Plant Coagulant Research Articles

Exploring the Milk‐Clotting Properties of a Plant Coagulant from the Berries of S. elaeagnifolium var. Cavanilles

Solanum elaeagnifolium (trompillo or silverleaf nightshade) is an endemic plant from the northeast of Mexico and southwest of United States. This plant is considered as a weed with negative impact on agriculture and livestock production. Nevertheless, in some places of Chihuahua, Mexico, the berries of this plant have been used for decades in the manufacture of artisanal filata-type asadero cheese. The milk-clotting enzyme of S. elaeagnifolium has been scarcely studied; for this reason, the aim of this work was to explore some properties of this plant coagulant. Protein extracts (PEs) from ripe berries of S. elaeagnifolium were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assessed for milk-clotting activity. In addition, milk gels and miniature fresh-type cheeses manufactured with the plant coagulant were analyzed for their texture properties. The PE from the berries of S. elaeagnifolium contained 8 proteins with molecular weights from 22 to 62 kDa. Some bands observed in the PE had similar molecular weights as reported for aspartic proteinases such as chymosin. The extracts from the berries of S. elaeagnifolium had lower milk-cloting activities than observed with rennin or chymosin, but this plant coagulant produced firm gels under acidic conditions. The mini-cheeses manufactured with this coagulant were softer than cheeses manufactured with rennin or chymosin. For this reason, the coagulant from the ripe berries of S. elaeagnifolium could be suitable not only for the manufacture of filata-type cheeses but also for the manufacture of soft cheeses such as cream cheese. Silverleaf nightshade (trompillo) is a plant that grows in northern Mexico and the southwestern United States. This plant is considered a weed with negative impact on agriculture and livestock production. However, the ripe berries of this plant have been used by ancient Pima Indians as a substitute of rennin in making cheese. In this work, it was observed that this plant coagulant had lower activity and produced softer cheeses than did rennin or chymosin. For this reason, the coagulant from berries of S. elaeagnifolium could be used for the manufacture of soft cheeses such as cream cheese.

Characterization of the purified actinidin as a plant coagulant of bovine milk

In this work, actinidin was characterized in view of its possible suitability as a coagulant enzyme in the manufacturing process of cheese. The results show that actinidin does exhibit milk-clotting activity, which is correlated with the enzyme concentrations. The combined use of urea and SDS–PAGE led to the conclusion that the milk clot is clearly separated from the whey proteins and corresponds to casein coagulum. Moreover, both the enzyme dependence on pH and temperature and the stability profiles are fully suitable with the chemical–physical conditions adopted during the cheese-making procedure. The analysis of the kinetic constants as well as the electrophoretic pattern of the hydrolysis products suggests that β-casein is the preferred substrate of actinidin, whereas κ-casein seems to be hydrolyzed only in a few large fragments.

Effect of Plant Originated Coagulants and Chymosin on Ovine Milk Coagulation

The coagulation of ewe's milk was studied by using plant source coagulants namely the artichoke, Cynara scolymus L. cv. Blanca, and latex from the fig tree (Ficus carica L.). A turbidimetric method was used to evaluate and compare the coagulation properties of the novel coagulants with chymosin treated samples. Syneresis capacity and sensory evaluation of resultant cheese samples were studied and it was found that both cynara and chymosin produced sigmoidal increase in turbidity to the milk with three distinct phases. The coagulation kinetics was affected substantially by both coagulants. Plant coagulant induced shorter gelation time compared to chymosin however required longer time for restructuration (end of coagulation). The coagulum obtained with the latex of Ficus carica had a higher yield, owing to its high water retention capacity. With the exception of color, the overall sensory attributes did not show significant differences among coagulants.

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

SummaryPowdered 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.

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 αs-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.

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 C18 : 1.

Terpenoids and benzenoids in La Serena cheese made at different seasons of the year with a Cynara cardunculus extract as coagulant

The objective was to determine the influence of the season of manufacture and the ripening time on the volatile terpenoids and benzenoids of La Serena cheese made in summer, winter and spring, using a plant coagulant. Ewes were fed with formulated feed in summer, grazing on natural pastures in winter and spring. Most terpenoids and benzenoids present in cheese were observed in the formulated feed (mainly terpenes), and in the Cynara cardunculus extract (mainly benzenoids). The enantiomeric α(+)⧸ α(−)−pinene ratio changed during ripening. γ-Curcumene, α-humulene, ethyl benzene and propyl benzene and all xylene isomers decreased, while α-terpineol, verbenone, benzyl alcohol, 2-phenyl-ethanol, benzoic acid methyl ester, and the phenolic compounds increased significantly during ripening. Safranal, geranyl acetone, γ-curcumene and α-curcumene were more abundant in spring than in winter cheeses. Alkyl benzenes and other benzenoids were significantly more abundant in summer than in winter or spring cheeses.

Proteolysis in goats’ milk cheese made with calf rennet and plant coagulant

Primary proteolysis, secondary proteolysis and changes in hydrophilic (HI) and hydrophobic (HO) peptides during ripening of a goats’ milk cheese (Murcia al Vino) manufactured with either plant coagulant (PC) or calf rennet (AR) were studied. In the Murcia al Vino cheese, intense proteolytic activity took place during ripening, and after a 60-day ripening period of the cheeses made with animal rennet, values of 19.04, 9.61, 0.55 and 0.66 for water-soluble nitrogen (WSN) at pH 4.6, non-protein nitrogen (NPN), ammonia nitrogen (NH 3N) and amino acid nitrogen (AAN), were obtained. In the cheeses produced with PC, proteolysis was more intense, and after 60 days of ripening, values of 34.79, 11.16, 0.59 and 0.88 for WSN, NPN, NH 3N and AAN were obtained. Casein (CN) proteolysis was more intense in cheeses manufactured with PC, compared with those manufactured with AR. β-CN degradation products ( γ-CN fractions) were higher in cheeses manufactured with AR. HI and HO concentrations and the HO/HI ratio was higher in cheeses made with PC. These results were further confirmed by principal component analysis (PCA). The use of PC for goat cheese manufacture might accelerate ripening and, thus, a cheese with different sensory characteristics caused by increased cyprosin proteolytic activity may be obtained.

Clotting and proteolytic properties of plant coagulants in regular and ultrafiltered bovine skim milk

Regular and ultrafiltered (UF; 1×, 2× and 4× concentrated) skim milk samples were treated with a range of enzymes including calf rennet, ficin and papain. The clotting properties, curd casein profiles and free amino acid (FAA) contents were determined. In general, UF milk samples coagulated faster and formed firmer curds irrespective of protein concentration. Furthermore, both ficin and papain had a more significant effect on proteolysis in curd formed from regular and 1× UF milk than on 2× or 4× UF milk. Cardoon extract and calf rennet had very similar clotting properties, although the former caused both the capillary electrophoresis profile of caseins and FAA measurements to show slightly more extensive hydrolysis in the curd. The results suggest that the UF process may cause structural changes to proteins or other milk constituents with a resultant change in clotting properties and proteolysis of the casein molecules.

Effect of pH on the Gelation Properties of Skim Milk Gels Made From Plant Coagulants and Chymosin

The effect of three milk pH values, 6.0, 6.3 and 6.7, on gelation properties was monitored by small amplitude oscillatory rheology as well as a large deformation (yield) test for gels induced by the plant coagulants, Cynara cardunculus L. and Cynara humilis L., and chymosin. Gel microstructure was studied using confocal scanning laser microscopy. For each coagulant, a decrease in pH of milk resulted in a decrease in gelation time (tg), and an increase in the rate of increase in storage modulus (G′). At pH 6.0 and 6.3, plant coagulant-induced gels reached a maximum value in G′ (G′max) followed by a decrease in G′ values during the rest of the experiment, reflecting higher proteolytic activity of plant coagulants towards caseins as determined by SDS-PAGE. Gels produced at pH 6.0 and 6.3, exhibited a minimum in loss tangent (tan δ) followed by slight increase in tan δ during gel aging, that may have been associated with faster rearrangements of the gel network structure. In gels aged for ∼6h, the values for G′, tan δ at low frequency (0.006Hz) and yield stress were higher for chymosin than for plant-induced gels. For gels with the same pH value, no major differences were observed in microstructure between coagulants. Gels made at low pH values (6.3 and 6.0) appeared to have a denser or more interconnected structure than gels made at pH 6.7. Our results suggest that, at a low pH, the type of coagulant used in gelation is likely to have a considerably impact on gel/cheese structure.

Effect of gelation temperature on the properties of skim milk gels made from plant coagulants and chymosin

Reconstituted skim milk was gelled at 25–40°C with the plant-origin coagulants from Cynara cardunculus L. or Cynara humilis L. or with fermentation-produced chymosin. Gel formation and ageing were monitored by low amplitude oscillatory rheology and confocal scanning laser microscopy. Arrhenius plots for the rate of milk gelation were also determined. Plant coagulants had shorter gelation time (tg) at 25°C, 35°C and 40°C, and higher initial rate of increase in G′ values at all temperatures tested. The firmest gels at long ageing times were produced by chymosin at 30°C and 32°C. At a gelation temperature of 25°C, the differences in rheological and microstructural characteristics between plant coagulants and chymosin were considerable; plant coagulants had shorter tg and higher G′ values. For the lowest gelation temperatures, plant coagulants had smaller activation energy values for gelation. Most of the gelation results were similar between plant coagulants, but some differences were found in the values of tg, the rate of increase in G′ and loss tangent parameter. The characteristics of gels produced with plant coagulants were influenced less by the changes in temperature compared with chymosin-produced gels, which may be an important consideration in using plant-origin coagulants in the production of cheeses with a wider range of gelation temperatures.

Amino acid and soluble nitrogen evolution throughout ripening of Serra da Estrela cheese

Abstract Four batches of Serra da Estrela cheese originating from as many dairy farms were sampled throughout the ripening period, and assayed for the evolution of free amino acid (FAA) content, total nitrogen content (TN), water-soluble nitrogen content (WSN), trichloroacetic acid-soluble nitrogen content (TCASN) and phosphotungstic acid-soluble nitrogen content (PTASN). The WSN content increased from 1% (on the day of manufacture) up to 43% of TN by 180 d of ripening, thus reflecting the intense proteolytic activity of the enzymes contributed by the plant coagulant utilized. The TCASN was also found to be high in this cheese by the end of ripening (16–20%), which suggests a high extent of FAA release throughout maturation. The major FAA by 180 d of ripening were Glu, Val, Leu and Lys, representing 56–70% of the total in all four dairies sampled. Cheeses produced from refrigerated milk possessed higher amounts of γ-amino-n-butyric acid (Gaba) and lower amounts of Glu when compared with those manufactured with non-refrigerated milk.

Hydrolysis of Caseins By Extracts of Cynara Cardunculus Precipitated by Ammonium Sulfate

ABSTRACT: Polyacrylamide gel electrophoresis (in the presence of urea) and gel permeation chromatography were employed to assess the profile of hydrolysis of caseins and the activity of enzymes contributed by the flowers of the plant Cynara cardunculus on bovine caseins, after previous precipitation with ammonium sulfate or in a plain crude aqueous extract. Results indicated that the qualitative degradation profile of bovine caseins by plant enzymes (cardosins) remains essentially unchanged upon extraction, and quantitative analysis showed that the precipitated fractions had a higher coagulant‐to‐proteolytic activity ratio; hence, the results showed that inexpensive precipitation with ammonium sulfate can successfully be used as a purification method in the production of that plant coagulant in standardized form.

Advances in the role of a plant coagulant (Cynara cardunculus) in vitro and during ripening of cheeses from several milk species

Extracts from dried flowers of Cynara cardunculushave been employed successfully for many centuries in Portugal and some regions of Spain for the manufacture of traditional cheeses. Sev- eral studies have been performed in vitro and in actual cheeses focusing on the activity and specificity of proteinases from C. cardunculus, in attempts to further characterise this plant coagulant as a proteolytic agent in cheese ripening. In vitro studies encompassed extraction conditions, storage of extracts, proteolysis and specificity on caseins using experimental conditions that parallel milk and cheese during ripening. In vivo studies encompassed the effect of the type of milk (bovine, ovine and caprine) in terms of proteolysis, specificity of those plant proteinases during ripening, comparison of microbiological, chemical, proteolytic and lipolytic characteristics in ovine milk cheeses relative to animal rennet counterparts, and effect of native microflora, thermal processing and addition of starter cultures in ovine cheeses. milk clotting enzyme / plant coagulant / cheese / ewe's milk / cow's milk / goat's milk / ripening Resume - Role de l'extrait coagulant de chardon (Cynara cardunculus) in vitro et au cours de l'affinage de fromages fabriques a partir de laits de plusieurs especes. L'extrait de fleurs sechees de Cynara cardunculusa ete employe avec succes depuis plusieurs siecles au Portugal et dans quel- ques regions d'Espagne pour la fabrication de fromages traditionnels. De nombreuses etudes ont ete realisees in vitro et en fabrication fromagere sur l'activite et la specificite des proteases de C. cardun- culus, dans le but de mieux caracteriser ce coagulant vegetal comme agent proteolytique dans l'affi- nage du fromage. Les etudes in vitro ont concerne les conditions d'extraction, la conservation des extraits, la proteolyse et la specificite sur les caseines en utilisant des conditions experimentales qui

Rheological properties of milk gels made with coagulants of plant origin and chymosin

The rheological properties of milk gels made using coagulants obtained from the plants Cynara cardunculus L. and Cynara humilis L. were compared with those of fermentation-produced chymosin, using dynamic low amplitude oscillation as well as large strain (yield) testing. Gelation experiments were performed at 321C using skim milk powder that had been reconstituted for 2 or 16 h at 321C. The storage modulus (G 0 ), loss tangent (tand) at low frequency (0.002 Hz) andyieldstress were higher for chymosin-induced gels than those of plant coagulants, when tested B6 h after coagulant addition. Plant coagulants were slightly more proteolytic than chymosin, andcasein hydrolysis may have resultedin lower gel firmness. Most of the rheological properties were similar for the two plant coagulants, in agreement with their similar enzyme contents. Gelation properties were different in milk reconstituted for 2 or 16 h. This behaviour was probably due to casein hydrolysis by plasmin, as milk reconstituted for 16 h at 321C hadsignificant levels of degradation of both as- and b-caseins. The addition of soybean trypsin inhibitor which inhibits plasmin activity resulted in similar gelation profiles for gels made from milk reconstituted for 16 and 2 h. r 2002 Publishedby Elsevier Science Ltd .

Nutritional and Rheological evaluation of West African Soft cheese made from plant coagulant (Calotropis procera) during storage

Storage of West African soft cheese “Waranski” for up to 15 weeks in a refrigerator was examined for the composition, protein fractions (non casein nitrogen, non protein nitrogen, casein nitrogen, total nitrogen) hardness, cohesiveness gumminess, elasticity, chewiness, adhesiveness and pH. Whereas cheese hardness, gumminess, elasticity, chewiness and adhesiveness decreased as the storage day progressed, the protein fractions (NCN, NPN, CN, and total nitrogen) increased slightly. Each of the textural measurement correlated significantly (p Abbrev. NCN - Non Casein nitrogen, CN= casein nitrogen, NPN - Non protein nitrogen The Journal of Food Technology in Africa Volume 6 No.3, 2001, pp. 93-95 KEY WORDS: west African soft cheese, casein nitrogen, protein fractions and rheological properties.

Chemical and microbiological characteristics of ewes' milk cheese manufactured with extracts from flowers of Cynara cardunculus and Cynara humilis as coagulants.

The chemical and microbial characteristics as well as the flavor and aroma of Los Pedroches cheese made using aqueous extracts of Cynara cardunculus L. flowers were compared with those of cheeses manufactured with extracts of Cynara humilis L. throughout ripening. The two thistle species assayed were found to have no appreciable effect on the moisture, fat, protein, and NaCl contents of the cheese or on its water activity, flavor, and aroma; however, the use of C. humilis resulted in reduced lactic acid content (p < 0.001) and higher pH values (p < 0.05) relative to those of cheese specimens produced with C. cardunculus. The protein breakdown of the cheeses was assessed in terms of soluble nitrogen (SN), nonprotein nitrogen (NPN), and amino acid nitrogen (AAN). Proteolysis was more marked and rapid in cheese containing C. cardunculus as coagulant, the SN and NPN contents of which were significantly higher (p < 0. 01) than those of the cheese obtained with the species C. humilis; AAN contents were similar in both species of Cynara throughout ripening. Although total viable, coliform, and lactobacilli counts were similar in cheeses produced with both types of plant coagulant throughout ripening, enterobacteria and yeasts counts (p < 0.01) and molds counts (p < 0.05) were higher in cheese produced with C. humilis than in cheese obtained with C. cardunculus.

Optimizing physical parameters affecting coagulation of turbid water with Morninga oleifera seeds

Moringa oleifera is a plant coagulant and coagulant aid which has potential for use on a large scale for treatment of turbid waters in tropical developing countries. This paper presents results of optimization studies on physical factors including rapid mix velocity gradient and time, slow mix velocity gradient and time, and dosage of M. oleifera for low, medium and high raw water turbidities (50–750 NTU). Regression analysis was also carried out to study the effect of multiple factor interaction in the coagulation of turbid water with M. oleifera. This was carried out using the results of experimental runs involving varying initial turbidity, M. oleifera dosage, rapid mix velocity gradient, slow mix velocity gradient, and slow mix time.

Rennet: Current trends and future research

Abstract A worldwide shortage of calf rennet for cheese production has existed for several decades. Bovine pepsin and to a certain extent porcine pepsin and plant coagulants have been used as rennet substitutes, but these have not been commercially successful owing to their extensive proteolytic nature and other inherent drawbacks. Investigations to develop other alternatives have resulted in the introduction of various microbial coagulants which have found markets in several countries despite certain shortcomings when compared with traditional calf rennet. However, in some countries, microbial coagulants have not been accepted for regular cheese manufacture because they are believed to result in a reduced yield and a lower quality product; this is particularly applicable to cheddar cheese manufacture. Immobilization of proteases for milk coagulation has received renewed thrust; this may convert renneting of milk to a continuous operation. However, this technique has yet to be applied on an industrial scale; such a process is not likely to be successful since immobilized enzyme technology is dependent on the ability of a small, rapidly diffusing substrate to move quickly around the immobilized enzyme. In contrast, casein micelles are large and diffusion is slow, so the proteolysis rate is very slow. Microbial rennets offer an attractive target for genetic engineers since it may be possible to alter their structure/function characteristics to match those of calf rennet; application of site‐directed mutagenesis could be particularly rewarding. The term “calf rennet,” in the cheese industry, generally refers to an enzyme extract obtained from the fourth stomach (abo‐masum) of 10‐ to 30‐day‐old calves and used to coagulate milk for cheese production. The purified milk‐clotting enzyme present in crude rennet preparations is known as “rennin” or “chymosin.” The designation “chymosin” is now recommended in the international enzyme nomenclature (renin is associated with hypertension and is derived from the kidney). Calf rennet is referred to as “animal rennet” frequently, the term “chymase” appears in the older literature as a synonym for rennet. In more general usage, however, any milk‐clotting enzyme preparation yielding a relatively stable curd is designated as rennet. Christian Hansen is credited with the first industrial production of rennet in 1874 (1). This enzyme was called chymosin (EC 3.4.23.4); it belongs to the group of aspartic proteases and is the standard against which all other types of milk‐clotting enzymes are compared. Since the origin of cheese production, the manufacturing process has always needed soluble enzymes to clot the milk; it has been adapted to the properties of calf rennet, an essential enzyme for this purpose. Rennet coagulates milk rapidly at its natural pH with little further degradation of the milk proteins. The zymogen, prorennin, is converted to chymosin at pH below 5.0, but optimally at pH 2.0 (2). Chymosin, a strong protease, has been crystallized (3) and detailed crystallographic studies of the enzyme have been reported by Bunn et al. (4). Besides a slightly high proteolytic activity, another disadvantage of this enzyme is that it is extracted from the abomasum of the unweaned calf. As the calf ages, chymosin is replaced by pepsin, although in cattle, the secretion of chymosin never comes to a complete stop. Although pepsin can clot milk, it has a tendency to result in higher fat losses because the curd formed has a more open, looser structure than that formed with chymosin; the cheese produced also has a softer body than desired. Pepsin and a commercial product under the trade name Metroclot (5, 6) have, however, been used for the production of a variety of cheeses in the past. The kinetic properties and amino acid composition of chymosin and pepsin have been extensively studied. There is more or less a consensus of opinion in favor of chymosin as the enzyme for cheese making; presently three companies are producing calf chymosin through recombinant DNA technology. The cloned chymosin preparations produced by different microorganisms have been tested in various countries for cheese manufacture. No major differences could be detected among cheeses made with cloned chymosin and those made with the natural enzyme. This review describes the past, present, and future status of rennet substitutes utilized as milk coagulants and applications of modern biological tools to strain improvement and process development.

Relation of Ash/Moisture Quotients in Some Cheeses to Hydrolysis of αs- and β-Casein

In Bola cheese (made from cows’ milk clotted by rennet) ash/moisture quotients did not influence the relative proteolysis of the major caseins. The quotients were related inversely in a similar way to the extent of hydrolysis of αs- and β-casein. In Manchego cheese (made from sheeps’ milk clotted by rennet) the ash concentration was related inversely to the enzymic hydrolysis of β-casein but not to the proteolysis of αs-casein. In Serena cheese (also made from sheeps’ milk but clotted by a plant coagulant from a cardoon Cynara sp.) the ash concentration was related inversely to the hydrolysis of αs-casein but not to the proteolysis of β-casein.