[Improvement of Nitrite Analysis Method in Food Products].

The conventional analysis method has problems with extraction efficiency, operability, and reproducibility. In this study, we attempted to solve these problems and improve the analytical method to obtain sufficient extraction efficiency and good operability and accuracy. The conventional method was able to get sufficient extraction in dried meat products, where the extraction efficiency of the conventional method was low, by increasing the concentration of sodium hydroxide solution at the time of homogenization. Suction filtration after adding the defoaming agent was added allowed for accurate volume adjustment. The turbidity of the extract caused by insufficient addition of zinc acetate solution was removed by increasing the amount of zinc acetate solution that was added. Turbidity caused by starch was removed by adding pancreatin. The RSD of the quantitative values was improved by adding sodium hydroxide solution and 80-90℃ water and immediately homogenizing. Furthermore, by changing the dilution factor of the extract solution in the colorimetric method, the inhibition of coloration by reducing substances was suppressed, and more accurate quantitative values could be obtained than with the conventional method. The recovery rate was 78.5-105% (RSD 0.7-5.8%), which was a good result. This method was considered to be a useful analytical method that can contribute to improving the inspection accuracy of nitrite ion analysis.

Estimation of daily intake of food additives by Japanese young children using the market basket method in 2018.

To estimate the daily intake of food additives by young children aged 1-6 years in Japan, an intake survey was conducted in 2018 using the market basket method for food additives, including twelve types of colourants, three kinds of preservatives, three kinds of sweeteners and two kinds of food manufacturing agents. A list of the daily consumption of processed foods was prepared based on a special survey (MHLW 2011) and used for the estimation. The results of the survey showed that the food additives with the highest daily intake were phosphorus compounds (phosphoric acid and its salts; 11.2 mg/kg bw/day, expressed as phosphorus), followed by propylene glycol (0.80 mg/kg bw/day). The daily intake of other food additives ranged from 0 to 0.20 mg/kg bw/day. The estimated daily intake of each food additives by young children was compared with the acceptable daily intake (ADI) or maximum tolerable daily intake (MTDI). The highest ratio of the estimated daily intake to ADI was 3.2% for propylene glycol, whereas the ratios of the estimated daily intake to ADI for colourants, preservatives and sweeteners ranged from 0 to 1.1% (benzoic acid). The ratio of the estimated daily intake to MTDI for phosphorus compounds was 16%.

[Validation of Method for Nitrite Determination in Foods].

In general, nitrite in food is extracted under slightly alkaline conditions, deproteinized, and analyzed by a colorimetric method using color development by diazotization. However, depending on the sample, the sample solution may become cloudy and difficult to filter by the deproteinization treatment of the analytical method. Recently, an improved analytical method that solves these problems has been reported. Therefore, a validation study was performed on the improved analytical method was performed. The concentrations of sodium nitrite added to cod roe, fish sausage, and ham, which were not labeled with sodium nitrite, were set at the upper limits of the standards for use. We set the target values of 70-120% for trueness, less than 15% for intralaboratory reproducibility, and less than intralaboratory reproducibility for repeatability. As a result, the target values were met for the three samples verified: 88-92% for trueness, 2.0-3.0% for repeatability, and 3.2-4.3% for intralaboratory reproducibility. In addition, an interlaboratory study was conducted by eight institutes on the improved analytical method for nitrite. At each institution, sodium nitrite was added to the same three samples as in the validation study, at concentrations equivalent to twice the lower limit of quantification and the upper limit of the standards for use and analyzed in triplicate. The estimated trueness from the obtained analyses ranged from 82 to 95%, the repeatability ranged from 2.3 to 5.8%, and the inter-room reproducibility ranged from 3.5 to 11%. Thus, the improved analytical method could be useful for determining nitrite in foods.

[Storage Stability of Nitrite in Nitrite Analysis of Meat Products].

For the analysis of nitrite ions in food, the stabilities of nitrite ions in meat products and their standard solutions were evaluated. Nitrite is easily oxidized or reduced; hence, products with standard solutions or colour retention agent must be carefully handled. To assess the stability and decreasing trend of nitrite, we examined the storage stability of standard solutions using calibration curves, the time course of nitrite in chopped meat products stored under different conditions, and the time course of nitrite in the sample solutions. Regarding calibration curves, the storage stability was determined for standard solutions that were prepared with ultrapure water at concentrations of 0.025 and 0.4 µg/mL and were stored at 5℃ for one year. The results revealed no changes in concentration of any solution over time, suggesting that no readjustments to the standard solution concentration were necessary before testing until one year after their preparation. Time course of nitrite in chopped meat products stored under different conditions showed a significant decrease in nitrite in refrigerated storage (5℃), whereas stability of nitrite was maintained for up to 1 day in frozen storage (-20℃) and for 14 days in frozen storage (-40℃). The time course of nitrite in the sample solutions showed that the quantitative values of nitrite in the extract remained unchanged within one week of extraction for the meat products tested in the study.

[Determination of Nitrate Ion in Cheese and Sake].

The Japanese official analysis method for determination of nitrate ions in food products used as food additives is associated with various challenges. In some kinds of cheese, the extract becomes suspended. The volume of extracted solution is often not accurate owing to the presence of residues in the solution. Moreover, the determination with liquid chromatography-ultraviolet detection (HPLC-UV) is difficult owing to the influence of impurities. Sake usually does not contain lipids or proteins ; therefore, its analysis can be simplified by omitting the co-precipitation steps to remove them. In the present study, for cheese, the amount of sodium hydroxide solution that causes suspension was reduced, and the influence of residues was removed by adjusting the volume after suction filtration. Whereas, sake was diluted with water and centrifuged. Furthermore, solid-phase extraction (SPE) method using cartridge containing carbon molecular sieve to remove the influence of impurities on the chromatogram was successfully established. The recoveries of the nitrate ions were good outcomes of 91.3-99.6% (CV 0.9-4.5%) (n=5). The analysis range was 0.010-0.20 g/kg for cheese, 0.010-0.20 g/L for milk, and 0.010-0.10 g/kg for sake. The developed analysis methods are considered useful, because various challenges of the official analysis method can be solved and the operation are efficient.

Development of a highly sensitive liquid chromatography with tandem mass spectrometry method for the qualitative and quantitative analysis of high-intensity sweeteners in processed foods.

A new method for the simultaneous determination of two sweeteners (Advantame and Neotame) in processed foods using liquid chromatography (LC) with tandem mass spectrometry(MS/MS) was developed herein. Chromatographic separations were performed using an ACQUITY UPLC CSH C18 column at 40 °C via a mobile phase comprising 10-mmol/L ammonium formate and methanol. Samples were prepared via rapid dialysis using 30% methanol solution in a thermostatic shaker set at 160 rpm and 50 °C for 1 h. The matrix in the test solution had no effect on the identification and quantification of the compound without a clean-up step using solid-phase extraction (SPE). This method satisfied all validation criteria with a limit of quantification (LOQ) of 0.01 µg/g for all samples. Using this method, the amounts of Advantame and Neotame in 24 processed foods were subsequently investigated, with the results indicating their detection beyond the lower LOQ. Moreover, a multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) method was developed and described to further enhance product-identification ability.

[Determination of Aspartame, Acesulfame Potassium, and Sucralose in Chewing Gum by Dialysis Extraction].

The three sweeteners, aspartame, acesulfame potassium, and sucralose, in chewing gum were determined by using dialysis and direct extraction methods. The results revealed that the previously reported dialysis method tended to show poor extraction of aspartame in comparison with the direct-extraction method. The direct extraction also caused operational problems, such as the gum base adhering to the instruments. Therefore, we attempted to improve the dialysis method by changing the dialysate, to which the three sweeteners were extracted while the sample stayed inside the dialysis tube. By changing the dialysate to 60% methanol and dialyzing for 24 hr at room temperature or 2 hr with shaking while heating at 50℃, all three sweeteners were extracted as good as those with the direct-extraction method.

[A Screening Analysis Method for Stevia Sweetener in Processed Foods by LC-MS/MS].

A LC-MS/MS-based screening method was developed for stevia sweetener in processed foods. After extraction of stevia sweetener from processed foods by dialysis, the dialysate was diluted with water, and stevia sweetener was measured by LC-MS/MS. Recovery from 5 kinds of processed foods spiked with 10 mg/kg of stevioside （SS）, 10 mg/kg of rebaudioside A （RS）, or 100 mg/kg of α-glu-cosyltransferase-treated stevia （Gts） product was excellent, and no interfering peak was observed. Thirty-six commercial processed foods indicated as containing "stevia" were analyzed using this established method. Among them, 33 contained SS, 33 contained RS, and 11 contained Gts. Five products contained both stevia extract and Gts.

[Determination of Nitrite in Meat and Fish Products using Dialysis Extraction].

A simple and useful method for the determination of nitrite in meat and fish products was developed. The sample (2.5 g) was extracted and cleaned up by dialysis in tris hydroxymethyl aminomethane solution with shaking at 80℃ for 2.5 hr. Nitrite in the dialysate was quantified by colorimetric analysis. Furthermore, the dialysate was cleaned up with SPE under cooling, and nitrite in the resulting solution was determined using LC-UV with an anion exchange column for confirmation. The recoveries of nitrite from frankfurter and fish sausage, fortified at the levels of 0.002 g/kg and the maximum usage dose (0.070 g/kg for meat products, 0.050 g/kg for fish sausage) ranged from 82.6 to 104.8% in colorimetric analysis and from 88.3 to 97.6% in LC-UV confirmation analysis. The values determined in various meat and fish products by the developed method and by the Japanese official method were approximately equivalent.

[Method of Quantitative Analysis Using Enzymatic Hydrolysis of α-Glucosyltransferase-Treated Stevia in Foods].

A quantitative analysis by HPLC of α-glucosyltransferase-treated stevia in foods was considered. This analysis is the way which hydrolyzed α-glucosyltransferase-treated stevia in the stevioside (SS) and the rebaudioside A (RS) using a glucoamylase. Recovery (%) of α-glucosyltransferase-treated stevia, spiked at 200 mg/kg in various foods, were more than 80% and the relative standard deviations were less than 5.0% as SS and RS for the rate of collection. A qualitative analysis by LC-MS/MS was performed 36 products of commercial foods containing stevia. We quantified of 11 products in which α-glucosyltransferase-treated stevia was detected. Quantitative value was at most 180 mg/kg as SS, at most 70 mg/kg as RS.

[Analysis of Acidic Tar Dyes in High-Protein Foods: Achievement of Improved Recoveries and Shortened Operation Time by Loading Sample Extract onto Polyamide Columns at pH 8.5].

The recoveries of xanthene dyes in the analysis of acidic tar-dyes in high-protein foods were improved by loading them onto polyamide columns at pH 8.5, instead of using the conventional pH 3-4 solution. The experimental scale was reduced to approximately half that of the conventional method. Furthermore, instead of eliminating the organic solvent in the extract by evaporation, the extract was diluted with water prior to PA column cleanup in order to reduce the ratio of organic solvent so that acidic tar-dyes would be better retained on the column. The above two procedures shortened the operation time and allowed for a simpler protocol. With this method, the recoveries of erythrosine, phloxine, and rose bengal from salted cod roe were 82, 88, and 74%, respectively. The recovery percentages were greatly improved compared to those achieved by conventional column loading at pH 3.5 (26, 44, and 18%, respectively). The recoveries of azo-dyes (Amaranth, New Coccine, Allura Red AC, Tartrazine, Sunset Yellow FCF) were also improved from 41-66 to 79-99%.

A Rapid Dialysis Method for Analysis of Artificial Sweeteners in Foods (2nd Report).

Following the previous report, a rapid dialysis method was developed for the extraction and purification of four artificial sweeteners, namely, sodium saccharide (Sa), acesulfame potassium (AK), aspartame (APM), and dulcin (Du), which are present in various foods. The method was evaluated by the addition of 0.02 g/kg of these sweeteners to a cookie sample, in the same manner as in the previous report. Revisions from the previous method were: reduction of the total dialysis volume from 200 to 100 mL, change of tube length from 55 to 50 cm, change of dialysate from 0.01 mol/L hydrochloric aqueous solution containing 10% sodium chloride to 30% methanol solution, and change of dialysis conditions from ambient temperature with occasional shaking to 50℃ with shaking at 160 rpm. As a result of these revisions, the recovery reached 99.3-103.8% with one hour dialysis. The obtained recovery yields were comparable to the recovery yields in the previous method with four hour dialysis.

Analyses of Acidic Tar Dyes in High-Protein Foods and Examination of Extraction and Clean-Up Methods for Various Foods.

Extraction and clean-up methods were examined for the analysis of acidic tar dyes in various high-protein foods. 1% Aqueous ammonia followed by ethanol, 1% aqueous ammonia-ethanol (1 : 1) mixture, and 1% aqueous ammonia-tetrahydrofuran (1 : 1) mixture were used in sequence for boiled fish paste (kamaboko), pounded fish cake (hanpen), and sausage. The sausage extract was centrifuged at low temperature to solidify and remove the contained fat. Salted cod roe with red pepper was extracted twice with 1% aqueous ammonia-ethanol (1 : 1) mixture, followed by extraction with 1% aqueous ammonia-tetrahydrofuran (1 : 1) mixture. A divinylbenzene-N-vinylpyrrolidone copolymer column was used for the clean-up of xanthen dyes. In the case of clogging-prone samples, the same type of large-particle-size column was used. A polyamide column was used for clean-up of the other dyes. When each dye was added at 5 µg/g in the foods, recoveries from kamaboko, hanpen, and sausage ranged from 76 to 102%, and the average recovery from the two types of salted cold roe with red pepper ranged from 45 to 98%.

Analysis of Acidic Tar Dyes in High Protein Food: Effect of pH of Column Loading Solution for Clean-Up.

The effect of pH of the clean-up process in the analysis of 11 permitted tar dyes in high protein food was investigated by using a handmade polyamide column (PA column) and Oasis HLB. Boiled fish paste spiked with the 11 dyes was extracted with appropriate solvents and the pH of the extract was adjusted to 3.0-7.0 in increments of 0.5, followed by clean-up with the PA column. At pH 3.0-5.5, precipitate formed in the extract clogged the column, and the recoveries of R3, R104 and R105 were 26-68%. At pH 6.0-7.0, clogging was not observed and the recoveries of the 3 dyes were somewhat higher, at 38-79%. The recoveries of other dyes were more than 80% at pH 3.0-7.0. With Oasis HLB, column loading was conducted at pH 11.0, and the recoveries of the 3 dyes improved to 70-83%. In conclusion, all 11 dyes could be cleaned-up with the PA column and Oasis HLB and the recoveries exceeded 70%.

A Rapid Dialysis Method for Analysis of Stevioside and Rebaudioside A in Foods.

A rapid dialysis method for the analysis of stevioside (SS) and rebaudioside A (RS) in foods was developed. Minced samples (10 g) were packed into 30 cm net length dialysis tubing with 30% methanol to increase the dialysis efficiency. The dialysis tubing was put in a 100 mL centrifuge tube, and the total fluid volume was made up to 100 mL with 30% methanol. Dialysis was done with shaking while heating at 50℃. The dialysis times were reduced from 48-72 hr in the conventional method to 2 hr under these conditions. The dialysate was loaded on a C18 solid- phase extraction cartridge, and the cartridge was washed with 40% methanol. SS and RS were eluted from the cartridge with 80% methanol, and separated by reversed-phase HPLC. Recovery yields (%)of SS and RS, spiked at 0.02 g/kg in various foods, were 83.0-105.1% and the relative standard deviations were mostly less than 5%.

Use of Dialysis and Liquid-Liquid Extraction for the Determination of Propionic Acid by Gas Chromatography.

A simple and efficient method for the determination of propionic acid (PA) in foods was developed. The sample was cleaned up by dialysis, and PA in the resulting solution was extracted into ethyl acetate for GC analysis. Sodium sulfate was used as a salting-out agent in the extraction process, and GC-FID and GC-MS were successfully applied to the determination and confirmation of PA, respectively. The recoveries were in the range of 98.9-104.4% at the addition level of 0.2 g/kg from 6 foods, bread, cake, cheese, worcester sauce, vinegar-pickles and yogurt. To evaluate the performance of the developed method, recoveries from bread, cake and cheese were compared with those of the notified method at the maximal allowable addition level of PA as a preservative for each food. Recoveries of 98.2-99.5% for the developed method and 91.2-92.0% for the notified method were obtained. The analytical limit was 0.1 g/kg in samples for both determination and confirmation.

[A rapid dialysis method for analysis of artificial sweeteners in food].

A simple and rapid dialysis method was developed for the extraction and purification of four artificial sweeteners, namely, sodium saccharin (Sa), acesulfame potassium (AK), aspartame (APM), and dulcin (Du), which are present in various foods. Conventional dialysis uses a membrane dialysis tube approximately 15 cm in length and is carried out over many hours owing to the small membrane area and owing to inefficient mixing. In particular, processed cereal products such as cookies required treatment for 48 hours to obtain satisfactory recovery of the compounds. By increasing the tube length to 55 cm and introducing efficient mixing by inversion at half-hour intervals, the dialysis times of the four artificial sweeteners, spiked at 0.1 g/kg in the cookie, were shortened to 4 hours. Recovery yields of 88.9-103.2% were obtained by using the improved method, whereas recovery yields were low (65.5-82.0%) by the conventional method. Recovery yields (%) of Sa, AK, APM, and Du, spiked at 0.1 g/kg in various foods, were 91.6-100.1, 93.9-100.1, 86.7-100.0 and 88.7-104.7 using the improved method.

Lifespan extension in the spontaneous dwarf rat and enhanced resistance to hyperoxia-induced mortality.

Lifespan extension has been demonstrated in dwarfism mouse models relative to their wild-type. The spontaneous dwarf rat (SDR) was isolated from a closed colony of Sprague-Dawley (SD) rats. Growth hormone deficiencies have been indicated to be responsible for dwarfism in SDR. Survival time, the markers of oxidative stress, antioxidant enzymes, and resistance to hyperoxia were compared between SDR and SD rats, to investigate whether SDR, a dwarfism rat model, also extends lifespan and has an enhanced resistance to oxidative stress. SDRs lived 38% longer than SD rats on average. This is the first report to show that dwarf rats exhibit lifespan extensions similar to Ames and Snell mice. Decreased 8-oxo-2'-deoxyguanosine (8-oxodG) content, a marker of oxidative DNA damage, indicated suppressed oxidative stress in the liver, kidney, and lung of SDRs. Increased glutathione peroxidase enzyme activity was consistent with decreased 8-oxodG content in the same tissues. The heart and brain showed a similar tendency, but this was not significant. However, the catalase and superoxide dismutase enzyme activities of SDRs were not different from those of SD rats in any tissue. This was not what the original null hypothesis predicted. SDRs had potent resistance to the toxicity associated with high O2 (85%) exposure. The mean survival time in SDRs was more than 147% that of SD rats with 168h O2 exposure. These results suggest that the enhanced resistance to oxidative stress of SDRs associated with enhanced hydrogen peroxide elimination may support its potential role in lifespan extension.

SOD1 (copper/zinc superoxide dismutase) deficiency drives amyloid ß protein oligomerization and memory loss in mouse model of Alzheimer disease.

Oxidative stress is closely linked to the pathogenesis of neurodegeneration. Soluble amyloid ß (Aß) oligomers cause cognitive impairment and synaptic dysfunction in Alzheimer disease (AD). However, the relationship between oligomers, oxidative stress, and their localization during disease progression is uncertain. Our previous study demonstrated that mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn-SOD, SOD1) have features of drusen formation, a hallmark of age-related macular degeneration (Imamura, Y., Noda, S., Hashizume, K., Shinoda, K., Yamaguchi, M., Uchiyama, S., Shimizu, T., Mizushima, Y., Shirasawa, T., and Tsubota, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 11282-11287). Amyloid assembly has been implicated as a common mechanism of plaque and drusen formation. Here, we show that Sod1 deficiency in an amyloid precursor protein-overexpressing mouse model (AD mouse, Tg2576) accelerated Aß oligomerization and memory impairment as compared with control AD mouse and that these phenomena were basically mediated by oxidative damage. The increased plaque and neuronal inflammation were accompanied by the generation of N(ε)-carboxymethyl lysine in advanced glycation end products, a rapid marker of oxidative damage, induced by Sod1 gene-dependent reduction. The Sod1 deletion also caused Tau phosphorylation and the lower levels of synaptophysin. Furthermore, the levels of SOD1 were significantly decreased in human AD patients rather than non-AD age-matched individuals, but mitochondrial SOD (Mn-SOD, SOD2) and extracellular SOD (CuZn-SOD, SOD3) were not. These findings suggest that cytoplasmic superoxide radical plays a critical role in the pathogenesis of AD. Activation of Sod1 may be a therapeutic strategy for the inhibition of AD progression.

Age-related increase of reactive oxygen generation in the brains of mammals and birds: is reactive oxygen a signaling molecule to determine the aging process and life span?

Since Harman proposed the "free-radical theory of aging", oxidative stress has been postulated to be a major causal factor of senescence. The accumulation of oxidative stress-induced oxidatively modified macromolecules, including protein, DNA and lipid, were found in tissues during the aging process; however, it is not necessarily clear which factor is more critical, an increase in endogenous reactive oxygen and/or a decrease in anti-oxidative defense, to the age-related increase in oxidative damage. To clarify the increasing production of reactive oxygen with age, we examined reactive oxygen-dependent chemiluminescent (CL) signals in ex vivo brain slices prepared from different-aged animal brains during hypoxia-reoxygenation treatment using a novel photonic imaging method. The CL signal was intensified during reoxygenation. The signals in SAMP10 (short-life strain) and SAMR1 (control) brain slices increased with aging. The slope of the increase of CL intensity with age in P10 was steeper than in R1. Age-dependent increase of CL intensity was also observed in C57BL/6 mice, Wistar rats and pigeons; however, superoxide dismutase (SOD) activity in the brain did not change with age. These results suggest that reactive oxygen production itself increased with aging. The rate of age-related increases of CL intensity was inversely related to the maximum lifespan of animals. We speculate that reactive oxygen might be a signaling molecule and its levels in tissue might determine the aging process and lifespan. Decelerating age-related increases of reactive oxygen production are expected to be a potent strategy for anti-aging interventions.