| Perspective |
|
|
|
The UW–Madison
has released 32 new faculty positions in a campus-wide competition, with
the goal of enhancing already existing high-priority campus programs. Based
on FRI’s proposal, four of these new faculty positions have been consigned
to food safety. The preliminary announcement follows:. |
|
Faculty Positions in Food Safety
The University of Wisconsin–Madison has announced the development of a
cluster of tenure-track faculty positions in food safety and will invite
applications from qualified individuals. Successful candidates will develop
innovative, responsive and highly competitive research programs that interact
closely with other faculty at U.W.– Madison who conduct research in food
safety. Successful candidates will have primary or joint faculty appointments
as appropriate in participating departments where they will provide instructional
and research leadership, and will coordinate their research/outreach activities
through the Food Research Institute of the University of Wisconsin–Madison.
Contact: Dr. Michael W. Pariza, Director, Food Research Institute, University
of Wisconsin–Madison, 1925 Willow Drive, Madison, WI 53706.
Four positions are available for immediate recruitment, as follows:
-
Emphasis on mycotoxins (70% research; 30% instruction)—The incumbent
will investigate the biochemistry and genetics of mycotoxin synthesis by
toxigenic molds, and the detection of mycotoxins in foods, feeds, and other
biological materials, and provide undergraduate/graduate instruction in
area(s) of expertise as appropriate.
-
Emphasis on toxigenic molds (70% research; 30% instruction)—The
incumbent will investigate the physiology and ecology of toxigenic molds,
and factors that influence plant susceptibility and/or resistance to infection
by toxigenic molds, and provide undergraduate/graduate instruction in area(s)
of expertise as appropriate.
-
Emphasis on immune response to foodborne pathogens and their toxins
(70% research; 30% instruction)—The incumbent will investigate the host
response to foodborne pathogens and their toxins, and the roles of foodborne
pathogens and their toxins on immune modulation, and provide undergraduate/graduate
instruction in area(s) of expertise as appropriate.
-
Emphasis on novel engineering applications (70% research; 30% instruction)—The
incumbent will investigate novel approaches to detecting and controlling
foodborne pathogens and their toxins in foods, food processing equipment,
and food manufacturing facilities, and provide undergraduate/graduate instruction
in area(s) of expertise, for example in the food engineering/food science
programs, as appropriate. Possible areas of specialty include surface modification,
biosensors, and applicable instrumentation.
| Comparative Survival
of Salmonella typhimurium DT104, Listeria monocytogenes,
and Escherichia coli O157:H7 in Preservative-free Apple Cider and
Simulated Gastric Fluid |
return to top |
We compared the survival of three-strain mixtures (ca. 107
CFU/ml each) of Salmonella typhimurium DT104, Listeria monocytogenes,
and Escherichia coli O157:H7 in pre-pasteurized and unpasteurized
preservative-free apple cider (pH 3.3–3.5) during storage at 4 and 10°C
for up to 21 d. Salmonella typhimurium DT104 populations decreased
by <4.5 log10 CFU/ml during 14 d storage at 4 and 10°C
in pasteurized cider, and by >5.5 log10 CFU/ml during
14 d in unpasteurized cider stored at these temperatures. However, after
7 d at 4°C, the S. typhimurium DT104 populations had decreased
by only about 2.5 log10 CFU/ml in both pasteurized and unpasteurized
cider. Listeria monocytogenes populations decreased below the plating
detection limit (10 CFU/ml) within 2 d under all conditions tested. Survival
of E. coli O157:H7 was similar to that of S. typhimurium
DT104 in pasteurized cider at both 4 and 10°C over the 21 d storage
period, but E. coli O157:H7 survived better (ca. 5.0 log10
CFU/ml decrease) than S. typhimurium DT104 (>7.0 log10
CFU/ml decrease) after 14 d at 4°C in unpasteurized cider. In related
experiments, when incubated in simulated gastric fluid (pH 1.5) at 37°C,
S.
typhimurium DT104 and L. monocytogenes were eliminated (5.5–6.0
log10 CFU/ml decrease) within 5 and 30 min, respectively, whereas
E. coli O157:H7 concentrations decreased only 1.60–2.80 log10
CFU/ml within 2 h. These results highlight the inherent differences in
acid tolerance between these three pathogens and suggest that if S.
typhimurium DT104, or even L. monocytogenes survive in apple
cider, they are less likely than E. coli O157:H7 to survive passage
through the stomach.
—Ann M. Roering, John B. Luchansky, Anne M. Ihnot,
Susan E. Ansay, Charles W. Kaspar, and S. C. Ingham
| Recovery of Escherichia
coli Biotype I during Refrigerated Storage of Beef Carcasses Inoculated
with a Fecal Slurry |
|
The survival of Escherichia coli Biotype I on beef carcasses was
investigated during a 10-day storage period at 4°C. Three beef carcasses
were inoculated with a fresh slurry of cattle manure. Two sponge samples
from each of 3 sites were taken from each of the three carcasses on days
0, 1, 3, 7, and 10 after inoculation and tested for E. coli Biotype
I. The initial numbers ranged from an average of 171 cfu/cm2
to 405 cfu/cm2, depending on the counting method used (Petrifilm
and MPN). In general, an appreciable reduction in the numbers occurred
during the first 24 hours of storage, with an average reduction of 164
to 400 cfu/cm2, depending on the counting method. E. coli
were not detected on day 7 of the storage period on 2 of the 3 carcasses,
though viable E. coli were recovered from these 2 carcasses after
a 24-hr enrichment at 37°C. Viable E. coli cells were detected
on the third carcass after 7 days at 4°C. On day 10 viable cells were
recovered from 2 of the 3 carcasses, but were not recovered from the third
carcass. No significant difference in recovery of viable cells was observed
between the two methods of enumeration on days 0, 1 and 3, though viable
E.
coli were recovered from all 3 carcasses on day 7. These findings suggest
that i) refrigerated storage for more than 3 days may inactivate contaminating
bacteria and improve safety, and ii) meat plants employing long term dry
aging storage may test negative for E. coli, indicating such products
are free of fecal contamination after 7 days, but such carcasses may still
contain other bacterial indicators of fecal contamination, including enterococci.
— Mehmet Calicioglu, D. R. Buege, S. C. Ingham, and
J. B. Luchansky
| Growth and Biocontrol of Enterotoxigenic
Bacillus
cereus in Infant Formula |
|
We initiated a study to determine the potential for growth and enterotoxin
production by Bacillus cereus in re-hydrated infant formula and
to evaluate the effectiveness of various bacteriocins in controlling growth
of the organism during storage at various temperatures. We inoculated 3
different rehydrated infant formulas (low iron and iron-fortified with
and without maltodextrin) with 103 spores/ml of a 3-strain cocktail
of B. cereus and stored the formulas at refrigeration (4, 8, and
12°C) and abuse (25°C) temperatures for up to 10 days. No growth
occurred at any of the refrigeration temperatures. At 25°C growth began
within 6 hours, and cell numbers reached 107 cfu/ml within 24
hr in all 3 formulations. Similar growth patterns occurred at lower inoculum
levels (10 and 100 cfu/ml). When formula was inoculated with 103 spores/ml
and stored at room temperature (25°C) for 6 hr before being refrigerated,
outgrowth of spores occurred within 12 hr at 12°C and 8°C. Growth
continued at 12°C over 48 hr, with cell counts reaching 105
cfu/ml. We found that 103 spores/ml of certain strains grew
within 4 days in all 3 formulas at 25°C and 12°C. One strain grew
at 8°C in the iron-fortified formula without maltodextrin, and another
grew at 8°C in both of the iron-fortified formulas. We have developed
an ELISA (enzyme-linked immunosorbent assay) that can detect as little
as 0.25 ng/ml of each component of the tripartite B. cereus enterotoxin,
hemolysin BL, and are assaying samples from the infant formula studies
for the presence of toxin. So far we have observed that all 3 toxin components
can be detected in formula after 24 hr of growth at 25°C. We are investigating
the use of bacteriocins active against B. cereus. We tested five
bacteriocins of lactic acid bacteria origin against a battery of B.
cereus strains, using a spot-on-lawn in vitro assay. Results indicate
that nisin is the most widely effective against the B. cereus strains
tested, and is also the easiest to use, since it is commercially available
in a standardized formulation. We have begun testing the efficacy of nisin
in infant formulas against our 3-strain B. cereus cocktail and strain
HRM44. Preliminary results show that 0.05% nisin (w/v) can inhibit the
growth of strain HRM44 spores inoculated at 103 cfu/ml in formula
at 8, 12, and 25°C for up to 10 days. Nisin also inhibits growth of
the 3-strain cocktail at these temperatures, although the spores in the
cocktail are less sensitive, and 0.10% nisin may be needed to provide inhibition.
Results obtained to date indicate a potential for B. cereus to grow
in re-hydrated infant formula during storage at 8°C or higher, and
that the use of nisin can provide a hurdle to prevent outgrowth.
—Amy Wong and John Luchansky
| Can Food Allergies Be Prevented
or Cured? |
|
Food allergies affect 6–8% of children and 1–2% of adults in the United
States. Reactions to foods can vary from mild episodes of hives to fatal
anaphylaxis. Food allergic reactions are due to the development of IgE
antibodies directed against specific proteins in the food. These antibodies
circulate in the bloodstream and attach to the surface of mast cells in
the tissues of the respiratory system, gastrointestinal system, and skin.
When the food protein(s) come in contact with the IgE on the mast cell,
histamine is released which leads to the allergic symptoms — hives, swelling
in the skin, respiratory distress, vomiting, and shock. The propensity
of an individual to produce IgE antibodies to food proteins is dependent
upon genetic factors. Why certain foods, such as peanuts or fish, are more
likely to cause allergy than other foods is unknown. Currently, the only
ways to deal with food allergy are to avoid exposure, and administration
of medications (epinephrin and antihistamines) when reactions occur. Since
reactions can occur unexpectedly and with exposure to even small quantities
of the food, new approaches are needed. Attempts to desensitize peanut
allergic patients using conventional immunotherapy (“allergy shots”) have
not been successful largely due to severe reactions to the treatment. Although
improvements occurred in the threshold level of peanut exposure, reactions
were not totally prevented. A new approach is the use of anti-IgE monoclonal
antibodies which reduce the circulating levels of IgE. This treatment has
been successfully used in the treatment of allergic rhinitis (“hay fever”)
and allergic asthma by the Allergy Section at the University of Wisconsin–Madison.
This treatment is far safer than traditional immunotherapy. Although this
treatment has not been applied to food allergies, it will be investigated
in the near future. Molecular biology studies of peanuts and other allergens
indicate that certain amino acid sequences on the protein are necessary
for the development of the IgE antibodies. Immunization with these peptides
can shift the immune response away from the production of IgE. Molecular
cloning of food allergens has led to the recognition that vaccination of
animals with the DNA encoding for allergic proteins will also abrogate
the IgE antibody response. Whether DNA-based vaccines can be used to prevent
the development of allergy to specific food proteins has not been evaluated.
—Robert K. Bush, M.D.
| Transgenic Alfalfa: A New System |
|
Countless examples of gene transfer have been published, but finding applicable
products has been slow in development. A team of scientists with both basic
and applied skills was organized to apply concepts of gene transfer to
a working system of production. The team included Richard Burgess (former
director of the Biotechnology Center), Sandra Austin-Phillips (gene transfer),
Richard Straub (plant fractionation), Richard Koegel (harvest engineer),
Edwin Bingham (alfalfa geneticist) and Mark Cook (animal scientist, FRI).
Alfalfa was selected for gene transfer. UW–Madison has a long history of
fractionation of juice proteins from alfalfa. In addition, alfalfa represents
a significant crop raised in Wisconsin, and the unique color of alfalfa
serves as a marker in a predominantly corn/soybean meal feeding system.
Juices of alfalfa, expressing the desirable gene product, can be sprayed
on foods post-pelleting, preserving gene product activity. Phytic acid
is an inositol molecule that chelates plant phosphorus, rendering more
than 60% of dietary phosphorus unavailable for digestion. Due to the unavailability
of phytic phosphorus, animal diets must be supplemented with inorganic
phosphorus which increases the cost of animal production in the US over
$100 million per year. The enzyme phytase breaks down phytic acid and liberates
the phosphorus, making it available for use in animal metabolism. It was
theorized that the addition of phytase to animal feed would make phytic
phosphorus available, thereby reducing the need to add inorganic phosphorus.
Using phytic phosphorus would also decrease the levels of fecal phosphorus
in animal excreta, and improve its value as a fertilizer. The phytase gene
was successfully transferred from a fungus to alfalfa. The juice collected
from alfalfa contained 1% of the soluble protein expressing phytase activity.
When applied to chicken feed, the need for additional inorganic phosphorus
was eliminated. Excreted phosphorus was reduced more than 50%. Seed stock
is currently being generated in preparation for technology transfer. Studies
on stability, fractionation, and animal performance continue. Other valuable
genes are being inserted and expressed. This illustrates the ability of
gene transfer to generate valuable products at a low cost. It is estimated
that only 6% of the Wisconsin alfalfa crop will provide enough enzyme to
meet the phytase needs of 7 billion broilers and 100 million pigs raised
in the US. This illustration also demonstrates environmentally clean methods
to advance the food production system.
—Mark E. Cook
| Breakthrough in Transgenic Animal
Technology |
|
Research reported in the 24 November 1998 edition of the Proceedings
of the National Academy of Sciences (PNAS) may herald a new era in
biopharmaceutical production. Work performed in the laboratory of former
University of Wisconsin–Madison dairy science professor Robert Bremel,
and by Gala Design LLC, a Sauk City, WI, company with which Bremel is now
associated, resulted in new efficiency in the production of transgenic
livestock. Dairy livestock are seen as key players in the production of
genetically engineered protein drugs, such as monoclonal antibodies, hormones,
vaccine proteins and enzymes. Introducing new genes into the animals allows
them to produce specific pharmaceutical proteins in their milk, from where
it can be extracted as a drug component. Costs of producing proteins by
this route are far lower than traditional pharmaceutical production methods.
The PNAS article describes a new method of gene introduction that increases
the efficiency of production of transgenic cattle. The transgameticTM
method inserts a gene into the unfertilized oocyte or egg, which stably
incorporates the gene into the maternal germline. Once the egg is fertilized,
all cells of the resulting embryo carry the new gene, and the calf is born
with the capability to secrete a new protein in milk. Subsequent generations,
offspring of each founder animal, will also carry the desired gene. Older
production methods made transgenic livestock very costly. Cloning and pronuclear
microinjection typically lead to only 1 percent of animals born carrying
the new gene. The new technology also sidesteps problems of gene stability
and mosaicism seen with microinjection. When DNA is microinjected into
a fertilized embryo, the DNA is often not taken up until cell division
has occurred. As a result, only some cell lineages carry the new gene.
If the germ or sex cells don’t carry the new gene, the gene isn’t reliably
transferred to offspring. This technology paves the way to applications
of biotechnology in agricultural livestock, similar to those that have
changed crop agriculture in recent years. The technology may also have
applications in other mammals. The scientists believe the technology described
also points to a key process in evolution. Transposable elements, or transposons,
are known to jump to new locations in the genome (the cell’s hereditary
material) and bring about changes in the genome that confer selective advantage.
The experimental introduction of genes by a vector — which is structurally
very similar to a transposable element — into the exposed genome of the
oocyte results in uptake of new, fully heritable, genetic characteristics.
—UW–Madison Agricultural and Consumer Press Service
Agnes R. Denes, Ph.D., Research Associate
|
Shortly after getting my B.S. in physical
chemistry at the “Al. I. Cuza” University in Iasi, Romania, I came to the
University of Wisconsin–Madison for my graduate studies. I received the
Ph.D. in the Forestry Department in August 1998, working on cold-plasma-induced
surface modification of natural and synthetic polymeric materials. I was
involved in multiple research projects, one of them in collaboration with
Dr. Amy Wong at the Food Research Institute. After finishing my graduate
studies, I joined Dr. Wong’s research group. We are currently working on
plasma-induced modification of organic and inorganic substrates to develop
surfaces that inhibit biofilm deposition.
Plasma-enhanced chemical processing is an excellent approach
for surface modification of various biomaterials used in the food industry,
and we hope that our work will bring us closer to creating materials that
prevent the attachment of bacteria and the formation of biofilms. Our work
also involves characterization of newly developed surface chemistries and
properties using analytical tools available throughout the UW campus (e.g.,
electron spectroscopy for chemical analysis, atomic force microscopy, attenuated
total reflection Fourier transform infrared spectroscopy).
My husband is also in Madison, and he is a graduate student
working on his Ph.D. in the Electrical Engineering Department. Aside from
our common interest in the field of plasmas, the gaseous mixture of electrons,
ions and neutral particles, we love traveling and the outdoors. When time
permits, we are skating, swimming, playing tennis or walking our dog, Muki.
Although we still miss the mountains from back home, we love Madison and
its surroundings; we have met a lot of nice people and made good friends
during our four years here. |
Dr. Denes at electron spectroscope for chemical analysis. |
|
| Cook |
Presentations by Mark Cook:
“Conjugated linoleic
acid on swine health and development.” Mark Cook at the North Carolina
Swine Conference. Nov. 17, 1998. Raleigh, N.C.
“Conjugated linoleic
acid on poultry production.” Mark Cook at the North Carolina Poultry Conference.
Dec. 16, 1998. Raleigh, N.C. |
| Luchansky |
Presentations by John B. Luchansky:
“Practical examples of using PFGE to subtype bacteria
from a variety of sources.” Invited speaker and participant at the CDC
Workshop on Molecular Subtyping of Listeria monocytogenes and Escherichia
coli O157:H7 by Pulsed-field Gel Electrophoresis. Centers for Disease
Control and Prevention, Atlanta, Georgia. November 18, 1998.
“Molecular tracking and biocontrol of bacteria associated
with food.” Invited speaker at the Agricultural Research Service of the
United States Department of Agriculture. The Eastern Regional Research
Center, Wyndmoor, Pennsylvania. December 15, 1998.
“Perceptions and prescriptions-Can Escherichia coli
O157:H7 be controlled on farms or in foods?” Invited speaker at the 2nd
Annual American Meat Science Association, National Meat Association Western
Science Research Update. Doubletree Hotel and Conference Center, Monterey,
California. February 17, 1999.
Luchansky also traveled to Chicago in January to serve
as the Technical Presentations Subcommittee Representative for the Biotechnology
Division of the Institute of Food Technologists.
In February, Luchansky and Senior Research Specialists
Alan
J. Degnan and Nan G. Faith hosted a group of undergraduate students
for an afternoon at the FRI. This is the third year running that John,
Al, and Nan gave students first-hand experience with genomic fingerprinting
and biocontrol of E. coli O157:H7 associated with fermented meats.
It was an enjoyable and productive experience for the mentors and the students. |
| Pariza |
Presentation by M. W. Pariza:
Co-organizer of the first “International Workshop on
Conjugated Linoleic Acid Analysis,” held in Washington DC, January 1999. |
A recent article published by the Ministry of Health of New Zealand cautioned
parents and medical professionals against the indiscriminate use of soy-based
infant formulae. Isoflavone compounds, naturally present in soybean products,
have been shown to adversely affect reproduction in some animal species
and to have ill effects on some cells in culture. These compounds are weak
estrogens and it has been suggested that they may have physiological effects
on the neuroendocrine systems of infants who consume soy-based infant formulae.
Today, soy-based infant formulae account for approximately 25% of the
formula sold in the U.S., and thousands of infants have consumed it with
no overt signs of toxicity. Because of added mineral supplements which
may contain incidental aluminum, soy formulae contain relatively high concentrations
of aluminum (10–300 times the concentration of aluminum in human milk).
Healthy full-term infants are not at substantial risk for aluminum toxicity
but preterm and sick infants, particularly those with reduced renal function,
may absorb excessive aluminum which can interfere with bone formation and
nerve function.
Soy proteins are also allergenic. One of the main reasons that soy formula,
rather than a cow’s milk-based formula, is fed to infants in the U.S. is
the belief that it is less likely to provoke allergic reactions. However,
a recent study of 263 Canadian infants, with a genetic predisposition to
allergy, who were fed formulae either soy-based, cow’s milk-based or partial
whey hydrolysate-based, compared to breast milk for at least the first
4 months, revealed that the incidence of asthma and eczema was similar
in the infants fed the cow’s milk formula and the soy formula. In comparison,
children who had consumed breast milk or the whey hydrolysate had about
half the incidence of allergic reactions up to 5 years of age.
Phytoestrogens (isoflavones) are the latest safety concern associated
with soy formulae. While some studies on carcinogenesis have demonstrated
a protective effect of these compounds for certain cancers, other studies
in animals and cell cultures have demonstrated deleterious effects. Plasma
concentrations of isoflavones in soy-fed infants average 0.98 ug/ml compared
to .005 ug/ml in infants on breast milk or cow’s milk formula. On a body
weight basis, soy-fed infants consume about the same quantity of phytoestrogens
as adults who consume moderate amounts of soy foods. However, Japanese
adults who traditionally eat a variety of soy foods have plasma concentrations
of isoflavones considerably lower than that measured in the soy-fed infants.
How biologically active are the plasma isoflavones? Isoflavones in soy-based
infant foods are predominantly in the glucoside form which is unreactive.
In the adult digestive tract, these conjugated forms can be readily hydrolyzed
to form active compounds. However, we do not know how efficient infants
are at hydrolysis of the glucosides.
Soy-based infant formulae, fortified with certain minerals and amino
acids, have not been definitely implicated in any human illness or disorders
with the exception of allergic reactions and aluminum toxicity in some
preterm infants, but there does appear to be a potential for some adverse
neuroendocrine effects caused by phytoestrogens. One concern is that these
effects may not be manifested until many years later, for example, when
the soy-fed infants reach puberty.
Reference: Food Chem News, Nov 9, 1998, p4.
—Ellin Doyle
Since potatoes are a staple food in many countries, it has recently been
proposed that varieties of potatoes be developed which could act as vehicles
to induce immunity to food and waterborne pathogens. If this were technically
feasible, it might be an effective means for vaccinating populations, particularly
in underdeveloped areas, against epidemic diseases such as cholera. Potatoes
could perhaps induce immunity to foodborne infections from Salmonella
or E. coli. Several research groups are now working to achieve these
goals.
The gene for a cholera toxin B subunit pentamer, which binds to cell
surface receptors in the mucosa of the intestinal tract, has been introduced
into potato tissues. When raw tuber tissue from these transgenic plants
was fed to mice, it was found to induce both mucosal and systemic (serum)
antibodies. When the mice were challenged with cholera toxin, there was
a 60% reduction in the production of diarrheal fluid in the intestine.
The antibodies induced by the transgenic potatoes in the mucosal tissue
inhibited binding of the cholera toxin and significantly reduced symptoms
of cholera. Since humans don’t normally eat raw potatoes, potatoes were
cooked to determine whether cooking would destroy their ability to induce
anti-cholera antibodies. About 50% of the immunogenic activity remained
after cooking, hence the researchers suggested that future experiments
might be aimed at finding a suitable fruit, consumed raw, as a foodborne
vector for the vaccine.
In another series of experiments, the gene for the heat-labile enterotoxin
subunit produced by E. coli was introduced into potato tissues.
Mature tubers were harvested from plants grown from these transformed tissues
and were found to produce this toxin subunit which binds to the intestinal
mucosa. As with the previous experiments, mice fed these raw potatoes developed
mucosal and serum antibodies which provided partial immunity to the effects
of the E. coli enterotoxin. Further experiments with 14 brave and
healthy human volunteers, fed 50–100 g raw transgenic potatoes containing
an average of 0.75 mg of the E. coli enterotoxin subunit, demonstrated
that humans also develop both mucosal and serum antibodies after eating
these transgenic potatoes.
Fears over the safety of genetically engineered potatoes have been aroused
in England by results of some toxicity tests in rats. In these experiments,
genes coding for a lectin from snowdrop bulbs were introduced into potatoes
in an attempt to make them more pest resistant. Some of the rats fed these
potatoes exhibited adverse effects on their immune systems and on some
of their organs, including kidney, spleen, and brain. These results have
been disputed by other workers in the field and pending confirmation, should
be considered as preliminary. Lectins are considered antinutritional for
humans, though they are denatured or destroyed by heat, so cooked potatoes
may be safe.
—Ellin Doyle
return to:
| FRI Newsletter, Spring 1999
index | FRI Communications
| FRI home page |
Dept.
Food Microbiology & Toxicology | UW–Madison
|
Copyright © 1999
Food Research Institute
Last modified: 5 October
1999
Questions or Comments? e-mail
Barbara Cochrane