Yeasts as adjunct starters in matured Cheddar cheese

Debaryomyces hansenii and Yarrowia lipolytica are typical foodborne yeast species frequently associated with dairy products and capable of predominating the yeast composition in such systems. The two species fulfil a number of criteria to be regarded as co-starters for cheesemaking. They are known for their proteolytic and lipolytic activity as well as their compatibility and stimulating action with the lactic acid starter cultures when co-inoculated. Recent studies indicated that yeasts could be included as part of starter cultures for the manufacturing of cheese, enhancing flavour development during the maturation. The potential of D. hansenii and Y. lipolytica as agents for accelerated ripening of matured Cheddar cheese has been evaluated during four cheese treatments. The interaction between the two yeast species and the lactic acid bacteria was surveyed incorporating (i) D. hansenii, (ii) Y. lipolytica, (iii) both species as adjuncts to the starter culture and (iv) a control cheese without any additions for the production of matured Cheddar cheese. The physical and chemical properties of the cheeses were monitored in order to evaluate the contribution of the yeasts to cheese maturation. The yeasts grew in association with the lactic acid bacteria without any inhibition. The yeasts species when individually added contributed to the development of bitter flavours despite accelerated development of strong Cheddar flavours. When both species were incorporated as part of the starter culture, the cheese, however, had a good strong flavour after a reduced ripening period. The cheese retained this good flavour and aroma after 9 months of production. The simultaneous application of D. hansenii and Y. lipolytica as part of the starter culture for the production of matured Cheddar cheese is proposed.     

Characterization of yeasts involved in the ripening of Pecorino Crotonese cheese

The aims of this work were to identify and characterize for some important technological properties the yeast species present throughout the ripening process of Pecorino Crotonese, a traditional cheese produced in a well defined area of Southern Italy. In particular, the strain technological properties considered include fermentation/assimilation of galactose and lactose, assimilation of lactate and citrate in the presence of different NaCl concentrations, hydrolysis of butter fat, skim milk, gelatine and casein, production of brown pigments in cheese agar and ability to produce biogenic amines. High yeast levels were recorded in cheese samples already after 5 h of brining (about 5 log cfu/g) and these concentration remained constant during ripening. The yeast isolates belonged to restrict number of yeast species. While Kluyveromyces lactis and Saccharomyces cerevisiae were isolated prevalently in the first stages of Pecorino Crotonese production, Yarrowia lipolytica and Debaryomyces hansenii dominated during the later stages of maturation. Otherwise, the latter two were very NaCl resistant species. In fact, D. hansenii strains conserved the ability to assimilate lactose and galactose in the presence of 10% NaCl, while almost all the strains of Y. lipolytica isolated assimilated citrate and lactate up to 7.5% NaCl. Y. lipolytica isolates evidenced also the highest proteolytic and lipolytic activities and the capability to catabolize tyrosine producing brown pigment. In addition they resulted in the highest aminobiogenic potential decarboxylating ornithine, phenylalanine, tyrosine and lysine. However, they were not able to produce histamine, biogenic amine produced by three strains of D. hansenii.     

DNA typing methods for differentiation of Debaryomyces hansenii strains and other yeasts related to surface ripened cheeses

The discriminative power of ITS-PCR, ITS-PCR RFLP and mitochondrial (mt)-DNA RFLP were evaluated for differentiation of yeasts of importance for surface ripened cheeses. In total 60 isolates were included. Of these, 40 strains of the following species, Debaryomyces hansenii var. hansenii, D. hansenii var. fabryi, Saccharomyces cerevisiae, Candida zeylanoides, Kluyveromyces lactis and Yarrowia lipolytica, were obtained from culture collections and 20 isolates of D. hansenii representing six different phenotypes were collected from seven Danish producers of surface ripened cheeses. ITS-PCR was evaluated for differentiation at species level on the 40 strains obtained from culture collections. Ten strains of each variety of D. hansenii and five strains of each of the above mentioned species were analysed. For each of the investigated species, a specific ITS1-5.8S rDNA-ITS2 region size was observed. Accordingly ITS-PCR was found valuable for differentiation at species level of yeasts of importance for surface ripened cheeses. ITS-PCR RFLP was investigated for the purpose of strain typing of D. hansenii. Ten CBS strains of each variety of D. hansenii were analysed. Only one enzyme (TaqI) out of several investigated (BamHI, DpnI, Fnu4HI, HaeIII, HindIII, HpaII, NlaII, Sau3AI, TaqI) demonstrated genetic diversity within the strains. This enzyme divided the 20 strains in three groups. Sequence analysis of the ITS1-5.8S rDNA-ITS2 region for the type strains of each variety of D. hansenii showed an identity of 99.84%, corresponding to a difference in one basepair. Based on these results, ITS-PCR RFLP was found ineffective for strain typing of D. hansenii. MtDNA RFLP using HaeIII and HpaII was evaluated for strain typing of D. hansenii on the 20 CBS strains of D. hansenii. The CBS strains were divided into 16 groups according to their restriction profiles, which proved the method useful for typing of D. hansenii at subspecies level. The 20 dairy isolates showed a lower genetic variability than the CBS strains as they were divided into eight groups. Cluster analysis of the 20 CBS strains and the 20 dairy isolates based on their mtDNA restriction profiles showed (max. similarity level = 52%) that the dairy isolates only clustered with the CBS strains of D. hansenii var. hansenii. For some of the dairies more than one strain of D. hansenii were found to be involved in the ripening process, indicating that the method could be useful for subspecies typing and investigation of the microbial succession between strains of D. hansenii during the ripening process of surface ripened cheeses.     

Yeasts associated with Sardinian ewe's dairy products

In the present work, the occurrence of yeasts in different types of typical Sardinian ewe's cheeses (32 samples of pecorino, 32 of caciotta, 40 of feta, 56 of ricotta) was determined. For the strains isolated the following properties were studied: proteolytic and lipolytic activities, the ability to grow at different temperatures, different concentrations of salt, and to assimilate and/or ferment compounds like lactate, citrate, lactose, glucose, galactose, lactic acid. Of 160 samples analysed, 76.2% yielded growth of yeasts. Yeast counts showed a certain variability among the samples. The highest levels were observed in caciotta and feta cheeses. A total of 281 strains belonging to 16 genera and 25 species were identified. In general, Debaryomyces hansenii was the dominant species, representing 28.8% of the total isolates. Other frequently appearing species were Geotrichum candidum, Kluyveromyces lactis and K. marxianus. Other genera encountered were Pichia, Candida, Dekkera, Yarrowia and Rhodotorula. With regard to the biochemical and technological properties of the yeasts, only K. lactis, K. marxianus and Dek. anomala assimilated and fermented lactose, whereas the majority of the species assimilated lactic acid. The assimilation of citrate was a characteristic of D. hansenii, R. rubra and Y. lipolytica. On the whole, the yeasts were weakly proteolytic while lipolytic activity was present in several species. A high percentage of strains showed a certain tolerance to low temperatures while only some strains of D. hansenii and K. lactis were able to grow at a 10% NaCl concentration.     

Microbiological and chemical characterization of Fiore Sardo, a traditional Sardinian cheese made from ewe's milk

The purpose of this study was to assess the chemical and microbial characteristics of 12 batches of artisanal Fiore Sardo, a protected designation of origin (PDO) hard cheese made from raw ewe's milk without addition of starters, during maturation. High standard deviations were observed for moisture percentage, total solids percentage and NaCl percentage content, possibly owing to differences in manufacturing processes and/or milk composition. Total mesophilic bacteria varied between 10 log₁₀ cfu/g in 48-h-old cheese samples and 3 log₁₀ cfu/g in 9-month-old samples. Total coliforms and staphylococci showed the highest counts at 48 h of ripening then decreased significantly, dropping to levels below 2 log₁₀ cfu/g at 3 months of maturation. Lactic acid bacteria and enterococci were the dominant micro-organisms throughout maturation. They were mainly represented by the species Lactococcus lactis ssp. lactis, Enterococcus faecium, Lactobacillus plantarum and Lactobacillus casei group. Low levels of yeasts were detected throughout the maturation period of the cheese. Debaryomyces hansenii and Kluyveromyces lactis var. lactis were the prevalent yeast species isolated.     

Presence and changes in populations of yeasts on raw and processed poultry products stored at refrigeration temperature

A study was undertaken to determine populations and profiles of yeast species on fresh and processed poultry products upon purchase from retail supermarkets and after storage at 5 degrees C until shelf life expiration, and to assess the potential role of these yeasts in product spoilage. Fifty samples representing 15 commercial raw, marinated, smoked, or roasted chicken and turkey products were analyzed. Yeast populations were determined by plating on dichloran rose bengal chloramphenicol (DRBC) agar and tryptone glucose yeast extract (TGY) agar. Proteolytic activity was determined using caseinate and gelatin agars and lipolytic activity was determined on plate count agar supplemented with tributyrin. Populations of aerobic microorganisms were also determined. Initial populations of yeasts (log10 cfu/g) ranged from less than 1 (detection limit) to 2.89, and increased by the expiration date to 0.37-5.06, indicating the presence of psychrotrophic species. Highest initial populations were detected in raw chicken breast, wings, and ground chicken, as well as in turkey necks and legs, whereas roasted chicken and turkey products contained less than 1 log10 cfu/g. During storage, yeast populations increased significantly (P < or = 0.05) in whole chicken, ground chicken, liver, heart and gizzard, and in ground turkey and turkey sausage. Isolates (152 strains) of yeasts from poultry products consisted of 12 species. Yarrowia lipolytica and Candida zeylanoides were predominant, making up 39 and 26% of the isolates, respectively. Six different species of basidiomycetous yeasts representing 24% of the isolates were identified. Most Y. lipolytica strains showed strong proteolytic and lipolytic activities, whereas C. zeylanoides was weakly lipolytic. Results suggest that yeasts, particularly Y. lipolytica, may play a more prominent role than previously recognized in the spoilage of fresh and processed poultry stored at 5 degrees C.     

Effectiveness of immersion treatments with acids, trisodium phosphate, and herb decoctions in reducing populations of Yarrowia lipolytica and naturally occurring aerobic microorganisms on raw chicken

Yarrowia lipolytica, one of the predominant yeasts in raw poultry, is believed to play a role in spoilage. This study was undertaken to investigate treatments to control the growth of Y. lipolytica on raw chicken stored at refrigeration temperature. Raw chicken wings inoculated with a mixture of five strains of Y. lipolytica isolated from raw poultry were dipped in solutions containing 2, 5, or 8% lactic acid, 2% lactic acid containing 0.2, 0.4, or 0.8% potassium sorbate or sodium benzoate, and 4, 8, or 12% trisodium phosphate solution. Populations of the yeast and total aerobic microorganisms were determined before and after treatment. Immersion of wings in 2% lactic acid (with or without 0.2% potassium sorbate or sodium benzoate) or 4% trisodium phosphate caused a significant (alpha = 0.05) reduction in numbers of Y. lipolytica and aerobic microorganisms. Treatment with 2% lactic acid containing 0.4 or 0.8% preservative did not result in additional significant reductions. Treatment of chicken wings with 2% lactic acid or 8% trisodium phosphate significantly reduced numbers of Y. lipolytica by 1.47 and 0.65 log10 cfu/g, respectively, and aerobic microorganisms by 2.60 and 1.21 log10 cfu/g, respectively, compared to controls. Growth of Y. lipolytica on wings stored at 5 degrees C for up to 9 days, however, was not affected by these treatments. Significant reductions in the population of Y. lipolytica occurred when the yeast was inoculated into 100% basil, marjoram, sage, and thyme decoctions, but not in 100% oregano or rosemary decoctions, held at 5 degrees C for 24 h. Treatment of chicken wings with 100% sage or thyme decoctions significantly reduced populations of Y. lipolytica but did not control its growth during storage at 5 degrees C for up to 9 days. The small, temporary decreases in numbers of Y. lipolytica and aerobic microorganisms resulting from immersion treatment of chicken wings with sage and thyme decoctions render these treatments of questionable value as preservation interventions.     

Microbiological characteristics of "androlla", a Spanish traditional pork sausage

Counts of total aerobic mesophilic microflora, lactic acid bacteria, salt-tolerant microflora, Enterobacteriaceae, enterococci, moulds and yeasts, and staphylococci, and some physico-chemical parameters (total solids, NaCl and nitrate contents and pH and aw values) were determined in 20 units of "androlla", a traditional dry-fermented sausage made in the NW of Spain. In general, high counts of all the investigated microbial groups were observed, with average values of 8.99 +/- 0.46 log cfu/g for the total aerobic mesophilic microflora, 9.11 +/- 0.16 log cfu/g for the lactic acid bacteria, 6.87 +/- 0.68 log cfu/g for the salt-tolerant microflora, 2.80+/-1.85 log cfu/g for the Enterobacteriaceae, 3.25 +/- 1.86 log cfu/g for the enterococci, 4.30 +/- 1.73 log cfu/g for the moulds and yeasts, and 3.62 +/- 0.60 log cfu/g for the staphylococci. From MRS agar, SPC agar + 7.5% NaCl, VRBG agar, and KAA agar, 10 colonies were randomly taken from each androlla unit and from each culture medium. A total of 200 strains per culture medium were then identified using the classical methods. Among the isolates from MRS agar, Lactobacillus sakei predominated, followed by Lactobacillus curvatus, Lactobacillus alimentarius and Lactobacillus plantarum. Of the 200 isolates obtained from SPC agar + 7.5% NaCl, only 56 strains belonged to the Staphylococcaceae or Micrococcaceae families. Among the Staphylococcaceae, Staphylococcus xylosus was the main species, followed by Staph. epidermidis; Staph. equorum, Staph. capitis and Staph. saprophyticus were isolated in very low proportions. Among the Micrococcaceae, Micrococcus luteus predominated, followed by Micrococcus lylae, Kocuria varians and Kocuria kristinae. Of the 150 isolates obtained from VRBG agar, Hafnia alvei was the main species, followed by Serratia liquefaciens and Enterobacter amnigenus; six isolates were identified as Salmonella. Among the 190 isolates obtained from KAA agar, 122 were considered enterococci; 20 isolates were identified as Enterococcus faecium, one as Enterococcus faecalis and 101 as Enterococcus inter faecalis-faecium.     

Effects of restricted feeding and antioxidant supplementation on pig performance and quality characteristics of longissimus dorsi muscle from Landrace and Duroc pigs

The microbial flora of naturally fermented sausages was studied. Lactic acid bacteria were the dominant species at the end of fermentation in all 3 batches (ca. 10(8) cfu g(-1)). Enterobacteria, Pseudomonas, yeasts and aerobic spore-formers decreased during fermentation and the ripening process and were below the detection limit in the end product. Enterococci exceeded 10(4)-10(5) cfu g(-1) during fermentation and remained constant at this level during ripening. Gram-positive, catalase-positive cocci exceeded 10(5) cfu g(-1), except for batch 1, during the first days of fermentation and then decreased until the end of ripening (10(2)-10(4) cfu g(-1)). No pathogenic staphylococci, sulfite reducing clostridia or Salmonella spp. were detected. Listeria spp. occurred in the first days of fermentation but were eliminated by the end of whole process in all batches. Identification showed that the majority of lactobacilli isolated from MRS agar strains were assigned to the species of Lactobacillus plantarum and Lb. plantarum/pentosus. All the isolated strains from the mannitol salt agar belonged to the genus of Staphylococcus. The predominant species were Staphylococcus saprophyticus, Staphylococcus xylosus and Staphylococcus simulans. The tests used to characterize the lactic acid bacteria and staphylococci as well as their distribution on the three batches were also discussed.     

L-methionine degradation potentialities of cheese-ripening microorganisms

Volatile sulphur compounds are major flavouring compounds in many traditional fermented foods including cheeses. These compounds are products of the catabolism of L-methionine by cheese-ripening microorganisms. The diversity of L-methionine degradation by such microorganisms, however, remains to be characterized. The objective of this work was to compare the capacities to produce volatile sulphur compounds by five yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, Saccharomyces cerevisiae and five bacteria, Brevibacterium linens, Corynebacterium glutamicum, Arthrobacter sp., Micrococcus lutens and Staphylococcus equorum of technological interest for cheese-ripening. The ability of whole cells of these microorganisms to generate volatile sulphur compounds from L-methionine was compared. The microorganisms produced a wide spectrum of sulphur compounds including methanethiol, dimethylsulfide, dimethyldisulfide, dimethyltrisulfide and also S-methylthioesters, which varied in amount and type according to strain. Most of the yeasts produced methanethiol, dimethylsulfide, dimethyldisulfide and dimethyltrisulfide but did not produce S-methylthioesters, apart from G. candidum that produced S-methyl thioacetate. Bacteria, especially Arth. sp. and Brevi. linens, produced the highest amounts and the greatest variety of volatile sulphur compounds includling methanethiol, sulfides and S-methylthioesters, e.g. S-methyl thioacetate, S-methyl thiobutyrate, S-methyl thiopropionate and S-methyl thioisovalerate. Cell-free extracts of all the yeasts and bacteria were examined for the activity of enzymes possibly involved in L-methionine catabolism, i.e. L-methionine demethiolase, L-methionine aminotransferase and L-methionine deaminase. They all possessed L-methionine demethiolase activity, while some (K. lactis, Deb. hansenii, Arth. sp., Staph. equorum) were deficient in L-methionine aminotransferase, and none produced L-methionine deaminase. The catabolism of L-methionine in these microorganisms is discussed.     

Development of primers for detecting dominant yeasts in smear-ripened cheeses

PCR primers were developed for the specific detection of Clavispora lusitaniae, Debaryomyces hansenii var hansenii, Geotrichum candidum, Kluyveromyces lactis and K. marxianus and Yarrowia lipolytica, yeast species commonly found on the surface of smear cheese. Forty eight representative strains frequently found in smear cheeses or taxonomically related to the target yeasts were used as templates, to validate the designed primers. The specific and selective detection of these yeasts was effective in situ, in Livarot smear, without yeast isolation and culture and was comparable with data obtained with a conventional method. The primers described here have thus potential for PCR studies applied to cheese. It should also be possible to use some of these primers with other substrates.     

Evaluation of the ability of Yarrowia lipolytica to impart strain-dependent characteristics to cheese when used as a ripening adjunct

Four Yarrowia lipolytica strains were tested as cheese-ripening adjuncts with milk culture in cheese production to evaluate their effects on the microbiological and biochemical features of the cheeses. The Y. lipolytica strains were able to overcome the other naturally occurring yeasts and were compatible with lactic acid bacteria (LAB). Fourier transform infrared (FTIR) profiles and analysis of free fatty acids (FFAs) released during ripening showed that the strains induced a marked lipolysis and gave rise to different FFAs accumulation over time with respect to the control. Strain-dependent protein breakdown patterns were identified and these biochemical differences resulted in different cheese organoleptic characteristics.     

Seasonal diversity of yeasts associated with white-surface mould-ripened cheeses

Yeasts present in Camembert and Brie cheeses during processing were monitored in a single cheese factory during summer and winter, to determine the seasonal diversity of yeasts over a ripening period of 56 days. Lactic acid bacteria predominate during cheese making, but yeasts play a significant role during maturation reaching counts of 6 log units at the later stages of ripening. Sources of yeast contamination that may lead to contamination of the curd were also determined. A diverse variety of 20 yeast species representing 10 genera were present during the winter period associated with the factory environment, during processing and ripening, whereas only seven yeast species representing six genera were isolated during summer. Although a broad spectrum of yeasts was isolated from both cheeses, Debaryomyces hansenii and Yarrowia lipolytica were the most abundant yeast species isolated. Other species encountered were Torulaspora delbrueckii, Rhodotorula mucilaginosa, Rhodotorula minuta and various species of Candida.     

Antimicrobial activity of cinnamon and clove oils under modified atmosphere conditions

Mixtures of cinnamon and clove oils were tested for inhibitory activity against important spoilage microorganism of intermediate moisture foods. Four fungal species (Aspergillus flavus, Penicillium roqueforti, Mucor plumbeus and Eurotium sp.), four yeasts species (Debaryomyces hansenii, Pichia membranaefaciens, Zygosaccharomyces rouxii and Candida lipolytica), and two bacteria species (Staphylococcus aureus and Pediococcus halophilus) inoculated separately on agar plates were sealed in a barrier pouch and exposed to essential oil volatiles under a modified atmosphere of low O2 (<0.05-10%) and high CO2 (20% or 40%), with the balance being N2. A. flavus and Eurotium sp. proved to be the most resistant microorganisms. Cinnamon and clove oils added between 1000 and 4000 microL at a ratio of 1:1 were tested for minimum inhibitory volume (MIV) against molds and yeasts. The gas phase above 1000 microL of the oil mixture inhibited growth of C. lipolytica and P. membranaefaciens; 2000 microL inhibited growth of A. flavus, P. roqueforti, M. plumbeus, Eurotium sp., D. hansenii, and Z. rouxii, while inhibition of A. flavus required the addition of 4000 microL. Higher ratios of cinnamon oil/clove oil were more effective for inhibiting the growth of A. flavus.     

Occurrence of antibiotic-resistant enterobacteria in agricultural foodstuffs

Antibiotic-resistant bacteria or their corresponding resistance determinants are known to spread from animals to humans via the food chain. We screened 20 vegetable foods for antibiotic-resistant coliform bacteria and enterococci. Isolates were directly selected on antibiotic-containing selective agar (color detection). Thirteen "common vegetables" (tomato, mushrooms, salad) possessed 10(4)-10(7) cfu/g vegetable of coliform bacteria including only few antibiotic-resistant variants (0-10(5) cfu/g). All seven sprout samples showed a some orders of magnitude higher contamination with coliform bacteria (10(7)-10(9) cfu/g) including a remarkable amount of resistant isolates (up to 10(7) cfu/g). Multiple resistances (up to 9) in single isolates were more common in sprout isolates. Resistant bacteria did not originate from sprout seeds. The most common genera among 92 isolates were: 25 Enterobacter spp. (19 E. cloacae), 22 Citrobacter spp. (8 C. freundii), and 21 Klebsiella spp. (9 K. pneumoniae). Most common resistance phenotypes were: tetracycline (43%), streptomycin (37%), kanamycin (26%), chloramphenicol (29%), co-trimoxazol (9%), and gentamicin (4%). The four gentamicin-resistant isolates were investigated in molecular details. Only three (chloramphenicol) resistant, typical plant-associated enterococci were isolated from overnight enrichment cultures. In conclusion, a contribution of sprouts contaminated with multiresistant, Gram-negative enterobacteria to a common gene pool among human commensal and pathogenic bacteria cannot be excluded.     

Changes in the microflora of manouri, a traditional Greek whey cheese, during storage

Six batches of manouri cheese were manufactured, and selected microbial groups were counted throughout storage for 20 days at 4°C. The counts of all the microbial groups increased ( P  < 0.05) throughout storage and reached higher levels in cheeses made in summer than in those made in spring. Moreover, the micro-organisms developed better on the cheese surfaces than in the interiors, especially in summer. The pH (6.78–7.33) and salt-in-moisture content (2.53–3.72) of the cheeses did not seem to affect the growth of the bacteria and yeasts present. The isolates of enterobacteriaceae were mainly Hafnia (68.75%), while the isolates from the Baird–Parker medium were mainly staphylococci. There was a great diversity of yeast species, but Debaryomyces hansenii and Pichia membranefasciens predominated. The isolates of enterobacteriaceae, staphylococci and—to a lesser extent—yeasts were proteolytic, but the free amino acid (N-NH2) content of the cheese did not increase significantly during storage (136.4–225.2 mg/kg glycine equivalent). It also seemed that milk fat was not degraded to any great extent (acid degree value 0.09–0.19) by the lipolytic activity of the strains. The main enzymes detected in selected isolates of enterobacteriaceae were leucine aminopeptidase and phosphohydrolase.     

Influence of Australian Native Herbs on the Maturation of Vacuum-packed Cheese

The composition, microbiology and biochemistry of semi-hard cheeses flavoured with native mint, lemon myrtle and bush tomato (BT) were compared with unflavoured (control) cheese during a 90-day period of maturation. Moisture, protein and salt levels of all cheeses were similar and did not change during maturation. However, the fat content of control cheese was significantly higher than that of the flavoured cheeses while the pH of cheese flavoured with BT was consistently lower throughout maturation. Total viable organisms, Lactobacillus and Lactococcus counts were between 10⁶ and 10⁷ colony forming units (cfu)/g cheese for all cheeses. Yeast and mould count was <10² cfu/g cheese throughout the maturation of all cheeses except in the cheese flavoured with BT which was >10³ cfu/g cheese. Biochemical indices of proteolysis and lipolysis increased with the extent of maturation in all cheeses but were most pronounced in the BT-flavoured cheese. The capillary electrophoretic profile of this cheese also indicated a more extensive hydrolysis of both α s- and β -caseins. The microbiological quality of BT appeared to have exerted a very significant influence on cheese properties.     

Mini soft cheese as a simple model for biochemical studies on cheese-making and ripening

A new miniature cheese model obtained under controlled microbiological conditions was proposed, characterized and tested for reproducibility. Optimal heat treatment of cheesemilk was defined, as well as maximal ripening time. Miniature cheeses were obtained with batch pasteurized milk (65 °C, 30 min) and ripened at 5 °C. Lactic and nonlactic microbial populations were monitored by plate counts. Proteolysis was assessed by nitrogen fractions, electrophoresis and liquid chromatography, and a sniffing test was applied to evaluate aroma. Coliform bacteria decreased during ripening but moulds and yeasts increased up to 10⁴ cfu/g after 60 d, which defined the end of ripening period. Starter population remained constant during all ripening (10⁹ cfu/g), while nonstarter lactic acid bacteria increased from ∼10² to 10⁴ cfu/g. Soluble nitrogen levels at pH 4.6, in trichloracetic acid (0.73 mol/l) and in phosphotungtic acid (0.009 mol/l) were 151, 67, and 10 g/1000 g of the total nitrogen, respectively, after 60 d of ripening, which are usual values for soft cheeses. Proteolytic patterns as measured by electrophoresis were also similar to those of standard cheeses, as well as the aroma of the products. Peptide profiles revealed that the areas of most peaks increased with ripening time. The proposed model showed to be suitable for the production of mini cheese specimens for laboratory testing of cultures and enzymes in similar conditions to their real environment in the food matrix.     

Dynamics and characterization of yeasts during ripening of typical Italian dry-cured ham

The evolution of the yeast population during manufacturing and ripening of dry-cured Parma ham was investigated. Contamination levels ranged from 10(5) to 10(7) cfu/g on muscle surface, 10(4) to 10(6) cfu/g on covering fat and exceeded 10(7) cfu/g on spreadable fat mince ("sugna"). Two hundred and sixty one yeast isolates underwent identification test, showing that the predominant species of yeast population during the whole maturing process were Debaryomyces hansenii, Candida zeylanoides, Debaryomyces maramus, and to a lesser extent, Candida famata and Hyphopichia burtonii. The species Candida catenulata, Candida guilliermondii, Candida edax and other genera like Cryptococcus and Wingea were occasionally found. The yeast counts and species distribution changed according to the stage of processing and to the ham sampling location. At the end of the cold phase, the washing procedure was effective in lowering the yeast count in muscle and fat surface layers, but during the next ageing stages, yeast colonization of unskinned ham muscle increased again, though species distribution changed if compared to previous manufacturing phases. The ripening steps taken into account from the end of the cold phase to the final outcome, were always characterized by more than one yeast species, suggesting that yeasts other than Debaryomyces spp. could play a remarkable role on the sensory and safety properties of typical Italian dry-cured ham.     

The occurrence and growth of microorganisms during the fermentation of fish sausage

Minced fish (mullet) sausage mixes containing added sugar, salt, nitrate, nitrite and spices were fermented (48 h, 30 degrees C) by indigenous flora or by a starter culture (Pediococcus acidilactici) and the microbial ecology and behaviour of various bacteria was monitored. Pediococcus pentosaceus and Lactobacillus plantarum dominated the indigenous fermentation, achieving populations of 10(7)-10(8) cfu/g by 48 h, and decreasing the pH of the mix to 4.5-4.7. Significant growth (10(5)-10(7) cfu/g) of Staphylococcus warneri, Staphylococcus aureus, Staphylococcus epidermidis. Micrococcus varians and Micrococcus luteus also occurred during this fermentation. Less growth was exhibited by Bacillus megaterium and yeasts. Pediococcus acidilactici dominated the fermentation when it was inoculated as a starter culture, but indigenous lactic acid bacteria (P. pentosaceus and L. plantarum) also grew to 10(7)-10(8) cfu/g. The growth of other bacteria and yeasts was restricted during fermentation with starter culture. Inoculated Escherichia coli, Salmonella typhimurium, Salmonella sofia, and Staphylococcus aureus grew to 10(6)-10(7) cfu/g in the sausage mix during indigenous fermentation. Lesser growth occurred for Bacillus cereus, Clostridium perfringens and Vibrio parahaemolyticus. Growth of these bacteria was significantly inhibited in sausage mix fermented with P. acidilactici.