Non-starter Lactic Acid Bacteria Research Articles

Autochthonous cultures to improve the quality of PGI Castellano cheese: Impact on proteolysis, microstructure and texture during ripening

The aim of this research was to find out the effect of different combinations of starter and non-starter cultures on the proteolysis of Castellano cheese during ripening. Four cheese batches were prepared, each containing autochthonous lactobacilli and or Leuconostoc, and were compared with each other and with a control batch, that used only a commercial starter. To achieve this, nitrogen fractions (pH 4.4-soluble nitrogen and 12 % trichloroacetic acid soluble nitrogen, polypeptide nitrogen and casein nitrogen), levels of free amino acids and biogenic amines were assessed. Texture and microstructure of cheeses were also evaluated. Significant differences in nitrogen fractions were observed between batches at different stages of ripening. The free amino acid content increased throughout the cheese ripening process, with a more significant increase occurring after the first 30 days. Cheeses containing non-starter lactic acid bacteria exhibited the highest values at the end of the ripening period. Among the main amino acids, GABA was particularly abundant, especially in three of the cheese batches at the end of ripening. The autochthonous lactic acid bacteria were previously selected as non-producers of biogenic amines and this resulted in the absence of these compounds in the cheeses. Analysis of the microstructure of the cheese reflected the impact of proteolysis. Additionally, the texture profile analysis demonstrated that the cheese's hardness intensified as the ripening period progressed. The inclusion of autochthonous non-starter lactic acid bacteria in Castellano cheese production accelerated the proteolysis process, increasing significantly the free amino acids levels and improving the sensory quality of the cheeses.

Brazilian indigenous nonstarter lactic acid bacteria enhance the diversification of volatile compounds in short-aged cheese.

There is growing interest in using autochthonous lactic acid bacteria (LAB) that provide unique sensory characteristics to dairy products without affecting their safety and quality. This work studied the capacity of three Brazilian indigenous nonstarter LABs (NSLAB) to produce biogenic amines (BAs) and evaluated their effect on the volatile organic compounds (VOCs), microbial LAB communities, and physicochemical profile of short-aged cheese. Initially, the strain's potential for biosynthesis of BAs was assessed by PCR and in vitro assays. Then, a pilot-scale cheese was produced, including the NSLAB, and the microbial and VOC profiles were analyzed after 25 and 45 days of ripening. As a results, the strains did not present genes related to relevant BAs and did not produce them in vitro. During cheese ripening, the Lactococci counts were reduced, probably in the production of alcohols and acid compounds by the NSLAB. Each strain produces a unique VOC profile that changes over the ripening time without the main VOCs related to rancid or old cheese. Particularly, the use of the strain Lacticaseibacillus. paracasei ItalPN16 resulted in production of ester compounds with fruity notes. Thus, indigenous NSLAB could be a valuable tool for the enhancement and diversification of flavor in short-aged cheese.

The microbial and metabolite composition of Gouda cheese made from pasteurized milk is determined by the processing chain

Gouda cheeses of different production batches and ripening times often differ in metabolite composition, which may be due to the starter culture mixture applied or the growth of non-starter lactic acid bacteria (NSLAB) upon maturation. Therefore, a single Gouda cheese production batch was systematically investigated from the thermized milk to the mature cheeses, ripened for up to 100 weeks, to identify the main bacterial species and metabolites and their dynamics during the whole production and ripening. As this seemed to be starter culture strain- and NSLAB-dependent, it requested a detailed, longitudinal, and quantitative investigation. Hereto, microbial colony enumeration, high-throughput full-length 16S rRNA gene sequencing, and a metabolomic approach were combined. Culture-dependently, Lactococcus lactis was the most abundant species from its addition as part of the starter culture up to the first two months of cheese ripening. Afterward, the NSLAB Lacticaseibacillus paracasei became the main species during ripening. The milk was a possible inoculation source for the latter species, despite pasteurization. Culture-independently, the starter LAB Lactococcus cremoris and Lc. lactis were the most abundant species in the cheese core throughout the whole fermentation and ripening phases up to 100 weeks. The cheese rind from 40 until 100 weeks of ripening was characterized by a high relative abundance of the NSLAB Tetragenococcus halophilus and Loigolactobacillus rennini, which both came from the brine. These species were linked with the production of the biogenic amines cadaverine and putrescine. The most abundant volatile organic compound was acetoin, an indicator of citrate and lactose fermentation during the production day, whereas the concentrations of free amino acids were an indicator of the ripening time.

Discriminative power of DNA-based, volatilome, near infrared spectroscopy, elements and stable isotopes methods for the origin authentication of typical Italian mountain cheese using sPLS-DA modeling

Origin authentication methods are pivotal in counteracting frauds and provide evidence for certification systems. For these reasons, geographical origin authentication methods are used to ensure product origin. This study focused on the origin authentication (i.e. at the producer level) of a typical mountain cheese origin using various approaches, including shotgun metagenomics, volatilome, near infrared spectroscopy, stable isotopes, and elemental analyses. DNA-based analysis revealed that viral communities achieved a higher classification accuracy rate (97.4 ± 2.6 %) than bacterial communities (96.1 ± 4.0 %). Non-starter lactic acid bacteria and phages specific to each origin were identified. Volatile organic compounds exhibited potential clusters according to cheese origin, with a classification accuracy rate of 90.0 ± 11.1 %. Near-infrared spectroscopy showed lower discriminative power for cheese authentication, yielding only a 76.0 ± 31.6 % classification accuracy rate. Model performances were influenced by specific regions of the infrared spectrum, possibly associated with fat content, lipid profile and protein characteristics. Furthermore, we analyzed the elemental composition of mountain Caciotta cheese and identified significant differences in elements related to dairy equipment, macronutrients, and rare earth elements among different origins. The combination of elements and isotopes showed a decrease in authentication performance (97.0 ± 3.1 %) compared to the original element models, which were found to achieve the best classification accuracy rate (99.0 ± 0.01 %). Overall, our findings emphasize the potential of multi-omics techniques in cheese origin authentication and highlight the complexity of factors influencing cheese composition and hence typicity.

Dissecting the Genetic Basis of the Technological, Functional, and Safety Characteristics of Lacticaseibacillus paracasei SRX10.

Nonstarter lactic acid bacteria (NSLAB) are major contributors to the unique characteristics (e.g., aroma, flavor, texture) of dairy and nondairy fermented products. Lc. paracasei SRX10 is an NSLAB strain originally isolated from a traditional Greek cheese and previously shown to exhibit favorable biotechnological characteristics. More specifically, the strain showed tolerance to simulated gastrointestinal conditions, exopolysaccharide (EPS) biosynthetic capacity, and lack of hemolytic activity and was used in the production of yoghurt and feta cheese with distinct organoleptic characteristics. The aim of the present study was to investigate these traits at the genome level through whole-genome sequencing (WGS), annotation, and comparative genomics. Functional annotation of the genome revealed that Lc. paracasei SRX10 can utilize different carbon sources, leading to the generation of flavor compounds, including lactic acid, acetate, ethanol, and acetoin. Similarly, full clusters for fatty acid biosynthesis, protein and peptide degradation, as well as genes related to survival under extreme temperatures, osmotic shock, and oxidative stress were annotated. Importantly, no transferable antibiotic resistance genes or virulence factors were identified. Finally, strain-specific primers based on genome-wide polymorphisms were designed for the efficient and rapid identification of Lc. paracasei SRX10 via multiplex PCR in fermented products.

Comparative analysis of microbial succession and proteolysis focusing on amino acid pathways in Asiago-PDO cheese from two dairies

In this study, a comprehensive and comparative analysis was conducted on Italian Asiago-PDO cheese obtained from two different dairies named Dairy I and Dairy II using industrial and natural fermented milk, respectively. The analysis encompassed the evaluation of chemical composition, the succession of the microbiota during manufacture and ripening, and proteolysis mainly focusing on free individual amino acid (FAA) profiles. A metagenomic approach was used to investigate the cheese microbiome functionality. Differences in gross chemical composition were more evident during ripening, with Dairy II showing higher variability within batches. The microbiota varied significantly between the two dairies and ripening stages. The choice of starter culture shaped the microbiota during production and affected the microbial diversity of non-starter lactic acid bacteria (NSLAB) originated from the raw milk during ripening. Peptide chromatographic profiles and FAA concentrations increased as ripening progressed, with Dairy I showing higher production of FAA. Functional analysis of the metagenomes linked species to specific amino acid metabolism/catabolism pathways. The amino acid metabolism pathways, particularly those related to aromatic amino acids, lysine, and branched-chain amino acids, were affected by the presence of specific NSLAB species, which differed between the two dairies. The results obtained in this study reveal the impact of starter culture on peculiar cheese microbiota assemblies, which selectively targets amino acid pathways, providing insights into the potential flavor and aroma characteristics of Asiago-PDO cheese.

Metabarcoding analysis reveals a differential bacterial community profile associated with ‘Torta del Casar’ and ‘Queso de la Serena’ PDO cheeses

A detailed understanding of the microbial ecology of artisanal cheeses is a crucial factor for implementation practices that can help to update their quality. In this study, the bacterial communities of Protected Designation of Origin (PDO) ‘Queso de la Serena’ and ‘Torta del Casar’ cheeses were characterised by 16S rRNA gene metabarcoding sequencing over 60 days of ripening at three dairies for each cheese variety. Both PDO cheeses presented a distinctive and unique bacterial community, suggesting that the manufacturing practices of each PDO shaped the microbiota. Lactococcus lactis and, to a lesser extent, Leuconostoc mesenteroides were the key lactic acid bacteria (LAB) species that drove the ripening process of PDO ‘Torta del Casar’, whereas in PDO ‘Queso de la Serena’, Leu. mesenteroides was the main LAB species, followed by Lactococcus raffinolactis and Lc. lactis, depending on the dairy. Furthermore, a wide range of non-starter LAB, Gram-negative bacteria and Gram-positive catalase-positive cocci were found, with a variable presence and abundance between dairies. In this regard, the dairy was a strong contributing factor to the variability in the composition of the bacterial community in each cheese type. In contrast, the impact of the ripening process was limited. Overall, this study enhances the understanding of the bacterial dynamics that occur during the ripening of these traditional PDO cheeses, highlighting the need to improve the standardisation of the raw materials and manufacturing practices to minimise inter-dairy variability, ensuring final quality and their unique sensory traits.

The Controversial Nature of Some Non-Starter Lactic Acid Bacteria Actively Participating in Cheese Ripening.

This mini review deals with some controversial non-starter lactic acid bacteria (NSLAB) species known to be both human and animal pathogens but also health-promoting and probiotic. The focus is on Lactococcus garvieae, two Streptococcus species (S. uberis and S. parauberis), four Weissella species (W. hellenica, W. confusa, W. paramesenteroides, and W. cibaria), and Mammalicoccus sciuri, which worldwide, are often found within the microbiotas of different kinds of cheese, mainly traditional artisanal cheeses made from raw milk and/or relying on environmental bacteria for their ripening. Based on literature data, the virulence and health-promoting effects of these bacteria are examined, and some of the mechanisms of these actions are reviewed. Additionally, their possible roles in cheese ripening are also discussed. The analysis of the literature data available so far showed that, in general, the pathogenic and the beneficial strains, despite belonging to the same species, show somewhat different genetic constitutions. Yet, when the safety of a given strain is assessed, genomic analysis on its own is not enough, and a polyphasic approach including additional physiological and functional tests is needed.

Non-starter lactic acid bacteria and citrate fermenting bacteria in milk supply chain: Are they easily controlled?

The frequency and possible sources of non-starter lactic acid bacteria (NSLAB) and citrate fermenting bacteria (CFB) at four points of a milk supply chain were evaluated. The relationship between the distance from farms (G1 and G2) to industry with NSLAB, CFB and psychrotrophic bacteria populations was also evaluated. A positive correlation between psychrotrophic and CFB was verified, which points to the need for the correct use of good milking practices to control these bacteria. NSLAB showed a significant increase in the population in raw milk samples collected in industrial silos (4.4 log cfu mL−1). CFB populations showed a significant increase in milk from tank trucks (4 log cfu mL−1). There was no difference between G1 and G2 in microorganisms counts. Even after the pasteurisation process, NSLAB and CFB populations remained high, which points to the occurrence of post-heat treatment recontamination, formation of biofilms and/or the presence of thermoduric microbiota.

Associations between the Bacterial Composition of Farm Bulk Milk and the Microbiota in the Resulting Swedish Long-Ripened Cheese.

The maturation of a traditional Swedish long-ripened cheese has shown increasing variation in recent years and the ripening time is now generally longer than in the past. While the cheese is reliant on non-starter lactic acid bacteria for the development of its characteristic flavour, we hypothesised that the observed changes could be due to variations in the microbiota composition and number of bacteria in the raw milk used for production of the cheese. To evaluate associations between microbiota in the raw milk and the resulting cheese, three clusters of commercial farms were created to increase variation in the microbiota of dairy silo milk used for cheese production. Cheese production was performed in three periods over one year. Within each period, milk from the three farm clusters was collected separately and transported to the cheese production facility. Following pasteurisation, the milk was processed into the granular-eyed cheese and matured at a dedicated cheese-ripening facility. For each cheese batch, farm bulk and dairy silo milk samples, a starter culture, early process samples and cheese samples from different stages of maturation (7-20 months) were collected and their microbiota characterised using 16S rRNA amplicon sequencing. The microbiota in the farm bulk milk differed significantly between periods and clusters. Differences in microbiota in dairy silo milk were observed between periods, but not between farm clusters, while the cheese microbiota differed between periods and clusters. The top 13 amplicon sequence variants were dominant in early process samples and the resulting cheese, making up at least 93.3% of the relative abundance (RA). Lactococcus was the dominant genus in the early process samples and, together with Leuconostoc, also dominated in the cheese samples. Contradicting expectations, the RA of the aroma-producing genus Lactobacillus was low in cheese during ripening and there was an unexpected dominance of starter lactic acid bacteria even at the later stages of cheese ripening. To identify factors behind the recent variations in ripening time of this cheese, future studies should address the effects of process variables and the dairy environment.

Study of dairy heterofermentative lactic acid bacilli for cereal-based matrices

Forty obligate heterofermentative lactic acid bacilli (HB) isolated from dairy environment as non-starter lactic acid bacteria (NSLAB) were studied regarding their potential as novel starter cultures for fermented foods. Preliminary genotipic and phenotypic tests allowed the selection of 16 strains, identified as Limosilactobacillus fermentum (12 strains), Levilactobacillus brevis (2), Lentilactobacillus buchneri (1) and Weissella confusa (1). They were initially studied at the technological level, focusing on stress factors (low temperature, heat, salt and acidic media), growth in milk, viability after freezing and thawing and growth in cereal-based matrices. Based on this, six strains were chosen for further studies: L. brevis (LB61 and LB66), L. buchneri (LBu39), L. fermentum (LF22 and LF68) and W. confusa (W20). The selected strains grew well in wheat and gluten-free (GF) doughs, produced exopolysaccharides, acetic and lactic acid in a ratio (<1:5) that allowed the inhibition of undesirable organisms without causing off-flavours, and had good salt resistance. Among them, Weissella confusa W20 was used as starter culture for sourdough fermentation to manufacture gluten-free breads, showing noticeable improvements at the organoleptic level in comparison to GF breads fermented only with yeast. This work remarks the importance of studying the potential of wild strains to find alternative matrices beyond those from where they were isolated. With this regard, our investigation allowed the selection of six dairy strains with potential as starter cultures for bread making, which reaffirms that allochthonous strains can be suitable for sourdough preparation.

Assessment of bacteriocin producing Enterococcus faecium HZ as adjunct culture to improve the aroma formation and antimicrobial activity in white-brined cheese

AbstractCultures used in dairy products make it possible to obtain standard industrial products. However, they all provide a uniform taste and aroma. Generally, non-starter lactic acid bacteria (NSLAB) isolated from raw-milk or artisanal cheeses offer varied sensory characteristics when integrated in cheese provided that biosafety criteria are met. Enterococcus faecium HZ was previously isolated from traditional Turkish cheese and determined to have strong antibacterial activity as well as no gelatinase and hemolysis activities. In this study, this strain was used as adjunct culture in white-brined cheese to improve the physicochemical, textural, and aromatic properties, as well as antimicrobial activity. Cheeses with E. faecium HZ had a higher sensory score, which could be due to the aroma-active compounds produced by this strain. The incorporation of E. faecium HZ also improved the microbial quality of cheeses and showed an inhibitory effect via a stable enterocin production on indicator microorganisms.

The characterization of the non-starter lactic acid bacteria and yeast microbiota and the chemical and aromatic properties of traditionally produced Turkish White Cheese.

Turkish White Cheese is a brined (or pickled) cheese with a salty, acidic flavor and a soft or semi-hard texture. It is the most produced and consumed type of cheese in Turkey. The purpose of this study was to determine the non-starter lactic acid bacteria and yeast microbiota of traditionally produced Turkish White Cheese and analyze the chemical properties and the aroma profile of the cheese. The results of the study identified 27 distinct strains belonging to 14 the non-starter lactic acid bacteria species and 49 different strains belonging to 11 yeast species. Lactobacillus plantarum was found to be the dominant species among the lactic acid bacteria, while Candida zeylanoides was the dominant yeast species in the White Cheese samples. In addition, Kluyveromyces lactis and Debaryomyces hansenii were prominent yeast species in cheese samples. Turkish White Cheese samples had different aromatic properties. The study is highly significant as it anaylzed both non-starter lactic acid bacteria and yeast microbiota of traditionally produced Turkish White Cheese through molecular methods. It also determined and analyzed a number of chemical and aromatic properties of White Cheese.

How starter cultures affect the peptidomic profile and bioactive activities of the Asiago-PDO cheese throughout ripening

Our study investigated the chemical, microbiological, and bioactive peptide profiles of Asiago Protected Designation of Origin (PDO) cheese from two dairies (Dairy I and II) produced over two consecutive days (batches) and analysed during three months of ripening. The effect of different starter cultures was evaluated. The microbiome varied between the dairies and batches, with curds post-salting dominated by the starter culture-associated genera. During ripening, there was an increasing trend in the Lactobacillus genus, especially for Dairy I, which used an industrial starter. Bioactive peptide intensities differed throughout ripening due to the extent of proteolysis, and their intensity or concentration evolved, modifying, and differentiating profiles. The industrial starter used in Dairy I had the highest relative intensity (average value 76.50%) of bioactive peptides after three months of ripening. In contrast, the cheeses made with natural milk starter (Dairy II) had lower total relative intensity (average value 47.75%) but produced ACE-inhibitory peptides through sub-dominant strains and non-starter lactic acid bacteria. The importance of autochthonous strains of each micro-region even within a delimited PDO production area was highlighted.

The rotation of primary starter culture mixtures results in batch-to-batch variations during Gouda cheese production.

Industrial production of Gouda cheeses mostly relies on a rotated use of different mixed-strain lactic acid bacteria starter cultures to avoid phage infections. However, it is unknown how the application of these different starter culture mixtures affect the organoleptic properties of the final cheeses. Therefore, the present study assessed the impact of three different starter culture mixtures on the batch-to-batch variations among Gouda cheeses from 23 different batch productions in the same dairy company. Both the cores and rinds of all these cheeses were investigated after 36, 45, 75, and 100 weeks of ripening by metagenetics based on high-throughput full-length 16S rRNA gene sequencing accompanied with an amplicon sequence variant (ASV) approach as well as metabolite target analysis of non-volatile and volatile organic compounds. Up to 75 weeks of ripening, the acidifying Lactococcus cremoris and Lactococcus lactis were the most abundant bacterial species in the cheese cores. The relative abundance of Leuconostoc pseudomesenteroides was significantly different for each starter culture mixture. This impacted the concentrations of some key metabolites, such as acetoin produced from citrate, and the relative abundance of non-starter lactic acid bacteria (NSLAB). Cheeses with the least Leuc. pseudomesenteroides contained more NSLAB, such as Lacticaseibacillus paracasei that was taken over by Tetragenococcus halophilus and Loigolactobacillus rennini upon ripening time. Taken together, the results indicated a minor role of leuconostocs in aroma formation but a major impact on the growth of NSLAB. The relative abundance of T. halophilus (high) and Loil. rennini (low) increased with ripening time from rind to core. Two main ASV clusters of T. halophilus could be distinguished, which were differently correlated with some metabolites, both beneficial (regarding aroma formation) and undesirable ones (biogenic amines). A well-chosen T. halophilus strain could be a candidate adjunct culture for Gouda cheese production.

Lactic acid bacteria in cow raw milk for cheese production: Which and how many?

Lactic Acid Bacteria (LAB) exert a fundamental activity in cheese production, as starter LAB in curd acidification, or non-starter LAB (NSLAB) during ripening, in particular in flavor formation. NSLAB originate from the farm and dairy environment, becoming natural contaminants of raw milk where they are present in very low concentrations. Afterward, throughout the different cheesemaking processes, they withstand chemical and physical stresses becoming dominant in ripened cheeses. However, despite a great body of knowledge is available in the literature about NSLAB effect on cheese ripening, the investigations regarding their presence and abundance in raw milk are still poor. With the aim to answer the initial question: "which and how many LAB are present in cow raw milk used for cheese production?," this review has been divided in two main parts. The first one gives an overview of LAB presence in the complex microbiota of raw milk through the meta-analysis of recent taxonomic studies. In the second part, we present a collection of data about LAB quantification in raw milk by culture-dependent analysis, retrieved through a systematic review. Essentially, the revision of data obtained by plate counts on selective agar media showed an average higher concentration of coccoid LAB than lactobacilli, which was found to be consistent with meta-taxonomic analysis. The advantages of the impedometric technique applied to the quantification of LAB in raw milk were also briefly discussed with a focus on the statistical significance of the obtainable data. Furthermore, this approach was also found to be more accurate in highlighting that microorganisms other than LAB are the major component of raw milk. Nevertheless, the variability of the results observed in the studies based on the same counting methodology, highlights that different sampling methods, as well as the "history" of milk before analysis, are variables of great importance that need to be considered in raw milk analysis.

Queijo Serra da Estrela PDO Cheese: Investigation into Its Morpho-Textural Traits, Microbiota, and Volatilome.

Queijo Serra da Estrela is a PDO Portuguese cheese produced through coagulation of raw ewe’s milk using cardoon (Cynara cardunculus L.) flowers. The present research was aimed at depicting an up-to-date and comprehensive overview of the microbiota of Queijo Serra da Estrela cheese. To this end, viable counting and metataxonomic analysis were carried out on cheeses sampled from four Portuguese artisan producers. Physico-chemical and morpho-textural analyses were also performed, together with the analysis of volatile organic compounds (VOCs). Finally, non-starter lactic acid bacteria (NSLAB) isolated from the cheeses were characterized for their enzymatic activities using a semi-quantitative method. According to the metataxonomic analysis, Lactococcus lactis and Lactococcus piscium were the species occurring at the highest relative abundance. The isolates collected from the cheeses were assigned to Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus lactis, Levilactobacillus brevis, Latilactobacillus graminis, Leuconostoc mesenteroides, and the Lacticaseibacillus casei group. The enzymatic characterization of these cultures highlighted esterase, aminopeptidase, acid phosphatase, beta-galactosidase, alpha-glucosidase, and beta-glucosidase among the major enzymatic activities. Fungal populations were dominated by Debaryomyces hansenii and Kurtzmaniella zeylanoides; however, species rarely found in cheese (e.g., Candida boidinii, Vishniacozyma victoriae, and Starmerella) were also detected. The volatile compounds characterizing the analyzed cheeses were carboxylic acids and esters, followed by carbonyl compounds and alcohols.

Relationship between the dynamics of volatile aroma compounds and microbial succession during the ripening of raw ewe milk-derived Idiazabal cheese

Cheese microbiota contributes to various biochemical processes that lead to the formation of volatile compounds and the development of flavour during ripening. Nonetheless, the role of these microorganisms in volatile aroma compounds production is little understood. This work reports for the first time the dynamics and odour impact of volatile compounds, and their relationship to microbial shifts during the ripening of a raw ewe milk-derived cheese (Idiazabal). By means of SPME-GC-MS, 81 volatile compounds were identified, among which acids predominated, followed by esters, ketones and alcohols. The ripening time influenced the abundance of most volatile compounds, thus the moments of greatest abundance were determined (such as 30–60 days for acids). Through Odour Impact Ratio (OIR) values, esters and acids were reported as the predominant odour-active chemical families, while individually, ethyl hexanoate, ethyl 3-methyl butanoate, ethyl butanoate, butanoic acid or 3-methyl butanal were notable odorants, which would provide fruity, rancid, cheesy or malt odour notes. Using a bidirectional orthogonal partial least squares (O2PLS) approach with Spearman's correlations, 12 bacterial genera were reported as key bacteria for the volatile and aromatic composition of Idiazabal cheese, namely Psychrobacter, Enterococcus, Brevibacterium, Streptococcus, Leuconostoc, Chromohalobacter, Chryseobacterium, Carnobacterium, Lactococcus, Obesumbacterium, Stenotrophomonas and Flavobacterium. Non-starter lactic acid bacteria (NSLAB) were highly related to the formation of certain acids, esters and alcohols, such as 3-hexenoic acid, ethyl butanoate or 1-butanol. On the other hand, the starter LAB (SLAB) was related to particular ketones production, specifically 3-hydroxy-2-butanone; and environmental and/or non-desirable bacteria to certain ketones, hydrocarbons and sulphur compounds formation, such as 2-propanone, t-3-octene and dimethyl sulphone. Additionally, the SLAB Lactococcus and Psychrobacter, Brevibacterium and Chromohalobacter were described as having a negative effect on aroma development caused by NSLAB and vice versa. These results provide novel knowledge to help understand the aroma formation in a raw ewe milk-derived cheese.

​Identification of Lactic Acid Bactria in Dairy Products using Culture-independent Methods: A Review

The microbiota of dairy products is composed of starter and non-starter lactic acid bacteria, as well as other bacteria, yeasts and filamentous fungi that form secondary microbiota and play an important role in cheese ripening. The use of combined LAB strains based on compatibility and/or coexistence tests plays an important role in food production and preservation, while combinations of strains that exhibit mutually inhibitory properties must be avoided. For centuries, researchers have focused on the exploration of autochthonous LAB biodiversity from various ecological dairy niches in order to address its role in texture and flavour formation and to select strains for use as starter cultures. Recent advanced technological tools like next-generation sequencing machines have played a significant role in the fast and accurate characterization and isolation of microbial communities from dairy products. However, the characterization and isolation of LAB using culture-independent methods is also a reliable and cost-effective method for studying a bacterial community without prior cultivation. These methods include single-strand conformation polymorphism (SSCP-PCR), denaturing gradient gel electrophoresis (DGGE), thermal gradient gel electrophoresis (TGGE), real-time PCR (qPCR), terminal restriction fragment length polymorphism (T-RFLP), length heterogeneity PCR (LH-PCR), automated ribosomal intergenic spacer analysis (ARISA), and fluorescence in situ hybridization (FISH). Therefore the goal of this review is to compare and describe the most recent culture-independent molecular-based methods for the characterization and isolation of LAB in dairy products.

Relationship between the Dynamics of Gross Composition, Free Fatty Acids and Biogenic Amines, and Microbial Shifts during the Ripening of Raw Ewe Milk-Derived Idiazabal Cheese.

This study reports for the first time the relationship between bacterial succession, characterized by high-throughput sequencing (sequencing of V3-V4 16S rRNA regions), and the evolution of gross composition, free fatty acids (FFAs) and biogenic amines (BAs) during cheese ripening. Specifically, Idiazabal PDO cheese, a raw ewe milk-derived semi-hard o hard cheese, was analysed. Altogether, 8 gross parameters were monitored (pH, dry matter, protein, fat, Ca, Mg, P and NaCl) and 21 FFAs and 8 BAs were detected. The ripening time influenced the concentration of most physico-chemical parameters, whereas the producer mainly affected the gross composition and FFAs. Through an O2PLS approach, the non-starter lactic acid bacteria Lactobacillus, Enterococcus and Streptococcus were reported as positively related to the evolution of gross composition and FFAs release, while only Lactobacillus was positively related to BAs production. Several environmental or non-desirable bacteria showed negative correlations, which could indicate the negative impact of gross composition on their growth, the antimicrobial effect of FFAs and/or the metabolic use of FFAs by these genera, and their ability to degrade BAs. Nonetheless, Obesumbacterium and Chromohalobacter were positively associated with the synthesis of FFAs and BAs, respectively. This research work provides novel information that may contribute to the understanding of possible functional relationships between bacterial communities and the evolution of several cheese quality and safety parameters.