Free amino acid content of goat's milk cheese made with animal rennet and plant coagulant

BACKGROUND: Enzymes present in the flowers of Cynara cardunculus (cyprosins) are used in the production of some traditional Spanish and Portuguese cheeses, replacing animal rennet. The aim of this work was to study the changes that take place in free amino acids during the ripening of a goat's milk cheese (Murcia al Vino) manufactured with plant coagulant (PC) or animal rennet (AR). RESULTS: The total free amino acid (TFAA) concentration increased during ripening, with Ile, Val, Ala, Phe, Gaba, Arg and Lys representing more than 50% of the TFAA content at 60 days in both types of cheese. The TFAA concentration was significantly higher in cheeses made with PC (854 mg 100 g^?1 total solids (TS)) than those made with AR (735 mg 100 g^?1 TS). The concentration of most free amino acids, especially His, Ser, Gln, Thr, Ala, Met and Ile, was higher in the PC cheese. CONCLUSION: Cheese made using PC as coagulant presented higher contents of free amino acid throughout the ripening period than cheese made using AR. Therefore we can conclude that the use of PC to produce Murcia al Vino goat's cheese would accelerate the ripening process as a result of increased cyprosin proteolytic activity. Copyright © 2011 Society of Chemical Industry     

Compositional characteristics of Murcia al Vino goat's cheese made with calf rennet and plant coagulant

The aims of the present study were to determine compositional characteristics of Murcia al Vino goat's cheese and to evaluate the effect of different types of coagulants (animal rennet and powdered vegetable coagulant obtained from the cardoon Cynara cardunculus ) on the improvement of the technological quality of Murcia al Vino goat's cheese. Only the pH, TA and ash and Ca contents were affected in a significant way by the type of coagulant used. There was a decrease in the moisture and an increase in the sodium chloride during ripening, while other parameters were unchanged. The pH and titratable acidity (TA) values of the cheeses were affected significantly by the time of ripening. There was an increase of the Na as well as a significant decrease of the Ca, P, Zn, Cu and Fe components, as a consequence of the increase of the acidity of the cheese during ripening. The esterified fatty acids were not modified significantly by the type of coagulant or by the time of ripening. Those most abundant in this type of cheese were C₁₆ and the C_18 : 1.     

Influence of residual milk-clotting enzyme and proteolysis on melting properties of soft cheese

In this work, we assessed the influence of coagulant residual activity and primary proteolysis on Cremoso Argentino cheese melting properties. For that purpose, we made Cremoso soft cheeses using different amounts of coagulant, and also obtained samples in which milk-clotting enzyme was inactivated. Primary proteolysis correlated with residual activity of coagulant in early stages of cheese ripening; however, it was similar in all cheeses after 30 days. The hydrolysis of caseins did not significantly affect the melting ability of the cheeses, expressed as the area increase after heating samples under standardized conditions. Samples with similar proximate composition showed some changes in meltability; those seemed related to pH evolution during ripening.     

The effect of natural cheddar cheese ripening on the functional and textural properties of the processed cheese manufactured therefrom

ABSTRACT:  Cheddar cheese ripened at 8 °C was sampled at 7, 14, 28, 56, 112, and 168 d and subsequently used for the manufacture of processed cheese. The cheddar cheese samples were analyzed throughout ripening for proteolysis while the textural and rheological properties of the processed cheeses (PCs) were studied. The rate of proteolysis was the greatest in the first 28 d of cheddar cheese ripening but began to slow down as ripening progressed from 28 to 168 d. A similar trend was observed in changes to the texture of the PC samples, with the greatest decrease in hardness and increase in flowability being in the first 28 d of ripening. Confocal scanning laser microscopy showed that the degree of emulsification in the PC samples increased as the maturity of the cheddar cheese ingredient increased from 7 to 168 d. This increased emulsification resulted in a reduction in the rate of softening in the PC in samples manufactured from cheddar cheese bases at later ripening times. Multivariate data analysis was performed to summarize the relationships between proteolysis in the cheddar cheese bases and textural properties of the PC made therefrom. The proportion of α _{s 1}-casein (CN) in the cheddar cheese base was strongly correlated with hardness, adhesiveness, fracturability, springiness, and storage modulus values for the corresponding PC. Degradation of α _{s 1}-CN was the proteolytic event with the strongest correlation to the softening of PC samples, particularly those manufactured from cheddar cheese in the first 28 d of ripening.     

Study of macromolecular interactions in low‐fat brined cheese modified with Zedu gum

The functionality of Zedu gum as a fat mimetic in low‐fat brined cheese was studied. The physicochemical, textural, rheological, microstructural and sensory properties of cheese samples modified with 0.1% and 0.25% of Zedu gum were compared to those of control cheeses (low‐fat and full‐fat cheeses with no fat mimetic) during ripening. To obtain further information about the cheeses' structure and interactions between macromolecules (casein protein and Zedu gum), other parameters were analysed by differential scanning calorimetry and Fourier transform infrared (FTIR) spectroscopy. Incorporation of Zedu gum into low‐fat cheese caused an open microstructure and softer texture in comparison with the control low‐fat cheese. The thermal properties and FTIR spectra of the cheeses were influenced by both fat mimetic and ripening time. On days 1 and 60 of ripening time, the lower value of enthalpy of the low‐fat cheese with 0.25 g of Zedu gum/kg of milk (AS 0.25) in comparison with control low‐fat cheese could have been due to the electrostatic nature of the interactions between Zedu gum and casein protein. On both days, the FTIR spectrum of AS 0.25 showed a well separated absorption at 1746 cm^?1 possibly due to the formation of ester groups as a result of the interaction of the carbonyl groups in Zedu gum with the hydroxyl groups of some amino acids in casein.     

Residual clotting activity and ripening properties of vegetable rennet from Cynara cardunculus in La Serena cheese

Vegetable rennet extracted from Cynara cardunculus flowers is traditionally used in the manufacture of La Serena cheese. High levels of proteolytic enzymes of the flowers are responsible for its clotting activity and strong proteolytic action. The presence of residual coagulant in cheese and whey was measured by adding known amounts of vegetable rennet as internal standard. We found no differences between the residual coagulant activity of La Serena cheese compared with other types of cheese. The coagulant content detected at the end of four cheesemakings (vat of 830 l) in cheese and whey represented 27 and 78%, respectively, of the total amount added to milk. When measurements were carried out in 16 different cheeses, vegetable rennet appeared to be highly stable during cheese ripening. Cheese composition (moisture, pH, NaCl, fat and protein) was kept relatively constant during ripening, which seems to contribute to stability of residual activity. Electrophoretic analyses of water insoluble fractions from cheeses manufactured with vegetable rennet showed that α_s-casein was less susceptible to proteolysis than β-casein. The water soluble nitrogen/total nitrogen (WSN/TN) exhibited higher levels only during the first 30 days of ripening although non-protein nitrogen/total nitrogen (NPN/TN) ratio and amino acid nitrogen (NH₂-N) increased with ripening time.     

Inhibition of Residual Coagulant in Cheese using Pepstatin

Pepstatin A, an inhibitor of acid proteases, was added (7.5, 15 or 30 μmol L^-1) to the curds/whey mixture at the start of cooking to inhibit residual coagulant in miniature (20 g) Cheddar-type cheeses. No degradation of _s1-casein was observed by urea–polyacryl amide gel electrophoresis (PAGE) in the pepstatin-treated cheeses, indicating that all the concentrations of pepstatin used in this study effectively inhibited residual coagulant throughout ripening. The level of water-soluble N (WSN) as % of total N increased very slowly in the pepstatin-treated cheeses, while there was a steady increase in WSN in the control cheeses; after 4 months of ripening, the level of WSN in the control cheese was nearly three times as high as in the cheese treated with 30 μmol L^-1 pepstatin. Urea–PAGE of water-soluble fractions (WSF) showed marked differences between pepstatin-treated cheeses and their respective controls throughout ripening. Reverse-phase HPLC of the WSF of the cheeses showed that the peptides _s1-CN f1-9/13, which are formed from the chymosin-produced peptide, _s1-CN f1-23, by the action of the cell envelope-associated proteinase of Lactococcus, were not present in pepstatin-treated cheeses. Levels of total free amino acids (as determined by the Cd–ninhydrin method) were higher in controls than in pepstatin-treated cheeses throughout ripening. The results of this study demonstrated that pepstatin is a very effective inhibitor of residual coagulant in cheese.     

Seasonal variations in the free fatty acid composition of Manchego cheese and changes during ripening

 This paper examines the evolution of the free fatty acid concentration in Manchego cheese during ripening (up to 150 days) and in two different cheesemaking seasons (autumn and winter). Factorial analysis of the principal components showed that short-chain fatty acids were the variables that best correlated (81% of the total variance explained) with the samples when distributed according to ripening time. These fatty acids likewise differentiated autumn and winter samples. Received: 30 April 1999 / Revised version: 5 July 1999     

Study of organic acids, volatile fraction and caseins of a new Halloumi-type cheese during ripening in whey brine

Organic acids, fat hydrolysis, volatile compounds and sensory characteristics of a new brine cheese which combines characteristics of Halloumi and Feta cheeses during its ripening in whey brine (100g NaCl L⁻¹) were studied. Thermotolerant protease of Mucor miehei as a coagulant enzyme and a mixture of thermotolerant starter cultures Enterococcus faecium 0165 (0.5% w/w) and Lactobacillus casei 80 10D were used. Good quality new Halloumi-type cheese was produced with higher proteolysis than traditional Halloumi cheese kept in whey brine. The volatile compounds identified comprised alcohols, aldehydes, ketones, acids, esters, hydrocarbons and sulphur compounds. Ethanol was the dominant volatile compound determined. Lactic acid was the dominant acid produced; its concentration increased during ripening, reaching a maximum value of 9929 mg kg⁻¹ at day 30. Acetic acid was also found in high amounts, which increased during cheese ripening. Lipolysis of cheese was not intense. The most abundant acids of the mature cheese were palmitic, oleic and acetic acid. The Halloumi-type cheese scored higher in the sensory analysis when fresh than did the mature cheese.     

Biochemistry of cheese ripening

Rennet-coagulated cheeses are ripened for periods ranging from about two weeks to two or more years depending on variety. During ripening, microbiological and biochemical changes occur that result in the development of the flavour and texture characteristic of the variety. Biochemical changes in cheese during ripening may be grouped into primary (lipolysis, proteolysis and metabolism of residual lactose and of lactate and citrate) or secondary (metabolism of fatty acids and of amino acids) events. Residual lactose is metabolized rapidly to lactate during the early stages of ripening. Lactate is an important precursor for a series of reactions including racemization, oxidation or microbial metabolism. Citrate metabolism is of great importance in certain varieties. Lipolysis in cheese is catalysed by lipases from various source, particularly the milk and cheese microflora, and, in varieties where this coagulant is used, by enzymes from rennet paste. Proteolysis is the most complex biochemical event that occurs during ripening and is catalysed by enzymes from residual coagulant, the milk (particularly plasmin) and proteinases and peptidases from lactic acid bacteria and, in certain varieties, other microorganisms that are encouraged to grow in or on the cheese. Secondary reactions lead to the production of volatile flavour compounds and pathways for the production of flavour compounds from fatty acids and amino acids are also reviewed.     

Use of principal component analysis to evaluate the physical properties of Mahon cheese

 The texture, colour and water activity of the seven existing types of Mahon cheese, manufactured under the Mahon cheese Appellation of Origin, were studied using eight physical variables. Four of the types are industrially manufactured [fresh (less than 10 days of ripening), half-ripened (2–5 months of ripening), ripened (5–10 months of ripening) and old-ripened (more than 10 months of ripening)] and three are traditionally manufactured (half-ripened, ripened and old-ripened). ANOVA, Tukey's multiple range test and principal component analysis (PCA) were used. Mahon cheeses from different ripening periods exhibited significant differences in water activity and yellowness values (P<0.001). PCA reduced the eight physical variables to two independent components, which accounted for 84.4% of the total variance. The first principal component (PC1) separated cheeses characterised by high water activity, high springiness values and slight yellowness from those with high hardness and puncture force; PC1 therefore distinguished among samples from different ripening periods. Cohesiveness, chewiness and gumminess were highly correlated in the second principal component, which could be interpreted as an index of cohesiveness. Received: 8 February 1999     

处理发酵剂对促熟干酪中生物胺和游离氨基酸的影响

以0、30、60、90 d促熟干酪中生物胺（组胺、色胺、苯乙胺、尸胺、酪胺）和游离氨基酸含量为指标,研究了添加复合处理发酵剂对干酪产生游离氨基酸和生物胺量的影响。结果表明：不同处理发酵剂添加量对各种生物胺产生的影响不同;添加处理发酵剂干酪中生物胺和游离氨基酸含量增加,其含量随着复合处理发酵剂添加量增加而增加。     

The effect of elevated temperature on ripening of Dutch type cheese

The aim of this study was to explore the effect of elevated temperature (16 °C) on ripening of Dutch-type cheese. Three slices of each cheese block were further divided into three layers. The processes in the control samples of cheese ripening at 10 °C were also monitored. The contents of free amino acids in accelerated cheese were two times higher than were those in control samples. The highest contents of free amino acids were observed in the cores of all slices of cheeses ripening at both temperatures. The contents of tyramine, in layers of the studied slices, reached almost 500 mg kg⁻¹ during 56 days of the experiment. The contents of biogenic amines in the edges grew even higher. Accelerated cheese showed faster equalisation of hardness than did control samples. The increase of temperature by 6 °C can reduce the ripening time in cellars by approximately one half.     

Metabolomics-based component profiling of hard and semi-hard natural cheeses with gas chromatography/time-of-flight-mass spectrometry, and its application to sensory predictive modeling

Gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS) was used to analyze hydrophilic low molecular weight components, including amino acids, fatty acids, amines, organic acids, and saccharides, in cheese, and the sensometric application for practical metabolomic studies in the food industry is described. Derivatization of target analytes was conducted prior to the GC/TOF-MS analysis. Data on 13 cheeses, six Cheddar cheeses, six Gouda cheeses and one Parmigiano-Reggiano cheese, were analyzed by multivariate analysis. The uniqueness of the Parmigiano-Reggiano cheese metabolome was revealed. Principal component analysis (PCA) showed no grouping of the Cheddar cheeses and Gouda cheeses according to production method or country of origin. The PCA loading plot confirms that many amino acids contribute positively to PC1, suggesting that PC1 is closely related to degradation of proteins, and that lactic acid contributed positively to PC2, whereas glycerol contributed negatively to PC2, suggesting that factors regarding degradation of carbohydrates and fats were expressed in PC2. Partial least squares (PLS) regression models were constructed to predict the relationship between the metabolite profile and two sensory attributes, "Rich flavor" and "Sour flavor", which were related to maturation. The compounds that play an important role in constructing each sensory prediction model were identified as 12 amino acids and lactose for "Rich flavor", and 4-aminobutyric acid, ornithine, succinic acid, lactic acid, proline and lactose for "Sour flavor". The present study revealed that metabolomics-based component profiling, focusing on hydrophilic low molecular weight components, was able to predict the sensory characteristics related to ripening.     

The effect of three different ripening/storage conditions on the distribution of selected parameters in individual parts of Dutch‐type cheese

The aim of the study was (i) to detect changes of dry matter, NaCl and twenty‐two free amino acids contents, pH and levels of selected microorganisms in four layers of cheese (from edge to core) during ripening and storage period and (ii) to describe the changes of the above‐mentioned parameters caused by early relocation of cheese from optimum ripening conditions to refrigeration temperatures. The number of mesophilic aerobic and facultative anaerobic bacteria and lactic acid bacteria differed significantly (P < 0.05) during the experiment dependent on the analysed layer and ripening/storage conditions. The free amino acid content differed significantly in individual analysed layers of cheese and also according to individual ripening/storage conditions. The highest content of free amino acids was found in samples stored at optimal ripening temperatures. Cheese hardness was also analysed and the lowest one was detected in samples ripened under optimal temperatures for the whole period. Early release of cheeses into storage rooms with lower temperature significantly affected properties of these products.     

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.     

Proteolysis during the ripening of goats’ milk cheese made with plant coagulant or calf rennet

Powdered plant coagulant (PPC) obtained from the cardoon (Cynara cardunculus) was compared with calf rennet (CR) for the manufacture of goats’ milk cheese, by determining difference in the proteolysis throughout ripening. There were no substantial differences between the compositions of cheeses made using the two types of coagulants. However, cheeses manufactured with PPC exhibited higher levels of pH 4.6-SN than cheese made using CR. The extent of breakdown of α_s-casein, as measured by urea-PAGE, was greater in cheese made using PPC than cheese made using CR. The formation of hydrophobic peptides and the ratio of hydrophobic/hydrophilic peptides throughout the ripening were higher in cheeses made with PPC than in cheeses made with CR. Principal component analysis (PCA) of peak heights of RP-HPLC peptide profiles of the ethanol-soluble and ethanol-insoluble fractions distributed the samples according to the coagulant used in their manufacture. Quantitative differences in several peptides were evident between the two types of cheese.     

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).     

Reduced-fat Cheddar cheese from a mixture of cream and liquid milk protein concentrate

Reduced-fat Cheddar cheese (RFC) was manufactured from standardized milk (casein/fat, C/F ˜ 1.8), obtained by (1) mixing whole milk (WM) and skim milk (SM) (control) or (2) mixing liquid milk protein concentrate (LMPC) and 35% fat cream (experimental). The percentage yield, total solid (TS) and fat recoveries in the experimental RFC were 22.0, 63.0 and 89.5 compared to 9.0, 50.7 and 87.0 in the control RFC, respectively. The average % moisture, fat, protein, salt and lactose were 40.7, 15.3, 32.8, 1.4 and 0.07%, respectively, in the experimental cheese and 39.3, 15.4, 33.0, 1.3 and 0.10%, respectively, in the control cheese. No growth of nonstarter lactic acid bacteria (NSLAB) was detected in the control or the experimental cheeses up to 3 months of ripening. After 6 months of ripening, the experimental cheese had 10⁷ cfu NSLAB/g compared to 10⁶ cfu/g in the control. The control cheese had higher levels of water-soluble nitrogen (WSN) and total free amino acids after 6 months of ripening than the experimental cheese. Sensory analysis showed that the experimental cheeses had lower intensities of milk fat and fruity flavours and decreased bitterness but higher intensities of sulphur and brothy flavours than in the control cheese. The experimental cheeses were less mature compared to the control after 270 days of ripening. It can be concluded from the results of this study that LMPC can be used in the manufacture of RFC to improve yield, and fat and TS recovery. However, proteolysis in cheese made with LMPC and cream is slower than that made with WM and SM.     

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.