Yield,changes in proteolysis,and sensory quality of Prato cheese produced with different coagulants

The objective of this research was to compare the effect of 2 fungal proteases, one that is already commercially established as a milk-clotting agent and another produced at the laboratory scale, on Prato cheese composition, protein and fat recovery, yield, and sensory characteristics. Cheeses were produced according to the traditional protocol, using protease from the fungus Thermomucor indicae-seudaticae N31 and commercial coagulant from Rhizomucor spp. as clotting agents. A 2 × 6 factorial design with 3 replications was performed: 2 levels of coagulants and 6 levels of storage time. After 5, 12, 19, 33, 43, and 53 d of refrigerated storage (12°C), cheeses were monitored for proteolysis, firmness, and casein degradation by capillary electrophoresis. Sensory acceptance was evaluated after 29 d of manufacturing. The different coagulants did not statistically affect Prato cheese composition, protein and fat recovery, and yield. Both cheeses presented good sensory acceptance. Proteolysis increased and firmness decreased for both cheeses during the storage time, as expected for Prato cheese. Caseins were well separated by capillary electrophoresis and the results showed, with good resolution, that the cheeses exhibited similar protein hydrolysis profile. Both cheeses presented good sensory acceptance. The gathered data showed that the protease from T. indicae-seudaticae N31 presented similar action compared with the commercial enzyme, indicating its efficiency as clotting agent for Prato cheese manufacture.     

How three adventitious lactic acid bacteria affect proteolysis and organic acid production in model Portuguese cheeses manufactured from several milk sources and two alternative coagulants

Model cheeses were manufactured according to a full factorial experimental design to help shed light on the individual and combined roles played by 3 native lactic acid bacteria (Lactococcus lactis ssp. lactis, Lactobacillus brevis, and Lactobacillus plantarum) upon proteolysis and organic acid evolution in cheese. The model cheeses were manufactured according to a generally representative Portuguese artisanal protocol, but the (ubiquitous) adventitious microflora in the cheesemaking milk were removed via sterilization before manufacture; therefore, the specific effects of only those lactic acid bacteria selected were monitored. In addition, 2 types of coagulant (animal and plant) and 3 types of cheesemaking milk (cow, sheep, and goat) were assessed to determine their influence on the final characteristics of the model cheeses. The nature of the coagulant appeared to be essential during the first stage of proteolysis as expected, whereas the contribution of those bacteria to the pools of total free AA and organic acids was crucial afterward. This was especially so in terms of the differences observed in the metabolisms of lactic acid (in the case of Lactococcus spp.) as well as acetic and citric acids (in the case of Lactobacillus spp.).     

Microbial and sensory changes throughout the ripening of Prato cheese made from milk with different levels of somatic cells

The objective of this research was to evaluate the effects of 2 levels of raw milk somatic cell count (SCC) on the composition of Prato cheese and on the microbiological and sensory changes of Prato cheese throughout ripening. Two groups of dairy cows were selected to obtain low-SCC (<200,000 cells/mL) and high-SCC (>700,000 cells/mL) milks, which were used to manufacture 2 vats of cheese. The pasteurized milk was evaluated according to the pH, total solids, fat, total protein, lactose, standard plate count, coliforms at 45°C, and Salmonella spp. The cheese composition was evaluated 2 d after manufacture. Lactic acid bacteria, psychrotrophic bacteria, and yeast and mold counts were carried out after 3, 9, 16, 32, and 51 d of storage. Salmonella spp., Listeria monocytogenes, and coagulase-positive Staphylococcus counts were carried out after 3, 32, and 51 d of storage. A 2 × 5 factorial design with 4 replications was performed. Sensory evaluation of the cheeses from low- and high-SCC milks was carried out for overall acceptance by using a 9-point hedonic scale after 8, 22, 35, 50, and 63 d of storage. The somatic cell levels used did not affect the total protein and salt:moisture contents of the cheeses. The pH and moisture content were higher and the clotting time was longer for cheeses from high-SCC milk. Both cheeses presented the absence of Salmonella spp. and L. monocytogenes, and the coagulase-positive Staphylococcus count was below 1 × 10² cfu/g throughout the storage time. The lactic acid bacteria count decreased significantly during the storage time for the cheeses from both low- and high-SCC milks, but at a faster rate for the cheese from high-SCC milk. Cheeses from high-SCC milk presented lower psychrotrophic bacteria counts and higher yeast and mold counts than cheeses from low-SCC milk. Cheeses from low-SCC milk showed better overall acceptance by the consumers. The lower overall acceptance of the cheeses from high-SCC milk may be associated with texture and flavor defects, probably caused by the higher proteolysis of these cheeses.     

Characterization of lactic acid bacteria isolated from raw cows’ milk cheeses currently produced in Galicia (NW Spain)

Twenty cheeses belonging to the four Protected Designations of Origin manufactured in Galicia (NW Spain) (6 Arzúa-Ulloa, 4 Tetilla, 6 Cebreiro and 4 San Simón da Costa) were selected from a total of 60 cheeses on basis of their (typical) sensorial profiles. A total of 218 lactic acid bacteria (LAB) isolates were obtained from the predominant microflora of the selected cheeses and were identified as Lactococci (98 isolates), Leuconostocs (56), Mesophilic Lactobacilli (54), Pediococci (8) and Enterococci (2). Eighty-four of the isolates produced mainly malty, spicy or sulfide flavours in pasteurised whole milk, and were not characterized further. Some good producers of diacetyl-acetoin in milk (>100 mg/L) were found among a total of 129 LAB selected, although the isolates were generally less acidifying and less proteolytic than many of those obtained 10-15 years ago. The results suggest that the microflora in cheese-making environments have undergone changes, with the most evident difference being the practical absence of Enterococcal strains among the current isolates.     

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.     

Prevalence and characterization of antibiotic resistant Enterococcus faecalis in French cheeses

Prevalence of enterococci and antibiotic resistance profiles of Enterococcus faecalis was analyzed in 126 French cheeses from retail stores. Forty-four percent of pasteurized or thermised-milk cheeses, and up to 92% of raw-milk cheeses contained detectable enterococci. A total of 337 antibiotic resistant enterococci were isolated in 29% and 60% of pasteurized-milk and raw-milk cheeses, respectively. E. faecalis was the predominant antibiotic resistant species recovered (81%), followed by Enterococcus faecium (13%), and Enterococcus durans (6%). The most prevalent antibiotic resistances were tetracycline (Tet) and minocycline (Min), followed by erythromycin (Ery), kanamycin (Kan) and chloramphenicol (Cm). The most common multiple antibiotic resistance phenotype was Cm Ery Kan Min Tet. The occurrence of antibiotic genes, as searched by PCR, was 100 % for aph3′IIIa, 96 % for ermB, 90 % for tetM and 80 % for catA in isolates resistant to Kan, Ery, Tet or Cm, respectively. MLST analysis of 30 multidrug resistant E. faecalis revealed that ST19, CC21, CC25 and CC55 isolates were the most common in cheeses. In conclusion, as in many other European countries, French cheeses do contain enterococci with multiple antibiotics resistances. However, low occurrence of high-level gentamicin resistant or sulfamethoxazole/trimethoprim-resistant enterococci and absence of vancomycin- or ampicillin- resistant enterococci indicate that cheeses cannot be considered as a major reservoir for nosocomial multi-drug resistant enterococci.     

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.     

Coalho Cheese Made with Protease from Thermomucor indicae‐seudaticae N31: Technological Potential of the New Coagulant for the Production of High‐Cooked Cheese

The aim of this study was to explore the use of a new coagulant from Thermomucor indicae‐seudaticae N31 for the manufacture of a high‐cooked starter‐free cheese variety, by evaluating its physicochemical and functional characteristics in comparison to cheeses made with a traditional commercial coagulant. Coalho cheese was successfully produced with the new protease as it exhibited comparable characteristics to the one produced using the commercial enzyme: pH behavior during manufacture; cheese composition; protein and fat recovery; and cheese yield. In addition, during storage, melting was low and not affected by storage time; the increase of TCA 12% soluble nitrogen (% of total nitrogen) was lower than half of that of pH 4.6 soluble nitrogen (% of total nitrogen); concentration of β‐CN significantly decreased, whereas α_s1‐CN concentration was not affected by storage time.     

Prato cheese as suitable carrier for Lactobacillus acidophilus La5 and Bifidobacterium Bb12

To assess Prato cheese as suitable carrier for probiotic bacteria, four cheeses were produced. Control cheese contained only starter culture, whereas the others contained starter culture and the probiotic cultures Lactobacillus acidophilus La5 and Bifidobacterium Bb12 either separately or in combination. Bacterial viability, physicochemical composition, proteolysis, and texture profile were assessed over 60 days of storage. The addition of microorganisms together or separately did not affect the characteristics of Prato cheese. On storage, the cheeses showed increased proteolysis, lower firmness, and the probiotic cheeses presented counts higher than 10⁶ cfu g⁻¹. The viability of probiotics during in vitro gastrointestinal simulation, including the effect of the cheese matrix, was also assessed. The probiotic bacteria showed resistance to loss of viability during in vitro gastrointestinal simulation.     

Influence of starters on chemical, biochemical, and sensory changes in Turkish White-brined cheese during ripening

Turkish White-brined cheese was manufactured using Lactococcus strains (Lactococcus lactis ssp. lactis NCDO763 plus L. lactis ssp. cremoris SK11 and L. lactis ssp. lactis UC317 plus L. lactis ssp. cremoris HP) or without a starter culture, and ripened for 90 d. It was found that the use of starters significantly influenced the physical, chemical, biochemical, and sensory properties of the cheeses. Chemical composition, pH, and sensory properties of cheeses made with starter were not affected by the different starter bacteria. The levels of soluble nitrogen fractions and urea-PAGE of the pH 4.6-insoluble fractions were found to be significantly different at various stages of ripening. Urea-PAGE patterns of the pH 4.6-insoluble fractions of the cheeses showed that considerable degradation of α_s1-casein occurred and that β-casein was more resistant to hydrolysis. The use of a starter culture significantly influenced the levels of 12% trichloroacetic acid-soluble nitrogen, 5% phosphotungstic acid-soluble nitrogen, free amino acids, total free fatty acids, and the peptide profiles (reverse phase-HPLC) of 70% (vol/vol) ethanol-soluble and insoluble fractions of the pH 4.6-soluble fraction of the cheeses. The levels of peptides in the cheeses increased during the ripening period. Principal component and hierarchical cluster analyses of electrophoretic and chromatographic results indicated that the cheeses were significantly different in terms of their peptide profiles and they were grouped based on the use and type of starter and stage of ripening. Levels of free amino acid in the cheeses differed; Leu, Glu, Phe, Lys, and Val were the most abundant amino acids. Nitrogen fractions, total free amino acids, total free fatty acids, and the levels of peptides resolved by reverse phase-HPLC increased during ripening. No significant differences were found between the sensory properties of cheeses made using a starter, but the cheese made without starter received lower scores than the cheeses made using a starter. It was found that the cheese made with strains NCDO763 plus SK11 had the best quality during ripening. It was concluded that the use of different starter bacteria caused significant differences in the quality of the cheese, and that each starter culture contributed to proteolysis to a different degree.     

Enterococcus populations in artisanal Manchego cheese: biodiversity, technological and safety aspects

Enterococci represent a considerable proportion of the microbiota in Manchego cheeses. In this study, a total of 132 enterococci isolated from good quality Manchego cheeses from two dairies at different ripening times were genotypically characterized and identified using molecular techniques. Representative isolates from the clusters obtained after genotyping were assayed for some enzymatic activities considered to have a potential role in cheese ripening, and for 2,3-butanedione and acetoin production, evaluation of odor intensity and appearance in milk and safety evaluation. Enterococcus faecalis was the predominant specie, accounting for 81.8% of the total isolates, while Enterococcus faecium, Enterococcus hirae and Enterococcus avium were present in low proportions. The number of genotypes involved at each ripening time varied both between dairies and with the ripening times; genotype E. faecalis Q1 being present in almost all the samples from both dairies. Eight isolates showed a higher proteolytic activity and 3 isolates produced high quantities of acetoin-diacetyl, for which reason they are interesting from a technological standpoint. A low antibiotic resistance was found and almost all the strains were susceptible to clinically important antibiotics. On the contrary, only four isolates (E. faecalis C4W1 and N0W5, and E. faecium N32W1 and C16W2) did not harbor some of the virulence genes assayed.     

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.     

Selection, application and monitoring of Lactobacillus paracasei strains as adjunct cultures in the production of Gouda-type cheeses

Raw milk cheeses have more intense flavours than cheeses made from pasteurized milk and harbour strains with potential adjunct properties. Two Lactobacillus paracasei strains, R-40926 and R-40937, were selected as potential adjunct cultures from a total of 734 isolates from good quality artisan raw milk Gouda-type cheeses on the basis of their prevalence in different cheese types and/or over several production batches, safety and technological parameters. Conventional culturing, isolation and identification and a combined PCR-DGGE approach using total cheese DNA extracts and DNA extracts obtained from culturable fractions were employed to monitor viability of the introduced adjuncts and their effect on the cheese microbiota. The control cheese made without adjuncts was dominated by members of the starter, i.e. Lactococcus lactis and Leuconostoc pseudomesenteroides. In the cheeses containing either R-40926 or R-40937, the respective adjuncts increased in number as ripening progressed indicating that both strains are well adapted to the cheese environment and can survive in a competitive environment in the presence of a commercial starter culture. Principal component analysis of cheese volatiles determined by steam distillation-extraction and gas chromatography-mass spectrometry could differentiate cheeses made with different concentrations of adjunct R-40926 from the control cheese, and these differences could be correlated to the proteolytic and lipolytic properties of this strain. Collectively, results from microbiological and metabolic analyses indicate that the screening procedure followed throughout this study was successful in delivering potential adjunct candidates to enrich or extend the flavour palette of artisan Gouda-type cheeses under more controlled conditions.     

Inhibition of Residual Coagulant in Cheese using Pepstatin

Pepstatin A, an inhibitor of acid proteases, was added (7.5, 15 or 30 μmol L^-1) to the curds/whey mixture at the start of cooking to inhibit residual coagulant in miniature (20 g) Cheddar-type cheeses. No degradation of _s1-casein was observed by urea–polyacryl amide gel electrophoresis (PAGE) in the pepstatin-treated cheeses, indicating that all the concentrations of pepstatin used in this study effectively inhibited residual coagulant throughout ripening. The level of water-soluble N (WSN) as % of total N increased very slowly in the pepstatin-treated cheeses, while there was a steady increase in WSN in the control cheeses; after 4 months of ripening, the level of WSN in the control cheese was nearly three times as high as in the cheese treated with 30 μmol L^-1 pepstatin. Urea–PAGE of water-soluble fractions (WSF) showed marked differences between pepstatin-treated cheeses and their respective controls throughout ripening. Reverse-phase HPLC of the WSF of the cheeses showed that the peptides _s1-CN f1-9/13, which are formed from the chymosin-produced peptide, _s1-CN f1-23, by the action of the cell envelope-associated proteinase of Lactococcus, were not present in pepstatin-treated cheeses. Levels of total free amino acids (as determined by the Cd–ninhydrin method) were higher in controls than in pepstatin-treated cheeses throughout ripening. The results of this study demonstrated that pepstatin is a very effective inhibitor of residual coagulant in cheese.     

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.     

Evaluation of lactic acid bacteria and yeast diversity in traditional white pickled and fresh soft cheeses from the mountain regions of Serbia and lowland regions of Croatia

The goal of this study was the characterisation of indigenous lactic acid bacteria (LAB) and yeasts isolated from nine white pickled (BG) and nine fresh soft (ZG) artisanal cheeses collected in Serbia and Croatia. While LAB were present in all of the cheeses collected, yeasts were found in all BG cheeses but only in three ZG cheese samples. High LAB and yeast species diversity was determined (average H′_L = 0.4 and H′_Y = 0.8, respectively). The predominant LAB species in white pickled (BG) cheeses were Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides, while in fresh soft (ZG) cheeses the most dominant LAB species were L. lactis, Enterococcus faecalis, and Leuconostoc pseudomesenteroides. Among the 20 yeast species found, Debaryomyces hansenii, Candida zeylanoides, and Torulaspora delbrueckii were found to be predominant in BG cheeses, while Yarrowia lipolytica was predominant in ZG cheeses. The characterisation of metabolic and technological potentials revealed that 53.4% of LAB isolates produced antimicrobial compounds, 44.3% of LAB strains showed proteolytic activity, while most of the yeast species possessed either lipolytic or proteolytic activity. In conclusion, the results obtained in this study showed that the composition of LAB and yeast populations in white pickled and fresh soft cheeses is region specific. The knowledge gained in this study could eventually be used to select region specific LAB and yeast strains for the production of white pickled and fresh soft artisanal cheeses with geographically specific origins under controlled conditions.     

Short communication: Carnobacterium maltaromaticum: the only Carnobacterium species in French ripened soft cheeses as revealed by polymerase chain reaction detection

The PCR technique using Cb1-Cb2R and species-specific primers was applied to various French soft flowered or washed rind cheeses to identify Carnobacterium species. Thirty cheeses made from cow's, ewe's, or goat's milk (raw or pasteurized), of which 20 were Appellation d’Origine Contrôlée, were analyzed in the autumn and spring. The results revealed that, irrespective of the season, the Carnobacterium genus was initially detected in 5 cheeses and was detected in 5 others after an enrichment period. Polymerase chain reaction results using species-specific primers of Carnobacterium showed that these 10 cheeses contained only the species Carnobacterium maltaromaticum. Six different patterns of fermentation were found, and 3 of the 10 cheeses contained C. maltaromaticum isolates with anti-Listeria activity.     

Use of powdered vegetable coagulant in the manufacture of ewe's milk cheeses

A powdered vegetable coagulant obtained from the cardoon Cynara cardunculus and characterised as free of viable micro‐organisms, soluble and stable without the need for preservatives was evaluated, and compared with crude aqueous extract, in batches of Los Pedroches cheese, by determining various chemical, biochemical, microbiological and sensory parameters. Parameters were monitored over 3 months of ripening. High casein hydrolysis was observed after 2 days of ripening. The soluble nitrogen values reached at the end of ripening were over 34% of the total nitrogen in cheeses produced with both types of coagulant. For most parameters studied, no differences were observed between the two types of coagulant, although higher counts were observed for some microbiological groups in cheeses produced with crude aqueous extracts. The sensory quality of cheeses was practically identical with both types of coagulant. © 2002 Society of Chemical Industry     

Genotypic identification of some lactic acid bacteria by amplified fragment length polymorphism analysis and investigation of their potential usage as starter culture combinations in Beyaz cheese manufacture

In this study, 2 different starter culture combinations were prepared for cheesemaking. Starter culture combinations were formed from 8 strains of lactic acid bacteria. They were identified as Lactococcus lactis ssp. lactis (2 strains), Lactobacillus plantarum (5 strains), and Lactobacillus paraplantarum (1 strain) by amplified fragment length polymorphism analysis. The effects of these combinations on the physicochemical and microbiological properties of Beyaz cheeses were investigated. These cheeses were compared with Beyaz cheeses that were produced with a commercial starter culture containing Lc. lactis ssp. lactis and Lc. lactis ssp. cremoris as control. All cheeses were ripened in brine at 4°C for 90 d. Dry matter, fat in dry matter, titratable acidity, pH, salt in dry matter, total N, water-soluble N, and ripening index were determined. Sodium dodecyl sulfate-PAGE patterns of cheeses showed that α_S-casein and β-casein degraded slightly during the ripening period. Lactic acid bacteria, total mesophilic aerobic bacteria, yeast, molds, and coliforms were also counted. All analyses were repeated twice during d 7, 30, 60, and 90. The starter culture combinations were found to be significantly different from the control group in pH, salt content, and lactobacilli, lactococci, and total mesophilic aerobic bacteria counts, whereas the cheeses were similar in fat, dry matter content, and coliform, yeast, and mold counts. The sensory analysis of cheeses indicated that textural properties of control cheeses presented somewhat lower scores than those of the test groups. The panelists preferred the tastes of treatment cheeses, whereas cheeses with starter culture combinations and control cheeses had similar scores for appearance and flavor. These results indicated that both starter culture combinations are suitable for Beyaz cheese production.