Application and method of Leuconostoc mesenteroides in preparing fermented food or beverage

Abstract

The invention discloses the application and method of Leuconostoc mesenteroides in the preparation of fermented food or beverage, and relates to the field of microbial fermentation technology. Leuconostoc mesenteroides I1 / 53 produces antibacterial components such as hydrophilic esters, thioaminophenol C, cerulenin and faecalis in fermented sugarcane juice beverage, which can effectively inhibit foodborne pathogens, including Staphylococcus aureus, Escherichia coli, Salmonella, Listeria monocytogenes, Staphylococcus lentus, Bacillus subtilis, Bacillus cereus and Micrococcus luteus; at the same time, it can also enhance the flavor characteristics of sugarcane juice beverage.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to an application and method of Leuconostoc mesenteroides in preparing fermented food or beverage. Background Art

[0002] Pathogens and spoilage microorganisms contamination is an important cause of food safety. Preservatives are a type of food additive that can inhibit the growth of microorganisms, prevent food from spoiling, and extend the shelf life. At present, the commonly used methods to prevent spoilage in the food industry mainly include pasteurization, ultraviolet sterilization, and chemical preservatives. However, a large number of research reports show that no matter which of the above methods, there are certain shortcomings or hazards; for example, pasteurization can cause the degradation of nutrients in food, especially vitamins and antioxidants; chemical preservatives can cause environmental pollution and microbial resistance. In addition, excessive consumption of chemical preservatives has potential carcinogenicity, teratogenicity, or food poisoning and other safety hazards. Therefore, finding new healthy and safe "preservatives" is a new way to control food pathogens and spoilage microorganisms in the future. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes the use of Leuconostoc mesenteroides with a preservation number of GDMCC No: 61720 in the preparation of fermented food or beverage, which can effectively improve the antibacterial property of the fermented food or beverage and improve the flavor characteristics.

[0004] The invention also provides a method for improving the antibacterial ability and flavor of fermented food or beverage.

[0005] The invention also provides a fermentation product.

[0006] The present invention also provides the use of the above-mentioned Leuconostoc mesenteroides in a bacterial agent for fermentation production for preparing fermented food or beverage.

[0007] The present invention also provides the use of the above-mentioned Leuconostoc mesenteroides or the method or the fermentation product

[0008] According to the first aspect of the present invention, an embodiment of the present invention provides a use of Leuconostoc mesenteroides with a preservation number of GDMCC No: 61720 in the preparation of fermented food or beverage.

[0009] According to some embodiments of the invention, the fermented beverage comprises fermented cane juice.

[0010] According to a second aspect of the present invention, a method for improving the antibacterial ability and flavor of fermented food or beverage comprises: inoculating Leuconostoc mesenteroides with a preservation number of GDMCC No: 61720 into a raw material to be fermented for fermentation.

[0011] According to some embodiments of the present invention, the fermentation temperature is 25°C to 40°C, preferably 35°C to 40°C, more preferably 34°C to 37°C, and even more preferably 36°C to 37°C.

[0012] According to some embodiments of the present invention, the fermentation time is 12 hours to 96 hours, preferably 24 hours to 72 hours, and more preferably 36 hours to 60 hours.

[0013] According to some embodiments of the present invention, the inoculation density of Leuconostoc mesenteroides is 10 5 CFU / mL~10 9 CFU / mL. Preferably 10 6 CFU / mL~10 8 CFU / mL.

[0014] A fermented product according to a third aspect of the present invention is prepared by the method described in the second aspect of the present invention.

[0015] According to some embodiments of the present invention, the fermented product comprises fermented cane juice.

[0016] According to some embodiments of the present invention, the fermentation product includes at least one of hydrophilic esters, thioaminophenol C, cerulenin, and sordin.

[0017] According to some embodiments of the present invention, the fermented product further comprises at least one of Schisandra saponin C2, 1-(methylthio)propyldisulfidebutyl, glucosyl isomalt, deoxyfructosamine, and spinachin 3-gentioside.

[0018] According to the fourth aspect of the present invention, an embodiment of the present invention is the use of Leuconostoc mesenteroides with the accession number of GDMCC No: 61720 in a fermentation production bacterial agent for preparing fermented food or beverage.

[0019] According to some embodiments of the invention, the fermented beverage comprises fermented cane juice.

[0020] According to some embodiments of the present invention, the method for using the bacterial agent for fermentation production includes the method described in the embodiment of the second aspect above.

[0021] According to the fifth aspect of the present invention, the Leuconostoc mesenteroides with the accession number of GDMCC No: 61720, or the method described in the second aspect, or the fermentation product described in the third aspect in the preparation of an antibacterial product.

[0022] According to some embodiments of the present invention, the inhibitory target of the antibacterial product is bacteria, specifically food-borne bacteria.

[0023] According to some embodiments of the present invention, the bacteria include Gram-positive bacteria and Gram-negative bacteria.

[0024] According to some embodiments of the present invention, the Gram-positive bacteria include at least one of Staphylococcus aureus, Listeria monocytogenes, Staphylococcus lentus, Bacillus subtilis, Bacillus cereus and Micrococcus luteus.

[0025] According to some embodiments of the present invention, the Gram-negative bacteria include at least one of Escherichia coli and Salmonella.

[0026] According to the fifth aspect of the present invention, the method described in the second aspect of the present invention, or the use of the fermented product described in the third aspect of the present invention in the preparation of food, health products, additives, and feed

[0027] The beneficial effects of the present invention are:

[0028] The intestinal membrane leuconostoc I1 / 53 of the embodiment is obtained after separation, purification and screening from sugarcane juice, and the whole genome analysis of the strain is combined to confirm that the intestinal membrane leuconostoc I1 / 53 is safe and pathogen-free. The sugarcane juice obtained by fermentation of intestinal membrane leuconostoc I1 / 53 contains antibacterial substances such as water-absorbing esters, thioaminophenol C, cerulin and faecal spores, which can effectively inhibit a variety of gram-negative bacteria and gram-positive bacteria, including common food-borne pathogens such as Staphylococcus aureus, Escherichia coli, Salmonella, Listeria monocytogenes, Staphylococcus lentus, Bacillus subtilis, Bacillus cereus and Micrococcus luteus, and has a broad-spectrum antibacterial effect. In addition, after fermentation of intestinal membrane leuconostoc I1 / 53, the flavor characteristics of sugarcane juice beverage can also be enhanced and its quality can be improved. In short, compared with the prior art, intestinal membrane leuconostoc I1 / 53 has high safety, antibacterial effect and stability, and has broad application prospects in the fields of food, feed, breeding, etc.

[0029] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 are four secondary metabolic gene clusters related to antibacterial effects of Leuconostoc mesenteroides I1 / 53 according to an embodiment of the present invention;

[0031] Figure 2 The results of the antibacterial experiment on the concentrate of cane juice fermented by Leuconostoc mesenteroides I1 / 53 according to one embodiment of the present invention are shown. DETAILED DESCRIPTION

[0032] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0033] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0034] In the description of the present invention, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products or apparatuses.

[0035] The Leuconostoc mesenteroides I1 / 53 in the following example was deposited at the Guangdong Provincial Microbiological Culture Collection Center of the Guangdong Provincial Institute of Microbiology, Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou on June 11, 2021, with the deposit number GDMCC No: 61720.

[0036] Example 1

[0037] This example measures and analyzes the whole genome of Leuconostoc mesenteroides I1 / 53, as follows:

[0038] The DNA of Leuconostoc mesenteroides I1 / 53 was extracted using the Hipure Bacterial DNA Kit (provided by Shanghai Meiji Biotechnology Co., Ltd.), and the concentration and purity of the DNA were analyzed by Nanodrop2000 and Qubit 3.0 fluorometers; the DNA library was constructed using the SQK-LSK109 kit (provided by Shanghai Meiji Biotechnology Co., Ltd.), and the whole genome of Leuconostoc mesenteroides I1 / 53 was determined using the PromethlON and NovaSeq6000 platforms; the sequencing results were then quality controlled to remove low-quality fragments, genome assembly, and structural analysis, and the high-quality sequences obtained were then compared and analyzed in the antiSMASH database.

[0039] like Figure 1As shown in the data, Leuconostoc mesenteroides I1 / 53 contains four secondary metabolic gene clusters related to antibacterial effects, namely ribosomally-synthesized and post-translationally modified peptides (RiPP), terpenes, type III polyketide synthase (T3PKS) and betalactone, indicating that Leuconostoc mesenteroides I1 / 53 has the potential to synthesize a variety of antibacterial components.

[0040] Example 2

[0041] This example explores the effect of Leuconostoc mesenteroides I1 / 53 on fermentation of sugarcane juice, and the specific steps are as follows:

[0042] Live bacteria and inactivated bacteria Leuconostoc mesenteroides I1 / 53 (1×10 9 CFU) were inoculated into 10mL fresh cane juice (provided by Huizhou Boluo County Guanyin Pavilion Sugar Co., Ltd.), fermented at 37℃ for 48h, then centrifuged at 4℃, 8000rpm for 10min, 50μL of supernatant was mixed thoroughly in 750μL pre-cooled acetonitrile, ultrasonicated for 3min, centrifuged at 4℃, 13000rpm for 15min, and 600μL of supernatant was used for non-target metabolomics analysis by ultra-high pressure liquid chromatography triple quadrupole tandem time-of-flight mass spectrometry. Chromatographic and mass spectrometric information was collected by Progenesis QI V2.0 software, and through a series of quality control operations, including noise reduction, peak alignment, and zeroing, the high-quality chromatographic and mass spectrometric information obtained was compared and analyzed in the public database, and accurately identified based on the secondary mass spectrometric information.

[0043] The chromatographic column was ACQUITY UPLC BEH C18 (100×2.1mm, 1.7μm); the parameters were set as follows: column temperature 40°C, mobile phase flow rate 0.35mL / min, injection volume 2.0μL. Mobile phase A was pure water, mobile phase B was acetonitrile, elution gradient: 0min 98.0% A, 6.5min 50% A, 10min 98% A, and the total detection time was 12.5min.

[0044] The mass spectrometer parameters were set as follows: in positive ion mode, M / Z was 50-1200, collision voltage was 35 V, desolvation and conical gas was nitrogen, capillary pressure was 2.5 kilowatt-volts, conical gas flow rate was 40 L / h, and ion source temperature was 120°C.

[0045] The results are shown in Table 1.

[0046] Among the differential metabolites, 4 metabolites with antibacterial activity were detected, including hydrospermella, thioaminophenol C, cerulenin and faeciumin; 7 flavor substances were detected, including schisandrin C2, 1-(methylthio)propyl disulfide butyl, glucosyl isomalt, deoxyfructoseamine, rhamnoside 3-rutin, spinachin 3-gentioside and 3,4-dicaffeoylquinic acid methyl ester, of which 5 metabolites increased in abundance and 2 metabolites decreased in content. This indicates that Leuconostoc mesenteroides I1 / 53 can enhance the antibacterial effect of sugarcane juice beverages, and also enhance the flavor and characteristics of sugarcane juice beverages.

[0047] Table 1 The main changes in antibacterial and flavor components of sugarcane juice fermented by Leuconostoc mesenteroides I1 / 53

[0048]

[0049]

[0050] Example 3

[0051] In this example, the antibacterial effect of Leuconostoc mesenteroides I1 / 53 sugarcane juice fermentation concentrate was tested, and the specific steps were as follows:

[0052] 1) Leuconostoc mesenteroides I1 / 53 (1×10 9 CFU) were inoculated into 10 mL of fresh cane juice, fermented at 37°C for 48 h, centrifuged at 4°C and 8000 rpm for 15 min, 10 mL of supernatant was taken, protease was added and the mixture was warmed at 33°C for 1 h, and then centrifuged at 4°C and 13000 rpm for 15 min. The supernatant was concentrated to 2 mL using liquid nitrogen to obtain a concentrate.

[0053] The protease was composed of trypsin and proteinase K (provided by Shanghai MacLean Biochemical Technology Co., Ltd., with product numbers T6325 and P816054, respectively), and the enzyme concentration was 5 mg / L.

[0054] 2) The antibacterial effect of the concentrate on various foodborne pathogens was investigated by the Oxford cup method:

[0055] In a sterile clean bench, draw 0.2 mL of indicator bacterial solution (bacterial concentration is 1×10 7 CFU / mL) was spread on LB solid medium. Under sterile conditions, a sterilized Oxford cup (Oxford cup size: outer diameter 8mm, inner diameter 6mm, height 10mm) was placed vertically on the surface of LB solid medium and gently pressed. 200 μL of the concentrate from step 1) was added to the Oxford cup and cultured at 37°C for 24 hours. The antibacterial test was repeated 5 times.

[0056] Among them, indicator bacteria include Staphylococcus aureus, Escherichia coli, Salmonella, Listeria monocytogenes, Staphylococcus lentus, Bacillus subtilis, Bacillus cereus and Micrococcus luteus.

[0057] The test results are as follows Figure 2 shown.

[0058] Leuconostoc mesenteroides I1 / 53 has good antibacterial effect against Staphylococcus aureus, Escherichia coli, Salmonella, Listeria monocytogenes, Staphylococcus lentus, Bacillus subtilis, Bacillus cereus and Micrococcus luteus, and the diameters of the inhibition zones are 27.17±0.12, 30.97±0.47, 21.30±0.46, 20.67±0.31, 24.57±0.35, 20.13±0.21, 21.33±0.25 and 19.52±0.28 mm, respectively.

[0059] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A method for increasing the content of cerulenin and sordin in sugarcane juice or reducing the content of 3, 4-dicaffeoylquinic acid methyl ester in sugarcane juice, characterized in that: The method comprises: inoculating Leuconostoc mesenteroides with a preservation number of GDMCC No: 61720 into a raw material to be fermented for fermentation; the raw material to be fermented is sugarcane juice.

2. The method according to claim 1, characterized in that The fermentation temperature is 25°C to 40°C.

3. The method according to claim 1, characterized in that The fermentation time is 12 h to 96 h.

4. The method according to claim 1, characterized in that The inoculation density of the enteric Leuconostoc mesenteroides is 10 5 CFU / mL~10 9 CFU / mL.

5. Use of the method according to any one of claims 1 to 4 in the preparation of food, health products and feed.

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