Japan Food Research Laboratories

Western honeybee (Apis mellifera) α-glucosidase III (HBG-III), which is secreted from the hypopharyngeal glands of honeybees, plays a role in converting nectar into honey. Consequently, hypothesizing that HBG-III is a suitable marker of honey authenticity, we developed an analytical method to determine the HBG-III content and investigated its applicability to various commercial products. Following extraction from honey using phosphate-buffered saline, HBG-III was concentrated using an ultrafiltration membrane and subsequently fragmented with trypsin and lysyl endopeptidase mixture. The specific peptide fragments were used for quantitation by liquid chromatography-tandem mass spectrometry. The established method was validated for linearity, accuracy, precision, and the limit of quantitation (LOQ). As a result, the calibration curve was linear in the range of 0.01-0.3 μM, the mean recovery ranged from 73.8 to 89.2 %, the within-laboratory reproducibility (RSDwr) ranged from 3.9 to 6.5 %, and the LOQ was 1.9 mg/kg. An investigation of HBG-III concentrations in 65 honey products available on the Japanese market revealed that the HBG-III content of 15 low-priced honey products was below the LOQ. This suggested that these products may be adulterated with non-honey syrups. Therefore, this method can serve as an effective tool to verify the authenticity of honey products.

A quantitative method for determining resorcylic acid lactones (zeranol, taleranol, zearalanone, and zearalenone) in bovine urine using liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and evaluated for use in the monitoring of Japanese beef exports to the European Union. This method involves the hydrolysis of conjugates by ß-glucuronidase/aryl sulfatase, clean-up with an immunoaffinity cartridge, and analysis using LC–MS/MS. The developed method was evaluated for zeranol, taleranol, zearalanone, and zearalenone in bovine urine at concentrations of 1 and 2 μg/L. The trueness ranged from 92 to 101%, with intra-day precision of <9% and inter-day precision of <13%. No interfering peaks were observed in the chromatogram near the analyte retention times. This analytical method is valuable for monitoring taleranol, zearalanone, and zearalenone in beef exports to the European Union. The proposed method is also applicable for screening whether zeranol was illegally administered or ingested via feed contaminated with Fusarium toxins, particularly in cases in which taleranol was detected.

Injectable anesthesia is widely used in laboratory animals because of its ease of administration and minimal equipment requirements. However, it necessitates careful monitoring as well as thermal and oxygen support. This study evaluated the efficacy of medetomidine-alfaxalone-butorphanol (MAB) anesthesia in male rabbits using a dual-route administration protocol. The anesthetic doses were as follows: medetomidine, 0.2 mg/kg; alfaxalone, 2.0 mg/kg; and butorphanol, 2.0 mg/kg. MAB anesthesia, administered via intravenous and subcutaneous routes, induced rapid and smooth induction, achieving anesthetic scores comparable to those of medetomidine-midazolam-butorphanol (MMB) anesthesia. MAB anesthesia resulted in mild hypothermia during the procedure. Upon atipamezole administration, rabbits under MAB anesthesia exhibited faster recovery of the righting reflex and respiration rate than those under MMB. Importantly, no abnormal behaviors, such as jumping or agitation, were observed during induction or recovery, as reported with alfaxalone use in other species. Both protocols maintained spontaneous breathing, although transient hypoxemia was observed in all rabbits. The dual-route MAB protocol provided effective anesthesia while addressing the limitations of conventional MMB anesthesia in rabbits, suggesting its potential as a refined anesthetic method for this species. Unlike mice, which showed weaker anesthetic effects with MAB compared to MMB, MAB demonstrated superior anesthetic properties in rabbits. This study highlights the importance of species-specific anesthetic protocols and the potential benefits of MAB anesthesia in rabbits, particularly its smooth induction and recovery profile, without adverse behaviors often associated with alfaxalone in other species.

Diethyl ether and chloroform have been widely used in fat analysis of foods. However, there are risks associated with their use or human health hazards. In this report, we investigated safer alternative solvents aiming to reduce the amount of these solvents used. Compared to diethyl ether, the results of fat analysis using hexane were equivalent, with good analytical precision. In addition, the hexane-isopropanol mixture also showed similar results compared to the chloroform-methanol mixture. The results of this study suggested that the use of hexane and the hexane-isopropanol mixture as alternative solvents may reduce the use of diethyl ether, a highly flammable material, and chloroform, specified chemical substance.

Diethyl ether and chloroform have been widely used in fat analysis of foods. However, there are risks associated with their use or human health hazards. In this report, we investigated safer alternative solvents aiming to reduce the amount of these solvents used. Compared to diethyl ether, the results of fat analysis using hexane were equivalent, with good analytical precision. In addition, the hexane-isopropanol mixture also showed similar results compared to the chloroform-methanol mixture. The results of this study suggested that the use of hexane and the hexane-isopropanol mixture as alternative solvents may reduce the use of diethyl ether, a highly flammable material, and chloroform, specified chemical substance.

To accurately quantify the phospholipids in infant formula by subclass, we developed an analytical method using phosphorus-31 nuclear magnetic resonance spectroscopy (³¹P NMR). We performed heated extraction method using a mixture of ethanol and water to extract phospholipids from infant formula and replace the highly toxic chloroform traditionally used for extraction. In the ³¹P NMR measurement, we also avoided using chloroform by dispersing the extracts in surfactants with a strong affinity for phospholipids. Although polar lipids in milk are characterized by a high content of sphingomyelin, the separation of sphingomyelin and phosphatidylethanolamine signals was insufficient to accurately determine their signal areas. To overcome this issue, we applied and evaluated two different methods, integration and deconvolution, for calculating the signal areas. During method validation in a spiked recovery test, the deconvolution method gave a recovery rate closer to 100% than the integration method. The main phospholipid subclasses found in infant formula were phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and sphingomyelin. However, when the formula contained soy lecithin, additional soy-derived phospholipids, such as phosphatidic acid, were detected. Using equipment with a phosphorus resonance frequency of 202 MHz and a measurement time of approximately 4 h, the quantification limit was 5 mg/100 g. The developed method will be useful for analysis of phospholipids in infant formula. graphical abstract Fullsize Image

A collaborative study among 11 different Japanese laboratories was undertaken to devise an analytical method for determining the fatty acid composition of fats and oils by gas chromatography (GC) using hydrogen (H2) or nitrogen (N2) as carrier gases alternative to helium (He). This collaborative study was organized by the Alternative Gas for the GC Subcommittee of the Japan Oil Chemists’ Society and was evaluated by analyzing a standard mixture of fatty acid methyl esters (Supelco 37 Component FAME Mix), soybean, and sardine oils. On a 50% cyanopropyl siloxane capillary column, the resolution of 37 FAME mixtures using He, H2, or N2 gas was greater than 1.0. For the area% of the 37 FAME mixtures, the reproducibility relative standard deviations ranged from 0.59 to 4.72% for He gas, 0.39 to 4.96% for H2 gas, and 0.60 to 4.91% for N2 gas. No significant difference was observed in the area% for the 37 FAME mixtures among the three carrier gases. Furthermore, no significant differences in the percentages of major fatty acids in the soybean and sardine oils were detected between the different carrier gases. This study also determined that the analysis time was prolonged when N2 gas was used compared to when H2 or He gas was used. However, a cyanopropyl siloxane capillary column for fast and selective separation could improve the analysis time using N2 gas. Therefore, we conclude that the present method using H2 or N2 gas qualifies as a tentative official method of the Japan Oil Chemists’ Society. graphical abstract Fullsize Image

Chloropropanols and related substances have received a great deal of attention in the world. Regulation values have already been established in the EU for infant formula. There are several validated analytical methods for these substances, such as the AOAC official method. However, no analytical official method has been established for infant formula in Japan. In this study, the AOAC official method was improved to a more versatile splitless injection method in order to make it feasible for many laboratories. The results of the validation data for the improved method with optimized injection conditions were as follows. The limit of quantification was 10 μg/kg, the recovery rate ranged from 81.6 to 114.5%, and the intermediate precision ranged from 1.6 to 7.6%. These results were adapt to AOAC and EU requirement and demonstrated the good validity of the improved method.

Thiouracil (2-thiouracil) is a thyrostat used to promote weight gain in cattle. However, its use is prohibited within the European Union (EU), necessitating the monitoring of its presence in bovine urine for beef exports to the EU. In this study, we present the development and validation of a quantitative method for the determination of 2-thiouracil, 4-thiouracil, and 6-methyl-2-thiouracil in bovine urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method involves stabilizing the analytes by adding hydrochloric acid and ethylenediaminetetraacetic acid to the sample, followed by derivatization with 3-iodobenzyl bromide, cleanup with a divinylbenzene-N-vinylpyrrolidone copolymer cartridge, and subsequent LC-MS/MS analysis. The developed method was validated for determination of 2-thiouracil, 4-thiouracil, and 6-methyl-2-thiouracil in bovine urine at a concentration of 10 μg/L. The trueness ranged from 94 to 97%, with intra-day precisions below 5% and inter-day precisions below 8%. No chromatographic interference was observed near the analytes’ retention times. This analytical method is particularly valuable because it can determine whether 2-thiouracil was illicitly administered or ingested via feed containing plants of the Brassicaceae family, by confirming the presence of 6-methyl-2-thiouracil or 4-thiouracil alongside 2-thiouracil in bovine urine.

Human milk oligosaccharides (HMOs) have been positively associated with child neurodevelopment in some cohort studies. However, there is a lack of consistency in the association between HMOs and benefits to infants’ brains. Moreover, the quantification methods for HMOs have not yet been standardized. In this study, we developed a quantification method for evaluating eight HMOs (2′‐fucosyllactose [2′‐FL], 3′‐fucosyllactose [3′‐FL], 3′‐sialyllactose [3′‐SL], 6′‐sialyllactose [6′‐SL], lactosialyltetrasaccharide a [LSTa], lactosialyltetrasaccharide b [LSTb], lactosialyltetrasaccharide c [LSTc], and disialyllacto‐N‐tetraose [DSLNT]) in breast milk. After validating the method, we applied it to 1‐month breast milk samples (n = 150) from the Tohoku Medical Megabank Project Birth and Three‐Generation Cohort Study to assess HMO profiles in breast milk and their possible association with changes in head circumference z‐score (ΔHCZ) and neurodevelopmental scores of children (as measured by the Ages and Stages Questionnaire, Third Edition). The validation demonstrated that the method had relative standard deviation ≤ 12.7% of precision and 79.5–110.9% of accuracy. Using this method, eight HMO levels (2′‐FL, 0–4.74 mg/mL; 3′‐FL, 0.02–1.52 mg/mL; 3′‐SL, 0.07–0.32 mg/mL; 6′‐SL, 0.01–0.70 mg/mL; LSTa, 0.002–0.043 mg/mL; LSTb, 0.02–0.31 mg/mL; LSTc, 0.001–0.47 mg/mL; and DSLNT, 0.09–0.71 mg/mL [min–max, all participants]) and the ratio of low secretors (16.0%) in the Japanese cohort were obtained. The obtained HMO levels in breast milk were subjected to multivariate analysis to screen for HMOs showing a positive association with ΔHCZ and neurodevelopmental scores. The results proposed that ΔHCZ was positively associated with LSTb and 2′‐FL levels, whereas neurodevelopmental scores were positively associated with 2′‐FL levels (among all participants) and 3′‐SL and DSLNT levels (among secretor participants). This study showed that the developed method provides HMO profiles in Japanese breast milk, as well as additional information on the associations between specific HMOs and neurodevelopment, reinforcing the sum of evidence for the role of HMOs in the brain.

Medetomidine, midazolam, and butorphanol (MMB) anesthesia is the preferred choice for rodents but requires excess volume of intramuscular injection in rabbits, which can lead to muscular damage. This study aimed to evaluate a dual-route MMB administration via the intravenous and subcutaneous routes in rabbits. MMB was administered to male Kbs:JW rabbits with an intravenous injection of 0.2 mL/kg followed by a subcutaneous injection of 0.8 mL/kg, totaling 0.2 mg/kg medetomidine, 2.0 mg/kg midazolam, and 2.0 mg/kg butorphanol. We compared the anesthetic effects of this dual-route method with those of intramuscular administration. The dual-route method resulted in a shorter induction time and similar anesthetic duration compared with those of the intramuscular route. While it induced a temporary decrease in body temperature within 30 min post-injection, other vital signs, such as respiration rate, heart rate, and O2 saturation, remained similar. Notably, unlike intramuscular administration, dual-route administration did not increase tissue injury marker levels. This dual-route MMB administration provided sufficient anesthetic depth during surgery, eliminating pain reflexes. Double-dose administration extended anesthetic duration but resulted in rare fatalities, indicating room for protocol improvement. In conclusion, the novel anesthetic method is preferable for injectable anesthesia in rabbits, providing rapid induction and sufficient anesthetic duration, while potentially minimizing muscle injury. This technique may be beneficial for both laboratory and companion animals and significantly enhance animal welfare in anesthesia by reducing the pain associated with injectable anesthesia.

Opantimycin A, a rare antimycin-class antibiotic without the macrolide core, was isolated from Streptomyces sp. RK88-1355 in 2017. In this study, we explored the total synthesis and stereochemical assignment of...

Low-dose aspirin for primary prevention is determined by the balance of risks of cardiovascular events and adverse effects. We assessed the long-term gastrointestinal symptoms or bleeding with low-dose aspirin in diabetic patients. The Japanese Primary Prevention of Atherosclerosis with Aspirin for Diabetes (JPAD) trial was a randomized clinical trial to evaluate the efficacy and safety of low-dose aspirin in patients with type 2 diabetes. As a post hoc analysis, we investigated the incidence of upper gastrointestinal symptoms or bleeding in aspirin (100 mg enteric-coated aspirin or 81 mg buffered aspirin daily) and no-aspirin groups within and beyond 3 years. Of 2535 patients (mean age 65 years, 55% male) followed for a median of 11.2 years, 1258 were included in the aspirin group (951 enteric-coated, 208 buffered, 99 unknown) and 1277 were included in the no-aspirin group. The cumulative incidence of upper gastrointestinal symptoms or bleeding was higher in the aspirin group than the no-aspirin group (8.8% vs. 5.7% at 18 years; p < 0.0001). The increased risk in the aspirin group was prominent within 3 years, and the hazard ratio (HR) [95% confidence interval (CI)] of the aspirin group was 7.10 [3.21–15.7], but attenuated beyond 3 years (HR 1.20 [0.76–1.89]). In 1159 patients in the aspirin group, the cumulative incidence was lower in the enteric-coated than in the buffered aspirin groups (2.9% vs. 7.3%; p = 0.003) within 3 years, and the adjusted HR of enteric-coated aspirin was 0.38 [0.20–0.72] compared with the buffered aspirin group. The upper gastrointestinal symptoms or bleeding of low-dose aspirin within 3 years, and the aspirin formulations, were relevant for decision making of initiation and continuation of low-dose aspirin for primary prevention.

The ionosphere is the Earth’s upper atmosphere with sufficiently charged particles that influence the electromagnetic wave propagation, thereby impeding spaceborne synthetic aperture radar (SAR) observations. High precision imaging and the measurements of geographic and geophysical processes using interferometric SAR (InSAR) and polarimetric SAR (Pol-SAR) are hampered. This study presents a detailed and comprehensive review of the research on ionospheric effects on SAR, InSAR, and Pol-SAR, their correction approaches, and reverse guidance for ionospheric sounding. The prospect is raised at the end of the paper, and the ionosphere will continue to be a research hotspot in the remote sensing community due to the development of low-frequency SAR satellites. In addition, applying spaceborne SAR to ionospheric sounding is a promising issue for space weather and geospace physics due to high spatiotemporal resolution and abundant parameter information.

Yogurt is a widely consumed dairy product that is obtained through the fermentation of lactic acid bacteria. During fermentation, these bacteria produce lactic acid, which lowers the pH and causes the coagulation of milk proteins. Metabolites of lactic acid bacteria, such as carbonyl compounds, nonvolatile or volatile acids, and exopolysaccharides, significantly affect the quality of yogurt. The production method also greatly influences yogurt characteristics. Yogurt can be produced in various forms, including solid, fluid, and soft-drinking types, depending on the manufacturing method employed. In addition, the raw materials, sterilization, homogenization, fermentation, and other conditions in each manufacturing method greatly affect yogurt’s physical properties and flavor. This chapter summarizes common and modern methods of yogurt production.

Background: Reproducibility has been well-studied in the field of food analysis; the relative standard deviation is said to follow the Horwitz curve with certain exceptions. However, little systematic research has been done on predicting repeatability or intermediate precision. Objective: We developed a regression method to estimate within-laboratory standard deviations using hierarchical Bayesian modeling and analyzing duplicate measurement data obtained from actual laboratory tests. Methods: The Hamiltonian Monte Carlo method was employed and implemented using R with Stan. The basic structure of the statistical model was assumed to be a chi-squared distribution, the fixed effect of the predictor was assumed to be a nonlinear function with a constant term and a concentration-dependent term, and the random effects were assumed to follow a lognormal distribution as a hierarchical prior. Results: By analyzing over 300 instances, we obtained regression results that fit well with the assumed model, except for moisture, which was a method-defined analyte. The developed method applies to a wide variety of analytes measured using general principles, including spectroscopy, GC, and HPLC. Although the estimated precisions were within the HorRat(r) criteria, some cases using high-sensitivity detectors, such as mass spectrometers, showed standard deviations below that range. Conclusion: We propose utilizing the within-laboratory precision predicted by the model established in this study for internal quality control and measurement uncertainty estimation without considering the sample matrices. Highlights: Performing statistical modeling on data from double analysis, which is conducted as a part of internal quality controls, will simplify the estimation of the precision that fits each analytical system in a laboratory.