Group Title: Agricultural research (Washington, D.C.)
Title: Agricultural research
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Title: Agricultural research
Uniform Title: Agricultural research (Washington, D.C.)
Physical Description: v. : ill. ; 25-28 cm.
Language: English
Creator: United States -- Science and Education Administration
United States -- Agricultural Research Administration
United States -- Agricultural Research Service
Publisher: Science and Education Administration, U.S. Dept. of Agriculture :
Science and Education Administration, U.S. Dept. of Agriculture :
Supt. of Docs., U.S. G.P.O., distributor
Place of Publication: Washington D.C
Publication Date: November 1996
Frequency: monthly[1989-]
bimonthly[ former jan./feb.-may/june 1953]
monthly[ former july 1953-198]
Subject: Agriculture -- Periodicals   ( lcsh )
Agriculture -- Research -- Periodicals   ( lcsh )
Agriculture -- Periodicals -- United States   ( lcsh )
Agriculture -- Research -- Periodicals -- United States   ( lcsh )
Genre: federal government publication   ( marcgt )
periodical   ( marcgt )
Statement of Responsibility: U.S. Department of Agriculture.
Dates or Sequential Designation: Began with vol. 1, no. 1 (Jan. 1953).
Issuing Body: Vols. for Jan./Feb.-Nov. 1953 issued by: Agricultural Research Administration; Dec. 1953-<Sept. 1976> by: Agricultural Research Service; <June 1979>-June 1981 by: the Science and Education Administration; July 1981- by: the Agricultural Research Service.
General Note: Description based on: Vol. 27, no. 7 (Jan. 1979).
General Note: Latest issue consulted: Vol. 46, no. 8 (Aug. 1998).
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Bibliographic ID: UF00074949
Volume ID: VID00002
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: ltuf - ABP6986
oclc - 01478561
alephbibnum - 000271150
lccn - agr53000137
issn - 0002-161X

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Pennies for Research
Can Cut Dollars
From Health Care

The annual outlay for treatment
and care of diseases linked to diet
exceeds $200 billion. For cardiovas-
cular disease alone, estimated costs
are more than $56 billion annually.
That's 1,000 times the USDA budget
for human nutrition research.
In spite of our ability to reduce
human suffering through dietary
changes, the federal investment in
human nutrition research has stag-
nated in real dollars since the early
1980's. I am working to change that.
Agricultural Research Service nutri-
tion program leaders have spent long
hours planning the agency's future
direction for research aimed at
reducing the toll of heart disease,
cancer, and other diseases of aging.
Nutrition scientists are only begin-
ning, for example, to understand the
relationship of plant foods, their phy-
tonutrients, and their role in health.
ARS has already taken a lead role
in studying one class of phyto-
nutrients-the carotenoids-measur-
ing and cataloging their levels in U.S.
foods and in the human body since the
early 1980's. Recent studies at ARS's
Western Human Nutrition Research
Center found that when women ate
too few fruits and vegetables contain-
ing beta carotene and other caro-
tenoids, they experienced adverse
hormone changes and tissue damage,
even though they got ample vitamin A
in a supplement.
Researchers at the Human Nutri-
tion Research Center on Aging at
Tufts University in Boston are collab-
orating with Harvard University
investigators to determine the effec-
tiveness of carotenoids in breast
cancer prevention. And, as you'll read
in this issue, research on volunteers at
the Beltsville (Maryland) Human

Nutrition Research Center indicates
that five reasonable-size servings of
carotenoid-rich foods daily can raise
levels of several carotenoids in blood
serum and colon cells and signifi-
cantly improve immune capacity.
In other studies at Beltsville,
researchers have discovered that two
carotenoids-lutein and zeaxan-
thin-are strong antioxidants. And
they are the only carotenoids found
in the retina of the human eye,
suggesting that they may protect
against a condition that leads to
blindness in many elderly people.
But carotenoids are only one class
of compounds among the more than
600 phytonutrients in fruits, vegeta-
bles, beans, grains, and seeds.
These compounds include a wide
range of chemical structures and
protect us by different modes of
action. In addition to acting as
antioxidants, they may boost the
immune system or encourage en-
zymes that detoxify carcinogens or
bind to excess estrogen that might
otherwise promote cancers in the
breast and other tissues.
Identifying individual phytonutri-
ents that are most beneficial to the
human body and determining their
modus operandi are major issues for
future study. But a group of research-
ers at the Boston center has taken a
different approach. Using a sensitive
chemical assay they developed, they
measured the total antioxidant
capacity of whole foods. It turned out
that the most potent foods have
dozens to hundreds of active com-
pounds, many belonging to another
class of pigments known as fla-
vonoids. Animal studies are now in
progress to see if this potency in the
laboratory translates to antioxidant
protection in living systems.
With such a vast array of phytonu-
trients, many of which are still
unknown, it becomes obvious why it

is so important to eat a variety of
plant foods rather than rely on supple-
ments. ARS research will continue to
identify the most beneficial com-
pounds, how they function and how
to measure that function, how much
we need for optimal protection, what
combinations of foods enhance their
absorption by the human body, and
how this bioavailability is affected by
cooking or storage.
As more of this information
becomes available, other sectors of
ARS can increase the amounts of
phytonutrients we consume. One way
is through plant breeding: We've
already produced varieties of toma-
toes, sweetpotatoes, corn, carrots, and
cantaloupes with increased total
carotene content. Our scientists have
also identified carotene-containing
germplasm for vegetables that typi-
cally contain no carotene, including
cauliflower, cucumbers, and potatoes.
Soil nutrient levels and availability
play a role in phytonutrient levels.
Food processing is another potential
route for increasing intake-either by
adding phytonutrients as food ingredi-
ents or by preventing their loss. And
postharvest research can find ways of
maintaining high levels through
shipping and storage.
With extensive research in all these
areas, ARS researchers are in a
pivotal position to conduct, collabo-
rate in, and coordinate studies aimed
at improving the nutritional content of
our food supply. In the agricultural
sector, history has shown that dollars
spent on ARS research have multi-
plied in productivity and products. I
have no doubt that we can continue
our record and help cut health-care
costs through adequately funded
research into phytonutrients and
nutrition in general.

Floyd P. Horn
ARS Administrator

Agricultural Research/November 1996

November 1996
Vol. 44, No. 11
ISSN 0002-161X

Agricultural Research is published monthly by
the Agricultural Research Service, U.S.
Department of Agriculture, Washington, DC
The Secretary of Agriculture has determined
that this periodical is necessary in the transac-
tion of public business required by law.
Dan Glickman, Secretary
U.S. Department of Agriculture
Catherine Woteki, Acting Under Secretary
Research, Education, and Economics
Floyd P. Horn, Administrator
Agricultural Research Service
Robert W. Norton, Director
Information Staff
Editor: Lloyd McLaughlin (301) 344-2514
Assoc. Editor: Linda McElreath (301) 344-2536
Art Director: William Johnson (301) 344-2561
Photo Editor: John Kucharski (301) 344-2900
Assoc. Photo Ed.: Anita Daniels (301) 344-2956
Information in this magazine is public property
and may be reprinted without permission. Non-
copyrighted photos are available to mass media
in color transparencies. Order by photo number
and date of magazine issue.
Subscription requests should be placed with
New Orders, Superintendent of Documents,
P.O. Box 371954, Pittsburgh, PA 15250-7954.
See back cover for order form.
Complimentary I-year subscriptions are
available to public libraries, schools, employees
of the U.S. Department of Agriculture, and the
news media. Send requests or comments to:
Editor, Agricultural Research Magazine, Room
408, 6303 Ivy Lane, Greenbelt, MD 20770. E-
To visit Agricultural Research magazine on the
Internet, go to and select
News and Information.
This magazine may report research involving
pesticides. It does not contain recommendations
for their use nor does it imply that uses dis-
cussed herein have been registered. All uses of
pesticides must be registered by appropriate
state and/or federal agencies before they can be
Reference to any commercial product or service
is made with the understanding that no discrimi-
nation is intended and no endorsement by the
U.S. Department of Agriculture is implied.
USDA prohibits discrimination in its programs
on the basis of race, color, national origin, sex,
religion, age, disability, political beliefs, and
marital or familial status. (Not all prohibited
bases apply to all programs.) Persons with
disabilities who require alternative means for
communication of program information
(Braille, large print, audiotape, etc.) should
contact the USDA Office of Communications
at (202) 720-2791. To file a complaint, write
the Secretary of Agriculture, U.S. Department
of Agriculture, Washington, DC 20250, or call
(202) 720-7327 (voice) or (202) 720-1127
(TDD). USDA is an equal employment
opportunity employer.

Agricultural Research/November 1996

Agricultural Research

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Cover: Researchers are discovering that the
hundreds of colorful pigments in fruits and
vegetables provide crucial nutrition in the human
diet. Photo by Scott Bauer. (K7246-1)


Plant Pigments

Paint a Rainbow ofAntioxidants

.; : or millions of years, insects
I:) have relied on flower color to
find the right food source.
Now nutrition researchers are discov-
/,' ering that the rainbow of color
pigments in fruits and vegetables
may do more than simply attract
attention or please the eye.
The orange pigment, beta caro-
tene-best known of the plant color
compounds-first caught researchers'
eyes when population studies linked
low rates of certain cancers with a
high intake of fruits and vegetables
containing lots of beta carotene.
A A current theory holds that cancer,
heart disease, stroke, and other dis-
eases of aging result from cumulative
damage to cells by free radicals-
most of which our cells generate
through ordinary metabolism. So nu-
trition and medical researchers are
dissecting the fruits and vegetables
consumed by healthy populations,
looking for the best combinations to
prevent such damage.
What they are finding is that fruits
and vegetables contain hundreds of
other pigments besides beta caro-
.: tene-as well as nonpigment com-
pounds-that may play a role in
preventing oxidative damage.
Studies to date suggest certain
plant chemicals, or phytochemicals,
appear to maintain health by boosting
the immune system, reducing inflam-
mation and allergies, detoxifying
contaminants and pollutants, and/or
activating enzymes that block unbri-
died cell division.
Fred Khachik, a chemist with
ARS, points out that the diets of most
Using high-performance liquid chromatography, chemist Frederick Khachik analyzes an people supply more of two other
extract of human blood for dietary carotenoids and their metabolites. (K7496-1) carotenoids-lutein and lycopene-
than they do beta carotene.

Agricultural Research/November 1996

"The results from epidemiological
and human studies can be better
interpreted if the bioavailability,
metabolism, and potential cancer-
preventive effects of these other
major dietary carotenoids are also
studied," he says.
And that's what Khachik has been
doing for the past 13 years. He and
colleague Gary Beecher, who heads
food composition research at ARS'
Beltsville (Maryland) Human Nutri-
tion Research Center (BHNRC), have
tailored several analytical processes
to search for carotenoids in human
blood, tissue, and breast milk, as well
as in foods.
Of the 50 or 60 carotenoids in
foods, 7 had been found in blood
serum and breast milk before
Khachik began his studies. He
identified eight more. Presumably,
they have some useful function or the
body wouldn't expend the effort to
selectively absorb them, he says.
In addition, Khachik has found
eight metabolic byproducts, or
metabolites, made by three of these
carotenoids. Most of these metabo-
lites are oxidized forms of lutein and
its alter ego, zeaxanthin, which is in
foods in much lower concentrations.
Two are oxidized forms of lycopene.
"We know these oxidized metabo-
lites aren't in the foods," he says.
"And we're not creating them in the
The fact that they are in blood
suggests that lutein and lycopene
behave as antioxidants, counteracting
the assaults of free radicals and thus
sparing crucial biological chemicals
from damage. And there's some
evidence that carotenoids and their
metabolites activate enzymes in-
volved in detoxifying contaminants

Agricultural Research/November 1996

or blocking the unrestrained increase
in cancer cells, Khachik adds.
What's more, lutein may help
prevent the leading cause of blind-
ness in the elderly-macular degen-
eration-which results from long-
term deterioration of the center of the
retina, known as the macula.
Khachik says that a study reported
in 1994 by Harvard researchers found
that people with the highest intake of
fruits and vegetables, especially those
rich in lutein, had half the risk of
macular degeneration as those with
the lowest intake. In his own analysis
of extracts from human retinas, he
found lutein, zeaxanthin, and several
of their oxidized metabolites-but no
other carotenoids.



Research technician John McEwen
prepares fruits and vegetables for the
chromatographic separation of antioxidant
components. (K7495-2)

Lutein-along with several other
carotenes-gives summer squash,
apricots, peaches, and oranges their
yellow to light-orange color. But its
richest sources are kale, spinach, and
collard greens, in which the yellow
pigment is masked by the more
abundant green chlorophyll pigments.
Lycopene gives tomatoes their
bright red hue and invariably stains
our clothes when we indulge in a
plate of spaghetti. High intakes may
protect against prostate cancer. In a
study of men 40 years and older,
those who consumed more than 10
servings of tomato products per week
had two-thirds the risk of prostate
cancer as those who ate less than 1.5
servings weekly.
"Lutein and lycopene possess
exceptionally high antioxidant
capacity compared to other caro-
tenoids and may be useful in prevent-
ing cancer," says Khachik.

How Much Gets in Us?
Khachik has shown that blood
levels of lutein go up when people
take pure lutein supplements. But
how much do they rise when the
digestive tract has to sort through the
fiber and thousands of other com-
pounds found in carotenoid-rich
foods? That question inspired the
formation of the BHNRC Caro-
tenoids Research Unit headed by
nutritionist Bev Clevidence.
In a recent study, she and 11
colleagues lunched on kale, tomato
juice, and sweetpotatoes every day
for 3 weeks to test how well lutein,
lycopene, and beta carotene are
absorbed from foods rich in these
antioxidants and to look at some of
their effects in the body.


They looked for changes in both
oxidative damage to blood lipids and
DNA and the immune system's
ability to mobilize its defenses.
"We wanted to know if people
could get enough of these
carotenoids from realistic food
portions to have any benefit," says
In a study of 1,300 elderly people
living in Massachusetts, she notes,
the subjects who reported the highest
intakes of dark-green and deep-
yellow vegetables were half as likely
to die from cardiovascular disease 5
years later and one-third as likely to
die from cancer as those with the
lowest intakes.
"The highest intakes averaged
slightly more than two servings
daily, and the lowest intakes aver-
aged less than one," she says. "So
adding two more servings of these
dark-green and yellow vegetables
each day may confer significant
health benefits."
By selecting vegetables rich in
lutein (kale), lycopene (tomato
juice), and beta carotene (sweetpota-
to), the Beltsville researchers man-
aged to consume 20 milligrams (mg)
of each carotenoid, or 60 mg total.
"This is a tenfold increase over
typical U.S. carotenoid intakes,"
says Clevidence, "and it was
achieved with very tolerable serv-
ings." Lunch consisted of less than 5
ounces of steamed kale, about 5.5
ounces of mashed sweetpotato, and 6
to 8 fluid ounces of tomato juice.
Beta carotene proved to be the
most bioavailable, increasing 116
percent in blood plasma during the
treatment period. Lutein increased
67 percent, on average, but blood
levels were quite variable among the
12 volunteers. Lycopene had the
lowest bioavailability, with a 26-
percent increase. Khachik also saw a
small increase in the oxidized
metabolites of lutein and lycopene in

Vietitian Evelyn Lashley (left) and food
service worker Sara Mayock weigh kale,
tomato juice, and sweetpotatoes for a
carotenoid study with human volunteers.

blood, indicating that the carotenoids
were taking hits from free radicals.
A close inspection of colon cells
by Beltsville colleague Padmanabhan
Nair suggests that the bulk of these
carotenoids may be providing
protection within cells, rather than
circulating in the plasma. He found
huge increases ranging from 1,000
percent for lycopene to 4,000 percent
for beta carotene.
Despite these increases, the re-
searchers found no change in oxida-
tion of blood lipids, suggesting that
protection may be occurring in tis-
sues rather than blood. And they saw
no more protection of DNA, based on
breakdown products in the urine.
They did, however, find a signifi-
cant improvement in immune func-
tion. Immunologist Tim Kramer, also
a study volunteer, looked at the
ability of T-cells to divide when
stimulated with a mitogen. Compared

to a prestudy evaluation, T-cell
proliferation increased by about one-
third at the time the researcher-
volunteers stopped consuming the
carotenoid-rich vegetables, he says,
and stayed at that level for 3 weeks
before beginning to drop.
T-cell proliferation is a good
measure of the overall function of the
immune system, he explains, because
the cells act as a kind of player
coach. They not only attack invading
organisms, they also coordinate the
activity of other immune cells.
Adult Americans average 1.2
servings of fruit each day and 3.1
servings of vegetables-according to
USDA's 1989-91 nationwide food
consumption survey-with the dark-
green and yellow vegetables eaten
much less frequently. Bananas and
white potatoes are the number-one
fruit and vegetable, and both are low
in carotenoids, says Clevidence.
"Increasing consumption of fruits
and vegetables is an effective strate-
gy for preventing diseases of aging,"
she says. "But, for the greatest health
benefits, we need to identify those
that provide optimum protection and
emphasize their consumption."

The Fruit, the Whole Fruit, and
Nothing but...
Another group of ARS researchers
is taking a different approach to
assaying protective effects of fruits
and vegetables. Led by Ron Prior at
the Jean Mayer USDA Human
Nutrition Research Center on Aging
at Tufts in Boston, Massachusetts,
this group is finding that blueberries,
strawberries, red bell peppers,
Concord grapes, and beets-along
with several deep-green vegetables-
are workhorses at disarming free
radicals, at least in the test tube.
Recent large clinical trials have
focused on specific antioxidant
compounds in fruits and vegetables,
such as beta carotene, vitamin C, or

Agricultural Research/November 1996

Scientific program officer Ronald L. Prior
(left) and visiting scientist Emin Sofic
evaluate antioxidant components of
strawberries. (K7495-1)

The ORAC Assay Automated
Several methods have recently been developed to measure the total antioxi-
dant capacity of biological samples. But the ORAC (Oxygen Radical Absor-
bance Capacity) method is unique, says developer Guohua Cao.
ORAC measures the degree to which a sample inhibits the action of an
oxidizing agent and how long it takes to do so. Then it integrates the two
measurements into a single one. This provides an accurate and reproducible
measurement for different types of antioxidants having different strengths.
The assay combines a chemical marker that fluoresces with the te.t sample
and an oxidizing agent, such as the peroxyl radical, the hydroxyl radical, or
metal ions. The test sample can be a biological sample, such as blood serum,
tissue, or a food extract; or a chemical sample, such as \ vitamin E.
As long as the antioxidant in the test sample disarms the free radicals, the
marker stays intact and continues to fluoresce. A detector in the analyzer
measures the strength of the light emitted from the marker. As the antioxi-
dants become spent, more and more of the marker is destroyed, and fluores-
cence eventually drops to zero. Unlike other methods, ORAC accounts for all
inhibition-from the beginning of fluorescence, to zero.
In 1994, Cao brought his method to the Boston center to work with Ron
Prior. But it w as time-consuming, particularly for the analyses of large
numbers of samples. So they automated the ORAC assay bN adapting it to
work in a COBAS FARA II analyzer and linking the anal zer to a computer
to store the data on a hard disk. The anal) zer uses robotics to precisely add
the samples and control the reaction timing.
Now, the researchers can determine the total antioxidant capacity of a
dozen samples in little more than an hour. ORAC. as no\w automated, is a
relatively simple but sensitive technique that affords nutritionists, epidemiolo-
gists, and clinicians a reliable and convenient tool for studying the role of
oxygen free radicals in disease processes or for assessing the protective
effects of antioxidants.
However, the COBAS FARA II analyzer is no longer available, so the
researchers are working with other equipment manufacturers to develop an
instrument suitable for wide applications-and one that can analyze more
samples at the same time.-By Judy McBride, ARS.

Hlgn-pertormance liquni
chromatographs-like this one, for kale-
show the wide range of potential
antioxidants in selected fruits and
vegetables. Each needlelike peak
represents a compound that can be
oxidized and reduced. Foods with many
peaks score high in total antioxidant
capacity. (K7496-2)

Test foods, like this frozen kale being checked by nutritionist Beverly Clevidence, were
selected from single lots to ensure consistent amounts of carotenoids. (K7496-4)

Agricultural Research/November 1996


vitamin E. But "fruits and vegetables
have many other antioxidant com-
pounds," says Prior. "Much of the
protection they confer against
cancer, heart disease, and stroke may
be from compounds other than these
So he and colleagues Guohua
Cao, Hong Wang, and Emin Sofic
are determining the total antioxidant
capacity of the fruit or vegetable
itself, using a chemical assay devel-
oped by Cao (see page 7). And
they're finding that a large group of
usually colorful compounds, some of
which are flavonoids, may be
responsible for much of the antioxi-
dant protection against peroxyl
radicals produced in the test tube.
The peroxyl radical is an oxidiz-
ing agent akin to the type our cells
generate day in and day out during
ordinary metabolism, says Cao, who
is a chemist and medical doctor.
Prior's group ran the assay for
total antioxidant capacity on more
than a dozen commonly eaten fruits,
five fruit juices, and more than a
score of vegetables and other foods
that they bought from Boston area
supermarkets on three different
occasions. They also tested green
and black teas. Blueberries, straw-
berries, and Concord grape juice
scored highest among the fruits and
juices tested.
Three and one-half ounces of
blueberries-about two-thirds of a
cup worth-disarmed as many
peroxyl radicals as 1,773 Internation-
al Units (IU) of vitamin E or 1,270
mg of vitamin C did. Strawberries
were more than half as potent as
blueberries. Plums scored third,
having nearly two-thirds the total
antioxidant capacity of strawberries.
Among the 22 vegetables assayed,
kale led the bunch with a total anti-
oxidant capacity a little above that of
strawberries, followed by spinach
with about the same potency as

To determine total antioxidant activity,
visiting scientist Guohua Cao loads
extracts of fruit and vegetable samples
onto an automated centrifugal analyzer.

plums. Three and one-half ounces of
kale-an easily edible portion--dis-
armed as many free radicals as 837
IU of vitamin E or 599 mg of vitamin
C did. Total antioxidant capacity of
kale was about twice as potent as
beets and broccoli flowers, 8 to 9
times more potent than carrots and
string beans, and 29 to 35 times more
potent than celery and cucumber.
Cao says that garlic tested higher
in antioxidant capacity than fresh
kale because it has so little water.
But this favorite of herbal medicine
dropped to the low end of the pack
when the vegetables were compared
on a dry-weight basis. And few of us
but the most stouthearted can eat 3.5
ounces of garlic at a sitting.
The Boston researchers also tested
strawberries and all the vegetables
against two other oxidizing agents
generated via normal metabolism-
the hydroxyl radical and copper ions.

In earlier studies, Cao had found
that vitamins C and E can actually be
turncoats while in the company of
transition metals-such as copper or
iron ions-and become oxidizing
agents themselves. That did not
happen when the whole fruit or
vegetable extract was pitted against
copper ions, says Cao.
"Whole fruits and vegetables
contain a mixture of natural antioxi-
dants that can protect the vulnerable
Which of these compounds get
absorbed by the body and increase
protection from oxidative stress or
improve health in some other fash-
ion? The researchers are currently
conducting studies on rodents to get
some preliminary answers.
"If the animal studies produce
positive results," says Prior, "it gives
us a measure of the quality of foods
other than the common nutrients bio-
medical science has been looking at."
Future research will evaluate the
potential of this method to add to the
understanding of how phytochemi-
cals convey protective effects.-By
Judy McBride, ARS.
Frederick Khachik, Beverly A.
Clevidence, Tim R. Kramer, and
Padmanabhan Nair are at the
USDA-ARS Beltsville Human Nutri-
tion Research Center, 10300 Balti-
more Ave., Beltsville, MD 20705-
2350; Khachik phone (301) 504-
8830, fax (301) 504-8314; Clevi-
dence phone (301) 504-8367, fax
(301) 504-9098; Kramer and Nair
phone (301) 504-8351, fax (301)
Ronald L. Prior and Guohua Cao
are at the Jean Mayer USDA Human
Nutrition Research Center on Aging
at Tufts University, 711 Washington
St., Boston, MA 02111; Prior phone
(617) 556-3310; Cao phone (617)
556-3141; fax for both (617) 556-
3299. *

Agricultural Research/November 1996


S satisfying both
your sweet tooth and
your nutritional goals D
could soon be easier than
ever. That's because
ARS scientists have de-
veloped a way to add
more fruit to confections,
baked goods, and frozen
"Many candies and
baking ingredients already
include some fruit, but they
often contain mostly sugar. B\
creating products with fruit as
the main ingredient, consumers
can get a more nutritious end
product," says ARS research tood
technologist Tara H. McHugh
Fruit growers could also benefit
from an expanded market for frultI
like peaches, pears, and apricot,. ,he
says. And the fresh fruit could be
pureed during the growing sedaon
and then processed year-round.
McHugh and colleagues combine
fruit puree with unique gelling agents
and then form the mixture into shapes
using molds or twin-screw extruders.
So far, they've included up to 30 per-
cent fruit in the molded pieces, with
moisture, starch, and sugar making
up the other ingredients. With the ex-
trusion process, the product can be
made entirely of fruit puree.
Agricultural engineer Charles C.
Huxsoll and technicians Julie Hsu
and Joseph Carlos also work on the
project. All are in the ARS Process
Chemistry and Engineering Unit at
the Western Regional Research
Center in Albany, California.
"Restructuring fruit by using
gelling agents with twin-screw
extrusion is new, and it may take the
industry process to a new level of
sophistication," says Huxsoll. The
gelling agents, which include new
combinations of starch and gelatin,
make the texture of the fruit mixture
adaptable to a wide variety of uses.

Agricultural Research/November 1996

Molded and
e.iruded fruil
puree. can enhance
the nutritional
quality of candie..
buked goods. and
Iroien treats.
I k7493-1 I


ine on the ..
inLrcdicnts used ..
with the puree, textures
could range from soft and
chewy to hard and crunchy.
The puree mixture can be poured
into molds to make virtually any
shape. The twin-screw extruder com-
bines the ingredients and pushes the
mixture through a die. The long rope
of fruit product is then cut into pieces
such as stars, squares, or circles, de-
pending on the shape of the die.
"Restructured fruit pieces that
would be developed by confectioners
into candy alternatives would require
different functional properties than
pieces to be incorporated into baked
or frozen foods. By using different
gelling agents, the properties can be
developed to meet the needs of these
varied applications," says McHugh.
One potential limiting factor to
molding the puree mixture is its
"If the viscosity of the fruit
mixture is too high, it won't flow
smoothly into the mold. The resulting

pieces would be
irregularly shaped and less appealing
to consumers," says McHugh.
The extrusion process overcomes
this limitation, because the mixture is
blended and shaped within the
extruder and doesn't require pouring.
It's also faster, taking only minutes to
produce the final product. Molded
pieces may take about 1 day to set.
Researchers are ready to move the
technology from the lab to commer-
cial applications. The confectionery,
or candy, industry would likely be the
first and largest user.-By Kathryn
Barry Stelljes, ARS
Tara H. McHugh, Charles C.
Huxsoll, and other scientists named
are in the USDA-ARS Process
Chemistry and Engineering Research
Unit, Western Regional Research
Center, 800 Buchanan St., Albany,
CA 94710; phone (510) 559-5864,
fax (510) 559-5777, e-mail *

Rings shown on the plastic plate examined by ARS chemist Betty Burri reveal the amount
of a vitamin-A-related protein in blood samples from volunteers. (K5755-3)

What Carotenes Do for You

C arotenes-members of the
family of orange, yellow,
and red pigments-may be
essential nutrients that we require for
the best health, two preliminary ARS
studies suggest.
"Right now, carotenes aren't
among the list of 23 vitamins and
minerals that are known to be essen-
tial," says Betty J. Burri, a chemist at
the ARS Western Human Nutrition
Research Center in San Francisco,
Neither are the many other caro-
tenoids that make carrots a vivid
orange, turn squashes a colorful
yellow, or tint tomatoes an attractive
"It might come as a surprise to
most people," says Burri, "that even
beta carotene, perhaps the best known
of the carotenes, doesn't have its own
RDA, or Recommended Dietary

Allowance. The only proven func-
tion for carotenes is as precursors-
called provitamins-to vitamin A."
To find more about the interrela-
tion of carotenes and vitamin A,
Burri conducted two experiments,
each with about a dozen healthy
women aged 18 to 42. She designed
the tests to explore the question, "If
people eat adequate amounts of
vitamin A, do they need to eat
She explains, "If the only ac-
knowledged role of carotenes is to
serve as precursors of vitamin A,
then, in theory at least, if you have
enough vitamin A, you don't need
the carotenes."
Our bodies require vitamin A for
good eyesight, proper growth and
reproduction, and prevention of
some diseases.
Burri and coworkers are now

publishing results of the second
study, completed in 1994. Many of
the findings agree with those of the
first experiment, which ended in
1992. Today, both experiments stand
as the longest and most rigidly
controlled beta carotene studies done
with human volunteers.
Burri's volunteers lived at the
nutrition center for 14 to 17 weeks,
so scientists and other staffers could
control all food and exercise.
Both studies tracked many key
indicators of general health. In each
experiment, volunteers ate meals that
featured familiar foods-but were
low in carotenes and other caro-
tenoids. In one test, all volunteers ate
only meals low in beta carotene.
They followed this stint with a
repletion phase in which they
downed beta carotene supplements in
addition to low-carotene meals.
In the other test, all volunteers ate
low-carotene meals. Some took
carotene supplements throughout the
study; the others added supplements
only during the repletion phase.
Importantly, all meals provided an
ample supply of preformed vitamin A
from such sources as beef, chicken,
turkey, pork, and fish. One day's
menu, for example, included bagels,
cream cheese, and low-fat milk at
breakfast; pork chow mein, apple
juice, and macaroon cookies for
lunch; and beef-potato casserole,
pasta salad, and grape juice for
dinner, with vanilla ice cream as a
snack before bedtime. Meals exclud-
ed all colorful fruits and veggies.
The low-carotene stints may have
been the culprit behind two kinds of
symptoms noted in some volunteers
in both studies: changes in the level
of thyroid hormones and an increase
in oxidative damage. Too, the low-
carotene regimen may have been a
contributing factor to disrupted
menstrual cycles.

Agricultural Research/November 1996

Burri says she and colleague Zisca
R. Dixon checked levels of thyroid
hormones because the linkages
between vitamin A and thyroid
hormones are well established. "But
links are less well-established," Burri
explains, "between thyroid hormones
and carotenoids. Our results suggest
that low-carotene regimens may
stimulate increases in a key thyroid
hormone, thyroxine."
Some-but not all-carotenes can
protect the body from oxygen dam-
age. "Oxidation," Burri says, "leads
to the release of rogue electrons,
called free radicals, that have been
implicated in increased incidence of
cancer, stroke, arteriosclerosis, and
cataracts. Many carotenes act as
antioxidants in lab tests, but more
evidence is needed on how important
they are to human health."
The researchers found that several
indicators of oxidative damage were
higher during depletion than reple-
tion. Concentrations of compounds in
the breath and blood known as
carbonyls, for instance, were about
50 percent higher during the low-
carotene phases. An indicator of
oxidative damage called thiobarbitu-
ric acid reactive substances-or
TBARS-increased in plasma during
depletion by about four times. And
the activity of superoxide dismutase,
a well-known antioxidant, decreased
by about 30 percent during depletion.
In both tests, most of the women
also developed abnormal menstrual
cycles. However, most participants
reported normal cycles within 3
months after leaving the center.
Physiologist Mary J. Kretsch at the
center led this part of the studies and
a series of four shorter tests that
tracked effects of low-carotenoid
meals on menstrual cycles of 35
healthy women.
She says her findings suggest that
regimens low in carotenes and other

Agricultural Research/November 1996

carotenoids may cause menstrual
cycle dysfunction. Further, supple-
menting the women with 95 retinol
equivalents a day of beta carotene-
about the same amount as in 2/3 cup
green beans-wasn't enough to
ensure normal ovulatory function.
"Perhaps a higher amount of beta
carotene," she says, "is required to
prevent these menstrual cycle abnor-
malities. Or, other carotenoids or
other compounds found in highly


For studies to determine the importance of
carotene, certified nursing assistant
Rowena Mallari (left) samples a low-
carotene lunch served by dietitian Doris
DeLeon. Colorful, high-carotene foods are
in the foreground. (K5758-1)

pigmented fruits and vegetables
might be needed."
Results from the carotene studies,
says Burri, "suggest that we need
carotenes-at least in small
amounts-not only as vitamin A
precursors but also as antioxidants.
"There's some evidence from
epidemiological studies that car-

otenes may have important functions
beyond serving as vitamin A precur-
sors, particularly as protectors against
oxidative damage. If carotenes turn
out to be important to human nutri-
tion in their own right, their ability to
defend the body against oxidative
damage will likely be the most
convincing factor.
"For now," Burri says, "we have
important new facts about carotenes
from our studies, but we still don't
know what amount of carotenes is
needed for optimum health. Nor do
we know which carotenes are best for
us. However, if our findings hold up
in future studies at our lab and
elsewhere, we will have established
that carotenes are required for
optimal human health.
"Meanwhile, the experiments are a
reminder of how important it is to eat
vegetables-even some of the less
popular ones-that are good sources
of carotenes."
Those veggies include carrots,
sweetpotatoes, spinach, collards,
parsley, kale, pumpkin, mustard
greens, beet greens, and winter
squash. Several fruits are also good
sources, such as apricots, canteloupe,
and mangoes.
In addition to ARS colleagues at
San Francisco and the Beltsville
(Maryland) Human Nutrition Re-
search Center, Burri collaborated
with researchers from the University
of California at Davis and Los
Angeles, University of Illinois at
Champaign-Urbana, Centers for
Disease Control and Prevention,
University of Nevada at Reno,
University of Connecticut, and
Florida International University.-By
Marcia Wood, ARS.
Betty J. Burri is at the USDA-ARS
Western Human Nutrition Research
Center, P.O. Box 29997, San Fran-
cisco, CA 94129; phone (415) 556-
6285, fax (415) 556-1432. *

Ky Be an, `` .



When Brad Morris walks
into the seed storage room
at his U.S. Department of
Agriculture lab, he often wonders
what pharmaceutical secrets may be
hidden inside the velvet beans, jack
beans, and winged beans tucked
away in paper bags in the 40"F air.
Those are only a few of the
legumes waiting for outside research-
ers to take a peek at their potential as
future sources of drugs and other
medicines, says Morris.
"I see the collection as an un-
opened medicine chest," he says.
Morris is curator of the special-
purpose legume collection main-
tained by USDA's Agricultural
Research Service in Griffin, Georgia.
Curators often develop an attachment
to their collections, and Morris is no
exception. He likes to talk about the
potential of his offbeat, largely
unknown assortment of legumes you
never heard of-at least not yet.
Morris notes that legumes are the
third-largest family of flowering
plants, with an estimated 20,000
known species worldwide. But only a
handful-peanuts, soybeans, peas,
lentils, beans, alfalfa, clover, and
vetch, for example-are grown on a
wide scale. Many of the rest are in
sealed bags in a cold-storage phar-
macy, so to speak.
The Griffin collection contains
more than 4,000 accessions. Winged
bean, jack bean, and velvet bean (also
called cowitch) sound odd enough-
but how about snout bean, ringworm
bush, and fish poison bean?
"Most of the accessions are from
tropical countries, which is one
reason why they aren't as well known
in this country," Morris says. "But
many of these legumes are valuable
sources of everything from insecti-
cides to brain chemicals such as
dopamine and serotonin."
Take, for example, the winged
bean, Psophocarpus tetragonolobus,

Agricultural Research/November 1996

a plant with high levels of proteins
called lectins, which medical re-
searchers use as diagnostic tools
because they bind to certain blood
cells and specialized transport cells.
Mich Hein, a scientist at Scripps
Research Institute in La Jolla,
California, was looking for a source
of winged beans for his research to
determine how vaccines might be
delivered in edible foods. The Griffin
collection had the winged beans he
was looking for.
Once he received the seed, Hein
grew the plants in greenhouses and
within several months tested them for
lectins. He found several that pro-
duced high levels of a specific lectin
he wanted.
In his lab studies, Hein fed the
lectin to mice and
found that it binds SCOTT BAUER
to M-cells in the
animal's small
intestine. M-cells
are "lookout" cells
that sample pro-
teins in the gut and
then transport them
to the immune
may then send out
antibodies in
response. Hein says
the winged bean
lectin, when fed to
mice, stimulates
their immune
system to produce At Griffin, Georgi
antibodies that Desmodium being
recognize the other researchers.
reclamation and s
lectin-the same
response that a
vaccine elicits.
"We're trying to determine if plant
lectins cause a similar response in
humans," Hein says. "The winged
bean collection has been important in
our preliminary studies, which we
hope will allow us to design edible
vaccines in the future."

Morris notes that winged beans,
aside from being a source of lectin,
also contain erucic acid (an antitumor
medication) and polyunsaturated fatty
acids that can be used to treat acne
and eczema.
Another special-purpose legume
that has drawn interest is kudzu,
Pueraria montana var. lobata. The
plant was originally brought into the
United States from Asia as a cattle
feed and ground cover. Now kudzu is
considered a noxious weed in certain
states, having overgrown many a
roadside landscape.
Michael Byrne, president of
Propagation Technologies, based
near Kalamazoo, Michigan, ordered
kudzu seed from Morris last year
after receiving permission from

a, agronomist Braa Morris inspects
grown to regenerate seed for distribution to
Desmodium is a legume used for land
oil conservation. (K7488-1)

Michigan authorities to grow the
weed in the greenhouse to evaluate
its pharmaceutical potential.
Byrne says he saw a television re-
port that the plant may contain chem-
icals that could help treat alcoholism.
The roots are used as a food starch/

thickener, so supposedly it was
through the use of the root as food
that the reduction of alcohol craving
was first noticed as a side effect.
In the greenhouse, Byrne is
growing out kudzu seeds from
Griffin and other
sources to evaluate
their potential. SCOTT BAUER
"The active
ingredient could be
found in the
highest concentra-
tion in the roots,
seeds, or leaves.
We don't know
yet," Byrne says.
"But we think it's
worth investigat-
ing. There are a lot
of fairly innocu-
ous-looking plants
around that can do
some amazing
Morris says
kudzu, which
should be studied
in indoor contain-
ment facilities in
areas where it's Agronomist Brad
considered a weed, technician Geral
is a source of a field observation:
number of chemi- valuable genetic
breeders and scie
cals including (K7486-1)
daidzein (an anti-
inflammatory and
daidzin (a cancer preventive), genis-
tein (an antileukemic), and others.
Norberta Schoene, a chemist with
ARS' Beltsville (Maryland) Human
Nutrition Research Center, has
conducted rat studies showing that
genistein, a plant estrogen, reduced
aggregation of blood clots by 57
percent. Such clotting, she explains,
contributes to clogged arteries and
eventually to heart attacks.

d Wea
s to id

She also notes that in Asian
regions-where people eat tofu,
miso soup, and other soybean
products as a dietary staple-there
are lower levels of breast and
prostate cancer and heart disease
than in Western
countries, where
soybeans are a
much smaller part
of the diet.
Soybeans and
Scblack beans are
rich sources of
genistein, which
is also being
examined for
fighting the
leukemia that
strikes bone
marrow and other
Soybeans also
contain high
levels of daidzein,
which the Chinese
have long used as
an herbal medi-
cine and to cut the
ris (left) and craving for
itherly conduct alcohol. As
lentify potentially Morris noted,
for research use by kudzu-which
Byrne is investi-
gating as an
contains daidzein.
"It doesn't hurt to investigate folk
remedies, because they may have a
scientific basis," Schoene says.
Some of the special-purpose
legumes are also excellent sources of
alkaloids, which the plants produce
in their leaves to protect them
against insects, diseases, and herbi-
vores, explains Prakash Kadkade, a
scientist with Phyton, Inc., a bio-
pharmaceutical company based in

Ithaca, New York. Examples of plant
alkaloids are nicotine, quinine,
cocaine, and morphine.
"We are screening many plants in
search of compounds that we think
may have therapeutic potential,"
Kadkade says.
Morris has also received requests
from researchers in Paraguay for
guar, Cyamopsis tetragonoloba,
which yields galactomannan gum
used as a stabilizer in confectioneries.
And scientists in Italy have
requested velvet bean, Mucuna
pruriens, a source of the dopa that's
converted by the brain to the neuro-
transmitter dopamine. Reductions in
dopamine have been associated with
Parkinson's disease, which occurs
when brain cells that produce dopam-
ine are destroyed.
Velvet bean also contains bufote-
nine (a cholinesterase inhibitor) and
serotonin (a brain neurotransmitter
that may be involved in learning,
sleep, and control of moods).
Along with their pharmaceutical
potential, the special-purpose le-
gumes also fix nitrogen, enriching the
soil and making them ideal candidate
crops for sustainable agriculture.
Some legumes can add up to 500
kilograms of nitrogen per hectare to
the soil-alleviating the need for
fertilizer and lessening the chance of
water pollution.
"We've only scratched the surface
on the potential of these legumes,"
Morris says.-By Sean Adams, ARS.
Brad Morris is in the USDA-ARS
Plant Genetic Conservation Resourc-
es Unit, Regional Plant Introduction
Station, 1109 Experiment Street,
Griffin, Georgia 30223; phone (770)
229-3253, fax (770) 229-3324, e-mail *

Agricultural Research/November 1996

Potential Phytochemicals from the ARS Special-
Purpose Legume Collection


Coffee senna
(Senna occidentalis)

Common indigo
(Indigofera tinctoria)

Fish poison bean
(Tephrosia vogelii)

Hyacinth bean
(Lablab purpureus)

Jack bean
(Canavalia ensiformis)

(Pueraria montana var. lobata)

Ringworm bush
(Senna alata)

Snout bean
(Rhynchosia minima)

Sunn hemp
(Crotalaria juncea)

Sunn hemp
(Crotalaria Retusa)

Tephrosia purpurea

Velvet bean (Cowitch)
(Mucuna pruriens)

White tephrosia
(Tephrosia candida)

Source of-















Winged bean Erucic acid
(Psophocarpus tetragonolobus) PUFAs*

* Polyunsaturated fatty acids

Agricultural Research/November 1996

Used to/as-

Fight leukemia, tuberculosis, tumors; antiseptic; pesticide
Arrest bleeding; antiseptic; pesticide
Fight inflammation, cellular mutation, breast cancer
Antiseptic; pesticide
Fight tumors, carcinomas; antiseptic

Antiseptic; wound-cleaner; pesticide

Kill unwanted cells; pesticide
Fight tumors; pesticide
Kill unwanted cells; pesticide

Lower blood pressure

Arrest bleeding
Fight flu, viruses, bacteria, fungi
Fight cervical, liver cancer; lower blood sugar

Fight inflammation, microbial infections; dilate coronary arteries;
promote estrous cycle; relax muscles

Fight tumors, leukemia, tuberculosis; antiseptic; pesticide
Arrest bleeding; antiseptic; pesticide
Fight inflammation, cellular mutation, breast cancer; antiseptic
Fight tumors, carcinomas; antiseptic


Fight tumors; lower blood pressure
Fight tumors

Fight tumors, leukemia; lower vital heart function, blood pressure;
Fight tumors

Fight tumors, rheumatic problems; kill unwanted cells
Fight tumors; pesticide
Kill unwanted cells; pesticide

Reduce cholinesterase enzyme
Treat Parkinson's disease
Treat parasitic intestinal worms; pesticide
Reduce cholinesterase enzyme, intestinal gas; relax muscles;
clotting agent

Fight tumors; pesticide
Kill unwanted cells; pesticide

Fight tumors
Treat muscular sclerosis, acne, eczema; fight nerve inflammation





For shippers, it may ease
fruit fly restrictions.

K : .-

. 9.






ow do you suffocate
Mexican fruit flies that may
be hitchhiking in a ship-
ment of grapefruit-without smother-
ing the fruit's quality before it
reaches grocery stores?
A simple answer may be to take
just enough oxygen out of the
atmosphere in the shipping container
so the flies are starved of it.
The fruits should fare well. Be-
cause they would respire or "breathe"
more slowly during shipping, they
would hold on longer to their fresh-
picked appearance and to their flavor,
sugars, moisture, and nutrients.
The principle works, but the
answer is far from simple. For
example, solid proof is required that
air with less than 1 percent oxygen
instead of the normal 20.9 percent
would reliably kill all the flies.
Ninety-nine percent mortality isn't
good enough.
But tough federal requirements
like this have not prevented progress
on a new project led by Robert
Mangan. An Agricultural Research
Service entomologist, he heads the
Crops Quality and Fruit Insects
Research Unit at ARS' Subtropical
Agricultural Research Laboratory in
Weslaco, Texas.
Mangan attempts to kill the bugs
with a range of gas mixtures that
plant physiologist Krista Shellie
determines don't damage the fruit.
Mangan, Shellie, and colleagues Guy
Hallman and Sam Ingle began the
new project this year. They aim to
see if technology known as con-
trolled atmosphere, or CA-long
used commercially to maintain
quality of shipped produce during 1
to 3 weeks of transport in sealed
containers-could be adapted for
double duty.

Mexican fruit flies laying eggs in
grapefruit before a test of the reduced-
oxygen treatment. (K7500-1)

Agricultural Research/November 1996

Weslaco's CA studies are part of a
3-year cooperative research and
development agreement (CRADA)
with TransFRESH Corp. of Salinas,
California. "TransFRESH is the
world leader in transporting fresh
produce in refrigerated containers
under a CA environment," says
TransFRESH laboratory manager
Susan Ajeska.
In 1995, the company supplied its
patented CA system for shipping
more than 400,000 tons of perish-
ables from 16 terminals, including 6
in the United States. Customers are


Biological technician Eleazar Moreno
artificially infects a mango with a fruit
fly larva in one of the controlled
atmosphere (CA) tests. (K7500-2)

mostly exporters, importers, transpor-
tation companies, and supermarket
chains. Commodities include avoca-
dos, cherries, asparagus, lettuce,
melons, peaches, nectarines, plums,
and other perishables.
"Ordinary refrigeration will take
shipped produce a long way," Ajeska

notes. But "CA can take it further,
and it will arrive in better shape. If a
controlled atmosphere quarantine
treatment comes from the CRADA, it
could help expand U.S. export
markets in the Far East and Pacific
Rim countries," she says.
The Weslaco researchers want CA
to provide shippers with a new, in-
transit insurance policy: a quarantine
treatment for citrus when Mexican
fruit flies infest a production area.
Fruit fly invasions can trigger costly
eradication programs requiring
repeated aerial sprays of insecticide,
and they can devastate agricultural
production and trade.
The flies, native to Mexico,
triggered a quarantine in part of
Texas as recently as this year.
Fortunately, the disruption was
minor, since the outbreak was near
the end of the citrus harvest. But
Mexican fruit fly invasions are an
annual threat in Texas and other fruit-
producing states, such as California
and Florida.

A Nondamaging Alternative?
Mangan, Ingle, and Shellie have
developed quarantine treatments
using hot water, air, or water vapor.
But fruit quality sometimes suffers.
Plus, the Weslaco treatments-like
all other approved ones, including
methyl bromide-must be done
before fruit can be shipped. The CA
approach could eliminate the threat
of the flies without delaying shipping
or damaging the fruit.
Another issue driving the Weslaco
project is an environmental one: the
scheduled phaseout of methyl bro-
mide by 2001. This chemical, besides
its quarantine applications for killing
insects in infested fruits and vegeta-
bles, is used to protect crops from
soilborne pests and diseases. It's also
used to shield harvested commodities
from pests during storage.

The phaseout results from an
international accord signed by the
United States and other countries in
Montreal, Canada, in 1991. This
came about because methyl bro-
mide-along with other synthetic
chemicals-contributes to depleting
the ozone layer of Earth's atmos-
phere. The ozone layer helps protect
the planet's inhabitants from exces-
sive solar radiation.
"The Agricultural Research
Service is a world leader in
developing methyl bromide alter-
natives," Mangan says. Congress
increased the agency's research funds
for alternatives for methyl bromide-
pre-planting and postharvest uses-
from $7 million in 1993 to $13.7
million in 1996.
Mangan emphasizes that the CRA-
DA's short-term CA goal for citrus is
limited to finding an alternative to
only one of methyl bromide's many
uses. This goal is to see if CA can
kill any and all Mexican fruit flies-
adults, larvae, and eggs-infesting
sealed containers of citrus for a ship-
ping duration of 1 to 3 weeks.
Methyl bromide is the most com-
mon treatment now available for
citrus. A hot-air treatment developed
at Weslaco is also approved. But
Mangan says it is not used yet, large-
ly because of the cost of developing
equipment at a time when the cheaper
methyl bromide is still available.
Plus, Shellie notes, "uneven heat-
ing or other problems may cause loss
of flavor in late-season citrus."

CA-For Better Ripening, Less
For mangoes, Weslaco's search is
not for an alternative to methyl
bromide, known to damage the fruits.
Instead, shippers want an alternative
that works better than the available
one, heat treatment, which acceler-
ates ripening and can shorten shelf

"The controlled atmosphere
treatments can result in slower, more
uniform ripening. They may also
delay certain postharvest diseases,"
Shellie says.
Weslaco's research strategy for
CA includes control of temperature
and humidity, as well as of oxygen,

Plant physiologist Krista Shellie takes color rea
CA-treated mangos. Circled areas allow resear
measure fruit color, hue, and intensity at the sa
before and after treatment. (K7500-3)

nitrogen, and carbon dioxide gases.
Specific gas combinations tested are
proprietary information of Trans-
FRESH, but oxygen levels are below
1 percent. By contrast, air is about
20.9 percent oxygen, 78.1 percent
nitrogen, and 0.03 percent carbon

Outside a Weslaco laboratory sit a
couple of TransFRESH's 30,000
multi-ton shipping containers.
Twenty feet long, they are "the small
ones," says TransFRESH lab
technician Jaya Cummaragunta.
"Most of our containers are twice that
length." Cummaragunta works in
Weslaco alongside ARS
For experiments, the
containers' built-in
refrigeration units keep
the fruit cool. But the
scientists can run several
tests at a time inside
them. Gas for a test
comes through a tangle of
thin plastic tubes that
travel underground from a
nearby utility building
and disappear into the
containers. There, the
tubes deliver precise CA
gas mixes under very low
pressure into each of
several large plastic
buckets. Each bucket
holds several fruits under
a tight lid, with only a
tiny escape hole to keep
gas pressure steady.
Inside the utility
building, the scientists
have turned a walk-in
chamber into a container
surrogate. It's populated
dings from with more fruit-filled
hers to buckets and gas tubes.
me spot All the tubes run to a
"mixing board." The
scientists use dials and
gauges on this device to set as many
as six different combinations of gas
for delivery through the tubes from
supply hoses on nearby tanks.
For each test, the scientists record
changes in the treated fruit's skin
color, firmness, and sugar and acid
contents. A panel of tasters rates fruit

Agricultural Research/November 1996

flavor, sweetness, texture, and other
qualities important to consumers.
Setting up CA tests using artifi-
cially fly-infested fruit can be a
challenge, says entomologist Sam
Ingle. "With grapefruit, we remove a
small core of the peel, like a plug,
from the stem end. Then we place
several fly eggs or larvae inside the
small, natural cavity JACK DYKINGA
beneath the peel and
replace the core, sealing it
with hot glue."
To infest the mangoes,
the scientists need more
cooperation from the flies,
because taking a plug ..'
would damage the fruit's
skin and flesh. So, after
mangoes begin ripening,
Ingle places some atop
screened cages used as
laboratory homes and
nurseries for tens of
thousands of fruit flies at a
time. Upside down, adult
female flies manage to
deposit 20 to 30 eggs
through the screen and
beneath a mango's skin.
"By the end of August,"
Mangan says, "we'd run
tests with more than
70,000 lab-reared Mexican
fruit flies, about 3,500
grapefruit, and some 5,000
mangoes. Those tests
examined a half-dozen
A maze of
different combinations of sored fri
stored frui
gases at five different TransFRE
temperatures. In some mangoes fi
tests using artificially
infested fruit, all the flies
died in a matter of 1 to 3 days,
without compromising the quality of
the grapefruit or mangoes."
According to Shellie, "low oxy-
gen, rather than high carbon dioxide,
seems to be the key in fruit quality.
We have found no damage at all to
grapefruit or mangoes chilled at oxy-

Agricultural Research/November 1996

gen concentrations below 1 percent.
But elevated carbon dioxide causes
appearance and flavor problems."
"I'm heartened by what's hap-
pened so far," Ajeska says. "The
CRADA maximizes resources of
both organizations. Bob Mangan and
his team design the experiments and
cover the details of quarantine

hoses delivers precise CA gas mixtures to
t. Here, entomologist Robert Mangan and
SH technician Jaya Cummaragunta examine
rom test containers. (K7500-4)

requirements. They frequently
suggest, on the basis of a test result,
what a potential treatment might look
like. Then we at TransFRESH can
evaluate whether it may be economi-
cally feasible. We want to do com-
mercial tests as soon as Bob con-

cludes from the lab trials that the
time is right."
That time hasn't yet arrived,
Mangan says.
"Results from some kinds of trials
have been steady; other results have
been tantalizing but inconsistent. So
we know we have more work to do.
Still, out of this may come applica-
tions for other fruits and insects, as
well as Mexican fruit flies. But each
insect and fruit is different."
This fall, the researchers are
beginning tests with oranges, along
with a second round of grapefruit
studies.-By Jim De Quattro, ARS.
Robert Mangan, Krista Shellie,
Guy Hallman, and Sam Ingle are in
the USDA-ARS Crop Quality and
Fruit Insects Research Unit, Subtrop-
ical Agricultural Research Laborato-
ry, 2301 S. International Blvd.,
Weslaco TX 78596; phone (210) 565-
2647, fax (210) 565-6652, e-mail
Susan Ajeska is with TransFRESH
Corp., Salinas, CA; phone (408) 772-
7269. For more information about
TransFRESH, contact Gwen Peake,
Fineman Associates, San Francisco,
CA: phone (415) 777-6933. *

What's America's favorite fresh
fruit (on a pounds-per-person
basis)? Answer below.

1 Apples
Q Bananas
1 Oranges
1 Strawberries

a!iAJaS qajrasasy O!uouooa/vaSnf
:a3jnoS sa!uJaqMyMls jo spunod t pun 'saUuJo Jo
spunod sal3idd o jo spunod 61[ al Osl| aM 'I661
u! spunod gZ utql a.jou lr I 'ON aOIM sneuuni

Better Vaccines for Healthier Catfish

Like the old joke says,
there's good news and bad
news about Edwardsiella
ictaluri, the bacterial cul-
prit behind $20-million annual losses
to enteric septicemia of catfish (ESC).
First, the good news: Despite
conventional wisdom, plummeting
water temperatures don't necessarily
have a chilling effect on a vaccinated
fish's ability to fend off E. ictaluri.
"It's long been thought that if you
immunized a fish against E. ictaluri
and subsequently put it in water that
was 66.2F or cooler, the fish would
lose its immunity after about 3
months," says microbiologist Phillip
H. Klesius. He heads the ARS Fish

Pond-raised catfish. (K5328-10)
Diseases and Parasites Research
Laboratory at Auburn, Alabama.
"People believed that the fish's
immune system became dormant in
the colder water."
Not so, two studies by Klesius
suggest. In the first, conducted in
1980, Klesius showed that immuno-
globulin production-the manufacture
of protective antibodies by the fish's

immune system-is not influenced by
water temperature.
In more recent tests, Klesius
immunized catfish with a live E.
ictaluri vaccine, then grew the fish in
water temperatures of either 66.2F or
78.80F. For 4 months, the fish were
challenged with exposure to E.
ictaluri once a month. The result:
Immunized fish in the colder water
were no more likely to become
infected than their counterparts in
warmer waters.
"This shows that acquired immuni-
ty against ESC is long-lasting at ei-
ther 78.8 degrees or 66.2 degrees," he
says. "We don't know what would
happen if water temperature dropped
as low as 41 F, for example, but it

works at 660F. This gives the farmer a
wider window of opportunity in
which to vaccinate against ESC."
Now for the bad news: Choosing a
vaccine against E. ictaluri may not be
as simple as believed in the past.
"Although E. ictaluri isolated from
various situations have different
names, it was not thought that they
were actually different," Klesius
explains. "For example, the name

AL-93-58 simply meant it was the E.
ictaluri isolated in 1993 from fish in
Alabama in clinical case number 58-
not that it was actually very different
from the isolate known as AL-93-75."
The concept of all E. ictaluri being
equal was a comforting one because it
meant vaccinating with one isolate
should protect against any E. ictaluri
that came along. Studies by Klesius
and by Craig A. Shoemaker in early
1996 shattered that illusion.
"We immunized channel catfish
with one of five isolates of E. ictaluri,
then challenged them with other
isolates," Klesius recalls. "We found,
for example, that immunizing with
isolates AL-93-75, EILO, AL-93-58,
or S-94-1017 induced immunity
against AL-93-75 but that neither
ATCC-33202 nor S-94-1051 did.
"It was believed that all E. ictaluri
produced essentially the same anti-
gens, or proteins that stimulate the
fish's body to produce an immune
response," says Klesius. "Our results
show for the first time that differences
exist between E. ictaluri isolates in
their ability to induce protective
immunity against ESC."
Since vaccination with some iso-
lates does protect against others,
Klesius thinks it's possible certain
isolates share so-called antigen
patterns. This might mean a vaccine
that carries a specific pattern would
protect against isolates that share it.
"Our next step is to work out the
predominant antigen patterns among
the E. ictaluri isolates and work out a
vaccine from that," Klesius says. "Just
because we've discovered there are
differences in the isolates doesn't
mean we have to reinvent the wheel
here."-By Sandy Miller Hays, ARS.
Phillip H. Klesius is at the USDA-
ARS Fish Diseases and Parasites
Laboratory, 990 Wire Road, Auburn,
AL 36831-0952; phone (334) 887-
4526, fax (334) 887-2983, e-mail *

Agricultural Research/November 1996

Oceanographic Instruments Monitor

Fishpond Algae

Cat Fishery in Portland,
Arkansas, a fast-growing
company that sells
400,000 pounds of catfish
weekly. The Catfish Farmers of
America named him Arkansas'
Catfish Farmer of Year for 1995. But
he wants to do more.
"If we could find an economical
way to control off-flavors and the
blue-green algae that produce them, it
would be a tremendous boon to the
industry, no question," he says.
Many producers would agree.
Currently, a major obstacle to catfish
sales is the off-flavor compounds
produced by microscopic algae.
These natural algae byproducts are
absorbed into the fishes' meat, giving
them a muddy taste. The fish are still
safe to eat, but they just don't taste
good anymore.
It's estimated that during the late
summer and fall, as much as 80
percent of U.S. catfish loses some
flavor quality.
As a result, many are unmarket-
able, causing an extra $5.8 to $12
million in overhead costs. That's why
three times a week, Jeffers takes fish
from harvest-ready ponds for flavor
testing by processors.
Catfish ponds are an ideal habitat
for algae, which are often beneficial
to the farmer. They provide oxygen
for the fish and help stabilize pond
temperatures. But it's not possible to
predict when the algae will produce
the off-flavor compounds.
Recently, Agricultural Research
Service scientists David F. Millie and
Chris P. Dionigi joined forces with
Oscar M. Schofield, a professor of
oceanography at Rutgers University,
to develop ways to monitor algae in
catfish ponds.
The studies focus on the relation-
ship between the algae present, their
health, and the presence of off-

Agricultural Research/November 1996

In a series of experiments at
Stoneville, Mississippi, the research
team explored using oceanographic
instruments to see if they might
provide an efficient means to moni-
tor algae in catfish ponds. These
instruments, which measure the
optical properties of water, might be
used to estimate how much and what
kinds of algae are present in a pond.
Algae require light for growth.
But some absorbed light is re-emitted
as fluorescence in a process that can
provide basic biological information
about algae cells.
Researchers are looking at an
instrument called the SAfire (Spec-
tral Absorption and Fluorescence
Instrument), which can measure the

algae cells, which allows researchers
to estimate how healthy the algae are.
"What we hope to do is differenti-
ate algae by their fluorescence
properties and determine whether
there is a connection between algae
health and the production of off-
flavor compounds," says Millie, a
microbiologist at ARS' Southern
Regional Research Center in New
Orleans, Louisiana.
"While initial results were promis-
ing, we need to finish analyzing all
the results," says Millie's colleague,
plant physiologist Dionigi. "The
instruments were designed for clear,
blue ocean water and might not
provide reliable information in murky
catfish ponds."-By Jill Lee, ARS.

Catfish ponds showing algae growth. (K4724-13]

color of light that algae fluoresce.
Scientists have long speculated this
information could be used to assess
the kinds of algae in a pond-
particularly the blue-green algae
associated with off-flavors. A second
instrument, the pulse amplitude
modulated fluorometer, measures
how much light is fluoresced by

David F. Millie is in the USDA-
ARS Food and Feed Processing
Research Unit, Southern Regional
Research Center, P.O. Box 19687,
New Orleans, LA 70179; phone (504)
286-4457, fax (504) 286-4419, e-mail *

Suppressing a

Serious Citrus Pest

Citrus root weevils can cost growers more than
$1,200 per acre. Already, in 1996, they have caused an
estimated $72-million loss in Florida, creating economic
havoc for some of that state's citrus growers. This
weevil has also become a significant pest of ornamental
and vegetable crops.
"No part of the citrus tree is safe from this insect,"
says William J. Schroeder, an ARS entomologist who
recently retired but is still working as KEITH WELLER
an ARS consultant. "The adult citrus
root weevil feeds on leaves, but its
larvae do the most damage by attacking
tree roots."
Root damage interferes with the
transport of nutrients and water to the
rest of the tree, resulting in fewer and
smaller fruit and eventually tree death.
The citrus root weevil, Diaprepes
abbreviatus, first appeared in Florida in
1964. Growers then controlled its
spread with strong chemicals, many of
Adult citrus root
which have since been taken off the Ab lt citus roo(
abbreviatus. (K7,
market because of environmental and
human health concerns.
For 2 years, Schroeder and ARS
colleagues at the U.S. Horticultural Research Laboratory
in Orlando have been controlling this pest with a tiny
parasitic worm, the Steinernema riobravis nematode.
Sprayed on the soil, these nematodes burrow down to
tree roots, hungrily seeking weevil larvae. Once inside
the larvae, they release a bacterium that kills the larvae
within 48 hours.
"The nematodes then reproduce themselves, and the
offspring feed on the bacte- KEITH WELLER
ria, nourishing another gen-
eration of juvenile nema-
todes," Schroeder explains.
But not much is yet
known about the bacterium
"We know that a symbi-
otic relationship exists be-
tween it and the nema-
tode," says Heather Smith,
a graduate student working
with Schroeder. "The nem-
atode acts as both a host
and a vector, since the bac-
terium can't survive out- Entomologist William Schroed
side a host for any length nematodes to citrus foliage to s
of time." when the leaves dry. (K7458-9)


It gives the bacterium a way to live, and it recipro-
cates by multiplying itself and becoming food for the
nematode. Once the bacterium kills a larva, it releases
antimicrobial agents that prevent the growth of other
bacteria, so that it can then reproduce without
"The nematodes can survive in a host for up to three
generations," says Smith. "Then they leave, with their
bacteria ensconced in their gut, to look
for a new host."
Schroeder and Smith are working to
identify and characterize the bacterium.
"We expect it to be of the genus
Xenorhabdus," says Smith, "because
all other symbiotic bacteria associated
with the nematode family are of this
genus. Also, preliminary results from
carbohydrate fermentation tests, fatty
acid analyses, protein comparisons, and
several other basic microbiological
studies point to Xenorhabdus."
I, Diaprepes Smith is doing a final comparison to
check similarities in DNA profiles of
four other species of bacteria in this
genus. "We're also conducting patho-
genicity tests to see just how this bacterium kills the
citrus root weevil larvae," she says.
Schroeder says that Florida citrus growers routinely
use nematodes in an integrated pest management pro-
gram for root weevil control. "We want to learn more
about this bacterium so that we can help growers opti-
mize control of citrus root weevil." He thinks that timing
applications might help.
"We're also investigating
other vectors that might
develop a symbiotic rela-
tionship with the bacterium,
giving us another potential
biological agent."-By
Doris Stanley, ARS.
William J. Schroeder and
Heather L. Smith can be
reached at the USDA-ARS
Horticultural Research
Laboratory, 2120 Camden
Road, Orlando, FL 32803;
phone (407) 897-7300, fax
(407) 897-7309. *

er applies Steinernema riobravis
see how well nematodes survive

Agricultural Research/November 1996


Science Update

Older Hens: Fasting, Eggs, and
ARS scientists in Georgia, in a
series of studies, found new clues for
coping with two problems in the egg
industry: egg contamination with
Salmonella and low egg production
by older hens. In one study, scientists
found these birds' natural resistance
to Salmonella infection plummets
after they are put on a fast for a week
or so. Fasting, a common industry
practice, causes the hens to molt.
This triggers hormonal and other
changes, soon restoring up to 90
percent of a hen's previous egg-
laying capacity. In tests, fasted
chickens became infected with
Salmonella after ingesting as few as
10 bacterial cells. Typically, a
chicken that is not being fasted
becomes infected-through what's
called the oral-fecal route-only after
ingesting 10,000 or more Salmonella
cells shed by an infected bird. The
increased susceptibility of fasted
hens leaves them more vulnerable to
contracting the infection by a less
common route-through the air. Plus,
fasted hens' increased susceptibility
to infection raises the odds of egg
contamination. On average, the odds
are low-less than 3 in 10,000. But
they could be lowered further with an
effective alternative to fasting. One
method with potential is an experi-
mental low-calcium diet developed at
the University of Georgia. Joint
studies by the university and ARS
found that birds molted with this diet
instead of by fasting were 100 times
less susceptible to infection. Peter
Holt, USDA-ARS Southeast Poultry
Research Laboratory, Athens,
Georgia, phone (706) 546-3442.

International Germplasm Could
Help Lay Down a Bunt
ARS scientists confirmed that
wheats from India and Mexico hold
genetic resistance to Karnal bunt, a
fungal disease that threatens U.S.
wheat exports. The resistant germ-
plasm was selected from 40,000 lines
from collections at Punjab Agri-
cultural University in India and
CIMMYT (International Maize and
Wheat Improvement Center) in
Mexico. In lab tests, scientists found
resistant germplasm from Mexico
that also resisted Asian forms of
Tilletia indica fungi. And vice versa:
they identified resistant Indian lines
that ward off Mexican strains of the
fungus. By incorporating such
resistant lines in breeding programs,
U.S. breeders might better protect the
American crop from future out-
breaks-such as the one that began in
durum wheat last March in Arizona.
Countries that don't have the fungus
won't buy wheat from countries that
do, so keeping the disease out of
American wheat is a top priority.
Morris Bonde/Gary Peterson, USDA-
ARS Foreign Disease/Weed Science
Research Laboratory, Frederick,
Maryland, phone (301) 619-2860/

Interleukin-4 for Deworming?
Nature lends a hand to help
animals rid themselves of gastrointes-
tinal worms. This finding by ARS
scientists could lead to a new way to
treat parasite-infected people as well
as animals. Worm infection stimu-
lates an animal's immune cells to
make a natural substance, interleu-
kin-4. IL-4 starts the worm-expulsion
process, though all the mechanics

aren't yet known. In lab tests, re-
searchers injected IL-4 into healthy,
uninfected mice. The animals'
intestinal muscles began to contract
more often and to secrete more
fluids. Together, these responses
could dislodge worms from the
intestine. This discovery could lead
to using synthetic copies of IL-4 to
treat the infections. Joseph F. Urban,
Jr., USDA-ARS Immunology and
Disease Resistance Laboratory,
Beltsville, Maryland, phone (301)

Probing Irrigation Needs
Automated irrigation for cotton
and other field crops is the objective
of a cooperative research and devel-
opment agreement between ARS and
Dynamax, Inc., of Houston, Texas.
ARS researchers designed a system
that turns on water pumps when
there's no longer enough water in the
soil for plant roots to use. An elec-
tronic pulse travels by cable to
stainless steel probes in the soil at
various depths to several feet. Soil
water is computed by how long a
pulse takes to pass through the probe.
Dynamax is manufacturing the
system and will develop it further
with help from ARS and Texas A&M
University. The system's cost may be
offset by labor savings and lower
expenses from pumping only when
water is needed. Steve Evett, USDA-
ARS Water Management Research
Unit, Bushland, Texas, phone (806)

Agricultural ResearchlNovember 1996

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