1. Introduction to Trehalose
Trehalose is a naturally occurring disaccharide composed of two glucose molecules linked by an α,α-1,1-glycosidic bond. It is widely distributed in nature and found in various organisms, including bacteria, fungi, yeast, algae, plants, and invertebrates such as insects and crustaceans. Its chemical name is α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside, and it has the molecular formula C₁₂H₂₂O₁₁ with a molecular weight of 342.30 g/mol. The compound’s CAS number is 99-20-7.
Trehalose has attracted considerable attention in the fields of chemistry, biochemistry, food science, pharmaceuticals, and materials engineering due to its remarkable physicochemical stability, non-reducing nature, and ability to protect biological structures against stress such as desiccation, oxidation, and temperature extremes. From a chemical engineering standpoint, trehalose represents an interesting molecule both in terms of synthetic pathways and industrial processing technologies for large-scale manufacture.
This article provides an in-depth discussion of the chemical structure and properties, production methods (including enzymatic, microbial, and chemical synthesis routes), and diverse applications of trehalose across industries.
2. Chemical Structure and Physicochemical Properties
2.1 Molecular Structure
Trehalose is a non-reducing disaccharide in which two α-D-glucose units are joined by a 1,1-α linkage. Unlike maltose (α-1,4 linkage) or sucrose (α-1,β-2 linkage), trehalose’s unique 1,1 bond confers exceptional stability. Because neither anomeric carbon is free, trehalose does not exhibit reducing properties and is resistant to Maillard browning reactions. The glycosidic oxygen bridge provides conformational rigidity that enhances its thermal and acid stability compared with other disaccharides.
The major structural form in aqueous solution is α,α-trehalose, although under certain crystallization conditions other anomeric conformations may transiently appear. In the crystalline state, trehalose exists in dihydrate or anhydrous forms, depending on humidity and temperature.
2.2 Physical Properties
| Property | Value / Description |
| Molecular Formula | C₁₂H₂₂O₁₁ |
| Molecular Weight | 342.30 g/mol |
| Appearance | White, crystalline powder |
| Solubility | Freely soluble in water (approx. 68 g/100 mL at 20 °C); insoluble in most organic solvents |
| Melting Point | Decomposes at ~203–210 °C |
| Optical Rotation | [α]D = +199° (in water, c = 10%) |
| Sweetness | Approximately 45% of sucrose |
| pH (10% solution) | Neutral (~5.0–6.5) |
| Hygroscopicity | Moderate; forms a stable dihydrate |
| Stability | Highly stable to acid hydrolysis and heat compared to maltose or sucrose |
Trehalose’s hydration behavior is particularly noteworthy. It can form a dihydrate crystal that binds two water molecules in a stable lattice, contributing to its superior moisture retention capacity. When dehydrated, it can transition into an amorphous glass state, which has been associated with its bioprotective effects — the ability to stabilize proteins, membranes, and cells during desiccation or freezing.
2.3 Chemical Reactivity
Because trehalose lacks a free anomeric hydroxyl group, it is chemically inert toward most reducing or condensation reactions typical of reducing sugars. It does not participate in Maillard reactions with amino compounds, making it highly desirable in foods and pharmaceuticals that are sensitive to browning or degradation. Under strong acidic or enzymatic conditions, trehalose can be hydrolyzed into two molecules of D-glucose:
Trehalose+H2→2D-glucose (acid or trehalase)
This hydrolysis is relatively slow under mild conditions, which contributes to trehalose’s stability and controlled energy release when used as a dietary carbohydrate.
3. Natural Occurrence and Biological Function
Trehalose plays a vital role as an energy reserve and stress-protectant in many organisms. It is accumulated in yeast and fungi as a storage carbohydrate and as a stabilizer during dehydration. In insects and nematodes, it serves as a blood sugar regulating energy source. Certain bacteria and algae synthesize trehalose as a compatible solute, providing protection against osmotic shock, desiccation, and high temperature. In plants, trehalose and its metabolic intermediates contribute to carbohydrate signaling pathways influencing growth and stress tolerance.
These biological insights have inspired industrial chemists and engineers to explore biotechnological production routes that mimic or harness natural trehalose metabolism.
4. Industrial Production of Trehalose
Commercial production of trehalose has evolved significantly over the last several decades. Early methods relied on extraction from natural sources such as yeast or fungi, but these were inefficient and costly. Modern manufacturing is primarily based on enzymatic conversion of starch or maltodextrins, utilizing microbial enzymes that can specifically convert α-1,4 linkages of glucose polymers into the α,α-1,1 linkage of trehalose.
4.1 Early Chemical Synthesis
Chemical synthesis of trehalose was first achieved in the 20th century through glycosylation reactions involving protected glucose derivatives. Although these methods are of theoretical significance, they are not economically feasible for large-scale production due to complex multi-step procedures, low yields, and environmental concerns associated with protecting group chemistry. However, they remain important for producing isotopically labeled or modified trehalose analogs used in biochemical research.
4.2 Enzymatic and Biocatalytic Production
The enzymatic route revolutionized trehalose manufacturing, primarily driven by advances in microbial enzymology and immobilized biocatalyst technology.
4.2.1 Two-Enzyme Conversion System
The most successful industrial process — pioneered by Hayashibara Co. in Japan — uses a two-enzyme system derived from Arthrobacter sp. or Thermus aquaticus:
- Maltooligosyltrehalose synthase (MTSase, EC 5.4.99.15)
Catalyzes intramolecular transglycosylation of α-1,4-linked glucosides (from starch or maltodextrins) to produce maltooligosyltrehalose (mainly trehalose units within an oligosaccharide chain). - Maltooligosyltrehalose trehalohydrolase (MTHase, EC 3.2.1.141)
Hydrolyzes the α-1,4 linkage adjacent to the trehalose unit, releasing free trehalose.
The overall process converts partially hydrolyzed starch or liquefied corn starch into trehalose with high efficiency.
Starch (α-1,4 glucan) →Trehalose+Glucose/Oligosaccharides (Bioconversion, MTSase/MTHSase)
4.2.2 Process Flow
- Raw material preparation:
Starch (from corn, cassava, or potato) is liquefied using α-amylase to produce maltooligosaccharides (DP 4–10). - Enzymatic conversion:
The liquefied starch slurry (typically 40–50% solids) is treated sequentially with MTSase and MTHase at 55–60 °C, pH 6.0. Reaction time ranges from 12–24 h. - Purification:
The resulting mixture contains trehalose (up to 45% of total sugars), glucose, and residual oligosaccharides. Ion-exchange resins, activated carbon, and chromatographic separation are used to purify trehalose. - Crystallization:
Concentrated trehalose syrup is crystallized under controlled temperature and humidity to yield trehalose dihydrate crystals. Drying under reduced pressure or at moderate heat produces the anhydrous form if desired.
This enzymatic route offers high yield (≈80% conversion), mild reaction conditions, and an environmentally friendly process with minimal by-products.
4.2.3 Alternative Enzymatic Pathways
Some bacteria synthesize trehalose via trehalose phosphorylase (TPase) or trehalose synthase (TreS) systems that convert maltose or glucose-1-phosphate into trehalose. For example:
Maltose →Trehalose (Tres)
Although this single-enzyme process is conceptually simpler, the equilibrium often limits yield, requiring optimized bioreactor conditions or coupled reactions to remove glucose. Research continues into genetically engineered microbes capable of overproducing trehalose from inexpensive substrates.
4.3 Fermentation-Based Microbial Production
In addition to enzymatic conversion, some microorganisms can directly accumulate trehalose during fermentation. Yeasts such as Saccharomyces cerevisiae, Candida utilis, and bacteria like Corynebacterium glutamicum naturally produce trehalose intracellularly as a stress metabolite. Engineering these strains for extracellular trehalose secretion could simplify downstream recovery, though yields currently remain lower than enzymatic conversion processes.
4.4 Chemical Engineering Considerations
From a process engineering perspective, key design considerations for trehalose production include:
- Bioreactor design: Ensuring proper mixing, pH control, and temperature regulation to maximize enzymatic activity and minimize substrate degradation.
- Enzyme immobilization: Enhances enzyme stability and reusability, reducing operational costs.
- Downstream purification: The separation of trehalose from glucose and maltose requires high-performance chromatography or membrane filtration systems.
- Crystallization kinetics: Control of supersaturation and cooling rate affects crystal morphology, purity, and hydration state.
- Energy and water balance: Concentration and drying stages are energy-intensive; process integration and waste heat recovery improve sustainability.
Modern plants typically operate continuous or semi-batch systems with integrated enzymatic reactors and simulated moving bed (SMB) chromatography, achieving high throughput and purity (>99%).
5. Chemical Stability and Functional Properties
5.1 Thermal and Acid Stability
Trehalose exhibits extraordinary stability under heat and acidic conditions relative to other sugars. Its degradation onset temperature exceeds 200 °C, and it resists hydrolysis in mild acid (pH > 3) for extended periods. These features make it suitable for high-temperature food processes (baking, extrusion, sterilization) and pharmaceutical formulations requiring autoclaving.
5.2 Glass Formation and Water Replacement Hypothesis
A distinctive feature of trehalose is its ability to form an amorphous glass matrix upon dehydration. This glass immobilizes biomolecules and cellular components, reducing molecular motion and preventing denaturation. The “water replacement hypothesis” suggests that trehalose substitutes hydrogen bonds normally formed by water, preserving the structure of proteins and membranes during drying. This mechanism explains the survival of “anhydrobiotic” organisms that can withstand complete desiccation, such as Artemia cysts and Tardigrades.
5.3 Oxidative and Free Radical Protection
Trehalose also demonstrates antioxidant behavior indirectly, by stabilizing metal ions and preventing Fenton-type reactions. Its high hydration shell and glassy state reduce the mobility of reactive oxygen species, providing protective effects in both food preservation and biomedical applications.
6. Industrial and Commercial Applications
Trehalose’s unique combination of chemical inertness, thermal stability, moderate sweetness, and bioprotective functionality has led to its widespread use across multiple industries.
6.1 Food and Beverage Industry
Trehalose is classified as a safe food ingredient (GRAS in the United States, approved by EFSA in Europe, and by Japan’s MHLW). It is valued for the following functions:
- Stabilizer and texture modifier: Maintains moisture in baked goods, noodles, and confectionery; prevents starch retrogradation and protein denaturation.
- Sweetener: Offers 40–50% sweetness of sucrose with a clean taste and low hygroscopicity.
- Color and flavor protection: Because it is non-reducing, it prevents Maillard browning and retains natural color and aroma during heat processing.
- Cryoprotectant: Reduces ice crystal formation in frozen desserts and preserves the quality of seafood, meats, and fruits during freezing.
- Energy ingredient: Provides sustained energy release and has a low glycemic index compared to glucose or sucrose.
Its ability to mask bitterness and stabilize flavors makes it popular in coffee, tea, and beverage formulations.
6.2 Pharmaceutical and Biotechnological Applications
Trehalose has proven invaluable in pharmaceutical and medical contexts due to its protein- and membrane-stabilizing properties:
- Protein stabilization: Prevents aggregation or denaturation of therapeutic proteins, enzymes, and antibodies during lyophilization or storage.
- Vaccine preservation: Used as an excipient in freeze-dried vaccines and biologics, maintaining antigen integrity.
- Cell and tissue preservation: Enhances survival of cells, liposomes, and membranes during cryopreservation or dehydration.
- Ophthalmic and dermal formulations: Incorporated into eye drops and skincare products to protect cells from oxidative and dehydration stress.
- Drug delivery systems: The glassy matrix of trehalose can encapsulate active ingredients, enabling controlled release and improved stability.
In 2008, trehalose was notably used in biopharmaceutical formulations for monoclonal antibodies and viral vectors, due to its compatibility and inertness under sterilization conditions.
6.3 Cosmetics and Personal Care
Trehalose functions as a moisture-retaining and protective agent in cosmetics. It helps maintain skin hydration, protects keratin structure in hair, and stabilizes emulsions. In sunscreens and anti-aging creams, it helps prevent oxidative stress caused by UV exposure.
6.4 Agriculture and Animal Feed
In agriculture, trehalose is studied for enhancing plant stress tolerance to drought and salinity. Exogenous application or genetic engineering to enhance trehalose biosynthesis can improve crop resilience. In animal nutrition, trehalose can serve as an energy source and feed stabilizer, protecting probiotic bacteria and enzymes in feed additives.
6.5 Chemical and Material Sciences
In materials engineering, trehalose’s glass-forming ability makes it useful in biopolymer stabilization, bioplastics, and biosensor preservation. It is employed to protect enzymes and DNA immobilized on surfaces or incorporated into polymer matrices. Its compatibility with hydrophilic polymers like poly(vinyl alcohol) and dextran enables the formation of stable biocomposites.
7. Toxicological and Safety Aspects
Trehalose is generally recognized as safe, with low toxicity and no mutagenic or carcinogenic effects. The LD₅₀ (oral, rat) exceeds 15 g/kg, similar to other carbohydrates. It is digested in humans by trehalase, an enzyme located in the small intestinal brush border, which hydrolyzes trehalose into two glucose molecules for absorption.
However, individuals with trehalase deficiency (a rare genetic condition) may experience mild gastrointestinal discomfort upon consuming large amounts of trehalose. Regulatory agencies worldwide have approved trehalose for use in food and pharmaceuticals without restriction, subject to good manufacturing practices.
8. Market Overview and Economic Considerations
Commercial trehalose production has expanded significantly since its industrial debut in the early 1990s. The largest producers are located in Japan, China, and South Korea, with production capacities exceeding several thousand metric tons per year.
Key economic factors include:
- Raw material cost: Corn or tapioca starch is the main feedstock; price fluctuations directly influence production cost.
- Enzyme cost and stability: Immobilized enzyme systems help reduce enzyme consumption and improve cost efficiency.
- Purification and crystallization energy: Optimization of chromatography and drying reduces energy demand.
- Market demand: The global trehalose market is driven by applications in food, cosmetics, and pharmaceuticals, with steady annual growth (estimated CAGR of 5–7%).
Although trehalose remains more expensive than sucrose or maltose, its functional advantages justify its use in high-value products.
9. Future Developments and Research Directions
Trehalose continues to attract research interest for both fundamental and applied science:
- Metabolic Engineering:
Genetically modified microorganisms (e.g., E. coli, Corynebacterium) are being engineered to overexpress trehalose biosynthetic genes for direct fermentation production. - Green Manufacturing:
Advances in enzyme immobilization, bioreactor design, and continuous processing are improving yield and sustainability. Integration with biorefinery systems may allow trehalose co-production with bioethanol or lactic acid. - Biomedical Innovations:
Trehalose is being explored as a therapeutic molecule for neurodegenerative diseases (such as Huntington’s and Alzheimer’s), where it induces autophagy and reduces protein aggregation. - Advanced Materials:
Trehalose-based polymers and nanocomposites are under development for applications in bioprinting, tissue engineering, and controlled drug release. - Synthetic Derivatives:
Chemical modification of trehalose (e.g., acylated, phosphorylated, or fluorinated derivatives) expands its chemical versatility and compatibility with new applications in materials science and biotechnology.
10. Environmental and Sustainability Aspects
From a sustainability perspective, trehalose manufacturing aligns well with green chemistry principles:
- Renewable feedstocks: Starch from plants serves as a renewable and biodegradable carbon source.
- Mild reaction conditions: Enzymatic catalysis avoids harsh chemicals and high energy input.
- Biodegradability: Trehalose and its by-products are environmentally benign and non-toxic.
- Waste valorization: Process residues (e.g., glucose or oligosaccharides) can be recycled into bioethanol or used as feed additives.