Application of volatile compound generated based on serratia rubra in postharvest disease control and storage and preservation of fruits and vegetables
By utilizing the volatile compounds 2-octanone, 2-nonanone, and 2-nonanol produced by Serratia marcescens, the problem of controlling postharvest Penicillium rot in fruits and vegetables has been solved, achieving safe and environmentally friendly biological control, significantly reducing the rate of fruit and vegetable decay while maintaining quality, and expanding the application scope of biological control.
Patent Information
- Application Number
- CN202511354724.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-22
- Publication Date
- 2025-12-26
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Existing technologies for the prevention and control of postharvest diseases in fruits and vegetables are limited. Physical methods require sophisticated equipment and are difficult to industrialize, while chemical methods are prone to drug resistance and environmental pollution. Biological control methods are not effective enough, especially in controlling postharvest Penicillium wilt in fruits and vegetables.
Volatile compounds produced by Serratia rubidaea B11, including 2-octanone, 2-nonanone, and 2-nonanol, were used to control diseases in fruits and vegetables and inhibit the growth of Penicillium expansum through inoculation and fumigation.
It significantly reduces the incidence of postharvest blue mold in fruits and vegetables, reduces natural decay, maintains the quality of fruits and vegetables, provides a safe and environmentally friendly biological control method, enriches the means of storing and preserving fruits and vegetables, and has broad market application prospects.
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Figure CN121196008A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biological control technology for postharvest diseases, specifically relating to a volatile substance of Serratia marcescens and its use in controlling postharvest Penicillium rot in fruits and vegetables and for storage and preservation. Background Technology
[0002] Fruits and vegetables are susceptible to rotting and spoilage due to infection by pathogens such as *Penicillium expansum* during post-harvest storage and transportation, leading to reduced commercial value and economic losses. Currently, the main methods for controlling post-harvest diseases in fruits and vegetables include physical, chemical, and biological control. Physical methods require sophisticated equipment and are difficult to industrialize; chemical methods can easily lead to drug resistance in pathogens and pollute the environment; biological control methods have advantages such as safety and environmental friendliness, but current research needs improvement in terms of effectiveness, and there is limited research on using antagonistic bacteria to control post-harvest diseases in fruits and vegetables. Summary of the Invention
[0003] The present invention aims to provide a volatile substance of Serratia marcescens and its use in postharvest disease control and storage preservation of fruits and vegetables, so as to effectively solve the problems existing in the prior art.
[0004] The Serratia rubidaea B11 used in this invention is from the China Center for Type Culture Collection, with accession number CCTCC NO: M20241377, and is a publicly available strain.
[0005] The application of the volatile substances of Serratia marcescens provided by this invention for postharvest disease control in fruits and vegetables is carried out according to the following steps:
[0006] (1) Serratia marcescens was inoculated into LB medium for the first activation culture to obtain the activation solution; then the activation solution was transferred to a new LB medium for the second activation to obtain the bacterial culture solution. After centrifugation, the bacterial sludge was obtained, washed with sterile physiological saline, and then prepared into a Serratia marcescens bacterial suspension; PDA medium was injected into a sterile sealed box, and after solidification, the bacterial suspension was inoculated and spread evenly with a sterile spreader to obtain the treated sterile sealed box.
[0007] Perforations can be made on the surface of fruits and vegetables using a sterile punch, inoculated with a suspension of Penicillium spores, placed in a treated sterile sealed box, and cultured under constant temperature conditions to verify the control of VOCs on postharvest Penicillium rot in fruits and vegetables; or untreated, naturally grown fruits and vegetables can be placed in a treated sterile sealed box and sealed to achieve the prevention and control of postharvest Penicillium rot in fruits and vegetables using VOCs.
[0008] (2) VOCs produced by Serratia marcescens include 2-octanone, 2-nonanone and 2-nonanol. 2-octanone, 2-nonanone or 2-nonanol are further used to control postharvest Penicillium rot in fruits and vegetables. Sterilized filter paper sheets that completely cover the bottom are placed in a sterile sealed box, and 2-octanone, 2-nonanone or 2-nonanol is added to obtain a treated sterile sealed box. Untreated fruits and vegetables in their natural state are placed in the treated sterile sealed box and sealed, so that they are in the same space as 2-octanone, 2-nonanone or 2-nonanol, thereby achieving the control of postharvest Penicillium rot in fruits and vegetables by volatile substances.
[0009] The fruits and vegetables mentioned include pears, apples, peaches, and apricots;
[0010] The components and preparation method of the LB culture medium in step (1) are as follows: 10g tryptone, 5g yeast extract, 10g sodium chloride, distilled water to 1000mL, natural pH, sterilized at 115℃ for 20min.
[0011] The components of the PDA culture medium mentioned in step (1) are as follows, per 1L: 200g of diced potato, boiled in double-distilled water and kept boiling for 10 minutes, filtered through gauze to obtain the filtrate, 20g of glucose, and double-distilled water to make up to 1000mL, pH set to natural, 20g of agar added, and autoclaved at 121℃ for 20 minutes.
[0012] In step (1), the inoculum size for activation culture was 0.5-2%, and the culture conditions were 25℃, 180rpm, and shake culture for 24h.
[0013] The centrifugation conditions in step (1) are 4℃, 8000rpm, and 10-15min.
[0014] The mass concentration of the sterile saline solution in step (1) is 0.85%.
[0015] The concentration of the Serratia marcescens suspension in step (1) is 1×10⁻⁶. 8 The inoculum size of *Serratia marcescens* suspension was 0.5-2% (cells / mL).
[0016] The constant temperature condition in step (1) is 25℃, and the culture time is 1-5 days.
[0017] In step (2), the concentration of 2-octanone, 2-nonanone, or 2-nonanol used is 0.02 to 0.16 μL / mL, and 0.5 mL to 2 mL of 2-octanone, 2-nonanone, or 2-nonanol is used for every 1000 to 10000 square centimeters of sterile sealed box.
[0018] Preferably, the volatile substance is 2-octanone at a concentration of 0.08 μL / mL.
[0019] The main volatile component in the filtrate was 2-octanone, which showed significant inhibitory effects on Penicillium expansum with a minimum inhibitory concentration (MIC) of 0.08 μL / mL.
[0020] Beneficial effects:
[0021] (1) The volatile substances of Serratia marcescens used in this invention can significantly reduce the incidence of postharvest blue mold in fruits and vegetables, reduce the natural decay rate, have no significant adverse effects on the quality of fruits and vegetables, and can alleviate the quality deterioration of fruits and vegetables during storage to a certain extent, which is beneficial to the storage and preservation of fruits and vegetables.
[0022] (2) Compared with chemical bactericides, the biological control method of the present invention has the advantages of safety and environmental protection, and will not cause harm to consumers’ health and the environment, which meets the needs of green development.
[0023] (3) This invention provides a new and effective means for the prevention and control of postharvest diseases of fruits and vegetables, enriches the application scope of biological control, and has broad market application prospects. Attached Figure Description
[0024] Figure 1 The effect of VOCs from S. rubidaea B11 on the control of Penicillium rot in pear fruit is shown in Figure A. A shows actual images of treated pear fruit, and Figure B shows statistical data. Note: CK represents sterile saline treatment (control group); B11 represents the S. rubidaea B11 treatment group; the numbers on the horizontal axis represent treatment time; different lowercase letters indicate significant differences between different treatments (P<0.05).
[0025] Figure 2 The in vitro inhibitory effects of various substances on P. expansum are shown; where A represents the colony growth of P. expansum, and B represents the colony diameter data of P. expansum; CK represents the control group without any added substances, and different lowercase letters indicate significant differences between groups (p<0.05).
[0026] Figure 3 The control effect of 2-octanone fumigation on Penicillium rot in pear fruit is shown in Figure A, B, and C. CK represents the control group. Different lowercase letters indicate significant differences between groups (p<0.05).
[0027] Figure 4The effect of 2-octanone treatment on spore germination and germ tube length of P. expansum; where A: visual image under a microscope, B: spore germination rate and germ tube length; CK represents the control group, and different lowercase letters indicate significant differences between groups (p<0.05). Detailed Implementation
[0028] The present invention will be further described in detail below through specific embodiments: The Serratia rubidaea B11 used in the present invention is from the China Center for Type Culture Collection, with accession number CCTCC NO: M20241377, and is a publicly disclosed strain. The specific fruit and vegetable selected is pear.
[0029] Serratia marcescens was inoculated at a 1% inoculum into LB medium for the first activation culture at 25°C and 180 rpm for 24 h to obtain the activated solution. Then, the activated solution was transferred to fresh LB medium at a 1% inoculum for a second activation culture at 25°C and 180 rpm for 24 h to obtain the bacterial culture broth. After centrifugation (4°C, 8000 rpm, 10 min), bacterial sludge was obtained, washed with sterile physiological saline, and then prepared into a Serratia marcescens bacterial suspension with a concentration of 1×10⁻⁶. 8 CFU / mL.
[0030] The composition and preparation method of LB medium are as follows: per 1L, 10g tryptone, 5g yeast extract, 10g sodium chloride, distilled water to a final volume of 1000mL, natural pH, sterilized at 115℃ for 20min.
[0031] The components of PDA medium are as follows (per 1L): 200g of diced potato, boiled in double-distilled water for 10 minutes, filtered through gauze to obtain the filtrate, 20g of glucose added, double-distilled water added to a final volume of 1000mL, pH set to natural, 20g of agar added, and autoclaved at 121℃ for 20 minutes.
[0032] Example 1: Determination of the effect of VOCs from S. rubidaea B11 on postharvest Penicillium mold control in pear fruit
[0033] Pear fruit pretreatment: Select healthy pears with uniform color, ripeness, free from pests and diseases, and of uniform size and without mechanical damage. Soak the selected pears in a 0.5% (v / v) sodium hypochlorite solution for 2 minutes, then rinse off the residual sodium hypochlorite with tap water and place them in a basket to air dry naturally for later use.
[0034] Using a sterile punch, make three evenly spaced holes (5mm x 4mm) at the equator of each pear fruit and allow them to air dry at room temperature. Inoculate the holes with 1×10⁻⁶ ppm of the solution. 5Prepare 30 μL of a *P. expansum* spore suspension (spores / mL) in a sterile container. Pour PDA medium into a sterile 6 L (6000 cm³) container, ensuring the bottom is completely covered. After the medium has solidified, spread 1 mL of a 1×10⁻⁶ m³ solution onto the container. 8 A CFU / mL suspension of *S. rubidaea* B11 was prepared, with sterile physiological saline as a control. Pears inoculated with *P. expansum* were then placed on the grid partition of a food storage container, ensuring the pears did not directly contact the culture medium. The container was then covered and incubated at 20°C, and the extent of pear decay was observed.
[0035] S. rubidaea B11 can produce VOCs, which can be used to fumigate pears, such as... Figure 1 As shown, fumigation with VOCs produced by S. rubidaea B11 significantly inhibited the growth of pear fruit lesion diameter. On the 3rd, 4th, and 5th days of storage, the diameters of rotten pear fruit after fumigation with VOCs produced by S. rubidaea B11 were 9.51 mm, 10.48 mm, and 14.23 mm, respectively, all significantly lower than the rotten diameters of pear fruit in the control group (p<0.05), with values of 11.29 mm, 14.98 mm, and 20.43 mm, respectively.
[0036] Example 2: Determination of the in vitro inhibitory effect of VOCs on P. expansum
[0037] Based on the identified VOCs and their relative content and safety, three compounds—2-octanone, 2-nonanone, and 2-nonanol—were initially selected for further testing to assess their inhibitory effects on P. expansum.
[0038] The specific procedure is as follows: Add PDA medium to one compartment of a bipartite plate. After solidification, punch holes (8 mm in diameter) and inoculate 30 μL of *P. expansum* spore suspension into each hole. Place an 8 mm diameter filter paper disc in the other compartment and add 30 μL of pure 2-octanone, 2-nonanone, and 2-nonanol to the disc, respectively. A control without any added compounds is used. Then, cover the plate and seal it with sealing film. Incubate at 25°C for 4 days, observing the growth of *P. expansum* and recording the colony diameter.
[0039] Of the 26 substances detected, three substances with relatively high safety and relatively high content—2-octanone, 2-nonanone, and 2-nonanol—were selected to study their in vitro inhibitory effects on *P. expansum*. Figure 2As shown, compared with the control group, the growth of *P. expansum* was inhibited to varying degrees after treatment with the three substances, and the colony diameter was significantly reduced. Specifically, treatment with 2-octanone resulted in only a small amount of hyphae growth, with a colony diameter of only 0.87 mm, and there was no significant difference between 2-octanone and 2-nonanol (the measured diameters have been adjusted to exclude the hole diameter). Given the low cost and high safety of 2-octanone, and considering both the inhibitory effect and economic factors, 2-octanone was ultimately chosen for subsequent experiments.
[0040] Example 3: Determination of the effect of 2-octanone fumigation on postharvest Penicillium mold control in pear fruit
[0041] Filter paper was evenly placed at the bottom of a sterile food storage container. 2-Octanone was then added to the filter paper at concentrations of 0, 0.02, 0.04, 0.08, and 0.16 μL / mL. Three holes (5 mm × 4 mm) were evenly punched at the equatorial region of each pear using a sterile punch. The pears were then air-dried at room temperature. 1 × 10⁻⁶ ppm of 2-octanone was inoculated into each hole. 5 Prepare 30 μL of P. expansum spore suspension (spores / mL) in a sterile container for later use. Place pears pre-inoculated with P. expansum on a grid partition in a sterile plastic box, ensuring the pears do not directly contact the filter paper. Store in a sealed container, and record the rate and diameter of decay after 4 days.
[0042] like Figure 3 As shown, 2-octanone inhibited the growth of *Penicillium expansum* on pear fruit, consistent with the concentration used to control its growth on agar plates. On day 4 of storage, pears in both the control and 0.02 μL / mL treatment groups were completely rotten, but the rot diameter in the 0.02 μL / mL treatment group was significantly smaller than that in the control group. Treatment with 0.04 μL / mL significantly reduced both the rot rate and rot diameter (p < 0.05). When the 2-octanone concentration was increased to 0.08 μL / mL and 0.16 μL / mL, the disease was completely inhibited. These results indicate that fumigation with different concentrations of 2-octanone significantly reduced the rot rate and rot diameter of pear fruit with increasing concentration, and high concentrations completely inhibited rot. However, excessively high 2-octanone concentrations (0.16 μL / mL) caused a slight change in the surface color of the pear fruit; therefore, the concentration should not be too high.
[0043] Example 4: Determination of the effect of 2-octanone on P. expansum spore germination and germ tube length
[0044] Add P. expansum spore suspension to an Erlenmeyer flask containing PDB medium, adjusting the final spore concentration to 1×10⁻⁶. 6The concentrations of spores / mL were measured, and then 2-octanone was added to the bottles to achieve volume concentrations of 0, 0.02, 0.04, 0.08, and 0.16 μL / mL, with no 2-octanone added serving as a control group. The bottle openings were sealed with plastic film and incubated at 25°C and 75 rpm in a shaker for 16 h. Spore germination was observed under a microscope, and the germination rate and germ tube length were measured.
[0045] like Figure 4 As shown, different concentrations of 2-octanone significantly inhibited the spore germination rate and germ tube length of *P. expansum*, with the inhibitory effect increasing with increasing concentration. In the control group, the spore germination rate and germ tube length of *P. expansum* after 16 h of culture were 84.70% and 34.00 μm, respectively. When treated with 0.02 μL / mL of 2-octanone for 16 h, the germination rate and germ tube length significantly decreased to 5.71% and 11.45 μm, respectively. At other higher concentrations, the growth of *P. expansum* spores was completely inhibited.
[0046] In summary, this study fully demonstrates that VOCs produced by *S. rubidaea* B11 can significantly inhibit the growth of *P. expansum*, exhibiting good control effects against Penicillium rot. Furthermore, GC-MS analysis revealed that the key component of the VOCs, 2-octanone, possesses significant antibacterial activity. It exerts its antibacterial effect primarily by inhibiting spore germination, disrupting the cell morphology of the pathogen, and damaging the cell membrane integrity, thus demonstrating promising market application prospects.
[0047] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. The use of volatile compounds produced by *Serratia marcescens* for postharvest disease control and storage preservation of fruits and vegetables, characterized in that... The specific steps are as follows: (1) Serratia marcescens was inoculated into LB medium for the first activation culture to obtain the activation solution; then the activation solution was transferred to a new LB medium for the second activation to obtain the bacterial culture solution. After centrifugation, the bacterial sludge was obtained, washed with sterile physiological saline, and then prepared into a Serratia marcescens bacterial suspension; PDA medium was injected into a sterile sealed box, and after solidification, the Serratia marcescens bacterial suspension was inoculated and spread evenly with a sterile spreader to obtain the treated sterile sealed box; Perforations can be made on the surface of fruits and vegetables using a sterile punch, inoculated with a suspension of Penicillium spores, placed in a treated sterile sealed box, and cultured under constant temperature conditions to verify the control of VOCs on postharvest Penicillium rot in fruits and vegetables; or untreated, naturally grown fruits and vegetables can be placed in a treated sterile sealed box and sealed to achieve the prevention and control of postharvest Penicillium rot in fruits and vegetables using VOCs. (2) VOCs produced by Serratia marcescens include 2-octanone, 2-nonanone and 2-nonanol; a sterile filter paper sheet completely covering the bottom is placed in a sterile sealed box, and volatile compounds 2-octanone, 2-nonanone or 2-nonanol are added to obtain a treated sterile sealed box; untreated fruits and vegetables in their natural state are placed in the treated sterile sealed box and sealed, so that they are in the same space as 2-octanone, 2-nonanone or 2-nonanol, thereby achieving the control of postharvest blue mold of fruits and vegetables by volatile substances; The fruits and vegetables mentioned include pears, apples, peaches, and apricots; The aforementioned Serratia marcescens is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M20241377.
2. The use of volatile compounds produced by *Serratia marcescens* according to claim 1 for postharvest disease control and storage preservation of fruits and vegetables, characterized in that... The components and preparation method of the LB culture medium mentioned in step (1) are as follows: 10g tryptone, 5g yeast extract, 10g sodium chloride, distilled water to a final volume of 1000mL, natural pH, sterilized at 115℃ for 20min.
3. The use of volatile compounds produced by *Serratia marcescens* according to claim 1 for postharvest disease control and storage preservation of fruits and vegetables, characterized in that... The components of the PDA culture medium mentioned in step (1) are as follows, per 1L: 200g of diced potato, boiled in double-distilled water for 10 minutes, filtered through gauze to obtain the filtrate, 20g of glucose, and double-distilled water to a final volume of 1000mL, pH set to natural, 20g of agar added, and autoclaved at 121℃ for 20 minutes.
4. The use of volatile compounds produced by *Serratia marcescens* according to claim 1 for postharvest disease control and storage preservation of fruits and vegetables, characterized in that... In step (1), the inoculum size for activation culture was 0.5-2%, and the culture conditions were 25℃, 180rpm, and shake culture for 24h.
5. The use of volatile compounds produced by *Serratia marcescens* according to claim 1 for postharvest disease control and storage preservation of fruits and vegetables, characterized in that... The centrifugation conditions described in step (1) are 4℃, 8000rpm, and 10-15min.
6. The use of volatile compounds produced by *Serratia marcescens* according to claim 1 for postharvest disease control and storage preservation of fruits and vegetables, characterized in that... The mass concentration of the sterile saline solution mentioned in step (1) is 0.85%.
7. The use of volatile compounds produced by *Serratia marcescens* according to claim 1 for postharvest disease control and storage preservation of fruits and vegetables, characterized in that... The concentration of the Serratia marcescens suspension in step (1) is 1×10⁻⁶. 8 The inoculum size of *Serratia marcescens* suspension was 0.5-2% (cells / mL).
8. The use of volatile compounds produced by *Serratia marcescens* according to claim 1 for postharvest disease control and storage preservation of fruits and vegetables, characterized in that... The constant temperature condition described in step (1) is 25℃, and the culture time is 1-5 days.
9. The use of volatile compounds produced by *Serratia marcescens* according to claim 1 for postharvest disease control and storage preservation of fruits and vegetables, characterized in that... The concentration of 2-octanone, 2-nonanone, or 2-nonanol used in step (2) is 0.02 to 0.16 μL / mL, and 0.5 mL to 2 mL of 2-octanone, 2-nonanone, or 2-nonanol is used for every 1000 to 10000 square centimeters of sterile sealed box.
10. The use of volatile compounds produced by *Serratia marcescens* according to claim 9 for postharvest disease control and storage preservation of fruits and vegetables, characterized in that... The volatile substance selected was 2-octanone, with a concentration of 0.08 μL / mL.