Levibacillus zyma capable of producing characteristic flavor substance p-cresol of sour bamboo shoots through fermentation
By screening and identifying Lactobacillus CCFM1420, the problem of unclear metabolic pathway in sour bamboo shoots was solved, and the efficient conversion of para-hydroxybenzyl alcohol to sour bamboo shoots was achieved, which improved the flavor and production standardization of sour bamboo shoots, and had wide application prospects.
Patent Information
- Application Number
- CN202510441694.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-09
- Publication Date
- 2025-07-25
AI Technical Summary
The metabolic pathway of p-cresol in sour bamboo shoots in the prior art is not clear, which limits the optimization and flavor regulation of the sour bamboo shoots industry, and lacks functional microorganisms that can produce p-cresol.
A strain of Lactobacillus zymae (Levilactobacillus zymae) CCFM1420 was screened and identified, which could convert parabenzyl alcohol in fresh bamboo shoots into paracresol in sour bamboo shoots under facultative anaerobic conditions, providing efficient strain resources.
It has achieved nearly 100% conversion of para-hydroxybenzyl alcohol into para-cresol, which has improved the flavor regulation and standardized production potential of sour bamboo shoots. It has acid and salt resistance characteristics, and is suitable for fermented vegetables and food additives.
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Abstract
Description
Technical Field
[0001] The present invention relates to Levilactobacillus zymae which can ferment to produce p-cresol, a characteristic flavor substance of pickled bamboo shoots, and belongs to the field of microbial technology. Background Art
[0002] p-Cresol (p-methylphenol), a chemical substance with a leathery and pit mud odor, has a very low threshold of only 55 μg / kg. At the same time, p-cresol is the compound with the highest content of flavor substances in pickled bamboo shoots and is also the characteristic flavor substance of the unique sour and stinky smell of pickled bamboo shoots (see the papers "Characterization of physicochemical properties and flavor profiles of fermented Chinese bamboo shoots (suansun) from Liuzhou and Guilin" and "Optimization and comparative analysis of extraction methods for odor substances in fermented pickled bamboo shoots in Guangxi"). Pickled bamboo shoots are traditional fermented foods in southern China and are very popular among the public because of their unique sour and stinky smell. However, the metabolic pathway of p-cresol in pickled bamboo shoots remains undetermined, which greatly limits the further optimization of the pickled bamboo shoot industry.
[0003] Microorganisms are the core of pickled bamboo shoot fermentation. Research shows that during the fermentation of pickled bamboo shoots, dominant fermenting strains such as Leuconostoc mesenteroides, Weissella spp., Lactobacillus brevis, Lactobacillus plantarum, and Lactobacillus fermentum will produce flavor substances such as organic acids, aldehydes, and esters (see the paper "Research progress on microorganisms and flavor formation in pickled bamboo shoots"). However, there is currently no research report on the functional microorganisms involved in the formation of p-cresol.
[0004] Therefore, obtaining bacterial species resources that can produce p-cresol is very important for targeted regulation of the unique flavor of pickled bamboo shoots and the standardization construction work of pickled bamboo shoots. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide a Levilactobacillus zymae that can convert p-hydroxybenzyl alcohol in bamboo shoots into p-cresol, so as to provide high-quality bacterial species resources for targeted regulation of the flavor of pickled bamboo shoots and the standardized production of pickled bamboo shoots.
[0006] The present invention provides a strain of Levilactobacillus zymae CCFM1420, which was deposited at the Guangdong Provincial Culture Collection of Microorganisms on October 31, 2024, with the deposit number GDMCC No: 65378 and the deposit address being the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.
[0007] In one embodiment, the Levilactobacillus zymae CCFM1420 is isolated from a pickled bamboo shoot sample sourced from Liuzhou City, and its 16S rDNA sequence is as shown in SEQ ID NO: 1.
[0008] The present invention also provides a method for preparing p-cresol by transforming p-hydroxybenzyl alcohol using Levilactobacillus zymae CCFM1420. The method is to first inoculate the above-mentioned Levilactobacillus zymae CCFM1420 into a fermentation medium containing p-hydroxybenzyl alcohol for fermentation to obtain a fermentation broth, and then extract p-cresol from the fermentation broth.
[0009] In one embodiment, the fermentation medium includes but is not limited to MRS medium.
[0010] In some embodiments, the method comprises the following steps:
[0011] (1) Cultivation of the strain: Inoculate Levilactobacillus zymae CCFM1420 into a medium and culture for 16 - 24 h;
[0012] (2) Transformation of p-hydroxybenzyl alcohol: Collect the bacterial suspension prepared in step (1), transfer it to an MRS medium containing p-hydroxybenzyl alcohol, and culture at 35 - 37 °C for at least 14 - 18 h.
[0013] In some specific embodiments, the concentration of p-hydroxybenzyl alcohol in the medium in step (2) is 0.02 mmol / L.
[0014] The present invention also provides a method for fermenting pickled bamboo shoots using Levilactobacillus zymae CCFM1420.
[0015] The present invention also provides the fermentation broth obtained by fermenting the Levilactobacillus zymae CCFM1420.
[0016] The present invention also provides the application of the above-mentioned Levilactobacillus zymae CCFM1420 in transforming p-hydroxybenzyl alcohol to prepare p-cresol.
[0017] The present invention also provides the application of the Levilactobacillus zymae CCFM1420 in fermenting to prepare p-methylphenol or products containing p-methylphenol.
[0018] In one embodiment, the products include but are not limited to food additives or spices.
[0019] Beneficial effects:
[0020] The present invention screens out a strain of Lactobacillus fermentans CCFM1420, which has the characteristics of acid resistance and salt resistance and has the application potential of fermented vegetables. The Lactobacillus fermentans CCFM1420 of the present invention can convert p-hydroxybenzyl alcohol to generate p-cresol, and the conversion rate is close to 100%. The strain is used for sour bamboo shoot fermentation, and p-hydroxybenzyl alcohol in fresh bamboo shoots can be used to generate p-cresol, which has extremely high application prospects in the standardized production of sour bamboo shoots and the like.
[0021] Biomaterial Deposit
[0022] Levilactobacillus zymae CCFM1420, taxonomically named Levilactobacillus zymae, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 31, 2024, with the deposit number GDMCC No: 65378, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 These are the colony characteristics of Levilactobacillus zymae CCFM1420.
[0024] Figure 2 These are the bacterial characteristics of Levilactobacillus zymae CCFM1420.
[0025] Figure 3 This is the growth curve of Levilactobacillus zymae CCFM1420.
[0026] Figure 4 The survival rate of Levilactobacillus zymae CCFM1420 in MRS liquid culture medium containing different concentrations of NaCl.
[0027] Figure 5 The contents of p-hydroxybenzyl alcohol and p-cresol before and after fermentation of Levilactobacillus zymae CCFM1420. DETAILED DESCRIPTION
[0028] The present invention is further described below in conjunction with specific embodiments and drawings.
[0029] The culture medium involved in the following examples is as follows:
[0030] MRS solid medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast powder 5 g / L, diammonium hydrogen citrate 2 g / L, K2HPO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4·H2O 0.05 g / L, Tween 80 1 mL / L, agar powder 15 g / L, cysteine hydrochloride 0.5 g / L.
[0031] MRS liquid medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast powder 5 g / L, diammonium hydrogen citrate 2 g / L, K2HPO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4·H2O 0.05 g / L, Tween 80 1 mL / L, cysteine hydrochloride 0.5 g / L.
[0032] MRS - p - hydroxybenzyl alcohol medium: p - hydroxybenzyl alcohol is added to the MRS medium at a final concentration of 0.02 mmol / L. Example 1: Screening of Levilactobacillus zymae CCFM1420
[0033] Using the pickled bamboo shoot juice from Guangxi Zhuang Autonomous Region as a sample, it was serially diluted 10 - fold with sterile normal saline to 10-3, and then 100 μL of the diluted solutions with dilution factors of 10 -1 、10 -2 、10 -3 were respectively spread on the MRS solid medium and cultured in an inverted position at 37 °C for 48 h. The colony morphology was observed and recorded; different - shaped colonies on the MRS solid medium were picked for streak isolation. After static culture at 37 °C for 48 h, different - shaped single colonies on the MRS solid medium were picked again for streak isolation until pure single colonies with consistent morphology were obtained; the colony morphology of the single colonies was observed, and the strain with small, round, milky - white, convex, smooth colony morphology, spherical cell morphology, and positive Gram - staining result was selected. Here, a strain that might be Levilactobacillus zymae was obtained and named Levilactobacillus zymae CCFM1420.
[0034] The microscopic examination method is as follows: Take a clean glass slide, heat it slightly on the flame and then cool it; after cooling, add a small drop of sterile water in the central part of the clean glass slide, and use an inoculation loop to pick a small amount of bacteria from a single colony beside the flame and mix it with the water; burn off the excess bacteria on the inoculation loop, and then use the inoculation loop to spread the bacteria into a uniform thin layer with a diameter of 1 cm; after the uniform thin layer on the clean glass slide dries naturally, heat it 3 - 4 times on a gentle fire for fixation; add 1 - 2 drops of crystal violet solution to the smear, stain for 1 min; gently wash off the stain with water, and gently blot the water on the glass slide with absorbent paper; add 1 - 2 drops of iodine solution to the smear, stain for 1 min, gently wash off the stain with water, and gently blot the water on the glass slide with absorbent paper; add 1 - 2 drops of 95% (v / v) ethanol solution to the smear, decolorize for 5 - 15 s, gently rinse with water, and gently blot the water on the glass slide with absorbent paper; add 1 - 2 drops of safranin solution to the smear, stain for 2 min, and after drying, conduct microscopic examination with a microscope to observe the bacterial morphology;
[0035] The Gram staining method refers to the textbook "Industrial Microbial Breeding" (author: Zhuge Jian); the method for investigating physiological and biochemical characteristics refers to the textbook "Handbook for the Systematic Identification of Common Bacteria" (author: Dong Xiuzhu).
[0036] The method for measuring the growth curve is as follows: Dispense the test medium into 100 mL blue-capped bottles, inoculate the test strain at an inoculation amount of 2%, mix well, and immediately dispense the mixture into test tubes, and incubate in a constant temperature incubator at 37°C. Take out 3 test tubes every 2 h, shake them well, and measure OD600. Zero with the medium as a blank tube. Draw a growth curve with the growth time on the abscissa and OD600 on the ordinate.
[0037] The colony characteristics of this strain are shown in Figure 1 , and the bacterial characteristics are shown in Figure 2 , and the growth curve is shown in Figure 3 .
[0038] Example 2: Identification and preservation of Levilactobacillus zymae CCFM1420
[0039] Extract the genome of the strain Levilactobacillus zymae CCFM1420, amplify and sequence the 16S rDNA of the strain Levilactobacillus zymae CCFM1420 (sequencing was performed by BGI. Among them, the nucleotide sequence of the 16S rDNA amplification of the strain Levilactobacillus zymae CCFM1420 is shown in SEQ ID NO: 1. The primers used for amplification are 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT). Compare the sequenced sequence with the nucleic acid sequence in NCBI. The results show that the strain Levilactobacillus zymae CCFM1420 is Levilactobacillus zymae, named Levilactobacillus zymae CCFM1420).
[0040] Pick a single colony of Levilactobacillus zymae CCFM1420 and inoculate it into MRS liquid medium, and culture it statically at 37°C for 24 h to obtain a bacterial solution; take 1 mL of the bacterial solution and put it into a bacteria preservation tube, centrifuge at 6000 rpm for 3 min, then discard the supernatant, and resuspend the bacterial pellet in 30% (v / v) glycerol solution and store it at -80°C.
[0041] Example 3: Study on the fermentation characteristics of Levilactobacillus zymae CCFM1420
[0042] 1. Acid tolerance
[0043] Streak the Levilactobacillus zymae CCFM1420 obtained in Example 2 on MRS solid medium and culture it at 37°C for 48 h to obtain single colonies; pick single colonies and inoculate them into MRS liquid medium respectively, and culture them at 37°C for 18 h for activation, and activate them continuously for three generations. Adjust the pH of the MRS liquid medium to 3.0 with HCl. Inoculate the activated Levilactobacillus zymae CCFM1420 into the MRS liquid with a pH of 3.0 at an inoculation amount of 2% (V / V), mix well, and then culture it at a constant temperature of 37°C for 3 h, dilute it with physiological saline, take 1 mL of the bacterial solution with an appropriate dilution gradient and pour it, and measure the viable bacteria count. Take the viable bacteria count at 0 h as the control group, and calculate the survival rate of the strain at pH 3.0. The formula for calculating the survival rate of the strain is as follows:
[0044] Acid tolerance survival rate = N2 / N1 × 100%;
[0045] Among them, N1 is the viable count (CFU / mL) at 0 h; N2 is the viable count (CFU / mL) after 3 h of treatment.
[0046] The results showed that the survival rate of Levilactobacillus zymae CCFM1420 after 3 h of treatment in acidic MRS medium with a pH of 3 was 90.72%, indicating good acid tolerance.
[0047] 2. Salt tolerance
[0048] The activated Levilactobacillus zymae CCFM1420 was inoculated into MRS liquid medium containing 3 g / 100 mL, 6 g / 100 mL, and 9 g / 100 mL NaCl at an inoculation amount of 2% (V / V), and cultured at 37 °C for 3 h. Then it was diluted with normal saline, 1 mL of the bacterial liquid with an appropriate dilution gradient was taken, and the viable count was determined by the plate pouring method. The formula for calculating the survival rate of the strain is as follows:
[0049] Salt tolerance survival rate = N2 / N1 × 100%;
[0050] Among them, N1 is the viable count (CFU / mL) at 0 h; N2 is the viable count (CFU / mL) after 3 h of salt treatment.
[0051] The results showed ( Figure 4 ) that the survival rates of Levilactobacillus zymae CCFM1420 in MRS liquid medium containing 3 g / 100 mL, 6 g / 100 mL, and 9 g / 100 mL NaCl were 125.38%, 41.51%, and 66.11%, respectively.
[0052] Example 4 Comparative experiment on the preparation of p-cresol by transforming p-hydroxybenzyl alcohol using Levilactobacillus zymae CCFM1420
[0053] The Levilactobacillus zymae CCFM1420 obtained in Example 2 was streaked on MRS solid medium and cultured at 37 °C for 24 h to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium respectively, and cultured at 37 °C for 24 h for activation. After continuous activation for two generations, the activated solution was obtained; the activated solution was inoculated into MRS liquid medium at an inoculation amount of 2% (v / v) respectively, and cultured at 37 °C for 24 h to obtain the bacterial liquid.
[0054] Dissolve p - hydroxybenzyl alcohol in ultrapure water. After filtering through a 0.22 - μm filter in a laminar flow hood, add it to the sterilized culture medium to a final concentration of 0.02 mmol / L. After mixing, aliquot 5 mL of the MRS culture medium containing p - hydroxybenzyl alcohol into test tubes. Inoculate the activated bacterial solution into the MRS culture medium containing p - hydroxybenzyl alcohol at a ratio of 2% (v / v), and incubate at 37 °C for 14 days.
[0055] Filter 1 mL of the supernatant through a microporous filter membrane with a pore size of 0.45 μm, and then use the obtained filtrate for reverse - phase high - performance liquid chromatography (RP - HPLC) analysis. Use a BEH C18 chromatographic column. Mobile phase A: glacial acetic acid: water (v / v = 1:99); Mobile phase B: glacial acetic acid: acetonitrile: aqueous solution (v / v = 1:30:69). Gradient elution: 80%A + 20%B (0 min - 16 min); 40%A + 60%B (16 min - 23 min); 25%A + 75%B (23 min - 34 min); 100%B (34 min - 50 min).; Set the fluorescence detection wavelength gradient: 0 min, excitation wavelength is 284 nm, emission wavelength is 333 nm; 5 min, excitation wavelength is 275 nm, emission wavelength is 315 nm; 8 min, excitation wavelength is 277 nm, emission wavelength is 319 nm; 12 min, excitation wavelength is 272 nm, emission wavelength is 310 nm; 25 min, excitation wavelength is 273 nm, emission wavelength is 312 nm. Column temperature: 30 °C; Column flow rate: 1 mL / min; Injection volume: 20 μL.
[0056] At 14 days of fermentation, take samples of the fermentation supernatant and detect the contents of p - hydroxybenzyl alcohol and p - cresol before and after fermentation of the supernatant (the detection results are shown in Figure 5 ). From Figure 5 it can be seen that in the fermentation supernatant of Levilactobacillus zymae CCFM1420, the content of p - cresol increased from 0 mmol / L to 0.018 ± 0.001 mmol / L at 14 days of fermentation, while the content of p - hydroxybenzyl alcohol decreased from 0.02 mmol / L to 0.001 ± 0 mmol / L.
[0057] Example 5: Fermenting pickled bamboo shoots with Levilactobacillus zymae CCFM1420
[0058] Since the presence of p-hydroxybenzyl alcohol can be detected in fresh bamboo shoots, the content is 0.032±0.021mmol / L, the strain CCFM1420 obtained by screening was activated in MRS medium for three generations, expanded in 100mL MRS medium, cultured at 37℃ to the logarithmic phase and immediately taken out, centrifuged at 8000r / min for 20min at 4℃, and the supernatant was discarded. After that, the bacterial sludge was washed with 0.9% (w / v) physiological saline for 3 times, and centrifuged at 8000r / min for 5min after each washing and the supernatant was discarded. The fresh bamboo shoots were peeled and washed, and then put into the kimchi jar, and purified water was added to cover the fresh bamboo shoots, and the strain CCFM1420 was inoculated into it, so that the bacterial concentration after inoculation was 1×10 7 CFU / mL, stir evenly, cover with a kimchi cover and ferment in a closed manner at room temperature. At the same time, according to the above process, the strain inoculation is not carried out, and an equal amount of pure water is directly added to the closed fermentation of sour bamboo shoots as a clear water fermentation control group. After 30 days of fermentation, the sour bamboo shoot blocks and sour bamboo shoot juice are collected respectively, mixed and homogenized according to a mass ratio of 1:1, and the hydroxybenzyl alcohol and p-cresol contents therein are detected according to the method of Example 4. The results showed that in the system with the addition of strain CCFM1420 for enhanced fermentation, p-hydroxybenzyl alcohol was reduced to 0.004±0.001mmol / L, and the p-cresol content was increased from 0mmol / L to 0.019±0.001mmol / L.
[0059] Comparative Example 1:
[0060] The natural fermentation method for preparing sour bamboo shoots was used as a control, and the specific implementation method was the same as that in Example 5. The results showed that the content of p-cresol in the fermented mature sour bamboo shoot system was 263.58±35.81 μg / kg.
[0061] Although the present invention has been disclosed as above in the form of a 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. Levilactobacillus zymae CCFM1420 was deposited at the Guangdong Microbial Culture Collection Center on October 31, 2024, with the deposit number GDMCC No: 65378.
2. A method for preparing p-methylphenol, characterized in that, Inoculate the Levilactobacillus zymae CCFM1420 described in claim 1 into a fermentation medium containing p - hydroxybenzyl alcohol for fermentation to obtain a fermentation broth, and then extract p - cresol from the fermentation broth.
3. The method according to claim 2, wherein The fermentation medium includes, but is not limited to, MRS medium.
4. The method according to claim 2 or 3, characterized in that, The method includes the following steps: (1) Inoculate Levilactobacillus zymae CCFM1420 into a medium and culture for 16 - 24 h; (2) Collect the bacterial suspension prepared in step (1), transfer it to a medium containing p - hydroxybenzyl alcohol, and culture at 35 - 37 °C for at least 14 - 18 h.
5. The method according to claim 4, wherein In step (2), the concentration of p - hydroxybenzyl alcohol in the medium is 0.02 mmol / L.
6. A microbial preparation containing the Levilactobacillus zymae CCFM1420 described in claim 1.
7. Use of the Levilactobacillus zymae CCFM1420 described in claim 1 in fermenting pickled bamboo shoots.
8. Use of the Levilactobacillus zymae CCFM1420 described in claim 1 in the conversion of p - hydroxybenzyl alcohol to prepare p - cresol.
9. Use of the Levilactobacillus zymae CCFM1420 described in claim 1 in the fermentation preparation of p - methylphenol or products containing p - methylphenol.
10. The application according to claim 9, wherein The products include, but are not limited to, food additives or fragrances.