Comparison of Detection Limits for Allergenic Foods between Total Adenylate (ATP+ADP+AMP) Hygiene Monitoring Test and Several Hygiene Monitoring Approaches.

ABSTRACT: Validation and verification of cleaning and inspection methods are essential to prevent the spread of allergens via cross-contact. Among the hygiene monitoring tests used on-site, the ATP test is rapid and provides quantifiable results. Nevertheless, because a wide variety of foods contain significant amount of ADP and/or AMP due to the degradation of ATP, the ATP+ADP+AMP (A3) test is preferred for detecting food debris. Hence, the A3 test may be valuable in screening food debris that may contain residual allergens. In this study, the detection limits of the A3 test for 40 foods that are regulated in several countries as allergenic were compared with those of the other hygiene monitoring tests used on-site: the conventional ATP test with similar sensitivity for ATP, the protein swab test that detects as little as 50 µg of protein, and the lateral flow immunoassay (LFI). The A3 test demonstrated lower detection limits than did the ATP test. The detection sensitivity of the A3 test was greater than that of the protein swab test except for its use on gelatin (extracted protein). The cleaning validation performance using a stainless steel model in fish and meat revealed that the A3 test is efficient in verifying the levels of remaining food debris. Although LFI displayed the best sensitivities for 10 of 14 foods, it is not commercially available for some specific allergens; however, the A3 test can detect such food debris. Moreover, the detection limits of the A3 test were preferable or comparable to those of LFI for crustacean shellfish and for processed grains, with the exception of wheat flour and buckwheat. A field study in a food processing plant demonstrated that the amount of both A3 and milk protein (enzyme-linked immunosorbent assay) considerably decreased as the cleaning steps progressed. Therefore, the A3 test is effective in detecting the risk for allergen cross-contact after inadequate cleaning.

Microbe participation in aroma production during soy sauce fermentation.

Soy sauce is a traditional Japanese fermented seasoning that contains various constituents such as amino acids, organic acids, and volatiles that are produced during the long fermentation process. Although studies regarding the correlation between microbes and aroma constituents have been performed, there are no reports about the influences of the microbial products, such as lactic acid, acetic acid, and ethanol, during fermentation. Because it is known that these compounds contribute to microbial growth and to changes in the constituent profile by altering the moromi environment, understanding the influence of these compounds is important. Metabolomics, the comprehensive study of low molecular weight metabolites, is a promising strategy for the deep understanding of constituent contributions to food characteristics. Therefore, the influences of microbes and their products such as lactic acid, acetic acid, and ethanol on aroma profiles were investigated using gas chromatography/mass spectrometry (GC/MS)-based metabolic profiling. The presence of aroma constituents influenced by microbes and chemically influenced by lactic acid, acetic acid, and ethanol were proposed. Most of the aroma constituents were not produced by adding ethanol alone, confirming the participation of yeast in aroma production. It was suggested that lactic acid bacterium relates to a key aromatic compound, 2,5-dimethyl-4-hydroxy-3(2H)-furanone. However, most of the measured aroma constituents changed similarly in both samples with lactic acid bacterium and acids. Thus, it was clear that the effect of lactic acid and acetic acid on the aroma profile was significant.

Influence of yeast and lactic acid bacterium on the constituent profile of soy sauce during fermentation.

Soy sauce is a Japanese traditional seasoning composed of various constituents that are produced by various microbes during a long-term fermentation process. Due to the complexity of the process, the investigation of the constituent profile during fermentation is difficult. Metabolomics, the comprehensive study of low molecular weight compounds in biological samples, is thought to be a promising strategy for deep understanding of the constituent contribution to food flavor characteristics. Therefore, metabolomics is suitable for the analysis of soy sauce fermentation. Unfortunately, only few and unrefined studies of soy sauce fermentation using metabolomics approach have been reported. Therefore, we investigated changes in low molecular weight hydrophilic and volatile compounds of soy sauce using gas chromatography/mass spectrometry (GC/MS)-based non-targeted metabolic profiling. The data were analyzed by statistical analysis to evaluate influences of yeast and lactic acid bacterium on the constituent profile. Consequently, our results suggested a novel finding that lactic acid bacterium affected the production of several constituents such as cyclotene, furfural, furfuryl alcohol and methional in the soy sauce fermentation process.

Metabolic profiling approach to explore compounds related to the umami intensity of soy sauce.

A metabolic profiling approach was employed to explore the compounds that affect the intensity of umami taste in soy sauce. Twenty-five kinds of soy sauces were analyzed using GC-MS and LC-MS, wherein measurement data for 427 compounds were obtained. The umami taste intensity of each soy sauce sample was also quantitated by sensory evaluation and a projection to latent structure (PLS) regression analysis was conducted using the compounds' measurements and umami taste intensity data. Variable importance for the projection (VIP) value obtained via PLS was used for the estimation of the relevance for umami taste intensity. N-(1-Deoxyfructos-1-yl)glutamic acid (Fru-Glu) had the highest VIP value, and addition of Fru-Glu to soy sauce increased umami taste intensity better than glutamic acid at the same concentration as confirmed by sensory evaluation. This study showed that the combination of metabolic profiling approach and sensory evaluation can be used effectively to determine compounds related to taste.

Analysis of the correlation between dipeptides and taste differences among soy sauces by using metabolomics-based component profiling.

Characterizing the relationships between the components and taste differences among soy sauces can help evaluate and improve the quality of soy sauces. Although previous studies have reported that certain taste-active dipeptides, the relationships between taste differences and dipeptides of soy sauces are unknown. Therefore, our objective in this study was to investigate the correlations between the dipeptides and the taste differences among soy sauces. To analyze the dipeptides, we constructed an analytical method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) in multiple reaction monitoring mode. Based on this method, we detected 237 dipeptides, the largest number ever detected in soy sauce research. Next, orthogonal projections to latent structures regressions were performed. The data matrix of components, including dipeptides and other low-molecular-weight hydrophilic components obtained from gas chromatography/mass spectrometry (GC/MS), served as explanatory variables (366 in total), whereas a sensory data matrix obtained using quantitative descriptive analysis served as the response variable. The accuracy of models for the sweetness and saltiness differences constructed using the LC/MS/MS and GC/MS data matrix were higher than did models constructed using only the GC/MS data matrix. As a result of investigation of the correlation between the dipeptides and taste differences among soy sauces by using variable importance in the projection (VIP) score, many dipeptides showed the high correlation with taste differences. Specifically, Ile-Gln, Pro-Lys, Ile-Glu, Thr-Phe, and Leu-Gln showed the high VIP score on sweet differences. This study is the first report that reveals the correlations between the dipeptides and taste differences among soy sauces.

Discrimination of methicillin-resistant Staphylococcus aureus from methicillin-susceptible Staphylococcus aureus or coagulase-negative staphylococci by detection of penicillin-binding protein 2 and penicillin-binding protein 2' using a bioluminescent enzyme immunoassay.

For the discrimination of methicillin-resistant S. aureus (MRSA) from methicillin-susceptible S. aureus (MSSA) or coagulase-negative staphylococci (CNS), we developed a bioluminescent enzyme immunoassay (BLEIA) for detecting penicillin-binding protein 2 (PBP2) and penicillin-binding protein 2' (PBP2') using biotinylated firefly luciferase. The BLEIA was able to detect recombinant PBP2 at 50 pg/ml and recombinant PBP2' at 500 pg/ml. PBP2 and PBP2' present in the membranes of S. aureus were extracted by acid and detergent treatment. The method was able to detect PBP2 or PBP2' extracted from 10(6) colony forming units of S. aureus because of efficient extraction and the high sensitivity of luciferase. In a study of clinical isolates previously characterized as either MRSA or MSSA by antibiotic susceptibility testing, all 34 specimens identified as MRSA were both PBP2 and PBP2' positive. The 34 MSSA specimens were PBP2 positive and PBP2' negative. Moreover, the BLEIA could detect PBP2' extracted from four species of methicillin-resistant CNS, but not PBP2 extracted from four species of methicillin-resistant and methicillin-susceptible CNS. This result suggested that PBP2 could be a unique marker for discrimination of S. aureus from CNS. A BLEIA that is able to detect PBP2 and PBP2' may be useful in clinical diagnostics.

Tensile mechanics of alanine-based helical polypeptide: force spectroscopy versus computer simulations.

In nature, an alpha-helix is commonly used to build thermodynamically stable and mechanically rigid protein conformations. In view of growing interest in the mechanical rigidity of proteins, we measured the tensile profile of an alanine-based alpha-helical polypeptide on an atomic-force microscope to investigate the basic mechanics of helix extension with minimal interference from side-chain interactions. The peptide was extended to its maximum contour length with much less force than in reported cases of poly-L-Glu or poly-L-Lys, indicating that chain stiffness strongly depended on the physicochemical properties of side chains, such as their bulkiness. The low tensile-force extension originated presumably in locally unfolded parts because of spontaneous structural fluctuations. In 50% trifluoroethanol, the well-known helix-promoting agent, the rigidity of the sample polypeptide was markedly increased. Computer simulations of the peptide-stretching process showed that a majority of constituent residues underwent a transition from an alpha-helical to an extended conformation by overcoming an energy barrier around psi approximately 0 degrees on the Ramachandran plot. The observed lability of an isolated helix signified the biological importance of the lateral bundling of helices to maintain a rigid protein structure.

Ingestion of difructose anhydride III, a non-digestible disaccharide, improves postgastrectomy osteopenia in rats.

OBJECTIVE: Total gastrectomy produces osteopenia with calcium malabsorption. We previously demonstrated that difructose anhydride III (DFAIII), a non-digestible disaccharide, stimulates intestinal calcium absorption in normal and ovariectomized rats. In the present study, we examined the effects of feeding DFAIII on gastrectomy-induced calcium malabsorption and osteopenia in rats. The potential of DFAIII to promote large intestinal calcium absorption was also evaluated through comparison with that of fructooligosaccharides (FOS). MATERIAL AND METHODS: Male Sprague-Dawley rats were divided into two groups: totally gastrectomized and sham-operated rats. After a postoperative recovery period, rats from each group were divided into three subgroups and fed the control, DFAIII (30 g/kg), or FOS (30 g/kg) diet for 28 days. RESULTS: Total gastrectomy severely reduced net calcium absorption, femoral calcium content and bone mineral density, resulting in fragility of the femur. DFAIII or FOS feeding partly and similarly restored the lowered calcium absorption and femoral variables, with an increase in the total short-chain fatty acid pool in the cecum. In gastrectomized rats, net calcium absorption was correlated with several cecal parameters, suggesting that cecal fermentation of DFAIII is associated with the improvement in gastrectomy-induced calcium malabsorption. Urinary excretion of deoxypyridinoline (D-Pyr) as a marker of bone resorption was increased by gastrectomy, and the elevated D-Pyr excretion was suppressed by feeding DFAIII. CONCLUSIONS: Supplemental feeding of DFAIII partly prevents postgastrectomy osteopenia as a result of an improvement in calcium absorption. Our results suggest that the promotive effects of DFAIII on calcium absorption in the large intestine are comparable to those of FOS.

Ingestion of difructose anhydride III, a non-digestible disaccharide, prevents gastrectomy-induced iron malabsorption and anemia in rats.

OBJECTIVE: Total gastrectomy produces iron malabsorption and anemia, and several non-digestible carbohydrates promote mineral absorption. In this study, we examined the effects of feeding difructose anhydride III (DFAIII), a non-digestible disaccharide, on gastrectomy-induced iron malabsorption and anemia in rats in comparison with those of feeding fructo-oligosaccharides (FOS). METHODS: Sham-operated and totally gastrectomized male Sprague-Dawley rats were fed the control, DFAIII (30 g/kg), or FOS (30 g/kg) diet for 4 wk. Feces and tail blood were collected at 2 and 4 wk to evaluate body iron status and iron absorption. RESULTS: Gastrectomy severely decreased net iron absorption, hemoglobin concentration, and hematocrit in the control dietary group. The decreased absorption in gastrectomized rats was restored to the sham control level by feeding the DFAIII or FOS diet. Iron absorption in sham rats was higher in the FOS and DFAIII groups than in the control group. Hemoglobin concentration and hematocrit in gastrectomized rats fed the DFAIII diet, but not the FOS diet, returned to levels comparable to the effects in sham rats fed the control diet. Feeding DFAIII increased short-chain fatty acid pools and decreased pH of cecal contents. These parameters for cecal fermentation correlated with iron absorption. CONCLUSIONS: DFAIII feeding restores gastrectomy-induced iron malabsorption, resulting in complete prevention of iron-deficiency anemia in rats. Cecal fermentation of DFAIII may contribute to the improvement in these gastrectomy-induced defects. Feeding with low level of FOS did not fully improve postgastrectomy anemia.

Effects of difructose anhydride III (DFA III) administration on rat intestinal microbiota.

The effects of difructose anhydride III (di-D-fructofuranose-1,2':2,3'-dianhydride; DFA III) administration (3% DFA III for 4 weeks) on rat intestinal microbiota were examined using denaturing gradient gel electrophoresis (DGGE). According to DGGE profiles, the number of bacteria related to Bacteroides acidofaciens and uncultured bacteria within the Clostridium lituseburense group decreased, while that of bacteria related to Bacteroides vulgatus, Bacteroides uniformis and Ruminococcus productus increased in DFA III-fed rat cecum. In the cecal contents of DFA III-fed rats, a lowering of pH and an increase in short chain fatty acids (SCFAs), especially acetic acid, were observed. The DFA III-assimilating bacterium, Ruminococcus sp. M-1, was isolated from the cecal contents of DFA III-fed rats. The strain had 98% similarity with R. productus ATCC 27340T (L76595), and mainly produced acetic acid. These results confirmed that the bacteria harmful to host health were not increased by DFA III administration. Moreover, DFA III stimulated the growth of Ruminococcus sp. M-1 producing acetic acid, which may alter the intestinal microbiota towards a healthier composition. It is expected that DFA III would be a new candidate as a prebiotic.

Reducing effect of ingesting tannic acid on the absorption of iron, but not of zinc, copper and manganese by rats.

Interest in the beneficial effects of polyphenols, including tannic acid (TA), is increasing, although, these compounds also have adverse effects; for example, on the absorption of iron (Fe), and possibly other trace minerals. We examined the effect of a graded dose of TA on the absorption of Fe and compared with that of zinc (Zn), copper (Cu) and manganese (Mn) in rats. We also investigated the effect of TA on cecal fermentation which plays a role in absorption. In Experiment 1, to set the optimum dose of Fe, male Sprague-Dawley rats (weighing 70-90 g) after acclimatization were fed with different levels of dietary Fe (5, 10, 20, 30 and 35 mg/kg). We observed that the hematocrit (Ht), serum Fe concentration and transferrin saturation (%) were each reduced in those rats fed less than 20 mg/kg Fe in a dose-dependent manner. In Experiment 2, the rats were fed with test diets containing the minimum required level of Fe, 30 mg/kg diet, with (5, 10, 15 and 20 g/kg diet) or without TA for a period of three weeks. Feeding a diet containing more than 10 g TA/kg diet, but not 5 g TA/kg diet, reduced the hemoglobin concentration (Hb), Ht and serum Fe concentration due to decreased Fe absorption. In contrast, the Zn, Cu and Mn absorption was not affected by TA feeding. It is also demonstrated that liver Fe, but not the Zn, Cu and Mn contents, were lower in the TA groups than in the TA-free control group. Feeding TA slightly decreased the pH value of the cecal contents with an increase in the major short-chain fatty acid pool. About 15% of the ingested TA were recovered in the feces of each TA-fed group. Our results demonstrate that more than 10 g TA/kg diet induced anemia by reducing the Fe absorption, although there was no effect on the absorption of other important trace minerals. Our findings suggest that the usual intake of polyphenols is relatively safe, but that a high intake by supplementation or by dietary habit of tannin affects only the Fe level.

Ingestion of difructose anhydride III and voluntary running exercise independently increase femoral and tibial bone mineral density and bone strength with increasing calcium absorption in rats.

Difructose anhydride III (DFAIII), a nondigestible disaccharide, promotes intestinal calcium absorption. Exercise-induced mechanical stimuli are essential for bone growth. In the present study, we examined the effects of consuming DFAIII and voluntary running exercise on calcium absorption and bone characteristics using male Sprague-Dawley rats (4 wk old). The study was designed in a 2 x 2 factorial arrangement with two conditions (sedentary or exercised) and two diets [AIN-93G diet with or without DFAIII (30 g/kg diet)]. Both consuming DFAIII and running exercise increased net calcium absorption, and the effects of DFAIII and exercise were additive. Both consuming DFAIII and exercise also increased femoral variables such as calcium content and total bone mineral density (BMD); however, only consuming DFAIII increased bone strength in the femur. Conversely, running exercise augmented tibial calcium content, total BMD and bone strength, but consuming DFAIII did not. We conclude that consuming DFAIII and running exercise additively enhance calcium absorption and differentially stimulate femoral and tibial BMD and mechanical properties in rats.

Ingestion of an indigestible saccharide, difructose anhydride III, partially prevents the tannic acid-induced suppression of iron absorption in rats.

Dietary tannic acid (TA) inhibits iron absorption and some indigestible oligosaccharides have been shown to promote mineral absorption. In this study, we examined whether difructose anhydride III (DFA III) or fructooligosaccharide (FOS) stimulate iron absorption in TA-fed rats. Two experiments were conducted using male Sprague-Dawley rats (weighing 90-110 g) in a randomized block design. Rats were fed control, DFA III or FOS (30 g/kg) diets in expt. 1, and control, TAcontrol, TAFOS or TADFA III (TA, 15 g/kg) diets in expt. 2 for 3 wk during which blood sampling was performed weekly and fecal collection twice. In expt. 1, apparent iron absorption was higher (P < 0.001) in the DFA III-fed (65.7 and 55.9%, d 8-10 and 19-21) and FOS-fed (59.9%, d 19-21) groups than in the control group (48.4 and 45.4%, d 8-10 and 19-21) without differences in blood hemoglobin concentrations or hematocrits. TA feeding reduced hemoglobin concentrations and hematocrits (119.1 g/L, 0.360; P < 0.001), and the feeding of TADFA III partially improved this anemic condition (129.6 g/L, 0.403), whereas TAFOS feeding did not influence these variables (120.6 g/L, 0.342; expt. 2). Iron absorption was lower in the TA-fed groups (19.8%; P < 0.001) than in the control group (49.4%), whereas the absorption in both TA-fed indigestible sugar groups was higher (DFA III, 43.2 and 38.2%, d 8-10 and 19-21; FOS, 39.4%, d 8-10; P < 0.001) than in the TA-control group except for the TAFOS-fed group (25.1%, d 19-21). Serum iron concentrations, unsaturated iron-binding capacities, total iron-binding capacities and transferrin saturations (%) were not improved by the feeding of TADFA III or TAFOS. Furthermore, liver iron concentrations were decreased by TA feeding (P < 0.001) and were not increased by the feeding of indigestible sugars. The feeding of DFA III or FOS decreased the pH of the cecal contents (P < 0.001) while increasing major organic acid pools. In all groups fed TA, approximately 18% of the ingested TA was recovered in the feces. Our results demonstrate that TA reduces iron absorption and induces anemia, conditions that are partially prevented by the feeding of DFA III, but not FOS.

Ingestion of water-soluble soybean fiber prevents gastrectomy-induced iron malabsorption, anemia and impairment of voluntary running exercise performance in rats.

We examined the effects of feeding water-soluble soybean fiber (WSSF), a highly fermentable dietary fiber, on gastrectomy-induced iron malabsorption, anemia and impairment of exercise performance in rats in two separate experiments. The study was designed as a 2 x 2 factorial arrangement with operation (total gastrectomy) and diet (WSSF) under sedentary (Experiment 1) and exercised (Experiment 2) conditions. In Experiment 1, gastrectomy reduced net iron absorption, hemoglobin concentration, hematocrit and hemoglobin regeneration efficiency (P < 0.01). However, in rats fed a WSSF diet (50 g/kg diet), iron absorption and the hematological variables of the gastrectomized rats were comparable to those in the sham-operated rats, demonstrating that ingestion of WSSF promotes iron absorption and prevents anemia after gastrectomy. Feeding WSSF increased pools of organic acids and soluble iron in cecal contents and decreased the pH of the cecal contents (P < 0.001). Of the many cecal variables measured, net iron absorption in gastrectomized rats was most closely correlated (r = 0.614, P < 0.01) with the short-chain fatty acid pool in the cecum. Cecal fermentation of WSSF may contribute to improvements in gastrectomy-induced nutritional defects. In Experiment 2, we examined voluntary running exercise performance in totally gastrectomized rats fed diets with or without WSSF. Total gastrectomy severely impaired running performance (P < 0.001), and WSSF feeding largely restored the lowered performance. We conclude that feeding WSSF improves anemia and impaired voluntary running performance in totally gastrectomized rats.

Ingestion of water-soluble soybean fiber improves gastrectomy-induced calcium malabsorption and osteopenia in rats.

OBJECTIVE: Total gastrectomy produces calcium malabsorption and osteopenia. We examined the effects of feeding water-soluble soybean fiber (WSSF), a highly fermentable dietary fiber with low viscosity, on calcium absorption and bone characteristics in totally gastrectomized rats. METHODS: Male Sprague-Dawley rats were assigned to two groups: sham operation and gastrectomy. After the postoperative recovery period, rats of each group were fed diets with or without WSSF (50 g/kg diet) for 4 wk. RESULTS: Net calcium absorption and femoral dry weight and calcium content and maximum breaking force were lower in the gastrectomy groups than in the sham groups. In the gastrectomy groups, calcium absorption and these femoral parameters were higher in rats fed the WSSF diet than in rats fed the WSSF-free diet. Feeding WSSF increased the concentrations of short-chain fatty acids and soluble calcium and decreased the pH in cecal contents. Net calcium absorption in gastrectomized rats correlated positively with the concentration of total short-chain fatty acids (the sum of acetic, propionic, and butyric acids) in cecal contents (r = 0.545, P < 0.05) and negatively with cecal pH (r = -0.628, P < 0.05). CONCLUSIONS: These findings indicate that ingestion of WSSF partly prevents the diminished calcium absorption after total gastrectomy in rats, resulting in the improvement of postgastrectomy osteopenia. The increase of calcium absorption may be associated with cecal fermentation of WSSF.