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.     

Changes in the free amino acid content during ripening of Idiazabal cheese: influence of starter and rennet type

The effect of starter and rennet type on free amino acid release during ripening of Idiazabal cheese was studied. Four batches of cheeses were manufactured depending on the rennet used, commercial calf rennet or artisanal lamb rennet, and the addition or not of starter culture. Cheese samples contained 24 individual free amino acids Leu, Glu, Val and Phe showing the highest contents during the ripening. The results indicated that the release of the free amino acids during ripening was strongly affected by starter added to the cheeses, and that this effect varied markedly with the rennet used for cheesemaking. Total amounts of free amino acids were higher for the cheeses made with commercial calf rennet than for those made with artisanal lamb rennet, regardless of starter addition. Likewise, the highest total free amino acid levels were found in the cheeses made with starter, regardless of the type of rennet used.     

Biochemical patterns in ovine cheese: Influence of probiotic strains

This study was undertaken to evaluate the effect of lamb rennet paste containing probiotic strains on proteolysis, lipolysis, and glycolysis of ovine cheese manufactured with starter cultures. Cheeses included control cheese made with rennet paste, cheese made with rennet paste containing Lactobacillus acidophilus culture (LA-5), and cheese made with rennet paste containing a mix of Bifidobacterium lactis (BB-12) and Bifidobacterium longum (BB-46). Cheeses were sampled at 1, 7, 15, and 30 d of ripening. Starter cultures coupled with probiotics strains contained in rennet paste affected the acidification and coagulation phases leading to the lowest pH in curd and cheese containing probiotics during ripening. As consequence, maturing cheese profiles were different among cheese treatments. Cheeses produced using rennet paste containing probiotics displayed higher percentages of α_S1-I-casein fraction than traditional cheese up to 15 d of ripening. This result could be an outcome of the greater hydrolysis of α-casein fraction, attributed to higher activity of the residual chymosin. Further evidence for this trend is available in chromatograms of water-soluble nitrogen fractions, which indicated a more complex profile in cheeses made using lamb paste containing probiotics versus traditional cheese. Differences can be observed for the peaks eluted in the highly hydrophobic zone being higher in cheeses containing probiotics. The proteolytic activity of probiotic bacteria led to increased accumulation of free amino acids. Their concentrations in cheese made with rennet paste containing Lb. acidophilus culture and cheese made with rennet paste containing a mix of B. lactis and B. longum were approximately 2.5 and 3.0 times higher, respectively, than in traditional cheese. Principal component analysis showed a more intense lipolysis in terms of both free fatty acids and conjugated linoleic acid content in probiotic cheeses; in particular, the lipolytic pattern of cheeses containing Lb. acidophilus is distinguished from the other cheeses on the basis of highest content of health-promoting molecules. The metabolic activity of the cheese microflora was also monitored by measuring acetic, lactic, and citric acids during cheese ripening. Cheese acceptability was expressed for color, smell, taste, and texture perceived during cheese consumption. Use of probiotics in trial cheeses did not adversely affect preference or acceptability; in fact, panelists scored probiotic cheeses higher in preference over traditional cheese, albeit not significantly.     

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.     

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.     

Influences of rennet and container types on proteolysis of traditional Kurdish cheese during the ripening

The effect of rennet and container types was evaluated on proteolysis of traditional Kurdish cheese during 60 days ripening. The enzymes involved were commercial chymosin and traditional rennet from lamb abomasum. Goat skin (traditional container) and plastic containers were used as storage containers. The trend of proteolysis was determined by measuring the content of nitrogen (N) in compounds soluble in water, 12% trichloroacetic acid and 5% phosphotungstic acid along with the urea–polyacrylamide gel electrophoresis method. The results showed that the nitrogen in compounds soluble in water, 12% trichloroacetic acid and 5% phosphotungstic acid was higher in ripened cheeses into plastic containers; however, the containers had no significant effect on the breakdown of α‐ and β‐caseins (P < 0.05). Using commercial rennet caused the breakdown of α‐ and β‐caseins and the level of nitrogen in compounds soluble in water to increase. Finally, however, the amount of α‐ and β‐caseins breakdown was trivial, and α‐casein was decreased more than β‐casein in all samples.     

Casein breakdown during ripening of Idiazabal cheese: influence of starter and rennet type

The objective of this work was to determine the effect of starter and rennet type on casein breakdown during Idiazabal cheese ripening. Four batches of cheeses were manufactured with two rennets, commercial calf rennet and artisanal lamb rennet, and the use of natural flora or a commercial starter. Electrophoretic analysis of cheese samples showed six bands identified as α_s1‐, α_s2 + β‐, α_s1‐I‐, γ₁‐, β‐I‐ and para‐κ‐casein. As expected, the casein breakdown during cheese ripening was considerably affected by rennet type and the use of a commercial starter. The artisanal lamb rennet produced a higher hydrolysis of casein fractions than the commercial calf rennet, probably owing to its high percentage of chymosin (around 78%). The effect of addition of starter on proteolysis was dependent on the casein fractions generated by artisanal lamb rennet or commercial calf rennet. © 2000 Society of Chemical Industry     

Proteolysis on Reggianito Argentino cheeses manufactured with natural whey cultures and selected strains of Lactobacillus helveticus

Reggianito Argentino cheese is traditionally manufactured with whey starter cultures that provide typical and intense flavor but can cause poor quality standardization. In this study, the influence of natural and selected starters on Reggianito Argentino cheese proteolysis was investigated. Cheeses were manufactured with three strains of Lactobacillus helveticus (SF133, SF138 and SF209) cultured individually in sterile whey and used as single or mixed starters. Control cheeses were made with natural whey starter culture. Cheeses were analyzed to determine gross composition, as well as total thermophilic lactic flora. Proteolysis was assessed by N fractions, electrophoresis and liquid chromatography. Gross composition of the cheeses did not significantly differ, while viable starter cell counts were lower for cheeses made with strain SF209 alone or combined with other strains. Soluble N at pH 4.6 was the same for cheeses made with natural or selected starters, but soluble N in 12% trichloroacetic acid and 2.5% phosphotungstic acid was significantly higher in cheeses made with starters containing strain SF209. Nitrogen fractions results indicated that natural whey starter cultures could be replaced by several starters composed of the selected strains without significant changes to proteolysis patterns. Starter cultures prepared only with SF209 or with the three selected L. helveticus strains produced cheese products with significantly more proteolysis than control cheeses. Chromatographic profiles analyzed by principal components showed that three main peaks on chromatograms, presumptively identified as Tyr, Phe, and Trp, explained most of variability. Principal component scores indicated that cheese samples were grouped by ripening time, which was confirmed by linear discriminant analysis. On the contrary, samples did not cluster by Lactobacillus strain or type of starter.     

凝乳酶对低脂干酪微观结构和功能特性的影响

研究了低脂干酪成熟过程中蛋白质水解程度对干酪本身的微观结构和功能特性的影响.以低脂乳为原料,添加不同剂量的凝乳酶制备低脂干酪,测定干酪不同成熟期的熔度及质量分数为12%的TCA-SN并观察干酪的微观结构.结果表明,在低脂干酪中添加双倍的凝乳酶时可以减少干酪的硬度、增加熔度和改善其感官状态;当添加3倍凝乳酶时将会导致产品比添加正常凝乳酶量时更有胶弹性.因此,添加双倍凝乳酶时能有效改善低脂干酪的质构、功能特性及感官状态.     

Influence of calcium and phosphorus, lactose, and salt-to-moisture ratio on Cheddar cheese quality: proteolysis during ripening

Proteolysis in cheese is influenced by the state of proteins (protein-calcium-phosphate interactions), level of indigenous milk enzymes (plasmin), externally added milk-clotting enzymes (chymosin), and endogenous and exogenous enzymes from starter and non-starter lactic acid bacteria (NSLAB). The objective of this study was to determine how different levels of calcium (Ca) and phosphorus (P), residual lactose, and salt-to-moisture ratio (S/M) in cheese influence proteolysis during ripening. Eight cheeses with 2 levels of Ca and P (0.67 and 0.47% vs. 0.53 and 0.39%, respectively), 2 levels of lactose at pressing (2.4 vs. 0.78%), and 2 levels of S/M (6.4 vs. 4.8%) were manufactured. The cheeses were analyzed for changes in pH 4.6-soluble N, and starter and NSLAB counts during 48 wk of ripening. Cheeses at d 1 were also analyzed for residual chymosin, plasmin, and plasminogen activity. A significant increase in soluble N was observed during ripening for all the treatments. Cheeses with low Ca and P, low lactose, and low S/M treatments exhibited higher levels of proteolysis as compared to their corresponding high treatments. Differences in the rate of proteolysis for cheeses with different levels of Ca and P might be due to changes in protein conformation and differences in residual chymosin in the cheeses. Cheeses with low Ca and P were manufactured by lowering the pH at set and drain, which led to higher chymosin retention in cheeses with low Ca and P compared with high Ca and P. Differences in proteolysis between treatments with different levels of lactose were also partly attributed to residual chymosin activity. In all treatments, a major fraction of plasmin existed as plasminogen, indicating minimal contribution of plasmin to proteolysis in Cheddar cheeses. The number of starter bacteria, in all treatments, decreased significantly during ripening. However, the decrease was larger in the case of high S/M treatments compared with low S/M treatments. In contrast, the number of NSLAB increased during ripening, and low S/M cheeses had higher counts compared with high S/M cheeses. The differences in proteolysis due to S/M were partially attributed to changes in protein conformation or bacterial proteolytic activity.     

Influence of the starter culture on the microbiological and sensory characteristics of ewe's cheese

Changes which take place in the sensory characteristics of cheeses during ripening are influenced by different factors, involving rennet, starter culture and adventitious contamination of the cheese by non-starter lactic acid bacteria. The objective of this work was to study the influence of the starter on sensory and microbiological ewe's cheese properties during ripening time. Four batches (two with starter added and two without) were manufactured. Milk and cheeses at different stages of ripening were analysed. Cheeses manufactured without adding starter showed a significantly higher level of mesophilic aerobic microflora, lactobacilli, facultatively heterofermentative lactobacilli and enterococci (indigenous microflora) than cheeses manufactured with starter. This study has also shown that adding or not adding starter affects the flavour profile of the cheese. Cheeses with starter added showed greater intensity of the following attributes: refreshing, astringent, sweet; and received lower scores on bitterness. With respect to texture, the said cheeses develop a more homogenous texture and greater elasticity throughout ripening.     

酪蛋白降解对牦牛乳硬质干酪苦味的影响

针对牦牛乳硬质干酪的苦味缺陷,分别以小牛皱胃酶、微生物凝乳酶和木瓜蛋白酶制作的牦牛乳硬质干酪为研究对象,利用尿素聚丙烯酰胺凝胶电泳,研究牦牛乳硬质干酪pH 4.6水不溶性酪蛋白的降解程度,且对成熟过程中的牦牛乳硬质干酪苦味进行感官评价,探究牦牛乳硬质干酪pH 4.6水不溶性酪蛋白降解对其苦味的影响。结果表明：牦牛乳硬质干酪在成熟期间酪蛋白发生了明显的降解,且αs-酪蛋白均比β-酪蛋白降解速率快。经尿素聚丙烯酰胺凝胶电泳分离后,发现木瓜蛋白酶制作的牦牛乳硬质干酪pH 4.6水不溶性酪蛋白在Pre-αs-酪蛋白区域有较强的蛋白带。木瓜蛋白酶制作的牦牛乳硬质干酪pH 4.6水不溶性酪蛋白中αs-酪蛋白和β-酪蛋白降解程度均显著或极显著高于微生物凝乳酶和小牛皱胃酶制作的牦牛乳硬质干酪（P＜0.05或P＜0.01）,木瓜蛋白酶制作的牦牛乳硬质干酪的苦味值极显著高于微生物凝乳酶和小牛皱胃酶制作的牦牛乳硬质干酪的苦味值（P＜0.01）,通过主成分分析得出3 种凝乳酶制作牦牛乳硬质干酪的苦味值和未降解β-酪蛋白和αs-酪蛋白含量成极显著负相关。这为控制牦牛乳硬质干酪品质提供了理论参考。     

Influence of lamb rennet paste containing probiotic on proteolysis and rheological properties of pecorino cheese

Pecorino cheeses made from heat-treated ewes’ milk using traditional lamb rennet paste (RP), lamb rennet paste containing Lactobacillus acidophilus (LA-5; RPL), and lamb rennet paste containing a mix of Bifidobacterium lactis (BB-12) and Bifidobacterium longum (BB-46; RPB) were characterized for proteolytic and rheological features during ripening. Consumer acceptance of cheeses at 60 d of ripening was evaluated. Lactobacillus acidophilus and Bifidobacterium mix displayed counts of 8 log₁₀ cfu/g and 9 log₁₀ cfu/g, respectively, in cheese during ripening. The RPB cheese displayed a greater degradation of casein (CN) matrix carried out by the enzymes associated to both Bifidobacterium mix and endogenous lactic acid microflora, resulting in the highest values of non-CN N and water-soluble N and the highest amount of α_s-CN degradation products in cheese at 60 d of ripening. The RPL cheese displayed intermediate levels of lactic acid bacteria and of N fractions. The percentage of γ-CN in RP and RPL cheeses at 60 d was 2-fold higher than in the cheese curd of the same groups, whereas the mentioned parameter was 3-fold higher in RPB cheese than in the corresponding fresh curd according to its highest plasmin content. The lower hardness in RPB at the end of ripening could be ascribed to the greater proteolysis observed in cheese harboring the Bifidobacterium mix. Although differences in proteolytic patterns were found among treatments, there were no differences in smell and taste scores.     

Ripening of Camembert-type cheese made from caprine milk using calf rennet or kid rennet as coagulant

Camembert-type cheese was made from caprine milk using either calf rennet or kid 'Grandine' rennet as coagulant. The pH of all cheeses increased throughout ripening and levels of pH 4.6-soluble nitrogen increased from 8.1 to 18.2% of total nitrogen (TN) and from 6.9 to 20% TN for the cheeses made using calf rennet and kid rennet, respectively. Degradation of β-casein, measured by urea–polyacrylamide gel electrophoresis, and total and free amino acids were greater in the cheese made using kid rennet. Production of peptides, analysed by high performance liquid chromatography (HPLC), was slightly more extensive in the Camembert-type cheese made using calf rennet as coagulant. In general, a higher degree of proteolysis was found in Camembert-type cheese made from caprine milk using kid rennet than in cheese made using calf rennet as coagulant.     

Proteolysis texture and microstructure of low‐fat Tulum cheese affected by exopolysaccharide‐producing cultures during ripening

The objective of the study was to determine the effects of exopolysaccharide (EPS)‐producing or non‐EPS‐producing starters on proteolysis, physical and microstructural characteristics of full‐fat or low‐fat Tulum cheeses during ripening. For this purpose, Tulum cheese was manufactured using full‐ or low‐fat milk with EPS‐producing and non‐EPS‐producing starter cultures. Chemical composition, proteolysis, texture profiles and microstructure of the cheeses were studied during 90 days of ripening. Urea‐PAGE of water‐insoluble and RP‐HPLC peptide profiles of water‐soluble fractions of the cheeses showed that the use of starters resulted in different degradation patterns in all cheeses during ripening. Although β‐casein exhibited similar degradation patterns in all cheeses, small differences are present in α_s1‐casein degradation during ripening. Reducing fat in Tulum cheese changed the RP‐HPLC peptide profile of the cheeses. The use of EPS‐producing cultures improved the textural characteristics and changed the microstructure and proteolysis of low‐fat Tulum cheese.     

Effects of Penicillium roqueforti and whey cheese on gross composition,microbiology and proteolysis of mould‐ripened Civil cheese during ripening

Four different types of mould‐ripened Civil cheese were manufactured. A defined (nontoxigenic) strain of a Penicillium roqueforti (SC 509) was used as the secondary starter with and without addition of the whey cheese (Lor); in parallel, secondary starter‐free counterparts were manufactured. Chemical composition, microbiology and proteolysis were studied during the ripening. The incorporation of whey cheese in the manufacture of mould‐ripened Civil cheese altered the gross composition and adversely affected proteolysis in the cheeses. The inoculated P. roqueforti moulds appeared to grow slowly on those cheeses, and little proteolysis was evident in all cheese treatments during the first 90 days of ripening. However, sharp increases in the soluble nitrogen fractions were observed in all cheeses after 90 days. Microbiological analysis showed that the microbial counts in the cheeses were at high levels at the beginning of ripening, while their counts decreased approximately 1–2 log cfu/g towards the end of ripening.     

Effect of a hygienized rennet paste and a defined strain starter on proteolysis,texture and sensory properties of semi-hard goat cheese

A hygienized rennet paste (HRP) and a defined strain starter culture, including Lactobacillus casei subsp. casei IFPL as adjunct, were considered for manufacturing Majorero cheese, a Spanish traditional variety made from goat milk. Influence of both factors on physicochemical characteristics, proteolysis, rheological and sensory properties, was evaluated throughout the ripening. Cheeses produced either industrially (IL) or in artisanal manner (AL) were compared with the experimental lot (EL), which included HRP and IFPL starter in its manufacture. Results showed a low level of primary proteolysis, expressed by a low content of non-casein nitrogen (NCN), in experimental cheeses. Despite the slightly poor texture (hard and crumbly) related to the high TS and salt contents, a good general acceptability was attained for EL, with the best scores for aroma and flavour intensities achieved at 30 ripening days. In fact, the sensory panel detected the “piquant” flavour (typical of the artisanal cheese variety) in EL after 15 days of ripening.     

Proteolysis in Manchego-type cheese salted by brine vacuum impregnation

A new salting procedure based on the brine vacuum impregnation of porous products was tested on Manchego-type cheese and compared with conventional brine immersion. Its effect on cheese proteolysis throughout a 90-d ripening period was determined. Three cheese regions were evaluated (the rind, the middle, and the internal regions). The parameters analyzed were total N, water-soluble N, soluble N in trichloroacetic acid and soluble N in phosphotungstic acid by using the Kjeldahl method, casein profile by urea-PAGE, and peptide profile of the water soluble nitrogen extract by reverse-phase HPLC. Free amino acid formation was monitored with a spectrophotometric method by using a Cd-ninhydrin reagent. Globally, proteolysis was significantly affected by ripening stage (increasing throughout all the maturation period studied) and cheese region (rind showed a proteolysis pattern different from the middle and internal regions). The salting procedure only affected cheese proteolysis in the rind, whereas conventional brine-salted cheeses showed lower proteolysis than vacuum-impregnated cheeses.     

Use of high pressure treatment to attenuate starter bacteria for use as adjuncts for Cheddar cheese manufacture

Attenuated starter bacteria cannot produce acid during cheese manufacture, but contain enzymes that contribute to cheese ripening. The aim of this study was to investigate attenuation of starter bacteria using high pressure treatment, for use in combination with a primary starter for Cheddar cheese manufacture, and to determine the effect of such adjunct cultures on secondary proteolysis during ripening. Lactococcus lactis ssp. cremoris HP and L. lactis ssp. cremoris 303 were attenuated by pressure treatment at 200 MPa for 20 min at 20 °C. Cheddar cheese was manufactured using untreated cultures of both these starter strains, either alone or in combination with their high pressure-treated equivalents. High pressure-treated starters did not produce acid during cheese manufacture and starter counts in cheeses manufactured using high pressure-treated starter did not differ from those of the controls. Higher levels of cell lysis were apparent in cheese manufactured using high pressure-treated strains than in the controls after 26 d of ripening. Small differences were observed in the peptide profiles of cheeses, analysed by reversed-phase HPLC; cheeses manufactured using high pressure-treated starters also had slightly higher levels of amino acids than the relevant controls. Overall, addition of high pressure-treated starter bacteria as a secondary starter culture accelerated secondary proteolysis in Cheddar cheese.

Industrial relevance

Attenuated starters provide extra pool of enzymes, which can influence cheese ripening, without affecting the cheese making schedule. This paper presents an alternative method for attenuation of starter bacteria using high pressure treatment and their subsequent use to accelerate secondary proteolysis in Cheddar cheese during ripening.