Quality & Substantiation: ZBiotics® Pre-Alcohol

Engineered Probiotic Ingredient: Bacillus subtilis ZB183™

About ZBiotics® Pre-Alcohol and Bacillus subtilis ZB183™

ZBiotics® Pre-Alcohol is a beverage containing a patented, genetically engineered probiotic (B. subtilis ZB183™) that helps you land on your feet the day after drinking alcohol. It’s engineered to break down an unwanted metabolic byproduct of alcohol consumption called acetaldehyde – the main culprit behind rough mornings after drinking. This probiotic was fully developed by ZBiotics’s in-house team of microbiologists and is available exclusively from ZBiotics.

Quality Testing

At ZBiotics, we pride ourselves on our relentless focus on quality in our products. While quality starts in the lab when we build each strain, it doesn’t stop there. We extensively test every manufacturing batch of both our probiotic ingredients and our final products to ensure that we are not only delivering clean and unadulterated products, but also delivering functional and viable products.

The testing standards we apply to Pre-Alcohol and its primary ingredient Bacillus subtilis ZB183™ are listed below.

Please note that the following information is subject to change without notice and may be out of date. While we endeavor to keep this information updated, our quality systems evolve over time, and we cannot guarantee that this page may not reflect outdated information from time to time.

Testing of Bacillus subtilis ZB183™ probiotic biomass

This is our hero ingredient and what makes Pre-Alcohol special. We thoroughly test every single batch, so we know for sure we have clean, viable, and safe probiotic going into every final product.

Activity

Validates that the probiotic ingredient is producing the enzyme acetaldehyde dehydrogenase (ALDH) and that the enzyme is active. Because the production of active ALDH is the key function that makes B. subtilis ZB183™ perform the task of converting acetaldehyde into acetate, it’s critical to ensure that this function is preserved in every new batch we produce.

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Strain Activity Acetaldehyde dehydrogenase can drive the formation of a coenzyme called NADH from the oxidized form of the coenzyme, called NAD+ in the presence of Acetaldehyde. We can measure the activity of the ALDH enzyme by monitoring the production of the NADH from NAD+. Limit / Target >9 nmol NADH/min/mg total protein Method ALDH NAD+ Plate Assay (as described in Hassan-Casarez et al, 2024) Testing frequency Every batch

Viability

We run germination and flow cytometry tests to help ensure that the manufacturing process has not caused damage to the probiotic bacterial spores in a way that could impact their functionality.

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Germination We simulate spore germination with an amino acid trigger to ensure each batch is viable and can germinate as expected. Limit / Target Cells respond to germination trigger with a drop in OD600 in less than 1 hour Method OD600 Germination Assay (as described in Tehri et al., 2018) Testing frequency Every batch
Dormant Spores Limit / Target High (>85%) Method Flow Cytometry Testing frequency Every batch
Germinated Cells Limit / Target Low (<10%) Method Flow Cytometry Testing frequency Every batch
Injured Cells Limit / Target Low (<10%) Method Flow Cytometry Testing frequency Every batch
Dead Cells Limit / Target Low (<10%) Method Flow Cytometry Testing frequency Every batch

Probiotic Quantity

We measure the quantity of viable probiotic bacteria per gram of finished ingredient in order to specifically calculate how much powder needs to go into the final product to ensure we hit our delivered CFU target.

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Enumeration (CFU/g) Limit / Target As tested‡ Method CMMEF (5th Ed., Chpt 20), mod. Testing frequency Every batch

Purity

We ensure every lot is a single strain of the intended probiotic. We sequence to ensure no mutations have arisen during fermentation that could impact performance

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Spore Microscopy Limit / Target Spherical/elongated‡ Method 1000X Phase Contrast Testing frequency Every batch
Cell Microscopy (after germination) Limit / Target Rod-shaped‡ Method 1000X Phase Contrast Testing frequency Every batch
NGS (next-generation sequencing) Genotype Limit / Target <4 SNP (single-nucleotide polymorphism) >80% occurrence, 90% max coverage† Method Map to reference, SNP analysis Testing frequency Every batch
NGS Purity Running NGS on every single batch helps ensure that the only microbe that was grown in any given batch was our target microbe—B. Subtilis ZB183™. Note that standard microbial contamination testing only tests for known pathogens, but doesn’t actually check whether there are other non-pathogenic microbes in the batch. We go the extra mile with NGS sequencing to ensure that it’s not just pathogens we’re testing for, but all non-ZBiotics microbes. Limit / Target >99%† Method Map to Reference Testing frequency Every batch

Organoleptics & Stability

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Color Limit / Target Light to dark tan† Method Organoleptic Testing frequency Every batch
Visual inspection Limit / Target Visually free from foreign material† Method Organoleptic Testing frequency Every batch
Texture Limit / Target Crystalline, free flowing powder† Method Organoleptic Testing frequency Every batch
Odor Limit / Target Strong fermentation† Method Organoleptic Testing frequency Every batch
Moisture content Limit / Target <10%† Method AOAC 925.09 Testing frequency Every batch
Water activity Limit / Target <0.5† Method No reference Testing frequency Every batch

Contamination – Microbial

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Yeast and mold Limit / Target ≤ 300 cfu/g† Method US Pharmacopeia Ch 2021 Testing frequency Every batch
Salmonella Limit / Target Negative/10g† Method US Pharmacopeia Ch 2021 Testing frequency Every batch
Coliforms Limit / Target ≤30 cfu/g† Method CMMEF Ch 8.7 Testing frequency Every batch
E. coli Limit / Target Negative/10g† Method USP <2022> Testing frequency Every batch
L. monocytogenes Limit / Target Negative/25g† Method FDA BAM Ch 10 Testing frequency Every batch
S. aureus Limit / Target <10 cfu/g† Method USP <2022> Testing frequency Every batch
Presumptive B. cereus Limit / Target <100 cfu/g† Method FDA BAM Ch 14 Testing frequency Every batch

Contamination – Heavy Metals

We applaud the FDA Closer to Zero Initiative and support this standard being set for all foods. We proactively comply by setting our heavy metal production specifications not more than 10x of median production values.

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Lead Limit / Target <900 ppb† = <0.0018 ug/serving† Method AOAC 2011.19, 993.14, 2015.01 Testing frequency Every batch
Mercury Limit / Target <10 ppb† = <0.00002 ug/serving† Method AOAC 2011.19, 993.14, 2015.01 Testing frequency Every batch
Cadmium Limit / Target <400 ppb† = <0.0008 ug/serving† Method AOAC 2011.19, 993.14, 2015.01 Testing frequency Every batch
Arsenic Limit / Target <600 ppb† = <0.0012 ug/serving† Method AOAC 2011.19, 993.14, 2015.01 Testing frequency Every batch

†Release Specification (i.e. we won’t release product if it doesn’t meet this specification)

‡Key Performance Indicator (used to track general product performance and quality, but not a release specification)

    • Testing of Pre-Alcohol final product

    We run these tests on our final formulated product when they are being filled into the final sellable units. The formulation includes B. subtilis ZB183™ as well as other ingredients like flavors and stabilizing fillers.

    Probiotic Quantity

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    Enumeration (CFU/g) Limit / Target Meets ALDH performance standard Method CMMEF (5th Ed., Ch. 20), mod. Testing frequency Every batch

    Physical & Organoleptic

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    Appearance (includes color) Limit / Target Cloudy light tan liquid Method Visual inspection Testing frequency Every batch
    Taste Limit / Target Acidic, sour, citrus Method Sample taste Testing frequency Every batch

    Contamination – Microbial

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    Yeast and mold Limit / Target ≤ 100 cfu/g† Method AOAC 997.02; BioLumix Testing frequency Every batch
    Salmonella Limit / Target ≤ 100 cfu/g† Method AOAC 997.02; BioLumix Testing frequency Every batch
    E. coli Limit / Target Negative/25g (or Absent/10g)† Method AOAC 997.02; BioLumix Testing frequency Every batch
    S. aureus Limit / Target Not Detected/10g† Method AOAC 997.02; BioLumix Testing frequency Every batch
    B. cereus Limit / Target ≤ 100 cfu/g† Method BioLumix Testing frequency Every batch
    L. monocytogenes Limit / Target Not Detected/25g† Method BioLumix Testing frequency Every batch

    Contamination – Heavy Metals

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    Lead Limit / Target < 0.00001 ug/serving Method STM-ICP1000 Testing frequency Annually
    Mercury Limit / Target < 0.00001 ug/serving Method STM-ICP1000 Testing frequency Annually
    Cadmium Limit / Target < 0.00001 ug/serving Method STM-ICP1000 Testing frequency Annually
    Arsenic Limit / Target < 0.00001 ug/serving Method STM-ICP1000 Testing frequency Annually

    †Release Specification

    ‡Key Performance Indicator

    • Substantiation Studies

    At ZBiotics, we benefit from the incredible work of the scientists and institutions who have spent years studying alcohol, acetaldehyde, and how they affect the way you might feel the day after drinking.

    Acetaldehyde concentrations build up in the colon

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    Jokelainen, K., Roine, R. P., Vaananen, H., Farkkila, M. & Salaspuro, M. In vitro acetaldehyde formation by human colonic bacteria. Gut 35, 1271–1274 (1994). Go to study Study Type In vitro Research Institution Research Unit of Alcohol Diseases and Department of Gastroenterology, University Central Hospital of Helsinki, Helsinki, Finland
    Salmela, K. S. et al. Characteristics of Helicobacter pylori alcohol dehydrogenase. Gastroenterology 105, 325–330 (1993). Go to study Study Type In vitro Research Institution Research Unit of Alcohol Diseases, Helsinki University Central Hospital, Helsinki, Finland
    Konkit, M., Choi, W. J. & Kim, W. Aldehyde dehydrogenase activity in Lactococcus chungangensis: Application in cream cheese to reduce aldehyde in alcohol metabolism. Journal of Dairy Science 99, 1755–1761 (2016). Go to study Study Type In vitro and in vivo  Research Institution Department of Microbiology, Chung-Ang University College of Medicine, Seoul, Republic of Korea

    The role of acetaldehyde in discomfort

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    Mackus, M., Stock, A.-K., Garssen, J., Scholey, A. & Verster, J. C. Alcohol 121, 9–18 (2024). Go to study Study Type Mix of in vitro and in vivo studies Research Institution Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, The Netherlands
    Eggleton, M. G. The diuretic action of alcohol in man. J Physiol 101, 172–191 (1942). Go to study Study Type Mix of in vitro and in vivo studies Research Institution Department of Pharmacology, University College, London, UK
    Penning, R., Nuland, M. van, Fliervoet, L. A. L., Olivier, B. & Verster, J. C. Curr. Drug Abuse Rev. 3, 68-75 (2010). Go to study Study Type Mix of in vitro and in vivo studies Research Institution Utrecht Institute for Pharmaceutical Sciences, Division of Pharmacology, Utrecht University, Utrecht, The Netherlands

    The minimal impact of alcohol on dehydration

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    Quertemont, E. & Didone, V. Role of Acetaldehyde in Mediating the Pharmacological and Behavioral Effects of Alcohol. Alcohol Res Health 29, 258–265 (2006). Go to study Study Type Mix of in vitro and in vivo studies Research Institution Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, The Netherlands
    Jung, T. W. et al. Alcohol and Alcoholism 41, 231–235 (2006). Go to study Study Type In vivo Research Institution Division of Endocrinology and Metabolism, and Department of Internal Medicine,Yonsei University College of Medicine, Seoul, Korea