Lipolysis in probiotic and synbiotic cheese: The influence of probiotic bacteria, prebiotic compounds and ripening time on free fatty acid profiles

The influence of probiotic bacteria (Lactobacillus casei-01, Bifidobacterium lactis B94), prebiotic compounds (FOS and inulin) and ripening time (0-60 days) on the free fatty acid (FFA) profile of cheese, with special emphasis on the conjugated linoleic acid (CLA) content, was investigated. After 60 days of ripening, 10⁹-10¹⁰ cfu g⁻¹ cheese were recorded in both probiotic and synbiotic cheeses, despite harsh conditions of low pH values (4.1-5.1) and low moisture content (<30%, w/w). Increases in total FFA and CLA were observed throughout the ripening period, especially in synbiotic cheeses containing FOS and inulin (50:50) inoculated with B. lactis B94. The addition of FOS alone or combined with inulin did not significantly affect probiotic strain growth and viability during the ripening period; however, the advantage of the addition of prebiotic compounds in probiotic cheese manufacture is that it may allow the production of cheeses with improved performance as far as functional CLA compounds are concerned, as well as an improved nutritional quality reflected in a lower atherogenicity index.     

Survival of micro‐organisms and organic acid profile of probiotic Cheddar cheese from buffalo milk during accelerated ripening

The study aimed to assess the impact of ripening at elevated temperatures on the survival of probiotic micro‐organisms and production of organic acids in Cheddar cheese. Cheese was manufactured from buffalo milk using lactococci starters along with different probiotic bacteria (Lactobacillus acidophilus LA‐5, Bifidobacterium bifidum Bb‐11 and Bifidobacterium longum BB536) as adjunct cultures. The cheeses were ripened at 4–6 °C or 12–14 °C for 180 days and examined for composition, organic acids and microbial survival. The production of organic acids was accelerated at 12–14 °C when compared to normal ripening temperatures. The probiotic bacteria increased production of lactic and acetic acids, compared to cheese made with lactococci alone. The survival of the mesophilic starters was significantly (P < 0.05) reduced in all the cheese samples ripened at the higher temperature. However, the probiotic bacteria remained viable (>7.0 log₁₀ cfu/g) throughout the 180 days of ripening, irrespective of temperature. It was concluded that Cheddar containing additional probiotic cultures can effectively be ripened at elevated temperatures without any adverse effects.     

Assessment of the microbiological safety of salad vegetables and sauces from kebab take-away restaurants in the United Kingdom

The purpose of this study was to establish the microbiological safety of salad vegetables and sauces served in kebab take-away restaurants. Comparison with published microbiological guidelines revealed that 4.7% of 1213 salad vegetable samples were of unsatisfactory microbiological quality due to Escherichia coli and/or Staphylococcus aureus levels at ≥10² cfu g⁻¹. Another 0.3% of salad samples were of unacceptable quality due to S. aureus at ≥10⁴ cfu g⁻¹ (2 samples) or the presence of Salmonella Kentucky (1 sample). Cucumber was the most contaminated salad vegetable with regards to unsatisfactory levels of E. coli (6.0%) or S. aureus (4.5%). Five percent of 1208 sauce samples were of unsatisfactory microbiological quality due to E. coli, S. aureus at ≥10² cfu g⁻¹ and/or Bacillus cereus and other Bacillus spp. at ≥10⁴ cfu g⁻¹. A further 0.6% of sauce samples were of unacceptable quality due to Bacillus spp. (Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis) at ≥10⁵ cfu g⁻¹ or the presence of Salmonella Agbeni (1 sample). More samples of chilli sauce (8.7%) were of unsatisfactory or unacceptable microbiological quality than any other sauce types. The results emphasize the need for good hygiene practices in kebab take-away restaurants handling these types of ready-to-eat products.     

Influence of lamb rennet paste containing probiotic on proteolysis and rheological properties of pecorino cheese

Pecorino cheeses made from heat-treated ewes’ milk using traditional lamb rennet paste (RP), lamb rennet paste containing Lactobacillus acidophilus (LA-5; RPL), and lamb rennet paste containing a mix of Bifidobacterium lactis (BB-12) and Bifidobacterium longum (BB-46; RPB) were characterized for proteolytic and rheological features during ripening. Consumer acceptance of cheeses at 60 d of ripening was evaluated. Lactobacillus acidophilus and Bifidobacterium mix displayed counts of 8 log₁₀ cfu/g and 9 log₁₀ cfu/g, respectively, in cheese during ripening. The RPB cheese displayed a greater degradation of casein (CN) matrix carried out by the enzymes associated to both Bifidobacterium mix and endogenous lactic acid microflora, resulting in the highest values of non-CN N and water-soluble N and the highest amount of α_s-CN degradation products in cheese at 60 d of ripening. The RPL cheese displayed intermediate levels of lactic acid bacteria and of N fractions. The percentage of γ-CN in RP and RPL cheeses at 60 d was 2-fold higher than in the cheese curd of the same groups, whereas the mentioned parameter was 3-fold higher in RPB cheese than in the corresponding fresh curd according to its highest plasmin content. The lower hardness in RPB at the end of ripening could be ascribed to the greater proteolysis observed in cheese harboring the Bifidobacterium mix. Although differences in proteolytic patterns were found among treatments, there were no differences in smell and taste scores.     

Microbiological and physicochemical properties of dry‐cured neck inoculated with probiotic of Bifidobacterium animalis ssp. lactis BB‐12

The effect of inoculum level of Bifidobacterium animalis ssp. lactis BB‐12 probiotic strain and ripening period on the quality of dry‐cured neck was studied. The microbiological parameters (Enterobacteriaceae, LAB and TVC) and physicochemical attributes (pH value, a_w, TBARS index, colour) were determined directly after fermentation at 15 °C for 3 weeks, after 6 and 12 months of ripening at 4 °C. The highest LAB count and a lower pH value were found in the meat inoculated with probiotic strain at 6.6 log cfu g^?1 (B2) followed by inoculation with probiotic strain at 6.3 log cfu g^?1 (B1). Level of inoculation had not had an influence on water activity, TBARS index and total colour parameters. Changes of fat oxidation during half‐year of ripening were limited in probiotic meat samples compared to naturally fermented control meat (C). Based on the results, it can be concluded that the most favourable physicochemical and microbiological parameters of the dry‐cured neck were obtained after 6 months of ripening. At that time, the Bifidobacterium BB‐12 at both levels is a good potential starter for meat fermentation.     

Inhibitory effect of clove oil on Listeria monocytogenes in meat and cheese

The antilisteric activity of clove oil was examined in meat and cheese at both 30°C and 7°C. At concentrations of 0·5% and 1% clove oil restricted the growth of Listeria monocytogenes in the food items at both temperatures. The inhibitory activity of clove oil was more pronounced at a concentration of 1%.Listeria counts in treated samples were 1–3 log₁₀cfu g⁻¹less compared to controls at different intervals during storage. The results revealed the potential of clove oil as a natural preservative in meat and cheese.     

Behaviour of Escherichia coli strains during semi-hard and hard raw milk cheese production

The behaviour of two Escherichia coli strains, one with a high and the other with a low thermotolerance phenotype, was investigated during production and ripening of Swiss semi-hard and hard raw milk cheese. In semi-hard cheese, counts of E. coli increased during production, before a log-linear decrease occurred during ripening, with a faster rate of reduction in core than in rind samples, and faster reduction of the more heat-sensitive strain in rind samples. Nevertheless, at the end of semi-hard cheese ripening, E. coli were present at least at 1.3 log₁₀ cfu g⁻¹ in rind samples and remained detectable by enrichment of core samples. During the first day of hard cheese production, both E. coli strains were almost completely inactivated. Detection by enrichment was possible in one of twelve spiked cheeses after 16 weeks, indicating the potential of a thermotolerant E. coli strain to survive until the end of ripening.     

The fate of Listeria monocytogenes during the manufacture of Camembert-type cheese: A comparison between raw milk and milk treated with high hydrostatic pressure

Camembert-type cheese was produced from: raw bovine milk; raw milk inoculated with 2 or 4 log CFU/ml Listeria monocytogenes; raw milk inoculated with L. monocytogenes and subsequently pressure-treated at 500 MPa for 10 min at 20 °C; or uninoculated raw milk pressure-treated under these conditions. Cheeses produced from both pressure-treated milk and untreated milk had the typical composition, appearance and aroma of Camembert. Curd and cheese made from inoculated, untreated milk contained large numbers of L. monocytogenes throughout production. An initial inoculum of 1.95 log CFU/ml in milk increased to 4.52 log CFU/g in the curd and remained at a high level during ripening, with 3.85 log CFU/g in the final cheese. Pressure treatment inactivated L. monocytogenes in the raw milk at both inoculum levels and the pathogen was not detected in any of the final cheeses produced from pressure-treated milk. Therefore high pressure may be useful to inactivate L. monocytogenes in raw milk that is to be used for the production of soft, mould-ripened cheese.

Industrial relevance

This paper demonstrates the potential of high pressure (HP) for treatment of raw milk to be used in the manufacture of soft cheeses. HP treatment significantly reduced the level of Listeria monocytogenes in the raw milk and so allowed the production of safer non-thermally processed camembert-like soft cheese.     

Bacterial diversity of Darfiyeh, a Lebanese artisanal raw goat's milk cheese

In order to contribute to the preservation of the Lebanese dairy heritage, the aim of this study was to characterize the Darfiyeh cheese, a traditional variety made from raw goat's milk and ripened in goat's skin. Three independent batches of Darfiyeh production were analyzed after 20, 40 and 60 days of ripening. Mesophilic lactobacilli, thermophilic coccal-shaped lactic acid bacteria (LAB) and thermophilic lactobacilli were enumerated. In order to explore the Darfiyeh natural ecosystem, a combination of phenotypical and molecular approaches was applied. The latter included Polymerase Chain Reaction-temporal temperature gel electrophoresis (PCR-TTGE), classical PCR and quantitative PCR. These methods revealed the presence of Streptococcus thermophilus, Enterococcus faecium, Enterococcus durans, Enterococcus faecalis, Enterococcus malodoratus, group D Streptococcus sp., Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris, Lactobacillus plantarum, Lactobacillus curvatus, Staphylococcus haemolyticus, Escherichia coli, Clostridium sp./Eubacterium tenue. Real-time PCR enabled quantification of E. faecium, with a detection of 10⁷–10⁹ cfu g⁻¹ of product. The present molecular approaches combined with phenotypic method allowed describing the complex natural ecosystem of Darfiyeh, giving useful information for the preservation of Lebanese artisanal dairy products.     

Isomaltooligosaccharide increases the Lactobacillus rhamnosus viable count in Cheddar cheese

Five batches of Cheddar cheese were manufactured containing different levels of isomaltooligosaccharide (IMO) and a probiotic strain of Lactobacillus rhamnosus to study the effect of IMO on the survival of starter lactococci and probiotic micro‐organisms, on proteolytic patterns, cheese composition and sensory properties. The cheese was exposed to conditions simulating those found in the gastrointestinal tract to evaluate the survival of Lb. rhamnosus. Results demonstrated that the addition of Lb. rhamnosus and IMO did not affect the main compositional variables of Cheddar cheese. The counts of starter culture and probiotic organisms increased in cheese which contained Isomaltooligosaccharide (Batches 3, 4 and 5) more than in the control (Batches 1 and 2) during the fermentation. The probiotic counts in fresh cheese (B‐4) was 9.23 log₁₀ cfu/g which was more than one log cycle greater than in the control (B‐2). The probiotic counts remained above 8 log₁₀ cfu/g at the end of the manufacturing process. Primary proteolysis was not affected by the addition of probiotic bacteria and IMO, but the level of secondary proteolysis was slightly higher compared with the control group. The addition of IMO improved the texture and sensory quality of the cheese and the probiotic bacterium had the same effect. Under conditions that simulated the gastrointestinal tract, the probiotic bacteria in cheese (B‐4) exhibited good survival and remained above the recommended 6–7 log₁₀ cfu/g.     

Inhibition of Clostridium sporogenes growth in mascarpone cheese by co-inoculation with Streptococcus thermophilus under conditions of temperature abuse

The ability of a biological control system to inhibit the outgrowth of Clostridium sporogenes spores during storage of mascarpone cheese under temperature-abuse conditions was investigated. Challenge studies were carried out on mascarpone cheese artificially contaminated with spores of C. sporogenes (10 cfu g⁻¹), and with or without the coinoculum of a Streptococcus thermophilus strain (10⁵cfu g⁻¹). During storage at 4, 12, and 25°C, the outgrowth of clostridia spores, the growth of S. thermophilus, and the pH changes were evaluated at 10, 20, 30, and 40 days. In mascarpone cheese stored at 4° and 12°C, S. thermophilus and C. sporogenes did not show any growth. The initial pH (6·14) of the product also remained unchanged. During storage at 25°C S. thermophilus grew up to about 10⁷cfu g⁻¹after 10 days, resulting in a pH decrease of mascarpone cheese to values close to 4·5. The cell number decreased progressively during storage reaching values near to 10¹cfu g⁻¹after 40 days, whereas product acidity remained constant. C. sporogenes, when inoculated alone, also grew at 25°C. The cell number increased to levels of about 10⁷cfu g⁻¹after 20–40 days of storage according to the different mascarpone cheese lots used. No growth was found when C. sporogenes was co-inoculated in mascarpone cheese with S. thermophilus and stored at 25°C. The study on the behaviour of C. sporogenes, known as a non-toxigenic variant of Clostridium botulinum, allowed us to obtain useful information for setting up an effective biological control system to inhibit growth of the toxigenic species as well. The use of an additional barrier, besides refrigerated storage, may help to maintain the safety of mascarpone cheese in the event it was exposed to elevated temperatures.     

Changes in the Microflora of Valdeteja Raw Goat's Milk Cheese throughout Manufacturing and Ripening

Changes in the microbiological flora of Valdeteja, a ripened home-made raw goat's milk cheese produced in northwest Spain, were studied during the manufacture and ripening processes using the spiral plating method. High counts of all microbial groups were observed in milk (7.66, 7.57, 6.13, 6.91, 2.47, 5.18, 3.27 and 3.85 log₁₀ cfu/g for aerobic mesophilic bacteria, lactococci, lactobacilli, leuconostocs, enterococci, Enterobacteriaceae,Micrococcaceae and moulds and yeasts, respectively). Counts increased between 0.76 and 1.96 log units from milk to curd. Lactic acid bacteria (lactococci, lactobacilli and leuconostocs) were the dominant microorganisms throughout ripening. Lactococci dominated over the lactobacilli up to day 27 of ripening. Leuconostocs were stable from day 2 of ripening. The final levels of typical enterococci were 3.62 log₁₀ cfu/g. Aerobic mesophilic bacteria, Enterobacteriaceae andMicrococcaceae reached their maximum in curd and decreased significantly throughout ripening. The decrease ofEnterobacteriaceae was very marked, although they did not completely disappear at the end of the ripening process (2.74 log₁₀ cfu/g). Moulds and yeasts counts increased significantly during ripening, giving final levels of 6.09 log₁₀ cfu/g. A strong correlation (P<0.001) was found between pH and lactobacilli (R=−0.87), leuconostocs (R=−0.82) and moulds and yeasts (R=−0.74) and between a_w and various microbial groups (e.g. EnterobacteriaceaeR=0.83).     

Probiotic Cheddar cheese: Influence of ripening temperatures on survival of probiotic microorganisms, cheese composition and organic acid profiles

Bifidobacterium longum 1941, Bifidobacterium animalis subsp. lactis LAFTI^®B94 (B94), Lactobacillus casei 279, Lb. casei LAFTI^®L26 (L26), Lactobacillus acidophilus 4962 or Lb. acidophilus LAFTI^®L10 (L10) were used as an adjunct in the production of Cheddar cheeses which were ripened for 24 wk at 4 and 8 °C. Effects of ripening temperatures on survival of starter lactococci and probiotic microorganisms, pH and composition of cheeses and production of organic acids were examined. The counts of starter lactococci in cheeses produced with B. animalis B94, Lb. casei L26 or Lb. acidophilus 4962 ripened at 8 °C were significantly lower than those ripened at 4 °C (P < 0.05) at 24 wk. Probiotic microorganisms remained viable (>7.50 log₁₀ CFU/g) at the end of 24 wk and their viability was not affected by the ripening temperatures. There were significant effects of the type of probiotic microorganisms used, ripening time, ripening temperatures and their interactions on the concentration of lactic and acetic acids in the cheeses (P < 0.05). The acetic acid concentration in cheeses made with Bifidobacterium sp. or Lb. casei sp. was significantly higher than that of the control cheese (P < 0.05). Citric, propionic and succinic acids contents of the cheeses were not significantly affected by the type of probiotic microorganisms or ripening temperatures (P > 0.05).     

Effect of X-ray treatments on inoculated Escherichia coli O157: H7, Salmonella enterica, Shigella flexneri and Vibrio parahaemolyticus in ready-to-eat shrimp

This study was conducted to evaluate the inactivation effect of X-ray treatments on Escherichia coli O157: H7, Salmonella enteric (S. enterica), Shigella flexneri (S. flexneri) and Vibrio parahaemolyticus (V. parahaemolyticus) artificially inoculated in ready-to-eat (RTE) shrimp. A mixed culture of three strains of each tested pathogen was used to inoculate RTE shrimp. The shrimp samples were inoculated individually with selected pathogenic bacteria then aseptically placed in sterile plastic cups and air-dried at 22 °C for 30 min (to allow bacterial attachment) in the biosafety cabinet prior to X-ray treatments. The inoculated shrimp samples were then placed in sterilized bags and treated with 0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 2.0, 3.0 and 4.0 kGy X-ray at ambient temperature (22 °C and 60% relative humidity). Surviving bacterial populations were evaluated using a non-selective medium (TSA) with the appropriate selective medium overlay for each bacterium; CT-SMAC agar for E. coli O157: H7, XLD for S. enterica and S. flexneri and TCBS for V. parahaemolyticus. More than a 6 log CFU reduction of E. coli O157: H7, S. enterica, S. flexneri and V. parahaemolyticus was achieved with 2.0, 4.0, 3.0 and 3.0 kGy X-ray, respectively. Furthermore, treatment with 0.75 kGy X-ray significantly reduced the initial microflora on RTE shrimp samples from 3.8 ± 0.2 log CFU g⁻¹ to less than detectable limit (<1.0 log CFU g⁻¹).     

Effect of lactic acid on Listeria monocytogenes and Edwardsiella tarda attached to catfish skin

This study evaluated the use of lactic acid to decontaminate Listeria monocytogenes andEdwardsiella tarda attached to catfish skin with or without mucus. At the highest inoculum levels (10⁴–10⁵cfu skin⁻¹), lactic acid (0·5–2·0%) exposure for 10 min reduced counts of L. monocytogenes firmly attached to catfish skin by 0·9–>1·9 log₁₀cfu skin⁻¹and cells loosely attached by 2·7–>3·7 logs. Counts of E. tarda firmly attached to catfish skin were reduced by 0·9–>3·0 logs and cells loosely attached by 1·5–>3·5 logs. Overall bacterial numbers of lactic acid-treated cells that were firmly attached to skin with mucus were higher than on skin without mucus. Firmly attached L. monocytogenes was more resistant to lactic acid than was firmly attached E. tarda. Catfish skin mucus decreased the antimicrobial effect of lactic acid against attached L. monocytogenes and E. tarda.     

Effect of post-ripening processing on the histamine and histamine-producing bacteria contents of different cheeses

Histamine is the most active biogenic amine and the one most commonly involved in food-borne intoxications. In this work, the effect of the post-ripening processing (cutting, slicing and grating) of different types of cheese on their histamine and histamine-producing bacteria contents was analysed. The average histamine content detected in Emmental cheeses was 220.8 mg kg⁻¹; the highest concentrations (up to 734.1 mg kg⁻¹) were recorded in grated samples. Significantly more histamine-producing bacteria were detected by real-time quantitative PCR (RT-qPCR) in the post-ripening-processed samples than in entire cheeses in all the cheese types analysed. A good association was obtained between the number of histamine-producing bacteria determined by RT-qPCR and histamine concentrations as determined by HPLC. Results for the analysis of entire Emmental cheeses and their post-ripening processed products suggested that processing had an important influence on the presence of histamine in cheese.     

Effect of calcium alginate and resistant starch microencapsulation on the survival rate of Lactobacillus acidophilus La5 and sensory properties in Iranian white brined cheese

Two types of probiotic cheese, with free and microencapsulated bacteria, were manufactured in triplicate under the same conditions. The number of viable cells during 182 days of storage in refrigerated conditions was evaluated. The number of viable cells of Lactobacillus acidophilus was reduced significantly from day 28 to day 182 of storage period in both types of cheese, but reduction in the cheese containing free cells (5.1 ± 0.67 log cfu g⁻¹) was significantly p < 0.05 higher than the cheese containing microencapsulated cells (11.00 ± 0.58 log cfu g⁻¹). The results showed that, microencapsulation in calcium alginate gel and resistant starch was able to increase the survival rate of L. acidophilus La5 in Iranian white brined cheese after 6 months of storage.     

Release and identification of angiotensin-converting enzyme-inhibitory peptides as influenced by ripening temperatures and probiotic adjuncts in Cheddar cheeses

The aim of the study was to examine the release of angiotensin-converting enzyme (ACE)-inhibitory peptides in Cheddar cheeses made with starter lactococci and Bifidobacterium longum 1941, B. animalis subsp. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. casei LAFTI^® L26, Lb. acidophilus 4962 or Lb. acidophilusLAFTI^® L10 during ripening at 4 and 8 °C for 24 weeks. ACE-inhibitory activity of the cheeses was maximum at 24 weeks. Cheeses made with the addition of Lb. casei 279, Lb. casei LAFTI^® L26 or Lb. acidophilus LAFTI^® L10 had significantly higher (P < 0.05) ACE-inhibitory activity than those without any probiotic adjunct after 24 weeks at 4 and 8 °C. The IC₅₀ of cheeses ripened at 4 °C was not significantly different (P > 0.05) to that ripened at 8 °C. The lowest value of the IC₅₀ (0.13 mg mL⁻¹) and therefore the highest ACE-inhibitory activity corresponded to the cheese with the addition of Lb. acidophilus LAFTI^® L10. Several ACE-inhibitory peptides were identified as κ-CN (f 96–102), α_s1-CN (f 1–9), α_s1-CN (f 1–7), α_s1-CN (f 1–6), α_s1-CN (f 24–32) and β-CN (f 193–209). Most of the ACE-inhibitory peptides accumulated at the early stage of ripening, and as proteolysis proceeded, some of the peptides were hydrolyzed into smaller peptides.     

Repair and growth of heat- and freeze-injured Escherichia coli O157:H7 in selective enrichment broths

Two Escherichia coli O157:H7 strains, ATCC 35150 and 43894, were heat injured in a beef infusion at 53°C for 40 and 50 min, respectively (1· 5–2·0 log₁₀cfu ml⁻¹of injury) and freeze injured at −25°C for 30 days (1 log₁₀cfu ml⁻¹of injury) as determined by plating on MacConkey agar with 0·60% bile salts #3 (Mac-BS) as the selective medium and on Brain Heart Infusion agar (BHIA) as the non-selective medium. Repair of injury was measured in five selective enrichment broths [buffered peptone water supplemented with vancomycin, cefsulodin, and cefixime (BPW-VCC), modified EC broth with novobiocin (mEC+n), enterohaemorrhagic E. coli enrichment broth (EEB), double modified TSB (dmTSB), and BCM^®E. coli enrichment broth (BCM^®-EB)] versus TSB as the non-selective control broth over 3 h incubation at 37°C and 42°C. Repair was measured as the increase in cfu ml⁻¹enumerated on Mac-BS with time vs the total cfu ml⁻¹(injured and uninjured cells) enumerated on BHIA. In mEC+n, EEB, and dmTSB some death of both heat- and freeze-injured cells occurred immediately during the 3 h incubation (decrease on BHIA plates), and there was either minimal or no repair of the injured cells at both temperatures. Efficient repair of heat injury was obtained with both BPW-VCC and BCM^®-EB, but the latter produced a growth rate and final cell concentration closer to TSB. In freeze-injury repair however, BPW-VCC gave poor results while repair in BCM^®-EB was equal to TSB. Both BCM^®-EB and BPW-VCC inhibited the growth of all Gram-positive and a select number of Gram-negative bacteria tested. The ability of the selective enrichment broth BCM^®-EB to resuscitate heat- and freeze-injured E. coli O157:H7 efficiently within 3 h, warrants further testing with other types of stress in both artificially and naturally contaminated foods.