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. 2025 Mar 19;27:102402. doi: 10.1016/j.fochx.2025.102402

Phytochemical profiling of Thai plant-based milk alternatives: Insights into bioactive compounds, antioxidant activities, prebiotics, and amino acid abundance

Nonthiwat Taesuk a, Aidong Wang b, Manmanut Srikaew c, Theeraphan Chumroenphat d, Daniela Barile b, Sirithon Siriamornpun c,e, Apichaya Bunyatratchata c,e,⁎
PMCID: PMC11984580  PMID: 40213326

Abstract

This study compared the bioactive compounds, antioxidant activities, presence of prebiotic oligosaccharides, and amino acid profiles of six Thai plant-based milk alternatives: oat, yellow corn, tamarind seed, jackfruit seed, germinated red rice, and red rice milk. Among these six plant-based milks, oat milk exhibited the highest concentrations of amino acids and β-glucan. Jackfruit seed milk showed the closest resemblance to oat milk in terms of amino acid composition, suggesting its potential as a plant-based milk alternative. Tamarind seed milk demonstrated high levels of total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity (measured by DPPH and FRAP), and presence of prebiotic oligosaccharides including raffinose, stachyose, and verbascose. Germinated red rice milk displayed significantly higher raffinose content compared to red rice milk. This investigation provides the first comprehensive report on the compositions of various Thai plant-based milks, highlighting their potential use in functional beverage development.

Keywords: Plant-based milk alternatives, Amino acids, Prebiotics, Bioactive compounds, Antioxidant activities

Highlights

  • •

    Comprehensive analysis of phytochemicals in six Thai plant-based milks.

  • •

    Jackfruit seed milk showed amino acid profiles closely resembling oat milk.

  • •

    Tamarind seed milk showed high TPC, TFC, antioxidant activity and oligosaccharides.

  • •

    Prebiotic raffinose, stachyose, and verbascose varied across plant-based milks.

  • •

    Germinated red rice milk contained significantly higher raffinose than red rice milk.

1. Introduction

Over the past few years, climate change and the environmental impact on human beings have been a huge concern across the globe. These issues have led to a growing trend toward sustainability to reduce pollution and environmental harm. Food production is one of the major contributors to environmental impact, accounting for over 26 % of global greenhouse gas emissions (Ritchie et al., 2022). Plant-based food, when compared to animal-based alternatives, generally leads to lower pollution levels and causes less environmental damage (McClements, 2020; Ritchie et al., 2022). For example, 1 kg of beef produces 60 kg of greenhouse gas emissions, whereas 1 kg of peas emits only 1 kg of greenhouse gases (Ritchie et al., 2022). Additionally, public perception associates plant-based diets with health benefits due to lower consumption of saturated fat and higher fiber intake (Lea et al., 2006). These trends in healthy diet consumption and sustainability have driven the expansion of plant-based diet markets.

Plant-based food is composed of “all minimally processed fruits, vegetables, whole grains, legumes, nuts and seeds, herbs, and spices and excludes all animal products, including red meat, poultry, fish, eggs, and dairy products”(Ostfeld, 2017). According to the USDA report, the market of plant-based food in Thailand is expected to grow annually from the current 2–10 % to 10–35 %, reaching the value of U.S. $1.5 billion in 2024 (Sirikeratikul, 2021). One of the major plant-based diet categories is plant-based milk alternatives, which account for about 35 % of the total plant-based food markets (Gaan et al., 2020). Over the past few years, plant-based milk alternatives have grown rapidly with several products being commercially available. The sale of dairy alternative products in Thailand increased from approximately U.S $600 million in 2016 to around U.S $800 million in 2020 (Sirikeratikul, 2021). The plant-based beverage industry, particularly in the functional beverage segment, has experienced significant growth, expanding by 20 % with numerous commercial products now available (Gaan et al., 2020; Sirikeratikul, 2021).

Plant-based milk alternatives are fluids made by breaking down the plant materials to small size, extracted by water, and homogenized, mimicking the appearance of cow's milk (Sethi et al., 2016). Plant-based milk alternatives are free of lactose and cholesterol compared to dairy milk and generally low in calories and saturated fat (Mäkinen et al., 2016; Sethi et al., 2016). Due to concerns regarding calorie intake, hypercholesterolemia, lactose intolerance and allergy to some diary proteins, many consumers would prefer to choose plant-based milk alternatives over cow milk (Sethi et al., 2016; Yu et al., 2023). Even though from a nutritional point of view, the value of plant-based beverages can be inferior compared to cow's milk, plant-based milk alternatives contain bioactive compounds and prebiotics that could provide health benefits to the consumers (Sethi et al., 2016).

Plant-based milk alternatives are rich in antioxidants including phenolic compounds and flavonoids (Aydar et al., 2020). A study by Silva et al. (2020) showed that the total phenolic compounds of plant-based beverages ranged from 0.20 to 12.4 mg GAEq/L (Silva et al., 2020). The antioxidant activity was also reported in the range from 3.07 to 306.46 μmol TE/L (Silva et al., 2020). Moreover, potential prebiotics have been found in several plant-based milks including oat milk, almond milk, and soy milk (Huang et al., 2023; Sethi et al., 2016). A prebiotic is “a substrate that is selectively utilized by host microorganisms conferring a health benefit” (Gibson et al., 2017). Prebiotic oligosaccharides such as stachyose, raffinose and are known to be present soymilk in significant quantities (Singh & Vij, 2018). β-glucan, a potential prebiotic, has also been identified in oat milk (Deswal et al., 2014; Sethi et al., 2016). These prebiotics might also be found in other plant-based milk alternatives, which deserved further investigation.

Apart from the popular plant-based beverages, plant-based raw materials that are widely available in Thailand, rich in antioxidants and/or prebiotics could be a potential candidate for production of Thai-plant based milk alternatives. Thai pigmented rice was reported to contain bioactive compounds (Vichit & Saewan, 2015). Total phenolic content was found in the range of 0.05–0.54 mg GAE/mL for black rice and 0.21–0.99 mg GAE/mL for red rice (Vichit & Saewan, 2015). Some by-products of tropical fruits such as kernel seeds, which are often discarded as food waste, also contain nutritional and functional values (Kumoro et al., 2020). These seeds might potentially be good to produce plant-based milk alternatives. Jackfruit seeds were about 18–25 % of total fruit weight (Kumoro et al., 2020) and also contained polyphenols, flavonoids, and tannins with the value of 243 mg GAE/100 g dry seeds, 2.03 mg CE/100 g dry seeds and 0.06 mg/100 g seed, respectively (Kumoro et al., 2020). Tamarind seed was reported to have high antioxidant activity (Natukunda et al., 2015). Moreover, some of these plant materials contain prebiotic oligosaccharides. For example, jackfruit seeds were found to have raffinose and stachyose (Jayus et al., 2016). Tamarind seed was reported to contain verbascose, stachyose, and raffinose (Pugalenthi et al., 2004). These plant-based raw materials are a source of bioactive compounds/prebiotics and they are major agricultural crops in Thailand. Plant-based milk alternatives that produce from these raw materials deserved to be investigated in detail of bioactive compounds, prebiotics, or other important aspects for the potential use as a candidate for Thai plant-based milk alternative production in the future.

To the best of our knowledge, there have not been an in-depth analysis conducted on the individual profile and abundance, particularly focusing on prebiotics and amino acids, in less popular plant-based milk alternatives such as jackfruit seed milk or tamarind seed milk. This study aimed to analyze the bioactive compounds, antioxidant activities, prebiotics, and amino acid abundance of plant-based milk alternatives produced from seeds/cereals of major agricultural crops in Thailand and compare them with other more popular seed/cereal-based milk alternatives to assess their potential as future functional beverages.

2. Materials and methods

2.1. Preparation of plant-based milks from raw materials

Plant-based milk alternatives were prepared following previously published methods (Jiang et al., 2013) with some modification. All plant-based milk samples were prepared using the same procedure and raw materials (oat, yellow corn, tamarind seed, jackfruit seed, germinated red rice and red rice) were obtained from a single batch to reduce lot-to-lot differences. Briefly, the seed materials (Fig. 1) were washed and soaked in deionized water (DI) in the ratio of 1:9 w/v (raw material: water) for 24 h at ambient temperature. The samples were ground and the slurry was filtered through cheesecloth to obtain plant-based milks. These milks were thermally treated at 80 °C for 5 min and then stored at −20 °C until they were analyzed.

Fig. 1.

Fig. 1

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Appearance, color and pH of the raw ingredients and final plant-based milk alternatives.

2.2. Color and pH measurement of plant-based milk alternatives

The color of plant-based milks was measured using a Chroma meter CR-400 (Konica Minolta Inc., Osaka, Japan) following the manufacturing protocol. The color results were expressed as CIE L*a*b*. The color difference (ΔE) was calculated following the equation below (Chung et al., 2015):

ΔE∗=ΔL∗2+Δa∗2+Δb∗2½

The pH was determined using a pH meter (FiveEasy Plus Mettler toledo, Greifensee, Switzerland). The experiment was performed in three replicates.

2.3. Characterization of bioactive compounds on plant-based milk alternatives

2.3.1. Sample preparation

Plant-based milk samples (30 mL) were defatted by centrifugation at 7000 rpm for 10 min at 4 °C (Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany). Once the samples were clearly separated into layers, the skim plant-based milks were carefully collected in a separate vial for subsequent analysis of total phenolic content, total flavonoid content, and antioxidant activity.

2.3.2. Determination of total phenolic content and total flavonoid content

Total phenolic content (TPC) was quantified using Folin–Ciocalteu's method according to a previously published method (Kubola et al., 2011) with some modification. Briefly, 0.5 mL of extracted samples were mixed with 2.5 mL of 10 % Folin–Ciocalteu reagent, left for 4 min, and added 2 mL of 7.5 % sodium carbonate solution. The samples were incubated at ambient temperature for 2 h in the dark and the absorbance were read at 725 nm using a spectrophotometer. The experiment was performed in three replicates. The total phenolic content was expressed in μg of gallic acid equivalents per mL sample (μg GAE/mL).

Total flavonoid content (TFC) was determined according to a published method (Chumroenphat et al., 2021) with some modification. Briefly, 500 μL of extracted samples were mixed with 2.25 mL of DI water and 150 μL of 5 % NaNO2 solution. The samples were votexed and left in the dark for 6 min. Then 300 μL of 10 % AlCl3 was added and left for 6 min in the dark before adding 1000 μL of 1 M NaOH and 550 μL of DI water. The absorbance was read at 510 nm using a spectrophotometer. The experiment was carried out in triplicates. Total flavonoid content was expressed as μg quercetin equivalents per mL sample (μg QE/mL).

2.3.3. Determination of antioxidant activity by DPPH and FRAP

Antioxidant activity were performed using DPPH (2,2-Diphenyl-1-Picrylhydrazyl) Scavenging Assay according to the published method (Marinova & Batchvarov, 2011) with some adjustment. Briefly, 0.5 mL of extracted samples were mixed with 4.5 mL of 0.06 mM DPPH solution. The samples were shaken and left in the dark at ambient temperature for 30 min. The absorbance was measured at 517 nm using UV–Vis Spectrophotometer. The assay was carried out in triplicates and the results were expressed as μg Vitamin C Equivalent per mL sample (μg VCE /mL).

Antioxidant activity were measured by FRAP (ferric reducing antioxidant power) assay according to the published method (Hosseinzadeh & Sadeghnia, 2005) with some modification. Briefly, 0.1 mL of extracted samples were combined with 4.5 mL of FRAP reagent. The samples were mixed, left in the dark at ambient temperature for 10 min and the absorbance was measured at 593 nm. The assay was performed in three replicates and the results were expressed as μg Ferrous sulfate (FeSO4) per mL sample (μg FeSO4/mL).

2.4. Quantification of total amino acids by LC-MS/MS

The sample preparation for the LC-MS/MS analysis of total amino acids were modified from a previous published methods (Mazhitova & Kulmyrzaev, 2016) with some adjustments. Briefly, 0.7 g of plant-based milk samples were mixed with 5 mL of 6 N HCl. The hydrolysis was carried out in a hot-air oven (WTB Binder ED115/E2, Germany) at 110 °C for 23 h. The acid solution was removed using rotary evaporation (Buchi R-210, Switzerland). Subsequently, the samples were redissolved in 10 mL of distilled water, filtered through a 0.22 μm nylon membrane prior to analysis by LC–MS/MS.

The samples were analyzed by LC/MS/MS (Shimadzu LCMS-8030 triple quadrupole mass spectrometer) with electrospray ionization (ESI) and HPLC system (Shimadzu, Kyoto, Japan). The gradient elution was performed on an InertSustain® C18 (2.1 × 150 mm, 3 μm) column, coupled with a guard column. The LC/MS/MS condition was operated following the method as described previously (Chumroenphat et al., 2021). The analysis was performed in three replicates and the results were expressed as μg/g sample.

2.5. Characterization of prebiotics

2.5.1. Quantification of β-glucan

Prebiotic β-glucan in twelve plant-based milk were quantified using the β-glucan assay kit (Megazyme, Bray Co., Wicklow, Ireland). The experiment was performed following the manufacturing protocol and the analysis was carried out in triplicate.

2.5.2. Quantification of oligosaccharides

Six plant-based milks were dried into powders using a freeze dryer (Martin Christ beta 1–8 LSCbasic, Germany). The samples were frozen at −30 °C overnight and then subjected to the freeze-drying process. The ice condenser was set at −55 °C and the vacuum pressure of 0.128 mbar was applied during the process. The samples were stored at −20 °C prior to analysis.

For oligosaccharide quantification, 20 mg of each plant-based milk powder was aliquoted into 1.5 mL tubes and suspended in 900 μL nanopure water. 50 μL of Carrez I (85 mM K4[Fe(CN)6]) and II solutions (250 mM ZnSO4) were added into the tubes to make the total liquid volume of each tube 1 mL. The samples were vortexed and shaken at 400 rpm for 5 min on a thermomixer and centrifuged at 13,000 rcf at room temperature for 15 min. The supernatants were transferred to 2 mL volumetric flasks. 1 mL of water was added into the 1.5 mL tubes to resuspend the pellet and extract the remaining oligosaccharides at 400 rpm for 5 min on a thermomixer. The mixture was centrifuged at 13,000 rcf at room temperature for 15 min, and the supernatants were transferred to their corresponding 2 mL volumetric flask to combine with the previous supernatants. Additional water was added to the mark to bring the final volume to 2 mL. Samples were filtered with 0.2 μm syringe filter before analysis.

Filtered samples were diluted and injected onto a ThermoFisher Dionex ICS-5000 + high-performance anion-exchange chromatography-pulsed amperometric detection (HPAE-PAD) system equipped with a CarboPac PA200 guard column (3 × 50 mm) and a CarboPac PA200 analytical column (3 × 250 mm). Mobile phase solvents consisted of nanopure water (A) and 200 mM NaOH (B). Chromatographic separation was carried out using the following gradient: 0–12 min (0.6–10 % B), 12–19 min, (10–30 % B) at a flow rate of 0.5 mL/min. The column was flushed with 100 % B for 5 min at the end of each run and equilibrated with 0.6 % B for 10 min before the next injection. Calibration curves were constructed by injecting standard solutions with concentrations of 0.0001–0.01 g/L. Raffinose (product 95,068, purity 99.0 %), stachyose (product S4001, purity 98 %), and verbascose (product 56,217, purity 97.3 %) were purchased from MilliporeSigma (St. Louis, MO, USA). The concentration of raffinose, stachyose, and verbascose in samples was calculated with the calibration curves. The analysis was performed in duplicate.

2.6. Data analysis

The results were presented as mean ± standard deviation of the three replicates (SD). For bioactive compounds (TPC, TFC) and antioxidant activities measured by DPPH and FRAP were log transformed by applying the logarithm function to the data values to achieve a normal distribution prior to statistical analyses. One-way analysis of variance (ANOVA) and Tukey's multiple comparison tests were performed to indicate the differences in bioactive compounds and antioxidant activities of the six plant-based milk alternatives using R version 4.3.1. A p-value less than 0.05 (p < 0.05) was considered statistically significant and indicated by different superscript letters among the samples. For individual amino acid profiling, the amino acid contents were analyzed using a heatmap and Ward's hierarchical clustering method via the “heatmaply” package (Galili et al., 2018). This analysis was performed on the raw data to preserve the original content and maintain the interpretability of the clustering patterns. Clustering was based on the similarity in amino acid content. For principal component analysis (PCA), all data were log-transformed to equalize variance, ensuring that variables with larger variance did not disproportionately influence the principal components. This analysis was performed using the “factoextra” package (Kassambara & Mundt, 2020) in R software.

3. Results and discussion

The results and discussion section has been subdivided into five parts, encompassing: (1) Color and pH measurement of plant-based milk alternatives, (2) Bioactive compounds and antioxidant activities, (3) amino acid profile and concentrations, (4) Prebiotic characterization, and (5) principal component analysis (PCA) of plant-based milk composition.

3.1. Color and pH measurement of plant-based milk alternatives

The pH level of plant-based milks significantly impacts their physicochemical, biochemical, and sensory properties. It influences solubility, microbial growth, as well as texture and flavor of the product (McClements & Grossmann, 2022). Previous studies have reported that plant-based milk alternatives generally have a pH ranging from 6.0 to 7.5 (Daszkiewicz et al., 2024; Jemaa et al., 2021). While pH can be adjusted using food-grade acids or bases (McClements & Grossmann, 2022), this study measured the natural pH without adjustment. The pH levels of six plant-based milk alternatives ranged approximately from 4.6 to 6.2, with oat milk exhibiting the lowest pH at 4.6 and red rice milk presenting the highest pH value at 6.2 (Fig. 1). The lower pH compared to previous reports may be due to differences in raw material composition, processing methods, or storage conditions. When comparing color measurements to oat milk as the control, jackfruit seed milk alternative closely resembled oat milk in color compared to other alternatives. Color attributes were expressed in term of lightness (L*), greenish–reddish color coordinate (a*) and bluish-yellowish color coordinate (b*) within the CIELAB color space (Sant'Anna et al., 2013). Both lightness (L*) and yellowness (b*) showed no significant difference between oat and jackfruit milk (Fig. 1). Based on the color difference (△E*), jackfruit seed milk alternative demonstrated the smallest deviation at 4.3, making it the closest color to oat milk. Regarding color, tamarind seed milk appeared to be the most distinct from oat milk, a difference that is clearly observable in the milk's appearance (Fig. 1).

3.2. Bioactive compounds and antioxidants activities

The variations in levels of bioactive compounds and antioxidant activities of six plant-based milk alternatives were shown in Fig. 2. According to the statistical analyses, tamarind seed milk alternative contained significantly higher levels of TPC, TFC, and antioxidant activity than other plant-based milk alternatives (p < 0.05). The average TPC, TFC, and antioxidant activities measuring by DPPH and FRAP of tamarind seed milk alternative were 2488.44 ± 23.57 μg GAE/mL, 8037.04 ± 128.30 μg QE/mL, 9270.86 ± 161.16 μg VCE/mL, and 24954.17 ± 260.21 μg FeSO4/mL, respectively (Table S1). Although some milk samples exhibited higher TFC than TPC (see Table S1), similar findings have been reported in other studies (Ahmed et al., 2019; Azieana et al., 2017). This discrepancy may result from the Folin-Ciocalteu method's sensitivity to specific flavonoid structures. Flavonoids, with varying reducing capacities based on their hydroxylation and glycosylation patterns (Pérez et al., 2023), may not react as strongly with the Folin-Ciocalteu reagent, potentially leading to an underestimation of their concentration. Nevertheless, tamarind seed consistently exhibited the highest values for both TPC and TFC among the samples. The seed coat of tamarind has been identified as a rich source of phytochemicals with antioxidant properties, including tannins, procyanidin, epicatechin, catechin, 2-hydroxy-3′,4′-dihydroxyacetophenone, methyl 3,4-dihydroxybenzoate, and 3,4 dihydroxyphenyl acetate (Wandee et al., 2022). The results of this study therefore demonstrate that despite being processed into a liquid for milk alternative, tamarind seeds retain significant bioactive compounds and antioxidant properties, highlighting their potential utility in functional beverages.

Fig. 2.

Fig. 2

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Analysis of total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activities.

The antioxidant activity results were higher in the FRAP assay compared to the DPPH method, which may be attributed to their differing mechanisms. The DPPH assay evaluates antioxidant activity based on the ability of antioxidants to stabilize the DPPH radical by donating electrons or hydrogen atoms, whereas the FRAP assay assesses it through the electron transfer mechanism (Munteanu & Apetrei, 2021; Zielińska & Turemko, 2020). Interestingly, the antioxidant activities measured by DPPH and FRAP exhibited a consistent trend with TFC rather than TPC. For the TFC, DPPH, and FRAP analyses, the red rice milk alternative showed the second highest values, followed by germinated red rice, jackfruit seed, oat, and yellow corn. It was suggested that procyanidins, the predominant natural flavonoid pigments found in colored red rice grains (Vichit & Saewan, 2015), contribute to these high antioxidant activities. Cereals/seeds containing dark-colored pigments generally possess the higher bioactive compounds and antioxidant activities compared to those with light-colored pigments. Among cereal/seed samples with light-colored pigments (jackfruit seed, oat, and corn), jackfruit seed milk alternatives showed significantly higher values of TPC, TFC, DPPH, and FRAP (p < 0.05) compared to oat and corn milk alternatives. Jackfruit seeds, often discarded as waste or by-products of the food processing industry, have been reported to contain significant bioactive compounds, including phenolics, flavonoids and tannins (Kumoro et al., 2020). See Table S1 for measuring variable dataset.

3.3. Amino acid profile and concentrations

The amino acid profile and concentrations of 20 amino acids, including both essential and non-essential amino acids, in six plant-based milk samples were quantified using LC-MS/MS. Nine amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) are classified as essential, as they cannot be synthesized by humans and must be obtained through the diet (Lopez & Mohiuddin, 2024). Arginine is considered conditionally essential, as the body may not synthesize adequate amounts during specific physiological conditions (Lopez & Mohiuddin, 2024). In this study, essential amino acids were reported as the nine listed above plus arginine, while the remaining amino acids were classified as non-essential, as they are synthesized by the body and not primarily derived from the diet (Lopez & Mohiuddin, 2024). Overall, the concentration of essential amino acids was higher than the non-essential amino acids in all milk alternatives. Oat milk exhibited the highest total, essential, and non-essential amino acid contents followed by jackfruit seed, yellow corn, tamarind seed, red rice and germinated red rice (Fig. 3). There were significant differences in the total, essential, and non-essential amino acid contents among the milk alternatives investigated (p < 0.05), excepted for essential amino acids between jackfruit seed milk and yellow corn milk (Fig. 3). The individual amino acid content in the six plant-based milk alternatives is provided in Table S2.

Fig. 3.

Fig. 3

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Characterization of amino acid content using LC-MS/MS.

For amino acid profiling, the individual amino acid concentrations (μg/g) and the clustering of samples are shown in Fig. 4. The clustering was determined based on the similarity in their amino acid content. Using Ward's method through the ‘heatmaply’ R package, the plant-based milk alternatives were categorized into three distinct groups. The first group comprised only oat milk, the second group included jackfruit seed milk and yellow corn milk and the third group consisted of red rice, germinated red rice, and tamarind seed. Regarding essential amino acids, the amino acid profiles of jackfruit seed milk and yellow corn milk are more similar to oat milk compared to other samples. Red rice and germinated red rice milks are the most similar to each other and both are closest to tamarind seed milk. Among all six plant-based milk alternatives, arginine was the predominant amino acid, ranging from 168.01 μg/g in germinated red rice milk to 367.65 μg/g in oat milk (Fig. 4 and Table S2). Phenylalanine, isoleucine and leucine were more abundant in jackfruit seed, yellow corn, and oat milk alternatives compared to red rice, germinated red rice and tamarind seed milks (Fig. 4). Histidine was present at intermediate levels compared to the other essential amino acids, while methionine, valine, lysine and threonine were generally detected in lower abundance (below 100 μg/g) across all samples, except for valine in oat milk, methionine in yellow corn milk and lysine in jackfruit seed milk (Fig. 4 and Table S2). Arginine has been reported to play important roles in cellular regeneration, wound healing, immunity and the regulation of nutrient metabolism (Sudar-Milovanovic et al., 2015; Wu et al., 2021). Phenylalanine is also essential for the formation of neurotransmitters and cognitive development (Akram et al., 2020). Isoleucine and leucine are branched chain amino acids (BCAAs) involved in protein synthesis and regulation of various key signaling pathways, connecting diverse physiological and metabolic processes (Zhang et al., 2017). Given the variability in amino acid composition among plant-based milk alternatives, some essential amino acids were present in lower concentrations. Consumers relying on these alternatives should consider incorporating complementary protein sources to ensure a balanced amino acid intake.

Fig. 4.

Fig. 4

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Heat map and hierarchical clustering analysis using Ward's method based on amino acid contents; plot generated using the “heatmaply” package in R version 4.3.1. Abbreviations: Ala: Alanine, Arg: Arginine, Asp: Aspartic acid, Glu: Glutamic acid, Gln: Glutamine, Gly: Glycine, His: Histidine, Ile: Isoleucine, Leu: Leucine, Lys: Lysine, Met: Methionine, Phe: Phenylalanine, Pro: Proline, Ser: Serine, Thr: Threonine, Tyr: Tyrosine, and Val: Valine.

For non-essential amino acids, the profile of jackfruit seed milk alternative showed the most similarity to that of oat milk, particularly in tyrosine and aspartic acid concentrations (Fig. 4). While tyrosine is used in protein synthesis across all organisms, aspartic acid participate in various metabolic pathways (Alfosea-Simón et al., 2021; Schenck & Maeda, 2018). Although jackfruit seed milk alternative contained lower amino acid content compared to oat milk, their amino acid profiles were closely resembled each other. This discovery suggests that jackfruit seed milk could emerge as a promising candidate among plant-based milk alternatives. Utilizing jackfruit seeds, which are typically considered waste or by-products of the food processing industry, as a plant-based milk alternative also contributes to the sustainability and resilience of the global food system. In addition, the findings of this study offer important insights into the amino acid profiles of various plant-based milk alternatives, suggesting their potential use in formulating nutritionally rich plant-based milk products for human consumption.

3.4. Prebiotic characterization

β-Glucan was detected in three milk alternatives derived from oat, jackfruit seed, and germinated red rice. The oat milk exhibited the highest concentrations of β-glucans (180.46 ± 2.47 mg/100 mL) followed by germinated red rice milk (3.00 ± 0.00 mg/100 mL) and jackfruit seed milk (2.02 ± 0.19 mg/100 mL). Oat naturally contains a high level of β-glucan (Burton & Fincher, 2012), with other studies reporting concentrations of 9.84 mg/mL (Zhou et al., 2023) and 0.5 g/100 g of β-glucan (Onning et al., 1999). Variations in β-glucan levels between this study and others may be attributed to differences in oat variety, beverage composition, and processing conditions. By comparing with oat milk, β-glucan in geminated red rice and jackfruit seed milks were present in trace amount. The concentrations of prebiotic raffinose, stachyose and verbascose, determined through HPAE-PAD analysis, are presented in Table 1. A peak matching the elution retention time of raffinose was detected in all six milk alternatives, ranging from 0.04 % to 0.60 % of dry weight. Tamarind seed showed the highest level (0.60 %) followed by germinated red rice (0.36 %). A peak for stachyose was also present in all plant-based milk alternatives except for yellow corn, which fell below the detection limit. Stachyose levels ranged from 0.01 % to 0.98 % of dry weight, with the highest level found in Tamarind seed milk (0.98 %) followed by oat milk alternative (0.11 %). Regarding verbascose, a peak matching the elution time was observed in all six milk samples, but only germinated red rice fell below the detection limit. In the other samples, verbascose content ranged from 0.01 % to 0.54 % of dry weight, with Tamarind seed exhibiting the highest content (0.54 %) followed by yellow corn milk (0.20 %). Interestingly, it was found that the prebiotic raffinose content in germinated red rice milk was nine times higher than that in red rice milk. Further investigation is necessary to understand whether there is an impact of germination on prebiotic content. The identification of the three oligosaccharides in plant-based milk alternatives was based on the comparison of retention time with authentic standards. Raffinose, stachyose and verbascose have not previously been reported in oat milk but the raffinose-family oligosaccharides is known to be present in cereals (Bachmann et al., 1994). A spiking experiment was conducted using authentic standards of raffinose, stachyose and verbascose in oat milk to confirm presence (Fig. S1).

Table 1.

Analysis of oligosaccharides using HPAE-PAD.

Samples Raffinose
(% dry weight) 		Stachyose
(% dry weight) 		Verbascose
(% dry weight)
Oat 0.04 ± 0.01c 0.11 ± 0.00b 0.03 ± 0.00d
Yellow corn 0.06 ± 0.00c bld 0.20 ± 0.01b
Tamarind seed 0.60 ± 0.01a 0.98 ± 0.03a 0.54 ± 0.01a
Jackfruit seed 0.06 ± 0.00c 0.09 ± 0.00b 0.07 ± 0.00c
Germinated red rice 0.36 ± 0.00b 0.02 ± 0.00c bld
Red rice 0.04 ± 0.00c 0.01 ± 0.00c 0.01 ± 0.00d
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Results are expressed as mean ± SD of duplicate measurements. Mean values in the same row with different superscript letters are significantly different (p < 0.05).

Overall, oat exhibited the highest level of β-glucan, while tamarind seed had the highest levels of prebiotic raffinose, stachyose, and verbascose. Other plant-based milk alternatives contained various prebiotics at different levels, depending on the type of milk. To the best of our knowledge, this study is the first to report detailed profiles and abundance of prebiotics in various Thai plant-based milk alternatives. This information is invaluable for the development of functional beverages containing diverse prebiotic oligosaccharides, facilitating optimal formulations made for specific applications in the future.

3.5. PCA plant-based milk composition

The PCA biplot in Fig. 5 illustrates the variation among six different plant-based milk alternatives based on their bioactive compounds, antioxidant activities, amino acid contents and prebiotics. The first two principal components (PC1 and PC2) collectively explain 83.45 % of the total variance, with PC1 contributing 62.92 % and PC2 contributing 20.53 %. PC1 demonstrates a strong separation among the samples, with oat, jackfruit seed, and yellow corn positioned on the left side of the plot, whereas tamarind seed, germinated red rice, and red rice are distributed on the right side. According to Fig. 5, the PCA biplot suggests that amino acid contents primarily contribute to PC1, while bioactive compounds, antioxidant activities, and prebiotics have a stronger influence on PC2.

Fig. 5.

Fig. 5

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Principal component analysis (PCA) biplot displaying the overall composition of the six plant-based milk alternatives.

Among the six milk alternatives, oat milk exhibited the highest concentrations of amino acid and β-glucan, whereas, tamarind seed milk contained the highest levels of bioactive compounds, antioxidant activities, and prebiotic raffinose, stachyose and verbascose. In terms of amino acid profiles, jackfruit seed milk displayed the close similarity to yellow corn, as evidenced by the proximity of their points in the PCA plot. Overall, both milks exhibited the highest similarity to oat milk alternatives when compared to other plant-based milks. Germinated red rice and red rice milk alternatives appeared to have comparable profiles. These PCA results are consistent with the data on amino acid profiles and bioactive compounds presented in Fig. 2 and Fig. 4.

4. Conclusion

Our study reveals several key findings regarding the composition and potential of various plant-based milk alternatives. Firstly, oat milk stands out with the highest levels of amino acids and β-glucan among the six alternatives assessed. Jackfruit seed milk shows the closest resemblance to oat milk in terms of amino acid composition, highlighting its potential as a nutritious alternative. Secondly, tamarind seed milk emerges as a promising candidate due to its significantly higher content of TPC, TFC, DPPH and FRAP antioxidant activities and prebiotic oligosaccharides including raffinose, stachyose, and verbascose. Furthermore, germinated red rice milk exhibits significantly higher raffinose content compared to red rice milk. This emphasizes the need for further investigation into the potential impact of germination on prebiotic content. In the future, additional research is required to develop functional beverages using these plant-based milk alternatives and, in particular, the novel beverages made from using jackfruit and tamarind seeds should be evaluate for safety before launching in the market. Moreover, it is important to consider factors such as industrial scale-up, thermal processing, seed varieties and genotypes, environmental influences and agricultural practices to standardize plant-based milk formulations. Shelf life evaluation should also be assessed to ensure product quality overtime. Additionally, comparing plant-based milks with cow's milk could further clarify the nutritional differences and bioactive properties of these alternative milks, providing valuable insights into their potential health benefits. Our study represents a pioneering effort in quantifying bioactive compounds and profiling less commonly used raw materials for plant-based milk production, providing a valuable foundation for innovation in functional beverage development.

CRediT authorship contribution statement

Nonthiwat Taesuk: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Resources, Methodology, Formal analysis, Data curation. Aidong Wang: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation. Manmanut Srikaew: Writing – review & editing, Investigation, Formal analysis, Data curation. Theeraphan Chumroenphat: Writing – review & editing, Investigation. Daniela Barile: Writing – review & editing, Validation, Supervision, Resources, Funding acquisition, Conceptualization. Sirithon Siriamornpun: Writing – review & editing, Supervision, Resources, Conceptualization. Apichaya Bunyatratchata: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used ChatGPT in order to improve language and readability. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Declaration of competing 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.

Acknowledgments

This work (Grant No. RGNS 65-142) was supported by Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (OPS MHESI), Thailand Science Research and Innovation (TSRI) and Mahasarakham University. The authors also thank the Laboratory Equipment Center of Mahasarakham University for their collaboration and provision of research facilities during this study. The quantification of oligosaccharides at the Barile Lab, UC Davis, was supported by a Hatch project CA-D-FST-2744-H.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2025.102402.

Appendix A. Supplementary data

Supplementary material.

mmc1.docx (817.5KB, docx)

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material.

mmc1.docx (817.5KB, docx)

Data Availability Statement

Data will be made available on request.

Articles from Food Chemistry: X are provided here courtesy of Elsevier

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