M. Medina

Inhibition of Listeria monocytogenes by enterocin 4 during the manufacture and ripening of Manchego cheese.

The inhibitory effect of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4, on Listeria monocytogenes strains Ohio and Scott A during themanufacture and ripening of Manchego cheese was investigated. Raw ewe’s milk wasinoculated with ca 105 cfu ml−1 of L.monocytogenes and with 1% of a commercial lactic starter, 1% of an Ent. faecalis INIA 4 culture, or 1% of each culture. Manchego cheeses were manufactured according tousual procedures. Listeria monocytogenes Ohio counts decreased by 3 log units after8 h and by 6 log units after 7 d in cheese made from milk inoculated with Ent. faecalisINIA 4 or with both cultures, whereas no inhibition was recorded after 60 d in cheese made frommilk inoculated with commercial lactic starter. Listeria monocytogenes Scott A wasnot inhibited by enterocin 4 during cheese manufacture, but decreases of 1 log unit after 7 d andof 2 log units after 60 d were achieved in cheese made from milk inoculated with bothcommercial lactic starter and Ent. faecalis INIA 4.

Accelerated Decrease of Enterobacteriaceae Counts During Ripening of Raw Milk Manchego Cheese by Lactic Culture Inoculation

Fourteen vats of Manchego cheese were manufactured from uncooled raw sheep milk; 7 experimental vats inoculated with 1% Streptococcus lactis culture in skim milk and 7 non-inoculated control vats were simultaneously manufactured. Experimental cheeses showed significantly lower pH values than control cheeses throughout the curing period, average differences exceeding 0.2 pH unit at all stages of ripening. Mean log counts of vat milks were 5.90 for Enterobacteriaceae , 5.64 for coliforms and 4.00 for fecal coliforms. After 60 d, log counts of these three microbial groups in experimental cheeses averaged 1.83, 1.46 and 1.02, respectively, while their mean values in control cheeses were 3.78, 3.60 and 2.64; differences between experimental and control cheeses were all significant at the 5% level. Enterobacter cloacae , Escherichia coli and Hafnia alvei were the only Enterobacteriaceae species detected in 60-d-old cheeses. Sensory evaluation data, salt and water content of experimental and control cheeses showed no significant differences.

Diversity among lactococci isolated from ewes raw milk and cheese

P. GAYA, M. BABÍN, M. MEDINA and M. NUÑEZ.1999.The technological and genetic characteristics of lactococci present in ewes’ raw milk and 1‐d‐old ewes’ raw milk cheeses sampled over a 1‐year period were investigated. The proportion of lactic acid bacteria isolates from milk samples able to decrease milk pH by more than 1·25 units after 6u2003h incubation at 30u2003°C reached 14·5% in spring vs 10·7% in summer, 8·3% in autumn and 3·0% in winter. In 1‐d‐old cheese samples, the proportion of lactic acid bacteria able to lower milk pH by more than 1·25 units increased up to 32·3% in spring vs 23·4% in summer, 8·0% in autumn and 10·3% in winter. Fast acid‐producing lactic acid bacteria mainly belonged to the genus Lactococcus. Using polymerase chain reaction protocols, fast acid‐producing lactococci were grouped as 61u2003Lactococcus lactis subsp. lactis, 13u2003L. lactis subsp. cremoris and 14u2003L. lactis subsp. lactis biovar diacetylactis. Randomly amplified polymorphic DNA (RAPD) fingerprinting of fast acid‐producing lactococci, using two primers, resulted in 21 different RAPD patterns for L. lactis subsp. lactis isolates, nine RAPD patterns for L. lactis subsp. cremoris isolates and three RAPD patterns for L. lactis subsp. lactis biovar diacetylactis isolates. Up to 19 different RAPD patterns were found for L. lactis isolates from cheeses made in a particular month.

The Effects of Cultivating Lactic Starter Cultures with Bacteriocin-Producing Lactic Acid Bacteria

The effects of bacteriocins produced by six strains of lactic acid bacteria on 9 mesophilic and 11 thermophilic commercial starter cultures were investigated in mixed cultures of commercial starters with bacteriocin-producing strains in milk. The bacteriocins produced by the test organisms were nisin A, nisin Z, lacticin 481, enterocin AS-48, a novel enterocin, and a novel plantaricin. Mesophilic commercial starters were in most cases tolerant of bacteriocins, with only two of the starters being partially inhibited, one by four and the other by two bacteriocins. The aminopeptidase activities of mesophilic starters were generally low, and only one of the combinations of mesophilic starter-bacteriocin producer gave double the aminopeptidase activity of the starter culture without the bacteriocin producer. Thermophilic commercial starters were more sensitive to bacteriocins than mesophilic starters, with six thermophilic starters being partially inhibited by at least one of the bacteriocins. Their aminopeptidase activities were generally higher than those of the mesophilic starters. The aminopeptidase activities of seven thermophilic starters were increased in the presence of bacteriocins, by factors of up to 9.0 as compared with the corresponding starter cultures alone. Bacteriocin-producing strains may be used as adjunct cultures to mesophilic starters for the inhibition of pathogens in soft and semihard cheeses, because mesophilic starters are rather tolerant of bacteriocins. Bacteriocin producers may also be used as adjunct cultures to thermophilic starters of high aminopeptidase activity, more sensitive to lysis by bacteriocins than mesophilic starters, for the acceleration of ripening in semihard and hard cheeses.

Behavior of Salmonellae During Manufacture and Ripening of Manchego Cheese

Six Salmonella strains were inoculated into 12 vats (2 vats/strain) of pasteurized sheep milk at a level of 104 cells/ml, and Manchego cheese was manufactured by usual procedures, with 1% of a Streptococcus lactis culture as starter. Growth of Salmonella occurred during the first 6-9 h, with mean increases in log counts of 1.67, 1.49 and 1.71 respectively for Salmonella enteritidis , S. typhi and S. typhimurium ; data inversely correlated to pH decrease. Mean numbers of Salmonella declined during the first week by 4.43, 1.18 and 3.97 log cycles for the three serotypes, respectively, with a significant correlation between decreases in pH and in Salmonella log counts. Salmonella survived for 4 weeks in 9 vats, for 6 weeks in 3 vats and was absent from all lots of 8-week Manchego cheese. Brilliant green agar yielded the highest productivity among five selective agars used for the enumeration of Salmonella by direct-plating procedures, while enrichment in selenite cystine broth followed by streaking to bismuth sulfite agar gave the highest Salmonella recovery of all eight broth-agar combinations tested.

Hydrolysis of caseins and formation of hydrophilic and hydrophobic peptides by wild Lactococcus lactis strains isolated from raw ewes’ milk cheese

Aims: To investigate the hydrolysis of αS1‐, αS0‐, βB‐, βA1‐ and βA2‐caseins by 32 wild lactococci of different randomly amplified polymorphic DNA (RAPD) patterns, isolated from raw ewes’ milk cheese, and the production of hydrophilic and hydrophobic peptides from whole casein by those strains.

Effect of pH, temperature and culture medium composition on the production of an extracellular cysteine proteinase by Micrococcus sp. INIA 528

Growth and proteinase production by Micrococcus sp. INIA 528 in a batch‐operated laboratory fermentor were investigated, with trypticase soy broth as the basal medium for studies on optimum temperature, pH and medium composition. Maximum growth was recorded at 34°C and pH 715, whereas optimum temperature and pH for proteinase production were 31°C and pH 6.25. Maximum rate of enzyme production occurred during the late log and early stationary phases of growth. Addition of 5.0 g 1‐1 yeast extract, 1.0 g 1‐1 glucose, 1.0 g 1‐1 MgSO4 or 1.0 g 1‐1 K2HPO4 to basal medium resulted in a lower enzyme yield, but supplementation of basal medium with 2.5 g 1‐1 (NH4)2SO4 increased enzyme production by 45%. A high initial biomass added to fresh broth supplemented with 2.5 g 1‐1 (NH4)2SO4 only increased enzyme activity by 19%, compared to the maximum enzyme activity achieved with the standard inoculum.

Production of PR Toxin and Roquefortine by Penicillium roqueforti Isolates from Cabrales Blue Cheese

PR toxin production in yeast extract-sucrose broth by 33 Penicillium roqueforti isolates from Cabrales blue cheese was quantified by a disc assay technique with Bacillus megaterium NRRL B-1368 as the test organism. Isolates from the interior of the cheese reached an average production of 1,89 mg PR toxin/100 ml, whereas the mean level of isolates from the surface was 1.64 mg/100 ml. Roquefortine production in the same broth by these isolates was quantified by a similar technique, with Bacillus stearothermophilus DSM 22 as the test organism. Mean production of roquefortine was 0.18 mg/100 ml for P. roqueforti isolates from the interior and 0.09 mg/100 ml for isolates from the surface of the cheese. If lactose or sodium lactate replaced sucrose in the growth medium, levels of both toxins decreased considerably. The identity of PR toxin and roquefortine in crude extracts was confirmed by thin-layer chromatography.

Purification and characterization of three extracellular proteinases produced by Pseudomonas fluorescens INIA 745, an isolate from ewe's milk.

Three proteinases were isolated from culture medium of Pseudomonas fluorescens INIA 745 and purified to homogeneity by a combination of Phenyl-Sepharose, DEAE-Sepharose, and Sephadex G-100 chromatography. Optimal temperature for enzymatic activity was 45 degrees C for all three proteinases. The pH optimum of proteinases I and II was found to be 7.0, while that of proteinase III was 8.0. Divalent metal ions like Cu2+, Co2+, Zn2+, Fe2+, and Hg2+ were inhibitory to proteinase activity while Ca2+, Mg2+, and Mn2+ had little or no inhibitory effect. The three enzymes were strongly inhibited by EDTA and 1,10-phenantroline and partially by cysteine. The three enzymes are metalloproteinases since they were inhibited by chelators and reactivated by Co2+, Mn2+, Cu2+, and Zn2+. The Km values of proteinases I, II, and III for casein were calculated to be 3.2, 2.6, and 5.2 mg/ml, respectively. Proteinases II and III rapidly degraded beta-casein, with preference to alphas1-casein, whereas proteinase I hydrolyzed both casein fractions at a slow rate.