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Effect of Acid-Precipitated Casein in Calf Milk Replacer on Rennet Clot1
E f f e c t o f A c i d - P r e c i p i t a t e d Casein in Calf M i l k Replacer on R e n n e t C l o t 1 K. J. JENKINS Animal Research Centre Research Branch, Agriculture Canada
Ottawa, Ontario K1A 0C6
ABSTRACT
teristics of rennet clots formed by the skim casein.
An in vitro experiment demonstrated that substitution of skim milk powder in calf milk replacer with acid-precipitated casein and whey (constant 23.5% protein in dry matter) resulted in extended coagulation time with rednet (pH 6.1, 39°C) and weaker curd strength. If resuits are translatable to conditions in calf abomasum after feeding, reduced" curd strength from acid-precipitated casein might promote quicker stomach emptying and reduce reported beneficial effect of clot formation on nutrient digestibility.
MATERIALS AND METHODS
INTRODUCTION
The development of a firm abomasal curd in the young calf appears to have a beneficial effect in promoting nutrient digestibility (3). As calves up to 3 to 4 wk of age still are undergoing development of their digestive enzyme system (6), this is likely due to a prolonged retention of nutrients in the abomasum resulting in more efficient gastrointestinal digestion. The formation of a rennet curd in milk or skim milk solutions is due chiefly to a reaction between casein and the chymosin (E.C. 3.4. 23.4) in rennet. In the preparation of calf milk replacers, skim milk powder is replaced sometimes in part with acid-precipitated casein (APC) and whey to reduce costs. However, the harsh treatments used in preparing APC destroy its coagulating properties with rennet. The present in vitro work was to determine how inclusion of APC in milk replacer containing skim powder affects formation and charac-
Received January 22, 1982. I Contribution No. 1056. 1982 J Dairy Sci 65:1652-1654
Milk replacers were prepared by dispersing the A P e (vitamin-free casein, United States Biochemicals, Cleveland) in water at room temperature, adding .1 N NaOH until the APC dissolved, and then reducing pH to neutrality with .1 N HCI. The nonlipid ingredients then were added and dissolved, the mixture heated to 55°C, and the fats and emulsifiers blended in for 2 min by Waring Blendor. After the mixture was cooled to 39°C, pH was adjusted to 6.1 with .1 N HCI. This pH was chosen as the pH of abomasal contents is approximately 6.1 (5) in the young preruminant calf immediately after feeding, when clotting is initiated. Four complete calf milk replacers were prepared (at 14% total solids), their compositions differing only as a result of stepwise replacement of the skim milk powder with A P e plus whey and glucose added as filler (Table 1). The ingredients common to all milk replacers and not shown in Table 1 were (% of dry matter): soy lecithin .90, Tween 8 0 . 0 6 , MgSO4 1, CaC12 .71, micromineral premix .30, vitamin p r e m i x . 30, choline chloride .10. The composition of micromineral and vitamin premixes were as in (4). The skim milk powder was a low-temperature, spraydried product. Protein and fat i n a l l milk replacers were maintained at 23.5 and 20% of dry matter. Curd firmness was measured in triplicate sample runs (with a 1:20 dilution of standard rennet at 39°C) as described by Emmons and Lister (2). After the curd firmness measurement, the curd was cut from the sides of the beaker by a thin spatula and allowed to stand for an additional 60 min. The clot plus absorbed liquid was analyzed for dry matter and total lipids by standard AOAC methods (1).
1652
TECHNICAL NOTE
1653
TABLE 1. Composition of milk replacers and effect of acid-precipitated casein (APC) on rennet clot from skim powder, a Control, nil APC
Low APC
Med APC
High APC
Skim milk powder Casein, acid-precipitated Whey, sweet Tallow, edible Coconut oil Glucose Others
67 0 10 17 2
47 5 23 17 2 2.6 3.4
33 9 29 17 2 6.6 3.4
20 15 15 17 2 27.6 3.4
Reconstitution, DM %
14
14
14
14
Rennet curd Coagulation time, sec pH at formation Dry matter, g Dry matter, % of replacer DM Fat, g Fat, % of replacer fat Curd firmness (CF), g CF, g per g MR skim casein CF, g per g MR total casein
34 d 6.1 6.6 b 47 b 2.2 b 79 b 50.1 b 20.1 20.1 b
45 c 6.1 6.4 b 46 b 2.1 bc 75 bc 29.7 c 16.5 11.9 c
72 b 6.1 5.8 c 42 c 1.8 c 64 c 20.8 cd 17.3 8.4 d
69 b 5.9 5.4 c 39 c 1.2 d 43 d 13.8 d 18.4 4.7 e
Ingredients in DM (%)
.6 3.4
SE
4.1 ... .16 1.2 .14 4.3 4.0 1.6 1.2
aMean of triplicate determinations. Other ingredients (3.4%) described in Materials and Methods. Casein in skim (33% protein) estimated as 80% of protein• b'c'd'eMeans within lines not bearing common superscripts differ (P<.05).
RESULTS A N D DISCUSSION
In a p r e l i m i n a r y e x p e r i m e n t acid-precipit a t e d casein ( A P e ) did n o t f o r m a r e n n e t curd over a wide range o f c o n c e n t r a t i o n s (.5, 1.0, 2.0, 3.0, 4.2 g / 1 0 0 m l ) a n d pH (5.0 t o 6.1). These increasing c o n t e n t s o f A P e a d d e d to a c o n s t a n t 5% o f r e c o n s t i t u t e d s k i m m i l k p o w d e r progressively d e l a y e d f o r m a t i o n o f r e n n e t curd f r o m t h e skim p o w d e r . In t h e e x p e r i m e n t r e p o r t e d here, in w h i c h skim p o w d e r was r e p l a c e d w i t h A P e plus w h e y to m a i n t a i n a c o n s t a n t p r o t e i n (N × 6 . 2 5 ) conc e n t r a t i o n in m i l k replacer (23.5% o f d r y m a t t e r ) r a t h e r t h a n a d d i n g A P e t o a c o n s t a n t perc e n t o f s k i m p o w d e r , r e n n e t c l o t t i n g at p H 6.1 also was d e l a y e d (Table 1). T h e c o m b i n a t i o n s o f skim p o w d e r a n d A P e in t h e f o u r m i l k replacers were ( g / 1 0 0 ml): 9.4 a n d 0, 6.6 a n d .7, 4.6 a n d 1.3, 2.8 a n d 2.1. A t t h e h i g h e s t A P e t h e r e n n e t clot w o u l d n o t f o r m unless t h e p H was r e d u c e d t o 5.9. As A P e was increased (and skin p o w d e r l o w e r e d ) t h e clots were n o t f o r m e d as well a n d h a d a m o r e gray appear-
ance. R e p l a c e m e n t of skim p o w d e r w i t h A P e plus w h e y r e d u c e d t h e clot dry m a t t e r w e i g h t o n l y slightly b u t caused a m o r e m a r k e d decrease in t h e p e r c e n t a g e o f m i l k replacer fat o c c l u d e d in t h e clot. No a t t e m p t was made t o d e t e r m i n e h o w m u c h A P e in t h e m i l k replacer b e c a m e p a r t o f t h e clot. However, m u c h of t h e A P e m u s t have e n d e d in t h e clot, b e c a u s e w i t h m a r k e d r e d u c t i o n o f skim m i l k p o w d e r t h e clots m a i n t a i n e d similar d r y m a t t e r weights, fat o c c l u s i o n was n o t increased, a n d m o s t o f w h e y is expelled f r o m r e n n e t clots in t h e syneresis fluid• N o t a b l y , curd f i r m n e s s decreased as t h e skim p o w d e r was l o w e r e d (and A P e plus w h e y increased), d e m o n s t r a t i n g t h a t a l t h o u g h A P e m a y have b e e n o c c l u d e d in t h e clot, it did n o t c o n t r i b u t e to curd s t r e n g t h . T h e curd f i r m n e s s was similar to t h a t r e p o r t e d b y E m m o n s a n d Lister (2) for c o m p a r a b l e c o n c e n t r a t i o n s of r e c o n s t i t u t e d s k i m m i l k p o w d e r alone. If t h e s e results are t r a n s l a t a b l e to c o n d i t i o n s in t h e calf a b o m a s u m immediately a f t e r f e e d i n g (pH 6.1), Journal of Dairy Science Vol. 65, No. 8, 1982
1654
JENKINS
it t h e n w o u l d b e e x p e c t e d t h a t r e p l a c e m e n t o f skim p o w d e r in milk replacer w i t h APC plus w h e y w o u l d l o w e r curd s t r e n g t h , causing t h e clots to b r e a k u p a n d leave t h e a b o m a s u m m o r e quickly. A c c o r d i n g l y , this m i g h t r e d u c e t h e rep o r t e d (3) beneficial e f f e c t of a b o m a s a l clot f o r m a t i o n o f m i l k replacer o n n u t r i e n t digestibility. REFERENCES
1 Association of Official Agricultural Chemists. 1970. Official methods of analysis, l l t h ed. Assoc. Offic. Agric. Chem., Washington, DC. 2 Emmons, D. B., and E. E. Lister. 1976. Quality of protein in milk replacers for young calves I. Fac-
Journal of Dairy Science Vol. 65, No. 8, 1982
tors affecting in vitro curd formation by rennet from reconstituted skim milk powder. Can. J. Anita. Sci. 56:317. 3 Frantzen, J. F., R. Toullec, and C. M. Mathieu. 1971. Influence de la coagulation des proteins sur l'utilisation digestive d'un lait de remplacement par le veau pr+ruminant. Pages 1 - 5 in 10th Congr. lnt. Zootech., Versailles. 4 Jenkins, K. J., and D. B. Emmons. 1979. Effect of fat dispersion method on performance of calves fed high-fat milk replacers. Can. J. Anim. Sci. 59: 713. 5 Porter, J.W.G. 1979. Digestion in the preruminant animal. Proc. Br. Nutr. Soc. 28:115. 6 Radostits, O. M., and J. M. Bell. 1970. Nutrition of the preruminant dairy calf with special reference to the digestion and adsorption of nutrients: A review. Can. J. Anim. Sci. 50:405.
Ottawa, Ontario K1A 0C6
ABSTRACT
teristics of rennet clots formed by the skim casein.
An in vitro experiment demonstrated that substitution of skim milk powder in calf milk replacer with acid-precipitated casein and whey (constant 23.5% protein in dry matter) resulted in extended coagulation time with rednet (pH 6.1, 39°C) and weaker curd strength. If resuits are translatable to conditions in calf abomasum after feeding, reduced" curd strength from acid-precipitated casein might promote quicker stomach emptying and reduce reported beneficial effect of clot formation on nutrient digestibility.
MATERIALS AND METHODS
INTRODUCTION
The development of a firm abomasal curd in the young calf appears to have a beneficial effect in promoting nutrient digestibility (3). As calves up to 3 to 4 wk of age still are undergoing development of their digestive enzyme system (6), this is likely due to a prolonged retention of nutrients in the abomasum resulting in more efficient gastrointestinal digestion. The formation of a rennet curd in milk or skim milk solutions is due chiefly to a reaction between casein and the chymosin (E.C. 3.4. 23.4) in rennet. In the preparation of calf milk replacers, skim milk powder is replaced sometimes in part with acid-precipitated casein (APC) and whey to reduce costs. However, the harsh treatments used in preparing APC destroy its coagulating properties with rennet. The present in vitro work was to determine how inclusion of APC in milk replacer containing skim powder affects formation and charac-
Received January 22, 1982. I Contribution No. 1056. 1982 J Dairy Sci 65:1652-1654
Milk replacers were prepared by dispersing the A P e (vitamin-free casein, United States Biochemicals, Cleveland) in water at room temperature, adding .1 N NaOH until the APC dissolved, and then reducing pH to neutrality with .1 N HCI. The nonlipid ingredients then were added and dissolved, the mixture heated to 55°C, and the fats and emulsifiers blended in for 2 min by Waring Blendor. After the mixture was cooled to 39°C, pH was adjusted to 6.1 with .1 N HCI. This pH was chosen as the pH of abomasal contents is approximately 6.1 (5) in the young preruminant calf immediately after feeding, when clotting is initiated. Four complete calf milk replacers were prepared (at 14% total solids), their compositions differing only as a result of stepwise replacement of the skim milk powder with A P e plus whey and glucose added as filler (Table 1). The ingredients common to all milk replacers and not shown in Table 1 were (% of dry matter): soy lecithin .90, Tween 8 0 . 0 6 , MgSO4 1, CaC12 .71, micromineral premix .30, vitamin p r e m i x . 30, choline chloride .10. The composition of micromineral and vitamin premixes were as in (4). The skim milk powder was a low-temperature, spraydried product. Protein and fat i n a l l milk replacers were maintained at 23.5 and 20% of dry matter. Curd firmness was measured in triplicate sample runs (with a 1:20 dilution of standard rennet at 39°C) as described by Emmons and Lister (2). After the curd firmness measurement, the curd was cut from the sides of the beaker by a thin spatula and allowed to stand for an additional 60 min. The clot plus absorbed liquid was analyzed for dry matter and total lipids by standard AOAC methods (1).
1652
TECHNICAL NOTE
1653
TABLE 1. Composition of milk replacers and effect of acid-precipitated casein (APC) on rennet clot from skim powder, a Control, nil APC
Low APC
Med APC
High APC
Skim milk powder Casein, acid-precipitated Whey, sweet Tallow, edible Coconut oil Glucose Others
67 0 10 17 2
47 5 23 17 2 2.6 3.4
33 9 29 17 2 6.6 3.4
20 15 15 17 2 27.6 3.4
Reconstitution, DM %
14
14
14
14
Rennet curd Coagulation time, sec pH at formation Dry matter, g Dry matter, % of replacer DM Fat, g Fat, % of replacer fat Curd firmness (CF), g CF, g per g MR skim casein CF, g per g MR total casein
34 d 6.1 6.6 b 47 b 2.2 b 79 b 50.1 b 20.1 20.1 b
45 c 6.1 6.4 b 46 b 2.1 bc 75 bc 29.7 c 16.5 11.9 c
72 b 6.1 5.8 c 42 c 1.8 c 64 c 20.8 cd 17.3 8.4 d
69 b 5.9 5.4 c 39 c 1.2 d 43 d 13.8 d 18.4 4.7 e
Ingredients in DM (%)
.6 3.4
SE
4.1 ... .16 1.2 .14 4.3 4.0 1.6 1.2
aMean of triplicate determinations. Other ingredients (3.4%) described in Materials and Methods. Casein in skim (33% protein) estimated as 80% of protein• b'c'd'eMeans within lines not bearing common superscripts differ (P<.05).
RESULTS A N D DISCUSSION
In a p r e l i m i n a r y e x p e r i m e n t acid-precipit a t e d casein ( A P e ) did n o t f o r m a r e n n e t curd over a wide range o f c o n c e n t r a t i o n s (.5, 1.0, 2.0, 3.0, 4.2 g / 1 0 0 m l ) a n d pH (5.0 t o 6.1). These increasing c o n t e n t s o f A P e a d d e d to a c o n s t a n t 5% o f r e c o n s t i t u t e d s k i m m i l k p o w d e r progressively d e l a y e d f o r m a t i o n o f r e n n e t curd f r o m t h e skim p o w d e r . In t h e e x p e r i m e n t r e p o r t e d here, in w h i c h skim p o w d e r was r e p l a c e d w i t h A P e plus w h e y to m a i n t a i n a c o n s t a n t p r o t e i n (N × 6 . 2 5 ) conc e n t r a t i o n in m i l k replacer (23.5% o f d r y m a t t e r ) r a t h e r t h a n a d d i n g A P e t o a c o n s t a n t perc e n t o f s k i m p o w d e r , r e n n e t c l o t t i n g at p H 6.1 also was d e l a y e d (Table 1). T h e c o m b i n a t i o n s o f skim p o w d e r a n d A P e in t h e f o u r m i l k replacers were ( g / 1 0 0 ml): 9.4 a n d 0, 6.6 a n d .7, 4.6 a n d 1.3, 2.8 a n d 2.1. A t t h e h i g h e s t A P e t h e r e n n e t clot w o u l d n o t f o r m unless t h e p H was r e d u c e d t o 5.9. As A P e was increased (and skin p o w d e r l o w e r e d ) t h e clots were n o t f o r m e d as well a n d h a d a m o r e gray appear-
ance. R e p l a c e m e n t of skim p o w d e r w i t h A P e plus w h e y r e d u c e d t h e clot dry m a t t e r w e i g h t o n l y slightly b u t caused a m o r e m a r k e d decrease in t h e p e r c e n t a g e o f m i l k replacer fat o c c l u d e d in t h e clot. No a t t e m p t was made t o d e t e r m i n e h o w m u c h A P e in t h e m i l k replacer b e c a m e p a r t o f t h e clot. However, m u c h of t h e A P e m u s t have e n d e d in t h e clot, b e c a u s e w i t h m a r k e d r e d u c t i o n o f skim m i l k p o w d e r t h e clots m a i n t a i n e d similar d r y m a t t e r weights, fat o c c l u s i o n was n o t increased, a n d m o s t o f w h e y is expelled f r o m r e n n e t clots in t h e syneresis fluid• N o t a b l y , curd f i r m n e s s decreased as t h e skim p o w d e r was l o w e r e d (and A P e plus w h e y increased), d e m o n s t r a t i n g t h a t a l t h o u g h A P e m a y have b e e n o c c l u d e d in t h e clot, it did n o t c o n t r i b u t e to curd s t r e n g t h . T h e curd f i r m n e s s was similar to t h a t r e p o r t e d b y E m m o n s a n d Lister (2) for c o m p a r a b l e c o n c e n t r a t i o n s of r e c o n s t i t u t e d s k i m m i l k p o w d e r alone. If t h e s e results are t r a n s l a t a b l e to c o n d i t i o n s in t h e calf a b o m a s u m immediately a f t e r f e e d i n g (pH 6.1), Journal of Dairy Science Vol. 65, No. 8, 1982
1654
JENKINS
it t h e n w o u l d b e e x p e c t e d t h a t r e p l a c e m e n t o f skim p o w d e r in milk replacer w i t h APC plus w h e y w o u l d l o w e r curd s t r e n g t h , causing t h e clots to b r e a k u p a n d leave t h e a b o m a s u m m o r e quickly. A c c o r d i n g l y , this m i g h t r e d u c e t h e rep o r t e d (3) beneficial e f f e c t of a b o m a s a l clot f o r m a t i o n o f m i l k replacer o n n u t r i e n t digestibility. REFERENCES
1 Association of Official Agricultural Chemists. 1970. Official methods of analysis, l l t h ed. Assoc. Offic. Agric. Chem., Washington, DC. 2 Emmons, D. B., and E. E. Lister. 1976. Quality of protein in milk replacers for young calves I. Fac-
Journal of Dairy Science Vol. 65, No. 8, 1982
tors affecting in vitro curd formation by rennet from reconstituted skim milk powder. Can. J. Anita. Sci. 56:317. 3 Frantzen, J. F., R. Toullec, and C. M. Mathieu. 1971. Influence de la coagulation des proteins sur l'utilisation digestive d'un lait de remplacement par le veau pr+ruminant. Pages 1 - 5 in 10th Congr. lnt. Zootech., Versailles. 4 Jenkins, K. J., and D. B. Emmons. 1979. Effect of fat dispersion method on performance of calves fed high-fat milk replacers. Can. J. Anim. Sci. 59: 713. 5 Porter, J.W.G. 1979. Digestion in the preruminant animal. Proc. Br. Nutr. Soc. 28:115. 6 Radostits, O. M., and J. M. Bell. 1970. Nutrition of the preruminant dairy calf with special reference to the digestion and adsorption of nutrients: A review. Can. J. Anim. Sci. 50:405.