IngredientsFungal protease is a collective term for proteolytic enzymes — enzymes that catalyze the hydrolysis of peptide bonds in protein molecules — derived from filamentous fungi. Proteases are obtained from plants, animals, and microorganisms. When sourced from fungi, particularly from food-grade mold species, they are referred to as fungal proteases or fungal peptidases. In the dietary supplement context, the term almost exclusively refers to enzyme preparations derived from Aspergillus oryzae, Aspergillus niger, and, less commonly, species of Rhizopus, Penicillium, Mucor, and Trichoderma.
Primary species of commercial and supplemental importance:
Commercial fungal protease is typically a fungal protease/peptidase complex produced by submerged fermentation of a selected strain of Aspergillus oryzae and contains both endoprotease and exopeptidase activities. Proteases belong to the group of hydrolases and exist as acid, neutral, and alkaline proteases.
Common names and product identifiers include:
Production methods: Manufacturers can use A. oryzae to produce enzymes in multiple media, including both solid-state fermentation (SSF) or submerged fermentation (SmF). In solid-state fermentation, filamentous fungi grow on grain or legume substrates under controlled humidity and temperature, closely replicating the traditional koji-making process. In submerged fermentation, the fungus is grown in liquid medium, which allows for more tightly controlled and scalable industrial production.
Available forms and preparations:
Proteolytic fungal enzyme food supplement compositions are designed for use as tablets, capsules, powder, or liquid food supplements, to be taken with protein-containing foods in order to convert ingested dietary proteins into free amino acids.
The history of human use of fungal proteases is inseparable from the history of koji-mold fermentation in East Asia. Koji mold (Aspergillus oryzae) is the only known domesticated fungal species, playing a central role in traditional East Asian fermented foods such as soy sauce, miso, and sake. The A. oryzae-based preparation of koji, which has a long tradition of more than 1,000 years, is used in the production of sake (rice wine), shoyu (soy sauce), amazake (rice koji beverage), osu (rice vinegar), kurosu (black rice vinegar), shochu (distilled alcoholic beverage fermented with koji), and miso (soybean paste).
Aspergillus oryzae is an asexual, ascomycetous fungus used for hundreds of years in the production of soy sauce, miso, and sake without recorded incidents. Some authors place the earliest domestication of koji molds far earlier: Aspergillus oryzae was domesticated over 2,000 years ago in East Asia, transforming grains and soybeans into umami-packed delights through powerful enzymes like amylases and proteases. Even earlier archaeological evidence has been interpreted to suggest fungal fermentation practices in China dating back approximately 9,000 years.
The central role of A. oryzae in traditional fermentation rests almost entirely on its protease production. The mold's digestive enzymes — proteases and amylases — break down the proteins and starches into simpler molecules that will then be fermented by yeasts. In soy sauce production, fungal proteases liberate free amino acids — especially glutamate — that are responsible for the characteristic umami flavor. During A. oryzae-mediated soy sauce fermentation, diverse enzymes are leveraged by the fungus to break down proteins and carbohydrates under high-salt conditions.
The koji molds produce many enzymes, including amylases, proteases, lipases, and tanninase, that break down (hydrolyse) macromolecules like starches, proteins, and fats into their constituent parts, such as dextrin, glucose, peptides, amino acids, and fatty acid chains. These cultures are ancient technologies used to produce a variety of fermented food products, including soy sauces, jiang/miso, fermented black soybeans, and grain-based wines like sake, amazake, and li.
Proteolytic enzymes have been used extensively as therapeutic agents for decades. The earliest studies used pancreatic enzymes in the treatment of cancer. Later, proteolytic enzymes from non-animal sources such as the plant enzymes bromelain and papain and proteases derived from fungi such as Aspergillus sp. were investigated. The industrial transition from traditional fermentation to purified enzyme supplements occurred through the 20th century, with microbial enzyme production for food and pharmaceutical uses being described in scientific literature as early as the 1950s.
Proteases from Aspergillus oryzae are commercially used in the production of sake and soy sauce as well as in flavoring of other food products. The isolation and formal characterization of protease enzymes from Aspergillus species for pharmaceutical and nutritional applications began in earnest in the mid-20th century, with the earliest clinical trials on products such as "Brinase" (an A. oryzae protease preparation) and "Asperkinase" dating to the 1960s and 1970s.
Fungal protease preparations used in dietary supplements are not single enzymes but complex mixtures containing multiple protease types distinguished by their catalytic mechanism, optimal pH range, and specificity. The four main classes recognized in fungal sources are:
The distinction between endoproteases and exoproteases is also functionally significant. Endopeptidases are characterized by their preferential action at the peptide bonds in the inner regions of the polypeptide chain. Aminopeptidases act at a free N-terminus of the polypeptide chain and liberate a single amino acid residue, a dipeptide, or a tripeptide. Carboxypeptidases act at C-terminals of the polypeptide chain and liberate a single amino acid or a dipeptide.
Aspergillus oryzae is genomically remarkable in its protease-coding capacity. According to Machida et al. (2005), A. oryzae has the largest expansion of hydrolytic genes with 135 proteinase genes including both endo- and exoproteases. Specifically, A. oryzae possesses 65 endopeptidase and 69 exopeptidase genes, supporting efficient protein degradation.
Key individual proteases within A. oryzae-derived preparations include:
The mechanism of action of fungal proteases involves the formation of an intermediate acyl-enzyme that covalently links the enzyme to the N-terminal of the substrate. In the second step, the water molecule completes the hydrolysis by attacking the intermediate. It results in the release of the second-half product with the regeneration of free enzyme.
A key advantage of fungal protease blends in supplemental applications is their ability to remain catalytically active across the wide pH range encountered throughout the gastrointestinal tract. Only blends of highly pH- and temperature-stable proteases are active in the pH range of the entire digestive tract, which ranges from an acidic pH of 2 in the stomach to a semi-alkaline pH of 8 in the small intestine. This contrasts with animal-derived digestive enzymes such as pepsin, which have a narrow acidic optimum, and pancreatic proteases, which function primarily in the near-neutral small intestine.
Fungal protease enzymes rapidly produce amino acids in free form, in contrast to the enzymes papain, bromelain, and pepsin, which produce short-chain peptides from protein substrates. This was confirmed by tests which showed that the fungal proteolytic enzyme food supplement composition produced over 40 percent more amino acids in free form than the enzymes pepsin and pancreatin in comparative tests conducted in a gastrointestinal simulator.
Throughout the fermentation process, endoproteases primarily create numerous free termini, facilitating the action of exoproteases. In the digestive tract, this coordinated action — endoproteases fragmenting large protein molecules internally, followed by exoproteases releasing individual amino acids from the resulting peptide termini — ensures that dietary protein is broken down into a mixture of small peptides and free amino acids suitable for intestinal absorption.
This is the area with the most direct clinical evidence for fungal protease supplementation in humans.
Open-label study (Oben et al., 2008): The purpose of this study was to determine if Aminogen®, a patented blend of digestive proteases from Aspergillus niger and Aspergillus oryzae, would significantly increase the in-vivo absorption rate of processed whey protein concentrate (WPC) over control values. It also investigated if any increase would be sufficient to significantly alter nitrogen (N2) balance and C-reactive protein (CRP) levels over control values as further evidence of increased WPC absorption rate. Two groups of healthy male subjects were assigned a specified balanced diet before and after each of two legs of the study. Subjects served as their own controls. In the first leg, each control group was dosed with 50 g of WPC following an overnight fast. The conclusion was that a patented blend of digestive proteases (Aminogen®) increased the absorption rate of processed WPC over controls, as measured by statistically significant increases in AUC, total serum amino acid (TSAA) levels, individual serum amino acid (ISAA) levels, and N2 balance.
Reported dosages in this study used 2.5 g and 5 g of Aminogen® co-ingested with 50 g of WPC. All individual serum amino acid levels in the 2.5 g treatment group except glycine, histidine, methionine, and serine were significantly higher than in the control group at 4 hours. All individual serum amino acid levels in the 5 g treatment group except methionine and serine were significantly higher than in the control group at 4 hours. The N2 balance was significantly higher in each treatment group compared to the corresponding control group. Significant differences in CRP levels were reported between each treatment group compared to the corresponding control group.
This was an open-label, non-randomized study in which subjects served as their own controls. The study was sponsored by the manufacturer of Aminogen® and was not independently replicated. These design features limit the strength of the conclusions.
More recent crossover trial (2025): Another human trial by Oben et al. using 2.5 g and 5 g of Aminogen (proteases from Aspergillus niger and A. oryzae) reported increased serum amino acid concentration following co-ingestion with 50 g WPC compared with 50 g WPC alone in young healthy male participants.
In vitro and simulated digestion evidence: A study tested the hydrolytic efficacy of 6 fungal enzymes in the INFOGEST static in vitro simulation of gastrointestinal (GI) digestion. A dose-response study of 3 individual fungal proteases, a lipase, and an amylase with glucoamylase demonstrated improved dietary protein, lipid, and carbohydrate hydrolysis, respectively, from an oral nutritional supplement under simulated gastric or GI conditions, compared to pepsin and pancreatin-based control conditions. A combination of the 6 enzymes improved macronutrient digestion, including enhanced release of individual amino acids from oral nutritional supplement and mixed meal substrates. The study also validated digestive models of aging and proton pump inhibitor use, and showed that the enzyme blend improved gastric digestion under these compromised digestive conditions. This is in vitro evidence and does not directly demonstrate clinical outcomes in humans.
Evidence in exocrine pancreatic insufficiency (animal model): One study investigated the activity of aspergillus protease given in combination with a novel lipase and fungal amylase, as well as commercially available pancrelipase, in exocrine pancreatic insufficient (EPI) pigs. After a high-fat diet plus a standardized dietary whey substrate, blood was withdrawn at intervals and analyzed for amine groups using a modified ninhydrin reaction. Plasma peptide-derived amino acid concentration was significantly increased in response to aspergillus protease as follows: 50 mg dose (137% increase; p = 0.05), 75 mg dose (154% increase; p = 0.008) compared to no enzyme. Administration of 75 mg of aspergillus protease led to significantly higher peptide-derived amino acid AUC and Cmax when compared to 600 mg pancrelipase. This study was conducted in an animal model, and results cannot be directly extrapolated to humans.
Overall evidence strength for protein digestion/amino acid absorption: Preliminary to moderate. The available human clinical data are from small studies, some lacking blinding or independent replication, and with potential for industry bias. The mechanistic and in vitro evidence base is strong. Independent, large-scale randomized controlled trials are lacking.
One study of a blend of 5 microbial enzymes including protease, lipase, amylase, cellulase, and lactase in patients with functional dyspepsia showed a significant reduction in GI symptoms after 60 days of three times daily supplementation, compared to placebo (Majeed et al., 2018). However, this multienzyme blend included protease of bacterial origin (Bacillus subtilis), with other fungal enzymes (amylase and lactase from A. oryzae; lipase from Rhizopus oryzae); the contribution of fungal protease specifically cannot be isolated from these results.
In recent years, several well-designed clinical studies have shown that pancreatic or digestive enzyme supplements could be promising alternative approaches in managing functional dyspepsia syndrome. Postmarketing surveillance studies of a multienzyme formulation to evaluate efficacy and tolerability revealed that treatment was able to decrease frequency and severity of various dyspeptic symptoms in patients with functional dyspepsia.
Overall evidence strength for functional dyspepsia: Preliminary. The available evidence involves multienzyme preparations, making it impossible to attribute effects specifically to fungal protease. More targeted trials isolating fungal protease are needed.
Acute fungal protease supplementation was shown to lower post-prandial duodenal gluten concentration following administration of a porridge with gluten in a cross-over study of gluten-sensitive subjects (König et al., 2017). Related, a combination of 3 fungal proteases and a plant protease reduced GI symptoms in individuals with non-celiac gluten sensitivity (Ido et al., 2018).
DPP-IV (dipeptidyl peptidase IV) may help some people with gluten sensitivity break down trace amounts of gluten, though it is not a substitute for a gluten-free diet in celiac disease. Importantly, the evidence that fungal proteases can degrade gluten sufficiently to render gluten-containing food safe for individuals with celiac disease has not been established in rigorous clinical trials.
Overall evidence strength for gluten sensitivity: Preliminary. A small number of human studies show reductions in duodenal gluten concentrations and GI symptoms, but evidence from large, well-powered RCTs is absent. These preparations are not established treatments for celiac disease.
Several older clinical investigations and patent literature have cited anti-inflammatory properties of Aspergillus oryzae protease preparations. Proteases from Aspergillus oryzae have been shown to be potent anti-inflammatory mediators in some older studies. The clinical evidence supporting this use was generated largely in the 1960s using preparations such as "Asperkinase," with limitations typical of the research methodology of that era.
The study by Oben et al. noted that treatment groups showed significantly reduced C-reactive protein (CRP) levels compared to controls when Aminogen® was co-ingested with whey protein. Acute dosing of Aminogen®, a patented blend of digestive protease enzymes isolated from Aspergillus and blended with whey protein concentrate, increased the rate of protein absorption. The results indicated a faster rate of amino acid absorption reflected in significantly higher blood levels of amino acids, increased nitrogen retention, and significantly reduced levels of C-reactive protein. It is not established whether this CRP reduction reflects direct anti-inflammatory action by fungal protease or an indirect effect of improved protein nutrition.
In an animal (weaned piglet) study, protease addition reduced serum levels of inflammatory markers TNF-α, IL-1β, and IL-6, suppressed mRNA expression of pro-inflammatory factors in the jejunum, and inhibited MAPK and NF-κB signaling pathways. This represents animal data that cannot be directly extrapolated to human supplementation outcomes.
Overall evidence strength for anti-inflammatory effects: Weak to preliminary in the human context. The bulk of mechanistic evidence is from in vitro and animal studies. The CRP reduction seen in human studies may be confounded by protein absorption improvements rather than direct anti-inflammatory enzyme activity.
Peptides generated by fungal protease digestion of whey protein may help facilitate lower blood levels of total cholesterol and LDL cholesterol than peptides produced from whey protein by endogenous proteases, leading to greater cardiovascular effects.
Whey protein containing 3% Aminogen® was well tolerated by subjects and resulted in no significant changes in hemodynamic parameters or markers of clinical safety for cardiovascular, calcium, liver, and kidney function when compared to whey protein alone.
There were surprising findings regarding significant main and interactive effects reported for total cholesterol and LDL cholesterol. Similarly, interactive effects for diastolic blood pressure strongly approached significance (p = 0.06). The authors noted that the whey protein-only group showed increases in total cholesterol and LDL cholesterol that were not seen in the fungal protease + whey group, but these findings require independent confirmation.
Overall evidence strength for cardiovascular effects: Very preliminary. The available evidence is from a single small trial and is insufficient to draw conclusions about fungal protease supplementation and cardiovascular outcomes.
Animal research has produced preliminary findings suggesting that dietary protease supplementation may modulate intestinal microbiota composition. Microbiota compositions were significantly altered by protease addition, with increased abundance of beneficial bacteria (Lachnospiraceae AC2044 group and Prevotellaceae UCG-001) and reduced harmful Terrisporobacter. These findings are from a weaned piglet study and have not been replicated in human clinical trials.
Fungal protease activity in supplements is measured in enzyme activity units rather than mass alone. The principal activity units used in the food supplement industry and the Food Chemicals Codex (FCC) are:
Activity units measure how much digestive work the enzyme can actually perform. Two products can both contain "200 mg protease blend," but one may have 10 times the enzymatic activity of the other depending on the enzyme source and potency.
Dosages reported in clinical and preclinical studies:
Aspergillus oryzae is an important filamentous fungus that is widely applied in the traditional fermentation and food processing industries, such as soy sauce, soybean paste, and sake brewing. This long history of its widespread use in the food industry has led to A. oryzae being recognized by the Food and Drug Administration (FDA) of the United States as Generally Recognized as Safe (GRAS) organisms. Its safety is also supported by the World Health Organization (FAO/WHO).
Carbohydrase and protease from Aspergillus oryzae are listed by FDA as "substances derived from microorganisms recognized by FDA as Generally Recognized as Safe in Opinion Letters."
The main difference that decides their fates for industrial applications is that even though they have a similar gene cluster responsible for production of aflatoxin, only A. flavus is capable of producing aflatoxin whereas A. oryzae does not, no matter the cultivation conditions.
Although supplementing with digestive enzymes is becoming increasingly popular, there is little clinical data available regarding the safety of oral fungal proteases as digestive aids or dietary supplements.
The primary findings from the 30-day double-blind safety study demonstrated that 40 g doses of whey protein twice a day containing 3% Aminogen® are well tolerated, as none of the parameters evaluated including the clinical chemistry profiles were negatively affected. This is also supported by no adverse events being reported throughout the 4-week study period.
Digestive enzyme supplementation may also simply be presumed to be safe, since enzymes have been used in food processing and as food additives for almost a century.
The koji strain of Aspergillus oryzae has been consumed in foods for many years worldwide. While the A. oryzae interventions did not always report benefits in animal feed supplement experiments, there were no reports of adverse effects.
A significant, source-backed safety distinction must be made between ingested fungal protease supplements and the respiratory hazard posed by Aspergillus spore-derived protease allergens through inhalation. These are mechanistically separate exposure routes with different risk profiles.
Asthma affects more than 250 million people worldwide, and nearly 10% of people with asthma have severe, treatment-resistant disease, which is frequently associated with IgE sensitization to ubiquitous fungi, typically Aspergillus fumigatus. A major Aspergillus fumigatus allergen, Asp f13, which is a serine protease (alkaline protease 1), promotes airway hyper-responsiveness by infiltrating the bronchial submucosa and disrupting airway smooth muscle cell–extracellular matrix interactions.
Aspergillus secretes a serine protease — Alkaline Protease 1 or Asp f 13 — that drives IgE antibody production in animal models and persons with asthma, and the quantity of Asp f 13 detected in the lungs of asthmatics directly correlates with disease severity.
This allergenic concern relates primarily to inhalational exposure (e.g., environmental mold exposure or occupational exposure to enzyme dusts), not to oral ingestion of purified, food-grade enzyme preparations. Nevertheless, individuals with documented Aspergillus hypersensitivity should be aware that oral fungal protease preparations derive from the same genus, and cross-reactivity has been documented in the allergy literature. Cross-reactivity has been reported among fungal species.
A distinguishing feature of protease allergens is their intrinsic ability to overcome host tolerance. Without additional adjuvants, allergenic house dust mite extracts, papain, or fungal allergens can induce allergen-specific IgE, initiate gene expression and mediator release, and recruit eosinophils and antigen-presenting cells to challenged airways or skin. This process is prevented by specific and non-specific protease inhibitors.
These mechanisms, extensively characterized in the context of inhaled allergens, have not been directly demonstrated for orally ingested, purified food-grade fungal protease enzyme supplements. The oral route involves gastrointestinal degradation of proteins before systemic absorption, substantially altering the immunological exposure compared to inhalation.
In addition to anti-inflammatory effects, proteases are administered orally for digestive enzyme replacement (digestive aids, cystic fibrosis) and for potentiation of drug effects, especially of antibiotics. The potentiation of antibiotic activity by oral proteases is a documented pharmacological interaction for systemic protease preparations; however, whether food-grade fungal protease supplements at typical supplemental doses produce clinically meaningful drug interactions has not been established in rigorous human trials.
The systemic absorption of active fungal protease enzyme has been a subject of investigation. The prevailing finding of some studies is that proteases can be partially absorbed intact, with activity preserved, from the digestive tract and subsequently distributed systemically. However, whether this occurs to a clinically meaningful extent with orally dosed food-grade fungal protease supplements — rather than with pharmaceutical protease preparations — has not been established.
Certain strains of A. oryzae have been shown to produce the mycotoxins aspergillic acid, kojic acid, cyclopiazonic acid, and B-nitropropionic acid, and maltoryzine. For this reason, commercial-grade fungal enzyme preparations intended for food and supplement use must be produced from qualified, tested strains that have been verified not to produce toxins at biologically relevant levels, and are subject to quality controls under GRAS frameworks.
Health conditions that Fungal protease may help support.
Blends of microbial digestive enzymes including fungal protease have shown significant reductions in GI symptoms such as gas, bloating, and indigestion in clinical trials involving functional dyspepsia and general upper GI discomfort. Fungal proteases' acid stability allows activity early in the gastric phase, which is mechanistically favorable for reducing incompletely digested protein fragments.
Clinical research on Aminogen®, a fungal protease blend derived from Aspergillus niger and Aspergillus oryzae, shows that co-ingestion with whey protein significantly increases postprandial plasma amino acid levels and nitrogen retention in active men. Enhanced amino acid bioavailability from supplemental protein is a mechanistic pathway relevant to training adaptations. Evidence is preliminary and product-specific rather than broadly generalizable to all fungal protease preparations.
Fungal proteases from multiple Aspergillus species exhibit documented fibrinolytic activity in vitro and in early human/animal studies, degrading fibrin and activating hemostasis-related proteins. Aspergillus oryzae-derived CA-7 was used clinically as a thrombolytic agent in case reports. Most current evidence for fibrinolytic activity of supplemental-grade oral fungal protease is indirect.
Proteolytic enzymes, including microbial/fungal-derived proteases, have shown the capacity to reduce systemic inflammatory markers such as CRP and ESR in controlled clinical trials. The proposed mechanism involves degradation of pro-inflammatory protein mediators and immune complexes. Evidence is primarily from multi-enzyme formulas rather than single-ingredient fungal protease products.
Fungal proteases derived from Aspergillus oryzae and related species are active across a broad pH range (3–9), making them effective throughout the GI tract. Clinical studies show fungal protease supplementation reduces duodenal gluten concentration in gluten-sensitive subjects and reduces GI symptoms in functional dyspepsia. A broad-spectrum clinical RCT confirmed significant dyspepsia symptom reduction.
Aspergillus-derived fungal proteases, particularly AN-PEP and Aspergillus oryzae/melleus enzyme blends, have demonstrated in clinical trials the ability to degrade immunogenic gluten peptides and reduce associated symptoms in gluten-sensitive individuals. Reduction of intact immunogenic peptide load is the primary mechanistic basis for attenuated allergic/sensitivity responses.
Fungal proteases derived from Aspergillus species have demonstrated the ability to degrade gluten peptides in the stomach before they reach the small intestine, reducing post-prandial gluten concentrations and symptom scores in gluten-sensitive individuals. A prolyl endopeptidase from Aspergillus niger (AN-PEP) and multi-fungal protease blends are the best-studied preparations. Evidence comes from multiple randomized crossover trials.
Digestive enzyme blends containing fungal protease have been employed as adjunct therapies in functional GI disorders including IBS, with clinical evidence from dyspepsia trials supporting symptom reduction. Protease activity may help reduce the protein fermentation load reaching the colon, which can contribute to IBS symptoms. Evidence specific to IBS vs. functional dyspepsia remains limited.
Enhanced amino acid bioavailability from fungal protease co-ingestion with dietary protein is mechanistically linked to muscle protein synthesis and recovery. Clinical trials with Aspergillus-derived protease blends show significantly increased postprandial amino acid levels and nitrogen balance in resistance-trained individuals. Evidence for direct reduction of DOMS or muscle damage biomarkers specifically attributable to fungal protease is not yet robustly established.
Multi-enzyme formulations containing Aspergillus-derived microbial proteases have been tested in randomized controlled trials for post-surgical inflammation and wound healing, demonstrating significant reductions in CRP, ESR, pain scores, and analgesic use versus placebo. Both general surgical and orthopedic post-operative settings have been studied.
Body systems that Fungal protease may help support.
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