Metabolite profiling of Cheonggukjang, a fermented soybean paste,during fermentation by gas chromatography-mass spectrometry and principal component analysis

Metabolite profiling by applying principal component analysis (PCA) to a GC–MS data set was performed to assess changes in a select number of pre-defined metabolites during the fermentation of Cheonggukjang. A total of 20 amino acids, 12 organic acids, and nine fatty acids were found as targeted metabolites in Cheonggukjang. As fermentation proceeded, the levels of most amino acids decreased during the early stage of fermentation, but increased in the later part of fermentation. On the other hand, amounts of fatty acids increased throughout the fermentation process, while those of most organic acids, except for tartaric acid, decreased. A PCA score plot illustrated good separation of the Cheonggukjang samples according to fermentation period by combining principal component 1 (PC 1) (36.8%) with PC 2 (30.3%). Tryptophan, citric acid, β-alanine, itaconic acid, 2-hydroxyglutaric acid, γ-aminobutyric acid, leucine, malic acid, and tartaric acid were the major components that differentiated the various samples.     

Production of volatile compounds by Lactobacillus sakei from branched chain α-keto acids

Lactobacillus sakei belongs to the main flora of raw fermented sausages and is used as starter culture. Bacterial starter cultures can convert amino acids to α-keto acids by aminotransferases. These α-keto acids are the precursors of aroma active aldehydes, alcohols and carboxylic acids. In this study the formation of aldehydes, alcohols and carboxylic acids from leucine, isoleucine, valine and the corresponding α-keto acids are analysed in model fermentations with two different strains of L sakei. In the absence and upon addition of leucine, isoleucine and valine they produced 1 μg/ml 3-methylbutanoic, 0.2 μg/ml 2-methylbutanoic acid and 3 μg/ml 2-methylpropanoic acid, respectively. Upon addition of α-ketoisocaproic acid, α-keto-3-methyl-pentanoic acid or α-ketoisovaleric acid the amount of the corresponding carboxylic acid was increased to 40 μg/ml 3-methylbutanoic acid, 20 μg/ml 2-methylbutanoic acid and 35 μg/ml 2-methylpropanoic acid. The response patterns of the strains and amounts of carboxylic acids produced were similar. This behaviour was typical when compared with other strains of L. sakei and suggests general lack of transaminase activity and a limit in the transport of branched chain amino acids and their conversion to volatiles, some of which can contribute to the aroma of fermented sausages.     

蛇龙珠葡萄酒中挥发性有机酸风味的研究

采用液-液微萃取和气相色谱串联质谱的方法分析了不同挥发性有机酸对蛇龙珠葡萄酒风味的贡献,并分析了蛇龙珠葡萄酒发酵过程中主要挥发酸的变化规律。研究结果表明,蛇龙珠葡萄酒中浓度高于其阈值的挥发酸有:乙酸、异丁酸、丁酸、异戊酸、己酸和辛酸,这几种挥发酸主要来源于酵母的酒精发酵过程,其中异戊酸是蛇龙珠葡萄酒中香气强度最大的挥发酸。      

Effects of time of grape pomace fermentation and distillation cuts on the chemical composition of grape marcs

 The effects of fermentation time and distillation cuts on the composition of distillates in terms of ethanal, 1,1-diethoxyethane, methanol, 1-butanol, 2-butanol, 1-propanol, 2-methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, ethyl acetate, ethyl 2-hydroxypropanoate, 3-methylbutyl acetate, hexyl acetate, 2-phenylethyl acetate, hexanol, trans-2-hexenol, trans-3-hexenol, cis-3-hexenol, 2-phenylethanol, ethyl butyrate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate, 2-methylpropanoic acid, 3-methylbutanoic acid, hexanoic acid, octanoic acid, decanoic acid, and dodecanoic acid were assessed through data generated according to a factorial design using analysis of variance and principal component analysis. Four times of storage of pomace obtained following winemaking of two grape varieties of white Verde wine (Alvarinho and Loureiro) and three distillation cuts were considered; volatile compounds in the 24 samples generated were analyzed directly, and indirectly after extraction and concentration, by capillary gas chromatography. The results generated have suggested clear differences (P<0.05) between distillate cuts obtained throughout fermentation times for each grape variety. The major differences between the different distillate fractions analyzed were accounted for by the contents of diethyl butanoate, ethyl 2-hydroxypropanoate, and the sum of 3-methylbutanoic and 2-methylpropanoic acids for Loureiro, whereas the main differences were accounted for by the contents of diethyl butanoate and the sum of the carboxylic acids for Alvarinho.     

Effects of time of grape pomace fermentation and distillation cuts on the chemical composition of grape marcs

 The effects of fermentation time and distillation cuts on the composition of distillates in terms of ethanal, 1,1-diethoxyethane, methanol, 1-butanol, 2-butanol, 1-propanol, 2-methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, ethyl acetate, ethyl 2-hydroxypropanoate, 3-methylbutyl acetate, hexyl acetate, 2-phenylethyl acetate, hexanol, trans-2-hexenol, trans-3-hexenol, cis-3-hexenol, 2-phenylethanol, ethyl butyrate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate, 2-methylpropanoic acid, 3-methylbutanoic acid, hexanoic acid, octanoic acid, decanoic acid, and dodecanoic acid were assessed through data generated according to a factorial design using analysis of variance and principal component analysis. Four times of storage of pomace obtained following winemaking of two grape varieties of white Verde wine (Alvarinho and Loureiro) and three distillation cuts were considered; volatile compounds in the 24 samples generated were analyzed directly, and indirectly after extraction and concentration, by capillary gas chromatography. The results generated have suggested clear differences (P<0.05) between distillate cuts obtained throughout fermentation times for each grape variety. The major differences between the different distillate fractions analyzed were accounted for by the contents of diethyl butanoate, ethyl 2-hydroxypropanoate, and the sum of 3-methylbutanoic and 2-methylpropanoic acids for Loureiro, whereas the main differences were accounted for by the contents of diethyl butanoate and the sum of the carboxylic acids for Alvarinho. Received: 23 January 1998 / Revised version: 5 June 1998     

Volatile profiles of thua nao, a Thai fermented soy product

Thua nao, a Thai traditional fermented soybean has a unique odour due to the presence of several volatile components. In this investigation, the cooked non-fermented soybeans and soybean (thua nao) fermented by pure Bacillus subtilis TN51 (TNB51) and naturally fermented soybean (TNMX) were analysed for odour components by GC–MS, using the PDMS-SPME head space technique. The quantities of total volatiles in both TNB51 and TNMX were analogous, but the major volatile classes present in TNB51 and TNMX were pyrazines and ketones, respectively. The predominant volatiles in pure Bacillus-fermented thua nao included 2,5-dimethylpyrazine, 2-methylbutanoic acid, 2,3,5-trimethylpyrazine, 2-methylpropanoic acid and acetic acid, whereas the major volatiles in the naturally fermented soybean included 2,5-dimethylpyrazine, benzaldehyde, 5-methyl-3-hexanone, 2-butanone and 3-methyl-2-pentanone. In total, 85 volatile compounds were detected.     

Comparison of volatile metabolic profiles in fermented milk of Streptococcus thermophilus during the postripening period at different incubation temperatures

Streptococcus thermophilus has been extensively applied in fermented milk. This study used gas chromatography-ion mobility spectroscopy to determine and evaluate the volatile metabolites in raw milk, milk fermented at 37°C, and milk fermented at 42°C. Ten discriminatory volatile metabolites were identified at different incubation temperatures: acetone, 2-heptanone, 2-pentanone, 2-hexanone, butanal, hexanal, ethyl acetate, 3-methylbutanal, 3-methylbutanoic acid, and 2-methylpropanoic acid, indicating that fermentation temperature affected the spectrum of volatiles in milk fermented by different strains of S. thermophilus. Specifically, fermentation at 37°C led to accumulation of short-chain fatty acids, whereas fermentation at 42°C enriched ketones and other flavor substances in the fermented milk, enhancing the flavor of the product. This work examined the differences between the volatile metabolites produced by different S. thermophilus strains fermented at different temperatures to evaluate the effect of temperature on the metabolic pathways.     

The influence of environmental parameters on the catabolism of branched-chain amino acids by Staphylococcus xylosus and Staphylococcus carnosus

Degradation of the amino acids leucine, isoleucine and valine into branched flavour compounds by Staphylococcus xylosus and Staphylococcus carnosus was studied using resting cell cultures added to a defined reaction medium under different environmental conditions relevant to sausage fermentation. The environmental conditions studied were temperature (12–28°C), NaCl concentration (4.0–12.0% (w/w)) acidity (pH 4.8–5.8) and addition of manganese (0–2.5 mg Mn/kg). Flavour compounds were sampled by automatic static headspace collection and separated/quantified using gas chromatography/flame ionization detection (GC/FID). Main volatile catabolic products of leucine, isoleucine and valine were 3-methylbutanoic, 2-methylbutanoic and 2-methylpropanoic acids, respectively. The generation of branched flavour compounds was influenced significantly by most of the investigated environmental parameters, but in particularly by temperature and NaCl.Raising the temperature increased the production of branched-chain acids by both S. xylosus and S. carnosus, whereas increasing NaCl concentration had a clear negative effect, reducing the concentrations of branched-chain acids for both species but in particularly for S. carnosus. The acidity of the medium was also important for both Staphylococcus spp., but with opposite effects. Lowering pH decreased the generation of branched-chain acids by S. carnosus but increased the generation by S. xylosus. In addition, several two-factor interactions between temperature, NaCl concentration and pH were significant. Manganese addition increased the branched-chain acid concentration slightly for S. xylosus but not for S. carnosus.     

Changes in organic acid contents during mead wort fermentation

Organic acids contained in honey influence the fermentation rate and frequently cause mead fermentation to halt. The purpose of this study was to determine the changes that occur in a group of selected organic acids during the fermentation of mead worts and their impact on the process. Mead worts (1:2 and 1:3 (honey:water)) were fermented using Saccharomyces cerevisiae yeast of the Johannisberg-Riesling breed (?OCK 105). The worts were supplemented with diammonium hydrogen phosphate (0.4 g/l) and citric acid (0.25 g/l). During fermentation, the contents of selected carboxylic acids were determined using gas chromatography. Mead worts contain relatively high amounts of medium-chain fatty acids, which are believed to inhibit fermentation. The dominant compounds of this group are decanoic (42 mg/l), dodecanoic (31 mg/l) and octanoic (26 mg/l) acids. The experiments demonstrated that during the early days of fermentation, the main acids to form are the acetic and succinic acids, which reduce the wort pH, while the content of fatty acids drops by 70–80%. During fermentation, the amounts of the formic, hexadecanoic and octadecanoic acids also fall.     

Methane fermentation of a mixture of seaweed and milk at a pilot-scale plant

In this study, a pilot-scale plant was built to examine the practicality of producing biogas from seaweeds, widely available in Japan. Laminaria sp. and Ulva sp. seaweeds were mixed with other organic waste (milk) and used as fermentation materials. Though quantities and ratios of the materials were varied, the ratio of generated methane to input chemical oxygen demand (COD) was largely stable (0.2–0.3 m³ methane/kg COD) and the organic acid concentration in the methane fermentation solution was low (< 1200 ppm) during prolonged operation. These findings indicate that stable methane fermentation was achieved and that mixing with other organic material was effective in suppressing fluctuations in material amounts caused by the variable supply of seaweeds. Our results demonstrate the practical feasibility of biogas generation using seaweeds.     

Analysis of organic acids in Chinese raisin tree (Hovenia dulcis) peduncle and their changes in liquid fermentation process

Chinese raisin tree (Hovenia dulcis) peduncle is a wonderful food material for fermented food for high contents of sugars and a variety of organic acids. The organic acids in the fresh peduncle, fermented wine, and vinegar were identified and quantified by HPLC, and changes in biochemical parameters and organic acids in the fermentation process were studied. Skin-on fermentation was carried out in the alcoholic fermentation, and 9 identified organic acids fluctuated by physical or biochemical reactions during the alcoholic fermentation. Submerged fermentation was carried out in the acetic fermentation. It took 72 h to finish the 1^st cycle of acetification with the titratable acidity 5.42%(w/v), and 30 h to finish the 2^nd cycle. The contents of acetic acid, formic acid, and α-ketoglutaric acid increased, while the rest identified organic acids decreased in the acetification process. The fermentation developed in this study appeared to be a practical processing method for Chinese raisin tree peduncles.     

Metabolic footprint analysis of volatile metabolites by gas chromatography-ion mobility spectrometry to discriminate between different fermentation temperatures during Streptococcus thermophilus milk fermentation

Streptococcus thermophilus is widely used in the dairy industry to produce fermented milk. Gas chromatography-ion mobility spectrometry–based metabolomics was used to discriminate different fermentation temperatures (37°C and 42°C) at 3 time points (F0: pH = 6.50 ± 0.02; F1: pH = 5.20 ± 0.02; F2: pH = 4.60 ± 0.02) during S. thermophilus milk fermentation, and differences of fermentation physical properties and growth curves were also evaluated. Fermentation was completed (pH 4.60) after 6 h at 42°C and after 8 h at 37°C; there were no significant differences in viable cell counts and titratable acidity; water-holding capacity and viscosity were higher at 37°C than at 42°C. Different fermentation temperatures affected volatile metabolic profiles. After the fermentation was completed, the volatile metabolites that could be used to distinguish the fermentation temperature were hexanal, butyraldehyde, ethyl acetate, ethanol, 3-methylbutanal, 3-methylbutanoic acid, and 2-methylpropionic acid. Specifically, at 37°C of milk fermentation, branched-chain AA had higher levels, and leucine, isoleucine, and valine were involved in growth and metabolism, which promoted accumulation of some short-chain fatty acids such as 3-methylbutanoic acid and 2-methylpanprooic acid. At 42°C, at 3 different time points during fermentation, ethanol from glycolysis all presented higher levels, including acetone and 3-methylbutanal, producing a more pleasant flavor in the fermented milk. This work provides detailed insight into S. thermophilus fermented milk metabolites that differed between incubation temperatures; these data can be used for understanding and eventually predicting metabolic changes during milk fermentation.     

Fatty acid composition of seed oil of different Sorghum bicolor varieties

In order to find out new sources of premium quality edible oil in the country, seeds of ten varieties of Sorghum bicolor were initially analyzed for their total oil contents. The seed oil was later fractionated into eight fatty acids including two new saturated fatty acids. The oil contents were determined by Soxhlet method and compared with the results obtained by NMR analysis. The total oil contents in the seeds of sorghum ranged from 5.0 to 8.2 % (w/w), indicating non significant difference obtained by two different techniques. The results revealed that oleic acid (31.12–48.99%), Palmitoleic acid (0.43–0.56%), linoleic acids (27.59–50.73%), linolenic acid (1.71–3.89%), stearic acid (1.09–2.59%) and palmitic acid (11.73–20.18%) was present in the seed oil of different sorghum varieties when analyzed by GC–MS. It was observed that in most of the varieties polyunsaturated fatty acids (PUFA) were higher than monounsaturated fatty acids (MUFA). The two atypical SFAs, octanedioic (C8:0) and azelaic acid (C9:0) were found in some varieties. These results suggest that these S. bicolor varieties could be additional sources of edible oil due to presence of clinically important saturated and high concentration of unsaturated fatty acids. A large scale production of the seed oil after refining process can contribute towards alleviation of edible oil shortage in the country with increased use of premium quality oil.     

Buckwheat honeys: Screening of composition and properties

The quality of 10 buckwheat honeys, collected from Italian and est European beekeepers declaring to produce monofloral honey, were evaluated by means of their pollen, physicochemical, phenolic and volatile composition data. The results of the traditional analyses and in particular electrical conductivity, optical rotation, pH and sugar composition revealed some poorly pure samples that could not fit in the buckwheat tipology. Honey volatiles, analysed by solid phase microextraction (SPME) and gas chromatography–mass-spectrometry (GC/MS), showed more than 100 volatile compounds, most of them present in all honey samples but with quantitative variation. Besides many furfural derivates, 3-methylbutanoic acid was the main volatile compound found in most of honeys. Also the presence of 2- and 3-methylbutanal and pheynalcetaldehyde confirmed the typical buckwheat aroma of some studied samples, corroborating physicochemical data. The HPLC phenolic profile was similar across the samples and p-hydroxybenzoic and p-coumaric acids proved to be the main components.     

Nickel–boron alloy catalysts reduce the formation of Trans fatty acids in hydrogenated soybean oil

Soybean oil was hydrogenated by a group of amorphous alloy nanocatalysts of nickel and boron (Ni–B). The Ni–B alloy catalysts showed activities dependent on synthesis conditions. The trans-fat contents in products hydrogenated by one Ni–B catalyst were approximately half of those when a commercial nickel catalyst was used. The Ni–B catalyst had a lower selectivity towards formation of trans-fatty acids but a higher selectivity towards formation of stearic acid than the commercial nickel catalyst. The increases in trans-fatty acids correlated linearly with the increases in C18:1 (oleic and elaidic acids) for nickel-containing catalysts. The incorporation of boron in nickel-containing catalysts increased the selectivity ratio (for forming stearic acid) but did not change the linolenic selectivity. Because of the simplicity to synthesize amorphous catalysts, this work may lead to future development of novel alloy nanocatalysts to synergistically reduce the formation of both trans- and saturated fatty acids in hydrogenated products.     

Metabolomic analysis of meju during fermentation by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF MS)

Changes in the water-soluble metabolites of meju during fermentation were analysed by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF MS), and the resultant data were statistically processed by partial least squares-discriminant analysis (PLS-DA). Various metabolites, including amino acids, small peptides, nucleosides, urea cycle intermediates, and organic acids, which are responsible for the unique taste and nutritional and functional quality of fermented soy foods, were clearly altered by increasing the fermentation period. Changes in these metabolites allowed discrimination among meju samples with different fermentation periods (0, 10, 20, 40, and 60 d) on a PLS-DA score plot, and the fermentation was mainly processed between 10 and 40 d of fermentation. Twenty-two metabolites (phenylalanine, glutamic acid, leucine, adenine, citrulline, arginine, glutamine, γ-aminobutyric acid, proline, acetylornithine, valine, pipecolic acid, methionine, citric acid, xanthine, tyrosine, isoleucine, Glu-Tyr, Ser-Pro, tryptophan, Glu-Phe, and Leu-Val-Pro-Pro) with high PLS-DA values of over 1.00 were determined as the major compounds contributing to the discrimination of meju samples. These metabolites, which were positively related to the sensory quality of meju, can be used as fermentation biomarkers for the production of meju and to construct the meju fermentation metabolic pathway. Therefore, our results indicate that monitoring the changes in metabolites during meju fermentation might be important for producing meju-related foods with good nutritional and sensory quality.     

Characterisation of volatile and non-volatile metabolites in etiolated leaves of tea (Camellia sinensis) plants in the dark

Aroma is an essential factor affecting the quality of tea (Camellia sinensis) products. While changes of volatile compounds during tea manufacturing have been intensively studied, the effect of environmental factors on volatile contents of fresh tea leaves has received less attention. We found that C. sinensis var. Yabukita kept in darkness by shading treatment for 3 weeks developed etiolated leaves with significantly increased levels of volatiles, especially volatile phenylpropanoids/benzenoids (VPBs). Upstream metabolites of VPBs, in particular shikimic acid, prephenic acid, and phenylpyruvic acid, showed lower levels in dark treated than in control leaves, whereas the contents of most amino acids including l-phenylalanine, a key precursor of VPBs, were significantly enhanced. In addition, analysis by ultra performance liquid chromatography-time of flight mass spectrometry, capillary electrophoresis–time of flight mass spectrometry, high performance liquid chromatography, and gas chromatography–mass spectrometry indicated that volatile and non-volatile metabolite profiles differed significantly between dark treated and untreated leaves.     

Variations in essential oil and fatty acid composition during Myrtus communis var. italica fruit maturation

The essential oil and fatty acid composition of Myrtus communis var. italica fruit during its ripening was determined. The effect of the harvesting time on some physical properties of Myrtus fruits, fruit weight and moisture content, were significant. The increase of fruit weight (from 2.54 to 8.79 g% fruits) during ripeness was correlated positively with that of moisture content (from 28% to 72%). Fruit essential oil yields varied from 0.003% to 0.01% and showed a remarkable increase at 60 days after flowering to reach a maximum of 0.11%. Forty-seven volatile compounds were identified in fruit essential oils; 1,8-cineole (7.31–40.99%), geranyl acetate (1.83–20.54%), linalool (0.74–18.92%) and α-pinene (1.24–12.64%) were the main monoterpene compounds. Total fatty acid contents varied from 0.81% to 3.10% during fruit maturation and the predominant fatty acids were linoleic (12.21–71.34%), palmitic (13.58–37.07%) and oleic (6.49–21.89%) acids. The linoleic acid proportions correlated inversely with palmitic and oleic acids during all the stages of ripening.     

Seasonal variation in the fatty acid composition of three Mediterranean fish – sardine (Sardina pilchardus), anchovy (Engraulis encrasicholus) and picarel (Spicara smaris)

The total fat contents and the fatty acid compositions of three common Mediterranean fish, namely sardine (Sardina pilchardus), anchovy (Engraulis encrasicholus) and picarel (Spicara smaris) were determined at bimonthly intervals for a one-year period. The purpose of this work was to study the seasonal variation of the fatty acids in the three fish that are some of the best sources of n − 3 fatty acids. The fat and fatty acid content of the investigated fish species show a significant seasonal dependency. Two of the fish (anchovy and picarel) have the highest fat content during the late winter – spring period. On the other hand, sardine shows the highest fat concentrations during the spring-early summer period. The fish that showed the highest variation in fatty acid composition was the anchovy. The sardine was found to be the best source of n − 3 fatty acids during the one-year period (35.35 g/100 g fatty acids). Finally the picarel had the highest oleic acid content (on average, 13.89/100 g fatty acids).     

Formation of vinylphenolic pyranoanthocyanins by Saccharomyces cerevisiae and Pichia guillermondii in red wines produced following different fermentation strategies

The strains of two species of yeast, Saccharomyces cerevisiae and Pichia guillermondii, both with high hydroxycinnamate decarboxylase (HCDC) activity (56% and 90% respectively), were used in the fermentation of musts enriched with grape anthocyanins, to favour the formation of highly stable vinylphenolic pyranoanthocyanin pigments. The different strains were used to ferment the must separately, simultaneously, or sequentially, the latter involving an initial period using the yeast with the greatest HCDC activity (P. guillermondii). The must was made from concentrated grape juice diluted to 220 g/l of sugar, and enriched with grape anthocyanins to 100 mg/l and with p-coumaric acid to 120 mg/l. The pH was fixed to 3.5. All 50 ml micro-fermentations were done in triplicate. The development of anthocyanin-3-O-glucoside precursors, the decarboxylation of p-coumaric acid, and the formation of 4-vinylphenol and vinylphenolic pyranoanthocyanin derivatives were studied during the fermentation. The fermentation strategy used and the yeast HCDC activity significantly influenced the formation of vinylphenolic pyranoanthocyanins. The latter molecules were separated, identified, and quantified using high performance liquid chromatograph with diode array detection and electrospray-mass spectrometry (HPLC-DAD-ESI/MS). The volatile compounds profile was screened during fermentation using gas cromatogrphy-flame ionisation detection (GC-FID), in order to detect and quantify the main molecules. The best results were reached with the sequential fermentation (3.74 mg/l of malvidin-3-O-glucoside-4-vinylphenol). This work shows that during mixed or sequential fermentations carried out with non-Saccharomyces or highly fermentative Saccharomyces strains, with high HCDC activity, the content of stable pigments can be increased without sensorial modifications.