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.     

Proteolytic and lipolytic changes during the ripening of León raw cow's milk cheese, a Spanish traditional variety

Proteolytic and lipolytic changes were studied throughout ripening of five batches of León cow's milk cheese, a traditional variety made in the north of Spain. Total soluble nitrogen, non-protein nitrogen, oligopeptides nitrogen, amino nitrogen and ammonia nitrogen fractions increased slightly during the ripening process. The final values of these nitrogen fractions indicate that this cheese undergoes a very slight proteolysis as much in extent as in depth. This weak protein degradation is corroborated when the caseins and their degradation products were quantified by electrophoresis. β-Casein stayed practically intact throughout the ripening process and only 10% of α_s-casein became degraded. The content of total free amino acids increased progressively but in a slightly increased way during ripening, reaching final average values of 592 mg (100 g)⁻¹ of total solids. The most abundant free amino acid at the end of ripening was lysine, followed by leucine, glutamic acid, tryptophan, valine and phenylalanine. The acidity index of the fat values increased during ripening by a factor of 4.39. The final values of this parameter are in the range of those observed in other cow's milk cheeses ripened by bacteria. The content in total free fatty acids underwent an increase throughout ripening reaching final average values of 6669 ppm. The most abundant free fatty acid at the end of ripening was oleic acid followed by butyric and palmitic acids. The high content of short-chain fatty acids is outstanding, specially that of butyric acid.     

SURFACE PROPERTIES OF PROTEIN LAYERS ADSORBED FROM MIXTURES OF GELATIN WITH VARIOUS CASEINS

We report surface tensions and surface shear viscosities of protein layers adsorbed from mixtures of gelatin +α_s11-casein, β-casein, k-casein or sodium caseinate. Under conditions where the two protein components carry opposite net charges, the combined experimental data at 25C and neutral pH are consistent with a two-layer model of the mixed protein film. The surface properties of sodium caseinate lie intermediate between those for the two major individual caseins (α_s1 and β).     

Proteolysis in ultra-filtered and conventional Iranian white cheese during ripening

Conventional and ultra-filtered (UF) Iranian white cheeses were made with almost identical gross chemical composition and the extent and characteristics of proteolysis were studied during ripening. UF cheeses exhibited a lower rate of development of pH 4.6-soluble nitrogen than conventional cheeses. The rates of degradation of α_s1-casein and particularly β-casein were lower in UF cheeses than in conventional cheeses. Plasmin activity was lower in UF cheeses than that in conventional cheese, whereas coagulant activity was higher in the former. Noticeable qualitative and quantitative differences were observed in reverse-phase high performance liquid chromatography (RP-HPLC) peptide profiles between UF and conventional white cheeses and chemometric analysis of peak height data distributed the cheeses into two separate groups. The levels of free amino acids in UF cheeses were lower than in conventional cheeses. Lower peptide degradation and production of amino acids suggested slower ripening, which may have been associated with the weak aroma development characteristic of UF cheeses.     

Production of hard cheese from caprine milk by the use of two types of probiotic cultures as adjuncts

Hard cheeses (Kefalotyri-like) were manufactured from caprine milk with yoghurt as a starter (A), and with its partial replacement with the probiotic adjuncts Lactobacillus rhamnosus LC 705 (B) and/or Lactobacillus paracasei ssp. paracasei DC 412 (C). Both adjuncts retarded the growth of enterococci, and the environment in cheese B did not favour the recovery of lactic acid bacteria (LAB) on Rogosa agar. However, better recovery of the LAB population on M17 agar from cheeses B and C made with adjuncts was recorded early in ripening, and this was accompanied by a greater decrease in pH. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell protein demonstrated that cheese C, made with Lb. paracasei ssp. paracasei as adjunct, is a better vehicle for delivery of live probiotic cells (10 ⁷   cfu/g) to the gastrointestinal tract than cheese B, made with Lb. rhamnosus ; the latter did not belong to the predominant microflora of one out of the two B cheeses. Urea-PAGE electrophoresis results indicated that adjunct lactobacilli enhanced the degradation of both α _S -casein (α _S -CN) and β-casein (β-CN). In the fresh cheese, hydrolysis of α _S -CN was more rapid than β-CN, and the free amino acid content of B and C was higher than in A. Lipolysis products were also higher in B and C than in A as ripening progressed, and the organoleptic characteristics of these cheeses resulted in higher scores, in the order C > B > A. Thus, making Kefalotyri-like cheese from caprine milk with probiotic lactobacilli, particularly Lb. paracasei ssp. paracasei, as adjunct can be considered an effective way of producing a cheese with a large number of probiotic cells.     

The effect of natural cheddar cheese ripening on the functional and textural properties of the processed cheese manufactured therefrom

ABSTRACT:  Cheddar cheese ripened at 8 °C was sampled at 7, 14, 28, 56, 112, and 168 d and subsequently used for the manufacture of processed cheese. The cheddar cheese samples were analyzed throughout ripening for proteolysis while the textural and rheological properties of the processed cheeses (PCs) were studied. The rate of proteolysis was the greatest in the first 28 d of cheddar cheese ripening but began to slow down as ripening progressed from 28 to 168 d. A similar trend was observed in changes to the texture of the PC samples, with the greatest decrease in hardness and increase in flowability being in the first 28 d of ripening. Confocal scanning laser microscopy showed that the degree of emulsification in the PC samples increased as the maturity of the cheddar cheese ingredient increased from 7 to 168 d. This increased emulsification resulted in a reduction in the rate of softening in the PC in samples manufactured from cheddar cheese bases at later ripening times. Multivariate data analysis was performed to summarize the relationships between proteolysis in the cheddar cheese bases and textural properties of the PC made therefrom. The proportion of α _{s 1}-casein (CN) in the cheddar cheese base was strongly correlated with hardness, adhesiveness, fracturability, springiness, and storage modulus values for the corresponding PC. Degradation of α _{s 1}-CN was the proteolytic event with the strongest correlation to the softening of PC samples, particularly those manufactured from cheddar cheese in the first 28 d of ripening.     

EFFECT OF ADDITION OF α-, β-, AND κ-CASEIN, AND Na-CASEINATE ON VISCOELASTIC PROPERTIES OF SKIM MILK CURD

Creep compliance was used to determine the effects of the addition of α-, β-, and κ-casein, and Na-caseinate on the viscoelastic properties of skim milk curd. The results of all measurements can be represented by a six-element mechanical model. Addition of α- and β-casein, and Na-caseinate (1.80g/L) to raw skim milk reduced the instantaneous modulus of rigidity and final viscosity of the curd, while κ-casein addition at the same level increased both viscoelastic parameters. Shielding of κ-casein and depletion of serum Ca⁺⁺ ions by α- and β-casein is thought to have caused the reduction of curd rigidity and viscosity. Subsequent experiments indicated that the addition of β-casein before and after rennet hydrolysis produced different curd strength with the latter producing a stronger curd.     

RHEOLOGICAL AND CHEMICAL PROPERTIES OF MOZZARELLA CHEESE

Dynamic viscoelastic parameters and chemical properties of Mozzarella cheese produced using a "no-brine" cheese making method with 3 different cooking temperatures (38, 41, and 44C) were determined. Samples were stored for 3 weeks at 4C before dynamic mechanical analysis at 22C. G', G" and tan δ were 5.8 – 6.4 × 10⁵ dyne/cm², 1.9 – 2.1 × 10⁵ dyne/cm², and 0.33 – 0.35, respectively, at 1% strain and 10 rad/s. The percentage of intact α_s-casein and β-casein were 38–40% and 33–35% of total protein in the cheese, respectively. The range of cooking temperatures used in this experiment had little effect on dynamic viscoelastic properties or the amount of intact protein for the cheese.     

Sodium caseinates — composition and properties of different preparations

A number of samples of commercially available spray-dried and roller-dried caseinates have been compared with freshly prepared caseinate from the laboratory. The compositions of the materials were measured using chromatography, and their calcium susceptibilities were determined using turbidity measurements. The chromatographic analysis showed that the commercial caseinates differed substantially from the fresh material, especially in the regions where α-casein and α_s2-caseins were eluted. The fresh caseinate was more affected by the presence of calcium ions than the others, and there were also differences between the individual spray-dried caseinates in this respect. The roller-dried caseinates were very insensitive to the presence of calcium ions.     

Changes in the microbiological and chemical characteristics of an artisanal, low-fat cheese made from raw ovine milk during ripening

Changes in the microbial flora of batzos cheese made from raw ovine milk were studied during ripening. Lactic acid bacteria and Enterobacteriaceae were the predominant groups of micro-organisms. Cheeses manufactured in summer had higher microbial counts than those made in spring, with the exception of staphylococci. Nevertheless, Enterobacteriaceae and coliforms decreased more rapidly in cheese made in summer and counts at the end of storage were lower than those in spring cheese.
Enterococci predominated in the ripened curd of cheese made in spring, whereas lactobacilli were the most abundant lactic acid bacteria in cheese made in summer. Enterococcus faecium was the predominant species in spring, and Lactobacillus paracasei ssp. paracasei predominated in cheese made in summer. The pH of the cheeses was > 5.0 throughout ripening, and NaCl-in-moisture content (> 8.0%) permitted the growth and survival of salt-tolerant micro-organisms. α_s1-Casein degraded at a faster rate than β-casein; both caseins were hydrolysed more rapidly in spring than in summer. The free amino acid content became higher in summer cheese (566.24–3460.25 µg/g of glycine equivalent) than in spring cheese because of the progress of ripening. Moreover, the milk fat of the cheese was degraded more in the summer than in the spring. The results suggest that there could be advantages to using starter cultures and improving the level of hygiene during milk and cheese production in order to eliminate undesirable micro-organisms and standardize cheese quality.     

Stability of β-lactoglobulin/micellar casein mixtures on heating in simulated milk ultrafiltrate at pH 6.0

The stability of β-lactoglobulin (β-lg)/micellar casein (MC) mixtures was examined on heating at pH 6.0 in increasing levels of lactose-free simulated milk ultrafiltrate (SMUF). Heated β-lg associated with MC to form stable particles (up to 771 nm in size in SMUF × 0.5). Higher levels of SMUF induced reductions in the charge on particles, resulting in greater aggregation and precipitation. Results indicated that the nonthiol-containing α_s1- and β-casein fractions showed greater interaction with β-lg on heating than the thiol-containing fractions (α_s2-casein and κ-casein). Casein proteins and their fractions have potential application in the development of heat-stable dairy-based beverages.     

Isolation and Identification of Low-Molecular-Weight Peptides from Emmentaler Cheese

ABSTRACT: Four peptides, derived from the N-terminal fragment of α_sl -casein [CN (1–9), CN (1–12), CN (1–13) and CN (1–14)] were isolated from a low-molecular-weight extract of a commercial Emmentaler cheese (age: approx. 5.5 mo) by gel filtration and RP-FPLC. Capillary zone electrophoresis was used to check the purity of the peptides. Using automatic Edman degradation and MALDI-TOF mass spectrometry, amino acid sequence and molecular mass of the peptides were determined to identify them. The peptide α_sl -CN (1–13) was identified for the first time in Emmentaler cheese. The peptide fraction was shown not to contribute to the characteristic flavor of Emmentaler cheese.     

微胶囊化蛋白酶在干酪制备中的应用及干酪成熟过程中电泳分析

将采用冷冻干燥工艺得到的明胶-阿拉伯胶-凝乳酶微胶囊用于干酪制备过程中,在排乳清之后向干酪中添加5.00 g微胶囊化蛋白酶,以促进干酪的成熟。在干酪成熟过程中通过三羟甲基氨基甘氨酸-尿素-十二烷基磺酸钠聚丙烯酰胺凝胶电泳以及毛细管电泳分析监测干酪中pH 4.6不溶性氮部分的变化情况。结果表明：干酪在成熟初期,αs1-酪蛋白较β-酪蛋白先水解,生成αs1-酪蛋白（f 102～199）和αs1-I-酪蛋白;干酪成熟21 d时,β-酪蛋白开始水解,αs1-酪蛋白继续保持水解,生成的大的水解产物同时也发生二次水解;干酪成熟90 d时干酪中β-酪蛋白比αs1-酪蛋白具有更广泛的水解程度,酪蛋白降解的同时,生成大量新的小分子肽。     

Lipolysis and proteolysis profiles of fresh artisanal goat cheese made with raw milk with 3 different fat contents

The objective of this study was to describe the proteolysis and lipolysis profiles in goat cheese made in the Canary Islands (Spain) using raw milk with 3 different fat contents (0.5, 1.5, and 5%) and ripened for 1, 7, 14, and 28 d. β-Casein was the most abundant protein in all cheeses and at all ripening times. Quantitative analysis showed a general decrease in caseins as ripening progressed, and degradation rates were higher for α_S1-casein than for β-casein and α_S2-casein. Furthermore, the degradation rate during the experimental time decreased with lower fat contents. The α_S2-casein and α_S1-casein levels that remained in full-fat and reduced-fat cheeses were less than those in low-fat cheese. In contrast, β-casein also showed degradation along with ripening, but differences in degradation among the 3 cheese types were not significant at 28 d. The degradation products increased with the ripening time in all cheeses, but they were higher in full-fat cheese than in reduced-fat and low-fat cheeses. The free fatty acid concentration per 100 g of cheese was higher in full-fat cheese than in reduced- and low-fat cheese; however, when the results were expressed as milligrams of free fatty acids per gram of fat in cheese, then lipolysis occurred more rapidly in low-fat cheese than in reduced- and full-fat cheeses. These results may explain the atypical texture and off-flavors found in low-fat goat cheeses, likely the main causes of non-acceptance.     

Effect of technological parameters on the characteristics of kasseri cheese made from raw or pasteurized ewes' milk

Two groups of kasseri cheese (pasta filata type) were manufactured from raw or pasteurized ewes' milk, without starter cultures. Cheeses of each group were divided into two subgroups: the first was ripened and stored at 4°C and packaged in plastic film; the second ripened and stored at 15°C and coated with paraffin wax. Milk pasteurization and technological parameters had a significant effect on the pH ( P  < 0.05), while only technological parameters had an effect on the total solids content. At day 120, the range of mean cfu/g counts for the mesophilic aerobic flora was 9.5 × 10⁷−1.4 × 10⁸; for the thermophilic streptococci, the range was 2.6 × 10⁷−7.6 × 10⁷; and for the thermophilic bacilli, 9.8 × 10⁶−1.7 × 10⁷. Changes in the N fractions became significant after 30 days of ripening. For mature 120-day-old cheeses, the percentage of total N soluble at pH 4.6 was 22.7%–22.9% in raw milk cheeses and 19.0%–21.7% in pasteurized milk cheeses. The percentage of total N soluble at 12% TCA was 10.1%–12.2% in raw milk cheeses and 7.3%–11.5% in pasteurized milk cheeses; the percentages of total N soluble at 5% PTA were 3.1%–4.0% and 2.6%–3.6%, respectively. The residual α_s-casein percentages at day 120 ranged between 63% and 78% of the respective area at day 1; the residual β-casein ranged between 67% and 75%. There were some characteristic differences in the reverse phase-HPLC peptide profiles of the four cheeses. In general, the effect of the different ripening conditions was more pronounced in cheeses made from pasteurized milk.     

Maribo cheese manufactured with concentrated milk: characteristics, maturation and yield

Research was carried out to study the feasibility of making maribo cheese using milk fortified by the addition of skim milk powder. A control (T-C) with 82 g l^-1 solids-non-fat (SNF) and 32 g l^-1 milk fat was included, along with three treatments with 11.7 (T-1), 14.6 (T-2) and 16.6 g l^-1 SNF (T-3) and standardization of the milk fat. Some chemical characteristics of the cheese milks and of the endproducts were studied and, in addition, cheese yield and the progress of maturation were monitored. It was observed that, as maturation proceeded in all treatments, there was a steady increase in the ripening index (soluble nitrogen/total nitrogen %), which indicates a progressive advance of proteolysis. Nevertheless, there were significant differences (p <.05) between the ripening indices of the control and the rest of the treatments. Furthermore, as the extent of maturation increased, α_sl-casein was degraded more than β-casein. The yield of cheese increased proportionally as the concentration of non-fat-solids in the milk increased.     

Study of the biochemical changes during ripening of Ahumado de Áliva cheese: a Spanish traditional variety

The changes in chemical composition, main physico-chemical parameters, classical nitrogen fractions, caseins and their degradation products, and some fat characteristics were studied during the ripening of ten batches of Ahumado de Áliva cheese, a traditional variety made in the north of Spain. The values of the different compositional and physico-chemical parameters at the end of the ripening did not differ significantly from those found in other cows' milk cheeses elaborated by similar technology. The low pH values are outstanding. The presence of residual lactose at the end of ripening is also relevant. Total soluble nitrogen and non-protein nitrogen increased very little during ripening. The evolution of the values of the different nitrogen fractions show that this cheese undergoes very little proteolysis and that the rennet is the main proteolytic agent. Using PAGE, it was possible to show that, throughout ripening, only 22% of α_s-casein and 9% of β-casein were degraded. The TBA value indicated that the fat of Ahumado de Áliva cheese does not undergo noticeable autooxidation during ripening. The acidity index of the fat also indicated that this cheese underwent little lipolysis during ripening.     

STARCH MOBILIZATION AND SUCROSE ACCUMULATION IN THE PULP OF KEITT MANGOES DURING POSTHARVEST RIPENING

Sugar is a determinant for the quality of mangoes, but information about its accumulation is scarce. Although starch can contribute to sugar production during ripening, not much is known about the enzymes involved. This work presents the changes in carbohydrate and enzymes during the development and ripening of Keitt mangoes. Starch disappearance was concomitant to a fivefold increase of sucrose, the most abundant sugar of the ripe fruits. The activities of α-amylase, β-amylase, phosphorylase and isoamylase were detected in the pulp, and while α-amylase increased parallel to the starch content, β-amylase presented a 20-fold increase during ripening. On the other hand, high phosphorylase activity was observed when fruits were still accumulating starch, and lowered during ripening. Isoamylase was detected during development and increased slightly during ripening, which would be in agreement to the expected role for isoamylases as acting on both subproduct of starch synthesis and degradation.

PRACTICAL APPLICATIONS

The present work reinforces our previous works that Keitt mangoes do not ripen when attached to the tree. This fact allowed us to study all the starch degradation after mango harvesting which does not occur with the other cultivars. Data obtained in this work reinforce the role of α-amylase, β-amylase and isoamylase rather than the starch phosphorylases on starch granule degradation in mangoes, and the subsequent soluble sugar accumulation.     

Changes in rheological and viscoelastic properties and protein breakdown during the ripening of 'Port Salut Argentino' cheese

Changes in the rheological behaviour and viscoelastic properties during ripening at 10°C of a soft cheese (Port Salut Argentino) packaged in a plastic film (EVA-EVA) were analysed. Casein degradation was measured by electrophoresis slab gels; α_s1 casein degradation was rapid and striking compared with that of β casein. Rheological parameters obtained from uniaxial compression tests changed during ripening: hardness decreased, adhesiveness and cohesiveness increased.
An exponential decay equation with two maxwellian elements and one elastic in parallel was fitted to characterize stress relaxation curves. Viscoelastic parameters (elastic moduli and relaxation times) were obtained using non-linear regression analysis. The elastic equilibrium modulus decreased 80% during the ripening period and the viscosity of the element with the highest relaxation time decreased 20%. These parameters represented the changes observed in cheese hardness and elasticity during ageing time and were related to the extent of casein breakdown.     

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Method for the Quantification of β-Casein Fragment (f 193-209)

ABSTRACT: Bitter cheese extract was incubated with cell-free extract of Lactobacillus helveticus strain WSU19. Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) analysis showed an intense peak at m/z-1880 that was markedly reduced after a 24-h incubation, as was bitterness. The m/z-1880 peptide was identified as β-casein (f193-209). Synthetic β-casein (f193-209) was mixed with internal standards, asparagine or deuterated glutamine substitution in β-casein (f193-209). Concentration ratios of β-casein (f193-209) to internal standard were plotted against signal ratios. Plots were linear for both internal standards. This study shows that MALDI-TOF is a rapid method for measuring a specific peptide in cheese and may be useful for assessing cheese ripening and screening cultures for debittering activity.