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Co nt ro l o f Lac tose Crys ta l l iza t ion in Du lce de Lech e by Be ta-D-Galac tos idaseAc t iv i ty f rom Permeabilized l u y v e r o m y c e s la c t i sCells

J O SE G E R A L D O S A B I O N I ~ D ~ .O JO SI~ R E Z E N D E P I N H E i R O ~ D A I S O N O L Z A N Y S I LVAJO SI~ B E N I C I O PA E S C H AV E S 2 a nd A R N A L D O C H A E R B O R G E S

Depar tamento de Bio logia Gera lUniversidade Federal de Vi~osa

3 6 5 7 0 Vi § o s a M G B r a zi l

A B S T R A C T

F e a s i b i l i t y o f c o n t r o l l i n g l a c t o s e c r y s -t a l l i z a t i o n i n d u l c e d e l e c h e b y p e r -m e a b i l i z e d Kluyv erom yces lactisce l l s wass t u d i e d. O p t i m u m t e m p e r a t u r e f o r l a c ta s ea c t i v i t y w a s 4 5 ° C . S t a b i l i t y o f t h e e n z y m es y s t e m r e m a i n e d p r a c t i c a l l y u n a l t e r e dd u r i n g s t o r ag e a t r o o m t e m p e r a t u r e o rb e l o w f o r u p t o 7 m o . D e g r e e o f la c t o s eh y d r o l y s i s i n m i l k w a s d e t e r m i n e d a s af u n c t i o n o f th e c o n t e n t o f p e r m e a b i l i z e dc e l ls . A d d i t i o n o f 4 0 0 o r 8 0 0 r a g / l i t e r o fp e r m e a b i l i z e d c el ls t o m i l k u s e d t o m a k e

d u l c e , f o l l o w e d b y 2 .5 o r 3 .5 h in -c u b a t i o n a t 3 5 ° C , r e s u l t e d i n h y d r o l y s i so f 4 5 . 0 2 a n d 6 9 . 1 3 % o f th e l a c t o s e .T h e r e w a s n o l a c t o s e c r y s t a l l i z a t i o n a f t e r1 2 0 d a y s s t o r a g e in p r o d u c t s t r e a t e d b yp e r m e a b i l i z e d c e ll s . T h e s e n s o r y q u a l i t i e so f t h e d u l c e s w e r e s i m i l ar t o t h o s e o ft h e c o n t r o l .

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

L a c t o s e c r y s t a ls in c o n c e n t r a t e d d a i r yp r o d u c t s s u c h a s c o n d e n s e d m i l k a n d d u l c e d el e c h e m a y c a u s e a s a n d y te x t u r e a n d r e d u c ec o n s u m e r a c c e p t a b i l i t y. C r y s t a l s t e n d t oa g g r e g a t e a n d a l t e r t h e p h y s i c a l c h a r a c t e r o f t h ep r o d u c t ( 2, 4 ). U n d e r n o r m a l c o n d i t i o n s f o rd a i r y p r o d u c t s , a l p h a - l a c t o s e i s t h e m a j o rd e t e r m i n a n t o f t h e n a t u r e a n d d e g r e e o f c ry s t a l -l i z a t i on (11 ) .

Va r i o u s s t u d i e s h a v e b e e n d i r e c t e d t o w a r dc o n t r o l l i n g la c t o s e c r y s t a l l i z a t i o n i n d u l c e d el ec he (1 , 4 , 9, 13 , 14 , 15 , 18 ). Va rg as (18 )t e s t e d d i f f e r e n t c o n c e n t r a t i o n s o f m i l k f a t,

Received September 2, 1983.1D epartam ento de Nutri~:~'o, UFO P, 35400,

Ouro P reto,MG Brazil.Depa rtamento de Tecnologia de Alimentos, U FV,

36570, Viffosa, MG , Brazil.

g l u c o s e , a n d s ta r c h i n f o r m u l a t i o n s o f d u l c e d el e c h e . S h e f o u n d t h a t 1 . 28 % f a t a n d a d d e ds t a rc h r e d u c e d c r y s t a l l iz a t i o n b u t d i d n o t a l t e rn o r m a l c h a r a c t e r i s t ic s o f t h e p r o d u c t . A d d i t i o no f g l u c o s e i n c r e a s e d i t s b r i g h t n e s s .

H o s k e n ( 4 ) , h o w e v e r, t e s t e d s e v en d u l c ef o r m u l a t i o n s a n d o b t a i n e d b e s t r e s u l t s w i t h

a d d i t i o n o f 2 % g l u c o s e ; a d d i t i o n o f s t a r c hr e d u c e d c r y s t a l l iz a t i o n b u t a l t e re d o t h e rc h a r a c t e r i s t i c s o f t h e p r o d u c t . Ve l a s c o , c i t e d b yS i l v a ( 1 4 ) , r e d u c e d n e t l a c t o s e i n d u l c e d el e c h e w i t h a d d i t i o n o f c a s e i n ( s o d i u m sa l t) .T h e p r o d u c t w a s s i m i la r t o t h e t r a d i t i o n a l o n e ,e v e n t h o u g h i t h ad l e s s l a c t o s e . S t u d i e s b yS o u z a ( 1 5 ) o n t h e e f f e c t o f a c i d i t y a n d h e a tt r e a t m e n t o f t h e m i l k i n d i c a t e d t h a t a c i d i t ya l o n e w a s n o t s u f f i c i e n t to p r e v e n t c r y s t a l -l i z a ti o n , w h e r e a s h e a t t r e a t m e n t r e d u c e d t h e

p r o b l e m .P a r t i a l h y d r o l y s i s o f l a c t o s e w i t h b e t a -

D - g a l a c t o s i d a s e w i l l p r e v e n t c r y s t a l l i z a t i o n i nc o n c e n t r a t e d d a i r y p r o d u c t s ( 1 , 9 , 1 4 ). T h eg a l a c t o se a n d g l u co s e p r o d u c e d b y h y d r o l y s i sa r e s o l u b l e in w a t e r a t c o n c e n t r a t i o n s o f 3 2 a n d5 0 % w t / v o l , a t 2 5 ° C ( 8 ) . To g e t h e r t h e y h a v eh i g h e r r e d u c i n g p o w e r t h a n l a c t o s e an d c a u s ea c c e l e r a t e d n o n e n z y m a t i c b r o w n i n g d u r i n g t h em a n u f a c t u r e o f d u l c e ( 8, 9 ).

C a s t e l o e t a l ., c i t e d b y S i l v a ( 1 4 ) , u s e d t h e

e n z y m e l a ct a se i n t h e m a n u f a c t u r e o f d u l c e d el e c h e a n d o b s e r v e d t h a t a b o u t 2 0% la c t o s eh y d r o l y s i s g u a r a n t e e d p h y s i c a l s t a b i l i ty o f t h ep r o d u c t f o r m o r e t h a n 1 2 0 d a y s a t r o o mt e m p e r a t u r e . T h e p r o d u c t h a d a d a r k e r c o lo rt h a n t h e c o n t r o l . C o e l h o ( 1 ) , i n a s i m i l a re x p e r i m e n t , u s i n g l a c t a s e a t 2 0 to 2 0 0 0 m g / l i t e ro f m i l k , o b s e r v e d b e s t r e s ul ts w i t h a d d i t i o n o f4 0 m g / l i t e r f o ll o w e d b y i n c u b a t i o n a t 3 4 °C f o r4 h . E x c e p t f o r c o l o r c h a n g e s, a l t e r a t i o n o fn o r m a l c h a r a c t e r is t i c s o f t h e d u l c e w e r e n o t

o b s e r v e d . S i l v a ( 1 4 ) , b y a d d i t i o n o f 4 0 m g / l i t e re n z y m e t o m i l k a t 8 ° C , f o l l o w e d b y in c u b a t i o na n d c o n t i n u o u s a g i t a t io n , o b t a i n e d a h o m o g -e n i z e d d u l c e d e l e c h e . T h i s p r o d u c t w a s

1984 J Dai ry Sc i 67 :22 10-22 15 221 0

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LACTOSE HYDROLYSIS 2211

similar to the tradit ional Brazilian dulce deleche in color, texture, and flavor and showedno sandiness after 90 days storage.

Luuvrema (9) described the advantages oflactose h ydrolysis and how en zymat ical ly totreat milk for its potential use in manufactureof dulce de leche .

Beta-D-galactosidase is marketed under thetrade nam e Maxilact R , (Gist Brocades,Holland). The enzyme is obtained from aspecial strain of the yeast Kluyveromyces lactis(3). Its high cost and Brazilian impor tingrestrictions make utilization by dairy industriesdifficult (4, 14).

Van-Dam et al. (17), treated cells of Saccbaromyces fragilis with ethyl alcohol andobtained a product with beta-D-galactosidaseactivity that was denot ed as permea bilizedcells .

The objective of this experiment was toinvestigate control of lactose crystalliz ation in

dulce de leche with beta-D-galactosidaseactivity in permeabilized Kluyveromyces lactiscells.

M T E R I L S N D M E T H O D S

P r e p a r a t i o n o f Kluyveromyces lactisP e r m e a b i l i z e d C e l ls

Yeast stock culture (1.0% peptone; 2.0%lactose; .5% yeast extract; 1.5% of agar; pH7.0), was used to inoculate .5 liter sterilizedskim milk in a 1000 ml Erlen meyer flask,followed by 24-h aerobic incuba tion at 30°C.This mother culture was inoculated into afer ment or cont aini ng 7.5 liters sterilized skimmilk. The fermentor, constructed by BiasinoxIndust ria e Com~rcio Ltda, Lambari, MG-Brazil,was equipped with aeration and agitationsystems. The skim milk was incubated for 12 hat 30°C with the agitator run ning at 170 rpmand aeration rate corresponding to one volumeof air per volume of media per minute (1vol/vol/min). After incubation the 8 liters ofculture was transferred to a 200-1iter stainlesssteel tank containing 92 liters of skim milk,previously heated to 90°C, then cooled to 30°C.The t ank was equipped with an agitation systemand an aeration system con necte d to an air com-pressor (Douat type - 40 liters) for injection offiltered air. To m inimiz e bacterial growth 5btg/ml each of streptomycin and chloram-phenicol were added. After 12-h incubation,

cells had acquired high growth and elevatedbeta-D-galactosidase activity. The medium wascooled to 2°C and maintained at that tem-perature until cells were harvested. Yeast cellswere separated from the milk by centrif ugatio nat 2800 x g for 5 min and maintaine d underrefrigeration for fur ther per meabil izatio n. Cell-ular permeabilization was achieved by 96%ethyl alcohol added to attain a final concen-tratio n of 80% in the mixture (17). The resulting}'east cell suspension was passed through aBuchner funnel. The precipitate was dried at40°C for 1 h in an oven and then stored in arefrigerator at 4°C. In this way the yeastcellular membrane became permeabilized tolactose.

Enzymatic properties of the permeabilizedcells were studied with respect to optimumtemperature for hydrolytic activity, temper-ature stability during storage, and degree oflactose hydrolysis as a function of variousconcentrations of permeabilized cells.

Optim um t emperature for hydrolytic activitywas determined by our suspending 1 mg/ml ofpermeabilized cells in distilled water. In cub ati ontemperatures were 2, 10, 22, 28, 35, 40, 45, 50,and 60°C. Three test tubes con taini ng 4.4 ml ofa Z buffer (10), .1 ml of the cell suspension,and .5 ml of 2.5 mM ortho-nitrophenyl-beta-D-galactoside (ONPG), were used for eachtemperature. Tubes were incubated for 15 rainat the chosen temperature, and the reaction wasstopped by additi on of 1.0 ml of 1 M Na2CO3to each tub e. Color intensit y was deter mined at420 nm with a Spectroni c 20. spec trop hotom eterwith 4.9 ml of a Z buffer solution and .1 ml cellsuspension as control. By concentration oforthonitrophenol (/lmoles/ml) plotted versusincubation temperature, optimum temperatureof hydrolytic activity was estimated.

Enzyme stability was determined as afunction of storage temperature and time.Three sealed test tubes, each containi ng abo ut3 g of permeabilized cells, were stored at 25°C(environmental), 5°C (refrigerator), and at-20°C (freezer). Weekly, and later monthly,the hydrolytic activity at 30°C was determinedas described.

Degree of lactose hydro lysis was deter minedin skim milk heated to 90°C and cooled to 2°C.Permeabilized cells at 0, 200, 400, 600, and1000 mg/liter were added. Suspensions for eachconcentration were divided into seven portions

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2212 SABIONI ET AL.

of 20 ml, on e for each incuba tio n period,transferred to 50-ml Erlenmeyer flasks con-taining the substrate and inc ubate d for .5, 1.0,1.5, 2.0, 2.5, 3.0, and 4.0 h at 35°C in a waterbath with slight agitation. After incubation, .3ml of acetic acid (1 + 9) was added. Eachmixture was agitated and then left in a steambath at 80°C for 5 min to precipitate milkproteins and inactivate the enzyme. Precipitatedprotei ns were separated by centrif ugatio n at2500 × g for 2 rain. One milliliter of thesupernatant fraction was transferred to a 50-mlvolumetric flask and the volume brought to 50ml with distilled water. The sample was storedat 4°C until analyzed. Lactose hydrolysis wasdetermined according to a method described byPotter (12). Lactose conte nt before hydrolysiswas determined as described by Teles et al.(16).

L a c to s e H y d r o l y s i s a n d P r o d u c t i o n o f D u l c e

Permeabilized cells were used to hydrolyzelactose in milk used for produ ctio n of dulcede l eche . Three hu ndre d and fifty liters ofmilk were skimmed by centrifugal separation,and the resulting skim milk and cream warmedto 90°C, cooled, and maintained at 2°C.Permeabilized cells at 400 to 800 rag/liter wereadded to the milk. For each concentration,hydrolysis was observed at the end of 2.5 and3.5 h at 35°C with a slight and continuousagitation to avoid ceil sedim entat ion. Afterhydrolysis, the treated mi lk (50 liters each) washeated to 80°C to inactivate the enzyme.Lactose hydrolysis was determined as describedby Potter (12).

Milk for the pro duc tion of dul ce wasstandardized to 2.5% fat (5) and acidity neu-tralized to .13% lactic acid (5)by additionof sodium bicarbonate. Dulce was made byadding 8 kg sucrose (commercial grade) to 50liters of milk and the mixture concentrated inan op en conc entr ator to 65 to 70 ° Brix asdetermined by a densitometer. The product wasplaced in 250-g glass cups, sealed with metallids, heated in live steam for 20 min, cooled bywater spray to about 50°C, and stored at roomtemperature.

S e n s o r y E v a l u a t i o n f o r F l a v o ra n d C r y s t a l l i z a t i o n

Dulce de leche's flavor accepta bility wasevaluated by a 10-membe r sensory panel using a

9-po int hedonic scale as follows: 1 - horribl e, 2- bad, 3 - obje ctionable , 4 - less than ac-ceptable, 5 - acceptable, 6 - more thanacceptab le, 7 - good, 8 - very good, and 9 -excellent according to the ASTM (AmericanSociety for Testing and Materials), cited bySilva (14).

Evaluat ion of degree of cr ystallizati on wasafter 30, 60, 90, and 120 days storage by fivetrained, experienced panelists, according to thefollowing scale: 1 - wit hou t perceptiblecrystallizati on, 2 - min or fine crystallization, 3- sandy, 4 - very sandy, 5 - excessively sandy.

S t a t i s t i c a l A n a l y s i s

Permeabilized cells and time of hydrolysiswere studied in a fractional factorial scheme inrandomized blocks. The transformed squareroots of the panelist scores were used foranalysis of variance.

R E S U L T S A N D D I S C U S S IO N

Beta-D-glactosidase activity of permeabilizedK. lactis cells was determined as a function ofincubation temperature (Figure 1). The greatestenzyme activity was at 40 to 45°C, and minim um

at 2°C or 60°C. Results by Wierzbicki andKosikowski (19) were similar. Van-Dam et al.(17) showed that the optimum temperature for

i

ooE . 2

o

O

ro .I

°a

z

o ' ~ b 2 o ' ~ o ' 4o ~ ;o '

Te r n p e r o t u r e ( * C )

Figure 1. Beta-D-galactosidase activity as measuredby ortho-nitrophenyl-beta-D-galactoside (ONPG) hy-drolysis by Ktuyveromyces lactis permeabilizedcells as function of temperature.

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LACTOSE HYDROLYSIS 2213

U )q )

~ 1 . 3 '

2q }

i •, e , -

ZI

o

~ - 2 0 C5 C2 5 C

S t o r a g e t im e ( M o n t h s )Figure 2. Beta-D-galactosidase activity of Kluy

veromyces lactispermeabilized ceils as a function of

temperature and time of storage before use as mea-sured by ortho-nitrophenyl-beta-D-galactoside (ONPG)hydrolysis.

beta-D-galactosidase activity in permeabilizedcells of S a c c h a r o m y c e s f r a g i l i s was 35°C,although the ideal temperature for growth was28°C. Even though optimum growth temper-atures for both K. lactis and S. fragil is wereidentical (7), the same did not apply to the

beta-D-galactosidase hydrolytic activity, thatwas near 45°C for K. lactis. This is of micro-biological significance, because it allows utiliza-tion of permeabilized cells at temperaturesdetrimental for growth of pathogenic micro-organisms.

Enzyme activity essentially was unchangedafter storage for 7 mo at -20, 5, or 25°C(Figure 2).

A higher concentration of permeabilizedcells (up to 1000 rag/liter) resulted in increased

hydrolysis rate with behavior similar to a firstorder reaction (Figure 3). According to Leh-ninger (6), it is necessary not only to select theproper enzyme concentration but also toprovide other factors such as substrate con-centration, temperature, and pH to reach theregion of direct proportionality• These resultsalso indicated that hydrolysis does not increasein direct proportion to permeabilized cellconcentrations, because substrate is the limitingfactor. One of the objectives in this experiment

was to provide 40 to 70% lactose hydrolys is inmilk for use in dulc e produ ction . Such aproduct would have a final lactose concentr ationbelow the maxi mum limit of solubility and

would not crystallize. Addition of 400 or 800mg/liter of permeabilized cells to milk followedby 2.5 or 3.5-h inc uba tion at 35°C resulted in45 to 69% lactose hydrolysis (Table 1). Thiswas obtained withou t any risk of conta minat ionand with reasonable permeabilized cell cost.

Analysis of variance of sensory evaluationscores on effect of permeabilized cells on theproperties of dulce de leche indicated thatpanelists did not detect significant differences(P< .05) among cell addition rates and hydrolysistimes (Table 1). The dulc e's flavor was notaltered by 400 or 800 mg/liter of permeabilizedcells added to milk as compared with thecontrol (Table 1).

Dulce de Ieche produced without additionof permeabilized cells (control), was the onlyproduct that developed lactose crystals (Table1). Crystallization increased in the controlduring storage as indicated by panelist scores,which ranged from 1.7 at 30 days to 3.8 after120 days. Addition of permeabilized cellsprevented crystal formation for 120 days atlactose hydrolysis rates that varied from 45.02to 69.13% (Table 1).

Luursema (8) showed that hydrolysisincreased the reducing power of milk carbo-hydrates because of the probable effect of

P e r m e o b i liz e d c e l l s m g . l )I 0 0 0

o 6 0 0• 4 0 0• 2 0 0= z e r o

t O 0

• 0

i 8 0 ' o0

6 0

L -- 4 0 • •

~ 2 0

_J

i 3 4

H y d r o l y s i s t i m e ( h )Figure 3. Percent hydrolysis of lactose in milk

at 35°C as a function of time and permeahilizedKluyveromyces lactiscells addition•

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22 14 SABIONI ET AL.

TABLE 1. Ext ent of lactose hydrolysi s and mean sensory scores for flavor and crystallization in dul ce withvarious concentration s of permeabilized luyveromyces lact iscells and reaction times.

Mean scores b

Amo unt of Time of Lactose Mean Storage time (days)

permeabilized cells hydrolysis hydrolysis flavor scores a 30 60 90 120

(mg/liter)

Zero (control)400

800

(h) (%)

7.05 1.70 2.93 3.86 3.80215 4;102 7.16 1.00 1.00 1.00 1.003.5 52.81 7.16 1.00 1.00 1.00 1.002.5 61.87 7.15 1.00 1.00 1.00 1.003.5 69.13 7.81 1.00 1.00 1.00 1.00

al, Horrible, 2, bad, 3, objectionable, 4, less than acceptable, 5, acceptable, 6, more than acceptable, 7,good, 8, very good, and 9, excellent.

bl, Without perceptible crystallization, 2, minor or fine crystallization, 3, sandy, 4, very sandy, and 5,excessively sandy.

galactose. The same was observed in a morerap id nonenz ymat i c da rken ing dur ing con-cen t ra t ion o f mi lk t r ea t ed wi th pe rmeab i l i zedcel ls . S imilar resul ts were obta ined by Hosken(4), Lu uvr ema (9), Coelh o (1), and Silva (14).

C O N C L U S I O N

Lactose hydrolys is by permeabi l ized K.l ac t i s cei ls to contro l crys ta l l iza t ion in dulcede lech e offers a s imple econ omi c a l terna-t ive to t he use of pur i f ied enzymes. Becauseenz yme act iv i ty in dr ied , permeab i l ized cel lsremained s table for up to 7 mo a t room tem-perature , s torage and handl ing proce dures arenot cr i t ica l .

C K N O W L E D G M E N T

The authors are indebted to J . Warren Stul l ,Nu t r i t i on and Food Sc ience Depar tmen t ,Univers i ty of Arizona, for h is k indness incareful ly reviewing th is paper and for h is mosthelpful remarks .

R E F E R E N C E S

1 Coelho, E.B.B. 1980. Utiliza(ffo da beta-D-galac-tosidase no controle da cristalizaq~o do doce deleite. M.S. Thesis, U.F.V., Imprensa Universit~ria,Viqosa, MG, Brazil.

2 Doan, F .J. 1958. Problems of lactose crystallization

in concentrated milk products. J. Dairy Sci.41:325.3 Gist Brocades. 1978. Industrial Products Division.

Maxilact a dairy yeast lactase. Technical data sheet.MIL 01-01-78. O5EMO3, Delft Holland.

4 Hosken, F. S. 1969. Doce de leite - Durabil idade ecristalizag~'o. Rev. Inst. Lat. Cand. Tostes. Brazil24:10.

5 Lara, A.B.W.H.; G. Nazi.rio; M.E.W. Almeida, andW. Pregno lato. 1976. Pages 151--178 in Normasanaliticas do Instituto Adolfo Lutz. 2nd ed. Vol. 1.Sec. Est. Saude, S~b Paulo, Brazil.

6 Lehninge r, A. L. 1970. Pages 183 -2 12 in Bio-

chemistry. 2nd ed. Worth Publ. Inc. New York,NV.7 Lodder, J. 1974. Pages 349-351 in The yeasts, a

taxonomic study. 2nd ed. North-Holland Publ.Co., Amsterdam, Holland.

8 Luursema, J. O. 1973. Dulce de leche non cristal-izable. Ind. Lechera 57:2.

9 Luuvrema, I. J. 1978. Aplicaciones de lactase parael dulce de leche. Ind. Lacteas 27:25.

10 Miller, J. H. 1974. Experi men ts in molecula rgenetics. Cold Spring Harbor Laboratory. NewYork, NY.

11 Nickerson, T. A., and E. E. Moore. 1974. Alpha-lactose and crystallization rate. J. Dairy Sci.57: 160.

12 Potter, F. E. 1950. A colorimetric met hod forquantitative determination of degree of lactosehydrolysis. J. Dairy Sci. 33:803.

13 Santos, D. M. 1976. Arenosidade no doce de leite.Rev. Inst. Lat. Canal. Tostes. 31:3.

14 Silva, T.J.P. 1980. Utiliza~'o dabeta-D-glactosidaseno processO continuo de fabrica~'o do doce deleite homogeneiz ado. (M.S. Thesis), UFV, Impr ensaUniv., Vi~osa, MG, Brazil.

15 Souza, L.R.P. 1979. Efeit o da acidif ica(~o e dotratamento t~rmico do leite na qualidade do docede leite. M.S. Thesis, UFV, Imprensa Univ., Viqosa,

MG, Brazil.16 Teles, F.F.F., C. K. Young, and J. W. Stull. 1978.A method for rapid determination of lactose. J.Dairy Sci. 61:506.

17 Van-Dam, B., J. G. Revallier-Warffemius, and L. C.

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LACTOSE HYDROLYSIS 221 5

Van-Darn Schermerhorn. 1950. Preparation oflactase from Saccbaromyces fragilisNeth. MilkDairy J. 4:96.

18 Vargas, M. M. 1979. InfluOncia da gordura, daglucose e do amido na cristaliza~ o do doce de

leite. M.S. Thesis, U.F.V., Imprensa Universit~ria.Viqosa, MG, Brazil.

19 Wierzbicki, L. E., and F. V. Kosikowski . 1973.Lactase potenti al of various microorganisms grownin whey. J. Dairy Sci. 56:26.

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