Determination of 4(5)-methylimidazole in soy sauce and other foods by LC-MS/MS after solid-phase extraction

A method for the determination of 4(5)-methylimidazole (4MeI) in naturally brewed soy sauce was developed for the first time using solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). SPE on silica-based reversed-phase cartridges with heptafluorobutyric acid as an ion-pairing reagent was used for the efficient cleanup of 4MeI. A multimode ODS column was employed for the chromatographic separation. To subtract the matrix effect during LC-MS/MS analysis, a standard addition method was used. The levels of 4MeI found in naturally brewed soy sauce were extremely low (ranging from <0.002 to 0.023 μg/g), whereas those in soy sauces containing caramel color were generally high (ranging from 0.43 to 4.8 μg/g). The method proved to be useful for the analysis of 4MeI in other foods such as caramel colors, drinks, and Worcestershire sauce.     

大豆酱油电渗析脱盐工艺参数对其脱盐率及品质的影响

为了降低大豆酱油的含盐量,该文利用电渗析技术对大豆酱油进行脱盐处理,通过单因素试验探讨了电压、流速、pH值对电渗析脱盐效果的影响,确定了较佳电渗析工艺。结果表明在电压为9 V,流速为2.4 cm/s,pH值为4.2,电渗析50 min时脱盐效果较好,氨基酸态氮损失较小,氨基酸态氮损失19.4%,脱盐率达到81.6%。利用氨基酸自动分析仪和气相色谱-质谱联用仪（gas chromatography-mass spectrometry）分析了电渗析前后游离氨基酸及挥发性风味物质的变化规律,结果表明电渗析过程中氨基酸及风味物质均有损失。几种苦味氨基酸如组氨酸、精氨酸、赖氨酸损失较大,分别损失了23.79%、26.39%和28.5%;电渗析过程中,酱油风味物质中醇类、酚类损失较大,尤其是4-乙基愈创木酚损失最多,但因其气味阈值较低,因此对酱油风味影响较小。研究结果为减盐酱油系列产品的生产提供了参考和依据。     

Transcriptome profiling of heat-resistant strain Bacillus licheniformis CGMCC3962 producing Maotai flavor

Although Maotai flavor liquor is exclusive due to its soy sauce flavor, knowledge of its key compound and production mechanism is still scarce until now. To gain insight into the production mechanism of soy sauce flavor, a soy sauce flavor producing strain with high efficiency and heat-resistant capability was obtained, and the metabolic mechanism of the strain was investigated with the technique of microarray profiling. Because high temperature was a key factor for soy sauce flavor production, the global gene expression of this heat-resistant strain fermented at 55 °C was analyzed. Except for the responsive increase of heat shock proteins, which maintained cell survival during heat stress, biosynthesis of cysteine was also up-regulated. In addition, some metabolites were significantly increased when cysteine was added to the fermentation medium, such as 2,3-butanediol, 3-hydroxy-2-butanone, and tetramethylpyrazine, which were important flavor compounds in soy sauce flavor liquor and might be related with soy sauce flavor. The results indicated that cysteine might play an important role in the formation of soy sauce flavor compound, and it might act as an indirect precursor or stimulator of soy sauce flavor formation. This was the first use of the microarray profiling tool to investigate the fermentative strains for Chinese traditional liquor, which would allow a deeper insight into the mechanism of the formation of soy sauce flavor compound.     

Comprehensive analytical method for the determination of hydrophilic metabolites by high-performance liquid chromatography and mass spectrometry

A method for the comprehensive analysis of hydrophilic metabolites, based on a combination of high-performance liquid chromatography and mass spectrometry, is described. We evaluated three types of stationary phases to achieve the separation of highly hydrophilic metabolites. Good chromatographic retention and separation of these metabolites were achieved on a pentafluorophenylpropyl-bonded silica column with gradient elution, using 0.1% aqueous formic acid and acetonitrile as the mobile phase. The optimized conditions allowed the comprehensive determination of the standard 49 kinds of amino acids, 6 kinds of amines, 45 kinds of organic acids, 18 kinds of nucleic bases, 5 kinds of nucleosides, and 14 kinds of nucleotides, and then the linearity, dynamic range, detection limit, and precision of the retention time and the peak area were validated. We applied this method for the targeted analysis of the components in soy sauce. The results from the quantitative determination of amino acids were compared to those obtained with an amino acid analyzer, and the accuracy was in the range between 85 and 119%. The accuracy of other detected components was confirmed to be 105-133% by the recovery rate after the addition of standard compounds. We also applied the method for the nontargeted metabolic profiling of the components in several kinds of soy sauces with the principal component analysis. They were classified by the manufacturing methods, and the components that corresponded to the differences were identified. This method could be useful for the targeted analysis of hydrophilic metabolites as well as their nontargeted metabolic profiling.     

Formation and inhibition of cholesterol oxidation products during marinating of pig feet

Cholesterol oxidation products (COPs), formed during the heating of cholesterol-rich foods, have been shown to cause cancer and coronary heart disease. The objectives of this study were to develop a GC-MS method for the determination of COPs in pig feet meat, skin, and juice during marinating and to study the formation and inhibition of COPs as affected by the incorporation of soy sauce and sugar. Results showed that an HP-5MS column could provide an adequate separation of cholesterol, 5α-cholestane (internal standard), and seven COPs, including 7α-OH, 7β-OH, 5,6β-OH, 5,6α-OH, triol, 25-OH, and 7-keto, within 15 min with a temperature-programming method. Most COPs in pig feet meat were generated at a larger amount than in pig feet skin and marinating juice over a 24 h heating period at about 100 °C. The Maillard browning index rose with increasing heating time, whereas the pH showed a slight change in marinated juice. Both reducing sugar and free amino acid contributed to the formation of Maillard reaction products. The incorporation of soy sauce and crystal sugar into fresh juice was effective in inhibiting COPs formation in pig feet, skin, and juice over a 30 min preheating period.     

Antiplatelet activity of soy sauce as functional seasoning.

In seeking the functionality of foodstuffs applicable to medicine, soy sauce was found to show antiplatelet activity. Therefore, the active components in soy sauce were purified, structurally identified, and studied for their inhibitory effects on the aggregation of human platelets. Aqueous 2-fold diluents of soy sauce inhibited platelet aggregation induced by collagen and epinephrine depending on the dilution factor. Since a basic extract with diethyl ether completely inhibited collagen-induced aggregation, it was subjected to serial extractions and multistep HPLC fractionations for purifying antiplatelet components. The finally obtained isolates were identified as 1-methyl-1,2,3,4-tetrahydro-beta-carboline and 1-methyl-beta-carboline on the basis of EI-MS, (1)H NMR, diode array, and fluorescence spectra. Their spectral data and chromatographic behaviors were the same as those of synthetic ones. 1-Methyl-1,2,3, 4-tetrahydro-beta-carboline showed mean concentrations (n = 5-6) of 4.6, 4.2, 28.6, 11.6, and 65.8 microgram/mL to produce 50% inhibition of the maximal aggregation response induced by epinephrine, platelet-activating factor, collagen, adenosine 5'-diphosphate, and thrombin, respectively. Its inhibitory effect was much greater than that of 1-methyl-beta-carboline on platelet aggregation by all the tested inducers. The quantitative HPLC analysis revealed that the significant amounts of both antiplatelet compounds were uniformly contained in commercially available soy sauce. From these results, soy sauce may be referred to as functional seasoning containing alkaloidal components with the potent preventive effect on thrombus formation.     

Differential effects of powdered whole soy milk and its hydrolysate on antiobesity and antihyperlipidemic response to high-fat treatment in C57BL/6N mice

This study was performed to investigate the beneficial effects of powdered whole soy milk and its hydrolysate, compared to the processed soy milk and its hydrolysate, on the alteration of lipid metabolism and their possible effects on antiobesity in C57BL/6N mice fed a high-fat and -cholesterol diet. The mice were divided into a control group (20% casein) and four test groups for 5 weeks: soy milk (SM, 20% soy milk protein), soy milk hydrolysate (SMH, 20% hydrolyzed soy milk protein), whole soy milk (WSM, 20% whole soy milk protein), and whole soy milk hydrolysate (WSMH, 20% whole soy milk hydrolysate protein). The body weight and adipose tissue weights were significantly lowered in SMH, WSM, and WSMH groups compared to the control group despite providing an isoenergetic diet. Plasma lipid concentrations and hepatic fatty acid synthase (FAS) and glucose-6-phosphate dehydrogenase (G6PD) activities were significantly lowered in all soy milk groups; however, the hepatic lipid contents and malic enzyme (ME) activity were only significantly lowered in the WSM and WSMH groups, compared to the control group. Data suggest that powdered WSM or WSMH appears to be more beneficial than SM or SMH in overall antiobesity and antihyperlipidemic properties following in the order WSMH/WSM, SMH, SM, and casein.     

Effects of temperature and sodium chloride concentration on the activities of proteases and amylases in soy sauce koji

This study investigated the effects of temperature and sodium chloride concentration on the proteolytic and amylolytic activities of soy sauce koji. The optimal temperatures for both protease and amylase were found in the range of 50-55 degrees C. The protease was not stable at 55 degrees C and retained only approximately 20% residual activity after incubation at 55 degrees C for 4 h. The protease was labile in sodium chloride solution, whereas the amylase was quite stable. The residual protease activity in an 18% NaCl solution was only approximately 3%. The harvested koji was mixed with 1.5 volumes of water (v/w) and incubated at 45 degrees C for 48 h; the total nitrogen and amino nitrogen contents were 1.3 and 0.56%, respectively. The results indicated that the hydrolysis of koji at the critical temperature of 45 degrees C could be employed as a rapid fermentation method to reduce the time for soy sauce manufacturing. According to this study, the combination of 5% sodium chloride and fermentation at 45 degrees C was considered as the best condition for the prohydrolysis of koji for making soy sauce. In addition, the critical temperature of 45 degrees C was very important when used in the preparation of protein hydrolysates for the flavoring industry and for the preparation of biologically active peptides.     

Enzymatic formation of ether linkage producing shoyuflavones from genistein and (+/-)-trans-epoxysuccinic acid

The production mechanism of shoyuflavones, conjugated ethers of isoflavones with tartaric acid and isolated from fermented soy sauce, was studied. In the high molecular weight fraction of the culture extract of Aspergillus oryzae, genistein was transformed into shoyuflavone B in the presence of (+/-)-trans-epoxysuccinic acid but not in the low molecular one. Asp. sojae and Asp. tamarii showed high activity similar to Asp. oryzae but none of Asp. niger, Rhizopus oligosporus, and Mucor praini did. The contents of epoxysuccinic acids in the starting materials of soy sauce and the cultures of various Asp. fungi were determined as dimethyl 2-chloro-3-hydroxysuccinate derivatives by GC-MS. Although epoxysuccinic acids were contained in Asp. oryzae, Asp. sojae, and Asp. tamarii cultures, they were not found in soybeans and wheat. A possible producing mechanism for shoyuflavones by enzymatically conjugating isoflavones to (+/-)-trans-epoxysuccinic acid with ether linkage was suggested.     

Ultraviolet spectrophotometric determination of benzoic acid in soy sauce.

Proteins and other interfering substances are precipitated from soy sauce, using sodium tungstate under acidic conditions. After centrifugation, the supernate is successively extracted with ethyl ether to isolate the benzoic acid in the organic solvent. The ethyl ether extract is washed with dilute HCl. Benzoic acid is then quantitatively determined by ultraviolet spectrophotometry. Recovery of sodium benzoate added to soy sauce ranged from 94 to 104%.     

Low molecular weight compounds responsible for savory taste of Indonesian soy sauce

Indonesian soy sauce is made using only soybeans as the nitrogenous source. Moromi obtained from fermentation of yellow soybeans using Aspergillus sojae as the starter was investigated. The fraction with molecular weights of less than 500 Da obtained by stepwise ultrafiltration was then fractionated by several chromatographic procedures, including gel filtration chromatography and RP-HPLC. Several chemical analyses, CE profiles, and taste profiles were performed to obtain the most intense umami fraction. The main components eliciting or enhancing the umami taste present in the fraction were purified and identified by protein sequencing, ESI-MS, and (1)H NMR at 400 MHz. Besides free l-glutamic acid and aspartic acid, free aromatic amino acids such as l-phenylalanine and l-tyrosine may also play an important role in impressing savory or umami taste of Indonesian soy sauce at their subthreshold concentrations and in the presence of salt and free acidic amino acids. This is reported as a new phenomenon of the so-called bitter amino acids.     

Characterization of the key aroma compounds in soy sauce using approaches of molecular sensory science

Application of aroma extract dilution analysis (AEDA) to the volatiles isolated from a commercial Japanese soy sauce revealed 30 odor-active compounds in the flavor dilution (FD) factor range of 8-4096, among which 2-phenylethanol showed the highest FD factor of 4096, followed by 3-(methylsulfanyl)propanal (methional), the tautomers 4-hydroxy-5-ethyl-2-methyl- and 4-hydroxy-2-ethyl-5-methyl-3(2H)-furanone (4-HEMF), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (4-HDF), and 3-hydroxy-4,5-dimethyl-2(5H)-furanone (sotolone), all showing FD factors of 1024. Thirteen odorants were quantified by stable isotope dilution assays, and their odor activity values (OAVs) were calculated as ratio of their concentrations and odor thresholds in water. Among them, 3-methylbutanal (malty), sotolone (seasoning-like), 4-HEMF (caramel-like), 2-methylbutanal (malty), methional (cooked potato), ethanol (alcoholic), and ethyl 2-methylpropanoate (fruity) showed the highest OAVs (>200). An aqueous model aroma mixture containing 13 odorants, which had been identified with the highest OAVs, in concentrations that occur in the soy sauce showed a good similarity with the overall aroma of the soy sauce itself. Heat treatment of the soy sauce resulted in a clear change of the overall aroma. Quantitation of selected odorants revealed a significant decrease in sotolone and, in particular, increases in 2-acetyl-1-pyrroline, 4-HDMF, and 4-HEMF induced by heating.     

Evaluation of precision estimates for fiber-dimensional and electrical hygrometers for water activity determinations

The precision of instruments used in 3 collaborative studies conducted within the Food and Drug Administration over a 4-year period (1981, 1982, 1984) for water activity (aw) determinations according to the official AOAC method is evaluated. Calibration responses of the instruments were tested for linearity over the aw range from 0.75 to 0.97. Average absolute percent difference between predicted and assigned aw values for the linear model ranged from 0.3 to 0.7% for a fiber-dimensional hygrometer (Abbeon) and 3 electrical hygrometers (Beckman, Rotronics, and Weather Measure). The calibration responses for another electrical hygrometer (Hygrodynamics) were nonlinear. The fiber-dimensional hygrometer yielded mean aw values and precision estimates that did not differ significantly from those obtained with the electrical hygrometers for (NH4)2SO4slush, KNO3 slush, sweetened condensed milk, pancake syrup, and cheese spread. However, the mean aw value for a soy sauce was 0.838 for the electrical hygrometers compared with 0.911 for the fiber-dimensional hygrometer. The fiber-dimensional hygrometer was affected by a volatile component(s) in the soy sauce that caused an erroneously high aw value. Pooled estimates of reproducibility (Sx) in the 3 studies were 0.008 for the fiber-dimensional hygrometer and 0.010 for the electrical hygrometers; these values were not significantly different from those reported in the study that verified the current official AOAC method.     

Formation of cholesterol oxidation products in marinated foods during heating

The objectives of this study were to develop a gas chromatography-mass spectrometry (GC-MS) method to analyze the contents of cholesterol oxidation products (COPs) in marinated eggs, pork, and juice and to compare the effect of heating time and soy sauce or sugar on the formation of COPs. By using a silica cartridge for purification and GC-MS with selected ion monitoring for detection, seven COPs, including 7alpha-hydroxycholesterol, 7beta-hydroxycholesterol, 5,6alpha-epoxycholesterol, 5,6beta-epoxycholesterol, 5alpha-cholestane-3beta, 5,6beta-triol, 5-cholesten-3beta-25-diol, and 7-ketocholesterol, as well as internal standard 5alpha-cholestane, were resolved within 16 min by using a HP-5MS capillary column. During marinating, the levels of most COPs followed an increasing trend with increasing heating time. However, a higher amount of COPs was generated for ground pork as compared to eggs. The incorporation of soy sauce or sugar (1 and 10%) was effective in inhibiting COPs formation, with the latter being more pronounced than the former in both marinated eggs and pork.     

Identification of 4-methylspinaceamine, a pictet-spengler condensation reaction product of histamine with acetaldehyde, in fermented foods and its metabolite in human urine

Previous study demonstrated that 4-methylspinaceamine (4-methyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine), a Pictet-Spengler condensation reaction product of histamine with acetaldehyde, is present in human urine. The current study sought to determine whether 4-methylspinaceamine is present in fermented foods; its presence might be expected since both histamine and acetaldehyde are often present in these foods. Soy sauce, fish sauce, cheese, and shao hsing wine (Chinese wine) were found to contain 4-methylspinaceamine. The concentration of 4-methylspinaceamine excreted in human urine was greatly elevated after ingestion of a meal containing soy sauce as a dietary source of 4-methylspinaceamine, demonstrating that the level of 4-methylspinaceamine in human urine was affected by dietary foods. In addition, a metabolite of 4-methylspinaceamine in human urine was investigated. An enhanced peak in the HPLC chromatogram of human urine samples after ingestion of 4-methylspinaceamine-containing foods was observed. A peak at the same retention time was also observed from a human urine sample after administration of 4-methylspinaceamine, suggesting that the peak was due to a metabolite. By comparison with the newly synthesized authentic compound, the metabolite was identified as 1,4-dimethylspinaceamine.     

Determination of fluazifop-butyl and fluazifop acid in soybeans and soybean oil using liquid chromatography with oxidative amperometric detection

A new method is described for the determination of the herbicide fluazifop-butyl, and its metabolite fluazifop acid, in soybeans and soybean oil as fluazifop acid. Liquid chromatography with amperometric detection (LC/AD) is used to determine fluazifop acid produced from the metabolism or base hydrolysis of fluazifop-butyl in soybeans and soybean oil. These foods were spiked with fluazifopbutyl at 0.05, 0.10, and 0.50 ppm and hydrolyzed with 0.2N NaOH in methanol. The hydrolysate (adjusted to pH less than or equal to 1) is extracted with dichloromethane and the extract is washed with 1.0% NaHCO3. The NaHCO3 is acidified to pH less than or equal to 1 and extracted with dichloromethane; the partitioning is repeated 2 more times. The dichloromethane is removed, mobile phase solvent is added, and aliquots are injected onto a PRP-1 liquid chromatographic column; fluazifop acid is separated from coextracted compounds and detected at an applied potential of + 1.25 V, using an amperometric electrochemical detector in the oxidation mode. Recoveries ranged from 69 +/- 6.5 to 101 +/- 18% and from 72 +/- 7.5 to 88 +/- 11% for soybeans and soybean oil, respectively. Accuracy of these recoveries was confirmed by use of 14C-radiolabeled fluazifop-butyl and by liquid scintillation spectrometry of the 14C-fluazifop acid released.     

Reaction of tryptophan with carbohydrates: identification and quantitative determination of novel beta-carboline alkaloids in food

The formation of various carbohydrate-derived beta-carbolines was observed when model reactions of tryptophan with glucose were studied by means of HPLC with diode array detection, as well as by means of HPLC-MS. Isolation of these compounds and subsequent characterization by tandem mass spectrometry and NMR spectroscopy led to the identification of diastereomeric 1-(1,3,4,5-tetrahydroxypent-1-yl)-9H-pyrido[3,4-b]indoles (1a/b), 1-(1,4,5-trihydroxypent-1-yl)-9H-pyrido[3,4-b]indoles (2a/b), and E/Z isomers of 1-(1,5-dihydroxypent-3-en-1-yl)-9H-pyrido[3,4-b]indole (3a/b). HPLC-MS was used to prove the presence of these novel beta-carboline alkaloids in various food samples. In addition, quantitative determination of beta-carbolines 1a, 1b, and 2a/b in numerous products was achieved by means of HPLC with fluorometric detection. Concentrations ranged from 12 to 1922 microg/L for 1a and 1b and from 3 to 644 microg/L for 2a/b. The highest concentrations of all carbohydrate-derived beta-carbolines under study were found in ketchup, soy sauce, and fish sauce.     

Hydrophobicity of bitter peptides from soy protein hydrolysates

Soy peptides were characterized for flavor, chemical properties, and hydrophobicity to investigate their relationships with bitterness. Five peptide fractions ranging in average molecular mass from 580 to 11300 Da were fractionated by ultrafiltration from two commercial soy protein hydrolysates. The bitterness of fractionated peptides was related to molecular mass, with maximum bitterness observed at approximately 4000 Da for one hydrolysate and 2000 Da for the other. The bitterness increased as the peptide M(w) decreased to 3000 Da for the first hydrolysate and to 2000 Da for the second one and then decreased as the peptide M(w) decreased below 1000 Da. The peptide fraction with molecular mass of <1000 Da showed the lowest bitterness for both. The hydrophobicity data based on Q values do not support Ney's Q rule as a predictor of bitterness for soy peptides.     

Measurement of conjugated linoleic acid (CLA) in CLA-rich potato chips by ATR-FTIR spectroscopy

A conjugated linoleic acid (CLA)-rich soy oil has been produced by photoisomerization of soy oil linoleic acid. Nutritional studies have shown that CLA possesses health benefits in terms of reducing certain heart disease and diabetes risk factors. Potato chips are snacks that are readily produced in the CLA-rich soy oil containing CLA levels similar to those of the oil used for frying. The objective of this study was to develop an FTIR method to rapidly determine the CLA content of oil in potato chips. Photoirradiated soy oil samples with ～25% total CLA were mixed with control soy oil, and 100 soy oil samples with total CLA levels ranging from 0.89 to 24.4% were made. Potato chips were fried using each of these 300 g CLA rich soy oil mixtures at 175 °C for approximately 3 min. Duplicate GC-FID fatty acid analyses were conducted on oil extracted from each batch of potato chips. The chip samples were ground and then scanned using ATR-FTIR spectroscopy with the aid of a high-pressure clamp, and duplicate spectra of each sample were averaged to obtain an average spectrum. Calibration models were developed using PLS regression analysis. These correlated the CLA isomer concentrations of potato chips obtained by GC-FID fatty acid analysis with their corresponding FTIR spectral features. The calibration models were fully cross validated and tested using samples that were not used in the calibration sample set. Calibrations for total CLA, trans,trans CLA, trans-10,cis-12 CLA, trans-9,cis-11 CLA, cis-10,trans-12 CLA, and cis-9,trans-11 CLA had coefficients of determinations (R2v) between 0.91 and 0.96 and corresponding root-mean-square error of prediction (RMSEP) ranging from 0.005 to 1.44. The ATR-FTIR technique showed potential as a method for the determination of the CLA levels in unknown potato chip samples.