Application of Ligilactobacillus salivarius SP36, a Strain Isolated from an Old Cheese Seal, as an Adjunct Culture in Cheesemaking.

Adjunct cultures originating from artisanal cheese environments may play an important role in recreating and developing traditional cheese flavours, thanks to their enzymatic activities, involved in different metabolic pathways that occur during cheese ripening. In this work, Ligilactobacillus salivarius SP36, a strain isolated from an old cheese seal, was added as an adjunct culture to the cheese's raw milk, and its effect on the microbiological, physical-chemical and sensory characteristics of the cheese was studied. The use of L. salivarius SP36 in cheese manufacturing had no significant (p > 0.05) effect on the cheese microbiota, gross composition (fat percentage, protein, total solids, moisture and NaCl concentration), colour or texture of the cheese. However, L. salivarius SP36 increased (p < 0.01) the formation of 25 volatile compounds, including 10 esters, 1 aldehyde, 8 alcohols and 6 carboxylic acids. In addition, cheeses made with L. salivarius SP36 received higher scores (p < 0.01) for aroma intensity and quality than control cheeses. L. salivarius SP36 proved to be a good candidate as an adjunct culture for cheesemaking, since it improved the cheese flavour by making it more intense and recovering typical sensorial notes of traditional cheeses.

Selection of starter lactic acid bacteria capable of forming biofilms on wooden vat prototypes for their future application in traditional Sicilian goat's milk cheese making.

In this study, 327 presumptive lactic acid bacteria (LAB) were isolated from goats' milk acid curds produced at a Sicilian dairy farm with the aim to identify potential starter cultures for traditional cheeses. All isolates were first processed by randomly amplified polymorphic DNA (RAPD)-PCR analysis. This approach identified 63 distinct strains which were evaluated for their acidifying capacity. Only 15 strains specifically stood out for their acidification capacity and were identified through 16S rRNA gene sequencing as Lactococcus lactis (11 strains) Enterococcus faecalis (three strains), and Ligilactobacillus animalis (one strain). Notably, all 15 LAB isolates produced bacteriocin-like inhibitory substances and anti-biofilm compounds, against both planktonic and biofilm forms of Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli, and Staphylococcus aureus, albeit at varying levels. Among these 15 LAB, En. faecalis RGM25 and Lc. lactis RGM55, susceptible to five antibiotics tested, were put in contact with wooden vat prototypes, because all equipment used in traditional cheese production in Sicily are made of wood. Scanning electron microscopy and bacterial plate counts of the wooden vat prototypes showed the development of biofilms at levels of approximately 6.0 log CFU/cm2. Overall, this study contributes to establishing a custom-made LAB starter cultures with bio-preservatives properties for Sicilian cheese productions.

In vitro assessment of biofunctional properties of Lactiplantibacillus plantarum strain Jb21-11 and the characterization of its exopolysaccharide / Evaluación in vitro de las propiedades biofuncionales de Lactiplantibacillus plantarum cepa Jb21-11 y caracterización de su exopolisacárido

The microbiota of traditional food provides a rich reservoir of biodiversity to find new strains with interesting features for novel functional food formulation. Therefore, this study aimed to investigate the biofunctional potential of the lactic acid bacteria (LAB) strain Jb21-11 isolated from Jben, a traditional Algerian fresh cheese. This isolate was selected out of a collection of 154 LAB based on its exopolysaccharide (EPS) phenotype and was preliminarily identified by polyphasic characterization as Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum) and its biofunctional properties were then assessed in vitro. The tested strain demonstrated good resistance to gastric juice, acidity around pH 2, and 2% (v/v) bile salts, which are important characteristics for potential biofunctional LAB candidates. It also showed a good production of ropy EPS with 674 mg/L on MRS medium. However, this ability appears to compromise the adhesion of the strain to Caco-2 cells (less than 1%), which according to our results, seems not to be related to autoaggregation and hydrophobicity (44.88 ± 0.028% and 16.59 ± 0.012%). Furthermore, promising antimicrobial activity against three pathogenic bacteria (Escherichia coli, Staphylococcus aureus, and Salmonella) was detected probably due to antimicrobial metabolites excreted during fermentation process into the medium. Moreover, the strain L. plantarum Jb21-11 displayed a therapeutic functionality with both anti-inflammatory and immunomodulatory action using RAW 264.7 cells. The chemical features of the novel ropy Jb21-11-EPS were also investigated revealing the presence of three monosaccharides, namely, mannose, galactose, and glucose, with a molar ratio of 5.42:1.00:4.52 linked together by α- and β-glycosidic bonds, presenting a relatively high molecular weight of 1.08 × 105 Da of interest for a texturing potential. Therefore, the new producing EPS strain Jb21-11 is a promising candidate for use as an adjunct culture for improving the texture of functional food.(AU)

In vitro assessment of biofunctional properties of Lactiplantibacillus plantarum strain Jb21-11 and the characterization of its exopolysaccharide.

ABSTACT: The microbiota of traditional food provides a rich reservoir of biodiversity to find new strains with interesting features for novel functional food formulation. Therefore, this study aimed to investigate the biofunctional potential of the lactic acid bacteria (LAB) strain Jb21-11 isolated from Jben, a traditional Algerian fresh cheese. This isolate was selected out of a collection of 154 LAB based on its exopolysaccharide (EPS) phenotype and was preliminarily identified by polyphasic characterization as Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum) and its biofunctional properties were then assessed in vitro. The tested strain demonstrated good resistance to gastric juice, acidity around pH 2, and 2% (v/v) bile salts, which are important characteristics for potential biofunctional LAB candidates. It also showed a good production of ropy EPS with 674 mg/L on MRS medium. However, this ability appears to compromise the adhesion of the strain to Caco-2 cells (less than 1%), which according to our results, seems not to be related to autoaggregation and hydrophobicity (44.88 ± 0.028% and 16.59 ± 0.012%). Furthermore, promising antimicrobial activity against three pathogenic bacteria (Escherichia coli, Staphylococcus aureus, and Salmonella) was detected probably due to antimicrobial metabolites excreted during fermentation process into the medium. Moreover, the strain L. plantarum Jb21-11 displayed a therapeutic functionality with both anti-inflammatory and immunomodulatory action using RAW 264.7 cells. The chemical features of the novel ropy Jb21-11-EPS were also investigated revealing the presence of three monosaccharides, namely, mannose, galactose, and glucose, with a molar ratio of 5.42:1.00:4.52 linked together by α- and ß-glycosidic bonds, presenting a relatively high molecular weight of 1.08 × 105 Da of interest for a texturing potential. Therefore, the new producing EPS strain Jb21-11 is a promising candidate for use as an adjunct culture for improving the texture of functional food.

Reduction of PDO Pecorino Siciliano cheese making duration: Microbial dynamics and quality attributes deriving from replacing whey permeate with hot water during cooking.

This work was carried out with the aim to reduce the transformation duration of Protected Designation of Origin (PDO) Pecorino Siciliano cheese. To this purpose, the cooking in hot water (experimental production, EXP) was compared to the traditional cheese cooking under whey permeate (control production, CTR). The microbiological composition of under rind (UR) and core (Co) section of CTR and EXP cheeses was determined by a combined culture-dependent and -independent approach. Total mesophilic microorganisms and lactic acid bacteria (LAB) present in raw ewes' milk (5.0 log CFU/mL) increased during cheese making and reached values of about 8.0 log CFU/g in both sections (UR and Co) of 5-month ripened cheeses of both productions (CTR and EXP) monitored. The identification of the viable LAB populations in ripened cheeses showed that Enterococcus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Limosilactobacillus and Streptococcus dominated UR and Co sections of all cheeses. MiSeq Illumina analysis demonstrated that LAB populations (lactobacilli, lactococci and streptococci) dominated the bacterial community of cheeses at 95.63-98.41 % of relative abundance. The two different cooking operations did not influence the physicochemical characteristics of PDO Pecorino Siciliano cheeses. Sensory evaluation performed by artificial senses analysis and trained panelists confirmed that the modification of PDO Pecorino Siciliano cheese production protocol did not significantly affect product characteristics and overall acceptance. Thus, data of this work confirmed that cooking under hot water allowed to reduce transformation duration and safeguard typicality of PDO Pecorino Siciliano cheese.

Microbial screening of animal skin bags used in traditional cheesemaking.

Bouhezza is a traditional Algerian cheese produced and ripened in goatskin bags called Djeld. The aim of this study was to characterize the microbial ecosystem from Djeld (fresh and dried Djeld for making Bouhezza cheese) and the changes introduced by Lben microflora during its preparation and to identify its role in cheesemaking and its safety. Two replicates of fresh and dried skin bags (FS and DS) were sampled and analyzed before and after contact with Lben. The microbiological results showed no pathogens. Skins observed before the addition of Lben were less populated 2.86 and 3.20 log CFU cm-2 than skins examined after the addition of Lben (approximately 6.0 log CFU cm-2), suggesting a potential role of Lben in releasing some microorganisms into the skin during its time in the Djeld. However, an increase in mesophilic lactic acid bacteria and yeasts was observed in Lben after different periods of interaction with the skin. PCR-TTGE revealed the predominance of lactic acid bacteria (Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Staphylococcus equorum subsp. linens, Lactococcus cremoris, Streptococcus thermophilus) and a few high-GC-content bacteria (Lacticaseibacillus paracasei, Brevibacterium casei). Transfer of several microbial species was observed between the goatskin bag biofilm and Lben during the overnight interaction. Bands corresponding to Lacticaseibacillus paracasei, Brevibacterium casei, and Lactobacillus delbrueckii subsp. lactis were detected in the fresh skin profile and in Lben after contact with the fresh skin. Lacticaseibacillus paracasei was found in dried skin and Lben after contact with dry skin. Lactobacillus helveticus and Enterococcus faecalis appeared in the Lben profile and persisted in Lben and the biofilm-covered dry skin after interaction. These results demonstrate an exchange of specific microbial populations between goatskin bag biofilm and Lben during the traditional preparation method, suggesting that the diversity of goatskin biofilm contributes to the microbial diversity of Lben used in the production of Bouhezza cheese.

Postprandial Inflammatory and Metabolic Responses Induced by Authentic Mytilinis Cheese: A Preliminary, Crossover, Nutritional Intervention in Healthy Adults.

Several Mediterranean traditional cheeses may present a beneficial effect on postprandial metabolic and inflammatory modulation due to the presence of bioactive components. The objective of the present preliminary nutritional intervention was the investigation of the postprandial metabolic responses after the intake of traditional Authentic Mytilinis cheese in olive oil with herbs, compared to the corresponding responses after consumption of Italian Parmesan cheese, in healthy participants. A pilot crossover, randomized, single-blinded, intervention clinical trial was conducted in 10 healthy men and women subjects, aged 18-30 years, after random allocation into the control and the intervention groups. The participants received a high-fat carbohydrate meal containing either Authentic Mytilinis cheese (the authentic nonrefrigerated recipe) or Italian Parmesan PDO cheese. After a washout week, the participants consumed the same meals conversely. Differences in the postprandial responses of glucose, triglycerides, uric acid and serum total, HDL and LDL cholesterol levels, as well as of the plasma total antioxidant capacity according to the FRAP method, were determined between groups for fasting, 30 min, 1.5 h, and 3 h after meal intake. The results suggested that meals did not significantly affect postprandial metabolic and inflammatory responses. However, Authentic L Mytilinis cheese resulted in a lower increase of LDL cholesterol (p > 0.05) and induced a significant decrease of serum triglycerides (p < 0.05) in the last 1.5 h after a meal, compared to Italian Parmesan cheese. Further investigation with large prospective studies is necessary to validate the current findings.

An Insight into Goat Cheese: The Tales of Artisanal and Industrial Gidotyri Microbiota.

The purpose of this study was to determine for the first time the microbiota in artisanal-type and industrial-type Gidotyri cheeses and investigate the influence of the cheese-making practices on their composition using culture-independent techniques. The microbiota present in artisanal with commercial starters (Artisanal_CS, n = 15), artisanal with in-house starters (Artisanal_IHS, n = 10) and industrial (Ind., n = 9) Gidotyri cheese samples were analyzed using a targeted metagenomic approach (16S rRNA gene). The Ind. Gidotyri cheese microbiota were less complex, dominated by the Streptococcaceae family (91%) that was more abundant compared to the artisanal Gidotyri cheeses (p < 0.05). Artisanal cheeses were more diverse compositionally with specific bacterial species being prevalent to each subtype. Particularly, Loigolactobacillus coryniformis (OTU 175), Secundilactobacillus malefermentans (OTU 48), and Streptococcus parauberis (OTU 50) were more prevalent in Artisanal_IHS cheeses compared to Artisanal_CS (p ≤ 0.001) and Ind. (p < 0.01) Gidotyri cheeses. Carnobacterium maltaromaticum (OTU 23) and Enterobacter hormaechei subsp. hoffmannii (OTU 268) were more prevalent in Artisanal_CS cheeses compared to Artisanal_IHS cheeses (p < 0.05) and Ind. cheeses (p < 0.05). Hafnia alvei (OTU 13) and Acinetobacter colistiniresistens (OTU 111) tended to be more prevalent in Artisanal_CS compared to the other two cheese groups (p < 0.10). In conclusion, higher microbial diversity was observed in the artisanal-type Gidotyri cheeses, with possible bacterial markers specific to each subtype identified with potential application to traceability of the manufacturing processes' authenticity and cheese quality.

Screening of Wild Lactic Acid Bacteria from Algerian Traditional Cheeses and Goat Butter to Develop a New Probiotic Starter Culture.

Twenty-five lactic acid bacterial (LAB) strains have been isolated from traditional goat butter and three types of cheese (dry Klila, frech Klila, and Bouhezza) and evaluated for technological abilities, probiotic properties, and potentials as starter cultures. The twenty-five LAB strains comprised eight strains belonging to Lactobacillus, four strains belonging to Lactococcus, eleven strains belonging to Enterococcus, and two strains belonging to Leuconostoc. A non-hierarchical cluster analysis was performed in order to select the performing strains. After carrying out the preliminary phenotypic characterizations and the probiotic potential, three strains designated as BM10, B15, and C30 belonging to the genus Lactobacillus and Enterococcus with good tolerance to acidity were selected. The strains showed a significant resistance to 0.5% bile salts and 0.4% phenol. Hemolytic activity was not detected; in addition, good hydrophobicity and autoaggregation was obtained. A significant antimicrobial activity was exhibited by all selected strains against Listeria innocua. Genotypic identification by 16S rRNA allowed the identification of B15, BM10, and C30 as Lactobacillus plantarum, Lactobacillus casei, and Enterococcus durans, respectively. The results of the current study suggest that the strains isolated from Algerian fermented dairy products have high potential as probiotic starter cultures in the goat butter and cheese industry.

Bio-production of lactic and lactobionic acids using whey from the production of cow's milk Wagashi cheese in Benin.

Traditional cheese is the main milk derivative in Bénin. This traditional process is not efficient and generate a lot of whey which has no real use until now. It is just disposed without being environmentally treated. Its use as a source for lactobionic and lactic acids production by Pseudomonas taetrolens and Lactobacillus casei is studied in this work, being also a proposal that can greatly boost economically the dairy sector in the country and reduce the end-of-cycle impact of the residue. To our knowledge, no data is available in the metabolization of Bénin's traditional cheese whey and its potential transformation into commercially valuable products such as lactobionic and lactic acids. With bulk filtration, non-controlled pH batch fermentations and without nutrients supplementation, 66 and 22% of lactose in the traditional cheese whey have been converted into lactobionic acid and lactic acid using Pseudomonas taetrolens and Lactobacillus casei, respectively. Those are important results that encourage to enhance the bioprocesses used in a cost-effective way in order to scale up an industrial production.

Processing and preservation methods of Wagashi Gassirè, a traditional cheese produced in Benin.

Purpose: Wagashi Gassirè (WG) is a traditional cheese produced from cow milk following local processing methods in Benin. The aim of this study was to describe the milk processing methods and the preservation practices with the objective of improving WG production and sanitary quality. Methods: A survey was carried out among 390 actors (84 dairy farmers, 165 producers, 53 traders, and 88 consumers) from two municipalities (Dassa and Nikki) in Benin. Results: WG is highly preferred by consumers for its whiteness (63.0%), softness (24.7%), smoothness (19.2%), and firmness (13.7%). WG production is based on the coagulation of milk using Calotropis procera extracts as coagulant. Six milk processing methods, including three new WG production methods were identified, depending on how the C. procera extracts were pre-treated and used during WG production. Boiling (67%) was the most widely used as WG preservation method. The use of aluminium cooking pots (100% of WG producers), WG open-air production (66.7% of producers) and antibiotic misuse (59.3% of dairy farmers) may lead to the chemical or microbiological contamination of WG. Conclusions: Six WG production and six preservation methods were identified after the survey among WG producers and traders. Future studies should assess the sanitary and physico-chemical quality of WG from the identified processing and preservation methods. The next step of research should be also focused on the development of specific standards to produce a better quality of WG.

Prevalence, Antibiotic Resistance and Virulence of Enterococcus spp. Isolated from Traditional Cheese Types.

Background: Enterococci are naturally found in the gastrointestinal (GI) tract of animals and humans, as well as animal-derived foods and vegetables. We here aimed to determine the prevalence, antibiotic resistance, and virulence determinants of E. faecium and E. faecalis in traditional cheese in the North-west of Iran. Materials and Methods: Fifty specimens of popular traditional cheese from dairy stores of Urmia and Tabriz, Iran, were collected. Identification of the genus and species of enterococci was done using molecular and phenotypic techniques. Results: Forty-eight (96 %) of 50 traditional cheese samples were harboring Enterococcus spp, including Enterococcus faecalis (n= 40; 83.33 %) and E. faecium (n= 8; 16.67 %). The prevalence of enterococci ranged from 1.1×105 to 9.7×104 CFU/g, and 1.1×103 to 9.8×103 CFU/g in Urmia and Tabriz samples, respectively. Rifampicin resistance (n= 38; 79.2 %) was the most common pattern observed in the susceptibility test, which was followed by quinupristin/dalfopristin (n= 33; 68.75 %). Among E. faecalis isolates, cpd (100 %), ace (92.5 %) and gelE (87.5 %), and among E. faecium isolates, gelE (100 %) and asa1 (75 %) were found to have the most common virulence genes. Conclusion: E. faecalis was the predominant species, displaying more virulence determinants. It also had high antibiotic resistance, as compared to E. faecium. The enterococci identified here commonly expressed virulence and antibiotic resistance determinants. So, it is required to improve the maintenance and production quality of traditional cheese to avoid enterococci contamination.

The Role of the Emotional Sequence in the Communication of the Territorial Cheeses: A Neuromarketing Approach.

Over the past few years, many studies have shown how territoriality can be considered a driver for purchasing agri-food products. Products with certification of origin are perceived as more sustainable, safer and of better quality. At the same time, producers of traditional products often belong to small entities that struggle to compete with large multinational food corporations, having less budget to allocate to product promotion. In this study, we propose a neuromarketing approach, showing how the use of these techniques can help in choosing the most effective commercial in terms of likeability and ability to activate mnemonic processes. Two commercials were filmed for the purpose of this study. They differed from each other in terms of emotional sequence. The first aimed primarily at eliciting positive emotions derived from the product description. The second aimed to generate negative emotions during the early stages, highlighting the negative consequences of humans' loss of contact with nature and tradition and then eliciting positive emotions by presenting cheese production using traditional techniques as a solution to the problem. Based on the literature on the emotional sequences in social advertising, we hypothesised that the second commercial would generate an overall better emotional reaction and activate mnemonic processes to a greater extent. Our results partially support the research hypotheses, providing useful insights both to marketers and for future research on the topic.

Prevalence and antimicrobial resistance profiling of Staphylococcus aureus isolated from traditional cheese in Yunnan, China.

The prevalence of staphylococcal infection and the emergence of multidrug resistance of Staphylococcus aureus (S. aureus) are major concerns in food safety and public health. This study aimed to investigate the prevalence of S. aureus isolated from traditional Chinese Rubing and Rushan cheese, antimicrobial resistance profiles, genomic characteristics, and predict antimicrobial resistance genes (ARGs). From 124 samples, 18 of 62 (29.03%) of Rubing and 5 of 62 (8.06%) of Rushan cheese were confirmed to be S. aureus positive by standard culture-based methods. Twenty-three coagulase-positive staphylococci isolates were grouped into 16 clusters by pulsed-field gel electrophoresis and subjected to routine susceptibility testing to 12 antibiotics. Those isolates exhibited high resistance to penicillin (100%), erythromycin, trimethoprim-sulphamethoxazole (34.78%), oxacillin, clindamycin, and cefoxitin (21.74%). Multidrug-resistant (MDR) S. aureus was found in 34.78% (8 of 23) of isolates. Further, S. aureus strain DC.RB_015 isolated from Rubing cheese, recognized as the most resistant to six antibiotics, was selected for whole-genome sequencing (WGS), continued with in silico approaches. S. aureus DC.RB_015 had a single chromosome size of 2,794,578 bp and a plasmid size of 22,961 bp. The strain harbored 18 predicted ARGs, including eight efflux pump genes (mepA, tet(K), arlR, arlS, norA, mgrA, tet(38), LmrS), one peptidoglycan biosynthesis gene (bacA), two ß-lactams resistance genes (mecA, blaZ), and seven genes conferring other antimicrobial resistance (APH(3')-IIIa, aad(6), ErmB, SAT-4, mecR1, GlpT, murA). The results of this study expand the knowledge of S. aureus strain DC.RB_015, increase food safety awareness, and will be helpful in establishing therapeutic therapy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03072-4.

Control of Listeria monocytogenes growth and virulence in a traditional soft cheese model system based on lactic acid bacteria and a whey protein hydrolysate with antimicrobial activity.

"Torta del Casar" is a Spanish soft-ripened cheese made with sheep's raw milk and subjected to a short ripening process, which favors the growth of pathogenic microorganisms including Listeria monocytogenes. The development of strategies to control pathogens and minimize health risks associated with the presence of L. monocytogenes in these products is of great interest. In this regard, the anti-Listeria activity of a whey protein hydrolysate (ProH) alone or combined with six lactic acid bacteria strains isolated from cheese was evaluated in this study as a biocontrol strategy using a "Torta del Casar" cheese-based medium. The most active combinations of lactic acid bacteria assayed induced a reduction higher than two logarithmic units in the growth of L. monocytogenes (serotype 4b) compared to their respective control when they were co-inoculated in "Torta del Casar" cheese-based medium at 7 °C for 7 days. In addition, the observed downregulation of some key virulence genes of L. monocytogenes suggests that the strain Lactiplantibacillus plantarum B2 alone and combined with the strain Lactiplantibacillus spp. B4 are good candidates to be used as biocontrol agents against L. monocytogenes growth in traditional soft cheeses based on raw milk during their storage at refrigeration temperatures.

Genetic Diversity of Leuconostoc mesenteroides Isolates from Traditional Montenegrin Brine Cheese.

In many dairy products, Leuconostoc spp. is a natural part of non-starter lactic acid bacteria (NSLAB) accounting for flavor development. However, data on the genomic diversity of Leuconostoc spp. isolates obtained from cheese are still scarce. The focus of this study was the genomic characterization of Leuconostoc spp. obtained from different traditional Montenegrin brine cheeses with the aim to explore their diversity and provide genetic information as a basis for the selection of strains for future cheese production. In 2019, sixteen Leuconostoc spp. isolates were obtained from white brine cheeses from nine different producers located in three municipalities in the northern region of Montenegro. All isolates were identified as Ln. mesenteroides. Classical multilocus sequence tying (MLST) and core genome (cg) MLST revealed a high diversity of the Montenegrin Ln. mesenteroides cheese isolates. All isolates carried genes of the bacteriocin biosynthetic gene clusters, eight out of 16 strains carried the citCDEFG operon, 14 carried butA, and all 16 isolates carried alsS and ilv, genes involved in forming important aromas and flavor compounds. Safety evaluation indicated that isolates carried no pathogenic factors and no virulence factors. In conclusion, Ln. mesenteroides isolates from Montenegrin traditional cheeses displayed a high genetic diversity and were unrelated to strains deposited in GenBank.

Survival rate of Escherichia coli O157 in artificially contaminated raw and thermized ewe milk in different Pecorino cheese production processes.

Pecorino is a typical Italian cheese, mostly produced in central and southern Italy regions using ewe raw milk and following traditional procedures. The use of raw milk constitutes a risk linked to the potential survival or multiplication of pathogenic microorganisms, as Shiga toxin-producing Escherichia coli (STEC). The aim of this study was to compare different Italian traditional Pecorino production methods to determine if there were any phases that could influence the Escherichia coli O157 survival rate, but also if they could negatively influence lactic acid bacteria survival rate, during the phases of production and ripening. Therefore batches of Pecorino cheese were prepared using different production methods, representing the real and typical cheese production in southern and central Italy regions: 1) heating the milk at 37 °C for about 40 min before curding, 2) heating the milk at 60 °C (thermization) for 13 min, so that the alkaline phosphatase reaction is still positive before curding, 3) cooking curd at 41 °C and 4) at 45 °C, both for 5 min. Our results demonstrated that traditional milk treatments different from pasteurization can help but do not eliminate serious microbiological treats, as E. coli O157, especially if the raw milk is heavily contaminated. The heat treatment at 60 °C applied to raw milk was able to decrease the concentration of E. coli O157 of 1.7 log10CFU/ml and, according to the inactivation slope, it would be further reduced prolonging the heating treatment. The results obtained also showed that, during the Pecorino cheese ripening, E. coli O157 was always enumerable for 60 days, remaining detectable after 90 days of ripening.

Improvement of Raw Milk Cheese Hygiene through the Selection of Starter and Non-Starter Lactic Acid Bacteria: The Successful Case of PDO Pecorino Siciliano Cheese.

This review article focuses on the technological aspects and microbiological critical points of pressed-cooked cheeses processed from raw ewe's milk without the inoculation of starter cultures, in particular "Pecorino" cheese typology produced in Italy. After showing the composition of the biofilms adhering to the surface of the traditional dairy equipment (mainly wooden vat used to collect milk) and the microbiological characteristics of PDO Pecorino Siciliano cheese manufactured throughout Sicily, this cheese is taken as a case study to develop a strategy to improve its hygienic and safety characteristics. Basically, the natural lactic acid bacterial populations of fresh and ripened cheeses were characterized to select an autochthonous starter and non-starter cultures to stabilize the microbial community of PDO Pecorino Siciliano cheese. These bacteria were applied at a small scale level to prove their in situ efficacy, and finally introduced within the consortium for protection and promotion of this cheese to disseminate their performances to all dairy factories. The innovation in PDO Pecorino Siciliano cheese production was proven to be respectful of the traditional protocol, the final cheeses preserved their typicality, and the general cheese safety was improved. An overview of the future research prospects is also reported.

Adjunct Culture of Non-Starter Lactic Acid Bacteria for the Production of Provola Dei Nebrodi PDO Cheese: In Vitro Screening and Pilot-Scale Cheese-Making.

The present study aimed at selecting non-starter lactic acid bacteria strains, with desirable technological and enzymatic activities, suitable as adjunct culture for the Provola dei Nebrodi cheese production. One hundred and twenty-one lactic acid bacteria, isolated from traditional Provola dei Nebrodi cheese samples, were genetically identified by Rep-PCR genomic fingerprinting, using the (GTG)5-primer, and by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Twenty-seven strains, included in the qualified presumption of safety (QPS) list, were tested for technological and proteinase/peptidase activities. Results showed that technological features and flavour formation abilities were strain-dependent. Among the selected strains, Lacticaseibacillus paracasei PN 76 and Limosilactobacillus fermentum PN 101 were used as adjunct culture in pilot-scale cheese-making trials. Data revealed that adjunct cultures positively affected the flavour development of cheese, starting from 30 days of ripening, contributing to the formation of key flavour compounds. The volatile organic compound profiles of experimental cheeses was significantly different from those generated in the controls, suggesting that the selected adjunct strains were able to accelerate the flavour development, contributing to a unique profile of Provola dei Nebrodi cheese.

Molecular detection, phylogenetic analysis, and antibacterial performance of lactic acid bacteria isolated from traditional cheeses, North-West Iran.

Lactic acid bacteria (LAB) are candidate probiotic bacteria that can provide health benefits when delivered via functional foods. The purpose of this study was to isolate and characterize LAB from traditional cheeses consumed in north-west regions of Iran. A number of sixty traditional cheeses samples were collected and initially screened as LAB using biochemical and molecular methods. A fragment of 1,540 bp in size of 16s rRNA gene was amplified from 70 bacterial isolates. Restriction fragment length polymorphism (RFLP) was employed to differentiate LAB isolates. LAB isolates generated three different RFLP patterns using HinfI restriction enzyme. Phylogenetic analysis revealed that LAB isolates belonged to three genera including Enterococcus, Lactobacillus, and Lactococcus. Most of the isolated LAB strains belonged to Enterococcus spp. The antimicrobial performance of eight LAB isolates with different RFLP patterns ranged from 6.72 to 14.00 mm. It was concluded that molecular characterization of LAB strains in traditional cheeses will enhance our understanding of traditional food microbiota and will help to find bacterial strains with probiotic potential with great benefit both in health and industry.