Enzymatic Digestion for Improved Bacteria Separation from Leafy Green Vegetables

Abstract

An effective and rapid method for the separation of bacteria from food matrix remains a bottleneck for rapid bacteria detection for food safety. Bacteria can strongly attach to a food surface or internalize within the matrix, making their isolation extremely difficult. Traditional methods of separating bacteria from food routinely involve stomaching, blending, and shaking. However, these methods may not be efficient at removing all the bacteria from complex matrices. Here, we investigate the benefits of using enzyme digestion followed by immunomagnetic separation to isolate Salmonella from spinach and lettuce. Enzymatic digestion using pectinase and cellulase was able to break down the structure of the leafy green vegetables, resulting in the detachment and release of Salmonella from the leaves. Immunomagnetic separation of Salmonella from the liquefied sample allowed an additional separation step to achieve a more pure sample without leaf debris that may benefit additional downstream applications. We have investigated the optimal combination of pectinase and cellulase for the digestion of spinach and lettuce to improve sample detection yields. The concentrations of enzymes used to digest the leaves were confirmed to have no significant effect on the viability of the inoculated Salmonella. Results reported that the recovery of the Salmonella from the produce after enzyme digestion of the leaves was significantly higher (P <0.05) than traditional sample preparation methods to separate bacteria (stomaching and manually shaking). The results demonstrate the potential for use of enzyme digestion prior to separation can improve the efficiency of bacteria separation and increase the likelihood of detecting pathogens in the final detection assay.

Human pathogens in plant biofilms: Formation, physiology, and detection

“…This approach has been very effective when treating meat and egg products, and may have the potential same effect when treating plant related extracts, for degradation of polymeric sugars, proteins etc, especially when using harsher mechanical treatment (blender, stomacher), although this needs to be validated further. Our initial data indicate that enzymes with potential for biofilm disintegration including plant cell wall degrading enzymes such as cellulases, hemicellulases, pectinases do not affect microbial viability, in agreement with the literature (Wang et al, ).…”

“…can be used to degrade the biofilm matrix (Furukawa et al, ; Lequette et al, ), and a mixture of them is generally necessary due to the variety of polymers composing the matrix. Wang et al (), recently reported that enzyme digestion (using cellulase and pectinase commercial enzyme preparations) improved separation of bacteria (in this case, Salmonella ) and recovery from leafy green vegetables. They report the enzymes did not affect microbial viability and the resultant extract did not have significant antimicrobial activity.…”

Siccibacter turicensis from Kangaroo Scats: Possible Implication in Cellulose Digestion

“…are established as effective degraders of similar cellulosic wastes for the production of bioethanol, which may have a crucial role in fulfilling current and future energy demands [ 4 ]. In some recent studies, cellulases have been evaluated successfully for decontaminating the cellulosic biofilm-forming microbes like Salmonella spp., when used in processing vegetables and meat [ 5 , 6 ]. In a similar study, cellulases have been used to improve the feedstock for broiler chickens [ 7 ].…”