ABSTRACT

OAKES, AARON JEFFREY. Development of Process to Add Value to Aflatoxin Contaminated

Peanut Meal. (Under the direction of Jack P. Davis.)
Plants are inexpensive sources of protein and make good candidates for protein extraction. Processes have been developed to extract protein from corn, wheat, oats, barley, and
peanut meal. Enzymatic hydrolysis increases protein extraction and creates peptides shown
to have bio-active properties. Peanut meal is the material that remains after commercial oil
extraction from peanuts. Aflatoxin content limits the value of peanut meal and this material
is not currently considered to be food grade by the Food and Drug Administration. Recently
a novel process was developed that simultaneously extracted protein and removed aflatoxin
from peanut meal [Seifert, 2009]. A terminal product of this process was an aqueous protein/peptide extract with negligible levels of aflatoxin. The purpose of this study was to
generate similar protein/peptide extracts and convert them to powders using industrially
relevant larger scale processing techniques.
Naturally contaminated peanut meal containing 52 g/kg aflatoxin B1 was dispersed in
water to create a 4 kg batch at 10% peanut meal to water. The pH was adjusted to either
pH 2.1 or 9.1 using 2N HCl or NaOH. The enzymes pepsin and Alcalase were added at
enzyme to protein levels of 19000 units/g and 0.6 Anson units (AU)/g of peanut meal protein
respectively, similar to the used by Seifert and others [Seifert et al., 2010].
A sodium bentonite clay was added at 0.2% (w/w) as a processing aid to bind aflatoxin.
Each treatment was also prepared without the addition of clay. Pepsin control treatments
were prepared by adjusting the pH to 2.0 but omitting the addition of enzyme, both with and
without clay. Similarly Alcalase controls were prepared by adjusting pH 9.1, but omitting
Alcalase, both with and without the addition of clay. All eight treatments were performed in
triplicate for a total of 24 samples. Dispersions were stirred for one hour and then allowed
to settle for 40 minutes before the soluble supernatant was collected. The clay with bound
aflatoxin and peanut meal particulates were then physically removed from the water soluble
fractions by gravitational separation.
Maltodextrin with a dextrose equivalent of 12 was added to the collected supernatant
at (8% w/w). After stirring for 45 minutes the supernatant with maltodextrin was spray
dried using a Bchi B-290 lab scale spray dryer. The spray dryer inlet temperature was
185 C and the outlet temperature was maintained at 90 C by adjusting the feed flow rate.
Powder yields from the spray dryer were affected by the treatments and ranged from 56 to
84%. Yields were calculated as the ratio of solids collected to solids sprayed. The treatment
that yielded the most protein was at pH 9.1 with the addition of Alcalase. This treatment

had good spray drying yields at 60%, the powders contained 30% protein and no detectable
aflatoxin on a dry weight basis. The addition of clay reduced the aflatoxin content of all
powders, regardless of pH or presence of enzyme, to below 20 g/kg. Powders made from
treatments with enzyme had higher oxygen radical absorbing capacity than those without
enzyme addition. All powders contained less than 20 g/kg aflatoxin. Moderately aflatoxin
contaminated peanut meal can be used to produce protein powder containing undetectably
low levels of aflatoxin and 30% protein.

Development of Process to Add Value to

Aflatoxin Contaminated Peanut Meal

by
Aaron Jeffrey Oakes

A thesis submitted to the Graduate Faculty of

North Carolina State University
in partial fulfillment of the
requirements for the Degree of
Master of Science

Food Science
Raleigh, North Carolina
2011

APPROVED BY:

Timothy H. Sanders

Jack P. Davis
Chair of Advisory Committee

G. Keith Harris

BIOGRAPHY

Aaron Jeffrey Oakes was born during a snow storm on a cold night in December of 1982 in the
small coastal town of Brunswick, Maine. Considering his start, it is fitting that Aaron would
spend ten years of his life playing ice hockey. Growing up, Aaron was also active in Boy
Scouts of America eventually earning the Eagle Scout award. His scouting experience evinced
in his high school hockey career when the team elected Aaron, a junior, to be team captain.
By the conclusion of the season, Aaron earned the conferences Sportsmanship Award and
won, by popular vote, the teams Most Valuable Player Award.
After high school, Aarons athletic focus shifted to cycling while his academic focus became the science behind athletics. At the University of Massachusetts - Amherst, Aaron majored in exercise science while racing for, and eventually presiding over, the UMass Bicycle
Racing Club. With graduation only months away, he discovered food science and promptly
added it as a major. In 2006, Aaron graduated from UMass, earning a Bachelors of Science
with a dual concentration in Food Science and Exercise Science.
Shortly after graduation, Aaron worked for Decas Cranberries Inc. grading and assuring
quality of harvested cranberries. Aaron left Decas to work as a product developer in the
R&D department at Unilever North America in Englewood Cliffs, NJ. While at Unilever,
Aaron developed a product that showcased on the supermarket shelves within 18 months,
which proved a very gratifying experience. That same year, Aaron finished 4th place in
the Mountain Bike National Championship race in the Semi-Pro category, another equally
gratifying experience given his years of hard work and dedication. The result qualified Aaron
for an upgrade to the Pro category.
In early 2008 Aaron left Unilever to race mountain bikes full time and prepare for graduate
school at North Carolina State University. By Fall 2008, he had a home in the USDA ARS
peanut lab at NCSU working under the direction of Dr. Jack Davis. Aaron is currently
juggling roles as a professional-level mountain bike and cyclocross racer, a full-time graduate
student, a part-time researcher, and a part-time coach with Wenzel Coaching.

ii

TABLE OF CONTENTS

List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vi

List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

Chapter 1 Review of the Literature . . . . . . . . . . . .

1.1 Introduction to the peanut . . . . . . . . . . . . . .
1.2 Nutrition . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1 Health benefits of peanut consumption . .
1.2.2 Bioactive compounds . . . . . . . . . . . .
1.2.3 Peanut protein . . . . . . . . . . . . . . . .
1.3 Peanut Meal . . . . . . . . . . . . . . . . . . . . . .
1.3.1 Introduction . . . . . . . . . . . . . . . . . .
1.3.2 Value . . . . . . . . . . . . . . . . . . . . . .
1.3.3 Potential . . . . . . . . . . . . . . . . . . . .
1.4 Aflatoxin . . . . . . . . . . . . . . . . . . . . . . . .
1.4.1 Introduction . . . . . . . . . . . . . . . . . .
1.4.2 Prevalence in crops . . . . . . . . . . . . . .
1.4.3 Health concerns . . . . . . . . . . . . . . . .
1.4.4 Prevalence in food and feed . . . . . . . . .
1.4.5 Destruction and management of aflatoxin
1.4.6 Prevention and management . . . . . . . .
1.5 Peanut allergens . . . . . . . . . . . . . . . . . . . .
1.5.1 Mitigation strategies . . . . . . . . . . . . .
1.5.2 Therapies . . . . . . . . . . . . . . . . . . .
1.6 Hydrolysis . . . . . . . . . . . . . . . . . . . . . . .
1.6.1 Introduction . . . . . . . . . . . . . . . . . .
1.6.2 Uses . . . . . . . . . . . . . . . . . . . . . .
1.6.3 Nutritional considerations . . . . . . . . . .
1.7 Spray Drying . . . . . . . . . . . . . . . . . . . . .
1.7.1 Introduction . . . . . . . . . . . . . . . . . .
1.7.2 Drying bioactive compounds . . . . . . . .
1.7.3 Drying method comparisons . . . . . . . .
1.7.4 Stickiness . . . . . . . . . . . . . . . . . . .
1.7.5 Strategies to reduce stickiness . . . . . . .
1.7.6 Maltodextrin as a carrier . . . . . . . . . .
1.7.7 Protein denaturation during drying . . . .
1.7.8 Outlet temperature . . . . . . . . . . . . . .

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Chapter 2 Amino Acid Analysis of Hydrolyzed Peanut Meal Proteins . . . . . . . .

2.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2.3
2.4

Materials & Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Results & Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

25
25

Chapter 3 Process Development . . . . . . . . . .

3.1 Abstract . . . . . . . . . . . . . . . . . . . .
3.2 Introduction . . . . . . . . . . . . . . . . . .
3.3 Materials & Methods . . . . . . . . . . . . .
3.3.1 Protein extraction from peanut meal
3.3.2 Insoluble fraction . . . . . . . . . . .
3.3.3 Water soluble fraction . . . . . . . .
3.4 Results & Discussion . . . . . . . . . . . . .
3.4.1 Protein extraction from peanut meal
3.4.2 Separation . . . . . . . . . . . . . . .
3.4.3 Insoluble fractions . . . . . . . . . .
3.4.4 Soluble fraction . . . . . . . . . . . .

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Chapter 4 Process Flow . .

4.1 Abstract . . . . . . .
4.2 Introduction . . . . .
4.3 Materials & Methods
4.3.1 Extraction . .
4.3.2 Spray drying
4.4 Results & Discussion
4.4.1 Extraction . .
4.4.2 Spray drying

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Chapter 5 Finished Material Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3 Materials & Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.1 Aflatoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.2 Turbidity of powder solutions . . . . . . . . . . . . . . . . . . . . . . . .
5.3.3 Fiber, and ash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.4 Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.5 Interfacial tension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.6 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
5.3.7 Protein solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.8 Oxygen radical absorbance capacity (ORAC) . . . . . . . . . . . . . . . .
5.3.9 Hygroscopicity of powders . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4 Results & Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.1 Aflatoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.2 Turbidity of powder solutions . . . . . . . . . . . . . . . . . . . . . . . .
5.4.3 Fiber and ash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.4 Sugar content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.5 Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iv

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5.4.6
5.4.7
5.4.8
5.4.9
5.4.10

Surface tension . . . . . . . . . . . .
SDS-PAGE . . . . . . . . . . . . . . .
Protein solubility . . . . . . . . . . .
Oxygen radical absorbance capacity
Hygroscopicity of powders . . . . .

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Chapter 6 Concluding remarks & future work . . . . . . . . . . . . . . . . . . . . . .

6.1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2 Future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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LIST OF TABLES

Table 1.1

Nutritional Properties of Raw Peanuts [America, 2008,Ahmed and Young,

1982] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table 1.2 Mineral Content of Raw Peanuts [Ahmed and Young, 1982] . . . . . . .
Table 1.3 Amino acids composition of peanut protein adapted from [Lusas, 1979]
Table 1.4 Peanut meal pricing adapted from [Seifert, 2009] . . . . . . . . . . . . . .
Table 1.5 LD50 of AFB1 for several species adapted from [Newberne and Butler,
1969, Wogan, 1966]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table 1.6 Levels of total aflatoxin contamination permissible by the FDA adapted
from [FDA, 2008] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table 1.7 Legal limits of aflatoxin in peanuts: adapted from [Reddy et al., 2010] .
Table 1.8 AFB1 contamination levels and frequency in market products adapted
from [Li et al., 2009, Reddy et al., 2010, Turner et al., 2002, Williams et al.,
2004] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table 1.9 Spray dried heat-sensitive or bioactive compounds . . . . . . . . . . . . .
Table 1.10 Temperatures at which microbial death or enzyme inactivation rate changes
Table 1.11 Spray dried protein products . . . . . . . . . . . . . . . . . . . . . . . . . .
Table 3.1

2
3
5
7
8
9
10

11
18
22
23

Table 3.2

Yields as ratio of solubles collected to total batch size, solids, and protein
contents of water soluble fractions collected by centrifugation and settling 35
Dry peanut meal particle size distribution . . . . . . . . . . . . . . . . . .
35

Table 4.1

Treatment names and summary of conditions . . . . . . . . . . . . . . . .

Table 5.1

Concentration of sugars in soluble fractions and feed stocks: mean of 3

replicates 1 standard error . . . . . . . . . . . . . . . . . . . . . . . . . .
Hygroscopicity of powders expressed as g water uptake per 100 g powder solids after 1 week at 20 C 82% relative humidity: means of 3 replicates 1 standard error . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Table 5.2

vi

45
66

73

LIST OF FIGURES

Figure 1.1

Figure 2.1

Figure 3.1
Figure 3.2
Figure 3.3
Figure 3.4
Figure 3.5
Figure 3.6
Figure 3.7

Figure 3.8
Figure 4.1
Figure 4.2
Figure 4.3
Figure 4.4
Figure 4.5
Figure 4.6
Figure 4.7
Figure 5.1
Figure 5.2

Geography of populations at risk of chronic exposure to unregulated

aflatoxin exposure LAC, Latin America and the Caribbean: Adapted
from [Williams et al., 2004] . . . . . . . . . . . . . . . . . . . . . . . . . .

Amino acid composition of water soluble controls (A), hydrolysates

after 240 minutes (B), and unhydrolyzed peanut meal (C) Points are
means of 3 1 standard deviation Adapted from [Seifert, 2009] . . . .

26

Soluble solids content of water soluble fraction collected as affected by

meal to solvent ratio of dispersion . . . . . . . . . . . . . . . . . . . . . .
Soluble protein content of supernatant as affected by meal to solvent ratio
Soluble protein content of water supernatant per gram peanut meal as
affected by meal to solvent ratio . . . . . . . . . . . . . . . . . . . . . . .
Volume of soluble fraction with increasing settling time for 5, 10, 15,
and 20% dispersions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Vacuum oven dial setting and corresponding measured air or water
temperature inside the oven . . . . . . . . . . . . . . . . . . . . . . . . . .
Bchi B-290 spray dryer components weighed for yield and hold-up
calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of spray dryer inlet temperature and maltodextrin content on
powder yield during spray drying of water soluble fractions collected
from 10% dispersions at pH 8.2 . . . . . . . . . . . . . . . . . . . . . . . .
Effects of spray dryer inlet temperature on powder yield for pepsin and
Alcalase treated peanut meal solubles . . . . . . . . . . . . . . . . . . . .
pH of dispersions before (pHi ), and after (pHf ) the application of treatments acid/base, enzyme, and clay . . . . . . . . . . . . . . . . . . . . .
Mass of solubles collected after settling . . . . . . . . . . . . . . . . . . .
Solids content of solubles collected after settling (%) . . . . . . . . . . .
Solids collected in the soluble fraction as % solids * mass of soluble
fraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Solids content of insoluble fraction . . . . . . . . . . . . . . . . . . . . . .
Solids collected in each section of spray dryer as percentage of total
solids sprayed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quantity of solids fed to spray dryer as feed stock quantity * percent
solids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Aflatoxin content of insoluble materials, powders, and feed stocks on
solids basis by treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Absorbance of hydrated powder solutions (15, 1, 0.5, and .1% w/v) at
500 nm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

32
33
34
36
38
40

41
42
48
49
50
51
52
54
55
61
63

Figure 5.3
Figure 5.4
Figure 5.5
Figure 5.6
Figure 5.7
Figure 5.8
Figure 5.9

Visual appearance of pH2 & clay, Pep & clay, pH8 & clay, Alcalase &
clay soluble fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fiber and ash content of insoluble fractions on dry weight basis . . . .
Protein content of powders and feed stocks by treatment on solids basis
(g/ 100g) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Interfacial tension of feed stocks over time, the blue line represents value
for water at 25 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SDS-PAGE hydrated powders and feed stocks at the same solids contents for pH2, Pep, pH8, and Alc treatments . . . . . . . . . . . . . . . .
BCA protein solubility of powder protein in McIlvaine buffers at pH 3.0,
5.0, 7.0 in g/mL: means of 3 replicates 1 standard error . . . . . . .
Oxygen radical absorbance capacity of powders and soluble fractions
(mMol TE): means of 3 replicates 1 standard error . . . . . . . . . . .

viii

64
65
67
68
70
71
72

CHAPTER

ONE
REVIEW OF THE LITERATURE

1.1

Introduction to the peanut

The earliest recorded history of Arachis hypogaea L., the plant we know as the peanut, dates
back to the early 1500s [Hammons, 1982]. South America is credited as the origin of wild
peanuts, and peanut cultivation originates in the area that is now southern Bolivia - northwestern Argentina [Hammons, 1982]. Cultivated peanuts are generally divided into four
market types: Spanish, runner, Virginia, and Valencia [Henning et al., 1982]. These types are
characterized by the number and arrangement of branches on the plant [Wynne and Coffelt,
1982]. In the United States peanuts are grown in Virginia, North Carolina, South Carolina,
Texas, Oklahoma, New Mexico, Mississippi, and Arkansas, but the most productive region
includes Georgia, Florida, and Alabama [Henning et al., 1982]. Most of these peanuts are
grown in Georgia, and 25% of the US crop is grown in Texas [APC, 2010]. Finished peanut
production exceeds 3 109 pounds annually [NAS, 2009]. The majority (80%) of these peanuts
are runner type, which are primarily used for peanut butter production [APC, 2010]. Peanuts
as a snack nut are the most commonly consumed of all snack nuts [Mintel, 2009]. The virginia
type peanuts are the largest seed size and are used as a snack nut [APC, 2010]. A far less
prevalent snack nut is the valencia type, which only accounts for about 1% of the US peanut
crop [APC, 2010]. Four percent of the US crop are spanish type peanuts which are used for
candy and confections [APC, 2010]. The popularity of peanuts is due to their pleasing flavor,
texture, and favorable nutritional properties [Mintel, 2009].
Roasting and salting are done to improve the flavor of peanuts and this is the most widely
used method of preparation [Singh and Singh, 1991]. Whole peanuts are also prepared by
boiling, fermented and fried, coated with flour and fried, and as peanut butter [Singh and
Singh, 1991].

1.2

Nutrition

Raw peanuts contain a healthy balance of micro and macro-nutrients [Ahmed and Young,
1982]. Peanuts are composed of 15-21% carbohydrates by weight [America, 2008]. The principle carbohydrates in peanuts are fructose, glucose, inositol, and sucrose, which typically
comprise 2.7, 1.9, 1.3, and 149mg per 100 g of peanuts respectively [Ahmed and Young, 1982].
Lipids make up 44-56% of peanuts by weight and this oil is primarily composed of mono- and
poly-unsaturated fatty acids [USDA, 2010]. Only about 6.8% of the weight of raw peanuts
is saturated fatty acids [USDA, 2010]. The relative amounts of each fatty acid in peanuts
varies depending on cultivar, growing location, and maturity level of the peanut [Ahmed
and Young, 1982]. Peanuts also contain a variety of vitamins and minerals [USDA, 2010].
Nutritional content varies by peanut cultivar and generally these nutritive differences are
small [Ahmed and Young, 1982]. Protein content of raw peanuts ranges from 22-30% [USDA,
2010].

Table 1.1. Nutritional Properties of Raw Peanuts [America, 2008, Ahmed and Young, 1982]
Nutrient

Contentmg per 100g

Vitamin A
Vitamin E
Niacin
Pyridoxine
Riboflavin
Thiamin
Vitamin C

1.2.1

26
46
12.8-16.7
0.30
0.13
0.99
5.8

Health benefits of peanut consumption

The American Heart Association (AHA) estimates that one in three Americans has some
type of cardiovascular disease (CVD), which was involved in 56% of all deaths in 2005 [AHA,
2009]. Links between diet and risk for developing CVD are well established [Hu and Willett,
2002].
Increasing intake of fruits and vegetables decreases the risk of developing coronary heart
disease (CHD) [Liu, 2001]. Similarly, the relative risk of mortality from stroke, ischemic heart
disease, and CVD is significantly reduced by consuming greater than 3 serving of fruits and
vegetables daily [Bazzano et al., 2002, Joshipura, 2001].

Table 1.2. Mineral Content of Raw Peanuts [Ahmed and Young, 1982]
Nutrient

Content mg per 100g

Calcium
Copper
Iron
Magnesium
Manganese
Phosphorus
Potassium
Sodium
Zinc

82.6
1.2
1.6
174.1
1.8
438.1
626.3
6.3
6.1

Foods other than fruits and vegetables have similar health protective benefits. Diets low
in saturated fatty acids and high in unsaturated fatty acids reduce total blood cholesterol concentrations while improving the ratios of high density lipoprotein to low density lipoproteins,
thus reducing risks for CVD and CHD [Hu and Willett, 2002,Wu et al., 2009]. The lowest rates
of mortality were strongly associated with high consumption of cereals, legumes, fish, oils,
and wine [Menotti, 1999]. Peanuts are about 50% lipid, the ratio of saturated to unsaturated
fatty acids are consistent with blood lipid profile improving diets. Moreover, Kris-Etherton
and others [Kris-Etherton et al., 1999] reviewed equations used to predict the blood cholesterol and lipoprotein levels in subjects consuming nuts and found that the actual reductions
in cholesterol were 25% greater than predicted. This indicates that non-lipid components in
nuts are likely responsible for part of the reductions. The same authors later confirmed that
indeed bioactive compounds in nuts positively affect blood lipids and can prevent CHD [KrisEtherton et al., 2008]. Hamsters that consumed diets containing peanut oil, peanut flour, or
whole peanuts had lower instances of risk factors for CVD than those consuming the control
diet [Stephens et al., 2010]. This study was the first to indicate non-lipid peanut components
have a favorable impact on risk factors for CVD.

1.2.2

Bioactive compounds

Bioactive compounds, also called phytochemicals, are found in plant foods and provide
health benefits greater than those provided by the basic nutrition of the food in which they are
found [Leonora et al., 2008]. Examples of bioactive compounds found in foods are phenolic
compounds, which include flavonoids, resveratrol, and phytoestrogens [Kris-Etherton et al.,
2002]. Bioactive compounds have many benefits to human health, and can serve many functions within the body including antigens, enzyme inhibitors, and hormones which can reduce

both hypertension and inflammation [Havsteen, 2002]. A survey of the diet of Finnish people
indicated that those with the highest intakes of flavonoids had lower mortality rates [Knekt
et al., 2002]. Specifically, high levels of quercetin and kaempferol were associated with decreased mortality from ischemic heart disease [Knekt et al., 2002]. Rates of death from cerebrovascular disease and cancer were lowest among those with highest kaempferol, herperetin,
and naringenin intakes, while reduction of rheumatoid arthritis, type 2 diabetes, cataract, and
asthma all relate to high levels of flavonoid intakes [Knekt et al., 2002]. Whole peanuts contain several classes of bioactive compounds like flavonoids, phenolic acids, plant sterols and
stilebens [Leonora et al., 2008]. Peanut kernels contain, trans-resveratrol, dihydroquercetin,
p-hydroxybenzoic acid, p-coumaric acid; the skins, stems, and other parts not typically consumed, are also concentrated sources of bioactive compounds [Leonora et al., 2008]. Recent
research had demonstrated that fat-free peanut flour can reduce the incidence of atherosclerosis in hamsters [Stephens et al., 2010]. This suggests that non-lipid components found in
peanuts may have heart health benefits.

1.2.3

Peanut protein

Raw peanuts contain 22-30% protein depending on peanut variety [USDA, 2010]. The vast
majority (90%) of this protein is globular and classified as either arachin or conarachin
[Cherry, 1990,Lusas, 1979]. Globular means that in water the tertiary structure of the proteins
is roughly spherical [Damodaran, 1996]. Arachin proteins are storage proteins whereas the
conarachin proteins are present in the cellular cytoplasm of the peanut [Cherry, 1990]. The
other 10% of peanut proteins are albumins, meaning water soluble [Lusas, 1979, Damodaran,
1996].
Proteins are polymers of amino acids, and peanut proteins are composed of mostly glutamic acid, arginine, and aspartic acid (Table 1.3). A nutritionally complete protein is one
containing all the essential amino acids: isoluecine, methionine, tryptophan, leucine, phenylalanine, valine, lysine, threonine, and histidine [Manore and Thompson, 2000]. Peanuts
contain all the essential amino acids, but they are not present in large enough quantities for
peanut protein to be considered a complete protein (Table 1.3). In peanuts lysine, methionine,
and threonine are considered the limiting amino acids because they are found in insufficient
quantities [McOsker, 1962]. Supplementing diets with these amino acids improves protein exchange ratio and provides sufficient amino acids for optimal growth [McOsker, 1962, Manore
and Thompson, 2000].
One third of all children under 5 years of age in the world are malnourished [Latham,
1997]. Protein deficiency is the major contributing factor to this malnourishment affecting
approximately 200 million people [Latham, 1997]. Inexpensive sources of protein are needed

Table 1.3. Amino acids composition of peanut protein adapted from [Lusas, 1979]

Amino Acid

gram per 16 g Nitrogen

Glutamic Acid
Aspartic Acid
Arginine
Leucine
Glycine
Phenylalanine
Serine
Valine
Proline
Alanine
Isoleucine
Tyrosine
Lysine
Threonine
Histidine
Cystine
Tryptophan
Methionine

19.9
14.1
11.3
6.7
5.6
5.2
4.9
4.5
4.4
4.2
4.1
4.1
3.0
2.5
2.3
1.3
1.0
0.9

to ensure adequate nutrition for populations in these parts of the world. The proteins available from various plant sources can provide balanced and adequate quantities of amino acids
for human health [Young and Pellett, 1994]
Peanuts can be processed to a variety of protein-rich food ingredients [Diarra et al.,
2005, Cherry, 1990]. Many commercially available food ingredients are derived from peanuts
and peanut protein like flours, flakes, concentrates, and isolates [Lusas, 1979]. Peanut flour
for example, contains 60% protein and can be used to fortify breads, cereals, corn chips, and
donuts [Ayres and Davenport, 1977]. Several processes can be used to produce peanut milk
which can be consumed as a beverage or further processed into cheese, yogurt and spread
type products [Diarra et al., 2005]. Peanuts are abundant with roughly four billion pounds
produced annually in the US, and relatively inexpensive [Ash and Dohlman, 2008]. Unfortunately many peanuts, especially those grown in developing countries, are often contaminated
with aflatoxin.

1.3

Peanut Meal

1.3.1

Introduction

World-wide, peanuts are produced primarily for oil; however, in the US peanuts are primarily consumed directly and generally only substandard peanuts are used for oil production.
Aflatoxin contaminated peanuts are discolored, and less dense than uncontaminated peanut
seeds and subsequently discarded during peanut sorting [Whitaker, 1997]. These discarded
substandard nuts are generally pressed then solvent extracted for oil. The virtually fat-free
material remaining after the oil is extracted from peanuts is called peanut meal [Mcwatters
and Cherry, 1982]. Peanut meal is effectively twice as concentrated in water soluble compounds compared to whole seeds because by weight roughly half of a peanut is oil. This
means peanut meal contains 45 to 60 percent protein and the whole seed only contains 22 to
30 percent protein [Mcwatters and Cherry, 1982].

1.3.2

Value

Peanut meal is used as a protein source in animal feeds and the quality as a feed ingredient
has been studied. The quality of eggs improves with protein supplementation from peanut
meal [Pesti et al., 2003]. For broiler chickens however, soy protein meal is a better protein
source than peanut meal [Costa et al., 2001]. Pigs fed nine diets had no differences in growth
when on a peanut meal based diet compared to the control soy bean meal diet [Shelton et al.,
2001].

1.3.3

Potential

Cottonseed, soybean, pea, cashew, flax, pecan, and even tomato seed meals can all be used to
produce high protein food ingredients [Lawhon et al., 1972,Liadakis et al., 1995,Manak et al.,
1980, McWatters and Cherry, 1977, Oomah et al., 1994, Piva et al., 1971].
Currently, peanut meal is not considered a food grade material by the US Food and Drug
Administration (FDA), likely due to the aflatoxin contamination commonly found in peanut
meal. Because peanut meal is not food grade, literature on protein extraction from peanut
meal for food purposes is limited; however, protein extraction from whole peanuts has been
investigated [Rustom et al., 1991, Cherry, 1990]. Protein extraction yields can be improved
by manipulating the solids to solvent ratio, pH of the solvent, temperature of solvent, and
duration of the extraction [Rustom et al., 1991]. Enzymatic hydrolysis can further increase the
amount of solids extracted from peanut [Rustom et al., 1993]. Protein can be extracted from
whole peanuts and subsequently spray dried to create high protein food ingredients [Lawhon

Table 1.4. Peanut meal pricing adapted from [Seifert, 2009]

Use
Dairy Cattle Feed
Animal Feed
Fertilizer

Aflatoxin Level (g/kg)

Price ($/ton)

< 20
20 300
> 300

210
175
95

et al., 1981]. However, the value and potential uses of peanut meal are limited by the aflatoxin
contamination levels (Table 3.2).

1.4
1.4.1

Aflatoxin
Introduction

Toxic metabolites of fungi known as mycotoxins are prevalent in a variety of crops, feeds, and
foods [Diener et al., 1982]. Aspergillus flavus is the most common mycotoxin producing fungus
found in peanuts; this is true across various climates and geographic regions [Diener et al.,
1982]. Aflatoxin is produced by the fungi Aspergillus flavus and Aspergillus parasiticus: hence
the name afla-toxin [Eaton and Gallagher, 1994]. Aflatoxin B1 (AFB1) is the most common of
the four forms of aflatoxin, B1, B2, G1, and G2 [Diener et al., 1982].

1.4.2

Prevalence in crops

Many agricultural commodities are often contaminated with aflatoxin producing fungi including coffee, corn, wheat, rice, soybean, sunflower, cotton, almond, pistachio, and peanut
[Soliman, 2002, Bhat et al., 2010, Yazdanpanah et al., 2005]. Humidity, temperature, and time
influence growth of A. parasiticus and A. flavus [Rustom, 1997,Diener et al., 1982]. On peanuts
between 6 and 46 C A. parasiticus can produce aflatoxin, and between 12 and 42 C A. flavus
produces aflatoxin as long as a minimum moisture content of 8% is met [Rustom, 1997, Diener et al., 1982]. Drought stresses also make peanuts more susceptible to contamination
likely due to decreased physiological activity within the peanut [Diener et al., 1982]. Peanut
cultivar also effects contamination levels; newer cultivars are more resistant to fungal growth
while native, or unimproved, cultivars are more susceptible to contamination [Mutegi et al.,
2009].

Table 1.5. LD50 of AFB1 for several species adapted from [Newberne and Butler, 1969,Wogan,
1966].
Animal

LD50 (mg/kg)

Cat
Dog
Duckling
Guinea Pig
Hamster
Mouse
Pig
Rabbit
Rat
Trout

1.4.3

0.6
0.5-1.0
0.3-0.4
1.4
10.2
9.0
0.6
0.3
1.0-17.9
0.5

Health concerns

The carcinogenic effects of aflatoxin in animals are well established and highly species dependent [Eaton and Gallagher, 1994]. The lethal orally administered dose of aflatoxin B1
required to kill 50% of a test population (LD50) for several species is summarized in table 1.5.
Since aflatoxin, specifically aflatoxin B1 (AFB1), is one of the most carcinogenic chemicals
studied, the FDA, World Health Organization (WHO), and European Union (EU) limit concentrations in foods intended for animal feed (Table 1.6) [Eaton and Gallagher, 1994, FDA,
2008].
Numerous epidemiological studies have established the connection between aflatoxin
consumption and incidence of liver cancer in humans [Eaton and Gallagher, 1994, Rustom,
1997, Wild and Gong, 2010, Williams et al., 2004]. Acute aflatoxin poisoning is rare, 25% of
these cases are fatal, and chronic exposure rates are high, especially in developing counties [Williams et al., 2004]. As such, many countries established legal limits for aflatoxin concentrations allowed in foods, specifically peanuts, intended for human consumption (Table
1.7) [Reddy et al., 2010]. Developing countries have especially high incidences of aflatoxicosis
because regulation and testing is prohibitively expensive, and food is scarce so uncontaminated alternatives are not available [Williams et al., 2004].

1.4.4

Prevalence in food and feed

There is a high prevalence of contaminated food being sold (Table 1.8) [Li et al., 2009, Reddy
et al., 2010, Turner et al., 2002]. Consequently, levels of exposure are especially high in the

Table 1.6. Levels of total aflatoxin contamination permissible by the FDA adapted from [FDA,
2008]
Peanut products intended for

Action level
g/kg

Dairy animals
Immature animals
Breeding beef cattle, breeding swine, or mature poultry
Finishing swine of 100 pounds or greater

20
20
100
200

Figure 1.1. Geography of populations at risk of chronic exposure to unregulated aflatoxin

exposure LAC, Latin America and the Caribbean: Adapted from [Williams et al., 2004]

Table 1.7. Legal limits of aflatoxin in peanuts: adapted from [Reddy et al., 2010]

Agency/Country

Aflatoxin

Upper limit
g/kg

Australia
Brazil
Bulgaria
Canada
China
Egypt
Hungary
India
Japan
Kenya
Korea
Russia
Taiwan
Turkey
United States

Total
B1 and G1
Total
Total
B1
Total
Total
Total
B1
Total
B1
B1
Total
B1
Total

15
30
15
15
20
10
15
30
10
20
10
5
15
5
20

affected countries with rates of exposure exceeding 90% of those populations [Williams et al.,
2004].

1.4.5

Destruction and management of aflatoxin

The destruction of aflatoxin in contaminated food and feeds has been thoroughly investigated [Bullerman and Bianchini, 2007, Ciegler, 1978, Piva et al., 1995]. The treatments can be
classified by mode of action as either chemical, mechanical, or radiative.
Chemical
Various chemical treatments of naturally and artificially contaminated foodstuffs effectively
reduce aflatoxin contamination levels [Piva et al., 1995, Rhee et al., 1977]. During processing
of peanut protein isolate or concentrate from contaminated peanuts, most (85%) aflatoxin
remains with the protein fraction [Rhee et al., 1977]. However, the addition of methylamine
at levels up to 1.25%, hydrogen peroxide, or ammonia gas reduces the aflatoxin remaining
in the resultant protein concentrates [Rhee et al., 1977]. Similarly, aflatoxin can be completely destroyed during processing of protein isolate/concentrate with the addition of acetone at >65%, isopropyl alcohol at 80%, benzoyl peroxide at 0.5%, or sodium hypochloride

10

Table 1.8. AFB1 contamination levels and frequency in market products adapted from [Li
et al., 2009, Reddy et al., 2010, Turner et al., 2002, Williams et al., 2004]
Country
Bangladesh
Botswana
Botswana
Brazil
Brazil
China
Cyprus
Egypt
Gambia
Ghana
Guinea
India
Japan
Nepal
Senegal
Sudan
Thailand
Uganda

Commodity

Frequency of aflatoxin
%

Peanuts
Peanuts
Peanut Butter
Peanut Products
Peanuts
Peanut butter
Peanut butter
Peanut and watermelon seeds
Peanuts
Peanuts
Peanuts
Groundnut
Peanut butter
Peanuts
Peanut Oil
Peanut and peanut butter
Peanut oil
Peanut, cassava

11

67
27
82
56.7
82

Contamination level
g/kg
65
12-329
0.3-23
43-1100
43-1100
1 - 79
> 10
162

13-32
61
21

85

12

1-112
> 30
2.6
> 30
40
87.4-197.3
102
> 100

at 0.2% [Natarajan et al., 1975, Rhee et al., 1977, Stoloff et al., 1976]. In addition to reducing
contamination as processing aids, hydrogen peroxide, sodium hypochlorite, and ammonia
are effective as treatments when applied directly to foodstuffs [Piva et al., 1995,Paulsen et al.,
1976]. Calcium hydroxide, formaldehyde, and sodium bisulfite also effectively destroy aflatoxin [Piva et al., 1995]. Treatment with ozone is effective, but dependent on temperature and
treatment time; in some foods, like corn, undesirable oxidation of fatty acids can limit the
potential use of this treatment [Dollear, 1968, Jr. and King, 2002, Proctor et al., 2004]. Indeed,
while many chemical treatments are effective, safety and health concerns, as well as cost,
prohibit commercial application of these treatments [Piva et al., 1995].
Mechanical
Commonly used physical food processing methods also reduce aflatoxin levels [Bullerman
and Bianchini, 2007]. Many processes used in food preparation are moderately effective at
destroying aflatoxin: washing wheat, washing and cooking rice, pressure cooking beans,
frying tortilla chips, and baking bread will reduce the aflatoxin levels in the finished product
[Bullerman and Bianchini, 2007]. Aflatoxin contamination in coffee beans is reduced during
the traditional roasting process which subjects beans to 180 C for 18 min [Soliman, 2002].
Similarly, cooking rice, or steaming wheat prior to bread baking reduces aflatoxin levels;
wet heating methods are generally more effective than dry heating [Hwang and Lee, 2006, Je
et al., 2005]. The efficacy of thermal destruction of AFB1 in peanut and cotton seed meals
is strongly dependent on moisture content of the meals [Mann, 1967]. Longer cook times
and higher temperatures, more effectively reduce aflatoxin levels [Yazdanpanah et al., 2005].
Aflatoxin B1 begins to decompose at 150 C, but dry heating as high as 237 to 306 C are
required to completely decompose AFB1 [Rustom, 1997].
Radiative
Gamma radiation applied to infested cereals completely destroys mycotoxin producing fungi
at doses as low as 6 kGy and prevents regrowth for at least 100 days after treatment [Aziz
et al., 2006]. This treatment is effective for contaminated or potentially contaminated foods
and feeds. Even at a lower dose of 4 kGy, fungi are nearly eliminated; such a treatment could
provide extra assurance of safety for potentially contaminated food and feeds [Aziz et al.,
2006]. Similarly, a 5 kGy dose will reduce, and 10 kGy will completely remove, the fungal
load in herbs and prevent regrowth after 30 days of storage [Aquino et al., 2010]. The same 10
kGy dose administered to peanuts completely inhibits fungal growth, while higher doses (15
- 30 kGy) will destroy the aflatoxin produced by those fungi in peanuts [Prado et al., 2003].
Ultraviolet (UV) radiation degrades AFB1, and contaminated peanut oil treated with UV

12

light for 2h destroys 40 to 45% of AFB1 present [Rustom, 1997]. Ultraviolet radiation from
sunlight degrades 50% of AFB1 in naturally contaminated peanut product [Rustom, 1997].
While these chemical and physical methods to mitigate aflatoxin contamination occurrence and levels are effective, their implementation is difficult due to cost and safety concerns [Piva et al., 1995].

1.4.6

Prevention and management

Preventing growth and aflatoxin production in food reduces total contamination levels and
provides an alternative strategy for ameliorating potential contamination. Where fungal
growth is unavoidable, due to a drought for example, aflatoxin contamination can be reduced
by applying to the soil a strain of A. flavus that does not produce mycotoxins [Dorner, 2008].
The atoxigenic A. flavus out-competes the naturally occurring toxin producing A. flavus, thus
reducing overall contamination [Dorner, 2009].
Segregation
Peanuts infested with fungi may have a dark and wrinkled skin; however, these nuts comprise
only a small percentage of peanuts in a typical lot [Cucullu et al., 1966]. These contaminated
nuts may have very high (6,000 g/kg) concentrations of aflatoxin [Cucullu et al., 1966].
Another smaller subset, usually less than one percent, of nuts do not show visible wrinkling
or discoloration and can have even higher levels of aflatoxin up to 1,100,000 g/kg [Cucullu
et al., 1966]. Both subsets typically represent less than ten percent of a total lot, but contain
nearly all of the aflatoxin and are less dense than intact kernels [Cucullu et al., 1966]. Indeed,
removing this subset of highly contaminated nuts is one of the most effective strategies to
reduce aflatoxin contamination. After harvest, peanuts are sorted by kernel size and density
[Dorner, 2008, Whitaker and Dickens, 1979]. The sorting process allows testing of individual
lots, which can then be rejected if contamination is detected [Dorner, 2008, Whitaker and
Dickens, 1979]. Sorting is generally effective, and the chance of accepting a contaminated lot
is often less than 5% [Whitaker, 1997, Whitaker and Dickens, 1979, Dickens, 1977]. Sorting
peanuts by color can remove the majority (70 to 90%) of aflatoxin contaminated peanuts
[Whitaker, 1997, Dorner, 2008]. Since seed discoloration is strongly associated with AFB1
contamination rates, color sorting blanched peanuts is even more (up to 91%) effective than
sorting unblanched peanuts by color [Dorner, 2008].
Sequestrants
Good farming practices, sorting, and processing may not eliminate 100% of aflatoxins present.
Human exposure to aflatoxin can be limited by: improving post-harvest storage conditions

13

for susceptible food, and using sequestrant clays to mitigate absorption [Groopman et al.,
2008]. Many types of sequestrants effectively absorb mycotoxins, especially AFB1, in vivo
and in vitro [Ramos et al., 1996]. Among the most effective seqrestrants are activated charcoal, bentonite, zeolite, diatomaceous earth, and hydrated sodium calcium aluminosilicates
(HSCAS) [Ramos et al., 1996, Modirsanei et al., 2008]. The most extensively studied sequestrants are the HSCAS [Phillips, 1999], which have a very high capacity to tightly bind aflatoxins in vitro [Grant and Phillips, 1998].
Despite extensive study, the discrepancy between sorbent sequestering capacity for AFB1
in vitro, and the in vivo applications is limiting [Moschini et al., 2008, Diaz et al., 2004]. Nevertheless, sorbent clays, in particular sodium bentonite, effectively limit deleterious effects
of aflatoxin consumption in poultry and livestock [Ramos et al., 1996]. Broiler chickens are
commonly used to evaluate in vivo efficacy of sequestrants [Ramos et al., 1996]. Even in
the absence of added aflatoxin, broiler chickens consuming sodium bentonite clay improved
weight gain, feed consumption, protein efficiency ratio, and protein digestibility [Pasha et al.,
2008]. Inclusion of only 0.3 or 0.5% sodium bentonite in feed can prevent the negative health
effects associated with high levels (1,000 g/kg) of aflatoxin consumption fed to broilers [Kermanshahi et al., 2009,Rosa et al., 2001]. Bentonite clays also protect piglets and sheep from the
deleterious effects of aflatoxin ingestion [Schell et al., 1993, Thieu et al., 2008, Khadem et al.,
2007]. Sodium bentonite clays are widely used because they are effective and inexpensive
to use [Khadem et al., 2007, Diaz et al., 2004]. Sodium bentonite can be used to effectively
remove aflatoxin during protein extraction from peanut meal [Seifert et al., 2010]. This novel
approach allows utilization of peanut meal as an inexpensive source of protein.

1.5

Peanut allergens

Many methods for removal or destruction of aflatoxin are available, but peanut allergies pose
a growing concern for many people. Peanut allergy is one of the most common and severe
food allergies in the United States affecting more than 3 million Americans [Sicherer and
Sampson, 2009,Sicherer et al., 2003]. Children with peanut allergic parents are ten times more
likely to be allergic than the general population [Sicherer and Sampson, 2009]. An allergy is
characterized by an "adverse immune reaction", usually to a protein [Sicherer and Sampson,
2009]. The major food allergens are water-soluble glycoproteins between 10 and 70 kDa, and
relatively stable to heat, acid, and proteases making them difficult to destroy [Sicherer and
Sampson, 2009]. The major peanut allergen is the 63 kDa glycoprotein known as Ara h1
[Maleki et al., 2000b]. A second major peanut allergen, Ara h2, may effect more people than
Ara h1 and is a smaller glycoprotein approximately 15 kDa [Burks et al., 1992, Koppelman
et al., 2004].

14

1.5.1

Mitigation strategies

Heating typically reduces the allergenicity of proteins by denaturing them [Wal, 2003]. It
is however, exceedingly difficult to destroy the major allergenic proteins in peanuts because
they have a high resistance to heat and a specific tertiary structure which protects the binding
sites from degradation [Kopper et al., 2005, Maleki et al., 2000b]. Furthermore, the roasting
process, which is used extensively in the US, increases the IgE-binding of peanuts [Maleki
et al., 2000a]. Conversely, boiling peanuts apparently decreases allergenicity, which is likely
due to the migration of proteins from the nuts to the cooking water [Mondoulet et al., 2005].
Because protein destruction by heating is ineffective, enzymatic hydrolysis may be used to
reduce allergenicity of peanut proteins by altering structure [Sen et al., 2002]. Unfortunately,
allergenic peanut proteins, in particular Ara h1, resist enzymatic hydrolysis by pepsin [Maleki
et al., 2000b].

1.5.2

Therapies

The most commonly employed strategy for patients with peanut allergy is to simply avoid
contact with peanuts [Sicherer and Sampson, 2009]. This strategy is limited because it is impossible to completely control the environment and incidental contact with peanut protein is
nearly unavoidable. Sub-lingual immunotherapy (SLIT) is the gradual deliberate exposure of
patients to minute amounts of the allergenic proteins to build a general tolerance to avoid an
adverse acute allergic response [Sicherer and Sampson, 2009]. This strategy is limited by the
varying responses to the treatment as some patients have an adverse reaction to even minute
doses and never develop a tolerance [Sicherer and Sampson, 2009]. Another approach is peptide immunotherapy which involves creating a vaccine composed of small peptides [Sicherer
and Sampson, 2007]. The peptides generically contain all amino acid sequences so antigen
presenting cells are provided with information to create antibodies for all possible peanut allergens, and without binding to the mast cells responsible for adverse reaction [Sicherer and
Sampson, 2007]. The therapeutic peanut peptides can be generated by enzymatic hydrolysis
of peanut proteins.

1.6

Hydrolysis

1.6.1

Introduction

Hydrolysis is the chemical process by which a molecule is cleaved into two parts by the
addition of a molecule of water. One fragment of the parent molecule gains a hydrogen
ion (H+ ) from the additional water molecule. The other group reacts with the remaining

15

hydroxyl group (OH ). This reaction is catalyzed by strong acids, strong bases, or enzymes.
Hydrolysis of proteins produces peptides and free amino acids. The degree of hydrolysis
(DH) refers to the percentage of peptide bonds broken and is often measured by the amount
of acid or base required to maintain a given pH [Panyam and Kilara, 1996]. Another way to
determine DH is a spectrophotometric trinitrobenzenesulfonic (TNBS) acid method [AdlerNissen, 1976] More extensively hydrolyzed proteins have higher DH values and a greater
proportion of lower molecular weight peptides than the same unhydrolyzed protein.

1.6.2

Uses

Industrially, proteins are hydrolyzed to improve functionality and nutritional quality or to

improve the value of waste proteins, as in slaughterhouse waste for example [Bhaskar et al.,
2007]. Hydrolysis typically increases protein solubility across a pH range, while decreasing the functionality associated with protein unfolding [Beuchat et al., 1975, Ruz-Henestrosa
et al., 2007]. This is probably due to the loss of aliphatic structure of the protein molecules.
Many functional properties are highly dependent on DH; foaming, gelation, and emulsification properties are generally hindered by smaller peptides and improved by larger ones [Panyam and Kilara, 1996]. For example, the hydrolysis of whey proteins improves heat stability,
reduces allergenicity, and produces bioactive peptides [Foegeding et al., 2002]. Using hydrolysis to create a specific DH can be used for tailoring amounts and size of peptides for special
diets, and altering the functional properties of gelation, foaming and emulsification [Foegeding et al., 2002]. Despite reducing emulsifying and foaming capacity, hydrolysis improves free
radical scavenging ability of quinoa and soy protein [Monu, 2003, Peta Ramos and Xiong,
2002].

1.6.3

Nutritional considerations

Limited hydrolysis of soy protein isolates results in increasing antioxidant activity [Peta
Ramos and Xiong, 2002]. Hydrolyzed proteins, especially small (di- and tri-peptides) peptides are more quickly absorbed through the small intestine than free amino acids [Webb,
1990]. Hydrolysis can be used to improve the amino acid profile of mixtures of proteins
from several sources, providing an opportunity to inexpensively create high quality protein
foods [Radha et al., 2008]. Peanut protein hydrolysates have balanced amino acid profiles
consistent with the United Nations Food and Agriculture Organization and World Health
Organization (FAO/WHO) guidelines for human health [Kain et al., 2009]. In addition to improving the nutritional properties of proteins, hydrolysis can also be used to create bioactive
compounds and nutraceutical ingredients.

16

Enzymatic hydrolysis is the most commonly used method to create angiotensin 1 converting enzyme (ACE) inhibitory peptides, which play an important role in regulating blood
pressure, a major health concern [Guang and Phillips, 2009a]. ACE inhibitory peptides can
be isolated from peanut flour using the enzyme Alcalase [Guang and Phillips, 2009b]. Peanut
protein hydrolysates can be used as natural antioxidants with reported free radical scavenging properties similar to butylated hydroxy-toluene and ascorbic acid [Chen et al., 2007]. The
excellent nutritional properties of hydrolyzed peanut proteins provide incentives to further
investigate the production and properties of these hydrolysates.

1.7

Spray Drying

1.7.1

Introduction

Dry food materials are less expensive to transport and store because they are more compact,
and weigh less than their full-moisture counterparts. Spray drying is one of the most widely
used drying processes in the food and pharmaceutical industries [Masters, 1991]. Spray
drying is a commonly used drying process because it can be operated continuously with high
throughput [Masters, 1991]. The drying process consists of three main steps: atomization of
the liquid feed material, dehydration of atomized droplets, and separation of the dry particles
from the drying gas [Masters, 1991].
In spray drying, atomization refers to the process by which liquid feed material is sprayed
into a fine mist, which greatly increases the surface area of the solution to be dried. Droplets
are typically between 5 m and 200 m, and upon contact with heated drying gas the droplets
dry very quickly. The dry particles are separated from the drying medium by centrifugal
force in an apparatus, typically called a cyclone. Outlet temperature is the temperature of the
drying air measured at or just before the cyclone [Masters, 1991].
Heated air is pumped into the drying chamber and the inlet is the point at which the
atomizer enters the drying chamber [Masters, 1991]. The inlet air temperature can exceed 400
C,

but the outlet temperature is lower and primarily a function of feedstock moisture content

and feed flow rate [Masters, 1991, Samborska et al., 2005]. Changing spray dryer process parameters or feedstock properties will influence the finished powder characteristics [Masters,
1991,Maa, 1998,Goula and Adamopoulos, 2004]. Thermal and evaporative efficiencies as well
as product yields are influenced by spray dryer parameters [Goula and Adamopoulos, 2003].
Yield can be expressed as the ratio of solid material collected to the amount of solid material fed into the spray dryer. Spray drying conditions must be carefully chosen to maximize
yields for each dryer layout. By manipulating process parameters according to feed liquid
characteristics and dryer layout, many heat sensitive and even bioactive compounds can be

17

Table 1.9. Spray dried heat-sensitive or bioactive compounds

Compound
ACE inhibitory peptides (alfalfa)
ACE inhibitory peptides (shrimp)
-galactosidase
Insulin
Lysozyme
Lysozyme and Tripsin
Soy protein isolate
Tripsin

Reference
[Kapel et al., 2006]
[He et al., 2008]
[Branchu et al., 1999]
[Sthl et al., 2002]
[Elkordy et al., 2002]
[Hulse et al., 2009]
[Boatright and Hettiarachchy, 1995]
[Millqvist-Fureby et al., 1999]

successfully spray dried.

1.7.2

Drying bioactive compounds

Spray dried bioactive compounds can retain the same functional properties from lab scale to
pilot scale [He et al., 2008]. This consistency across scales may explain the popularity of spray
drying. Model enzymes and proteins like tripsin and insulin can be successfully spray dried
and they are commonly used in pharmaceutical research [Sthl et al., 2002, Millqvist-Fureby
et al., 1999, Hulse et al., 2009]. A variety of other heat sensitive food and pharmaceutical
materials have been successfully spray dried (Table 1.9).

1.7.3

Drying method comparisons

Spray drying has been compared to other commonly used drying methods. Freeze drying is
used to dry heat sensitive materials because of the low temperatures used. Spray drying can
be 30 to 50 times less expensive than freeze drying and an acceptable alternative [Masters,
1991]. In many cases the dry product produced by spray drying has better functional properties than when produced by freeze drying, for example faba bean powder has better solubility
and emulsion capacity when produced by spray drying than by freeze drying [Cepeda et al.,
1998]. Spray drying reduces the anti-nutritional trypsin inhibitors, tannins, and phytates in
faba bean protein powder, as well as freeze drying does [Otegui et al., 1997]. Most of the
functional properties of those protein powders were comparable, but the spray dried powder
had better gelation properties [Otegui et al., 1997]. Spray dried potato protein powder has
comparable quality to vacuum freeze dried powder, but with a lighter color that is known to
improve consumer acceptability [Claussen et al., 2007,Lkra et al., 2008]. Shrimp hydrolysate

18

paste produced by vacuum drying at 80 C had a lower concentration of luecine tyrosine

serine, but more aspartic acid, glutamic acid, arginine than the spray dried powder [BuenoSolano et al., 2009]. Freeze dried soy protein isolates are typically less soluble than those
produced by spray drying [Boatright and Hettiarachchy, 1995]. Similarly, peanut protein
powders produced by spray drying have better water holding, oil binding, emulsion and
foaming capacity compared to peanut protein powder prepared by vacuum oven drying [Yu
et al., 2007]. Despite the fact that spray drying preserves functionality of the dried material,
this method has some limitations.

1.7.4

Stickiness

Sticky foods are particularly difficult to spray dry and this problem has been investigated
[Chen et al., 2007]. Despite the difficulty, many sticky foods can be successfully dried including: sweet potatoes, honey, tomato pulp, and sugar solutions [Goula and Adamopoulos,
2008, Truong et al., 2005, Grabowski, 2006]. Stickiness during drying is due to the dried material having a critical viscosity decrease from 1010 1012 Pas to 106 108 Pas, or exceeding the
glass transition temperature (Tg ) by more than 20 C [Downton, 1982, Bhandari et al., 1997].
The presence of organic acids and small molecular weight carbohydrates in feedstocks leads
to stickiness during spray drying [Jayasundera et al., 2009]. Stickiness is difficult to measure
objectively; several methods have been developed for both powders and carbohydrate solutions [Boonyai et al., 2004, Adhikari et al., 2007, Aguilera et al., 1995]. Powder stickiness is the
main cause of decreased yields during spray drying [Jayasundera et al., 2009, Adhikari et al.,
2007].

1.7.5

Strategies to reduce stickiness

The two main causes of low yields are dripping, indicating insufficient drying, or caking due
to over-drying [Meng et al., 2007]. The latter refers to stickiness and both causes are remedied
by either adjusting spray dryer conditions or modifying the feed material [Meng et al., 2007].
Spray dryer conditions like temperatures, air and feed flow rates, as well as the physical
design of the dryer can be adjusted to reduce stickiness [Maa, 1998, Meng et al., 2007]. For
example, using dehumidified air improves the yield of spray dried tomato pulp, an otherwise
prohibitively sticky material [Goula and Adamopoulos, 2008]. Over-drying can be prevented
by maintaining droplet temperatures less than 20 C above Tg of the feed stock [Truong et al.,
2005]. Optimal drying conditions for spray drying can be predicted based on estimated Tg of
the feed stock [Truong et al., 2005].
Additionally, the composition of the feed stock can be modified to improve yields by
adding carrier agents [Jayasundera et al., 2009]. The carrier agent either modifies the sur-

19

face composition, or changes the bulk Tg [Jayasundera et al., 2009]. For some products the
quantity of carrier agent necessary would alter bulk composition so much that consumer
acceptability would decrease or the standard of identity would no longer be met; for example, spray dried honey is only 50% honey [Wang and Langrish, 2009]. Consequently surface
active compounds have been investigated as carrier agents which can be used in very low
concentrations [Wang and Langrish, 2009]. These compounds work by forming a non-sticky
surface layer on the surface of the drying droplets [Wang and Langrish, 2009]. Adding surface active proteins to sucrose solution in a ratio of 0.5 : 99.5 (0.5% of bulk on solids basis)
protein to sucrose causes spray drying yields to improve from zero to as much as 85% [Adhikari et al., 2009a]. The protein forms a non-glassy skin around the sucrose droplet reducing
stickiness resulting in successful spray drying [Adhikari et al., 2009a]. Similarly, tripsin was
predominant the surface of droplets mostly composed of carbohydrates [Millqvist-Fureby
et al., 1999]. Other surface active compounds like small molecular weight surfactants can
also be used to selectively control the surface composition of drying droplets to improve
yields [Adler et al., 2000, Bosquillon et al., 2004]. However, in sugar rich solution containing
surface active protein, small molecular weight surfactants can drastically reduce yields by
displacing the non-sticky forming protein layer from the surface of the droplets [Adhikari
et al., 2009c].
Another strategy for increasing yields of sticky solutions is to increase the Tg of the feed
material [Bhandari et al., 1997]. High molecular weight additives effectively increase the Tg so
hotter drying temperatures can be used without stickiness problems [Bhandari and Howes,
1999]. That is, for example, rather than lowering the dryer temperature to match the feed, the
feed can be modified to by increasing Tg .

1.7.6

Maltodextrin as a carrier

Maltodextrin can be used as an additive to increase Tg of feed solutions containing sticky

materials [Adhikari et al., 2004].
Typically, maltodextrins with low dextrose equivalent (DE) values are used because low
DE maltodextrins have the highest molecular weight, which increases Tg of the feed stock
more than would the same mass of a maltodextrin with a high DE [Avaltroni et al., 2004,
Bhandari and Howes, 1999]. Powder yields and properties such as solubility, and solids
content, of many food powders are increased by the addition of maltodextrin to feed stock
prior to spray drying [Goula and Adamopoulos, 2008, Grabowski, 2006, Namaldi et al., 2006,
Abdul-Hamid et al., 2002, Kurozawa et al., 2009]. Food powders with low hygroscopicity are
desirable and the addition of maltodextrin to feed solutions prior to spray drying decreases
hygroscopicity of the resultant powders [Goula and Adamopoulos, 2008, Kurozawa et al.,

20

2009]. In both spray dried chicken hydrolysates and tomato pulp adding more maltodextrin
to the feed solution prior to spray drying resulted in powders that were less hygroscopic
[Goula and Adamopoulos, 2008, Kurozawa et al., 2009]. Similarly, the DE of the maltodextins
used affected hygroscopicity of the tomato powder such that DE 6, 12, and 21 were least,
middle, and most hygroscopic respectively, and in chicken hydrolysate maltodextrins reduced
hygroscopicity more than gum arabic at the same usage levels [Goula and Adamopoulos,
2008, Kurozawa et al., 2009]. Maltodextrin is commonly used for microencapsulation because
it provides stability to sensitive materials during spray drying and storage [Gharsallaoui
et al., 2007, Gibbs et al., 1999].

1.7.7

Protein denaturation during drying

Maltodextrin inhibits protein denaturation during thus preserving enzyme activity after
spray drying [Namaldi et al., 2006, Baeza and Pilosof, 2002, Anandharamakrishnan et al.,
2007]. Carbohydrates reduce protein denaturation during drying possibly by forming hydrogen bonds with the proteins to replace the water-protein hydrogen bonds [Carpenter and
Crowe, 1989]. A more likely mechanism is that the protein loses conformational freedom due
to the high solute concentration caused by the addition of carbohydrates [Prestrelski et al.,
1993]. Because denaturation is a loss of tertiary structure, the lack of conformational freedom
preserves the tertiary structure of the proteins [Prestrelski et al., 1993]. Another way to reduce conformational freedom is to increase the concentration of protein in solution. Protein
solutions with high protein concentrations retain tertiary structure after spray drying better
than solution with low protein concentrations [Millqvist-Fureby et al., 1999]. In summary the
factors total solids content, outlet temperature, and carrier concentration have the greatest
impact on protein stability during spray drying [Sloth et al., 2008].

1.7.8

Outlet temperature

The maximum temperature drying particles are assumed to reach during spray drying is
the measured outlet temperature [Masters, 1991]. Outlet temperature is a function of other
process variables and is the best predictor of residual enzyme activity and microbial inactivation during spray drying [Samborska et al., 2005, Sthl et al., 2002, Elizondo and Labuza,
1974, Labuza et al., 1970]. Microbial inactivation or microbial death rate can be described by
the pseudo-z value defined as the change in temperature required to cause a 10-fold increase
in microbial death [Teixeira et al., 1995]. During spray drying of lactate dehydrogenase outlet
temperatures above 89 C resulted in a decrease in pseudo-z value. This means above 89 C(the
pseudo-z) a small increase in outlet temperature causes a greater loss of activity than the same
temperature increase when below 89 C [Matzinos and Hall, 1993]. This critical outlet temper-

21

Table 1.10. Temperatures at which microbial death or enzyme inactivation rate changes

Material

Outlet
( C)

Effect

Reference

-amylase
Whey protein
Insulin
Lactate dehydrogenase
L. bulgaricus
S. cerevisiae
S. cerevisiae

> 80
> 90
> 120
> 89
< 70
< 84
< 80

denaturation
denaturation
denaturation
change pseudo-z
kill
< 4 log kill
4 log kill

[Samborska et al., 2005]

[Anandharamakrishnan et al., 2007]
[Sthl et al., 2002]
[Matzinos and Hall, 1993]
[Teixeira et al., 1995]
[Elizondo and Labuza, 1974]
[Labuza et al., 1970]

ature can be identified for microbial death rates and protein denaturation rates [Samborska
et al., 2005,Teixeira et al., 1995,Sthl et al., 2002,Anandharamakrishnan et al., 2007,LiCari and
Potter, 1970, Costa et al., 2002]. The critical temperatures are those above which denaturation
inactivation of a desirable compound are too great, or below which there is a sharp decline
in microbial death rate (Table 1.10). Similar breaks in death rates of microbes have been
found for various microorganisms, (Table 1.10). An outlet temperature of 90 C is commonly
used to spray dry proteins (Table 1.11). An outlet temperature of 90 C is expected to cause a
minimum 4 log reduction of microorganisms without reducing solubility or denaturing the
protein. These considerations perhaps explain why this temperature is commonly used.

22

Table 1.11. Spray dried protein products

Product

Inlet
( C)

Outlet
( C)

Reference

Alfalfa protein hydrolysate

Bovine plasma
Bovine serum albumin
Chicken hydrolysate
Faba bean
Faba bean protein
Herring hydrolysates
Insulin
Milk emulsions
Shrimp peptides
Sweet potato puree
Pea protein
Potato protein
Soy protein isolate
Tilapia hydrolysate
Whey protein

170

90
60-110
90
91-102
100
122
100
50-150
90
90
100
86
90
80-85
90
80,100

[Kapel et al., 2006]

[Matzinos and Hall, 1993]
[Adler et al., 2000]
[Kurozawa et al., 2009]
[Cepeda et al., 1998]
[Otegui et al., 1997]
[Hoyle and Merritt, 1994]
[Sthl et al., 2002]
[Sliwinski et al., 2003]
[He et al., 2008]
[Grabowski, 2006]
[Sumner et al., 1981]
[Claussen et al., 2007]
[Boatright and Hettiarachchy, 1995]
[Abdul-Hamid et al., 2002]
[Anandharamakrishnan et al., 2007]

150
180
180
190
180
100-220
150
170
150-220
190
210-215
180
180,200

23

CHAPTER

TWO
AMINO ACID ANALYSIS OF HYDROLYZED PEANUT MEAL
PROTEINS

2.1

Abstract

This work was a contribution to a larger study accepted for publication to the Journal of
Food Biochemisty. The amino acid content of proteins influences the solubility and thus
functionality of those proteins. Peanut meal protein was hydrolyzed for 240 min using three
commercially available food grade proteases. The amino acid contents of the water soluble
hydrolysates were compared to each other, control samples, and unhydrolyzed peanut protein. Enzymatic hydrolysis increased solubility of all hydrolysates and negligibly effected the
relative solubility of individual amino acids.

2.2

Introduction

Amino acids are largely responsible for the functional properties and nutritional quality
of proteins. The sequence and quantity of amino acids determines the tertiary structure.
Tertiary structure determines the solubility of the protein. Solubility is the basis of other
functional properties. Antioxidant activity is one such functional property. All 20 amino
acids can potentially quench free radicals. The most reactive amino acids (AAs) contain
nucleophillic-sulfur containing or aromatic side chains. Among the most reactive AAs are
cysteine and methionine which are sulfur containing AAs, and tryptophan, tyrosine, and
phenylalanine which have aromatic side chains. Amino acids are classified as either essential
or non-essential to human health. The relative amounts of these essential amino acids in a
protein can be used to calculate the nutritional quality of that protein containing those amino
acids.

24

2.3

Materials & Methods

Samples of interest were completely hydrolyzed in triplicate for 18 h at 110 C in the presence of 6 N HCl containing 0.1% phenol. Aliquots of the final hydrolysates were then derivatized using AccQFluorTM reagent (Waters Corp., Milford, MA) as outlined in the manual
(WAT052874, Rev 0). Derivatives were analyzed using a Summit Model HPLC (Dionex Corp.,
Sunnyvale, CA) equipped with a Waters AccQTag (C18, 4m, 150 mm x 3.9mm) column. Eluant A was an aqueous phosphate buffer solution containing triethylamine (purchased from
Waters Corp.) and diluted with water. Eluant B was Acetonitrile:water at a ratio of 60:40,
v:v. The gradient for the analysis started at 0% B then increased to 2.5% in 0.5 min, to 7%
in 15 min, to 10% in 19 min, to 33% in 32 min and held for 1 min. Eluant B was increased
in 1 min to 100%, held for 3 min, and then decreased to 0% in 3 min. The column was then
re-equilibrated at 0% B for 13 min. Total run times were 50 min at 37 C with a flow rate of 1.0
mL/min. The injection volume was 20 mL. Fluorescence detection with excitation/emission
wavelengths set to 250/395 nm was used.
A standard mixture of amino acids was spiked with an internal standard of -aminobutyric
acid and hydrolyzed using the same methods as the samples. Values from this standard runs
were used to calculate response factors for each amino acid. Samples were spiked with
an equivalent amount of internal standard and response factors were used to quantify the
amount of each amino acid in the samples. As a control, a standard reference sample of
peanut butter, SRM 2387 (NIST, Washington, DC) was treated as a sample and hydrolyzed
and derivatized with every sample set.
Means of individual amino acid concentrations were compared using Tukey Tests for both
unhydrolyzed control samples and samples hydrolyzed for 240 min. Statistical analyses were
performed using SAS (Cary, NC).

2.4

Results & Discussion

Amino acid composition of the unhydrolyzed controls were compared to those of the 4 h hydrolysates for each of the three proteases, and the amino acid composition of the dry, unhydrolyzed, defatted peanut meal (Figure 2.1). Cysteine, methionine, and tryptophan were not
measured. The oxidative conditions for this analysis normalized glutamine and asparagine
with glutamic acid and aspartic acid respectively. Alcalase and flavourzyme controls had
higher concentrations of each amino acid than pepsin, but this difference was not significant
(Figure 2.1A). For the hydrolyzed samples (240 min) Alcalase produced significantly (p <
0.05) higher concentrations of many amino acids than pepsin or flavourzyme (Figure 2.1 B).
The sum of amino acid contents for each sample correlated well (R

25

= 0.82) with total

solids contents for each corresponding sample. This correlation suggests that apparent differences in amino acid contents are due to solubility differences between treatments. The
samples of each hydrolysate with greater total solids also had higher concentrations of each
amino acid.
The amino acid content of each hydrolysate was normalized to a percent basis by dividing
each individual amino acid content by the sum of all amino acids for that sample. With
the differences in solubility removed the relative amounts of each amino acid were then
compared between treatments. Comparing normalized contents of time 0 controls and 240
min treatments demonstrated that differences between individual amino acid contents varied
slightly (only 2.7%) and most varied less than 1%. Compared to the peanut meal protein
sample, the relative amino acid contents of each hydrolysate were roughly the same. This
indicates that the relative concentrations of amino acids were minimally changed by these
hydrolysis treatments.

0.6

(A)

(B)

(C)

0.4

g/100 g

g/100 mL

0.5

Alcalase
pepsin
Flavourzyme
peanut meal

0.3

0.2

2
0.1

Glu
t

art
As
p

ic
Ac
S id
am erin
ic e
Ac
Gly id
His cine
ti
Arg dine
Th inin
reo e
n
Ala ine
n
Pro ine
Ty line
ros
i
Va ne
line
L
Iso ysin
leu e
Ph Le cine
en uc
yla ine
lan
ine

As
pa
rt

Glu
t

As
pa
rtic
Ac
Glu
id
tam Serin
ic e
Ac
Gly id
His cine
ti
Arg dine
Th inin
reo e
n
Ala ine
n
Pro ine
Ty line
ros
i
Va ne
line
Iso Lysin
leu e
Ph Le cine
en uc
yla ine
lan
ine

ic
Ac
S id
am erin
ic e
A
Gly cid
His cine
ti d
Ar ine
Th ginin
reo e
n
Ala ine
n
Pr ine
ol i
Ty ne
ros
in
Va e
li
Ly ne
Iso sin
l eu e
c
Ph Le ine
en uc i
yla ne
l an
ine

0.0

Figure 2.1. Amino acid composition of water soluble controls (A), hydrolysates after 240
minutes (B), and unhydrolyzed peanut meal (C)
Points are means of 3 1 standard deviation
Adapted from [Seifert, 2009]

26

CHAPTER

THREE
PROCESS DEVELOPMENT

3.1

Abstract

Aflatoxin contaminated peanut meal is an inexpensive source of high quality protein. Previous research has demonstrated that sodium bentonite clay can be used to sequester aflatoxin
during aqueous extraction of peanut protein [Seifert et al., 2010]. This novel approach was
applied to the development of a pilot scale process to generate aflatoxin-free protein powder from aflatoxin-contaminated peanut meal. The remaining insoluble material can be oven
dried and used as an animal feed ingredient. A series of preliminary experiments were
conducted to determine the processing steps and equipment necessary to produce peanut
meal protein powder on a scale suitable to process 10 kg batches. Twelve dispersions were
evaluated for solids, protein, and protein content of the water soluble fractions per gram of
peanut meal used in each dispersion. Gravitational separation was used to separate water
soluble and insoluble fractions. Soluble fractions contained 9-12% solids after the addition
of maltodextrin. Maltodextrin was used to facilitate spray drying and minimized differences
between treatments in spray drying performance. Several spray dryers and drying conditions
were evaluated. Using a Bchi B-290, an inlet temperature of 185 C and outlet temperature
of 90 C produced the highest yields after the addition of 8% maltodextrin to soluble fractions
collected using gravitational separation.

3.2

Introduction

Malnutrition, specifically protein deficiency, is a growing problem in developing countries

[Latham, 1997]. Vegetable proteins are a relatively inexpensive source of essential amino
acids. Increasing interest in previously underutilized protein sources has lead to develop-

27

ment of processes to extract protein from plant sources including tea, wheat, barley, corn,
rice, and peanuts [Cagampang et al., 1966, Celus et al., 2007, Guan and Yao, 2008, Shen et al.,
2008, Rustom et al., 1991]. Enzymatic hydrolysis is often used to facilitate the extraction of
protein from plant materials. The non-protein components of these plant protein sources
include fiber, lignin, and others. For effective aqueous extraction of soluble proteins the insoluble components must be separated from the soluble fraction. Centrifugation or filtration
are used to separate the protein fraction from the insoluble materials. Soluble protein fractions are then concentrated using isoelectric precipitation, filtration, drying, or a combination
of these methods. Several processing techniques to extract and concentrate protein from
peanut meal were evaluated. The following work presents a series of experiments used to
evaluate individual processing steps in isolation. The results of these experiments were used
to construct a complete processing scheme.

3.3

Materials & Methods

Peanut meal was donated by Golden Peanut, LLC (Alpharetta, GA), and Aflatoxin content
was determined by AOAC method 991.31. Proximate analysis was performed by BarrowAgee Laboratories, LLC (1555 Threeplace Memphis, TN 38116; (901) 332-1590).The peanut
meal used in these experiments contained 52 g/kg aflatoxin B1 (AFB1), 2.9% fat, 47.8 %
protein, 5.4% fiber, and 5.2% ash all on wet weight basis. On a dry weight basis this meal
contained: 59 g/kg aflatoxin, 3.3% fat, 53.9% protein, 6.1% fiber, and 5.9% ash. To estimate
the particle size range of the dry peanut meal a 400 g sample of dry peanut meal was poured
over a series of standard sieves (Fisher Scientific Co.), including US No. 12, 30, 35, and 80
sieves. These sieves have openings of: 1680, 600, 500, and 175 m respectively. A pan was
placed under the No. 80 sieve to catch any particles passing through the 175 m opening.
Empty sieves were weighed prior to, and after, peanut meal addition. Percent size distribution was calculated as weight of meal on a sieve divided by the total weight of meal. The
frequency distribution histogram was converted to a density function and plotted using R: A
language and environment for statistical computing [R Development Core Team, 2010]. Line
plots were made using the sciplot package for factorial designs, error bars indicate 1 standard
error [Morales et al., 2010].

3.3.1

Protein extraction from peanut meal

Dispersions
Several dispersions of peanut meal in deionized (di), 20 C water were made at concentrations of 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, and 21% (w/w). Using a Wheaton overhead

28

stirrer (Wheaton Instruments Millville, NJ) the dispersions were stirred for 20 min before the
addition of acid or base to adjust the pH to 2.0 or 8.2. Dispersions were transferred into
1600 mL tubes after 1 h and centrifuged at 25,412 x g for 20 min at 20 C. Soluble fractions
were decanted and protein content was determined using the bicinchoninic acid (BCA) assay
(Pierce, Rockford, IL) using bovine serum albumin as the reference protein. Approximately
2 g of solubles were analytically weighed in an aluminum dish and heated in a vacuum oven
(VWR Scientific, Inc., West Chester, PA) at 115 C for 16 h to determine solids content. The
dried samples in aluminum dishes were then cooled to room temperature in a desiccator
before their mass was determined.
Separation
Soluble and insoluble fractionation was assessed by making 700 g peanut meal dispersions at
concentrations of 5, 10, 15, and 20% (w/w) peanut meal in 20 C di water. Dispersions were
stirred for 20 min using a Wheaton overhead stirrer. The pH of the dispersion was adjusted
to 8.2 using 2 N NaOH. Stirring was resumed for an additional 1 h. Immediately after final
stirring, 100 mL of dispersion were added to graduated cylinders. At 60, 120, 180, and 240
min the volume of insolubles was recorded and the density of a 1 mL aliquot was determined
at 20 C using a DMA 5000 density meter (Anton Paar).
The following experiment was conducted to determine how soluble fractions collected
after centrifugation compared to those collected after gravitational settling. A 6 kg 10% (w/w)
peanut meal in di water, dispersion was prepared. Following a 20 min equilibration period of
stirring at 20 C, 180 mL of 2 N NaOH was added to adjust the pH to 8.08, from an initial pH
of 6.07. After 1 h, while still stirring, a 1 L aliquot (1690 g) was siphoned into a beaker and
centrifuged in a Sorvall RC-5 refrigerated centrifuge at 25412 x g for 20 minusing the Sorvall
GSA rotor (Sorvall Newton, CT). Stirring was ceased and the dispersion was allowed to
settle. After 4 h the supernatant was siphoned from the dispersion and analyzed for protein
and solids content. Immediately after centrifugation the soluble fractions were decanted and
analyzed for protein and solids content using the BCA and oven drying methods previously
described.

3.3.2

Insoluble fraction

Insoluble fractions were oven dried to determine solids content. Yield was calculated as mass
of solubles collected divided by the difference between total batch dispersion mass and the
mass of the soluble fraction collected.

29

3.3.3

Water soluble fraction

Vacuum oven drying

Vacuum oven drying was investigated as a method to increase the solids content of soluble
fractions prior to spray drying. A custom drying vessel made with 308 grade 16 gage stainless
steel, dimensions: width 25 cm, depth 45.7 cm, and height 12.7 cm was used to contain water
for determining the relationship between the dial setting and air temperature in a vacuum
oven. Filled to a depth of 10 cm, the vessel had a capacity of 11.4 L.
The oven dial setting was adjusted and temperatures of water in the vessel and air in the
oven equillibrated for 30 min. Two thermometers were positioned in the oven such that the
bulb of one was immersed in water while the other was exposed to the air.
Water flux (N) was calculated for solubles concentration (Equation 3.1). Water content of
1 mL aliquots was estimated by measuring the density using the DMA 5000 density meter.

W
At
W is water content in kg water per kg dry material
N=

(3.1)

A is surface area of sample

t is evaporation time in seconds
Density and solids content of soluble fractions correlated very well (R

= 0.95) and density

was used to estimate solids using equation 3.2.

Solids = 375.43 + 374.80 Density

(3.2)

Spray drying
R SD-Basic, UK) was used to dry peanut meal extracts.
A lab scale spray dryer (LabPlant

Water was pumped for 1 min through spray dryer nozzle into a graduated cylinder. After one
minute the volume of water was recorded and volumetric flow rate as mL/h was calculated
for each pump speed measured. Aliquots of soluble peanut extracts with added maltodextrin
(feed stocks) were spray dried across a broad range of inlet temperatures and feed flow rates.
The outlet temperature and observations were recorded.
A pilot scale spray dryer (Anhydro Laboratory Spray Dryer S-1, Anhydro Inc., Attleboro
Falls, MA, U.S.A.) equipped with a 2-fluid nozzle for atomization and a mixed-flow airproduct pattern was evaluated.
Four different feed stocks from 10% dispersions at pH 8.2 were sprayed at 180, 200, and
R M100) was donated by Grain Processing Corp. (Muscatine,
220 C. Maltodextrin (Maltrin

30

IA; Lot number M0910530). The dextrose equivalents and moisture content were given as 10.7
and 4.7% respectively. The maltodextrin was added to the solubles so the final solids content
of the feed stocks were 10, 15, and 20%. A control feed stock containing no maltodextrin was
also spray dried. Yields were calculated as the ratio of solids collected in the collection vessel
(Figure 3.6) Solids collected in the collection vessel (Figure 3.6 component C), divided by the
total amount of solids fed into the dryer was termed yield. Solids collected in components A
and B (Figure 3.6) divided by solids fed to the dryer were termed hold-up and expressed as
a percentage. Overall thermal efficiency was estimated using equation 3.3 [Masters, 1991].

overall =

T1 T2
T1 T0

100

(3.3)

Two 10 kg peanut meal dispersions were made at 10% w/w in di water at 20 C. One
dispersion was adjusted to pH 8.01 with 2 N NaOH, and hydrolyzed with 92 g liquid Alcalase
from Bacillus licheniformis (batch 056K1213, EC 232-752-2), which is 2.4 Anson Units per g of
peanut meal protein. The second dispersion was adjusted to pH 2.0 and hydrolyzed with
10 g pepsin dissolved in 90 mL 0.1 N HCl. Approximately 800 g aliquots of each dispersion
were spray dried after adding maltodextrin at a concentration of 8%. Each feedstock was
spray dried at 135, 160, 185, 210 C. Yields, hold up, and powder solids were measured after
each run and runs were done in duplicate for a total of 16 runs.

3.4
3.4.1

Results & Discussion

Protein extraction from peanut meal

In general, increasing the amount of solvent used to extract protein from the same amount of
peanut meal increased the amount of protein extracted; however, the concentration of protein
in solvent decreased. The highest meal to solvent ratios produced the greatest total soluble
solids content in the soluble fractions after centrifugation. The protein content of soluble
fractions also increased with increasing meal to solvent ratios (Figure 3.2). Using less meal to
solvent resulted in more protein extracted per amount of meal used (Figure 3.3). Increasing
the ratio of peanut meal to solvent increased solids and protein content of the soluble material
collected after centrifugation. However, the amount of soluble protein per gram of peanut
meal remained unchanged or decreased slightly with increasing meal to solvent ratio. This
experiment indicated that 10% was the point at which solids extracted per gram of peanut
meal dispersed decreased. For future work it was determined that 10% (w/w) was best.
Other authors have shown 8% and 10% are most efficient for protein extraction [Rustom
et al., 1991].

31

10
8

Soluble Solids Content of Supernatant (%)

10

11

12

13

14

15

16

17

18

19

20

21

Concentration of Peanut Meal Dispersions at pH8 (% w/w)

Figure 3.1. Soluble solids content of water soluble fraction collected as affected by meal to
solvent ratio of dispersion

Solvent system
Two normal (N) HCl and 2 N NaOH were used to adjust the pH of the peanut meal-water
dispersions to pH 2.0 and 8.2 respectively. The initial pH of peanut meal dispersions ranged
from 5.83 to 6.37 with a mean value of 6.04. Immediately after the addition of acid or base
to the dispersion the pH value changed rapidly. Adjusting the pH of the dispersion to the
target pH by drop-wise addition of acid or base was difficult and inconsistent. Data were
compiled from 19 previous pH adjustments and averaged to determine the volume of acid
or base required to adjust a specific volume of peanut meal dispersion at 10% (w/w) to a

32

40000

30000

10

11

10000

20000

Protein content of supernatant (g/mL)

12

13

14

15

16

17

18

19

20

21

Concentration of Peanut Meal Dispersions at pH8 (% w/w)

Figure 3.2. Soluble protein content of supernatant as affected by meal to solvent ratio

given pH. The addition of 0.4 mL of 2 N HCl for each gram of peanut meal, or 0.31mL/g of
2 N NaOH in 10% (w/w) dispersions resulted in pH of 2.0 and 8.2 respectively. This method
of adjusting the pH more closely represents industrial practices and eliminates the lag time
between acid/base addition and pH reading as a source of experimental error.

3.4.2

Separation

In previously published work by Seifert and others [Seifert et al., 2010] small batches were
utilized and a refrigerated batch centrifuge was used to separate insoluble peanut material
from the protein rich soluble hydrolysates. These previous experiments used centrifugation

33

250
50

100

150

200

Protein Collected per Gram of Peanut Meal (g/mL/g)

10

11

12

13

14

15

16

17

18

19

20

21

Concentration of Peanut Meal Dispersions at pH8 (% w/w)

Figure 3.3. Soluble protein content of water supernatant per gram peanut meal as affected by
meal to solvent ratio

at 25,412x g with a pH8 solvent system. In the current experiments, for which large amounts
of material were required, separation was achieved by gravitational settling. No additional
separation was observed for settling times longer than 1 h (Figure 3.4). Despite the apparent
advantages of higher meal to solvent ratios, the amount of separation rapidly diminished
with increasing meal to solvent ratio when gravitational settling was used as the means of
separation (Figure 3.4). More peanut meal to solvent increased the solids extracted, but
decreased the absolute amount of soluble material collected. Considering the equipment
limitations, economic value of peanut meal, and total processing time 10% (w/w) dispersions
of peanut meal were chosen and 1 h was used as the settling time.

34

Table 3.1. Yields as ratio of solubles collected to total batch size, solids, and protein contents
of water soluble fractions collected by centrifugation and settling

Centrifugation
Settling

Protein
g/mL

Solids
%

Yield
%

15900
16200

3.5
3.8

63
34

Table 3.2. Dry peanut meal particle size distribution

Percentage of Sample
%

Particle Size Range

m

4.4
53.4
9.5
27.6
5.1

> 1680
1680-600
600-500
500-175
< 175

Soluble fractions collected by centrifugation and settling had slightly different solids and
protein contents. The biggest difference between separation methods was yield, which was
defined as the ratio of soluble material collected to the total batch size (Table 3.1). The
protein and solids contents of the soluble fractions collected by gravitational separation and
centrifugation were very similar; however, the absolute amount of protein, as the product of
yield and protein or solids, was much greater when centrifugation was used. This suggests
that using settling as a method to separate insoluble from soluble fractions will generate
material analogous to the material that would be generated if high through put continuous
centrifugation equipment were used. However, in an industrial setting, centrifugation should
be used to increase the amount of soluble solids collected.
Peanut meal particle size distribution was determined to help with the selection of equipment for industrial scale separation. The majority of particles in this sample of peanut meal
passed through openings 600 m but not 1680 m in size. The apparent bi-modality of the
size distribution is likely due to the large differences between opening sizes with the screens
used when passing from one screen to another (Table 3.2).

35

100

50

75

25

5
10
15
20

Volume of Soluble Material (mL)

Settling Time (hours)

Figure 3.4. Volume of soluble fraction with increasing settling time for 5, 10, 15, and 20%
dispersions

3.4.3

Insoluble fractions

Due to the scale and equipment used in processing the recovery of insoluble material was
nearly 100%. The moisture content of the insoluble fractions ranged from 10.4 to 12.7%. The
insoluble material containing hydrolyzed proteins can potentially be used as a value added
animal feed ingredient.

36

3.4.4

Soluble fraction

Drying soluble fractions increased concentration of active/soluble components namely proteins and peptides. Though not specifically investigated in this work, hydrolysis of peanut
proteins can be used to generate ACE inhibitory peptides. For these and other bio-active
peptides to have value as food ingredients they must be concentrated. Spray drying is a fast,
continuous, high volume, and relatively inexpensive method to create powders. Commercial spray drying typically utilizes liquid feeds between 30 and 50% solids [Masters, 1991].
The total soluble solids content of the soluble fractions ranged from 3 to 6%, so additional
concentration prior to spray drying was needed.
Vacuum oven drying
Vacuum oven drying was investigated as an inexpensive method to concentrate soluble solids
prior to spray drying. For better repeatability, oven dial settings and resultant air temperature
and water temperatures were first modeled (Figure 3.5). The advantage of using a vacuum
oven is that low temperatures can be used. However, the rate of evaporation, termed water
flux, was limited by air flow. Water flux is defined as mass of water loss per surface area per
second (Equation 3.1). Mean water flux (N) of all trails was 3 104 kg/m2 s. High oven and
sample temperatures increased N, but this plateaued at about 6 104 kg/m2 s. The plateau
of N was attributed to the formation of a film at the surface of the drying liquid. Film formation was only observed during drying of samples which did not have protease added. This
difference between protease hydrolyzed treatments and control treatments without protease
would be enough to warrant the use of an alternative method of drying because the process
should be the same for all treatments in order to evaluate the treatment effects. This observation aside, N was too low to sufficiently concentrate the soluble material quickly enough to
finish a single processing run in a 24 hour period.
Spray drying
A LabPlant spray dryer was evaluated as a spray dryer to dry the soluble fractions collected.
Feed stock flow rate and inlet temperature were adjustable on this dryer. Outlet temperature
(Tout ) is dependent on inlet temperature (Tinlet ) and feed flow rate [Masters, 1991]. High Tinlet
cause higher Toutlet [Masters, 1991]. Preliminary dual scanning calorimetric (DSC) studies and
the literature (Table 1.10) suggest the maximum Toutlet to preserve protein functionality with
reduced microbial load in the finished powder is 90 C. This maximum Toutlet limits which
Tinlet are possible for a given spray dryer. The atomizing gas flow rate was not adjustable
on the LabPlant dryer. To dry the soluble materials with an Toutlet of 90 C a low Tinlet was

37

110
100

Air
Water

80

60

70

Degrees C

90

50

5.5

6.5

Dial Setting
Figure 3.5. Vacuum oven dial setting and corresponding measured air or water temperature
inside the oven

required. The feed flow rate required to maintain these Toutlet and Tinlet was prohibitively
slow. Furthermore the low Tinlet chosen would not be practical from a thermal efficiency
standpoint (Equation 3.3). Instead, when a high Tinlet was used the feed flow rate had to be
increased to maintain the 90 C Toutlet . These conditions were more thermally efficient, but
the increased feed flow without proportionally increasing spray gas flow caused an increase
in droplet size. These larger droplets were too large to dry prior to contacting the drying
tube, resulting in wet-spotting.
Under optimal conditions the surface of the droplets dry rapidly enough that upon incidental collision with another droplet or surface inside the spray dryer, the droplets do not

38

adhere. Low yields and fouling inside the spray dryer occurs when droplets are too large.
The lab plant dryer was inadequate for the peanut meal solubles due to the lack of adjustability of spray gas flow rate. At feed flow rates necessary to maintain Toutlet at 90 C the
atomization was insufficient to prevent wet spotting.
The Anhydro spray dryer S-1, located in the North Carolina State University Food Science
Department pilot plant, was the next spray dryer evaluated. The peristaltic pump used to
supply feed solutions to the dryer caused non-uniform flow rate (pulsing) into the dryer.
Pulsing could cause scorching of some droplets and under-drying of other droplets. To
overcome pulsing a restriction valve was placed between the pump and the dryer. The valve
restricted flow causing constant back pressure on the pump, but a uniform flow rate into the
dryer. Unfortunately this valve had no markings or setting making it difficult to repeat the
same feed flow rate accurately. This dryer was not suitable because the feed flow rate could
not be controlled in a repeatable manner. Furthermore the dryer had leaky gaskets sealing
the drying chamber, which could lead to product loss.
Another major limitation of this dryer was the mixed flow air feed flow. The initial
counter-current flow is harsh on heat sensitive proteins. In counter-current flow the heated
drying air flows in the opposite direction of liquid feed spray, so as particles dry they flow
into warmer air. The driest particles are dispersed in the hottest air. Other limitations include
the lack of repeatability of feed flow rate. The implications of changing feed flow rate on
particle size would make good replication impossible.
The way in which drying air and atomized feed droplets mix effects thermal efficiency as
well as the finished powder properties. Three types of drying air and feed mixing are commonly used: co-current, counter-current, and mixed. In co-current mixing sprayed droplets
and heated drying air flow in the same direction. This is the best type of mixing for thermally
liable materials because as the droplet dries the air in which it is suspended cools. The driest
droplets are therefore suspended in the coolest air. Counter-current mixing is the opposite
of co-current and the most thermally efficient. This type of dryer is often used for industrial
material production like grit for sandpaper. The drops and hot air flow in opposite directions
so the driest drops contact the hottest air. Counter-current mixing causes the droplets for
any given moisture content to contact hotter air than during co-current mixing, even when
the same Tinlet and Toutlet are used. Mixed flow mixing is the combination of counter-current
and co-current. Mixing is initially counter-current and finished co-current. This is achieved
by spraying droplets against the drying air flow. Dry air velocity is greater than that of the
suspended droplets which change direction and flow with the drying air. This mode allows
the thermal efficiency advantages of counter-current, and is gentler on the drying material
than true counter-current mixing. Drier size and design influences thermal efficiency and
particle heating.

39

The Bchi B-290 lab scale spray dryer utilized co-current mixing and offered adjustable
spray gas flow rate. Three components of the Bchi B-290 were labeled and weighed before
and after each spray drying run (Figure 3.6). Yield was the primary criterion by which spray
drying success was measured. Only material collected in section C (Figure 3.6) was measured
for moisture content and the material in the other sections was assumed to be the same as that
in section C. The sum of yield and hold-up was nearly 100%, the difference is attributed to
differing moisture contents of the material collected in each section. The discrepancy between
the sum of yield and hold-up and 100% may also be explained by the loss of fine particles
through the exhaust air. The cyclone (Figure 3.6 component B) in the Bchi B-290 separates
particles from drying air when the particle size is > 2 m.

Figure 3.6. Bchi B-290 spray dryer components weighed for yield and hold-up calculations

Solids content and inlet temperatures were the greatest determinants of spray drying
yields for these materials (Figure 3.7). The addition of maltodextrin to achieve total soluble
solids content of 10 and 15% greatly increased yields at 180, 200, and 220 C. Increasing
solids to 20% with the addition of maltodextrin caused a decrease in yield due to increased

40

fouling in the dryer. To maintain the same Toutlet when using a feed solution with high total
soluble solids (TSS), the feed flow rate had to be increased because the amount of water
being evaporated decreased. Less water undergoing phase change during drying left more
thermal energy to increase particle and air temperature thus increasing Toutlet . Increasing
the feed flow rate provided more water to keep Toutlet down, but also increased the ratio of
feed flow rate to atomizing gas flow rate. The increased ratio caused an increase in droplet
size which created larger particles causing incomplete drying in the drying tube and more
fouling. The apparent trend of lower inlet temperatures and increasing yields warranted

100

further investigation.

80

Control
10% Malto
15% Malto
20% Malto

60

40

Powder Yield (%)

20

180

200

220

Spray Dryer Inlet Temperature (C)

Figure 3.7. Effects of spray dryer inlet temperature and maltodextrin content on powder yield
during spray drying of water soluble fractions collected from 10% dispersions at pH 8.2

41

The experiment to determine Tinlet at which to dry Alcalase and pepsin hydrolyzed soluble fractions indicated that lower Tinlet produced greater yields (Figure 3.8). The solids
content of the powders ranged from 9.1 to 11.7%. The means solids content of all Alcalase
powders was 11.56 0.06 and 9.41 0.29 for all pepsin treatments. The mean solids content for Alcalase and pepsin treatments varied only slightly at different inlet temperatures
suggesting that outlet temperature and feed solution affected powder solids more than inlet

55

50

45

Powder Yield (%)

60

65

70

temperature.

40

35

135

Alcalase
Pepsin

160

185

210

Spray Dryer Inlet Temperature (C)

Figure 3.8. Effects of spray dryer inlet temperature on powder yield for pepsin and Alcalase
treated peanut meal solubles

42

The feed flow rate necessary to maintain Toutlet of 90 C with an Tinlet of 135 C was about
200 mL/h which was prohibitively slow. A 1600 mL run would take 8 hours and use more
than a whole tank of nitrogen. Another limitation to using 135 C Tinlet was that the reduced
feed flow rate, with out compensation of the spray gas flow rate, resulted in excessive material
loss through the exhaust air. The material loss caused rapid filter fouling and loss of pressure
in the dryer. The loss of pressure in the dryer triggered a protective dryer shutdown circuit,
causing the dryer to cease functioning.
It was therefore determined that spray drying using the Bchi B-290 with inlet temperature of 185 C would yield similar amounts of powder for both Alcalase and pepsin hydrolyzed feed stocks. It was also concluded that maltodextrin should be added to produce
feed stocks of about 15% TSS to increase powder yield from the spray dryer.

43

CHAPTER

FOUR
PROCESS FLOW

4.1

Abstract

A process has been developed to extract and hydrolyze protein from aflatoxin contaminated
peanut meal while simultaneously removing aflatoxin [Seifert et al., 2010]. The aflatoxin-free
liquid hydrolysate produced using this process has potential food applications. This novel
process was adapted to process enough liquid hydrolysate to then spray dry to generate a
protein/peptide powder. The solvent system used for protein extraction was similar to that
described by Seifert and others [Seifert et al., 2010]. Protein extraction and hydrolysis was
conducted in 4 kg batches in order generate enough soluble material for subsequent spray
drying. A total of 8 treatments were performed in triplicate.
Maltodextrin was added to the soluble fractions at 8% and the resultant feed stocks were
spray dried at Tinlet of 185 C and the Toutlet was maintained at 90 C. Powder yield from the
spray dryer ranged from 56 to 84%.

4.2

Introduction

An increased interest in protein extraction from plant materials has lead to the development
of a novel process to extract protein from peanut meal [Seifert et al., 2010]. On a bench scale,
the process described effectively extracts and hydrolyzes proteins from aflatoxin contaminated peanut meal. The process utilizes commercially available feed additives as a processing
aid to remove aflatoxin. The terminal product of this process is an aqueous solution containing hydrolyzed protein with aflatoxin levels below 20 g/kg. Large scale implementation of
this process has yet to be demonstrated.
For commercial application of the peanut meal extract as a potential food ingredient, it

44

Table 4.1. Treatment names and summary of conditions

Treatment Name
pH2
pH2 & clay
Pep
Pep & clay
pH8
pH8 & clay
Alc
Alc & clay

Treatment No.

pH

Enzyme

Clay

1
2
3
4
5
6
7
8

2
2
2
2
8
8
8
8

0
0
1
1
0
0
1
1

0
1
0
1
0
1
0
1

must be dried to a powder. Powders are less costly to transport and store because they weigh
less and require less space than liquids. Large scale drying processes typically used for thermally liable materials are freeze drying or spray drying. This work describes development of
a process to create aflatoxin-free hydrolyzed protein powder on a larger scale (4kg).

4.3
4.3.1

Materials & Methods

Extraction

A 400 g sample of peanut meal was added to 3600 g di water at 20 C and stirred for 20 min
with a wheaton stirrer on dial setting 2.8. After 20 min of stirring the pH of the dispersion
was recorded as initial pH. One of eight treatments was then applied to the dispersion (Table
4.1). Treatments labeled pH 2 (No. 1-4) were mixed with 160 mL 2 N HCl, and treatments
labeled pH 8 (No. 5-8) were mixed with 124 mL 2 N NaOH. The pH 2 treatments with
enzyme addition were named Pep and were treated with 4 g pepsin [enzyme commission
(EC) number 232-629-3] dispersed in 396 mL of 0.1 N HCl. This amount of enzyme is 21,000
units per mg of protein which is similar to previous peanut meal hydrolysis in which 19,000
units per gram were used [Seifert et al., 2010]. The pH 8 treatments with enzyme addition
were mixed with 46 g liquid Alcalase from Bacillus licheniformis (batch 056K1213, EC 232752-2), at 2.4 Anson Units per gram. The enzyme to substrate ratio was 0.6 Anson units
per gram of peanut meal protein, also similar to previously reported values [Seifert et al.,
2010]. Treatments with clay (No. 2, 4, 6, 8), were mixed with 8 g (0.2% w/w) of AB20,
a sodium bentonite clay commercially available as a feed additive, provided by Prince Agri
Products, Inc. (Quincy, IL). All treatments were performed in a completely randomized order

45

in triplicate for a total of 24 dispersions. All statistical analyses were performed using R: A
Language and Environment for Statistical Computing [R Development Core Team, 2010].
Two minutes after the addition of acid/base, enzyme, and/or clay the pH was recorded as
final pH. The dispersions were then stirred for 1 h. After 1 h the stirrer was stopped, and the
dispersions were allowed to settle for 1 h. After settling a peristaltic pump was used to collect
the soluble fractions. The soluble fraction was identified as the particulate-free solution above
the topmost layer of compacted peanut meal particles, and tubing was positioned 1 cm above
this layer. Solubles fractions collected were filtered through 2 layers of cheesecloth and the
mass of the solubles was determined. Aliquots of solubles were retained for solids, density,
and other subsequent analyses. Solids and density analysis were performed as described in
a previous section (3.3).
Feed stock
R M100) was donated by Grain Processing Corp. (Muscatine, IA; Lot
Maltodextrin (Maltrin

number M0910530) and was added to the solubles at a concentration of 8% by weight. The
dextrose equivalents and moisture content of the maltodextrin were given as 10.7 and 4.7%
respectively. With a magnetic stir bar maltodextrin was stirred into solubles for 40 minat 20
CAfter

stirring the solubles with maltodextrin, aliquots of the feed stock were collected for
solids, density and other analyses.

4.3.2

Spray drying

Feed stocks were weighed prior to and after spray drying, the mass of feed stock fed to the
spray dryer was calculated. The spray dryer was operated at Tinlet of 185 C and the Toutlet
was maintained at 90 C by adjusting the feed flow rate. A peristaltic pump was used to
supply feed stock to a Bchi B-290 lab scale spray dryer at volumetric flow rates of 620 to 780

mL/hNitrogen
was used as the atomizing gas and supplied at a flow rate of approximately
660 mL/h, which corresponded to a height of 40 mm on the rotameter. The atomizing gas
flow rate remained constant for all treatments.

4.4
4.4.1

Results & Discussion

Extraction

The initial pH (pHi ) of the peanut meal dispersions ranged from 6.3 to 6.5 (Figure 4.1). Two
minutes after application of treatments the final pH (pHf ) for all treatments in the pH 2
group ranged from 1.9 to 2.3 with a mean of 2.1. The pHf of the pH 8 treated set ranged

46

from 8.8 to 9.3 with a mean of 9.1. After settling, the mass of solubles collected ranged from
1,366 mL to 2,008 mL and was significantly (p < 0.001) greater for all treatments at pH 2,
than those treated with pH 8. The addition of enzyme significantly (p < 0.001) increased
solubles collected regardless of pH (Figure 4.3). For each pH with or without the addition
of enzyme, clay had no effect on solubles yielded. The solids content of the soluble fractions
collected ranged from 3.0 to 6.2%. This difference is likely due to hydrolysis increasing
protein solubility. The addition of enzyme significantly (p < 0.001) increased solids content,
and the addition of clay had no effect. The pH 8 treatments yielded soluble fractions with
significantly (p < 0.001) higher solids contents than pH 2 treatments. Accordingly, the pair
of treatments at pH 8 had significantly higher solids contents than all four treatments at pH
2, and the pair of treatments with Alcalase had significantly higher solids contents than all
other treatments (Figure 4.3).
To compare efficacy of treatments to extract solubles from peanut meal, total solids extracted was calculated for each treatment as the product of % soluble solids in soluble fractions collected and the mass of solubles collected. Pepsin treatments yielded slightly more
solids than pH 2 treatments, though not significantly (p = 0.07). Alcalase treatments yielded
significantly (p < 0.001) more soluble solids than all other treatments (Figure 4.4). Pepsin
treatments yielded the most soluble material, but the relatively low solids content of this material limits the efficacy of this treatment because collection of solids is the absolute goal. This
is likely due to the addition of the 396 mL of 0.1 N HCl in which the pepsin was dissolved
prior to addition. Higher solids contents are preferable because less subsequent drying will
be required to produce a powdered material from this soluble fraction. Using this criterion
to assess treatments, Alcalase treatments are the best.
The solids contents of insoluble fractions ranged from 10.4 to 12.7 % (Figure 4.5). Insoluble fractions treated with clay had slightly higher solids contents presumably because the
additional clay settled out of solution with the other insoluble peanut meal materials. As
expected, the treatments in which soluble solids were increased has lower solids contents in
the insoluble fractions. Enzymatically hydrolyzed treatments resulted in insoluble fractions
with lower solids contents, and pH 8 treatments also had lower solids contents. Only the pH
effect was significant at (p < 0.001).
Feed stocks after the addition of 8% (w/w) maltodextrin had solids contents ranging from
9.8 to 12.6%. The absolute difference (2.8%) in the feed stocks was smaller than the difference
between solids content of soluble fractions (3.2%) prior to the addition of maltodextrin. On
a relative basis the difference was much smaller; for the soluble fractions prior to the addition of maltodextrin, the lowest solids content extracted was only 48% of the highest solids
content. However in the case of the feed stocks, the lowest solids content was still 78% of the
largest, meaning the solids contents of the feed stocks were more similar than those of the

47

10

pHf
pHi

Alc

Alc & clay

pH2

pH2 & clay

Pep

Pep & clay

pH value

pH8

pH8 & clay

Treatment

Figure 4.1. pH of dispersions before (pHi ), and after (pHf ) the application of treatments
acid/base, enzyme, and clay

48

2000
1900
1800

1600

1700

1500

1400

Mass of Soluble Fraction Collected After Settling (g)

pH2

pH2 & clay

Pep

Pep & clay

pH8

pH8 & clay

Alc

Treatment

Figure 4.2. Mass of solubles collected after settling

49

Alc & clay

5.5
5.0
4.5
4.0

pH8

pH8 & clay

3.5

3.0

Mass of Soluble Fraction Collected After Settling (g)

6.0

pH2

pH2 & clay

Pep

Pep & clay

Alc

Alc & clay

Treatment

Figure 4.3. Solids content of solubles collected after settling (%)

50

100
70

80

90

60

Solids Yielded as Percent Solids * Mass of Solution (g)

50

pH2

pH2 & clay

Pep

Pep & clay

pH8

pH8 & clay

Alc

Alc & clay

Treatment

Figure 4.4. Solids collected in the soluble fraction as % solids * mass of soluble fraction

51

13.0
12.5
12.0

11.5

11.0

10.5

Solids Content of Insoluble Fractions(%)

pH2

pH2 & clay

Pep

Pep & clay

pH8

pH8 & clay

Treatment

Figure 4.5. Solids content of insoluble fraction

52

Alc

Alc & clay

solubles. The addition of maltodextrin minimized the differences in solids contents between
treatments.
While the difference in total solids content of each treatment was minimized by the addition of maltodextrin, the proportion of peanut solids to maltodextrin solids decreased. Clay
had no effect on solids content of the soluble fractions and the ratio of maltodextrin to peanut
solids in the feed stocks was 2.2, 2.2, 1.8, and 1 to 1 for pH2, Pep, pH8, and Alc treatments
respectively. Assuming only water is lost during drying, the powders would have 31, 31, 36,
and 50% peanut material for pH2, Pep, pH8, and Alc treatments respectively. This dilution
of solids between treatments influenced powder properties.

4.4.2

Spray drying

Powder yield was the primary criterion by which spray drying performance was evaluated.
Since spray drying conditions were determined as discussed in the previous chapter, differences between powder yields and powders were attributed to treatment effects rather than
spray drying conditions. Powder yields as the ratio of solids collected to solids sprayed
ranged from 56.1 to 83.5 % and the mean was 66.5% (Figure 4.6). The factors pH, enzyme,
and the pH enzyme interaction were mildly significant (p < 0.05). Ignoring the effects of the
combination enzyme and pH, the lumped mean yield of all pH 2 samples was significantly
(p < 0.001) larger than pH 8 samples. Similarly all enzyme treated samples regardless of
pH yielded significantly (p < 0.05) more powder solids than those samples without enzyme.
The pH2 treatment yielded significantly (p < 0.05) more powder than the pH8 treatments
and more than Alcalase, although not significantly at p < 0.05. Pepsin treatment yielded
significantly (p < 0.05) more than all other treatments.
The pH2 sets, both with and without the addition of enzyme, yielded more powder solids
than the pH8 treatments. Within each pH treatment the addition of enzyme increased yields.
The increased yields may be due to the increased solids content of the soluble fractions for
those treatments (Figure 4.7). The solids sprayed and percent powder yield did not correlate
well (R

= 0.05) indicating that the solids content of the feed stock did not affect powder

yield. The absolute amount of feedstock sprayed correlated much better (R 2 = 0.16), but only
explains a small amount of the variation between treatments. Yields were better when larger
quantities of feed stock were sprayed. It is possible that a certain amount of fouling within
the dryer is inevitable, and a thin coating of spray dried material coats all surfaces within the
dryer. After this initial coating has occurred the material no longer sticks, perhaps because
the particles are more adhesive than cohesive. If this theory were true, spraying more feed
stock would result in less hold up and therefore higher yields because as a percent of the
total feed stock sprayed, the fixed amount of initial material to adhere to the spray dryer

53

100
80
60
40

Powder Yields Solids Basis (%)

Dryer Section

B
C

20

Pep

Pep & clay

Alc

Alc & clay

pH2

pH2 & clay

pH8

pH8 & clay

Treatment

Figure 4.6. Solids collected in each section of spray dryer as percentage of total solids sprayed

54

160
150

140

130

120

110

Solids Fed to Spray Dryer (g)

100

90

pH2

pH2 & clay

Pep

Pep & clay

pH8

pH8 & clay

Alc

Alc & clay

Treatment

Figure 4.7. Quantity of solids fed to spray dryer as feed stock quantity * percent solids

55

decreases. In future work speculation of this nature will be avoided by spraying the same
amount of solids for each treatment.
Hold up was defined as the mass of material accumulated in sections A and B of the
spray dryer as shown in figure 3.6 and described in section 3.4. As a percentage of total
solids sprayed the total hold up calculated as the sum of sections A and B ranged from
18.8 to 47.2%. One limiting assumption in calculating hold up this way was that the solids
content of the material in spray dryer sections A and B was the same as the solids content of
the material in section C. Section C was the collection vessel in which finished dry powders
were deposited, the solids content of these powders was determined as previously described.
It was assumed that the hold up material had the same solids content as collected powders.
The material adhered to the walls of the drying chamber (section A) likely had a lower solids
content than the dried powder in section C. This assumption was made because material
in sections A and B is a thin film or crust which would be difficult to collect analytically,
and not plentiful enough to accurately dry for solids determination. The sum of hold up
and yield was not 100%, but it was often close. The discrepancy may be explained by the
aforementioned assumptions and the loss of small particles through exhaust air. The cyclone
(component B) was capable of separating particles between 2 and 25 m in diameter from
the drying air. Particles larger than 25 m failed to flow with drying air to the cyclone and
collect in section A and particles smaller than 2 m remained with drying air and exited the
cyclone through the exhaust.
Powders were further characterized to explain differences in yields between treatments,
and help assess potential uses as food ingredients.

56

CHAPTER

FIVE
FINISHED MATERIAL ANALYSIS

5.1

Abstract

Soluble protein-rich extracts from peanut meal were spray dried into powders that each had
different functional properties. Powders made from peanut meal extracts were assessed for
composition, solubility, antioxidant capacity, and hygroscopicity. Powder compositions and
properties varied by treatments. Powders produced with Alcalase contained the most protein, the highest proportion of small molecular weight peptides, and the greatest antioxidant
capacity. All powders were moderately hygroscopic, with potentially limited storage stability.
Powders produced with pH8 treatments were least hygroscopic which was attributed to these
powders containing the lowest proportion of mono- and di-saccharides. Enzymatic hydrolysis improved protein solubility and antioxidant capacity of all powders. Treatments including
the clay had nearly no aflatoxin present in the soluble fractions. All powders contained below the allowable limit (20 g/kg)for AFB1 in foods as defined by the FDA. Therefore these
powders have potential as food ingredients.

5.2

Introduction

Many criteria can be used to assess the value of a potential food ingredient. Of primary importance are composition and solubility. During spray drying the composition of feed stocks
can affect spray drying performance and finished powder properties. The drying droplets
can behave in unexpected ways when the bulk composition of feed stock and surfaces of
drying droplet are not identical. Surface tension of feed stocks is the energy at the interface
of feed stock and drying air. Materials with different surface tensions indicate differences in
composition between the bulk and at the interface. Feed stocks made with similar compo-

57

sitions can have different surface tensions caused by the addition of small amounts (< 1%)
of surface active compounds. Many proteins are surface active, meaning they adsorb to the
air-water interface, lowering the surface tension.
A spray dried protein powder must be soluble to be functional. Protein solubility is
affected by the pH of the system in which is dissolved, as well as the molecular weight of
proteins. Low molecular weight di- and tri-peptides are among the most reactive proteins,
often possessing bio-active health promoting properties, and antioxidant capacity [Elias et al.,
2008,Kitts and Weiler, 2003]. This work characterizes the products produced from the process
described in chapter 4.

5.3
5.3.1

Materials & Methods

Aflatoxin

Aflatoxin content of soluble fractions, spray dried powders, and insoluble fractions collected
was determined using AOAC method 991.31. Aflatoxin content was converted to solids basis.

5.3.2

Turbidity of powder solutions

The light absorbance of powders solutions was measured at 500 nm. Approximately 0.16 g
of powder were accurately weighed into 1 mL microfuge tubes and 800 mL pH 7.0 McIlvaine
buffer was added. Tubes were mixed by vortex for 2 min then transferred to a 96 well plate.
Additional buffer was added to the wells to create dilutions of, 15, 1 ,0.5, and 0.1% (w/v). The
plate shook for 30 s and absorbance was read 10 times by SAFIRE2 monochromator based
microplate reader equipped with Magellan (v. 6.1) reader software (Tecan USA, Raleigh, NC).

5.3.3

Fiber, and ash

Proximate analysis including fiber and ash determination was performed on insoluble fractions by Barrow-Agee labs (Memphis, TN).

5.3.4

Protein

Insoluble fractions were analyzed for %N by Barrow-Agee labs using Kjeldahl method.

5.3.5

Interfacial tension

Interfacial tension of soluble fractions and feed stocks was measured using an automated
contact angle goniometer (Rame-Hart Inc, Mountain Lakes, NJ) in combination with the

58

DROPimage computer program. Samples were diluted 1:50 in di water and the surface
tension of freshly formed pendant drops at room temperature was measured for 60 s.

5.3.6

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

Twenty g solutions of powders produced by treatments pH2 & clay, Pep & clay, pH8 & clay,
and Alc & clay were prepared. The solids content of the solutions matched the solids content
of the feed stocks from which the powders were made. The amount of powder and water
mix was calculated using equation 5.1.
Powder =

feed stock solids g/g 20g

powder solids g/g

Water = 20g Powder

(5.1)
(5.2)

Powder solutions were stirred for 90 min at 20 C prior to dilution.

Feed stocks were mixed by vortex prior to dilution.
R Tricine SDS Sample Buffer (2X), NuPAGE
R
All samples were diluted using Novex

Reducing Agent (10X), and deionized water according to manufacturers directions. Ten L
R 1 mm by 10 well pre-cast 16% Tricine
of each solution were loaded per well in Novex

Gels (Invitrogen, Carlsbad, CA) which are used for resolving low molecular weight proteins
R Plus2 Prestained Standard molecular weight marker was used as
and peptides. See Blue

a reference and loaded into the first well. Lanes 3 through 10 were loaded with feed stocks
then powder solutions in pairs by treatment. Electrophoresis conditions were 130 V for 90
min. SimplyBlueTM SafeStain was used to stain the gel for 60 min. The gels were destained
in deionized water overnight and then dried using Gel-DryTM Drying Solution.

5.3.7

Protein solubility

A sample of 0.16 g of each powder was added to 800 L of McIlvaine buffer at pH 3.0, 5.0, and
7.0. The newly formed dispersions were mixed by vortex for 10 s, then inverted and mixed
by vortex another 10 s and finally inverted again and mixed by vortex 10 s. Dispersions
were allowed to settle for 1 h and then were centrifuged at 10,000 x g for 10 min. After
fractionation, 40 L of each soluble fraction were transferred to a 96 well plate and 320 L
working reagent. BCA protein standards were plated, absorbance was measured at 420 nm
and soluble protein content was calculated using the standard curve.

59

5.3.8

Oxygen radical absorbance capacity (ORAC)

A 70 nmol sodium salt solution of Fluorescein was prepared in 75 mol phosphate buffer.
Trolox standards were prepared from 50 to 3.12 mol in phosphate buffer. Fluorescence
was measured using the SAFIRE2 monochromator based microplate reader at excitation and
emission filter wavelengths of 483 +/- 8 and 525 +/- 12 nm, respectively. The reaction was
carried out in 75 mmol phosphate buffer at pH 7.4 with a final volume of 250 L. Diluted
samples and Trolox standards, both at 130 L were added to the wells. 60 L of the Fluorescein solution, was then rapidly added. The plate was incubated in the SAFIRE2 for 15 min at
37 C. After incubation, 60 L of AAPH solution was rapidly added. Before each measurement the plate shook for 5 s. Measurements were made once each minute for 80 minutes.
Net area under the fluorescence decay curve was correlated with Trolox concentration using
a linear model to build a standard curve. Antioxidant absobance of the samples were then
calculated as Trolox equivalents (TE) based on the standard curve.

5.3.9

Hygroscopicity of powders

The method to determine hygroscopicity was adapted from [Cai and Corke, 2000]. Approximately 2 g of dry powder were accurately weighed into clean dry aluminum pans. Powders
were stored at room temperature for 7 days in a desiccator. The desiccator contained 1 L
of saturated Na2 SO4 solution creating 81 % relative humidity environment. After one week
hygroscopicity was calculated as grams of water absorbed per 100 grams of powder solids.

5.4
5.4.1

Results & Discussion

Aflatoxin

The peanut meal used contained 59 g/kg aflatoxin B1 (AFB1) on a dry weight basis. Without
reducing the aflatoxin content through processing, this work fails to improve the value of
peanut meal. Accordingly, the aflatoxin contents of the two terminal products of this process
were determined. Treatments were compared by the addition of clay (Figure 5.1).
Clay had a very strong (p < 0.001) effect on aflatoxin content of powders. The addition of
enzyme or the pH had little to no effect.
The aflatoxin content of insoluble materials was significantly (p < 0.001) effected by both
pH and clay. Treatments at pH 8 had less residual aflatoxin contamination. This is consistent with reports of weak bases causing destruction of the ring structure of the aflatoxin B1
molecule [Beckwith et al., 1975].
For both powder and insoluble materials clay had the strongest effect on aflatoxin. The

60

60
40
20
0

Alfatoxin Content on Solids Basis (g/kg)

80

Feed Stocks
Insolubles
Powders

pH2

pH2 & clay

Pep

Pep & clay

pH8

pH8 & clay

Alc

Alc & clay

Treatment

Figure 5.1. Aflatoxin content of insoluble materials, powders, and feed stocks on solids basis
by treatment

61

addition of clay had a negligible effect on the other properties discussed. Feed stocks were
also evaluated for aflatoxin content, pH had a small (p < 0.1) effect on aflatoxin content, and
clay had a very strong (p < 0.001) effect as expected. It is interesting to note that pepsin
treatments appear to have no aflatoxin in the feed stock, but after spray drying aflatoxin
appears. The detection limit of the equipment used for this analysis is 1 g/kg, and the
AFB1 content of feed stocks were below this threshold. After the removal of water by spray
drying, the aflatoxin is concentrated enough to be detected.
Nearly all powders contained less than 20 g/kg AFB1, which is below the FDA threshold
for AFB1 in foods. The potential for food applications of peanut meal protein extract greatly
increases the value of commercial peanut meal which is currently relegated to animal feed
markets.

5.4.2

Turbidity of powder solutions

The turbidity of feed stocks was determined by absorbance at 500 nm and indicated that
concentration affected turbidity (Figure 5.2). Solutions with more TSS were more turbid.
The treatments pH 8 and Alcalase were more turbid than all pH 2 and pepsin treatments
at each dilution. The addition of clay clarified the solubles for pH8 and Alcalase treatments
which was observed as the lower absorbance across all dilutions. Clays, including AB20, are
often used as clarifying agents in beverage processing; namely wine and apple juice. The
differences in clarity were more evident upon visual inspection. Soluble fractions differed
greatly in appearance and clarity between treatments (Figure 5.3).

5.4.3

Fiber and ash

The fiber content of the insoluble fractions ranged from 6.4 to 9.4% with none of the treatments varying significantly (Figure 5.4).
The ash content ranged from 3.8 to 7.4%. Clay added to the dispersions remained with the
insoluble fractions as evident by the increase in ash value for each treatment that included
clay. Surprisingly, pH 8 treatments had significantly (p < 0.001) more ash in the insoluble
fractions. Perhaps salts and minerals found in peanut meal are more soluble at pH 2 than
pH 8 so the insoluble fractions at pH8 retained more of these.

5.4.4

Sugar content

The sugar content of soluble materials and feed stocks were quantified (Table 5.1). The addition of maltodextrin to soluble fractions increased the total sugars content in solubles compared to feed stocks. The maltodextrin added contained 0.8, 2.9, 4.4, and 3.8% mono-, di-, tri-,

62

0.8

0.4

pH2
pH2 & clay
Pep
Pep & clay
pH8
pH8 & clay
Alc
Alc & clay

0.2

Absorbance at 500nm

0.6

15

0.5

0.1

0.0

Concentration of Powder in Solution (% w/v)

Figure 5.2. Absorbance of hydrated powder solutions (15, 1, 0.5, and .1% w/v) at 500 nm

and tetra-saccharides, while 88.1% was pentasaccharides and larger carbohydrates. Increases
in the total sugars were likely caused primarily by the breakdown of the pentasaccharides
present in the maltodextrin, rather than by direct addition of those mono- and di-saccharides.

63

pH2

Pep

pH8

Alc

Figure 5.3. Visual appearance of pH2 & clay, Pep & clay, pH8 & clay, Alcalase & clay soluble
fractions

5.4.5

Protein

The protein content of the insoluble material ranged from 5.6 to 7.1% on wet weight basis.
The mean protein content was 6.2%. Treatments caused reductions in protein content of the
insoluble fractions, but since the range was so small these differences were unimportant from
a practical standpoint. As expected, insolubles with low protein matched the solubles with
higher protein contents. Treatments that resulted in more extracted protein show increased
protein contents in the soluble fraction and decreased protein remaining in the insolubles
from which the protein was extracted.
The pH of the dispersion significantly (p < 0.001) affected protein content in the soluble
fraction, as did enzyme addition. Furthermore there was a highly significant (p < 0.001)
interaction between pH and enzyme.
Protein content of the powders was significantly different for all treatment, on which clay
had no effect (Figure 5.5). Soluble fractions high in protein were sprayed into powders with
high protein, on a solids basis there was little change between the protein contents of the
two materials. The first two treatments pH2 and pH2 & clay increased in protein content
from feed stock to powder (Figure 5.5). For most treatments the powder and soluble fractions
have nearly identical protein contents on a dry weight basis, which was expected. This
similarity indicates that there was no change in the proportions of solids as a result of spray

64

10

Concentration on Solids Basis (g/100g)

Ash
Fiber

pH2

pH2 & clay

Pep

Pep & clay

pH8

pH8 & clay

Alc

Alc & clay

Treatment

Figure 5.4. Fiber and ash content of insoluble fractions on dry weight basis

drying. An increase in protein from feed stock to powder indicated a loss of non-protein
solids during the spray drying process because the protein contents were presented on a
dry weight basis. It has been shown that adding small amounts of surface active protein
to feed stocks with high concentrations of sugar causes a drastic increase in spray drying
yields [Adhikari et al., 2009b]. The mechanism responsible is the migration of proteins to the
droplet surface where they rapidly dry forming a non-sticky film. The migration of protein
to the surface may create droplets (therefore dried particles) with varying compositions. Very

65

Table 5.1. Concentration of sugars in soluble fractions and feed stocks: mean of 3 replicates
1 standard error

Treatment

Glucose

Fructose

Sucrose

Total

material

g/mL

g/mL

g/mL

g/mL

pH2 feed stock

Pep feed stock
pH8 feed stock
Alc feed stock

544 8
551 6
531 50
675 22

172 0
156 7
150 12
169 8

6056 49
3821 9
6237 538
6077 129

8565 87
6331 17
8006 282
9720 360

pH2 solubles
Pep solubles
pH8 solubles
Alc solubles

90 7
81 4
44 1
94 6

182 7
161 5
140 4
153 9

6562 434
3974 240
6753 623
6812 706

6885 450
4800 245
7598 639
8348 935

small droplets have higher surface area to volume ratios than large droplets. If the droplet
surface is predominantly protein then the ratio of protein to non-protein materials is much
greater in the smallest droplets than the largest droplets when those proteins are surface
active. Assuming the proteins in the feed solutions sprayed are surface active, and the small
droplets or particles are lost through the exhaust, the ratio of protein to other solids collected
will be biased towards protein. This would result in an apparent decrease in protein content
on a solids basis as a result of spray drying. The feed solution would have more protein than
the powder for the same treatment. If the proteins had no surface activity then small droplets
should contain the same proportions of protein to non-protein material as large droplets.

5.4.6

Surface tension

Surface tension is denoted is defined as force (N) per unit length (m) and describes the
energy at an interface between two phases [Walstra, 2003]. In the context of spray drying,
interfaces are important in droplet formation during the atomization processes when spray
droplets are formed. More energy for atomization is required to produce droplets of the
same size when spraying feed stocks with high interfacial tension than is required for a feed
stock with low interfacial tension.
The surface tension of freshly formed droplets of feed stock decreased for treatments over
one minute (Figure 5.6). Alcalase treatments caused a greater reduction in surface tension
than all other treatments. Under similar conditions, Alcalase has been shown to more extensively hydrolyze peanut meal protein than pepsin [Seifert et al., 2010] . The creation of

66

40
30
20
10
0

Protein Content on Solids Basis (g/100g)

Feed Stocks
Powders

pH2

pH2 & clay

Pep

Pep & clay

pH8

pH8 & clay

Alc

Alc & clay

Treatment

Figure 5.5. Protein content of powders and feed stocks by treatment on solids basis (g/ 100g)

new small molecular weight peptides is likely responsible for the decrease in surface tension.
The ratio of spray gas to feed stock mass flow rates was assumed to remain constant for all
treatments. The energy supplied for the creation of droplets was therefore also assumed to
remain constant. Lower surface tension and the same amount of force caused the creation of
smaller droplet. Smaller droplets with more protein at the surface may explain the apparent
loss of protein during spray drying as seen in figure 5.5. Conversely, the smallest droplets
for pH2 treatments would more closely match the composition of the bulk feed stock, which

67

75

70

65

55

60

50

Surface tension mN/m

45

pH2
Pep
pH8
Alc
10

20

30

40

50

60

Time (seconds)

Figure 5.6. Interfacial tension of feed stocks over time, the blue line represents value for water
at 25 C

was 31% peanut material and 69% maltodextrin on a solids basis. So a loss of specifically
the smallest droplets would cause the loss of maltodextrin at more than double the rate of
peanut material loss. The result for these treatments would be an apparent increase in protein caused by spray drying. Effects on powder composition may be more pronounced in a
large scale spray drying operation in which droplets have longer residence times. The Bchi
B-290 has a mean particle residence time of only 1 to 1.5 s. Large scale spray dryers can have
drying tubes several times larger resulting in residence times of 30 sfor drying droplets. This

68

additional time allows partitioning or migration of solutes prior to skin formation which can
lead to the particle surface having very different composition than the bulk.

5.4.7

SDS-PAGE

The relative molecular weight distribution of proteins and peptides remained unchanged by
the process of spray drying (Figure 5.7). Alcalase hydrolyzed samples clearly resulted in a
greater proportion of smaller molecular weight proteins and peptides than the pH8, pH2, and
pepsin samples. The larger proportion of low molecular weight material indicates Alcalase
more extensively hydrolyzed the peanut meal proteins than the other treatments.

5.4.8

Protein solubility

Powder protein solubility was improved by enzymatic hydrolysis (Figure 5.8). Regardless of
the pH at which enzymatic hydrolysis occurred, the solubility of the proteins was improved
by the addition of protease. Peanut powder protein was most soluble at pH 7.0 and least
soluble at pH 3.0. The solubility of protein in powders produced without protease addition
was very pH dependent. Treatments at pH2 were more soluble in the pH 3.0 buffer and less
so in pH 7.0 buffer, while the pH8 treatments showed the opposite trend. Clay had no effect
on protein solubility so treatments are presented by pH and enzyme effects only.
The addition of enzyme to dispersions created a finished powder with increased protein
solubility across the range of pH values. These powders therefore have a broader range of
potential applications.

5.4.9

Oxygen radical absorbance capacity

Oxygen radical absorbance capacity (ORAC) was measured for both soluble fractions and
powders created using all treatments with clay. The inclusion of clay has no effect on properties of the powders or soluble fractions so only treatments with clay were evaluated. The pH8
and Alc treatments had higher ORAC than pH2 or Pep treatments. In each case the addition
of protease increased ORAC, but this effect was much more apparent at pH8 (Figure 5.9).
The increased proportion of small molecular weight peptides as shown in Figure 5.7, be
have caused the increased ORAC for those hydrolyzed samples. All proteins can potentially
function as antioxidant, but small molecular weight peptides, and hydrolyzed proteins with
loss of tertiary structure have the greatest antioxidant capacities [Elias et al., 2008].

69

pH2 Pep Alc pH8

+clay +clay +clay +clay
188
98
62
49
38
28
17
14
6
3

r
de
w
Po les
b
lu
So er
d
w
Po les
b
lu
So er
d
w
Po les
b
lu
So er
d
w
Po les
b
lu
So

Figure 5.7. SDS-PAGE hydrated powders and feed stocks at the same solids contents for pH2,
Pep, pH8, and Alc treatments

5.4.10

Hygroscopicity of powders

The hygroscopicity of the powders was almost the same for all treatments (Table 5.2). The
only treatment with a meaningful difference in hygroscopicity was the pH8 set. Treatments
at pH 8 with and without clay were nearly identical. The dextrose equivalents of maltodextrins has been shown to change the hygroscopicity of spray dried powders [Cai and Corke,
2000, Goula and Adamopoulos, 2008]. Low molecular weight maltodextrins contain more
hydrophillic groups, causing an increase in hygroscopicity.
The powders produced using pH8 treatments were least hygroscopic. The feed stocks

70

50000
40000
30000

20000

10000

Protein Content of Supernatant (g/mL)

pH2
pH8
Alc
Pep

3.0

5.0

7.0

Buffer pH

Figure 5.8. BCA protein solubility of powder protein in McIlvaine buffers at pH 3.0, 5.0, 7.0
in g/mL: means of 3 replicates 1 standard error

from which these powders were made contained the lowest concentrations of glucose and
sucrose (Table 5.1). The low concentration of sugars which would cause high hygroscopicity
may have caused the powder to have lower hygroscopicity. Alcalase treatments however have
much higher concentrations of glucose and fructose, but do not show higher hygroscopicity
values. It is possible that all treatments, except for pH8, reached the maximum moisture
saturation for that material.
All peanut protein powders were much more hygroscopic than the maltodextrin used as

71

35000

Feed Stock
Powder

14000

21000

7000

ORAC (mM Trolox Equivalents)

28000

pH2

Pep

pH8

Alc

Treatment

Figure 5.9. Oxygen radical absorbance capacity of powders and soluble fractions (mMol TE):
means of 3 replicates 1 standard error

the carrier agent. Acidic conditions and Alcalase are known to hydrolyze starches like maltodextrin [Pirt and Whelan, 1951]. Pepsin and pH2 treatments were acidic and it is likely
the maltodextrin was hydrolyzed causing the increased glucose and fructose contents for
these treatments. Similarly the Alcalase treated samples had increased glucose and fructose
concentrations compared to pH8 treatments. Monosaccharides are known to be more hygroscopic than low DE maltodextrins. The differences in hygroscopicity were likely due to the
hydrolysis of the maltodextrin during stirring while the maltodextrin was dissolved into the

72

Table 5.2. Hygroscopicity of powders expressed as g water uptake per 100 g powder solids
after 1 week at 20 C 82% relative humidity: means of 3 replicates 1 standard error

Treatment

Hygroscopicity
g/ 100g solids

pH2
pH8
Pep
Alc
Maltodextrin

53.1
40.1
55.9
52.8
31.3

solubles to create the feed stocks.

73

1.2
0.8
1.3
1.6
0.1

CHAPTER

SIX
CONCLUDING REMARKS & FUTURE WORK

6.1

Conclusions

Commercially available peanut meal with moderate AFB1 contamination was used to produce virtually AFB1-free protein powder. The used involved alkaline aqueous extraction
of proteins with the addition of the commercially available protease Alcalase. A sodium
bentonite clay AstraBen20 was added and effectively bound to aflatoxins in solution. The
clay-AFB1 complex and other insoluble peanut material were allowed to settle out of solution. This insoluble fraction may have increased value as an animal feed ingredient because
of the clay addition and protein hydrolysis.
Maltodextrin was added to the soluble fraction to facilitate spray drying. Spray drying
with an inlet temperature of 185 C and outlet temperature of 90 C provided good powder
yields (70%). The powders were 28% protein on a dry weight basis. Powder solubility
was good under acidic, neutral, and basic pH values. Powders produced with Alcalase
contained a large proportion of low molecular weight peptides which potentially have bio
active properties. The antioxidant capacity of the Alcalase powders also indicated that they
may have enhanced food or nutritional functionality.

6.2

Future work

The level of aflatoxin contamination in the peanut meal was limiting. Contamination levels
higher than 52 g/kg should be used to confirm the efficacy of clay in this process to remove
AFB1 from more highly contaminated peanut meal. Aflatoxin contamination is not limited to
peanuts, but affects a large variety of crops. This processing scheme should be applied to a
variety of other commodities and clays to confirm that the process can be used to effectively

74

remove AFB1.
Additional work should be done to optimize the amount of maltodextrin needed to facilitate good drying. For the current study maltodextrin addition severed dual roles in promoting good drying and minimizing differences between treatments such that all treatments
yielded enough powder for further analysis. Future work should be conducted to optimize
spray drying conditions and feed stock preparation for the Alc treatments.
Hydrolysis work can be tailored to generate peptides for peptide immunotherapy, and
possibly spray dried to generate peanut protein based nutraceuticals.

75

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