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Alliance of Crop, Soil and Environmental Science Societies
Vol. 94 (5), 2016, Pages: 263-264

0552 Evaluation of microbial enzymes for degradation of exopolymeric substances (EPS) within biofilm matrices for more effective cleaning

N. Garcia-Fernandez, A. Hassan and S. Anand

Dairy Science Department, South Dakota State University, Brookings.

Abstract

This study was conducted to determine whether biofilms on reverse osmosis (RO) membranes could be disrupted by natural crude enzymes (CE) obtained from microorganisms with the ability to degrade exopolymeric substances (EPS). The first enzyme extract (termed CE1) was obtained from a bacteriophage with infectivity for the EPS producing Lactococcus lactis ssp. cremoris strain JFR. The phage was isolated from a dairy plant environment and propagated overnight in liquid cultures of its host. The pH was then neutralized (pH 7.0) and phage particles were removed by centrifugation (75,600 × g for 60 min). The second extract (CE2) was obtained from supernatants of JFR cultures in 10% whey protein concentrate (WPC35), showing a reduction in its viscosity after 72 h incubation (suspected to produce hydrolases to degrade its own EPS). The third extract (CE3) was derived from supernatants of overnight tryptic soy broth (TSB) cultures of Bacillus mojavensis strain Bc, which hydrolyzed amylopectin and reduced viscosity of milk fermented with ropy yogurt cultures. All supernatants were microfiltered (0.22 μm) and then concentrated by ultrafiltration (10 KDa). Standard static biofilm assays were conducted by developing biofilms on 2 by 2 cm pieces of RO polyamide membrane for 24 h. The CE1 preparation was applied to JFR biofilms that were formed using M17 as the suspension medium. The CE2 was applied to biofilms formed on membranes by slime producing Bacillus cultures (Bmojavensis strain Bc and B. licheniformis strain K1) using TSB. The CE3 was applied to biofilms formed by a ropy strain of Streptococcus thermophilus (ST3534) with M17. The biofilms were treated with CE at 1% (v/v) in phosphate buffer saline (PBS) or just PBS as control at 37°C for 30 min under agitation. The viable cell counts were determined by standard culture techniques after detaching cells with stomacher. All experiments were repeated 3 times. The CE extracts obtained from the bacteriophage (CE1), JFR (CE2), and Bc (CE3) cultures reduced the counts of the respective single species biofilms by 0.5 log CFU/cm2 (JFR), 0.3 and 0.7 log CFU/cm2 (Bc or K1), and 0.2 log CFU/cm2 (ST3534), respectively. These results provide an early indication that natural crude enzymes from microbial sources have the potential to selectively degrade the resistant biofilms matrices. This work also suggests that cleaning applications must contain a diverse mixture of hydrolytic enzymes to degrade the complex matrix of multiple species biofilms of dairy separation membranes.

 

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