Compositional,biochemical and textural changes during ripening of Tulum cheese made with different coagulants

In the present study, biochemical, chemical and texture changes in Tulum cheeses made using calf rennet and microbial rennets (Aspergillus niger protease and Rhizomucor miehei protease) were compared during ripening for up to 90 days. A total of 15 free fatty acids (FFAs) were detected in the cheese samples. The peroxide values (PV) of the cheeses increased significantly (P < 0.05) during ripening and the cheese made with calf rennet had the highest PV. Proteolysis in the cheeses increased as the ripening time increased. α_s1‐casein and β‐casein degradation was higher in cheeses manufactured with R. miehei protease. Cheeses made with calf rennet were significantly (P < 0.05) harder, more adhesive, more cohesive and more resilient than those made with microbial rennet.     

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     

Biochemical and microbiological characterization of artisan kid rennet extracts used for Cabrales cheese manufacture

Four samples of artisanal kid rennet extracts from four different Cabrales cheese producers were biochemically and microbiologically characterized. Most samples had a very acidic pH (around pH 4.0), which may condition their biochemical and microbial variables. The milk-clotting activity of the extracts was assessed using a Formagraph apparatus. The properties of these artisanal rennets were found to be comparable to a 1/10 dilution of 1:10,000 commercial calf rennet; their enzymatic potentials, measured with a semi-quantitative commercial system (API ZYM), showed only very slight differences. A large population of lactobacilli was found in all artisanal kid rennet samples, whereas coliforms, enterococci, staphylococci and leuconostocs were only occasionally encountered. Sixty-four representative colonies were classified by PCR amplification and the sequencing of a stretch of their 16S rDNA genes. Strains of Lactobacillus plantarum completely dominated one of the extracts. In all others samples, strains of this homofermentative species and of the heterofermentative Lactobacillus brevis were present in similar amounts.     

Evaluation of Harp Seal Gastric Protease as a Rennet Substitute for Cheddar Cheese

A crude preparation of gastric proteases from Harp Seal (Pagophilus groenlandicus) was found to coagulate milk over a wider pH range than porcine pepsin and had a higher ratio of milk clotting to proteolytic activity with hemoglobin at pH 1.8. Cheddar cheese prepared with seal gastric protease (SGP) gave significantly higher sensory scores than cheese made with calf rennet. Chemical analysis of the cheeses revealed a lower concentration of citrate-HCl soluble nitrogen and less free and peptide-bound amino acids in SGP cheese than in the cheeses made with calf rennet and Mucor miehei protease.     

Effect of the use of three different lamb paste rennets on lipolysis of the PDO Pecorino Romano Cheese

Two rennet pastes were prepared from lambs on different diets, and then, together with a commercial lamb rennet paste, were used to produce protected designation of origin (PDO) Pecorino Romano cheeses. Chymosin activity and lipolytic activity were higher in the prepared rennets than in the commercial rennet. Lipolysis was greatest in cheese produced with one of the prepared rennets. Both quantitative and qualitative differences in free fatty acids were found in the cheeses. The taste of cheeses made with the prepared rennets was preferred by an expert sensory panel and also considered more piquant than that of cheese made with commercial rennet. The results indicate that both the diet of the lambs and slaughtering conditions should be regulated to produce rennet that preserves the traditional characteristics of PDO Pecorino Romano cheese. Both lipolytic and proteolytic properties of rennet pastes should be standardised for the preparation of such cheeses.     

Diffuse Reflectance Profiles of Eight Milk-Clotting Enzyme Preparations

Eight milk-clotting enzyme prepartations were standardized to equal clot time and used to coagulate pasteurized whole milk. Diffuse reflectance profiles were monitored for 60-min using a fiber optic sensor sensitive to infrared light at 950 nm. Modified M. miehei and M. pusillus protease, recombinant chymosin and calf rennet produced similar profiles. Rates of increase in diffuse reflectance were E. parasitica recombinant chymosin > calf rennet > modified M. miehei, M. pusillus var. Lindt > 50:50 blend of calf rennet and bovine pepsin > unmodified M. miehei > pepsin. Monitoring milk coagulation as described may be useful during cheese making and allow setting optimal conditions for milk-clotting enzyme preparations.     

Effects of Enzyme Choice and Fractionation of Commercial Enzyme Preparations on Protein Recovery in Curd

Five different commercial milk clotting preparations (bovine rennet, calf rennet, calf rennet-porcine pepsin mixture, Mucor miebei protease, and modified Mucor miehei protease) were adjusted to equivalent milk clotting activities and then used to clot milk. Percentages of protein in the resulting wheys were compared. Calf rennet, bovine rennet, or modified Mucor miehei protease caused less loss of protein to the whey than Mucor miehei protease or calf rennet-porcine pepsin mixture. The five enzyme preparations were then fractionated by gel filtration. Fractions with milk clotting activity were pooled. Original enzyme preparations and the pooled fractions made from them were standardized to the same clotting activity, then used to coagulated milk to compare their effect on protein loss to the whey. Fractionation significantly improved protein recovery when bovine rennet and calf rennet-porcine pepsin mixture were used as coagulants but not when calf rennet, Mucor miehei protease, or modified Mucor miehei protease were used.     

The influence of the enzymatic composition of lamb rennet paste on some properties of experimentally produced PDO Fiore Sardo cheese

An experiment on PDO (Protected Designation of Origin) Fiore Sardo cheese making was carried out to assess the influence of rennet paste on cheese quality. Four different preparations of lamb rennet paste (A, B, C and D) were used. Rennet A came exclusively from suckling lambs slaughtered immediately after suckling, rennet B from suckling lambs not fed for 12 h, and rennets C and D from suckling lambs allowed to graze and slaughtered in the same way as A and B, respectively. In vitro lipolysis and multiple cheese-making trials were carried out for each rennet. The enzymatic composition of the prepared rennets was different. Lipolytic activity was assessed in vitro by measuring the quantity of free fatty acids released by hydrolysis of a sheep milk cream substrate. Rennet A had the greatest lipolytic activity, which had a strong preference for esters of short-chain fatty acids. The cheeses made using the different rennets did not differ significantly in their moisture, protein and fat content or in the nitrogen fractions which resulted from proteolysis. The cheese produced with rennet A had the highest lipolysis. Short-chain fatty acids, in particular butyric acid, were prevalent, as they were in the in vitro trials. The diet and the presence of milk in the abomasa was responsible for the enzymatic peculiarities of rennet A. Suckling stimulated the secretion of pre-gastric lipase from the pharingeal tissue. Thus, if one wishes to produce a rennet with the particular enzymatic composition necessary for a particular cheese, the diet and the slaughtering conditions of the lambs should be controlled.     

Influence of rennet milk-clotting activity on the proteolytic and sensory characteristics of an ovine cheese

Roncal cheese (regulated by an Apellation of Origin) is a traditional hard cheese manufactured from raw ewe's milk in the region of Navarre in Spain. Roncal cheeses, manufactured using two lamb rennets with different milk-clotting activity levels, were evaluated to compare their chemical, proteolytic, and sensory characteristics. A preliminary study of samples of lamb rennets indicated that a large proportion of such rennets did not fulfil current microbiological requirements and likewise revealed considerable variation in the milk-clotting activity of the samples examined. Trends in the overall physicochemical parameter values (pH, dry matter, fat, and protein) were similar in both cheese batches. Proteolysis of the nitrogen fractions was observed to take place at a faster rate in the cheeses made using the rennet with the higher milk-clotting activity (soluble nitrogen, non-protein nitrogen, and amino acid nitrogen values around 13–20% higher than in the cheeses made using the rennet with the lower milk-clotting activity after 180 days of ripening). Urea-PAGE electrophoretic analysis of the caseins from the cheeses manufactured using both types of rennet showed that the β-caseins were less susceptible to proteolysis than the α_s-caseins. The effect of the different milk-clotting activity levels was most pronounced on the α_s-caseins, in which the rennet with the higher milk-clotting activity gave higher breakdown. Nevertheless, the differences in the proteolysis rates did not yield any appreciable sensory differences.     

Rennet and rennet substitutes

The use of calf rennet substitutes in cheese manufacture will increase because of costs linked with the availability of calf vells on the world market and the development of new cheese making processes such as ultrafiltration. The development of recombinant DNA techniques for chymosin production will also have an impact on the traditional calf rennet market when rennet manufacture by this method has been perfected. Further research into the secondary action of rennet during cheese maturation will also lead to a better understanding of how rennet can influence the flavour and textural characteristics of various cheese.     

Proteolysis of bovine αs2-casein by chymosin

Proteolysis of bovine α_s2-casein by chymosin (E. C. 3.4.23.4) in solution in 100mm Na phosphate buffer, pH 6.5, at 30 °C was studied by reversed-phase (RP)-HPLC and urea-polyacrylamide gel electrophoresis (PAGE). Chymosin hydrolyzed α_s2-casein in solution to eight peptides detectable by urea-PAGE. Peptides soluble in acetate buffer, pH 4.6, were isolated by RP-HPLC on a C₁₈ column using an acetonitrile/water gradient and identified from their N-terminal amino acid sequence. The chymosin cleavage sites were at the bonds Phe₈₈-Tyr₈₉, Tyr₉₅-Leu₉₆, Gln₉₇-Tyr₉₈, Tyr₉₈-Leu₉₉, Phe₁₆₃-Leu₁₆₄, Phe₁₇₄-Ala₁₇₅ and Tyr₁₇₉-Leu₁₈₀. Chymosin cleavage sites were restricted to the hydrophobic regions of the molecule. The bond-type in α_s2-casein cleaved by chymosin was in agreement with that found to be susceptible to chymosin in other caseins. The primary site of chymosin action on α_s2-casein appeared to be at Phe₈₈-Tyr₈₉.     

Residual Milk Coagulants in Whey — Their Inactivation and Effects on Ultrafiltration Flux and Nitrogen Yields

Ten commercial milk coagulants were used to produce Cheddar cheese wheys. Wheys were then concentrated fourfold by ultrafiltration following pasteurization, separation, and prewarming. Fluxes were monitored. Wheys, final concentrates, and pooled permeates were tested for total nitrogen, nonprotein nitrogen, and fat. Wheys and concentrates were also tested for residual milk clotting activity. Residual activity was observed in the wheys and concentrates originating from the unmodified proteases of Mucor miehei and Mucor pusillus but not from the two modified proteases of Mucor miehei, an Endothia parasitica protease, two bovine pepsins, a bovine pepsin-swine pepsin blend, a Mucor pusillus-swine pepsin blend, or calf rennet, which served as the control. No significant differences in flux or nitrogen yields were observed.     

Evaluation of Thiol Activated Proteases from Clam Viscera as a Rennet Substitute for Cheese-Making

Clam rennet, which is a crude enzyme preparation of cathepsin B-like protease from clam viscera was characterized and compared to porcine pepsin and calf rennet for its suitability as a milk coagulant in cheesemaking. Clam rennet was more proteolytic and produced a softer curd than the other two coagulants. However, influences of the pH and temperature on milk clotting with clam rennet were very similar to those of calf rennet. The cheddar cheese made from clam rennet was not inferior to the Cheddar cheese made from calf rennet. Quality enhancement occurred despite the view that high ratio of proteolytic to clotting activity is generally considered to be unfavorable for cheese-making. The higher proteolytic activity appeared to accelerate the ripening process. A small yield loss occurred as a result of excessive proteolysis during cheese-making.     

Proteolytic and Milk Clotting Fractions in Milk Clotting Preparations

Five different milk clotting preparations were fractionated on Sephadex G-100 and then tested for milk clotting activity and for proteolysis of denatured hemoglobin. Two preparations were also tested for proteolytic activity on casein. Proteolytic activities on hemoglobin were correlated with clotting ability of bovine rennet, calf rennet, and rennin-pepsin mixture at pH 1.6 and with Mucor miehei protease at pH 5.2. Modified Mucor miehei protease activities on hemoglobin correlated equally well at pH 1.6 and 5.2. Gel filtration through Sephadex G-100 and elimination of nonclotting fractions reduced the proteolytic activities on hemoglobin at pH 5.2 of calf rennet, bovine rennet, Mucor miehei protease, modified Mucor miehei protease, and rennin-pepsin mixture by 68.6, 88.5, 3.7, 53.7, and 91.2%, respectively. At pH 1.6, proteolysis was reduced by 54.2, 41.2, 51.8, 59.5, and 60.8%. Proteolytic activities of bovine rennet and renninpepsin mixture on casein were reduced by 59.8 and 72%, respectively.     

Evolution of lipolysis during the ripening of traditional Feta cheese

Lipolysis was studied during ripening of traditional Feta cheese produced in two small dairies, A and B. The cheeses were made from a thermized mixture of ewes’/goats’ milk by using yoghurt as starter and artisanal rennet from lambs’ and kids’ abomasa (cheese A) or mixed artisanal rennet with calf rennet (cheese B).The acid degree value and the free fatty acids (FFA) contents in both cheeses increased sharply up to 18 d (pre-ripening period at 15 °C) and continued to increase throughout ripening. In both mature cheeses, acetic acid was found at high levels (13–18% of the total FFAs). However, except for this, all FFA contents differed significantly (P < 0.05) between the two cheeses throughout ripening. The levels of individual and total C2:0–C8:0, C10:0–C14:0 and C16:0–C18:2 fatty acids were significantly higher (P < 0.05) in cheese A than in cheese B. Presumably the difference, especially in the C2:0–C8:0 content, was due mainly to the type of the rennet used. Butyric acid was the dominant FFA in cheese A (20% of the total FFAs at 120 d), while the most abundant FFAs in cheese B were capric (18%) and lauric acid (18%). In general, the lipolysis degree of the two cheeses was higher than those reported for the industrially-made Feta cheese.In organoleptic evaluation, cheese A had a piquant taste that was attributed to its high content of butyric acid and showed a significantly (P < 0.05) higher total score than cheese B.     

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.     

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.     

凝乳酶的研究进展

传统干酪制作使用的凝乳酶来源于小牛皱胃,目前小牛皱胃酶的供应量仅能满足世界干酪产量所需凝乳酶的20%～30%。小牛皱胃酶的供需矛盾和价格高昂等因素,使得寻求其替代物成为乳品领域科学研究的热点之一。本文首先介绍了小牛皱胃酶的研究现状,其次从动物、植物、基因重组以及微生物来源,综述了不同凝乳酶之间酶性质差异以及凝乳酶在干酪应用中的研究,旨在为凝乳酶的研究提供一定的理论参考,为寻找凝乳酶替代物提供思路。     

Influence of the technological properties of vegetable rennet (Cynara cardunculus) on the physicochemical,sensory and rheological characteristics of ‘Torta del Casar’ cheese

The purpose of this work was to investigate the influence of technological properties of vegetable rennet (Cynara cardunculus) on the characteristics of ‘Torta del Casar’ cheese to establish the parameters that can be useful for quality control of this coagulant. The physicochemical, microbial, texture and sensory properties of the cheeses were evaluated and correlated to clotting and proteolytic activities of the vegetable rennets used for their manufacture. The level of in vitro degradation of α‐casein and κ‐casein/β‐casein degradation ratio was found to be good tools to predict the effect of vegetable rennet on the rheological properties of these cheeses.