analyses of tannins in regional specific ceylon teas...

ANALYSES OF TANNINS IN REGIONAL SPECIFIC CEYLON TEAS

N.L.W.S.A. Karunaratne

GS / MSC / FOOD / 2799 / 06

Thesis is submitted to the University of Sri Jayewardenepura as the partial fulfillment requirement for the award of

The degree of Masters of Science

In

Food Science and Technology

Department of Food Science & Technology

Faculty of Applied Sciences University of Sri Jayewardenepura

Dedicated to

My ever loving late fcit her.

It/ly most Precious Mother

Who nurtured and groomed me

To be a Valued Citizen.

Also to

My loving wiJ

&

Little daughter

DECLARATION

I do hereby declare that the work described in this thesis was carried out by me under the supervision of Dr. K.K.D.S. Ranaweera and Mr. Chaminda Munasinghe and a report on this has not been submitted in whole or in part to any University or any other institution for another Degree/diploma.

N.L.W.S.A.Karunaratne

Date

Certified by:

External Su ervisor:

Mr. c/ui inda unasinghe Laboratory Manager, Unilever Sri Lanka Ltd., 258, Vincent Perera Mawatha, Colombo 14.

Internal S7 rvisor:

Dr. K.K.D. Ranaweera, Co-ordinato of Msc. in Food Science & Technology I-lead Department of Food Science & Technology University of Sri Jayewardenepura

TABLE OF CONTENT

Page No. Acknowledgement 05

Abstract 06

Chapter 01

1- Introduction 09 IHistory of tea 09

1.2 History of tea plant 09 1.3. Agriculture of tea 10 1.4. Manufacturing process of black tea. 11 1.5 Objectives of the study 13

Chapter 02

Literature review 14 2.1 Chemistry and Biochemistry of tea 14 2.2 Polephenols in tea 14 2.3 Composition of a typical tea beverage 16 2.4 Physiological effects of tea 16 2.5 Polephenols as antioxidants 16 2.6 Sri Lankan tea industry 17 2.7 Different regions and sub-districts where teas are grown

In Sri Lanka 18

Chapter 03

3.1 Experimental / Methodology 20 3.2 Methodology used 22 3.3 Measurement of sample 23 3.4 Preparation of standard solution series 24 3.5 Calculation of tea polephenols 25 3.6 Calculation of the dilution of tea extracts 25 3.7 Relating tea extract absorption values with the standard series 26

4.0 Results and Discussion 26

4.1 Analyses and Interpretation of results 27 4.2 Statistical analyses of results 34 4.3 Significance of relationship between variables and predictors 42

5. Conclusion and Recommendations 43

References 44 Appendices 45

4

ACKNOWLEDGEMENT

I would wish to express my sincere gratitude to my external supervisor

Dr. K.K.D.S. Ranaweera, Co-ordinator of MSc. in Food Science &

Technology and Head Department of Food, for all the valuable guidelines and

insights given to me in completing this project.

Also, I would wish to express my heart full thank to my internal supervisor

Mr. Charninda Munasinghe, for assisting and guiding me towards a successful

completion of the project.

Special thank is extended to Dr. Tissa Amarakoon - Head of Biochemistry,

Tea Research Institute, Sri Lanka, for his valuable contribution made, Mr.

Mohan Lokuge and all the staff members at laboratory and Mr. Buddhi

Somaweera in the Quality Assurance department Unilever Sri Lanka Ltd., who

assisted me in numerous ways and means in completion of this report.

5

ABSTRACT

Tea in general is considered as a health beverage, although varying claims are

still being made concerning the health benefits of tea.

Sri Lanka is the third largest exporter of tea, with the widest variety offered to

international customers. Japan is a larger importer of Ceylon teas with a

percentage share of 5.94, with a consumption of 130,000 metric tonnes to

2,188,000 metric tonnes of black tea in total in 1993. Majority of its

customers focus on the health benefits provided by same, claims that the levels

of tannin content in Ceylon teas is having a declining trend with time since of

late. They being the customer, are interested in understanding the probable

cause(s) for same.

They are also concerned on how the Tannin content varies in regional specific

Ceylon black tea and also in understanding the factors contributing to the said

variation such as the Agro climatic conditions, soil conditions etc...

Therefore, they proposed an analysis to be carried out in order to verify same.

Certain concerns could be noted in this study, such as the differences in the

methodologies adopted in analysing the tannin contents, which could most

probably be the cause for varying results and also the analysis method below

adopted by the Tea Research Institute has analysed for total polyphenols and

catechines and the method adopted in this study has focussed only on tannins.

1-lowever, owing to Unilever safety guidelines, the methanol extraction of tea

could not be carried out. 1-lence, had to carryout only the hot water extraction

method.

Method used by the Tea Research Institute :- ISO method for total polyphenols - ISO 14502-1 (2005) is as follows:

Procedure

Tea sample has to be grinded.

Extraction into Methanol/water (70%)

Folin-Ciocalteu reagent added.

Spectroscopic meaurement at 765 nm.

Standard graph is prepared using gallic acid as standard.

Analysis results for Total polyphenols and individual catechins according to the said method.

Region % of Total

Polyphenols

% of Total catecliins

NuwaraEliya/

Udapussellawa

18.47 6.41

Western Region 15.06 2.12

Uva 13.82 1.9

Mid Country 18.34 3.68

Low Country 19.69 4.59

Some interesting observations risen from the results could he seen in

experiment carried out are as follows:-

There is no direct co-relation of temperature to the variation in tannin

content in any of the agro climatic subdivisions chosen for the study.

There has been a positive co-relation with the rain fall in the 04 month

period in two regions.

viz.:- Uva region and Nuwaraeliya / Udapussellawa region

7

There has been a negative co-relation with the rain fall in the 04 month

period in the other three regions,

viz.:- Mid grown, Western region and Low grown region.

These are further discussed under 'RESULTS AND DISCUSION' below

CHAPTER 01

1. INTRODUCTION

Tea is the most popular beverage in the world, second only to water. Tea

(Ccimellici sinensis) (L). 0. Kuntze is considered to be the most popular non

alcoholic beverage next to water. What are plucked for processing are the

tender leaves and the bud of the tea plant, which is referred to as the flush.

What are Tea Tannins?

Tannins are a group of very astringent compounds which bind themselves to

protein. Tannins also combine with enzymes because these are proteins. The

tanning of enzymes leads to inactivation or reduction of enzyme activity

I.I. HISTORY OF TEA

Tea has been discovered by the Chinese emperor Shen Nung where he claimed

that it was able to detoxify 72 kinds of poison (Teranishi et al., 1995)

Tea became very popular in both east and west and the present tradition of the

afternoon tea established particularly as a British habit. Cultivation of tea was

introduced to several other countries, with the growing popularity of tea.

1.2. HISTORY OF TEA ILANT

Tea plant was classified as Thea sinensis in 1753, in the binomial system

originated by Linnaeus. Many synonyms have bee given, but now it is

generally accepted that the tea plant is classified in the family Theaceae and in

Camel/ia species.

Commercially cultivated tea consist of natural hybrids of 03 main types. Viz-

Camel/ia sinensis var sinensis for small leaves China plants.

1.3. AGRICULTURE OF TEA

Tea grows best in tropical and sub-tropical areas where adequate rain fall of

approximately 2,000 mm, good drainage and slightly acidic soils prevail. In

hot tropical areas quality is improved by planting at higher altitudes as

practiced in Sri Lanka, where tea cultivation is done at elevations upto 2.000

meters.

10

1.4. MANUFACTURING PROCESSOF BLACK TEA

The Tea Plant Degree 'fermentation'

Plucking

The tea flush

"White" tea

Withering

ng (green tea)

Withered Flush

Rolling Firing

"Yellow" tea (ren tea)

Dhool

'Fermentation'

"Red" tea

Fermented Dhool

Black Tea

11

When oolong and black teas are to be produced, the fresh leaves are allowed

to be produced, the fresh leaves are allowed to wither until the moisture

content of the leaves is reduced to 55% to 72% of the leaf weight.

1.4.1 Withering

Harvested teas are handled in a manner to prevent bruising and to promote

dissipation of heat generated during continued respiration, The moisture

content of tea is reduced from 75% to 55% approximately, by a flow of cool

air.

1.1.2 Rolling

Rolling helps to establish proper conditions for enzymatic oxidation of the

flavanols, by atmospheric oxygen. Rolling is accomplished by disruption of

the cell structure which facilitate enzyme - substrate contact. The leaf mass is

also maintained in a physical state

1.4.3 Fermentation (Oxidation)

During this process the green leaf is converted to black tea. Although this is

referred to as fermentation. it became recognised around 1901 as an oxidation

process initiated by the tea enzymes. This process actually starts at the onset of

maceration and it allowed continuing under ambient conditions. Temperature

of the leaf is maintained around 25 - 30 C.

1.4.4 Firing

At this stage of processing, fermenting enzymes are deactivated by passing hot

forced air over the tea leaves. Many organochemical processes are accelerated

during this period, as are the enzymatic reactions before thermal inactivation.

12

Firing also reduces the moisture level to 2% - 3%. This is critical as

incomplete inactivation can cause accelerated deterioration during storage.

1.4.5 Sorting

The dried teas are sorted into different grades, by passing it over a series of

vibrating screens of different mesh sizes. Electro statically charged rollers

preferentially attract and remove fibre and stalks in the processed black tea.

1.5 OBJECTIVES OF THE STUDY:

To study the variation of Tannin content in regional specific Ceylon

black tea.

To understand the factors contributing to the said variation such as the

Agro climatic conditions, soil conditions etc...

To recommend tea drinking consumer segments, the means of

extracting the maximum tannin content to the brew, in order to obtain

maximum possible health benefits.

CHAPTER 02

2. LITERATURE REVIEW

2.1 CHEMISTRY & BIOCHEMISTR OF TEA

Tea is the subject of many biochemical investigations as it produces a number

of unique natural products. in contrast to the isolation and quantification of the

Tea flush, consisting of the terminal bud and two adjacent leaves of the tea

plant

Tea is rich in high amounts of anti oxidant flavonoids, which help clean

harmful compounds from the blood.

2.2 POLYPHENOLS IN TEA:

The term polyphenol is an inclusive descriptor referring to the millions of

natural and synthetic aromatic molecules that are substituted with multiple

hydroxyl groups. Polyphenols of Black tea are poorly understood but

seemingly well defined group of compounds. These are principally responsible

for the colour and astringency and partially responsible for flavour. The most

abundant class of chemical compounds in fresh tea flush is the polephenols.

(made up of many phenol molecules) The phenol molecule has a single

hydroxyl group attached to an aromatic ring; the polyphenols have a mumber

of these - UI-I groprs. Of the polyphenolic categories, the flavonoids are the

most abundant in tea flush. Flavonoids are 2- phenyl benzopyran based

compounds, classified into six groups:

Flavones.

F lavaii ones

Isofiavones.

Flavanols (including catechines or flavan - 3- ols)

Flavonols

Anthocyanidins

Flavanols and Flavonols are the main components in fresh leaf. Catechines are

the major flavanols in tea. EGCG, EGC, ECG. EC,GC and C and they undergo

oxidative dimerisationn or polyrnerisation during black tea manufacture.

14

Flavonols (such as quercetin, kaempferol, myricetin and their glycosides) and

anthocyanidins are also found in the flush in appreciable amounts, but they do

not undergo much change during black tea manufacture The tea leaves contain

two enzymes, plephenol oxidase and peroxidase, which are involved I the

oxidation of polephenols.

These may be subdivided by several chemical backbone structures comprising

of: -

Sinple lea polyphenols

Gallic Acid and its quinic acid ester (or depside, as quinic acid esters are

commonly referred). theogallin, have been identified in tea.

Other polyphenols Flavones and their glycosides such as Apigenin have been

detected in tea but represent a very small fraction of the polypheenols present.

2.2.1 Tannins:-

Although it is commonly stated that there are no tannins (meaning

Hydrolyzable tannins such as pentagalloylglucose ) in tea, this statement is not

strictly true. In addition to the gallic acid esters of the catechins and their

oxidation products (which can be hydrolyzed to produce gallic acid readily

and precipitate proteins), there is also a small quantity of hydrolysable tannin

(Nonaka et al.. 1984; Yoshida et.al 1990; Hatano et.al., 1994.) The unique

hydrolysabie tannins in tea are typically "hybrid" tannins such as

camelliatannin A. which is a galloylglucose derivative with pendant catechines.

The tailic acid derivatives common to gall- nuts and tree hark are not present

in significant quantities in tea infusions.

2.2.2 Catechins:

Of the total catechines consumed by humans, a large percentage passes out

unchanged in the faeces (Hara, 1997) and of that absorbed into the body,

approximately 60% are excreted in the urine and the rest in the bile (Brown

15

and Griffiths. 198 1) Catechines are absorbed into the blood in the portal vein

of humans (1-lara, 1 997) and the rats (Okushio et al., 1996)

2.3 Composition of a Typical Tea Beverage, %wt/wt Solids

Green Tea Black Tea

Catechins 30% 9%

Theaflavins 4% Simple Polephenols 2% 3%

Flavonols 2% 1%

Other Polephenols 6% 23% Theanine 3% 3%

Aminoacids 3% 3%

Peptides/Proteins 6% 6% Organic Acids 2% 2%

Sugars 7% 7%

Other Carbohydrates 4% 4%

Lipids

Caffeine 3% 3% Other methylxanthines <1% <1%

Potassium 5% 5% Other minerals/Ash 5% 5%

Aroma Trace Trace

2.4 Physiological Effects of Tea:

This is mainly because epidemiological studies carried out I different parts of

the world are difficult to compare, as the chemical composition of green and

black teas are vastly different. In addition, the manner of consumption and

beverage temperature may have some impact on the pharmacology of tea.

A beneficial effect of tea is the caffeine in tea, that is essential for the

characteristic good taste of tea. Caffeine gets readily bonded to some of the

oxidized polyphenols and as a consequence,

2.5 Polyplienols as Antioxidents

Tea contains an infusion of the leaves from the Camel/ia sinensis plant rich in

polyphenolic compounds known as catechins, the most abundant of which is

(-)-EGCG. Although tea has been consumed for centuries, it has only recently

been studied extensively as a health-promoting beverage that may act to

16

prevent a number of chronic diseases and cancers. Research has progressed

extensively on the chemical and biological properties and functionality of tea

polyphenols. Characterization of the antioxidant properties of tea fiavonoids is

a prime example of the newer research initiatives. The antioxidant activity of

tea extracts and tea polyphenols has been studied in a variety of model

systems. It is clear that the polephenols from green and black tea are effective

antioxidants when evaluated in both chemically and biochemically based test

systems. A major challenge of cancer prevention is to integrate new molecular

findings into clinical practice. Therefore, identification of more molecular

targets and biomarkers for tea polyphenols is essential for improving the

design of tea trials and will greatly assist in a better understanding of the

mechanisms underlying its anti-cancer activity.

2.6 Sri Lankan Tea industry:

Tea sector in Sri Lanka has always been a vital sector in national economy. It

is also the country's largest employment provider, proving employment

directly and indirectly to over one million of people. Tea also contributes

significant amount to the Sri Lanka government revenue and also to the Gross

Domestic Product (GDP)

Ceylon tea from Sri Lanka, acclaimed as the best tea in the world has its

inherent characteristics and reputation for more than a century. The influence

of climatic conditions of its plantation imparts to the product a variety of

flavours and aromas synonymous with quality.

Sri Lanka, as the 3Id tea producing country globally has a production share of

9% in the international market sphere and has around 19% share of the global

demand.

Sri Lanka produces tea throughout the year and the growing areas are mostly

concentrated in the central highlands and southern inland areas of the island.

17

2.7 Different regions where teas are grown in Sri Lanka and sub districts

Uva Region:-

Grown 3,000 to 5,000 feet up, on the eastern slopes of Sri Lanka's central

mountains. Teas from UVA districts have quite a distinct flavour and

pungency and are widely used in many blends.

UVA teas can be enjoyed as a self drinking tea, especially during UVA quality

season, from July to September each year.

Uva subdistricts:

Malwatta/Welimada

Demodera / Haliela/Badulla

Passara/Lunugala

Madulsima

Nuwaraeliya/Udapussellawa Region:-

Nuwaraeliya is an oval shapen mountain valley, the plateau being 6,240 feet

above sea level.

Nuwaraeliya offers a combination of attractions such as:-

- Healing climate

- Scenic beauty

- Wooded wilderness

- Flowery meadows

- I-ugh plateau

Produces tea with unique flavour. Tea when brewed is light, but has an

exquisite flavour and aroma. It has truly being said that NuwaraEliya to

Ceylon tea what champagne is to French wine.

18

Teas grown in Medium region:

Tea plantations around Kandy, the ancient capital of Ceylon, supply what are

known as Mid-country teas. These are full bodied strong teas, which appeal to

everyone, who like a good thick coloury brew. Tea plantations are located

2,000 feet to 4.000 feet where tea is first grown in Sri Lanka.

Main Sub Districts:-

Matale

Pussellawa/I-Iewaheta

Low grown Teas

Southern part of the country has an exclusive condition in soil which gives

blackness to the leaves as well as strength and character in the cup. The unique

feature of these teas, grown from sea level to around 2,000 feet are their

appearance and special taste. The stylish range of whole leaf teas are enhanced

by attractive golden and silver tips produced from a range of tea hushes that

thrive in fertile soils and warm conditions, which are ideal for those who like a

thick, sweet brew or without milk.

Main Sub Districts:-

- Ratnapura/Balangoda

- Deniyaya

- Galle

- Matara

19

ClIAPTER 03

3.1 Experimentatal / Methodology

3.1.1 Location

Experiment was carried out at Unilever Sri Lanka Ltd. Laboratory.

3.1.2 Sampling Methodology:

Following garden marks of each of the agro climatic subdivision is sampled

and analysed, each sample in dupliates, during the four month period from

March to June 2008

Region/Agroclimatic Sub-Division

Garden Mark sampled

lJva Region

Craig

S arnia

Canavarella

Dambatenne

El teb

Low Grown Region

Lumbini

Pothatuwa

S uduwelipothahena

Kal ubowitiyana

S uni Is

NuwaraEliya Udapussellawa Region

Pedro

Inverness

Alma Gonapitiya

Liddesdale

Highforest

Medium Region

Craighead

Coolbawn

Kenilworth

Doombagastalawa

Hellebodde

Westerns

Brunswick

C am pion

Bogawantalawa

Stonycliff

Adisham

20

Apparatus and Reagents

Spectrophotorneter.

Home Key

Oi

000 000

21

25 nil Measuring Flask

Pipetter

Tip for micro pipet

Tartaric acid iron reagent

3.2 Methodology used:

UV spectrophotometric analyses.

Analyses Method: (iron / tartaric acid reaction)

1.Measure 100mg of Iron(II) Sulfate Heptahydrate (FeS0471-120 FW

278.02) and 5 00mg of Potassium Sodium (+)-Tartrate Tetrahydrate

(C4H4KNaO4H2O FW :282.22) in a 100 ml measuring flask.

2.Make up to volume with distilled water in a lOOml measuring flask.

Phosphoric acid buffer (SORENSEN Buffer):

Dissolve 5.93g of Sodium hydrogen phosphate dihydrate (Na2H PO4

2H20 FW : 177.99) with 500rn1 of distilled water in a 1000rnl beaker.

(A)

Dissolve 2.26- of Potassium Dihydrogenphosphate (KI-l2PO4 FW

163.09) with 250m1 of distilled water in a SOOml beaker. (B)

Adjust pH to 7.5 as mix little by little B to A

Cold storage after remove to special beaker.

22

3.2.1 Preparation of Tea extract:

Weigh 5g of tea blend into a 300m1 beaker.

Add 250m1 of water at a temperature of 90-95 deg centigrade into the beaker. Keep for 6 minutes covered with a watch a glass.

Stir for lOsecs, each 5 times in clockwise and counter clockwise direction with a spoon and keep for 3 minutes.

Filter the extract through a mesh. (Aperture size 0.125 - 0.150mm)

Cool the sample to 25°C-35°C with rulming water.

Adjust the volume of the tea extract to 250m1 in a volumetric flask.

Take 25m1 of the extract to prepare the final solution in a 250m1 volumetric flask.

Take 5m1 of the above solution.

3.3 Measurement of sample

1 Room temperature storage the regents in advance.

2 Sample dilution

2.1 Extract

2.1 .1 Diluted to beverage base.

2.1.2 TakeS mL of sample in a25 ml Measuring Flask.*

2.2 Beverage

2.2.1 Take 5 mL of sample in a 25 ml Measuring Flask.*

3 Take S mL of diluted sample* in a 25 ml Measuring Flask.

4 Add 51n1 of Tartaric acid iron reagent in a 25 ml Measuring Flask.

5 Make up to volume with Phosphoric acid buffer to 25 ml

6 Put a lid on a 25 ml Measuring Flask.

7 Give a good shake for coloring.

8 Measure by the UV absorbance at 540nm. (This must be 1 .000>)

23

9 Read absorbance at 540nm against a pure blank (substitute 51-n1 of

distilled water for sample)

Tannin Content (mg%) = 52 x Abs. (540 nm) - 0.48

Note:- AFTER USE THE EQUIPMENTS MUST BE THOROUGHLY

CLEANED TO REMOVE ALL DR[NK DEPOSITS AND CHEMICALS.

Source:- (Pepsi/Lipton), Method of analysing Tea tannins

3.4 Preparation of standard solution series

By taking lml,2m1,3m1,4m1,5m1 from the above stock solution and making

the volume upto lOOmI the standard solution

series 5mg/I OOml, I 0mg/i OOmJ,l 5mg/i OOml,2Omg/1 OOmI,25rng/ 1 OOml can be

prepared respectively.

From each of these standard solution series 5m1 is taken and diluted upto

25ni1.(5m1 sample or standard + tartaric + phosphoric)

24

3.5 Calculation of milligrams of polyphenols present in the standard

solution series

No. of grams of tannin(polyphenols)present in each of the ethly gallic acid

standard solution series is;

5mg/100rn1. 5mg/lOOrni x 5/25rn1 = img/lOOrni

10mg/i 00m1, 10mg/1 OOmix5/25rn1 = 2mg/I OOmi

c)lSmg/iOOmi, 15rng/100mlx5/25rn1 = 3mg/I00rni

d)20mg/100m1, 20mg/100mlx5/25m1 = 4mg/100mi

e)25mg/1 OOmi, 25mg/1 OOmlxS/25rn1 = 5mg/1 OOml

3.6 Calculation of the Dilution of the tea extract

5g of tea/250m1 water x 25m1/250m1 water x 5/25m1 (sample -F tartaric +

phosphoric buffer)

Therefore, the number of mg of tea present in the final 25 ml = 0.01g(lOmg)

Which can be written as 0.0Ig/25m1(lOmg/25m1)

For ease of calculation we multiply both the top and bottom values by 4 which

gives 0.04g/I00ml (40mg/100ml)

25

3.7 Relating the tea extract absorption values with the standard series

The equation for the standard curve is y = 0.1 151x - 0.0241

For example when you take the average absorption value of B738,339 189 i.e

0.2175 and replaced withy the value is 1.6802mg of polyphenols

The tannin percentage is expressed as a percentage to the final mg of tea

present in the tea extract ie:40mg

Therefore The Tannin % is:

1.6802mg/40mg x 1.5 x 100 = 6.30075

4.0 RESULTS AND DISCUSION:

Results of the variation in tannin content in the five regions with rainfall and temperature over the four month period analysed, is summarised in the table be low.

Region ______________ Month _________

MacI: April May Jui,e

Uva

Rainfall/mm 290.25 136 43.8 63.1

Temperature/°C 20.9 21.5 21.4 21.2

AverageTannin 9.3 6.30 6.30 8.52

Low Grown Rainfall/mm 366.5 293.5 205.3 155.9

Temperature/°C 27.7 27.6 27.1 26.7

AverageTannin % 7.3 8.9 7.62 7.13

NuwaraEliya Udapussellawa

Rainfall/mm 158.4 175.9 171.9 164.9

Temperature/°C 17.1 17.1 16.1 15.7

AverageTannin % 5.96 7.23 7.15 6.03

Medium Region (Mid country)

Rainfall/mm 182.85 197.15 217.75 224.55

Temperature/°C 26.1 25.6 24.8 24.5

AverageTannin 11.17 10.21 8.72 8.09

Western Region

Rainfall/mm 237.1 335.3 504.65 444.3

Temperature/°C 17.1 17.1 16.1 15.7

A ye rage Tannin % 10.56 10.01 9.64 6.99

26

4.1 ANALYSES AND INTERPRETATION OF RESULTS

Over the four month period the research was carried out, the following trends

in the agro-clirnatic conditions and variation in the tannin content could he

observed.

Uva Region.

Rainfall has been on the decline over the said period except from May to June

where there has been a slight increase. Temperature on the other hand has

been more or less static during the period of analyses with 210C on average.

Variation in the tannin content was from 9.3% to 8.52%, with 6.3% recorded

both in the months of April and May.

Lou Grown Region:

As in the Uva region, trend of rainfall has been on the declining over the said

period except from May to June, where there has been a slight increase in

rainfall. Temperature on the other hand has been more or less static during the

period of analyses, average temperature being 270C. Variation in the tannin

content was a slight reduction from 7.3% to 7.13%, with an increase to 8.9%

in April and a reduction to 7.62% in May and a further reduction to 7.62 in

June.

iVuwaraEliya Udapussellat'a Region:

Rainfall has been on the decline over the said period except from March to

April, where there has been a slight increase on average of 170mm.

Temperature on the other hand has been more or less static during the period

of analyses, average temperature being 170C. with a slight reduction to 15.7°C

in the month of June. Variation in the tannin content was a slight reduction

from 5.96 to 7.23%, with a fluctuation, slightly increasing and decreasing over

the four month analyses period.

27

Medium Region:

Rainfall has been on the increase over the said period except from May to June

from 182.85 to 224.55mm, while the temperature has been more or less

constant with a gradually slight reduction from 26.1 to 24.5°C.

Variation in the tannin content was a slight reduction from 11 .7% to 7.09%,

with decreasing over the four month analyses period..

Western Region.

Rainfall has been on the decline over the said period except from May to June

where there has been a slight increase.237.lmm to 504.65mm, with the

highest rainfall in May. Temperature has varied slightly from 15.70C to 17.1°C

Variation in the tannin content was a slight reduction from 6.99% to 10.56%,

with a significant reduction from May to June.

UVA teas:

Variation of Tannin content in teas in UVA region with Rainfall

I..

I. Mach April May June

Months

-•-- RainfalVrrvri —w-- Tannin %

28

Variation of Tannin content in teas in the UVA region with temperature

100

10 a

1 Mach April May June

Months

Temperature/OC a Tannin %

Low Grown teas

Variation of Tannins in Low Grown teas with temperature

100 -

U,

> I .- 0 -J

Mach April May June

Month

-+-- Temperature/OC ---- Tannin %

29

WE

1

Variation of Tannin content in Low Grown teas with Rainfall

1000

100

10

Mach April May June

Months

RainfalVrrrn —e— Tannin %

NuwaracLiya /IJdapussellawa:

Variation of Tannin content of teas in in Nuwarael

/UdaDussellawa Reaion with rainfall

Mach April May June

Months

Rainfall/rnni —a-- Tannin %

30

Variation of Tannin content in

NuwaraEliya/Udapussellawa Region with tern perature

------------

1

Mach April May

June

Months

Temperature/0C---Tannin%

Medium Grown teas:

Variation in Tannin content in Medium teas with rainfall

1000 ............................................................................................................................................

G) 100

>

10

Mach April May June

Month

—s-- Rainfall/mm -- Tannin %

n 31

Variatio of Tannin content in Medium teas with temperature

100 ....-.--....,.,.-.,--.--.----

w

10 -

Mach April April May June

Month

Terr'perature/OC _.—TanninJ

Teas grown in Western Region:

Variation of Tannin content in teas grown in the western region with rainfall

1000 . - --.

100 C,

C,

.3 10 .---.-----.

Mach April May June

Month

Rainfall/mm ---- Tannin %

32

Variation of Tannin content in teas grown in the western region with

temperature

100

.2 6 10 ---------- -.

0 -J

Mach April May June

Month

—.--Temperature/OC --•-----Tannin

By referring the variation of Tannin content with rainfall and temperature in

each agro climatic sub-division ca be summarised as follows:-

In the Uva region there seems to be a positive relationship existing with the

rain fall in the 04 month period for which the analysis was carried out.

However, there does not seem to be a direct relationship existing, with the

temperature in this time period.

There seems to be a negative relationship existing with the rain fall over the 04

month period in the Low Grown region.

In this region as in the Uva region, there does not seem to be a direct

relationship existing, with the temperature in the aforesaid 04 month period.

In the Nuwaraeliya/Udapussellawa region, there seems to be a positive

relationship existing with the rain fall in the 04 month period.

Like in the previous regions, there does not seem to be a direct relationship

existing, with the temperature in the aforesaid 04 month period.

In the Medium region too. there seems to be a negative relationship existing

with the rain fall in the 04 month period.

3.)

As in the case with other regions, there does not seem to be a direct

relationship existing, with the temperature in the aforesaid 04 month period.

In the Western region too, there seems to be a negative relationship existing

with the rain fall in the 04 month period, where the analyses was carried out.

However, there does not seem to be a direct relationship existing, with the

temperature in the aforesaid 04 month period.

There is no direct co-relation of temperature to the variation in tannin content

in any of the agro climatic subdivisions chosen for the study.

4.2 STATISTICAL ANALYSES OF RESULTS USING MINITAB FRIEDMANN TEST:

UVA PROVINCE

I. Regression Analysis: UVA Tannin versus UVA Temperature, Uva RF I

The regression equation is UVA Tannin = 140 - 6.22 UVA Temperature - 0.00226 UVA RF 1

Predictor Coef SE Coef T P Constant 140.10 53.32 2.63 0.232 UVA Temperature -6.221 2.483 -2.51 0.242 UVA RF 1 -0.002258 0.005869 -0.38 0.766

S = 0.786698 R-Sq = 91.3% R-Sq(adj) = 74.0%

Analysis of Variance

Source DF SS MS F P Regression 2 6.5158 3.2579 5.26 0.295 Residual Error 1 0.6189 0.6189 Total 3 7.1347

Source DF Seq SS UVA Temperature 1 6.4242 UVA RF 1 1 0.0916

34

Regression Analysis: UVA Tannin versus UVA Temperature

The regression equation is UVA Tannin = 125 - 5.53 UVA Temperature

Predictor Coef SE Coef T P Constant 125.14 27.64 4.53 0.045 UVA Temperature -5.531 1.301 -4.25 0.051

S = 0.596018 R-Sq = 90.0% R-Sq(adj) = 85.1%



Regression Analysis: UVA Tannin versus UVA Rain Fall

The regression equation is UVA Tannin = 6.49 + 0.00837 UVA RF 1

Predictor Coef SE Coef T P Constant 6.492 1.276 5.09 0.037 UVA RF 1 0.008366 0.007742 1.08 0.393

S = 1.50080 R-Sq = 36.9% R-Sq(adj) = 5.3%



Low Grown 1. Regression Analysis: Low Grown Ta versus Low Grown RF, Low Grown Tea

15

The regression equation is Low Grown Tannin = - 144 - 0.0254 Low Grown RF + 5.79 Low Grown Temperature

Predictor Coef SE Coef T P Constant -143.69 60.61 -2.37 0.254 Low Grown RF -0.02538 0.01156 -2.19 0.272 Low Grown Temperature 5.789 2.326 2.49 0.243

S = 0.497309 R-Sq = 87.2% R-Sq(adj) = 61.5%



Source DF Seq SS Low Grown RF 1 0.1468 Low Grown Temperature 1 1.5316

2.Regrcssion Analysis: Low Grown Tannin versus Low Grown Rain Fall

The regression equation is Low Grown Tannin = 7.13 ± 0.00237 Low Grown RF

Predictor Coef SE Coef T P Constant 7.133 1.560 4.57 0.045 Low Grown RF 0.002367 0.005826 0.41 0.724

S 0.943 107 R-Sq = 7.6% R-Sq(adj) = 0.0%



36

3. Regression Analysis: Low Grown Tannin versus Low Grown Temperature

The regression equation is Low Grown Tannin = - 15.9 + 0.87 Low Grown Temperature

Predictor Coef SE Coef T P Constant -15.93 28.75 -0.55 0.635 Low Grown Temperature 0.868 1.054 0.82 0.497

S = 0.848033 R-Sq = 25.3% R-Sq(adj) = 0.0%


Source DF SS MS F P Regression 1 0.4874 0.4874 0.68 0.497 ResidualError 2 1.4383 0.7192 Total 3 1.9257

NUWARAELIYA I UDUPUSSELLAWA

I. Regression Analysis: NEUP Tannin versus NEUP Temperature, NEUP R F

The regression equation is NEUP Tannin = - 10.1 + 0.157 NEUP Temperature + 0.0839 NEUP RF

Predictor Coef SE Coef T P Constant -10.079 6.790 -1.48 0.377 NEUP Temperature 0.1568 0.2994 0.52 0.693 NEUP RF 0.08395 0.02757 3.04 0.202

S = 0.369080 R-Sq = 90.5% R-Sq(adj) = 7 1.5%



37

Source DF Seq SS NEUP Temperature 1 0.0348 NEUP RF 1 1.2627

Regression Analysis: NEUP Tannin versus NEUP Temperature

The regression equation is NEUP Tannin = 4.1 + 0.151 NEUP Temperature

Predictor Coef SE Coef T P Constant 4.10 11.20 0.37 0.750 NEUP Temperature 0.1513 0.6783 0.22 0.844

S = 0.836323 R-Sq = 2.4% R-Sq(adj) = 0.0%


Source DF SS MS F P Regression 1 0.0348 0.0348 0.05 0.844 Residual Error 2 1,3989 0.6994 Total 3 1.4337

Regression Analysis: NE/UP Tannin versus NE/UP Rain Fall

The regression equation is NEUP Tannin = - 7.48 + 0.0839 NEUP RF

Predictor Coef SE Coef T P Constant -7.477 3.696 -2.02 0.180 NEUP RF 0.08386 0.02201 3.81 0.062

S = 0.294626 R-Sq = 87.9% R-Sq(adj) = 8 1.8%



38

Medium

Regression Analysis: Medium Tannin versus Medium Temperature, Medium RF

The regression equation is Medium Tannin = - 54.8 + 2.40 Medium Temperature + 0.0 187 Medium RF

Predictor Coef SE Coef I P Constant -54.83 26.06 -2.10 0.282 Medium Temperature 2.3975 0.7868 3.05 0.202 Medium RF- 0.01867 0.03016 0.62 0.647

S 0.03490 15 R-Sq = 100.0% R-Sq(adj) = 99.9%



Source DF Seq SS Medium Temperature 1 5.8788 Medium RF 1 0.0005

Regression Analysis: Medium Tannin versus Medium Temperature

The regression equation is Medium Tannin = - 38.7 + 1.91 Medium Temperature

Predictor Coef SE Coef T P Constant -38.7020 0.5778 -66.99 0.000 Medium Temperature 1.91087 0.02287 83.54 0.000

S = 0.0290240 R-Sq 100.0% R-Sq(adj) = 100.0%



39

3. Regression Analysis: Medium Tannin versus Medium Rain Fall

The regression equation is Medium Tannin = 24.6 - 0.0732 Medium RF

Predictor Coef SE Coef I P Constant 24.5917 0.4932 49.86 0.000 Medium RF -0.073181 0.002391 -30.60 0.001

S = 0.0791524 R-Sq = 99.8% R-Sq(adj) = 99.7%


Source DF SS MS F P Regression 1 5,8679 5.8679 936.61 0.001 Residual Error 2 0.0125 0.0063 Total 3 5.8805

Western

1. Regression Analysis: Western Tannin versus Western Temperature, Western Rain Fall

The regression equation is Western Tannin = - 42.2 + 2.95 Western Temperature + 0.0074 Western RF

Predictor Coef SE Coef T P Constant -42.16 32.57 -1.29 0.419 Western Temperature 2.948 1.763 1.67 0.343 Western RF 0.00743 0.01059 0.70 0.611

S = 1.16057 R-Sq = 82.1% R-Sq(adj) = 46.2%



Source DF Seq SS Western Temperature 1 5.502 Western RF 1 0.662

Regression Analysis: Western Tannin versus Western Temperature

The regression equation is Western Tannin = - 22.1 + 1.90 Western Temperature

Predictor Coef SE Coef T P Constant -22.09 13.42 -1.65 0.242 Western Temperature 1.9026 0.8129 2.34 0.144

S = 1.00227 R-Sq = 73.3% R-Sq(adj) = 59.9%



Regression Analysis: Western Tannin versus Western Rain Fall

ftc regression equation is Western Tannin = 12.2 - 0.00755 Western RF

Predictor Coef SE Coef T P Constant 12.176 3.070 3.97 0.058 Western RF -0.007549 0.007793 -0.97 0.435

S=l.59885 R-Sq31.9% R-Sq(adj)0.0%



41

4.3 Significance of Relationship between the variables and predictors:

Agro climatic Variables F value Table F value Existence of a Subdivision Reading relationship

between the variable and predictor

Temperature 526 5.0503 No va

Rainfall 1.17 5.0503 No

Temperature 0.68 5.0503 No Low Grown

Rainfall 0.17 5.0503 No

NuwaraEliya Temperature 0.05 5.0503 No

Rainfall 14.52 5.0503 Yes /Udapussellawa Temperature 6978.69 5.0503 Yes

Mediums Rainfall 936.61 5.0503 Yes

Temperature 5.48 5.0503 Yes Westerns

Rainfall 0.94 5,0503 No

42

5. CONCLUSION AND RECOMMENDATIONS

It could be concluded that further analyses and investigations are required to

be carried out in order to conclude of a relationship of the probable factors

identified to be contributing to the lower tannin content in made black Ceylon

tea, as the results herein are insufficient to come to such a conclusion.

A possible reason for the tannin content in Sri Lankan teas to have come down

as highlighted by customers is the poor leaf plucking standards practiced by

the tea pluckers, by plucking more than 02 leaves and a bud which has been

the norm in the industry. Another possible factor affecting the tea tannin

content is Soil organic carbon content

Soil organic carbon content in up country should be above 3% and in low

country 2%

Two leaves and one bud

41,

As mentioned above, tea pole

phenols/tannins have shown to be

health benefits. As a result, customers who are mostly in the health conscious

segment could be advised to adhere to the following procedure, in order to

extracting the maximum tannin content to the brew, thereby to have its derived

health benefits.

I. Weigh 51, of tea blend into a 300rnl cup.

Add 250rnl of water at a temperature of 90-95 deg centigrade into the

cup. Keep for 6 minutes covered.

Stir for lOsecs. each 5 times in clockwise and counter clockwise direction

with a spoon and keep for 3 minutes.

Filter the extract through a mesh. (Aperture size 0.125 - 0.150mm)

REFERENCES:

Matthew E. Harowy and Douglas A. Balentine (1997), Tea Chemistry

Wickremasinghe, R.L. Monographs on Tea production in Sri Lanka - No. 7 Facets of tea research in practice.

Modder W.W.D. and Amarakoon A.M.T. Tea and Health, 2002

Harler, C.R.Culture and Marketing of tea

pat B, Kirthisinghe, D.Ranasinghe, DIP! 1,484,54 Tea Research Institute, September 1997.

http://www.tea.co.uk/healthresearch.php

httj2://www.pureceylontea.com

Retrieving project from file: 'E:\MINITAB.MPJ'

APPENDICES

F Table for a1pha05. 1 F (.05,df1,df2)

I 2 3 4 S 6 7 8 9 10 12 IS 20 24 30 40 60 120 (IF

161 199 215 224 230 233 236 238 240 241 243 245 248 249 250 21 252 23 254.314

4476 .50 .70 58 .16 .98 .76 .88 .54 .88 .90 .94 . .01 .05 .09 .14 .19

00 73 32 19 60 84 27 33 17 1 60 99 31 18 51 32 57

- 19. 19. 19. 19. 9. 19. 19. 19. 19. 19. 19. 19. 19. 19. 19. 19. 1918

2 000 164 246 296 329 353 371 384 395 412 429 445 454 462 470 479 9.4957

0 129

3 8 4 5 2 0 8 9 5 I 8 I 4 7 I 74

101 9.5 9.2 9.1 9.0 8.9 8.8 8.8 8.8 8.7 8.7 8.7 8.6 8.6 8.6 8.5 8.5 8.549 8.5264

280 521 766 172 135 406 867 452 123 855 446 029 602 385 166 944 720 4

7.70 6.9 6.5 6.3 6.2 6.1 6.0 6.0 5.9 5.9 5.9 5.8 5.8 5.7 5.7 57 5.6 5.658 -

86 443 914 882 561 631 942 410 988 644 117 578 025 744 459 70 877 I .628I

660 5.7 5.4 5.1 50 49 48 48 47 47 46 46 4 45 44 44 44 4398 4,j6O

79 861 095 922 503 503 759 183 725 351 777 188 581 272 957 638 314 5

5.98 5.1 4.7 4.5 4.3 4.2 4.2 . 4.1 4.0 4.0 3.9 3.9 3.8 3.8 3.8 3.7 3.7 3.704 6 3668)

433 571 337 874 839 067 468 990 600 999 38! 742 41 (182 74j 198 7

7 5.59 47 43 41 39 38 37 37 36 36 35 1 34 3.4 33 31 31 3267 398

14 374 468 203 715 660 870 257 767 365 747 107 445 105 758 404 043 4

5.31 4.4 4.0 3.8 3.6 3.5 3.5 3.4 3.3 3.3 3.2 3.2 3.1 3.1 3.0 3.0 3.0 2.966 927

77 590 662 379 875 806 005 381 881 472 839 184 503 152 794 428 053 9 -.

5.11 4.2 3.8 3.6 3.4 3.3 3.2 3.2 3.1 3.1 . 3.0 3.0 2.9 2.9 2.8 2.8 2.7 2.747 27067

74 565 625 331 817 738 927 296 789 373 729 061 365 005 637 259 872 5

4.96 4.1 3.7 3.4 3.3 3.2 3.1 3.0 3.0 2.9 2.9 2.8 2.7 2.7 2.6 2.6 2.6 2.581) 2.5379

46 028 083 780 258 172 355 :717 204 782 130 450 740 372 996 609 211 1

4. 3.9 3.5 3.3 3.2 3.0 3.0 2.9 2.8 2.8 2.7 2.7 2.6 2.6 2.5 2.5 2.4 2-118 -

43 823 874 567 ((39 946 123 480 962 536 876 186 464 (190 705 309 9(11 2.404

12 4.

3.8 3.4 3.2 3.1 2.9 2.9 2.8 2.7 2.7 2.6 . 2.6 2.5 2.5 2.4 2.4 2.3 2.341

72 83 901 592 09 961 134 486 964 54 866 169 436 0' 663 259 842 0 2.2962

4. 38 34 31 30 29 28 27 27 26 26 25 24 24 23 23 22 2252 13 2.2064

056 105 791 254 153 321 669 144 710 037 331 589 202 803 392 966 4 72

4. 3.7 3.3 3.1 2.9 2.8 2.7 2.6 2.6 2.6 2.5 2.4 2.3 2.3 2.3 2.2 2.2 2.177 14 60 389 439 122 582 477 642 987 458 022 342 630 879 487 ((82 664 229

8 2.1307

4. IS

3.6 3.2 3.0 2.9 2.7 ' 2.7 2.6 2.5 2.5 2.4 2.4 2.3 2.2 2.2 2.2 2.1 2.1 0658

31 823 874 556 013 905 066 408 876 437 753 034 275 878 468 043 601 I ' -

1( 4.

3.6 3.2 3.0 2.8 2.7 2.6 2.5 2.5 2.4 2.4 2.3 2.2 2.2 2.1 2.1 2 1 2.058

10337

389 069 524 413 572 911 377 935 247 522 756 354 938 507 058 9 _,00)6

3.5 3.! 2.9 2.8 2.6 2.6 2.5 2.4 2.4 2.3 2.3 . 2.2 2.1 2.! 2.1 2.0 2.110

45 915 968 647 100 987 143 480 943 499 807 077 304 898 477 040 584 7 1.9604 17

45

13

4.

18 3.5 3.1 2.9 2.7 2.6 2.5 2.5 2.4 2.4 2.3 2.2 2.1 2.1 2.1 2.0 2.0 1968

I 9168

3 9 546 599 277 729 613 767 102 •563 117 421 686 906 497 071 629 166 I

19 383. 1 2.8 2.7 2.6 2.5 2.4 2.4 2.3 2.3 2.2 2.1 2.1 2.0 2.0 1.9 1,930

1.9780

07 219 274 951 401 283 435 768 227 779 080 341 555 141 712 264 795 2

4. 3.4 3.0 2.8 2.7 2.5 2.5 2.4 2.3 2.3 2.2 2.2 2.1 2.0 2.0 1.9 1.9 1.896 84P 20

35 12 928 984 661 109 990 140 471 928 479 776 03 242 82 391 938 464 -

34 30 28 26 25 24 244. ] 23 23 22 21 20 20 20 (9 19 I86 18117

21 48 668 72 401 848 727 876 205 1660 210 504 757 960 '540 102 64 16 7

22

4. 3.430 28 26 25 ] 24 23 23 22 22 21 20 20 19 19 18 1838

17831

09434 491 167 613 491 638 ]. 965 419 967 258 508 707 283 842 380 894 0

23 4 3.4 3.0 2.7 2.6 2.5 2.4 2.3 2.3 2.2 2.2 2.1 2.0 20 1.9 1.9 1.8 1.812 7570

91 221 280 955 400 277 422 748 201 747 036 282 476 050 605 139 648 8

4.

3.4 3.0 2.7 2.6 2.5 2.4 2.3 2.3 2.2 2.1 2.1 2.0 1.9 1.9 1.8 1.8 1.789 24 028 088 763 207 082 226 551 002 547 834 077 267 838 390 920 424 . 6

97

4.

1.733(1

3.3 2.9 2.7 2.6 2.4 2.4 2.3 . 2.2 2.2 2.1 2.0 2.0 1.9 ] 1.9 1.8 1.8 1.768 17110

25 24 852 912 587 030 904 047 371 821 365 649 889 075 643 192 718 217 4 17

4.

26 33 29 27 25 24 23 23 22 22 21 20 19 19 19 18 18 1748 16906

690 72 426 868 741 883 205 655 197 479 716 898 464 010 533 027 8 52

4.

27 i 33 29 27 25 24 23 23 22 22 21 20 19 19 18 18 17 1730 16717

00 541 604 278 719 591 732 ], 053 501 . 043 323 558 736 299 842 36! 851 6

4. 3.3 2.9 2.7 2.5 2.4 2.3 2.2 2.2 2.1 2.1 2.0 1.9 1.9 1.8 1.8 1.7 1.713 -

28 404 467 141 581 453 593 913 360 900 179 411 586 147 687 203 689 8

4.

1.641

3.3 2.9 2.7 2.5 2.4 2.3 2.2 2.2 ' 2.1 2.1 2.0 1.9 1.9 1.8 1.8 1.7 1.698 29 1 8 277 340 014 454 324 463 783 229 768 045 275 446 005 543 055 537 I

1.6376

303.3 2.9 2.6 2.5 2.4 2.3 2.2 . 2.2 2.1 2.0 2.0 1.9 1.8 1.8 1.7 1.7 1.683

1.6223 (1)9 158 223 896 336 205 343 662 107 646 921 148 317 874 409 918 396 . 5

4.

3.2 2.8 2.6 2.4 2.3 ' 2.2 2.1 2.1 2.0 ' 2.0 1 1.9 1.8 1.7 1.7 1.6 1.6 1.576 40 08 1.5089

387 060 49 359 490 802 240 772 035 245 389 929 444 928 373 647

4.

089

31 27 2 23 2221 2.0 2.0 19 19 18 17 17 16 1.5 I 1467 3893 60 00

(2 504 581 252 683 541 665 970 401 '926 174 364 480 001 491 943 343 3 I.

120 92 30 26 24 22 21 20 20 1.9 19 1.8 17 16 16 1 14 14 1 3 1

01 718 802 472 899 750 868 . 164 588 lOS 337 '505 587 084 543 952 290 9

- 2.9 2.6 2.3 2.2 2.0 2.0 1.9 1.8 1.8 1.7 = 1.6 1.5 .5 1.4 1.3 1.3 1.221

' 957 = 049 719 141 986 096 384 799 307 522 664 705 173 591 940 180 4

1.000))

15

46

analyses of tannins in regional specific ceylon teas...

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