*2.1. Materials*

Skimmed milk powder (SMP, fat 1.2%, protein 33.0%, carbohydrate 54%, sodium 0.39%, calcium 1.24%, Pams, Auckland, New Zealand) and bagged black tea (premium Ceylon tea, Dilmah Co., Peliyagoda, Sri Lanka) were purchased from local supermarket (Christchurch, New Zealand). DPPH (2,2-Diphenyl-1-picrylhydrazyl), ABTS (2,2-Diphenyl-1-picrylhydrazyl), TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine), gallic acid were purchased form Sigma-Aldrich Co., New York, NY, USA). Trolox (6-hydroxy-2,5,7,8-tetramethylchloromane-2-carboxylic acid) was purchased from Acros Organic (NS, New York, NY, USA). Phosphate buffered saline was purchased from Oxoid Ltd. (Hampshire, England). Methanol was purchased from ECP-Laboratory Reagent (Auckland, New Zealand). Ultrapure water was generated by a Milli-Q water purification system.

#### *2.2. Preparation of Black Tea Infusion (BTI)*

Bagged black tea was used to make tea infusion. One tea bag (2 g-equivalent of tea leaves) per 100 mL RO (Reverse osmosis) water was used to extract polyphenol rich infusions, with the mixture being soaked at 85 ◦C for 15 min in a water bath. After the extraction, the bags were removed and the solution was cooled to 40 ◦C in ice water for further experiments [34].

#### *2.3. (Stirred) ADG Preparation*

In general, tea-enriched ADG samples were made by acidification of tea-enriched reconstituted skimmed milks (T-RSM). Regarding the preparation of a T-RSM, SMP was dissolved in the water-BTI mixture solvent; the volume fraction of BTI in the water-BTI mixture solvent was manipulated for achieving the targeted proportion of BTI in the overall T-RSM system. The targeted BTI proportions in individual T-RSM samples were 15%, 30%, 45% and 60% (*w*/*w*). Also, each of the yield T-RSM sample should contain 10% (*w*/*w*) milk dry matter. Then, ADG samples was prepared directly from the T-RSM samples containing different proportions of tea infusion. RSM samples without the supplement of BTI were used to prepare the negative control ADG samples. The ADG samples were prepared following Vega and Grover [35] method with slight modification. Briefly, for instance, for each replicate milk sample, 200 g of SMP was dissolved in water-BTI mixture solvent to yield 2000 g T-RSM under vortex condition for 30 min and then refrigerated at 4 ± 1 ◦C overnight before further use. As aforementioned, the volume fraction of BTI in T-RSM was controlled for achieving the targeted proportion. The T-RSM was heated in water bath at 85 ◦C for 20 min and then rapidly cooled to 40 ◦C in an ice-water bath.

GDL (1.3% *w*/*w*) was added and dissolved in the T-RSM system with gentle stir. Different T-RSM (including RSM control) samples were then incubated at 35 ◦C until pH reached pH 4.55. At this point, the set ADG samples were prepared. Twelve replicate set gels (batches) were made for each sample. Subsequently, the individual set gels were homogenized (using IKA T25 Digital Ultra-Turrax, Werke GmbH & Co. KG, Staufen, Germany, at 4500 rpm, for 1 min) [36,37] for making stirred gels. These stirred gels containing 0%, 15%, 30%, 45% and 60% (*w*/*w*) of black tea infusion were the key samples studied in this research and these samples are named as ADG0% (negative control), ADG15%, ADG30%, ADG45% and ADG60% respectively. The stirred gels were stored at 4 ± 1 ◦C for further analysis. Related measurements were carried out on day 1, 7, 14, 21 and 28 of cold storage.

#### *2.4. pH and Ca2*+ *Content Measurement*

The pH of ADGs was determined at 20 ± 2 ◦C using a digital pH meter after calibration (SevenEasy pH, Mettler-toledo GmbH, Schwerzenbach, Switzerland).

The free Ca2+ content in ADG system was determined by a portable Calcium Ion-Selective Electrode (B-751, LAQUA Twin Calcium Ca++ Ion Meter, Horiba, Japan) according to Kosasih, et al. [38] and the instruction of the Ca2+ meter. The Ca2+ meter was calibrated by a 2-point calibration using 150 ppm and 2000 ppm Ca2+ standards before use and the error was below 20%. Samples stayed for 1 h at ambient temperature and temperature was measured by a thermometer right before pH and free Ca2+ content were measured.

pH and free Ca2+ content of stirred ADG samples were carried out on the day 1, day 7, day 14, day 21 and day 28 of cold storage. Measurements were carried out in triplicate.

#### *2.5. Texture Characteristics*

Textural Attributes of Stirred Gel. The textural properties of stirred gel were characterized by back-extrusion method described by Ciron, et al. [14]. A 5-kg load cell was used and the samples were tested in cylinder pot (50 mm internal diameter) at 15 ◦C, using an extrusion disc (Φ = 35 mm) operating at a set-speed of 1.0 mm/s to a 30 mm depth. Firmness and cohesiveness values were calculated from the obtained profiles using the software provided by Stable Microsystems. Textural profile of stirred ADG samples were carried out on the day 1, day 7, day 14, day 21 and day 28 of cold storage. Textural profiles were tested in triplicate for stirred gel samples.

#### *2.6. Phase Stability*

The water-holding capacity (WHC) and syneresis rate of ADGs were measured according to the method of Ciron et al. [32] with minor modifications using a centrifuge (Heraeus® Multifuge X3R, Heraeus Co., Hanau, Germany). For measuring WHC, the stirred ADG samples (1.0 g) in 1.5-mL microtubes (Axygen®) were centrifuged at 15,000× *g* for 15 min at 25 ◦C. The WHC was expressed as % (pellet/sample, *w*/*w*). The extent of syneresis (EOS) was determined by centrifuging stirred ADG samples (1.0 g) in 1.5 mL tubes at 101× *g* for 60 min at 5 ◦C. The EOS was expressed as % (supernatant/sample, *w*/*w*). WHC and EOS measurements of the stirred ADGs were conducted in quadruplicate. WHC and EOS measurements of stirred ADG samples were carried out on the day 1, day 7, day 14, day 21 and day 28 of cold storage.

#### *2.7. Antioxidant Capacity Evaluation*

Sample Extract. The sample (1 g for both tea infusion and gel samples) was placed in a 50-mL plastic pot with 20 mL 50% methanol solution and stirred overnight (speed 3, RT 15 Power 15-position analogue hotplate stirrer, IKA, Staufen im Breisgau, Germany) at ambient temperature. Extracts were stored at −20 ◦C until required.

Total Phenolic Content (TPC). The total phenolic content (TPC) of the sample extracts was determined by a spectrophotometer in triplicate using Folin-Ciocalteu (F-C) reagen<sup>t</sup> according to the method described in previous studies [39,40] with slight modifications. 500 μL of sample was added to the test tubes followed by 2.0 mL of sodium carbonate (7.5 g/100 mL) and 2.5 mL of 0.2 mol/L Folin-Ciocalteu reagent. The samples were mixed thoroughly and stored in the dark for 2 h before the absorbance was measured at 760 nm using VWR V-1200 Spectrophotometer (VWR International Co., Pennsylvania, USA). TPC was expressed as mg gallic acid equivalents (GAE) per 100 g of fresh material.

DPPH Assay. The DPPH radical-scavenging activity was assayed by the method reported by Al-Dabbas et al. [41]. DPPH was dissolved in methanol to ge<sup>t</sup> a concentration of 0.1 mM. To 500 μL of sample extract, 1 mL 0.1 mM DPPH solution and 1.5 mL of methanol were added and mixed by vortex. The absorbance was measured at 517 nm (VWR International Co., Pennsylvania, USA) after the solution was kept at room temperature for 30 min in the dark, methanol was used as the control. The DPPH radical-scavenging activity was expressed as mg Trolox equivalents (TE) per 100 g of fresh material. Each sample was analysed in triplicate.

Ferric Reducing/Antioxidant Power (FRAP) Assay. FRAP was assessed according to Khanizadeh, Tsao, Rekika, Yang and DeEll [40] with slight modifications. A fresh working solution of FRAP reagen<sup>t</sup> was prepared each time by mixing acetate buffer (300 μM, pH 3.6), a solution of 10 mM TPTZ in 40 mM HCL, and 20 mM FeCl3•7H2O at 10:1:1 (*v*/*v*/*v*). 250 μL of standards of iron (II) sulphate (FeSO4•7H2O) or sample extracts were added to 2.5 mL of the FRAP reagen<sup>t</sup> and the absorbance at 593 nm recorded immediately after the addition of the sample and after 2 h incubation at 37 ◦C [42] (p.39). The results were expressed as μmol Fe2+/g sample. Each sample was analysed in triplicate.

ABTS radical scavenging capacity. The ABTS radical scavenging assay was based on the method of Elfalleh, et al. [43]. ABTS working solution was prepared by mixing colourless ABTS stock solution (7 mM in water) with 2.45 mM potassium persulfate (1:1) and then maintain the reaction for 16 h in the dark at room temperature. Before analysis, the working solution was diluted to an absorbance of 0.70 (±0.02) at 734 nm with PBS (pH 7.4) and 3 mL of ABTS·+ transferred to a cuvette. After the addition of 300 μL Trolox or sample extract, the mixture was well mixed by blowing with pipette, allow to stand 6 min and absorbance read at 734 nm. All samples were assayed in triplicate. The results were expressed as Trolox equivalents (TE).

TPC, DPPH, FRAP and ABTS measurements of stirred ADG samples were carried out on the day 1, day 7, day 14, day 21 and day 28 of cold storage.

#### *2.8. Microstructure of ADG*

Scanning Electron Microscope (SEM). The bulk microstructure characterization method was developed according to Kalab [44]. Approximately 3 mm<sup>3</sup> cubes of chilled stirred gel samples were coated in a thin layer of low melting point agarose (3%) before being placed into primary fixative (3% glutaraldehyde 0.1 M sodium cacodylate buffer, pH 7.2) for at least 8 hours at ambient temperature. The samples were then washed three times (10–15 min each) in sodium cacodylate buffer (0.1 M, pH 7.2) followed by post fixation in 1% osmium tetroxide (in sodium cacodylate buffer) for 1 hour at room temperature and another three buffer washes. Finally, the samples were dehydrated in gradient ethanol series (25%, 50%, 75%, 95% and 100%) for 10–15 min each before a final 100% ethanol wash for 1 h.

Samples were critical point dried using liquid CO2 as the CP fluid and 100% ethanol as the intermediary (Polaron E3000 series II critical point drying apparatus, Quorum Technologies, Laughton, UK). Samples were torn to expose structure, mounted on to aluminium stubs, sputter coated with approximately 200 nm of gold (Baltec SCD 050 sputter coater, Schalksmühle, Germany) and viewed in the FEI Quanta 200 Environmental Scanning Electron Microscope with the EDS Detector (EDAX Genesis, Sydney, Australia) at an accelerating voltage of 20 kV. Analyses of ADG microstructure were conducted at Massey University, Palmerston North, New Zealand [45–48].

Confocal Laser Scanning Microscopy (CLSM). The planar microstructure of the protein arrangemen<sup>t</sup> in stirred ADG was investigated using CLSM. The method was adapted from Ciron, Gee, Kelly and Auty [32] with modifications. A drop of stirred gel sample was placed in a concave slide and 40 μL each of 0.2 g/<sup>L</sup> Nile red (in methanol) and fast green (in water) were added before being covered with a coverslip.

Imaging was carried out using the Leica DM6000B SP5 confocal laser scanning microscope system with LAS AF software (version 2.7.3.9723; Leica Microsystems Berlin, CMS GmbH, Berlin, Germany). Images were acquired with a HCX PL APO CS 10x (N.A. 0.40), HCX PL FLUOTAR 40x (N.A. 0.75) and HCX PL APO CS 100x oil (N.A. 1.40). Nile red and fast green were sequentially imaged through excitation at 488 nm (argon laser) and 633 nm (HeNe 633 laser) (respectively) and emission collection at 498–569 nm and 643–787 nm (respectively).

#### *2.9. Statistical Analysis*

One-way analysis of variance (ANOVA) (with Fisher comparison) and principle component analysis (PCA – as of Figure 1) were carried out using Minitab 17.0 (Minitab, State College, PA, USA) and the significance level was set at *p* ≤ 0.05.

**Figure 1.** Score plots of principle component analysis (PCA). ADG0%: plain acidified dairy gel, ADG15%: acidified dairy gel containing 15% black tea infusion, ADG30%: acidified dairy gel containing 30% black tea infusion, ADG45%: acidified dairy gel containing 45% black tea infusion, ADG60%: acidified dairy gel containing 60% black tea infusion. PC1 strongly associated with TPC, DPPH (2,2-Diphenyl-1-picrylhydrazyl), ABTS (2,2-Diphenyl-1-picrylhydrazyl) and ferric reducing antioxidant power (FRAP); PC2 strongly associated with values of EOS, pH and Ca2+. (**a**) is PCA plot based on different storage time; (**b**) based on different formula.

#### **3. Results and Discussion**

#### *3.1. Physicochemical Characteristics*

The physicochemical characteristics of ADGs are shown in Table 1. As it is shown in Table 1, plain ADG0% showed the highest values in EOS in comparison with other ADG samples on day 1. Throughout the cold storage period, in general, decrease trends were observed in pH, Ca2+ concentration, and EOS for all ADG samples. Significant decreases of Ca2+ concentration were only observed in ADG45% and ADG60% on day 1 (in comparison to ADG0%). The decrease of Ca2+ concentration may due to the interaction between calcium ion and polyphenols or oxalate in tea. Charrier and coworkers [49] stated that the oxalate in teas may bind with calcium ion. Yamada et al. [50] used infrared spectroscopy (IR) and matrix-assisted laser desorption ionization technique with a time-of-flight mass spectrometer (MALDI-TOF-MS) found out that the calcium ion increased the amount of tea stain greatly due to the combination of phenolic compounds and calcium ions. Such finding provided an evidence that calcium-bridged polyphenols complex may be formed. Another study [51] also evidenced occurrence of EGCG-Ca2+-EGCG bridging e ffect. These Ca2<sup>+</sup>-phenol interaction mechanisms help explained our results regarding the observed decrease of Ca2+ from a molecular point of view. The slight decrease trend of pH upon increasing the incorporation amount of tea infusion (Table 1) is attributed to the relatively lower pH of black tea infusion (pH 5.18) [21].


**Table 1.** Physicochemical characteristics between di fferent formulations of black tea enriched acidified dairy gel (ADG) during a cold storage of 28 days (4 ◦C).

A–E Means ± SD within a column with different superscripts differ (*p* ≤ 0.05). a–e Means ± SD within a row with different superscripts differ (*p* ≤ 0.05). 1 Formulation: ADG0%: acidified dairy gel without supplementation of tea infusion; ADG15%: acidified dairy gel containing 15% (*w*/*w*) black tea infusion; ADG30%: acidified dairy gel containing 30% (*w*/*w*) black tea infusion; ADG45%: acidified dairy gel containing 45% (*w*/*w*) black tea infusion; ADG60%: acidified dairy gel containing 60% (*w*/*w*) black tea infusion; 2 EOS: Extend of syneresis; 3 WHC: Water holding capacity.

Stirred ADGs are viscoelastic semi-solid material, poor gel structure and poor stability are associated with higher risk of shrinkage and subsequent expulsion of whey solution [32], so a relatively phase stable ADG should be able to retain a certain amount water over a storage time [52]. Interestingly, by including tea infusion in the stirred ADG, the syneresis rate tended to decrease when the stirred gel was just made (day 1, Table 1). A study demonstrated an opposite phenomenon, in which the authors found that polyphenols especially at higher concentrations are able to reduce the elasticity of acidified milk gels and induce extensive shrinkage of gel system, therefore, more syneresis [53]. Such mechanism can explain our EOS results on day 28. Larger polyphenols with more aromatic rings

and hydroxyl groups have stronger affinity to casein for forming protein–phenol complex [1]. Such type of complex may result in tighter gel network then cause relatively more release of whey fraction [27]. In the same study [27], the coworkers found two types of protein–phenol complex systems when milk proteins interact with phenolic compounds from either green tea or Pu-erh tea. These two different complex systems resulted in different texture and syneresis rate. Green tea infusion was able to reduce syneresis; however, Pu-erh tea promoted syneresis in acidified dairy gel [27]. The current research showed that black tea had even stronger ability than green tea (as shown in the reference [27]) in terms of reducing the syneresis rate of ADG on day 1 (Table 1). After 28 days of storage time, ADG0% and ADG15% were not significantly different for EOS; however, for gels with higher levels of tea infusion inclusion the EOS were significantly increased indicating time is an important factor for developing the impact of protein–phenol complex on gel structure (Table 1). Such explanation is confirmed by (SEM) microstructure images (e.g., Figure 2e vs. Figure 3e) which will be discussed later.

(**c**) (**d**) 

**Figure 2.** SEM images of plain ADG0% (**a**), ADG with 15% black tea infusion (**b**), ADG with 30% black tea infusion (**c**), ADG with 45% black tea infusion (**d**), ADG with 60% black tea infusion (**e**) (day 1).

**Figure 3.** *Cont*.

**Figure 3.** SEM images of plain ADG0% (**a**), ADG with 15% black tea infusion (**b**), ADG with 30% black tea infusion (**c**), ADG with 45% black tea infusion (**d**), ADG with 60% black tea infusion (**e**) (day 28).

As for WHC, ADG15% and ADG30% acquired the highest values for day 1; however, after 28 days ADG15% had the highest WHC than other gel samples including the control gel without inclusion of tea infusion (*p* < 0.05), thus the relatively lower amount of tea infusion (15%) may help to maintain the gel structure of ADGs. Reports have shown that exopolysaccharides (EPSs) can act as an agen<sup>t</sup> which is capable of thickening, stabilizing, emulsifying and gelling as well as water-binding in the food system and EPSs can be produced by certain strains, such as *Lactobacillus paracasei*, therefore an increase in EOS was observed through cold storage [14,52]. However, in this research, no starter culture was used and the gelation was induced by (slow) direct acidification due to the hydrolysis of GDL. Such results sugges<sup>t</sup> that not only starter culture and its products but also physicochemical interactions between dairy components and tea phenolic compounds can alter gel structure and texture, therefore, the gel physical stability over a period. The same mechanism has been discussed previously elsewhere [27], although, in that research the dairy gel was induced by fermentation of starter culture, the author attributed the texture and stability features to the protein–phenol interactions.

#### *3.2. Texture Characteristics*

The textural characteristics of stirred ADGs are shown in Table 2. In general, firmness describes the force needed for causing a certain deformation. Cohesiveness represents for the threshold of extent of a material deformation for triggering an irreversible rupture [54]. The former parameter provides good indication about how hard or how soft a gel material, whereas, the later indicates the tenacity of a gel material (e.g., brittleness). On day 1, incorporation of tea infusion regardless the addition level resulted no change in firmness in comparison to the control (Table 2). Over the storage time (> day 1), all gel samples showed an increase in gel firmness in comparison to day 1, this is due to the reformation of gel structure after shearing the gel on day 0 and ongoing fusion of casein particles such as dissociation of casein micelles at relatively low pH and rearrangemen<sup>t</sup> of bonds and strands [55]. The similar phenomenon was observed in a previous work in which the authors observed significant increase of storage modulus of stirred yogur<sup>t</sup> gel over a period of storage (up to 35 days) [56]. Apparently, the current results showed that the higher addition amount of tea infusion (ADG60%, Table 2) retarded the fusion process of casein particles in the gel system over the 28 days storage time. In summary, including tea infusion at extremely high level (e.g., 60%) tends to make dairy gel relatively softer over storage time. The reason is not clear that why ADG45% had slightly higher firmness than ADG30% after 28 days of storage. The impact of tea infusion on dairy gel firmness is not fully clear and the published data is lack of consistency. Opposite observations were reported, for instance, one group found that green tea extracts were able to increase the firmness of yogurt. And the phenomenon was attributed to flavonoid–protein cross-linking [57]. However, the authors did not mention the details of the gel preparation procedure. It is important to point out that the shearing process of set gel for the preparation of stirred is a critical step, which determines the firmness of the final sample. If the gel shearing process was not consistent, consequently, it is impossible to make relevant discussion about the causality for experimental observations. Moreover, it was found that green tea extracts significantly increased hardness of cheese gel but decreased its cohesiveness and this is due to the net effect of moisture reduction and alter microstructure of cheese gel in which green tea extract was included [30]. Similar to our findings, Najgebauer-Lejko et al. [27] also found that tea infusion may be able to decrease firmness of acidified dairy gel, although green tea (rather than black tea) and lower addition levels were used (5–15%) in that research.


**Table 2.** Texture characteristics of stirred gel samples.

A–E Means ± SD within a column with different superscripts differ (*p* ≤ 0.05). a–e Means ± SD within a row with different superscripts differ (*p* ≤ 0.05). 1 Gel samples: ADG0%: acidified dairy gel without supplementation of tea infusion; ADG15%: acidified dairy gel containing 15% (*w*/*w*) black tea infusion; ADG30%: acidified dairy gel containing 30% (*w*/*w*) black tea infusion; ADG45%: acidified dairy gel containing 45% (*w*/*w*) black tea infusion; ADG60%: acidified dairy gel containing 60% (*w*/*w*) black tea infusion.

Unlike the situation for firmness, incorporation of tea infusion (15–60%) in ADG significantly reduced the cohesiveness on day 1 (Table 2). Such results indicated that addition of tea infusion may make the acidified dairy gel slightly more brittle. The opposite results were reported for green tea and Pu–erh tea enriched yogur<sup>t</sup> systems, in which cohesiveness was increased upon addition of tea infusion (up to 15%) [27]. However, it is important to point out that not only were different teas used in the two studies, but also relatively lower tea infusion addition levels were investigated in the mentioned reference. Najgebauer–Lejko et al. believed that the increase of cohesiveness is due to the existence of protein–phenol interactions and such interactions strengthened the food internal bonds [27]. However, such deduced mechanism does not explain the contradictory trends between firmness and cohesiveness as found in the mentioned study. In the current research, the changing trend of cohesiveness over 28 days of storage was similar to the firmness-changing trend. The change of firmness and cohesiveness upon addition of tea infusion may be attributed to the alteration of microstructure during storage period [30]. The mechanism will be elaborated later in the microstructure section.

#### *3.3. Antioxidant Capacity*

To determine the antioxidant abilities of the obtained stirred ADGs and tea infusion, we chose three methods which allowed us to measure both the ability to reduce pro-oxidant metal ions (FRAP assay) and radical scavenging activity (DPPH assay and ABTS assay) along with TPC measurement. The results are shown in Table 3.


**Table 3.** Total phenolic content and antioxidant capacity of stirred ADGs.

A–E Means ± SD within a column with different superscripts differ (*p* ≤ 0.05). a–e Means ± SD within a row with different superscripts differ (*p* ≤ 0.05). 1 Formulation: ADG0%: acidified dairy gel; ADG15%: acidified dairy gel containing 15% (*w*/*w*) black tea infusion; ADG30%: acidified dairy gel containing 30% (*w*/*w*) black tea infusion; ADG45%: acidified dairy gel containing 45% (*w*/*w*) black tea infusion; ADG60%: acidified dairy gel containing 60% (*w*/*w*) black tea infusion; EOS: Extend of syneresis; WHC: Water holding capacity.

Based on the results from FRAP, DPPH and ABTS radical assays, the strongest antioxidant capacity appeared in tea infusion (TPC: 1632.56 ± 9.99 GAE μg/g; FRAP: 17.20 ± 0.09 Fe2<sup>+</sup> equivalent μmol/g; DPPH: 9.26 ± 0.08 TE μmol/g and ABTS: 9.30 ± 0.04 TE μmol/g) compared to dairy gel samples. The difference in antioxidant activity is due to the different supplement volume of the original tea infusion. Based on the original antioxidant capacity of black tea infusion and the proportion of tea infusion supplementation, we calculated the theoretical antioxidant capacities for tea-enriched ADGs. By comparing the theoretical and practical values, we found that the acidified dairy gels did not reduce the antioxidant activity from the tea infusion. The higher proportion of tea infusion presented in ADGs the more antioxidant capacity and TPC. Interestingly, the increasing rate in TPC was much lower than the increasing rate of tea infusion supplementation (e.g., comparison between ADG0%, ADG15%, and ADG30%) which indicated that the polyphenols in tea were binding with calcium ion or protein as reported by Tanizawa, et al. [58] and Spiro and Chong [59], respectively. These previous findings are in a good agreemen<sup>t</sup> with our results in which we observed that the free Ca2+ were decreased upon the increase of the addition level of tea infusion (Table 1). Surprisingly, some antioxidant parameters were remained at higher level at day 28 compared to results from day 7, 14 and 21. Although the mechanism has not ye<sup>t</sup> been clear, similar results were reported by Najgebauer-Lejko, et al. [15]. The authors studied yogur<sup>t</sup> gels with incorporation of different levels of either green tea infusion or Pu-erh tea infusion, and their results showed that the antioxidant activities decreased until day 21 for both types of tea but such activity increased back to nearly the initial value on day 28.

Overall, the incorporation of black tea infusion in stirred ADG significantly enhanced the antioxidant potential of ADG as a 4 to 12-fold increase in DPPH, an 8 to 40-fold in FRAP and a 6 to 18-fold in ABTS value were observed in comparison to plain ADG0%. The antioxidant activities were stable over the 28 days shelf life. Moreover, such activities may be effectively increased by incorporation of increased proportion of black tea infusion.

#### *3.4. Principle Component Analysis (PCA)*

Plots of PCA is shown in Figure 1. PC1 and PC2 including physicochemical properties and antioxidant properties explained 79.1% of the variation with 54.9% and 24.2% for the two PCs respectively. The main di fference between PC1 and PC2 is that PC1 strongly associated with TPC (PC1 0.394; PC2 0.037), DPPH (PC1 0.385; PC2 0.156), ABTS (PC1 0.395; PC2 0.191) and FRAP (PC1 0.398; PC2 0.217) but values of EOS (PC1 0.145; PC2 −0.355), pH (PC1 0.100; PC2 −0.559) and Ca2+ (PC1 −0.234; PC2 −0.490) were the dominant variables for PC2. Furthermore, Figure 1a. showed the sample di fferentiation-based storage time (shelf life); Figure 1b. showed that gels with di fferent proportions of tea infusion are clearly separated when taking both physicochemical and antioxidant activity associated factors into account. As it is shown in the biplot figure (in the Supplemental Material, Supplement Figure S1), the antioxidant properties were mainly determined by the incorporation rate of tea infusion in stirred ADGs, but the textural and physicochemical properties were mainly influenced by storage period.
