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LIBRARY
Michigan State
University

 

 

 

This is to certify that the

thesis entitled

Zinc Bioavaiiability in a Semolina/Soy Protein
Mixture Was Not Affected by Extrusion Processing

presented by

Soo-Young Kang

has been accepted towards fulfillment
of the requirements for

Masters Human Nutrition

degree in

 

 

Wanda L. Chenoweth

Major professor

 

Date December 9, 1996

 

0-7639 MS U i: an Affirmative Action/Equal Opportunity Institution

PLACE ll RETURN BOX to removethle checkout horn your record.
TO AVOID FINES return on or before date due.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU IeAn Nflnnetlve ActionEqueI OpportmIty Inetltulon
Wane-o1

ZINC BIOAVAILABILITY IN
A SEMOLINA/SOY PROTEIN MIXTURE
WAS NOT AFFECTED BY EXTRUSION PROCESSING
By

Soo-Young Kang

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1996

ABSTRACT

ZINC BIOAVAILABILITY IN A SEMOLINA/SOY PROTEIN MIXTURE
WAS NOT AFFECTED BY EXTRUSION PROCESSING

By

Soo-Young Kang

Effects of extrusion processing on zinc bioavailability in a mixture of 85%
semolina and 15% soy protein concentrate was determined using in vitro and in vivo
techniques. Soluble and dialyzable zinc content of the extruded product was 15.3 and
13.0 ug/g, respectively, compared to 15.6 and 15.0 ug/g in the raw ingredients. After a
zinc deficient diet period, zinc bioavailability in diets in which all of the zinc (14 ppm)
was provided either by the extruded product or by the raw ingredients was determined in
two groups of male rats. No differences were found in concentrations of zinc in plasma,
liver and femur between rats fed the extruded and raw diets after 3 weeks. Apparent zinc
digestibility was similar in the two groups. These results are consistent with the in vitro

results showing no effect of extrusion processing on zinc bioavailability.

To my family

ACKNOWLEDGMENTS

I am thankful to my advisor, Dr. Wanda L. Chenoweth for her guidance,
encouragement, and valuable advice throughout the entire course of the study. My
appreciation is also extended to the members of the committee, Dr. Maurice R. Bennink
and Dr. Dale R. Romsos.

I am also grateful to Kim F usee for her assistance during the animal study, and
Yong-Soo Chung and Ritu Ummadi for their friendship and supports.

Finally, I am particularly grateful to my mother and husband whose love and

support have always been the source of my inspiration to achieve and excel.

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................ vii
LIST OF FIGURES ......................................................................................................... viii
INTRODUCTION ............................................................................................................... i
LITERATURE REVIEW .................................................................................................... 3
1. Zinc metabolism ........................................................................................................... 3

2. Assessment of zinc bioavailability ............................................................................... 6

a. In vitro studies .......................................................................................................... 6

b. In vivo studies .......................................................................................................... 7

1) Human studies ..................................................................................................... 7

2) Animal studies ..................................................................................................... 8

3. Dietary factors affecting zinc bioavailability ............................................................. 10

a. Copper-zinc interactions ........................................................................................ 10

b. Iron-zinc interactions ............................................................................................. 11

c. Protein-zinc interactions ........................................................................................ 13

(1. Amino acid-zinc interactions ................................................................................. 16

4. Phytate-mineral interactions ...................................................................................... 1 8

a. Chemical characteristics of phytic acid .................................................................. 18

b. Interactions with minerals ...................................................................................... 19

c. Interactions with protein and mineral ..................................................................... 21

5. Extrusion processing ................................................................................................. 22

a. Effect of extrusion processing on zinc bioavailability .......................................... 23

b. Effect of extrusion processing on wheat and soybean protein ............................... 24

6. Summary ................................................................................................................... 29
MATERIALS AND METHODS ....................................................................................... 31
1. Preparation of extruded and raw products ................................................................. 31

2. Materials .................................................................................................................... 32

3. Research design ......................................................................................................... 32

a. In vitro studies ........................................................................................................ 32

1) Soluble zinc analysis ......................................................................................... 33

2) Dialyzable zinc analysis .................................................................................... 33

b. In vivo rat experiments .......................................................................................... 34

4. Diets ........................................................................................................................... 35

5. Analytical methods .................................................................................................... 37

a. Phytate .................................................................................................................... 37

b. Protein determination ............................................................................................. 39
c. Mineral analyses ..................................................................................................... 39
1) Wet ashing ......................................................................................................... 39
2) Products and diets .............................................................................................. 4O
3) Tissues ............................................................................................................... 4O
4) Feces .................................................................................................................. 41
5) NIST standard .................................................................................................... 41
d. Statistical analyses ................................................................................................. 41
RESULTS. ....................................................................................................................... 42
1. Analyses of products and diets .................................................................................. 42
a. Zinc, copper, iron and phytic acid content of products .......................................... 42
b. Zinc, copper, iron, phytic acid and protein content of diets ................................... 42
2. In vitro experiments ................................................................................................... 45
a. Soluble and dialyzable zinc content of products .......... '. ......................................... 45
3. In vivo animal experiment ......................................................................................... 45
a. Body weight, weight gain, food intake, efficiency of food utilization and total
mineral intake ........................................................................................................ 45
b. Organ wet weights, dry weights and % body weight of organs ............................. 48
c. Plasma concentration of zinc .................................................................................. 51
(1. Tissue concentration of zinc .................................................................................. 51
e. Tissue concentration of copper ............................................................................... 55
f. Tissue concentration of iron ................................................................................... 57
g. Feces weights and concentrations of zinc, copper and iron ................................... 57
h. Apparent digestibility of protein, zinc, copper and iron ........................................ 60
DISCUSSION .................................................................................................................... 62
CONCLUSION .................................................................................................................. 69
FUTURE RESEARCH ............................................................... 70
LIST OF REFERENCES ................................................................................................... 71

vi

LIST OF TABLES

TABLE 1: Composition of diets ...................................................................................... 36

TABLE 2: Analyzed phytate, zinc, copper, and iron content of extruded and raw
products ........................................................................................................... 43

TABLE 3: Estimated phytate and analyzed protein, zinc, copper, iron content of diets .44

TABLE 4: Soluble, percent soluble, dialyzable and percent dialyzable zinc in extruded

and raw products ............................................................................................. 46
TABLE 5: Body weights, food intake and food efficiency ratios ................................... 47
TABLE 6: Zinc, copper and iron intake .......................................................................... 49
TABLE 7: Organ wet weights and relative organ weights .............................................. 50
TABLE 8: Organ dry weights .......................................................................................... 52
TABLE 9: Plasma concentration of zinc ......................................................................... 53
TABLE 10: Concentration and total content of zinc in tissues ......................................... 54
TABLE 11: Concentration and total content of copper in tissues ..................................... 56
TABLE 12: Concentration and total content of iron in tissues .......................................... 58

TABLE 13: The weight of feces and concentrations and total contents of zinc, copper,

iron in feces ..................................................................................................... 59
TABLE 14: Apparent digestibility of protein, zinc, copper and iron ................................ 61

vii

LIST OF FIGURE

FIGURE 1: Possible mechanism for transcellular zinc absorption in the intestinal cells....5

viii

INTRODUCTION

Zinc is a trace mineral nutrient essential to the growth and health of all mammals,
including humans. In general, meat, eggs, shellfish, legumes, and whole grain cereals
have a high content of zinc. Dietary factors which influence the absorption of zinc include
“extrinsic factors”, that is, other chemical components of the diet that may inhibit or
enhance zinc absorption, and “intrinsic factors”, related to the chemical nature of the zinc
itself, which may increase or decrease its biological availability (Solomons, 1982).

Fairweather—Tait (1992) defined bioavailability as the proportion of the total
mineral in a food, meal or diet that is utilized for normal body function. Zinc
bioavailability of various foods in a mixed meal may not be the same as that
demonstrated in single food studies because of a variety of potentially complex nutrient-
nutrient interactions (Wood and Zheng, 1990).

Extrusion processing, a procedure used in mass production of cereal and legume
derivatives, cookies and other preshaped products, has become of major industrial
importance (Harper, 1979). Interactions among food components during extrusion
processing may have positive or negative effects on the bioavailability of nutrients,
including bioavailability of zinc. Although processing and cooking have little effect on
zinc itself in most food, zinc is a divalent ion and, as such, can form a wide variety of

inorganic salts and organic zinc complexes of varying stability during processing. It is the

latter form of zinc, particularly its association with a variety of zinc proteins and possible
increased risk of deficiency and toxicity, that has received the greatest share of attention
in zinc biochemistry to date (Smith, 1988). Formation of stable phytate-protein-zinc
complexes also has been suggested as an explanation of observed differences in zinc
absorption with different methods for processing (Erdman et al, 1980).

This research will investigate the effect of extrusion processing on zinc

bioavailability in a semolina product containing soy protein concentrate.

REVIEW OF LITERATURE

ZINC METABOLISM

Absorption of Zn is largely a function of the presence or absence of substances in
the food or meal that alter the solubility or availability of Zn compounds at the absorption
site. Zn is present in all organs, tissues, fluids, and secretions of the body. Zn is primarily
an intracellular ion, with well over 95% of the total-body Zn found within cells. Zn is
associated with all organelles of the cell, but about 60-80% of the cellular Zn is found in
the cytosol (King and Keen, 1994).

Zn is absorbed all along the small intestine; only small amounts are absorbed in
the stomach and large intestine. Most of Zn is probably absorbed in the jejunum.
Sandstrom and Lonnerdal (1989) reported that fiee Zn is able to form coordination
complexes with various exogenous and endogenous ligands, such as amino acids,
phosphates, and other organic acids

The mechanism by which Zn enters mucosa] cells is unknown. Zn uptake across
the brush border surface occurs by both a carrier-mediated mechanism and diffusion.
Research to elucidate the mechanism of Zn absorption (Cousins, 1989) has consistently
suggested the involvement of one or more carrier molecules in the absorption process, but
viable candidates have not been identified. Hempe and Cousins (1992) showed that
cysteine-rich intestinal protein (CRIP) binds Zn during transmucosal Zn transport, and

that CRIP functions as an intracellular Zn carrier. CRIP is a 77 -amino acid, 86-kDa

protein with seven cysteine residues (Birkenmeir and Gordon, 1986). Hempe and Cousins
(1992) also suggested that CRIP is a saturable, intracellular Zn transport protein.
Metallothionein (MT), a cysteine-rich, low-molecular-weight protein, has high affinity
for Zn, Cu and other heavy metals. MT is also suggested to inhibit Zn absorption in
competition with CRIP. They present the hypothetical model of Zn absorption shown in
Figure l to describe the envisioned roles of CRIP and MT in transcellular Zn transport.
Following a meal, Zn moving from the lumen into the enterocyte may associate with a
variety of intracellular constituents, including CRIP, MT and various nonspecific Zn-
binding constituents (N SB). Zn bound to CRIP would thus increase the intracellular
concentration of Zn that can diffuse across the cell for exchange and export at the
basolateral membrane, thereby enhancing the rate of Zn absorption. The possible
involvement of a basolateral membrane-bound acceptor/carrier molecule is indicated (=).
MT inhibits Zn absorption by limiting Zn binding to CRIP. When the luminal Zn
concentration is high, Zn bound to MT would be free to associate with NSB. NSB could
thus serve as a labile pool of Zn in the intestinal cell. The model indicates that
transcellular Zn absorption could be regulated by dietary or physiological factors that
alter either CRIP or MT gene expression. The number of Zn-binding sites in CRIP is not
known.

Zn trapped within the intestinal cell is eventually lost in the feces in the normal
course of mucosal cell turnover. Transport of Zn across the serosal membrane is can'ier
mediated and occurs by an adenosine triphosphate (ATP)-driven mechanism. Zn is
released by the intestinal cells at the basolateral-serosal surface into the mesenteric

capillaries and is carried by the portal blood to the liver. About 0.05 mo] (3 mg) of Zn

lumen Plasma

 

 

 

 

 

 

 

 

 

NSRIn
Albunin
i cm
In ++ Zn 4+
(RIP-7n
\ Albumin-Zn
NIT-2n m

 

CRIP: cysteine-rich intestinal protein
MT: metallothionein
NSB: nonspecific zinc-binding constituents

== : possible involvement of a basolateral membrane-bound acceptor/carrier molecule

Figure 1: Possible mechanism for transcellular zinc absorption in the intestinal cells.

( Adapted from Hempe and Cousins, J. Nutr. 1992)

is normally circulating in the plasma at any given moment. Harris and Keen (1989)
reported that this Zn is portioned among albumin (57%), a-Z-macroglobulin (40%), and
amino acids (3%). These loosely bound albumin and amino acid fractions of circulating
Zn provide the transport and delivery of Zn to tissues.

There is no specific Zn “store.” Nonetheless, some sources of endogenous Zn are
retained preferentially in certain tissues such as bone in response to a decrease in dietary
Zn. The major route for endogenous Zn excretion is into the gastrointestinal tract with
ultimate loss in the feces. Fecal losses are a combination of unabsorbed dietary Zn and
endogenous Zn secretions. Pancreatic secretions are the major source of endogenous Zn
losses. Other sources include biliary and gastroduodenal secretions, transepithelial flux of
Zn from the mucosal cells, and Sloughing of old mucosal cells into the gut. Surface losses
through desquamation of skin, outgrowth of hair, and sweat contribute up to 15 umol
(1 mg) of Zn daily. Other sources of loss include the semen and menstrual secretions

(Hambidge et al, 1986).

ASSESSMENT OF ZINC BIOAVAILABILITY

In Vitro Studies

In the past few years in vitro methods to assess the bioavailability of minerals and
trace elements have gained popularity because of their accuracy, speed of analysis and
relatively low costs. In vitro methods offer an appealing alternative to human and animal
studies. Most of the in vitro methods simulate gastrointestinal conditions. In the small

intestine several components of our food form soluble or insoluble complexes with

minerals and trace elements. The solubility of minerals and trace elements afler
Simulation of the conditions in the stomach (pH 1-2, 37 °C) and small intestine (pH 6.5-8,
37 °C) as a measure of in vivo bioavailability has been studied by Narasinga Rao and
Prabhavathi (1978), Wien and Schwartz (1983), Hunt et a1 (1987), Sandberg et a1 (1989),
Schwartz and Nevins (1989) and Tumland et a1 (1990). The authors reported
contradictory results with respect to the correlation between in vitro and in vivo
bioavailability.

Miller et al (1981) used the dialyzability of Fe under simulated conditions of the
stomach and the small intestine as an index for its bioavailability. This method has been
modified by some other researchers for estimation of the bioavailability of Fe and Zn.
Promising correlations between in vitro dialyzability and in vivo bioavailability were
reported (Schricker et al, 1981; Hazell and Johnson, 1987; Hurrell et al, 1988; Sandstrom
and Alrngren, 1989). However, it is concluded that the prediction of bioavailability with
in vitro methods is relative rather than absolute because not all important physiological
factors can be simulated in vitro. Thus, in vitro methods may be useful primarily for

ranking purposes.

In Vivo Studies

Human studies. In vivo experiments with experimental subjects are the best way
to study the bioavailability of minerals and trace elements to man. Estimation of apparent
absorption using the balance study technique requires long periods on a constant diet
(Schwartz et al, 1986) to achieve equilibrium and is not a sensitive method for the

identification of individual factors that influence Zn absorption. For example, Tumlund et

a1 (1982) showed that fecal Zn samples collected from 1 day’s Zn intake can also contain
non-absorbed fractions of Zn from the previous days’ meals as well as fractions of
endogenous Zn excreted over the same period of time. Using a radionuclide, Zn
absorption can be determined from measurements of the whole-body retention of the
isotope. The meals are labeled with radioisotope extrinsically or intrinsically. In recent
years there has been interest in the use of stable isotopes such as 67Zn as a safe alternative
to study Zn metabolism, particularly in infants and pregnant women. The increase in
plasma Zn after a Zn load also has been used as a method to identify dietary and
physiological factors affecting Zn absorption. One of major limitations of this method is
that the dose of Zn needed to produce detectable increases of plasma Zn concentration is
much higher than the normal dietary level of Zn. Human studies, however, are time-
consuming and very expensive, and often quite variable results are obtained which are

difficult to interpret. As an alternative, the rat is often used as a model for man.

Animal studies. In many studies in experimental animals, stock diets are fed for
long periods prior to the test period. These diets are usually formulated to provide at least
a minimum supply of most nutrients. The use of Zn—deficient diets in such experimental
models could also induce systemic changes in the intestine that may influence the
outcome of Zn absorption studies. Sandstrom and Lonnerdal (1989) summarized
intestinal uptake models for assessing Zn bioavailability: 1) Animals in which a segment
of the small intestine is tied off or a loop of intestine is extemalized. The intestine is then
perfused with a solution of Zn and by measurements of changes in Zn concentration,

transfer rates and kinetics of Zn transport can be determined. 2) Another approach is to

use everted gut sacs; intestinal segments are surgically removed, everted. 3) Brush border
membrane vesicles (BBMV) prepared by physically removing the mucosa and selectively
precipitating the BBMVS. These methods have potential drawbacks including effects of
anesthesia on metabolism and absence of intact innervation and blood supply. The
advantage of the above approaches is, of course, that many conditions can be closely
controlled and, thus, effects of pH, ionic strength, concentrations of ions, ligands,
competitors and stimulatory factors can be determined. Radioisotope studies in animals
have been used in a way not possible in human subjects. Information can be gained about
the metabolic fate of the absorbed Zn; tissue can be homogenized and the molecular
localization of the element can be determined. Fox et a1 (1994) reported that the isotOpe
such as 65Zn was more likely to behave in the same manner as the intrinsic Zn than the
stable isotope 67Zn.

Total femur Zn and body weight of weaning rats were shown to be suitable
parameters for a Zn bioassay ( Momcilovic et al, 1975). Femur Zn was compared to body
weight as a suitable parameter for the determination of Zn bioavailability. They
concluded that a slope-ratio bioassay was appropriate for the determination of the
bioavailability of Zn, when the logarithm of total femur Zn was plotted against the level
of dietary Zn. Forbes and Parker (1977) were able to successfully measure Zn
bioavailability from soy flour utilizing male weaning rats in a slope ratio assay procedure
patterned after Momcilovic et al (1975). Hettiarachchy and Erdman (1984) also
determined the bioavailability of Zn in mature winged bean seed flour in a slope ratio

assay by measuring weight gain and total tibia Zn.

10

DIETARY FACTORS AFFECTING ZINC BIOAVAILABILITY

A theoretical framework for interactions between minerals and trace elements was
set by the studies by Hill and Metrone (1970). These investigators postulated that
elements with similar physicochemical properties may compete for common pathways
and act antagonistically to each other biologically. In biologic systems Zn is virtually
always in the divalent state. Zn typically forms complexes with a coordination number of
4 with a tetrahedral disposition of ligands around the metal (King and Keen, 1994). These
parameters are identical to those for Cu2+ and Cd“, and therefore these elements would be
expected to interact with Zn. Another interaction, less readily applicable in the basis of
common structural features of the ions involved, is that between Zn and Fe (Solomons,
1986). In this case, the mutual affinity of Zn and Fe for a biological carrier such as
transferrin, capable of binding many different elements, explains the interaction. Zn also
readily complexes to amino acids, peptides, proteins, and nucleotides. Zn has an affinity

for thiol and hydroxy groups and for ligands containing electron-rich nitrogen as a donor.

Copper-Zinc Interactions

Cu-Zn interactions have been of particular interest. Large quantities of ingested
Zn can interfere with Cu absorption. Because of the necessity of Cu for Fe metabolism,
anemia may result (Patterson et al, 1985). The apparent Cu retention and fecal excretion
are clearly modulated by the concentration and duration of dietary Zn ingestion. Festa et

al (1985) showed that an intake of 150 mg Zn per day for 2 weeks reduced apparent Cu

ll

retention in a group of young men. There is some evidence that even relatively low levels
of dietary Zn may interfere with Cu absorption. In adult males, it was shown that an
intake of Zn only 3.5mg/day above the RDA reduced apparent Cu retention when Cu was
ingested at the level of 2.6mg/day (F esta et al, 1985). Studies in rats using isolated,
vascularly perfused rat intestines and dietary concentrations of 5, 30 and 180 mg Zn/kg
and 1, 6, 36 mg Cu/kg have not, however, shown mutually interactive effects on their
absorption ( Oestreicher and Cousins, 1985). A high intake of Cu does not inhibit Zn
absorption. Valberg et a1 ( 1984) found no effect of 5 mg Cu on the absorption of 0.5 mg
Zn in water in humans. In human diets the relation between Zn and Cu is almost always a
higher Zn than Cu content, and Cu is practically not a serious inhibitor of Zn absorption.

It is understood that the key to the interaction between Zn and Cu lies in the
induction of intracellular MT. MT is a Cu- and Zn- binding protein that is induced by
high levels of Zn and Cu in the diet (Bremmer and Beattie, 1990). MT concentration in
intestine responds to an increased absorption of Zn, Cu and Cd but seems to be more
influenced by dietary Zn than by Cu (Oestreicher and Cousins, 1985). A high intake of
Zn induces the synthesis of MT in the mucosal cell. This protein sequesters Cu, making it
unavailable for serosal transfer and thus decreases Cu absorption. This property has been
put into therapeutic use as a means to limit the absorption of Cu in Wilson’s disease. The
long-term Zn therapy, at a dose of 100 to 200 mg/day of elemental Zn, suffices to prevent
the continued accumulation of Cu in the liver or the reaccumulation after a period of

penicillamine therapy (Brewer et al, 1987 ; Sandstead, 1987).

12

Iron-Zinc Interactions

The interaction between Fe and Zn is still controversial. Pharmacological doses of
Fe are often given to groups of subjects with high Zn requirements such as pregnant
women and teenagers. Furthermore, diets like infant formulas are often enriched with Fe.
A negative effect of Fe on Zn absorption could therefore have serious implications for
supplying Zn to vulnerable groups. High levels of Fe in the diet have been shown to
inhibit Zn absorption by competition with endogenous low molecular weight ligands
(Evans and Johnson, 1981; Gordon, 1983). Ferrous iron, but not heme iron, has been
known to inhibit Zn absorption when the molar proportion of Fe is two or three times that
of Zn. The effects of ferric ion on Zn absorption are less marked than those of the
divalent form (Solomons et a1, 1983). The interaction of Fe and Zn and competitive
inhibition of Zn uptake by excess Fe in the ratio of 2:1 or greater appear to have a
measurable effect on human Zn nutriture. Pregnant women and artificially fed infants
consuming Fe supplements or Fe-fortified formulae may develop Zn deficiency,
suggesting a negative impact of excessively high Fe:Zn ratios in diet (Solomons and
Cousins, 1984). Using body retention of 65Zn as an index of absorption, Valberg et al
(1984) found that both inorganic Fe and heme Fe inhibited Zn absorption in humans from
6-mg doses of Zn as Zn chloride. Inorganic Fe had no effect, however, on 65Zn absorption
from an extrinsically labeled turkey test meal. Similar differences in the effects of Fe on
Zn absorption depending upon whether the Zn is supplied in aqueous solution or from a
meal have been observed by Sandstrom et al (1985). The absorption of Zn from infant
formulas by adults was not influenced by differences in the Fe content of the two

formulas (Lonnerdal et al, 1984). Solomons (1986) has reviewed studies of the interaction

13

between Fe and Zn and suggested that the total amount of ionic species determines the
effect on absorption of Zn. He claimed that when the total amount of ions given as a
single oral dose in solution is greater than 25 mg, a measurable effect on human Zn
nutriture can be expected. The different effect of Fe on Zn absorption from a solution and
from a food exemplifies the complex interactions between promoters and antagonists of
absorption. The practical implication of these results could be that higher amount of Fe
than that of Zn can reduce the efficacy of a Zn supplement while normal levels of Fe used
in enrichment of food do not seem to significantly impair Zn supply. However, most
studies of Zn absorption have been performed in healthy non-pregnant adults, and the
effect of Fe could be different in groups with higher Zn or Fe needs. Most, but not all, of
the direct Zn absorption studies in human subjects, in which the total acute oral dose has
exceeded 25 mg of Zn, have shown an inhibition of Zn uptake by pharmacological doses
of ferrous iron (Solomons and Jacobs, 1981; Solomons et al, 1983; Meadows et al, 1983).
However, Castillo-Duran and Solomons (1991) measured Zn absorption in healthy
volunteers following ingestion of 400 g of cooked beef containing 16 mg of Zn. Fe/Zn
ratios of 1:1, 4: 1-but not 7: l-achieved by addition of 16, 64, and 112mg of ferrous iron in

the form of ferrous sulfate produced a significant reduction in the plasma Zn response.

Protein-Zinc Interactions

The effect of dietary protein on Zn absorption in humans is also controversial.
Both the type and the amount of protein appear to affect the degree of Zn absorption.
Significantly lower fecal Zn losses were observed in adult males fed a high protein diet

(150g/d), achieved by intake of a bread enriched with purified protein sources, as

14

compared with an intake of 50 g protein/d and similar Zn intakes ( Greger and Snedeker,
1980). Urinary Zn excretion, however, was increased on the higher protein intake and the
apparent retention of Zn was not statistically different. Much research has been done on
the absorption of the Zn present in milk itself. Zn in cow’s milk is less available than in
human milk. Many observations suggest that the protein content and composition of milk
diets to a large extent influence the intestinal handling of Zn. In cow’s milk, Zn is found
predominantly bound to the high-molecular-weight protein, casein ( Cousins and Smith
1980; Harzer and Kauer, 1982; Blakeborough et al, 1983), and it was suggested early on
that Zn bound to casein may not be utilized by the young infant ( Lonnerdal et al, 1980).
From in vitro dialysis experiments a Zn-binding capacity of 8.4 ug Zn/mg bovine casein
has been observed at slightly alkaline pH ( Harzer and Kauer, 1982). This study also
indicated that Zn is complexed to the negatively charged phosphate groups of casein.
Human milk contains a lower concentration of casein which is also less phosphorylated
than bovine casein. Casein separated from human milk by ultracentrifugation was found
to contain 14% of the total Zn content; albumin bound 28% and the remaining Zn was
equally distributed between fat and low-molecular-weight ligands (Lonnerdal et al, 1982).
In human adults, dietary proteins are digested to 80-90% in the small intestine where the
protein-bound Zn is probably rendered available for absorption. Incomplete hydrolysis of
the casein in cow’s milk may lead to decreased absorption of Zn in infants (Lonnerdal et
al, 1985). Cow’s milk and lactose-free cow’s milk significantly reduced Zn absorption in
lactose-tolerant and lactose-intolerant postmenopausal women when fractional Zn
absorption was studied with a dual-isotope technique using “Zn and (”Zn (Wood and

Hanssen, 1988). Greger et a1 (1989) reported that rats fed supplemental lactose or

15

unhydrolyzed cow’s milk retained more Zn and Mg in bone. Wood and Zheng (1990)
reported no significant effect of milk or calcium phosphate supplementation on either Zn
absorption or retention in postmenopausal women. They concluded that increased milk
consumption with meals has no detrimental consequences on Zn nutriture in elderly
women.

In view of the high affinity of Zn for undigested protein, even a minor impairment
in protein digestion could affect Zn absorption (Wapnir, 1990). Istfan et a1 (1983)
measured Zn absorption, with the aid of the stable isotope 70Zn, from a formula diet with
either egg protein or a soy protein concentrate as the sole source of protein and with 8.5
or 12.8 mg of added Zn as Zn chloride. No significant difference in Zn absorption
between the diets was observed.

The most questioned protein source, in terms of its effects on mineral absorption,
has been soy isolate. Forbes and Parker (1977) showed that Zn in a soy protein product
was less available than that from a highly available source, ZnSO,, though the presence of
soy protein did not affect availability of Zn from other sources in the diet. Lo et a1 (1981)
suggested that this inhibitory action in isolated soy protein may relate to 1) the residual
amount of phytate remaining after the isolation and purification procedure, which may
produce an elevated phytate to Zn ratio, 2) the structure and nature of soy protein itself. A
much lower absorption of Zn from a soy-based infant formula compared with human
milk or cow’s milk formula has been observed in adults ( Sandstrom et al, 1983), and
growth studies of children recovering from protein-energy malnutrition also imply a poor
availability of Zn from soy-based formulas (Golden and Golden, 1981). Zn in soy

formula is associated with the large-molecular-weight proteins and the solubility of Zn

l6

bound to this soy protein fraction at physiological pH is low ( Sandstrom et al, 1983).
Although a lower small intestinal protein digestion of a 25% soy protein diet compared
with a meat diet has been observed in ileostomates (Sandstrom et al, 1986), this had no
significant effect on apparent Zn absorption. When all meat protein was replaced by soy
protein, a lower fractional absorption than expected from the dietary Zn content was
observed in studies using the extrinsic labeling radionuclide technique to measure Zn

absorption from a composite meal (Sandstrom et al, 1987).

Amino Acids-Zinc Interactions

Competition between Zn and other elements for absorption binding sites can
influence absorbability. Meat, liver, eggs, and seafood are considered to be good sources
of Zn because of the relative absence of chemical constituents that inhibit Zn absorption
and because of certain amino acids that improve Zn solubility. Cysteine and histidine
enhance Zn absorbability by forming stable complexes with Zn. Greger and Snedeker
(1980) also observed that supplementing a low protein diet with sulphur-containing
amino acids increased urinary Zn excretion in adult males. While this could indicate a
higher absorption of Zn, it could also be due to other changes in Zn metabolism induced
by the changed amino acid intake ( Snedeker and Greger, 1981). Snedeker and Greger
(1983) studied the effects on Zn absorption of a variety of dietary protein levels with and
without supplements of histidine and cysteine. Apparent absorption of Zn was higher
when rats were fed a 45% lactalbumin diet or a 15% lactalbumin diet supplemented with
histidine and cysteine than when fed the unsupplemented 15% protein lactalbumin diet.

An improvement in Zn absorption was also observed in rats when cysteine and

l7

methionine were added to a soy protein diet; absorption was similar to that from a
lactalbumin-containing diet ( Greger and Mulvaney, 1985). Magee and Lugeye (1985)
showed that chicks that received an amino acid supplement, without additional mineral
reinforcement, had an improvement in their Zn status, indicating that amino acids
sufficed to improve the mineral balance of birds without extra doses of the element. The
greater Zn absorption achieved by amino acids in the small intestine appeared to be due
to both mediated and nonmediated transport mechanisms for amino acids. Greger (1988)
suggested that amino acids may improve absorption by forming soluble complexes with
Zn.

The amino acid with the highest affinity toward Zn, histidine, may be involved in
Zn homeostasis. There have been attempts to titrate the optimum concentration of
histidine in rodent diets in order to maintain normal Zn level in serum. Increases in this
amino acid from 0 to 0.40% correlated with an increase in serum Zn and Cu (Koo et al,
1986). However, diets with 8% histidine in the diet produced low plasma Zn and severe
tissue Zn deficiency in rats (Wensink and Van den Hamper, 1988). Moderate amounts of
histidine have given clear results in human. Ingestion of Zn complexed with histidine at a
ratio of 1 :2 or 1:12 increased serum Zn concentration 25% more than ingestion of Zn
sulfate. Zinc histidine complexes are better absorbed than zinc sulfate in humans
(Scholrnerich et al, 1987). Wapnir et a] (1986) suggested that proline and tryptophan
have affinities toward Zn almost as great as that of histidine in humans. It has been
postulated that the increased availability of Zn from animal (meat) sources is caused by a
“ meat factor”. Sulflrydryl compounds such as cysteine and glutathione that are high in

meat may bind Zn and increase absorption efficacy (Hortin et al, 1991).

l8

PHYTIC ACID-MINERAL INTERACTIONS

Chemical Characteristics of Phytic Acid

Phytic acid, myoinositol hexaphosphate (l,2,3,4,5,6 hexakis dihydrogen
phosphate), is a naturally occurring compound formed during the maturation of seeds and
cereal grains. In the seeds of legumes, it accounts for about 70% of the phosphate content
and is structurally integrated with the protein bodies as phytin, a mixed potassium,
magnesium, and calcium salt of inositol (Erdman, 1979). Phytic acid is the most abundant
form of phosphorous in plants. Sandberg et al (1987) showed that during food processing
and digestion, inositol hexaphosphate can be partially dephosphorylated to produce
degradation products, such as penta-, tetra-, and triphosphate, by the action of
endogenous phytases, which are found in most phytic acid-containing seeds.

Phytic acid has been considered as an antinutrient due to its inhibitory effect on
mineral bioavailability. The conformation of this highly polar compound explains its high
affinity toward di- and trivalent cations (Wapnir, 1990). The most striking chemical
impact of phytic acid is its strong chelating ability with multivalent cations, especially di-
and trivalent cations, to form cation-phytic acid complexes. Zhou and Erdman (1995)
reviewed that the complexes are usually soluble at acidic pH, but they have limited
solubility at neutral pH, a pH near to that in the small intestine. The insolubility of the
complexes is regarded as the major reason for the reduced bioavailability of phytic acid-
mineral complexes. Minerals of concern would include Zn, Fe, Ca, Cu as well as
phosphorous itself from phytic acid. In particular, phytic acid-induced Zn deficiency has

been extensively reported.

l9

Interactions with Minerals

The phytate to Zn molar ratio has been suggested as a parameter to predict Zn
bioavailability. Oberleas and Harland (1981) proposed that in humans adequate dietary
Zn can be provided by a daily diet having a phytate to Zn molar ratio of 10 or less. In rats,
low-phytate wheat brans with phytate to Zn molar ratios of 8 or less were equivalent to
Zn sulfate as dietary sources of Zn for growth (Monis and Ellis, 1980b). Ellis and Morris
(1981) also reported that reduction of the soybean phytate:Zn molar ratio from 33 to 13
improved the bioavailability of soybean Zn to rats. Thus they found that femur Zn values
of rats fed soybean at a phytate:Zn molar ratio of 13 were significantly greater than values
of rats fed untreated soybean as the Zn source. Lo et a1 (1981) found that phytate has a
marked effect on Zn absorption when the Zn status of the animal is low but has little
effect when the phytate-Zn molar ratio is nine or less and the physiological Zn status is
adequate. It has been suggested that this difference in absorption relates to the fact that
phytate is hydrolyzed in the intestinal lumen by the Zn-dependent enzyme, phosphatase
or phytase, in Zn-adequate but not in Zn-deficient animals. On the basis of animal
experiments to date, a daily phytate: Zn molar ratio of 10 or less is thought to be
acceptable in providing adequate dietary Zn, and daily ratios consistently above 20 may
jeopardize Zn status (Zhou and Erdman, 1995).

Phytate also forms complexes with more than one mineral in a complex. The Ca
content of the diet is of vital importance to the negative impact of phytic acid on Zn
bioavailability. Ca exaggerates the inhibitory effect of phytic acid on Zn bioavailability
by forming insoluble Ca-phytic acid-Zn complexes. The interaction is so strong that a

[phytic acid] x [Ca] / [Zn] molar ratio has been found in some studies to be a better

20

predictor of Zn bioavailability than [phytic acid]/ [Zn] molar ratio alone (Bindra et al,
1986; Davies et al, 1985). In an in vitro study, Gifford-Steffen and Clydesdale (1993)
observed that at pH 5.5, there was a significant increase in soluble Zn and soluble phytate
at the level of 0.25 mmol Ca compared to the higher levels (0.49-4.94 mmol) due to the
possible formation of a soluble Ca-phytate-Zn complex. Various combinations of Ca
(4.94 mmol), Zn (0.0071 mmol) and phytate (0.284 mmol) were added either to soy
concentrate, casein or torula yeast. The presence of phytate in the protein+Zn+phytate
treatment of soy concentrate resulted in a highly significant reduction in solubility of both
protein and Zn. CaCO3 significantly reduced Zn solubility in all proteins, both with and
without phytate. Ca solubility also was significantly reduced but only when phytate was
present. Phytate solubility decreased at pH 5.5 when Ca was present. This work supported
the hypothesis that increasing amounts of Ca in a diet containing Zn and phytate might
reduce the bioavailability of Zn. However, in vitro solubility does not necessarily
correlate with bioavailability.

Dietary levels of Ca used in animal experiments are frequently much higher than
those in human diets. Addition of milk and cheese to a phytic acid-containing bread
improved the absorption of Zn (Sandstrom et 31,1980). However, the content of many
other nutrients also was increased by the addition of milk, and it was concluded that the
increase in protein content of the meal was counterbalancing the negative effect of phytic
acid. When calcium chloride was added to a phytic acid-containing soy isolate formula,
an improvement of Zn absorption was also observed (Lonnerdal et al, 1984). These

results indicate that at the levels of Ca and phytic acid found in most human diets,

21

the Ca-phytate—Zn interaction observed in animal studies has limited relevance with

regard to Zn absorption in humans.

Interactions with Protein and Mineral

According to Cheryan (1980), phytic acid has an electrostatic interaction with
proteins. At a low pH, below the isoelectric point of proteins, terminal lysyl, histidyl, or
arginyl groups could be positively charged. Any of these groups can directly form a
complex with a negatively charged phytate anion. At the same time, the protein molecule
could bind with additive phytate anions, depending on the number of positively charged
groups and conformational conditions. At intermediate pH values, only the lysyl and
arginyl groups are still positively charged, so that a slight possibility of electrostatic
interactions exists. If the pH is very high, the interaction between phytic acid and protein
is diminished (Lésztity and Lasztity, 1990).

The presence of other components in the food system gives additional possibilities
for protein-phytic acid interactions. If polyvalent cations, such as Zn or Ca are present,
the cations may form a bridge between the phytate anion and a negatively charged group
of protein, thus allowing the binding of phytic acid by proteins at neutral and high pH.
Formation of stable protein-phytic acid-Zn complexes has been suggested as an
explanation of observed differences in Zn absorption with different methods for
processing of soya protein (Erdman et al,1980). Forbes and Erdman (1983) suggested that
these complexes could be thermodynamically stable and resistant to proteolytic digestion
in the gastrointestinal tract. Phytate has been suggested to influence protein digestibility.

Sandstrom et al (1986) observed in ileostomy patients that complete recovery of phytic

22

acid from a soy protein-based diet in small intestinal digesta was associated with a
concomitant reduction in protein digestibility. Ritter et a1 (1987) reported that in vitro
digestibility (determined by equilibrium dialysis method) of soy protein with lower
phytate content (0.07%) was greater than soy protein with a higher content (1.41%).
Honig and Wolf (1991) investigated the associations of essential minerals with specific
proteins or phytic acid by gel filtration techniques, and they found associations of protein
with phytic acid and minerals in defatted soybean flakes. There was a significant negative
correlation between phytic acid and in vitro protein digestibility of soybean and other
legumes (Chitra et al, 1995). Singh et al (1991) claimed that phytic acid reduced protein

digestibility in groundnut.

EXTRUSION PROCESSING

Harper (1989) defined extrusion cooking as a high-temperature short-time
(HTST) technique for production of a range of foods and food ingredients-especially
plant proteins, starches, and cereal products. Extrusion cooking has some unique features
compared to other heat processes in that the material is subjected to intense mechanical
shear. It is capable of breaking covalent bonds in bipolymers.

Food extruders are generally divided into two classifications, single-screw and
twin-screw. Because of its improved pumping action, the corotating twin-screw extruder
is finding increased use to produce products which require precise process control
(Huber, 1991). The operation of an extruder has been summarized by Harper (1989).

Ingredients are released from the feed hopper into the preconditioner at a controlled rate.

23

Raw granular food ingredients are uniformly moistened or heated by contact with water
or steam in the preconditioner before entering the extruder. When the food enters the
extruder, the extrusion screw sequentially conveys and heats food ingredients and works
them into a continuous plasticized mass while rotating in a tightly fitting barrel. As the
flights on the extruder screw convey the food materials down the barrel, the mechanical
energy used to turn the screw is dissipated causing a rapid rise in the temperature of the
food ingredients. The resulting plasticized feed ingredients are then forced through a die.
The pressure drop across the die and rapidly converts the high-temperature water in the
product to steam and causes puffing to occur. The advantages of extrusion processing
over conventional cooking procedures are its relatively low cost, high productivity,
energy efficiency, ability to produce high quality products, ability to process dry, viscous

materials, and ease of production of new foods and product shapes (Harper, 1989)

Effect of Extrusion Processing on line Bioavailability

Because the bioavailability of minerals is influenced by phytate, the effect of
processing on the phytate content of extruded products is therefore of interest. Anderson
et al (1981) reported a 13-3 5% reduction in phytate content in an extrusion-cooked,
bran-enriched product. Because Zhou et a1 (1992) showed phytate reduction in soybean
protein increased Zn bioavailability in rats, one might postulate that extrusion processing
would increase Zn bioavailability. Kivisto et al (1986), however, suggested that Zn
bioavailability was lower in extruded than in unprocessed bran due to a processing

induced deactivation of the phytase naturally present in cereals and legumes.

24

In contrast, mineral bioavailability was not affected by extrusion processing in
some studies. There was no alteration in the absorption of Fe from potato and maize
containing low levels of phytate as a result of extrusion cooking at 120-140 °C in rats

(Fairweather-Tait et al., 1987). Other studies conducted with normal adults and using the

stable isotopes, ’8 Fe and ’7 Zn, revealed that extrusion cooking of a bran flour mixture at
a maximum temperature of 140 °C produced no effect on the retention of these two
elements (F airweather-Tait et al 1989). The test meals were comprised of 100 ml
pasteurized full-fat milk plus 40 g of a 1:1 mixture of wheat bran and wheat flour.
Because Platt et al (1987) reported that milk exerted a positive effect on the phytate
induced precipitation of Zn and Fe and thus their potential bioavailability, any changes in
Zn availability caused by extrusion processing may have been counteracted by the milk.
Moreover, there were differences in the composition of diets and variations in extrusion
conditions. Therefore the conflicting results on the effects of extrusion on Zn availability
reported by various researchers may be partially explained by the differences in

temperature, pressure, and moisture conditions used during extrusion processing.

Effects of Extrusion Processing on Wheat and Soy Protein

The traditional classes of wheat endospenn proteins as separated by
fractional extraction using the procedure of Osborne (1907) include: 1) the gluten
proteins (gliadins, LMW glutenins, and residue proteins or HMW glutenins) and 2) the
nongluten or soluble proteins ( albumins and globulins). Wheat flour and gluten are

important feedstocks. The importance of chemical and physical reactions on mass

25

transport and heat transfer during extrusion has been recognized, and the importance of
chemical reactions in both starch and protein fi'actions of commonly extruded food
materials has been recognized. Wheat flour has a unique composition compared to other
feedstocks. Though protein comprises 10-14% (wt) of the total flour, it is responsible for
the viscosity and elasticity of dough to a great extent. Gluten is comprised off glutenin
and gliadin. Glutenin, a linear, asymmetric protein with molecular weights > a million
Daltons, is responsible for the viscoelastic properties of gluten (Strecker et al, 1995).
Edwart (1988) determined the influence of disulfide bonds on gluten viscoelastic
properties and structure. Glutenin was hypothesized as a trifirnctional monomer capable
of polymerizing via disulfide bond formation. Schofield et al (1983) analyzed the effects
of heat on sulfhydryl-disulfide interchange reactions and concluded they were primary
bonds responsible for network formation upon thermosetting. The reaction mechanism in
gluten during extrusion processing was dominated by polymerization providing a buffer
to shear forces (Strecker et al, 1995).

Soybeans are high in protein but also contain a number of minor constituents
traditionally considered to be antinutritional factors. These include phytic acid, trypsin
inhibitors, saponins and isoflavones. Soybean processing changes the content of these
minor constituents in various ways. During the course of soybean processing to change
physicochemical properties, the chemical composition may change as well. Most
texturized soy proteins are produced from soy flours or soy protein concentrates by
extrusion. Extruders compress and work soy ingredients into a flowing, hot, plastic mass

(Rockey et al. 1993).

26

Soy protein products are mainly used as ingredients in formulated foods and
seldom are seen by the public. Most soy proteins are derived from “ white flakes,” made
by dehulling, flaking and defatting soybeans by hexane extraction. These may then be
milled into defatted flours or grits containing ~50-54% protein; extracted with ethanol or
acidic waters to remove flavor compounds and flatulence sugars, producing soy protein
concentrates containing 65-70% protein ( Lucas and Riaz, 1995). The main objective in
producing soy protein concentrates is to remove strong-flavor components and the
flatulence sugars (stachyose and raffinose), but other soluble compounds and some
minerals also are extracted. In turn, both the protein and dietary fiber contents are
increased. Approximately 20% of total nitrogen is lost as albumins when preparing
isoelectric-leached soy protein concentrates. Most soy proteins are globulins, whose
solubility and dissociation are greatly affected by pH and salt content of the solution. In
native soybean protein molecules, most amino acid residues responsible for the chemical
reactions during processing of soybean protein foods-such as cysteine(-SH), cystine
(S-S), and hydrophobic amino acid residues- are buried in the inside region of the
molecule, inaccessible to water. These residues become reactable with each other through
the exposure from the inside by heat denaturation during extrusion processing. The
unique texture of texturized soybean products produced by extrusion is the result of both
the intermolecular interchange reaction between the exposed -SH and S-S groups and the
intermolecular hydrophobic reaction among the exposed hydrophobic amino acid
residues (Fukushima, 1991).

Guj ska and Khan (1991) showed that a high degree of protein insolubility was

found in pinto and navy beans after extrusion at 110-150°C. The results suggested

27

formation of ternary phytate-protein-mineral complexes during extrusion processing and
these complexes decrease the solubility of protein. Prudencio-Ferreira and Areas (1993)
Showed that disulfide linkages, and hydrophobic and electrostatic interactions were the
main stabilizing mechanisms for the 3-dimensional structure and moisture contents of soy
protein. Infrared spectra showed the presence of B-sheet anti-parallel structures. Peptide
bonds were of negligible importance in extrusion texturization of soy protein. It is well
established that extrusion processing decreases the solubility of semolina proteins.
Dexter and Matsuo (1977) previously reported that extrusion processing decreased the
solubility of semolina proteins. Various studies have also reported that high-temperature
drying of pasta resulted in decreased protein solubility (De-Stefanis and Sgrulletta, 1990;
Aktan and Khan, 1992). Ummadi et al (1995) Showed that extrusion processing at 50 and
96 °C caused a marked decrease in the percentage of total protein present as albumin,
globulin, gliadin, and glutenin fractions with a corresponding increase in the insoluble
residue fraction. Extrusion at a higher temperature (96 °C) caused a greater increase in
insoluble residue than did the lower temperature (50 °C). The results also suggested that
extrusion may have promoted the formation of additional disulfide bonds, although
Dexter and Matsuo (1977 ) reported that extrusion of semolina at 50 °C did not produce
any significant changes in disulfide bond levels. The role of disulfide bonds in thermal
extrusion effects on protein solubility has been studied previously. Hager (1984) showed
that disulfide bonds contribute to the new, extended protein networks produced by
extrusion of soy concentrate. Rhee et a1 (1981) reported that extrusion at 138 °C reduced
protein solubility; an increase in protein solubility was achieved by the use of 2-

mercaptoethanol and SDS, suggesting disulfide linkages may be responsible for the

28

decrease in protein solubility after extrusion. Li and Lee (1994) showed that the solubility
of protein in wheat flour extrudate increased at an extrusion temperature of 60 and 90 °C
but not at 120 and 160 °C. They reported that disulfide bond and hydrophobic
interactions occurring during the extrusion processing caused changes in solubility of
protein in extruded wheat flour and egg white. Extrusion processing imparts thermal and
mechanical energy to dough and causes unfolding and reassociation of the protein
molecules, forming new linkages. Wheat flour protein was more highly crosslinked by
disulfide bonds with extrusion cooking, and some covalent bond cross-linking was
formed during extrusion (Koh and Schaich, 1994).

Extrusion may also influence protein digestibility of products. Dahlin and Lorenz
(1993) reported that in vitro protein digestibility of extruded wheat was improved by
extrusion. Products extruded at 100-150 °C showed the highest protein digestibility.
However, Rudolph et al (1983) and Knabe et a1 (1989), in studies with growing and
finishing pigs, reported lower apparent ileal digestibility values of crude protein and
amino acids in extruded soybean than in soybean meal. In a study by Fan et al (1995),
pigs were fed on maize starch-based diets formulated to contain 160 g/kg of crude protein
from soybean meal, soybean meal plus extruded soybean. The apparent ileal digestibility
values of crude protein and most of the amino acids were higher in soybean meal than in
extruded soybean. They found that high residual trypsin inhibitor activity in extruded
soybean was 2.5-4.0 times higher. In contrast, Godinez et a1 (1992) reported that
digestibility of bean protein is related to the presence of trypsin inhibitors. Inhibition of

trypsin, an enzyme actively involved in proteolysis, could possibly impair protein

29

digestibility, and inhibitors are inactivated by thermal treatment such as extrusion

processing.

SUMMARY

The purpose of this research is to determine the effect of extrusion processing on
Zn bioavailability. High temperature short time extrusion processing has become
increasingly used for texturization of plant proteins (Harper,1986). To date there have
been limited studies to determine bioavailability of Zn in extruded products. According to
Sandberg and her colleagues (1986), extrusion processing of a high-fiber cereal causes a
loss of endogenous phytase activity, the phytate hydrolytic enzyme present in whole
grains, but does not alter the availability of the protein, fat or starch. The apparent
absorption of certain minerals in a high fiber cereal product was altered, as determined in
studies with seven ileostomy patients (Kivisto et al, 1986). Although absorption of Fe
was unaltered, that of Zn decreased by 41%. In addition, significant reductions in
absorption of Mg (-31%) and P (-l 1%) were observed. Anderson at al (1981) showed a
13 to 35% reduction in phytate content in an extrusion-cooked, bran-enriched product.
Other studies reported that reduction in phytate content in soybean products improved Zn
bioavailability in rats (Zhou et al, 1992). Thus, one rrright expect improvement of Zn
bioavailability in extruded products containing phytate. However, the studies by
Fairweather-Tait et al (1989) revealed that extrusion cooking of a bran flour mixture

produced no effect on the retention of Fe and Zn in humans.

30

The heating of proteins during processing has been Shown to modify their ternary
and quaternary structures. Extrusion processing has the potential to alter protein structure,
solubility, and digestibility by heat, pressure, and shear (Phillips, 1989). Denaturation
could, under some circumstances, facilitate digestion (Wapnir, 1990). However, in
extrusion cooking, some unique features are present because the product is subjected to
high pressure in combination with severe shear. The degree of mixing and
homogenization during extrusion cooking might decrease diffusion barriers or break
chemical bonds and thus, increase reactivity in a food system (BjOrk and Asp, 1983).
Interactions among food components such as phytate, protein and Zn during extrusion
cooking may have positive, negative or no effects on the bioavailability of Zn. Since
extrusion cooking is used increasingly for production of weaning food and breakfast

cereals, thorough knowledge of the effects on nutritional value is essential

MATERIALS AND METHOD

PREPARATION OF EXTRUDED AND RAW PRODUCTS

Semolina is wheat flour milled from durum wheat. Semolina (30 mesh) with
approximately 65% extraction and enriched with niacin, iron, thiarnin and riboflavin, was
obtained from the North Dakota Mill & Elevator, Grand Folks, ND. Enriched semolina is
known to have added amounts of 3.13 mg iron, 0.53 mg thiarnin, 0.49 mg riboflavin and
2.86 mg niacin per 100 g (U SDA,1989). Semolina (85%) and soy protein concentrate
(15%) (Procon 2000, Central Soya , Fort Wayne, IN) were used to make a product in the
shape of small “O’s”. A corotating twin-screw extruder (Creusot-Loire, Model 45) was
used to extrude the semolina /soy product at the temperature of 92 to 96°C. Water was
injected into the feed at the rate of O. 12 lein. The screw was operated at 900 rpm, and
the feeder was set to deliver 2.06 kg of the semolina/soy mixture per a minute. The final
product was dried in a vat dryer/ blower to a moisture content of 8 to 9%. The raw
ingredients and the extruded product were put in plastic bags and stored in a cubicle

(18°C). The extruded product was ground using a blender prior to analyses.

31

32

MATERIALS

The following chemicals were purchased from J. T. Baker Chemical
(Phillipsburg, NJ): potassium hydroxide, sodium hydroxide, sodium sulfite, sodium
bisulfite, EDTA (disodium salt), 30% hydrogen peroxide, concentrated sulfuric acid,
zinc, copper, and iron standard solutions (1000 ppm). Bovine liver (No 1557a) and wheat
flour (No 1567a) were purchased from National Institute of Standards and Technology
(NIST) ( Gaithersburg, MD). Methoxyflurane was fiom Pitman-Moore (Mundelein, IL);
hydrochloric acid and ether were from Mallinckrodt (Paris, Kentucky); ultrapure
concentrated nitric acid and ammonium molybdate were from Fisher Scientific
(Pittsburgh, PA). Glycerol, pepsin (fi'om porcine stomach mucosa), pancreatin (from
porcine pancreas) and bile extract (porcine) were from Sigma Chemical Co. (St. Louis,

MO).

RESEARCH DESIGN

In Vitro Studies

Quantitative determination of soluble zinc was done by the assay described by
Gifford-Steffen and Clydesdale (1993) with modifications. Dialyzable zinc was estimated
using a modification of an in vitro method developed by Miller et a1 (1981). Mineral
content (zinc, copper and iron) of the extruded and raw ingredients was determined using

atomic absorption spectrophometry (AAS) (Perkin-Elmer Model 2380, Norwalk, CT).

33

Soluble Zinc Analysis. Triplicate samples (2 g) of ground extruded or raw product

were added to 100 ml of 0.01 N HCl. The mixture was placed on a magnetic stirrer at
medium speed for 30 min. The pH was adjusted to 2.0 i 0.05 with HCl and stirred an
additional 30 min. The pH was then adjusted to 5.5 i 0.05 with NaOH followed by a 30
minute period of mixing. Two 30-ml aliquots of each sample were centrifuged at 3335 x
g for 20 min (Sorvall RC-SB, Dupont ). A 10-ml aliquot of the supernatant of each

sample was pipetted into a 25-ml Erlenmyer flask and wet ashed using HNO3 and H202,

Dialyzable Zinc Analysis The method involves simulated gastrointestinal
digestion followed by measurement of soluble, low molecular weight zinc. It differs from
soluble zinc analysis method in two important ways: 1) pH adjustment from gastric to
intestinal levels is gradual and 2) only low molecular weight, soluble zinc is used in the
estimation of dialyzable zinc. The method involves a two-stage digestion: stage 1 (pepsin
digestion) and stage 2 (pancreatin digestion). The amount of zinc which passes into a
dialysis sac (dialyzable zinc) during stage 2 is used as an indicator of zinc availability.
Specifics of the method follow:

Triplicate samples (5 g) of extruded or raw product were placed in a 125-ml
Erlenmyer flask. Fifty ml of 0.1 N HCl was added to the product to form a slurry, and the
pH was adjusted to 2.0 i 0.05 by dropwise addition of 6 N NaOH with rapid stirring. A
blank containing no sample but otherwise prepared as above was also run to determine
the contribution of zinc in the enzymes to dialyzable zinc. A 5 ml aliquot of pepsin

solution (10g pepsin/ 100ml 0.1N HCl) was added to each flask. The flasks were inverted

several times to mix and incubated for 2 h in a 37 °C shaking water bath. The contents of

34

each flask were mixed with 5 ml of the pancreatin-bile suspension (0.4g pancreatin, 2.5g
bile extract! 100ml 0.1M NaHCO3) and titrated to pH 7.5 with 0.5 N KOH. The aliquots
were then incubated in a 37°C shaking water bath for 30 min. A dialysis bag with a
molecular weight cutoff of 6000-8000 Da was prepared and put into each sample. The
bag contained a volume of 0.5 N NaHCO3 equal to the volume of 0.5 N KOH required to
titrate the pancreatin-bilc-sample mixture to pH 7.5, plus double deionized water (DDW)
to bring the total volume to 20 ml. Incubation was continued for 2h. A 2 m1 aliquot of

the dialysate was taken and wet ashed prior to zinc analysis by AAS.

In Vivo Animal Experiment

The in vivo bioavailability of zinc in the extruded product and the raw ingredients
was determined in weaning Sprague-Dawley male rats (Harland , Indianapolis, IN). Rats
were housed individually in suspended stainless steel cages in a temperature- and
humidity- regulated room with alternating 12-hour light/dark cycle with lights on at 7:00
am. Temperature was maintained at 20-22 °C, humidity at 68-70%. Food consumption
and body weights were measured at least twice weekly. Rats were housed in metabolic
cages during the last week of the experimental diet period for daily collections of feces.
Food intake was measured daily during the fecal collection period. Distilled water was
supplied ad libitum throughout the experiment using glass water bottles with plastic caps
and stainless steel drinking tubes. Care and treatment of rats were approved by the All
University Committee on Animal Use and Care at Michigan State University.

During 8 days of adaptation the rats were fed rat chow containing adequate zinc.

A zinc deficient basal diet was then fed for 10 days to maximize the efficiency of zinc

35

absorption. At the end of the zinc deficient basal diet period, six rats were killed to get
baseline data. Rats were then assigned to the following three experimental diet groups:
extruded diet (n=13); raw diet (n=6); control diet (n=13). At the end of wk 1 of the
experimental diet period, six rats each in extruded and control groups were killed. During
week 3, daily fecal samples were collected for seven days. The fecal samples were wiped
to remove attached hair and food. The fecal samples were combined and dried in a drying
oven to a constant weight. At the end of three weeks, rats were anesthetized with
methoxyflurane and killed by drawing blood from the heart using 3ml syringes rinsed
with 1000 units/ml sodium heparin solution. Blood samples were centrifuged
(International Centrifuge, Model UV) at 1500 x g for 20 minutes; plasma was removed
and frozen. Livers, kidneys, and femurs were removed, rinsed in saline, cleaned of

adhering matter, weighed and frozen.

DIETS

The diets were formulated in accordance with the general guidelines prepared by
the American Institute of Nutrition ( Reeves et al, 1993) with modifications. Composition
of the basal zinc deficient diet and three test diets is listed in Tablel. The protein source
in the basal diet was spray dried egg white. An AIN-93 mineral mix without zinc, copper
and iron was purchased from Dyets (Bethlehem, PA). Cupric carbonate and ferric citrate
were premixed with Dyetrose and then incorporated into the basal diet to provide
recommended levels of copper and iron. Because of the relatively low phosphorous

content of egg white, potassium phosphate was added in order to provide an adequate

36

 

TABLE 1

Composition of diets '

 

 

Component Basal Extruded Raw Control
g/kg

Extruded product -- 5 16 -- --
Raw product -- -- 543 --
Egg white2 220 107 95 234
Cornstarch 380.5 -- -- 366.5
Dyetrose3 132 109.5 94.5 132
AIN-93G mineral mix4 35 35 35 35
(Zn, Cu and Fe free)

mg/kg
Zinc carbonate’ -- -- -- 27.0
Cupric carbonate6 9.6 -- -- 9.2
Ferric citrate7 269.5 -- -- 208.7
Potassium phosphate8 5461 -- -- 5403

 

1The following ingredients (g/kg) were added to all diets: sucrose, 100; soybean oil
(0.02% TBHQ, Dyets, Inc., Bethlehem, PA), 70; fiber (Cellulose, FCC, Dyets, Inc.,
Bethlehem, PA), 50; AIN-93 vitamin mix (Dyets, Inc., Bethlehem, PA), 10; and

choline bitartrate, 2.5.

2Egg white, Spray dried (Dyets, Inc., Bethlehem, PA)

3Dyets, Inc., Bethlehem, PA

4Contained (g/kg mineral mix): Sucrose, 221; CaCO,, 357; KH2P04, 196; K3C6H507-H20,
70.8; NaCl, 74; KZSO.” 46.6; MgO, 24; MnC03, 0.63; K103, 0.01; NaZSeO3-O.Ol;
(NH,)6M0702,-4H20, 0.008; Na203Si-9H20, 1.45; CrK(SO,)'12H20, 0.275; LiCl, 0.02;
H3BO3, 0.08; NaF, 0.06; NiCO3, 0.03; NH,VO3, 0.007.

’Fisher Scientific, Fair Lawn, NJ

°J.T. Baker Chemical Co. Phillipsburg, NJ

7FeC6H507-HZO, Matheson Coleman & Bell, Norwood, OH

8Monobasic, Mallinckrodt, Paris, KE

37

amount of phosphorous. The control diet was similar to the basal diet except for an
increase in the amount of zinc and minor adjustments in the protein and iron to be similar
to amounts in the extruded and raw diets. The ground extruded product and the raw
product ingredients were used to provide all of the zinc (14 ppm) in the extruded and raw
experimental diets, respectively. This level of zinc is adequate but low compared to the
recommended level in the AIN-93 diet. Approximately 50% of the total protein was
supplied by egg white, and the remainder by the extruded product or raw product
ingredients. Adj ustrnents in the amounts of cornstarch and Dyetrose were made to

compensate for carbohydrate in the semolina.

ANALYTICAL METHODS

Phytate

The phytate content of the extruded and raw products was determined using the
anion-exchange method of Harland and Oberleas (1986). The procedures were as follows:
Triplicate samples (2.000 g) of ground extruded and raw products were accurately
weighed and placed in 125ml Erlenmyer flasks. Forty ml of 2.4% HCl was added and the
flasks were covered with parafihn and shaken vigorously for 3 hr at room temperature.
The mixture was centrifuged and then filtered through Whatrnan No. 1 filter paper.
Plastic columns (4.0 ml with 2 frits ) were washed with 3ml of DDW and packed with
0.5 g of chloride exchange resin (AG l-X8, 200-400 mesh) in a water slurry. Before use,
the packed columns were washed with 10 ml of 0.7 M NaCl to assure chloride saturation

of the resin and then with 15 ml of DDW. A blank for one batch was prepared by mixing

38

1 ml 2.4% HCl with 1 ml EDTA-NaOH reagent (0.11 M EDTA and 0.75M NaOH),
diluted to 25 ml with DDW, and then poured onto the column. One milliliter of the
sample extract was mixed with 1 ml of the EDTA-NaOH reagent, diluted to 25 ml with
DDW and transferred onto the column to elute most of the inorganic phosphate. The
remaining inorganic phosphate was eluted with 15 ml of 0.1 M NaCl. The phytate was
eluted from the column with 15 ml of 0.7 M NaCl and collected in a 25 ml Erlenmyer
flask. Concentrated HZSO, (0.5 ml) and concentrated IrINO3 (3.0 ml) were added, and then
the mixture was digested on low heat until all acid was evaporated. DDW (10 ml) was
added to the flask and heated on low temperature for 10 minutes and then allowed to
cool. The content of the flask was quantitatively transferred to a 50-ml volumetric flask.
Two ml of molybdate solution ( 2.5% ammonium molybdate in 1 N H2S04) was added,
and the contents mixed; 1 ml of sulfonic acid reagent (0.16% 1-amino-2-naphthol-4-
sulfonic acid, 1.92% Na2S03, and 9.60% NaHSO3 in DDW) was added, and the flask was
made to volume with DDW. After standing for 15 minutes, the absorbance was read at
640 nm. The phosphorous values were calculated from a standard curve and converted to
phytate using a conversion factor of 3.55.

The standard curve was prepared by pipetting 1, 3 or 5 ml aliqouts of phosphorous
standard solution (80 ug P/ml) into 50 ml volumetric flasks and then adding 20 ml of
DDW. After the flask was mixed thoroughly, 2 ml of molybdate solution was added, and
the content mixed. One ml of sulfonic acid reagent was then added, and the contents
mixed well. The flask was made to volume with DDW and mixed well. The absorbance

was read at 640 nm after 15 min (Spectronic 21, Baush & Lomb).

39

Protein Analysis

The protein content of the diets and dried feces was analyzed by micro kjeldahl
nitrogen analysis using a modification of the AOAC (1984) method. Concentrated
sulfuric acid (5 ml) and one catalyst tablet (potassium sulfate and selenium; Tecator Co.,
England) were added to each of the digestion tubes containing preweighed samples.
Triplicate samples were digested using gradually increased temperatures until digestion
was complete (Tecator 1016 Digestor, Tecator Co., England). The samples were distilled
using a Buchi 323 distillation unit ( Switzerland) and then titrated (716 DMS Titrino,
Switzerland ). The protein content was calculated on a dry weight basis using a nitrogen

conversion factor of 5.70.

Mineral Analyses

Wet ashing. Triplicate 0.5 g samples of ground samples were weighed and placed
in 25 ml Erlenmyer flasks with 10 ml concentrated HN03. The flask was placed on a hot
plate at low heat for 1 day and then allowed to go to dryness. Five ml of 30% H202 was
added. If a white ash was not obtained, more H202 ( 5 to 10 ml) was added. When
digestion was complete, samples were diluted with acid and DDW prior to analyzing
minerals by AAS.

Products and gigs. The extruded product, raw product ingredients and rat diets
were wet ashed. For copper analysis, the ashed samples were diluted with lml 0.1N HCl
and 9m] DDW for products and lml 0.1N HCl and 4m] DDW for diets. Diluted product
samples were further diluted 1:10 with DDW for zinc and iron analyses. Diluted diet

samples were diluted 1:10 or 1:5 with DDW for zinc and iron analyses.

40

M. Livers and kidneys were freeze-dried ( Unitrap II, Virtis Co., Gardiner,
NY) to a constant weight. Liver samples were crushed manually. Duplicate 0.5 g liver
samples and one kidney (approximate 0.16g) were soaked overnight in 10 ml
concentrated HNO3 to allow tissues to dissolve in the acid before wet ashing. If a white
ash was not obtained or particles were present in the flask when diluted with 0.1 N HCl,
repeat ashing was necessary. Ashed samples were diluted with 5 ml 0.1N HCl for copper
analysis. Diluted ashed liver and kidney samples were further diluted (1: 26 and 1:16,
respectively) for analyses of zinc and iron.

F emur samples (one from each rat) were soaked in ether for 24 hours to extract fat
and allowed to dry briefly at room temperature. The femur samples were placed in acid-
washed ceramic crucibles and dried in a drying oven (Blue M, Blue Island, IL ) to a
constant weight and then dry ashed in a muffle fumace (Temco, Model No 293C, Barber-
Coleman, Rockford, IL) at 600 °C for 40 hours. When the femur samples were cool, 2 ml
of 3 N HCl was added. Diluted samples were further diluted (1: 1) for c0pper analysis.
For femur zinc and iron, 0.1 ml of samples was diluted with 5.9 ml of DDW.

Feces. The dried feces were ground in a blender, weighed and wet-ashed. Ashed
samples were diluted with 10 ml 1 N HCl. Further dilution (1:30) was made for fecal zinc
and iron.

NIST Standard. Standard reference materials of bovine liver and wheat flour were
digested and analyzed using the same procedures as for tissues samples to check the
validity of the methodology.

PM. Plasma samples were diluted 1: 10 with deionized water for analysis of

zinc by AAS. Standard solutions were made by diluting stock standards with a 5%

41

glycerol solution. The glycerol solution was used to compensate for the viscosity of the

serum.

Statistical Analyses

The Student’s t-test was used to compare data obtained at the end of week 1 of the
experimental period. Data at the end of experiment (week 3) were subjected to a one-way
analysis of variance at the 95% confidence level to determine the significance of
differences between means. Significant differences among the treatments were
determined by Fischer’s least significant differences (LSD) test when appropriate. All

percentage data were log transformed prior to statistical analyses.

RESULTS

Analyses of products and diets

Zing, copper, irpn and phyaic acid pontent of products. The analyzed amounts of

zinc, copper and iron were similar in the extruded and raw products (Table 2). The
accuracy of analytical procedures were verified by analysis of NIST wheat flour. The
analyzed zinc, copper and iron concentrations in wheat flour (13.3i0.5, 21:04 and
12.5:09 ug/g, respectively) were within the range of certified values. Literature values
for the phytate content of wheat flour and soy protein concentrate are 0.28% and 1.25-
2.7%, respectively (Oberleas and Harland, 1981; Anderson and Wolf, 1995). We
estimated that the mixture of 85% semolina/15% soy protein concentrate had 0.43-0.64%
of phytate in it. The analyzed values for the products used in this experiment were low
(0.34% and 0.37% for the extruded and the raw semolina/soy protein product ) compared

to literature values and also similar to each other.

Zinc, copper, irgn, phytjp acid and protein content of diets. The analyzed amounts

of zinc, copper and iron were similar in the extruded and raw diets (Table 3). However
concentrations of zinc and iron in the control diet were 14% and 27% higher than in the

extruded diet and 17% and 15% higher than in the raw diet.

42

 

43

 

TABLE 2

Analyzed phytate, zinc, copper, and iron content of extruded and raw productsl

 

Analyzed mineral Content (uglg)

 

Phytate(mg/ g) Zinc Copper Iron
Extruded product 3.42:0.79 27.2:08 7.08i0.20 57.5i1.5
Raw PTOdUCt 3.73:0.39 25.8i0.4 6.25i0.07 54.0i1.8

 

1Each value represents mean i SEM of six determinations.

 

44

 

TABLE 3

Estimated phytate and analyzed protein, zinc, copper, iron content of diets'

 

 

Diets Phytate Protein Anal zed Mineral ontent m diet
(mg/ g diet)2 (%) Zinc Copper Iron

Basal -- 16.9:02 38:03 5.07:0.2 60.1:t1.3

Extruded 1.76 18.5iO.1 14.8:05 5.46:0.2 40.7:26

Raw 2'07- 17.8:03 14.5:03 5.67:0.4 44.7:22

Com” " 17.6iO.1 169:0.8 5.48:0.3 51.6i2.5

 

1Each value represents mean i SEM of six determinations.

2 Phytate values are obtained by calculation.

 

45

Concentrations of phytate in the diets containing the extruded and raw product was
calculated to be 0.18% and 0.20%, respectively based on the analyzed amount of phytate
in the extruded product or raw ingredients. It was assumed that basal and control diets

contained no phytate.

In Vitro Experiments

Soluble and dialyzable zinc content of products. The analyzed amount of soluble

zinc and percent soluble zinc were similar in extruded and raw products (Table 4). The
soluble zinc content of the extruded product was 15.3 ug zinc/ g compared to 15.6 ug/g in
the raw ingredients, respectively. These amounts represent approximately 56% of the
total zinc. The extruded product contained 13.0 ug dialyzable zinc/ g compared to 15.0
ug/ g in the raw ingredients or 47.8 and 58.1 % of total zinc, respectively. These results

indicate that extrusion processing had little effect on the bioavailability of zinc as

evaluated by these in vitro techniques.

In Vivo Animal Experiment

Body weights, weight gain, food intake, effipiency of food utilization and total

mineral intake. Rats fed the basal zinc deficient diet consumed an average of 8.0 g
food/day and gained 1.4 g/day (Table 5). During the first week of the experimental diet
period, rats fed the extruded diet consumed more food than rats fed the control egg white

diet, although total zinc intakes were similar. Rats fed the extruded diet also consumed

46

 

TABLE 4

Soluble, percent soluble, dialyzable and percent dialyzable zinc in extruded and
raw products‘

 

Product Soluble Zn Soluble Zn Dialyzable Zn Dialyzable Zn

 

(pg/g) (%) (pg/g) (%)
Extruded 153:3.0 56.5i10.9 13.0:05 47.8:1.7
Raw 15.6:1.7 56.6: 6.5 150:0.2 58.1108

 

1Each value represents mean :1: SEM of six determinations.

 

47

 

TABLE 5

Body weights, food intake and food efficiency ratios“

 

 

 

 

Diets Initial Final Weight Food Food
Weight Weight Gain Intake Efficiency
(8) (g) (g/d) I g/d) Ratios (%)3

Zn deficient

Basal 108:3 125:4 1.4:0.2 8.0:0.2 17.4:2.7
Week 1"

Extruded 125:4 172:3 66:05 1 16:03“ 57.1:4.5

Control 124:5 163:6 5.5:0.4 10.1:0.3b 54.6:3.8
Week 35

Extruded 122:6 236:7° 5.4:0.1° 14.2:0.4b 38.5:1.0b

Raw 126:5 248:3° 58:0.3b 15.8:0.3° 37.0:o.9ab

Control 123:5 211:9a 4.2:o.3a 12.4:05' 34.0:1.3a

 

1Each value represents mean : SEM (after lwk, n=6 in extruded and control;

after 3wk, n=7 in extruded, control and n=6 in raw)

2Food Efficiency Ratio = (weight gain, g/ total food intake, g) x 100 %

3Data were log-transformed prior to statistical analysis.

4Different letters within a column indicate significant differences at p<0.05 as determined
by Student’s t-test.

SDifferent letters within a column indicate significant differences at p<0.05 as determined
by ANOVA and LSD.

48

more copper but less iron than those fed the control diet. At the end of week 3 of the
experimental diet period, the mean body weight (211:9 g) of rats in the control group
was significantly lower than that of the extruded (236:7 g) and raw (248:3 g) groups.
There was no Si grrificant difference in the body weight of the extruded and raw groups.
The lower total weight gain of rats in the control group, which was 89% of that in the
extruded group and 85% of that in the raw group, can be explained by their significantly
lower intake of food. Food efficiency ratio was lower in rats fed the control diet
compared to the extruded or raw diet.

The total dietary intakes of zinc, copper and iron are shown in Table 6 . Because
of differences in food intake and slight differences in mineral content of the diets, the
total copper intake during the experimental diet period was lowest in rats fed the control
diet and highest in rats fed the raw diet. However, the total zinc intake was not
statistically different in all three groups. The total iron intake in the control group was

higher than that in the extruded group because of the higher content of iron in the diet.

ghgan wet weights, m weights gd % hody weight of orggs. After consuming

the experimental diets for one week, rats fed the control diet had higher femur relative
weight compared to rats fed the extruded diet (Table 7). At the end of the basal diet
period, rats had similar wet and relative weights to those in rats that were killed at wk 1
except for liver wet weight and relative femur weight. At the end of wk 3 of the
experimental diet period, rats fed the raw diet had Significantly higher liver weight than
rats fed the control diet. Rats fed the semolina/soy protein diets had significantly higher

mean kidney weight than rats fed the control diet. However, there was no Significant

49

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51

difference between the mean wet weight of kidneys in rats fed the extruded and raw diets.
Mean femur wet weight was not statistically different in rats fed the three diets whereas
relative weight of femur was significantly different only between the raw and control
groups. The same trend was observed in the mean liver, kidney and femur dry weight
values for rats fed the three diets (Table 8). These results could be related to differences
in body weights. Body weights of rats in the extruded and raw diet were also significantly

higher than those fed the control diet.

Plasma concentration of zinc. Plasma zinc concentration in rats fed the control
was higher compared to value at the end of basal zinc deficient diet period, and was
similar to that in rats fed extruded diet at the end of wk 1 (Table 9). Plasma concentration
of zinc was significantly higher in rats fed the control diet than in rats fed extruded and
raw diets at the end of wk 3. There was no significant difference in plasma zinc
concentrations between rats fed the extruded and raw diets suggesting no effect of

extrusion processing on zinc bioavailability.

Tissue concentrgjgn of zinc, At the end of the zinc deficient basal diet period,
zinc concentrations in liver and kidney were 73.2 and 82.7 ug/g and similar to those in
rats fed the extruded or the control diet afier wk 1 (Table 10). Concentration of zinc in the
femur and total zinc in liver, kidney and femur were lower compared to rats fed the two
experimental diets for 1 week. After 1 week, the concentrations of zinc and total content
of zinc in the femur of rats in the control group were significantly higher than those in

rats fed the extruded diet. Rats fed the control diet, however, had significantly higher total

52

 

 

 

 

 

TABLE 8
Organ dry weights'
Diets Liver Kidneys Femur
(g) (g) (g defatted dry wt)
Zn deficient
Basal 1.67i0.14 O.28i0.01 0.24:0.01
Week 12
Extruded 2.10i0.13 O.34i0.02 0.29:0.00
Control 2.36i0.13 0.33:0.01 0.30:0.01
Week 33
Extruded 2.88:0.12ab 0.43:0.01b O.40i0.01
Raw 3.07i0.06b O.46i0.01" 0.41:0.01
Control 2.61:0.17‘ 0.38i0.01‘ 0.38:0.01

 

1Each value represents mean i SEM (afier lwk, n=6 in extruded and control;
after 3wk, n=7 in extruded, control and n=6 in raw)
2Different letters within a column indicate significant differences at p<0.05 as determined

by Student’s t-test.

3Different letters within a column indicate significant differences at p<0.05 as determined
by ANOVA and LSD.

53

 

TABLE 9

Plasma concentration of zinc1

 

 

 

 

Diets Plasma Zn (umol/L)
Zn deficient

Basal 10.3.t1.40
Week 12

Extruded 21.7i-l.25

Control 22.5i1.84
Week 33

Extruded 23.7i0.93"

Raw 25.1il.01‘

C°mr°1 27.6i1.04b

 

1Each value represents mean i SEM (after lwk, n=6 in extruded and control;

afier 3wk, n=7 in extruded, control and n=6 in raw)

2Different letters within a column indicate significant differences at p<0.05 as determined
by Student’s t-test.

3Different letters within a column indicate significant differences at p<0.05 as determined
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femur zinc than rats fed the extruded and the raw diet possibly due to absence of phytate
in the diet and different source of protein. Afier week 3 of the experimental diet period,
the concentration of zinc in kidneys of rats fed the extruded or raw diets was significantly
lower than in rats fed the control diets. Total liver zinc was significantly higher in the
extruded diet-fed and in the raw diet-fed rats than in the control diet—fed rats. The only
difference between the extruded and the raw groups was higher total kidney zinc in the
raw diet-fed rats. These results confirmed in vitro results showing extrusion processing
had no effect on zinc bioavailability. The results also indicated that phytate and

differences in protein source may have influenced zinc bioavailability.

Tissue concentration of copper. Tissue total copper in liver and kidney at the end
of the zinc deficient basal period was lower compared to that at wk 1 (Table 11). The
concentration of copper and total copper in tissues were unaffected by the two dietary
treatments afler 1 week of the experimental period. Inconsistent changes in tissue copper
were observed at the end of week 3. Rats fed the extruded and the raw diets had
significantly higher concentration of copper in liver and significantly lower concentration
in kidney than rats fed the control diet at wk 3. Rats fed the extruded diet had
significantly higher femur copper concentration than in rats fed the raw diet. Total liver
copper was statistically higher in rats fed the extruded and raw diets than in rats fed the
control diet, and the total kidney copper was statistically higher in rats fed the extruded
diet than in rats fed the raw diet. The femur total copper was statistically higher in rats fed

the extruded diet than in rats fed the control diet.

56

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57

Tissue concentration of iron. Concentration of iron in tissues at the end of the zinc
deficient diet period was higher but tissue total iron was lower compared to values at wk
1 (Table 12). After 1 week of the experimental diet period, the concentration and the
total content of iron in the femur were significantly higher in rats fed the control diet than
rats fed the extruded diet. After wk 3, there were no significant differences in the
concentration and the total content of iron in liver and femur. Kidney iron concentration
was significantly higher in the control diet-fed rats than in the extruded diet-fed rats,
whereas the total kidney iron was significantly lower in the control diet-fed rats than in

the raw diet-fed rats.

Feces weights and concentrations of zinc, copper and iron. Fecal dry weights were

significantly different among groups : raw > extruded > control (Table 13). The fecal
concentration of zinc was higher in the extruded diet-fed group but total fecal zinc
excretion was lower compared to the raw diet-fed group because of lower total fecal
weight. Significant differences in fecal zinc concentration and total zinc were found only
between the raw and the control diet group. Rats fed the raw diet had a significantly
lower zinc concentration compared to control rats. However, total zinc was significantly
higher in rats fed the raw diet compared to control rats. Fecal copper concentration was
significantly higher in rats fed the control diet than rats fed the extruded and raw diets
while total copper was significantly higher in rats fed the raw diet than rats fed the
extruded and control diets. A similar trend was found in fecal iron concentration and total
iron. Control rats had a significantly higher iron concentration but raw diet-fed rats had a

significantly higher total iron.

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60

Apparent digestibilig of protein, zinc, copper and iron. Apparent protein

digestibility was lower in rats fed the raw diet compared to rats fed the extruded or
control diet (Table 14). Although the mean apparent digestibility of zinc was 30 to 50%
higher in rats fed the control egg white diet compared to the semolina/soy protein diets,
the differences were not significant due to large individual variations. In contrast, copper
digestibility was significantly higher in the semolina/soy diet-fed groups than in the
control diet-fed group. However, iron digestibility was higher in rats fed the control diet

than in rats fed the extruded diet.

61

 

TABLE 14

Apparent digestibility of protein, zinc, copper and iron" 1‘ 3

 

 

Diets Protein (%)4 Zinc (%)5 Copper (%)5 Iron (%)5
Extruded 91 .2i0.6b 34.4:73 24.8:15b 8.8i1.7a
Raw 89.3iO.6‘ 29.9i4.3 20.4:1 .4b 12.6i2.6“b
Control 92.7:04b 44.6zt5.5 1 1.25:2.7a 242:3.1b

 

1Each value represents mean i SEM (n=7 in extruded, control; n=6 in raw)

2All data were log-transformed prior to statistical analysis

3Different letters within a column indicate significant differences at p<0.05 as determined
by ANOVA and LSD.

4Apparent protein digestibility = (N consumed/d - N feces/d ) / N consumed/d x 100
sApparent mineral digestibility

= ( pg mineral consumed/d - pg mineral in feces/d ) / ug mineral consumed/d x 100

DISCUSSION

Limited data for the effects of extrusion processing on zinc bioavailability have
been reported. Kivisto et a1 (1986) reported an adverse effect of extrusion processing on
zinc absorption. Extrusion cooking of a high fiber cereal product decreased apparent
absorption of zinc in humans, but the mechanism of action was not clearly defined.
Fairweather-Tait (1989), however, showed no effect of extrusion processing on zinc
isotope retention from a mixed meal of wheat bran and flour in human subjects.
Undoubtedly there are chemical changes associated with extrusion processing and these
may alter mineral bioavailability. Anderson et a1 (1981) reported a reduction in phytate
content in an extruded bran product. Because phytate reduction in soy protein has been
shown to increase zinc bioavailability in rats (Zhou et al, 1992), one might speculate that
extrusion processing of a product containing phytate would enhance zinc bioavailability.

Our results in rats showing no differences in plasma, liver and femur zinc
concentrations between the extruded- and raw diets-fed groups suggest that the extrusion
conditions used in the present study did not affect zinc bioavailability. These results are
consistent with results in the study in humans by Fairweather—Tait et a1 (1989). Because
the extruded product was fed in their study as part of a test meal containing milk, the
possible modifying effect of milk on zinc bioavailability may have influenced their

results. There also was little change in phytate content between extruded and nonextruded

62

63

(raw) products. Kivisto et a1 (1986) argued that the lower absorption of zinc in ileostomy
patients given an extruded cereal compared with a nonextruded product may have been
indirectly due to differences in phytase levels in the cereals. They suggested that
extrusion processing may deactivate the phytase that is naturally present in cereal thereby
leading to higher intestinal phytate levels and hence greater zinc binding. Sandberg et al
(1986) also showed no change in phytate on extrusion cooking of a high fiber cereal
product. Anderson and Wolf (1995) in a recent review concluded that phytic acid is
generally stable to processing of soybeans including extrusion processing. Therefore, it
should be noted that the extruded and raw diet had similar amount of phytic acid, which
is thought to form insoluble complexes with proteins and zinc. Formation of phytic acid-
protein-zinc complexes has been suggested as an explanation for observed differences in
zinc absorption with different methods for processing of soy protein (Erdman et al, 1980).
Even though total phytic acid content may not be altered by extrusion processing,
interactions among phytic acid, protein and zinc may still occur. Conflicting results in the
various studies can be explained in terms of variations in extrusion conditions together
with the use of different raw materials.

Zinc was less bioavailable in the semolina/soy protein diet that contained phytate
than in the control egg white diet that did not contain phytate. These results are in
agreement with the observations of numerous investigators (Zhou and Erdman, 1995).
The phytate to zinc molar ratio has been suggested as a parameter to predict zinc
bioavailability. Values greater than 12 have been reported to reduce accumulation of zinc
in femurs in rats but not depress grth (Davies and Olpin, 1979; Mon'is and Ellis,

1980a). Estimated phytate to zinc molar ratio of the extruded diet and raw diet were 11.7

64

and 13.9, respectively, and the egg white-based control diet was assumed to contain no
phytate. It is concluded that zinc bioavailability is not inhibited when the phytate to zinc
molar ratio is less than 12. The extent of formation of phytate-protein-zinc complexes is
dependent upon the presence of other minerals, particularly calcium. Graf and Eaton
(1984) indicated that a low calcium content in the diet (0.75%) had no effect on zinc
bioavailability when the phytate to zinc molar ratio was 12 or less, whereas when the
calcium content of the diet was increased to 1.75%, grth of rats was significantly
depressed. In the present study, estimated calcium concentrations were lower than the
level shown to have a negative effect on zinc bioavailability, and similar in all diets: 0.55,
0.55 and 0. 52% in the extruded, raw and control diets, respectively.

Another dietary component which influences zinc absorption is type and amount
of protein. Davisson et a1 (1996) found no significant differences in the zinc absorption in
adult humans from semi-synthetic liquid meals containing 30% egg albumin when
compared with those without added protein. However, when dephytinized soybean-
protein isolate ( < 0.01 g phytic acid/kg) was added instead of albumin, a significant
reduction in the absorption of zinc was observed. These results demonstrate that zinc
absorption is inhibited by certain protein sources such as dephytinized soybean-protein
isolate, while other proteins have little or no effect.

When food intakes were not controlled, rats fed the semolina/soy protein diets
tended to ingest more food than the control group fed the egg white-based diet. The
factor(s) responsible for this apparent preference for the semolina/soy diets are not clear,
but may have included differences in palatability of the diets. Although all diets

contained egg white, the amount in the extruded and raw diets was approximately half

65

that in the control diet. Zhou et al. (1992) showed that soy protein-fed rats consumed
greater amounts of diet than egg white-fed rats. In our experiment, higher total weight
gains in the semolina/soy diets were also observed, and this difference was related to
higher food intakes. If food intakes are controlled, theoretically the total weight gains
should be the same in all groups. Although diets were planned to be as similar as
possible, small compositional differences in the diets may have contributed in part to the
differences in weight gain.

A feeding trial of 1 week following a 10 d depletion period was found to be long
enough to show significant differences in femur zinc content in response to differences in
zinc bioavailability of the extruded semolina/soy and the control egg white diets.
Concentrations of zinc and total zinc in the femur of rats in the control group were
significantly higher than in rats fed the extruded diet after 1 week. Plasma zinc
concentrations also were higher in the control group but the difference was not
statistically significant. After week 3 of the experimental period, there was a significant
difference in plasma zinc between rats fed the extruded and the control diet. Therefore, a
feeding trial more than three weeks may be needed to show significant differences in
plasma concentrations of zinc in response to differences in bioavailability.

Zinc digestibility was higher in the extruded diet-fed group than in the raw diet-
fed and higher in the control diet-fed group than the semolina/soy protein diet-fed group,
but the differences were not statistically significant. Apparent protein digestibility for all
three groups was ~90%. Godinez et a1 (1992) reported that the values for apparent protein
digestibility in rats are considerably higher than the ones obtained in humans. Apparent

protein digestibility of black beans cooked at 120 °C with or without seedcoat varied

66

from 67-73% in humans and 70-81% in rats. There was a significant negative correlation
between phytic acid and in vitro protein digestibility of soybean and other legumes
(Chitra et al, 1995). It appeared that phytic acid reduced the protein digestibility by
interfering with protease enzymes (Singh et al, 1991). However, in our study, apparent
protein digestibility was low in rats fed the raw diet containing phytate whereas
digestibility in rats fed the extruded diet was similar to that in rats fed the control diet.
This result may be related to the heating of protein during processing, which has been
shown to cause protein denaturation, destruction of protease inhibitors and consequently
increased digestibility (Wapnir, 1990). Rats fed the raw diet also had higher fecal protein
output. This result is consistent with a report by Wu et a1 (1995). They observed high
fecal protein (based on Kjeldahl N) relative to low protein digestibility in a rat balance
study.

Results of the in vitro measures to assess zinc bioavailability showed no
differences in soluble and dialyzable zinc between extruded and raw products. These
results are consistent with our in vivo results showing that extrusion processing has no
effect on zinc bioavailability. Other researchers reported contradictory results with
respect to the correlation between in vitro and in vivo bioavailability when they
investigated the solubility of minerals as a measure of in vivo bioavailability (N arasinga
Rao and Prabhavathi, 1978; Wien and Schwartz, 1983; Hunt et al, 1987; Sandberg et al,
1989; Schwartz and Nevins, 1989; Tumland et a1, 1990). The in vitro method developed
by Miller et al (1981), which is a so-called “ equilibrium” in vitro method, has been
modified by several investigators for estimation of the bioavailability of iron and zinc.

The equilibrium method is based on equilibrium dialysis of minerals across a

67

semipermeable membrane as a model for the passage across the intestinal wall .
Promising correlations between in vitro dialyzability and in vivo bioavailability were
reported (Schricker et al, 1981; Hazell and Johnson, 1987; Hurrell et al, 1988; Sandstrom
and Ahngren, 1989). Because the absorption of minerals and trace elements is taking
place in the complex environment of the small intestine, simulation of the conditions
prevailing in the small intestine is probably the most critical step for in vitro methods
aiming at prediction of the bioavailability of minerals and trace elements. Therefore,
Wolters et al (1993) developed a continuous in vitro method by which dialyzable
components are continuously removed fiom pancreatic digestion mixture. They compared
the results of the continuous in vitro method with the results of the equilibrium in vitro
method with respect to the bioavailability of zinc and other minerals from different types
of bread. The dialyzability of zinc was comparable for the two methods.

No studies determining the effect of extrusion processing on copper
bioavailability were identified. Data for effects of extrusion processing on iron
bioavailability are conflicting. Absorption of iron was not altered in the same experiment
by Kivisto et al (1986) that showed absorption of zinc was decreased by 41%. Extrusion
processing had no effect on the amount of iron absorbed from potato or maize products
by rats (Fairweather-Tait, 1987 ). There also was no effect of extrusion on the retention of
iron as well as zinc using test meals in human subjects (Fairweather-Tait et al, 1989).
However, results in a study by Ummadi et a1 (1993) showed decreased bioavailability of
iron in an extruded semolina product. Our results showed a pattern that total contents of
copper and iron in liver and kidney were higher in rats fed the raw diet due to higher

total copper and iron intake. This pattern was not found in total copper and iron in femur.

 

68

Copper digestibility was significantly higher in semolina/soy diet-fed groups compared to
the control diet-fed group, whereas iron digestibility was higher in rats fed the control
diet compared to rats fed the extruded semolina/soy diet. Consequently, no conclusion
can be made related to the effect of extrusion processing on copper and iron

bioavailability in this product.

CONCLUSION

The overall results of this study indicate that zinc bioavailability is not affected by
extrusion processing. Using in vitro techniques to evaluate zinc bioavailability, when
soluble and dialyzable zinc were measured there was no differences between the extruded
and raw products. In vitro results were confirmed in the animal study showing extrusion
processing had no effect on the bioavailability of zinc. There were no significant
differences in plasma and tissue zinc concentrations between the two groups. The fecal
concentration of zinc was higher in the extruded diet-fed groups but total fecal zinc
excretion was lower because of lower total fecal excretion. Zinc digestibility was similar
for the two group. Apparent protein digestibility was lower in rats fed the raw diet.
Results for copper and iron showed minor and inconsistent differences.

Compared to the rats fed semolina/soy protein diets, rats consuming the control
diet based on egg white showed a lower total food intake and lower body weight. Plasma
and femur zinc concentrations were higher than those in rats fed the semolina/soy protein
diet. The apparently better utilization of zinc by rats fed this diet might be explained by
the absence of phytate in the diet, although the phytate/zinc molar ratios in the extruded
and raw diets were only 11.7 and 13.9, respectively. Differences in protein source may
also have influenced zinc bioavailability. Although all diets contained egg white, the

amount in the extruded and raw diets was approximately half that in the control diet.

69

FUTURE RESEARCH

The nutritional consequences of extrusion processing as a food preparing process
are not fully elucidated. Extrusion processing under conditions we used did not lead to
negative or positive effects on the bioavailability of zinc. The possibility of negative or
positive effects cannot be excluded, however, if products were extruded under conditions
of different pressure and temperature. Therefore, more research is needed to examine the
effects of extrusion processing on mineral bioavailability under different processing
conditions. These efforts could contribute to determine optimum extrusion processing
conditions required for improving mineral bioavailability in a wide variety of cereals and
legumes.

Another aspect of zinc bioavailability is the adverse effect of phytic acid in
forming phytate-protein-zinc complexes. Different compositions of test diets, e. g. higher
content of phytic acid, may induce different results in zinc bioavailability because
extrusion processing may cause changes in phytate-protein-zinc interactions. Future study
to identify and develop a procedure to quantify phytate-protein-zinc complexes in foods

is also recommended.

70

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