A pre-harvest spraying fresh-keeping agent for kiwifruit normal temperature storage and a preparation method and application thereof

Abstract

The present application relates to the field of agricultural product storage and preservation technology, in particular to a pre-harvest spraying preservative for kiwi fruit normal temperature storage and a preparation method and application thereof. The pre-harvest spraying preservative comprises the following components in the amount: 100-200 mg / L of dihydromyricetin, 1x10 7 -1x10 9 CFU / mL of Sphingobium multivorum J8. It is found that dihydromyricetin has no significant inhibitory effect on Sphingobium multivorum J8 in a specific concentration range, but can reduce the damage of environmental stress to Sphingobium multivorum J8 by absorbing ultraviolet rays and scavenging free radicals, which is conducive to the survival of Sphingobium multivorum J8 on the fruit surface, thereby producing a synergistic effect in prolonging the normal temperature shelf life of kiwi fruit and maintaining the organic acid content.

Description

Technical Field

[0001] This invention relates to the field of agricultural product storage and preservation technology, specifically to a pre-harvest spray preservative for room temperature storage of kiwifruit, its preparation method, and its application. Background Technology

[0002] kiwi( Actinidia (spp.) are rich in vitamin C and organic acids, but are climacteric fruits, making them highly dependent on the cold chain. After harvesting and leaving the cold chain, the fruit is prone to softening and rotting at room temperature, especially susceptible to soft rot (mainly caused by *Botrytis cinerea* and gray mold), leading to a shortened shelf life and increased storage and logistics costs.

[0003] Existing kiwifruit preservation technologies include post-harvest preservation methods, such as Chinese patents CN115820490A and CN116941665A. However, post-harvest preservation technologies still have the following problems: (1) Post-harvest treatment is difficult to control latent infection in the field: Pathogens of diseases such as kiwifruit soft rot often invade the fruit through the style and lenticels during the full bloom and young fruit stages and remain in a latent state. Post-harvest treatment can only act on the surface of the fruit peel, and its control effect on pathogens that have invaded deep into the pulp is limited. (2) Pre-harvest spraying of biocontrol bacteria is prone to inactivation: When live bacteria are directly sprayed on the surface of the fruit in the field, environmental stresses such as ultraviolet radiation, high temperature and drought will cause the bacteria to die rapidly, and the field control effect is unstable. (3) Limited effect of single component: Antibacterial agents are easily photo-oxidized and degraded under strong light in the field; at the same time, it is generally believed that substances with broad-spectrum antibacterial activity (such as dihydromyricetin) may inhibit the growth and activity of bacteria when used in combination with live bacteria, thus limiting the combined application of the two.

[0004] In view of the above-mentioned problems, the present invention provides a pre-harvest spray preservative for room temperature storage of kiwifruit, its preparation method and application. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a pre-harvest spray preservative for room temperature storage of kiwifruit, its preparation method, and its application. The aim is to address the problems in existing technologies where post-harvest treatment cannot effectively control latent infection, pre-harvest spraying of biocontrol bacteria is prone to inactivation, and single-component agents have limited effectiveness.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: Firstly, a pre-harvest spray preservative for room-temperature storage of kiwifruit, wherein the pre-harvest spray preservative comprises the following components in appropriate amounts: a concentration of 100-200 mg / L dihydromyricetin, and a viable bacteria concentration of 1×10⁻⁶. 7 -1×10 9 CFU / mL Sphingomyelin-J8

[0007] Based on the above technical solution, the present invention can be further improved as follows.

[0008] Furthermore, the pre-harvest spray preservative includes the following components in appropriate amounts: a concentration of 100-200 mg / L dihydromyricetin and a viable bacteria concentration of 1×10⁻⁶. 8 CFU / mL Sphingomyelin-J8

[0009] Furthermore, the pre-harvest spray preservative also includes the following component in terms of dosage: Tween-80 at a volume percentage of 0.05% (v / v).

[0010] Secondly, a method for preparing a pre-harvest spray preservative for room temperature storage of kiwifruit includes the following steps: first, dissolving dihydromyricetin in an alcohol solvent, then diluting it with water, and then adding Sphingomyelin-J8 suspension and Tween-80 to obtain the pre-harvest spray preservative.

[0011] Furthermore, the alcohol solvent includes ethanol; The volume fraction of the alcohol solvent in the pre-harvest spray preservative is ≤0.5%.

[0012] Furthermore, the *Sphingospora polyphaga* J8 suspension is prepared through the following steps: *Sphingospora polyphaga* (… Sphingobacterium multivorum After activation, Sphingomyelin-J8 strain was cultured with shaking, centrifuged to collect Sphingomyelin-J8, and resuspended in sterile water to obtain a suspension of Sphingomyelin-J8.

[0013] Thirdly, a method for preserving kiwifruit at room temperature by spraying a preservative before harvest includes the following steps: (1) During the critical growth period of kiwifruit, spray the leaves and fruits of the plant with the pre-harvest preservative for normal temperature storage of kiwifruit; the critical growth period includes the flowering period, the young fruit period, the fruit enlargement period and the ripening period. (2) Manage the fruit according to routine procedures until it matures, then harvest and store it at room temperature. Here, managing the fruit according to routine procedures until it matures is the conventional management method in this field.

[0014] Furthermore, the spraying in step (1) specifically involves: choosing to carry out the spraying on a sunny evening or in a rainless weather, so that the pre-harvest preservative for room temperature storage of kiwifruit remains on the plant leaves and fruit surface for at least 4 hours.

[0015] Furthermore, the harvesting time in step (2) is when the soluble solids content of the kiwifruit reaches 6.8%-7.5%.

[0016] Furthermore, the storage at room temperature in step (2) specifically refers to storing the harvested product at a temperature of 20-25℃ and a relative humidity of 85%-95%.

[0017] This invention found that dihydromyricetin has no significant inhibitory effect on Sphingomyelin-J8 within a specific concentration range. On the contrary, it can reduce the damage of environmental stress to J8 by absorbing ultraviolet light and scavenging free radicals, which is conducive to its survival on the fruit surface. This results in a synergistic effect in extending the shelf life of kiwifruit at room temperature and maintaining the organic acid content.

[0018] The beneficial effects of this invention are: (1) Comparative advantages of pre-harvest and post-harvest treatment: The present invention utilizes pre-harvest spraying of preservative compound treatment to intervene during the critical window period of pathogen invasion, especially during the flowering period and young fruit period when pathogens are prone to invade. The compound spraying can kill free pathogens in the field and occupy the ecological niche on the fruit surface, which is more effective than post-harvest soaking treatment.

[0019] (2) Synergistic reduction of decay rate: The present invention utilizes pre-harvest spraying of preservative compound treatment to kill free pathogens in the field and occupy ecological niches on the fruit surface, thus blocking latent infection; the data from the examples show that the overall effect of compound treatment is better than any single treatment.

[0020] (3) Significantly delays the respiratory climax: The present invention utilizes pre-harvest spraying of preservative compound treatment to not only reduce the respiratory intensity, but also significantly delay the respiratory peak of kiwifruit at room temperature from the 3rd day to the 9th day, thus prolonging the fruit firmness maintenance time from a physiological mechanism perspective.

[0021] (4) Maintaining flavor quality during normal storage: This invention utilizes pre-harvest spraying of preservative compound treatment to delay the consumption of titratable acid (TA) and better maintain the sweet and sour flavor of kiwifruit. Attached Figure Description

[0022] Figure 1 The effects of different treatments on the weight loss and decay rate of 'Jinyan' kiwifruit during postharvest storage at room temperature were investigated; where A represents the change in weight loss and B represents the change in decay rate. Figure 2 The effects of different treatments on the quality of 'Jinyan' kiwifruit during postharvest storage at room temperature were investigated. Among them, A represents the change in firmness, B represents the change in respiration intensity, C represents the change in soluble solids (SSC), and D represents the change in titratable acid (TA). Detailed Implementation

[0023] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0024] Description of the source of materials and reagents: Polyphagocytosine bacillus ( Sphingobacterium multivorum J8, with accession number CGMCC No. 25711, details can be found in patent application CN115820490A regarding *Sphingosine monocytogenes* (J8). Sphingobacterium multivorum J8 records.

[0025] Example 1: Preparation of preservative sprayed before harvest.

[0026] A method for preparing a pre-harvest preservative for room temperature storage of kiwifruit includes the following steps: (1) Preparation of strains and reagents: Polyphagocytosine bacillus ( Sphingobacterium multivorum J8, dihydromyricetin (DMY), purity ≥98%.

[0027] (2) Preparation of preservatives for pre-harvest spraying: Sphingosporobacter pylori J8 was inoculated into LB liquid medium and cultured with shaking. The cells were collected by centrifugation and resuspended in sterile water to obtain a Sphingosporobacter pylori J8 suspension. DMY was weighed, dissolved in a small amount of anhydrous ethanol, diluted with sterile water, and then added to the Sphingosporobacter pylori J8 suspension and 0.05% (v / v) Tween-80. The mixture was thoroughly mixed to obtain a composite solution, which is the pre-harvest preservative. The final concentration of dihydromyricetin in the composite solution was 100 mg / L, and the viable cell concentration of Sphingosporobacter pylori J8 was 1×10⁻⁶. 8 CFU / mL, ethanol volume fraction ≤0.5%.

[0028] Example 2 Compared with the example, everything is the same as in Example 1 except for the following: the final concentration of dihydromyricetin in the composite solution is 200 mg / L, and the viable bacterial concentration of Sphingosine monocytogenes J8 is 1×10⁻⁶. 7 CFU / mL, ethanol volume fraction ≤0.5%.

[0029] Example 3 Compared with the example, everything is the same as in Example 1 except for the following: the final concentration of dihydromyricetin in the composite solution is 150 mg / L, and the viable bacterial concentration of Sphingomyelin-J8 is 1×10⁻⁶. 9 CFU / mL, ethanol volume fraction ≤0.5%.

[0030] Comparative Example 1 Compared with the example, everything is the same as in Example 1 except for the following: the final concentration of dihydromyricetin in the composite solution is 50 mg / L, and the viable bacterial concentration of Sphingosine monocytogenes J8 is 1×10⁻⁶. 8 CFU / mL, ethanol volume fraction ≤0.5%.

[0031] Comparative Example 2 Compared with the example, everything is the same as in Example 1 except for the following: the final concentration of dihydromyricetin in the composite solution is 500 mg / L, and the viable bacterial concentration of Sphingomyelin-J8 is 1×10⁻⁶. 8 CFU / mL, ethanol volume fraction ≤0.5%.

[0032] Experimental Example 1: Comparison of the effects of different treatments on the room temperature preservation of 'Jinyan' kiwifruit.

[0033] 1. Experimental design.

[0034] (1) Select 'Jinyan' kiwifruit plants with uniform vigor and set up the following 5 treatment groups: ①CK (control group): Sterile water (containing 0.05% Tween-80) was sprayed at four pre-harvest stages. ② Single DMY pre-harvest group: DMY solution (100 mg / L + 0.05% Tween-80) was sprayed at four pre-harvest stages. ③ Single J8 pre-harvest group: Spray with Sphingomyelin-Polyphagenin J8 solution (1×10⁻⁶) at four pre-harvest stages. 8 CFU / mL + 0.05% Tween - 80); ④ Postharvest composite group: No preharvest treatment, postharvest soaking in a composite solution (DMY 100 mg / L + viable concentration of Sphingomyelin-J8 1×10⁻⁶). 8 CFU / mL + 0.05% Tween (80°C) for 5 minutes; ⑤ Pre-harvest compound group I (this invention): Spraying a compound solution (DMY 100 mg / L + viable concentration of Sphingosine monocytogenes J8 of 1×10⁻⁶) at four pre-harvest stages. 8 CFU / mL + 0.05% Tween - 80).

[0035] Specifically, the four pre-harvest spraying stages are: during the full bloom, young fruit, fruit enlargement, and ripening stages of kiwifruit, choose a sunny evening or rainless day to evenly spray the above compound solution onto the plant leaves and fruits until dripping, ensuring that the solution remains on the surface for at least 4 hours.

[0036] (2) Storage and testing: Harvesting was carried out without damage when the soluble solids content of the fruit reached 6.8%–7.5%. After harvesting, all fruits in all treatment groups were stored at room temperature (20–25℃), and relevant indicators were measured periodically. Each group had 3 replicates, with 30 fruits in each replicate.

[0037] 2. Experimental results.

[0038] (1) Weight loss and decay rate during normal temperature storage.

[0039] The softening, juice leakage, or rotting of kiwifruit was used as the criteria for judgment. The rotting rate was calculated using the formula: Rotting rate % = Number of rotten fruits / Total number × 100. The weight loss was calculated by weighing, with 20 randomly selected kiwifruits labeled sequentially. The weight of the fruits was measured every 3 days, and the weight loss rate was calculated using the formula: Weight loss rate % = (Weight before storage - Weight after storage) / Weight before storage × 100.

[0040] like Figure 1 As shown in Figure A, the weight loss rate of each group increased with prolonged storage time. From day 6 of storage, the weight loss rate of the pre-harvest composite group I was lower than that of the control group (CK), and also lower than that of each single treatment group and the post-harvest composite group. On day 15 of storage, the weight loss rate of the control group was 4.7%, and that of the pre-harvest composite group I was 3.9%.

[0041] like Figure 1 Table B and Table 1 show the decay rate of each treatment group on day 15 of storage.

[0042] Table 1. Effects of different treatments on the decay rate of kiwifruit. As shown in Table 1, the decay rate of the pre-harvest composite group I was lower than that of each single treatment group and the post-harvest composite group.

[0043] (2) Changes in hardness.

[0044] like Figure 2 As shown in Figure A, the fruit firmness of all treatment groups decreased with prolonged storage time. The CK group experienced the fastest decrease in fruit firmness, dropping from approximately 14 kg / cm² on the third day of storage. 2 Reduced to 4.0 kg / cm 2 The following groups showed the following hardness reduction rates: single J8 pre-harvest group, single DMY pre-harvest group, and post-harvest composite group; composite pre-harvest group I showed the slowest hardness reduction, maintaining approximately 10 kg / cm² on the third day of storage. 2 The above levels remained at 6 kg / cm² on day 6. 2 The results on day 12 were still significantly higher than those of other treatment groups, effectively delaying the fruit softening process.

[0045] (3) Changes in respiratory intensity.

[0046] The respiration rate of fruits and vegetables was measured using a GHX-3051H fruit and vegetable respiration analyzer, calibrated with a standard CO2 concentration of 1040 μL / L. The gas flow rate was 0.5 L / min, and the carrier gas was de-CO2-treated air. The results are expressed in mg CO2 / (kg·h). One-way ANOVA was used to statistically analyze the differences in the means of each treatment.

[0047] like Figure 2As shown in Figure B, the respiration intensity of the fruit in the control group (CK group) showed a significant peak on the 3rd day of storage (peak value approximately 88 mg CO2·kg). －1 ·h －1 The peak respiratory rate in the single J8 pre-collection group occurred on day 3, with a peak value of approximately 75 mg CO2·kg⁻¹. －1 ·h －1 The overall respiratory intensity in the DMY-only pre-collection group was lower than that in the CK group, with a peak value of approximately 55 mgCO2·kg. －1 ·h －1 The post-harvest respiratory peak in the combined group occurred on day 3, with a peak value of approximately 90 mgCO2·kg⁻¹. －1 ·h －1 The peak respiratory rate in the pre-harvest group I was significantly delayed until day 9 of storage, with a peak value of only about 52 mg CO2·kg⁻¹. －1 ·h －1 The concentration was significantly lower than that of other treatment groups, indicating that the combined pre-harvest treatment can effectively suppress fruit respiration intensity and delay the respiration climacteric.

[0048] (4) Changes in soluble solids (SSC) and titratable acid (TA).

[0049] Soluble solids (TSS) content was determined using an RA250-WE handheld digital refractometer. Juice was extracted by squeezing the fruit pulp, mixed thoroughly, and two drops were used for measurement, expressed as a percentage (%). Titratable acid content was determined using acid-base titration. One-way ANOVA was used to statistically analyze the differences in the means of each treatment.

[0050] like Figure 2 As shown in Figure C, the soluble solids content of each group showed a slow upward trend. The SSC content of the compound pre-harvest group I was consistently higher than that of the CK group during the middle and late storage period (days 6-15). On day 15 of storage, the SSC content of the compound pre-harvest group I reached about 13.5%, which was significantly higher than that of the CK group (12.0%), indicating that the compound treatment could maintain a good sugar content level in the fruit.

[0051] like Figure 2 As shown in Figure D, the titratable acid content of each group decreased with prolonged storage time, with the CK group showing the fastest rate of decrease. At harvest, the initial TA content of each group was approximately 1.4%. After 15 days of storage at room temperature, the CK group was 0.15%, the single J8 pre-harvest group was 0.18%, and the single DMY pre-harvest group was 0.17%. The TA content in the post-harvest composite group was 0.20%, and that in the pre-harvest composite group I was 0.28%; this indicates that the TA content in the pre-harvest composite group I was higher than that in the control groups.

[0052] Experimental Example 2: Preservation effect of composite solutions of different concentrations.

[0053] ① Pre-harvest compound group II (Example 2): Spraying a compound solution (DMY 200 mg / L + viable concentration of Sphingospora polyphaga sinensis J8 of 1×10⁻⁶) at four pre-harvest stages 7 (CFU / mL + 0.05% Tween - 80). The spoilage rate was 26.3% after 15 days of storage at room temperature.

[0054] ② Pre-harvest compound group III (Example 3): Spraying a compound solution (DMY 150 mg / L + viable concentration of Sphingomyelin-J8 of 1×10⁻⁶) at four pre-harvest stages. 9 (CFU / mL + 0.05% Tween - 80). The spoilage rate was 25.9% after 15 days of storage at room temperature.

[0055] ③ Low concentration group (Comparative Example 1): DMY 50 mg / L + viable bacterial concentration of Sphingosine monocytogenes J8 was 1×10 8 CFU / mL + 0.05% Tween-80 was sprayed at four pre-harvest stages. The decay rate was 28.5% after 15 days of storage at room temperature, and the synergistic effect was not significant.

[0056] ④ High concentration group (Comparative Example 2): DMY 500 mg / L + viable bacterial concentration of Sphingosine monocytogenes J8 was 1×10 8 CFU / mL + 0.05% Tween-80 was sprayed at four pre-harvest stages. The decay rate on day 15 of storage at room temperature was 30.8%, which was higher than that of the compound pre-harvest group I in Example 1.

[0057] The results showed that when the DMY concentration was too low (50 mg / L), the preservation effect was not significant, while when the concentration was too high (500 mg / L), it actually aggravated decay. Excessive concentration may also cause osmotic pressure stress or physiological and biochemical interference to the fruit tissue, thus weakening the preservation effect. Therefore, the optimal DMY concentration range is 100–200 mg / L.

[0058] Experimental Example 3: The preservation effect of combined treatment on 'Hongyang' kiwifruit.

[0059] The cultivar 'Hongyang', highly susceptible to soft rot, was selected and divided into a control group (sterile water + 0.05% Tween-80) and a pre-harvest compound group I (DMY 100 mg / L + viable concentration of Sphingomyelin-J8 at 1×10⁻⁶). 8 CFU / mL + 0.05% Tween-80), store at room temperature (20-25℃) after harvest.

[0060] On day 15 of storage at room temperature, the decay rate of the CK group was 41.3%, and the decay rate of the pre-harvest composite group I was 28.2%. This indicates that this method also has a significant effect on room temperature preservation for susceptible varieties.

[0061] Experimental Example 4: Effects of combined treatment on the activity of fruit defense enzymes.

[0062] On the day of harvest and on the 5th day of storage at room temperature, the activity of phenylalanine ammonia-lyase (PAL) in the fruit was measured using a phenylalanine ammonia-lyase kit. The results are shown in Table 2. Table 2 Effects of different treatments on PAL activity in kiwifruit (U·g) －1 ) The results showed that pre-harvest compound spraying could improve PAL activity in fruits and maintain a high level during storage at room temperature.

[0063] In summary, the present invention has the following advantages: (1) Intervention during the critical window period before harvest to effectively block latent infection: When the pathogens are easily invaded during the flowering period and the young fruit period, compound spraying can kill the free pathogens in the field and occupy the ecological niche on the fruit surface. The effect is significantly better than post-harvest treatment; (2) Synergistic effect to significantly reduce the decay rate: Dihydromyricetin has no significant inhibitory effect on Sphingomyelin J8 within a specific concentration range. Instead, it can reduce the damage of environmental stress to J8 by absorbing ultraviolet rays and clearing free radicals, and improve its survival rate on the fruit surface. The synergistic effect of the two makes the decay rate of 15 days of storage at room temperature more than 6 percentage points lower than that of the control; (3) Significantly delay the respiratory climacteric and extend the shelf life: The compound treatment can delay the respiratory peak of kiwifruit at room temperature from the 3rd day to the 9th day, effectively delaying the softening of the fruit and maintaining its firmness; (4) Maintain the flavor quality during storage at room temperature: The compound treatment can slow down the consumption of titratable acid and better maintain the sweet and sour flavor of kiwifruit.

[0064] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A pre-harvest preservative for room-temperature storage of kiwifruit, characterized in that, The pre-harvest spray preservative includes the following components in appropriate dosages: dihydromyricetin at a concentration of 100-200 mg / L, and live bacteria concentration of 1×10⁻⁶. 7 -1×10 9 CFU / mL Sphingomyelin-J8 2. The pre-harvest preservative for room-temperature storage of kiwifruit according to claim 1, characterized in that, The pre-harvest spray preservative includes the following components in appropriate dosages: dihydromyricetin at a concentration of 100-200 mg / L, and live bacteria concentration of 1×10⁻⁶. 8 CFU / mL Sphingomyelin-J8 3. A pre-harvest preservative for room-temperature storage of kiwifruit according to claim 1 or 2, characterized in that, The pre-harvest preservative also includes the following components: 0.05% Tween-80 by volume.

4. A method for preparing a pre-harvest preservative for room-temperature storage of kiwifruit according to any one of claims 1 to 3, characterized in that, The process includes the following steps: first, dissolve dihydromyricetin in an alcohol solvent, then dilute with water, and finally add Sphingomyelin-J8 suspension and Tween-80 to obtain a pre-harvest spray preservative.

5. The method for preparing a pre-harvest preservative for room-temperature storage of kiwifruit according to claim 4, characterized in that, The alcohol solvent includes ethanol; The volume fraction of the alcohol solvent in the pre-harvest spray preservative is ≤0.5%.

6. The method for preparing a pre-harvest preservative for room-temperature storage of kiwifruit according to claim 4, characterized in that, The Sphingomyelin-J8 suspension was prepared by the following steps: after activating the Sphingomyelin-J8 strain, it was cultured by shaking, the Sphingomyelin-J8 strain was collected by centrifugation, and then resuspended in sterile water to obtain the Sphingomyelin-J8 suspension.

7. A method for preserving kiwifruit at room temperature by spraying a preservative before harvest, characterized in that, The steps include the following: (1) During the critical growth period of kiwifruit, the leaves and fruits are sprayed with the pre-harvest preservative for kiwifruit storage at room temperature as described in any one of claims 1 to 3; the critical growth period includes the full bloom period, the young fruit period, the fruit enlargement period and the ripening period; (2) Manage the fruit according to the usual procedure until it is ripe, then harvest it and store it at room temperature.

8. The method for preserving kiwifruit at room temperature by spraying a preservative before harvest according to claim 7, characterized in that, The specific spraying in step (1) is carried out in the evening of a sunny day or in a rainless weather, so that the pre-harvest preservative for room temperature storage of kiwifruit stays on the leaves and fruit surface for at least 4 hours.

9. A method for preserving kiwifruit at room temperature by spraying a preservative before harvest according to claim 7, characterized in that, The harvesting time in step (2) is when the soluble solids content of the kiwifruit reaches 6.8%-7.5%.

10. A method for preserving kiwifruit at room temperature by spraying a preservative before harvest according to claim 7, characterized in that, In step (2), the storage at room temperature specifically means that after harvesting, the product is stored at a temperature of 20-25℃ and a relative humidity of 85%-95%.

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