Influence of green tea-enriched fermentation and cold storage on yogurt’s probiotic viability and metabolomic signatures

Abstract

This study investigated the synergistic effects of varying green tea powder concentrations and storage conditions on total phenolic content, antioxidant activity, microbial viabilityand the metabolite profile of yogurt formulations. The total phenolic content and antioxidant activity increased proportionally to the concentration of green tea powder. Enhanced acidity, faster fermentation, and higher Lactobacillus spp. viability was observed.Multivariate analysis demonstrated distinct group clustering, indicating clear metabolic divergence driven by the incorporation of green tea and fermentation in cold storage. A total of 86 metabolites were identified, which predominantly comprise polyphenols derived from green tea powder. Pathway enrichment analysis revealed that the synergistic interaction between LAB (lactic acid bacteria) and tea polyphenols during cow’s milk fermentation significantly modulated multiple metabolic pathways, including central carbon metabolism, nucleotide metabolism, redox regulation, amino acid metabolism, and lipid metabolism, which are responsible for contributing enhanced nutritional and functional properties of yogurt.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-026-02138-4.

Introduction

Bos taurus’s (cow) milk, which has been an essential ingredient, is frequently turned into fermented goods like kefir and yogurt. As a complex food matrix, yogurt contains a wide range of biomolecules providing its nutritional and sensory qualities, including proteins, bioactive peptides, carbohydrates, fats, amino acids, organic acids, fatty acids, nucleotides, minerals, vitamins, and aromatic compounds (Abedin et al., 2024; Shori and Baba, 2023). Lactic acid bacteria (LAB) such as Streptococcus thermophilus, Lactobacillus species, Lactococcus lactis, and Bifidobacterium species, ferment milk to produce yogurt. These live cultures must be present in sufficient amounts when consumed, approximately 10⁶–10⁷ CFU/g, to offer health benefits, particularly in maintaining gut health (Brishti et al., 2025; Shori et al., 2022).

Green tea has more than 500 bioactive substances, particularly polyphenols, which are a major class of phytochemicals. The most abundant polyphenols are catechins, especially epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), and epicatechin (EC). It also includes amino acids, flavonoids, phenolic acids, alkaloids, and vitamins (Kurnianto and Rini, 2024; Zhao et al., 2022). Green tea's polyphenols function as metabolic prebiotics, influencing microbial communities to improve probiotic viability and gut health. While green tea water extracts usually only separate water-soluble phytochemicals, green tea powder offers the whole range of phytochemicals, including fiber, minerals, flavonoids, and bound polyphenols that are absent from steeped tea. This distinction could have a major impact on the yogurt's metabolite composition as well as the dynamics of LAB fermentation (Jeong et al., 2018; Zonus et al., 2025). With the rise of nutritional metabolomics, untargeted mass spectrometry (MS)-based metabolomic analysis allows deeper investigation of how plant polyphenols interact with probiotic metabolism, potentially generating novel bioactive metabolites. However, there is currently limited integration of metabolomics with microbiological analyses in green tea yogurt research (Kurnianto and Rini, 2024; Zhang et al., 2021).

Therefore, a clear knowledge gap exists in understanding how whole green tea powder affects LAB viability, antioxidant activity, and the overall metabolome of yogurt. Thus, the objective of this study is to investigate the fermentation effect of milk fortified with green tea powder on total phenolic content, antioxidant activity, sensory property, metabolite composition, and microbial profile of yogurt. Addressing this gap will provide mechanistic insights into how tea-derived polyphenols interact during probiotic fermentation, offering evidence for developing next-generation functional yogurts with improved qualities that promote health.

Material and methods

Chemicals and reagents

All chemicals used were analytical grade. Phenolphthalein, Sodium Hydroxide (NaOH), Hydrochloric Acid (HCl), Ethanol, Folin–Ciocalteau reagent. Sodium Carbonate (Na₂CO₃), Gallic Acid, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), Methanol, 2,2’-azino-bis (3-ethylbenzo-thiazoline-6-sulphonic acid (ABTS), Potassium Persulphate, Acetate buffer, 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) reagent, Iron (III) chloride (FeCl₃), and Sulfate Heptahydrate (FeSO₄_7H₂O) solution were purchased from Chemiz, UK. Peptone Water Buffer, M17 media, MRS medium were purchased from Oxoid, UK. Acetonitrile and Formic Acid were purchased from Sigma-Aldrich, USA. Green tea (Camellia sinensis) with matcha powder was bought from the local market, brand Ito En, manufactured by Ito En, Ltd, headquartered in Shibuya, Tokyo.

Preparation of starter culture

1 L of fresh, pasteurized full-cream milk was heated to 90˚C for two minutes before being cooled to 42 °C. A capsule containing a probiotic mixture consisting of Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, Bifidobacterium infantis, and Bifidobacterium longum was added and incubated at 42 °C for 12 h until it reached pH 4.5.

Preparation of plain yogurt and green tea powder yogurts

Plain yogurt was prepared by mixing 90 mL of milk and 10 mL of starter culture. Different concentrations of green tea yogurt were prepared with green tea (Camellia sinensis) powder: 0.5 g (0.5% w/v), 1 g (1% w/v), 1.5 g (1.5% w/v), and 2 g (2% w/v) to each 10 mL of the starter culture, and milk was added to reach a total volume of 100 mL. Samples were analyzed before fermentation (BF), then fermented at 42 °C ± 0.5℃ in a water bath until the pH reached 4.5 and analyzed immediately after fermentation (AF). The fermented yogurts were stored at 4 °C (cold storage) and further analyzed on days 1, 7, 14, 21, and 28.

Physicochemical analysis: measurement of pH and determination of total titratable acid (TTA)

The pH was measured using a digital pH meter. 1 mL of the yogurt sample was diluted with 9 mL of distilled water (ratio 1:9), and 5 drops of 0.1% phenolphthalein were added. The yogurt suspension was titrated with 0.1 N NaOH. The volume of NaOH was recorded, and total titratable acidity was calculated using the following formula:

$$\text{TTA}\left(\%\right)=10\times {\text{V}}_{\text{NaOH}}\times 0.1\times 0.009\times 100\%$$

where 10 = dilution factor; V_NaOH = volume of NaOH; 0.1N = normality of NaOH; 0.009 = conversion factor whereby 1 mL of 0.1N NaOH neutralizes 0.009 g of lactic acid.

Preparation of yogurt water extract

Yogurt water was extracted by homogenizing 10 g of yogurt with 2.5 mL of distilled water, followed by acidification to pH 4 using 0.1 M HCl. The mixture was incubated at 45 °C for 10 min and then centrifuged at 5000 × g for 10 min at 4 °C. The supernatant was collected, neutralized to pH 7 with 0.1 N NaOH, and centrifuged under the same conditions.

Total phenolic content (TPC) assay

TPC was measured as described by Muniandy et al. (2016). 1 mL of yogurt water extract was mixed with 5 mL of distilled water, 1 mL of 95% ethanol, and 0.5 mL of 1 M Folin–Ciocalteau. The mixture was allowed to stand at room temperature for 5 min. Subsequently, 1 mL of 5% (w/v) Na₂CO₃ was added, and the reaction was incubated in the dark at room temperature for 1 h. The absorbance was measured at 725 nm using distilled water as a blank. Total phenolic content was calculated based on a gallic acid standard curve and expressed as (μg gallic acid equivalents per milliliter, (GAE)/mL) (Muniandy et al., 2016).

Antioxidant assays

Radical scavenging rate by 2,2-diphenyl-1-picrylhydrazyl (DPPH)

The DPPH radical scavenging experiment was determined following the procedure described by Muniandy et al. (2016), with minor modifications. Briefly, 100 μL yogurt water extract was mixed with 3.9 mL of 0.1 mM DPPH solution and incubated in the dark at room temperature for two hours. The absorbance was then measured at 515 nm using distilled water as the blank. A mixture containing 100 μL of methanol and 3.9 mL of DPPH solution served as the control. The radical scavenging activity was calculated using:

$$\text{Scavenging}\text{activity}\left(\%\right)\text{=}\text{1 -}\left[\left(\text{Absorbance}\text{of}\text{sample}\right)/\left(\text{Absorbance}\text{of}\text{control}\right)\right]\times \text{100}\%$$

Radical Scavenging Rate by 2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulphonic acid) (ABTS)

The ABTS•⁺ radical scavenging activity was determined according to the method Sah et al. (2014), A 20 µL aliquot of the yogurt water extract was added to 2 mL of ABTS reagent, and the mixture was incubated at 30 °C for 6 min. Absorbance was then measured at 734 nm, with the blank prepared by substituting the sample with 20 µL of distilled water. The radical-scavenging activity was calculated using:

$$\text{Scavenging}\text{activity}\left(\%\right)\text{=}\text{1 -}\left[\left(\text{Absorbance}\text{of}\text{sample}\right)\text{/}\left(\text{Absorbance}\text{of}\text{control}\right)\right]\times \text{100}$$

Ferric reducing antioxidant potential (FRAP) assay

The FRAP assay was performed according to the method of Muniandy et al. (2016). 300 mM acetate buffer, 10 mM 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) reagent and 20 mM FeCl₃ solutions at a ratio of 10:1:1 was mixed to prepare FRAP reagent. 400 µL of yogurt water extract or a standard iron (II) sulfate heptahydrate (FeSO4 · 7H2O) solution was added to 3.6 mL of FRAP reagent. The mixtures were incubated at 37 °C for 10 min in a water bath, after which the absorbance was measured at 593 nm using distilled water as the blank. A standard curve of FeSO₄ˑ7H₂O (0.3–1.0 mM) was used to quantify antioxidant capacity, expressed as mmol Fe²⁺ equivalent per liter (mmol Fe²⁺E/L).

Microbial viable cell count

Enumeration of Streptococcus thermophilus

The spread plate method was used to count S. thermophilus as described by Shori et al. (2018). Samples were serially diluted in peptone water buffer to a dilution of factor (10^–6). A sterilized petri dish was then filled with sterilized M17 media, and a sterile L-shaped spreader was used to evenly distribute 0.1 mL of the diluted sample onto the solid agar surface. The plate was incubated at 37 °C for two days, and the following formula was used to express the number of colonies that developed following incubation as colony-forming units per milliliter sample (CFU/mL):

CFU/mL = No. of colonies formed X dilution factor of the sample/0.1 mL of sample.

Enumeration of lactobacillus spp.

Lactobacillus species were enumerated using the pour plate method as described by Shori et al. (2018). Samples were serially diluted in peptone water (10^–5). The MRS medium was maintained in a molten state at 45 to 50 °C. A sterile petri dish was inoculated with 1.0 mL of the diluted sample, followed by the addition of 10 mL of sterile MRS medium and allowed to solidify. Another 10 mL of MRS medium was poured and left to solidify. The plates were then sealed with parafilm and incubated at 37 °C for 48 h. The colony counts were determined using the following formula, and results were expressed as colony-forming units per milliliter (CFU/mL):

$$\text{CFU/mL}\text{=}\text{No.}\text{of}\text{colonies}\text{formed}\times \text{dilution}\text{factor}\text{of}\text{sample/1.0 mL}\text{of}\text{sample}$$

All microbial counts were conducted in triplicate, and method precision was assessed based on the repeatability of log CFU measurements. The relative standard deviation (RSD) was calculated as the ratio of the standard deviation to the mean log CFU value, expressed as:

$$\text{RSD}\left(\%\right)\text{=}[\left(\text{Mean/Standard}\text{Deviation}\left(\text{SD}\right)\right]\times \text{100}$$

Across all samples and storage periods, the RSD values remained below 5% (Table S9), indicating that the microbial enumeration method demonstrated acceptable precision.

Metabolite analysis

Green tea powder yogurt (1% w/v) and plain yogurt were analyzed before fermentation and after fermentation, as described by Li et al. (2020) with slight modification. 10 g of each yogurt sample was centrifuged at 10,000 × g for 30 min at 4 °C. 1 mL of supernatant was mixed with 7 mL of acetonitrile (ACN) for 5 min. The samples were centrifuged at 10,000 × g at 4 °C for 30 min. The samples were concentrated using a Speed Vacuum Concentrator (Scanvac), reconstituted in 500 μL of 40% acetonitrile, and filtered through 0.22-μm microporous membranes (Sigma-Aldrich). Metabolite separation was performed using a Thermo Scientific C18 column (Acclaim ™ Polar Advantage II, 3 × 150 mm, 3 μm particle size) on an UltiMate 3000 UPHLC system (Dionex) using electrospray ionization. A 5 μL injection volume was used, with a total run time of 22 min. Gradient elution was carried out using H₂O/0.1% Formic Acid (buffer A) and 100% ACN (buffer B) at a flow rate of 0.4 mL/min and a column temperature of 40 °C. The gradient started at 5% B (0–3 min), 80% B (3–10 min), 80% B (10–15 min), and 5% B (15–22 min). MS and MS/MS data acquisition in negative ion modes. Caffeine was spiked into each analytical run as a system suitability and quality control (QC) compound to monitor instrument stability, retention time reproducibility, and mass accuracy.

Statistical analysis and data processing

Three biological replicates (n = 3) were performed, and Two-way ANOVA and multiple comparisons by Tukey's test were carried out using the IBM-SPSS Statistics package version 21 (SPSS Inc., Chicago, IL, USA). Statistically significant was defined at p < 0.05. Superscript letters indicating significant differences and Spearman correlation analysis were assigned using RStudio (version 2024.12.0 + 467).

Mass spectral data were acquired using Bruker Daltonics software (Bruker Daltonics, version 4.0). Raw data files (.d) were imported into the Profile Analysis program (Bruker Daltonics) for further processing. Peak alignment and normalization were performed using a custom technique based on extracted ion chromatograms (EIC). Analytical parameters included a retention time (RT) range of 0–20 min, mass range of 100–1000 Da, and a mass tolerance of 0.02 Da. Spectral acquisition was conducted at a rate of 1 spectrum per second across an m/z range of 50–1500. Fragmentation spectra for data-dependent annotation were obtained using Auto MS/MS mode.

The metabolic difference was analyzed using Principal Component Analysis (PCA), Partial Least Squares-Discriminant Analysis (PLS-DA), and hierarchical clustering to analyze LC–MS data. SIMCA-P version 18.0 (Sartorius Stedim Data Analytics AB, Umeå, Sweden) was used to do multivariate data analysis (MVDA). Unless otherwise specified, data were Pareto-scaled and log-transformed before analysis. Model performance was evaluated using R²X, R²Y, and Q² metrics, with validation conducted via cross-validation and permutation testing (n = 200). Significant metabolites were identified based on Variable Importance in Projection (VIP) scores > 1.0 and annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database via the MetFrag Web tool (accessed September 6–12, 2025). Pathway enrichment and topology analyses were performed in MetaboAnalyst 6.0 using the Hypergeometric Test and Relative-betweenness Centrality, respectively (accessed September 20, 2025).

Results and discussion

pH and TTA% changes

Before fermentation (BF), the pH of all yogurt samples was within 5.95 to 6.01 and after fermentation (AF), pH dropped significantly in all samples, reaching values between 4.47 and 4.53 in green tea yogurts and 4.51 in plain yogurt, as shown in Fig. 1(A) and Table S1. TTA% increased significantly (p < 0.05) in all samples during fermentation, as shown in Fig. 1(B) and Table S2. TTA% rose significantly after fermentation (AF), with higher values in green tea yogurts reaching up to 0.96% (2% green tea yogurt). Green tea yogurts consistently exhibited higher acidity than plain yogurt at each time point. A progressive drop in pH and a corresponding increase in TTA% are attributed to the metabolic activity of LAB, primarily S. thermophilus and L. bulgaricus, which catabolize milk lactose into lactic acid and other organic acids. The collection of lactic acids and organic acids such as acetic, formic, citric, and butyric acids, and volatile compounds like acetaldehyde, caused the pH to decrease and the acidity to rise significantly in the yogurts during fermentation and cold storage. These substances together give yogurt its distinctive sourness and flavor profile (Cheng et al., 2025; Punia Bangar et al., 2022).

Fig. 1.

(A) pH changes.( B) Total Titratable Acidity (TTA) % changes of plain yogurt and different concentrations of green tea powder yogurts before fermentation (BF), during fermentation (2 h and 4 h), after fermentation (AF), and during refrigerated storage (4˚C) on day 1, day 7, day 14, day 21, and day 28

The drop in pH and increase in TTA% were faster during fermentation, driven by optimal incubation temperature and the metabolic activity of highly active probiotic cultures. In contrast, changes during cold storage were more gradual. A synergistic interaction between S. thermophilus and L. bulgaricus happens that stimulates the growth and acidification (Hoxha et al., 2023; Yang et al., 2025). Yogurts supplemented green tea powder exhibited faster reduction in pH and higher acidity. This enhanced acidification is attributed to increased bacterial activity, the presence of additional nutrients, and bioactive compounds in green tea that collectively enhance fermentation kinetics and support probiotic viability (Muniandy et al., 2016; Xu et al., 2025). The phenolic constituents of green tea are also known to enhance the buffering capacity of yogurt, mitigating pH fluctuations caused by the accumulation of organic acids. As a result, the buffering capacity tends to increase with higher green tea concentration (Muniandy et al., 2016). However, a slight rise in pH observed toward the end of storage may be attributed to ammonia accumulation resulting from S. thermophilus urease activity, as well as the buffering effects of amino-nitrogen compounds generated during protein degradation or a decline in LAB viability over time (Yang et al., 2025). Previous studies have shown that the incorporation of polyphenol-rich plant-based compounds can enhance yogurt acidification by stimulating the metabolic activity of LAB (Xu et al., 2025). These findings are consistent with the present study, suggesting that green tea powder enhances fermentation efficiency and accelerates acidification by promoting microbial activity.

Total phenolic content (TPC)

The total phenolic content (TPC) of all yogurt samples is shown in Fig. 2(A) and Table S3. TPC increased significantly in all yogurt samples during fermentation and the first week of storage. This rise is partly attributed to the release of tyrosine and other amino acids with phenolic side chains during milk proteolysis. Additionally, microbial activity during post-acidification may enhance TPC by degrading aromatic ring structures and liberating phenolic groups (Amirdivani and Baba, 2011; Li et al., 2019). The significant amount of TPC observed in plain yogurt can be ascribed to naturally occurring milk phenols, likely derived from ruminant feed, amino acid catabolism, and small peptides released through bacterial proteolysis of milk proteins (Muniandy et al., 2016; Shori et al., 2022). Phenolic compounds are typically secondary metabolites essential for defense, adaptation, and protection against damage or infection. These compounds, which can be simple molecules or complex polymers and typically feature one or more hydroxyl groups attached to aromatic rings (Sharma et al., 2022). The consistently high TPC in green tea–fortified yogurts throughout fermentation and storage is attributed to the rich phytochemical profile of green tea. Key contributors include catechins and other flavonoids, anthocyanins, and phenolic acids. Previous studies done with green tea water extract have shown a similar trend (Amirdivani and Baba, 2015; Zhao et al., 2022).

Fig. 2.

(A) TPC changes. ( B) DPPH radical scavenging activity. ( C) ABTS radical scavenging activity. ( D) FRAP of plain yogurt and different concentrations of green tea powder yogurts before fermentation (BF), after fermentation (AF), and during refrigerated storage (4˚C) on day 1, day 7, day 14, day 21, and day 28

In the second week of storage, the TPC declined across all yogurt types, likely due to microbial transformation, oxidative degradation triggered by exposure to oxygen, light, and low pH, as well as the binding of phenolic compounds to proteins and other matrix constituents (Amirdivani and Baba, 2015; Rocchetti et al., 2022). Yogurt can effectively deliver polyphenols through milk protein–polyphenol complexes to reach the gut by enhancing the bioavailability of phenolic chemicals while also enhancing their stability during processing and storage. This interaction supports the fortification of yogurt with polyphenol-rich substances, including green tea, resulting in elevated TPC levels (Zhang et al., 2024; Zonus et al., 2025). Beyond potential health benefits and improved product quality, these complexes also contribute to yogurt’s structural integrity and functional properties (Feng et al., 2023; Zonus et al., 2025).

Antioxidant activity

Green tea–fortified yogurts showed significantly higher antioxidant activity as shown in Fig. 2(B) and Table S4 for DPPH, Fig. 2(C) and Table S5 for ABTS, and Fig. 2(D) and Table S6 for FRAP, compared to plain yogurt throughout fermentation and storage, with activity increasing proportionally to green tea concentration. The 2% green tea yogurt consistently exhibited the highest values across all assays, while plain yogurt remained the lowest at every time point. The metabolic activity of the yogurt bacteria, which remain viable and active during fermentation and cold storage, may be responsible for the increase in antioxidant activity of all yogurts during fermentation and the first few weeks of storage. The proteolysis by LAB and the enzymatic hydrolysis that releases bioactive peptides with antioxidant properties are the causes of the increased antioxidant activity in fermented milk products. Usually composed of 5–11 amino acids, these antioxidative peptides are abundant in hydrophobic residues, proline, histidine, tyrosine, or tryptophan. Proteolytic enzymes found in probiotic microbes, especially those from the Lactobacillus, Bifidobacterium, and Bacillus strains, hydrolyze dietary proteins into smaller peptides during fermentation. These bioactive peptides have anti-inflammatory, anti-cancer, antihypertensive, and antioxidant qualities, among other health-promoting qualities (Li et al., 2019; Sah et al., 2014; Singh, 2025).

The chemical structures of plant-derived polyphenolic chemicals, namely the quantity and arrangement of hydroxyl groups on aromatic rings, are inherently linked to their antioxidant efficiency. These structural characteristics support several antioxidant activity mechanisms, including electron transfer, metal ion chelation, and hydrogen atom donation. These interactions have the potential to elevate the total antioxidant capacity of yogurt systems enriched with polyphenol-rich substances, such as green tea (Olszowy, 2019). The good number of phytochemicals in green tea adds to the antioxidant ability of the yogurts. Additives like fruits (Priyashantha et al., 2025), plants (Shori and Baba, 2023), herbs (Amirdivani and Baba, 2011), and anything edible are always on researchers’ list to enhance the antioxidant ability of yogurts in creating functional foods. The added polyphenols and flavonoids support probiotic bacterial viability and strengthen antioxidant activity (Brishti et al., 2025). Muniandy et al. (2016) made yogurts with several types of teas, and it was found that compared to other tea types, green tea has better free radical scavenging and ferric reduction properties because of its high polyphenol content, especially its catechins. Degradation of phenolic compounds by oxidation and polymerization processes, as well as a decrease in viable probiotics, may be the cause of the decrease in DPPH, ABTS radical scavenging activity, and FRAP of yogurts in the subsequent weeks of storage. During storage, probiotic bacteria in yogurt can break down polyphenols into less active forms, antioxidants can be degraded by enzymes such as polyphenol oxidases or peroxidases, low pH can destabilize some phenolics and result in precipitate or structural alterations that diminish antioxidant efficacy (Shori and Baba, 2023; Yu and Hu, 2025). The combination of dairy and tea increases the antioxidant capacity of yogurt, exhibiting how phenolics from tea and naturally occurring antioxidants from dairy products enhance the functional value of yogurt.

Microbial analysis

During fermentation, S. thermophilus cell counts increased significantly (p < 0.05), with green tea yogurts showing slightly higher counts than plain yogurt, as shown in Fig. 3(A) and Table S7. Lactobacillus spp. cell counts increased significantly (p < 0.05) across all treatments, with green tea-fortified yogurts showing slightly higher levels than plain yogurt, as shown in Fig. 3(B) and Table S8. During cold storage, a decrease in microbial viability was observed across all treatments. S. thermophilus and L. bulgaricus, are the main LAB, function synergistically to drive acidification and texture development. Rapid microbial proliferation occurs during fermentation, initiated by S. thermophilus, which metabolizes lactose into lactic acid, formic acid, and carbon dioxide. This lowers the pH and creates an environment conducive to the growth of L. bulgaricus. In turn, L. bulgaricus enhances lactic acid production and proteolytic activity, hydrolyzing milk proteins (casein) into peptides and amino acids, some of which are reutilized by S. thermophilus to sustain its growth (Abedin et al., 2024; Yang et al., 2025). The increase in TTA and drop in pH during the first week in all yogurts supported the rise in S. thermophilus counts, suggesting that the growing bacterial population enhanced lactic acid production during storage. Post-acidification contributed to the decrease in viable counts of lactobacilli and streptococci in the subsequent storage weeks. This reduction is primarily attributed to the accumulation of organic acids, which progressively lowers the pH and creates a more acidic environment that may inhibit bacterial growth or harm cells. Additionally, nutrient depletion in the milk matrix restricts bacterial metabolism and replication. Environmental stressors during cold storage, including exposure to oxygen, low storage temperatures, can weaken bacterial cell membranes and reduce microbial viability (Muniandy et al., 2017).

Fig. 3.

(A) Changes in viable cell counts of Streptococcus thermophilus and ( B) Lactobacillus spp. of plain yogurt and different concentrations of green tea powder yogurts before fermentation (BF), after fermentation (AF), and during refrigerated storage (4˚C) on day 1, day 7, day14, day 21, and day 28

Prebiotics are added to enhance the viability of probiotics, and research has shown that tea polyphenols can be utilized as a prebiotic source to promote yogurt bacteria (Pramanik et al., 2025). Higher Lactobacillus spp. viability was also encouraged by green tea yogurts, likely due to the presence of powder's prebiotic-like ingredients, as it was added as powder and not just water extract. Green tea extracts as prebiotic agents can beneficially modulate gut microbiota, leading to protective effects against inflammatory conditions like colitis (Liu et al., 2020). These polyphenols have been shown to have positive impacts because of the glucosidase action of bacteria that generate energy by using sugars from glycosylated flavonoids (Singh et al., 2025).

The correlation analysis shown in Table 1 demonstrated that interactions between TPC and LAB viability are highly dependent on bacterial strain, phenolic concentration, and storage duration. Strong positive correlations observed for S. thermophilus in plain and moderately fortified green tea yogurts suggest that phenolic compounds and fermentation-induced products may support microbial activity under favorable conditions. The weaker and inconsistent correlations observed for Lactobacillus spp. further highlight strain-specific responses to phenolic environments. In previous yogurt studies utilizing green tea water extract, high levels of these beneficial bacteria were consistently maintained throughout storage, preserving the probiotic integrity and functional quality (Amirdivani and Baba, 2015; Muniandy et al., 2017). Rahmani et al. (2021) reported that the incorporation of green tea extract at different concentrations did not significantly impact the viability of Lactobacillus acidophilus and Bifidobacterium bifidum during the storage period. Celik et al. (2023) have demonstrated that incorporating black and green tea into yogurt enhances its bioactive properties without compromising texture or microbiological viability (> 10⁷ cfu/g during storage) while preserving sufficient sensory acceptability. Unal et al. (2018) also found that the yogurts with moderate concentrations of green and black tea improved the survival of the starter culture. Green tea powder can be incorporated into yogurts without compromising the probiotic viability required for exerting health benefits.

Table 1.

Spearman correlation coefficients (ρ) and p-values between Total Phenolic Content (TPC) and Lactic Acid Bacteria (LAB) viability of yogurts across different concentrations and storage times

Category	Condition	S. thermophilus	Lactobacillus spp.
		Coefficients (ρ)	p-value	Coefficients (ρ)	p-value
Yogurt type	Plain	0.84*	0.02	0.64	0.14
	0.5% green tea	0.79	0.05	0.39	0.40
	1% green tea	0.29	0.56	− 0.29	0.56
	1.5% green tea	0.90*	0.01	0.04	0.96
	2% green tea	0.71	0.09	− 0.04	0.96
Storage time	BF	− 0.67	0.22	0.82	0.09
	AF	− 0.90	0.08	0.82	0.09
	Day1	− 1.00*	0.02	− 0.40	0.52
	Day7	− 0.87	0.05	0.10	0.95
	Day14	− 0.87	0.05	0.30	0.68
	Day21	-0.90	0.08	0.60	0.35
	Day28	− 0.70	0.23	0.60	0.35

Spearman’s correlation coefficients (ρ) were calculated to assess the association between TPC and LAB viability. Values in *indicate statistically significant correlations (p < 0.05). BF = Before Fermentation, AF after fermentation

Metabolomics

Multivariate analysis

Green tea-fortified and plain yogurt samples exhibited distinct separation along PC1 and PC2, as shown in PCA plot in Fig. 4(A), suggesting significant biochemical changes resulting from both fermentation and the incorporation of green tea powder. The two principal components (PC1 and PC2 explained 45.7% and 27.4% of the total variance, respectively. These group separations were further supported by the supervised PLS-DA model Fig. 4(B) which showed improved class discrimination with R2X(cum) = 0.731, indicating strong model reliability. Additionally, the minimal metabolic shifts observed between PA and PB groups suggest that fermentation alone, in the absence of green tea, contributes minimally to metabolite diversification, highlighting the role of green tea powder as a functional modulator in yogurt systems.

Fig. 4.

(A) Score Scatter Plot PCA (B) Score Scatter Plot PLS-DA. GB (Green Tea Powder Yogurt Before Fermentation), GA (Green Tea Powder Yogurt After Fermentation), PB (Plain Yogurt Before Fermentation), PA (Plain Yogurt After Fermentation)

LAB fermentation primarily facilitates lactose hydrolysis into lactic acid and related organic acids, alongside proteolysis that liberates free amino acids and bioactive peptides. These biochemical processes enhance yogurt’s flavor, nutritional value, and functional qualities, though their effects were relatively modest in plain yogurt formulations (Cheng et al., 2025). In contrast, green tea supplementation introduced a diverse array of polyphenols, including catechins (EGCG, EGC, EC, and ECG), flavonol glycosides, and phenolic acids, which underwent extensive microbial biotransformation. Enzymes such as tannase, esterase, and β-glucosidase catalyze the hydrolysis of complex catechins into simpler, more bioavailable compounds, including gallic acid, quercetin, kaempferol, and caffeic acid, thereby enhancing bioavailability (Muniandy et al., 2016; Xu et al., 2025). This transformation promoted LAB growth, viability, antioxidant activity, and acidification rates, as seen in the results above. Green tea fortification thus significantly enriched the metabolic diversity of yogurts, introducing novel classes of bioactive metabolites while concurrently supporting microbial viability (Amirdivani and Baba, 2015; Sharma et al., 2022). The ability of certain LAB to thrive in the presence of polyphenolic compounds supports the development of functional foods aimed at fostering a healthy gut ecosystem (Pramanik et al., 2025).

Metabolite identification and pathway enrichment analysis

86 metabolites were successfully identified having a Variable Importance in Projection (VIP) score > 1.0, as shown in Table S10. Most of the identified metabolites were polyphenols, including flavonoids, phenolic acids, and their conjugate forms, derived from the incorporation of green tea powder. Green tea catechins, EGCG, EGC, EC, and ECG, had high VIP score. Additional tea-derived flavonol glycosides are also prominent, such as myricoside, isovitexin 2''''-O-arabinoside, p-coumaroyl quinic acid, and quinic acid. In contrast, several metabolites were common across all yogurt formulations (PB, PA, GB, and GA), encompassing tricarboxylic acid cycle (TCA) metabolites such as citrate and cis-aconitate; amino-sugar phosphate (N‑acetyl-galactosamine 6-phosphate) and folate derivatives (10-Formyltetrahydropteroyldiglutamate); and glutathione disulfide, guanosine 3''-diphosphate 5''-triphosphate, carboxyphosphonoenolpyruvate, and L-xylo-Hexulonolactone.

Pathway analysis for milk-derived metabolites was conducted using Bos taurus (cow) to ensure accurate representation of dairy-related pathways. In the absence of a dedicated KEGG library for Camellia sinensis (green tea), tea-derived metabolites were analyzed using Arabidopsis thaliana (thale cress), the most widely utilized plant model organism. Figure 5(A, B) display all matched pathways from the pathway enrichment analysis and pathway impact values from the pathway topology analysis for Bos taurus and Arabidopsis thaliana, respectively. The meta-analysis identified 33 pathways using the Bos taurus library (Table S11), with 10 pathways exhibiting statistical significance (p < 0.05). In comparison, the Arabidopsis thaliana library yielded 43 pathways (Table S12), of which 12 were significant at the same threshold. The most significantly impacted pathways are annotated in the corresponding plots.

Fig. 5.

Scatter plot of identified pathways using ( A) Bos taurus library and ( B) Arabidopsis thaliana library. Each bubble represents a pathway, where highly enriched routes appear as larger circles because node size correlates to the enrichment ratio, while bubble color reflects significance (yellow = less significant, red = highly significant). Large red bubbles highlight the most biologically meaningful pathways in the analysis

The synergistic action of LAB and tea polyphenols during cow’s milk fermentation modulated several metabolic pathways. Significant pathways influenced by both milk- and plant-derived metabolites included glycolysis or gluconeogenesis, the TCA cycle, starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, and pyruvate metabolism. Galactose metabolism was significantly impacted by milk metabolites but not by those from tea. These reflect the core microbial catabolism of milk carbohydrates into lactic acid and associated intermediates, facilitating downstream energy generation. Additionally, purine metabolism (significantly enriched in the milk pathway) suggests active nucleotide turnover during bacterial proliferation (Yang et al., 2025). Purine metabolism, amino sugar and nucleotide sugar metabolism, and starch and sucrose metabolism pathways were significantly modulated in yogurt. The biosynthesis of phenylalanine, tyrosine, and tryptophan reflects the availability of aromatic amino acids derived from milk proteins and their microbial transformation during fermentation. Subsequent phenylalanine metabolism involves microbial catabolism of free phenylalanine into downstream metabolites such as phenylpyruvate, phenylacetate, and phenylethyl alcohol, which contribute fruity, floral, or malty notes to both yogurt types (Sharma et al., 2022; Yang et al., 2025).

Pathway analysis using a plant-based library revealed the distinct metabolic contribution of green tea phytochemicals. Strong enrichment was observed in flavonoid biosynthesis as well as flavone and flavonol biosynthesis pathways, consistent with the presence of catechins, flavonols, and other polyphenolic derivatives introduced by green tea powder (Amirdivani and Baba, 2015; Zhao et al., 2022). Collectively, the cow and plant pathway libraries offer complementary insights into the metabolic landscape of yogurt fermentation and demonstrate that green tea fortification not only preserves classical fermentation-associated pathways but also expands the metabolic landscape through the incorporation of plant-derived bioactive metabolites. In summary, the findings of our study support the use of green tea powder as a functional ingredient in fermented dairy matrices, offering a natural strategy to enhance antioxidant activity, probiotic stability, and the delivery of bioactive compounds and probiotics through consumption of yogurt with health-promoting properties.

Electronic supplementary material

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Glossary

PLS-DA

Partial least squares-Discriminant Analysis

PCA

Principal Component Analysis

VIP

Variable Importance in Projection

PCA

lactic acid bacteria

EGCG

epigallocatechin gallate

ECG

epicatechin gallate

EGC

epigallocatechin

EC

epicatechin

NaOH

Sodium Hydroxide

HCL

Hydrochloric Acid

(Na₂CO₃)

Sodium Carbonate

LAB

Lactic Acid Bacteria

S. thermophilus

Streptococcus thermophilus

L. bulgaricus

Lactococcus bulgaricus

TTA

Total Titratable Acid

(GAE)/MI

gallic acid equivalents per milliliter

TPC

Total Phenolic Content

CFU/MI

colony-forming units per milliliter

ACN

acetonitrile

QC

quality control

EIC

extracted ion chromatograms

VIP

After fermentation before cold storage

KEGG

Kyoto Encyclopedia of Genes and Genomes

BF

Before fermentation

AF

After fermentation before cold storage

PB

plain yogurt before fermentation

PA

plain yogurt after fermentation

GB

green tea yogurt before fermentation

GA

green tea yogurt after fermentation

Funding

Open access funding provided by The Ministry of Higher Education Malaysia and Universiti Malaya. This work was funded by Ministry of Higher Education, Malaysia, FRGS 2019-1, Saiful Anuar Karsani.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.