Method for inhibiting meiqx production by oyster umami peptide

Abstract

This invention provides a method for inhibiting MeIQx formation using oyster umami peptides, belonging to the field of bioactive peptide technology. The method includes the following steps: S1, cleaning, peeling, chopping, and homogenizing tilapia, cuttlefish, and shrimp meat to prepare fish paste; adding oyster umami peptides to the fish paste and then frying to obtain a sample; S2, dissolving a certain amount of threonine, creatinine, and glucose in phosphate buffer to obtain solution A; mixing a certain amount of soybean oil with diglycerides containing oyster umami peptides to obtain solution B; thoroughly mixing solutions A and B in a sealed high-temperature reaction flask, cooling after an oil bath, to obtain a MeIQx formation simulation system; S3, detecting the MeIQx content, carbonyl value, intermediate substances, and precursor substances in the MeIQx formation simulation system solution. This invention inhibits MeIQx formation by blocking precursor degradation and intermediate product generation at multiple targets, as well as stabilizing protein structure during thermal processing.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and in particular to a method for inhibiting the formation of MeIQx by oyster umami peptides. Background Technology

[0002] Heterocyclic aromatic amines (HAAs) are a class of highly mutagenic and carcinogenic compounds formed through the Maillard reaction during the high-temperature processing of protein-rich foods (such as fish and meat products). More than 30 heterocyclic amines have been identified, among which 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is one of the compounds with the highest carcinogenic risk. MeIQx exhibits extremely strong mutagenic activity in the Ames test and has been classified as a Group 2A carcinogen (probably carcinogenic to humans) by the International Agency for Research on Cancer (IARC). Epidemiological studies have shown a significant positive correlation between long-term consumption of high-temperature processed meat products containing MeIQx and the risk of developing various cancers, including colorectal cancer, gastric cancer, and breast cancer. Therefore, effectively inhibiting the formation of MeIQx in high-temperature processed foods has become a crucial issue that urgently needs to be addressed in the field of food safety.

[0003] The formation of MeIQx primarily depends on the Maillard reaction pathway. Under high-temperature conditions, reducing sugars (such as glucose) in food undergo a Strecker degradation reaction with amino acids (especially threonine), generating reactive α-dicarbonyl intermediates such as glyoxal, acetone aldehyde, and formaldehyde. These dicarbonyl compounds further condense with creatine to form an imidazoquinoxaline skeleton, ultimately generating MeIQx. Therefore, regulating the Maillard reaction pathway and eliminating reaction intermediates are key strategies for inhibiting MeIQx formation. Based on this mechanism, numerous studies have focused on finding MeIQx inhibitors with high safety and good thermal stability from natural sources for application in food processing.

[0004] In recent years, bioactive peptides derived from food proteins have gradually attracted researchers' attention due to their high safety, easy absorption by the human body, and diverse functions. Oysters are rich in high-quality protein, and their enzymatic hydrolysates contain a variety of flavor peptides with good umami characteristics and processing adaptability. Among them, a tripeptide CF3 (sequence Cys-Phe-Phe) derived from oysters has been reported to have significant MeIQx inhibitory activity. Its mechanism of action may involve cysteine ​​residues in the peptide acting through active thiol groups (…). The oyster umami peptide undergoes a nucleophilic addition reaction with an α-dicarbonyl intermediate to form a stable thioacetal adduct, thereby blocking the synthetic pathway of MeIQx. However, a systematic and scientific evaluation method for the MeIQx inhibitory effect of this oyster umami peptide is currently lacking. Therefore, a method for inhibiting MeIQx formation by oyster umami peptide is needed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for inhibiting the formation of MeIQx by oyster umami peptides, thereby solving the existing technical problems.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for inhibiting MeIQx generation by oyster umami peptides includes the following steps: S1. Sample preparation: Tilapia, cuttlefish and shrimp meat were cleaned, skinned, chopped and homogenized to prepare fish paste. Oyster umami peptides were added to the fish paste and then fried to obtain the sample. S2. Construction of the simulation system: A certain amount of threonine, creatine and glucose were dissolved in phosphate buffer to obtain solution A. A certain amount of soybean oil was mixed with diglyceride containing oyster umami peptide to obtain solution B. Solution A and solution B were thoroughly mixed in a sealed high-temperature reaction flask, cooled after an oil bath, to obtain the MeIQx generation simulation system. S3. Substance detection: The MeIQx content, carbonyl value, intermediate substances and precursor substances in the simulated system solution generated by MeIQx are detected.

[0007] Furthermore, in step S1, 1g of oyster umami peptide is added for every 500g of fish paste.

[0008] Furthermore, in step S1, the frying temperature is 165°C and the frying time is 8 minutes.

[0009] Further, in step S2, the amount of threonine added is 0.4 mmol, the amount of creatinine added is 0.4 mmol, the amount of glucose added is 0.2 mmol, and the amount of phosphate buffer added is 5 mL.

[0010] Furthermore, in step S2, the amount of soybean oil added is 200 μL, and the amount of diglyceride containing 10 mg of oyster umami peptide added is 5 mL.

[0011] Furthermore, in step S2, the oil bath temperature is 185℃ and the oil bath time is 2-8 minutes.

[0012] Furthermore, in step S3, the MeIQx content is detected using ultra-high performance liquid chromatography-tandem mass spectrometry, and the carbonyl valence is detected using the 2,4-dinitrophenylhydrazine colorimetric method.

[0013] Furthermore, in step S3, the intermediate substances include formaldehyde, glyoxal, acetaldehyde, 2,5-dimethylpyrazine, and acrolein.

[0014] Furthermore, the contents of formaldehyde and glyoxal were detected using derivatization-high performance liquid chromatography, the contents of acetaldehyde were detected using headspace gas chromatography, the contents of 2,5-dimethylpyrazine were detected using headspace solid-phase microextraction-gas chromatography, and the contents of acrolein were detected using DNPH derivatization-high performance liquid chromatography.

[0015] Furthermore, in step S3, the precursor substances are detected using high-performance liquid chromatography, and the precursor substances include glucose, threonine, and creatine.

[0016] During heat processing, frying disrupts the secondary structure of surimi proteins, degrades polysaccharides, and triggers lipid oxidation, leading to the release of MeIQx precursors and the accumulation of carbonyl intermediates, creating favorable conditions for MeIQx formation. Oyster umami peptides first establish an inhibitory basis through structural protection. By reconstructing the intermolecular hydrogen bond network and maintaining the integrity of the protein conformation, they reduce the transition from α-helices to β-sheets and random coils. This invention revealed differences in these changes in protein secondary structure through FTIR spectroscopy. Simultaneously, oyster umami peptides target the characteristics of different surimi species, protecting cuttlefish surimi polysaccharides, inhibiting the degradation of shrimp chitin, and slowing lipid oxidation in tilapia and cuttlefish surimi, thereby reducing the release of precursors and the formation of intermediates. This aligns with the protective mechanism of natural bioactive peptides on heat-processed protein systems. Furthermore, oyster umami peptides enhance the stability of the surimi colloidal system by regulating intermolecular interactions, significantly improving protein-water interactions, hydrophobic interactions, and hydrogen bond strength, increasing the absolute value of the zeta potential of surimi after frying, and improving the hydrophilicity of the protein interface. The increase in the absolute value of the zeta potential enhances the stability of the protein colloidal system, reduces protein aggregation, and thus reduces abnormal protein degradation, further blocking the generation of precursors and intermediates. Ultimately, oyster umami peptides directly intervene in the chemical reaction pathway of MeIQx generation, inhibiting the degradation of precursor substances, reducing carbonyl oxidation levels, and reducing the generation of key intermediates, thereby blocking the condensation cyclization process of MeIQx at multiple targets.

[0017] The present invention, by adopting the above-described technical solution, has the following beneficial effects: This invention constructs a multi-dimensional evaluation system covering MeIQx content, carbonyl value, intermediate substances, and precursor substances. Through the simultaneous measurement and correlation analysis of the above multiple indicators, it can not only accurately evaluate the inhibitory effect, but also systematically reveal the target and inhibitory pathway of oyster umami peptides.

[0018] In this invention, oyster umami peptides are selected as natural inhibitors, which have a significant inhibitory effect on the formation of MeIQx, the degradation of intermediate substances and the formation of precursor substances, thereby reducing the impact of MeIQx generated during high-temperature food processing on the human body. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the MeIQx content detection results in the simulation system of this invention; Figure 2 This is a schematic diagram of the carbonyl valence content detection results in the simulation system of this invention; Figure 3 This is a schematic diagram of the detection results of intermediate substance content in the simulation system of this invention; Figure 4 This is a schematic diagram of the detection results of precursor substance content in the simulation system of this invention; Figure 5 This is a schematic diagram showing the detection results of MeIQx content in the fish paste of this invention; Figure 6 This is a schematic diagram showing the detection results of carbonyl content in fish paste according to the present invention; Figure 7 This is a schematic diagram showing the detection results of intermediate substances in the fish paste of the present invention; Figure 8 This is a schematic diagram showing the detection results of precursor substance content in the fish paste of the present invention; Figure 9 This is a schematic diagram of the molecular interaction detection results of the present invention; Figure 10 This is a schematic diagram of the zeta potential detection results of the present invention; Figure 11 This is a schematic diagram of the contact angle detection results of the present invention; Figure 12 This is a schematic diagram of the FTIR detection results of the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are merely to provide the reader with a thorough understanding of one or more aspects of the present invention, and these aspects of the invention can be implemented even without these specific details.

[0021] Example A method for inhibiting MeIQx generation by oyster umami peptides includes the following steps: S1. Sample Preparation: Tilapia, cuttlefish, and shrimp meat were cleaned, skinned, chopped, and homogenized to prepare fish paste. Each portion was weighed at 500 g, and three groups were established: Unprocessed group: No flavoring agents were added and no frying was performed; these were named Unprocessed-Tilapia, Unprocessed-Cuttlefish, and Unprocessed-Shrimp. Control group (CK group): No flavoring agents were added; the fish paste was fried at 165℃ for 8 min; these were named CK-Tilapia, CK-Cuttlefish, and CK-Shrimp. Oyster umami peptide (CF3) group: 1 g of oyster umami peptide (CF3) was added to each portion of fish paste, and then the mixture was fried at 165℃ for 8 min; these were named CF3-Tilapia, CF3-Cuttlefish, and CF3-Shrimp.

[0022] S2. Construction of the simulated system: Dissolve 0.4 mmol threonine, 0.4 mmol creatinine, and 0.2 mmol glucose in 5 mL phosphate buffer to obtain solution A. Mix 200 μL soybean oil with 5 mL of diglyceride containing 10 mg CF3 to obtain solution B. Mix solutions A and B thoroughly in a 50 mL sealed high-temperature reaction flask, and incubate at 185°C in an oil bath for 2, 4, 6, and 8 min, then immediately terminate the reaction by incubating in an ice-water bath.

[0023] S3, Substance Detection MeIQx content detection Take 3 mL of the reaction mixture, add 3 mL of ethyl acetate, vortex for 5 min, centrifuge at 4000 r / min for 10 min, collect the upper organic phase, repeat the extraction 3 times, combine the extracts and concentrate under vacuum at 40℃ to near dryness, dissolve the residue in 1 mL of methanol, filter through a 0.22 μm nylon filter membrane and analyze using a Waters ACQUITY UPLC I-Class-Xevo TQ-S ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) (Waters Corporation, Massachusetts, USA). Chromatographic conditions: C18 column (2.1 mm × 100 mm, 1.7 μm, Waters Corporation, USA), column temperature 40℃, mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile, gradient elution program was 0-2 min (5% B), 2-8 min (5%-30% B), 8-10 min (30%-95% B), 10-12 min (95% B), 12-12.5 min (95%-5% B), 12.5-15 min (5% B), flow rate 0.3 mL / min, injection volume 5 μL. Mass spectrometry conditions: Electrospray ionization (ESI) source, positive ion mode, capillary voltage 3 kV, ion source temperature 150℃, desolvation gas temperature 450℃, desolvation gas flow rate 800 L / h; in multiple reaction monitoring (MRM) mode, the precursor ion of MeIQx was m / z 214.1, the quantitative daughter ion was m / z 131.1, the qualitative daughter ion was m / z 199.1, the cone voltage was 40 V, and the collision energy was 25 eV for quantitative ion and 20 eV for qualitative ion.

[0024] Carbonyl valence detection The carbonyl valence content (X) was determined using the 2,4-dinitrophenylhydrazine (DNPH) colorimetric method. 1 mL of the simulated system solution was taken and diluted to 10 mL with deionized water. 1 mL of the diluted solution was mixed with an equal volume of DNPH reaction solution, and the mixture was reacted at 40 °C for 20 min. After cooling, 8 mL of a 2.5% n-butanol solution containing 0.02% KOH was added, and the mixture was centrifuged at 3000 r / min for 5 min. The supernatant was then measured at 420 nm to determine the absorbance. The carbonyl valence content X was calculated using the formula: X(meq / kg)=A(1545×m×V1×V2)Eq.(7) In the formula: A is the absorbance value; m represents the mass (g); 1545 is the average value of the equivalent absorption coefficients of various carbonyl compounds per milligram; V1 is the volume of the diluent (mL); and V2 is the total volume of the diluted sample (mL).

[0025] Intermediate substance detection Formaldehyde and glyoxal were determined by derivatization-high performance liquid chromatography (HPLC). One mL of sample was added to an equal volume of 0.05 mol / L DNPH acetonitrile-phosphoric acid solution. The mixture was derivatized in a 60℃ water bath in the dark for 30 min. Then, 2 mL of n-hexane was added to remove lipid-soluble impurities. The mixture was centrifuged at 8000 r / min for 10 min. The supernatant was filtered through a 0.22 μm organic phase filter membrane. A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used with methanol-water (70:30, v / v) as the mobile phase. The flow rate was set to 1 mL / min, and the detection wavelength was 360 nm. Standard curves were plotted using formaldehyde and glyoxal standard derivatization solutions, respectively.

[0026] Acetaldehyde content was determined by headspace gas chromatography (HS-GC). 2 mL of sample was placed in a 20 mL headspace vial, sealed, and equilibrated at 70 °C for 30 min. The chromatographic conditions were as follows: DB-23 column (60 m × 0.32 mm × 0.25 μm, Agilent Technologies, Inc., Santa Clara, USA); vaporization chamber temperature 200 °C; flame ionization detector (FID) temperature 250 °C; hydrogen flow rate 40 mL / min; air flow rate 400 mL / min; column flow rate 1.3 mL / min; split ratio 1:1; temperature program: 40 °C held for 5 min, then increased to 180 °C at 10 °C / min and held for 4 min.

[0027] The content of 2,5-dimethylpyrazine was determined by headspace solid-phase microextraction-gas chromatography (HS-SPME-GC). 2 mL of sample was placed in a headspace vial, and a 50 / 30 μm DVB / CAR / PDMS solid-phase microextraction head (Supelco, Bellefonte, USA) was used for extraction in a 55℃ water bath for 40 min, followed by injection and elution for 3 min. The chromatographic conditions were as follows: PEG-20M column (30 m × 0.32 mm × 0.25 μm, Dalian Zhonghuida Scientific Instruments Co., Ltd., China); vaporization chamber and detector temperature: 250℃; hydrogen flow rate: 40 mL / min; air flow rate: 400 mL / min; column flow rate: 2 mL / min; split ratio: 1:1; temperature program: 40℃ held for 5 min, increased to 80℃ at 2℃ / min held for 2 min, then increased to 210℃ at 8.1℃ / min held for 5 min. A standard curve was plotted using 2,5-dimethylpyrazine standards.

[0028] The acrolein content in the simulated system was determined using DNPH derivatization-high performance liquid chromatography (HPLC), with the same procedures as for formaldehyde and glyoxal derivatization and pretreatment. Chromatographic conditions: C18 reversed-phase column (250 mm × 4.6 mm, 5 μm), methanol-water (60:40, v / v) as the mobile phase, flow rate set at 1 mL / min, and detection wavelength at 360 nm. A standard curve was plotted using acrolein standards following the same derivatization method.

[0029] Precursor substance detection The contents of glucose, threonine, and creatinine in the simulated system were determined by high performance liquid chromatography (HPLC). 1 mL of sample was taken, and 2 mL of acetonitrile was added to precipitate the protein. After vortexing for 2 min, the mixture was centrifuged at 10000 r / min for 10 min. The supernatant was filtered through a 0.22 μm filter before analysis. Glucose was detected using a refractive index detector (RID). An Agilent ZORBAX NH2 column (4.6 mm × 250 mm, 5 μm, Agilent Technologies, Inc., Santa Clara, USA) was used, with acetonitrile-water (75:25, v / v) as the mobile phase, a flow rate of 1 mL / min, and a column temperature of 30 °C. Threonine and creatinine were analyzed using o-phthalaldehyde derivatization-fluorescence detection (OPA) with a C18 column (4.6 mm × 250 mm, 5 μm). Mobile phase A was 0.02 mol / L sodium acetate buffer, and mobile phase B was methanol-acetonitrile (1:1, v / v). The gradient elution program was 0–15 min (10%–40% B), 15–20 min (40%–100% B), 20–25 min (100% B), and 25–30 min (100%–10% B), at a flow rate of 1 mL / min. The excitation wavelength was 340 nm, and the emission wavelength was 450 nm. Standard curves were plotted using glucose, threonine, and creatinine standard solutions for quantitative analysis.

[0030] MeIQx generation simulation results showed that oyster umami peptide (CF3) inhibited MeIQx by 65.5%. Figure 1 As shown, the inhibition rate of carbonyl value is 50%, as Figure 2 As shown, the inhibition rates for the key intermediates 2,5-dimethylpyrazine and acrolein were 57.3% and 60.1%, respectively. Figure 3 As shown, the inhibition rates of creatinine and threonine degradation reached 48.9% and 81.5%, respectively. Figure 4 As shown, this means that CF3 can directly inhibit the formation of MeIQx through the chemical reaction pathway.

[0031] Experimental studies on fish paste showed that MeIQx accumulated significantly after frying, following a pattern of shrimp paste > cuttlefish paste > tilapia paste; CF3 could reduce the MeIQx content in the above-mentioned systems, such as... Figure 5 As shown, the inhibition rates of key intermediates exceeded 47%, 53%, and 62%, respectively. Figure 6 As shown. Meanwhile, CF3 inhibited the carbonyl values ​​of cuttlefish surimi and shrimp surimi by 48.6% and 59.5%, respectively. Figure 7 As shown, it can delay the degradation and transformation of precursor substances, and can still maintain a high residual amount of precursor substances after frying for 8 minutes, such as Figure 8 As shown, this indicates that CF3 can block the MeIQx generation pathway from multiple targets.

[0032] From the perspective of molecular interactions, CF3 enhances protein-water interactions, such as... Figure 9 As shown, this increases the absolute value of the Zeta potential, such as Figure 10 As shown, this reduces the contact angle and improves the hydrophilicity of the protein interface, such as... Figure 11 As shown, the release of precursors and carbonyl intermediates was suppressed, such as Figure 6-8 As shown.

[0033] FTIR results showed that the untreated group of fish paste had a stable hydrogen bond structure (3300 cm⁻¹). - 1 ) and typical α-helical characteristic peak (1650 cm⁻¹) - 1 ),like Figure 12 As shown in A, D, and G; the secondary structure of the protein in group CK was disrupted, with α-helices transforming into β-sheets and random coils, resulting in a significant increase in transmittance, as shown in Figures 1-3. Figure 12 As shown in B, E, and H; while CF3 maintained a stable hydrogen bond network, inhibiting the conversion of α-helices to β-sheets and random coils, thus reducing the infrared transmittance of fried fish paste, as shown in Figures B, E, and H; Figure 12 As shown in C, F, and I, intermolecular hydrogen bonds are reconstructed to restore hydration capacity. Amide II band analysis indicates that the peak positions in the CK group show a blue shift compared to the untreated group, as shown in... Figure 12 As shown in B, E, and H, the peak positions of group CF3 all exhibit a redshift, such as Figure 12 As shown in C, F, and I, CF3 enhances intermolecular interactions and maintains the integrity of the protein conformation. CF3 exhibits differential absorption peaks in different types of cuttlefish surimi, such as the CO stretching peak (1014 cm⁻¹) caused by CF3 in cuttlefish surimi polysaccharides. -1 The transmittance of ) decreases, such as Figure 12 As shown in F, the chitin COC stretching peak of shrimp surimi (1030-1100 cm⁻¹) is... -1 The transmittance of ) drops to 20%, such as Figure 12 As shown in Figure I, the fatty ester bond peaks of tilapia paste and cuttlefish (1740 cm⁻¹)-1 Enhancement, such as Figure 12 As shown in C and F.

[0034] The above results indicate that oyster umami peptides inhibit the degradation of polysaccharides and chitin, as well as lipid oxidation, by stabilizing protein structure, reconstructing hydrogen bond networks, and enhancing molecular interactions. This reduces the release and accumulation of MeIQx precursors and key carbonyl intermediates, thereby blocking the MeIQx generation pathway at the molecular structure level and achieving highly efficient inhibition. The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for inhibiting the formation of MeIQx by oyster umami peptides, characterized in that: Includes the following steps: S1. Sample preparation: Tilapia, cuttlefish and shrimp meat were cleaned, skinned, chopped and homogenized to prepare fish paste. Oyster umami peptides were added to the fish paste and then fried to obtain the sample. S2. Construction of the simulation system: A certain amount of threonine, creatine and glucose were dissolved in phosphate buffer to obtain solution A. A certain amount of soybean oil was mixed with diglyceride containing oyster umami peptide to obtain solution B. Solution A and solution B were thoroughly mixed in a sealed high-temperature reaction flask, cooled after an oil bath, to obtain the MeIQx generation simulation system. S3. Substance detection: The MeIQx content, carbonyl value, intermediate substances and precursor substances in the simulated system solution generated by MeIQx are detected.

2. The method for inhibiting MeIQx generation by oyster umami peptides according to claim 1, characterized in that: In step S1, 1g of oyster umami peptide is added for every 500g of fish paste.

3. The method for inhibiting MeIQx generation by oyster umami peptides according to claim 1, characterized in that: In step S1, the frying temperature is 165℃ and the frying time is 8 minutes.

4. The method for inhibiting MeIQx generation by oyster umami peptides according to claim 1, characterized in that: In step S2, the amount of threonine added is 0.4 mmol, the amount of creatinine added is 0.4 mmol, the amount of glucose added is 0.2 mmol, and the amount of phosphate buffer added is 5 mL.

5. The method for inhibiting MeIQx generation by oyster umami peptides according to claim 1, characterized in that: In step S2, the amount of soybean oil added is 200 μL, and the amount of diglyceride containing 10 mg of oyster umami peptide added is 5 mL.

6. The method for inhibiting MeIQx generation by oyster umami peptides according to claim 1, characterized in that: In step S2, the oil bath temperature is 185℃ and the oil bath time is 2-8 minutes.

7. The method for inhibiting MeIQx generation by oyster umami peptides according to claim 1, characterized in that: In step S3, the MeIQx content is detected by ultra-high performance liquid chromatography-tandem mass spectrometry, and the carbonyl valence is detected by 2,4-dinitrophenylhydrazine colorimetric method.

8. The method for inhibiting MeIQx generation by oyster umami peptides according to claim 1, characterized in that: In step S3, the intermediate substances include formaldehyde, glyoxal, acetaldehyde, 2,5-dimethylpyrazine, and acrolein. The content of formaldehyde and glyoxal is detected by derivatization-high performance liquid chromatography, the content of acetaldehyde is detected by headspace gas chromatography, the content of 2,5-dimethylpyrazine is detected by headspace solid-phase microextraction-gas chromatography, and the content of acrolein is detected by DNPH derivatization-high performance liquid chromatography.

9. The method for inhibiting MeIQx generation by oyster umami peptides according to claim 1, characterized in that: In step S3, the precursor substances are detected by high performance liquid chromatography, and the precursor substances include glucose, threonine and creatine.

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