Fermentation of carbohydrates from cheese sources by non-starter lactic acid bacteria isolated from semi-hard Danish cheese

Carbohydrate fermentation of 45 isolates of non-starter lactic acid bacteria from Danish semi-hard cheeses was studied using BioScreen C equipment. Thirty-nine of the isolates were identified as Lactobacillus paracasei/casei/rhamnosus, 2 as Lb. curvatus and 4 as a new species, Lb. danicus, using ITS-PCR. A specially designed carbohydrate-restricted medium supplemented with one of nine carbohydrates was used to evaluate potential carbohydrate sources in cheese–milk-fat globule membrane (MFGM), glycomacropeptide (GMP), or lysed cells. Lb. paracasei strains grew well on the carbohydrates from MFGM, GMP and bacterial cell wall peptidoglycan. The highest growth rates were observed on N-acetylglucosamine (NAG) (0.32–0.56 h⁻¹) and the lowest on ribose (0.12–0.23 h⁻¹, if ribose was fermented at all). Lb. danicus strains grew better on carbohydrates from lysed bacterial cells than on carbohydrates from MFGM or GMP, and it was the only species with a shorter lag-phase on ribose or NAG after being pre-incubated on ribose.     

Effect of temperature on growth and metabolism of probiotic bacteria in milk

The growth and metabolism of six probiotic strains with documented health effects were studied in ultra-high temperature (UHT) treated milk supplemented with 0.5% (w/v) tryptone or 0.75% (w/v) fructose at different temperatures. The probiotic strains were Lactobacillus acidophilus La5, Lb. acidophilus 1748, Lb. johnsonii LA1, Lb. rhamnosus GG, Lb. reuteri SD 2112 and Bifidobacterium animalis BB12. Fermentation was followed for 48 h at 20, 30, 37 and 45 °C and the samples were analysed for pH, log cfu mL⁻¹, volatile compounds, organic acids and carbon dioxide. All six probiotic strains showed very different profiles of metabolites during fermentation, however, the two Lb. acidophilus strains were the most alike. All strains, except Lb. reuteri SD 2112, showed viable cell numbers above 6.5 log cfu mL⁻¹ after 48 h fermentation at 30, 37 and 45 °C. The probiotic strains produced different amounts of metabolic products according to temperature and fermentation time illustrating the importance of controlling these parameters.     

Identification of cultivable lactic acid bacteria isolated from Algerian raw goat's milk and evaluation of their technological properties

One hundred and fifty-eight strains of lactic acid bacteria isolated from Algerian raw goat's milk were identified and technologically characterized. Five genera were found: Lactobacillus (50.63%), Lactococcus (25.94%), Streptococcus (14.56%), Leuconostoc (7.59%) and Pediococcus (1.26%). The predominant species were Lactococcus lactis (32 strains), Streptococcus thermophilus (23 strains), Lactobacillus bulgaricus (19 strains), Lb. helveticus (16 strains) and Lb. plantarum (14 strains).Approximately 39% of the lactic acid bacteria isolated produced more than 0.6% lactic acid (w/v) after 18 h of incubation, and belonged to the Lactococcus and Lactobacillus genera. The highest proteolytic activity was approximately 3 mg tyrosine l⁻¹ for mesophilic strains and nearly 5 mg tyrosine l⁻¹ for thermophilic lactobacilli after 72 h. High aromatic activity (more than 0.8 mg diacetyl l⁻¹ after 16 h) was detected in 14% of the strains.Nine strains were used to make dairy products (a yoghurt-like product and Edam-type cheese) on a pilot scale in the laboratory. The best-liked organoleptic characteristics were noted in a yoghurt produced with a mixed culture made up of S. thermophilus (strain 16TMC+) and Lb. helveticus (strain 20TMC) and in a cheese made with a starter composed of Lc. lactis subsp. lactis (strain 10MCM) and L. lactis subsp. lactis (V.P. +) (strain 19MCM).     

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.     

Effective valorisation of distillery stillage by integrated production of lactic acid and high quality feed

Utilization of distillery stillage from bioethanol production for lactic acid and feed production was studied. The lactic acid fermentation of the stillage was performed by Lactobacillus rhamnosus ATCC 7469 and maximal lactic acid concentration of 50.18 g L^− 1, yield of 0.90 g g^− 1, productivity of 1.48 g L^− 1 h^− 1 and viable cell number of 5 × 10⁹ CFU mL^− 1 were achieved. Solid residues with biomass remains after lactic acid fermentation were assessed for animal consumption. The content of proteins and ash decreased in the residues after the fermentation, whilst the content of oil and nitrogen free extract was higher when compared to unfermented samples. The digestible (17480.64 kJ kg^− 1) and metabolisable (17389.08 kJ kg^− 1) energies as well as digestibility (966.95 g kg^− 1) of the fermentation residue were very high. The in vitro assessment of L. rhamnosus ATCC 7469 survival in simulated gastric conditions has shown high survival rate (87%). In addition, this bacterium has shown good antimicrobial activity against the most important pathogens and capability to produce exopolysaccharide on different sugars present in animal diet. After effective lactic acid fermentation, the residues could be recommended as a high quality feed for monogastric animals.     

Mesophilic lactobacilli in Fiore Sardo cheese: PCR-identification and evolution during cheese ripening

The mesophilic lactobacilli colonizing Fiore Sardo ewe's milk cheese were characterized. They seemed to be the dominant non-starter lactic acid bacteria composing its natural microflora, with a viable cell number varying from 10⁵ CFU g⁻¹ (1-day-old cheese) to 10⁸ CFU g⁻¹ (30-day-old cheese) and then slowly decreasing up to 10⁴ CFU g⁻¹ after 7 months’ ripening. Considering the relevance of mesophilic lactobacilli in affecting the cheese ripening, a PCR-based taxonomic identification of the Lactobacillus species isolated was performed. Cheese samples were collected from 3 farms and 457 isolates from cheeses at different ripening times were analysed with species-specific primers for L. plantarum, L. casei group, L. paracasei, L. casei, L. rhamnosus, L. pentosus, L. paraplantarum, L. curvatus, L. graminis and L. sake. L. plantarum and L. paracasei were the most frequently detected species. Moreover, the development and the evolution during ripening of the facultatively heterofermentative Lactobacillus species (FHL) were different in the three batches of cheese.     

Heat resistance of Lactobacillus paracasei isolated from semi-hard cheese made of pasteurised milk

Nine strains of non-starter Lactobacillus paracasei isolated from semi-hard cheese made of pasteurised milk were selected for their anticlostridial activity. Resistance to thermisation (60 °C, 5 min) and pasteurisation (73 °C, 15 s) was investigated using a submerged-coil apparatus. MRS broth-grown cultures of all nine strains survived thermisation in buffer. The level of resistance to thermisation was strain dependent and lower for freshly grown cells (stationary phase cells) than for resting cells (freshly grown cells kept diluted 10-fold in MRS broth at 17 °C for 6 days). None of the nine Lb. paracasei strains survived or recovered after pasteurisation in buffer when grown in MRS broth, while seven of the nine strains survived pasteurisation in UHT whole milk when grown in milk. Identity of the strains was successfully confirmed during the experiments using repetitive-PCR analysis. The potential of Lb. paracasei strains to survive pasteurisation of cheese milk was demonstrated.     

Milk fermentation by functional mixed culture producing nisin Z and exopolysaccharides in a fresh cheese model

A nisin Z-producing strain, Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 and two nisin-sensitive cultures, Lactobacillus rhamnosus RW-9595 M producing exopolysaccharide (EPS), and Lc. lactis subsp. cremoris for acidification, were tested in pure and mixed cultures during milk fermentation. The mixed culture of the three strains showed a higher acidifying capacity at 34°C and 38°C, even though populations of Lc. cremoris were largely reduced compared with pure cultures. Bacteriocin production was 3.1–4.6-fold higher in mixed cultures than for pure cultures of Lc. diacetylactis UL719. These data can be explained by commensalism behavior relying on high proteolytic activity of Lc. cremoris and autolysis and nisin Z-induced lysis. In mixed culture, EPS production was 3-fold lower than for Lb. rhamnosus RW-9595 M pure culture. Our data showed that this strain combination, with nisin-producing and sensitive strains, can be used in mixed cultures for manufacture of fresh cheese with improved functional properties.     

Nonstarter lactic acid bacteria biofilms and calcium lactate crystals in Cheddar cheese

A sanitized cheese plant was swabbed for the presence of nonstarter lactic acid bacteria (NSLAB) biofilms. Swabs were analyzed to determine the sources and microorganisms responsible for contamination. In pilot plant experiments, cheese vats filled with standard cheese milk (lactose:protein = 1.47) and ultrafiltered cheese milk (lactose:protein = 1.23) were inoculated with Lactococcus lactis ssp. cremoris starter culture (8 log cfu/mL) with or without Lactobacillus curvatus or Pediococci acidilactici as adjunct cultures (2 log cfu/mL). Cheddar cheeses were aged at 7.2 or 10°C for 168 d. The raw milk silo, ultrafiltration unit, cheddaring belt, and cheese tower had NSLAB biofilms ranging from 2 to 4 log cfu/100 cm². The population of Lb. curvatus reached 8 log cfu/g, whereas P. acidilactici reached 7 log cfu/g of experimental Cheddar cheese in 14 d. Higher NSLAB counts were observed in the first 14 d of aging in cheese stored at 10°C compared with that stored at 7.2°C. However, microbial counts decreased more quickly in Cheddar cheeses aged at 10°C compared with 7.2°C after 28 d. In cheeses without specific adjunct cultures (Lb. curvatus or P. acidilactici), calcium lactate crystals were not observed within 168 d. However, crystals were observed after only 56 d in cheeses containing Lb. curvatus, which also had increased concentration of d(−)-lactic acid compared with control cheeses. Our research shows that low levels of contamination with certain NSLAB can result in calcium lactate crystals, regardless of lactose:protein ratio.     

Characterization of bacteriocin ST8KF produced by a kefir isolate Lactobacillus plantarum ST8KF

Lactobacillus plantarum ST8KF, isolated from kefir, produced a 3.5 kDa bacteriocin (bacST8KF) active against Lb. casei, Lb. salivarius, Lb. curvatus and Listeria innocua. BacST8KF was sensitive to proteolytic enzymes, but stable between pH 2.0 and 10.0, and heat resistant (20 min at 121 °C). BacST8KF did not adsorb to the surface of the producer cell. Maximum activity (25,600 AU mL⁻¹) was recorded in MRS broth with glucose, in MRS broth with glucose replaced by sucrose, and in MRS broth with glucose, supplemented with KH₂PO₄ after 24 h at 30 °C. Tri-ammonium citrate and glycerol in excess of 5.0 g L⁻¹ repressed bacST8KF production. Production of bacST8KF increased from 800 AU mL⁻¹ after 3 h of fermentation in MRS broth at 30 °C to 12,800 AU mL⁻¹ after 9 h and to 51,200 AU mL⁻¹ after 27 h. These results suggest that bacST8KF may be a secondary metabolite and shows that its mode of activity is bacteriostatic.     

Production of organic acids by Lactobacillus strains in three different media

Ten strains of Lactobacillus (Lb). casei, Lb. rhamnosus, Lb. plantarum, Lb. paracasei and Lb. curvatus species were chosen to determine the production of organic acids after cultivation in skimmed milk, MRS broth and Jerusalem artichoke (JA) medium. The highest acidity was obtained in MRS broth and the weakest acidification was found in skimmed milk. Lb. casei Shirota produced the highest amount and Lb. rhamnosus VT1 the lowest amount of substances being estimated as titratable acidity. All strains produced lactic acid in the investigated broth and most of the strains produced acetic acid in MRS broth except Lb. curvatus 2768 and Lb. casei Shirota, in JA broth except Lb. paracasei SF1 and in skimmed milk except Lb. casei 2750, Lb. curvatus 2768, Lb. curvatus 2775 and Lb. casei Shirota. All strains, except Lb. plantarum 01, produced butyric acid in MRS broth. Beside the lactic and acetic acids, formic, citric, succinic and glutamic acids were also produced in MRS broth; formic and succinic acids were produced in skimmed milk and succinic acid in JA broth. Some strains showed change in their fermentation profile from homofermentative to mix-acid fermentation in milk. The antifungal efficiency of the lactic and acetic acid in the amount produced by lactobacilli was investigated. None of the investigated aspergilli were inhibited. The inhibitory effect of acids against Fusarium increased unequivocally with the increasing concentration. The study pointed at the dissimilarity of organic acid production of Lactobacillus strains, which was considerably influenced by the media.     

Variability of the species and strain phenotype composition of the non-starter lactic acid bacterial population of cheddar cheese manufactured in a commercial creamery

The non-starter lactic acid bacteria (NSLAB) present in cheddar cheese manufactured in a commercial creamery was monitored phenotypically to the strain level over a period of 12 months to examine the effects of maturity status and manufacturing practices on the composition of the population. Five Lactobacillus spp. and Leuconostoc lactis were identified among the 459 isolates selected. The predominant NSLAB, Lactobacillus paracasei and Lactobacillus brevis, were present in 59 and 31% of the cheeses examined and represented 52.7 and 25.8%, respectively, of the isolates identified. Among the NSLAB screened 71 different phenotypic profiles were identified and these included 26 biotypes of Lb. paracasei, 14 Lb. brevis, 11 Lactobacillus plantarum, 10 Lactobacillus curvatus and 7 Leuc. lactis. The average number of strains recovered from a cheese was 3.9±2.1 and ranged from 1 to 11. Although approximately 70% of the cheese samples were dominated by three or less strains the NSLAB populations were heterogeneous and the majority (61.5%) were comprised of four or more strains of one or more species. Only 30 of the biotypes were recovered from more than one population. There was no evidence for the repeated recurrence of any of the strains isolated although some of the Lb. paracasei strains were present intermittently in cheeses throughout the 12-month manufacturing period. Six Lb. brevis strains also recurred in some of the cheeses produced in a limited period during the autumn. Pronounced shifts in the species complement and strain profile occurred during maturation, while the average number of strains present in the cheese decreased with increasing maturity. Microbiological examination of the NSLAB population of cheese either produced in different vats during the same production run or manufactured in the same vat but in different production runs (vat fills) indicated that the number of strains common to paired samples from two vats or a single vat in successive production runs was only 1.7±1.4 and 1.5±1.2, respectively, and confirmed the inherent variability that exists, both within and between production runs, in the non-starter population of cheese manufactured in a commercial creamery.     

Proteolytic pattern and organic acid profiles of probiotic Cheddar cheese as influenced by probiotic strains of Lactobacillus acidophilus,Lb. paracasei,Lb. casei or Bifidobacterium sp.

Cheddar cheeses were produced with starter lactococci and Bifidobacterium longum 1941, B. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. paracasei LAFTI^® L26, Lb. acidophilus 4962 or Lb. acidophilus LAFTI^® L10 to study the survival of the probiotic bacteria and the influence of these organisms on proteolytic patterns and production of organic acid during ripening period of 6 months at 4 °C. All probiotic adjuncts survived the manufacturing process of Cheddar cheese at high levels without alteration to the cheese-making process. After 6 months of ripening, cheeses maintained the level of probiotic organisms at >8.0 log₁₀ cfu g⁻¹ with minimal effect on moisture, fat, protein and salt content. Acetic acid concentration was higher in cheeses with B. longum 1941, B. lactis LAFTI^® B94, Lb. casei 279 and Lb. paracasei LAFTI^® L26. Each probiotic organism influenced the proteolytic pattern of Cheddar cheese in different ways. Lb. casei 279 and Lb. paracasei LAFTI^® L26 showed higher hydrolysis of casein. Higher concentrations of free amino acids (FAAs) were found in all probiotic cheeses. Although Bifidobacterium sp. was found to be weakly proteolytic, cheeses with the addition of those strains had highest concentration of FAAs. These data thus suggested that Lb. acidophilus 4962, Lb. casei 279, B. longum 1941, Lb. acidophilus LAFTI^® L10, Lb. paracasei LAFTI^® L26 and B. lactis LAFTI^® B94 can be applied successfully in Cheddar cheese.     

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

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

Diversity and probiotic potentials of lactic acid bacteria isolated from gilaburu,a traditional Turkish fermented European cranberrybush (Viburnum opulus L.) fruit drink

The aim of the present study was to characterize lactic acid bacteria (LAB) strains isolated from traditional fermented gilaburu fruit juice and their probiotic potential. The LAB counts of the fermented gilaburu fruit juice were in the range of 3.92–8.30 log cfu/g. Total of 332 isolates belonging to Lactobacillus and Leuconostoc species were characterized from traditional fermented gilaburu juice by genotypic methods. It was also determined that the major LAB strains belong to Lactobacillus plantarum (173 isolates), Lactobacillus casei (52 isolates) and Lactobacillus brevis (24 isolates), while Lactobacillus buchneri, Lactobacillus parabuchneri, Lactobacillus pantheris, Leuconostoc pseudomesenteroides and Lactobacillus harbinensis were the least in isolated LAB strains. In terms of the probiotic potentials, Lb. plantarum strains were able to grow at pH 2.5, but 3 of Lb. casei strains, one of each Lb. brevis and Lb. buchneri strains could not grow at the same pH. All selected LAB stains were resistant to bile salt at ≤ 0.3% concentration. While all the LAB species grew at 15 °C, two Lactobacillus hordei strains could also grow at 45 °C. The highest cell hydrophobicity degrees were for Lb. casei (G20a) and Lb. plantarum (G19e) as 87.5 and 86.0%, respectively. Listeria monocytogenes and Bacillus cereus were the most sensitive bacteria against the selected LAB strains, while Escherichia coli and Staphylococcus aureus were the most resistant. Again all the isolated LAB species were resistant to three antibiotics; kanamycin, streptomycin and vancomycin. Characterization and probiotic potentials of the LAB isolated from fermented gilaburu (Viburnum opulus) juice were studied first time, and further research needs to be done on their behaviors in similar food formulations as a probiotic.     

Evolution of lipolysis during the ripening of traditional Feta cheese

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

Optimization of the associative growth of novel yoghurt cultures in the production of biomass, β-galactosidase and lactic acid using response surface methodology

The associative growth of Streptococcus thermophilus 95/2 (St 95/2) and Lactobacillus delbrueckii ssp. bulgaricus 77 (Lb 77) isolated from the Toros mountain region of Turkey was investigated with respect to lactic acid, biomass and β-galactosidase enzyme production using response surface methodology (RSM). The ratio (St 95/2:Lb 77) of the strains and media formulation had significant effect on all responses (p < 0.001). The predicted enzyme activity (2.14 U mL^?1), lactic acid (22.50 g L^?1) and biomass (7.11 g L^?1) production at optimum conditions were very close to the actual experimental values (2.14 U mL^?1, 22.94 g L^?1 and 7.86 g L^?1, respectively). The optimum conditions were to use these cultures in a ratio of 1.66:1.62 (St 95/2:Lb 77) in a medium containing whey (5%), corn steep liquor (4%), potassium phosphate (2%) and peptone (2%) at 43 °C for 8 h. The associative growth provided 6.4% and 39% more β-galactosidase activity and 8.73% and 44% more lactic acid compared with the results obtained using pure St 95/2 and Lb 77 strains, respectively.     

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

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

Extension of Tosèla cheese shelf-life using non-starter lactic acid bacteria

Six strains of non-starter lactic acid bacteria (NSLAB) were used to extend the shelf-life of the fresh cheese Tosèla manufactured with pasteurised cows’ milk. The acidification kinetics of three Lactobacillus paracasei, one Lactobacillus rhamnosus and two Streptococcus macedonicus were studied in synthetic milk medium. Lb. paracasei NdP78 and NdP88 and S. macedonicus NdP1 and PB14-1 showed an interesting acidifying capacity and were further characterised for growth in UHT milk and production of antimicrobial compounds. Lb. paracasei NdP78 and S. macedonicus NdP1 grew more than 2 log cycles in 6 h. Lb. paracasei NdP78 was also found to produce a bacteriocin-like inhibitory substance (BLIS) active against Listeria monocytogenes. The four NSLAB strains (singly or in combination) were used to produce experimental pilot-scale cheeses which were compared by a panel. The cheese manufactured with the mixed culture Lb. paracasei NdP78 - S. macedonicus NdP1 was the most appreciated for its sensory properties. The cheeses produced at factory-scale showed higher concentrations of lactobacilli (7.90 log CFU/g) and streptococci (6.10 log CFU/g), but a lower development of coliforms (3.10 log CFU/g) and staphylococci (2.78 log CFU/g) than control cheese (4.86, 4.89, 4.93 and 5.00 log CFU/g of lactobacilli, streptococci, coliforms and staphylococci, respectively) processed without NSLAB addition. The food pathogens Salmonella spp. and Listeria monocytogenes were never detected. The dominance of the species inoculated was demonstrated by denaturing gradient gel electrophoresis (DGGE), whereas strain recognition was evaluated by randomly amplified polymorphic DNA (RAPD)-PCR. From the results obtained, Lb. paracasei NdP78 and S. macedonicus NdP1 were able to persist during the storage of Tosèla cheese and their combination influenced positively the sensory characteristics and shelf-life of the final product.