Artisanal Cheese Research Articles

Evaluation of traditional semi‐hard Brazilian cheeses using chemometric tools to identify possible denomination fraud

This study aimed to evaluate and classify some of the main traditional Brazilian semi‐hard cheeses, using multivariate tools to determine possible denomination fraud. Fifty‐six samples of Minas Artisanal (MA), Colonial (AC), Minas Padrão (MP) and Meia Cura (MC) cheeses were analysed. All the evaluated parameters indicated high variability. PCA and HCA confirmed two groupings (AC and MP; and MA and MC) with high similarity. The DD‐SIMCA method classified the cheeses with 83% accuracy. AC (48%) and MA (20%) cheeses were the most well classified in their respective denomination. These results were efficient in classifying groups and demonstrating the lack of authenticity regarding the denominations of these cheeses.

Meta-analysis of the global occurrence of S. aureus in raw cattle milk and artisanal cheeses

Meta-analysis of the global occurrence of S. aureus in raw cattle milk and artisanal cheeses

Selective enrichment of the raw milk microbiota in cheese production: Concept of a natural adjunct milk culture.

In cheese production, microorganisms are usually added at the beginning of the process as primary starters to drive curd acidification, while secondary microorganisms, with other pro-technological features important for cheese ripening, are added as selected cultures. This research aimed to investigate the possibilities of influencing and selecting the raw milk microbiota using artisanal traditional methods, providing a simple method to produce a natural supplementary culture. We investigated the production of an enriched raw milk whey culture (eRWC), a natural adjunct microbial culture produced from mixing an enriched raw milk (eRM) with a natural whey culture (NWC). The raw milk was enriched by spontaneous fermentation for 21 d at 10°C. Three milk enrichment protocols were tested: heat treatment before incubation, heat treatment plus salt addition, and no treatment. The eRMs were then co-fermented with NWC (ratio of 1:10) at 38°C for 6 h (young eRWC) and 22 h (old eRWC). Microbial diversity during cultures' preparation was evaluated through the determination of colony forming units on selective growth media, and next-generation sequencing (16S rRNA gene amplicon sequencing). The enrichment step increased the streptococci and lactobacilli but reduced microbial richness and diversity of the eRMs. Although the lactic acid bacteria viable count was not significantly different between the eRWCs, they harbored higher microbial richness and diversity than NWC. Natural adjunct cultures were then tested in cheese making trials, following the microbial development, and assessing the chemical quality of the 120 d ripened cheeses. The use of eRWCs slowed the curd acidification in the first hours of cheese making but the pH 24 h after production settled to equal values for all the cheeses. Although the use of diverse eRWCs contributed to having a richer and more diverse microbiota in the early stages of cheese making, their effect decreased over time during ripening, showing an inferior effect to the raw milk microbiota. Even if more research is needed, the optimization of such a tool could be an alternative to the practice of isolating, geno-pheno-typing, and formulating mixed-defined-strain adjunct cultures that require knowledge and facilities not always available for artisanal cheese makers.

Underexplored Potential of Lactic Acid Bacteria Associated with Artisanal Cheese Making in Brazil: Challenges and Opportunities

Artisanal cheeses are prepared using traditional methods with territorial, regional and cultural linkages. In Brazil, there is a great diversity of artisanal cheeses (BAC), which have historical, socioeconomic and cultural importance. The diversity of the BAC between producing regions is due to the different compositions of raw milk, the steps involved in the process and the maturation time. The crucial step for cheese differentiation is the non-addition of starter cultures, i.e., spontaneous fermentation, which relies on the indigenous microbiota present in the raw material or from the environment. Therefore, each BAC-producing region has a characteristic endogenous microbiota, composed mainly of lactic acid bacteria (LAB). These bacteria are responsible for the technological, sensory and safety characteristics of the BAC. In this review, the biotechnological applications of the LAB isolated from different BAC were evidenced, including proteolytic, lipolytic, antimicrobial and probiotic activities. In addition, challenges and opportunities in this field are highlighted, because there are knowledge gaps related to artisanal cheese-producing regions, as well as the biotechnological potential. Thus, this review may provide new insights into the biotechnological applications of LAB and guide further research for the cheese-making process.

Evaluation of antifungal activity of lactic acid bacteria against fungi in simulated cheese matrix

This study aimed to investigate the antagonist activity of lactic acid bacteria (LAB) isolated from Brazilian artisanal cheese and dairy environments against filamentous fungi commonly found in dairy products. In the in vitro test, a total of fifty-two LAB isolates were screened for their inhibitory effects against Aspergillus niger IOC 207 and Penicillium chrysogenum IOC 132. Among them, five strains (Weissella confusa W5, W8; Weissella paramesenteroides W9; Weissella cibaria W25; Lactiplantibacillus plantarum Q4C3) were selected for further in situ evaluation using a cheese matrix. The selected LAB strains exhibited excellent antifungal effect in simulated cheese matrix against both fungi targets; however, when combined with a commercial starter culture, their antifungal activity was reduced. Nevertheless, the results of this study suggest that these LAB strains may have potential as bioprotective cultures in food applications; they have the ability to produce antifungal compounds that could be characterized and potentially used as adjunct cultures in future food processing. Further research in this direction could shed light on their metabolic capabilities and pave the way for their potential use as biopreservatives in the food industry.

Diversity analysis of the yeast and fungal community structure in Kazak cheese from the Yili Pastoral area in Xinjiang

The production of Kazak cheese, one of the traditional fermented dairy products of Kazak people in Xinjiang, China, depends mainly on fermentation by lactic acid bacteria, but these are often mixed with some yeasts; both play an important role in determining cheese quality. Yeast and fungal communities in 14 artisanal cheeses from 3 pastoral areas of Yili were analysed by culturing and Illumina MiSeq technology. Culture results classified 63 yeast isolates into 10 genera and 11 species. Illumina MiSeq sequencing results showed that 103 fungal genera and 140 fungal species were present in the 14 cheese samples; Pichia (26.43%) and Pichia sporocuriosa (23.13%) were, respectively, the dominant genera and species. Illumina MiSeq sequencing identified 18 yeast species. Only 3 yeast species were identified by both methods, indicating significant differences between the two methods in yeast identification ability. Yeast and fungal community compositions in the samples varied with geographical origin.

Probiotic Properties, Safety Assessment, and Aroma-Generating Attributes of Some Lactic Acid Bacteria Isolated from Iranian Traditional Cheese

Artisanal cheeses are known as the source of beneficial lactic acid bacteria (LAB). Therefore, this study aimed to isolate and characterize LAB with different proteolytic activities from Iranian artisanal white cheeses. The isolates were classified into low, medium, and high proteolytic activity clusters via K-means clustering and identified as Lactiplantibacillus (Lpb.) pentosus L11, Lpb. plantarum L33, and Enterococcus faecium L13, respectively. Some safety tests (such as resistance to antibiotics, hemolytic activity, and biogenic amine production), probiotic properties (including cell surface hydrophobicity, auto/co-aggregation, and antibacterial activity), and production of volatile compounds were evaluated. These were non-hemolytic and non-biogenic amine producers, and showed no irregular antibiotic resistance. Lpb. plantarum L33 had the highest hydrophobicity (30.55%) and auto-aggregation (49.56%), and the highest co-aggregation was observed for Lpb. pentosus L11 with Staphylococcus aureus (61.51%). The isolates also showed a remarkable antibacterial effect against pathogenic bacteria. Moreover, Lpb. pentosus L11 and Lpb. plantarum L33 with low and medium proteolytic activity produced a wider range of volatile compounds in milk compared to the strain with a high proteolytic effect. The results showed that a probiotic strain with low or medium proteolytic activity could improve the flavor characteristics of fermented milk.

Technological and safety assessment of selected lactic acid bacteria for cheese starter cultures design: Enzymatic and antimicrobial activity, antibiotic resistance and biogenic amine production

Twenty lactic acid bacteria (LAB) isolated from artisanal cheeses and previously selected for their technological properties were screened for their enzymatic activities, antimicrobial activity and safety. The aim was to select those LAB strains that were safe and showed advantageous properties for the development of starter cultures for cheese making, discarding those that could transfer antibiotic resistance or produce any toxic biogenic amines. Aminopeptidase activities were detected in most strains, particularly high for the substrate leucine arylamidase, and most lactobacilli and Leuconostoc strains showed high β-galactosidase activity. Glutamate dehydrogenase (GDH) activity was detected in 13 strains, although the activity values varied widely. All strains showed antimicrobial activity against the indicator microorganisms due to acid production. However, only one of the Lactiplantibacillus plantarum strains showed an inhibitory activity against Enterococcus faecalis due to bacteriocin-like compounds. In particular, Levilactobacillus brevis TAUL1567 and Lactiplantibacillus paraplantarum TAUL1399 showed resistance to tetracycline and ampicillin, respectively, above the cut-off values and were therefore excluded. Tyramine was only produced by L. brevis TAUL1567 (193.15 μg ml−1), while putrescine was produced by this strain and two strains of Lactococcus lactis. Finally, 14 strains produced γ-aminobutyric acid (GABA), five of them at concentrations around or above 100 μg ml−1.

Prevention of Fungal Contamination in Semi-Hard Cheeses by Whey-Gelatin Film Incorporated with Levilactobacillus brevis SJC120.

Cheese whey fermented by lactic acid bacteria (LAB) was used to develop an edible film with antifungal properties. Five LAB strains isolated from artisanal cheeses were screened for antifungal activity and incorporated into a whey-gelatin film. Of the strains tested, Levilactobacillus brevis SJC120 showed the strongest activity against five filamentous fungi isolated from cheese and cheese-making environment, at both 10 °C and 20 °C. The cell-free supernatant from L. brevis inhibited fungal growth by more than 80%. Incorporation of bacterial cells into the film did not alter the moisture content, water vapor permeability, or mechanical and optical properties. The whey-gelatin film was also able to maintain the viability of L. brevis cells at 107 log CFU/g after 30 days at 10 °C. In cheeses wrapped with L. brevis film, the size of fungal colonies decreased by 55% to 76%. Furthermore, no significant differences (p > 0.05) were observed in cheese proteolysis or in the moisture, fat, and protein content of the cheese wrapped with films. The results showed that whey-gelatin film with L. brevis SJC120 can reduce the contamination of cheese with filamentous fungi and could be used as an alternative to conventional cheese preservation and packaging.

Microbiological quality and cultivable bacterial community of fresh and ripened Minas cheeses made from raw and pasteurised milk

The microbiological and physicochemical characteristics of 56 samples of Brazilian fresh (Minas frescal cheese, MFC) and ripened (Minas padrão cheese, MPC; Minas artisanal cheese, MAC) cheeses produced from pasteurised (MFC and MPC) or raw milk (MAC) were evaluated. Significant differences were observed between samples, with a positive emphasis on microbiological quality for MPC. MALDI-TOF MS identified a diverse bacterial community from the 808 colonies isolated from different culture media. Staphylococcus aureus (34.32%) and Escherichia coli (35.59%) were the most isolated species. The eae gene, which confers virulence on E. coli strains, was observed in 23 samples (41.07%), and in 21.05%, 27.78%, and 73.68% of MFC, MPC and MAC samples, respectively. There was no correlation between the evaluated indicators and the presence of eae-positive cheese samples. The choice of the ideal microbiological indicator must be specific, considering the physicochemical characteristics and the raw milk of the different types of cheese.

Natural rennet sterilisation by non-thermic methods for fresh cheese manufacture

Natural rennet is widely used in artisanal cheese shops to produce fresh cheese with particular characteristics such flavour, in comparison to artificial rennet. This rennet is produced by the cheesemakers under non-hygienic conditions. The use of non-thermal technologies such as ultrasound and UV light can be an alternative of rennet sanitisation. The objective of the present work was to evaluate the effect of applying of different times of ultrasound or ultraviolet on the microbial growth in natural rennet for fresh cheese manufacture, its curd yield and curd hardness texture. Natural rennet was prepared and divided into three samples, one of which was exposed to ultrasound for 5, 10 and 15 min, the second one received UV radiation for 30, 60 and 90 min, and the last one was considered as the control treatment. A microbial test was conducted to analyse total coliforms, Escherichia coli, Staphylococcus aereus and Salmonella spp. Fresh cheeses were manufactured using the different rennets and the coagulant effect was tested by way of with a texture test (hardness). The results showed the highest microbial growth in the control treatment; none of the US treatments allowed microbial growth and the UV radiation was effective for all microorganisms at a 90 min exposure. All rennet treatments maintained their coagulant effect. In conclusion, the use of non-thermal methods was effective for rennet sterilisation, although it affected curd yield (2% less). Highlights Ultrasound inhibits pathogenic microorganisms growing in natural rennet. Sterilisation by non-thermal methods does not affect the coagulation process. A 42 kHz ultrasound at, 70 W power caused the best reduction of pathogenic bacteria.

The effect of seasonality on the biogenic amines, free amino acids, and physico-chemical composition of raw milk Fiore Sardo cheese produced in Sardinia (Italy)

The effect of seasonality on the content of biogenic amines (BA), compounds that could threaten food safety at a high level, were evaluated in Fiore Sardo, a typical artisanal raw sheep's milk cheese produced in Sardinia. The cheese samples collected from six farms, after four months of ripening, were manufactured from January to June. The relationship between the BA content, the free amino acid (FAA) concentration, and the physico-chemical composition (pH, a w , NaCl, humidity, and the fat and protein content) was considered. The FAA and BA contents were determined by HPLC-FL. The BA content and the other parameters analysed were significantly affected (p ≤ 0.000) by seasonality and the farm of origin. Despite the differences attributable to the producers, the seasonal variations of all the parameters considered followed the same trend. As the season changed, the level of total BA grew, reaching a mean content of 1402 ± 207 mg/kg in the samples produced in June, almost five times the mean value of those manufactured in January. The most represented biogenic amine was tyramine, followed by putrescine and cadaverine. Histamine was determined almost exclusively in some samples produced in the warmer months, while β-phenylethylamine and tryptamine were found in a low concentration or not detected. The winter products did not exceed the threshold of 900 mg/kg proposed for the total BA in food, while most of the samples manufactured in the hottest months exceeded this limit, posing a potential risk to consumers' health. The total FAA and BA content were correlated (p = 0.01), indicating that with the change of the season, proteolysis phenomena increased and made the precursor amino acids available for microbial decarboxylase activity. In addition, from January to June, the pH and fat levels grew, while those of a w , humidity and NaCl tended to decline. The study highlighted that as the season changed, the rise in temperature, probably combined with the increment of fat and decrement of NaCl content, could favour proteolysis phenomena and bacterial decarboxylase activity and, as a result, the escalation of BA production. Interventions in the production protocol to reduce the negative effect of seasonality on cheese safety are needed. In addition, in light of these results, it is possible to speculate that the rising temperature and the predicted lengthening of the warm season due to climate change, which also seems to affect the spring periods, could create the ideal conditions for the production of biogenic amines for an increasingly extended phase of the year.

Incidence and Levels of Aflatoxin M1 in Artisanal and Manufactured Cheese in Pernambuco State, Brazil.

Cheese is one of the most susceptible dairy foods to accumulating aflatoxins due to their high affinity to caseins. The consumption of cheese contaminated with high levels of aflatoxin M1 (AFM1) can be highly harmful to humans. The present work, based on high-performance liquid chromatography (HPLC), highlights the frequency and levels of AFM1 in coalho and mozzarella cheese samples (n = 28) from the main cheese-processing plants in Araripe Sertão and Agreste in the state of Pernambuco, Brazil. Of the evaluated cheeses, 14 samples were artisanal cheeses and the remaining 14 were industrial (manufactured) cheeses. All samples (100%) had detectable levels of AFM1, with concentrations ranging from 0.026 to 0.132 µg/kg. Higher levels (p < 0.05) of AFM1 were observed in artisanal mozzarella cheeses, but none of the cheese samples exceed the maximum permissible limits (MPLs) of 2.5 µg/kg established for AFM1 in cheese in Brazil and 0.25 µg/kg in the European countries by the European Union (EU). The high incidence of low levels of AFM1 found in the evaluated cheeses underscores the need for stringent control measures to prevent this mycotoxin in milk used for cheese production in the study area, with the aim of protecting public health and reducing significant economic losses for producers.

THE MEDIATING EFFECT OF PERSONAL VALUES ON THE INFLUENCE OF HUMAN CAPITAL ON THE PRODUCTION OF ARTISANAL CHEESES

This study proposed the identification of personal values and their role as mediation between human capital and the expression of quality. The relationship between these factors in artisanal food production in small companies is still little explored, so this work has the objective of bringing advances in this theme. For the analysis of the results, a quantitative method was used, structural model equations, through the partial least squares regression method (PLS). The results found were satisfactory, demonstrating that investments in human capital must be the object of public policies, as their influence overlaps the influence of the personal values of each entrepreneur for the expression of quality conventions. The assumptions about the effect of mediation of values in the relationship between human capital and quality worlds are positive and significant, demonstrating that the values had a complementary partial mediation effect between human capital and quality, but with less importance than the direct impact of human capital in quality. It was possible to verify the importance of personal experience, combined with constant training for the development of human capital in the company. In small companies, as is the case with the production of artisanal Brazilian cheeses, in which production and handling are under the control of the family, the contribution of human capital to quality is even more evident. At the same time, personal values have a strong influence on small companies, where the characteristics, behavior, attitudes and beliefs of the entrepreneur are decisive for the results of the enterprise.

Analysis of total, thermotolerant and Salmonella spp. in artisanal cheeses with the ARTE Seal sold in Ribeirão Preto – SP

Analysis of total, thermotolerant and Salmonella spp. in artisanal cheeses with the ARTE Seal sold in Ribeirão Preto – SP

Novel Peptides in Fermented Milk with Specific Lactobacillus Strains Potential Antiobesity Effect: In Vitro and in Silico Analysis

The objective was to investigate the potential antiobesity effect of fermented milks (FMs) with 11 strains of specific Lactobacillus strains isolated from artisanal Mexican cheeses. First, pancreatic lipase inhibitory activity (PLIA) was determined in all FMs. Then, the FM with the most PLIA was selected for peptide identification by high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS–MS) and for adipogenesis studies. FMs with Limosilactobacillus fermentum J20 (J20) displayed the highest (p < 0.05) PLIA (81.70 ± 2.24%) and the lowest IC50 (0.05 0.05 mg/mL) of all. Also, FM J20 inhibited lipid accumulation in a 3T3-L1 cell line. Eight novel identified peptides released by in silico digestion from the fraction with the most PLIA (p < 0.05) presented sequences that strongly bind enzyme active sites and may be responsible for lipid accumulation inhibition during adipogenesis. Thus, FM J20 may be used for the development of a dairy product with potential antiobesity effects.

Seasonal variation in the Canastra cheese mycobiota.

Canastra cheese is the most well-known artisanal cheese produced in Brazil. Although its production includes a step to remove fungi from the cheese surface, in recent years some cheesemakers have preserved the autochthonous fungi grown during ripening due to an interest in the sensory characteristics attributed to these microorganisms. In this work, the mycobiota of artisanal cheeses produced in the Canastra region was characterized based on ITS marker gene analysis. A total of 96 artisanal cheeses from 16 different farms across 9 cities were collected during two different periods (dry and wet seasons). The Canastra cheese mycobiota was significantly impacted by the season, the city of production and the farm but altitude did not affect the fungal community of the cheeses analyzed. Debaryomyces prosopidis was most abundant in the majority of samples across both seasons. During the wet season, Trichosporon asahii, Kluyveromyces lactis and Fusarium solani were the next most abundant species, followed by Torulaspora delbrueckii and Acremonium citrinum. These results highlight the importance of manufacturing practices and seasonality on the fungal composition of Canastra cheeses. These insights are particularly important in light of recent new regulation in Brazil, removing previous obstacles for surface fungi to persist on cheese. These new regulations will allow new approaches to cheese production, and ultimately, novel products.

Comparative Genomic Analysis Reveals the Functional Traits and Safety Status of Lactic Acid Bacteria Retrieved from Artisanal Cheeses and Raw Sheep Milk.

Lactic acid bacteria (LAB) are valuable for the production of fermented dairy products. We investigated the functional traits of LAB isolated from artisanal cheeses and raw sheep milk, assessed their safety status, and explored the genetic processes underlying the fermentation of carbohydrates. Lactiplantibacillus plantarum had the largest and more functional genome compared to all other LAB, while most of its protein-encoding genes had unknown functions. A key finding of our analysis was the overall absence of acquired resistance genes (RGs), virulence genes (VGs), and prophages, denoting that all LAB isolates fulfill safety criteria and can be used as starter or adjunct cultures. In this regard, the identified mobile genetic elements found in LAB, rather than enabling the integration of RGs or VGs, they likely facilitate the uptake of genes involved in beneficial functions and in the adaptation of LAB in dairy matrices. Another important finding of our study was that bacteriocins and CAZymes were abundant in LAB though each species was associated with specific genes, which in turn had different activity spectrums and identified applications. Additionally, all isolates were able to metabolize glucose, lactose, maltose, and sucrose, but Lactiplantibacillus plantarum was strongly associated with the fermentation of rhamnose, mannose, cellobiose, and trehalose whereas Levilactobacillus brevis with the utilization of arabinose and xylose. Altogether these results suggest that to fully exploit the beneficial properties of LAB, a combination of strains as food additives may be necessary. Interestingly, biological processes involved in the metabolism of carbohydrates that are not of direct interest for the dairy industry may yield valuable metabolites or activate pathways associated with beneficial health effects. Our results provide useful information for the development of new probiotic artisanal cheeses and probiotic starter cultures.

Evaluation of gamma-aminobutyric acid content in Portuguese cheeses with protected designation of origin status.

Health-conscious consumers are increasingly paying attention to healthy diets and focusing on natural bioactive compounds in foods and their effects on mental health. This opens new opportunities for the study of artisanal cheeses as biofunctional foods. In the work described in this Research Communication, the gamma-aminobutyric acid (GABA) content of seven different Portuguese cheeses produced from unpasteurized cow, sheep, and goat milk and granted with protected designation of origin (PDO) status was analysed. The PDO cheeses made from cow milk analysed in this study were São Jorge (3, 4, 7, 12 and 24 months of maturation) and Pico cheeses. PDO cheeses made from sheep milk were Serra da Estrela, Serpa, Nisa and Azeitão. Cheeses made from sheep and goat milk included Beira Baixa yellow cheese. The GABA content in the Azorean PDO cheeses (made from cow milk) ranged from 1.23 to 2.64 g/kg of cheese. Higher variations in GABA content were observed in cheeses made from sheep and goat milk (0.73-2.31 g/kg). This study provides information on the GABA content in different Portuguese PDO cheeses and shows that hard or semi-hard ripened cheeses are a suitable matrix for GABA production by lactic acid bacteria.

Characterisation and sensory appraisal of fresh 'molido' cheese from cows fed different levels of sunflower silage inclusion in small-scale dairy systems.

Small-scale dairy systems require feeding strategies to improve their productivity and sustainability. These systems in central Mexico mostly sell their milk to local artisan cheese makers. Cow diets influence milk and dairy products composition; therefore, the objective was to evaluate the effect on physicochemical characteristics, sensory appraisal, and economic performance of cheese made with milk from cows fed different levels of sunflower silage (SFSL). Treatments were the inclusion of SFSL in the forage DM component of diets (T0 = 0% SFSL; T20 = 20%; T40 = 40%; and, T60 = 60%). Physicochemical composition was analysed by analysis of variance in a completely randomised design and the sensory assessment of the cheese was with a hedonistic five-point scale and the results were analysed by means of descriptive statistics and a radar graph. Partial budgets were used for economic analyses. There were significant differences (P < 0.05) for all physicochemical variables (moisture content, cheese yield, protein, fat content, and pH) of the cheese, with higher values for yield, protein, and fat content in the treatments that included SFSL. Sensory assessment values were in the average perception range of judges. Treatments that included SFSL had lower production costs, higher margins over costs, and higher returns/costs ratios. The inclusion of sunflower silage at 40 and 60% of the forage component of diets for dairy cows in small-scale dairy systems had positive effects. Yields of cheese as well as protein and fat content were higher. There were positive sensory attributes that meant acceptance by consumers, lower production costs, and higher incomes from cheese making.