A strain of high yield p-toluenol fermentation lactobacillus limosus and its application in the production of acid bamboo

Abstract

The application discloses a fermentation Lactobacillus mucosus with high yield of p-cresol and application of the fermentation Lactobacillus mucosus in production of acid bamboo shoots, and belongs to the field of microbial fermentation. The application separates a fermentation Lactobacillus mucosus with a preservation number of GDMCC No: 65785. The fermentation Lactobacillus mucosus has strong acid resistance and osmotic pressure resistance, and the acid yield reaches 10.75 g / L in 48 hours, and the degradation rate of nitrite can reach 74.5%. The fermentation Lactobacillus mucosus is used for acid bamboo shoot fermentation, and experimental results show that, compared with natural fermentation, inoculation of the fermentation Lactobacillus mucosus can significantly improve the content of p-cresol, a key flavor substance in acid bamboo shoots, further improves the flavor quality of the acid bamboo shoots, makes the flavor of the acid bamboo shoot products stable, shortens the fermentation period of the acid bamboo shoots, and provides new technical support for efficient, standardized and large-scale fermentation production of the acid bamboo shoots.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation, and in particular to a high-yield p-cresol-producing fermenting Lactobacillus mucilaginosus and its application in the production of pickled bamboo shoots. Background Technology

[0002] Pickled bamboo shoots are favored by consumers for their tangy taste and unique fermented flavor. Their rich fermented aroma is also an important indicator of their sensory quality. Research shows that p-cresol is a characteristic flavor compound of pickled bamboo shoots. However, Guangxi pickled bamboo shoots are produced using natural fermentation methods, which result in a long fermentation cycle, complex and uncontrollable microbial flora, and unstable quality, greatly limiting the large-scale development of my country's fermented vegetable industry. Summary of the Invention

[0003] The purpose of this invention is to provide a high-yield fermenting *Lactobacillus mucilaginosus* strain for producing p-cresol and its application in the production of pickled bamboo shoots, in order to solve the problems existing in the prior art. The p-cresol content in pickled bamboo shoots produced by fermentation using the *Lactobacillus mucilaginosus* strain provided by this invention is significantly higher than that in pickled bamboo shoots produced by natural fermentation. Using the *Lactobacillus mucilaginosus* strain provided by this invention can shorten the fermentation cycle of pickled bamboo shoots and improve the quality of pickled bamboo shoots.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides a high-yield p-cresol-producing fermenting Lactobacillus fermentum strain, which has been deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 65785) on January 14, 2025, at the address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] This invention provides an application of the aforementioned fermenting *Lactobacillus mucilaginosus* in the preparation of vegetable fermentation agents.

[0007] The present invention also provides a vegetable fermentation agent, including the above-mentioned fermenting Lactobacillus mucilaginosus.

[0008] The present invention also provides the application of the aforementioned fermenting Lactobacillus mucilaginosus or the aforementioned vegetable fermentation agent in the production of fermented vegetables.

[0009] Preferably, the fermented vegetable is pickled bamboo shoots.

[0010] The present invention also provides a method for producing pickled bamboo shoots by fermentation, comprising the steps of inoculating the aforementioned fermentation liquid of Lactobacillus mucilaginosus or the aforementioned vegetable fermentation agent, and carrying out sealed fermentation.

[0011] Preferably, the OD of the fermented *Lactobacillus mucilaginosus* culture is... 600nmThe inoculum size is 0.8, and the inoculum size of the fermented *Lactobacillus mucilaginosus* culture is 2% by volume.

[0012] Preferably, the sealed fermentation time is 30 days.

[0013] This invention provides a method for producing pickled bamboo shoots according to the method described.

[0014] This invention provides an application of the aforementioned fermented Lactobacillus mucilaginosus or the aforementioned vegetable fermentation agent in the production of p-cresol.

[0015] The present invention discloses the following technical effects:

[0016] The *Lactobacillus fermentans* isolated in this invention exhibits strong acid and osmotic pressure resistance, and its acid production reaches 10.75 g / L after 48 hours, with lactic acid and acetic acid as the main components; the degradation rate of nitrite can reach 74.5%. When the *Lactobacillus fermentans* provided by this invention was used for fermenting pickled bamboo shoots, experimental results showed that, compared with natural fermentation, inoculation with the *Lactobacillus fermentans* provided by this invention can significantly increase the content of p-cresol, a key flavor compound in pickled bamboo shoots, further improving the flavor quality of pickled bamboo shoots, stabilizing the flavor of the pickled bamboo shoot product, and shortening the fermentation cycle of pickled bamboo shoots. This provides new technical support for the efficient, standardized, and large-scale fermentation production of pickled bamboo shoots. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Flowchart for the isolation, purification, and preservation of lactic acid bacteria;

[0019] Figure 2 Figure (a) shows the colony morphology of lactic acid bacteria (A) and Gram staining.

[0020] Figure 3 Phylogenetic tree diagram of strain N-11;

[0021] Figure 4 This is a standard curve for nitrite. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Example 1

[0028] 1. Isolation and identification of strains

[0029] 1.1 Strains Isolation

[0030] Starting from day 0 of fermentation of pickled bamboo shoots, samples were taken every 6 days until the end of fermentation on day 30. The samples were mixed thoroughly, and the fermentation broth was transferred to 50 mL centrifuge tubes for immediate isolation of lactic acid bacteria. Figure 1 The method shown was used to isolate, purify and preserve lactic acid bacteria; the lactic acid bacteria was named ZFSS14.

[0031] 1.2 Molecular biological identification

[0032] Genomic DNA was extracted from strain ZFSS14 using a DNA extraction kit. Universal primers 27F and 1492R were used for gene amplification. The amplification conditions were: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, repeated 30 times, followed by a final extension at 72℃ for 5 min, and storage at 4℃. The 16S rRNA sequence was compared with the NCBI database, and a phylogenetic tree was constructed using MEGA 11.0.

[0033] 2. Results and Discussion

[0034] 2.1 Morphological identification of lactic acid bacteria

[0035] Samples were taken from naturally fermented pickled bamboo shoots. After dilution and spreading on MRS solid medium, single colonies of different morphologies and sizes were picked, purified, and Gram-stained. The color was milky white, and the colonies were round, with neat edges, smooth surfaces, and moist opacities. Microscopic examination showed that strain ZFSS14 was rod-shaped or short rod-shaped. The results of Gram staining and microscopic examination of lactic acid bacteria colonies are as follows: Figure 2 As shown.

[0036] 2.2 Molecular biological identification of lactic acid bacteria

[0037] 16S rRNA sequencing was performed on the high-yield p-cresol lactic acid bacteria ZFSS14, and the strain's gene sequence was uploaded to the NCBI system. ZFSS14 was ultimately identified as a fermenting *Lactobacillus mucilaginosus*. Figure 3 Phylogenetic tree of Lactobacillus fermentans ZFSS14.

[0038] 3. Strain preservation

[0039] Limosilactobacillus fermentum ZFSS14 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65785, deposited on January 14, 2025, at the address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0040] Example 2

[0041] 1. Experimental Methods

[0042] 1.1 Determination of the acid-producing capacity of fermenting *Lactobacillus mucilaginosus*

[0043] One mL of *Lactobacillus fermentatus* preserved in glycerol tubes was inoculated into MRS liquid medium and cultured at 37°C for 24 h. The culture was then transferred to MRS solid medium and cultured for 36 h. Single bacteria were then picked and cultured in MRS liquid medium for another 24 h, for a total of three generations of activation. The activated *Lactobacillus fermentatus* culture was then inoculated into MRS liquid medium at a volume fraction of 2% and cultured at 37°C for 48 h.

[0044] 1.1.1 pH determination

[0045] The pH value of the MRS liquid culture medium was measured using a pH meter after 48 hours of culture.

[0046] 1.1.2 Determination of Organic Acids

[0047] Take 1 mL of fermentation broth, centrifuge at 10000 r / min for 2 min, take the supernatant, filter it through a 0.22 μL filter membrane and inject it into the sample. Determine the content of organic acids (citric acid, malic acid, succinic acid, lactic acid, acetic acid) using HPLC.

[0048] The chromatographic conditions were based on the method disclosed in the literature "Xiao Yangsheng: Study on the metabolic regulation mechanism of functional microorganisms in Sichuan pickled vegetables. 2020." with slight modifications. An Aminex HPX-87H column was used, with a mobile phase of 0.005M dilute sulfuric acid solution and isocratic elution; the column temperature was 35℃, the flow rate was 0.5 mL / min; the detector was a UV detector with a detection wavelength of 210 nm, and the injection volume was 10 μL. Three replicates were performed for each group, and the mean and standard deviation were calculated.

[0049] 1.2 Determination of acid tolerance of fermenting *Lactobacillus mucilaginosus*

[0050] To ensure that *Lactobacillus fermentatus* maintains its viability during the fermentation of pickled bamboo shoots, its acid resistance needs to be determined. Meanwhile, while stomach acid can prevent contamination by harmful external microorganisms, it affects the survival of exogenous probiotics in the gastrointestinal tract. Therefore, the selected probiotics need to have strong resistance to stress conditions within the gastrointestinal tract.

[0051] The strain preserved in glycerol was inoculated into 150 ml LMR S liquid medium and incubated at 37°C for 24 h, for a total of three generations of activation. The culture was centrifuged at 4°C and 6000 rpm for 5 min, the supernatant was discarded, and the culture was washed twice with sterile physiological saline to adjust the OD. 600 =0.8±0.02. Add 5 mL of bacterial suspension to 45 mL of liquid culture medium at the corresponding pH concentration (pH=2.5) to resuspend, and shake well. Spread the bacterial suspension on MRS solid medium, incubate at 37℃ for 24 h, and count the colony growth.

[0052] The acid resistance of the strains was tested, and the acid resistance rate was calculated using the following formula:

[0053]

[0054] In the formula:

[0055] X represents acid tolerance performance;

[0056] B represents the colony count over 4 hours, expressed in CFU / mL.

[0057] A represents the colony count at 0h, expressed in CFU / mL.

[0058] 1.3 Determination of the ability of fermenting *Lactobacillus mucilaginosus* to degrade nitrite

[0059] The fermentation performance of *Lactobacillus fermentatus* in fermented vegetables was evaluated by determining its ability to degrade nitrite. After activation, *Lactobacillus fermentatus* was inoculated at a volume fraction of 2% into liquid medium containing 200 mg / L NaNO₂MRS and incubated at 37°C for 24 h. 5 g of the fermentation broth was weighed and placed in a 250 mL stoppered conical flask. 12.5 mL of 50 g / L saturated borax solution was added, along with 150 mL of water at 70°C. The mixture was stirred and heated in a boiling water bath for 15 min. The flask was then removed, cooled in a cold water bath, and allowed to reach room temperature. The extract was quantitatively transferred to a 200 mL volumetric flask, and 5 mL × 10⁻⁶ mol / L water was added. 6 Mix 1000 g / L potassium ferrocyanide solution with water, shake well, and then add 5 mL of 220 g / L zinc acetate solution to precipitate the protein. Add water to the mark, shake well, let stand for 30 min, remove the upper layer of fat, filter the supernatant through filter paper, discard the first 30 mL of filtrate, and keep the filtrate for later use.

[0060] Pipette 40.0 mL of the above filtrate into a 50 mL volumetric flask. Separately, pipette 0.0 mL, 2.5 mL, 5.0 mL, 7.5 mL, 10.0 mL, 12.5 mL, 20.0 mL, and 25.0 mL of 10 mg / L sodium nitrite standard solution into separate 50 mL volumetric flasks. Add 2 mL of 4 g / L p-aminobenzenesulfonic acid solution to each standard and test tube, mix well, and let stand for 3-5 minutes. Then, add 1 mL of 2 g / L nylondiamine hydrochloride solution to each, dilute with water to the mark, mix well, and let stand for 15 minutes. Adjust the zero point using the zero tube and measure the absorbance at a wavelength of 538 nm. Plot a standard curve. Perform a reagent blank simultaneously.

[0061] Degradation rate formula:

[0062]

[0063] Where: N0 represents the NaNO2 nitrite content in the blank group, N t This indicates the nitrite content at the end of incubation. Each group has 3 replicates, and the average value is taken.

[0064] 1.4 Determination of the osmotic pressure resistance of fermenting *Lactobacillus mucilaginosus*

[0065] After activation, *Lactobacillus fermentatus* was inoculated at a volume fraction of 2% into MRS liquid medium with NaCl concentrations of 0%, 2%, 4%, 6%, and 8%, respectively, and incubated at 37°C for 24 hours. After 24 hours, the viable cell count was determined using the colony plate count method. Each group was tested in triplicate, and the average value was taken.

[0066] 1.5 Application of *Lactobacillus mucilaginosus* in the production of pickled bamboo shoots

[0067] The strain preserved in glycerol was inoculated into 150 mL of MRS liquid medium and incubated at 37°C for 24 h, for a total of three generations of activation. The culture was centrifuged at 4°C and 6000 rpm for 5 min, the supernatant was discarded, and the culture was washed twice with sterile physiological saline to adjust the OD. 600 =0.8±0.02.

[0068] The preparation method of the pickled bamboo shoot samples was determined with reference to the current Guangxi local standard DB45 / T 2548-2022 Technical Specification for Pickled Bamboo Shoot Processing.

[0069] Peel the outer husks and remove the woody parts from the roots of fresh bamboo shoots. Wash and dry them, then cut them into even pieces. Place them in a 3L food-grade fermentation tank, add 2L of spring water to completely submerge the raw materials, and add 2% (by volume) of fermenting lactobacillus to the fermentation tank. Seal the tank. Produce three parallel tanks and ferment them for 30 days. After fermentation, mix the three batches of pickled bamboo shoots evenly to form one sample, and then divide it back into three equal samples.

[0070] 1.6 Determination of p-cresol in pickled bamboo shoots

[0071] The method disclosed in the reference "Li SB, TianYF, Sun MH, Liu JJ, Bai YX, Liu XL, Guo Y: Characterization of Key Aroma Compounds in Fermented Bamboo Shoots Using Gas Chromatography-Olfactometry-Mass Spectrometry, Odor Activity Values, and Aroma Recombination Experiments. Foods 2022, 11(14)" was modified slightly to determine the p-cresol content in pickled bamboo shoots.

[0072] 1.6.1 Headspace Solid-Phase Microextraction

[0073] Weigh 50g of pickled bamboo shoot sample and mix with an equal mass of ultrapure water at a 1:1 ratio, then homogenize thoroughly using a homogenizer. Accurately weigh 6.0g of the pickled bamboo shoot homogenate into a 20mL headspace vial, and equilibrate for 30min under sealed conditions at 50℃. Insert the aged extraction head to a depth of 1cm, and after adsorption in a 50℃ water bath for 30min, immediately insert the fully adsorbed extraction head into the GC-MS inlet and desorb at 250℃ for 3min.

[0074] 1.6.2 GC-MS determination

[0075] Volatile components in pickled bamboo shoot samples were separated using an Agilent HP-INNOWAX capillary column (60 m × 0.25 mm, 0.25 μm). The injection port temperature was 250 °C; high-purity helium was used as the carrier gas; and the flow rate was 1 mL / min. The temperature program was as follows: initial column temperature 40 °C, held for 4 min; then increased to 140 °C at 6 °C / min, held for 5 min; increased to 150 °C at 3 °C / min, held for 1 min; increased to 200 °C at 5 °C / min, held for 1 min; and finally increased to 240 °C at 8 °C / min, held for 10 min.

[0076] MS method conditions are as follows: ionization mode EI, electron energy 70eV, filament emission current 0.25mA, detector temperature 230℃, ion source temperature 230℃, quadrupole temperature 150℃, mass scan range 40-400u, scan time 250ms, scan mode: full scan; tuning file is standard tuning.

[0077] 1.6.3 Qualitative and quantitative analysis of volatile compounds

[0078] (1) Qualitative Analysis

[0079] Qualitative analysis of volatile compounds in the sample was performed using mass spectrometry (MS) and retention index (RI). Mass spectrometry (MS): During the NIST 14.0 database search, background subtraction was performed to select compounds with a matching degree greater than 80%. Retention index (RI): Under the same operating conditions as the sample, a mixture of n-alkane standards (C6-C30) was injected. The retention index (RI) of each volatile compound in the sample was calculated based on the retention time of each alkane and compared with the RI reported in the literature for qualitative analysis. The formula for calculating the retention index is as follows:

[0080]

[0081] In the formula: x is the analyte;

[0082] n and n+1 are the number of carbon atoms in the two adjacent n-alkanes before and after the peak of the analyte, respectively.

[0083] RI is the retention index of the corresponding component;

[0084] TR represents the retention time of the corresponding component.

[0085] (2) Semi-quantitative analysis

[0086] Using 2-methyl-3-heptanone (1.5 μL, 1.65 g / L) as an internal standard, the concentration of volatile compounds in the sample was calculated by comparing the peak area of ​​the compound with that of the internal standard.

[0087] 2. Experimental Results

[0088] 2.1 Acid-producing capacity of fermenting Lactobacillus mucilaginosus

[0089] Lactic acid and acetic acid are the main sources of the sour flavor of pickled bamboo shoots, while a low pH environment can effectively inhibit the growth of harmful microorganisms and extend the shelf life of pickled bamboo shoots. The acid-producing capacity of fermenting *Lactobacillus mucilaginosus* is shown in Table 1.

[0090] Table 1. Acid production and pH value after fermentation with *Lactobacillus mucilage*.

[0091]

[0092] 2.2 Acid resistance of fermenting *Lactobacillus mucinus*

[0093] The excellent acid resistance of *Lactobacillus fermentatus* ensures the continued activity of the strain during vegetable fermentation, while also reducing fermentation time and inhibiting various pathogens. During the fermentation of pickled bamboo shoots, as fermentation progresses, the acidity gradually increases, causing less acid-resistant strains to become inactive, and the dominant strains to gradually become moderately and strongly acid-resistant. The acid resistance results of *Lactobacillus fermentatus* are shown in Table 2.

[0094] Table 2 Acid resistance of fermenting Lactobacillus mucinus

[0095]

[0096] 2.3 Nitrite-reducing ability of fermenting *Lactobacillus mucilaginosus*

[0097] Nitrite standard curve as follows Figure 4 As shown in Table 3, the degradation capacity of fermenting *Lactobacillus mucinus* is as follows.

[0098] Nitrite is a common substance in kimchi and a harmful substance that affects food safety. Research on fermenting Lactobacillus strains that degrade nitrite is of great significance for kimchi production.

[0099] Table 3. Ability of fermenting *Lactobacillus mucilage* to degrade nitrite.

[0100]

[0101] 2.4 Osmotic pressure resistance of fermenting *Lactobacillus mucinus*

[0102] The osmotic pressure resistance of fermenting *Lactobacillus mucinus* is shown in Table 4.

[0103] Table 4 Osmotic pressure resistance of Lactobacillus fermentum

[0104]

[0105] 2.5 Ability of fermenting *Lactobacillus mucilaginosus* to produce p-cresol

[0106] p-Cresol is a key flavor compound in pickled bamboo shoots, contributing a pungent and putrid odor. Table 5 shows the results of p-cresol production capacity during the fermentation of *Lactobacillus mucilaginosus* in the production of pickled bamboo shoots.

[0107] Table 5. Content of p-cresol and OVA value in pickled bamboo shoots

[0108]

[0109] The threshold data for p-cresol in water in Table 5 are taken from the literature "Leo van Heimert: A Compilation of Aroma Thresholds for Compounds; 2015."

[0110] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A high-yield fermenting *Lactobacillus mucilaginosus* strain producing p-cresol ( Limosilactobacillus fermentum ), characterized in that, The fermenting Lactobacillus mucinus has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65785, deposit date of January 14, 2025, and address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The application of the fermenting *Lactobacillus mucilaginosus* as described in claim 1 in the preparation of vegetable fermentation agents.

3. A vegetable fermentation inoculant, characterized in that, Includes the fermenting myxobacterium as described in claim 1.

4. The application of the fermenting *Lactobacillus mucilaginosus* of claim 1 or the vegetable fermentation agent of claim 3 in the production of fermented vegetables.

5. The application as described in claim 4, characterized in that, The fermented vegetable is pickled bamboo shoots.

6. A method for producing pickled bamboo shoots through fermentation, characterized in that, The process includes inoculating the fermented Lactobacillus mucilaginosus liquid of claim 1 or the vegetable fermentation agent of claim 3, followed by sealed fermentation.

7. The method as described in claim 6, characterized in that, The OD of the fermented Lactobacillus mucin culture 600nm The inoculum size is 0.8, and the inoculum size of the fermented *Lactobacillus mucilaginosus* culture is 2% by volume.

8. The method as described in claim 6, characterized in that, The sealed fermentation time is 30 days.

9. The use of the fermenting *Lactobacillus mucilaginosus* of claim 1 or the vegetable fermentation agent of claim 3 in the production of p-cresol.

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