Angiotensin-converting enzyme inhibitory activity of milk fermented by wild and industrial Lactococcus lactis strains

Angiotensin I-converting enzyme inhibitory (ACEI) activity was evaluated and compared in <3 KDa water-soluble extracts (WSE) isolated from milk fermented by wild and commercial starter culture Lactococcus lactis strains after 48 h of incubation. The highest ACEI activities were found in WSE from milk inoculated with wild L. lactis strains isolated from artisanal dairy products and commercial starter cultures. On the other hand, the lowest ACEI activities were found in WSE from milk inoculated with wild strains isolated from vegetables. Moreover, the IC₅₀ values (concentration that inhibits 50% activity) of WSE from artisanal dairy products were the lowest, indicating that these fractions were the most effective in inhibiting 50% of ACE activity. In fact, a strain isolated from artisanal cheese presented the lowest IC₅₀ (13 μg/mL). Thus, it appears that wild L. lactis strains isolated from artisanal dairy products and commercial starter cultures showed good potential for the production of fermented dairy products with ACEI properties.     

Kluyveromyces lactis but not Pichia fermentans used as adjunct culture modifies the olfactory profiles of Cantalet cheese

Yeasts are commonly detected in cheese. Two yeast species, Kluyveromyces lactis and Pichia fermentans, were isolated at high populations from raw-milk Cantal cheese, a French Protected Denomination of Origin hard cheese. To investigate the interest of these 2 species as adjunct cultures to promote flavor development of Cantalet cheese, they were added at 10⁵ cfu/mL to microfiltered milk. The global microbiological, biochemical, and flavor changes induced by the presence of the yeasts in cheese were determined. Adjunct yeasts were present at 10⁶ cfu/g in curd, declined to 10⁴ to 10⁵ cfu/g in cheese, and did not influence gross composition, content of free amino acids, or content of free fatty acids. By using 8-way gas chromatography-olfactometry in parallel with gas chromatography-mass spectrometry, 30 odorous compounds of Cantalet cheese were identified. The olfactory profiles of K. lactis cheeses contained significantly greater levels of 8 odorous compounds (ethanol, ethyl hexanoate, 4 aldehydes, and 2 branched-chain acids) compared with the control and P. fermentans cheeses. Sensory analysis of cheeses flavor discriminated K. lactis cheeses on only 2 attributes (acetaldehyde and alcohol odors). This study shows that yeast contribution is species-specific and that K. lactis, at a population of 10⁶ viable cells/g, can influence Cantalet cheese flavor.     

Combined pH and high hydrostatic pressure effects on Lactococcus starter cultures and Candida spoilage yeasts in a fermented milk test system during cold storage

The combined effects of high pressure processing (HPP) and pH on the glycolytic and proteolytic activities of Lactococcus lactis subsp. lactis, a commonly used cheese starter culture and the outgrowth of spoilage yeasts of Candida species were investigated in a fermented milk test system. To prepare the test system, L. lactis subsp. lactis C10 was grown in UHT skim milk to a final pH of 4.30 and then additional samples for treatment were prepared by dilution of fermented milk with UHT skim milk to pH levels of 5.20 and 6.50. These milk samples (pH 4.30, 5.20 and 6.50) with or without an added mixture of two yeast cultures, Candida zeylanoides and Candida lipolytica (10⁵ CFU mL⁻¹ of each species), were treated at 300 and 600 MPa (≤20 °C, 5 min) and stored at 4 °C for up to 8 weeks. Continuing acidification by starter cultures, as monitored during storage, was substantially reduced in the milk pressurised at pH 5.20 where the initial titratable acidity (TA) of 0.40% increased by only 0.05% (600 MPa) and 0.10% (300 MPa) at week 8, compared to an increase of 0.30% in untreated controls. No substantial differences were observed in pH or TA between pressure-treated and untreated milk samples at pH 4.30 or 6.50. The rate of proteolysis in milk samples at pH values of 5.20 and 6.50 during storage was significantly reduced by treatment at 600 MPa. Treatment at 600 MPa also reduced the viable counts of both Candida yeast species to below the detection limit (1 CFU mL⁻¹) at all pH levels for the entire storage period. However, samples treated at 300 MPa showed recovery of C. lipolytica from week 3 onwards, reaching 10⁶–10⁷ CFU mL⁻¹ by week 8. In contrast, C. zeylanoides did not show any recovery in any of the pressure-treated samples during storage.     

How three adventitious lactic acid bacteria affect proteolysis and organic acid production in model Portuguese cheeses manufactured from several milk sources and two alternative coagulants

Model cheeses were manufactured according to a full factorial experimental design to help shed light on the individual and combined roles played by 3 native lactic acid bacteria (Lactococcus lactis ssp. lactis, Lactobacillus brevis, and Lactobacillus plantarum) upon proteolysis and organic acid evolution in cheese. The model cheeses were manufactured according to a generally representative Portuguese artisanal protocol, but the (ubiquitous) adventitious microflora in the cheesemaking milk were removed via sterilization before manufacture; therefore, the specific effects of only those lactic acid bacteria selected were monitored. In addition, 2 types of coagulant (animal and plant) and 3 types of cheesemaking milk (cow, sheep, and goat) were assessed to determine their influence on the final characteristics of the model cheeses. The nature of the coagulant appeared to be essential during the first stage of proteolysis as expected, whereas the contribution of those bacteria to the pools of total free AA and organic acids was crucial afterward. This was especially so in terms of the differences observed in the metabolisms of lactic acid (in the case of Lactococcus spp.) as well as acetic and citric acids (in the case of Lactobacillus spp.).     

Quantitative PCR for the specific quantification of Lactococcus lactis and Lactobacillus paracasei and its interest for Lactococcus lactis in cheese samples

The first objective of this work was to develop real-time quantitative PCR (qPCR) assays to quantify two species of mesophilic lactic acid bacteria technologically active in food fermentation, including cheese making: Lactococcus lactis and Lactobacillus paracasei. The second objective was to compare qPCR and plate counts of these two species in cheese samples. Newly designed primers efficiently amplified a region of the tuf gene from the target species. Sixty-three DNA samples from twenty different bacterial species, phylogenetically related or commonly found in raw milk and dairy products, were selected as positive and negative controls. Target DNA was successfully amplified showing a single peak on the amplicon melting curve; non-target DNA was not amplified. Quantification was linear over 5 log units (R² > 0.990), down to 22 gene copies/μL per well for Lc. lactis and 73 gene copies/μL per well for Lb. paracasei. qPCR efficiency ranged from 82.9% to 93.7% for Lc. lactis and from 81.1% to 99.5% for Lb. paracasei. At two stages of growth, Lc. lactis was quantified in 12 soft cheeses and Lb. paracasei in 24 hard cooked cheeses. qPCR proved to be useful for quantifying Lc. lactis, but not Lb. paracasei.     

Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation

The potential impact of aromatic and pectinolytic yeasts on cocoa flavour was investigated using two defined mixed starter cultures encompassing strains of Pichia kluyveri and Kluyveromyces marxianus for inoculating cocoa beans in small scale tray fermentations. Samples for microbial and metabolite analysis were collected at 12–24 hour intervals during 120 h of fermentation. Yeast isolates were grouped by (GTG)₅-based rep-PCR fingerprinting and identified by sequencing of the D1/D2 region of the 26S rRNA gene and the actin gene. Pulsed Field Gel Electrophoresis (PFGE) was conducted on isolates belonging to the species P. kluyveri and K. marxianus to verify strain level identity with the inoculated strains. Furthermore, Denaturing Gradient Gel Electrophoresis (DGGE) was performed to follow yeast and bacterial dynamics over time including the presence of the bacterial inoculum consisting of Lactobacillus fermentum and Acetobacter pasteurianus. Yeast cell counts peaked after 12 h of fermentation with the predominant species being identified as Hanseniaspora opuntiae and Hanseniaspora thailandica. P. kluyveri and K. marxianus were found to compose 9.3% and 13.5% of the yeast population, respectively, after 12 h of fermentation whilst PFGE showed that ~ 88% of all P. kluyveri isolates and 100% of all K. marxianus isolates were identical to the inoculated strains. Despite never being the dominant yeast species at any stage of fermentation, the un-conched chocolates produced from the two inoculated fermentations were judged by sensory analysis to differ in flavour profile compared to the spontaneously fermented control. This could indicate that yeasts have a greater impact on the sensory qualities of cocoa than previously assumed.     

Mycotoxin production by isolates of Fusarium lactis from greenhouse sweet pepper (Capsicum annuum)

Internal fruit rot, caused by Fusarium lactis, is an important disease of sweet pepper (Capsicum annuum) in Canadian greenhouses. Production of the mycotoxins fumonisin B₁ (FB₁), moniliformin (MON) and beauvericin (BEA) by F. lactis (17 isolates) and the related species F. proliferatum (three isolates) and F. verticillioides (one isolate), which are also associated with internal fruit rot, was evaluated on rice medium. All 21 isolates examined were found to produce BEA, at concentrations ranging from 13.28 to 1674.60 ppm, while 13 of 17 F. lactis isolates and two of three F. proliferatum isolates produced MON (0.23 to 181.85 ppm). Only one isolate of F. lactis produced detectable levels of FB₁ in culture, whereas all three F. proliferatum isolates and the F. verticilloides isolate produced this mycotoxin (0.28 to 314 ppm). Production of FB₁, MON and BEA was also evaluated in inoculated pepper fruits showing mild or severe symptoms of infection. FB₁ could be detected in both lightly and heavily diseased fruit tissue after inoculation with F. lactis, F. proliferatum or F. verticilloides, at concentrations ranging from 0.61 to 8.04 ppm. BEA was also detected in lightly and heavily diseased fruit tissue inoculated with F. lactis, as well as in heavily diseased tissue inoculated with F. proliferatum (3.00 to 19.43 ppm), but not in tissue inoculated with F. verticilloides. MON was detected in all tissues inoculated with F. proliferatum or F. verticilloides, and in heavily diseased tissue inoculated with F. lactis (0.03 to 0.27 ppm). The three mycotoxins were also found in naturally infected sweet pepper fruits exhibiting symptoms of internal fruit rot and collected from a commercial greenhouse. The production of MON, BEA and FB₁ alone or in combination by isolates of F. lactis suggests that development of internal fruit rot of sweet pepper is an important food safety concern, and that every effort should be made to cull infected fruit before it makes it to market.     

Production of flavour-active methionol from methionine metabolism by yeasts in coconut cream

Yeasts Candida kefyr NCYC143, Candida utilis CUM, Kluyveromyces lactis KL71, Saccharomyces bayanus SB1, Saccharomyces cerevisiae EC1118, Saccharomyces chevalieri CCICC1028, Candida famata (previously Torulopsis candida) CCICC1041 and Williopsis saturnus var. saturnus CBS254 were screened for their ability to produce flavour-active methionol (3-methylthio-1-propanol) in coconut cream supplemented with l-methionine. The yeasts varied with their ability to produce methionol from methionine with Saccharomyces cerevisiae EC1118 producing the most, followed by Kluyveromyces lactis KL71. Little methionol was produced by the other yeasts. Methionol production by Kluyveromyces lactis KL71 was subjected to further studies under different conditions of initial pH (4.0-6.3), temperature (20-33 °C), l-methionine concentration (0.05-0.25%) and yeast extract concentration (0-0.50%); optimal conditions were established at pH 5.0, 33.0 °C, 0.15% l-methionine and 0.05% yeast extract. CharmAnalysis™ using SPME-GC-MS was conducted on the coconut cream ferment; methional (3-methylthio-1-propanal), methionol and 2-phenylethyl acetate were found to be the most potent aroma-active compounds. The product of coconut cream fermentation by Kluyveromyces lactis KL71 may be considered as a novel, plant-based, natural and complex flavoring bioingredient in food applications.     

Yeast biocontrol of fungal spoilage of pears stored at low temperature

To reduce the use of fungicides, biological control with yeasts has been proposed in postharvest pears. Most studies of antagonists selection have been carried out at room temperature. However, in regions like North Patagonia where fruits are stored at − 1/0 °C during 5-7 months the selection of potential antagonist agents must be carried out at low temperature. In this study, 75 yeast cultures were isolated from healthy pears from two Patagonian cold-storage packinghouses. Aureobasidium pullulans, Cryptococcus albidus, Cryptococcus difluens, Pichia membranifaciens, Pichia philogaea, Rhodotorula mucilaginosa and Saccharomyces cerevisiae yeast species were identified. Additionally, 13 indigenous isolates of Penicillium expansum and 10 isolates of Botrytis cinerea were obtained from diseased pears, characterized by aggressiveness and tested for sensitivity to postharvest fungicides. The yeasts were pre-selected for their ability to grow at low temperature. In a first biocontrol assay using the most aggressive and the most sensitive isolate of each pathogen, two epiphytic isolates of A. pullulans and R. mucilaginosa were the most promising isolates to be used as biocontrol agents. They reduced the decay incidence by P. expansum to 33% and the lesion diameter in 88% after 60 days of incubation in cold. Foreign commercial yeast used as a reference in assays, only reduced 30% of lesion diameter in the same conditions. Yeasts were not able to reduce the incidence of B. cinerea decay. The control activity of the best two yeasts was compared with the control caused by the fungicides in a second bioassay, obtaining higher levels of protection against P. expansum by the yeasts. These two regional yeasts isolates could be promising tools for the future development of commercial products for biological control.     

Fingerprint of lactic acid bacteria population in beef carpaccio is influenced by storage process and seasonal changes

We have investigated the population structure of lactic acid bacteria (LAB) for several beef carpaccio available on the market with the purpose of comparing the effect of storage process (modified-atmosphere packaging and vacuum-packaging) and of seasonal changes on this microbial population. Out of 60 samples we have characterised 214 isolates accounting for 10 LAB species and 35 isolates accounting for 11 non-LAB species. Lactobacillus sakei, Leuconostoc carnosum and Leuconostoc mesenteroides were the most prevailing LAB species with a frequency of identification within 66%, 62% and 52% of the samples respectively. These 3 species were also characterised by a phenotypic intra-species diversity of isolates based on colony morphology. We showed that the prevalence was increased 1.5 fold for L. sakei and L. mesenteroides during the summer sampling in comparison to the spring or the fall sampling suggesting an environmental origin of these two species. Seasonal variations were also observed for the prevalence of Lactobacillus fuchuensis and L. carnosum in spring (2- and 1.5-fold increase, respectively) and of Brochothrix thermosphacta in fall (6-fold increase). Finally, we demonstrated that the growth potential after the sell-by-date was favourable of 1.25 log₁₀ cfu g⁻¹ to Leuconostoc spp. in modified-atmosphere packaging and of 1.38 log₁₀ cfu g⁻¹ to Lactobacillus spp. in vacuum-packaging. In conclusion, we show that important and unsuspected traits in bacterial population dynamics can be unravelled by large sampling strategies. We discuss about the need to take this assessment into account for further studies on bacterial ecosystems of meat.     

Characterization of lactic acid bacteria isolated from raw cows’ milk cheeses currently produced in Galicia (NW Spain)

Twenty cheeses belonging to the four Protected Designations of Origin manufactured in Galicia (NW Spain) (6 Arzúa-Ulloa, 4 Tetilla, 6 Cebreiro and 4 San Simón da Costa) were selected from a total of 60 cheeses on basis of their (typical) sensorial profiles. A total of 218 lactic acid bacteria (LAB) isolates were obtained from the predominant microflora of the selected cheeses and were identified as Lactococci (98 isolates), Leuconostocs (56), Mesophilic Lactobacilli (54), Pediococci (8) and Enterococci (2). Eighty-four of the isolates produced mainly malty, spicy or sulfide flavours in pasteurised whole milk, and were not characterized further. Some good producers of diacetyl-acetoin in milk (>100 mg/L) were found among a total of 129 LAB selected, although the isolates were generally less acidifying and less proteolytic than many of those obtained 10-15 years ago. The results suggest that the microflora in cheese-making environments have undergone changes, with the most evident difference being the practical absence of Enterococcal strains among the current isolates.     

The efficacy of nisin can drastically vary when produced in situ in model cheeses

Nisin, a bacteriocin produced by strains of Lactococcus lactis, has a broad inhibitory effect against Gram-positive bacteria. This study investigated the efficacy of nisin Z against Lactobacillus sakei when produced by a nisin-producing strain L. lactis in model cheeses manufactured with ultrafiltrated milk. These cheeses, containing 0, 4 or 10% of gelatin in their dry matter, were inoculated with both strains. Measurement of Lb. sakei loss of viability was an indirect indicator of nisin in situ efficacy. After 24 h, the loss of viability of Lb. sakei was from 0.73 ± 0.14 to 3.30 ± 0.60 log₁₀ cfu g⁻¹ in the cheeses with 0 and 10% of gelatin, respectively, indicating a better in situ efficacy of nisin when gelatin was incorporated. However, the concentration of nisin produced by Lactococcus was similar (3.5 μg g⁻¹) in all model cheeses when measured using an enzyme-linked immune sorbent assay (ELISA). The growth of Lactococcus was slightly improved when gelatin was incorporated, leading to a higher lactate concentration, which is one of the factors explaining the increased nisin efficacy. These results reinforced previous observations that prediction of nisin efficacy in complex food systems remains difficult.     

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.     

Novel Streptococcus infantarius subsp. infantarius variants harboring lactose metabolism genes homologous to Streptococcus thermophilus

The biodiversity and growth dynamics of Lactic Acid Bacteria (LAB) in farm-house Ossau-Iraty cheeses were investigated from vat milk to 180 days of ripening in six independent batches made from six raw ewe’s milks using five typical cheese-making methods. Commercial starter S1 was used for three batches, starter S1 combined with S2 for one batch and no starter for two batches.Up to ten LAB species from five genera and up to two strains per species were identified per milk; up to eleven species from five genera and up to three strains per species were identified per cheese. Lactococcus lactis, Lactobacillus paracasei, Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, and Leuconostoc mesenteroides were detected in all cheeses. Lactococci reached the highest counts irrespective of the milk and starter used. Lactococci and enterococci increased during manufacture, and mesophilic lactobacilli increased during ripening. Strain and species numbers, the percentage of isolates originating from the raw milk, maximum counts of each genus/species and time for reaching them, all varied according to whether or not a starter was used and the composition of the starter. The genotypes of strains within species varied according to the raw milk used. This generated distinct LAB microbiotas throughout manufacture and ripening that will certainly impact on the characteristics of the ripened cheeses.     

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.     

Production of wara, a West African soft cheese using lemon juice as a coagulant

As an important protein source for West African consumers, wara cheese made from the leave extract of Calotropis procera has extremely short shelf life of only 2-3 days [Adegoke, G. O., Nse, E. N., & Akanni, A. O. (1992). Effects of heat, processing time, and pH on the microflora, aflatoxin content, and storability of wara, a soft white cheese. Die Nahrung, 36(3), 259-264; Umoh, V. J., & Solomon, O. (2001). Safety assessment and critical control point of milk product and some cereal beverages in Northern Nigeria. In: Proceedings of USDA/USAID/NIGERIA international conference on food safety and security, August 1-3 (pp. 122-127). Ibadan, Nigeria: IITA; Belewu, M. A., Belewu, K. Y., & Nkwunonwo, C.C. (2005). Effect of biological and chemical preservatives on the shelflife of West African soft cheese. African Journal of Biotechnology, 4, 1076-1079; Adetunji, A. O., Alonge, D. O., & Chen, J. (Unpublished). Microbial quality of wara, a southwestern Nigerian soft cheese]. Lemon juice was used in this study as a substitute coagulant during wara manufacture in order to improve the microbial quality of wara. The cheese was manufactured from pasteurized milk inoculated with 10¹ or 10² CFU ml⁻¹ of Listeria monocytogenes. Samples of the milk or cheese were taken along the manufacturing steps and during a 5 d storage period at 15 and 28 °C in order to determine the populations of L. monocytogenes, total aerobes, Enterobacteriaceae, and psychrotrophs, as well as mold and yeast. On the 4th day of storage, portions of the un-inoculated control cheese from 28 °C were deep fried in vegetable oil, mimicking the practice of West African local cheese processors. The results showed that L. monocytogenes, at both inoculation levels, did not survive the manufacture of wara. In samples initially inoculated with 10¹ CFU ml⁻¹ of L. monocytogenes, the Enterobacteriaceae counts decreased from the initial 1.78 to 1.00 Log₁₀ CFU g⁻¹ with the addition of lemon juice, and became undetectable (<1.00 Log₁₀ CFU g⁻¹) at the curdling point as well as during the 5 d storage period at both temperatures. The total aerobic counts increased from the undetectable level on the 1st day of storage to 7.65 and 3.39 Log₁₀ CFU g⁻¹, respectively at 28 or 15 °C on the 5th day of storage. The psychrotrophic, as well as the yeast and mold counts increased from the undetectable levels on the 1st day of storage to 7.11 and 5.03 Log₁₀ CFU g⁻¹, respectively at 28 °C. At 15 °C however, the population of pyschrotrophs remained undetectable throughout the 5 d storage period whereas, the yeast and molds count increased to 3.08 Log₁₀ CFU g⁻¹ on day 3 before quickly decreasing to the undetectable levels on the 5th day of storage. A similar trend was observed in cheese made from the milk with an initial Listeria inoculation level of 10² CFU ml⁻¹. The results of this study showed that lemon juice significantly reduced the populations of the sampled microorganisms, especially the populations of Enterobacteriaceae.     

Evaluation of the functional potential of Weissella and Lactobacillus isolates obtained from Nigerian traditional fermented foods and cow's intestine

The characterisation of 24 lactic acid bacteria (LAB) isolates from Nigerian traditional fermented dairy foods, including some cow's intestine isolates, was conducted in order to select isolates for potential use as probiotics. LAB isolates were identified by partial sequencing the 16S rRNA gene as belonging to the species Lactobacillus paracasei, Lactobacillus brevis and mainly Weissella confusa. At the end of a characterisation process, 2 L. paracasei and 2 W. confusa isolates were selected, and their resistance to a simulated gastrointestinal digestion and their ability to adhere to eukaryotic cell lines were assessed. The survival to the simulated gastrointestinal passage was higher when bacterial suspensions were made in skimmed milk (2.0 ± 0.8 log units reduction) or at the simulated gastric juice pH 3 (2.7 ± 0.9 log units reduction) than at pH 2.0 (5.5 ± 0.7 log units reduction). Adhesion of LAB to both intestinal and vaginal epithelial models was comparable or higher than that of the reference Lactobacillus rhamnosus GG. However, some of the isolates increased the adhesion of the pathogen Escherichia coli LMG2092 to HT-29 and HeLa monolayers. Overall, isolates L. paracasei UI14 and W. confusa UI7 are good candidates for further studying potential benefits that support their use as probiotics. This is one of the few articles reporting the characterisation and the probiotic potential of Weissella, although more studies are needed in order to establish their safety for potential probiotic applications.     

Evaluation of microbial survival post-incidence on fresh Mozzarella cheese

Commercial fresh Mozzarella cheese is made by direct acidification and is stored dry or in water without salt addition. The cheese has a shelf life of 6 wk, but usually develops an off-flavor and loses textural integrity by 4 wk, potentially due to the lack of salt and high moisture that allow the outgrowth of undesirable bacteria. To understand how microbial incidence affects cheese quality and how incident pathogen-related bacteria are limited by salt level during refrigerated storage, we made fresh Mozzarella cheese with high (2%) and low (0.5%) salt. The high-salt cheese was packaged and stored dry. The low-salt cheese was packaged and stored either dry or in 0.5% salt brine. One portion of cheeses was evaluated for surviving incident microbes by aerobic plate counts, coliform counts, and psychrophilic bacterial counts, of which coliforms and psychrophiles were not detected over 9 wk. Aerobic plate counts remained at 100 to 300 cfu/g up to 2 wk but increased by 1,000- to 10,000-fold between 4 and 6 wk at all salt levels and storage conditions. Other portions of cheeses were inoculated with either Escherichia coli or Enterococcus faecalis, both of which increased by 100-fold over 90 d of storage. Interestingly, E. coli added to the cheese brine first grew in the brine by 100-fold before attaching to the cheese, whereas Ent. faecalis attached to the cheese within 24 h and grew only on the cheese. We conclude that incident bacteria, even from similar environments, may attach to cheese curd and survive differently in fresh Mozzarella cheese than in brine. Overall, 2% salt was insufficient to control bacterial growth, and slow-growing, cold- and salt-tolerant bacteria may survive and spoil fresh Mozzarella cheese.     

A survey on distribution and toxigenicity of Aspergillus section Flavi in poultry feeds

Thirty-five samples of poultry feeds and corresponding raw materials (maize, soybean and meat meal) from a processing plant were analyzed to evaluate the distribution and toxigenicity of Aspergillus section Flavi isolates. Mycological analysis of the samples indicated the presence of five fungal genera (Aspergillus, Penicillium, Fusarium, Cladosporium, and Eurotium). Aspergillus flavus was the predominant species being present in 48.5% of the analyzed samples. Ninety-one isolates belonging to Aspergillus section Flavi were isolated; ninety were identified as A. flavus and only one as A. parasiticus. Fifty-seven isolates were capable of producing sclerotia, 41 were identified as L-type strains and 16 as type S. Fifty-seven percent of the isolates produced AFB₁ levels ranging from 0.05 μg/kg to 27.7 μg/kg whereas 86.8% produced CPA from 1.5 μg/kg to 137.8 μg/kg. L-strains produced from 0.05 to 14.8 μg/kg of aflatoxin and type S produced levels from 0.05 to 1.65 μg/kg. No significant differences in CPA production among S- and L-strains were observed. Sclerotial isolates produced AFB₁ levels ranging between 0.05 and 27.7 μg/kg and CPA levels from 3.8 to 47.3 μg/kg. More than half of the A. flavus isolates were able to produce AFB and CPA simultaneously. Twenty percent of the 35 samples were contaminated with aflatoxin B₁ whereas 34.3% were contaminated with CPA. The high rate of CPA producing isolates represents a potential risk of contamination with this toxin in poultry feeds.     

Characterization of autochthonal yeasts isolated from Spanish soft raw ewe milk protected designation of origin cheeses for technological application

The yeasts involved in the ripening process of artisanal soft raw ewe milk Protected Designation of Origin (PDO) Torta del Casar and Queso de la Serena cheeses produced in Extremadura, Spain, were isolated throughout their ripening process, strain typed, and characterized for some important technological properties. A total of 508 yeast isolates were obtained and identified by inter-single sequence repeat anchored PCR amplification analysis and subsequent sequencing of the internal transcribed spacer ITS1/ITS2 5.8S rRNA. A total of 19 yeast species representing 8 genera were identified. Debaryomyces hansenii, Pichia kudriavzevii, Kluyveromyces lactis, and Yarrowia lipolytica were the predominant species. We selected 157 isolates, by genotyping and origin, for technological characterization. The evaluation of yeast isolates' growth under stress conditions of cheese ripening showed that 87 presented better performance. Among them, 71 isolates were not able to catabolize tyrosine to produce a brown pigment. Principal component analysis of the biochemical features of these isolates showed that 9 strains stood out, 3 K. lactis strains (2287, 2725, and 1507), 2 Pichia jadinii (1731 and 433), 2 Yarrowia alimentaria (1204 and 2150), Y. lipolytica 2495 and P. kudriavzevii 373. These strains displayed strong extracellular proteolytic activity on skim milk agar as well as an adequate enzymatic profile (strong aminopeptidase and weak protease activity), suggesting their great potential for cheese proteolysis. Extracellular lipolytic activity was mainly restricted to Yarrowia spp. isolates and weakly present in P. kudriavzevii 373 and K. lactis 2725, although enzymatic characterization by API-ZYM (bioMérieux SA) evidenced that all may contribute, at least in part, to the lipolysis process. Moreover, these strains were able to assimilate lactose, galactose, and glucose at NaCl concentrations higher than that usually found in cheese. However, lactate and citrate assimilation were limited to Y. lipolytica 2495, P. kudriavzevii 373, and P. jadinii 433, and may contribute to the alkalinizing process relevant to biochemical processes that take place in the last stages of ripening. By contrast, K. lactis strains showed acidifying capacity and β-galactosidase activity and may take part in the initial stages of ripening, together with lactic acid bacteria. Thus, considering the technological characteristics studied, the 9 selected strains presented biochemical features well suited to their potential use as adjunct cultures, alone or in combination with autochthonous starter bacteria in the cheesemaking process, to overcome the heterogeneity of these PDO cheeses, preserving their unique sensory characteristics.