Dipeptidyl peptidase IV inhibitory dipeptides contained in hydrolysates of green tea grounds

Abstract

Peptides inhibiting human dipeptidyl peptidase IV (hDPP-IV) have potential as functional food ingredients to help prevent type 2 diabetes. In the present study, we analyzed the hDPP-IV inhibitory dipeptides contained in the hydrolysates of green tea grounds. The IC₅₀ values of 337 standard dipeptides were calculated for the hDPP-IV inhibition. Next, to reveal whether the top 17 dipeptides with IC₅₀ values less than 0.1 mM were present in the hydrolysates, the tea grounds hydrolysates were analyzed by LC-MS. Among the 17 dipeptides, Val-Leu, Leu-Trp, Trp-Ile, Asn-His, Thr-His, Thr-Trp, Trp-Ala, and Trp-Val were identified in the hydrolysates as novel hDPP-IV inhibitory dipeptides. Val-Leu particularly inhibited hDPP-IV (IC₅₀: 0.074 mM) and was the most abundant (558 µg/g). These results suggest that green tea grounds are a promising dietary source for generating inhibitory peptides of hDPP-IV.

Introduction

Human dipeptidyl peptidase-IV (hDPP-IV) is necessary for prompt degradation of incretin hormones, glucagon-like peptide-1 (GLP-1), and gastric inhibitory polypeptide (GIP) in the blood (Ahrén, 2008; Mentlein, 1999). Incretins potentiate insulin secretion from the pancreas in a glucose-dependent manner (Kim and Egan, 2008). Therefore, hDPP-IV inhibitors (gliptins) are frequently prescribed for the management of type 2 diabetes (Caironi et al., 2020). Inhibitory peptides of hDPP-IV also can prevent type 2 diabetes in the same manner as gliptins, and thus show promise as a functional food ingredient. To date, several studies have found hDPP-IV inhibitory peptides in a variety of dietary proteins including milk, soybeans, rice, salmon, tuna cooking juice, and dry-cured ham (Gallego et al., 2014; Gao et al., 2020; Hatanaka et al., 2012; Hikida et al., 2013; Huang et al., 2012; Jin et al., 2020; Lacroix and Li-Chan 2013; Lacroix and Li-Chan 2014; Lan et al., 2015; Li-Chan et al., 2012; Nongonierma and FitzGerald 2014; Rivero-Pino et al., 2020; Tulipano et al., 2011; Worsztynowicz et al., 2020). In particular, milk proteins are considered to be a suitable source of hDPP-IV inhibitory peptides.

The purpose of the present study was to analyze hDPP-IV inhibitory peptides contained in the hydrolysates of green tea grounds, which form a residual paste after tea is extracted. Beverage companies that manufacture tea products generate large amounts of tea grounds as a by-product. Therefore, developing effective usage of tea grounds could solve a considerable problem in the tea industry (Ozbayram et al., 2020; Yang et al., 2015; Zheng et al., 2017). Tea grounds contain approximately 30% (w/w) residual (insoluble) proteins such as denatured ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). Because of the high protein content, tea grounds would be available as a source of functional peptides, including hDPP-IV inhibitory peptides.

Identifying functional peptides from protein hydrolysates is a major focus of current food peptide research (Imai et al., 2019; Ito et al., 2013; Li et al., 2020; Ogawa et al., 2019). However, the purification of functional peptides from protein hydrolysates is complicated by the complex composition of various peptides. The relative quantity of peptides in protein hydrolysates and the ease of purification from the hydrolysates often lead to biases in the discovery of functional peptides. As a result, previous reports have described only a limited range of hDPP-IV inhibitory peptides (Liu et al., 2019; Nongonierma and FitzGerald, 2014). To avoid such biases, in this study, we adapted an approach of “reverse-type peptidomics.” In this approach, hDPP-IV inhibitory peptides identified by analyzing a synthetic dipeptide library were queried against the peptides contained in the hydrolysates of tea grounds. As a result, we determined that the green tea grounds hydrolysates contained at least 8 novel hDPP-IV inhibitory dipeptides.

Materials and Methods

Materials and chemicals    Glycyl-proline-4-methyl-coumaryl-7-amide (Gly-Pro-MCA) was purchased from Peptide Institute Inc. (Osaka, Japan). Standard dipeptides (337) were purchased from Anaspec (CA, USA). hDPP-IV cDNA (GenBank: AB590646) was purchased from Promega (WI, USA). Food-processing proteases were kindly provided by Amano Enzyme Inc. (Nagoya, Japan). Proteases used in this study were as follows: (1) Protease A “Amano” 2 SD, (2) Protease M “Amano” SD, (3) Protease P “Amano” 6 SD, (4) ProteAX, (5) PROTIN SD-AY10, (6) PROTIN SD-NY10, (7) THERMOASE PC10F, and (8) Peptidase R. Green tea leaf was purchased from Chikumeidou (Shizuoka, Japan).

Preparation of tea grounds hydrolysates    Green tea leaves were extracted using hot water at 80 °C 10 times. The residual wet paste (tea grounds) was suspended in 10 mM sodium phosphate buffer (pH 8.0). Food-processing proteases were added to the tea grounds, and the slurry was stirred at 42 °C for 3 h. The reaction was stopped by heating the slurry at 95 °C for 3 min. The insoluble residue was removed by centrifugation at 8 000 rpm for 10 min, and the supernatant was freeze-dried. In a typical example, when 200 g of green tea leaves (900 g of wet paste) was treated with 0.002% (w/w to wet paste) PROTIN SD-NY10, 10 g of lyophilized powder of the hydrolysates was obtained.

Analysis of hDPP-IV inhibitory effects    The heterologous expression and analysis of hDPP-IV inhibition was performed as previously reported (Hikida et al., 2013; Lan et al., 2014; Lan et al., 2015). The enzymatic analysis of hDPP-IV was performed at 37 °C. The composition of the reaction mixture was as follows: 100 mM sodium phosphate buffer (pH 8.0), 0.04 mU hDPP-IV, 50 µM Gly-Pro-MCA, and an appropriate amount of dipeptide or tea grounds hydrolysates. The hydrolysis of the synthetic substrate by hDPP-IV was quantified by fluorescence (excitation and emission at 360 nm and 440 nm, respectively) using a Flexstation II (Molecular Devices, CA, USA). IC₅₀ values were calculated using nonlinear regression analysis with GraphPad Prism 6 (GraphPad Software, Inc., CA, USA).

LC-MS analysis of 17 dipeptides contained in the hydrolysates of tea grounds    The hydrolysates extracted from green tea grounds were dissolved in Milli-Q water (1 000 ppm) and filtered through a 0.2 µm PTFE filter. Preparations of 17 standard dipeptides were dissolved in dimethyl sulfoxide and diluted with methanol for quantitative analysis. LC-MS analysis was performed at 40 °C with an Accela LC system (Thermo Fisher Scientific, MA, USA) in conjunction with a quadrupole mass spectrometer, Q Exactive (Thermo Fisher Scientific), equipped with a TOSOH TSKgel ODS-100V column (3.0 × 150 mm, 5 µm; Tosoh Bioscience Japan, Tokyo, Japan). Xcalibur software (Thermo Fisher Scientific) was used for system control and data analysis. Gradient elution was performed using a two-solvent system (solvent A: 0.1% formic acid; solvent B: acetonitrile containing 0.1% formic acid). The gradient program was as follows: (1) 0–3 min, 2% B hold; (2) 3–20 min, linear gradient to 98% B; and flow rate, 0.4 mL/min. The injection volume was 5 µL of the sample solution. The MS instrument was operated in the electrospray ionization positive mode. Spectra were recorded over m/z 100–1 000, and the resolution was 70 000.

Results and Discussion

Preparation of tea grounds hydrolysates    Thirty-six types of hydrolysates of green tea grounds were prepared by using combinations of 8 different proteases, and the hDPP-IV inhibitory effects were evaluated (Fig. 1). The extracted sample from unprocessed tea grounds did not show hDPP-IV inhibition. The hDPP-IV inhibitory effects were related to the peptide contents (A220) of the hydrolysates. These results indicate that the hDPP-IV inhibitory peptides were generated through hydrolysis of residual proteins by proteases. The hydrolysates of green tea grounds prepared using PROTIN SD-NY10, a bacterial metalloprotease (EC 3.4.24.28) derived from Bacillus amyloliquefaciens, showed the highest hDPP-IV inhibitory effect (27.9% inhibition) (Fig. 1A). Therefore, this hydrolysate was used for the subsequent analysis (Fig. 1C).

Fig. 1.

hDPP-IV inhibitory effects and relative peptide contents of the green tea grounds hydrolysates.

A) The hDPP-IV inhibitory effects of the hydrolysates. Inhibition ratio (%) compared to control (distilled water) are shown. B) Absorbances at 220 nm to reflect peptides contents of the tea grounds hydrolysates. Proteases used in A) and B) are as follows: (1) Protease “Amano” A SD, (2) Protease M “Amano” SD, (3) Protease P “Amano” 6 SD, (4) ProteAX, (5) PROTIN SD-AY10, (6) PROTIN SD-NY10, (7) THERMOASE PC10F, and (8) Peptidase R, respectively. C) A picture of lyophilized powder of green tea grounds hydrolysates treated with PROTIN SD-NY10.

hDPP-IV inhibitory effects of dipeptides    Dipeptides show a relatively high hDPP-IV inhibitory effect compared to single amino acids or other oligopeptides (Hikida et al., 2013; Lan et al., 2014). In addition, dipeptides are more frequently produced from proteins than other oligopeptides; e.g., one dipeptide appears for every 400 amino acids, while one tripeptide appears for every 8 000 amino acids. Therefore, we focused on dipeptides as a potential hDPP-IV inhibitor. In a previous study, we conducted a large-scale analysis of a synthetic dipeptide library (337 dipeptides) to reveal the hDPP-IV inhibitory effect, the inhibition rate (%) at 1 concentration (Lan et al., 2015). In this study, we determined the IC₅₀ values of 206 dipeptides to further evaluate the efficacy with which they inhibited hDPP-IV activity. The IC₅₀ values were broadly distributed, ranging from 0.04 mM to greater than 1 mM. The ranking of IC₅₀ values calculated in this study is largely consistent with the ranking of inhibition intensity of hDPP-IV inhibitory dipeptides that we previously reported (Lan et al., 2015). Therefore, the characteristics of hDPP-IV inhibitory dipeptides have been described in detail elsewhere (Lan et al., 2015). We identified the 17 top-ranking dipeptides with strong inhibition of hDPP-IV (IC₅₀ < 0.1 mM): Trp-Arg, Trp-Pro, Asn-His, Trp-Ala, Trp-Val, Ile-Pro, Val-Leu, Thr-His, Ile-Ala, Met-Met, Leu-Ala, Phe-Ala, Met-Leu, Trp-Ile, Val-Pro, Thr-Trp, and Leu-Trp.

Quantitative analysis of 17 dipeptides in the hydrolysates extracted from green tea grounds    To reveal whether the 17 identified hDPP-IV inhibitory dipeptides were present in the hydrolysates extracted from green tea grounds, qualitative analysis was conducted by LC-MS using synthetic dipeptides as a standard; namely, the 17 dipeptides were individually analyzed based on the m/z specific to each dipeptide. This demonstrated that 12 dipeptides (Val-Leu, Phe-Ala, Ile-Ala, Leu-Ala, Leu-Trp, Trp-Ile, Asn-His, Thr-His, Thr-Trp, Trp-Ala, Ile-Pro, and Trp-Val) were contained in the hydrolysates. Subsequently, the content of each dipeptide was determined by LC-MS analysis using the calibration curves from the synthetic dipeptides. Representative data for Val-Leu are shown in Fig. 2. The contents in the hydrolysates and IC₅₀ values for hDPP-IV inhibition of the 17 dipeptides are shown in Table 1. Among them, the 12 dipeptides generated by protease treatment were identified as prime candidates for inhibition of hDPP-IV. To the best of our knowledge, Val-Leu, Leu-Trp, Trp-Ile, Asn-His, Thr-His, Thr-Trp, Trp-Ala, and Trp-Val are novel hDPP-IV inhibitory dipeptides discovered from dietary protein hydrolysates. Val-Leu inhibited hDPP-IV particularly well (ranking 7 of 337 dipeptides) and was the most abundant dipeptide evaluated here (558 µg/g).

Fig. 2.

Quantification of dipeptides in hydrolysates extracted from green tea grounds.

Analysis of Val-Leu is shown as a representative result. A) LC-MS chromatogram (m/z 231.1703 ± 5 ppm) for standard Val-Leu. B) Calibration curve calculated from the analysis data for standard Val-Leu. C) Total ion chromatogram (m/z 150–400) of the tea grounds hydrolysates. D) LC-MS chromatogram to analyze Val-Leu in the hydrolysates (m/z 231.1703 ± 5 ppm).

Table 1. Top 17 hDPP-IV inhibitory dipeptides contained in the green tea grounds hydrolysates

Dipeptides	Contents in the hydrolysates of green tea grounds (µg/g)	IC50(mM)
Val-Leu *	558±11	0.074±0.010
Phe-Ala	304±3	0.091 ±0.002
lle-Ala	254±11	0.089±0.003
Leu-Ala	191 ±6	0.090±0.007
Leu-Trp *	70.4±3.3	0.098±0.001
Trp-lle *	40.9±2.0	0.092±0.008
Asn-His*	35.4±7.6	0.061±0.003
Thr-His *	28.8±1.6	0.081 ±0.002
Thr-Trp *	28.7±1.1	0.097±0.004
Trp-Ala *	17.3±0.6	0.063±0.001
lle-Pro	2.93±0.12	0.068±0.012
Trp-Val *	2.42±0.03	0.063±0.005
Trp-Arg	n.d.	0.040±0.004
Trp-Pro	n.d.	0.049±0.003
Met-Met	n.d.	0.089±0.005
Met-Leu	n.d.	0.091 ±0.004
Val-Pro	n.d.	0.092±0.006

The contents in the tea grounds hydrolysates and the hDPP-IV inhibition data are presented as means ± standard error (n = 3). Asterisk indicates novel hDPP-IV inhibitory dipeptides discovered as food ingredients in this study.

Identification of the parent protein of hDPP-IV inhibitory dipeptides    The protease that produced the most hDPP-IV inhibitory peptides from the tea grounds was PROTIN SD-NY10. The enzyme is a bacterial neutral protease, bacillolysin, derived from B. amyloliquefaciens. As the substrate specificity of this enzyme has only been partially elucidated (Cho et al., 2003), it is not possible to accurately identify the hydrolysis site of tea leaf proteins. On the other hand, large amounts of nitrogen (15–35% of total nitrogen in green leaves) are present as Rubisco in C3 plants including tea plants (Envans, 1989). Therefore, to deduce the parent protein of the hDPP-IV inhibitory dipeptides, amino acid sequences of Rubisco were used to identify possible instances of our dipeptides (Fig. 3). Among the dipeptides detected in the hydrolysates, the sequences of 9 dipeptides (Val-Leu, Phe-Ala, Ile-Ala, Leu-Ala, Trp-Ile, Asn-His, Thr-Trp, Ile-Pro, and Trp-Val) were found in the amino acid sequence of Rubisco, while Leu-Trp, Thr-His, and Trp-Ala were not found. Notably, the Val-Leu and Leu-Ala sequences were found in 4 and 6 instances, respectively. This is consistent with the relatively high content of Val-Leu in the tea grounds hydrolysate (Table 1). As expected, it is possible that the hDPP-IV inhibitory dipeptides identified in this study were generated from Rubisco. Identifying the position of the dipeptide sequence in the parent protein may aid in selecting the appropriate protease used for hydrolysis of the tea grounds (Liu et al., 2019; Nongonierma et al., 2014).

Fig. 3.

Loci of identified TOP17 hDPP-IV inhibitory dipeptides in Rubisco sequences. The hDPP-IV inhibitory dipeptides identified in the tea grounds hydrolysates are underlined. The amino acid sequences of both the Rubisco large chain [(rbcL uniport: Q8WIZ5)] and small chain [(rbcS A0FIP7)] from Camellia sinensis are shown.

Conclusions

The reverse-type peptidomics approach adapted in this study led to the discovery of several hDPP-IV inhibitory dipeptides, in particular Val-Leu, which has not been previously identified in dietary protein hydrolysates by conventional methods. This approach, which prioritizes activity over content of ingredients, can provide a new and complementary perspective on identifying functional peptides. The results obtained in this study suggest that green tea grounds are a promising source of dietary protein for generating hDPP-IV inhibitory peptides.

Acknowledgements    The authors thank Dr. Y. Ohhara (Univ. Shizuoka) for useful suggestions and discussions in the present study. This work was supported in part by a Grant-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (grant numbers: 18K19753, 18H03195, and 19K15792), and the Fuji Foundation for Protein Research. The authors would like to thank Enago (www.enago.jp) for the English language review.

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