Selective enumeration and survival of bifidobacteria in fresh cheese

Five species of bifidobacteria (15 strains), two strains of Lactococcus lactis ssp. lactis, two strains of L. lactis ssp. cremoris, and one strain of L. lactis ssp. lactis var diacetylactis were included in a study to develop a selective medium for enumeration of bifidobacteria from fresh cheese. Viable counts of bifidobacteria or lactococci on modified Columbia agar base (CAB with 0.05% cysteine-HCl) plus raffinose (0.5%) containing various selective agents were compared with non-selective media. The mCAB plus raffinose with lithium chloride (2 g L⁻¹) and sodium propionate (3 g L⁻¹) with pH adjusted to 5.1 was used successfully as a selective medium for the enumeration of bifidobacteria from fresh cheese. Using this medium, it was determined that bifidobacteria could survive up to 15 days at a level higher than 10⁶ cfu g⁻¹ in a fresh cheese stored at 4 or 12°C. The decrease in the viable counts of bifidobacteria was faster during storage at 4°C than at 12°C.     

Growth energetics of Lactococcus lactis subsp. lactis biovar diacetylactis in cometabolism of citrate and glucose

Certain strains of lactic acid bacteria present in commercial cheese starters, characterized by faint transparent colonies on an agar plate containing 1 mg kg ⁻¹ crystal violet (CVT), were identified as Lactococcus lactis subsp. (ssp) lactis biovar diacetylactis. The effect of citrate on the growth of these strains (CVT strains) in the presence of glucose was studied, in comparison with L. lactis strains. Molar growth yield from glucose (Y_G, g dry weight/mole of glucose consumed) for CVT strains grown on glucose plus citrate was significantly higher than the control (i.e. without citrate), but not for other L. lactis strains tested. Enhanced Y_G was also observed at a pH-controlled experiment, indicating that enhanced Y_G did not result from a buffering effect of citrate. CVT strains, in contrast to other strains of the same species, were shown to obtain enough energy to enhance Y_G on glucose–citrate mixtures.     

The diversity of potential cheese ripening characteristics of lactic acid starter bacteria: 1. Resistance to cell lysis and levels and cellular distribution of proteinase activities

By reference to subcellular fraction markers, the resistance to lysis of 23 strains of Lactococcus lactis subsp. cremoris, 30 strains of L. lactis subsp. lactis and five strains of Streptococcus thermophilus and the levels and distribution of proteinase activity in the strains were determined. Strains of L. lactis subsp. cremoris were readily lysed by transfer to hypotonic buffer after treatment with lysozyme alone, whilst strains of L. lactis subsp. lactis and S. thermophilus could be efficiently lysed in this way only after treatment with a combination of lysozyme and mutanolysin. With a few notable exceptions, those strains which gave the fastest rates of acid production also generally presented higher levels of cell surface proteinase, as determined by activity on fluorescein isothiocyanate-labelled β-casein. The highest levels of cell surface proteinase detected were found for strains of L. lactis subsp. cremoris. However, the levels of total proteinase activity in the lactococcal strains did not correlate with the rate of acid production in milk, some slow acid-producers yielding similar or greater total proteinase levels than fast acid-producers. Homology to DNA probes for the lactococcal cell surface proteinase gene and to the conserved region encoding the serine proteinase active site was shown by the fast acid-producing lactococcal strains, but not by most of the slow acid-producing lactococcal strains or by the strains of S. thermophilus. A significant proportion of the total proteinase activity was recovered in the subcellular fractions in which high levels of cytoplasmic marker enzyme activity were found. The total proteinase levels detected in strains ofL. lactis subsp. lactis showed a greater range of variation than in the strains of L. lactis subsp. cremoris. High levels of total proteinase activity were found in the slow acid-producers despite the strains having been grown in the presence of yeast extract. For many of the strains, the levels of proteinase released from the cell surface during cell wall degradation with lytic enzyme treatment were higher than those found using whole cells, suggesting that a significant amount of proteolytic activity was either inaccessible to substrate or present in an inactive form.     

Incorporation of Lactobacillus acidophilus in Minas fresh cheese and its implications for textural and sensorial properties during storage

The effect of Lactobacillus acidophilus on instrumental texture profile and related properties of Minas fresh cheese during storage at 5 °C and on sensory performance was investigated. Four cheese-making trials were prepared, two supplemented with a mesophilic type O culture (T1, T2) and two with lactic acid (T3, T4). L. acidophilus was added in T2 and T3. The viability of L. acidophilus, instrumental texture profile analysis and related properties were monitored during storage for up to 21 days. Probiotic cheeses T3 were firmer by the end of storage, due to higher values of pH and hardness. Differences detected were attributed to the starter, rather than to L. acidophilus. Viability of L. acidophilus during storage ranged from 6.04 to 6.93 for T2 and from 5.46 to 6.53 log cfu g⁻¹ for T3, which performed better in sensory evaluation. Minas fresh cheese is a suitable food system for the delivery of L. acidophilus.     

Phenotypic and genetic diversity of Lactococcus lactis and Enterococcus spp. strains isolated from Northern Spain starter-free farmhouse cheeses

To evaluate a previous phenotypic classification of lactococci, 39 presumed lactococcal strains were classified by molecular techniques. The strains were also subjected to several typing techniques to estimate the phenotypic and genetic diversity present in original populations from starter-free farmhouse cheeses. Partial Amplified rDNA Restriction Analysis (partial ARDRA) with either restriction enzyme MboII or HhaI divided these isolates into four distinctive groups. Sequencing of representative amplicons identified 29 isolates as belonging to Lactococcus lactis subsp. lactis (24) and Lactococcus lactis subsp. cremoris (5). The remaining 10 isolates were shown to be Enterococcus durans (8) and Enterococcus faecalis (2), which were misclassified by the traditional tests. Thus, partial ARDRA was successfully used to classify wild Lactococcus-like strains into Lactococcus and Enterococcus species. The technique also allowed differentiation of L. lactis strains at subspecies level. The 29 strains of L. lactis showed five different fermentation profiles, four distinct Random Amplification of Polymorphic DNA (RAPD) profiles, and 14 unrelated profiles by both Restriction Fragment Length Polymorphism analyzed by Pulsed Field Gel Electrophoresis (RFLP-PFGE) and Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Using the same techniques, the 10 enterococcal strains showed four fermentation profiles, four RADP, and six by RFLP-PFGE and SDS-PAGE, respectively. Several typing techniques, especially RFLP-PFGE and SDS-PAGE, revealed wide phenotypic and genetic variability in both the lactococcal and enterococcal isolates. Two simple, rapid and cheap techniques (partial ARDRA and SDS-PAGE) are proposed as reliable tools for the classification and typing of new lactococcal-like isolates.     

The diversity of potential cheese ripening characteristics of lactic acid starter bacteria: 2. The levels and subcellular distributions of peptidase and esterase activities

The levels and subcellular distributions of various peptidase and esterase activities in a range of lactococcal and Streptococcus thermophilus strains were investigated. There was no correlation between the levels of the enzymes in the different strains and the ability of the strains to produce acid when grown in milk. While considerable differences between individual strains were apparent, average levels of X-prolyldipeptidyl aminopeptidase, dipeptidase and tripeptidase were similar in the Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris strains studied, while that of lysylaminopeptidase (i.e. activity assayed using lysine p-nitroanilide as substrate) in the L. lactis subsp. cremoris strains was approximately double that in the L. lactis subsp. lactis strains. The average levels of lysylaminopeptidase and X-prolyldipeptidyl aminopeptidase in the S. thermophilus strains studied were similar to those in the L. lactis subsp. cremoris strains, while the average levels of dipeptidase and tripeptidase were considerably lower. All peptidases studied were recovered predominantly in the cytoplasmic fraction, although in a few strains there was some evidence to suggest that a part of the tripeptidase activity may be associated with cell structures comprising the particulate fraction. The levels of esterase activity in the strains were considerably different between strains. However, the average level of esterase activity detected in the two lactococcal subspecies was similar, while that in the S. thermophilus strains was more than double the lactococcal average. The subcellular distribution of the esterase in all strains studied showed that a significant proportion of the activity is located on the cell surface.     

Effect of the lactoperoxidase system on the activity of mesophilic cheese starter cultures in goat milk

The effect of goats’ milk lactoperoxidase (LP) system on the activity of commercially available mesophilic cheese starter cultures was investigated. The growth and acid production of the starter cultures were measured at 2 h intervals for 8 h in goats’ milk kept at 30°C. Most of the starter cultures examined were found to be sensitive to the LP system, but varied in their susceptibility to inhibition. The activity of the mixed starter cultures CHN11, CHN22, CHN19, DCC240 and Flora Danica Normal was strongly inhibited by the LP system. However, the mixed starter culture LL 50C showed resistance to the LP system. The single strain culture Lactococcus lactis subsp. lactis NCDO 605 was inhibited by the LP system. However, the cultures Lactococcus lactis subsp. diacetylactis NCDO 176 and Leuconostoc mesenteroides subsp. cremoris ATCC 33313 were insensitive to the LP system. The results of this study indicate the need for routine screening of starter cultures for resistance to the LP system before using them for cheesemaking from goats’ milk preserved by the LP system.     

Lactic acid starter and probiotic bacteria: a comparative “in vitro” study of probiotic characteristics and biological barrier resistance

Probiotic characteristics (deconjugation of bile salts, hydrophobicity and β-galactosidase activity) and the resistance to biological barriers (gastric juice and bile salts) of 24 strains of lactic acid starter bacteria (Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Lactococcus lactis) and 24 strains of probiotic bacteria (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus and bifidobacteria) were compared. Among the probiotic bacteria tested, Lactobacillus acidophilus was the most interesting species since it showed high values of resistance to gastric juice and bile, hydrophobicity and β-galactosidase and bile salts deconjugation activities. Bifidobacterium bifidum strains showed the same behavior, although the values of the parameters investigated were slightly lower than those obtained for Lactobacillus acidophilus. On the other hand, it was demonstrated that Lactobacillus delbrueckii subsp. bulgaricus was the lactic acid starter species with the best probiotic characteristics among the starter species assessed. It was resistant to gastric juice and bile, and showed high values for β-galactosidase activity. On the other hand, lactic acid starter bacteria showed hydrophobicity values similar to or higher than those obtained for the strains of the Lactobacillus casei . According to the results found, the total probiotic value of a fermented dairy product should take into account not only the intestinal probiotic cultures used in the formulation but also the probiotic contribution of the lactic acid starter microflora.     

Effect of some technological parameters on microbiological, chemical and sensory qualities of Civil cheese during ripening

The objective of this research was to determine the effects of different ripening methods [brine salting, dry salting, incorporating with Lor cheese (LR) and vacuum packaging] of Civil cheeses on its microbiological, chemical and sensory properties. Civil cheeses were analysed on the 2nd, 30th, 60th and 90th day of ripening. Chemical compositions of the cheeses were significantly different. While the highest dry matter and titratable acidity values were determined on dry salted cheeses, the highest fat and fat in dry matter contents were found in Civil cheese ripened together with LR. The water-soluble nitrogen and ripening index values were lower in cheese ripened incorporating with LR. Excessive proteolysis was not seen in any of cheese samples. The ripening in different methods affected microbiological and sensory properties of Civil cheese. Panellists preferred vacuum packaging and dry-salting cheeses compared to the other samples on the 90th day of ripening.     

Antimicrobial activity of neutralized extracellular culture filtrates of lactic acid bacteria isolated from a cultured Indian milk product (‘dahi’)

Neutralized extracellular culture filtrate obtained from isolates of Lactobacillus acidophilus, Lactobacillus delbruecki ssp. bulgaricus, Lactobacillus salivarius and Lactococcus lactis ssp. lactis from ‘dahi’ showed weal to moderate inhibition of Staphylococcus aureus, Bacillus cereus, Escherichia coli, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus laterosporus, Bacillus subtilis and Pseudomonas aeruginosa when tested by the diffusion agar well assay method. The effective minimum quantity of lactic culture filtrates required to obtain complete inhibition of an inoculum of 10³ cfu/ml of the bacteria tested was between 20 and 26% (vol/vol), as determined by the agar incorporation method. Neutralized extracellular culture filtrate of these lactic cultures added at a level of 10% in sterile, 10% reconstituted non-fat dry milk was able to either suppress or retard growth of selected bacterial cultures when incubated at 37°C for 24 h. This study indicated the antimicrobial activity of dahi and the potential of using neutralized extracellular culture filtrate of lactic acid bacteria in the biopreservation of foods.     

Accelerated ripening of low fat Ras cheese by attenuated lactobacilli cells

Thirteen Ras cheese were made from 4% fat raw milk; 3% raw and heat treated; 2% raw and heat treated milks in order to study the effect of freeze-shocked or heat-shocked L. casei NIH 334 or L. helveticus CNRZ 53 on the quality of the resultant cheeses. The soluble nitrogen, soluble tyrosine, soluble tryptophan, total volatile fatty acids, titratable acidity and organoleptic evaluation scores increased as ripening period progressed, while moisture decreased. Neither strain nor the heated lactobacilli had significant effects on moisture content of cheeses, while increasing their acidity. Cheeses with freeze-shocked L. casei or L. helveticus had higher titratable acidity than cheeses in which heat-shocked cells were added. However, cheeses added L. helveticus had higher acidity than those with L. casei. Ripening indices (soluble nitrogen, soluble tyrosine, soluble tryptophan and total volatile fatty acids) and organoleptic evaluation scores had similar trends. Cheeses with attenuated lactobacilli had higher ripening indices and cheese scores than cheeses without lactobacilli. Addition of either freeze-shocked L. casei or L. helveticus yielded cheeses having higher ripening indices and organoleptic scores than cheeses made with heat-shocked lactobacilli. The best cheeses were made from 3% fat milk heated to 70 °C, and containing freeze-shocked L. helveticus followed by cheeses made from 2% fat milk heated to 75 °C and containing freeze-shocked L. helveticus.     

Development of bioactive food packaging materials using immobilised bacteriocins lacticin 3147 and nisaplin

Immobilisation of the bacteriocins nisin and lacticin 3147 to packaging materials was investigated. Stability of both cellulose-based bioactive inserts and anti-microbial polyethylene/polyamide pouches was examined over time. Anti-microbial activity against the indicator strain Lactococcus lactis subsp. lactis HP, in addition to Listeria innocua DPC 1770 and Staphylococcus aureus MMPR3 was observed for all bacteriocin-adsorbed materials. Activity retention of the inserts showed an initial decrease in the first week of storage but remained stable for the remaining 3 months of the trial. However, adsorption of lacticin 3147 to plastic film was unsuccessful, nisin bound well and the resulting film maintained its activity for 3-month period, both at room temperature and under refrigeration. When applied to food systems, the anti-microbial packaging reduced the population of lactic acid bacteria in sliced cheese and ham stored in modified atmosphere packaging (MAP) at refrigeration temperatures, thus extending the shelf life. Nisin-adsorbed bioactive inserts reduced levels of Listeria innocua by ≥2 log units in both products, and Staphylococcus aureus by 1.5 log units in cheese, and 2.8 log units in ham. Similar reductions were observed in cheese vacuum-packaged in nisin-adsorbed pouches.     

Biogenic amines in Iranian white brine cheese: modelling and optimisation of processing factors

The simultaneous effects of processing factors such as ripening time (25–75 days), ripening temperature (4–14 °C) and brine concentration (10–13%) on biogenic amines content, proteolysis and sensory score of Iranian white brine cheese were studied, in 12 cheeses. Response surface methodology (RSM) was used to minimise biogenic amines content. At low level of ripening time, biogenic amines content decreased with increasing levels of brine concentration but at high level of ripening time, brine concentration had inverse effect. Ripening time showed quadratic effect on biogenic amines content. Based on biogenic amines content and sensory score, the optimum conditions were 13% brine and ripening at 9–14 °C for 43–65 days.     

Proteolysis in Manchego-type cheese salted by brine vacuum impregnation

A new salting procedure based on the brine vacuum impregnation of porous products was tested on Manchego-type cheese and compared with conventional brine immersion. Its effect on cheese proteolysis throughout a 90-d ripening period was determined. Three cheese regions were evaluated (the rind, the middle, and the internal regions). The parameters analyzed were total N, water-soluble N, soluble N in trichloroacetic acid and soluble N in phosphotungstic acid by using the Kjeldahl method, casein profile by urea-PAGE, and peptide profile of the water soluble nitrogen extract by reverse-phase HPLC. Free amino acid formation was monitored with a spectrophotometric method by using a Cd-ninhydrin reagent. Globally, proteolysis was significantly affected by ripening stage (increasing throughout all the maturation period studied) and cheese region (rind showed a proteolysis pattern different from the middle and internal regions). The salting procedure only affected cheese proteolysis in the rind, whereas conventional brine-salted cheeses showed lower proteolysis than vacuum-impregnated cheeses.     

Behaviour of Listeria monocytogenes in raw sausages (merguez) in presence of a bacteriocin-producing lactococcal strain as a protective culture

The effectiveness of a bacteriocin produced by Lactococcus lactis subsp. lactis M in reducing population level and growth of Listeria monocytogenes ATCC 7644 in fermented merguez sausage was examined. Two different formulas (with or without added nitrites) were assayed and predetermined numbers of Listeria (ca 10⁶ cfu g⁻¹) were added to sausage mixture. The effect of in situ production of the bacteriocin by Lactococcus lactis M on Listeria monocytogenes ATCC 7644 during fermentation and storage of merguez sausages at room (ca 22 °C) or at refrigeration (ca 7 °C) temperature was tested. Results indicated that counts of Listeria monocytogenes were decreased during fermentation of merguez samples fermented with either the bacteriocin-producing Lactococcus lactis M (Bac⁺) or a nonbacteriocin-producing Lactococcus lactis J (Bac⁻). However, reduction in Listeria cfu's was greater in samples fermented with the Bac⁺ than in those fermented with the Bac⁻ starter. In merguez sausage made without nitrites addition, the Bac⁺ starter induced further decrease in Listeria counts by 1.5 log cycles compared with that induced by the Bac⁻ starter. While in merguez samples with added nitrites (0.4%), the effect of the bacteriocin produced in situ was less important than in those made without nitrites addition.     

Effect of fermented milks on humoral immune response in mice

Eight groups of 20 CD-1 mice were fed for eight days with UHT milk fermented by one of the following bacteria: Bifidobacterium longum, Lactobacillus acidophilus, L. delbrueckii spp. bulgaricus, L. casei spp.rhamnous, L. helveticus, Lactococcus lactis spp. cremoris, Lactococcus lactis spp. lactis, Streptococcus salivarius spp. cremoris. No significant differences were observed in the serum IgG and IgA level, nor in the bronchoalveolar IgG level. Only mice fed milk fermented with L. delbrueckii spp. bulgaricus showed a significant increase (P < 0·05) in their bronchol-alveolar IgA level after eight days. The extent of proteolysis of the fermented milks was not correlated with the bronchoalveolar IgA level.     

The potential of immobilized cell technology to produce freeze-dried, phage-protected cultures of Lactococcus lactis

Lactococcus lactis cells were immobilized in calcium alginate beads and added to growth media to enable biomass production in the gel. The immobilized population was then freeze-dried. Survival during freeze-drying (FD), stability upon high-temperature storage and residual humidity were evaluated. This culture was compared to a classical liquid starter and fresh beads of immobilized L. lactis in simulated quarg manufacture. Acidifying characteristics, proteolytic activity, sensitivity to bacteriophage and sensory properties of quarg products were evaluated.

The population in the beads prior to FD was 7 × 10¹⁰ CFU/g. The best survival rate to FD (62–79%) was obtained when the beads were mixed with a milk-based protective solution. In the absence of protective ingredients (milk solids, sucrose and vitamin C) the alginate beads had high water content following FD. Survival of the immobilized cells during FD was as high for immobilized cells as that of free cells. Stability of the immobilized cells at high-temperature storage (45–55°C) was higher than for the free cells. Quarg cheese was succesfully produced in 5 h with the immobilized freeze-dried (IFD) culture, but sensory evaluation confirmed a significant texture difference between cheeses made with free or IFD cultures. Higher inoculation rates were required with the IFD culture to obtain the same acidifying activity as classical fresh liquid starters. The IFD culture performed well under phage contamination; following a 6-h fermentation, 30% of the cells remained viable and active in the phage-contaminated sample. Increase in non-protein amino compounds (0·2 g/100 g cheese) over a 30-day storage period at 4°C was similar in quarg cheeses made with fresh or IFD starters, despite the higher inoculation rate used with the IFD culture.     

In vitro adherence and other properties of lactobacilli used in probiotic yoghurt-like products

This study compared selected probiotic properties of 9 strains of the Lactobacillus acidophilus group (6 L. acidophilus and 3 L. johnsonii) and 9 strains of the L. casei group, isolated from probiotic dairy products sold on the German market or supplied by a producer of starter cultures. Using an in vitro system simulating gastric transit, strains of the L. acidophilus group were more tolerant to the low pH 2.0 than strains of L. paracasei and L. rhamnosus which rapidly lost their viability after exposure to the simulated gastric juice containing pepsin. Milk showed a protective effect on strains less resistant to the gastric acidity. Bile salt hydrolase activity was detected in all strains of L. acidophilus/L. johnsonii but not in strains of the L. casei group. All lactobacilli were able to adhere to mucus secreting HT29 MTX cells and to human collagen type IV, fibrinogen and fibronectin although differences in the percentage of adhering bacteria were observed between different strains.     

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

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