Lactobacillus pentosus HM-417, complex microbial inoculant and application of complex microbial inoculant

Abstract

The invention relates to the technical field of microorganisms, and discloses lactobacillus pentosus HM-417, a complex microbial inoculant and application of the lactobacillus pentosus HM-417, the lactobacillus pentosus HM-417 is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 65105. The lactobacillus pentosus HM-417 has been preserved in Guangdong Microbial Culture Collection Center. Experiments prove that the lactobacillus pentosus has the effect of producing GABA at high yield after fermentation, and also has the effect of producing delicious amino acids. And when being used for preparing the hotpot soup base, not only is the content of GABA increased, but also the content of flavor amino acid is increased, the nutritional value is higher, and the taste is more delicious.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus pentosus HM-417, a composite bacterial agent and applications thereof. Background Art

[0002] γ-aminobutyric acid (GABA) is a small molecular weight non-protein amino acid. It is not only an important nutrient, but also functional. It is an important inhibitory neurotransmitter in the central nervous system. It has many physiological functions such as improving sleep quality, promoting metabolism, lowering blood pressure and blood sugar, and is widely used in the food field. At present, the main methods for producing GABA are chemical synthesis, plant enrichment and microbial fermentation. The chemical synthesis method has a violent reaction and low safety. The plant enrichment method has great limitations and low content in the production of GABA, which is not suitable for large-scale production. The microbial fermentation method has the advantages of high yield, low cost and high safety.

[0003] Lactic acid bacteria can produce GABA, which has high safety and production efficiency and is widely used in food production. However, the current lactic acid bacteria GABA production is low. At the same time, food not only needs to meet nutritional needs, but also needs to taste delicious. Umami, as the fifth basic taste, is pleasant and can increase appetite. However, the current lactic acid bacteria fermented products have a bland taste and cannot take into account both nutritional properties and umami requirements. Summary of the invention

[0004] In view of this, the present invention provides a Lactobacillus pentosus HM-417, a composite bacterial agent and applications thereof, to solve the problem that existing fermented products have a bland taste and cannot take into account both nutritional properties and umami taste.

[0005] In a first aspect, the present invention provides a Lactobacillus pentosus (Lactobacillus pentosus) HM-417.

[0006] In an optional embodiment, the Lactobacillus pentosus HM-417 has been deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC No: 65105.

[0007] In an optional embodiment, the nucleotide sequence of 16s rDNA of Lactobacillus pentosus HM-417 is shown in SEQ ID NO:1.

[0008] In a second aspect, the present invention provides a Lactobacillus pentosus ( Lactobacillus pentosus ) Screening method of HM-417.

[0009] In an optional embodiment, the screening method of Lactobacillus pentosus HM-417 comprises: enriching and culturing the broth of the fermented product, and then spreading and culturing it in a culture medium containing calcium carbonate; selecting colonies with calcium-dissolving rings and inoculating them into a fermentation medium for fermentation to obtain a fermentation broth; and screening Lactobacillus pentosus with high γ-aminobutyric acid production according to the γ-aminobutyric acid content in the fermentation broth.

[0010] In a third aspect, the present invention provides a composite bacterial agent.

[0011] In an optional embodiment, the composite bacterial agent includes the Lactobacillus pentosus HM-417 and / or Lactobacillus plantarum producing umami amino acids or their culture, fermentation product or metabolite; Optionally, the umami amino acids include one or a combination of two or more of glycine, alanine, tyrosine, phenylalanine, aspartic acid and glutamic acid.

[0012] In an optional embodiment, the plant lactobacillus producing umami amino acids is ( Lactiplantibacillus plantarum )JCM 1149.

[0013] In an optional embodiment, in the composite bacterial agent, the ratio of the number of live bacteria of Lactobacillus pentosus HM-417 and Lactobacillus plantarum is (1-3): (1-2); Optionally, the composite bacterial agent is composed of a Lactobacillus pentosus HM-417 preparation and a Lactobacillus plantarum preparation, and the volume ratio of the Lactobacillus pentosus preparation to the Lactobacillus plantarum preparation is 3:2; The number of viable Lactobacillus plantarum in the Lactobacillus plantarum preparation is 4×10 8 -5×10 8 CFU / mL, and the number of viable Lactobacillus pentosus in the Lactobacillus pentosus preparation is 4×10 8 -5×10 8 CFU / mL.

[0014] In a fourth aspect, the present invention provides a use of the Lactobacillus pentosus HM-417 or its culture or fermentation product, or the composite bacterial agent in fermented food or in the preparation of fermented food; Optionally, the application is application in fermented food with probiotic function and / or umami taste or in the preparation of fermented food with probiotic function and / or umami taste.

[0015] In a fifth aspect, the present invention provides a hot pot soup base, wherein the raw materials of the hot pot soup base include the composite bacterial agent; Optionally, the ingredients of the hot pot soup also include peppers and tomatoes; Optionally, the mass ratio of pepper to tomato is (40-60):(40-60); And / or, the ratio of the added amount of the composite bacterial agent to the total mass of the pepper and the tomato is (2-8) ml:100 g.

[0016] In an optional embodiment, the raw materials of the hot pot soup also include glutinous rice flour, salt and sugar; Among the raw materials for hot pot soup, the added amount of glutinous rice flour is 8wt%-12wt% of the total mass of the peppers and tomatoes, the added amount of salt is 4wt%-7wt% of the total mass of the peppers and tomatoes, and the added amount of white sugar is 5wt%-8wt% of the total mass of the peppers and tomatoes.

[0017] In an optional embodiment, the composite bacterial agent and other raw materials of hot pot soup are mixed, sealed and fermented to obtain the hot pot soup; Optionally, the temperature of the sealed fermentation is 30°C-37°C, and the time of the sealed fermentation is 5 days-8 days.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a Lactobacillus pentosus HM-417, with a deposit number of GDMCC No: 65105. Experiments have shown that the fermentation product of the Lactobacillus pentosus HM-417 with a deposit number of GDMCC No: 65105 contains a large amount of GABA and has more umami amino acids. When the fermentation product is used to prepare hot pot soup, not only the content of GABA is increased, but also the content of umami amino acids is increased, the nutritional value is higher, and the taste is more delicious.

[0019] The present invention also provides a composite bacterial agent, comprising the Lactobacillus pentosus HM-417 and Lactobacillus plantarum producing umami amino acids. The Lactobacillus plantarum producing umami amino acids has a synergistic effect with the Lactobacillus pentosus HM-417, can further increase the yield of GABA and the yield of umami amino acids, and is used for preparing hot pot soup, which has higher nutritional value and more delicious taste.

[0020] Biological Deposit Lactobacillus pentosus ( Lactobacillus pentosus ) has been deposited in Guangdong Microbiological Culture Collection Center, with the deposit number GDMCC No: 65105. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code 511356, and the deposit date is September 6, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 This is the initial screening result of GABA-producing lactic acid bacteria in Example 1 of the present invention; Figure 2 is the GABA yield of different strains in Example 1 of the present invention; Figure 3 is the HPLC test result of the GABA standard substance in Example 1 of the present invention; Figure 4 is the HPLC test result of strain HM-417 in Example 1 of the present invention; Figure 5 It is the detection result of strain HM-417 in Example 1 of the present invention; the left picture is the colony morphology picture, and the right picture is the microscopic examination result; Figure 6 is the umami amino acid content in the seven fermentation broths of Example 2 of the present invention; Figure 7 It is the plant lactobacillus in Example 2 of the present invention Lactiplantibacillus plantarum Morphological observation results of JCM 1149; the left picture is the colony morphology, and the right picture is the microscopic examination result; Figure 8 is the GABA content in the hot and sour hot pot soup ingredients D1-D8 in Test Example 1 of the present invention; Fig. 9 It is the umami amino acid content in the hot and sour hot pot soup base D1-D8 in Test Example 1 of the present invention. DETAILED DESCRIPTION

[0023] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0024] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings: In the present application, the selection scope involving "and / or" includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, wherein the any and all combinations include any combination of two related listed items, any more related listed items, or all related listed items.

[0025] In the present application, the percentage content, unless otherwise specified, refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0026] In this application, when it comes to percentage concentration, unless otherwise specified, it refers to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0028] Lactobacillus pentosus HM-417 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 6, 2024, with the address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and the deposit number is GDMCC No: 65105.

[0029] Lactic acid bacteria can produce organic acids such as lactic acid, so they can regulate the pH value in the intestine, promote the growth of probiotics and inhibit the reproduction of harmful bacteria. Therefore, proper intake of lactic acid bacteria products helps maintain intestinal health. The production of GABA by lactic acid bacteria has high safety and production efficiency. In the prior art, enterococci are also used to produce GABA, but enterococci can survive in the human intestine together with other types of bacteria, and may have certain pathogenicity.

[0030] In order to solve the problems existing in the above-mentioned related technologies, according to a first aspect of the present invention, the present invention provides a Lactobacillus pentosus, and the preservation number of the Lactobacillus pentosus is GDMCC No:65105.

[0031] In some embodiments, the nucleotide sequence of 16s rDNA of Lactobacillus pentosus is shown as SEQ ID NO:1.

[0032] The lactobacillus pentosus HM-417 of the present invention, which is deposited as GDMCC No: 65105, has the effect of high production of GABA after fermentation, and also has the effect of producing umami amino acids. The GABA content of its fermentation product is as high as 900.3 μg / mL, and the umami amino acid content of its fermentation product is 1261.98 μg / mL; using it to prepare hot pot soup, not only the content of GABA is increased, but also the content of umami amino acids is increased, the nutritional value is higher, and the taste is more delicious. The content of GABA in the prepared hot pot soup is 506.6 μg / mL, and the content of umami amino acids is 789.7 μg / mL.

[0033] In some embodiments, the screening method of Lactobacillus pentosus comprises: enriching and culturing the soup of the fermented product, and then spreading and culturing it in a culture medium containing calcium carbonate; selecting a colony with a calcium-dissolving ring for separation and purification, and inoculating the purified single colony into a fermentation medium for fermentation; and screening the Lactobacillus pentosus that produces γ-aminobutyric acid according to the content of γ-aminobutyric acid in the fermentation broth; Exemplarily, the enrichment culture specifically comprises: sealing and culturing the broth of the fermented product at 28-35° C. for 20-28 hours.

[0034] And / or, the preparation method of the fermented product comprises: mixing pepper, kimchi liquid and kimchi mother water and then sealing and fermenting to form a fermented product; the kimchi liquid is formed by mixing and boiling raw materials including spices, salt, white wine, white sugar and water; the kimchi mother water can be homemade or commercially available.

[0035] In the screening method of Lactobacillus pentosus of the present application, MRS medium can be used to enrich lactic acid bacteria, and TYG fermentation medium can be used for fermentation. TYG fermentation medium can promote the proliferation of lactic acid bacteria and the generation of metabolites.

[0036] Optionally, the fermentation medium is a TYG fermentation medium, and the components of the TYG fermentation medium include: peptone, sodium L-glutamate, yeast extract and sugars; In a second aspect, the present invention provides a composite bacterial agent, which includes Lactobacillus plantarum and the Lactobacillus pentosus that produce umami amino acids; the umami amino acids include one or a combination of two or more of glycine, alanine, tyrosine, phenylalanine, aspartic acid and glutamic acid.

[0037] In order to increase the content of umami amino acids and γ-aminobutyric acid (GABA) in the fermentation broth, this application chooses to use a composite bacterial agent for fermentation, and determines that Lactobacillus plantarum that produces umami amino acids and Lactobacillus pentosus that produces γ-aminobutyric acid have a synergistic effect. The two lactic acid bacteria together increase the production of umami amino acids and GABA, avoiding the introduction of pathogenic bacteria. Adding the above two lactic acid bacteria to hot pot soup can increase the production of GABA and umami amino acids at the same time, improve the taste and nutritional value of the product; especially for hot pot soup containing peppers and tomatoes, it has a better effect of improving the taste and nutritional value of the product.

[0038] In some embodiments, in the composite bacterial agent, the ratio of the number of viable Lactobacillus pentosus and Lactobacillus plantarum is (1-3): (1-2); Optionally, the composite bacterial agent is composed of a Lactobacillus pentosus preparation and a Lactobacillus plantarum preparation, and the volume ratio of the Lactobacillus pentosus preparation to the Lactobacillus plantarum preparation is 3:2; The number of viable Lactobacillus plantarum in the Lactobacillus plantarum preparation is 4×10 8 -5×10 8 CFU / mL, and the number of viable Lactobacillus pentosus in the Lactobacillus pentosus preparation is 4×10 8 -5×10 8 CFU / mL.

[0039] In some embodiments, the Lactobacillus plantarum is Lactiplantibacillus plantarum JCM 1149.

[0040] In a third aspect, the present invention provides a use of the plant lactobacillus or its culture or fermentation product, or the composite bacterial agent in the preparation of fermented food with probiotic function and / or umami taste.

[0041] In a third aspect, the present invention provides a hot pot soup base, wherein the hot pot soup base comprises the composite bacterial agent; in terms of mass percentage, the addition amount of the composite bacterial agent is 2%-4% of the total mass of the hot pot soup base; Optionally, the hot pot soup also includes peppers and tomatoes; the mass ratio of peppers to tomatoes in the hot pot soup is (40-60): (40-60).

[0042] In some embodiments, the hot pot soup base further includes glutinous rice flour, salt and sugar; In the hot pot soup, the added amount of glutinous rice flour is 8wt%-12wt% of the total mass of the peppers and tomatoes, the added amount of salt is 4wt%-7wt% of the total mass of the peppers and tomatoes, and the added amount of white sugar is 5wt%-8wt% of the total mass of the peppers and tomatoes.

[0043] In a fourth aspect, the present invention provides a method for preparing the hot pot soup base, comprising mixing the composite bacterial agent with other raw materials of the hot pot soup base, and sealing and fermenting the mixture to obtain the hot pot soup base; Optionally, the temperature of the sealed fermentation is 30°C-37°C, and the time of the sealed fermentation is 5 days-8 days.

[0044] The present invention is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed by the present invention.

[0045] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0046] Example 1 Screening of GABA-producing lactic acid bacteria HM-417 This embodiment provides a GABA-producing Lactobacillus pentosus, and the screening method is as follows: (1) Production of naturally fermented pickled peppers Preparation of kimchi liquid: Pour 1L of mineral water into an oil-free pot, add spices (including 20-40g garlic, 2-4 slices of ginger, 3-5 star anise, 10-12g pepper) and boil; weigh 30g salt, 10g white wine and 15g white sugar into the pot, continue to cook for 5 minutes, and cool thoroughly to room temperature.

[0047] Wash and dry 100g of fresh peppers, sterilize the kimchi jar with hot water, and put the peppers into the kimchi jar. Add 500ml of kimchi mother water to all the cooled kimchi liquid, mix well and put it into the kimchi jar. Seal and ferment for 7 days to form fermented pickled peppers.

[0048] (2) Isolation and purification of lactic acid bacteria 100 ml of the fermented pickled pepper juice obtained in step (1) was placed in a ziplock bag and sealed at 30°C for 24 h to enrich the bacteria. 1 mL of the enriched sample was inoculated into 100 mL of MRS liquid culture medium (10 g of peptone, 10 g of meat extract, 5 g of yeast extract, K 2 HPO 4 2g, diammonium hydrogen citrate 2g, sodium acetate 5g, glucose 20g, calcium carbonate 20g, soil temperature 80 1ml, MgSO 4 7H 2 O0.58g, MnSO 4 ·H 2 O 0.189g, add distilled water to 1000mL, pH 6.2-6.4), incubate at 30℃ for 24h, and enrich again. Take 1mL of the enriched bacterial solution and add 9ml of physiological saline to dilute to form 10-1 Concentration dilution, gradient dilution to 10 -7 , take 10 -6 and 10 -7 0.5 mL of each dilution was spread on MRS agar medium (10 g peptone, 10 g meat extract, 15 g agar, 5 g yeast extract, K 2 HPO 4 2g, diammonium hydrogen citrate 2g, sodium acetate 5g, glucose 20g, calcium carbonate 20g, soil temperature 801ml, MgSO 4 7H 2 O 0.58g, MnSO 4 ·H 2 O 0.189g, add distilled water to 1000mL, pH 6.2-6.4), culture at 30℃ for 48h, pick lactic acid bacteria colonies with calcium dissolving rings and streak culture for purification, pick out the purified single colonies and save them for later use; (3) Initial screening of GABA-producing lactic acid bacteria The purified single colony was inoculated into 50 mL of TYG fermentation medium (5 g of tryptone, 10 g of sodium L-glutamate, 5 g of yeast extract, 10 g of glucose, distilled water to 1000 mL, pH 6.2-6.6) for fermentation, and cultured at 30°C for 72 h. The GABA in the fermentation broth was qualitatively detected by thin layer chromatography; the test results were as follows: Figure 1 As shown; 4 kinds of lactic acid bacteria were screened, Figure 1 The 3rd track (named HHJ-417), the 6th track (named WC-417), the 7th track (named HM-417) and the 8th track (named BM-417) are shown in the figure.

[0049] (4) Rescreening of GABA-producing lactic acid bacteria The single strains (HHJ-417, WC-417, HM-417 and BM-417) that were initially screened and capable of producing GABA were inoculated into 50 mL of TYG fermentation medium and fermented again. The culture was kept at 37 °C for 72 h, and the GABA content in the fermentation broth was quantitatively detected by HPLC. The HPLC method used o-phthalaldehyde (OPA) pre-column derivatization-ultraviolet detection to compare the five strains. The results are as follows: Figure 2-4 As shown, according to the test results, a lactic acid bacteria strain with a GABA content of 900.3 mg / L in the fermentation broth was screened and identified as Lactobacillus pentosus by molecular biology and named Lactobacillus pentosus HM-417. Figure 2 This is a comparison of GABA production among five strains; Figure 3 This is the HPLC test result of GABA standard; Figure 4This is the HPLC test result of strain HM-417.

[0050] (5) Detection of umami amino acids produced by Lactobacillus pentosus HM-417 The amino acid analyzer was used to detect the content of umami amino acids in the fermentation broth fermented again by Lactobacillus pentosus HM-417 in step (4). The result showed that the content of umami amino acids in the fermentation broth was 1261.98 μg / mL, including 238.64 μg / mL of glutamic acid, 187.51 μg / mL of aspartic acid, 238.82 μg / mL of phenylalanine, 176.12 μg / mL of alanine, 329.28 μg / mL of tyrosine, and 91.61 μg / mL of glycine.

[0051] (6) Morphological characteristics Preliminary identification: The colony morphology of the isolated and purified Lactobacillus pentosus HM-417 was observed under a microscope. Figure 5 , Figure 5 The middle left picture shows the colony morphology of Lactobacillus pentosus HM-417, and the right picture shows the microscopic examination results. The cells are rod-shaped with round ends, most of them are single, and a few are arranged in pairs or chains.

[0052] (7) 16S rDNA sequencing of single strains After testing by the scientific compass test platform, the details are as follows: 7.1 Lysis buffer preparation Prepare bacterial lysis buffer: 1 μL 0.2moL / L NaOH, 2.5 μL 1% SDS.

[0053] 7.2 Genomic DNA extraction Take 5 μL of bacterial lysate in a 1.5 mL centrifuge tube, add a certain amount of the test bacterial precipitate to the centrifuge tube, mix well (be careful to avoid a large number of bubbles), react for 5 min, add 200 μL of dd H 2 O Stop the reaction (mix well).

[0054] 7.3 Genomic PCR Amplification Primer sequences 27F (SEQ ID NO:2): 5'-AGAGTTTGATCCTGGCTCAG-3' 1492R (SEQ ID NO:3): 5'-GGTTACCTTGTTACGACTT-3' The PCR reaction system is shown in Table 1: Table 1 PCR reaction system Components volume <![CDATA[ddH 2 The]]> 10 μL 27F 0.5 μL 1492R 0.5 μL Taq enzyme 12.5μL DNA 1.5 μL Total volume 25 μL PCR reaction conditions are shown in Table 2: Table 2 PCR reaction conditions

[0055] 7.4 Gel electrophoresis conditions U=120 V, A=120 mA, t=20 min.

[0056] 7.5 Gene sequencing The PCR products were sequenced using the Sanger method. One-way primer sequencing was performed first. If there was no overlapping peak in the one-way sequencing, the sequence at the other end was detected. The sequencing results were spliced ​​using DNAMAN software.

[0057] The sequencing result is shown in SEQ ID NO:1.

[0058] SEQ ID NO: 1: Multiple reference strain sequences were obtained from the NCBI database, and the full-length 16SrDNA sequences of the isolated strain and the reference strain were analyzed using the software BioEdit and MEGA11 to construct a phylogenetic tree of the isolated strain and the reference strain. The strain was determined to be of the genus Lactobacillus pentosus and was named Lactobacillus pentosus HM-417.

[0059] Example 2 Screening of lactic acid bacteria producing umami amino acids Lactobacillus plantarum Lactiplantibacillus plantarum Six different single colonies of Lactobacillus plantarum (labeled as WGX, WGH, WGM, SJ-3, SJ-6 and SJ-9) that produced umami amino acids screened from JCM 1149 (labeled as SJ-8) and sour porridge (a kind of porridge made from high-quality glutinous rice or millet as raw materials after fermentation) were inoculated into 50 mL of TYG fermentation medium (5 g of tryptone, 10 g of sodium L-glutamate, 5 g of yeast extract, 10 g of glucose, distilled water to 1000 mL, pH 6.2-6.6), and cultured at 37°C for 72 h to obtain fermentation broth. The umami amino acid content in the fermentation broth was detected by amino acid analyzer. The results are as follows: Figure 6 As shown in Figure 2, the lactic acid bacteria with the highest umami amino acid content are Lactobacillus plantarum. Lactiplantibacillus plantarum JCM 1149, morphological observations are shown in Figure 7 shown.

[0060] Example 3 Composite bacterial agent This embodiment provides a composite bacterial agent, and the preparation method is as follows: The Lactobacillus pentosus HM-417 obtained by screening in Example 1 and the commercially available Lactiplantibacillus plantarum JCM 1149 were inoculated into 100 mL of MRS liquid medium for culture, and cultured at 30°C for 48 h. The cultured bacterial solution was placed in 100 mL of MRS liquid medium for culture again, and cultured at 30°C for 24 h. The two bacterial solutions were centrifuged at 5000 r / min for 15 min. After removing the supernatant, the bacteria were washed three times with sterile saline, and then mixed with sterile saline to obtain bacterial suspensions, and Lactobacillus pentosus preparations and Lactobacillus plantarum preparations were obtained, respectively. The number of viable Lactobacillus pentosus in the Lactobacillus pentosus preparation was 5×10 8 CFU / mL; the obtained Lactobacillus plantarum preparation Lactiplantibacillus plantarum The viable count of JCM 1149 is 5×10 8CFU / mL. The two preparations were then mixed into five composite bacterial agents at a volume ratio of 1:3, 3:1, 1:1, 2:3, and 3:2, each of which was 40 ml; among them, the composite bacterial agent with a volume ratio of 1:3 was marked as J3; the composite bacterial agent with a volume ratio of 3:1 was marked as J4; the composite bacterial agent with a volume ratio of 1:1 was marked as J5; the composite bacterial agent with a volume ratio of 2:3 was marked as J6; and the composite bacterial agent with a volume ratio of 3:2 was marked as J7, and refrigerated at 0-4°C for later use.

[0061] Example 4 Hot Pot Soup This embodiment provides a method for preparing hot pot soup, comprising: (1) Remove the stems of the peppers, wash and dry them, then chop them. Wash and mash the tomatoes into a paste to make tomato sauce. (2) Mix 2400 g of crushed chili pepper and tomato sauce in a mass ratio of 1:1, add 192 g of glutinous rice flour, 96 g of salt and 144 g of sugar; (3) 48 ml of lactic acid bacteria agent (J5 prepared in Example 3) was added to the raw material mixture in (2), and the mixture was sealed and fermented at 30° C. for 8 days to prepare hot and sour hot pot soup.

[0062] Example 5 Hot Pot Soup This embodiment provides a method for preparing hot pot soup, comprising: (1) Remove the stems of the peppers, wash and dry them, then chop them. Wash and mash the tomatoes into a paste to make tomato sauce. (2) Mix 2400 g of crushed chili pepper and tomato sauce in a mass ratio of 40:60, add 288 g of glutinous rice flour, 168 g of salt and 120 g of sugar; (3) 192 ml of lactic acid bacteria agent (J5 prepared in Example 3) was added to the raw material mixture in (2), and the mixture was sealed and fermented at 37° C. for 5 days to prepare hot and sour hot pot soup.

[0063] Example 6 Hot Pot Soup This embodiment provides a method for preparing hot pot soup, comprising: (1) Remove the stems of the peppers, wash and dry them, then chop them. Wash and mash the tomatoes into a paste to make tomato sauce. (2) Mix 2400 g of crushed chili pepper and tomato sauce in a mass ratio of 60:40, add 240 g of glutinous rice flour, 120 g of salt and 192 g of sugar; (3) 240 ml of lactic acid bacteria agent (J5 prepared in Example 3) was added to the raw material mixture in (2), and the mixture was sealed and fermented at 306° C. for 7 days to prepare hot and sour hot pot soup.

[0064] Test Example 1 The plant lactobacillus preparation obtained in Example 3 ( Lactiplantibacillus plantarum JCM 1149 is marked as J1), the Lactobacillus pentosus preparation obtained in Example 3 (HM-417 is marked as J2) and the composite bacterial agents J3-J7 are used to prepare hot and sour hot pot soup, and the preparation method is as follows: (1) Remove the stems of the peppers, wash and dry them, then chop them. Wash and mash the tomatoes into a paste to make tomato sauce. (2) Mix 2400 g of crushed chili pepper and tomato sauce in a mass ratio of 1:1, add 240 g of glutinous rice flour, 96 g of salt and 144 g of sugar, mix evenly and divide into 8 portions, each 345 g, numbered D1-D8; (3) D8 was a blank control group, in which no lactic acid bacteria were added and the mixture was sealed and fermented at 30-37°C for 6 days to produce hot and sour hot pot soup; D1-D7 were inoculated with 12 ml of the above-mentioned lactic acid bacteria agent, respectively, and fermented at 30-37°C for 6 days to obtain hot and sour hot pot soup (see Table 3 for details).

[0065] Table 3 Hot pot soup recipe Hot and Sour Hot Pot Soup Ingredients Compound microbial agent number Lactobacillus pentosus preparation: Lactobacillus plantarum preparation (volume ratio) Addition amount of lactic acid bacteria agent / ml D1 J1 0:4 12 D2 J2 4:0 12 D3 J3 1:3 12 D4 J4 3:1 12 D5 J5 1:1 12 D6 J6 2:3 15 D7 J7 3:2 12 D8 - 0:0 0 Note: HM-417:SJ-8 is the volume ratio of Lactobacillus pentosus preparation to Lactobacillus plantarum preparation in the lactic acid bacteria agent, and 0:0 means that no lactic acid bacteria agent is added as the blank control group.

[0066] The hot and sour hot pot soup ingredients D1-D8 were filtered with gauze to obtain the soup, and the supernatant was taken for detection after centrifugation at 10000r / min for 10min. The GABA content in the fermentation broth was quantitatively detected by high performance liquid chromatography. The high performance liquid chromatography method used o-phthalaldehyde (OPA) pre-column derivatization-ultraviolet detection method, as follows: Derivatization reaction: 400 μL borate buffer, 80 μL OPA derivatizing agent, and 80 μL GABA standard solution and sample solution were measured and mixed evenly. The derivatization reaction lasted for 5 min. The mixture was filtered through a 0.22 μm organic filter membrane and 20 μL was injected for analysis and detection.

[0067] Chromatographic conditions: chromatographic column: C18 (5 μm, 250×4.6 mm); column temperature: 40°C; flow rate: 0.8 mL / min; mobile phase: acetonitrile and 20 mmol / L sodium acetate in a ratio of 21:79.

[0068] Test results such as Figure 8 The amino acid analyzer was used to detect the umami amino acid content in the soup. The results are shown in Table 4 and Fig. 9 shown.

[0069] Table 4 Comparison of umami amino acid content

[0070] from Figure 8 and Fig. 9 It can be seen that the content of GABA and the content of umami amino acids in the hot and sour hot pot soup prepared by using Lactobacillus pentosus or Lactobacillus plantarum alone are both low; the content of GABA and the content of umami amino acids in the hot and sour hot pot soup prepared by using the composite bacterial agent are both higher than those in the hot and sour hot pot soup prepared without using any bacterial agent, and the content of umami amino acids in the hot and sour hot pot soup using the composite bacterial agent is higher than that using Lactobacillus plantarum alone, indicating that the addition of Lactobacillus pentosus HM-417 can promote the production of umami amino acids by Lactobacillus plantarum; at the same time, the content of GABA in the hot and sour hot pot soup using the composite bacterial agent is higher than that using Lactobacillus pentosus alone, indicating that the addition of Lactobacillus plantarum Lactiplantibacillus plantarum JCM 1149 can promote the production of GABA by Lactobacillus pentosus HM-417; in short, the use of composite bacterial liquid has the effect of increasing the production of GABA and umami amino acids, and Lactobacillus pentosus and Lactobacillus plantarum have a synergistic effect; when the volume ratio of Lactobacillus pentosus preparation to Lactobacillus plantarum preparation is 3:2, the production of GABA and umami amino acids are the highest.

[0071] Comparative Example 1 This comparative example provides a composite bacterial agent, and the preparation method is as follows: After activation, the number of viable bacteria reached 5×10 8 CFU / mL of Lactobacillus pentosus 36mL (No. ATCC8041, purchased from Beijing Baozang Biotechnology Co., Ltd.); add 24ml of the Lactobacillus plantarum preparation obtained in Example 3 to obtain 60ml of composite bacterial agent J8; Prepare hot and sour hot pot soup according to the method of Experimental Example 1, as follows: (1) Remove the stems of the peppers, wash and dry them, then chop them. Wash and mash the tomatoes into a paste to make tomato sauce. (2) Mix 300 g of crushed chili pepper and tomato sauce in a mass ratio of 1:1, add 15 g of glutinous rice flour, 12 g of salt and 18 g of sugar, and mix well; (3) The composite bacterial agent J8 was added and the mixture was sealed and fermented at 30-37°C for 6 days to obtain hot and sour hot pot soup base D9.

[0072] The hot and sour hot pot soup D9 of comparative example 1 was filtered through gauze to obtain a soup, and the content of GABA and the content of umami amino acids were tested. The results are as follows: The content of GABA in hot and sour hot pot soup D9 was detected by high performance liquid chromatography, which was 433.93 μg / mL. The content of umami amino acids in hot and sour hot pot soup D9 was detected by amino acid analyzer, which was 981.62 μg / mL.

[0073] The yield of GABA and the yield of umami amino acids in the hot and sour hot pot soup D9 of Comparative Example 1 were lower, but far less than the composite bacterial agent J7 of the present application.

[0074] The above experiments show that the composite bacterial agent of the present application has a good effect of increasing the production of GABA and the production of umami amino acids.

[0075] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A Lactobacillus pentosus (Lactobacillus pentosus) HM-417, characterized in that The Lactobacillus pentosus HM-417 has been deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 65105.

2. The Lactobacillus pentosus HM-417 according to claim 1, characterized in that The nucleotide sequence of 16s rDNA of the Lactobacillus pentosus HM-417 is shown in SEQ ID NO:

1.

3. A method for screening Lactobacillus pentosus HM-417 according to claim 1 or 2, characterized in that: include: After the soup of the fermented product is enriched and cultured, it is spread and cultured in a culture medium containing calcium carbonate; a colony with a calcium-dissolving ring is selected and inoculated into a fermentation culture medium for fermentation to obtain a fermentation liquid; and according to the content of gamma-aminobutyric acid in the fermentation liquid, a lactobacillus pentosus with high gamma-aminobutyric acid production is screened and obtained.

4. A composite bacterial agent, characterized in that: The method comprises the Lactobacillus pentosus HM-417 and / or Lactobacillus plantarum producing umami amino acids according to claim 1 or 2, or a culture, fermentation product or metabolite thereof; Optionally, the umami amino acids include one or a combination of two or more of glycine, alanine, tyrosine, phenylalanine, aspartic acid and glutamic acid.

5. The composite bacterial agent according to claim 4, characterized in that: The plant lactobacillus producing umami amino acids is ( Lactiplantibacillus plantarum )JCM 1149.

6. The composite bacterial agent according to claim 4 or 5, characterized in that: In the composite bacterial agent, the ratio of the number of live bacteria of Lactobacillus pentosus HM-417 and Lactobacillus plantarum was (1-3): (1-2); Optionally, the composite bacterial agent is composed of a Lactobacillus pentosus HM-417 preparation and a Lactobacillus plantarum preparation, and the volume ratio of the Lactobacillus pentosus preparation to the Lactobacillus plantarum preparation is 3:2; The number of viable Lactobacillus plantarum in the Lactobacillus plantarum preparation is 4×10 8 -5×10 8 CFU / mL, and the number of viable Lactobacillus pentosus in the Lactobacillus pentosus preparation is 4×10 8 -5×10 8 CFU / mL.

7. Use of the Lactobacillus pentosus HM-417 or its culture or fermentation product according to any one of claims 1 to 2, or the composite bacterial agent according to any one of claims 4 to 6 in fermented food or in the preparation of fermented food; Optionally, the application is application in fermented food with probiotic function and / or umami taste or in the preparation of fermented food with probiotic function and / or umami taste.

8. A hot pot soup base, characterized in that: The raw materials of the hot pot soup contain the composite bacterial agent according to any one of claims 4 to 7; Optionally, the ingredients of the hot pot soup also include peppers and tomatoes; Optionally, the mass ratio of pepper to tomato is (40-60):(40-60); And / or, the ratio of the added amount of the composite bacterial agent to the total mass of the pepper and the tomato is (2-8) ml:100 g.

9. The hot pot soup base according to claim 8, characterized in that: The raw materials of the hot pot soup also include glutinous rice flour, salt and sugar; Among the raw materials for hot pot soup, the added amount of glutinous rice flour is 8wt%-12wt% of the total mass of the peppers and tomatoes, the added amount of salt is 4wt%-7wt% of the total mass of the peppers and tomatoes, and the added amount of white sugar is 5wt%-8wt% of the total mass of the peppers and tomatoes.

10. The method for preparing hot pot soup base according to claim 8 or 9, characterized in that: The composite bacterial agent and other raw materials of hot pot soup are mixed, sealed and fermented to obtain the hot pot soup; Optionally, the temperature of the sealed fermentation is 30°C-37°C, and the time of the sealed fermentation is 5 days-8 days.

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