A kind of acid-producing Klebsiella strain and its application

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to an acid-producing Klebsiella strain and its application. The present invention provides an acid-producing Klebsiella strain YS-pp-01, which is classified as: Klebsiella acidophilus Klebsiella oxytoca, The deposit number is CGMCC No. 32447. The microbial fermentation provided by the present invention realizes the efficient synthesis of EPSFs compounds, which are quality components in tea leaves. This breaks the rate-limiting path that the compound can only be obtained as a natural product through tea storage. Compared with other endophytes isolated, its function of converting catechins is significant. After microbial cultivation, the yield of star components in tea leaves such as theabrownin and theaflavins is also greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to an acid-producing Klebsiella strain and an application thereof. Background Art

[0002] N-ethyl-2-pyrrolidone-substituted flavanols (EPSFs) are flavanone alkaloids that have garnered significant attention in recent years for their significant nutritional and health potential. Formed in tea leaves, these compounds are derived from the catechin component and theanine as precursors. While retaining the anti-inflammatory and antioxidant bioactivities of the catechin component, they also possess enhanced structural stability and human biocompatibility, promoting their effective nutritional and health benefits. EPSFs hold great promise for biomedical and healthcare applications. However, EPSFs are present in extremely low concentrations in fresh leaves of the currently widely cultivated tea plant. They are only found in significant quantities in long-stored tea, where their concentration increases with storage time. This suggests that the tea plant's metabolic capacity for EPSF synthesis is limited. Endophytes are considered key potential regulators of this limitation. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned existing technologies and provide an acid-producing Klebsiella strain and its application. The strain is obtained by isolating endophytes from tea leaves in the laboratory and can utilize catechin components and theanine in tea leaves to achieve efficient synthesis of EPSFs compounds.

[0004] A Klebsiella oxytoca strain YS-pp-01, classified as Klebsiella oxytoca Klebsiella oxytoca , The deposit number is CGMCC No.32447.

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

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

[0007] The present invention also provides a microbial preparation containing Klebsiella oxytoca strain YS-pp-01, which is classified as: Klebsiella oxytoca Klebsiella oxytoca , The deposit number is CGMCC No.32447.

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

[0009] Furthermore, the present invention provides a use of any of the aforementioned Klebsiella oxytoca strains YS-pp-01 or the aforementioned microbial preparation in the preparation of a product for increasing the content of EPSFs compound components in tea.

[0010] Furthermore, the present invention provides the use of any of the aforementioned Klebsiella oxytoca strains YS-pp-01 or the aforementioned microbial preparations in the preparation of products that improve tea quality. 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 theabrownins, theaflavins, and anthocyanins.

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

[0012] Furthermore, the present invention provides a tea fermentation method, wherein any one of the aforementioned Klebsiella oxytocaine strains YS-pp-01 or the aforementioned microbial preparation is added during the tea fermentation process.

[0013] Furthermore, the present invention provides a tea tree cultivation method, wherein any one of the aforementioned Klebsiella oxytoca strains YS-pp-01 or the aforementioned microbial preparation is applied during the growth of the tea trees.

[0014] The present invention has the following advantages over the prior art:

[0015] Efficient synthesis of EPSFs compounds, the quality components in tea, can be achieved through microbial fermentation. There have been no previous reports on the functions of this microorganism in synthesizing EPSFs compounds, which has broken the rate-limiting pathway that this compound can only be obtained as a natural product through tea storage. Compared with other endophytes isolated, its function of converting catechins is significant, and after microbial cultivation, the production of star components in tea such as theabrownins and theaflavins has also been greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Figure 3. Blank tea soup and S. marcescens and K. oxytoca cultured in tea soup.

[0017] Figure 2 Statistical diagram of the difference in catechin content in tea soup treated with different strains;

[0018] Figure 3 Statistical diagram of differential metabolites in tea soup treated with different strains;

[0019] Figure 4 Statistical graph of the changes in L-theanine (A) and EGCG (B) content during the growth of K. oxytoca;

[0020] Figure 5 Peak graphs of EGCG-cThea (A) and EC-cThea (B) after 15 h of culture of K. oxytoca;

[0021] Figure 6 Statistical graph of theanine and EGCG content in K. oxytoca cultured for 9h and 15h after exogenous addition;

[0022] Note: CK, blank tea soup control with exogenous addition of theanine and EGCG; YS, K.oxytoca tea soup culture treatment with exogenous addition of theanine and EGCG;

[0023] Figure 7 The difference in theanine content in tea soup treated with different strains. Note: The data in the figure are the results of log2 normalization of the original data;

[0024] Figure 8 Abundance changes of tea plant endophyte g__Klebsiella at different stages of black tea processing;

[0025] Figure 9 Colony morphology of the tea plant endophyte Klebsiella acidophilus strain YS-pp-01. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the embodiments of the present invention. For purchased commodities in the test method, if the specific conditions are not indicated, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they can all be conventional products purchased from the market.

[0027] The present invention provides a strain of Klebsiella oxytoca YS-pp-01, which is classified as: Klebsiella oxytoca Klebsiella oxytoca , The deposit number is CGMCC No.32447, the depositor is the General Microbiology Center of China Culture Collection Administration (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is October 31, 2024.

[0028] In the present invention, Klebsiella oxytoca Klebsiella oxytoca Referred to as " K. oxytoca ”.

[0029] Serratia marcescens Serratia marcescens Abbreviation "S. marcescens" .

[0030] “g_ Klebsiella ” refers to Klebsiella spp. Klebsiella ).

[0031] Example 1

[0032] 1. Correlation analysis between tea plant endophytes and quality components

[0033] Fresh leaves of the tea variety Yunkang No. 10 were collected from the Menghai Experimental Base of the Yunnan Institute and processed into black tea. 16s rRNA sequencing was performed on the fresh leaves and samples collected at each processing stage. The sequencing work was carried out at Meiji Company, and the original data was submitted to NCBI (SUB15108778). The sequencing data was used to analyze the microbial population structure in the tea samples, and the physiological and biochemical indicators such as water extract, total free amino acids, caffeine, tea polyphenols, and catechin components in the tea leaves at each stage of the tea processing process (including withering, rolling, fermentation and drying) including fresh leaves were analyzed. The structural composition data of endophytes were correlated with the content of bioactive ingredients in tea, and information on microorganisms that are significantly correlated with the formation of tea quality components was obtained. Among them, g_ Klebsiella There was a significant negative correlation between the catechin components in fresh tea leaves and during tea processing (Table 1, Figure 8 ).

[0034] Table 1 g_Klebsiella Correlation analysis with tea quality components

[0035]

[0036] 2. Isolation and identification of endophytes from tea plants

[0037] Medium preparation stage: LB solid medium (10.0 g / L peptone, 5.0 g / L sodium chloride, 1.0 g / L glucose, 5.0 g / L yeast extract powder, 15.0 g / L agar, balance water, final pH 7.0 ± 0.2) was used as the growth medium for endophytic bacteria and fungi. No antibiotics were added to the medium. After weighing according to the formula, the volume was adjusted to 500 mL with deionized water, sterilized at 121°C for 20 min, cooled to 50-60°C, and poured into a plate on a clean bench for later use.

[0038] Material preparation stage: Collect freshly picked tea leaves and tea samples in the fermentation stage of black tea, place them on ice, and bring them back to the laboratory. Before isolating the strains, the tea samples were surface sterilized (wash the samples with sterile water for 0.5 minutes, then wash them in 75% ethanol for 1 minute, then wash them with 2% NaClO for 3 minutes, transfer them to 75% sterile ethanol for 1 minute, and finally wash the plant tissue with sterile water for 0.5 minutes). Take 10 g of surface-sterilized tea leaves and place them in a sterilized small blender with 50 mL of sterile water. Press and blend them alternately for 1 minute. The blended stock solution was filtered with sterile medical gauze. The filtered stock solution was then sterilized with sterile water for 10 minutes. 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 The tea leaves were sterilized and the working solution was prepared in a clean bench.

[0039] Inoculation stage: Take 100 uL of the working solution and spread it on LB solid medium and place it in a 28-degree incubator for 2-3 days. The grown bacterial colonies are transferred and purified. The bacteria are purified by streak dilution.

[0040] Strain identification and preservation: The purified microorganisms were sequenced and identified using 16s-36F / 16S-1492R sequences. Multiple endophytic bacteria of different genera were obtained. The 16s-36F sequence was shown in SEQ ID NO. 1, and the 16S-1492R sequence was shown in SEQ ID NO. 2. The strain was named Klebsiella oxytoca strain YS-pp-01 and classified as Klebsiella oxytoca. Klebsiella oxytoca The deposit number is CGMCC No. 32447. The fungus was isolated and cultured from tea leaves during the fermentation stage of black tea.

[0041] Laboratory strains were cultured in LB broth (10.0 g / L peptone, 5.0 g / L sodium chloride, 1.0 g / L glucose, 5.0 g / L yeast extract powder, with the remainder being water, to a final pH of 7.0 ± 0.2) at 28°C with constant temperature shaking for 16 h to obtain a bacterial solution. The bacterial solution was then mixed with 50% glycerol (1:1 v / v) and frozen at −80°C. The 50% glycerol was prepared from 0.9% NaCl and 100% glycerol (1:1 v / v).

[0042] Identification of the activity of endophytic bacteria YS-pp-01 from tea plants

[0043] Based on the results of the previous association analysis, the obtained endophytic bacteria were subjected to in vitro bioactivity analysis.

[0044] First, the strain must be activated. The stored strain is streaked and diluted using LB solid medium and cultured in a 28°C incubator for 1-2 days. After activation, a single clone is picked and placed in 20 mL LB broth medium. After shaking culture at 28°C and 200 rpm for 12-16 hours (OD600 = 0.95-1.05), it is placed on a clean bench for use.

[0045] Endophyte activity testing was performed using tea broth as the culture medium. The preparation method for the tea broth followed the national standard "GB-T8305-2013, Determination of Tea Extracts": 5g of tea powder was weighed into a 500mL Erlenmeyer flask, 300mL of boiling distilled water was added, and the flask was immediately transferred to a boiling water bath for 45 minutes (shaking every 10 minutes). After extraction, the flask was immediately filtered under reduced pressure while still hot. The tea residue was washed several times with approximately 150mL of boiling distilled water and transferred to a 500mL volumetric flask. After cooling to room temperature, the flask was brought to volume with deionized water. The broth was transferred to a reagent bottle, sterilized at 120°C for 20 minutes, and then placed in a laminar flow hood for later use. Tea powder can be fresh tea leaves or samples from any stage of tea processing (including withering, rolling, fermentation, and drying) that have been freeze-dried and then ground. The reason for using fresh leaves to prepare tea in the experiment is that the raw materials are easy to obtain and, compared with the tea processing process, the catechin content in fresh leaves is the highest, making it easier to observe differences.

[0046] The activated bacterial suspension was transferred to sterilized tea at a ratio of 1:20 v / v. Uninoculated blank tea served as a control. Three replicates were set for each group, and the cultures were shaken at 28°C and 200 rpm for 16 hours. After incubation, the bacterial suspension / tea in both the experimental and control groups was centrifuged at 8000 rpm for 3 minutes, and the supernatant was collected for analysis of the catechin content in the tea. This determination was based on the method specified in the current national standard, "GB-T8313-2018, Determination of Tea Polyphenols and Catechins in Tea."

[0047] The results of the analysis showed that compared with the blank tea soup control, the cultured endophytes K. oxytoca The catechin components in the tea soup decreased significantly or even disappeared, and there was a significant difference in the cultured endophytes. S. marcescens There was no significant change in the catechin content in the tea soup ( Figure 1 , 2). Indicates that endophytes K. oxytoca It can make the catechin components in the culture environment disappear in large quantities, and S. marcescens The remaining endophytes, represented by , lack similar functions.

[0048] Differential metabolite analysis

[0049] Tea soup was extracted from fresh tea leaves, and blank tea soup was used as a control condition to isolate two endophytes that showed significant differences in the previous experiment. K. oxytoca and S. marcescens Cultivate. The preparation of tea soup and the activation of strains were the same as described above. Cultured at 200 rpm at 28 ° C for 16 h, centrifuged at 8000 rpm for 3 min, took 4 mL of supernatant and placed it in a 10 mL volumetric flask, fixed to volume with 70% methanol or pure methanol (chromatographic grade), filtered with a 0.45 μm organic membrane, and analyzed for differential components in the tea soup. Each treatment was repeated three times. The differences in catechin components between groups were analyzed using high performance liquid chromatography (LC5090); the differences in tea metabolite components between groups were analyzed using liquid chromatography-tandem mass spectrometry (6546LC / Q-TOF), and scanned in positive and negative ion modes, respectively. The compounds were compared and analyzed using the online website GNPS (https: / / gnps.ucsd.edu) and the self-built database. The results showed that ( Figure 2 , 3), compared with blank tea and control bacteria S. marcescens Compared with the culture environment of functional bacteria K. oxytoca The catechin components in the culture environment were significantly reduced, and the content of EPSFs compounds (EGCG-cThea, EGC-cThea) was significantly increased; at the same time, the control bacteria S. marcescens and functional bacteria K. oxytoca The content of theanine in the culture environment was significantly reduced. K. oxytoca Existence S. marcescens The tea leaves contain a functional component that promotes the binding of theanine and catechins in the tea soup. Furthermore, based on the analysis results, it is speculated that the color differences between the different treatment groups may be due to differences in the content of theaflavins and anthocyanins.

[0050] Study on catechin components and theanine conversion rules

[0051] To further confirm the K. oxytoca It is speculated that it can promote the combination of theanine and catechin components in tea soup. This time, a group of experiments were set up. K. oxytoca During the incubation process, the changes in the reaction raw materials (catechin components and theanine) and products (EPSFs compounds) were obtained by monitoring the tea soup every 3 hours, so as to explore the correlation between the three and prove the function of the endophyte. Therefore, consistent with the previous experiments, this experiment still used tea soup prepared from fresh tea leaves for the treatment of K. oxytocaThe tea soup was cultured, with blank tea soup as the control group, at 28°C with shaking at 200 rpm. Samples were collected at 0h, 3h, 6h, 9h, 12h, and 15h, centrifuged at 8000 rpm for 3 min, and 4 mL of the supernatant was placed in a 10 mL volumetric flask. The volume was adjusted with 70% methanol or pure methanol (chromatographic grade). After filtration with a 0.45μm organic membrane, the 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 using a high-performance liquid chromatography (LC5090), the changes in theanine between groups were analyzed using a fully automatic amino acid analyzer (SVKAM / S-433), and the EPSFs compounds between groups were quantitatively analyzed using a liquid chromatography-mass spectrometry instrument (6546LC / Q-TOF). The results showed that the catechin components, especially EGCG ( Figure 4 A) and theanine ( Figure 4 B) in K. oxytoca During the first 9 hours of culture, no significant changes in content occurred. Taking the 9th hour as the turning point, the contents 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.

[0052] To explore endophytes K. oxytoca The potential of exogenous theanine and catechin components is transformed by exogenously adding theanine and catechin components to K. oxytoca The analysis was conducted 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 theanine and EGCG added was used as a blank control (denoted as CTth-CK). The same culture method and detection method were then 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 trend of change. 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 ).

[0053] Detection of the content of three theazolam elements

[0054] Tea soup was extracted from fresh tea leaves, and blank tea soup was used as the control condition. After incubation at 28℃ for 16 hours, the difference in the three thea compounds between the groups was analyzed by UV spectrophotometer. The results showed that ( Figure 7), K. oxytoca The contents of theabrownin and theaflavins in the culture environment of (BAC2) were significantly higher than those in the blank control, and the theabrownin content was 17.26 times that of the blank control (CK) and 2.91 times that of the control bacteria (BAC1), while the theaflavins content was 1.86 times that of the blank control.

[0055] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

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

2. A microbial preparation, characterized in that The microbial preparation contains the acid-producing Klebsiella strain YS-pp-01, classified as: Klebsiella acidophilus Klebsiella oxytoca , the deposit number is CGMCC No.32447.

3. Use of the Klebsiella oxytoca strain YS-pp-01 according to claim 1 or the microbial preparation according to claim 2 in preparing a product for degrading catechin components in tea soup.

4. Use of the oxytoca Klebsiella strain YS-pp-01 according to claim 1 or the microbial preparation according to claim 2 in tea fermentation.

5. A tea fermentation method, characterized in that: The acid-producing Klebsiella strain YS-pp-01 according to claim 1 or the microbial preparation according to claim 2 is added during the tea fermentation process.

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