Lactobacillus buchneri for fermentation of red vinasse pickled cowpeas and application of lactobacillus buchneri for fermentation of red vinasse pickled cowpeas

Abstract

The invention discloses lactobacillus buchneri for fermentation of red vinasse acid beans and application of the lactobacillus buchneri, and belongs to the field of microorganisms and food processing. According to the lactobacillus buchneri 36, the lactobacillus buchneri 36 with high acetic acid yield is screened from naturally-fermented red vinasse acid vigna unguiculata, fermentation characteristics of the lactobacillus buchneri 36 are detected, and it is found that the lactobacillus buchneri 36 is good in growth characteristic and high in acid resistance and salt resistance, the nitrite degradation rate reaches 100%, and the acetic acid yield reaches 23538 mg / L within 24 hours; when the lactobacillus buchneri 36 provided by the invention is used for fermenting and producing the red vinasse pickled beans, acetic acid and various organic acids can be efficiently produced, and a foundation is laid for the application of the lactobacillus buchneri 36 in the fermented pickles in the future. The method is of great significance in standardizing the production process of the red vinasse acid, improving the product quality and promoting the development of the Wuxuan red vinasse acid industry in Guangxi province.

Description

Technical Field

[0001] The invention relates to the field of microorganisms and food processing, and in particular to a lactobacillus lentus used for fermentation of red glutinous rice and sour beans and an application thereof. Background Art

[0002] Lentilactobacillus buchneri belongs to the kingdom of bacteria, the phylum Firmicutes, the class Bacilli, and the genus Lentilactobacillus. Lentilactobacillus buchneri is known for its strong antibacterial ability and can produce isomerase to isomerize glucose into fructose, which can be used to make mixed syrups to increase sweetness. Currently, most of the reports on Lentilactobacillus buchneri are about the fermentation of silage by Lentilactobacillus buchneri, while there are few reports on the probiotic properties and fermentation properties of Lentilactobacillus buchneri.

[0003] Red yeast sour is a traditional and special intangible cultural heritage food in Wuxuan, Guangxi. Its production method is to put red yeast rice, various vegetables, garlic, and salt into a pickling container in layers, and then fill it with rice wine and seal it for fermentation. The unique flavor of red yeast sour comes from the joint action of multiple complex microorganisms. During the fermentation process of red yeast sour beans, Lactobacillus lentus can produce a large amount of acetic acid to increase the content of organic acids. According to the traditional pickling method in the past, high-concentration white vinegar can be added to shorten the pickling time. Lactobacillus lentus can produce 2500mg / L acetic acid in two days of fermentation, which is equivalent to adding half a bottle of commercial white vinegar. However, the production of red yeast sour is currently concentrated in small workshops, and the production process is not standardized, resulting in unstable product quality and susceptibility to contamination, and urgent technical improvement is needed. Summary of the invention

[0004] The purpose of the present invention is to provide a Lactobacillus lentus of buchneri for fermenting red glutinous rice sour beans and an application thereof, so as to solve the problems existing in the above-mentioned prior art. The Lactobacillus lentus of buchneri 36 provided by the present invention is acid and salt resistant and can also degrade sodium nitrite with a degradation rate of up to 100%. Moreover, when fermenting and producing red glutinous rice sour beans, the Lactobacillus lentus of buchneri can quickly produce a variety of organic acids such as acetic acid, with high production efficiency and good product flavor.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a strain of Lactobacillus buchneri 36, which has been deposited in the General Microbiology Center of China National Committee for the Preservation of Microorganisms, with a deposit number of CGMCC NO.32919, a deposit time of December 6, 2024, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention also provides an application of the Lactobacillus lentus 36 in preparing a bacterial agent for fermenting red glutinous rice and sour beans.

[0008] The present invention also provides a bacterial agent, comprising the Lactobacillus lentus 36.

[0009] Preferably, in the bacterial agent, the live bacteria concentration of the Lactobacillus lentus 36 is not less than 10 7 CFU / mL.

[0010] The present invention also provides an application of the Lactobacillus lentus 36 or the bacterial agent in the fermentation production of red glutinous rice sour beans.

[0011] The present invention also provides a method for producing red glutinous rice sour beans by fermentation, comprising the step of inoculating the lactobacillus lentus 36 or the bacterial agent into cowpeas for fermentation.

[0012] Preferably, the method comprises the following steps:

[0013] The Lactobacillus lentus 36 was expanded and cultured, centrifuged, and resuspended to a viable bacterial concentration of 10 7 CFU / mL, bacterial solution was obtained;

[0014] The bacterial liquid is mixed with rice wine, added into red yeast rice, and stirred thoroughly to obtain modified red yeast rice;

[0015] Wash the cowpeas, expose them to the sun, and tie them into knots after drying to obtain dry beans;

[0016] Put the modified red yeast rice, dried beans and garlic into the jar in layers in order, add rice wine to the jar mouth, and seal the jar mouth with the modified red yeast rice;

[0017] Keep sealed and ferment at 30℃ for 15 days.

[0018] Preferably, in the step of preparing the modified red yeast rice, the volume ratio of the bacterial liquid to the rice wine is 3:200; the volume mass ratio of the bacterial liquid to the red yeast rice is 3 mL:40 g.

[0019] The invention also provides red lees sour beans produced according to the method.

[0020] The present invention also provides an application of the Lactobacillus lentus 36 or the bacterial agent in degrading nitrite.

[0021] The present invention discloses the following technical effects:

[0022] The present invention screens a strain of Lactobacillus buchneri 36 with high acetic acid production from naturally fermented red glutinous rice sour beans, detects its fermentation characteristics, and finds that the strain has good growth characteristics, strong acid resistance and salt resistance, a degradation rate of nitrite of 100%, and a 24-hour acetic acid production of 23538 mg / L; the Lactobacillus buchneri 36 provided by the present invention is used for fermentation and production of red glutinous rice sour beans, which can efficiently produce acetic acid and a variety of organic acids, and lays a foundation for the application of Lactobacillus buchneri in fermented kimchi in the future. The present invention is of great significance for standardizing the production process of red glutinous rice acid, improving product quality, and promoting the development of the red glutinous rice acid industry in Wuxuan, Guangxi. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The HPLC chromatograms of 11 organic acid standards are shown in Figure 1. The chromatographic peaks of the 11 organic acids are, from left to right, 1. oxalic acid, 2. tartaric acid, 3. quinic acid, 4. formic acid, 5. malic acid, 6. ascorbic acid, 7. lactic acid, 8. acetic acid, 9. citric acid, 10. fumaric acid, and 11. succinic acid.

[0025] Figure 2 The figure is a high performance liquid chromatogram of the fermentation broth of Lactobacillus lentus 36 culture medium; wherein, the chromatographic peaks of organic acids are 5. quinic acid, 6. ascorbic acid, 7. lactic acid, 8. acetic acid, 11. citric acid from left to right, and the remaining chromatographic peaks are impurity peaks generated by non-organic acid products;

[0026] Figure 3 This is a bar chart showing the acetic acid production of 14 lactic acid bacteria;

[0027] Figure 4 is a growth curve of Lactobacillus lentus 36;

[0028] Figure 5 is the acid production curve of Lactobacillus lentus 36;

[0029] Figure 6 This is the standard curve diagram for the determination of nitrite content;

[0030] Figure 7 This is a bar graph for testing the sodium chloride resistance of Lactobacillus lentus 36;

[0031] Figure 8This is a curve diagram for detecting the acid resistance of lactic acid bacteria Lactobacillus lentus 36;

[0032] Fig. 9 This is the acid production curve of red glutinous rice acid fermented by Lactobacillus lentus 36;

[0033] Fig.10 This is the HPLC chromatogram of organic acids of red glutinous rice acid fermented by Lactobacillus lentus 36 for 15 days; Note: The chromatographic peaks of organic acids are 5. oxalic acid, 6. quinic acid, 7. ascorbic acid, 8. lactic acid, 9. acetic acid from left to right, and the remaining chromatographic peaks are impurity peaks produced by non-organic acid products;

[0034] Fig.11 This is the appearance of the colony of Lactobacillus lentus 36. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Example 1 Isolation, purification and identification of strains

[0041] 1. Isolation and purification of strains

[0042] (1) Collection of red glutinous rice sour beans samples: In July 2023, red glutinous rice sour beans samples were collected from farmers in Wuxuan County, Laibin City, Guangxi Zhuang Autonomous Region.

[0043] (2) Isolation of lactic acid bacteria strains: Use a pipette to draw 100 μL of red glutinous rice and sour beans fermentation liquid, and use sterile saline to perform gradient dilution. Select the bacterial liquid with the appropriate dilution gradient, evenly spread it on MRS medium containing 0.5% calcium carbonate, and culture it at 37°C for 1-2 days. Select a single colony that forms a transparent circle, transfer it to a new MRS medium with an inoculation loop, and purify it by streaking. Repeat the culture three times, each time at 37°C for 1-2 days, until a single colony with consistent morphology is obtained (such as Fig.11 shown).

[0044] The isolated strains were preliminarily screened by catalase test and Gram staining, and the screened lactic acid bacteria were numbered and preserved.

[0045] 2. Strain Identification

[0046] (1) Extraction of strain genome: Use an inoculation loop to pick a single colony from the plate and add 200 μL of 2% chelex100 to a 1.5 mL centrifuge tube, seal with sealing film, boil in a water bath for 30 min, 12000 r / min, and centrifuge for 10 min. The 16S rRNA gene of the strain was amplified by PCR (Table 1) and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing analysis. The primers used to amplify the 16S rRNA gene are as follows:

[0047] (2) 16S rDNA sequence primers are: 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO. 1), 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO. 2).

[0048] Table 1 Addition amount of each component in PCR amplification system

[0049] Reaction components Dose / μL Tap 25μL <![CDATA[ddH2O]]> 22μL 27F 1μL 1492R 1μL Strain DNA 1μL

[0050] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 4 min; denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 2 min, 32 cycles; and extension at 72°C for 10 min.

[0051] (3) Strain identification: By using the BLAST function of NCBI and performing sequence comparison in the GenBank database, it was found that the strain obtained in the present invention had the highest homology with Lentilactobacillus buchneri, which was 100%. Thus, this strain was determined to be Lentilactobacillus buchneri and was numbered 36.

[0052] (4) Strain preservation: Lentilactobacillus buchneri 36 has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration with the deposit number CGMCC NO.32919. The deposit date is December 6, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0053] Example 2 Screening of high-yielding acetic acid lactic acid bacteria

[0054] (1) Strain collection: The strains were collected from farmer samples in Wuxuan County, Laibin City, Guangxi Zhuang Autonomous Region in July 2023. The isolation details are shown in Table 2.

[0055] Table 2 Information of tested strains

[0056]

[0057] (2) Preparation of acetic acid standard curve: Weigh 0.1 g of acetic acid and prepare 1000 mg / L acetic acid mother solution in a 100 mL volumetric flask. Then dilute it into 75 mg / L, 100 mg / L, 125 mg / L, 150 mg / L, and 200 mg / L standard solutions in sequence, and use Shimadzu high performance liquid chromatograph to draw the standard curve. Prepare standard samples of oxalic acid, tartaric acid, quinic acid, formic acid, malic acid, ascorbic acid, lactic acid, citric acid, fumaric acid, and succinic acid in the same way.

[0058] (3) HPLC conditions (Table 3): Chromatographic column, OAA organic acid chromatographic column. Mobile phase A: 0.01 mol / L potassium dihydrogen phosphate solution. Mobile phase B: methanol.

[0059] Table 3 Lactic acid bacteria organic acid elution procedure

[0060]

[0061] (4) Sample processing: 1 mL of fermentation liquid was taken, diluted 20 times with ultrapure water, and then filtered through a 0.22 μm filter membrane for determination. Figure 3 The acetic acid production of each test strain was rounded.

[0062] (5) The results of acetic acid production are as follows Figure 3, Lactobacillus lentus 36 has the strongest acetic acid production capacity, which can reach 23538 mg / L.

[0063] The HPLC results of the above 11 organic acid standards are as follows: Figure 1 And as shown in Table 4.

[0064] Table 4 HPLC results of 11 organic acid standards

[0065]

[0066] According to the chromatographic results ( Figure 2 and Table 5), it can be seen that in addition to high production of acetic acid, Lactobacillus lentus 36 can also produce organic acids such as ascorbic acid, lactic acid, quinic acid, and citric acid.

[0067] Table 5 HPLC results of fermentation broth of Lactobacillus lentus 36 culture medium

[0068]

[0069] Example 3 Determination of fermentation characteristics of Lactobacillus lentus 36

[0070] (1) Growth curve determination of Lactobacillus lentus 36

[0071] The strain was inoculated into MRS broth medium for activation twice, inoculated into MRS broth at an inoculum of 2% (v / v), cultured at 37°C, and the OD was measured every 1 h using a microplate reader. 600nm The absorbance was measured and the growth curve was drawn. Figure 4 As shown, Lactobacillus lentus 36 entered the logarithmic phase after 8 h of culture and reached the stable phase after 15 h.

[0072] (2) Determination of acid production curve of Lactobacillus lentus 36

[0073] The strain was inoculated into MRS broth medium for activation twice, and then inoculated into MRS broth at an inoculum of 2% (v / v) and cultured at 37°C for 24 h. During this period, the pH value of the fermentation broth was measured every 2 h using a pH meter, and the acid production curve was drawn. Each treatment was calculated based on the average result of 3 parallel samples. The results are shown in Figure 5 As shown, Lactobacillus lentus 36 reached the lowest pH value at 10 h.

[0074] (3) Determination of nitrite degradation rate of Lactobacillus lentus 36

[0075] Take 1mL of 1000mg / L nitrite stock solution and add it to a 100mL volumetric flask to dilute it to 100mg / L stock solution, and then dilute it to 0.5mg / L, 1.0mg / L, 1.5mg / L, 2.0mg / L, and 2.5mg / L standard solutions. Draw a standard curve after determination using an ion chromatograph. The standard curve is as follows: Figure 6 shown.

[0076] Ion chromatography conditions: Chromatographic column, Shenghan SH-AP-1 anion chromatography column. Eluent: 15mmol potassium hydroxide. Elution time: 30min, flow rate: 0.7mL / min.

[0077] The strain was inoculated into MRS broth medium for activation twice, and then inoculated into MRS broth containing 125 mg / L sodium nitrite at an inoculum size of 2% (v / v). After culturing at 37°C for 24 h, the nitrite content was determined using an ion chromatograph.

[0078] Sample treatment: Pipette 1 mL of fermentation broth, dilute 100 times with ultrapure water, treat with RP pretreatment column, discard the first 3 mL of filtrate, collect the filtrate and filter through 0.22 μm filter membrane for standby use.

[0079] Determination of nitrite content in samples: Take 10 mL of the filtered filtrate, transfer it to a 15 mL centrifuge tube, and place it in an automatic injection tower for determination. Ion chromatography conditions: Chromatographic column, Shenghan SH-AP-1 anion chromatography column. Eluent: 15 mmol potassium hydroxide. Elution time: 30 min, flow rate: 0.7 mL / min.

[0080] Nitrite degradation rate (%) = (nitrite content before culture - nitrite content after culture) / nitrite content before culture.

[0081] The results showed that Lactobacillus lentus 36 could completely degrade sodium nitrite in the culture medium within 24 hours, with a degradation rate of 100%.

[0082] (4) Sodium chloride tolerance test of Lactobacillus lentus 36

[0083] The strain was inoculated into MRS broth medium for activation twice, and then inoculated into MRS broth containing 0%, 1%, 3%, 5%, 7%, and 9% sodium chloride at an inoculum of 2% (v / v). The culture was carried out at 37°C for 24 h, and the OD was measured using an enzyme reader. 600nm The absorbance is measured and a bar graph is drawn. Figure 7 As shown, Lactobacillus lentus 36 can grow well at a sodium chloride concentration of 5%, its growth begins to be inhibited at 7%, and its growth is completely inhibited at 11%.

[0084] (5) Acid resistance test of Lactobacillus lentus 36

[0085] The strain was inoculated into MRS broth medium for activation twice, and then inoculated into MRS broth containing 0%, 1%, 3%, 5%, 7%, and 9% sodium chloride at an inoculum of 2% (v / v), cultured at 37°C for 24 h, and the OD was measured using an enzyme reader. 600nm Absorbance and draw a bar graph. Figure 8 As shown, the results showed that Lactobacillus lentus 36 could grow normally at a sodium chloride concentration of 5%, the growth began to be inhibited at a concentration of 7%, and the growth was completely inhibited at a concentration of 11%.

[0086] Example 4 Application of Lactobacillus lentus 36 in fermentation of red glutinous rice and sour beans

[0087] (1) Artificial inoculation and fermentation to prepare red glutinous rice sour beans:

[0088] Lactobacillus lentus 36 was inoculated into MRS liquid medium and cultured in a 37°C incubator for 12 h. After centrifugation, the bacterial solution was prepared into 10 7 The concentration of CFU / mL.

[0089] Wash the fresh beans, remove the old parts, and expose them to the sun for 12 hours.

[0090] Tie the dried beans into a knot and set aside.

[0091] After mixing the bacterial liquid with rice wine, add it into the red yeast rice, stir well and set aside.

[0092] Place red yeast rice, sour beans and garlic into the jar in layers until it reaches the top of the jar, then add rice wine until it reaches the top of the jar, and finally seal the jar with red yeast rice.

[0093] Seal the jar and keep it at about 30℃ for fermentation for about 15 days.

[0094] (2) Changes in pH of red glutinous rice sour beans fermented by Lactobacillus lentus 36

[0095] A pH meter was used to measure the pH value of the fermented liquor of red glutinous rice and sour beans at 1, 2, 4, 7, 15 and 30 days of fermentation, and the acid production curve was drawn. The average value of 3 parallel samples was taken for each treatment result. Fig. 9 As shown, the results showed that the pH of the system decreased rapidly after the strain was introduced, reaching the lowest pH value within 1 day. After 5 days, the pH value of red glutinous rice acid remained basically stable and no longer changed significantly.

[0096] (3) Determination of organic acids in red glutinous rice sour beans fermented with Lactobacillus lentus 36

[0097] According to the method in Example 2, the organic acid content in the red glutinous rice sour beans after fermentation with Lactobacillus lentus 36 for 15 days was determined.

[0098] like Fig.10 As shown in Table 6, the results show that red glutinous rice acid contains a variety of organic acids such as oxalic acid, quinic acid, ascorbic acid, lactic acid and acetic acid, showing a relatively rich variety of organic acids.

[0099] Table 6 High performance liquid chromatography of organic acids in red glutinous rice fermented by Lactobacillus lentus 36 for 15 days

[0100]

[0101] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Lactobacillus buchneri 36, characterized in that: The Lactobacillus lentus 36 has been deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit number CGMCCNO.32919, the deposit time is December 6, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A use of the Lactobacillus lentus 36 as claimed in claim 1 in the preparation of a bacterial agent for fermentation of red glutinous rice and sour beans.

3. A bacterial agent, characterized in that: It includes the Lactobacillus lentus 36 described in claim 1.

4. The bacterial agent according to claim 3, characterized in that In the bacterial agent, the live bacteria concentration of the Lactobacillus lentus 36 is not less than 10 7 CFU / mL.

5. Use of the Lactobacillus lentus 36 of claim 1 or the bacterial agent of claim 3 or 4 in the fermentation production of red glutinous rice sour beans.

6. A method for producing red glutinous rice sour beans by fermentation, characterized in that: The method comprises the steps of inoculating the lactobacillus lentus 36 described in claim 1 or the bacterial agent described in claim 3 or 4 into cowpea for fermentation.

7. The method according to claim 6, characterized in that The following steps are involved: The Lactobacillus lentus 36 was expanded and cultured, centrifuged, and resuspended to a viable bacterial concentration of 10 7 CFU / mL, bacterial solution was obtained; The bacterial liquid is mixed with rice wine, added into red yeast rice, and stirred thoroughly to obtain modified red yeast rice; Wash the cowpeas, expose them to the sun, and tie them into knots after drying to obtain dry beans; Put the modified red yeast rice, dried beans and garlic into the jar in layers in order, add rice wine to the jar mouth, and seal the jar mouth with the modified red yeast rice; Keep sealed and ferment at 30℃ for 15 days.

8. The method according to claim 7, characterized in that In the step of preparing the modified red yeast rice, the volume ratio of the bacterial liquid to the rice wine is 3:200; the volume mass ratio of the bacterial liquid to the red yeast rice is 3mL:40g.

9. A sour beans made from red glutinous rice produced according to the method according to any one of claims 6 to 8.

10. Use of the Lactobacillus lentus 36 of claim 1 or the bacterial agent of claim 3 or 4 in degrading nitrite.

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