Slowed gastrointestinal transit is associated with an altered caecal microbiota in an aged rat model.

Gastrointestinal (GI) motility is largely dependent upon activity within the enteric nervous system (ENS) and is an important part of the digestive process. Dysfunction of the ENS can impair GI motility as is seen in the case of constipation where gut transit time is prolonged. Animal models mimicking symptoms of constipation have been developed by way of pharmacological manipulations. Studies have reported an association between altered GI motility and gut microbial population. Little is known about the changes in gut microbiota profile resulting specifically from pharmacologically induced slowed GI motility in rats. Moreover, the relationship between gut microbiota and altered intestinal motility is based on studies using faecal samples, which are easier to obtain but do not accurately reflect the intestinal microbiome. The aim of this study was to examine how delayed GI transit due to opioid receptor agonism in the ENS modifies caecal microbiota composition. Differences in caecal microbial composition of loperamide-treated or control male Sprague Dawley rats were determined by 16S rRNA gene amplicon sequencing. The results revealed that significant differences were observed at both genus and family level between treatment groups. Bacteroides were relatively abundant in the loperamide-induced slowed GI transit group, compared to controls. Richness and diversity of the bacterial communities was significantly lower in the loperamide-treated group compared to the control group. Understanding the link between specific microbial species and varying transit times is crucial to design interventions targeting the microbiome and to treat intestinal motility disorders.

Association of Single Nucleotide Polymorphism in the DGAT1 Gene with the Fatty Acid Composition of Cows Milked Once and Twice a Day.

A single nucleotide polymorphism (SNP) rs109421300 of the diacylglycerol acyltransferase 1 (DGAT1) on bovine chromosome 14 is associated with fat yield, fat percentage, and protein percentage. This study aimed to investigate the effect of SNP rs109421300 on production traits and the fatty acid composition of milk from cows milked once a day (OAD) and twice a day (TAD) under New Zealand grazing conditions. Between September 2020 and March 2021, 232 cows from a OAD herd and 182 cows from a TAD herd were genotyped. The CC genotype of SNP rs109421300 was associated with significantly (p < 0.05) higher fat yield, fat percentage, and protein percentage, and lower milk and protein yields in both milking frequencies. The CC genotype was also associated with significantly (p < 0.05) higher proportions of C16:0 and C18:0, higher predicted solid fat content at 10 °C (SFC10), and lower proportions of C4:0 and C18:1 cis-9 in both milking frequencies. The association of SNP with fatty acids was similar in both milking frequencies, with differences in magnitudes. The SFC10 of cows milked OAD was lower than cows milked TAD for all three SNP genotypes suggesting the suitability of OAD milk for producing easily spreadable butter. These results demonstrate that selecting cows with the CC genotype is beneficial for New Zealand dairy farmers with the current payment system, however, this would likely result in less spreadable butter.

High throughput screening of technological and biopreservation traits of a large set of wild lactic acid bacteria from Brazilian artisanal cheeses.

This study aimed to evaluate technological (acidification, proteolysis, lipolysis, resistance to low pH, NaCl, and bile salts) and biopreservation (antimicrobial activity against foodborne pathogens) features of 1002 LAB by high throughput screening (HTS) methods. The LAB was isolated from 11 types of Brazilian artisanal cheeses (BAC) marketed in the main 5 producing regions. Remarkable intra-species variability in acidification rates have been found, which was most pronounced between isolates from Mina's artisanal cheeses, Caipira and Coalho cheeses. Lacticaseibacillus paracasei and Levilactobacillus brevis showed the fastest acidification rate; however, all isolates showed slower acidification rates than a lactococcal control strain (4.3 × lower). When testing inhibitory effects, > 75% of LAB isolates could inhibit the growth of Staphylococcus aureus ATCC 19095 and Listeria monocytogenes ATCC 7644. Two of these isolates, identified as Lactiplantibacillus plantarum and Lentilactobacillus buchneri, the sterile and neutral supernatants alone, were sufficient to inhibit L. monocytogenes growth. Principal component analysis (PCA) allowed the identification of functional groups based on proteolytic and lipolytic activity, osmotic stress resistance, and inhibition of L. monocytogenes. The type of cheese the isolates were recovered from influenced properties such as anti-listerial compounds and lipolytic enzyme production. The use of HTS and multivariate statistics allowed insights into a diverse set of LAB technological and biopreservation properties. These findings allow a profound knowledge of the heterogeneity of a large set of isolates, which can be further used to design starter cultures with varied and combined properties, such as biopreservation and technological features. Besides that, HTS makes it possible to analyze a vast panel of LAB strains, reducing costs and time within laboratory analysis, while avoiding the loss of information once all LAB are tested at the same time (differently from the traditional labor-intensive approach, in which a few numbers of strains is tested per time).

A centrifugation-based clearing method allows high-throughput acidification and growth-rate measurements in milk.

The turbidity of milk prohibits the use of optical density measurements for strain characterizations. This often limits research to laboratory media. Here, we cleared milk through centrifugation to remove insoluble milk solids. This resulted in a clear liquid phase, termed milk serum, in which optical density measurements can be used to track microbial growth until a pH of 5.2 is reached. At pH 5.2 coagulation of the soluble protein occurs, making the medium opaque again. We found that behavior in milk serum was predictive of that in milk for 39 Lactococcus lactis (R2 = 0.81) and to a lesser extent for 42 Lactiplantibacillus plantarum (formerly Lactobacillus plantarum; R2 = 0.49) strains. Hence, milk serum can be used as an optically clear alternative to milk for comparison of microbial growth and metabolic characteristics. Characterization of the growth rate, specific acidification rate for optical density at a wavelength of 600 nm, and the amount of acid produced per unit of biomass for all these strains in milk serum, showed that almost all strains could grow in milk, with higher specific acidification and growth rates of Lc. lactis strains compared with Lb. plantarum strains. Nondairy Lc. lactis isolates had a lower growth and specific acidification rate than dairy isolates. The amount of acid produced per unit biomass was relatively high and similar for Lc. lactis dairy and nondairy isolates, as opposed to Lb. plantarum isolates. Lactococcus lactis ssp. lactis showed slightly lower growth and acidification rates when compared with ssp. cremoris. For Lc. lactis strains a doubling of the specific acidification rate occurred with a doubling of the maximum growth rate. This relation was not found for Lb. plantarum strains, where the acidification rate remained relatively constant across 39 strains with growth rates ranging from 0.2 h-1 to 0.3 h-1. We conclude that milk serum is a valuable alternative to milk for high-throughput strain characterization during milk fermentation.

Validation of a High-Throughput Sausage Casing Model for the Assessment of Bacterial Inactivation Affected by Salt Concentration, pH, and Temperature.

Previous studies have shown the efficacy of high concentrations of salt as the main preservative against vegetative bacteria present on natural sausage casings. These studies were limited in the number of variables and the interactions between these variables that were assessed. To remedy this situation, a MicroCasing high-throughput model was developed and validated to study the inactivation kinetics of various combinations of parameters (salt concentration, pH, and temperature) on eight bacterial isolates of Salmonella enterica, Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes over a prolonged period. A Weibullian power model was the best fit to show the trends in sensitivity of each bacterial isolate to salt, pH, and temperature over time. The inactivation kinetics generated with this novel approach could serve as a predictive model for the required salting period for casings. The actual bacterial contamination of the product can vary with the respective production step during processing from animal intestine into sausage casings (initial level, ∼105 CFU/g; level after salting, <102 CFU/g). Subsequent selection and grading of these casings will require complete removal of all salt, and upon completion of this production step, the casings will be resalted. By determining the actual contamination level before the salting process, the minimum storage period in salt can be calculated and potentially optimized by adjusting the pH and temperature. As a result, a standard holding period of at least 30 days may no longer be necessary to produce salted natural casings in accordance with validated quality and food safety criteria.

Importance of intact secondary protein structures of cell envelopes and glass transition temperature of the stabilization matrix on the storage stability of probiotics.

Lactobacillus reuteri LR6 cells were stabilized using a novel combination of wet granulation and fluidized-bed-drying techniques. The stabilized cells were stored at 37 °C and at two water activity (aw) levels (0.11 & 0.30). Superior storage stability was recorded in the lower aw environment, supported by a stronger glassy matrix when skim milk powder was used as the excipient. The initial viable cell populations of the samples stabilized in different matrices ranged from 8.3 to 9.1 log CFU/g. At the end of the storage period, the viable cell populations were reduced to 6.7 to 7.3 log CFU/g at aw 0.11 and to 6.1 to 6.6 CFU/g when the aw was maintained at 0.30. Fourier transform infrared spectroscopic examination of the cell envelopes revealed substantial dissimilarities between samples at the beginning and at the end of the storage period, which indicated alteration in the secondary protein structures of the cell envelope and also correlated well with the loss in cell viability. In milk-powder-based matrices, adjusting the aw to 0.30 resulted in a weaker or no glassy state whereas the same matrices had a high glass transition temperature at aw 0.11. This strong glassy matrix and low aw combination was found to enhance the bacterial stability at the storage temperature of 37 °C. Scanning electron microscopy revealed the formation of corrugated surfaces and blister-type deformations on the cell envelopes during the stabilization process.

Heat resistance of spores of 18 strains of Geobacillus stearothermophilus and impact of culturing conditions.

In this study, different methods were evaluated for enumeration of spores of G. stearothermophilus, different sporulation methods were assessed for yields and wet heat resistances of obtained spores, and subsequently, the variation in heat resistances of spores was determined. Overall, tryptone soya agar (TSA) was the most suitable medium for enumeration of spores of this thermophilic bacterium. Sporulation on different media both at 55 and at 61 °C led to considerable variation in spore heat resistance. The heat resistance of spores was highest upon sporulation on medium supplemented with free ions of calcium, potassium, magnesium and manganese (CaKMgMn). For 18 different G. stearothermophilus strains that were isolated from various sources, spores were subsequently produced on nutrient agar supplemented with CaKMgMn at 55 °C. Strain ATCC 12980T, also known as 9A20, which is commonly used in steam sterilization tests was included. The survival of spores of all strains was assessed at 125 °C and 130 °C using two independent spore batches per strain. The mean D125°C for spores of the 18 strains was 1.1 min (95% PI 0.48-2.3 min) and the mean D130°C was 0.37 min (95% PI 0.17-0.82 min). For spore inactivation of these 18 strains, a z-value of 11.1 °C was estimated, resulting in an estimated D-value of 2.4 min (95% PI 1.1-5.2) at the reference temperature 121.1 °C. Based on the data sets obtained in this study, it was found that the variability in spore heat resistance could largely be attributed to strain variability and conditions used during sporulation (especially the sporulation medium); reproduction and experimental variabilities were much smaller. The established variabilities were compared with the overall variability in spore heat resistance of G. stearothermophilus based on a meta-analysis of reported D-values. The data presented indicate that strain variability and history of sporulation each account for approximately half of the overall variability observed with respect to the heat resistance of spores of G. stearothermophilus. The findings presented in this study allow for optimal recovery of G. stearothermophilus spores from foods and a better understanding of factors that determine the heat resistance properties of spores of G. stearothermophilus. Moreover, this study once more underlines the limited effects of heat treatments used in the food industry on inactivation of spores of this bacterium.

Cell Surface Properties of Lactococcus lactis Reveal Milk Protein Binding Specifically Evolved in Dairy Isolates.

Surface properties of bacteria are determined by the molecular composition of the cell wall and they are important for interactions of cells with their environment. Well-known examples of bacterial interactions with surfaces are biofilm formation and the fermentation of solid materials like food and feed. Lactococcus lactis is broadly used for the fermentation of cheese and buttermilk and it is primarily isolated from either plant material or the dairy environment. In this study, we characterized surface hydrophobicity, charge, emulsification properties, and the attachment to milk proteins of 55 L. lactis strains in stationary and exponential growth phases. The attachment to milk protein was assessed through a newly developed flow cytometry-based protocol. Besides finding a high degree of biodiversity, phenotype-genotype matching allowed the identification of candidate genes involved in the modification of the cell surface. Overexpression and gene deletion analysis allowed to verify the predictions for three identified proteins that altered surface hydrophobicity and attachment of milk proteins. The data also showed that lactococci isolated from a dairy environment bind higher amounts of milk proteins when compared to plant isolates. It remains to be determined whether the alteration of surface properties also has potential to alter starter culture functionalities.

Ex vivo motility in the base of the rabbit caecum and its associated structures: an electrophysiological and spatiotemporal analysis

We examined the coordination between contractile events at different sites in the basal portion of the rabbit caecum and its associated structures that were identified by electrophysiological recordings with simultaneous one-dimensional, and a novel two-dimensional, spatiotemporal mapping technique. The findings of this work provide evidence that the caecum and proximal colon/ampulla coli act reflexly to augment colonic outflow when the caecum is distended and mass peristalsis instituted, the action of the latter overriding the inherent rhythm and direction of haustral propagation in the adjacent portion of the proximal colon but not in the terminal ileum. Further, the findings suggest that the action of the sacculus rotundus may result from its distension with chyme by ileal peristalsis and that the subsequent propagation of contraction along the basal wall of the caecum towards the colon may be augmented by this local distensión (AU)

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Ex vivo motility in the base of the rabbit caecum and its associated structures: an electrophysiological and spatiotemporal analysis.

We examined the coordination between contractile events at different sites in the basal portion of the rabbit caecum and its associated structures that were identified by electrophysiological recordings with simultaneous one-dimensional, and a novel two-dimensional, spatiotemporal mapping technique. The findings of this work provide evidence that the caecum and proximal colon/ampulla coli act reflexly to augment colonic outflow when the caecum is distended and mass peristalsis instituted, the action of the latter overriding the inherent rhythm and direction of haustral propagation in the adjacent portion of the proximal colon but not in the terminal ileum. Further, the findings suggest that the action of the sacculus rotundus may result from its distension with chyme by ileal peristalsis and that the subsequent propagation of contraction along the basal wall of the caecum towards the colon may be augmented by this local distension.

Characterisation of the contractile dynamics of the resting ex vivo urinary bladder of the pig.

OBJECTIVES: To characterise the area and movements of ongoing spontaneous localised contractions in the resting porcine urinary bladder and relate these to ambient intravesical pressure (Pves ), to further our understanding of their genesis and role in accommodating incoming urine. MATERIALS AND METHODS: We used image analysis to quantify the areas and movements of discrete propagating patches of contraction (PPCs) on the anterior, anterolateral and posterior surfaces of the urinary bladders of six pigs maintained ex vivo with small incremental increases in volume. We then correlated the magnitude of Pves and cyclic changes in Pves with parameters derived from spatiotemporal maps. RESULTS: Contractile movements in the resting bladder consisted only of PPCs that covered around a fifth of the surface of the bladder, commenced at various sites, and were of ≈6 s in duration. They propagated at around 6 mm/s, mainly across the anterior and lateral surface of the bladder by various, sometimes circular, routes in a quasi-stable rhythm, and did not traverse the trigone. The frequencies of these rhythms were low (3.15 cycles/min) and broadly similar to those of cyclic changes in Pves (3.55 cycles/min). Each PPC was associated with a region of stretching (positive strain rate) and these events occurred in a background of more constant strain. The amplitudes of cycles in Pves and the areas undergoing PPCs increased after a sudden increase in Pves but the frequency of cycles of Pves and of origin of PPCs did not change. Peaks in Pves cycles occurred when PPCs were traversing the upper half of the bladder, which was more compliant. The velocity of propagation of PPCs was similar to that of transverse propagation of action potentials in bladder myocytes and significantly greater than that reported in interstitial cells. The size of PPCs, their frequency and their rate of propagation were not affected by intra-arterial dosage with tetrodotoxin or lidocaine. CONCLUSIONS: The origin and duration of PPCs influence both Pves and cyclic variation in Pves . Hence, propagating rather than stationary areas of contraction may contribute to overall tone and to variation in Pves . Spatiotemporal mapping of PPCs may contribute to our understanding of the generation of tone and the basis of clinical entities such as overactive bladder, painful bladder syndrome and detrusor overactivity.

Determination of villous rigidity in the distal ileum of the possum (Trichosurus vulpecula).

We investigated the passive mechanical properties of villi in ex vivo preparations of sections of the wall of the distal ileum from the brushtail possum (Trichosurus vulpecula) by using a flow cell to impose physiological and supra-physiological levels of shear stress on the tips of villi. We directly determined the stress applied from the magnitude of the local velocities in the stress inducing flow and additionally mapped the patterns of flow around isolated villi by tracking the trajectories of introduced 3 µm microbeads with bright field micro particle image velocimetry (mPIV). Ileal villi were relatively rigid along their entire length (mean 550 µm), and exhibited no noticeable bending even at flow rates that exceeded calculated normal physiological shear stress (>0.5 mPa). However, movement of villus tips indicated that the whole rigid structure of a villus could pivot about the base, likely from laxity at the point of union of the villous shaft with the underlying mucosa. Flow moved upward toward the tip on the upper portions of isolated villi on the surface facing the flow and downward toward the base on the downstream surface. The fluid in sites at distances greater than 150 µm below the villous tips was virtually stagnant indicating that significant convective mixing in the lower intervillous spaces was unlikely. Together the findings indicate that mixing and absorption is likely to be confined to the tips of villi under conditions where the villi and intestinal wall are immobile and is unlikely to be greatly augmented by passive bending of the shafts of villi.

Characterisation of mixing in the proximal duodenum of the rat during longitudinal contractions and comparison with a fluid mechanical model based on spatiotemporal motility data.

The understanding of mixing and mass transfers of nutrients and drugs in the small intestine is of prime importance in creating formulations that manipulate absorption and digestibility. We characterised mixing using a dye tracer methodology during spontaneous longitudinal contractions, i.e. pendular activity, in 10 cm segments of living proximal duodenum of the rat maintained ex-vivo. The residence time distribution (RTD) of the tracer was equivalent to that generated by a small number (8) of continuous stirred tank reactors in series. Fluid mechanical modelling, that was based on real sequences of longitudinal contractions, predicted that dispersion should occur mainly in the periphery of the lumen. Comparison with the experimental RTD showed that centriluminal dispersion was accurately simulated whilst peripheral dispersion was underestimated. The results therefore highlighted the potential importance of micro-phenomena such as microfolding of the intestinal mucosa in peripheral mixing. We conclude that macro-scale modeling of intestinal flow is useful in simulating centriluminal mixing, whereas multi-scales strategies must be developed to accurately model mixing and mass transfers at the periphery of the lumen.

The partitioning of water in aggregates of undigested and digested dietary particles.

The hydration of fibre particles derived from wheat and wood was quantified, before and after in vitro digestion, and compared with fibre particles from the colonic digesta of pigs and from human faeces. Total water and the extra- and intra-particulate water components were determined using a combination of centrifugation, drying, gas pycnometry and image analysis. The water of saturation (WS) of wood particles and AllBran® measured after in vitro digestion was up to double that of wheat fibres after in vitro digestion, and increased with particle size and loss of soluble material, but was not associated with the chemical composition of the fibres. Fibre that had undergone in vitro gastric digestion and that had been recovered from the colon or faeces, sequestered about 3% of the Ws into intra-particulate spaces, the remainder occupying extra-particulate spaces. The authors speculate that large quantities of fibre must be eaten to sequester toxins that locate into the intra-particulate space.

Multifactorial diversity sustains microbial community stability.

Maintenance of a high degree of biodiversity in homogeneous environments is poorly understood. A complex cheese starter culture with a long history of use was characterized as a model system to study simple microbial communities. Eight distinct genetic lineages were identified, encompassing two species: Lactococcus lactis and Leuconostoc mesenteroides. The genetic lineages were found to be collections of strains with variable plasmid content and phage sensitivities. Kill-the-winner hypothesis explaining the suppression of the fittest strains by density-dependent phage predation was operational at the strain level. This prevents the eradication of entire genetic lineages from the community during propagation regimes (back-slopping), stabilizing the genetic heterogeneity in the starter culture against environmental uncertainty.

The bioelectrical basis and validity of gastrointestinal extracellular slow wave recordings.

Gastrointestinal extracellular recordings have been a core technique in motility research for a century. However, the bioelectrical basis of extracellular data has recently been challenged by claims that these techniques preferentially assay movement artifacts, cannot reproduce the underlying slow wave kinetics, and misrepresent the true slow wave frequency. These claims motivated this joint experimental-theoretical study, which aimed to define the sources and validity of extracellular potentials. In vivo extracellular recordings and video capture were performed in the porcine jejunum, before and after intra-arterial nifedipine administration. Gastric extracellular recordings were recorded simultaneously using conventional serosal contact and suction electrodes, and biphasic and monophasic extracellular potentials were simulated in a biophysical model. Contractions were abolished by nifedipine, but extracellular slow waves persisted, with unchanged amplitude, downstroke rate, velocity, and downstroke width (P>0.10 for all), at reduced frequency (24% lower; P=0.03). Simultaneous suction and conventional serosal extracellular recordings were identical in phase (frequency and activation-recovery interval), but varied in morphology (monophasic vs. biphasic; downstroke rate and amplitude: P<0.0001). Simulations demonstrated the field contribution of current flow to extracellular potential and quantified the effects of localised depolarisation due to suction pressure on extracellular potential morphology. In sum, these results demonstrate that gastrointestinal extracellular slow wave recordings cannot be explained by motion artifacts, and are of a bioelectrical origin that is highly consistent with the underlying biophysics of slow wave propagation. Motion suppression is shown to be unnecessary as a routine control in in vivo extracellular studies, supporting the validity of the extant gastrointestinal extracellular literature.

Fluid mechanical consequences of pendular activity, segmentation and pyloric outflow in the proximal duodenum of the rat and the guinea pig.

We conducted numerical experiments to study the influence of non-propagating longitudinal and circular contractions, i.e. pendular activity and segmentation, respectively, on flow and mixing in the proximal duodenum. A lattice-Boltzmann numerical method was developed to simulate the fluid mechanical consequences for each of 22 randomly selected sequences of high-definition video of real longitudinal and radial contractile activity in the isolated proximal duodenum of the rat and guinea pig. During pendular activity in the rat duodenum, the flow was characterized by regions of high shear rate. Mixing was so governed by shearing deformation of the fluid that increased the interface between adjacent domains and accelerated their inter-diffusion (for diffusion coefficients approx. less than 10(-8) m² s(-1)). When pendular activity was associated with a slow gastric outflow characteristic of post-prandial period, the dispersion was also improved, especially near the walls. Mixing was not promoted by isolated segmentative contractions in the guinea pig duodenum and not notably influenced by pylorus outflow. We concluded that pendular activity generates mixing of viscous fluids 'in situ' and accelerates the diffusive mass transfer, whereas segmentation may be more important in mixing particulate suspensions with high solid volume ratios.

Spatiotemporal mapping of ex vivo motility in the caecum of the rabbit.

We used high definition radial, strain rate and intensity spatiotemporal mapping to quantify contractile movements of the body and associated structures of the rabbit caecum when the terminal ileum was being perfused with saline at a constant rate. This perfusion caused gradual distension of the caecum as a result of relative restriction of outflow from the ampulla caecalis. The body of the caecum exhibited two patterns of motility that appeared autonomous, i.e. occurred independently of any contractile activity at the inlet or outlet. Firstly, the pattern that we termed ladder activity consisted of an orderly sequential contraction of bundles of axially oriented circular muscle between the spiral turns of longitudinal muscle and proceeded either from base to tip or from tip to base at a similar frequency and velocity. Secondly, less-localised, rapidly propagating synchronous contractions of both circular and longitudinal muscle, which were more common when the caecum was distended, that were termed mass peristalsis. Movements of the ileum and sacculus rotundus occurred at the same frequency and were broadly coordinated. Distension of the distal sacculus occurred synchronously with contraction of the ileum and did not propagate in an orderly manner across the structure, i.e. was instantaneous. This pattern was consistent with hydrostatic distension. Contractions propagated through the ampulla caecalis in either an orad or an aborad direction at a similar frequency to, and broadly correlated with, those in the ileum. The frequencies of distension of the sacculus and of contraction in the ileum and ampulla were momentarily augmented during mass peristalsis. The authors conclude that there was some coordination between the contractile activity of the terminal ileum and the caecal ampulla during periods of ongoing inflow from the ileum and between these structures and the caecum during mass peristalsis.

Volatile compound fingerprinting of mixed-culture fermentations.

With the advent of the -omics era, classical technology platforms, such as hyphenated mass spectrometry, are currently undergoing a transformation toward high-throughput application. These novel platforms yield highly detailed metabolite profiles in large numbers of samples. Such profiles can be used as fingerprints for the accurate identification and classification of samples as well as for the study of effects of experimental conditions on the concentrations of specific metabolites. Challenges for the application of these methods lie in the acquisition of high-quality data, data normalization, and data mining. Here, a high-throughput fingerprinting approach based on analysis of headspace volatiles using ultrafast gas chromatography coupled to time of flight mass spectrometry (ultrafast GC/TOF-MS) was developed and evaluated for classification and screening purposes in food fermentation. GC-MS mass spectra of headspace samples of milk fermented by different mixed cultures of lactic acid bacteria (LAB) were collected and preprocessed in MetAlign, a dedicated software package for the preprocessing and comparison of liquid chromatography (LC)-MS and GC-MS data. The Random Forest algorithm was used to detect mass peaks that discriminated combinations of species or strains used in fermentations. Many of these mass peaks originated from key flavor compounds, indicating that the presence or absence of individual strains or combinations of strains significantly influenced the concentrations of these components. We demonstrate that the approach can be used for purposes like the selection of strains from collections based on flavor characteristics and the screening of (mixed) cultures for the presence or absence of strains. In addition, we show that strain-specific flavor characteristics can be traced back to genetic markers when comparative genome hybridization (CGH) data are available.

Mixed-culture transcriptome analysis reveals the molecular basis of mixed-culture growth in Streptococcus thermophilus and Lactobacillus bulgaricus.

Many food fermentations are performed using mixed cultures of lactic acid bacteria. Interactions between strains are of key importance for the performance of these fermentations. Yogurt fermentation by Streptococcus thermophilus and Lactobacillus bulgaricus (basonym, Lactobacillus delbrueckii subsp. bulgaricus) is one of the best-described mixed-culture fermentations. These species are believed to stimulate each other's growth by the exchange of metabolites such as folic acid and carbon dioxide. Recently, postgenomic studies revealed that an upregulation of biosynthesis pathways for nucleotides and sulfur-containing amino acids is part of the global physiological response to mixed-culture growth in S. thermophilus, but an in-depth molecular analysis of mixed-culture growth of both strains remains to be established. We report here the application of mixed-culture transcriptome profiling and a systematic analysis of the effect of interaction-related compounds on growth, which allowed us to unravel the molecular responses associated with batch mixed-culture growth in milk of S. thermophilus CNRZ1066 and L. bulgaricus ATCC BAA-365. The results indicate that interactions between these bacteria are primarily related to purine, amino acid, and long-chain fatty acid metabolism. The results support a model in which formic acid, folic acid, and fatty acids are provided by S. thermophilus. Proteolysis by L. bulgaricus supplies both strains with amino acids but is insufficient to meet the biosynthetic demands for sulfur and branched-chain amino acids, as becomes clear from the upregulation of genes associated with these amino acids in mixed culture. Moreover, genes involved in iron uptake in S. thermophilus are affected by mixed-culture growth, and genes coding for exopolysaccharide production were upregulated in both organisms in mixed culture compared to monocultures. The confirmation of previously identified responses in S. thermophilus using a different strain combination demonstrates their generic value. In addition, the postgenomic analysis of the responses of L. bulgaricus to mixed-culture growth allows a deeper understanding of the ecology and interactions of this important industrial food fermentation process.