IngredientsAcid protease is the collective name for a class of proteolytic enzymes defined by their optimal catalytic activity under acidic conditions. An acid protease is a protein-digesting enzyme that exhibits maximum activity and stability in acid conditions (pH 2.0–5.0) and is inactivated at pH values above 6.0. Acid proteases have a low isoelectric point and are low in basic amino acids.
According to the optimal pH value of the reaction, proteases are divided into acid protease, neutral protease, and alkaline protease. The general term covers a class of enzymes that hydrolyze protein peptide bonds; according to the way they hydrolyze polypeptides, they can be divided into endopeptidases and exopeptidases. Aspartic proteases, which constitute the predominant biochemical class of acid proteases, consist of 380–420 long chains of amino acid residues constituting the active site for catalytic activity. These acidic proteases are endopeptidases and grouped into three families: pepsin (A1), retropepsin (A2), and enzymes from Para retroviruses (A3).
Acid proteases are mainly aspartic proteases and are distributed across all forms of life, including vertebrates, plants, fungi, bacteria, and viruses. In official biochemical nomenclature, the aspartic protease class is designated EC 3.4.23 by the International Union of Biochemistry. In terms of optimal pH for enzymatic action, there are three major families of carboxyl proteinases which act at acidic pH: aspartic peptidases, serine-carboxyl peptidases, and glutamic peptidases.
Key synonyms and related terms include: acid proteinase, aspartic protease, aspartyl protease, aspartic endopeptidase, and — for the principal endogenous mammalian representative — pepsin. Acid proteases are a well-established group of proteolytic enzymes which digest proteins and peptides in an acidic solution. Some well-known acid proteases are pepsin, gastricsin, chymosin, and cathepsin D. Most of these enzymes share similar amino acid sequences, three-dimensional structures, active-site structures, and catalytic mechanisms. The primary acid protease of commercial and dietary supplement importance is derived from fungal fermentation, principally from species of Aspergillus and Rhizopus/Mucor. Fungus-derived acid proteases such as Aspergillopepsins I and II from Aspergillus niger are most commonly used in the food and beverage industries.
Fungal Protease A from Aspergillus oryzae carries CAS# 9025-49-4; Acid Stable Protease A from Aspergillus niger also carries CAS# 9025-49-4; Protease AM from Aspergillus melleus carries CAS# 9074-07-1.
The sources of microbial acid proteases are extensive and may originate from any type of microorganism. Fungal proteases have been used in the food industry due to their safety and enzymatic characteristics. Two types are widely used in the food and beverage industries: those from Aspergillus, which resemble pepsin, and those from Mucor, which resemble rennin.
In comparison with alkaline proteases, extracellular acid proteases are mostly produced by fungal species, such as Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, Aspergillus fumigatus, and Aspergillus saitoi. Additional commercially relevant species include:
In vertebrates, pepsin is the primary endogenous acid protease. An acid protease called pepsin is released into the stomach by "chief cells" in the stomach lining. Pepsin is present in gastric juice as pepsinogen, which in the presence of HCl is converted into pepsin; pepsin then partially hydrolyzes proteins into proteoses and peptones. In addition to fungal and mammalian sources, the archeal thermophile Sulfolobus acidocaldarius produces a heat-stable acid protease (thermopsin), and select bacterial strains of Bacillus sp. also produce acid-active proteases.
Acid protease preparations used in dietary supplements are derived almost exclusively by microbial fermentation, followed by extraction, purification, concentration, and drying. They are commercially available as:
Activity units specific to acid protease supplements include the SAP (Spectrophotometric Acid Protease) unit and the SAPU variant. A SAP unit is defined as that proteolytic activity that will liberate one micromole of tyrosine per minute under the conditions of the assay stated in the Food Chemicals Codex; a HUT unit (Hemoglobin Unit, Tyrosine Basis) is defined as the amount of enzyme that produces, in one minute, a hemoglobin hydrolysate whose absorbance at 275 nm is the same as that of a solution containing 1.10 micrograms per milliliter of tyrosine in 0.006N hydrochloric acid.
While the term "acid protease" is a modern biochemical designation, acid protease-producing microorganisms and the fermented foods that rely on their activity have been embedded in culinary and medicinal traditions for millennia.
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 Japanese dynasty and nation have domesticated and utilized koji fermented with non-pathogenic fungus Aspergillus oryzae for more than 1,300 years. Since the earliest historical age of Japan, Japanese people isolated the fungus Aspergillus oryzae, belonging to ascomycetes, and domesticated and utilized it to produce koji as a catalyst for starch in rice; during the process that lasted for 1,300 years, non-pathogenic strains were genetically selected by specialized technicians of koji.
Aspergillus oryzae is a filamentous micro-fungus that has been used for centuries in the fermentation of different foods in many countries all over the world and is also a rich source of many bioactive secondary metabolites. Key enzymes expressed during koji fermentation include alkaline protease (optimal at pH 9.0 and 40°C), neutral protease I (broad specificity), and acid protease (optimal at pH 3.7, 39 kDa).
The acid protease gene (pepA) of Aspergillus oryzae is specifically expressed in solid-state rice-koji culture. Acid protease produced by A. niger can retain activity in the low-pH environment formed at the late stages of soy sauce brewing. Koji making is one of the most important steps in soy sauce brewing: soybeans are steamed as pretreatment, then mixed with flour and wheat, followed by fermentation; the most commonly used industrial koji-making process involves fermentation with Aspergillus oryzae to yield soy sauce.
Aspergillopepsin I, formerly known as aspergillopeptidase A, was isolated from Aspergillus saitoi, a microorganism used in fermentation of the traditional Japanese liquors awamori and shochu.
Tempeh, an Indonesian traditional fermented food lasting for more than 400 years, is produced by fermenting soybeans with Rhizopus oligosporus or R. oryzae and has several health benefits including improvement of cognitive function, gut immunity, intestinal microbial flora, hyperlipidemia, and anemia. The acid proteases secreted by these organisms are directly responsible for the partial hydrolysis of soy proteins during fermentation.
Recent research has elucidated that koji contains medicinal substances such as Taka-diastase, acid protease, koji glycosylceramide, kojic acid, oligosaccharides, ethyl-α-D-glucoside, ferulic acid, ergothioneine, pyroglutamyl leucine, pyranonigrin A, resistant proteins, deferriferrichrysin, polyamines, Bifidobacterium-stimulating peptides, angiotensin I-converting enzyme inhibitor peptides, 14-dehydroergosterol, beta-glucan, biotin, and citric acid. The acid protease from A. oryzae (Taka-protease) was among the first microbial enzymes to be isolated and studied in the late 19th century by Japanese chemist Jokichi Takamine, whose preparation of digestive enzymes from koji laid the groundwork for the modern enzyme supplement industry.
Commercial acid protease supplements are typically comprised of one or more of the following biochemically distinct enzyme types:
Most aspartic proteases have a bilobal structure. The cleft where the substrate binds is deep and situated between the two lobes. The two catalytic aspartate residues (Asp32 and Asp215 in the pepsin numbering) lie in each of the two lobes and are exquisitely aligned at the bottom of the cleft. This structural feature is the basis for their catalytic activity. Eukaryotic aspartic proteases further include conserved disulfide bridges, which can assist in identification of the polypeptides as aspartic acid proteases.
Aspergillopepsins are extracellular proteinases secreted by fungal mycelia. The enzymes are of practical importance for fungal nutrition in an acidic environment. The optimal pH of Aspergillopepsin I for milk casein digestion is in the pH range of 2.5–3.0 and the proteinase is fairly stable over the range of 2.5–6.0.
Pepsin A (EC 3.4.23.1) shows particularly broad specificity; although bonds involving phenylalanine and leucine are preferred, many others are also cleaved to some extent. Preferential cleavage involves hydrophobic, preferably aromatic, residues in P1 and P1' positions. Aspergillopepsin I from A. saitoi generally favors hydrophobic amino acid residues in P1 and P1', but it also accepts Lys in P1, which leads to activation of trypsinogen at acidic pH. The broad specificity of acid-stable protease enables the enzyme to easily and efficiently hydrolyze most soluble proteins.
The most widely accepted mechanism for aspartyl proteases is a general acid-base mechanism involving coordination of a water molecule between the two highly conserved aspartate residues. One aspartate activates the water by abstracting a proton, enabling the water to perform a nucleophilic attack on the carbonyl carbon of the substrate scissile bond, generating a tetrahedral oxyanion intermediate stabilized by hydrogen-bonding with the second aspartic acid. Rearrangement of this intermediate leads to protonation of the scissile amide, which results in the splitting of the substrate peptide into two product peptides.
The two aspartyl residues are in close geometric proximity in the active molecule: one aspartate is ionized whereas the second one is unionized at the optimum pH range of 2–3. In contrast to serine and cysteine proteases, catalysis by aspartic protease does not involve a covalent intermediate, though a tetrahedral intermediate exists. The nucleophilic attack is achieved by two simultaneous proton transfers: one from a water molecule to the diad of the two carboxyl groups, and a second from the diad to the carbonyl oxygen of the substrate with concurrent CO–NH bond cleavage. This general acid-base catalysis, which may be called a "push-pull" mechanism, leads to the formation of a non-covalent neutral tetrahedral intermediate.
Different proteases require different pH levels to be activated and function optimally. Acid proteases such as pepsin work best in the stomach, as pepsin requires activation by stomach acid. The partially digested proteins produced in the stomach are then acted on by pancreatic enzymes in the small intestine.
Because acid-stable fungal protease (Protease 3.0) has an effective pH range of 2.75 to 4.7, it is uniquely suited to work synergistically with endogenous pepsin to provide protein digestion in the stomach. A unique combination of at least one acid protease fungal enzyme and at least one semi-alkaline protease fungal enzyme allows enzymatic activity to occur throughout the gastrointestinal pH spectrum associated with a human digestive system, converting ingested dietary protein into free amino acids and short chain peptides.
Research, inspired by the proteases of human breast milk, has demonstrated that exogenous microbial proteases can activate within the human digestive tract and substantially increase the digestion of targeted proteins that are otherwise difficult to fully digest.
Nearly all known aspartyl proteases are inhibited by pepstatin, a natural pentapeptide produced by various Streptomyces species. Aspergillopepsin I from A. saitoi is resistant to inhibitors of ordinary pepsin-type aspartic proteinases such as pepstatin, DAN, and l,2-epoxy-3-(p-nitrophenoxy)propane (EPNP); no naturally occurring inhibitor is known for this enzyme, though the propeptide is a strong inhibitor.
Evidence level: Moderate for in vitro; Preliminary to moderate for human clinical data; results are mixed and limited by small sample sizes and industry funding in several trials.
Research has demonstrated that exogenous microbial proteases can activate within the human digestive tract and substantially increase the digestion of targeted proteins. Using the INFOGEST 2.0 in vitro protocol, an acid-active family of bacterial proteases (S53 family) substantially improved the digestibility of an array of animal and plant-derived proteins — soy, pea, chickpea, rice, casein, and whey. On average, this protease elevated protein digestibility by 115% during the gastric phase and by 15% in the intestinal phase, based on the degree of hydrolysis. This study was conducted in vitro, not in humans, and was funded in part by Digestiva, Inc., and Mars Inc.
A key series of clinical investigations examined Aminogen®, a proprietary blend of fungal proteases derived from Aspergillus species. A patented oral fungal protease blend (Aminogen®) maintains activity after oral dosing. Acute dosing of Aminogen® with whey protein concentrate was effective for increasing the rate of protein absorption; forty healthy males were divided into two groups and received either 50 g whey protein concentrate (80% protein) or 50 g of whey protein concentrate with Aminogen®. The results indicated a faster rate of amino acid absorption reflected in significantly higher blood levels of amino acids and increased nitrogen retention in the Aminogen® group.
A double-blind clinical study investigated a fungal protease enzyme system over 30 days in active, healthy men. 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; 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. However, few studies have examined the safety of repeated dosing of oral enzymes with an appropriate substrate.
A 2025 randomized, double-blind, placebo-controlled crossover clinical trial published in a peer-reviewed journal evaluated the effects of a mixture of three microbial protease preparations (P3) when co-ingested with whey protein concentrate. The aim was to assess the effects of a mixture of 3 microbial protease preparations (P3) on postprandial plasma amino acid concentration when co-ingested with whey protein concentrate (WPC) in healthy young adults; P3 was first tested in vitro for proteolytic effects in a static simulation of orogastric digestion. In a subsequent randomized, double-blinded, placebo-controlled crossover study, 12 males and 12 females (mean BMI: 23.6; mean age: 25 years) consumed WPC (25 g protein) containing P3 or placebo. Co-ingestion of WPC with P3 was found to enhance early postprandial plasma aminoacidemia and alter select indices of appetite and satiety in young adults. This trial was funded by BIO-CAT, Inc.
In vivo human research using microbial proteases as a nutritional strategy to enhance the postprandial increase in blood amino acid concentration has either shown a benefit or no difference when compared with a placebo. Using 2.5 g and 5 g of Aminogen (proteases from Aspergillus niger and A. oryzae), increased serum amino acid concentration was reported following co-ingestion with 50 g WPC compared with WPC alone in young healthy male participants. Overall, these results suggest that the source, type, and/or dose of each microbial protease and/or dietary protein may influence the capacity of proteolytic enzymes to augment postprandial blood amino acid concentration.
Earlier studies have shown that the co-ingestion of an exogenous enzyme blend along with plant protein increases protein digestibility and reduces the difference between animal and plant protein in terms of nutritive benefits. Several clinical studies have demonstrated a positive impact of enzyme supplementation on protein digestion and absorption.
Limitation note: The majority of human trials in this area are small (fewer than 50 participants), short-duration (acute or ≤30 days), use heterogeneous enzyme products, and a number of published trials have been funded by the manufacturers of the proprietary enzyme blends tested. Independent replication with pre-registered, adequately powered trials remains limited.
Evidence level: Preliminary (in vitro and rationale-based); limited direct clinical evidence for fungal acid protease specifically in this context.
High protein consumption and over-processing inhibit the activity of endogenous proteases, resulting in incomplete digestion of protein; the addition of digestive enzymes increases the digestion and possibly enhances the absorption rate. The widespread adoption of acid-active proteases has the potential to enhance nutritional value and contribute to food security and sustainability; this approach could complement ongoing efforts to improve proteins in the food supply, increase the quality of more sustainable protein sources, and aid in the nourishment of patients with clinically compromised, fragile intestines and individuals such as older adults and high-performance athletes who have elevated protein needs.
Data suggest that protease supplementation for enhanced protein digestion and amino acid absorption may best be directed toward individuals with compromised endogenous digestive capacity, such as older adults or those with partial pancreatic insufficiency. However, for frank exocrine pancreatic insufficiency (EPI), pharmaceutical-grade prescription pancreatic enzyme replacement therapy (PERT) — rather than dietary supplement-grade acid proteases alone — remains the standard of care. Steatorrhea is the most important digestive manifestation in EPI; the current treatment includes enzyme supplementation with porcine pancreatic enzyme concentrate, consisting mainly of lipase, amylase, and protease.
Evidence level: In vitro only; no direct clinical evidence in humans as a supplement.
In vitro digestion studies using acid protease supplements such as PepzymeAG (75,000 HUT/g, acid proteases from Aspergillus niger) have explored the generation of bioactive peptides with antioxidant and antidiabetic properties from dietary proteins. Effects on protein have been evaluated in terms of degree of hydrolysis, peptide pattern, and biological activities of the peptides under simulated gastrointestinal conditions using the INFOGEST harmonized method. These findings are entirely preliminary and cannot be extrapolated to clinical outcomes in humans.
Acid proteases, among other functions, may be used as a substitute for activities associated with renin, papain, and pepsin. Species of Aspergillus and Mucor are important acid protease sources. While this is not a health/supplement application, the coagulation of milk proteins by acid proteases from Mucor miehei and Rhizomucor pusillus has been practiced industrially for decades as a vegetarian rennet substitute in cheese production.
Important note: Acid proteases are measured in enzyme activity units rather than mass alone; a given mass of product with low enzyme activity is not equivalent to the same mass at higher activity.
The long history of safe use of A. oryzae by the food fermentation industry and the lack of aflatoxin production has earned it GRAS (generally recognized as safe) status from the FDA. Aspergillus oryzae and Aspergillus niger are on the generally recognized as safe (GRAS) list of the Food and Drug Administration (FDA) in the United States. 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."
A. oryzae has a long history of safe use in the production of food ingredients and has been used to produce numerous food ingredients that are GRAS. Unlike some members of the genus Aspergillus, there is no record of A. oryzae producing mycotoxins, and finished products derived from it have shown non-detectable levels of T-2 toxin, zearalenone, ochratoxin A, sterigmatocystin, and aflatoxins B1, B2, G1, and G2.
Protease enzyme supplements are generally considered safe with a low risk of adverse effects. Common side effects include mild gastrointestinal symptoms such as bloating or discomfort, but these are uncommon. Few studies have examined the safety of repeated dosing of oral enzymes with an appropriate substrate, and robust long-term safety data from controlled human trials remain limited.
While A. oryzae interventions in animal studies did not always report benefits, there were no reports of adverse effects.
Individuals with known mold sensitivities or documented allergies to Aspergillus species represent the principal population at elevated risk. Aspergillus fumigatus and related pathogenic aspergilli produce proteases that act as potent allergens when inhaled, but the dietary supplement species A. oryzae and A. niger are not pathogenic and are classified as GRAS by the FDA. Nonetheless, the documented allergenic potential of Aspergillus-family proteases via the inhalation route is important context for sensitive individuals handling enzyme powders in bulk form.
A secreted protease of Aspergillus oryzae was shown to elicit Th2 immunity and allergic inflammation in mice through proteolytic cleavage of fibrinogen. This finding derives from an airway exposure model and is not directly applicable to the oral dietary supplement route; however, it underscores the need for caution in occupational or bulk-handling contexts.
Because of economic and food safety issues, A. oryzae continues to be classified as a taxon separate from the closely related A. flavus, a predominant producer of aflatoxins. The long history of safe use of A. oryzae by the food fermentation industry and the lack of aflatoxin production has earned it GRAS status from the FDA. Quality-assured production from reputable manufacturers involves testing finished enzyme preparations for mycotoxin absence.
The heat generated in food processing and cooking typically destroys enzyme activity before ingestion. Conversely, activity of enzymes for dietary supplementation should be intact to aid in digestion when taken orally. Clinical research has shown that a patented oral fungal protease blend maintains activity after oral dosing.
No formally documented or clinically established drug interactions specific to orally ingested fungal acid proteases are reported in the reviewed literature. Nearly all known aspartyl proteases are inhibited by pepstatin, suggesting that concurrent use of pepstatin (a naturally occurring metabolite occasionally present in certain supplements) could theoretically reduce acid protease activity, though this interaction has not been documented clinically at supplement doses.
Individual humans differ in protein digestive capacity. Excess amounts of protein intake could reduce the percentage of protein absorption by the intestine. Populations with reduced endogenous protease capacity — including older adults, individuals post-bariatric surgery, and those with conditions associated with hypochlorhydria — represent a potential target population for acid protease supplementation; however, controlled trials in these specific groups have not been extensively published as of the time of writing.
The biochemistry of acid proteases — their structure, catalytic mechanism, pH optima, and endogenous physiological roles — is very well established in peer-reviewed literature. The commercial and industrial use of fungal acid proteases in East Asian fermentation is documented over centuries and corroborated by modern microbiological and biochemical research.
The evidence for exogenous acid protease supplementation improving protein digestion and amino acid bioavailability in healthy humans is preliminary to moderate. Several small randomized controlled trials and acute crossover studies have reported significant increases in postprandial serum amino acids and nitrogen retention when fungal protease blends (including acid-active components) are co-ingested with dietary protein. However, many of these studies have been conducted by or funded by manufacturers, involve small samples, use proprietary blends (making it difficult to isolate the contribution of the acid-active fraction specifically), and have not been independently replicated at scale. In vitro data are more consistent in demonstrating enhanced protein hydrolysis, but the translation to clinically meaningful human health outcomes requires further investigation.
Health conditions that Acid protease may help support.
Acid protease (fungal acid protease) is active at gastric pH levels (pH 2.0–4.0), complementing the limited pH range of pancreatic proteases. It is included in broad-spectrum digestive enzyme supplements to ensure protein digestion across the full pH spectrum of the GI tract. Acid-stable fungal proteases have been studied for gluten hydrolysis.
Body systems that Acid protease may help support.
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