Fresh and Fermented Vegetables as a Source of Proteolytic Bacteria

. KOTLAR CE, ; AGÜERO MV, ; PONCE AG, ; PÉREZ BORLA O, ; ROURA SI.

Handbook of plant-based fermented food and beverage technology

Publishers: CRC Press

Lugar: Florida; Año: 2011; p. 731 - 756

Microbial proteases are among the most important hydrolytic enzymes and have been studied extensively since the advent of enzymology. There is renewed interest in the study of proteolytic enzymes, which not only play an important role in the cellular metabolic process but have also gained considerable attention in the industrial community. Moreover, proteases are the most important category of industrial enzymes, accounting for more than 65% of the total industrial enzyme market (Banik and others 2004). Microbial proteases are a group of enzymes that can have application in numerous industries (Gulrajani and others 2000; Gupta and others 2002; Najafi and others 2005; Prakash and others 2005) and they are important tools in medical and pharmaceutical process (Bhaskar and others 2007; In and others 2002). In food processing industry the proteolytic activity of microorganisms is associated with a wide variety of process such as fermented foods (Casaburi and others 2008; Hayashi and others 1990; Mauriello and others 2002; Oneca and others 2007), oil extraction (Kashyap and others 2007), bakery (Lauer and others 2000), clarification of juices (Dawes and others 1994) and waste treatments (Sudeepa and others 2007; Wang and others 1997). Bacteria, moulds and yeast are some of the microorganisms that are able to produce proteases. Microorganisms elaborate a large array of proteases, which are intracellular and/or extracellular. Intracellular proteases are important for various cellular and metabolic processes, such as sporulations, differentiation protein turnover, maturation of enzymes and hormones and maintenance of cellular protein pool. The extracellular ones are important for proteins hydrolysis in cell free environments and able the cell to absorb and utilize hydrolytic products. At the same time, these extracellular proteases have also been commercially exploited to assist protein degradation in various industrial processes (Salem and others 2009). Microbes represent an excellent protease source due to their broad biochemical diversity (Godfrey and others 1996), their rapid growth, the limited space and the low cost that growing substrates required for their cultivation (Gupta and others 2002). Each organism or strain has its own special conditions for maximum enzyme production. Extracellular protease production by microorganisms is highly influenced by media components, variation in carbon/nitrogen ratio, presence of some easily metabolizable sugars, such as glucose (Beg and others 2003) and presence of metal ions (Secades and others 1999). Several others factors, such as aeration, inoculums density, pH, temperature and incubation time also affect the amount of protease production and their interaction plays an important role in the synthesis of these enzymes (Puri and others 2002). Vegetables are renewable raw materials associated with diverse microbial populations (Gómez and others 2002; Ponce and others 2008), therefore they could be an interesting and cheap source of proteolytic bacteria. Among vegetables cabbage (Brassica oleracea var capitata) is a biennial sturdy and inexpensive leafy plant. It is widely cultivated throughout the world and stored so well that it is available all the year round. The aim of the present chapter was to describe the isolation and characterization of extracellular producing bacteria from fresh and fermented cabbage. In order to select profitable bacteria to apply on different industries, the proteolytic substrate specificity of the isolated bacteria was assayed using different protein sources. Taken into account the uses and the high demand of proteases, there is a need for the research of new strains of bacteria that produce enzymes with novel properties and the development of low cost industrial medium formulations. In commercial practice, the optimization of medium composition is done to maintain a balance between the various medium components, minimizing the amount of unutilized components at the end of fermentation. In the light of the above facts, another objective of this study was to optimize the fermentation medium in order to enhance the protease production from two of the strains previously isolated from fresh and fermented cabbage. The optimization of culture medium and cultivation was performed using both conventional methods and the Plackett-Burman (PB) experimental design that is usually applied as a pre-optimization stage looking for the study of the statistical factors with significance on the dependent variable. Once the critical components and operating conditions to the protease production were screened, the second stage of the optimization procedure was to find the optimum for maximum product formation. Response Surface Methodology (RSM) was employed in this second stage to optimize the fermentation medium for enhancing protease production.