Ewe's Milk Cheese Research Articles

Biochemical and physicochemical characteristics and volatile profiles of Erzincan Tulum cheese coagulated by herb-fortified artisanal lamb rennet

The characteristics of Erzincan Tulum cheese (ETC), an artisanal ewe-milk cheese and the first cheese with the designation of origin in Türkiye, are closely related to the geographical location of production and the human factors influencing every stage of the production process. This study aims to determine the influence of different coagulants (namely, herb-fortified artisanal lamb rennet (ALR), artisanal calf rennet (ACR), and commercial calf rennet (CCR)) on the biochemical, physicochemical characteristics, and volatile compounds of ETC, produced on the Heybeli plateau of the Erzincan province. Using artisanal rennet increased total solids, fat content, and acidity while decreasing pH and aw values. The highest PTA-SN (soluble nitrogen in 5 % phosphotungstic acid, w/v) and TCA-SN (soluble nitrogen in 12 % trichloroacetic acid w/w) values at the end of the ripening period (day 150 of storage) were observed in the ALR-Cheese (ALR-C). Furthermore, this cheese exhibited the highest rate of lipolysis (P < 0.01). Electrophoretograms revealed that αs-1 and β-casein degradation rates were the most significant in ALR-C. Following the 30th day of storage, the highest counts of lactic acid bacteria (LAB) were found in ALR-C. The most rapid reduction of coliforms, E. coli, and yeast-mold was observed in this sample throughout the storage. Eighty-nine volatile compounds were identified in the samples. Particularly noteworthy, ALR-C exhibited the highest ratios of hexanoic, octanoic, and decanoic acids after 90 days of storage. Additionally, steroid compounds were exclusively detected in this sample. The findings of this research make a substantial contribution to the existing literature concerning rennet types and the aromatic profile of ETC. These aspects are crucial for further investigations to enhance the cheese's quality and safety.

Physicochemical and microbiological characterisation of a typical Italian raw ewe's milk cheese: Pecorino bagnolese

Pecorino Bagnolese is a typical Italian cheese made from raw ewe's milk in province of Avellino (southern Italy). The aim of this study was to evaluate the physicochemical and microbiological parameters during cheese-making and ripening (30, 90 and 180 d). Results showed that the cheese-making and ripening influenced the pH, water activity, moisture, fat content and levels of Enterobacteriaceae. Changes in the fatty acids profile of ripened cheeses compared to milk were observed, suggesting the influence of ripening on the fatty acids profile. Nutritional indices demonstrated positive health attributes. The concentration of malondialdehyde remained consistently low, while free fatty acids showed an increasing trend during ripening. Coagulase-positive staphylococci complied with the legislative threshold value, but colonies of Staphylococcus aureus were found. Pathogens were not detected. In conclusion, the data collected provided a better characterisation of this traditional cheese and can be used for broadening knowledge of raw ewe's milk cheeses.

Description of Ewiss cheese, a new ewe milk cheese processed by Swiss cheese manufacturing techniques: Microbiological, physicochemical, and sensory aspects

Typically, Swiss-type cheese is made from cow milk. However, in the present work an attempt to expand the sheep supply chain and product offering in this field was made by developing a new type of cheese using Swiss-type cheese technology. The cheese was manufactured under industrial conditions, and fermentations were carried out using freeze-dried commercial starters that are traditionally used in the production of Swiss cheese. Two experimental "Ewiss cheese" (EC) products were produced using raw milk (RM) and pasteurized milk (PM), respectively. Fourteen microbial groups were investigated by plate counts from curd until ripened cheeses. According to microbiological analyses, no statistically significant differences were found between the 2 productions with respect to the group of lactic acid bacteria (LAB). The curds were mainly characterized by mesophilic LAB cocci (7.45 log10 cfu/g in RM-EC and 7.33 log10 cfu/g in PM-EC). However, at the end of the ripening period (9 mo), the cheeses exhibited a higher presence of mesophilic LAB rods. Undesired microbiological groups were found only in the curd of raw milk cheese in the range of 104 to 105 cfu/g, but they were reaching undetectable levels by plate count in the cheese at the end of ripening. The RM-EC and PM-EC were characterized by 76% and 68% of DM, respectively. These cheeses contained 29.30% and 34.36% of protein, and 51.31% and 50.38% of fat, respectively. Textural analysis showed differences in terms of hardness, chewiness, and gumminess between the experimental cheeses and Swiss cheese sold on the market. These differences could be attributed to the higher protein content of ewe milk. The main fatty acids in the cheeses were palmitic acid, myristic acid, oleic acid, and capric acid. Among the organic acids, RM-EC had higher concentrations of lactic acid, whereas PM-EC was higher in propionic acid. The ewe cheeses emitted 46 volatile compounds, including acids, aldehydes, ketones, esters, alcohols, and other compounds. The PM-EC was characterized by the main compounds of Swiss-type cheese: acetic acid, butyric acid, ethyl butyrate, ethyl caproate, propanoic acid, and tetramethylpyrazine. Sensory evaluation showed that the new dairy products were generally appreciated, and PM-EC was the most preferred by the judges. This research has enabled the development of new ewe milk products, which could stimulate the valorization of a sector that has been long neglected and still has a large margin of improvement.

Phenotypic and genotypic characterization of antimicrobial resistances reveals the effect of the production chain in reducing resistant lactic acid bacteria in an artisanal raw ewe milk PDO cheese

Antimicrobial resistance (AMR) is a significant public health threat, with the food production chain, and, specifically, fermented products, as a potential vehicle for dissemination. However, information about dairy products, especially raw ewe milk cheeses, is limited. The present study analysed, for the first time, the occurrence of AMRs related to lactic acid bacteria (LAB) along a raw ewe milk cheese production chain for the most common antimicrobial agents used on farms (dihydrostreptomycin, benzylpenicillin, amoxicillin and polymyxin B). More than 200 LAB isolates were obtained and identified by Sanger sequencing (V1-V3 16S rRNA regions); these isolates included 8 LAB genera and 21 species. Significant differences in LAB composition were observed throughout the production chain (P ≤ 0.001), with Enterococcus (e.g., E. hirae and E. faecalis) and Bacillus (e.g., B. thuringiensis and B. cereus) predominating in ovine faeces and raw ewe milk, respectively, along with Lactococcus (L. lactis) in whey and fresh cheeses, while Lactobacillus and Lacticaseibacillus species (e.g., Lactobacillus sp. and L. paracasei) prevailed in ripened cheeses. Phenotypically, by broth microdilution, Lactococcus, Enterococcus and Bacillus species presented the greatest resistance rates (on average, 78.2 %, 56.8 % and 53.4 %, respectively), specifically against polymyxin B, and were more susceptible to dihydrostreptomycin. Conversely, Lacticaseibacillus and Lactobacillus were more susceptible to all antimicrobials tested (31.4 % and 39.1 %, respectively). Thus, resistance patterns and multidrug resistance were reduced along the production chain (P ≤ 0.05). Genotypically, through HT-qPCR, 31 antimicrobial resistance genes (ARGs) and 6 mobile genetic elements (MGEs) were detected, predominating Str, StrB and aadA-01, related to aminoglycoside resistance, and the transposons tnpA-02 and tnpA-01. In general, a significant reduction in ARGs and MGEs abundances was also observed throughout the production chain (P ≤ 0.001). The current findings indicate that LAB dynamics throughout the raw ewe milk cheese production chain facilitated a reduction in AMRs, which has not been reported to date.

Sensory Attributes and Instrumental Chemical Parameters of Commercial Spanish Cured Ewes' Milk Cheeses: Insights into Cheese Quality Figures.

The external appearance of some of the Protected Designation of Origin (PDO) cured cheeses is similar to other cheese samples made in Spain: 1 kg and 2.5-3 kg formats, cylindrical, and with or without a pleita mark on the surface. In this work, commercial cured ewe's milk cheese samples with a similar external appearance were analyzed, including five PDO and five non-PDO samples. The parameters analyzed were color, texture, pH, humidity, water activity, and the volatile profile. Additionally, a descriptive and consumer-sensory analysis of the cheese samples was carried out. Statistical analysis of the results showed that luminosity, color coordinates a* and b*, percentage of deformation, humidity, water activity, and acid contents were significantly higher in non-PDO cheese samples. The breaking force, maximum force, and the content of esters were significantly higher in those cheese samples with PDO. In addition, PDO cheese samples showed higher scores for all attributes evaluated by consumers, except for color. These results suggest that PDO cheeses are placed on the market with a higher degree of ripening than non-PDO ones and that consequently they are more positively valued by consumers.

Reduction of Beta Cyclodextrin by Curd Washing in Low-Cholesterol Manchego Cheese.

Beta-cyclodextrin (β-CD) is a cyclic oligosaccharide consisting of seven glucose units. β-CD is increasingly used in food research to reduce cholesterol due to its affinity for non-polar molecules such as cholesterol and as a natural additive. The purpose of this study was to evaluate the effect of curd washing in ewe's milk cheese on the reduction in cholesterol by β-CD from pasteurized ewe's milk Manchego cheese and the characteristics of its main components: milk, lipids, and flavor. An approximately 98.45% cholesterol reduction was observed in washed experimental cheeses that were treated by using β-CD. The remaining residual β-CD from the effect of curd washing was 0.15% in mature cheese, of the initial 1% β-CD treatment of the milk. The chemical properties (fat, moisture, and protein) did not change as a result of the curd washing with or without β-CD. The curd washing with or without β-CD on the levels of the various lipid fraction (fatty acids, triglycerides, and phospholipids) were comparable in treated and untreated cheeses. The effects of curd washing and the β-CD treatment did not significantly affect flavor components or short chain free fatty acids. The β-CD molecules were edible and nontoxic; as a result, they could be used safely in cholesterol removal processing in cheese manufacturing, improving the reduction in residual β-CD by curd washing by 85%. Therefore, the present study suggests that curd washing combined with β-CD is an effective process for cholesterol removal in Manchego cheese, preserving its desirable properties.

Microbiological Biodiversity of Regional Cow, Goat and Ewe Milk Cheeses Produced in Poland and Antibiotic Resistance of Lactic Acid Bacteria Isolated from Them.

(1) Unique sensory values of traditional and regional dairy products made them more and more popular among consumers. Lactic acid bacteria naturally occurring in these products can express antibiotic resistance and be a reservoir of antibiotic resistance genes (ARG) in the environment. The aim of the study was to characterize the microbial diversity of twenty regional cheeses produced from non-pasteurized cow, goat and ewe milk, and investigate the phenotypic and genotypic antibiotic resistance (AR) of lactic acid bacteria isolated from these products. (2) Conventional microbiological methods were applied for the enumeration of lactic acid bacteria (lactobacilli and lactococci) and their isolation, and for the enumeration of Enterococcus, Staphylococcus, Enterobacteriaceae and spores. The disc diffusion method was applied for phenotypic AR. The PCR-based methods were used for strain identification, microbiological diversity of cheeses (PCR-DGGE), and for AR gene detection. (3) Among 79 LAB isolates the most frequent species were L. plantarum (n = 18), Leuc. lactis (n = 17), Lc. lactis (n = 11), Leuc. mesenteroides (n = 9) and L. pentosus (n = 8). Additionally, by using the PCR-DGGE method, DNA of L. casei was found in nine products. Lactobacilli (5.63-8.46 log cfu/g) and lactococci (6.15-8.41 log cfu/g) predominated over Enterococcus (max. 4.89 log cfu/g), Staphylococcus (max. 4.18 log cfu/g), and Enterobacteriaceae (mostly up to 4.88 log cfu/g). Analysis of phenotypic resistance to tetracycline (30 µg), erythromycin (15 µg), and chloramphenicol (30 µg) showed that 29% of LAB isolates were resistant to one antibiotic, 8%-to two, and 12%-to all tested antibiotics. Antibiotic resistance genes (AGR) for tetracycline (tet(M), tet(L), tet(W)), erythromycin (erm(B)) and chloramphenicol (cat-TC) were detected in 30 (38%), 29 (36.7%) and 33 (43.4%) LAB isolates, respectively. Among 31 LAB isolates phenotypically susceptible to all tested antibiotics, only 5 (16%) had no ARGs. (4) The results obtained in our work shed light on the potential threat posed by the widespread presence of ARGs in LAB present in regional cheeses.

Low-frequency ultrasound as a tool for quality control of soft-bodied raw ewe's milk cheeses

The relationship between the quality attributes and the ultrasound parameters of Protected Designation of Origin (PDO) soft-bodied cheeses from “Torta del Casar” and “Queso de la Serena” was studied. Cheese samples were taken from six different dairy industries (three for each PDO) in two different seasons. Ultrasound parameters related to velocity, attenuation and frequency were calculated and correlated with the physico-chemical, microbiological, rheological and sensorial properties of the cheeses. Several ultrasonic parameters showed a high correlation with changes in the physico-chemical, textural and sensorial properties of this product, which were partially associated with microbial activity. Among these, stand out the negative correlation of the sensorial descriptor “firmness” with bulk modulus ( κ ). The predictive models achieved by the multivariate linear regression method (MLR) showed the highest correlation coefficients for firmness by texture compression analysis (TCA) and the sensory descriptor “intensity”, including as the explanatory variables for both models, fast Fourier transform (FFT) frequencies and attenuation parameters. This work highlights the on-line capability of ultrasound for non-destructive quality assessment of this traditional and heterogeneous soft cheese based on the predictive potential of several secondary ultrasonic parameters. • Soft-bodied raw ewe's milk cheeses granted the PDO label were evaluated. • Relationships among cheese quality attributes and ultrasound parameters were studied. • Ultrasonic parameters showed correlations with physico-chemical, textural and sensorial properties. • A predictive model was developed by the multivariate linear regression method. • Ultrasound parameters are useful tools for quality control of soft-bodied cheeses.

Short communication: To what extent do environmental or technological conditions affect the sensory differentiation of raw ewe milk cheeses produced in valley or mountain farms?

This work discusses the extent to which differences in technological or environmental conditions affect the sensory properties of cheeses made in mountain or valley farms. The work is focused on Idiazabal cheese, frequently made with lamb rennet paste and with milk from sheep flocks managed under extensive grazing systems. Fourteen sensory attributes were assessed, and free fatty acid content and rennet lipase activity were analyzed in cheeses from mountain and valley farms. Sensory differences between cheeses were mainly dependent on the type and amount of rennet used for cheesemaking, particularly on the level of rennet lipase activity. The sensory attributes did not appear to be influenced by pasture type, grazing practices, or environmental conditions associated with farm location. Rennet lipase activity was responsible for the high scores of strong sensory attributes such as pungent mouthfeel, and these attributes masked the potential sensory differences that could be found due to farm location.

The characteristic of sheep cheese “Bryndza” from different regions of Slovakia based on microbiological quality

The aim of our study was to describe microorganisms which occur in the traditional Slovak cheese „Bryndza“. There were a total of 60 cheese samples collected from ten different farms during May 2019. The microbiota studies included the total bacterial count, coliforms, enterococci, lactic acid bacteria, yeasts and microscopic fungi. The total bacterial counts were cultivated on plate count agar at 30 °C in aerobic conditions, lactic acid bacteria on MRS at 37 °C in anaerobic conditions, coliform on VRBL and VRBG at 37 °C in aerobic condition, yeasts and microscopic fungi on MEA at 25 °C under aerobic condition. Gram-positive, Gram-negative and yeasts isolates were identified with MALDI-TOF MS Biotyper. Totally, a number of 1175 isolates of G-, G+ and yeast were identified with score higher than 2 and moulds. Escherichia coli and Stenotrophomonas maltophilia were the most frequently identified species of Gram-negative and Leuconostoc mesenteroides ssp. mesenteroides and Lactococcus lactis ssp. lactis from Gram-positive bacteria. Yarrowia lipolitica and Kluyveromyces lactis were the most distributed yeasts. Lactic acid bacteria group was represented by Lactobacillus, Lactococcus, Leuconostoc and Pediococcus. The most abundant genera of lactic acid bacteria were Lactobacillus with 11 species. This study describes the indigenous microbiota of the traditional ewe's milk cheeses from Slovakia.

Effects of adding solid and molten chocolate on the physicochemical, antioxidant, microbiological, and sensory properties of ewe's milk cheese.

A novel dairy product, namely "chocolate cheese", was produced with two typical Sicilian food products: Pecorino cheese, processed from ewe's milk, and Modica chocolate. The cheese, manufactured with 0%, 5%, 10%, and 15% (w/w) solid or molten chocolate, was evaluated after 0, 2, 4, and 6 weeks of vacuum storage for its nutritional and health properties. The addition of chocolate reduced the pH, protein, fat, and ash; the addition of 5% or 10% molten chocolate reduced hardness (N/mm2 ). The addition of either solid or molten chocolate resulted in a slight increase (P <0.1038) in the total polyphenol content, a higher oleic acid content, and less oxidative stability. The microbiological profile showed that the total mesophilic count and the number of mesophilic coccus lactic acid bacteria (LAB) were approximately equal (about 108 CFU/g) in all cheese. The survival of the microorganisms was affected by both the chocolate added and the storage time. Chocolate cheese stored for 6 weeks had less Enterobacteriaceae than control cheese, whereas yeasts were detected at higher cell densities in the former cheese. Filamentous fungi were undetectable in some cheese. Differences were also observed in the number of mesophilic rod LAB, which increased progressively over time in all cheese, and in Enterobacteriaceae, yeasts, and filamentous fungi, which decreased during storage. Descriptive and hedonic sensory tests and principal component analysis showed that fresh cheese and cheese stored for 2 weeks, including 5% molten chocolate, were the most preferred by evaluators. Based on these results, chocolate cheese has the potential to be appreciated in the market for its nutritional, health, and sensory properties. PRACTICAL APPLICATION: Chocolate cheese, made by combining two typical Sicilian foods, Pecorino cheese and Modica chocolate, is proposed as a novel dairy product. The highest sensory acceptance was obtained with the addition of 5% molten chocolate and storage for 2 weeks. Given its improved antioxidant properties, healthier fat, and sensory properties, chocolate cheese has the potential to be appreciated in the market, especially by young consumers.

Volatile compounds and sensory changes after high pressure processing of mature “Torta del Casar” (raw ewe's milk cheese) during refrigerated storage

Abstract The main objective of this paper was to study the changes during refrigerated storage in the volatile compounds and the sensory characteristics of raw ewe's milk mature Torta del Casar cheese High Pressure Processing-treated (200 or 600 MPa for 5 or 20 min) on day 60 after manufacture. Most of the volatile compounds and sensory characteristics were significantly affected by the refrigerated storage and to a lesser extent by the HPP treatment. The 600 MPa HPP treatments caused a decrease in the levels of the most abundant volatile compounds (acids and esters) and in bitterness scores on days 120 and 180. A significant effect was also found for some compounds, the overall flavour, and saltiness on day 240 after manufacture. Industrial relevance text High pressure processing treatments at 600 MPa applied to mature raw ewe's milk cheese such as “Torta del Casar” cheese could be an interesting option for the cheese industry to prevent some of the changes that occur during refrigerated storage that are related to over-ripening and excessive bitterness development. In addition, compared to the application of the treatment before maturation is completed, treating mature cheeses is convenient for the cheese industry because it provides the advantage of not requiring any unpackaging steps, which allows the food industry the commercialization of the cheeses without any additional packaging.

Application of multiple factor analysis for the descriptive sensory evaluation and instrumental measurements of bryndza cheese as affected by vacuum packaging

ABSTRACTMultiple factor analysis was used to examine naturally white, mature and spreadable bryndza cheese varieties. Sensory and physiochemical properties were evaluated during storage in vacuum and non-vacuum packed conditions at 4 ± 2°C. The results of statistical method showed similarity in varieties made from pasteurized ewe milk and mixed bryndza cheese before and after storage at both package conditions (p < 0.05). The statistical method used in the study was a suitable tool for assessing differences in food quality and provided an opportunity to visualize and categorize the results with regard to shelf life and packaging.

Effect of high-pressure treatments on proteolysis, volatile compounds, texture, colour, and sensory characteristics of semi-hard raw ewe milk cheese

High pressure (HP) offers potential industrial applications in cheese preservation, but it is essential to provide knowledge concerning their effects on the ripening process and sensory characteristics. In this study, we investigated the effect of different HP treatments (200–500MPa at 14°C for 10min on day 7) on proteolysis, texture, colour, volatile compounds and sensory characteristics of semi-hard raw ewe milk cheese. HP treatments did not affect pH or dry matter values of 60-day-old cheeses. Treatments at pressure levels up to 400MPa led to significant (P<0.01) increases in the total free amino acids (FAA) content at 60days, compared to control cheese, although the cell-free aminopeptidase activity was lower. HP retarded the formation of some volatile compounds in cheeses, the number of compounds affected by HP being higher as the pressure level increased. Cheeses pressurized at 300–500MPa had lower levels of 2-butanone, 2-butanol, 2-propen-1-ol, 1-butanol and acetic acid than control cheese, cheeses pressurized at 400–500MPa lower levels of 1-propanol, 2-pentanol, and butyric and hexanoic acids, and cheeses pressurized at 500MPa lower levels of ethanol, 3-methyl-1-butanol and 3-methyl-2-buten-1-ol. All HP-treated cheeses showed higher fracturability values, and higher Hue angle and lower a* values than control cheese. Despite the differences detected by instrumental analyses between HP-cheeses and control cheese, few significant differences were found between the sensory characteristics of HP-cheeses and control cheese. Only the pressurization of cheese at 500MPa affected significantly (P<0.01) some of the sensory characteristics, with a negative effect on taste intensity but a positive effect on aroma quality.In summary, HP treatments at 200 and 300MPa showed the mildest effects on the characteristics of semi-hard raw ewe milk cheese. HP treatment of this cheese variety at 300, 400 and 500MPa prevented late blowing defect caused by Clostridium tyrobutyricum (Ávila et al., 2016, Food Microbiol. 60, 165–173). Thus, it may be concluded that HP treatment at 300MPa is the most adequate procedure, able to prevent late blowing with minimum changes in cheese characteristics.

Rapid detection of Lactococcuslactis isolates producing the lantibiotics nisin, lacticin 481 and lacticin 3147 using MALDI-TOF MS

The aim of the study was to evaluate the potential use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for fast and reliable detection of strains producing the lantibiotics nisin, lacticin 481 and lacticin 3147 in a large collection of lactococci. A total of one hundred lactococcal isolates from traditional ewe's and goat's raw milk cheeses were identified to the species level as Lactococcuslactis by MALDI-TOF MS based on comparison with lactococcal entries in the BioTyper database. Mass spectra in the range 2000–4000Da of the identified isolates were compared to reference spectra of three lactococcal strains producing lacticin 481 (IFPL 330), lacticin 3147 (IFPL 105) and nisin (IFPL 503). Only eight isolates had mass spectra with peaks that could be unequivocally identified as lacticin 481 (2900.47Da) or nisin (3330.31Da). None of the assayed isolates matched the mass spectra corresponding to the two-peptide lacticin 3147 (2847.97 and 3306.29Da). The results obtained by MALDI-TOF MS were genetically validated by amplification of the corresponding structural gene coding for lacticin 481, nisin and lacticin 3147. MALDI-TOF MS can be used as a fast and reliable technique to screen a large number of lactococcal isolates for the ability to produce the lantibiotics nisin, lacticin 481 and lacticin 3147.

Industrial-scale application of Lactobacillus reuteri coupled with glycerol as a biopreservation system for inhibiting Clostridium tyrobutyricum in semi-hard ewe milk cheese

The suitability of the biopreservation system formed by reuterin-producing L. reuteri INIA P572 and glycerol (required for reuterin production) to prevent late blowing defect (LBD) was evaluated in industrial sized semi-hard ewe milk cheese contaminated with Clostridium tyrobutyricum INIA 68, a wild strain isolated from a LBD cheese. For this purpose, six batches of cheese were made (three with and three without clostridial spores): control cheeses with lactococci starter, cheeses with L. reuteri as adjunct, and cheeses with L. reuteri and 30 mM glycerol. Spores of C. tyrobutyricum INIA 68 germinated during pressing of cheese curd, causing butyric acid fermentation in cheese after 30 d of ripening. The addition of L. reuteri, without glycerol, enhanced the symptoms and the formation of volatile compounds associated with LBD. When glycerol was added to cheese milk contaminated with C. tyrobutyricum, L. reuteri was able to produce reuterin in cheese resulting in cheeses with a uniform cheese matrix and a volatile profile similar to cheese made with L. reuteri and glycerol (without spores). Accordingly, L. reuteri INIA P572 coupled with glycerol seems a novel biopreservation system to inhibit Clostridium growth and prevent LBD by means of in situ reuterin production.

Influence of reuterin-producing Lactobacillus reuteri coupled with glycerol on biochemical, physical and sensory properties of semi-hard ewe milk cheese

The biochemical, physical and sensory characteristics of ewe milk cheeses made with reuterin-producing Lactobacillus reuteri and glycerol (substrate for reuterin production) were assessed. Cheese made with lactococci starter (CTRL), cheese made with starter and L. reuteri (SLR), and cheese made with starter, L. reuteri and 30mM glycerol (SLR-G) were manufactured. L. reuteri reached counts above 7logcfu/g on day 1. Lactococci survival was enhanced in SLR cheese without affecting cheese pH, dry matter, proteolysis, concentration of most free amino acids (FAA), textural and most color parameters, or sensory characteristics. In situ production of reuterin by L. reuteri was only detected in SLR-G cheese, decreasing LAB counts although acidification remained unaffected. SLR-G cheese showed higher values of cell free aminopeptidase activity, overall proteolysis and FAA, particularly glutamic acid, than CTRL and SLR cheeses. The addition of L. reuteri-glycerol resulted in lower hardness and elasticity values in SLR-G cheese and influenced its L*, a* and b* color parameters. However, these changes, which were detected by instrumental analysis, did not affect the sensory scores for texture and color quality of SLR-G cheese, and it received the highest scores for taste quality. Our results suggest that L. reuteri-glycerol may provide a suitable system to release the antimicrobial reuterin in cheese without affecting negatively its sensory characteristics.

Protease and lipase activities of fungal and bacterial strains derived from an artisanal raw ewe's milk cheese

We previously identified the microbiota present during cheese ripening and observed high protease and lipase activity in Divle Cave cheese. To determine the contribution of individual isolates to enzyme activities, we investigated a range of species representing this microbiota for their proteolytic and lipolytic ability. In total, 17 fungal, 5 yeast and 18 bacterial strains, previously isolated from Divle Cave cheese, were assessed. Qualitative protease and lipase activities were performed on skim-milk agar and spirit-blue lipase agar, respectively, and resulted in a selection of strains for quantitative assays. For the quantitative assays, the strains were grown on minimal medium containing irradiated Divle Cave cheese, obtained from the first day of ripening. Out of 16 selected filamentous fungi, Penicillium brevicompactum, Penicillium cavernicola and Penicillium olsonii showed the highest protease activity, while Mucor racemosus was the best lipase producer. Yarrowia lipolytica was the best performing yeast with respect to protease and lipase activity. From the 18 bacterial strains, 14 and 11 strains, respectively showed protease and lipase activity in agar plates. Micrococcus luteus, Bacillus stratosphericus, Brevibacterium antiquum, Psychrobacter glacincola and Pseudomonas proteolytica displayed the highest protease and lipase activity. The proteases of yeast and filamentous fungi were identified as mainly aspartic protease by specific inhibition with Pepstatin A, whereas inhibition by PMSF (phenylmethylsulfonyl fluoride) indicated that most bacterial enzymes belong to serine type protease. Our results demonstrate that aspartic proteases, which usually have high milk clotting activity, are predominantly derived from fungal strains, and therefore fungal enzymes appear to be more suitable for use in the cheese industry. Microbial enzymes studied in this research might be alternatives for rennin (chymosin) from animal source because of their low cost and stable availability. Future studies will aim to purify these enzymes to test their suitability for use in similar artisanal cheeses or in large scale commercial cheeses.

Effect of reuterin-producing Lactobacillus reuteri coupled with glycerol on the volatile fraction, odour and aroma of semi-hard ewe milk cheese

The effect of the biopreservation system formed by Lactobacillus reuteri INIA P572, a reuterin-producing strain, and glycerol (required for reuterin production), on the volatile fraction, aroma and odour of industrial sized semi-hard ewe milk cheese (Castellano type) was investigated over a 3-month ripening period. The volatile compounds were extracted and analyzed by SPME-GC-MS and cheese odour and aroma profiles were studied by descriptive sensory analysis. Control cheese was made only with a mesophilic starter and experimental cheeses with L. reuteri were made with and without glycerol. The addition of L. reuteri INIA P572 to milk enhanced the formation of six volatile compounds. Despite the changes in the volatile compounds profile, the use of L. reuteri INIA P572 did not noticeably affect the sensory characteristics of cheese. On the other hand, the addition of L. reuteri INIA P572 coupled with 30mM glycerol enhanced the formation of twelve volatile compounds, but decreased the formation of five ones. The use of the biopreservation system did not affect overall odour and aroma quality of cheese although it resulted in a significant decrease of the odour intensity scores. In addition, this cheese received significant higher scores for “cheesy” aroma and significant lower scores for the aroma attributes “milky”, “caramel” and “yogurt-like”. The first two axes of a principal component analysis (PCA) performed for selected volatile compounds and sensory characteristics, accounting for 75% of the variability between cheeses, separated cheeses made with L. reuteri INIA P572 and glycerol from the rest of cheeses, and also differentiated control cheese from cheeses made with L. reuteri INIA P572 from day 60 onward. Our results showed that the reuterin-producing L. reuteri INIA P572 strain, when coupled with glycerol, may be a suitable biopreservation system to use in cheese without affecting odour and aroma quality.

Suitability of a new mixed-strain starter for manufacturing uncooked raw ewe's milk cheeses

Most raw milk Ossau-Iraty cheeses are currently manufactured on-farm using the same commercial streptococcal-lactococcal starter (S1). One way to enhance the microbial diversity that gives raw milk its advantages for cheese-making is to formulate new starters combining diverse, characterized strains. A new starter (OI) combining 6 raw milk strains of lactococci, recently isolated and characterized, was tested in parallel with the current starter by making 12 Ossau-Iraty raw milk cheeses at 3 farmhouses under the conditions prevailing at each farm. Compliance of the sensory characteristics with those expected by the Ossau-Iraty professionals, physicochemical parameters and coliforms were quantified at key manufacturing steps. The new starter OI gave cheeses having proper compliance but having lower compliance than the S1 cheeses under most manufacturing conditions, while managing coliform levels equally well as starter S1. This lower compliance relied more on the absence of Streptococcus thermophilus in starter OI, than on the nature of the lactoccocal strains present in starter OI. The study also shows that variations in 5 technological parameters during the first day of manufacture, within the range of values applied in the 3 farmhouses, are powerful tools for diversifying the scores for the sensory characteristics investigated.