"Defense Technology Journal （防衛技術ジャーナル

8 月号）"

1 August 2021

Advanced Study for Defense Medicine

Bioshell Calcium Oxides (BiSCaO) Water used by
National Defense Medical College to protect healthpersonnel against COVID-19 infection.
Professor emeritus, National Defense Medical College

Masayuki ISHIHARA

Chief Scientific Officer, Plus Lab Co., Ltd.
NPO corporation, NBCR Countermeasure Promotion Mechanism

Shinichi SAWADA

Chief Executive Officer, Plus Lab Co., Ltd.

Summary

1.

All health care workers from National Defense Medical College and its hospital who were dispatched
to attend to COVID-19 patients on the Diamond Princess cruise ship and at the Osaka Corona
Intensive Care Center were well protected from COVID-19 infections with the help of BiSCaO Water.

2.

BiSCaO Water is produced from scallop shell and is the first in the world to combine strong
sterilization abilities and high levels of safety for humans. BiSCaO is a designated food additive.

3.

BiSCaO Water has stronger microbicidal and virucidal activities than ethanol andhypochlorous acid (HOCI).

4.

BiSCaO Water can be used by humans for hand sterilization and mouth cleaning without
apparent harmful side effects. BiSCaO Water is kind to hands and skin.

5.

This article focuses on BiSCaO Water’s sterilization ability and safety for infectious
cleaning/disinfectant, and to compare with ethanol and HCIO on effectiveness and safety to theenvironment and on humans.

1１．Introduction
A division of Biomedical engineering in the Research Institute of National Defense Medical College
has been undertaking research and development on a safe and highly versatile protection/
decontamination agents against CBRN (C: hazardous chemicals, B: pathogenic microorganisms,N/R:
nuclear/radioactive materials) for more than ten years. The agents researched include metal
nanoparticles s uch as silver, copper, zinc oxide, and titanium oxide; weakly acidic hypochlorous acid
(HOCI) solution,alcohol, and heated bioshell calcium oxide (BiSCaO®).

ear

ateria s ra ia on ontro

a arn iron enta sa et

Biosa ete i a ontro C

Figure 1. Protection/decontamination agents against CBRN studied in division of Biomedical
engineering in Research Institute of National Defense Medical College.

In this current pandemic of coronavirus disease (SARS-CoV-2 which is now called COVID-19),
it is evident that some antiseptics/disinfectants, such as ethanol and sodium hypochlorite(NaClO), show significant activity with broad microbicidal and antiviral activities; notably, activity against
COVID-19 resulting from disruption of the viral envelope. However, ethanol is flammable and can causerough hands and rashes. Furthermore, ethanol degrades various chemical fibers, plastic, and non-woven
fabrics. Hypochlorous acid (HOCI) solution (pH 6, 200 ppm) is much more superior to sodium hypochlorite(NaClO) solution (pH = 10) in terms of higher microbicidal activity against a broad range of2

microorganisms. However, HOCl is less stable to various environmental factors compared to NaClO1,2).
Notably, the presence of various organic compounds and in-organic ions easily resultsin rapid deterioration of HOCl. Therefore, low concentration of HOCl (> 50 ppm) under organic
matter contaminated environments, high temperature (> 25 ℃), and/or sun shine including UVlight rapidly diminishes the microbicidal and antiviral activities of HOCI. Usage of higher concentrations of
HOCl (> 100 ppm) are restricted as they cause the generation of hazardous substances including
chloramines, trihalomethane, and chlorine (Cl2); and de-colorize and corrodes metal by their oxidizing
action. Therefore, disinfectants that can decrease the bacterial and viral bio-burden without
harmful side effects and environmental disruption are essential for environmental hygiene andpublic health.
Calcium oxide (CaO; quicklime) and calcium hydroxide (Ca (OH)2; slaked lime) produced
from limestone is readily available and is an important in-organic compound used in various
industries, for example, as an absorbent, toxic-waste remediation agent, and an alkalization agent.
However, calcined CaO and Ca(OH)2 with fossil fuel contain harmful impurities such as particulatematters, and quicklime has a dangerously high heat of hydration 3,4）For that reason, direct use to foods
and living body is prohibited.
On the other hand, heated scallop or egg shell powder is a readily available source of CaO
and Ca(OH)2 which are used as a food additive and environmental hygiene materials. Most scallop
shells are considered to be industrial waste, and the shells accumulate on the shores of scallop harvesting
districts in Japan, causing serious environmental problems such as offensive odors and soil pollution due
to harmful materials such as shellfish poisons and heavy metals leaching from internal organs of discarded
scallop shells5).
The main component of the most commercially available heated shell powder products asfood additives is calcium hydroxide (Ca(OH)2) produced by hydration of CaO, and the effect of
Ca(OH2)-based products are lower than other CaO-based products. Bioshell calcium oxide (BiSCaO®)
is now commercially available from Plus Lab Co. Ltd., Kanagawa, Japan. Plus Lab performed
research and development for BiSCaO® for more than 3 years. According to the productinstr tions, BiSCaO® ontains o er 99.6% CaO an 6 μof average diameter, which is an
6,7

unprecedented product as cleaning agent /disinfectants ).
Moreover, BiSCaO Water is a highly concentrated Ca2+ ion solution, is colorless andtransparent with pH >12.7. BiSCaO Water is first commercialized for minimizing the spread of
infectious diseases following the outbreak of COVID-19 in 2020. This article aimed to
introduce BiSCaO ®and BiSCaO Water as a cleaning agent /disinfectant, which we studied and3

developed as a subject for advanced study by Defense Medicine in Ministry of Defense during
2017~2019, and to compare it to ethanol and HClO on effectiveness and safety to the environment andthe living body.

2．BiSCaO® and BiSCaO Water as useful and safe
cleaning agent/disinfectant
２－１．Preparation and characterization of BiSCaO® and BiSCaO Water.
We are studying Biomaterials including suspensions based on BiSCaO® as shown in Figure 2 inDivision of Biomedical Engineering of Research Institute in National Defense Medical College.
BiSCaO® itself is poorly water-soluble under strongly alkaline conditions. Consequently,
BiSCaO® suspensions containing a high concentration of BiSCaO® tend to plug spray nozzles
due to precipitation. We previously reported that production and characterization of BiSCaO®
dispersion, colloidal dispersion, and BiSCaO Water to prevent the inconvenience 6,7).
BiSCaO Water showed higher deodorization and microbicidal activities than BiSCaO® suspension6,7).
BiSCaO® is authorized to produce as a food additive for food preservation8) and calcium enhancer as wellas measures of infectious disease protection and decontamination including COVID-19 by
their cleaning/disinfectant and deodorizing properties. Furthermore, we confirmed that BiSCaO®
is able to remove coagulation sediment and to decompose and detoxify hazardous chemicals
including dioxin such as PCB and water-soluble organophosphorus pesticides.

On the other hand, although in general it is said to be difficult to produce a highly
concentrated Ca2+ ion solution under strong alkaline condition, Plus Lab successfully produced a novel
BiSCaO Water, prepared by repeatedly (more than 50 times) adding 10 wt％ BiSCaO® Powder tochilled clean water and gently decanting the supernatant into a separate container 9,10). BiSCaO
Water is a highly concentrated Ca2+ ion solution, and is colorless and transparent with pH >12.7.
BiSCaO Water is commercialized for minimizing the spread of infectious diseases during the
COVID-19 outbreak in early 2020. Since then it is widely used by National Defense Medical College
and its hospital, elderly care facilities, restaurants, medical institutes, food related companies, andlocal governments. In fact, a large amount of BiSCaO Water was used for health care workers
dispatched from National Defense Medical College and its hospital to the Diamond Princess cruise ship,
Osaka Corona Intensive Care Center, and Fever patient center. The result - all dispatched health care4

workers were healthy and safe. BiSCaO Water was also used when a corona cluster occurred in a student
dormitory of National Defense Medical College. Again, the health workers involved were well protectedfrom being infected with COVID-19 and no side effects were reported to have been suffered. Thus, it has
been confirmed that BiSCaO Water is effective and safe.

Figure 2. Forms and application of BiSCaO® containing liquids and BiSCaO Water

２－２．Comparison of disinfectant ability between BiSCaO Water, ethanol, andHypochlorous acid
Disinfection effects of BiSCaO Water9,10) against contamination suspensions including 51 ±
14 (× 107) CFU (Colony-Forming Units) /mL of total viable cells (TC) and 45 ± 25 (×10 6)
6 CFU/mL of coliform bacteria (CF) were compared to ethanol and Hypochlorous acid (HClO)1,2).
Undiluted BiSCaO Water (pH 12.7), ethanol (99.5%), and 200 ppm HCIO withcommercially available highest concentration (pH 6.2) are defined as 100%, and the mixtures ofeach disinfectant and contamination suspension with the final concentrations (75, 50, 25, and12.5%) were tested for microbicidal activities.
The CFU/mL for floating TC and CF with 75 and 50% of BiSCaO Water, ethanol, and HClO
were below the detection limit, indicating those three disinfectants have strong microbicidal

5

activities. The microbicidal activities decreased with lower concentrations of each disinfectant
with less than 25% and all the three disinfectants with 6.25% have little microbicidal activities.
BiSCaO Water with 25, 12.5, and 6.25% exhibited slightly higher microbicidal activity thanethanol and HClO with no significant difference.

BiSCaO Watern i teCO,6.

t ano 99. %Figure 3. . Bactericidal activity of disinfectants against a contaminated suspension comprisingnormal bacterial flora. TC (left) and CF (right) released from the contaminated suspension in eachsample were measured as CFU/mL. BiSCaO Water, ethanol, and HClO had strong microbicidal
activities. Plating and counting were performed as a set of 3 technical replicates (n = 3).

２－３．Comparison of cleaning abilities of BiSCaO Water, ethanol, and HClO toenvironmental surfaces
Six items - Toilet sandals, flat wood of old wooden box, cowhide sofa, television screen, metal
door knob, and acrylic plate for splash prevention (referred to as “environmental surfaces”) were washedwith tap water, BiSCaO Water (undiluted), ethanol (75%), and HClO solution (pH 6.2、200 ppm), and thentested with ATP rapid hygiene monitoring (Kikkoman Biochemifa Company, Tokyo) for cleaning effect6and simple wiping. General viable bacteria colony formation test (Nissui Pharmaceutical Co., Ltd.)
were performed for cleaning effect and for microbicidal effect, respectively. In this, it is said that
optimal concentration of ethanol for disinfection is 70－80%, and commercially available highest
concentration of HClO for antiseptic solution is 200 ppm. Therefore, undiluted BiSCaO Water,
75% ethanol, and 200 ppm HClO solution were used in this study.

ATP rapid hygiene monitoring test is to quantify total amount of ATP (adenosine
triphasphate), ADP (adenosine diphasphate), and AMP (adenosine monophasphate) as dirt index,
based on relative value of chemiluminescence induced with luciferase catalyst (Relative Light
Unit or “RLU”). According to the instructions, less than 500 RLU, 501～1000 RLU, and more than
1

1 LU stanor “C ean Pass ”, “Be are”, an “Dirt Fai re ” respectively. As shownin Figure 4, all six items of environmental surfaces before cleaning were Dirty, and all the six
items remained as Dirty after cleaning with tap water although RLU values were decreased on
television screen, metal door knob, and acrylic plate with tap water. On the other hand,
after cleaning television screen, metal door knob, and acrylic plate with BiSCaO Water,
ethanol, and HClO - RLU levels decreased to below the detection limit. In other words, they are clean.
However, toilet sandals, flat wood of old wooden box, cowhide sofa remained as Dirty (Failure).
From the results of ATP rapid hygiene monitoring, it proven that it is insufficient towash porous materials such as toilet sandals, flat wood of old wooden box, and cowhide sofa witheither BiSCaO Water, ethanol, or HClO. There are no significant differencesin RLU values between BiSCaO Water, ethanol, and HClO. Since ATP rapid hygiene monitoring
test is to quantify total amount of adenosine phosphate, the data do not reflect effect on removing
pathogenic virus and bacteria. Therefore, simple wiping general viable bacteria colony formation
test (Nissui Pharmaceutical Co., Ltd.) were performed for microbicidal effect.

7

a

ater

t ano
Be oreeanin

BiSCaO Watern i ete%CO
6. ,Figure 4. Cleaning effect of BiSCaO Water, ethanol, and HClO by ATP rapid hygienemonitoring test. Each environmental surface after cleaning with either BiSCaO Water, ethanol, or
HClO were measured Relative Light Unit (RLU) using ATP rapid hygiene monitoring. The
counting was performed as a set of 3 technical replicates (n = 3) and error bars represent means ± S.D.

２－4．Comparison of disinfectant abilities of BiSCaO Water, ethanol, and HClO toenvironmental surfaces
Simple wiping general viable bacteria colony formation test exhibited that CFU (ColonyForming Units) /mL of total viable cells (TC) after washing all six environmental surfaces with
BiSCaO Water (undiluted), ethanol (75%), and HClO (200 ppm) decreased to less than 100 CFU(Figure 5). Those results suggested that BiSCaO Water, ethanol, and HClO have sufficiently highmicrobicidal activities in spite of insufficient cleaning ability to porous environmental surfaces
by ATP rapid hygiene monitoring. There are no significant differences in RLU values between
BiSCaO Water, ethanol, and HCIO.

8a

BiSC aO Watern i te

ater

t ano

%

CO
6. ,

Be ore eanin

Figure 5. Disinfection effect of BiSCaO Water, ethanol, and HClO by simple wiping general
viable bacteria colony formation test. Each environmental surface after cleaning with either
BiSCaO Water, ethanol, or HClO were measured CFU (Colony-Forming Units) /mL of total
viable cells (TC). The counting was performed as a set of 3 technical replicates (n = 3) and error
bars represent means ± S.D.

２－5．Comparison of deterioration in non-woven fabric with BiSCaO Water, ethanol,and HClO12)
Surgical masks (surgical mask ST; Utsunomiya Seisaku Co. Ltd., Osaka, Japan) and N95
masks (GIKO 1400, NIOSH N95, Fitlife Corp., Tokyo, Japan) were sprayed with BiSCaO Water,
ethanol (75%), and hypochlorous acid (200 ppm, pH 6.2) twice per day over 3 days. The Scanninge tron Mi ros o e ‘SEM” image of inner sheet in the two BiSCaO Water-treated masks was identical tothat of control, which suggests that BiSCaO Water did not cause damage in both masks. In contrast,
the inner sheets of both masks were melted by ethanol, and filtration efficiency reduced withethanol (data not shown). HClO cause minor damages in both masks (Figure 6).

9Figure 6. Non-woven fabric deterioration with BiSCaO Water, ethanol, and HClO. The SEM
image of inner sheet of surgical and N95 masks, which submerged in enough amount of BiSCaO
Water, ethanol, and hypochlorous acid twice per every day for 3 days.

２－6．Effect of Sterilization of fingers and changes of pH on BiSCaO Water

Microbicidal effects of BiSCaO Water spray (1.5 mL × 3) to contaminated fingers of three
volunteers (a, b, c) were evaluated using Palm Check (Nikken Bio Medical Laboratory Inc.
Kyoto) incubated at 37℃ for 24 h (Figure 7 (A)). Although many general viable bacterialcolonies were observed before spraying BiSCaO Water, there were no colonies after spraying it.
BiSCaO Water was sprayed onto palm, and the pH of wet palm was measured over thecourse of the next 5 min using a bench pH meter (LAQUA pH meter, F-74, HORIBA, Ltd.)equipped with a Micro Tough electrode (9618S-10D; HORIBA, Ltd.). The pH immediately

10decreased after spraying - the pH of the palm fell to 12 after 1 min, and continued falling to weakalkaline and reached to pH 10 after 5 min. Thus, when BiSCaO Water was sprayed onto palm,
strong alkaline condition (pH > 12) is maintained for only 1 min. Furthermore, nobody complained ofside effects such as rough hands. On the other hand, it has been observed that Sodium Hydroxide (NaOH)
solution (pH = 12.7) causes disorders like burns and deteriorates chemical fiber, plastic, and metal such asaluminum and copper. Therefore, NaOH solution may not apply as cleaning/disinfectants.

aaFigure 7. Effect of Sterilization of palms and changes of pH on BiSCaO Water. A: total viable
cells before and after spraying BiSCaO Water. B: Changes of pH after spraying BiSCaO Wateron palm.

11

３．Discussion

BiSCaO Water which has strong microbicidal and virucidal activities with strong alkaline(pH 12.7) is produced from scallop shells without alcohol and chlorine-based substances, and is ahighly concentrated calcium ion solution. Evaluation by Japan Food Research Laboratory (JFRL)
demonstrated that BiSCaO Water by using the fifty-percent tissue culture infectious dose (TCID50)
method9 eliminated influenza A (H1N1), an enveloped virus, and Feline calicivirus, a non-enveloped virus
within 1 min. Also, by using in vitro assays method, BiSCaO Water eliminated 99% of bacteria suchas Escherichia coli strains NBRC 3972 and O-157:H7, Pseudomonas aeruginosa, Salmonella, andStaphylococcus aureus within 5 mins 9,10 and eliminated 99.9% within 15 mins. Figure 8 summarizes
on drug resistances of pathogenic microbes and biological significance of disinfection.

so te e reasei ro esro t sression resi

Stren t oi ro i i a arinsin a aai ro esitre o a o n trient so r es

er or e a er eanin

Figure 8. Drug resistances of pathogenic microbes and biological significance of disinfection.

12However, since those evaluations were to perform against only solution floating
microorganisms, effectiveness of BiSCaO Water against environmental surfaces containing
biofilm was not clear. Therefore, we performed study on cleaning and sterilization effects of
BiSCaO Water compared/examined to ethanol and HClO. First of all, BiSCaO Water hasbactericidal activities identical to ethanol and HClO by the study using contamination suspension(Figure 3). Furthermore, BiSCaO Water exhibited cleaning and sterilization effect against various
environmental surfaces identical to ethanol and HClO (Figures 4, and 5). Notably, BiSCaO Water,
ethanol, and HClO have sufficiently high microbicidal activities (CFU reduced to tenths of a few)
in spite of insufficient cleaning ability (RLU reduced to a fraction) to porous environmental
surfaces such as toilet sandals, flat wood of old wooden box, and cowhide sofa by ATP rapid
hygiene monitoring. Although there are no significant differences in RLU and CFU values
between BiSCaO Water, ethanol, and HClO, BiSCaO Water has slightly higher microbicidal
activity than ethanol and HClO (Figures 4, 5).
Some antiseptics/disinfectants, such as ethanol and HClO, show significant activity withbroad microbicidal and virucidal activities, notably, activity against COVID-19 resulting in
disruption of the viral envelope. However, ethanol has little virucidal activities to non-envelop
type virus such as norovirus. On the other hand, chlorine-derived disinfectants are effective to
non-envelop type virus, but decolorization and metal corrosive are caused with the effective
concentration (> 50 ppm). Furthermore, high concentrations of ethanol and chlorine-derived
disinfectant have unique pungent odor. When they directly touch the skin, rough skin and allergies
are caused in association with reducing water retention. The direct application of ethanol or HClO
to cloth and non-woven masks causes decoloization, melt, and damage. Therefore, disinfectants
that can decrease the bacterial and viral bioburden without harmful side effects and environmental
disruption are essential for defense and environmental hygiene and public health. Although BiSCaO Water
was not directly examined on inactivation of COVID-19, BiSCaO Water can very quickly inactivate both
non-enveloped virus such as Feline calicivirus and enveloped virus such as influenza A (H1N1) without
harmful side effects and environmental disruption. Since COVID-19 is non-enveloped virus, it can be said
that BiSCaO Water is effective to inactivate COVID-19. Furthermore, our study demonstrated BiSCaO
Water has identical virucidal activity as cleaning agen/disinfectant for environmental surfaces to ethanol
and HClO.
Concerns have been raised on the safety of BiSCaO Water when applied to the living bodyin view of the strong alkalinity of those reagent. Recent study showed that the high initial pH of
BiSCaO Water following application to various environmental surfaces and palm, rapidly
decreases to weak alkaline (pH＜10) with the generation of CaCO3 by the interaction between

13Ca2+ ions in BiSCaO Water and CO2 in the air5. We infer that the ingestion of a small amount of
BiSCaO Water would not cause problem if swallowed or inhaled, given that CaCO3 powder will
be converted to a safe and soluble compound (calcium bicarbonate (Ca(HCO3)2)) upon further
interaction between CaCO3 and CO2.
Moreover, BiSCaO Water has deodorant effect (reduce odor, without masking with fragrance),
since it is composed of CaO and Ca ion which are non-volatile1,2). Thus, BiSCaO Water is expected to be
widely used as an effective and safe decontamination agent for cleaning and sterilizing for various
environmental surfaces and finger/palm. In summary, comparisons of BiSCaO Water, ethanol, and HClO
on impact to environmental and living body have been described in Table 1 below. Thus, this study on
BiSCaO Water suggests a potential application of BiSCaO Water as antiseptic/disinfectant forbroad pathological microbes including fungi, bacteria, and viruses, especially for COVID-19
instead of alcohol and chlorine-based disinfectant, and expect to widely apply and use for hygiene
activities in Japan Self-Defense Force.

14

Acknowledgement
A special note of acknowledgement and deep appreciation to Dr Hideaki WATANABE (Former
Commissioner of Acquisition, Technology and Logistics Agency in the Ministry of Defense) who
recommended the writing of this article and who provided substantial comments.

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