Measuring Dissolved Oxygen in Miso: Implications for Forensic Science and Semisolid Food Analysis

Measuring Dissolved Oxygen in Miso: Implications for Forensic Science and Semisolid Food Analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Measuring Dissolved Oxygen in Miso: Implications for Forensic Science and Semisolid Food Analysis Yoshiro Koda, Mikiko Soejima This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6581404/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract In a retrial, the prosecutor sought a scientific opinion from one of the authors on whether blood stains on cotton clothes placed in miso , a traditional Japanese fermented food, for 1 year and 2 months, would remain reddish, and asked him to testify. As a first step, we searched the literature for information on the levels of dissolved oxygen (DO) in miso that might affect the discoloration of blood stains, but found no relevant information. Then, we investigated whether the DO concentration in miso could be measured using an optical oxygen sensor by analyzing freshly prepared and commercially available ripened miso . The DO concentration in miso decreased to below the detection limit (0.002%) within 9.5 to 23.2 h after preparation and remained low, even after the miso ripened. Thus, the low DO concentration may have affected the redness of the bloodstain. The amount of oxygen in foods must be controlled because it affects essential factors, such as taste, nutritional value, color, and aroma; our results suggest that an optical oxygen sensor could serve as a useful tool for measuring DO concentration in semisolid fermented foods, such as miso . Biological sciences/Biochemistry/Biophysical chemistry Biological sciences/Biotechnology/Assay systems Biological sciences/Microbiology/Fungi/Fungal biology miso dissolved oxygen optical oxygen sensor blood stains Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The Hakamata case involved the murder of four family members of a managing director who worked at a miso processing factory in Shizuoka Prefecture in 1966, along with theft and arson. A factory employee was arrested and denied the charges, but his death penalty conviction was finalized in 1980. After two requests for retrial, the Shizuoka District Court decided to start the retrial in 2014, suspending his execution and detention. Although the Tokyo High Court overturned this decision in 2018, the Supreme Court reinstated it in 2020, leading to further hearings. In March 2023, the Tokyo High Court supported the Shizuoka District Court's retrial decision, and a retrial began in 2024. On September 26, 2024, the defendant was declared not guilty, with the prosecution choosing not to appeal. Fourteen months after the incident, five blood-stained cotton clothes were found in a tank over a meter deep, with approximately eight tons of miso (fermented soybean paste). Testimonies and photographs taken at the time indicated that the blood stains were reddish. At the initial trial 45 years ago, the redness of the blood stains was not considered, and the clothing items were identified as the ones the defendant had been wearing at the time of the crime. As a result, the defendant was convicted. However, prior to the trial at Shizuoka District Court in 2014, the defense argued that blood stains would turn dark brown in a short period and would no longer remain red if placed in miso for over one year. Finally, in 2023, the Tokyo High Court decided to retry the case because it was impossible for the blood stains to have remained red. Thus, the prosecutor asked one of the authors to prepare a scientific opinion on this issue and testify at the retrial, which began in 2024. Since it is challenging to determine whether the redness of a bloodstain remains when placed in miso for > 1 year, we considered the formation of methemoglobin, which is a result of hemoglobin oxidation, and the subsequent degradation of hemoglobin to be the key events in the discoloration of blood stains 1 – 3 . Therefore, as a first step in our evaluation, we searched the literature on dissolved oxygen (DO) concentration in miso , but did not find relevant information. However, we found measurements of DO concentrations in other Japanese fermented foods, such as shoyu (soy sauce) and sake (Japanese rice wine) 4 – 6 . Koji is a fermented product prepared by growing Aspergillus species in soybeans and/or grains, such as rice and barley 7 , 8 . Miso is a traditional Japanese food made by mixing soybean pastes with rice koji , barley koji , soybean koji , and salt. It is then ripened for several months to over a year 7 , 8 . Aspergillus oryzae is most commonly used as a starter culture to prepare koji for making miso , and lactic acid bacteria and salt-tolerant yeasts, mainly Zygosaccharomyces rouxii , play important roles in miso maturation 7 , 8 . The koji mold in miso dies within a short time after preparation due to the high salt concentration (approximately ≥ 10%); however, enzymes present in koji , such as proteases, aminopeptidases, carboxypeptidases, L-glutaminases, amylases, and cellulases, remain functional over a long period 7 , 8 . These enzymes digest the starch and proteins found in grains and soybeans 7 , 8 . On the other hand, sake is fermented using rice koji made by growing A. oryzae ; however, the type of yeast ( Saccharomyces cerevisiae ) used for fermentation is different from that used to make miso and shoyu 9 . Shoyu is produced by growing Aspergillus sojae or A. oryzae using barley koji exclusively; however, the lactic acid bacteria and yeasts are almost the same as those used in miso 10 . Previous studies indicated that the DO concentration in the supernatant of shoyu moromi , a soft solid formed during the fermentation process of multiple ingredients in the production of sake , shoyu , miso , and other fermented products, and a water-diluted sake moromi dropped below the detection limit of the devices within a few days 4 – 6 . The detection limit was 0.1% O 2 for the supernatant of shoyu moromi and 1 ppb for water-diluted sake moromi. However, in such studies, the DO concentrations in shoyu and sake were measured during brewing using the diaphragm electrode method 4 – 6 , which is suitable for stirrable liquids, but not se miso lid pastes such as miso 11 . One report on the measurement of the redox potential of miso 12 revealed that the redox potential values were affected by DO. Other reports have indicated that the redox potential of miso was close to those of shoyu and sake 13 , 14 . The redox potential of miso is merely the sum of those of various redox substances, and the redox state of hemoglobin in miso is affected by the type and amount of these reducing substances, as well as the DO concentration in the miso . In any case, this value alone does not necessarily allow us to predict the redox state of hemoglobin. Conversely, if the DO in miso is low, oxygenation of hemoglobin is reduced, decreasing the likelihood of hemoglobin auto-oxidation and the subsequent formation of methemoglobin. Taken together, the findings of these studies indicate that, although measured indirectly, the DO concentration of miso is estimated to be extremely low, similar to those of shoyu and sake during brewing. Seven forensic scientists, including one of the authors, prepared an expert report stating this fact before the retrial began, and one of the authors testified as such at the retrial. Alternatively, the DO concentration can be measured using optical oxygen sensors, which have been relatively recently developed and mainly based on the principle of fluorescence quenching 15 . This method has been used to measure the DO concentrations in the liquid and gas phases of wine, orange juice, and processed foods, as well as strawberry puree and mayonnaise, which are semisolid pastes 16 – 22 . Therefore, in the present study, we aimed to determine whether the DO concentration in miso can be measured using an optical oxygen sensor immediately after its preparation and in already-ripened miso . 2. Results 2.1 DO concentration in handmade miso First, the DO concentration was measured at a depth of 5 cm in each of the five laboratory-made miso samples (430 g) immediately after preparation. As shown in Fig. 1 , the DO concentration at the time of preparation ranged from 14.8–17.9% (16.8 ± 1.2%); however, these initial values were outside the measurement range of the sensor (0–5% v/v O 2 ). Values above and below the measurement range are displayed by this sensor; however, these are not considered accurate. The device also indicated that the O 2 concentration in the air was approximately 17.2–17.9%; however, it may have been approximately 20.9% 23 . Furthermore, the O 2 concentration (or DO concentration) in 450 mL of distilled water placed in a 500-mL beaker and left in the atmosphere was almost the same as that of the air, with a DO concentration of approximately 8 ppm. In any case, the actual DO concentration in miso at the time of preparation is considered to be slightly higher than these indicated values. Thereafter, the DO concentration gradually decreased and fell below the detection limit (at 0.002% O 2 ) between 9.5 and 23.2 h after preparation (average = 14.8 h, n = 5). As this method measures the partial pressure of oxygen at the tip of the probe, the value of the oxygen partial pressure of liquids placed in the atmosphere will be the same as that of the air. Thus, the detection limit of an oxygen concentration of 0.002% or less indicates that the oxygen partial pressure at that area is 0.015 mmHg or less. Therefore, this optical sensor can accurately measure DO concentrations in miso in the range of 0.002–5% O 2 . The DO concentration in miso appeared to decrease to < 0.002% within one day of preparation; however, it is unclear how low it actually was, that is, how close it was to zero. In addition, because the DO concentration decreases at the same oxygen partial pressure as salinity increases, the salinity of the homemade miso (12%) was incorporated into the software to correct the DO concentration values. At a salt concentration of 12%, the DO concentration was approximately 36% of the value without salt. After correcting for salinity, the DO concentration was calculated to be less than 0.4 ppb, which was below the detection limit. Miso also contains relatively high concentrations (15–20%) of sugars, including glucose 24 . Sugar, as well as salt, reduces the DO concentration 25 . Because the software used in this study does not allow for adjustments based on the sugar concentration, the actual DO concentration (ppb) is expected to be even lower than the displayed value. Additionally, O 2 %, which is proportional to the oxygen partial pressure, is not affected by the salinity or sugar concentration. Therefore, in this experiment, the DO concentration was expressed in O 2 %, rather than in ppb. The DO concentration remained low for 2 to 36 h after falling below the detection limit. When the probe was removed and reinserted in a different location for measurement (from 1 to 19 days), it took 46 to 129 min (average = 76.6 min, n = 5) for the DO concentration to fall below the detection limit. Hence, the DO concentration in the miso was still considered to be below the detection limit. Next, the miso was fermented in a plastic container at room temperature (controlled at 25 ± 1°C) for approximately 1 month, then transferred to a glass beaker and fermented for 2 more months (a total of 3 months), after which the DO concentration in the same miso was measured thrice at 3-min intervals. The results showed that, even for the same miso , the time required for the DO concentration to reach the detection limit varied between experiments, ranging from 24 to 132 min (average 81.0 min, n = 3) (Fig. 2 ). Nonetheless, it was concluded that the DO concentration in miso could be measured using the probe, and the value was considered extremely low. 2.2 DO concentration in commercially available miso We measured the DO concentrations in five commercially available miso packages (Fig. 3 ). Miso A and B, made from rice koji, along with miso E, made from a combination of rice and barley koji, were raw miso containing live yeast. In contrast, miso C and D, made from rice koji, contained inactivated yeast. The DO concentration of each type of miso was measured twice. During the first measurement, we left them at room temperature until we opened the package, and all samples, except for Miso D, were packed with an oxygen absorber (a small package containing particles primarily made of iron used to prevent the degradation of miso caused by oxidation resulting from oxygen being trapped in the gaps of the container). During the second measurement, the miso packages were stored in a refrigerator for 3 to 6 days and then returned to room temperature before the measurement. In the first round of measurements, it took 3.7 to 7.1 h for the DO concentration to fall below the detection limit, whereas during the second round, the DO concentrations, except for that of Miso E, took longer to fall below the detection limit (Table 1 ). Although it is unknown when these commercially available misos were made, we found that the DO concentration was extremely low, even in already-ripened miso . Furthermore, it was below the detection limit in raw miso containing live yeast (Fig. 3 A, B, E) and in those heated (or treated) with ethanol to inactivate the yeast (Fig. 3 C, D). This observation suggested that the oxygen consumed around the probe was not solely due to yeast respiration. In addition, the DO concentration in miso was below the detection limit, regardless of oxygen absorber use or koji type. Table 1 Time for DO concentration to fall below the detection limit. Time for DO concentration (%) to fall below the detection limit (hours) 1st measurement 2nd measurement Miso A 4.1 12.4 Miso B 3.7 12.1 Miso C 5.7 8.6 Miso D 7.1 8.3 Miso E 3.7 3.5 Note: Miso A–D were rice koji miso , and miso E was a rice and barley koji miso . Miso A, B, and E were raw miso with live yeast, while miso C and D contained inactivated yeast. Only miso D was packed without an oxygen absorber. 3. Discussion In this study, we measured the DO concentration in miso using an optical oxygen sensor to estimate whether the redness of bloodstains in miso would be maintained for more than a year. Our results confirmed that the DO concentration in miso fell below the detection limit of the device immediately after the miso was prepared. Previous reports have suggested that the DO concentration in miso reaches nearly zero within 20 to 30 days, aligning with the redox potential stabilization timeline 12 . The redox potential change in miso is greatly affected by DO concentration as well as the total amount of redox substances, such as amino acids. Hence, it is difficult to accurately measure the degree of decrease in DO concentration from the redox potential of miso . Notably, the findings of this study, which used an optical oxygen sensor, revealed that the DO concentration in miso reached ≤ 0.002% in less than 1 day, which is much shorter than previous estimates. In addition, the DO concentration at 5-cm depth in a 430-g sample may differ from that measured at a depth of 1 m or more in larger eight-ton tanks, where the blood-stained clothes were found. Although potentially lower in the latter situation, the DO concentration could not be verified as it was beyond the detection limit of the currently available oxygen sensors. Conversely, during shoyu brewing, the DO concentration at a depth of 40 cm was estimated to be nearly zero, whereas at a depth of 10 cm, the oxygen absorption rate was 8% of that at the surface 26 . This is consistent with the fact that the amount of yeast per unit volume is much higher in shoyu moromi than in miso 27 . The DO concentration in sake is reportedly higher when brewed on a small scale than when brewed on a large scale 28 . This phenomenon is attributable to the differences in the viscosities of miso (130,000 mPa·s), a semisolid substance, and sake and shoyu (< 10 mPa·s), which are both liquids ( https://tokisangyo.co.jp/wp-content/uploads/2022/01/syokuhin-data.pdf ). Higher viscosity leads to minimal diffusion of oxygen (from the air on the surface) inside miso compared with that in soy sauce or sake. Therefore, the DO concentration in miso is typically lower than those in shoyu and sake moromi , especially near the surface, where the substance is in contact with air. By the time of the retrial, we had not yet measured the DO concentration in miso with an optical oxygen sensor; therefore, we could not conclusively testify that it was extremely low based on our findings. Based on previous findings that showed that yeast did not grow anywhere in miso except on the surface 29 , 30 , one of the authors testified that the DO concentration in miso , especially near the bottom of large-scale tanks, may be extremely low, potentially close to zero. This opinion was rejected by the judges, who ruled that there was no scientific basis for extrapolating data obtained from studies involving shoyu to a semisolid like miso to prove that the DO concentration was that low. Meanwhile, the prosecutors had tested multiple samples by preparing blood stains on cotton clothes and placing them in miso in plastic containers. Some of these containers contained oxygen absorbers, and some of these appeared to retain a reddish tinge after several months, regardless of the use of oxygen absorbers. Before the appeal trial at the Tokyo High Court in 2023, a defense witness submitted an expert report stating that the prosecutor’s experiments showed that the oxygen concentration in the miso was reduced to approximately 0.1% (absolute value of 0.8 mm Hg) of the atmospheric oxygen by the oxygen absorber, which caused the blood stains to remain red for several months. This witness was later called to appear at the Tokyo High Court of Appeals in 2023 on remand to testify. He said before the court that the oxygen concentration in sake is around 0.1% during brewing, but it is not that low in miso , and this opinion was adopted. The Tokyo High Court finally ordered the case to be retried at the Shizuoka District Court. Before the retrial began, the prosecutor asked the defense witness “What does it mean that the oxygen concentration in sake is around 0.1% during brewing?” to which the witness replied in writing, “It is a relative concentration in the air, not an absolute concentration, which is 0.1% of the atmosphere, 0.02% in absolute concentration, which translates to approximately 5 ppb in DO concentration.” The defense witness, however, stated that the possibility of the DO concentration in sake during brewing being < 5 ppb could not necessarily be ruled out. Furthermore, when asked to provide evidence in support of the above statement by the prosecutor, the defense witness responded that they were referring to the reference in the expert report that the prosecution witnesses (including one of the authors) presented prior to the retrial 5 . However, the reference did not mention that the DO concentration in sake during brewing was approximately 5 ppb. Instead, it stated that it was below the detection limit of the instrument (1 ppb), and the tables in the reference indicated “<2 ppb” 5 . The court concluded, based on defense testimony, that even with a DO concentration as low as 5 ppb, oxygen persists in miso post-preparation, causing rapid heme oxidation during hemoglobin decomposition. The Shizuoka District Court in 2024 as well as the Tokyo High Court judges in 2023 concluded that the blood stains on cotton rags did not retain any redness after being placed in miso for > 1 year in tune with the experiment conducted by the aforementioned prosecutor. Thus, although redness is subjective, and objectively determining whether blood stains retain redness over a given period is difficult, the Shizuoka District Court judges concluded that if the five pieces of clothing that the defendant was allegedly wearing at the time of the crime were placed in miso for 1 year and 2 months, the bloodstains would not retain any redness. Subsequently, they found the defendant not guilty because they determined that the clothes were not worn at the time of the crime and therefore could not have been placed there by the defendant. In other words, it was determined that the evidence was fabricated by investigators. Previously, DO concentrations in shoyu and sake during brewing were measured using the diaphragm electrode method 4 – 6 . However, a direct measure of DO concentration in shoyu moromi was not possible because shoyu moromi is semisolid; hence, the supernatant of moromi was used 6 . In addition, sake moromi is not sufficiently liquefied until 5 days after brewing; therefore, a water-diluted moromi (dilution ratio of 2.2) was used for measurement 5 . These observations confirm that the diaphragm electrode method does not accurately measure the DO concentration of shoyu and sake immediately after brewing. In addition, the time taken for the DO concentrations in both shoyu and sake immediately after brewing to reach values below the detection limit cannot be exactly determined by this method. Our results showed that an optical oxygen sensor can effectively measure the DO concentration, even in semisolid pastes such as miso , and is superior to the diaphragm electrode method—especially when measuring DO concentration in shoyu and sake moromi immediately after their preparation. Controlling the amount of oxygen in foods is necessary because it affects essential factors, such as taste, nutritional value, color, and aroma. Based on our results, we believe that optical oxygen sensors can serve as effective tools for measuring the DO concentration in several foods, regardless of their viscosity. Given the significant impact of oxygen on food deterioration, employing this method to measure the oxygen concentration in food across different physical states— solid, semisolid, and liquid— will grow increasingly important in the future. To the best of our knowledge, this study is the first to measure the DO concentration in miso . 4. Methods 4.1 Miso preparation Handmade miso was made using a miso kit (yield: 2 kg, consisting of 650 g of raw rice koji and 1.34 kg of mashed soybeans with salt) purchased from Suzuki Koji Store (Shizuoka, Japan). In this study, the raw materials were dispatched by courier; therefore, 3–6 days had already passed (since the koji was completed) by the time the miso was prepared. The composition of the miso in which the clothes were placed was unclear and could not be accurately reproduced using a commercial miso kit. However, we mixed the koji and mashed soybeans with salt in a proportion similar to the one described in the written report. We prepared miso by combining 110 g of raw rice koji and 320 g of mashed soybeans with salt (the final salinity was approximately 12%), mixing them well by hand, and removing the maximum amount of air. This mixture was then placed in a 500-mL glass beaker at room temperature (controlled at 25 ± 1 ℃). The surface of the miso was covered with plastic wrap to prevent it from drying out. The DO concentration was measured immediately and approximately 3 months after preparation ( miso was fermented in a plastic container for approximately 1 month and then transferred to a glass beaker, where it continued to ferment for another 2 months. In addition, five types of commercially available miso (four rice koji miso , Fig. 3 A–D, and one rice and barley koji miso , Fig. 3 E) were purchased from a local supermarket, and their DO values were measured. Two rice koji miso ( miso A: salinity 11.6%, 650 g and miso B: salinity 8.3%, 650 g) and one rice and barley koji miso ( miso E: salinity 8.1%, 500 g) were raw miso containing live yeasts, and the other two rice koji miso ( miso C: salinity 10.3%, 700 g and miso D: salinity 9.4%, 800 g) contained inactivated yeasts. 4.2 Oxygen measurements The oxygen-sensing probe DP-PSt6 (PreSens-Precision Sensing GmbH, Regensburg Germany) with a measurement range of 0–5% v/v O 2 or 0–2 mg dissolved O 2 /L was inserted 5-cm deep into each of the miso samples (Fig. 4 ). According to the manufacturer’s instruction manual, DP-PSt6 consists of a polymer optical fiber, one end of which is coated with an oxygen-sensitive foil. The end of the fiber is covered with a high-grade steel tube to protect both the sensor material and the fiber. The steel tube has an outer diameter of 4 mm and a length of 10 cm. The DO concentration was measured every 3 to 10 min by connecting the probe to the OXY-1 SMA Trace, operated by the PreSens Measurement Studio 2 software (PreSens-Precision Sensing GmbH) 19 – 22 . The OXY-1 SMA Trace device can be used for oxygen monitoring in several different measurement setups, including air, water, and sediment samples, such as soil. Declarations Competing interests The author(s) declare no competing interests. Author Contribution Y.K. contributed to planning and conducting experiments, supervision, data analysis, writing of the original draft, reviewing and editing the manuscript. M.S. contributed to planning and conducting experiments, data analysis, reviewing and editing the manuscript. Y.K. prepared figures 1-4. All authors reviewed the manuscript. Acknowledgement We thank Dr. Hideaki Satou (Department of Medical Biochemistry, Kurume University School of Medicine) and Dr. Kwesi Teye (Kurume University Institute of Cutaneous Cell Biology) for their useful comments and discussions, and Ms. Katherine Ono and Editage (www.editage.com) for their assistance with the English language editing of this manuscript. Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Tsuruga, M., Matsuoka, A., Hachimori, A., Sugawara, Y. & Shikama, K. The molecular mechanism of autoxidation for human oxyhemoglobin. Tilting of the distal histidine causes nonequivalent oxidation in the beta chain. J. Biol. Chem. 273 , 8607–8615 (1998). Yasuda, J. et al. The alpha 1 beta 1 contact of human hemoglobin plays a key role in stabilizing the bound dioxygen. Eur. J. Biochem. 269 , 202–211 (2002). Shikama, K. & Matsuoka, A. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6581404","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":457543360,"identity":"f8344179-ab13-4353-a6b3-e41cfcca7377","order_by":0,"name":"Yoshiro Koda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYDACHgYGCYYKIOMAG4o4G3blcC1nSNbC2IapBTcwOHP44I2f87bJ8R1gS5OubNsmxyCRwPjhBwNfHk4tZ9uSLXu33TaWPMB2TPJs221joBZmyR4GtmKcWs7zmEnwbruduOEAe5tkY9vtxP03EhikgX5JbMCjRfLvnNv1MC31DUBbfuPVcrbHTJq34XaCAchhQC0JQIex4bVF8syxZGuZY7cNZx5mS7ZsOHfbsIHnYZtljwFuv/CdST54803NbXm+422GNxvKbsszsCcfvvGj4hjOEFM4AGMxw8UYgU4yOJaAS4s8LhfX4NQyCkbBKBgFIw4AAESPWGb4KcIYAAAAAElFTkSuQmCC","orcid":"","institution":"Kurume University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yoshiro","middleName":"","lastName":"Koda","suffix":""},{"id":457543361,"identity":"3641a8ba-7200-4368-91d5-1689b15fd42c","order_by":1,"name":"Mikiko Soejima","email":"","orcid":"","institution":"Kurume University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mikiko","middleName":"","lastName":"Soejima","suffix":""}],"badges":[],"createdAt":"2025-05-03 02:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6581404/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6581404/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-17556-3","type":"published","date":"2025-08-28T15:56:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83028005,"identity":"a7ab7955-656d-474e-a2a3-c5a4dc1a27dd","added_by":"auto","created_at":"2025-05-19 08:42:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":66512,"visible":true,"origin":"","legend":"\u003cp\u003eThe DO concentration in \u003cem\u003emiso\u003c/em\u003e immediately after preparation. DO concentration was measured five times by connecting the oxygen dipping probe DP-PSt6 to the OXY-1 SMA Trace device. The probe operated by PreSens Measurement Studio 2 software was inserted inside the \u003cem\u003emiso\u003c/em\u003e (to a depth of approximately 5 cm). DO measurements were taken every 3 min. The results of five experiments are displayed every hour.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6581404/v1/ef0d1c5091d3a57763a0f650.png"},{"id":83028928,"identity":"95452a08-148b-4fc9-885d-853ff151f8f8","added_by":"auto","created_at":"2025-05-19 08:50:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":65459,"visible":true,"origin":"","legend":"\u003cp\u003eThe DO concentration in \u003cem\u003emiso\u003c/em\u003e approximately 3 months after preparation. DO concentration was measured thrice using the oxygen dipping probe DP-PSt6 connected to the OXY-1 SMA Trace. The probe, operated by PreSens Measurement Studio 2 software, was inserted inside the \u003cem\u003emiso\u003c/em\u003e (at a depth of approximately 5 cm). DO concentration measurements were taken every 3 min, and the results of three experiments are displayed.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6581404/v1/92d7e5cfc1d86325f885d2fd.png"},{"id":83028013,"identity":"51c5c08a-59ad-4e79-9195-5228167f67ae","added_by":"auto","created_at":"2025-05-19 08:42:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":247102,"visible":true,"origin":"","legend":"\u003cp\u003eThe DO concentration evaluation in five commercially available \u003cem\u003emiso \u003c/em\u003esamples. \u003cem\u003eMiso\u003c/em\u003e A–D were rice \u003cem\u003ekoji\u003c/em\u003e \u003cem\u003emiso\u003c/em\u003e, and \u003cem\u003emiso\u003c/em\u003e E was a rice and barley \u003cem\u003ekoji\u003c/em\u003e \u003cem\u003emiso\u003c/em\u003e. \u003cem\u003eMiso\u003c/em\u003e A (salinity 11.6%, 650 g), \u003cem\u003emiso\u003c/em\u003e B (salinity 8.3%, 650 g), and \u003cem\u003emiso\u003c/em\u003e E (salinity 8.1%, 500 g) were raw \u003cem\u003emiso\u003c/em\u003e with live yeast, and \u003cem\u003emiso\u003c/em\u003e C (salinity 10.3%, 700 g) and D (salinity 9.4%, 800 g) were \u003cem\u003emiso\u003c/em\u003econtaining inactivated yeast.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6581404/v1/dc01e4363888543c480bccc9.png"},{"id":83028929,"identity":"467af58b-019f-48cb-98f7-5b262e77c858","added_by":"auto","created_at":"2025-05-19 08:50:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":348502,"visible":true,"origin":"","legend":"\u003cp\u003eHandmade \u003cem\u003emiso\u003c/em\u003e and the oxygen dipping probe DP-PSt6, OXY-1 SMA Trace for measuring DO concentration. The probe was inserted into the \u003cem\u003emiso\u003c/em\u003e (at a depth of approximately 5 cm).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6581404/v1/83f6ad944cf294e63ef0a465.png"},{"id":90345593,"identity":"b2c8406a-43e1-4c8b-afdb-108c44dc9bf1","added_by":"auto","created_at":"2025-09-01 16:10:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1391150,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6581404/v1/d1d9baa6-b9e3-4a10-8d1e-252fbb6fd4a9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Measuring Dissolved Oxygen in Miso: Implications for Forensic Science and Semisolid Food Analysis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe Hakamata case involved the murder of four family members of a managing director who worked at a miso processing factory in Shizuoka Prefecture in 1966, along with theft and arson. A factory employee was arrested and denied the charges, but his death penalty conviction was finalized in 1980. After two requests for retrial, the Shizuoka District Court decided to start the retrial in 2014, suspending his execution and detention. Although the Tokyo High Court overturned this decision in 2018, the Supreme Court reinstated it in 2020, leading to further hearings. In March 2023, the Tokyo High Court supported the Shizuoka District Court's retrial decision, and a retrial began in 2024. On September 26, 2024, the defendant was declared not guilty, with the prosecution choosing not to appeal.\u003c/p\u003e \u003cp\u003eFourteen months after the incident, five blood-stained cotton clothes were found in a tank over a meter deep, with approximately eight tons of \u003cem\u003emiso\u003c/em\u003e (fermented soybean paste). Testimonies and photographs taken at the time indicated that the blood stains were reddish. At the initial trial 45 years ago, the redness of the blood stains was not considered, and the clothing items were identified as the ones the defendant had been wearing at the time of the crime. As a result, the defendant was convicted. However, prior to the trial at Shizuoka District Court in 2014, the defense argued that blood stains would turn dark brown in a short period and would no longer remain red if placed in \u003cem\u003emiso\u003c/em\u003e for over one year. Finally, in 2023, the Tokyo High Court decided to retry the case because it was impossible for the blood stains to have remained red. Thus, the prosecutor asked one of the authors to prepare a scientific opinion on this issue and testify at the retrial, which began in 2024.\u003c/p\u003e \u003cp\u003eSince it is challenging to determine whether the redness of a bloodstain remains when placed in \u003cem\u003emiso\u003c/em\u003e for \u0026gt;\u0026thinsp;1 year, we considered the formation of methemoglobin, which is a result of hemoglobin oxidation, and the subsequent degradation of hemoglobin to be the key events in the discoloration of blood stains\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, as a first step in our evaluation, we searched the literature on dissolved oxygen (DO) concentration in \u003cem\u003emiso\u003c/em\u003e, but did not find relevant information. However, we found measurements of DO concentrations in other Japanese fermented foods, such as \u003cem\u003eshoyu\u003c/em\u003e (soy sauce) and \u003cem\u003esake\u003c/em\u003e (Japanese rice wine)\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eKoji\u003c/em\u003e is a fermented product prepared by growing \u003cem\u003eAspergillus\u003c/em\u003e species in soybeans and/or grains, such as rice and barley\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eMiso\u003c/em\u003e is a traditional Japanese food made by mixing soybean pastes with rice \u003cem\u003ekoji\u003c/em\u003e, barley \u003cem\u003ekoji\u003c/em\u003e, soybean \u003cem\u003ekoji\u003c/em\u003e, and salt. It is then ripened for several months to over a year\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eAspergillus oryzae\u003c/em\u003e is most commonly used as a starter culture to prepare \u003cem\u003ekoji\u003c/em\u003e for making \u003cem\u003emiso\u003c/em\u003e, and lactic acid bacteria and salt-tolerant yeasts, mainly \u003cem\u003eZygosaccharomyces rouxii\u003c/em\u003e, play important roles in \u003cem\u003emiso\u003c/em\u003e maturation\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003ekoji\u003c/em\u003e mold in \u003cem\u003emiso\u003c/em\u003e dies within a short time after preparation due to the high salt concentration (approximately\u0026thinsp;\u0026ge;\u0026thinsp;10%); however, enzymes present in \u003cem\u003ekoji\u003c/em\u003e, such as proteases, aminopeptidases, carboxypeptidases, L-glutaminases, amylases, and cellulases, remain functional over a long period\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These enzymes digest the starch and proteins found in grains and soybeans\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. On the other hand, \u003cem\u003esake\u003c/em\u003e is fermented using rice \u003cem\u003ekoji\u003c/em\u003e made by growing \u003cem\u003eA. oryzae\u003c/em\u003e; however, the type of yeast (\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e) used for fermentation is different from that used to make \u003cem\u003emiso\u003c/em\u003e and \u003cem\u003eshoyu\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eShoyu\u003c/em\u003e is produced by growing \u003cem\u003eAspergillus sojae\u003c/em\u003e or \u003cem\u003eA. oryzae\u003c/em\u003e using barley \u003cem\u003ekoji\u003c/em\u003e exclusively; however, the lactic acid bacteria and yeasts are almost the same as those used in \u003cem\u003emiso\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Previous studies indicated that the DO concentration in the supernatant of \u003cem\u003eshoyu moromi\u003c/em\u003e, a soft solid formed during the fermentation process of multiple ingredients in the production of \u003cem\u003esake\u003c/em\u003e, \u003cem\u003eshoyu\u003c/em\u003e, \u003cem\u003emiso\u003c/em\u003e, and other fermented products, and a water-diluted \u003cem\u003esake moromi\u003c/em\u003e dropped below the detection limit of the devices within a few days\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The detection limit was 0.1% O\u003csub\u003e2\u003c/sub\u003e for the supernatant of \u003cem\u003eshoyu moromi\u003c/em\u003e and 1 ppb for water-diluted \u003cem\u003esake moromi.\u003c/em\u003e However, in such studies, the DO concentrations in \u003cem\u003eshoyu\u003c/em\u003e and \u003cem\u003esake\u003c/em\u003e were measured during brewing using the diaphragm electrode method\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, which is suitable for stirrable liquids, but not se\u003cem\u003emiso\u003c/em\u003elid pastes such as \u003cem\u003emiso\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOne report on the measurement of the redox potential of \u003cem\u003emiso\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e revealed that the redox potential values were affected by DO. Other reports have indicated that the redox potential of \u003cem\u003emiso\u003c/em\u003e was close to those of \u003cem\u003eshoyu\u003c/em\u003e and \u003cem\u003esake\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The redox potential of \u003cem\u003emiso\u003c/em\u003e is merely the sum of those of various redox substances, and the redox state of hemoglobin in \u003cem\u003emiso\u003c/em\u003e is affected by the type and amount of these reducing substances, as well as the DO concentration in the \u003cem\u003emiso\u003c/em\u003e. In any case, this value alone does not necessarily allow us to predict the redox state of hemoglobin. Conversely, if the DO in \u003cem\u003emiso\u003c/em\u003e is low, oxygenation of hemoglobin is reduced, decreasing the likelihood of hemoglobin auto-oxidation and the subsequent formation of methemoglobin. Taken together, the findings of these studies indicate that, although measured indirectly, the DO concentration of \u003cem\u003emiso\u003c/em\u003e is estimated to be extremely low, similar to those of \u003cem\u003eshoyu\u003c/em\u003e and \u003cem\u003esake\u003c/em\u003e during brewing. Seven forensic scientists, including one of the authors, prepared an expert report stating this fact before the retrial began, and one of the authors testified as such at the retrial.\u003c/p\u003e \u003cp\u003eAlternatively, the DO concentration can be measured using optical oxygen sensors, which have been relatively recently developed and mainly based on the principle of fluorescence quenching\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This method has been used to measure the DO concentrations in the liquid and gas phases of wine, orange juice, and processed foods, as well as strawberry puree and mayonnaise, which are semisolid pastes\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20 CR21\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Therefore, in the present study, we aimed to determine whether the DO concentration in \u003cem\u003emiso\u003c/em\u003e can be measured using an optical oxygen sensor immediately after its preparation and in already-ripened \u003cem\u003emiso\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 DO concentration in handmade miso\u003c/h2\u003e \u003cp\u003eFirst, the DO concentration was measured at a depth of 5 cm in each of the five laboratory-made \u003cem\u003emiso\u003c/em\u003e samples (430 g) immediately after preparation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the DO concentration at the time of preparation ranged from 14.8\u0026ndash;17.9% (16.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%); however, these initial values were outside the measurement range of the sensor (0\u0026ndash;5% v/v O\u003csub\u003e2\u003c/sub\u003e). Values above and below the measurement range are displayed by this sensor; however, these are not considered accurate. The device also indicated that the O\u003csub\u003e2\u003c/sub\u003e concentration in the air was approximately 17.2\u0026ndash;17.9%; however, it may have been approximately 20.9%\u003csup\u003e23\u003c/sup\u003e. Furthermore, the O\u003csub\u003e2\u003c/sub\u003e concentration (or DO concentration) in 450 mL of distilled water placed in a 500-mL beaker and left in the atmosphere was almost the same as that of the air, with a DO concentration of approximately 8 ppm. In any case, the actual DO concentration in \u003cem\u003emiso\u003c/em\u003e at the time of preparation is considered to be slightly higher than these indicated values. Thereafter, the DO concentration gradually decreased and fell below the detection limit (at 0.002% O\u003csub\u003e2\u003c/sub\u003e) between 9.5 and 23.2 h after preparation (average\u0026thinsp;=\u0026thinsp;14.8 h, n\u0026thinsp;=\u0026thinsp;5). As this method measures the partial pressure of oxygen at the tip of the probe, the value of the oxygen partial pressure of liquids placed in the atmosphere will be the same as that of the air. Thus, the detection limit of an oxygen concentration of 0.002% or less indicates that the oxygen partial pressure at that area is 0.015 mmHg or less. Therefore, this optical sensor can accurately measure DO concentrations in \u003cem\u003emiso\u003c/em\u003e in the range of 0.002\u0026ndash;5% O\u003csub\u003e2\u003c/sub\u003e. The DO concentration in \u003cem\u003emiso\u003c/em\u003e appeared to decrease to \u0026lt;\u0026thinsp;0.002% within one day of preparation; however, it is unclear how low it actually was, that is, how close it was to zero. In addition, because the DO concentration decreases at the same oxygen partial pressure as salinity increases, the salinity of the homemade \u003cem\u003emiso\u003c/em\u003e (12%) was incorporated into the software to correct the DO concentration values. At a salt concentration of 12%, the DO concentration was approximately 36% of the value without salt. After correcting for salinity, the DO concentration was calculated to be less than 0.4 ppb, which was below the detection limit.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMiso\u003c/em\u003e also contains relatively high concentrations (15\u0026ndash;20%) of sugars, including glucose\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Sugar, as well as salt, reduces the DO concentration\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Because the software used in this study does not allow for adjustments based on the sugar concentration, the actual DO concentration (ppb) is expected to be even lower than the displayed value. Additionally, O\u003csub\u003e2\u003c/sub\u003e%, which is proportional to the oxygen partial pressure, is not affected by the salinity or sugar concentration. Therefore, in this experiment, the DO concentration was expressed in O\u003csub\u003e2\u003c/sub\u003e%, rather than in ppb.\u003c/p\u003e \u003cp\u003eThe DO concentration remained low for 2 to 36 h after falling below the detection limit. When the probe was removed and reinserted in a different location for measurement (from 1 to 19 days), it took 46 to 129 min (average\u0026thinsp;=\u0026thinsp;76.6 min, n\u0026thinsp;=\u0026thinsp;5) for the DO concentration to fall below the detection limit. Hence, the DO concentration in the \u003cem\u003emiso\u003c/em\u003e was still considered to be below the detection limit. Next, the \u003cem\u003emiso\u003c/em\u003e was fermented in a plastic container at room temperature (controlled at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) for approximately 1 month, then transferred to a glass beaker and fermented for 2 more months (a total of 3 months), after which the DO concentration in the same \u003cem\u003emiso\u003c/em\u003e was measured thrice at 3-min intervals. The results showed that, even for the same \u003cem\u003emiso\u003c/em\u003e, the time required for the DO concentration to reach the detection limit varied between experiments, ranging from 24 to 132 min (average 81.0 min, n\u0026thinsp;=\u0026thinsp;3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Nonetheless, it was concluded that the DO concentration in \u003cem\u003emiso\u003c/em\u003e could be measured using the probe, and the value was considered extremely low.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 DO concentration in commercially available miso\u003c/h2\u003e \u003cp\u003eWe measured the DO concentrations in five commercially available \u003cem\u003emiso\u003c/em\u003e packages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Miso A and B, made from rice koji, along with miso E, made from a combination of rice and barley koji, were raw miso containing live yeast. In contrast, miso C and D, made from rice koji, contained inactivated yeast. The DO concentration of each type of \u003cem\u003emiso\u003c/em\u003e was measured twice. During the first measurement, we left them at room temperature until we opened the package, and all samples, except for \u003cem\u003eMiso\u003c/em\u003e D, were packed with an oxygen absorber (a small package containing particles primarily made of iron used to prevent the degradation of \u003cem\u003emiso\u003c/em\u003e caused by oxidation resulting from oxygen being trapped in the gaps of the container). During the second measurement, the \u003cem\u003emiso\u003c/em\u003e packages were stored in a refrigerator for 3 to 6 days and then returned to room temperature before the measurement. In the first round of measurements, it took 3.7 to 7.1 h for the DO concentration to fall below the detection limit, whereas during the second round, the DO concentrations, except for that of \u003cem\u003eMiso\u003c/em\u003e E, took longer to fall below the detection limit (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although it is unknown when these commercially available \u003cem\u003emisos\u003c/em\u003e were made, we found that the DO concentration was extremely low, even in already-ripened \u003cem\u003emiso\u003c/em\u003e. Furthermore, it was below the detection limit in raw \u003cem\u003emiso\u003c/em\u003e containing live yeast (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B, E) and in those heated (or treated) with ethanol to inactivate the yeast (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). This observation suggested that the oxygen consumed around the probe was not solely due to yeast respiration. In addition, the DO concentration in \u003cem\u003emiso\u003c/em\u003e was below the detection limit, regardless of oxygen absorber use or \u003cem\u003ekoji\u003c/em\u003e type.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTime for DO concentration to fall below the detection limit.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTime for DO concentration (%) to fall below the detection limit (hours)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1st measurement\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2nd measurement\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMiso\u003c/em\u003e A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMiso\u003c/em\u003e B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMiso\u003c/em\u003e C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMiso\u003c/em\u003e D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMiso\u003c/em\u003e E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote: \u003cem\u003eMiso\u003c/em\u003e A\u0026ndash;D were rice \u003cem\u003ekoji miso\u003c/em\u003e, and \u003cem\u003emiso\u003c/em\u003e E was a rice and barley \u003cem\u003ekoji miso\u003c/em\u003e. \u003cem\u003eMiso\u003c/em\u003e A, B, and E were raw \u003cem\u003emiso\u003c/em\u003e with live yeast, while \u003cem\u003emiso\u003c/em\u003e C and D contained inactivated yeast. Only \u003cem\u003emiso\u003c/em\u003e D was packed without an oxygen absorber.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eIn this study, we measured the DO concentration in \u003cem\u003emiso\u003c/em\u003e using an optical oxygen sensor to estimate whether the redness of bloodstains in \u003cem\u003emiso\u003c/em\u003e would be maintained for more than a year. Our results confirmed that the DO concentration in \u003cem\u003emiso\u003c/em\u003e fell below the detection limit of the device immediately after the \u003cem\u003emiso\u003c/em\u003e was prepared. Previous reports have suggested that the DO concentration in \u003cem\u003emiso\u003c/em\u003e reaches nearly zero within 20 to 30 days, aligning with the redox potential stabilization timeline\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The redox potential change in \u003cem\u003emiso\u003c/em\u003e is greatly affected by DO concentration as well as the total amount of redox substances, such as amino acids. Hence, it is difficult to accurately measure the degree of decrease in DO concentration from the redox potential of \u003cem\u003emiso\u003c/em\u003e. Notably, the findings of this study, which used an optical oxygen sensor, revealed that the DO concentration in \u003cem\u003emiso\u003c/em\u003e reached\u0026thinsp;\u0026le;\u0026thinsp;0.002% in less than 1 day, which is much shorter than previous estimates. In addition, the DO concentration at 5-cm depth in a 430-g sample may differ from that measured at a depth of 1 m or more in larger eight-ton tanks, where the blood-stained clothes were found. Although potentially lower in the latter situation, the DO concentration could not be verified as it was beyond the detection limit of the currently available oxygen sensors.\u003c/p\u003e \u003cp\u003eConversely, during \u003cem\u003eshoyu\u003c/em\u003e brewing, the DO concentration at a depth of 40 cm was estimated to be nearly zero, whereas at a depth of 10 cm, the oxygen absorption rate was 8% of that at the surface\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. This is consistent with the fact that the amount of yeast per unit volume is much higher in \u003cem\u003eshoyu moromi\u003c/em\u003e than in \u003cem\u003emiso\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The DO concentration in \u003cem\u003esake\u003c/em\u003e is reportedly higher when brewed on a small scale than when brewed on a large scale\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This phenomenon is attributable to the differences in the viscosities of \u003cem\u003emiso\u003c/em\u003e (130,000 mPa\u0026middot;s), a semisolid substance, and \u003cem\u003esake\u003c/em\u003e and \u003cem\u003eshoyu\u003c/em\u003e (\u0026lt;\u0026thinsp;10 mPa\u0026middot;s), which are both liquids (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tokisangyo.co.jp/wp-content/uploads/2022/01/syokuhin-data.pdf\u003c/span\u003e\u003cspan address=\"https://tokisangyo.co.jp/wp-content/uploads/2022/01/syokuhin-data.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Higher viscosity leads to minimal diffusion of oxygen (from the air on the surface) inside \u003cem\u003emiso\u003c/em\u003e compared with that in soy sauce or \u003cem\u003esake.\u003c/em\u003e Therefore, the DO concentration in \u003cem\u003emiso\u003c/em\u003e is typically lower than those in \u003cem\u003eshoyu\u003c/em\u003e and \u003cem\u003esake moromi\u003c/em\u003e, especially near the surface, where the substance is in contact with air.\u003c/p\u003e \u003cp\u003eBy the time of the retrial, we had not yet measured the DO concentration in \u003cem\u003emiso\u003c/em\u003e with an optical oxygen sensor; therefore, we could not conclusively testify that it was extremely low based on our findings. Based on previous findings that showed that yeast did not grow anywhere in \u003cem\u003emiso\u003c/em\u003e except on the surface\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, one of the authors testified that the DO concentration in \u003cem\u003emiso\u003c/em\u003e, especially near the bottom of large-scale tanks, may be extremely low, potentially close to zero. This opinion was rejected by the judges, who ruled that there was no scientific basis for extrapolating data obtained from studies involving \u003cem\u003eshoyu\u003c/em\u003e to a semisolid like \u003cem\u003emiso\u003c/em\u003e to prove that the DO concentration was that low.\u003c/p\u003e \u003cp\u003eMeanwhile, the prosecutors had tested multiple samples by preparing blood stains on cotton clothes and placing them in \u003cem\u003emiso\u003c/em\u003e in plastic containers. Some of these containers contained oxygen absorbers, and some of these appeared to retain a reddish tinge after several months, regardless of the use of oxygen absorbers. Before the appeal trial at the Tokyo High Court in 2023, a defense witness submitted an expert report stating that the prosecutor\u0026rsquo;s experiments showed that the oxygen concentration in the \u003cem\u003emiso\u003c/em\u003e was reduced to approximately 0.1% (absolute value of 0.8 mm Hg) of the atmospheric oxygen by the oxygen absorber, which caused the blood stains to remain red for several months. This witness was later called to appear at the Tokyo High Court of Appeals in 2023 on remand to testify. He said before the court that the oxygen concentration in \u003cem\u003esake\u003c/em\u003e is around 0.1% during brewing, but it is not that low in \u003cem\u003emiso\u003c/em\u003e, and this opinion was adopted. The Tokyo High Court finally ordered the case to be retried at the Shizuoka District Court. Before the retrial began, the prosecutor asked the defense witness \u0026ldquo;What does it mean that the oxygen concentration in \u003cem\u003esake\u003c/em\u003e is around 0.1% during brewing?\u0026rdquo; to which the witness replied in writing, \u0026ldquo;It is a relative concentration in the air, not an absolute concentration, which is 0.1% of the atmosphere, 0.02% in absolute concentration, which translates to approximately 5 ppb in DO concentration.\u0026rdquo; The defense witness, however, stated that the possibility of the DO concentration in \u003cem\u003esake\u003c/em\u003e during brewing being \u0026lt;\u0026thinsp;5 ppb could not necessarily be ruled out. Furthermore, when asked to provide evidence in support of the above statement by the prosecutor, the defense witness responded that they were referring to the reference in the expert report that the prosecution witnesses (including one of the authors) presented prior to the retrial\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, the reference did not mention that the DO concentration in \u003cem\u003esake\u003c/em\u003e during brewing was approximately 5 ppb. Instead, it stated that it was below the detection limit of the instrument (1 ppb), and the tables in the reference indicated \u0026ldquo;\u0026lt;2 ppb\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The court concluded, based on defense testimony, that even with a DO concentration as low as 5 ppb, oxygen persists in miso post-preparation, causing rapid heme oxidation during hemoglobin decomposition. The Shizuoka District Court in 2024 as well as the Tokyo High Court judges in 2023 concluded that the blood stains on cotton rags did not retain any redness after being placed in \u003cem\u003emiso\u003c/em\u003e for \u0026gt;\u0026thinsp;1 year in tune with the experiment conducted by the aforementioned prosecutor. Thus, although redness is subjective, and objectively determining whether blood stains retain redness over a given period is difficult, the Shizuoka District Court judges concluded that if the five pieces of clothing that the defendant was allegedly wearing at the time of the crime were placed in \u003cem\u003emiso\u003c/em\u003e for 1 year and 2 months, the bloodstains would not retain any redness. Subsequently, they found the defendant not guilty because they determined that the clothes were not worn at the time of the crime and therefore could not have been placed there by the defendant. In other words, it was determined that the evidence was fabricated by investigators.\u003c/p\u003e \u003cp\u003ePreviously, DO concentrations in \u003cem\u003eshoyu\u003c/em\u003e and \u003cem\u003esake\u003c/em\u003e during brewing were measured using the diaphragm electrode method\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, a direct measure of DO concentration in \u003cem\u003eshoyu moromi\u003c/em\u003e was not possible because \u003cem\u003eshoyu moromi\u003c/em\u003e is semisolid; hence, the supernatant of \u003cem\u003emoromi\u003c/em\u003e was used\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In addition, \u003cem\u003esake moromi\u003c/em\u003e is not sufficiently liquefied until 5 days after brewing; therefore, a water-diluted \u003cem\u003emoromi\u003c/em\u003e (dilution ratio of 2.2) was used for measurement\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These observations confirm that the diaphragm electrode method does not accurately measure the DO concentration of \u003cem\u003eshoyu\u003c/em\u003e and \u003cem\u003esake\u003c/em\u003e immediately after brewing. In addition, the time taken for the DO concentrations in both \u003cem\u003eshoyu\u003c/em\u003e and \u003cem\u003esake\u003c/em\u003e immediately after brewing to reach values below the detection limit cannot be exactly determined by this method. Our results showed that an optical oxygen sensor can effectively measure the DO concentration, even in semisolid pastes such as \u003cem\u003emiso\u003c/em\u003e, and is superior to the diaphragm electrode method\u0026mdash;especially when measuring DO concentration in \u003cem\u003eshoyu\u003c/em\u003e and \u003cem\u003esake moromi\u003c/em\u003e immediately after their preparation. Controlling the amount of oxygen in foods is necessary because it affects essential factors, such as taste, nutritional value, color, and aroma. Based on our results, we believe that optical oxygen sensors can serve as effective tools for measuring the DO concentration in several foods, regardless of their viscosity. Given the significant impact of oxygen on food deterioration, employing this method to measure the oxygen concentration in food across different physical states\u0026mdash; solid, semisolid, and liquid\u0026mdash; will grow increasingly important in the future. To the best of our knowledge, this study is the first to measure the DO concentration in \u003cem\u003emiso\u003c/em\u003e.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Miso preparation\u003c/h2\u003e \u003cp\u003eHandmade \u003cem\u003emiso\u003c/em\u003e was made using a \u003cem\u003emiso\u003c/em\u003e kit (yield: 2 kg, consisting of 650 g of raw rice \u003cem\u003ekoji\u003c/em\u003e and 1.34 kg of mashed soybeans with salt) purchased from Suzuki \u003cem\u003eKoji\u003c/em\u003e Store (Shizuoka, Japan). In this study, the raw materials were dispatched by courier; therefore, 3\u0026ndash;6 days had already passed (since the \u003cem\u003ekoji\u003c/em\u003e was completed) by the time the \u003cem\u003emiso\u003c/em\u003e was prepared. The composition of the \u003cem\u003emiso\u003c/em\u003e in which the clothes were placed was unclear and could not be accurately reproduced using a commercial \u003cem\u003emiso\u003c/em\u003e kit. However, we mixed the \u003cem\u003ekoji\u003c/em\u003e and mashed soybeans with salt in a proportion similar to the one described in the written report. We prepared \u003cem\u003emiso\u003c/em\u003e by combining 110 g of raw rice \u003cem\u003ekoji\u003c/em\u003e and 320 g of mashed soybeans with salt (the final salinity was approximately 12%), mixing them well by hand, and removing the maximum amount of air. This mixture was then placed in a 500-mL glass beaker at room temperature (controlled at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃). The surface of the \u003cem\u003emiso\u003c/em\u003e was covered with plastic wrap to prevent it from drying out. The DO concentration was measured immediately and approximately 3 months after preparation (\u003cem\u003emiso\u003c/em\u003e was fermented in a plastic container for approximately 1 month and then transferred to a glass beaker, where it continued to ferment for another 2 months.\u003c/p\u003e \u003cp\u003eIn addition, five types of commercially available \u003cem\u003emiso\u003c/em\u003e (four rice \u003cem\u003ekoji miso\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;D, and one rice and barley \u003cem\u003ekoji miso\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) were purchased from a local supermarket, and their DO values were measured. Two rice \u003cem\u003ekoji miso\u003c/em\u003e (\u003cem\u003emiso\u003c/em\u003e A: salinity 11.6%, 650 g and \u003cem\u003emiso\u003c/em\u003e B: salinity 8.3%, 650 g) and one rice and barley \u003cem\u003ekoji miso\u003c/em\u003e (\u003cem\u003emiso\u003c/em\u003e E: salinity 8.1%, 500 g) were raw \u003cem\u003emiso\u003c/em\u003e containing live yeasts, and the other two rice \u003cem\u003ekoji miso\u003c/em\u003e (\u003cem\u003emiso\u003c/em\u003e C: salinity 10.3%, 700 g and \u003cem\u003emiso\u003c/em\u003e D: salinity 9.4%, 800 g) contained inactivated yeasts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Oxygen measurements\u003c/h2\u003e \u003cp\u003eThe oxygen-sensing probe DP-PSt6 (PreSens-Precision Sensing GmbH, Regensburg Germany) with a measurement range of 0\u0026ndash;5% v/v O\u003csub\u003e2\u003c/sub\u003e or 0\u0026ndash;2 mg dissolved O\u003csub\u003e2\u003c/sub\u003e/L was inserted 5-cm deep into each of the \u003cem\u003emiso\u003c/em\u003e samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). According to the manufacturer\u0026rsquo;s instruction manual, DP-PSt6 consists of a polymer optical fiber, one end of which is coated with an oxygen-sensitive foil. The end of the fiber is covered with a high-grade steel tube to protect both the sensor material and the fiber. The steel tube has an outer diameter of 4 mm and a length of 10 cm. The DO concentration was measured every 3 to 10 min by connecting the probe to the OXY-1 SMA Trace, operated by the PreSens Measurement Studio 2 software (PreSens-Precision Sensing GmbH)\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The OXY-1 SMA Trace device can be used for oxygen monitoring in several different measurement setups, including air, water, and sediment samples, such as soil.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe author(s) declare no competing interests.\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.K. contributed to planning and conducting experiments, supervision, data analysis, writing of the original draft, reviewing and editing the manuscript. M.S. contributed to planning and conducting experiments, data analysis, reviewing and editing the manuscript. Y.K. prepared figures 1-4. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Dr. Hideaki Satou (Department of Medical Biochemistry, Kurume University School of Medicine) and Dr. Kwesi Teye (Kurume University Institute of Cutaneous Cell Biology) for their useful comments and discussions, and Ms. Katherine Ono and Editage (www.editage.com) for their assistance with the English language editing of this manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTsuruga, M., Matsuoka, A., Hachimori, A., Sugawara, Y. \u0026amp; Shikama, K. The molecular mechanism of autoxidation for human oxyhemoglobin. Tilting of the distal histidine causes nonequivalent oxidation in the beta chain. \u003cem\u003eJ. Biol. 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Jpn\u003c/em\u003e. \u003cb\u003e70\u003c/b\u003e, 893\u0026ndash;898 (1975).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"miso, dissolved oxygen, optical oxygen sensor, blood stains","lastPublishedDoi":"10.21203/rs.3.rs-6581404/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6581404/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn a retrial, the prosecutor sought a scientific opinion from one of the authors on whether blood stains on cotton clothes placed in \u003cem\u003emiso\u003c/em\u003e, a traditional Japanese fermented food, for 1 year and 2 months, would remain reddish, and asked him to testify. As a first step, we searched the literature for information on the levels of dissolved oxygen (DO) in miso that might affect the discoloration of blood stains, but found no relevant information. Then, we investigated whether the DO concentration in \u003cem\u003emiso\u003c/em\u003e could be measured using an optical oxygen sensor by analyzing freshly prepared and commercially available ripened \u003cem\u003emiso\u003c/em\u003e. The DO concentration in \u003cem\u003emiso\u003c/em\u003e decreased to below the detection limit (0.002%) within 9.5 to 23.2 h after preparation and remained low, even after the \u003cem\u003emiso\u003c/em\u003e ripened. Thus, the low DO concentration may have affected the redness of the bloodstain. The amount of oxygen in foods must be controlled because it affects essential factors, such as taste, nutritional value, color, and aroma; our results suggest that an optical oxygen sensor could serve as a useful tool for measuring DO concentration in semisolid fermented foods, such as \u003cem\u003emiso\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Measuring Dissolved Oxygen in Miso: Implications for Forensic Science and Semisolid Food Analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-19 08:42:33","doi":"10.21203/rs.3.rs-6581404/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-21T11:15:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-14T08:53:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"239492068564557713057332478827851334959","date":"2025-07-07T09:14:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-16T08:05:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165159151363818011026097537100062004915","date":"2025-05-15T18:16:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-15T17:17:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-15T17:08:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-15T16:56:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-14T12:18:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-03T02:20:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bc15a157-810e-4f0e-8b28-222f9e53fcc2","owner":[],"postedDate":"May 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48626957,"name":"Biological sciences/Biochemistry/Biophysical chemistry"},{"id":48626958,"name":"Biological sciences/Biotechnology/Assay systems"},{"id":48626959,"name":"Biological sciences/Microbiology/Fungi/Fungal biology"}],"tags":[],"updatedAt":"2025-09-01T16:09:14+00:00","versionOfRecord":{"articleIdentity":"rs-6581404","link":"https://doi.org/10.1038/s41598-025-17556-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-28 15:56:54","publishedOnDateReadable":"August 28th, 2025"},"versionCreatedAt":"2025-05-19 08:42:33","video":"","vorDoi":"10.1038/s41598-025-17556-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-17556-3","workflowStages":[]},"version":"v1","identity":"rs-6581404","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6581404","identity":"rs-6581404","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}