Evaluation of Microbial Safety of Dairies using Bacterial Proteomic Profiling via MALDI Approach

Summary

This protocol enables rapid, cost-effective evaluation of microbial composition in dairy products by combining culturomics with MALDI-TOF MS. It delivers taxonomy results comparable to 16S rRNA sequencing with a shorter turnaround time than traditional culture methods.

Abstract

We present a protocol using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, hereinafter referred to as "MS") identification method for monitoring the presence of microorganisms in different dairy products (butter, mozzarella cheese, milk, cream) using a proteomic approach, modern culturomics, and various sample preparation procedures that could be incorporated to the food safety quality control laboratories as a fast, reliable, and robust tool for monitoring microbial safety. It includes a microbial isolation step, picking grown colonies, sample preparation procedures (intact cells, on-spot and in-tube extraction), setting MS analysis parameters, generating and collecting MS spectra (protein fingerprints), and finally getting taxonomy using the MS platform, followed by data interpretation and visualization. This method, validated by numerous studies, offers a more reliable and cost-effective identification of microorganisms from milk compared to commercial biochemical test systems. It stands out as a promising advancement in food microbiology, meeting the diverse requirements of laboratories through its simple protocols and significantly reduced analysis time. The presented protocol addresses the needs of the food industry by providing reliable microbial identification in a proper turnaround time (TAT) that is well-suited to manufacturing processes.

Introduction

The overall goal of this protocol is to provide a comprehensive methodology for monitoring the microbial safety of various dairy products, including butter, cheese, milk, and cream, by employing MS for bacterial proteomic profiling. This approach, which combines modern culturomics with optimized sample preparation procedures, is intended to be a fast, reliable, and robust tool for food safety and quality control laboratories. The rationale behind creating and applying this methodology comes from the limitations of standard microbial identification methods. For many years, veterinary and food diagnostic laboratories have relied on conventional biochemical tests that ex....

Protocol

The reagents and the equipment used in this study are listed in the Table of Materials.

1. Isolation of dairy-related bacteria

1. Obtaining bacterial cultures from liquid or solid dairy products
   1. Culture media selection
      1. For bacterial isolation from dairy samples, select the culture media according to the expected microbial groups, including general growth media, media for Gram-negative bacteria, lactic acid bacteria (LAB), spore-forming bacteria, as well as slow-growing or fastidious microorganisms.
         NOTE: To maximize recovery of both dominant and rare species, multip....

Results

Culturomic approach
The culturomic approach is a strategy aimed at maximally diversifying the conditions of microorganism cultivation in order to increase the chances of isolating, among others, rare, difficult to cultivate, or previously undescribed species¹². This makes it possible to obtain a much broader picture of the biodiversity of microflora present in various samples, e.g., food samples. Increased diversity of microorganisms can be observed already at the stage of vis.......

Discussion

The protocol dedicated to microbial identification of dairy products offers an important advancement over traditional methods by ensuring optimal consensus on speed, reliability, and cost-effectiveness. Nevertheless, some crucial steps within the protocol demand careful attention. Firstly, the isolation and culturing phase is foundational. The culture media and incubation conditions must match the expected microbial diversity and load. Obtaining pure cultures is paramount, often requiring subculturing to avoid mixed prof.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
70% formic acid	Chempur	115676307
Acetonitrile	Merck KGaA	1.00014.1011
APT agar	Merck KGaA	1.10453.0500
Bacterial Test Standard	Bruker Daltonik GmbH	8255343
CBL Agar (China Blue Lactose Agar)	Merck KGaA	1.02348.0500
Columbia Blood agar	Oxoid	CM0331B
M17 agar
Milk Plate Count Agar (MPCA)	Oxoid	cm0681
MRS agar (de Man, Rogosa and Sharpe Agar)	Merck KGaA	1.10660.0500
MSP 96 target polished steel BC	Bruker Daltonik GmbH	8280800
Peptone water	Sigma-Aldrich	70179
Scheadler agar	Sigma-Aldrich	91019
Sheep blood	Biomaxima	SL0160-100
trifluoroacetic acid	Sigma-Aldrich	T6508
Tryptic Soy Agar (TSA)	Sigma-Aldrich	22091
Water	Merck KGaA	1.15333.2500
α-Cyano-4-hydroxycinnamic acid	Bruker Daltonik GmbH	8201344