Fermented Dairy Research Articles

Comparative genomics and phenotypic assessment of lactic acid bacteria isolated from artisanal cheese as potential starter cultures

Novel starter cultures are coveted by the dairy industry for enhancing the sensory, quality, and safety attributes of fermented dairy products. In this study, artisanal cheeses were investigated as a source of novel starter cultures. Lactic acid bacteria (LAB) were isolated from the cheese and underwent an antimicrobial activity screening. Six potential antimicrobial producers were selected for whole genome sequencing and were confirmed to be new LAB strains belonging to the genera Lacticaseibacillus, Lactococcus, Leuconostoc, and Enterococcus. Genes associated with carbohydrate utilization, proteolytic enzyme production, and exopolysaccharide synthesis were identified. Additionally, in silico analysis revealed safety-related traits including absence of antibiotic resistance genes, intact prophage regions, and biogenic amine production genes. Genome-guided phenotypic confirmation of important identified traits was completed, which included utilization of multiple carbohydrates, hydrolysis of casein, coagulation of milk, and susceptibility to antibiotic. Among these LAB strains, Lactococcus lactis OSY-92 contained and expressed the necessary genes for a dairy starter culture. The strain displayed anti-Gram-positive antimicrobial activity, a strong preference for lactose and galactose, limited casein hydrolysis, and ability to coagulate milk in 7.5 h. When all these findings were considered, L. lactis OSY-92 qualified as a potential novel and safe starter culture for dairy fermentations.

Probio-Ichnos: A Database of Microorganisms with In Vitro Probiotic Properties.

Probiotics are live microorganisms that, when consumed in adequate amounts, exert health benefits on the host by regulating intestinal and extraintestinal homeostasis. Common probiotic microorganisms include lactic acid bacteria (LAB), yeasts, and Bacillus species. Here, we present Probio-ichnos, the first manually curated, literature-based database that collects and comprehensively presents information on the microbial strains exhibiting in vitro probiotic characteristics (i.e., resistance to acid and bile, attachment to host epithelia, as well as antimicrobial, immunomodulatory, antiproliferative, and antioxidant activity), derived from human, animal or plant microbiota, fermented dairy or non-dairy food products, and environmental sources. Employing a rigorous methodology, we conducted a systematic search of the PubMed database utilizing the keyword 'probiotic' within the abstracts or titles, resulting in a total of 27,715 studies. Upon further manual filtering, 2207 studies presenting in vitro experiments and elucidating strain-specific probiotic attributes were collected and used for data extraction. The Probio-ichnos database consists of 12,993 entries on the in vitro probiotic characteristics of 11,202 distinct strains belonging to 470 species and 143 genera. Data are presented using a binary categorization approach for the presence of probiotic attributes according to the authors' conclusions. Additionally, information about the availability of the whole-genome sequence (WGS) of strains is included in the database. Overall, the Probio-ichnos database aims to streamline the navigation of the available literature to facilitate targeted validation and comparative investigation of the probiotic properties of the microbial strains.

Temperature-Dependent Metabolic Interactions Between Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus in Milk Fermentation: Insights from GC-IMS Metabolomics

Streptococcus (S.) thermophilus and Lactobacillus (L.) delbrueckii ssp. bulgaricus are widely used as a combined starter culture for milk fermentation, often at temperatures of 37°C and 42°C. To investigate the metabolic interplay between these 2 species during the fermentation process, this study examined the growth and fermentation characteristics of different S. thermophilus strains cocultured with L. delbrueckii ssp. bulgaricus ND02 at these 2 temperature conditions. Gas chromatography-ion mobility spectrometry (GC-IMS) metabolomics was employed to analyze changes in the milk metabolome during 3 key fermentation stages: initiation (F0, pH 6.50 ± 0.02), curdling (F1, pH 5.20 ± 0.02), and endpoint (F2, pH 4.50 ± 0.02). The results showed that 42°C fermentation promoted rapid bacterial growth, with significantly reduced fermentation time compared with 37°C. Interestingly, 37°C fermentation favored the enrichment of volatile fatty acids like 2-methylpropanoic acid, 3-methylbutanoic acid, and ethyl acetate. In contrast, 42°C fermentation led to increased levels of ketones such as acetone, 2-hexanone, 2-pentanone, and 2-heptanone. Sensory evaluation indicated that the 42°C fermented milk had higher overall scores. Discriminatory flavor metabolites were more abundant during the later fermentation stage (F1 to F2), while the underlying metabolic pathways became increasingly active. These findings provide insights into the dynamic changes in volatile metabolite profiles of fermented milk produced under different temperature and time conditions using varied starter culture combinations. The results are valuable for optimizing dairy fermentation processes and product quality.

Phage defence loci of Streptococcus thermophilus-tip of the anti-phage iceberg?

Bacteriapossess (bacterio)phage defence systems to ensure their survival. The thermophilic lactic acid bacterium, Streptococcus thermophilus, which is used in dairy fermentations, harbours multiple CRISPR-Cas and restriction and modification (R/M) systems to protect itself against phage attack, with limited reports on other types of phage-resistance. Here, we describe the systematic identification and functional analysis of the phage resistome of S. thermophilus using a collection of 27 strains as representatives of the species. In addition to CRISPR-Cas and R/M systems, we uncover nine distinct phage-resistance systems including homologues of Kiwa, Gabija, Dodola, defence-associated sirtuins and classical lactococcal/streptococcal abortive infection systems. The genes encoding several of these newly identified S. thermophilus antiphage systems are located in proximity to the genetic determinants of CRISPR-Cas systems thus constituting apparent Phage Defence Islands. Other phage-resistance systems whose encoding genes are not co-located with genes specifying CRISPR-Cas systems may represent anchors to identify additional Defence Islands harbouring, as yet, uncharacterised phage defence systems. We estimate that up to 2.5% of the genetic material of the analysed strains is dedicated to phage defence, highlighting that phage-host antagonism plays an important role in driving the evolution and shaping the composition of dairy streptococcal genomes.

Functional and practical insights into three lactococcal antiphage systems.

The persistent challenge of phages in dairy fermentations requires the development of starter cultures with enhanced phage resistance. Recently, three plasmid-encoded lactococcal antiphage systems, named Rhea, Aristaios, and Kamadhenu, were discovered. These systems were found to confer high levels of resistance against various Skunavirus members. In the present study, their effectiveness against phage infection was confirmed in milk-based medium, thus validating their potential to ensure reliable dairy fermentations. We furthermore demonstrated that Rhea and Kamadhenu do not directly hinder phage genome replication, transcription, or associated translation. Conversely, Aristaios was found to interfere with phage transcription. Two of the antiphage systems are encoded on pMRC01-like conjugative plasmids, and the Kamadhenu-encoding plasmid was successfully transferred by conjugation to three lactococcal strains, each of which acquired substantially enhanced phage resistance against Skunavirus members. Such advances in our knowledge of the lactococcal phage resistome and the possibility of mobilizing these protective functions to bolster phage protection in sensitive strains provide practical solutions to the ongoing phage problem in industrial food fermentations.IMPORTANCEIn the current study, we characterized and evaluated the mechanistic diversity of three recently described, plasmid-encoded lactococcal antiphage systems. These systems were found to confer high resistance against many members of the most prevalent and problematic lactococcal phage genus, rendering them of particular interest to the dairy industry, where persistent phage challenge requires the development of starter cultures with enhanced phage resistance characteristics. Our acquired knowledge highlights that enhanced understanding of lactococcal phage resistance systems and their encoding plasmids can provide rational and effective solutions to the enduring issue of phage infections in dairy fermentation facilities.

Fortification of Goat Milk Yogurts with Encapsulated Postbiotic Active Lactococci.

The species Lactococcus lactis is a bacterium extensively used in the dairy industry. This bacterium is Generally Recognized as Safe and was added to the European Food Safety Authority's Qualified Presumption of Safety list. The major functions of this species in dairy fermentation are the production of lactic acid from lactose, citric acid fermentation, and the hydrolysis of casein. But, the representatives of this species that produce bacteriocin substances can also exert an inhibitory effect against spoilage bacteria. The aims of this study were to test three lactococcal strains isolated from raw goat milk for their postbiotic activity and to test their stability in goat milk yogurts after their application in encapsulated form for their further application. To achieve these aims, validated methods were used. Three Lactococcus lactis strains (identified by Blastn 16S rRNA analysis) produced bacteriocin substances/postbiotics. These concentrated postbiotics inhibited the growth of enterococci and staphylococci (by up to 97.8%), reaching an inhibitory activity of up to 800 AU/mL. The encapsulated (freeze-dried) lactococci survived in the goat milk yogurts with sufficient stability. Strain MK2/8 fortified the yogurts in the highest amount (8.1 ± 0.0 cfu/g log 10). It did not influence the pH of the yogurt.

Unveiling the genetic basis and metabolic rewiring behind the galactose-positive phenotype in a Streptococcus thermophilus mutant

Streptococcus thermophilus (S. thermophilus) is a widely used starter culture in dairy fermentation, but most strains are galactose-negative and only metabolize glucose from lactose hydrolysis. In this study, we aimed to uncover the mechanisms underlying the acquisition of a stable galactose-positive (Gal+) phenotype in a mutant strain of S. thermophilus IMAU10636. By treating the wild-type strain with the mutagenic agent N-methyl-N-nitro-N-nitrosoguanidine, we successfully isolated a Gal+ mutant, S. thermophilus IMAU10636Y. Comparative enzyme activity assays revealed that the mutant exhibited higher β-galactosidase and galactokinase activities, but lower glucokinase and pyruvate kinase activities compared to the wild-type. High-performance liquid chromatography analysis confirmed the mutant’s enhanced ability to utilize lactose and galactose, leading to increased glucose secretion. Integrated genome and transcriptomics analyses provided deeper insights into the underlying genetic and metabolic mechanisms. We found that the metabolism regulatory network of the glycolysis / Leloir pathway was altered in the mutant, possibly due to the upregulation of the gene expression in the galR-galK intergenic region. This likely led to increased RNA polymerase binding and transcription of the gal operon, ultimately promoting the Gal+ phenotype. Additionally, we identified a mutation in the scrR gene, encoding a LacI family transcriptional repressor, which also contributed to the Gal+ phenotype. These findings offer new perspectives on the metabolic rewiring and regulatory mechanisms that enable S. thermophilus to acquire the ability to metabolize galactose. This knowledge can inform strategies for engineering and selecting Gal+ strains with desirable fermentation characteristics for dairy applications.

Dairy Intake in Relation to Prediabetes and Continuous Glycemic Outcomes: A Systematic Review and Dose-Response Meta-Analysis of Prospective Cohort Studies

BackgroundModest inverse associations have been found between dairy intake, particularly yogurt, and type 2 diabetes risk. Investigating associations of dairy intake with early onset of type 2 diabetes offers opportunities for effective prevention of this condition. ObjectivesThis study aims to investigate the relationships between the intake of different dairy types, prediabetes risk, and continuous glycemic outcomes. MethodsSystematic literature searches across multiple databases were performed of studies published up to September 2023. Included were prospective cohort studies in healthy adults that examined the association between dairy intake and prediabetes risk according to diagnostic criteria, or continuous glycemic markers. A dose-response random-effects meta-analysis was used to derive incremental relative risks (RRs) for associations of total dairy, fermented dairy, milk, yogurt, cheese (all total, high-fat, and low-fat), cream, and ice cream with prediabetes risk adjusted for sociodemographic, health and cardiometabolic risk factors, and dietary characteristics. ResultsThe meta-analyses encompassed 6653 prediabetes cases among 95,844 individuals (age range 45.5–65.5 y) including 6 articles describing 9 cohorts. A quadratic inverse association was observed for total dairy intake and prediabetes risk, with the lowest risk at 3.4 servings/d (RR: 0.75; 95% confidence interval: 0.60, 0.93; I2 = 18%). Similarly, total, and high-fat cheese exhibited nonlinear inverse associations with prediabetes risk, showing the lowest risk at 2.1 servings/d (0.86; 0.78, 0.94; I2 = 0%, and 0.90; 0.81, 0.99; I2 = 12%), but a higher risk at intakes exceeding 4 servings/d. Ice cream intake was linearly associated with prediabetes risk (0.85; 0.73, 0.99; I2 = 0% at the highest median intake of 0.23 servings/d). Other dairy types showed no statistically significant associations. The systematic review on dairy intake and glycemic outcomes showed considerable variabilities in design and results. ConclusionsThe findings suggest an inverse association between moderate dairy and cheese intake in preventing prediabetes. The potential for reverse causation and residual confounding highlights the need for studies with comprehensive repeated measurements. Trial registration numberPROSPERO 2023 CRD42023431251.

Food Consumption and Risk of Islet Autoimmunity and Type 1 Diabetes in Children at Increased Genetic Susceptibility for Type 1 Diabetes

BackgroundProspective longitudinal evidence considering the entire childhood food consumption in relation to the development of islet autoimmunity (IA or) type 1 diabetes is lacking. ObjectivesWe studied the associations of consumption of various foods and their combinations with IA and type 1 diabetes risk. MethodsChildren with genetic susceptibility to type 1 diabetes born in 1996–2004 were followed from birth up to ≤6 y of age in the prospective birth cohort type 1 diabetes prediction and prevention study (n = 5674). Exposure variables included 34 food groups covering the entire diet based on repeated 3-d food records at ages 3 mo to 6 y. Endpoints were islet cell antibodies plus biochemical IA (n = 247), multiple biochemical IA (n = 206), and type 1 diabetes (n = 94). We analyzed associations between longitudinally observed foods and risk of IA/type 1 diabetes using a Bayesian approach to joint models in 1-food and multi-food models adjusted for energy intake, sex, human leukocyte antigen genotype, and familial diabetes. ResultsThe final multi-food model for islet cell antibodies plus biochemical IA included oats [hazard ratio (HR): 1.09; 95% credible interval (CI): 1.04, 1.14], banana (HR: 1.07; 95% CI: 1.03, 1.11), and cruciferous vegetables (HR: 0.83; 95% CI: 0.73, 0.94). The final model for multiple biochemical IA included, in addition to the above-mentioned foods, fermented dairy (HR: 1.42; 95% CI: 1.12, 1.78) and wheat (HR: 1.10; 95% CI: 1.03, 1.18). The final multi-food model for type 1 diabetes included rye (HR: 1.27; 95% CI: 1.07, 1.50), oats (HR: 1.15; 95% CI: 1.03, 1.26), fruits (HR: 1.05; 95% CI: 1.01, 1.09), and berries (HR: 0.67; 95% CI: 0.50, 0.93). ConclusionsHigher consumption of oats, gluten-containing cereals, and fruits was associated with increased that of cruciferous vegetables with decreased risk of several type 1 diabetes-related endpoints when considering all the foods in combination. Further etiological and mechanistic studies are warranted.

Serial fermentation in milk generates functionally diverse community lineages with different degrees of structure stabilization.

Microbiome applications require approaches for shaping and propagating microbial communities. Shaping allows the selection of communities with desired taxonomic and functional properties, while propagation allows the production of the biomass required to inoculate the engineered communities in the target ecosystem. In top-down community engineering, where communities are obtained from a pool of mixed microorganisms by acting on environmental variables, a major challenge is to master the balance between shaping and propagation. However, the ecological factors that favor high dynamics of community structure and, conversely, those that favor stability during propagation are not well understood. In this work, short-term dairy backslopping was used to investigate the key role of the taxonomic composition and structure of bacterial communities on their dynamics. The results obtained open up interesting prospects for the biotechnological use of microbiomes, particularly in the field of dairy fermentation, to diversify approaches for injecting microbial biodiversity into cheesemaking processes.

Draft genome sequences of 53 Lactococcus and Leuconostoc strains isolated from two undefined DL-starter cultures.

This study presents the complete genomes of 53 strains of Lactococcus and Leuconostoc isolated from two undefined DL-starter cultures originating from Denmark, Tistrup, and P. The genomes were reconstructed using long-read, nanopore-based DNA sequencing, delivering comprehensive data set for comparative genomics and taxonomic classification, with potential utility in dairy fermentation processes.

The Microbial Community of Natural Whey Starter: Why Is It a Driver for the Production of the Most Famous Italian Long-Ripened Cheeses?

The remarkable global diversity in long-ripened cheese production can be attributed to the adaptability of the cheese microbiota. Most cheese types involve intricate microbial ecosystems, primarily represented by lactic acid bacteria (LAB). The present study aims to review the microbial community’s diversity in dairy fermentation processes, focusing on two famous Italian cheeses, Grana Padano and Parmigiano Reggiano, produced using natural whey starter (NWS). NWS, created by retaining whey from the previous day’s cheese batches, forms a microbiological connection between daily cheese productions. Through this technique, a dynamic microbiota colonizes the curd and influences cheese ripening. The back-slopping method in NWS preparation ensures the survival of diverse biotypes, providing a complex microbial community in which interactions among microorganisms are critical to ensuring its technological functionality. As highlighted in this review, the presence of microbial cells alone does not guarantee technological relevance. Critical microorganisms can grow and colonize the curd and cheese. This complexity enables NWS to adapt to artisanal production technologies while considering variations in raw milk microbiota, inhibitory compounds, and manufacturing conditions. This critical review aims to discuss NWS as a key factor in cheese making, considering microbial communities’ ability to evolve under different selective pressures and biotic and abiotic stresses.

Comparative genomics of four lactic acid bacteria identified with Vitek MS (MALDI-TOF) and whole-genome sequencing

Lactic acid bacteria (LAB) can be used as a probiotic or starter culture in dairy, meat, and vegetable fermentation. Therefore, their isolation and identification are essential. Recent advances in omics technologies and high-throughput sequencing have made the identification and characterization of bacteria. This study firstly aimed to demonstrate the sensitivity of the Vitek MS (MALDI-TOF) system in the identification of lactic acid bacteria and, secondly, to characterize bacteria using various bioinformatics approaches. Probiotic potency-related genes and secondary metabolite biosynthesis gene clusters were examined. The Vitek MS (MALDI-TOF) system was able to identify all of the bacteria at the genus level. According to whole genome sequencing, the bacteria were confirmed to be Lentilactobacillus buchneri, Levilactobacillus brevis, Lactiplantibacillus plantarum, Levilactobacillus namurensis. Bacteria had most of the probiotic potency-related genes, and different toxin-antitoxin systems such as PemIK/MazEF, Hig A/B, YdcE/YdcD, YefM/YoeB. Also, some of the secondary metabolite biosynthesis gene clusters, some toxic metabolite-related genes, and antibiotic resistance-related genes were detected. In addition, Lentilactobacillus buchneri Egmn17 had a type II-A CRISPR/Cas system. Lactiplantibacillus plantarum Gmze16 had a bacteriocin, plantaricin E/F.

Ribosome profiling reveals downregulation of UMP biosynthesis as the major early response to phage infection.

The ribosome profiling technology has provided invaluable insights for understanding cellular translation and eukaryotic viral infections. However, its potential for investigating host-phage interactions remains largely untapped. Here, we applied ribosome profiling to Lactococcus cremoris cultures infected with sk1, a major infectious agent in dairy fermentation processes. This revealed a profound downregulation of genes involved in pyrimidine nucleotide synthesis at an early stage of phage infection, suggesting an anti-phage program aimed at restricting nucleotide availability and, consequently, phage propagation. This is consistent with recent findings and contributes to our growing appreciation for the role of nucleotide limitation as an anti-viral strategy. In addition to capturing rapid alterations in gene expression levels, we identified translation occurring outside annotated regions, as well as signatures of non-standard translation mechanisms. The gene profiles revealed specific changes in ribosomal densities upon infection, reflecting alterations in the dynamics of the translation process.

Fermentation Practices Select for Thermostable Endolysins in Phages.

Endolysins are produced by (bacterio)phages and play a crucial role in degrading the bacterial cell wall and the subsequent release of new phage progeny. These lytic enzymes exhibit a remarkable diversity, often occurring in a multimodular form that combines different catalytic and cell wall-binding domains, even in phages infecting the same species. Yet, our current understanding lacks insight into how environmental factors and ecological niches may have influenced the evolution of these enzymes. In this study, we focused on phages infecting Streptococcus thermophilus, as this bacterial species has a well-defined and narrow ecological niche, namely, dairy fermentation. Among the endolysins found in phages targeting this species, we observed limited diversity, with a singular structural type dominating in most of identified S. thermophilus phages. Within this prevailing endolysin type, we discovered a novel and highly conserved calcium-binding motif. This motif proved to be crucial for the stability and activity of the enzyme at elevated temperatures. Ultimately, we demonstrated its positive selection within the host's environmental conditions, particularly under the temperature profiles encountered in the production of yogurt, mozzarella, and hard cheeses that rely on S. thermophilus.

Thiamine-Starved Lactococcus lactis for Producing Food-Grade Pyruvate.

Lactococcus lactis is a safe lactic acid bacterium widely used in dairy fermentations. Normally, its main fermentation product is lactic acid; however, L. lactis can be persuaded into producing other compounds, e.g., through genetic engineering. Here, we have explored the possibility of rewiring the metabolism of L. lactis into producing pyruvate without using genetic tools. Depriving the thiamine-auxotrophic and lactate dehydrogenase-deficient L. lactis strain RD1M5 of thiamine efficiently shut down two enzymes at the pyruvate branch, the thiamine pyrophosphate (TPP) dependent pyruvate dehydrogenase (PDHc) and α-acetolactate synthase (ALS). After eliminating the remaining enzyme acting on pyruvate, the highly oxygen-sensitive pyruvate formate lyase (PFL), by simple aeration, the outcome was pyruvate production. Pyruvate could be generated by nongrowing cells and cells growing in a substrate low in thiamine, e.g., Florisil-treated milk. Pyruvate is a precursor for the butter aroma compound diacetyl. Using an α-acetolactate decarboxylase deficient L. lactis strain, pyruvate could be converted to α-acetolactate and diacetyl. Summing up, by starving L. lactis for thiamine, secretion of pyruvate could be attained. The food-grade pyruvate produced has many applications, e.g., as an antioxidant or be used to make butter aroma.

A multifaceted investigation of lactococcal strain diversity in undefined mesophilic starter cultures.

This study reports on the development of a combined culture-based analysis and metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, a novel qPCR-based genotype detection assay, capable of discerning nine distinct lactococcal genotypes (based on lactococcal cell wall polysaccharide biosynthesis gene clusters), was used to monitor compositional changes in an undefined starter culture following phage attack. These analytical approaches facilitate a multifaceted assessment of starter culture compositional stability during milk fermentation, which has become an important QC aspect due to the increasing demand for consistent and high-quality dairy products.

The effect of seed germination and Bacillus spp. on the ripening of plant cheese analogs.

The development of novel plant-based fermented food products for which no traditional templates exist requires the development of starter cultures. Although the principles of microbial flavor formation in plant-based analogs partially overlap with dairy fermentations, the composition of the raw materials and thus likely the selective pressure on the activity of starter cultures differs. Experiments that are described in this study explored the use of seed germination, the use of lactic acid bacteria, and the use of bacilli to reduce hygienic risks, to acidify plant milk, and to generate taste-active compounds through proteolysis and fermentative conversion of carbohydrates. The characterization of fermentation microbiota by culture-dependent and culture-independent methods also confirmed that the starter cultures used were able to control microbial communities throughout 90 d of ripening. Taken together, the results provide novel tools for the development of plant-based analogs of fermented dairy products.

Lactococcus lactis in Dairy Fermentation—Health-Promoting and Probiotic Properties

The use of lactic acid bacteria (LAB) in the fermentation process to produce fermented foods has a long history. Furthermore, LAB are beneficial microorganisms known for their health-promoting characteristics. During fermentation, LAB have the capacity to produce significant amounts of bioactive substances, such as peptides, bacteriocins, lactic acid, exopolysaccharides (EPSs), enzymes, and others. Lactococcus lactis as one of the best-known and well-characterized species of LAB serves as a model organism for studying LAB. For a very long time, L. lactis has been used in milk fermentation, both in well-monitored industrial settings and on a small scale in traditional operations. Furthermore, L. lactis is a vital microorganism in the dairy food fermentation industry due to its role in acidification, flavor development, and the creation of various dairy products, including cheese, fermented butter, and others. The novelty of this review is the comprehensive and organized presentation of the main benefits of the use of L. lactis in milk fermentation processes including technological and safety features relevant for the dairy industry, probiotic potential, the ability to produce bioactive compounds (e.g., bacteriocins, GABA), and the recent development of such bacteria research methods like whole genome sequencing (WGS).

A Metagenomics Approach to Enumerate Bacteriophages in a Food Niche.

Dairy fermentation relies on the activity of starter cultures composed primarily of lactic acid bacteria such as Lactococcus and Streptococcus thermophilus strains to produce consistent, high-quality products. Bacteriophages are a constant threat to the industry, often causing slowed or failed fermentation resulting in significant economic losses. To ensure the continuation of reliable fermentation practices, it is important to detect and monitor the phage populations impacting different starter cultures. This has traditionally been done primarily through culture-dependent methods but has since expanded into viral metagenomics. Here we outline a protocol for a targeted virome extraction from a dairy whey sample, followed by subsequent sequencing and phageome analysis of the sample.