Quality & Substantiation: ZBiotics® Sugar-to-Fiber

Engineered Probiotic Ingredient: Bacillus subtilis ZB423™

About ZBiotics® Sugar-to-Fiber and Bacillus subtilis ZB423™

ZBiotics® Sugar-to-Fiber is a powder drink mix that contains the genetically engineered probiotic strain Bacillus subtilis ZB423™. This probiotic was fully developed by ZBiotics’ in-house team of microbiologists and is available exclusively from ZBiotics. Its function is to create levan fiber using dietary sucrose it encounters as it passes through the intestinal tract. This function is important for microbiome health, which creates a series of downstream benefits for overall health.

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 Bacillus subtilis ZB423™ and to Sugar-to-Fiber are listed below.

Testing of Bacillus subtilis ZB423™ probiotic biomass

This is our hero ingredient and what makes Sugar-to-Fiber 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 levansucrase and that the enzyme is active. Because the production of active levansucrase is the key function that makes B. subtilis ZB423™ perform the task of converting sugar to fiber, it’s critical to ensure that this function is preserved in every new batch we produce.

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Strain Activity The levansucrase enzyme cleaves sucrose into glucose and fructose, and then stacks the fructoses together to make levan fiber.  This leaves the glucose free.  To quantify the activity of the enzyme, we measure the amount of free glucose released over time after inputting a known quantity of sucrose. Limit / Target >8 mg glucose/dL/min‡ Method Levansucrase Assay (similar to that described in van Hijum et al, 2004) 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 Hassan-Casarez et al, 2024) 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 ZB423™. 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 2022 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†  = <0.059 ug/serving† Method AOAC 2011.19, 993.14, 2015.01 Testing frequency Every batch
Mercury Limit / Target <10 ppb†  = <0.001 ug/serving† Method AOAC 2011.19, 993.14, 2015.01 Testing frequency Every batch
Cadmium Limit / Target <150 ppb†  = <0.010 ug/serving† Method AOAC 2011.19, 993.14, 2015.01 Testing frequency Every batch
Arsenic Limit / Target <600 ppb†  = <0.039 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 Sugar-to-Fiber 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 ZB423™ as well as other ingredients like flavors and stabilizing fillers.

    Probiotic Quantity

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

    Physical & Organoleptic

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    Appearance (includes color) Limit / Target Light tan to beige powder† Method Visual inspection Testing frequency Every batch
    Odor Limit / Target Neutral† Method Olfactory inspection Testing frequency Every batch
    Taste Limit / Target Slightly sweet/tart† Method Sample taste Testing frequency Every batch
    Moisture content Limit / Target Report Only† Method STM-WC1005 Testing frequency Every batch
    Water activity Limit / Target < 0.5† Method AOAC  OMA 978.18 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 Not Detected/10g† Method AOAC 991.14; BioLumix Testing frequency Every batch
    E. coli Limit / Target Negative/25g (or Absent/10g)† Method AOAC 080601; BioLumix Testing frequency Every batch
    S. aureus Limit / Target Not Detected/10g† Method AOAC 2003.07; BioLumix Testing frequency Every batch
    B. cereus Limit / Target Not Detected/25g† Method BioLumix Testing frequency Every batch
    Listeria sp. Limit / Target ≤ 100 cfu/g† Method BioLumix Testing frequency Every batch

    Contamination – Heavy Metals

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    Arsenic Limit / Target ≤ 1.77 mcg/day† Method STM-ICP1000 Testing frequency Annually
    Cadmium Limit / Target ≤ 0.14 mcg/day† Method STM-ICP1000 Testing frequency Annually
    Mercury Limit / Target ≤ 0.14 mcg/day† Method STM-ICP1000 Testing frequency Annually
    Lead Limit / Target ≤ 0.41 mcg/day† 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 the effects of fiber and levan on the microbiome and the impact of the microbiome on the rest of the body.

    Levan supports the gut microbiome

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    Xu, M. et al. Simulated Digestion and Fecal Fermentation Behaviors of Levan and Its Impacts on the Gut Microbiota. J. Agric. Food Chem. 71, 1531–1546 (2023). Go to study Study Type In vitro Research Institution School of Chemical Engineering and Technology, Tianjin University, PR, USA
    Bahroudi, S., Shabanpour, B., Combie, J., Shabani, A. & Salimi, M. Levan Exerts Health Benefit Effect through Alteration in Bifidobacteria Population. Iran Biomed J 24, 54–59 (2020). Go to study Study Type In vivo (mice) Research Institution Physiology and Pharmacology Department, Pasteur Institute of Iran
    Liu, C., Kolida, S., Charalampopoulos, D. & Rastall, R. A. An evaluation of the prebiotic potential of microbial levans from Erwinia sp. 10119. Journal of Functional Foods 64, 103668 (2020). Go to study Study Type In vitro but with gut samples from healthy donors Research Institution Department of Food and Nutritional Sciences, University of Reading, UK

    Adding fiber to your diet

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    Mysonhimer, A. R. & Holscher, H. D. Gastrointestinal Effects and Tolerance of Nondigestible Carbohydrate Consumption. Adv Nutr 13, 2237–2276 (2022). Go to study Study Type A summary of 103 clinical trials of varying fiber types and the resulting level of discomfort based on dose Research Institution Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL

    Fiber supports your health

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    Zhao, L. et al. Gut bacteria selectively promoted by dietary fibers…. Science 359, 1151–1156 (2018). Go to study Study Type Clinical Trial Research Institution New Jersey Institute for Food, Nutrition, and Health, NJ USA
    Wastyk, H. C. et al. Gut-microbiota-targeted diets modulate human immune status. Cell 184, 4137-4153.e14 (2021). Go to study Study Type Clinical Trial Research Institution Department of Bioengineering, Stanford School of Medicine, Stanford, CA, USA

    Fiber diversity is important

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    Ranaivo, H. et al. Increasing the diversity of dietary fibers in a daily-consumed bread modifies gut microbiota and metabolic profile in subjects at cardiometabolic risk. Gut Microbes 14, 2044722 (2022). Go to study Study Type Clinical Trial Research Institution University of Lyon, France

    Impacts of a healthy microbiome

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    Seethaler, B. et al. Short-chain fatty acids are key mediators of the favorable effects of the Mediterranean diet on intestinal barrier integrity: data from the randomized controlled LIBRE trial. The American Journal of Clinical Nutrition 116, 928–942 (2022). Go to study Study Type Clinical Trial Research Institution Institute of Nutritional Medicine, University of Hohenheim, Stuttgart, Germany

    Benefits of making Levan in the gut

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    Yamamoto, Y. et al. In vitro digestibility and fermentability of levan…. The Journal of Nutritional Biochemistry 10, 13–18 (1999). Go to study Study Type In vitro and in vivo (rats) Research Institution Department of Food and Nutrition, Faculty of Human Life Science, Osaka City University, Osaka, Japan