# Screening, Safety Assessment, and Process Optimization of Lactic Acid Bacteria from Traditional Yak Yogurt as Adjunct Cultures

## TL;DR

Researchers isolated a lactic acid bacteria strain from yak yogurt that can speed up cheese ripening while being safe and efficient.

## Contribution

A novel, safe, and efficient adjunct lactic acid bacteria strain (Limosilactobacillus fermentum 270) is developed for accelerated cheese ripening.

## Key findings

- Limosilactobacillus fermentum 270 showed high tolerance to harsh cheese-making conditions.
- The strain has strong proteolytic and flavor-enhancing properties with no biogenic amine synthesis genes.
- Optimized cheese-making conditions improved efficiency and viability of the adjunct culture.

## Abstract

Cheese ripening is slow and costly, driving interest in accelerating maturation. This study aimed to isolate a safe, efficient adjunct starter from traditional Sichuan yak yoghurt, a niche rich in stress-adapted lactic acid bacteria. From 295 isolates, 15 strains tolerant to high salt, low pH, and low temperature were selected. Using acidification, autolysis, proteolysis, and peptidase activity as indices, principal component analysis identified Limosilactobacillus fermentum 270 as the best candidate. Phenotypic assays showed no haemolysis, gelatin liquefaction, indole production, or amino acid decarboxylase activity. Whole-genome sequencing confirmed species identity and revealed 52 protease/peptidase genes, complete pathways for diacetyl/acetoin biosynthesis and branched-chain amino acid conversion, and no functional biogenic amine synthesis genes. Stress-related genes (F-ATPase, glycine-betaine transport, cold-shock proteins) support cheese adaptability. Antibiotic resistance gene homologs were mainly chromosomal and unlinked to mobile genetic elements; a functional CRISPR-Cas system lowers horizontal transfer risk. The strain was developed as a freeze-dried direct-vat starter (97.3% viability). Orthogonal optimisation of yak Gouda cheese-making defined best conditions: 0.018% adjunct, 45 min acidification, pH 5.8, and 30% curd washing. L. fermentum 270 thus combines proteolytic, flavour-enhancing, genetic safety, and processing traits, offering a promising adjunct for accelerated cheese ripening.