Lactobacillus mucilaginosus for high-yield production of p-cresol and application of lactobacillus mucilaginosus in production of sour bamboo shoots

Abstract

The invention discloses a high-yield p-cresol fermentation lactobacillus mucilaginosus strain and application thereof in production of sour bamboo shoots, and belongs to the field of microbial fermentation. According to the invention, a strain of fermentation mucus lactobacillus is obtained through separation, and the preservation number is GDMCC No: 65785. The lactobacillus mucus has relatively strong acid resistance and osmotic pressure resistance, the acid yield in 48 hours reaches 10.75 g / L, and the degradation rate of nitrite can reach 74.5%. The lactobacillus mucus provided by the invention is used for fermenting sour bamboo shoots, and experimental results show that compared with natural fermentation, the lactobacillus mucus provided by the invention is inoculated, so that the content of a key flavor substance p-cresol in the sour bamboo shoots can be remarkably increased, the flavor quality of the sour bamboo shoots is further improved, and the flavor of sour bamboo shoot products tends to be stable; the fermentation period of the sour bamboo shoots is shortened, and a new technical support is provided for high-efficiency, standardized and large-scale fermentation production of the sour bamboo shoots.

Description

Technical Field

[0001] The present invention relates to the field of microbial fermentation, and particularly to a Limosilactobacillus fermentum with high p-cresol productivity and its application in the production of pickled bamboo shoots. Background Art

[0002] Pickled bamboo shoots are deeply favored by consumers for their sour taste and unique fermentation flavor. Its strong fermentation aroma is an important index for evaluating the sensory quality of pickled bamboo shoots. Research shows that p-cresol is a characteristic flavor substance of pickled bamboo shoots. The pickled bamboo shoots in Guangxi use the natural fermentation method, which has a long fermentation cycle, complex and uncontrollable flora, and unstable quality, greatly restricting the large-scale development of the fermented vegetable industry in China. Summary of the Invention

[0003] The purpose of the present invention is to provide a Limosilactobacillus fermentum with high p-cresol productivity and its application in the production of pickled bamboo shoots, so as to solve the problems existing in the above-mentioned prior art. The content of p-cresol in the pickled bamboo shoots fermented by using the Limosilactobacillus fermentum provided by the present invention is significantly higher than that of the pickled bamboo shoots produced by natural fermentation. Using the Limosilactobacillus fermentum provided by the present invention can shorten the fermentation cycle of pickled bamboo shoots and improve the quality of pickled bamboo shoots.

[0004] To achieve the above purpose, the present invention provides the following solutions:

[0005] The present invention provides a Limosilactobacillus fermentum with high p-cresol productivity. The Limosilactobacillus fermentum has been deposited in the Guangdong Provincial Culture Collection of Microorganisms, with the deposit number of GDMCC No: 65785, the deposit date of January 14, 2025, and the deposit address of the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0006] The present invention provides an application of the Limosilactobacillus fermentum in the preparation of a vegetable fermentation inoculant.

[0007] The present invention also provides a vegetable fermentation inoculant, including the above-mentioned Limosilactobacillus fermentum.

[0008] The present invention also provides an application of the Limosilactobacillus fermentum or the vegetable fermentation inoculant in the production of fermented vegetables.

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

[0010] The present invention also provides a method for fermenting and producing pickled bamboo shoots, including the step of inoculating the Limosilactobacillus fermentum bacterial liquid or the vegetable fermentation inoculant and performing sealed fermentation.

[0011] Preferably, the OD of the Limosilactobacillus fermentum bacterial liquid 600nmIt is 0.8, and according to the volume fraction, the inoculation amount of the fermented Lactobacillus mucosae bacterium solution is 2%.

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

[0013] The present invention provides a pickled bamboo shoot produced according to the described method.

[0014] The present invention provides an application of the described Lactobacillus mucosae or the described vegetable fermentation inoculant in the production of p-cresol.

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

[0016] The isolated Lactobacillus mucosae of the present invention has strong acid and osmotic pressure tolerance properties, and the acid production amount of this Lactobacillus mucosae reaches 10.75 g / L in 48 h, and the main components are lactic acid and acetic acid; the degradation rate of nitrite can reach 74.5%. Using the Lactobacillus mucosae provided by the present invention for pickled bamboo shoot fermentation, the experimental results show that, compared with natural fermentation, inoculating the Lactobacillus mucosae provided by the present invention can significantly increase the content of the key flavor substance p-cresol in pickled bamboo shoots, further improve the flavor quality of pickled bamboo shoots, make the flavor of pickled bamboo shoot products tend to be stable, shorten the fermentation period of pickled bamboo shoots, and provide new technical support for efficient, standardized and large-scale fermentation production of pickled bamboo shoots. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 It is a flow chart for the isolation, purification and preservation of lactic acid bacteria;

[0019] Figure 2 It is a diagram of lactic acid bacteria colonies (A) and Gram staining morphology (a);

[0020] Figure 3 It is a phylogenetic tree diagram of strain N-11;

[0021] Figure 4 It is a standard curve diagram of nitrite. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

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

[0024] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0025] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0026] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0027] Example 1

[0028] 1. Isolation and identification of strains

[0029] 1.1 Isolation of strains

[0030] Starting from 0 d of pickled bamboo shoot fermentation, samples were taken regularly every 6 d until the fermentation ended on the 30th d. The samples were mixed evenly, and the fermentation broth was placed in a 50 mL centrifuge tube, and lactic acid bacteria were immediately isolated. The isolation, purification, and preservation of lactic acid bacteria were carried out according to the method shown in Figure 1 The lactic acid bacteria were named ZFSS14.

[0031] 1.2 Molecular biology identification

[0032] The genomic DNA of strain ZFSS14 was extracted using a DNA extraction kit. Universal primers 27F and 1492R were selected to amplify the genes of the strain. The amplification conditions were as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 30 cycles, extension at 72°C for 5 min, and storage at 4°C. The 16S rRNA sequence was compared with the NCBI database, and a phylogenetic tree was constructed using MEGA11.0.

[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 with different colony morphologies and sizes were picked. After purification, Gram staining was performed separately. Their color was milky white, the colonies were round, with neat edges, smooth surfaces, moist and opaque. It can be seen from the microscopic examination results that strain ZFSS14 was rod-shaped or short rod-shaped. The lactic acid bacteria colonies and the results of Gram staining microscopic examination were as Figure 2 shown.

[0036] 2.2 Molecular Biological Identification of Lactic Acid Bacteria

[0037] 16S rRNA sequencing was performed on the high-yield p-cresol-producing lactic acid bacterium ZFSS14. The gene sequence of the strain was uploaded to the NCBI system. Finally, strain ZFSS14 was identified as Limosilactobacillus fermentum, Figure 3 and the phylogenetic tree of Limosilactobacillus fermentum ZFSS14 is shown.

[0038] 3. Strain Preservation

[0039] Limosilactobacillus fermentum ZFSS14 has been deposited in the Guangdong Provincial Microbial Culture Collection Center, with the deposit number GDMCC No: 65785. The deposit date was January 14, 2025, and the deposit address was the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou.

[0040] Example 2

[0041] 1. Experimental Method

[0042] 1.1 Determination of the Acid-Producing Ability of Limosilactobacillus fermentum

[0043] 1 mL of Lactobacillus mucosae fermentum preserved in a glycerol tube was inoculated into MRS liquid medium and cultured at 37 °C for 24 h, then inoculated into MRS solid medium and cultured for 36 h, and then a single colony was picked and cultured in MRS liquid medium for 24 h, for a total of three generations of activation. The activated Lactobacillus mucosae fermentum bacterial liquid was inoculated into MRS liquid medium at an inoculation amount of 2% (v / v) and cultured at 37 °C for 48 h.

[0044] 1.1.1 Determination of pH

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

[0046] 1.1.2 Determination of organic acids

[0047] 1 mL of the fermentation broth was taken, centrifuged at 10000 r / min for 2 min, and the supernatant was taken. After filtering through a 0.22 μL filter membrane, it was injected for analysis, and the contents of organic acids (citric acid, malic acid, succinic acid, lactic acid, acetic acid) were determined by HPLC.

[0048] The chromatographic conditions were referred to the method disclosed in the literature "Xiao Yangsheng: Research on the Metabolic Regulation Mechanism of Functional Microorganisms in Sichuan Pickles. 2020." and slightly modified. The chromatographic column used was Aminex HPX-87H, the mobile phase was 0.005 M dilute sulfuric acid solution, isocratic elution; the column temperature was 35 °C, the flow rate was 0.5 mL / min; the detector was an ultraviolet detector, the detection wavelength was 210 nm, and the injection volume was 10 μL. Each group was done in 3 parallels, and the average value and standard deviation were calculated.

[0049] 1.2 Determination of the acid tolerance of Lactobacillus mucosae fermentum

[0050] In order to ensure that Lactobacillus mucosae fermentum can maintain its viability during the pickled bamboo shoot fermentation process, it is necessary to determine the acid tolerance of Lactobacillus mucosae fermentum. At the same time, the gastric acid in the body can prevent the contamination of external harmful microorganisms, but they affect the survival of exogenous probiotics in the gastrointestinal tract. Therefore, the selected probiotics need to have strong resistance to the stress conditions in the gastrointestinal tract.

[0051] The strain preserved in glycerol was inoculated into 150 mL of MRS liquid medium, placed in a 37 °C constant temperature incubator, and cultured for 24 h, for a total of three generations of activation. The culture solution was centrifuged at 4 °C and 6000 r / min for 5 min, the supernatant was discarded, and it was washed twice with sterile normal saline to adjust OD 600 = 0.8 ± 0.02. 5 mL of the bacterial liquid was added to 45 mL of the liquid medium with the corresponding pH concentration (pH = 2.5) and resuspended, and shaken evenly. The bacterial liquid was spread on MRS solid medium and cultured at 37 °C for 24 h, and the growth of colonies was counted.

[0052] To detect the acid tolerance of the strain, the acid tolerance rate was calculated according to the following formula:

[0053]

[0054] In the formula:

[0055] X is the acid tolerance performance;

[0056] B is the number of colonies at 4 h, with the unit of CFU / mL;

[0057] A is the number of colonies at 0 h, with the unit of CFU / mL.

[0058] 1.3 Determination of the ability of Lactobacillus mucosae to degrade nitrite

[0059] The fermentation performance of Lactobacillus mucosae in fermented vegetables was evaluated by measuring its ability to degrade nitrite. After activation, Lactobacillus mucosae was inoculated into MRS liquid medium containing 200 mg / L of NaNO 2 at an inoculum size of 2% (v / v) and incubated at 37 °C for 24 h. Weigh 5 g of the fermentation broth and place it in a 250 mL stoppered conical flask. Add 12.5 mL of 50 g / L saturated borax solution and 150 mL of water at 70 °C, mix well, heat in a boiling water bath for 15 min, take out, cool in a cold water bath, and let it stand to room temperature. Quantitatively transfer the above extract to a 200 mL volumetric flask, add 5 mL × 10 6 g / L potassium ferrocyanide solution, shake well, then add 5 mL of 220 g / L zinc acetate solution to precipitate proteins. Add water to the mark, shake well, let it stand for 30 min, remove the upper layer of fat, filter the supernatant with filter paper, discard the first 30 mL of the filtrate, and reserve the filtrate.

[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 50 mL volumetric flasks. Add 2 mL of 4 g / L sulfanilic acid solution to both the standard tubes and the test tubes, mix well, let it stand for 3 min - 5 min, then add 1 mL of 2 g / L N-(1-Naphthyl)ethylenediamine dihydrochloride solution to each, add water to the mark, mix well, let it stand for 15 min, adjust the zero point with the zero tube, measure the absorbance at a wavelength of 538 nm, and plot the standard curve. At the same time, do a reagent blank.

[0061] Degradation rate formula:

[0062]

[0063] Among them: N 0 represents the nitrite content in the blank group of NaNO 2 nitrite, N tIndicates the nitrite content at the end of incubation. There are 3 parallels in each group, repeated 3 times, and the average value is taken.

[0064] 1.4 Determination of the osmotic pressure tolerance of Lactobacillus mucosae fermentum

[0065] After activation, Lactobacillus mucosae fermentum was inoculated into MRS liquid medium with NaCl concentrations of 0%, 2%, 4%, 6%, and 8% respectively at an inoculation amount of 2% (volume fraction), and cultured at a constant temperature of 37°C for 24 h. After 24 h, the colony plate counting method was used to determine the viable cell count. There were 3 parallel experiments in each group, repeated 3 times, and the average value was taken.

[0066] 1.5 Application of Lactobacillus mucosae fermentum in the production of pickled bamboo shoots

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

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

[0069] The fresh bamboo shoots were peeled and the lignified parts at the roots were removed, washed, dried, and evenly cut into pieces. They were placed in a 3 L food-grade fermentation barrel, 2 L of mountain spring water was added to completely immerse the raw materials, and added to the fermentation barrel at an addition amount of 2% (volume fraction) of Lactobacillus mucosae fermentum, and then sealed with a lid. 3 barrels were made in parallel and fermented for 30 days uniformly. After fermentation, the 3 barrels of pickled bamboo shoots obtained were evenly mixed into 1 sample, and then evenly divided into 3 samples again.

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

[0071] The method disclosed in the reference "Li SB, Tian YF, 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 slightly modified to determine the p-cresol content in pickled bamboo shoots.

[0072] 1.6.1 Headspace solid-phase microextraction

[0073] Weigh 50 g of pickled bamboo shoot samples and mix them with an equal mass of ultrapure water at a ratio of 1:1, and use a homogenizer to fully homogenize the mixture. Accurately weigh 6.0 g of the pickled bamboo shoot homogenate into a 20 mL headspace vial, stir and equilibrate it for 30 min under sealed conditions at 50 °C, and insert the aged extraction head. The insertion depth is 1 cm. After adsorbing for 30 min in a 50 °C water bath, immediately insert the extraction head after adsorption into the GC-MS injection port and desorb it at 250 °C for 3 min.

[0074] 1.6.2 GC-MS determination

[0075] Use an Agilent HP-INNOWAX capillary column (60 m × 0.25 mm, 0.25 μm) to separate the volatile components in the pickled bamboo shoot samples. The injection port temperature is 250 °C; the carrier gas is high-purity helium; the flow rate is 1 mL / min. The temperature programming: the initial column temperature is 40 °C, hold for 4 min, then increase to 140 °C at a rate of 6 °C / min, hold for 5 min, increase to 150 °C at a rate of 3 °C / min and hold for 1 min, increase to 200 °C at a rate of 5 °C / min and hold for 1 min, and finally increase to 240 °C at a rate of 8 °C / min and hold for 10 min.

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

[0077] 1.6.3 Qualitative and quantitative analysis of volatile compounds

[0078] (1) Qualitative analysis

[0079] Qualitatively analyze the volatile compounds in the samples according to mass spectrometry (MS) and retention index (RI). Mass spectrometry (MS): During the retrieval process of the NIST14.0 database, compounds with a matching degree greater than 80% are selected after deducting the spectral background. Retention index (RI): Under the same operating conditions as the samples, inject a mixture standard of n-alkanes (C6 - C30), and calculate the retention index RI of each volatile compound in the samples based on the retention time of each alkane, and compare it with the RI reported in the literature for qualitative analysis. The retention index calculation formula is as follows:

[0080]

[0081] In the formula: x is the component to be measured;

[0082] n and n + 1 are the carbon atom numbers of two adjacent n-alkanes before and after the peak emergence of the component to be measured, respectively.

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

[0084] TR is 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 the internal standard, the concentration of volatile compounds in the sample is calculated by comparing the peak area of the compound with that of the internal standard.

[0087] 2. Experimental results

[0088] 2.1 Acid production ability of Lactobacillus mucosae

[0089] Lactic acid and acetic acid are the main sources of the sour flavor of pickled bamboo shoots, and a low pH environment can effectively inhibit the growth of harmful microorganisms and extend the shelf life of pickled bamboo shoots. The acid production ability results of Lactobacillus mucosae are shown in Table 1.

[0090] Table 1 Acid production amount and pH value after fermentation of Lactobacillus mucosae

[0091]

[0092] 2.2 Acid tolerance ability of Lactobacillus mucosae

[0093] The excellent acid tolerance of Lactobacillus mucosae can ensure the activity of the strain during vegetable fermentation, and at the same time has the effects of reducing the fermentation and ripening time and inhibiting various pathogenic bacteria. During the fermentation of pickled bamboo shoots, as the fermentation progresses, the acidity gradually increases, and the strains with poor acid tolerance gradually become inactivated, and the dominant bacteria gradually become medium-acid-tolerant and strong-acid-tolerant strains. The acid tolerance results of Lactobacillus mucosae are shown in Table 2.

[0094] Table 2 Acid tolerance of Lactobacillus mucosae

[0095]

[0096] 2.3 Nitrite reduction ability of Lactobacillus mucosae

[0097] The nitrite standard curve is as Figure 4 shown, and the degradation ability of Lactobacillus mucosae is shown in Table 3.

[0098] Nitrite is a common substance in pickled vegetable foods and also a harmful substance affecting food safety. Studying Lactobacillus mucosae strains that degrade nitrite is of great significance for pickled vegetable production.

[0099] Table 3 Nitrite degradation ability of Lactobacillus mucosae during fermentation

[0100]

[0101] 2.4 Osmotic pressure tolerance of Lactobacillus mucosae

[0102] The osmotic pressure tolerance of Lactobacillus mucosae is shown in Table 4.

[0103] Table 4 Osmotic pressure tolerance of Lactobacillus mucosae

[0104]

[0105] 2.5 p-Cresol production ability of Lactobacillus mucosae

[0106] p-Cresol is a key flavor substance in pickled bamboo shoots, adding a pungent smell and putrid smell to pickled bamboo shoots. The results of p-cresol production ability of Lactobacillus mucosae during pickled bamboo shoot production are shown in Table 5.

[0107] Table 5 p-Cresol content and OVA value in pickled bamboo shoots

[0108]

[0109] Among them, the threshold data of p-cresol in water in Table 5 are excerpted from the literature "Leo van Hemert: Compilation of Compound Aroma Thresholds; 2015."

[0110] The embodiments described above are only for describing the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Limosilactobacillusfermentum that produces high levels of p-cresol, characterized in that: The fermented mucus lactobacillus has been deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 65785, a deposit date of January 14, 2025, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. Use of the fermented mucus lactobacillus as claimed in claim 1 in preparing vegetable fermentation agent.

3. A vegetable fermentation agent, characterized in that: The invention comprises the fermented mucus lactobacillus according to claim 1.

4. Use of the fermented mucus lactobacillus according to claim 1 or the vegetable fermentation agent according to claim 3 in producing fermented vegetables.

5. The use according to claim 4, characterized in that The fermented vegetables are sour bamboo shoots.

6. A method for producing sour bamboo shoots by fermentation, characterized in that: The method comprises the steps of inoculating the fermented mucus lactobacillus liquid according to claim 1 or the vegetable fermentation bacterial agent according to claim 3, and sealing and fermenting.

7. The method according to claim 6, characterized in that The OD of the fermented Lactobacillus mucilaginosus liquid 600nm is 0.8, and according to the volume fraction, the inoculation amount of the fermented mucus lactobacillus liquid is 2%.

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

9. A sour bamboo shoot produced according to the method according to any one of claims 6 to 8.

10. Use of the fermented mucus lactobacillus according to claim 1 or the vegetable fermentation agent according to claim 3 in the production of p-cresol.

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