Klebsiella oxytoca strain and application thereof

Abstract

The invention belongs to the technical field of microorganisms, particularly relates to an acid-producing Klebsiella oxytoca strain and application thereof, and provides the acid-producing Klebsiella oxytoca strain YS-pp-01 which is classified and named as Klebsiella oxytoca and has the preservation number of CGMCC No.32447. Efficient synthesis of a quality component EPSFs compound in tea leaves is achieved through fermentation of microorganisms provided by the invention; compared with other separated endophytes, the endophyte disclosed by the invention has a remarkable catechin conversion function, and the yield of theabrownin, theaflavin and other star components in tea is also greatly increased after microbial culture.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Klebsiella oxytoca strain and its application. Background Art

[0002] N-ethyl-2-pyrrolidone-substituted flavanols (EPSFs) are flavonoid alkaloid compounds that have received extensive attention in recent years due to their great potential for nutritional and health benefits. This compound is further formed by combining catechin components and theanine in tea leaves as precursors. On the basis of retaining the anti-inflammatory, antioxidant and other biological activities of catechin components, it further enhances the structural stability and human affinity, promotes the effective exertion of its nutritional and health functions, and has broad application prospects in the fields of biomedicine and health care. However, the content of EPSFs compounds in the fresh tea leaves widely planted at present is extremely low, and they only exist in large quantities in long-term stored tea, and their content increases continuously with the extension of storage time, which implies that the level of EPSFs compounds synthesized by the tea tree's own metabolic system is restricted to a certain extent. Endophytes are considered to be important potential regulatory factors to relieve this restriction. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a Klebsiella oxytoca strain and its application in view of the above-mentioned deficiencies of the prior art. This strain is obtained by isolating endophytes from tea tree leaves in the laboratory and can efficiently synthesize EPSFs compounds by using catechin components and theanine in tea leaves.

[0004] A Klebsiella oxytoca strain YS-pp-01, classified and named: Klebsiella oxytoca Klebsiella oxytoca , The preservation number is CGMCC No. 32447.

[0005] Further, the 16s-36F sequence of the Klebsiella oxytoca strain YS-pp-01 is as shown in SEQ ID NO.1.

[0006] Further, the 16S-1492R sequence of the Klebsiella oxytoca strain YS-pp-01 is as shown in SEQ ID NO.2.

[0007] The present invention also provides a microbial preparation, and the microbial preparation contains the Klebsiella oxytoca strain YS-pp-01, classified and named: Klebsiella oxytoca Klebsiella oxytoca , The preservation number is CGMCC No. 32447.

[0008] Furthermore, the present invention provides the use of any one of the Klebsiella oxytoca strains YS-pp-01 or the microbial preparation in the preparation of a product for degrading catechin components in tea soup.

[0009] Furthermore, the present invention provides the use of any one of the Klebsiella oxytoca strains YS-pp-01 or the microbial preparation in the preparation of a product for promoting the content of EPSFs compounds in tea leaves.

[0010] Furthermore, the present invention provides the use of any one of the Klebsiella oxytoca strains YS-pp-01 or the microbial preparation in the preparation of a product for improving the quality of tea leaves. Specifically, the Klebsiella oxytoca strain YS-pp-01 provided by the present invention can be used for the efficient synthesis of tea quality components such as tea brown pigment, theaflavin, and anthocyanin.

[0011] Furthermore, the present invention provides the use of any one of the Klebsiella oxytoca strains YS-pp-01 or the microbial preparation in tea fermentation.

[0012] Furthermore, the present invention provides a tea fermentation method, which adds any one of the Klebsiella oxytoca strains YS-pp-01 or the microbial preparation during the tea fermentation process.

[0013] Furthermore, the present invention provides a tea tree cultivation method, which applies any one of the Klebsiella oxytoca strains YS-pp-01 or the microbial preparation during the growth process of the tea tree.

[0014] The present invention has the following advantages compared with the prior art: The efficient synthesis of the quality component EPSFs compound in tea leaves can be achieved through microbial fermentation. There has been no previous report on the related function of the microbial synthesis of EPSFs compound, breaking through the rate-limiting path that this compound can only obtain natural products through tea storage. Compared with other isolated endophytes, its function of transforming catechin is significant, and after microbial culture, the yields of star components in tea leaves such as tea brown pigment and theaflavin have also been greatly improved. Description of the Drawings

[0015] Figure 1 Diagram of blank tea soup and the situation of S. marcescens and K. oxytoca cultured in tea soup; Figure 2 Statistical chart of the difference in catechin component content in tea soup under different strain treatments; Figure 3 Statistical chart of differential metabolites in tea soup under different strain treatments; Figure 4Statistical chart of the changes in the contents of L-theanine (A) and EGCG (B) during the growth of K. oxytoca; Figure 5 Chromatogram of EGCG-cThea (A) and EC-cThea (B) after culturing K. oxytoca for 15 h; Figure 6 Statistical chart of the contents of theanine and EGCG in the culture of K. oxytoca with exogenous addition at 9 h and 15 h; Note: CK, blank tea soup control with exogenous addition of theanine and EGCG; YS, treatment of K. oxytoca tea soup culture with exogenous addition of theanine and EGCG; Figure 7 Difference chart of theaflavins content in tea soup under different strain treatments. Note: The data in the figure are the results after log2 normalization of the original data; Figure 8 Abundance change chart of the endophytic bacterium g__Klebsiella of tea plants at each stage of black tea processing; Figure 9 Colony morphology chart of the endophytic bacterium Klebsiella oxytoca strain YS-pp-01 of tea plants. Detailed implementation manners

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. For purchased commodities in the test methods, if the specific conditions are not specified, they shall be carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they can all be obtained as conventional products through commercial purchase.

[0017] The Klebsiella oxytoca strain YS-pp-01 provided by the present invention, classified and named: Klebsiella oxytoca Klebsiella oxytoca , The preservation number is CGMCC No. 32447, the preservation unit is the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation date is October 31, 2024.

[0018] In the present invention, the Klebsiella oxytoca strain Klebsiella oxytoca is simply referred to as " K. oxytoca ".

[0019] Serratia marcescens Serratia marcescens is simply referred to as "S. marcescens” ".

[0020] "g_ Klebsiella " refers to the genus Klebsiella ( Klebsiella ).

[0021] Example 1 1. Correlation analysis between endophytes in tea plants and quality components Fresh leaf raw materials of the tea variety Yunkang 10 were collected from the Menghai Experimental Base of the Yunnan Institute and processed into black tea. Samples were collected from the fresh leaf raw materials and each processing stage for 16S rRNA sequencing. The sequencing work was carried out by Majorbio Co., Ltd., and the raw data was submitted to NCBI (SUB15108778). The microbial population structure in the tea samples was analyzed using the sequencing data, and the physiological and biochemical indexes such as water extract, total free amino acids, caffeine, tea polyphenols, and catechin components in the tea samples at each stage (including withering, rolling, fermentation, and drying) during the tea processing process including fresh leaves were analyzed. The correlation analysis was carried out between the endophyte structure composition data and the content of bioactive components in the tea, and the information of microorganisms significantly related to the formation of tea quality components was obtained. Among them, g_ Klebsiella showed a significant negative correlation with catechin components in fresh tea leaves and during the tea processing process (Table 1, Figure 8 ).

[0022] Table 1 g_Klebsiella Correlation analysis with tea quality components

[0023] 2. Isolation and identification of endophytes in tea plants Culture medium preparation stage: LB solid medium (peptone 10.0 g / L, sodium chloride 5.0 g / L, glucose 1.0 g / L, yeast extract powder 5.0 g / L, agar 15.0 g / L, the balance is water, final pH 7.0 ± 0.2) was selected as the growth medium for endophytic bacteria and fungi. No antibiotics were added to the medium. After weighing according to the formula, it was made up to 500 mL with deionized water, sterilized at 121 °C for 20 min, cooled to 50 - 60 °C, and poured into plates in a laminar flow hood for standby.

[0024] Material preparation stage: Freshly picked tea leaves and tea samples at the black tea fermentation stage were collected and placed on ice, and then brought back to the laboratory. The tea samples were surface sterilized before strain isolation (washed the samples with sterile water for 0.5 min, then washed in 75% ethanol for 1 min, then washed with 2% NaClO for 3 min and transferred to 75% sterile ethanol for 1 min, and finally washed the plant tissue with sterile water for 0.5 min). 10 g of surface-sterilized tea leaves were placed in a sterilized small blender, 50 mL of sterile water was added, and it was alternately pressed and beaten for 1 min. The crushed stock solution was filtered with sterile medical gauze, and the filtered stock solution was diluted with sterile water by 10 0 、10 -1 、10 -2 、10-3 , 10 -4 , 10 -5 Dilutions of 6 levels such as these are made, and the undiluted solutions at each dilution level are used as working solutions for subsequent inoculation. The entire process of surface sterilization of tea leaves and preparation of working solutions is carried out in a laminar flow hood.

[0025] Inoculation stage: Take 100 μL of the working solution and spread it onto LB solid medium respectively, then place it in an incubator at 28 °C for 2 - 3 days, and transfer and purify the grown bacterial colonies. The bacteria are purified by the method of streaking dilution.

[0026] Species identification and preservation: The purified microorganisms are subjected to sequence amplification and microbial species identification using 16s - 36F / 16S - 1492R. After identification, multiple endophytic bacteria of different genera and species are obtained. Among them, the strain with the 16s - 36F sequence shown in SEQ ID NO.1 and the 16S - 1492R sequence shown in SEQ ID NO.2 is named Klebsiella oxytoca strain YS - pp - 01, and the taxonomic name is Klebsiella oxytoca Klebsiella oxytoca , and the preservation number is CGMCC No.32447. This strain is isolated and cultured from tea leaf samples during the black tea fermentation stage.

[0027] For the strains in the laboratory, use LB broth medium (peptone 10.0 g / L, sodium chloride 5.0 g / L, glucose 1.0 g / L, yeast extract powder 5.0 g / L, the balance is water, and the final pH is 7.0 ± 0.2) in an environment at 28 °C, and obtain the bacterial solution after constant temperature shaking culture for 16 h. Mix the bacterial solution with 50% glycerol (1:1 v / v) and store it at - 80 °C. The 50% glycerol is prepared from 0.9% NaCl:100% glycerol (1:1 v / v).

[0028] Activity identification of the tea tree endophytic bacterium YS - pp - 01 Based on the results of the previous correlation analysis, in vitro biological activity analysis of the obtained endophytic bacteria is carried out.

[0029] First, strain activation is required. Streak - dilute the preserved strain using LB solid medium, place it in an incubator at 28 °C for 1 - 2 days. After activation, pick a single colony and inoculate it into 20 mL of LB broth medium, and culture it at 28 °C and 200 rpm for 12 - 16 hours (OD600 = 0.95 - 1.05), then place it in the laminar flow hood for standby.

[0030] The activity detection of endophytes uses tea soup as the culture condition. The preparation method of the tea soup refers to the method provided in the national standard "GB-T8305-2013 Determination of water extract in tea": Weigh 5 g of tea powder into a 500 mL conical flask, add 300 mL of boiling distilled water, and immediately transfer it to a boiling water bath for extraction for 45 min (shake it once every 10 min). Immediately after the extraction, perform vacuum filtration while it is hot. Wash the tea residue several times with about 150 mL of boiling distilled water and then transfer it to a 500 mL volumetric flask. After cooling to room temperature, make up the volume with deionized water. Transfer the made-up tea soup to a reagent bottle, sterilize it at 120 °C for 20 min, and then place it in a laminar flow bench for standby. The tea powder can be selected from fresh tea leaves of tea trees or samples at any stage of the tea processing process (including withering, rolling, fermentation, and drying), and then freeze-dried and ground. The reason for using fresh tea leaves to prepare the tea soup in the experiment is that on the one hand, the raw materials are easily obtained, and on the other hand, compared with the tea processing process, the content of catechin components in fresh tea leaves is the highest, and it is easier to observe the difference phenomenon.

[0031] Transfer the activated bacterial liquid to the sterilized tea soup at a ratio of 1:20 v / v. The un-inoculated blank tea soup is used as a control. Each group has 3 replicates and is cultured with shaking at 28 °C and 200 rpm for 16 hours. After the culture is completed, centrifuge the bacterial liquid / tea soup of the experimental group and the control group at 8000 rpm for 3 min, and then take the supernatant to detect the content of catechin components in the tea soup. The detection method is carried out in combination with the detection method provided in the current national detection standard "GB-T8313-2018 Detection method for the content of tea polyphenols and catechins in tea".

[0032] Analysis of the detection results found that compared with the blank tea soup control, the catechin components in the tea soup for culturing endophytes K. oxytoca decreased significantly or even disappeared, and significantly different from this, the content of catechin components in the tea soup for culturing endophytes S. marcescens did not change significantly ( Figure 1 , 2). It shows that endophytes K. oxytoca can cause a large amount of catechin components in the culture environment to disappear, while the other endophytes represented by S. marcescens lack similar functions.

[0033] Differential metabolite analysis Extract the tea soup from fresh tea leaves of tea trees, and use the blank tea soup as the control condition to analyze two endophytes K. oxytoca and S. marcescensCultivate. The method for preparing the tea soup and activating the strains is the same as described above. Cultivate with shaking at 200 rpm at 28 °C for 16 h, centrifuge at 8000 rpm for 3 min, take 4 mL of the supernatant and place it in a 10 mL volumetric flask, dilute to the mark with 70% methanol or pure methanol (chromatographically pure), filter through a 0.45 μm organic membrane, and then analyze the differential components in the tea soup. Each treatment is repeated 3 times technically. Use a high performance liquid chromatograph (LC5090) to analyze the differences in catechin components between groups; use a liquid chromatography - time - of - flight mass spectrometer (6546LC / Q - TOF) to analyze the differences in the composition of tea metabolites between groups, scan in positive and negative ion modes respectively, and compare and analyze the compounds using the online website GNPS (https: / / gnps.ucsd.edu) and the self - built database. The results show ( Figure 2 , 3), compared with the culture environment of the blank tea soup and the control bacteria S. marcescens , in the culture environment of the functional bacteria K. oxytoca , the catechin components are significantly reduced, and the contents of EPSFs compounds (EGCG - cThea, EGC - cThea) are significantly increased; at the same time, the contents of theanine in the culture environments of the control bacteria S. marcescens and the functional bacteria K. oxytoca are both significantly reduced. It is speculated that K. oxytoca there are functional components in S. marcescens that are not present in and can promote the combination of theanine and catechin components in the tea soup. In addition, based on the analysis results, it is speculated that the difference in color between different treatment groups may be caused by the differences in the contents of theaflavin components and anthocyanin components.

[0034] Study on the transformation rules of catechin components and theanine To further confirm the speculation that " K. oxytoca has the function of promoting the combination of theanine and catechin components in the tea soup", a set of experiments was set up in this study. During the cultivation of K. oxytoca , by monitoring the theanine, catechin components, and EPSFs compounds in the tea soup every 3 hours, the change rules of the reaction raw materials (catechin components and theanine) and the products (EPSFs compounds) were obtained, so as to explore the correlation among the three and prove the function of this endophyte. Therefore, consistent with the previous experiment, in this experiment, the tea soup prepared from fresh tea leaves was still used to K. oxytocaCultivation was carried out, with blank tea soup as the control group, and shaking culture was carried out at 28℃ and 200 rpm. Samples were taken at 6 time points, including 0h, 3h, 6h, 9h, 12h and 15h, and centrifuged at 8000 rpm for 3 min. 4 mL of supernatant was taken and placed in a 10 mL volumetric flask, and the volume was fixed with 70% methanol or pure methanol (chromatographic grade). After filtration with a 0.45μm organic membrane, catechin components, theanine and EPSFs compounds in the tea soup were detected. Each treatment was repeated three times. The differences in catechin components between groups were analyzed by high performance liquid chromatography (LC5090), the changes in theanine between groups were analyzed by fully automatic amino acid analyzer (SVKAM / S-433), and the EPSFs compounds between groups were quantitatively analyzed by liquid chromatography-mass spectrometry (6546LC / Q-TOF). The analysis results showed that catechin components, especially EGCG ( Figure 4 A) and theanine ( Figure 4 B) K. oxytoca There was no significant change in the content during the first 9 hours of the culture process. Taking the 9th hour as the turning point, the content of EGCG and theanine decreased rapidly during the 9-15th hour. At the same time, the presence of EGCG-cThea was detected at 9h, 12h and 15h, and the presence of EC-cThea was detected at 15h. Figure 5 ), indicating that endophytes in tea trees K. oxytoca It does have the function of catalyzing the combination of catechin components in fresh leaves with theanine to form EPSFs compounds.

[0035] To explore endophytes K. oxytoca The potential of exogenous theanine and catechin components is transformed by adding exogenous theanine and catechin components to K. oxytoca The analysis was carried out in a culture environment. 30 mg of theanine and EGCG were added to 150 mL of tea soup to form an exogenous addition with a final concentration of 200 mg / L. The tea soup with added theanine and EGCG was used as a blank control (denoted as CTth-CK), and then the same culture method and detection method were used to detect the content of theanine and EGCG at 9h and 15h. The test results showed that the content of theanine and EGCG at the two time points showed a consistent change trend. The two contents at 15h of culture were significantly lower than those at 9h, and there were also significant differences compared with CTth-CK ( Figure 6 ).

[0036] Thea three elements content detection Tea soup was extracted from fresh tea leaves, and blank tea soup was used as the control condition. After incubation at 28°C for 16 h, the difference in the contents of the three thea elements between the groups was analyzed using an ultraviolet spectrophotometer. The results showed that ( Figure 7 ),K. oxytoca The contents of theabrownine and theaflavin in the culture environment of (BAC2) were significantly higher than those in the blank control, and the content of theabrownine was 17.26 times that of the blank control (CK) and 2.91 times that of the control bacteria (BAC1), and the content of theaflavin was 1.86 times that of the blank control.

[0037] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Klebsiella oxytoca strain YS-pp-01, characterized in that, Classification name: Klebsiella oxytoca Klebsiella oxytoca , The preservation number is CGMCC No. 32447.

2. The Klebsiella oxytoca strain YS-pp-01 according to claim 1, wherein The 16s-36F sequence of the strain YS-pp-01 is shown in SEQ ID NO.

1.

3. The Klebsiella oxytoca strain YS-pp-01 according to claim 1, characterized in that, The 16S-1492R sequence of the strain YS-pp-01 is shown in SEQ ID NO.

2.

4. A microbial preparation, characterized in that, The microbial agent contains Klebsiella oxytoca strain YS-pp-01, and the taxonomic name is Klebsiella oxytoca Klebsiella oxytoca , and the preservation number is CGMCC No. 32447.

5. Use of the Klebsiella oxytoca strain YS-pp-01 according to any one of claims 1-3 or the microbial preparation according to claim 4 in the preparation of a product for degrading catechin components in tea soup.

6. Use of the Klebsiella oxytoca strain YS-pp-01 according to any one of claims 1-3 or the microbial preparation according to claim 4 in the preparation of a product for promoting the content of EPSFs compounds in tea leaves.

7. Use of the Klebsiella oxytoca strain YS-pp-01 according to any one of claims 1-3 or the microbial preparation according to claim 4 in the preparation of a product for improving the quality of tea leaves.

8. Use of the Klebsiella oxytoca strain YS-pp-01 according to any one of claims 1-3 or the microbial preparation according to claim 4 in tea fermentation.

9. A method for tea fermentation, characterized in that, Add the Klebsiella oxytoca strain YS-pp-01 according to any one of claims 1-3 or the microbial preparation according to claim 4 during the tea fermentation process.

10. A method for cultivating tea trees, characterized in that, During the growth process of tea trees, apply the Klebsiella oxytoca strain YS-pp-01 according to any one of claims 1-3 or the microbial preparation according to claim 4.

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