Chuguas (Ullucus Tuberosus)

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Nutritional composition of ulluco (Ullucus

tuberosus) tubers
Article · October 2000
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Proceedings of the Nutrition Society of New Zealand, 2000, Vol. 25 55
Nutritional composition of ulluco (Ullucus tuberosus) tubers
J.M. BUSCH and G.P. SAVAGE

Food Group, Animal and Food Sciences Division, Lincoln University,
Canterbury
ABSTRACT
Ulluco have recently been re-introduced into New Zealand from South America and
are currently undergoing growing trials with a view to release as a new vegetable crop. This
crop produces highly variegated coloured small tubers which can been eaten both cooked and
raw. Colours include yellow, red, magenta, and green, sometimes in the same tuber. The leaves
can also be eaten. Proximate analysis in New Zealand has shown that the tubers contain high
levels of carbohydrate (84-94%), moderate levels of protein (9-12%), low levels of fat (0.7-
0.9%), and some vitamin C (5-9 mg/100 g). They compare well with other staple crops eaten in
this country. Some tubers contain mucilage which can either been removed by soaking or left
specifically to thicken the food.
BACKGROUND AND HISTORY IN SOUTH AMERICA
Ulluco (Ullucus tuberosus Loz.) is a staple food crop grown by subsistence farmers
living at high elevations (2,400-4,000 metres) in the Andean regions of countries such as Peru,
Ecuador, Colombia, Venezuela and northwestern Argentina (Arbizu et al., 1997). It is still
largely unknown outside South America.
Ulluco was probably brought into cultivation from the wild in the central Andes of
Peru and Bolivia (Arbizu et al., 1997). Botanical material from several coastal Peruvian
archaeological sites has been identified as containing starch grains, vessels and xylem of ulluco.
Illustrations of ulluco have been found on wooden vessels, ceramic urns and sculptures from
the same region, which are dated from about 2,250 – 2,050 BC (Sperling and King, 1990). The
Incas cultivated a wide variety of root crops including ulluco, which became less important as
they were forced to grow European vegetables after the Spanish invasion in 1531 (Popenoe,
1992). The name ulluco is derived form the Quechua word ulluku. The large number of local
names including ulluma, illaco, melloco, papalisas, michur and rubas (Arbizu et al., 1997)
show the importance of ulluco in these regions of South America.
It is common for farmers to grow a large number of different cultivars of ulluco
together in the same fields, and interspersed with other traditional root crops such as oca and
mashua (NRC, 1989). Farmers chose to plant particular cultivars based on their characteristics
such as sweet tasting, ability to be stored before eating, less mucilage and greater yield
(Vietmeyer, 1984; King, 1986). Plants with red tubers are also thought to be more frost resistant
than others and yellow tubers are the most common ones sold in markets in Ecuador (Pietelä
and Jokela, 1988).
In contrast to some other commonly grown root crops from South America, such as
mashua and oca, this crop has recently moved to being a popular cash crop sold in markets of a
number of South American cities (Dionis, 1998).
There are few reports on the nutritional composition of ulluco in English. Table 1
summarises the data from five authors.
56
Table 1: Comparison of nutritional analyses (g/100 g DM) of ulluco grown in South
American countries.
Author(s) Moisture Protein Fat Carbohydrate Ash Fibre

Anon, (1998)b 85.9 7.1 0.7 88.6 4.3 4.3c
Gross et al, (1989) 87.6 8.5 1.4 84.2 5.4 4.0a
Kingb, (1988) 85.7 13.7 1.03 73.8 4.2 4.7a
King and Gershoff, 86.1 13.2 0.7 77.3 3.5 4.3c
(1987)
Sperling and King, 85.6 13.2 0.7 75.7 4.2 4.9a
(1990)
a = crude fibre: b = average of cultivars :c method of analysis not given
Figure 1: Ulluco tubers and a small part of the trailing plant.

Ulluco are small, potato shaped, (2-10 cm) (Figure 1) or sometimes elongated (2-15
cm) multicoloured tubers that are produced in an extremely wide range of colours. The skin can
be one colour or multicoloured with small discrete spots or larger patches of different colours.
Colours can be red, yellow, green, white, pink, burgundy, yellow and red, yellow and green,
yellow with red spots and red with yellow spots (Pietilä and Jokela, 1988; Flores and Flores,
1997; Brown, 1997). Tubers also show variation in colours among the same cultivar. They have
waxy skins and the bright colours mean they almost look like plastic decorations (Popenoe,
1992). Inside, the tubers are yellow or white, with a clear distinction between skin and inside
(NRC, 1989).
Tubers are cooked in a variety of different ways in South America with many
countries having regional dishes. Principally the tubers are boiled and served with sauces or
used in stews with meat and other vegetables (NRC, 1989). Contemporary dishes incorporate
ulluco tubers in salads. Ulluco leaves (which taste like spinach) are also eaten in soups and
salads (Martin et al., 1997). Dehydrated ulluco are usually eaten in soups and stews in the
Peruvian highlands. (Arbizu et al., 1997) They can be eaten fresh or dehydrated by freeze
drying (Collins, 1993) and can be stored for years (Peitielä and Jokela, 1988).
POTENTIAL USE IN NEW ZEALAND
Ulluco have recently been introduced into New Zealand and are being evaluated as a
potential new addition to the range of vegetables consumed in this country. This tuber has the
potential to be popular with New Zealand consumers. It is reported that ulluco tubers have a
long shelf life (Flores and Flores, 1997). Because of the large variety of colours, they could be
sold as a mixed blend rather than as a uniform product (NRC, 1989). The skin of ulluco tuber is
soft and does not need to be peeled before eating. The white to lemon-yellow flesh has a
smooth silky texture with a nutty taste (Popenoe, 1992). A major appeal of ulluco is its crisp
texture, which remains even when cooked (NRC, 1989). Ulluco tubers are reported to have a
long shelf life (Flores and Flores, 1997). A small number of tubers contain high mucilage levels
which could be seen as a negative feature because of the resulting gumminess in texture (NRC,
1989). Mucilage is a non-starch polysaccharide so contributes to the total carbohydrate content
of the tuber. In South America ulluco with high mucilage levels are often used to thicken stews.
Ulluco tubers with high mucilage content are gummy when raw, but after cooking this
characteristic is usually reduced or lost (Brown, 1997). The mucilage can easily be removed by
soaking the tubers in water or parboiling before use.
Ulluco tubers are usually cooked whole and take about the same time as potatoes to
cook. This may discourage some people from eating them in New Zealand where there is a
growing emphasis on quick preparation and cooking methods. The use of the microwave may
reduce this cooking time. Sensory evaluation and colour analysis in New Zealand showed that
panellists liked the red tubers best (Busch et al., 2000). This is in contrast to a study done by
Espinoza and Crissman (1997) in Ecuador which showed that consumers preferred the yellow
and red tubers. Pietalä and Jokela (1988) also reported that the yellow cultivar was the most
popular one eaten from the markets they sampled in Peru, Bolivia and northern Argentina.
Table 2 lists the nutritional composition of a number of staple foods commonly eaten in New
Zealand.
58
Table 2: Proximate analysis of other staple root crops commonly eaten in New Zealand
(g/100 g DW), King, (1988).
Moisture Crude Total fat Total carbohydrate

(fresh weight) Protein
Corn 63.5 11.2 3.6 83.0
(Zea mays)
Oca or NZ yam 79.0 4.3 1.0 88.1
(Oxalis tuberosa)
Potato 78.0 9.5 0.4 84.1
(Solanum tuberosum)
Sweet potato 70.2 4.7 1.3 91.9
(Ipomoea batatas)
True yam 72.0 7.9 0.7 86.4
(Dioscorea spp.)
Busch et al., (2000) showed that consumers prefer particular cultivars, and that there is
potential to promote ulluco as an exotic and organic crop for urban dwellers. It has been shown
for similar root and tuber crops that colour is an important sensory attribute for consumer
acceptability. There has also been interest expressed by chefs who recognise the appeal of the
colours of the ulluco in the visual appearances of the dishes they create.
This paper reports on the nutritional composition of ulluco grown in New Zealand and
compares these results with ulluco grown in South America and other staple, high carbohydrate
crops commonly consumed in New Zealand. The results of sensory evaluation as it relates to
CIE colour are also included.
METHODS
All chemical analyses on the tuber samples were carried out in triplicate. Moisture,
ash, total fat, protein (Nx6.25) and total dietary fibre were determined in accordance with
AOAC (1995) methods.
Starch
Starch content was determined using a Sigma assay kit (Sigma Chemical Company, St
Louis, USA). Triplicate samples (0.1-0.2 g) were weighed into 100 mL conical flasks and
hydrolysed with 20 mL deionised water and 5 mL 8 M HCl for 30 minutes at 60°C in a shaking
waterbath. Deionised water was used in preference to dimethyl sulfoxide because the high
mucilage content appeared to interfere with the extraction of the starch. After cooling, 5 mL of
8 M NaOH was added and the samples made up to 100 mL in a volumetric flask with 0.112 M
citrate buffer, pH 4.0. The glucose of the filtered solution was determined following incubation
of the sample with amyloglucosidase to degrade the hydrolysed starch into glucose. The
released glucose was assayed using hexokinase and glucose-6-phosphate deydrogenase to
release NADPH, which was measured at 340 nm. Free sugar (glucose) present in the original
sample was determined at the same time by measuring the glucose content.
Total dietary fibre

The total dietary fibre content of the ulluco were determined using a Sigma assay kit
(TDFAB-2) (Sigma Chemical Company, St Louis, USA). The results were not corrected for
residual protein or ash.
Total mucilage
The preliminary extraction procedures of Fedenuik and Biliaderis (1984) were used to
extract the mucilage. Twenty g of finely crushed ulluco were placed in 400 mL of deionised
water in a concial flask, stirred for 10 minutes, covered with plastic clingwrap and then placed
in a shaking waterbath for 2 hours at 80°C. The hot samples were filtered (Whatman No 1) into
a weighed container, dried at 105°C, and reweighed. Only one extraction was carried out on
each cultivar.
Data analysis
Statistical analysis was performed using the Analysis of Variance (ANOVA)
procedure available in SAS (Version 6, SAS Institute INC, Cary, NC, USA). Means were
separated at the 5 % level of significance using the LSD procedure.
Colour
Tubers from each cultivar were placed on a white background until it was completely
obscured with tubers. Colour readings of the skin and flesh colour of the raw and cooked ulluco
were taken with the CIELAB system using a Minolta Chroma meter CR-210. The meter was
calibrated to a standard white tile (L* 98.07, a* -0.23, b* 1.88) and readings were taken by
placing the detector (aperture area, 50 mm) over the top of the grouped ulluco (2o to observer).
Readings for each cultivar were made in triplicate.
NUTRITIONAL CONTENT OF ULLUCO TUBERS

Moisture
Table 3 shows that the average moisture content of ulluco grown in New Zealand
ranged from 79-84%. The moisture content of the New Zealand grown ulluco (78-92%) was
lower than those given for South American grown ulluco (Table 1). This may be due to the
smaller tuber size which occurred in response to the low rainfall during the 1998-99 growing
season and the late maturity of ulluco in Canterbury, New Zealand. Results from the 1999-2000
growing season (data not shown) are on average 4 % higher at 83-88%, which is similar to
other authors (Table 1).
Protein
The protein content of the New Zealand grown ulluco ranged from 9-12%. Two of the
cultivars grown in New Zealand had a significantly higher protein content (12 %) than the other
cultivars, while one cultivar had a significantly lower protein (9%). Previous authors also report
that there is a range of protein levels for different cultivars (King and Gershoff, 1987). The
protein content of ulluco grown in South America ranges from 7-14 %, (Table 1).
Carbohydrate
It is important to note that both the carbohydrate and fibre analyses used by in this
paper were different from that used by other authors so direct comparisons cannot be made. In
this paper the carbohydrate contents are reported separately as dietary fibre, starch, free glucose
and mucilage. Adding these separate analyses together, but omitting mucilage, to calculate the
total carbohydrate gives somewhat lower carbohydrate figures (about 15%) than reported by
other researchers with one cultivar being considerably lower than the others. Including the
mucilage in the total gives carbohydrate results which are similar to those reported by other
authors who, however, did not report whether their samples were soaked to remove the
mucilage before analysis.
60
Table 3: Proximate analysis of five ulluco cultivars (g/100 g DM)1 grown in New
Zealand (Busch et al., 2000).
Moisture Protein Total fat Mucilage

Cultivar (g/100 g fresh weight) (N x 6.25)
U2 81.12 ± 2.15b 9.05 ± 0.49b 0.85 ± 0.03a 17.41
U3 79.08 ± 2.60b 9.67 ± 0.36b 0.70 ± 0.01b 19.88
U9 81.07 ± 1.97b 12.19 ± 0.50a 0.86 ± 0.04a 10.46
U13 84.41 ± 1.58a 11.83 ± 0.89a 0.95 ± 0.07a 14.74
U15 82.01 ± 2.31b 9.84 ± 0.42b 0.92 ± 0.04a 41.30
LSD p<0.05 3.60 1.54 0.12
Starch Free glucose Dietary fibre Vitamin C
U2 37.11 ± 1.95a 0.47 ± 0.13 34.75 ± 2.00 4.8

U3 36.56 ± 2.29a 0.76 ± 0.20 34.85 ± 1.01 5.2
U9 38.78 ± 1.27a 0.77 ± 0.25 34.29 ± 1.11 5.9
a
U13 35.00 ± 2.14 1.36 ± 0.12 37.57 ± 0.72 5.3
b
U15 16.70 ± 2.98 1.46 ± 0.27 34.66 ± 1.66 8.8
LSD p<0.05 7.38 NS NS
1
each value represents a mean ± standard error of 3 replicates, except for mucilage (only one
sample was analysed because insufficient material was available). Values with a different letter
are significantly different within each cultivar (p< 0.05)
NS = not significantly different (p > 0.05)
The carbohydrate content of four of the New Zealand grown ulluco ranged from 77-86
% (excluding mucilage), with the other cultivar having a significantly lower carbohydrate level
(66 %) than the other cultivars. This latter cultivar, appeared, from the limited analysis done, to
have considerably higher mucilage content at 41.3% than the others, which ranged from 10.4 to
19.8%. Table 1 shows that the average carbohydrate content of ulluco grown in South America
ranged from 73-88%.
Mucilage
Busch et al., (2000) were to first to report in an English language journal on the
mucilage content of ulluco. Mucilages are carbohydrate polymers of high molecular weight,
consisting of two or more different kinds of monosaccharide joined by glycosidic links (Aurand
and Woods, 1973). They are formed in the plant cells, can be extracted in hot water and do not
normally form gels (Considine, 1982). In South America, some authors reported that variation
in mucilage content may influence the choice of cultivar, with some cultivars being grown for
specific uses (Collins, 1993). Tubers in South America are cut very finely before use and the
mucilage becomes apparent exuding from the cut surfaces of the slices (Busch et al., 2000).
Mucilage can be a positive feature because, like potato starch, it can be used successfully in
cooked dishes as a thickener.
The New Zealand grown ulluco were not soaked to remove mucilage before cooking
and the mucilage extracted was not completely pure. This means that the reported mucilage
content may be an overestimation. Further work needs to be done on purifying and
characterising the mucilage content.
Lipids
The fat content of ulluco grown in New Zealand ranged from 0.8-0.9% for four of the cultivars
while one had a fat content at 0.7%, than the other cultivars, Table 1. Other authors reported a
low fat content for ulluco grown in South America (0.7-1.5%), (Table 1).
Minor constituents of ulluco
Vitamin C
The vitamin C content of the raw ulluco was determined using the AOAC Official
Method 967.21 (AOAC, 1995), which involves the reduction of the dye DCPIP (2,6-
dichloroindophenol) by vitamin C. For samples U2 and U15, which are already coloured red,
the end point of the titration was to a consistent red colour. Because of the difficulty in
establishing an accurate end point further work should involve the use of an automatic titrator
as this method does not rely on a colour change to measure the end point. Results for all
samples are given in Table 3.
Ulluco tubers are reported as having a high vitamin C content of 28 mg/100 g (NRC,
1989) but it is not indicated whether this is on a dry weight or a wet weight basis. Further work
will analyse the vitamin C content of cooked ulluco.
The vitamin C content of these ulluco are similar to other commonly eaten vegetables
such as carrots (6 mg/100 g), celery (7.5 mg/100 g), but lower than kumara (85 mg/100 g) and
yams (17 mg/100 g)(Burlingame and Milligan, 1997).
Saponins
After sensory evaluation was undertaken by Busch et al., (2000) some panellists
reported that they developed a rather unusual mouth sensation and after taste about ten minutes
after sampling the ulluco. They did not however specify which particular cultivar caused it.
Dini et al (1991) reported that triperpenoid saponins are found in ulluco and explained that their
presence may account at least in part for the unpalatable taste of some tubers and may explain
why the crop is not more widely cultivated. Saponins in other foods are know to give a bitter
taste (Savage, 1993). It is possible that the panellists may have been reporting on the effect of
the saponin content because mucilage does not appear to have a sharp taste.
Colour
Raw skin and flesh

The skin of cultivars U2 and U15 had red-based colours because their hue angles (hab)
(Table 4) were close to pure red-purple (0°) and the skin colour of cultivars U3 and U9 had a
yellow colour base (hab close to 90°). The skin colour of U13 was between red and yellow
(42°). U9 had the most intense yellow (highest b* and c*) and was brighter than cultivars U3
and U13. The skin of U2 had the same colour as U15 as there was no significant differences in
L*, c* and hab values between these two cultivars. Cultivars with yellow-based colours were
lighter (higher L*) than the red-based cultivars. The raw flesh of all cultivars were in yellow
shades with little difference in lightness and intensity.
Cooked skin and flesh

The skins of cooked U2 and U15 still had shades of red after cooking, but the colour
was more yellow (higher b* and hab) (Table 4). The flesh colour of all cultivars, except U9, was
redder (higher a* and lower hab) after cooking, and the flesh colour of U2 and U15 had changed
from yellow to red shades. This may have been caused by diffusion of red colour from the skin
to the flesh tissues during boiling. Cultivar U9, both raw and cooked, had the yellowest and
62
lightest skin and flesh colours, and the negative a* values for U9 flesh colour indicated that it
was more green than the other cultivars. U13, the cultivar with the most visual colour
variation, gave a red colour reading with the colorimeter.
After the cooked and raw tubers were cut the skin outside skin colour showed as a
coloured band around the outside of the cut slice. This band was discrete for raw tubers and
more diffuse for cooked tubers. The colorimeter recorded the flesh of cooked tubers as having a
higher red value than for raw flesh.
Table 4. The CIE colour values for skin and flesh of raw and cooked ulluco
(Busch et al., 2000).
Skin colour
Raw Cooked
Cultivar
L* A* b* c* hab L* a* b* c* Hab
U2 30.8d 27.6 a 1.2 d 27.6 b 2.4 d 31.4 17.5 12.4 21.5d 79.0 d
c b d
b c b b b d
U3 45.7 11.2 26.5 28.7 67.0 48.0 1.5 36.8 36.9b 95.2
a b ab
a d a a a e a
U9 53.5 1.1 42.7 42.7 88.6 50.1 -0.3 43.6 43.6 96.1 a
a a
U13 41.6 c 20.9 b 18.7 c 28.1 b 41.8 c 43.1 6.5 c 30.1 30.7 c 88.9 c
b c
U15 30.9 d 29.6 a 2.3 d 29.7 b 4.5 d 32.5c 23.8a 14.5 27.9c 91.3
d bc
2
LSD 3.3 2.4 3.5 4.1 2.7 4.4 1.8 5.2 4.3 4.7
Flesh colour
Raw Cooked
Cultivar
L* a* B* C* hab L* a* b* c* Hab
U2 51.1 b 6.6 a 34.5 b 35.1 b 79.0 d 37.4 c 18.9 b 14.3 23.7 37.1
c c c
a c a a ab b c
U3 60.5 -4.9 52.4 52.7 95.2 45.5 12.7 24.7 27.8 62.7
b bc b
a c a a a a d
U9 58.0 -5.2 48.3 48.3 96.1 51.3 -3.8 42.0 42.2 95.2
a a a
b b b b c a c
U13 53.7 0.8 38.8 38.8 88.9 50.0 13.6 27.6 30.8 63.8
b b b
b b b b bc d a d
U15 52.8 -1.1 34.5 34.5 91.4 31.1 27.8 7.9 28.9 15.8
b d
LSD 3.2 3.4 6.0 6.1 4.7 2.4 1.2 4.1 4.2 1.0
1
each value represents a mean of 3 replicates; means within a column with different
superscripts are significantly different (p < 0.05).
2
LSD = Least significant difference (p< 0.05)
Comparison with other staple crops grown in New Zealand

The nutritional value of ulluco is good when compared with staple root and tubers
crops commonly eaten in New Zealand (Table 2). Ulluco have about the same protein content
as potatoes and corn, but higher than sweet potato. The carbohydrate content of ulluco is
comparable with potato, sweet potato and corn. Ulluco have a lower fat content than corn and
sweet potato, but higher than potatoes. This shows that ulluco compares favourably in
composition content with the other commonly eaten, starchy staple crops.
While there are a large number of different varieties of vegetables grown in New
Zealand at present no other vegetable crop has the extensive colour range offered by ulluco The
most common cultivars of oca (Oxalis tuberosus)(known as yams in New Zealand), and sweet
potato (Ipomoea batatas)(known as kumara in New Zealand) are bright red. Some other
vegetables, such as peppers are yellow and green, but there are no mulitcoloured (yellow with
red spots or red with yellow spots, or yellow with green spots) vegetables like ulluco available.
It is known that consumers choose vegetables on the basis of their colour when raw (Sangketkit
et al, 2000).
Overall, the panellists preferred cultivars U2 and U15, which have red skins. This may
be because the panel members were unfamiliar with ulluco and their decision on colour
preference may have been influenced by their familiarity with the red cultivars of oca and sweet
potato crops grown in New Zealand. Espinosa and Crissman (1999) reported that in the three
sites studied in Ecuador, the most favoured ulluco colours were yellow and red. However
Pietela and Jokela (1988) reported that the yellow cultivar was the most popular one eaten as
food in the markets they sampled in Peru, Bolivia and northern Argentina. Consumers in
southern Colombia liked ulluco, which had magenta spots speckled on a yellow background
(Vietmeyer, 1984).
Panellists from the sensory evaluation of Busch et al., (2000) did not like the colour of
raw U3, U9 and U13, because they were multicoloured and/or had different coloured spots and
they thought they were diseased. Consumers from some cities in Peru also do not like
multicoloured, multishaped tubers (Dionis, 1999). The panellists in this study also assumed that
green or partially green cultivars were “bad” and may contain high levels of glycoalkaloids.
Ulluco however do not contain glycoalkaloids even in the “green” areas of the tuber. Panellists
were probably aware of the danger of eating potatoes that have turned green (Considine, 1982).
The preference for one of the ulluco tubers changed from second least preferred to
second most preferred when it was cooked. This is most likely because the panellists appeared
to like the change in flesh colour from the yellow raw flesh to the red cooked flesh that
happened because of the diffusion of the red skin colour through to the flesh on cooling. It is
likely that the red colour pigment in the ulluco is anthocyanin. This pigment is water soluble,
and so would easily leach out of the skin into the flesh as well as into the cooking water
(Vietmeyer, 1984; Potter and Hotchkiss, 1995).
CONCLUSIONS
Ulluco is a colourful root crop that has the potential to be a popular addition to the diet
of people living in New Zealand. In common with other high carbohydrate root crops grown in
New Zealand it also contains moderate levels of protein and has a low fat content. It will be a
versatile crop because it can be eaten both cooked and raw. In New Zealand the preferred
ulluco colour was the red tubers over the yellow, yellow and green, and spotted multicoloured
tubers. While these earlier studies showed some consumers did not like its unusual and
unfamiliar colours it is likely that it will soon become popular because of the visual appeal of
the tubers, particularly when whole and cut, and its versatility of use. It can be regarded as a
potentially a nutritious addition to the New Zealand diet.
ACKNOWLEDGEMENTS
The authors wish to acknowledge the contribution to this paper from a number of staff
of Crop and Food Research Ltd., Lincoln, in particular Dr R. J. Martin.
64
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Flannery, K.V. (1973). The origins of agriculture. Annual Review of Antrhopology 2, 271-310.
Flores, H.E.and Flores, T. (1997). Biology and biochemistry of underground plant storage
organs, in Functionality of food phytochemicals, Ed. by Johns T. Plenum Publishing
Corporation, New York, pp 113-132.
Gross, R., Koch, F., Malaga, I., de Mirando, A.F., Schoeneberger, H. and Trugo, L.C. (1989).
Chemical Composition and Protein Quality of Some Local Andean Food Sources.
Food Chemistry 34, 25-34.
King, S.R. (1986).Tubers from the Andes Extinction or Propagation Vol.10, No.6, pp 6-11,
Nov/Dec.
King, S.R. (1988). Economic Botany of the Andean Tuber Crop Complex: Lepidium meyenii,
Oxalis tuberosa, Tropaeolum tuberosum and Ullucus tuberosus. PhD Dissertation,
The City University of New York.
King, S.R. and Gershoff, S. (1987). Nutritional Evaluation of Three Underexploited Andean
Tubers Oxalis tuberosa (Oxalidaceae), Ullucus tuberosus (Baselacease), and
Tropaeolum tuberosum (Topaecolaceae). Econonic Botany 41, 503-511.
Martin, R.J., Halloy, S. and Deo B. (1997). Preliminary Assessment of South AmericanTuber
Crops, in Proceedings of the First Australasian New Crops Conference, Ed. .by BC
Imrie Research Paper No 97/21, Rural Industries Research and Development
Corporation, Canberra, Australia 2, 155-161.
National Research Council. (1989). Lost Crops of the Incas:Little-Known Plants of the Andes
with Promise for Worldwide Cultivation, National Academy Press, Washington DC,
USA.
Pietilä, L. and Jokela, P. (1988). Cultivation of minor tuber crops in Peru and Bolivia. Journal
of .Agricultural. Science of Finland 60, 87-92.
Popenoe, H.L.(1992). New Crops for South America’s Farmers. Acta. Hort. 128: 209-210.
Potter, N.N. and Hotchkiss, J.H., Food Science 5th ed., Chapman and Hall, New York. 415-412
(1995).
Sangketkit, C., Savage, G.P., Martin, R.J., Searle, B.P. and Mason, S.L., (2000). Sensory
evaluation of new lines of oca (Oxalis tuberosa) grown in New Zealand. Food Quality
and Preference 11, 189-199.
Savage, G.P. (1993). Saponins, in Encyclopaedia of Food Science, Food Technology and
Nutrition, Ed, by R. Macrae R.K. Robinson and M.J. Sadler, Academic Press, London.
3998-4001.
Sperling, C.R. and King, S.R. (1990). Andean Tuber Crops: Worldwide Potential. In Advances
in New Crops. First National Symposium new crops: research developments,
economics, Indianapolis, USA October 23-26, 1988, Ed. by J Janick and J E Simon.
Timber Press, Portland, OR, USA, pp. 425-435.
Vietmeyer, N. (1984). Lost Crops of the Incas. Ceres 17, 3, 37-39.
66
Effect of cooking on the soluble oxalate content of three

cultivars of oca (Oxalis tuberosa)
PERNILLA ALBIHN1 and G.P. SAVAGE2

1
Department of Food Science, Swedish University of Agricultural Science,
Uppsala, Sweden and
2
Food Group, Animal and Food Sciences Division, Lincoln University,
Canterbury
ABSTRACT
Soluble oxalate analysis was carried out on three commercially grown New Zealand
cultivars of oca (Oxalis tuberosa Mol.), the traditional pinkish-red cultivar and two newly
selected cultivars “Mellow Yellow” and “Apricot Delight”. The three cultivars of oca (known
as yam in New Zealand) were cooked by boiling, steaming and baking in an oven and the
soluble oxalate content was determined by HPLC analysis following a hot water extraction of
the macerated tubers. The mean soluble oxalate content of the tubers, on a fresh weight basis,
was raw (2.80 g/kg), boiled (2.34 g/kg), steamed (2.94 g/kg) and baked (4.93 g/kg). Overall,
baking appeared to increase the oxalate concentration, on a fresh weight basis, in the whole
cooked tubers in all cultivars.
INTRODUCTION
Oca is the common name for the starchy tuber crop that in New Zealand is called yam
(Veitmeyer, 1991). The oca tuber has normally a cylindrical or turbinate shape (Kay, 1973).
The skin is smooth, bearing many scales that cover the deep eyes, and the colour of flesh and
skin varies from white to deep red over a range of cream, yellow, apricot, orange and pink
(Kay, 1973; King 1988). The more commonly grown pink-red New Zealand cultivar of oca has
a narrow genetic base and is thought to originate from Central or Southern Chile (Veitmeyer,
1991; King, 1988). In New Zealand, oca tubers are served steamed, boiled or baked like
potatoes (Veitmeter, 1991). The flavour varies from very bitter to sweet. Sangketkit et al.,
(2000) suggested that New Zealand consumers prefer the bright red coloured oca that cooks to a
slightly mealy texture, has bright yellow flesh colour and a sweet taste. This study also showed
that the colour of the skin was the most important factor affecting the purchase of oca by New
Zealand consumers (Sangketkit et al., 2000). Nevertheless, different coloured cultivars of oca
have been introduced to the market during the last few years.
While the nutritional value of oca is very good when compared to other staple root and
tuber crops the tuber does contain moderately high levels of oxalates (Sangketkit et al., 1999).
While a number of reasons have been proposed for the production of oxalates in plants (Libert
and Franceschi, 1987; Franceschi and Loewus, 1995) the main reason appears to be as a
protectant against predatory insects (Yoshihara et al., 1980). When consumed by humans
oxalates are an antinutritive factor that affects calcium availability and metabolism (Noonan
and Savage, 1999).
Ross et al., (1999) investigated the oxalate content of 14 cultivars of oca. The results
from this research show that the oxalate level in oca tubers is moderately high and consists of
only soluble oxalate. This is most unusual and a review of the literature (Noonan and Savage,
1999) suggests that oca may be the only common food in which oxalate occurs only in the
soluble form. This finding is important if, as is currently assumed, soluble oxalate is more
bioavailable than insoluble oxalate (Brinkley et al., 1981; Liebman and Chai, 1997; Holmes
and Kennedy, 2000).
The preliminary studies of Sangketkit et al., (1999) on non-commercial cultivars of
oca showed that the level of soluble oxalate remains relatively unchanged when the tuber is
steamed or boiled but increases markedly when the tuber is baked. Three possibilities have
been suggested to explain the apparent rise in oxalate levels on baking; the first possibility is
that loss of moisture during baking may well explain most of the apparent increase in oxalate
content of the baked tubers. Another possibility is the effect of sampling, as oxalate may not be
uniformly distributed in the tuber. It is also possible that ascorbic acid could breakdown to
oxalate in the skin of the tuber as a result of heating the skin during baking. This study focuses
on the effect of cooking on oxalate content of three different commercial cultivars of oca to
confirm these earlier observations.
MATERIALS AND METHODS
Sample collection and cooking

Three different cultivars of known origin (Feilding, New Zealand) were bought from
two different supermarkets in Christchurch in July 2000. Analysis was carried out on average
sized tubers (length 5-7.5 cm, diameter 2.5-3.75 cm) for each cultivar. The oca were either
boiled or steamed for 20 minutes or baked for 60 minutes. The raw or cooked whole tubers
were sampled in triplicate by taking longitudinally cut segments from three representative
tubers.
Dry matter determination

Representative samples of fresh and cooked tubers were dried to constant weight in an
oven set at 105°C.
Oxalate analysis
Five-gram samples of raw or cooked oca were extracted using the methods described
by Ross et al., (1999). HPLC analysis of extracted oxalate was carried out on a 300 × 7.8 mm
ion exclusion column (HPX-87H, Bio-Rad, Hercules, CA, USA). The solvent used was 0.0125
M sulphuric acid and the equipment consisted of a Spectra-Physics (SP 8800, CA, USA) pump,
a uv/vis detector (Spectra-Physics, SP8450, CA, USA) set at 210 nm and a Cromatopac
integrator (Shimadzu, C-R3A, Japan). Ten µL samples were injected onto the column and
eluted at a flow rate of 0.7 mL/min. The results were calculated as g/kg fresh weight (FW).
RESULTS AND DISCUSSION
The mean values for the dry matter content of the raw and cooked oca are shown in
Table 1. The dry matter content of the boiled or steamed tubers did not appear to change when
compared to the raw values. The dry matter content of the baked oca was increased by 56%
when compared to the % dry matter of the raw tubers.
68
Table 1: Dry matter of three different cultivars of oca (g/100g).
Cultivar Raw Boiled Steamed Baked

Red 13 11 14 24
Apricot Delight 13 12 13 19
Mellow Yellow 13 14 14 18
Mean 13.0 12.3 13.5 20.3
The mean values of oxalate concentration as a result of the different cooking methods
are shown in Table 2. Overall, baking increased the oxalate concentration on a wet matter basis
when compared to the values for the boiled and steamed tubers. Boiling decreased oxalate
concentration in Mellow Yellow and Apricot Delight, but increased the concentration in the red
cultivar. Apart from the red cultivar, steaming the tubers appeared to have little effect on the
final oxalate content of the cooked tubers when compared to the original raw tubers.
Table 2: Soluble oxalate content (mean ± SD) of three different cultivars of oca expressed
on a fresh weight basis (g/kg FW).
Cultivar Raw Boiled Steamed Baked

Red 2.65 ± 0.20 3.39 ± 0.33 3.53 ± 0.07 4.62 ± 0.18
Apricot Delight 3.41 ± 0.36 1.69 ± 0.08 2.94 ± 0.18 4.65 ± 0.77
Mellow Yellow 2.33 ± 0.07 1.95 ± 0.36 2.35 ± 0.26 5.53 ± 1.07
Mean 2.80 ± 0.56 2.34 ± 0.92 2.94 ± 0.59 4.93 ± 0.52
The mean oxalate content of the baked oca expressed on a fresh weight basis increased
by 76% when compared to the oxalate content of the raw tubers. These results confirm the
earlier study by Sangketkit et al., (1999), that baking gives an apparent increase in the oxalate
content in the tuber due to a loss of moisture during baking. These results suggest that the
increase in oxalate content appears to be more than can be explained by a concentrating effect
of moisture loss during baking. Further studies are needed to understand the processes
involved.
After boiling, soluble oxalate could be detected in the cooking water, which indicates
a leaking from the tubers and explains the overall loss, although this did not occur in the red
cultivar. Steaming caused some rupture in the skin and leaching could have occurred
(particularly in the cultivar Apricot Delight) as the steam condensed on the tuber and dripped
back into the cooking pot below.
Comparison between the different cultivars in this study indicates that the new
cultivars, Mellow Yellow and Apricot Delight, do not show generally lower oxalate levels
when baked when compared to the traditional red cultivar. These two cultivars do, however,
appear to lose more oxalate by leaching into the cooking water when compared to the red
cultivar. The oxalate content of the red cultivar (mean 2.65 g/kg FW) analysed in this study
was higher than for similar samples collected and analysed in 1997 by Ross et al., (1999)
which ranged from 1.37 to 1.71 g/kg FW and a similar sample collected and analysed in 1998
by Sangketkit et al., (1999) which contained 1.45 g/kg FW. This variation in results may be
due to different sampling procedures, but could also be a result of variations in growing
conditions from year to year.
CONCLUSIONS
Boiling and steaming are recommended methods to cook oca in order to keep oxalate
intake as low as possible. Oca is consumed in New Zealand as an additional vegetable not as a
staple so it is unlikely that a harmful dose of oxalate would be consumed in one day, even from
baked oca. Overall oca is a nutritious and tasty food, and since it is not an everyday food item
in New Zealand there seems to be no need for healthy persons to restrict their consumption.
The levels of soluble oxalate found in oca are higher than the soluble oxalate levels found in a
number of foods recognised as containing oxalates such as spinach and rhubarb stalks but these
foods contain much higher levels of total oxalates when cooked (Noonan and Savage, 1999;
Savage et al., 2000).
In a diet where oca is more frequently consumed or served in greater quantities overall
oxalate intake might be of some concern. Noonan and Savage (1999) found that the lowest
lethal dose of oxalate reported is two grams. It could therefore be recommended that the intake
in one meal does not exceed 2/3 of this dose. The highest amount of oxalate (5.5 g/kg) was
found in baked Mellow Yellow therefore, a maximum intake of 200 g of baked oca per day
would be an appropriate recommendation. The maximum daily intake of boiled and steamed
oca based on these results would be 350 g. Since all the oxalate in oca exists in its soluble form,
the effect on mineral bioavailability should also be taken into consideration when oca is
consumed on a regular basis.
REFERENCES
Brinkley, L. MgGuire, J. Gregory, J. and Pak, C.Y.C. (1981). Bioavailability of oxalate in

foods. Urology 17, 534-538.
Francesschi, V.R. and Loewus, F.A. (1995). Oxalate biosynthesis and function in plants and
fungi. Calcium Oxalate in Biological Systems, Chapter 6 (pp. 113-130), CRC Press.
Holmes, R.P. and Kennedy, M. (2000). Estimation of the oxalate content of foods and daily
oxalate intake. Kidney International 57, 1662-1667.
Kay, D.E. (1973). Root crops. The Tropical Products Institute, England, pp 96-99.
King, S.R. (1988). Economic Botany of the Andean Tuber Crop Complex: Lepidium meyenii,
Oxalis tuberosa and Ullucus tuberosus. PhD Dissertation, The City University of New
York.
Libert, B. and Franceschi, V.R. (1987). Oxalate in Crop Plants. Journal of Agricultural and
Food Chemistry 35, 926-938.
Liebman, M. and Chai, W. (1997). Effect of dietary calcium on urinary oxalate excretion after
oxalate loads. American Journal of Clinical Nutrition 65, 1453-1459.
Noonan, S.C. and Savage, G.P. (1999). Oxalate content of foods and its effect on humans. Asia
Pacific Journal of Clinical Nutrition 8, 64-74.
Ross, A.B. Savage, G.P. Martin, R.J. and Vanhanen, L. (1999). Oxalates in oca (New Zealand
yam) (Oxalis tuberosa Mol.). Journal of Agricultural and Food Chemistry 47, 5019-
5022.
Sangketkit, C., Savage, G.P., Martin, R.J. Mason, S. and Vanhanen, L. (1999). Food contains
more than nutrients, Oxalates in oca: A negative feature? 2nd South West Pacific
Nutrition and Dietetic Conference, Nutrition Society of New Zealand and New Zealand
Dietetic Association, Auckland, pp. 44-50.
Sangketkit, C., Savage, G.P., Martin, R.J., Searl, B.P. and Mason, S.L. (2000). Sensory
evaluation of new lines of oca (Oxalis tuberosa) grown in New Zealand. Food Quality
and Preference 11, 189-199.
70
Savage, G.P., Vanhanen, L., Mason, S.L. and Ross, A.B. (2000). Effects of cooking on the
soluble and insoluble oxalate content of some New Zealand foods. Journal of Food
Composition and Analysis 13, 201-206.
Veitmeyer, N. (1991). Lost Crops of the Incas. New Zealand Geographic 10, 49-67.
Yoshihara, T., Sogawa, K. Pathak, M.O., Julianov, B.O. and Sakamura, S. (1980). Oxalic acid
as a sucking inhibitor of the brown planthopper in rice (Delphacidae homoptera).
Entomological and Experimental AppIications 27, 149-155.
Vitamin B12 - An Undervalued Vitamin

TINI M. GRUNER
Animal and Food Sciences Division, Lincoln University, Canterbury
ABSTRACT
Vitamin B12 is not commonly thought of as causing deficiency as it is present

abundantly in most people’s diet. Dietary sources include meat, seafood and dairy products.
However, the vitamin does not always reach the target organs for use in metabolism. Factors
affecting its uptake, transport in the blood and storage in tissues include the presence and
efficacy of Intrinsic Factor, the plasma transport proteins transcobalamin II and haptocorrin,
and the tissue storage proteins methylmalonyl-CoA mutase and methionine synthase. Further,
currently employed blood tests fail to diagnose the deficiency accurately. More specific tests,
such as homocysteine and methylmalonic acid, have been developed which are direct indicators
of the metabolic activity of the vitamin. Hence a number of people suffering from vitamin B12
deficiency go undiagnosed. Vegans and people who have impaired absorption, such as in
pernicious anaemia, after stomach or small intestine resection, on antacid treatment, and the
elderly are most at risk of deficiency. The most efficacious treatment is by intramuscular
injection.
HISTORY OF VITAMIN B12
Vitamin B12 is one of the more recent vitamins to be discovered and structurally
analysed. However, as early as the 19th century a Scottish physician suspected that a certain
type of anaemia in humans was probably due to a malfunctioning of the digestive and
assimilative organs. It was not until 1926, though, that it was discovered that large amounts of
raw liver or its juice taken daily could cure this anaemia that would, if left untreated, lead to
death. In 1929, Castle suggested the existence of an 'intrinsic factor' in gastric juice which was
necessary in combination with the 'extrinsic factor' found in raw liver to avoid this anaemia.
STRUCTURE OF VITAMIN B12
Vitamin B12 (Figure 1) is a water-soluble vitamin and is unique among the vitamins in
that it contains an essential mineral: Cobalt (Co). It belongs to a class of compounds called
corrinoids. The corrinoid ring of the vitamin is not unlike the porphyrin ring of haemoglobin or
chlorophyll. In addition, ‘true’ vitamin B12 or cobalamin (cbl) has a ribonucleotide, 5,6-
dimethylbenzimidazole (DMB), as its lower ligand (α-face and either a methyl-, 5’-
deoxyadenosyl-, cyano- or hydroxo-group as its upper ligand (β-face). Vitamin B12-like
compounds with modifications in either the upper or lower ligand, or both, lack biological
activity in mammals and are termed ‘analogues’. Only methylcobalamin (me-cbl) and 5’-
deoxyadenosylcobalamin (ado-cbl) are active as coenzymes in mammals.
72
Figure 1: The structure of cobalamins (vitamin B12).
H2N
NH2 O
O X = OH OH-cbl
O CN CN-cbl
- NH2 CH3 me-cbl
corrin ring
X
H2N N
N O OH OH
β-face
- N
Co N
N
O N N NH2
O
O N
H2N
ado-cbl
O NH2
N
NH
- O
O
P O HON
α-face
OH
VITAMIN B12 COENZYMES
5’-deoxyadenosylcobalamin
In mammals, ado-cbl is needed for the conversion of propionate to succinate. The
crucial step involves methylmalonyl-CoA mutase (E.C. 5.4.99.2) in the isomerisation of
methylmalonyl-CoA to succinyl-CoA. The mutase is vitamin B12 dependent, therefore in
vitamin B12 deficiency this conversion cannot proceed. Methylmalonic acid (MMA) is formed
and accumulates in the tissues, blood and urine. The pathway is outlined in Figure 2. Succinate
enters the TCA cycle to yield malate which is exported to the cytoplasm. There it is further
metabolised via triose phosphate to glucose.
Figure 2: The role of 5’-deoxyadenosyl-cobalamin in propionic acid metabolism.
CH3CH2COOH Propionic acid
Propionyl-CoA synthase
CH3CH2COSCoA Propionyl-CoA
Propionyl-CoA carboxylase
H
HOOC-C-COSCoA (D)-Methylmalonyl-CoA
CH3
Methylmalonyl-CoA racemase
(L)-Methylmalonyl-CoA
hydrolase
CH3
HOOC-C-COSCoA (L)-Methylmalonyl-CoA Methylmalonic acid
H
Methylmalonyl-CoA mutase
ado-cbl
HOOCCH2CH2COSCoA Succinyl-CoA
Succinic thiokinase
CH2COOH
Succinic acid
CH2COOH
Methylcobalamin
Me-cbl, the other coenzyme form of vitamin B12, is required for the synthesis of
methionine from homocysteine (Figure 3).
74
Figure 3: The role of methylcobalamin in methyl transfer.
CH3
S-adenosyl-homocysteine S-adenosyl-methionine
Multiple transferases
S-adenosyl- Methionine
homocysteine Adenosine Adenosine adenosyl-
hydrolase transferase
THF N5-methyl-THF
SH S-CH3
CH2 CH2
CH2 CH3-B12 B12 CH2
H-C-NH3+ H-C-NH3+
COO- COO-
Homocysteine Methionine synthase Methionine

(methyl-cobalamin)
THF: Tetrahydrofolate
With the aid of methionine synthase (E.C. 2.1.1.13) me-cbl transfers its methyl group
to homocysteine to yield methionine. Methionine is converted to S-adenosyl methionine which
is a methyl group donor for other metabolic reactions. S-adenosyl-homocysteine is formed
which hydrolyzes to form homocysteine, which can either be degraded to form cysteine or
remethylated to methionine. Therefore vitamin B12 deficiency leads to elevated levels of plasma
homocysteine due to the failure of me-cbl to convert homocysteine to methionine. There is also
a link between folate metabolism and vitamin B12 through the methyltetrahydrofolate-
homocysteine transmethylase reaction which regulates tetrahydrofolic acid (Figure 4).
Figure 4: Metabolic pathways involving methylcobalalmin.
Deoxyuridylate Thymidylate Thymine-DNA

Thymidylate
synthetase
5-Formyl THF
(Folinic acid)
Dihydrofolate Folic acid
5, 10 Methylene THF Dihydrofolate
reductase
5-Formimino-THF THF
Glutamate FIGLU Methionine

me-cbl Methionine synthetase
Homocysteine
MeTHF
THF: Tetrahydrofolate FIGLU: Formiminoglutamic acid
ABSORPTION, RETENTION AND ELIMINATION OF COBALAMINS
Absorption, retention and elimination of cbl is influenced by the interplay and

availability of dietary intake, digestive secretions, and saturation of blood and tissue stores
(Gräsbeck, 1984; Guéant and Gräsbeck, 1990). In vivo, the vitamin is normally found attached
to one of a number of binding proteins. Free (unbound) vitamin B12 is found only in negligible
amounts. The binding proteins are quite distinctive in their function and site of action. Figure 5
gives an overview of the path of vitamin B12 from the uptake in the stomach through to the final
metabolic sites: the cells.
76
Figure 5: Pathway of vitamin B12 in the body.
HC (from saliva)
gastric
mucosa LIVER
STOMACH
B12 from food use and storage
IF
(as synthase and
HCl mutase) other
tissues
IF
biliary B12 proteins
excretion TC II HC
B12 - HC (0.1%-0.2% lost)
PYLORUS B12 - IF
free B12 (1%)
HCO3- B12 - IF HCdeg. ILEUM
PANCREAS
HC: Haptocorrin IF: Intrinsic Factor TC: Transcobalamin
Absorption
Vitamin B12, once liberated from food, binds to a protein called haptocorrin (HC)
(Nexø et al., 1994). HC is secreted by salivary and gastric glands and therefore is present in
gastric juice. At the acid pH of the stomach vitamin B12 preferentially binds to HC (Carmel,
1995).
The secretion of intrinsic factor (IF) is stimulated by the presence of food, primarily
protein, in the stomach and the presence of hydrochloric acid and pepsin. IF secretion is
increased by insulin, gastrin and histamine. Histamine H2-receptor antagonists have been
known to reduce IF secretion in humans (Jacob et al., 1980; Guéant and Gräsbeck, 1990). IF
combines with cbl in the upper small intestines, where HC is degraded by pancreatic enzymes
(chymotrypsin, trypsin and elastase working together) and bile in an alkaline milieu (Nicolas
and Guéant, 1995), and delivers the vitamin to the distal ileum for absorption (Booth, 1967).
The IF-B12 complex is absorbed only in the terminal ileum by very few specialised
membrane lipoproteins (Jacob et al., 1980). The reaction between receptor and IF-B12 complex
depends on the presence of calcium ions, and to a lesser extent magnesium ions (Beesley and
Bacheller, 1980). The reaction is optimal between pH 6.4 and 8.4 (Jacob et al., 1980; Nicolas
and Guéant, 1994). Bile acids seem to be necessary for the IF-cbl complex to bind to the
surface receptors in the terminal ileum (Seetharam et al., 1992).
Transit through the ileal enterocyte occurs against a concentration gradient
(Lindenbaum, 1979) and thus requires energy. In humans, the time from ingestion to
appearance in the blood has been estimated at 5 to 8 hours (Linnell and Matthews, 1984; Allen,
1976). This process is a limiting factor in cbl absorption (Scott, 1997).
Retention
Once vitamin B12 has been absorbed into the bloodstream, it is then carried by serum
proteins termed transcobalamins (Katz and O’Brian, 1979; Hall, 1979). Transcobalamin II (TC
II) takes up vitamin B12 from IF in the ileal enterocyte and therefore binds most of the absorbed
cbl. The TC II-B12 complex (holo-TC II) as well as free TC II (apo-TC II) are then released into
the portal blood (Rothenberg and Quadros, 1995). Still, approximately 90% of TC II is present
in plasma as apo-TC II (Hall, 1989).
In humans, TC II appears to be specific for 'true' vitamin B12. It also has been found to
bind 6 to 20 % of the endogenous vitamin B12 (Allen, 1976). Only TC II is considered to
facilitate the uptake of vitamin B12 into the cells. Absence of TC II manifests itself in the first
three to six weeks of life in the form of megaloblastic anaemia, failure to thrive, repeated
infections and neurological changes. In vitamin B12 deficiency, cbl appears to be depleted from
TC II first, therefore the degree of saturation of TC II is the earliest sign of an impending
deficiency (Herzlich and Herbert, 1988; Herbert et al., 1990).
The other major cbl binder, HC, is not only found in saliva and digestive juices but
also in other body fluids such as tears, milk, amniotic fluid, cerebro-spinal fluid, blood and bile
(Hall, 1989). The majority of HC is found in the upper digestive tract. In plasma, it is
sometimes called TC I or TC III, to differentiate it from TC II (Kumar and Meyer, 1980).
However, the complete range of functions of HC has probably not yet been fully elucidated
(Nexø et al., 1994). Its role in metabolism is still rather unclear as humans with congenital
defects in HC production do not necessarily seem to have any abnormality in cbl metabolism or
storage (Allen, 1976).
Whereas the role of TC II is to carry exogenous vitamin B12 to the cells for
metabolism or storage, HC apparently carries about 90% of the endogenously derived vitamin
B12 in the plasma but contributes very little to the overall movement of vitamin B12 in humans
(Jacob et al., 1980; Seetharam and Alpers, 1994). HC is the most abundant binding protein in
plasma (Seetharam and Alpers, 1994). Around 80% is found as HC-B12 (Hall, 1989). It binds
cbl more tightly than TC II. However, since TC II is present in blood mainly in the apo form,
this will bind cbl first. HC does not seem to have any transport function; it may act more like a
blood storage of cbl, releasing cbl sparingly as and when the necessity arises (Herbert, 1968).
The role of TC II is to carry vitamin B12 to the various tissues and to promote its
uptake. There are specific receptors for the TC II-B12 complex on the cell surface which allow it
to cross the cell membrane intact by pinocytosis. Once inside the cell, TC II is broken down by
lysosomes after they fuse with the pinocytotic vesicles, freeing the vitamin B12 and thus making
it available for cell metabolism (Hall, 1989). This step is magnesium dependent (Idriss and
Jonas, 1991). The liberated cbl can then be either returned to the plasma for transport to other
metabolic sites or for elimination from the system, or it can be converted to the active
coenzyme forms and stored (Jacob et al., 1980).
Significant amounts of vitamin B12 are converted to ado-cbl (in mitochondria) or to
me-cbl (in cytosol) (Kolhouse and Allen, 1977). This occurs particularly in the liver where the
vitamin accumulates in significant amounts. Ado-cbl, the major portion of biologically active
vitamin B12 in the cells (Linnell and Matthews, 1984; Linnell, 1975), becomes associated with
the mitochondria where it is bound to methylmalonyl-CoA mutase. OH-cbl and me-cbl are
mainly found in the cytoplasm, the latter being attached to methionine synthase. Therefore, the
mutase and the synthase have been called intracellular cobalamin-binding proteins and play a
major role in the intracellular retention and compartmentalisation of cbl (Kolhouse and Allen,
1977).
78
Excretion
The majority of surplus cbl, bound and unbound, appears to be eliminated through the
kidneys and this only occurs when the total binding capacity of TC II is exceeded (Cooksley et
al., 1974). This is the basis of the Schilling test, where patients are given radiolabeled Co
orally, followed by unlabeled vitamin B12 as intramuscular (i.m.) injection to flush the 57Co out.
If radioactivity in a 24 hour urine collection is less than 8 %, the oral dose has not been
absorbed and malabsorption of vitamin B12 is suspected (Clementz and Schade, 1990). Urine,
therefore, eliminates the immediate excess after administration of supra-physiological amounts.
Under normal metabolic conditions less than 250 ng/day of cbl are eliminated in the urine
(Beck, 1982).
Okuda et al., (1958) postulated another route of excretion of unwanted cbl - via bile
and faeces - as the vitamin B12 content of urine did not reflect the amount of unmetabolised
vitamin B12. Gräsbeck et al., (1958) confirmed that this was the main route of cbl elimination in
humans and postulated that there must be an entero-hepatic circulation, reabsorbing some of the
cbl initially excreted by bile. The entero-hepatic circulation could serve to regulate cbl balance
in three ways: 1) the elimination of unwanted analogues, 2) maintaining normal cbl balance
(Green et al., 1981), and 3) retention of cbl in times of shortage.
ASSESSING VITAMIN B12 STATUS
Since it takes several years for humans, after intake or absorption of cbl has stopped,
to develop deficiency symptoms, early and reliable diagnostic tools are essential (Lindenbaum
et al., 1990).
Radioisotope dilution assay

The Radio Isotope Dilution Assay for vitamin B12 in serum or liver is the most
commonly used assay. However, it depends on the binder used in the assay whether only ‘true’
vitamin B12 or the vitamin plus its analogues are measured. If pure IF is used as a binder only
cbl is assayed. However, binders often contain various amounts of HC which will also bind
analogues too. Therefore, if analogues are measured as well, erroneously high results for
vitamin B12 can be obtained and deficiency can be missed. Hence the assay lacks specificity.
Commonly, vitamin B12 concentrations are measured in plasma to assess cbl status.
Concentrations of around 300 pmol/l are considered normal in humans (Nexø et al., 1994).
However, since vitamin B12 plays no active role in blood, concentrations reflecting only dietary
intake and acting as a short-term storage at best, no information can be gained about the
metabolically active vitamin. Kolhouse et al., (1978) found that around 20 % of patients with
cbl deficiency can be missed that way. Savage et al., (1994) reported up to 50 % false positive
results and Norman and Morrison (1993) as much as 40 % false negatives.
Determination of liver vitamin B12 concentrations gives a more accurate indication of
the amount of vitamin B12 the body has at its disposal. However, the problems inherent in the
method still apply. In addition, it is more difficult to obtain liver biopsies and not without pain
and risk to the patient.
Methylmalonic acid
In both humans and animals, MMA is now increasingly used in research and routine
diagnosis of vitamin B12 deficiency. Elevated levels of MMA have been claimed to be the first
sign of vitamin B12 deficiency (Norman et al., 1982) and specific for it (Ho et al., 1987).
Normal serum MMA concentrations in humans range from 53 nmol/l as the lower limit to 640
nmol/l as the upper limit (Stabler et al., 1986; Savage et al., 1994). However, the earlier
analyses focused on measuring MMA in urine since urinary MMA is much greater than serum
MMA. In normal human urine MMA ranges from 0.85 to 43 µmol/l (Marcell et al., 1985).
Norman (1996) reported that the urinary MMA spot test was the most accurate test for vitamin
B12 deficiency with a sensitivity of 100% and a specificity of 99%. Given normal renal
function, plasma and urinary MMA (adjusted to creatinine or collected over 24 hours) correlate
well for humans (Rasmussen et al., 1989). After parenteral vitamin B12 therapy, elevated MMA
levels return to normal.
Homocysteine
Homocysteine has created much interest in the last decade as an independent risk
factor for cardiovascular disease in humans (Ueland et al., 1993). It is elevated in vitamin B12
as well as in folate deficiency, therefore other tests need to be considered when homocysteine is
to be used as a screening for cbl status. Savage et al., (1994) suggested the combined use of
MMA and homocysteine: if both are normal, vitamin B12 deficiency is ruled out with virtual
certainty. Although they found MMA to be the better indicator, there are occasions, such as in
renal dysfunction or disturbed bowel flora (e.g. after antibiotics), when MMA appears normal
despite deficiency. When Stabler et al., (1996) compared MMA and homocysteine in 60 normal
patients, 60 with cbl deficiency and 60 with folate deficiency, all cbl deficient and all folate
deficient patients had raised homocysteine (> 15 µmol/l), whereas only cbl deficient patients
had raised MMA. The two tests in conjunction were optimal in distinguishing between vitamin
B12 and folate deficiency (Stabler et al., 1996).
Transport proteins
Adaptive changes in transport proteins and their degree of saturation is being used to
assess cbl status in humans (Herbert et al., 1990). Since holo-TC II represents the metabolically
active form in which tissues take up the vitamin, Herbert et al., (1990) showed that the earliest
indication of a negative vitamin B12 balance in people with pernicious anaemia and AIDS is
low serum holo-TC II. If vitamin B12 deficiency is supected but other markers, such as serum
and liver vitamin B12, MMA and homocysteine, are still within the normal range assaying holo-
TC II becomes important (Kapel et al., 1988).
SOURCES OF VITAMIN B12
Apart from vitamin D and A, vitamin B12 is the only other vitamin not found in plants,
with the exception of fermented foods, like seaweeds and tempeh, where it has been detected in
trace amounts. Vitamin B12 synthesis from Co is accomplished by only a few species of micro-
organisms. Anaerobic bacteria found in sewage sludge, manure, soil and dried estuarine mud
produce this vitamin. Rumen bacteria produce the vitamin which the host animal is then able to
absorb and utilise.
In monogastrics, the vitamin is synthesised from Co by bacteria in the intestinal tract
but this is lost in the faeces because it is past the absorption site for the vitamin, hence in
humans vitamin B12 needs to be obtained from the diet. Only animal products (meat or dairy)
contain sufficient vitamin B12 to meet metabolic demands. Food sources high in vitamin B12
include products from those animals that ingest large numbers of micro-organisms or store
accumulated cbl (shellfish, fish, egg yolk and organ meat).
80
DIETARY REQUIREMENTS OF VITAMIN B12
In Western countries, the daily absorption of vitamin B12 in humans has been
estimated at around 1 to 5 µg (Clementz and Schade, 1990) with minimum physiological
requirements considered to be 0.6 to 1.2 µg (Schneider and Stroinski, 1987). Up to 100 µg of
vitamin B12 per 100 g of wet weight can be found in food of which only the first 2 µg are
absorbed actively. A very small amount is also absorbed by passive diffusion (Scott, 1997). The
rest is eliminated. The Recommended Daily Allowance for most countries and endorsed by the
WHO is 1 to 2 µg per day (Gräsbeck, 1984; Scott, 1997). Cooking and the action of digestive
enzymes help to free the vitamin from food and make it available for absorption (Nicolas and
Guéant, 1995; Carmel, 1994).
The human body contains about 3 to 5 mg vitamin B12. It is generally widely
distributed throughout the body with about half of it stored in the liver (Clementz and Schade,
1990), the rest is mainly found in kidneys and bones. There is little variation between different
animal species and humans in respect to the amount of vitamin B12 in the various tissues
(Underwood, 1971). The amount stored in the body would last several years before reserves
became depleted if vitamin B12 intake stopped (Belaïche and Cattan, 1989). Total body half-life
has been estimated to be 1 to 4 years (Schneider and Stroinski, 1987).
DEFICIENCY SIGNS AND SYMPTOMS
Deficiency sets in gradually as the vitamin is depleted from the tissues. Symptoms can
be vague initially, such as lack of energy, anorexia and lowered resistance to infection
(Clementz and Schade, 1990). In general, symptoms fall into two categories: haematological or
neurological. Haematological changes lead to megaloblastic anaemia (which is virtually the
same as in folic acid deficiency) with macrocytosis, hypersegmentation of neutrophil nuclei,
bone marrow changes, and often pancytopenia (Carmel, 1990).
Nervous system symptoms include soreness and weakness in arms and legs,
diminished reflex response and sensory perception, difficulty in walking (gait ataxia) and
speaking, and jerking of limbs. This leads to irreversible damage of the central nervous system
in humans if left untreated. As the depletion progresses, brain damage occurs, manifesting in
sore mouth, numbness, shooting pains or pins-and-needles in limbs, memory defects, to
psychotic episodes and dementia (Kirschmann, 1979). If the condition is treated within the first
year of ocurrence these symptoms, including dementia (Swain, 1995), can be reversed.
CAUSES OF VITAMIN B12 DEFICIENCY AND PEOPLE AT RISK
Since the vitamin is only obtainable from animal sources people who do not consume
animal products, such as vegans, are therefore at risk of deficieny. Children of vegans are even
more at risk (van Dusseldorp et al., 1999) and can show impaired vitamin B12 metabolism
already at birth. Vegetarians, even if they consume some dairy products in their diet, could be
at risk of deficiency since these foods are not as high in the vitamin as meat (Duo et al., 2000).
Therefore, people who consume meat in their diet are not generally thought of as
suffering from a lack of the vitamin. However, causes of vitamin B12 deficiency do not only
include inadequate ingestion but also inadequate absorption, increased requirements and
increased excretion.
Inadequate absorption occurs when a transport protein, notably IF, is reduced or
lacking. The production of IF is dependent on the presence of hydrochloric acid and pepsin in
the stomach. Therefore, gastric atrophy, hypochlorhydia or achlorhydia, gastric resection or
removal, all result in loss of IF (Nicolas and Guéant, 1995). Malabsorption of cbl has also been
found in 40 % of people with chronic pancreatitis and in 100 % of people with cystic fibrosis
which is corrected by administration of pancreatic extracts (Belaïche and Cattan, 1989). Anti-
histamine treatment and Helicobacter pylori infection also lead to reduced vitamin B12 uptake
(Nicolas and Guéant, 1995).
Reduced hydrochloric acid, and therefore IF, is common amonst the elderly as part of
the slowing down of the metabolism (Stabler, 1995), in people who use antacids regularly
(Termanini et al., 1998), and sometimes in people with allergies. If antibodies are produced
against IF, pernicious anaemia results and represents an auto-immune disease. Bacterial
overgrowth in the small intestines or parasitic infestation can deplete the host of ingested
vitamin B12 and lead to deficiency (Nicolas and Guéant, 1995), as does removal of the terminal
ileum (the site of vitamin B12 absorption), e.g. due to Crohn’s disease.
In situations of additional nutritional demands, such as during pregnancy, when under
stress or if suffering from hyperthyroidism requirements for the vitamin increase and may not
be met by daily intake. Likewise, daily needs increase with increased excretion. This mainly
occurs when the liver is damaged through disease such as hepatitis, or through exposure to
toxins, drugs (including prescription medicines) or regular use of alcohol (Baker et al., 1998).
SUPPLEMENTATION
Obviously, not only people with pernicious anaemia need to be supplemented with
vitamin B12. Particularly at risk are the elderly, vegans, regular consumers of antacids, drugs
and alcohol, people with gut and liver infections and neurological symptoms, and gastric and
ileal resections. Those with allergies, familial neurological problems, and with depressed
immunity and energy would most likely benefit from additional vitamin B12 as well.
Daily oral vitamin B12 supplementation is only of use if the vitamin is lacking in the
diet. Therefore, injections should be given in all other cases to ensure adequate vitamin B12
status. Injections have the advantage that they are safe, efficient and effective to replenish the
tissues, bypassing the IF binding phase of absorption. Vitamin B12, administered as hydroxo-
cobalamin, has no known toxicity even in large doses. Maintenance dosage is usually 1000 µg
OH-cbl, given in monthly to three monthly intervals.
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Conjugated linoleic acid (CLA)
A.K.H. MACGIBBON and A.M. ROWAN

New Zealand Dairy Research Institute, Palmerston North
ABSTRACT
The consumption of CLA has been shown to have a wide range of health benefits in
animal models, including inhibition of carcinogenesis and atherosclerosis, inhibition of weight
loss induced by immune stimulation, inhibition of diabetes and increase in the percentage of
lean body mass. CLA is found naturally in foods derived from ruminant animals. Dairy
products, especially New Zealand milkfat, are excellent sources of CLA. These foods provide
a source of CLA in the human diet. It is now known that some CLA can be produced
endogenously by humans and is present in human breast milk.
CLA is a fatty acid and so is associated with the fat phase of products, being generally
higher in full fat products than low fat products. It occurs as a range of isomers of linoleic acid
which possess a conjugated double bond. While chemically there are a wide range of possible
isomers, naturally, the predominant isomer in milkfat is the cis-9, trans-11 isomer. While the
presence of the CLA fatty acids in milkfat has been known for decades, the health interest is
new. Most nutritional studies to date have been carried out with chemically prepared CLA
which contains a range of isomers so it has been unclear as to the biopotency and the
importance of the individual isomers. Currently nutritional studies are being carried out on the
individual isomers.
INTRODUCTION
It is intriguing that there are potential health benefits from a fatty acid, in fact, an
animal-derived fatty acid and an animal-derived trans fatty acid at that. In recent years there
has been a sustained interest in the health benefits of conjugated linoleic acid (CLA). The
initial studies isolated CLA as an anticarcinogenic component from grilled hamburger meat
(minced beef) (Ha et al., 1987), as outlined by Parodi (1999) in a history of CLA contained in
the book edited by Yurawecz et al., (1999). From this initial work, the health implications of
CLA have widened to include inhibition of carcinogenesis and atherosclerosis, inhibition of
weight loss induced by immune stimulation, inhibition of diabetes and increase in the
percentage of lean body mass. These will be discussed in more detail later. It is of interest that
the presence of CLA in bovine milkfat had been known for many years (Parodi, 1977), but it
was only after these studies that any interest was taken in the potential health benefits of milkfat
CLA. The aim of this review is to detail some of the properties of CLA, especially from
milkfat, and to relate these to the potential health benefits.
PROPERTIES OF CLA
CLA is a fatty acid and so is associated with the fat phase of products. It contains two
double bonds, but unlike the majority of fatty acid double bonds found in nature it is conjugated
(that is one carbon separation rather than the typical two carbon separation, methylene
interrupted double bonds). Usually at least one of the double bonds is a trans double bond
rather than the more common cis double bond.
86
Figure 1: Chemical structure of CLA isomers.
10 12
HO
trans-10, cis-12
C
O 9
HO
11
cis-9, trans-11 CLA
The parent fatty acid is the linoleic fatty acid that is in the cis-9, cis-12 configuration.
Isomerisation of the double bonds can give a range of isomers including the cis-9, trans-11
CLA and trans-10, cis-12 CLA (the two which have been linked to health benefits). The range
of possible isomers is important as the CLA isomers are not separated on typical fatty acid
analysis, requiring specialised gas chromatography or high performance liquid chromatography
to observe the different species. Thus, unless specifically analysed, the range of the isomers of
CLA is not determined. Synthetic CLA fatty acids can be produced by alkali isomerisation, at
high temperature, of commercial oils high in linoleic acid (e.g. safflower, sunflower or
soyabean). However, if the reaction is uncontrolled a wide range of isomers can be produced
and the difficulty for biochemical researchers working with syntheic CLA has been in knowing
whether the observed effects are due to the entire CLA class, or a particular isomer within that
class (Stanley and Hunter, 2000). In general, amount of c-9,t-11 and t-10,c-12 CLA isomers is
similar (about 1:1) but there are varying amounts of other isomers to make up the total CLA.
Yurawecz et al., (1999a) found that commercial CLA products contained between 20-90% total
CLA and 60-100% of the sum of c-9,t-11 and t-10,c-12 CLA isomers in the total CLA. Recent
work has used CLA with less of the other isomers and more recently pure isomers have been
available in small quantities enabling researchers to actually define the isomers with the
specific bioactivity.
While commercial oils can be used for the synthesis of CLA there is minimal natural
CLA present in vegetable oils. In fact ruminant fats are the best dietary source of CLA (beef,
lamb, goats and especially dairy products) (Chin, 1992). CLA is present in both the depot fat
and the milk, however, in general, the values are higher in the milk on a fat basis. In these
natural products the major isomer is the cis-9, trans-11 isomer (about 70-90%) with only trace
amounts of trans-10, cis-12 CLA. In the rumen the CLA is formed by microorganisms as an
intermediate in the biohydrogenation of unsaturated fatty acids. The pathway for the
biohydrogenation of linoleic acid is as follows. The linoleic acid is isomerised by linoleic
isomerase from rumen bacteria to CLA, which in turn is hydrogenated to a trans monoene
(vaccenic acid), and finally the fully saturated stearic acid. A recent finding is that the reverse
pathway from vaccenic acid to CLA with a desaturase is an important step in both ruminant and
human metabolism (Griinari et al, 2000). Thus CLA can be formed from linoleic acid (in the
rumen) or vaccenic acid.
We have been interested in the concentration of CLA in New Zealand milkfat and how
it changes through the dairy season (Creamer and MacGibbon, 1996; MacGibbon, 1999). New
Zealand dairy farming is based on year-round pasture feeding and so is quite different from the
substantial supplement feeding which is typical of much of the world’s dairy production.
Studies of the changes in the CLA content in the milkfat extracted from commercial butters
from different regions in New Zealand has shown two features.
Firstly, there is a seasonal variation in the CLA content of the milk, with a mean of 11
mg/g fat and a range of 7-15 mg/g fat (MacGibbon and Hill, 1998). Note that the changes
mimic the changes in the rate of grass growth with CLA lower in the summer.
Secondly, these CLA values are higher than those generally reported for CLA from
dairy products sourced from other countries (Chin, 1992). The New Zealand CLA values are
two to three times higher than those reported for countries where supplementary feeding is
dominant (such as the USA and parts of Europe). Other countries that use pasture feeding such
as Ireland and parts of Australia also show high levels of CLA.
Milkfat is made up of triglycerides, three fatty acids attached to a glycerol backbone.
Robinson and MacGibbon (2000) have shown that the CLA fatty acid in milkfat is distributed
widely through the range of milkfat triglycerides and so is not easy to concentrate from the bulk
milkfat by extraction techniques.
There is considerable herd-to-herd and cow-to-cow variation in CLA. MacGibbon and
Hill (1998) showed that there was a significant difference between the CLA content of Friesian
and Jersey cows: Freisian being typically 50% higher. In addition the feeding of the cows can
play a significant part. The difference between supplement and grass feeding has already been
discussed however specific feeding practices can be adopted to maximise the CLA levels.
Though increasing the polyunsaturated fatty acid content, especially linoleic acid, can increase
the CLA levels, the outcome is not certain. Obviously changing the milk composition of the
cow is affected by a wide range of factors including the type of feed, the form of the feed, the
condition and the rate of passage in the rumen as well as the metabolic pathways. Thus caution
is required in transplanting procedures from one trial to another.
However there are obviously opportunities to enhance the levels of CLA in dairy
products, should the potential health benefits be realised.
HEALTH BENEFITS OF CLA
The literature on purported health-promoting attributes of CLA is extensive and

growing. The largest research effort to date has been in the anti-cancer area, with mainly in
vitro and animal studies reported to date, however work in the areas of cardiovascular health,
immune function, body composition, diabetes, bone health and muscle strength has also been
carried out. Much of the research is now focussing on the mechanisms of CLA action, as well
88
as the relative activities of the different isomers of CLA, and defining the effective dose for
humans.
Cancer
Over the last 15 years or so, several key research groups have systematically
investigated the effects of synthetic mixtures of CLA isomers on tumourigenesis and cancer
progression, in vitro and using animal models. CLA is effective in inhibiting cancer cell growth
in vitro. Studies have used cancer cell lines of the colon, skin, liver, ovary, prostate and other
tissues, to investigate the effect of synthetic CLA on suppressing cell growth (Cornell et al.,
1997; Yoon et al., 1997; Visonneau, 1996). Other studies have demonstrated that CLA may be
effective at all stages of the cancer process – initiation, promotion, progression and metastasis
(Pariza et al., 1999). The mechanisms of action of CLA in the cancer process have been
reviewed by Pariza et al., (2000).
Animal models have been used to determine dose responses, effects of different forms
of CLA and diet composition, at different cancer sites. CLA, at doses of up to 1% w/w of the
diet, reduced tumour incidence, weight and development by up to 60% in animal models of
forestomach cancer (Ha et al., 1990), mammary cancer (Ip et al., 1995; 1991), and skin cancer
(Belury et al., 1995). Furthermore, CLA effectively reduced mammary tumour growth when
fed to rats prior to carcinogen exposure, and when given after cancer initiation (Ip et al.,1995).
It was suggested that the greatest effect of CLA in reducing mammary tumour development is
during the critical period of mammary tissue development.
Most of the research to date has investigated the effects of CLA using synthetic free
fatty acid isomers, however in food CLA exists as part of a triglyceride. One study has
confirmed that triglyceride-bound CLA has the same anti-cancer properties as the free fatty
acid (Ip et al., 1995). More recently food sources of CLA have also been investigated. Butter,
with enhanced natural CLA (4.1% of fatty acids), reduced tumour incidence and number by up
to 50% compared to a control diet (Table 1). The effect on tumour growth was similar for
natural CLA and synthetic CLA (Ip et al., 1999), confirming that the natural CLA was
biologically active.
Table 1: Mammary cancer prevention in rats fed different sources of CLA. Control was typical
butter (containing 0.5% CLA) and CLA isomers were from commercial chemical
products.
Group CLA in diet Tumour Total no.

incidence tumours
Control 0.1% 28/30 (93%) 92

Control + c-9, t-10 isomer 0.8% 16/30 (53%) 46
Control + mixed isomers 0.8% 17/30 (57%) 48
Butter high in natural CLA 0.8% 15/30 (50%) 43
From Ip et al., (1999).
There is little evidence to date for a role of CLA in reducing the risk of human cancer.
Epidemiological studies allow some information on the relationship between CLA consumption
and cancer risk, although these types of studies are limited by a lack of accurate information on
the CLA content of a foods. Breast cancer incidence was lower in women with the highest
tissue levels of CLA (Lavillonniere and Bougnoux, 1999), which largely reflects CLA intake,
although trans vaccenic acid from milk products will also contribute to tissue CLA levels. It is
still difficult to estimate the dose required for the same effect in humans as has been reported
for animals.
Cardiovascular disease
CLA is a trans fatty acid, and despite some suggestion that trans fatty acids may have
cholesterol raising effects, CLA appears to have beneficial properties for heart health. Synthetic
CLA isomers have been shown to reduce LDL-cholesterol and triglycerides by up to 20% (Lee
et al., 1994; Nicolosi et al 1997), although differential effects of the isomers have been reported
(Gavino et al., 2000; de Deckere et al., 1999). Other studies have reported no effect of CLA on
blood lipids (Stangl et al., 1999; Sugano et al., 1997). Rats fed butter with low or high synthetic
CLA levels had lower liver cholesterol levels than animals fed control butter (Wright et al.,
2000). CLA intakes of 1% w/w of the diet reduced rodent aorta fatty streaks and reduced
atherosclerotic thickening (Nicolosi et al., 1997), however, studies using lower CLA intakes
(0.25% w/w) found no reduction in fatty streaks in animal models (Paigen et al., 1987; Munday
et al., 1999).
CLA may have other biological effects that are protective for cardiovascular disease.
In vitro studies have also suggested that both cis-9, trans-11 CLA and trans-10, cis-12 CLA
isomers may have anti-thrombotic properties. CLA reduced platelet aggregation, and the
formation of thromboxane B2 compared to linoleic acid (Truitt et al., 1999). Further, CLA
appears to directly alter the cell membrane function of heart tissue (Sugano et al., 1997; Xiao et
al., 1998).
Recent human studies have provided conflicting evidence for the effect of synthetic
CLA in heart health. Blankson et al. (2000) reported small but statistically significant
reductions in total and LDL-cholesterol after 12 weeks intervention with both 1.7g or 3.4 g/day.
Higher doses were not effective. Nelson et al (2000) reported the results of a controlled trial in
which healthy women received 2.4 g/day of synthetic CLA for two months. Plasma and platelet
CLA levels were increased, but there were no effects on blood lipids or indicators of thrombotic
tendency such as platelet aggregation. No studies have been reported for an effect of natural
CLA in heart health. As more human studies are reported the role of CLA in cardiovascular
health may become clearer.
Body composition
The effect of CLA on body composition changes and weight loss has been investigated
in several animal species and humans. Diets containing between 0.5% and 3% w/w of the diet
have been shown to reduce body fat in mice (Hayman, 2000; Park et al., 1997), rats and
chickens (Pariza et al., 1996) and pigs (Dugan et al., 1997) by up to 70%, although the
reduction varies between species. One study also suggested the effect of synthetic CLA was
more pronounced in female animals (Park et al., 1997). A recent randomised controlled study
involving otherwise healthy overweight and obese adults, investigated the dose response of 1.7
g to 6.8 g/day of synthetic CLA taken for 12 weeks (Blankson et al., 2000). At doses of 3.4 and
6.8 g/day there were significant reductions in body fat mass compared to the control. There
were however, no effects on body weight or BMI. Conversely other human studies reported no
effect of around 3 g/day in overweight subjects (Zambell et al., 2000; Atkinson, 1999). In
subjects with normal weight, small reductions in body weight have been reported with 4.2
g/day CLA (Vessby and Smedman, 1999). Natural CLA from ruminant fat, consumed as CLA-
enriched cheese, was less effective in lowering body fat in mice compared to synthetic CLA-
enriched cheese (Dhiman et al., 2000). This may be because of the different biological
potencies of the different isomers found in natural sources of CLA versus synthetic sources.
90
Indeed, recent studies suggest that the most effective CLA isomer in affecting body
composition is trans-10, cis-9 CLA (Park et al., 1999). Animals were fed either diets containing
no CLA, a mixture of CLA isomers, cis-9, trans-11 CLA or trans-10, cis-12 CLA for 4 weeks
(Park et al., 1999). All CLA treatments reduced weight gain in growing animals, however, the
greatest effect was for the trans-10, cis-12 CLA isomer. Gavino et al., (2000) fed hamsters 1%
mixed isomers, 0.2% cis-9, trans-11 CLA or 0.2% w/w linoleic acid. The highest CLA intake
of mixed isomers resulted in the greatest reduction in weight gain, also demonstrating that the
trans-10, cis-9 CLA isomer is biologically active.
CLA also increased body ash content, assumed to relate to bone mineral content
(Hayman, 1999; Park et al., 1997). A human study showed that CLA supplementation during
resistance training tended to increase bone mineral content, but not bone mineral density after
four weeks (Kreider et al., 1998). From the same study (Ferreira et al., 1998) non-significant
increases in muscle strength and reduced catabolic status were observed after supplementation
of around 6 g/day CLA for four weeks during resistance training. Longer term studies may be
required to see significant effects on bone and muscle metabolism.
Immune function
It has been suggested that much of the explanation for the biological effects of CLA is
due to its effect on the immune system. In particular, CLA inhibits the production of the
cytokine, tumour necrosis factor alpha or TNF-α (Pariza et al., 1999). TNF-α is involved in
inflammatory processes which may play a role in many pathological conditions such as
atherosclerosis, cancer progression and obesity. Synthetic CLA also enhances macrophage
phagocytosis, lymphocyte proliferation, NK cell cytotoxicity and nitric oxide production in
vitro (Zhao, 1999; Cook et al., 1993; Michal et al., 1992). In addition to immune enhancement,
CLA can modulate allergic response parameters, such as IgE (Sugano et al., 1998), lipid
mediators such as PGE2 and leukotrienes, in animal models of type 1 hypersensitivity
(Whigham et al., 2000).
However, a recent human study of healthy women found no improvement in immune
status after 63 days supplementation with CLA capsules containing 2.5 g/day CLA (Kelley et
al., 2000). Immune parameters measured included lymphocyte proliferation, blood cell
numbers, antibody responses, and delayed type hypersensitivity reactions.
Diabetes
CLA may also play a role in type 2 diabetes and has been shown to normalise blood
glucose responses in genetically predisposed animal models of diabetes (Houseknecht et al.,
1998). Zucker rats fed synthetic CLA (1.5% w/w) had significantly improved glucose response
after two weeks compared to controls. A recent human study (Belury, et al., 2000; Mahon et
al., 2000) has reported that 64% of type 2 diabetic subjects had improved insulin levels after
eight weeks of CLA treatment (6 g/day). Further work will reveal the mechanisms involved, but
it has been suggested that CLA activates peroxisome proliferator-activated receptors, which are
associated with tissues involved in lipid metabolism.
Quality of life
A recent dose reponse study in overweight adults, included a survey on changes in
quality of life parameters (Blankson et al., 2000). Subjects completed self-assessment
questionnaires at the beginning and end of the 12 weeks of supplementation with doses of CLA
from 1.7 g/day to 6.8 g/day. All CLA doses improved sleep and stress levels relative to the
control group. The maximum benefits were achieved with doses of CLA of 1.7 or 3.4 g/day.
These findings require further substantiation, however it is suggested that moderate doses of
CLA may offer an even wider range of benefits to those already studied to date.
CONCLUSION
There are potentially very significant health benefits to be gained from CLA, if these
effects are confirmed by current research, especially in human studies. As dairy products are
an excellent source of CLA, there is the potential for the continued consumption of fat-
containing dairy products to be seen in a positive light. Specifically, it is a positive attribute for
milkfat. While at present the CLA target values for significant benefits are unclear, the work
presented shows the potential to increase the CLA levels in milkfat.
ACKNOWLEDGEMENTS
We acknowledge financial support from the New Zealand Foundation for Research,
Science and Technology (DRI X001) and the New Zealand Dairy Board.
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Flavour components in the rhizome of soil-grown wasabi

TAMANNA SULTANA1, G.P. SAVAGE1, D.L. McNEIL1, N.G. PORTER2
and R.J. MARTIN2
1
Food Group, Animal and Food Sciences Division, Lincoln University, Canterbury and
2
New Zealand Institute for Crop & Food Research Ltd, Christchurch
ABSTRACT
Wasabi, Japanese horseradish (Wasabia japonica (Miq) Matsum) is grown to prepare

a green paste which is eaten with traditional Japanese dishes. The plant is grown as a perennial
crop in Japan and is now also grown in New Zealand. The best quality wasabi products are
produced from the rhizomes (stems) although other parts of the plant such as petioles and
leaves also possess some pungency and are also used as raw materials. The characteristic
flavour of wasabi comes from the volatile isothiocyanates (ITCs), which are evolved from
glucosinolates by enzymatic hydrolysis when tissues are macerated. In this study the total
isothiocyanate (ITC) and six different ITCs were measured in the rhizome of wasabi grown at
Lincoln under four different soil treatments. The level of total ITC ranged from 2425 to 2810
mg/kg fresh weight, which was significantly higher than the values reported in the literature for
wasabi grown in Japan (mean 1659 mg/ kg). Allyl isothiocyanate (AITC) was the main ITC and
it contributed between 86 to 92% of the total ITC measured in the rhizomes. Overall, there were
small changes in the individual ITCs as a result of the different treatments (control, lime,
manure, lime and manure), however no correlation between ITC concentration and yield of
plants was found. The total ITC contents quantified by GCMS were marginally higher than the
total ITC’s measured by UV spectrometry after approximately one year storage of paraffin oil
extract at 0 to -4ºC.
INTRODUCTION
Flavour does not add nutritional value to food but it can improve the taste of a dish.
Wasabi (Wasabia japonica (Miq) Matsum) is an aromatic herb that gives a hot taste with a
pungent-greenish smell. Due to its unique flavour it has established a strong demand in
traditional and modern food dishes in Japan and is becoming well known in western cuisine.
Wasabi has been developed as a new crop in New Zealand since 1982 (Douglas and Follett,
1992). Wasabi is a member of the cruciferae family, which also contains other vegetables like
cabbage, broccoli and mustard. All plant parts of wasabi exhibit flavour though a rhizome
(stem) from the mature plant provides maximum pungency (Sultana et al., 2001a). Thus,
wasabi is cultivated primarily for the rhizomes and it is the most demanded and expensive
tissue. Traditionally in Japan, wasabi paste is a popular condiment with raw fish (sashimi) and
sushi. It is also used as a spice during cooking. However, modern technology allows wasabi to
be used as an ingredient to prepare a range of sauces and mayonnaises. The other wasabi plant
parts are used mainly to make pickles in sake brine or soy sauce, cheese and crackers
(Chadwick et al., 1993).
The characteristic flavour of wasabi comes from isothiocyanates (ITCs) (McGregor et
al., 1983; Delaquis and Mazza, 1995; Masuda et al., 1996), which are evolved from plant
tissues when they are disrupted. However, plant tissues do not contain natural ITCs but contain
glucosinolates (GSLs), which are precursors of the isothicyanates. GSLs are a group of
glucosides, stored within the cell vacuoles of all cruciferae plants (Delaquis and Mazza, 1995).
These GSLs are biosynthesised from protein amino acids by three major steps shown in Figure
96
1 (modified from Fahey et al., 2001). The GSLs have a common structure consisting of a
central thiocyanate group, the sulphur atom with a β-link to D-glucose and the carbon atom of a
thiocyanate group attached to a side group (R). A wide variety of side groups (R) can produce
different GSLs and therefore different ITCs. GSLs coexist in plants, but are not in contact with
the hydrolytic enzyme myrosinase (thioglucoside glycohydrolase EC 3.2.2.1) in the intact cell.
Figure 1: Biosynthesis of glucosinolates (GSLs).

CO2
[O]
1. R CH COOH R CH COOH R CH
NH2 NHOH NOH
Protein amino acid N-Hydroxylated Aldoxime

oxy amino acid
CH2OH
O H
Cysteine UDP-glucose UDP
OH
S
OH H
2. Aldoxime R C SH R C
Through
intermediate NOH NOH
steps
Thiohydroxamic acid Desulfoglucosinolate
CH2OH
PAPS O H
PAP
OH
S
3. Desulfoglucosinilate R C OH H
N OSO3-
Glucosinolate
Steps
1. Conversion of protein amino acids to aldoxime is initiated by N- hydroxylation of an
amino acid followed by decarboxylation to form aldoxime. This step is catalysed by
cytochrome p450.
2. a) Introduction of sulphur to aldoxime from cysteine to produce thiohydroxamic acid
through unknown intermediate steps (Fahey et al., 2001, McGregor, 1993).
b) S-glycosylation of thiohydroxamic acid to form desulfoglucosinolate is catalysed by
uridine diphosphate derivative of glucose.
3. Final step is glucosinolate formation by sulphation of desulfoglucosinolate calalysed
by high energy sulphate donor, 3-phosphoadenosine 5-phosphosulphate (PAPS).
When plant tissues are mechanically disrupted or injured (e.g. by chewing, crushing or
grating in the preparation of food) the enzyme is released from the cell wall, and in the presence
of adequate moisture myrosinase rapidly hydrolyses the GSLs (Figure 2) to yield glucose and
an aglucone (McGregor, 1993). The organic aglucone is unstable and undergoes Lossen
Rearrangement (Fenwick et al., 1983) to produce sulphate and a variety of products. The nature
of products is dependent on a number of factors, including the structure of the GSL side chain,
the reaction conditions (e.g. pH), the presence of cofactors (eg. metal ions, specific protein),
temperature and duration, the age and condition of plant tissues. Isothiocyanates (ITCs) are
formed from GSLs under neutral and alkaline conditions. However, GSLs, which contain a β-
hydroxyl group (I in Figure 2) in their side chain, give rise to ITCs that spontaneously cyclise to
form oxazolidinethiones. Some aromatic and heterocyclic GSLs (II in Figure 2) produce ITCs,
which are unstable at pH 7 or higher and break down to release the corresponding alcohol and
inorganic thiocyanate ions. However, once formed ITCs are more stable under acidic
conditions. In a weakly acidic pH or in the presence of Fe+2 and/or endogenous nitrile factor,
nitriles are produced from the aglucone by autolysis instead of ITC, with the liberation of
elemental sulphur (McGregor, 1993). The relative proportion of ITC to nitriles can vary widely
depending upon the conditions of autolysis (Mcgregor, 1993). Thiocyanate formation is
believed to involve a cofactor, which may also be a protein, since it been shown to be labile to
both heat and polar organic solvents. Most of the sulphur containing end products formed by
enzymatic and non-enzymatic reactions of GSLs are volatile (Delaquis and Mazza, 1995). A
summary of different ITCs reported from previous investigations in wasabi are listed in Table
1. The complete taste of wasabi derives from a combination of tastes or odours of all the ITCs
combined together. Naturally Allyl ITC (2-propenyl ITC, CH2=CH-CH2-N=C=S) has the first
effect on the overall taste because it is found in highest concentration in wasabi rhizomes and
other plant tissues (Sultana et al., 2002a). Allyl ITC is also found in the highest concentration
in horseradish (Sultana et al., 2001b). While AITC is the main component of wasabi due to its
pungent flavour other ITCs e.g. 6-methylthiohexyl ITC and 7- methylthioheptyl ITC, by giving
their characteristic fresh greenish flavour, may contribute significantly to the total taste profile
of wasabi (Ina et al., 1989; Masuda et al., 1996).
Natural ITCs are volatile anti-nutritional compounds, are toxic at high intakes and
possesses strong pungent smells. Thus, the overall palatability of cruciferae plants undoubtedly
limits their intake by animals (Fenwick et al., 1984). However, the most common reason of
cruciferae plants for making ITCs is believed to be due to its important role in protecting these
plants from attack by insects and animals.
98
Figure 2: Conversion of glucosinolates to different products by enzymic
and nonenzymic reaction.
Table 1: Isothiocyantes in wasabi.
Isothiocyanates Concentration mg/kg fresh weight Odour descriptionb,d,e

(ITCs) Wasabi Wasabi Wasabi Mean
rhizomea rhizomeb rootc published
values
Isopropyl ITC - 7.6 - 7.6 Chemical, weak mustard like
Allyl ITC 1282 1880 1110 1424 Strongly pungent, mustard-like,
lacrymatory, bitter
n-butyl ITC - - 17.4 17.4 ---
Sec-butyl ITC 11.4 13 - 12.2 Chemical, weak mustard like
Isobutyl ITC 0.6 3.9 - 2.3 Sweet, chemical
3-butenyl ITC 123.5 25 18.3 55.6 Green, pungent, aroma
4-pentenyl ITC 65.2 31 39.0 45.1 Green, pungent, acrid, fragrant
leaf
5-hexenyl ITC 16.9 8.0 10.2 11.7 Green, pungent, fatty
6-heptenyl ITC 1.0 0.6 - 0.8 Green, pungent, fatty
3-methylthio 2.4 Tr - 2.4 Strongly raddish-like, pungent

propyl ITC
Benzyl ITC - - - - Chemical, pungent
2-phenylethyl ITC - Tr - - Strongly radish-like, pungent,
strong watercress aroma, tingling
sensation
4-methylthio 0.2 - - 0.2 -
butyl ITC
5-methylthio 9.9 1.5 4.8 5.4 Radish-like, pickle-like
pentyl ITC
6-methylthio 35.0 4.8 18.9 19.6 Radish-like, sweet, fatty
hexyl ITC
7-methylthio 3.2 0.9 14.4 6.2 Sweet, fatty, radish-like, pickle-
heptyl ITC like
5-methyl - - 21.7 21.7 -
sulphinylpentyl ITC
6-methyl - - 78.0 78 -
sulphinylhexyl ITC
7-methyl - - 14.1 14.1 -
sulphinylheptyl ITC
Total ITC 1653.9 1976.3 1346.8 1659
a
Data from Kumagai et al., (1994); Data from Masuda et al., (1996); cWasabi root in this
b
reference presumably refers to either the rhizome or the total root plus rhizome mass, data from
Etoh et al., (1990); dData from Fenwick et al., (1983); eIna et al., (1981); Tr, less than
0.5mg/kg; -, not reported
In this study, a commercial cultivar of wasabi in New Zealand was grown with
different organic and inorganic fertiliser treatments to evaluate the effect of soil fertilisation on
yield and quality (ITC concentration) of wasabi. The individual ITC (isopropyl, sec-butyl, allyl,
3-butenyl, 4-pentenyl and 5-hexenyl ITCs) and the total ITC concentrations from the sum of
individual components are presented here, only for the rhizomes the most used and valuable
part of wasabi. Detailed discussion of plant yield and AITC compositions in other plant tissues
have been presented elsewhere (Sultana et al., 2002a).
100
The objectives of this study were:
1) To compare the total ITC concentration quantified by two analytical methods (UV and
GCMS) on the same samples.
2) To examine how flavour components are affected by lime and manure treatments especially
when manure containing sulphur as an ingredient was used
3) Comparison of results from New Zealand grown wasabi with published reports.
MATERIALS AND METHODS
Location
‘Daruma’ the current commercial cultivar of wasabi grown in Canterbury was used in
this study. The wasabi was grown in a 1324 m2 shade house at the Lincoln research farm of
Crop & Food Research, Canterbury, New Zealand (43 39-S, 172 29-E, 11 m above sea level).
The soil type was a Templeton Silt Loam. The growth experiment followed a previous wasabi
crop described by Martin and Deo (2000). After that crop had been removed, the trial area was
fallowed over winter then ploughed on 16th October 1997.
Experimental design
A split plot design with four replications was used with four main plot treatments and
six sub plot treatments. Details about crop establishment, management, experimental design,
allyl isothiocyanate yield and concentration in the above ground tissues have been presented by
Sultana et al., (2002a).
Only the main plot treatments mentioned below are reported in this paper (Table 2):
Control (no lime or manure)
Lime (10 t CaCO3/ha)
Manure (poultry manure 30 t/ha, pig manure 400m3/ha)
Lime and manure (10 t CaCO3/ha, poultry manure 30 t/ha, pig manure 400 m3/ha)
The main plot treatments were applied on 29th October 1997. The plots were then
moulded into beds 1m wide and 0.3m high. Ten-week old seedlings were transplanted into the
beds on 25th November1997. The plots were irrigated, weeded and sprayed to ensure no other
limitation to growth. The trial was harvested in May 1999. Fifteen plants were harvested and
weighed. A 50-100 g sub sample of rhizomes from two plants was retained for ITC analysis.
Analytical methods
Two methods were used for analysis these were:
1) UV Spectrophotometer for total ITC concentration
2) Gas chromatography Mass Spectroscopy (GC-MS) for individual ITC and
total ITC concentration assay.
Method 1: UV method
Extraction
Frozen rhizome was homogenized in a domestic juicer and 2 g of pasted rhizome was
mixed with 20 g light paraffin oil. Samples were incubated at room temperature for one hour
with occasional shaking. The solids were removed by centrifugation and the paraffin oil extract
stored in a cold room (0°C) to assay for isothicyanates. The remainder of the wasabi paste was
used for dry matter analysis.
Conversion of ITC to thioureas

Extracted paraffin oil (6 g) was added with methanol (5 ml) and ammonia solution (1
ml) and incubated at 45°C with continual mixing by inversion for 5 hours. The isothiocynate is
converted to allyl thiourea by this process using a modification of the Doun and Hougen (1976)
method. After separation, an aliquot (0.05 ml) of the methanol phase was diluted to 4 ml with
methanol for measurement of UV absorbance by spectrophotometer. A standard curve was
established with allyl thiourea in methanol.
Calibration
Standards and extracts were measured against a methanol blank using a UV
absorbancespectrophotometer (set at 244 nm) for calculation of total ITC present in wasabi
rhizomes.
Method 2: GC-MS method

Partition
The stored paraffin oil extract (1ml) from Method 1 was partitioned into methanol
(1ml) by incubation at 20°C and continual inversion for 30 minutes.
Identification
A Hewlett Packard (HP) 6890 gas chromatograph fitted with flame ionisation detector
(FID) and a HP 6890 automatic injector was used with the following operating conditions:
Column: HP –INNOWax, 30 m, 0.25 mm internal diameter, 0.25 µm film thickness, capillary
column. Carrier gas: Hydrogen, 85 Kpa inlet pressure, flow rate 2.3 ml/min, linear velocity 57
cm/sec. Injection: Splitless mode, 2 µl of methanolic extract. Temperature (°C): Inlet 180°C,
detector 250°C, column 50 to 100°C at 5°C /min, 100 to 200°C at 10°C /min, holds at 200°C
for 2 minutes. Detection: Flame ionization detector. Peak areas were recorded and calculated
using HP chemstation software (version A.06.03).
Butyl and phenyl ITCs were used as external standards and demonstrated significant
differences in FID response. The levels of individual ITCs were quantified from the peak area
using a butyl ITC calibration curve. Isopropyl, sec-butyl, Allyl, 3-butenyl, 4-pentenyl, 5-
hexenyl ITCs were identified by GCMS on a Carlo Erba MFC500 GC and a Kratos MS80RFA
mass spectrometer. Other ITCs (e.g. isobutyl and 6-methyl thiohexyl ITCs) were tentatively
identified from wasabi extract by GC-MS but it was not possible to quantify these ITCs in these
samples. Total and individual ITC tissue concentrations are expressed as mg/kg and the total
concentration was calculated from sum of six ITCs.
Statistical Analysis
The effects of lime and manure treatments on the production of individual ITCs in
wasabi rhizomes were analysed with analysis of variance (ANOVA) and least significant
difference of means (LSD at 5%) using Genstat statistical package (Genstat 5, 2000).
Significant and non-significant responses are presented in Table 2 with associated P values to
give an indication of trends.
RESULTS
Different ITCs in wasabi

Six isothiocyanates were identified from wasabi rhizomes; isopropyl ITC, sec-butyl
ITC, Allyl ITC, 3-butenyl ITC, 4-pentenyl ITC and 5-hexenyl ITC. Mean concentration of each
ITC in the rhizomes are summarised in Table 2. Allyl ITC (AITC) ranged from 2136 to 2570
102
mg/kg of the fresh rhizomes and was the highest ITC among other ITCs mentioned here. AITC
contributed between 86 to 92% of the total ITC. 3-Butenyl ITC and 4-pentenyl ITC contributed
approximately 5% and 3% respectively of the total ITC concentration. Isopropyl, sec-butyl and
5-hexenyl ITCs contributed only a very small proportion to the total ITC level.
Table 2: Effect of treatments on isothiocyanates in wasabi.
Main plot treatments
P value
Control Lime Manure Lime and manure LSD
Composition of 0 kg Ca/ha 3640 kg 2.5 t N/ha 3640 kg ca/ha -

the fertilizer 0 kg N/ha Ca/ha 64 kg S/ha 2.5 t N/ha
treatments 0 kg S/ha 64 kg S/ha
ITC content of
the wasabi
rhizomes
Iso-propyl ITC 34.3 34.8 39.7 34.8 NS
(mg/kg)
Sec-butyl ITC 39.2 40.3 43.9 41.4 NS
(mg/kg)
Allyl ITC 2136.1b 2570.0a 2364.3ab 2180.9b 0.018
(mg/kg) 265.2
3-butenyl ITC 131.9 146.1 137.7 131.2 NS
(mg/kg)
4-Pentenyl ITC 76.9 72.1 74.7 70.4 NS
(mg/kg)
5-Hexenyl ITC 23.5 21.9 22.3 22.9 NS
(mg/kg)
Total ITC 2425 2810 2577 2551 NS
GC-MS method
(mg/kg)
Total ITC 2202b 2648a 2429ab 2094b 0.017
UV method 326.7
(mg/kg)
% Difference 10% 6% 6% 22%
between the
two methods
Mean values followed by the same letter are not significantly different at a LSD of 5%;
NS = not Significant
The effect of treatments on individual ITC concentrations

The lime treatment had a positive effect (P=0.018*), which resulted in a 20% increase
of AITC (2570 mg of AITC per kg of rhizome) relative to the control level. However addition
of lime did not significantly affect the other isothiocyanates, 3-butenyl ITC did show a
marginal increase (11%). The response to manure of ITCs was relatively minor though the soil
received a high proportion of organic N (2.5 t/hectare) and organic S (64 kg/hectare) from this
treatment. AITC concentration increased 11% with manure but this was not significant
(P<0.05). This treatment tended to increase the concentration of other ITCs e.g. isopropyl and
sec-butyl ITC concentrations in the rhizomes. Application of lime and manure together
(treatment 4) had no significant effect on ITC concentration.
Comparison of Total ITC concentrations by UV and GC methods

The total ITC concentration quantified by the GC method from the sum of individual
ITC concentrations showed a 6-22% higher value than when measured by the UV absorbance
spectrophotometer method from the same samples in this trial. However, the responses of the
treatments assayed by the two different methods were very similar. The lime treatment
increased total ITC concentration by 20% over the control value for both methods. The manure
treatment increased total ITC by 6% and 10% for the GC and UV methods respectively.
However lime and manure together showed a different response, showing a 5% increase by the
GC method but a negative response measured by the UV method.
Comparison of ITC concentration in wasabi rhizomes grown in New Zealand with Japan
The levels of ITCs found in this study from New Zealand grown wasabi rhizomes
were much higher for individual as well as total ITC concentrations than published results for
wasabi rhizomes grown and analysed in Japan. Comparing the mean value of each ITC from
published results (Table 1) with results from the manure (Table 2) and lime treatments gave 4
and 3.6 times higher concentrations respectively for isopropyl ITC. For other ITCs the results
found were 2.6 and 2.3 times higher for sec-butyl; 66% and 80% higher for allyl ITC; 1.4 and
1.6 times higher for 3-butenyl ITC; 66% and 60% higher for 4-pentenyl and 55%, 87% higher
for 5-hexenyl ITCs. Total Isothiocyante concentrations found in the New Zealand analyses also
showed higher values compared to the mean values of published data (Table 1) e.g. 55% and
69% (by the GC method) and 46% and 60% increase (by the UV method) of total ITC level
respectively from the manure and lime treatments.
DISCUSSION
AITC was the main flavour component due to having the highest content in wasabi. It
was the major contributor to the total ITC concentrations of the rhizomes. Since each ITC has a
unique odour characteristic (Table 1) minor ITCs are also important to consider in the overall
flavour profile of wasabi, which derives from the combined flavour characteristics of individual
ITCs. The increased level of total ITC resulting from calcium application indicated that wasabi
plants may do better soils with a higher pH or the lime may have increased S availability in
soil. The soil pH level was 5.9 to 6.4 before planting a little lower than recommended for
cruciferae (Wood et al., 1986). However, there was no significant total dry matter increase in
the rhizome due to the lime treatment. Sultana et al., (2002a) reported that there was no
significant effect of calcium on overall fresh yield or AITC yield in different plant parts for this
study but the fresh yield of rhizome was reduced by lime application which might cause an
increase in the AITC concentration. Wang et al., (1996; 1999) reported that application of
dolomite powde increased soil pH and the fresh weight yield of wasabi rhizomes.
104
In the manure treatments a higher proportion of organic N (2.5 t/hectare) than organic
S (64 kg/hectare) was applied from a combination of poultry and pig manure but the response
of ITC concentration was not significant. The reason for this change could be complex and it
may be that the soil pH was reduced compared to the control level and this may have made
organic sulphur more unavailable for plants. The 11% AITC response in this treatment is much
lower than the 52% increase in AITC obtained with the applicationof 228 kg inorganic S/ha and
200 kg N/ha as ammonium sulphate (Sultana et al., 2001a). Thus either the total amount of S
or ratio of S and N may need to be higher to get an increase in ITC tissue concentration. The
inorganic sulphur may also be more available than organic S and thereby more effectively used
by the plant to increase the concentration of flavour components (Follett et al., 1981).
The two methods of measuring ITCs gave slightly different values for total ITC. This
is acceptable when it is considered how the samples were stored and analysed. The individual
ITC assay by GC was performed after approximately a year storage of paraffin extract at 0 to -
4°C after the UV assay was done. Very small losses of ITC may have occurred during storage
of paraffin extract at 0 to -4°C but the GC results were always marginally higher than the
results obtained from the UV assay. It is also possible that alkyl isothiocyantes (isopropyl, sec-
butyl ITCs) may behave slightly different during storage than ω-alkenyl ITCs and this needs to
be investigated.
Differences in ITCs in published reports may result from a number of reasons e.g. soil
type, soil pH, application of fertiliser or other treatments, methods of analysis, rhizome size and
plant age etc. However, the higher levels of individual and total ITCs investigated in this study
from New Zealand grown material when compared with material grown in Japan indicates an
excellent potential for the production of high quality wasabi exists in New Zealand.
CONCLUSIONS
In this study six isothiocyanates were measured in wasabi rhizomes and among them
allyl ITC was the major component because of its highest concentration in the tissue. Since
each ITC has slightly different odour profiles (Ina et al., 1981; Fenwick et al., 1983; Masuda et
al., 1996) their contribution to the total ITC concentration is important to the unique of flavour
of wasabi. Overall the three main soil treatments had no significant positive effect on ITCs
concentration in wasabi. However, a small positive effect of lime on AITC concentration and
total ITC was observed but Sultana et al., (2001a) reported that though calcium increased the
concentration of AITC overall the effect of lime was not significant on plant yield and AITC
yield. The trial was not established to measure the effect of a single component like nitrogen,
sulphur or calcium from organic treatments because at least two of these components were
combined in the treatments. Thus, a further study is needed to determine the effects of nitrogen
and sulphur both alone and relative to each other with higher rates of S in manure on the
production of ITCs. Higher levels of ITCs from New Zealand grown wasabi indicated that it
was a high quality raw material for the food industry.
ACKNOWLEDGEMENTS
The authors wish to thank Dr Nigel Perry (Crop and Food Research, Dunedin) and
Bruce Clark (University of Canterbury) for their assistance in GCMS analysis of individual
ITCs in wasabi. The assistance of Bas Deo (Crop & Food Research, Lincoln) in growing
wasabi is greatly appreciated. Special Thanks to Jonathan Depree for his support in this writing
by providing the total ITC data measured by UV method.
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Nutritional composition of ulluco (Ullucus

Article · October 2000

Janette Busch Geoffrey Peter Savage

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Nutritional composition of ulluco (Ullucus tuberosus) tubers

J.M. BUSCH and G.P. SAVAGE

BACKGROUND AND HISTORY IN SOUTH AMERICA

Author(s) Moisture Protein Fat Carbohydrate Ash Fibre

Figure 1: Ulluco tubers and a small part of the trailing plant.

POTENTIAL USE IN NEW ZEALAND

Moisture Crude Total fat Total carbohydrate

Total dietary fibre

NUTRITIONAL CONTENT OF ULLUCO TUBERS

Moisture Protein Total fat Mucilage

Starch Free glucose Dietary fibre Vitamin C

U2 37.11 ± 1.95a 0.47 ± 0.13 34.75 ± 2.00 4.8

Minor constituents of ulluco

Raw skin and flesh

Cooked skin and flesh

Comparison with other staple crops grown in New Zealand

Anonymous, (1998). Andean tubers. Potato Research Program of the National

Effect of cooking on the soluble oxalate content of three

PERNILLA ALBIHN1 and G.P. SAVAGE2

MATERIALS AND METHODS

Sample collection and cooking

Dry matter determination

RESULTS AND DISCUSSION

Cultivar Raw Boiled Steamed Baked

Cultivar Raw Boiled Steamed Baked

Brinkley, L. MgGuire, J. Gregory, J. and Pak, C.Y.C. (1981). Bioavailability of oxalate in

Vitamin B12 - An Undervalued Vitamin

Vitamin B12 is not commonly thought of as causing deficiency as it is present

HISTORY OF VITAMIN B12

STRUCTURE OF VITAMIN B12

Figure 1: The structure of cobalamins (vitamin B12).

VITAMIN B12 COENZYMES

CH3CH2COOH Propionic acid

Homocysteine Methionine synthase Methionine

Deoxyuridylate Thymidylate Thymine-DNA

Glutamate FIGLU Methionine

THF: Tetrahydrofolate FIGLU: Formiminoglutamic acid

ABSORPTION, RETENTION AND ELIMINATION OF COBALAMINS

Absorption, retention and elimination of cbl is influenced by the interplay and

HC: Haptocorrin IF: Intrinsic Factor TC: Transcobalamin

ASSESSING VITAMIN B12 STATUS

Radioisotope dilution assay

SOURCES OF VITAMIN B12

DIETARY REQUIREMENTS OF VITAMIN B12

DEFICIENCY SIGNS AND SYMPTOMS

CAUSES OF VITAMIN B12 DEFICIENCY AND PEOPLE AT RISK

Conjugated linoleic acid (CLA)

A.K.H. MACGIBBON and A.M. ROWAN

Figure 1: Chemical structure of CLA isomers.

cis-9, trans-11 CLA

HEALTH BENEFITS OF CLA

The literature on purported health-promoting attributes of CLA is extensive and

Group CLA in diet Tumour Total no.

Control 0.1% 28/30 (93%) 92