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J Sci Food Agric 1996,71,449-458
Free and Total Ammo Acid Composition of
Edible Parts of Beans, Kale, Spinach, Cauliflower
and Potatoes as Influenced by Nitrogen
Fertilisation and Phosphorus and Potassium
Deficiency
Wilfried H Eppendorfer*
Section of Soil, Water and Plant Nutrition, Department of Agricultural Sciences, The Royal Veterinary and
Agricultural University, 40, Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark
and Sarren W Bille
Ministry of Agriculture and Fisheries, 10, Slotsholmsgade, DK-1216 Copenhagen K , Denmark
(Received 7 July 1995; revised version received 17 November 1995; accepted 26 February 1996)
Abstract: Vegetables were grown in pots at widely differing nutrient levels, which
greatly affected dry matter (DM) yields and total-N concentrations in all crops.
Nitrate-N contents were low and little affected in cauliflower and potatoes, and
highest, and strongly affected, in spinach and kale. The sum of free amino acid-N
as percentage of total-N of bean pods, kale, spinach, cauliflower curds and
potato tubers varied between 12 and 27%, 10 and 21%, 5 and 12%, 7 and 36%
and 34 and 56%, respectively. In beans and potatoes asparagine was the dominant free amino compound (29-55% and 33-59% free amino-N as percentage of
total free amino-N, respectively), whereas in kale, spinach and cauliflower free
glutamine was dominant (17-52%, 31-48% and 14-54%, respectively). Free
essential amino acids were generally found in very low concentrations, especially
cysteine (which can partly replace essential methionine in nutrition), tryptophan
and phenylalanine. With some exceptions in beans, the concentrations of all total
amino acids in D M increased linearly with increasing total-N content, and with
correlation coefficients very close to 1.00 in most cases. P- and K-deficiency
affected free and total amino acid composition mainly through their effects on
total-N content but had some specific effect on arginine concentrations. Generally, chemical scores of the crude protein decreased with increasing N content of
DM, which was mainly due to low contents of S-amino acids.
Key words : free amino acids, amides, nitrate, protein, beans, kale, spinach,
cauliflower, potatoes, fertiliser effects.
INTRODUCTION
studied (Nowakowski and Byers 1972; Rendig 1984).
Their importance for all aspects of plant metabolism is
generally recognized and metabolic studies of different
N-compounds of plants abound (Miflin and Lea 1977).
Besides their significance in plant metabolism, such Ncompounds are also of great importance in the nutrition
of animals and man whose N-metabolism may be considerably influenced by amounts and forms of N ingested with different plant materials (Kofranyi 1967; Eggum
1973). In monogastric animals and man the nutritional
It is well known that environmental as well as genetic
factors may greatly influence the chemical composition
of plants (Wild and Jones 1988; Martin del M o h o et a1
1989). Of various plant components which may be
affected, nitrogen fractions, particularly amino acids,
amides and nitrate, are quite prominent and widely
* To whom correspondence should be addressed.
449
J Sci Food Agric 0022-5142/96/$09.000 1996 SCI. Printed in Great Britain
450
W H Eppendorfer, S W Bille
value of N-compounds or of crude protein (totalN x 6-25) is dependent on their content of essential
amino acids, which have to be present in the feed or
food in certain amounts and combinations, to allow for
an optimal utilisation of the protein (FAO/WHO 1973).
Plant proteins and non-protein N-fractions also contain
non-essential amino acids, the amounts of which have a
bearing on the protein quality of plants (FAO/WHO
1973). In earlier studies of effects of nutrient supply on
amino acid composition and protein quality of vegetables (Eppendorfer 1978; Eppendorfer et al 1979) only
results of total amino acids analyses were published.
However, for a more detailed understanding of nutrient
effects on variations of nitrogen fractions in plants, free
amino acids, amides and nitrate have also to be considered.
Thus, the aim of the present investigation has been to
study the influence of varying levels particularly of
nitrogen and of phosphorus and potassium on free
amino acid, amide and nitrate content of various vegetables at a marketable stage of development. In order to
be able to estimate the contribution made by individual
free amino acids to corresponding total amino acid contents (free + protein amino acids), the latter were also
determined. To allow a comparison with the nutritional
requirement as given by the composition of a
FAO/WHO reference protein (FAO/WHO 1973),
results of total amino acid analyses are also expressed
as g per 16 g N which is equivalent to the proportion of
crude protein. These results are also used to calculate
chemical scores (CS, Block and Mitchell 1946) which
express the proportion of the first limiting essential
amino acid in relation to the corresponding recommended value of the above FAO/WHO reference
protein.
MATERIALS AND METHODS
The plants were grown in the open in PVC pots with a
cross-section of 500 cm2 and a height of 40 cm. To
obtain a wide variation in nitrogen concentration, a
nutrient-deficient, virgin, sandy soil from Sdr Omme,
Jutland, mixed with 25% by volume of sphagnum peat,
was used. Before addition of CaCO, the pH (H,O)of
the soil mixture was 4.0. After harvest pH (H,O) values
were 5-6, depending on treatments. Except for varying
nutrient doses in respective experimental treatments,
each pot received a basal dressing of 2.5 g P, 6.0 g K
(spinach 4.0 g), 1.5 g Mg, 50 g CaCO,, 0.25 g Mn,
0.25 g Cu, 8 mg B and 3 mg Mo. The rates of N-, Pand K-application are given in the tables. Nitrogen,
phosphorus, potassium and magnesium were applied as
Ca(NO,),, KH,PO,, KCl and MgS0,. 7 H,O, respectively. All nutrients were thoroughly mixed with the
soil. The largest rates of nitrogen were split and partly
applied in liquid form. The plants used were French
beans (Phaseolus oulgaris L cv Darkskinned Perfection),
kale (Brassica oleracea acephala, DC cv Grena), spinach
(Spinacea oleracea L cv Dorema), cauliflower (Brassica
oleracea botrytis L cv Idol) and potatoes (Solanum
tuberosum L cv Bintje). Growing conditions were
described earlier (Eppendorfer 1978). The crops were
harvested at a marketable stage of growth. Except for
potato tubers, part of which were used directly for
extraction, the other crops were, after harvest, at once
placed between layers of dry-ice, deep-frozen and freezedried as soon as possible.
Analytical methods
The dry matter content was determined by freeze-drying
and further drying at 100°C for 16 h. Total-N was
determined by a micro-Kjeldahl method; the salicylic
acid-thiosulphate modification was used to include
nitrate-N. Nitrate was determined according to
Bremner and Keeney (1965). For free amino acid
analysis 15 g deep-frozen spinach, 1.5 g freeze-dried
kale, 2 g freeze-dried beans or cauliflower or 40 g fresh
potatoes were extracted with 80 ml 5g litre-' picric
acid, according to a slightly modified method by Stein
and Moore (1954). Free amino acids and amides in the
extracts were determined on a Beckman Amino Acid
Analyser, Model 120C, by using lithium buffer solutions
on the exchange columns, which allowed a clear separation of amino acids and of asparagine and glutamine.
For total amino acid analysis, 0.5 g freeze-dried,
finely ground plant material was hydrolysed under
reflux with 500 ml 6 M HCl for 24 h with addition of
0.5 ml thioglycolic acid, to prevent oxidation of tyrosine
(Eppendorfer and Bille 1973). Amino acid in the hydrolysates were determined on a Beckman Amino Acid
Analyser, Model 120°C, by using sodium-based buffer
solutions. Methionine and cystine were determined as
methionine sulphone and cysteic acid after oxidising the
samples with performic acid at 50°C for 15 min and
subsequent hydrolysis according to a method by Moore
(1963) as modified by Weidner and Eggum (1966).
RESULTS AND DISCUSSION
Tables 1-5 present data for crops of beans, kale,
spinach, cauliflower and potatoes, respectively. In each
table the three sets of columns give the free and total
amino acids (pmol g-l), and the total amino acids per
16 g non-nitrate N as is customary in animal nutrition
studies. Also in each table the first row gives the nutrient levels of treatments and the subsequent rows give
the results obtained for the crop under these treatments:
firstly dry matter (DM) yield (g per pot and kg-'), total
N and nitrate N (g kg-' DM); thereafter follow the
amino acids, etc abbreviated conventionally or as
shown in the footnotes to Table 1; the bottom row
0.50
3.26
2.00
17.16
13-67
62.36
8-20
3-84
29.04
5.73
2.17
1-56
0-39
0.60
150-47
3.38
3.44
0.45
19-66
0-42
2.00
2540
0.37
13.23
0.61
7.60
14.91
23-34
4.72
2.61
12-a4
6.05
8.04
2.5 1
0.36
0-38
31.67
5-62
3.55
0.64
10.33
0.45
2.66
12.23
LYS
His
Arg
ASP
Thr
Ser
Glu
Pro
GlY
Ala
Val
Ile
10.0
8.9
26.4
21.01
0.73
3.77
27.43
4-09
38.23
7.00
2.41
1.20
0.49
1-20
211.02
49.59
4.52
18.1
1.2
3-3
5.8
8.2
7.2
30.4
4-1
2.3
3.5
3.7
10.1
7.6
6-7
3.9
14.0
25.0
5.9
0.51
5.03
2.79
21-98
18.88
8 1*23
10.26
12.0
53.8
84,O
39.1
2.7
CV (%)(I
17.1
16.4
72.1
63.4
30.2
42.1
112-7
60.9
80.9
107.7
60.9
85.1
82.2
71-3
52.9
83.0
31.1
38.7
25.0
21.0
21.8
61.0
74.4
29,8
49.1
285.4
65.1
126.1
169.0
62.8
95.5
120.4
79-4
54.2
90.9
32.5
43.9
39.1
Jm
20.2
18.7
64.5
64.4
23.4
42.7
207.7
57.1
105.6
136.0
57.3
82.6
101.4
69.7
48.0
79.4
26.8
37.5
30.8
Total aa (pmol g - ' DM)
8.3
5.9
1.9
2.5
2.9
1.4
1.8
2-9
1.6
2.3
1.6
1-8
2.6
1.8
1.9
2.2
8.5
CV (%)a
cs
1-32
1.57
83
10.15
4.48
4.09
4-68
5.35
4.43
6.97
3-60
4-09
5.44
5.94
3.00
4.69
9.60
4.64
25.0
1.34
1.51
81
5.11
1.97
4.03
15.00
3.70
6.02
10.86
3.58
3.36
4-90
4.43
3.42
5.65
2.64
3.37
29.5'
1.13
1*43
73
4.78
2.03
3-76
16.69
3.41
5.83
10.95
3.18
3.15
4.73
4.09
3.13
5.24
2.58
3.18
36.4'
Total aa (g per 16 g N ( - NO,))
3.5
6.0
4.0
7.0
5.0
4.0
5-5
FAOIWHO
Ref prot
'
Coefficient of variation for all five crops (Tables l-s), based on standard errors s =
for a mean, where d is the difference between duplicate determinations and p is
the number of samples.
Sum of aa N as % of total-N.
Total-N without NO,-N.
In all tables Asn = asparagine, Gln = glutamine, E-am = ethanolamine, 7-AB = y-aminobutyric acid.
TYr
Phe
Asn
Gln
E-am
Trp
7-AB
CYS
Met
Caa-Nb
Leu
8.0
85.1
84.5
30-8
1.3
2.0
27.3
81.5
25.0
< 0.05
g N per Pot
g DM per pot
DM (g k g - 7
Total-N (g kg-' DM)
NO,-N (g kg-' DM)
Free aa (pmol g - ' DM)
TABLE 1
Nutrient effects on concentrations in dry matter (DM) of total and nitrate nitrogen, free and total amino acids (aa), amides, and on chemical score (CS) of green bean pods
(Phaseolus vulgaris L cv Carlos Favorit)
7
R
g.
$
!L
2
c1
n
cp
cp
452
W H Eppendorfer, S W Bille
TABLE 2
Nutrient effects on concentrations in dry matter (DM) of total and nitrate nitrogen, free and total amino acids (aa), amides, and on
chemical score (CS) of kale (Erassica oleracea acephala DC cv Grena)
Free aa (pmol g-' D M )
g N per Pot
g DM per pot
DM (g kg-')
Total-N (g kg-' DM)
NO,-N (g kg-' DM)
LYS
His
Arg
ASP
Thr
Ser
Glu
Pro
GlY
Ala
Val
Ile
Leu
TYr
Phe
Asn
Gln
E-am
TrP
y-AB
CYS
Met
Eaa-N"
1.o
64.7
134
22.9
0.4
6.0
132.6
125
31.6
2.6
1.46
2.82
5.05
5.72
4.78
8.60
9.88
26.02
0.81
7.34
3.97
1.75
1.10
0.63
0.71
7.24
13.81
3.91
1.74
2.53
8.10
6.70
6.44
16.59
20.32
40.88
1.97
12.56
4.52
1.70
1.38
0.68
0.87
10.51
63.20
3.64
17.39
tr
tr
10.17
23.04
tr
tr
14.73
Total aa ( p o l 9 - l D M )
Total aa (g per 16 g N( - NO,))
+
8 6
102.4
129
47.1
9.0
1.94
4.22
18.00
10.40
7.83
22.98
33.36
64.77
5.58
15.36
5.46
2.23
1.37
0.92
0.80
22.18
179.69
3.84
0.20
20.82
2.12
2.19
20.60
22.9
31.6
47.1
60.7
21.7
45.9
89.2
52.7
60.5
117.9
91.5
92.6
89.2
66.8
44.4
81.4
27.8
41.3
72.9
25.2
58.7
110.7
67.2
78.6
210.6
124.8
117.1
116.3
83.0
54.4
103.4
36.0
53.2
15.4
16.6
76.9
19.4
20.0
73.2
22~5~
29*Ob
38.1b
88.4
32.6
80.6
150.6
86.6
99.9
333.4
167.3
150.7
143.5
102.9
66.6
124.6
45.5
66.3
6.31
2.40
5.68
8.44
4.47
4.52
12.34
7.49
4.94
5.66
5.56
4.14
7.59
3.58
4.83
7.87
2.16
5.64
8.14
4.41
4.55
17.11
7.93
4.85
5.72
5.36
3.93
7.48
3.61
4.85
5.43
2.13
5.90
8.42
4.34
4.40
20.61
8.16
4.75
5.38
5.06
3.67
6.87
3.45
4.60
23.5
25.0
65.0
1.33
1.77
89
1.31
1.66
85
1.19
1.56
79
CS
Sum of aa N as YOof total-N.
Total-N without NO,-N.
gives the total amino acid N (YOtotal N) in the first two
sets of columns and chemical scores in the third set.
Bean pods
In Table 1 the three N-levels represent N-deficiency,
-optimum and -surplus, which is also reflected in the
DM yields. Our results show an exceptionally wide
range in total-N content. Increasing N applications
increased total free amino acid-N as % of total-N from
12% to 27% which agrees very well with the value of
16.5% found by Wunsch (1975) at a pod N content of
2.62%. The increases, as well as their level in DM, differed greatly between different amino acids. At all three
N-levels the largest contributions to the free amino acid
pool were made by asparagine and serine, and the Ieast
by tyrosine, lysine and cystine. In contrast, isoleucine
and leucine decreased with increasing N content, and
the concentration of histidine was inexplicably high at
the lowest N-level. Total amino acid contents were generally less affected by nitrogen applications than free
amino acids or amides. The decrease of the sum of total
amino acid-N as % of total-N from 72% at the low to
61% at the high N-level was larger than could be
explained by the concomitant increase in nitrate nitrogen (from <0.05 to 2.7 g kg-' DM). Table 1 also contains values of desirable essential amino acid
concentrations as recommended in human nutrition by
FAO/WHO (1973). As a comparison at higher N contents in D M shows, all values are increasingly falling
short. This applies particularly to methionine cystine,
whose combined concentration generally is first limiting
in legume seeds, whereac the concentration of lysine
normally is quite high in legume foliage and seeds.
Despite a roughly five-fold higher concentration of
methionine than of cystine/cysteine in the free amino
acid pool, the content of methionine is only slightly
higher in the protein (g per 16 g N). Similarly, it can be
seen that free cysteine only represents 2-3.5% of total
cysteine whereas free methionine amounts to 11-17% of
the total methionine. Of the other free essential amino
acids threonine in particular, represented a rather large
+
2.40
8.21
27.06
2.61
1.45
7.30
3.01
1-63
2-73
0.93
1-31
4.51
25-70
0
tr
11.19
tr
tr
5.3 1
5.88
1-40
1-20
5.82
7-78
3.22
14.97
38.52
3.74
10-90
14-34
4.69
2-80
3.10
1.35
2-22
6-20
48-78
0
tr
20-05
tr
tr
6.72
1-46
0.97
5.33
1.53
1-02
3.24
6-03
2.52
8.12
28-73
4.75
1.51
9-57
4.02
2.42
4.32
1.35
1.66
3-82
28-44
0
tr
11.85
0-14
0.19
6431
Sum of aa N as % of total-N.
0 g P + 4.0 g K.
' 2.5 g P + 0 g K.
' Total-N without N03-N.
Ile
Leu
TYr
Phe
Asn
Gln
E-am
Trp
7-AB
CYS
Met
Zaa-N"
Val
His
Arg
ASP
Thr
Ser
Glu
Pro
GlY
Ala
LYS
3.0
42.0
69.2
55.7
12.2
0.07
0-14
11.04
5-60
0.56
1.42
8.63
10.37
3.58
10-54
32.98
3.22
13.52
10.00
3.88
3.54
6.15
8.07
3.90
8.53
80.21
0
tr
3(-P)b
5.1
120-9
40.3
1.5
( p o l g-' D M )
1.o
45.3
69.0
43.1
4-1
a0
0-25
37.9
68.6
35.1
1-6
Free
1*79
2-97
31-71
4.91
5-56
29.64
58.54
6.04
21-87
14-74
9.34
3.96
7.25
2.47
2.51
12.27
117-28
0
tr
15.65
tr
tr
11.56
3(-K)'
22.8
79.7
67-2
11.6
97.7
33.5
79.1
156.9
84.3
95.5
248.4
99-8
170.9
153.2
120.9
79.3
142.6
50.8
71.2
32.2
30-1
69.9
27.7
24-7
73-3
43.1
84.8
28.4
65.4
138.1
73.2
82.9
206.9
85.7
145.9
131.5
103.9
68.5
124.6
44.7
61.5
35.1
32-3
35.9
60.9
110-7
37.0
87.8
179.6
95.9
110.7
292.0
106.6
195.7
173.5
134.6
87.7
159.7
56.2
78.4
55.7
32-8
35.1
71.7
88-1
30.4
73.0
154-4
81-9
90-2
277.4
94.7
166-4
136.8
107.6
72.3
132.3
55.1
67.3
40-3
Total aa (pmolg-' D M )
35.7
41.6
62.1
125.5
43.7
131.7
201.2
108.7
135.2
407.1
120.7
229.3
193.2
156.0
100.6
182.4
65.8
90.0
67.2
CS
1.61
1-76
96
4.30
7.81
3.86
4.86
5-60
5.82
5.92
2-11
5-44
8.78
4.16
4.16
14-54
4-72
5-23
33.5'
1-59
1-85
98
5.95
2.11
5.62
8-78
4.20
4.28
15.80
4.51
5.86
2-13
5.65
8.58
4-12
4-12
15.01
4-71
5-26
5.60
5.81
4.28
7-68
3.78
4.84
1-45
1.79
91
5.40
5.69
5.79
4.23
7.71
3.74
4.76
43.5'
39.0''
55.6'
5-28
1-95
6.60
7-70
3.73
4-09
17.25
4.00
5.02
4.96
5.26
3.81
6.88
3.43
4.29
1-24
1.79
87
38.8'
5.31
1.95
5.25
8-47
4.01
3.91
16.83
4.50
5.15
5.02
5.19
3-91
7.16
4.12
4.59
1.64
2.16
98
Total aa (g per 16 gN ( - NO,))
TABLE 3
Nutnent effects on concentrations in dry matter (DMj of total and nitrate nitrogen, free and total amino acidb (aa), amides, and on chemical scare (CS) of spinach (Spinacea
oleracea L cv Dorema)
*rl
P
w
VI
c
P
m
C
3
3
...
I,
5
E
0
%5.
%
&
-
2m
454
part of the total (24-29%), whereas only 0.6-0.7% of
total lysine was found as free amino acid. The other
extreme is represented by free aspartic acid +
asparagine which amounted to 34.8, 80.7 and 86.6%
of the total at increasing N contents.
Kale
Increasing N-applications first increased and then
decreased DM yields of kale (Table 2), thus indicating
deficiency, sufficiency and surplus or toxicity of nitrogen, respectively. Total-N and NO,-N concentrations
were raised from 22.9 to 47.1 and from 0-4 to
9.0 g kg- ' DM, respectively. Increasing the N-level
from 1 g to 8 + 6 g per pot doubled the sum of free
amino N as 'KOof total-N from 10% to 21%, which is
close to the magnitude for bean pods. Effects of increasing N-Jevels on individual free amino acid concentrations of kale were most pronounced for glutamine,
followed by proline, glutamic acid, serine and asparagine. Generally, their proportion of corresponding total
amino acid concentrations were quite high, especially
glutamine and proline, which increased from 12 to 54%
and from 28 to 38%, respectively, at the 1 g and 8 + 6 g
N level. Practically at all three N-levels, the largest contributions to the free amino acid pool of kale were made
by glutamine and proline, whereas tyrosine, phenylalanine, leucine, lysine and isoleucine contributed the least.
Total amino acid contents of kale were highest, and
increases resulting from N-applications most pronounced, for glutamic acid (glutamine), followed by
proline, glycine, aspartic acid (asparagine), alanine and
leucine. The sum of total amino acid-N as % of total-N
decreased from 76.9% to 65% at the 8 + 6 g N level,
which was entirely due to the corresponding increase in
NO,-N. After correction for the nitrate content there
were only small changes. A number of amino acids
slightly decreased with increasing N-content, only glutamic acid increased greatly and a few others slightly,
thereby more than compensating the decreases of the
others. In contrast to what was found with bean pods,
most essential amino acids concentration met the
FAO/WHO requirement (Table I), except for
methionine + cystine, and for lysine, isoleucine and
leucine at the highest N-level. Thus, methionine
(+cystine) is the most limiting essential amino acid in
kale as well as in bean pods and in most other vegetative plant material. Above results generally agree with
findings by Byers (1971) and Eppendorfer (1978).
Spinach
Increasing levels of N caused large increases in both
total- and nitrate-N of spinach (Table 3). Strong Pdeficiency caused a reduction in the concentration of
total-N and, particularly, of NO,-N, whereas K-
W H Eppendorfer, S W Bille
deficiency resulted in an exceptionally high concentration of total-N, although DM yield was much less
reduced than by P-deficiency. Increasing N-applications
hardly affected the sum of free amino acid-N, which is
in contrast to corresponding changes in beans, kale and
potatoes (Tables 1, 2 and 5 (see below)). P- and Kdeficiency, however, both almost doubled the sum of
free amino acid-N, despite a large difference in total-N
content of DM. N-applications increased free glycine,
glutamine and serine considerably. K-deficiency particularly increased the concentrations of glycine, arginine,
serine, glutamine, asparagine and glutamic acid. Similar,
but generally less pronounced changes were found with
P-deficiency, which, on the other hand, especially
increased the concentration of free tyrosine. At all Nconcentrations of DM glutamine and glutamic acid
contributed most to the free amino acid pool; combined
they represented between 46 and 53% of free amino N.
The lowest concentrations were represented by cysteine,
methionine, lysine and histidine. From total amino acid
composition in Table 3 it can be seen that at all five
treatments the highest concentration was found for glutamic acid, followed by glycine, aspartic acid, alanine,
leucine and valine, whereas cystine, methionine and histidine were lowest. In accordance with results by
Wenzel and Michael (1967) with spinach leaves and by
Byers (1971) with barley, lupin and Chinese cabbage
leaves, there were only minor changes in amino acid
composition of spinach crude protein with increasing
N-applications, when nitrate-N was excluded from
total-N. This is due to the low level of the free amino
acid pool (about 6% of total-N) which, in contrast to
the practically unchangeable amino acid composition
of protein fractions, may be considerably affected by
N-applications. Under P- and, particularly, under
K-deficiency conditions, changes in amino acid
composition of crude protein are somewhat more pronounced, which is also evident from the higher proportion of the sum of free amino N as YOof total-N in DM.
A comparison with the FAO/WHO reference protein
requirement (given in Table 1) shows that, except for the
highest N-content, the concentrations of most essential
amino acids practically reach that requirement. Total
tryptophan, however, was not determined in the present
investigation.
Cauliflower
Results in Table 4 show that besides nitrogen, Kdeficiency in particular caused a great increase in
total-N content of cauliflower, whereas nitrate content
was hardly affected. The sum of free amino acid-N as a
percentage of total-N was of about the same size as for
spinach, whereas P- and K-deficiency four- or five
doubled its proportion, respectively. With moderate Pdeficiency and a fairly high N-content by far the most
prominent free amino compound was glutamine, whose
455
Free and total amino acids in vegetables
TABLE 4
Nutrient effects on concentrations in dry matter (DM) of total and nitrate nitrogen, free and total amino acids (aa), amides, and on
chemical score (CS) of cauliflower (Brassica oleracea botrytis L cv Idol)
Free aa (pmol g-' D M )
8.0
g N per pot
62.7
g DM per pot
92.5
DM (g kg- I )
Total-N (g kg-' DM) 31.6
NO,-N (g kg-' DM)
0.4
LYS
His
Arg
ASP
Thr
Ser
Glu
Pro
GlY
Ala
Val
Ile
Leu
TYr
Phe
Asn
Gln
E-am
TrP
+
8 4
57.9
93.0
42.2
0.8
8(-P)b
45.1
94.4
40.4
S(-K)c
16.6
77.8
68.0
I .o
Total aa (pmol g-' DM)
31.6
42.2
8(-P)d
7.8
108
48.4
0.5
7.08 86.4 91.8
84.2 132.4
1.83
1.56
4.34
14.54 25.1 29.7
30.4 52.1
2.62
0.59
13.44 28.54 55.6 71.5 149.7 109.4
61.79 74.14 172.27 127.1 152.4 168.4 306.5
4.13
86.8 106.1
12.44 17.07 65.1 75.4
22.81 37.16 59.94 87.5 119.1 148.3 171.7
27.85 35.98 52.89 183.6 324.3 362.4 679.3
3.40
2.84
5.89
1.27
7.14
8.74 118.2 132.9 117.9 172.2
6.17 115.0 162.5 154.7 217.2
12.19 32.16
6.24 20.35 35.54 93.6 117.1 111.2 160.6
12.57 58.8 66.2
1.91
6.20
63.4 93.0
1.04
2.60
4.80 101.0 107.4 98.1 145.8
1-71
6.27 32.6 36.8
1.17
36.9 55.5
1.14
1.90 11.30 46.4 50.3 49.4 79.7
6.24 28.19 97.19
15.66 191.39 478.68
6.25
7.63
19.35
0.97
0.50
2.09
6.93 10.00 10.82 18.70
tr
tr
0.46
0.26 22.9 27.2
30.8 39.0
tr
tr
0.99
2.33 25.6 27.5
24.4 34.6
7.44
7.33 26.50 36.41 68.6 65.0
66.6 70.6 CS
1.68
1.61
0.72
11.86
4.54
11.49
31.72
2.14
1.94
13.03
5.36
1.73
1.42
0.72
0.74
4.47
23.97
6.17
7-AB
CYS
Met
Zaa-N"
68-0
Total aa (g per 16 g N ( - N O , ) )
-
31.2"
41.4"
47.9'
67.0'
6.48
2.00
4.97
8.67
3.98
4.72
13.86
5.19
1.78
4.82
7.85
3.48
4.84
18.43
4.11
1.59
8.71
7.49
3.05
5.20
17.81
4.62
1.93
4.55
9.74
3.02
4.31
23.88
4.55
5.26
5.62
3.95
6.80
3.04
4.00
3.85
5.60
5.30
3.36
5.45
2.58
3.21
2.95
4.60
4.35
2.77
4.29
2.23
2.73
3.09
4,62
4.50
2.92
4.58
2.40
3.14
1.42
1.95
96
1.27
1.59
78
1.25
1.22
61
1.13
1.23
65
Sum of aa N as % of total-N.
' 0.75 g P + 6.0 g K.
2.5 g P + 0.25 g K.
0.15 g P + 6.0 g K.
Total-N without NO,-N
proportion, together with free glutamic acid, amounted
to 62.7% of total glutamic acid, followed by aspartic
acid, which, together with asparagine amounted to
60.7% of total aspartic acid. With strong K-deficiency,
resulting in an extremely high total-N content of
68.0 g kg-' DM, the glutamine concentration also was
extremely high, and together with free glutamic acid,
they amounted to 78.2% of total glutamic acid. Free
aspartic acid as the second most important amino compound under K-deficiency conditions, together with
asparagine, even represented 87.9% of total aspartic
acid content of cauliflower.
When expressed as g per 16 g N the effect of higher
N-contents, generally, has been a decline in the concentration of most amino acids, except glutamic acid,
which increased considerably. For several amino acids
the pattern is somewhat different under strong P- and,
particularly, K-deficiency. Only with the lowest N-
content do all essential amino acid concentrations of
cauliflower crude protein reach, or come close to the
FAO/WHO requirement values as given in Table I . At
higher N-concentrations most values are considerably
below requirements.
Potatoes
In Table 5 the free and total amino acid composition of
fresh potatoes are given. Increasing the N-level beyond
the optimum amount of 6 g N per pot resulted in
decreases in both DM yield and content, whereas
total-N and NO,-N content increased. Both P- and Kdeficiency greatly reduced DM yield but increased
total-N concentration, when compared with the same
N-level (12 g N) without nutrient deficiency. Nitrate-N
contents were only of significance at very high N-levels,
and even then amounted to only 5-6% of total-N,
+
+
2.18
1.99
10.61
13.64
5.09
7-10
19.92
34-92
1-81
8.51
9.90
1a68
0.69
0.33
0-87
138.59
91-24
0-33
0.13
20.30
0.40
2.15
39-34
5-79
2.82
4.37
11.11
4.32
5.54
12.89
1-43
1.89
6-12
14.03
6-17
2-26
3-74
4.32
48-06
30.70
0.26
1*89
17-61
0.43
2.06
33.92
Sum of aa N as % of total-N.
0.15 g P 8.0 g K.
4-0 g P 0.15 g K.
* Total-N without NO,-N.
His
k g
ASP
Thr
Ser
Glu
Pro
GlY
Ala
Val
Ile
Leu
TYr
Phe
Asn
GlU
E-am
Trp
1-AB
CYS
Met
Caa-No
LYS
12.0
266.4
185
22-8
0.9
6.0
483.1
215
12.1
0.1
13-06
0-38
3-64
55.82
20-24
0.61
2-74
49-09
21-48
0-86
1.66
45-76
5.10
8.12
12-81
16.61
5-69
11.47
18-49
3-74
2.13
9-64
17.52
5-44
0.93
1.84
1.83
276.57
92.64
0.52
2.13
1.61
9.47
21-79
6-17
8-67
30.80
35.64
1-56
6-42
9.00
1*42
0.54
0.24
0.62
257.27
131.28
0.24
2-85
3.38
15.59
18.18
7-52
11-52
24-78
72-09
2-46
13-31
13-03
2-39
0.96
0.54
1a 5 0
204.14
173-89
0.36
'
12(-K)'
50.6
208
28.9
0.7
12(-P)b
65-8
167
24.2
1-6
12 + 8
170-0
168
29.3
1-6
Free aa (pmol g-' DM)
8.0
9.4
71.7
29.3
8.8
17-7
93-2
23.0
26.8
79.3
24.4
34-4
32-4
39.3
24.1
33.9
15-4
20.3
12.1
12.7
13.8
68.2
44.7
12.9
32.5
198.7
38.8
44.1
165.8
72.4
56.4
51.1
55-4
34.1
58.4
21-2
28.7
22.8
14.7
16.2
67.9
52-5
16.6
41-3
275.7
46.4
53.7
221.3
115.3
64.2
63.4
65.1
39.6
67.4
25-2
33.5
29.3
13.6
12.3
66.3
42.8
12.1
30.0
252.4
37-8
42-3
175-6
73.3
53.3
47-6
52-1
33.1
58.0
22.7
29-4
24.2
Total M (junol g- D M )
'
14-5
16.9
65.5
52.0
20.3
37.4
328.3
43.8
51.8
180.9
46.6
55.4
57.0
66.4
42.4
66.7
25.7
33.5
28.9
CS
1.29
1.88
85
5.71
1.81
4.11
16.54
3.65
3.76
14-54
3-75
3.44
3.85
6.14
4-19
5-94
3-71
4-49
12.0''
1.12
1.50
75
2-82
3.46
5-60
4.77
1.46
4.14
19.32
3-37
3-38
17.81
6.08
3.10
3.33
4-74
3-28
21.9d
1-03
1.39
69
4-43
1-48
4.16
21.19
3.19
3.26
18.81
7.66
2.71
3-26
4.40
2-99
5.10
2.62
3.19
27.7'
71
1.17
1.30
4.42
1-32
3.69
23.78
3.19
3.15
18.29
5.96
2.83
3.01
4.33
3.08
5.39
2.90
3.44
22.6''
Total aa (g per 16 g N (-NO,))
69
0.99
1-42
4.31
1-78
3.70
24-80
2.95
3.08
15.09
3.04
2.62
2.88
4.42
3-15
4-97
2-65
3-16
28*2d
TABLE 5
Nutrient effects on concentrations in dry matter (DM) of total and nitrate nitrogen, free and total amino acids (aa),amides, and on chemical score (CS) of potato tubers (Solanum
tuberosum L cv Bintje)
$
2
%
3
457
Free and total amino acids in vegetables
which is in agreement with other investigations (Carter
and Bosma 1974). At normal, moderate N-levels, NO,N represents only 1% of total-N or less. In boiled
potato tubers the nitrate content is much less
(Eppendorfer et a f 1979). Increasing N-contents of DM
increased the sum of free amino acid-N, as a percentage
of total-N, from 34% to 49% with the largest Napplication rate (12 + 8 g/pot), and even higher, to
56%, under strong P-deficiency conditions. These
results are in agreement with those of Mulder and
Bakema (19S6), who determined free and protein amino
acid by paper chromatography. In experiments with 12
potato varieties they found a variation in soluble nonprotein-N as a percentage of total-N between 43.0 and
56.9%. As ‘Table 5 shows, in all five treatments the
dominant compounds were asparagine and glutamine,
the first of which was found in much higher concentration than glutamine, especially under K- and, also,
under P-deficiency conditions. As a percentage of total
free amino acid-N, asparagine- and glutamine-N
content varied between 33 and 59% and between 20
and 34%, respectively. Corresponding figures for free
aspartic and glutamic acids varied from 1.8 to 3.9% and
from 2.0 to 4.4%, respectively. Free proline in particular but also glutamine was negatively affected by Kdeficiency, whereas histidine increased. Of all free amino
compounds determined in potato tubers, ethanolamine
was generally found in lowest concentrations (Table 5),
followed by cysteine, tyrosine, leucine, isoleucine and
phenylalanine. Total amino acids were positively correlated with M content of DM. Due to the large amount
contributed by the amides, aspartic and glutamic acids
represented by far the largest parts of the sum of total
amino acids. K-deficiency resulted in a higher concentration of aspartic acid but a lower one of proline and
glutamic acid than expected from equal total-N contents in treatments 12 + 8 g N and 12(-K) g N. When
values for ammonia-N found in the hydrolysates were
added to the sum of total amino acid-N in Table 5
(excluding tryptophan, which was not determined), the
resulting sums came close to 86% of total-N in all five
treatments. Increasing N-concentrations of DM
decreased the concentrations in crude protein of most
amino acids, whereas aspartic acid (asparagine) and glutamic acid (glutamine) increased. These results are in
agreement with earlier findings (Eppendorfer et a1 1979),
and also with those of Rexen (1976) and Baerug et a1
(1979). The unusual increase, from 3-75 to 7-76 g per
16 g N of proline. is entirely due to the corresponding
increase in free proline, which increased from 5.9 to
62.5% of total proline. It appears that free proline is
easily affected by changing growing conditions, which is
also evident from the low content with K-deficiency.
A comparison with the FAO/WHO reference amino
acid pattern given in Table 1 shows that at the lowest
N-content of DM, all essential amino acids, except
leucine, were present in sufficient amounts for their
optimal utilisation in nutrition. At the higher Ncontents, which, however, rarely are found under
normal growing conditions, practically all essential
amino acids became limiting.
In order to obtain a single value for the protein
quality of the vegetables, the tables also contain chemical scores (CS, Block and Mitchell 1946), which express
the percentage value of the first limiting essential amino
acid in relation to the corresponding recommended
value of the FAO/WHO reference protein. Although
total tryptophan was not determined in the present
investigation,
earlier
studies
with
vegetables
(Eppendorfer 1978) have shown that it was not a limiting essential amino acid. An examination of the tables
shows that with the exception of spinach, there is a clear
decrease in CS with increasing N-content of DM. Apart
from cauliflower, where leucine was generally the first
limiting essential amino acid, in the other crops it was
mostly the lack of sufficient methionine cystine in
crude protein which resulted in less than optimal values
of CS. In vegetative plant material the S-containing
amino acids are often found to be first limiting, whereas
lysine normally is present in more than sufficient
amounts compared with the FAO/WHO amino acid
pattern (Wilson and Tilley 1965; Byers 1971). However,
it should be remembered that these purely chemical
evaluations of protein quality may give somewhat different results from biological evaluations in, for
instance, N-balance trials with rats. Discrepancies
between results from chemical and biological evaluations of protein quality might possibly be connected
with varying parts of crude protein being made up by
free amino acids and amides, thus influencing the
digestion and absorption of N-compounds.
+
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