Influence of lamb rennet paste containing probiotic on proteolysis and rheological properties of pecorino cheese

Pecorino cheeses made from heat-treated ewes’ milk using traditional lamb rennet paste (RP), lamb rennet paste containing Lactobacillus acidophilus (LA-5; RPL), and lamb rennet paste containing a mix of Bifidobacterium lactis (BB-12) and Bifidobacterium longum (BB-46; RPB) were characterized for proteolytic and rheological features during ripening. Consumer acceptance of cheeses at 60 d of ripening was evaluated. Lactobacillus acidophilus and Bifidobacterium mix displayed counts of 8 log₁₀ cfu/g and 9 log₁₀ cfu/g, respectively, in cheese during ripening. The RPB cheese displayed a greater degradation of casein (CN) matrix carried out by the enzymes associated to both Bifidobacterium mix and endogenous lactic acid microflora, resulting in the highest values of non-CN N and water-soluble N and the highest amount of α_s-CN degradation products in cheese at 60 d of ripening. The RPL cheese displayed intermediate levels of lactic acid bacteria and of N fractions. The percentage of γ-CN in RP and RPL cheeses at 60 d was 2-fold higher than in the cheese curd of the same groups, whereas the mentioned parameter was 3-fold higher in RPB cheese than in the corresponding fresh curd according to its highest plasmin content. The lower hardness in RPB at the end of ripening could be ascribed to the greater proteolysis observed in cheese harboring the Bifidobacterium mix. Although differences in proteolytic patterns were found among treatments, there were no differences in smell and taste scores.     

Comprehensive analysis of proteolysis during 8 months of ripening of high-cooked Old Saare cheese

We applied capillary electrophoresis, liquid chromatography coupled with tandem mass-spectrometry (MS/MS), and ultra-performance liquid chromatography to determine the composition of water-insoluble and water-soluble proteinaceous fractions of the cheese and to study in detail the degradation of caseins during 8 mo of ripening of Estonian high-temperature cooked hard cheese Old Saare. The application of high-resolution and high-accuracy MS/MS enabled identification of more than 3,000 small peptides, representing a fairly full casein peptidome containing peptides of 4 to 25 AA in length: 1,049 from β-casein (CN), 944 from α_S1-CN, 813 from α_S2-CN, and 234 from κ-CN. The majority of β-CN- and α_S1-CN-derived peptides originated from the N-terminal parts of the molecule, f6-93 and f1-124, respectively; peptides from α_S2-CN arose predominantly from the C-terminal end f100-162. At the beginning of ripening, we found a relatively high amount of peptides originating from the glycomacropeptide part of κ-CN, whereas peptides from para-κ-CN prevailed during the later stages of ripening of the cheese. The cleavage patterns of β-CN, α_S2-CN, as well as α_S1-CN, showed that primary proteolysis was started mainly by plasmin, although a low proteolytic activity of chymosin was also evident. Based on the analysis of cleavage sites, we observed a significant participation of proteolytic enzymes, including amino- and carboxypeptidases, of both mesophilic and thermophilic starter bacteria in further hydrolysis of oligopeptides during the ripening. Several new phosphopeptides were detected in the result of MS/MS data analysis. The profiles of the estimated concentrations of phosphopeptides revealed that those originating from β-CN and α_S1-CN accumulated during cheese maturation. In contrast, we did not notice any generation of phosphopeptides from the highly phosphorylated part of α_S2-CN, f25-80, presumably due to the inaccessibility of this region to the action of plasmin and chymosin. The analysis of cleavage sites and the combination of principal component and clustering analyses provided a characterization of the complex dynamics of formation and degradation of peptides during cheese maturation. We made an attempt to obtain a comprehensive picture of proteolysis during Old Saare cheese ripening on the basis of the detailed peptidomic data, including also the less abundant peptides determined by MS/MS, and complemented by the data on intact caseins and free AA and reported the results in the paper.     

Identification of casein peptides in plasma of subjects after a cheese-enriched diet

Despite several studies support the functionality of casein peptides in vitro, a gap of knowledge still exist about their bioavailability.In this pilot study the bioavailability of phosphopeptides (CPPs) was investigated in four healthy subjects who consumed for one week 100 g/day of Parmigiano Reggiano cheese after one week of a dairy products-free diet. CPPs were detected in plasma samples from fasting subjects after the cheese-enriched diet and peptides were detected variously in almost all the 4 samples. Some α_s1- and α_s2-CN-derived CPPs, such as α_s1-CN (f43–52 and f43–50) and α_s2-CN (f8–12), (f7–12) and (f6–12) as well as four non-phosphorylated peptides belonging to the C-terminal end of β-CN (f193–209, f194–209, f200–209) were detected in plasma samples submitted to extraction and enrichment by hydroxyapatite (HA) chromatography followed by MALDI-TOF and nano LC-ESI/MS/MS analysis.Data indicated that casein oligopeptides are bioavailable after a continued intake of cheese. Future studies are warranted to ascertain this finding on a wider population and to clarify the mechanisms behind CPPs bioaccessibility and absorption.     

Investigation of the ability of a purified protease from Pseudomonas fluorescens RO98 to hydrolyze bitter peptides from cheese

The ability of a purified protease from Pseudomonas fluorescens RO98 to hydrolyze bitter peptides found in Cheddar cheese was investigated. The purified protease was incubated with α_s1-casein f1–9 and β-casein f193–209 in a model system (pH 6.8, 30°C) to determine hydrolysis. Residual substrate and hydrolysis products were determined by capillary electrophoresis. Both peptides were hydrolyzed by the protease during the 90-min assay. α_s1-Casein f1–9 was hydrolyzed into two products and β-casein f193–209 was degraded completely in 90 min to three hydrolytic products. This protease hydrolyzed bitter peptides that are known to accumulate in Cheddar and Gouda cheese during aging, suggesting a possibility to debitter Cheddar cheese.     

Ripening of extra-hard cheese made with mesophilic DL-starter

Extra hard cheese is commonly made with thermophilic starters using high temperatures to stimulate expulsion of whey. In this work, microflora, proteolysis and volatiles were investigated in an extra-hard cheese made with mesophilic DL-starter, produced using challenging cooking temperatures for the starter bacteria over several hours. Cheese from six commercially produced vats was investigated over 56 weeks. The number of starter bacteria decreased after three weeks of ripening. Casein breakdown was characterised by chymosin and plasmin activity on α_s1- and β-caseins, respectively. Peptide profiles showed accumulation of Lactococcus derived peptides from α_s1-CN f1–23, and the peptide β-CN 29–93 as a result of joint plasmin and chymosin activity and absence of highly proteolytic thermophilic Lactobacillus, commonly present in extra-hard cheese. The composition of amino acids depended mainly on starter during the first 26 weeks of ripening. The content of volatiles depended both on ripening time and the starter used.     

Effect of protein composition on the cheese-making properties of milk from individual dairy cows

The objective of this study was to evaluate the effect of variations in milk protein composition on milk clotting properties and cheese yield. Milk was collected from 134 dairy cows of Swedish Red and White, Swedish Holstein, and Danish Holstein-Friesian breed at 3 sampling occasions. Concentrations of α_S1-, β-, and κ-casein (CN), α-lactalbumin, and β-lactoglobulin (LG) A and B were determined by reversed phase liquid chromatography. Cows of Swedish breeds were genotyped for genetic variants of β- and κ-CN. Model cheeses were produced from individual skimmed milk samples and the milk clotting properties were evaluated. More than 30% of the samples were poorly coagulating or noncoagulating, resulting in weak or no coagulum, respectively. Poorly and noncoagulating samples were associated with a low concentration of κ-CN and a low proportion of κ-CN in relation to total CN analyzed. Furthermore, the κ-CN concentration was higher in milk from cows with the AB genotype than the AA genotype of κ-CN. The concentrations of α_S1-, β-, and κ-CN and of β-LG B were found to be significant for the cheese yield, expressed as grams of cheese per one hundred grams of milk. The ratio of CN to total protein analyzed and the β-LG B concentration positively affected cheese yield, expressed as grams of dry cheese solids per one hundred grams of milk protein, whereas β-LG A had a negative effect. Cheese-making properties could be improved by selecting milk with high concentrations of α_S1-, β-, and κ-CN, with high κ-CN in relation to total CN and milk that contains β-LG B.     

Occurrence,origin and fate of pyroglutamyl-γ3-casein in cheese

During plasmin-catalysed proteolysis of β-casein (β-CN) in cheese, pyroglutamyl-γ₃-casein (pγ₃-CN), originates from the cyclisation of the N-terminal Glu of γ₃-CN (β-CN f108–209). This peptide progressively increased in content during ripening of Grana Padano and Parmigiano-Reggiano Protected Designation of Origin cheeses. A preliminary survey revealed pγ₃-CN in both hard and extra-hard cheese varieties; in semi-hard cheeses it was present only in mature samples. To understand the mechanisms of pγ₃-CN formation/degradation, in vitro trials (real cheese, model cheeses and buffered γ₃-CN solutions) were performed. Both the effect of cheese pH and the capacity of certain microorganisms to cyclise the N-terminal Glu residue of γ₃-CN were studied. The results suggested that pγ₃-CN formed spontaneously during cheese ripening, mainly at the acid pH characteristic of hard and extra-hard cheeses; the role of cyclase enzymes was negligible. Differences in pγ₃-CN degradation were observed due to the proteolytic activity of bacteria involved in the cheese ripening.     

Effect of different ventilation regimens on ewes' milk and Canestrato Pugliese cheese quality in summer

The influence of three different ventilation regimens on air pollution in sheep houses and on the quality of ewe milk and of Canestrato Pugliese cheese was investigated during the summer season. The experimental treatments were low ventilation regimen (VR=35 m3/h per ewe) split in 30-min ventilation cycles (LOV-30); moderate ventilation regimen (VR=70 m3/h per ewe) split in 30-min ventilation cycles (MOV-30); moderate ventilation regimen (VR=70 m3/h per ewe) split in 60-min ventilation cycles (MOV-60). The LOV-30 milk had higher microbial load and bulk milk somatic cell count (BMSCC) and resulted in a weaker casein matrix in the curd compared with the MOV-30 and MOV-60 treatments. At 45 d of ripening, the LOV-30 cheeses had a lower casein content and higher non-casein nitrogen (NCN) and water-soluble nitrogen (WSN) contents than the MOV-30 and MOV-60 cheeses. Urea-polyacrylamide gel electrophoresis (urea-PAGE) of the pH 4.6-soluble N extract showed that the MOV-60 cheeses had fewer bands derived from casein (CN) hydrolysis than the LOV-30 or MOV-30 cheeses, despite its having exhibited the highest plasmin (PL) activity levels. Our results suggest that the ventilation regimen is critical in dairy sheep housing for optimizing the hygienic quality of ewe milk and the proteolytic processes occurring in Canestrato Pugliese cheese during ripening.     

Effect of polymorphisms in the leptin, leptin receptor, and acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) genes and genetic polymorphism of milk proteins on cheese characteristics

Cheese production has increased worldwide during the last decade and is expected to increase within the coming decade as well. Despite this, the relations between cow genetics and cheese characteristics are not fully known. The aim of this study was to determine if polymorphisms in the leptin (LEP), leptin receptor (LEPR), and acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) genes as well as genetic variants of β-casein (β-CN), κ-CN, and β-lactoglobulin (β-LG) affect technological properties important for cheese production and, hence, could act as genetic makers for cheese quality. Individual milk samples from the Swedish Red and the Swedish Holstein breeds were analyzed for sizes of CN micelles and fat globules as well as rennet-induced gel strength, gelation time, and yield stress. Model cheeses were produced to study yield, hardness, and pH of the cheeses. The A1457G, A252T, A59V, and C963T single nucleotide polymorphisms (SNP) were analyzed on the LEP gene, the T945M SNP on the LEPR gene, and the Nt984+8(A-G) SNP on the DGAT1 gene. In addition, genetic variants of β-CN, κ-CN, and β-LG were determined. The results indicate that technological properties were influenced by the LEPR_T945M polymorphism, which had an association with gel strength, yield stress, and cheese hardness (T > C). However, also LEP_A252T was shown to affect gel strength (T > A), whereas the LEP_A59V had an effect on fat globule size (T > C). For the milk protein genes, favorable effects were found for the A and B variants of β-LG and κ-CN, respectively, on gel strength, gelation time, and yield stress. In addition, the B variant of κ-CN was shown to be associated with smaller CN micelles than the A variant. Thus, the results demonstrate potential genetic markers for cheese characteristics. However, milk composition traits also affected the obtained results, thus making it necessary to thoroughly assess the different aspects regarding the influence of gene effects on cheese characteristics before directly selecting for certain alleles or genetic variants to improve the processing and quality of cheese.     

Study on milk-clotting mechanism of rennet-like enzyme from glutinous rice wine: proteolytic property and the cleavage site on kappa-casein

Chinese Royal cheese, an ancient and attractive dairy product now in China, is made from milk coagulated with glutinous rice wine. In this paper, it was mainly studied on the proteolytic property toward proteins of bovine milk including caseins (CN) and whey proteins and the cleavage bond on the κ-CN of rennet-like enzyme purified from glutinous rice wine by ion-exchange chromatography. Compared with whey protein, the rennet-like protease has substrate specificity toward CN but with different hydrolysis degrees among κ-, α-, and β-CN, and the α-CN was almost completely degraded, whereas κ- and β-CN partly showed hydrolysis in 12 h. The analysis for enzyme digestion by electrospray tandem mass spectrometry, Q-TOF2, and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry revealed that the cleavage of protease from glutinous rice wine on κ-CN mainly happens at the Thr₉₄-Met₉₅ bond, which is different from the most chymosin-sensitive bond, Phe₁₀₅-Met₁₀₆.     

Analysis by capillary electrophoresis of the proteolytic activity of a Bacillus subtilis neutral protease on bovine caseins

Capillary electrophoresis using an untreated fused-silica capillary and a low-pH buffer containing urea and hydroxypropyl methyl cellulose was used to follow the proteolytic action of neutral protease of Bacillus subtilis on isolated casein fractions and whole milk casein (CN) samples. Electrophoretic analysis showed that this protease caused intense hydrolysis of α-CN, β-CN and κ-CN. The breakdown products due to the attack of the neutral protease on β-CN, γ₂-CN A, γ₁-CN B, γ₁-CN A¹, γ₃-CN B, γ₁-CN A², γ₃-CN A, β-CN-I A¹, β-CN-I A² and β-CN-I B, and on α_s-CN, α_s1-CN-I, α_s0-CN-I and α_s1-CN f(1–23) were separated.     

Chemical and fatty acid composition of Manchego type and Panela cheeses manufactured from either hair sheep milk or cow milk

This study compared the chemical composition and fatty acid (FA) profile of Manchego type cheese and Panela cheese made from hair sheep milk and compared these with both types of cheese manufactured with cow milk as a reference. In addition, this study aimed to determine differences in sensory characteristics between Manchego type cheeses manufactured with either hair sheep milk or cow milk. A total of 25 and 14 Manchego type cheeses from hair sheep milk and cow milk were manufactured, respectively. In addition, 30 and 15 Panela cheeses from hair sheep milk and cow milk were manufactured, respectively. The chemical composition and FA profile were determined in all cheeses. In addition, a sensory analysis was performed in Manchego type cheeses manufactured from either hair sheep milk or cow milk. Moisture content was lower in Manchego type cheeses (37.5 ± 1.26 and 37.5 ± 1.26 g/100 g in cheeses manufactured from hair sheep milk and cow milk, respectively) than in Panela cheeses (54.0 ± 1.26 and 56.1 ± 1.26 g/100 g in cheeses manufactured from hair sheep milk and cow milk, respectively). Ash, protein, and sodium contents were higher in Manchego type cheeses than in Panela cheeses. Manchego type cheese manufactured from hair sheep milk contained more C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, C18:2 cis-9,cis-12, total saturated FA, total short-chain FA, total medium-chain FA, total polyunsaturated FA, and de novo FA than Manchego type cheeses from cow milk. Total content of short-chain FA was higher in hair sheep cheeses (24.4 ± 1.30 and 19.6 ± 1.30 g/100 g in Manchego type and Panela cheeses, respectively) than in cow cheeses (8.89 ± 1.30 and 8.26 ± 1.30 g/100 g in Manchego type and Panela cheeses, respectively). Manchego type cheeses from hair sheep milk obtained higher scores for odor (7.05), texture (6.82), flavor (7.16), and overall acceptance (7.16) compared with those made from cow milk (6.37, 6.12, 6.17, and 6.83, respectively). In conclusion, both Manchego type cheese and Panela cheese manufactured with hair sheep milk had a similar chemical composition and contained higher levels of short-chain FA, total polyunsaturated FA, and de novo FA than those manufactured with cow milk.     

In vitro gastrointestinal digestion of bovine αS1- and αS2-casein variants gives rise to different IgE-binding epitopes

The occurrence and differences of resistant regions containing IgE-binding epitopes of α_S1-casein (α_S1-CN) variants B and C, as well as α_S2-CN A and B, after in vitro gastrointestinal digestion was investigated using mass spectrometry. The amino acid substitutions characterising the genetic variants affected the peptide pattern arising from the caseins and thus modifications in their allergenic epitopes occurred. Peptides f174–193 in α_S1-CN B and f179–198 in α_S1-CN C correspond to the IgE-binding epitope f173–194, which has been reported as one of the major epitopes in α_S1-CN B. Within α_S2-CN, the two variant-specific peptides, f7–29 from variant A and f1–22 from variant B, contain the previously identified IgE-binding epitope f1–20. These peptides, and in consequence the protein variants, may exhibit different immunoreactions, which could be significant in the production of milk with improved nutritional properties, such as hypoallergenic quality, by selection and breeding of cows with particular milk protein genotypes.     

SDS-PAGE of Proteins in Goat Milk Cheeses Ripened under Different Conditions

Proteolytic characteristics of five varieties of commercial goat milk cheeses and a cow milk Cheddar aged under different conditions were evaluated by SDS-PAGE and an advanced densitometry system. All fresh goat cheeses had distinctively lower intensities of α_S1-casein (CN) bands than those of cow milk Cheddar, whereas intensities of β-CN were much greater in the goat cheeses. The PAGE patterns clearly displayed α_S2-CN in all goat cheese, but it was negligible in cow milk Cheddar. The greater protein degradation in hard goat cheeses than cow milk Cheddar at 4°C and 22°C strongly correlated with water-soluble nitrogen compound concentrations and densitometric values of corresponding cheeses.     

Peptides inhibitory to endopeptidase and aminopeptidase fromLactococcus lactis ssp.lactis MG1363, released from bovineβ-casein by chymosin,trypsin or chymotrypsin

Peptides inhibitory to the 70-kDa endopeptidase (PepO) from the cytoplasm ofLactococcus lactis ssp.lactis MG1363 were isolated from the supernatant (pH 4.6) of chymosin, tryptic and α-chymotryptic hydrolysates ofβ-casein (β-CN) by reversed-phase HPLC and identified by sequencing and mass spectrometry. Chymosin releasedβ-CN f193–209, kinetic constant (K _i) of which for inhibition of PepO was 60 μM. This peptide also inhibited (K _i=1700 μM) the 95-kDa aminopeptidase (PepN) fromL. lactis ssp.lactis MG 1363. Trypsin released two PepO-inhibitory peptides: one,β-CN f69–97, was not degradable by PepO (K _i=4.7 μM), while the other,β-CN f141–163, was degradable by PepO but competitively inhibited hydrolysis of methionine enkephalin by PepO. A peptide,β-CN f69–84, which inhibited PepO with aK _i of 8.1 μM, was isolated from the α-chymotryptic hydrolysate. Peptides released fromβ-CN by trypsin or chymotrypsin had very little inhibitory activity against PepN. PepO degradedβ-CN f193–209 very slowly compared with the hydrolysis of methionine enkephalin. All four inhibitory peptides (β-CN f193–209, f69–97, f69–84, f141–163) were readily degraded by thermolysin.     

Chymosin-mediated proteolysis, calcium solubilization, and texture development during the ripening of cheddar cheese

Full fat, milled-curd Cheddar cheeses (2 kg) were manufactured with 0.0 (control), 0.1, 1.0, or 10.0 μmol of pepstatin (a potent competitive inhibitor of chymosin) added per liter of curds/whey mixture at the start of cooking to obtain residual chymosin levels that were 100, 89, 55, and 16% of the activity in the control cheese, respectively. The cheeses were ripened at 8°C for 180 d. There were no significant differences in the pH values of the cheeses; however, the moisture content of the cheeses decreased with increasing level of pepstatin addition. The levels of pH 4.6-soluble nitrogen in the 3 cheeses with added pepstatin were significantly lower than that of the control cheese at 1 d and throughout ripening. Densitometric analysis of urea-PAGE electro-phoretograms of the pH 4.6-insoluble fractions of the cheese made with 10.0 μmol/L of pepstatin showed complete inhibition of hydrolysis of α_S1-casein (CN) at Phe₂₃-Phe₂₄ at all stages of ripening. The level of insoluble calcium in each of 4 cheeses decreased significantly during the first 21 d of ripening, irrespective of the level of pepstatin addition. Concurrently, there was a significant reduction in hardness in each of the 4 cheeses during the first 21 d of ripening. The softening of texture was more highly correlated with the level of insoluble calcium than with the level of intact α_S1-CN in each of the 4 cheeses early in ripening. It is concluded that hydrolysis of α_S1-CN at Phe₂₃-Phe₂₄ is not a prerequisite for softening of Cheddar cheese during the early stages of ripening. We propose that this softening of texture is principally due to the partial solubilization of colloidal calcium phosphate associated with the para-CN matrix of the curd.     

Contribution of autochthonous non-starter lactobacilli to proteolysis in Caciocavallo Pugliese cheese

Caciocavallo Pugliese cheese was manufactured according to a traditional protocol and with added autochthonous Lactobacillus paracasei subsp. paracasei and L. parabuchneri strains, isolated from a well-flavoured Caciocavallo Pugliese cheese, to evaluate their contribution to the cheese biochemical characteristics. Using a “two step REP-PCR” protocol, L. paracasei subsp. paracasei strains were shown to sustain high viability during ripening, while the L. parabuchneri strains were not recovered. The inoculated cheese showed higher levels of free amino acids, as well as differences in the profiles of individual free amino acids in comparison with the control cheese. The addition of autochthonous Lactobacillus strains with interesting technological properties in Caciocavallo Pugliese cheese manufacturing could make it feasible to improve cheese processing, while still maintaining the sensory characteristics of this typical pasta-filata cheese.     

Textural changes of Canestrello Pugliese cheese measured during storage

The changes in the fundamental textural properties of the Canestrello Pugliese cheese, during ripening, were studied, accounting for the internal and external portions of the cheese. The cheese was ripened at 12–14 °C and 80% relative humidity and analyzed at 0, 15, 30, 45, 60, 95, and 120 days to determine the moisture content, water activity (A_w), pH values and some fundamental-mechanical and dynamic-mechanical properties such as the elastic modulus (E_c), storage modulus (G′), loss modulus (G″) and relaxation times distribution curve. Significant differences in the moisture content, A_w, pH and the elastic modulus were observed for the inside and outside portions of the investigated cheese. In particular, for the first 60 days of ripening the elastic modulus of the outer region was higher than that of the inner region. As the ripening process approached the end of the fourth month the contrary was true. Moreover, results show that, above all the pH and A_w play a major role in determining the mechanical properties of the Canestrello Pugliese cheese during ripening.     

Effect of the use of three different lamb paste rennets on lipolysis of the PDO Pecorino Romano Cheese

Two rennet pastes were prepared from lambs on different diets, and then, together with a commercial lamb rennet paste, were used to produce protected designation of origin (PDO) Pecorino Romano cheeses. Chymosin activity and lipolytic activity were higher in the prepared rennets than in the commercial rennet. Lipolysis was greatest in cheese produced with one of the prepared rennets. Both quantitative and qualitative differences in free fatty acids were found in the cheeses. The taste of cheeses made with the prepared rennets was preferred by an expert sensory panel and also considered more piquant than that of cheese made with commercial rennet. The results indicate that both the diet of the lambs and slaughtering conditions should be regulated to produce rennet that preserves the traditional characteristics of PDO Pecorino Romano cheese. Both lipolytic and proteolytic properties of rennet pastes should be standardised for the preparation of such cheeses.