Lactic acid bacteria and application thereof

Abstract

The invention provides pediococcus pentosaceus and an application of the pediococcus pentosaceus. The pediococcus pentosaceus is pediococcus pentosaceus LQQ-1-R with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.30627, and the pediococcus pentosaceus LQQ-1-R is preserved in the China General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms. After the pickles are inoculated with the pediococcus pentosaceus, the acid production capacity of fermentation is weak, the aroma production capacity is high, and the sensory quality is good. The pediococcus pentosaceus LQQ-1-R disclosed by the invention has a good application prospect in shallow fermented pickles.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology. The present invention specifically relates to the screening of a strain of Pediococcus pentosaceus ( Pediococcus pentosaceus ) and its application in the shallow fermentation process of kimchi. Background Art

[0002] Kimchi is mainly fermented by lactic acid bacteria. A large number of studies have confirmed that Leuconostoc, Lactobacillus, and Weissella are the dominant lactic acid bacteria in the kimchi fermentation process. Lactobacillus has a strong acid resistance, which causes kimchi to continue to slowly ferment and produce lactic acid during the storage, transportation and sales of kimchi, resulting in the problem of "over-acidification", that is, kimchi "post-acidification". Post-acidification can easily lead to a decrease in kimchi flavor and a softening of texture. Shallow fermentation can not only improve product quality, but also solve the problem of post-acidification of kimchi, and can also comply with the requirements of low acid and low salt advocated by the international community today. At present, most of the kimchi on the market are sterilized kimchi produced by industrial production, and traditional kimchi at home is prone to excessive fermentation-post-acidification. Relatively speaking, the innovation of shallow fermented kimchi is to solve the problem of over-acidification in traditional kimchi and the sterility of industrial kimchi. Therefore, the acid production ability of lactic acid bacteria is an important factor in controlling the quality of kimchi.

[0003] At present, there are many inventions about high-acid-producing lactic acid bacteria. Patent CN202311198623.X invented a high-acid-producing lactic acid bacteria suitable for low-temperature sauerkraut fermentation in the north, which provides a good strain resource for sauerkraut fermentation production in high latitudes and cold areas. Patent CN201910404204.4 provides a high-acid-producing lactic acid bacterium Enterococcus faecium, which is suitable for low-temperature straw micro-storage fermentation conditions in the north and has a high acid production. The applicant found that high-acid-producing lactic acid bacteria can cause the phenomenon of "post-acidification" in kimchi. Therefore, the purpose of the present invention is to provide a low-acid and high-fragrance lactic acid bacteria that meets the international requirements of low acid and low salt on the basis of ensuring the taste, solves the post-acidification phenomenon, and at the same time ensures that kimchi contains live bacteria. Summary of the invention

[0004] The purpose of the present invention is to provide a Pediococcus pentosaceus and application thereof.

[0005] The present invention provides a Pediococcus pentosaceus, which is a Pediococcus pentosaceus deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on May 14, 2024, with a deposit number of CGMCC No. 30627 ( Pediococcus pentosaceus )LQQ-1-R, the storage address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The present invention also provides an application of Pediococcus pentosaceus in shallow fermentation of kimchi, comprising the following steps:

[0007] Inoculate the lactic acid bacteria suspension into fresh vegetables, add salt water and spice juice, mix well, and ferment to obtain fresh, fragrant, crispy, tender and refreshing kimchi.

[0008] Wherein, the lactic acid bacteria is the CGMCC No. 30627 Pediococcus pentosaceus ( Pediococcus pentosaceus )LQQ-1-R.

[0009] Furthermore, the concentration of the Pediococcus pentosaceus suspension in the kimchi needs to reach 10 5 -10 7 CFU / g.

[0010] Furthermore, the Pediococcus pentosaceus suspension is prepared according to the following method: a certain amount of Pediococcus pentosaceus inoculum is taken and activated with 10-20 mL of 2-5% sterile glucose water at 25-30° C. for 20-30 minutes.

[0011] Furthermore, the kimchi in step (1) is made from fresh white radish, carrots and green bamboo shoots, which are washed, peeled and cut into pieces, and an equal amount of drinking water is added. After balance, 4-6% of salt is added and 3-5% of spice juice is added.

[0012] Furthermore, the preparation of the spice juice (calculated as water) in step (1) is to add 3-5% dried chili pepper, 0.3-0.5% prickly ash, 1-3% ginger, 1-3% garlic, 0.3-0.5% star anise, and 0.1-0.3% fennel, crush the above spices, and extract them in a closed manner in a 75-85°C water bath for 2-3h, and then add 3-5% white wine after cooling.

[0013] In step (2), the fermentation condition is constant temperature fermentation at 20-30°C for 3-5 days.

[0014] The experimental results show that the present invention provides a new strain of Pediococcus pentosaceus. The experimental verification shows that the Pediococcus pentosaceus inoculated pickles have weak acid production ability and high aroma production ability compared with natural fermentation, and have good sensory quality. The Pediococcus pentosaceus of the present invention is a Pediococcus pentosaceus (CGMCC No. 30627) deposited by the General Microbiological Center of the China Microbiological Culture Collection Administration. Pediococcus pentosaceus The Pediococcus pentosaceus LQQ-1-R of the present invention has good application prospects in shallow fermented kimchi.

[0015] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0016] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the single colony morphology of the LQQ-1-R strain.

[0018] Figure 2 This is the microscopic morphology of the LQQ-1-R strain.

[0019] Figure 3 This is the phylogenetic tree of the LQQ-1-R strain.

[0020] Figure 4 This is a comparison chart of the types of volatile flavor substances in kimchi fermented by inoculation with LQQ-1-R and naturally fermented kimchi. DETAILED DESCRIPTION

[0021] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0022] MRS broth medium: peptone 10 g, beef extract 10 g, yeast powder 5 g, potassium dihydrogen phosphate 2 g, diammonium citrate 2 g, sodium acetate 5 g, glucose 20 g, Tween 80 1 mL, magnesium sulfate heptahydrate 0.58 g, manganese sulfate tetrahydrate 0.25 g, distilled water 1000 mL, pH 6.2 to 6.4. Add 1.5% (w / v) agar powder and 1.5% (w / v) calcium carbonate to the solid medium. Sterilize at 121℃ for 15 min.

[0023] Plate count agar: 5.0 g of tryptone, 2.5 g of yeast extract, and 1.0 g of glucose are dissolved in 1000 mL of distilled water, boiled to dissolve, and the pH is adjusted to 7.0±0.2. Sterilize at 121℃ for 15 min.

[0024] Vegetable juice medium: After processing fresh vegetables (white radish, cucumber, tomato), mix the vegetables at a ratio of 7:2:1, add 0.05% ginger, chili pepper and 0.01% garlic, break the juice wall at a ratio of 1:1, filter through gauze, add 2% salt, 2% glucose, 1% yeast extract and 1% soy protein powder. Sterilize at 115 ℃ for 15 min.

[0025] Example 1: Isolation and identification of Pediococcus pentosaceus of the present invention

[0026] 1. Experimental Methods

[0027] 1. Separation

[0028] (1) Isolation of lactic acid bacteria from homemade kimchi water and preliminary screening of their acid-producing capacity

[0029] After the kimchi sample was shaken and mixed, 0.5 mL of liquid was pipetted into 4.5 mL of MRS broth medium. After being cultured at 37°C overnight, the culture was diluted 10 times in a gradient manner, and 200 μL of the appropriate gradient dilution solution was spread on MRS solid medium and cultured at 37°C for 48 h. Colonies with small calcium dissolution circles, round, slightly flat or convex, white or yellow were selected for streaking purification three times. 22 lactic acid bacteria were isolated and purified from 15 homemade kimchi water samples, and the strain numbers were LQQ-1-R~LQQ-22-R. Single colonies of the purified strains were picked and inoculated into 10 mL of MRS broth medium, cultured at 37°C overnight, and the culture was mixed with equal volumes of 60% (v / v) sterile glycerol and stored at -20°C for later use.

[0030] Rescreening of lactic acid bacteria for acid production

[0031] ① Determination of acid production capacity in MRS medium

[0032] Pick the above-preserved glycerol, streak it on MRS solid medium, and culture it at 37℃ for 48 hours. Pick a single colony and inoculate it in 10 mL MRS medium. Culture it at 37℃ for 12 hours as seed liquid. Inoculate the seed liquid into 50 mL MRS medium at a 3% inoculation rate, and test the acidity and pH value after culturing it at a constant temperature of 37℃ for 24 hours. Take samples for each strain of bacteria 3 times for testing, and take the average value of the results. pH value detection: Use a pH meter to directly measure the reading. After turning on the acidometer and preheating it for 30 minutes, calibrate it with standard buffers of pH 6.86 and pH 4.00. Total acid detection: Determine it according to the method in GB 12456-2021 "National Food Safety Standard Determination of Total Acid in Food".

[0033] The experimental results are shown in Table 1. The pH values ​​of LQQ-4-R~LQQ-22-R are all less than 3.9, and the total acid content is higher than 1.20 g / L. In contrast, the acid production capacity of LQQ-1-R~LQQ-3-R is weaker and the difference is significant, so LQQ-1-R~LQQ-3-R were selected for subsequent determination of acid production capacity in vegetable juice medium.

[0034] Table 1 Acid production results of strains in MRS medium Strain number pH Total acid (g / L) Strain number pH Total acid (g / L) LQQ-1-R 3.94 <![CDATA[1.02±0.04 a ]]> LQQ-12-R 3.70 <![CDATA[1.32±0.01 ef ]]> LQQ-2-R 4.65 <![CDATA[0.33±0.02 b ]]> LQQ-13-R 3.64 <![CDATA[1.32±0.01 f ]]> LQQ-3-R 4.23 <![CDATA[0.79±0 c ]]> LQQ-14-R 3.61 <![CDATA[1.34±0.02 f ]]> LQQ-4-R 3.82 <![CDATA[1.22±0 d ]]> LQQ-15-R 3.76 <![CDATA[1.32±0 fg ]]> LQQ-5-R 3.71 <![CDATA[1.22±0.01 d ]]> LQQ-16-R 3.64 <![CDATA[1.32±0 fg ]]> LQQ-6-R 3.74 <![CDATA[1.21±0.01 d ]]> LQQ-17-R 3.70 <![CDATA[1.31±0.02 fg ]]> LQQ-7-R 3.76 <![CDATA[1.24±0.02 d ]]> LQQ-18-R 3.66 <![CDATA[1.32±0.04 fgh ]]> LQQ-8-R 3.77 <![CDATA[1.24±0.03 d ]]> LQQ-19-R 3.62 <![CDATA[1.37±0.01 fghj ]]> LQQ-9-R 3.77 <![CDATA[1.24±0.01 d ]]> LQQ-20-R 3.68 <![CDATA[1.36±0.01 fghj ]]> LQQ-10-R 3.80 <![CDATA[1.24±0.03 de ]]> LQQ-21-R 3.64 <![CDATA[1.39±0.04 ghj ]]> LQQ-11-R 3.77 <![CDATA[1.27±0.01 de ]]> LQQ-22-R 3.68 <![CDATA[1.39±0.01 hj ]]>

[0035] ② Determination of acid production capacity in vegetable juice culture medium

[0036] According to the results of the acid production ability test in the above MRS medium, several strains of lactic acid bacteria with weak acid production were selected and inoculated into the vegetable juice medium at an inoculation volume of 10 7 CFU / mL, fermented at 25 ℃ for 7 days, measured pH and acidity, and further screened. The pH and acidity determination methods are the same as above.

[0037] The experimental results are shown in Table 2. The total acid content of the vegetable juice medium inoculated and fermented with LQQ-1-R was significantly lower than that inoculated and fermented with LQQ-2-R and LQQ-3-R, and there was a significant difference in acid production capacity between LQQ-1-R and LQQ-2-R, LQQ-3-R.

[0038] Table 2 Acid production results of strains in vegetable juice medium Strain number pH Total acid LQQ-1-R 3.67 <![CDATA[0.58±0 a ]]> LQQ-2-R 3.37 <![CDATA[1.05±0.01 b ]]> LQQ-3-R 3.39 <![CDATA[1.04±0.01 c ]]>

[0039] From the above results, it can be seen that the acid production ability of LQQ-1-R was weak after 7 days of vegetable juice culture. Based on the detection indicators of primary screening and rescreening - acid production ability, strain LQQ-1-R was selected as the subsequent functional bacteria for identification.

[0040] 2. Identification of strains

[0041] ① Morphological identification:

[0042] Streak the screened strains on MRS medium, culture at 37°C for 48 h, observe and record the colony morphology. Add 1 drop of sterile saline in the center of the slide, then use a sterile inoculation loop to pick up a ring of lactic acid bacteria colonies and mix them evenly with the saline to disperse them into a thin, cloudy layer. Take another clean coverslip, put one side in contact with the bacterial solution, and slowly cover the bacterial solution at a 45° angle (to avoid bubbles that affect observation). Observe with a low-power microscope first, and then use a high-power microscope to observe the shape and size of the lactic acid bacteria. The colony morphology is as follows: Figure 1 As shown in the figure, the colonies are round, white, opaque, convex, smooth on the surface, and have neat edges. The shape and size of lactic acid bacteria were observed under a high-power microscope. Figure 2 shown.

[0043] ②Molecular identification:

[0044] The DNA of excellent lactic acid bacteria was extracted using a bacterial genomic DNA extraction kit, and the DNA was used as a template to perform polymerase chain reaction (PCR) amplification using the universal primers 27F (5'-AACTGAGTTTGATCCTGGCTC-3') and 1492R (5'-TACGGTTACCTTGTTACGACTT-3') of 16SrDNA. The products obtained by PCR amplification were sent to Sangon Biotech (Chengdu) Co., Ltd. for sequencing.

[0045] The 16S rDNA gene sequence obtained after sequencing was submitted to the GenBank database of the National Center for Biotechnology Information (NCBI) of the United States. The basic local alignment search tool (BLAST) was used for homology search and comparison. The 16S rDNA gene sequence of the model strain with higher homology was selected, and the neighbor joining (NJ) method in MEGA7.0 software was used to construct a phylogenetic tree. The results are shown in Figure 2. Figure 3 As shown, strain LQQ-1-R and Pediococcus pentosaceus The homology of Pediococcus pentosaceus reached 99.86%. Combined with the morphological characteristics and physiological and biochemical identification results, strain LQQ-1-R was identified as Pediococcus pentosaceus. Pediococcus pentosaceus The strain LQQ-1-R was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on May 14, 2024. The deposit location is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30627.

[0046] The beneficial effects of the strain LQQ-1-R of the present invention are demonstrated by experimental examples below.

[0047] Experimental Example 1 Laboratory simulated kimchi fermentation of strain LQQ-1-R.

[0048] Experimental group: Assorted kimchi was made using white radish, carrots, and green bamboo shoots as raw materials. Fresh vegetables were washed, peeled, cut, dried, and put into a glass kimchi jar. An equal amount of drinking water was added. After balance, 4% salt was added and 5% spice juice was added (5% dried chili, 0.5% pepper, 2% ginger, 1% garlic, 0.5% star anise, 0.2% fennel were added. The above spices were crushed and sealed in a water bath at 75-85℃ for 3h. After cooling, 5% liquor was added. Lactic acid bacteria (Pediococcus pentosaceus with a preservation number of CGMCC No.30627) were inoculated at the same time. Pediococcus pentosaceus ) to make the concentration of bacterial suspension in kimchi reach 10 7 CFU / mL. Control group: The same conditions as the inoculated kimchi fermentation except that no inoculation was performed. Two parallel fermentations were performed for each fermentation method, and the fermentation was performed at a constant temperature of 25°C. On the 4th day of fermentation, samples were taken to detect volatile flavor substances and conduct sensory evaluation.

[0049] (1) Determination of volatile flavor substances

[0050] Extraction of volatile flavor substances: 4 mL of fermentation liquid was transferred into a 15 mL gas phase bottle, and 1 g of NaCl was added at the same time. After preheating at 43 °C for 30 min, the extraction head was inserted into the headspace bottle and extracted at 43 °C for 30 min.

[0051] GC-MS determination:

[0052] ① Gas chromatography conditions: HP-5MS capillary column (30 m×250 μm×0.25 μm); helium as carrier gas at the inlet, operated in splitless mode at a flow rate of 1 mL / min; auxiliary heater temperature of 280 °C; column initial temperature of 35 °C, maintained for 3 min, increased to 160 °C at 6 °C / min, and increased to 250 °C at 10 °C / min; inlet temperature: 230 °C.

[0053] ②Mass spectrometry conditions: The mass spectrometer was operated at 70 eV in the scanning range of 50 u to 550 u; the quadrupole was operated in electron ionization mode at 150 °C.

[0054] A total of 66 volatile substances were detected in the experimental group, with a total concentration of 0.471505 mg / mL, including 12 alcohols, 26 alkenes, 10 alkanes, 5 aldehydes, 5 aromatic compounds, 3 ketones, 2 ethers, 1 sulfide, 1 ester and 1 other substance. Alcohols were the most abundant substance in the kimchi of the experimental group, followed by esters and alkenes. The sum of the contents of alcohols, esters and alkenes accounted for 50.77%, 21.21% and 16.97% of the total, respectively. Among them, alcohol substances mainly come from the raw materials themselves or are obtained through microbial activities. Alcohol substances are mainly ethanol, linalool and 2-menthol. Ethanol is mainly produced by the conversion of sugars by heterolactate bacteria through the pentose phosphate pathway. Its content accounts for a large proportion of the flavor substances in kimchi, providing the kimchi with a wine aroma. Linalool has a strong, sweet, lily of the valley and citrus aroma, and is an important aroma substance in fermented radish

[20] . In addition to the camphor smell, 2-terpenoids also have a strong pine aroma. Aldehydes are provided by raw materials on the one hand, and on the other hand, they come from reactions such as fatty acid oxidation and amino acid degradation. The aroma of aldehydes can act on the senses to make the aroma more obvious. Among them, nonanal has rose, citrus, and fat aromas. Among olefins, α-curcumene (lemon aroma), α-gingerene (sandalwood aroma), watercressene (black pepper and holly aroma), and camphene (camphor aroma) are mainly derived from ginger. Ketones have floral and minty aromas, among which 2-terpenoids have the highest content.

[0055] A total of 57 volatile substances were detected in the control group with a total concentration of 0.45788309 mg / mL, including 12 alcohols, 22 alkenes, 7 alkanes, 3 aldehydes, 4 aromatic compounds, 2 ketones, 3 ethers, 2 sulfides, 3 esters and 1 other substance.

[0056] The types of volatile substances in the experimental group were higher than those in the control group, among which 30 volatile flavor substances, including 3-methyl-1-butanol, dextrorotatory terpenoids, 2-camphenol, 2,4,6-trimethylbenzaldehyde, (-)-caryophyllene, isocaryophyllene, trans-α-bisabolene, (+)-cyclophane, β-sesquiphellandrene, and (-)-isocaryophyllene, were unique to the control group. The comparison results of the types of volatile flavor substances in the experimental group and the control group are as follows Figure 4 shown.

[0057] (3) Sensory evaluation

[0058] On the 4th day of fermentation, sensory evaluation was performed on the experimental group and the control group. Evaluation was performed according to the following criteria: According to the sensory scoring criteria, the color and shape, aroma, texture, and taste of kimchi were scored (overall score = color and shape × 20% + aroma × 20% + texture × 30% + taste × 30%). The sensory scoring criteria are shown in Table 3.

[0059] Table 3 Sensory scoring criteria for kimchi

[0060] The sensory evaluation results are shown in Table 4

[0061] Table 4 Sensory quality of kimchi

[0062] ① In terms of color and morphology, the pickle juice of the experimental group was clear, free of impurities and foreign matter, and the pickles without mold and floating film were fresh and shiny, while the pickle juice of the control group was relatively clear and the pickles were dark yellow in color;

[0063] ② In terms of aroma, the experimental group and the control group have fermentation aroma and compound aroma after the addition of auxiliary materials (such as sauce aroma, ester aroma, etc.), without bad smell or other odors;

[0064] ③ In terms of texture, the experimental group had fine texture, crisp texture, and pleasant taste, while the control group had fine texture, average crispness, and was softer than the experimental group;

[0065] ④ In terms of taste, the kimchi in the experimental group was delicious, refreshing and pleasant, with a moderate sour taste, while the sour taste in the control group was stronger than that in the experimental group.

[0066] The experimental results show that the pickles inoculated with Pediococcus pentosaceus of the present invention have weak acid production ability and high aroma production ability compared with natural fermentation, and have good sensory quality. The Pediococcus pentosaceus of the present invention is a Pediococcus pentosaceus with a preservation number of CGMCC No. 30627 preserved by the General Microbiological Center of the China Microbiological Culture Collection Administration Committee ( Pediococcus pentosaceus The Pediococcus pentosaceus LQQ-1-R of the present invention has good application prospects in shallow fermented kimchi.

[0067] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A lactic acid bacterium, characterized in that: It is a Pediococcus pentosaceus (CGMCC No. 30627) preserved by the General Microbiological Center of the China Microbiological Culture Collection Administration. Pediococcus pentosaceus )LQQ-1-R.

2. The Pediococcus pentosaceus LQQ-1-R according to claim 1, which can be used for shallow fermentation of kimchi, is characterized by: Inoculate the fresh vegetables with the Pediococcus pentosaceus LQQ-1-R bacterial agent, mix well, and ferment to obtain fresh, fragrant, crispy, tender and refreshing kimchi.

3. The method for preparing the bacterial agent according to claim 2 comprises: The Pediococcus pentosaceus according to claim 1 ( Pediococcus pentosaceus ) LQQ-1-R was cultured in MRS medium at 37°C for 18 h to obtain bacterial cells, which were centrifuged and added with 15% (m / v) sterilized skim milk to obtain a bacterial suspension. The bacterial suspension was freeze-dried to obtain a bacterial agent.

4. The method according to claim 2, characterized in that: Pediococcus pentosaceus should be treated according to 10 5 -10 7 CFU / g were inoculated into kimchi.

5. The method according to any one of claims 2 to 4, characterized in that: The lactic acid bacteria suspension is prepared according to the following method: a certain amount of lactic acid bacteria agent is taken and activated with 10-20 mL of sterile glucose water with a mass concentration of 2-5% at 25-30° C. for 20-30 minutes.

6. The method according to claim 2, characterized in that: The kimchi is made of fresh white radish, carrots, and green bamboo shoots, which are washed, peeled, cut, and an equal amount of drinking water is added. After balance, 4-6% salt is added and 3-5% spice juice is added. The spice juice (in terms of water) is prepared as follows: 3-5% dried chili peppers, 0.3-0.5% prickly ash, 1-3% ginger, 1-3% garlic, 0.3-0.5% star anise, and 0.1-0.3% fennel. The above spices are crushed and sealed in a water bath at 75-85°C for 2-3 hours. After cooling, 3-5% white wine is added. The prepared bacterial agent is added according to 10 5 -10 7 CFU / g was added to kimchi.

7. The method according to claim 6, characterized in that: The fermentation conditions are constant temperature fermentation at 20-30℃ for 3-5 days.

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