Process Optimization of GABA Instant White Tea Based on Response Surface Methodology and Analysis of Key Flavor Substances

Abstract

This study developed an optimized processing strategy for γ-aminobutyric acid (GABA) instant white tea (GABA-IT) using GABA-enriched white tea as raw material, systematically characterizing its chemical composition and volatile profile. In contrast to the conventional instant tea production process, this work integrates response surface methodology with spray-drying parameter optimization. This integrated approach enables the simultaneous enhancement of functional components and sensory quality. A response surface design was employed to refine the extraction and spray-drying variables following preliminary single-factor trials, and the optimal parameter combination was subsequently determined (40% ethanol concentration, material-to-liquid ratio of 1:15, extraction time of 3 days, atomization speed of 300 rpm, and inlet temperature of 120 °C); the resulting GABA-IT exhibited significantly improved quality characteristics. Specifically, the GABA content increased by 209% (reaching 4.42 mg/g), and theanine, catechins, and caffeine were enriched by 200–300%. Regarding volatile profiles, processing led to a reduction in esters but an increase in aldehydes and hydrocarbons. Relative odor activity value (rOAV) analysis revealed that epoxy-β-ionone and linalool were the key contributors to the characteristic aroma of GABA-IT. Collectively, this study demonstrates the technical feasibility of producing GABA-rich instant tea with enhanced functional components and improved sensory quality, providing practical guidance for the large-scale industrial production of functional tea beverages.

1. Introduction

γ-Aminobutyric acid (GABA) is a naturally occurring, non-proteinogenic amino acid widely distributed across plant, animal, and microbial systems [1]. In mammalian nervous systems, GABA functions as the predominant inhibitory neurotransmitter, contributing to the regulation of excitatory–inhibitory homeostasis [2] and the modulation of neuroplastic processes [3]. Consequently, it is crucial in the pathogenesis and management of neurological disorders, such as epilepsy [4]. Beyond its neurological functions, accumulating evidence highlights GABA’s broad physiological bioactivities, including antihypertensive, anxiolytic, sleep-enhancing, and mood-regulating effects [5,6,7]. These multifaceted health benefits have positioned GABA as a high-value neuro-nutraceutical compound, driving significant research interest within food science and neuroscience.

Tea (Camellia sinensis) serves as an important natural matrix for dietary GABA enrichment. Since the pioneering development of “Gabaron tea” via anaerobic incubation [8], GABA-enriched teas have emerged as promising functional beverages that retain the bioactive profile of traditional tea, such as polyphenols and theanine, while offering enhanced neuroprotective properties [9]. However, the processing of GABA tea presents a complex challenge: balancing functional enrichment with sensory quality. Anaerobic stress, while effectively stimulating GABA accumulation via the GABA shunt, often induces alterations in volatile and non-volatile metabolites, potentially leading to the loss of characteristic freshness or the formation of off-flavors [10,11]. Studies have shown that optimizing parameters such as vacuum treatment, shaking, and withering can mitigate these issues. For instance, combining vacuum with shaking significantly boosts GABA levels in white tea while imparting desirable sweet, fruity, and floral notes [12]. Furthermore, metabolomic investigations indicate that precise control over anaerobic conditions can promote the biosynthesis of important aroma compounds, including linalool and geraniol, thereby harmonizing the flavor profile with functional enhancement [13,14,15]. Taken together, these studies indicate that processing methods substantially influence both volatile and non-volatile components, emphasizing the critical role of processing techniques in shaping the sensory characteristics of GABA-enriched tea products.

Instant teas are increasingly favored for their convenience, stability, and high bioavailability of functional components. According to market analyses reported by Grand View Research, the global instant tea market has demonstrated steady growth in recent years, driven by rising demand for convenient and health-oriented beverages. Similarly, statistics from FAO highlight the continuous expansion of value-added tea products in international trade. These trends underscore the commercial potential of developing functional instant tea products with differentiated health benefits. Despite these advances in leaf tea, the development of GABA-enriched instant white tea remains largely unexplored. Unlike green or black teas, white tea is minimally processed, preserving a unique profile of high amino acids and a delicate, refreshing floral aroma [10]. Notably, several studies have reported that white tea exhibits comparatively higher endogenous GABA accumulation potential than other tea types, and anaerobic treatment can further elevate its GABA concentration. However, such anaerobic processing often induces a perceptible sour taste and alters the delicate floral aroma of white tea, thereby compromising its sensory acceptance. Therefore, compared with other tea types, GABA-enriched white tea requires separate optimization when converted into instant powder. The inherently high GABA level, combined with acidity developed during anaerobic treatment, necessitates precise control of extraction and drying parameters to rebalance taste attributes and preserve characteristic aroma compounds. Converting GABA-enriched white tea leaves into an instant powder introduces additional processing challenges related to extraction and drying, which may cause thermal degradation of heat-sensitive aroma compounds and bioactive ingredients. While optimization studies exist for GABA instant green and black teas [16,17], there is a lack of systematic research addressing how the extraction and spray-drying variables influence the GABA retention and, crucially, the characteristic aroma profile of instant white tea.

Therefore, the present study aimed to optimize the processing conditions for GABA-enriched instant white tea and to systematically characterize its chemical composition and aroma profile. Headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME-GC-MS) was employed to elucidate the changes in volatile compounds induced by processing. This work seeks to provide mechanistic insights into flavor formation during instant tea processing and to establish a technical basis for the industrial development of functional tea beverages that integrate enhanced neurofunctional value with desirable sensory quality.

2. Materials and Methods

2.1. Materials and Reagents

Fresh leaves of Camellia sinensis var. assamica (one bud with two leaves) were harvested in September 2019 from the Guangmang Mountain area, Menghai County, Yunnan Province, China (100°28′54.001″ E, 22°0′55.537″ N).

Methanol and acetonitrile (HPLC grade) were procured from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade and obtained from Shanghai McLean Biochemical Technology Co., Ltd. (Shanghai, China), including acetic acid, chloroform, Folin–Ciocalteu reagent, disodium hydrogen phosphate, potassium dihydrogen phosphate, stannous- chloride, ninhydrin, o-phthalaldehyde (OPA), boric acid, and β-mercaptoethanol. Authentic reference standards were used for analysis, including γ-aminobutyric acid (GABA), theanine (purity ≥ 98%), and caffeine (CA), which were purchased from the National Institute for Food and Drug Control (Beijing, China). Amino acid standards included aspartic acid (Asp), glutamic acid (Glu), serine (Ser), histidine (His), glycine (Gly), threonine (Thr), arginine (Arg), alanine (Ala), tyrosine (Tyr), cysteine (Cys), and methionine (Met). Polyphenol and flavonoid standards, included gallic acid (GA), catechin (C), epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin gallate (EGCG), 1,4,6-tri-O-galloyl-β-D-glucose (GG), ellagic acid (EA), myricetin (My), quercetin (Qu), and luteolin (Lu), were also obtained from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Preparation of GABA-Enriched White Tea and Instant Tea

GABA enrichment was performed using vacuum anaerobic technology following the method described by Duan [18]. Briefly, fresh leaves were withered for 8 h and then subjected to anaerobic treatment in a vacuum oven at 34 °C for 20 h. Following treatment, the leaves were dried at 50 °C to obtain GABA-enriched white tea (RM). The RM was subsequently used as the raw material for the preparation of GABA-enriched instant white tea (GABA-IT).

2.3. Optimization of GABA-IT Processing Conditions

2.3.1. Single-Factor Experiments

Preliminary single-factor experiments were implemented to evaluate and refine both extraction and spray-drying parameters. Sensory scores were considered the primary evaluation criterion, and powder yield was adopted as a secondary comprehensive indicator for the selection of optimal experimental conditions.

Optimization of Extraction Conditions

The effects of ethanol concentration, material-to-liquid ratio, and extraction time on GABA yield were investigated. The GABA yield was calculated using the following equation: GABA Yield (%) = (GABA content in instant powder × powder yield)/(GABA content in raw material × raw material weight) × 100%.

The effects of ethanol concentration (0%, 20%, 40%, 60%, and 80%, v/v), material-to-liquid ratio (1:5, 1:10, 1:15, 1:20, 1:25, and 1:30, g/mL), and extraction time (1, 2, 3, 4, and 5 days) were systematically evaluated through single-factor experiments. During each trial, the remaining variables were kept constant, with 40% (v/v) ethanol, a 1:10 (g/mL) ratio, and a 2-day extraction period applied as baseline conditions where appropriate.

Each experimental condition was carried out in duplicate, and the optimal level for each factor was determined based on the highest GABA yield.

Optimization of Spray-Drying Parameters

Spray-drying conditions were optimized using the extract obtained under the following conditions: 35% ethanol, 1:10 material-to-liquid ratio, and 2 days of extraction. The effects of atomization speed (150, 200, 250, 300, and 350 r/min) and inlet drying temperature (80, 100, 120, 140, and 160 °C) were investigated. The resulting powders were evaluated, and the parameters yielding the highest sensory score were chosen for further investigation.

2.3.2. Statistical Optimization Using Response Surface Methodology (RSM)

According to the outcomes of single-factor extraction experiments for instant tea, response surface methodology (RSM) was applied using Design-Expert software (version 8.0.6) following the Box-Behnken experimental design. GABA yield (Y) was selected as the response variable, while ethanol concentration (A), material-to-liquid ratio (B), and extraction time (C) were the independent variables (Table S1). The optimized conditions derived from the model were validated by preparing GABA-IT in duplicate and comparing the experimental GABA yield with the predicted value.

2.4. Sensory Evaluation

Sensory evaluation was conducted by a trained panel consisting of five tea tasters (three females and two males, aged 20–45 years) with prior experience in tea sensory assessment. The evaluation was performed in accordance with the Chinese National Standard GB/T 23776-2018 [19] (Methodology for Sensory Evaluation of Tea), with minor modifications to accommodate the characteristics of instant tea products.

Given the exploratory nature of this study, the assessment was carried out descriptively, focusing on appearance, infusion clarity, color, aroma, taste, and wet leaf attributes. No quantitative scoring or statistical analysis was applied, and the results are presented as qualitative observations.

2.5. Characteristic Chemical Components of Tea and Instant Tea

Moisture content was determined by the direct drying method (GB 5009.3-2016) [20]. Water extract content was measured using the total extraction method (GB/T 8305-2013) [21]. Total free amino acids were determined via the ninhydrin colorimetric method (GB/T 8314-2013) [22], and tea polyphenols were analyzed according to GB/T 8313-2018 [23]. High-performance liquid chromatography (HPLC) was used to quantify catechins, gallic acid, caffeine, flavonoids, and individual amino acids following the protocols described by [24,25].

2.6. Determination of Volatile Compounds

2.6.1. Volatile Compound Extraction by HS-SPME

Volatile compounds were extracted using headspace solid-phase microextraction (HS-SPME). Briefly, 1 g of instant tea powder was accurately weighed into a 20 mL headspace vial (Agilent Technologies, Santa Clara, CA, USA). Subsequently, 2 mL of saturated NaCl solution was added to dissolve the sample and enhance the release of volatile compounds via the salting-out effect. The vial was immediately sealed with a crimp-top cap fitted with a PTFE-silicone septum (Agilent). Prior to extraction, the sample was equilibrated at 60 °C for 10 min. A 65 μm DVB/CAR/PDMS fiber (Supelco, Bellefonte, PA, USA) was then exposed to the sample headspace for 20 min at 60 °C to adsorb volatile analytes.

2.6.2. Analytical Conditions for GC–MS Determination

Volatile compounds were thermally desorbed from the SPME fiber in the injection port of an Agilent 7890B gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) at 250 °C for 5 min under splitless conditions. Chromatographic separation was achieved using a DB-5MS capillary column (30 m × 0.25 mm × 1.0 μm, Agilent Technologies, Santa Clara, CA, USA) with helium as the carrier gas at a constant flow rate of 1.0 mL/min. The temperature program was initiated at 40 °C (held for 5 min), increased to 280 °C at 6 °C/min, and maintained at the final temperature for an additional 5 min. Mass detection was performed on an Agilent 7000D system (Agilent Technologies, Santa Clara, CA, USA) operating in electron ionization mode (70 eV). The ion source, quadrupole, and transfer line temperatures were maintained at 230 °C, 150 °C, and 280 °C, respectively. Mass spectra were recorded in full-scan mode within an m/z range of 30–350. Volatile compounds were tentatively identified by comparing their mass spectra with the NIST library and by calculating linear retention indices (LRIs) relative to a homologous series of n-alkanes (C7–C40).

The identification of volatile compounds was based on mass spectral matching and retention index comparison and should be considered tentative. Relative quantification was performed without an internal standard; therefore, the results represent semi-quantitative comparisons rather than absolute concentrations.

2.6.3. rOAV Calculation

The relative abundance (C_i) of each volatile compound was calculated via an area normalization method, expressed as the ratio of the individual chromatographic peak area to the total peak area. After determining the relative contents of the volatile compounds, odor threshold values in water (T_i) were collected from published reports, and the relative odor activity value (rOAV) was subsequently calculated using the following equation. Compounds with rOAV values ≥ 1 were considered key aroma-active compounds that play a decisive role in the overall aroma profile of the sample, whereas compounds with 0.1 < rOAV values < 1 were regarded as aroma-modifying components that contribute to the aroma in a supplementary manner.

$$\mathrm{rOAV}={\displaystyle \frac{C_i}{C_{max}}}\times {\displaystyle \frac{T_{max}}{T_i}}\times 100$$

where rOAV represents the relative odor activity value; C_max is the relative content (%) of the compound with the highest aroma contribution; T_max is the odor threshold (μg·kg⁻¹) of the compound with the highest aroma contribution in water; Ci is the average relative content (%) of each volatile compound in the sample; and Ti is the odor threshold (μg·kg⁻¹) of each volatile compound in water.

The odor thresholds used for rOAV calculation were derived from literature values determined in aqueous systems. Given the complexity of the tea matrix, these thresholds may not fully reflect actual sensory perception in the product. Therefore, rOAV values should be interpreted as indicative rather than definitive measures of aroma contribution.

2.7. Statistics Analysis

All experiments were performed using three independent biological replicates (i.e., independently prepared extraction or processing batches). For each biological replicate, measurements were conducted in triplicate as analytical replicates. Results are expressed as mean ± standard deviation (SD). Statistical comparisons among groups were performed using one-way analysis of variance (ANOVA) implemented in SPSS Statistics 26 (IBM Corp., Armonk, NY, USA). When significant differences were detected, Tukey’s multiple comparison test was applied to determine differences between means. A significance level of p < 0.05 was considered statistically significant. Hierarchical clustering analysis was conducted using MetaboAnalyst 6.0, and graphical representations were prepared using Origin 2022 (OriginLab, Northampton, MA, USA). Error bars in the figures represent the standard deviation derived from biological replicates.

3. Results

3.1. Optimization of Processing Conditions for GABA-Enriched Instant White Tea (GABA-IT)

To establish a rational and industrially applicable processing framework, a stepwise optimization strategy was adopted in this study. The extraction stage was first optimized using Response Surface Methodology (RSM) to maximize GABA yield as the primary functional target, followed by evaluation and selection of spray-drying parameters to balance sensory quality, powder yield, and GABA retention.

3.1.1. Single-Factor Optimization of Extraction Conditions

Ethanol concentration, material-to-liquid ratio, and extraction time were identified as key factors influencing the extraction efficiency. The effects of these parameters on GABA yield are presented in Figure 1A–C. The preliminary single-factor experiments indicated that suitable extraction conditions included approximately 40% ethanol, a material-to-liquid ratio of about 1:15, and an extraction period of roughly 3 days. These ranges were subsequently used to define the levels for the response surface methodology (RSM) design.

Figure 1.

Optimization results of processing technology for GABA-IT. (A) GABA yield at different ethanol concentrations, (B) solid–liquid ratios, (C) and extraction times. (D) Sensory evaluation scores at different atomization speeds (E) and spray drying temperatures. Different letters indicate significant differences among groups (p < 0.05).

3.1.2. Optimization of Spray-Drying Conditions

Spray-drying parameters significantly influence the sensory quality and powder yield of instant tea. Given that drying conditions primarily affect product quality preservation rather than GABA biosynthesis, a single-factor evaluation approach was employed to identify practical operating conditions compatible with industrial spray-drying systems. As shown in Figure 1D, atomization speeds of 300 and 350 rpm resulted in the highest sensory scores, producing instant teas with clear, bright liquor and optimal taste. Although there was no significant difference in sensory quality between these two speeds, the powder yield at 300 rpm (7.5 g) was notably higher than at 350 rpm (6.1 g). Consequently, 300 rpm was selected as the optimal atomization speed, achieving both superior sensory quality and maximum GABA retention (3.84 ± 0.30 mg/g).

The inlet air temperature also played a critical role (Figure 1E). Lower temperatures (80 °C and 100 °C) resulted in low powder yields and poor sensory quality, characterized by bitterness and astringency. Conversely, while higher temperatures (140 °C and 160 °C) improved the flavor, they led to a reduction in powder yield, likely due to the sticking of powder to the chamber walls. The optimal balance was achieved at 120 °C, which yielded the highest sensory score, the best clarity, the highest powder yield (10.4 g), and a high GABA content (3.71 ± 0.15 mg/g). Thus, 120 °C was established as the optimal inlet temperature for GABA-IT production. It should be noted that, unlike the extraction stage, the drying parameters were optimized based on practical performance indicators (sensory attributes, yield, and GABA retention) rather than through a multi-factorial RSM design.

3.1.3. Optimization of Extraction by Response Surface Methodology (RSM)

Based on the single-factor results, a Box–Behnken design was employed to optimize the extraction parameters. Based on the single-factor results, a Box–Behnken design was employed to optimize the extraction parameters. RSM was specifically applied at this stage to quantitatively model and maximize GABA yield, which was considered the key functional attribute of the developed product.

To quantitatively assess the effects of the experimental factors, a quadratic polynomial regression model was formulated to characterize the relationship between GABA yield (Y) and the selected independent variables: ethanol concentration (X₁), material-to-liquid ratio (X₂), and extraction time (X₃). The regression equation is as follows: Y = 43.13 + 0.14X₁ + 0.91X₂ − 1.75X₃ − 0.67X₁X₂ − 0.62X₁X₃ + 0.38X₂X₃ − 3.92X₁² − 3.55X₂² − 4.00X₃².

The analysis of variance (ANOVA) for the regression model is shown in Table 1. The model was highly significant (p < 0.01), and the lack-of-fit was not significant (p > 0.05), indicating that the model adequately accurately predicted the experimental data. The coefficient of determination (R² = 0.9386) further confirmed the reliability of the polynomial model. Among the three factors, extraction time exerted the most significant influence on GABA yield (p < 0.05), followed by the material-to-liquid ratio and ethanol concentration. The quadratic terms (A², B², C²) were all highly significant (p < 0.01), suggesting a curvilinear relationship between the variables and the response.

Table 1.

Analysis of variance (ANOVA) for the response surface model of GABA extraction yield.

Source of Variation	Sum of Squares	df	Mean Square	F-Value	p-Value	Significance
Model	241.83	9	26.87	11.90	0.0018	**
A: Ethanol concentration	0.16	1	0.16	0.07	0.7975	ns
B: Material-to-liquid ratio	6.55	1	6.55	2.90	0.1323	ns
C: Extraction time	24.42	1	24.42	10.81	0.0133	*
AB	1.78	1	1.78	0.79	0.4040	ns
AC	1.51	1	1.51	0.67	0.4399	ns
BC	0.57	1	0.57	0.25	0.6299	ns
A2	64.69	1	64.69	28.65	0.0011	**
B2	53.07	1	53.07	23.50	0.0019	**
C2	67.37	1	67.37	29.83	0.0009	**
Residual	15.81	7	2.26	—	—	—
Lack of fit	9.14	3	3.05	1.83	0.2820	ns
Pure error	6.67	4	1.67	—	—	—
Total	257.64	16	—	—	—	—

** Highly significant (p < 0.01); * significant (p < 0.05); ns not significant (p ≥ 0.05).

The interactions among variables were illustrated through three-dimensional response surfaces and corresponding contour projections (Figure 2). The steep slopes of the response surfaces confirm that all three factors substantially affected the GABA yield. Moreover, the elliptical shape of the contour lines in Figure 2B,D,F suggests significant interactions between the variables. Specifically, strong interaction effects were observed between ethanol concentration and extraction time, as well as between extraction time and the material-to-solvent ratio.

Figure 2.

Response surface and contour plots of different variables on GABA yield. (A,B) Ethanol concentration and material-to-liquid ratio; (C,D) ethanol concentration and extraction time; (E,F) material-to-liquid ratio and extraction time.

According to the regression model, the theoretical optimal conditions for maximum GABA yield were an ethanol concentration of 40.50%, material-to-liquid ratio of 1:15.57, and extraction time of 2.79 days, with a predicted yield of 64.25%. For practical feasibility, the parameters were adjusted to 40% ethanol, 1:15 ratio, and 3 days. Under these verified conditions, the actual GABA yield was 65.52%, which aligned well with the predicted value, confirming the validity of the model.

Although a multi-response desirability function combining all quality attributes was not implemented, this stepwise optimization strategy allowed targeted functional enhancement (via RSM) while ensuring practical drying performance and sensory acceptability.

3.2. Sensory Evaluation and Characteristic Physicochemical Composition of RM and GABA-IT

3.2.1. Comparative Sensory Evaluation of RM and GABA-IT Samples

A sensory evaluation was conducted on RM and its processed product, GABA-IT, as summarized in Table 2. The RM exhibited a bud-and-leaf appearance with an orange-red glossy color and good uniformity. The liquor showed a bright orange-yellow hue; the aroma was characterized by a sweet and fruity profile; and the infusion exhibited a mellow, sweet, and smooth taste profile. The infused leaves appeared orange-red, tender, and glossy, reflecting a high overall sensory quality.

Table 2.

Sensory evaluation of RM and GABA-IT.

Samples	Appearance	Infusion Color	Aroma	Taste	Wet Leaf
RM	Connected bud-leaves, orange-red and lustrous, uniform	Bright orange-yellow	Sweet with distinct fruity notes	Sweet-mellow, smooth texture	Orange-red, soft and bright, uniform
GABA-IT	Fine powdery texture, excellent solubility	Bright orange-red	Pronounced sweet fragrance	Fresh-mellow, refreshing briskness	—

After undergoing the instant processing procedure, GABA-IT showed significant changes in sensory characteristics. The appearance transformed into a fine, uniform powder with excellent solubility. The liquor color became a brighter orange-red. The aroma simplified into a dominant sweet note, while the taste shifted to a fresh, mellow, and brisk profile, possibly due to enhanced Maillard reactions during processing, which contributed to a stronger umami-like freshness. The evaluation of infused leaves was not applicable, due to the powdered form.

GABA-IT exhibited comparable overall sensory characteristics to RM in terms of appearance, infusion clarity, color, aroma, taste, and wet leaf attributes, based on the descriptive evaluation conducted by the panel. The processing into instant tea did not result in noticeable deterioration of key sensory features. Instead, GABA-IT maintained the fundamental sensory profile of RM, with some panelists noting subtle differences in aroma intensity and taste perception. Given the limited panel size and the qualitative nature of the assessment, these observations should be considered preliminary.

3.2.2. Characteristic Physicochemical Composition of RM and GABA-IT

Characteristic physicochemical components of RM and the processed GABA-IT were analyzed (Table 3). The contents of nearly all amino acids and catechins in GABA-IT were significantly higher than those in RM. Specifically, the γ-aminobutyric acid (GABA) content increased from 1.43 mg/g to 4.42 mg/g, and the theanine content rose from 11.18 mg/g to 34.25 mg/g, both more than tripling their original levels. As reported in previous studies, GABA-enriched green tea, obtained through MSG treatment and vacuum processing, contains 1.481 mg/g of GABA. Further processing of this tea into instant green tea powder results in an approximate increase to 3.4 mg/g. This demonstrates a similar concentration-driven enrichment during instant processing [26]. Additionally, several other amino acids, including arginine, glutamic acid, histidine, and methionine, as well as major catechin components such as EGCG, ECG, EC, and CA, exhibited similar upward trends, demonstrating a pronounced enrichment effect of the instant tea processing on these functional compounds. Similarly, caffeine (CA) content increased markedly from 18.09 mg/g in RM to 51.34 mg/g in GABA-IT, representing nearly a threefold elevation after processing. This indicates that caffeine was effectively extracted and concentrated during instant tea production.

Table 3.

Characteristics and chemical composition of RM and GABA-IT.

Compound	RM (mg/g)	GABA-IT (mg/g)
GABA	1.43 ± 0.06	4.42 ± 0.13 *
Thean	11.18 ± 0.30	34.25 ± 0.75 *
Ala	0.27 ± 0.01	0.81 ± 0.02 *
Arg	1.63 ± 0.06	4.92 ± 0.08 *
Asp	0.31 ± 0.01	0.95 ± 0.03 *
Cys	0.24 ± 0.01	0.78 ± 0.02 *
Glu	0.81 ± 0.02	2.42 ± 0.04 *
Gly	0.18 ± 0.01	0.44 ± 0.01 *
His	0.71 ± 0.02	2.24 ± 0.05 *
Ile	0.29 ± 0.01	0.9 ± 0.03 *
Leu	0.38 ± 0.01	1.07 ± 0.03 *
Met	0.76 ± 0.02	2.25 ± 0.06 *
Phe	0.67 ± 0.02	1.95 ± 0.04 *
Pro	0.16 ± 0.01	0.42 ± 0.01 *
Ser	0.71 ± 0.02	1.89 ± 0.04 *
Thr	0.14 ± 0.01	0.42 ± 0.01 *
Tyr	0.58 ± 0.02	1.83 ± 0.04 *
C	9.03 ± 0.46	3.99 ± 0.26 *
CA	18.09 ± 0.49	51.34 ± 7.76 *
CG	0.36 ± 0.04	0.68 ± 0.06 *
EC	6.61 ± 0.34	17.25 ± 0.73 *
ECG	11.65 ± 0.55	31.24 ± 2.73 *
EGC	1.91 ± 0.24	4.58 ± 0.12 *
EGCG	3.26 ± 0.18	9.54 ± 0.89 *
GA	2.22 ± 0.15	6.60 ± 0.96 *
GC	0.94 ± 0.04	2.05 ± 0.24 *
GCG	2.49 ± 0.16	9.06 ± 0.82 *
GG	0.66 ± 0.03	1.86 ± 0.15 *

Note: Data are expressed as mean ± SD. Statistical comparisons between RM and GABA-IT were performed using Welch’s t-test. * denotes the presence of a significant difference (p < 0.05).

This significant compositional change is primarily attributed to the preparation process of instant tea, which involves hot water extraction, concentration, and drying. These steps not only disrupt tea leaf cellular structures, facilitating the release and enrichment of water-soluble components like amino acids and catechins, but also concentrate soluble solids during evaporation and spray drying, thereby increasing the measured content per unit mass in the final powder. Consequently, the observed differences in overall component distribution mainly reflect a concentration and enrichment effect associated with instant tea processing.

The optimal extraction parameters determined by response surface methodology, including a 40% ethanol concentration, a material-to-liquid ratio of 1:15, and an extraction time of 3 days, significantly improved the powder yield to 65.52%, representing an increase of nearly 15% compared with the initial process (56.83%). During the spray-drying stage, an atomization rate of 300 r/min combined with an inlet temperature of 120 °C resulted in a relatively high powder yield of 10.4 g, effectively retaining the main functional components. In GABA-IT, the contents of GABA (4.42 mg/g), theanine (34.25 mg/g), EGCG (9.54 mg/g), and caffeine (51.34 mg/g) were increased by approximately 2–3 times compared to the raw tea. This enrichment effect can be attributed to the selective release of water-soluble bioactives during ethanol extraction and the protective solidification of thermolabile compounds during rapid spray-drying.

3.3. Comparative Analysis of Volatile Profiles Between RM and GABA-IT

3.3.1. Identification and Classification of Volatile Compounds

GC-MS analysis was employed to characterize the volatile profiles of RM and GABA-IT. In total, 138 volatile constituents were characterized, including 20 esters, 22 alcohols, 19 ketones, 17 aldehydes, 7 acids, 27 hydrocarbons, 14 heterocyclic or aromatic compounds, and 12 compounds classified as others.

Figure 3A illustrates the relative content distribution of these chemical classes. Significant variations were observed between the two samples. In the raw material (RM), the volatile profile was dominated by esters (23.00%), alcohols (21.09%), and hydrocarbons (16.96%). However, following the instant tea processing, the proportions of esters and alcohols decreased to 18.64% and 6.51%, respectively. Conversely, hydrocarbons became the predominant class in GABA-IT, increasing to 23.56%. Additionally, the proportions of aldehydes (15.96%) and “other” compounds (21.03%) were markedly elevated in the instant product. These shifts suggest that while processing facilitates the release or formation of certain volatiles (e.g., aldehydes via lipid oxidation or Strecker degradation), it concurrently leads to the loss of heat-sensitive or water-soluble compounds, particularly alcohols and low-molecular-weight esters.

Figure 3.

Composition analysis of volatile compounds in RM and GABA-IT. (A) Volatile compound substance classification stacked chart. (B) Principal component analysis of peak area of identified compounds.

Principal component analysis (PCA) was applied to evaluate and visualize the overall variation among samples (Figure 3B). The first two principal components (PC1 and PC2) accounted for 89.04% of the total variance (74.4% and 15.8%, respectively). The clear separation between RM and GABA-IT clusters on the score plot confirms that the instant processing induces a fundamental restructuring of the volatile profile.

3.3.2. Multivariate Characterization of Differential Volatile Profiles

To further distinguish variations in aroma profiles, an OPLS-DA model was established (Figure 4A). The model showed a tendency toward separation between the two groups, and permutation testing (n = 200, Figure S1) was performed to assess model stability. Although the R²X, R²Y, and Q² values were high, the relatively limited sample size compared to the number of variables suggests that the model should be interpreted with caution, and the results should be considered exploratory rather than confirmatory.

Figure 4.

Multivariate and univariate analysis of differential volatile compounds between RM and GABA-IT. (A) OPLS-DA score plot. (B) Volcano plot of differential volatile compounds.

Using screening thresholds of VIP > 1, p < 0.05, and fold change (FC) ≥ 2, a total of 87 volatile compounds were recognized as significantly differential. The volcano plot (Figure 4B) visualizes these changes. These differential compounds represent potential markers contributing to the observed aroma variation. Compounds that were down-regulated in GABA-IT were predominantly associated with green, fresh, fruity, and floral sensory attributes. Representative examples included ethyl heptanoate, trans-3-hexen-1-ol, hexanoic acid, phenylacetaldehyde, and β-phellandrene, indicating a pronounced reduction in freshness-related aroma components. This decrease may be associated with thermal volatilization or degradation of relatively low-boiling-point compounds during spray drying; however, direct mechanistic confirmation was not performed in the present study.

In contrast, a smaller group of volatile compounds was significantly up-regulated in GABA-IT, including linalool, dihydroactinidiolide, β-cyclocitral, methyl palmitate, pentadecane, and 2-methylnaphthalene. These compounds are typically associated with sweet, caramel-like, woody, or waxy aroma notes. Their increased relative abundance may reflect preferential retention of higher-boiling-point volatiles and/or the formation of thermally derived aroma compounds during drying. Possible pathways include carotenoid degradation or Maillard-type reactions; however, these interpretations remain hypothetical and warrant further targeted investigation.

3.3.3. Key Aroma-Active Compounds (rOAV Analysis)

To evaluate the sensory contribution of these volatiles, relative odor activity values (rOAVs) were calculated (Table 4). Volatile compounds identified by GC–MS were further evaluated using relative odor activity values (rOAVs), and those with rOAV ≥ 1 were defined as aroma-active compounds. In RM, the overall aroma profile was predominantly driven by fresh and fruity esters and aldehydes. Ethyl heptanoate exhibited the highest rOAV (100.02), imparting intense fruity and wine-like notes and acting as the primary aroma-active compound. Other important contributors included trans-2-nonenal (rOAV = 11.51), associated with green and cucumber-like aromas, and phenylacetaldehyde (rOAV = 5.46), contributing floral and honey-like characteristics. Together, these compounds accounted for the fresh and vibrant sensory profile of the raw tea.

Table 4.

rOAVs of key aroma-active compounds in RM and GABA-IT.

Classification	Compound	CAS	Threshold (μg/kg) 1	Flavor Discription	rOAV
					RM	GABA-IT
Esters	Ethyl salicylate	118-61-6	84	caramel, pepperminty	0.03 ± 0.00 *	nd
	2,2,4-trimethyl-1,3-pentanediol diisobutyrate	6846-50-0	14	musty	nd	0.14 ± 0.01 *
	Ethyl heptanoate	106-30-9	2	fruity, pineapple, cognac, rummy, wine	100.02 ± 3.06 *	10.42 ± 0.52
	Methyl palmitate	112-39-0	2	Oily, waxy, fatty	0.20 ± 0.03	26.52 ± 3.35 *
Alcohols	Linalool	78-70-6	6	Floral and citrus-like aroma	1.64 ± 0.09	8.93 ± 0.73 *
Ketones	Jasmone	488-10-8	0.26	woody, herbal, floral, spicy, jasmine, celery	3.22 ± 0.31	9.40 ± 0.54 *
	2-Heptanone	110-43-0	140	pear, apple	0.02 ± 0.01	0.01 ± 0.00
Aldehydes	2-Heptenal	57266-86-1	16	Green	0.06 ± 0.02 *	nd
	β-cyclocitral	432-25-7	3	Sweet, tropical fruity, green, floral	0.08 ± 0.01	0.53 ± 0.03 *
	Phenylacetaldehyde	122-78-1	6.3	Sweet, honey-like, floral (rose), green and slightly fruity aroma	5.46 ± 0.14 *	1.01 ± 0.11
	trans,trans-2,4-Heptadienal	4313-03-5	10	Citrus, herbal, terpene, camphor	0.39 ± 0.05	3.84 ± 1.35 *
	trans-2-nonenal	18829-56-6	0.08	green, cucumber, aldehydic, citrus	11.51 ± 0.10 *	5.67 ± 0.66
Hydrocarbons	(+)-α-phellandrene, (+)-(4S)-α-phellandrene	2243-33-6	200	dill	0.05 ± 0.00 *	nd
	(E)-β-Farnesene	28973-97-9	87	citrus, green	0.02 ± 0.00 *	nd
	β-phellandrene	555-10-2	36	terpenic, herbal	0.17 ± 0.01 *	0.04 ± 0.00
Heterocyclic and aromatic compound	Toluene	108-88-3	87	sweet	0.04 ± 0.00 *	nd
	Irisone	14901-07-6	5.9	floral, sweet, violet	0.38 ± 0.03	1.17 ± 0.04 *
	M-cymene	535-77-3	800	Woody, herbal, spicy	0.02 ± 0.00 *	nd
others	2-Methylnaphthalene	91-57-6	4	sweet, floral, woody	nd	0.34 ± 0.02 *

Note: Only compounds with rOAV > 0.1 in at least one sample are presented for brevity. “nd” indicates not detected or rOAV < 0.01. Data are expressed as mean ± SD. Statistical comparisons between RM and GABA-IT were performed using Welch’s t-test. * denotes the presence of a significant difference (p < 0.05). ¹ odor thresholds in water. Odor thresholds in water were obtained from [27,28,29].

In contrast, the contribution of freshness-related odorants in GABA-IT decreased markedly. For example, the rOAV of ethyl heptanoate declined sharply to 10.42, indicating a substantial attenuation of fruity notes. Concurrently, the aroma profile shifted toward sweeter, heavier, and more stable odorants. Methyl palmitate (waxy/oily) showed a pronounced increase in rOAV from 0.20 to 26.52, becoming one of the dominant aroma-active compounds in GABA-IT. In addition, jasmone (woody/floral) increased from 3.22 to 9.40, while linalool (floral/citrus) rose from 1.64 to 8.93. β-Cyclocitral and irisone also exhibited enhanced rOAVs, further contributing to the altered aroma profile.

Overall, the rOAV results are consistent with the sensory evaluation outcomes, demonstrating a clear transition from a “fresh and fruity” aroma profile in RM to a “sweet, mellow, and stable” profile in GABA-IT. This shift reflects the combined effects of volatile loss and selective retention or formation of thermally stable aroma compounds during instant tea processing.

3.3.4. Aroma Wheel Analysis

The aroma wheel (Figure 5) provides an integrated visualization of the sensory characteristics associated with key differential volatile compounds, categorizing the aroma profile into fruity, floral, sweet, green, woody, herbal, waxy, and chemical attributes.

Figure 5.

Flavor wheel illustrating the characteristic volatile compounds of GABA instant white tea.

Fruity and floral notes were primarily associated with esters and alcohols, such as ethyl heptanoate and linalool, which contributed to the fresh and vibrant aroma of the raw tea. Green and herbal attributes were mainly derived from aldehydes (e.g., trans-2-nonenal) and terpenes, including β-phellandrene, and were characteristic of freshness-related sensory impressions.

In contrast, sweet, woody, and waxy notes became more pronounced in GABA-IT. These attributes were closely linked to compounds such as phenylacetaldehyde, jasmone, methyl palmitate, and long-chain hydrocarbons, reflecting the shift toward heavier and more stable aroma components induced by instant tea processing.

4. Discussion

4.1. Overcoming the Flavor–Function Trade-Off in GABA Tea Processing

Previous studies have established that while anaerobic stress effectively stimulates GABA accumulation in tea leaves via the glutamate decarboxylase (GAD) pathway, it frequently compromises sensory quality. Specifically, the process often leads to the degradation of freshness-related volatiles and the development of “stuffy” or fermented off-flavors [11,30]. This creates a significant technological barrier: the trade-off between functional enrichment and flavor preservation.

In the present study, this limitation was effectively addressed by combining RSM-guided aqueous-ethanolic extraction with optimized spray-drying. This integrated process yielded an instant white tea (GABA-IT) with a GABA content of 4.42 mg/g. This value is notably higher than those typically reported for conventional GABA tea infusions and commercial instant tea products [31], suggesting that the optimized extraction acted as a concentration step for polar bioactive compounds.

4.2. Functional Intensification and Bioactive Profile Reconstruction

A distinguishing feature of the developed GABA-IT is its chemical profile characterized by markedly increased levels of GABA, theanine, catechins, and caffeine compared with the raw material. Importantly, these increases do not result from de novo formation during processing but are primarily attributable to the combined effects of efficient aqueous extraction and subsequent concentration during evaporation and spray drying. The disruption of cellular structures during extraction facilitates the release of water-soluble compounds, while moisture removal during drying concentrates soluble solids in the final powder, leading to higher measured contents per unit mass.

As a consequence of this concentration-driven intensification, GABA-IT exhibits elevated levels of both stimulant- and relaxation-associated compounds. Caffeine is widely recognized as a central nervous system stimulant, whereas GABA and theanine have been reported in previous studies to be involved in neuromodulatory and relaxation-related processes. The concurrent increase in these bioactive components suggests that GABA-IT may present a compositionally balanced profile, potentially contributing to a differentiated sensory–physiological positioning. This compositional interplay is consistent with the literature indicating that theanine may modulate certain caffeine-related excitatory responses under specific experimental conditions [32]. However, it should be noted that no direct biological or clinical evaluations were conducted in the present study, and therefore these interpretations remain compositional in nature rather than experimentally validated functional outcomes. In addition, the increased levels of catechins, particularly EGCG, may enhance the antioxidant-related compositional characteristics of the product based on previously reported bioactivities [9]. Nevertheless, confirmation of physiological relevance, bioavailability, and health-related effects would require further in vitro, in vivo, or human intervention studies. Therefore, rather than reflecting selective enhancement of a single compound class, the preparation of GABA-enriched white tea into instant powder results in a concentration-driven enrichment of multiple bioactive constituents, potentially modifying the compositional profile of the final product. The functional implications discussed herein should be regarded as indicative of potential rather than demonstrated efficacy.

4.3. Sensory Transformation and Acceptance

Although the instant processing restructured the volatile profile, shifting the dominant aroma from fresh and green (esters and alcohols) to sweet, woody, and waxy (aldehydes and hydrocarbons), the sensory acceptance was surprisingly enhanced. The observed decrease in ester compounds may be attributed to their relatively low thermal stability and susceptibility to hydrolysis under heating conditions. Esters such as ethyl derivatives can undergo thermal degradation or transesterification during spray drying. In contrast, the increase in certain aldehydes may result from lipid oxidation pathways or carotenoid degradation reactions, which are known to generate aroma active compounds such as β-ionone derivatives during moderate thermal treatment [33]. Furthermore, heat-induced Maillard reactions between reducing sugars and amino acids may have generated low-threshold heterocyclic compounds, including pyrazines, contributing to roasted or caramel-like nuances [34]. These compounds often exhibit strong odor activity even at low concentrations, which may explain why rOAV analysis identified specific aroma active volatiles as dominant contributors despite overall changes in volatile composition.

These findings suggest that the proposed processing strategy was able to retain the essential sensory attributes of the original raw material while achieving substantial enrichment of bioactive compounds. The concentration and extraction steps involved in instant tea production may have contributed to modifications in flavor-related compounds, potentially influencing aroma and taste perception. However, given that the sensory evaluation was conducted descriptively with a small panel, further studies employing a larger trained panel and quantitative statistical analysis are necessary to confirm these observations and to substantiate claims regarding sensory improvement.

4.4. Industrial Feasibility and Scalability of the Optimized Processing Approach

From an industrial perspective, the combination of response surface optimization and spray drying offers operational feasibility, reproducibility, and compatibility with existing instant tea production lines. The defined solvent ratio, extraction time, and inlet temperature provide scalable parameters that can be readily translated into pilot- and commercial-scale systems. Compared with conventional instant tea processes that rely solely on aqueous extraction, the present approach allows more precise control over functional compound enrichment while maintaining acceptable sensory characteristics, thereby enhancing its industrial applicability.

Beyond processing feasibility, the compositional profile of GABA-IT suggests a clear potential market positioning as a functional “relaxation tea”. The simultaneous enrichment of GABA and theanine, compounds widely associated with stress modulation and neuromodulatory balance, together with moderate caffeine content, supports a product concept aimed at promoting calm alertness rather than strong stimulation. This balanced functional profile may appeal to consumers seeking stress relief, mental focus, or improved work–life balance in fast-paced environments. Therefore, the optimized processing strategy not only demonstrates technical scalability but also enables differentiation within the competitive instant tea market through a clearly defined functional identity.

4.5. Study Limitations

Despite the promising findings, several limitations of this study should be acknowledged. First, the sensory evaluation was conducted by a small trained panel and was descriptive in nature without quantitative scoring or statistical analysis. Therefore, conclusions regarding sensory acceptability should be considered preliminary and require validation through larger-scale, statistically supported consumer studies.

Second, the functional relevance of GABA-IT was inferred from its enriched bioactive composition rather than from in vivo or clinical assessments. Although the elevated levels of GABA, theanine, catechins, and caffeine suggest potential physiological benefits, further biological and human intervention studies are necessary to substantiate these functional claims.

Thirdly, this study did not implement an integrated multi-response desirability optimization combining all bioactive compounds and quality attributes. Future work may adopt multi-objective RSM modeling to achieve holistic optimization.

Finally, while the optimized extraction and spray-drying parameters demonstrate industrial feasibility at the laboratory scale, pilot-scale and long-term production stability assessments are needed to confirm scalability and economic viability under commercial conditions. Future studies addressing these aspects would further strengthen the translational value of the proposed processing strategy.

5. Conclusions

This study established an optimized processing strategy for GABA-enriched instant white tea (GABA-IT) using response surface methodology combined with controlled spray drying. Under the optimal conditions (extraction with 40% ethanol at a 1:15 ratio for 3 days; spray-drying at 120 °C and 300 rpm), the product exhibited a concentration-driven enrichment of multiple bioactive constituents. Specifically, the GABA content increased by 209% (reaching 4.42 mg/g), and theanine, catechins, and caffeine were enriched by 200–300%. The processing also induced a marked reconfiguration of the volatile profile. Fresh and green-associated compounds were relatively reduced, whereas several thermally stable or heat-derived volatiles, including linalool, dihydroactinidiolide, and β-cyclocitral, were relatively enriched, suggesting a shift from fruity-green notes toward sweeter and warmer aroma characteristics. Given the semi-quantitative nature of volatile analysis, these changes should be interpreted as compositional trends rather than definitive sensory outcomes. Overall, the transformation from leaf tea to instant powder resulted in the simultaneous intensification of key chemical constituents and restructuring of the aroma profile. These findings provide a scientific and technical basis for the industrial development of GABA-enriched white tea instant products and highlight their compositional potential as multifunctional tea beverages. Further studies on bioavailability, sensory validation, and biological activity are required to substantiate potential health-related effects.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/foods15050967/s1, Figure S1: Permutation test of the OPLS-DA model for volatile compounds; Table S1: Response surface experimental design for manufacture of GABA instant tea; Table S2: Experimental results of response surface design; Table S3: The relative contents (%) of the volatile compounds.

Author Contributions

Conceptualization, W.J.; methodology, W.J. and B.J.; formal analysis, D.Y., W.J., B.J., T.W., Q.C., C.Z., N.B., Y.G., H.G., F.Y., X.T., P.L., B.C., G.P. and Y.Z.; resources, M.Z.; writing—original draft preparation, D.Y. and W.J.; writing—review and editing, M.Z. and T.W.; supervision, M.Z.; project administration, M.Z.; funding acquisition, M.Z. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Ethical review and approval were waived for this study by the Ethics Committee of Yunnan Agricultural University, as the study involved the sensory evaluation of tea products which are safe for human consumption. The evaluation followed standard protocols, and informed consent was obtained from all participants.

Informed Consent Statement

Informed consent was obtained from all participants involved in the sensory evaluation.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed towards the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by the Project of the Science and Technology Department of Yunnan, Grant No. 202502AE090028; the Basic Research Key Project of Yunnan, Grant No. 202501AS070039; and the Yunnan Provincial Joint Project of Basic Agricultural Research, Grant No. 202401BD070001-007.