1. Introduction
Depending on the degree of leaf oxidation, the most common types of teas are green, oolong, and black tea [
1,
2]. Matcha tea is a variety of powdered Tencha green tea (
Camellia sinensis) that differs from traditional green tea in conditions during cultivation, when it is characteristically prepared from green tea leaves grown under the shade technique that are steamed, dried, and finally ground on several millstones [
3,
4,
5,
6,
7,
8]. The processes of preparing and consuming matcha tea also differ from other teas. Generally, green tea is consumed as a water extract of the leaves, whereas in matcha tea, the ground leaves are covered with water, and the whole leaves are consumed [
9].
The effect of the different processing and consumption methods leads to the ingestion of more significant amounts of bioactive substances occurring in tea, e.g., polyphenols, amino acids, saponins, chlorophyll, L-theanine, and caffeine. Most of these compounds have been declared to positively affect human health [
5,
10,
11]. The health effects associated with drinking green tea have been reported to comprise protection against cancer, lowering the risk of cardiovascular disease and stroke, cognitive dysfunction, allergy relief effects, and preventive effects on metabolic syndrome, reducing the development of mortality, and three important aspects of death, such as heart disease, respiratory diseases, and cerebrovascular disease [
5,
9,
11,
12,
13].
Generally, carbohydrates are produced in plant cells through the process of photosynthesis. In particular, carbohydrates serve plants for the growth and development of cellular structures; they are a source of energy, and many of them are used by cells for the biosynthesis of lipids, proteins, and polysaccharides [
14].
Camellia sinensis raw leaves are composed of carbohydrates in the range of 4–7% of dry weight (DW) [
15]. It is a generally known fact that green tea shoots constitute 4–10% of polysaccharides, 6–8% of cellulose, and 3–5% of low molecular weight carbohydrates [
15]. Monosaccharides such as D-arabinose, D-galactose, D-glucose, L-rhamnose, D-fructose, and oligosaccharides such as maltose, sucrose, raffinose, lactose, and stachyose were noticeably determined in green tea [
12,
16]. Moreover, the α-galactooligosaccharides such as raffinose and stachyose are well known to serve as prebiotics [
12], and myo-inositol contributes to female reproductive health [
15].
Organic acids are intermediate products of carbohydrates in metabolic pathways, such as the Krebs cycle and the shikimic acid pathway, and they play a role in maintaining cell osmotic pressure [
17]. Tea leaves are a significant source of malic and oxalic acids along with citric, isocitric, and succinic acids. For instance, oxalic and citric acids are co-responsible for the quality of green tea leaves, and succinic and citric acids enhance the umami taste of glutamic acid in matcha [
8,
17,
18]. More profoundly, shade treatment decreased the content of quinic and succinic acid but increased the content of malic and citric acids in matcha tea [
11]. Both shikimic and quinic acids enter the biosynthesis of the polyphenols. The content of quinic acid present in the stems has also been reported to be one of the predictor factors prolonging tea quality and is recognized to be used as an anti-depressive and cognition-improving substance [
11]. On the contrary, the total amount of organic acids such as succinic, oxalic, malic, lactic, and citric acids, etc., increases dominantly after withering, rolling, and fermentation, when black tea is produced [
17].
Metabolic processes in the intestinal tract affect the release and absorption of individual nutrients of the food matrix. Therefore, consuming food rich in individual nutrients does not necessarily mean that more of these compounds will be absorbed into the digestive tract. The effect of matcha tea polyphenols on human organism has been addressed, including the digestibility of phenolics [
19]. Both bioavailability and bioaccessibility are important factors for determining the efficiency of the nutrient intake from meals and its further total nutritional value [
20,
21]. In addition, innovative research has been conducted to analyze the percentage of solid undigested proportions of plant food to evaluate the rest of the bioactive compounds after the in vitro digestion process. This factor represents the amount of analyte retained in the undigested matrix expressed as percentage [
19,
22].
To the best of our knowledge, we are unaware of any studies that have addressed the influence of digestibility under in vitro conditions on low molecular weight carbohydrates and organic acids after digestion in the stomach, and subsequently in the small intestine simulated under in vitro conditions. Therefore, a simulated in vitro digestion process provided by pepsin and pancreatin enzymes under human body temperature was applied. The aim of this research was to assess the contents of individual low molecular weight carbohydrates and organic acids in the native and undigested portions of matcha powders using a high-performance liquid chromatography method with the spectrophotometric and refractometric detection. Moreover, the appropriate retention factors (RF) of the analyzed compounds in the undigested portions were expressed as well.
2. Materials and Methods
2.1. Chemicals
Pepsin with an enzymatic activity of 2000 FIP-U/g, and a mixture of pancreatic enzymes such as protease, amylase, and lipase with activities of 350, 7500, and 6000 FIG-U/g, respectively, were purchased from Carl Roth and Merck (Karlsruhe and Darmstadt, Germany). HPLC standards in purity of ≥99.5% (L-arabinose, L-rhamnose, D-glucose, D-fructose, xylose, maltose, saccharose, trehalose, oxalic, succinic, malic and citric acids) were acquired from Sigma-Aldrich (St. Louis, MI, USA). Acetone, HCl, KH2PO4, and Na2HPO4 12 H2O were provided from Sigma-Aldrich (St. Louis, MI, USA). In addition, KH2PO4, methanol, acetone, HCl, and Na2HPO4×12 H2O were supported by Penta (Prague, Czech Republic). Redistilled water was supplied by a Purelab Classic Elga water system (Labwater, London, UK).
2.2. Matcha Tea Samples
The matcha samples tested consisted of five high-quality organic Japanese and Chinese powders, and a matcha tea (Camellia sinensis), produced from Tencha leaves. Five matcha tea samples were selected for analysis as follows: premium Organis matcha tea, premium Allnature organic matcha tea, Imbio matcha tea (all originating in China), Iswari organic matcha powder, and Natu matcha tea (both originating in Japan). All were sold with expiration dates in 2024. They were kept in the original sacks (0.070 to 0.25 kg) without access to sunlight under the control temperature of about 23 ± 2 °C for a maximum of two months.
2.3. Digestion Process Simulated In Vitro
The gastrointestinal process, simulated under in vitro conditions, including gastric and intestinal phases and determinations of ash and dry matter content, were determined according to Koláčková [
19] with a slight modification (
Figure 1).
The in vitro digestibility value of the powder form of matcha tea was firstly determined using pepsin and pancreatin enzymes in an incubator (DaisyII, Ankom Technology, Macedon, NY, USA). The matcha tea sample (0.25 g) was weighed and sealed by impulse sealer (KF-200H, Penta Servis, Holice, Czech Republic) in digestion sacks (F57 type, Ankom Technology, Macedon, NY, USA).
To model the gastric condition, the experimental flask was infused by 1.7 L of 0.1 M HCl containing pepsin (0.63 g). Consequently, the samples were exposed for 2 h at 37 °C and then washed by redistilled water. To simulate small intestine conditions, phosphate buffer (consisting of Na2HPO4×12 H2O and KH2PO4, pH 7.45) and a mixture of pancreatin enzymes (3.0 g) were dissolved in 1.7 L of redistilled water and added to the incubation flask. After a twenty-four hours incubation at 37 °C, the samples were treated with redistilled water followed by drying at 105 °C for 24 h, and weighting.
In the final step of the analysis, the sacks containing the rests of the matcha tea were burned in a muffle oven (LM112.10, Veb Elektro, Berlin, Germany) at 550 °C for 5 h, so they could be cooled in an exicator and weighed. All assessments were repeated as three independent experiments. In vitro dry matter digestibility value (DMD) was calculated using Formulas (1)–(5):
where the appropriate values indicate the following: DMD (dry matter digestibility, %), DMR (amount of the sample without the sack after the digestion, g), DM (dry weight of the sample, g), DW (dry weight of the sample presented in %), m
s (the sample amount, g), c
1 (the correction of the weight of the sack after the incubation, g), c
2 (the correction of the weight of the sack after the burning, g), m
p (the amount of ash from the empty correction sack, g), m
1 (the weight of the empty bag, g), m
2 (the sample amount, g), m
3 (the weight of the dried bag with the sample after the incubation, g).
Preparation of Undigested Portions of Matcha Leaves
To obtain both forms of undigested portions of matcha, the digestibility assessment was determined as follows: (i) after stomach incubation, when the undigested part of the matcha sample was dried at 30 °C for 24 h, (ii) after stomach and small intestine digestion, when the undigested part of the matcha sample was dried at 30 °C for 24 h (
Figure 1). These experiments were repeated three times. The undigested residue of matcha obtained in solid form was extracted following the methodology described in
Section 2.4.
2.4. Extraction of Low Molecular Weight Saccharides and Organic Acids
Extracts from matcha tea samples were prepared in the same way to determine free carbohydrates and organic acids. The sample extracts were divided into three groups as follows: (i) native matcha sample, in which the digestion process was not carried out; (ii) undigested part of the matcha sample, in which in vitro digestion was terminated in the stomach; (iii) undigested part of the matcha sample, in which in vitro digestion was performed in both the stomach and the small intestine. A sample of 0.2 g of powder matcha from each treatment group was weighed in 2 mL plastic Eppendorf tubes with 2 mL of redistilled water, and the microtube content was mixed. Subsequently, the tube was placed in a TS-100 thermoshaker (Biosan, Riga, Latvia) for 10 min at 60 °C to extract carbohydrates and organic acids. Consequently, the sample was centrifuged for 10 min at 23,000×
g (Velocity 13μ, Dynamica Scientific Ltd., London, UK) and filtered through a nylon syringe filter (13 mm × 0.45 μm). The filtrate thus prepared was processed for HPLC analysis [
11,
23].
2.5. Determination of Individual Organic Acids Using High-Performance Liquid Chromatography
The organic acid profile (oxalic, succinic, and malic and citric acids) was tested using an HPLC Dionex Ultimate 3000 liquid chromatogram system equipped by DAD detector (DAD-3000 RS, Thermo Scientific Waltham, MA, USA) with minor modifications [
24]. Organic acids were separated on a Phenomenex Synergi Hydro-RP C18 column (250 × 4.6 mm; 4 μm, Phenomenex; Torrance, CA, USA). Twenty µL of sample extract was applied into the column. Regarding the gradient mode, two mobile phases were used: (A) 20 mM of potassium dihydrogen phosphate and (B) methanol. The gradient elution had the following settings: 0% of B at 0–2.5 min; 0–30% of B between 2.5 and 2.6 min; 30% of B between 2.6 and 2.9 min; 30–0% of B between 2.9 and 3.0 min; 0% of B between 3.0 and 10 min. The mobile phase flow rate was set at 1 mL/min, the column temperature was adjusted at 60 °C, and the wavelength of 210 nm for recording the chromatogram was used. Concerning the calibration range of 5.0–50.0 μg/mL, the detector response was linear for organic acids whereas correlation coefficients exceeded 0.9992. The individual compounds were identified according to the time of retention and the method of standard addition.
2.6. Determination of Low Molecular Weight Saccharides Using High-Performance Liquid Chromatography
The presentation of individual carbohydrates (L-rhamnose, L-arabinose, D-fructose, D-glucose, xylose, trehalose, maltose, and saccharose) in appropriate extracts was analyzed using the HPLC equipment consisting of Thermo Scientific DionexUltimate 3000 coupled to a detector ERC RefractoMax 520, Ultimate 3000 autosampler, binary pump HPG-3xRS and solve selector valve HPG-30400RS (Waltham, MA, USA). The carbohydrates’ profile was measured using a Phenomenex Rezex RCM-Monosaccharide Ca
+2 column (100 × 7.8 mm; 8 μm, Phenomenex, Torrance, CA, USA) in isocratic elution mode whereas redistilled water as a mobile phase with the rate of flow of about 0.4 mL/min for 15 min was applied. The injected volume was adjusted to 45 µL. The column chamber and cell of the RI detector were adjusted at 80 and 35 °C, respectively. Chromatograms with linear responses were recorded within the calibration ranges of 0.5–10.0 µg/mL with correlation coefficients exceeding 0.9990. Individual analytes were evaluated tentatively according to the time of retention for standards and the method of standard addition. Data signals were evaluated by LC ChromeleonTM 7.2 software (Thermo Scientific, Waltham, MA, USA) [
25].
2.7. Effect of In Vitro Digestion Process on the Saccharide and Organic Acid Content
The amounts of each saccharide and organic acid that retained in the undigested portion of the tea leaves were expressed as retention factors (RF, %). The RF value was calculated using Equation (6):
where the appropriate values indicate the following: RF (the retention factor of the compound in the undigested part of the leaves, %), CUWF (the concentration of the compound in the undigested part of the leaves, mg/g), DMD (the dry matter digestibility value, %), and CNWF (the concentration of the compound in the raw form of the matcha, mg/g) [
19].
2.8. Statistical Analysis
All analyses were replicated 3–5 times and their results were reported as mean ± standard deviation on a dry weight basis. The results of all analyses were statistically reported using a one-way analysis of variance (ANOVA). Subsequently, Tukey’s test was applied to identify the differences among means. The significance level of all statistical tests was set at 0.05.
4. Conclusions
This research work provides data on the organic acid, low molecular weight carbohydrates, and dry matter digestibility values in matcha teas and their undigested portions. This unique study also evaluates the effect of the in vitro digestion process on retention factors of individual analytes. The organic acid contents were established in this order: citric acid (up to 44.8 mg/g) > malic acid > oxalic acid ≥ succinic acid (up to 4.44 mg/g). Taking into account the low molecular weight carbohydrates in native matcha samples, the sequence of the highest amounts is as follows: trehalose > L-arabinose > L-rhamnose > D-glucose, while L-rhamnose was evaluated only in two of the five samples, and D-fructose, xylose, maltose, and saccharose were not detected in any of the matcha teas. It can be assumed that plants synthesize trehalose as a stress protectant, and it may be interesting in the future to study the carbohydrate content depending on the shading technique. The highest retention factor (RF) after in vitro digestion in the stomach was found for succinic acid (15–34%), followed by citric, malic, and oxalic acids (6–13%). Regarding gastric and intestinal digestion, malic acid was completely released from the matrix of the tea leaves; contrarily, succinic acid (RF up to 7%) was still bounded in matcha tea leaves and theoretically it should pass into the large intestine. To evaluate the RF value for saccharides after gastric digestion, D-glucose (23–50%) followed by trehalose (1–18%) was retained in the matcha tea leaf in the highest proportions, while L-arabinose was completely released. D-glucose was completely released from green tea leaves only after simulating both gastric and intestinal phases of digestion; in contrast, trehalose was not completely released from matcha, and even 13% of trehalose can still remain in tea leaves, and it theoretically seems to pass to the large intestine.
Results can facilitate a better understanding of the chemical composition, reinforcing consumers’ perception of matcha tea as a health-promotion beverage prepared in the form of the whole leaf parts. In the measurement of oxalic acid, considering that the process of shading tea leaves increases the concentration of this acid and its RF value is too small, it would be appropriate in the future to evaluate the recommended maximum daily intake of matcha tea for people sensitive to the formation of urinal stones, for example. In addition, for the industry, our knowledge could mainly contribute to the identification of matcha tea quality markers in the future, and the elucidation of the chemical reactions that occur during the shading processing of green tea leaves into matcha tea.