J . Sci. Food Agric. 1986,37, 1001-1010

Nutritional Evaluation of Soya Beans (Glycine max): Nitrogen Balance and Fractionation Studies

George Grant, Norma H. McKenzie, William B. Watt, James C. Stewart, Patricia M. Dorward and Arpad Pusztai

Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen A B 2 9SB

(Manuscript received 19 March 1986)

The poor nutritional performance of rats fed raw soya-bean-containing diets appeared to be due to reduced apparent digestion and absorption of dietary protein, coupled to changes in systemic metabolism leading to a poor overall nitrogen balance. Aqueous heat treatment greatly reduced but did not eliminate the antinutritional effect. Heat treatment with aqueous ethanol was more effective. The whey protein (pH 4.8 soluble extract) fraction contained the bulk of the trypsin inhibitor and haemagglutination activity and gave the poorest net protein utilisation value. However, an eight-fold increase in trypsin inhibitor content did not significantly effect apparent nitrogen digestibility. Therefore inhibition of gut proteolytic enzyme activity in v ivo by soya bean trypsin inhibitors did not account fully for the poor nutritional performance. The problems elicited by soya bean are not transitory and thus there was a cumulative deterioration in overall performance for 16 weeks as a result of continuous exposure to soya bean.

Keywords: Soya bean; antinutritional factors; fractionation; trypsin inhibitors; lectin.

1. Introduction

The poor nutritive value of soya bean meal is now well established and is usually ascribed to the presence of a number of heat labile antinutritional factors’ and to the refractory nature of the native protein.’ Trypsin inhibitors, which should cause reduced digestion and absorption of dietary protein have been generally believed to be the main antinutritional f a ~ t 0 r . I . ~ However, the experimental evidence is somewhat contradictory.

Rackis4 and Kakade et a1.’ estimated that 3@50% of the growth depression was due to trypsin inhibitors whereas Gertler et al.’ found that they played only a minor role. Turner and Liener6 found that the soya bean lectin contributed little to toxicity whereas Sambeth et a1.’ showed that whilst there was no correlation between intraperitoneal toxicity and trypsin inhibitor content the highest levels of intraperitoneal toxicity were associated with fractions containing large amounts of lectin. Naim et al.’ have also found considerable growth depression with a protein fraction of low trypsin inhibitor content. Similar studies carried out on kidney bean (Phaseolus v u l g ~ r i s ) ~ indicate that trypsin inhibitors have little or no role in the toxicity of those beans.

In order to clarify the position with respect to the effect of various components of soya bean meal upon nutritional performance a comprehensive fractionation study of the constituents of soya bean combined with nutritional evaluation of the fractions was carried out. In the present paper an account of the nutritional evaluation of some of these fractions and of nitrogen balance studies on soya bean-fed rats is given.

1001

1002 G . Grant el al.

2. Experimental

2.1. Materials Samples of whole soya bean were purchased from Health and Dietary Food Stores (Aberdeen). Casein was supplied by BDH Chemicals (Poole, Dorset). lactalbumin and egg albumin by Sigma Chemical Co. (Poole, Dorset).

Diets for the net protein utilisation (NPU) and nitrogen balance studies were prepared as previously' and contained a total of 100 g protein kg-' diet comprising casein, egg albumin or seed protein or a combination of casein and seed protein. Most protein sources were fed without supplementation with any individual amino acids. Some were however supplemented to bring the concentrations of individual amino acids to the target requirements for rats.'"

For the long term (28 week) feeding studies the raw soya bean and lactalbumin diets were formulated such that they had similar protein (100 g kg-') lipid (220 g kg-I) and gross energy (4.6 kcal g-I) contents. The soya bean diet contained per kg: 270 g raw soya bean meal, 230 g maize starch, 100 g potato starch, 150 g glucose, 150 g corn oil, 50 g vitamin mixture and 50 g mineral mixture. The lactalbumin diet contained per kg: 130 g lactalbumin, 300 g maize starch, 100 g potato starch, 150 g glucose, 220 g corn oil, 50 g vitamin mixture and 50 g mineral mixture. The soya bean diet was supplemented with L-tryptophan and L-methionine to bring it to the target requirements for rats."

The vitamin mixture used in the long term feeding trials contained 200 mg thiamine, 200 mg pyridoxine, 200 mg riboflavin, 200 mg p-amino benzoic acid, 600 mg nicotinic acid, 400 mg Ca pantothenate, 100 mg fo lk acid, 100 mg biotin, 8000 mg inositol, 240 mg vitamin A, 50 mg vitamin D, 1200 mg vitamin E, 2 mg vitamin K, 500 mg vitamin B12 and 16 g choline chloride made up to 1 kg with maize starch. The mineral mixture contained 400 mg CuS04.5H20. 5 g FeS04.7H20, 4 g MnS04.4H20, 3600 mg ZnS04.7H20, 60 mg KI, 120 mg NaF, 10 mg NH4 V 0 3 , 80 mg NiCI2.6H20, 120 mg SnCl4.SH20, 6 mg NaSe03, 960 mg (chrome alum) CrK(S04)2.12H20. 410 g CaC03, 314 g KH2P04, 22 g KCI, 102 g MgS04.7H20 and 142 g Na2HP04 in approximately 1 kg. In addition to the mineral mixture 400 mg silicic acid kg-' diet was also added.

2.2. Methods

2.2. I . Nutritional evaluation NPU, digestibility and biological value were measured as previously.' Seed protein fractions were included in the diets either in proportion to their relative amounts in the original beans with the total protein content of the diet being adjusted t o 100 g kg-' by the addition of casein or at a concentration of 100 g protein kg-'.

2.2.2. Nitrogen balance experiments These studies were carried out as previously."

2.2.3. Long-term feeding trials The 100 g protein kg-' soya bean diet was fed ad lib. The amount of the 100 g protein kg-' lactalbumin diet made available was adjusted to equal the daily ad lib intake of rats fed the soya bean diet.

2.2.4. Chemical analysis Diets, carcass and ground faeces samples were analysed for moisture content and total N, and urine samples were analysed for total N, amino-N, urea, creatinine, NH3 and allantoin as previously." Lipid content of the dried faeces, carcass and diet samples was estimated by difference in weight following extraction for 24 h at room temperature with 2 : 1 (by vol) chloroform :methanol at a sample to solvent ratio of 1 : 200 (wt : vol). Occasionally the chloroform :methanol extract was recovered by rotary evaporatien. It was then re-extracted with

Nutritional evaluation of soya beans 1003

2: 1 (by vol) chloroform: methanol refiltered and recovered. There were no significant differences between the values obtained by either method.

2.2.5. Haemagglutination Samples were tested for haemagglutinating activity towards rabbit erythrocytes as previously. l2

One unit of haemagglutinin activity was defined as the amount of material per ml in the last dilution giving 50% agglutination. For comparison all values were expressed as specific activity (H.U. mg-I).

2.2.6. Trypsin inhibitor activity Commercial trypsin (type 111S, Sigma Chemical Co.), in which the true trypsin content was estimated after active site titration with p-nitrophenyl p'-guanidin~benzoate,'~ was added to solutions of the soya bean protein fractions and allowed to react. The level of uninhibited trypsin activity was then assessed by the method of Fahrney and Gold.14 By comparison with tests done using a commercial soya bean trypsin inhibitor preparation (type IS, Sigma Chemical Co.), it was possible to assess the equivalent inhibitor content of each fraction. Trypsin inhibitor content was then expressed as the gram equivalent of commercial soya bean trypsin inhibitor.

2.2.7. Amino acid analyses These were carried out as previously.15 The limiting amino acids were cysteine and methionine and the chemical score was calculated by comparison with the combined target requirement of 4.5 g kg-' diet."

2.2.8. Fractionation of raw soya bean Procedure 1-aqueous heating: Air dried soya bean seeds (1 kg) were soaked for 16 h in distilled water and then heated in distilled water (300 g wet weight of seed litre-') at 100°C for 20 min. Both the soaking and cooking water extracts were discarded. The heated seeds were freeze dried (815 g) and ground in a Moulinex Junior Coffee Grinder.

Procedure 2-ether extraction: Air dried soya bean flour (1 kg) was extracted by stirring with 2 litres petroleum ether for 2 h at room temperature followed by filtration. This was repeated four times with the final extraction being for 16 h at room temperature. After filtration, the final residue was air dried (765 8). The ether soluble fractions were combined and recovered by rotary evaporation (153 ml).

Procedure k x t r a c t i o n of defatted meal: Defatted soya bean meal (procedure 2) (1 kg) was extracted by stirring with water (pH adjusted to 8 with 1 M sodium hydroxide) at +1"C for 8 h at a meal to water ratio of 1: 10 (wt:vol). After centrifuging (24 OOOxg for 1 h) the residue was re-extracted for 16 h at a flour to water ratio of 1 : 15 (wt : vol). After centrifuging, the insoluble residue was recovered by freeze drying (318 8). The supernatants were combined, dialysed exhaustively against water and recovered 5y freeze drying (390 8). The diffusate from the dialysis was recovered by rotary evaporation and freeze drying (183 g). Whilst the proportions of insoluble residue and pH 8 soluble non-diffusable extract recovered were similar to that obtained in a small scale pilot experiment, the proportion of diffusable material recovered was only 60% of that recovered in the pilot experiment. This difference is probably due to losses incurred during recovery of the very large volumes (over 35 litres) of diffusate.

Procedure &aqueous ethanol extraction: One kilogram of defatted (procedure 2) soya bean meal was extracted by stirring with 2 litres of 70% ethano1:water (by vol) at 60°C for 4 h. The extraction was repeated two times with the final extraction being 16 h at 60°C. After filtration, the residue was washed with 1 litre of acetone and air dried (799 g). The acetone wash was combined with the 70% ethanol :water (by vol) extracts and recovered by rotary evaporation and freeze drying (210 8).

Procedure 5-extraction of ether and aqueous ethanol extracted meal: Defatted and aqueous ethanol extracted soya bean meal (procedure 4) (1 kg) was extracted under similar conditions to

66

1004 G . Grant ef al.

procedure 3. The insoluble residue (825 g), pH 8 soluble supernatant (68 g) and diffusate (33 g) were recovered.

Procedure &isolation of major soya bean proteins: These were prepared essentially by the method of Thanh and Shibasaki.16 Defatted soya bean meal (procedure 2) (1 kg) was extracted twice with 0.03 M Tris HCl pH 8.0 containing 0.01 M 2-mercaptoethanol at a meal to buffer ratio of 1: 10 (wt:vol) for 1 h at 20°C. After centrifuging (24 OOOxg 20 min 20"C), the pH of the combined supernatants was adjusted to 6.4. The 11s globulin fraction was then recovered by centrifuging (24 OOOxg 20 min 4°C). The pH was further lowered to 4.8 and the resultant precipitate (7s globulins) were collected. The supernatant (whey proteins) was adjusted to pH 8.0 and kept at +1"C overnight to precipitate phytate. After clarification by centrifuging, the whey proteins were concentrated by full saturation with ammonium sulphate and recovered by dialysis and freeze drying (57 g). The 11s globulin preparation was washed in Tris-HC1 pH 6.4 buffer and then dispersed in that buffer (protein concentration 2-3% (wt:vol)). The pH of the solution, was raised by addition of 1 M sodium hydroxide until the protein dissolved (pH 8). The solution was then kept overnight at +l"C. A trace of precipitate was removed by centrifuging and the 11s globulin was recovered by dialysis and freeze drying (112 g). The 7 s globulin preparation was dissolved in Tris-HC1 buffer (pH 7.8), the pH lowered to 6.2 and the solution kept overnight at +1"C. The precipitate (7s polymerised globulins) was spun off. The precipitate was redissolved in pH 7.8 buffer, the pH of the supernatant was adjusted to 7.8 and both fractions, 7s polymerised globulins (73 g) and 7s globulins (137 g) were recovered by dialysis and freeze drying.

3. Results

Inclusion of fully supplemented raw soya bean meal as sole source of dietary protein for rats led to poor growth and the weight gain per gram of food intake obtained was only 60% of that with rats fed an egg albumin-containing diet (Table 1).

Whilst the nitrogen intake was lower than that of rats fed on the egg albumin diet, both the faecal nitrogen and urinary nitrogen (primarily urea nitrogen) outputs were considerably elevated and were approximately twice of those of rats fed on the egg albumin diet. The resultant poor nitrogen-balance and apparent NPU values closely reflected the weight changes observed. Although apparent nitrogen digestibility was reduced by 15% in soya bean-fed rats, the apparent lipid digestibility was not affected.

Heat treatment of soya bean (procedure 1) prior to incorporation into diets for rats, led to an improvement in growth rate and apparent nitrogen digestibility (Table 1). However faecal nitrogen and urinary nitrogen output was still higher than that of rats fed the egg albumin diet and consequently the nitrogen-balance and apparent NPU values obtained were lower than those obtained with egg albumin.

Ether extraction (procedure 2) did not appear to improve the nutritional quality of soya bean meal (Table 2). However the antinutritional factor/s could be solubilised from defatted (procedure 2) soya bean meal by aqueous extraction (procedure 3) and consequently the nutritional performance of rats fed diets containing a dialysed pH 8 soluble extract (procedure 3) was similar to that obtained with rats fed raw soya bean-containing diets. Neither the ether extract (procedure 2) nor the insoluble residue (procedure 3) nor the diffusate (procedure 3) impaired rat growth.

Aqueous ethanol extraction at 60°C (procedure 4) of defatted (procedure 2) soya bean meal considerably improved performance (Table 2). Rats fed diets containing this pretreated meal had food intakes 50% higher than those on the original raw soya bean meal gained in weight and gave NPU values (45) close to that expected from the chemical score (50). Apparent digestibility was also improved. Aqueous ethanol pretreatment considerably reduced the amount of material which could be solubilised from the meal by aqueous extraction (procedure 5) (Table 2) indicating that most proteins had been denatured at 60°C in aqueous ethanol. Neither the

Nutritional evaluation of soya beans 1005

Table 1. Results of nitrogen balance experiments obtained with rats fed on diets containing raw soya bean or heated soya bean (procedure 1)

Heated soya bean Egg albumin Raw soya bean (procedure 1)

Diet control kg-7 supplemented' supplemented' Non-protein (100 g protein (100 g protein kg-l) (100 g protein kg-l)

Starting weight (9)

Weight change (g)'

Intake'

Food nitrogen (mg) Food lipid (g) Water (ml)

Fcmd (9)

Outpuf Faeces (9) Faeces nitrogen (mg) Faeces lipid (9)

Urine volume (ml) Urine nitrogen (mg) Urea nitrogen (mg) Ammonia nitrogen (mg) Creatinine nitrogen (mg) Allantoin nitrogen (mg) a-Amino nitrogen (mg)

Nitrogen-balance Apparent NPUd Apparent nitrogen digestibility' Apparent lipid digestibility/

Nitrogen (%) (dried carcass) Lipid (%) (dried carcass)

82.4f4.3

- 10.8k 1.6 (7)b

41.7f2.7 27.4f4.6

8.4-CO.l 29.2f 5.6

2.57k0.57 119. I f 13.7 0.69f0.05 9.7 f 2.0

108.2t10.5 53.2f10.8 8.8f1.7 4.5f0.8

27.8f18.6 2.2f0.3

-199.8k34.3 -

-

91.7f0.2

11.27k0.23 18.45f1.00

8 1 . 5 f 4 . 5

+32.8f8.5 (Wb

91.9k2.3 1540.9k39.3

18.9k0.7 120.7k16.4

4.31k0.91 172.6f 11.6 0.64f0.03

52.8f10.0 152.6f8.9 57.0f22.3 10.6f2.9 7.0f1.0

53.0k9.0 3.7k0.8

+1215.8f29.1 93.0f5.0 96.4f0.2 96.6k0.2

10.03f0.25 22.30f 1 .00

86.1 f 5 . 2

+17.3+3.3 09Ib

78.5k1.7 1376.8k29.3

21.1k0.7 77.5f6.6

8.25k 1.38

1.27fO.05

25.6k4.2 283.0f6.6 185.9f 38.8 11.6f2.4 7.8f1.5

56.6f9.9 3.3f0.5

373.6f8.6

+720.2+21.7 68.0f5.0 81.1f0.5 94.0k0.2

10.22f0.65 26.00f0.85

78.9f5.2

+29.3+5.2 ( I l l b

91.8f2.3 1602.4f40.1

24.8f0.7 90.4f5.8

8.70k1.38 275.1 k 11.8

1.29f0.04

27.1k3.8 201.3k0.9 95.5f33.1 16.3f2.9 6.8k0.9

57.9f7.6 3.9f0.5

+1106.1 f29.7 83.0f5 .O 90.0k0.4 94.8f0.2

10.16f0.78 26.75f0.80

"Diets supplemented with individual amino acids to meet the target requirement'* for rats. bNumber of observations. 'Values are calculated per rat over an 8-day period. dCalculated from nitrogen balance data. 'Estimated from faecal nitrogen. 'Estimated from faecal lipid.

dialysed pH 8 soluble extract, nor the diffusate impaired nutritional performance and whilst the growth of rats on diets containing the insoluble residue was poorer than would be expected the NPU values obtained (50) were quite close to that predicted from the chemical score (58).

Although supplementation of raw soya bean meal-containing diets with tryptophan and methionine improved nutritional quality, the food intake, growth and NPU values obtained were still poor when compared to those of rats fed diets containing an 'ideal' protein such as egg albumin (Table 3) . Replacement of soya bean meal by a pH 8 extract (procedure 3 ) of defatted (procedure 2) soya bean meal further reduced performance. When this extract was separated into its major protein components (procedure 7) it was found that the whey (pH 4.8 soluble extract) fraction depressed food intake and growth more than did either the pH 8 extract or the original soya bean meal (Table 3) and only 48% of the ingested dietary whey protein was utilised by rats. However although the bulk of the trypsin inhibitor and haemagglutination activity was found to be associated with the whey fraction the apparent nitrogen digestibility was similar to that observed in the original meal. With the 11s globulin, 7s globulin and 7s polymerised globulin fractions, the growth rate, NPU and nitrogen digestibility values obtained were much higher than those of the original meal. However they were still slightly below that obtained with egg albumin-fed rats.

Tab

le 2

. The

nut

ritio

nal

prop

ertie

s of

soy

a be

an m

eal b

efor

e an

d af

ter

trea

tmen

t with

eth

er (

proc

edur

e 2)

or e

ther

and

aqu

eous

eth

anol

(pr

oced

ure

4) a

nd o

f aq

ueou

s ex

trac

ts o

f de

fatte

d m

eal

(pro

cedu

re 3

) or

defa

tted

and

aque

ous

etha

nol

trea

ted

mea

l (p

roce

dure

5)

Def

atte

d so

ya b

ean

mea

l D

efat

ted

and

etha

nol e

xtra

cted

soy

a be

an m

eal

Raw

Pr

oced

ure

2 Pr

oced

ure

3 Pr

oced

ure

4 Pr

oced

ure

5 ~

Cas

ein

soya

bea

n ~

prot

ein

prot

ein

prot

ein

Def

atte

d N

on-

(100

g

(100

g

Die

t co

ntro

l kg

-’)

kg-’

) m

eal

Frac

tion

wei

ght (

9)

from

1 k

g of

air

dr

ied

soya

bea

n m

eal

- -

lo00

76

5 Pe

rcen

tage

nitr

ogen

-

14.4

4 5.

96

7.87

C

ompo

sitio

n Fr

actio

n -

- 26

9 21

2

-

-

111

-

193

317

246

242

343

343

344

342

I of

Die

t (g

kg-

’) C

asei

n Fo

od in

take

(g)

“ St

artin

g w

eigh

t (g

)’ W

eigh

t cha

nge

(g)”

-8

3 +4

3 -3

5 -3

3 C

hem

ical

sco

reb

-

68

53

53

Net

pro

tein

util

isat

ion

-

70

35

36

100

81

84

Nitr

ogen

dig

estib

ility

-

Bio

logi

cal v

alue

-

70

43

43

PH 8

Et

her

Inso

lubl

e so

lubl

e ex

trac

t re

sidu

e pr

otei

ns

Dif

fusa

te

Extr

acte

d Et

hano

l m

eal

extr

act

Inso

lubl

e re

sidu

e

PH 8

so

lubl

e pr

otei

ns

Dif

fusa

te

153 ml

42.5

ml

111

292

343

+36 68

70

93

76

- 24

1 5.

98

68

82

326

342

+so

66

70

95

74

298

13.6

4 82

33

265

341

-21 57

48

92

52

140

1.30

40

108

337

342

+65 67

75

97

78

61 1

161

9.03

1.

02

162

43

10

107

360

347

346

346

+34

+ 60

50

66

45

69

90

99

50

70

504

11.0

7 13

5 8 34

2 34

6 +2

2 58

50

90

56

42

20

11.4

5 1.

51

11

6

102

110

342

352

347

345

+46

+57

66

68

66

68

97

99

68

68

“Val

ues

are

give

n pe

r gr

oup

of 4

rats

ove

r the

10

day

expe

rim

enta

l pe

riod

. bC

hem

ical

scor

e w

as c

alcu

late

d on

the

basi

s of

the

sulp

hur

amin

o ac

id c

onte

nt o

f th

e di

et c

ompa

red

to t

he ta

rget

req

uire

men

t” o

f 4.

5 g

kg-’

die

t.

Tabl

e 3.

The

nut

ritio

nal

prop

ertie

s, tr

ypsi

n in

hibi

tor

cont

ent a

nd h

aem

aggl

utin

atio

n ac

tiviti

es o

f is

olat

ed p

rote

in f

ract

ions

from

def

atte

d so

ya b

ean

mea

l (pr

oced

ure

6)

~~

~ ~

Def

atte

d so

ya b

ean

mea

l

Die

t

Egg

Pr

oced

ure

3 Pr

oced

ure

6 N

on-

albu

min

R

aw

prot

ein

(100

g p

rote

in

soya

bea

n pH

8 so

lubl

e 7s

pol

ymer

ised

W

hey

cont

rol

kg-7

m

eal"

pr

otei

ns"

11s

glob

ulin

s"

7s g

lobu

lins'

glob

ulin

s"

prot

eins

'

Frac

tion

wei

ght

(8)

from

1 k

g of

air

-

44

drie

d so

ya b

ean

mea

l -

lo00

29

8 86

10

5 56

Pe

rcen

tage

nitr

ogen

-

- 5.

96

13.6

4 16

.20

14.8

0 13

.80

13.6

3

activ

ity (

HU

mg-

') -

50

200

1 1

1 lo00

Hae

mag

glut

inat

ion

Tryp

sin

inhi

bito

r co

nten

t (g

kg-

' so

ya b

ean

mea

l)

15.4

0 15

.40

0.31

2.

29

0.38

12

.40

Food

inta

ke (g

)' 20

7 42

8 36

0 32

0 43

6 43

9 41

6 31

3 St

artin

g w

eigh

t (g

)' 31

5 31

7 31

5 31

5 31

5 31

7 31

6 31

5 W

eigh

t ch

ange

(g)b

-5

4 +1

72

+ 90

+ 40

+146

+1

44

+ 123

+3

3 C

hem

ical

sco

reb

100

100

100

100

100

100

100

Net

pro

tein

util

isat

ion

90

65

57

82

77

84

48

Nitr

ogen

dig

estib

ility

10

0 87

85

99

96

96

85

B

iolo

gica

l val

ue

90

76

67

83

80

88

56

- -

-

-

- - -

'Raw

, so

ya b

ean

mea

l an

d pr

otei

n fr

actio

ns w

ere

inco

rpor

ated

int

o di

ets

as s

ole

prot

ein

sour

cce

at a

con

cent

ratio

n of

100

g pr

otei

n kg

-' d

iet.

The

die

ts w

ere

supp

le-

'Foo

d in

take

, sta

rtin

g w

eigh

t an

d w

eigh

t ch

ange

are

giv

en p

er g

roup

of

four

rat

s ov

er 1

0 da

ys.

men

ted

with

ind

ivid

ual a

min

o ac

ids

to m

eet t

he ta

rget

req

uire

men

ts f

or r

ats.

1008 G. Grant et al.

Figure 1. The growth curves for groups of four rats fed either (0). soya bean (1oOg protein kg-') diet ad lib or (0). lactalbumin (100 g protein kg-') diet pair fed over a 28 week period. Initial food intake was 10 g rat-' day-' rising to 18 g rat-' day- ' at 28 weeks.

Time (weeks)

The poor food conversion rates observed with rats fed soya bean diets over a short (10 day) period continued in animals tested in a much longer trial (Figure 1). Consequently over a 16 week period rats fed the soya bean-containing diet fell progressively behind their counterparts which were pair-fed a lactalbumin diet with a similar total protein, lipid and gross energy content to that of the soya bean-containing diet. The soya bean-fed rats weighed 16% less than their lactalbumin-fed controls after 28 weeks.

4. Discussion

The poor nutritional performance of rats fed raw soya bean diets was due to a poor overall nitrogen balance resulting from elevated outputs of both faecal and urinary (primarily urea nitrogen) nitrogen. Faecal nitrogen output, when corrected for endogenous nitrogen losses, accounted for only 18% of the ingested nitrogen and thus, whilst this indicates a reduction in digestion and absorption of dietary protein, changes in systemic metabolism may also contribute to the poor performance of soya bean-fed rats.

Some soya bean antinutritional factors would appear to be resistant to rigorous heat treatment and therefore whilst aqueous heat treatment greatly improved food intake and weight gain the overall performance was still below that expected. Such heat stable antinutritional factors may have contributed to the diarrhoea and weight loss or poor growth problems observed in preruminant calves fed diets containing pretreated soya bean meals.",18 Heating with aqueous ethanol at 60°C was a more effective pretreatment procedure.

The net protein utilisation of soya bean fractions can be inversely correlated with both haemagglutinin and trypsin inhibitor content and thus the whey protein fraction, which contains the bulk of the haemagglutinin and trypsin inhibitor activity, gave the poorest NPU value. This agrees with previous findings of Rackis et al.,I9 Garlich and Nesheirn2' and de Muelenaere" but is contrary to that of Naim et a/.' who found the poorest growth to be associated with the acid precipitated protein fraction which had a low trypsin inhibitor content. This discrepancy may be due to the different fractionation procedures used.

Despite the inverse correlation between NPU value and trypsin inhibitor content, no direct correlation between dietary trypsin inhibitor content and apparent nitrogen digestibility was found. Diets based upon raw soya bean meal, a pH 8 soluble extract (procedure 3) or whey proteins (procedure 6) (Table 3) containing the equivalent of 4, 6 or 33 g of trypsin inhibitor kg-' diet were digested equally well (85-87%) by rats. Indeed for an apparent nitrogen digestibility of 85% to have been achieved with rats fed diets containing whey protein (procedure 6 ) a minimum of 50% of the constituent trypsin inhibitors would have had to be degraded and absorbed. This would be an absolute minimum value since the other major whey protein component the lectin, is probably like many other seed lectins partially resistant to proteolytic

Nutritional evaluation of soya beans 1009

degradati~n.''-'~ These findings contradict those of Rackis3 and Liener and Kakade25 but are consistent with those of Palmer et al.9 with regard to kidney bean trypsin inhibitors and indicate that inhibition of gut proteolytic enzyme activity in vivo by soya bean trypsin inhibitors does not fully account for the poor nutritional performance of soya bean-fed rats. However soya bean trypsin inhibitors have been shown in vivo to effect pancreatic cell turnover26 and to stimulate pancreatic exocrine sectionz7 and the implications of these changes for overall nutritional performance need now to be more closely examined. The observations of this study, coupled with those of Naim et al.* and Sambeth et aL7 also highlight the need to study far more closely the effects of other whey components such as lectins upon nutritional performance.

Continuous exposure to soya bean diets over a long period led to a cumulative deterioration in overall performance for 16 weeks and thus the anti-nutritional problems elicited by consumption of raw soya bean or soya bean products are not a transitory response to first exposure to soya bean-containing diets. With the increasing usage of soya bean or soya bean-based food products this may have considerable implications for both human and animal nutrition.

5. Conclusions

The poor nutritional performance of rats fed diets containing raw soya bean appears to be due to reduced apparent digestion and absorption of dietary protein coupled' possibly to changes in systemic metabolism leading to a poor overall nitrogen balance. The antinutritional effects involved can be reduced but not eliminated by rigorous aqueous heat treatment. Aqueous ethanol heat treatment which combined the beneficial effects of removing tannins, saponins etc., with mild denaturation of most proteins was more effective.

The whey protein fraction (soluble at pH 4.8), which contained the bulk of the haemagglutinin and trypsin inhibitor activity, gave the poorest NPU value when incorporated into diets for rats. However an eight-fold increase in trypsin inhibitor content did not significantly affect apparent nitrogen digestibility and therefore inhibition of gut proteolytic enzyme activity in vivo by soya bean trypsin inhibitors does not fully account for the poor nutritional performance. Conse- quently, the implications of trypsin inhibitor induced pancreatic changes and the effects of other whey components upon overall nutritional performance now need to be examined.

Continuous intake of soya bean-containing diets for up to 16 weeks leads to a cumulative deterioration in growth and thus the problems associated with their consumption are not a transitory response to first exposure to soya bean.

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18.