Blakeslea trispora

Identity and Classification

Taxonomy and Nomenclature

Blakeslea trispora Thaxter (1914) is a non-photosynthetic, filamentous, aerobic mold. It is a zygomycete fungus classified in the family Choanephoraceae, Order Mucorales. Its full taxonomic placement is: Kingdom Fungi; Phylum Zygomycota; Class Zygomycetes; Order Mucorales; Family Choanephoraceae; Genus Blakeslea; Species trispora. The genus Blakeslea was named in honor of American botanist Albert Francis Blakeslee, based on B. trispora as the type species.

A bisexual mold from the Zygomycetes family, Blakeslea trispora stands out as an important microbial producer of natural β-carotene, a valuable compound with significant nutritional and industrial applications. It is the only mucoral fungus of this group used commercially to produce β-carotene and lycopene.

Natural Habitat and Growth

It is isolated from soils in subtropical and tropical regions of the world (e.g., southeastern United States, Southeast Asia) and is reported as a pathogen of multiple plant species. In vivo pathogenicity testing using animal models suggests this fungus is not a cause of animal or human disease.

It produces two types of sporangia — a major form of asexual reproduction among zygomycetes — including large sporangia with many sporangiospores and sporangioles with only a few or one sporangiospore. Sporangiospores are pigmented, striate, and fusiform, typically with polar appendages.

Common Forms and Preparations

β-Carotene is obtained from B. trispora by co-fermentation of the two sexual types of the fungus in specific proportions. Both types are stable in cultures maintained under conditions consistent with good manufacturing practice. These source organisms are neither pathogenic nor toxinogenic. The compound is isolated from the fungal biomass by solvent extraction and crystallized. The main articles of commerce are suspensions in food-grade vegetable or plant oil and water-dispersible powders. These formulations are made for ease of use and in order to improve stability, as carotenes easily oxidize.

The colouring principle consists predominantly of trans β-carotene together with variable amounts of cis isomers of β-carotene. Minor amounts of other carotenoids, of which γ-carotene accounts for the major part, may also be present. The only organic solvents used in the extraction and purification are ethanol, isopropanol, ethyl acetate, and isobutyl acetate.

The final β-carotene product appears as red to brownish-red crystals or a crystalline powder. For lycopene, the lycopene product is formulated into a 20% or 5% sunflower oil suspension.

Historical and Traditional Use

Blakeslea trispora has no documented history of traditional use in food or medicine by any culture in the way that many botanical dietary supplements do. The organism is a soil-dwelling plant pathogen, not a food traditionally consumed by human populations. Its significance as a source ingredient emerged entirely from 20th-century industrial microbiology research.

Traditionally, carotenoids are produced by extraction from natural plant sources and by chemical synthesis for use as food colorants, animal feed supplements, and nutraceuticals for cosmetic and pharmaceutic purposes. β-Carotene's chemical synthesis has been known since 1956. The identification of B. trispora as a microbial production platform for β-carotene developed in subsequent decades; due to the increasing importance of β-carotene, biotechnological methods of production have been sought, with the green alga Dunaliella salina and the fungus Blakeslea trispora emerging as the microorganisms of choice for industrial production.

Several fungi of the order Mucorales synthesize β-carotene, including Phycomyces blakesleeanus, Choanephora cucurbitarum, and Blakeslea trispora. Industrial production of β-carotene from fungi has focused on B. trispora because of its inherently higher β-carotene content and the ability to grow well in standard fermentations of liquid media. Commercially, Vitan Ltd. (Ukraine) and Vitatene (Spain) use the heterothallic zygomycete Blakeslea trispora, producing, after intensive classical strain improvement by random mutagenesis and selection, up to 7 g/L β-carotene in large-scale fermentations according to patent literature.

Key Constituents and Active Compounds

β-Carotene

β-Carotene has a molecular weight of 536.9 and the molecular formula C₄₀H₅₆ with eleven conjugated double bonds. β-Carotene is a molecule which displays a red-orange pigment. Synthetic β-carotene has the all-trans isomeric configuration, whereas β-carotene from various natural sources has a variety of forms: all-trans, mono-cis, di-cis, and poly-cis. β-Carotene is the most active carotenoid, known for its ability to convert into vitamin A.

Lycopene

Lycopene is an intermediate in the biosynthesis of all dicyclic carotenoids including β-carotene. The predominant lycopene isomer produced in material from B. trispora is all-trans lycopene. Lycopene is one of the most important carotene molecules because it is capable of producing both β-carotene and other carotenoids, well known for their potent antioxidant activities.

Phytoene and Other Minor Carotenoids

In unmated cultures of B. trispora, the major carotene is phytoene (7,8,11,12,7′,8′,11′,12′-octahydro-ψ,ψ-carotene). In addition, β-carotene and traces of lycopene (ψ,ψ-carotene) and γ-carotene (β,ψ-carotene) are present. The non-photosynthetic carotenogenic fungus Blakeslea trispora is important for the production of β-carotene and lycopene due to its legal status and capacity to synthesize elevated levels of certain compounds that support elevated carotenoid output.

Trisporic Acid (Trisporoids)

Trisporic acid (TA) is secreted from the mycelium of B. trispora during mating between heterothallic strains and is considered as a mediator of the regulation of mating processes and an enhancer of carotene biosynthesis. Carotenoids are precursors of many apocarotenoids that contain very important sex-specific precursors, trisporic acid (TSA) for the sexual reproduction of Blakeslea trispora. Carotenes produced from carotenoids are further processed by carotene oxygenase to synthesize trisporic acid. TSA produced from carotene stimulates both sexually complementary cells to make contact with each other, and TSA is considered an important signalling molecule for the initiation and control of sexual reproduction.

Mechanisms of Action and Biosynthetic Pathway

Carotenoid Biosynthetic Pathway

β-Carotene biosynthesis in B. trispora occurs via the mevalonate pathway. The biosynthesis pathway involves key enzymes such as phytoene synthase, lycopene cyclase, and phytoene dehydrogenase, which are encoded by genes like carRA and carB.

The pathway contains three enzymatic activities: (i) phytoene synthase, which links two molecules of geranylgeranyl pyrophosphate to form phytoene; (ii) phytoene dehydrogenase, which introduces four double bonds in the phytoene molecule to yield lycopene; and (iii) lycopene cyclase, which sequentially converts the ψ acyclic ends of lycopene to β rings to form γ-carotene and β-carotene.

More specifically, acetyl-CoA is first condensed to 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA), then converted to mevalonate (MVA) by HMG-CoA reductase (encoded by hmgr), and then to isopentenyl pyrophosphate (IPP), a precursor of isoprenoid biosynthesis. IPP is converted to farnesyl pyrophosphate (FPP) and then to geranylgeranyl pyrophosphate (GGPP). Condensation of two GGPP molecules by phytoene synthase results in phytoene; lycopene is then produced by phytoene dehydrogenase (CarB). Lycopene can also be cyclized into γ-carotene and β-carotene by lycopene cyclase.

Genetic Basis of Biosynthesis

The carB and carRA genes are the key genes involved in β-carotene biosynthesis in B. trispora. The carB gene has a length of 1,955 bp, including two introns of 141 and 68 bp, and encodes a protein of 66.4 kDa with phytoene dehydrogenase activity. The carRA gene contains 1,894 bp, with a single intron of 70 bp, and encodes a protein of 69.6 kDa with separate domains for lycopene cyclase and phytoene synthase.

The carRA genes have two domains: (i) the R domain, located near the 5′ end, which encodes lycopene cyclase activity, and (ii) the A (or P) domain, which is downstream of the R domain and encodes phytoene synthase.

Role of Mating and Trisporic Acid in Carotenogenesis

The accumulation of β-carotene in B. trispora is associated with sexual interaction. Mating of two sexually compatible strains increases mycelial β-carotene accumulation 5- to 15-fold, as does the addition of trisporic acids, compounds with hormonal activity formed upon mating.

Stimulating both mating types by TSA promotes synthesis of β-carotene. As β-carotene is produced, it becomes a precursor of trisporoid, which is a pheromone for B. trispora. Production of β-carotene promotes a positive feedback process that further stimulates carotenogenesis and the production of trisporoid, which serves as a β-carotene–increasing substance.

Pigment production and accumulation is stimulated by sex-specific pheromones, for example trisporic acids, the synthesis of which commences after inoculation of the production fermenters with both B. trispora mating partners. The high β-carotene accumulation of 4% or more within the B. trispora biomass may be facilitated by the cell's high triglyceride content.

Industrial Lycopene Production

For industrial production of lycopene, Blakeslea trispora is grown with a lycopene cyclase inhibitor which can be introduced into the fermentation process. This blocks the enzymatic conversion of lycopene to β-carotene, allowing lycopene to accumulate. The zygomycete Blakeslea trispora is used on an industrial scale to produce β-carotene, and a semi-industrial process has also been developed for lycopene production.

Antioxidant Mechanisms of the Carotenoid Products

Carotenoids have highly efficient antioxidant scavenging activities against reactive oxygen species (ROS), such as singlet oxygen and free radicals. Therefore, they have the ability to prevent chronic diseases such as cancer, cerebrovascular and cardiovascular diseases, and myocardial infarction.

The industrial importance of β-carotene results from its provitamin A, antioxidant, and immunomodulatory activity as well as its protective effect against cancer and cardiovascular diseases.

Regulatory and Safety Status

This microorganism is recognized as safe for consumption by the Food and Drug Administration (FDA), and is known for its high production of both beta-carotene and lycopene.

The European Union has granted multiple regulatory authorizations for carotenoids derived from B. trispora. The European Commission's Scientific Committee on Food (SCF) considered that β-carotene produced by co-fermentation of Blakeslea trispora DS 30627 and DS 30628 is equivalent to the chemically synthesised material used as food colorant and is therefore acceptable for use as a colouring agent for foodstuffs.

The SCF concluded that β-carotene from B. trispora, produced via a similar biosynthetic route and process as lycopene from B. trispora, is acceptable for use as a colouring agent for foodstuffs. A similar position regarding β-carotene from B. trispora was taken by the JECFA (Joint FAO/WHO Expert Committee on Food Additives), who concluded that β-carotene isolated from different sources, including B. trispora, is acceptable for food additive use.

Decision 2009/365/EC authorised the placing on the market of lycopene from Blakeslea trispora as a novel food ingredient in the European Union. This followed an earlier authorization, Decision 2006/721/EC, also authorising the marketing of lycopene from B. trispora as a novel food ingredient.

Under Commission Implementing Regulation (EU) 2017/2470, lycopene from Blakeslea trispora was authorized as a novel food with specified maximum levels across food categories, including 2.5 mg/100 g in fruit/vegetable juice-based drinks, 8 mg/meal in total diet replacements for weight control, 5 mg/100 g in breakfast cereals, 10 mg/100 g in fats and dressings, 3 mg/100 g in bread, and 1 mg/100 g in soups other than tomato soups.

At the international level, the JECFA monograph for β-carotene from Blakeslea trispora was most recently updated at the 87th meeting in 2019, published as FAO JECFA Monograph 23. The substance is assigned the synonym CI Food Orange 5 and INS No. 160a(iii), and is defined as being obtained by a fermentation process using the two sexual mating types (+) and (−) of the fungus Blakeslea trispora.

Scientific Evidence by Area of Use

Antioxidant Activity

Evidence level: Primarily in vitro and animal models; limited direct human evidence for B. trispora-derived products specifically.

Most antioxidant evidence for B. trispora-derived carotenoids has been generated using the purified compounds (β-carotene, lycopene) rather than the whole organism or its biomass. β-Carotene, as a potent antioxidant compound, has gained extensive attention. Blakeslea trispora, a filiform aerobic fungus, has been proposed as a natural source of β-carotene for commercial exploitation. However, investigation into whether β-carotene extracted from Blakeslea trispora specifically can attenuate oxidative stress has been limited.

One zebrafish (Danio rerio) study evaluated β-carotene extracted from B. trispora at concentrations of 0, 10, 20, and 40 μg/mL. The results indicated that β-carotene reduced migration of neutrophils and released liver damage. β-Carotene was found to reduce index levels of oxidative stress response (HMOX-1, ROS, NADPH, MDA), inflammatory factors (IL-1β, IL-6, IL-8, TNF-α), and liver function proteins (AST, ALT) increased by copper sulfate. β-Carotene also promoted the activities of SOD, GSH-Px, ACP, AKP, and LZM and increased the protein of immune-related factors, IgM and IFN-γ. These results demonstrate that β-carotene has antioxidant, anti-inflammatory, and hepatoprotective activity and participation in immunoregulation. This was a preclinical animal study; findings cannot be directly extrapolated to humans.

A 2013 study cited in published literature assessed administration of B. trispora powder, which contains high amounts of lycopene, in adult mice. Researchers reported that administration of Blakeslea trispora powder, which contains high amounts of lycopene, has the potential to protect the liver, brain, kidney, and skin against oxidative stress. This is done by reducing the concentration of ROS and by enhancing the activities of antioxidant enzymes. Again, this was an animal study.

Cancer-Related Research

Evidence level: Epidemiological associations are mixed; clinical trial results are inconclusive to potentially adverse for supplementation in high-risk populations. No clinical trials have specifically tested B. trispora preparations as such.

Human clinical evidence in this domain pertains to the constituent carotenoids (β-carotene, lycopene), not to B. trispora fermentation products specifically. Several epidemiological studies have linked increased lycopene consumption with decreased prostate cancer risk. These findings are supported by in vitro and in vivo experiments showing that lycopene not only enhances the antioxidant response of prostate cells, but is even able to inhibit proliferation, induce apoptosis, and decrease the metastatic capacity of prostate cancer cells. However, there is still no clearly proven clinical evidence supporting the use of lycopene in the prevention or treatment of prostate cancer, due to the limited number of published randomized clinical trials and the varying quality of existing studies.

A dose-response meta-analysis of observational studies found that dietary lycopene intake and blood lycopene levels were associated with reduced risk of prostate cancer (RR for dietary lycopene intake: 0.86, 95% CI: 0.75–0.98; RR for blood lycopene levels: 0.81, 95% CI: 0.69–0.96). Dose-response analysis indicated that risk of prostate cancer was reduced by 3% per 1 mg/day increment of dietary lycopene intake. However, both α-carotene and lycopene were not shown to lower the risk of advanced prostate cancer, and the results, if replicated, suggest a need for clinical research into the health benefits of supplementation.

A large prospective nested case-control study within the PLCO Cancer Screening Trial found different results. No association was observed between serum lycopene and total prostate cancer (OR 1.14; 95% CI 0.82–1.58) or aggressive prostate cancer (OR 0.99; 95% CI 0.62–1.57). β-Carotene was associated with an increased risk of aggressive prostate cancer (OR 1.67; 95% CI 1.03–2.72 for highest vs. lowest quintile), in particular regional or distant stage disease (OR 3.16; 95% CI 1.37–7.31).

For lung cancer specifically, large-scale randomized controlled trials generated critical safety findings. The ATBC Study was a randomized, double-blind intervention trial conducted in southwest Finland, in which 29,133 male smokers aged 50–69 years were enrolled and randomly assigned to one of four groups (α-tocopherol, β-carotene, both, or placebo). The ATBC Study reported an 18% excess in cumulative lung cancer incidence and an 8% excess in overall mortality in the β-carotene arm of the trial, whereas the CARET study showed 28% more lung cancer cases and a 17% increase in overall mortality in the active intervention group.

The dose of β-carotene in CARET was 30 mg/day. In the ATBC study, more than 29,000 middle-aged male smokers were randomly assigned to 50 mg of alpha-tocopherol, 20 mg of beta-carotene, both, or a placebo daily for five to eight years.

A meta-analysis, based on data from 109,394 subjects, conclusively demonstrated a 24% increase in the risk of lung cancer among smokers who received high-dose beta-carotene supplements. Intervention studies in smokers have unexpectedly reported increased lung tumor rates after high, long-term, beta-carotene supplementation. The ATBC study, the CARET study, the Antioxidant Polyp Prevention trial, and the E3N study provide evidence that the adverse effects of beta-carotene supplementation are correlated with the smoking status of the study participants.

The reported mechanisms of lycopene action in vivo include regulation of oxidative and inflammatory processes, induction of apoptosis, and inhibition of cell division, angiogenesis, and metastasis formation. There is a recognized need to look for predictive factors to identify a population that may benefit from lycopene supplementation. The potential candidates appear to be race, single nucleotide polymorphisms in carotene-cleaving enzymes, some genetic abbreviations, and insulin-like growth factor-dependent and inflammatory diseases.

Cardiovascular Health

Evidence level: Epidemiological and mechanistic data are promising; robust randomized controlled human trials specifically using B. trispora-derived preparations are absent.

Lycopene and β-carotene, two kinds of important fat-soluble carotenoids, are considered essential nutrients in human diet because they prevent cardiovascular diseases, regulate the immune system, and are considered as anti-carcinogenic agents and antioxidants. β-Carotene and other carotenoids play crucial roles for oxidative stress reduction and cardiovascular protection. These assertions are supported primarily by epidemiological and mechanistic data, not by dedicated interventional trials.

Vitamin A Precursor Activity (Provitamin A)

Evidence level: Well-established biochemical mechanism; β-carotene from B. trispora is internationally recognized as a provitamin A food colorant.

β-Carotene is a precursor of vitamin A and has multiple physiological functions. Its conversion to retinol is a well-characterized and internationally accepted biochemical pathway. The International Numbering System classification of β-carotene from B. trispora as INS No. 160a(iii) reflects its established function as both a colorant and a provitamin A source. This carotenoid is widely used in the production of foods, beverages, animal feed, cosmetics, and pharmaceuticals.

Immune System Modulation

Evidence level: Preliminary; primarily mechanistic and animal/in vitro data.

Animal studies report that β-carotene extracted from B. trispora promoted the activities of SOD, GSH-Px, ACP, AKP, and LZM, and increased immune-related factors IgM and IFN-γ, demonstrating antioxidant, anti-inflammatory, and hepatoprotective activity and participation in immunoregulation in a zebrafish model. Translation of these findings to human clinical outcomes has not been established in controlled clinical trials.

Anti-Aging and Skin Protection

Evidence level: Speculative to preliminary; primarily in animal models.

Researchers have been investigating whether the fungus Blakeslea trispora could be a potent effector of anti-aging because of its ability to efficiently mass-produce amounts of lycopene. No controlled human clinical trials have been published specifically evaluating B. trispora-derived preparations for anti-aging or skin protection endpoints.

Body Systems and Health Areas

The carotenoids produced by Blakeslea trispora are associated with the following body systems and health domains, primarily based on evidence relating to their constituent molecules:

• Visual system and eye health: Via provitamin A (β-carotene → retinol), which is essential for rhodopsin synthesis and maintenance of visual epithelia.
• Immune system: Provitamin A, antioxidant, and immunomodulatory activities have been described for β-carotene.
• Cardiovascular system: β-Carotene and other carotenoids play crucial roles in oxidative stress reduction and cardiovascular protection, as assessed epidemiologically.
• Gastrointestinal/hepatic system: Animal studies suggest hepatoprotective potential of B. trispora-derived β-carotene.
• Oncology (preventive context): Lycopene is a carotenoid nutraceutical that has been associated with enhanced protection against cancer, cardiovascular disease, hypertension, and neurodegenerative and inflammatory diseases, though clinical evidence from randomized trials is inconsistent.
• Skin: Carotenoids accumulate in the skin and epidermis, where they may modulate oxidative stress; however, clinical evidence in this domain for B. trispora-derived materials specifically is minimal.

Dosage Forms and Dosages Reported in Studies

No clinical trials have tested defined doses of Blakeslea trispora biomass or extract directly in humans as a dietary supplement formulation. Dosage information therefore pertains to the regulatory frameworks for its carotenoid outputs.

Regulatory dose frameworks for lycopene from B. trispora:

• An α-tocopherol-containing oil suspension of lycopene, obtained from B. trispora, for use as a novel food ingredient in foodstuffs leading to an additional intake of up to about 2 mg/day was concluded not to be of concern from the safety point of view.
• The EFSA AFC Panel derived an Acceptable Daily Intake (ADI) of 0.5 mg/kg body weight per day for lycopene from all sources.

Regulatory dose frameworks for β-carotene from B. trispora:

• The JECFA Committee established a group ADI of 0–5 mg/kg body weight for synthetic beta-carotene and beta-carotene derived from B. trispora. This ADI applies to use of beta-carotene as a colouring agent and not to its use as a food supplement.

Doses used in clinical trials of constituent carotenoids (not B. trispora-specific):

• The dose of beta-carotene in the CARET trial was 30 mg/day.
• In the ATBC study, male smokers received 20 mg of beta-carotene daily for five to eight years.

Fermentation yields reported in biotechnology studies:

• An adapted strain (A278) produced 54 ± 1.95 mg/L of β-carotene, compared to 21.6 ± 2.11 mg/L for the wild type, representing a 45% increase in yield.
• In olive processing wastewater-based fermentations enriched with nutrients, β-carotene production of 61.2 mg/L (lye) and 64.1 mg/L (washing waters), accounting for 82–88% of total carotenoid content, was reported.

Safety Considerations and Interactions

Safety of the Source Organism

The source organisms (B. trispora mating strains) are neither pathogenic nor toxinogenic. In vivo pathogenicity testing using animal models suggests this fungus is not a cause of animal or human disease. Co-fermentation of Blakeslea trispora DS 30627 and DS 30628 complies with EC specifications and is free of mycotoxins or other toxic metabolites.

Allergenicity

There are no reports on allergic reactions due to Blakeslea trispora. In addition, the amount of protein detected in lycopene preparations made from this fungus is very small. The EFSA Panel concluded that it is currently unlikely that the final product poses a risk of triggering allergic reactions.

Genotoxicity

The JECFA Committee concluded that, on the basis of the source organisms, the production process, and composition characteristics, β-carotene from B. trispora does not raise specific concerns and from a toxicological point of view should be considered equivalent to chemically synthesized β-carotene. This opinion was supported by the negative results in two tests for genotoxicity (mutagenesis and chromosomal aberration).

β-Carotene derived from B. trispora was tested for mutagenic activity in Salmonella typhimurium TA1535, TA1537, TA98, and TA100, with and without an exogenous metabolic activation system from rat liver. No mutagenic activity was observed.

Critical Safety Signal: High-Dose β-Carotene Supplementation in Smokers

This is the most clinically significant safety concern associated with β-carotene from any source, including B. trispora. The Alpha-Tocopherol Beta-Carotene (ATBC) cancer prevention study and the Beta-Carotene and Retinol Efficacy Trial (CARET) reported an increase in lung cancer risk and total mortality in heavy smokers (defined as a smoking history of 20+ pack-years) and asbestos workers who fortified their diet with synthetic β-carotene supplements at daily doses of 20 or 30 mg.

A meta-analysis, based on data from 109,394 subjects, conclusively demonstrated a 24% increase in the risk of lung cancer among smokers who received high-dose beta-carotene supplements.

Importantly, the JECFA Committee reaffirmed that rats are not an appropriate model for deriving an ADI for β-carotene due to the relatively low bioavailability of β-carotene in rats compared with humans. Therefore, the Committee withdrew the group ADIs of 0–5 mg/kg bw for synthetic β-carotene and β-carotene derived from Blakeslea trispora. The Committee was unable to establish a group ADI for these substances because a group ADI is applicable to the general population, which includes heavy smokers.

The biological explanation remains to be elucidated for the discrepancy between the potential protective effects against lung cancer from dietary levels of β-carotene in the general population (including heavy smokers) and the apparent increase in lung cancer risk for certain populations exposed to high amounts of lung carcinogens with concurrent use of β-carotene supplementation.

Lycopene ADI Exceedance Concerns in Children

The EFSA AFC Panel derived an ADI of 0.5 mg/kg body weight per day for lycopene from all sources. However, it was indicated that this ADI may be exceeded by pre-school and school children under certain food additive use scenarios.

Equivalence to Synthetic Carotenoids

Synthetic β-carotene has the all-trans isomeric configuration, whereas β-carotene from B. trispora as a natural source has a variety of forms: all-trans, mono-cis, di-cis, and poly-cis. The production of carotenoids by microbial biosynthesis is a classic example of competition between chemical and biological processes. Advantages of the biotechnological processes include the possibility of obtaining, in a simple way, the carotenoids of more complex structure, as well as the conformational isomers that only occur naturally.

Lycopene is biosynthesized by the fungus B. trispora through the same pathway as lycopene produced in the tomato. This structural identity underpins regulatory evaluations that have treated them as equivalent for safety purposes.

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