Adaptation and Validation of Food Product Specific Analytical Methods for Monitoring Prebiotics Present in Different Types of Processed Food Matrices

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Rebbeca M. Duar University of Nebraska-Lincoln, [email protected]

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Duar, Rebbeca M., "Adaptation and Validation of Food Product Specific Analytical Methods for Monitoring Prebiotics Present in Different Types of Processed Food Matrices" (2011). Dissertations, Theses, & Student Research in Food Science and Technology. 21. https://digitalcommons.unl.edu/foodscidiss/21

This Article is brought to you for free and open access by the Food Science and Technology Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Dissertations, Theses, & Student Research in Food Science and Technology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. ADAPTATION AND VALIDATION OF FOOD PRODUCT SPECIFIC ANALYTICAL

METHODS FOR MONITORING PREBIOTICS PRESENT IN DIFFERENT TYPES OF

PROCESSED FOOD MATRICES

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.

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

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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 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 25. 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 ...... 70

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

A. LITERATURE RIVIEW

Background of Prebiotics

The Prebiotic concept was introduced by Gibson & Roberfoid (1995) as “nondigestible 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).

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

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 nondigestible 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)).

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).

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.

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)).

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).

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)).

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))

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).

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)).

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).

Figure 8. Stability of Vivinal ® GOS at low pH and 85 °C for 5 minutes. Source: (Friesland Foods Domo, GRASS notice FDA (2007)).

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.

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).

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.

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.

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, 33 USP in 150 ml water) 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 250 barley flour, niacin, iron, thiamin, mononitrate, riboflavin, folic acid) Granulated sucrose 75 Baking powder (Baking soda, corn starch, 15 sodium aluminium sulfate, calcium sulfate, monocalcium phosphate) 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.

Table 4. Nutritional Bar Formulation

Component g/batch Rolled oats (whole) 420 Granola cereal (whole grain rolled oats, 420 evaporated cane juice, expeller pressed canola oil, defatted wheat germ, oat flour, brown rice syrup, molasses, salt, natural flavor, soy lecithin) 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.

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.

Accuratly weigh a sub sample and combine with water, at the Mix 30 min at room temperature. appropiate proportions.

Centrifuge at 10000 g for 20- Filter supernates to remove 30 min at room temperatute. large particles.

Collect filtrate and pass an Dry sample under vacuum at aliquot through a 0.45 µm 50 °C to obtain a dry residue. filter.

Resuspend sample in nanopure water at a final volume Analyze with HPLC. resulting in 1% solution.

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

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.

Cool solution to Accurately weigh Add hot distilled room temperature, 1.0±0.05 g of a test water at 80°C; stir adjust volume to 100 portion. 80°C for 15 min. ml with distilled water, and mix.

Filter aliquot of Centrifuge at 1000 g solution through Transfer 0.2 ml of for 20 min ,or until paper Whatman No. filtrate. pellet is formed. 1, or equivalent.

Add 0.2 ml of Add 0.2 ml alkaline sucrase/amylase borohydride solution solution, and to each tube and Add 0.5 ml acetic incubate at 40°C for incubate at 40°C for acid and mix. 30 min. 30 min.

Add 0.1 ml of 0.1 M Add 0.1 mL of sodium acetate Transfer four 0.2 ml fructanase to 3 of buffer to the 4th aliquots. these samples. sample (sample blank).

Add 5.0 ml Incubate in boiling Incubate at 40°C for PAHBAH working water bath for 6 min. 20 min. reagent.

Remove all tubes from boiling water bath and Measure absorbance immediately place in of all solutions at cold water (18–20°C) 410 nm. for ca 5 min.

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

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. Prepare a solution of Prepare a solution myo-inositol at the same Accurately weigh a of a galactose and glucose concentration of the test portion and combine and the internal standard 34 expected concentration of with to achive 1mg/ml to (myo-inositol) each at the sugar in sample GOS concentration . 1mg/ml (STD). (ISTD)

Mix 30 min at room temp Transfer 250 µl of Add 50 µl of 2.8M sample (S) to a 35 ml sulfuric acid and 50 µl Centrifuge to obtain open test tube with screw pellet at room. cap. of ISTD to sample (S).

Add 40 µl of freshly Hydrolyze: Pressure cook Add 75 µl of 12 M prepared 150 mg/ml in High (15 psi and ammonium hydroxide and alkaline sodium 80 °C for 1 h). mix. borohydride.

Add 40 µl of glacial acetic acid; mix. Add 0.5 ml of 1- Let stand 10 min at room Incubate at 40 °C for 1 h. methylimidazole; mix. temperature. Add 5 ml of acetic anhydride (slowly); mix.

Move tubes to a cooler Add 1 ml absolute Let stand 10 min at room with ice up to shoulder of ethanol. temperature. tube

Slowly add 5 ml of well Let stand 5 min at room Add another 5 ml of 7.5 mixed 7.5 M sodium temperature. M sodium hydroxide. hydroxide; mix.

Analyze the STD at as the Transfer ethyl acetate Analyze by GC. first and last injection of (top) layer to a fresh tube. the runs.

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

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.

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).

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.

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.

Table 6. Optimization conditions for extracting FOS in the different food products Further procedures Amount of Extraction Solvent for extraction Food Product sub-sample analyzed optimization.

Extruded cereal 10.00 ± 0.05 g 20.0 ± 0.1 ml of 1:1 Concentrated to a water: ethanol final volume of 1 ml.

Muffins 1.00 ± 0.05 g 2.0 ± 0.1 ml of water -

Cookies 10.00 ± 0.05 20.0 ± 0.1 ml of water -Pellet extracted an additional time with 10 ml of water. Concentrated to a Nutrition bar 10.00 ± 0.05 g 10.0 ± 0.1 ml of water 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 Volume Volume Amount of sub- of extraction solvent of extraction solvent Food sample analyzed (water) indicated by used for assay Product the protocol 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

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.

Table 7. Optimization conditions for extracting GOS from sports drink and breakfast cereal Further procedures Amount of sub- Extraction Solvent for extraction Food Product sample analyzed (water) optimization Mix immediately after adding water to Extruded cereal 0.50 ± 0.05 g 50.0 ± 0.1 ml 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).

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).

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).

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.

Table 8. Method performance characteristics for FOS in different food products Breakfast Nutrition cereal Cookie Muffin bar Sports drink Linearity (regression analysis y = 103688x y = 258 152x y = 272931 x y = 285392x y = 3123539x Kestose : -26321 +186097 +80203 +128321 + 16253 0.25 - 7.5 mg/ml(n=3) r=0.9996 r=0.9753 r=0.9970 r2=0.9998 r= 0.9999 y = 136427x y = 273142x y = 272931x y = 273098x y= 2722490x Nystose: - 36158 – 112449 + 80203 + 48825 + 21160 0.25 - 7.5 mg/ml (n=3) r = 0.9996 r = 0.9973 r = 0.9976 r = 0.9987 r= 0.9999

1-fructofuranosyl nystose: 0.25-7.5 mg/ml y = 32660x y = 272058x y = 268210x y = 276210x y= 2615410x - 1093.5 – 60167 - 6532.1 – 11172 + 28631 (n=3) r = 0.9988 r = 0.9751 r = 0.9973 r = 0.9986 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

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.

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

Breakfast Nutrition cereal Cookie Muffin bar Sports drink Linearity (regression analysis

Inulin:3-30 mg/ml y = 0.0149x y= 0.0081x y = 0.0157x y = 0.0157x y = 0.0148x (n=5) + 0.0013 + 0.0044 +0.0058 +0.0058 - 0.0001 r = 0.9989 r = 0.9994 r =0.9994 r = 0.9994 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

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.

Table 11. Method performance characteristics for GOS in different food products Breakfast cereal Sports drink Linearity (regression analysis y = 361610x - 30357 y = 361610x – 30357 Galactose: 1- 20 mg/ml (n=3) r = 0.9985 r = 0.9985 y = 306002x + 59647 y = 306002x + 59647 Glucose 1- 20 mg/ml (n=3) r = 0.9992 r = 0.9992 y = 461702x - 61489 y = 461702x - 61489 myo-inositol: 1- 20 mg/ml (n=3) r = 0.9984 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

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.

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

FOS content

Sample Before After % Recovered correction factor correction factor

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).

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

Inulin content

Sample Base Supplemented Supplemented After applying % formula with 1% Inulin minus base correction Recovered formula factor

Extruded 1.03 ± 0.12 2.22 ± 0.106 1.25 ± 0.04 1.62 ± 0.03 106% cereal 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).

Table 12. Percent recoveries of 1% GOS supplemented food products after processing GOS content Sample Supplemented with 1% % Recovery GOS

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).

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.

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.

Figure 23. Inulin recovery in breakfast cereal supplemented with 1% Inulin 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.

Figure 24. GOS recovery in breakfast cereal supplemented with 1% GOS 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.

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.

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.

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.

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.

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

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.

References

AACC Int. (1994). Total Dietary Fiber-Determined as Neutral Sugar Residues, Uronic Acid Residues, and Klason Lignin (Uppsala Method) Method 32-25. In A. International, AACC International. Approved Methods of Analysis (11 ed.). St. Paul, MN, U.S.A. AACC Int. (2001). Measurement of Total Fructan in Foods by an Enzymatic/Spectrophotometric Method (Method 32-32.01). In 2001, AACC International Aproved Methods of Analysis (11 ed.). St. Paul, MN, USA. Alander, M., Matto, J., Kneifel, W., Johansson, M., Kogler, B., Crittenden, R., et al. (2001). Effect of galactoologosaccharide suplementation on human fecal microflora and on survival and persistance of Bifidobacterium lactis Bb-12 in the gastrointestinal tract. International Dairy Journal, 11, 817-825. Albayrak, N., & Yiang, S. (2002). Production of galacto-oligosaccharides from Lactose by Aspergillus orizae β-galactosidase immobilized on cotton cloth. Biotechnology and Bioengineering, 77, 8-19. Albrecht, S., Schols, H., Klarenbeek, B., Voragen, A., & Gruppen, H. (2010). Introducing capillary electrophoresis with laser-indced fluorescence (CE-LIF) as a potential analysis and quantification tool for galactooligosaccharides extracted from complex food matrices. Journal of Agricultural and Food Chemistry , 58, 2787- 2794. Alles, M., Hartemink, R., Meyboom, S., Harryvan, J., Van Laere, J., Nagengast, F., et al. (1999). Effect of trasgalactooligosaccharides on the composition of the human intestinal microflora and on putative risk markers for colon cancer. American Journal of Clinical Nutrition, 69, 980 991. Alsaffar, A. A. (2011). Effect of food processing on the resistant starch content of cereals and cereal products-A review. International Journal of Food Science and Technology, 46, 455-462. 74

Antosova, M., Polakovic, M., & Bales, V. (1999). Separation of fructoologosaccharides on a cation-exange HLPC column in silver form with refractometric detection. Biotechnology Techniques, 13, 889-882. AOAC. (1998). AOAC peer verifies methods program; Method on policies and procedures,. Retrieved from www.aoac.org AOAC. (2002). AOAC Official Method 999.03 Measurement of the total fructan in foods. Archer, B., Johnson, S., Devereux, H., & Baxter, A. (2004). Effect of fat replacement by inulin or lupin-kernel fibre on sausage patty acceptability, post-meal perceptions of satiety and food intake in men. British Journal of Nutrition , 91, 591-599. Bakker-Zierikzee, A., Tol, E., Kroes, H., Alles, M., & Kok, F. (2006). Faecal SIgA secretion in infants fed on pre- or probiotic infant formula. Pediatric Allergy Immunology, 17, 134-140. Bankova, E., Bakalova, N., Petrova, S., & Kolev, D. (2006). Enzymatic Synthesis of Oligosaccharides and Alkilglycosides in Water-Organic Media via Transglycosylation of Lactose. Biotechnology and Biotechnological Equipment, 114-119. Ben, X., Zhou, X., Zhao, W., Yu, W. P., Zhang, W., Wu, S., et al. (2004). Supplementation of milk formula with galactooligosaccharides impoves intestinal microflora and fermantation in term infants. Chinese Medical Journal, 117, 927- 931. Berreteau, H., Delattre, C., & Michaud, P. (2006). Production of Oligosaccharides as promising New Food Aditive Generation. Food Technology and Biotechnology, 44, 323-333. Birkett, A., & Francis, C. (2009). Short-Chain Fructo-oligosaccharides: A low Molecular Weight Fructan. In S. Cho, Handbook of Prebiotics and Probiotics Ingredients: Health Benefits and Food. (pp. 14-38). CRC Press. Bjorck, I., Nyman, M., & Asp, N.-G. (1984). Extrusion cooking and dietary fiber: effects on dietary fiber content and on degradation in the rat intestinal tract. Cereal Chemistry, 61, 175-179. 75

Blecker, C. F., Van Herck, J., Chevelier, J., & Paquot, M. (2002). Kinetic Study of the Acid Hydrolysis of Various Oligofructose Samples. Journal of Agricultural Food Chemistry, 50, 1602-1607. Böhm, A., Kaise, I., Trebstein, A., & Henle, T. (2005). Heat-induced degradation of inulin. European Food Research and Technology, 220, 466-471. Bouhnik, Y., Achour, L., Paineau, D., Riottot, A., & B., F. (2007). Four-week short chain fructo-oligosaccharides ingestion leads to increasing fecal bifidobacteria and cholesterol excretion in healthy elderly volunteers. Nutrition Journal , 42, 42-46. Bouhnik, Y., Raskine, L., Simoneau, G., Vicaut, E., Neut, C., Flourié, B., et al. (2004). The capacity of nondigestible carbohydrates to stimulate fecal bifidobacteria in healthy humans: a double-blind, randomised, placebo-controlled, parallel-group, dose-response relation study. American Journal of Clinical Nutrition , 80, 1658- 1664. Bruzzese, E., Volpicelli, M., Squeglia, V., Bruzzese, D., Salvini, F., Bisceglia, M., et al. (2009). A formula containing galacto- and fructo-oligosaccharides prevents intestinal and extra-intestinal infections: an observational study. Clinical Nutrition , 28, 156-161. Buddington, R., Williamsn, C., Chen, S., & Wintherly, S. (1996). Dietary supplement of neosugar alters the fecal flora and decreases activities of some reductive enzymes in human subjects. American Journal of Nutrition , 63, 709-716. Bunce, T., Howard, M., Kerley, M., Alle, G., & Pace, L. (1995). Protective effect of fructooligosaccharide (FOS) in prevention of mortality and morbidity from infectious E coli K:88 challenge. American Journal of Animal Science, 63, 1784- 1788. Campbell, J. M., Bauer, L., Fahey, G. C., H. A., Wolf, B., & Hunter, D. E. (1997). Selected Fructooligosaccharide (1-kestose, Nystose, and 1- Fructofunarosylnystose) Composition of Food and Feeds. Journal of Agricultural and Food Chemistry , 45, 3076-3082. Cani, P., Joly, E., Horsmans, Y., & Delzene, N. (2006). Oligofructose promotes satiety in healthy human: a pilot study. European Journal of Clinical Nutrition, 60, 567- 572. 76

Casellas, F., Borruel, N., Torrejón, A., Varela, E., Antolin, M., Guarner, F., et al. (2007). Oral oligofructose-enriched inulin supplementation in acute ulcerative colitis is well tolerated and associated with lowered faecal calprotectin. Alimentary Pharmacology & Therapeutics, 9, 1061-10670. Causey, J., Feirtag, J., Gallaher, D., Tubgland, B., & Slavin, J. (2000). Effects of dietary inulin on serum lipids, blood glucose and the gastrointestinal environment in hypercholesterolemic men. Nutrition Research , 20, 141-201. Cherbut, C. (2002). Inulin and oligofructose in the dietary fiber concept. British Journal of Nutrition , 159-162. Chonan, O., & Watanuki, M. (1995). Effect of galactooligosaccharides on calcium absorption in rats. Journal of Nutrition Science and Vitaminology., 41, 95-104. Chonan, O., & Watanuki, M. (1996). The effect of 6'-galactooligosaccharides on bone mineralization of rats adapted to different levels of dietary calcium. International journal for vitamin and nutrition research, 66, 244-249. Correa-Matos, N., Donovan, S., Isaacson, R., Gaskins, H. W., & Tappenden, K. (2003). Fermentable fiber reduces recovery time and improves intestinal fuction in piglets following Salmonella typhimurium infection. Journal of Nutrition , 133, 1845- 1852. Coundray, C., Bellanger, J., Castilglia-Delavaud, C., Remesy, C., Vermorel, M., & Rayssignuier, Y. (1997). Effect of soluble or partly soluble dietary fibres supplementation on absorption and balance of calcium, magnesium, iron and zinc in healthy young men. European Journal of Clinical Nutrition, 51, 375-380. Courtin, C., Swennen, K., Verjans, P., & Delcour, J. (2009). Heat and pH stability of prebiotics arabinoxylooligosaccharides, xylooligosaccharides and fructooligosaccharides. Food Chemistry, 112, 831-837. Courtin, C., Van den Broek, H., & Delcour, J. (2000). Determination of reducing end sugar residues in oligo- and polysaccharides by gas -liquid chromatography. Journal of Chromatography A, 866, 97-104. Crittenden, R., & Playne, M. (2006). Modifying the Human Intestinal Microbiota with Prebiotics. In A. Ouwehand, & E. Vaugha, Gastrointestinal Microbiology (pp. 285-302). CRC Press / ITPS. 77

Davis, L., Martinez, I., Walter, J., & Hutkins, R. (2010). A dose dependent impact of prebiotic galactooligosaccharides on the intestinal microbiota of healthy adults. International Journal of Food Microbiology, 144, 285-292. de Slegte, J. (2002). Determination of trans-galactooligosaccharides in selected food products by ion-exchange chromatography: colaborative study. Journal of AOAC international, 85, 417-423. Delcour, J. A. (2010). Principles of Cereal Science and Tecnology. St Paul, MN, U.S.A: AACC International , Inc. Delzenne, N., & Kok, N. (2001). Effects of fructans-type prebiotics on lipid metabolism. American Journal of Clinical Nutrition, 73, 456s-458s. Den Hond, E., Geypens, B., & Ghoos, Y. (2000). Effect of high performance chicory inulin on constipation. Nutrition Research, 20, 731-736. Dias, L., Veloso, A., Correia, D., Rocha, O., Torres, D., Rocha, I., et al. (2008). UV spectrophotometry method for the monitoring of galacto-oligosaccharides production. Food Chemistry, 113, 246-252. Drakoularakou, A., Tzortzis, G., Rastall, R., & Gibson, G. (2010). A double-blind, placebo-controlled, randomized human study assessing the capacity of a novel galacto-oligosaccharide mixture in reducing travellers' diarrhoea. European Journal of Clinical Nutrition, 64, 146-152. FDA. (2007). GRAS Notification for Galacto-oligosaccharides (GOS). Frank, A. (2002). Technological functionality of inulin and oligofructose. British Journal of Nutrition, 87, S287-S291. Fukushima, Y., Jun, C., Kegai, K., Ohta, A., Sakai, K., Uenishi, K., et al. (2002). Calcium absorption of malt drinks containing fructooligosaccharides and safety in humans. Journal of Nutritional Food, 5, 49-60. Furrie, E., Macfarlane, S., Kennedy, A., Cummings, J., Walsh, S., O'Neil, D., et al. (2005). Synbiotic therapy (Bifidobacterium longum/Synergy 1) initiates resolution of inflammation in patients with activeulcerative colitis: a randomised controlled pilot trial. Gut, 54, 242-249. Gibson, G. (2000). Dietary Modulation of the Human Gut Microflora Using the Prebiotics Oligofructose and Inulin. Journal of Nutrition, 129, 1438S- 1441S. 78

Gibson, G. R., Beatty, E. R., Wang, X., & Cummings, J. (1995). Selective stimulation of bifidobacteria in the human colon by oligofructose and inulin. Gastroenterology, 108, 975-982. Gibson, G., & Roberfoid, M. B. (1995). Dietary modulation of the human colonic miciobiota: Introducing the concept of prebiotics. Journal of Nutrition, 1401- 1412. Gibson, G., Beatty, E., Wang, X., & Cummings, J. (1995). Selective stimulation of bifidobacteria in the human colon by oligofructose and inulin. Gastroenterology, 103, 975-982. Gibson, G., Probert, H., Van Loo, J., Rastall, R., & Roberfroid, M. (2004). Dietary Modulation of the Human Colonic Microbiota: Updating the Concept of Prebiotics. Nutrition Research Reviews, 17, 259-275. Guadalberto, D. B., & Kazemzadeh, M. a. (1997). "Effect of extrusion processing on the soluble and insoluble fiber, and phytic acid contents of cereal brans." . Plants Foods for Human Nutrition, 51, 187-198. Guigoz, Y., Rochat, F., Perruisseau-Carrier, G., Rochat, I., & Schiffrin, E. (2002). Effects of oligosaccharide on the faecal flora and non-specific immune system in elderly people. Nutrition Research, 22, 13-25. Guy, R. (2001). Extrusion Cooking - Technologies and Applications. Woodhead Publishing. Hayashi, S., Yoshiyama, T., & Fuji, N. S. (2000). Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum. Biotechnology Letters , 22, 1465-1469. Hidaka, H., Eida, T., Takizawa, T., & Tashiro, Y. (1986). Effects of fructooligosaccharides on intestinal flora and human health. Bifidobacteria Microflora , 5, 37-50. Hofer, K., & Jeneweing, D. (1999). Enzymatic determination of inulin in food and dietary supplements. European Food Research and Technolgy, 209, 423-427. Huebner, J., Wehling, R., Parkhurts, A., & Hutkins, R. (2008). Effec of processing conditions on the prebiotic activity of commercial prebiotics. International Dairy Journal, 18, 287-293. 79

Hussein, H., Campbell, J., Bauer, L. F., Hogarth, L., & Wolf, B. H. (1998). Ingredients, Selected Fructooligosaccharide Composition of Pet-Food. Journal of Nutrition , 128, 28035-28055. IPCS. (1999). Sucrose International Chemical Safety Card 1507. Retrieved November 14, 2011, from International Programme on Chemical Safet: http://www.inchem.org/documents/icsc/icsc/eics1507.htm ISAPP. (2008). 6th Meeting of the International Scientific Association of Probiotics and Prebiotics. International Scientific Association of. London. Ito, M., Kimura, M., Deguchi, Y., Miyamori-Watabe, A., Yajima, T., & T., K. (1993). Effects of transgalactosylated disaccharides on the human intestinal microflora and their metabolism. Journal of Nutrition Science and Vitaminology, 39, 279- 278. IUPAC. (1997). Compendium of Chemical Terminology (the "Gold Book"). (2nd ed.). Oxford: Blackwell Scientific Publications. Jenkins, D., Kendall, C., & Vuksan, V. (1999). Inulin, Oligofructose and intestinal function. 1431S- 1433S. Kaplan, H., & Hutkins, R. (2003). Metabolism of Fructooligosaccharides by Lactobacillus paracasei 1195. Applied and Environmental Microbiology., 69, 2217-2222. Kewicki, R. (2007). The stability of gal-polyols and oligosaccahrides during pasteurization at low pH. LWT - Food Science and Technology, 40, 1259-1265. Kleessen, B., Sykura, B., Zunft, H., & Blaunt, M. (1997). Effects of inulin and lactose on fecal microflora, microbial activity, and bowel habit in elderly constipated persons. American Journal of Clinical Nutrition, 65, 1397-1402. Kok, N., Roberfroid, M., & Delxenne, N. (1996). Involvement of lipogenesis in the lower VLDL secretion induced by oligofuctose in rats. Brithish Journal of Nutrition , 76, 881-890. Kolida, S., & Gibson, G. (2007). Prebiotic capacity if Inulin-type fructans. The Journal of Nutrition, 137, 2503S - 2506S. 80

Korakli, M., Hinrichs, C., Ehrmann, M., & Vogel, R. (2003). Enzymatic determination of inulin and fructooligosaccharides in food. European Food Research and Technology , 217, 530-234. Kukkonen, K., Savilahti, E., Haahtela, T., Juntunen-Backman, K., Korpela, R., Poussa, T., et al. (2008). Long-term safety and impact on infection rates of postnatal probiotic and prebiotic (synbiotic) treatment: randomized, double-blind, placebo- controlled trial. Pediatrics , 122, 122-128. Kuz, C., Rudloff, S., & Baier, W. S. (2000). Oligosaccharides in human milk: Structural, Functional, and Metabolic Aspects. Annual Review of Nutrition , 20, 699-722. L'Homme, C., Arbelot, M., Paugserver, A., & Biagini, A. (2003). Kinetics of Hydrolysis of Fructooligosacharides in MIneral- Buffered Aqueous Solutions; Influence of pH and Temperture. Journal of Agricultural and Food Chemistry, 51, 224-228. L'homme, C., Arbelot, M., Puigserver, A., & Biagini, A. (2003). Kinetics of Hydrolysis of Fructooligosaccharides in Mineral-Buffered Aqueous Solutions: Influence of pH and Temperature. Journal of Agricultural and Food Chemistry, 51, 224-228. Lindsay, J., Whelan, K., Stagg, A., Gonin, P., Al-Hassi, H., Rayment, N., et al. (2005). Clinical, microbiological, and immunological effects of fructo-oligosaccharide in patients with Crohn’s disease. International Journal of Gastroenterology and Hepatology, 55, 348-355. Lopez, H., Coundray, C., Levrat-Verny, M., Feillet-Coudray, C., Demigne, C., & Remesy, C. (2000). Fructooligosaccharides enhance mineral apparent absorption and counteract the deleterious effects of phytic acid on mineral homeostasis in rats. Journal of Nutritional Biochemistry , 11, 500-508. Macfarlane, G., Steed, H., & and Macfarlane, S. (2008). Bacterial metabolism and health- related effects of galacto-oligosaccharides and other prebiotics. Journal of Applied Microbiology , 104, 305-3440. Matusek, A., Meresz, P., Diem Le, T., & örsi, F. (2009). Effect of temperature and pH on the degradation of fructo-oligosaccharides. European Food Research and Technology, 228, 355-365. 81

Menne, E., Guggenbuhl, N., & Roberfroid, M. (2000). Fn-type Chicory Inulin Hydrolysate Has a Prebiotic Effect in Humans. Journal of Nutrition , 130, 1197- 1199. Moro, G., minoli, I., Mosca, M., Fanaro, S., Jelinek, J., Stahl, B., et al. (2002). Dosage- Related Bifidogenic Effects of Galacto- and Fructooligosaccharides in Formula- Fed Term Infants. Journal of Pediatric Gastroenterology and Nutrition, 34, 291- 295. Mujoo, R., & Ng, P. (2003). Physicochemical Properties of Bread Baked from Flour Blended with Immature Wheat Meal Rich in Fructooligosaccharides. Food Chemistry and Toxicology , 68, 2448-2452. Napoli, J., Brand-Miller, J., & Conway, P. (2003). Bifidogenic effects of feeding infant formula containing galactooligosaccharides in healthy formula-fed infants. Asia Pacific Journal of Clinical Nutrition , 12, 60-64. Nauta, A., Bakker-Zierikze, A., & Schoterman, M. (2009). galacto-oligosaccharides. In S. Cho, Handbook of Prebiotics and Probiotics Ingredients: Health Benefits and Food Applications (pp. 14-38, 44-63, 75-88). CRC Press. Nguyeh, Q., Mattes, F., Hoschke, A., Rezessy-Szabo, J., & Bhat, M. (1999). Produccion purificacion and identificacion of fructooligosaccharides produces by beta- fructofuranosidase from Aspergillud Niger IMI 303386. Biotechnology Letters, 21, 183-186. Niness, K. R. (1999). Inulin and Oligofructose: What are They? Journal of Nutrition , 129, 1402S- 1406S. Nishizawa, K., Nakajima, M., & Nabetani, H. (2000). Kinetic Study on Transfructosylation by b-Fructofuranosidase from Aspergillus nigerATCC 20611 and Availability of a Membrane Reactor for Fructooligosaccharide Production. Food Science and Technology Research , 7, 39-44. Ohta, A., Motohashi, Y., Sakai, K., Hirayama, M., Adachi, T., & Sakuma, K. (1998). Dietary fructooligosaccharides increase calcium absorption and levels of mucosal calbindin-D9k in the large intestine of gastrectomized rats. Scandinavian Journal of Gastroenterology, 33, 1062-1068. 82

Ohta, A., Ohtsuki, M., Baba, S., Adachi, T., Sakata, T., & Sakaguchi, E. (1995). Calcium and magnesium absorption from the colon and rectum are increased in rats fed fructooligosaccharides. Journal of Nutrition, 125, 2417-2424. Osborn, D., & Sinn, J. (2007). Prebiotics in infants for prevention of allergic disease and food hypersensitivity. Cochrane Database of Systematic Reviews , 17, 1-25. Paineau, D., Payen, F., Panserieu, S., Coulombier, G., Sobazek, A., Lartigau, I., et al. (2008). The effects of regular consumption of short-chain fructo-oligosaccharides on digestive comfort of subjects with minor functional bowel disorders. British Journal of Nutrition , 99, 311-318. Park, J., Oh, T., & Yun, J. (2001). Purification and characterization of a novel transfructosylating enzyme from Bacillus macerans EG-6. Process Biochemistry, 37, 471-476. Piirainen, L., Kekkonen, R., Kajander, K., Ahlroos, T., Tynkkynen, S., Nevala, R., et al. (2008). In school-aged children a combination of galactooligosaccharides and Lactobacillus GG increases bifidobacteria more than Lactobacillus GG on its own. Annals of Nutrition and Metabolism , 52, 204-208. Pool- Zobel, B., van Loo, J., Rowland, I., & Roberfroid, M. (2002). Experimental evidences on the potential of prebiotic fructans to reduce the risk of colon cancer. British Journal of Nutrition, 87, S273–S281. Pranznik, W., E, C., & FIlipiak- Floriewiez, A. (2002). Soluble dietary fibres in Jerusalem artichoke powders: Composition and application in bread. Nahrung, 46, 151-157. Quintero, M., Maldonado, M., Perez-Munoz, M., Jimenez, R., Fangman, T., Rupnow, J., et al. (2011). Adherence Inhibition of Cronobacter sakazakii to Intestinal Epithelial Cells by Prebiotic Oligosaccharides. Current Microbiology , 62, 1448– 1454. Ramnani, P., Gaudier, E., Bingham, M., Bruggen, P., Touhy, K. M., & Gibson, G. R. (2010). Prebiotic effect of fruit and vegetables shots containing Jesusalem artichock inulin: a human intervention study. British Journal of Nutrition, 104, 233- 240. 83

Roberfoid, M., Van-Loo, J., & Gibson, G. (1998). The bifidogenic nature of chicory inulin and its hydrolysis products. Journal of Nutrition, 128, 9-11. Roberforid, M. (2007). Prebiotics: The Concept Revisited. The Journal of Nutrition , 137, 830S-837S. Roberfroid, M. (2002). Fuctional foods: concepts and application to inulin and oligofructose. British Journal of Nutrition, 87, 139-143. Roberfroid, M., Gibson, G., Hoyles, L., McCartney, A., Rastal, R., Rowland, I., et al. (2010). Prebiotic effects: metabolic and health benefits. British Journal of Nutrition, 104, 3-51. Rousseau, V., Lepargneur, J., Roques, C. R.-S., & Paula, F. (2005). Prebiotic effects of oligosaccharides on selected vaginal lactobacilli and pathogenic microorganisms. 11, 145-159. Rowland, I., & Tanaka, R. (1993). The effects of transgalactosylated oligosaccharides on gut flora metabolism in rats associated with a human fecal microflora,. Journal of Applied Bacteriology, 74, 667–674. Sangeethat, P., Ramesh, M., & Prapulla, S. (2005). Recent trends in the microbial producction, analysis and application of Fructooligosaccharides. Trends in Food Science & Technology, 16, 442-457. Sangwan, V., Tomar, S., Singh, R., Singh, A., & Ali, B. (2011). Galactooligosaccharides: Novel Components of Designer Foods. Journal of Food Science, 76, 103-111. Sanz, M., Cote, G., Gibson, G., & Rastall, R. (2005). Prebiotic Properties of Alternansucrase Maltose-Acceptor Oligosaccharides. Journal of Agricultural and Food Chemistry, 53, 5911-5916. Schley, P., & Field, C. (2002). The immune-enhancing effects of dietary fibres and prebiotics. British Journal of Nutrition , 87, s221-s230. Scholtens, P., Alliet, P., Raes, M., Alles, M., Kroes, H., Boehm, G., et al. (2008). Fecal Secretory Immunoglobulin A Is Increased in Healthy Infants Who Receive a Formula with Short-Chain Galacto-Oligosaccharides and Long-Chain Fructo- Oligosaccharides. Journal of Nutrition, 138, 1141-1147. Searle, L., Best, A., Nunez, A., Salguero, F., Johnson, L., Weyer, U., et al. (2009). A mixture containing galactooligosaccharide, produced by the enzymic activity of 84

Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium infection in mice. Jornal of Medical Microbiology , 58, 37-48. Sheu, D., Lio, P., Chen, S., Lin, C., & Duan, K. (2001). Production of fructooligosaccharides in high yield using a mixed enzyme system of β- fructofuranosidase and glucose oxidase. Biotechnology Letters, 23, 1499-1503. Shiomi, N., Onodera, S., Chatterron, J., & Harrison, P. (1991). Separation of Fructooligosaccharides Isomers by Anion-exchange Chromatography. 55, 1427- 1428. Shoaf, K., & Hutkins, R. (2008). Adherence, anti-adherence, and oligosaccharides preventing pathogens from sticking to the host. In S. L. Taylor, Advances in Food and Nutrition Research (Vol. 55, pp. 101-161). Academic Press. Shoaf, K., Mulvey, G., Armstrong, G., & Hutkins, R. (2006). Prebiotic Galactooligosaccharides Reduce Adherence of Enteropathogenic Escherichia Coli to issue Culture Cells. Infection and Inmunity, 74, 6920-6928. Silk, D., Davis, A., Vulevic, J., Tzortziss, G., & Gibson, G. (2009). Clinical trial: the effects of trans-galactooligosaccharide prebiotic on fecal microbiota and symptoms in irritable bowel syndrome. Alimentary Pharmacology Therapy, 29, 508-518. Splechtna, B., Nguyen, T., Steinbock, M., Kulbe, K., Lorenz, W., & Haltrich, D. (2006). Production of prebiotic galacto-oligosaccharides from lactose using β- galactosidases Lactobacillus reuteri. Journal of Agricultural and Food Chemistry, 54, 4999-5006. Tahiri, M., Tressol, J., Arnaud, J., Bornet, F., Bouteloup-Demange, C., Feillet-Coudray, C., et al. (2001). Five-week intake of short-chain fructo-oligosaccharides increases intestinal absorption and status of magnesium in postmenopausal women. Journal of Bone Mineral Research , 16, 2152-2160. Taper, H., & Roberfroid, M. (1999). Influence of inulin and oligofructose on breast cancer and tumor growth. Journal of Nutrition , 129, 1488-1491. Taper, H., & Roberfroid, M. (1999). Influence of inulin and oligofructose on breast cancer tumor growth. The Journal of Nutrition, 129, 1488S-1491S. 85

Thuoty, K., Kolida, S., Lustenberger, A., & Gibson, G. (2001). The prebiotic effects of biscuits containing partially hydrolysed guar gum and fructo-oligosaccharides – a human volunteer study. British Journal of Nutrition, 86, 341-348. Uenishi, K., Ohta, A., Fukushima, Y., & Kagawa, Y. (2002). Effect of a Malt Drink Containing Fructooligosaccharides on Calcium Absorption and Safety of Long- term Administration. Japanese Journal of Nutrition and Dietetics, 60, 11-18. Ueno, K., Ishiguro, Y., Yodoshida, M., Onodera, S., & Shiomi, N. (2011). Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in edible burdock (Arctium lappa L.). Chemistry Central Journal , 5, 1-9. van den Heuvel, E., Muijs, T., Brouns, F., & Hendriks, H. (2009). Short-chain fructooligosaccharides improve magnesium absorption in adolescent girls with a low calcium intake. Nutritional Research, 29, 229-237. Van den Heuvel, E., Schaafsma G, M. T., & W., v. D. (1998). Nondigestible oligosaccharides do not interfere with calcium and nonheme-iron absorption in young, healthy men. American Journal of Clinical Nutrition , 67, 45-451. van den Heuvel, E., Schoterman, M., & Muijs, T. (2000). Transgalactooligosaccharides stimulate calcium absorption in postmenopausal women. Journal of Nutrition , 13, 2938-2942. van den Heuvel, G., Schaafsma, G., Muys, T., & Dokkum, W. (1998). Nondigestible oligosaccharides do not interfere with calcium and noheme-iron absorbtion in young healthy men. American Journal of Clinical Nutrition, 67, 445-451. van Hoffen, E., Ruiter, B., Faber, J., M'Rabet, L., Knol, E., Stahl, B., et al. (2009). A specific mixture of short-chain galacto-oligosaccharides and long-chain fructooligosaccharides induces a beneficial immunoglobulin profile in infants at high risk for allergy. Allergy , 64, 484-487. Vendrell-Pascuas, S., Castellote- Bargallo, A., & Lopez- Sabater, M. (2000). Determination of inulin in meat ptoducts by high-preformance liquid chromatography with refractive index detection. Journal of Chromarography A, 881, 591-597. Voragen, A. (1998). Technological aspects of functional food-related carbohydrates. Trends in Food Science and Technology, 9, 328-335. 86

Vulevic, J., Drakoularakou, A., Yaqoob, P., Tzortzis, G., & Gibson, G. (2008). Modulation of the fecal microflora profile and immune function by a novel trans- galactooligosaccharide mixture (B-GOS) in healthy elderly volunteers. American Journal of Clinical Nutrition, 88, 1438-1446. Wang, H., Zhang, Z., Liang, L., Wen, S., Liu, C., & Xu, X. (2010). A comparative study of hugh-performance liquid chromatography and colorimetric method for inulin determination. European Food Research and Technology, 230, 701-706. Wang, H., Zhang, Z., Liang, L., Wen, S., Lui, C., & Xu, X. (2010). A comparative study of high preformnce liquid chromatography and colirimetruc method for inulin determination. European Food Research and Technology, 230, 701-706. Watzl, B., Girrbach, S., & Roller, M. (2005). Inulin, oligofructose and immunomodulation. British Journal of Nutrition, 93, S49-S55. Wijnands, M., Appel, M., Hollanders, V., & Woutersen, R. (1999). A comparison of the effects of dietary cellulose and fermentable galacto-oligosaccharide, in a rat model of colorectal carcinogenesis: Fermentable fibre confers greater protection than non-fermentable fibre in both high and low fat backgrounds,. 20, 651-656. Williams, C. (1999). Effects of Inulin on Lipid Parameters in Humans. The Journal of Nutrition , 129, 1471S-1473S. Yamashita, K., Kawai, K., & Itakura, M. (1984). Effects of fructo-oligosaccharides on blood glucose and serum lipids in diabetic subjects. Nutrition Research, 4, 229- 232. Younes, H., Coundray, C., Bellanger, J., Demigne, C., Rayssiguier, Y., & Remesy, C. (2001). Effects of two fermentable carbohydrates (inulin and resistant starch) and their combination on calcium and magnasium balance in rats. British Journal of Nutrition, 86, 479-485. Yun, J. (1996). Fruncooligosaccharides -Occurrence, preparation, and application. Enzyme and Microbial Technology , 19, 107-117. Zuleta, A., & Sambucetti, E. (2001). Inulin Determination for Food Labeling. Journal of Agricultural and Food Chemistry, 49, 4570-4572.

Appendix 1

Flow diagrams of the optimized extraction procedure and analysis of

FOS from the different food products

Accuratly weigh 10 ± 0.05 g Mix 30 min at room and combine with 20 ml of 1:1 temperature. ethanol :water

Centrifuge at 10000 g for 20- Filter supernatant to remove 30 min at room temperatute. large particles.

Collect filtrate and pass an Dry sample under vacuum at aliquot through a 0.45 µm 50 °C to obtain a dry residue. filter.

Resuspend sample in nanopure water at a final volume Analyze with HPLC. resulting in 1% solution.

Figure 1. Flow diagram of the optimized extraction procedure and analysis of FOS from the in the extruded cereal

Accurately weigh 1 ± 0.05 g and combine with 2 ml ± 0.1 of Mix 30 min at room temperature. water

Centrifuge at 10000 g for 20-30 Filter supernatant to remove large min at room temperatute. particles.

Collect filtrate and pass an Analyze with HPLC. aliquot through a 0.45 µm filter.

Figure 2. Flow diagram of the optimized extraction procedure and analysis of FOS from the muffins

Accurately weigh 10 ± 0.05 g Mix 30 min at room and combine with 20 ± 0.1 ml temperature. of water

Collect supernatant. Add 10 ± Centrifuge at 10000 g for 20- 0.1 ml of water to the pellet 30 min at room temperatute. and mix 30 min at room temperature

Filter supernatants to remove Collect filtrates eand pass an aliquot through a 0.45 µm large particles. filter.

Analyze with HPLC.

Figure 3. Flow diagram of the optimized extraction procedure and analysis of FOS from the cookies.

Figure 4. Flow diagram of the optimized extraction procedure and analysis of FOS from the nutrition bar

Accurately weigh 10 ± 0.05 g Mix 30 min at room and combine with 10 ml of 1:1 temperature. ethanol :water

Centrifuge at 10000 g for 20- Filter supernatant to remove 30 min at room temperatute. large particles.

Collect filtrate and pass an Dry sample under vacuum at aliquot through a 0.45 µm 50 °C to obtain a dry residue. filter.

Resuspend sample in nanopure water at a final volume Analyze with HPLC. resulting in 1% solution.

Accurately measure 1.0 ± 0.05 ml.

Filter sample through a 0.45 µm filter.

Analyze with HPLC.

Figure 5. Flow diagram of the optimized extraction procedure and analysis of FOS from the nutrition bar

Appendix 2

Flow diagrams of the optimized extraction procedure and analysis of

inulin from the different food products

Cool solution to Accurately weigh Add hot distilled room temperature, 1.0±0.05 g of a test water at 80°C; stir adjust volume to 50 portion. 80°C for 15 min. ml with distilled water, and mix.

Filter aliquot of Centrifuge at 1000 g solution through Transfer 0.2 ml of for 20 min ,or until paper Whatman No. filtrate. pellet is formed. 1, or equivalent.

Add 0.2 ml of Add 0.2 ml alkaline sucrase/amylase borohydride solution solution, and to each tube and Add 0.5 ml acetic incubate at 40°C for incubate at 40°C for acid and mix. 30 min. 30 min.

Add 0.1 ml of 0.1 M Add 0.1 mL of sodium acetate Transfer four 0.2 ml fructanase to 3 of buffer to the 4th aliquots. these samples. sample (sample blank).

Add 5.0 ml Incubate in boiling Incubate at 40°C for PAHBAH working water bath for 6 min. 20 min. reagent.

Remove all tubes from boiling water bath and Measure absorbance immediately place in of all solutions at cold water (18–20°C) 410 nm. for ca 5 min. Figure 1. Flow diagram of the optimized extraction procedure and analysis of inulin from the extruded cereal.

Cool solution to Accurately weigh Add hot distilled room temperature, 1.0±0.05 g of a test water at 80°C; stir adjust volume to 50 portion. 80°C for 15 min. ml with distilled water, and mix.

Filter aliquot of Centrifuge at 1000 g solution through Transfer 0.2 ml of for 20 min ,or until paper Whatman No. filtrate. pellet is formed. 1, or equivalent.

Add 0.2 ml of Add 0.2 ml alkaline sucrase/amylase borohydride solution solution, and to each tube and Add 0.5 ml acetic incubate at 40°C for incubate at 40°C for acid and mix. 30 min. 30 min.

Add 0.1 ml of 0.1 M Add 0.1 mL of sodium acetate Transfer four 0.2 ml fructanase to 3 of buffer to the 4th aliquots. these samples. sample (sample blank).

Add 5.0 ml Incubate in boiling Incubate at 40°C for PAHBAH working water bath for 6 min. 20 min. reagent.

Remove all tubes from boiling water bath and Measure absorbance immediately place in of all solutions at cold water (18–20°C) 410 nm. for ca 5 min. Figure 2 Flow diagram of the optimized extraction procedure and analysis of inulin from the muffins.

Cool solution to Accurately weigh Add hot distilled room temperature, 1.0±0.05 g of a test water at 80°C; stir adjust volume to 50 portion. 80°C for 15 min. ml with distilled water, and mix.

Filter aliquot of Centrifuge at 1000 g solution through Transfer 0.2 ml of for 20 min ,or until paper Whatman No. filtrate. pellet is formed. 1, or equivalent.

Add 0.2 ml of Add 0.2 ml alkaline sucrase/amylase borohydride solution solution, and to each tube and Add 0.5 ml acetic incubate at 40°C for incubate at 40°C for acid and mix. 30 min. 30 min.

Add 0.1 ml of 0.1 M Add 0.1 mL of sodium acetate Transfer four 0.2 ml fructanase to 3 of buffer to the 4th aliquots. these samples. sample (sample blank).

Add 5.0 ml Incubate in boiling Incubate at 40°C for PAHBAH working water bath for 6 min. 20 min. reagent.

Remove all tubes from boiling water bath and Measure absorbance immediately place in of all solutions at cold water (18–20°C) 410 nm. for ca 5 min. Figure 3. Flow diagram of the optimized extraction procedure and analysis of inulin from the nutrition bar.

Cool solution to Accurately measure Add hot distilled room temperature, 1.0 ±0.05 ml of a water at 80°C; stir adjust volume to 50 test portion. 80°C for 15 min. ml with distilled water, and mix.

Add 0.2 ml of Filter aliquot of sucrase/amylase solution through Transfer 0.2 ml of solution, and paper Whatman No. filtrate. incubate at 40°C for 1, or equivalent. 30 min.

Add 0.2 ml alkaline borohydride solution Transfer four 0.2 ml to each tube and Add 0.5 ml acetic aliquots. incubate at 40°C for acid and mix. 30 min.

Add 0.1 ml of 0.1 M Add 0.1 mL of sodium acetate fructanase to 3 of buffer to the 4th Incubate at 40°C for these samples. sample 20 min. (sample blank).

Remove all tubes from Add 5.0 ml boiling water bath and Incubate in boiling PAHBAH working immediately place in water bath for 6 min. reagent. cold water (18–20°C) for ca 5 min.

Measure absorbance of all solutions at 410 nm.

Figure 3. Flow diagram of the optimized extraction procedure and analysis of inulin from the sports drink.

Appendix 3

Flow diagrams of the optimized extraction procedure and analysis of

GOS from different food products M

Prepare a solution Prepare a solution of Accurately weigh 0.5 ± galactose and glucose myo-inositol at the Vortex inmediatly 0.05 g and combine and the internal same concentration of after adding water with 50.0 ± 0.1 ml of standard to (myo- the expected water to achive 1mg/ml to prevent gel inositol) each at concentration of the GOS concentration . formation. 1mg/ml (STD). sugar in sample (ISTD)

Mix 30 min at room Hydrolyze: Pressure temp Transfer 250 µl of Add 50 µl of 2.8M sample (S) to a 35 ml cook in High (15 psi sulfuric acid and 50 µl Centrifuge at 4500 rpm open test tube with and for 20 min to obtain screw cap. of ISTD to sample (S). pellet 80 °C for 1 h).

Add 40 µl of freshly Add 40 µl of glacial Add 75 µl of 12 M prepared 150 mg/ml Incubate at 40 °C for 1 acetic acid; mix. ammonium hydroxide alkaline sodium h. Add 0.5 ml of 1- and mix. borohydride. methylimidazole; mix.

Add 5 ml of acetic anhydride (slowly); Let stand 10 min at Add 1 ml absolute Let stand 10 min at mix. room temperature. ethanol. room temperature.

Move tubes to a cooler Slowly add 5 ml of Let stand 5 min at room Add another 5 ml of 7.5 with ice up to shoulder well mixed 7.5 M temperature. M sodium hydroxide. of tube sodium hydroxide; mix.

Transfer ethyl acetate Analyze the STD at as (top) layer to a fresh Analyze by GC. the first and last tube. injection of the runs.

Figure 1. Flow diagram of the optimized extraction procedure and analysis of GOS from the extruded cereal. Prepare a solution Prepare a solution of Accurately measure N galactose and glucose myo-inositol at the same 1.0 ± 0.05 ml and and the internal concentration of the combine with 100.0 ± standard to (myo- expected concentration 0.1 ml of water to inositol) each at of the sugar in sample achive 1mg/ml GOS 1mg/ml (STD). (ISTD) concentration .

Transfer 250 µl of Add 50 µl of 2.8M Hydrolyze: Pressure sample (S) to a 35 ml sulfuric acid and 50 cook in High (15 psi open test tube with µl of ISTD to sample and screw cap. (S). 80 °C for 1 h).

Add 40 µl of freshly Add 75 µl of 12 M prepared 150 mg/ml Incubate at 40 °C for ammonium hydroxide alkaline sodium 1 h. and mix. borohydride.

Add 40 µl of glacial acetic acid; mix. Add 5 ml of acetic Let stand 10 min at Add 0.5 ml of 1- anhydride (slowly); room temperature. methylimidazole; mix. mix.

Move tubes to a Add 1 ml absolute Let stand 10 min at cooler with ice up to ethanol. room temperature. shoulder of tube

Slowly add 5 ml of Add another 5 ml of well mixed 7.5 M Let stand 5 min at 7.5 M sodium sodium hydroxide; room temperature. hydroxide. mix.

Transfer ethyl acetate Analyze the STD at as (top) layer to a fresh Analyze by GC. the first and last tube. injection of the runs.

Figure 2. Flow diagram of the optimized extraction procedure and analysis of GOS from the extruded cereal.