Tolerase G® - Clinical Research Data Summary

Tolerase G® AN-PEP Prolyl Endoprotease (Aspergillus niger)

Tolerase G® is a clinically studied ingredient used in the following Silver Fern™ Brand product:

  • Gluten Comfort

Introduction

Tolerase G® is a targeted digestive enzyme designed to support the breakdown of gluten early in the digestive process. It contains a specialized prolyl endoprotease derived from Aspergillus niger (AN-PEP), an enzyme capable of cleaving the proline-rich gluten proteins that are difficult for typical human digestive enzymes to fully break down. Unlike many proteases, Tolerase G® remains active in the acidic environment of the stomach and works alongside the body's natural digestive enzymes to begin breaking down gluten proteins during the early stages of digestion.*

Human clinical and mechanistic studies show that AN-PEP can accelerate the breakdown of gluten during digestion, including under conditions that reflect typical meal consumption and small amounts of incidental gluten exposure. Tolerase G® supports digestive comfort and helps the body process gluten more efficiently — it is intended to complement, not replace, a gluten-conscious diet.*

The following studies evaluate the digestive activity of the AN-PEP enzyme. These summaries are provided for educational purposes and do not imply that this product treats or prevents any disease.

These are the studies for Tolerase G®. Below, we provided a summary of each key data for each study, along with a link to the complete clinical research.

  1. Summary of Study 1 – Full Study: https://pubmed.ncbi.nlm.nih.gov/26040627/
  2. Summary of Study 2 – Full Study: https://pubmed.ncbi.nlm.nih.gov/29026170/
  3. Summary of Study 3 – Full Study: https://pubmed.ncbi.nlm.nih.gov/17494108/
  4. Summary of Study 4 – Full Study: https://pubmed.ncbi.nlm.nih.gov/16690904/
  5. Summary of Study 5 – Full Study: https://www.frontiersin.org/journals/pediatrics/articles/10.3389/fped.2019.00193/full

Ingredient Manufacturer Link: https://www.dsm-firmenich.com/en/businesses/health-nutrition-care/news/talking-nutrition/digestive-comfort-gluten-sensitive-adults.html



Study 1 Summary

Complete Study Information: https://pubmed.ncbi.nlm.nih.gov/26040627/

Randomized, Double-Blind, Placebo-Controlled Crossover Trial of AN-PEP and Gluten Protein Breakdown During Human Digestion
Salden, B.N. et al. Alimentary Pharmacology & Therapeutics. 2015. 42:273–285.

This randomized, double-blind, placebo-controlled crossover trial evaluated whether AN-PEP could accelerate the breakdown of gluten proteins during human digestion in 12 healthy adults (ages 18–45) over 4 test days with ≥1-week washouts. A 4 g gluten meal was administered intragastrically to standardize digestive conditions under both low-calorie and high-calorie meal conditions.

Study Design Highlights

  • Study design: Randomized, double-blind, placebo-controlled crossover
  • Participants: 12 healthy adults (ages 18–45)
  • Gluten dose: 4 g gluten protein per test meal
  • Meal conditions: Low-calorie and high-calorie
  • Measurements: ELISA-based gluten detection, Western blot, PEG-3350 dilution marker, gastric emptying tracking

Key Findings (What the Study Showed)

1. Gluten Protein Breakdown in the Stomach

  • Low-calorie meal — AN-PEP: 35 µg·min/mL vs. placebo: 389 µg·min/mL (p < 0.001)
  • High-calorie meal — AN-PEP: 53 µg·min/mL vs. placebo: 386 µg·min/mL (p < 0.001)

2. Gluten Protein Reaching the Upper Small Intestine (Duodenum)

  • Low-calorie meal — AN-PEP: 7 µg·min/mL vs. placebo: 168 µg·min/mL (p < 0.001)
  • High-calorie meal — AN-PEP: 4 µg·min/mL vs. placebo: 32 µg·min/mL (p < 0.001)

3. Total Gluten Protein Delivery to the Duodenum (Adjusted for Dilution)

  • Low-calorie meal — AN-PEP: 2,813 µg·min vs. placebo: 31,952 µg·min (~91% reduction; p < 0.001)
  • High-calorie meal — AN-PEP: 2,553 µg·min vs. placebo: 13,095 µg·min (~80% reduction; p = 0.013)

AN-PEP broke down gluten proteins rapidly within approximately one hour even under low-calorie meal conditions. Adverse events were mild, infrequent, and comparable between AN-PEP and placebo groups.

Benefits of Tolerase G® Demonstrated by This Study

Based directly on the clinical outcomes, AN-PEP was shown to:
✓ Substantially reduce detectable gluten protein concentrations in the stomach vs. placebo
✓ Reduce gluten protein reaching the upper small intestine by ~80–91% vs. placebo
✓ Act rapidly within approximately 1 hour during digestion
✓ Work consistently across low-calorie and high-calorie meal conditions
✓ Be well tolerated in healthy adults

Why This Study Matters

This study provides direct human evidence that AN-PEP can significantly accelerate gluten protein breakdown during digestion. By acting early in the stomach, the enzyme helps break down gluten proteins that are typically difficult for standard digestive enzymes to process — and does so across different meal types.*

Reference Link: https://pubmed.ncbi.nlm.nih.gov/26040627/



Study 2 Summary

Complete Study Information: https://pubmed.ncbi.nlm.nih.gov/29026170/

Randomized, Double-Blind, Placebo-Controlled Crossover Trial of AN-PEP During Realistic Solid Meal Conditions
König, J. et al. Scientific Reports. 2017. 7:13100.

This randomized, double-blind, placebo-controlled crossover trial evaluated AN-PEP tablets (high-dose and low-dose) vs. placebo in 16 completing adults (ages 18–70) during consumption of a complex oat porridge meal containing ~0.5 g gluten — reflecting small amounts of gluten present in everyday foods. Gastric and duodenal samples were collected over 180 minutes.

Study Design Highlights

  • Study design: Randomized, double-blind, placebo-controlled crossover
  • Participants: 18 enrolled; 16 completed all visits (ages 18–70)
  • Meal: Complex oat porridge (~0.5 g gluten) — realistic everyday meal conditions
  • Groups: High-dose AN-PEP tablets, low-dose AN-PEP tablets, placebo tablets
  • Measurements: Gluten-specific ELISA, AUC analysis over 180 minutes

Key Findings (What the Study Showed)

1. Gluten Protein Breakdown in the Stomach

  • Placebo: 176.9 µg×min/mL (median)
  • High-dose AN-PEP: 22.0 µg×min/mL — 88% reduction (p = 0.001)
  • Low-dose AN-PEP: 25.4 µg×min/mL — 86% reduction (p = 0.001)

2. Gluten Protein Levels in the Duodenum

  • Placebo: 14.1 µg×min/mL
  • High-dose AN-PEP: 6.3 µg×min/mL — 56% reduction (p = 0.019)
  • Low-dose AN-PEP: 7.4 µg×min/mL — 48% reduction (p = 0.015)

3. Success Rates (≥50% Gluten Reduction vs. Placebo)

  • Stomach — High dose: 80.0% of participants; Low dose: 86.7% of participants
  • Duodenum — High dose: 76.9% of participants; Low dose: 58.3% of participants
  • No significant difference between dose levels; adverse events minimal with only 1 event possibly related to the study product

Benefits of Tolerase G® Demonstrated by This Study

Based directly on the clinical outcomes, AN-PEP tablets were shown to:
✓ Reduce gastric gluten protein concentrations by ~86–88% vs. placebo
✓ Reduce duodenal gluten protein levels by ~48–56% vs. placebo
✓ Achieve ≥50% gluten reduction success in 80–87% of participants (stomach)
✓ Work effectively in tablet form during a realistic solid food meal
✓ Demonstrate consistent activity at both tested doses
✓ Be safe and well tolerated

Why This Study Matters

This study confirms that AN-PEP is effective when delivered in a tablet format during typical eating conditions — including complex solid foods and small amounts of incidental gluten — extending the clinical evidence from Study 1 to real-world meal scenarios.*

Reference Link: https://pubmed.ncbi.nlm.nih.gov/29026170/



Study 3 Summary

Complete Study Information: https://pubmed.ncbi.nlm.nih.gov/17494108/

AN-PEP Gluten Protein Breakdown in a Dynamic Gastrointestinal Model
Mitea, C. et al. Gut. 2008. 57:25–32.

This study evaluated AN-PEP using the TIM (TNO dynamic multi-compartment gastrointestinal simulation) system, testing a white bread meal (~5 g gluten) and a complex fast-food meal (bread, hamburger, fries, soda; ~5 g gluten) with and without AN-PEP, sampling at stomach, duodenum, jejunum, and ileum levels.

Key Findings (What the Study Showed)

  • Without AN-PEP: Significant amounts of gluten proteins remained detectable in stomach samples after 120 minutes of digestion
  • With AN-PEP: Breakdown occurred as early as 45 minutes; intact gluten proteins undetectable at approximately 90 minutes
  • Only minimal amounts of intact gluten proteins progressed beyond the stomach with AN-PEP present
  • Complex fast-food meal: Gluten proteins disappeared from stomach samples within ~60 minutes with AN-PEP vs. still detectable at same time point without
  • AN-PEP maintained activity at low gastric pH, resisted pepsin degradation, and functioned throughout the typical ~2-hour gastric digestion period

Why This Study Matters

This gastrointestinal model study provides mechanistic evidence that AN-PEP promotes early gluten protein degradation in both simple and complex food matrices, maintaining strong activity under physiologically relevant digestive conditions including stomach acid and pepsin — providing the foundational mechanistic rationale for Tolerase G®.*

Reference Link: https://pubmed.ncbi.nlm.nih.gov/17494108/



Study 4 Summary

Complete Study Information: https://pubmed.ncbi.nlm.nih.gov/16690904/

AN-PEP Stability and Activity Under Gastric Conditions vs. Earlier Gluten-Digesting Enzymes
Stepniak, D. et al. American Journal of Physiology – Gastrointestinal and Liver Physiology. 2006. 291:G621–G629.

This in vitro and ex vivo mechanistic study compared AN-PEP (Aspergillus niger prolyl endoprotease) vs. FM-POP (Flavobacterium meningosepticum prolyl oligopeptidase) under gastric conditions including pH 2–5 and pepsin exposure, evaluating gluten peptide degradation rates, intact gluten protein breakdown, and stability throughout simulated digestion.

Key Findings (What the Study Showed)

1. AN-PEP Acid Stability vs. Earlier Enzymes

  • AN-PEP: Detectable activity from pH 2–8; optimal at pH 4–5; stable at pH 2.0; resistant to pepsin degradation
  • FM-POP (comparator): Lost ~50% activity within 15 minutes at pH 2; rapidly degraded by pepsin

2. Speed of Gluten Peptide Degradation

  • AN-PEP half-life: 2.4–6.2 minutes
  • FM-POP half-life: 140–550 minutes
  • AN-PEP breaks down gluten peptides approximately 25–90x faster than the comparator enzyme

3. Extent of Gluten Protein Breakdown

  • Complete disappearance of detectable gliadin proteins under AN-PEP treatment
  • Near-complete degradation of glutenin proteins
  • Most resulting peptide fragments <1 kDa — substantially smaller than original gluten proteins
  • Activity maintained throughout simulated digestion including gastric acid, pepsin, and pancreatic enzyme phases

Why This Study Matters

This study identifies the key characteristic that makes AN-PEP uniquely suitable for gastric gluten digestion: its stability in stomach acid and resistance to pepsin. Unlike earlier enzymes that were inactivated before they could act, AN-PEP retains full enzymatic activity in the stomach — exactly where gluten protein digestion needs to begin.*

Reference Link: https://pubmed.ncbi.nlm.nih.gov/16690904/



Study 5 Summary

Complete Study Information: https://www.frontiersin.org/journals/pediatrics/articles/10.3389/fped.2019.00193/full

AN-PEP Activity in a Standardized In Vitro Simulated Gastrointestinal Digestion Model
Salden, B.N. et al. Frontiers in Pediatrics. 2019. 7:193.

This in vitro digestion study evaluated AN-PEP using a standardized simulated gastrointestinal model with a gastric phase (low pH + pepsin) and intestinal phase (pancreatic enzymes), testing whole gluten proteins and proline-rich gluten peptide fragments.

Key Findings (What the Study Showed)

  • AN-PEP rapidly degraded gluten proteins in acidic stomach-like conditions, with significant breakdown beginning in the gastric phase before intestinal digestion
  • Substantially reduced detectable proline-rich gluten fragments — the peptide type most resistant to normal digestion
  • Functioned effectively in the presence of pepsin and pancreatic enzymes without interfering with endogenous digestive enzyme activity
  • Maintained stability and activity across both gastric and intestinal phases of the simulated digestion model

Why This Study Matters

This in vitro model study provides mechanistic confirmation that AN-PEP works alongside the body's natural digestive enzymes — not instead of them — maintaining complementary activity through both gastric and intestinal digestion phases, and specifically targeting the proline-rich gluten fragments that are most resistant to normal digestion.*

Reference Link: https://www.frontiersin.org/journals/pediatrics/articles/10.3389/fped.2019.00193/full

Big-Picture Integration with Other Studies

  • Study 1: Direct human RCT evidence — AN-PEP reduced gluten protein delivery to the duodenum by ~80–91% vs. placebo under low-calorie and high-calorie meal conditions, with rapid activity within ~1 hour.
  • Study 2: Human RCT confirming AN-PEP effectiveness in tablet form during realistic solid meal conditions with ~0.5 g incidental gluten — achieving ~86–88% gastric reduction in 80–87% of participants.
  • Study 3: Dynamic gastrointestinal model demonstrating gluten protein breakdown as early as 45 minutes, undetectable intact gluten by ~90 minutes, and consistent activity in both simple and complex food matrices.
  • Study 4: Mechanistic comparison showing AN-PEP is uniquely stable at pH 2, resistant to pepsin, and 25–90x faster than earlier gluten-digesting enzymes — explaining its gastric efficacy.
  • Study 5: In vitro mechanistic confirmation that AN-PEP specifically targets proline-rich gluten fragments, works alongside endogenous digestive enzymes, and maintains activity through both digestive phases.

Tolerase G® Ingredient Summary & Real-World Relevance

AN-PEP — the active enzyme in Tolerase G® — is not a general protease. It is a highly specialized prolyl endoprotease designed to support the digestion of gluten proteins. Gluten contains proline-rich sequences that are difficult for many common digestive enzymes to fully break down. AN-PEP is uniquely suited for this task: it remains active in the acidic environment of the stomach, resists degradation by pepsin, and begins breaking down gluten proteins early in the digestive process — where protein digestion naturally starts.*

Human clinical studies confirm that AN-PEP can accelerate gluten protein breakdown during digestion under both controlled research conditions and realistic everyday meal scenarios, including solid foods and small amounts of incidental gluten. Mechanistic studies explain why: its acid stability and speed (25–90x faster than earlier enzymes) distinguish it from general digestive enzyme supplements.*

Tolerase G® works alongside the body's natural enzymes to support digestive comfort when consuming gluten-containing foods, and is intended to complement — not replace — a gluten-conscious diet.*

Ingredient Manufacturer Link: https://www.dsm-firmenich.com/en/businesses/health-nutrition-care/news/talking-nutrition/digestive-comfort-gluten-sensitive-adults.html

Tolerase G® is a clinically studied ingredient used in the following Silver Fern™ Brand product:

  • Gluten Comfort

*These statements have not been evaluated by the Food and Drug Administration. This product is not intended to diagnose, treat, cure, or prevent any disease.

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