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Adaptation and Validation of Food ProductSpecific Analytical Methods for MonitoringPrebiotics Present in Different Types of ProcessedFood MatricesRebbeca M. DuarUniversity of Nebraska-Lincoln, [email protected]

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ADAPTATION AND VALIDATION OF FOOD PRODUCT SPECIFIC ANALYTICAL

METHODS FOR MONITORING PREBIOTICS PRESENT IN DIFFERENT TYPES OF

PROCESSED FOOD MATRICES

by

Rebbeca M. Duar

A THESIS

Presented to the Faculty of

The Graduate College at the University of Nebraska

In Partial Fulfillment of Requirements

For the Degree of Master of Science

Major: Food Science and Technology

Under the Supervision of Professor Vicki L. Schlegel

Lincoln, Nebraska

December, 2011

ADAPTATION AND VALIDATION OF FOOD PRODUCT SPECIFIC ANALYTICAL

METHODS FOR MONITORING PREBIOTICS PRESENT IN DIFFERENT TYPES OF

PROCESSED FOOD MATRICES

Rebbeca M. Duar M.S

University of Nebraska 2011

Adviser: Vicki L. Schlegel.

Prebiotic carbohydrates are now added to a variety of processed foods to beneficially

affect the gut microbial composition and activities. However, published data remain limited on

the stability of prebiotics during food processes, such as baking, extrusion, pasteurization, high

temperature heating, low pH condition, etc. As the complexity of the food matrix may also affect

the ability to test for prebiotics, product specific analytical methods, including UV-vis

spectroscopy, GC and HPLC, were developed and validated to monitor the stability of FOS,

inulin and GOS in different types of processed food (breakfast cereal, cookie, muffin, sports

drink and a nutritional bar). The results showed satisfactory linearity (r>0.9) for all the validated

methods, however differences in the complexity of the ingredients were reflected in method

accuracy and precision. Method precision was determined by calculating the percent relative

standard deviation (% RSD) of spiked samples (n=5) below (0.5%) and above (2%) the target

amount to be supplemented in the food products (1%) with results ranging from 1-39%. Methods

for monitoring FOS and GOS resulted in low detection and quantitation limits allowing analysis

of prebiotics of less than 1% in the presence of complex matrices. On the other hand the inulin

method presented high detection and quantitation limits. Additionally, accuracy was affected by

compounds present in the food matrix, which was accessed by determining the percent

recoveries of spiked prebiotic in the control samples and applying corresponding correction

factors to the supplemented processed foods when the accuracy was below 90% or above 110%.

The chemical fate of the prebiotics was determined by applying the optimized and validated

extraction and analytical method to prototype food products with 1% supplemented prebiotic.

Recoveries ranged from 25-300% depending on method’s performance, complexity of the matrix

and the severity of the processing effects. Finally the fate of the prebiotics in a cereal and sports

drink that were prepared under various processing conditions indicated a high stability of GOS,

while FOS and inulin were affected by low pH and high temperatures.

iii

ACKNOWLEDGMENTS

First of all we thank USDA-NRI for funding this project. We also thank GTC nutrition,

Beneo-Orafti, for providing us with prebiotics.

This thesis wouldn’t be possible without the direction and dedication of my adviser, Dr

Vicki Schlegel, to whom I extend my most sincere thanks for more than two years of mentoring,

and support. My special thanks to Dr. Hutkins Dr. Wehling, Dr. Rose and Dr. Cuppett, for their

help and advice. Also, thanks to Richard and all my lab mates for their help and good times in

the lab.

My deepest gratitude goes to my mom and aunt, without them I would have never made

it this far in life. They have been there for me every step of the way, have always loved me

unconditionally, and have supported me through all of my tough decisions.

To my friends, my crowd, Felipe and Maria Isabel, thanks for your unconditional

friendships and for company and support, especially late in the evenings, and when I needed help

the most. Thanks guys for keeping me sane and happy through the journey of grad school. My

special thanks to my “favorite” friend Michal, you never let me give up.

Last but not least, thanks be to God for my life through all tests in the past two years.

Nobody said it was easy, but at in the end you get what you deserve, the amount of effort you put

determines the amount of joy that you receive.

iv

TABLE OF CONTENT

LIST OF FIGURES ...................................................................................................................... vii

LIST OF TABLES ......................................................................................................................... ix

A. LITERATURE RIVIEW ............................................................................................................ 1

Background of Prebiotics ............................................................................................................. 1

A.1 Chemistry of Prebiotics ....................................................................................................... 1

A.1.1 Fructooligosaccharides ............................................................................................. 2

A.1.2 Inulin ......................................................................................................................... 4

A.1.3 Galactooligosaccharides ........................................................................................... 5

A.2 Health Promoting Properties of Prebiotics ......................................................................... 6

A.2.1 FOS Health Promoting Properties ............................................................................ 8

A.2.2 Inulin Health Promoting Properties ........................................................................ 10

A.2.3 GOS Health Promoting Properties ......................................................................... 12

A.3 Prebiotics in Foods ............................................................................................................ 15

A.3.1 FOS in Foods .......................................................................................................... 15

A.3.2 Inulin in Foods ........................................................................................................ 15

A.3.3 GOS in Foods .......................................................................................................... 19

A.4 Prebiotic Analysis ............................................................................................................. 21

A.4.1 FOS Analysis .......................................................................................................... 21

A.4.2 Inulin Analysis ....................................................................................................... 22

A.4.3 GOS Analysis ........................................................................................................ 22

B. OBJECTIVES AND SPECIFIC AIMS.................................................................................... 23

C. MATERIALS AND METHODS ............................................................................................. 24

C.1 Specific aim 1: Develop and/or adapt extraction and analytical procedures. ..................... 24

C.1.1. FOS Analysis ........................................................................................................... 27

C.1.2 Inulin Analysis .......................................................................................................... 29

C.1.3 GOS Analysis ........................................................................................................... 32

C.2 Specific Aim 2: Method Validation .................................................................................... 35

v

C.2.1 Accuracy ................................................................................................................... 35

C.2.2 Precision ................................................................................................................... 35

C.2.3 Linearity of Calibration Curves ................................................................................ 35

C.2.4 Limit of Detection ..................................................................................................... 36

C.2.5 Limit of Quantitation ................................................................................................ 36

C.3 Specific Aim 3: Analysis of 1% prebiotic supplemented food products. ........................... 36

C.4 Specific aim 4: To assess the chemical fate of FOS, GOS and inulin during various

processing treatments. ................................................................................................................ 37

C.4.1 Chemical Fate of FOS, Inulin and GOS in Sports Drink ......................................... 37

C.4.2 Chemical Fate of FOS, Inulin, and GOS in Extruded ready-to-eat Breakfast Cereal.

............................................................................................................................................ 38

D. RESULTS AND DISCUSSION .............................................................................................. 40

D.1 Specific aim 1: Method Development or Adaptation ......................................................... 40

D.1.1 FOS ........................................................................................................................... 40

D.1.2 Inulin ......................................................................................................................... 42

D.1.3 GOS .......................................................................................................................... 45

D.2 Specific Aim 2: Method Validation ................................................................................... 49

D.2.1 FOS ........................................................................................................................... 49

D.2.2 Inulin ......................................................................................................................... 52

D.2.3 GOS .......................................................................................................................... 54

D.3 Specific Aim 3: Analysis of 1% prebiotic supplemented food products. ........................... 56

D.3.1 FOS ........................................................................................................................... 56

D.3.2 Inulin ......................................................................................................................... 57

D.3.3 GOS .......................................................................................................................... 57

D.4. Specific aim 4. Chemical fate of FOS, GOS and inulin during various processing

treatments. .................................................................................................................................. 61

D.4.1 Chemical fate of FOS in the breakfast cereal. .......................................................... 61

D.4.2 Chemical fate of inulin in breakfast cereal ............................................................... 62

vi

D.4.3 Chemical fate of GOS in breakfast cereal ................................................................ 62

D.4.4 Chemical fate of FOS in sports drink ....................................................................... 66

D.4.5 Chemical fate of inulin in sports drink ..................................................................... 66

D.4.6 Chemical fate of GOS in sports drink ...................................................................... 67

E. CONCLUSIONS ...................................................................................................................... 71

Bibliography ..................................................................................................................... 73

vii

LIST OF FIGURES

Figure 1. Structure of the commercial fructooligosacharides. (Source: Hussein et al. (1998)). ..... 3

Figure 2. Structure of inulin. (Source: Ueno et al. (2011)). ........................................................... 4

Figure 3. Structure of the commercial GOS (adopted from Dr Jens Walter, Department of Food

Science and Technology, University of Nebraska, Nutraceuticals notes). ......................... 5

Figure 4. Proposed mechanism by selective fermentation of prebiotics and subsequent production

of short chain fatty acids resulting in improved bowel habit, increased dietary mineral

absorption, and lower risks of colon cancer. (Source: Crittenden et al. (2006)). ............... 7

Figure 5. Schematic illustration of the adherence (A) and anti-adhesive agents: probiotics (B)

adhesion analogs (C), and receptor analogs (D). (Source: Shoaf et al. (2008)). ............. 14

Figure 6. Percentages of glycosidic linkages los by hydrolysis in FOS incubated for different

time periods (0, 5, 15, 20, 30 and 60 min) at 100 °C. (Source: (Courtin et al. (2009)) ... 16

Figure 7. Degradation of inulin from chicory during thermal treatment up to 60 minutes at

temperatures between 100 and 195 °C. (Adapted from Böhm et al. (2005)). .................. 18

Figure 8. Stability of Vivinal ® GOS at low pH and 85 °C for 5 minutes. Source: (Friesland

Foods Domo, GRASS notice FDA (2007)). ..................................................................... 20

Figure 9. Flow diagram of the extraction procedure and analysis of FOS from the food matrices

........................................................................................................................................... 28

Figure 10. Flow diagram of the extraction procedure and analysis of inulin from the food

matrices. ............................................................................................................................ 31

Figure 11. Flow diagram of the extraction procedure and analysis of GOS from the food

matrices. ............................................................................................................................ 34

Figure 12. 23

factorial design applied to the pasteurized sports drink. ......................................... 39

Figure 13. Different variables of screw speeds and temperatures while maintaining other

extrusion parameters. ........................................................................................................ 39

Figure 14. GC chromatogram of GOS (1mg/ml). Peaks: galactose (ret time 10.12 min), glucose

(ret time 10.83 min) myo-inositol (11.26 min). ................................................................ 46

Figure 15. GC chromatogram of sports drink control. Peaks: fructose (ret time 9.473 min),

glucose (11.31 min) myo-inositol (11.75 min). ................................................................ 47

viii

Figure 16. GC chromatogram of sports drink spiked with 1% GOS. Peaks: Fructose (ret time

9.495 min), galactose (10.32 min), glucose (11.21 min), myo-Inositol (11.26 min)....... 47

Figure 17. GC chromatogram of breakfast cereal control. Peaks: Fructose (ret time 9.19 min),

glucose (10.78 min), myo-inositol (11.23 min) ................................................................ 48

Figure 18. GC chromatogram of breakfast cereal spiked with 1% GOS. Peaks fructose (ret time

9.264 min), galactose (10.05 min), glucose (ret time 10.82 min) myo-inositol (11.308

min). .................................................................................................................................. 48

Figure 19. Percent recovery of 1% supplemented FOS in different prototype foods after

processing. Results shown as MEAN ± STD (n=3). ........................................................ 58

Figure 20. Percent recovery of inulin after processing of food products supplemented of 1%

prebiotic. Results shown as MEAN ± STD (n=3). ........................................................... 59

Figure 21. Percent GOS after processing of food products supplemented of 1% prebiotic.

Results shown as MEAN ± STD (n=3)............................................................................. 60

Figure 22. FOS recovery in breakfast cereal supplemented with 1% FOS and extruded under

different screw and temperature conditions. Results shown as MEAN ± STD (n=3). Bars

with different letters are statistically different (p>0.05) using Tukey HSD test. .............. 63

Figure 23. FOS recovery in sports drink supplemented with 1% FOS prepared and under

different pH and sweetener ratios. Results shown as MEAN ± STD (n=3). Bars with

different letters are statistically different (p>0.05) using Tukey HSD test. ...................... 68

Figure 24. Inulin recovery in sports drink supplemented with 1% inulin and prepared under


different letters are statistically different (p>0.05) using Tukey HSD test. ...................... 69

Figure 25. GOS recovery in sports drink supplemented with 1% GOS and prepared under


different letters are statistically different (p>0.05) using Tukey HSD test ....................... 70

ix

LIST OF TABLES

Table 1. Extruded Ready-to-eat Breakfast Cereal Formulation .................................................. 24

Table 2. Cookie Formulation ....................................................................................................... 25

Table 3. Muffin Formulation ....................................................................................................... 25

Table 4. Nutritional Bar Formulation ........................................................................................... 26

Table 5. Sports Drink Formulation ............................................................................................... 26

Table 6. Optimization conditions for extracting inulin in the different food products ................ 44

Table 7. Optimization conditions for extracting GOS from sports drink and breakfast cereal .... 46

Table 8. Method performance characteristics for FOS in different food products ...................... 51

Table 9. Method performance characteristics for inulin in different food products ..................... 53

Table 10. Percent recoveries of 1% FOS supplemented food products after processing. ............ 58

Table 11. Percent recoveries of 1% inulin supplemented food products after processing. .......... 59

Table 12. Percent recoveries of 1% GOS supplemented food products after processing ............ 60

1

A. LITERATURE RIVIEW

Background of Prebiotics

The Prebiotic concept was introduced by Gibson & Roberfoid (1995) as “non-

digestible food ingredients that beneficially affect the host by selectively stimulating the

growth and/or activity of one or a limited number of bacteria in the colon, and thus

improves host health”. Later the concept was updated to “a selectively fermented

ingredient that allows specific changes, both in the composition and/or activity in the

gastrointestinal microflora that confers benefits upon host well-being and health” (Gibson

et al., 2004). Lastly during the 2008 International Scientific Association for Probiotics

and Prebiotics (ISAPP) meeting, a prebiotic was defined as “a selectively fermented

ingredient that results in specific changes in the composition and/or activity of the

gastrointestinal microbiota, thus conferring benefit(s) upon host health” (ISAPP 2008).

The “Prebiotic effect” refers to the pathological and physiological effects both in

experimental and human intervention studies specifically linked-to or at least correlated

with selective changes in gut microbiota composition (Roberfroid et al. 2010).

A.1 Chemistry of Prebiotics

The human small intestine produces enzymes that are specific for α-glycosidic

linkages. Prebiotics are carbohydrates with molecular structures resistant to the digestive

enzymes due to the β-configuration of their glycosidic bonds, thus reaching the colon

where they serve as fermentation substrates for selected colonic microbiota. Not all

dietary carbohydrates are prebiotics, and resistance to digestion alone is not the sole

criteria to classify a carbohydrate as a prebiotic. Roberforid (2007) suggested certain

requirements for a prebiotic: 1) resist digestion and absorbtion in the upper

2

gastrointestinal tract; 2) be a suitable susbtrate for selected bateria in the colon i.e.,

bifidobacteria and lactobacilli; 3) contribute to the growth of selected gastrointestinal

bacteria towards a healthier composition contributing to host’s health and well-being.

The majority of scientific data regarding prebiotic effects include inulin-type fructans and

galactooligosaccharides (Roberfroid et al. 2010).

A.1.1 Fructooligosaccharides

Fructooligosaccharides (FOS) belong to a heterogeneous group of carbohydrates

with the characteristic of having fructose residues as the majority of their monomers.

Commercially available FOS are produced enzymatically from natural disaccharides or

polysaccharides and consist of molecular chains with a degree of polymerization (DP)

lower than 9 (Nguyeh et al., 1999). One type of FOS is derived from the partial

enzymatic hydrolysis of inulin using endo-inulinase but it can also be synthesized from

sucrose by a transfructosylation processes with the enzyme β-fructofuranosidase from

Aspergillus niger (Yun 1996; Frank 2002). The main FOS consist of 1-kestose (GF2),

nystose (GF3) and 1F β-fructofuranosylnystose (GF4), which are oligomers of one

glucose unit and two, three and four fructose units, respectively (Figure 1). The fructosyl-

glucose linkage is always α-(1→2) and the fructosyl-fructose linkages are -(2→1)

(Antosova et al. 1999; Birkett et al. 2009).

3

Figure 1. Structure of the commercial fructooligosacharides. (Source: Hussein et

al. (1998)).

4

A.1.2 Inulin

Inulin is a type of fructan widely found in nature for plant energy storage (Niness

1999). From a structural point of view, it is a polysaccharide of D-fructofuranose units β-

(2→1) linked with a D-glucosyl residue at the end of the chain (Figure 2) (Ueno et al.

2011). Depending on the source, the degree of polymerization ranges from 20 to 60

(Roberforid 2007; Böhm et al. 2005). These carbohydrates have been classified as non-

digestible oligosaccharides due to their resistance to human enzyme hydrolysis as

demonstrated in both in vitro and in vivo methods (Cherbut 2002; Roberfoid et al. 1998).

Figure 2. Structure of inulin. (Source: Ueno et al. (2011)).

5

A.1.3 Galactooligosaccharides

Galactooligosaccharides (GOS) are derived from soya beans and lactose and are

one of the most commonly produced prebiotics (Nauta et al. 2009). GOS are comprised

of different short chain polysaccharides, each of which contain galactose as monomers

bonded by β-(1→3), β-(1→4), or β-(1→6) linkages with a terminal glucose (Figure 3).

These oligosaccharides are enzymatically produced by transglycosylation of lactose using

β-galactosidase (Bankova et al. 2006; Berreteau et al. 2006).

Figure 3. Structure of the commercial GOS (adopted from Dr Jens Walter,

Department of Food Science and Technology, University of Nebraska, Nutraceuticals

notes).

6

A.2 Health Promoting Properties of Prebiotics

Several potential health benefits have been attributed to these non-absorbable

carbohydrates, which include increasing of fecal bulk, promoting growth of selective but

beneficial microorganisms of the human microflora, lowering of serum cholesterol,

improving mineral absorption and enhancing immune function (Crittenden et al. 2006;

Roberfroid et al. 2010). As an outcome to these benefits, there is interest in prebiotics as

preventative interventions for health related conditions or even serious diseases,

including ulcerative colitis, colon cancer, coronary heart disease, and osteoporosis. The

mechanisms by which these events occur are not entirely understood and are still under

scientific debate (Roberfroid et al. 2010). However some of the proposed health

promoting mechanisms of prebiotics are depicted in Figure 4, and include: 1.) growth

stimulation of endogenous beneficial bacteria in the gut, hence promoting colonization

resistance against pathogenic bacteria by nutrient and niche competition; 2.) selective

fermentation of the prebiotics to short chain fatty acids (SCFA) resulting in increased

absorption of essential minerals, and lipogenisis regulation in the host and lower

intestinal pH antigenic for some pathogenic bacteria; 3.) promotion of colonic peristalsis

by increasing stool weight and bulk, which in turn shortens transit time and alleviates

constipation.

7

Figure 4. Proposed mechanism by selective fermentation of prebiotics and

subsequent production of short chain fatty acids resulting in improved bowel habit,

increased dietary mineral absorption, and lower risks of colon cancer. (Source: Crittenden

et al. (2006)).

8

A.2.1 FOS Health Promoting Properties

FOS are recognized as a prebiotic because it meets all the required criteria. FOS

thus reaches the colon without being metabolized and are then fermented by the colonic

microbiota to lactate and SCFA (Niness 1999; Nguyeh et al. 1999). Several in vitro

studies have demostrated that FOS is selectively fermented by the health promoting

bacteria, bifidobacteria and lactobacilli (Bouhnik et al. 2007; Kapl (Kaplan & Hutkins,

2003)an et al. 2003; Macfarlane et al. 2008). In particular, a number of human studies

have shown the bifidogenic effect of FOS at doses ranging from 1 to 31 g/day (Thuoty et

al. 2001; Buddington et al. 1996; Menne et al. 2000; Bouhnik et al. 2007).

Studies have also shown that FOS are not utilized by harmful bacterial, e.g.,

Escherichia coli or Clostridium dificile (Hidaka et al. 1986). In a 48-hour incubation

study with mixed fecal flora, Rousseau et al. (2005) demostrated that the pathogenic

microorganisms Candida albicans and Gardnerella vaginalis, which are often causers of

urogenital infections, did not utilize FOS. The inhibition of such pathogenic bacterial

growth by FOS has been attributed to SCFA production and lower intestinal pH (Birkett

et al. 2009). Other studies involving animals fed FOS followed by exposure to pathogens

showed similar protective effects. For example, Brunce et al. (1995) determined that the

mortality and morbidity of piglets supplemented with FOS for 6 days were reduced

compared to a control group given enterotoxigenic E. Coli K88 alone. In addition,

piglets fed FOS (7.5 g/L) for 14 days resulted in a significant reduction in the severety of

Salmonella typhimuriumin infections (Correa-Matos et al. 2003).

9

FOS supplementation has further been associated with modulating and

strengthening the immune function. Guigoz et al. (2002) showed that consumption of 8 g

of FOS per day by elderly people reduced the inflammatory response. Moreover, the

addition of FOS and GOS (8 g/L in a 9:1 ratio) to an infant formula induced beneficial

antibody profiles by significantly reducing plasma levels of IgE, IgG1, IgG2 and IgG3, in

infants at a high risk for allergies (van Hoffen et al., 2009). Early exposure of non-

breast-fed infants to a formula of FOS-GOS (6 g/L) significantly increased secretory

immunoglobulin after 16 weeks, thereby enhancing the maturation and development of

the gastrointestinal immune defense (Bakker-Zierikzee et al. 2006; Scholtens et al. 2008).

Using a randomized, placebo-controlled trial with health infants, Bruzzese et al.

(2005) reported that supplementation of infant formula with FOS and GOS had a

prophylactic effect in the incidence of gastroenteritis and diarrhea. Likewise, Lindsay et

al. (2005) encountered a positive therapeutic effect by patients with Crohn’s disease after

receiving 15 g of FOS for 3 weeks. Treatment of 5 g of FOS in patients with minor

functional bowel disorders during a 6 week period significantly reduced the incidence of

digestive disorders by 46.3% and associated symptoms (Paineau et al. 2008).

Several research groups have shown that FOS fermentation in the colon increases

the colorectal absorption of calcium, iron, and magnesium in rats (Ohta et al. 1995;

Lopez et al. 2000). Similar effects have been reported in humans (Ohta et al. 1998;

Tahiri et al. 2001; Fukushima et al. 2002; Uenishi et al. 2002; van den Heuvel et al.

2009). In particular, Bouhnick et al. (2007) showed that FOS fermentation in the colon

produces SFCA resulting in a lower intestinal pH, thus increasing mineral solubility. It

has also been proposed that FOS aids in rebalancing lipid homeostasis and leads to higher

10

cholesterol excretion (Bouhnik et al. 2007). Although the mechanisms causing these

responses are not fully understood, it has been attributed to 1.) decreases in the daily

energy intakes due to lower calorie value of FOS when used as a sweetener; 2.) beneficial

changes in the intestinal microflora influencing lipid absorption and metabolism; 3.)

binding of FOS to lipids preventing cholesterol absorption at the intestinal mucosal; and

4.) increased synthesis of fermentation by products (SCFA), which may decrease

cholesterol synthesis in the liver (Yamashita et al. 1984; Kok et al. 1996; Delzenne &

Kok 2001).

Lastly, research in animal models has revealed that FOS has significant

anticarcinogenic properties, including prevention of colon cancer (Pool- Zobel et al.

2002), lower incidence of tumors, metastases and inhibition of malignant tumor growth

(Taper et al. 1999).

A.2.2 Inulin Health Promoting Properties

According to several research groups inulin is fermented in the colon producing

lactate and SCFA thereby evoking beneficial changes in the intestinal microflora and

improving mucosal integrity (Roberfoid et al. 1998; Jenkins et al. 1999; Gibson 2000).

The prebiotic effect of inulin has been shown to stimulate the growth of Bifidobacterium

and reduce the Enterobacteriacea populations in both human and animal studies (Gibson

et al., 1995; Kleessen et al. 1997; Kolida et al. 2007; Ramnani et al. 2010). It has also

been proposed that inulin aids in the survival and implantation of probiotic bacteria

providing a symbiotic effect (Niness 1999).

Recent data indicate that inulin consumption beneficially modulates the immune

system, including the gut-associated lymphoid tissues (Schley et al. 2002). Watzl et al.

11

(2005) fed rats an inulin enriched diet (10%) resulting in a significant higher levels of the

anti-inflammatory cytokine interlukin-10. A prospective randomized, placebo controlled

pilot trial performed by Casellas et al. (2007) showed beneficial effects on symptoms and

reduced intestinal inflammation in patients with ulcerative colitis when administrated 4 g

of inulin 3 times a day for 14 days. Additionally Furrie et al. (2005) tested the effects of

a symbiotic therapy on patients with ulcerative colitis by combining inulin (6 g) with the

probiotic bacteria Bifidobacterium longum (2 x 1011

) twice a day for 4 weeks. This study

indicated a reduction in the ulcerative colitis symptoms and a significant decrease in the

mucosal anti-inflammatory cytokines.

Other studies have shown anti-cancer responses to dietary treatment with inulin,

such as reducing the incidence and growth of malignant tumors and decreasing

metastases (Taper et al. 1999). Inulin consumption has been linked to promoting satiety

and lowering total energy intake, which has the potential of translating into a supplement

for weight management (Archer et al. 2004). For instance, consumption of 16 g/day of

inulin increased satiety and reduced hunger in humans (Cani et al. 2006). Research

groups have reported that inulin consumption resulted in increased fecal biomass and

water content in stools, thus improving laxation and reducing constipation (Gibson et al.

1995; Kleessen et al. 1997). Den Hond et al. (2000) investigated the effect of inulin on

bowel function in healthy volunteers with low stool frequency, and determined that

administration of 15 g/day for 2 weeks significantly increased stool frequency.

Furthermore, fermentation of inulin by the gastrointestinal microflora has been associated

with greater intestinal absorption of minerals. Coundray et al. (1997) confirmed that

ingestion of inulin (40 g/day) by young men for a 28 day period significantly increased

12

calcium. Similarly, Younes et al. (2001) showed a significant increase in the calcium and

magnesium absorption in rats fed a high-inulin diet (100 g/kg) for 21 days. Lastly,

improved blood lipid profiles were reported in response to inulin consumption (Williams

1999; Causey et al. 2000). For example, the latter group showed a significant reducction

in serum tryglycerides (by 40 mg/dl).

A.2.3 GOS Health Promoting Properties

GOS are completely fermented in the human colon producing SCFA (Alles et al.

1999) and their bifidogenic effect has been demonstrated throughout the literature

(Alander et al. 2001; Ben et al. 2004; Bouhnik et al. 2004; Vulevic et al. 2008). Davis et

al. (2010) tested the bifidogenic effect of different doses of GOS in healthy adults and

showed a significant increase at 5 g and 10 g. Silk et al. (2009) demonstrated that

consumption of GOS at doses ranging 3.5-7 g/day enhanced fecal bifidobacteria and

alleviated the symptoms of patients with irritable bowel syndrome. Additionally, GOS

intake prevented the incidence and symptoms of travelers’ diarrhea (Drakoularakou et al.

2010).

Studies have confirmed the anti-adherent activity of commercially available GOS

against several infectious bacteria in the gastrointestinal tract. It has been proposed that

GOS acts as molecular decoys that mimic the carbohydrate binding sites recognized by

the pathogen in epithelial cells (Figure 5). Specifically, pathogens bind to the GOS rather

than to the host cells and are thus displaced from the gastrointestinal tract without causing

infection (Shoeaf et al. 2008). Other researchers have shown that GOS inhibited the

adherence of Escherichia coli, Salmonella enterica serovar Typhimurium and two

13

different strains of Cronobacter sakazakii to tissue culture cells at a concentration of 16

mg/ml (Shoaf et al. 2006; Searle et al. 2009; Quintero et al. 2011).

Reports have shown that the consumption of GOS increases calcium absorption

using rats as the model system (Chonan et al. 1995; Chonan et al. 1996) but humans

subjects produced opposite outcomes. van den Heuvel et al. (1998) investigated the

effect on calcium and iron absorption in young healthy subjects fed 15 g/d GOS but

increases absorption of either mineral did not occur. In a second study, the same research

group reported that GOS consumption by postmenopausal women (20 g/d) for 9 days

resulted in higher true calcium absorption (van den Heuvel et al. 2000).

Vulevic et al. (2008) showed beneficial immune effects of GOS consumption with

44 elderly subjects who received a 5.5 g/day treatment. The results indicated a significant

increase in phagocytosis and natural killer activities, as well as elevated production of

anti-inflammatory cytokines with a concomitant reduction in pro-inflammatory

cytokines. In particular, GOS have attracted attention due to similarities to human milk

oligosaccharides (Alander et al. 2001; Moro & Arslanoglu 2005 Sangwan et al. 2011)

that play an important role in infants’ immune system development and modulation of

their intestinal microflora.

GOS has also been linked to lower bacterial enzyme activities involved in the

formation of carcinogenic compounds and to lower levels of secondary bile acids linked

to increased colon cancer risk (Ito et al. 1993; Rowland et al. 1993). Administration of

GOS has shown protective properties against colo-rectal cancer by reducing the number

of tumors in a rat model system (Wijnands et al. 1999).

14

Figure 5. Schematic illustration of the adherence (A) and anti-adhesive agents:

probiotics (B) adhesion analogs (C), and receptor analogs (D). (Source: Shoaf et al.

(2008)).

15

A.3 Prebiotics in Foods

Some prebiotics, such as inulin and oligofructose, occur naturally in foods

(Roberfroid 2002) but are increasingly used as functional ingredients for a variety of

processed foods (Roberfroid et al. 2010). However, during processing carbohydrates

undergo different changes, such as Maillard-reaction, caramelization and hydrolysis

(Matusek et al. 2008; Birkett et al. 2009). Certainly, oligosaccharides that have been

enzymatically or chemically hydrolyzed are not expected to retain prebiotic activity

considering that released sugars would be absorbed in the intestinal tract or metabolized

by the general commensal flora. Thus, the interaction of the prebiotic with the

surrounding matrix and the effect that processing might have on their structure must be

considered (Huebner et al. 2008).

A.3.1 FOS in Foods

FOS are extensively distributed in the plant kingdom, particularly in banana,

wheat, barley, onion, garlic, asparagus and Jerusalem artichoke (Campbell et al. 1997;

Roberfroid 2002). FOS has been used in the food industry to manufacture pastry, frozen

deserts and dairy products; contributing to both body, texture and as a low calorie

sweetener (Campbell et al. 1997; Niness 1999; Sangeethat et al. 2005). Although little

data exist on the physio-chemical characteristics and other stability properties of FOS in

processed foods (Yun 1996), Mujoo et al. (2003) showed that added kestose and nystose

levels decreased after the bread baking process. According to results reported by Courtin

et al. (2009), a reduction of 60-67% (w/w) of the glycosidic linkages β-(2→1) occurred

between fructose units at low pH (2.0 and 3.0) and after incubation at 100 °C for 60

minutes (Figure 6). Other experiments have shown significant degradation of FOS under

16

acidic conditions (pH 2-4) and high temperature 70-120 °C (L'homme et al. 2003;

Matusek et al. 2008).

Figure 6. Percentages of glycosidic linkages los by hydrolysis in FOS incubated

for different time periods (0, 5, 15, 20, 30 and 60 min) at 100 °C. (Source: (Courtin et al.

(2009))

17

A.3.2 Inulin in Foods

Inulin is found naturally in a variety of plants and in some bacteria and fungi, thus

inulin has always been part of the human diet. Common sources include chicory roots

(Cichorium intybus), Jerusalem artichoke, leaks, onion, garlic and asparagus (Frank,

2002). Inulin can also be enzymatically synthesized from sucrose (Niness 1999; Frank

2002; Roberfroid 2002)

In the food industry, inulin is widely used for both its nutritional and functional

properties (Frank 2002). It is often used in the production of low glycemic index foods

intended for diabetics and hydrolyzed to produce fructose syrups (Hofer et al. 1999).

Furthermore, inulin improves physical and organoleptic properties of low fat foods and

can be used as a fat replacer. Inulin is added to foods as a prebiotic ingredient and to

enrich the fiber content of products without sacrificing mouth feel and or producing off-

flavors (Niness 1999; Frank 2002). Nevertheless, Böhm et al. (2005) showed that dry

thermal treatment of inulin caused significant degradation of the long fructose chains

leading to the formation of smaller molecules such as di-D fructose and dianhydrides

(Figure 7). Losses of the initial inulin content in bakery products during the dough

development and the baking process has been reported (Pranznik et al. 2002).

18

Figure 7. Degradation of inulin from chicory during thermal treatment up to 60

minutes at temperatures between 100 and 195 °C. (Adapted from Böhm et al. (2005)).

19

A.3.3 GOS in Foods

Products containing GOS were first launched in Japan in the late 1980s (Nauta et

al. 2009). GOS has since been recognized as a stable, soluble and functional ingredient,

suitable as an ingredient for a number of foods and beverages, including dairy products,

bakery products, breakfast cereals, beverages, snack bars infant formulas, functional

foods, and clinical nutrition formulas (FDA 2007; Sangwan et al. 2011). GOS have a

very stable shelf life even at low pH and high temperature conditions (Figure 8) due to

the presence of β-type linkages (FDA 2007; Nauta et al. 2009; Sangwan et al. 2011). In

food products requiring heat treatment, GOS remains stable at temperatures up to 100 and

120 °C and pH values of 3-7 (FDA 2007).

GOS are increasingly applied as ingredients for infant formula and growing-up-

milks as a means to mimic the biological functions of human milk oligosaccharides

(Napol et al. 2003; Nauta et al. 2009; Sangwan et al. 2011). The amount of supplemented

GOS varies by product but current infant formulas typically contain up to 8 g GOS per l

(FDA 2007), which is based in part on the amount of complex oligosaccharides in mature

human milk (5-8 g/l) (Kuz et al. 2000). Results from studies by Savino et al. (2003) and

Moro et al. (2002) indicated that infant formula supplemented with up to 7.2 g GOS per l

was well tolerated by healthy term infants without causing any adverse effects. GOS are

also incorporated into symbiotic formulations to enhance the survival, colonization and

functionality of probiotics (Kukkonen et al. 2008; Piirainen et al. 2008).

20

Figure 8. Stability of Vivinal ® GOS at low pH and 85 °C for 5 minutes. Source:

(Friesland Foods Domo, GRASS notice FDA (2007)).

21

A.4 Prebiotic Analysis

As stated previously, carbohydrates can undergo different changes, including

Maillard-reactions, caramelization and hydrolysis; hence the supplemented prebiotics

could lose their effect by breaking down or changing during processing (Matusek et al.

2008). Although different analytical methods to detect and characterize non-digestible

carbohydrates are reported throughout the literature, the vast majority have only been

applied to the analysis of pure prebiotics and not to prebiotics supplemented in different

complex foods matrices. Therefore, it is necessary to develop selective analytical

methods, capable of accurately measuring prebiotics in the presence of complex food

matrices.

A.4.1 FOS Analysis

Several methods to detect and quantitate FOS have been discussed in the

literature, including thin layer chromatography (Park et al. 2001), gas chromatography

with mass spectrometry detection and nuclear magnetic resonance (Hayashi et al. 2000).

Nevertheless, high performance liquid chromatography (HPLC) using both polar and

non-polar resin based columns with refractive index detection is the prevalent technique

for FOS analyses (Sangeethat et al. 2005). Anion exchange chromatography methods

using an alkaline mobile phase and pulsed electrochemical detection (Shiomi et al. 1991;

Campbel et al. 1997) as well as, cation-exchange chromatography using water as the

eluent and Ca2+

as the counter ion (Antosova et al. 1999) have also been established.

22

A.4.2 Inulin Analysis

Presently inulin can be legally labeled as “dietary fiber”, but the official method

for analysis of “dietary fiber” is unable to detect it (Frank 2002). For this reason, liquid

chromatography methods with refractive index detection have been developed for its

detection and quantification, both directly and indirectly after hydrolysis to fructose

(Zuleta et al. 2001; Vendrell-Pascuas et al. 2000; Wang et al. 2010). However,

colorimetric methods for fructans based on acidic or enzymatic hydrolysis are more

commonly used due to their simplicity and low costs (Hofer et al. 1999; Korakli et al.

2003; Wang et al. 2010). The official AOAC method for the measurement of fructans is a

colorimetric method where fructans are hydrolyzed to fructose and then measured after

reaction with p-hydroxybenzoic acid hydrazide (AOAC 2002).

A.4.3 GOS Analysis

Several analytical methods have been published pertaining to the identification

and quantification of GOS, including UV spectroscopy (Dias et al. 2008), capillary

electrophoresis (Albrecht et al. 2010), HPLC with refractive index detection (Albayrak &

Yiang 2002), and thin layer chromatography (Sanz et al. 2005; Splechtna et al. 2006).

The existing official method for the determination of trans-galactooligosaccharides in

food products (AOAC 2001.2) is based on high performance anion-exchange

chromatography with pulsed amperometric detection of galactose after hydrolysis of

GOS with β-galactosidase (de Slegte 2002).

23

B. OBJECTIVES AND SPECIFIC AIMS

Stability of prebiotics during food processing is a very important requirement

given that prebiotics are biologically active compounds in terms of their health promoting

properties. As the complexity of the food matrix may also affect the ability to test for

prebiotics, product specific analytical methods are needed to monitor different types of

processed food. Therefore the objective of this project was to adapt or develop, validate,

and then apply analytical methods that are capable of monitoring prebiotics in different

types of processed food matrices, including breakfast cereal, cookie, muffin, sports drink

and a nutritional granola bar. The objective of this project was satisfied by completing

the following specific aims.

Specific Aim 1: To develop and/or adapt extraction and product specific

analytical procedures for measuring FOS, GOS and inulin in the different food matrices.

Specific Aim 2: To validate the methods established from Specific Aim 1 for

each food matrix.

Specific Aim 3: To apply the validated method to the food matrices prepared

with 1% of the prebiotic under standard unit preparation procedures.

Specific aim 4: To assess the chemical fate of FOS, GOS and inulin in prototype

of extruded cereal and a sports drink prepared under different processing / formulation

parameters.

24

C. MATERIALS AND METHODS

C.1 Specific aim 1: Develop and/or adapt extraction and

analytical procedures

Processed foods used for this study included extruded ready-to-eat breakfast

cereal, muffin, cookie, and nutritional bar, which were provided by Dr. Randy Wehling

(Department of Food Science and Technology, University of Nebraska). The

formulations of each product are shown in Tables 1-5. Supplemented products were

prepared with the following prebiotics: 1) Galactooligosacharides (GTC nutrition),

90%db. 2) Short-chain fructooligosaccharides powder (NutraFlora®) (GF2 33.8%, GF3

50.1%, and GF4 11.6%) 3) Inulin (ORAFTI) ≥ 92.2% oligofructose, DP between 2 and 8.

Table 1. Extruded Ready-to-eat Breakfast Cereal Formulation

Component g/batch

Oat flour 800

Corn flour 1010

Sucrose (granulated table sugar) 160

Salt 20

Calcium carbonate 10

Prebiotic 20

*Refer to Figure 13 for processing parameters.

25

Table 2. Cookie Formulation

Component g/batch

Shortening 64

Sugar 130

Salt, USP 2.1

Bicarbonate of Soda, USP 2.5

Dextrose solution (8.1g dextrose hydrous,

USP in 150 ml water)

33

Distilled water 16

Flour14% mb 225

*Baked in an industrial rotating oven at 204 ° C for 10 min.

Table 3. Muffin Formulation

Component g/batch

All-purpose flour (Bleached wheat flour, maltea

barley flour, niacin, iron, thiamin, mononitrate,

riboflavin, folic acid)

250

Granulated sucrose 75

Baking powder (Baking soda, corn starch,

sodium aluminium sulfate, calcium sulfate,

monocalcium phosphate)

15

Salt 3.1

Eggs (whole, slightly beaten) 50

Milk (fluid) 200

Butter(melted) 75

Distilled water 50

Prebiotic 7.5

*Baked in a conventional oven at 240 ° C for 20 min.

26

Table 4. Nutritional Bar Formulation

Component g/batch

Rolled oats (whole) 420

Granola cereal (whole grain rolled oats,

evaporated cane juice, expeller pressed canola oil,

defatted wheat germ, oat flour, brown rice syrup,

molasses, salt, natural flavor, soy lecithin)

420

Margarine 50

Honey (clover) 350

Peanut butter(creamy style) 100

Sucrose(granulated table sugar) 50

Salt 5

Roasted peanuts (chopped) 75

Prebiotics 14.7

* Ingredients mixed in a table top mixer, compressed until firmly packed

and allowed to set overnight.

Table 5. Sports Drink Formulation

Component g/L

Granulated sucrose 25

Corn syrup solids 25

Citric acid According to pH

Sodium chloride 1

Sodium citrate 0.1

Prebiotic - resistant starch 10

Red food color Small amount

* Refer to Figure 12 for processing parameters.

27

C.1.1. FOS Analysis

FOS was initially extracted according to the method shown in Figure 9 and

adjusted to achieve optimal recovery from each food based on spike recovery tests (Table

6) . Identification and quantification of FOS was determined by high performance liquid

chromatography using an amino bonded phase carbohydrate column (Waters

3.9x300mm) warmed at 35 °C and interfaced to a refractive index detector (Waters RI

2414) maintained at 35 °C and sensitivity 1. Acetonitrile in water (75:25 v/v) served as

the mobile phase and was set at a flow rate of 1.0 ml/min (Nishizawa et al. 2000; Sheu et

al. 2001). FOS standards of 1-kestose, nystose and 1-fructofuranosylnystose, (Wako,

Osaka, Japan) were injected at 5 concentration points ranging from 0.25 to 7.50 mg/ml.

Peak areas were recorded and plotted to construct calibration curves. FOS levels in the

samples were determined by using the calibration curve constructed from external

standards.

28

Figure 9. Flow diagram of the extraction procedure and analysis of FOS from the

food matrices.

Accuratly weigh a sub sample and combine with water, at the

appropiate proportions.

Mix 30 min at room temperature.

Centrifuge at 10000 g for 20-

30 min at room temperatute. Filter supernates to remove

large particles.

Collect filtrate and pass an aliquot through a 0.45 µm

filter.

Dry sample under vacuum at 50 °C to obtain a dry residue.

Resuspend sample in nanopure water at a final volume

resulting in 1% solution. Analyze with HPLC.

29

C.1.2 Inulin Analysis

Inulin was extracted from the processed foods using the basic protocol shown in

Figure 10 unless indicated otherwise in Section D. Inulin content in the samples was

measured according to the Total Fructan AOAC method 999.03, using an assay kit

purchased from Megazyme International (K-FRUC 5/2008,Ireland Ltd., County Wicklow

Ireland). The kit included sucrose/amylase and fructanase as freeze-dried powders. The

sucrase/amylase mixture contained 100 U of sucrase, 500 U of β-amylase from B. cereus,

100 U of pullulanase from K. pneumoniae and 1000 U of maltase from yeast. The

fructanase mixture included 10000 U of exo-inulinase and 100 U of endoinulinase. The

enzyme solutions were prepared by dissolving sucrase/amylase and the fructanase freeze-

dried powders into 22 ml of sodium maleate (100 mM, pH 6.5) and 22 ml of sodium

acetate buffer respectively. Enzyme solutions were divided into 5 ml aliquots and stored

at 20 °C in polypropylene containers. Solutions and buffers consisted of sodium maleate

buffer (100 mM, pH 6.5), sodium acetate buffer (100 mM, pH 4.5), sodium hydroxide

(50 mM), acetic acid (100 mM) and alkaline borohydride solution (10 mg/mL of sodium

borohydride in 50 mM sodium hydroxide). The p-hydroxybenzoic acid hydrazide

(PAHBAH) reducing sugar assay reagent was prepared by mixing 2 solutions

immediately before use. Solution A consisted in 10 g PAHBAH (Sigma Cat. No. H-9882,

Sigma Chemical Co. St. Louis, MO 63178, USA) and 10 ml concentrated HCl diluted to

200 ml with distilled water and stored at room temperature (ca 22°C). Solution B was

prepared by dissolving 24.9 g of trisodium citrate dihydrate, 2.20 g of calcium chloride

dihydrate and 40.0 g sodium hydroxide into 2 l of distilled water stored at room

temperature.

30

Briefly, the method consisted of extracting the inulin from the food products with

hot water, followed by a treatment with sucrase and with a mixture of starch-degrading

enzymes. The sugars were then reduced to sugar alcohols with alkaline borohydride.

Finally, inulin was hydrolyzed to fructose with purified fructanase (exo-inulinase plus

endo-inulinase), The reducing sugars were measured with a spectrophotometer (Beckman

Coulter DU 800) at 410 nm after reaction with para-hydroxybenzoic acid hydrazide.

(PAHBAH).

Inulin was quantitated against a 5 point calibration curve constructed from

standards (ORAFTI, Pennsylvania, USA) ranging from 0.3 to 30 mg/ml. Total inulin

content was determined by subtracting the fructan value obtained for the control from the

fructan value of the spiked sample.

31

Figure 10. Flow diagram of the extraction procedure and analysis of inulin from

the food matrices.

Accurately weigh

1.0±0.05 g of a test

portion.

Add hot distilled

water at 80°C; stir

80°C for 15 min.

Cool solution to

room temperature,

adjust volume to 100

ml with distilled

water, and mix.

Centrifuge at 1000 g

for 20 min ,or until

pellet is formed.

Filter aliquot of

solution through

paper Whatman No.

1, or equivalent.

Transfer 0.2 ml of

filtrate.

Add 0.2 ml of

sucrase/amylase

solution, and

incubate at 40°C for

30 min.

Add 0.2 ml alkaline

borohydride solution

to each tube and


30 min.

Add 0.5 ml acetic

acid and mix.

Transfer four 0.2 ml

aliquots.

Add 0.1 mL of

fructanase to 3 of

these samples.

Add 0.1 ml of 0.1 M

sodium acetate

buffer to the 4th

sample

(sample blank).

Incubate at 40°C for

20 min.

Add 5.0 ml

PAHBAH working

reagent.

Incubate in boiling

water bath for 6 min.

Remove all tubes from

boiling water bath and

immediately place in

cold water (18–20°C)

for ca 5 min.

Measure absorbance

of all solutions at

410 nm.

32

C.1.3 GOS Analysis

GOS was extracted and analyzed by the AACC method 32-25 (AACC Int 1994)

with some modifications, as shown in Figure 11. Residues of GOS were determined as

alditol acetates by gas liquid chromatography as described by Courtin et al. (2000). In

brief, samples were hydrolyzed with 2.8 M sulfuric acid into galactose and glucose,

followed by a reduction to sugar alcohols with alkaline borohydride (150 mg/mL of

sodium borohydride in 3 M ammonium hydroxide) and a final acetylation to alditol

acetates (Courtin et al. 2000). The derivatized sugars (1.0 µl) were resolved on an Elite

225 column (Perkin Elmer N9316177, 30 m x 0.25 mm x 0.25 µm film) interfaced to a

gas chromatograph (Agilent Technologies 7820A) with splitter injection port (split ratio

1:20) and flame ionization detector. The carrier gas was helium while the separation and

detection temperatures were 220 °C and 240 °C, respectively.

Correction factors (CFms), accounting for sugar losses during hydrolysis and

derivatization and for different GC responses were calculated for each individual

monosaccharide relative to an internal standard (myo-inositol) using the following

correction factor obtained from known standards (glucose, galactose).

Where Ams = peak area for individual monosaccharide, Astd = peak area for

internal standard (myo-inositol), Wms = weight (milligrams) of monosaccharide in the

sample, and Wstd = weight (milligrams) of internal standard in the solution.

Levels of galactose and glucose as anhydrosugars (AS) from GOS were

calculated from the equation:

33

Where Fm = recalculation factor for individual monosaccharides to

polysaccharide residues (0.90 for hexoses), and S = weight (mg dry matter) of original

sample.

Furthermore GOS in the food samples was calculated based only on the amount

of galactose recovered. Percent of galactose derived from GOS was calculated by

analyzing standards of known concentrations with the same procedure as for the samples.

Finally to determine the presence of free galactose in the sample, and/or if degradation of

GOS occurred during processing, the content of free galactose in both the control and

supplemented (1% GOS) samples was quantified by reducing the monosaccharides

present in the samples prior to the hydrolysis.

34

Figure 11. Flow diagram of the extraction procedure and analysis of GOS from

the food matrices.

Prepare a solution galactose and glucose

and the internal standard to (myo-inositol) each at

1mg/ml (STD).

Prepare a solution of myo-inositol at the same

concentration of the expected concentration of

the sugar in sample (ISTD)

Accurately weigh a of a test portion and combine with to achive 1mg/ml GOS concentration .

Mix 30 min at room temp

Centrifuge to obtain pellet at room.

Transfer 250 µl of sample (S) to a 35 ml

open test tube with screw cap.

Add 50 µl of 2.8M

sulfuric acid and 50 µl

of ISTD to sample (S).

Hydrolyze: Pressure cook

in High (15 psi and

80 °C for 1 h).

Add 75 µl of 12 M

ammonium hydroxide and

mix.

Add 40 µl of freshly

prepared 150 mg/ml

alkaline sodium

borohydride.

Incubate at 40 °C for 1 h.

Add 40 µl of glacial

acetic acid; mix.

Add 0.5 ml of 1-

methylimidazole; mix.

Add 5 ml of acetic

anhydride (slowly); mix.

Let stand 10 min at room

temperature.

Add 1 ml absolute

ethanol.


temperature.

Move tubes to a cooler

with ice up to shoulder of

tube

Slowly add 5 ml of well

mixed 7.5 M sodium

hydroxide; mix.


temperature.

Add another 5 ml of 7.5

M sodium hydroxide.

Transfer ethyl acetate

(top) layer to a fresh tube. Analyze by GC.

Analyze the STD at as the first and last injection of

the runs.

35

C.2 Specific Aim 2: Method Validation

Method validation was performed according to the statistical design presented in

the United States Pharmacopia and the AOAC Peer verified methods: Manual for policies

and procedures (AOAC 1998) based upon accuracy, precision, linearity of the standard

curve, limit of detection, limit of quantitation, and specificity / selectivity.

C.2.1 Accuracy

Processed finished products that had been prepared without a given prebiotic were

extracted / analyzed via the cited extraction / analysis methods (S1), which served as the

control. These samples were spiked with the prebiotic at known concentrations above and

below and at the target levels expected in the treated products (S2). The products were

again extracted and analyzed based upon the optimized analysis methodologies. Method

accuracy was thus be accessed by determining % recoveries as follows:

% Recovery = (Conc. S2 –Conc. S1/Known increment conc) X 100.

C.2.2 Precision

The relative standard deviation of individual results (% RSD) was determined by

analyzing replicate samples (n = 5) prepared according to the conditions of the tests

(Specific Aim 1).

C.2.3 Linearity of Calibration Curves

Standards with different but known concentrations (4-5 different concentrations)

were analyzed in triplicate and the response was correlated vs. concentration. The

regression curve y= mx + b, was calculated by the method of least squares of the standard

responses vs. concentration, where “m” is the slope of the line and “y” is the y intercept.

36

The correlation coefficient (R) was determined to access degree of linearity (AOAC

1998).

C.2.4 Limit of Detection

Limits of detection were determined by calculating the mean value of the matrix

blank response (n ≥10) times 3 standard deviation of the mean.

C.2.5 Limit of Quantitation

Limits of quantitation will be determined by calculating the mean value of the

matrix blank response times (n ≥10) times 10 standard deviation of the mean.

C.2.6 Specificity / Selectivity

Matrix blanks were analyzed to ensure that no interfering compounds were

present or that a given carbohydrate was not indistinguishable from the corresponding

standard material in the appropriate matrix.

C.3 Specific Aim 3: Analysis of 1% prebiotic supplemented

food products

Based upon the results generated in Specific Aims 1 and 2, the extraction and

analysis procedures were applied to the final products (Table 1-5) containing 1% of the

prebiotic in the initial formulation. Percent recovery of the different formulated

prototypes was determined and corrected based on the recovery from the validation

studies, i.e., if % recoveries of a given method were low a corresponding correction

factor was applied to the test results.

37

C.4 Specific aim 4: To assess the chemical fate of FOS, GOS

and inulin during various processing treatments

The chemical fate of FOS, inulin and GOS was determined by applying the

validated extraction and analytical methods in the sports drink and extruded ready-to-eat

cereal prepared under different processing conditions as described in the next sections.

Means of the prebiotic content were calculated with ANOVA tests followed by the Tukey

HSD test set at a 5% level of significance.

C.4.1 Chemical Fate of FOS, Inulin and GOS in Sports Drink

The pasteurized sports drink was prepared according to the formulation shown in

Table 5 and Figure 12. All ingredients were mixed with distilled water to a final volume

of 1 l. A pH of 3.5 was adjusted accordingly for each batch using citric acid. The mixed

ingredients were then stirred and heated to a minimum temperature of 175 ˚C using a stir/

hot plate. The drink product was hot-filled into PET bottles and allowed to cool. Upon

cooling, the final sports drink products were stored at ambient temperatures prior to

analysis.

To determine the effects of pH and sweetener composition on prebiotic stability,

the following treatments were implemented: 1) varied sucrose: Corn syrup solids

(DE=60) ratios (1:2, 1:1, 2:1), and 2) pH values 3.0, 3.5, and 4.0. The experiment was

completely randomized as a 23

factorial with a split plot design. Each trial held at constant

sweetener ratio while adjusting pH to 3.0, 3.5 and 4.0. Figure 12 shows the 23 factorial

design.

38

C.4.2 Chemical Fate of FOS, Inulin, and GOS in Extruded ready-to-eat

Breakfast Cereal

Extruded ready-to-eat breakfast cereal samples were prepared using different

screw speed (120, 170, and 220 rpm) and temperature (110, 140, and 170 ˚C) as provided

by Dr. Randy Wehling (Department of Food Science and Technology, University of

Nebraska Lincoln). A conical twin screw laboratory extruder (C.W. Brabender Model

2003 GR-8) with a barrel diameter of 1.9 cm and a length: diameter ratio of 20:1 was

used for the extrusion process. The mix was equilibrated overnight with appropriate

additions of distilled water to obtain a final moisture content of 17% prior to extrusion.

Trials run were conducted to determine optimum feed-mix moisture content, barrel

temperature, and screw speed for cereal model expansion. The screw speed (170 rpm)

and barrel temperature (140 °C) combination that provided optimum expansion was

selected. A complete randomized design with varying two additional screw speeds (120

rpm and 220 rpm) and two additional temperatures (120 °C and 170 °C) were conducted

while holding the other parameter constant at the optimum value (Figure 13).

39

Figure 12 . 23

factorial design applied to the pasteurized sports drink.

Figure 13. Different variables of screw speeds and temperatures while

maintaining other extrusion parameters.

40

D. RESULTS AND DISCUSSION

Methods for extracting and quantitating inulin, FOS and GOS from different

processed food products were developed by spiking known amounts (0.5, 1 and 2%) of a

given prebiotic into the product base formula (without supplemented prebiotic) and

determining the amount recovered. This approach is not typically applied to food based

ingredients and thus has not been reported for method development of any prebiotic

supplemented food. Method performance was then validated based on precision,

linearity, detection limit, quantitation limits and accuracy. Moreover the validated

methods for each prebiotic were applied to the food matrices prepared with 1% of the

prebiotic under standard unit preparation procedures. Finally the chemical stability of

prebiotics in terms of the total supplemented levels was evaluated for the breakfast cereal

and the sports drink when prepared under different processing conditions.

D.1 Specific aim 1: Method Development or Adaptation

D.1.1 FOS

FOS were initially extracted according to the steps shown in Figure 9. This

method was selected based on excellent separation, precise quantititation and high

percent recoveries of the FOS standards, as completed in our lab. Furthermore, separation

was achieved under isocratic conditions in aproximately 30 min. and often fitration was

the only sample clean-up step needed prior to HPLC analysis. Nevertheless, further

adjustments to the extraction method were needed for some of the food matrices to

improve the overall accuracy (Table 6).

41

Because the extruded breakfast cereal had a high water absorption capacity, most

likely due to the high content of soluble fibers and starch present in the oat and corn

flours (Delcour 2010); this characteristic precluded the use of water as the only extraction

solvent. FOS was thus extracted from the breakfast cereal using a mixture of water and

ethanol at a 1:1 (v/v). A similar approach but with 25% boiling ethanol was successfully

used to extract FOS from bread (Mujoo et al. 2003). After extraction, the samples were

placed under vacuum until completely dried. Finally, the samples were re-dissolved to 1

ml of nanopure water to achieve a FOS concentration (mg/ml) above the detection and

quantitation limits of the method. Alternatively, the muffins exhibited low water

absorption and 2 ml of water was sufficient to achieve percent recoveries of ~100%.

A second extraction was needed for 1% spiked cookie to completely extract FOS.

It was hypothesized that because the cookies contained high levels of sucrose (27 mg per

gram of cookie), which is highly soluble in water (IPCS 1999), the extraction solvent was

saturated by this simple sugar preventing complete extraction of the prebiotic in a single

step approach. This hypothesis was confirmed by the higher percent recoveries obtained

from an additional extraction (<80-100%). In the case of the nutritional bar, the

ingredients, such as roasted chopped peanuts and whole rolled oats, together with the

higher viscosity of honey and peanut butter prevented homogeneous distribution of FOS

in the matrix (Table 4). A larger amount of test portion (10 g) combined with a water 1:2

(w/v) ratio was required to achieve suitable FOS recoveries (66-113%). Finally, the

sports drink did not require any extraction procedures and only limited sample handling

procedures, which consisted of passing the drink through a 0.45 µm syringe filter to

42

remove large particles prior to HPLC analysis. Table 6 summarizes the optimization

conditions for the FOS spiked food products.

D.1.2 Inulin

Inulin was initially extracted and analyzed with the official AACC method 32-32

as shown in Figure 10. This method was chosen because it is widely used due its

simplicity and economy. Additionally, it can be used to measure fructans in a wide range

of plant and food materials (AACC Int 2001; Wang et al. 2010). However, food product

specific adjustments to the original extraction steps (Figure 10) were needed to improve

method performance (Table 7). For example, according to the official method, total

fructan content is calculated by measuring free fructose. In brief, it compares the

absorbance of the test sample with the absorbance from the reaction of a fructose

standard (54.4 µg) with the coloring agent PAHBAH. Using a conversion factor, the

amount of free fructose is then converted to anhydrofructose as occurs in fructan. For

this project, the use of a calibration curve was the preferred method to determine the

concentration of inulin in the food samples. A set of inulin standards were used to

construct the curve and the response of the test sample was then interpolated to determine

the concentration. The latter proved to be a reliable approach, as the curve was linear

within the working range of the spiked sample and had a strong correlation (r>0.99)

allowing the use of inulin standards for the remaining experiments. Furthermore, overall

precision of the method performance improved (< 75-100%) when 50 ml of water were

used as the extraction solvent instead of the 100 ml indicated in the method’s original

protocol (data not shown).

43

One important characteristic of this method is that the color response is the same

for fructose and glucose (AACC Int 2001). Hence, the response obtained from a

product’s base formula (control) had to be subtracted from the response obtained from

the spiked samples. The high sucrose content in the cookie (Table 2) probably

overwhelmed the capacity of the coloring agent (PAHBAH) thus affecting the ability of

the method to differentiate fructose from the inulin from glucose and fructose derived

from sucrose. A possible resolution of this problem would be to increase the incubation

time with sodium borohydride to ensure that the monosaccharides resulting from the

sucrase incubation step are completely reduced to sugar alcohols, thereby preventing any

non-selective interaction with the PAHBAH coloring agent. Wang et al. (2010) also

concluded that the colorimetric method is prone to interfering compounds such as

glucans, hexoses and sucrose resulting in inaccuracies and overestimation. The authors

proposed the use of an HPLC method to address this issue; however, sample preparation

and analysis were more laborious. The colorimetric method was still preferred due to

effective preliminary results. In addition a large number of samples could be analyzed in

a shorter time, as was required for this project.

44

Table 6. Optimization conditions for extracting FOS in the different food products

Food Product

Amount of

sub-sample analyzed

Extraction Solvent

Further procedures

for extraction

optimization.

Extruded cereal

10.00 ± 0.05 g

20.0 ± 0.1 ml of 1:1

water: ethanol

Concentrated to a

final volume of 1 ml.

Muffins

1.00 ± 0.05 g

2.0 ± 0.1 ml of water

-

Cookies

10.00 ± 0.05


-Pellet extracted an

additional time with

10 ml of water.

Nutrition bar

10.00 ± 0.05 g


Concentrated to a

final volume of 1 ml.

Sports drink

1.00 ± 0.05 ml

No extraction needed.

-

Table 6. Optimization conditions for extracting inulin in the different food products

Food

Product

Amount of sub-

sample analyzed

Volume

of extraction solvent

(water) indicated by

the protocol

Volume

of extraction solvent

used for assay

optimization.

Extruded cereal 1.00 ± 0.05 g 100 ± 0.1 ml 50 ± 0.1 ml

Muffins

1.00 ± 0.05 g

100 ± 0.1 ml

50 ± 0.1 ml

Cookies

1.00 ± 0.05 g

100 ± 0.1 ml

Optimization was not

achieved.

Nutrition bar

1.00 ± 0.05 g

100 ± 0.1 ml

50 ± 0.1 ml

Sports drink

1.00 ± 0.05 ml

100 ± 0.1 ml

50 ± 0.1 ml

45

D.1.3 GOS

The official AOAC method for GOS suffers from poor selectivity due to

interfering compounds present in the matrix, especially when measuring small quantities.

In order to accurately analyze GOS in the complex processed food products, a gas-

chromatography (GC) approach was selected. An example of a GC-chromatogram of

galactose, fructose, and myo-inositol is shown in Figure 14. Additionally, chromatograms

are also presented of the controls and spiked samples for the extruded cereal and sports

drink. It must be noted that in the control formula the galactose peak (ret time ~10 min) is

not present (Figures 15, 17) whereas it appears in the 1% GOS supplemented samples

(Figures 16, 18).

Courtin et al. (2000) had effectively used this method to measure and

characterize polysacharides but it has has never been adapted for GOS standards or

supplemented processed foods. Therefore, the GOS extraction approach was developed

as it applied to extruded product and sports drink only, i.e., the products subjected to

several different processing parameters (Specific Aim 4, refer to Section D.4).

As shown in Table 7, a one-time extraction of 0.5 g of sample for 30 minutes and

50 ml of water as the solvent, followed by centrifugation was sufficient to obtain 98% of

the spiked GOS from the breakfast cereal. To optimize the extraction steps, the samples

had to be mixed immediately after adding water to prevent gel formation. No additional

extraction steps was needed for the sports drink to recover 91% of the spiked GOS;

however, samples were diluted 10 fold with water to stay within the linear range of the

calibration curve.

46

Table 7. Optimization conditions for extracting GOS from sports drink and

breakfast cereal

Food Product

Amount of sub-

sample analyzed

Extraction Solvent

(water)

Further procedures

for extraction

optimization

Extruded cereal

0.50 ± 0.05 g

50.0 ± 0.1 ml

Mix immediately after

adding water to

prevent gel formation.

Mix for 30 min,

followed by

centrifugation at 4500

rpm for 20 min

Sports drink 1.00 ±.05 ml 10.0 ± 0.1 ml Mix, no extraction

needed.

Figure 12. GC chromatogram of GOS (1mg/ml). Peaks: galactose (ret time 10.12

min), glucose (ret time 10.83 min) myo-inositol (11.26 min).

47

Figure 13. GC chromatogram of sports drink control. Peaks: fructose (ret time

9.473 min), glucose (11.31 min) myo-inositol (11.75 min).

Figure 14. GC chromatogram of sports drink spiked with 1% GOS. Peaks:

Fructose (ret time 9.495 min), galactose (10.32 min), glucose (11.21 min), myo-Inositol

(11.26 min).

48

Figure 15. GC chromatogram of breakfast cereal control. Peaks: Fructose (ret

time 9.19 min), glucose (10.78 min), myo-inositol (11.23 min)

Figure 16. GC chromatogram of breakfast cereal spiked with 1% GOS. Peaks

fructose (ret time 9.264 min), galactose (10.05 min), glucose (ret time 10.82 min) myo-

inositol (11.308 min).

49

D.2 Specific Aim 2: Method Validation

Selective and sensitive analytical methods for the quantitative evaluation of

prebiotics in different food matrices are critical to evaluate their stability during different

food preparation procedures. Therefore, the analytical methods optimized in Specific

Aim 1 were validated to ensure reliable and reproducible measurement of each prebiotic

in each food matrix. Method validation included accuracy, precision, linearity, limit of

detection, limit of quantitation and selectivity (AOAC, 1998).

D.2.1 FOS

Linearity of the FOS assay was evaluated with each food product to confirm the

performance of the method and the equipment. FOS standards ranging from 0.25-7.5

mg/ml resulted in consistently linear calibration curves with strong correlations (r > 0.90)

for kestose, nystose and 1-fructofuranosyl nystose (Table 9). These calibration curves

were then used to determine limits of detection and quantitiation. The results showed that

both parameters were lower than 1% for all the food products. The sports drink exhibited

the lowest limits of detection and quantitation at 0.008% and 0.014%, respectively. On

the other hand, the remaining food products produced detection limits from 0.14-0.21%

and quantitation limits from 0.43- 0.75%.

Effects from differences in food matrices were predominantly reflected in method

accuracy and precision. Method precision was determined by calculating the percent

relative standard deviation (% RSD) of spiked samples (n=5) below (0.5%) and above

(2%) the target amount to be supplemented in the food products (1%) (Table 9). The

sports drink showed the highest precision (1-4%) while the breakfast cereal exhibited the

lowest precision (18-27%). The complexity of the matrix and the presence of interfering

50

compounds in the breakfast cereal, as opposed to a liquid matrix requiring minimum

clean-up and handling steps, might have contributed to the differences in precision

between the breakfast cereal and the sports drink. Moreover, variability decreased as the

% prebiotic increased for all the food products with the notable exception of the cookie.

In this case, the lowest %RSD (2%) occurred for the 0.5% spiked prebiotic sample as

opposed to a %RSD of 19% when spiked with 2% prebiotic. The high sucrose content in

the cookie probably increased the amount of solutes in the extraction solvent allowing

lower amounts of prebiotic (i.e. 0.5%) to be extracted and dissolved. Higher amounts

(i.e. 2%) might have caused the solvent to reach or to be close to the saturation point

resulting in partial extractions of the prebiotic. One possible solution would be to increase

the volume and/or the temperature of solvent to prevent saturation. Moreover, method

accuracy, as determined by percent recoveries of spiked prebiotic in the control samples,

was between 80 and 100% for the cookie, sports drink and muffin. For food products

with less than 90% recovery, as is the case of breakfast cereal and nutritional bar, a

correction factor was applied to the final results when analyzing prebiotic supplemented

food products after processing.

51

Table 8. Method performance characteristics for FOS in different food products

Breakfast

cereal Cookie Muffin Nutrition

bar Sports drink Linearity (regression analysis

Kestose : 0.25 - 7.5 mg/ml(n=3)

y = 103688x -26321

r=0.9996

y = 258152x

+186097

r=0.9753

y = 272931x

+80203

r=0.9970

y = 285392x

+128321

r2=0.9998

y = 3123539x

+ 16253 r= 0.9999

Nystose: 0.25 - 7.5 mg/ml (n=3)

y = 136427x - 36158

r = 0.9996

y = 273142x

– 112449 r = 0.9973

y = 272931x

+ 80203 r = 0.9976

y = 273098x

+ 48825 r = 0.9987

y= 2722490x

+ 21160 r= 0.9999

1-fructofuranosyl

nystose: 0.25-7.5 mg/ml

(n=3)

y = 32660x - 1093.5

r = 0.9988

y = 272058x

– 60167 r = 0.9751

y = 268210x - 6532.1

r = 0.9973

y = 276210x

– 11172 r = 0.9986

y= 2615410x

+ 28631 r² = 0.9995

Precision (%RSD)

Standard:0.5% (n=5) 26.98 2.37 38.70 24.15 3.88 Standard: 1% (n=5) 20.25 22.80 16.64 8.16 3.88 Standard: 2% (n=5) 18.44 18.88 3.85 8.27 0.73 Detection limit (%)

Kestose

0.046 0.050 0.003 0.048 0.001

Nystose 0.078 0.090 0.000 0.000 0.003

1-fructofuranosyl nystose 0.088 0.050 0.143 0.093 0.004

Total FOS 0.213 0.190 0.146 0.141 0.008

Quantitation limit (%)

Kestose 0.090 0.270 0.140 0.274 0.003

Nystose 0.150 0.104 0.000 0.000 0.006

1-fructofuranosyl nystose 0.230 0.058 0.560 0.320 0.005

Total FOS 0.467 0.432 0.700 0.594 0.014 Accuracy/specificity

(%recovery) Standard:0.5%(n=5) 106.99 92.02 107.53 90.98 100.62 Standard: 1% (n=5) 81.62 103.7 103.41 82.07 102.19

Standard: 2% (n=5) 84.01 86.75 100.65 79.41 94.08

52

D.2.2 Inulin

As shown in Table 10, method linearity of the inulin assay was evaluated by

constructing a 5 point calibration curve with standards ranging from 3-30 mg/ml.

Calibration curves were re-evaluated with each food product analysis generating

regression lines with strong correlation coefficients (r > 0.99). In addition, method

precision was determined by again analyzing the %RSD of the control samples (n=5)

spiked with 0.5%, 1%, and 2% inulin (w/v). For the 1% spiked samples, %RSD ranged

between 5-18% for all the products. The sports drink produced the lowest %RSD (3-8%)

whereas %RSD were the highest for the cookies (5-32%) and breakfast cereal (7-30%).

The lowest limit of detection (0.14%) resulted for sports drink while the

nutritional bar exhibited the highest limit of detection (2.55%). A similar trend occurred

for the limit of quantification with the lowest values produced for the sports drink

(0.24%) and the highest for the nutrition bar (4.64%). With the exception of sports drink,

both limits of detection and quantitation were over the target amount of prebiotic to be

supplemented in the food products (1%). However, this problem was addressed during

the method optimization as the matrix response was taken into account when calculating

the amount of inulin present in the samples (Table 6). Because the percent recoveries

were above 110% for both the spiked breakfast cereal and the muffin, a correction factor

was applied when calculating the amount of inulin present in the supplemented products

after processing.

53

Table 9. Method performance characteristics for inulin in different food products

Breakfast

cereal Cookie Muffin Nutrition

bar Sports drink Linearity (regression

analysis

Inulin:3-30 mg/ml (n=5)

y = 0.0149x

+ 0.0013 r = 0.9989

y= 0.0081x

+ 0.0044 r = 0.9994

y = 0.0157x

+0.0058 r =0.9994

y = 0.0157x

+0.0058 r = 0.9994

y = 0.0148x

- 0.0001 r² = 0.9994

Precision (RSD % )

Standard: 0.5% (n=5) 30.18 31.63 10.70 25.24 3.08

Standard: 1% (n=5) 14.74 4.97 16.88 9.06 5.84

Standard: 2% (n=5) 7.19 29.49 8.66 16.34 8.35

Detection limit (%) 1.44 2.14 1.20 2.55 0.144

Quantitation limit (%) 4.43 3.50 2.42 4.65 0.42

Accuracy/specificity

(%recovery) Standard: 0.5% (n=5) 107.20 125.41 160.09 103.31 106.65

Standard: 1% (n=5) 115.20 99.04 121.20 107.48 103.60

Standard: 2% (n=5) 117.43 109.45 104.79 113.30 99.49

54

D.2.3 GOS

The linearity of the assay and r values of the GOS method were confirmed by

constructing 5 point calibration curves with galactose, glucose, and myo-inositol each

ranging in concentration from 1-20 mg/ml (Table 11). Linear regions were obtained for

each standard at the cited concentration range with coefficient correlations greater than

0.99.

Additionally, overall method precision was high as indicated by low %RSD

values for samples (n=5) spiked with each of three levels of GOS (0.5%, 1%, and 2%

(w/w), and (w/v) in the sports drink. Method precision for the sport drink at 1% GOS

was 2%, whereas a %RSD of 7% and 6% resulted for the 0.5% and 2% GOS spiked

drink, respectively. Likewise the breakfast cereal exhibited a %RSD of 8% for the 0.5%

and 1% spiked base formula with a slightly lower %RSD (5%) for the 2% GOS spiked

sample.

Limits of detection for GOS were 0.024% in the sports drink and 0.032% in the

breakfast cereal. The lowest quantitatable percent of GOS was 0.024% for the sports

drink and 0.093% for the breakfast cereal. As these values were below the amount of

prebiotic supplemented in the food matrices, the method was considered suitable to apply

to the supplemented food systems. Furthermore, the method presented high percent

recoveries for both the breakfast cereal (98%) and sports drink (91%) indicating a high

accuracy and specificity of the assay. A summary of all the method performance results

are shown in Table 11.

55

Table 11. Method performance characteristics for GOS in different food products

Breakfast cereal Sports drink

Linearity (regression analysis

Galactose: 1- 20 mg/ml (n=3) y = 361610x - 30357

r = 0.9985

y = 361610x – 30357

r = 0.9985

Glucose 1- 20 mg/ml (n=3) y = 306002x + 59647

r = 0.9992

y = 306002x + 59647

r = 0.9992

myo-inositol: 1- 20 mg/ml (n=3) y = 461702x - 61489

r = 0.9984

y = 461702x - 61489

r = 0.9998

Precision (RSD % )

Standard: 0.5% (n=5) 7.68 6.99 Standard: 1% (n=5) 8.32 1.63 Standard: 2% (n=5) 4.7 6.45

Detection limit (%)

GOS 0.032 0.024

Quantitation limit(%) 0.093 0.024 GOS

Accuracy/specificity (%recovery)

Standard: 0.5% (n=5) 132.60 86.68 Standard: 1% (n=5) 97.71 91.42 Standard: 2% (n=5) 101.54 91.59

56

D.3 Specific Aim 3: Analysis of 1% prebiotic supplemented

food products

Published data remain limited about the stability of prebiotics exposed to different

food processes such as baking, extrusion, pasteurization, high temperature heating, low

pH condition, etc., despite the food industry’s interest in supplement products. The

objective of Specific Aim 3 was to determine the prebiotic content after processing food

products supplemented with 1% (w/w) FOS, inulin, and GOS. The optimized and

validated method for each matrix (Specific Aim 1-2) was then applied to the

supplemented prototype foods. The muffin was prepared and kept in the freezer at -20 ˚C

whereas the extruded cereal, cookie, and nutritional bar were prepared and maintained at

ambient temperature prior to analysis. The sports drink, with equal amounts of sucrose

and corn syrup solids, was adjusted to pH 3.5, pasteurized at 79 ˚C and stored at room

temperature.

D.3.1 FOS

As shown in Table 12 and Figure 19, recoveries were 100%, 91% and 113% for

the supplemented muffin, cookie and nutrition bar, respectively. For the breakfast cereal,

only 47% of the supplemented FOS remained after extrusion at optimal conditions (170

rpm and 140 °C). Similarly, only 54% of the FOS supplemented into the sports drink was

recovered for a formulation pH of 3.5 and a pasteurization temperature of 79 ˚C.

According to L’Homme et al. (2003) and Voragen (1998), FOS are unstable at elevated

temperatures and low pH, which may have contributed to the low recoveries for the

cereal and sports drink. Extruded materials are also exposed to intense mechanical shear

and high pressure in addition to high temperature (Bjorck et al. 1984). Redistribution

57

of soluble and insoluble fiber occurs during extrusion of whole grains, meaning structural

changes occur at the molecular level (Bjorck et al., 1984; Guadalberto & Kazemzadeh

1997); this effect may have also contributed to the degradation FOS in the breakfast

cereal.

D.3.2 Inulin

Percent recoveries of inulin were determined by subtracting the fructan content in

the base formula and applying the corresponding correction factor. Recoveries of 106%,

103% and 107% and 126% were obtained for the supplemented extruded cereal, nutrition

bar, sports drink and muffins, respectively (Table 13 and Figure 20). On the other hand,

the percent recovery of inulin in the cookies was approximately 300%. This outcome

was expected as the optimization of the method was not achieved most likely due the

high sucrose content in the matrix.

D.3.3 GOS

Stability of the GOS was assessed by applying the validated method to the

extruded cereal and sports drink. As shown in Table 14 and Figure 19, percent recoveries

for the extruded cereal and the sports drink were ~ 100% indicating that the cited process

parameters used did not affect GOS stability.

58

Table 10. Percent recoveries of 1% FOS supplemented food products after

processing.

FOS content

Sample Before

correction factor

After

correction factor

% Recovered

Extruded cereal

0.39 ± 0.01

0.47 ± 0.01

47%

Muffins 0.91 ± 0.07 - 91%

Cookie 1.01± 0.12 - 100%

Nutrition bar 0.94 ± 0.05 1.13 ± 0.63 113%

Sports Drink 0.54 ±0.08 - 54%

Results shown as MEAN ± STD (n=3)

Figure 17. Percent recovery of 1% supplemented FOS in different prototype foods after

processing. Results shown as MEAN ± STD (n=3).

59

Table 11. Percent recoveries of 1% inulin supplemented food products after processing.

Sample

Inulin content

Base

formula

Supplemented

with 1% Inulin

Supplemented

minus base

formula

After applying

correction

factor

%

Recovered

Extruded

cereal

1.03 ± 0.12

2.22 ± 0.106

1.25 ± 0.04

1.62 ± 0.03

106%

Muffins 1.07 ± 0.31 2.65 ± 0.35 1.58 ± 044 1.26 ±0.35 126%

Cookies 1.51 ± 0.31 4.64 ± 1.10 3.13 ± 1.31 - 300%

Nutrition Bar 2.03 ± 0.26 3.06 ± 0.09 1.04 ± 0.09 - 103%

Sports Drink 0.052 ± 0.004 11.24 ± 0.79 1.07 ± 0.08 - 107%

Results shown as MEAN ± STD (n=3)

Figure 18. Percent recovery of inulin after processing of food products

supplemented of 1% prebiotic. Results shown as MEAN ± STD (n=3).

60

Table 12. Percent recoveries of 1% GOS supplemented food products after processing

Sample

GOS content

Supplemented with 1%

GOS

% Recovery

Extruded cereal

1.06 ± 0.034

106%

Sports drink

1.07 ±0.078

107% Results shown as MEAN ± STD (n=3)

Figure 19. Percent GOS after processing of food products supplemented of 1%

prebiotic. Results shown as MEAN ± STD (n=3).

61

D.4. Specific aim 4. Chemical fate of FOS, GOS and inulin

during various processing treatments

To further evaluate the chemical stability of prebiotics in the breakfast cereal,

different screw speeds and barrel temperatures were used during the extrusion.

Specifically, 120 °C and 170 °C were used for the low and high barrel temperatures; and

120 rpm and 220 rpm were used for the low and high screw speeds. (Refer to Figure 13

for d). Three trials for each prebiotic were conducted using a split plot design where one

optimal condition was held (i.e. barrel temperature or screw speed), while the other

condition was varied.

To determine the effects of pH and reducing sugar content on stability of the

prebiotics in sports drinks, various sweetener ratios of sucrose: corn syrup solids (1:1,

1:2, 2:1) were tested at different pH values (3.0, 3.4 and 4.0). The experiment was

completely randomized with a split-plot design, where the sweetener composition was

held and pH was varied for each trial (n=3).

D.4.1 Chemical fate of FOS in the breakfast cereal

As shown in Figure 20, extrusion in general reduced the levels of supplemented

FOS in the breakfast cereal. During optimum extruding conditions less than 50% of the

initial supplemented FOS was recovered. A similar tendency occurred regardless of the

extruding condition but the lowest FOS levels were recovered when extrusion was held at

the highest temperature (170 °C).

Several mechanisms could be involved in the degradation of FOS. For example,

the higher screw speed exposes the FOS to sheer stress. At lower screw speeds the

pressure within the extruder is increased due to higher screw fill, hence that product is

62

exposed to high temperature and pressure for a longer time, resulting in chemical bond

breakage of the macromolecules (Guadalberto et al. 1997). Furthermore, liability of FOS

to heat hydrolysis has been well demonstrated in previous studies (L'Homme et al. 2003;

Matusek et al. 2009; Kewicki 2007).

D.4.2 Chemical fate of inulin in breakfast cereal

Optimal and low temperature extruding conditions did not significantly affect

inulin levels in the supplemented breakfast cereal. However, variations in screw speeds

resulted in recoveries of 25% and 34% at 120 rpm and 170 rpm, respectively (Figure 23).

Additionally, low levels of inulin (35%) were recovered after high temperature (170 °C)

extruding conditions. Although Böhm et al. (2005) showed that thermal treatment of

inulin at temperatures between 135 and 195 °C can induce significant degradation, there

remains a significant lack of scientific information on the thermal stability of inulin

during extrusion.

D.4.3 Chemical fate of GOS in breakfast cereal

Based on the results obtained from this study, GOS was stable at all extrusion

conditions with 99% recoveries for the product processed under the optimal processing

conditions. As shown in recoveries of 115%, 105% and 102% occurred at high screw

speed, low screw speed and low barrel temperatures, respectively. The lowest levels

(81%) were recovered when the breakfast cereal was extruded at the highest temperature

but the results were not significantly different (p>0.05) from the optimal conditions.

63

Figure 20. FOS recovery in breakfast cereal supplemented with 1% FOS and

extruded under different screw and temperature conditions. Results shown as MEAN ±

STD (n=3). Bars with different letters are statistically different (p>0.05) using Tukey

HSD test.

64

Figure 23. Inulin recovery in breakfast cereal supplemented with 1% Inulin and

extruded under different screw and temperature conditions .Results shown as MEAN ±


HSD test.

65

Figure 24. GOS recovery in breakfast cereal supplemented with 1% GOS and

extruded under different screw and temperature conditions. Results shown as MEAN ±


HSD test.

66

D.4.4 Chemical fate of FOS in sports drink

FOS was more stable as the pH increased in the sports drink, regardless of the

sweeter composition (Figure 23). Although a percent recovery of 97% resulted for the

supplemented FOS at pH 4.0 and lower reducing sugar content (S:CSS 2:1), the results

were not significantly different (p>0.05) from the other sweetener ratios. At pH 3 and pH

3.5, recoveries of less than 70% independent of the sweetener composition were

obtained. Nevertheless, percent recovery (37%) was significantly (p<0.05) decreased at

pH 3 and equal amounts of sucrose and corn syrup solids. Previous studies have reported

low FOS stability at elevated temperatures and low pH (Blecker et al. 2002; L'Homme et

al. 2003; Matusek et al. 2009; Kewicki 2007), fructose furanosyl residues in the FOS are

believed to be prone to the acid hydrolysis (Voragen 1998).

D.4.5 Chemical fate of inulin in sports drink

Compared to FOS, inulin was more stable regardless of the pH or sweetener

composition (Figure 24.). The lowest inulin recovered (90%) resulted at pH 3 with added

sucrose and corn syrup solids at 1:1 ratio. This result was statistically different (p>0.05)

form those obtained for the pH 3.5 and pH 4.0 formulations at the same sweetener

compositions but no significant differences occurred for the other treatments. Christian

et al. (2000) reported degradation of inulin at high temperature (160 ᵒC) in the presence

of citric acid. According to L'Homme et al. (2003) hydrolisis is caused by the

protonation of oxygen in the glycosidic bond.

67

D.4.6 Chemical fate of GOS in sports drink

As shown in Figure 23, supplemented GOS was stable at all pH values and/or

sweetener conditions. Overall, GOS had the greatest stability in the sports drink among

the studied prebiotics with recoveries > 95% at pH 3 and pH 3.5. Lower levels were

recovered at pH 4 but were not significantly different (p>0.05) from the other treatments.

Stability of GOS at high temperatures and acid conditions has been evaluated and

confirmed by several authors. Kewicki (2007) reported that GOS had not undergone

hydrolysis at pH 3 with heating at 100 ᵒC for 10 min, and only low losses occurred (5%)

when the pH was dropped to 2.0. The presence of the different β-bonds was attributed to

GOS thermostability at low pH (Voragen 1998). Hence, the results obtained from this

study are in very good agreement with the literature.

68

Figure 21. FOS recovery in sports drink supplemented with 1% FOS prepared

and under different pH and sweetener ratios. Results shown as MEAN ± STD (n=3). Bars

with different letters are statistically different (p>0.05) using Tukey HSD test.

69

Figure 22. Inulin recovery in sports drink supplemented with 1% inulin and

prepared under different pH and sweetener ratios. Results shown as MEAN ± STD (n=3).

Bars with different letters are statistically different (p>0.05) using Tukey HSD test.

70

Figure 23. GOS recovery in sports drink supplemented with 1% GOS and

prepared under different pH and sweetener ratios. Results shown as MEAN ± STD (n=3).

Bars with different letters are statistically different (p>0.05) using Tukey HSD test

71

E. CONCLUSIONS

There is an increasing interest in the use of prebiotics as food ingredients capable

of modifying the intestinal microbiota as a means to confer beneficial health effects.

Therefore, it is essential that prebiotics remain stable under typical food manufacturing

and processing conditions. In this project, methods for extracting and analyzing 3

commonly studied prebiotics (inulin, FOS and GOS) were initially developed and

validated for different processed prototype food products. Depending on the complexity

of the food matrix, optimized extraction procedures were required. When the method

accuracy was below 90% or above 110%, correction factors were applied for the analysis

of some supplement products. Method accuracy was determined via a spiked recovery

approach, which is not a typical step for food based ingredients. Results indicated that

method’s precision (%RSD) decreased as the matrix complexity increased but linear

calibration curves with strong correlation coefficients (r > 0.90) were generated for each

method.

Optimized methods for monitoring FOS and GOS presented low detection and

quantitation limits, resulting in reliable techniques to analyze low amounts of the

prebiotics in the presence of complex matrices. On the other hand the enzymatic-

spectrophotometric method to analyze inulin presented high detection and quantitation

limits and accuracy was affected by compounds present in the food matrix. Hence,

additional research is needed to develop a robust and high throughput method to monitor

inulin in the presence of complex food matrices.

72

Finally, degradation of FOS and inulin must be considered when added as a

prebiotic ingredient into thermally treated foods, especially in presence of acid. On the

other hand, this study showed that GOS was stable to high temperature and low pH.

73

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A

Appendix 1

Flow diagrams of the optimized extraction procedure and analysis of

FOS from the different food products

B

Figure 1. Flow diagram of the optimized extraction procedure and analysis of

FOS from the in the extruded cereal

Accuratly weigh 10 ± 0.05 g and combine with 20 ml of 1:1

ethanol :water



30 min at room temperatute. Filter supernatant to remove

large particles.


filter.




C


FOS from the muffins

Accurately weigh 1 ± 0.05 g and combine with 2 ml ± 0.1 of

water Mix 30 min at room temperature.

Centrifuge at 10000 g for 20-30

min at room temperatute. Filter supernatant to remove large

particles.

Collect filtrate and pass an aliquot through a 0.45 µm filter.

Analyze with HPLC.

D


FOS from the cookies.

Accurately weigh 10 ± 0.05 g and combine with 20 ± 0.1 ml

of water



30 min at room temperatute.

Collect supernatant. Add 10 ± 0.1 ml of water to the pellet

and mix 30 min at room temperature

Filter supernatants to remove

large particles.

Collect filtrates eand pass an aliquot through a 0.45 µm

filter.

Analyze with HPLC.

E


FOS from the nutrition bar

Accurately weigh 10 ± 0.05 g and combine with 10 ml of 1:1

ethanol :water



30 min at room temperatute. Filter supernatant to remove

large particles.


filter.




F


FOS from the nutrition bar

Accurately measure 1.0 ± 0.05 ml.

Filter sample through a 0.45 µm filter.

Analyze with HPLC.

G

Appendix 2


inulin from the different food products

H


inulin from the extruded cereal.

Accurately weigh


portion.

Add hot distilled


80°C for 15 min.

Cool solution to

room temperature,

adjust volume to 50

ml with distilled

water, and mix.



pellet is formed.

Filter aliquot of

solution through

paper Whatman No.

1, or equivalent.

Transfer 0.2 ml of

filtrate.

Add 0.2 ml of

sucrase/amylase

solution, and


30 min.

Add 0.2 ml alkaline


to each tube and


30 min.

Add 0.5 ml acetic

acid and mix.


aliquots.

Add 0.1 mL of

fructanase to 3 of

these samples.

Add 0.1 ml of 0.1 M

sodium acetate

buffer to the 4th

sample

(sample blank).


20 min.

Add 5.0 ml

PAHBAH working

reagent.

Incubate in boiling






for ca 5 min.

Measure absorbance

of all solutions at

410 nm.

I

Figure 2 Flow diagram of the optimized extraction procedure and analysis of

inulin from the muffins.

Accurately weigh


portion.

Add hot distilled


80°C for 15 min.

Cool solution to

room temperature,

adjust volume to 50

ml with distilled

water, and mix.



pellet is formed.

Filter aliquot of

solution through

paper Whatman No.

1, or equivalent.

Transfer 0.2 ml of

filtrate.

Add 0.2 ml of

sucrase/amylase

solution, and


30 min.

Add 0.2 ml alkaline


to each tube and


30 min.

Add 0.5 ml acetic

acid and mix.


aliquots.

Add 0.1 mL of

fructanase to 3 of

these samples.

Add 0.1 ml of 0.1 M

sodium acetate

buffer to the 4th

sample

(sample blank).


20 min.

Add 5.0 ml

PAHBAH working

reagent.

Incubate in boiling






for ca 5 min.

Measure absorbance

of all solutions at

410 nm.

J


inulin from the nutrition bar.

Accurately weigh


portion.

Add hot distilled


80°C for 15 min.

Cool solution to

room temperature,

adjust volume to 50

ml with distilled

water, and mix.



pellet is formed.

Filter aliquot of

solution through

paper Whatman No.

1, or equivalent.

Transfer 0.2 ml of

filtrate.

Add 0.2 ml of

sucrase/amylase

solution, and


30 min.

Add 0.2 ml alkaline


to each tube and


30 min.

Add 0.5 ml acetic

acid and mix.


aliquots.

Add 0.1 mL of

fructanase to 3 of

these samples.

Add 0.1 ml of 0.1 M

sodium acetate

buffer to the 4th

sample

(sample blank).


20 min.

Add 5.0 ml

PAHBAH working

reagent.

Incubate in boiling






for ca 5 min.

Measure absorbance

of all solutions at

410 nm.

K


inulin from the sports drink.

Accurately measure

1.0 ±0.05 ml of a

test portion.

Add hot distilled


80°C for 15 min.

Cool solution to

room temperature,

adjust volume to 50

ml with distilled

water, and mix.

Filter aliquot of

solution through

paper Whatman No.

1, or equivalent.

Transfer 0.2 ml of

filtrate.

Add 0.2 ml of

sucrase/amylase

solution, and


30 min.

Add 0.2 ml alkaline


to each tube and


30 min.

Add 0.5 ml acetic

acid and mix.


aliquots.

Add 0.1 mL of

fructanase to 3 of

these samples.

Add 0.1 ml of 0.1 M

sodium acetate

buffer to the 4th

sample

(sample blank).


20 min.

Add 5.0 ml

PAHBAH working

reagent.

Incubate in boiling






for ca 5 min.

Measure absorbance

of all solutions at

410 nm.

L

Appendix 3


GOS from different food products

M


GOS from the extruded cereal.


and the internal standard to (myo-inositol) each at 1mg/ml (STD).

Prepare a solution of myo-inositol at the

same concentration of the expected

concentration of the sugar in sample (ISTD)

Accurately weigh 0.5 ± 0.05 g and combine

with 50.0 ± 0.1 ml of water to achive 1mg/ml

GOS concentration .

Vortex inmediatly after adding water

to prevent gel formation.

Mix 30 min at room temp

Centrifuge at 4500 rpm for 20 min to obtain

pellet



Add 50 µl of 2.8M

sulfuric acid and 50 µl

of ISTD to sample (S).

Hydrolyze: Pressure

cook in High (15 psi

and

80 °C for 1 h).

Add 75 µl of 12 M

ammonium hydroxide

and mix.


prepared 150 mg/ml

alkaline sodium

borohydride.

Incubate at 40 °C for 1

h.


acetic acid; mix.

Add 0.5 ml of 1-

methylimidazole; mix.

Add 5 ml of acetic anhydride (slowly);

mix.

Let stand 10 min at

room temperature.

Add 1 ml absolute

ethanol.

Let stand 10 min at

room temperature.

Move tubes to a cooler

with ice up to shoulder

of tube

Slowly add 5 ml of

well mixed 7.5 M

sodium hydroxide; mix.


temperature.

Add another 5 ml of 7.5

M sodium hydroxide.


(top) layer to a fresh

tube.

Analyze by GC. Analyze the STD at as

the first and last injection of the runs.

N


GOS from the extruded cereal.


and the internal standard to (myo-inositol) each at 1mg/ml (STD).

Prepare a solution of myo-inositol at the same

concentration of the expected concentration of the sugar in sample

(ISTD)

Accurately measure 1.0 ± 0.05 ml and

combine with 100.0 ± 0.1 ml of water to

achive 1mg/ml GOS concentration .



Add 50 µl of 2.8M

sulfuric acid and 50

µl of ISTD to sample

(S).

Hydrolyze: Pressure

cook in High (15 psi

and

80 °C for 1 h).

Add 75 µl of 12 M

ammonium hydroxide

and mix.


prepared 150 mg/ml

alkaline sodium

borohydride.

Incubate at 40 °C for

1 h.


acetic acid; mix.

Add 0.5 ml of 1-

methylimidazole;

mix.

Add 5 ml of acetic anhydride (slowly);

mix.

Let stand 10 min at

room temperature.

Add 1 ml absolute

ethanol.

Let stand 10 min at

room temperature.

Move tubes to a

cooler with ice up to

shoulder of tube

Slowly add 5 ml of

well mixed 7.5 M

sodium hydroxide;

mix.

Let stand 5 min at

room temperature.

Add another 5 ml of

7.5 M sodium

hydroxide.


(top) layer to a fresh

tube.

Analyze by GC. Analyze the STD at as

the first and last injection of the runs.

adaptation and validation of food product specific analytical

Documents