High-simulation umami substance detection method based on tongue organs and application of high-simulation umami substance detection method

Abstract

The invention discloses a high-simulation umami substance detection method based on tongue organs and an application of the high-simulation umami substance detection method. The method comprises the following steps: culturing tongue organs in vitro in a 3D manner to obtain taste cells with a taste recognition function, dyeing by using a calcium ion fluorescent probe, exogenously adding a to-be-detected umami solution, and determining the signal change of calcium ions in the taste cells based on flow cytometry, thereby recognizing the umami strength of umami substances and the synergistic effect of the umami substances. The platform can directly measure the strength of the umami substances and the interaction effect of various umami substances, and compared with the existing electronic tongue and biological electrode sensor technology, the platform is closer to the real taste perception of human beings, and a more accurate umami detection method is provided.

Description

Technical Field

[0001] The present invention relates to the technical field of taste detection, and particularly relates to a method and application for detecting high-fidelity umami substances based on tongue organoids. Background Art

[0002] Currently, the electronic tongue is a commonly used taste detection technology that simulates human taste perception through chemical sensors and is widely used in fields such as food. It can quickly analyze basic taste components, such as sweet, sour, bitter, salty, etc. However, when measuring umami substances, especially in the synergistic effects between umami substances, there are significant deviations. The electronic tongue usually cannot accurately reflect the enhancement effect when multiple umami substances act together, and this synergistic effect is often ignored or cannot be recognized. In addition, there are also errors when the electronic tongue measures umami intensity and taste changes, and it cannot fully simulate the complex taste experience of humans. Although technologies based on biological sensors such as taste cells have been developed, which detect through electrode signal conversion, they essentially cannot directly capture intracellular signals, so there are still deficiencies in sensitivity and reproducibility. Summary of the Invention

[0003] In view of the above problems existing in the prior art, the present invention provides a method and application for detecting high-fidelity umami substances based on tongue organoids. The present invention is based on mouse tongue organoids, and the change of calcium ion signal is measured by flow cytometry to evaluate the effect of taste stimulation.

[0004] The technical solution of the present invention is as follows:

[0005] The first object of the present invention is to provide a method for culturing tongue organoids, which comprises the following steps:

[0006] (1) Cut the lingual papilla region into small pieces, mechanically dissociate them into minced meat, digest with trypsin, centrifuge and filter through a sieve after terminating the digestion to obtain a cell suspension;

[0007] (2) Inoculate the cells of the cell suspension prepared in step (1) into an organoid culture medium for culture for 7 - 14 days; Harvest the organoids or passage them at 7 - 14 days according to the organoid state. When passing the generation, collect the organoids by centrifuging at 1200 rpm for 3 minutes, then incubate them in 0.25% trypsin-EDTA at 37°C for 10 minutes; After terminating the digestion, filter through a 300-mesh sieve to prepare a single-cell suspension, and inoculate the cells into the taste organoid culture medium in a 24-well plate; Repeat the passage culture in a cell incubator;

[0008] (3) Identify the maturely cultured organoids, perform cell identification with antibodies corresponding to the umami receptors Krt8, T1R1, T1R3, mGluR1, and / or mGluR4. After determining the expression of umami receptors, prepare tongue organoids with the expression of umami receptors.

[0009] In one embodiment of the present invention, in step (1), the cells are digested with 0.25% trypsin for 15 min and passed through a 300-mesh sieve.

[0010] In one embodiment of the present invention, in step (2), the culture medium comprises a basal medium and additives. The basal medium is DMEM / F12 medium. The additives include 200 - 400 ng / mL of R-spondin-1, 100 - 200 ng / mL of Noggin, 1 - 5% of B27, 50 - 100 ng / mL of epidermal growth factor, 1 - 2% of N 2 , 100× penicillin-streptomycin, 1 - 2% Glutamax, 1 - 2 mmol / L of HEPES, 10 - 20 μM of Y-27632, and 3 - 10% of frozen Matrigel. (Note that the percentages mentioned above are volume percentages).

[0011] Factors that can promote the development of taste cells, such as specific growth factors or hormones, are added during the culture of tongue tissues.

[0012] In one embodiment of the present invention, the cultivation of tongue organoids should not only be morphologically similar to real tongue tissues but also meet certain standards in terms of function, especially in detecting umami substances and expressing taste receptors. The tongue organoids need to maintain an active taste perception function and be able to respond to various taste stimuli. Therefore, the cultivated tongue organoids should have structural maturity, such as taste bud-like structures, correctly arranged taste receptor cells, and a simulation of neural networks. Only the tongue organoids that meet these standards can be used for subsequent taste detection and functional analysis.

[0013] The second object of the present invention is to provide a tongue organoid prepared by the above cultivation method.

[0014] The third object of the present invention is to provide a high-fidelity umami substance detection method based on the above tongue organoid, comprising the following steps:

[0015] (1) Digest the tongue organoid with trypsin to obtain a taste cell suspension.

[0016] (2) Stain the taste cell suspension with the Flur-4 calcium ion fluorescent probe to obtain a taste cell suspension loaded with the calcium ion fluorescent probe.

[0017] (3) Prepare a sodium glutamate standard solution and a solution of the umami substance to be measured. Mix the sodium glutamate standard solution and the solution of the umami substance to be measured with the suspension of taste cells loaded with the calcium ion fluorescent probe obtained in step (2) respectively. Immediately after mixing, perform cell fluorescence detection by flow cytometry, and compare the umami intensity of the umami substance by counting the fluorescence signals of the target cell population.

[0018] In one embodiment of the present invention, in step (1), the tongue-like organ is incubated in 0.25% trypsin-EDTA at 37 °C for 10 minutes. After terminating the digestion, it is passed through a 300-mesh sieve to obtain a suspension of taste cells.

[0019] In one embodiment of the present invention, in step (2), the suspension of taste cells is resuspended in 1 ml of Fluo-4 staining solution and incubated at 37 °C for 30 - 60 minutes under light-shielded conditions.

[0020] In one embodiment of the present invention, the volume concentration of the Fluo-4 fluorescent probe in the Fluo-4 staining solution is 0.05 - 0.5%.

[0021] In one embodiment of the present invention, in step (3), the umami substances include amino acid substances with umami, nucleotide substances, peptides, and amino acid derivatives.

[0022] In one embodiment of the present invention, the amino acid substances are one or more of L-glutamic acid, L-aspartic acid, L-proline, L-tyrosine, L-glycine, L-alanine, L-phenylalanine, L-valine, L-leucine, and L-isoleucine; the nucleotide substances are one or more of 5'-guanylic acid (GMP), 5'-inosinic acid (IMP), 5'-adenylic acid (AMP), 5'-uridylic acid (UMP), and 5'-cytidylic acid (CMP); the peptides are one or more of fish peptides, meat peptides, and peptides in fermentation products; the amino acid derivatives are one or more of N-acetylglutamic acid derivatives, γ-glutamine derivatives, L-cysteine derivatives, L-methionine derivatives, and L-tryptophan derivatives.

[0023] The beneficial technical effects of the present invention are as follows:

[0024] By detecting the responses of taste cells in the organoids to different taste substances, the present invention can effectively measure the intensity of umami substances and their synergistic effects. Compared with the electronic tongue technology, it has obvious advantages in terms of accuracy and closeness to human taste perception. By directly measuring the calcium ion signal, the present invention realizes the rapid and accurate detection of taste responses and is applicable to fields such as food and condiment development. The present invention has the following advantages:

[0025] 1. Detection results are closer to human true taste perception: By using mouse tongue organoids to simulate the human taste system, the detection results of the present invention are more consistent with human taste perception, overcoming the problem of large differences between existing electronic tongue technologies and actual perception, especially being more accurate when detecting the intensity and synergistic effect of umami substances.

[0026] 2. High sensitivity and precision: By directly measuring the changes in calcium ion signals of taste cells using flow cytometry, the present invention provides a highly sensitive and precise measurement method for taste responses, capable of quickly and directly reflecting the degree of response of taste cells to taste stimuli.

[0027] 3. Wide applicability: The present invention is not only applicable to the detection of single umami substances, but also can effectively evaluate the synergistic effect between multiple umami substances, meeting the R & D needs in fields such as food and seasonings, and having broad application prospects.

[0028] 4. Potential to replace electronic tongue technology: Compared with existing electronic tongues, the umami detection sensor of the present invention has greater physiological relevance and more reference value for detection results, so it has the potential to replace electronic tongue technology and become a new generation of taste detection technology. Brief Description of the Drawings

[0029] Figure 1 It is the umami detection principle based on tongue organoids.

[0030] Figure 2 It is a real - shot picture of the growth of tongue organoids.

[0031] Figure 3 Receptor identification of tongue organoids by immunofluorescence.

[0032] Figure 4 It is the standard curve for umami detection based on tongue organoids.

[0033] Figure 5 It is the detection results of umami substances and synergistic umami enhancement based on tongue organoids.

[0034] Figure 6 It is the detection results of umami substances and synergistic umami enhancement by a professional sensory evaluation panel.

[0035] Figure 7 It is the detection results of umami substances and synergistic umami enhancement by a voltammetric electronic tongue.

[0036] Figure 8 It is the detection results of umami substances and synergistic umami enhancement by a potentiometric electronic tongue.

[0037] Figure 9 It is the PCA analysis results of four detection methods. Detailed Embodiments

[0038] The present invention will be specifically described below in conjunction with the accompanying drawings and embodiments.

[0039] For those embodiments in which specific experimental steps or conditions are not indicated, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments whose manufacturers are not indicated, they are all conventional reagent products that can be obtained through commercial purchase.

[0040] The mice were from Liaoning Changsheng Biotechnology Co., Ltd., with the variety of C57 and 3 weeks old.

[0041] The formulation of the MRS liquid medium in the following embodiments is as follows: The basal medium includes DMEM / F12 medium, and the added factors include 200 ng / mL of R-spondin-1, 100 ng / mL of Noggin, 2% of B27, 50 ng / mL of epidermal growth factor, 1% of N 2 , 100× penicillin-streptomycin, 1% Glutamax, 1 mmol / L of HEPES, 10 μM of Y-27632, and 5% of cryo Matrigel (the percentages shown are volume percentages).

[0042] The umami substances in the following embodiments can be selected from one or several of the commonly used umami substances, such as sodium glutamate, disodium inosinate, umami peptide (sequence: EP: Glutamic acid Glu - Proline Pro).

[0043] Example 1

[0044] A high-fidelity umami substance detection method based on tongue organoids includes the following steps:

[0045] (1) Cell separation of tongue tissue extraction. Cut the tongue papilla area into small pieces, mechanically dissociate the tissue into minced meat, digest it with 0.25% trypsin for 15 minutes, centrifuge after terminating digestion, and pass through a 300-mesh sieve to obtain a cell suspension.

[0046] (2) Organoid culture. Inoculate the cell suspension in a 24-well plate and culture it with organoid medium; according to the density of the growing organoids, change the medium every two days and culture it in a cell incubator for 14 days; harvest the organoids at 14 days, take some organoids for identification and umami detection, and use the remaining part for passage; during passage, collect the organoids by centrifuging at 1200 rpm for 3 minutes, then incubate them in 0.25% trypsin-EDTA at 37 °C for 10 minutes; after terminating digestion, pass through a 300-mesh sieve to prepare a single-cell suspension, and inoculate the cells in the taste organoid medium in a 24-well plate; repeat the passage culture in a cell incubator.

[0047] (3) Identification of organoids, such as Figure 2-3As shown, the matured cultured organoids are identified. After cell identification is performed using antibodies corresponding to umami receptors such as Krt8, T1R1, T1R3, mGluR1, and mGluR4, and the expression of umami receptors is confirmed, the detection of umami substances is carried out.

[0048] (4) Preparation of a suspension of taste cells loaded with a calcium ion fluorescent probe. The organoids that have undergone umami receptor identification are collected by centrifugation at 1200 rpm for 3 minutes, and incubated in 0.25% trypsin-EDTA at 37 °C for 10 minutes; after terminating the digestion, they are passed through a 300-mesh sieve to prepare a single-cell suspension, which is stained with the Flur-4 calcium ion fluorescent probe to obtain a suspension of taste cells loaded with the calcium ion fluorescent probe.

[0049] (5) Detection and evaluation of umami substances. Standard solutions of sodium glutamate, disodium inosinate, and EP umami peptide at 0, 5, and 10 mM are prepared respectively. By mixing the standard solution of the umami substance to be tested with the suspension of taste cells loaded with the calcium ion fluorescent probe obtained in step (4), cell fluorescence detection is immediately carried out by flow cytometry after mixing. The umami intensity of umami substances is compared by counting the fluorescence signals of the target cell population.

[0050] Example 2

[0051] A highly simulated umami substance detection method based on tongue organoids includes the following steps:

[0052] (1) Extraction of tongue tissue and cell separation. The tongue papilla area is cut into small pieces, the tissue is mechanically dissociated into a minced meat-like state, digested with 0.25% trypsin for 15 minutes, and after terminating the digestion, centrifuged and passed through a 300-mesh sieve to obtain a cell suspension.

[0053] (2) Organoid culture. The cell suspension is inoculated in a 24-well plate and cultured with an organoid medium; according to the density of the growing organoids, the medium is changed every two days and cultured in a cell incubator for 14 days; at 14 days, the organoids are harvested, part of the organoids are used for identification and umami detection, and the remaining part is used for passage; during passage, the organoids are collected by centrifugation at 1200 rpm for 3 minutes, then incubated in 0.25% trypsin-EDTA at 37 °C for 10 minutes; after terminating the digestion, they are passed through a 300-mesh sieve to prepare a single-cell suspension, and the cells are inoculated in the taste organoid medium in a 24-well plate; repeated passage culture is carried out in a cell incubator.

[0054] (3) Identification of organoids. The matured cultured organoids are identified. After cell identification is performed using antibodies corresponding to umami receptors such as Krt8, T1R1, T1R3, mGluR1, and mGluR4, and the expression of umami receptors is confirmed, the detection of umami substances is carried out.

[0055] (4) Preparation of taste cell suspension loaded with calcium ion fluorescent probe: The organoids identified for umami receptors were collected by centrifugation at 1200 rpm for 3 minutes, and incubated in 0.25% trypsin-EDTA at 37°C for 10 minutes; after terminating the digestion, passed through a 300-mesh sieve to prepare a single-cell suspension, and stained with Flur-4 calcium ion fluorescent probe to obtain a taste cell suspension loaded with calcium ion fluorescent probe.

[0056] (5) Detection and evaluation of synergistic umami enhancement: Standard solutions of sodium glutamate, disodium inosinate, and EP umami peptide mixed in pairs at equal concentrations of 5 mM and 10 mM were prepared respectively. By mixing the standard solution of the umami substance to be tested with the taste cell suspension loaded with calcium ion fluorescent probe obtained in step (4), cell fluorescence detection was immediately carried out by flow cytometry after mixing. The umami intensity of the umami substances was compared by counting the fluorescence signals of the target cell population.

[0057] Comparative Example 1

[0058] (1) Sample preparation: Standard solutions of sodium glutamate (MSG), disodium inosinate (IMP), and EP umami peptide at 0 mM, 5 mM, and 10 mM were prepared, and single solutions and mixed solutions of pairwise combinations were prepared respectively. The samples were numbered in random order to avoid preconception of the evaluators.

[0059] (2) Sensory evaluation: A trained sensory evaluation panel was selected, and the umami intensity of each sample was evaluated using a 9-point scale method (1 is extremely weak, 9 is extremely strong). After the evaluation was completed, the data was collected and the umami differences between different samples were analyzed, focusing on evaluating whether the pairwise mixed solutions showed significant synergistic umami enhancement effects.

[0060] Comparative Example 2

[0061] (1) Sample preparation: Standard solutions of sodium glutamate (MSG), disodium inosinate (IMP), and EP umami peptide at 0 mM, 5 mM, and 10 mM were prepared, and single solutions and mixed solutions of pairwise combinations were prepared respectively.

[0062] (2) Voltammetric electronic tongue determination: Detection was carried out using a voltammetric electronic tongue instrument. Each sample was injected into the sensor system in turn. The electronic tongue recorded the umami signals of each sample through different taste sensors to generate sensing data related to human taste perception. Each sample was measured at least 3 times repeatedly to ensure the accuracy and repeatability of the data.

[0063] (3) Data analysis:

[0064] By analyzing the signal output generated by the electronic tongue, the umami intensity and taste patterns of different samples are compared. The focus is on analyzing whether the pairwise mixed solutions exhibit a synergistic umami-enhancing effect between umami substances, and evaluating the umami-enhancing effects of different combinations.

[0065] Comparative Example 3

[0066] (1) Sample preparation: Prepare standard solutions of sodium glutamate (MSG), disodium inosinate (IMP), and EP umami peptide at 0 mM, 5 mM, and 10 mM, respectively, and make single solutions and pairwise combined mixed solutions.

[0067] (2) Potentiometric electronic tongue measurement: Use a potentiometric electronic tongue instrument for detection. Inject each sample into the sensor system in turn. The electronic tongue records the umami signals of each sample through different taste sensors, generating sensing data related to human taste perception. Each sample is measured at least 3 times to ensure the accuracy and repeatability of the data.

[0068] (3) Data analysis:

[0069] By analyzing the signal output generated by the electronic tongue, the umami intensity and taste patterns of different samples are compared. The focus is on analyzing whether the pairwise mixed solutions exhibit a synergistic umami-enhancing effect between umami substances, and evaluating the umami-enhancing effects of different combinations.

[0070] To evaluate the sensitivity and response characteristics of the sensor, the taste organoids are stimulated with MSG solutions of different concentrations, and the concentration range includes 10 -7 M, 10 -5 M, 10 -3 M, 5×10 -3 M, 10 -2 M, 5×10 -2 M, 10 -1 M, and 2×10 -1 M. Flow cytometry is used to record the changes in the intracellular calcium ion signal intensity in taste organoids at different concentrations. By analyzing the relationship between the signal intensity and the concentration of umami substances, it is found that the change in signal intensity shows a good correlation with the concentration of umami substances, showing a concentration-dependent response. According to the experimental results as Figure 4 shown, in the concentration range from 10 -7 M to 2×10 -1 M, the change in signal intensity is linearly correlated with the concentration of umami substances, and the correlation coefficient reaches 99.05%. This result indicates that the umami detection sensor based on tongue organoids can efficiently and accurately detect umami substances and has excellent detection performance.

[0071] The umami and synergistic umami enhancement results of sodium glutamate (MSG), disodium inosinate (IMP), and EP umami peptide at 0 mM, 5 mM, and 10 mM in Examples 1-2 were analyzed, and the results are as Figure 5 shown, Figure 5 A corresponds to Example 1, Figure 5 B-C correspond to Example 2; the results show that in a single-substance system, the signal feedback of the taste biosensor at a higher concentration (10 mM) is significantly stronger than that at a lower concentration (5 mM), indicating that the sensor has good detection performance in a single-substance system. For the detection of the synergistic umami effect, at high and low concentrations, a synergistic umami enhancement effect was exhibited between IMP and MSG and EP, while no synergistic effect was observed between MSG and EP. These results indicate that there is a significant synergistic umami effect between IMP and MSG, and EP. The umami detection sensor based on the tongue-like organ of the present invention can effectively analyze the synergistic effect of umami substances and has good sensitivity and accuracy.

[0072] In Comparative Example 1, as Figure 6 shown, the umami and synergistic umami enhancement results of sodium glutamate (MSG), disodium inosinate (IMP), and EP umami peptide at 0 mM, 5 mM, and 10 mM were analyzed by sensory evaluation. With the increase in concentration, the umami intensity of the three substances increased significantly. A significant umami enhancement effect was observed between IMP and MSG. In the concentration range of 5 mM and 10 mM, the umami of the mixture of IMP and MSG was enhanced by 366.7±72.1% and 288.9±28.4% compared with that of MSG alone. Similarly, a significant umami enhancement effect was also observed between IMP and EP. In the concentration range of 5 mM and 10 mM, the umami of the mixture of IMP and EP was enhanced by 328.2±88.1% and 289.5±42.5% compared with that of EP alone. No synergistic umami effect was observed between MSG and EP. The results showed a similar trend to those detected by the umami detection sensor based on the tongue-like organ.

[0073] In Comparative Examples 2 and 3, as Figure 7-8 shown, for the umami substances in a single system, both the voltammetric electronic tongue and the potentiometric electronic tongue could detect obvious umami signals. However, for the synergistic umami enhancement effect of two umami substances, neither of these two electronic tongues could recognize the synergistic umami enhancement effect.

[0074] In addition, through PCA analysis of the four detection methods, the results are as Figure 9 shown, and the results show that the umami detection sensor based on the tongue-like organ has a high similarity to human sensory evaluation, indicating that the umami detection sensor based on the tongue-like organ has the ability to be closer to the real taste perception of humans compared with the electronic tongues on the market.

[0075] The embodiments provided above are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit the order of their execution. Obvious improvements made by those skilled in the art in combination with the existing well-known general knowledge also fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for culturing tongue organoids, characterized in that: The steps include: (1) Cut the papilla area of ​​the tongue into small pieces and mechanically dissociate it into minced meat, digest it with trypsin, and after terminating the digestion, centrifuge and sieve to obtain a cell suspension; (2) inoculating the cells obtained from the cell suspension in step (1) into an organoid culture medium and culturing for 7-14 days; harvesting the organoids or subculturing them at 7-14 days depending on the state of the organoids; during subculturing, collecting the organoids by centrifugation at 1200 rpm for 3 minutes, and then incubating them in 0.25% trypsin-EDTA at 37°C for 10 minutes; after terminating the digestion, passing through a 300-mesh sieve to prepare a single-cell suspension, and inoculating the cells in a taste organoid culture medium in a 24-well plate; and repeating the subculturing in a cell culture incubator; (3) The mature organoids are identified by using antibodies corresponding to Krt8, T1R1, T1R3, mGluR1 and / or mGluR4 umami taste receptors to identify the cells. After confirming the expression of umami taste receptors, tongue organoids expressing umami taste receptors are prepared.

2. The culture method according to claim 1, characterized in that In step (1), 0.25% trypsin was used for digestion for 15 min; and the mixture was passed through a 300-mesh sieve.

3. The culture method according to claim 1, characterized in that In step (2), the culture medium includes a basal culture medium and added factors; the basal culture medium is DMEM / F12 culture medium; the added factors include 200-400 ng / mL of R-spondin-1, 100-200 ng / mL of Noggin, 1-5% by volume of B27, 50-100 ng / mL of epidermal growth factor, 1-2% of N2, 100× penicillin-streptomycin, 1-2% Glutamax, 1-2 mmol / L of HEPES, 10-20 μM of Y-27632 and 3-10% frozen Matrigel.

4. A tongue organoid produced by the culture method according to any one of claims 1 to 3.

5. A highly simulated umami substance detection method based on the tongue organoid according to claim 4, characterized in that: The steps include: (1) Digesting the tongue organoids with trypsin to prepare a taste cell suspension; (2) staining the taste cell suspension with Flur-4 calcium ion fluorescent probe to obtain a taste cell suspension loaded with calcium ion fluorescent probe; (3) preparing a sodium glutamate standard solution and a solution of a umami substance to be tested, and mixing the sodium glutamate standard solution and the solution of the umami substance to be tested with the taste cell suspension loaded with the calcium ion fluorescent probe obtained in step (2), respectively, and immediately performing cell fluorescence detection by flow cytometry after mixing, and comparing the umami intensity of the umami substance by counting the fluorescence signals of the target cell population.

6. The detection method according to claim 5, characterized in that: In step (1), the tongue organoids are incubated in 0.25% trypsin-EDTA at 37°C for 10 minutes, and after terminating the digestion, the organoids are passed through a 300-mesh sieve to obtain a taste cell suspension.

7. The detection method according to claim 5, characterized in that: In step (2), the taste cell suspension is resuspended in 1 ml of Fluo-4 staining solution and incubated at 37° C. for 30-60 minutes in the dark.

8. The detection method according to claim 7, characterized in that: The volume concentration of Fluo-4 fluorescent probe in Fluo-4 staining solution is 0.05-0.5%.

9. The detection method according to claim 5, characterized in that: In step (3), the umami substances include amino acid substances, nucleotide substances, peptides and amino acid derivatives having umami taste.

10. The detection method according to claim 9, characterized in that: The amino acid substances are one or more of L-glutamic acid, L-aspartic acid, L-proline, L-tyrosine, L-glycine, L-alanine, L-phenylalanine, L-valine, L-leucine, and L-isoleucine; the nucleotide substances are one or more of 5′-guanylic acid, 5′-inosinic acid, 5′-adenylic acid, 5′-uridylic acid, and 5′-cytidylic acid; the peptides are one or more of fish peptides, meat peptides, and peptides in fermentation products; the amino acid derivatives are one or more of N-acetylglutamate derivatives, γ-glutamine derivatives, L-cysteine ​​derivatives, L-methionine derivatives, and L-tryptophan derivatives.

Images