A lactobacillus buchneri strain capable of fermenting p-hydroxybenzyl alcohol in bamboo shoots to form an acid bamboo characteristic flavor substance

Abstract

The application discloses a lactobacillus buchneri capable of fermenting p-hydroxybenzyl alcohol in bamboo shoots to form characteristic flavoring substances of sour bamboo shoots, and belongs to the field of probiotic fermentation.The lactobacillus buchneri CCFM1421 is screened, and has been preserved in the Guangdong Microbial Culture Collection Center on October 31, 2024, with a preservation number of GDMCC No: 65377.The lactobacillus buchneri CCFM1421 screened in the application has the characteristics of acid and salt resistance, can utilize p-hydroxybenzyl alcohol to ferment p-cresol, and provides high-quality strain resources for targeted regulation of the flavor of sour bamboo shoots and the development of sour bamboo shoot fermentation agents.

Description

Technical Field

[0001] This invention relates to a strain of Lactobacillus brunelli that can ferment and transform p-hydroxybenzyl alcohol in bamboo shoots to form characteristic flavor substances of sour bamboo shoots, belonging to the field of microbial technology. Background Technology

[0002] p-Cresol (p-methylphenol) is the main flavor compound responsible for the pungent odor of pickled bamboo shoots, possessing a burnt, animal-like smell. Pickled bamboo shoots, a traditional delicacy, are not only a common cooking ingredient in southern China but also the soul of Liuzhou river snail rice noodles. To date, pickled bamboo shoots are widely favored by consumers for their unique flavor and crisp texture. However, the precursors and metabolic pathways of p-cresol in pickled bamboo shoots have not been clearly reported.

[0003] Microorganisms are the main factors affecting the quality and flavor of pickled bamboo shoots. Studies have shown that during the fermentation process of pickled bamboo shoots, dominant fermentation strains such as Weissella, Lactobacillus fermentum, Lactobacillus plantarum, and Pediococcus pentosaceus produce organic acids, amino acids, and other substances through a series of biochemical processes. At the same time, they produce characteristic flavor substances that play a decisive role in the unique flavor of pickled bamboo shoots, such as p-cresol (see the paper "Changes in Volatile Flavor Substances During Bamboo Shoot Fermentation").

[0004] Some literature reports that p-cresol may be produced by the conversion of tyrosine by Clostridium butyricum, while others have shown a positive correlation between p-cresol and lactic acid bacteria. Furthermore, the abundance of Clostridium butyricum in pickled bamboo shoots is extremely low (see the papers "The microbials succession and their correlation with the dynamics of flavor compounds involved in the natural fermentation of suansun, a traditional Chinese fermented bamboo shoots" and "Characterization of physicochemical properties and flavor profiles of fermented Chinese bamboo shoots (suansun) from Liuzhou and Guilin"). Moreover, Clostridium butyricum is inedible, while lactic acid bacteria are safe fermentation agents for consumption.

[0005] Therefore, obtaining the key edible lactic acid bacteria that can produce p-cresol is of great significance for the standardized production of pickled bamboo shoots. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a strain of Lactobacillus brunelli and its use in the fermentation and conversion of p-hydroxybenzyl alcohol to p-cresol.

[0007] This invention provides a strain of Lactobacillus buchneri, CCFM1421, which was deposited on October 31, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNo: 65377, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] In one embodiment, the Lactobacillus brunetti CCFM1421 was derived from a pickled bamboo shoot sample from Liuzhou City, and its 16S rDNA sequence is shown in SEQ ID NO: 1.

[0009] The present invention also provides a microbial preparation containing the aforementioned Lactobacillus brunelli CCFM1421.

[0010] In one embodiment, the viable count of Lactobacillus bruneri CCFM1421 in the microbial preparation is ≥1×10⁻⁶. 7 CFU / mL or 1×10 7 CFU / g.

[0011] The present invention provides a method for preparing p-cresol, which involves fermenting Lactobacillus bryonicus CCFM1421 in an environment containing p-hydroxybenzyl alcohol to prepare p-cresol.

[0012] In one embodiment, the method includes the following steps:

[0013] (1) Bacterium brevicornu CCFM1421 was cultured in a culture medium to prepare a bacterial suspension;

[0014] (2) Collect the bacterial suspension prepared in step (1) and transfer it to MRS medium containing p-hydroxybenzyl alcohol, and incubate at 35-37°C for at least 14-18 hours.

[0015] In one embodiment, the bacterial suspension is prepared by the following steps: Lactobacillus bruneri CCFM1421 is inoculated into liquid MRS medium, incubated at 37°C for 14-18 hours, the bacterial cells are collected by centrifugation, washed with physiological saline, and resuspended to obtain a Lactobacillus bruneri CCFM1421 bacterial suspension.

[0016] In one embodiment, the concentration of p-hydroxybenzyl alcohol in the culture medium in step (2) is 0.02 mmol / L.

[0017] The present invention also provides a fermentation broth obtained by fermenting the Lactobacillus brunelli CCFM1421.

[0018] This invention also provides the application of the above-mentioned Lactobacillus brunelli CCFM1421 in the conversion of p-hydroxybenzyl alcohol to p-cresol.

[0019] The present invention also provides the application of the Lactobacillus bryonicus CCFM1421 in the fermentation preparation of p-methylphenol or products containing p-methylphenol.

[0020] In one embodiment, the product includes, but is not limited to, food additives or flavorings.

[0021] Beneficial effects:

[0022] This invention screened and obtained a strain of Lactobacillus brunelli CCFM1421, which has acid and salt resistance and can convert p-hydroxybenzyl alcohol to p-cresol. The conversion product has a high content and can be used to ferment and prepare p-cresol, a characteristic flavor substance of pickled bamboo shoots. This provides a high-quality strain resource for targeted regulation of pickled bamboo shoot flavor and development of pickled bamboo shoot fermentation agents.

[0023] Preservation of biological materials

[0024] Lactobacillus buchneri (CCFM1421), classified as Lentilactobacillus buchneri, was deposited on October 31, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 65377). The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0025] Figure 1 This is a colony characteristic of Lactobacillus brunelli CCFM1421.

[0026] Figure 2 This is a bacterial characteristic of Lactobacillus brunelli CCFM1421.

[0027] Figure 3 The growth curve of Lactobacillus brunelli CCFM1421 is shown.

[0028] Figure 4 This is the conversion route of p-cresol; where C05357 refers to oxidized azurin and C05358 refers to reduced azurin.

[0029] Figure 5 The content of p-hydroxybenzyl alcohol and p-cresol obtained by fermentation of Lactobacillus bruneri CCFM1421.

[0030] Figure 6 The survival rate of Lactobacillus brunetti CCFM1421 in MRS liquid medium containing different concentrations of NaCl is shown. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0032] The culture media involved in the following examples are as follows:

[0033] MRS solid medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 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 ​​0.5 g / L.

[0034] MRS liquid culture medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 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 ​​0.5 g / L.

[0035] MRS-p-hydroxybenzyl alcohol medium: p-hydroxybenzyl alcohol was added to MRS medium at a final concentration of 0.02 mmol / L. Example 1: Screening of Lactobacillus bruneri CCFM1421

[0036] Using pickled bamboo shoot juice from Guangxi Zhuang Autonomous Region as a sample, it was serially diluted 10-fold with sterile physiological saline to 100%. -3 Then take 100 μL of each diluted 10. -1 10 -2 10 -3 The diluted solution was plated on MRS solid medium and incubated upside down at 37°C for 48 hours. The colony morphology was observed and recorded. Colonies of different morphologies were picked from the MRS solid medium and streaked for isolation. After incubation at 37°C for 48 hours, single colonies of different morphologies were picked from the MRS solid medium again and streaked for isolation until pure single colonies with consistent morphology were obtained. The colony morphology of single colonies was observed. Strains with small, round, milky white, raised, and smooth colonies, spherical cell morphology, and Gram-positive results were selected.

[0037] The microscopic examination method is as follows: Take a clean glass slide, heat it slightly over a flame and then cool it; after cooling, add a small drop of sterile water to the center of the clean glass slide, and use an inoculation loop to pick up a small amount of bacterial cells from a single colony near the flame and mix it with the water; burn off the excess bacterial cells on the inoculation loop, and then use the inoculation loop to spread the bacterial cells 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 over a low flame 3-4 times to fix it; add 1-2 drops of crystal violet solution to the smear and stain for 1 min; Gently wash away the stain with water and gently blot the slide with absorbent paper. Add 1-2 drops of iodine solution to the smear and stain for 1 minute. Gently wash away the stain with water and gently blot the slide with absorbent paper. Add 1-2 drops of 95% (v / v) ethanol solution to the smear and decolorize for 5-15 seconds. Rinse gently with water and gently blot the slide with absorbent paper. Add 1-2 drops of safranin solution to the smear and stain for 2 minutes. After drying, examine under a microscope to observe the morphology of the bacteria.

[0038] The Gram staining method is based on the textbook "Industrial Microbial Breeding" (author: Zhuge Jian); the methods for examining physiological and biochemical characteristics are based on the textbook "Handbook of Systematic Identification of Common Bacteria" (author: Dong Xiuzhu).

[0039] The growth curve was determined as follows: The test culture medium was dispensed into 100mL blue-capped bottles, and the test strain was inoculated at a 2% inoculum rate. After thorough mixing, the mixture was immediately dispensed into test tubes and incubated at 37℃. Every 2 hours, three test tubes were removed, thoroughly shaken, and the OD600 was measured, using the culture medium as a blank tube for zeroing. The growth curve was plotted with growth time on the x-axis and OD600 on the y-axis.

[0040] After screening, a strain of Lactobacillus was obtained and named CCFM1421. The colony characteristics of this strain are as follows: Figure 1 As shown, the bacterial cell characteristics are as follows Figure 2 The growth curve of strain CCFM1421 is as follows: Figure 3 .

[0041] Example 2: Identification and preservation of Lactobacillus bruneri CCFM1421

[0042] The genomes of laboratory-grown strain CCFM493 and strain CCFM1421 (screened in Example 1) were extracted and amplified by PCR using primers consisting of 16sF and 16sR. The amplified products were then sequenced, and the 16S rDNA sequence is shown in SEQ ID NO:1. Homology comparison analysis of the 16S rDNA sequences in the GenBank library was performed using the BLAST program. The results showed that strains CCFM493 and CCFM1421 shared 99.9% homology with *Lactobacillus buchneri*. Therefore, based on these identification results, the extracted strains CCFM493 and CCFM1421 were identified as *Lactobacillus buchneri*.

[0043] The primer pair sequence information is as follows:

[0044] 16sF: 5′-AGAGTTTGATCCTGGCTCAG-3′;

[0045] 16sR: 5'-AGAAAGGAGGTGATCCAGC-3'. The PCR amplification conditions used were as follows: pre-denaturation: 95℃ for 5 min; denaturation: 95℃ for 15 s, annealing: 55℃ for 15 s, extension: 72℃ for 1.5 min, for a total of 35 cycles; final extension: 72℃ for 7 min, stored at 4℃.

[0046] Single colonies of Lactobacillus bruneri CCFM493 and Lactobacillus bruneri CCFM1421 were picked and inoculated into MRS liquid medium and cultured statically at 37°C for 24 h to obtain bacterial suspension. 1 mL of bacterial suspension was taken into a preservation tube, centrifuged at 6000 rpm for 3 min, and the supernatant was discarded. The bacterial sludge was resuspended in 30% (v / v) glycerol solution and stored at -80°C.

[0047] Example 3: Preparation of Lactobacillus bromide suspension

[0048] The obtained Lactobacillus brunetti CCFM493 and CCFM1421 were inoculated into liquid MRS medium, incubated at 37°C for 24 h, centrifuged at 4°C and 6000 rpm for 8 min, and the bacterial pellet was collected. After washing three times with physiological saline and shaking, the bacterial suspension of the strain was obtained.

[0049] Example 4: Preparation of p-cresol by fermentation of p-hydroxybenzyl alcohol with Lactobacillus brycete CCFM1421

[0050] Dissolve p-hydroxybenzyl alcohol in ultrapure water, filter through a 0.22 μm filter in a clean bench, and add to sterile culture medium to a final concentration of 0.02 mmol / L. Mix well, and dispense 5 mL of the MRS medium containing p-hydroxybenzyl alcohol into test tubes. Administer the activated bacterial culture at a concentration of 1 × 10⁻⁶. 7 CFU / ml was inoculated into MRS medium containing p-hydroxybenzyl alcohol and cultured at 37°C for 14 days. The supernatant was then collected by centrifugation at 8000 rpm for 5 min.

[0051] 1 mL of the supernatant was filtered through a 0.45 μm microporous membrane, and the filtrate was then used for reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. A BEH C18 column was used, with 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 was used: 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). Fluorescence detection wavelength settings gradient: 0 min, excitation wavelength 284 nm, emission wavelength 333 nm; 5 min, excitation wavelength 275 nm, emission wavelength 315 nm; 8 min, excitation wavelength 277 nm, emission wavelength 319 nm; 12 min, excitation wavelength 272 nm, emission wavelength 310 nm; 25 min, excitation wavelength 273 nm, emission wavelength 312 nm. Column temperature: 30℃; column flow rate: 1 mL / min; injection volume: 20 μL.

[0052] In the process of preparing p-cresol by fermentation of Lactobacillus brunelli suspension, the biotransformation and synthesis route is as follows: Figure 4 As shown: Possible pathways for p-cresol were identified using KEGG. Figure 4 It is possible that the strain uses a certain dehydroxylase to remove the hydroxyl group from p-hydroxybenzyl alcohol, turning it into p-cresol.

[0053] After 14 days of fermentation, samples of the fermentation supernatant were taken to determine the levels of p-hydroxybenzyl alcohol and p-cresol before and after fermentation (results are shown in [link to sample]). Figure 5Analysis of the changes in p-hydroxybenzyl alcohol and p-cresol content in the fermentation products before and after fermentation revealed that, compared with Lactobacillus Brucella CCFM493, the p-cresol content in the fermentation supernatant of Lactobacillus Brucella CCFM1421 increased from 0 mmol / L to 0.015±0.001 mmol / L after 14 days of fermentation, while the p-hydroxybenzyl alcohol content decreased from 0.02 mmol / L to 0.004±0.0005 mmol / L. In contrast, the p-hydroxybenzyl alcohol content in the fermentation supernatant of Lactobacillus Brucella CCFM493 remained unchanged, and p-cresol was not detected.

[0054] Example 5: Characterization of Lactobacillus bruneri CCFM1421

[0055] 1. Acid resistance

[0056] Lactobacillus Brucella CCFM1421 obtained in Example 2 was streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and activated by culturing at 37°C for 18 h, for three consecutive generations. The pH of the MRS liquid medium was adjusted to 3.0 with HCl. The activated Lactobacillus Brucella CCFM1421 was inoculated into MRS liquid at pH 3.0 at a 2% (v / v) inoculation rate, mixed well, and cultured at 37°C for 3 h. The culture was then diluted with physiological saline, and 1 mL of the appropriate dilution gradient was decanted to determine the viable cell count. The viable cell count at 0 h was used as the control group, and the survival rate of the strain at pH 3.0 was calculated. The formula for calculating the strain survival rate is shown below:

[0057] Acid resistance survival rate = N2 / N1 × 100%;

[0058] N1 is the number of viable bacteria (CFU / mL) at 0 h; N2 is the number of viable bacteria (CFU / mL) after 3 h of treatment.

[0059] The results showed that the survival rate of Lactobacillus brunetti CCFM1421 after 3 hours of treatment in acidic MRS medium at pH 3 was 12.86%.

[0060] 2. Salt tolerance

[0061] Activated *Lactobacillus brunetti* CCFM1421 was inoculated at 2% (V / V) into MRS liquid medium containing 3 g / 100 mL, 6 g / 100 mL, and 9 g / 100 mL NaCl, respectively, and incubated at 37°C for 3 h. The culture was then diluted with physiological saline, and 1 mL of each diluted culture was used to determine the viable cell count using the pour plate method. The formula for calculating the survival rate of the strain is shown below:

[0062] Salt tolerance survival rate = N2 / N1 × 100%;

[0063] Wherein, N1 is the number of viable bacteria (CFU / mL) at 0h; N2 is the number of viable bacteria (CFU / mL) after 3h of salt treatment.

[0064] The results show (e.g.) Figure 6 The survival rates of Lactobacillus brunetti CCFM1421 in MRS liquid medium containing 3 g / 100 mL, 6 g / 100 mL, and 9 g / 100 mL NaCl were 143.12%, 45.07%, and 42.69%, respectively.

[0065] The above results indicate that Lactobacillus brunelli CCFM1421 has acid and salt resistance, and has the potential to be used in the preparation of fermented condiments.

[0066] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Lactobacillus brunelli ( Lentilactobacillus buchneri CCFM1421 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 31, 2024, with accession number GDMCC No: 65377.

2. A microbial preparation containing the Lactobacillus brunelli CCFM1421 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The viable count of Lactobacillus bruneri CCFM1421 is ≥1×10⁻⁶. 7 CFU / mL or 1×10 7 CFU / g.

4. A method for preparing p-cresol, characterized in that, The Lactobacillus brunelli strain CCFM1421 of claim 1 was fermented in an environment containing p-hydroxybenzyl alcohol, and then p-cresol was extracted from the fermentation broth.

5. The method according to claim 4, characterized in that, The method includes the following steps: (1) Prepare a bacterial suspension by culturing Lactobacillus bruneri CCFM1421 in a culture medium; (2) Collect the bacterial suspension prepared in step (1), transfer it to MRS medium containing p-hydroxybenzyl alcohol, and incubate at 35-37°C for at least 14-18 h.

6. The method according to claim 5, characterized in that, The bacterial suspension was prepared by the following steps: Lactobacillus bruneri CCFM1421 was inoculated into liquid MRS medium, cultured statically at 30-37°C for 14-18 h, the bacterial cells were collected by centrifugation, washed with physiological saline, and resuspended to obtain the Lactobacillus bruneri CCFM1421 bacterial suspension.

7. The method according to claim 5, characterized in that, In step (2), the concentration of p-hydroxybenzyl alcohol in the culture medium is ≥0.02 mmol / L.

8. The application of Lactobacillus brunelli CCFM1421 as described in claim 1 in the fermentation of pickled bamboo shoots.

9. The use of Lactobacillus brunelli CCFM1421 as described in claim 1 in the fermentation preparation of p-methylphenol or food additives containing p-methylphenol.

10. The use of Lactobacillus brunelli CCFM1421 as described in claim 1 in the fermentation preparation of p-methylphenol or flavorings containing p-methylphenol.

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