Gas production by Paucilactobacillus wasatchensis WDCO4 is increased in Cheddar cheese containing sodium gluconate.

Paucilactobacillus wasatchensis can use gluconate (GLCN) as well as galactose as an energy source and because sodium GLCN can be added during salting of Cheddar cheese to reduce calcium lactate crystal formation, our primary objective was to determine if the presence of GLCN in cheese is another risk factor for unwanted gas production leading to slits in cheese. A secondary objective was to calculate the amount of CO2 produced during storage and to relate this to the amount of gas-forming substrate that was utilized. Ribose was added to promote growth of Pa. wasatchensis WDC04 (P.waWDC04) to high numbers during storage. Cheddar cheese was made with lactococcal starter culture with addition of P.waWDC04 on 3 separate occasions. After milling, the curd was divided into six 10-kg portions. To the curd was added (A) salt, or salt plus (B) 0.5% galactose + 0.5% ribose (similar to previous studies), (C) 1% sodium GLCN, (D) 1% sodium GLCN + 0.5% ribose, (E) 2% sodium GLCN, (F) 2% sodium GLCN + 0.5% ribose. A vat of cheese without added P.waWDC04 was made using the same milk and a block of cheese used as an additional control. Cheeses were cut into 900-g pieces, vacuum packaged and stored at 12°C for 16 wk. Each month the bags were examined for gas production and cheese sampled and tested for lactose, galactose and GLCN content, and microbial numbers. In the control cheese, P.waWDC04 remained undetected (i.e., <104 cfu/g), whereas in cheeses A, C, and E it increased to 107 cfu/g, and when ribose was included with salting (cheeses B, D, and F) increased to 108 cfu/g. The amount of gas (measured as headspace height or calculated as mmoles of CO2) during 16 wk storage was increased by adding P.waWDC04 into the milk, and by adding galactose or GLCN to the curd. Galactose levels in cheese B were depleted by 12 wk while no other cheeses had residual galactose. Except for cheese D, the other cheeses with GLCN added (C, E and F) showed little decline in GLCN levels until wk 12, even though gas was being produced starting at wk 4. Based on calculations of CO2 in headspace plus CO2 dissolved in cheese, galactose and GLCN added to cheese curd only accounted for about half of total gas production. It is proposed that CO2 was also produced by decarboxylation of amino acids. Although P.waWDC04 does not have all the genes for complete conversion and decarboxylation of the amino acids in cheese, this can be achieved in conjunction with starter culture lactococcal. Adding GLCN to curd can now be considered another confirmed risk factor for unwanted gas production during storage of Cheddar cheese that can lead to slits and cracks in cheese. Putative risk factors now include having a community of bacteria in cheese leading to decarboxylation of amino acids and release of CO2 as well autolysis of the starter culture that would provide a supply of ribose that can promote growth of Pa. wasatchensis.

Survival of probiotic adjunct cultures in cheese and challenges in their enumeration using selective media.

Various selective media for enumerating probiotic and cheese cultures were screened, with 6 media then used to study survival of probiotic bacteria in full-fat and low-fat Cheddar cheese. Commercial strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, or Bifidobacterium lactis were added as probiotic adjuncts. The selective media, designed to promote growth of certain lactic acid bacteria (LAB) over others or to differentiate between LAB, were used to detect individual LAB types during cheese storage. Commercial strains of Lactococcus, Lactobacillus, and Bifidobacterium spp. were initially screened on the 6 selective media along with nonstarter LAB (NSLAB) isolates. The microbial flora of the cheeses was analyzed during 9 mo of storage at 6°C. Many NSLAB were able to grow on media presumed selective for Lactococcus, Bifidobacterium spp., or Lb. acidophilus, which became apparent after 90 d of cheese storage, Between 90 and 120 d of storage, bacterial counts changed on media selective for Bifidobacterium spp., suggesting growth of NSLAB. Appearance of NSLAB on Lb. casei selective media [de man, Rogosa, and Sharpe (MRS)+vancomycin] occurred sooner (30 d) in low-fat cheese than in full-fat control cheeses. Differentiation between NSLAB and Lactococcus was achieved by counting after 18 to 24h when the NSLAB colonies were only pinpoint in size. Growth of NSLAB on the various selective media during aging means that probiotic adjunct cultures added during cheesemaking can only be enumerated with confidence on selective media for up to 3 or 4 mo. After this time, growth of NSLAB obfuscates enumeration of probiotic adjuncts. When adjunct Lb. casei or Lb. paracasei cultures are added during cheesemaking, they appear to remain at high numbers for a long time (9 mo) when counted on MRS+vancomycin medium, but a reasonable probability exists that they have been overtaken by NSLAB, which also grow readily on this medium. Enumeration using multiple selective media can provide insight into whether it is the actual adjunct culture or a NSLAB strain that is being enumerated.

Metabolism of Escherichia coli injured by copper.

Escherichia coli injured by copper in carbonate buffer simulating the drinking water environment showed decreased oxygen utilization. Oxygraph measurements revealed that copper-injured bacteria had a rate of oxygen utilization that was less than 25% of that of control cells. Respirometry experiments measured rates over a longer period of time and showed similar trends. Nuclear magnetic resonance spectroscopy (13C nmr) and gas chromatography were used to identify differences in metabolism between healthy and injured populations of E. coli. The rate of glucose utilization by injured cells under anaerobic conditions was 64% of that of healthy cells. The rates of lactate and ethanol accumulation were 88 and 50% of the control, respectively. The 13C nmr studies of oxygenated cultures revealed differences in the accumulation of acetate and glutamine. Aerobic utilization of glucose and succinate by injured cells were 87 and 21% of the rate of the controls, respectively. Additional studies revealed injured cells had a decreased ability to reduce 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) with a variety of carbohydrate substrates. Injured cells reduced greater quantities of INT than healthy cells when NADH was used as a substrate. A comparison of metabolic end products suggested that injured cells also had considerable differences in carbon flow compared with healthy cells.

Mechanism of gastric hyperemia induced by intragastric hypertonic saline in rats.

BACKGROUND: Intragastric hypertonic (2 mol/L) saline produces injury in the gastric mucosa and a significant increase in gastric blood flow (hyperemia) in anesthetized rats. We studied the mechanism of this hyperemia. METHODS: Rats were treated with intravenous boluses of NG-nitro-L-arginine methyl ester (3 mg/kg) to block synthesis of endogenous nitric oxide, pyrilamine (1 mg/kg) to inhibit H1 receptors, or indomethacin (5 mg/kg) to block synthesis of endogenous prostaglandins during blood flow studies or with subcutaneous capsaicin (125 mg/kg) 10-14 days before blood flow studies to ablate capsaicin-sensitive afferent nerves. Gastric mucosal blood flow was measured by hydrogen gas clearance before and during intragastric administration of 2 mol/L saline. RESULTS: The gastric hyperemia induced by intragastric 2 mol/L saline was completely blocked only by indomethacin. The associated gastric mucosal damage was increased significantly. CONCLUSIONS: In the rat stomach, the gastric hyperemia induced by intragastric 2 mol/L saline is mediated by endogenous prostaglandins and plays a protective role. Endogenous nitric oxide, H1 receptors, and capsaicin-sensitive afferent nerves are not involved in this protective hyperemia.

Evidence for the role of copper in the injury process of coliform bacteria in drinking water.

Low levels of copper in chlorine-free distribution water caused injury of coliform populations. Monitoring of 44 drinking water samples indicated that 64% of the coliform population was injured. Physical and chemical parameters were measured, including three heavy metals (Cu, Cd, and Pb). Copper concentrations were important, ranging from 0.007 to 0.54 mg/liter. Statistical analyses of these factors were used to develop a model to predict coliform injury. The model predicted almost 90% injury with a copper concentration near the mean observed value (0.158 mg/liter) in distribution waters. Laboratory studies with copper concentrations of 0.025 and 0.050 mg/liter in an inorganic carbon buffer under controlled conditions of temperature and pH caused over 90% injury within 6 and 2 days, respectively. Studies of the metabolism of injured Escherichia coli cells indicated that the respiratory chain is at least one site of damage in injured cells.

Helicobacter pylori culture supernatant inhibits binding and proliferative response of human gastric cells to epidermal growth factor: implications for H.pylori interference with ulcer healing?

Helicobacter pylori (H. pylori) infection of gastric mucosa is strongly associated with gastritis and peptic ulcer disease. However, the mechanisms of the ulcerogenic action of H. pylori and/or the interference of H. pylori with ulcer healing are unknown. Through binding to its receptor, epidermal growth factor (EGF) accelerates cells migration and triggers epithelial cell proliferation which are both important for the healing of gastroduodenal ulcers. H. pylori seems to interfere with ulcer healing, but the cellular and molecular targets and mechanisms of these actions have not been elucidated. In the present study, we tested the effect of H. pylori culture supernatant (dialyzed to remove molecules smaller than 10 kD) on EGF binding to its receptor and on the proliferative response of human gastric Kato III cells to EGF. H. pylori culture supernatant significantly reduced specific binding of EGF to its receptor and reduced EGF-stimulated gastric cell proliferation. Since ulcer healing requires epithelial cell proliferation and cell migration (re-epithelialization), which are both triggered by EGF binding to its receptor, the alteration in these mechanisms by H. pylori product may be the basis of H. pylori-induced interference with ulcer healing.

Anti-neutrophil serum attenuates dextran sulfate sodium-induced colonic damage in the rat.

BACKGROUND: The role of neutrophils in experimental colonic damage induced by dextran sulfate sodium is uncertain. We test the hypothesis that neutrophils are of pathogenic significance and anti-neutrophil serum will attenuate the colonic damage induced by oral dextran sulfate sodium in rats. METHODS: Rabbit anti-rat neutrophil serum (anti-neutrophil serum) or control rabbit serum was administered to, and circulating neutrophil count was monitored in, rats before and during feeding of dextran sulfate sodium or regular rat diet for 2 weeks. Histologic features of mucosal damage were evaluated in hematoxylin and eosin-stained proximal and distal colonic sections by a blinded observer. RESULTS: Oral dextran sulfate sodium induces weight loss, diarrhea, peripheral neutrophilia, and colonic damage. Anti-neutrophil serum induced neutropenia and significantly attenuated the weight loss, the neutrophil infiltration in the colon, and the mucosal necrosis and pathologic index in the distal colon. CONCLUSION: The data showing that anti-neutrophil serum attenuates distal colonic mucosal injury induced by dextran sulfate sodium support the hypothesis that neutrophils play a pathogenic role in this model of colonic mucosal damage.

Helicobacter pylori culture supernatant interferes with epidermal growth factor-activated signal transduction in human gastric KATO III cells.

The mechanisms by which Helicobacter pylori infection leads to gastroduodenal ulceration remain poorly understood. Previous studies have shown that H. pylori vacuolating cytotoxin (VacA) inhibits proliferation of gastric epithelial cells, which suggests that H pylori may interfere with gastric mucosal repair mechanisms. In this study, we investigated the effects of H. pylori broth culture supernatants on epidermal growth factor (EGF)-mediated signal transduction pathways in a gastric carcinoma cell line (KATO III). Exposure of these cells to EGF resulted in increased expression and phosphorylation of the EGF receptor (EGF-R), increased ERK2 activity and phosphorylation, and increased c-fos protein levels. Preincubation of cells with broth culture supernatant from VacA (+) H. pylori strain 60190 inhibited the capacity of EGF to induce each of these effects. In contrast, preincubation of cells with broth culture supernatant from an isogenic VacA-mutant strain (H. pylori 60190-v1) failed to inhibit the effects of EGF. These results suggest that the H. pylori vacuolating cytotoxin interferes with EGF-activated signal transduction pathways, which are known to be essential for cell proliferation and ulcer healing.

Functional ablation of afferent nerves aggravates dextran sulphate sodium-induced colonic damage in rats.

Dextran sulphate sodium (DSS) is an oral agent capable of inducing chronic diarrhoea and colonic inflammation and necrosis in rats. The role of the afferent nerves in this model of colonic mucosal damage is not known. The hypothesis that functional ablation of the capsaicin-sensitive afferent nerves will aggravate DSS-induced colonic damage in rats was tested. Capsaicin pretreatment was used to ablate afferent nerve function and DSS was administered in the drinking water. Control rats received vehicle pretreatment and water without DSS. There were significant correlations between diarrhoea score, mucosal neutrophil infiltration, mucosal necrosis, and anaemia. Capsaicin pretreatment increased diarrhoea score and colonic mucosal neutrophil infiltration in the rats with colonic damage after 2 or 14 days of DSS. In addition, it induced anaemia and mortality in rats after 14 days of DSS. The data supports the hypothesis that functional ablation of the capsaicin-sensitive afferent nerves aggravates the colonic damage induced by DSS.

Helicobacter pylori induces apoptosis in human epithelial gastric cells by stress activated protein kinase pathway.

BACKGROUND: The pathway by which Helicobacter pylori induces apoptosis in gastric epithelial cells is not known. The aim of this study was to determine whether H. pylori-induced apoptosis is associated with SAPK/JNK activity in human gastric cancer KATO III cells. MATERIALS AND METHODS: H. pylori VacA toxin positive strain was incubated with KATO III cells for 0.5, 1, 2 or 24 hours. The SAPK/JNK protein was harvested from the KATO III cell lysate by precipitation with a C-jun fusion protein and its activity was measured by C-jun phosphorylation utilizing transblotting and phosphoserine antibody. Cellular apoptosis was demonstrated by DNA fragmentation. In addition, cell growth in coculture with H. pylori was determined over 72 hours. RESULTS: H. pylori significantly stimulated SAPK/JNK activity in KATO III cells with a peak at the 0.5 hour time point (3.6-fold vs. control, p <.05), but a return to basal levels by 2 hours. In addition, significant DNA fragmentation was observed after 24 hours in these cells but not in the control KATO III cells. Cell growth was inhibited in a dose dependent fashion in coculture with H. pylori. CONCLUSION: These results show that H. pylori triggers an increase in apoptosis in KATO III cells as reflected by DNA fragmentation. This effect was preceded and correlated with an increase in SAPK/JNK activity suggesting that the H. pylori-induced apoptosis in human gastric epithelial cells may be mediated by the SAPK/JNK pathway.