Nematophilic symbiotic bacteria segmented temperature control fermentation method, product and application thereof
By using a segmented temperature-controlled fermentation method with nematode symbiotic bacteria in combination with insulin, the antibacterial activity of the fermentation products was improved, solving the problem of post-harvest rotting of chives, extending the storage period of chives and maintaining their quality.
Patent Information
- Application Number
- CN202510951521.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-10
- Publication Date
- 2025-12-09
AI Technical Summary
Existing technologies are not ideal for preventing rotting during the post-harvest storage of chives, especially for controlling the terrestrial Raoult bacterium, resulting in a short storage period for chives.
A segmented temperature-controlled fermentation method using nematode symbiotic bacteria was adopted, which included controlling the fermentation temperature at different stages and adding insulin during the logarithmic growth phase. This optimized the growth of the nematode symbiotic bacteria and the antibacterial activity of the fermentation products, resulting in the preparation of effective fermentation products.
It significantly inhibits the growth of *Laurella multocida*, the pathogen that causes leeks to rot, prolongs the storage period of leeks, and maintains the freshness and nutritional components of leeks.
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Figure CN121086901A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a segmented temperature-controlled fermentation method for nematode symbiotic bacteria, the product, and its applications. Background Technology
[0002] Chives are a perennial herbaceous plant, a nutritious vegetable with medicinal value, and are favored by many consumers. However, chives have a high water content, making them prone to wilting and rotting after harvesting and during storage and transportation, thus limiting their marketability. Therefore, post-harvest preservation techniques for chives are crucial.
[0003] Pathogenic microbial infection is one of the main causes of vegetable spoilage during post-harvest storage. The inventors collected leeks from Pingdingshan, Henan Province, and isolated a rot-causing pathogen from the rotting leeks. Molecular identification confirmed it to be *Raoultella terrigena*. This bacterium is a major rot-causing pathogen in leeks, and existing drugs are not very effective against it.
[0004] Entomopathogenic nematode symbiotic bacteria have attracted significant attention both domestically and internationally due to their rapid insecticidal action, safety for humans and animals, wide host range, and strong resistance. With the deepening research into entomopathogenic nematode symbiotic bacteria, various biological functions have been discovered. Their secondary metabolites possess multiple biological activities, including insecticidal, antibacterial, antitumor, and nematicidal effects, making them a novel biological resource with development potential and application prospects, and showing good commercial potential in agriculture.
[0005] Insulin is a protein hormone secreted by pancreatic β-cells in response to the induction of endogenous or exogenous substances. It regulates carbohydrate, fat, and protein metabolism through cellular signal transduction, thereby influencing reproductive and aging processes in organisms. Currently, there are numerous reports on the effects of insulin on nematodes, but fewer studies on its symbiotic bacteria in entomopathogenic nematodes. Research using *Caenorhabditis elegans* as a model is particularly clear. Recent studies have also confirmed that the ILP and IGF signaling pathways in insulin can affect the regulation of metabolism, growth and development, reproductive capacity, stress resistance, and lifespan in insects and nematodes. Summary of the Invention
[0006] One of the technical problems to be solved by the present invention is to provide a segmented temperature-controlled fermentation method for nematode symbiotic bacteria, so as to improve the prevention and control of rotting diseases in chives and extend the storage period of chives.
[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:
[0008] A method for staged temperature-controlled fermentation of nematode symbiotic bacteria includes the following steps:
[0009] S1. Take the preserved nematode symbiotic bacteria and streak them onto NA medium plates, then incubate at 28°C.
[0010] S2. After 24 hours of incubation, pick a single colony and streak it onto an NBTA medium plate, then incubate at 28°C.
[0011] S3. After 48 hours of culture, a single colony of primary type I bacteria was inoculated into LB liquid medium and cultured at 28℃ and 150r / min for 24 hours to obtain seed culture solution.
[0012] S4. Inoculate the seed culture medium with an inoculation amount of 6% by volume and carry out segmented temperature-controlled fermentation. The segmented temperature control is as follows: the fermentation temperature is controlled at 30-35℃ from the start of fermentation to 18 hours, and the fermentation temperature is controlled at 25-28℃ from 18 to 36 hours. The rotation speed is 130-160 r / min throughout the fermentation process.
[0013] In the segmented temperature-controlled fermentation method of nematode symbiotic bacteria provided by the present invention, preferably, the segmented temperature control is as follows: the fermentation temperature is controlled at 33°C from the start of fermentation to 18 hours of fermentation, and the fermentation temperature is controlled at 28°C from 18 to 36 hours of fermentation.
[0014] In the segmented temperature-controlled fermentation method of nematode symbiotic bacteria provided by the present invention, insulin is preferably added 5 to 8 hours after the start of fermentation, that is, insulin is added during the logarithmic growth phase of the nematode symbiotic bacteria.
[0015] In the segmented temperature-controlled fermentation method of nematode symbiotic bacteria provided by the present invention, more preferably, the amount of insulin added is 0.001 to 0.01 mg / L (equivalent to adding 0.5 to 5 mL / L of insulin solution).
[0016] Insulin is added as an insulin solution, which is prepared by mixing a 1 mg / mL insulin stock solution with Ringer's solution at a volume ratio of 1:500.
[0017] In the segmented temperature-controlled fermentation method of nematode symbiotic bacteria provided by the present invention, more preferably, the amount of insulin added is 0.004 mg / L (equivalent to 2 mL / L insulin solution).
[0018] In the segmented temperature-controlled fermentation method for nematode symbiotic bacteria provided by this invention, the nematode symbiotic bacteria is Enterobacter nematode NK, which was provided by the laboratory of the School of Life Sciences and Engineering, Henan University of Urban Construction (frozen at -80℃). This strain was isolated by the inventors from the intestines of *Heterobacter* entomopathogenic nematodes collected in Pingdingshan City, Henan Province, and was first disclosed in "Development of Aqueous Suspension of *Entomopathogenic Nematode Symbiotic Bacteria NK*", Journal of Henan University of Urban Construction, Vol. 30, No. 1, pp. 80-86, Publication Date: February 28, 2021. The public can request this strain from the School of Life Sciences and Engineering, Henan University of Urban Construction.
[0019] In the segmented temperature-controlled fermentation method of nematode symbiotic bacteria provided by the present invention, more preferably, the fermentation medium is: Na2SO4 1.5g / L, MgSO4 1.3g / L, (NH4)2SO4 2.4g / L, peptone 20g / L, KH2PO4 0.7g / L, glucose 9g / L, K2HPO4 0.5g / L, pH=7.2~7.4.
[0020] In the segmented temperature-controlled fermentation method of nematode symbiotic bacteria provided by the present invention, insulin is most preferably added 6 hours after the start of fermentation.
[0021] Another problem to be solved by the present invention is to provide a segmented temperature-controlled fermentation product of nematode symbiotic bacteria to prevent leek rot and extend the storage period of leeks.
[0022] This invention also provides the application of a segmented temperature-controlled fermentation product of nematode symbiotic bacteria in the prevention and control of leek rot disease.
[0023] By employing the above technical solution, segmented temperature control during fermentation enhances the growth of nematode-symbiotic bacteria and the antibacterial activity of the fermentation product. Adding insulin during the logarithmic growth phase of the nematode-symbiotic bacteria further increases the growth of these bacteria and the antibacterial activity of the fermentation product. The fermentation product obtained by this invention exhibits significant antibacterial activity against *Laurella terrestris*, the pathogen causing rotting in chives, effectively preventing rotting and extending the storage period of chives. Attached Figure Description
[0024] Figure 1 The images show the morphological characteristics and Gram staining of the pathogen causing rot in chives. a) shows the morphological characteristics from the front, b) shows the morphological characteristics from the back, and c) shows the Gram-stained bacterial morphology under an optical microscope.
[0025] Figure 2 A phylogenetic tree of leek rot pathogens constructed based on 16S rDNA sequence homology;
[0026] Figure 3The diagram shows the pathogenicity of putrefactive bacteria. a represents the control group, b represents the "injured" group, and c represents the "uninjured" group.
[0027] Figure 4 The inhibition zone diagrams of the products obtained in each example against the putrefactive pathogens (first test);
[0028] Figure 5 The inhibition zone diagrams of the products obtained in each example against the putrefactive pathogens (second test);
[0029] Figure 6 The inhibition zone diagrams of the products obtained in each example against the putrefactive pathogens (third test);
[0030] Figure 7 The growth of pathogenic bacteria causing rot in leek leaves was observed on the 5th day after spraying the fermentation products. a was the blank control group, b was the 6th group of Example, and c was the 1st group of Comparative Example.
[0031] Figure 8 Line graph showing the changes in vitamin C content in each group of chives;
[0032] Figure 9 Line graph showing the changes in chlorophyll content in chives for each group;
[0033] Figure 10 Line graph showing the changes in malondialdehyde content in each group of chives;
[0034] Figure 11 Line graph showing the changes in peroxidase activity in each group of chives;
[0035] Figure 12 Line graph showing the changes in catalase activity in each group of chives;
[0036] Figure 13 Line graph showing the changes in ascorbic acid peroxidase activity in each group of chives. Detailed Implementation
[0037] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0038] Culture medium preparation:
[0039] PCA medium: tryptone 5 g / L, yeast extract 2.5 g / L, glucose 1 g / L, agar 20 g / L, pH=7.2.
[0040] NA medium: beef extract 3 g / L, peptone 5 g / L, nutrient agar 20 g / L, pH=7.2~7.4.
[0041] NBTA medium: NA medium, triphenyltetrazolium chloride (TTC) 0.04 g / L, bromothymol blue (BTB) 0.025 g / L.
[0042] LB medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH = 7.2–7.4.
[0043] Fermentation medium: Na₂SO₄ 1.5 g / L, MgSO₄ 1.3 g / L, (NH4)2SO4 2.4 g / L, peptone 20 g / L, KH2PO4 0.7 g / L, glucose 9 g / L, K2HPO4 0.5 g / L, pH=7.2~7.4.
[0044] Insulin solution preparation:
[0045] Ringer's solution: NaCl 8.6 g / L, KCl 0.3 g / L, CaCl2 0.28 g / L.
[0046] Insulin solution: Insulin stock solution: Ringer's solution = 1:500 (volume ratio), the concentration of insulin stock solution is 1 mg / mL.
[0047] Example 1
[0048] This embodiment provides a segmented temperature-controlled fermentation method for nematode symbiotic bacteria, including the following steps:
[0049] S1. Take the preserved Enterobacter nematodes NK and streak it onto NA medium plates, then incubate at 28°C.
[0050] S2. After 24 hours of incubation, pick a single colony and streak it onto an NBTA medium plate, then incubate at 28°C.
[0051] S3. After 48 hours of culture, a single colony of primary type I bacteria (blue-green) was inoculated into LB liquid medium and cultured at 28℃ and 150r / min for 24 hours to obtain seed culture solution.
[0052] S4. Inoculate the seed culture medium with an inoculum of 6% by volume and carry out segmented temperature-controlled fermentation. The segmented temperature control is as follows: the fermentation temperature is controlled at 35℃ from the start of fermentation to 18 hours, and at 25℃ from 18 to 36 hours. The rotation speed is 150 r / min throughout the fermentation process. Collect the fermentation broth after fermentation to obtain the fermentation product.
[0053] Example 2
[0054] This embodiment provides a segmented temperature-controlled fermentation method for nematode symbiotic bacteria, including the following steps:
[0055] S1. Take the preserved Enterobacter nematodes NK and streak it onto NA medium plates, then incubate at 28°C.
[0056] S2. After 24 hours of incubation, pick a single colony and streak it onto an NBTA medium plate, then incubate at 28°C.
[0057] S3. After 48 hours of culture, a single colony of primary type I bacteria (blue-green) was inoculated into LB liquid medium and cultured at 28℃ and 150r / min for 24 hours to obtain seed culture solution.
[0058] S4. Inoculate the seed culture medium with an inoculum of 6% by volume and carry out segmented temperature-controlled fermentation. The segmented temperature control is as follows: the fermentation temperature is controlled at 30℃ from the start of fermentation to 18 hours, and at 28℃ from 18 to 36 hours. The rotation speed is 150 r / min throughout the fermentation process. Collect the fermentation broth after fermentation to obtain the fermentation product.
[0059] Example 3
[0060] This embodiment provides a segmented temperature-controlled fermentation method for nematode symbiotic bacteria, including the following steps:
[0061] S1. Take the preserved Enterobacter nematodes NK and streak it onto NA medium plates, then incubate at 28°C.
[0062] S2. After 24 hours of incubation, pick a single colony and streak it onto an NBTA medium plate, then incubate at 28°C.
[0063] S3. After 48 hours of culture, a single colony of primary type I bacteria (blue-green) was inoculated into LB liquid medium and cultured at 28℃ and 150r / min for 24 hours to obtain seed culture solution.
[0064] S4. Inoculate the seed culture medium with an inoculum of 6% by volume and carry out segmented temperature-controlled fermentation. The segmented temperature control is as follows: the fermentation temperature is controlled at 33℃ from the start of fermentation to 18 hours, and at 28℃ from 18 to 36 hours. The rotation speed is 160 r / min throughout the fermentation process. Collect the fermentation broth after fermentation to obtain the fermentation product.
[0065] Example 4
[0066] This embodiment provides a segmented temperature-controlled fermentation method for nematode symbiotic bacteria, including the following steps:
[0067] S1. Take the preserved Enterobacter nematodes NK and streak it onto NA medium plates, then incubate at 28°C.
[0068] S2. After 24 hours of incubation, pick a single colony and streak it onto an NBTA medium plate, then incubate at 28°C.
[0069] S3. After 48 hours of culture, a single colony of primary type I bacteria (blue-green) was inoculated into LB liquid medium and cultured at 28℃ and 150r / min for 24 hours to obtain seed culture solution.
[0070] S4. Inoculate the seed culture medium with 6% (v / v) of the seed culture and carry out segmented temperature-controlled fermentation. The segmented temperature control is as follows: the fermentation temperature is controlled at 33℃ from the start of fermentation to 18 hours; 5 mL / L insulin solution is added 5 hours after the start of fermentation; the fermentation temperature is controlled at 28℃ from 18 to 36 hours. The fermentation speed is 140 r / min throughout the entire fermentation process. Collect the fermentation broth after fermentation to obtain the fermentation product.
[0071] Example 5
[0072] This embodiment provides a segmented temperature-controlled fermentation method for nematode symbiotic bacteria, including the following steps:
[0073] S1. Take the preserved Enterobacter nematodes NK and streak it onto NA medium plates, then incubate at 28°C.
[0074] S2. After 24 hours of incubation, pick a single colony and streak it onto an NBTA medium plate, then incubate at 28°C.
[0075] S3. After 48 hours of culture, a single colony of primary type I bacteria (blue-green) was inoculated into LB liquid medium and cultured at 28℃ and 150r / min for 24 hours to obtain seed culture solution.
[0076] S4. Inoculate the seed culture medium with 6% (v / v) of the seed culture and carry out segmented temperature-controlled fermentation. The segmented temperature control is as follows: the fermentation temperature is controlled at 33℃ from the start of fermentation to 18 hours; 0.5 mL / L insulin solution is added 8 hours after the start of fermentation; the fermentation temperature is controlled at 28℃ from 18 to 36 hours. The fermentation speed is 160 r / min throughout the entire fermentation process. Collect the fermentation broth after fermentation to obtain the fermentation product.
[0077] Example 6
[0078] This embodiment provides a segmented temperature-controlled fermentation method for nematode symbiotic bacteria, including the following steps:
[0079] S1. Take the preserved Enterobacter nematodes NK and streak it onto NA medium plates, then incubate at 28°C.
[0080] S2. After 24 hours of incubation, pick a single colony and streak it onto an NBTA medium plate, then incubate at 28°C.
[0081] S3. After 48 hours of culture, a single colony of primary type I bacteria (blue-green) was inoculated into LB liquid medium and cultured at 28℃ and 150r / min for 24 hours to obtain seed culture solution.
[0082] S4. Inoculate the seed culture medium with 6% (v / v) of the seed culture and carry out segmented temperature-controlled fermentation. The segmented temperature control is as follows: the fermentation temperature is controlled at 33℃ from the start of fermentation to 18 hours; 2 mL / L insulin solution is added 6 hours after the start of fermentation; the fermentation temperature is controlled at 28℃ from 18 to 36 hours. The fermentation speed is 150 r / min throughout the entire fermentation process. Collect the fermentation broth after fermentation to obtain the fermentation product.
[0083] Comparative Example 1
[0084] This comparative example provides a segmented temperature-controlled fermentation method using nematode symbiotic bacteria, including the following steps:
[0085] S1. Take the preserved Enterobacter nematodes NK and streak it onto NA medium plates, then incubate at 28°C.
[0086] S2. After 24 hours of incubation, pick a single colony and streak it onto an NBTA medium plate, then incubate at 28°C.
[0087] S3. After 48 hours of culture, a single colony of primary type I bacteria (blue-green) was inoculated into LB liquid medium and cultured at 28℃ and 150r / min for 24 hours to obtain seed culture solution.
[0088] S4. Inoculate the seed culture medium with an inoculum of 6% by volume and incubate at 28℃ and 150 r / min for 36 hours. Collect the fermentation broth after fermentation to obtain the fermentation product.
[0089] Comparative Example 2
[0090] This comparative example provides a segmented temperature-controlled fermentation method using nematode symbiotic bacteria, including the following steps:
[0091] S1. Take the preserved Enterobacter nematodes NK and streak it onto NA medium plates, then incubate at 28°C.
[0092] S2. After 24 hours of incubation, pick a single colony and streak it onto an NBTA medium plate, then incubate at 28°C.
[0093] S3. After 48 hours of culture, a single colony of primary type I bacteria (blue-green) was inoculated into LB liquid medium and cultured at 28℃ and 150r / min for 24 hours to obtain seed culture solution.
[0094] S4. Inoculate the seed culture medium with 6% (v / v) of the seed culture and incubate at 28°C and 150 rpm for 36 hours. Add 2 mL / L insulin solution 6 hours after fermentation begins. Collect the fermentation broth after fermentation to obtain the fermentation product.
[0095] Test case
[0096] This study used Pingjiu No. 4 chive variety from Pingdingshan Jiudu Agricultural Technology Co., Ltd. as the material.
[0097] 1. Isolation, purification, morphological and molecular biological identification, and pathogenicity identification of the pathogen causing spoilage in chives.
[0098] (1) Isolation of rotten pathogens: After rinsing the rotten leek leaves with sterile water and drying them, the junction of healthy and rotten leek leaves was cut into small squares using the tissue isolation method. The squares were placed on PCA medium and cultured at 37°C for 2 days.
[0099] (2) Purification of putrefactive pathogens: After the putrefactive pathogens to be isolated grow into single colonies with a diameter of 1 cm, the single colonies are cut off and recultured in new PCA medium using the three-point inoculation method, and repeated 3 times.
[0100] (3) Morphological and microscopic observation: The purified putrefactive pathogens were inoculated onto PCA medium and cultured at 37°C for 48 h. The morphological characteristics and color of the colonies were recorded. The cultured putrefactive pathogens were Gram-stained and prepared into slides for observation under a microscope. The characteristics were recorded. The results are as follows: Figure 1 .Depend on Figure 1 It can be seen that the colonies of the putrefactive pathogen are nearly circular, with a distinct white protrusion in the center and a lighter, almost transparent edge. Gram staining confirms that the putrefactive pathogen is a Gram-negative bacterium.
[0101] (4) Molecular biological identification of the putrefactive pathogen: DNA was extracted using the Sangon Biotech bacterial genomic DNA kit. Universal primers for bacterial identification were used: the upstream primer was 5'-AGTTTGATCMTGGCTCAG-3', and the downstream primer was 5'-GGTTACCTTGTTACGACTT-3'. The amplification system followed the PCR reaction procedure as per the kit instructions. The products were detected by electrophoresis and sent to the Scientific Compass testing institution for sequencing. The results were compared and analyzed in the NCBI database, and a phylogenetic tree was constructed using MEGA software (e.g., ...). Figure 2 As shown in the figure, homology analysis was performed.
[0102] PCR amplification of the causative pathogen yielded a band of approximately 1500 bp. The obtained 16S rDNA sequence of the causative pathogen was compared with the NCBI database, showing a 99% homology with the sequence of *Raoultella terrigena*. Figure 2 It can be seen that the causative pathogen is most closely related to Raoultella terrigena. Based on the morphological characteristics of the causative pathogen and the 16S rDNA sequence comparison results, the causative pathogen of postharvest rot in chives was identified as Raoultella terrigena.
[0103] (5) Pathogenicity identification of the rot-causing pathogen: The purified rot-causing pathogen was inoculated onto healthy leek leaves using the "needle prick" and "no treatment" methods to evaluate the pathogenicity of the pathogen to leeks. The pathogenicity of the rot-causing pathogen was identified according to Koch's postulates. Figure 3 As shown, the leaves of chives inoculated with the rot-causing pathogen were more severely rotten than those of the control group. The chives inoculated with "damage" showed the most severe rot and rotted faster than those inoculated with "undamaged" pathogens, indicating that the pathogen has a strong pathogenicity on post-harvest chives.
[0104] 2. The effect of fermentation products on the prevention and control of pathogens that cause spoilage in chives.
[0105] Pick the septic pathogen with an inoculation loop and inoculate it into PCA liquid culture medium. The liquid culture medium is placed in a constant temperature shaking incubator and cultured at 28℃ and 150r / min for 48 h.
[0106] 1) Plate test of fermentation products inhibiting the growth of putrefactive pathogens
[0107] Thirty-three PCA culture plates were prepared, with putrefactive pathogens added to each plate after 48 hours of incubation. The plates were spread evenly and divided into 11 groups of three plates each. These groups were: blank control group, fermentation medium group, insulin group, Example 1 group, Example 2 group, Example 3 group, Example 4 group, Example 5 group, Example 6 group, Comparative Example 1 group, and Comparative Example 2 group. In the blank control group, 1cm diameter filter paper soaked in sterile water was placed on the PCA culture plates. In the fermentation medium group, 1cm diameter filter paper soaked in fermentation medium was placed on the PCA culture plates. In the insulin group, 1cm diameter filter paper soaked in insulin solution was placed on the PCA culture plates. In Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Comparative Example 1 group, and Comparative Example 2 group, 1cm diameter filter paper soaked in the fermentation products obtained in Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Comparative Example 1 group, and Comparative Example 2 group were respectively placed on PCA culture plates containing 1cm diameter filter paper soaked in the fermentation products obtained in Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Comparative Example 1 group, and Comparative Example 2 group.
[0108] The PCA culture plates were incubated at 28℃ for 48 hours, and the results are as follows: Figure 4 , 5 As shown in Figure 6. The diameter of the inhibition zone was measured using the cross-sectional method, and the average diameter of the inhibition zone from three parallel experiments was calculated. The results are shown in Table 1.
[0109] Table 1. Inhibition zones of each group against putrefactive pathogens
[0110]
[0111] From Table 1 and Figure 4 , 5 As shown in Figures 6 and 7, the size of the inhibition zones in the fermentation medium group and the insulin group was not different from that in the blank control group, indicating that the fermentation medium and insulin had no direct inhibitory effect on the putrefactive pathogens. Compared with the blank control group, the inhibition zones in groups 1 to 6, and groups 1 and 2 of Examples 1 to 6, and groups 1 and 2 of Comparative Examples 1 and 2 were significantly larger than those in the blank control group; the inhibition zones in groups 1 to 3 of Examples 1 were significantly larger than those in Comparative Example 1, which did not undergo segmented temperature control fermentation or add insulin during the fermentation process; the inhibition zones in groups 4 to 6 of Examples 4 were significantly larger than those in Comparative Example 2, which did not undergo segmented temperature control fermentation but added insulin during the fermentation process, indicating that segmented temperature control is beneficial for promoting the production of substances that inhibit putrefactive pathogens by the nematode symbiotic bacteria; the inhibition zones in groups 4 to 6 of Examples 4 to 6 were larger than those in groups 1 to 3, indicating that insulin was added to the fermentation medium during the fermentation stage in Examples 4 to 6, suggesting that the addition of insulin can promote the production of substances that inhibit putrefactive pathogens by the nematode symbiotic bacteria.
[0112] 2) Experiment on the inhibition of the growth of rot-causing pathogens on leek leaves and changes in leek quality by fermentation products.
[0113] A quantity of fresh chives was collected, and the following groups were established: a blank control group, a fermentation medium group, Example 1 group, Example 2 group, Example 3 group, Example 4 group, Example 5 group, Example 6 group, Comparative Example 1 group, and Comparative Example 2 group. After inoculating the chives with the rot-causing pathogen via needle prick for 48 hours, the blank control group was sprayed with sterile water, the fermentation medium group was sprayed with fermentation medium, and Example 1 group, Example 2 group, Example 3 group, Example 4 group, Example 5 group, Example 6 group, Comparative Example 1 group, and Comparative Example 2 group were sprayed with the fermentation products obtained from Examples 1, 2, 3, 4, 5, 6, 1, and 2, respectively. The spraying amount was based on a chive weight: spraying volume ratio of 25:1, and the fermentation product bacterial concentration was 10. 6 CFU / mL. Spray once every 24 hours for 5 consecutive days. Observe the changes in the chive leaves and detect relevant indicators of chive quality changes.
[0114] Figure 7 The condition of the chive leaves on day 5 after spraying was recorded in the blank control group, Example 6 group, and Comparative Example 1 group. Figure 7 It can be seen that by inoculating the rot-causing pathogens on the "wounded" leek leaves and then spraying the fermentation product, the fermentation product obtained in Example 6 has a very good antibacterial effect on the rot-causing pathogens. The leaves do not have obvious rot-causing pathogens and are not rotten, and are very fresh.
[0115] The quality indicators of each group of chives were then measured using the following method (measured on days 1, 3, 5, 7 and 10 respectively).
[0116] 1> Vitamin C content determination: Weigh 2g of leek sample from each group, chop it, add 10mL of 10% HCl, grind the leek into a slurry, centrifuge at 4500r / min for 20min, and collect the supernatant as the leek extract. Take 0.2mL of the leek extract, add 0.4mL of 10% HCl and 9.4mL of distilled water, shake well, transfer to a quartz cuvette, set the wavelength of the UV spectrophotometer to 243nm, and measure the absorbance of the leek extract (A1); take a test tube and add 1.2mL of 1mol / L NaOH solution, 3mL of distilled water, and the corresponding 0.3mL of leek extract, add 1.2mL of 10% HCl and 9.3mL of distilled water, and measure the absorbance of the extract at 243nm (A2). Therefore, the absorbance of the leek extract = A1. A2. Calculation of Vitamin C Content: The vitamin C content of leeks was calculated based on the standard curve equation of vitamin C and the following formula. The results are shown in Table 2.
[0117] Vitamin C content =
[0118] In the formula: c is the vitamin C concentration of chives (μg / mL); V 待测总 The total volume (mL) of the leek vitamin C extract; V 总 V1 is the total volume (mL) of the homogenized leek paste after grinding; V2 is the volume (ml) of the leek VC extract taken for absorbance measurement; W 总 The weight of chives is (g); 100 means 100g of chives.
[0119] Table 2. Results of Vitamin C Content Changes in Each Group of Leeks
[0120]
[0121] 2. Chlorophyll Content Determination: Weigh an appropriate amount of leek sample and grind it until the leek tissue turns white. Pour the mixture into a funnel, filter, and dilute to a final volume. Measure the absorbance of the leek chlorophyll extract at 665 nm and 649 nm, and calculate the absorbance using the formula. The results are shown in Table 3.
[0122] Chlorophyll content (mg / g)
[0123] In the formula: C is the pigment content (mg / mL); V is the total volume of the extract (mL); N is the dilution factor of the extract; W is the mass of the chives (g).
[0124] Table 3. Results of chlorophyll content changes in leek leaves in each group
[0125]
[0126] From Table 2 and Figure 8 Table 3 and Figure 9 It can be seen that the changes in vitamin C and chlorophyll content in the fermentation medium group and the insulin group were basically similar to those in the blank control group, indicating that the fermentation medium and insulin had no direct effect on delaying the decline in vitamin C and chlorophyll content in leeks. The rate of decline in vitamin C and chlorophyll content in Examples 1 to 6 and Comparative Examples 1 and 2 was significantly lower than that in the blank control group. The rate of decline in vitamin C and chlorophyll content in Examples 1 to 3 was significantly lower than that in Comparative Example 1. The rate of decline in vitamin C and chlorophyll content in Examples 4 to 6 was significantly lower than that in Examples 1 to 3 and Comparative Example 2. Examples 1 to 3 underwent segmented temperature-controlled fermentation without the addition of insulin. Comparative Example 1 did not undergo segmented temperature-controlled fermentation or the addition of insulin. Comparative Example 2 did not undergo segmented temperature-controlled fermentation but added insulin. This shows that segmented temperature-controlled fermentation and the addition of insulin can promote the production of substances that inhibit leeks' rot by nematode symbiotic bacteria, thus enabling the fermentation products to delay the decline in vitamin C and chlorophyll content in leeks.
[0127] 3. Determination of Malondialdehyde (MDA) Content: A suitable amount of leek sample was ground into a paste with 10% TCA solution and centrifuged at 5500 r / min. The supernatant was the MDA extraction supernatant. In a test tube, 0.67% TCA and the MDA extraction supernatant were added, mixed, and heated to boiling. After cooling, the absorbance was measured at wavelengths of 450 nm, 532 nm, and 600 nm. The MDA content in the leek was calculated using the following formula. The results are shown in Table 4.
[0128] MDA (μmol / g)
[0129] Where: OD 450 OD 532 OD 600 Vs represents the absorbance of the test mixture at wavelengths of 450 nm, 532 nm, and 600 nm; Vs is the volume of the malondialdehyde extract of the leek (mL); V is the total volume of the extract (mL); and m is the mass of the leek (g).
[0130] Table 4. Results of malondialdehyde (MDA) content changes in each group of chives
[0131]
[0132] From Table 4 and Figure 10 It can be seen that the changes in malondialdehyde (MDA) content in the fermentation medium group and the insulin group were basically similar to those in the blank control group, indicating that the fermentation medium and insulin had no direct effect on delaying the increase in MDA content in leeks. Compared with the blank control group, the increase rate of MDA content in Examples 1 to 6 and Comparative Example 2 was significantly lower. The increase rate of MDA content in Examples 4 to 6 was lower than that in Examples 1 to 3. The increase rate of MDA content in Comparative Examples 1 and 2 was significantly higher than that in Examples 1 to 6. Comparative Example 1 did not undergo segmented temperature-controlled fermentation or add insulin during the fermentation process, while Comparative Example 2 did not undergo segmented temperature-controlled fermentation but added insulin. It can be seen that segmented temperature-controlled fermentation and the addition of insulin have a promoting effect on the production of substances that inhibit leeks' rot caused by nematode symbiotic bacteria.
[0133] 4. Peroxidase (POD) Activity Assay: The sample was chopped and placed in a cold mortar. Phosphate buffer was added, and the mixture was ground into a slurry. The slurry was then transferred to a centrifuge tube and centrifuged at 4 °C and 5500 r / min for 25 min. The supernatant was collected as the crude POD extract. Three test tubes were filled with 1.8 mL of 0.2% guaiacol, 2 mL of phosphate buffer, 2 mL of 0.3% H2O2, and 0.2 mL of the crude POD extract, respectively. The absorbance at 470 nm was measured, and the ΔOD per gram of sample per minute was recorded. 470Add 1 to become 1 POD activity unit, and calculate the POD activity according to the following formula. The results are shown in Table 5.
[0134] U
[0135] Where: ΔOD 470 V represents the change in absorbance of the mixture during the reaction time. s V is the volume of crude extract (mL); V is the total volume of leek POD extract (mL); m is the mass of leek (g); and t is the reaction time (min).
[0136] Table 5. Results of peroxidase activity changes in each group of chives
[0137]
[0138] From Table 5 and Figure 11 It can be seen that the changes in peroxidase activity in the fermentation medium group and the insulin group were basically similar to those in the blank control group, indicating that the fermentation medium and insulin had no direct effect on delaying the increase in peroxidase activity in leeks. Compared with the blank control group, the increase rate of peroxidase activity in leek groups 1 to 6, comparative example 1, and comparative example 2 was significantly lower than that in the blank control group. The increase rate of peroxidase activity in leek groups 4 to 6 was significantly lower than that in groups 1 to 3. The increase rate of peroxidase activity in leek groups 1 and 2 was significantly higher than that in groups 1 to 6. Comparative example 1 did not undergo segmented temperature-controlled fermentation or add insulin during the fermentation process, while comparative example 2 did not undergo segmented temperature-controlled fermentation but added insulin. It can be seen that segmented temperature-controlled fermentation and the addition of insulin have a promoting effect on the production of substances that inhibit leeks rot caused by nematode symbiotic bacteria.
[0139] 5. Catalase (CAT) Activity Assay: Leek samples were ground into a slurry in a cold mortar with buffer solution. The slurry was centrifuged at 4℃ and 5500 rpm for 25 min. The supernatant was collected and stored at low temperature; this supernatant is the CAT extract. The reaction system consisted of 0.15 mL of crude CAT extract, 2.8 mL of 0.1 mol / L PBS, and 1 mL of 0.3% H2O2 in a test tube. The absorbance was measured at 240 nm as ΔOD per gram of sample per minute. 240 A decrease of 0.1 in the value represents one CAT activity unit. CAT activity was calculated using the following formula, and the results are shown in Table 6.
[0140] U
[0141] Where: ΔOD 240 V represents the change in absorbance of the mixture at a wavelength of 240 nm. SV is the volume of CAT extract of chives added (mL); V is the total volume of CAT extract of chives (mL); m is the mass of chives (g); and t is the reaction time (min).
[0142] Table 6. Results of catalase activity changes in each group of leeks
[0143]
[0144] From Table 6 and Figure 12 It can be seen that the changes in catalase activity in the fermentation medium group and the insulin group were basically similar to those in the blank control group, indicating that the fermentation medium and insulin had no direct effect on delaying the decrease in catalase activity in leeks. The rate of decrease in catalase activity in Examples 1 to 6 was lower than that in the blank control group, Comparative Example 1, and Comparative Example 2, demonstrating that the fermentation products obtained in Examples 1 to 6 can effectively delay the decrease in catalase activity in leeks.
[0145] 6. Ascorbate peroxidase (APX) activity assay: Leek samples were added to buffer solution and ground into a slurry under ice bath conditions. The slurry was then centrifuged at 4℃ and 5500 r / min for 25 min, and the supernatant was used as the crude extract. The reaction system consisted of 2.6 mL PBS buffer, 0.1 mL crude extract, and 0.3 mL 2 mmol / L H2O2. The absorbance was measured at 290 nm, and expressed as ΔOD per gram of leeks per minute. 290 A reduction of 0.01 represents one unit of enzyme activity. APX activity is calculated using the following formula:
[0146] U
[0147] Where: ΔOD 290 V represents the change in absorbance of the extract; S V is the volume of crude extract during the determination (mL); V is the total volume of extract (mL); m is the mass of leek (g).
[0148] Table 7. Results of ascorbate peroxidase activity assay in each group of leeks
[0149]
[0150] From Table 7 and Figure 13It can be seen that the changes in ascorbic acid peroxidase activity in the fermentation medium group and the insulin group were basically similar to those in the blank control group, indicating that the fermentation medium and insulin had no direct effect on delaying the decrease in ascorbic acid peroxidase activity in leeks. The rate of decrease in ascorbic acid peroxidase activity in the groups of Examples 1 to 6 was significantly lower than that in the blank control group and Comparative Example 1, indicating that the fermentation products obtained in Examples 1 to 6 had a significant inhibitory effect on the leeks rot-causing pathogen. Comparative Example 1 did not undergo segmented temperature-controlled fermentation or add insulin during the fermentation process, while Comparative Example 2 did not undergo segmented temperature-controlled fermentation but added insulin. This shows that segmented temperature-controlled fermentation and the addition of insulin promoted the production of substances by the nematode symbiotic bacteria that inhibit the leeks rot-causing pathogen.
[0151] Vitamin C is an important nutrient and antioxidant in leeks; malondialdehyde (MDA) is the main product of membrane peroxidation in plant tissues, and its content is an important indicator for measuring lipid peroxidation and membrane oxidative damage; changes in chlorophyll content in leeks can indirectly reflect the aging status of leeks; peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) are all related to preventing fruit and vegetable aging and maintaining plant antioxidant capacity.
[0152] In summary, the fermentation broth of the nematode symbiotic bacterium Enterobacter NK of the present invention can effectively delay the decline in vitamin C and chlorophyll content in chives, slow down the loss of nutritional quality of chives, and maintain the color of chive leaves; it can also effectively inhibit the increase of malondialdehyde content, reduce the degree of cell membrane lipid peroxidation, slow down the damage to chive cell membranes, maintain the activity of ascorbic acid peroxidase and catalase, reduce the damage of reactive oxygen species to chive tissues, maintain the antioxidant capacity of chives, delay the aging of chive tissues, and improve the storage quality of chives.
[0153] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.
Claims
1. A method for staged temperature-controlled fermentation of nematode symbiotic bacteria, characterized in that, During the fermentation stage, the temperature is controlled in stages: from the start of fermentation to 18 hours, the fermentation temperature is controlled at 30-35℃, and from 18 to 36 hours, the fermentation temperature is controlled at 25-28℃.
2. The method for segmented temperature-controlled fermentation of nematode symbiotic bacteria according to claim 1, characterized in that, The segmented temperature control is as follows: the fermentation temperature is controlled at 33℃ from the start of fermentation to 18 hours of fermentation, and at 28℃ from 18 to 36 hours of fermentation.
3. The method for segmented temperature-controlled fermentation of nematode symbiotic bacteria according to claim 1 or 2, characterized in that, Add insulin 5–8 hours after fermentation begins.
4. The segmented temperature-controlled fermentation method for nematode symbiotic bacteria according to claim 3, characterized in that, The amount of insulin added is 0.001 to 0.01 mg / L.
5. The segmented temperature-controlled fermentation method for nematode symbiotic bacteria according to claim 4, characterized in that, The amount of insulin added was 0.004 mg / L.
6. The method for segmented temperature-controlled fermentation of nematode symbiotic bacteria according to claim 5, characterized in that, The nematode symbiotic bacterium is Enterobacter nematode NK.
7. The method for segmented temperature-controlled fermentation of nematode symbiotic bacteria according to claim 6, characterized in that, The fermentation medium consists of: Na2SO4 1.5 g / L, MgSO4 1.3 g / L, (NH4)2SO4 2.4 g / L, peptone 20 g / L, KH2PO4 0.7 g / L, glucose 9 g / L, K2HPO4 0.5 g / L, and pH 7.2–7.
4.
8. The method for segmented temperature-controlled fermentation of nematode symbiotic bacteria according to claim 7, characterized in that, Insulin was added 6 hours after fermentation began.
9. A segmented temperature-controlled fermentation product of a nematode symbiotic bacterium.
10. Application of a segmented temperature-controlled fermentation product of a nematode symbiotic bacterium in the prevention and control of leek rot disease.