Volatile compounds and aroma of Hispánico cheese manufactured using lacticin 481-producing Lactococcus lactis subsp. lactis INIA 639 as an adjunct culture

Lacticin 481-producing Lactococcus lactis subsp. lactis INIA 639 (BP), bacteriocin-nonproducing L. lactis subsp. lactis INIA 437 (BNP), or a combination of both strains were used as mesophilic cultures for Hispánico cheese manufacture. A Lactobacillus helveticus (LH) culture of high aminopeptidase activity, sensitive to lacticin 481, was used as thermophilic culture. Three batches (BP+LH, BNP+LH, and BNP+BP+LH cheeses) were made in duplicate experiments. Ethanol, 1-propanol, and three ethyl esters reached their highest levels in BP+LH cheese, whereas acetic acid, five ketones, and two alkanes were at their maximum levels in BNP+LH cheese. Higher levels of acetaldehyde and three branched chain aldehydes were found in BNP+BP+LH cheese than in the other two cheeses. Aroma quality and aroma intensity scores were higher for BP+LH and BNP+BP+LH cheeses than for BNP+LH cheese.     

Outgrowth inhibition of Clostridium beijerinckii spores by a bacteriocin-producing lactic culture in ovine milk cheese

In the manufacture of model cheeses, ovine milk was deliberately contaminated with spores of Clostridium beijerinckii INIA 63, a wild isolate from Manchego cheese with late blowing defect, and inoculated with nisin- and lacticin 481-producing Lactococcus lactis subsp. lactis INIA 415 as starter, to test its potential to prevent the late blowing defect, or with L. lactis subsp. lactis INIA 415-2, a spontaneous mutant not producing bacteriocins. Cheeses made individually with the lactococcal strains, without clostridial spores, served as controls. Cheese made with clostridial spores and L. lactis subsp. lactis INIA 415-2 showed late blowing defect after 120 days of ripening. Spoilt cheese also showed lower concentrations of lactic acid, and higher levels of acetic, propionic and butyric acids, and of other volatile compounds such as 2-propanol and 1-butanol, than control cheese. In addition, cheese made with the bacteriocin producer did not show any late blowing symptoms, despite its spore counts similar to those of blown cheese, pointing to outgrowth inhibition of C. beijerinckii spores by bacteriocins. Besides, cheese made with the bacteriocin producer showed similar concentrations of lactic acid and volatile compounds than control cheese. Inclusion of L. lactis subsp. lactis INIA 415 in starter cultures seems a feasible method to prevent late blowing defect in cheese without altering its sensory characteristics.     

Effect of high-pressure treatment and a bacteriocin-producing lactic culture on the proteolysis, texture, and taste of Hispánico cheese

The effects of high-pressure treatment, by itself or in combination with a bacteriocin-producing culture added to milk, on the proteolysis, texture, and taste of Hispánico cheese were investigated. Two vats of cheese were manufactured from a mixture of cow and ewe milk. Milk in one vat was inoculated with 0.5% Lactococcus lactis ssp. lactis INIA 415, a nisin Z and lacticin 481 producer; 0.5% L. lactis ssp. lactis INIA 415-2, a bacteriocin-nonproducing mutant; and 2% of a commercial Streptococcus thermophilus culture. Milk in the other vat was inoculated with 1% L. lactis ssp. lactis INIA 415-2 and 2% S. thermophilus culture. After ripening for 15 d at 12°C, half of the cheeses from each vat were treated at 400 MPa for 5 min at 10°C. Ripening of high-pressure-treated and untreated cheeses continued at 12°C until d 50. High-pressure treatment of cheese made from milk without the bacteriocin producer accelerated casein degradation and increased the free AA content, but it did not significantly influence the taste quality or taste intensity of the cheese. Addition of the bacteriocin producer to milk lowered the ratio of hydrophobic peptides to hydrophilic peptides, increased the free AA content, and enhanced the taste intensity. The combination of milk inoculation with the bacteriocin producer and high-pressure treatment of the cheese resulted in higher levels of both hydrophobic and hydrophilic peptides but had no significant effect on the free AA content, taste quality, or taste intensity.     

Nisin and lacticin 481 coproduction by Lactococcus lactis strains isolated from raw ewes’ milk

Bacteriocin-producing lactococci were isolated from raw ewes’ milk samples obtained from 5 different Protected Designation of Origin Zamorano cheese manufacturers. Thirteen isolates with antimicrobial activity against Lactococcus lactis HP were selected. Eleven were identified by a PCR technique as L. lactis ssp. lactis and 2 were identified as L. lactis ssp. cremoris. They were grouped under 4 different pulsed-field gel electrophoresis patterns. The presence of structural genes of both nisin and lacticin 481 was detected in 10 L. lactis ssp. lactis isolates belonging to 2 different pulsed-field gel electrophoresis patterns. Coproduction of nisin and lacticin 481 was confirmed after semipurification by using selective indicators. The production of 2 bacteriocins by the same strain is an uncommon property, with relevance in food safety. Nisin and lacticin 481 L. lactis-producing strains might be used as adjunct cultures to the commercial starter in the manufacture of dairy products.     

Influence of a bacteriocin-producing lactic culture on proteolysis and texture of Hispánico cheese

Hispánico cheese was manufactured in duplicate experiments, each consisting of two 50-L vats, and ripened for 75 days. Lactic cultures for experimental cheese were 0.5% Lactococcus lactis subsp. lactis INIA 415 (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain), a bacteriocin-producing (Bac⁺) strain harbouring the structural genes of nisin Z and lacticin 481, 0.5% L. lactis subsp. lactis INIA 415-2, a Bac⁻ mutant and 2% TA052, a commercial Streptococcus thermophilus culture. Lactic cultures for control cheese were 1% L. lactis subsp. lactis INIA 415-2 and 2% TA052. S. thermophilus counts were lower, and levels of cell-free intracellular aminopeptidases higher, from day 1 in cheese made with the bacteriocin producer, indicating early lysis of the thermophilic culture. Experimental cheese showed reduced proteolysis of α_s-casein and lower levels of hydrophilic and hydrophobic peptides than control cheese. However, proteolysis as estimated by the o-phthaldialdehyde method and total level of free amino acids were in experimental cheese 1.38- and 2.47-fold, respectively, those in control cheese on day 25, and 1.49- and 2.34-fold, respectively, on day 75. Higher values of fracturability, elasticity and hardness were recorded from day 50 for cheese made with the bacteriocin producer, which were related to its higher residual α_s-casein content. The use of a bacteriocin-producing culture, though retarding α_s-casein proteolysis and softening of texture, enhanced considerably secondary proteolysis during cheese ripening.     

Lysis of starters in UF cheeses: Behaviour of mesophilic lactococci and thermophilic lactobacilli

The objective of this work was to study the autolytic behaviour of strains of mesophilic (Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris) and thermophilic lactic acid bacteria (Lactobacillus helveticus, Lb. delbrueckii subsp. lactis and Streptococcus thermophilus) in UF cheese. Cheeses were made from a UF-retentate (milk concentrated by a factor of 6) of microfiltered milk (0.8 μm pore size membrane) using the following starter systems: (1) single inocula of autolytic strains of L. lactis (US3, AM2 or AM1), non-autolytic strains of L. lactis (AM2-C or CNRZ-144), (2) a co-inocula of strains of Lb. helveticus (ITG-LH1, CNRZ-32 or CNRZ-303), Lb. delbrueckii subsp. lactis (ITG-LL14 or ITG-LL51) with the same strain of S. thermophilus CNRZ-1358. Cell viability was monitored over a 28 day ripening period by enumeration on selective media. Degree of lysis was determined by the measurement of the intracellular marker lactate dehydrogenase (LDH) activity, and also by immunodetection of intracellular proteins with species specific antibodies. In UF cheeses, lysis of autolytic strains of L. lactis was significantly delayed, showing release of intracellular components after 21 days of ripening. No lysis was observed for non-autolytic L. lactis strains or for S. thermophilus. Lysis of thermophilic lactobacilli (Lb. helveticus, Lb. delbrueckii), was observed from the start of ripening, but the onset and the level of lysis observed was strain and species dependent.     

An application in Gouda cheese manufacture for a strain of Lactobacillus helveticus ND01

Gouda cheese was manufactured with Lactococcus lactis ssp. lactis IMAU60010, L. lactis ssp. cremoris IMAU40136 and L. helveticus ND01 isolated from the naturally fermented milk in China. Starter cultures added with L. helveticus ND01 produced Gouda cheese with dramatically more proteolysis than control cheeses. Compared with control cheese, experimental cheese with L. helveticus ND01 adjunct revealed dramatic increase in both Angiotensin I‐converting enzyme (ACE)‐inhibitory activity and γ‐aminobutyric acid content. The ACE‐inhibitory activity of Gouda cheeses with the addition of 1 × 10⁵ CFU/mL L. helveticus ND01 increases from 53.7 to 83.1% at 6 weeks of ripening.     

Lactic acid bacteria with cholesterol-lowering properties for dairy applications: In vitro and in situ activity

Cholesterol-lowering activity is one of the most promising properties of lactic acid bacteria with probiotic characteristics. In the present study, 58 potentially probiotic lactic acid bacteria were tested for their ability to survive in vitro digestion and reduce cholesterol in a medium containing cholesterol and bile acids. The best-performing strains (Lactobacillus casei VC199, Lactobacillus paracasei ssp. paracasei SE160 and VC213, Lactobacillus plantarum VS166 and VS513, Enterococcus faecium VC223, and Enterococcus lactis BT161) resulted in a 42 to 55% reduction of the cholesterol level in broth and were further tested in cheese manufacture. The cholesterol content in all the cheeses decreased with ripening. All the strains were present in the cheese at levels higher than 10⁷ cfu/g until 60 d of ripening, the highest reductions (up to 23%) being obtained when Lb. paracasei ssp. paracasei VC213 and E. lactis BT161 were added during the cheese-making. The adjunct cultures had no negative effect on the sensory characteristics of the cheese. Thus, these strains with proven in vitro properties are good candidates for novel probiotic-containing formulations and could be used to functionalize foods such as dairy fermented products.     

Employment of autochthonous microflora in Pecorino Sardo cheese manufacturing and evolution of physicochemical parameters during ripening

The isolation and identification of lactic acid bacteria (LAB) from raw ewes’ milk and traditional Pecorino Sardo cheese made from this milk without the addition of starter culture was carried out to define the autochthonous lactic microflora present in milk and the evolution of LAB during cheese ripening. Isolation of 275 strains belonging to different Lactococcus, Lactobacillus, Streptococcus and Enterococcus species was achieved. Coccal-shaped LAB were found to predominate during cheese fermentation, while lactobacilli were preponderate during the latter phase of ripening. The technological selection of a total of 174 LAB strains belonging to the species Lactococcus lactis, Streptococcus thermophilus, Lactobacillus helveticus and Lb. casei allowed an experimental starter to be prepared, in which a potentially probiotic species, Lb. casei was used. The suitability of the autochthonous starter culture was tested in cheese-making trials, using thermised ewes’ milk, by comparing experimental Pecorino Sardo cheese with a control cheese produced at industrial scale using a whey starter culture from previous batches of manufacture. In particular, microbiological and physicochemical parameters were determined over 210 days of cheese ripening. Although sensory evaluation did not show any significant difference between experimental and control Pecorino Sardo cheeses, the use of the selected autochthonous starter allowed the production of experimental cheese with a significantly higher level of free amino acids, in particular essential amino acids, in comparison with the Pecorino Sardo control cheeses.     

Invited review: Lactobacillus helveticus—A thermophilic dairy starter related to gut bacteria

The strain Lactobacillus helveticus DPC4571 has emerged as a promising flavor adjunct culture for Cheddar cheese given that it is consistently associated with improved flavor. The availability of the complete genome sequence of Lb. helveticus DPC4571 has enabled the search for the presence or absence of specific genes on the genome, in particular those of technological interest. Indeed, this analysis has facilitated a greater understanding into the functioning of lactic acid bacteria as a whole. The biochemical pathways of Lb. helveticus responsible for producing flavor compounds during cheese ripening are poorly understood but now with the availability of a complete genomic sequence are ripe for exploitation. Bioinformatic analysis of the genome of Lb. helveticus DPC4571 has revealed a plethora of genes with industrial potential including those responsible for key metabolic functions that contribute to cheese flavor development such as proteolysis, lipolysis, and cell lysis. In addition, it has been demonstrated that Lb. helveticus has the potential to produce bioactive peptides such as angiotensin converting enzyme inhibitory activity in fermented dairy products, demonstrating the therapeutic value of this species. A most intriguing feature of the genome of Lb. helveticus DPC4571 is the remarkable similarity in gene content with many intestinal lactobacilli, although originating from considerably different environments. Bioinformatic analysis demonstrated that 65 to 75% of genes were conserved between the commensal and dairy lactobacilli, which allowed key niche-specific gene sets to be described. This review focuses on the isolation, characterization, and exploitation of the Lb. helveticus species with particular emphesis on taking into consideration recent genome sequence data for Lb. helveticus and other Lactobacillus species.     

Applicability of bacteriocin‐producing Lactobacillus plantarum,Enterococcus faecium and Lactococcus lactis ssp. lactis as adjunct starter in Minas Frescal cheesemaking

The antimicrobial and technological characteristics of three bacteriocinogenic cultures used as adjunct starters in Minas Frescal cheese were investigated. The cheeses were manufactured with 1% commercial lactic starter and 0.5%Lactococcus lactis ssp. lactis ATCC 11454, Lactobacillus plantarum ALC 01 or Enterococcus faecium FAIR‐E 198. The cheeses were then artificially inoculated with Listeria monocytogenes Scott A, Staphylococcus aureus ATCC 27154 and Bacillus cereus K1‐B041 and stored for 21 days at 8°C. The results show that there was no significant difference in the counts of L. monocytogenes and S. aureus between the cheeses made with added bacteriocinogenic cultures and the control cheese. On the other hand, B. cereus exhibited susceptibility to Lb. plantarum ALC 01 and E. faecium FAIR‐E 198 from the seventh day of storage. However, these cultures increased the proteolysis of the Minas Frescal cheese.     

Biochemical patterns in ovine cheese: Influence of probiotic strains

This study was undertaken to evaluate the effect of lamb rennet paste containing probiotic strains on proteolysis, lipolysis, and glycolysis of ovine cheese manufactured with starter cultures. Cheeses included control cheese made with rennet paste, cheese made with rennet paste containing Lactobacillus acidophilus culture (LA-5), and cheese made with rennet paste containing a mix of Bifidobacterium lactis (BB-12) and Bifidobacterium longum (BB-46). Cheeses were sampled at 1, 7, 15, and 30 d of ripening. Starter cultures coupled with probiotics strains contained in rennet paste affected the acidification and coagulation phases leading to the lowest pH in curd and cheese containing probiotics during ripening. As consequence, maturing cheese profiles were different among cheese treatments. Cheeses produced using rennet paste containing probiotics displayed higher percentages of α_S1-I-casein fraction than traditional cheese up to 15 d of ripening. This result could be an outcome of the greater hydrolysis of α-casein fraction, attributed to higher activity of the residual chymosin. Further evidence for this trend is available in chromatograms of water-soluble nitrogen fractions, which indicated a more complex profile in cheeses made using lamb paste containing probiotics versus traditional cheese. Differences can be observed for the peaks eluted in the highly hydrophobic zone being higher in cheeses containing probiotics. The proteolytic activity of probiotic bacteria led to increased accumulation of free amino acids. Their concentrations in cheese made with rennet paste containing Lb. acidophilus culture and cheese made with rennet paste containing a mix of B. lactis and B. longum were approximately 2.5 and 3.0 times higher, respectively, than in traditional cheese. Principal component analysis showed a more intense lipolysis in terms of both free fatty acids and conjugated linoleic acid content in probiotic cheeses; in particular, the lipolytic pattern of cheeses containing Lb. acidophilus is distinguished from the other cheeses on the basis of highest content of health-promoting molecules. The metabolic activity of the cheese microflora was also monitored by measuring acetic, lactic, and citric acids during cheese ripening. Cheese acceptability was expressed for color, smell, taste, and texture perceived during cheese consumption. Use of probiotics in trial cheeses did not adversely affect preference or acceptability; in fact, panelists scored probiotic cheeses higher in preference over traditional cheese, albeit not significantly.     

Influence of starters on chemical, biochemical, and sensory changes in Turkish White-brined cheese during ripening

Turkish White-brined cheese was manufactured using Lactococcus strains (Lactococcus lactis ssp. lactis NCDO763 plus L. lactis ssp. cremoris SK11 and L. lactis ssp. lactis UC317 plus L. lactis ssp. cremoris HP) or without a starter culture, and ripened for 90 d. It was found that the use of starters significantly influenced the physical, chemical, biochemical, and sensory properties of the cheeses. Chemical composition, pH, and sensory properties of cheeses made with starter were not affected by the different starter bacteria. The levels of soluble nitrogen fractions and urea-PAGE of the pH 4.6-insoluble fractions were found to be significantly different at various stages of ripening. Urea-PAGE patterns of the pH 4.6-insoluble fractions of the cheeses showed that considerable degradation of α_s1-casein occurred and that β-casein was more resistant to hydrolysis. The use of a starter culture significantly influenced the levels of 12% trichloroacetic acid-soluble nitrogen, 5% phosphotungstic acid-soluble nitrogen, free amino acids, total free fatty acids, and the peptide profiles (reverse phase-HPLC) of 70% (vol/vol) ethanol-soluble and insoluble fractions of the pH 4.6-soluble fraction of the cheeses. The levels of peptides in the cheeses increased during the ripening period. Principal component and hierarchical cluster analyses of electrophoretic and chromatographic results indicated that the cheeses were significantly different in terms of their peptide profiles and they were grouped based on the use and type of starter and stage of ripening. Levels of free amino acid in the cheeses differed; Leu, Glu, Phe, Lys, and Val were the most abundant amino acids. Nitrogen fractions, total free amino acids, total free fatty acids, and the levels of peptides resolved by reverse phase-HPLC increased during ripening. No significant differences were found between the sensory properties of cheeses made using a starter, but the cheese made without starter received lower scores than the cheeses made using a starter. It was found that the cheese made with strains NCDO763 plus SK11 had the best quality during ripening. It was concluded that the use of different starter bacteria caused significant differences in the quality of the cheese, and that each starter culture contributed to proteolysis to a different degree.     

Keto acid decarboxylase and keto acid dehydrogenase activity detected during the biosynthesis of flavor compound 3-methylbutanal by the nondairy adjunct culture Lactococcus lactis ssp. lactis F9

3-Methylbutanal is one of the primary substances that contribute to the nutty flavor in cheese. Lactococcus strains have been shown to have higher aminotransferase and α-keto acid decarboxylase activities compared with other microbes, indicating that they might form a higher amount of 3-methylbutanal by decarboxylation. Several dairy lactococcal strains have been successfully applied as adjunct cultures to increase the 3-methylbutanal content of cheese. Moreover, compared with dairy cultures, the nondairy lactococci are generally metabolically more diverse with more active AA-converting enzymes. Therefore, it might be appropriate to use nondairy lactococcal strains as adjunct cultures to enrich the 3-methylbutanal content of cheese. This study thereby aimed to select a nondairy Lactococcus strain that is highly productive of 3-methylbutanal, identify its biosynthetic pathway, and apply it to cheese manufacture. Twenty wild nondairy lactococci isolated from 5 kinds of Chinese traditional fermented products were identified using 16S rRNA sequence analysis and were found to belong to Lactococcus lactis ssp. lactis. The nondairy strains were then screened in vitro for their production of 3-methylbutanal and whether they met the criteria to become an adjunct culture for cheese. The L. lactis ssp. lactis F9, isolated from sour bamboo shoot, was selected because of its higher 3-methylbutanal production, suitable autolysis rate, and lower acid production. The enzymes involved in the catabolic pathway of leucine were then evaluated. Both α-keto acid decarboxylase (6.96 μmol/g per minute) and α-keto acid dehydrogenase (30.06 μmol/g per minute) activities were detected in nondairy L. lactis F9. Cheddar cheeses made with different F9 levels were ripened at 13°C and analyzed after 90 d by a combination of instrumental and sensory methods. The results showed that adding nondairy L. lactis F9 significantly increased 3-methylbutanal content and enhanced the nutty flavor of the cheese without impairing its textural properties. Thus, nondairy L. lactis F9 efficiently enhanced the biosynthesis of 3-methylbutanal in vitro and in manufactured cheese.     

Effects of exopolysaccharide-producing cultures on the viscoelastic properties of reduced-fat Cheddar cheese

The objective was to study the influence of different exopolysaccharide (EPS)-producing and nonproducing lactic cultures on the viscoelastic properties of reduced-fat Cheddar cheese. Changes in the viscoelastic properties were followed over a ripening period of 6 mo. Results showed that the elastic, viscous, and complex moduli were higher in reduced-fat cheeses made with EPS-nonproducing cultures than in full-fat cheese. No differences in the viscoelastic properties were found between young reduced-fat cheese made with a ropy strain of Lactococcus lactis ssp. cremoris (JFR1) and its full-fat counterpart. Interestingly, the changes in viscoelastic moduli in both full-fat cheese and reduced-fat cheese made with JFR1 during ripening followed the same pattern. Whereas the moduli increased during the first month of ripening in those 2 cheeses, a dramatic decrease was observed in all other cheeses. Slopes of the viscoelastic moduli as a function of frequency were lower in the full-fat than in reduced-fat cheeses. The creep test showed that fresh reduced-fat cheese made with JFR1 was less rigid and more deformable than that made with EPS-nonproducing cultures. The creep and recovery properties of young reduced-fat cheese made with JFR1 and the full-fat type were similar. No differences were found in the viscoelastic properties between reduced-fat cheese made with no EPS and those made with EPS-producing adjunct cultures of Streptococcus thermophilus. After 6 mo of ripening, cheeses made with EPS-producing cultures maintained lower elastic and viscous moduli than did those made with no EPS.     

Preliminary observations on proteolysis in Manchego cheese made with a defined-strain starter culture and adjunct starter (Lactobacillus plantarum) or a commercial starter

Different authors have demonstrated the potential of adding lactobacilli as adjunct cultures to pasteurized milk used in cheese manufacture. The aim of this work was to observe the effect of the use of a defined-strain starter culture and the addition of an adjunct culture (Lactobacillus plantarum) to cheesemilk in order to determine their effect on the ripening of Manchego cheese. Manchego cheeses were manufactured using one of the following starter culture systems: a defined starter consisting of Lactococcus lactis ssp. lactis and Leuconostoc mesenteroides ssp. dextranicum; a defined starter, as described above, and Lb. plantarum, which were isolated from a good quality Manchego cheese made from raw milk, or a commercial starter comprised of two strains of Lc. lactis. The cheeses were sampled at 15, 45, 90 and 150 d of ripening. Principal component analysis of peak heights of reversed-phase HPLC chromatograms of 70% (v/v) ethanol-insoluble and -soluble fractions distributed the samples according to the starter used in their manufacture. Quantitative differences in several peptides were evident between the three cheeses. Cheeses made with the defined-strain starters received higher scores for the flavour quality and intensity and for overall impression than the cheeses made with the commercial starter.     

The effect of sodium reduction with and without potassium chloride on the survival of Listeria monocytogenes in Cheddar cheese

Sodium chloride (NaCl) in cheese contributes to flavor and texture directly and by its effect on microbial and enzymatic activity. The salt-to-moisture ratio (S/M) is used to gauge if conditions for producing good-quality cheese have been met. Reductions in salt that deviate from the ideal S/M range could result in changing culture acidification profiles during cheese making. Lactococcus lactis ssp. lactis or Lc. lactis ssp. cremoris are both used as cultures in Cheddar cheese manufacture, but Lc. lactis ssp. lactis has a higher salt and pH tolerance than Lc. lactis ssp. cremoris. Both salt and pH are used to control growth and survival of Listeria monocytogenes and salts such as KCl are commonly used to replace the effects of NaCl in food when NaCl is reduced. The objectives of this project were to determine the effects of sodium reduction, KCl use, and the subspecies of Lc. lactis used on L. monocytogenes survival in stirred-curd Cheddar cheese. Cheese was manufactured with either Lc. lactis ssp. lactis or Lc. lactis ssp. cremoris. At the salting step, curd was divided and salted with a concentration targeted to produce a final cheese with 600 mg of sodium/100 g (control), 25% reduced sodium (450 mg of sodium/100 g; both with and without KCl), and low sodium (53% sodium reduction or 280 mg of sodium/100 g; both with and without KCl). Potassium chloride was added on a molar equivalent to the NaCl it replaced to maintain an equivalent S/M. Cheese was inoculated with a 5-strain cocktail of L. monocytogenes at different times during aging to simulate postprocessing contamination, and counts were monitored over 27 or 50 d, depending on incubation temperature (12 or 5°C, respectively). In cheese inoculated with 4 log₁₀ cfu of L. monocytogenes/g 2 wk after manufacture, viable counts declined by more than 3 log₁₀ cfu/g in all treatments over 60 d. When inoculated with 5 log₁₀ cfu/g at 3 mo of cheese age, L. monocytogenes counts in Cheddar cheese were also reduced during storage, but by less than 1.5 log₁₀ cfu/g after 50 d. However, cheese with a 50% reduction in sodium without KCl had higher counts than full-sodium cheese at the end of 50 d of incubation at 4°C when inoculated at 3 mo. When inoculated at 8 mo postmanufacture, this trend was only observed in 50% reduced sodium with KCl, for cheese manufactured with both cultures. This enhanced survival for 50% reduced-sodium cheese was not seen when a higher incubation temperature (12°C) was used when cheese was inoculated at 3 mo of age and monitored for 27 d (no difference in treatments was observed at this incubation temperature). In the event of postprocessing contamination during later stages of ripening, L. monocytogenes was capable of survival in Cheddar cheese regardless of which culture was used, whether or not sodium had been reduced by as much as 50% from standard concentrations, or if KCl had been added to maintain the effective S/M of full-sodium Cheddar cheese.     

Microbiological,compositional, biochemical and textural characterisation of Caciocavallo Pugliese cheese during ripening

A microbiological, compositional, biochemical and textural characterisation of the pasta filata Caciocavallo Pugliese cheese during ripening is reported. Fully ripened cheese contained a total of ca. log 8.0 cfu g⁻¹ mesophilic bacteria and ca. log 6.0 cfu g⁻¹ presumptive staphylococci, while the number of thermophilic and mesophilic rod and coccus lactic acid bacteria varied during ripening. A two-step RAPD-PCR protocol was used to differentiate biotypes. The natural whey starter was composed mainly of Lactobacillus delbrueckii, Lb. fermentum, Lb. gasseri, Lb. helveticus and Streptococcus thermophilus strains. After day 1 of ripening, Lb. delbrueckii became dominant and some strains of Enterococcus durans and E. faecalis appeared. Non-starter lactic acid bacteria, such as Lb. parabuchneri and Lb. paracasei subsp. paracasei formed a large part of the lactic microflora at 42 and 60 d of ripening. The level of pH 4.6-soluble nitrogen increased from the outer to the inner of the cheese and also increased in each section as ripening progressed, attaining values of 18–15%. Urea-PAGE electrophoresis showed that degradation of α_s1-casein was more rapid than that of β-casein throughout ripening and the rates at which both caseins were degraded greatly increased from the outside to the inside of the cheese. Based on the primary proteolysis products, both chymosin and plasmin appeared to be active. RP-HPLC profiles of the 70% ethanol-soluble, pH 4.6-soluble nitrogen, showed a large number of peaks, indicating a heterogeneous mixture of proteolytic products. There were both age- and section-related changes in the area of the different peptide peaks. Butyric (C4:0), caproic (C6:0), palmitic (C16:0) and oleic (C18:1) acids were the free fatty acids found at the highest concentrations. The level of short chain fatty acids (e.g., butyric and caproic) decreased from the middle and inner to outer sections of the cheese. Peptidase activity in the curd was pronounced, increased during ripening and varied with the cheese section. The greatest increase of the peptidase activity coincided with a change in the lactic microflora and with the prevalence of non-starter lactic acid bacteria. Microbial esterases were supposed to be active together with rennet paste. Little change in the firmness and fractures stress during maturation were found by textural analyses of the raw cheese. The flowability was similar to that of typical low-moisture Mozzarella cheese, while stretchability was lower. The heat-induced changes in phase angle of Caciocavallo Pugliese cheese indicated a phase transition from largely elastic rheological characteristics in unheated cheese to a more viscous and fluid character in melted cheese.