Cheese Ripening Research Articles

Characterization of Microbial Shifts during the Production and Ripening of Raw Ewe Milk-Derived Idiazabal Cheese by High-Throughput Sequencing.

Simple SummaryIdiazabal is a traditional cheese produced from raw ewe milk in the Basque Country (Southwestern Europe). The sensory properties of raw milk cheeses have been attributed, among other factors, to microbial shifts that occur during the production and ripening processes. In this study, we used high-throughput sequencing technologies to investigate the microbiota of Latxa ewe raw milk and the dynamics during cheese production and ripening processes. The microbiota of raw milk was composed of lactic acid bacteria (LAB), environmental bacteria and non-desirable bacteria. Throughout the cheese making and ripening processes, the growth of LAB was promoted, whereas that of non-desirable and environmental bacteria was inhibited. Moreover, some genera not reported previously in raw ewe milk were detected and clear differences were observed in the bacterial composition of raw milk and cheese among producers, in relation to LAB and environmental or non-desirable bacteria, some of which could be attributed to the production of flavour related compounds.In this study, we used high-throughput sequencing technologies (sequencing of V3–V4 hypervariable regions of 16S rRNA gene) to investigate for the first time the microbiota of Latxa ewe raw milk and the bacterial shifts that occur during the production and ripening of Idiazabal cheese. Results revealed several bacterial genera not reported previously in raw ewe milk and cheese, such as Buttiauxella and Obesumbacterium. Both the cheese making and ripening processes had a significant impact on bacterial communities. Overall, the growth of lactic acid bacteria (LAB) (Lactococcus, Lactobacillus, Leuconostoc, Enterococcus, Streptococcus and Carnobacterium) was promoted, whereas that of non-desirable and environmental bacteria was inhibited (such as Pseudomonas and Clostridium). However, considerable differences were observed among producers. It is noteworthy that the starter LAB (Lactococcus) predominated up to 30 or 60 days of ripening and then, the growth of non-starter LAB (Lactobacillus, Leuconostoc, Enterococcus and Streptococcus) was promoted. Moreover, in some cases, bacteria related to the production of volatile compounds (such as Hafnia, Brevibacterium and Psychrobacter) also showed notable abundance during the first few weeks of ripening. Overall, the results of this study enhance our understanding of microbial shifts that occur during the production and ripening of a raw ewe milk-derived cheese (Idiazabal), and could indicate that the practices adopted by producers have a great impact on the microbiota and final quality of this cheese.

Effect of ripening and in vitro digestion on free amino acids and Angiotensin I converting enzyme inhibitory (ACE-I) potential of cow and Buffalo milk cheddar cheeses

ABSTRACT Cheddar cheese is a source of a variety of biologically active peptides and free amino acids (FAAs), which contribute to the cheese aroma, flavors, and textures. The FAA liberation and angiotensin-converting enzyme inhibition potential of cheddar cheese made from cow and buffalo milk before and after in vitro digestion by gastric and duodenal enzymes with ripening were investigated. The cheese samples were stored under refrigeration condition (4°C) for ripening for 3, 6, and 9 months. The possible differences existing in the level of FAAs were explored before and after in vitro digestion of ripened cheddar cheese made from cow and buffalo milk. The lower level of angiotensin-converting enzyme inhibition value 62.4 ± 1.04% was observed in gastric digested cheese samples after 9 months of ripening, while the highest angiotensin-converting enzyme inhibition value 93.1 ± 1.29% was observed in duodenal digested cheese samples after 6 months of ripening. This study showed that before and after in vitro digestion, highest angiotensin-converting enzyme inhibitory potential of cheddar cheese was observed between 6 and 9 months of ripening. In 9-month ripened cheese, a significant increase in the contents of glutamic, glutamine, valine, leucine, and lysine was observed after duodenal digestion in case of both cow and buffalo milk Cheddar cheeses. The contents of valine, ornithine, and tryptophan were negligible before digestion and significantly increased afterward.

Characterization of autochthonal Hafnia spp. strains isolated from Spanish soft raw ewe's milk PDO cheeses to be used as adjunct culture

The present work was performed to study the enterobacteria involved in the ripening of the artisanal raw ewe's milk PDO cheeses ‘Torta del Casar’ and ‘Queso de la Serena’ produced in Extremadura (Spain). These isolates were strain-typed, safety tested and characterized for some important technological properties. A total of 485 enterobacterial isolates were clustered by RAPD-PCR and subsequently identified by partial sequencing of the 16S rRNA gene. Among the 17 different species identified, Hafnia paralvei was the predominant species; H. alvei and Lelliottia amnigena were present to a lesser extent. Therefore, 55 Hafnia spp. strains, selected according to their genetic profile and dairy origin, were tested for the safe application. Overall, they were able to produce the biogenic amines putrescine and cadaverine under favourable conditions, presented α-haemolytic activity and did not produce cytolytic toxin active against HeLa cells or contain virulence genes. In addition, antibiotic susceptibility profiles showed that 17 Hafnia spp. strains were less resistant to the 33 antibiotics tested; subsequently, they were further technologically characterized. Although they showed differences, in general, they were well adapted to the stress conditions of cheese ripening. Among them, two strains, H. alvei 544 and 1142, are highlighted mainly due to their proteolytic activity at refrigeration temperatures and their low or null gas production. Although further studies are necessary before industrial application, these two strains are proposed for potential use as adjunct cultures to favour the homogeneity of these PDO cheeses, preserving their unique sensory characteristics.

Do 'cheese factory-specific' mites (Acari: Astigmata) exist in the cheese-ripening cabinet?

To determine whether the mites used in the ripening process of traditional cheeses are genetically unique to cheese factories, we investigated mites from three types of traditional cheeses, that use mites in the ripening process: 'Würchwitzer Milbenkäse' from Germany and 'Mimolette' and 'Artisou' from France. In addition, traditional ripened cheeses were purchased from cheese specialty stores in France (Mimolette) and Japan ('Laguiole' from France) as well as stores in temporary markets in France ('Salers' and 'Cantal vieux') and the mites obtained from those cheeses were analyzed in this study. Partial sequences of the 28S rRNA gene (28S) were determined and used to reconstruct a phylogenetic tree. Tyrolichus casei, the dominant cheese mite species from the ripening cabinets of three traditional cheese producers and two cheese specialty stores in France and Japan, had identical partial 28S sequences. All specimens from Cantal vieux from a store in the temporary market in France had an identical sequence with Acarus siro and Acarus immobilis in the determined region of the 28S sequences. Mite individuals from Salers from a store in the temporary markets in France shared the same haplotype as Acotyledon paradoxa. For the T. casei individuals from five different localities (19 individuals in total), the nuclear loci were obtained using MIG-seq. More than several thousand genomic regions are amplified simultaneously by multiplex PCR, and targeting regions surrounded by inter-simple sequence repeats (ISSRs) in the genome were sequenced using the MiSeq system (Illumina). SNPs extracted from this genome-wide analysis showed that no genetic structure existed in the populations from any region. Among the five samples from the three regions, which were more than 500km apart and from completely different environments, the mites had no geographic bias, but all mite individuals were genetically nearly identical. Thus, we found no evidence to support the existence of 'cheese factory-specific' T. casei mites, at least in terms of genetic analysis.

Investigation of the Effect of Technological Processing on the Quality of Goat's Milk Cheese

In this article, the possibility of using goat's milk in the production of cheeses is investigated and justified. In order to improve the functional and technological properties of cheeses, the influence of technological processes on the quality indicators of cheeses has been studied. Taking into account the conducted research and practical experience, it is advisable to carry out the maturation of thermized goat's milk at 65°C, followed by its pasteurization at 72-75°C. The use of partial salting of cheese in grain in an amount of 0.5-0.7 kg of table salt per 100 kg of milk increases the moisture content of the product by 2.2-2.8%, provides a more uniform salting of cheese heads and reduces the duration of salting by 1-2 days. Reducing the duration of salting, maturation in brine and carrying out brine-free storage of cheeses helps to reduce the content of sodium chloride to 4-5% versus 6-7% in control. Thus, it was found that reducing the duration of salting brine cheeses in brine 20-22% concentration to 4-5 days instead of 20-30 and carrying out without brine maturation increases the moisture content in the finished product by 2.5-3.5% and reduces the salt content by 2.0-2.5%, which intensifies the process of cheese maturation. Production modes have been optimized and the expediency of using bacterial starter culture and a preparation for brine cheeses consisting of salt-resistant strains of mesophilic lactic acid streptococci and L.casei sticks has been proved. This makes it possible to intensify microbiological and biochemical processes and accelerate the maturation of cheeses by 2 times.

The identification of antioxidant and ACE-I peptides in different turkish ripened cheeses.

The online version contains supplementary material available at 10.1007/s13197-022-05414-3.

Effect of sake lees on cheese components in cheese ripened by Aspergillus oryzae and lactic acid bacteria

More than 2,000 varieties of cheese currently exist in the world, and cheese manufacture continues to flourish. To develop the cheese ripening process, additional ingredients are used during cheese production. In this study, the effect of sake lees as an additional ingredient on the fermentation of cheese using Aspergillus oryzae (koji mold), known as koji cheese, was investigated. Aspergillus oryzae is used in the fermentation of Japanese traditional foods, such as sake and soy sauce, given its strong enzymatic activities, as well as in cheese production (i.e., koji cheese). Sake lees, a by-product of the fermentation of rice with A. oryzae and yeasts in the sake brewing process, contains various metabolites, such as amino acids. Here, supplementation with sake lees enhanced the activities of lactic acid bacteria and affected the color of the cheese. Metabolome analysis revealed that sake lees altered the balance of carbohydrates and fatty acids in the cheese. Remarkably, supplementation with sake lees enhanced the production of umami-enhancing γ-glutamyl (kokumi-active) peptides. This study suggests that a new type of cheese can be produced using A. oryzae and sake lees, and information on the synergistic effects of A. oryzae and sake lees will aid the development of cheese production.

Cloning, purification, and characterization of branched-chain α-keto acid decarboxylases from Lactococcus lactis strains with different 3-methylbutanal production abilities

Lactococcus lactis is an important food-grade microorganism that has been successfully applied as a starter to increase the level of 3-methylbutanal produced during the ripening of cheese. Three variants of branched-chain α-keto acid decarboxylase (KADC) were discovered in L. lactis strains with different 3-methylbutanal production abilities. Three genes encoding KADCs of varying lengths (KADC-long, KADC-middle, and KADC-short) were cloned and heterologously expressed into Escherichia coli. KADC activity was only detected in the E. coli cloned with the KADC-long-encoding gene. Homology modeling of the three KADC recombination proteins showed that an active-site residue (Glu462) and an S-pocket structure were necessary for the ability to catalyze substrates. KADC-long showed maximum activity at pH 7.0 and 30 °C. The substrate hydrolysis and kinetic parameters demonstrated that KADC-long efficiently produces 2-methylbutanal and 3-methylbutanal. The heterologous expression of the full-length kdcA in low-3-methylbutanal-yield L. lactis strains increased their production yields. The results of this study demonstrate the function of the complete KADC in 3-methylbutanal production.

Determination of probiotic and some technological properties of lactic acid bacteria isolated from cheeses sold in the Kilis region, Turkey

Aims: The purpose of this study is to evaluate technological properties and probiotics potential of lactic acid bacteria (LAB) isolated from 15 raw milk Turkish Beyaz fresh cheese made in the Kilis region, Turkey.Methods and Results: 287 colonies from 15 cheese samples were selected and 91 of them were analyzed as Gram-positive and catalase-negative. 19 strains of 91 colonies were accepted as potential probiotics since they stayed alive in pH 3.5 and 0.3% bile salt. They were identified as Lactobacillus sp. (11 strains), Leuconostoc sp. (3 strains), Streptococcus sp. (2 strains), Lactococcus sp. (2 strains), and Enterococcus sp. (1 strain) by biochemical tests and API test kit. It was determined that 19 strains with high acidification rate, 15 strains demonstrated weak proteolytic activity, 3 ones moderate, and 1 strong proteolytic activity. It was determined that 1 of the strains were resistant to chloramphenicol, 2 to tetracycline, and 6 to vancomycin. No strains resistant to penicillin and erythromycin could be detected. In the artificial gastric juice resistance test, although no viability was detected at pH 2.0. But it was determined that the viability values varied between 89.49-111.79% at pH 3.0. Also, these strains showed growth at bile salt. According to plasmid profiles of strains, 13 of 19 potential probiotic strains were determined to have plasmid DNA in the range of 1-5, while other strains were determined to not have plasmid DNA. The molecular sizes of the plasmid DNA of the isolated strains ranged from 2-16 kb.Conclusions: In the research, it was determined that LABs isolated from Turkish white fresh cheese produced in Kilis have probiotic potential and can be used as starters in various fermented foods.Significance and Impact of the Study: According to the results obtained, it was concluded that some strains can be used as probiotic starter culture in cheese production and others can be used for ripening of cheese.

The Reduction of Salt in Different Cheese Categories: Recent Advances and Future Challenges.

Public awareness about excessive sodium intake and nutrition claims related to salt content entail the need for food industries to carefully reconsider the composition and processing of high sodium foods. Although in some products the reformulation with alternative ingredients is commonly practiced, in cheese the reduction of salt is still a challenging task, as sodium chloride exerts multiple and fundamental functions. Salt favors the drainage of the residual whey, enhances the taste and the aroma profile, regulates the texture, the final pH, the water activity, and affects the microbial growth. Ultimately, salt content modulates the activity of starter and non-starter lactic acid bacteria (NSLAB) during cheese manufacturing and ripening, influencing the shelf-life. Any modification of the salting procedure, either by reducing the level of sodium chloride content or by replacing it with other salting agents, may affect the delicate equilibrium within the above-mentioned parameters, leading to changes in cheese quality. The decrease of Na content may be differently approached according to cheese type and technology (e.g., soft, semi-hard, hard, and mold-ripened cheeses). Accordingly, targeted strategies could be put in place to maintain the overall quality and safety of different cheeses categories.

Analysis of antimicrobial whey protein‐based biocomposites with lactic acid, tea tree (Melaleuca alternifolia) and garlic (Allium sativum) essential oils for Edam cheese coating

To protect fermented cheese from spoilage during the cheese maturation process, polymeric films are used. However, aiming for sustainability, interest in protein‐based biodegradable and antimicrobial coatings in the dairy industry has rapidly increased. Therefore, the objective of this study was to improve and analyse whey protein‐based coatings with different polysaccharides such as pectin, sodium alginate and carboxymethylcellulose, and different antimicrobial agents such as montmorillonite, garlic essential oil, tea tree essential oil and lactic acid. The results show that sodium alginate could be combined with lactic acid and used for further application.

Impact of an Omega-3-Enriched Sheep Diet on the Microbiota and Chemical Composition of Kefalograviera Cheese.

Kefalograviera is a well-known hard Greek cheese. The aim of this study was to determine how milk produced from ewes fed omega-3-enriched diets could influence the microbiota as well as the chemical composition of Kefalograviera cheese. At the start of the trial, 30 dairy ewes (Lesvos and Chios crossbreed) were selected and fed a conventional diet, based on alfalfa hay, straw and concentrate feed that contained soybean meal for a period of thirty days. Then, for a period of sixty days the same ewes were fed an omega-3-enriched concentrate feed with a lower level of soybean meal that contained 10% flaxseed and 10% lupins. Milk yield was collected individually on Days 30, 60 and 90 and used to produce three different batches of Kefalograviera cheeses, at the same cheese factory, by using a traditional recipe and identical preparation conditions (pasteurization of milk, salt, rennet and culture). Sample analysis was done after six months of Kefalograviera cheese ripening. MALDI-TOF-MS (matrix-assisted laser desorption/ionization time of flight mass spectrometry) identification was performed by contrasting the samples’ mass spectra with the corresponding reference database. The correlation between the different Kefalograviera cheeses revealed the predominant species being Lactococcus lactis, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus paracasei, Enterococcus faecium and Enterococcus faecalis, with significant quantitative differences between the experimental groups and the controls. Pediococcus spp. was isolated only from the experimental groups’ cheeses and Staphylococcus spp. only from the controls’ cheese, suggesting—among other differences—a bacterial microbiota distinction between the groups. Moreover, increased levels of alpha-linolenic acid and total polyunsaturated omega-3 fatty acids were noted in the enriched Kefalograviera cheeses. These promising findings suggest that enriched Kefalograviera cheese could be manufactured via enriching the ewes’ diets, with potential benefits for the consumers’ health.

The influence of milk protein cross-linking by transglutaminase on technology, composition and quality properties of Gouda-type cheese

The influence of TGase addition simultaneously with rennet on technology, ripening process, sensory properties, texture, microstructure and yield of Gouda-type cheese without inactivation of the enzyme was evaluated. Samples of control (C) and modified (M) Gouda-type cheeses were examined 1 day after brining and after 2, 4 and 6 weeks of ripening. TGase addition resulted in increased coagulation time and formation of a more delicate curd, difficult to process. The enzymatic modification of milk proteins resulted in significantly increased water content in cheese, causing changes in the content of free fatty acids and nitrogen fractions during cheese ripening, and had a negative impact on sensory properties, especially cheese structure, which was crumbly and brittle. Microstructure of ripened M was clearly layered and flaky. TGase modification caused formation of macromolecular protein aggregates. TGase application during production of Gouda-type cheese showed no positive influence on its technology, composition and most quality properties.

Antioxidant and antihypertensive activity of Gouda cheese at different stages of ripening.

In Mexico, local ripened cheeses such as Chihuahua, Ranchero, and Cotija are produced, being consumed in great quantities together with imported cheeses. Proteolysis that takes place during ripening generates bioactive peptides; in this way the cheese acquires potential as a functional food. The ripening process of Gouda cheese was studied based on its bromatological and sensorial properties, bioactivity, and peptide profile. Ripened cheese met bromatological standard parameters and showed higher overall acceptability. After 90days, bioactivity reached maximum values for radical scavenging (6.6%), ferric reducing power (11.2%), metal chelating effect (49%), and angiotensin I-converting enzyme inhibitory activity (66.2%). Eight peptides were identified, four from αS1-casein, f(1-9, 1-13, 1-14, and 25-36), and four from β-casein, f(11-28, 60-63, 193-209, and 197-205). Ripening of Gouda cheese results in a product with functional potential due to the presence of peptides with biological activity. Additionally, the methodology proposed in this work could be used by the dairy industry to monitor the manufacturing process and ripening of other types of cheeses.

Chemical and Flavor Characteristics of Enzyme-Modified Cheese Made by Two-Stage Processing.

(1) Background: to date, a clear description of the impact of specific enzymes on the enzyme-modified cheese (EMC) flavor is lacking. Moreover, comparative studies on the aroma compounds’ intensity of EMC have been rarely investigated. Therefore, this study was done to determine the influence of incubating substrates with proteases and different lipases on cheese ripening index and aroma compounds. (2) Methods: two-stage processing was adopted; proteolysis followed by lipolysis. (3) Results: results showed that the usage of Flavourzyme may improve the value of pH 4.6-WSN/TN%. Butanoic acid and hexanoic acid have a significant influence on the overall flavor of EMCs. In particular, the ethenyl acetate compound was detected in all products and was perceived as a fruity and sweet aroma, which has not been reported in previous literature. The concentration of short-chain fatty acids of EMCs made by Lipase MER was higher than EMCs made by Palatase, while the total content of medium and long-chain fatty acids of EMCs made by Lipase MER was lower than EMCs made by Palatase. The percentage of esters compounds in EMCs made by Lipase AY 30G was higher than the other two lipases, except EMC1. (4) Conclusions: Flavourzyme may be used to speed up the ripening of cheeses that need extensive proteolysis. The ability of Lipase MER to hydrolyze short-chain fatty acids was stronger than that of Palatase, while the ability of Lipase MER to hydrolyze medium and long-chain fatty acids was weaker than that of Palatase. The use of Lipase AY 30G was accompanied by the production of some other flavor esters, which made the final hydrolysates more fragrant and may be a good choice to produce fruity cheese flavor EMC. While Lipase MER may barely contain ester activity. This study may provide a reference for the selection of incubated enzymes for specific flavor EMC.

Metatranscriptomic Analyses Unravel Dynamic Changes in the Microbial and Metabolic Transcriptional Profiles in Artisanal Austrian Hard-Cheeses During Ripening.

Vorarlberger Bergkäse (VB) is an artisanal Austrian washed-rind hard cheese produced from alpine cows’ raw milk without the addition of ripening cultures. Ripening time is a key factor in VB, as it strongly influences the microbial communities present in the cheeses and the organoleptic properties of the product. In this study, the microbial and metabolic transcriptional profiles in VB rinds at different ripening times were investigated. VB products before (30 days of ripening) and after (90 days of ripening) selling were selected, RNA was extracted and subjected to shotgun metatranscriptomic sequencing. The analysis revealed some of the previously described abundant bacterial taxa of Brevibacterium, Corynebacterium, Halomonas, Psychrobacter, and Staphylococcus to be highly active in VB rinds. Additionally, the investigation of most important metabolic pathways in cheese ripening clearly showed differences in the gene transcription profiles and the active microbiota between the two ripening points investigated. At 30 days of ripening, metabolic events related with the degradation of residual lactose, lactate, citrate, proteolysis, and lipolysis were significantly more transcribed and mainly associated with Staphylococcus. On the other hand, genes involved in the degradation of smaller compounds derived from previous metabolism (i.e., metabolism of free amino acids and fatty acids) were significantly more expressed in VB rinds with 90 of ripening, and mainly associated with Brevibacterium and Corynebacterium. These latter metabolic activities are responsible of the generation of compounds, such as methanethiol and 2,3-butanediol, that are very important for the flavor and aroma characteristics of cheeses. This study shows the dynamic changes in the gene transcriptional profiles associated with energy substrates metabolism and the generation of organoleptic compounds during VB ripening and uncovers bacterial taxa as key drivers of the ripening process. These taxa might be the target for future studies toward an accelerated cheese ripening and the enhancement of its organoleptic properties.

Determination of Virulence Factors and Antibiotic Resistances of Enterococcus spp. Identified from Different Stages of Ripened (Classical) White Cheese Production

The objective of this research was to determine the presence of virulence genes (asa1, gelE, cylA, ace, esp, hyl and efaA), and vancomycin resistance genes (vanA, vanB, vanC2/C3) and the resistance to some antibiotics of Enterococcus spp. isolates previously identified from different stages of ripened white cheese production. In addition, gelatinase, β-hemolytic and DNase activity, and biofilm formations were examined phenotypically. In this study, efaA in 95.9%, asa1 in 89%, ace in 68.5%, esp in 52.1%, gelE in 78.1%, cylA in 16.4% and hyl in 23.3% of isolates were detected. Also, vanA in 31.5%, vanB in 8.2%, and in vanC2/C3 23.3% resistance genes were determined. β-hemolytic and DNase activity were detected in 23.2% and 16.4% of the isolates, while gelatinase activity and biofilm formation could not be detected phenotypically. Moreover, streptomycin and erythromycin resistances were found in 73.9% and in %43.8 of isolates. As a result, it was concluded that Enterococcus spp. may pose a risk for public health and food safety in terms of their virulence factors and antibiotic resistance. For this reason, it was suggested that the strains to be selected as starter cultures should be used after evaluating their virulence factors and resistance to antibiotics.

Formation and dynamics of aroma compounds during manufacturing-ripening of Bouhezza goat cheese

The aromatic profile development of Bouhezza goat cheese during manufacturing-ripening in traditional goatskin bags was assessed using solid-phase microextraction (SPME) and steam distillation (SD) coupled to gas chromatography-olfactometry. Sixty different odour-active compounds (OACs) were revealed. The volatile profile of Bouhezza goat cheese changed during cheese ripening, but ester and terpene compounds dominated throughout manufacturing-ripening. During ripening the number of alcohols, esters, heterocyclic compounds, ketones, sulphurs, terpenes and tiazole increased gradually. Cheese aroma results differed between the extraction methods; thiazole and sulphurs were better extracted by SPME, but heterocyclic compounds were exclusively extracted using SD. SD revealed a more complete volatile and aromatic profile of Bouhezza compared with SPME; however, the low level of common OACs showed that the methods are complementary to each other and SPME cannot be neglected. The application of different extraction methods helps to achieve a complete description of the aromatic profile of traditional cheeses.

Characterization of autochthonal yeasts isolated from Spanish soft raw ewe milk protected designation of origin cheeses for technological application

The yeasts involved in the ripening process of artisanal soft raw ewe milk Protected Designation of Origin (PDO) Torta del Casar and Queso de la Serena cheeses produced in Extremadura, Spain, were isolated throughout their ripening process, strain typed, and characterized for some important technological properties. A total of 508 yeast isolates were obtained and identified by inter-single sequence repeat anchored PCR amplification analysis and subsequent sequencing of the internal transcribed spacer ITS1/ITS2 5.8S rRNA. A total of 19 yeast species representing 8 genera were identified. Debaryomyces hansenii, Pichia kudriavzevii, Kluyveromyces lactis, and Yarrowia lipolytica were the predominant species. We selected 157 isolates, by genotyping and origin, for technological characterization. The evaluation of yeast isolates' growth under stress conditions of cheese ripening showed that 87 presented better performance. Among them, 71 isolates were not able to catabolize tyrosine to produce a brown pigment. Principal component analysis of the biochemical features of these isolates showed that 9 strains stood out, 3 K. lactis strains (2287, 2725, and 1507), 2 Pichia jadinii (1731 and 433), 2 Yarrowia alimentaria (1204 and 2150), Y. lipolytica 2495 and P. kudriavzevii 373. These strains displayed strong extracellular proteolytic activity on skim milk agar as well as an adequate enzymatic profile (strong aminopeptidase and weak protease activity), suggesting their great potential for cheese proteolysis. Extracellular lipolytic activity was mainly restricted to Yarrowia spp. isolates and weakly present in P. kudriavzevii 373 and K. lactis 2725, although enzymatic characterization by API-ZYM (bioMérieux SA) evidenced that all may contribute, at least in part, to the lipolysis process. Moreover, these strains were able to assimilate lactose, galactose, and glucose at NaCl concentrations higher than that usually found in cheese. However, lactate and citrate assimilation were limited to Y. lipolytica 2495, P. kudriavzevii 373, and P. jadinii 433, and may contribute to the alkalinizing process relevant to biochemical processes that take place in the last stages of ripening. By contrast, K. lactis strains showed acidifying capacity and β-galactosidase activity and may take part in the initial stages of ripening, together with lactic acid bacteria. Thus, considering the technological characteristics studied, the 9 selected strains presented biochemical features well suited to their potential use as adjunct cultures, alone or in combination with autochthonous starter bacteria in the cheesemaking process, to overcome the heterogeneity of these PDO cheeses, preserving their unique sensory characteristics.

Possible inhibitory effect of microbial transglutaminase on the formation of biogenic amines during Trappist cheese ripening

ABSTRACT Trappist cheese (semi-hard, rennet-coagulated cheese with round eyes) was manufactured and matured for 4 weeks at 12 ± 1°C, 85% relative humidity (RH). The effect of microbial transglutaminase (MTGase) was followed by measuring the levels of free amino acids (FAAs) and biogenic amines (BAs) every 2 weeks during 4 weeks of cheese ripening. Results show that MTGase can decrease the cadaverine production by 30%, but only at the initial stage of ripening. Application of MTGase results in 49% less putrescine, 12% less tyramine production at the end of 4 weeks ripening time, and can decrease histamine levels by 8% after 2 weeks of ripening time in the examined semi-hard cheese type.