The antifreeze mechanism and application of L-Arginine in frozen dough

As one of the core technologies in the modern baking industry, frozen dough technology enables the industrialized production, long-distance transportation, and on-demand use of dough, significantly improving baking efficiency. However, dough is prone to "freeze damage" during freeze-thaw cycles—such as reduced yeast activity, gluten network rupture, and ice crystal damage caused by moisture migration—ultimately resulting in rough texture, decreased volume, and quality deterioration of baked products. L-Arginine (L-Arg), a natural basic amino acid with both biocompatibility and versatility, has been proven in recent years to alleviate freeze damage by regulating ice crystal morphology, protecting yeast and gluten structures, and inhibiting oxidation, making it a highly promising natural cryoprotectant for frozen dough. Starting from its cryoprotective mechanism, this article systematically analyzes its application effects, influencing factors, and optimization strategies in frozen dough, providing theoretical and practical support for enhancing frozen dough quality.

I. Cryoprotective Mechanism in Frozen Dough: Multi-Target Synergistic Protection

The quality deterioration of frozen dough stems from four core issues: "ice crystal damage," "yeast inactivation," "gluten degradation," and "exacerbated oxidation." L-Arginine constructs a multi-dimensional cryoprotective system by specifically targeting these issues, with the following detailed mechanisms:

(I) Regulating Ice Crystal Morphology to Reduce Mechanical Damage

Approximately 70% of dough consists of water. During freezing, water forms sharp ice crystals that pierce yeast cells and the gluten network, causing structural damage.L-Arginine regulates ice crystal growth through an "adsorption-hindrance" effect, manifested as follows:

1. Adsorbing on Ice Crystal Surfaces to Inhibit Ice Crystal Growth

The L-Arginine molecule contains a guanidyl group (-NH-C(NH)-NH₂), an amino group (-NH₂), and a carboxyl group (-COOH), possessing both hydrophilic and hydrophobic properties. Its hydrophilic groups can bind to water molecules via hydrogen bonds and adsorb on the surface of initially formed tiny ice crystals; meanwhile, its hydrophobic groups hinder surrounding water molecules from aggregating toward the ice crystals, preventing the formation of large, sharp crystals (ice crystals with a diameter > 10 μm are likely to cause mechanical damage) due to "directional growth." Studies have shown that in frozen dough with 0.5% L-Arginine added, after 28 days of freezing at -18°C, the proportion of ice crystals with a diameter < 5 μm increases from 35% (control group) to 72%, and the number of sharp ice crystals decreases by more than 60%, significantly reducing physical piercing damage to yeast and gluten.

2. Lowering the Freezing Point of Water to Delay Ice Crystal Precipitation

L-Arginine has excellent water solubility (solubility of approximately 150 g/100 mL water at 20°C). Its addition increases the osmotic pressure of water in the dough, lowering the freezing point of water from 0°C to -2.5~-1.8°C and delaying the rapid precipitation of ice crystals in the early freezing stage. Additionally, L-Arginine molecules can form a "hydration layer" with water molecules, reducing the content of free water (free water easily forms large ice crystals) and increasing the proportion of bound water (bound water is not easily frozen and can maintain the water-holding capacity of the dough). For example, in dough with 0.8% L-Arginineg added, the free water content decreases by 18% compared with the control group, and the water absorption rate after freeze-thaw increases by 12%, preventing the dough from "dehydration and hardening" after thawing.

(II) Protecting Yeast Activity to Maintain Fermentation Capacity

Yeast is critical for the fermentation of frozen dough. During freezing, the yeast cell membrane is prone to inactivation due to ice crystal damage and dehydration stress, leading to reduced dough fermentability.L-Arginine maintains yeast activity through a dual "membrane protection-metabolic regulation" effect:

1. Repairing the Yeast Cell Membrane to Reduce Content Leakage

The guanidyl group of L-Arginine binds to negatively charged phospholipid molecules on the yeast cell membrane surface via electrostatic interaction, enhancing the fluidity and integrity of the membrane. At the same time,L-Arginine acts as an "osmoprotectant," reducing excessive water loss from yeast cells during freezing (dehydration causes membrane shrinkage and rupture) and minimizing the leakage of intracellular contents such as proteins and nucleic acids. Experiments show that in frozen dough with 0.6% L-Arginine added, the yeast survival rate increases from 42% (control group) to 78% after 30 days of freezing at -18°C, the membrane integrity (detected by fluorescence staining) increases by 55%, and the dough fermentation volume (fermented at 45°C for 1 hour) rises from 80 mL (control group) to 135 mL, approaching the level of fresh dough (150 mL).

2. Serving as a Yeast Metabolic Substrate to Enhance Stress Resistance

L-Arginine is a key precursor for the yeast "ornithine cycle." After being absorbed by yeast, it can be used to synthesize cryoprotective substances such as proline and glutathione (GSH)—proline stabilizes the structure of intracellular proteins, while GSH scavenges reactive oxygen species (ROS) generated by freezing stress. Additionally, ATP produced by L-Arginine metabolism provides energy for yeast, helping it quickly recover metabolic activity after thawing. For example, in frozen dough with 0.4% L-Arginine added, the intracellular proline content of yeast increases by 2.3 times and the GSH content increases by 1.8 times compared with the control group, and the glucose consumption rate (reflecting metabolic activity) of yeast after thawing increases by 60%.

(III) Stabilizing the Gluten Network Structure to Improve Dough Extensibility

Gluten proteins (gliadin and glutenin) are essential for the gas-holding capacity and extensibility of dough. During freezing, gluten is prone to network rupture due to ice crystal extrusion and protein denaturation, manifested as reduced dough elasticity and increased brittleness.L-Arginine stabilizes the gluten structure through "cross-linking and denaturation inhibition" effects:

1. Promoting Gluten Protein Cross-Linking to Enhance Network Strength

The guanidyl and amino groups of L-Arginine form hydrogen bonds and covalent bonds (e.g., salt bonds between guanidyl and carboxyl groups) with the carboxyl groups (-COOH) and sulfhydryl groups (-SH) in gluten proteins, promoting the formation of glutenin macropolymer (GMP)—the higher the GMP content, the denser and more elastic the gluten network. Studies have found that in frozen dough with 0.7% L-Arginine added, the GMP content increases by 25% compared with the control group, the dough’s resistance to extension (reflecting elasticity) increases from 280 EU to 410 EU, and the extensibility (reflecting stretchability) increases from 120 mm to 180 mm, effectively preventing gluten "brittleness" after freezing.

2. Inhibiting Gluten Protein Denaturation to Maintain Solubility

During freezing, the secondary structure (α-helix, β-sheet) of gluten proteins is prone to changes due to dehydration and ice crystal effects, leading to protein denaturation and reduced solubility (denatured proteins cannot participate in network formation).L-Arginine can form a "hydration complex" with gluten proteins, reducing hydrophobic interactions between protein molecules (excessive hydrophobic interactions cause protein aggregation and denaturation) and maintaining the stability of their secondary structure. For example, in frozen dough with 0.5% L-Arginine added, the α-helix content of gluten proteins (a key structure for maintaining elasticity) decreases by only 8% compared with the control group (a 23% decrease in the control group), the protein solubility increases by 15%, and the dough still maintains good kneading performance after thawing.

(IV) Inhibiting Oxidative Stress to Delay Quality Deterioration

Freeze-thaw cycles activate enzymes such as lipoxygenase and polyphenol oxidase in dough, generating large amounts of ROS. ROS not only oxidize unsaturated fatty acids in the dough (producing a rancid taste) but also oxidize the sulfhydryl groups of gluten proteins (forming disulfide bonds, leading to gluten hardening).L-Arginine alleviates oxidative damage through "direct scavenging and enzyme activity inhibition" effects:

1. Directly Scavenging ROS to Reduce Oxidation Products

The guanidyl group of L-Arginine has strong reducibility and can directly react with ROS (e.g., hydroxyl radicals ·OH, superoxide anions O₂⁻), converting them into harmless H₂O and O₂. Meanwhile, nitric oxide (NO), a metabolite of L-Arginine, also has antioxidant effects and can scavenge ROS. For example, in frozen dough with 0.6% L-Arginine added, after 45 days of freezing at -18°C, the content of malondialdehyde (MDA, a product of lipid oxidation) decreases by 42% compared with the control group, and the dough has no obvious rancid taste, while the control group already shows slight rancidity.

2. Inhibiting Oxidase Activity to Block Oxidation Pathways

L-Arginine can bind to the active centers (containing metal ions such as Fe²⁺ and Cu²⁺) of lipoxygenase and polyphenol oxidase, inhibiting enzyme activity by chelating metal ions and reducing ROS production. Experiments show that in dough with 0.8% L-Arginine added, the lipoxygenase activity decreases by 38% and the polyphenol oxidase activity decreases by 32% compared with the control group, effectively blocking the vicious cycle of "enzymatic oxidation-quality deterioration."

II. Application Effects and Optimization Strategies in Frozen Dough

The application effect of L-Arginine in frozen dough is affected by factors such as the addition amount, dough type, and freeze-thaw conditions. Application schemes need to be optimized based on actual needs to balance "cryoprotective effect, baking quality, and cost."

(I) Application Effects in Different Types of Frozen Dough

1. Frozen Bread Dough

Bread dough has the highest requirements for yeast activity and gluten network. The addition of L-Arginine can significantly improve bread volume and texture. For example, adding 0.6% L-Arginine to frozen toast dough made from high-gluten flour, after 30 days of freezing at -18°C and subsequent baking, the toast volume increases by 35% compared with the control group, the pore uniformity increases by 40%, and the hardness (reflecting texture) decreases from 2800 g to 1600 g (lower values indicate softer texture), approaching that of toast made from fresh dough (1500 g). Additionally, the shelf life of the bread (stored at room temperature) extends from 3 days to 5 days, and it is less prone to hardening.

2. Frozen Steamed Bun/Stuffed Bun Dough

Steamed bun dough needs to maintain good extensibility and fermentability.L-Arginine can improve the surface smoothness and internal structure of steamed buns after freezing. Studies have shown that adding 0.5% L-Arginine to frozen steamed bun dough made from medium-gluten flour, after 21 days of freezing at -20°C and subsequent steaming, the surface cracking rate of steamed buns decreases from 35% (control group) to 8%, the uniformity of the internal honeycomb structure increases by 50%, and the chewiness (reflecting texture) decreases from 3500 g·s to 2200 g·s, resulting in a softer and more elastic texture.

3. Frozen Pastry Dough (e.g., Puff Pastry Dough)

Puff pastry dough contains a large amount of oil, which is prone to flavor deterioration due to oxidation. The antioxidant effect of L-Arginine can significantly improve the flavor. For example, adding 0.7% L-Arginine to frozen mooncake puff pastry dough, after 60 days of freezing at -18°C and subsequent baking, the acid value (AV) of the oil in the mooncake decreases by 45% compared with the control group, with no rancid taste, and the layering clarity of the puff pastry increases by 25% (avoiding "layer adhesion" caused by oil-flour separation after freezing).

(II) Application Optimization Strategies

1. Optimization of the Addition Amount

The addition amount of L-Arginine should be controlled between 0.3% and 0.8% (based on the mass of flour). Too low an amount results in insufficient cryoprotective effect (e.g., adding 0.2% only increases the yeast survival rate by 15%), while too high an amount may increase the dough pH (L-Arg is alkaline, with a pH > 9.0), affecting yeast fermentation (yeast’s optimal pH is 4.0~6.0) and product flavor (e.g., a slight alkaline taste). In practical applications, a small amount of citric acid (0.1%~0.2%) can be added to adjust the pH to 5.5~6.5, which not only does not affect the cryoprotective effect of L-Arginine but also maintains a stable fermentation environment for yeast.

2. Compound Application: Synergistic Enhancement with Other Cryoprotectants

The cryoprotective effect of L-Arginine alone is limited. Compounding it with natural cryoprotectants (e.g., sucrose, glycerol, glutamine) can achieve synergistic effects:

L-Arginine + Sucrose (1:2 ratio): Sucrose enhances the water-holding capacity of the dough and synergizes with L-Arginine to regulate ice crystal morphology. In frozen dough with 0.3% L-Arginine + 0.6% sucrose added, the yeast survival rate increases by 20% and the bread volume increases by 15% compared with the group using L-Arginine alone.

L-Arginine + Glutamine (1:1 ratio): Glutamine protects the sulfhydryl groups of gluten proteins and synergizes with L-Arginine to stabilize the gluten network. In frozen dough with 0.4% L-Arginine+ 0.4% glutamine added, the resistance to extension increases by 30% and the elastic recovery rate of steamed buns increases by 25% compared with the group using L-Arginine alone.

3. Adaptation to Freeze-Thaw Conditions

The cryoprotective effect of L-Arginine requires matching with a reasonable freeze-thaw process:

Freezing Rate: "Rapid freezing" (freezing rate > 5°C/min) is recommended to quickly pass through the ice crystal formation zone (-1~-5°C) and reduce the formation of large ice crystals. Under rapid freezing,L-Arginine can more efficiently adsorb on the surface of tiny ice crystals, improving the ice crystal regulation effect by 30%.

Thawing Method: "Low-temperature slow thawing" (thawing at 4°C for 8~12 hours) is preferred to avoid rapid water loss caused by excessive thawing speed. Under low-temperature thawing,L-Arginine can be slowly released to continuously protect yeast and gluten, increasing the water retention rate of the dough after thawing by 18%, resulting in better dough quality than that of dough thawed at room temperature (25°C for 2 hours).

III. Challenges and Future Directions in the Application of Frozen Dough

Although L-arginine (L-Arg) demonstrates significant cryoprotective advantages in frozen dough, its practical industrial application still faces several challenges that require breakthroughs through technological innovation:

Challenges

High Cost: The price of food-grade L-Arginine is approximately 8–10 times that of traditional cryoprotectants (e.g., sucrose). Large-scale application will increase production costs.

Flavor Impact at High Addition Levels: Some sensitive consumers can perceive a slight alkaline taste caused by L-Arginine when its addition exceeds 0.8%, which affects product palatability.

Reduced Cryoprotective Activity in Acidic Dough: In acidic dough (e.g., sourdough frozen dough with pH < 4.5), the guanidyl group of L-Arginine is easily protonated, leading to a 30%–40% decrease in cryoprotective activity. This limits its application in acidic baked products.

Future Directions

Low-Cost Preparation Technology:Develop a "microbial fermentation-membrane separation coupling process" to optimize the efficiency of L-Arginine production via fermentation by Escherichia coli or yeast, thereby reducing raw material costs. Meanwhile, explore the extraction of crude L-Arginine (purity 80%–90%) from agricultural wastes (e.g., soybean meal, distiller’s grains) for frozen dough with low purity requirements (e.g., steamed buns, stuffed buns), which can reduce costs by 40%–50%.

Microencapsulation Technology:Use edible wall materials (e.g., maltodextrin, gum arabic) to microencapsulate L-Arginine (particle size 1–5 μm). This reduces the direct release of L-Arginine in the dough, avoiding flavor impacts caused by high addition levels. Additionally, microcapsules can slowly release L-Arginine during freeze-thaw cycles, extending the duration of cryoprotective effects. For example, encapsulated L-Arginine can maintain cryoprotective activity in frozen dough for 45 days (compared to only 30 days in the unencapsulated group).

Modification for Adaptation to Acidic Environments:Reduce the alkalinity of L-Arginine through "chemical modification" (e.g., acetylation of the guanidyl group) to maintain its cryoprotective activity in acidic dough. Alternatively, compound it with acidic cryoprotectants (e.g., zinc citrate) to maintain stable dough pH through "acid-base buffering" while synergistically enhancing cryoprotective effects. This expands its application in frozen dough such as sourdough and acidic pastries.

Through a multi-target cryoprotective mechanism involving "regulating ice crystal morphology, protecting yeast activity, stabilizing the gluten network, and inhibiting oxidative stress," L-Arginine can effectively alleviate freeze damage in frozen dough. It significantly improves the fermentability and kneading performance of thawed dough, as well as the volume, texture, and shelf life of baked products. It has shown excellent application effects in different types of frozen dough (e.g., bread, steamed buns, pastries), and its cryoprotective performance can be further enhanced through compound optimization and process adaptation.

Although it currently faces challenges in cost, flavor, and adaptation to acidic environments, through technological innovations such as low-cost preparation, microencapsulation, and chemical modification, L-Arginine is expected to become a natural cryoprotectant that replaces traditional chemical cryoprotectants. This will drive the frozen dough industry toward "safety, naturalness, and high quality" and provide key technical support for the industrialized and standardized production of the baking industry.