The stability of L-Arginine in functional beverages

As a core functional ingredient in functional beverages that improves blood circulation and relieves fatigue, L-Arginine (L-Arg) stability directly determines a product’s efficacy retention rate, shelf life, and drinking safety. In the complex system of functional beverages (containing acidic components, electrolytes, vitamins, essences, and external factors such as oxygen and light), L-Arginine is prone to chemical degradation (guanidine group cleavage, oxidation reactions) and physical instability (decreased solubility, crystallization and precipitation). These issues lead to efficacy loss, flavor deterioration (e.g., off-flavors from putrescine), and other quality problems. Focusing on three dimensions—"key factors affecting stability", "stability evaluation methods", and "stabilization technology pathways"—this article systematically analyzes the stability patterns of L-Arginine in functional beverages, providing a basis for formula design and production process optimization.

I. Key Factors Affecting the Stability of L-Arginine in Functional Beverages

The stability of L-Arginine is regulated by both "intrinsic system characteristics" and "external storage conditions". Different factors act on its molecular structure (amino and guanidine groups) or dissolution environment, triggering different types of degradation reactions. These factors can be categorized into four core types:

(I) System pH: The Core Trigger Regulating Degradation Reactions

The L-Arginine molecule contains basic groups (amino group -NH₂, guanidine group -NH-C(=NH)-NH₂), and its chemical stability is extremely sensitive to pH. The degradation mechanism and rate vary significantly across different pH ranges:

Acidic environment (pH < 5.0): Most functional beverages are acidic (e.g., lemon or grapefruit-flavored drinks with pH 3.0–4.5). In this environment, the amino and guanidine groups of L-Arginine undergo protonation (forming -NH₃⁺), which temporarily improves solubility (solubility reaches 180 g/L at pH 4.0 and 20°C). However, long-term storage triggers guanidine group hydrolysis—protonated guanidine groups are easily attacked by water molecules, breaking down to form urea and putrescine (H₂N-(CH₂)₄-NH₂). Putrescine not only has a pungent "amine-like off-flavor" but also further oxidizes to form aldehydes, exacerbating flavor deterioration. Experimental data shows: Under storage conditions of pH 3.5 and 37°C, the degradation rate of L-Arginine (0.8% concentration) reaches 5%–8% per week, with a retention rate of only 62% after 3 months; at pH 6.0, the degradation rate drops to 1%–2% over the same period, and the retention rate reaches 94%.

Strongly alkaline environment (pH > 8.0): While this avoids guanidine group hydrolysis, the amino groups of L-Arginine are easily oxidized by oxygen in the air to form imine compounds. Additionally, the beverage system is prone to browning (Maillard reaction), which affects appearance and taste. Therefore, strongly alkaline formulations are rarely used in functional beverages.

Neutral to slightly alkaline environment (pH 6.0–7.0): The molecular structure of L-Arginine is most stable in this range—amino and guanidine groups are in a "partially protonated" state, making them resistant to both hydrolysis and oxidation. This is the optimal pH range for balancing solubility and stability, especially for bottled functional beverages requiring long-term storage (over 12 months).

(II) Oxidation: A Critical Driver Accelerating Functional Ingredient Loss

Both the guanidine and amino groups of L-Arginine contain active hydrogen, making them susceptible to oxidation by oxygen, free radicals, or metal ions in the beverage system. This oxidation produces non-functional products (e.g., NO₂⁻, NO₃⁻, ureido derivatives), with two main oxidation pathways:

Direct oxidation: Residual oxygen (headspace oxygen, dissolved oxygen) during beverage filling or singlet oxygen excited by light (ultraviolet rays) can directly attack the C=N bond of the guanidine group, breaking it down to form NO₂⁻ and releasing ammonia-like gases. In beverages containing vitamin C (ascorbic acid), ascorbic acid free radicals generated by vitamin C oxidation further accelerate L-Arginine oxidation, forming an "oxidative chain reaction". Experiments show that the oxidation rate of L-Arginine in beverages containing 8 mg/100 mL vitamin C is 30% faster than in groups without vitamin C.

Metal ion-catalyzed oxidation: Electrolytes added to functional beverages (e.g., Fe²⁺, Cu²⁺, derived from raw materials or leached from metal filling equipment) can generate hydroxyl radicals (・OH) through the "Fenton reaction". ・OH has extremely high oxidative activity toward L-Arginine, causing a 15%–20% degradation rate of L-Arginine within 24 hours—far higher than the 3%–5% rate in groups without metal ions. Additionally, metal ions can form insoluble chelates with L-Arginine (e.g., Fe²⁺-L-Arginine complexes), leading to crystallization and precipitation. This not only affects appearance but also reduces the content of functional ingredients.

(III) Temperature and Light: Synergistic Effects of External Storage Conditions

Temperature and light accelerate the degradation of L-Arginine by increasing molecular kinetic energy and triggering chemical reactions, making them the main external factors contributing to stability decline during shelf life:

Temperature: Following the "Arrhenius equation", the degradation rate of L-Arginine increases by approximately 2–3 times for every 10°C rise in temperature. In high-temperature environments (e.g., summer transportation compartment temperatures exceeding 40°C), the guanidine group hydrolysis rate of L-Arginine in acidic beverages accelerates significantly. For example, in beverages at pH 4.0 and 37°C, L-Arginine degrades by 8%–10% per month; at 45°C, the degradation rate rises to 18%–22% over the same period, with obvious off-flavors appearing within 1 month. Low-temperature storage (4–10°C) can significantly delay degradation, controlling the degradation rate within 5% over 3 months, but it requires consideration of product circulation costs (e.g., higher cold chain transportation fees).

Light: Ultraviolet rays (especially the UV-B band at 280–320 nm) can excite electron transitions in L-Arginine molecules, destroying the conjugated structure of the guanidine group. At the same time, light promotes the conversion of oxygen to ozone, intensifying oxidation reactions. For beverages packaged in transparent PET bottles, the retention rate of L-Arginine is only 70% after 1 month of outdoor light exposure; in contrast, brown PET bottles (blocking over 90% of ultraviolet rays) or aluminum foil composite packaging can increase the retention rate to over 90%, with significantly reduced browning.

(IV) Interactions with Other Components: Cumulative Effects of System Complexity

Functional beverages often contain blended electrolytes, sweeteners, essences, and other components. Some of these components interact physically or chemically with L-Arginine, affecting its stability:

Electrolytes: In addition to the catalytic oxidation by metal ions (Fe²⁺, Cu²⁺), high-concentration sodium chloride (>0.1%) reduces the solubility of L-Arginine. For example, in beverages containing 0.2% sodium chloride at 20°C, the solubility of L-Arginine drops from 150 g/L to 120 g/L, making it prone to crystallization and precipitation (especially during low-temperature storage), which affects product uniformity.

Acidic flavoring agents: Acidic components such as citric acid and malic acid are key for taste adjustment, but excessive addition (e.g., citric acid concentration >0.5%) can cause the system pH to drop excessively (<3.5), accelerating guanidine group hydrolysis. Additionally, the carboxyl groups of citric acid may form amide bonds with the amino groups of L-Arginine, generating non-functional peptide derivatives and further reducing efficacy retention.

Essences and pigments: Some artificial essences (e.g., terpene components in citrus essences) or natural pigments (e.g., β-carotene) easily generate free radicals under light. These free radicals indirectly attack L-Arginine, intensifying oxidative degradation. Experiments show that the oxidation rate of L-Arginine in beverages added with 0.2% orange essence is 15% higher than in groups without essence.

II. Stability Evaluation Methods for L-Arginine in Functional Beverages

To accurately quantify the stability of L-Arginine, a multi-dimensional evaluation system combining "content determination", "degradation product analysis", and "sensory evaluation" must be established, covering full-cycle monitoring from production to shelf life. Common evaluation methods are as follows:

(I) High-Performance Liquid Chromatography (HPLC): Accurate Determination of L-Arginine Content

HPLC is the gold standard for determining L-Arginine content. It directly reflects degradation levels through separation and quantification, with key operating points as follows:

Chromatographic conditions: Use an amino column (e.g., Zorbax NH₂ column, 4.6 mm × 250 mm) or a C18 column (requiring derivatization). The mobile phase is a mixture of acetonitrile and 0.05 mol/L potassium dihydrogen phosphate solution (volume ratio 65:35, pH 6.0), with a detection wavelength of 210 nm (ultraviolet end absorption), flow rate of 1.0 mL/min, and column temperature of 30°C.

Sample pretreatment: Take 5 mL of the beverage sample and filter it through a 0.22 μm filter membrane (to remove impurities and particles). If the sample contains pigments or essences, purify it with a solid-phase extraction column (e.g., C18 solid-phase extraction column) to avoid interference peaks affecting quantification.

Stability indicators: Determine the peak area of L-Arginine at different storage times (Day 0, Day 7, Day 30, Day 90) and calculate the retention rate (content after storage / initial content × 100%). A retention rate ≥85% is generally considered to meet shelf life requirements (12 months). This method has a recovery rate of 98%–102% and a relative standard deviation (RSD) <2%, with excellent accuracy and repeatability.

(II) Analysis of Degradation and Oxidation Products: Assessing Safety and Flavor Deterioration

The degradation of L-Arginine produces harmful off-flavor substances such as putrescine and cadaverine, while oxidation generates NO₂⁻. Specific methods are required to detect these products and assess product safety:

Putrescine detection: Use high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in multiple reaction monitoring (MRM) mode. Quantify putrescine using its characteristic ion pair (m/z 89→30), with a detection limit of 0.01 mg/L. This method can accurately detect trace amounts of putrescine in beverages (the safety limit is usually below 0.5 mg/L).

NO₂⁻ detection: Use spectrophotometry (Griess reagent method). NO₂⁻ reacts with Griess reagent (sulfanilamide + N-(1-naphthyl)ethylenediamine dihydrochloride) to form a pink azo compound. Measure the absorbance at a wavelength of 540 nm and calculate the content using a standard curve. This method is easy to operate and suitable for rapid on-line detection (detection limit 0.02 mg/L).

(III) Sensory Evaluation and Physical Stability Monitoring: Reflecting Product Market Acceptance

In addition to chemical indicators, physical stability (solubility, clarity) and sensory characteristics (flavor, color) directly affect consumer acceptance. These require a combination of intuitive evaluation and instrumental measurement:

Physical stability: Store beverage samples at 4°C, 25°C, and 45°C, and regularly observe for crystallization, stratification, or precipitation. Simultaneously, use a turbidimeter to measure turbidity (unit: NTU). For beverages with an initial turbidity <5 NTU, the increase in turbidity during shelf life should be ≤3 NTU; otherwise, the product is considered physically unstable.

Sensory evaluation: Form a panel of 10–15 professional evaluators and score the beverage’s flavor (bitterness, metallic taste, amine taste), color (browning degree), and mouthfeel (smoothness) according to the Food Sensory Evaluation Standards (1–9 point scale, with 9 being the best). A flavor score ≥7 and no obvious off-flavors indicate sensory qualification.

III. Technical Pathways to Improve the Stability of L-Arginine in Functional Beverages

To address the aforementioned influencing factors, a stabilization technology system must be established from three aspects—"formula optimization, process improvement, and packaging selection"—to preserve the efficacy and quality of L-arginine throughout the product shelf life.

(I) Formula Optimization: Building a Stable Chemical Environment

Precise Regulation of pH and Buffering SystemsLock the beverage system’s pH within the stable range of 6.0–7.0. If an acidic taste (e.g., pH 4.0–4.5) is required, adopt a "segmented buffering technology": first dissolve L-arginine in a sodium bicarbonate solution at pH 6.5 (to ensure molecular stability), then slowly adjust the pH to the target value using citric acid to avoid degradation caused by direct exposure to an acidic environment. Additionally, add 0.1%–0.3% citrate buffer pairs (citric acid-sodium citrate) to stabilize pH fluctuations during storage (controlling fluctuations within ±0.2).

Adding Compound Antioxidants and Metal ChelatorsUse a combined system of "primary antioxidant + auxiliary antioxidant + metal chelator":

Primary antioxidant: Add 0.02%–0.05% ascorbyl palmitate (fat-soluble, forms an antioxidant film at the liquid-gas interface to block oxygen contact) or 0.01%–0.03% vitamin E (synergistically inhibits free radicals).

Auxiliary antioxidant: Add 0.01%–0.02% EDTA-2Na (disodium ethylenediaminetetraacetate) to chelate Fe²⁺ and Cu²⁺ in the system, breaking the metal-catalyzed oxidation chain.

Synergistic effect: This system can reduce the oxidation rate of L-arginine by more than 60% while protecting other functional ingredients (such as vitamin C) from oxidation.

Selecting High-Stability L-Arginine DerivativesFor beverages requiring long-term storage or acidic environments (pH < 5.0), replace free L-arginine with L-arginine hydrochloride (L-Arg·HCl) or microencapsulated L-arginine:

L-Arg·HCl: Has higher solubility in acidic conditions (up to 250 g/L at 20°C) and its guanidine group is resistant to hydrolysis. At pH 3.5, its retention rate reaches 88% after 3 months of storage (compared to only 72% for free L-arginine). Note: It increases the saltiness of the beverage, so the dosage of sodium chloride should be reduced by 0.02%–0.03% to balance taste.

Microencapsulated L-arginine: Use β-cyclodextrin (0.5%–1.0%) or maltodextrin (1%–2%) for spray-drying encapsulation of L-arginine (encapsulation efficiency ≥ 85%). The microcapsule wall isolates L-arginine from oxygen, acidic environments, and metal ions, while masking bitterness. Its retention rate can exceed 95% after 3 months of storage.

(II) Process Improvement: Reducing Degradation During Processing

Optimizing Sterilization and Filling ProcessesL-arginine is sensitive to high temperatures, so the timing of addition must be adjusted based on the sterilization method:

Pasteurization (65–75°C, 15–30 minutes): Suitable for adding L-Arg·HCl. Control the sterilization temperature below 70°C and duration within 20 minutes to ensure an L-arginine retention rate ≥ 90%.

Ultra-High Temperature (UHT) Sterilization (135–150°C, 2–5 seconds): Adopt "aseptic cold filling"—after UHT sterilization of the beverage base, cool it to 25–30°C, then aseptically add the L-arginine solution (pre-sterilized via a 0.22 μm filter) to avoid high-temperature degradation. The retention rate can reach over 98%.

Meanwhile, use "nitrogen displacement technology" during filling: fill food-grade nitrogen (purity ≥ 99.9%) into the bottle to expel headspace oxygen, reducing the headspace oxygen content to ≤ 1% and dissolved oxygen content to ≤ 0.5 mg/L, significantly lowering oxidation risks.

Step-by-Step Dissolution and HomogenizationTo prevent degradation or crystallization of L-arginine due to localized high concentrations, adopt a "step-by-step dissolution process": first add L-arginine (or its derivatives) to 50%–60% purified water (water temperature 30–40°C to improve solubility), stir at 300–500 rpm for 5–10 minutes until fully dissolved, then add other ingredients such as electrolytes and sweeteners. If microencapsulated L-arginine is used, perform homogenization (pressure 20–30 MPa, temperature 30–40°C) to ensure uniform particle size of microcapsules (≤ 5 μm), avoiding stratification and precipitation, and ensuring consistent L-arginine content in each sip.

(III) Packaging Selection: Blocking External Adverse Factors

Choose packaging materials with "high barrier properties" to block light and oxygen intrusion:

Light-Proof Packaging: Prioritize brown PET bottles (colored with carbon black or iron oxide) or transparent PET bottles with UV absorbers (e.g., UV-327, added at 0.01%–0.02%). These can block over 90% of ultraviolet rays, preventing light-induced oxidation and degradation.

Oxygen-Barrier Packaging: For Tetra Pak or pouch beverages, use "PET/aluminum foil/PE composite film" packaging. Aluminum foil completely blocks oxygen and light, ensuring an L-arginine retention rate ≥ 92% during 12 months of storage. For bottled beverages, select screw caps with sealing gaskets to reduce oxygen infiltration during transportation.

The stability of L-arginine in functional beverages is the result of the synergistic effect of multiple factors. System pH, oxidation, temperature, light, and component interactions are core influencing factors, and stabilization must be achieved through systematic optimization of "formula, process, and packaging". In practical applications, technical pathways should be selected based on the beverage type (sports drinks, energy drinks) and shelf life requirements: for acidic sports drinks, prioritize L-arginine hydrochloride + compound antioxidant systems; for energy drinks, select microencapsulated L-arginine + aseptic cold filling processes, combined with brown barrier packaging. Through scientific stability evaluation methods (HPLC content determination, degradation product analysis, sensory evaluation), the efficacy retention rate of L-arginine can be ensured to be ≥ 85% during the shelf life, providing guarantees for the quality and safety of functional beverages.