Biodiversity of spoilage lactobacilli: phenotypic characterisation.

Preventing food spoilage is a challenge for the food industry, especially when applying mild preservation methods and when avoiding the use of preservatives. Therefore, it is essential to explore the boundaries of preservation by better understanding the causative microbes, their phenotypic behaviour and their genetic makeup. Traditionally in food microbiology, single strains or small sets of selected strains are studied. Here a collection of 120 strains of 6 different spoilage related Lactobacillus species and a multitude of sources was prepared and their growth characteristics determined in 384-well plates by optical density measurements (OD) over 20 days, for 20 carbon source-related phenotypic parameters and 25 preservation-related phenotypic parameters. Growth under all conditions was highly strain specific and there was no correlation of phenotypes at the species level. On average Lactobacillus brevis strains were amongst the most robust whereas Lactobacillus fructivorans strains had a much narrower growth range. The biodiversity data allowed the definition of preservation boundaries on the basis of the number of Lactobacillus strains that reached a threshold OD, which is different from current methods that are based on growth ability or growth rate of a few selected strains. Genetic information on these microbes and a correlation study will improve the mechanistic understanding of preservation resistance and this will support the future development of superior screening and preservation methods.

The effect of calcium on the transcriptome of sporulating B. subtilis cells.

Bacterial spores formed in the presence of high concentrations of minerals are a major problem in the food industry because of their extreme heat resistance. In order to enhance our insight in the molecular mechanisms underlying this phenomenon we have performed a detailed time-resolved analysis of the genome-wide transcriptome pattern of Bacillus subtilis sporulated both in the absence and presence of high calcium concentrations. The data was analysed in two ways. First, we determined the influence of the presence of high calcium levels during sporulation on the expression of gene groups as defined in Subtilist and KEGG pathways database. Second, we assessed the differential expression at the level of individual genes. When analysing groups and pathways, we found that those annotated as being involved in sporulation were significantly affected. Also, groups and pathways involved in flagella formation and biofilm matrix production were affected by the presence of calcium in the sporulation medium. When we analysed the behaviour of individual genes we found 305 genes influenced by calcium, including all known spore coat polysaccharide biosynthesis genes (10 induced and 1 repressed). A number of the calcium affected genes were also involved in biofilm formation. Minimal overlap with other stress outputs like sigma B activation and weak acid stress response was noted. Those genes that did overlap were unique to that combination which corroborates the notion that the cells sense these conditions differently.

The characterisation of Bacillus spores occurring in the manufacturing of (low acid) canned products.

Spore-forming bacteria can be a problem in the food industry, especially in the canning industry. Spores present in ingredients or present in the processing environment severely challenge the preservation process since their thermal resistance may be very high. We therefore asked the question which bacterial spore formers are found in a typical soup manufacturing plant, where they originate from and what the thermal resistance of their spores is. To answer these questions molecular techniques for bacterial species and strain identification were used as well as a protocol for the assessment of spore heat stress resistance based on the Kooiman method. The data indicate the existence and physiological cause of the high thermal resistance of spores of many of the occurring species. In particular it shows that ingredients used in soup manufacturing are a rich source of high thermal resistant spores and that sporulation in the presence of ingredients rich in divalent metal ions exerts a strong influence on spore heat resistance. It was also indicated that Bacillus spores may well be able to germinate and resporulate during manufacturing i.e. through growth and sporulation in line. Both these spores and those originating from the ingredients were able to survive certain thermal processing settings. Species identity was confirmed using fatty acid analysis, 16SrRNA gene sequencing and DNA-DNA hybridisation. Finally, molecular typing experiments using Ribotyping and AFLP analysis show that strains within the various Bacillus species can be clustered according to the thermal resistance properties of their spores. AFLP performed slightly better than Ribotyping. The data proofed to be useful for the generation of strain specific probes. Protocols to validate these probes in routine identification and innovation aimed at tailor made heat processing in soup manufacturing have been formulated.

The impact of functional genomics on microbiological food quality and safety.

In the food processing industry, unwanted occurrence and growth of spoilage and pathogenic microorganisms is a key concern. A prime example is the extremely heat resistant bacterial endospores, microbial survival structures, that create problems due to their ability to survive classical thermal treatments and their ability to subsequently germinate and form actively growing vegetative cells. Research on food spoilage Bacillus subtilis isolates using the Amplified Fragment Length Polymorphism (AFLP) technology and micro-array technology has identified a number of genomic factors correlated to the level of spore heat resistance. Strains could be classified according to these genomic markers. In addition, it was shown with the sequenced B. subtilis laboratory strain that sporulation in the presence of in particular calcium ions in a cocktail of calcium, magnesium, iron, manganese and potassium promotes thermal resistance of developing spores. This physiological observation correlated with an increased expression during sporulation of genes encoding small acid soluble spore proteins. Screening of ingredients using DNA-chip based techniques identifying the above indicated molecular markers, should allow in the future the identification of the occurrence of spoilage and pathogenic bacteria and prediction of their thermal preservation stress resistance. Currently various projects aiming at the integration of genomics data and micro(nano)-technology, a prerequisite if the alluded to ingredient Quality Control is going to succeed, are running or are being set-up. Information from these projects will be used together with the requirements of product organoleptic quality to derive robust integrated food safety and food quality processing parameters. Such parameters should form the basis of future food Quality Assurance systems.

A new tool for studying the molecular architecture of the fungal cell wall: one-step purification of recombinant trichoderma beta-(1-6)-glucanase expressed in Pichia pastoris.

The fungal cell wall is a supramolecular network of glycoproteins and polysaccharides. Its analysis is seriously hampered by the lack of easily available hydrolytic enzymes in a pure form. Here we describe a simple and efficient purification procedure of a recombinant beta-(1-6)-glucanase from Trichoderma harzianum expressed in Pichia pastoris. Transformed cells efficiently secreted the enzyme into the induction medium. We purified the enzyme using a one-step method based on hydrophobic interaction chromatography. The yield was 80%. SDS-PAGE of the purified enzyme revealed a single band with an apparent molecular mass of 43 kDa. The isoelectric point of the enzyme was 5.8, and it showed maximal enzyme activity and stability at pH 5.0. As beta-(1-6)-glucan is an important component of fungal cell walls, the easy availability of pure beta-(1-6)-glucanase will highly facilitate studies of the molecular organization of the fungal cell wall.

A model for the combined effects of temperature and salt concentration on growth rate of food spoilage molds.

We modeled mold growth on a solid culture medium at various temperatures and NaCl concentrations by using five common food spoilage molds (Penicillium roqueforti, Trichoderma harzianum, Paecilomyces variotii, Aspergillus niger, and Emericella nidulans). For the description of the growth rate (expressed as the increase in colony diameter per unit of time) as a function of temperature and NaCl concentration, a six-parameter model has been developed. The model combines either the Rosso-type or the Ratkowsky-type temperature dependence with the NaCl concentration dependence derived from the relationship between the growth rate and square root of (1 - water activity), as proposed by Gibson and coworkers (A. M. Gibson, J. Baranyi, J. I. Pitt, M. J. Eyles, and T. A. Roberts, Int. J. Food Microbiol. 23:419-431, 1994). The model will be of use to food microbiologists whose aim is to predict the likelihood of fungal spoilage.