Hydroxytyrosol is produced by microbial fermentation

As a highly active natural phenolic compound, the microbial fermentation production of hydroxytyrosol has become an important alternative to chemical synthesis and plant extraction, thanks to its advantages such as wide raw material sources, mild reaction conditions, and strong sustainability. The core of microbial fermentation lies in screening high-efficiency strains with the ability to synthesize or convert hydroxytyrosol, and improving product yield by optimizing culture conditions. Relevant research mainly focuses on strain screening strategies, functional verification, and regulation of culture parameters, as detailed below:

I. Screening Strategies for High-Yield Strains

Strains used for microbial fermentation of hydroxytyrosol are mainly divided into two categories: one is naturally occurring strains capable of synthesizing or converting precursors (such as tyrosol, oleuropein, p-coumaric acid, etc.) into hydroxytyrosol; the other is engineered strains with functional capabilities obtained through genetic engineering modification. The screening process needs to combine the metabolic characteristics of microorganisms with the synthetic pathway of the target product, conducting precise screening in stages.

(1) Source and Enrichment of Strains

Screening of natural strains should focus on environmental samples that may be exposed to phenolic substances, as long-term adaptation can lead to the evolution of metabolic pathways for degrading or converting phenolics:

Environmental samples: Rhizosphere soil of olive trees, compost of olive processing waste (pomace, leaf residue), winemaking wastewater, etc. These environments are rich in phenolic precursors, which easily enrich microorganisms capable of converting phenolics (such as lactic acid bacteria, yeasts, Pseudomonas, Bacillus, etc.). For example, Lactobacillus plantarum isolated from olive pomace compost may carry glycosidase genes due to long-term exposure to oleuropein (a glycosidic precursor of hydroxytyrosol), enabling it to hydrolyze oleuropein into hydroxytyrosol.

Known functional strain libraries: Screening from preserved microbial strains, such as Saccharomyces cerevisiae (with strong phenolic metabolic capacity), Escherichia coli (easy for genetic modification), Aspergillus niger (secreting various glycosidases and oxidoreductases), etc., to determine their transformation potential through functional verification.

(2) Screening Methods and Functional Verification

Screening should proceed step-by-step from "primary screening - secondary screening - functional confirmation", combining rapid detection and precise quantitative techniques:

Primary screening: Design rapid detection methods based on the chemical properties of hydroxytyrosol. For example, using the color reaction of phenolic hydroxyl groups with FeCl₃ purple, screening colored colonies on precursor-containing medium plates; or preliminarily judging products by thin-layer chromatography (TLC), using hydroxytyrosol standards as controls to initially screen high-yield strains through spot position and color intensity.

Secondary screening: Conduct shake-flask fermentation of primary-screened strains, and quantitatively analyze hydroxytyrosol content in fermentation broth by high-performance liquid chromatography (HPLC) or ultra-performance liquid chromatography (UPLC) to exclude false-positive strains. Meanwhile, determine strain growth (e.g., OD₆₀₀) and calculate the ratio of "product yield / bacterial biomass" to screen strains with high metabolic efficiency.

Functional confirmation: Verify whether strains contain key metabolic enzyme genes (e.g., glycosidase genes can hydrolyze the glycosidic bond of oleuropein, and phenolic monooxygenase genes can catalyze tyrosol hydroxylation) through genome sequencing or transcriptome analysis; or detect the transformation ability of intracellular/extracellular enzymes of strains on precursors through in vitro enzyme activity assays to clarify their metabolic pathways.

II. Optimization of Strain Culture Conditions

Strains obtained through screening require optimization of culture conditions (medium composition and environmental parameters) to improve hydroxytyrosol yield, with the core being to promote bacterial growth and efficient operation of target metabolic pathways.

(1) Optimization of Medium Composition

Medium components directly affect the metabolic flow of strains, requiring focused regulation of carbon sources, nitrogen sources, precursor substances, and cofactors:

Carbon sources: It is necessary to select carbon sources that can support bacterial growth without inhibiting phenolic metabolism. Rapidly utilizable carbon sources such as glucose and sucrose can promote rapid bacterial proliferation, but excessive amounts may inhibit the expression of precursor-transforming enzymes through "carbon catabolite repression"; slowly utilizable carbon sources such as maltose and lactose can reduce repression and are more conducive to hydroxytyrosol accumulation. For example, when oleuropein is used as a precursor, the hydroxytyrosol yield of Lactobacillus plantarum in 2% maltose medium is 18% higher than that in glucose medium.

Nitrogen sources: Nitrogen sources provide raw materials for bacterial synthesis of enzyme proteins. Organic nitrogen (yeast extract, peptone) is more conducive to enzyme activity expression than inorganic nitrogen (NH₄Cl, (NH₄)₂SO₄) due to its rich amino acid content. Studies have shown that when yeast extract and peptone are added at a 1:1 ratio (total concentration 1.5%), the phenolic monooxygenase activity of Pseudomonas is the highest, and hydroxytyrosol yield increases by 25%.

Precursor substances: Precursor concentration needs to be controlled within the range of "not inhibiting strain growth and meeting transformation needs". For example, when tyrosol is used as a precursor, concentrations exceeding 5 mM will inhibit E. coli growth, while the conversion rate is the highest (up to 60%) at 2-3 mM; when oleuropein is used as a precursor, due to its glycosidic bond, its concentration should be adjusted according to the glycosidase activity of the strain, usually 1-2 g/L is appropriate, as excessively high concentrations will cause glycosidase saturation.

Cofactors: Adding metal ions (such as Fe²⁺, Mg²⁺) can activate oxidoreductase activity (e.g., Fe²⁺ is a coenzyme of monooxygenase); adding vitamins (such as vitamin C) can reduce oxidative degradation of hydroxytyrosol and improve product stability.

(2) Regulation of Culture Environment Parameters

Environmental parameters indirectly affect product synthesis by influencing bacterial activity and enzyme stability:

Temperature: It needs to match the optimal growth temperature and enzyme activity temperature of the strain. The optimal temperature for bacteria (such as Pseudomonas) is mostly 30-37℃, for yeasts (such as Saccharomyces cerevisiae) is 25-30℃, and for molds (such as Aspergillus niger) is 28-32℃. For example, the glycosidase activity of Aspergillus niger is the highest at 30℃, with an oleuropein conversion rate of 75%, while enzyme activity decreases by 30% when the temperature rises to 35℃.

pH: Acidic environments are conducive to the growth of lactic acid bacteria (pH 5.0-6.0), and neutral environments are suitable for Pseudomonas (pH 6.5-7.5). Deviation from the optimal pH range will cause enzyme denaturation. For example, the hydrolysis rate of oleuropein by Lactobacillus plantarum is 68% at pH 5.5, but only 23% when pH drops to 4.0.

Aeration and agitation: Aerobic strains (such as Pseudomonas, Aspergillus niger) require sufficient oxygen (shaker rotation speed 150-200 rpm, fermenter aeration rate 1-2 vvm) to promote redox reactions (e.g., tyrosol hydroxylation requires oxygen participation); anaerobic or facultative anaerobic strains (such as lactic acid bacteria) require low-oxygen environments to avoid oxidative stress inhibiting growth.

Culture time: It is necessary to coordinate the cycle of bacterial growth and product accumulation. Bacteria proliferate rapidly in the logarithmic growth phase, and enzyme synthesis increases; products begin to accumulate in large quantities in the stationary phase, and excessive culture may lead to decreased yield due to nutrient depletion or product degradation. For example, when Saccharomyces cerevisiae ferments tyrosol, the hydroxytyrosol yield reaches a peak after 48 hours of culture at 28℃ and 180 rpm, and decreases by 12% when cultured for 72 hours.

III. Strain Modification and Application Potential

The yield of natural strains is often limited, requiring breeding technologies to improve performance:

Mutagenesis breeding: Treating strains with mutagens such as ultraviolet (UV) and nitrosoguanidine to screen high-yield mutants. For example, after UV mutagenesis of Pseudomonas, the obtained mutant strain has a 40% higher hydroxytyrosol yield than the original strain, with good genetic stability (yield fluctuation < 5% after 10 passages).

Genetic engineering modification: Enhancing target metabolic flux by overexpressing key enzyme genes (e.g., introducing oleuropein glycosidase genes into E. coli) or knocking out genes of competitive metabolic pathways (e.g., reducing precursor conversion to other phenolics). For example, recombinant E. coli can produce more than 3 times the hydroxytyrosol yield of natural strains due to overexpression of tyrosol monooxygenase genes.

The core of microbial fermentation for hydroxytyrosol production lies in the synergy of "high-efficiency strain screening - culture condition optimization - strain performance improvement". By directionally screening environment-adaptable strains or modifying engineered strains, combined with precise regulation of medium and environmental parameters, the yield and production efficiency of hydroxytyrosol can be significantly improved. This technology not only provides a green pathway for the industrial production of hydroxytyrosol but also offers a reference for the microbial synthesis of other natural phenolic compounds, promoting the sustainable utilization of natural active ingredients.