Assessment of the response of indigenous microflora and inoculated Bacillus licheniformis endospores in reconstituted skim milk to microwave and conventional heating systems by flow cytometry.

Heat treatment is one of the most widely used processing technologies in the dairy industry. Its primary purpose is to destroy microorganisms, both pathogenic and spoilage, to ensure the product is safe and has a reasonable shelf life. In this study microwave volumetric heating (MVH) was compared with a conventional tubular heat exchanger (THE), in terms of the effects of each at a range of temperatures (75°C, 85°C, 95°C, 105°C, 115°C, and 125°C) on indigenous microflora viability and the germination of inoculated Bacillus licheniformis endospores in reconstituted skim milk. To assess the heat treatment-related effects on microbial viability, classical agar-based tests were applied to obtain the counts of 4 various microbiological groups including total bacterial, thermophilic bacterial, mesophilic aerobic bacterial endospore, and thermophilic aerobic bacterial endospore counts, and additional novel insights into cell permeability and spore germination profiles post-heat treatment were obtained using real-time flow cytometry (FC) methods. No significant differences in the plate counts of the indigenous microorganisms tested, the plate counts of the inoculated B. licheniformis, or the relative percentage of germinating endospores were observed between MVH- and THE-treated samples, at equal temperatures in the range specified above, indicating that both methods inactivated inoculated endospores to a similar degree (up to 70% as measured by FC and 5 log reduction as measured by plate counting for some treatments of inoculated endospores). Furthermore, increased cell permeability of indigenous microflora was observed by FC after MVH compared with THE treatment of uninoculated skim milk, which was reflected in lower total bacterial count at a treatment temperature of 105°C. This work demonstrates the utility of FC as a rapid method for assessing cell viability and spore inactivation for postthermal processing in dairy products and overall provides evidence that MVH is at least as effective at eliminating native microflora and inoculated B. licheniformis endospores as THE.

The effect of buttermilk or buttermilk powder addition on functionality, textural, sensory and volatile characteristics of Cheddar-style cheese.

The influence of buttermilk or buttermilk powder addition to cheese milk or cheese curds respectively on cheese functional properties, free fatty acid profiles and subsequent volatile and sensory characteristics was investigated. Buttermilk addition to cheese milk resulted in a softer cheese compared to other cheeses, with a significantly reduced flowability, while buttermilk powder addition had no influence on cheese firmness but cheese flowability was also reduced compared to the control cheese. Larger pools of free fat, higher levels of free fatty acids, volatile compounds and significant differences in sensory profiles associated with off-flavour were also observed with the addition of buttermilk to cheese milk. Application of light microscopy, using toluidine blue stain, facilitated the visualisation of fat globule structure and distribution within the protein matrix. Addition of 10% buttermilk powder resulted in significant increases in volatile compounds originating from proteolysis pathways associated with roasted, green aromas. Descriptive sensory evaluation indicated few differences between the 10% buttermilk powder and the control cheese, while buttermilk cheeses scored negatively for sweaty, barnyard aromas, oxidized and off flavors, correlating with associated volatile aromas. Addition of 10% buttermilk powder to cheese curds results in cheese comparable to the control Cheddar with some variations in volatile compounds resulting in a cheese with similar structural and sensory characteristics albeit with subtle differences in overall cheese flavor. This could be manipulated to produce cheeses of desirable quality, with potential health benefits due to increased phospholipid levels in cheese.

Redefining the effect of salt on thermophilic starter cell viability, culturability and metabolic activity in cheese.

This study investigated the differential effect of salt concentration in the outside and inside layers of brine salted cheeses on viability, culturability and enzyme activity of starter bacteria. The high-salt environment of the outside layer caused a sharp decrease in L. helveticus viability as measured by traditional plate counts. Remarkably, this was associated with lower release of intracellular enzymes (LDH), reduced levels of proteolysis and larger membrane integrity as measured by flow cytometry (FC) following classical Live/Dead staining. FC analysis of light scattering properties highlighted a significant reduction in size and granularity of the microbiota located in the cheese surface, suggestive of cell shrinkage and condensation of internal macromolecules probably due to hyperosmotic stress. The microbiota of the cheese surface were found to experience greater oxidative stress, as measured by FC analysis of the total levels of reactive oxygen species, compared to that of the interior layer. These results lead us to postulate that the physiology and health status of the microbiota were significantly different in the outer and inner layers of the cheese. The hyperosmotic environment of the outer layer resulted in reduced cell lysis, as measurable by assays based upon membrane integrity, but rather triggered cell death via mechanisms involving cell shrinkage and ROS-mediated damage of vital intracellular components. This study challenges the current thinking on how salt controls microbial activity in ripening cheese, especially in cheeses which are brine salted as local variations in biochemical ripening indices can differ significantly from the outside to the inside of a ripening cheese.

Influence of buttermilk powder or buttermilk addition on phospholipid content, chemical and bio-chemical composition and bacterial viability in Cheddar style-cheese.

The effect of buttermilk powder addition post-curd formation or buttermilk addition to cheese milk on total and individual phospholipid content, chemical composition, enzyme activity, microbial populations and microstructure within Cheddar-style cheese was investigated. Buttermilk or buttermilk powder addition resulted in significant increases in total phospholipid content and their distribution throughout the cheese matrix. Addition of 10% buttermilk powder resulted in higher phospholipid content, moisture, pH and salt in moisture levels, and lower fat, fat in dry matter, L. helveticus and non-starter bacteria levels in cheeses. Buttermilk powder inclusion resulted in lower pH4.6/Soluble Nitrogen (SN) levels and significantly lower free amino acid levels in 10% buttermilk powder cheeses. Buttermilk addition provided a more porous cheese microstructure with greater fat globule coalescence and increased free fat pools, while also increasing moisture and decreasing protein, fat and pH levels. Addition of buttermilk in liquid or powdered form offers potential for new cheeses with associated health benefits.

Small data set analysis in surface metrology: an investigation using a single point incremental forming case study.

A new method for applying statistical techniques with small data sets in surface metrology is demonstrated. This method allows for surfaces or surface-creation processes to be differentiated with as few as six measurement regions. A case study in surface roughness of single point incremental forming is used to demonstrate this method because previous work in this area has not provided quantitative statistical testing to support conclusions. The results from the case study indicate that surface roughness parameters Sz and relative length at scales less than 200 nm are greater when the roll marks on the surface are oriented perpendicular rather than parallel to the forming direction.

The potential of flow cytometry in the study of Bacillus cereus.

Flow cytometry (FCM) is a rapid method allowing the acquisition of multiparametric data from thousands of individual cells within a sample. As well as measuring the intrinsic light scattering properties of cells, a plethora of fluorescent dyes may be employed to yield information on macromolecule content, surface antigens present or physiological status. Despite FCM's indispensability within other fields e.g. immunology, it is underutilized within microbiological research. In this review, a strong case is presented for the potential of FCM in the study of Gram-positive spore-former, Bacillus cereus. Previous reports where FCM was successfully used in the study of B. cereus are reviewed along with relevant studies involving other members of the genus. Under headings reflecting common research themes associated with B. cereus, specific instances where FCM has generated novel data, providing a unique insight into the organism, are discussed. Further applications are posited, based on the authors' own research with FCM and B. cereus and work extant in the broader field of microbial cytometry. The authors conclude that, while the expense of equipment and reagents is an undeniable disadvantage, FCM is a technique capable of generating significantly novel data and allows the design and execution of experiments that are not possible with any other technique.

Lipolysis in cheddar cheese made from raw, thermized, and pasteurized milks.

The evolution of free fatty acids (FFA) was monitored over 168 d of ripening in Cheddar cheeses manufactured from good quality raw milk (RM), thermized milk (TM; 65 degrees C x 15 s), and pasteurized milk (PM; 72 degrees C x 15 s). Heat treatment of the milk reduced the level and diversity of raw milk microflora and extensively or wholly inactivated lipoprotein lipase (LPL) activity. Indigenous milk enzymes or proteases from RM microflora influenced secondary proteolysis in TM and RM cheeses. Differences in FFA in the RM, TM, and PM influenced the levels of FFA in the subsequent cheeses at 1 d, despite significant losses of FFA to the whey during manufacture. Starter esterases appear to be the main contributors of lipolysis in all cheeses, with LPL contributing during production and ripening in RM and, to a lesser extent, in TM cheeses. Indigenous milk microflora and nonstarter lactic acid bacteria appear to have a minor contribution to lipolysis particularly in PM cheeses. Lipolytic activity of starter esterases, LPL, and indigenous raw milk microflora appeared to be limited by substrate accessibility or environmental conditions over ripening.

Production of ingredient-type cheddar cheese with accelerated flavor development by addition of enzyme-modified cheese powder.

Fast-ripened Cheddar cheeses for ingredient purposes were produced by addition of a dried enzyme-modified cheese (EMC; 0.25 and 1 g/100 g of milled curd) at the salting stage during a standard Cheddar cheese-making procedure. Populations of starter and nonstarter lactic acid bacteria (NSLAB), levels of proteolysis and lipolysis, volatile analysis, and flavor development (by quantitative descriptive sensory analysis) were monitored over a 6-mo ripening period. Levels of free AA and free fatty acids were elevated in the experimental cheeses on d 1 because of inclusion of the EMC. Counts of NSLAB were also elevated in the experimental cheeses compared with the control cheese from the start of ripening. Levels of free AA were slightly elevated in the experimental cheeses at 1, 2, and 4 mo, but significantly greater accumulations were detected by 6 mo of ripening, with His, Leu, and glutamate reflecting the greatest increases. Levels of long-chain free fatty acids increased up to 2 mo, indicating an initial stimulation of lipolysis, but had decreased by 6 mo, indicating greater catabolism, probably caused by NSLAB and increased starter lysis. Principal component analysis of the volatile compounds showed few differences in the aroma profiles among the cheeses up to 4 mo of ripening, but a large separation of the cheeses supplemented with EMC relative to the control was observed by 6 mo. Sensory analysis of the cheeses with added EMC showed an acceleration of 2 mo in flavor development compared with the control cheese with the addition of 1 g/100 g of EMC developing a flavor profile at 4 mo similar to the control cheese at 6 mo of ripening. However, atypical Cheddar flavors developed on prolonged storage. This study shows the potential of adding EMC during Cheddar production to produce a fast-ripened ingredient-type Cheddar cheese.

Proteolytic enzyme activities in Cheddar cheese juice made using lactococcal starters of differing autolytic properties.

AIMS: To determine proteolytic enzyme activities released in Cheddar cheese juice manufactured using lactococcal starter strains of differing autolytic properties. METHODS AND RESULTS: The activities of residual chymosin, cell envelope proteinase and a range of intracellular proteolytic enzymes were determined during the first 70 days of ripening when starter lactococci predominate the microbial flora. In general, in cell free extracts (CFE) of the strains, the majority of proteolytic activities was highest for Lactococcus lactis HP, intermediate for L. lactis AM2 and lowest for L. lactis 303. However, in cheese juice, as ripening progressed, released proteolytic activities were highest for the highly autolytic strain L. lactis AM2, intermediate for L. lactis 303 and lowest for L. lactis HP. CONCLUSIONS: These results indicate that strain related differences in autolysis influence proteolytic enzyme activities released into Cheddar cheese during ripening. No correlation was found between proteolytic potential of the starter strains measured in CFE prior to cheese manufacture and levels of activities released in cheese juice. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings further support the importance of autolysis of lactococcal starters in determining the levels of proteolytic activities present in cheese during initial stages of ripening.

Cheddar cheese cooking temperature induces differential lactococcal cell permeabilization and autolytic responses as detected by flow cytometry: implications for intracellular enzyme accessibility.

AIMS: To determine the influence of cheese cooking temperature on autolysis and permeabilization of two lactococcal starter strains in broth and in Cheddar cheese juice during ripening. METHODS AND RESULTS: Flow cytometry (FCM) was used to identify and enumerate intact and permeabilized cells in broth and in Cheddar cheese juice. Levels of intracellular enzyme activities were quantified concurrently. Permeabilized cell numbers increased for both strains in broth following a temperature shift from 32 to 38 degrees C and was accompanied by an increase in the level of accessible intracellular enzyme activities. The relative proportions of intact and permeabilized cell populations, as detected by FCM in cheese juice, changed during 42-day ripening. Permeabilized cell populations increased during ripening for both strains; however, an increase in accessible intracellular enzyme activity was observed only for the highly autolytic strain Lactococcus lactis AM2. CONCLUSIONS: Differences in the autolytic and permeabilization response induced by cooking temperature in two lactococcal strains affects intracellular enzyme accessibility in Cheddar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the importance of the autolytic and permeabilization properties of lactic acid bacteria starter strains and their impact on cheese ripening.

A survey of lipolytic and glycolytic end-products in commercial Cheddar enzyme-modified cheese.

The concentrations of L- and D-lactic acid and free fatty acids, C4:0 to C18:3, were quantified in a range of commercial enzyme-modified Cheddar cheeses. Lactic acid in Cheddar enzyme-modified cheeses varied markedly depending on the manufacturer. Differences in the ratio of L- to D-lactic acid indicate that cheeses of different age were used in their manufacture or contained varying levels of nonstarter lactic acid bacteria. The level of lipolysis in enzyme-modified cheese was higher than in natural Cheddar cheese; butyrate was the predominant free fatty acid. The addition of exogenous acetate, lactate, and butyrate was also indicated in some enzyme-modified cheeses and may be used to confer a specific flavor characteristic or reduce the pH of the product. Propionate was also found in some enzyme-modified cheese products and most likely originated from Swiss-type cheese used in their manufacture. Propionate is not normally associated with natural Cheddar cheese flavor; however, it may be important in the flavor and aroma of Cheddar enzyme-modified cheese. Levels of lipolysis and glycolysis appear to highly controlled as interbatch variability was generally low. Overall, the production of enzyme-modified Cheddar cheese involves manipulation of the end-products of glycolysis (lactate, propionate, and acetate) and lipolysis to generate products for specific applications.

Control of the eukaryotic cell cycle by MAP kinase signaling pathways.

In an often rapidly changing environment, cells must adapt by monitoring and reacting quickly to extracellular stimuli detected by membrane-bound receptors and proteins. Reversible phosphorylation of intracellular regulatory proteins has emerged as a crucial mechanism effecting the transmission and modulation of such signals and is determined by the relative activities of protein kinases and phosphatases within the cell. These are often arranged into complex signaling networks that may function independently or be subject to cross-regulation. Recently, genetic and biochemical analyses have identified the universally conserved mitogen-activated protein (MAP) kinase cascade as one of the most ubiquitous signal transduction systems. This pathway is activated after a variety of cellular stimuli and regulates numerous physiological processes, particularly the cell division cycle. Progression through the cell cycle is critically dependent on the presence of environmental growth factors and stress stimuli, and failure to correctly integrate such signals into the cell cycle machinery can lead to the accumulation of genetic damage and genomic instability characteristic of cancer cells. Here we focus on the MAP kinase cascade and discuss the molecular mechanisms by which these extensively studied signaling pathways influence cell growth and proliferation.

Sin1: an evolutionarily conserved component of the eukaryotic SAPK pathway.

The fission yeast Sty1/Spc1 mitogen-activated protein (MAP) kinase is a member of the eukaryotic stress-activated MAP kinase (SAPK) family. We have identified a protein, Sin1, that interacts with Sty1/Spc1 which is a member of a new evolutionarily conserved gene family. Cells lacking Sin1 display many, but not all, of the phenotypes of cells lacking the Sty1/Spc1 MAP kinase including sterility, multiple stress sensitivity and a cell-cycle delay. Sin1 is phosphorylated after stress but this is not Sty1/Spc1-dependent. Importantly, Sin1 is not required for activation of Sty1/Spc1 but is required for stress-dependent transcription via its substrate, Atf1. We find that in the absence of Sin1, Sty1/Spc1 appears to translocate to the nucleus but Atf1 is not fully phosphorylated and becomes unstable in response to environmental stress. Sin1 is also required for effective transcription via the AP-1 factor Pap1 but does not prevent its nuclear translocation. Remarkably chimaeric fusions of sin1 with chicken sin1 sequences rescue loss of sin1 function. We conclude that Sin1 is a novel component of the eukaryotic SAPK pathway.

The Win1 mitotic regulator is a component of the fission yeast stress-activated Sty1 MAPK pathway.

The fission yeast Sty1 mitogen-activated protein (MAP) kinase (MAPK) and its activator the Wis1 MAP kinase kinase (MAPKK) are required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress. Like the mammalian JNK/SAPK and p38/CSBP1 MAPKs, Sty1 is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, UV light, menadione, heat shock, and the protein synthesis inhibitor anisomycin. We have recently identified two upstream regulators of the Wis1 MAPKK, namely the Wak1 MAPKKK and the Mcs4 response regulator. Cells lacking Mcs4 or Wak1, however, are able to proliferate under stressful conditions and undergo sexual differentiation, suggesting that additional pathway(s) control the Wis1 MAPKK. We now show that this additional signal information is provided, at least in part, by the Win1 mitotic regulator. We show that Wak1 and Win1 coordinately control activation of Sty1 in response to multiple environmental stresses, but that Wak1 and Win1 perform distinct roles in the control of Sty1 under poor nutritional conditions. Our results suggest that the stress-activated Sty1 MAPK integrates information from multiple signaling pathways.

The Mcs4 response regulator coordinately controls the stress-activated Wak1-Wis1-Sty1 MAP kinase pathway and fission yeast cell cycle.

The fission yeast Sty1 MAP kinase is required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress. Like the mammalian JNK/SAPK and p38/CSBP1 MAP kinases, Sty1 is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, menadione, heat shock, and the protein synthesis inhibitor anisomycin. We have identified an upstream regulator that mediates activation of the Sty1 MAP kinase by multiple environmental stresses as the product of the mitotic catastrophe suppressor, mcs4. Mcs4 is structurally and functionally homologous to the budding yeast SSK1 response regulator, suggesting that the eukaryotic stress-activated MAP kinase pathway is controlled by a conserved two-component system. Mcs4 acts upstream of Wak1, a homolog of the SSK2 and SSK22 MEK kinases, which transmits the stress signal to the Wis1 MEK. We show that the Wis1 MEK is controlled by an additional pathway that is independent of both Mcs4 and the Wak1 MEK kinase. Furthermore, we demonstrate that Mcs4 is required for the correct timing of mitotic initiation by mechanisms both dependent and independent on Sty1, indicating that Mcs4 coordinately controls cell cycle progression with the cellular response to environmental stress.

The Atf1 transcription factor is a target for the Sty1 stress-activated MAP kinase pathway in fission yeast.

The atf1+ gene of Schizosaccharomyces pombe encodes a bZIP transcription factor with strong homology to the mammalian factor ATF-2. ATF-2 is regulated through phosphorylation in mammalian cells by the stress-activated mitogen-activated protein (MAP) kinases SAPK/JNK and p38. We show here that the fission yeast Atf1 factor is also regulated by a stress-activated kinase, Sty1. The Sty1 kinase is stimulated by a variety of different stress conditions including osmotic and oxidative stress and heat shock. Deletion of the atf1+ gene results in many, but not all, of the phenotypes associated with loss of Sty1, including sensitivity to environmental stress and inability to undergo sexual conjugation. Furthermore, we identify a number of target genes that are induced rapidly in a manner dependent upon both the Sty1 kinase and the Atf1 transcription factor. These genes include gpd1+, which is important for the response of cells to osmotic stress, the catalase gene lambda important for cells to combat oxidative stress, and pyp2+, which encodes a tyrosine-specific MAP kinase phosphatase. Induction of Pyp2 by Atf1 is direct in that it does not require de novo protein synthesis and results in a negative feedback loop that serves to control signaling through the Sty1/Wis1 pathway. We show that Atf1 associates stably and is phosphorylated by the Sty1 kinase in vitro. Taken together, these results indicate that the interaction between AM and Sty1 is direct. These findings highlight a remarkable level of conservation in transcriptional control by stress-activated MAP kinase pathways between fission yeast and mammalian cells.

Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast.

Simultaneous inactivation of pyp1 and pyp2 PTPases in fission yeast leads to aberrant cell morphology and growth arrest. Spontaneous recessive mutations that bypass the requirement for pyp1 and pyp2 and reside in two complementation groups were isolated, sty1 and sty2. sty1- and sty2- mutant cells are substantially delayed in the timing of mitotic initiation. We have isolated the sty1 gene, which encodes a MAP kinase that is closely related to a subfamily of MAP kinases regulated by osmotic stress including Saccharomyces cervisiae HOG1 and human CSBP1. We find that sty2 is allelic to the wis1 MAP kinase kinase and that delta sty1 and delta wis1 cells are unable to grow in high osmolarity medium. Osmotic stress induces both tyrosine phosphorylation of Sty1 and a reduction in cell size at division. Pyp2 associates with and tyrosine dephosphorylates Sty1 in vitro. We find that wis1-dependent induction of pyp2 mRNA is responsible for tyrosine dephosphorylation of Sty1 in vivo on prolonged exposure to osmotic stress. We conclude that Pyp1 and Pyp2 are tyrosine-specific MAP kinase phosphatases that inactivate an osmoregulated MAP kinase, Sty1, which acts downstream of the Wis1 MAP kinase kinase to control cell size at division in fission yeast.

Gastric schwannoma. Clinical and pathologic considerations.

Solitary gastric schwannomas are rare clinical entities. Presented are three cases encountered in a single hospital in a 4.5-year period. Solitary gastric schwannomas represent a small percentage of schwannomas and a small percentage of gastric tumors. These tumors are usually asymptomatic and may present as upper gastrointestinal bleeding or as mass lesions. Upper endoscopy is important in the initial evaluation of these patients. Pathologic diagnosis is based on features of palisading nuclei, spindle-cell morphology, and hyalinized vessels. Immunohistochemical stains and electron microscopy may be helpful for diagnosis. Complete excision of the tumor is adequate treatment. The risks of coexistent malignancies have not been stressed, but this association may be important; therefore, we recommend a diligent search for other tumors. Prognosis following treatment is good.