Improvement of tea leaves fermentation with Aspergillus spp. pectinase

JOURNALOFBIOSCIENCE AND BIOENGINEERING Vol. 94, No. 4,299-303.2002

Improvement of Tea Leaves Fermentation with Aspergillus spp. Pectinase JAYARAMAN ANGAYARKANNI,‘* MUTHUSAMY SUBBAIYAN MURUGESAN,’ AND KRISHNASAMY

PALANISWAMY,* SWAMINATHAN’

Department of Biotechnology, Bharathiar University, Coimbatore-641 046, India’ and Department of Microbiologv/Bioinformatics, Karpagam Arts and Science College, Coimbatore-MI 021, India2 Received 13 February 2002/Accepted 17 June 2002

The pectinase enzymes isolated from Aspergillus spp., A. indicus, A. jluvus and A. niveus were used for fermentation of tea leaves. The enzymes were purified and characterized. The effect of both crude enzyme preparation and purified pectinase enzymes on the improvement of tea leaf fermentation were determined in terms of theaflavin, thearubigin, high polymerized substances, total liquor colour, dry matter content and total soluble solids of the tea produced. The crude enzyme preparations obtained from ethanol precipitation were found to be more effective in improving tea leaf fermentation than the purified pectinase enzymes. [Key words: Aspergillus spp., pectinase enzyme, tea fermentation]

ing in incomplete oxidation of oxidizable matters. But the application of external enzymes that degrade the cell walls of tea leaves will result in complete maceration of cells of tea leaves (11). Hence, tissue maceration using enzymes will improve the fermentation by releasing the reactants. It has been reported that endotypes of pectinase enzymes, both hydrolase and lyase, are the major factors responsible for plant tissue maceration (12, 13). Pectinase enzymes from fungal origin, especially Aspergillus niger are industrially produced to be used as processing aids for extraction, clarification and maceration purposes (14). In the present study, pectinase enzymes obtained from Aspergillus inducus, A. flaws and A. niveus are used for maceration of tea leaves and the improvement in the tea quality due to pectinase treatment is compared with the tea processed by conventional method and with tea fermented with commercially available pectinase preparation. The quality parameters studies were theaflavin (TF), thearubigin (TR), highly polymerized substances (HPS), total liquor colour (TLC), dry matter content (DMC) and total soluble solids (TSS).

Tea is classified as black tea and green tea based on the manufacturing process. The essential difference in the processing is that the black tea is allowed to ferment before firing, while the green tea is rapidly dried (1). During the fermentation process, the tea catechins are oxidized to orthoquinones which condense to form theaflavins (TF). These theaflavins act as oxidizing agents for the substances like gallic acid. Epitheaflavic acids, formed by the oxidation of gallic acid, condense with theaflavins to produce the polymeric thearubigins (TR). These thearubigins are responsible for colour, body and taste while theaflavins content in tea determines the briskness, brightness and quality of the liquor. An ideal fermentation process results in a proper balance of TF and TR (2). Among the various components estimated and evaluated for the quality of black tea, only TF content was reported to play a major role in tea quality (36). Catechins, TR and caffeine are being tested for inhibiting the cancer formation in animal models (7). The TF in black tea are found to inhibit lung and Oesophagal carcinogenesis (8, 9). In another report Yang et al. (10) have reported that the black tea polyphenol, theaflavin-3-3’-digallate inhibited the growth of Harastransformed 21BES cells. Hence, any increase in tea phenolic compounds could improve the quality and therapeutic value of tea. During fermentation, the colour change associated with the development of characteristic aroma takes place. The rate of tea fermentation will depend upon the degree of contact between enzyme and the substrate since they are not in homogenous system. All the reactants of fermentation except oxygen are endogenous to the tissues of the tea shoot tips but separated by cell membranes. It is known that the enzyme, polyphenol oxidase, is located in the cell sap. Maceration of tea leaves by rolling will cause only partial rupture of the cells result-

MATERIALS

AND METHODS

Pectinase enzyme preparation For pectinase enzyme production, the fungi were grown in Singh and Wood medium (15). The medium was inoculated with the fungal spore suspension (lo%, v/v) and incubated on a rotary shaker at 125 rpm for 4 d at 27f2’C. At the end of the incubation period, the culture medium was filtered on a Whatman no. 1 filter paper (Whatman International, Maidstone, UK). The culture filtrate was centrifuged at 10,000 rpm for 20 min at 4°C and the clear supernatant was dialyzed and used as the enzyme preparation. Pectinase activity in the dialyzed sample was determined by the method recommended by Sherwood (16). The estimation recommended by Sherwood was used to estimate pectin lyase. Enzyme purification The dialyzed clear supernatant (200

* Corresponding author. e-mail: [email protected] phone: +91-422-424652 fax: +91-422-422387 299

300

ANGAYARKANNI

ml) was mixed with three volumes of cold ethanol with continuous stirring and kept undisturbed for overnight at 4°C. The resulting precipitate was collected by centrifugation at 9000xg for 30 min at 4°C. The precipitate was dissolved in McIlvaine’s buffer (0.2 M; pH 5.0), dialyzed overnight against distilled water and used for tea processing. For further purification, the dialyzed sample was lyophilized to 5 ml and passed through Sephadex G-100 column (I .5 x45 cm; Sigma Aldrich, MO, USA) equilibrated with citrate buffer (0. I M; pH 5.0) and eluted with the same buffer. Fractions of 10 ml/h were collected. The protein content (17) and the pectinase enzyme activity were assayed in each fraction. The active fractions were pooled together and used for determination of enzyme properties like optimum pH, temperature, optimum substrate concentration (V,, and K,,,) (18) and molecular weight (19). Tea processing Tea clone representing ‘Assam’ (UPASI-3) cultivar was selected for the study. The plucked apical bud and two leaves were withered for 18 h and made into cut dhool by CTC (crush, tear and curl) process. The cut dhool (750 g) was taken in plastic trays and sprayed with the following enzyme preparations: (i) Mcllvaine’s buffer (25 ml; 0.2 M, pH 5.0, control), (ii) crude enzyme preparation (25 ml; 0.1 IU/ml pectinase activity), (iii) purified enzyme (25 ml; 1.O W/ml pectinase activity), and (iv) commercial enzyme biopectinase (1 ml diluted to 25 ml with McIlvaine’s buffer; 0.2 M, pH 5.0). Commercial enzyme biopectinase was purchased from Biocon Pvt. Ltd., Bangalore, Kamataka, India and the diluted enyzme had an activity of 1.O IU/ml pectinase activity. The treated dhool was allowed to ferment at room temperature for 1 h. After an hour, the tea samples were fired (97°C to IOS’C) to terminate the enzyme activity and to restrict the moisture content by 4% to 6%. The tired tea was sifted and the biochemical constituents of the tea leaf, TF, TR, HPS and TLC were estimated by solvent extraction method of Takeo and Ozawa (20); TSS content was estimated by ISS (21) method. Solvent extraction method The solvent extraction of tea was carried out in separating funnels with adequate shaking at every stage as depicted in the flow chart (Fig. 1). The parameters TF, TR, HPS and TLC were calculated from the absorbance values as given below. TF (%)=4.313xC TR (%)= 13.643 (B+D-C) HPS (% as TR)=13.643 xE TLC (%)= 10 x A The multiplication factors mentioned in the equations were derived from molar extinction co-efficients of pure compounds (22) and dilution factors. HPS was represented as TR. In case of TLC, the dilution factor was 10. The accuracy of the contents was tested by conducting the analysis thrice. The tea extract was taken thrice and the parameters were determined in triplicates. The average of the readings were taken as the final value.

RESULTS

J. Broscr. BIOENG..

ET AL.

AND DISCUSSION

Enzyme production The results revealed that Aspergillus spp., A. indicus, A. flavus and A. niveus are capable of producing pectinase. A. indicus produced 0.460 IU/ml of pectinase; A. flavus produced 0.410 IU/ml of pectinase; A. niveus produced 0.430 m/ml of pectinase. Friedrich et al. (23) reported that A. niger mutant Al38 produced 0.03 to 0.14 EZ3,, of pectin lyase. Tubercularia vulgaris produced 0.184 U/ml of pectin lyase in submerged culture (24). Acuna-Arguelles et al. (25) reported that a strain of A. niger CH4 produced O.O08U/ml/h of pectin lyase in liquid and

Black

tea, 4 g + Boiling

water,

200 ml

1

i IBMK

i

layer

Aqueous

layer

Aqueous Eth.,

45%, 9 ml L-L

layer discarded

+ Aqueous layer,

lml + Eth.,

45 %, 9 ml + OD, 380 nm

(E)

FIG. I. Flow chart for Solvent extraction method. IBMK, Isobutyl-methyl ketone; Eth., Ethanol.

solid state fermentations. When compared to these reports, it was evident that the fungi used in the present study are capable of producing good amounts of pectinase. Pectinase enzyme obtained from Enzyme purification A. indicus culture filtrate was purified by 11.20-fold with a specific activity of 13.33 IU/mg protein; percent recovery was 13.04. A. flaws pectinase was purified by 23.08-fold with a specific activity of 2 1.OOIU/mg protein and enzyme recovery of 15.37%. A. niveus pectinase was purified by 14.41-fold with a specific activity of 16.00 IU/mg protein and recovery of 11.16% (Table 1). Houdenhoven (Ph.D. theEnzyme characterization sis, Agricultural University, Wageningen, The Netherlands, 1975) reported that pectin lyases isolated from A. niger I and II had an optimum pH of 6 with Km values of 5 and 0.9 g/ml respectively; the molecular weights were 35.4 and 33.1 kDa respectively. Pectin lyase from A. niger CH4 showed an optimum temperature of 50°C and pH optimum of 7; the Km value of the enzyme was 12.8 and 2.28 mg/ml in liquid and solid state fermentation (25). The pectin lyase enzymes of A. niger, after purification had a specific activity of 20 U/mg protein; molecular weight of about 38,000 with an optimum pH of 6.4; the K,,, value was 0.2 mM (26). In the present study, the purified enzyme of A. indicus showed an optimum pH of 6 and temperature of 50°C for its activity. The enzyme was stable at 37°C for 2 d and at 50°C for 60 min. V,, and K,,, values were 10.02 IU/mg and 10

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301

TABLE 1. Purification of pectinase enzyme from culture filtrates Fungal species

Culture filtrate Ethanol precipitation Column chromatographySephadex G 100 (Fraction 5) Culture filtrate Ethanol precipitation Column chromatographySephadex G 100 (Fraction 5) Culture filtrate Ethanol precipitation Column chromatographySephadex G 100 (Fraction 5)

A. indicus

A. jlavus

A. niveus

Optimum pH Optimum temperature (“C) Thermostability 37°C (d) 50°C (min) V,, (IU/mg protein) K, (mg/ml) Molecular weight (kDa) (SDS-PAGE)

A. indicus

A. ,jlavus

205.00 72.00 31.50

0.45 0.50 0.10

215.00 44.00 24.00

0.39 0.70 0.10

0.46 1.60 2.00

500 40 15

0.41 1.80 2.10

500 40 15

0.43 1.10 1.60

Activity (IWml)

TABLE 2. Properties of pectinase enzyme Properties

0.39 0.60 0.15

Specific activity Wmg) 1.19 2.67 13.33

Total activity (IU) 230.00 64.00 30.00

Total volume (ml) 500 40 15

Sample

A. niveus

6 50

5 50

6 50

2 60 10.02 10 28.5

3 60 33.42 6.28 22

3 60 12.37 9.54 23

205 kDa

116kDa

66.0LDa

36.OkDa 29.OkDa

24.0kDa

2O.lkDa 14.2kDa

6.5kDa

FIG. 2. SDS-PAGE gel showing the molecular weights of A. indicus, A. j7avus, and A. niveus pectinases. Lane 1, Markers; lane 2, A. indicus; lane 3, A. flaws; lane 4, A. niveus.

Protein (mg/ml)

Purification fold

Percent recovery

1.oo 2.24 11.20

100.00 27.83 13.04

1.91 3.60 21 .oo

1.oo 3.00 23.08

100.00 35.12 15.37

1.11 1.57 16.00

1.oo 1.41 14.41

100.00 20.47 11.16

mg/ml respectively against pectin substrate. The molecular weight of the enzyme was 28.5 kDa (Table 2 and Fig. 2). A. j7uvus enzyme had an optimum pH of 5 and temperature of 50°C. The enzyme was stable for 3 d at 37°C and for 60 min at 5O’C; I’,,,, and K,,, values were 33.42 IU/mg and 6.28 mg/ml respectively for pectin and the molecular weight of the enzyme was 22 kDa (Table 2 and Fig. 2). For A. niveus pectinase, the optimum pH was 6 and the optimum temperature was 5O’C. The enzyme was stable for 3 d at 37°C and for 60 min at 50°C; V,, and K,,, values of the enzyme against pectin were 12.37 IU/mg and 9.54 mg/ml respectively and the molecular weight was 23 kDa (Table 2 and Fig. 2). Tea processing Studies have been carried out only on isolation, purification and characterization of various enzymes in tea leaves and their role in tea processing (Mahantas, Abstr. Int. Symp. on Tea and Human Health, TRA, Calcutta, p. 44-45, 1993; [27-29]), but the effect of added enzymes on tea quality was not much studied. Marimuthu et al. (30) reported that use of biopectinase and biocellulase (commercially available enzymes purchased from Biocon Pvt. Ltd.) at 0.6% concentration could improve tea quality with an increase of 24.77% TF, 21.52% TR, 21.54% HPS, 23.33% and 17.49% TSS. In another study, they have reported that commercially available biopectinase, when used for tea leaf fermentation could increase TF by 5.8%, TR by 5.72%, HPS by 4.96% and TSS by 9.29% (31). Mixed enzyme extract of A. oryzae, A. wentii, A. tamari, A. japonicus, A. awamori and Trichoderma koningii was reported to enhance the final tea quality with an increase in TF by 45%, TR by 48%, HPS by 33%, TLC by 19% and TSS by 3% (32). The results obtained in the present study (Table 3) revealed that the exogenous application of fungal enzymes improve the tea quality. Especially, the crude enzyme preparations of the test fungi were more effective than the purified enzymes and the commercial biopectinase in increasing the tea quality parameters. The crude enzymes extract from fungi comprises of all enzymes, cellulase, hemicellulase (xylanase), proteinase, pectinase, etc. (datas not shown), whereas the purified enzyme solution contains only pectinase. The tea leaf is composed of cellulose, hemicellulose and pectin. When the crude enzyme extract (enzyme com-

302

J. Broscr. BIOENG.,

ANGAYARKANNI ET AL. TABLE 3. Effect of pectinase enzyme treatment on tea quality DMRT ranking

(Conventional method) Control 1.05 g, 8 SD: 0.012 Crude enzyme (2.5 ILV750 g tea leaf) A. indicus 1.51 c,3 SD: 0.013 A. fravus 1.71 a, 1 SD: 0.009 A. niveus 1.67 b,2 SD: 0.009 Purified enzyme (25 IU/750 g tea leaf) A. indicus 1.45 d, 5 SD: 0.013 A. j7avus 1.47 d,4 SD: 0.005 A. niveus 1.41 e, 6 SD: 0.013 Commercial enzyme 1.37 f, 7 SD: 0.013 cv: 0.9% 0.010 P: SED: 0.011 LSD (%): 0.032

TR (%)

DMRT ranking

HPS (%)

9

e, 8 0.014

8.28

a, 2 0.013 a, 1 0.008 a, 3 0.009

9.93

10.08 10.12 10.06

9.64 9.85 9.61 9.36 0.4% 0.010 0.030 0.088

c, 5 0.019 b, 4 0.009 c, 6 0.021 d, 7 0.021

DMRT ranking &

7

DMRT ranking

DMC (%)

3.12

e, 7 0.005

96.88

a, 1 0.014 b, 2 0.025 bc, 3 0.008

96.69

0.015

8.63 11.42

8.83 9.3 9.12 7.98 0.8% 0.010 0.058 0.170

___-

TLC (%)

b, 2 0.008 f, 6 0.005 a, 1 0.005

3.71

e, 5 0.025 c, 3 0.016 d, 4 0.033 h, 8 0.033

3.5

3.58 3.56

3.48 3.47 3.48 1.0% 0.010 0.030 0.087

cd, 4 0.016 d, 5 0.022 d, 6 0.013 d, 5 0.013

96.77 96.75

96.52 96.47 96.53 96.32 0.0% 0.010 0.024 0.008

DMRT ranking

TSS (%)

DMRT ranking

a, 1

C6

0.005

0.005

d, 4 0.016 b, 2 0.005 c, 3 0.013

37.87

c, 6 0.001 f, 7 0.022 e, 5 0.009 g>8 0.009

38.02

37.02 39.86

37.92 39.04 37.01

c, 5 0.010 g>7 0.014 a, 1 0.013 c, 3 0.013 d, 4 0.008 b, 2 0.008 g, 8 0.008

0.0% 0.010 0.026 0.009

The ranking based on the highest value of the parameter taken is given in numbers and the different alphabetical letters indicate that each value differs significantly from the other value at 5% level. TF, Theaflavin; TR, thearubigin; HPS, highly polymerised substances; TLC, total liquor colour; D-MC, dry matter content; TSS, total soluble solids.

plex) was sprayed on tea leaf during fermentation, all the polymeric compounds, cellulose, hemicellulose and pectin, are hydrolyzed by the complex action of all enzymes in the extract. Hence results in higher maceration of tea leaves and in turn fermentation. In case of purified enzyme, only pectin in the tea leaves was hydrolyzed and less maceration was achieved. The use of crude enzyme preparation of A. indicus, A. flavus and A. niveus resulted in the maximum increase in TF content by 43.8 l%, 62.86% and 59.05% respectively, whereas the purified enzymes increased the TF content by 38.10%, 40% and 34.29% and the commercial enzyme increased the TF content only by 30.48%. DMRT analysis indicated that comparatively the crude enzyme preparations of the test fungi enhanced the TF content to the maximum. Likewise the maximum increase in TR content was observed when treated with crude enzyme of A. indicus (12%), A. jhus (12.44%) and A. niveus (11.78%). While, the purified enzyme improved the TR content by 7.1 l%, 9.44% and 6.78% and the commercial enzyme by only 4%. DMRT analysis showed that the crude enzyme preparations of the test fungi significantly (at 5% level) increased the TR content than the purified and commerical enzymes. In case of HPS, the crude enzyme of A. indicus, A. jlavus and A. niveus showed an increase by 19.93%, 4.23% and 37.92% respectively. The purified enzyme of the fungi exhibited 6.64%, 12.32% and 10.15% enhancement of HPS and the commercial enzyme exhibited only 3.62% increase. DMRT analysis indicated that the crude enzyme of the test fungi significantly (at 5% level) increased the HPS content than the other enzyme preparations. The TLC was enhanced to maximum of 18.9 l%, 14.74% and 14.10% by the crude enzyme of A. indicus, A. flaws and A. niveus. Whereas, the

purified enzyme resulted in an increase of 12.18%, 11.54% and 11.22% and the commercial enzyme increased the TLC by only 11.54%. DMRT analysis of the data clearly showed that the crude enzyme of the test fungi improved the TLC to a significant level (at 5% level) than the purified enzyme of the test fungi and the commercial enzyme. DMC was decreased by 0.20%, 0.11% and 0.13% by the crude enzyme of A. indicus, A. jlavus and A. niveus, respectively. Whereas the purified enzymes decreased the DMC by 0.37%, 0.42% and 0.36% and the commercial enzyme decreased the DMC by 0.58% only. Similarly TSS was increased by 0.91% and 6.21% by the crude enzyme of A. indicus and A. niveus, respectively. While, the purified enzymes of A. indicus, A. flavus and A. niveus increased the TSS by 1.3 l%, 1.04% and 4.02% and the commercial enzyme decreased the TSS by 1.39%. These results clearly indicated the crude enzyme of the test fungi was the most effective in improving the fermentation of tea leaves. Comparatively, the improvement in tea leaves fermentation observed in the present study due to enzyme treatment was better than in the earlier reports (30, 3 1). On comparison, the results of Senthilkumar et al. (32) was higher than the presented results because in that work mixed crude enzyme extract of six fungi was used wherein the concentration of the hydrolytic enzymes might be more and also the enzyme concentration was not adjusted to the 0.1 IU/ml pectinase as it was done in this present work. DMRT analysis was done mainly to find out the effect of addition of external enzymes in crude and purified form on the improvement of fermentation of tea leaves. Even though there was no significant difference between the enzymes of three strains, there was significant difference between the control and experimental samples (tea obtained by fermentation with addition of external enzymes), which established

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the fact that external addition of enzymes does have an effect on improving tea leaf fermentation. The statistical analysis done by Duncan’s mutiple range test (DMRT) clearly indicated that the addition of external enzymes in crude form during tea leaf fermentation significantly increased the tea quality parameters at 5% level. The results reveal that the crude enzyme preparations, obtained after ethanol precipitation in enzyme purification step, could be used for fermentation of tea leaves in CTC process to improve the tea leaf fermentation. REFERENCES Stuart, M. A.: Chinese materia medica, p. 536. International Book Distributors, India (1985). 2. Sanderson, G. W.: The chemical composition of fresh tea 1.

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