Development and application of new nucleic acid-based technologies for microbial community analyses in foods

Several challenges still persist in the analysis of microorganisms in foods, particularly in studies of complex communities. Nucleic acid-based methods are promising tools in addressing new questions concerning microbial communities. We have developed several new methods in the field of nucleic acid-based microbial community analyses. These methods cover both sample preparation and detection approaches. The sample preparation method involves simplified DNA purification using paramagnetic beads. As an extension of this method, the same paramagnetic beads are used for both cell separation and DNA purification. This enables full automation. The separate detection of viable and dead bacteria is a major issue in nucleic acid-based diagnostics. We have applied a living/dead dye that binds covalently to DNA and inhibits the PCR from dead cells. In addition, a DNA array-based detection assay has been developed. The assay combines the specificity obtained by enzymatic labeling of DNA probes with the possibility of detecting several targets simultaneously by DNA array hybridization. In combination with 16S rDNA amplification, this is a promising tool for community analyses. Also, we have developed a novel approach for multiplex quantitative PCR. The multiplex PCR has been combined with our DNA array-based detection method. Finally, we are now in the process of adapting a system for monitoring microbial growth and death in real-time through the tagging of bacteria with green fluorescent protein (GFP) combined with fluorescence detection using a high-resolution confocal laser scanner.     

Impact of ‘oxidizing’ and ‘reducing’ buckwheat sourdoughs on brown rice and buckwheat batter and bread

The influence of reducing and oxidizing buckwheat sourdoughs on the rheological, protein, and bread properties of buckwheat and brown rice flour was investigated. Batters and breads prepared with chemically acidified doughs, fresh pre-doughs, and fresh pre-doughs containing glutathione (3 mM) were used as controls. No significant differences were observed after the addition of reducing and oxidizing sourdoughs in all trials. Proteolysis was observed after proofing time in buckwheat and brown rice batters, respectively. Acidified doughs increased the elasticity and the gelatinization temperature of buckwheat batters. No notable microstructure changes were detected in brown rice batters. The extension of fermentation time in sourdough caused a slight decrease in bread volumes in all trials. Sourdoughs increased the bread volume and decreased the crumb hardness of buckwheat breads. In trials with brown rice flour, the addition of sourdough did not show relevant volume differences as compared to the controls, except big voids in sourdough bread crumb. Linear correlations between hardness, volume, and cells’ density were observed. However, no clear correlations among rheological parameters and bread characteristics could be detected. These results indicated that the applied strains were responsible for the leavening capacity of the yeast during the proofing time and for crumb structure in trials with buckwheat and brown rice flour. Applied sourdoughs were able to change the molecular, and bread properties of buckwheat and brown rice bread.     

Quality changes during refrigerated storage of MA-packaged pre-rigor fillets of farmed Atlantic cod (Gadus morhua L.) using traditional MAP, CO2 emitter, and vacuum

ABSTRACT:  Quality changes during 3 wk of refrigerated storage (1.3 °C) were studied on pre -rigor filleted farmed Atlantic cod packed in modified atmosphere (MAP, 60% CO₂ and 40% O₂) or vacuum. The packages of MAP contained either a CO₂ emitter and low gas volume to product volume (g/p ratio) of 1.3, or a 3.9 g/p ratio and no emitter. The CO₂ level remained stable or increased in the packages with CO₂ emitter, whereas the CO₂ level in the packages with no CO₂ emitter decreased to 40% after 4 d of refrigerated storage. High levels of oxygen in the gas mixture prevented formation of trimethyl amine (TMA) during storage of the MA-packed fish, whereas the TMA content increased significantly after 10-d storage in vacuum. MA-packed samples had the highest values of 1-penten-3-ol. Sensory scores of sour, sulfur, and pungent odors were significantly higher for vacuum-packed cod compared to the 2 MA-packaging methods measured 14 d after slaughtering. No differences in sensory scores were observed between the 2 methods of MAP, and shelf life of these samples seemed to be 14 to 21 d. Cod samples packaged in vacuum packages had higher pH values compared to ordinary MAP and packages containing a CO₂ emitter. Bacterial growth was inhibited by MAP and resulted at the end of the storage period in dominance of Carnobacterium and some Photobacterium . In MA packages with high O₂ levels the Photobacterium was inhibited. It is concluded that CO₂ emitters are well suited for reduction of transport volume for MA-packaged farmed cod.     

Identification and characterization of a glucan-producing enzyme from Lactobacillus hilgardii TMW 1.828 involved in granule formation of water kefir

Water kefir is a home made fermented beverage based on a sucrose solution with fruit extracts. The inoculum of such fermentations consists of macroscopic granula containing lactic and acetic acid bacteria, and yeasts, which are embedded in an exopolysaccharide (EPS) matrix. In this work, a strain of Lactobacillus hilgardii producing large amounts of the granule-forming dextran could be isolated. The glycosyltransferans (Gtf) commonly called glucansucrase responsible for the production of this dextran was purified from L. hilgardii. Characteristic enzyme kinetic data were obtained. Optimum activity was observed between pH 4.3 and 4.6 and temperatures between 40 °C and 45 °C. A Michaelis–Menten kinetic could be fit to the experimental data and a K_M of 0.0385 M was calculated. The corresponding gtf gene was identified and characterized. It encodes a 1448 amino acid protein with higher homologies to Gtfs of Lactobacillus parabuchneri, Lactobacillus sakei and Lactobacillus fermentum followed by lower homologies to Lactobacillus reuteri Gtfs. By knockout experiments the role of this gene in granule dextran production was demonstrated.     

Induction of Gushing with Recombinant Class II Hydrophobin FcHyd5p from Fusarium culmorum and the Impact of Hop Compounds on its Gushing Potential

Malt‐induced gushing is a problem that has been known for many years. Mechanisms and inducing agents are still not fully understood and identified. Hydrophobins produced by various filamentous fungi are currently under discussion as biological gushing‐inducing compounds. In the current study the class II hydrophobin FcHyd5p, from the cereal pathogen Fusarium culmorum, was employed in beer and other carbonated beverages for gushing experiments and the influence of hop compounds on gushing potential was examined. It was demonstrated that this protein strongly induces gushing in various carbonated beverages, including beer. It was further demonstrated that the resulting gushing volume is susceptible to certain hop compounds and can be decreased significantly by the addition of these substances.     

Strain-level characterization of nonstarter lactic acid bacteria in Norvegia cheese by high-resolution melt analysis

The nonstarter lactic acid bacteria (NSLAB) constitute an important microbial group found during cheese ripening and they are thought to be fundamental to the quality of cheese. Rapid and accurate diagnostic tests for NSLAB are important for cheese quality control and in understanding the cheese ripening process. Here, we present a novel rapid approach for strain-level characterization through combined 16S rRNA gene and repetitive sequence-based high-resolution melt analysis (HRM). The approach was demonstrated through the characterization of 94 isolates from Norvegia, a Gouda-type cheese. The HRM profiles of the V1 and V3 variable regions of the 16S rRNA gene of the isolates were compared with the HRM profiles of 13 reference strains. The HRM profile comparison of the V1 and V3 regions of the 16S rRNA gene allowed discrimination of isolates and reference strains. Among the cheese isolates, Lactobacillus casei/paracasei (62 isolates) and Lactobacillus plantarum/Lactobacillus pentosus (27 isolates) were the dominant species, whereas Lactobacillus curvatus/Lactobacillus sakei were found occasionally (5 isolates). The HRM profiling of repetitive sequence-based PCR using the (GTG)(5) primer was developed for strain-level characterization. The clustering analysis of the HRM profiles showed high discriminatory power, similar to that of cluster analysis based on the gel method. In conclusion, the HRM approach in this study may be applied as a fast, accurate, and reproducible method for characterization of the NSLAB microflora in cheese and may be applicable to other microbial environments following selective plate culturing.     

The effect of crowding stress on bacterial growth and sensory properties of chilled Atlantic salmon fillets

Abstract: Atlantic salmon were subjected to minimal preslaughter crowding stress (Control), short‐term crowding for 20 min (SS‐group), or long‐term crowding for 24 h (LS‐group). The fish were filleted prerigor, cut into 270 g pieces, and packaged in modified atmosphere (60% CO₂ and 40% N₂). Fillet quality analyses were determined during 22 d of storage at 0.3 °C. Bacterial growth and unpleasant sensory properties increased earlier in the LS‐group. The negative effects of long‐term preslaughter stress were more pronounced for raw than cooked samples, and more pronounced for odor than flavor. Sequence analyses of bacterial DNA at the end of storage revealed that 100% of the bacteria were comprised by Photobacterium phosphoreum of the SS‐ and LS‐group, whereas the Control group also contained 21% of Carnobacterium maltaromaticum (lactic acid bacteria, LAB). Counting of LAB, using Man‐Rogosa‐Sharke agar, similarly showed higher numbers of the Control group after 15 d of storage. A total bacterial count of log 6 CFU/g was observed after 15 d of storage of the LS‐group, which was 3 and 7 d earlier compared with the Control and SS‐group, respectively. Fillet color, texture, and liquid losses were not negatively affected by preslaughter crowding stress. From the sensory and bacterial analyses, it is concluded that long‐term crowding stress accelerates bacterial growth and development of unpleasant sensory properties, hence reduces the shelf life of prerigor modified atmosphere packaged (MAP) salmon. Practical Application: Stressful handling of Atlantic salmon before slaughter resulted in faster reduction of fresh taste and smell, faster bacterial growth, and hence shorter shelf life. The deteriorative effects were more pronounced of raw compared to cooked salmon. Therefore, salmon should be handled carefully in connection with slaughter to avoid impaired welfare and fillet quality, in particularly for fish that is consumed raw, such as sushi.     

Interdependent Regulation of Polycystin Expression Influences Starvation-Induced Autophagy and Cell Death

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by deficiency of polycystin-1 (PC1) or polycystin-2 (PC2). Altered autophagy has recently been implicated in ADPKD progression, but its exact regulation by PC1 and PC2 remains unclear. We therefore investigated cell death and survival during nutritional stress in mouse inner medullary collecting duct cells (mIMCDs), either wild-type (WT) or lacking PC1 (PC1KO) or PC2 (PC2KO), and human urine-derived proximal tubular epithelial cells (PTEC) from early-stage ADPKD patients with PC1 mutations versus healthy individuals. Basal autophagy was enhanced in PC1-deficient cells. Similarly, following starvation, autophagy was enhanced and cell death reduced when PC1 was reduced. Autophagy inhibition reduced cell death resistance in PC1KO mIMCDs to the WT level, implying that PC1 promotes autophagic cell survival. Although PC2 expression was increased in PC1KO mIMCDs, PC2 knockdown did not result in reduced autophagy. PC2KO mIMCDs displayed lower basal autophagy, but more autophagy and less cell death following chronic starvation. This could be reversed by overexpression of PC1 in PC2KO. Together, these findings indicate that PC1 levels are partially coupled to PC2 expression, and determine the transition from renal cell survival to death, leading to enhanced survival of ADPKD cells during nutritional stress.