A Lactobacillus casei with the activity of inhibiting Fusarium dimerum and its application in the storage of green peppers

Abstract

The present invention discloses a Lactobacillus casei strain with the activity of inhibiting Alternaria alternata and its application in the storage of green peppers. The present invention provides Lactobacillus casei ( Lactobacillus casei ) GHY-4, with the preservation registration number of CGMCC No. 31925. Further, the present invention provides a bacterial agent of Lactobacillus casei GHY-4. The present invention also protects the application of Lactobacillus casei GHY-4 or the said bacterial agent in inhibiting Alternaria alternata. The present invention also protects the application of Lactobacillus casei GHY-4 or the said bacterial agent in the fresh-keeping of green peppers. The present invention can be used for the fresh-keeping of green peppers and has application and popularization value for the green pepper industry.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a Lactobacillus casei with the activity of inhibiting Alternaria alternata and its application in the storage of green peppers. Background Art

[0002] Green peppers are highly nutritious and are vegetables commonly liked by people all over China. Every year, a large amount of fresh green peppers are transported from one region to another. The rot caused by post-harvest infectious diseases of green peppers accounts for about 50%-90% of the total loss. There are many types of infectious diseases that cause a large amount of fruit rot during the storage of green peppers. Some diseases not only cause a large amount of infection and harm to the growth period of green pepper fruits, but also have a stronger infectivity and higher incidence on stored green peppers. Green pepper black spot disease is a disease that mainly harms green peppers caused by Alternaria alternata. Its symptoms mainly show that small round slightly sunken spots initially appear at the infected part, and then the spots rapidly expand into sunken lesions. Gray to black molds can form at the lesions. At the same time, the inner wall of the fruit is infected by the molds, and the serious rot of the fruit is more obvious during transportation and storage. How to extend the storage and sales period of green peppers and reduce post-harvest losses through appropriate post-harvest treatment technologies is an urgent problem to be solved.

[0003] At present, the preservation technology of green peppers mainly relies on chemical preservatives. Common chemical preservatives currently used in fruits and vegetables include ClO 2 , 1-methylcyclopropane (1-MCP), salicylic acid (SA), etc. However, continuous use of chemical preservatives will have potential problems such as the generation of drug resistance in pathogenic bacteria, environmental pollution, and residues in agricultural products that endanger health. Physical antibacterial preservation technologies mainly include heat treatment, low-temperature storage, modified atmosphere packaging, and irradiation storage. Although physical antibacterial preservation technologies can avoid the risk of harm caused by chemical antibacterial preservation, physical preservation technologies such as low-temperature storage, modified atmosphere storage, heat treatment, etc. have disadvantages such as high energy consumption in operation, high investment in equipment, and low applicability, and have certain limitations in use. Biogenic antibacterial preservation technologies have the characteristics of strong targeting, low preparation cost, wide source channels, small regional differences, less pollution, low drug resistance, and low toxic and side residues. They are considered a sustainable, safe, and environmentally friendly modern antibacterial preservation technology. Exploring new and highly efficient antagonistic bacteria has important theoretical and practical significance, and can also accelerate the commercialization process of microbial control of fruits, which is of great significance for promoting the rapid and healthy development of the vegetable industry. Summary of the Invention

[0004] The purpose of the present invention is to provide a Lactobacillus casei with the activity of inhibiting Alternaria alternata and its application in the storage of green peppers.

[0005] The present invention provides Lactobacillus casei ( lactobacillus casei ) GHY-4.

[0006] Lactobacillus casei ( Lactobacillus casei ) GHY-4 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on September 10, 2024, and the deposit registration number is CGMCC No. 31925.

[0007] Furthermore, the present invention provides an inoculant of Lactobacillus casei GHY-4.

[0008] Specifically, the inoculant is a liquid inoculant.

[0009] The active ingredient provided by the inoculant is Lactobacillus casei GHY-4.

[0010] In the inoculant, the content of Lactobacillus casei GHY-4 is 1×10 6 cfu / mL - 1×10 10 cfu / ml.

[0011] Specifically, the preparation method of the inoculant is as follows: inoculate Lactobacillus casei GHY-4 into MRS liquid medium, and culture it by shaking at 30°C and 150 rpm until the bacterial concentration reaches 1×10 10 cfu / mL.

[0012] Specifically, the preparation method of the inoculant is as follows: inoculate Lactobacillus casei GHY-4 into MRS liquid medium, and culture it by shaking at 30°C and 150 rpm until the bacterial concentration reaches 1×10 6 cfu / mL.

[0013] The present invention also protects the application of Lactobacillus casei GHY-4 or the inoculant in inhibiting Alternaria.

[0014] The present invention also protects the application of Lactobacillus casei GHY-4 or the inoculant in preparing an Alternaria inhibitor.

[0015] The present invention also protects the application of Lactobacillus casei GHY-4 or the inoculant in the fresh-keeping of green peppers.

[0016] The present invention also protects the application of Lactobacillus casei GHY-4 or the inoculant in preparing a green pepper fresh-keeping agent.

[0017] The present invention also protects the application of Lactobacillus casei GHY-4 or the inoculant, which is any one of the following (a) to (i):

[0018] (a) Inhibiting the rot of green pepper fruits;

[0019] (b) Delaying the ripening process of green pepper fruits after picking;

[0020] (c) Retarding the decrease in hardness of green pepper fruits after harvesting;

[0021] (d) Inhibiting the increase in cell membrane permeability of green pepper fruits after harvesting;

[0022] (e) Reducing the respiration intensity of green pepper fruits;

[0023] (f) Increasing the soluble solid content of green pepper fruits;

[0024] (g) Inhibiting the degradation of titratable acid in green pepper fruits;

[0025] (h) Increasing the ascorbic acid content of green pepper fruits;

[0026] (i) Increasing the chlorophyll content of green pepper fruits.

[0027] The present invention also protects the application of Lactobacillus casei GHY-4 or the said bacterial agent in the preparation of products;

[0028] The present invention also protects a product, which comprises Lactobacillus casei GHY-4 or the said bacterial agent.

[0029] Any of the above-mentioned products is a reagent or a kit.

[0030] Any of the above-mentioned products is an Alternaria inhibitor.

[0031] Any of the above-mentioned products is a green pepper preservative.

[0032] The use of the said product is any one of the following (a) to (i):

[0033] (a) Inhibiting the rot of green pepper fruits;

[0034] (b) Retarding the ripening process of green pepper fruits after harvesting;

[0035] (c) Retarding the decrease in hardness of green pepper fruits after harvesting;

[0036] (d) Inhibiting the increase in cell membrane permeability of green pepper fruits after harvesting;

[0037] (e) Reducing the respiration intensity of green pepper fruits;

[0038] (f) Increasing the soluble solid content of green pepper fruits;

[0039] (g) Inhibiting the degradation of titratable acid in green pepper fruits;

[0040] (h) Increasing the ascorbic acid content of green pepper fruits;

[0041] (i) Increasing the chlorophyll content of green pepper fruits.

[0042] Specifically, any of the above-mentioned Alternaria is Alternaria viticola.

[0043] Exemplarily, any of the above-mentioned green peppers is a yellow-skinned hot pepper.

[0044] The present invention provides a new strain of Lactobacillus casei, which has an antibacterial effect against Alternaria, and after soaking the green pepper fruits with the bacterial liquid of this strain, it can delay the ripening process of the green pepper fruits and inhibit or delay their decay. The present invention can be used for the preservation of green peppers and has application and promotion value for the green pepper industry.

[0045] Deposition description

[0046] Species name: Lactobacillus casei

[0047] Latin name: Lactobacillus casei

[0048] Taxonomic naming: Lactobacillus casei ( Lactobacillus casei )

[0049] Strain number: GHY-4

[0050] Depository: General Microbiological Center of China Committee for Culture Collection of Microorganisms

[0051] Abbreviation of depository: CGMCC

[0052] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0053] Deposition date: September 10, 2024

[0054] Registration number in the depository center: CGMCC No. 31925 Description of the drawings

[0055] Figure 1 It is a morphological photograph of Lactobacillus casei GHY-4 in Example 1.

[0056] Figure 2 It is a photograph of the antibacterial test of Lactobacillus casei GHY-4 against Alternaria in Example 2.

[0057] Figure 3 It is an exemplary photograph of green peppers at each sampling time point in Example 3.

[0058] Figure 4 It is the statistical result of the decay rate in Example 3.

[0059] Figure 5 It is the statistical result of the hardness in Example 3.

[0060] Figure 6 It is the statistical result of the cell membrane permeability in Example 3.

[0061] Figure 7It is the statistical result of the breathing intensity in Example 3.

[0062] Figure 8 It is the statistical result of the soluble solid content in Example 3.

[0063] Figure 9 It is the statistical result of the titratable acid content in Example 3.

[0064] Figure 10 It is the statistical result of the VC content in Example 3.

[0065] Figure 11 It is the statistical result of the chlorophyll content in Example 3. Detailed implementation manners

[0066] The present invention will be further described in detail below in conjunction with the specific implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0067] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged. Excel 2013, SPSS 26.0 and Hiplot software are used to organize, analyze and plot the data. When performing variance analysis and multiple significant difference analysis, p < 0.05 indicates a significant difference. The Alternaria used in Example 2 is Alternaria viticola (China Center for Agricultural Culture Collection, strain number ACCC 36134).

[0068] MRS liquid medium (pH 6.2 ± 0.2): containing 10.0 g of peptone, 5.0 g of beef extract powder, 4.0 g of yeast extract powder, 20.0 g of glucose, 1.0 ml of Tween-80, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 2.0 g of ammonium citrate, 15.0 g of agar, 0.2 g of MgSO 4 •7H 2 O, 0.05 g of MnSO 4 •4H 2 O, and the balance is distilled water.

[0069] Example 1, Isolation, Identification and Preservation of Lactobacillus casei GHY-4

[0070] Strain GHY-4 was screened from traditional Gansu sour water.

[0071] Morphological characteristics of strain GHY-4 (see Figure 1 ): Round-ended straight bacilli, about 0.85 μm × 4.1 μm, single; surface colony diameter is about 2.4 mm, convex, round, smooth, fine, white in color.

[0072] Physiological and biochemical characteristics of strain GHY-4: Gram-positive, producing lactic acid.

[0073] Genomic DNA of strain GHY-4 was extracted, and PCR amplification was carried out using the primer pair composed of primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 2) and primer 1492R (5'-TACGGCTACCTTGTTACGACTT-3', SEQ ID NO: 3). Then the amplified product was recovered and sequenced. Primer 27F and primer 1492R are universal primers targeting the coding gene of 16S rRNA. The sequencing result is shown in SEQ ID NO: 1. The sequencing result was subjected to homology comparison in NCBI.

[0074] Based on the above morphological characteristics, physiological and biochemical characteristics, and molecular identification results, strain GHY-4 belongs to Lactobacillus casei, and it is named Lactobacillus casei GHY-4.

[0075] Lactobacillus casei ( Lactobacillus casei ) GHY-4 was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on September 10, 2024, and the deposit registration number is CGMCC No. 31925.

[0076] Example 2. In vitro inhibition evaluation of Lactobacillus casei GHY-4 against Alternaria

[0077] I. Preparation of GHY-4 bacterial liquid

[0078] The activated Lactobacillus casei GHY-4 was inoculated into MRS liquid medium and cultured with shaking at 30 °C and 150 rpm until the bacterial concentration reached 1×10 10 cfu / mL, which is the GHY-4 bacterial liquid.

[0079] II. Detection of the inhibitory effect of Lactobacillus casei GHY-4 on Alternaria

[0080] 1. Pick a loopful of Alternaria colony, transfer it to 10 ml of physiological saline and shake for 2 min, then filter through a 400-mesh gauze to remove spores, and collect the filtrate (detected by hemocytometer, the Alternaria content in the filtrate is 8×10 5cfu / ml), coat the LB solid medium plate. The plate coated with 100 μl filtrate / plate is called the 100 μl plate, and the plate coated with 200 μl filtrate / plate is called the 200 μl plate.

[0081] 2. Group treatment:

[0082] 100 μl experimental group: Dip the inoculation loop into the GHY-4 bacterial liquid prepared in step 1 and streak on the 100 μl plate (2 μl bacterial liquid / plate), and then culture at 30 °C for 48 h.

[0083] 200 μl experimental group: Dip the inoculation loop into the GHY-4 bacterial liquid prepared in step 1 and streak on the 200 μl plate (2 μl bacterial liquid / plate), and then culture at 30 °C for 48 h.

[0084] CK group: Take the 100 μl plate without any treatment and culture at 30 °C for 48 h.

[0085] Perform three repeated treatments with consistent results. Exemplary photos are shown in Figure 2 . It can be clearly seen that Lactobacillus casei GHY-4 can effectively inhibit the growth of Alternaria alternata. As the addition amount of Alternaria alternata increases, the inhibitory effect of Lactobacillus casei GHY-4 on Alternaria alternata weakens.

[0086] Example 3. Application of Lactobacillus casei GHY-4 in the preservation of green peppers

[0087] The variety of green pepper fruits used is yellow-skinned hot pepper ( Capsicum annuum L.), purchased from Shenxian County, Liaocheng, Shandong. All fruits only take 24 h from harvest to transportation to the laboratory, and are transported under cold chain throughout the process. After the fruits arrive at the laboratory, they are left standing for 12 h before the experiment. Green pepper fruits with uniform size, no mechanical damage on the surface, and no pests and diseases are selected for the experiment.

[0088] I. Preparation of GHY-4 bacterial liquid

[0089] Inoculate the activated Lactobacillus casei GHY-4 into MRS liquid medium and culture it with shaking at 30 °C and 150 rpm until the bacterial concentration reaches 1×10 6 cfu / mL, which is the GHY-4 bacterial liquid.

[0090] II. Group treatment

[0091] Take green pepper fruits and divide them into two groups, and treat them as follows:

[0092] Experimental group (GHY-4 group): Immerse 300 green pepper fruits in the GHY-4 bacterial liquid for 15 s, then take them out and drain until no water drips, and then lay them flat in a cardboard box with compartments (1 green pepper fruit is placed in each compartment).

[0093] Control group (CK group): Soak 300 green pepper fruits in sterile water for 15 s, then take them out and drain until no water drips, and then lay them flat in a cardboard box with compartments (one green pepper fruit is placed in each compartment).

[0094] Storage conditions: humidity 80% ± 0.5%, temperature 25 ± 1°C.

[0095] Set 4 sampling time points, namely 0 hour of storage, 4 days later, 8 days later, and 12 days later.

[0096] III. Evaluation of fresh-keeping effect

[0097] 1. Influence on the decay rate

[0098] After harvesting, green peppers are extremely prone to water loss and decay, resulting in a decline in fruit quality and affecting the edible value and commercial value. Samples are taken at four sampling time points, and 10 are randomly selected from each group each time for detecting the fruit decay rate.

[0099] Decay rate (%) = number of rotten green peppers ÷ total number of green peppers × 100%.

[0100] Exemplary photos of green peppers at each sampling time point are shown in Figure 3 . The statistical results of the decay rate are shown in Figure 4 . Compared with the CK group, the fruit decay rate of the GHY-4 group is significantly reduced (p < 0.05). During the whole storage period, the decay rate of green peppers in the GHY-4 group remains at a low level, because Lactobacillus casei GHY-4 can effectively inhibit the large-scale reproduction of green pepper pathogenic bacteria.

[0101] 2. Influence on color and luster

[0102] The L* value represents the brightness of the fruit. The a* value represents the red-green value, and from negative to positive values indicates the change of the pericarp color from green to red or yellow. The b* value represents the yellow-blue value, and from negative to positive values indicates the change of the pericarp color from blue to yellow.

[0103] Samples are taken at four sampling time points, and 10 are randomly selected from each group each time. The color difference meter is used to measure the color parameters L*, a*, and b* of the fruit respectively, and the L*, a*, and b* values are recorded.

[0104] The results are shown in Table 1. The a* value of the green peppers in the GHY-4 group was lower than that of the green peppers in the CK group, indicating that the treatment with Lactobacillus casei GHY-4 could better maintain the green color of green peppers during postharvest storage. Compared with the green peppers in the CK group, the change range of L* in the GHY-4 group was smaller, indicating that the treatment with Lactobacillus casei GHY-4 could better maintain the fruit brightness of green peppers during postharvest storage. The b* value of the green peppers in the GHY-4 group was significantly lower than that of the green peppers in the CK group (P < 0.05). The results showed that the treatment with Lactobacillus casei GHY-4 could better maintain the color quality of green peppers and maintain the color.

[0105]

[0106] 3. Effect on hardness

[0107] Hardness can be used to judge the texture of fruits and is a major indicator reflecting fruit storability and measuring storage effects. As fruits age, various quality indicators of fruits also decline. For example, the deterioration of postharvest quality of vegetables is often accompanied by fruit softening. After the hardness decreases, fruits will develop bruises when subjected to mechanical impact or compression, and then the fruits will mildew and rot.

[0108] Samples were taken at four sampling time points, and 10 samples were randomly selected from each group each time. A texture analyzer was used to measure the fruit hardness.

[0109] The statistical results of hardness are shown in Figure 5 . The hardness of the green peppers in the GHY-4 group was significantly higher than that of the green peppers in the CK group (p < 0.05). Thus, it can be seen that the treatment with Lactobacillus casei GHY-4 can maintain a relatively high hardness of green peppers during postharvest storage, thereby maintaining the storage quality of green peppers and extending the shelf life.

[0110] 4. Effect on cell membrane permeability

[0111] The cell membrane permeability of fruit tissues reflects the integrity and aging degree of tissue cell membranes.

[0112] Sampling samples of the experimental group and the control group at 4 sampling time points were taken respectively. Weigh 3 g of fruits, cut them into 1-mm-thick slices, make up to 25 mL with deionized water, and shake at 110 rpm for 30 min (the conductivity measured at this time is conductivity P1), then boil for 10 min and quickly cool, and add deionized water to 25 mL (the conductivity measured at this time is conductivity P2). Measure the conductivity of deionized water, which is conductivity P0.

[0113] Calculate the cell membrane permeability (P).

[0114]

[0115] The statistical results of cell membrane permeability are shown in Figure 6The cell membrane permeability of the GHY-4 group of green peppers was significantly lower than that of the CK group of green peppers (p < 0.05), indicating that the treatment with Lactobacillus casei GHY-4 could effectively maintain the integrity of the cell membrane of green peppers, protect the cell tissue structure from being damaged, and reduce the membrane permeability of the fruits.

[0116] 5. Effects on Respiration Intensity

[0117] The change in respiration intensity is one of the important indicators of fruit and vegetable ripening. The higher the respiration intensity, the faster the fruit metabolism rate and the faster the decay rate. Green peppers are typical non-climacteric fruits, and the respiration rate shows a downward trend with the increase of storage time.

[0118] Take the sampling samples of the experimental group and the control group at 4 sampling time points (10 samples are taken for each group at each time point). Weigh 10 fruits and place them in a 2L airtight glass container for 20 minutes. Collect 1ml of the headspace gas in the glass container, which is the test gas. Use gas chromatography to detect the CO 2 content, and calculate the CO 2 production amount (in mg) per unit fresh weight (i.e., per kilogram of fresh weight) of the fruits in the unit time (i.e., per hour) in the glass container, which is the respiration intensity. The unit of respiration intensity is mg / (kg·h).

[0119] The statistical results of respiration intensity are shown in Figure 7 . Compared with the CK group of green peppers, the respiration intensity of the GHY-4 group of green peppers was significantly reduced (p < 0.05), indicating that the treatment with Lactobacillus casei GHY-4 could delay the ripening and senescence of green peppers.

[0120] 6. Effects on Soluble Solids and Titratable Acids

[0121] Soluble solids refer to the total of all compounds dissolved in water in liquid or fluid foods, including sugars, acids, vitamins, minerals, etc. Their content determines the edible taste of fruits. The content of titratable acids (TA) is an important factor affecting the flavor quality of green peppers and is also one of the important constituent traits of green pepper quality.

[0122] Take the sampling samples of the experimental group and the control group at 4 sampling time points (10 samples are taken for each group at each time point). Take the fruits, freeze them in liquid nitrogen and then crush them. Weigh 5g of the frozen sample, place it in a mortar and grind it thoroughly. Transfer all the fruit homogenate to a 100mL volumetric flask, dilute it to the mark with distilled water, shake well, let it stand for 30 minutes and then filter to collect the filtrate. Take the filtrate and use a PAL-1 digital refractometer to measure. The conversion coefficient of titratable acid content is calculated as malic acid (%), and the conversion coefficient of soluble solids content is calculated as soluble sugar (%).

[0123] The results are shown in Figure 8 andFigure 9 Compared with the green peppers in the CK group, the soluble solid content of the green peppers in the GHY-4 group was significantly increased, that is, the treatment with Lactobacillus casei GHY-4 could keep the green peppers with a relatively high soluble solid content. Compared with the green peppers in the CK group, the titratable acid content of the green peppers in the GHY-4 group was significantly increased.

[0124] 7. Effects on Ascorbic Acid Content and Chlorophyll Content

[0125] The ascorbic acid (VC) content is one of the important factors to measure the nutritional value of green peppers. The chlorophyll content directly affects the appearance quality of green pepper fruits. The sampling samples of the experimental group and the control group at 4 sampling time points were taken respectively (10 samples were taken for each group at each time point).

[0126] (1) Detection of Ascorbic Acid Content (Ammonium Molybdate Assay Method)

[0127] ① Solution Preparation:

[0128] Oxalic Acid-EDTA Solution: Take 6.3 g of oxalic acid and 0.75 g of disodium ethylenediaminetetraacetate, and make up to 1000 ml with distilled water.

[0129] 5% Ammonium Molybdate Solution: Take 5 g of ammonium molybdate and make up to 100 ml with distilled water.

[0130] 5% Sulfuric Acid Solution: Add 19 volumes of distilled water to 1 volume of 98% sulfuric acid.

[0131] 20% Glacial Acetic Acid Solution: Add 5 volumes of distilled water to 1 volume of glacial acetic acid.

[0132] Metaphosphoric Acid-Acetic Acid Solution: Take 15 g of metaphosphoric acid and 40 ml of 20% glacial acetic acid solution, add 250 ml of distilled water, dissolve and make up to 500 ml with distilled water.

[0133] ② Preparation of Sample Extract:

[0134] Weigh 2 g of fresh weight sampling samples, add 4 ml of oxalic acid-EDTA solution, grind into a homogenate, then rinse with 25 ml of distilled water and collect the liquid, then ultrasonically extract at 40 °C for 15 min, then make up to 50 ml with distilled water, fully shake and mix evenly, filter, and collect the filtrate, which is the sample extract.

[0135] ③ Determination:

[0136] Take a 10 ml test tube, add 5 mL of sample extract, 2 mL of oxalic acid-EDTA solution, 0.5 mL of metaphosphoric acid-acetic acid solution, 1 mL of 5% sulfuric acid solution and 2 mL of 5% ammonium molybdate solution, mix well and water bath at 35 °C for 30 min, then measure the absorbance at 700 nm with a spectrophotometer, and calculate the vitamin C content in the sample extract through the standard curve equation.

[0137] Standard curve equation: ;

[0138] y is the absorbance and x is the vitamin content (g) in the test solution.

[0139] Calculate the vitamin C content (Vc) of the sampled sample.

[0140] ;

[0141] C: Vitamin C content in the sample extract;

[0142] V t : Total volume of the sample extract (50 ml);

[0143] W: Fresh weight of the sampled sample (2 g);

[0144] V m : Volume of the sample extract added during measurement (5 ml).

[0145] (2) Detect chlorophyll content (keep away from light throughout the process)

[0146] Take 0.5 g of the sampled sample with fresh weight, add pre-cooled acetone-ethanol at 4°C (obtained by mixing 1 volume part of acetone and 1 volume part of ethanol), homogenize, then filter with filter paper, collect the filtrate with a 50 mL brown volumetric flask, let it stand until the filtrate turns white, and then make up to the 50 mL graduation line with sterile water to obtain the sample extract. Measure the absorbance values of the sample extract at 663 nm and 645 nm. Calculate the chlorophyll a concentration (ρ a ), chlorophyll b concentration (ρ b ), and chlorophyll concentration (ρ t ) in the sample extract. Calculate the chlorophyll content of the sampled sample in mg / g.

[0147] .

[0148] The results are shown in Figure 10 and Figure 11 . During storage, the VC content of green pepper fruits showed an overall downward trend. The treatment with Lactobacillus casei GHY-4 delayed the decline rate of VC in green pepper fruits, keeping its content at a relatively high level, with a significant difference from the CK group (p < 0.05). As the storage time extended, the chlorophyll content in green peppers showed a downward trend, and the chlorophyll content of green peppers in the GHY-4 group was significantly higher than that of green peppers in the CK group (p < 0.05).

[0149] The results of this example show that Lactobacillus casei GHY-4 has the effects of delaying post-ripening and inhibiting rot in the early stage of green pepper storage.

[0150] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, in accordance with the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made using conventional techniques known in the art. The application of some basic features can be made within the scope of the following appended claims.

Claims

1. Lactobacillus casei ( lactobacillus casei ) GHY-4, whose deposit registration number is CGMCC No.31925.

2. the bacterial agent of Lactobacillus casei described in claim 1.

3. Application of Lactobacillus casei described in claim 1 or the bacterial agent described in claim 2 in inhibiting Alternaria alternata; the application is an application for non-disease treatment purposes.

4. Use of the Lactobacillus casei described in claim 1 or the bacterial agent described in claim 2 in the preparation of an Alternaria inhibitor.

5. Application of the Lactobacillus casei described in claim 1 or the bacterial agent described in claim 2 in green pepper preservation.

6. Use of the Lactobacillus casei described in claim 1 or the bacterial agent described in claim 2 in the preparation of a green pepper preservative.

7. The use of the Lactobacillus casei according to claim 1 or the bacterial agent according to claim 2, which is any one of the following (a) to (i): (a) Inhibit the rot of green pepper fruit; (b) delaying the ripening process of green pepper fruits after they are picked; (c) delaying the loss of firmness of green pepper fruits after picking; (d) inhibiting the increase in cell membrane permeability after picking of green pepper fruits; (e) Reduce the respiration rate of green pepper fruits; (f) reducing the decrease in soluble solids content of green pepper fruits after picking; (g) inhibiting the degradation of titratable acid in green pepper fruits after picking; (h) reducing the decrease in ascorbic acid content in green pepper fruits after picking; (i) Reduce the decrease in chlorophyll content in green pepper fruits after picking.

8. Use of the Lactobacillus casei according to claim 1 or the bacterial agent according to claim 2 in preparing a product; The use of the product is any of the following (a) to (i): (a) Inhibit the rot of green pepper fruit; (b) delaying the ripening process of green pepper fruits after they are picked; (c) delaying the loss of firmness of green pepper fruits after picking; (d) inhibiting the increase in cell membrane permeability after picking of green pepper fruits; (e) Reduce the respiration rate of green pepper fruits; (f) reducing the decrease in soluble solids content of green pepper fruits after picking; (g) inhibiting the degradation of titratable acid in green pepper fruits after picking; (h) reducing the decrease in ascorbic acid content in green pepper fruits after picking; (i) Reduce the decrease in chlorophyll content in green pepper fruits after picking.

9. A product comprising the Lactobacillus casei according to claim 1 or the bacterial agent according to claim 2.

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