In-depth characterization of Lactobacillus delbrueckii subsp. lactis 313 for growth and cell-envelope-associated proteinase production

Abstract

The effect of process conditions on the growth and production of cell-envelope-associated proteinase (PrtL) by Lactobacillus delbrueckii subsp. lactis ATCC^® 7830™ (LDL 313) was studied. Cell growth was profuse under the temperature conditions of 37 °C, 40 °C and 45 °C, with cell growth rates, μ_max increasing with temperatures. Initial culture pH of 6 displayed the highest PrtL yield of 2.33 U. For each growth temperature the cell growth rates under anaerobic conditions were markedly higher than microaerophilic conditions, attributable to efficient sugar utilization under anaerobic fermentation. Overall, the proteolytic activities of cell-bound PrtL (bPrtL) were found to be higher than that of released PrtL (rPrtL). Higher rPrtL activities were displayed by anaerobic cultures over the entire fermentation period whereas the converse was true for bPrtL with microaerophilic cultures in the mid-late exponential phase displaying higher bPrtL activities than anaerobic cultures. Further, the proteinases had caseinolytic specificity for β-casein and κ-casein placing them in the cell-envelope-associated proteinases (CEPs) class I (CEP_I) of the lactococcal CEPs grouping. This study provides insights into conditions for profuse growth and proteinase production by LDL 313 for subsequent technological applications in fermented foods, the dairy industry and bioactive peptide production.

Introduction

Lactic acid bacteria (LAB) are ubiquitous and fastidious microorganisms with several technological, health and industrial significance. They are auxotrophic for several amino acids and their requirement for amino acids is not fully satisfied by the low concentration of free amino acids in milk; hence, they synthesize several proteolytic enzymes in order to grow and survive in milk [1], [2], [3]. The proteolytic system of several LAB species has been well characterized and is composed of cell wall-bound proteinases, responsible for the first step of casein hydrolysis, as well as several peptide- and amino acid-transport systems, and various intracellular peptidases (mainly aminopeptidases and dipeptidases) responsible for the release of single amino acids [4], [5], [6]. As a consequence of their proteolytic ability, LAB enhances the organoleptic properties of dairy foods, and facilitates the release of bioactive peptides from native proteins when used as starters in dairy foods [7], [8].

However, unlike the genera Lactococcus, the proteolytic system of the genera Lactobacillus has not yet been characterized systematically [2], [8]. Among the lactobacilli, the proteinase system of Lactobacillus casei is the best studied [2], [5], [9]. The proteolytic systems have also been studied each for Lactobacillus delbrueckii subsp. bulgaricus [10], Lactobacillus sanfrancisco CB1 [11], Lactobacillus helveticus [12] and L. delbrueckii subsp. lactis strain ACA-DC 178 and CRL 581 [2], [8]. However, to the best of our knowledge the proteolytic system of L. delbrueckii subsp. lactis strain 313 (ATCC^® 7830™) has not been studied.

L. delbrueckii subsp. lactis strain 313 (LDL 313) is employed as biological material in certain vitamin B₁₂ assays [13], [14] because it is auxotrophic for vitamin B₁₂. The species is also known for its ability to produce hydrogen peroxide [15] as well as its use in the production of sour bread such as the German sour rye due to its high acid tolerance [16]. Further, like other lactobacilli species, LDL 313 putatively produces proteolytic enzymes and finds use in the dairy industry for the production of hard cheeses, such as Emmenthal, Provolone and Grana [8] and yogurt [15]. Proteases produced by LDL 313 strains may also release bioactive peptides from proteins and thus represent new sources for biotechnological use. Despite the wideness in the utility of LDL 313, extensive studies on the growth and product formation, as influenced by process conditions, is largely lacking. The scope of technological and industrial application of LDL 313 is expansible considering the peculiarities of biochemical properties of this species. For example, extensive biochemical studies on its proteolytic system will help unravel the role of LDL 313 enzymes in proteolysis and subsequent use of the hydrolytic products. Once an application is established for the enzymes, the necessary process conditions (such as culture conditions) that enhance enzyme synthesis can be duly optimized to increase product yield. A major advantage of fermentation is that conditions such as media composition, temperature, pH, dissolved oxygen and build-up of waste metabolites, which influence cell growth and product synthesis, can be examined and controlled [17]. Elucidating the best conditions for the profuse synthesis of bio-products is therefore necessary to improve fermentation processes for useful technological applications. This work seeks to understand the growth profile of LDL 313 under different fermentation conditions in order to optimize proteinase production and yields from this species. The Gompertz model was used to interpret the kinetic growth data obtained from microbial growth.