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Gamma-aminobutyric acid (GABA)-producing lactic acid bacteria (LAB) can be used as starters in the development of GABA-enriched functional fermented foods. In this work, four GABA-producing strains each of Lactococcus lactis and Streptococcus thermophilus species were isolated from cow's milk, and their phenotypic, technological, and safety profiles determined. Genome analysis provided genetic support for the majority of the analyzed traits, namely, GABA production, growth in milk, and the absence of genes of concern. The operon harboring the glutamate decarboxylase gene ( gadB ) was chromosomally encoded in all strains and showed the same gene content and gene order as those reported, respectively, for L. lactis and S. thermophilus . In the latter species, the operon was flanked (as in most strains of this species) by complete or truncated copies of insertion sequences (IS), suggesting recent acquisition through horizontal gene transfer. The genomes of three L. lactis and two S. thermophilus strains showed a gene encoding a caseinolytic proteinase (PrtP in L. lactis and PrtS in S. thermophilus ). Of these, all but one grew in milk, forming a coagulum of good appearance and an appealing acidic flavor and taste. They also produced GABA in milk supplemented with monosodium glutamate. Two L. lactis strains were identified as belonging to the biovar. diacetylactis, utilized citrate from milk, and produced significant amounts of acetoin. None of the strains showed any noticeable antibiotic resistance, nor did their genomes harbor transferable antibiotic resistance genes or genes involved in toxicity, virulence, or pathogenicity. Altogether these results suggest that all eight strains may be considered candidates for use as starters or components of mixed LAB cultures for the manufacture of GABA-enriched fermented dairy products.

In food, bacteria carrying antibiotic resistance genes could play a prominent role in the spread of resistance. Staphylococcus equorum populations can become large in a number of fermented foods, yet the antibiotic resistance properties of this species have been little studied. In this work, the resistance/susceptibility (R/S) profile of S. equorum strains ( n = 30) from cheese to 16 antibiotics was determined by broth microdilution. The minimum inhibitory concentration (MIC) for all antibiotics was low in most strains, although higher MICs compatible with acquired genes were also noted. Genome analysis of 13 strains showed the S. equorum resistome to be composed of intrinsic mechanisms, acquired mutations, and acquired genes. As such, a plasmidic cat gene providing resistance to chloramphenicol was found in one strain; this was able to provide resistance to Staphylococcus aureus after electroporation. An msr (A) polymorphic gene was identified in five strains. The Mrs(A) variants were associated with variable resistance to erythromycin. However, the genetic data did not always correlate with the phenotype. As such, all strains harbored a polymorphic fosB / fosD gene, although only one acquired copy was associated with strong resistance to fosfomycin. Similarly, a plasmid-associated blaR1-blaZI operon encoding a penicillinase system was identified in five ampicillin- and penicillin G-susceptible strains. Identified genes not associated with phenotypic resistance further included mph (C) in two strains and norA in all strains. The antibiotic R/S status and gene content of S. equorum strains intended to be employed in food systems should be carefully determined.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

High throughput sequencing has recently revealed the presence of Tetragenococcus-related DNA sequences in dairy environments such as brine and cheeses. In the present work, a selective medium was developed to isolate Tetragenococcus spp. from two ripened, traditional, Spanish, blue-veined cheese varieties made from raw milk. The strains recovered belonged to either Tetragenococcus koreensis or Tetragenococcus halophilus species. Twenty of these isolates (15 of T. koreensis and 5 of T. halophilus) were then subjected to a battery of phenotypic and genetic tests, and six strains (4 T. koreensis and 2 T. halophilus) to genome sequencing. Wide genetic and phenotypic diversity was noted. All strains grew poorly in milk, producing small quantities of lactic and acetic acids. Most strains used lactose as a carbon source and ferment milk citrate. In agreement, genome analysis detected in the genome of the six strains analyzed gene clusters harboring several lactose/galactose-related genes and genes encoding citrate metabolic enzymes (permease, citrate lyase, and oxaloacetate decarboxylase). Most of the tested strains were resistant to erythromycin and clindamycin, and a few to other antimicrobial agents, but neither known mutations nor acquired genes conferring resistance to antibiotics were identified in their genomes. Neither were genes coding for pathogenicity or virulence factors detected. Decarboxylase-encoding genes involved in biogenic amine production were not identified, in keeping with the strains' negative biogenic amine-producer phenotype. Genome comparison revealed vast arrays of genes (similar in number to those described in other lactic acid bacteria) coding for components of proteolytic and lipolytic systems. Tetragenococcus strains showing desirable traits plus the absence of detrimental features might be exploitable in the form of secondary, adjunct or ripening cultures to ensure the typical bouquet of traditional blue-veined cheeses is obtained, or to diversify the final flavor in other varieties., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The aim of this study was to use local LAB cultures for the production of organic acid-rennet cheeses from unpasteurized cow's milk. Under industrial conditions, three types of cheese were produced, i.e., traditionally with acid whey (AW), with starter culture L. brevis B1, or with starter culture L. plantarum Os2. Strains were previously isolated from traditional Polish cheeses. Chemical composition, physico-chemical, microbiological, and sensory studies during 2 months of storage were carried out. As a result of this research, it was found that the basic composition was typical for semi-hard, partially skimmed cheeses. Mainly saturated fatty acids were detected. The cheeses were rich in omega-3, -6, and -9 fatty acids and conjugated linoleic acid (CLA), and were characterized by good lipid quality indices (LQI). All of the cheeses were characterized by a high number of lactic acid bacteria, with Enterobacteriaceae , yeast, molds, and staphylococci contaminants, which is typical microbiota for unpasteurized milk products. Water activity, pH, and total acidity were typical. A lower oxidation-reduction potential (ORP) of cheeses with the addition of strains and stability of the products during storage were observed. The B1 and Os2 cheeses were lighter, less yellow, had a more intense milk and creamy aroma, were softer, moister, and more elastic than AW cheese. The research results indicate the possibility of using environmental LAB strains in the production of high-quality acid-rennet cheeses, but special attention should be paid to the production process due to the microbiological quality of the cheeses.

The importance of starter cultures to cheese manufacture and ripening is well known. Starters are inoculated into cheese milk at a level of ∼10 6 cfu/mL either from a bulk culture or using commercial direct-to-vat cultures. Before ripening, starters grow in the milk to reach populations of 10 7 to 10 9 cfu/g of curd depending on processing variables such as cook temperature, inclusion of washing steps, degree of partitioning with curds and whey, and importantly salt addition rate. Inherent strain-related properties also determine final populations in the curd following manufacture and include temperature sensitivity, salt sensitivity, presence of prophage, autolytic and permeabilization properties (which are influenced by processing steps), presence and type of cell envelope proteinase, and metabolic activity. Ripening of important industrial cheese varieties such as Cheddar, Dutch, Swiss, and Italian-type cheese varieties is characterized by extended storage under temperature-controlled conditions enabling characteristic flavor and texture development to occur. Over ripening, microbiological, biochemical and enzymatic changes occur with a decline in starter viability, release of intracellular enzymes, hydrolysis of proteins, carbohydrates and lipids, and formation of a range of volatile and nonvolatile flavor components. Recent reports suggest that starter strains may be present during the later stages of ripening and therefore their potential role needs to be reconsidered. This review will focus on our current understanding of starter viability and vitality during cheese ripening and will also review the area of starter permeabilization, autolysis, and enzyme release., (© 2020, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Kopanisti is a Greek PDO cheese, which is traditionally produced by the addition of an amount of over-mature Kopanisti, called Mana Kopanisti, to initiate cheese ripening. The aim of this study was the production of four types of Kopanisti cheese (A–D) using pasteurized cow milk, and a combination of the following starters/adjuncts in order to test their ability to be used in Kopanisti cheese production: A: Lactococcus lactis subsp. lactis and Lacticaseibacillus paracasei, B: L. lactis and Lc. paracasei/Mana Kopanisti, C: L. lactis and Lc. paracasei/Ligilactobacillus acidipiscis and Loigolactobacillus rennini, D: Lig. acidipiscis and Loig. rennini. Throughout production and ripening, classical microbiological, metataxonomics and physicochemical analyses were employed, while the final products (Day 35) were subjected to sensory analysis as well. Most interestingly, beta-diversity analysis of the metataxonomics data revealed the clusters constructed among the Kopanisti types based on the different inoculation schemes. On day 35, Kopanisti A–C types clustered together due to their similar 16S microbiota, while Kopanisti D was highly differentiated. On the contrary, ITS data clustered Kopanisti B and C together, while Kopanisti A and D were grouped seperately. Finally, based on the sensory evaluation, Kopanisti C appeared to have the most suitable bacteria cocktail for the Kopanisti cheese production. Therefore, not only were the conventional starters used, but also the Lig. acidipiscis and Loig. rennini strains could be used in a standardized Kopanisti cheese production that could lead to final products of high quality and safety. [ABSTRACT FROM AUTHOR]

In this article, we question to what extent origin-food labels, namely Geographical Indications (GIs) and Slow Food Presidia, may effectively account for cultural biodiversity (CB). Building on Foucault's discourse theory, we question how the Slow Food movement and GI promoters have developed their own discourse and practice on CB, how these discourses contrast, and how they inform projects. Focusing on the practices to cultivate the microbiological life of three origin labeled cheeses (from France and Italy), we have revealed the gap between these institutional discourses and what happens on the ground. We argue that how actors' relationships in the marketplace unfold, from public authorities to the collectives of producers to consumers, may threaten the effects that these experiences of alternative food productions may have in the defense of biodiversity, causing, for instance, the loss of diversity of the invisible microbial ecosystems of artisan raw milk cheese. However, we conclude that, despite limitations, the mediatized institutional narrative on CB can amplify the political voice of local actors by fostering community and social relationships between the farmers. [ABSTRACT FROM AUTHOR]

Consumers show interest in preservation technologies that are not based on heating. This review examines the use of nonthermal technologies such as pulsed electric field (PEF) in cheesemaking. The effects of PEF on the destruction of pathogens in cheesemaking milk, as well as in brines, are examined. Additionally, PEF affects milk proteins and lipids, as well as starters, with potential consequences for the sensory properties of cheese. The challenges of scaling up the PEF in cheesemaking are addressed. In the aim of promoting durable agri‐food systems, applications of PEF in processes of cheese whey valorisation are also discussed. [ABSTRACT FROM AUTHOR]

The cheese microbiota comprises a consortium of prokaryotic, eukaryotic and viral populations, among which lactic acid bacteria (LAB) are majority components with a prominent role during manufacturing and ripening. The assortment, numbers and proportions of LAB and other microbial biotypes making up the microbiota of cheese are affected by a range of biotic and abiotic factors. Cooperative and competitive interactions between distinct members of the microbiota may occur, with rheological, organoleptic and safety implications for ripened cheese. However, the mechanistic details of these interactions, and their functional consequences, are largely unknown. Acquiring such knowledge is important if we are to predict when fermentations will be successful and understand the causes of technological failures. The experimental use of “synthetic” microbial communities might help throw light on the dynamics of different cheese microbiota components and the interplay between them. Although synthetic communities cannot reproduce entirely the natural microbial diversity in cheese, they could help reveal basic principles governing the interactions between microbial types and perhaps allow multi-species microbial communities to be developed as functional starters. By occupying the whole ecosystem taxonomically and functionally, microbiota-based cultures might be expected to be more resilient and efficient than conventional starters in the development of unique sensorial properties.

In this work, the technological behaviour in milk of a set of Lactococcus lactis strains, alone or in combination with strains of Leuconostoc spp. and Lactobacillus spp. isolated from traditional, raw milk cheeses made without commercial starters, was investigated. Small, mixture-specific differences during milk fermentation were recorded for growth, milk acidification and production of organic acids, volatile compounds, free amino acids and biogenic amines. Four combinations appropriate for use as dairy starters were tested in pilot-scale cheese trials. Two mixtures produced cheeses of high flavour and taste quality; these could be confidently used as starter cultures. [ABSTRACT FROM AUTHOR]

Fermented foods are within the food products more often complained of having caused biogenic amines poisoning. Concerning milk-based fermented foods, cheese is the main product likely to contain significant levels of biogenic amines, specially tyramine, histamine and putrescine. Prompted by the increasing awareness of the risks related to dietary uptake of high biogenic amine loads, in this review we report about cheese elaboration and processing technological aspects affecting biogenic amines levels. Synthesis of biogenic amines is possible only when three conditions converge: i) availability of the substrate amino acids; ii) presence of microorganisms with the appropriate catabolic pathway activated; and iii) environmental conditions favorable to the decarboxilation activity. These conditions depend on several factors such as milk treatment (pasteurization), use of starter cultures, NaCl concentration, time and temperature of ripening and preservation, pH… which will be discussed in this chapter.

Selected Leuconostoc strains are required as aroma producers in dairy starters. In this work, 42 Leuconostoc isolates from different raw milk traditional cheeses made without the addition of commercial starters were identified by molecular methods. These isolates were assigned to the species Leuconostoc citreum (24), Leuconostoc mesenteroides (13), and Leuconostoc lactis (5). Typing the isolates by repetitive extragenic profiling (Rep-PCR) identified 22 strains, out of which 14 were selected for further characterization based on their behavior in milk. The biochemical, technological, and food safety variables recorded were examined to select appropriate candidates for use as adjunct cultures. In agreement with the wide genetic diversity recorded by Rep-PCR, wide phenotypic biodiversity was seen among the species and strains in terms of enzyme and carbohydrate fermentation profile, acidification rate, the production of volatile compounds in milk, and growth under different pH, salt, and temperature conditions. No production of biogenic amines was recorded even when suitable amino acid precursors were present in the growth medium. All 14 strains proved to be susceptible or intrinsically resistant to a set of 16 antibiotics except for one Leuc. citreum strain that showed an atypical resistance to ciprofloxacin. The remaining 13 strains could be confidently used as components of adjunct cultures in the dairy industry. [ABSTRACT FROM AUTHOR]

Autochthonous starters are thought to increase the quality and safety of traditional cheeses while preserving their typicity. In order to select appropriate ripening cultures, 12 Penicillium roqueforti and 10 Geotrichum candidum strains isolated from a Spanish, traditional, blue-veined cheese were subjected to different biochemical and technological assays. All strains were weakly proteolytic, whereas all P. roqueforti and four G. candidum strains proved to be lipolytic. None of the strains produced biogenic amines in broth media containing the required amino acid precursors. Under simulated cheese conditions (10°C, pH=4.5, 5% NaCl), test strains showing technological performances superior to those of commercial strains currently in use were readily identified. [ABSTRACT FROM AUTHOR]

Abstract: Genetic diversity of Lactococcus lactis subsp. cremoris provides an important reservoir of industrial functions. Knowledge of strain diversity is an important step for the selection of starter cultures, because technological and sensorial attributes are strain-dependent and it may help to distinguish strains with particular technological properties and performances. In the present study, microarray-based comparative genomic hybridization (CGH) and multilocus sequence analysis (MLSA) were used to investigate the genetic variation among eight strains of Lactococcus lactis subsp. cremoris. The CGH analysis allows strain grouping and identification of absent or divergent genes involved in metabolism, amino acid biosynthesis, osmoregulation and proteolysis. The MLSA clustering of strains based on the partial sequence of eight genes shows good correlation with the CGH grouping. Strains HP, ATCC 19257 and Wg2 were clustered together, followed by E8, and finally SK11 was in a separate cluster. The combined information provides genetic markers for distinguishing between strains and their variants. By selecting strains according to their genetic profiles, compatible and complementary mixed starters can be formulated, opening new avenues for industrial applications. [Copyright &y& Elsevier]

Abstract: Classical culturing and denaturing gradient gel electrophoresis (DGGE) techniques have been used for studying the microbial diversity and dynamics of the traditional Spanish Casín cheese during manufacturing and ripening. As with other starter-free cheeses made from raw milk, the microbial diversity of Casín was shown to be high by both culturing and DGGE analyses. The culture technique showed that lactic acid bacteria (LAB) species constituted the majority of the microbial populations. Of the 14 bacterial species identified, Lactococcus garvieae was predominant in the three-day-old cheese sample, although it was replaced by Lactococcus lactis subsp. lactis at day 30. As expected, the DGGE profiles obtained were complex, consisting, depending on the sample, in five to ten different amplification bands. Among these, a band corresponding to Streptococcus thermophilus was observed throughout the whole manufacturing process. This species had never been identified from traditional Spanish cheeses previously. Culturing and molecular methods showed high populations of undesirable microorganisms, arguing for a required improvement in the hygiene of Casín manufacture. Random amplification of polymorphic DNA (RAPD) profiling suggested that the L. garvieae and L. lactis populations were composed of one and five strains, respectively. In addition, only a single L. lactis RAPD pattern was stably maintained from day three to day 30, indicating high succession of strains along ripening. After a thoroughly characterisation, strains of the two Lactococcus species could be used in designing specific starter cultures for Casín. Additional species (such as Lactobacillus plantarum and Corynebacterium variabile) might be included as adjunct cultures. [Copyright &y& Elsevier]

Cheeses are frequently made with natural whey starters (NWS). The whey from the previous cheese making is cultured and used for the next day. This practice, although essential for the development of typical sensory characteristics, can sometimes lead to acidification defects. In this work, the ability of Lactococcus lactis subsp. lactis biovar. diacetylactis to dominate over the other lactic acid bacteria (LAB) was tested in a controlled system as a possible explanation for these acidification breakdowns. A starter made of two Lc lactis subsp. lactis strains (LL), one Lc lactis subsp. cremoris strain (LC), and one Lc lactis subsp. lactis biovar. diacetylactis (LD) was added to sterilized milk. After incubation, the whey was removed and used to re-seed sterilized milk, the next day. This process was made during a five and twelve days' period. During the eight first days, the proportion of LD population increased, while the other LAB remained rather stable. Thereafter, LD strains dominated. At the same time, the diversity of LD population diminished considerably. If acidification ability of these LAB is not altered, this simplification was particularly hazardous in case of phages attack. LC and LL behavior was tested in milk containing increasing diacetyl concentrations. As long as diacetyl did not exceed a 5 ppm level - frequently measured in dairy products - its influence was impossible to detect. The selective advantage conferred by the citrate metabolism was proposed as a possible explanation for the LD population dominance. Other potential factors were also examined. [ABSTRACT FROM AUTHOR]

Abstract: Reggianito Argentino cheeses were made at pilot plant scale using a natural whey starter (control) and different combinations of strains of selected Lactobacillus helveticus (experimental cheeses). Free fatty acids (FFAs) were extracted from cheese samples at different ripening times, and FFAs from C6:0 to C18:2 were analysed by gas chromatography. The characteristics of the chromatographic profiles were studied by principal components and linear discriminant analysis were performed. Levels of individual FFAs from caproic (C6:0) to linoleic (C18:2) acids increased significantly during ripening in control and experimental cheeses. Palmitic (C16:0) and oleic (C18:1) acids were the most abundant FFAs throughout ripening in all cheeses. No significant differences were found between FFA profiles of control and experimental cheeses. The possibility of using selected strains of Lactobacillus to replace traditional “natural whey starter” in the production of the cheese is discussed. [Copyright &y& Elsevier]

The objective of the present study was to improve the characteristics of Graviera Kritis cheese. The influence of lactic acid and propionic acid starters was studied and then the effect of washing and salting the curd combined with the use of starters was investigated. Although the traditional technology without starters resulted in the highest organoleptic characteristics, it was shown that the use of starters could improve texture characteristics of the cheeses if it is combined with a curd wash to control acidity development. Lower pH values and higher contents of low molecular weight nitrogenous substances were consistent with texture defects. Moreover, the ripening at higher temperatures increased proteolysis levels and decreased cheese quality. [ABSTRACT FROM AUTHOR]

The cheese microbiota comprises a consortium of prokaryotic, eukaryotic and viral populations, among which lactic acid bacteria (LAB) are majority components with a prominent role during manufacturing and ripening. The assortment, numbers and proportions of LAB and other microbial biotypes making up the microbiota of cheese are affected by a range of biotic and abiotic factors. Cooperative and competitive interactions between distinct members of the microbiota may occur, with rheological, organoleptic and safety implications for ripened cheese. However, the mechanistic details of these interactions, and their functional consequences, are largely unknown. Acquiring such knowledge is important if we are to predict when fermentations will be successful and understand the causes of technological failures. The experimental use of "synthetic" microbial communities might help throw light on the dynamics of different cheese microbiota components and the interplay between them. Although synthetic communities cannot reproduce entirely the natural microbial diversity in cheese, they could help reveal basic principles governing the interactions between microbial types and perhaps allow multi-species microbial communities to be developed as functional starters. By occupying the whole ecosystem taxonomically and functionally, microbiota-based cultures might be expected to be more resilient and efficient than conventional starters in the development of unique sensorial properties. [ABSTRACT FROM AUTHOR]