Natural fermented flavor enhancer as well as preparation method and application thereof

Abstract

The invention relates to the technical field of food microorganisms, fermentation and seasonings, in particular to a natural fermentation flavor enhancer as well as a preparation method and application thereof. The natural fermentation flavor enhancer comprises 3-40 wt% of glutamic acid of a natural source and 0.2-25 wt% of IMP and / or GMP of a natural source. Wherein the natural fermented flavor enhancer is obtained by a method comprising fermentation by using microorganisms, and the microorganisms are selected from two or more of yeast, lactic acid bacteria, brevibacterium ammoniacum and corynebacterium glutamicum and at least comprise one of yeast or lactic acid bacteria. The natural fermented flavor enhancer is obtained through mixed fermentation of microorganisms, the flavor and taste of the flavor enhancer are improved, and astringency and peculiar smell of the product are eliminated; the natural substrate is adopted for fermentation, no additive is contained, and the requirements for greenness, safety and health are met.

Description

Technical Field

[0001] The invention relates to the technical field of food microorganisms, fermentation and condiments, and in particular to a natural fermented flavor enhancer and a preparation method and application thereof. Background Art

[0002] Food flavor enhancers can also be called flavor enhancers or umami agents. Flavor enhancers are substances that supplement or enhance the original flavor substances of food. After adding flavor enhancers, some foods have a delicious taste, increase appetite and enrich nutrition. Food umami agents do not affect the taste stimulation of the four basic tastes such as sour, sweet, bitter and salty and other flavor substances, but enhance their respective flavor characteristics, thereby improving the palatability of food. According to my country's "National Food Safety Standard Food Additives Use Standard", flavor enhancers are defined as supplements or enhances the original flavor substances of food. According to chemical composition, flavor enhancers include amino acids, nucleotides and orthocarboxylic acids.

[0003] MSG is a chemical compound composed of monosodium glutamate, and is the most commonly used flavor enhancer. There is no limit on the amount of consumption for adults, but it is not suitable for infants. With the demand for healthy food, people in developed or developing countries are more concerned about whether the food ingredients in the end products are natural. In Mintel's latest survey of first-, second- and third-tier cities in 2018, 42% of consumers said they tried to reduce their intake of MSG, so the growth rate of the market has slowed down significantly. This proportion has exceeded the number of people who try to control the intake of sugar (39%), salt (35%), and oil (35%). Compared with MSG manufacturers, the catering and hotel industries are the fastest to respond to this trend. At present, most foreign-funded high-end hotels in the Chinese market are determined not to use MSG.

[0004] In the past five years, the consumption of MSG has dropped from 1.146 million tons in 2013 to 922,000 tons in 2017. In the next five years, it will continue to decline and is expected to drop to 789,000 tons in 2022. In recent years, changes in consumer concepts have led to a shrinking market for MSG. At present, some families, mainly young people, refuse to use MSG. South Korea, Pakistan and other countries have also gradually introduced relevant regulations to prohibit the addition of MSG to food.

[0005] Yeast extract naturally contains nucleic acid and is a natural food ingredient with a rich and umami taste, mostly used for seasoning salty foods. However, it may have an adverse effect on the inherent taste of processed foods due to the odor and stink caused by its special fermentation smell, and its nucleic acid content is limited.

[0006] US Patent No. 6838100B2 discloses a method for preparing a fermented umami base, which comprises hydrolyzing a protein-containing material using a combination of at least one enzyme and at least one thermotolerant lactic acid strain, the strain having glutaminase activity, thereby providing glutamate or glutamate in the matrix sufficient to enhance the body taste. The umami obtained according to this process is not high enough. Therefore, it is necessary to add pure MSG (monosodium glutamate) and nucleotides (IMP and GMP) to these products, but the problem is that it is unnatural. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a natural fermented flavor enhancer with high content of flavor components, delicious and mellow taste and natural properties.

[0008] In view of the shortcomings of the prior art, one of the purposes of the present invention is to provide a natural fermented flavor enhancer; the second purpose of the present invention is to provide a method for preparing the above-mentioned natural biological fermented flavor enhancer, using natural raw materials as nutrient sources, using a mixture of multiple microorganisms to inoculate and ferment simultaneously or in batches, the flavor is improved, and the product has no peculiar smell; the third purpose of the present invention is to provide an application of the above-mentioned natural biological fermented flavor enhancer in food.

[0009] The technical solution of the present invention:

[0010] The present application provides a natural fermented flavor enhancer, which comprises 3-40wt% of glutamic acid from natural sources and 0.2-25wt% of IMP and / or GMP from natural sources; wherein the natural fermented flavor enhancer is obtained by a method including fermentation using microorganisms, wherein the microorganisms are selected from two or more of yeast, lactic acid bacteria, Brevibacterium ammoniagenes and Corynebacterium glutamicum, and at least one of yeast or lactic acid bacteria is included.

[0011] In some embodiments of the present application, the natural fermented flavor enhancer comprises 0.1-10 wt % of IMP from natural sources and 0.1-15 wt % of GMP from natural sources.

[0012] In some embodiments of the present application, the natural fermentation flavor agent comprises 3-40wt% of glutamic acid from natural sources and 2-25wt% of IMP and / or GMP from natural sources, preferably the natural fermentation flavor agent comprises 4-40wt% of glutamic acid from natural sources and 9-22wt% of IMP and / or GMP from natural sources, more preferably the natural fermentation flavor agent comprises 6-34wt% of glutamic acid from natural sources and 9-22wt% of IMP and / or GMP from natural sources;

[0013] Preferably, the natural fermented umami flavor agent comprises 2-10 wt%% of natural source IMP and 2-15 wt% of natural source GMP; preferably, the natural fermented umami flavor agent comprises 4-10 wt%% of natural source IMP and 4-12 wt% of natural source GMP.

[0014] In some embodiments of the present application, the yeast is Saccharomyces cerevisiae or Kluyveromyces marxianus; and / or, the lactic acid bacteria is Lactococcus lactis and / or Streptococcus lactis;

[0015] Preferably, the microorganism comprises Saccharomyces cerevisiae and one selected from the group consisting of Streptococcus lactis, Brevibacterium ammoniagenes and Corynebacterium glutamicum.

[0016] The present application provides a method for preparing the above-mentioned natural fermented flavor enhancer, which comprises obtaining the natural fermented flavor enhancer using a microbial fermentation method, wherein the microorganisms are selected from two or more of yeast, lactic acid bacteria, Brevibacterium ammoniagenes and Corynebacterium glutamicum, and at least one of yeast and lactic acid bacteria is included.

[0017] In some embodiments of the present application, the yeast is Kluyveromyces marxianus and / or Saccharomyces cerevisiae; and / or, the lactic acid bacteria is Lactococcus lactis and / or Streptococcus lactis;

[0018] Preferably, the microorganism comprises Saccharomyces cerevisiae and one selected from the group consisting of Streptococcus lactis, Brevibacterium ammoniagenes and Corynebacterium glutamicum.

[0019] In some embodiments of the present application, the fermentation inoculation method is simultaneous inoculation or batch inoculation, preferably batch inoculation.

[0020] In some embodiments of the present application, the preparation method further includes the step of performing cell disruption on the fermentation broth. Preferably, the cell disruption method is enzymatic cell disruption. More preferably, the enzyme used for enzymatic cell disruption is selected from one or more of cellulase, hemicellulase, glucanase or mannanase. Preferably, the enzyme added is 0.5-5wt% based on the dry matter mass of the fermentation broth.

[0021] In some embodiments of the present application, the preparation method further comprises a step of removing cell debris by centrifugation or filtration after cell disruption.

[0022] In some embodiments of the present application, the fermentation temperature is 28-32°C, preferably, the fermentation time is 6-12h; more preferably, the fermentation pH is 5-7;

[0023] Preferably, the fermentation inoculum amount is 1-5 v / v%.

[0024] In some embodiments of the present application, the fermentation medium includes a carbon source or a nitrogen source of natural origin;

[0025] Preferably, the carbon source is selected from one or more of glucose, fructose, sucrose, maltose, enzymatic hydrolysis products of starch, corn syrup and molasses, preferably, based on the mass of the fermentation medium, the content of the carbon source is 3-8%, more preferably, the starch is selected from one or more of wheat starch, cereal starch and cassava starch; more preferably, the carbon source is corn syrup and sucrose, preferably, based on the mass of the fermentation medium, the content of the corn syrup is 2-5%, and the content of the sucrose is 1-3%.

[0026] And / or, the nitrogen source is selected from one or more of yeast extract, amino acids and proteins. Preferably, the content of the nitrogen source is 3-8% based on the mass of the fermentation medium. More preferably, the nitrogen source is yeast extract.

[0027] The present application also provides the use of the above-mentioned natural fermentation flavor enhancer or the natural fermentation flavor enhancer prepared by the above-mentioned preparation method in food. Preferably, the addition amount of the natural fermentation flavor enhancer is 1-20% based on the weight of the food.

[0028] Beneficial effects of the present invention:

[0029] The invention adopts mixed fermentation of microorganisms to obtain a natural fermented flavor enhancer, thereby improving the flavor and mouthfeel of the flavor enhancer and eliminating the astringency and peculiar smell of the product; and adopts a substrate of natural origin for fermentation, does not contain additives, and meets the requirements of being green, safe and healthy. DETAILED DESCRIPTION

[0030] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0031] The term "off-flavor" as used herein refers to the unpleasant taste and aftertaste that umami flavor enhancers develop over time, such as a yeasty flavor.

[0032] The present invention uses two or more microorganisms to ferment natural protein-containing raw materials, with the aim of providing a natural food composition (rich in glutamic acid and / or flavor nucleotides), providing a strong umami taste to the food without using additives, while eliminating the peculiar smell of the umami agent and improving the flavor and mouthfeel of the composition.

[0033] In a specific embodiment of the present invention, the present invention provides a natural fermented flavor enhancer, which comprises 3-40wt% of glutamic acid from natural sources and 0.2-25wt% of IMP and / or GMP from natural sources; wherein the natural fermented flavor enhancer is obtained by a method including fermentation using microorganisms, and the microorganisms are selected from two or more of yeast, lactic acid bacteria, Brevibacterium ammoniagenes and Corynebacterium glutamicum, and at least one of yeast or lactic acid bacteria is included.

[0034] In some embodiments of the present application, the natural fermentation flavoring agent comprises 4-40wt% of glutamic acid from natural sources. In some embodiments of the present application, the natural fermentation flavoring agent comprises 6-34wt% of glutamic acid from natural sources. In some embodiments of the present application, the natural fermentation flavoring agent comprises 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 12wt%, 14wt%, 16wt%, 18wt%, 20wt%, 25wt%, 30%, 34wt%, 35wt% or 40wt% of glutamic acid from natural sources or glutamic acid from natural sources within the numerical range consisting of any two of the above-mentioned specific numerical values ​​as endpoints.

[0035] In some embodiments of the present application, the natural fermented flavor agent comprises 2-25wt% of natural source IMP and / or GMP. In some embodiments of the present application, the natural fermented flavor agent comprises 9-22wt% of natural source IMP and / or GMP. In some embodiments of the present application, the natural fermented flavor agent comprises 0.2wt%, 0.5wt%, 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 16wt%, 18wt%, 20wt% or 25wt% of natural source IMP and / or GMP or natural source IMP and / or GMP within the numerical range consisting of any two specific values ​​as endpoints.

[0036] In some embodiments of the present application, the natural fermentation flavor agent comprises 0.1-10wt% of IMP of natural origin. In some embodiments of the present application, the natural fermentation flavor agent comprises 2-10wt% of IMP of natural origin. In some embodiments of the present application, the natural fermentation flavor agent comprises 4-10wt% of IMP of natural origin. The natural fermentation flavor agent comprises 0.1wt%, 0.5wt%, 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt% or 10wt% of IMP of natural origin or IMP of natural origin within a numerical range consisting of any two of the above specific numerical values ​​as endpoints.

[0037] In some embodiments of the present application, the natural fermentation flavor agent comprises 0.1-15wt% of GMP of natural origin. In some embodiments of the present application, the natural fermentation flavor agent comprises 2-15wt% of GMP of natural origin. In some embodiments of the present application, the natural fermentation flavor agent comprises 4-12wt% of GMP of natural origin. In some embodiments of the present application, the natural fermentation flavor agent comprises 0.1wt%, 0.5wt%, 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt% or 15wt% of GMP of natural origin or GMP of natural origin within the numerical range consisting of any two specific numerical values ​​as endpoints.

[0038] The composition of the natural fermentation preservative in the present application is very complex. In addition to glutamic acid and / or IMP and / or GMP, it also contains other nutritional components, such as other amino acids, peptides, vitamins, fats and carbohydrates other than glutamic acid.

[0039] For the product of the present application, different forms of existence can be considered, and it can exist in any physical form, such as powder, paste, granules or liquid.

[0040] According to the present invention, all the natural fermentation flavor enhancer components (glutamic acid, IMP, GMP and nutritional components, etc.) mentioned herein are of natural origin and exist due to the raw material products used in the method of the present invention. In the present invention, glutamic acid, IMP and GMP and nutritional components of natural origin are understood to mean that these compounds are obtained by at least one of the following methods: (1) extraction from raw materials such as plants, animals or microbial materials; (2) fermentation; (3) biocatalysis.

[0041] The present application also provides a method for preparing the above-mentioned natural fermented flavor enhancer, which comprises obtaining the natural fermented flavor enhancer using a microbial fermentation method, wherein the microorganisms are selected from two or more of yeast, lactic acid bacteria, Brevibacterium ammoniagenes and Corynebacterium glutamicum, and at least one of yeast and lactic acid bacteria is included.

[0042] In some embodiments of the present application, the yeast is Kluyveromyces marxianus and / or Saccharomyces cerevisiae; and / or the lactic acid bacteria is Lactococcus lactis and / or Streptococcus lactis; preferably, the microorganism comprises Saccharomyces cerevisiae and one selected from the microbial group consisting of Streptococcus lactis, Brevibacterium ammoniagenes and Corynebacterium glutamicum. The present application selects Saccharomyces cerevisiae and one of the microbial groups consisting of Streptococcus lactis, Brevibacterium ammoniagenes and Corynebacterium glutamicum for fermentation, and the umami agent obtained by fermentation has better umami taste, mellow taste, and no astringency, yeasty taste or other peculiar smell.

[0043] In some embodiments of the present application, the fermentation temperature is 28-32°C, preferably, the fermentation time is 6-12h; more preferably, the fermentation pH is 5-7.

[0044] In some embodiments of the present application, the inoculation amount of the microbial fermentation is 1-5 v / v%.

[0045] In some embodiments of the present application, the fermentation substrates are all selected from substrates of natural origin, and the substrates of natural origin used can come from any type of carbon source or nitrogen source, as long as they are available to the strain used. For example, the carbon source can be glucose, fructose, sucrose, maltose, enzymatic hydrolysate of starch, corn syrup and molasses, and combinations thereof, preferably corn syrup and sucrose; the nitrogen source can be yeast extract, amino acid and protein products, and combinations thereof, preferably yeast extract. The starch is selected from one or more of wheat starch, cereal starch and cassava starch.

[0046] In some embodiments of the present application, the content of the carbon source is 3-8% based on the mass of the fermentation medium. In some embodiments of the present application, the content of the carbon source is 4-6% based on the mass of the fermentation medium. In some embodiments of the present application, the carbon source is corn steep liquor and sucrose. In some embodiments of the present application, the content of the corn steep liquor is 2-5% and the content of the sucrose is 1-3% based on the mass of the fermentation medium.

[0047] In some embodiments of the present application, the content of the nitrogen source is 3-8% based on the mass of the fermentation medium. In some embodiments of the present application, the content of the nitrogen source is 4-6% based on the mass of the fermentation medium.

[0048] In some embodiments of the present application, the preparation method further comprises the step of crushing the microorganisms in the fermentation broth. The cell crushing method can use methods known to those skilled in the art to treat the cells chemically, mechanically or enzymatically, preferably enzymatically. The enzymatic treatment adopts the following steps: adding enzymes to the fermentation broth, incubating at 50-65°C for 4-8h, and finally heating to 90-95°C for 1-3h to inactivate all enzyme activities to obtain an autolysis product.

[0049] It should be noted that the enzymes used for enzymatic cell wall breaking in the present application include but are not limited to one or more of cellulase, hemicellulase, glucanase or mannanase. Preferably, the amount of enzyme added is 0.5-5% based on the dry matter mass of the fermentation broth; more preferably, the enzymatic cell wall breaking temperature is 40-70°C, and further preferably, the enzymatic cell wall breaking time is 1-16h.

[0050] In some embodiments of the present application, the inoculation method of microbial fermentation can be simultaneous inoculation or batch inoculation, preferably batch inoculation, for example, after inoculating Corynebacterium glutamicum or Streptococcus lactis for fermentation for a period of time, Saccharomyces cerevisiae is inoculated to continue fermentation. In some embodiments of the present application, the inoculation method of microbial fermentation is batch inoculation. In some embodiments of the present application, the inoculation amount of Corynebacterium glutamicum or Streptococcus lactis is 1-5v / v%, the fermentation temperature is 28-32°C, preferably, the fermentation time is 6-12h; more preferably, the fermentation pH is 5-7. In some embodiments of the present application, the inoculation amount of Saccharomyces cerevisiae is 1-5v / v%, the fermentation temperature is 28-32°C, preferably, the fermentation time is 6-12h; more preferably, the fermentation pH is 5-7.

[0051] In some embodiments of the present application, the preparation method further comprises removing the broken cell fragments by filtration and / or centrifugation after the disruption treatment to obtain a supernatant.

[0052] In some embodiments of the present application, the preparation method further comprises concentrating the fermentation liquid or supernatant to obtain a paste product or continuing to dry to obtain a powder product.

[0053] The present application also provides the use of the above-mentioned natural fermented flavor enhancer in food. The added amount of the natural fermented flavor enhancer is 1-20% based on the weight of the food.

[0054] The beneficial effects of the present invention will be further illustrated by means of specific examples below.

[0055] The sources of raw materials and equipment used in the examples and comparative examples of the present invention are shown in Table 1.

[0056] Table 1 Raw materials used in the examples of the present invention and the comparative examples

[0057] Use of raw materials or equipment Model\Purity Manufacturers for sale Saccharomyces cerevisiae CICC1222 China Industrial Microbiological Culture Collection Management Center Lactococcus lactis CICC6242 China Industrial Microbiological Culture Collection Management Center Streptococcus lactis ATCC8014 Beijing Biological Collection Center Brevibacterium ammoniagenes CICC20104 China Industrial Microbiological Culture Collection Management Center Corynebacterium glutamicum CICC10212 China Industrial Microbiological Culture Collection Management Center Kluyveromyces marxianus CICC1276 China Industrial Microbiological Culture Collection Management Center Yeast Extract FM802 Angel Yeast Co., Ltd. sucrose Nanning Sugar Industry Co., Ltd. Corn Syrup Y042C Beijing Hongrun Baoshun Technology Co., Ltd. Cellulase 10000U / g Nanning Pangbo Bioengineering Co., Ltd. α-Mannanase 25000U / g Nanning Pangbo Bioengineering Co., Ltd. β-Glucanase 18000U / g Angel Yeast Co., Ltd.

[0058] Example 1

[0059] Preparation of fermentation medium: Take 1L of pure water and weigh the raw materials to prepare the medium according to the following mass concentrations: 5wt% yeast extract, 3wt% corn steep liquor and 2wt% sucrose, and adjust the pH to 6.8 with 0.1mol / LNaOH, and then sterilize at 115℃ for 20min.

[0060] Preparation of Lactococcus lactis seed activation medium: Take 1 L of pure water and weigh the following raw materials to prepare the medium according to the following mass concentrations: 1.5% tryptone, 1.5% yeast extract, 1.5% beef extract, 1% glucose, 1% sodium acetate, 0.2% dipotassium hydrogen phosphate, 0.2% triammonium citrate, 1% light calcium carbonate and 2% agar, and adjust the pH to 6.8 with 0.1 mol / L NaOH.

[0061] Saccharomyces cerevisiae seed activation medium: Take 1 L of pure water and weigh the following raw materials to prepare the medium according to the following mass concentrations: 2% glucose, 2% peptone and 1% yeast extract powder.

[0062] Lactococcus lactis seed liquid culture: Lactococcus lactis CICC 6242 was inoculated into a slant culture medium, and cultured in a 30° C. incubator for 24 hours for activation. The activated Lactococcus lactis was inoculated into a Lactococcus lactis seed activation culture medium, and cultured at 30° C. for 24 hours to obtain a Lactococcus lactis seed liquid.

[0063] Seed liquid culture of Saccharomyces cerevisiae: Saccharomyces cerevisiae CICC 1222 was inoculated into a slant culture medium, and cultured in a 30°C incubator for 24 hours for activation. The activated Saccharomyces cerevisiae was inoculated into a Saccharomyces cerevisiae seed activation culture medium, and cultured at 30°C for 24 hours to obtain a seed liquid of Saccharomyces cerevisiae.

[0064] The Lactococcus lactis seed solution and the Saccharomyces cerevisiae seed solution were mixed in equal amounts until the OD600 value reached 0.1, and inoculated into 50 mL of sterilized fermentation medium at a rate of 1 v / v%, and cultured at 30°C and 200 r / min with shaking for 12 h;

[0065] After the fermentation, the temperature was raised to 60°C for sterilization, and then 0.5% (based on the dry matter mass of the fermentation broth) of cellulase (based on the dry matter mass of the fermentation broth) was added, and the fermentation broth was incubated at 60°C for 6 hours, and finally the temperature was raised to 90°C and heated for 1.5 hours to inactivate all enzyme activity to obtain an autolysis product.

[0066] The autolysis product is separated by centrifugation to obtain the supernatant, which is concentrated to a paste product with a solid content of 60%, and then the concentrate is freeze-dried to obtain a powder product.

[0067] The content of glutamate, IMP and GMP was determined by the following method:

[0068] 1. Glutamate test method

[0069] Instrument: 2700 biochemical analyzer (YSI, USA), YSI2754 glutamate determination membrane.

[0070] Reagents: sodium glutamate, YSI2357 buffer solution (dissolve one bag of YSI2357 in 500 mL of high-purity water), L-glutamic acid standard (5 mmol / L).

[0071] 1) Prepare 5 mmol / L glutamic acid standard solution and YSI2357 buffer solution with high-purity water and put them into the special bottles equipped with YSI biochemical analyzer respectively;

[0072] 2) Mold installation: Remove the probe of the YSI biochemical analyzer, drop a drop of sodium chloride solution dedicated to the YSI biochemical analyzer in the center of the probe, then carefully open the membrane box, align it with the center of the probe, install the membrane into the probe, drop a drop of sodium chloride solution in the center of the membrane, install the probe and tighten it. Press the RUN button, rinse the membrane 3 times with buffer solution, and then rinse the membrane 3 times with standard solution;

[0073] 3) Procedure and parameter setting for L-glutamic acid determination: refer to the corresponding description of the L-glutamic acid determination chapter in the YSI biochemical analyzer operation manual to set the procedure and parameters;

[0074] 4) Membrane activation: It takes about 24 hours. The current value is used to determine whether the membrane activation is complete. Generally, when the current value is below 6, the membrane activation is considered complete. The smaller the current value, the better the membrane activation. When the membrane activation is complete, you can enter the sample determination program to start testing the sample.

[0075] 5) Sample determination: dissolve yeast extract in high-purity water and control the glutamic acid content in the solution to 0-5mmol / L. Pour the prepared yeast extract solution into the sample tank (1# workbench) or small beaker (2# workbench), press the sample key on the display screen, and the probe will immediately take a sample for determination. After 30 seconds, the instrument will display the test results on the display screen and prompt the end of the test signal. At this time, press the sample key again to start the test of a new sample.

[0076] 2. Test methods for IMP and GMP

[0077] IMP and GMP determinations are carried out according to the method for determining nucleotides in yeast extracts in GB / T23530-2009, which includes the following steps:

[0078] 2.1 Instruments and reagents

[0079] High performance liquid chromatograph (equipped with a low pressure quaternary pump, UV detector and column oven system) (Waters, E2695).

[0080] Chromatographic column: C18 column (e.g. intersil C18 250mm×4.6mm, other analytical columns with equivalent performance may also be used.)

[0081] Filtration device: 1000mL vacuum filter, 0.2um or 0.45um filter membrane.

[0082] Vacuum filtration and degassing device.

[0083] Volumetric flask: 100mL.

[0084] Analytical balance: sensitivity 0.1 mg.

[0085] Ammonium dihydrogen phosphate solution: 0.02mmol / L, pH = 5.4. Accurately weigh 2.30g of ammonium dihydrogen phosphate, dissolve it in high-purity water, add water to 900mL, adjust the pH to 5.40±0.10 with 0.1mol / L sodium hydroxide, and make up to 1L.

[0086] 5'-Guanylic acid disodium salt (GMP): molecular formula is C10H12N5Na2O8P, purity ≥98%. Sigma product number: G8377

[0087] 5'-Inosinic acid disodium salt (IMP): molecular formula is C10H11N4Na2O8P, purity ≥98%. Sigma product number: I4625

[0088] Nucleotide standard stock solution: Accurately weigh 0.0500 g of IMP and GMP dried at (105±2)℃ for 4 h, dissolve them in high-purity water, and make the volume to 100 mL to prepare standard stock solutions with a concentration of 500 ug / mL.

[0089] 2.2 Analysis steps

[0090] (1) Chromatographic conditions: Chromatographic column: intersil C18 250 mm × 4.6 mm;

[0091] Mobile phase: 0.02 mol / L ammonium dihydrogen phosphate solution, pH = 5.40;

[0092] Flow rate: 1.0ml / min;

[0093] Detection wavelength: 254nm;

[0094] Column oven temperature: 30°C

[0095] (2) Sample preparation

[0096] Weigh 1g to 2g of the sample (accurate to 0.0001g), dissolve it in water, transfer it into a 100mL volumetric flask and dilute it to the mark with water. Dilute the solution appropriately according to the nucleotides in the sample (the dilution factor is F, and the last dilution is fixed to the volume with the mobile phase). Filter it with a 0.45μm aqueous microporous membrane and set the filtrate aside.

[0097] (3) Preparation of standard solution

[0098] Table 2: Preparation of standard solutions

[0099]

[0100] Solution for making up the volume of standard series solutions: mobile phase

[0101] (4) Determination

[0102] The standard series and sample solutions were injected separately with an injection volume of 20 μL, and the chromatographic peaks of IMP and GMP in the samples were qualitatively determined according to the retention time of the standard. The percentage of each component was calculated by the external standard method according to the peak area of ​​the sample.

[0103] (5) Result calculation

[0104] IMP / GMP content calculation:

[0105] X=C×V×F / (M×(100-X1) / 100) / 10 6 ×100%

[0106] in:

[0107] X—IMP / GMP content in the sample, %;

[0108] C—Check the standard curve to obtain the content of IMP / GMP / UMP / CMP in the injection solution, μg / ml;

[0109] V—fixed volume, ml;

[0110] F—dilution factor;

[0111] M—sample mass, g;

[0112] X1—water content of sample, %;

[0113] The result is rounded to two decimal places.

[0114] Ratio calculation instructions: According to GB / T23530-2007 standard, IMP and GMP results need to be converted to dry value when calculating the ratio.

[0115] Precision: The absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.

[0116] After testing, the contents of glutamate, IMP and GMP are shown in Table 2.

[0117] Example 2

[0118] The preparation of the fermentation medium is the same as that in Example 1.

[0119] Preparation of Lactococcus lactis seed activation medium: Take 1 L of pure water, weigh the following raw materials to prepare the medium according to the following mass concentrations: 3% peptone, 3% yeast extract powder, 5% sucrose, 2% corn steep liquor, 0.2% dipotassium hydrogen phosphate, 0.2% sodium chloride, 0.02% magnesium sulfate heptahydrate, 0.005% manganese sulfate monohydrate, 0.5% triammonium citrate and 0.6% light calcium carbonate, and adjust the pH to 6.8 with 0.1 mol / L NaOH.

[0120] Preparation of Brevibacterium ammoniagenes seed activation culture medium: Take 1 L of pure water and weigh the raw materials to prepare the culture medium according to the following concentrations: glucose 22 g / L, succinic acid 1 g / L, urea 10 g / L, magnesium sulfate heptahydrate 0.4 g / L, methionine 0.5 g / L, potassium hydrogen phosphate 2.4 g / L, biotin 0.3 mg / L, manganese sulfate monohydrate 10 mg / L, vitamin B1 0.2 mg / L.

[0121] Lactococcus lactis seed liquid culture: Lactococcus lactis CICC 6242 was inoculated into a slant culture medium, and cultured in a 30° C. incubator for 24 hours for activation. The activated Lactococcus lactis was inoculated into a Lactococcus lactis seed activation culture medium, and cultured at 30° C. for 24 hours to obtain a Lactococcus lactis seed liquid.

[0122] Brevibacterium ammoniagenes seed liquid culture: Brevibacterium ammoniagenes CICC 20104 was inoculated into a slant culture medium, and cultured in a 30°C incubator for 24 hours for activation; the activated Brevibacterium ammoniagenes was inoculated into a Brevibacterium ammoniagenes seed activation culture medium, and cultured at 30°C for 24 hours to obtain Brevibacterium ammoniagenes seed liquid.

[0123] The Lactococcus lactis seed solution and the Brevibacterium ammoniagenes seed solution were mixed in equal amounts until the OD600 value reached 0.1, and inoculated into 50 mL of sterilized fermentation medium at 1 v / v%, and cultured at 30°C and 200 r / min with shaking for 10 h;

[0124] After the fermentation, the temperature was raised to 60°C for sterilization, and then 0.5% (based on the dry matter mass of the fermentation broth) of cellulase (based on the dry matter mass of the fermentation broth) was added, and the fermentation broth was incubated at 60°C for 6 hours, and finally the temperature was raised to 90°C and heated for 1.5 hours to inactivate all enzyme activity to obtain an autolysis product.

[0125] The autolysis product is separated by centrifugation to obtain the supernatant, which is concentrated to a paste product with a solid content of 60%, and then the concentrate is freeze-dried to obtain a powder product.

[0126] The same test method as in Example 1 was used, and the main components are shown in Table 2.

[0127] Example 3

[0128] The preparation of the fermentation medium is the same as that in Example 1.

[0129] Preparation of lactic acid streptococcus seed activation medium: take 1L of pure water, weigh the raw materials according to the following mass concentrations to prepare the medium: 3% peptone, 3% yeast extract powder, 5% sucrose, 2% corn steep liquor, 0.2% dipotassium hydrogen phosphate, 0.2% sodium chloride, 0.02% magnesium sulfate heptahydrate, 0.005% manganese sulfate monohydrate, 0.5% triammonium citrate, 0.6% light calcium carbonate, and adjust the pH to 6.8 with 0.1mol / LNaOH.

[0130] The preparation of the activating medium for Saccharomyces cerevisiae seeds is the same as that in Example 1.

[0131] Lactococcus seed liquid culture: Lactococcus ATCC 8014 was inoculated into a slant culture medium, and cultured in a 30°C incubator for 24 hours for activation. The activated Lactococcus was inoculated into a Lactococcus seed activation culture medium, and cultured at 30°C for 24 hours to obtain a Lactococcus seed liquid.

[0132] The culturing of the saccharomyces cerevisiae seed solution was the same as in Example 1.

[0133] The lactic acid Streptococcus seed solution and the cerevisiae yeast seed solution were mixed in equal amounts until the OD600 value reached 0.1, and inoculated into 50 mL of sterilized fermentation medium at 1 v / v%, and cultured at 30°C and 200 r / min for 10 h;

[0134] After the fermentation, the temperature was raised to 60°C for sterilization, and then 0.5% (based on the dry matter mass of the fermentation broth) of cellulase (based on the dry matter mass of the fermentation broth) was added, and the fermentation broth was incubated at 60°C for 6 hours, and finally the temperature was raised to 90°C and heated for 1.5 hours to inactivate all enzyme activity to obtain an autolysis product.

[0135] The autolysis product is separated by centrifugation to obtain the supernatant, which is concentrated to a paste product with a solid content of 60%, and then the concentrate is freeze-dried to obtain a powder product.

[0136] The same test method as in Example 1 was adopted, and the main components are shown in Table 2.

[0137] Example 4

[0138] The preparation of the fermentation medium is the same as in Example 1

[0139] The preparation of Brevibacterium ammoniagenes seed activation culture medium is the same as that in Example 2.

[0140] The preparation of the saccharomyces cerevisiae seed culture medium is the same as that in Example 1.

[0141] The cultivation of Brevibacterium ammoniagenes seed solution is the same as in Example 2.

[0142] The culture of saccharomyces cerevisiae seed solution is the same as in Example 1.

[0143] Mix equal amounts of Brevibacterium ammoniagenes seed liquid and Saccharomyces cerevisiae seed liquid until the OD600 value reaches 0.1, inoculate 1v / v% into 50mL sterilized fermentation medium, and culture at 30°C and 200r / min shaking for 10h;

[0144] After the fermentation, the temperature was raised to 60°C for sterilization, and then 2% β-glucanase (based on the dry matter mass of the fermentation broth) was added. The fermentation broth was incubated at 60°C for 6 hours, and finally the temperature was raised to 90°C and heated for 1.5 hours to inactivate all enzyme activities to obtain an autolysis product.

[0145] The autolysis product is separated by centrifugation to obtain the supernatant, which is concentrated to a paste product with a solid content of 60%, and then the concentrate is freeze-dried to obtain a powder product.

[0146] The same test method as in Example 1 was used, and the main components are shown in Table 2.

[0147] Example 5

[0148] The preparation of the fermentation medium is the same as that in Example 1.

[0149] Preparation of Corynebacterium glutamicum seed activation medium: Take 1 L of pure water, weigh each raw material to prepare the medium according to the following concentrations: 22 g / L glucose, 1 g / L succinic acid, 10 g / L urea, 0.4 g / L magnesium sulfate heptahydrate, 0.5 g / L methionine, 2.4 g / L dipotassium hydrogen phosphate, 0.3 mg / L biotin, 10 mg / L manganese sulfate monohydrate, 0.2 mg / L vitamin B1, and adjust the pH to 6.8 with 0.1 mol / L NaOH.

[0150] The preparation of the Saccharomyces cerevisiae seed activation medium is the same as in Example 1.

[0151] Cultivation of Corynebacterium glutamicum seed solution: Corynebacterium glutamicum CICC 10212 was inoculated into a slant culture medium and cultured in a 30° C. incubator for 24 h for activation; the activated Corynebacterium glutamicum was inoculated into a Corynebacterium glutamicum seed activation culture medium and cultured at 30° C. for 24 h to obtain Corynebacterium glutamicum seed solution.

[0152] The culturing of the saccharomyces cerevisiae seed solution was the same as in Example 1.

[0153] The seed solution of Corynebacterium glutamicum and the seed solution of Saccharomyces cerevisiae were mixed in equal amounts until the OD600 value reached 0.1, and inoculated into 50 mL of sterilized fermentation medium at a rate of 1 v / v%, and cultured at 30°C and 200 r / min with shaking for 10 h;

[0154] After the fermentation, the temperature was raised to 60°C for sterilization, and then 0.5% (based on the dry matter mass of the fermentation broth) of cellulase (based on the dry matter mass of the fermentation broth) was added, and the fermentation broth was incubated at 60°C for 6 hours, and finally the temperature was raised to 90°C and heated for 1.5 hours to inactivate all enzyme activity to obtain an autolysis product.

[0155] The autolysis product is separated by centrifugation to obtain the supernatant, which is concentrated to a paste product with a solid content of 60%, and then the concentrate is freeze-dried to obtain a powder product.

[0156] The same test method as in Example 1 was adopted, and the main components are shown in Table 2.

[0157] Example 6

[0158] The preparation of the fermentation medium is the same as that in Example 1.

[0159] The preparation of the Corynebacterium glutamicum seed activation medium is the same as that in Example 5.

[0160] The preparation of the activation medium for Saccharomyces cerevisiae seeds is the same as that in Example 1.

[0161] The cultivation of Corynebacterium glutamicum seed solution is the same as in Example 5.

[0162] The culturing of the saccharomyces cerevisiae seed solution was the same as in Example 1.

[0163] The seed liquid of Corynebacterium glutamicum was inoculated into 50 mL of sterilized fermentation medium at a rate of 1 v / v%, and cultured at 30° C. and 200 r / min with shaking for 10 h; then the seed liquid of Saccharomyces cerevisiae was inoculated into the fermentation liquid at a rate of 1 v / v%, and cultured at 30° C. and 200 r / min with shaking for 10 h.

[0164] After the fermentation, the temperature was raised to 60°C for sterilization, and then 0.5% (based on the dry matter mass of the fermentation broth) of cellulase (based on the dry matter mass of the fermentation broth) was added, and the fermentation broth was incubated at 60°C for 6 hours, and finally the temperature was raised to 90°C and heated for 1.5 hours to inactivate all enzyme activity to obtain an autolysis product.

[0165] The autolysis product is separated by centrifugation to obtain the supernatant, which is concentrated to a paste product with a solid content of 60%, and then the concentrate is freeze-dried to obtain a powder product.

[0166] The same test method as in Example 1 was adopted, and the main components are shown in Table 2.

[0167] Example 7

[0168] The preparation of the fermentation medium is the same as that in Example 1.

[0169] The preparation of the lactic acid streptococcus seed activation culture medium is the same as that in Example 3.

[0170] The culturing of the saccharomyces cerevisiae seed solution was the same as in Example 1.

[0171] The culture of lactic acid streptococcus seed solution is the same as in Example 3.

[0172] The lactic acid streptococcus seed liquid was inoculated into 50 mL of sterilized fermentation medium at a rate of 1 v / v%, and cultured at 30° C. and 200 r / min for 10 h; then the cerevisiae yeast seed liquid was inoculated into the fermentation liquid at a rate of 1%, and cultured at 30° C. and 200 r / min for 10 h.

[0173] After the fermentation, the temperature was raised to 60°C for sterilization, and then 0.5% cellulase (based on the dry matter mass of the fermentation liquid) was added. The fermentation liquid was incubated at 60°C for 6 hours, and finally the temperature was raised to 90°C and heated for 1.5 hours to inactivate all enzyme activities to obtain an autolysis product.

[0174] The autolysis product is separated by centrifugation to obtain the supernatant, which is concentrated to a paste product with a solid content of 60%, and then the concentrate is freeze-dried to obtain a powder product.

[0175] The same test method as in Example 1 was adopted, and the main components are shown in Table 2.

[0176] Example 8

[0177] The preparation of the fermentation medium is the same as in Example 1

[0178] The preparation of Brevibacterium ammoniagenes seed activation culture medium is the same as that in Example 2.

[0179] The preparation of Kluyveromyces marxianus seed culture medium is the same as that in Example 1.

[0180] The cultivation of Brevibacterium ammoniagenes seed solution is the same as in Example 2.

[0181] The cultivation of Kluyveromyces marxianus seed solution was the same as in Example 1.

[0182] The seed liquid of Brevibacterium ammoniagenes and the seed liquid of Kluyveromyces marxianus were mixed in equal amounts until the OD600 value reached 0.1, and inoculated into 50 mL of sterilized fermentation medium at 1 v / v%, and cultured at 30°C and 200 r / min for 10 h;

[0183] After the fermentation, the temperature was raised to 60°C for sterilization, and then 3% α-mannanase (based on the dry matter mass of the fermentation broth) was added. The fermentation broth was incubated at 60°C for 6 hours, and finally the temperature was raised to 90°C and heated for 1.5 hours to inactivate all enzyme activities to obtain an autolysis product.

[0184] The autolysis product is separated by centrifugation to obtain the supernatant, which is concentrated to a paste product with a solid content of 60%, and then the concentrate is freeze-dried to obtain a powder product.

[0185] The same test method as in Example 1 was used, and the main components are shown in Table 2.

[0186] Comparative Example 1

[0187] The difference from Example 1 is that Comparative Example 1 only uses Saccharomyces cerevisiae for fermentation. The test results are shown in Table 2.

[0188] Comparative Example 2

[0189] The difference from Example 1 is that Comparative Example 1 only uses Lactococcus lactis for fermentation. The test results are shown in Table 2.

[0190] Comparative Example 3

[0191] The difference from Example 5 is that Comparative Example 3 only uses Corynebacterium glutamicum for fermentation. The test results are shown in Table 2.

[0192] Comparative Example 4

[0193] The difference from Example 2 is that Comparative Example 4 only uses Brevibacterium ammoniagenes for fermentation. The test results are shown in Table 2.

[0194] Comparative Example 5

[0195] The difference from Example 3 is that Comparative Example 5 only uses lactic acid streptococci for fermentation. The test results are shown in Table 2.

[0196] Table 2 Component content test results

[0197]

[0198]

[0199] As shown in Table 2, the content of glutamic acid in the natural fermented flavor enhancers prepared in Examples 1-8 is 3.5-34%, the content of IMP is 0.1-10%, and the content of GMP is 0.1-12%. Among them, the content of IMP in the natural fermented flavor enhancers prepared in Examples 1-7 is 2.4-10%, and the content of GMP is 2.4-12%; further, the content of glutamic acid in the natural fermented flavor enhancers prepared in Examples 3-7 is 4.4-34%, the content of IMP is 4.4-10%, and the content of GMP is 5.4-12%. Compared with Example 5, Example 6 adopts a batch inoculation method, and the content of glutamic acid is significantly increased; compared with Example 3, Example 7 adopts a batch fermentation method, and the contents of IMP and GMP are significantly increased.

[0200] Compared with Example 3, Comparative Example 1 uses only Saccharomyces cerevisiae for fermentation, and the contents of glutamic acid, IMP and GMP in the natural fermented flavor enhancer obtained are significantly lower than those in Example 3; while Comparative Example 5 uses only Streptococcus lactis for fermentation, and its fermentation product does not contain glutamic acid, IMP and GMP. It can be seen that Example 3 ferments Streptococcus lactis and Saccharomyces cerevisiae at the same time, which promotes the fermentation of Saccharomyces cerevisiae to produce more umami substances such as glutamic acid, IMP and GMP.

[0201] Experimental example

[0202] The flavor and taste of the examples and comparative examples were evaluated by sensory evaluation, wherein the samples of examples 1-8 and comparative examples 1-5 were prepared into 1% solutions. 20 professional sensory evaluators were randomly invited to set up a single-person tasting room in accordance with the requirements of GB / T13868-2009. The evaluators performed sensory evaluation on the numbered samples and qualitatively described the flavor and taste of the samples through descriptive sensory analysis. The experimental results are shown in Table 3.

[0203] Table 3 Sensory evaluation test results

[0204]

[0205] As can be seen from Table 3, Examples 1-8 of the present application use two kinds of microorganisms for fermentation, and the natural fermented flavor enhancers produced therefrom have no yeast taste and no astringency; among them, the natural fermented flavor enhancers produced in Examples 3-7 have umami, sourness and a mellow taste.

[0206] Compared with the embodiment, the comparative examples 1-5 only use one microorganism for fermentation, and the umami agents produced therefrom all have astringent taste. The embodiment uses two microorganisms for fermentation, eliminates astringent taste and other foreign smells, and improves the flavor and taste of the umami agent.

[0207] Among them, compared with Example 5, Comparative Examples 1 and Comparative Examples 3 respectively use Saccharomyces cerevisiae and Corynebacterium glutamicum for fermentation, and the umami agents obtained therefrom have astringent and yeasty taste, while Example 5 uses Saccharomyces cerevisiae and Corynebacterium glutamicum for simultaneous fermentation, which eliminates peculiar smells such as astringent and yeasty taste, and improves the flavor and mouthfeel of the umami agent. Compared with Example 3, Comparative Examples 1 and Comparative Examples 5 respectively use Saccharomyces cerevisiae and Streptococcus lactis for fermentation, and the umami agents obtained therefrom have astringent taste, while Example 3 uses Saccharomyces cerevisiae and Streptococcus lactis for simultaneous fermentation, which eliminates peculiar smells such as astringent taste, and improves the flavor and mouthfeel of the umami agent.

[0208] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A natural fermented flavor enhancer, characterized in that: It contains 3-40wt% of glutamic acid from natural sources and 0.2-25wt% of IMP and / or GMP from natural sources; wherein the natural fermented flavor enhancer is obtained by a method including fermentation using microorganisms, wherein the microorganisms are selected from two or more of yeast, lactic acid bacteria, ammonia-producing Brevibacterium and glutamicum, and at least one of yeast or lactic acid bacteria is included.

2. The natural fermented flavor enhancer according to claim 1, characterized in that: The natural fermented flavor enhancer comprises 0.1-10 wt % of IMP of natural origin and 0.1-15 wt % of GMP of natural origin.

3. The natural fermented flavor enhancer according to claim 1 or 2, characterized in that: The natural fermentation flavoring agent comprises 3-40wt% of glutamic acid from natural sources and 2-25wt% of IMP and / or GMP from natural sources, preferably the natural fermentation flavoring agent comprises 4-40wt% of glutamic acid from natural sources and 9-22wt% of IMP and / or GMP from natural sources, more preferably the natural fermentation flavoring agent comprises 6-34wt% of glutamic acid from natural sources and 9-22wt% of IMP and / or GMP from natural sources; Preferably, the natural fermented umami flavor agent comprises 2-10 wt%% of natural source IMP and 2-15 wt% of natural source GMP; preferably, the natural fermented umami flavor agent comprises 4-10 wt%% of natural source IMP and 4-12 wt% of natural source GMP.

4. The natural leavening agent according to any one of claims 1 to 3, characterized in that The yeast is Saccharomyces cerevisiae or Kluyveromyces marxianus; and / or the lactic acid bacteria is Lactococcus lactis and / or Streptococcus lactis; Preferably, the microorganism comprises Saccharomyces cerevisiae and one selected from the group consisting of Streptococcus lactis, Brevibacterium ammoniagenes and Corynebacterium glutamicum.

5. The method for preparing the natural fermented flavor enhancer according to any one of claims 1 to 4, characterized in that: The preparation method comprises obtaining the natural fermented flavor enhancer by using a microbial fermentation method, wherein the microorganisms are selected from two or more of yeast, lactic acid bacteria, Brevibacterium ammoniagenes and Corynebacterium glutamicum, and at least one of yeast and lactic acid bacteria is included.

6. The preparation method according to claim 5, characterized in that: The yeast is Kluyveromyces marxianus and / or Saccharomyces cerevisiae; and / or the lactic acid bacteria is Lactococcus lactis and / or Streptococcus lactis; Preferably, the microorganism comprises Saccharomyces cerevisiae and one selected from the group consisting of Streptococcus lactis, Brevibacterium ammoniagenes and Corynebacterium glutamicum.

7. The preparation method according to any one of claims 4 to 6, characterized in that: The fermentation inoculation method is simultaneous inoculation or batch inoculation, preferably batch inoculation.

8. The preparation method according to any one of claims 4 to 7, characterized in that: The preparation method further comprises the step of cell disruption of the fermentation broth. Preferably, the cell disruption method is enzymatic cell disruption. More preferably, the enzyme used for the enzymatic cell disruption is selected from one or more of cellulase, hemicellulase, glucanase or mannanase. Preferably, the enzyme is added in an amount of 0.5-5wt% based on the dry matter mass of the fermentation broth.

9. The preparation method according to claim 8, characterized in that: The preparation method further comprises a step of removing cell debris by centrifugation or filtration after cell disruption.

10. The preparation method according to any one of claims 4 to 9, characterized in that: The fermentation temperature is 28-32°C, preferably, the fermentation time is 6-12h; more preferably, the fermentation pH is 5-7; Preferably, the fermentation inoculum amount is 1-5 v / v%.

11. The preparation method according to any one of claims 4 to 10, characterized in that: The fermentation medium includes a carbon source and / or a nitrogen source of natural origin; Preferably, the carbon source is selected from one or more of glucose, fructose, sucrose, maltose, enzymatic hydrolysis products of starch, corn syrup and molasses, preferably, based on the mass of the fermentation medium, the content of the carbon source is 3-8%, more preferably, the starch is selected from one or more of wheat starch, cereal starch and cassava starch; more preferably, the carbon source is corn syrup and sucrose, preferably, based on the mass of the fermentation medium, the content of the corn syrup is 2-5%, and the content of the sucrose is 1-3%. And / or, the nitrogen source is selected from one or more of yeast extract, amino acids and proteins. Preferably, the content of the nitrogen source is 3-8% based on the mass of the fermentation medium. More preferably, the nitrogen source is yeast extract.

12. Use of the natural fermented flavor enhancer according to any one of claims 1 to 4 or the natural fermented flavor enhancer prepared by the preparation method according to any one of claims 5 to 11 in food, preferably, the addition amount of the natural fermented flavor enhancer is 1-20% based on the weight of the food.

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