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Characterization of microflora in homemade semi-hard white Zlatar cheese.

The Zlatar cheese belongs to the group of traditionally homemade cheeses, which are produced from nonpasteurized cow's milk, without adding of any bacterial starter culture. Changes were followed in lactic acid bacteria population and chemical composition during the ripening period of cheese up to 60 days. Results showed that the percentage of lactic acid cocci was higher in raw milk and one day old cheese and their percentage was gradually decreasing, whereas the number of lactobacilli was increasing. After 30 days of cheese ripening the number of cocci increased again, reaching the number of lactobacilli. The results of API 50 CH system and rep-PCR analysis showed that Lactobacillus paracasei subsp. paracasei, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcuus faecium and Enterococcus faecalis were the main groups present during the ripening of Zlatar cheese. Results revealed that in older cheeses (45 and 60 days old) enterococci were the main group present. It was also demonstrated that 57 isolates showed antimicrobial activity. The number of bacteria showing antimicrobial activity slowly decreased during the ripening period and in samples of 60 days old cheese producers of antimicrobial activities were not detected.

Cheese↗

Membrane inlet mass spectrometric measurement of O2 and CO2 gradients in cultures of Lactobacillus paracasei and a developing Cheddar cheese ecosystem.

Membrane inlet mass spectrometry was used to measure O2 and CO2 as depth profiles in stab cultures of 0.1% agar Man Rogosa Sharpe medium inoculated with Lactobacillus paracasei CI3. Diffusion of CO2 from the central column of growth into the medium was observed to show lower concentrations where bacteria were absent. CO2 profiles developed in a manner similar to those in Cheddar cheese and O2 was undetectable at similar depths. Gases were analysed in Cheddar cheese over a maturation period of 200 d. O2 was detectable to depths of 13, 6 and 2.5 mm on days 2, 9 and 15, respectively, but then became undetectable at depths of 2.5-3 mm. CO2 concentrations measured within the cheese increased 10-fold from day 2 to day 200 to reach a value of around 15 mM. The progress of measured CO2 concentration over time at a given depth in cheese shows a hyperbolic type increase. Coefficient of regression values increase with depth to a maximum value of R2 = 0.93. In both systems, reductions and increases in CO2 were due to the absence or presence of bacterial growth, respectively. Confocal scanning laser and scanning electron microscopy was used to show spatial heterogeneity of microcolonies within the cheese ecosystem. This information can potentially be used as a non-sensory evaluation of cheese maturity status. Measurement of gases in a cheese ecosystem provides the first description of mass spectrometry being used to monitor the processes of microbial gaseous exchange with respect to O2 and CO2 in a cheese ecosystem.

Carbon Dioxide↗

Identification of yeasts and coryneform bacteria from the surface microflora of brick cheeses.

Coryneform bacteria and yeasts of 21 brick cheeses from six German dairies, produced by using undefined ripening cultures, were identified. Arthrobacter nicotianae, Brevibacterium linens, Corynebacterium ammoniagenes, Corynebacterium variabilis and Rhodococcus fascians were found in significant numbers. Out of 148 coryneform isolates 36 could not be identified at the species level. With the exception of a large rennet cheese, the coryneform microflora of rennet and acid cured cheeses were similar, but the cheeses had clearly different yeast populations. Debaryomyces hansenii and Galactomyces geotrichum prevailed in rennet cheeses while Kluyveromyces marxianus and Pichia membranaefaciens were the main species found in acid cured cheese. The dominance of Yarrowia lipolytica probably indicates an improper yeast population, resulting in poor cheese quality. Some of the species identified are potential candidates for designing a defined ripening culture for rennet red smear cheese.

Actinomycetales↗

Effect of added proteinases and level of starter culture on the formation of biogenic amines in raw milk Manchego cheese.

The influence of two proteinases (Bacillus subtilis neutral proteinase and Micrococcus sp. cysteine proteinase) and two starter culture levels (0.1% and 1%) on biogenic amine formation has been studied in raw ewes' milk Manchego cheese. Amino acid decarboxylating micro-organisms were determined on tyrosine enriched selective media. Biogenic amines were analysed by capillary electrophoresis in citrate buffer at pH 3.6. Addition of proteinases and level of starter culture did not influence the population of micro-organisms with amino acid decarboxylating activity, which represented on average 1% of the bacterial population in 30-day-old cheeses. Tyramine and histamine were detected in all batches of cheese from day 30. Concentrations of tyramine and histamine were higher in cheeses made from milk with neutral proteinase (up to 356 and 284 mg kg(-1), respectively, after 90 days) than in cheeses made from milk with cysteine proteinase (up to 269 and 189 mg kg(-1), respectively) or with no proteinase added (up to 305 and 226 mg kg(-1), respectively). Formation of tyramine and histamine was also favoured in cheeses made with 1% starter culture with respect to cheeses made with only 0.1% starter culture, probably due to the higher pH values of the former cheeses. After 90 days of ripening, concentrations of 10-20 mg kg(-1) phenylethylamine were observed in 9 of the 12 batches, and levels < 10 mg kg(-1) tryptamine were only detected in 3 batches, with no significant relationship between the concentration of these amines and proteinase addition or level of starter culture.

Animals↗

Proteolysis and formation of volatile compounds in cheese manufactured with a bacteriocin-producing adjunct culture.

Hispánico cheese, a semi-hard Spanish variety, was manufactured from a mixture of pasteurized cows' and ewes' milks (4:1) using a commercial mesophilic LD-type starter comprising Lactococcus lactis subsp. cremoris, Lc. lactis subsp. lactis, Lc. lactis subsp. lactis var diacetylactis and Leuconostoc mesenteroides subsp. cremoris. Varying amounts (0-1.0 g/kg) of an Enterococcus faecalis INIA 4 culture in milk were added as a bacteriocin-producing adjunct. Differences in pH between cheeses manufactured with and without the bacteriocin producer did not exceed 0.11 pH units. Starter lactococci lost viability more rapidly in cheeses made with the bacteriocin producer, which reached counts of up to 6 x 10(7) cfu/g during ripening. Aminopeptidase activity in 1-d-old cheese made from milk inoculated with 1.0 g bacteriocin-producing culture/kg was twice that in control cheese. Degrees of overall proteolysis and levels of total free amino acids in 45-d-old cheese made with 1.0 g bacteriocin-producing culture/kg were 1.80-fold and 2.17-fold those in control cheese of the same age. Inoculating milk with 1.0 g/kg bacteriocin-producing culture reduced the level of hydrophobic peptides in the resultant cheese, increased the concentrations of 3-methyl-1-butanal, diacetyl and acetoin, and resulted in the highest scores for flavour quality and flavour intensity throughout ripening.

Aminopeptidases↗

High-fat spreadable processed cheese for people with high blood cholesterol.

Part of the raw cheese base used for manufacturing high-fat processed cheese spread was replaced by cows' (C) or buffalo (B) butter residue at levels of up to 20%. These residues were produced during the conversion of butter to butter oil by traditional moderate (T) or severe (M) heat treatments. The hypocholesterolaemic properties of butter residues incorporated into processed cheese were tested with rats (serum and liver cholesterol) and human beings (serum cholesterol). When rats were given the control cheese with no butter residue there were significant increases in both serum and liver cholesterol compared with rats given stock diet. However, when part of the raw cheese base was replaced by butter residue these rises were reduced by amounts proportional to the level of butter residue. The hypocholesterolaemic properties of the residues were in order CT > BT, CM > BM, and incorporations of 10% CT, 15% BT, 15% CM and 20% BM were found to nullify the hypercholesterolaemic effects of control cheese and restore cholesterol levels to their normal values. A small human trial gave similar results. Incorporation of butter residue slightly affected the chemical composition of the cheeses. Fat content and pH were little different; however, cheeses with butter residue tended to have higher total protein but lower soluble protein, tyrosine and tryptophan, and total volatile fatty acids. Rheological properties were not altered significantly. Incorporation of butter residue improved the organoleptic properties, particularly flavour intensity. Storage at 5 degrees C for 2 months did not alter the organoleptic properties and changes in chemical composition followed the normal pattern.

Animals↗

Effects of including silage in the diet on volatile compound profiles in Montasio cheese and their modification during ripening.

Effects of including silage in the diet on volatile compound profiles and their modification during ripening of Montasio cheeses were examined. Twelve farms were selected and grouped according the type of forage in the ration: hay-based diets (four farms); hay and corn silage-based diets (four farms); and diets based on hay, corn silage and grass silages (four farms). For cheesemaking, 1000 kg of milk collected from two consecutive milkings was sampled from each farm and processed in the same cheese factory. Cheeses were ripened in the same cellar, in controlled humidity (78-85% relative humidity) and temperature (9-12 degrees C), until analysis. After 68 (62-74), 200 (194-206) and 360 (354-366) d of ripening, a cheese from each batch was analysed for moisture, protein and fat, volatile fatty acids (VFA) and volatile compounds. These latter were analysed by dynamic reverse carrier gas headspace gas chromatographic mass spectrometry (GC-MS) technique, scanning from m/z 29 to m/z 300 at 0.5 s cycle time. Total and individual VFA contents of cheeses did not differ between the different dietary treatments and increased linearly (P<0.01) during ripening, indicating that there were no appreciable defects of fermentation. Sixty-two volatile compounds were identified in the cheese samples: 12 aldehydes, 9 ketones, 16 alcohols, 17 esters, 2 hydrocarbons, 4 sulphur-containing compounds and 2 terpenes. Diet significantly affected the amount of total alcohols, ethanol, isobutanol, 1-penten-3-ol, 2-methyl-1-butanol and the sum of all the volatile compounds. Significant variations of 33 volatile compounds (9 aldehydes, 4 ketones, 6 alcohols, 10 esters, 1 hydrocarbons, 2 sulphur containing compounds, and 1 terpene) were observed during ripening. These results suggest that the influence of diet composition on volatile compounds of matured cheeses are related more to effects on microbial and chemical fermentations in cheese during ripening rather than to a direct transfer of molecules from milk.

Alcohols↗

Changes in lipid fractions and sensory properties of Idiazabal cheese induced by lipase addition.

This work studied the addition of an adequate lipase to enhance lipolysis reactions and the development of piquant flavour and sharp odour in Idiazabal cheese, as an alternative to the use of lamb rennet paste. Cheeses were manufactured from bulk raw ewes' milk in 50 l vats with commercial bovine rennet and 80 lipase units of pregastric or 180 lipase units of fungal lipase and ripened for 180 days. A higher lipolytic activity was induced by lipase addition promoting strong changes in odour and flavour attributes. Both fungal and pregastric lipases increased the content of total free fatty acids (FFA), but the fungal lipase released mainly medium- and long-chain FFA. In contrast, the pregastric lipase preferably released short-chain FFA. Diglyceride (DG) content was considerably higher in cheeses made with added pregastric lipase compared with those made with fungal lipase or with no lipase. Monoglycerides (MG) were detected only in cheeses made with either lipase added, reaching comparable concentrations after ripening for 180 days. The cheeses made with pregastric lipase had the highest scores for odour and flavour intensity, and sharp and rennet odours, desirable attributes for the Idiazabal cheese made with lamb rennet paste. None of the texture attributes were significantly influenced by the concentrations of MG and DG in the cheeses made with either lipase. Thus, the pregastric lipase was more appropriate than the fungal lipase to develop a more traditionally-flavoured Idiazabal cheese.

Animals↗

Evaluation of biogenic amines and microbial counts throughout the ripening of goat cheeses from pasteurized and raw milk.

The effect of the hygienic quality of milk on changes in microbial counts and biogenic amine content was evaluated during ripening of goat cheeses manufactured from pasteurized and raw milks at 1, 14, 30, 60 and 90 d. The original milk, rennet, curd and whey were also included in the study. The pH, salt content and extent of proteolysis in the cheese were also evaluated. Spermidine and spermine were the main amines in raw milk, while they were minor amines in cheeses. Other amines increased markedly during ripening, tyramine being the main amine in cheese made from raw milk and cadaverine and putrescine in those produced from pasteurized milk. Enterobacteriaceae counts decreased during ripening whereas those of lactic acid bacteria increased, especially lactobacilli and enterococci. Cheese made from raw milk showed higher microbial counts during ripening than those made from pasteurized milk, especially for Enterobacteriaceae and enterococci, counts being 2 or 3 log units higher. Raw milk cheese showed remarkably higher biogenic amines compared with pasteurized milk cheeses. Therefore, pasteurization of milk causes a decrease in final biogenic amine content of cheese as a result of the reduction of its microbial counts.

Animals↗

Starter strain related effects on the biochemical and sensory properties of Cheddar cheese.

A detailed investigation was undertaken to determine the effects of four single starter strains, Lactococcus lactis subsp. lactis 303, Lc. lactis subsp. cremoris HP, Lc. lactis subsp. cremoris AM2, and Lactobacillus helveticus DPC4571 on the proteolytic, lipolytic and sensory characteristics of Cheddar cheese. Cheeses produced using the highly autolytic starters 4571 and AM2 positively impacted on flavour development, whereas cheeses produced from the poorly autolytic starters 303 and HP developed off-flavours. Starter selection impacted significantly on the proteolytic and sensory characteristics of the resulting Cheddar cheeses. It appeared that the autolytic and/or lipolytic properties of starter strains also influenced lipolysis, however lipolysis appeared to be limited due to a possible lack of availability or access to suitable milk fat substrates over ripening. The impact of lipolysis on the sensory characteristics of Cheddar cheese was unclear, possibly due to minimal differences in the extent of lipolysis between the cheeses at the end of ripening. As anticipated seasonal milk supply influenced both proteolysis and lipolysis in Cheddar cheese. The contribution of non-starter lactic acid bacteria towards proteolysis and lipolysis over the first 8 months of Cheddar cheese ripening was negligible.

Animals↗

Effects of ventilation rate and of dietary protein level in an intensive dairy sheep system on the features of Canestrato Pugliese cheese.

Effects of ventilation rate and of dietary protein level in an intensive dairy sheep system on the features of Canestrato Pugliese cheese were studied. Cheeses were manufactured from the bulk milk obtained from ewes subjected to four different experimental treatments: (1) low dietary crude protein (CP) of 13% in dry matter (DM) at a low ventilation rate (23.5 m3/h per ewe) (LPLV); (2) low dietary CP at a moderate ventilation rate (47 m3/h per ewe) (LPMV), (3) moderate dietary CP of 16% DM at a low ventilation rate (MPLV); and (4) moderate dietary CP dietary at a moderate ventilation rate (MPMV). Bulk milk and cheeses (at 1, 15, 45 and 90 d of ripening) were analysed for chemical composition, N fractions and plasmin-plasminogen activities. The pH 4.6-soluble and insoluble N fractions were analysed by urea-PAGE. Bulk milk from ewes receiving the low CP diet displayed higher casein and lower urea contents. Ewes subjected to the low ventilation rate displayed a higher plasminogen activity in milk, whereas no differences emerged among treatments in the conversion of zymogen to plasmin. During ripening the plasmin-plasminogen system in cheese did not display significant changes across treatments. At 90 d of ripening, the cheese produced with milk from ewes receiving the low CP diet and exposed to the low ventilation rate displayed more markedly stained bands in the area of gamma-caseins in pH 4.6-insoluble N fraction. As from 15 d of ripening, the pH 4.6-soluble fraction showed a greater number of bands in the LPMV and MPMV than in the LPLV and MPLV cheeses. The results showed that Canestrato Pugliese cheese manufactured with milk from ewes fed the low CP diet and exposed to the moderate ventilation rate was characterized by higher protein and casein content in the fresh cheese and by a greater proteolysis after 90 d of ripening.

Animal Feed↗

Proteolysis in Hispánico cheese manufactured using a mesophilic starter, a thermophilic starter, and bacteriocin-producing Lactococcus lactis subsp. lactis INIA 415 adjunct culture.

Lactococcus lactis subsp. lactis INIA 415, a strain harboring the structural genes of bacteriocins nisin Z and lacticin 481, was used as adjunct culture in the manufacture of Hispánico cheese with a mesophilic starter and a thermophilic starter of high aminopeptidase activity. Addition of the bacteriocin producer promoted early lysis of mesophilic and thermophilic starter bacteria. Extracellular aminopeptidase activity in 7-day-old cheese made using mesophilic and thermophilic starters plus bacteriocin producer was 3.0-fold the level reached in cheese made without the bacteriocin producer. Proteolysis in cheese made with mesophilic and thermophilic starters plus bacteriocin-producing adjunct culture after 25 days of ripening was 1.5-fold the level reached in cheese made without the bacteriocin producer, and the level of total free amino acids was 2.9-fold the level found in cheese made without the bacteriocin producer. Cheese made with mesophilic and thermophilic starters plus bacteriocin producer received the highest scores for flavor quality and flavor intensity and reached in 25 days the flavor intensity score of a 75-day-old cheese made without the bacteriocin producer.

Amino Acids↗

Milk excretion of ivermectin and moxidectin in dairy sheep: assessment of drug residues during cheese elaboration and ripening period.

Ivermectin (IVM) and moxidectin (MXD) are broad-spectrum endectocide antiparasitic drugs extensively used in food-producing animals. The patterns of IVM and MXD excretion in milk were comparatively characterized following their subcutaneous administration (200 microg.kg(-1) of body weight) to lactating dairy sheep. The relationship between milk excretion and plasma disposition kinetics of both compounds was characterized. A pool of milk collected from all of the animals in each experimental group was used for cheese elaboration. IVM and MXD residual concentrations were assessed during the cheese-making process and ripening period. IVM and MXD concentrations were measured in plasma, milk, and milk product (whey, curd, and cheese) samples using an HPLC-based methodology with fluorescence detection. IVM and MXD were extensively distributed from the bloodstream to the mammary gland, and large quantities, particularly of MXD, were excreted in milk. Residual concentrations of both compounds were recovered in milk up to 30 (IVM) and 35 (MXD) days post-treatment. The total fraction of the administered dose excreted in milk for MXD was significantly higher than that of IVM. During cheese production, the highest residual concentrations of both molecules were measured in the curd. Thirty-four percent of the total drug residue measured in the pooled milk collected from treated sheep was lost during the cheese-making process. The lowest residual concentrations were measured in the whey. IVM and MXD concentrations in the elaborated cheese tended to increase during the ripening period, reaching the highest residual level at 40 days of cheese maturation. The long persistence of milk residual concentrations of MXD and IVM in lactating dairy sheep and the high concentrations found in cheese and other milk-related products should be seriously considered before recommendation of the extralabel use of these antiparasitic drugs in dairy animals.

Animals↗

Starter bacteria are the prime agents of lipolysis in cheddar cheese.

To assess the contribution of starter lactic acid bacteria (LAB) to lipolysis in Cheddar cheese, the evolution of free fatty acids (FFAs) was monitored in Cheddar cheeses manufactured from pasteurized milks with or without starter. Starter-free cheeses were acidified by a combination of lactic acid and glucono-delta-lactone. Starter cultures were found to actively produce FFAs in the cheese vat, and mean levels of FFAs were significantly higher in starter cheeses over ripening. The contribution of nonstarter LAB toward lipolysis appears minimal, especially in starter-acidified cheeses. It is postulated that the moderate increases in FFAs in Cheddar cheese are primarily due to lack of access of esterase of LAB to suitable lipid substrate. The results of this study indicate that starter esterases are the primary contributors to lipolysis in Cheddar cheese made from good quality pasteurized milk.

Animals↗

Influence of a probiotic adjunct culture of Enterococcus faecium on the quality of cheddar cheese.

Cheddar cheese has previously been shown to be an effective vehicle for delivery of viable cells of a probiotic Enterococcus faecium strain to the gastrointestinal tract. The particular strain, E. faecium PR88, has proven efficacy in the treatment of irritable bowel syndrome, and in this study it was evaluated for suitability as a starter adjunct for Cheddar cheese manufacture. When added to cheesemilk at an inoculum of 2 x 10(7) cfu/mL, the enterococcal adjunct maintained viability in Cheddar cheese at levels of up to 3 x 10(8) cfu/g during 9 months of ripening. Increased proteolysis and higher levels of some odor-active volatile compounds were observed in Cheddar cheeses containing the PR88 adjunct compared with the control throughout the ripening period. In addition, the enterococcal adjunct strain did not affect cheese composition. Although sensory evaluation showed no significant difference in flavor/aroma and body/texture scores between control and experimental cheeses, repeated comments by the commercial grader consistently described the cheeses containing PR88 as 'more advanced than the control' and as having 'better flavor'. These findings indicate that the presence of the PR88 adjunct strain in Cheddar cheese at levels of >/=10(8) cfu/g may positively influence Cheddar flavor.

Cheese↗

Investigation of fungal contamination in sheep and goat cheeses in southern Italy.

One hundred and twenty-two samples of cheeses made from goat and sheep milk and a mixture of the two types, produced in Southern Italy by industrial establishments or artisans, were analysed for the detection of fungal contamination and the presence of potentially toxigenic moulds. Only organoleptically acceptable cheeses without evident fungal contamination were studied. Among these, 40 were unripened, 30 medium and 52 long ripened cheeses. Moulds were found in 54 of the 122 analysed samples (44.3%). The most contaminated cheeses were the medium and long ripened ones (46.3% and 35.2%), and the industrial cheeses (59.1%). The artisan cheeses were the least contaminated (26.8%). Among the cheeses that tested positive, Penicillium species were the most frequently isolated (72.9%), followed by Geotrichum spp. (7.3%), Aspergillus spp. (4.2%) and Mucor spp. (4.2%). The potentially toxigenic species within the genera Penicillium, Aspergillus and Fusarium were mainly detected in sheep cheeses.

Animals↗

Biotechnological methods to accelerate cheddar cheese ripening.

Cheese is one of the dairy products that can result from the enzymatic coagulation of milk. The basic steps of the transformation of milk into cheese are coagulation, draining, and ripening. Ripening is the complex process required for the development of a cheese's flavor, texture and aroma. Proteolysis, lipolysis and glycolysis are the three main biochemical reactions that are responsible for the basic changes during the maturation period. As ripening is a relatively expensive process for the cheese industry, reducing maturation time without destroying the quality of the ripened cheese has economic and technological benefits. Elevated ripening temperatures, addition of enzymes, addition of cheese slurry, attenuated starters, adjunct cultures, genetically engineered starters and recombinant enzymes and microencapsulation of ripening enzymes are traditional and modern methods used to accelerate cheese ripening. In this context, an up to date review of Cheddar cheese ripening is presented.

Biotechnology↗

Inactivation of Staphylococcus aureus in raw milk cheese by combinations of high-pressure treatments and bacteriocin-producing lactic acid bacteria.

AIMS: To investigate the combined effect of high-pressure treatments (HPT) and milk inoculation with bacteriocin-producing lactic acid bacteria (BP-LAB) on the survival of Staphylococcus aureus during ripening of raw milk cheese. METHODS AND RESULTS: Cheeses were manufactured from raw milk artificially contaminated with S. aureus at ca 5 log CFU ml(-1), a commercial starter culture and one of seven strains of BP-LAB, added as adjuncts at 0.1%. HPT of cheeses were performed on days 2 or 50 at 300 MPa (10 degrees C, 10 min) or 500 MPa (10 degrees C, 5 min). On day 3, S. aureus counts were 6.46 log CFU g(-1) in control cheese. Milk inoculation with different BP-LAB lowered S. aureus counts on day 3 when compared with control cheese by up to 0.46 log CFU g(-1), HPT at 300 MPa on day 2 by 0.45 log CFU g(-1) and HPT at 500 MPa on day 2 by 2.43 log CFU g(-1). Combinations of BP-LAB with HPT at 300 and 500 MPa on day 2 lowered S. aureus counts on day 3 by up to 1.02 and 4.00 log CFU g(-1) respectively. CONCLUSIONS: The combined effect of milk inoculation with some of the BP-LAB tested and HPT of cheese on S. aureus inactivation was synergistic. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of HPT at lower pressures with BP-LAB inoculation is a feasible system to improve cheese safety in case of deleterious effects on cheese quality caused by HPT at higher pressures.

Antibiosis↗