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.     

Effects of riboflavin and fatty acid methyl esters on cholesterol oxidation during illumination

The effect of riboflavin or fatty acid methyl esters on cholesterol photooxidation was studied. Samples containing cholesterol, either alone or in combination with riboflavin or fatty acid methyl esters, were illuminated at 25 degrees C in an incubator for 28 days. The various cholesterol oxidation products (COPs) and cholesterol were analyzed by gas chromatography-mass spectrometry (GC-MS), and riboflavin was determined by HPLC. Results showed that the presence of riboflavin or fatty acid methyl esters facilitated production of COPs and degradation of cholesterol, and the degradation fits a first-order model. The COPs formed during light storage included 7 alpha-OH, 7 beta-OH, 7-keto, 3,5-cholestadien-7-one, 5,6alpha-EP, and 5,6beta-EP. The addition of riboflavin caused formation of 3,5-cholestadien-7-one through dehydration of 7-keto, whereas in the presence of docosahexaenoic acid methyl ester, the formation of 5,6alpha-EP or 5,6beta-EP was favored. Riboflavin was more effective for generation of COPs than fatty acid methyl esters.     

Inhibition of cholesterol oxidation products (COPs) formation in emulsified porcine patties by phenolic-rich avocado (Persea americana Mill.) extracts

The effect of phenolic-rich extracts from avocado peel on the formation of cholesterol oxidation products (COPs) in porcine patties subjected to cooking and chill storage was studied. Eight COPs (7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, 20α-hydroxycholesterol, 25-hydroxycholesterol, cholestanetriol, 5,6β-epoxycholesterol, and 5,6α-epoxycholesterol) were identified and quantified by GC-MS. The addition of avocado extracts (～600 GAE/kg patty) to patties significantly inhibited the formation of COPs during cooking. Cooked control (C) patties contained a larger variety and greater amounts of COPs than the avocado-treated (T) counterparts. COPs sharply increased in cooked patties during the subsequent chilled storage. This increase was significantly higher in C patties than in the T patties. Interestingly, the amount of COPs in cooked and chilled T patties was similar to those found in cooked C patties. The mechanisms implicated in cholesterol oxidation in a processed meat product, the protective effect of avocado phenolics, and the potential implication of lipid and protein oxidation are thoroughly described in the present paper.     

Kinetic studies of cholesterol oxidation as inhibited by stearylamine during heating

The formation of cholesterol oxidation products (COPs) during heating in the presence of stearylamine at 140 degrees C was analyzed by high-performance liquid chromatography (HPLC) and kinetically studied by use of nonlinear regression models. Results indicated that the COPs concentration increased with increasing heating time, and stearylamine was shown to reduce both oxidation and degradation rates of cholesterol. Without stearylamine, the highest rate constant (per hour) was observed for epoxidation (545.4), followed by free radical chain reaction (251.0), reduction (147.3), dehydration (95.8), triol dehydrogenation (4.7), degradation (0.34), triol formation (0.31), and dehydrogenation (0.13). With stearylamine, the epoxidation and free radical chain reaction rates could be reduced by about 800- and 3.4-fold, respectively, and triol formation during oxidation could be completely inhibited. In addition, the reactions for reduction, dehydration, degradation, and dehydrogenation could proceed slower in the presence of stearylamine. The kinetic model developed in this study can be used to predict the inhibition of COPs formation by stearylamine during heating of cholesterol.     

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.     

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.     

Kinetic model for studying the effect of quercetin on cholesterol oxidation during heating

Inhibition of the heat-induced cholesterol oxidation at 150 degrees C by incorporation of quercetin was kinetically studied. Results showed that without quercetin, the cholesterol oxidation products (COPs) concentration increased with increasing heating time. A low amount (0.002%, w/w) of quercetin was effective in inhibiting the formation of COPs during the initial heating period (< or =30 min) at 150 degrees C. However, after prolonged heating (30-120 min), a low antioxidant activity was observed because of the degradation of quercetin. When using nonlinear regression models for kinetic study of cholesterol oxidation in the absence of quercetin, the epoxidation showed the highest rate constant (h(-1) = 683.1), followed by free radical chain reaction (h(-1) = 453.5), reduction (h(-1) = 290.3), dehydration (h(-1) = 155.5), triol dehydrogenation (h(-1) = 5.35), dehydrogenation (h(-1) = 0.68), thermal degradation (h(-1) = 0.66), and triol formation (h(-1) = 0.38). However, in the presence of quercetin, the reaction rate constants (h(-1)) for epoxidation (551.4), free radical chain reaction (111.7), and thermal degradation (0.28) were reduced greatly. The kinetic model developed in this study can be used to predict the inhibition of COPs by quercetin during the heating of cholesterol.     

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.     

Generation of odorous acyloins by yeast pyruvate decarboxylases and their occurrence in sherry and soy sauce

Volatile acyloins (alpha-hydroxy ketones) were obtained by condensing either aldehydes with pyruvate or 2-keto acids with acetaldehyde in a reaction catalyzed by yeast pyruvate decarboxylases (EC 4.1.1.1). Odor qualities and threshold values of 34 acyloins were evaluated, and 23 of them possessed distinct flavor properties. Sherry and soy sauce flavors were analyzed: 2-hydroxy-3-pentanone and 3-hydroxy-2-pentanone were identified in soy sauce for the first time; these and 2-hydroxy-5-methyl-3-hexanone and 3-hydroxy-1-phenyl-2-butanone were isolated from sherry for the first time. The biocatalytic efficiencies of crude pyruvate decarboxylase preparations from Zygosaccharomyces bisporus, Saccharomyces cerevisiae, Kluyveromyces lactis, and Kluyveromyces marxianus were compared. Product yields comparable to those of conversions with purified pyruvate decarboxylase demonstrated the suitability of crude enzyme extracts as cost-effective biocatalysts in acyloin formation. Conversion rates of >50% showed that the potential of this type of enzyme to catalyze the formation of aliphatic acyloins has been underestimated before.     

Effect of soy milk characteristics and cooking conditions on coagulant requirements for making filled tofu

The amount of coagulant added to soy milk is a critical factor for tofu-making; particularly it affects the textural properties of tofu. Earlier research indicated that the critical point of coagulant concentration (CPCC) is a characteristic parameter of soy milk and could be used as an effective indicator of optimal coagulant concentration (OCC) for making filled tofu. The objective of this study was to investigate the possible correlations between CPCC and the characteristics of soy milk made from various soybean samples and the effect of soy milk cooking and dilution conditions on CPCC. CPCC was determined by a titration method. Calcium chloride and magnesium chloride were used as coagulants. Soy milk characteristics including solid, protein, phytate, pH, titratable acidity, mineral content, and 11S/7S protein and these characteristics as affected by heating rate, heating time, and sequence of dilution and heating were studied. The results showed that the CPCC was significantly (p < 0.05) positively correlated with phytate content (grams per gram of protein), pH, and 7S protein content but negatively correlated with protein content, 11S protein content, 11S/7S ratio, titratable acidity, and original calcium content. Within the same soybean material, more proteins required more coagulant, but higher protein concentration during cooking resulted in less coagulant required by each gram of protein during coagulation. The CPCC decreased with increasing soy milk heating time or decreasing heating rate. The sequence of heating and diluting for preparing soy milk also had an effect on CPCC.     

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.     

Identification and quantification of cholesterol and cholesterol oxidation products in different types of Iberian hams

Cholesterol and cholesterol oxidation products (COPs) were determined in four different groups of dry-cured Iberian hams, based on the feeding received by pigs and their degree of crossbreeding. After lipid extraction, GC-FID for cholesterol determination and GC-MS to analyze COPs were used. Cholesterol content ranged from 30 to 34 mg/100 g of muscle. Some of the COPs analyzed, such as 7alpha-hydroxycholesterol, 7beta-hydroxycholesterol, and 7-ketocholesterol, were detected in all of the samples. The major cholesterol oxide was 7-ketocholesterol; its concentration ranged from 57 to 71 microg/100 g of muscle. The content of cholesterol and cholesterol oxides in intramuscular lipids of hams was not affected by diet or crossbreeding of Iberian pigs.     

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。     

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.     

Analysis of early lipid oxidation in smoked, comminuted pork or poultry sausages with spices

Dynamic headspace/gas chromatography-mass spectrometry (GC-MS), front-face fluorescence spectroscopy, and a gas-sensor array technique (electronic nose) have previously detected lipid oxidation in pork back fat or mechanically recovered poultry meat earlier than or at the same time as a sensory panel. The present study was focused on measurement of early lipid oxidation in a more complicated product (freeze-stored, smoked sausages with spices). During the storage time, formation of components contributing to rancid odor and flavor (e.g., hexanal and 1-penten-3-ol) could be monitored with dynamic headspace/GC-MS. The GC-MS data also showed a decrease in 2-furancarboxaldehyde, which could indicate loss of Maillard type components often associated with acidic or meat odor and flavor. The fluorescence spectra were difficult to interpret, probably due to the simultaneous influence from increasing levels of lipid oxidation products and loss of fluorescent Maillard or spice components. The gas-sensor array responses were dominated by signals from, e.g., spice and smoke compounds.     

接种贝莱斯芽孢杆菌SW5菌株对发酵鳀鱼鱼露品质的影响

为探讨接种贝莱斯芽孢杆菌SW5菌株对发酵鳀鱼鱼露的影响,以贝莱斯芽孢杆菌SW5为唯一发酵菌株,以低值鳀鱼为原料,测定鱼露发酵过程中氨基酸态氮(AA-N)、挥发性盐基氮(TVB-N)、pH值、总酸含量,并使用顶空固相微萃取结合气相色谱-质谱联用仪(HS-SPME-GC-MS)测定发酵结束后发酵液的挥发性风味物质。结果表明,在发酵期间,利用SW5菌株发酵的处理组1在发酵第6天时AA-N含量最高,为0.76 g·100 mL^-1,达到市售二级鱼露标准。3种处理组和舟山商品鱼露中共检测出挥发性风味成分82种,主要是醇、酸、醛、酮、呋喃、烷烃及其他类化合物,接种组中的挥发性风味物质(40种)较未接种组(35种)有所增加。综上,接种贝莱斯芽孢杆菌SW5菌株发酵鱼露,能缩短发酵时间,增加风味物质种类,该菌株可用作海洋蛋白质源发酵精深加工的优良微生物菌株。     

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.     

基于电子鼻的鱼露香气品质识别

为了识别鱼露的品质,并为缩短发酵周期的工艺优选提供理论依据.利用电子鼻对7种鱼露样品的挥发性气味进行了分析,并与项空-气质联用( GC-MS)和感官分析结果进行比较.结果表明:鱼露香气成分复杂,工艺改良对气味影响很大,电子鼻18个金属传感器能很好地将不同样品的气味进行识别.以传统发酵原汁鱼露为标样,电子鼻分析结果表明,加曲改良工艺的4号样品与标样香气最为接近,相似系数达87.8％,该结果与GC-MS数据和感官分析结果一致,可为鱼露速酿工艺的优选提供参考.     

Influence of pH and ionic strength on heat-induced formation and rheological properties of soy protein gels in relation to denaturation and their protein compositions

The influence of pH and ionic strength on gel formation and gel properties of soy protein isolate (SPI) in relation to denaturation and protein aggregation/precipitation was studied. Denaturation proved to be a prerequisite for gel formation under all conditions of pH and ionic strength studied. Gels exhibited a low stiffness at pH >6 and a high stiffness at pH <6. This might be caused by variations in the association/dissociation behavior of the soy proteins on heating as a function of pH, as indicated by the different protein compositions of the dissolved protein after heating. At pH 3-5 all protein seems to participate in the network, whereas at pH >5 less protein and especially fewer acidic polypeptides take part in the network, coinciding with less stiff gels. At pH 7.6, extensive rearrangements in the network structure took place during prolonged heating, whereas at pH 3.8 rearrangements did not occur.     

Liquid chromatographic determination of protein acid hydrolysate content in blended soy sauce

Five methods were investigated for the determination of levulinic acid in soy sauce to determine the addition of protein hydrolysate, mainly acid hydrolysate of defatted soybeans. Best results were obtained by using liquid chromatography (LC) with 0.004 M HClO4 as the mobile phase and bromcresol purple as a post-column reagent. An innovative LC method with 0.1% H3PO4 as eluant was developed for determination of levulinic acid at 280 nm in soy sauce. This was the most time-saving method.