Luận văn thạc sĩ Công nghệ thực phẩm: Utilization of enzyme-treated wheat bran in making pasta with high fiber content

VIETNAM NATIONAL UNIVERSITY SYSTEM HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY FACULTY OF CHEMICAL ENGINEERING DEPARTMENT OF FOOD TECHNOLOGY MASTER’S THESIS UTILIZATION OF ENZYME-TREATED W

Dietary fiber

Definition, classification and physiological effects of dietary fiber

Dietary fibers are carbohydrate polymers with three or more monomeric units and not hydrolyzed by the endogenous enzymes of the human small intestine (Joint FAO/WHO Food Standards Programme, 2015) Based on their solubility in water, dietary fibers can be classified into insoluble and soluble dietary fibers Lignin, cellulose, and some hemicelluloses belong to the insoluble dietary fiber; while pectic polysaccharides, β-glucans, galactomannans, fructans, gums, and other non-starch polysaccharides are the representatives of soluble dietary fiber (Maphosa & Jideani, 2016)

Insoluble and soluble dietary fibers have different effects on normal gut activity The insoluble fibers are neither digested nor fermented in the gastrointestinal tract They are mainly responsible for absorption, swelling and holding of water molecules within their porous structures (Mudgil, 2017) The hydration of insoluble fibers may result in increased fecal bulk and softened stool, therefore improving proper bowel movement and facilitating laxation (Foschia et al., 2013) In contrast, the soluble fibers are fermented by the colonic microbiota in the large intestine with various extents Some of them are slowly fermented and exert laxative effect, whereas the others are rapidly fermented and serve as substrates for potentially beneficial bacteria (McRorie, 2015) Such soluble fibers selectively stimulate the growth and activity of these microorganisms, thus enhancing the host health This phenomenon is clinically designated as prebiotic effect (Slavin, 2013)

Certain soluble fibers such as β-glucan, psyllium and guar gum may increase the viscosity of the aqueous phase when ingested The consumption of this kind of fibers might lead to the decrease in the plasma cholesterol level and glycemic response (Stephen et al., 2017) The mechanism by which those fibers can lower the serum cholesterol level is the entrapment and elimination of bile via the stool (McRorie, 2015) In addition, the attenuation of glycemic response by soluble fibers could be explained by the limited contact between digestive enzymes and complex substrates due to the elevated viscosity, which in turn impedes the degradation of macromolecules into readily absorbed nutrients (McRorie, 2015) As a consequence, the absorption of glucose and other nutrients at the brush border of small intestine is slowed

It can be noted that more physiologically beneficial effects are associated with the consumption of soluble fiber compared to insoluble fiber The ratio of insoluble fiber to soluble fiber (IDF/SDF) provides an important information about the nutritional quality of foods The American Dietetic Association recommends a dietary fiber intake of 25–30 g/day for adults, in which an IDF/SDF ratio of 3:1 is advised to maximize potential health benefits of dietary fiber (Valdivia-López & Tecante, 2015) Other authors suggested that a source of dietary fiber should have an IDF/SDF ratio close to 2:1 to be accepted as a food ingredient (Rafiq et al., 2016).

Wheat bran–A potential source of dietary fiber

The wheat (Triticum spp.) kernel is made of 03 major components, i.e the bran, the germ and the endosperm During the conventional milling process of wheat grain, the majority of endosperm is separated from the germ and the bran Wheat bran occurs as a milling by-product Histologically, the bran comprises different tissue layers, namely the fruit coat, the seed coat, the hyaline layer, and the aleurone layer–a part of the starchy endosperm (Apprich et al., 2014) Wheat bran is considered as a concentrated source of dietary fiber The total dietary fiber content of this cereal material varies between 33.4 and 63.0% The proportion of each constituent in the total fiber content of wheat bran is shown in Table 2.1 The soluble portion of wheat bran fiber is less than 5% and consists of glucans and xylans (Onipe et al., 2015) The IDF/SDF ratio of wheat bran is apparently unbalanced and higher than the advised value Therefore, it is recommended to reduce this ratio by enzymatic treatment

Table 2.1 Dietary fiber components in wheat bran (Hemdane et al., 2015)

Dietary fiber Content (% dry basis)

Conversion of insoluble fiber into soluble fiber by enzymatic treatment

The use of specific endocarbohydrase could be a feasible strategy for conversion of insoluble dietary fibers into soluble fibers Concerning insoluble polysaccharides, the cleavage of internal glycosidic linkage by endocarbohydrases may lead to the formation of short-chain molecules, which could be soluble in water On the contrary, the action of exohydrolases may result in the formation of low molecular weight carbohydrates Thus, in order to minimize the transformation of fibers into undesired sugars, the presence of exocarbohydrase activity in the enzyme preparation with a large amount is not preferred

In cereal brans, xylan and cellulose are the most abundant insoluble fibers (Mudgil & Barak, 2013) Hence, utilization of xylanase and cellulase preparation may be useful for transforming insoluble fibers of brans into soluble fibers

Xylanases are a group of carbohydrase that catalyze the hydrolysis of xylan Xylanase preparation are mostly produced by bacteria and filamentous fungi Due to the heterogeneity and complicated chemical nature, the complete breakdown of xylan requires the action of several enzymes The xylanolytic enzyme system usually includes β-1,4-endoxylanase; β-xylosidase, acetyl xylan esterase, arabinase, α-glucuronidases, ferulic acid esterase, and p-coumaric acid esterase (X Liu & Kokare, 2017) All of these enzymes act cooperatively to hydrolyze xylan into constituent monomers Endoxylanase (EC 3.2.1.8) is of great importance since it can cleave the main backbone of xylan by catalyzing the hydrolysis of β-1,4 glycosidic linkages in xylan

Cellulases are multienzyme complexes with 03 different major components, namely endo-1,4-β-D-glucanase (EC 3.2.1.4), exo-glucanase/exo-cellobiohydrolase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21) All of these components act synergistically to hydrolyze cellulose completely into glucose Endo-1,4-β-D-glucanase attacks first and randomly to generate reducing and non-reducing ends, which are further cleaved by exo- cellobiohydrolase to release cellobiose Cellobiose is eventually cleaved by β-glucosidase (BGL) into 02 glucose molecules (Kuhad, Gupta, & Singh, 2011) BGL is the limiting factor in overall performance of cellulase complex since it is inhibited by its end-product, i.e glucose To convert insoluble fibers into soluble fibers, the BGLs which are sensitive to glucose inhibition are desired due to the restricted saccharification Normally, BGLs of Trichoderma reesei are subject to product inhibition (Singhania, Adsul, Pandey, &

Patel, 2017) Hence, cellulase preparations obtained from this fungus could be useful for converting cellulose into soluble fibers.

Application of cellulase and xylanase preparations in processing of cereal brans

Xylanase preparations have been applied in the cereal processing industry to improve the product quality (Singh et al., 2015) Endoxylanase preparations are used to solubilize arabinoxylan in rye bran (Figueroa-Espinoza, Poulsen, Borch Sứe, Zargahi, & Rouau, 2004) and also wheat bran (O Santala, Lehtinen, Nordlund, Suortti, & Poutanen, 2011; O K Santala, Nordlund, & Poutanen, 2013) Xylanase treatment is employed to produce feruloyl oligosaccharides by hydrolyzing xylan of wheat bran (Yuan, Wang, & Yao, 2006) Ingelbrecht, Moers, Abécassis, Rouau, and Delcour (2001) prepare pasta from durum semolina with the aid of an endoxylanase Treatment with endoxylanase is reported to lower extrusion pressure and increase the level of soluble fiber (i.e water- extractable arabinoxylans)

Cellulase preparations are commonly applied in processing cellulosic materials Treatment of wheat bran with cellulase preparation is applied to assist the extraction of xylo-ogliosaccharide (Zhao & Dong, 2016) Aktas-Akyildiz et al (2017) report that processing wheat bran with Cellulast 1.5L (a cellulase preparation) increases the soluble fiber content of bran In addition, the supplementation with enzyme-treated wheat bran significantly improves the soluble fiber content of bread samples Similarly, Vien and Le (2018) conclude that the cookie samples incorporated with cellulase-treated wheat bran exhibited higher soluble fiber content compared to those containing untreated bran These authors also establish the appropriate condition for the treatment of wheat bran with Viscozyme Cassava C (a commercial cellulase preparation) to transform insoluble fibers into soluble fibers, in which soluble fiber content increases by 38% and IDF/SDF ratio is reduced by 74%.

Pasta with high fiber content

Technology of making pasta with high fiber content

The conventional pasta made from durum wheat semolina has a low dietary fiber content, which is 1.2 g/100 g The Codex Alimentarius (2009) recommends that any products claiming to be a “source” of fiber should contain at least 3 g of fiber per 100 g of serving or 1.5 g of fiber per 100 kcal of serving; while to claim that a food is “high” in fiber, the product must contain at least 6 g of fiber per 100 g of serving or 3 g of fiber per

100 kcal of serving (Foschia et al., 2013) Generally, there are 02 types of materials for manufacture of pasta with improved fiber dietary content This kind of products can be prepared from the durum whole-wheat flour (whole-wheat pasta) or from the mixture of durum semolina and other fiber-rich ingredients (fiber-enriched pasta)

In the simplest production line, the whole-wheat flour or the mixture of semolina and fiber-rich materials are mixed with water to make a crumb-like dough before being kneaded The kneading and forming process can be done either manually or mechanically by laminators or extruders (Robin & Palzer, 2015) Lamination is used to produce linguini and square spaghetti while extrusion is used to make spaghetti and macaroni The formed dough sheets or extruded strands are then cut into appropriate shapes There are 02 main types of pasta manufactured: the fresh pasta, which is minimally dried or not dried, has a higher moisture content and therefore its shelf life is shorter; whereas the dried pasta has moisture content lower than 12% and has a longer shelf life.

Quality of fiber-enriched pasta

The incorporation of fiber-rich ingredients is found to improve the nutritional quality of pasta products The total dietary fiber and protein content of pasta is reported to be increased when introducing mushroom powders (Lu et al., 2018), deoiled chia flour (Aranibar et al., 2018), or wheat bran (Sobota, Rzedzicki, Zarzycki, & Kuzawińska, 2014) into the formulation The incorporation of some fiber-rich ingredients, namely β-glucan, resistant starch, and oat bran results in a lowered glycemic index of cooked pasta after in vitro digestion (Bustos, Perez, & León, 2011; Chillo, Ranawana, & Henry, 2011) In addition, certain fiber preparations are demonstrated to have high antioxidant activity, such as deoiled chia flour, carob fiber, and wheat bran Thus, the inclusion of such fibers into pasta causes an increase in the antioxidant activity of the final product (Aranibar et al., 2018; Aravind, Sissons, Egan, & Fellows, 2012; Biernacka, Dziki, Gawlik-Dziki, Różyło, & Siastała, 2017)

The incorporation of fibers is reported to affect the cooking quality of pasta, including optimal cooking time, cooking loss, and swelling index Aravind et al (2012) and Sobota et al (2014) figure out that the optimal cooking time is decreased when the level of insoluble fiber increases With regard to the cooking loss, this parameter is raised as the inclusion level of bran, β-glucan and inulin is elevated (Chillo et al., 2011; Kong et al., 2012; Tudoricǎ, Kuri, & Brennan, 2002) In terms of swelling index, this value is reported to vary with contrasting patterns in different studies The study of Foschia et al (2013) showed that all of the spaghetti samples fortified with fiber preparations (i.e oat bran, psyllium, β-glucan, and inulin) demonstrate a higher swelling index than that of the sample prepared from semolina only However, other publications report a decrease in the swelling index of pasta and rice noodle at increased enrichment level of durum bran (Aravind et al., 2012) or okra (Kang, Bae, & Lee, 2018)

The effects of fiber enrichment on the textural and sensory quality of pasta are reported in the literature In general, the addition of fiber-rich materials into pasta is found to negatively impact the texture of final product The texture profile of cooked pasta enriched with deoiled chia flour, wheat bran, and resistant starch shows a lower firmness compared to that of the sample without fiber (Aranibar et al., 2018; Bustos et al., 2011; Sobota et al., 2014) With respect to the sensory quality, the increase in the incorporation level of fiber generally causes a loss in consumer acceptance of pasta and noodle product (Biernacka et al., 2017; Bustos et al., 2011; B.-R Kim, Kim, Bae, Chang, & Moon, 2017) These findings also reveal that fiber-enriched pasta has a reduced score for appearance, firmness, color, taste, and overall acceptability Moreover, Aravind et al (2012) conclude that pasta samples incorporated with cereal brans exhibit a floury mouthfeel, coupled with a darker color and rougher surface compared to sample without bran.

Quality improvement of fiber-enriched pasta

Methods for improving textural quality of fiber-enriched pasta

It is known that the formation of starch–gluten network during the mixing and kneading step would determine the textural characteristics of pasta A proper pasta texture should be resistant to stretching and elastic in nature (C S Brennan, 2013) The addition of fiber may reduce the gluten content and physically disrupt the continuity of the starch– gluten network The weakened and disrupted starch–gluten matrix would exert negative effects on the textural integrity of pasta (Rakhesh, Fellows Christopher, & Sissons, 2014)

At the higher incorporation level of fiber, the gluten content of the dough would be lower and the disruptive impact of fiber on the starch–protein network would be more severe

According to the aforementioned changes, the enhancement in textural quality of pasta could be accomplished by increasing protein content of the dough and improving the continuity of the starch–protein matrix The augmentation in the protein content of dough could be delivered by the addition of other protein preparations, most importantly vital wheat gluten On the other hand, the formation of new covalent cross-links catalyzed by transglutaminase enzyme (EC 2.3.2.13) may strengthen the starch–protein network The reformed pasta texture is expected to improve the sensory quality of final product.

Improvement in pasta texture by addition of vital wheat gluten

Gluten is commonly found in wheat, rye, and barley It is defined as the protein fraction that is not soluble in water and NaCl 0.5 mol/L Gluten constitutes 80–90% the protein content in flour (Ortolan & Steel, 2017) The gluten is composed of monomeric gliadins (50–55%) and polymeric glutenins (45–50%) The gliadin fraction primely contains single polypeptide chains with MW range of 30,000–75,000 Da The gliadin chains associate with each other and with glutenins through hydrogen bond and hydrophobic interactions The polymeric glutenins are divided into high molecular weight (MW > 100,000 Da) and low molecular weight subunit (MW ≈ 45,000 Da) The high molecular weight subunit of the glutenin fraction is responsible for providing the viscoelastic property of gluten due to the ability to form an intermolecular network Meanwhile, the low molecular weight subunit is crucial for the formation of glutenin macropolymer by intermolecular disulfide bonds (Day, 2011)

Wheat gluten is a common food ingredient, produced by drying wet gluten after being separted from the starch slurry It is marketed into 02 types, “vital” and “nonvital” When in contact with water, vital wheat gluten can hydrate rapidly and restore its original functionality while nonvital wheat gluten does not (Esteller, Pitombo, & Lannes, 2005) Vital wheat gluten is widely used in the bakery industry to improve the crumb structure of baked product (Ortolan & Steel, 2017) The absence of gluten or reduction in its content may result in the lack of viscoelasticity in dough and therefore difficulty in obtaining the desired product texture and appearance (Moore, Juga, Schober, & Arendt, 2007)

The use of vital wheat gluten in the production of noodle and pasta product from low protein flours is reported in the litterature Raina, Singh, Bawa, and Saxena (2005) figure out at the base flour protein level of 16–18%, noodles prepared from broken rice flour and supplemented with vital gluten show similar texture to commercial semolina pasta The introduction of vital gluten is found to improve the cooking quality, firmness, tensile strength, and overall acceptability of oat noodle (Zhou, Zhu, Shan, Cai, & Corke, 2011) and white salted noodle (Park & Baik, 2009) Nevertheless, the findings of Cuicui, Qiyu, Zipeng, and Huili (2018) reveal that the inclusion of gluten with low disulfide bonds concentration causes an increase in the cooking loss of Chinese white noodle.

Improvement in pasta texture by the use of transglutaminase preparation

Transglutaminase preparations have been widely used in dairy, bakery, and meat processing (Kuraishi, Yamazaki, & Susa, 2001) The transglutaminase enzyme (protein- glutamine γ-glutamyl transferase) is found in microorganisms, plant, fish, and, mammals

It catalyzes the reaction between an ε-amino group in protein-bound lysine residues and a β-carboxyamide group in protein-bound glutamine residues This reaction leads to the formation of covalent cross-linking of proteins (Yokoyama, Nio, & Kikuchi, 2004) The generation of intermolecular cross-linkings induced by transglutaminase could enhance the rheological properties of the gluten network (Basman, Koksel, & Atli, 2006)

The utilization of transglutaminase preparations in noodle and pasta making is reported in several publications The addition of transglutaminase is found to lower the cooking loss of lupin flour noodle (Bilgiỗli & İbanoğlu, 2014), oat noodle (Wang, Huang, Kim, Liu, & Tilley, 2011), durum wheat pasta (Krisztina Takács, Gelencsér, & Kovács, 2007), and white salted noodle (Wu & Corke, 2005) In contrast, other authors conclude that the increase in transglutaminase dosage causes an elevation in cooking loss (Shiau & Chang, 2013) or does not change the cooking performance of noodle and pasta (Sissons, Aravind, & Fellows, 2010) On the other side, the addition of transglutaminase was found to improve the water absorption and swelling index of durum pasta (Krisztina Takács et al., 2007) and and gluten-free noodle (K Takács, 2007) Treatment with transglutaminase is reported to successfully enhance the textural quality of conventional pasta (Krisztina Takács et al., 2007), bran-enriched pasta (Basman et al., 2006) and whole wheat noodle (Niu, Hou, Kindelspire, Krishnan, & Zhao, 2017) Apart from the texture improvement, gluten-free noodle and pasta produced with the aid of transglutaminase also show a lower glycemic index (Gan, Ong, Wong, & Easa, 2009; Rosa-Sibakov et al., 2016).

Originality of this research

To the best of our knowledge, the effects of adding enzyme-treated wheat bran on the antioxidant activity and glycemic index of pasta have not been reported in any publications On the other hand, although some studies have used the vital gluten addition and transglutaminase treatment to improve the quality of noodle and pasta product, the effectiveness of both methods combined on quality of bran-enriched pasta is still unknown Our research would deal with the possibility of using vital gluten fornication, treatment with transglutaminase, and combination of gluten–transglutaminase addition to enhance the textural and sensory quality of our high fiber pasta product

Materials

Materials for making high fiber pasta

Wheat bran was provided by Binh Dong Milling Factory (Ho Chi Minh City, Vietnam) The proximate composition of wheat bran was as follows: moisture content ≤ 13%, lipid content ≤ 7%, and protein content ≥ 14% The obtained wheat bran was further milled and passed through a 35-mesh (0.5 mm) sieve The processed bran was packaged in PE (polyethylene) bags and stored at ‒18 o C

Durum wheat semolina was supplied by Vietnam Flour Mills Company (Ba Ria– Vung Tau Province, Vietnam) The proximate composition of durum semolina was as follows: moisture content ≤ 13%, lipid content ≤ 3%, protein content ≥ 12%, and total carbohydrate content ≥ 70% Durum semolina was kept in PE bags and stored at –18 o C

Vital wheat gluten was purchased from Roquette (Singapore) The vital gluten preparation has moisture content ≤ 12%, ash content ≤ 1%, protein content ≥ 80%, and gluten index ≥ 38 Gluten index is defined as the percentage of wet gluten remaining on a standardized sieve after centrifugation (Kaushik, Kumar, Sihag, & Ray, 2015) Gluten preparation was kept in PE bags and stored at 4 o C

Table salt was acquired from Southern Salt Group (Ho Chi Minh City, Vietnam) Table salt has moisture content ≤ 1% and sodium chloride content ≥ 99% Table salt was kept in PE bags and stored under ambient condition

Cellulase preparation under the trade name Viscozyme Cassava C was purchased from Novozyme (Denmark) This enzyme preparation is produced by Trichoderma reseii The determined cellulase activity was 233 U/mL One unit (U) of cellulase activity is defined as 1 μmol of reducing sugar released from CMC (carboxymethylcellulose) per minute under the assay condition (temperature of 50 o C, incubation time of 10 min, and pH 4.8) (Ghose, 1978) The optimal temperature and pH range of cellulase in this preparation are 45–60 o C and 4–6, respectively The enzyme preparation in liquid form was kept in HDPE (high density polyethylene) container and stored at 4 o C

Transglutaminase preparation under the trade name Protiact TG-GW was from Rama (Thailand) The determined transglutaminase activity was 109 U/g One unit (U) of transglutaminase activity is defined as the amount of enzyme that catalyzes the formation of 1 àmol hydroxamate per minute from N-carbobenzoxy-L-glutaminylglycine and under the assay condition (temperature of 37 o C and pH 6.0) (Ando et al., 1989) The optimal temperature and pH range of transglutaminase in this preparation are 40–55 o C and 5–8, respectively The enzyme preparation in powder form was kept in HDPE container and stored at –18 o C.

Chemicals

α-Amylase (trade name: Termamyl SC, α-amylase activity: 240 Kilo Novo α- amylase unit/mL), amyloglucosidase (AMG) (trade name: Dextrozyme GA, AMG activity: 270 AMG unit/mL) and protease preparation (trade name: Alcalase 2.5L, protease activity: 2.5 Anson unit/mL) was purchased from Novozyme (Denmark) Pepsin from porcine gastric mucosa (650 unit/mg protein) and pancreatin from porcine pancreas (4 × USP specifications) were acquired from Sigma-Aldrich (Saint Louis, Missouri, United States) Folin‒Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6- tri(2-pyridyl)-s-triazine (TBTZ), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and gallic acid were from Sigma-Aldrich (Saint Louis, Missouri, United States); 3,5-Dinitrosalicylic acid (DNS) and Nessler reagent were obtained from Merck (Darmstadt, Germany) Other chemicals were of analytical grade.

Methods

Procedures

i) Preparation of enzyme-treated pasta

Figure 3.1 shows the procedure for preparation of enzyme-treated wheat bran

Treated wheat bran Sieving Dilution

Figure 3.1 Preparation of enzyme-treated wheat bran

 Enzymatic treatment: The enzymatic treatment was carried out at 50 o C, 35 rpm for 2 h in a jacketed bioreactor with a U-shaped mixing blade (Model MD9501S, Malayan Daching Co., Singapore) The enzyme solution was slowly added to the wheat bran flour during mixing After incubation, the mixture was heated at 95 o C for 5 min for enzyme inactivation

 Drying: The treated bran sample were spread on a metallic tray and put into a dryer The height of the bran layer was about 1 cm The temperature and relative humidity of drying air was 50 o C and 55–65%, respectively The drying time was about 4 h

The final moisture content of dried bran was 10–11%

 Grinding: The dried bran sample was ground by a knife blender to reduce the size of agglomerated particles The size of each batch was 100 g and the grinding time was

 Sieving: The ground wheat bran particles that passed through a 35-mesh (0.5 mm) sieve were recovered and used to produce bran-enriched pasta

2i) Production of bran-enriched pasta

The procedure for making bran-enriched pasta at the laboratory scale is shown in Figure 3.2

Kneading Semolina, bran, table salt

Bran-enriched pasta Extrusion Distilled water

Figure 3.2 Production of bran-enriched pasta

 Mixing: The raw materials, including durum semolina, wheat bran flour, and table salt were mixed in the mixing bowl In the formulation of bran-enriched pasta, durum semolina was partially substituted by wheat bran (untreated or enzyme-treated) The substitution level of wheat bran varied according to each experiment The content of table salt was 0.5% on the basis of the base flour

 Hydration: Distilled water at 42 o C was slowly added, combined with mixing at ambient pressure to make a dough This process was performed in a stand mixer (Model SM8005, Ichiban Co., Japan) with a U-shape blade attached The mixing rate was 120 rpm The proportion of distilled water was 32% on the basis of the dough

 Kneading: The dough was kneaded for 20 min at ambient pressure This process was carried out in a stand mixer (Model SM8005, Ichiban Co., Japan) with a dough hook attached The mixing rate was 120 rpm

 Extrusion: The kneaded dough was fed to an extruder (Model HR2365/05, Philip Co., Netherlands) to form pasta strands The extrusion pressure was 720 kgf/cm 2

The diameter of the extrusion die was 1.6 mm The pasta strands with 50 cm length were cut and hang on metallic sticks

 Drying: Freshly extruded pasta strands were dried for 8 h in a convection dryer The temperature and relative humidity of the drying air were 50 o C and 55–65%, respectively The final moisture content of dried pasta was 10–12%

 The dried pasta samples were stored in sealed plastic bags at ‒18 o C until analysis For the evaluation of cooking qualities, dried pasta strands were broken into pieces of 5 cm in length prior to storage

3i) Preparation of gluten-fortified pasta

The procedure for making gluten-fortified pasta at the laboratory scale is shown in Figure 3.3

Kneading Semolina, bran, table salt, gluten

Gluten-fortified pasta Extrusion Distilled water

Figure 3.3 Production of gluten-fortified pasta

The raw materials, including durum semolina, enzyme-treated wheat bran flour, table salt, and vital gluten preparation were mixed in the mixing bowl The content of table salt was 0.5% on the basis of base flour Distilled water at 42 o C was slowly added to the base flour, combined with mixing to hydrate the ingredients The proportion of distilled water was 32% on the basis of the dough The remaining steps were performed as described in section 3.1.1.2i with the same technological parameters

4i) Preparation of transglutaminase-treated pasta

The procedure for making transglutaminase-treated pasta at the laboratory scale is shown in Figure 3.4

Kneading Semolina, bran, table salt

Figure 3.4 Production of transglutaminase-treated pasta

The raw materials, including durum semolina, enzyme-treated wheat bran flour, and table salt were mixed in a mixing bowl The content of table salt was 0.5% on the basis of base flour Transglutaminase preparation in powder form was dissolved in distilled water at 42 o C The enzyme solution was slowly added, combined with mixing to form a dough The proportion of distilled water was 32% on the basis of the dough The dough was kneaded for 20 min at ambient pressure, then covered in plastic wrap and incubated at 40 o C for 10 min The remaining steps were performed as described in section 3.1.1.2i with the same technological parameters

5i) Combination of vital gluten addition and transglutaminase treatment in pasta making

In this procedure, all of the raw materials (i.e durum semolina, enzyme-treated wheat bran flour, vital gluten, and table salt) were initially mixed The content of table salt was 0.5% on the basis of base flour Transglutaminase preparation in powder form was dissolved in distilled water at 42 o C The enzyme solution was slowly added, combined with mixing to form a dough The proportion of distilled water was 32% on the basis of the dough The remaining stages were performed as previously described in section 3.1.1.4i with the same technological parameters.

Experimental design

The experimental design of the research is summarized in Figure 3.5

1 Investigating the effects of adding untreated and treated wheat bran on pasta qualities

2 Investigating the effects of gluten fortification on pasta qualities

● antioxidant activity (total phenolic content, DPPH inhibition activity and FRAP)

3 Investigating the effects of transglutaminase treatment on pasta qualities

4 Investigating the effects of gluten fortification combined with transglutaminase treatment on pasta qualities

Figure 3.5 Experimental design i) Investigating the effect of adding untreated and treated bran on pasta qualities

 Varied parameter: The substitution levels of untreated and treated bran flour were 0,

 Salt and water content in the pasta formulation

 Kneading, extrusion and drying condition

 Total phenolic content of raw and cooked pasta

 Antioxidant activity of raw and cooked pasta: DPPH and FRAP assays

 Predicted glycemic index determined by in vitro digestion

2i) Investigating the effect of gluten fortification on pasta qualities

 Varied parameter: The addition levels of gluten were 0, 5, 10% and 15% on the basis of semolina‒bran mixture

 Substitution level of enzyme-treated wheat bran flour: 20%

 Salt and water content in pasta formulation

 Kneading, extrusion and drying condition

 Proximate composition of raw spaghetti: content of protein, lipid, starch, ash and dietary fiber (TDF, IDF and SDF)

 Cooking quality of spaghetti: optimal cooking time, cooking loss, swelling index, and water absorption index

 Antioxidant properties: total phenolic content, DPPH and FRAP assays

 Texture profile of cooked spaghetti: firmness, adhesiveness, springiness cohesiveness, gumminess, chewiness, tensile strength, and elongation rate

 Sensory quality of cooked spaghetti: overall acceptability

3i) Investigating the effect of transglutaminase treatment on pasta qualities

 Varied parameter: The dosages of transglutaminase were 0, 0.25, 0.50 and 0.75 U/g gluten

 Substitution level of enzyme-treated wheat bran flour: 20%

 Salt and water content in pasta formulation

 Kneading, extrusion and drying condition

 Proximate composition of raw pasta: content of protein, lipid, starch, ash and dietary fiber (TDF, IDF and SDF)

 Cooking quality of pasta: optimal cooking time, cooking loss, swelling index, and water absorption index

 Antioxidant properties: total phenolic content, DPPH and FRAP assays

 Texture profile of cooked pasta: firmness, gumminess, chewiness, adhesiveness, springiness, cohesiveness, tensile strength, and elongation rate

 Sensory quality of cooked pasta: overall acceptability

4i) Investigating the effect of gluten fortification combined with transglutaminase treatment on pasta qualities

 Replacement level of enzyme-treated wheat bran flour: 20%

 Salt and water content in pasta formulation

 Kneading, extrusion and drying condition

 Proximate composition of raw pasta: content of protein, lipid, starch, ash and dietary fiber (TDF, IDF and SDF)

 Cooking quality of pasta: optimal cooking time, cooking loss, swelling index, and water absorption index

 Antioxidant properties: total phenolic content, DPPH and FRAP assays

 Texture profile of cooked pasta: firmness, gumminess, chewiness, adhesiveness, springiness, cohesiveness, tensile strength, and elongation rate

 Sensory quality of cooked pasta: overall acceptability

The description of dough formulation and transglutaminase (TG) dosage used in pasta making is summarized in Table 3.1

Table 3.1 Dough formulation and transglutaminase (TG) dosage used in pasta making

Analytical methods

The moisture content was measured by drying at 105 o C in a moisture analyzer (ML-50, A&D, Tokyo, Japan) The protein content was determined by Kjeldahl method with a nitrogen-to-protein conversion factor of 5.8 for wheat (Fujihara, Sasaki, Aoyagi,

& Sugahara, 2008) The total lipid content was determined by a Soxhlet extraction method The ash content was determined by incineration at 600 o C The IDF and SDF content was determined according to Method 991.42 and 993.19 (AOAC, 2000), respectively The total starch content was quantified by Method 996.11 (AOAC, 2000) The gluten content of durum semolina was determined according to the method of Kaushik et al (2015)

Frozen pasta was defrosted for 20 min at room temperature prior to evaluation of cooking quality About 5 g of raw pasta is cooked in 50 mL of boiling distilled water The water level was kept at a constant level during the entire cooking period by continuously supplying the boiling water The optimal cooking time (OCT) of pasta is determined as the time when the white central core of the pasta disappeared after cross- cutting it with a razor blade (Aravind et al., 2012) To determine the cooking loss and swelling index, pasta samples are cooked to OCT The cooking water is collected from each sample and evaporated to constant weight at 105 o C in a convection dryer Cooking loss is calculated as the percentage of residual solid weight to raw pasta weight (Aravind et al., 2012) After cooking and draining, the pasta samples were analyzed for water absorption index and swelling index Water absorption index (WAI) was measured as follows: weight of cooked pasta weight ra pof w sta

WAI weig a r ht of a p taw as

Swelling index (SI) was determined by drying cooked pasta to constant weight at 105 o C in a convection dryer and calculated as follows: weight of asta weight of asta after drying

SI weight cooked p p of asta after drp ying

Five measurements of cooking quality in terms of cooking loss, WAI and SI were performed each pasta sample

In vitro digestion of cooked pasta was done following the method of M A

Brennan, Merts, Monro, Woolnough, and Brennan (2008) and Chillo et al (2011) with slight modifications Frozen pasta was defrosted at room temperature, cooked to OCT, and cut into pieces of 2‒5 mm (Foschia, Peressini, Sensidoni, Brennan, & Brennan, 2015) About 2 g of cooked and sliced pasta were mixed with 30 mL of distilled water and 0.8 mL of HCl 1M to achieve a pH of 2.5 After incubating at 37 o C for 10 min in a water bath, pepsin (1 mL of a 10% w/v solution in 0.05 M of aqueous HCl) was added and the incubation was maintained for a further 30 min at 37 o C The pH was subsequently adjusted to 6.9 by adding 1 mL of NaHCO3 1M, then 0.1 mL of amyloglucosidase and 5 mL of pancreatin (2.5% w/v solution in phosphate saline buffer, pH 6.9) were added The total volume was adjusted to 50 mL with phosphate saline buffer pH 6.9 and incubated at

37 o C with slow and constant mixing During the incubation, 1 mL of aliquot was withdrawn at 0, 30, 60, 90, 120, 150, and 180 min and then transferred into tubes containing 4 mL of absolute ethanol These were stored at ‒18 o C for analysis of reducing sugars using the spectrophotometric method with DNS reagent Before the analysis, the contents of the tubes were centrifuged at 3,000×g for 10 min to recover the supernatants

A calibration curve employing D-glucose was constructed The absorbance was read at

540 nm (CT-2300 Spectrophotometer, E-Chrom Tech, Taiwan) The amount of reducing sugars (expressed as glucose equivalent) was converted into the amount of hydrolyzed starch by multiplying by 0.9

A non-linear model established by Goủi et al (1997) was applied to describe the kinetics of starch hydrolysis The pseudo-first-order equation has the following formula:

CC  e  where C corresponds to the percentage of hydrolyzed starch at time t; C ∞ corresponds to the equilibrium percentage of hydrolyzed starch after 180 min; k is the kinetic constant and t is the time Non-linear regression was performed in ORIGIN PRO 8 to estimate the parameters

The rate of starch digestion was expressed as the percentage of hydrolyzed starch to the initial starch content at different time points (30, 60, 90, 120, 150 and 180 min) The area under the starch hydrolysis curve (AUC) was determined according to Hur, Lim, Decker, and McClements (2011):

The hydrolysis index (HI) was obtained by dividing the AUC of each sample by the AUC of the reference sample (fresh white bread)

HI AUC sample rof eference 100

The predicted glycemic index (pGI) was determined according to Ren et al (2016): pGI = 39.71 + 0.059HI

The extraction of antioxidant compounds were done as previously described by Biney and Beta (2014) with slight modifications To 1 g of sample, 10 mL of acidified methanol (concentrated HCl : methanol : water, 1:80:20) was added in 15 mL centrifuge tubes, vortexed and shaken at room temperature for 2 h The mixture was then centrifuged for 15 min at 4000 rpm The supernatant was collected and used for the total phenolic content, DPPH radical inhibition and ferric reducing ability (FRAP) assay

● Determination of total phenolic content

The Folin‒Ciocalteau method adapted by Beta, Nam, Dexter, and Sapirstein (2005) was used A portion of extract (0.2 mL) was added to 1.5 mL of a 10-fold freshly diluted Folin‒Ciocalteau reagent After vortexing, the mixture was allowed to equilibrate for 5 min, after which 1.5 mL of sodium carbonate solution (60 g/L) was added, combined with further vortexing The reaction was allowed to progress at room temperature and away from light for 90 min The absorbance was read at 725 nm Acidified methanol was used as blank A standard curve of versus absorbance was constructed using gallic acid The results were expressed as mg equivalent of gallic acid/100 g dry matter

● Determination of DPPH radical scavenging activity

Antioxidant activity was measured according to the method of Biney and Beta (2014) The method involves the use of the free radical 2,2-diphenyl-l-picrylhydrazyl (DPPH), where antioxidants were allowed to react with the stable radical in a methanol solution The discoloration of the DPPH radicals was followed by monitoring the decrease in its absorbance at a characteristic wavelength during the reaction The extract (0.1 mL) was reacted with 3.9 mL of a 60 μmol/L of DPPH solution (2.4 mg of DPPH in 100 mL of methanol) The absorbance (A) at 515 nm was read at 0 and 30 min Methanol was the blank Antioxidant activity was calculated as percentage of discoloration P discoloration:

A standard curve of different Trolox concentrations versus activity was constructed and results were expressed as μmol equivalent of Trolox/100 g dry matter

● Determination of reducing power (FRAP)

FRAP assay was performed according to Benzie and Strain (1996) with slight modifications In brief, the fresh working solution was prepared by mixing acetate buffer pH 3.6 with a 10 mM TPTZ solution in 40 mM HCl and a 20 mM FeCl3.6H2O solution (10:1:1, respectively) 20 μL of sample were added to 3.8 mL of FRAP working solution The mixture was then incubated in the dark for 15 min, and the absorbance was measured at 593 nm The results were expressed as μmol equivalent of Trolox/100 g dry matter

The color parameters of pasta (uncooked and cooked) were measured by Konica Minolta CR400 chroma meter (Osaka, Japan) using the CIELAB color space The results were expressed as L*, a*, and b* values L* value represents the lightness (0: darkest black, 100: brightest white), a* value describes the redness (positive) and greenness

(negative), and b* value describes the yellowness (positive) and blueness (negative)

Color parameters were read at different locations along the pasta strand The chroma C and hue angle h were calculated as follows:

  The total color difference ΔE was determined as follows:

       whereL a b * 0 , , * 0 0 * correspond to the values of the pasta without bran (100% semolina); while

L a b correspond to the values of the examined pasta sample

All samples were cooked to optimal cooking time on the day of determination

A texture analyzer (Stable Micro System TA-XT plus, United Kingdom) equipped with a Windows version of the Exponent Connect Lite software package was used to conduct the texture analysis The determined textural parameters were pasta firmness, springiness, cohesiveness, gumminess, tensile strength and elongation rate

In the Texture Profile Analysis (TPA), the probe compressed the samples at a speed of 1 mm/s to 30% strain The probe was subsequently retracted and followed by a second compression cycle after 1 s The results (springiness, cohesiveness, chewiness and gumminess) were recorded

In the tensile strength, 01 strand of cooked pasta was fixed to the arms of tensile grips The force (tensile strength) at the break point was measured at a speed of 1 mm/s The diameter of cooked pasta was determined to calculate the cross-sectional area The tensile strength TS and elongation rate ER were as follows:

TS = F / A where F is the force at the break point and A is the cross-sectional area of cooked pasta strand Elongation rate ER is calculated as follows:

ER = (L 1 ‒ L 2) / L 2 × 100% where L 1 is the stretch distance and L 2 is the set distance

Statistical analysis

All of the measurements were done in triplicate The results in this study were expressed as mean ± standard deviation One-way analysis of variance (ANOVA) and Turkey’s comparison test with significance level set at p < 0.05 were performed by using Minitab 17 software (Minitab Inc., United States)

Proximate composition, color, and antioxidant activity of raw materials

Proximate composition of raw materials

The proximate composition of raw materials is shown in Table 4.1

Table 4.1 Proximate composition of raw materials

– Values that do not share a lowercase letter within a row are significantly different (p < 0.05)

It is evident that both enzyme-treated and enzyme-treated wheat bran showed a remarkably higher protein, ash and total dietary fiber content in comparison with durum semolina This suggests that the incorporation of wheat bran into the conventional flour formulation can improve the nutritional quality of pasta The amino acid profile of wheat bran protein is of higher quality than that of endosperm (Balandrán-Quintana, Mercado- Ruiz, & Mendoza-Wilson, 2015) In addition, the minerals found in wheat bran are mainly

Fe, Zn, Mn, Mg and P (Onipe et al., 2015) On the other hand, durum semolina and gluten preparation had the highest starch and protein content, respectively

It should be noted that the cellulase treatment did not change the composition of wheat bran, except for the dietary fiber content After the enzymatic treatment, the soluble fiber content of wheat bran was increased by 14.5%, while the insoluble fiber content was decreased by 23.3% The IDF/SDF ratio of enzyme-treated wheat bran was 30.6% lower than that of untreated one The augmentation in soluble fiber content could be due to conversion of cellulose into soluble fragments by the action of cellulase The enzymatic hydrolysis also caused a slight reduction in the total fiber content of wheat bran This was probably due to generation of low molecular weight carbohydrates (LMWCs) In the determination of soluble fiber content, these LMWCs are hardly precipitated in 78% ethanol aqueous solution (McCleary et al., 2012) They could not be recovered and that results in the loss of total dietary fiber content.

Color parameters of raw materials

Table 4.2 depicts the color parameters of raw materials

Table 4.2 Color parameters of raw materials

Raw material Semolina Untreated wheat bran

‒4.89 ± 0.02 c 20.70 ± 0.20 b 21.27 ± 0.19 a 166.71 ± 0.16 c Values that do not share a lowercase letter within a row are significantly different (p < 0.05)

As can be seen from Table 4.2, durum semolina showed the highest L* value (lightness), followed by gluten preparation, untreated wheat bran and enzyme-treated wheat bran In contrast, the a* value (redness) of semolina was found to be the lowest, while the highest redness was attributed to enzyme-treated wheat bran The b*, C, and h value of semolina were generally lower than those of wheat bran and gluten preparation

It is noticeable that processing wheat bran with cellulase resulted in a 9.8% reduction in lightness and a 70.0% increase in redness The slightly darkened appearance of wheat bran could be explained by the formation and accumulation of Maillard reaction products during the enzymatic treatment procedure.

Antioxidant activity of raw materials

The antioxidant activity of raw materials is shown in Table 4.3

Table 4.3 Antioxidant activity of raw materials

Raw material Semolina Untreated wheat bran

854.1 ± 15.9 a Values that do not share a lowercase letter within a row are significantly different (p < 0.05)

The total phenolic content (TPC), DPPH antiradical activity and ferric reducing ability of the untreated wheat bran were 3.2, 7.6 and 45.1 times higher than those of the durum semolina, respectively It is reported that majority of antioxidants in wheat grain are the phenolic compounds located in the bran layer (Yu, Nanguet, & Beta, 2013) The TPC and ferric reducing power of both untreated and cellulase-treated wheat bran were not statistically significant (p < 0.05) L Liu et al (2017) report that a complex enzymatic hydrolysis employing α-amylase, glucoamylase, protease, and cellulase significantly increases the bioavailability of the antioxidative phenolics in rice brans In cereal brans, most of the phenolic compounds are conjugated with the cell wall components (Călinoiu

& Vodnar, 2018) Hence, the release of those phenolics required a combination of several enzymatic treatments

Additionally, the cellulase treatment led to a 18.7% increase in DPPH inhibition activity of wheat bran A partial explanation to this phenomenon could be the formation and accumulation of Maillard reaction products during the incubation with enzyme and drying process of treated bran The Maillard reaction generally involves the interaction between amino acids and reducing sugars or lipid oxidation products (Yilmaz & Toledo, 2005) The products derived from Maillard reaction were demonstrated to have various extents of in vitro radical scavenging activity (Nooshkam, Varidi, & Bashash, 2019).

Effects of wheat bran incorporation on the qualities of pasta

Antioxidant activity

The antioxidant activity of uncooked pasta samples is shown in Table 4.4

Table 4.4 Antioxidant activity of uncooked pasta samples

Sample code Pasta incorporated with untreated wheat bran Pasta incorporated with enzyme-treated wheat bran

S WB10 WB20 WB30 S EWB10 EWB20 EWB30

(mg GAE/100g d.m.) 108.4 ± 2.1 A,b 113.9 ± 4.3 A,b 139.5 ± 4.4 A,a 156.2 ± 5.9 A,a 108.4 ± 2.1 A,b 123.5 ± 3.6 A,b 146.8 ± 6.0 A,a 163.3 ± 6.8 A,a DPPH inhibition activity

(μmol TE/100 g d.m.) 185.9 ± 15.1 A,d 249.3 ± 12.9 A,c 374.5 ± 8.1 B,b 529.9 ± 16.2 B,a 185.9 ± 15.1 A,d 286.5 ± 8.2 A,c 460.1 ± 13.4 A,b 626.1 ± 24.0 A,a Ferric reducing power

Values that do not share a lowercase letter within a row are significantly different (p < 0.05)

Values that do not share an uppercase letter within a same level of bran are significantly different (p < 0.05)

In general, the addition of both untreated and enzyme-treated wheat bran led to the increase in antioxidant activity of raw pasta, including total phenolic content, DPPH radical scavenging activity and ferric reducing power At the enrichment level of 10%, pasta enriched with untreated bran did not show any significant differences (p > 0.05) in TPC and ferric reducing ability in comparison with the control However, as the content of untreated bran increased from 0 to 30%, the TPC, DPPH antiradical activity and FRAP of uncooked pasta increased by 44.1% The incorporation of antioxidant-rich materials into pasta to improve the antioxidant activity is reported in several publications (Aranibar et al., 2018; Biney & Beta, 2014; Lu et al., 2018) The antioxidants are suggested to involve in the protection against oxidative damages of cell and tissue The consumption of antioxidant plays an important role in the prevention of many diseases associated with the oxidative stress, such as breast cancer, cardiovascular diseases, and diabetes mellitus (R Dias, G Alves, Casal, F Oliveira, & M Silva, 2017)

A similar trend was also found in case of introducing enzyme-treated wheat bran into pasta formulation Particularly, pasta fortified with enzyme-treated bran exhibited a higher DPPH inhibition activity in comparison with pasta containing untreated bran at a same enrichment level This could be due to the fact that enzyme-treated wheat bran had a higher DPPH antiradical activity than untreated one It is postulated that the products of Maillard reactions formed during the enzymatic treatment procedure of wheat bran could be responsible for higher DPPH radical scavenging activity The furan ring and nitrogen containing brown compounds (i.e melanoidins) contributed to the antioxidant properties of baked grain products (Manzocco, Calligaris, Mastrocola, Nicoli, & Lerici, 2000).

In vitro starch digestion and predicted glycemic index of pasta

Figure 4.1 and Figure 4.2 illustrate the in vitro starch digestion and predicted glycemic index of pasta samples, respectively

Figure 4.1 In vitro starch digestion of pasta samples

Figure 4.2 Predicted glycemic index of pasta samples

Data that do not share a lowercase letter within a same type of bran are significantly different (p < 0.05) Data that do not share an uppercase letter within a same level of bran are significantly different (p < 0.05)

In our study, an in vitro method was used to evaluate the nutritional quality of high fiber pasta with regard to starch digestibility In this procedure, the percentage of hydrolyzed starch was monitored over a 3h digestion period and fitted to a pseudo-first- order model established by Goủi, Garcia-Alonso, and Saura-Calixto (1997) The selected model showed a good fit to our data with R 2 > 0.95 (Table 6.2) This would enable the calculation of predicted glycemic index of pasta samples

The highest rate of starch hydrolysis was observed from 0 to 30 min for all pasta samples (Figure 4.1) This could correspond to the digestion of starch granules at the outer layer of the pasta strand, since they were more swollen and physically accessible for the enzyme compared to that of the central layer (Camelo-Méndez, Agama-Acevedo, Rosell, de J Perea-Flores, & Bello-Pérez, 2018) After 30 min of the digestion time, the rate of

H y dro ly ze d st a rc h ( %)

H y dro ly ze d st a rc h ( %)

S WB10 WB20 WB30 S EWB10 EWB20 EWB30

P re dict ed g ly ce m ic ind ex starch hydrolysis was decreased This could be associated with the digestion of starch granules located at the intermediate and central region of pasta strand The dense protein network at the inner region of pasta restrict the amylolytic attack on starch granules (Zou, Sissons, Warren, Gidley, & Gilbert, 2016)

It is known that the rate of starch digestion governs the glycemic index of foods Foods with high glycemic index have the starch fractions digested and absorbed rapidly, leading to marked fluctuations in blood glucose (Foschia et al., 2015) It is evident that pasta incorporated with wheat bran showed a lower extent and rate of starch hydrolysis compared to sample without bran (Figure 4.1) The attenuated starch hydrolysis led to the reduction in the predicted glycemic index (pGI) of pasta (Table 4.2) At the enrichment level of 10%, there was no significant difference in pGI value of pasta enriched with untreated bran and bran-free pasta However, when the content of untreated and enzyme- treated wheat bran elevated from 0 to 30%, the pGI value of cooked pasta was significantly reduced by 14.3% and 20.0%, respectively

The rate and extent of starch digestion in food are controlled by several factors: structure of starch granules, structure of food, the amount of dietary fiber, protein and fat within the product (Dona, Pages, Gilbert, & Kuchel, 2010) In this research, the reduction in pGI of the wheat bran-enriched pasta could be due to the reduced availability of starch granules to α-amylase As the enrichment level of wheat bran elevated, the protein, dietary fiber and lipid content of pasta was also increased It is suggested that protein and dietary fiber might provide stearic hindrance, preventing α-amylase from being in contact with the starch granules (Hager, Czerny, Bez, Zannini, & Arendt, 2013) Additionally, the complexation between starch and lipid could play a certain role in the attenuation of starch hydrolysis (Lu et al., 2018)

On the other hand, the pGI of pasta enriched with enzyme-treated wheat bran was lower compared to pasta produced from untreated bran This was probably due to the high soluble content of pasta incorporated with enzyme-treated bran The presence of soluble fibers in the digesta could attenuate the glycemic response by the 02 following mechanisms: i) increase in the viscosity of the digesta, which may retard the enzymatic degradation of macromolecules (Chillo et al., 2011), 2i) complexation with α-amylase (e.g certain gums), forming gum–amylase complex that may lack of catalytic activity due to the competitive inhibition (Camelo-Méndez et al., 2018).

Effects of vital gluten addition and transglutaminase (TG) treatment on the qualities of

Proximate composition

The proximate composition of gluten-fortified and TG-treated pasta samples is shown in Table 4.5

The addition of gluten preparation generally led to an augmentation in protein content, however, a reduction in ash, starch and total dietary fiber content This is due to the fact that the gluten preparation has remarkably higher protein content but lower ash, starch and total dietary fiber content compared to wheat bran and durum semolina The lipid content among all pasta samples was found to be similar The pasta sample enriched with 15% vital gluten had 55.5% higher protein content and 13.1% lower total fiber content compared to the control Despite the marginal reduction in fiber content, all of the gluten-fortified pasta samples can be regarded as high fiber foods according to Codex Alimentarius (2009), since their total fiber content were higher than 6% (Foschia et al., 2013) On the other hand, the TG treatment did not change the proximate composition of pasta samples In addition, the pasta sample fortified with 5% and treated with TG showed a slightly reduced total dietary fiber content (4.4%) compared to the control All of the pasta samples showed a relatively balanced IDF/SDF ratio, about 4:1 In conclusion, the addition of vital gluten into flour formulation could slightly lower the total dietary fiber content, while treatment with TG did not produce any variation in fiber content

Table 4.5 Proximate composition of pasta samples

Sample code Protein (% d.m.) Lipid (% d.m.) Ash (% d.m.) Starch (% d.m.) TDF (% d.m.) IDF (% d.m.) SDF (% d.m.) IDF/SDF

EWB20 13.80 ± 0.44 d 3.95 ± 0.07 a 1.73 ± 0.06 a 74.46 ± 0.97 a 8.95 ± 0.15 a 7.16 ± 0.09 a 1.79 ± 0.06 a 4.00 ± 0.07 a G5 16.66 ± 0.30 c 3.98 ± 0.05 a 1.64 ± 0.05 ab 72.22 ± 0.88 ab 8.54 ± 0.05 b 6.82 ± 0.08 c 1.72 ± 0.03 a 3.96 ± 0.11 a G10 18.67 ± 0.27 b 3.99 ± 0.06 a 1.56 ± 0.07 b 69.45 ± 0.87 bc 8.17 ± 0.10 c 6.50 ± 0.07 d 1.66 ± 0.03 ab 3.91 ± 0.03 a G15 21.32 ± 0.72 a 4.02 ± 0.03 a 1.41 ± 0.06 c 66.41 ± 1.07 c 7.78 ± 0.07 d 6.24 ± 0.07 d 1.54 ± 0.04 b 4.05 ± 0.14 a T0.25 13.69 ± 0.50 d 3.97 ± 0.04 a 1.73 ± 0.06 a 73.05 ± 1.75 a 8.93 ± 0.06 a 7.13 ± 0.15 a 1.80 ± 0.09 a 3.98 ± 0.29 a T0.50 13.60 ± 0.59 d 3.93 ± 0.04 a 1.74 ± 0.06 a 73.22 ± 0.57 a 8.96 ± 0.13 a 7.14 ± 0.14 a 1.82 ± 0.08 a 3.93 ± 0.22 a T0.75 13.97 ± 0.13 d 3.96 ± 0.05 a 1.72 ± 0.02 a 73.67 ± 1.88 a 8.92 ± 0.05 a 7.11 ± 0.08 ab 1.81 ± 0.04 a 3.92 ± 0.12 a G5T0.25 16.53 ± 0.38 c 3.99 ± 0.04 a 1.64 ± 0.02 ab 72.65 ± 0.17 ab 8.54 ± 0.06 b 6.84 ± 0.07 bc 1.70 ± 0.04 a 4.02 ± 0.12 a

Values that do not share a letter within a column are significantly different (p < 0.05).

Cooking qualities

Table 4.6 demonstrates the cooking qualities of gluten-fortified and TG-treated pasta samples, namely optimal cooking time (OCT), cooking loss, water absorption index (WAI) and swelling index

Table 4.6 Cooking qualities of pasta samples

Sample code OCT (min) Cooking loss (%) WAI Swelling index

Values that do not share a letter within a column are significantly different (p < 0.05)

OCT and cooking loss are commonly used parameters to evaluate the cooking performance of pasta by consumers and producers (Tudoricǎ et al., 2002) It is expected that during cooking, pasta should release a minimal amount of matter into cooking water The supplementation of vital gluten into flour formulation caused an increase in OCT, nevertheless, a reduction in cooking loss As the vital gluten level raised from 0 to 15%, the OCT was elevated by 31.8% while the cooking loss was reduced by 19.3% These result is in agreement with Zhou et al (2011), who produce oat noodle with the aid of vital gluten The increased OCT could be attributed to the restricted water diffusion into pasta strand when cooking (Park & Baik, 2009) It is known that the incorporation of insoluble fibers into pasta could disrupt the gluten–starch matrix, which in turn providing paths for water to penetrate during cooking (Aravind et al., 2012) The addition of vital gluten would strengthen the gluten network, resulting in a more compact pasta structure The reinforced protein network could retard the leaching of starch and other exudates from pasta into cooking water Consequently, the cooking loss of pasta is reduced On the other hand, when the addition level of vital gluten elevated from 0 to 15%, the WAI and swelling index of pasta were reduced by 17.6% and 15.6%, respectively This could be explained by the reduction in the starch content of raw pasta (Rakhesh et al., 2014)

The TG-treated pasta samples exhibited slightly higher OCT and lower cooking loss compared to the control As the TG dosage increased from 0 to 0.75 U/g gluten, the OCT of pasta was raised by 9.1% whereas the cooking loss was decreased by 8.3% A similar trend is noted by Basman et al (2006) The authors report a decrease in total organic matter of cooking water when the TG dosage is increased The microstructural analysis shows that the introduction of cross-links catalyzed by TG improves the integrity of gluten network (Wu & Corke, 2005) As a consequence, the diffusion of water into pasta strand during cooking is limited Moreover, the strengthened protein network may act as a barrier and restrict the leaching of exudates from pasta to cooking water (Y Kim, Kee, Lee, & Yoo, 2014) On the contrary, other authors report that the cooking loss is not significantly affected by TG treatment (Wu & Corke, 2005) or even increased at high TG concentration (Aalami & Leelavathi, 2008) The intensified extent of cross-linking within gluten proteins at high TG dosage might weaken the protein–starch interactions, which facilitates the leaching of starch granule into cooking water (Aalami & Leelavathi, 2008)

On the other hand, the TG treatment did not produce any changes in the WAI and swelling index of pasta These results correspond to the similar starch content among TG-treated pasta samples Other publications also show an unchanged WAI and swelling index of noodle on the increasing TG dosage (Basman et al., 2006) However, Sissons et al (2010) reported a slight reduction in WAI of pasta It is hypothesized that the swelling of starch granules is prevented due to the compact and flexible protein network formed by TG treatment The differences in the results of the mentioned studies could be attributed to the variation in materials, pasta making process, dosage of TG, and incubation time

The pasta produced by the combination of vital gluten addition and TG treatment (G5T0.25 sample) exhibited a longer cooking time and a lower cooking loss compared to the pasta only enriched with vital gluten (G5 sample) or treated with TG (T0.25 sample) When vital gluten is supplemented into the base flour, the number of lysine and glutamine residues in the dough would be increased The augmentation in the quantity of acyl donor (glutamine) and acyl receptor (lysine) residues could facilitate the cross-linking induced by TG Consequently, the formation of cross-link would be more intensive, resulting in a further strengthened gluten network The protein network with high integrity could act as barrier to prevent the diffusion of water molecules into pasta as well as the leaching of exudates into cooking water.

Texture profile

Table 4.7 demonstrates the texture profile of gluten-fortified and TG-treated pasta samples

Table 4.7 Texture profile of pasta samples

Sample code Hardness (g) Gumminess (g) Chewiness (g) Adhesiveness

(g.s) Springiness Cohesiveness Tensile strength (kPa)

EWB20 805.1 ± 18.3 f 741.3 ± 64.3 f 515.7 ± 53.7 e 110.0 ± 14.8 a 0.70 ± 0.03 a 0.88 ± 0.01 a 33.6 ± 1.5 e 49.0 ± 3.6 e G5 1277.8 ± 117.9 d 1029.6 ± 84.8 de 801 ± 67.6 de 119.7 ± 18.4 a 0.74 ± 0.05 a 0.87 ± 0.02 a 44.9 ± 1.5 c 56.5 ± 4.9 de G10 1728.2 ± 53.2 b 1533.1 ± 38.4 b 1221.8 ± 52.5 b 125.4 ± 21.0 a 0.80 ± 0.03 a 0.89 ± 0.01 a 57.8 ± 2.7 b 78.2 ± 7.8 b G15 2320.1 ± 141.8 a 1994.9 ± 148.7 a 1650.6 ± 196.1 a 128.6 ± 20.5 a 0.83 ± 0.04 a 0.86 ± 0.02 a 78.7 ± 3.4 a 107.8 ± 5.2 a T0.25 932.8 ± 27.9 ef 801.4 ± 47.9 ef 551.4 ± 86.2 e 113.2 ± 14.5 a 0.69 ± 0.08 a 0.86 ± 0.03 a 34.8 ± 3.2 e 52.1 ± 6.2 de T0.50 1157.2 ± 148.7 de 1008.6 ± 147.3 def 748.5 ± 103.8 de 98.9 ± 17.5 a 0.74 ± 0.02 a 0.87 ± 0.01 a 38.4 ± 2.5 de 56.5 ± 6.5 cde T0.75 1361.7 ± 83.8 cd 1181.2 ± 57.3 cd 898.4 ± 143.7 cd 107.8 ± 16.9 a 0.76 ± 0.08 a 0.87 ± 0.02 a 43.8 ± 0.4 cd 65.7 ± 0.9 bcd G5T0.25 1582.4 ± 151.2 bc 1322.7 ± 115.3 bc 1134.8 ± 115 bc 102.4 ± 15.5 a 0.73 ± 0.11 a 0.89 ± 0.01 a 52.1 ± 1.2 b 70.9 ± 1.0b c

Values that do not share a lowercase letter within a column are significantly different (p < 0.05)

The texture profile of cooked pasta was assessed by the texture profile analysis (TPA) and tensile test It is evident that the supplementation of vital gluten resulted in a remarkably enhanced texture profile of cooked pasta At the incorporation level of 5%, TPA revealed that the hardness, gumminess, and chewiness of gluten-fortified pasta were 71.1%, 61.4%, and 71.5% respectively higher than those of pasta prepared from durum semolina and treated wheat bran (EWB20) Previous studies reveal that noodle prepared from flour with a higher protein content generally shows a firmer texture, perhaps due to the strengthened protein network (Raina et al., 2005; Shiau & Yeh, 2001) In addition, the pasta supplemented with 5% vital gluten had the tensile strength and elongation rate 44.4% and 49.4% higher respectively than those of EWB20 sample This indicates that the addition of vital gluten enhanced the elasticity When the enrichment level of vital gluten increased from 0 to 15%, the hardness, gumminess, chewiness, tensile strength, and elongation rate were dramatically raised by 188.2%, 169.1%, 220.1%, 134.2%, and 120.0%, respectively On the other hand, there was no significant difference (p > 0.05) in the adhesiveness, springiness, and cohesiveness among all cooked pasta samples

The TG treatment was shown to improve the texture profile of cooked pasta At the TG dosage of 0.25 U/g, the texture profile of TG-treated pasta was found to be similar to that of EWB20 sample However, as the TG dosage increased up to 0.75 U/g, the firmness, gumminess, and chewiness of cooked pasta were increased by 69.1%, 59.3% and 74.2%, respectively The augmentation in firmness and gumminess induced by TG treatment was also reported by Aalami and Leelavathi (2008) and Wu and Corke (2005), who worked on durum spaghetti and white salted noodle The enhanced firmness was attributed to the cross-linking function of TG, which could act directly on the proteins in flour and hence reinforcing the structure of the protein network (Wu & Corke, 2005) However, Sissons et al (2010) report that the firmness of pasta might be reduced after incubation for 40 min A partial explanation to this phenomenon is the high degree of cross-linking achieved after a long incubation time with TG, making the gluten network more elastomeric and allowing relaxation after extrusion This may result in the loss of a preferred orientation parallel to the extrusion direction that otherwise would improve the firmness (Sissons et al., 2010) Apart from this, the tensile test revealed that the tensile strength and elongation rate of pasta treated with TG at dosage of 0.75 U/g were 30.4% and 34.1% respectively higher than those of the EWB20 sample The study of Li, Tan,

Liong, and Easa (2014) also reveals an augmentation in the tensile strength of chili- layered noodle in case of using TG

In our study, it can be noted that the extent of texture improvement delivered by

TG treatment was generally lower compared to gluten fortification method Notably, the combination of gluten addition and TG treatment resulted in a further enhanced texture of cooked pasta compared to utilization of each method alone The firmness and tensile strength of pasta fortified with 5% gluten and treated with TG at dosage of 0.25 U/g gluten (G5T0.25 sample) were 22.1% and 11.5% respectively higher than those of pasta only enriched with 5% gluten, respectively Moreover, the G5T0.25 sample had firmness and tensile strength 80.4% and 55.5% higher than those of sample solely treated with TG at dosage of 0.25 U/g gluten The better texture obtained by fusion of vital gluten addition and TG treatment could due to the introduction of more lysine and glutamine residues from gluten This may lead to the increase in the number of cross-links catalyzed by TG, eventually strengthening the protein network.

Color parameters

Table 4.8 and Table 4.9 show the color parameters of uncooked and cooked pasta samples, respectively

Table 4.8 Color parameters of uncooked pasta samples

EWB20 79.79 ± 0.38 bc ‒2.77 ± 0.05 c 19.15 ± 0.08 bc 19.35 ± 0.08 bc 171.78 ± 0.16 de – G5 79.39 ± 0.30 cd ‒2.48 ± 0.05 b 19.29 ± 0.24 bc 19.45 ± 0.24 bc 172.68 ± 0.07 c 0.57 ± 0.23 bc G10 79.01 ± 0.11 d ‒2.36 ± 0.04 b 19.63 ± 0.13 b 19.77 ± 0.14 b 173.15 ± 0.07 b 1.01 ± 0.14 b G15 78.19 ± 0.21 e ‒2.17 ± 0.07 a 20.45 ± 0.23 a 20.57 ± 0.23 a 173.94 ± 0.16 a 2.16 ± 0.25 a T0.25 80.05 ± 0.15 b ‒2.66 ± 0.05 c 19.13 ± 0.14 c 19.31 ± 0.13 bc 172.08 ± 0.15 d 0.32 ± 0.11 c T0.50 80.35 ± 0.22 b ‒2.71 ± 0.03 c 18.39 ± 0.27 de 18.59 ± 0.27 de 171.61 ± 0.13 e 0.95 ± 0.34 b T0.75 80.99 ± 0.07 a ‒3.00 ± 0.02 d 17.97 ± 0.07 e 18.22 ± 0.07 e 170.51 ± 0.01 f 1.70 ± 0.06 a G5T0.25 80.11 ± 0.14 b ‒2.78 ± 0.02 c 18.80 ± 0.14 cd 19.01 ± 0.14 cd 171.60 ± 0.11 e 0.47 ± 0.19 bc

Values that do not share a lowercase letter are within a column significantly different (p < 0.05).

Table 4.9 Color parameters of cooked pasta samples

EWB20 54.00 ± 0.10 d ‒0.25 ± 0.19 a 18.54 ± 0.29 a 18.81 ± 0.30 a 179.24 ± 0.59 a – G5 56.01 ± 0.46 bc ‒0.76 ± 0.01 bc 18.59 ± 0.23 a 18.57 ± 0.28 a 177.65 ± 0.04 bc 2.03 ± 0.47 bc G10 56.95 ± 0.23 ab ‒0.96 ± 0.03 cd 18.80 ± 0.31 a 18.88 ± 0.46 a 177.07 ± 0.08 cd 2.97 ± 0.22 ab G15 57.98 ± 0.58 a ‒1.30 ± 0.15 d 18.86 ± 0.46 a 18.64 ± 0.23 a 176.05 ± 0.47 d 4.03 ± 0.57 a T0.25 54.46 ± 0.23 cd ‒0.35 ± 0.11 ab 18.46 ± 0.43 a 18.47 ± 0.43 a 178.93 ± 0.30 ab 0.78 ± 0.35 c T0.50 55.08 ± 0.02 cd ‒0.47 ± 0.05 ab 18.35 ± 1.02 a 18.36 ± 1.02 a 178.53 ± 0.10 ab 1.46 ± 0.31 bc T0.75 55.44 ± 0.42 bcd ‒0.64 ± 0.13 abc 18.04 ± 0.42 a 18.05 ± 0.42 a 177.97 ± 0.45 abc 1.67 ± 0.55 bc G5T0.25 56.93 ± 1.47 ab ‒0.49 ± 0.34 ab 19.30 ± 0.19 a 19.31 ± 0.19 a 178.54 ± 0.98 ab 3.00 ± 1.44 ab

Values that do not share a lowercase letter within a column are significantly different (p < 0.05).

In general, the fortification of vital gluten into flour formulation caused a slight reduction in the lightness (0.5–2.0%), coupled with an increase in redness (10.5–27.6%) and yellowness (0.7–6.8%) of the raw pasta The chroma and hue angle of uncooked pasta also experienced a marginal increase when vital gluten was added The slightly reduced

L* value of raw pasta may be attributed to the intensified formation of browning products from Maillard reaction at higher protein content On the other hand, the TG treatment was shown to slightly raise the lightness of both raw (0.9–1.5%) and cooked (0.9–2.7%) pasta This was probably due to the retarded development of Maillard reaction products, since the number of free amino groups (–NH2) in proteins could be reduced to form the cross- links under the catalysis of TG (Aalami & Leelavathi, 2008)

The addition of gluten and TG treatment with transglutaminase did not produce any noticeable variation in the color of pasta When the level of vital gluten and TG dosage elevated, the total color differences of both raw and cooked pasta were slightly raised Pasta sample fortified with gluten and treated with TG also showed a minute increase in the total color difference prior to cooking and after cooking However, the total color differences of all pasta samples were lower than 10, indicating that the variation in color among pasta samples could not be visually discriminated (Aalami & Leelavathi, 2008).

Overall acceptability

The sensory quality of pasta samples in terms of overall acceptability is shown in Table 4.10

Table 4.10 Overall acceptability of pasta samples

T0.25 5.48 ± 1.41 bc T0.50 5.83 ± 1.32 ab T0.75 6.15 ± 1.38 ab G5T0.25 5.75 ± 1.33 ab Values that do not share a letter within a column are significantly different (p < 0.05).

The incorporation of vital gluten was demonstrated to improve the consumer acceptance of cooked pasta At the fortification level of 5%, the overall acceptability of gluten-fortified pasta was similar to the control produced from durum semolina and 20% wheat bran (EWB20 sample) When the level of gluten increased up to 10%, the overall acceptability of pasta significantly was raised by 24.4% The pasta incorporated with 15% vital gluten showed the highest overall acceptability among all samples, which was 30.9% higher than that of the control The improvement in consumer acceptance appeared to correlate with the enhancement in texture of cooked pasta The study of Zhou et al (2011) also reveals that the overall acceptability of oat noodles is significantly increased by 60% when the level of gluten is increased from 0 to 10%

On the other side, TG treatment was also shown to enhance the sensory quality of pasta in terms of overall acceptability At the TG dosage of 0.25 U/g, TG-treated pasta sample showed a similar overall acceptability to the control (EWB20 sample) However, the acceptability was significantly improved as the TG dosage beyond 0.25 U/g The pasta treated with TG dosage of 0.75 U/g had the overall acceptability 25.0% higher than that of EWB20 sample The improved structure of pasta offered by TG treatment could be the reason for the enhancement in consumer acceptance Niu et al (2017) conclude that the integrity attribute of whole wheat noodle was improved when TG treatment was used The positive correlation between texture (firmness and elasticity) of TG-treated alkaline noodle with its sensory properties was also reported by Yeoh, Alkarkhi, Ramli, and Easa (2011) In our study, a positive and significant correlation (p < 0.05) was found between overall acceptability and firmness (r = 0.97), gumminess (r = 0.95), chewiness (r = 0.93) tensile strength (r = 0.97) and elongation rate (r = 0.95) of processed with TG

It is noticeable that the pasta enriched with vital gluten and processed the aid of

TG (G5T0.25 sample) exhibited a significantly higher overall acceptability in comparison with to EWB20 sample However, the overall acceptability of G5T0.25 sample was not statistically different (p > 0.05) compared to that of gluten-fortified pasta (G5 sample) or TG-treated pasta (T0.25 sample).

The incorporation of wheat bran into pasta led to an augmented total phenolic content, DPPH radical scavenging activity and ferric reducing power In particular, pasta incorporated with cellulase-treated wheat bran showed a higher DPPH antiradical activity compared the sample enriched with untreated bran Moreover, the addition of wheat bran into the flour formulation caused a reduction in the glycemic response during in vitro digestion The wheat bran-enriched pasta demonstrated a lower predicted glycemic index in comparison with semolina pasta On the other hand, the fortification of vital gluten or processing with TG lowered the cooking loss, as well as improving the firmness, tensile strength and overall acceptability of pasta Notably, the combination of gluten addition and TG treatment showed a higher effectiveness in enhancing cooking performance, firmness and tensile strength However, the overall acceptability of pasta enriched with gluten 5% and treated with TG at dosage of 0.25 U/g was similar to that of pasta enriched with gluten or treated with TG alone

Future research should focus on the in vivo glycemic response of bran-enriched pasta as well as the prebiotic potential of pasta incorporated with cellulase-treated bran

In addition, the study of adding other protein preparations rather vital gluten to improve quality of high fiber pasta is advised The investigation of producing bran-fortified pasta at the pilot scale is suggested Finally, it is also recommended to evaluate the consumer acceptability of high fiber pasta with a panel more diverse in age

Table 6.1 Percentage of starch hydrolyzed during in vitro digestion of pasta samples

(bread) S WB10 WB20 WB30 EWB10 EWB20 EWB30

Table 6.2 Kinetic parameters of starch hydrolysis during in vitro digestion of pasta samples

S WB10 WB20 WB30 EWB10 EWB20 EWB30

Table 6.3 Area under curve (AUC) and hydrolysis index (HI) of pasta samples

Table 6.4 Predicted glycemic index (pGI) of pasta samples

Sample code pGI Sample code pGI

Values that do not share a lowercase letter within a row are significantly different (p < 0.05)

Values that do not share an uppercase letter within a same level of bran are significantly different (p < 0.05)

Table 6.5 Proximate composition of bran-enriched pasta samples

Sample code Protein (% d.m.) Lipid (% d.m.) Ash (% d.m.) Starch (% d.m.) TDF (% d.m.) IDF (% d.m.) SDF (% d.m.) IDF/SDF

Values that do not share a lowercase letter within a column are significantly different (p < 0.05)

Values that do not share an uppercase letter within a same level of bran are significantly different (p < 0.05)

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