Chemical Profiles of Hydrolyzed Milk Samples after Treatment with Commercial Enzymes

ABSTRACT: Commercial enzymes (protease and lipase) were used to produce highly flavored cheese-like hydrolysates from fluid milk. Free fatty acids, free amino acids, degree of proteolysis, and volatile profiles were assessed to suggest the importance of proteolytic and lipolytic activity on cheese flavor development. Free fatty acid liberation was maximized with the combined Flavourzyme™ (protease) and Palatase ® (lipase) treatment incubated at 30°C, most likely due to synergism conferred by the protease. The Flavourzyme/Palatase samples incubated at 45°C generated the highest total concentration of volatile compounds. The addition of Flavourzyme generated free amino acids and low molecular weight peptides (< 1400 MW).     

Effects of blends of camel and calf chymosin on proteolysis,residual coagulant activity,microstructure, and sensory characteristics of Beyaz peynir

Beyaz peynir, a white brined cheese, was manufactured using different blends of camel chymosin (100, 75, 50, 25, and 0%) with calf chymosin and ripened for 90 d. The purpose of this study was to determine the best mixture of coagulant for Beyaz peynir, in terms of proteolysis, texture, and melting characteristics. The cheeses were evaluated in terms of chemical composition, levels of proteolysis, total free amino acids, texture, meltability, residual coagulant activity, microstructure, and sensory properties during 90 d of ripening. Differences in the gross chemical composition were statistically significant for all types of cheeses. Levels of proteolysis were highly dependent on the blends of the coagulants. Higher proteolysis was observed in cheeses that used a higher ratio of calf chymosin. Differences in urea-PAGE and peptide profiles of each cheese were observed as well. Meltability values proportionally increased with the higher increasing levels of calf chymosin in the blend formula. These coagulants had a slight effect on the microstructure of cheeses. The cheese made with camel chymosin had a harder texture than calf chymosin cheese, and hardness values of all cheese samples decreased during ripening. The cheeses with a high ratio of calf chymosin had higher residual enzyme activity than those made with camel chymosin. No significant difference in sensory properties was observed among the cheeses. In conclusion, cheeses made with a high level of calf chymosin had a higher level of proteolysis, residual coagulant activity, and meltability. The cheeses also had a softer texture than cheeses made with a high content of camel chymosin. Camel chymosin may be used as a coagulant alone if low or limited levels of proteolysis are desired in cheese.     

Impact of liposome-encapsulated enzyme cocktails on cheddar cheese ripening

Cheddar cheese proteolysis and lipolysis were accelerated using liposome-encapsulated enzymatic cocktails. Flavourzyme, neutral bacterial protease, acid fungal protease and lipase (Palatase M) were individually entrapped in liposomes and added to cheese milk prior to renneting. Flavourzyme was tested alone at three concentrations (Z1, Z2 and Z3 cheeses). Enzyme cocktails consisted of lipase and bacterial protease (BP cheeses), lipase and fungal protease (FP cheeses) or lipase and Flavourzyme (ZP cheeses). The resulting cheeses were chemically, rheologically and organoleptically evaluated during 3 months of ripening at 8 °C. Levels of free fatty acids and appearance of bitter and astringent peptides were measured. Certain enzyme treatments (BP and ZP) resulted in cheeses with more mature texture and higher flavor intensity in a shorter time compared with control cheeses. No bitter defect was detected except in 90-day-old FP cheese. A full aged Cheddar flavor was developed in Z3 and ZP cheeses, while treatment BP led to strong typical Cheddar flavor by the second month and did not exhibit any off-flavor when ripening was extended for a further month.     

Volatile compounds and proteolysis in traditional Beaten (Bieno sirenje) ewe's milk cheese

Beaten ewe's milk cheese is a traditional autochthonous type of cheese manufactured in Macedonia with a relatively high nutritional value and sensory characteristics. The objective of this study was to characterise the gross composition, proteolysis and volatile profiles of the cheese that is supplied from different retails. The ranges for gross composition were from 31.53% to 42.83% (w/w) for moisture, 41.99% to 50.98% (w/w) for fat‐in‐dry matter, 2.03% to 8.25% (w/w) for salt content and 20.74% to 33.35% (w/w) for protein. Proteolysis results showed various levels of soluble nitrogen ranged from 3.15 to 33.50 for water‐soluble nitrogen (WSN) and from 1.11% to 6.79% for nitrogen soluble in 12% trichloroacetic acid (TCA‐SN). The ranges for total free amino acids were from 1.65 to 8.06 mg Leu/g. Lower proteolysis was observed in the cheese samples due to high salt contents. In total, 65 volatile compounds were identified in Beaten cheese. As a conclusion, the variation of the peptide profile, electrophoresis and volatile contents of Beaten cheeses are due to the lack of standardised manufacture protocols.     

一种涂抹型核桃奶酪的研制

本研究以去皮核桃仁为原料,研究奶酪制作工艺,开发一种发酵涂抹型核桃奶酪。以感官评分、游离氨基酸为参考指标,筛选最适的酶解工艺条件;单因素试验结合响应面优化,研究发酵时间和奶油、乳清蛋白、蔗糖添加量对奶酪凝乳的影响,确定核桃奶酪工艺。结果表明:添加脂肪酶Palatase 20000 L、风味蛋白酶(添加比列为1:2,添加量为0.2%)复合酶解核桃乳时酶解效果最好,感官评分最高,游离氨基酸质量分数为(764±44)mg/100g;响应面优化结果显示乳清蛋白、奶油、蔗糖的添加量分别为1.56%、0.81%、6.37%,发酵时间24 h时,核桃奶酪感官评分达到最高(90.5分)。比较核桃发酵前后部分风味物质的含量差异,发现核桃奶酪中的游离氨基酸和游离脂肪酸含量均显著提高,其中游离氨基酸总量由发酵前的279.97 mg/kg增加到1262.02 mg/kg,对产品独特风味的形成贡献率较高。此工艺制备的核桃奶酪风味十足,营养价值高,具有较佳的感官品质,可为核桃产业的综合开发提供参考。     

Lipolysis,proteolysis and sensory properties of ewe’s raw milk cheese (Idiazabal) made with lipase addition

In this paper, we describe the effect of the addition of pregastric lipase on the composition and sensory properties of Idiazabal cheese. Free fatty acids (FFA), partial glycerides, free amino acids (FAA), gross composition and sensory characteristics were determined at different ripening times in cheeses manufactured with three different amounts of commercial animal lipase or with lipase-containing artisanal lamb rennet paste. The addition of lipase increased the content of total FFA, particularly of short-chain FFA, and that of total partial glycerides in cheeses. Unexpectedly, lipase utilization significantly affected total FAA concentration, which decreased in cheeses elaborated with high lipase amount. In general, Val, Glu and Leu were the major FAA, and their concentrations depended, mainly, on ripening time. Lipase addition had significant influence on the sensory characteristics of the cheeses, increasing scores for most of the flavour and odour attributes of the cheese. Principal component analysis (PCA) was done including dry matter, FFA, FAA, partial glycerides and odour and flavour attributes of the cheeses. It indicated that aroma and flavour parameters of Idiazabal cheese and the content of short-chain FFA and diglycerides were highly correlated to first principal component (PC1), while texture parameters, compositional variables and FAA were correlated to the second principal component (PC2).     

Chemometric analysis of proteolysis during ripening of Ragusano cheese

Chemometric modeling of peptide and free amino acid data was used to study proteolysis in Protected Denomination of Origin Ragusano cheese. Twelve cheeses ripened 3 to 7 mo were selected from local farmers and were analyzed in 4 layers: rind, external, middle, and internal. Proteolysis was significantly affected by cheese layer and age. Significant increases in nitrogen soluble in pH 4.6 acetate buffer and 12% trichloroacetic acid were found from rind to core and throughout ripening. Patterns of proteolysis by urea-PAGE showed that rind-to-core and age-related gradients of moisture and salt contents influenced coagulant and plasmin activities, as reflected in varying rates of hydrolysis of the caseins. Analysis of significant intercorrelations among chemical parameters revealed that moisture, more than salt content, had the largest single influence on rates of proteolysis. Lower levels of 70% ethanol-insoluble peptides coupled to higher levels of 70% ethanol-soluble peptides were found by reversed phase-HPLC in the innermost cheese layers and as the cheeses aged. Non-significant increases of individual free amino acids were found with cheese age and layer. Total free amino acids ranged from 14.3 mg/g (6.2% of total protein) at 3 mo to 22.0 mg/g (8.4% of total protein) after 7 mo. Glutamic acid had the largest concentration in all samples at each time and, jointly with lysine and leucine, accounted for 48% of total free amino acids. Principal components analysis and hierarchical cluster analysis of the data from reversed phase-HPLC chromatograms and free amino acids analysis showed that the peptide profiles were more useful in differentiating Ragusano cheese by age and farm origin than the amino acid data. Combining free amino acid and peptide data resulted in the best partial least squares regression model (R(2) = 0.976; Q(2) = 0.952) predicting cheese age, even though the peptide data alone led to a similarly precise prediction (R(2) = 0.961; Q(2) = 0.923). The most important predictors of age were soluble and insoluble peptides with medium hydrophobicity. The combined peptide data set also resulted in a 100% correct classification by partial least squares discriminant analysis of cheeses according to age and farm origin. Hydrophobic peptides were again discriminatory for distinguishing among sample classes in both cases.     

一种植物乳杆菌对干酪模型成熟中微生物及蛋白质水解的影响

采用无菌条件下生产的新鲜干酪凝块制作干酪模型,以WSN,12%TCASN和氨基酸含量等为指标测定植物乳杆菌SP-3对其的促熟作用。微生物测定表明SP-3在干酪模型的成熟过程中(12d)能保持较高的浓度;WSN结果表明SP-3对干酪中蛋白质的初级水解无显著影响;12%TCASN的结果表明SP-3影响了干酪中小肽的形成;5%PTA-SN和总游离氨基酸浓度测定结果表明植物乳杆菌SP-3促进了体系中游离氨基酸的产生;游离氨基酸的分析显示谷氨酸、亮氨酸、组氨酸的含量明显高于对照组,而丝氨酸、赖氨酸、脯氨酸的含量低于对照组。以上结果表明,植物乳杆菌SP-3能加快干酪体系中蛋白质的水解进程,具有促进干酪成熟的作用。     

Quality and ripening changes of Ras cheese made by direct acidification

Trials were carried out to produce Ras cheese of good quality without the use of starter. Cheese was made from pasteurized cow's milk acidified with lactic acid or citric acid to pH 5.8 alone or coupled with mixing the curd with glucono δ lactone (4.5 g/kg curd). Control cheese was made from milk ripened with a starter culture of S. lactis. Resultant cheeses showed poor body and texture, weak flavour intensity and low levels of soluble nitrogen compounds and free volatile fatty acids. Incorporation into the cheese curd of mixtures containing Fromase 100 (fungal protease) and Piccantase B (fungal lipase) or Fromase 100 and Capalase K (animal lipase) enhanced flavour intensity, improved body characteristics and accelerated the formation of both soluble nitrogen compounds and free volatile fatty acids. The organoleptic properties of the experimental cheeses with added enzymes were comparable to those of the control cheese.     

Enhancement of Blue cheese flavour using sodium dodecylsulphate and lipase

Accelerated Blue cheese ripening as affected by addition of sodium dodecylsulphate (SDS) in levels of 0.05 or 0.075% alone or SDS (0.05 or 0.075%) + lipase was studied. There were no effects on the cheese chemical composition by addition to the cheese curd of SDS or SDS/lipase combination, except for an increase in titratable acidity of cheese. Proteolysis, lipolysis and carbonyl formation were enhanced in Blue cheese treated with SDS alone. Addition of lipase with SDS greatly increased lipolysis and carbonyl formation but it did not increase proteolysis as SDS alone. Organoleptic evaluation indicated that addition of SDS alone or in combination with lipase enhanced the development of texture and flavour of cheese and improved Penicillium roqueforti distribution throughout the cheese. Cheese treated with SDS (0.075%) and lipase showed higher quality than the other cheeses.     

Impact of autolytic, proteolytic, and nisin-producing adjunct cultures on biochemical and textural properties of cheddar cheese

The effect of incorporating a highly autolytic strain (Lactobacillus delbrueckii subsp. bulgaricus UL12) a proteolytic strain (Lactobacillus casei subsp. casei L2A), or a nisin Z-producing strain (Lactococcus lactis, subsp. lactis biovar diacetylactis UL719) into Cheddar cheese starter culture (Lactococcus lactis KB and Lactococcus cremoris KB) on physicochemical and rheological properties of the resultant cheeses was examined. Cheeses were ripened at 7 degrees C and analyzed over a 6-mo period for viable lactococcal and lactobacilli counts, pH, titratable acidity (TA), lipolysis, proteolysis, and textural characteristics. The combination of the nisin-producing strain and autolytic adjuncts significantly increased the production of water-soluble nitrogen, free amino acids, and free fatty acids. The effect of Lc. diacetylactis UL719 alone or of Lb. casei L2A on water-soluble nitrogen and free amino acid contents were also significant, whereas their effect on free fatty acids was not. Viable counts of Lb. bulgaricus UL12 were significantly reduced in the presence of Lc. diacetylactis UL719. Lactobacilli-containing cheeses showed significantly lower values for hardness, fracturability, and springiness. It could be concluded that the addition of Lb. bulgaricus UL12 together with a nisin-producing strain produces a greater increase in cheese proteolysis and an improvement in Cheddar cheese texture.     

The effects of using a starter culture,lipase, and protease enzymes on ripening of Mihalic cheese

The purpose of this study was to determine the effects of fungal lipase from Mucor miehei and a bacterial neutral protease from Bacillus subtilis alone and combined with a starter culture on ripening properties of traditional Turkish Mihalic cheese. The use of protease with lipase (Cult + Prot + Lip) resulted in better flavour and texture with accelerated ripening. The obtained results pointed out that the gross compositions of the cheeses were changed by the type of enzymes and ripening time (P < 0.01). The acid degree value (ADV) of all cheeses showed a linear increase with ripening. The highest lipolysis rate was noted in lipase‐added cheese batch (as 5.56 ADV) with highest γ‐CN ratio and β‐CN degradation. At the end of ripening time, it was observed that αs‐CN ratios decreased in starter‐added (Cult), starter + protease–added (Cult + Prot), and protease‐added (Prot) cheese batches. The use of protease with lipase (Cult + Prot + Lip) resulted in better flavour and texture with accelerated ripening. Protease‐added cheeses, which were characterized by bitterness and crumbly textural properties owing to the intense breakdown of β‐casein, scored lower than lipase‐added cheeses. It was determined that the use of mesophilic aromatic starter culture with lipase and protease could be used to accelerate ripening of Mihalic cheese made from pasteurised milk.     

High-Pressure Treatment and Freezing of Raw Goat Milk Curd for Cheese Manufacture: Effects on Cheese Characteristics

High-pressure treatment of raw goat milk curd was investigated as an alternative to thermal treatment of milk in cheese manufacture, and curd freezing as a procedure to surmount the seasonality of goat milk production. Experimental cheeses were made by mixing (70:30) fresh cow milk curd with frozen curd from pasteurized goat milk (PGC), frozen curd from raw goat milk (RGC), or frozen pressurized curd from raw goat milk (PRGC). Control cheese was made from a mixture (70:30) of pasteurized cow and goat milk. RGC cheese showed the highest counts of staphylococci, Gram-negative bacteria and coliforms, whereas PRGC cheese had maximum aminopeptidase activity, esterase activity, and overall proteolysis. Control cheese exhibited the highest dry matter content and peptide levels, the lowest concentration of free amino acids, the highest concentration of volatile compounds such as free fatty acids, alcohols and esters, and the firmest texture. Differences in sensory characteristics between experimental and control cheeses were of minor importance. High-pressure treatment of curd allowed the production of cheese of bacteriological quality similar to that of control cheese made using pasteurized milk, while curd freezing did not alter the sensory characteristics of experimental cheeses with respect to control cheese.     

Effect of a food-grade enzyme preparation fromAspergillus oryzae on free fatty acid release in Manchego-type cheese from ovine and bovine milk

The addition of 50 mg Flavor Age (blend of lipase, proteinases and peptidases fromAspergillus oryzae) per kilogram of mixed ovine plus bovine milk for the manufacture of Manchego-type cheese caused the release of abundant free fatty acids (FFA), especially the short-chain fatty acids (C₄ to C₁₀). The effect accentuated with ripening time, the cheeses reaching a level of FFA around 2% at 120 days. The high concentration of FFA could have partially inhibited the growth of starter culture, as can be deduced from the higher residual lactose content of enzyme-treated cheeses. The results are compared and related to those obtained in a previous study on proteolysis and flavour development by the same enzyme preparation. They suggest that in the case of Man-chego-type cheese the liberation of FFA, although necessary, could be less important for flavour enhancement than the production of free amino acids.     

Study of the chemical composition,proteolysis, volatile compounds,and textural properties of industrial and traditional Beaten (Bieno sirenje) ewe milk cheese

The objective of this study was to determine the gross composition, proteolysis, and volatile and texture profiles during ripening of industrial (IND) and traditional (TRD) Beaten (Bieno sirenje) cheeses made by using ewe milk. In the course of the analyses, statistical differences were determined in some physicochemical parameters, nitrogen fractions, and total free amino acid levels between TRD and IND types of cheese. Higher levels of proteolysis were observed in IND cheeses than in TRD cheeses during ripening. Levels of residual β- and α_s-caseins were 72.2 and 48.7%, respectively, in 180-d-old TRD cheeses. However, the residual levels were 52.8% for β-casein and 18% for α_s-casein in IND cheeses. Similar differences were noted for the reversed-phase HPLC peptide profiles of 2 types of cheeses. Also, higher concentrations of peptides were eluted in IND cheeses than in TRD cheeses during ripening. A total of 73 volatile compounds, including alcohols (16), esters (17), acids (14), terpenes (7), ketones (5), aldehydes (4), and miscellaneous (10) were identified. The IND cheeses contained higher levels of carboxylic acids, esters, alcohols, and terpenes than the TRD cheeses; however, the same levels of methyl ketones were determined in the 2 types of cheeses at the end of ripening. These may be due to some differences (e.g., pasteurization and scalding temperature, among other factors) in the manufacture of the 2 types of Beaten cheeses. The textural profile of Beaten cheeses showed that TRD production method resulted in firmer, less fracturable, and stiffer cheeses than the IND production method. In conclusion, the results suggest that the use of industrial production method (pasteurization of cheese milk and curd scalding at 70°C) in the manufacture of Beaten ewe milk cheese enriched the volatile profile of the cheese.     

成熟温度和时间对Cheddar干酪成熟特性的影响研究

通过对不同成熟参数下Cheddar干酪蛋白分解、质构、pH及感官评价的分析,探讨了成熟参数对Cheddar干酪的影响,并得出研究条件下的最优成熟温度和时间,分析了Cheddar干酪在成熟过程中的游离氨基酸变化情况。     

Improving the quality of Ras cheese made without starter

Trials were carried out to produce Ras cheese of good quality without the use of starter. Cheese was made from cows' milk, pasteurized and acidified with lactic acid or citric acid to pH 5·8; cheese was also made after adding 4·5 g glucono-δ-lactone per kg to the acidified curd. Control cheese was made by the traditional method.The cheese had poor body and texture, weak flavour intensity, and low levels of soluble nitrogen compounds and free volatile fatty acids.Incorporation into the cheese curd of mixtures containing Fromase 100 (fungal protease) and Piccantase B (fungal lipase) or Fromase 100 and Capalase K (animal lipase) enhanced flavour intensity and body characteristics and accelerated the formation of both soluble nitrogen compounds and free volatile fatty acids. The organoleptic properties of the experimental cheese with added enzymes were comparable to the control cheese.     

Texture and flavour development in Ras cheese made from raw and pasteurised milk

Texture, proteolysis and flavour development in Ras cheeses made from raw or pasteurised milk with two different thermophilic lactic cultures were monitored during ripening. Results showed that at day 1 of manufacture, the moisture content and pH were lower in raw milk cheese than in pasteurised milk cheeses. Levels of water-soluble nitrogen, casein breakdown, free amino groups and free fatty acids were higher in cheese made from raw milk than in that made from pasteurised milk. Textural characteristics, such as hardness, cohesiveness and chewines, increased in all treatments during the first 60 days of ripening due to the reduction in the moisture level during the second stage of salting (dry salting during the first 60 days of ripening). Cheese made from raw milk received the highest texture and flavour scores by panellists.     

Proteolysis in ultra-filtered and conventional Iranian white cheese during ripening

Conventional and ultra-filtered (UF) Iranian white cheeses were made with almost identical gross chemical composition and the extent and characteristics of proteolysis were studied during ripening. UF cheeses exhibited a lower rate of development of pH 4.6-soluble nitrogen than conventional cheeses. The rates of degradation of α_s1-casein and particularly β-casein were lower in UF cheeses than in conventional cheeses. Plasmin activity was lower in UF cheeses than that in conventional cheese, whereas coagulant activity was higher in the former. Noticeable qualitative and quantitative differences were observed in reverse-phase high performance liquid chromatography (RP-HPLC) peptide profiles between UF and conventional white cheeses and chemometric analysis of peak height data distributed the cheeses into two separate groups. The levels of free amino acids in UF cheeses were lower than in conventional cheeses. Lower peptide degradation and production of amino acids suggested slower ripening, which may have been associated with the weak aroma development characteristic of UF cheeses.     

Effects of Commercial Food Grade Enzymes on Free Fatty Acid Profiles in Granular Cheddar Cheese

Commercial food grade enzymes (Neutrase, calf lipase and NaturAge) were incorporated into cheese at various concentrations and ripened at 7° and 13°C. Gas chromatographic analysis indicated that free fatty acid (FFA) production increased significantly (p<0.05) with lipase and high level NaturAge. Neutrase had little effect on FFA production but showed synergistic effects with lipase. The short-chain FFA profiles were similar among control, Neutrase- and NaturAge-treated cheeses. Significant correlations (p<0.05) between C₈ and C₁₄, C₁₆ or C_18:1 and total free fatty acids were observed in all tested samples. Total concentrations of C₄ and C₆ may be a good indicator of flavor development during cheese ripening. Cheese with a medium level of NaturAge, ripened at 13°C for 4 wk, showed profiles of FFA similar to those of the control cheese ripened at 7°C for 12 wk.