# Identification of Lactic Acid Bacteria From the Crust and Inner Part of Artisanally Produced Mihaliç Cheese Sold With Salty and Low Salty Label by Using MALDI‐TOF‐MS and 16S rDNA Sequencing

## TL;DR

This study identifies lactic acid bacteria in traditional Mihaliç cheese using two methods, revealing key species that contribute to its flavor and texture.

## Contribution

This is the first study to characterize the lactic microbiota of Mihalic cheese using MALDI-TOF MS and 16S rDNA sequencing.

## Key findings

- Lactococcus lactis was the predominant species in Mihaliç cheese isolates.
- Lacticaseibacillus casei/paracasei and Limosilactobacillus fermentum were the most common lactobacilli.
- SMC and LSMC samples showed distinct bacterial distributions, with Lim. fermentum more abundant in SMC.

## Abstract

Mihaliç is a traditional cheese manufactured using artisanal methods without adding starter cultures from a mixture of sheep's and cow's milk. The indigenous microbiota originating from raw milk and the surrounding environment, which adapt to the ripening conditions, play a crucial role in developing the artisanal cheeses' characteristic flavor, aroma, and texture. In this study, the lactic acid bacteria in both the crust and the inner part of Mihaliç sold under the label of Salty Mihaliç Cheese (SMC) and Low‐Salty Mihaliç Cheese (LSMC) were determined using MALDI‐TOF MS analysis and 16S rDNA sequencing. The enumeration of Streptococcaceae on M17 agar revealed approximately a 2‐log higher count compared to Lactobacillus and Enterococcus populations. Among the isolates recovered from M17 medium,
Lactococcus lactis
was identified as the predominant species, comprising 43.7% of the total. Notably, consistent with observations in many other artisanal raw milk cheeses,
Streptococcus gallolyticus
subsp. macedonicus and
Streptococcus infantarius
subsp. infantarius were also detected at high frequencies, with 41.2% and 7.5% prevalence rates, respectively. Among the enterococci,
Enterococcus faecium
and
Enterococcus faecalis
were the most frequently isolated species, accounting for 43.5% and 42.2% of the isolates, respectively. Regarding the distribution of lactobacilli, Lacticaseibacillus casei/paracasei and Limosilactobacillus fermentum emerged as the dominant species, exhibiting prevalence rates of 47.1% and 35.2%, respectively. Meanwhile, comparative analysis between SMC and LSMC cheese samples showed that Lim. fermentum was more abundant in SMC samples (55.1%), whereas Lcb. paracasei/casei was dominant in LSMC samples (55.9%). Furthermore, both Lim. fermentum and Lcb. rhamnosus were more frequently recovered from the inner part of the cheese matrix. These findings highlight the complex and diverse lactic microbiota of Mihaliç cheese, which is likely to play a significant role in the development of its unique flavor and textural characteristics.

Our results indicate that streptococci were predominant, particularly in the crust, and that Lacticaseibacillus paracasei/casei and Limosilactobacillus fermentum were the most common lactobacilli, with notable differences between SMC and LSMC samples. Furthermore, the presence of Streptococcus gallolyticus ssp. macedonicus and Str. infantarius ssp. infantarius as dominant species aligns with patterns seen in other raw milk cheeses. This is the first study to characterize the lactic microbiota of Mihalic cheese using this dual‐method approach.