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. 2025 Dec 17;33:103408. doi: 10.1016/j.fochx.2025.103408

Exploring bioactive and nutritional profiles in plant-based milk alternatives from green rice (Khao Dawk Mali 105) and its milling fractions

Apichaya Bunyatratchata a,b, Theeraphan Chumroenphat c, Nonthiwat Taesuk d, Sirithon Siriamornpun a,b,⁎
PMCID: PMC12775921  PMID: 41510130

Abstract

Rising demand for plant-based beverages drives interest in cereal-based milks, yet green rice (harvested before full maturity) remains underutilized. This study investigated bioactive compounds, nutritional composition, and amino acid profiles of milks from green rice and its milling fractions: green rice milk (GM), broken green rice milk (BGM), white (refined) broken green rice milk (WBGM), green rice bran milk (GBM), and white rice milk (WM). GBM showed the highest total phenolics, flavonoids, and antioxidant activities, followed by GM and BGM. All phenolics were detected in GM, whereas GBM had the highest phenolic acids but lacked chlorogenic acid. GBM showed superior nutrients except carbohydrates. Arginine and phenylalanine were predominant essential amino acids. Despite higher protein content, green rice milks showed lower amino acid levels, potentially due to poor extractability. PCA confirmed the nutrient- and bioactive-rich profile of GBM. These results underscore green rice and its fractions' potential as functional rice milks.

Keywords: Rice milk, Plant-based milk, Bioactive compounds, Nutritional composition, Amino acids, Rice milling fractions

Highlights

  • •

    Milk produced from green rice and its milling fractions surpassed white rice milk.

  • •

    Green rice bran milk (GBM) showed the highest TPC, TFC and antioxidant activity.

  • •

    GBM had the highest phenolic acids content but chlorogenic acid was absent.

  • •

    Green rice milks had more protein but less amino acid levels from poor extractability.

  • •

    Arginine and phenylalanine were the dominant essential amino acids in all samples.

1. Introduction

Plant-based milk alternatives are liquids created by breaking down plant materials into small particles, extracting them with water, and homogenizing the mixture to resemble the appearance of cow's milk (Sethi et al., 2016). These alternatives are lactose- and cholesterol-free, contain more fiber than cow's milk and are typically low in calories and saturated fat (Mäkinen et al., 2016; Sethi et al., 2016; Vagadia et al., 2018). According to the concerns of calorie intake, high cholesterol, lactose intolerance, and cow's milk allergies, many consumers prefer plant-based milk alternatives over cow's milk (Sethi et al., 2016). This preference has led to a rise in consumption and demand for plant-based milk alternatives as part of a healthier diet, a trend that is expected to continue rising.

Rice milk, a plant-based milk alternative, has grown in popularity due to its low allergic potential, making it suitable for individuals who are lactose intolerant or have nut or soy allergies. Rice (Oryza sativa) is a major cereal feeding nearly half of the global population (Wei & Huang, 2019). Unlike many other cereal grains, rice protein is hypoallergenic due to its gluten-free nature, making it ideal for gluten-free or hypoallergenic products. The development of rice grains proceeds through several stages: flowery, milky, mature, and fully mature (Ranathunga & Suwannaporn, 2022). After fertilization, the grain filling process begins. The milky stage is characterized by a liquefied endosperm. As the kernels achieve full grain fill and begin to dry, they transition to the dough stage, which is characterized by a soft, dough-like texture. The mature stage is reached once the grains achieve maximum dry matter accumulation and firmness. However, harvesting is not yet feasible at this stage, as the grain moisture content remains at or above 30 %. An additional two weeks are typically required for the moisture content to decrease to around 20 %, allowing the grains to reach harvest maturity (Chauhan et al., 2017; Smith, 1995).

Grains harvested before reaching full mature exhibit a green color and are referred to as “green rice” in East Asian countries (Chao et al., 2022). Green rice is typically harvested between 14 and 30 days before reaching full ripeness with optimum harvest time varying among rice varieties (Chao et al., 2022; Kim et al., 2016). The starting point for counting days can vary and is generally determined by the stakeholders. In Thailand, for example, the Rice Department considers the first day after flowering (DAF) to be the day on which around 80 % of the rice plants in a field are in bloom (Ranathunga & Suwannaporn, 2022). Green grains harvested before maturity have been reported to possess high nutritional value, be rich in bioactive compounds, and have improved digestibility (Chao et al., 2022; Kim et al., 2016; Pantoa, Kubota, et al., 2020; Ranathunga & Suwannaporn, 2022). Compared with brown rice, green rice contains higher levels of bioactive compounds, including γ-aminobutyric acid, γ-oryzanol, and α-tocopherol (Kim et al., 2016). Developing rice grains also exhibit significantly higher total phenolic and flavonoid contents, as well as higher protein content than mature grains (Ji et al., 2013; Lin & Lai, 2011). Consistent with these reports, green rice has been shown to possess higher total phenolic and flavonoid contents, as well as greater antioxidant activity than white rice (Bunyatratchata et al., 2025). These findings suggest that green rice represents a promising raw material for rice milk production. However, comprehensive analyses of rice milk derived from green rice remain limited, particularly regarding detailed profiling of individual bioactive compounds and amino acids, which deserves further investigation.

In addition to whole grains, the rice milling process generates several by-products, including bran and broken rice, which are often underutilized. Whole grain rice consists of the bran, germ and endosperm, whereas refined or white rice is polished to remove the bran and germ, leaving only the endosperm. Rice bran is a nutrient-rich material and contains numerous bioactive compounds (Sapwarobol et al., 2021). Broken rice results from mechanical tension during milling, causing some grains to fracture. After polishing, rice kernels are classified by size, and those measuring less than approximately three fourths of the whole kernel length are classified as broken rice (Bodie et al., 2019). Approximately 14 % of white rice milling produces broken rice (Moraes et al., 2014). Each ton of processed paddy rice yields 100 kg of rice bran, and 140 kg of broken rice (Moraes et al., 2014). These by-products, together with green rice, represent valuable raw materials for developing functional rice milk alternatives. Despite their potential, the bioactive and nutritional profiles of rice milks derived from green rice and its milling fractions remain largely unexplored, highlighting a critical knowledge gap and the need for systematic investigation.

To address this gap, the present study aimed to comprehensively analyze rice milks produced from green rice and its milling fractions, including broken green rice, white broken green rice (or refined broken green rice) and green rice bran, with white rice milk serving as the control. A schematic illustrating the products and by-products generated during the green rice milling process, which were used as raw materials for rice milk production, is presented in Fig. 1. Specifically, the study investigated the bioactive compound profiles, antioxidant activity, nutritional composition, and individual amino acid content. These samples are referred to as green rice milk (GM), broken green rice milk (BGM), white broken green rice milk (WBGM), green rice bran milk (GBM), and white rice milk (WM). The results provide valuable insights for the development of functional beverages and the improvement of existing rice milk products.

Fig. 1.

Fig. 1

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Schematic of products and byproducts generated during the green rice milling process, serving as raw materials for rice-based milk production. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2. Materials and methods

2.1. Preparation of rice milk alternatives from green rice and its milling fractions

Green rice and its milling fractions were obtained from a farm collaborator in Roi Et Province, Thailand. Jasmine green rice (Khao Dawk Mali 105 or KDML 105) was harvested 30 days after flowering (DAF). Whole grain green rice and its milling fractions, including broken green rice, white broken green rice, and green rice bran, were prepared under controlled milling conditions. A schematic illustrating the products and by-products generated during the green rice milling process, which were used as raw materials for rice-based milk production, is shown in Fig. 1.

Rice milk alternatives were prepared according to the published method (Taesuk et al., 2025), with all rice milk types processed using the same standardized procedure to ensure consistency across samples. Considering that processing steps can influence bioactive content, antioxidant release, and nutrient bioavailability (Tanwar et al., 2025), standardization was essential to minimize variation. In brief, green rice and its milling fractions of KDML 105, including whole grain green rice, broken green rice, white broken green rice, green rice bran with control white KDML 105 rice, were thoroughly washed and soaked in deionized water (DI) at a 1:9 w/v ratio of raw material to water for 24 h at ambient temperature. The soaked rice samples were then ground for 5 min using a blender (Panasonic MX-900M, Selangor Darul Ehsan, Malaysia), and the resulting slurry was filtered through cheesecloth to obtain different rice milks. The resulting milks included white rice milk (WM) from white Khao Dawk Mali 105, green rice milk (GM) from whole grain green rice, broken green rice milk (BGM) from broken green rice, white broken green rice milk (WBGM) from white broken green rice and green rice bran milk (GBM) from green rice bran. To prevent microbial fermentation, which could influence the nutritional composition of the samples (Wang & Wang, 2024), these milks underwent thermal treatment at 80 °C for 5 min and were stored at −20 °C until analysis.

2.2. Color measurement of green rice milk alternatives

The color of each rice milk sample was assessed using a Chroma meter CR-400 (Konica Minolta Inc., Osaka, Japan) in accordance with the manufacturer's instructions. The color measurements were performed in triplicate and reported in the CIE L*a*b* color space.

2.3. Analysis of total phenolic content and total flavonoid content and antioxidant activity measured by DPPH and FRAP

Rice milk samples (30 mL) were defatted by centrifuging at 7000 rpm for 10 min at 4 °C (Taesuk et al., 2025) to remove the fat (Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany). After the samples were separated into distinct layers, the skim rice milks were carefully transferred to separate vials for further analysis of total phenolic content, total flavonoid content, and antioxidant activity.

Total phenolic content (TPC) was determined using Folin–Ciocalteu's method as described in a previously published protocol (Taesuk et al., 2025). Briefly, 0.5 mL of the prepared samples was combined with 2.5 mL of 10 % Folin–Ciocalteu reagent and allowed to stand for 4 min before adding 2 mL of 7.5 % sodium carbonate solution. The samples were incubated at ambient temperature for 2 h in the dark and the absorbance was measured at 725 nm using a spectrophotometer. The experiment was conducted in triplicate and the TPC was expressed as μg of gallic acid equivalents per mL of sample (μg GAE/mL).

Total flavonoid content (TFC) was measured using a published method (Taesuk et al., 2025). In brief, 500 μL of prepared samples was mixed with 2.25 mL of DI water and 150 μL of 5 % NaNO2 solution. The samples were vortexed and kept in the dark for 6 min. Subsequently, 300 μL of 10 % AlCl3 was added, and the mixture was left in the dark for 6 min before adding 1000 μL of 1 M NaOH and 550 μL of DI water. Absorbance was then measured at 510 nm using a spectrophotometer. The experiment was performed in triplicate, and TFC was expressed as μg quercetin equivalents per mL of sample (μg QE/mL).

Antioxidant activity was assessed using DPPH (2,2-Diphenyl-1-Picrylhydrazyl) Scavenging Assay based on a published method (Taesuk et al., 2025). In summary, 0.5 mL of prepared samples was combined with 4.5 mL of 0.06 mM DPPH solution. The mixture was shaken and kept in the dark at ambient temperature for 30 min. Absorbance was recorded at 517 nm using UV–Vis spectrophotometer. The assay was conducted in triplicate, and the results were expressed as μg Vitamin C Equivalent per mL of sample (μg VCE/mL).

Antioxidant activity was analyzed by FRAP (ferric reducing antioxidant power) assay following a previously published method (Taesuk et al., 2025). In brief, 0.1 mL of prepared samples was mixed with 4.5 mL of FRAP reagent. The samples were left in the dark at ambient temperature for 10 min and absorbance was measured at 593 nm. The assay was conducted in triplicate, and the results were expressed as μg Ferrous sulfate (FeSO4) per mL of sample (μg FeSO4/mL).

2.4. Quantification and profiling of phenolic compounds

The analysis of phenolic acids was performed using High-Performance Liquid Chromatography (HPLC) (Agilent 1260, Agilent Technologies, USA), following a previously described method with some modifications (Chumroenphat et al., 2023). All rice milk alternatives were dried into powders using a freeze dryer (Martin Christ beta 1–8 LSCbasic, Germany). Samples were initially frozen at −30 °C overnight, then freeze-dried with the ice condenser set to −55 °C and the vacuum pressure of 0.128 mbar. The freeze-dried powder was extracted with 20 mL of water/methanol (80:20, v/v) and shaken at 37 °C at 150 rpm for 12 h. The extract was filtered through a 0.22 μm nylon membrane filter, and the clear extract was used for HPLC analysis with a PerkinElmer C18 column (4.6 mm × 150 mm, 5 μm particle size, Perkin Elmer, MA, USA). The mobile phase consisted of 1 % acetic acid (solvent A) and acetonitrile (solvent B). The gradient elution was performed as follows: 0–5 min, linear gradient from 5 % to 9 % B; 5–15 min, isocratic at 9 % B; 15–22 min, linear gradient from 9 % to11 % B; 22–38 min, linear gradient from 11 % to 18 % B; 38–43 min, linear gradient from 18 % to 23 % B; 43–44 min, linear gradient from 23 % to 90 % B; 44–45 min, linear gradient from 90 % to 80 % B; 45–55 min, isocratic at 80 % B; 55–60 min, linear gradient from 80 % to 5 % B. A 5-min re-equilibration at 5 % solvent B was used between individual runs. The analysis was conducted under gradient conditions at a column temperature of 38 °C, with an injection volume of 20 μL. Absorbance was measured using a photodiode array detector at specific wavelengths: 280 nm for gallic acid, protocatechuic acid, vanillic acid, cinnamic acid; and 320 nm for chlorogenic acid. The experiment was performed in triplicate, and the concentrations of phenolic compounds were expressed as micrograms per gram dry weight (μg/g DW).

2.5. Proximate analysis of green rice milk alternatives

Rice milk samples were assessed for moisture content, crude protein (using Kjeldahl method), crude fat content (via Soxhlet extraction), crude fiber content, ash content and carbohydrate content (calculated by difference). These parameters were quantified using standard analytical methods (AOAC, 2000). Additionally, the crude protein, crude fat, crude fiber, ash and carbohydrate contents were calculated based on a dry weight basis. The experiment was conducted in triplicate.

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

Amino acids were extracted and quantified from freeze-dried rice milk powders using a previously published method (Bunyatratchata et al., 2025). In brief, the freeze-dried samples were defatted with n-hexane, and the resulting defatted material was hydrolyzed using 6 mol/L HCl at 110 °C for 15 h. Following hydrolysis, the acid was removed by evaporation at 60 °C. The dried samples were redissolved in 10 mL of distilled water, centrifuged at 12,000×g for 10 min, and filtered through a 0.22 μm nylon membrane prior to LC-MS/MS analysis.

The analysis was performed using an LC/MS/MS system (Shimadzu LCMS-8030 triple quadrupole mass spectrometer) with electrospray ionization (ESI) and HPLC system (Shimadzu, Kyoto, Japan). Gradient elution was conducted on an InertSustain® C18 column (2.1 × 150 mm, 3 μm) coupled with a guard column. The method was operated as previously described (Bunyatratchata et al., 2025). The results, expressed as μg/g sample, were obtained from three replicates.

Amino acid profiles were visualized as heatmaps generated using MetaboAnalyst 6.0 (https://www.metaboanalyst.ca), an online platform for metabolomic data analysis. The raw data were analyzed by hierarchical clustering using Pearson distance and Ward's method. This approach enabled the comparison of amino acid distribution patterns across the various samples (Chumroenphat et al., 2025).

2.7. Data analysis

All experiments were performed in triplicate. Statistical differences among the samples were evaluated using one-way analysis of variance (ANOVA), followed by Duncan's multiple range test. Samples denoted by different letters (a, b, c, etc.) were considered significantly different at p < 0.05. Principal component analysis (PCA) was conducted to explore multivariate patterns in bioactive compounds and nutritional data using the “factoextra” package in R (Kassambara & Mundt, 2020).

3. Results and discussion

3.1. Color of green rice milk alternatives

The appearance and color of various rice milk alternatives including white rice milk (WM), green rice milk (GM), broken green rice milk (BGM), white broken green rice milk (WBGM) and green rice bran milk (GBM) are shown in Fig. 2. Color attributes were measured in terms of lightness (L*), green–red coordinate (a*) and blue-yellow color coordinate (b*) within the CIE L*a*b* color space. Compared to the control sample (WM), rice milks derived from green rice and its milling fractions (GM, BGM, WBGM and GBM) exhibited slightly higher L* values, indicating increased lightness. Rice milks produced from unrefined green rice (GM, BGM) or green rice bran (GBM) showed increased greenness (lower a* value) and reduced yellowness (lower b* value) compared with WM and WBGM. Although the b* values were lower, the reduced a* values of GM, BGM, and GBM indicate an increased greenish tone, which together contribute to a visually yellowish-green appearance, as shown in Fig. 2. The greenish color observed in rice milk derived from green rice is attributed to the presence of chlorophyll (Li et al., 2025). Among all samples, WBGM, obtained from white broken green rice, showed the most similar color to that of WM (Fig. 2). Such color characteristics may influence consumer acceptance; therefore, future studies should include sensory evaluation to further investigate this aspect.

Fig. 2.

Fig. 2

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Appearance and CIE L*a*b* color characteristics of different green rice milk alternatives (WM: white rice milk, GM: green rice milk, BGM: broken green rice milk, WBGM: white broken green rice milk, and GBM: green rice bran milk). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.2. TPC, TFC and antioxidant activity

Analysis of TPC, TFC, and antioxidant activity measured by DPPH and FRAP assays of various rice green milk alternatives is shown in Fig. 3. For TPC and TFC, the results reveal that GBM exhibited the highest contents followed by GM, BGM, WBGM, and WM respectively. These results reflect the composition of the source material, as rice bran is rich in phenolic acids and flavonoids with known antioxidant properties (Sapwarobol et al., 2021). The distribution of bioactive compounds in rice grain is highly localized, with the bran layer serving as the primary source of TPC, accounting for 60–86 % of total phenolics, significantly higher than the embryo (4–17 %) and endosperm (9–23 %) (Shao et al., 2014a). This localization is further supported by the observation that rice bran possesses significantly higher TPC than the endosperm (Ding et al., 2019). A previous study also demonstrated that green rice bran contains higher TPC and TFC compared to other green rice fractions (Bunyatratchata et al., 2025). As a result, milk derived from green rice bran exhibited the highest concentration of these bioactive compounds.

Fig. 3.

Fig. 3

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Analysis of total phenolic contents (TPC), total flavonoid contents (TFC) and antioxidant activities measured by DPPH and FRAP of various green rice milk alternatives (WM: white rice milk, GM: green rice milk, BGM: broken green rice milk, WBGM: white broken green rice milk, and GBM: green rice bran milk). Error bars represent the standard deviation of triplicate measurements. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Both whole grain green rice and broken green rice retain bran components, contributing to higher TPC and TFC in milks derived from these sources (GM, BGM) compared to milks produced from white (refined) rice (WBGM and WM). Interestingly, the BGM demonstrated substantial TPC (39.0 μg GAE/mL), slightly lower than GM (42.5 μg GAE/mL) (Table S1). This finding underscores the potential of broken rice, a byproduct of rice processing typically used as livestock feed (Gunha et al., 2023), as a valuable source for rice milk production. Furthermore, WBGM exhibited higher TPC and TFC than the control WM, indicating that green rice may offer more significant bioactive benefits than white rice. These findings are consistent with previous reports indicating that developing rice grains possess substantially higher total phenolic and flavonoid contents than mature grains (Ji et al., 2013; Lin & Lai, 2011). Similarly, green rice has been reported to exhibit higher total phenolic and flavonoid contents than white rice (Bunyatratchata et al., 2025). The high accumulation of these compounds in the developing grains may result from up-regulated structural and regulatory genes involved in phenolic biosynthesis (Ma et al., 2022). As grain development progresses, the relative abundance of phenolics declines, consistent with transcriptional profiles showing that gene expression mirrors the decreasing phenolic levels (Ma et al., 2022). Collectively, these findings demonstrate that green rice represents a promising raw material for rice milk production.

Antioxidant activities measured by both DPPH and FRAP assays also followed the trends observed in TPC and TFC results, as phenolic compounds and flavonoids are well known for their antioxidant properties (Sapwarobol et al., 2021). A significant correlation between phenolic and flavonoid contents and antioxidant activity further supports their roles as major contributors to the antioxidant capacity (Belew & Gebre, 2025). According to Fig. 3, GBM exhibited the highest antioxidant activities followed by GM, BGM, WBGM, and WM respectively. The consistent trends in antioxidant activities align with the TPC and TFC results, indicating that milk produced from green rice milling fractions contains high antioxidant activities, especially GBM.

3.3. Profile and content of phenolic compounds

The profiles and contents of five individual phenolic compounds (gallic acid, protocatechuic acid, chlorogenic acid, vanillic acid, and cinnamic acid) are presented in Table 1. Gallic acid was detected in all samples, with the highest concentration observed in GBM. The presence of the other phenolic compounds varied among the samples. For example, protocatechuic acid was detected in only three samples, with the highest content in BGM, followed by GM and WBGM. Interestingly, all five phenolic compounds were present in the GM sample, whereas GBM contained all except chlorogenic acid. This observation is consistent with a previous study reporting the absence of chlorogenic acid in green rice bran (Bunyatratchata et al., 2025). These differences in phenolic profiles are likely related to the structural parts of the rice grains used in the rice milk samples. Components such as bran, whole grain, broken rice, white (refined) broken rice or white rice influence the distribution and concentration of phenolic compounds. Previous studies have reported substantial differences in phenolic composition among different parts of the rice grain, including the bran, husk, brown rice, and milled rice (Butsat & Siriamornpun, 2010; Goufo & Trindade, 2014). Additionally, the developmental stage of the rice has also been shown to affect phenolic content (Giorni et al., 2020; Shao et al., 2014b). These findings suggest that rice milk produced from different rice milling fractions may exhibit distinct profiles and levels of phenolic compounds.

Table 1.

Phenolic acid contents in different rice milk alternatives produced from green rice and its milling fractions.

Phenolic acid contents (μg/g) Samples
WM GM BGM WBGM GBM
Gallic acid 80.19 ± 0.94d 86.88 ± 0.72cd 130.64 ± 1.63b 93.05 ± 9.83c 225.64 ± 5.85a
Protocatechuic acid ND 40.37 ± 0.19b ND 16.75 ± 3.90c 206.28 ± 6.25a
Chlorogenic acid ND 126.95 ± 5.35 ND ND ND
Vanillic acid ND 119.24 ± 9.95b ND ND 666.33 ± 24.94a
Cinnamic acid ND 11.12 ± 1.02b ND ND 23.96 ± 1.99a
Total 80.19 ± 0.94e 384.56 ± 4.18b 130.64 ± 1.63c 109.80 ± 4.19d 1122.21 ± 10.30a
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Values are expressed as mean ± SD of triplicate measurements (n = 3), Different letters (a–e) indicate significant differences (p < 0.05) within the same row, WM: white rice milk, GM: green rice milk, BGM: broken green rice milk, WBGM: white broken green rice milk, and GBM: green rice bran milk.

Overall, the highest total concentration of the five phenolic compounds was observed in GBM, followed by GM, BGM, WBGM, and WM. This pattern was consistent with the TPC values shown in Fig. 3. The lower phenolic content in WBGM and WM is likely due to the use of rice that has undergone removal of the bran layer during processing. Since the bran is rich in phenolic compounds (Shao et al., 2014a), its removal results in significantly reduced phenolic content. Interestingly, WBGM showed a higher phenolic content than WM. This difference may be attributed to variations in the parts of the rice grain used for milk production, as well as differences in the developmental stage of the rice.

3.4. Nutritional values of green rice milk alternatives

3.4.1. Proximate nutritional composition of green rice milk alternatives

The moisture content of various rice milks was in the range of 93–95 %. Other nutritional components including ash, crude protein, crude fat, crude fiber, and carbohydrate were calculated on a dry weight basis (Table 2). The results revealed that GBM exhibited the highest level of ash (18.7 %), crude protein (19.2 %), crude fat (20.6 %) and crude fiber (2.1 %) with the lowest carbohydrate content (39.4 %). Rice bran is known for its richness in lipids, proteins, vitamins, minerals and dietary fiber (Moraes et al., 2014; Sapwarobol et al., 2021), hence the high nutritional values also observed in green rice bran milk.

Table 2.

Proximate analysis of plant-based milk alternatives produced from green rice and its milling fractions.

Samples Moisture (%) Dry weight basis
Ash (%) Crude protein (%) Crude fat (%) Crude fiber (%) Carbohydrate (%)
WM 93.72 ± 0.24b 0.40 ± 0.03d 9.84 ± 0.12d 0.06 ± 0.01c 0.95 ± 0.01c 88.74 ± 0.07a
GM 94.74 ± 0.12a 2.02 ± 0.02b 10.83 ± 0.13b 0.25 ± 0.04b 1.54 ± 0.04b 85.36 ± 0.05d
BGM 94.43 ± 0.10a 1.97 ± 0.01b 10.32 ± 0.05c 0.30 ± 0.02b 1.56 ± 0.07b 85.84 ± 0.05c
WBGM 93.29 ± 0.17c 0.75 ± 0.02c 10.41 ± 0.12c 0.11 ± 0.01c 0.84 ± 0.05c 87.89 ± 0.06b
GBM 94.69 ± 0.37a 18.68 ± 0.24a 19.16 ± 0.11a 20.62 ± 0.13a 2.14 ± 0.12a 39.40 ± 0.15e
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Values are expressed as mean ± SD of triplicate measurements (n = 3), Different letters (a–e) indicate significant differences (p < 0.05) within the same column, WM: white rice milk, GM: green rice milk, BGM: broken green rice milk, WBGM: white broken green rice milk, and GBM: green rice bran milk. The crude protein, crude fat, crude fiber, ash and carbohydrate contents were calculated based on a dry weight basis.

GM and BGM exhibited similar ranking as the second highest in ash (2.0 %), crude fat (0.3 %), and crude fiber content (1.5–1.6 %). The retention of the bran layer from the green rice and broken green rice likely contributes to their higher fiber, fat, and ash contents compared to milks produced from refined rice. In terms of protein content, GM had the second highest level (10.8 %), while BGM (10.3 %) and WBGM (10.4 %) were not significantly different from each other, followed by WM (9.8 %). Despite not having particularly high protein content, its hypoallergenic nature makes it highly valued for human consumption (Moraes et al., 2014). The higher protein content in green rice milk compared to white rice milk adds to its nutritional benefits. These results align with previous reports showing that rice at early developmental stages contains higher protein levels than mature grains (Ji et al., 2013; Pantoa, Baricevic-Jones, et al., 2020). When comparing refined rice milks (WM and WBGM), WBGM showed higher ash and protein content than WM with no significant difference in crude fat and crude fiber. Generally white or refined rice milks (WM or WBGM) had higher carbohydrate content than green rice milks (GM or BGM). Interestingly, as mentioned earlier, BGM derived from broken green rice showed a nutritional profile similar to that of GM produced from green rice in terms of ash, crude fat and crude fiber, with slightly lower crude protein and slightly higher carbohydrate content. This result suggests that besides rice bran, green rice and even broken green rice could be potential candidates for rice milk production in terms of nutritional composition.

3.4.2. Amino acid contents in green rice milk alternatives

The amino acid profiles of rice milk alternatives (WM, GM, BGM, WBGM, and GBM) were quantified using LC-MS/MS, covering 20 amino acids, including essential and non-essential types. The analysis of amino acid profiles revealed variations among the samples (Fig. 4 and Table S2). However, arginine and phenylalanine were consistently the most abundant essential amino acids across all samples, with GBM showing the highest concentrations (Fig. 4). L-Arginine has been found to positively influence nutrient metabolism, promoting lean tissue growth and improving insulin resistance in humans (McNeal et al., 2016), while phenylalanine has potential as an anti-depressant (Akram et al., 2020). Phenylalanine is crucial for the central nervous system, especially in addressing chronic pain, depression, and various other disorders related to nervous system dysfunction (Akram et al., 2020). Among non-essential amino acids, glutamic acid and tyrosine were predominant in all samples (Fig. 4).

Fig. 4.

Fig. 4

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Heat map with hierarchical clustering (Ward's method) based on amino acid content in plant-based milk alternatives produced from green rice and its milling fractions (WM: white rice milk, GM: green rice milk, BGM: broken green rice milk, WBGM: white broken green rice milk, and GBM: green rice bran milk). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Interestingly, milk produced from white or refined rice (WM, WBGM) generally contained higher levels of amino acids than those from unrefined green rice samples (GM, BGM) (Fig. 4 and Table S2). This observation differs from the crude protein results, where WM showed the lowest protein content (Table 1). The discrepancy may be due to differences in protein extraction efficiency or hydrolysis during amino acid analysis. Proteins in rice bran form complex or bound structures through disulfide linkages, glycosylation, aggregation, and entanglements, which reduce solubility and make them more difficult to extract (Cho et al., 2022; Zheng et al., 2019). While acid hydrolysis releases protein-bound amino acids, certain peptide bonds, such as those involving valine, isoleucine, and leucine, are resistant to cleavage and may require extended hydrolysis, whereas acid-labile amino acids such as serine, threonine may degrade (Darragh & Moughan, 2005). As a result, proteins in milks derived from green rice and broken green rice, which retain the bran layer, may be less efficiently extracted and hydrolyzed, leading to lower measured amino acid levels. These samples may require optimized hydrolysis conditions to fully break down complex protein structures and release bound amino acids. In contrast, refined rice milks may contain a higher proportion of easily digestible and extractable proteins, resulting in higher amino acid yields during hydrolysis and LC-MS/MS analysis. Additionally, crude protein measurements using the Kjeldahl method capture total nitrogen, including non-protein nitrogen, which may overestimate actual protein content and should be considered when interpreting results.

3.5. PCA of the nutritional profiles in green rice milk alternatives

Principal component analysis was performed to explore the multivariate relationships among the nutritional composition, phenolic acids, antioxidant activities, and amino acid profiles of the five rice milk alternatives (WM, GM, BGM, WBGM, and GBM). The first two principal components (PC1 and PC2) explain a cumulative variance of 85.90 %, with PC1 accounting for 56.20 % and PC2 for 29.70 % (Fig. 5).

Fig. 5.

Fig. 5

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Principal component analysis (PCA) illustrating the multivariate variation in the total nutritional composition among five types of green rice milk (WM: white rice milk, GM: green rice milk, BGM: broken green rice milk, WBGM: white broken green rice milk, and GBM: green rice bran milk). PC1 (56.20 %) and PC2 (29.70 %) explain 85.90 % of the total variance. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The PCA plot revealed clear separation among the five rice milk alternatives, indicating distinct nutritional and phytochemical profiles. GBM was positioned at the far right of the PC1 axis, characterized by high levels of fat, ash, protein, TPC, TFC, gallic acid, vanillic acid, protocatechuic acid, cinnamic acid, antioxidant activities measured by DPPH and FRAP, as well as several amino acids, including glutamine, methionine, histidine, and lysine. These findings are consistent with the results in Fig. 3, Fig. 4, Table 1, Table 2, and Table S2, indicating that GBM is rich in bioactive compounds and several key nutrients. In contrast, WM and WBGM clustered in the lower left quadrant, showing negative associations with most phenolic compounds and antioxidant activities. GM was located in the upper left quadrant, distinguished by higher chlorogenic acid and tryptophan content. BGM was clustered closely to GM as they were in the same panel, indicating similar compositional characteristics.

Overall, PCA effectively discriminated the five rice milk alternatives based on integrated nutritional composition, antioxidant activities, and amino acid profiles. GBM exhibiting a nutrient-dense and bioactive-rich composition, whereas WM and WBGM showed lower levels of bioactive compounds, antioxidant activities, and amino acids. GM and BGM shared similar profiles, as their clusters were closely together in the same panel. These results highlight the potential of GBM as a promising functional beverage with higher nutritional and antioxidant properties.

4. Conclusion

This study comprehensively evaluated the bioactive compounds, antioxidant activity, nutritional composition, and amino acid profiles of rice milks produced from green rice and its milling fractions of Khao Dawk Mali 105, in comparison with conventional white rice milk. The results demonstrate the strong potential of green rice and its milling byproducts as valuable raw materials for developing functional plant-based beverages. Among the samples, green rice bran milk (GBM) and green rice milk (GM), particularly GBM, demonstrated superior bioactive content, antioxidant activities, and nutritional content, emphasizing the advantages of using green rice bran and whole grain green rice in rice milk production. Additionally, milk produced from broken green rice, despite being a byproduct of rice processing, generally contained higher bioactive compounds, antioxidant activities, and nutritional value than white rice milk, indicating its potential as a functional beverage ingredient. However, this study is limited to a single green rice variety and controlled processing conditions, which may affect generalizability. Processing techniques may also influence amino acid profiles; for instance, rice milk prepared with hydrogen-rich water has been reported to contain higher levels of certain essential amino acids (Zor et al., 2024), highlighting the need for further investigation. Future studies should explore multiple rice varieties, broader processing techniques, milk yield, economic assessment and storage stability to optimize product quality and maximize bioactive and nutritional benefits. Additionally, expanding sensory evaluation and consumer acceptance studies would also support commercialization. Collectively, this research establishes a strong foundation for developing high-value rice milk products with potential functional and nutritional benefits.

CRediT authorship contribution statement

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

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

In preparing this manuscript, the authors used ChatGPT to assist with grammar correction and improve 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

Acknowledgements

The authors acknowledge the financial support provided by Thailand Science Research and Innovation (TSRI) and Mahasarakham University. Appreciation is also extended to the Laboratory Equipment Center of Mahasarakham University for their technical assistance and scientific support. The bar graph was generated using GraphPad Prism.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2025.103408.

Appendix A. Supplementary data

Supplementary material
mmc1.docx (21.7KB, 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 (21.7KB, 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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