Volatilome of brine-related microorganisms in a curd-based medium.

The possible contribution of brine-derived microflora to the sensory attributes of cheese is still a rather unexplored field. In this study, 365 bacteria and 105 yeast strains isolated from 11 cheese brines were qualitatively tested for proteolytic and lipolytic activities, and positive strains were identified by sequencing. Among bacteria, Staphylococcus equorum was the most frequent, followed by Macrococcus caseolyticus and Corynebacterium flavescens. As for yeasts, Debaryomyces hansenii, Clavispora lusitaniae, and Torulaspora delbrueckii were most frequently identified. A total of 38% of bacteria and 59% of yeasts showed at least 1 of the metabolic activities tested, with lipolytic activity being the most widespread (81% of bacteria and 95% of yeasts). Subsequently 15 strains of bacteria and 10 yeasts were inoculated in a curd-based medium and assessed via headspace-solid phase microextraction coupled with gas chromatography-mass spectrometry to determine their volatilome. After a 30-d incubation at 12°C, most strains showed a viability increase of about 2 log cfu/mL, suggesting good adaptability to the cheese environment. A total of 26 compounds were detected in the headspace, carbonyl compounds and alcohols being the major contributors to the volatile profile of the curd-based medium. Multivariate analysis was carried out to elucidate the overall differences in volatiles produced by selected strains. Principal component analysis and hierarchical clustering analysis demonstrated that the brine-related microorganisms were separated into 3 different groups, suggesting their different abilities to produce volatile compounds. Some of the selected strains have been shown to have interesting aromatic potential and to possibly contribute to the sensory properties of cheese.

Potential of histamine-degrading microorganisms and diamine oxidase (DAO) for the reduction of histamine accumulation along the cheese ripening process.

Lentilactobacillus parabuchneri is the main bacteria responsible for the accumulation of histamine in cheese. The goal of this study was to assess the efficiency of potential histamine-degrading microbial strains or, alternatively, the action of the diamine oxidase (DAO) enzyme in the reduction of histamine accumulation along the ripening process in cheese. A total of 8 cheese variants of cow milk cheese were manufactured, all of them containing L. parabuchneri Deutsche Sammlung von Mikroorganismen 5987 (except for the negative control cheese variant) along with histamine-degrading strains (Lacticaseibacillus casei 4a and 18b; Lactobacillus delbrueckiisubsp. bulgaricus Colección Española de Cultivos Tipo (CECT) 4005 and Streptococcus salivariussubsp.thermophilus CECT 7207; two commercial yogurt starter cultures; or Debaryomyces hansenii), or DAO enzyme, tested in each cheese variant. Histamine was quantified along 100 days of cheese ripening. All the degrading measures tested significantly reduced the concentration of histamine. The highest degree of degradation was observed in the cheese variant containing D. hansenii, where the histamine content decreased up to 45.32 %. Cheese variants with L. casei, or L. bulgaricus and S. thermophilus strains, also decreased in terms of histamine content by 43.05 % and 42.31 %, respectively. No significant physicochemical changes (weight, pH, water activity, color, or texture) were observed as a consequence of the addition of potential histamine-degrading adjunct cultures or DAO in cheeses. However, the addition of histamine-degrading microorganisms was associated with a particular, not unpleasant aroma. Altogether, these results suggest that the use of certain histamine-degrading microorganisms could be proposed as a suitable measure in order to decrease the amount of histamine accumulated in cheeses.

Revealing the microbial heritage of traditional Brazilian cheeses through metagenomics.

Brazilian artisanal cheeses date from the first Portuguese settlers and evolved via local factors, resulting in unique products that are now part of the patrimony and identity of different Brazilian regions. In this study, we combined several culture-independent approaches, including 16S/ITS metagenetics, assembly- and deep profiling-metagenomics to characterize the originality of the microbiota of five varieties of Brazilian artisanal cheeses from the South and Southeast regions of Brazil. Their core microbiota contained mainly lactic acid bacteria (LAB), of which Lactococcus lactis subsp. lactis was the most frequent, followed by Streptococcus thermophilus in the South region. Moreover, several samples from the Southeast region contained, as dominant LAB, two other food Streptococci belonging to a new species of the salivarius group and S. infantarius. Rinds of samples from the Southeast region were dominated by the halotolerant bacterium Corynebacterium variabile, and the yeasts Diutina catenulata, followed by Debaryomyces hansenii and Kodamaea ohmeri. Rinds from the South region contained mainly LAB due to their short ripening time, and the predominant yeast was D. hansenii. Phylogenomic analysis based on L. lactis metagenome-assembled genomes (MAGs) showed that most Brazilian strains are closely related and form a different clade from those whose genomes are available at this time, indicating that they belong to a specific group. Lastly, functional analysis showed that S. infantarius acquired a âˆ¼ 26 kb DNA fragment from S. thermophilus starter strains that carry the LacSZ system, allowing fast lactose assimilation, an adaptation advantage for growth in milk. Finally, our study identified several areas of concern, such as the presence of somatic cell DNA and high levels of antibiotic resistance genes in several cheese microbiota, suggesting that milk from diseased animals may still be used occasionally. Overall, the data from this study highlight the potential value of the traditional and artisanal cheese production network in Brazil, and provide a metagenomic-based scheme to help manage this resource safely.

Multiple microbial cell-free extracts improve the microbiological, biochemical and sensory features of ewes' milk cheese.

This study used cell-free enzyme (CFE) extracts from Lactobacillus casei, Hafnia alvei, Debaryomyces hansenii and Saccharomyces cerevisiae to condition or accelerate Pecorino-type cheese ripening. Compositional, microbiological, and biochemical analyses were performed, and volatile and sensory profiles were obtained. Lactobacilli and cocci increased during ripening, especially in cheeses containing CFE from L. casei, H. alvei and D. hansenii (LHD-C) and L. casei, H. alvei and S. cerevisiae (LHS-C). Compared to control cheese (CC), several enzymatic activities were higher (P < 0.05) in CFE-supplemented cheeses. Compared to the CC (1907 mg kg-1 of cheese), the free amino acid level increased (P < 0.05) in CFE-supplemented cheeses, ranging from approximately 2575 (LHS-C) to 5720 (LHD-C) mg kg-1 of cheese after 60 days of CFE-supplemented ripening. As shown by GC/MS analysis, the levels of several volatile organic compounds were significantly (P < 0.05) lower in CC than in CFE-supplemented cheeses. All cheeses manufactured by adding multiple CFEs exhibited higher scores (P < 0.05) for internal structure, acid taste and juiciness than CC samples. This study shows the possibility of producing ewes' milk cheese with standardized characteristics and improved flavor intensity in a relatively short time.

The effect of reduced sodium chloride content on the microbiological and biochemical properties of a soft surface-ripened cheese.

Many health authorities have targeted salt reduction in food products as a means to reduce dietary sodium intake due to the harmful effects associated with its excessive consumption. In the present work, we evaluated the effect of reducing sodium chloride (NaCl) content on the microbiological and biochemical characteristics of an experimental surface-ripened cheese. A control cheese (1.8% NaCl) and a cheese with a reduced NaCl content (1.3% NaCl) were sampled weekly over a period of 27d. Reducing NaCl content induced microbial perturbations such as the lesser development of the yeast Debaryomyces hansenii and the greater development of the gram-negative bacterium Hafnia alvei. This was accompanied by changes in proteolytic kinetics and in profiles of volatile aroma compounds and biogenic amine production. Finally, the development of the spoilage microorganism Pseudomonas fragi was significantly higher in the cheese with a reduced salt content.

Microbial cell-free extracts as sources of enzyme activities to be used for enhancement flavor development of ewe milk cheese.

Freeze-dried cell-free extracts (CFE) from Lactobacillus casei LC01, Weissella cibaria 1XF5, Hafnia alvei Moller ATCC 51815, and Debaryomyces hansenii LCF-558 were used as sources of enzyme activities for conditioning the ripening of ewe milk cheese. Compared with control cheese (CC), CFE did not affect the gross composition and the growth of the main microbial groups of the cheeses. As shown through urea-PAGE electrophoresis of the pH 4.6-soluble nitrogen fraction and the analysis of free AA, the secondary proteolysis of the cheeses with CFE added was markedly differed from that of the CC. Compared with CC, several enzyme activities were higher in the water-soluble extracts from cheeses made with CFE. In agreement, the levels of 49 volatile compounds significantly differentiated CC from the cheeses made with CFE. The level of some alcohols, ketones, sulfur compounds, and furans were the lowest in the CC, whereas most aldehydes were the highest. Each CFE seemed to affect a specific class of chemical compounds (e.g., the CFE from H. alvei ATCC 51815 mainly influenced the synthesis of sulfur compounds). Apart from the microbial source used, the cheeses with the addition of CFE showed higher score for acceptability than the control cheese. Cheese ripening was accelerated or conditioned using CFE as sources of tailored enzyme activities.

Isolation and identification of the microbiota of Danish farmhouse and industrially produced surface-ripened cheeses.

For studying the microbiota of four Danish surface-ripened cheeses produced at three farmhouses and one industrial dairy, both a culture-dependent and culture-independent approach were used. After dereplication of the initial set of 433 isolates by (GTG)5-PCR fingerprinting, 217 bacterial and 25 yeast isolates were identified by sequencing of the 16S rRNA gene or the D1/D2 domain of the 26S rRNA gene, respectively. At the end of ripening, the cheese core microbiota of the farmhouse cheeses consisted of the mesophilic lactic acid bacteria (LAB) starter cultures Lactococcus lactis subsp. lactis and Leuconostoc mesenteorides as well as non-starter LAB including different Lactobacillus spp. The cheese from the industrial dairy was almost exclusively dominated by Lb. paracasei. The surface bacterial microbiota of all four cheeses were dominated by Corynebacterium spp. and/or Brachybacterium spp. Brevibacterium spp. was found to be subdominant compared to other bacteria on the farmhouse cheeses, and no Brevibacterium spp. was found on the cheese from the industrial dairy, even though B. linens was used as surface-ripening culture. Moreover, Gram-negative bacteria identified as Alcalignes faecalis and Proteus vulgaris were found on one of the farmhouse cheeses. The surface yeast microbiota consisted primarily of one dominating species for each cheese. For the farmhouse cheeses, the dominant yeast species were Yarrowia lipolytica, Geotrichum spp. and Debaryomyces hansenii, respectively, and for the cheese from the industrial dairy, D. hansenii was the dominant yeast species. Additionally, denaturing gradient gel electrophoresis (DGGE) analysis revealed that Streptococcus thermophilus was present in the farmhouse raw milk cheese analysed in this study. Furthermore, DGGE bands corresponding to Vagococcus carniphilus, Psychrobacter spp. and Lb. curvatus on the cheese surfaces indicated that these bacterial species may play a role in cheese ripening.

Bactérias do acido láctico e leveduras associadas com o queijo-de-minas artesanal produzido na região da Serra do Salitre, Minas Gerais / Lactic acid bacteria and yeasts associated with the artisanal Minas cheese produced in the region of Serra do Salitre, Minas Gerais

Em 10 fazendas da região da Serra do Salitre, MG, foram coletadas amostras de leite, soro fermentado (pingo), coalhada e queijo frescal para avaliar a microbiota de bactérias láticas e leveduras presentes. Uma diversidade menor de bactérias láticas foi observada durante a produção do queijo quando comparada à de leveduras. As espécies de bactérias láticas mais freqüentes foram Lactococcus lactis, Enterococcus spp., Enterococcus faecalis e Streptococcus agalactiae e de leveduras foram Debaryomyces hansenii e Kluyveromyces lactis. Apenas as populações de Enterococcus spp., Enterococcus faecalis e Leuconostoc mesenteroides apresentaram aumento significativo durante a produção do queijo. As espécies de bactérias láticas e leveduras encontradas nos diferentes substratos estudados podem ser responsáveis pelas características de aroma e sabor do queijo artesanal da Serra do Salitre.

Samples of milk, curd, cheese whey, and cheese were collected in 10 farms located at the region of Serra do Salitre, Minas Gerais state. These samples were studied in relation to their lactic acid bacteria and yeast populations. The diversity of lactic acid bacteria species was lower than the diversity of yeasts in these samples. The isolated lactic acid bacteria were Lactococcus lactis, Enterococcus spp., Enterococcus faecalis, and Streptococcus agalactiae; and the yeasts were Debaryomyces hansenii and Kluyveromyces lactis. Only the species Enterococcus spp., Enterococcus faecalis, and Leuconostoc mesenteroides showed an increase in their populations during the production of the artisanal cheese. Lactic acid bacteria and yeasts found in this study could be responsible by the sensorial characteristics of the artisanal cheese produced in the region of Serra do Salitre.

Growth and aroma contribution of Microbacterium foliorum, Proteus vulgaris and Psychrobacter sp. during ripening in a cheese model medium.

The growth and aroma contribution of Microbacterium foliorum, Proteus vulgaris and Psychrobacter sp., some common but rarely mentioned cheese bacteria, were investigated in a cheese model deacidified by Debaryomyces hansenii during the ripening process. Our results show that these bacteria had distinct growth and cheese flavour production patterns during the ripening process. P. vulgaris had the greatest capacity to produce not only the widest variety but also the highest quantities of volatile compounds with low olfactive thresholds, e.g. volatile sulphur compounds and branched-chain alcohols. Such compounds produced by P. vulgaris increased after 21 days of ripening and reached a maximum at 41 days. The three bacteria studied exhibited various degrees of caseinolytic, aminopeptidase and deaminase activities. Moreover, P. vulgaris had a greater capacity for hydrolysing casein and higher deaminase activity. Our results show that P. vulgaris, a Gram-negative bacterium naturally present on the surface of ripened cheeses, could produce high concentrations of flavour compounds from amino acid degradation during the ripening process. Its flavouring role in cheese cannot be neglected. Moreover, it could be a useful organism for producing natural flavours as dairy ingredients.

Stability of the biodiversity of the surface consortia of Gubbeen, a red-smear cheese.

A total of 1,052 bacteria and 828 yeasts were isolated from the surface flora of 6 batches of Gubbeen cheese made in 1996-1997 and 2002-2003. Stability of the microflora was evaluated over time and also during ripening at 4, 10, and 16 d (batches 4, 5, and 6) or at 4, 16, 23, and 37 d (batches 1, 2, and 3). Bacteria were identified using pulsed-field gel electrophoresis, repetitive extragenic palindromic-PCR, and 16S rRNA gene sequencing, and yeasts were identified by Fourier transform infrared spectroscopy. The bacteria included at least 17 species, of which the most common were Staphylococcus saprophyticus (316 isolates), Corynebacterium casei (248 isolates), Brevibacterium aurantiacum (187 isolates), Corynebacterium variabile (146 isolates), Microbacterium gubbeenense (55 isolates), Staphylococcus equorum/cohnii (31 isolates), and Psychrobacter spp. (26 isolates). The most common yeasts were Debaryomyces hansenii (624 isolates), Candida catenulata (135 isolates), and Candida lusitaniae (62 isolates). In all batches of cheese except batch 2, a progression of bacteria was observed, with staphylococci dominating the early stages of ripening and coryneforms the later stages. No progression of yeast was found. Pulsed-field gel electrophoresis showed that several different strains of the 5 important species of bacteria were present, but generally only one predominated. The commercial strains used for smearing the cheese were recovered, but only in very small numbers early in ripening. Four species, B. aurantiacum, C. casei, C. variabile, and Staph. saprophyticus, were found on all batches of cheese, but their relative importance varied considerably. The results imply that significant variation occurs in the surface microflora of cheese.

A model describing Debaryomyces hansenii growth and substrate consumption during a smear soft cheese deacidification and ripening.

A mechanistic model for Debaryomyces hansenii growth and substrate consumption, lactose conversion into lactate by lactic acid bacteria, as well as lactose and lactate transfer from the core toward the rind was established. The model described the first step (14 d) of the ripening of a smear soft cheese and included the effects of temperature and relative humidity of the ripening chamber on the kinetic parameters. Experimental data were collected from experiments carried out in an aseptic pilot scale ripening chamber under 9 different combinations of temperature (8, 12, and 16 degrees C) and relative humidity (85, 93, and 99%) according to a complete experimental design. The model considered the cheese as a system with 2 compartments (rind and core) and included 5 state evolution equations and 16 parameters. The model succeeded in predicting D. hansenii growth and lactose and lactate concentrations during the first step of ripening (curd deacidification) in core and rind. The nonlinear data-fitting method allowed the determination of tight confidence intervals for the model parameters. The residual standard error (RSE) between model predictions and experimental data was close to the experimental standard deviation between repeated experiments.

Deacidification by Debaryomyces hansenii of smear soft cheeses ripened under controlled conditions: relative humidity and temperature influences.

Model smear soft cheeses were prepared from pasteurized milk inoculated with Debaryomyces hansenii (304, GMPA) and Brevibacterium aurantiacum (ATCC 9175) under aseptic conditions. Debaryomyces hansenii growth and curd deacidification were studied in relation to ripening chamber temperature and relative humidity (RH). A total of 9 descriptors, mainly based on kinetic data, were defined to represent D. hansenii growth (2 descriptors), cheese deacidification (5 descriptors), and cheese ripening (2 descriptors). Regardless of the temperature, when the RH was 85%, D. hansenii growth was inhibited due to limitation of carbon substrate diffusions; consequently, cheese deacidification did not take place. Debaryomyces hansenii growth was most prolific when the temperature was 16 degrees C, and the RH was 95%. Kinetic descriptors of lactate consumption and pH increase were maximal at 16 degrees C and 100% RH. Under these 2 ripening conditions, on d 14 (packaging) the creamy underrind represented a third of the cheese; however, at the end of ripening (d 42), cheese was too liquid to be sold. Statistical analysis showed that the best ripening conditions to achieve an optimum between deacidification and appearance of cheeses (thickness of the creamy underrind) were 12 degrees C and 95 +/- 1% RH.

Comparison of volatile compounds produced in model cheese medium deacidified by Debaryomyces hansenii or Kluyveromyces marxianus.

The aroma of a deacidified cheese medium is the result of the overall perception of a large number of molecules belonging to different classes. The volatile compound composition of (60%) cheese medium (pH 5.8) deacidified by Debaryomyces hansenii (DCM(Dh)) was compared with the one deacidified by Kluyveromyces marxianus (DCM(Km)). It was determined by dynamic headspace extraction, followed by gas chromatography separation and quantification as well as by mass spectrometry identification. Whatever the media tested, a first class of volatile compounds can be represented by the ones not produced by any of the yeasts, but some of them are affected by K. marxianus or by D. hansenii. A second class of volatile compounds can be represented by the ones produced by K. marxianus, which were essentially esters. Their concentrations were generally higher than their thresholds, explaining the DCM(Km) global fruity odor. A third class can be represented by the ones generated by D. hansenii, which were essentially methyl ketones with fruity, floral (rose), moldy, cheesy, or wine odor plus 2-phenylethanol with a faded-rose odor. The impact of methyl ketones on the DCMDh global flavor was lower than the impact of 2-phenylethanol and even negligible. Therefore, the global faded-rose odor of D. hansenii DCM can be explained by a high concentration of 2-phenylethanol.

Yeasts as adjunct starters in matured Cheddar cheese.

Debaryomyces hansenii and Yarrowia lipolytica are typical foodborne yeast species frequently associated with dairy products and capable of predominating the yeast composition in such systems. The two species fulfil a number of criteria to be regarded as co-starters for cheesemaking. They are known for their proteolytic and lipolytic activity as well as their compatibility and stimulating action with the lactic acid starter cultures when co-inoculated. Recent studies indicated that yeasts could be included as part of starter cultures for the manufacturing of cheese, enhancing flavour development during the maturation. The potential of D. hansenii and Y. lipolytica as agents for accelerated ripening of matured Cheddar cheese has been evaluated during four cheese treatments. The interaction between the two yeast species and the lactic acid bacteria was surveyed incorporating (i) D. hansenii, (ii) Y. lipolytica, (iii) both species as adjuncts to the starter culture and (iv) a control cheese without any additions for the production of matured Cheddar cheese. The physical and chemical properties of the cheeses were monitored in order to evaluate the contribution of the yeasts to cheese maturation. The yeasts grew in association with the lactic acid bacteria without any inhibition. The yeasts species when individually added contributed to the development of bitter flavours despite accelerated development of strong Cheddar flavours. When both species were incorporated as part of the starter culture, the cheese, however, had a good strong flavour after a reduced ripening period. The cheese retained this good flavour and aroma after 9 months of production. The simultaneous application of D. hansenii and Y. lipolytica as part of the starter culture for the production of matured Cheddar cheese is proposed.