UNIVERSIDAD AUTONOMA DE NUEVO LEO FACULTAD DE CIENCIAS BIOLOGICAS

SUBD1RECCION DE POSTGRADO

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E X T R A C C I N Y C A R A C T E R I Z A C I O N Q U I M I C A , N U T R I C I O N A L Y F U N C I O N A L D E P R O T E I N A S D E

L A S E M I L L A D E Brassica campestris L .

TESIS

QUE COMO REQUISITO PARCIAL PARA OBTENER EL GRADO ACADEMICO DE DOCTOR EN CIENCIAS CON

ESPECIALIDAD EN ALIMENTOS

P R E S E N T A

MA. GUADALUPE DE JESUS ALANIS GUZMAN

MONTERREY N.L. FEBRERO DE 1993

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- -UUU / J 2 3 3

U N I V E R S I D A D A U T O N O M A D E N U E V O LEi FACULTAD DE CIENCIAS B I O L O G I C A ^

S I J B D I R F C C I O N D F P O S T G R A D O

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T R A C C I O N Y C A R A C I b R I 7 A C l N QU IM ICA , N U T H I C I U N A L Y F U N C I O N A L D E P H O l E I N A S Ub LA A L M I L L A E Brass ica campestris L.

T E S I S

QlJF C O M O R E Q U I S I T O PARCIAL P A R A O B T F N F R

EL G R A D O A C A D E M I C O D E

D O C T O R E N C I E N C I A S C O N E S P E C I A L I D A D EN A L I M E N T O S

P R E S E N T A

MA. G U A D A L U P E DE J E S U S A L A N I S G U Z M A N

M O N T E R R E Y N.L. F E B R E R O 1993

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U N I V E R S I D A D A U T O N O M A D E N U E V O L E O N FACULTAD DE CIENCIAS BIOLOGICAS

S U B D I R E C C I O N D E P O S T G R A D O

r R A C C I O N Y C A R A C T E R I Z A C I O N QUIM ICA, N U T R I C I O N A L Y F U N C I O N A L D E P R O T E I N A S D E LA S E M I L L A D E Brassica campsstris L.

Q U E C O M O R E Q U I S I T O P A R C I A L PARA O B T E N E R

EL G R A D O A C A D E M I C O D E

D O C T O R EN C I E N C I A S C O N E S P E C I A L I D A D E N A L I M E N T O S

P R E S E N T A

MA. G U A D A L U P E DE J E S U S A L A N I S G U Z M A N

T E S I S

C O M I S I O N D E T E S I S

Aprobada

DR. P E D R O W E S C H L E E L I N G (Director)

D R A MA. JUL IA V E R D E S T A R

DR RAT IKANTA MAITI

DR S E R G I O O . S E R N A SALD IVAR (Asesor Extemo)

NTERREY , N.L. M E X I C O F E B R E R O D E 1993

R H S U M I i N

Mn oste u a h . i j o su p l u m e el o b j e t i v o de c o n o c e r el p o t e n c i a l d e k> semi l l a d e

Bravien i. h i c o n t e n i d o de a m i n o c i d o s a f u t r a d o s y l is ina e s s u p e r i o r a

a n u l e s v l emimmosas . i . s m e j o r e s t arack-r .viieas h inc ionn l e s Fueron: c a p a c i d a d e s p u m a n t e ,

eNi.ibiUii.ul iU- !.i c \pu in . i \ ibsorcion d e a c e i t e , s i e n d o s u p e r i o r e s u u n a i s l a d a d e soya

A U S 1 k / \ ( I

T i l t tibjclive ol this research was to d e t e r m i n e the potent ia l of t he wild Brussica

cwnjic.sliis seed , as raw mater ial in the ob ten t ion ol' protein isolates, the chemica l , nutr i t ional

and toxicological character iza t ion of the seed and def fa ted f lour , finding in the seed a

content of 33% nil, 13% liber, 4 % ash and 2 6 % protein. The ylucosinolut.es w e r e found at

a low concent ra t ion of 0 .92% as well as the phyta tes of 32 n i g / l M g. T h e inact ivat ion of the

thioglucosidusc enzyme was made by heat ing ut 90 C for 18 seconds, ii was verif ied with

siniyrin as subs t ra te and de termining glucose as a final product . With the ex t rac t ion of oil

during a min imum period of 2 hours, the enzyme also lost its activity. T h e seeds f laked and

dehulled with sieve and air. were d e f f a t e n c d with ether , and the f lour o b t a i n e d had 3 .8%

crude f iber , 7 % ash and 48% prote in with a digestibility of 92.8%, and a nutri t ive va lue with

Tetriihyftwiic! tennophiUi (relative to casein), of 98%. The glucosinolates in t h e flour were

elevated to 2 .3% and the phytates to 67 nig/10 g. The minera les con ta ined in the seed as

well as in the f lour were Zn, Fe, Mn, P, Ca and Mg. Protein extract ion was m a d e from the

flour at d i f fe ren t pH (9. 6.5, 7, 7.5, 8 and 10.8) determinig the disolved p ro te in a n d the

phytic phosphorus in the spec t ropho tome te r , finding the maximum prote in ex t rac t ion (82,

80 y 7 4 % ) coinciding with a min imum quant i ty of phytates (70%) at p H 7, 7.5 a n d 8. T h e

extracts ob ta ined were analysed eJectroforetrcally in gels of polyacn'Janu'de with S D S (5 and

7.5%) f inding in each one of them 12 hands with a molecular weight of 10 to 74 K D a .

Extracts at p H 7 and 8. were precipi ta ted at p H 3.5, 4, 4.5, 5 and 5.5, ob ta in ing the

maximum prote in yield ( 5 ? % ) in the extract p H 7 al p H 3.5. Based on nut r i t ional

considerat ions in o rde r to select the p H of precipi ta t ion 4, with a yield of 3 6 % and 1.93%

of the initial phytates in the extract pH7 . T h e isolate ob ta ined had 9 3 % pro te in , with a

nutritive value relat ive to casein of 91%, ne i the r glucosinolates nor phyta tes w e r e d e t e c t e d

The content of su lpher aminoacids and lysine were grea ter than ce rea l a n d l eguminous

seeds. The best functional p r o b i t i e s were : whipping capacity and tbarn stability, and oil

ahsorption being grea te r than soy isolate. In conclusion the seed eva lua t ed had good

chemical composi t ion and that its prote in ic potential is very high nutr i t ionally.

I N D I C E .

Comalido Pgina

D E D I C A T O R I A

A G R A D E C I M I E N T O jj

R E C O N O C I M I E N T O iv

R E S U M E N v

A B S T R A C T vi

I N D I C E vii

I N D I C E D E C U A D R O S Y F I G U R A S r

I N T R O D U C C I O N 1

O B J E T I V O S 3

H I P O T E S I S D E T R A B A J O 3

O R I G I N A L I D A D 3

A N T E C E D E N T E S 5

I. Brassica campes tris. 5 la. Or igen y distribucin geogrf ica : 5 Ib. Or igen ci togeniico: 6 Ic. Es t ruc tura : 6 Id. Compos ic in qumica: 6 le. Aspec tos toxicolgicos: 9 11 C O N C E N T R A D O S Y A I S L A D O S P R O T E I N I C O S . 11 Ha. Usos: 11 Ilh. Potencia l idad de la p ro te na de Brassica

en tecnologa a l imentar ia : 12 lie. Funcional idad de las p ro te nas y

apl icaciones en a l imentos : 15

M E T O D O L O G I A : 17

I. C A R A C T E R I Z A C I O N F I S I C O Q U I M I C A D E LA S E M I L L A D E Brassica campes tris. 17

II. I N A C T I V A C I O N D E LA MIROS1NASA. 18 lia. Exper imen to 1 18 l lh. Exper imento 2. 19 [II T R I T U R A D O Y D E S C A S C A R I L L A D O . 19 IV. E X T R A C C I O N D E L A C E I T E Y D E S O L V E T I Z A D O . 19 V. S O L U B I L I Z A C I O N D E LAS P R O T E I N A S . 20 Va. Obtenc in J e las protenas. 20 VI. D E T E R M I N A C I O N E S Q U I M I C A S Y

E L F . C T R O F O R E T I C A S D E LOS E X T R A C T O S . 21 VII. P R E C I P I T A C I O N D E LAS P R O T E I N A S .

Seleccin de p H adecuado 21 VIH. L I O F I L 1 Z A C I O N . 2 2 IX. C A R A C T E R I Z A C I O N F I S I C O Q U I M I C A Y

N U T R I C I O N AL D E L A I S L A D O O B T E N I D O . 22

R E S U L T A D O S . 24

I . C A R A C T E R I Z A C I O N F I S I C O Q U I M I C A D E L A S E M I L L A Y P A S T A 24

II. I N A C T I V A C I O N DF. 1 A MIROS1NASA. 28 III. E X T R A C C I O N D E L A C E I T E Y D E S O L V E T I Z A D O . 28 IV. S O L U B I L I Z A C I O N D E LAS P R O T E I N A S . 29 V. E L E C T R O F O R E S I S D E LOS E X T R A C T O S . 30 VI. P R E C I P I T A C I O N D E LAS P R O T E I N A S . 32 VIL C A R A C T E R I Z A C I O N F I S I C O Q U I M I C A Y

N U T R I C I O N A L D E L A I S L A D O O B T E N I D O : 34

D I S C U S I O N E S . 37

I. C A R A C T E R I Z A C I O N F I S I C O Q U I M I C A D E L A S E M I L L A Y H A R I N A 37

II. I N A C T I V A C I O N DF. I A M I R O S I N A S A 43 III. T R I T U R A D O Y D E S C A S C A R I L L A D O . 45 IV. E X T R A C C I O N D E L A C E I T E Y D E S O L V E T 1 Z A D O . 45 V. S O L U B I L I Z A C I O N D E L A S P R O T E I N A S . 46 VI. E L E C T R O F O R E S I S D E LOS E X T R A C T O S . 47 Vil . P R E C I P I T A C I O N D E LAS P R O T E I N A S . 48 VIH. C A R A C T E R I Z A C I O N F I S I C O Q U I M I C A Y

N U T R I C I O N A L D E L A I S L A D O O B T E N I D O : 50

C O N C I . U S I O N K S

LH''IiRATURA CITADA

I N D I C E D E C U A D R O S Y F I G U R A S .

Pgina

C U A D R O 1 C A R A C T E R I Z A C I O N D E L A S E M I L L A D E B. ca ipes tris 26

CUADRO II C A R A C T E R I Z A C I O N D E LA H A R I N A D E S G R A S A D A DF. B. cumpcstris 27

GRAFICA 1 Proic ina y Fi tatos Solubles a Dit't'rt:me pH 29

FOTOGRAFIA I E L E C T R O F O R E S I S D E P R O T E I N A S N A T I V A S < E X T R A I D A S A D I F E R E N T E p H . 30

FOTOGRAFIA 2 E L E C T R O F O R E S J S D E P R O T E I N A S D E S N A T U -R A L I Z A D A S , E X T R A I D A S A D I F E R E N T E p H . 31

CUADRO III. P E S O S M O L E C U L A R E S ( K D a ) D E L A S P R O T E I N A S EN L O S E X T R A C T O S O B T E N I D O S 32

GRAFICA 2 Precipitacin de Prote nas a Di fe ren tes pH. 33

GRAFICA 3 Prutena y Flalos Precipi tados a p H 4. 34

CUADRO IV C A R A C T E R I Z A C I O N Q U I M 1 C A - N U T R I C I O N A L D E L A I S L A D O 35

CUADRO V P R O P I E D A D E S F U N C I O N A L E S D E A I S I A D O D E B. campesiris Y D E S O Y A 36

En iios recientes ha existido una