038: Prevalence and Diversity of Leuconostoc spp. From Raw Milk

Introduction

Leuconostoc spp. are heterofermentative, gram-positive lactic acid bacteria generally recognized as safe and nonpathogenic. They are often associated with raw milk and can contribute to spoilage, particularly in sweetened-flavored milks, manifesting as ropy-slimy products with lowered pH. Limited information is available on the prevalence and diversity of Leuconostoc spp. in raw milk. This study examines the prevalence and diversity of Leuconostoc spp. in raw milk silo samples from a Mid-Atlantic dairy processor over six months.

Methods

Raw milk samples were collected weekly from April to October 2024. Milk composition and microbial quality were analyzed. Leuconostoc spp. were detected by plating on De Man Rogosa and Sharpe medium supplemented with 10 µg/ml vancomycin, followed by incubation at 30°C for 48 hours. Suspected Leuconostoc isolates were verified by PCR and 16S rRNA gene sequencing and further assigned to species and genotype groups. Representative isolates were characterized for carbohydrate utilization (API 50 CH), exopolysaccharide (EPS) production, growth at different temperatures, and the ability to survive laboratory pasteurization.

Results

A total of 569 Leuconostoc isolates were obtained. Ribosomal RNA sequencing revealed six Leuconostoc species: L. lactis (48.68%), L. mesenteroides (33.74%), L. citreum (11.42%), L. falkenbergense (5.10%), L. pseudomesenteroides (0.88%), and L. holzapfelii (0.18%), which were further classified into ten different genotypes. Representative isolates from each genotype were further characterized. All selected isolates fermented galactose, glucose, fructose, N-acetylglucosamine, lactose, and sucrose. Mucoid substances (EPS) were produced by all L. citreum, three L. mesenteroides, and three L. falkenbergense isolates on sucrose (5% w/v) agar. All L. lactis and L. mesenteroides isolates grew well in unflavored milk after 15 days of incubation at 4°C, while all representative isolates grew at 10°C and 20°C. Additionally, three L. lactis isolates survived laboratory pasteurization in unflavored milk.

Significance

This study provides baseline information about the prevalence and diversity of Leuconostoc in raw milk. The identification of six different species and ten genotypes highlights the considerable diversity of this genus in raw milk. The ability of certain isolates to grow at refrigeration temperatures and survive pasteurization raises concerns about their persistence during processing and storage, potentially leading to quality defects.

Authors: Songyi Yoo, Laura Rolon, Robert F. Roberts