Production of biogenic amines during the ripening of Pecorino Abruzzese cheese

The production of biogenic amines (BA) during the manufacturing and ripening of sheep milk Pecorino Abruzzese cheeses prepared from raw milk without starter culture (A) and from pasteurized milk with added starter (B) were compared. At the end of ripening (60 days), the total BA contents of cheeses of batches A and B were 697 and 1086 mg kg⁻¹, respectively; the dominant BA were different. Single isolates of enterococci, pseudomonads and Enterobacteriaceae were screened for their potential to produce BA. Qualitative tests indicated a large spread of BA-forming cultures among the members of the Enterobacteriaceae and lactic acid bacteria (LAB). Differences among the levels of BA produced in UHT milk by representative isolates of coliforms, Pseudomonas and LAB were observed in relation to the microbial group or the isolate. The results emphasize the need to improve the general hygienic conditions of Pecorino Abruzzese cheese manufacture and control the indigenous bacterial population.     

Enterococcus populations in Pecorino Abruzzese cheese: biodiversity and safety aspects

The presence of enterococci in Pecorino Abruzzese cheese during ripening was evaluated. Counts were high, especially in fully ripened summer batches. Seventy strains were isolated and identified based on phenotypical and genotypical features as Enterococcus faecium (48.5%), Enterococcus faecalis (40%), and Enterococcus durans (11.5%), with the first species predominant in spring batches and the second predominant in summer batches. High biodiversity was revealed by random amplification of polymorphic DNA and a PCR assay, suggesting the presence of autochthonous strains. E. faecium isolates were the most resistant to the tested antibiotics, especially to erythromycin, chloramphenicol, and penicillin, but all strains were susceptible to vancomycin, as confirmed by the absence of vanA and vanB genes. The presence of some virulence determinants was investigated, revealing the diffusion of aggregation substance (asal) and gelatinase (gelE) genes in 37.5% of E. faecalis strains. However, none of the isolates produced gelatinase in vitro, suggesting the presence of silent genes. The virulence genes were absent in E. durans. Among E. faecium strains, only Lab 41/1 possessed gelE and asal, whose presence previously has been reported only in E. faecalis. Decarboxylating activity was revealed for phenylalanine (27% of the strains) and tyrosine (96%) but not histidine. The presence of a tyrosine decarboxylase-encoding gene was observed for all strains. A comparison of these results with those of previous studies of clinical and food isolates indicates that enterococci from Pecorino Abruzzese cheese have low pathogenic potential.     

Antimicrobial activity of enterococci strains isolated from white cheese

Twelve strains of enterococci isolated from white cheese samples obtained from different regions of Turkey were screened for their antimicrobial activity against some Gram-positive and Gram-negative food-borne pathogens and contaminating bacteria, also against themselves and some other lactic acid bacteria (LAB), including some species of lactococci, lactobacilli and Leuconostoc . Antimicrobial activity was confirmed for all 12 strains, including six Enterococcus faecium , two Enterococcus faecalis and four Enterococcus durans . Antimicrobial agents produced by these enterococci exhibited a spectrum of activity, which is mainly directed against food-borne pathogens, specially Listeria monocytogenes and Bacillus cereus and some other Bacillus spp. and also some enterococci. Tested LAB was insensitive to inhibitory agents produced by them.     

干酪中肠球菌种群的遗传差异及万古霉素抗性基因分析

通过种、属引物特异性扩增、重复序列PCR（rep-PCR）技术和万古霉素抗性基因检测对新疆北疆地区干酪样品中球菌的遗传结构差异进行分析。结果表明,15份样品共分离52株肠球菌,包括31株耐久肠球菌(Enterococcus durans)、18株粪肠球菌(Enterococcus faecalis)和3株屎肠球菌(Enterococcus faecium)。依据rep-PCR遗传指纹带谱分析,52株菌可以聚类成7个群,其中4个由E. durans构成。24株肠球菌检测到了万古霉素抗性基因,17株E. durans为VanC2/C3型,4株E. faecalis为VanC1型,2株E. faecium为VanB型,只有1株E. faecium为VanA型。新疆北疆地区干酪中肠球菌种群分布较为广泛,地域之间优势种群和基因型不同,同种肠球菌菌株之间存在广泛的遗传差异。     

Genotypic and technological characterization of enterococci isolated from artisanal Fiore Sardo cheese

A total of 118 enterococcal strains isolated from artisanal Fiore Sardo cheese were characterized technologically and genetically. The presence of potential virulence factors was also investigated. Strains were classified as Ec. faecium (84 strains), Ec. durans (24 strains) and Ec. faecalis (10 strains). RAPD-PCR analysis with two different primers (M13 and XD9) confirmed species identification and proved useful for the detection of interstrain variations, especially among Ec. faecium isolates. Most strains could hydrolyse casein and had weak acidifying activity in milk. None of the isolates produced lipolytic reactions. Gelatinase activity was observed in two strains of Ec. faecalis and one strain of Ec. durans. beta-Haemolysis on horses' blood was never detected in any of the strains, independently of species. Most strains produced tyramine from tyrosine but none decarboxylated lysine, histidine or ornithine. Overall, a wide spectrum of resistance was observed. Almost all strains were resistant to the aminoglycosides, gentamycin, kanamycin, streptomycin, neomycin, and to the semisynthetic penicillin, oxacillin, but resistance to vancomycin was not widespread among our strains: only one Ec. faecium and one Ec. durans strain were found to be vancomycin resistant. Our results show a certain diversity in technological traits of the enterococcal strains isolated from artisanal Fiore Sardo, together with a low incidence of some potentially pathogenic traits of health concern.     

Identification and first characterization of lactic acid bacteria isolated from the artisanal ovine cheese Pecorino del Poro

A total of 225 lactic acid bacteria, isolated from the artisanal raw ovine milk cheese Pecorino del Poro, were identified and given an initial characterization; the best 18 strains were tested for their antagonistic activity against autochthonous Escherichia coli . An intense antagonistic activity against E. coli growth, induced by strains of Lactobacillus paracasei ssp. paracasei or L. curvatus , was observed; the E. coli inhibition can be ascribed to the production of bacteriocin-like compounds. The lactobacilli seem able to improve the hygienic quality of other artisanal raw milk cheeses.     

Organic acids produced by lactobacilli, enterococci and yeasts isolated from Picante cheese

 Four species of bacteria (Enterococcus faecium and Enterococcus faecalis, Lactobacillus plantarum and Lactobacillus paracasei) and three species of yeasts (Debaryomyces hansenii, Yarrowia lipolytica and Cryptococcus laurentii), previously isolated from Picante cheese, were cultured in ovine and in caprine milk and assayed for sugar and organic acids metabolism for 6 days. The results indicated that both milk types can be coagulated by the four strains of lactic acid bacteria. Lb. paracasei led to a faster and greater reduction in pH. Production of lactic acid correlated to lactose degradation, and was highest for Lb. paracasei followed by E. faecium; citrate metabolism was apparent for E. faecalis and, to a lesser extent, for E. faecium, Lb. plantarum and Lb. paracasei. Relatively high contents of formic acid were found when inoculation was with Enterococcus and with Lb. plantarum. Received: 4 February 1999     

Antibiotic resistance and virulence traits of enterococci isolated from Baylough, an Irish artisanal cheese

Eight representative Enterococcus strains from a collection of over 600 previously isolated from an Irish artisanal cheese were subjected to phenotypic and genotypic analysis of antibiotic resistance and virulence properties. Genes encoding resistance to tetracycline (tet(M) and tet(L)) and/or erythromycin (erm(B)) were detected in five strains. In addition, all strains contained two or more of the virulence genes tested (agg, gel, cyl, esp, ace, efaAfs, and efaAfm). Further investigation into the transferability and environmental dissemination of these resistance and virulence traits will allow risk assessment and safety evaluation of artisanal cheeses.     

Evaluation of antimicrobial and proteolytic activity of enterococci isolated from fermented products

A collection of 26 enterococci isolated from dairy and meat products were tested for antimicrobial and proteolytic activity. Enterococcus faecium and E. faecalis were the most frequent species among tested enterococci, and 11 isolates produced antimicrobial compounds. Results revealed that 10 out of 11 enterococci synthesized enterocins showing antimicrobial activity against food-born pathogen such as Listeria monocytogenes and Staphylococcus aureus. The broadest spectrum of antimicrobial activity was detected in E. faecalis BGPT1-10P and BGPT1-78. E. faecalis BG221 showed antimicrobial activity that was not related to production of enterocin, H₂O₂ or organic acid. Twenty-five enterococci showed strong or moderate proteolytic activity towards β-casein. Two isolates, BGPT1-10P and BGPT1-78, showed the most intense hydrolysis of α_s1-, β-, κ-casein fractions, total casein as well as gelatin. Extracellular BGPT1-10P and BGPT1-78 proteinases have a molecular mass of about 29 kDa. Bacteriocin production and proteinase activity of natural isolates of enterococci may be of technological interest in dairy and meat-fermented products.     

Identification and preliminary characterization of strains of enterococci and micrococci isolated from Arzúa raw cows'-milk cheese

152 enterococcal isolates and 88 micrococcal isolates obtained from secondary microflora of Arzúa raw cows'-milk cheese at various stages of ripening were characterized. The most frequent Enterococcus specie was E. faecalis. The most frequent Micrococcus specie was M. varians, followed by M. luteus and M. lylae; 27 micrococcal isolates could not be assigned to species. Although most strains of micrococci did not cause appreciable acidification of milk, the proportions of micrococcal isolates displaying proteolytic and lipolytic activities were higher than those for enterococcal isolates. The biochemical and enzymatic characteristics of isolates from both groups displayed considerable variability, even within the same species. In screening microorganisms of this type for suitability as components of cheese starter cultures, it is therefore necessary, as has been pointed out by previous authors, to work with single strains.     

Hygienic and physicochemical quality characterisation of artisanal and industrial Pecorino Calabrese cheese

Samples of artisanal and industrial Pecorino Calabrese cheese were collected from 20 dairy farms located in Calabria (Italy) and analysed to evaluate the microscopic and microbiological quality. The number of yeasts and moulds, total coliforms and enterococci was significantly higher in the artisanal cheese. The presence of two samples in both farms exceeding the hygienic limit in terms of Escherichia coli count highlights incorrect hygienic procedures in this kind of production. The filth analysis showed that the amount of solid impurities was significantly higher in the artisanal cheese, while only arthropods were more frequent in the industrial one.     

Characterization of yeasts involved in the ripening of Pecorino Crotonese cheese

The aims of this work were to identify and characterize for some important technological properties the yeast species present throughout the ripening process of Pecorino Crotonese, a traditional cheese produced in a well defined area of Southern Italy. In particular, the strain technological properties considered include fermentation/assimilation of galactose and lactose, assimilation of lactate and citrate in the presence of different NaCl concentrations, hydrolysis of butter fat, skim milk, gelatine and casein, production of brown pigments in cheese agar and ability to produce biogenic amines. High yeast levels were recorded in cheese samples already after 5 h of brining (about 5 log cfu/g) and these concentration remained constant during ripening. The yeast isolates belonged to restrict number of yeast species. While Kluyveromyces lactis and Saccharomyces cerevisiae were isolated prevalently in the first stages of Pecorino Crotonese production, Yarrowia lipolytica and Debaryomyces hansenii dominated during the later stages of maturation. Otherwise, the latter two were very NaCl resistant species. In fact, D. hansenii strains conserved the ability to assimilate lactose and galactose in the presence of 10% NaCl, while almost all the strains of Y. lipolytica isolated assimilated citrate and lactate up to 7.5% NaCl. Y. lipolytica isolates evidenced also the highest proteolytic and lipolytic activities and the capability to catabolize tyrosine producing brown pigment. In addition they resulted in the highest aminobiogenic potential decarboxylating ornithine, phenylalanine, tyrosine and lysine. However, they were not able to produce histamine, biogenic amine produced by three strains of D. hansenii.     

Esterolytic and lipolytic activities of lactic acid bacteria isolated from ewe's milk and cheese

In the present work, we report on the esterase and lipase activities of lactic acid bacteria representing the genera Lactococcus, Leuconostoc, Lactobacillus, and Enterococcus isolated from ewe's milk and cheeses. Esterase activity was studied using alpha- and beta-naphthyl derivatives of 2 to 12 carbon atoms and postelectrophoretic detection. The lactic acid bacteria evaluated had intracellular esterase activities, which preferentially degraded the alpha- and beta-naphthyl derivatives of 2 to 6 carbon atoms. By studying postelectrophoretic patterns, it was found that some strains presented more than one esterase. Lactobacillus plantarum O236 showed four enzymes that hydrolyze carboxyl ester linkages with different specificity. Lipase activity was studied in intracellular and extracellular fractions using tributyrin, tricaprylin, triolein, and milk fat as substrates. The intracellular and extracellular fractions of Leuconostoc mesenteroides O257, Lactobacillus plantarum O236, and Lactobacillus acidophilus O177 were able to hydrolyze tributyrin. L. plantarum O186, L. acidophilus O252, Enterococcus faecium O174 and O426, and Enterococcus faecalis Ov409 showed lipase activity associated with the intracellular fraction on tributyrin. Lactococcus lactis O233, L. plantarum O155, and Lactobacillus casei O190 did not hydrolyze triglycerides. Not all strains that showed esterase activity exhibited high activity on triglycerides. Esterase and lipase activities were species- and strain-specific. Wide variations in activity between strains highlight the need for selecting appropriate starters to produce enzyme-modified cheese as well as accelerated ripened cheese.     

Biogenic amine content and microbiological profile of Pecorino di Farindola cheese

“Pecorino di Farindola” is a traditional ewes’ milk cheese produced in the Abruzzo region (Italy) and ripened for a minimum of 90 days. The main objective of this research was to characterize the microbiological and chemical composition of this cheese, manufactured in ten dairy farms during the winter cheese-making season (December through March). By using classical enumeration system on specific media variability was observed in the viable numbers of aerobic mesophilic bacteria, enterobacteria, coagulase-negative staphylococci, yeasts, enterococci, mesophilic and thermophilic lactobacilli, lactococci and thermophilic streptococci. Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 resulted to be absent in all the samples. Among compounds possibly impacting on human health, the isomer cis-9, trans-11 conjugated linoleic acid (CLA), was determined in high levels in all samples, ranging from 9.2 to 12.7 mg/g fat. Great diversity was also found in biogenic amine contents with a relevant presence of tyramine in all the cheeses. This work represents the first study on Pecorino di Farindola cheese and could contribute to deepen the knowledge on its microbiological and biochemical features, focusing on hygiene and consumer health aspects.     

Vancomycin-resistant enterococci isolated from animals and food

One hundred and one chicken products, boiled ham and turkey cold meat were acquired from 18 different supermarkets in Spain during October 1997 to June 1998 and were analyzed for vancomycin-resistant enterococci (VRE). In the same way, 50 intestinal chicken samples from a slaughterhouse were also studied. VRE were detected in 25 of 92 samples of food of chicken origin (27.2%), but no VRE were found in cooked pork or turkey products. VRE were also detected in 8 of 50 intestinal chicken samples from the slaughterhouse (16%). VRE were identified as Enterococcus durans (n = 11), Enterococcus faecalis (n = 10), Enterococcus faecium (n = 10) and Enterococcus hirae (n = 2). All these strains were characterized as belonging to the vanA genotype by polymerase chain reaction. Ampicillin, quinupristin/dalfopristin and high level aminoglycoside resistance were frequently found among these strains. Heterogeneity was observed in susceptibility patterns among VRE strains, even in those of the same species. The high rate of colonization of chicken products by vanA containing enterococci detected 6 months to 1 year after the banning of avoparcin as a growth promoter, supports other studies suggesting that the food chain could be a source of VRE colonization in humans and thus a source of VRE infections.     

Growth characteristics of enterococci isolated from Laban Rayeb

Eleven strains of Enterococcus (Streptococcus) faecalis isolated from Laban Rayeb, a type of fermented milk, had been studied when grew in milk. Enterococcus (Streptococcus) faecalis strains 19 and 22 were the most active strains in the production of lactic acid, acetaldehyde and diacetyl. Both strains exhibited a high proteolytic activity besides a good clean flavour. Based on the obtained results, Enterococcus (Streptococcus) faecalis 19 and 22 could be used to bring about the lactic fermentation during the manufacture of some cultured dairy products.     

Heat resistance of thermoduric enterococci isolated from milk

Enterococci are reported to survive pasteurisation but the extent of their survival is unclear. Sixty-one thermoduric enterococci isolates were selected from laboratory pasteurised milk obtained from silos in six dairy factories. The isolates were screened to determine log(10) reductions incurred after pasteurisation (63°C/30 min) and ranked from highest to lowest log(10) reduction. Two isolates each of Enterococcus faecalis, Enterococcus faecium, Enterococcus durans and Enterococcus hirae, exhibiting the median and the greatest heat resistance, as well as E. faecalis ATCC 19433, were selected for further heat resistance determinations using an immersed coil apparatus. D values were calculated from survival curves plotted from viable counts obtained after heating isolates in Brain Heart Infusion Broth at 63, 69, 72, 75 and 78°C followed by rapid cooling. At 72°C, the temperature employed for High Temperature Short Time (HTST) pasteurisation (72°C/15s), the D values extended from 0.3 min to 5.1 min, depending on the isolate and species. These data were used to calculate z values, which ranged from 5.0 to 9.8°C. The most heat sensitive isolates were E. faecalis (z values 5.0, 5.7 and 7.5°C), while the most heat resistant isolates were E. durans (z values 8.7 and 8.8°C), E. faecium (z value 9.0°C) and E. hirae (z values 8.5 and 9.8°C). The data show that heat resistance in enterococci is highly variable.     

Evaluation of the antioxidant properties and oxidative stability of Pecorino cheese made from the raw milk of ewes fed Rosmarinus officinalis L. leaves

The aim of this work was to study the effect of rosemary leaf dietary supplementation on the antioxidant activities and total phenolic content of Pecorino cheese. Three hundred and twenty‐four sheep were randomly assigned to two dietary groups, which received a standard diet based on lucerne hay and concentrate (400 g per day). The concentrate of the rosemary supplemented group contained 2.50% dried rosemary leaves. The trial lasted 7 weeks. Cheesemaking was performed 3, 5 and 7 weeks from the start of the trial. The Pecorino cheese antioxidant activity was modified by the diet. Rosemary supplementation increased the total phenolic content, enhanced the antioxidant properties and decreased the lipid oxidation of cheese. A slight decrease in flavour was detected in cheeses after 7 weeks of rosemary administration.     

Enterocin typing of enterococci isolated from dried infant foods

One hundred and fifty enterococcal isolates recovered from 16 market samples of infant foods and 35 from other sources were characterized and subjected to enterocin typing with 18 indicator strains. Among 150 enterococcal isolates, 114 (76%) were able to be typed by the indicator strains. Although 24 enterocin patterns were observed with these enterococci, the most prevalent types were X-9, 224, and 65-603. Occurrence of pattern X-9 either singly or in combination with many other types was most frequent. Many of the enterocin patterns in enterococcal isolates were recovered from samples of dairy water supply and hand washings of personnel working in a dairy plant that manufactured infant food; this suggests the possibility of these as sources of contamination. Enterocin typing of enterococci could prove useful in epidemiological studies.