Effect of high-pressure treatment on microbiology,proteolysis, lipolysis and levels of flavour compounds in mature blue-veined cheese

The effect of high-pressure (HP) treatment (400 MPa, 600 MPa) on ripening of mature 42-day-old Irish blue-veined cheese was studied. Counts of non-starter lactic acid bacteria, lactococci, yeasts, moulds, enterococci and total aerobic bacteria significantly decreased due to HP, with moulds being most sensitive and 600 MPa the most effective treatment. The levels of pH 4.6-soluble nitrogen and (12%) trichloroacetic acid-soluble nitrogen increased immediately after both HP treatments; however, after 28 days of storage, values were lower in HP-treated cheeses than in the control cheese. Urea-polyacrylamide gel electrophoresis showed increased breakdown of β-casein due to HP treatment at both 400 MPa and 600 MPa. Levels of free fatty acids were lower in HP-treated cheese than in the control, but not significantly so, and no significant changes could be observed in the level of flavour compounds of blue-veined cheese. Overall, HP treatment of blue-veined cheese reduced microbiological activity and decelerated proteolysis, with no statistically significant effects on development of flavour compounds.Industrial relevanceHigh-pressure treatment has been studied for the past 100 years; nevertheless, it was not applied in dairy industry, until recently, for a cheese spread. In this study, HP-induced inactivation of microbes and enzymes, which could arrest the ripening of high-quality mature (i.e., ripened) Irish farmhouse blue-veined cheese and thus extend shelf-life at optimal quality, was examined.     

Manufacture of Cheddar cheese from high-pressure-treated whole milk

The cheese-making characteristics of high-pressure (HP)-treated milk were examined. The rennet coagulation time of pasteurised milk decreased after HP treatment at 400 MPa but increased after treatment at 600 MPa. The L-value (whiteness) of milk decreased directly after HP treatment but, over the course of coagulation, whiteness of HP-treated milk increased to the same level as in the control. Cheddar cheese was then manufactured from raw whole milk or whole milk treated by high-pressure (HP) at 400 MPa (HP400) or 600 MPa (HP600) for 10 min at 20 °C. HP treatment of raw milk at 600 MPa resulted in a 3.66 log reduction in the initial counts of non-starter lactic acid bacteria (NSLAB), decreased protein and fat content, as well as a lower pH compared to the control. Furthermore, higher treatment pressures resulted in increased incorporation of β-lactoglobulin into the cheese curd, with parallel increases in yield by 1.23% and 7.78% for HP400 and HP600 cheeses, respectively. Overall, this study showed that the effects of HP treatment on milk proteins increased rennet coagulation times and changes in cheese composition at day 1.Industrial relevanceHigh-pressure treatment is a novel technology which has been applied to a number of commercial food products. In this study, HP-induced changes in milk proteins resulted in increased cheese yields and increased cheese whiteness. In addition, HP treatment significantly reduced the microflora of raw milk cheese. Those attributes could be of interest for both industry and consumer.     

Effect of high pressure treatment applied on starter culture or on semi-ripened cheese in the quality and ripening of cheese in brine

Cheese ripening acceleration is of continuous interest for the industry. High-pressure (HP) treatment of starter cultures used in cheese-manufacturing offers the potential to accelerate ripening by increasing the activity of their intracellular peptidases that contribute in the development of desired cheese organoleptic characteristics.The objective of the present research was the investigation of the effect of HP treatment (200 MPa-20 °C - 20 min) directly on white brined cheese or on the starter culture used for its manufacture (Str. thermophilus:L. lactis:L. bugaricus 2:1:1). For this purpose, the microbial, textural, physicochemical and organoleptic characteristics and proteolysis were assessed during the 2nd stage of ripening in cold stores. Control cheese without any treatment was also studied.Cheeses made with HP-treated starters had increased secondary proteolysis. Organoleptic scoring of these cheeses was higher during the whole storage period compared to control and HP-treated cheese. Their superiority was evident even at the early stages of ripening in cold stores, since no bitterness was detected. On the contrary, although HP treated cheeses showed the highest increase in aminopeptidases activities, this was not correlated with the studied ripening indices or the organoleptic characteristics.According to the results, HP-treated starter culture can accelerate proteolysis and potentially the ripening of cheese-in-brine.Industrial relevanceThe data obtained from this work suggest that application of HP treatment under optimized conditions on cheeses in brine starter cultures or on whole cheeses can be effectively used for the production of products with reduced ripening time. This is of great importance for the cheese industries, since the storage period for ripening is long (higher than two months), while applying HP treatment as suggested in this study, this time may be reduced to less than one month, producing cheeses of superior quality.     

Effect of combinations of high-pressure treatment and bacteriocin-producing lactic acid bacteria on the survival of Listeria monocytogenes in raw milk cheese

The combined effect of high-pressure (HP) treatment and bacteriocin-producing lactic acid bacteria (BP-LAB) on the survival of Listeria monocytogenes Scott A in cheeses made from raw milk that was inoculated with the pathogen at 4.80 log cfu mL⁻¹, a commercial starter and one of seven strains of BP-LAB was investigated. On day 3, the counts of L. monocytogenes were 7.03 log cfu g⁻¹ in a control cheese (without BP-LAB, not HP treated), 6.06–6.74 log cfu g⁻¹ in cheeses with BP-LAB, 6.13 log cfu g⁻¹ in a cheese without BP-LAB and treated on day 2 at 300 MPa, 2.01 log cfu g⁻¹ in a cheese without BP-LAB and treated on day 2 at 500 MPa, 3.83–5.43 log cfu g⁻¹ in cheeses with BP-LAB and treated on day 2 at 300 MPa, and 1.81 log cfu g⁻¹ or less in cheeses with BP-LAB and treated on day 2 at 500 MPa. HP treatment was more effective on day 51 than on day 2.     

Effect of high-pressure treatments on proteolysis and texture of ewes’ raw milk La Serena cheese

La Serena cheeses, made from Merino ewes’ raw milk, were high-pressure (HP)-treated at 300 or 400 MPa for 10 min at 10 °C, on days 2 or 50 of ripening. Cheeses treated by HP on day 2 showed higher pH values than control cheese on day 3, but cheeses treated by HP on days 2 or 50 and control cheese had similar pH values on day 60. Breakdown of caseins was delayed by HP treatment of cheeses on day 2. Cheeses treated by HP on day 2 showed higher levels of hydrophilic peptides, lower levels of hydrophobic peptides, lower hydrophobic peptides: hydrophilic peptides ratios, and higher total contents of free amino acids than those of control cheese. HP treatment of cheese on day 50 scarcely affected proteolysis of 60-day-old cheeses. Fracturability, hardness and elasticity values of cheeses treated by HP on day 2 were higher than those of control cheese and of cheeses treated on day 50. Cheeses treated at 400 MPa on day 2 received the lowest scores for quality of taste from panellists, whereas the rest of HP-treated cheeses did not differ from control cheese.     

Effect of high-pressure-processing on the microbiology,proteolysis, texture and flavour of Brie cheese during ripening and refrigerated storage

Brie cheeses were high pressure (HP)-treated at 400 or 600 MPa on days 14 or 21 after manufacture to prevent over-ripening. Lactic acid bacteria and Penicillium camemberti numbers declined markedly after HP treatment. In control cheese pH increased 2.0 units from day 21 to day 60, but less than 0.3 units in HP-treated cheeses. Cheeses treated at 600 MPa showed the maximum concentrations of residual caseins during refrigerated storage and control cheese the minimum concentrations. A 7.6-fold increase in hydrophobic peptides was recorded from day 21 to day 60 in control cheese and 0.8–1.6-fold increases in HP-treated cheeses. The maximum aminopeptidase activity was detected in control cheese, the highest free amino acid concentrations in cheeses treated at 400 MPa. The firmest texture was recorded for cheeses treated on day 14 at 400 or 600 MPa. HP-treated cheeses showed higher flavour quality scores than control cheese from day 60 onwards.     

Effect of a bacteriocin-producing Lactococcus lactis strain and high-pressure treatment on the esterase activity and free fatty acids in Hispánico cheese

The effect of milk inoculation with a bacteriocin-producing (BP) culture and of high-pressure (HP) treatment of 15-day-old Hispánico cheeses (400 MPa, 5 min, 10 °C), separately or combined, on the release of intracellular esterases and cheese lipolysis was investigated. Esterase activity and free fatty acids (FFAs) content increased during ripening of Hispánico cheese and palmitic, oleic and stearic acids being the most abundant FFAs. On day 15, the highest esterase activity was recorded for HP-treated BP cheese. The activity for HP-untreated BP cheese was the next highest. No difference in the activities was found between HP-treated and untreated cheeses made without BP culture. Total FFAs on day 15 were at a lower concentration in BP cheeses than in cheeses made without BP culture, probably due to the lower pH values of the former. The rate of total FFA accumulation from day 15 to day 50 was higher in BP cheeses (31.1–32.1% increase) than in cheeses made without BP culture (19.3–21.7% increase). The highest total FFA concentration on day 50 (612 mg kg⁻¹) was found for HP-untreated cheese made without BP culture.     

A preliminary study on the role of alkaline phosphatase in cheese ripening

A preparation of exogenous alkaline phosphatase (ALP), containing 17,500 mU L⁻¹, was added to pasteurized milk (PM) to study its role in cheese ripening. Three miniature Cheddar-type cheeses were made from PM containing no added ALP (control), PM plus 23 μL ALP (T1), to give ALP concentration similar to that in raw milk, and PM plus 46 μL ALP (T2). Milk, after addition of ALP, was held at 6 °C for 12 h before cheese manufacture and the experiment was replicated three times. The control, T1 and T2 milks contained ALP activity of 415, 2391 and 4705 mU L⁻¹, respectively. The addition of ALP to PM caused significant (P<0.05) changes in moisture content of miniature cheeses but did not cause any changes in protein content. Levels of water-soluble N during ripening of the cheeses were similar for control, T1 and T2 cheeses. The concentration of amino acids was not affected by the level of ALP present in milk. However, reversed-phase HPLC showed differences in the peptide patterns of control, T1 and T2 cheeses, suggesting a role of ALP in cheese ripening. The results suggest that ALP may play a role in cheese ripening, but further studies are needed to confirm this.     

Rheological properties of high-pressure-treated Gouda cheese

The rheological properties of high-pressure-treated (50–400 MPa, 1 h) and untreated Gouda cheese were compared. Immediately after pressure release, oscillation measurements gave lower storage and loss moduli from 50 MPa onwards. Simultaneously, tan δ was higher, indicating a relatively less solid-like behaviour of the pressurized samples. Creep measurements showed that samples treated at 400 MPa got less rigid, less solid-like, and more viscoelastic; from 50 MPa onwards, the samples had less resistance to flow at longer times. Texture profile analysis revealed that samples treated at 225 and 400 MPa showed no macroscopical breakage. Relaxation measurements gave a higher level of stress decay at long relaxation times and a higher rate at which the stress relaxes. During further ripening after pressure release, differences between pressure-treated and untreated samples became smaller. At 42 days of ripening, any or only a slight difference could still be observed. Dissolution experiments showed that hydrophobic interactions in Gouda cheese were weakened by pressure treatment. This possibly led to structural changes of the paracasein network causing the rheological property changes. These pressure effects on proteins in Gouda cheese are possibly reversible as hydrophobic interactions and rheological properties were restored during ripening.     

Effect of natamycin-containing coating on the evolution of biochemical and microbiological parameters during the ripening and storage of ovine hard-Gruyère-type cheese

The effect of a coating containing natamycin on the ripening course of the hard-Gruyère-type cheese Graviera Kritis was assessed. A single treatment at an early stage of ripening was carried out; samples from natamycin-treated (NT) and control cheeses (CTR) were then taken throughout a 12 month ripening and storage period. Coating gave a statistically significant reduction in the yeasts and moulds counts in the cheese rind. It did not influence the counts and the evolution of the thermophilic bacteria related to starter or of the propionic acid bacteria, nor did it affect the associated aminopeptidase activities. Gross composition of NT cheeses did not differ significantly from that of the control cheeses; the same was also true for proteolysis. Natamycin in the cheese rind after the removal of coating was lower than 0.1 mg dm⁻² at all stages of ripening and no migration to the cheese interior was observed.     

Effect of dairy farm and milk refrigeration on microbiological and microstructural characteristics of matured Serra da Estrela cheese

This work was aimed at enumerating the viable microorganisms in ripened Serra da Estrela cheeses, manufactured from both refrigerated and non-refrigerated milk, in various dairies located throughout the demarcated region. Scanning electron microscopy was used to analyze the microstructure, and thus aid in understanding possible differences in their microbiological profile. The cheeses were allowed to ripen under controlled conditions, and sampled at 60, 90, 120, 150 and 180 d following manufacture. Viable numbers of lactic acid bacteria, staphylococci, Enterobacteriaceae and yeasts were obtained following standard plate counting on a number of selective media. Lactococcus was the most abundant genus (above 10⁸ cfu g⁻¹ of cheese) up to 120 d of ripening. No significant microstructural differences were observed in cheeses manufactured in different dairies over the ripening process. However, microstructural differences were apparent between cheeses manufactured with refrigerated versus non-refrigerated milk.     

Proteolysis and biogenic amine buildup in high-pressure treated ovine milk blue-veined cheese

Penicillium roqueforti plays an important role in the ripening of blue-veined cheeses, mostly due to lactic acid consumption and to its extracellular enzymes. The strong activity of P. roqueforti proteinases may bring about cheese over-ripening. Also, free amino acids at high concentrations serve as substrates for biogenic amine formation. Both facts result in shorter product shelf-life. To prevent over-ripening and buildup of biogenic amines, blue-veined cheeses made from pasteurized ovine milk were high-pressure treated at 400 or 600 MPa after 3, 6, or 9 wk of ripening. Primary and secondary proteolysis, biogenic amines, and sensory characteristics of pressurized and control cheeses were monitored for a 90-d ripening period, followed by a 270-d refrigerated storage period. On d 90, treatments at 400 MPa had lowered counts of lactic acid bacteria and P. roqueforti by less than 2 log units, whereas treatments at 600 MPa had reduced lactic acid bacteria counts by more than 4 log units and P. roqueforti counts by more than 6 log units. No residual α-casein (CN) or κ-CN were detected in control cheese on d 90. Concentrations of β-CN, para-κ-CN, and γ-CN were generally higher in 600 MPa cheeses than in the rest. From d 90 onwards, hydrophilic peptides were at similar levels in pressurized and control cheeses, but hydrophobic peptides and the hydrophobic-to-hydrophilic peptide ratio were at higher levels in pressurized cheeses than in control cheese. Aminopeptidase activity, overall proteolysis, and free amino acid contents were generally higher in control cheese than in pressurized cheeses, particularly if treated at 600 MPa. Tyramine concentration was lower in pressurized cheeses, but tryptamine, phenylethylamine, and putrescine contents were higher in some of the pressurized cheeses than in control cheese. Differences in sensory characteristics between pressurized and control cheeses were generally negligible, with the only exception of treatment at high pressure level (600 MPa) at an early ripening stage (3 wk), which affected biochemical changes and sensory characteristics.     

Ripening of Cheddar cheese made from blends of raw and pasteurised milk

Cheddar cheeses were made from pasteurised milk (P), raw milk (R) or pasteurised milk to which 10 (PR10), 5 (PR5) or 1 (PR1) % of raw milk had been added. Non-starter lactic acid bacteria (NSLAB) were not detectable in P cheese in the first month of ripening, at which stage PR1, PR5, PR10 and R cheeses had 10⁴, 10⁵, 10⁶ and 10⁷ cfu NSLAB g⁻¹, respectively. After ripening for 4 months, the number of NSLAB was 1–2 log cycles lower in P cheese than in all other cheeses. Urea–polyacrylamide gel electrophoretograms of water-soluble and insoluble fractions of cheeses and reverse-phase HPLC chromatograms of 70% (v/v) ethanol-soluble as well as -insoluble fractions of WSF were essentially similar in all cheeses. The concentration of amino acids were pro rata the number of NSLAB and were the highest in R cheese and the lowest in P cheese throughout ripening. Free fatty acids and most of the fatty acid esters in 4-month old cheeses were higher in PR1, PR5, PR10 and R cheeses than in P cheese. Commercial graders awarded the highest flavour scores to 4-month-old PR1 cheeses and the lowest to P or R cheese. An expert panel of sensory assessors awarded increasingly higher scores for fruity/sweet and pungent aroma as the level of raw milk increased. The trend for aroma intensity and perceived maturity was R>PR10>PP5>PR1>P. The NSLAB from raw milk appeared to influence the ripening and quality of Cheddar cheese.     

Influence of high-pressure processing (HPP) on physico-chemical properties of fresh cheese

Freshly prepared rennet-coagulated soft cheese was high-pressure (HP) treated at up to 291 MPa and 29 min and using a full 2-factor central composite design of experiment, its physico-chemical properties (colour, fat, lipid oxidation, moisture and protein content, pH, and texture) were examined. HP treatment influenced significantly (p < 0.05) the colour, fat, moisture, lipid oxidation, hardness and adhesiveness of the fresh cheese. Fat content increased apparently as moisture decreased significantly after HP treatment of above 100 MPa. Increased pressures reduced lipid oxidation but increased yellowness although the latter showed more effect over redness in the HP-treated fresh cheese. Also, increased pressures increased hardness, decreased acidity and adhesiveness in HP-treated fresh cheese although increased exposure was found to increase acidity.Industrial relevanceHigh isostatic pressure for processing fresh cheese is yet to be adopted on an industrial scale. There is a need for research to provide evidence that improved properties of fresh cheese can be realized. The effects of HPP on rennet-coagulated soft Scottish cheese are investigated and the data from this study have provided points where optimized characteristic properties of HPP fresh cheese can be attained, which can serve as a lead for HPP users on fresh cheese.     

A sensitive HPLC method to detect hen's egg white lysozyme in milk and dairy products

Because of the lytic activity on the cell wall of bacteria like Clostridium tyrobutyricum, hen's egg white lysozyme is used in cheese manufacturing to prevent late blowing. A HPLC method capable to quantify as low as 0.8 ppm lysozyme in milk and cheese is proposed. Lysozyme was extracted with 1 m NaCl at pH 6.0 and the extract was deproteinized at low pH values, reaching a recovery up to 90%. Reversed-phase HPLC was performed on a polymeric column and monitoring lysozyme under fluorescence detection (excitation at 280 nm and emission at 340 nm). The repeatability of this determination tested on a 15-month-aged hard cheese and expressed as relative standard deviation was 1.47 (n=6) and no interference of peptides formed during ripening was observed. Four commercial preparations of egg white lysozyme gave a similar fluorescence response and the partitioning over cheese and whey was studied with one of them. About 80% of the lysozyme added to the cheesemilk at concentrations up to 80 ppm was retained in the cheese and the concentration factor of lysozyme from the cheesemilk to the cheese proved to be 8.2 on average. This HPLC method and the microbiological assay using Micrococcus luteus were compared in cheese analysis, proving the former to be more accurate and reliable than the latter. Eighteen commercial samples including both generic cheeses and cheeses having protected designation of origin, all of them not declared to contain lysozyme, showed concentrations of this enzyme ranging from 0 to 111 ppm.     

Use of high-pressure-treated milk for the production of reduced-fat cheese

Pasteurized (65°C, 30 min), pressurized (400 MPa, 22°C, 15 min) and pasteurized–pressurized milks were used for reduced-fat (approximately 32% of total solids) cheese production. Pressurization of milk increased the yield of reduced-fat cheese through an enhanced β-lactoglobulin and moisture retention. In addition, pressurisation of pasteurized skim milk improved its coagulation properties. The cheeses made from pasteurized–pressurized and pressurized milks showed a faster rate of protein breakdown than the cheese made from pasteurized milk, that might be mainly attributed to a higher level of residual rennet. Hardness of the experimental cheeses, as determined by both the sensory panel and instrumental analyses, decreased as the moisture content and proteolytic degradation of the cheese increased (pasteurized>pressurized>pasteurized–pressurized). In general terms, pressurization of reduced-fat milk prior to cheese-making improved cheese texture and thus accounted for a higher overall acceptability, except for the cheeses made from pasteurized–pressurized milk at 60 d of ripening, whose acceptability score was adversely affected by bitterness.     

Applicability of extracts from Centaurea calcitrapa in ripening of bovine cheese

Aqueous extracts obtained from cell suspension cultures of Centaurea calcitrapa were used as proteolytic additive in the manufacture of a commercial bovine cheese, coagulated with animal rennet and typically ripened for 28 d. The cheese was assessed in comparison to standard cheese for two levels of addition of said extract, viz. 0.61 and 1.22 mg of total protein mL⁻¹. The qualitative and quantitative evolutions of the nitrogen fractions were monitored in the experimental cheeses throughout the whole ripening period. In general, the chemical compositions of the cheeses were different depending on the amount of extract used, but no significant differences could be detected in the ripening index. With regard to electrophoretic profiles, the two types of cheese could be distinguished until up to ca. 7 d of ripening, but differences did essentially vanish by 28 d.     

Protocol for the manufacture of miniature washed-curd cheeses under controlled microbiological conditions

A protocol for the preparation of miniature washed-curd cheeses under controlled bacteriological conditions was designed and tested for reproducibility. The process was adapted from “Saint-Paulin” technology, and involves inoculation and renneting in autoclaved bottles, and cutting, stirring, curd washing and removal of whey by centrifugation. Pressing was simulated by low-speed centrifugation. All operations were performed using sterile techniques and autoclaved equipment. Forty miniature cheeses (approximately 40 g) were produced over 10 working days, and ripened for 28 days. Gross composition (dry matter, salt-in-moisture and pH) of the one-day-old cheeses did not differ significantly between cheesemaking days, and average values were 45.16 , 2.46 and 5.15%, respectively. Adventitious Lactobacillus population remained less than 200 CFU g⁻¹ all during ripening, and phages were absent. Nitrogen soluble at pH 4.4 and in phosphotungstic acid attained 21 and 3% of total nitrogen, respectively, in 28-day-old cheeses. The proposed model was shown to be suitable for the preparation of miniature cheese specimens for use in microbiological studies of cheese manufacture and ripening.     

Influence of lamb rennet paste on composition and proteolysis during ripening of Pecorino foggiano cheese

Rennet pastes produced by lambs subjected to three different feeding systems (mother suckling [MS], artificial rearing [AR], and artificial rearing with Lactobacillus acidophilus supplementation [ARLB]) and slaughtered at two different ages (20 and 40 d) were used for the manufacture of Pecorino foggiano cheese. Composition and proteolysis during ripening of Pecorino foggiano cheese (four replicates batches) were analyzed. Proteolysis was greater in cheeses made with rennet pastes from lambs slaughtered at 20 d, as shown by analysis of nitrogen fractions (water-soluble N and proteose peptones). Supplementation of milk substitute with L. acidophilus may have influenced the growth dynamics of lactic acid bacteria in the rennet pastes, with positive effects on levels of lactobacilli in cheese at the beginning of the ripening time. Lower pH values in ARLB cheese during ripening, together with higher cell loads, suggest that supplementation of milk replacer with L. acidophilus resulted in higher proteolytic activity, as also confirmed by the composition of the pH 4.6—insoluble nitrogen fraction. No differences were found in total concentration of free amino acids among the experimental cheeses; phenylalanine, isoleucine, leucine, lysine were found at the highest levels. The addition of probiotic bacteria to milk substitute in lamb rearing appears to give good-quality lamb rennet paste.     

Evolution of lipolysis during the ripening of traditional Feta cheese

Lipolysis was studied during ripening of traditional Feta cheese produced in two small dairies, A and B. The cheeses were made from a thermized mixture of ewes’/goats’ milk by using yoghurt as starter and artisanal rennet from lambs’ and kids’ abomasa (cheese A) or mixed artisanal rennet with calf rennet (cheese B).The acid degree value and the free fatty acids (FFA) contents in both cheeses increased sharply up to 18 d (pre-ripening period at 15 °C) and continued to increase throughout ripening. In both mature cheeses, acetic acid was found at high levels (13–18% of the total FFAs). However, except for this, all FFA contents differed significantly (P < 0.05) between the two cheeses throughout ripening. The levels of individual and total C2:0–C8:0, C10:0–C14:0 and C16:0–C18:2 fatty acids were significantly higher (P < 0.05) in cheese A than in cheese B. Presumably the difference, especially in the C2:0–C8:0 content, was due mainly to the type of the rennet used. Butyric acid was the dominant FFA in cheese A (20% of the total FFAs at 120 d), while the most abundant FFAs in cheese B were capric (18%) and lauric acid (18%). In general, the lipolysis degree of the two cheeses was higher than those reported for the industrially-made Feta cheese.In organoleptic evaluation, cheese A had a piquant taste that was attributed to its high content of butyric acid and showed a significantly (P < 0.05) higher total score than cheese B.