The performance of Streptococcus macedonicus ACA-DC 198 as starter culture in Kasseri cheese production

Streptococous macedonicus ACA-DC 198 was used both as an adjunct (Cheese B) and as sole starter (Cheese C) in Kasseri cheese production. Control cheese (Cheese A) was prepared using a commercial starter culture. In all cheeses, numbers of all microbial groups examined initially increased, but declined after Baski scalding and remained practically stable up to day 90. The presence of S. macedonicus was confirmed until the end of ripening (Cheeses B and C); this was also the case for the bacteriocin produced by S. macedonicus ACA-DC 198. X-propyl-dipeptidyl-aminopeptidase activity was detected in all cheeses after day 15. The physicochemical characteristics of the mature Cheese C were in agreement with those defined by the Greek legislation. The sensory properties of all mature cheeses corresponded with the ones characterizing the traditional Kasseri cheese. It was concluded that S. macedonicus ACA-DC 198 can be used as an adjunct in Kasseri cheese production.     

Survival of Listeria monocytogenes in Galotyri,a traditional Greek soft acid-curd cheese,stored aerobically at 4°C and 12°C

Galotyri is a traditional Greek soft acid-curd cheese, which is made from ewes’ or goats’ milk and is consumed fresh. Because cheese processing may allow Listeria monocytogenes post-process contamination, this study evaluated survival of the pathogen in fresh cheese during storage. Portions (0.5 kg) of two commercial types (<2% salt) of Galotyri, one artisan (pH 4.0±0.1) and the other industrial (pH 3.8±0.1), were inoculated with ca. 3 or 7 log cfu g⁻¹ of a five-strain cocktail of L. monocytogenes and stored aerobically at 4°C and 12°C. After 3 days, average declines of pathogen's populations (PALCAM agar) were 1.3–1.6 and 3.7–4.6 log cfu g⁻¹ in cheese samples for the low and high inocula, respectively. These declines were independent (P>0.05) of the cheese type or the storage temperature. From day 3, however, declines shifted to small or minimal to result in 1.4–1.8 log cfu g⁻¹ of survivors at 28 days of storage of all cheeses at 4°C, indicating a strong “tailing” independent of initial level of contamination. Low (1.2–1.7 log cfu g⁻¹) survival of L. monocytogenes also occurred in cheeses at 12°C for 14 days, which were prone to surface yeast spoilage. When ca. 3 log cfu g⁻¹ of L. monocytogenes were inoculated in laboratory scale prepared Galotyri of pH ≅4.4 and ≅3% salt, the pathogen died off at 14 and 21 days at 12°C and 4°C, respectively, in artisan type cheeses fermented with the natural starter. In contrast, the pathogen survived for 28 days in cheeses fermented with the industrial starter. These results indicate that L. monocytogenes cannot grow but may survive during retail storage of Galotyri despite its low pH of or slightly below 4.0. Although contamination of Galotyri with L. monocytogenes may be expected low (<100 cfu g⁻¹) in practice, that long-term survival of the pathogen in commercial cheeses was shown to be unaffected by the artificial contamination level (3 or 7 logs) and the storage temperature (4°C or 12°C), which should be a concern.     

Improving the viability of Bifidobacterium bifidum BB-12 and Lactobacillus acidophilus LA-5 in white-brined cheese by microencapsulation

The viability of Bifidobacterium bifidum BB-12 and Lactobacillus acidophilus LA-5 microencapsulated by either an extrusion or an emulsion technique and used in white-brined cheese was monitored. Both microencapsulation techniques were effective in keeping the numbers of probiotic bacteria higher than the level of the therapeutic minimum (>10⁷ cfu g^?1). While the counts of probiotic bacteria decreased approximately 3 log in the control cheese in which probiotics were used as free cells, the decrease was more limited in the cheeses containing microencapsulated cells (approximately 1 log). Medium- and long-chain free fatty acid contents of the cheeses with immobilized probiotics were much higher than in the control cheese. Similarly, cheeses made with immobilized probiotics contained higher acetaldehyde and diacetyl levels than the control. Experimental cheeses containing microencapsulated probiotics were not different from the control cheese in terms of sensory properties.     

A comparison of the chemical,microbiological and sensory characteristics of bovine and ovine Halloumi cheese

Commercial samples of fresh and mature Halloumi cheeses made from ovine or bovine milk were studied in order to establish their chemical, microbiological and sensory characteristics. Significant differences were observed between the two types of Halloumi cheese both when fresh and mature. The free volatile fatty acid (FVFA) content of the cheeses increased with maturation from 483 to 1356 mg kg⁻¹ for the ovine product, but lower values (380–1248 mg kg⁻¹) were found in the bovine cheese. During maturation for 40 days, Enterococcus faecium, which dominated the microflora of fresh ovine cheese, was replaced by lactobacilli, including a new species, Lactobacillus cypricasei, which was not found in the bovine samples. Fewer than 100 cfu g⁻¹ lactic acid bacteria (LAB) were present in the fresh bovine cheeses, but a microflora dominated by lactobacilli developed with time. Yeast counts in the mature ovine and bovine cheeses reached 2.3–2.8×10⁵ cfu g⁻¹ and, as some of the yeasts were proteolytic and/or lipolytic, it was assumed that they were having a positive impact of the flavour of the cheeses. The sensory panel distinguished significant differences in texture and flavour between the fresh and mature samples of both ovine and bovine cheeses and, overall, there was a significant preference for the ovine brand.     

Effect of commercial adjunct cultures on proteolysis in low-fat Kefalograviera-type cheese

The effect of two commercially available adjunct cultures, LBC 80 (Lactobacillus casei subsp. rhamnosus) and CR-213 (containing Lactococcus lactis subsp. cremoris and Lc. lactis subsp. lactis) on the proteolysis in low-fat hard ewes’ milk cheese of Kefalograviera-type was investigated. Two controls, a full-fat cheese (306 g kg⁻¹ fat, 378 g kg⁻¹ moisture) and a low-fat cheese (97 g kg⁻¹ fat, 486 g kg⁻¹ moisture, made using a modified procedure), were also prepared. The effect of adjunct culture on proteolysis, as examined by polyacrylamide gel electrophoresis of cheese and water soluble cheese extracts, was marginal. The reverse-phase HPLC peptide profiles of the water soluble extracts from low-fat cheeses were similar although some quantitative differences were observed between low-fat control cheese and experimental cheeses. The fat content as reflected by the differences in peptide profiles affected the pattern of proteolysis. Proteolysis, as measured by the percentage of total nitrogen soluble in water or in 120 g L⁻¹ trichloroacetic acid, was significantly (P<0.05) affected by the addition of adjunct cultures. Furthermore, the adjunct cultures enhanced the production of low molecular mass nitrogenous compounds; the levels of total nitrogen, soluble in 50 g L⁻¹ phosphotungstic acid, and of free amino acids were significantly (P<0.05) higher in the low-fat experimental cheeses than in the low-fat control cheese.     

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.     

Identification of bioactive peptides in commercial Cheddar cheese

This study examined the presence of antimicrobial, antioxidant and antihypertensive peptides in three commercially available Australian Cheddar cheeses. Peptide extracts as well as fractionated peptide extracts were examined. Commercial cheese A peptides exhibited the greatest inhibition against Bacillus cereus and also commercial cheese A fractionated peptides greater than 10 kDa showed the highest inhibition against B. cereus. Commercial cheese A peptides also showed the highest inhibition of 2,2-diphenyl-1-picrylhydrazyl (DPPH), a free radical used to measure antioxidant activity. All cheese fractionated peptides greater than 10 kDa demonstrated higher inhibition of DPPH after fractionation. Antihypertensive peptides were determined by inhibition of the angiotensin-converting enzyme (ACE). Overall, commercial cheese A had the lowest concentration required to inhibit ACE and commercial cheese A fractionated peptides lower than 5 kDa had the lowest inhibition after fractionation. These preliminary findings suggest that peptide extracts of three commercial Australian Cheddar cheeses exhibit antimicrobial, antihypertensive and antioxidant properties.     

Contribution of coagulant and native microflora to the volatile-free fatty acid profile of an artisanal cheese

The contributions of the coagulant Cynara cardunculus and of the microflora of raw milk to the volatile-free fatty acid profile of Serra da Estrela cheese were evaluated. The experimental design included both a model system and, dual cheeses. The study in the model system showed that isovaleric acid was the predominant volatile compound after 7 d of ripening. The systems inoculated with Enterococcus faecium produced the highest amount of this volatile (ca. 135.8 mg kg⁻¹ curd), while those inoculated with Lactobacillus plantarum produced the least (21.4 mg kg⁻¹ curd); Lactococcus lactis produced moderate amounts (ca. 34.2 mg kg⁻¹ curd) but a total amount of volatile-free fatty acids similar to those found in control samples. This is considered advantageous since this volatile fatty acid confers a harsh, piquant, mature flavour to cheese, coupled with the realisation that excess volatiles may result in off-flavours. The addition of cultures in experimental cheeses helped reduce ripening time to about one half. Inclusion of Lb. plantarum led to cheeses containing the highest amounts of volatiles, and exhibiting an aroma closest to that of typical Serra da Estrela cheese.     

Texture characteristics and pizza bake properties of low-fat Mozzarella cheese as influenced by pre-acidification with citric acid and use of encapsulated and ropy exopolysaccharide producing cultures

Low-fat Mozzarella cheeses containing 6% fat were made by pre-acidification of milk with citric acid to pH 6.1 and using encapsulated ropy or non-ropy exopolysaccharide (EPS) producing Streptococcus thermophilus. Moisture retention, changes in texture profile analysis (TPA), meltability and stretchability of cheese, and changes in colour, surface scorching and shred fusion were analysed after baking over 90 days (d). Control cheeses and those made from pre-acidified milk without EPS cultures had the lowest moisture content at 54.84% and 55.28%, respectively. Control cheeses were hardest and their meltability and stretchability were initially low. Hardness was reduced and the melt and stretch distances increased with time. When baked, control cheeses showed incomplete shred fusion. Pre-acidification reduced hardness and increased meltability. Capsular- and ropy-EPS were quantified at 30.42 and 30.55 mg g⁻¹ of cheese, respectively, and increased moisture retention in pre-acidified cheese to 56.67% and 56.21%, respectively. These cheeses were softer and exhibited lower springiness. Greater meltability was observed initially but became similar to control cheeses after 90 d of storage. When baked after 45 d of storage, cheeses containing EPS producing cultures showed improved shred fusion, meltability and a reduction in surface scorching.     

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.     

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.     

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.     

Effect of cook temperature on starter and non-starter lactic acid bacteria viability,cheese composition and ripening indices of a semi-hard cheese manufactured using thermophilic cultures

Semi-hard cheeses were manufactured using Streptococcus thermophilus and Lactobacillus helveticus cultures and their ripening was characterised. During cheese manufacture, curds were cooked to a maximum temperature of 47, 50 or 53 °C, pre-pressed under whey at pH 6.15, moulded, pressed and brined. Increased cook temperature resulted in increased manufacture time, a significantly reduced growth rate of S. thermophilus during manufacture in the order 47≈50 °C>53 °C and in significantly lower mean viable cell counts of S. thermophilus up to 56 d of ripening. Increasing cook temperature had no significant effect on mean viable cell numbers of L. helveticus or non-starter lactic acid bacteria (NSLAB). Cheeses produced from curds cooked to 47 °C had significantly higher levels of moisture in non-fat substances (MNFSs), salt-in-moisture and a significantly lower pH and levels of butyrate compared with cheeses produced from curds cooked to 50 or 53 °C.     

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.     

Activation energy measurements in rheological analysis of cheese

Activation energy of flow (E_a) between 30 and 44 °C was calculated from temperature sweeps of cheeses with contrasting characteristics to determine its usefulness in predicting rheological behavior upon heating. Cheddar, Colby, whole milk Mozzarella, low-moisture part-skim Mozzarella, Parmesan, soft goat, and Queso Fresco cheeses were heated from 22 to 70 °C, and E_a was calculated from the resulting Arrhenius plots. Protein and moisture content were highly correlated with E_a. The E_a values for goat cheese and Queso Fresco, which did not flow when heated, were between 30 and 60 kJ mol^?1. Cheddar, Colby, and the Mozzarellas did flow upon heating, and their E_a values were between 100 and 150 kJ mol^?1. Parmesan, the hardest cheese, flowed rapidly with heat and had an E_a > 180 kJ mol^?1. E_a provides an objective means of quantitating the flow of cheese, and together with elastic modulus and viscous modulus provides a picture of the behavior of cheese as it is heated.     

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.     

Lipolysis in reduced sodium Feta cheese made by partial substitution of NaCl by KCl

Feta cheese (five trials) of different sodium content was made from split lots of curd by varying the salting procedure, i.e. dry salting with NaCl (control) or mixtures of NaCl/KCl (3 : 1 or 1 : 1, w/w basis) and filling the cans with brine made with NaCl or the above NaCl/KCl mixtures, respectively. Lipolysis in cheese was monitored during aging by using the acid degree value (ADV) method and gas chromatography (GC). It was found that the ADVs of control and experimental cheeses were similar (P>0.05) at all sampling ages (3, 20, 40, 60, 120 and 240 d). Moreover, the results of GC showed that there were neither qualitative nor significant (P>0.05) quantitative differences in the individual free fatty acids (FFA) of the control and experimental cheeses at the ages of 40 and 120 d. These findings indicated that the partial substitution of NaCl by KCl in the manufacture of Feta cheese had no effect on lipolysis during cheese aging.     

A combined morphologic and molecular approach for characterizing fungal microflora from a traditional Italian cheese (Fossa cheese)

Phenotypic and genotypic methods were used to identify filamentous fungi that characterize traditional Italian Fossa cheese and its ripening environment. After ageing for 60 days at a dairy, it was ripened for an additional three months in a pit. In the fully ripened cheese, moulds ranged from 3 to 3.4 log cfu g^?1 and Penicillium was the prevalent species. Pit environmental fungi ranged from 530 to 750 cfu m^?3 (air) and from 130 to 340 cfu cm^?2 (surfaces). The dominant pit strains were Alternaria spp., Aspergillus spp., Cladosporium spp. and Penicillium spp. Phylogenetic analyses of 18S rRNA gene and ITS1-5.8S-ITS2 regions highlighted Penicillium camemberti, Aspergillus nidulans and Aspergillus versicolor as traceable species occurring in both the cheese and pit environment, suggesting their involvement in the development of typical Fossa cheese characteristics. This approach may be used for the identification of microflora on other cheese varieties to better understand the fungal contribution in cheese ripening.     

The influence of seasons and ripening time on yeast communities of a traditional Brazilian cheese

The occurrence and effects of the dry and rainy seasons on yeast populations in traditional Serro Minas cheese, one of the most popular cheeses produced from raw milk in Brazil, were studied over the course of 60 days of ripening. Enzymatic activity exhibited by these yeast isolates was also studied. A total of 19 yeast species were identified via sequence analysis of the D1/D2 domains of the large subunit of the rRNA gene. Fourteen yeast species were obtained from cheese produced during the dry season, and fifteen species were obtained from cheese produced during the rainy season. High diversity indices for the yeast species were determined for cheese manufactured during both seasons (average H′_D = 1.7 and H′_R = 1.5, respectively). The predominant species in Serro Minas cheese included Debaryomyces hansenii, Kodamaea ohmeri and Kluyveromyces marxianus. D. hansenii 28.12 showed low lipolytic and high proteolytic activity. K. marxianus 83F and 60P demonstrated lipolytic and β-galactosidase activity, respectively. K. ohmeri 88A displayed low lipolytic and β-galactosidase activity. Maximal lipase, β-galactosidase and protease activity was observed at 20 °C and pH 6.0, 30 °C and pH 7.0 and 50 °C and pH 6.0, respectively. Considering that D. hansenii 28.12, K. ohmeri 88A and K. marxianus 60P together showed protease, lipase and β-galactosidase activity in this study, further research on the possibility of including these yeasts as part of a starter culture and research on their effects on the sensory properties of Serro Minas cheese merit more study.     

Antimicrobial activity of pediocin-producing Lactococcus lactis on Listeria monocytogenes,Staphylococcus aureus and Escherichia coli O157:H7 in cheese

The antimicrobial activity of two pediocin-producing transformants obtained from wild strains of Lactococcus lactis on the survival of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 during cheese ripening was investigated. Cheeses were manufactured from milk inoculated with the three pathogens, each at approximately 6 log cfu mL⁻¹. Pediococcus acidilactici 347 (Ped⁺), Lc. lactis ESI 153, Lc. lactis ESI 515 (Nis⁺) and their respective pediocin-producing transformants Lc. lactis CL1 (Ped⁺) and Lc. lactis CL2 (Nis⁺, Ped⁺) were added at 1% as adjuncts to the starter culture. After 30 d, L. monocytogenes, S. aureus and E. coli O157:H7 counts were 5.30, 5.16 and 4.14 log cfu g⁻¹ in control cheese made without adjunct culture. On day 30, pediocin-producing derivatives Lc. lactis CL1 and Lc. lactis CL2 lowered L. monocytogenes counts by 2.97 and 1.64 log units, S. aureus by 0.98 and 0.40 log units, and E. coli O157:H7 by 0.84 and 1.69 log units with respect to control cheese. All cheeses made with nisin-producing LAB exhibited bacteriocin activity throughout ripening. Pediocin activity was only detected throughout the whole ripening period in cheese with Lc. lactis CL1. Because of the antimicrobial activity of pediocin PA-1, its production in situ by strains of LAB growing efficiently in milk would extend the application of this bacteriocin in cheese manufacture.