Propionibacterium metabolite containing gamma-Glu-ASP and preparation method of propionibacterium metabolite

Abstract

The invention discloses a propionibacterium metabolite containing gamma-Glu-ASP and a preparation method of the propionibacterium metabolite, and belongs to the technical field of food seasonings, the propionibacterium metabolite contains 0.3% or more of gamma-Glu-ASP per unit dry weight, according to the propionibacterium metabolite containing the gamma-Glu-ASP, propionibacterium is cultured by adopting a culture medium added with two compounds of Asp and Glu, and the propionibacterium metabolite containing the gamma-Glu-ASP is prepared by utilizing the obtained propionibacterium thallus. The bacteriostatic activity of the propionibacterium metabolite containing gamma-Glu-ASP disclosed by the invention is about 50% higher than that of a conventional propionibacterium metabolite without Glu and Asp; the propionibacterium metabolite containing gamma-Glu-ASP endows food with excellent strong taste, especially has a remarkable effect on the first taste and the second taste, and can be used in the industries of seasonings or food additives and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of food seasonings, and particularly relates to a propionibacterium metabolite containing gamma-Glu-ASP and a preparation method thereof. Background Art

[0002] The common function of Propionibacterium metabolites is to inhibit the proliferation of microorganisms, especially Gram-negative bacteria. They have a strong inhibitory effect on them. They can inhibit the division and proliferation of common food spoilage bacteria such as Pseudomonas putida, Pseudomonas aeruginosa, and Bacillus aeruginosa, and have the effect of extending the shelf life of food.

[0003] It is known that Propionibacterium can use trypsin as an amino acid source to synthesize propionibacterial antimicrobial peptides. The antimicrobial peptides that have been discovered so far mainly include propionibactin T1, propionibactin SM1, Jenseniin G, propionibactin PLG-1 and MicrogardTM. These antimicrobial peptides all have the effect of inhibiting the growth and proliferation of Gram-negative bacteria. However, the properties of these antimicrobial peptides in imparting a rich flavor to food have never been reported. In particular, the metabolites of Propionibacterium have the ability to impart an excellent front and middle flavor to food.

[0004] Aspartic acid (Aspartic acid) has a mild umami flavor and is one of the naturally occurring flavor-producing amino acids. Glutamic acid (Glutamic acid) is one of the most important and classic umami substances in nature. It is one of the 20 amino acids that make up protein and is the primary source of umami. When present in a free state (e.g., free glutamate), it can directly activate umami receptors (such as T1R1 / T1R3) on the human tongue, producing an umami sensation. However, existing preparation technologies are unable to produce combinations of Aspartic acid and Glu. In particular, existing technologies for the production of propionibacterial antimicrobial peptides, products of Propionibacterium, all inhibit the growth and proliferation of Gram-negative bacteria. However, these antimicrobial peptides' ability to impart a rich flavor to food has never been reported, particularly in the preparation of propionibacterial metabolites that impart an excellent front-mid flavor to food. Summary of the Invention

[0005] To address the technical challenges of the prior art, the present invention proposes a propionibacterium metabolite containing γ-Glu-ASP and a method for preparing the same. Propionibacterium is cultured in a culture medium supplemented with the compounds Asp and Glu, and the resulting Propionibacterium cells are used to prepare the propionibacterium metabolite containing γ-Glu-ASP. This propionibacterium metabolite containing γ-Glu-ASP imparts an excellent kokumi (rich flavor) to foods, particularly a pronounced front-mid flavor, and can be used in condiments, food additives, and other industries.

[0006] According to a first aspect of the technical solution of the present invention, 1. a propionibacterium metabolite containing γ-Glu-ASP is provided, characterized in that the propionibacterium metabolite contains more than 0.3% of the propionibacterium metabolite containing γ-Glu-ASP per unit dry weight.

[0007] Preferably, the propionibacterium in the propionibacterium metabolite is a propionibacterium capable of taking Glu and Asp into cells and accumulating γ-Glu-ASP in the cells.

[0008] Preferably, the propionibacterium in the propionibacterium metabolite is Propionibacterium sieversii, Propionibacterium acidogenicum, Propionibacterium terrei and Propionibacterium janaschii.

[0009] Preferably, the propionibacterium metabolite contains 0.003% or more of γ-Glu-ASP per unit dry weight.

[0010] Preferably, the Propionibacterium in the Propionibacterium metabolite containing γ-Glu-ASP may be a wild strain.

[0011] According to a second aspect of the technical solution of the present invention, a method for preparing the above-mentioned Propionibacterium metabolite containing γ-Glu-ASP is provided, which comprises the following steps: Step S1: Preparation of Propionibacterium culture medium and activation of Propionibacterium strains; Step S2: fermenting and culturing Propionibacterium based on the activated Propionibacterium strain obtained in step S1; Step S3: preparing propionibacterium metabolites from the propionibacterium fermentation broth prepared under the fermentation conditions of step S2.

[0012] Preferably, the culture medium in step S1 is a Propionibacterium culture medium that does not contain yeast powder.

[0013] Preferably, the carbon source of the culture medium is any one or more of lactose, glucose, sucrose, sodium lactate and acetic acid; the nitrogen source is any one or more of tryptone, soy peptone and fish meal peptone; the phosphate source, potassium source and manganese source are any one or a random combination of phosphoric acid, potassium dihydrogen phosphate, potassium chloride, potassium hydroxide, manganese sulfate and manganese chloride as the source of the phosphate source, potassium source and manganese source.

[0014] Preferably, Glu and Asp are present in the fermentation medium when the Propionibacterium species are cultivated.

[0015] Preferably, Glu and Asp are added at any time between 0 h and 144 h before the end of fermentation.

[0016] Compared with the prior art, the propionibacterium metabolite containing γ-Glu-ASP and the preparation method of the present invention have the following beneficial technical effects: The antibacterial activity of the propionibacterium metabolite containing γ-Glu-ASP is about 50% higher than that of the conventional propionibacterium metabolite without adding Glu and Asp.

[0017] The method for preparing the propionibacterium metabolite of γ-Glu-ASP provided by the present invention is simple to operate and has low cost. DETAILED DESCRIPTION

[0018] In order to make the technical problems solved by the present invention, the technical solutions adopted, and the beneficial effects obtained more clearly understood, the present invention is further described in detail below with reference to specific embodiments. The specific embodiments described herein are only used to illustrate the present invention and are not intended to constitute any limitation of the present invention. Unless otherwise defined, all terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs.

[0019] The following specific examples are provided to help understand the present invention, but it should be understood that the embodiments and test examples listed in the present invention are only used to illustrate the present invention and do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims.

[0020] The present invention discloses a propionibacterium metabolite containing γ-Glu-ASP and a preparation method thereof, belonging to the technical field of food seasonings. The present inventors discovered that by adding a certain amount of Glu and Asp to a propionibacterium culture medium, the fermented propionibacterium metabolite contains γ-Glu-Asp (γ-glutamyl-aspartic acid) and exhibits a kokumi flavor. γ-Glu-Asp belongs to the category of "flavor peptides" and can enhance umami flavor or improve mouthfeel in foods. It is formed in fermented products containing raw materials rich in glutamic acid and aspartic acid.

[0021] The propionibacterium metabolite of the present invention contains more than 0.3% of γ-Glu-ASP per unit dry weight, and further, the propionibacterium metabolite of the present invention contains more than 0.003% of γ-Glu-ASP per unit dry weight. The method for preparing the propionibacterium metabolite containing γ-Glu-ASP of the present invention is to culture propionibacterium with a culture medium added with two compounds, Asp and Glu, and use the obtained propionibacterium cells to prepare the propionibacterium metabolite containing γ-Glu-ASP. The propionibacterium metabolite containing γ-Glu-ASP has an antibacterial activity that is approximately 50% higher than that of conventional propionibacterium metabolites without the addition of Glu and Asp. At the same time, the propionibacterium metabolite containing γ-Glu-ASP gives food an excellent rich taste, especially a significant front and middle taste effect, and can be used in industries such as condiments or food additives.

[0022] In the present invention, when propionibacterium is inoculated into a propionibacterium culture medium without yeast powder, the propionibacterium grows normally and secretes propionibacterial antimicrobial peptides, but the resulting propionibacterial metabolites lack the ability to impart a kokumi top flavor to food. However, when Glu and Asp (Asp represents aspartic acid, and Glu represents glutamic acid) are added to the culture medium, a propionibacterial metabolite containing γ-Glu-ASP is obtained after the same fermentation time. This propionibacterial metabolite containing γ-Glu-ASP exhibits approximately 50% higher antibacterial activity than a conventional propionibacterial metabolite without Glu and Asp. Furthermore, the resulting propionibacterial metabolite containing γ-Glu-ASP exhibits an excellent kokumi flavor, particularly an excellent top-mid flavor within the kokumi flavor. Because this excellent kokumi flavor-imparting effect is further enhanced in the presence of salty or umami flavors, umami substances such as sodium L-glutamate and salty substances such as salt can be added to this propionibacterial metabolite, making it suitable for use in condiments and food additives. It's important to note that flavor patterns are primarily categorized into three types: top flavor, middle flavor, and aftertaste. These three flavor categories are relative concepts. Top flavor, middle flavor, and aftertaste generally refer to the flavors perceived 0-2 seconds after eating, 2-5 seconds after eating, and 5 seconds after eating, respectively. The "top flavor" mentioned above refers to the flavors perceived 0-5 seconds after eating, while the "middle flavor" described later refers to the flavors perceived 2 to approximately 30 seconds after eating.

[0023] In one embodiment, a Propionibacterium metabolite containing γ-Glu-ASP can be in the form of a paste, a powder, or a solution, or there is no particular limitation.

[0024] In one embodiment, a Propionibacterium metabolite containing γ-Glu-ASP contains 0.3% or more of Propionibacterium metabolite per unit dry weight. Preferably, a Propionibacterium metabolite containing γ-Glu-ASP contains 0.003% or more of γ-Glu-ASP per unit dry weight.

[0025] In the present invention, there are no specific limitations on Propionibacterium. Propionibacterium is limited to being able to take up Glu and Asp into its cells and accumulate γ-Glu-Asp within its cells. Propionibacterium can be a wild-type strain, a mutant, or a genetically recombinant strain. Mutants or genetically recombinant strains can be subjected to physical mutagenesis, such as ultraviolet light, or chemical mutagenesis, such as NTG, to enhance their ability to take up Glu and Asp.

[0026] In one embodiment, there is no particular limitation on the propionibacterium, as long as the propionibacterium can take up Glu into cells and accumulate it intracellularly, such propionibacterium include Propionibacterium sieversii, Propionibacterium acidogenicum, Propionibacterium tertii, Propionibacterium jannaschii, and the like. In a preferred embodiment, the propionibacterium is Propionibacterium sieversii, which is used to extract propionibacterial antimicrobial peptides.

[0027] In another embodiment, a method of preparing a Propionibacterium metabolite containing γ-Glu-ASP of the present invention comprises the following steps: Step S1: Preparation of Propionibacterium culture medium and activation of Propionibacterium strains; further comprising the following steps: Step S11: Prepare a Propionibacterium culture medium. A Propionibacterium culture medium without yeast powder is selected to eliminate the impact of yeast powder on the flavor of the final product. In a preferred embodiment, the carbon source of the culture medium is preferably lactose, glucose, sucrose, sodium lactate, and acetic acid. In a preferred embodiment, the nitrogen source of the culture medium is preferably a peptone such as tryptone, soy peptone, or fish meal peptone. In a preferred embodiment, the phosphate, potassium, and manganese sources of the culture medium are preferably a random combination of phosphoric acid, potassium dihydrogen phosphate, potassium chloride, potassium hydroxide, manganese sulfate, and manganese chloride, serving as the sources of the three ions. Glu and Asp are added to the prepared culture medium, where Asp represents aspartic acid and Glu represents glutamic acid. In a preferred embodiment, Glu and Asp can be present in the fermentation medium during culturing or added during the fermentation process. In a preferred embodiment, Glu and Asp are added to the culture medium between 0 and 144 hours before the end of fermentation. In a preferred embodiment, Glu and Asp are added to the culture medium between 0 and 72 hours before the end of fermentation. In a preferred embodiment, Glu and Asp are added to the culture medium 0.2 h to 14 h before the end of fermentation.

[0028] Step S12: Activate the Propionibacterium strain. Remove the ampoule tube and, after opening it, use a sterile pipette to draw 0.3-0.5 mL of sterile culture medium into the ampoule tube in a clean bench to dissolve the Propionibacterium cells. Incubate anaerobically at 37°C for 24-48 hours (an anaerobic environment, such as an anaerobic jar, is required) until visible colonies form.

[0029] Step S2: Fermenting and culturing the activated Propionibacterium strain obtained in Step S1; the culture conditions can be the same as those typically used to prepare Propionibacterium metabolites, and the culture conditions can be appropriately modified depending on the Propionibacterium used. Any method, such as fed-batch culture or continuous culture, can also be used. In a preferred embodiment, the Propionibacterium is any one of Propionibacterium sieversii, Propionibacterium acidogenicum, Propionibacterium tertii, and Propionibacterium jannaschii. In a preferred embodiment, the Propionibacterium sieversii is selected from the group consisting of Propionibacterium sieversii from which propionibacterial antimicrobial peptides are extracted. In a preferred embodiment, the Propionibacterium sieversii is placed in an incubator for submerged static culture at 20°C to 35°C. In a preferred embodiment, the Propionibacterium sieversii is placed in an incubator for submerged static culture at 28°C to 32°C. In a preferred embodiment, the Propionibacterium sieversii is placed in an incubator for submerged static culture at 29°C to 31°C.

[0030] The amount of Glu and Asp compounds added to the fermentation culture is generally 10 ppm or more of Glu, based on the final concentration in the culture medium at the time of addition. In a preferred embodiment, the amount of Glu added is 100 ppm or more. In a preferred embodiment, the amount of Glu added is 200 ppm or more. The amount of Asp added is generally 10 ppm or more. In a preferred embodiment, the amount of Asp added is 100 ppm or more. In a preferred embodiment, the amount of Asp added is 200 ppm or more. In a preferred embodiment, the amount of Asp added is 120 ppm. When adding Glu and Asp compounds, the added concentration can be determined according to the above range. There is no particular upper limit on the amount of Glu and Asp compounds used, but considering production costs, the amount of Glu and Asp compounds used can be set to less than 10,000 ppm. In a preferred embodiment, the amount of Glu and Asp compounds used is set to less than 1,000 ppm. In a preferred embodiment, the amount of Glu and Asp compounds used is set to less than 100 ppm.

[0031] Step S3: preparing propionibacterium metabolites from the propionibacterium fermentation broth prepared under the fermentation conditions of step S2; obtaining propionibacterium metabolites by centrifuging the propionibacterium fermentation broth prepared under the fermentation conditions of step S2.

[0032] The above operation yields a propionibacterium metabolite containing γ-Glu-Asp. In one embodiment, when the amount of Glu and Asp added is 200 ppm or greater, the propionibacterium metabolite contains 0.003% or greater of γ-Glu-Asp per dry weight. In another embodiment, the amount of γ-Glu-Asp added is 0.05% or greater of the dry weight of the propionibacterium metabolite starting material.

[0033] Glu and Asp added to the culture medium are taken up into the cells of Propionibacterium and converted, not only increasing the unit antibacterial activity of the Propionibacterium antimicrobial peptide by approximately 50%, but more importantly, generating the Propionibacterium metabolite γ-Glu-Asp, which has a strong top-flavor. The Propionibacterium metabolite can be an extract obtained by hot water extraction of Propionibacterium cells, or an extract obtained by digesting Propionibacterium cells with an enzyme preparation. Specifically, in a preferred embodiment, Propionibacterium cells are mixed with 50-100°C hot water in a ratio of 1:5-1:20 (w / v), extracted with stirring for 1-4 hours, and centrifuged to obtain an aqueous extract of the Propionibacterium metabolite. In another preferred embodiment, Propionibacterium cells are suspended in a pH 4.0-8.0 buffer, and 0.1-5% (w / w) protease or carbohydrase is added. Enzymatic hydrolysis is carried out at 30-60°C for 2-8 hours. The enzyme is inactivated and then centrifuged to obtain an enzymatic hydrolyzate of the Propionibacterium metabolite. Preferably, the enzyme preparation is selected from one or more combinations of neutral protease, alkaline protease, cellulase, and xylanase. More preferably, the enzyme is inactivated after the enzymatic hydrolysis at a temperature of 80-100° C. for 5-30 minutes.

[0034] In addition, the propionibacterium metabolite can be made into a paste or a powder by spray drying according to the needs of use.

[0035] The following further illustrates various details of the present invention in the form of embodiments.

[0036] Example 1: Preparation of Propionibacterium sieversii Metabolites Containing γ-Glu-ASP Step S1: Preparation of culture medium and activation of Propionibacterium sieversii strains; further comprising the following steps: Step S11: Prepare glucose culture medium; in this example, the composition of the glucose culture medium is as follows: 15 g glucose, 15 g tryptone, 6 mL sodium lactate (60% concentration), 0.25 g potassium dihydrogen phosphate, and 0.005 g manganese sulfate. Prepare the glucose culture medium according to the above recipe and dispense it into 150 mL screw-cap bottles. Autoclave the medium at 121°C for 15 minutes, cool to room temperature, and place on a sterile workbench. Adjust the initial pH of the medium to 6.5 using 1 M NaOH. Add Glu and Asp to prepare three different concentrations of glucose culture medium containing Glu and Asp: 10 ppm, 100 ppm, and 200 ppm, respectively.

[0037] Step S12: Activate the Propionibacterium bacteria. Remove the ampoule tube and, after opening it, use a sterile pipette to draw 0.3-0.5 mL of sterile culture medium into the ampoule tube in a clean bench to dissolve the Propionibacterium bacteria. Incubate the tube anaerobically at 37°C for 24-48 hours until visible colonies are formed.

[0038] Step S2: Fermentation and cultivation of Propionibacterium bacterial cell liquid; inoculating 7.5 mL of activated Propionibacterium sieversii bacterial liquid into 150 mL of glucose culture medium containing Glu and Asp compounds, and placing in an incubator at 30° C. for deep static cultivation for 144 h.

[0039] Step S3: Preparation of Propionibacterium Metabolites: The Propionibacterium sieversii fermentation broth prepared under the fermentation conditions of Step S2 is centrifuged to obtain Propionibacterium metabolites. Fermentation is continued until the pH of the Propionibacterium sieversii fermentation broth drops to approximately 4.5. The fermentation broth is then rapidly cooled to below 4°C to prevent metabolite degradation. The terminated Propionibacterium sieversii fermentation broth is placed in a centrifuge set to 4°C, a speed of ≥8,000 × g, and a centrifugation time of 15–30 minutes. After centrifugation, the supernatant is removed and carefully transferred using a sterile pipette to avoid disturbing the precipitate. The centrifuged P. shiehleri ​​cells were resuspended in 50 mL of ultrapure water to obtain the first P. shiehleri ​​cell. The cells were collected again by centrifugation and resuspended in 50 mL of ultrapure water to obtain the second P. shiehleri ​​cell. The cells were collected again by centrifugation and resuspended in 50 mL of ultrapure water to obtain the third P. shiehleri ​​cell. This process was repeated three or more times to ensure complete removal of culture medium components from the cells. The first, second, and third P. shiehleri ​​cells were resuspended in 2 mL of ultrapure water and heated at 75°C for 15 minutes. Extracts were obtained from the first, second, and third P. shiehleri ​​cells through the above process. Finally, the extracts were centrifuged to separate P. shiehleri ​​metabolites and cell residues.

[0040] Example 2 (Comparative Example) Preparation of Propionibacterium tertiotrophicum Propionibacterium metabolites.

[0041] Step S1: Preparation of culture medium and activation of Propionibacterium tertii strains; further comprising the following steps: Step S11: Prepare glucose culture medium; in this example, the composition of the glucose culture medium is as follows: 15 g glucose, 15 g tryptone, 6 mL sodium lactate (60% concentration), 0.25 g potassium dihydrogen phosphate, and 0.005 g manganese sulfate. Prepare the glucose culture medium according to the above recipe and dispense it into 150 mL screw-cap bottles. Sterilize the medium by autoclaving at 121°C for 15 minutes. Cool to room temperature and place on a sterile workbench. Adjust the initial pH of the culture medium to 6.5 using 1 M NaOH.

[0042] Step S12: Activate the Propionibacterium tertiolectans bacteria. Remove the ampoule tube and, after opening it, use a sterile pipette to draw 0.3-0.5 mL of sterile culture medium into the ampoule tube in a clean bench to dissolve the Propionibacterium tertiolectans bacteria. Anaerobically culture the tube at 37°C for 24-48 hours until visible colonies are formed.

[0043] Step S2: Fermentation culture of Propionibacterium bacterial cell liquid; 7.5 mL of activated Propionibacterium tertii bacterial liquid was inoculated into 150 mL of glucose culture medium containing Glu and Asp compounds, and placed in an incubator at 30° C. for deep static culture for 144 h.

[0044] Step S3: Preparation of Propionibacterium tert-efficiency metabolites: The Propionibacterium tert-efficiency fermentation broth prepared under the fermentation conditions of Step S2 is centrifuged to obtain Propionibacterium metabolites. Fermentation is terminated when the pH of the Propionibacterium tert-efficiency fermentation broth drops to approximately 4.5. The fermentation broth is rapidly cooled to below 4°C to prevent metabolite degradation. The terminated Propionibacterium tert-efficiency fermentation broth is placed in a centrifuge set to 4°C, a speed of ≥8,000 × g, and a centrifugation time of 15–30 minutes. After centrifugation, the supernatant is removed and carefully transferred using a sterile pipette to avoid disturbing the precipitate. The centrifuged P. tertiary cells were resuspended in 50 mL of ultrapure water to obtain the first P. tertiary cells. The P. tertiary cells were collected again by centrifugation and resuspended in 50 mL of ultrapure water to obtain the second P. tertiary cells. The P. tertiary cells were collected again by centrifugation and resuspended in 50 mL of ultrapure water to obtain the third P. tertiary cells. This process was repeated three or more times to ensure complete removal of culture medium components from the cells. The first, second, and third P. tertiary cells were resuspended in 2 mL of ultrapure water and heated at 75°C for 15 minutes. Extracts were obtained from the first, second, and third P. tertiary cells through the above process. Finally, the extracts were centrifuged to obtain P. tertiary metabolites and cell residues.

[0045] Test Example 1: Detection of γ-Glu-Asp in various Propionibacterium metabolites Determination of γ-Glu-Asp content in various Propionibacterium metabolites. This method involves fluorescently derivatizing the peptide with AQC (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) and detecting it by LC-MS, enabling determination of various amino acid contents.

[0046] To 5.0 μL of a Propionibacterium metabolite sample diluted to an appropriate concentration or 5.0 μL of a standard solution containing 1 μM γ-Glu-Asp, add 5.0 μL of ultrapure water, 5.0 μL of a 5 μM internal standard solution (labeled with a stable isotope), and 15 μL of borate buffer to form a mixture. Then, add 15 μL of the AQC reagent solution to this mixture. The mixture with the AQC reagent solution is heated at 50°C for 15 minutes, followed by the addition of 80 μL of a 0.2% formic acid solution to prepare the analytical sample.

[0047] Next, the analysis sample prepared as above was separated by reverse phase liquid chromatography and then loaded into a mass spectrometer. The separation conditions were as follows.

[0048] (1) HPLC: Agilent 2635 series; (2) Separation column: Unison UK-Phenyl, inner diameter 4.0 mm, length 200 mm, particle size 3 μm (manufactured by Imtakt); (3) Column temperature: 35°C; (4) Mobile phase A: 50 mM formic acid aqueous solution adjusted to pH 6.5 with sodium hydroxide; (5) Mobile phase B: acetonitrile; (6) Flow rate: 0.5 mL / min; (7) Elution conditions: Elution was performed using a mixture of mobile phase A and mobile phase B. The ratio of mobile phase B to the mixture was: 0 min (5%), 0 min to 15 min (5% to 45%), 15 min to 15.2 min (45% to 80%), 15.2 min to 17 min (80%), 17 min to 17.2 min (80% to 5%), and 17.2 to 25 min (5%).

[0049] Thereafter, the derivative of γ-Glu-Asp eluted under the above separation conditions is introduced into a mass analyzer and quantified based on the mass chromatogram.

[0050] (1) Mass analysis device: AB Sciex API3200 QTRAP; (2) Detection mode: Detection of selected ions (cationic mode); (3) Selected ions: Table 1.

[0051] γ-Glu-Asp derivatives were quantitatively analyzed using the analytical software Analyst Version 1.4.2. In the case of γ-Glu-Asp derivatives, Gly-d2 derivatives were used as internal standard substances for the quantitative analysis.

[0052] Test Example 2: Testing the Effect of Adding Glu and Asp to Propionibacterium sieversii In this test example 2, the content of γ-Glu-Asp contained in Propionibacterium shieldii was detected to verify the effect of adding Glu and Asp as precursors to the culture medium. The Propionibacterium shieldii tested was prepared according to Example 1.

[0053] 7.5 mL of activated Propionibacterium sieversii bacterial liquid was inoculated into 150 mL of glucose culture medium and placed in an incubator at 30° C. for deep static culture for 144 h.

[0054] Glucose culture medium composition: glucose 15 g, tryptone 15 g, sodium lactate 6 mL (concentration 60%), potassium dihydrogen phosphate 0.25 g, manganese sulfate 0.005 g.

[0055] Glucose culture medium was prepared according to the above formula and divided into 150 mL screw-cap bottles. The culture medium was sterilized by high-pressure steam at 121°C for 15 min. After cooling to room temperature, the medium was placed on a sterile operating table. The initial pH of the culture medium was adjusted to 6.5 using 1 M NaOH.

[0056] A 5% inoculum of pre-activated Propionibacterium shiehii seed liquid was inoculated into 150 mL of glucose medium, glucose medium containing Glu and Asp at a final concentration of 10 ppm, glucose medium containing Glu and Asp at a final concentration of 100 ppm, or glucose medium containing Glu and Asp at a final concentration of 200 ppm. The culture was then incubated in a submerged chamber at 30°C for 144 hours. The cells were harvested from the fermentation broth by centrifugation, the supernatant removed as much as possible, and the resulting cells resuspended in 50 mL of ultrapure water. The cells were then harvested by centrifugation again and resuspended in 50 mL of ultrapure water. This procedure was repeated three or more times to ensure complete removal of the culture medium components. The washed cells were resuspended in 2 mL of ultrapure water and heated at 75°C for 15 minutes. The above procedure yielded an extract from the Propionibacterium shiehii. The extract and cell residue were then separated by centrifugation.

[0057] An ultrafiltration membrane with a molecular weight cutoff of 10 kDa was used to remove cell debris from the Propionibacterium sieversii extract. The extracted components were subjected to the same procedures as in Example 1, first derivatized using AQC, and then the γ-Glu-Asp content in the extract was determined using LC-MS. The dry weight of the Propionibacterium sieversii cells was determined as follows: the Propionibacterium sieversii cells obtained by centrifugation were cleaned with ultrapure water and then dried in a forced air drying oven at 105°C for 2 hours. The above procedures were used to measure the γ-Glu-Asp content in a certain amount of culture medium and the weight of the Propionibacterium sieversii cells after drying, thereby calculating the γ-Glu-Asp content per unit weight of the Propionibacterium sieversii cells.

[0058] Table 2 γ-Glu-Asp content in the metabolites of Propionibacterium sieversii at different Asp addition levels

[0059] As can be seen from Table 2, when Glu and Asp are added to the culture medium, the γ-Glu-Asp content in the Propionibacterium sieversii bacteria will also increase, and at the same time, the Propionibacterium sieversii metabolites will be given a rich flavor, especially an excellent front and middle flavor.

[0060] Test Example 3: Sensory Evaluation of Propionibacterium Extract Containing γ-Glu-Asp First, a sample for sensory evaluation of a Propionibacterium extract containing γ-Glu-Asp was prepared according to the steps described below. The fermentation step was the same as in Test Example 2. 7.5 mL of activated Propionibacterium seeds were inoculated into 150 mL of glucose medium containing a final concentration of 100 ppm of γ-Glu-Asp, and then placed in a 30°C incubator for deep static culture for 144 hours. The Propionibacterium fermentation broth obtained by fermentation was then subjected to the same operation as in Test Example 2, and the γ-Glu-Asp concentration in the extract and the solid content in the extract were determined based on the components of the Propionibacterium cell extract.

[0061] The results were that the Propionibacterium extract without γ-Glu-Asp added to the culture medium was named Extract 1, in which the solid concentration was 1.2%; the Propionibacterium extract with Glu and Asp added had a γ-Glu-Asp concentration of about 900 ppm, in which the solid concentration was 0.69%, and was named Extract 2; the Propionibacterium extract with Glu and Asp added had a γ-Glu-Asp concentration of about 100 ppm, in which the solid concentration was 0.76%, and was named Extract 3.

[0062] Next, 10 professional panelists evaluated the kokumi of the samples.

[0063] Control group: Propionibacterium extract: aqueous solution containing 0.5% sodium chloride and 0.2% MSG; Sample 1: A solution containing Propionibacterium extract 1 added to the control sample to make the γ-Glu-Asp concentration reach 60 ppm; Sample 2: Add Propionibacterium extract 2 to the control sample to make the solid content the same as that in Sample 1; Sample 3: Propionibacterium extract 3 was added to the control sample to make the solid content the same as that in sample 1; It should be noted that the kokumi level of the control group was defined as 0.0, and the kokumi level of Sample 1 was defined as 4.0 during the sensory evaluation. The results are shown in the table below. The kokumi levels of Propionibacterium extracts with different γ-Glu-Asp contents were different. The evaluation results are shown in Table 3: Table 3: Sensory evaluation of kokumi taste of different samples

[0064] As shown in Table 3, a Propionibacterium extract having a kokumi taste can be prepared by adding Glu and Asp to a Propionibacterium culture medium without yeast powder. This Propionibacterium extract can impart an excellent top taste effect to food.

[0065] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple variations of the technical solution of the present invention can be made. These simple variations all fall within the scope of protection of the present invention.

[0066] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any manner unless there is any contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations. In addition, the various different embodiments of the present invention can also be combined in any manner, as long as they do not violate the concept of the present invention, and they should also be regarded as the contents disclosed by the present invention.

Claims

1. A propionibacterium metabolite containing γ-Glu-ASP, characterized in that The propionibacterium metabolite contains 0.3% or more of the propionibacterium metabolite containing γ-Glu-ASP per unit dry weight.

2. The propionibacterium metabolite containing γ-Glu-ASP according to claim 1, characterized in that The Propionibacterium in the Propionibacterium metabolite is a Propionibacterium that can take Glu and Asp into cells and accumulate γ-Glu-ASP in the cells.

3. The propionibacterium metabolite containing γ-Glu-ASP according to claim 2, characterized in that The propionibacterium in the propionibacterium metabolites are Propionibacterium sieversii, Propionibacterium acidogenicum, Propionibacterium terrei and Propionibacterium jensenii.

4. The propionibacterium metabolite containing γ-Glu-ASP according to claim 1, characterized in that The Propionibacterium metabolites contain more than 0.003% of γ-Glu-ASP per unit dry weight.

5. The propionibacterium metabolite containing γ-Glu-ASP according to claim 1, characterized in that The Propionibacterium in the Propionibacterium metabolite containing γ-Glu-ASP may be a wild strain.

6. A method for preparing the propionibacterium metabolite containing γ-Glu-ASP according to any one of claims 1 to 5, characterized in that: It includes the following steps: Step S1: Preparation of Propionibacterium culture medium and activation of Propionibacterium strains; Step S2: fermenting and culturing Propionibacterium based on the activated Propionibacterium strain obtained in step S1; Step S3: preparing propionibacterium metabolites from the propionibacterium fermentation broth prepared under the fermentation conditions of step S2.

7. The method for preparing a propionibacterium metabolite containing γ-Glu-ASP according to claim 6, characterized in that: In step S1, the culture medium is a Propionibacterium culture medium that does not contain yeast powder.

8. The method for preparing a propionibacterium metabolite containing γ-Glu-ASP according to claim 7, characterized in that: The carbon source of the culture medium is any one or more of lactose, glucose, sucrose, sodium lactate and acetic acid; the nitrogen source is any one or more of tryptone, soy peptone and fish meal peptone; the phosphate source, potassium source and manganese source are any one or a random combination of phosphoric acid, potassium dihydrogen phosphate, potassium chloride, potassium hydroxide, manganese sulfate and manganese chloride as the source of the phosphate source, potassium source and manganese source.

9. The method for preparing a propionibacterium metabolite containing γ-Glu-ASP according to claim 6, characterized in that: Glu and Asp are present in the fermentation medium when the Propionibacterium species are cultivated.

10. The method for preparing a propionibacterium metabolite containing γ-Glu-ASP according to claim 6, characterized in that: Glu and Asp were added at any time between 0 h and 144 h before the end of fermentation.

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