Lactobacillus for degrading glucosinolate and application of lactobacillus in radish fermentation

Abstract

The invention relates to the technical field of microbiology, in particular to lactobacillus for degrading glucosinolate and application of the lactobacillus to radish fermentation. The preservation number of the lactobacillus is CCTCC (China Center For Type Culture Collection) M20242737, and the invention also discloses an application of the lactobacillus in degradation of glucosinolate and an application of the lactobacillus in radish fermentation. The lactobacillus B10 disclosed by the invention can metabolize 4-methylthio-3-butenyl thioglycoside to produce acid in a relatively short time, so that the fermentation rate of the pickled radishes is increased; growth of other infectious microbes can be inhibited, acid can be produced in a large scale, and inhibition on breeding of infectious microbes which are not resistant to acid can be enhanced; the nitrite content can be effectively reduced, and the safety of the fermented radishes is ensured; under the condition of lactic acid bacteria fermentation, the salinity rise rate of the radishes is high, growth of microorganisms not resistant to salt is inhibited, the safety of the fermentation process is guaranteed, the accumulation speed of lactic acid and the consumption speed of reducing sugar can be increased, and the fermentation period is shortened; the lactobacillus B10 has good glucose metabolism capability, and can improve the fermentation rate of the pickled radish.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology, and particularly to Lactobacillus for degrading glucosinolate and its application in fermenting radishes. Background Art

[0002] Glucosinolates are sulfur-containing compounds unique to cruciferous vegetables. They are not biologically active themselves, but can be degraded by myrosinase to produce biologically active isothiocyanates (ITCs), nitriles, thiocyanates, and epithionitriles D. Changes in the content of glucosinolates, polyphenols and carotenoids during lactic-acid fermentation of cruciferous vegetables: A mini review[J]. Food Chemistry: X, 2022, 16: 100457]. There are four glucosinolates in the fleshy roots of white radish, among which 4-methylthio-3-butenyl glucosinolate (Glucoraphasatin, GRH) accounts for 87.95% of the total glucosinolates and is the most important glucosinolate in the fleshy roots of white radish. It can be degraded to produce the hydrolysis product 4-methylthio-3-butenyl isothiocyanate (4MTB-ITC), which not only provides unique flavor characteristics for fermented radish [Ishida M, Kakizaki T, Morimitsu Y, et al. Novel glucosinolate composition lacking 4-methylthio-3-butenyl glucosinolate in Japanese white radish (Raphanus sativus L.)[J]. Theoretical and Applied Genetics, 2015, 128(10): 2037-2046.], but also 4MTB-ITC has physiological functions such as liver protection and antagonism against pancreatic cancer [Yamaguchi Y, Sugiki M, Shimizu M, et al. Comparative analysis of isothiocyanates in eight cruciferous vegetables and evaluation of the hepatoprotective effects of 4-(methylsulfinyl)-3-butenyl isothiocyanate (sulforaphene) from daikon radish (Raphanus sativus L.) sprouts[J]. Food & Function, 2024, 15(9): 4894-4904.].

[0003] Meanwhile, during the fermentation of radishes, pH value and total acid are key indicators in the vegetable fermentation process. They may not only affect the growth of microorganisms but also influence the taste of pickled vegetables through the accumulation of metabolites, intuitively reflecting the changes in the quality and maturity of fermented radishes [Jing Qiyuan, Li Ting, Zeng Fankun, et al. Effects of Starter Cultures on the Quality of Pickled Radishes [J]. Food Science, 2021, 42(22): 171-177.]. The nitrite content is an important standard for measuring the safety and health of fermented radishes, with a limit standard of 20 mg / kg stipulated by the Chinese national standard [Mi T, Wang D, Yao S, et al. Effects of Salt Concentration on the Quality and Microbial Diversity of Spontaneously Fermented Radish Paocai [J]. Food Research International, 2022, 160: 111622.]. The total sugar content is an important indicator affecting the sensory quality of pickled vegetables, which can reflect the dynamic changes in the consumption of carbohydrate substances in radishes by lactic acid bacteria and the production of carbohydrate substances during the radish fermentation process [Yun Lin. Analysis of the Flavor Characteristics of Radish Pickles with Different Fermentation Methods and Screening of Starter Culture Strains [D]. Wuxi: Jiangnan University, 2020].

[0004] Therefore, obtaining a microorganism that can efficiently degrade GRH, meet the pH value and total acid indicators, reduce the nitrite content, and rapidly reduce the total sugar content has outstanding progressive significance for fermented radishes.

[0005] We obtained a lactic acid bacterium B10 that can efficiently degrade GRH and conducted its biological preservation. The preservation information is as follows:

[0006] The preservation number is: CCTCC M 20242737.

[0007] The name of the preserved biological material (Latin full name): Companilactobacillus futsaii B10; the full name of the unit preserving the sample of this biological material is: China Center for Type Culture Collection; the preservation address is: Wuhan University, Wuhan, China; the preservation time is: December 06, 2024.

[0008] This lactic acid bacterium B10 can effectively degrade GRH, accelerate the decrease in pH during the radish fermentation process, increase the total acid content during the radish fermentation process, reduce the nitrite content during the radish fermentation process; accelerate the decrease in the total sugar content during the radish fermentation process, etc., and can effectively improve the fermentation rate of radish pickles and increase the fermentation quality of radish pickles. Summary of the Invention

[0009] The object of the present invention is to provide Lactobacillus for degrading glucosinolate and its application in fermenting radishes, which can effectively degrade GRH, accelerate the decrease of pH during radish fermentation, increase the total acid content during radish fermentation, reduce the nitrite content during radish fermentation; accelerate the decrease of the total sugar content during radish fermentation, etc., and can effectively improve the fermentation rate of radish pickles and increase the fermentation quality of radish pickles.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] The present invention provides a Lactobacillus B10, and the preservation number of the Lactobacillus (Companilactobacillus futsaii) is: CCTCC M 20242737.

[0012] Further, the 16S rDNA sequence of the Lactobacillus B10 is as shown in SEQ ID NO:1.

[0013] The present invention provides the application of Lactobacillus B10 or its bacterial agent in degrading glucosinolate.

[0014] The present invention provides the application of Lactobacillus B10 or its bacterial agent in radish fermentation.

[0015] The present invention provides any one of the following applications of Lactobacillus B10 or its bacterial agent:

[0016] (1) Inhibiting the reproduction of acid-intolerant miscellaneous bacteria during radish fermentation;

[0017] (2) Accelerating the decrease of pH during radish fermentation;

[0018] (3) Increasing the total acid content during radish fermentation;

[0019] (4) Reducing the nitrite content during radish fermentation;

[0020] (5) Accelerating the increase of salinity during radish fermentation;

[0021] (6) Inhibiting the growth of salt-intolerant microorganisms during radish fermentation;

[0022] (7) Accelerating the decrease of the total sugar content during radish fermentation;

[0023] (8) Improving the fermentation rate of radish pickles;

[0024] (9) Increasing the isothiocyanate content of radish pickles;

[0025] (10) Increasing the nitrile and ester contents of radish pickles;

[0026] (11) Inhibiting the browning of radish color during fermentation.

[0027] The present invention has at least the following beneficial effects:

[0028] (1) During the fermentation of radish, Lactobacillus B10 of the present invention can metabolize GRH to produce acid in a relatively short time, improving the fermentation rate of radish pickles;

[0029] (2) Lactobacillus B10 of the present invention has good salt tolerance and can survive and grow in the radish fermentation environment (salt concentration is about 6%);

[0030] (3) During the fermentation of radish, Lactobacillus B10 of the present invention can inhibit the growth of other miscellaneous bacteria and produce a large amount of acid in the initial stage of fermentation. The rapid decrease in pH can also strengthen the inhibition of the reproduction of acid-intolerant miscellaneous bacteria;

[0031] (4) Lactobacillus B10 of the present invention can effectively reduce the nitrite content, ensuring the safety of fermented radish.

[0032] (5) During the fermentation of radish, the rising rate of the salinity of Lactobacillus B10 of the present invention is higher than that of natural fermentation. The increase in salinity is beneficial to inhibiting the growth of salt-intolerant microorganisms, ensuring the safety of the fermentation process, and can also accelerate the accumulation rate of lactic acid and the consumption rate of reducing sugar, shortening the fermentation cycle.

[0033] (6) During the fermentation of radish, Lactobacillus B10 of the present invention has good sugar metabolism ability, which can improve the fermentation rate of radish pickles.

[0034] (7) During the fermentation of radish, Lactobacillus B10 of the present invention detected more isothiocyanates, as well as volatile components such as nitriles and esters during the fermentation process than natural fermentation of radish. There are a total of 307 volatile components, which can increase the acceptance of fermented foods by consumers. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0036] Figure 1 Schematic diagram of the color development situation of B10 after growth on the improved MRS plate (left) and the blank plate (right);

[0037] Figure 2 Schematic diagram of the colony morphology of B10 (left) and Gram staining (right);

[0038] Figure 3 Schematic diagram of the acid production curve of B10 (left) and the GRH degradation curve (right);

[0039] Figure 4 Schematic diagram of the salt tolerance of B10;

[0040] Figure 5 Schematic diagram of the changes in pH and total acid content during natural fermentation and B10 fermentation of radish;

[0041] Figure 6 Schematic diagram of the changes in salinity and nitrite content during natural fermentation and B10 fermentation of radish;

[0042] Figure 7 Schematic diagram of the changes in total sugar content during natural fermentation and B10 fermentation of radish;

[0043] Figure 8 Schematic diagram of the inhibition of browning of radish color by B10 strain during fermentation. Specific implementation manners

[0044] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0045] The main objective of the present invention is to provide a strain of Lactobacillus paracasei that degrades glucosinolate and its application in fermenting radish. Specifically as follows:

[0046] 1 Materials and methods

[0047] 1.1 Materials and reagents

[0048] 1.1.1 Materials

[0049] Fresh white radishes, purchased from Yonghui Supermarket in Beibei District, Chongqing; pickled radishes, homemade by farmers in Beibei District, Chongqing.

[0050] 1.1.2 Reagents

[0051] 4-Methylthio-3-butenyl glucosinolate (HPLC, purity ≥ 99.8%) in white radish extract, Wuhan Tianzhi Biotechnology Co., Ltd.; peptone, beef extract, sodium chloride, yeast powder, sodium acetate, magnesium sulfate heptahydrate, manganese sulfate monohydrate, diammonium hydrogen citrate, dipotassium hydrogen phosphate, L-cysteine, Tween 80, bromocresol purple, agar powder, Beijing Aoboxing Biotechnology Co., Ltd.; Bacterial genomic DNA extraction kit, Tiangen Biotech Co., Ltd.

[0052] 1.1.3 Culture media

[0053] (1) Improved MRS solid medium (g / L): 4-methylthio-3-butenyl glucosinolate 6, peptone 10, beef extract 10, sodium chloride 5, yeast powder 5, sodium acetate 2, magnesium sulfate heptahydrate 0.1, manganese sulfate monohydrate 0.05, diammonium hydrogen citrate 2, dipotassium hydrogen phosphate 2.6, L-cysteine 1, Tween 80 0.1%, 0.5% bromocresol purple 1.5%, agar powder 1.5% - 2.0%, pH value = 6.2 - 6.4 (adjusted with NaOH)

[0054] (2) GRH liquid medium (g / L): 4-methylthio-3-butenyl glucosinolate 6, yeast powder 5, sodium chloride 5, L-cysteine 1, ascorbic acid 0.5

[0055] 1.2 Experimental methods

[0056] 1.2.1 LC / MS / MS analysis 1.2.1LC / MS / MS analysis of GRH (4-methylthio-3-butenyl glucosinolate)

[0057] Use LC / MS / MS to detect the content of 4-methylthio-3-butenyl glucosinolate in experimental samples. The UPLC system (Nexera X2, Shimadzu, Kyoto, Japan) is equipped with an SPD-20A UV detector and an Ultimate XB-C18 chromatographic column (2.1×100mm, 3μm, Welch Materials Inc., Ellicott, MD, USA). Mobile phases A and B are 10 mM ammonium formate solution and acetonitrile respectively. The flow rate is set at 0.40 mL / min, the detection wavelength is 234 nm, and the gradient program is as follows: 100% A, lasting for 0 - 1 min, from 100% to 95% of A lasting for 1 - 3 minutes, from 95% to 70% of A lasting for 3 - 6.2 minutes, 70% of A lasting for 6.2 - 7.2 minutes, from 70% to 0% of A lasting for 7.2 - 8 minutes, 0% of A lasting for 8 - 9 minutes, from 0% to 100% of A lasting for 9 - 10 minutes, and 100% of A lasting for 10 - 12 minutes.

[0058] Couple the UPLC system with a TOF mass spectrometer (AB Sciex, CA, USA) equipped with an electrospray ionization (ESI) source, and collect data in negative ion mode in the mass range of m / z 50 to 550. The spray voltage (ISVF) is -4500 V, the heater temperature (TEM) is 550 °C, the nebulizing gas (GS1) is 55 psi, the heating gas (GS2) is 55 psi, the curtain gas (CUR) is 35 psi, the collision energy is -40 eV, and the collision energy spread is 20 eV. Use TF 1.6.3 software to process the data to identify glucosinolates.

[0059] 1.2.2 Screening of lactic acid bacteria degrading GRH

[0060] Add 25 mL of radish pickle juice to a sterile homogenization bag containing 225 mL of sterile PBS buffer, mix well, and perform 10-fold serial dilutions (10 -1 ~10 -6 ). Then, take 100 μL of the diluted sample solutions with different concentrations and spread them evenly on the modified MRS solid medium, and incubate anaerobically at 37 °C for 24 - 48 h. Purify the lactic acid bacteria pure strains by streaking, and preserve the strains using 15% glycerol tubes and the freeze-drying method.

[0061] Secondary screening of lactic acid bacteria degrading glucosinolate GRH

[0062] Cultivate the test strains in MRS liquid medium, adjust the bacterial liquid concentration to OD 600nm = 0.1 with a sterile PBS solution, pipette 200 μL of the bacterial liquid and inoculate it into 5 mL of GRH liquid medium. Add 200 μL of sterile PBS solution to the blank group. Incubate anaerobically at 37 °C for 12 h, centrifuge at 8000 g for 5 min to obtain the supernatant, filter it through a 0.22 μm aqueous filter membrane, and determine the content of glucosinolate GRH by HPLC, and calculate the degradation rate of glucosinolate GRH.

[0063] Chromatographic conditions: 10 mM ammonium formate solution and acetonitrile are used as phase A and phase B respectively, the mobile phase flow rate is 0.40 mL / min, the column temperature is 40 °C, the detection wavelength is 234 nm, and the injection volume is 10 μL. The gradient elution program is as follows: 0.0 min, 100% A and 0% B; 2.0 min, 95% A and 5% B; 30.0 min, 30% A and 70% B; 33 min, 10% A and 90% B; 37.0 min, 10% A and 90% B; 37.1 min, 95% A and 5% B; 43 min, 95% A and 50% B; 48 min, 100% A and 0% B; 55 min, 100% A and 0% B. Prepare GRH standard solutions with concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 (g / L), plot the GRH standard curve equation with the concentration (g / L) as the abscissa and the peak area as the ordinate as y = 858737x + 562532, R 2 = 0.9997.

[0064] 1.2.3 Strain identification

[0065] Identify the strains with the activity of degrading glucosinolate GRH obtained by screening through 16S rDNA for molecular biology identification. Extract genomic DNA using a bacterial DNA extraction kit (Tiangen Biochemical). Amplify PCR with universal primers 27F and 1492R, and send the amplified product to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing, and perform BLAST alignment analysis on the sequencing results in NCBI.

[0066] Determination of the growth curve of strain 1.2.4

[0067] After the strain was activated, it was adjusted to OD600nm = 0.1 with sterile PBS buffer, and then added to MRS liquid medium at an inoculation amount of 1% (v / v). It was anaerobically cultured at 37 °C for 14 h. Samples were taken every 2 h to measure OD600nm and pH value. The growth time was taken as the abscissa, and OD600nm and pH value were taken as the ordinate to draw the curve.

[0068] Determination of the salt tolerance of strain 1.2.5

[0069] After the strain was activated, it was adjusted to OD600nm = 0.1 with sterile PBS buffer, and then added to MRS liquid medium with different salt concentrations (0, 30, 60, 90, 120, unit: g·L-1) at an inoculation amount of 1% (v / v). It was cultured at 37 °C for 24 h, and the absorbance of the bacterial solution at a wavelength of 600 nm at 0 h and 24 h was measured using a microplate reader.

[0070] Fermentation of radish by strain 1.2.6

[0071] 1.2.6.1 Preparation of fermented radish

[0072] Naturally fermented radish: The radish was washed clean to remove surface impurities, instantaneously heat-treated with boiling water, then washed 3 times with sterile water, cut into pieces about 1 cm × 1 cm × 1 cm, and salt water was added. It was packed into a jar and sealed.

[0073] Fermented radish inoculated with a single strain: The experimental strain was activated for 3 generations in the medium. Before inoculation, the cells were obtained by centrifugation at 6000g and washed 3 times with physiological saline. The initial inoculation amount was controlled at 10 7 CFU / mL. After all the raw materials were loaded into the fermentation jar, the strain for fermentation was added. Other fermentation processes were the same as the natural fermentation method.

[0074] Radish fermentation formula (g / 100 mL water): 100 g of white radish, 6 g of table salt.

[0075] 1.2.6.2 Determination of pH

[0076] Take 5 mL of pickled radish liquid and measure the pH of the radish pickle liquid with a pH meter.

[0077] 1.2.6.3 Determination of the total acid content

[0078] Determine the total acid content of pickled radish with reference to the national standard GB / T 12456—2008 "Determination of Total Acid in Foods". Weigh 5.000 g of pickled radish to make a homogeneous pulp, soak it in 50 mL of distilled water after boiling for half an hour, cool it, make up the volume to 100 mL and then filter. Accurately pipette 10.00 mL of the filtrate into a 150 mL conical flask, add 50 mL of distilled water, add 2 - 3 drops of 10 g / L phenolphthalein indicator (dissolved in 95% ethanol), titrate with 0.0500 mol / L NaOH standard titration solution until it turns slightly pink and does not fade in 30 s, which is the end point of titration. Record the volume of NaOH used and calculate.

[0079] 1.2.6.4 Determination of salinity

[0080] After weighing 5.000 g of pickled radish to make a homogeneous pulp, soak it in 50 mL of distilled water after heating to boiling for half an hour, cool it, filter and make up the volume to 100 mL. Pipette 2.00 mL of the filtrate into a conical flask, add distilled water to make up the volume to 50 mL, add 0.5 mL of 100 g / L potassium chromate indicator, titrate with 0.1000 mol / L AgNO3 standard solution until it just turns brick red, which is the end point. Record the volume of AgNO3 required and calculate.

[0081] 1.2.6.5 Determination of total sugar

[0082] Weigh 5.00 g of radish sample to make a homogeneous pulp, add 50 mL of distilled water and boil for 30 min, cool it, filter and make up the volume to 100 mL. Take 2 mL of the supernatant and determine the total sugar content by the sulfuric acid anthrone method.

[0083] Take 7 25 mL colorimetric tubes, accurately pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL and 1.2 mL of glucose standard stock solution (0.1 mg / mL) into the colorimetric tubes in sequence, add water to make up to 2.0 mL, shake well, then add 6 mL of sulfuric acid anthrone solution (accurately weigh 0.1 g of anthrone into a 100 mL volumetric flask, dissolve it with sulfuric acid and make up the volume to the scale with sulfuric acid), shake well, let it stand at room temperature for 20 min, place it in an ice - water bath for 15 min, take it out, adjust the zero point with the corresponding reagent, take the glucose content as the abscissa and the absorbance as the ordinate, measure the absorbance under the condition of wavelength 625 nm, and draw the standard curve as y = 3.07x + 0.074, R 2 = 0.9998.

[0084] 1.2.6.6 Determination of nitrite

[0085] Determine the nitrite content in pickled radish with reference to GB 5009.33 - 2016 "Determination of Nitrite and Nitrate in Foods".

[0086] 1.2.6.7 Determination of chromaticity

[0087] The color and luster was determined using the L*a*b* color system and directly measured with a color difference meter. The L*a*b* color system is established based on colorimetry. Among them, the L* value is the lightness value of the color, indicating the brightness or darkness of the color; the a* value and b* value are the chromaticity values of the color. The a* value gradually changes from red (+a*) to green (-a*), and the b* value gradually changes from yellow (+b*) to blue (-b*). Cubes with dimensions of 1.5 cm × 1.5 cm × 1.5 cm were cut from the middle part of the pickled radish for measurement. The upper, middle, and lower parts of the pickled radish cubes were selected for measurement, and the average value was taken after parallel measurement three times.

[0088] 1.2.6.8 Determination of Volatile Flavor Components

[0089] The headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) techniques were used to study the changes in volatile compounds of radish under different pickling conditions. SPME conditions: Transfer 5 g of pickled radish to a 20 mL headspace vial, add 2 μL of ethyl caprylate at 0.104 mg / mL as the internal standard. Insert the aged (250 °C, 30 min) DVB / CAR / PDMS extraction head into the sample vial for adsorption extraction, and adsorb at 60 °C for 30 min. Then insert the extraction head into the injection port of the gas chromatograph and desorb at 250 °C for 8 min. The column temperature was initially maintained at 40 °C for 10 min, then increased to 250 °C at a rate of 10 °C / min and held for 1 min. The flow rate was 1 mL / min, and the ion source temperature was 230 °C. The NIST17 library was used to screen volatile components with a similarity exceeding 70%. Under the same experimental conditions, n-alkanes (C7-C30) were used as an external reference to calculate the RI, which was compared with the RI of each volatile component. Volatile components with a difference in RI of <20 were retained [Zhao N, Ge L, Lai H, et al. Unraveling the contribution of pre-salting duration to microbial succession and changes of volatile and non-volatile organic compounds in Suancai (a Chinese traditional fermented vegetable) during fermentation [J]. Food Research International, 2022, 159: 111673]. The internal standard method was used to determine the absolute content of each volatile component.

[0090] 2. Results and Discussion

[0091] 2.1 Composition of Glucosinolates in White Radish

[0092] A total of 4 types of glucosinolates were detected in the fleshy roots of white radish, as shown in Table 1. Among them, 4-methylthio-3-butenyl glucosinolate (Glucoraphasatin, GRH) accounted for 87.95% of the total glucosinolates and was the most predominant glucosinolate in the fleshy roots of white radish. Therefore, 4-methylthio-3-butenyl glucosinolate was selected as the substrate to screen lactic acid bacteria with glucosinolate-degrading activity.

[0093] Table 1 Glucosinolates in White Radish

[0094]

[0095] 2.2 Screening of GRH-Degrading Lactic Acid Bacteria

[0096] Lactic acid bacteria were isolated and screened from radish pickle brine, and finally a lactic acid bacterium with high efficiency in degrading GRH was obtained, which was named B10. Figure 1 The colony morphology of B10 on the modified MRS plate is shown (left). The color of the medium turned yellow due to acid production, with the color of the blank plate (right) as the control. B10 was identified as Companilactobacillus futsaii by 16S rDNA, and the homology was 99.23%.

[0097] The 16S rDNA sequence of strain B10 is shown in SEQ ID NO:1 as follows:

[0098] SEQ ID NO:1 16S rDNA sequence of Companilactobacillus futsaii B10:

[0099]

[0100] We have deposited the B10 strain at the China Center for Type Culture Collection, and the deposit information is as follows:

[0101] The deposit number is: CCTCC M 20242737.

[0102] The name of the deposited biological material (full Latin name): Companilactobacillus futsaii B10;

[0103] The full name of the unit depositing the biological material sample is: China Center for Type Culture Collection;

[0104] The deposit address is: Wuhan, China, Wuhan University,

[0105] The deposit date is: December 06, 2024.

[0106] 2.3 Growth characteristics of the strain

[0107] Refer to Figure 2 , the B10 colony is round, milky white, and smooth on the surface (left in the figure). Gram staining (right in the figure) is negative, rod-shaped.

[0108] Inoculate B10 into GRH liquid medium and culture it at 37 °C for 14 h. The growth curve and acid production rate are as Figure 3 shown. As time changes, the pH value of the medium continuously decreases, and the OD600nm value continuously increases. The lag phase is from 0 - 4 h; the logarithmic growth phase is from 4 - 10 h, during which the pH decreases at the fastest rate; the stationary phase is from 10 - 14 h. The pH drops to 3.4 after 8 h, indicating that B10 can metabolize and produce acid in a short time and has the potential to ferment radishes.

[0109] 2.4 Salt tolerance

[0110] To evaluate the salt tolerance of B10, inoculate the B10 strain into GRH medium with different salt concentrations (0%, 3%, 6%, 9%, 12%) and culture it at 37 °C for 24 h. The OD of the bacterial liquid at 0 h and 24 h 600nm is as Figure 4 shown. At 0% - 3%, B10 shows good growth. Above 3% salt concentration, the growth of B10 begins to be inhibited but it can still grow. When the salt concentration is higher than 9%, the growth of B10 is almost completely inhibited, indicating that B10 can survive and grow in the radish fermentation environment (salt concentration is about 6%).

[0111] 2.5 Effects of Companilactobacillus futsaii on fermented radishes

[0112] 2.5.1 Changes in pH and total acid during natural fermentation and B10 fermentation of radishes

[0113] The pH value and total acid are key indicators during the vegetable fermentation process. They may not only affect the growth of microorganisms, but also influence the taste of pickled vegetables through the accumulation of metabolites, intuitively reflecting the changes in the quality and maturity of fermented radishes [Jing Qiyuan, Li Ting, Zeng Fankun, et al. Effects of starter cultures on the quality of pickled radishes [J]. Food Science, 2021, 42(22): 171-177.]. The changes in pH and total acid content during natural fermentation and B10 fermentation are as Figure 5 shown. The initial pH of both natural fermented pickled vegetables and B10 fermented pickled vegetables is around 5.5. In the first two days of fermentation, the pH of the radishes inoculated with lactic acid bacteria dropped rapidly from 5.5 to 3.2, and the total acid content increased rapidly to 0.25 g / 100 g, while the changes in pH and total acid content of the naturally fermented pickled radishes were slow. After seven days of fermentation, the total acid of the B10 fermented radishes reached 0.48 g / 100 g, higher than the total acid of 0.39 g / 100 g in natural fermentation. Lactic acid bacteria can inhibit the growth of other miscellaneous bacteria and produce a large amount of acid in the initial stage of fermentation. The rapid drop in pH can also strengthen the inhibition of the reproduction of acid-intolerant miscellaneous bacteria [Zhang Xiru, Guan Hui, Xing Shaohua, et al. Research progress on the microbial succession and flavor substance changes in pickled vegetables [J]. Food Science, 2021, 42(23): 294-305].

[0114] 2.5.2 Changes in salinity and nitrite during the natural fermentation and B10 fermentation of radishes

[0115] The nitrite content and salinity are important criteria for measuring the safety and health of fermented radishes. As Figure 6As shown in the figure, the nitrite content was relatively high in the first 2 days of fermentation, and the nitrite content in naturally fermented radish (13.11 μg / g) was much higher than that in B10-fermented radish (3.42 μg / g). This may be due to the growth and reproduction of some Gram-negative bacteria mainly present in the early stage of fermentation, which reduce nitrate in radish to nitrite [Yan P, Xue W, Tan S, et al. Effect of inoculating lactic acid bacteria starter cultures on the nitrite concentration of fermenting Chinese paocai [J]. Food Control, 2008, 19(1): 50-55.]. After the second day, the nitrite content began to decline and stabilized after 3 days. At the end of fermentation, the nitrite content in naturally fermented radish (4.60 μg / g) was higher than that in B10-fermented radish (2.25 μg / g), but lower than the limit standard of 20 mg / kg stipulated by the Chinese national standard [Mi T, Wang D, Yao S, et al. Effects of salt concentration on the quality and microbial diversity of spontaneously fermented radish paocai [J]. Food Research International, 2022, 160: 111622.]. This shows that inoculating Lactobacillus parafarraginis B10 can effectively reduce the nitrite content and ensure the safety of fermented radish.

[0116] The salinity in radish showed an increasing trend during the fermentation of both (natural fermentation and B10 fermentation), and the rising rate of salinity in B10-fermented radish was higher than that in natural fermentation. The increase in salinity is beneficial to inhibiting the growth of salt-intolerant microorganisms, ensuring the safety of the fermentation process, and can also accelerate the accumulation rate of lactic acid and the consumption rate of reducing sugar, shortening the fermentation cycle [Huang Daomei. Research on the changes of bacterial flora and flavor quality during the multi-strain co-fermentation of industrial salted radish with different salinities [D]. Sichuan Agricultural University, 2015.].

[0117] 2.5.3 Changes in total sugar during the fermentation of natural and B10-fermented radish

[0118] The total sugar content is an important indicator affecting the sensory quality of pickles, which can reflect the dynamic changes of the consumption of carbohydrate substances in radish by lactic acid bacteria and the production of carbohydrate substances during the fermentation of radish [Yun Lin. Analysis of the flavor characteristics of radish pickles with different fermentation methods and screening of starter cultures [D]. .无锡:江南大学,2020] . Such as Figure 7As shown, the total sugar in B10-fermented radish rapidly decreased to 10 mg / g on the 2nd day of fermentation and then stabilized, while the total sugar content in naturally fermented radish began to rapidly decrease only on the 3rd day, with a certain lag period. It can be seen that Lactobacillus parafarraginis B10 has good sugar metabolism ability. Lactobacillus parafarraginis is a homofermentative lactic acid bacterium that metabolizes sugars to produce lactic acid, explaining the rapid decrease in pH and the rapid increase in total acid in the initial stage of B10-fermented radish, indicating that B10 can improve the fermentation rate of radish pickles.

[0119] 2.5.4 Color change during the process of natural fermentation and B10 fermentation of radish

[0120] Due to the growth and metabolism of harmful microorganisms, red, pink, and gray may appear during the pickling process of radish. The changes in L*, a*, and b* values were measured respectively during natural fermentation and B10 fermentation to observe the color change situation. The results are shown in Table 2 and Figure 8 As shown, after 7 days of fermentation, the L* value of the naturally fermented group decreased, and the a* and b* values increased, indicating that the color of the radish turned brown during fermentation. The L* value of the radish in the B10-fermented group remained unchanged, and the a* and b* values decreased, indicating that the B10 strain can inhibit the browning of the radish color during fermentation. This may be due to the production of more acid or other antioxidants during the growth of the strain, which inhibits peroxidase.

[0121] Table 2 Color change during the process of natural fermentation and B10 fermentation of radish

[0122]

[0123]

[0124] 2.5.5 Volatile components during the process of natural fermentation and B10 fermentation of radish

[0125] Volatile components are important quality indicators affecting consumers' acceptance of fermented foods [Mi T, Wang D, Yao S, et al. Effects of salt concentration on the quality and microbial diversity of spontaneously fermented radish paocai [J]. Food Research International, 2022, 160: 111622]. The changes in volatile components during natural fermentation and B10 fermentation at 0, 1, 3, 5, and 7 days were analyzed by headspace solid-phase microextraction and gas chromatography-mass spectrometry. A total of 307 volatile components were detected and identified, as shown in Tables 3 and 4. Among them, there were 81 esters, 21 alcohols, 13 phenols, 14 nitriles, 20 aldehydes, 62 hydrocarbons, 32 ketones, 18 organic acids, 22 heterocycles, and 24 others. During the fermentation process, some volatile components increased, while others decreased or disappeared. Among all volatile components, esters had the highest proportion of 49.77%, followed by hydrocarbons (27.28%) and alcohols (9.08%). Different categories of volatile compounds showed different trends during fermentation. The contents of alcohols, phenols, nitriles, hydrocarbons, organic acids, heterocycles, and esters gradually increased, and anhydride compounds were also generated during B10 fermentation.

[0126] Glucosinolates are sulfur-containing compounds unique to cruciferous vegetables. They are not biologically active themselves but can be degraded by myrosinase to produce biologically active isothiocyanates (ITCs), nitriles, thiocyanates, and epithionitriles D. Changes in the content of glucosinolates, polyphenols and carotenoids during lactic-acid fermentation of cruciferous vegetables: A mini review[J]. Food Chemistry: X, 2022, 16: 100457]. Glucoraphanin (GRH) is the most abundant glucosinolate in white radish roots and can be degraded to produce the hydrolysis product 4-methylthio-3-butenyl isothiocyanate (4MTB-ITC), which not only provides unique flavor characteristics for fermented radishes [Ishida M, Kakizaki T, Morimitsu Y, et al. Novel glucosinolate composition lacking 4-methylthio-3-butenyl glucosinolate in Japanese white radish (Raphanus sativus L.)[J]. Theoretical and Applied Genetics, 2015, 128(10): 2037-2046], but 4MTB-ITC also has physiological functions such as liver protection and antagonism against pancreatic cancer [Yamaguchi Y, Sugiki M, Shimizu M, et al. Comparative analysis of isothiocyanates in eight cruciferous vegetables and evaluation of the hepatoprotective effects of 4-(methylsulfinyl)-3-butenyl isothiocyanate (sulforaphene) from daikon radish (Raphanus sativus L.) sprouts[J]. Food & Function, 2024, 15(9): 4894-4904.]. A total of five isothiocyanates were detected during natural fermentation and B10 fermentation, including 3-(methylthio)propyl isothiocyanate; 4-methylthio-3-butenyl isothiocyanate; 2-phenylethyl isothiocyanate; benzyl isothiocyanate; 1-isothiocyanatohexane, as shown in Table 5. Among them, both 3-(methylthio)propyl isothiocyanate and 4-methylthio-3-butenyl isothiocyanate are degradation products of GRH. During the fermentation process, the isothiocyanates showed a trend of first decreasing, then increasing, and then decreasing.The content of isothiocyanates in fresh radishes is relatively high. This may be because after the radish tissue is damaged, the physical isolation between glucosinolates and myrosinase is destroyed, resulting in the hydrolysis of a part of GRH. However, isothiocyanates may be degraded by microorganisms during the subsequent fermentation process, but this also promotes the formation of flavor components such as aldehydes and alcohols. More isothiocyanates, as well as volatile components such as nitriles and esters, were detected in the B10 fermentation process than in the natural fermentation of radishes.

[0127] Table 3 Types of Volatile Components during Natural Fermentation and B10 Fermentation

[0128]

[0129]

[0130] Table 4 Contents of Various Volatile Components during Natural Fermentation and B10 Fermentation (μg / kg)

[0131]

[0132] Table 5 Contents of Isothiocyanates during Natural Fermentation and B10 Fermentation (μg / kg)

[0133]

[0134] The above has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification is only the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A Lactobacillus B10, characterized in that, The deposit number of the Lactobacillus (Companilactobacillus futsaii) is: CCTCC M 20242737.

2. The Lactobacillus B10 according to claim 1, characterized in that, The 16S rDNA sequence of the Lactobacillus B10 is shown in SEQ ID NO:

1.

3. Use of the Lactobacillus B10 or its bacterial agent according to claim 1 in degrading glucosinolate.

4. Use of the Lactobacillus B10 or its bacterial agent according to claim 1 in radish fermentation.

5. Any one of the following uses of the Lactobacillus B10 or its bacterial agent according to claim 1: (1) Inhibiting the reproduction of acid-intolerant miscellaneous bacteria during radish fermentation; (2) Accelerating the decrease in pH during radish fermentation; (3) Increasing the total acid content during radish fermentation; (4) Reducing the nitrite content during radish fermentation; (5) Accelerating the increase in salinity during radish fermentation; (6) Inhibiting the growth of salt-intolerant microorganisms during radish fermentation; (7) Accelerating the decrease in total sugar content during radish fermentation; (8) Improving the fermentation rate of radish pickles; (9) Increasing the isothiocyanate content of radish pickles; (10) Increasing the nitrile and ester contents of radish pickles. (11) Inhibiting the browning of radish color during fermentation.

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