The development of a suitable manufacturing process for Benifuuki green tea beverage with anti-allergic effects

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1 Journal of the Science of Food and Agriculture J Sci Food Agric 85: (25) DOI: 1.12/jsfa.2215 The development of a suitable manufacturing process for Benifuuki green tea beverage with anti-allergic effects Hiroshi Nagai, 1 Mari Maeda-Yamamoto, 2 Yuko Suzuki, 1 Katsuhiko Sato 1 and Hiromichi Mitsuda 1 1 Beverage Research & Development Laboratory, Asahi Soft Drinks Co Ltd, Midori, Moriya City, Ibaraki 32-16, Japan 2 National Institute of Vegetable and Tea Science, National Agriculture and Bio-oriented Research Organization, 2769 Kanaya, Shimada City, Shizuoka , Japan Abstract: Epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3 Me) has been reported to inhibit type I allergy better than epigallocatechin gallate (EGCG), a major catechin in tea leaves (Camellia sinensis L). We examined the effects of extraction and sterilization on the catechin content and histamine release from mast cells, as a representative reaction of early phase allergy, in the manufacture of Benifuuki green tea beverage. Among various varieties of tea, the cultivar Benifuuki contains approximately 2% of EGCG3 Me. Ester-type catechins and their epimers increased with the increased extraction temperature of the tea. A tea infusion, extracted at 9 C, strongly inhibited histamine release from mast cells. Furthermore, sterilization affected the catechin content in the manufactured green tea beverage. Sterilization at high temperature promoted the isomerization of catechins and the sterilized green tea beverage had a strong inhibitory effect. When EGCG3 Me, EGCG, epicatechin-3-o-gallate (ECG) and their epimers, GCG3 Me (gallocatechin-3-o-(3-o-methyl) gallate), GCG (gallocatechin-3-o-gallate) and CG (catechin-3-o-gallate) were compared, the anti-allergic effect of GCG3 Me was strongest, and the order of activity was GCG3 Me > EGCG3 Me > GCG > EGCG. We consequently suggest that it was necessary to extract components from tea at the highest temperature possible, and to pasteurize under retort conditions (118.1 C, 2 min), to manufacture functional green tea beverage with an anti-allergic action. 25 Society of Chemical Industry Keywords: gallocatechin-3-o-(3-o-methyl) gallate (GCG3 Me); epigallocatechin-3-o-(3-o-methyl) gallate (EGCG3 Me); gallocatechin-3-o-gallate (GCG); epigallocatechin-3-o-gallate (EGCG); tea polyphenol; catechin; histamine release; anti-allergic activity; tea; retort; manufacturing process; epimerization INTRODUCTION Allergies are diseases of excess immunity. Mast cells play a critical role in the effector phase of IgEdependent immediate hypersensitivity and allergic diseases. Cross-linking of high-affinity IgE receptors (FcεRI) with IgE and allergens (cedar pollen, house dust, mites and food proteins) initiates the activation process, leading to the release of preformed and de novo synthesized vasoactive amines (histamine or serotonin), proteases, leukotrienes, cytokines, and chemokines. 1 3 These chemical and polypeptide agents elicit various allergy-associated pathophysiological changes locally and systemically: for instance, amines, histamine and serotonin enhance vascular permeability, and cytokines such as TNF-α recruit inflammatory cells to the site of allergen exposure. The chemical mediators may cause inflammation in the body and may trigger a critical allergic reaction. 4 The number of patients having allergosis, such as pollinosis, atopic dermatitis, allergic rhinitis, asthma and food allergies, has increased rapidly in recent years in Japan. It is said that more than 3% of Japanese have some allergy, 5 and 13 million people have pollinosis. Tea (Camellia sinensis L) is consumed all over the world, and in particularly large quantities in Japan and China where it has been used for medicinal purpose for centuries. It has been reported that tea has various bioregulatory activities, such as those that are anti-carcinogenic 6 11 and anti-metastatic, anti-oxidative, 17 2 antihypertensive, 21 anti-hypercholesterolemic, anticaries, 25,26 anti-bacterial 27 and intestinal flora amelioration. 28 The major mechanism for these activities has been shown to involve catechins, a group of polyphenolic compounds. Tea leaves contain approximately Correspondence to: Hiroshi Nagai, Beverage Research & Development Laboratory, Asahi Soft Drinks Co Ltd, Midori, Moriya City, Ibaraki 32-16, Japan hiroshi.nagai@asahiinryo.co.jp Contract/grant sponsor: Program for the Promotion of Research and Development for New Bioindustry Initiatives, Japan (Received 1 April 24; revised version received 28 October 24; accepted 23 February 25) Published online 9 June Society of Chemical Industry. J Sci Food Agric /25/$3. 166

2 Development of a manufacturing process for anti-allergic tea beverage 1 2% dry weight of catechins; ie ( )-epigallocatechin-3-o-gallate (EGCG; approximately 5% of total catechins), ( )-epigallocatechin (EGC; 25%), ( )-epicatechin-3-o-gallate (ECG; 12%) and ( )- epicatechin (EC; 12%). 29 In particular, EGCG is a rare catechin, found in no other plants. It has been reported that the flavonoids in 27 kinds of herb tea and green tea infusions prevent allergic disease. 3 Moreover, catechins have been reported to inhibit rat or mouse type I allergies or histamine release from rat basophils. 34 There is evidence that two O-methylated catechins, epigallocatechin 3-O-(3- O-methyl) gallate (EGCG3 Me) and epigallocatechin 3-O-(4-O-methyl) gallate (EGCG4 Me), in tea leaves in special tea varieties such as Benifuuki, Benifuji or Seishin-taipan, have stronger anti-allergic activities than EGCG. Furthermore, it was found that EGCG3 Me suppressed the FcεRI expression by inhibiting ERK1/2 phosphorylation 39 or mast cell activation by inhibiting multiple protein kinases. 4 The structural formulae of the catechins used are shown in Fig 1. Catechins have two asymmetric carbon atoms in the C-ring, and epimers of carbon at the C-2 position of the ring were detected in tea infusions. Analyses of catechins in tea infusions have shown that amounts of most catechins in tea infusions decreased by heat processing, but (+)-catechin was particularly increased, because of the epimerization of ( )-epicatechin. In Japan, canned and bottled teas are becoming remarkably popular. It was supposed that we could manufacture bottled tea with an anti-allergic action by using tea leaves containing anti-allergic substances. However, the relationship between the manufacturing process of this tea beverage and the anti-allergic activity was unclear. In this paper, we therefore tried to clarify the changes in catechin content and antiallergic action of Benifuuki green tea beverage during the manufacturing process. Figure 1. Structural formula of catechins. Arrows show the reversible relationship between catechins and their isomers. MATERIALS AND METHODS Reagents The catechins EGCG, gallocatechin-3-o-gallate (GCG), EGCG3 Me and GCG3 Me were kindly provided by Drs M Sano and T Miyase of the School of Pharmaceutical Science, University of Shizuoka, Japan. ECG and CG, o-phthalaldehyde, and N- acetylcysteine were purchased from Kurita Kougyo (Tokyo, Japan), Nacalai tesque (Kyoto, Japan) and Wako Chemical (Osaka, Japan), respectively. Preparation of green tea beverage 45 Benifuuki fresh tea leaves harvested in May 21 at the plantation of the National Research Institute of Vegetables and Tea Sciences in Kanaya, Shizuoka, Japan. Tea leaves were immediately steamed for 35 s and then dried using a 35-kg scale auto green tea manufacturing machine (Terada-Seisakusho, Shizuoka, Japan). To differentiate among the extraction conditions, Benifuuki green tea was extracted at 5, 7, 9 C for 6 min with 3 times the volume of distilled water. After filtration, 6 mg l 1 of L-ascorbic acid sodium salt and 7 mg l 1 of sodium bicarbonate (NaHCO 3 ) were added to these tea infusions, which were then sterilized with retort sterilization. 45 Another tea infusion extracted at 9 C was sterilized as follows: non-sterilized (hot pack; control), ultra-high temperature sterilization (UHT: 138 C, 3 s) or retort sterilization (118.1 C, 2 min). For the investigation, the concentration of polyphenols in each extract was standardized as follows. Analysis of polyphenol content Generally, the uniform quality of green tea beverage is controlled by the polyphenol concentration. To standardize each sample under various sterilized conditions to the same level of polyphenol, the polyphenol content in the infusion was measured by a colorimetric method using ferrous tartrate. 46 Briefly, an A solution (1 mg dl 1 of ferrous sulfate and 5 mg dl 1 of potassium sodium tartrate), a B solution (Sorensen buffer; 1/15 M Na 2 HPO 4 solution 1/15 M KH 2 PO 4 solution 84:16), and an ethyl gallate standard solution were prepared. Five milliliters of infusion sample (standard solution or distilled water as blank) was taken into a 25-ml flask, 5 ml of A solution was added and mixed completely, then B solution was added to make up to 25 ml. The absorbency of the reaction was measured by a spectrophotometer at the wavelength of 54 nm (SmartSpecPlus, Bio-Rad, CA, USA). Ethyl gallate was used as polyphenol standard. Standard solution was prepared, 2, 4, 6, 8 and 1 mg dl 1 to make a linear calibration curve. Polyphenol content (mg dl 1 ) was calculated by multiplying ethyl gallate content by a coefficient of Analysis of catechins 43,44 Tea beverage was diluted fivefold with distilled water and 2 µl of the sample was filtered through J Sci Food Agric 85: (25) 167

3 H Nagai et al a membrane filter (DISMIC-13HP-PTFE, pore size.45 µm, ADVANTEC, Tokyo, Japan) and injected with an autosampler (SIL-1Avp, Shimadzu, Kyoto, Japan) into the HPLC apparatus (Shimadzu class VP HPLC system). HPLC was performed with a Shimadzu LC-1A pump coupled with a photodiode-array detector (UV 28 nm) (SPD- M1Avp, Shimadzu, Kyoto, Japan) using a reversephase Mightysil RP-18 column (4.6 mm id 15 mm, particle size; 5 µm, Kanto Kagaku Chemical, Tokyo, Japan), which was eluted as described below, at a flow rate of 1 ml min 1 at 4 C. HPLC analysis was performed using a linear gradient system with mobile phase A (H 2 O acetonitrile H 3 PO 4 4:1:1) and mobile phase B (methanol mobile phase A 1:2). The linear gradient elution was maintained at 1% A for 2 min, programmed to 2% mobile phase A over 43 min, maintained at 2% mobile phase A for 5 min, and then returned to 1% mobile phase A for 1 min. Quantification was carried out using the external standard method. Quantification of catechins was performed using data acquisition and processing system of LC workstation (Class VP system, Shimadzu). Cells and culture Bone marrow cells from the femur of NC/Nga mice (Charles Liver Japan, Kanagawa, Japan) were cultured in 4 ng ml 1 murine recombinant IL-3 (Peprotec (NJ, USA)) containing RPMI164 medium supplemented with 1% heat-inactivated fetal bovine serum (Invitrogen Life Technologies, CA, USA), 2mM glutamine and 5 µm 2-mercaptoethanol in humidified 95% air 5% CO 2 at 37 C. 47 More than 95% pure mast cells were obtained as bone marrow derived mast cell (BMMC) after four weeks of culture. Degranulation by FcεRI cross-linking 4 BMMC cells were passively sensitized at a density of cells ml 1 with 1 µgml 1 anti-dinitrophenyl (DNP) mouse monoclonal IgE antibody Sigma- Aldrich (MO, USA) at 37 C overnight. After washing in Tyrode buffer (Ca 2+ -free; 1 mm HEPES ph 7.4 (Wako chemical, Osaka, Japan) with 8 mg dl 1 NaCl, 2 mg dl 1 KCl, 5.6mgdl 1 NaH 2 PO 4, 1 mg dl 1 glucose, 5 mg dl 1 gelatin and 1 µm MgCl 2 6H 2 O), they were re-suspended in Tyrode buffer at a density of cells ml 1, and incubated for 2 min with samples at 37 C, then stimulated by 3 ng ml 1 of DNP-HSA (human serum albumin) (LSL Cosmo Bio, Tokyo, Japan) with 3 µm CaCl 2 for 1 min at 37 C. The reactants were added to the 4mM EDTA/Tyrode solution and cooled on ice to stop the reaction. To measure the histamine, the reactions were centrifuged at 15 g for 5 min at 4 C, the equivalent volume of.1 M hydrochloride was added to the supernatant, and the released histamine was measured by on-column HPLC. 48 HPLC was performed using a Shimadzu LC-1A pump coupled with a fluorescent photometric detector (excitation 33 nm, emission 43 nm) (RF-1AxL, Shimadzu, Kyoto, Japan) with a reverse-phase Asahipak-ODP- 5-4E column (4.6 mm id 25 mm, particle size; 5 µm, Showa Denko, Tokyo, Japan). The reaction was eluted with 5 mm sodium borate acetonitrile (8/2) buffer containing 1 mm o-phthalaldehyde and 1mM N-acetylcysteine, at a flow rate of.5ml min 1 at 37 C. Statistics Theresultsareshownasthemeans± SD in triplicate experiments. Statistical analysis was performed using Tukey s multiple-range test. A value of p <.5 was considered statistically significant. RESULTS AND DISCUSSION We aimed to construct a more effective manufacturing process to commercialize an anti-allergic beverage made from Benifuuki tea cultivar. It was reported that heating process induces the epimerization of tea catechins, 42,43,49,5 and the inhibitory effect on histamine release of the epimerized catechins, GCG, is greater than that of EGCG. 33 There are two processes affecting the catechin content in a manufacturing process: the extraction from tea leaves and sterilization. To clarify the changes in catechin composition and anti-allergic activity (histamine release inhibitory effect) during the beverage manufacturing process, we used HPLC to measure the catechin content of tea extracted at various temperature. Tea infusions extracted at 5, 7, 9 C for 6 min were sterilized with retort sterilization, and the catechin content was analyzed. As shown in Fig 2, a rise in extraction temperature tended to increase Content of catechins (mg dl -1 ) EGCG GCG ECG CG 5 C 7 C 9 C Figure 2. Catechin content of tea samples prepared with extraction at various temperatures. Tea infusions extracted at 5, 7, 9 Cfor 6 min were sterilized with retort sterilization, and the catechin content was analyzed by HPLC. Each value represents the mean ± SD, n = 3. E 168 J Sci Food Agric 85: (25)

4 Development of a manufacturing process for anti-allergic tea beverage Histamine release inhibition (%) * * Temperature ( C) Figure 3. Histamine-release inhibitory effects of tea samples prepared with various temperature extractions. Tea infusions extracted at 5, 7, 9 C for 6 min were sterilized with retort sterilization, diluted twofold and added to BMMC assay. Each value represents the mean ± SD, n = 3. Significantly different from 5 C value, p <.5. Content of catechins (mg dl -1 ) Control UHT Retort the content of the catechins and their isomers. The catechin content extracted at 5 C was lower than that extracted at 7 or 9 C. It was clear that the increased extraction temperature resulted in a higher concentration of catechins. We then examined whether the extraction temperature affected the anti-allergic action. As shown in Fig 3, tea extracted at 9 C showed a strong inhibitory effect on histamine release. Extracts at 7 and 9 C significantly inhibited histamine release compared with 5 C extraction. It was reported that a temperature of more than 8 C caused an acceleration of catechin isomerization, 49 and thermal treatment at 12 Cfor 3 min in a ph 5 solution was effective in forming these isomers in quantity. 42,5 This study indicated that a high extraction temperature increased the level of catechins and their isomers and that inhibition of histamine release from BMMC was temperaturedependent. This result suggested that the extraction efficiency of catechins was promoted with a rise in extraction temperature and an accelerated anti-allergy effect. We next investigated the relationship between the sterilization condition and the amount of remaining catechins. Figure 4 shows the varying catechin content of tea under three sterilized conditions. Hot-pack (control; non-sterilization), the shortest heating time among the three conditions, produced 2% of epimers by isomerization. In contrast, approximately 5% or more isomers were produced by UHT or retort sterilization, respectively. There was a tendency for levels of EGCG3 Me to decrease and GCG3 Me to increase under retort sterilization compared with the UHT sterilization. It was therefore suggested that isomerization required longer heating time rather than higher heating temperature. Since it was surmised that the intensity of antiallergic activity of EGCG3 Me was different from the epi-form catechin, the effects of the sterilized conditions on histamine release from BMMC were EGCG GCG ECG Figure 4. Catechin content of Benifuuki tea samples prepared under different sterilized conditions Tea infusions extracted at 9 C for 6 min were sterilized as follows: Control: non-sterilization; UHT: ultra-high temperature sterilization (138 C, 3 s); Retort: retort sterilization (118.1 C, 2 min). Histamine release inhibition (%) Sterilized condition CG 2 µg 5 µg Polyphenol content E Control UHT Retort Control UHT Retort Figure 5. Histamine-release inhibitory effects of tea samples prepared with different sterilized conditions. Tea infusions extracted at 9 C for 6 min were sterilized under three conditions. These samples received 2 or 5 µgml 1 as polyphenol content in a BMMC assay. Control: non-sterilization; UHT: ultra-high temperature sterilization (138 C, 3 s); Retort: retort sterilization (118.1 C, 2 min). Each value represents the mean ±SD, n = 3. Significantly different from hot pack value, p <.5. investigated. Each tea sample was administered 2 or 5 µgml 1 as polyphenol content in a BMMC assay. As shown in Fig 5, the intensity of the inhibitory effect of these teas on histamine release from BMMC was: retort sterilization > UHT sterilization > control, in that order, and there was statistical significance * J Sci Food Agric 85: (25) 169

5 H Nagai et al (p <.5) between retort sterilization and control at 5 µgml 1 addition. At 5 µgml 1 addition, retort sterilization resulted in 1.4-fold inhibitory activity on histamine release compared with the control. These results indicated that thermal sterilization leads to the conversion of the epi-form catechins to catechins and the promotion of anti-allergic activity. Next, to clarify the relationship between the chemical structure of catechins and anti-allergic action, the inhibitory effects on histamine release at a concentration of 25 µgml 1 of CG, ECG, GCG, EGCG, EGCG3 Me and GCG3 Me were compared. As shown in Fig 6, O-methylated catechins had a stronger inhibitory effect than non-methylated catechins, EGCG, GCG, ECG or CG that had been reported to have an anti-allergic action, and isomerized catechins than the epi-form catechins. EGCG3 Me had approximately threefold inhibitory activity compared with EGCG, and it was suggested that EGCG3 Me was one of main active substances previously described. 35 Furthermore, GCG3 Me showed approximately 1.5- fold inhibitory activity of EGCG3 Me on histamine release. As shown in Fig 4, EGCG and GCG content was sixfold that of EGCG3 Me and GCG3 Me. To verify the contribution of O-methylated EGCGs and each isomerized catechin on the inhibitory activity, the actual inhibitory activity in Fig 5 was compared with the calculated inhibitory activity from Figs 4 and 6. The ratio of actual inhibitory activity control:uht:retort was 1:1.19:1.37. However, the ratio of calculated inhibitory activity of control:uht:retort was 1:1.17:1.2. From these results, it was assumed that O-methylated EGCGs and isomerized catechins mainly contributed to histamine release inhibitory activity. Figure 7 shows the EGCG3 Me and GCG3 Me content of these teas under each sterilized processing. The result indicated that half of epi-form catechins converted to their isomer by UHT processing and Histamine release inhibition (%) ECG CG EGCG Figure 6. Comparison of histamine-release inhibitory effects of ECG, CG, EGCG, GCG, EGCG3 Me and GCG3 Me. Each sample received 25 µgml 1. GCG E Content of E/ (mg dl -1 ) E Control UHT Retort Sterilized condition Figure 7. O-methylated catechins content under various sterilized conditions. These results show the HPLC analysis of tea samples with 5 µgml 1 as polyphenol. Control: non-sterilization; UHT: ultra-high temperature sterilization (138 C, 3 s); Retort: retort sterilization (118.1 C, 2 min). more than half of epi-form catechins converted to their isomer by retort processing. The intensity of the conversion rate of EGCG3 Me to GCG3 Me was retort > UHT sterilization > and control, in that order. In this paper, it has been shown that the process of sterilization was important for increased anti-allergic activity in the manufacture of the beverage. Retort or UHT sterilization caused a rise of inhibitory activity by the promotion of isomerization. Furthermore, it has been reported that GCG, an isomer of EGCG, had strong anti-allergic activity, 33 and this paper showed that GCG3 Me had a stronger inhibitory effect on histamine release. As a result, the epimers of epi-form catechins that were presented in tea leaves were more effective and the effect was GCG3 Me > EGCG3 Me > GCG > EGCG > CG = ECG. It has been reported that the inhibitory effect of GCG and EGCG on histamine release was stronger than that of ECG. ECG was also more active than EC and/or C. 34 When manufacturing green tea beverage, it is estimated that there is a 7 8% degradation of catechins by retort sterilization. However, it is interesting that high temperatures may result in efficient epimerization and promotion of anti-allergic activity. CONCLUSION This research indicated that the manufacturing process could promote the anti-allergic action of tea using Benifuuki green tea. To manufacture functional Benifuuki green tea with an anti-allergic effect, tea should be extracted at the highest temperature possible and sterilized under retort conditions (118.1 C, 2 min). ACKNOWLEDGEMENTS The authors wish to thank Satoshi Sunada for technical assistance. This work was supported by a grant from 161 J Sci Food Agric 85: (25)

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