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.     

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     

Amino acids evolution during ripening of goats' milk cheese manufactured with different coagulants

Powdered plant coagulant obtained from the cardoon flowers ( Cynara cardunculus ) was compared with calf rennet for the manufacture of traditional raw goats' milk cheese, by determining differences in the profiles of the amino acids throughout the ripening period. Derivatisation with o -phtaldialdehyde and a C₁₈ column were used for chromatographic separations. In order to establish the relationships between the different variables and to detect the most important causes of variability, principal component analysis was applied to the free amino acids data, which reduced to two dimensions where cheese samples from 2 to 60 days and cheeses from 90 to 120 days were distributed dependently of ripening time and coagulant used. Leu, Val, Lys, Gly, Tyr and Asp were correlated with PC1, which separated the samples according to their ripening time. Arg, His, Trp, Ser and Thr were correlated with PC2, which separated the samples according to the coagulant used.     

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.     

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.     

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.     

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.     

Preliminary observations of flavonoid glycosides from the vegetable coagulant Cynara L. in protected designation of origin cheeses

Aqueous extracts of Cynara cardunculus L. (cardoon) flowers have been used for centuries as vegetable coagulants for traditional cheesemaking in the Iberian Peninsula. However, the full chemical composition of the extract that is added to the milk has never been studied. In this study we investigated the phenolic components of the aqueous extracts from cardoon flowers and their detection in cheese. Analysis by HPLC-diode array detection (HPLC-DAD) revealed the presence of one flavonoid-7-glycoside (isorhoifolin) and the aglycones apigenin and luteolin. Another major flavonoid-7-glycoside (apigenin-7-O-glucuronide) was also identified and its structure elucidated by ¹³C-NMR. This represents the first report of these compounds in the aqueous extract of cardoon flowers. These compounds were also found in cheese curd made using this coagulant but were not present in the original milk, leading to the suggestion that these phenolic compounds could be used as authentication markers for cheeses made using cardoon extracts as the coagulant.     

Influence of vegetable and animal rennet on proteolysis during ripening in ewes’ milk cheese

The renewed interest in using enzymes from thistles of the genus Cynara in the making of traditional ewes’ milk cheese prompted us to investigate the effect of vegetable and animal rennet on proteolysis during ripening of Los Pedroches cheese. Casein hydrolysis was found to be much more extensive and faster in cheese made by using vegetable rennet (the amount of soluble nitrogen at 60, 80 and 100 days of ripening was more than 28% greater than that in cheese produced using animal rennet). The levels of insoluble Tyr and Trp were higher in cheese produced with vegetable rennet. PAGE, using gels containing 7 M urea, revealed decreased contents in residual α_s-CN and β-CN, as well as markedly increased levels of the more mobile components in cheese produced from vegetable rennet at the end of ripening. On the other hand, the degree of proteolysis in terms of NPN or its main components (peptides, amino acids and ammonia) was similar in cheese produced using animal or vegetable rennet.     

Influence of the Coagulant Level on Early Proteolysis in Ovine Cheese-like Systems Made with Sterilized Milk and Cynara cardunculus

ABSTRACT: The effect of coagulant level on the quality and quantity of protein breakdown during the first 24 h of ripening of cheese-like systems, manufactured with sterilized ovine milk using crude aqueous extracts of Cynara cardunculus as coagulant, was experimentally assessed. Urea-polyacrylamide gel electrophoresis was performed on both water-soluble and water-insoluble cheese extracts to monitor the casein degradation pattern; the ripening extension index and the ripening depth index were thus calculated. Peptides from the water-soluble fraction were isolated by reverse-phase, high-performance liquid chromatography and partially sequenced by Edman degradation. Higher residual coagulant levels in curdled milk led to earlier breakdown of caseins, as expected. The primary cleavage sites were Phe105-Met106 in k-casein, Phe23-Val24 in α_s1-casein, and Leu127-Thr128, Ser142-Trp143, Leu165-Ser166, and Leu190-Tyr191 in β-casein.     

Proteolysis in miniature Cheddar-type cheeses made using blends of chymosin and Cynara cardunculus proteinases as coagulant

Miniature (20 g) Cheddar-type cheeses were manufactured using blends of Cynara cardunculus proteinases and chymosin as coagulant (100 : 0, 50 : 50, 25 : 75 and 0 : 100 C. cardunculus proteinases : chymosin). There were no substantial differences between the compositions of cheeses made using any of the four coagulant blends. Cheeses manufactured with coagulant blends containing C. cardunculus proteinases exhibited higher levels of pH 4.6-soluble nitrogen than cheese made using chymosin as coagulant. The extent of breakdown of α _s1 -casein, as measured by urea-polyacrylamide gel electrophoresis (urea-PAGE), was greater in cheeses made using coagulant preparations containing C. cardunculus proteinases as a constituent than in cheese made using 100% chymosin as coagulant. Different reverse-phase high-performance liquid chromatography (RP-HPLC) peptide profiles of the ethanol-soluble and -insoluble fractions were obtained for cheeses made using either C. cardunculus proteinases or chymosin as coagulant. Principal component analysis and hierarchical cluster analysis of RP-HPLC data confirmed that the inclusion of even small proportions (25%) of C. cardunculus proteinases with chymosin in the coagulant blend greatly altered the pattern and extent of proteolysis in miniature Cheddar-type cheeses.     

Can the use of Australian cardoon (Cynara cardunculus L.) coagulant overcome the quality problems associated with cheese made from ultrafiltered milk?

Concentrated cow's milk, obtained by either limited ultrafiltration to arrive at a concentration factor of 1.4× (UF) or by mixing 4× UF milk with regular milk (MX) was used to manufacture cheeses coagulated with calf rennet or aqueous extract from Cynara cardunculus L. (cardoon). The manufactured cheeses were tested and compared with those made from regular milk for chemical and sensory properties, yield, textural and biochemical indices over a 60-day ripening period. There was no significant difference (P > 0.05) in the chemical properties with the type of coagulant but in general, a lower yield and greater bitterness were observed in the cheeses made using cardoon, while ultrafiltration led to reduced casein hydrolysis, less bitterness and harder, more crumbly cheeses irrespective of coagulant type. The MX process was successful in reducing the textural problems which occurred in cheese made with UF milk alone. The ultrafiltration process itself was apparently detrimental to the textural quality of cheeses, rather than the associated increase in concentration.     

Cheesemaking with vegetable coagulants—the use of Cynara L. for the production of ovine milk cheeses

This paper reviews the use of plant extracts as vegetable coagulants for cheesemaking. It covers the plants used as sources of coagulants, with a historical overview and particular emphasis on Cynara species. The genus Cynara  L., its composition, milk clotting and proteolytic enzymes (cardosins) and their specificity towards peptide linkages are also described. Cheeses produced in the Iberian Peninsula using Cynara  L. as coagulant are documented. Cynara  L. is still the most used vegetable coagulant in cheesemaking, and also the most investigated. However, much work remains to be done to understand its action during cheese maturation and further characterization.     

The hydration behaviour of rennet caseins in calcium chelating salt solution as determined using a rheological approach

Rennet casein is used in the manufacture of Mozzarella cheese analogues. Hot solutions of calcium chelating salts are used to facilitate hydration of the protein which subsequently acts as an emulsifier for the oil phase dispersed in the protein/aqueous phase of the cheese analogue. On cooling, a matrix resembling cheese in texture and other rheological properties is produced. Considerable variations in the functional performance of different rennet caseins during cheese analogue manufacture have been observed. Commercial rennet caseins have been obtained and are being studied to establish the nature and causes of these variations in performance. A simplified model system to study rennet casein hydration was developed. The method involves dispersing rennet caseins in solutions of chelating salt at 55°C with stirring. The changes in viscosity index that occur as the rennet casein hydrated were followed using a controlled shear rate rheometer equipped with a custom designed paddle stirrer. Hydration of the rennet casein particles was found to occur in a number of stages including particle swelling, clumping, network formation and subsequent breakdown of the network. Considerable variations in the times taken for different rennet caseins to swell, clump and reach maximum viscosity index and in the stability of the hydrated protein dispersions were found. The rheological properties of the rennet caseins during hydration were highly dependent on the concentration of the chelating salt in the hydrating solution.     

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.     

Proteolysis in rennet casein-based cheese analogues

Rennet caseins manufactured from mid- or late-lactation milk were used to prepare pilot- and commercial-scale Mozzarella-type cheese analogues. Mid-lactation rennet casein and analogues made from it contained low levels of plasmin; late-lactation rennet caseins and analogues made from them had higher levels of plasmin while pilot-scale analogues had higher levels of plasmin than commercial-scale analogues. The cheese analogues were stored at 8°C for 32 weeks during which time total bacterial counts increased from ∼10⁴ to 10⁷–10⁸ cfu g⁻¹, with non-starter lactic acid bacteria dominating the microflora. Proteolysis, as reflected by pH 4.6-soluble nitrogen and free amino acids, increased on storage of all the analogues. The increase was greater in the analogues manufactured from late-lactation rennet caseins than in those manufactured from mid-lactation rennet casein and in pilot-scale analogues than in commercial-scale analogues, reflecting the differences in plasmin levels as well as slight differences in composition. Polyacrylamide gel electrophoretograms of the cheese analogues showed that β-casein was hydrolysed more extensively than α_s1-casein and that the concentration of γ-caseins increased on storage. The degree of hydrolysis of β-casein was greater in the cheese analogues manufactured from late-lactation rennet caseins than in those manufactured from mid-lactation rennet casein and in the pilot-scale analogues than in the commercial-scale analogues reflecting the corresponding plasmin levels. Seventeen peptides in the pH 4.6-soluble extract of the cheeses were isolated and partially sequenced; all originated from the N-terminal region of β-casein; 9 peptides had Lys₂₉ of β-casein as the N-terminal, 6 peptides had Arg₁ of β-caesin as the N-terminal and the remaining two peptides had Ile₃₀ and Phe₃₃ of β-casein as the N-terminal, respectively. The pattern of casein hydrolysis in the cheese analogues suggests that plasmin was the primary proteolytic agent contributing to initial hydrolysis of the caseins while microbial proteinases and peptidases may have contributed to the high levels of free amino acids.     

Permeate Flux During Ultrafiltration of Whey: Influence of Milk Coagulant Used for Cheese Manufacture

A pilot-scale plate and frame UF system was used to fractionate Cheddar cheese whey and study the effects of different commercial milk coagulants on permeate flux. Coagulants used in this study were calf rennet, Mucor pusillus protease, and Mucor miebei protease. Whey UF performance studies were conducted at a commercial Cheddar cheese plant and at Cornell under controlled conditions. Ultrafiltration was done in a continuous mode and initial concentration factor was set at 2× to simulate the first stage of a multistage whey UF system.Permeate flux decline was rapid in the first 30 min of UF for all wheys studied. More important, the type of milk coagulant used in cheese making had a profound effect on permeate flux during whey UF. No differences in the gross composition of the various wheys were correlated with differences in permeate flux. The highest permeate flux was measured for UF of whey produced during manufacture of Cheddar cheese using coagulant derived from Mucor pusillus. Lowest permeate flux was measured for UF of whey produced during manufacture of Cheddar cheese using calf rennet. Whey from cheese manufactured using Mucor miebei coagulant had flux performance intermediate to Mucor pusillus and calf rennet. The impact of milk coagulants on whey UF process efficiency should be considered by cheese makers.     

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。     

解淀粉芽孢杆菌GSBa-1凝乳酶对羊奶干酪成熟特性的影响

为探究解淀粉芽孢杆菌GSBa-1凝乳酶制备的羊奶干酪(干酪B)成熟特性的变化,以采用商业凝乳酶和同批次羊奶制作的干酪(干酪A)为对照组,比较两组干酪在60d成熟期主要组分、质构特性、微生物指标及风味物质的变化。结果表明,两组干酪得率相差不大。成熟期间干酪的水分、蛋白质及脂肪含量呈先上升后下降趋势,干酪B始终高于干酪A;干酪游离氨基酸总量在成熟期间呈先下降后上升趋势,且干酪B中苯丙氨酸、天冬氨酸、异亮氨酸、甲硫氨酸、丝氨酸含量高于干酪A。成熟前期干酪B质构特性优于干酪A。干酪A成熟后乳酸乳球菌数量增加了(5.22±0.02)%,干酪B无显著变化(P>0.05)。成熟期内,两组干酪中挥发性风味物质种类和含量均增加,但干酪B中的壬酸、辛醇、2-庚酮、2-壬酮、二甲基砜使羊奶干酪风味独特、浓郁。因此,GSBa-1凝乳酶具备替代商业凝乳酶用于羊奶干酪生产的潜力,可对干酪风味的形成和品质的提升起到一定促进作用。     

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.