preparation of Salix musk liquid

[0023] Salisbury musk was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 20 mg / mL, and stored in a refrigerator at 4°C away from light for later use. When used, it was diluted with potato dextrose broth (PDB) to prepare the required concentration gradient.

[0024] Step 2: Preparation and cultivation of Botrytis cinerea

[0025] The autoclaved potato dextrose agar PDA medium was cooled to 55°C and poured onto a plate until the medium solidified. The Botrytis cinerea bacterial disc was inoculated onto the PDA plate and cultured in a constant temperature incubator at 28°C for later use.

[0026] Step 3: Preparation of Botrytis cinerea spore suspension

[0027] After 7 days of culture, the Botrytis cinerea plate was added with sterile distilled water, and the hyphae were scraped by streaking method. After filtering through four layers of sterile gauze, the spore suspension was obtained. The spore suspension was counted under an optical microscope, and the concentration of the spore suspension was adjusted to 1×10 6 Pieces / mL.

[0028] Example 2

[0029] Step 1: Salicornia inhibits the growth of Botrytis cinerea mycelium IC 50 Determination of value

[0030] The saliforn liquid was fully mixed with the PDA medium according to the concentration gradient. After the medium solidified, a 5mm diameter bacterial cake was inoculated into the center of the plate. The medium plate without the liquid was used as a blank control. The culture was cultured at 28°C until the mycelium of the blank control group covered the culture dish. Three parallel tests were performed for each group. The mycelium growth diameter was measured by the cross method, and the mycelium growth inhibition rate was calculated according to the formula: mycelium inhibition rate (%) = 1-(drug group diameter-bacteria cake diameter) / (control group diameter-bacteria cake diameter) × 100%. The IC50 value of saliforn inhibiting the mycelium growth of Botrytis cinerea was calculated using SPSS software to be 64.303ug / mL. Figure 1 The changes in the morphology of the fungus cake during the growth of Botrytis cinerea under the influence of different concentrations of salvinia were revealed. With the increase of salvinia concentration, the fungus disk significantly aggregated and shrank, and the growth was inhibited.

[0031] Example 3

[0032] Step 1: Observation on the inhibition of Botrytis cinerea hyphae growth by Salix salsa

[0033] The experimental process is the same as in Example 2. The changes in mycelial growth were observed under a microscope. Figure 3 ,according to Figure 1-3 It can be seen that the inhibitory effect of salvinia on the tested strains is very obvious. The higher the concentration, the greater the degree of inhibition on mycelial growth. When the concentration of salvinia increases to 64μg / mL, mycelium begins to tend to aggregate, the thickness of the cake gradually decreases, and the length of mycelium is shortened. When the concentration is 96ug / mL, the inhibition rate of mycelial growth of Botrytis cinerea reaches 63.07%. These results show that salvinia can damage the mycelium of Botrytis cinerea, thereby inhibiting its growth.

[0034] Step 2: Observation of the inhibition of spore germination of Botrytis cinerea by Salix sarmentosus

[0035] The drug and spore suspension were mixed, inoculated on a glass slide, covered with a cover slip, and cultured in a constant temperature incubator. The spore germination changes were observed under a microscope. Figure 5 .Depend on Figure 4It was found that as the drug concentration increased, the inhibition rate of spore germination increased significantly. When the drug concentration was 128ug / mL, the inhibition rate of spore germination was close to 100%. Figure 5 It can be seen that compared with the control group, after intervention with a concentration of 128ug / mL of the drug, the spores almost did not germinate after 8h.

[0036] Example 4

[0037] Step 1: Observation of the ultrastructure of Botrytis cinerea after treatment with Salix sargentii

[0038] Dilute the salvinia with PDA medium to a medium solution with a concentration of 96μg / mL and pour it into the culture dish. Use PBS buffer instead of the salvinia solution as a blank control. After the culture medium solidifies, insert a 5mm diameter bacterial cake into the center of the plate and culture it in a 28℃ biochemical incubator for 3 days. Cut the cultured bacterial cake and wash it 2-3 times with PBS buffer, centrifuge it at 3000r / min for 20min, and use a transmission electron microscope to observe the changes in the ultrastructure of the fungus surface after treatment. Figure 6 It can be seen that most of the Botrytis cinerea in the blank control group were in a normal state, with complete cell walls, complete and clear cell membranes, nuclei and other organelles. After being treated with salvinia, the edges of the hyphae cell membranes began to become rough and not smooth, and serious cytoplasm leakage occurred in the cells, the number of intracellular vacuoles increased, and cavitation occurred; the nuclei and organelles of some Botrytis cinerea dissolved and disappeared, and the cytoplasm became turbid. These results further illustrate from a microscopic level that salvinia treatment can destroy the cell structure of Botrytis cinerea, leading to cytoplasmic exudation, and thus produce an inhibitory effect on Botrytis cinerea.

[0039] Example 5

[0040] Step 1: Application of Salix sarmentosum to inhibit the growth of Botrytis cinerea and preserve freshness

[0041] In order to prove the specific preservation effect of Salix sarmentosum on Botrytis cinerea in strawberry, a strawberry model infected with Botrytis cinerea was established. Botrytis cinerea was inoculated in PDA medium and cultured in a biochemical incubator at 28°C for 7 days. Sterilized deionized water was added to the bacterial plate to obtain a spore suspension, and the concentration of the spore suspension was adjusted to 1×10 7 / mL. Select ripe strawberry fruits, soak the selected strawberries in sodium hypochlorite aqueous solution for 3 minutes, then soak them in distilled water for 5 minutes, and drain the water in a clean bench to ensure that no other bacteria grow on the surface of the strawberries. Stab the largest equatorial surface of the strawberry and inoculate 10μL of Botrytis cinerea spore suspension with a sterile needle. Each strawberry is inoculated with three wounds as the test strawberry. Carry out the following 4 groups of fresh-keeping tests, observe the strawberry conditions every 48 hours and take samples to measure the relevant evaluation indicators of strawberries.

[0042] Experimental group 1 (blank control group): The tested strawberries were not treated and stored at room temperature.

[0043] Experimental group 2 (negative control group): The strawberries were immersed in a 1% citric acid aqueous solution with a pH of 3, in order to adjust the pH to make it closer to the internal environment of the strawberries to ensure the preservation effect. After soaking for 60 seconds, the strawberries were drained and used as a negative control.

[0044] Test group 3 (64 μg / mL Salix musk group): Salix musk was dissolved in 1% citric acid aqueous solution (pH=3), and 1% glycerol was added to fully dissolve to prepare a Salix musk antibacterial agent with a concentration of 64 μg / mL. The tested strawberries were immersed in the prepared Salix musk antibacterial agent for 60 seconds, and then the strawberries were drained to dry their surfaces;

[0045] Experimental group 4 (128 μg / mL Salix musk group): Salix musk was dissolved in 1% citric acid aqueous solution (pH=3), and 1% glycerol was added to prepare a Salix musk antibacterial agent with a concentration of 128 μg / mL after sufficient dissolution. Strawberries were immersed in the prepared Salix musk antibacterial agent for 60 seconds and then the water was drained;

[0046] Record the state of the tested strawberries within 8 days, and record the appearance, smell and spoilage of each group of strawberries every 48 hours. Figure 7 The fruits of test groups 1 and 2 showed signs of mildew, darkening, and severe softening on the 4th day. The fruits of test groups 3 and 4 showed no signs of rot on the 4th day, but showed signs of mildew, darkening, and severe softening on the 6th day, indicating that the growth of Botrytis cinerea spores in the tested strawberries was significantly inhibited after treatment with Salix salicifolia.

[0047] The weight loss of each group of strawberries was measured by weighing method, and the measurement was carried out every 48 hours. The weight loss rate of strawberries was calculated according to the following formula: Strawberry weight loss rate (%) = (M0-M1) / M0×100% (M0: original weight of strawberries; M1: weight of strawberries during storage). Figure 8 It can be seen that the weight loss rate of strawberries showed an increasing trend with the extension of storage time. In contrast, the treatment with salsa muskrat played a role in inhibiting weight loss.

[0048] The change of strawberry hardness was measured by texture analyzer every 48 hours. The TPA texture analysis mode was selected in the texture analyzer. The probe model was TA44, the test speed was 1mm / s, the probe test distance was 5mm, and the trigger point load was 5g. During the fruit storage process, the hardness gradually decreased with the extension of time, which reflects the freshness of the fruit to a certain extent. Fig. 9The hardness of strawberries in the control group decreased the most, while the treatment with the drug-added group had a good delaying effect on the decrease in hardness of strawberries, among which the hardness of strawberries in test group 4 decreased the slowest.

[0049] The vitamin C content was mainly determined using the Nanjing Jiancheng Vitamin C (VC) / Ascorbic Acid (ASA) Assay Kit, with measurements taken every 48 hours. Fig.10 It clearly shows that the vitamin C content of each group showed a downward trend over time. Relatively speaking, the strawberries treated with Salix sarmentosum showed a higher level of vitamin C content.

[0050] The present invention observes the influence of salifon on the morphology of Botrytis cinerea hyphae and the spore germination state by an inverted microscope. The results show that with the increase of salifon concentration, the growth of Botrytis cinerea hyphae and spore germination are significantly inhibited. When the concentration of salifon is 96ug / mL, the inhibition rate of hyphae growth reaches 63.07%, and the IC50 value of the drug to Botrytis cinerea is 64.303μg / mL. Through transmission electron microscopy, it is further observed and explained from a microscopic level that salifon treatment can destroy the cell structure of Botrytis cinerea, leading to cytoplasmic exudation, thereby causing the decay and death of the fungus, and producing an inhibitory effect on Botrytis cinerea. And salifon is applied to the Botrytis cinerea infection model of strawberry, and the antibacterial and fresh-keeping effect of salifon in actual preservation is explored. The results show that compared with the blank group and the negative control group, the drug-added group has a significant inhibitory effect on the growth of Botrytis cinerea in strawberry, and delays the decline of the weight and hardness of strawberry, as well as the loss of nutritional value. The above experimental results show that salvinia is a compound with obvious inhibitory effect on fruit and vegetable fungi, and has the potential to become a new antibacterial agent that can replace chemical fungicides.

Claims

1. Application of a compound Salix salix in antibacterial and fresh-keeping of strawberries, characterized in that The compound salvinia is used in inhibiting the strawberry fungus Botrytis cinerea.

2. The use of a compound Salix salix in the antibacterial and fresh-keeping of strawberries according to claim 1, characterized in that The inhibitory concentrations of salvinia salicornia to Botrytis cinerea were 80ug / mL, 96ug / mL, 112ug / mL, and 128ug / mL.

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