Application of Lactobacillus plantarum and Kluyveromyces marxii in degrading fruit stone cells

Abstract

The present invention relates to the field of microbial technology, and in particular to the application of Lactobacillus plantarum JYLP‑326 and Kluyveromyces marxensis in degrading fruit stone cells. The present invention provides the application of a mixed bacterial agent comprising Lactobacillus plantarum JYLP‑326 and Kluyveromyces marxensis in degrading fruit stone cells, wherein the Lactobacillus plantarum comprises Lactobacillus plantarum JYLP‑326; the Kluyveromyces marxensis has a deposit number of CICC32015. After the present invention uses Lactobacillus plantarum JYLP‑326 and Kluyveromyces marxensis in combination, the cellulase production ability of the strain is significantly improved, the content of stone cells can be reduced, and the quality of pear juice is of great significance.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to application of plant lactobacillus and marxist Kluyveromyces in degrading fruit stone cells. Background Art

[0002] The quality of Chinese pears is not high, with high stone cell content, hardened flesh, thick skin and low sugar content. [3] This further leads to the poor taste of processed pear juice and the easy formation of precipitated particles. In addition, the precipitation of pear juice after long-term storage is because the pear pulp contains many hard particles clustered into stone cells. [1] Therefore, the number of stone cells has become an important indicator for judging the quality of pear juice. [2] . And the flavor and quality of pear juice need to be improved urgently. [4] .

[0003] Stone cells are thick-walled cells formed by the continuous deposition of secondary walls of thin-walled cells on the primary walls of stone cells. [5] The main components of stone cells are lignin, cellulose and hemicellulose. The formation of cellulose is closely related to the formation and development of stone cells. [6] Cellulose is a chain polymer composed of D-glucose linked by β-1,4 glycosidic bonds. Multiple cellulose molecules are arranged in parallel to form fiber bundles. The three major elements in cellulose are carbon (44.44%), hydrogen (6.17%) and oxygen (49.39%). [7] Generally, cellulose can be divided into crystalline and amorphous regions according to their aggregation state. The molecules in the crystalline region are closely arranged and difficult to degrade, while the molecules in the amorphous region are widely spaced and easily degraded. However, the crystalline and amorphous regions are staggered and have no obvious boundaries. [8] In addition, there are hydrogen bonds between cellulose, which seriously hinder the hydrolysis of cellulose. [9] This is also the reason why cellulose in pear juice is difficult to degrade, resulting in low juice quality and poor taste. Therefore, developing a mixed bacterial system of cellulose-degrading bacteria with high-efficiency cellulase activity is of great significance to reducing the formation of stone cells and thus improving the quality of pear juice. Summary of the invention

[0004] In order to solve the above problems, the present invention provides the use of Lactobacillus plantarum and Kluyveromyces marxii in degrading fruit stone cells. After the present invention uses Lactobacillus plantarum and Kluyveromyces marxii together, the cellulase production capacity of the strain is significantly improved, the content of stone cells can be reduced, and it is of great significance to improve the quality of pear juice.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The invention provides application of a mixed bacterial agent comprising plant lactobacillus and marxist Kluyveromyces marxist in degrading fruit stone cells. The plant lactobacillus comprises plant lactobacillus JYLP-326; the preservation number of the marxist Kluyveromyces marxist is CICC 32015.

[0007] The invention provides application of a mixed bacterial agent comprising plant lactobacillus and marxist Kluyveromyces in degrading fruit cellulose. The plant lactobacillus comprises plant lactobacillus JYLP-326; the preservation number of the marxist Kluyveromyces is CICC 32015.

[0008] Preferably, the fruit comprises pears.

[0009] The invention provides a biodegradation agent for degrading fruit stone cells. The effective components of the biodegradation agent include plant lactobacillus and kluyveromyces marxii. The plant lactobacillus includes plant lactobacillus JYLP-326. The preservation number of the kluyveromyces marxii is CICC 32015.

[0010] The invention provides a method for preparing a biodegradation agent according to the technical scheme, the method comprising the following steps: inoculating a plant lactobacillus suspension into an MRS broth culture medium for a first shaking culture to obtain a preculture medium; inoculating a marxianus kluyveromyces suspension into the preculture medium for a second shaking culture to obtain a biodegradation agent; the plant lactobacillus comprises plant lactobacillus JYLP-326; the marxianus kluyveromyces has a preservation number of CICC 32015.

[0011] Preferably, the bacterial activity of the Lactobacillus plantarum suspension is 6-7 lg CFU / mL; and the inoculation amount of the Lactobacillus plantarum suspension is 2% of the volume of the MRS broth culture medium.

[0012] Preferably, the bacterial activity of the Kluyveromyces marxianus suspension is 5-6 lg CFU / mL; and the inoculation amount of the Kluyveromyces marxianus suspension is 2% of the volume of the preculture medium.

[0013] Preferably, the first shaking incubation time is 24 hours, the temperature is 30° C., and the rotation speed is 120 r / min.

[0014] Preferably, the second shaking incubation time is 72 h, the temperature is 30° C., and the rotation speed is 120 r / min.

[0015] The present invention provides a method for degrading stone cells in fruit juice or preparing fermented juice, the method comprising the following steps:

[0016] The fruit juice is mixed with the biodegradation agent for fermentation.

[0017] Beneficial effects: The present invention provides an application of a mixed bacterial agent including plant lactobacillus and Marx Kluyveromyces in degrading fruit stone cells, wherein the plant lactobacillus includes plant lactobacillus JYLP-326; the Marx Kluyveromyces has a preservation number of CICC 32015. After the plant lactobacillus and Marx Kluyveromyces are used together, Marx Kluyveromyces has a promoting effect on plant lactobacillus, so that the cellulase production capacity of the two strains is significantly improved, and the content of stone cells can be reduced; thus, it can be seen that the co-fermentation of plant lactobacillus JYLP-326 and Marx Kluyveromyces achieves the effect of degrading stone cells. The results of the specific embodiment of the present invention show that the degradation rate of cellulose by mixing plant lactobacillus and Marx Kluyveromyces reaches 20.35%; and it is proved by scanning electron microscopy and Fourier infrared spectroscopy that the surface structure of stone cells can be damaged and the chemical structure of stone cells can be changed after the plant lactobacillus and Marx Kluyveromyces are mixed, so that the degradation effect of stone cells is more obvious. Therefore, the present invention not only provides theoretical support for constructing an efficient and fast cellulose degradation technology, but also can provide technical guidance for my country's pear and pear fruit product industry, thereby improving the quality of pear fruit products, promoting the prosperous development of the pear and pear juice industry, and helping fruit farmers increase production and income. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1 is the standard curve of glucose;

[0020] Figure 2 The disintegration of filter paper strips by different Lactobacillus plantarum;

[0021] Figure 3 is the standard curve of cellulose;

[0022] Figure 4 The changes of cellulose content in stone cells before and after fermentation;

[0023] Figure 5 This is the stone cell under a scanning electron microscope at 500 times after fermentation;

[0024] Figure 6 This is the stone cell under a scanning electron microscope at 2000 times after fermentation;

[0025] Figure 7 This is the stone cell under a scanning electron microscope at 5000 times after fermentation;

[0026] Figure 8 This is the stone cell under a scanning electron microscope at 500 times before fermentation;

[0027] Fig. 9 This is the stone cell under a scanning electron microscope at 2000 times before fermentation;

[0028] Fig.10 This is the stone cell under a scanning electron microscope at 5000 times before fermentation;

[0029] Fig.11 This is the Fourier infrared spectrum of stone cells before and after fermentation. DETAILED DESCRIPTION

[0030] Unless otherwise specified, the components and strains used in the present invention can be purchased by those skilled in the art.

[0031] The present invention provides the use of Lactobacillus plantarum and Kluyveromyces marxensis in degrading fruit stone cells, and further provides the use of Lactobacillus plantarum and Kluyveromyces marxensis in degrading fruit cellulose; the Lactobacillus plantarum includes Lactobacillus plantarum JYLP-326; the Kluyveromyces marxensis has a preservation number of CICC32015. The fruit of the present invention preferably includes pears. The present invention does not specifically limit the sources of the Lactobacillus plantarum JYLP-326 and Kluyveromyces marxensis, and they can be purchased conventionally by a person skilled in the art; in a specific embodiment of the present invention, the Lactobacillus plantarum JYLP-326 is preferably purchased from Shandong Zhongke Jiayi Bioengineering Co., Ltd., and the Kluyveromyces marxensis is preferably purchased from the China Industrial Microbiological Culture Collection Management Center.

[0032] The present invention uses Lactobacillus plantarum and Kluyveromyces marxii in combination, wherein Kluyveromyces marxii has a promoting effect on Lactobacillus plantarum; therefore, the two strains have stronger cellulase production capabilities, and the cellulose degradation rate reaches 20.35%, thereby achieving the effect of reducing stone cell formation.

[0033] Based on the advantages of the combined use of Lactobacillus plantarum and Kluyveromyces marxii, the present invention provides a biodegradation agent for degrading fruit stone cells, wherein the active ingredients of the biodegradation agent include Lactobacillus plantarum and Kluyveromyces marxii; the Lactobacillus plantarum includes Lactobacillus plantarum JYLP-326; the Kluyveromyces marxii has a preservation number of CICC 32015. The fruit of the present invention preferably includes pears.

[0034] The present invention provides a method for preparing the biodegradation agent described in the above technical solution, and the preparation method comprises the following steps:

[0035] The plant lactobacillus suspension is inoculated into the MRS broth medium for the first shaking culture to obtain the preculture medium; the marxian Kluyveromyces suspension is inoculated into the preculture medium for the second shaking culture to obtain the biodegradation agent.

[0036] The present invention inoculates the plant lactobacillus suspension into the MRS broth medium for the first shaking culture to obtain a preculture medium. The inoculation amount of the plant lactobacillus JYLP-326 suspension of the present invention is preferably 2% of the volume of the MRS broth medium; the bacterial activity of the plant lactobacillus JYLP-326 suspension is preferably 6.3lg CFU / mL; the first shaking culture time is preferably 24h; the temperature is preferably 30°C; and the rotation speed is 120r / min. There is no limitation on the preparation method of the plant lactobacillus suspension of the present invention, and a method well known to those skilled in the art can be used.

[0037] After obtaining the preculture medium, the present invention inoculates the Kluyveromyces marxianus suspension into the preculture medium for a second shaking culture to obtain a biodegradable agent. The inoculation amount of the Kluyveromyces marxianus suspension of the present invention is preferably 2% of the volume of the preculture medium; the viable count of the Kluyveromyces marxianus suspension is preferably 5.3 lg CFU / mL; the time of the second shaking culture is preferably 72h; the temperature is preferably 30°C; and the rotation speed is 120r / min. There is no limitation on the preparation method of the Kluyveromyces marxianus suspension of the present invention, and it can be prepared in a manner well known to those skilled in the art.

[0038] The present invention provides a method for degrading stone cells in fruit juice or preparing fermented juice, the method comprising the following steps:

[0039] The fruit juice is mixed with the biodegradation agent for fermentation.

[0040] The present invention mixes fruit juice with the biodegradation agent for fermentation. The present invention has already described the biodegradation agent in detail above, so it will not be repeated here. The present invention has no limitation on the ratio of the fruit juice to the biodegradation agent, and any ratio can be used. The present invention has no limitation on the fermentation method, and a method well known to those skilled in the art can be used.

[0041] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] The test strains and some reagents used in the following examples are as follows:

[0043] Test strains:

[0044] Lactobacillus plantarum JYLP-326 was purchased from Shandong Zhongke Jiayi Bioengineering Co., Ltd.;

[0045] Lactobacillus plantarum CICC 24435 and Lactobacillus plantarum CICC 24437 were purchased from China Industrial Microbiological Culture Collection Center;

[0046] Kluyveromyces marxii was purchased from China Industrial Microbiological Culture Collection Center with the collection number CICC32015.

[0047] Yali pears: farmers’ market in Baoding City, Hebei Province.

[0048] Test reagents:

[0049] DNS reagent: weigh 3.15 g of 3,5-dinitrosalicylic acid, add 500 mL of water and stir to dissolve, heat in a water bath to 45°C, then gradually add 100 mL of 200 g / L NaOH solution, stirring continuously until completely dissolved, then add 91 g of potassium sodium tartrate, 2.5 g of phenol and 2.5 g of sodium sulfite in sequence, stir until dissolved, cool to room temperature, dilute to 1000 mL, filter, store the filtrate in a brown bottle, protect from light, and use after 7 days;

[0050] 0.05 mol / L citrate buffer solution: weigh 10.5 g of citric acid, add 5.0 g of sodium hydroxide, then add 800 mL of water to dissolve, adjust the pH to 5.5 with 0.1 mol / L citric acid solution or 0.1 mol / L sodium citrate solution, and then dilute to 1000 mL with water;

[0051] Glucose standard solution with a concentration of 10 mg / mL: weigh about 1 g of anhydrous glucose that has been dried to constant weight at 105°C, add citrate buffer to dissolve, and make up to 100 mL.

[0052] Test medium:

[0053] MRS broth medium: glucose 20g, peptone 10g, yeast extract powder 5g, beef extract powder 10g, ammonium citrate 2g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, manganese sulfate 0.05g, magnesium sulfate 0.2g, Tween 80 1mL, distilled water 1000mL, pH 6.2±0.2, sterilized at 121℃ for 15min;

[0054] YPD medium: 20 g glucose, 20 g peptone, 10 g yeast extract powder, 1000 mL distilled water, sterilized at 121°C for 15 min;

[0055] Stone cell liquid fermentation medium: stone cells 20g, peptone 10g, beef extract powder 10g, yeast extract powder 5g, ammonium citrate 2g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween 80 1mL, distilled water 1000mL, pH 6.7±0.2, sterilization at 121℃ for 15min. The stone cells in this medium are extracted from duck pears according to the method of NY / T 1388-2007.

[0056] Example 1

[0057] The filter paper disintegration method was used to qualitatively identify the ability of the strain to degrade cellulose. According to the experimental method of Cheng Xin et al. (Cheng Xin. Study on the effect of lactic acid bacteria fermentation on the improvement of brown rice cooking quality [D]. Jiangnan University, 2018.), filter paper culture medium was prepared, and 0.2 mL of Lactobacillus plantarum JYLP-326 bacterial suspension with an OD value of 1.0 was added to the filter paper culture medium;

[0058] The preparation method of the plant lactobacillus JYLP-326 bacterial suspension with an OD value of 1.0 is as follows: the plant lactobacillus JYLP-326 strain stored at -80°C is inoculated into a sterilized MRS broth medium, and cultured in a biochemical incubator at 37°C for 24 hours to obtain activated plant lactobacillus JYLP-326; the activated plant lactobacillus JYLP-326 is centrifuged at 5000r / min for 10 minutes, the supernatant is removed, the bacterial strain is resuspended in a PBS buffer solution, mixed, centrifuged at 5000r / min for 10 minutes, the supernatant is taken, and the "resuspending-centrifuging-taking the supernatant" is repeated twice, and then the bacterial strain is resuspended in a PBS buffer solution, and the bacterial suspension is adjusted to an OD value of 1 with a PBS buffer solution, and the bacterial activity of the plant lactobacillus JYLP-326 bacteria in the bacterial suspension is 6.3lg CFU / mL.

[0059] Comparative Example 1

[0060] The steps are the same as those in Example 1, except that 0.2 mL of a Lactobacillus plantarum CICC24435 bacterial suspension having an OD value of 1.0 is used to replace the Lactobacillus plantarum JYLP-326 bacterial suspension;

[0061] The preparation method of the plant lactobacillus CICC 24435 bacterial suspension with an OD value of 1.0 is as follows: the activated plant lactobacillus CICC 24435 bacteria are centrifuged at 5000r / min for 10min, the supernatant is removed, the bacteria are resuspended in a PBS buffer, mixed, centrifuged at 5000r / min for 10min, the supernatant is taken, the step is repeated twice, the bacteria are then resuspended in a PBS buffer, and the bacterial suspension is adjusted to an OD value of 1 with a PBS buffer, and the bacterial activity of the plant lactobacillus CICC 24435 bacteria in the bacterial suspension is 6.3lg CFU / mL.

[0062] Comparative Example 2

[0063] The steps are the same as those in Example 1, except that 0.2 mL of a Lactobacillus plantarum CICC24437 bacterial suspension with an OD value of 1.0 is used to replace the Lactobacillus plantarum JYLP-326 bacterial suspension;

[0064] The preparation method of the plant lactobacillus CICC 24437 bacterial suspension with an OD value of 1.0 is as follows: the activated plant lactobacillus CICC 24437 bacteria are centrifuged at 5000 r / min for 10 min, the supernatant is removed, the bacteria are resuspended in a PBS buffer solution, mixed, centrifuged at 5000 r / min for 10 min, the supernatant is taken, and the "resuspending-centrifuging-taking the supernatant" is repeated twice, and then the bacteria are resuspended in a PBS buffer solution, and the bacterial suspension is adjusted to an OD value of 1 with a PBS buffer solution, and the bacterial activity of the plant lactobacillus CICC 24437 bacteria in the bacterial suspension is 6.3 lg CFU / mL.

[0065] Test Example 1

[0066] The filter paper culture medium without inoculation of strains was used as blank control. The blank control, the filter paper culture medium obtained in Example 1 and Comparative Examples 1-2 were shaken and cultured at 37°C and 120 r / min for 72 h to observe the filter paper disintegration effect. Since the filter paper strip disintegration experiment can comprehensively reflect the ability of the strain to secrete cellulase, the three strains of Lactobacillus plantarum were subjected to filter paper disintegration test to identify whether the strains have the ability to degrade cellulose. The test results are shown in Figure 2. Figure 1 As shown, Figure 1 The test tubes in the figure are the disintegration results of the blank control, Example 1, Comparative Example 1, and Comparative Example 2 from left to right.

[0067] Depend on Figure 1 It can be concluded that: compared with the blank control group without inoculation of strains, the test tubes of Example and Comparative Examples 1-2 became turbid and holes and breaks appeared in the filter paper strips, and all of them were able to degrade cellulose. Therefore, these three strains of Lactobacillus plantarum can be used for further research.

[0068] Example 2

[0069] The plant lactobacillus JYLP-326 bacterial suspension prepared in Example 1 was inoculated into an MRS broth medium, the inoculation amount being 2% of the volume of the MRS broth medium, the bacterial activity of the plant lactobacillus JYLP-326 bacterial suspension being 6.3 lg CFU / mL, and the suspension was cultured on a shaking table at 30°C for 24 h at a rotation speed of 120 r / min to obtain a preculture medium, and then the Kluyveromyces marxianus suspension was inoculated into the preculture medium, the inoculation amount being 2% of the volume of the preculture medium, the viable count of the Kluyveromyces marxianus suspension being 5.3 lg CFU / mL, and the suspension was cultured on a shaking table at 30°C at a rotation speed of 120 r / min, and a sample was taken every 24 h for a total of 3 times, i.e., samples were taken at 48 h, 72 h, and 96 h of fermentation, and the samples with different fermentation times were centrifuged at 8000 rpm and 4°C for 10 min, and the supernatant was taken, i.e., a crude enzyme solution, and the crude enzyme solution was stored at -20°C for standby use, recorded as 326 m;

[0070] The preparation method of the Kluyveromyces marxianus yeast suspension is as follows: inoculate the Kluyveromyces marxianus yeast strain stored at -80°C into a sterilized YPD medium, and culture it in a biochemical incubator at 28°C for 24 hours to obtain activated Kluyveromyces marxianus yeast; centrifuge the activated Kluyveromyces marxianus yeast at 5000r / min for 10 minutes, remove the supernatant, resuspend the bacteria in PBS buffer, mix well, centrifuge at 5000r / min for 10 minutes, take the supernatant, repeat "resuspend-centrifuge-take the supernatant" twice, then resuspend the bacteria in PBS buffer, and adjust the bacterial suspension to an OD value of 1 with PBS buffer. The preservation number of Kluyveromyces marxianus yeast is CICC 32015.

[0071] Comparative Example 3

[0072] The plant lactobacillus JYLP-326 bacterial suspension prepared in Example 1 was inoculated into MRS broth medium, the inoculation amount was 2% of the volume of the MRS broth medium, the bacterial activity of the plant lactobacillus JYLP-326 bacterial suspension was 6.3 lg CFU / mL, and the suspension was cultured on a shaking table at 30°C for 96 h. After culturing for 24 h, samples were taken every 24 h, for a total of 3 times, i.e., samples were taken at 48 h, 72 h and 96 h of fermentation. The samples with different fermentation times were centrifuged at 8000 rpm and 4°C for 10 min, and the supernatant was taken to obtain a crude enzyme solution for standby use, which was recorded as 326.

[0073] Comparative Example 4

[0074] The steps are the same as those in Comparative Example 3, except that the Lactobacillus plantarum CICC24435 bacterial suspension prepared in Comparative Example 1 is used instead of the Lactobacillus plantarum JYLP-326 bacterial suspension, and the bacterial activity of the Lactobacillus plantarum CICC24435 bacterial suspension is 6.3 lg CFU / mL, recorded as 435.

[0075] Comparative Example 5

[0076] The steps are the same as those in Example 2, except that the Lactobacillus plantarum CICC 24435 bacterial suspension prepared in Comparative Example 1 is used to replace the Lactobacillus plantarum JYLP-326 bacterial suspension, and the bacterial activity of the Lactobacillus plantarum CICC 24435 bacterial suspension is 6.31 g CFU / mL, recorded as 435 m.

[0077] Comparative Example 6

[0078] The steps are the same as those in Comparative Example 3, except that the Lactobacillus plantarum CICC24437 bacterial suspension prepared in Comparative Example 2 is used instead of the Lactobacillus plantarum JYLP-326 bacterial suspension, and the bacterial activity of the Lactobacillus plantarum CICC24437 bacterial suspension is 6.3 lg CFU / mL, recorded as 437.

[0079] Comparative Example 7

[0080] The steps are the same as those in Example 2, except that the Lactobacillus plantarum CICC 24437 bacterial suspension prepared in Comparative Example 2 is used to replace the Lactobacillus plantarum JYLP-326 bacterial suspension, and the bacterial activity of the Lactobacillus plantarum CICC 24437 bacterial suspension is 6.31 g CFU / mL, recorded as 437 m.

[0081] Test Example 2

[0082] The cellulase activity of the crude enzyme solution prepared in Example 2 and Comparative Examples 3 to 7 was measured, and the specific measurement method was as follows:

[0083] 1. Preparation of glucose standard curve:

[0084] Take 7 test tubes, add 0, 2, 3, 4, 6, 8, 10 mL of 10 mg / mL standard glucose solution into them respectively, and then dilute to 50 mL with citrate buffer solution to prepare glucose solutions with concentrations of 0-2 mg / mL;

[0085] Pipette 1.0 mL of the prepared glucose standard solution of different concentrations into a 25 mL volumetric flask, add 2 mL of water and 2 mL of DNS reagent, boil in a water bath for 5 min, cool to room temperature, dilute to 25 mL with water, measure the absorbance at a wavelength of 540 nm, and prepare a glucose standard curve with glucose concentration as the horizontal axis and absorbance as the vertical axis to obtain a linear regression equation, such as Figure 1 As shown, the regression equation is: y = 1.0954x + 0.0082, R 2 =0.9984.

[0086] 2. Cellulase activity determination. The indicators of cellulase determination include filter paper enzyme activity, endoglucanase activity, exoglucanase activity and β-glucosidase activity. The definition of enzyme activity unit is: the amount of enzyme required to catalyze the substrate to generate 1 μmol of glucose per milliliter of enzyme solution within 1 minute, that is, 1U / mL;

[0087] The determination method of filter paper enzyme activity was used for determination: the filter paper enzyme activity was determined according to GB / T 23881-2009, that is, 50 mg Whatman NO.1 filter paper was added to the test tube, 1.0 mL citrate buffer was added to soak the paper as a substrate, equilibrated at 37°C for 10 min, and then 0.5 mL crude enzyme solution and 5 mL distilled water were added in sequence, shaken for 3s to 5s, and kept in a constant temperature water bath at 50°C for 50 min. After taking it out, 2 mL DNS reagent was added. For the control group, 2.0 mL DNS reagent was first added to the crude enzyme solution and then kept in a constant temperature water bath. After shaking, it was boiled in a boiling water bath for 10 min, cooled to room temperature, and made up to 25 mL with distilled water. The control group was adjusted to zero, and the absorbance was measured at a wavelength of 540 nm. The corresponding glucose content was found from the glucose standard curve, and the corresponding enzyme activity value was obtained by combining the enzyme activity formula;

[0088] Determination of endoglucanase activity: refer to the method of Huang Chunkai (Huang Chunkai. Screening, identification and enzyme production characteristics of a cellulase-producing strain [D]. Changchun University of Technology, 2015.) to determine the endoglucanase activity and make slight modifications, that is, add 1.5mL of 0.05mol / L disodium hydrogen phosphate-citrate buffer solution with a pH of 6.8 to the test tube, prepare a CMC-Na substrate solution with a mass percentage of 1%, balance at 37℃ for 10min, then add 0.5mL of crude enzyme solution to the test tube, keep it in a constant temperature water bath at 50℃ for 50min, take it out and add 2mL of DNS reagent, the control group first adds 2mL of DNS reagent to the crude enzyme solution and then keeps it in a constant temperature water bath, shake it well and boil it in a boiling water bath for 10min, cool it to room temperature, dilute it to 20mL with distilled water, adjust it to zero with the control group, measure the absorbance at a wavelength of 540nm, find the corresponding glucose content from the glucose standard curve, and calculate the enzyme activity;

[0089] Determination of exoglucanase activity: Refer to Wu Jing's method (Wu Jing. Screening of high-yield cellulase molds and isolation and purification of cellulase systems [D]. Guizhou University, 2020.) to determine the exoglucanase activity and make slight modifications, that is, add 1.5mL of 0.05mol / L, pH 6.8 disodium hydrogen phosphate-citrate buffer into the test tube, prepare a 1% microcrystalline cellulose substrate solution by mass, balance at 37℃ for 10min, then add 0.5mL of crude enzyme solution to the test tube, bathe in 50℃ very warm water for 50min, take it out and add 2mL of DNS reagent. For the control group, first add 2mL of DNS reagent to the crude enzyme solution and then bathe in a constant temperature water bath. Shake well and boil in a boiling water bath for 10min. Cool to room temperature, dilute to 20mL with distilled water, adjust to zero for the control group, measure the absorbance at a wavelength of 540nm, find the corresponding glucose content from the glucose standard curve, and calculate the enzyme activity;

[0090] Determination of β-glucosidase activity: refer to Wu Jing's method (Wu Jing. Screening of high-yield cellulase molds and isolation and purification of cellulase systems [D]. Guizhou University, 2020.) to determine the β-glucosidase activity and make slight modifications, that is, add 1.5mL of 0.05mol / L disodium hydrogen phosphate-citric acid buffer solution with a pH of 6.8 into the test tube, prepare a 1% salicin substrate solution by mass, balance at 37°C for 10min, then add 0.5mL of crude enzyme solution to the test tube, and bathe in 50°C very warm water for 50min, then take it out and add 2mL of DNS reagent. For the control group, first add 2mL of DNS reagent to the crude enzyme solution and then bathe in a constant temperature water bath, shake well and boil in a boiling water bath for 10min, cool to room temperature, dilute to 20mL with distilled water, adjust to zero with the control group, measure the absorbance at a wavelength of 540nm, find the corresponding glucose content from the glucose standard curve, and calculate the enzyme activity;

[0091] Enzyme activity calculation formula:

[0092]

[0093] Wherein: U is the cellulase activity; m is the mass of glucose, mg; n is the dilution factor of the crude enzyme solution; 180.2 is the molecular weight of glucose; t is the reaction time, min; v is the volume of the enzyme solution, mL.

[0094] After fermentation for 48 h, 72 h and 96 h, the glucose content test results corresponding to the crude enzyme solutions obtained in Example 2 and Comparative Examples 3 to 7 are shown in Table 1, and the cellulase activity test results are shown in Table 2.

[0095] Table 1 Glucose content of crude enzyme solution in Example 2 and Comparative Examples 3 to 7 at different time periods

[0096]

[0097]

[0098] Table 2 The production of cellulase in crude enzyme solution of Example 2 and Comparative Examples 3 to 7 at different time periods

[0099]

[0100]

[0101] Note: CMCase is endoglucanase, PNPCase is exoglucanase, PNPGase is β-glucosidase, Fpase is filter paper enzyme, *=P<0.05, specifically: the data of CMCase, PNPCase, PNPGase, and Fpase in the table are compared with CMCase: 2.08U / mL, PNPCase: 4.58U / mL, PNPGase: 2.40U / mL, and Fpase: 2.04U / mL, respectively.

[0102] It can be concluded from Table 1 and Table 2 that at 96h of fermentation, except for the endoglucanase, the compound combination showed significant improvement in the enzyme activities of the other three enzymes compared with the combination of Lactobacillus plantarum alone; but compared with other compound combinations, the enzyme activity of 326m was the highest. The combinations with higher enzyme activities of cellulase, i.e. endoglucanase, exoglucanase and β-glucosidase were all the combination of Lactobacillus plantarum JYLP-326 and Kluyveromyces marxensis CICC32015, followed by the combination of Lactobacillus plantarum CICC 24435 and Kluyveromyces marxensis CICC 32015. In addition, we found that the exoglucanase and filter paper enzyme activities of the 326m group were significantly higher than those of other groups.

[0103] Since cellulose degradation is the result of the combined action of the cellulase system, we cannot unilaterally judge the degradation effect of exoglucanase on cellulose by its enzymatic activity. Therefore, in order to further screen out the most ideal strain combination for degrading cellulose in stone cells, we tested the cellulose content in stone cells before and after fermentation.

[0104] Example 3

[0105] The plant lactobacillus JYLP-326 bacterial suspension prepared in Example 1 was inoculated into a stone cell liquid fermentation medium, the inoculation amount was 2% of the volume of the stone cell liquid fermentation medium, the bacterial activity of the plant lactobacillus JYLP-326 bacterial suspension was 6.3 lg CFU / mL, and the culture was shaken at 30°C for 24 h at a speed of 120 r / min to obtain a preculture medium, and then the Kluyveromyces marxianus bacterial suspension prepared in Example 2 was inoculated into the preculture medium, the inoculation amount was 2% of the volume of the preculture medium, and the number of viable bacteria of the Kluyveromyces marxianus bacterial suspension was 5.3 lg CFU / mL, 30℃ shaker culture for 96h, speed of 120r / min, fermentation for 96h, sampling, that is, extracting the fermented stone cells, the specific steps are: the culture solution obtained after 96h of fermentation is centrifuged at 8000r / min and 4℃ for 15min, then the supernatant is poured out, the precipitate is retained, and then the precipitate is washed with 0.1M dilute hydrochloric acid to remove the bacteria on the surface of the stone cells, and then rinsed with distilled water twice, filtered, and the sample is obtained. The sample is dried in an oven at 50℃ to constant weight, recorded as 326m.

[0106] Comparative Example 8

[0107] The plant lactobacillus JYLP-326 bacterial suspension prepared in Example 1 was inoculated into a stone cell liquid fermentation medium, and the inoculation amount was 2% of the volume of the stone cell liquid fermentation medium. The bacterial activity of the plant lactobacillus JYLP-326 bacterial suspension was 6.3 lg CFU / mL. The suspension was cultured in a shaking table at 30°C with a rotation speed of 120 r / min. The fermentation was performed for 96 h. Samples were taken and dried in an oven at 50°C to constant weight, which was recorded as 326.

[0108] Comparative Example 9

[0109] The steps are the same as those in Comparative Example 8, except that the Lactobacillus plantarum CICC24435 bacterial suspension prepared in Comparative Example 1 is used instead of the Lactobacillus plantarum JYLP-326 bacterial suspension, and the bacterial activity of the Lactobacillus plantarum CICC24435 bacterial suspension is 6.3 lg CFU / mL, recorded as 435.

[0110] Comparative Example 10

[0111] The steps are the same as those in Example 3, except that the Lactobacillus plantarum CICC24435 bacterial suspension prepared in Comparative Example 1 is used instead of the Lactobacillus plantarum JYLP-326 bacterial suspension, and the bacterial activity of the Lactobacillus plantarum CICC24435 bacterial suspension is 6.3 lg CFU / mL, recorded as 435m.

[0112] Comparative Example 11

[0113] The steps are the same as those in Comparative Example 8, except that the Lactobacillus plantarum CICC24437 bacterial suspension prepared in Comparative Example 2 is used to replace the Lactobacillus plantarum JYLP-326 bacterial suspension, and the bacterial activity of the Lactobacillus plantarum CICC 24437 bacterial suspension is 6.3 lg CFU / mL, recorded as 437.

[0114] Comparative Example 12

[0115] The steps are the same as those in Example 3, except that the Lactobacillus plantarum CICC24437 bacterial suspension prepared in Comparative Example 2 is used instead of the Lactobacillus plantarum JYLP-326 bacterial suspension, and the bacterial activity of the Lactobacillus plantarum CICC24437 bacterial suspension is 6.3 lg CFU / mL, recorded as 437m.

[0116] Test Example 3

[0117] Preparation of cellulose standard curve:

[0118] A cellulose (CLL) content detection kit was selected to determine the cellulose content. The standard curve was operated according to the instructions of the kit. The specific process was to dilute the 10 mg / mL standard solution with distilled water to 0.09, 0.08, 0.07, 0.05, 0.025, 0.0125, and 0.00625 mg / mL standard solutions for use. 300uL of the diluted standard and 300uL of distilled water were added to 1.5mL centrifuge tubes, and then 70uL of working solution and 630uL of concentrated sulfuric acid were added to the centrifuge tubes, mixed well, and placed in a 95°C water bath for 10 minutes (cover tightly to prevent water loss), taken out and cooled to room temperature, and the absorbance value A at 620nm was measured, and recorded as A standard tube and A blank tube respectively. Calculate ΔA standard = A standard tube - A blank tube. According to the concentration of the standard tube (x, mg / mL) and the absorbance ΔA standard (y, ΔA standard), a standard curve was established to obtain the linear regression equation, see Figure 3 The specific regression equation is: y = 13.093x + 0.0416, R 2 =0.9965.

[0119] The stone cell liquid fermentation medium without the strain was used as the control group. The samples before fermentation of the control group, Example 3 and Comparative Examples 8 to 12 and the samples dried after fermentation for 96 hours were measured using a cellulose content detection kit (Beijing Solebow Biotechnology Co., Ltd., item number: BC 4280) to determine the changes in cellulose content in the stone cells before and after fermentation. The results are shown in Tables 3 and Figure 4 , Figure 4 Among them, 24435 is 435, 24437 is 437, 24435m is 435m, and 24437m is 437m.

[0120] Table 3 Changes in cellulose content in stone cells before and after fermentation

[0121]

[0122] The changes in cellulose content of stone cells before and after fermentation with Lactobacillus plantarum and Kluyveromyces marx are shown in Table 3 and Figure 3 As shown in the figure, the cellulose content of the stone cells without the strain was 28.49% after 96 hours of culture. Whether it was fermented by Lactobacillus plantarum alone or by mixed fermentation of Lactobacillus plantarum and Kluyveromyces marx for 96 hours, the cellulose content in the stone cells decreased. Among them, the cellulose content of the stone cells fermented by the combination of Lactobacillus plantarum JYLP-326 and Kluyveromyces marx CICC 32015 was the lowest, and the cellulose content dropped to 5.80%, and the degradation rate reached 20.35%. This result shows that the combination of Lactobacillus plantarum JYLP-326 and Kluyveromyces marx CICC 32015 can improve the degradation rate of cellulose in stone cells and play a certain role in the degradation of stone cells. Therefore, this combination of strains was determined as the ideal strain combination for degrading cellulose in stone cells.

[0123] At the same time, the apparent structure of the stone cells before and after fermentation in Example 3 was further observed, and the specific steps were as follows:

[0124] (1) Changes in the surface structure before and after stone cell fermentation

[0125] Samples were taken from Example 3, including samples before fermentation and samples after fermentation for 96 hours; the samples before and after fermentation were filtered and the bacteria were removed (the method for removing the bacteria was to remove the bacteria with dilute hydrochloric acid, and the specific steps were referred to "Construction of Rice Straw Degradation Microbial Community and Study on Its High-efficiency Degradation Mechanism"), and then dried in a 50°C oven to constant weight, and its surface structure was observed using a scanning electron microscope (SEM). Figures 5 to 10 It can be seen that the surface of the stone cells before degradation is relatively flat, smooth, and has a relatively complete structure with fewer pores. After fermentation, the surface of the stone cells becomes honeycomb-like, the surface becomes rough, and more obvious holes appear. This result shows that the cellulose structure in the stone cells has been eroded by the strains. This further illustrates that Lactobacillus plantarum and Kluyveromyces marxii have damaged the structure of the stone cells, making the degradation effect of the stone cells more obvious.

[0126] (2) Changes in chemical structure before and after stone cell fermentation

[0127] The chemical groups of the stone cells were analyzed by Fourier transform infrared spectrometer (FTIR) on the dried sample in step (1). The spectral absorption peak range was 4000-500cm -1 .

[0128] Fourier transform infrared spectroscopy explains the vibration caused by structural changes at the molecular level, and can qualitatively and semi-quantitatively reveal the transformation of functional groups. The Fourier transform infrared spectra of stone cells before and after fermentation are as follows: Fig.11 As shown in the figure, the spectra of the two groups are very similar, with no peaks appearing or disappearing. However, the peak intensity or peak width of several characteristic bands has changed, including 3000-3700cm -1 The peaks around 890-1100cm usually represent the stretching vibration of intramolecular or intermolecular hydrogen bonds. The peak intensity of the sample before fermentation is higher than that of the sample after fermentation, indicating that the hydrogen bond force is weakened, thereby increasing the OH stretching vibration of the hydroxyl group. The weakening of hydrogen bond force is conducive to the degradation of cellulose. -1 The absorption band of the fermented sample also showed a slight enhancement phenomenon. This is the vibration ring of cellulose and polysaccharides such as galactose and mannose, which indicates that cellulose is degraded into sugar substances. The absorption band of the fermented sample at 2900cm -1 The absorption band nearby showed an enhanced trend, which was caused by the methylation of the ester bonds, aromatic and ether bonds between the lignin polymers and the arabinose bonds after breaking. The above series of results show that the chemical structures of cellulose and lignin in the stone cells fermented by Lactobacillus plantarum JYLP-326 and Kluyveromyces marxensis CICC 32015 were damaged to a certain extent, which was conducive to the degradation of stone cells.

[0129] References:

[0130] [1] Jiaying Zhang, Jiaming Li, Cheng Xue, Runze Wang, Mingyue Zhang, Kaijie Qi, Jing Fan, Hongju Hu, Shaoling Zhang, Jun Wu. The Variation of Stone Cell Content in 236 Germplasms of Sand Pear (Pyrus pyrifolia) and Identification of Related Candidate Genes[J]. Horticultural Plant Journal, 2021, 7(02): 108-116.

[0131] [2]XUE Yong-song,XU Shao-zhuo,XUE Cheng,WANG Run-ze,ZHANG Ming-yue,LIJia-ming,ZHANG Shao-ling,WU Jun.Pearprocess:A new phenotypic tool for stonecell trait evaluation in pear fruit[J].Journal of Integrative Agriculture,2020,19(06):1625-1634.

[0132] [3]LászlóPolgár,Béla Kmellár,Péter Fodor.Evaluation of the Effect ofPear Stone Cells on the Recovery of Multi-Class Pesticides[J].AnalyticalLetters,2012,45(5-6):563-569.

[0133] [4]Wang Leyu,Zhang Hexin,Lei Hongjie.Phenolics Profile,AntioxidantActivity and Flavor Volatiles ofPear Juice:Influence ofLacticAcidFermentation Using Three Lactobacillus Strains in Monoculture and BinaryMixture[J].Foods,2021,11(1):11.

[0134] [5]Sheng Lixia et al.Analysis of transcriptome profiles of two Pyruspyrifolia cultivars reveals genes associated with stone cell development[J].Scientia Horticulturae,2021,288:110380.

[0135] [6]Wang Runze,Xue Yongsong,Fan Jing,Yao Jia Long,Qin Mengfan,Lin Tao,Lian Qun,Zhang Mingyue,Li Xiaolong,Li Jiaming,Sun Manyi,Song Bobo,ZhangJiaying,Zhao Kejiao,Chen Xu,Hu Hongju,Fei Zhangjun,Xue Cheng,Wu Jun.Asystemsgenetics approach reveals PbrNSC as a regulator of lignin and cellulosebiosynthesis in stone cells ofpear fruit[J].Genome Biology,2021,22(1):313-313.

[0136] [7]Turner Simon,Kumar Manoj.Cellulose synthase complex organizationand cellulose microfibril structure.[J].Philosophical transactions.Series A,Mathematical,physical,and engineering sciences,2018,376(2112):20170048.

[0137] [8]SETTERFIELD G,BAYLEY S T.Arrangement of Cellulose Microfibrils inWalls of Elongating Parenchyma Cells[J].The Journal of Biophysical andBiochemical Cytology,1958,4(4):377-382.

[0138] [9]Bengtsson Jenny,Olsson Carina,Hedlund Artur, Tobias,BialikErik.Understanding the Inhibiting Effect ofSmall-Molecule Hydrogen BondDonors on the Solubility of Cellulose in Tetrabutylammonium Acetate / DMSO.[J].The journal of physical chemistry.B,2017,121(50):11241-11248.

[0139] Although the present invention has been disclosed as above in the preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. The use of a mixed bacterial agent comprising plant lactobacillus and Kluyveromyces marxensis in degrading fruit stone cells, characterized in that: The plant lactobacillus includes plant lactobacillus JYLP-326; the deposit number of the Kluyveromyces marxii is CICC32015.

2. The use of a mixed bacterial agent comprising plant lactobacillus and Kluyveromyces marxii in degrading fruit cellulose, characterized in that: The plant lactobacillus includes plant lactobacillus JYLP-326; the deposit number of the Kluyveromyces marxii is CICC32015.

3. The use according to claim 1 or 2, characterized in that: The fruit includes pears.

4. A biodegradation agent for degrading fruit stone cells, characterized in that: The active ingredients of the biodegradation agent include Lactobacillus plantarum and Kluyveromyces marxii; the Lactobacillus plantarum includes Lactobacillus plantarum JYLP-326; the preservation number of the Kluyveromyces marxii is CICC32015.

5. The method for preparing the biodegradation agent according to claim 4, characterized in that: The following steps are involved: Inoculate the Lactobacillus plantarum suspension into the MRS broth medium for first shaking culture to obtain a preculture medium; The suspension of Kluyveromyces marxianus is inoculated into the preculture medium for second shaking culture to obtain a biodegradable agent; the plant lactobacillus includes plant lactobacillus JYLP-326; the deposit number of the Kluyveromyces marxianus is CICC32015.

6. The preparation method according to claim 5, characterized in that: The bacterial activity of the plant lactobacillus suspension is 6-7 lg CFU / mL; and the inoculation amount of the plant lactobacillus suspension is 2% of the volume of the MRS broth culture medium.

7. The preparation method according to claim 5, characterized in that: The bacterial activity of the Kluyveromyces marxianus suspension is 5-6 lg CFU / mL; the inoculation amount of the Kluyveromyces marxianus suspension is 2% of the volume of the preculture medium.

8. The preparation method according to claim 5, characterized in that: The first shaking incubation time is 24 hours, the temperature is 30° C., and the rotation speed is 120 r / min.

9. The preparation method according to claim 5, characterized in that: The second shaking incubation time was 72 h, the temperature was 30° C., and the rotation speed was 120 r / min.

10. A method for degrading stone cells in fruit juice, characterized in that: The method comprises the following steps: mixing fruit juice with the biodegradation agent according to claim 4 and fermenting the mixture.

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