Download Germination alters the chemical composition and protein quality of

J Sci Food Agric 1992, 60,419-423
Germination Alters the Chemical Composition
and Protein Quality of Lupin Seeds
Suzanne G Dagnia," David S Pettersoqb* Roma R Bellc and Frank V Flanagan"
a Edith
Cowan University, Western Australia, Australia
Department of Agriculture, Baron Hay Court, South Perth, Western Australia 61 5 1, Australia
Curtin University of Technology, Western Australia, Australia
(Received 13 August 1991 ; revised version received 29 May 1992; accepted 18 September 1992)
Abstract: The chemical composition and protein quality of the kernels from
Lupinus angustijolius seeds were compared with that for sprouts after 6 days
germination. Germination resulted in an apparent increase in protein content from
395 g kg-' to 435 g kg-' DM. Fat and carbohydrate contents decreased. The
oligosaccharide content of the sprouted lupin fell to a negligible level, while the
phytate and alkaloid concentrations fell from 4.7 g kg-' to 1.6 g kg-I and from
0.72 g kg-' to 0.16 g kg-'. respectively.
The quality of lupin kernel protein was poor with a protein efficiency ratio
(PER) of 1.45+0.15. Supplementation of kernel with DL-methionhe (2.0 g kg-')
increased the protein quality (PER = 2.87k0.17) to that of casein (PER =
2.86k0.18). Germination reduced protein quality (PER = 0.44-tO.I6), and did
not improve apparent protein digestibility (APD kernel = 80 f 4 YO; APD sprout
= 77 f5 YO).
Supplementation of sprout protein with DL-methionine (2.0 g kg-')
increased the protein quality (PER = 2.57 020). The total sulphur-containing
amino acid concentration of lupin kernel protein, 1.9 g per 16 g N was low. and
decreased further to 1.3 g per 16 g N in the sprout, a drop of 32%.
The results showed that germination of lupin seeds reduced the concentration
of the anti-nutritive factors; however, it also reduced protein quality.
Key words: lupin, germination, protein quality, nutritional quality, artinutrients.
el a1 1986). The cell wall material from the lupin kernel
has cholesterol lowering properties (Evans el al 1990).
Therefore, research into the use of lupin as a human food
seems warranted.
It is a generally held belief that sprouting increases the
nutritive value of legume seeds. Past research has
determined that germination improves the nutritional
value of Phaseolus radiatus, P angularis, Glycine hispida,
G m a s (Lee and Karunanithy 1990), red kidney beans
(El-Hag et a l l 9 7 8 ) and L albus (EJ-Habbal eta! 1987) by
increasing the proportions of proteins and ascorbic acid,
and/or by decreasing the content of anti-nutritive
factors. However, the nutritional value of navy and pinto
beans (P. uulgaris) was not improved following germination ; protein quality was unchanged, and trypsin
inhibitor activity persisted (Chang and Harrold 1988).
Although there was no information in the literature on
the effect of germination on the nutritive value of L
INTRODUCTION
Legumes are widely incorporated into human diets. The
soya bean has been used for many thousands of years in
the Orient as a vegetable, in beverages (especially as a
'milk') and as a base for fermented foods. It is also used
in a wide range of processed foods in Western nations
(Shurtleff and Aoyagi 1985). Seeds of Lupinus angusti$olius have an appearance and chemical composition
similar to the soya bean. The kernels are high in protein,
and Low in fat and anti-nutritive factors (Yanez ef al
1983; Lee 1986; Gross 1988; Petterson and Crosbie
1990). Lupin protein is low in the sulphur-containing
amino acids. However, when supplemented with DLmethionine (1.0-2.0 g kg-'), protein quality is high
(Sgarbieri and Galeazzi 1978; Yanez et al I983 ;Aguilera
* To whom correspondence should be addressed.
30
419
J F A 60
S G Dugniu, D S Petterson, R R Bell, F V Flunugun
410
TABLE 1
PER, PER ratio. apparent NPU and A P D scores for lupin kernels and sprouts"
Triwtiireiit
Divrorj. proii+i
grolrp
CIJllfC'Il l
PER
Ratio
NPU
.4 P D
507 (5.2)*
100.4 (5.8)**
100.0 (6.3)**
15.4 (5.6)***
89.9 (7.0)****
0.43 (005)*
0.58 (0.03)**
080 (0.04)*
0.63 (0.04)**
0.44 (0.13)*
0.92 (0.01)**
0.77 (0.05)***
048 (0.09)*
< 0.001
< 0~001
( N x 6.25) (g k g - ' )
I05
I10
I09
Kcrncls
Kerncls"
Casciii
Sprouts
Sprouts"
83
x4
ANOVA'
1.45 ( 0 1 5 ) *
2.87 (0.I7)**
2.86 (0. I8)**
0.44 (016)***
2.57 (020)****
< 0.001
< 0.001
I' Values arc means with standard deviations shown in brackets for 10 rats per group. Means for the same variable with a different
nuinbsr of superscript asterisks are significantly different by Scheffe's test ( P < 0.05).
I , Suppleincntcd with m-mcthionine (2 g kg I ) .
' ANOVA is one way analysis of variancc.
ringu.sri/dius. i t was thought likely that the concen-
trations of anti-nutritive factors would decrease and
protein content would increase, thus improving the
nutritional quality.
This study aimed to determine whether germination
would lead to a change in nutritive value of lupins with
respect to concentration of anti-nutritional factors and
to protein. in terms of both quantity and quality as
measured by amino acid profile and digestibility.
MATERIALS AND METHODS
Germination of seed
Lupin seeds ( L ungir.vt~foliuscv Gungurru) were soaked
in tap water for 24 h and germinated under natural
lighting at 30--25OC.The seeds were rinsed and drained
daily over the 6 day germination period. A kernel sample
was prepared by manually de-hulling lupin seeds. After
germination any seeds that had not already shed their
hulls were also de-hulled to provide a comparable sample
for analysis. The lupin sprout and kernel samples were
freeze-dried and milled prior to analysis. Seven batches
of sprouts were prepared and sub-samples of each
combined for analysis.
Analytical methods
Nitrogen. potassium, sodium and phosphorus were
determined by digesting the samples with sulphuric acid
and hydrogen peroxide, and analysing the diluted digest
through continuous flow colorimetry and flame photometry in a Leco four-channel autoanalyser. The fat
content was determined using a Tecator Soxtec apparatus. using Shell X4 as the solvent. Phytate extraction
and assay was by the rapid colorimetric procedure of
Latta and Eskin (1980). All of the above analyses were
conducted in duplicate. Amino acids were separated by
ion exchange chromatography and measured after
reaction with ninhydrin. The method of analysis is based
on Millipore-Waters manuals (Waters Associates 1984;
Walker 1986). The samples were oxidised with performic
acid, to ensure a good recovery of cystine and methionine, prior to hydrolysis at 110°C for 24 h in constant
boiling hydrochloric acid under nitrogen. The values
obtained for threonine, serine and isoleucine were
corrected for incomplete hydrolysis and partial decomposition using results from standard ANRC casein.
Tryptophan is not detected by this method. An extra
hydrolysis, without performic acid, enabled determination of phenylalanine, tyrosine and histidine. Protein
efficiency ratio (PER) followed AOAC methods (AOAC
1990). Apparent net protein utilisation (NPU) and
apparent protein digestibility (APD) were calculated
from nitrogen balance data obtained from metabolic
collections conducted during the third week of the PER
study.
Experimental diet
Milled lupin kernels or spouts (with or without the
addition of 2.0 g kg-' DL-methionhe) were incorporated
into four diets to provide about 100 g of protein per kg
of diet (the exact levels are shown in Table 1). The diets
were adjusted to provide equal amounts of fat
(80 g kg-'), crude fibre (10 g kg-I), moisture (100 g kg-I),
and ash (50 g kg-'), based on proximate analyses of
kernels and sprouts. Casein was used as the reference
protein in the fifth diet. The diets were supplemented
with vitamins and minerals according to AOAC (1990)
procedures.
Germination and nutritional quality of lupin seed
42 1
TABLE 2
Chemical analysis of lupin
and sprouts (g kg-') dry
basis)"
'(YO)
*
Analyte
sprouts (day
@
day adaptation period, experimental diets and water
were fed ad libiizcnz. Body weight and feed consumption
were recorded every 6 days, and on the 28th day of the
study. Faeces and urine were collected for four consecutive days during the third week of the experiment.
_ _ ___.____
Protein
Fat
Ash
Carbohydrate'
Oligosaccharide
Phytate
Calcium
Sodium
Potassi um
Phosphorus
Alkaloid (total)
I .8
<3
<3
-
3.8
2.9
2.2
1.4
2.2
1.8
10.7
395.0
82.0
25.5
385.5
59.0
4.7
1.o
0.6
8.9
2.9
0.72
453.1
41.0
39.8
332.1
3.0
I .6
2.6
2.7
8.4
3.5
0.16
Values are pooled samples analysed in duplicate.
CV--co-efficient of variation for assay.
' Carbohydrate by difference.
"
Data analysis
PER values were calculated from average weight gain
and nitrogen intake figures using the formula: PER =
change in body weight (g) per gram of protein consumed.
PER values were calculated by dividing the PER for the
test material by the PER for casein. NPU and APD
figures were determined from nitrogen (N) intake and
excretion during the 4-day metabolic collection, using
the following formulae: NPU = (N intake-(faecal
N + urinary N))/N intake; and AD = (N intake - faecal
N)/N intake. The data obtained were analysed using
one-way analysis of variance (ANOVA) and Scheffe's
test.
RESULTS
Experimental design
Biochemical evaluation
Fifty 21-day-old (Wistar) male laboratory rats were
obtained from the Animal Resource Centre (Murdoch
University, Australia). They were randomly allocated to
one of five groups, and housed in individual metabolic
cages in an environmentally controlled room (temperature, 20-22°C; relative humidity, 50%). After a 4
The concentrations of anti-nutritive factors (oligosaccharides, phytates and alkaloids), carbohydrate and fat
decreased during germination (Table 2). Ash and mineral
contents increased.
There was an apparent gain in protein concentration
in the sprouted lupin when expressed per kg DM.
TABLE 3
Amino acid profile of lupin kernels and sprouts (g per 16 g N)"
Atnino acidb
Cyscys as cysteic acid (cystine)
Methionine (as methionine sulphone)
Methionine +cystine
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
GIycine
Alanine
Valine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Lysine
Histidine
Arginine
Kernels
Sprouts (dav 6 )
Change
(Oh)
1.2
0.7
1.9
9.5
3.3
47
22.4
3.9
4.5
3.6
40
4.3
7.4
4.1
4.3
4.8
2.6
11.9
0.7
0.6
1.3
23.8
2.7
4.6
8.4
1.6
3.0
3.6
3.9
3.6
5.7
2.4
3.4
3.5
2.1
5.4
-42
- I4
- 32
+ 151
- 18
-2
- 63
- 59
- 33
0
-2
- I6
- 23
-41
-21
- 27
- 19
- 55
" Estimates based on pre-oxidation, 24 h and 72 h acid hydrolyses.
Note: tryptophan was not determined.
30-2
S G Dagnia, D S Petterson, R R Bell, F V Flanagan
422
TABLE 4
Body weights. average daily weight gain, food intake, dry matter digestibility and nitrogen excretion values for groups of rats used
in PER study of lupins"
Treatment
group
-
Kernels
Kernels"
Casein
Sprouts
Sprouts"
ANOVA'
Initial body
weight ( g )
Final body
weight ( g )
28 day food
intake ( g )
Dry matter
digestibility (YO)
354 (53)*
1.75 (0.30)*
565 (64)**
5.75 (0.90)**
516 (55)**
5.19 (0.60)**
034 (014)***
285 (37)*
3.73 (0.39)**** 536 (52)**
c 0.001
< 0001
93.2(1.6)*
93.4 (0.6)*
962 (0.5)**
909 (l.6)***
91.8 (0.7)*.***
< 0.001
Aijerage daily
weight gain (g)
_ _
66.7(7.4)
65.9(7.0)
65.3(8.4)
66.8(7.6)
65.2(7.2)
NS"
1 15.6(1 3.6)*
227.0(28.7)**
210.7(21.2)**
76.4 (94)***
169.6(15.9)****
< 0.001
Urinary N
Faecal N
k)
k)
0.30(0.06)*
0.28 (008)*
0.33 (@03)*
0.15 (0.05)*
0.22(0.03)**
0.09(0.01)***
0.15 (007)**
0.31 (008)*
< 0.001
0.10 (001)***
0.14 (0.02)*
< 0.001
" Values are means with standard deviations shown in brackets. Means for the same variable with a different number of superscript
asterisks are significantly different by Scheffe's test ( P < 005).
' Supplemented with DL-methionine (2 g kg-').
' ANOVA is one-way analysis of variance.
" NS is not significant.
Germination resulted in a decrease in the content of most
amino acids, with the exception of aspartic acid (Table
3). Most notably, the proportions of the total sulphurcontaining amino acids decreased by about one-third.
Protein quality
The results indicated that the quality of lupin kernel
protein was poor; however, supplementation with DLmethionine increased the quality to that of casein (Table
4). The PER study showed a substantial increase in
sprout protein quality after supplementation. However,
the quality of the supplemented sprout protein was
significantly less than the quality of the casein and
supplemented kernel proteins.
NPU was low in rats fed either kernels or sprouts, but
improved when diets were supplemented with DLniethionine (2.0 g kg-') (Table 4). Germination did not
improve protein digestibility since apparent digestibility
was 77*5% and 80?4% for sprouts and kernels,
respectively.
DISCUSSION
The concentration of oligosaccharides, alkaloids and
phytate decreased after 6 days germination. The reduction in the concentrations of anti-nutritive factors in
the sprout is of benefit for human nutrition. The
oligosaccharide content was reduced to a negligible level
therefore. flatulence, and other side-effects associated
with the consumption of high concentrations of oligosaccharides. will be greatly reduced when the germinated seeds are consumed. Sprouting lowered the
concentrations of the bitter alkaloids. In addition to
improving flavour, this would have also reduced the
already minimal risk of any untoward effects. The benefit
of a low phytate content in the sprout would be an
increase in mineral bioavailability, since phytate can
bind strongly with divalent ions, inhibiting their absorption. Phytate has also been shown to inhibit the
action of some digestive enzymes, by binding calcium
ions (necessary for trypsin and a-amylase activity) or
interacting with enzyme substrates (Liener 1989).
The protein quality of the lupin kernels and sprouts
was low. Supplementation of the kernel protein with DLmethionine (2 g kg-') increased the quality to that of
casein. Sprout protein quality also improved with
supplementation ; although not to the level of the casein
or supplemented kernels. The lower protein quality of
the sprouts is likely to be due to the reduction of the
content of sulphur-containing amino acids during germination. The proportions of other essential amino
acids, including threonine, lysine, leucine, isoleucine and
phenylalanine, decreased during germination. The fact
that the supplemented sprouts had a lower protein
quality than the supplemented kernels suggested that an
amino acid other than the sulphur amino acids became
limiting in the supplemented sprout diet. Further work
would be necessary to identify this second limiting amino
acid in sprouts.
The apparent increase in protein can be attributed to
the utilisation of fats and carbohydrates as energy
sources for the developing sprouts. The aspartic acid
concentration increased three-fold during germination
(Table 3). This change can be attributed to the release of
amino groups from other amino acids to oxaloacetate in
the shift from storage protein to functional protein
during the course of germination.
Consumption of lupin sprouts is most likely to be as
part of a mixed and varied diet, particularly in Western
countries where there is a wide range of dietary protein
sources. The loss of sulphur amino acids is unlikely to
Germination and nutritional quality of lupin seed
have an adverse effect on the quality of a mixed diet, and
would be outweighed by the benefits offered by the
reduction in the anti-nutritional factors, which also
means that they cannot have any adverse effect on the
rest of the dietary intake. A study of the time course for
the losses of sulphur amino acids and anti-nutrients
might indicate an optimal time for sprouting.
ACKNOWLEDGEMENTS
The authors thank the Grain Legumes Research Council
of Australia for financial support, Dr R L Davies and J
Baker for the amino acid analyses, and Dr D J Harris for
his assistance with other assays.
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