The Biodiversity of the Microbiota Producing Heat-Resistant Enzymes Responsible for Spoilage in Processed Bovine Milk and Dairy Products

Abstract

Raw bovine milk is highly nutritious as well as pH-neutral, providing the ideal conditions for microbial growth. The microbiota of raw milk is diverse and originates from several sources of contamination including the external udder surface, milking equipment, air, water, feed, grass, feces, and soil. Many bacterial and fungal species can be found in raw milk. The autochthonous microbiota of raw milk immediately after milking generally comprises lactic acid bacteria such as Lactococcus, Lactobacillus, Streptococcus, and Leuconostoc species, which are technologically important for the dairy industry, although they do occasionally cause spoilage of dairy products. Differences in milking practices and storage conditions on each continent, country and region result in variable microbial population structures in raw milk. Raw milk is usually stored at cold temperatures, e.g., about 4°C before processing to reduce the growth of most bacteria. However, psychrotrophic bacteria can proliferate and contribute to spoilage of ultra-high temperature (UHT) treated and sterilized milk and other dairy products with a long shelf life due to their ability to produce extracellular heat resistant enzymes such as peptidases and lipases. Worldwide, species of Pseudomonas, with the ability to produce these spoilage enzymes, are the most common contaminants isolated from cold raw milk although other genera such as Serratia are also reported as important milk spoilers, while for others more research is needed on the heat resistance of the spoilage enzymes produced. The residual activity of extracellular enzymes after high heat treatment may lead to technological problems (off flavors, physico-chemical instability) during the shelf life of milk and dairy products. This review covers the contamination patterns of cold raw milk in several parts of the world, the growth potential of psychrotrophic bacteria, their ability to produce extracellular heat-resistant enzymes and the consequences for dairy products with a long shelf life. This problem is of increasing importance because of the large worldwide trade in fluid milk and milk powder.

Keywords: Pseudomonas; Serratia; heat-resistant enzyme; lipase; microbial dynamics; peptidase; psychrotrophic; spoilage.

FIGURE 1

Hypothetic mechanism of UHT milk destabilization due to casein micelle proteolysis by heat-resistant peptidase during storage at ambient temperature. The different species and strains of proteolytic psychrotrophic bacteria may produce heat-stable peptidases, which hydrolyze different types of casein. Some heat-resistant peptidases have preferential cleavage sites in hydrophobic areas of casein (red areas) while others hydrolyze preferentially the κ-casein which makes the connection between the hydrophobic core and the hydrophilic medium (blue areas).

FIGURE 2

Clogging of the heating exchanger due to processing of milk spiked with Pseudomonas and stored for 5 days at 6.5°C. (A) UHT Process Pilot Plant (ILVO, Belgium), (B) heat exchanger, (C) detail of the heat exchanger with clotting of milk.

FIGURE 3

Correlation between off-flavors and protein hydrolysis by six different Pseudomonas peptidase groups in UHT-milk, as described by Marchand et al. (2009b). Raw milk was pasteurized, inoculated with one of the Pseudomonas strains to a final concentration of 3 log CFU/mL, and stored for 3 to 5 days at 6.5°C until skimming and indirect UHT-processing (5 s at 140°C). Processed milk was aseptically filled in high-density polyethylene bottles of 0.5 L and stored at 37°C to accelerate proteolysis. The six Pseudomonas peptidase milk samples were compared with the reference control milk by an experienced taste panel of 35 people at the Institute for Agricultural and Fisheries Research (ILVO), Belgium in a sensorial cabinet equipped with individually partitioned booths. Milk with off-flavor was diluted as follows: (A) Pseudomonas milk undiluted, (B) 2/3 Pseudomonas milk + 1/3 control milk, (C) 1/3 Pseudomonas milk + 2/3 control milk, (D) Control milk undiluted. The taste panel was asked to rank the milk samples (A–B–C–D) according to preference. Statistical evaluation of the results was based on the Rank Test to KRAMER (Kramer, 1960) for α = 0.05. Simultaneously, proteolysis (expressed as μmol glycine equivalents mL^-1 milk) was determined in each milk dilution (A–B–C–D) by the trinitrobenzenesulfonic acid (TNBS) method (Polychroniadou, 1988). No significant proteolysis off-flavors is indicated by black bars, the uncertainty range by light gray bars (the panel did not reject the milk samples; the lower limit of the bar is determined by the TNBS-value of the most diluted sample that was not rejected by sensory analysis) and the significant proteolysis off-flavors by dark gray bars (panel rejected the milk samples and tasted off-flavors; the lower limit of the bar is determined by the TNBS-value of the least diluted sample). This figure was reproduced from Marchand et al. (2017).