PART

Solid Waste Management

C H A P T E R 2

, ciil Studies oil jh, ^ cnols isolated *'•'•' ^'irtjieric spent: An a^io i/idiistriul ask:

Chapter II Bioanalytical agro industrial waste

J4£stract

Extracts rf tut menc (Cuicuntci lri^£^a) spent \v£r£ pt£p.?>£^^ ix refluKii^tsi th£

tunuenc sv£ptt with £thuiisi.c£t:^t£. \vat£K kK£t/:anri a^^^ c>cetri^£. •"••tyrui^ui-u. a^Hru.yit

cf trli^t/!£i'vrl.s in tun'-u.£nc sp£i'Vt eKtuict (T.SE) was ritaLn£i^ •^'tri'i^- £ttiutac£tat£.

Trtal phenrUc crnt£nts ivete ^£t£miLn£y( USLI/I^I FrUn anal CLrcait£u's i'U£t/:ra(.

AntLCKidant activLtia .rf T^E was assaui£at throu^^h Ln v-Urr mralels such as p££

iadicai sca\/£naHn(r' acti^Lti-i ustnsi Q,:::-a(Lph£i-vud-i'VLCtujihufJiazuiL (DPPH)

methrd, fi-carpt£n£'LLnri£ate yAral£l S!^st£^H ana/ antirxcafant cavacitia bu Lrcn(!/f)

r£ductLrn m£thr;^~. AaldLttrn r^'' in.''^^^ ppkPi rf T:SE tr /£'7n£j sunflrw£y rtl and

strraaie at taring tet'Hp£iatut£ i2Z: ± 3'C) fat S^- a/auis shcw£ai Lr\v£> v£yrKLa(£ valu£

(Pov) (113.9'^ m£q rKuc*£n "iza) and ft££ fattia aad crntent (FFA)

{1.11 m'l kiOH 'a } criMpaied tr cmtirl sampL£s POv iii'i.5^ ^^^^ rKia^£n 'iza>)

and FFA (1.2^ mri KOH d). Riefined Sunflrwer rd crntaLninC* lOO ppm rf

butiaiatid huidirKL'ttrui£n£ (T^HT) and id-l^r pp^A. c^ -a*t££n tea prliat-h£nrl (^iFP)

and id'dd ppku. r-f rrseyvian.1 shrived PO\' r-f s.5.r:-d: ii2.r:^''^ and lO'^.^r: t'nea

rKuir^en 'tzp and FFA crntent r-^dS'^, l.r2, i.de- mr^ i<OH d, tespectL\,elia a-^er SO

daMs rf stnaae at rrry^ ten^perature. Fhe minimui^A cmcenttatims r-^ FSE

teauired fr> LnhLbitirn rf Escherichia crli, staphuitrcrccus auteus. salntrnelLa

tLuvhi, and^alnimeUa entenca '.'.£r£ S'^r. 1200, iiod andi3dd tt,A. tespectivelia.

kteui'-vrt ds: Cuicuipia Irnpa: tui i'U.inz stf^t e^ti .l:t: Antioxidant astivitu; Anr.r^i-i rii^l act:\,itu:

{tnr^-ins^usti'U ivastf

Jrurnal r^FrrdAr'ticultui-e and EniirrnrPi£nt (Crmmunicated)

Chapter II Bioanalytical agro industrial waste

II. 1.Introduction

Turmeric rhizome {Curcuma longa, family Zingiberaceae) is a well-

established foodstuff. It is a main ingredient of curry powders. It is also well known

for its coloring, flavoring, curative and digestive properties. Tiumeric is a well-known

indigenous herbal medicine, which exhibits a wide range of biological activities, for

example, antibacterial [1], anticancer [2], anticoagulant [3], antiinflammatory [4,5],

antimutagenic [5,6], antioxidant [6,7], antiprotozoan [8], antispasmodic [4],

antitumour [9,10], antiviral [5], hepatoprotective [5], hypocholesterolaemic [11],

hypoglycaemic [12], hypolipemic [13], besides being effective oxygen species

scavengers and lipid peroxidase inhibitor [14].

The active components of turmeric is yellow pigment curcumin (0.5 - 6.0%).

The minor constituents are suggested to be geometrical isomers of the major

constituent [15] collectively called curcuminoids, which are extracted for its

nutraceutical and cosmaceutical preparations and the process produces a large portion

of turmeric spent which is considered as waste. As most of the food industries are

localized the waste generated may contribute to environmental pollution. During past

few years, there has been increasing interest in isolating and recovering important

components from food industrial waste to add value-addition to the residues. The

emergence of the waste management for sustainable development by encouraging

controlled exploitation of food industrial waste for economic gains will help to add

value-addition to the turmeric spent.

Turmeric is native of south and south East Asia. Turmeric probably originated

in the slopes of hills in the tropical forest of western ghatts of south India. Turmeric

can be grown in diverse tropical environment from sea level to height of 1500 mts in

hill slopes in temperature ranging from 20 to 30°C. A rainfall of 150 cm/annum or

more in the growing region, or an equivalent amount of irrigation is essential. India

remains the largest producer of turmeric, its average and production exceeding the

total of all other growing countries together [16].

Antioxidants are compounds that help to inhibit many oxidation reactions

caused by free radicals. Free radicals are imstable molecules which include hydrogen

atom, nitric oxide and molecular oxygen. They naturally occur in the body as a result

of chemical reaction during normal cellular processes. Reactive oxygen species

21

Chapter II Bioanalytical agro industrial waste

(ROS) sometimes called active oxygen species are various forms of activated oxygen,

which include free radicals such as superoxide (O2') and hydroxyl radical (OH), as

well as non free radical species such as hydrogen peroxide (H2O2) [17].

Spoilage of food due to the presence of bacterial and fungal infection has been

a major concern for decades and it causes a considerable loss world wide. The

demand for non toxic, natural antibacterials has been rising with increased awareness

[18,19].

The objective of this investigation was to determine the total phenolic content

of the turmeric spent resulting from its extraction and to evaluate the use of

polyphenols as a potential source of natural antioxidant and antimicrobial for

commercial applications.

II.2. Materials and methods

11.2.1. Chemicals

Methanol, ethyl acetate, ethanol, ethyl ether, sulphuric acid, acetic acid,

chloroform, Folin and Ciocalteu's reagent, sodium carbonate were from Ranbaxy,

India; gallic acid, 2,2-diphenyl-l-picrylhydrazyl (DPPH), butylated hydroxytoulene

(BHT), potassium iodide, potassium hydroxide, P-carotene, linolenic acid, Tween 40

were from Sigma Chemicals, USA; potassium ferric cyanide, trichloroacetic acid,

sodium phosphate, ammonium molybdate were from BDH, India; nutrient agar was

from HiMedia, Mumbai. Green tea polyphenols (GTP) and rosemary were from

Flavors and Essence Ltd. Mysore, India. Refined sunflower oil (Sun pure brand) was

procured from the local market.

11.2.2. Instrumentation

Visible spectra measurements were done using Specord 50 UV-Vis

spectrophotometer with 1.0-cm silica quartz matched cell.

11.2.3. Plant material

The residue after exhaustive extraction of oleioresin from turmeric rhizomes

were collected from food industry (Flavors and Essence Pvt. Ltd.,) Mysore, India.

The residue is termed as turmeric spent.

22

Chapter II Bioanalytical agro industrial waste

11.2.4. Sample preparation

The turmeric spent was dried in hot air oven at 60°C to remove moisture and

ground into fine powder. The material that passed through a 40-mesh sieve was

retained for further studies.

77.2.5. Preparation of extract

Fifty grams of turmeric spent prepared as above was transferred to a 1000 ml

round bottom flask, 200 ml of ethylacetate was added and refluxed for 45 min at 60''C

on a water bath. The extract was filtered through filter paper and the residue was

reextracted under the same conditions five times. The filtrate was combined and

evaporated in a rotary evaporator below 60''C. The procedure was repeated using

solvents like acetone, methanol and water. The extract obtained after evaporation of

organic solvents was analyzed for percentage of polyphenols.

77.2.6. Determination of total phenolic content

Phenolic compounds are very important plant constituent because of their

scavenging ability due to their hydroxyl groups. According to the latest report, a

highly positive relationship between total phenolic compounds and antioxidant

activity was found in many plant species [20], Phenolic compounds were associated

with antioxidant activity and play an important role in stabilizing lipid peroxidation.

Thus, first-ever attempt has been made to isolate the phenolic contents of turmeric

spent to provide value-addition to the food industrial residue.

The phenolic components were determined according to the procedure

described by Negi (2003) [21]. Twenty mg of extract were dissolved in 50 ml of 50 %

(v/v) methanol and filtered through Whatman no 44 filter paper. An aliquot of 0.4 ml

of sample was taken in a 25-ml standard flask and mixed with 1 ml of Folin and

Ciocalteu's reagent (1:10 diluted with water) and allowed to react for 5 min, 1 ml of

10 % sodium carbonate was added and allowed to stand for 90 min at room

temperature and the absorbance of the reaction mixture was read at 725 nm. Standard

graph was prepared using gallic acid as reference. The content of total polyphenols in

the extract was calculated firom the standard graph and expressed as percent

concentration in the extract.

23

Chapter 11 Bioanalytical agro industrial waste

II.2.7. Quenching of 2,2-diphenyl-l-picrylhydrazyl (DPPH) radical by TSE

The method described by Kitts [22] was used to assess the DPPH radical

scavenging capacity of TSE. A 0.1 mM DPPH solution in ethanol was mixed with

20, 40, 60, 80, 100, 120, 140 ng/ml of TSE and vortexed thoroughly. The mixtures

were allowed to stand at ambient temperature (22 ± 3°C) for 30 min. The absorbance

was measured at 519 nm using a spectrophotometer. The scavenging percentage was

calculated as per the following equation.

C . 0 / ( A b Control - A b sample ) X 1 0 0

Scavengmg, % = A " Control

II.2.8. Antioxidant assay using fi-carotene-linoleate model system

The antioxidant activity of TSE was evaluated using p-carotene-linoleate

model system as described by Jayaprakasha [23]. P-carotene (0.2 mg) in 0.2 ml of

chloroform, 20 mg of linolenic acid and 200 mg of Tween-40 (polyoxyethylene

sorbitan monopalmitate) were mixed. Chloroform was removed at 40*'C under

vacuum and the resulting mixture was diluted with 10 ml of water and mixed well. To

this 40 ml of oxygenated water was added. Four milliliter aliquots of the emulsion

were pipetted into different test tubes containing 0.2 ml TSE (200, 400, 600, 800 ^g)

and BHT (200, 400, 600, 800 ng) in ethanol. BHT was used for the comparative

study. Control contained 0.2 ml of ethanol and 4 ml of the above emulsion was

prepared. The tubes were placed in 50°C in water bath and the absorbance at 470 rmi

was taken at zero time (t = 0 min). Measurement of absorbance was continued till at

an interval of 15 min the color of P-carotene disappeared in the control tubes (t =120

min). A mixture prepared as above without P-carotene served as blank. All

determinations were carried out in triplicates. The antioxidant activity (AA) of the

extract was evaluated based on the extent of bleaching of the P-carotene using the

formula.

100[1-(A„-A.)] (A°o-A«t)

24

Chapter II Bioanalytical agro industrial waste

Where, Ao and Ao* are the absorbance values measured at zero time of the

incubation for test sample and control, respectively. At and A 't are the absorbance

measured in test sample and control respectively after incubation for 120 min.

11.2.9. Reducing power ofTSE

The reducing power of TSE was determined following the method of

Oyaizu [24]. Different amounts of TSE extract (0.00, 0.24, 0.60, 1.20, 1.80, 2.40,

3.00 mg/ml) in water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and

potassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1%). The mixture was incubated at 50°C

for 20 min. Trichloroacetic acid (10%, 2.5 ml) was added to the mixture and

centrifuged for 10 min. The upper layer of the solution (2.5 ml) was mixed with

distilled water (2.5 ml) and FeCb (0.5 ml, 0.1 %), and the absorbance was measured

at 700 nm. Absorbance of the reaction mixture increased with the increase in reducing

power.

11.2.10. Antioxidant activity assay using sunflower oil

Refined sunflower oil, fi"ee of additives was used as the substrate for oxidation

studies. Sunflower oil samples containing 1000 ppm of TSE and natural antioxidants

(GTP and rosemary) were separately prepared. Each 250 ml prepared oil sample was

placed in a 500 ml brown airtight glass bottle. Synthetic antioxidant (BHT) was also

mixed at its permitted limit of 100 ppm with sunflower oil for comparison [25].

Control sample of sunflower oil without the addition of antioxidant was stored under

identical conditions. All oil samples in each treatment were prepared in triplicates and

were stored at room temperature (22 ± 3°C) for 80 days. Oil sample from each

treatment was withdrawn periodically to assess the extent of oxidation.

II.2.10.1. Antioxidant activity measurement

The rate of oxidation was followed by periodic determination of POV of

stored sunflower oil subjected to different treatments. Each sample (1 g) was taken in

250-ml Erlenmeyer flask and dissolved in a mixture of 25 ml of acetic

acid/chloroform (3:2, v/v). Saturated solution of potassium iodide (1 ml) was added

and the flask was placed in a dark chamber for 5 min, after which 75 ml distilled

25

Chapter 11 Bioanafytical agro industrial waste

water was added. The liberated iodine was titrated against a sodium thiosulfate

solution (0.01 M) in the presence of starch indicator [26].

II.2.10.2. Free fatty acid content

Free fatty acid content was determined at regular intervals in stored sunflower

oil. 10 grams of each sample were weighed into an Erlermieyer flask and 50 ml of

ethanol/ethyl ether (1:1, v/v) mixture were added. This suspension was titrated

against O.IM potassium hydroxide (KOH), using 1 ml of phenolpthalein indicator,

until faint permanent pink color persisting for 30 Sec was obtained [26].

II.2.11. Antibacterial activity

Bacterial cultures of Escherichia coli. Staphylococcus aureus. Salmonella

typhi and Salmonella enterica were obtained from Defence Food Research

Laboratory, Mysore, India. The cultures were grown in nutrient agar at 37°C. Each

bacterial strain was transferred from slants at 4 -5°C to 10 ml broth and cultivated

overnight at 2)TC. A preculture was prepared by transferring 1 ml of this culture to

9 ml broth and cultivated for 48 h. The cells were harvested by centrifiigation

(1200 g, 5 min), washed and suspended in saline.

The TSE was tested against the growth of bacteria by the method of Negi

(1999)[27]. To flasks containing 20 ml melted cool agar, different concentrations of

test material in Tween 40 were added. Equivalent amounts of Tween 40 were used as

controls. One hundred microliter (about 10 cfii/ml) of each bacterium to be tested

were inoculated into the flasks imder aseptic conditions. The media were then poured

into sterilized petri plates in duplicate and incubated at 37°C for 20-24 h. The

colonies, after the incubation period were counted and expressed as colony forming

units per ml (cfii/ml) of culture. The minimum inhibitory concenfration (MIC) was

determined as the lowest concentration of the TSE required for inhibiting the

complete growth of the bacterium being tested.

26

Chapter II Bioanalytical agro industrial waste

II.3. Results and discussion

The % yields of TSE in ethyl acetate, acetone, methanol and water were 5.25,

7.13, 7.76 and 13.3 respectively. The total content of phenolics in TSE as determined

by Folin-Ciocalteu method are reported as gallic acid equivalents. Amongst the

various solvents tried ethylacetate gave the highest polyphenolic content. The order of

the efficiency of the different solvents in enhancing polyphenolics from TSE was

ethylacetate (6.75%) > acetone (4.11%) > methanol (3.91%)>water (3.12%).

The total phenolic contents in TSE determined are not absolute values but are

based on their chemical reducing capacity relative to gallic acid. In the present study

the responses of the TSE may arise from the variety and/or quantity of phenolics

found in extracts of turmeric spent.

Table II.l. Percent radical-scavenging activity on DPPH by TSE and gallic acid

Extract

concentration

20

40

60

80

100

120

140

TSE

(% scavenging)

20.36 ±0.37

37.4710.71

54.56 ±0.55

67.70 ± 0.67

78.31 ±0.48

82.81 ±0.77

84.71 ±0.28

Gallic acid

(% scavenging)

92.71 ±0.14

93.81 ±0.28

94.11 ±0.22

94.71 ±0.31

94.86 ± 0.48

94.46 ± 0.33

94.81 ±0.19

Mean ± Standard deviations (n=3)

II. 3.1. Free radical scavenging activity of TSE

The scavenging activity of TSE phenolics compared to gallic acid for DPPH

radical is shown in Table II.l. The results indicate a concentration-dependent

scavenging activity of the DPPH radical. Further, it was found that the scavenging

activity of TSE was found to be maximum at 140 |xg/ml and it was 84.71 %.

27

Chapter II Bioanalytical agro industrial waste

n.3.2. Antioxidant activity using fi-carotene-linoleate model system

Antioxidant activity of TSE in P-carotene-Iinoleate model system is shown in

Fig ILL Addition of TSE and BHT prevented the bleaching of P-carotene but to

different degrees. P-Carotene undergoes rapid discoloration in the absence of an

antioxidant. This is due to the coupled oxidation of P-carotene and linolenic acid,

which generate free radicals. The linolenic free radicals formed upon the abstraction

of a hydrogen atom from one of its diallylicmethylene groups attacks the highly

unsaturated P-carotene molecules. As a result, p-carotene gets oxidized and broken

down in part, subsequently the system looses its chromophore and characteristic

orange color; this can be monitored spectrophotometrically. In the present study, the

TSE was found to hinder bleaching of P-carotene by neutralizing the linoleate free

radical and other free radicals formed in the system. TSE showed a maximum of

92.74 % antioxidant activity when used at the highest concentration of 800 \x%.

120

SJ-^ > ^^ o 4->

C m

•D X o

4 ^

c <

100

80

60

40

?n

0

USE I BHT

200 400 600

Concentration (pg)

800

Fig.II.l. Antioxidant activity of extract from TSE and BHT by

p-carotene- linoleate model system

28

Chapter 11 Bioanalytkal agro industrial waste

11.3.3. Reducing power

Fig II.2 shows the reducing capacity of TSE and it is compared with BHT. For

this, we investigated the Fe /Fe transformation in the presence of TSE, using the

method of Oyaizu (1986)[24]. Earlier authors [28,29] have observed a direct

correlation between antioxidant activity and reducing power of certain plant extracts.

The reducing properties are generally associated with the presence of reductones, [28]

which have been shown to exert antioxidant action by breaking the free radical chain

by donating a hydrogen atom [30]. Reductones also react v^th certain precursors of

peroxide, thus preventing peroxide formation, hi Fig II.2 the reducing power of TSE

suggests that it contribute significantly towards the antioxidant effect. The reducing

power of TSE is closely related to the amount of the extract being used and increases

with increasing amovmt of the extract. However, the reducing power of BHT was

relatively greater than that of TSE.

1.6.

1.4-

r; S0.8

1 0 . 6

5 0.4

0.2 n)

( F 1 1 1 1

) 1 2 3 4

Concentration

-•—Series 1

-»-Series2

Fig.II.2. Reducing power of turmeric spent extract at different concentrations

II.3.4. Antioxidant activity of TSE in refined sunflower oil

Oxidation of lipids is imdesirable as it affects the flavor, aroma, nutritional

quality and even the texture of the product where it is used. The chemicals produced

by the oxidation of lipids are responsible for rancid flavor and aroma.

29

Chapter II Bioanalytical agro industrial waste

^ 180 1 o> ^ 160 a> w 140 -X f 120-

E 100-•^ 80-o •i 60 ra > « 40

•a

g 20 W

g. 0^ (

/ ,_^,-r^

1 1 1 1 1

) 20 40 60 80 100

Storage period (Days)

—•—Control

- * - B H T

GTP

—tr- Rosemary

-JK-TSE

Fig.II.3. Effect of synthetic antioxidant BHT (100 ppm), natural antioxidants

GTP (1000 ppm) and rosemary (1000 ppm) and TSE (1000 ppm) on

peroxide value (mmol oxygen /kg) during storage of sun flower

oil

1.4-

1.2

" SO.8

150-6 u. 0)0.4

^0.2

n -

( ) 20 40 60 80 100

Storage period (days)

—•—Control

GTP

K Rosemary

-)is-TSE

Fig.II.4. Effect of synthetic antioxidant BHT (100 ppm), natural

antioxidants GTP (1000 ppm), and rosemary (1000 ppm) and

TSE (1000 ppm) on free fatty acid (mg KOH/g sample) during

storage of sim flower oil.

30

Chapter II Bioanalytical agro industrial waste

BHT, GTP and rosemary are commonly used as antioxidants in oils. BHT is a

synthetic while; GTP and rosemary are natural antioxidants. The antioxidant property

of TSE was determined and compared with BHT, GTP and rosemary. The result

presented in Fig II.3 and II.4 manifest that TSE has antioxidant property, which is

comparable to GTP and rosemary. But TSE has lesser antioxidant activity when

compared to BHT. The FFA and POV did not increase much, which clearly indicates

that autooxidation of sunflower oil was greatly inhibited by the TSE. This study

revealed that the TSE can be used as a natural antioxidant at little higher

concentration compared to natural antioxidants like GTP and rosemary.

11.3.5. Antimicrobial activity

2000

1600-Minimum Inhibitory 1200

Concentration „ . . (ppm)

400

0 1^^ *

^ • 1

^v

<

-1 1 1 1

1 2 3 4 Bacteria

Fig. II.5. Effect of TSE on growth of Escherichia coli (1),

Staphylococcus aureus (2), Salmonella typhi (3) and

Salmonella enterica (4).

The effect of TSE on the growth of four different bacteria is dose dependent

as presented in Fig II.5. TSE inhibited growth of bacteria to variable extent,

depending on the bacterium in question. TSE completely inhibited the growth of

E.coli at 850 ppm concentration. But S. aureus, S. typhi and S.enterica required 1200,

1100, 1300 ppm of concentration respectively for the complete inhibition of growth.

31

Chapter 11 Bioanalytkal agro industrial waste

II.4. Conclusion

First-ever studies on turmeric spent have been carried out to recover the

polyphenols, which have demonstrated antioxidant and antimicrobial properties. The

study has been carried out to develop natural and cost-effective antioxidant material

from solid waste generated by food-industry. The extract from turmeric spent has

indicated high antioxidant and antibacterial activities. Further studies are in progress

to characterize individual phenolics and to elucidate the mechanism underlying

bioactive properties and existence of possible synergism, if any, among these

compounds.

In summary, the varied experience in industrial and agricultural sectors amply

proves that the crucial importance in management is in the realization of the benefits

of development in an optimum and sustainable manner. The problem is more acute

and wide spread in developing economics coupled with pressing social priorities.

Such problem acquires serious magnitude in polluting the environment. In such a

situation engineering of food-industrial waste for feed, food and functional foods

assumes paramount importance. This paper is a step in this direction.

32

Chapter II Bioanafytical agro industrial waste

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Chapter II Bioanalytical agro Industrial waste

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