Determination of taste-active compounds of a bitter Camembert cheese by omission tests

The taste-active compounds of a Camembert cheese selected for its intense bitterness defect were investigated. The water-soluble fraction (WSE) was extracted with pure water and fractionated by successive tangential ultrafiltrations and nanofiltration. The physicochemical assessment of these fractions led to the construction of a model WSE which was compared by sensory evaluation to the crude water-soluble extract, using a panel of 16 trained tasters. As no significant difference was perceived, this model WSE was then used directly or mixed with other cheese components for omission tests. Among the main taste characteristics of the WSE (salty, sour, umami and bitter), bitterness was found to be due to small peptides whose mass distribution was obtained by RPHPLC-MS (400-3000 Da) and whose taste properties are discussed.     

Sensory and mass spectrometric analysis of the peptidic fraction lower than one thousand daltons in Manchego cheese

A total of 107 different peptides, all derived from α_S1-, α_S2-, and β-casein, were identified in different fractions of artisan or industrial Manchego cheese at 4 and 8 mo of ripening, and their sequences were examined. Most of these peptides are described for the first time in Manchego cheese. Taste characteristics (umami and bitter) were assigned based on their AA sequence and the position of these AA within the sequence. The umami taste was predominant in all fractions analyzed by the panelists, and the peptides EQEEL, QEEL, and EINEL, containing a high number of glutamic residues, were found within the fractions. However, in several fractions described as having umami characteristics, no peptides responsible for this taste were detected. Therefore, compounds other than peptides seem to be involved in the umami properties of water-soluble extracts lower than 1,000 Da of Manchego cheese.     

Identification of Small Peptides Generated in Spanish Dry-cured Ham

ABSTRACT: A water‐soluble extract of dry‐cured ham was fractionated by gel filtration chromatography. Collected fractions (below 1200 Da) were sensory‐analyzed and subjected to amino acid analysis with and without previous acid hydrolysis, in order to determine the amino acid and peptide distribution. Those fractions with the highest peptide amount were further separated by reverse‐phase and cation‐exchange high‐performance liquid chromatography in order to isolate the peptides and sequence them. The results demonstrated the presence of small peptides, mainly dipeptides. The combination of these peptides and free amino acids may contribute to the characteristic taste in the savory fractions of dry‐cured ham.     

Evaluation of bitterness in Ragusano cheese

The appearance of undesirable bitter taste in Ragusano cheese was investigated by comparing the composition of 9 bitter cheeses with that of 9 reference cheeses of good quality by means of chemical, electrophoretic, and chromatographic analyses. Rates of proteolysis were significantly affected in cheeses of different quality. Primary proteolysis, as measured by pH 4.6-soluble N, was significantly greater in bitter cheeses compared with reference samples. Urea-PAGE profiles showed an almost complete breakdown of caseins in bitter cheeses and the further degradation of primary peptides into smaller compounds not detectable by this technique. Cheeses with defects had significantly lower levels of secondary proteolysis as reflected by the percentage of pH 4.6-soluble N soluble in 12% trichloroacetic acid and the amounts of total free amino acids. Peptides separated by reversed phase-HPLC revealed that the large and significant differences in peptide profiles of the soluble fractions between bitter and reference cheeses were mainly due to a much higher proportion of hydrophobic peptides in the former. The occurrence of bitterness in Ragusano cheese was therefore attributable to unbalanced levels of proteolysis and peptidolysis. Extensive degradation of caseins and primary peptides by activities of proteases produced large amounts of small- and medium-sized hydrophobic peptides that were not adequately removed by peptidases of microflora and therefore accumulated in cheese potentially contributing to its bitter taste. The presence of these compounds in bitter cheeses was related to high salt-in-moisture and low moisture contents that limited the enzymatic activities of microflora important in secondary proteolysis. Combining salt-in-moisture and the ratio of hydrophobic-to-hydrophilic soluble peptides resulted in the best logistic partial least squares regression model predicting cheese quality. Although bitterness is known to be rarely encountered in cheese at salt-in-moisture levels >5.0, all of the bitter cheeses analyzed in this study had salt-in-moisture levels much greater than this value. According to the logistic model, a risk of bitterness development may exist for cheeses with a midrange (5 to 10%) salt-in-moisture content but with an inadequate level of secondary proteolysis.     

Fractionation of the water-soluble extract of a cheese made from goats’ milk by filtration methods: behaviour of fat and volatile compounds

The intense aroma of goat cheese is mainly due to volatile polar compounds, particularly short- and medium-chain length free fatty acids, contained in its water-soluble extract (WSE). This fraction also contains the taste compounds of the cheese and has been fractionated in order to assess its chemical composition. The aim of the present work was to evaluate the behaviour of the most abundant volatile compounds and fat components in the WSE during multiple successive fractionation steps: i.e. fractionation by 10 and 1 kDa ultrafiltrations and 0.5 kDa nanofiltration. Lipid and volatile components were analysed by TLC-FID and GC–MS methods, respectively, at each filtration step. In the WSE, 43 volatile components were identified and short-chain free fatty acids were the most abundant. Volatile and fat compounds were largely retained during the 10 and 0.5 kDa filtration steps and to a lesser extent during the 1 kDa filtration step. The retention of various volatile compounds could be explained, beside possible interactions with the membranes used, by adsorption or interaction of these compounds with fat or protein material.     

Study of taste-active compounds in the water-soluble extract of mature Cheddar cheese

The chemical composition of the water-soluble extracts of mature Cheddar cheese were identified, with the emphasis on understanding the interplay of compounds contributing to the savoury taste in Cheddar. The ultra-filtered water-soluble extracts of two mature Cheddar cheeses were fractionated by gel permeation chromatography (GPC). By sensory evaluation, two taste-active GPC fractions were identified from each cheese. On the basis of chemical profiling of these fractions, aqueous model tastant mixtures were prepared and sensory omission tests carried out. Glutamic acid, organic acids and mineral salts were the main tastants, whereas the other amino acids had a limited impact on taste. The characteristic umami taste was explained by a synergistic effect of glutamic acid and salts. Matching umami taste intensities were obtained from different concentrations of glutamic acid and salts. Unmasking of a bitter or sweet taste from mixtures of sub-threshold concentrations of amino acids without glutamic acids was also observed.     

Angiotensin-converting enzyme inhibitory activity of water-soluble extracts of Asiago d'allevo cheese

The angiotensin-converting enzyme (ACE) inhibitory activity of water-soluble extracts (WSE) from Asiago cheeses was assayed in two cheese production systems and with different ripening times. The WSE were ultrafiltered through 10 kDa and 3 kDa cut-off membranes to evaluate the ACE inhibitory activity of long and short peptides, respectively. The cheese production systems had no significant effect on the ACE inhibitory activity, whereas 6-month-old cheeses had higher inhibitory potency than the more ripened ones. Moreover, the fraction containing peptides smaller than 3 kDa made a more considerable contribution to ACE inhibitory activity than the fraction smaller than 10 kDa, suggesting an inhibitory effect due to short peptides. The peptidic fraction was characterized using RP-HPLC coupled to mass spectrometric detection. Simulated gastrointestinal digestion was carried out to evaluate the effects of digestive enzymes on the generation of bioactive peptides, but this did not significantly affect the inhibitory activity.     

Fractionation and purification of antioxidant peptides from abalone viscera by a combination of Sephadex G-15 and Toyopearl HW-40F chromatography

Abalone viscera, a protein-rich by-products from the abalone processing industry, are normally discarded as wastes. In the present study, four different proteases were used to hydrolyse abalone viscera to prepare high-activity antioxidant peptides, and their hydrolysis effects were compared. Although the hydrolytic abilities of papain and trypsin were inferior to the alcalase and neutrase, their use resulted in significantly (P < 0.05) higher scavenging activities for DPPH. The hydrolysates mainly consisted of peptides with a molecular weight <3 kDa, and the fractionation was achieved using a combination of Sephadex G-15 and Toyopearl HW-40F gel filtration chromatography, which overcomes the disadvantages of traditional membrane separation technology in fractionating peptides with relatively similar molecular weight distribution. The DPPH scavenging activities of the components containing peptides <1 kDa (fractions E) were significantly (P < 0.01) higher than those containing peptides >1 kDa. The different fractions E were further purified by reversed-phase high-performance liquid chromatography, and a total of 16 target peptides, containing 4-8 amino acids, enriched in hydrophobic amino acids and C-terminal Arg, with molecular weights ranging from 500 to 850 Da, were identified.     

Isolation and identification of two novel umami and umami-enhancing peptides from peanut hydrolysate by consecutive chromatography and MALDI-TOF/TOF MS

Peanut hydrolysate produced by crude protease extract from Aspergillus oryzae HN 3.042 was found to elicit intense umami and umami-enhancing effect. Taste profiles, amino acid and organic acid composition of peanut hydrolysate and its separation fractions by ultrafiltration were evaluated. The results revealed that peanut hydrolysate was mainly low molecular weight compounds. Fractions of 1-3 kDa and below 1 kDa prominently contributed to the umami taste and umami-enhancing effect of the peanut hydrolysate. The two fractions were further purified, using gel filtration chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC), in combination with sensory evaluation, to obtain a umami peptide and umami-enhancing peptide. The active peptides were identified as Ser-Ser-Arg-Asn-Glu-Gln-Ser-Arg (SSRNEQSR, 963.9 Da) and Glu-Gly-Ser-Glu-Ala-Pro-Asp-Gly-Ser-Ser-Arg (EGSEAPDGSSR, 1091.1 Da), by MALDI-TOF/TOF MS, respectively.     

市售天然成熟干酪滋味特征差异性研究

利用气相色谱质谱联用仪、氨基酸分析仪对8种典型天然成熟干酪的主要滋味成分进行分析,通过滋味活力值和感官评定阐明8种干酪间的滋味差异情况。结果表明:除了山羊乳干酪和布里干酪,其他6种干酪中游离氨基酸对滋味均有贡献,特征滋味氨基酸有缬氨酸和赖氨酸,即干酪共同带有苦味和甜味,而蓝纹干酪特征性氨基酸种类最丰富,滋味最强;8种干酪共检测到11种游离脂肪酸,共有的特征滋味脂肪酸为己酸、壬酸、癸酸、月桂酸和肉豆蔻酸。其中酸味程度最强的为艾达姆干酪,帕玛森干酪的醇厚感最强;8种干酪氯化钠含量明显高于阈值,可显著感受到咸味。经感官分析,干酪的滋味中咸味和鲜味比较突出,蓝纹干酪滋味强度最强。     

不同处理方法对草菇呈味物质释放的影响

以草菇子实体为研究对象,以可溶性糖及糖醇、有机酸、5’-核苷酸、游离氨基酸组成和含量、分子质量分布为指标,研究常压蒸煮、高压蒸煮和酶解3?种处理方法对草菇呈味物质释放的影响,结果表明：常压蒸煮制备液可溶性糖及糖醇的总量显著降低,高压蒸煮制备液可溶性糖及糖醇的总量和原液基本相同,而酶解作用显著地提高了样品中可溶性糖的含量;和原液相比,常压蒸煮和高压蒸煮提取后有机酸总量显著增加,而复合酶解制备液有机酸总量显著降低;3?种处理方法作用后的游离氨基酸总量都有所增加,以高压蒸煮增加最显著,同时高压蒸煮制备液的鲜味氨基酸、甜味氨基酸以及苦味氨基酸的含量均显著增加;常压蒸煮和高压蒸煮制备后,大于3?000?Da组分的含量有所减少,高压蒸煮制备液中小于3?000?Da的组分含量最多,是制备呈味肽的重要来源。因此,不同处理方法对草菇呈味物质的释放有着明显差异,可以根据所需要的呈味物质采用不同的处理方法。     

不同蛋白酶对复合骨素酶解液呈味物质的影响

为研究不同蛋白酶对复合骨素（牛骨素和鸡骨素）酶解液呈味物质的影响,选取复合蛋白酶（P）、菠萝蛋白酶（B）和复合风味蛋白酶（F）,制备单一和组合酶解液,对酶解液肽分子质量分布、核苷酸含量、游离氨基酸含量等呈味物质指标进行综合评价。结果表明：P＋F组酶解液的水解度最大,酶解液中小分子质量肽（＜200 Da）分布比例最大;P＋F组酶解液的游离氨基酸含量最高,鲜味和甜味氨基酸是复合骨素酶解液的主要呈味成分;P＋F组的5’-核苷酸（5’-肌苷酸、5’-鸟苷酸、5’-腺苷酸）总含量较高。因此,复合蛋白酶和复合风味蛋白酶处理的复合骨素酶解液整体风味最佳。     

Transglutaminase cross-linking effect on sensory characteristics and antioxidant activities of Maillard reaction products from soybean protein hydrolysates

To improve the yield of Maillard peptides, a microbial transglutaminase (MTGase) was used to increase the content of 1000–5000 Da peptides in soybean protein hydrolysates by using a cross-linking reaction. The sensory characteristics and antioxidant activities of corresponding Maillard Reaction Products (MSPC) was then evaluated. After cross-linking treatment the content of 1000–5000 Da peptides in protein hydrolysates and the yield of Maillard peptides increased by 21.19% and 8.71%, respectively, which contributed to the improved mouthfulness of MSPC. The bitter amino acids were significantly decreased and the umami acids were markedly increased in MSPC. Volatile compounds identified by GC–MS analysis showed that the content of the important meaty flavour compounds (such as 2-methyl-3-furanthiol, bis(2-methyl-3-furyl)disulfide) of MSPC were dramatically higher than that of MRPs from uncross-linking peptides. Combined with sensory evalution, it was confirmed that MTGase cross-linking improved the flavour Characteristics and did not affect the antioxidant activity of MSPC.     

Evaluation of Peptide Contribution to the Intense Umami Taste of Japanese Soy Sauces

ABSTRACT: Koikuchi and tamari shoyu are 2 types of traditional Japanese soy sauce whose low-molecular-weight fractions (less than 500 Da) are known to have the most intense umami taste. Given that peptides were also abundant in some umami fractions of both shoyu, further investigation was conducted to evaluate the precise contribution of peptides to the soy sauce tastes. To locate and isolate peptide fractions, separation procedures using Sephadex G-25 SF, Sephadex G-10, and reversed-phase high-performance liquid chromatography (RP-HPLC) linked to sensory evaluation (by taste dilution analysis), as well as amino acid analysis were performed. Peptides having glutamyl residue were present in most peptide fractions. However, they all revealed negligible contributions to the intense umami taste of the soy sauces. On the other hand, free Glu, in the presence of sodium salt, as well as free Asp and several sweet-taste eliciting free amino acids were considered to be the main contributors to the umami taste of the soy sauces. This study of Japanese soy sauces gives a clear explanation of the frequently questioned peptide contribution to the taste of savory food product.     

FORMATION OF TASTE AND ODOR COMPOUNDS DURING PREPARATION OF DOUCHIBA, A CHINESE TRADITIONAL SOY-FERMENTED APPETIZER

Douchiba (DCB), a Chinese traditional soy‐fermented appetizer, is abundantly produced and widely consumed in Guizhou province of southwest China. In this study, an analysis of free amino acid (FAA) profiles, low‐molecular weight peptide fractions (LMWPFs), hydrophobic bitter peptide fractions (HBPFs) and volatile compounds was conducted to understand the changes in taste and odor compounds of DCB during five consecutive manufacturing stages: steamed soybean (SS), 5‐day incubated Koji (Koji), 6‐month fermented Douchi, semifinished Douchiba (sm‐DCB) and 6‐month ripened finished Douchiba (DCB). The total free amino acids (TFAAs) increased significantly from SS to DCB by about 11 times and amounted to 20.1% of the crude protein. Arg, Glu, Phe, Leu and Lys were the most abundant FAA at the end of ripening, and were 64.6% of TFAA. The most abundant taste active FAA classes were the bitter FAA, representing eight‐ and threefold increases over sweet and MSG‐like FAA, respectively. The final values of all bitter and monosodium glutamate‐like (MSG‐like) FAA at DCB stage were significantly higher than their respective thresholds. The ratio of oligopeptides (500～1000 Da) accounted for 13.8 and 2.5% of LMWPF and HBPF, respectively. However, the taste of DCB was predominated by saltiness, followed by moderate “umami” taste and slight bitterness, likely as a result of the balance and interaction among different taste components. A total of 122 volatile compounds were identified, including alcohols, acids, esters, aldehydes, ketones, phenols, sulfur‐containing compounds, heterocyclic compounds, alkines, benzenes and spice aromatics. The subtle aroma of the finished DCB seemed to depend not only on particular key compounds but also on a “critical balance” or a “weighted concentration ratio” of 60 compounds. These volatile compounds, mainly derived from lipid oxidation, amino acid degradation, carbohydrate fermentation, Maillard reaction and Strecker degradation during the microbial metabolism, enzymatic catalysis and spontaneous reactions.     

Soy Sauce and Its Umami Taste: A Link from the Past to Current Situation

ABSTRACT: Soy sauce taste has become a focus of umami taste research. Umami taste is a 5th basic taste, which is associated to a palatable and pleasurable taste of food. Soy sauce has been used as an umami seasoning since the ancient time in Asia. The complex fermentation process occurred to soy beans, as the raw material in the soy sauce production, gives a distinct delicious taste. The recent investigation on Japanese and Indonesian soy sauces revealed that this taste is primarily due to umami components which have molecular weights lower than 500 Da. Free amino acids are the low molecular compounds that have an important role to the taste, in the presence of sodium salt. The intense umami taste found in the soy sauces may also be a result from the interaction between umami components and other tastants. Small peptides are also present, but have very low, almost undetected umami taste intensities investigated in their fractions.     

Improvement of thermal,microwave and ultrasonication pretreatment on the production of antioxidant peptides from sweet potato protein via in vitro gastrointestinal digestion

Effects of thermal (boiling, steaming and autoclaving), microwave and ultrasonication pretreatments on the production of sweet potato protein hydrolysates (SPPH) through in vitro gastrointestinal digestion (GID) were investigated. All pretreatments significantly increased the degree of hydrolysis (DH), antioxidant activities and molecular weight (MW) <3 kDa peptide fractions contents of SPPH in the order of autoclaving > microwave, steaming > boiling > ultrasonication (P < 0.05). Correlation analysis between peptides content and antioxidant activity suggested that antioxidant activity of SPPH mainly attributed to MW <3 kDa peptides. Diverse peptides ranged from 487.24 to 1477.74 Da with 7–13 amino acids were identified in the MW <3 kDa peptides fraction with autoclaving pretreatment and matched sporamins A, A precursor and B sequences from LC–QTOF–MS/MS analysis. Conformational structures of nine peptides were predicted with well-known antioxidant amino acids. There is a high potential for SPPH used as a functional supplement in food system.     

不同分子质量大豆蛋白和鸡肉酶解物的美拉德反应产物风味特征分析

为了研究不同分子质量的肽在美拉德反应后的风味特征,以大豆蛋白和鸡肉为原料酶解得到蛋白酶解物。对酶解物超滤分离得到小于1000Da、1000~5000Da和大于5000Da的不同分子质量肽组分,并对大豆蛋白和鸡肉酶解物完全酸水解得到复合氨基酸。在得到的肽段和氨基酸中添加一定量的木糖,构建美拉德反应模型。对美拉德反应产物感官特性进行评价,再采用气相色谱-质谱联用、高效液相色谱对挥发性和不挥发性化合物进行分析。结果表明:在2种不同蛋白来源的酶解物的美拉德反应产物中,共鉴定出挥发性化合物69种,其中吡嗪和呋喃硫醇等肉香味化合物的含量最高。统计分析表明:不同分子质量的肽和游离氨基酸的挥发性化合物存在明显差异。小于1000Da的肽在美拉德反应过程促进了吡嗪类化合物的生成,其中包括了吡嗪、5-甲基-2-乙基吡嗪、2,5-二甲基-3-乙基吡嗪、三甲基吡嗪、2-乙酰基吡咯等挥发性化合物。与肽相比,游离氨基酸有利于含硫化合物如呋喃硫醇类和噻吩类物质的生成。有29种挥发性化合物的相对气味活性值大于1。偏最小二乘回归研究了气味化合物与感官评价之间的相关性,发现小于1000Da的肽与基本的肉味和浓厚味密切相关,而大于5000Da的肽与苦味和焦煳味密切相关。在美拉德反应后,分子质量大于5000Da的肽数量显著减少；分子质量1000~5000Da的肽降解可以补充消耗的游离氨基酸；分子质量小于1000Da的肽容易发生交联和聚合现象而含量增加,其产物对风味的影响明显增强,能更好地体现基本的肉味、鲜味和浓厚味。希望研究可以为利用蛋白质酶解物生产美拉德反应产物的应用提供理论支撑和指导。     

Short communication: Identification of the aroma compounds responsible for the floral/rose flavor in water-soluble fractions of Manchego cheese

Water-soluble fractions from Protected Denomination of Origin Manchego cheese, with molecular weight <1,000 Da, were fractionated using gel permeation chromatography and studied using both instrumental and sensorial analysis. In 2 of the fractions, panelists detected a floral, rose-like flavor. Analysis of these fractions by gas chromatography-mass spectrometry after simultaneous distillation extraction with dichloromethane identified 2-phenylethanol and phenylacetaldehyde as the compounds responsible for this flavor.     

Influence of starters on chemical, biochemical, and sensory changes in Turkish White-brined cheese during ripening

Turkish White-brined cheese was manufactured using Lactococcus strains (Lactococcus lactis ssp. lactis NCDO763 plus L. lactis ssp. cremoris SK11 and L. lactis ssp. lactis UC317 plus L. lactis ssp. cremoris HP) or without a starter culture, and ripened for 90 d. It was found that the use of starters significantly influenced the physical, chemical, biochemical, and sensory properties of the cheeses. Chemical composition, pH, and sensory properties of cheeses made with starter were not affected by the different starter bacteria. The levels of soluble nitrogen fractions and urea-PAGE of the pH 4.6-insoluble fractions were found to be significantly different at various stages of ripening. Urea-PAGE patterns of the pH 4.6-insoluble fractions of the cheeses showed that considerable degradation of α_s1-casein occurred and that β-casein was more resistant to hydrolysis. The use of a starter culture significantly influenced the levels of 12% trichloroacetic acid-soluble nitrogen, 5% phosphotungstic acid-soluble nitrogen, free amino acids, total free fatty acids, and the peptide profiles (reverse phase-HPLC) of 70% (vol/vol) ethanol-soluble and insoluble fractions of the pH 4.6-soluble fraction of the cheeses. The levels of peptides in the cheeses increased during the ripening period. Principal component and hierarchical cluster analyses of electrophoretic and chromatographic results indicated that the cheeses were significantly different in terms of their peptide profiles and they were grouped based on the use and type of starter and stage of ripening. Levels of free amino acid in the cheeses differed; Leu, Glu, Phe, Lys, and Val were the most abundant amino acids. Nitrogen fractions, total free amino acids, total free fatty acids, and the levels of peptides resolved by reverse phase-HPLC increased during ripening. No significant differences were found between the sensory properties of cheeses made using a starter, but the cheese made without starter received lower scores than the cheeses made using a starter. It was found that the cheese made with strains NCDO763 plus SK11 had the best quality during ripening. It was concluded that the use of different starter bacteria caused significant differences in the quality of the cheese, and that each starter culture contributed to proteolysis to a different degree.