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.     

The effect of varying casein/fat ratio on physicochemical and sensory qualities of Feta‐type cheese made using buffalo milk

The Feta‐type cheese was prepared with different casein/fat (C/F) ratios of buffalo milk using microbial rennet. The manufactured Feta cheeses were subjected to physicochemical and sensory quality at 15‐day interval up to 60 days of ripening. Sensory analysis discriminated the different level of C/F ratio of buffalo milk cheeses predominantly by age. There was no significant difference (P < 0.01) observed in cheese made from C/F ratio of 0.6–0.7 in terms of flavour. The titratable acidity (TA), soluble protein and free fatty acid appear to be age‐dependent and increased throughout the ripening in all experimental cheeses.     

Effects of coagulant type on the physicochemical and organoleptic properties of Kashar cheese

Kashar cheeses were manufactured using different coagulants (calf rennet, chymosin derived by fermentation and proteases from Rhizomucor miehei and Cryphonectria parasitica) and ripened for 90 days. Use of different coagulants did not influence the dry matter, fat, protein, salt, pH, titratable acidity, total free fatty acids and texture profile analyses. The levels of water‐soluble nitrogen, 12% trichloroacetic acid‐soluble nitrogen, and for 5% phosphotungstic acid‐soluble nitrogen, the sensory properties were significantly influenced by the use of different coagulants. β‐casein was more hydrolysed in the cheese manufactured using protease from Cryphonectria parasitica than the other cheeses during 90 d of 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。     

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.     

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     

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.     

Characteristics of Miniature Cheddar‐Type Cheese Made by Microbial Rennet from Bacillus amyloliquefaciens: A Comparison with Commercial Calf Rennet

Miniature Cheddar‐type cheeses were produced using microbial rennet from Bacillus amyloliquefaciens (milk‐clotting enzyme [MCE]) or calf rennet (CAR). With the exception of pH, there were no significant differences in gross composition between MCE‐cheese (MCE‐C) and CAR‐cheese (CAR‐C). The pH value of CAR‐C was significantly higher than that of MCE‐C at 40 and 60 d of ripening. The total nitrogen content of the pH 4.6‐soluble fraction obtained from MCE‐C was higher than that obtained from CAR‐C. However, nitrogen content of the 12% TCA‐soluble fraction was similar between CAR‐C and MCE‐C. The extent of α_s1‐casein and β‐casein hydrolysis, measured by urea‐PAGE, was similar in both cheese samples. The hydrolysis of β‐casein was lower than that of α_s1‐casein. Different reverse phase‐high‐performance liquid chromatography peptide profiles of ethanol‐soluble and ethanol‐insoluble fractions were obtained from CAR‐C and MCE‐C. The peptide content in the 2 cheese samples increased throughout ripening; the ratio of hydrophobic to hydrophilic peptides was lower in MCE‐C than in CAR‐C. Compared with CAR‐C, MCE‐C was softer as a result of higher protein hydrolysis. Microbial rennet from B. amyloliquefaciens contributed to higher proteolytic rates, which reduced ripening time.     

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.     

解淀粉芽孢杆菌GSBa-1凝乳酶在马苏里拉干酪中的应用

对分离自酒曲的1 株解淀粉芽孢杆菌GSBa-1发酵所产凝乳酶进行研究,该酶凝乳活力高而蛋白水解活力低,纯酶凝乳活力可达1.46×106 SU/g;使用该凝乳酶和商品凝乳酶制作马苏里拉干酪,并对干酪理化成分、成熟过程中pH值和微生物指标及干酪成熟前后质构特性、游离脂肪酸、可溶性蛋白、风味和干酪性能等指标进行对比分析。结果显示,理化成分上菌株凝乳酶与商品凝乳酶制作的干酪相接近（P＜0.05）。干酪在成熟过程中,发酵剂存活数先增加后减少,但其差异不大;菌株凝乳酶制作的干酪pH 4.6可溶性蛋白含量较多,干酪的游离氨基酸总量（76 mg/100 g）也高于商品凝乳酶制作的干酪游离氨基酸总量（55.3 mg/100 g）;菌株凝乳酶制作的干酪质构特性优于商品凝乳酶制作的干酪;电镜结果显示,菌株凝乳酶制作的干酪内部网状结构更充实;菌株凝乳酶具有稍强的蛋白水解活力,导致其制作的干酪风味物质种类多于商品凝乳酶制作的干酪,风味物质更加丰富。干酪样品的保形性和拉丝性实验测定结果显示,2 种凝乳酶制作的干酪性能差异不大（P＞0.05）;对2 种凝乳酶制作的干酪进行感官评定,其总评分相接近。以上结果表明,解淀粉芽孢杆菌GSBa-1凝乳酶在一定程度上可代替小牛凝乳酶应用于马苏里拉干酪的生产。     

Effect of artisanal kid rennet paste on lipolysis in semi-hard goat cheese

This study examined the use of hygienised kid rennet pastes in model cheese systems and also in goat milk semi-hard cheeses to promote lipolysis. The results obtained indicated that the use of rennet paste caused greater lipolysis and increased, mostly, the short-chain free fatty acid (FFA) content. The model systems made with whole goat’s milk using rennet paste and commercial rennet mixture exhibited a higher FFA content than did the rennet paste-free controls (31,600 vs. 25,600 μmol/kg cheese). For the pilot cheeses made with bovine rennet and rennet paste mixture, the increase in FFA level after 45 days of ripening compared with the cheeses prepared only with commercial rennet was as much as 6600 (μmol/kg cheese) and the increase in the butyric acid content was also 1650 (μmol/kg cheese). Moreover, after 15 days of ripening, industrially prepared cheeses made with rennet paste exhibited greater levels of FFA than did the cheeses made with commercial rennet (11,500 μmol/kg at 45 days of ripening). Their flavour was stronger and the organoleptic characteristics were better accepted, which implies less ripening time for commercial cheese manufacture.     

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.     

Characterization of wine rennet and its kinetics by gel electrophoresis

The rennet of glutinous rice wine (wine rennet) is an exclusive clotting agent for Chinese Royal cheese production. Some characterizations are reported herein in an attempt to provide evidence about the use of the protease as either a rennet substitute or an accelerator in cheese making and ripening. The results showed that wine rennet was a monomeric and unglycosylated protease. The N-sequencing indicated a high degree of similarity to other fungal rennets. The cleavage sites of wine rennet on oxidized insulin B chain identified by HPLC-mass spectrometry included Gln⁴-His⁵, Ala¹⁴-Leu¹⁵, Leu¹⁵-Tyr¹⁶, Tyr¹⁶-Leu¹⁷, and Phe²⁴-Phe²⁵ at pH 6.5, which were similar to those observed for Mucor rennet, but different from calf chymosin except for Leu¹⁵-Tyr¹⁶. A comparison study of the kinetic properties of wine rennet on bovine caseins with that of rennets from calf and Mucor miehei by gel electrophoresis showed that these rennets had similar coagulation efficiency but different reaction rates. Wine rennet exhibited a higher degree of degradation than the calf and Mucor enzymes at pH 6.5 and 40°C. Therefore, wine rennet would be an adjunct for calf rennet or an accelerator in cheese making.     

SENSORY CHARACTERISTICS OF EWE MILK CHEESE MADE WITH THREE TYPES OF COAGULANT: CALF RENNET, POWDERED VEGETABLE COAGULANT AND CRUDE AQUEOUS EXTRACT FROM CYNARA CARDUNCULUS

This article reports on a study of the sensorial characteristics of ewe milk cheese (Los Pedroches) made with animal rennet and two types of coagulant obtained from the cardoon Cynara cardunculus. The cheeses made with rennet displayed a less odor intensity: pungent odor and acid odor; a less acid taste; and a slightly lighter color. They were harder and firmer, but less creamy than those made with the vegetable coagulant (powdered vegetable coagulant [PVC] and crude aqueous extract [CAE] from C. cardunculus). However, the cheeses made with PVC showed organoleptic characteristics very similar to the cheeses made with CAE from C. cardunculus. After 90 days' ripening, the cheeses made with vegetable coagulant displayed a slightly more bitter taste than those made with rennet. In general terms, increased ripening time prompted the increased scores for most of the sensory attributes studied.     

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.     

Comparison of buffalo cottage cheese made from aqueous extract of Withania coagulans with commercial calf rennet

An aqueous extract of Withania coagulans was used to prepare cottage cheese from buffalo milk and its quality attributes were compared with cheese made from commercial rennet. Both cheeses contain satisfactory ranges of 49.6–54.7% moisture, 21.3–24.3% fat and 21.4–23.6% protein. The type of coagulant had no significant effect on acidity, protein and ash contents of both the cheeses. W. coagulans cheese showed a significantly (P < 0.05%) higher pH and moisture contents. Similarly, no marked differences were observed in their organoleptic evaluation, actual and theoretical yield. These results supported the fact that W. coagulans is a promising rennet substitute for cottage cheesemaking.     

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.     

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.     

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.     

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.