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J. Saudi Soc. for Food and Nutrition., Vol. 1, No. 1; 2006

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: ! "#$ %$ &' '( ) *+ ' +)'' ,- Citrus sinensis and

Citrus aurantifolia .'/ ) *+ 0+/' 12 )23Ocimum basillicum 2' 2 Melia

azedarach '' 1'( ) *+ 4 , 5"3 '/ '67 , 0+8 9)' ) :+ ;' <.=>/ ;'8 "#$ 1?11@ ! ' '2$AB 2

/ ' 4 C% D'=' >+ 9 -E F'$ ) '6 E= ) ;'/ C$' ':Ulocladium utrum, Trichoderma harzianum, Aspergillus ustus, and Penicillium ducaluxii ' *+ ' )/ ' F''6 '/ "3 4 12+ 2E% 98 ' ) 1' )' CE% U. utrum'A. ustus ' C% >=+ A/ 1)>3 ) / ))6' )

) *+ 5 '( ( 4 "3'/>! 4 0+8 ' D% A/ % J3 '2 )2 , 92E= + K@$< ' )3 @'2 >=+ -% )'' ,- '( / 1

E= ) 3' ,/ L@.' )/ E% P.ducaluxii ) E= A/ ' C% >=+ %>! 4*+ + .

*+ M 4 1>+' D'= N 4 - 2E= ' 2*+ 24 +2 )' 5 '( + + (@+ )/ J3 1,3 E= #= )' 2!

,+A1%>! A + (- 4 E= ' )' '3 ! .

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" INTRODUCTION

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A' ;'' 3 6 ) C ,' , )U% 1J' ) 3 6< E3V )1 - '1-*(.

( W ABC( ,3 :* C% 9- J3 C%X 7' 38 3 4 'E 6 # 1* ,*% C% *+ :J3 ,* 4/

YEP/ 7( C% 'E C% '7% 3 E' 4/ / 1E' ,/ / 1'' 7( C% '7% 3' 57( 1E= ' 4 + :'T "#$ 4

C' ,A -3 J',*3 46 )'=' >+D( C % C% N! C% C *R ) ! )6+ FA/' :E- 374 Z' J

E= *+' ' 7A'146 ,*3 '6P 4 - E= )/ T3' '8 9' (V ,<+ ) E= =* 9 , C% ' "#$ '-' 17P7/ N ) 6= 'A >/ [' C$ C ' 6%U E= 1: '

C'3 4 3* 4 'E+ 4 7 A' Q#X ' , 15E% \ ]7E= "#$ )' 1Aspergillus, Penicillium, Fusarium, Claviceps, Myrothecium,

Stachyobotrys, Alternaria and Claviceps ( Wieland, 1986) 1 C% ' Tournas

and Katsoudas (2005)'/ ,-3 C% &'' ' ' ,A 6E D'= J' FA/'/ ,- '/ *3 ]7 9 4 - E= ) ^!' ' 7> '

7'Cladosporium , Fusarium, Penicillium, Rhizopus, Botrytis, Aureobasidium,

Trichoderma, Alternaria and Yeasts "#$' 1 ' )E ,A N8 ) A


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* (' ,(' E= "#$ 4 F>-' 4 + E C 7%3* 3 ) ]7 < > 4 6 4 )13(___.(

,4 ' NE 4 :' C( E C% - ' 7= 9' ' )A3 ) A 4 ' 1<+VE '6 )

)' )/ )' 7 Q *+ )/ ' 1'' E= 4 F>- '- ]# 3>'/ >/Charleston et al. ( 2006) +CQ + Melia azedarach 'Azadirachta indica '- 4 Plutella xylostella $P' )%`1 + "#$ C% #*+ Q NT%3 E ' ' 1

+ ) 3,A ':.,-' )' '(Citrus saneness and Citrus aurantifolia : &' : C$'

.E C% ;6'%>! 4'$'( Q#X 74 T=3 4 , C' 'E )1)=' = 9 :4 &' )46 F'377(' 4

E% >(Sharma and Tripathi,2006; Kang et al. ,2006; Wu et al.,2007, Mi et al., 2007)

.)3Ocimum basillicum: )4 4 , D'/X1' >,A Q N 4 5'3'aglycones, linalool and methylchaviol'$P

) (Politeo, et al., 2007)1:47/ E= (Atanda et al., 2006).?. Melia azedarach:( )4 4 3 .'/ # 1( E C% +C

N8 ^<C$' 1' > 5' 7E A' )1)'+a' ,-4_b ( 17'6 )/ 3>'/, 3*' #X 4' 7 .(Rukmini, 1987)

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(Lopez-Caballero et al., 2005)

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()*+ Culture medium a 6' '' +(Sabouraud dextrose agar CM41, Oxoid,UK)

J3 #+/KC% 6$ Q ) 1 #R ' 1E- F ) , EX> 3 + - 67 C% D- ):<''V (4º - .

,#" & - Isolation and identification of study fungi

,64 E= -E +1984) Abdel-Hafez, (E-U8f' E/ C% 5 .)K (' C% D'=' >+ 9 -E F'$ )1

'3Y' Y( 6'' 4 (Glucose -Cazpek’s-Dox) E2' 2(Naguib, 1968):>/' ,6' 7! / ' 9 , (Smith

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Ulocladium utrum,, Trichoderma harzianum, Aspergillus ustus, and Penicillium ducaluxii

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) ' /! :Test of the water extracts on some fruits and vegetables infected by fungi

,>%/ + Q *+ C% C$')5 '( +7E+ '/ 3 4 +'('( 4 7>+' D'= N 3- A 1

E=+ 41A '( -' +3- >+' '=E= M )' +*+ 1- / %X 3 C% A'*

[Q.()0 #Statistical analysis

1'Q ! [Q ,'3 73 + Q*3! T.test 0 7TR ) 74 ,*3 .'= '<' 0' 4 E> ' ,

1)

,( C% [Q 3>'/)'( '( + )/ +' 5 " 4 ,"3" 5E- ' >=+ J3 + E= 9 ' 4 EA AB '# '

N=+V 6' CA 9 ' >/ 7 , C% E> - %>! % 16 @'@ / , 1Q#X Q ! 7 + 5/ ) + 4 '

5 '( E% C%U. utrum ?,b',@ ' ) CA ' 1 ! N=+ AK,K '?,@C ' ,= AB &>' #$' 19 ' '( 7'3

7' 5))6' ( 4 ,% )6' AB )/ ' J3 1 ' 4 T%3 )6' ,'3 )/ ' 1 4 :>/'] . 4

°' ° ;'/ (Lopez-Caballero et al., 2005;Wang et al., 2006) . 4 / + +6@'@%' N=+ ! 0/ - E% A. ustus

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'/ C%D 4 8 : % 6)(Azadirachtin <+ '> # 7*


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/ ! )m Q' ) $P' (3 %` ) ' 4 , D6= (Basak and Chakraborty, 1968) + + )/ '' 1

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+ E= 4 AB A8 )*+ %>! 4')5 '( + + !) (,(?(6 ) , C% N=+V C% 6 '@'@$'' )4

E% ' J3 1"3 4A. ustus ?,@ ! >=+ A CA ' ,@/ 1E% U. utrum ,%?,b@ ! >=+ A ?,K@' 9 '

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harzianum A ' ) CA ' 9 ' C% ' 9=' ' 4 7 .)' E= ' EA ' 4 5'3 )/ ) E ' *+ )/ ) #$'

#$EA AB '- )/ E= N ) J3 '= EA1D3>'/ #$' ,( C% [Q)(5E% )/U. utrum 'P. ducaluxii 4 )' '( + %>! 4

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T. harzianum A. ustus P. ducaluxii U. utrum,

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_, 'K,@1CA E= C% [Q E4/ ' C% N=+K, 'Kb,K@=+V 6 A 1 ! N,b 'K,b @1 ]# ='Mi et

al.(2007) *+' &' '(- CQ + )/ Citrus unshiu 4 5'3 narirutin 'hesperidin 6 C% +8 "#$' L?E% =$ ' 6=3 )' C% F6

Gigaspora margarita ']# ) Y 4 %) ,- N'3 '( C% '' '6 )B E% 'A.niger ,–? ' /*' Q 6' YEE Q C% ,

4 (Sharma and Tripati, 2006).! 4 %>@'@)"3 4 )3 + )/ ' 1)- )E= >=+

/>1J3,'8 E= ,K, 'K,b @ A ! N=+ ,? '_,b@.', CA E= , ',bb@ ! N=+' ,Kb ',@#$'D( Atanda et

al .( 2006) )/ 3>'/ C%>! )3 6 + @# '3 Q ! F> E= A. parasiticus'V ^! ) ,- ;' ) )'<%(G1+B1)

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/ A(Chah et al. 2006) D )3 .'/ )/ C% Z' 5' 5 > ' + D' ,( 4 ./)E=+` )A. ustus 'harzianum T.)# N=+ '$'' ) 7 1' 4 C%)' '( + 1N=+ C% '

E= ,'856 C% @'@ !,bK@',@C% 9= A ! 9 ' K,? 'K,@#' 1 ]C%CA E= 5#N=+ D ' ! K, 'K,b@ A

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b,b@;'/ ,<+ 1"#$ 4'*+ C% ) AB 7 C T%3 ',Q>+ E= ' 4 .

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EA AB)*+ 49 E= 1+ ^3 #$' A/ = ! '2*+ 4 $AB' E ' 7E+ 4 >8 7E(/' '

.' "#$ % 6'J3 0+8 *+ 9 % A8 + :>/

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+)3 .'/ +. C% + E= 9 ' [Q %>! 4 ' V N=+ E T%3 ' *+ ) =+ 6)1?11@(' Q !'

1a 6')'% "3 - '/ 6 "5B "AB 3 +V A/ '$' E' C% Al( 6 6 AB :+ 1;'' 74'' + +

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%X 3 C% ' )*+ ^61 / '* C% [Q 3>'/5 '( + % ++ @9 7+ !'

J3 146 ,*3"% ' E= ' )' '3' 7EX 4 ,.


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)3 4 1-*.)(.pC+ )' Y ,' #P8 < )> ) + 1#X CA C l 1#X' F#X .23 1)(_L

_= C% 1L_ L)3 .

5D C5

Abdel-Hafez S. I. (1984)· Survey of airborne fungus spores at Taif, Saudi Arabia. Mycopathol. 88 (1):39-44.

Atanda, O. O. ; Akpan, I. and Oluwafemi, F. (2006)· The potential of some spice essential oils in the control of A. parasiticus CFR 223 and aflatoxin production. Food Con. 18:601-607.


١٥

Basak, S. P. and Chakraborty, D. P. (1968)· Chemical investigation of azadirachta indica leaf (Melia azadirach). J. Indian Chem. Soc. 45 ( 5):466 - 467.

Bollen,G. L. (1972) · A comparison of the vitro antifungal spectra of thiophanates and benomyl. Neth. J. Plant Pathol. 78:5-64.

Chah, K. F.; Eze, C. A.; Emuelosi, C. E. and Esimone, C. O. (2006)· Antibacterial and wound healing properties of methanolic extracts of some Nigerian medicinal plants. J. Ethnopharmaco. 104(1-2):164-167.

Charleston, D. S.; Kfr, R.; Dicke, M. and Louise, E. M. (2006). Impact of botanical extracts derived from Melia azedarach and Azadirachta indica on populations of Plutella xylostella and its natural enemies: A field test of laboratory findings . Biol. Cont. 39( 1):105-114.

Ellis, M. B. (1971)· Dematiaceous Hyphomycetes. Common-Wealth Mycol. Institute, Kew, Surrey, England.

Ellis, M. B. (1976)· More Dematiaceous Hyphomycetes. Common-Wealth Mycol. Institute, Kew, Surrey, England.

Gilman, J. C. (1957)· A manual of soil fungi. Iowa, State Univ. Press. Ames. Iowa, U. S. A.

Gunjan, P. and Srivastva, A. K. (2007)· Azadirachtin production in stirred tank reactors by azadirachta indica suspension culture. Process Biochem. 42:93-97.

Kang, H. J.; Chawla,S. P.; Jo, C.; Kwon, J .H. and Byun, M. W. (2006) · Studies on the development of functional powder from citrus peel. Bior. Technol. 97( 4):614-620.

Lopez-Caballero,M. E.; Goamez-Guillen, M. C.; Perez-Mateos, M. and Montero, E. (2005) ·A functional chitosan-enriched fish sausage treated by high pressure. J. Food Sci. 70 (3):166 – 173.

Mi, R. K.; Won, C. K. ; Do, Y. L. and Chan, W. K. (2007)· Recovery of narirutin by adsorption on a non- ionic polar resin from water extract of Citrus unshiu peels. J. Food Engin.78:27-32.

Naguib, M. I. (1968)· Effect of various nitrogen sources and / or colchicine on the utilization of L-arabinose by Cunninghamella elegans. Acta Biol. Acad. Sci. Hung., 19:437- 444.

Politeo, O.; Jukic, M. and Milos, M. (2007)· Chemical composition and antioxidant capacity of free volatile aglycones from basil (Ocimum basilicum L.) compared with its essential oil. Food Chem. 10 (1):379-385.

Raper, K.B. and Thom, C. 1949· A Manual of the Penicillium. Williams & Wolkins, Baltimore. U.S.A.

Raper, K. B. and Fennell, D. I. (1965 )· The genus Aspergillus. Williams & Wolkins, Blatimore, U. S. A.

Rukmini, C. (1987)· Chemical and nutritional evaluation of neem oil. Food Chem.26 (2): 119-124.

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١٧

Effect of someNatural Extracts on the Growth of some Fungi

Rukaia Gashgari and Hessa Al- Semari

Girl’s College of Science Education Jeddah- Saudi Arabia

P.O.Box.45057 Jeddah 21512

ABSTRACT. The Aim of this study was to use materials extracted from the husks citrus orange and lemon (Citrus sinensis and Citrus aurantifolia), extracts from basil (Ocimum basillicum) and neem (Melia azedarach) leaves, and extracts (named chitosan) from the shell of some fish and shrimp. These extracts (used alone and/or mixed) made 15 different treatments. These materials were added at 1, 3, 4, and 5 % to a sabouraud dextrose agar to study the impact on the growth of four types of fungi isolated from the air of the vegetables and fruits markets in the city of Jeddah, Saudi Arabia namely: Ulocladium utrum,, Trichoderma harzianum, Aspergillus ustus, and Penicillium ducaluxii

Data showed that the extracts (alone or mixed) reduced the growth of all four tested fungi, U. utrum and A. ustus growth were decreased the most compared to the other fungi after 7 days of incubation. Chitosan alone or when mixed with other materials was more effective reducing the growth rate of fungi to 60%, followed by Ocimum basillicum and Melia azedarach leaves 40%, and the least reduction 10-20% was observed with Citrus sinensis and Citrus aurantifolia husks. Most inhibition of the growth was found in P. ducaluxii in all used extracts.

When spraying the previous extract (5% of mixed shrimp shell and neem extracts) on some vegetable and fruit, no fungal growth was observed.

Keywords: natural extracts, chitosan, Ocimum basillicum and Melia azedarach, Fungi.


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Weight control practices of U.S adolescents and adults. Ann. Inte. Med. 119 (7pt 2):

667 – 671.

Stamatakis, E.; Primatesta, P.; Chinn, S.; Rona, R. and Falascheti, E. (2005). Overweight

and obesity trends from 1974 to 2003 in English children: what is the role of

socioeconomic factors? Arch. Dis. Child. 90:999-1004.

Sturm, R. (2002). The effect of Obesity, smoking and drinking on medical problems and

costs. Health Affairs. 21(2):245-253.

SPSS. (1998). Statistical Package for Social Science. Computer Software.

Techernof, A.; Lamarche, B.; Prud'homme, D.; Nadeau, A.; Moorjani, S.; Labrie, F.;

Lupien, P. J. And Despres, J. P. (1996). The dense LDL phenotype: association with

plasma lipoprotein levels, visceral Obesity, and hyperinsulinanemia in men. Diabetes

Care. 19(6):629-637.

Tounian, P.; Aggoun, Y.; Dubern, B.; Varille, V.; Guy-Grand, B.; Sidi, D.; Girardet, J. P.

and Bonnet, D. (2001). Presence of increased stiffness of the common carotidartery

and endothelial dysfunction in severely obese children: a prospective study . The

Lancet 358 (929):1400-1404.

WHO. (1998). Preventing and managing the global Epidemic, WHO Geneva.


٣٤

Relationship between Nutritional Status and Blood-Lipids Levels in

School Children in Riyadh City

Latifah M. AL-Oboudi Girls University, College of Education for Home Economics, Department of Nutrition and Food Science,

Riyadh, Saudi Arabia.

ABSTRACT.. The aim of the study was to determine the relationship between nutritional status and plasma lipid levels in school children in Saudi school children (girls) aged 9-13.9 years. The study was conducted on 120 school girls randomly chosen. Height and weight were recorded and Body Mass Index (BMI) was calculated. Nutritional status was measured by (BMI) compared to standard tables. Fasting blood glucose (GLU), total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were measured. The results showed that the prevalence of obesity and overweight was 12.5% and 13.3%, respectively. The mean of TG was above cutoff points in obese girls. The prevalence of medium-risk values for HDL was recorded among obese, overweight and normal- weight girls. The correlation between obesity and TC was strongly negative and statistically significant. Overweight correlated negatively with level of TG, and underweight correlated negatively with LDL. The statistical significant was strong. The level of TC correlated negatively with under weight. A significant positive correlation was obtained between TG levels in blood and normal weight.

Journal of the Saudi Society for Food & Nutrition

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a) Abulfatih , H.A. (1979). Vegetation of higher elevation of Asir, Saudi Arabia. Proc. Saudi Biol. Soc. 3: 139-48.

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Subscription and Exchange The Saudi Society for Food & Nutrition, College of Food and Agricultural Sciences P.O. Box 2460, Riyadh 11451 Kingdom of Saudi

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Journal of the Saudi Society for Food and Nutrition

Published Biannually By

The Saudi Society for Food and Nutrition

Editorial Board

Prof. H. M. Abu-Tarboush Editor-in-Chief

Prof. H. A. Al-Mana Member Prof. B. H. Hassan Member Prof. M. M. Al-Dagal Member Dr. A. O. Musiger Member Dr. A. S. Bajaber Member

Office Address

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College of Food and Agricultural Sciences P.O. Box 2460, Riyadh 11451

Kingdom of Saudi Arabia

Table of Contents Journal of the Saudi Society for

Food and Nutrition Vol. 1, No. 1, 2006

Effect of some Natural Extracts on the Growth of some Fungi

Rukaia Gashgari and Hessa Al- Semari ………………………………………..

1

Relationship between Nutritional Status and Blood-Lipid Levels in School Children in Riyadh City

Latifah M. AL-Oboudi …………………………………………………………..

18 Bile Salts and Acid Tolerance and Cholesterol Removal from Media by some Lactic Acid Bacteria and Bifidobacteria

A. A Al-Saleh; A. A. M. Metwalli and H. M. Abu-Tarboush ………………. 1

Some Nutritional and Functional Characteristics of Mung Bean (Phaseolus aureus) Proteins

Saleh A. Alajaji and Tarek A. El-Adawy ………………………………………. 18

ICP-MS Simultaneous Determination of Some Essential Minerals and Heavy Metals in Some Commercial Drinks Consumed in Riyadh City

Ahmad H. Alghamdi, Ali F. Alghamdi and Abdulrahman A. Alwarthan ….. 31

Bile salts and acid tolerance and cholesterol removal from media ………


1

Bile Salts and Acid Tolerance and Cholesterol Removal from Media by some Lactic Acid Bacteria and Bifidobacteria

A. A Al-Saleh; A. A. M. Metwalli and H. M. Abu-Tarboush

Food Science and nutrition Dept. College of Food Science and Agric. King Saud Univ. Riyadh, Saudi Arabia

ABSTRACT. In this study three strains of Lactobacillus acidophilus (DSM 9126, DSM 20079 and DSM 20242), two strains of Bifidobacteria (infantis DSM 20088 and angulatum DSM 20098) and Streptococcus thermophilus DSM 20617 were tested for acid tolerance, bile salt tolerance, capability to remove cholesterol and to deconjugate sodium taurocholate from the culture medium. Results showed that a considerable variation existed among cultures in their growth viability in the presence of bile salt, deconjugation of sodium taurocholate and assimilation of cholesterol from the medium. Moreover, the two cultures of bifidobacteria (infantis DSM 20088 and angulatum DSM 20098) were shown to be the most bile salts tolerant culture. All bacterial strains tested in this study exhibited sensitivity to acidity at pH 2. However, increasing the pH of the medium to 3 had improved the acid tolerance of all strains except Streptococcus thermophilus. Addition of 1% skim milk powder into medium at pH 2 increased the viability of all strains especially bifidobacteria strains. All tested strains removed less cholesterol from the broth (ranged from 3.08-29.68%) compared to those grown in broth supplemented with 0.2% bile salts (from 36.07-55.43%). Furthermore, considerable amount of cholesterol was precipitated with cells obtained from broth enriched with 0.2% bile salts. Lactobacillus acidophilus DSM 20079 appeared to be more active in deconjugation of sodium taurocholate (2.38 µmol/ ml) compared to the other strains, as well as being able to remove up to 66.61 mg of cholesterol (95.6%) from the culture medium and therefore, is regarded as a suitable candidate probiotic and adjunct culture. _____________________________________________________________

INTRODUCTION

High serum cholesterol concentration is associated with the development of coronary heart disease (Usman and Hosono, 2000). Mann and Spoerry (1974) claimed that the consumption of fermented milk with Lactobacillus acidophilus reduced serum cholesterol level in Massai trip. Since then, the hypocholesterolemic effect of fermented dairy product has been observed in feeding studies either using humans (Harrison and Peat, 1975) or animals (Liong and Shah, 2006 and Daneilson et al., 1989). Many studies have reported the ability of Lactobacillus acidophilus (Gilliland et al., 1985 and Usman and Hosono, 1999) and bifidobacteria (Dambekodi and Gilliland, 1998) to assimilate cholesterol from laboratory media. Thus, both types of bacteria may have the potential to reduce serum cholesterol in humans. However, the ability to assimilate cholesterol from the media varied significantly amongst different bacterial strains. Many attempts have been made to elucidate the mechanism involved in the hypocholesterolemic action of lactic acid bacterial strains. One proposed mechanism is the assimilation of cholesterol by the cell wall during growth (Buck and Gilliland, 1994; Noh et al., 1997). Another mechanism is the deconjugation of bile salts by bacteria producing bile salt hydrolase. Most conjugated bile salts are recirculated through the enterohepatic circulation, while deconjugated bile salts are less soluble and excreted in the feces. The bile salts that are excreted must be replaced by new bile salts, which are formed from cholesterol in the body. Thus, the more bile salts



2

excreted, the more cholesterol is removed from the body. Furthermore, deconjugated bile salts do not stimulate the absorption of cholesterol and other lipids from the small intestine as well as do conjugated bile salts. Walker and Gilliland (1993) reported that some strains of Lactobacillus can deconjugate bile salts. While, Hill and Drasar (1968) suggested that Lactobacillus is incapable of deconjugated bile salts.

This study was conducted to compare bile salts and acid tolerance, assimilation of cholesterol and deconjugation of bile salts by different strain of lactic acid bacteria and bifidobacteria and to choose the most beneficial strain as adjunct culture in fermented dairy product.

MATERIALS AND METHODS Bacteria

Three strains of Lactobacillus acidophilus (DSM 9126, DSM 20079 and DSM 20242), two strains of Bifidobacterium (infantis DSM 20088 and angulatum DSM 20098)and one strain of Streptococcus thermophilus DSM 20617 were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen Gmbh (DSMZ, Germany). Stock cultures were stored in 40% glycerol at –20°C. The organisms were subcultured 3times before use in sterile de Man, Rogosa, Sharpe (MRS) broth using 1% inoculum and incubation for 20 h at 37°C. Acid Tolerance

Acid tolerance of the cultures was studied by incubating the organisms in MRS broth (in some experiments 1% skim milk powder was added to MRS broth). The pH was adjusted to 2.0 with HCl 1N and the cultures were incubated at 37°C for 3 h. Each of the bacterial strains was subcultured at least 3 times before experimental use MRS broth was inoculated (10% vol/vol) with bacterial strain, and growth was monitored using the plate count method as described by Pereira and Gibson (2002). A 1-mL sample was taken at zero, 1.5 and 3 h, and serial dilutions were made using peptone water diluent. Samples were plated onto MRS agar, and the plates were incubated at 37°C for 48 h in an anaerobic jar (Becton Dickinson Microbiology Systems, Sparks, MD) with a Gas Generating Kit (Oxoid, Ltd., Mitsubishi Gas Chemical Company) except for Streptococcus thermophilus, which was incubated under aerobic condition. Acid tolerance was determined by comparing the plate count after 1.5 and 3h with the initial plate count at 0 h (results were expressed as percentage). The experiments were repeated twice. Bile salts tolerance

Growth rate of bacterial cultures was determined in MRS broth containing different levels (0, 0.1, 0.3, 0.5 and 0.7%) of bile salts (oxgall). Freshly prepared cultures were inoculated (1%) into medium and incubated at 37°C for 24 h under anaerobic condition, except for Streptococcus thermophilus, which was incubated under aerobic condition. Optical densities were measured spectro-photometrically at 620 nm after 0, 3, 5 and 24 h. Cholesterol removal

Cholesterol solution (10 mg/ml in 96% ethyl alcohol) was prepared and filtered sterilized. For each culture to be tested, 70 µl of cholesterol solution was added to 10 of



3

MRS broth (final cholesterol concentration 70 µg/ml) containing 0.2% bile salts (oxgall) or not containing. To the MRS broth, 1% of freshly grown culture was added and incubated anaerobically at 37°C for 20 h except Streptococcus thermophilus which was incubated under aerobic condition. An uninoculated sample was used as control. After incubation the cells were removed by centrifugation at 10,000 g for 10 min at 4°C and cholesterol was determined in the supernatant using modified Rudel and Morris (1973) method in which three ml of supernatant, 2 ml of 33% (wt/vol) KOH and 3 ml 96% ethanol were placed in a capped test tube, vortexed for 20 second and incubated for 15 min at 60°C in a water bath. After incubation, the mixture was removed and cooled under tap water, then 5 ml of hexane and 3 ml of water were added and vortexed for one min. One milliliter of the hexane layer was transferred into a dry clean test tube and evaporated under nitrogen gas. One milliliters of cholesterol liquicolor enzymatic kit (Human-Gesellschaft fur Biochemica und Diagnostica mbh-Wiesbaden-Germany) was added. The solution was mixed and left for 5-10 min at 37°C and absorbance was measured at 500 nm with a spectrophotometer (LKB Biochrome ultrospec 11, Campridge, England). The ability of bacterial strain to remove cholesterol from media was calculated as percentage from the following equation:

A=100-(B/C)*100

Where A=% of cholesterol removed, B=absorbance of the sample containing the cells and C=absorbance of the sample without cells.

To measure the cholesterol removed with the cells, pellet cells obtained by centrifugation was resuspended in distilled water to the original volume of the culture and cholesterol was determined as mentioned above. Cholesterol remained with the pellet was calculated from the equation:

A= (B/C)*100

Where A= Cholesterol remained with the pellet (as percentage), B= absorbance of the sample containing the cells and C=absorbance of the sample without cells. It was observed that, sample containing no cells has no pellet and cholesterol was determined in the whole system. Deconjugation of bile salts

Deconjugation of bile salts by bacterial strains was tested qualitatively through the plate assay as described by Ahn et al., (2003). To MRS agar containing 0.5 g/l cysteine, 1 mM of sodium taurocholate (Sigma Chemical Co., USA) was added. After autoclaving and solidifying, the plates were incubated anaerobically for 48 h before use. The plates were inoculated with active culture (20 µl) and incubated for 72 h at 37°C. Precipitated cholic acid around colonies were observed. Deconjugation of bile salts was also measured quantitavely by measuring released cholic acid as described by Walker and Gilliland (1993).



4

RESULTS AND DISCUSSION

Acid tolerance The effect of pH on the viability of strains is presented in Fig. 1. Results showed

that, the viability of all tested bacterial strains markedly decreased on incubation at pH 2 for 1.5 h. Streptococcus thermophilus DSM 20617 and Bifidobacterium ifantis DSM 20088 were the most acid sensitive of all tested strains. These strains completely lost their viability after 1.5 h at pH 2. Bifidobacterium angulatum DSM 20098 retained about 26% of initial viability after 1.5 h compared to 8% for Lactobacillus acidophilus DSM 9126, 20079 and 20242. Addition of 1% of skim milk powder into MRS broth greatly improved acid tolerance of all strains, particularly bifidobacteria, which retained about 98 and 89% for B. infantis DSM 20088 and 95 and 42% for B. angulatum DSM 20098 after 1.5 and 2 h, respectively (Fig. 2). There were major differences between the viability of bacterial strains at pH 3 compared with that at pH 2. Bifidobacterium infantis DSM 2288 and B. angulatum DSM 20098 retained about 100% viability after 1.5 h and sharply decreased after 3 h. While L. acidophilus DSM 9126, 20079 and 20242 retained about 50, 63 and 47% viability after 1.5h and 9, 30 and 3% after 3h, respectively. However, S.thermophilus was the most acid intolerant strain (Fig. 3). Bile salts tolerance

In order to exert a beneficial effect in the digestive tract, probiotic culture must survive passage through the stomach and be tolerant to the bile salts concentrations in the small intestine (Sanders, 2000). Results from the comparison of different cultures for bile salts tolerance are shown in Figs (4-9). All strains exhibited considerable variations with regard to growth in control broth after 24h. The optical densities of Lactobacillus strains and Streptococcus reached to about 1.6 while it was about 1.1 for Bifidobacteria strains. With addition of bile salts to broth, the growth of strains also varied considerably. Bifidobacteria strains appeared to be the most resistant to bile salts while, the optical densities of treated samples increased than those of control after 24 h incubation. Moreover, the concentration of 0.5% bile salts had the highest enhancement for growth rate of bifidobacteria strains (Figs. 8 and 9). However, the growth rate of L. acidophilus DSM 9126 and DSM 20242 was greatly affected with addition of bile salts (higher than 0.1%), where L. acidophilus DSM 20079 showed an increase in optical densities up to 0.7% after 24 h incubation. Streptococcus thermophilus DSM 20617 also exhibited bile salts tolerance up to 0.5% bile salts. This finding is in good agreement with that observed by Pereria and Gipson (2002).



5

Strain 1 is Lactobacillus acidophilus DSM 9126, strain 3: Lac. Acid. DSM 20079, strain 10: Lac. acid. DSM 20242, strain 12: Strept. thermophilus 20617, strain 6: Bifidobacterium infantis DSM 20088 and strain 7: Bifido. angulatum DSM 20098.

Strain 1 is Lactobacillus acidophilus DSM 9126, strain 3: Lac. Acid. DSM 20079, strain 10: Lac. acid. DSM 20242, strain 12: Strept. thermophilus 20617,strain 6:Bifidobacterium infantis DSM 20088 and strain 7: Bifido. Angulatum DSM 20098.



6




7



8

Fig . 7. Bile salt tolerance of Streptococcus thermophilus DSM 20617 in MRS broth

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 5 10 15 20 25 30Incubation time (h)

Opt

ical

dens

ityat

620

nm

0 bile salts0.10.30.50.7



9

Cholesterol assimilation The percentage of cholesterol assimilated during 20 h of anaerobic growth at 37°C

in MRS broth (Fig.10) revealed a wide variation among strains. All tested strains were able to assimilate cholesterol to some extent; the assimilation ranged from 3.08-29.68% presenting around 2.16-20.77 µg/ml. Lactobacillus acidophilus DSM 9126 exhibited high cholesterol assimilation (29.68%) compared to the other strains. Liong and Shah (2005); Lin and Chen (2000) and Dambekodi and Gilliland (1998) reported that B. longum and L. acidophilus are able to uptake cholesterol into their cellular membrane. Therefore, residual cholesterol was determined with the pellet obtained by centrifugation. As shown in Fig. 11, about 40% (27.59 µg/ml) of the cholesterol was precipitated with mass cells of L. acidophilus DSM 20242. Pereria and Gipson (2002) observed that the uptake of cholesterol by lactic acid bacteria and Bifidobacteria was higher in the medium containing 0.4% oxgall. This concentration is high enough to inhibit some strains in this studies (i. e. Lactobacillus acidophilus DSM 9126 and DSM 20242) (Figs. 4 and 6), and hence, MRS



10


Strain 1 is Lactobacillus acidophilus DSM 9126, strain 3: Lac. Acid. DSM 20079, strain 10: Lac. acid. DSM 20242, strain 12: Strept. thermophilus 20617, strain 6: Bifidobacterium infantis DSM 20088 and strain 7: Bifido. angulatum DSM 20098



11

supplemented with 0.2% oxgall was used. Results in Fig. 12 represent the effect of addition of bile salts on the assimilation of cholesterol from the media. Results revealed that addition of bile salts greatly improved the uptake of cholesterol from the media. Lactobacillus acidophilus strains showed the highest activity to assimilate cholesterol from the media and it ranged from 49.57-55.43% (34.70-38.8 µg/ml). Brashwars et al. (1998) reported that L. casei able to assimilate about 16.9-73.3 µg/ml cholesterol in MRS supplemented with 6 mM sodium taurocholate. However, removal by bifidobacteria strains ranged from 41.93-44.19% (29.35-30.93 µg/ml). Removed cholesterol by Streptococcus thermophilus was the lowest (about 36.07% equivalent to 25.25 µg/ml). Moreover, the precipitated cholesterol with the pellet cells was enhanced with addition of bile salts and L. acidophilus DSM 20079 increased the precipitated cholesterol up to 45.70% (31.99 µg/ml) (Fig. 13). Total assimilated and precipitated cholesterol was depicted in Fig. 14. It seems that all tested strains exhibited activity (ranged from 74.7-96.6%) towards assimilation and precipitation of cholesterol. Lactobacillus acidophilus strains DSM 20079 and DSM 9126 were among the most active in removing cholesterol from the growth medium. Deconjugation of bile salts

Screening cultures for deconjugation of bile salts is shown in Fig.15. All cultures grown on sodium taurocholat-MRS agar plates formed fine precipitated white granules around and within the colonies to different extent. These white granules have been reported to be related to the solubility of bile salt at different pH (Dashkevicz and Feighner, 1989). The PKa of taurin-conjugated and unconjugated bile salts are 1.9 and 5.0, respectively (Ahn et al., 2003). Thus, at acidic pH, unconjugated bile salts are protonated and precipitated.

The amount of released cholic acid in the broth containing sodium taurocholate was also determined. Data in Table 1 revealed that L. acidophilus DSM 20079 and B. angulatum DSM 20098 and Bifidobacterium infantis DSM 20088 librated more free cholic acid (2.38, 2.23 and 2.02 µmol/ml, respectivly) than did L. acidophilus DSM 9126 and S.thermophilus DSM 20617 (1.97 and 1.91 µmol/ml in order). Pereira et al. (2003) found that release of cholic acid from sodium taurocholate depends on the production of bile salt hydrolase by bacterial strains. Kim et al. (2004) also purified bile salt hydrolase from bifidobacteri strains.



12

Strain 1 is Lactobacillus acidophilus DSM 9126, strain 3: Lac. Acid. DSM 20079, strain 10: Lac. acid. DSM 20242, strain 12: Strept. thermophilus 20617, strain 6: Bifidobacterium infantis DSM 20088 and strain 7: Bifido. angulat




13




14

20088 and strain 7: Bifido. angulatum DSM 20098.

Table1.Deconjugation of sodium taurocholate (6 mM) by bacterial cultures in MRS broth

Bacterial strain Free cholic acid (µmol/ ml) Lactobacillus acidophilus DSM 9126 1.97 Lactobacillus acidophilus DSM 20079 2.38 Bifidobacterium infantis DSM 20088 2.02 Bifidobacterium angulatum DSM 20098 2.23 Streptococcus thermophilus DSM 20617 1.91

Fig. 15 Deconjugation of bile salt by bacterial strains grown on bile salt-MRS agar plate

strain 1 is Lactobacillus acidophilus DSM 9126, strain 3: Lacto. acid. DSM 20079, strain 12:Strept. thermophilus DSM 20617, strain 6: Bifidobacterium infantis DSM

Strain 3Strain 1 Strain 12

Strain 6 Strain 7



15

REFERENCES Ahn, Y. T., Kim, G. B., Lim, K. S., Baek, Y. J. and Kim, H. U. (2003). Deconjugation of

bile salts by Lactobacillus acidophilus isolates. Int. Dairy J. 13, 303-311. Brashwars, M. M., Gilliland, S. E. and Buck, L. M. (1998). Bile salt deconjugation and

cholesterol removal from media by Lactobacillus casei. J. Dairy Sci. 81:2103-2110. Buck, L. M. and Gilliland, S. E. (1994). Comparison of freshly isolated strains of

Lactobacillus acidophilus of human intestinal origin for ability to assimilate cholesterol during growth. J. Dairy Sci. 77: 2925-2933.

Dashevicz, M. P. and Feighner, S. D. (1989). Development of a differential medium for bile salt hydrolase active Lactobacillus spp. Appl. and Environ. Microbiol. 55, 11-16

Dambekodi, P. C. and Gilliland, S. E (1998). Incorporation of cholesterol into the cellular membrane of Bifidobacterium longum. J. Dairy Sci. 81:1818-1824.

Daneilson, A. D., Peo, E. R., Shahani, K. M., Lewis, A. J., Whalen, P. J. and Amer, M. A. (1989). Anticholesterolemic property of Lactobacillus acidophilus yogurt fed to mature bears. J. Animal Sci. 67: 966.

Gilliland, S. E., Nelson, C. R. and Maxwell, C. (1985). Assimilation of cholesterol by Lactobacillus acidophilus. Appl. Environ. Microbiol. 49: 377-381.

Harrison, V. C. and Peat, G. (1975). Serum cholesterol and bowel flora in the newborn. Am. J. Clin. Nutr. 28: 1351-1355.

Hill, M. J. and Drasar, B. S. (1968). Degradation of bile salts by human intestinal bacteria. Gut. 9:22-27.

Kim, G.-B., Yi, S.-H. and. Lee, B. H. (2004). Purification and characterization of three different types of bile salt Hydrolases from Bifidobacterium strains. J. Dairy Sci. 87:258-266

Lin, M. Y. and Chen, T. W. (2000). Reduction of cholesterol by Lactobacillus acidophilus in culture broth. J. Food Drug Anal. :97-102..

Liong, M. T. and Shah, N. P. (2005). Acid and Bile tolerance and cholesterol removal ability of lactobacilli strains. J. Dairy Sci. 88:55-66

Liong, M. T. and Shah, N. P. (2006). Effects of a Lactobacillus casei synbiotic on serum lipoprotein, intestinal microflora, and organic acids in Rats. J. Dairy Sci. 89:1390-1399

Mann, G. V. and Spoerry, A. (1974). Studies of a surfactant and cholesteremia in the Massai. Am. J. Clin. Nutr. 27: 464-469.

Noh, D. O., Kim, S. H. and Gilliland, S. E. (1997). Incorporation of cholesterol into the cellular membrane of Lactobacillus acidophilus ATTCC 43121. J. Dairy Sci. 80: 3107-3113.

Pereira, D. I. A. and Gibson, G. R. (2002). Cholesterol assimilation by lactic acid bacteria and Bifidobacteria isolated from the human gut. Appl. and Environ. Microbiol. 68, (9) 4689-4693.

Pereira, D. I. A., McCartney, A. L. and Gibson, G. R. (2003). An in vitro study of probiotic potential of a bile salt hydrolyzing Lactobacillus fermentum strain and determination



16

of its cholesterol-lowering properties. Appl. and Environ. Microbiol. 69, (8) 4743-4752.

Rudel, L. L. and Morris, M. D. (1973). Determination of cholesterol using O-Phthalaldehyde. J. Lipid Res. 14: 364-366.

Sanders, M. E. (2000). Considerations for use of probiotic bacteria to modulate human health. J. Nutr. 130:3854-3905.

Usman, and Hosono, A. (1999). Bile tolerance, taurocholate deconjugation, and binding of cholesterol by lactobacillus gasseri strains. J. Dairy Sci. 82: 243-248.

Usman, and Hosono, A. (2000). Effect of administration of Lactobacillus gasseri on serum lipids and fecal steroids in hypercholesterolemic rats. J. Dairy Sci. 83: 1705-1711.

Walker, D. K. and Gilliland S. E. (1993). Relationships among bile tolerance, bile salt deconjugation and assimilation of cholesterol by Lactobacillus acidophilus. J. Dairy Sci. 76: 956-961.



17

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-)<,=E ,/,(N7 1 "% ; ? / , .% ( (O 4 GK,H%; /P + 7 " &' % (- () Q 4% ? + 7 R79 .

Saleh A. Alajaji and Tarek A. El-Adawy


18

Some Nutritional and Functional Characteristics of Mung Bean (Phaseolus aureus) Proteins


Food Process Technology Department, Buraydah Collage of Agriculture Technology, P. O. 266, Al-Qassim, Buraydah, Kingdom of Saudi Arabia

ABSTRACT. Mung bean seeds (Phaseolus aureus) were used to prepare flour (F), protein isolate (PI) and protein concentrate (PC). The chemical, nutritional quality and some functional properties of these products were studied. PI had higher (p < 0.05) contents of protein and ash than PC and F; while total fat and fiber contents of both PI and PC were nearly similar. Albumins was the major protein fraction in F. Both PI and PC are completely free from hemagglutinine activity and flatulence factors. Morever, the PI and PC had lower (p < 0.05) contents of tannins, phytic acid and trypsin inhibitor than F. In-vitro protein digestibility of PI and PC was also higher (p < 0.05) than F. PI had a greater proportion of leucine, lysine and tryptophan compared to F and PC. Total aromatic amino acids, leucine and isoleucine were high in globulin, prolamin and glutelin, respectively. Total sulphur amino acids is the first limiting amino acid in all protein products. The protein efficiency ratio of glutelin, prolamin and PI was almost same. The protein solubility pattern of F at different pH's showed a single isoelectric point at pH 4.5 and the solubility increased in both acidic and alkaline pH. Both PC and PI had highest emulsification capacity, oil absorption, foam capacity and stability than F.

Key words: Mung bean flour, protein isolate, protein concentrate, nutritional quality, functional properties.

INTRODUCTION Mung bean (Phaseolus aureus) is one of the edible legumes widely grown in different countries in Asia, Africa and USA. Some countries in the Middle East such as Egypt had been introduced recently mung beans by the Ministry of Agriculture to increase the supply of plants proteins requirement for human consumption. Mung bean contants about 20-27% protein and essential amino acid content comparable to that of soybean and kidney beans (Fan and Sosulski, 1974; Thompson et al., 1976 and Sobihah, 2004). Its low protein efficiency ratio (PER), however, has been attributed to the low methionine content and the presence of trypsin inhibitor (Bunce et al., 1970; Thompson et al., 1976 and El-Adawy et al., 2003).

Mung bean use as a protein supplement is limited by the beany flavor and dark color which it imparts on the final products. This problem was partly overcomed by sufficient dehulling of the beans before milling into flour (Thompson et al., 1976) Protein isolate and concentrate often had improved appearance, taste and nutritive value due to decreasing antinutritive materials compared with the original flour; therefore they can better be used as nutritional and functional ingredients in many food product (Mizrahi et

Some nutritional and functional characteristics of mung bean (Phaseolus aureus) proteins


19

al., 1967). The most widely used procedure to prepare legume and oilseed protein isolate is by isoelectric precipitation (Paredes-López et al., 1988 and Ordorica-Falomir et al., 1989). However, protein concentrate was prepared by extracting the proteins in 0.5% sodium carbonate, then dialyzed against distilled water for 48 hr (Sathe and Salunkhe, 1981). The purpose of the present investigation was to prepare mung bean protein isolate and concentrate and to evaluated some of the nutritional, physico-chemical and functional properties.

MATERIALS AND METHODS Mung bean sample Mature seeds of mung bean (Phaseolus aureus) variety Giza-1 (VC. 2010) were obtained from Agricultural Research Center, Seed Department, Giza, Egypt. The seeds cleaned by hand to remove the foreign materials. Preparation of mung bean flour (F) The seeds were cleaned by hand to remove the foreign materials then ground in an electric mill (Braun, model 1021, Germany) to pass a 80 mesh (British standard screen) nylon screen and referred as mung bean flour (F). Preparation of mung bean protein isolate (PI) The protein was extracted by 0.1N NaOH (pH 9) at room temperature using 1:20 (w/v) flour to solvent ratio, shacked for 1 hr and centrifuged for 15 min at 5000 rpm. Soluble proteins were precipitated from the obtained clear supernatant by adding acid (0.1N HCl) to pH 4.5. The protein isolate was recovered by centrifugation for 15 min at 5000 rpm then washing with distilled water and drying at 50°C for 16 hr in vacuum oven. Preparation of mung bean protein concentrate (PC)

The protein was extracted twice with 0.5% NaCO3 according to Sathe and Salunkhe (1981). The obtained supernatant was dialyzed using dialysis tube cutt-of (Molucular weight 2000 dalton) against distilled water for 72 hr with six changes of distilled water, then dried in vacuum oven at 50°C for 16 hr. Both PI and PC were ground to pass a 80 mesh sieve. Analytical methods Chemical composition Moisture (14.004), fat (14.018), ash (14.006), crude fiber (14.020) and protein N Χ6.25 (14.026) were determined as described by AOAC (1990). Protein fractionations The Osborne classification of protein was done according to the method of Abd El-Aal et al. (1986) using distilled water, 1 M sodium chloride, 70% ethanol and 0.2 M sodium hydroxide solutions to extract albumins, globulins, prolamins and glutelins, respectively. The obtained protein fractions were dried in vacuum oven at 50°C for 16 hr. Kjeldahl method (AOAC, 1990) was used to determine the protein in the extracted flour residue.



20

Antinutritional factors Total tannins (9.098) were determined calorimetrically as described in AOAC (1990). Phytic acid was determined according to the method of Wheeler and Ferrel (1971). Haemagglutinin activity was estimated according to the method of Liener and Hill (1953). Trypsin inhibitor activity was determined according to the method of Kakade et al. (1969) using benzoyl-DL-arginine-P-nitroanalide hydrochloric as the substrate. Flatulence factors (raffinose and verbascose) were determined according to Tanaka et al. (1975) using thin layer chromatography. Amino acids Amino acids were determined using a Mikrotechna AAA 881 automatic amino acid analyzer (Model 118 /119 CL, Czech Republic) according to method of Moore and Stein (1963). Hydrolysis of the samples was performed in the presence of 6 M HCl at 110°C for 24 hr under a nitrogen atmosphere. Sulfur-containing amino acids were determined after performic acid oxidation. Tryptophan was chemically determined by the method of Miller (1967). In-vitro protein digestibility (IVPD) In- vitro protein digestibility was determined as described by Salgó et al. (1984) by measuring the change in the sample solution pH after incubation at 37°C with trypsin-pancreatin enzyme mixture (Sigma Chem. Co.,St. Louis, Mo., USA) for 10 min. Computation of protein nutritional quality Computation of protein nutritional quality of mung bean products was determined on the basis of amino acid profile. Chemical score of amino acids was calculated using the FAO/WHO (1973) reference pattern. Essential Amino Acid Index (EAAI) was calculated according to Oser (1959) using the amino acid composition of the whole egg protein published by Hidvégi and Békés (1984). Protein efficiency ratio (PER) was estimated using the regression equation proposed by Alsmeyer et al. (1974): PER = - 0.468 + 0.454 (leucine) - 0.105 (tyrosine). Functional properties Protein -pH solubility profile One gram of F was dispersed in 20 ml of aqueous solvent and the pH adjusted in the range of 1 to 12 with 0.5 M HCl or 0.5 M NaOH. The suspension was shaken for 1 hr at room temperature (~ 25°C) then centrifuged at 4000 rpm for 20 min. The pH and nitrogen of clear supernatant were determined. Protein solubility index (PSI) in different solvents PSI was estimated in water at pH 9 and 12, 5%NaCl and 0.02M NaOH as described by Smith et al.(1959). Other functional properties Water and fat absorption were estimated according to the procedure of Sosulski (1962) and Sosulski et al. (1976), respectively. Emulsification capacity (ml oil/ gm protein at pH 9) was determined as described by Beuchat et al. (1975). The method of Lawhon et al. (1972) was used to determine the foam capacity and stability at pH 9.0 using 1% protein



21

solution. The percentage increase in volume after 30 sec was recorded as foam capacity. The change in volume of foam after 15, 30, 45 and 60 min of standing at room temperature (~ 25°C) was recorded as foam stability. Statistical analysis Results are expressed as the mean value ± standard deviation (SD) of three replicates, except for amino acid contents, which were determined in duplicate. Data were statistically analyzed using analysis of variance and least significant difference using SAS (1985). Significant differences were determined at the p < 0.05 level.

RESULTS AND DISCUSSION

Proximate composition of mung bean flour and proteins Table (1) shows that PI had a significantly higher (p<0.05) protein and ash contents than PC; while the fat and crude fiber contents of both PI and PC were similar (p>0.05). The protein content of PI was 6% higher than PC. Fan and Sosulski (1974) observed lower protein content (88%) of mung bean isolate preparations than that of PI (92.12%). On the other hand, the results of this study were in agreement with those reported by Thompson (1977 ).

Table (1) Chemical composition of mung bean flour (F), protein isolate (PI) and concentrate (PC).

Components F PI PC Total protein (N× 6.25) 27.21c±0.26* 92.12a±0.53 86.16b±0.51 Total fat 1.93a±0.10 0.21b±0.10 0.26b±0.10 Crude fiber 6.12a±0.30 0.19b±0.04 0.22b±0.03 Total ash 3.87b±0.10 4.94a±0.13 3.51c±0.10 Total carbohydrates** 60.87a±0.80 2.54c±0.20 9.85b±0.30 *Mean ±SD (n = 3). Means in the same row with no common superscript letter are significantly different (p < 0.05). ** By difference

Protein fractions Classification of mung bean flour proteins according to the method of Osborne (1924) is shown in Table (2). Significant differences (p<0.05) were observed among all protein fractions of mung bean flour. Albumins represented the major (p<0.05) protein fraction (47.34%) followed by globulins (19.52%) and glutilins (15.39%). These data indicated that mung bean flour has a protein of low molecular weight which could be functional. According to Adele (1975), the seed proteins are of two types, metabolic proteins which are normally of low molecular weight and storage proteins, mainly



22

globulins. According to this criterion metabolic protein represent about 50% of mung bean protein. Table (2) Protein content of mung bean fractions.

Protein fraction Protein (%) Protein recovery (gm/ 100gm total protein) Albumins 12.88a±1.73* 47.34 Globulins 5.31b±0.40 19.52 Prolamins 3.75d±0.31 13.78 Glutilins 4.19c±0.32 15.39 Residual protein 1.08e±0.16 3.97 *Mean ±SD (n = 3). Means in the same column with no common superscript letter are significantly different (p < 0.05). Antinutritional compoonents and in-vitro protein digestibility Table (3) shows that the F contains low level of trypsin inhibitor, hemagglutinine and phytic acid compared with those reported by Elkowicz and Sosulski (1982). These differences may be due to species variation. However, phytic acid content is higher than those noted by Tabekhia and Luh (1980). Isolation and concentration processes of proteins caused a significant reduction (p<0.05) in all antinutritive components. PI had lower (p<0.05) contents of tannin than that of PC. Both PI and PC were free from hemagglutinine activity and flatulence factors (raffinose and stachyose). Anderson et al. (1979) reported that protein isolates were free from flatulence factors.

In-vitro digestibility of PI and PC was significantly higher (p<0.05) than F. Generally, the high in-vitro digestibility of PI and PC may be due destruction of trypsin inhibitor or reduction of tannins. Barroga et al. (1985) reported that the tannins play an important role in the reduction of protein digestibility of mung beanflour.

Table (3) Antinutritional components and in-vitro digestibility of mung bean flour (F), protein isolate(PI) and concentrate (PC).

Property F PI PC Tannin (mg/ gm sample) 3.60a±0.51* 1.37c±0.40 1.96b±0.30

Reduction (%) ---- 61.94 45.56 Phytic acid (mg/ gm sample) 3.12a±0.32 1.41b±0.24 1.63b±0.28 Reduction (%) ---- 54.08 47.76 Trypsin inhibitor (TUI/ mg protein) 16.30a±1.10 4.10b±0.60 4.90b±0.95 Reduction (%) ---- 74.85 70.55 Hemagglutinine (HU/ gm sample) 1200a±0.0 0.00b±0.0 0.00b±0.0 Reduction (%) ---- 100 100 Raffinose (mg/ gm sample) 5.20a±0.60 0.00b±0.0 0.00b±0.0 Reduction (%) ---- 100 100 Stachyose (mg/ gm sample) 13.01a±1.60 0.00b±0.0 0.00b±0.0 Reduction (%) ---- 100 100 In-vitro protein digestibility (IVPD) 74.32b±1.30 79.32a±1.65 78.83a±1.73 *Mean ±SD (n = 3). Means in the same row with no common superscript letter are significantly different (p< .05)



23

Amino acid profile Amino acid composition of the F, protein fractions, as well as PI and PC are presented in Table (4). The F had the highest level of lysine, isoleucine and total aromatic amino acids compared with these reported by FAO/WHO (1973). Among all protein fractions, albumins contained the highest amounts of lysine and threonine, and lowest contents of isoleucine, leucine, tryptophan and valine. Total aromatic amino acid content was the highest in globulins, whears having the lowest isoleucine content. Leucine and isoleucine were the highest in prolamin and glutelin, respectively. PC was the highest in total sulphur amino acid and valine than F, but PI had greater proportion of leucine, lysine and tryptophan than F and PC. Relative to the FAO/WHO (1973) pattern, all samples especially PI and PC were rich in the total and most essential amino acids and poor in total sulphur amino acid. Nutritional quality

Table (5) shows that the chemical score (CS) value of the F (57.14%) was confirmed well with data reported by Khan et al. (1979), which found that the CS of mung bean varieties ranged from 55 to 58%. PC had the highest value of CS than that of the other mung bean protein products. Total sulphur amino acids (Cys + Met) was the first limiting amino acids in all mung bean protein fractions. The second limiting amino acid was threonine in most mung bean protein products and leucine in albumin. Khan et al. (1979) reported that the first and second limiting amino acids of mung bean flour were sulphur containing amino acid and threonine, respectively. The essential amino acid index (EAAI) was high for PI (70.43) and was low for glutelin and albumin (64.13, 64.14, respectively). The protein efficiency ratio (PER) of glutelin, prolamin and PI was almost the same and was higher than the other mung bean protein products. Functional properties

The protein solubility profile of mung bean vs pH is illustrated in Fig (1). It has a V-shaped pattern, with one sharp isoelectric point at pH 4.5. The high protein solubility at alkaline pH side and minimum at a pH of about 4.5. The solubility increased slowly between pH 8.0 and 12.0. The pH 9.0 was chosen as the suitable pH for future protein extractions to avoid the darkening of protein isolates at high alkaline pH. The present results confirmed well with those reported by Thompson (1977) and Sobihah (2004) for mung bean protein flour, which found that the high protein solubility of mung bean flour was at alkaline pH side and minimum at a pH of about 4.5.



24

Table (4) Amino acid profiles of mung bean flour (F), protein fraction, protein isolate (PI) and concentrate (PC).

Amino acid F PI PC Albumin Globulin Prolamin Glutelin FAO/WHO (1973)

Isoleucine 4.5 4.2 4.2 3.0 3.0 3.9 5.1 4.00 Leucine 7.8 8.0 7.0 5.6 6.7 8.1 8.0 7.00 Lysine 6.3 6.8 6.6 9.1 6.3 6.7 6.8 5.50 Cystine 0.6 0.5 0.9 0.8 0.6 0.8 0.4 - Methionine 1.4 1.7 1.7 1.6 1.5 1.1 1.0 - Total sulfur amino acids

2.0 2.2 2.6 2.4 2.1 1.9 1.4 3.5

Tyrosine 2.9 3.0 3.0 2.8 4.9 3.1 2.4 - Phenylalanine 5.6 5.5 5.5 4.8 5.9 5.4 4.7 - Total aromatic amino acids

8.5 8.5 8.5 7.6 10.8 8.5 7.1 6.00

Threonine 3.0 2.8 3.4 4.5 2.6 2.5 2.7 4.00 Tryptophan 1.0 1.7 0.9 0.9 1.6 1.1 1.1 1.00 Valine 5.0 5.2 5.4 4.5 4.6 4.9 4.9 5.00 Total essential amino acids

38.1 39.4 38.6 37.6 37.7 37.6 37.1 36.00

Histidine 2.6 2.2 2.3 2.1 2.7 3.4 2.4 Arginine 6.8 5.3 6.2 6.6 6.9 8.0 6.4 Aspartic acid 11.3 11.9 10.9 9.6 11.3 10.6 13.6 Glutamic acid 18.6 16.2 15.1 14.4 17.9 19.2 19.9 Serine 4.6 3.9 5.9 5.0 6.2 5.5 4.5 Proline 4.2 6.5 5.2 4.8 4.6 3.3 4.0 Glycine 3.7 3.5 4.9 5.4 3.2 3.8 3.4 Alanine 4.2 6.3 6.6 7.3 4.1 4.5 3.9 Total non- essential amino acids

56.0 55.8 57.1 55.2 56.9 58.3 58.1

Table (5): Nutritional evaluation of mung bean flour (F), protein fraction, protein isolate (PI) and concentrate (PC).

Nutritional parameter F Albumin Globulin Prolamin Glutelin PI PC Chemical Score (CS) 57.14 68.57 60.00 54.29 40.44 62.86 74.29

First limiting amino acid

Cys + Met

Cys + Met

Cys + Met

Cys + Met

Cys + Met

Cys + Met

Cys + Met

Second limiting amino acid

Thr (75) Leu (80) Thr (65) Thr (62.5)

Thr (67.5)

Thr (70) Thr (85)

Essential amino acid index (EAAI)

66.60 64.14 65.66 65.12 64.13 70.43 68.31

Protein efficiency ratio (PER)

2.77 1.78 2.06 2.88 2.91 2.85 2.30



25

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

0 2 4 6 8 1 0 1 2 1 4

p H

%So

lubl

epr

otei

n

Figure (1): Effect of pH on the protein solubility of mung bean flour.

Some functional properties of F, PI and PC are shown in Table (6). The protein solubility index of F was high in all solvents used for extraction. However, PI and PC solubility was significantly (p < 0.05) high at pH 9 and 12 as well as in 0.02% NaOH solutions and significantly low in 5% NaCl compared to F. Water absorption capacity of F was significantly higher (p < 0.05) than PI and PC. The high value of water absorption for F may be due to the presence of carbohydrates such as starch, which absorbs more water (Table 1). Fat absorption capacity of F was significantly lower (p<0.05) than PI and PC. The high values of fat absorption for both PC and PI may be due to alteration of the protein during isolation and concentration which could produce structure with more oil-binding sites. Both PI and PC performed better (p<0.05) than F in emulsification capacity at pH 9. Kinsella (1976) reported that emulsification capacity of proteins depends upon the solubilized protein in the solutions.



26

Table (6) Some functional properties of mung bean flour (F), protein isolate (PI) and concentrate (PC). Functional properties F PI PC

Protein solubility index: pH 9 90.02b±1.60* 95.17a±1.31 96.61a±1.41

pH 12 95.11b±1.30 97.15a±1.60 98.10a±1.50 5% NaCl solution 77.31a±1.40 8.19b±0.63 8.63b±0.70 0.02% NaOH solution 95.93b±1.20 97.91a±1.31 97.61a±1.42 Water absorption (%) 214.60a±2.81 144.50c±1.92 156.11b±1.81 Fat absorption (%) 94.10c±0.90 98.30b±0.60 145.20a±1.10 Emulsification capacity at pH 9 (ml oil/ gm protein)

210.61c±3.20 254.06b±0.06 269.30a±3.40

*Mean ±SD (n = 3). Means in the same row with no common superscript letter are significantly different (p<0.05).

Fig. (2) shows the foam capacity and stability of F, PI and PC at pH 9. F (60 ml) had lowest foam capacity than PI (116 ml) and PC (124 ml). El-Adawy et al. (2001) observed highest foam capacity of lupin seed protein isolate and concentrate than lupin seed protein flour. The same trend of foam capacity was observed for foaming stability, which was higher for both PC and PI than for F. In general, foam stability tend to decrease with time at room temperature, which may be due to collapsing and bursting of the formed air bubbles.

0

20

40

60

80

100

120

140

FC 15 30 45 60Standing time (min)

Foam

volu

me

(ml)

F

PI

PC

Figure (2): Foam capacity (FC) and stability of mung bean (F), protein isolate (PI) and concentrate (PC) at pH 9.



27

CONCLUSION This study revealed that both mung bean protein isolate and concentrate had a higher nutritive value due to decreasing the antinutritive materials compared to mung bean flour. The data obtained will be useful for using both mung bean protein isolate and concentrate as nutrient substitution or supplementation and as functional agent in food systems.

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Oser, B. L. (1959). An integrated essential amino acid index for predicting the biological value of proteins. In Protein and Amino Acid Nutrition, ed. Albanese, A. A. Academic Press, New York, pp. 295-311.

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Thompson, L. U. (1977). Preparation and evaluation of mung bean protein isolates. J. Food Sci. 42: 202-206.

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30

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Ahmad H. Alghamdi, Ali F. Alghamdi and Abdulrahman A. Alwarthan


31

ICP-MS Simultaneous Determination of Some Essential Minerals and Heavy Metals in Some Commercial Drinks Consumed in Riyadh City

Ahmad H. Alghamdi, Ali F. Alghamdi and Abdulrahman A. Alwarthan Department of Chemistry, College of Science, King Saud University

P.O Box 2455, Riyadh- 11451 Tel.: +966-14676001, Fax: +966-14675992.

[email protected] E-mail: ABSTRACT. Concentrations of several essential elements (Ca, Mg, Na, K, Zn, Mn, Fe, Cu, Co, Cr and Mo) and potentially toxic heavy metals (Pb, Cd, As and Sb) in different juice beverages and soft drinks purchased in Riyadh city were measured, primarily to assess whether their daily intakes through the consumption of these drinks comply with the recommended desired levels for essential minerals and permissible levels for toxic metals. These analytical data were obtained via the employment of an Inductively Coupled Plasma/Mass Spectroscopy (ICP-MS) technique. Data reported in the present study reveals that all toxic heavy metals are within or even considerably lower than the maximum authorized levels. Keyword: ICP-MS, Essential and heavy metals. Soft drinks, Fruit juice beverages. _________________________________________________________________________

INTRODUCTION In recent years, there has been a growing demand for evaluating the nutritional value and level of toxic and heavy metal contamination in various foodstuff, practically in soft drinks products, which are becoming customarily displacing other more nutritional food choices in people’s diets such as milk and fruit juice and even water (Calvadini et..al., 2000, Lytle et al., 2000). Accordingly, information on trace element concentrations in various food products is getting increasingly important in order to sustain the human health as well as from the food quality control point of view.

Today, soft drinks and beverages with added fruit juice are widely consumed in the diet by most segments of the population for modern societies. Thus, daily consumption of beverages contributes significantly to the requirements of the human organism of essential elements. Similarly, the daily excessive intakes of these beverages might also contribute to overall exposure (through ingestion) of human body to toxic heavy metals. As a result, the determination of levels of essential minerals and possible heavy metals in different kinds of foodstuffs for assurance of food nutritional quality and safety is becoming of vital importance.

The content levels of several elements in drinks (juice, carbonated soft drink, milk, tea, instant coffee .etc.) has been determined by various analytical techniques such as flame atomic absorption spectroscopy (Tripathi et al.,2002), electrothermal atomic absorption spectroscopy (Tripathi et al., 1999), cathodic stripping voltammetry (Colombo and Van den Berg , 1997), ion chromatography (Trifiro et al.,1996), capillary zone electrophoresis (Luque et al., 2006) and X-ray fluorescence technique (Haswell et al., 1998).

Inductively coupled plasma-mass spectroscopy (ICP-MS) technique can be applied in almost all the analytical chemistry areas. The wide use of this technique for analysis of different kinds of matrices may be attributed to its remarkable sensitivity, multi-elemental

ICP-MS Simultaneous Determination of Some Essential Minerals…………


32

capability and to the fact that the analytical curves are generally linear over 4-6 orders of magnitude (Broekaert 2002, Ebdon and Evans, 1998, Lajunen 1992). Accordingly, ICP-MS is one of the best analytical techniques for characterisation of the elemental composition of samples and offers a unique tool for food analysis. Hence, ICP-MS technique was a very effective analytical technique for trace and ultratrace determinations of numerous chemical elements in various foodstuffs ([de Sousa et al., 2005, Fernandez et al., 2002, Lante et al., 2006, Lara et al., 2005, Montesinos et al., 2005, Bratter et al., 1995, Mesallam, 1987).

The objective of this research study was to determine the content levels of both the various essential nutritional elements (Ca, Mg, Na, K, Zn, Mn, Fe, Cu, Co, Cr and Mo) and some heavy metals (Pb, Cd, As and Sb) in 12 commercial drinks available in Riyadh city local markets. Additional objective of this study was also to estimate the daily intakes of these elements in order to evaluate the nutritional value and toxic metal contamination status of the investigated foodstuffs.

EXPERIMENTAL Apparatus The analytical determination of the studied elements was carried out by ICP-MS: ELAN 9000 (PerkinElemer SCIEX Instruments, Concord, Ontario, Canada) equipped with a peristaltic pump used for sample introduction and \auto-sampler model AS 90 from Perkin Elmer used for auto sample transport. Samples were nebulized using a PE cross flow nebulizer. A Pentium digital computer was used to control the instrument and data acquisition, manipulation and storage. A solution having Mg, Rh, In, Pb and U (for most applications these elements are selected to cover the whole range of masses) at 10 ng g-1 was used to optimize the instrument in terms of sensitivity, resolution and mass calibration. Table (1) highlights the operating conditions of the instrument used in this study.

Table 1: Operating conditions of ELAN 9000 ICP-MS Description Value ICP RF Power 1300 W Nebulizer gas flow 0.76 L/min Lens voltage 13.0 V Sample flow rate 3 ml/min Analog stage voltage -2431.50 V Pulse stage voltage 1550 V Discrimination threshold 50.0 mV AC Rod offset -6.50 V Number of replicates 5 Readings/Replicates 1 Sweeps/Readings 30 Scan Mode Peak Hopping Dwell Time 40.0 ms Integration 1200 ms Internal standard Rhodium (103Rh)



33

Reagents, reference material and samples Analytical reagent grade chemicals were used in this study without further

purification. These include, nitric acid (69% v/v), Super Purity grade from Romil, England. Hydrochloric acid (37% v/v) and hydrofluoric acid (40% v/v) were super pure reagents from Merck, Germany. To evaluate the analytical performance of the utilized analytical technique a Standard Reference Material (SRM), namely SRM 1640 composed of natural fresh water was used. This standard reference material was purchased from the National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA. High purity water (DDW) (specific resistively 18 MΩ.cm-1) obtained from a Millipore Milli-Q water purification system was used throughout the work. Rhodium single element 1000 mg/L in 10% (v/v) HCl certified standard from Perkin Elmer, USA was used as an internal standard.

Different brands of very popular soft drinks and beverages with added fruit juice samples were purchased from local supermarkets in the city of Riyadh. Soft drinks mostly consist of carbonated water, sugar, coloring agents, citric acid, and other food additives such as flavors and preservatives. Beverages with added fruit juice contained the previous ingredients in addition to concentrated syrups, vitamin C, pectin and other additives. The total of 12 beverage samples investigated in this study were categorized as: soft drinks (8 samples) and beverages with added fruit juice (4 samples). Procedure

The analysed soft drinks and beverages with added fruit juices were subjected to mild clean-up procedures such as degassing with ultrasonic bath and paper filtration. For the soft drink samples, after dilution and acidification with nitric acid, part of each diluted solutions were aspirated into ICP-MS instrument for the direct analysis without any further preparation steps such as extraction or concentration. However, for the beverages with added fruit juice a pre-treatment microwave digestion method utilizing acid mixture was applied. All quantitative measurements for the studied elements were repeated five times. All the plastic and glassware were cleaned by soaking in dilute HNO3 and were rinsed with deionized water prior to use.

RESULTS AND DISCUSSION Validity of the applied analytical method Before the applied plasma emission spectroscopic method (ICP-MS) could provide useful analytical information for real samples, it was necessary to demonstrate that it is capable for providing acceptable analytical results. The analytical performance of the applied method can be verified via the evaluation of its precision and accuracy characteristic. The accuracy of the used analytical method was assessed via the analysis of standard reference material SRM 1640 and the obtained analytical results were compared with the certified values. As can be seen from Table (2), the obtained analytical result was generally in good agreement with NIST certified values. The accuracy of these analytical results was also checked via the F statistical test and since the calculated F values are less



34

than the critical value of F (6.4) at the 95% confidence level for five repeated measurements, there is no statistical difference between the measured and the certified values [Miller and Miller, 1994]. In terms of precision, the reproducibility of the employed analytical technique was demonstrated from estimation of the relative standard deviation (% RSD), which was obtained from five consecutive replicates measurements of SRM. The calculated % RSD values for the determined trace elements is also presented in Table (2).

Table 2: ICP-MS analytical results obtained for the certified reference material Precision (RSD%)**

F test Value*

Recovery% Certified value (ppb)

Measured value (ppb)

Element

1.23 5.2102.9%52.0 ±1.5 53.5 ±0.66 Al0.71 1.699.9%121.5 ± 1.1 121.4 ±0.86 Mn1.6 2.4102.9%29350 ±310 30200 ±481 Na1.9 3.5100.9%5820 ±60 5870 ±113 Mg4.3 2.7104%50.7 ±1.4 53.0 ±2.3 Li3.9 4.7103.4%26.7 ±0.51 27.6 ±1.1 As4.4 4.5103.3%148.2 ±3.2 153.2 ±6.8 Ba3.4 1.7102.5994 ± 271019 ±35 K0.8 2.2102.97450 ±89 7670 ± 60Ca3.3 5.2105.820.3 ± 0.31 21.5 ±0.71 Co5.4 4.395.4%85.2 ± 2.181.3 ±4.4 Cu

* The critical F value at 95% Confidence Level for five repeated measurements is (6.4) ** Relative Standard Deviation

Analysis of the selected commercial drinks After ensuring the accuracy and precision of the applied instrumental technique, it

was employed for the simultaneous determination of the investigated trace element in the selected commercial drinks. Although the employed instrumental technique (i.e ICP-MS) provided the analytical results for twenty eight different elements (Al, Be, Na, Mg, B, Li, As, Ba, Cd, Cr, Co, Fe, Pb, Mn, Mo, Se, Ag, Sr, V, Cu, Ni, K, Rb, Zn, Ca, Ti, Sn and Sb), only the concentration level of some essential minerals (Ca, Mg, Na, K, Zn, Mn, Fe, Cu, Co, Cr and Mo) and some heavy metals (As, Cd, Pb and Sb) will be represented and discussed. The analytical results obtained for the analysis of the selected elements in eight different commercial soft drinks available in Riyadh city local markets are presented in Table (3), whereas, Table (4) summarized the analytical results for these elements in four selected beverages manufactured in Riyadh city.



35

Table 3: Content levels of some minerals and heavy metals in some commercial soft drinks in Riyadh markets*.

Strawberry Berry Apple Orange Diet Lemon

Lemon Diet Cola

Cola Conce-ntration (ppb)

23443 ± 34

25596 ± 26

30181 ± 7.2

23682 ± 63

18140 ± 18

31338 ± 12

17781 ± 19.5

33052 ± 15.2

Ca

6693 ± 26

5426 ± 4.6

6136 ± 7.6

5148 ± 10

3506 ± 8.7

8267 ± 1.9

3215 ± 4.8

8720 ± 4.5

Mg

126736 ± 58

207459 ± 11

96675 ± 53

93250 ± 28

157989 ± 56

183136 ± 72

69767 ± 6.8

73224 ± 12

Na

14155 ± 16

9088 ± 9.7

100016 ± 43

27512 ± 41

28898 ± 42

10011 ± 18

17323 ± 46

10886 ± 21

K

266 ± 9

589 ± 0.6

312 ± 1.6

217 ± 0.74

375 ± 2.6

248 ± 0.71

1018 ± 2.9

591 ± 3.8

Zn

51 ± 0.9

100 ± 0.7

178 ± 0.8

451 ± 8

110 ± 1.2

25.9 ± 0.58

176 ± 1.5

336 ± 6.4

Mn

5687 ± 6

7484 ± 4.7

7591 ± 14

4988 ± 14

6590 ± 4.8

5343 ± 4.2

6746 ± 8.5

6153 ± 41

Fe

149 ± 4.1

621 ± 2.2

360 ± 0.9

110 ± 1.5

422 ± 2.5

220 ± 3.1

145 ± 0.9

587 ± 03.2

Cu

1.01 ± 0.01

2.4 ± 0.02

1.4 ± 0.1

1.08 ± 0.1

2.4 ± 0.09

0.74 ± 0.02

0.74 ± 0.03

1.55 ± 0.7

Co

302 ± 0.7

351 ± 1.4

354 ± 1.2

345 ± 6.3

22.1 ± 1.1

245 ± 1.3

0.0 ± 0

253 ± 1.8

Cr

37.9 ± 0.4

2.4 ± 0.3

41.1 ± 0.4

84 ± 0.58

7.2 ± 0.8

0.27 ± 0.03

12.1 ± 0.3

6.98 ± 0.54

Mo

127 ± 2.1

83.9 ± 1.2

37.7 ± 1.3

95.8 ± 0.1.4

76.2 ± 0.12

92.7 ± 1.14

74.2 ± 0.89

67.7 ± 0.48

Pb

14.1 ± 0.2

2.32 ± 08

11.9 ± 07

10.0 ± 0.03

80.9 ± 0.7

25.14 ± 0.76

5.1 ± .45

20 ± 1.4

Cd

0.0 ± 0.0

1.8 ± 0.08

0.0 ± 0.0

4.04 ± 0.04

0.0 ± 0.0

0.0 ± 0.0

1.35 ± 0.09

1.3 ± 0.5

As

0.51 ± 0.004

0.12 ± 0.004

0.3 ± 0.001

0.74 ± 0.04

0.147 ± 0.02

3.97 ± 0.08

0.75 ± 0.012

0.44 ± 0.032

Sb

* Means of five replications ± standard deviations.



36

Table 4: Content levels of some minerals and heavy metals in some commercial beverages in Riyadh markets*.

Tomato Cocktail Apple Orange Concentration (ppb)

54945 ± 1.8

18379 ± 18

33810 ± 52

29543 ± 34

Ca

59807 ± 26

12949 ± 34

12691 ± 25

15721 ± 56

Mg

2290339 ± 95

212613 ± 71

50529 ± 65

69486 ± 78

Na

1210484 ± 84

193591 ± 66

114105 ± 84

153487 ± 79

K

704 ± 0.5

608 ± 3

230 ± 2.8

351 ± 21

Zn

464 ± 11

1041 ± 14

259 ± 10

486 ± 32

Mn

11202 ± 42

6752 ± 11

14444 ± 29

14382 ± 46

Fe

546 ± 1

368 ± 7.8

280 ± 0.8

149 ± 2.6

Cu

5.5 ± 0.8

1.6 ± 0.02

1.9 ± 0.1

3.2 ± 0.9

Co

204 ± 0.8

340 ± 9

341 ± 30

355 ± 11

Cr

54.9 ± 3.2

0.46 ± 3

15.6 ± 0.9

62.4 ± 1.8

Mo

86 ± 1

61.2 ± 1.1

68.1 ± 7.3

53.4 ± 2.4

Pb

7.7 ± 0.7

4.1 ± 0.05

7.15 ± 1.8

10.7 ± 1.3

Cd

13.5 ± 0.5

5.8 ± 0.2

5.4 ± 0.5

3.6 ± 1.1

As

0.45 ±0.001 0.22 ± 0.01 0.23 ± 0.018.9 ± 0.8 Sb* Means of five replications ± standard deviations.

For all analysed commercial samples the concentration levels of the major elements (macroelements) such as K, Ca, Na and Mg were found to be much higher than the existed quantity of the trace elements (microelements) such as Zn, Mn, Fe, Cu, Cr, Mo and Co (Tables 3 and 4). In fact, potassium element was found in very substantial quantities in all fruit beverage samples. Its highest concentration level in these samples was 121,048,4 ppb (µg L-1) in Tomato beverage whereas its highest concentration in soft drink samples was 100,016 ppb concentration level for soft drink sample with apple flavour (i.e. this potassium content level in soft drink is nearly less than one tenth that in the juice beverage). However, with the exception of the tomato beverage, the sodium element



37

concentrations in the analyzed foodstuff samples varied less significantly between the fruit beverages (highest value is 212,613 ppb/Cocktail beverage) and soft drinks (highest value is 207,459 ppb/Berry soft drink). Similar concentration pattern was noticed for the concentration levels of calcium major element (highest concentrations were 338,10 ppb and 330,52 ppb for apple juice beverages and Cola soft drinks, respectively). Once again, with the omission of tomato beverage, it was found that the magnesium highest content (157,21 ppb/Orange beverage) in The analytical fruit beverages is nearly twice the highest content of this macroelement in soft drink samples (872,0 ppb/Cola soft drink ). In the case of iron element it was found to endure similar concentration pattern to that observed for Mg element. The iron highest content (144,44 ppb/Apple beverage) in juice beverages is nearly twice the highest content of this metal ion in soft drink samples (759,1 ppb/apple soft drink). With regard to the quantity levels of the trace heavy metals (Zn, Mn, Cu, Co, Cr and Mo) presented in the investigated commercial drinks it was concluded that on the whole there was no significant variation between the highest concentration levels of these heavy metals in fruit beverages and soft drinks. For examples, the highest cupper quantity in juice beverages was found in tomato beverage (546 ppb) whereas the highest copper concentration level in soft drink samples was in berry soft drink (621 ppb). In addition, the variation of the concentration levels of some toxic heavy metals (Pb, Cd, As and Sb) between the different analysed foodstuff sample was also evaluated. From these toxic elements, only Pb element was found to be present at relatively high concentration levels in most of the analysed commercial drinks especially in soft drink samples. The highest Pb content level (127 ppb) was found in Strawberry soft drink sample and the lowest Pb content level was detected in apple soft drink sample (37.7 ppb). For most of the analysed soft drink and juice beverages, Cd toxic metal concentrations were below 20 ppb level. However, both As and Sb were found to be present in very low concentration levels since their highest content level were 13.5 ppb (in tomato beverage) and 8.9 ppb (in orange beverage), respectively. Comparison of obtained results with reported values

In this study, potassium content levels were found in very considerable quantities (in comparison with other essential minerals) in all juice beverages. Similar high concentration levels for potassium mineral were detected in various canned juices commonly produced and consumed in Saudi Arabia (Al-Muhanna et al., 1989, Ewaidah, 1990). Unfortunately, to date few analytical research studies were devoted for evaluating food value for soft drinks in terms of their content levels of essential minerals. In contrary, most of the reported literature aimed to evaluate the extent of contamination of soft drinks and beverages by heavy metals (Al-Howaidy and Mohammed, 1997, Al-Swaidan, 1988, Onianwa, et al., 1999). The concentration levels of some trace heavy metals such as Zn, Fe, Cu, Co and Cr in one of the cocktail beverage brand name sample were previously investigated (Al-Mohemmed, 2001). Due to the differences in the manufacturing dates for the cocktail beverage sample analysed in this study (2004) and the previous study (2001), hence, the food and chemical compositions of the analyzed two samples might be fluctuated slightly. In general, the content levels of these trace heavy metals obtained in



38

our study were higher than those reported in the previous study. In addition, a brief comparison for the content levels for some of these essential minerals and heavy metals in juice beverages and soft drinks consumed in Saudi Arabia with reported levels (Onianwa et al., 1999, Luque et al., 2006, Oduoza, 1992) of these elements in similar commercial canned drinks in some other parts of the world was reveals that the content levels of some essential minerals particularly, K, Na and Ca are generally excited at higher levels in Saudi Arabian juice beverages. However, the overall variations of these essential minerals content levels between local and international soft drinks are less scattered. Maximum allowable limits

Table (5) illustrates a comparison between the maximum and average concentration levels of some heavy metals observed in this study with the maximum allowable limits (MAL) recommended by Saudi Arabian Standards Organization (SASO1997, SASO1978). The concentration levels for most elements were far below the recommended maximum allowable concentration limits except for iron heavy element in some samples. Based on the analytical results demonstrated above it can be conclude that the consumption of the commercial drink samples investigated in this study can be said to be very save regarding toxic trace metals content.

Table 5 : Concentrations (ppm) of heavy metals in fruit and soft drinks in Riyadh markets in comparison with the maximum allowable limits (MAL)*

maximum allowable limits Cocktail Juice

Apple Juice

Orange Juice

Soft drink

Maximum Concentration

Average concentration

Element

0.2 0.2 0.20.10.0140.004As0.3 0.3 0.30.20.1270.060Pb5.0 5.0 5.02.00.6210.330Cu5.0 5.0 5.02.01.020.459Zn15.0 10.0 15.00.514.4448.114Fe

* SASO (1997 and 1978).

ACKNOWLEDGEMENT The authors wish to express gratitude to Dr. Omar Al- Dayel from King Abdulaziz City for Science and Technology (KACST) for providing the support and cooperation to complete this work.



39

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Luque, S .S.; Mato, I.; Huidobro, J. F. and Lozano, J .S. (2006). Rapid capillary zone electrophoresis method for the determination of metal cations in beverages. Talanta. 68:1143-1147.

Lytle, L. A.; Seifert. S.; Greenstein, J. and McGovern, P. (2000). How do hildren’s eating patterns and food choices change over time?. Am. J. Health Promot. 14:222-228.

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Montesinos, P. C.; Cervera, M. L.; Pastor, A. and Guardia, M. (2005). Room temperature acid sonication ICP-MS multielemental analysis of milk. Anal. Chim Acta. 531:111-123.

Oduoza, C. F., (1992). Studies of food value and contaminations in canned foods. Food Chem. 44:9-12.

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Trifiro, A.; Saccani, G.; Zanotti, A.; Gherardi, S.; Cavalh, S. and Reschiotto, C. (1996). Determination of cations in fruit juices and purees by ion chromatography. J. Chromatog. A. 739:175-181

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