1. arginine, asparagine, glycine, lysine and threonine are double(Hemery

1.      AbstractRising thenutritional value of pastry products is important since the enriched productscan be used for special aims as developing countries or special diets (Majzoobi etal., 2012).

Wheat germ, ahighly nutritive part of wheat kernel is a milling industry by-product whichhas remarkable nutritional characteristics. However, it has limited usage dueto a high risk of rancidity, which can be reduced via usage of some thermaltreatments such as extrusion. The aim of this study was to check of how wheatgerm additive affects the changes to quality of dough. For this case, some differentamounts of extracted wheat germ are added to dough and then investigate thedifferences including: Rheological characteristics of the dough and qualitycharacteristics of the bread both physical and the sensory points of view. (Gómez et al.,2012).

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Wheat germhas a low shelf life. In order to raise its life quality, so that it can beused into different products. Different heat treatments are applied oncomposition of wheat germ. (Srivastava etal., 2007).2.      IntroductionWheathas a long history of cultivation, dating back to at least 6750 B.C.

(Khush, 1997), and is themost important grain food in the moderate temperate zones of the world. Wheatis classified into at least ten Triticum species, of which six are cultivated.Triticum aestivum L. includes five subspecies; most common wheat speciese are(Triticum aestivum L.

subspecies aestivum).Wheat germ is an important source ofseveral vitamins, minerals, and other phytonutrients, including apigenin andluteolin-based flavonoids(Liangli, 2008). Hence it isavailable as a separate entity because it is an important source of vitamin E.Wheat germ has only one half the glutamine and proline of flour, but the levelsof alanine, arginine, asparagine, glycine, lysine and threonine are double(Hemery etal., 2007). Thisby-product is characterized by its high protein content (over 20%), mainly inthe form of albumins and globulins, and its better-balanced amino acidcomposition, due partly to its higher lysine content than endosperm.

Furthermore,wheat germ has components with high antioxidant activity .In vitro analysis hasalso demonstrated that it has a beneficial effect on the growth of humanbifidobacteria. Consequently, wheat germ is a product with very interestingnutritional characteristics and with a significant potential for use as a supplementin specific formulations(Phillips andWilliams, 2009). Thisby-product of milling also has a considerable fat content of around 10 g/100 g,and a significant quantity of bioactive molecules related to its role as part ofthe embryo of a new plant. Among these bioactive molecules, the lipases and thelipoxygenases can hydrolyze lipids and initiate the oxidative rancidity process(Galli et al.,2017). This fact andthe high unsaturated fatty acid content of wheat germ lead to the short shelf-lifeof raw germ or even flours with germ. Furthermore, raw germ weakens bread doughdue to the presence of glutathione, a powerful reducer that weakens the glutennetwork by the breaking disulphide bonds(Kamel andStauffer, 1993).

A number oftreatments have been proposed to increase wheat germ shelf-life, includingconventional or microwave heating. Due to the nutritional interest of wheatgerm and the possibility of using a by-product from the milling industry, theaddition of raw wheat germ or its derivate to baking products has been studiedusing various approaches. Some of those studies were based on the addition ofraw wheat germ, but others incorporate defatted wheat germ, heated wheat germ,or a combination of the two to obtain products with a longer shelf life andbetter functional properties than raw wheat germ. In most cases, the heatingmethod was toasting or steaming.

Extrusion cooking is a rapid, high-temperaturetechnique for the production a wide range of food products. During extrusion ofth wheat germ oil to a combination of humidity, pressure, temperature andmechanical shear. This method has a number of advantages: a reduction of themicrobial content of the food; partial inactivation of enzymes and someanti-nutritional factors; modification of starch, lipid, protein, and fiberstructure; and, to some degree, a reduction in vitamin content, modifying thenutritional and functional properties of the product. As this process enablesthe wheat germ to be stabilized by applying a high temperature, it can be countedto be an alternative to other heating treatments. The aim of this study is todetermine how the extrusion process affects the ability of wheat germ to beused in bakery products.

This is done through an analysis of the influence ofadding different quantities of extruded and raw wheat germ on the rheologicalcharacteristics of dough and on the final quality of bread, from both thephysical and the sensory points of view(Majzoobi etal., 2012).2.

1             Benefits of wheat germThe bene?ts regardingto wheat germ and its derivatives include lowering plasma and livercholesterol, reducing cholesterol absorption, inhibiting platelet aggregation,improving physical endurance, retarding aging, improving fertility (Brandoliniand Hidalgo, 2012).Wheat germ hasa lot of essential nutrients, including potassium , riboflavin , iron , folicacid , phosphorous , thimin , zinc, magnesium and omega-3 fatty acids(NI, 2011). However, thehuman consumption of wheat germ is mostly limited by the presence of some anti-nutritionalmolecules (raf?nose, phytic acid, wheat germ agglutinins), although baking,mild thermal treatments and sourdough fermentation improve its digestibility.However, wheat germ can adversely affect ?our quality when left in the ?our,because highly unsaturated germ oil and oxidative and hydrolytic enzymes canpromote reactions leading to an increase in acidity and oxidative rancidity .Thus,an ef?cient separation of wheat germ from whole wheat is a signi?cantcommercial factor. For the milling industry, the ‘wheat germ’ which is theembryotic part of the wheat germ organ (Hui, 2006).

Nevertheless,morphological characteristic of the germ includes both embryo and scutellumfractions of the wheat kernel. Millers are unable to ef?ciently remove thescutellum and this fraction normally is carried over with the bran.Economically, the scutellum is even more valuable than the embryo fraction,because it has higher vitamin and total fat contents(Owens, 2001). In any case,the current germ separation technology gives yields of about 0.4–0.5% ofembryonic germ (Edwards, 2010), while thegerm as a whole represents 2.5–3.8% of the total kernel weight(Koehler andWieser, 2013).

2.2             Quality of doughIn recentyears, the baking industry has undergone very important changes in itsproductive processes. Some of the major changes have been brought about by anincreasing mechanization in its processing unit operations (Schmidhuberand Shetty, 2005). This fact hascontributed to increasing the demand for strong wheat flours, yielding doughwith high tolerance to handling and mixing, and able to remain stable duringfermentation. Functional properties of flours greatly depend on the glutenproteins. On the other hand, the quality of gluten is dependent on diversefactors such as wheat variety and growing conditions. For this reason, thecapacity of some countries to produce high-quality flours is limited.

In this context,the treatment of flour with functional additives is considered. Chemicalimprovers have been used for decades in bread making as a method of adjustingthe variations in flour properties and baking conditions. Nowadays, the bakingindustry is deeply involved in research for alternatives to chemical compoundsbecause of their potential hazards. The enzymatic treatment of wheat flours isan interesting alternative to create changes in the structure of the dough andin consequence, for improving functional properties of flours. They aregenerally recognized as safe and do not remain active in the final productafter baking. Therefore, enzymes do not have to appear on the label, which isan additional commercial advantage. The intentional inclusion of enzymes inbread formulas dates back to more than one century .

Today, a wide range ofenzymes produced especially for bread-making is available for bakers. A varietyof aims may be pursued by enzyme addition, for example, to achieve a partialgluten hydrolysis for improving machinability, to obtain enough sugars forfermentation by means of starch hydrolysis, to attain a certain amount of lipidperoxidation for dough strengthening, or to reduce retrogradation and crumbfirming through gelatinised starch hydrolysis. Gluten cross-linking enzymesplay an important role in current baking processes. Through differentbiochemical mechanisms (the oxidative coupling of thiol groups, the cross-linkof tyrosine residues due to the action of intermediate reactive compounds suchas hydrogen peroxide, the acyl-transfer reaction between amino acid residues),these enzymes promote the formation of covalent bonds between polypeptidechains within a protein or between different proteins, improving functionalbehaviour of dough during the bread-making process. Transglutaminase (TG) (EC2.3.2.13) is a transfer enzyme that is able to yield inter- and intramolecular?-N-(?-glutamyl)lysine cross-links.

Its addition causes structural changes ingluten proteins, being high molecular weight (HMW) glutenin subunits the mostaffected protein fraction. TG may also lead to the formation of disulfidebridges by oxidation due to the proximity of sulfur containing amino acids.Because of these effects, TG has been widely used to improve wheat doughfunctionality and bread quality.

The possibility of using this enzyme to reducesome of the detrimental effects of frozen storage of puff pastry and croissants,as well as to solve the damage promoted by the insect attack of wheat has beenproposed. The resultsobtained with wheat flour have been also assumed to other cereals, allowing animprovement in the viscoelastic properties of the rice dough and therefore inthe ability of rice flour to retain the carbon dioxide produced during proofing.Recently, the possibility that TG in wheat-based baked products may generatethe epitope associated with the coeliac response has been suggested, althoughthere is no experimental evidence to support this postulate. Glucose oxidase(EC 1.

1.3.4) (GO) is an oxidative enzyme that catalyses the oxidation of?-d-glucose to ?- d-gluconolactona and hydrogen peroxide.

Disulfide bondinterchange and the gelation of pentosans promoted by hydrogen peroxide actionare the most widespread theories to explain the strengthening effect of the GO.Furthermore, it has been related with the formation of non-disulfide covalentintermolecular bonds in the gluten proteins by GO treatment, either amongglutenins or between albumins and globulins. GO modifies the functionalproperties of dough, increasing its tenacity and elasticity. It is revealedeven an increase in the elastic and viscous moduli of rice flour dough. As aresult of such changes in dough behavior, GO showed positive effects on breadquality, yielding improved specific volume, bread texture and crumb grain.

Through a similar oxidative mechanism, hexose oxidase (EC (HO) hasbeen also suggested as an efficient bread improver.

When this enzyme is addedto dough model systems, it induces the formation of disulfide bridges betweenproteins and the gelation of pentosans, increasing dough strength and breadvolume. HO was found to be more effective than GO because of its ability forusing several monosaccharides and oligosaccharides as substrates and its higheraffinity for glucose. Since proposed laccase (LAC) (EC as dough andbread improver as a result of its oxidant effect on dough constituents,numerous studies have been developed to analyse the effects and applications ofthis oxidoreductase.

LAC is a type of polyphenol oxidase able to gel watersoluble arabinoxylans by coupling feruloyl esters of adjacent chains intodehydrodimers. The probable development of a protein–arabinoxylan network byLAC action has been hypothesized. Even it has concluded that gluten andarabinoxylans form two distinct networks, it is proposed a mechanism by whichtyrosine-containing proteins cross-link with arabinoxylans. Because of thesimultaneous arabinoxylans gelation and oxidative action, LAC additionsignificantly improves gluten quality and leads to changes in the rheologicalproperties of dough, slightly diminishing dough extensibility, increasing doughconsistency, reducing time to maximum consistency and accelerating doughbreakdown during mixing. Improvement in the quality of bread elaborated withLAC has been also reported. The functional properties of bread dough greatlydepend on the proteins forming the gluten network. Strengthening enzymes affectdifferent protein fractions (glutenins, gliadins, albumins or globulins)depending on their particular action mechanism.

The type of protein being cross-linkedappears to be more important than the cross-linking agent or type of cross-linkformed and it is highly correlated with the character of qualitative changes inthe final product. Thus, while HMW glutenin subunits are correlated withseveral macroscopic properties of dough and baked products (such as strength ofgluten network and volume), the albumins and globulins play an important rolein textural and crumb grain properties. For this reason, association ofdifferent gluten modifying enzymes could be an excellent option to improveoverall quality of baked products. Besides the gluten network, anothersecondary crosslinks among minor compounds of flour such as arabinoxylans andpentosans can be promoted. The combined use the aforementioned enzymes withnon-starch polysaccharide degrading enzymes could induce synergistic effects ondough behavior or product quality. Combinations of hemicellulase/GO/?-amylase,TG/amylase/hemicellulase and TG/pentosanase/?- amylase have been reported asbread quality enhancers. Amylolytic enzymes have been also proposed as activecontributors towards fresh bread quality and staling behavior during storage.The objective of this study is to analyze the individual and synergisticeffects of wheat germ oil currently used in increasing the nutritional value ofthe products in bread-making industries.

2.3             Extraction of wheat germAlthough it is possible to avoid organic solvent contamination whenusing pressure expulsion techniques, however it recovers only half of oilpresent in the germ. The solvent extraction method is more effective as 99% ofoil can be recovered. The solvents used for wheat germ oil extraction arehexane, 1, 2-dichloroethane, and ethanol. The catabolism of nutritionally significantcomponents of wheat germ oil can be minimized by avoiding higher extractiontemperatures and by using techniques such as cold pressing and supercriticalcarbon dioxide assisted extraction.

These techniques either avoid or reduce theuse of extraction temperature that might be detrimental of heat sensitivenutrients (Panfili etal., 2003). The oil yieldis 5.2–15% using petroleum hydrocarbon solvents or diethyl ether duringextraction whereas it may increase slightly (7.

2–15.5%) with use of more polarsolvents. The differences between these values results from oil loss duringmilling and limitations of some solvent extraction methods. Mechanicalpressing, aqueous, and enzymatic techniques have also been used for wheat germoil recovery. Mechanical procedures may yield solvent-free oil however yieldmay be lower than that from hexane extraction(Al-Obaidi etal., 2013).

Seed moisture and other operating conditions in respectiveextraction methods can dominantly affect oil yield. Seeds with too low moisturecannot be freed from oil whereas high moisture can result in the material inthe press. The maximum moisture for seeds varies depending on the type of seedand the method used for extraction such that a maximum oil yield (37%) can beobtained at 1.5% moisture content of germ by using a germ oil press. The wheat germ oil yield can be 92% during extraction with solventswhen innovating approaches such as supercritical carbon dioxide extraction isapplied which may also reduce the use of toxic solvent. Hence, taking intoaccount, the effects of various extraction methods and the respectiveextraction variables is crucial for wheat germ oil quality and itsindustrialization(ÖZCAN et al.,2013).

3.      MethodologyThe wheat germ ismilled with a refrigerated mill to produce a fine powder varying from about approximately20 nm average particle diameter(Panfili etal., 2003). The totalamount of extracts obtained from the wheat germ is washed by diethyl etherextraction with a Soxhlet apparatus for 16hr. Oil dissolved in thesupercritical CO2 is separated from the CO2 by decreasing the pressure to anear atmospheric level with the pressure regulating valve. The separated oilwas collected in a separator (about 350ml volume) made of glass. The CO2 waspassed through a filter to remove the entrained oils and then through arotameter and a wet-type gas meter before being exhausted. The amount of oilextracted with liquid or supercritical CO2 is gravimetrically determined.

Thedegree of oil extraction is expressed as a ratio to the amount of oil obtainedper gram of dry wheat germ. Oils are also extracted for comparison by recyclingeither a mixture of chloroform and methanol (CM-mixture) at a volume ratio of2:1 with a reflux condenser for 1.5hr, or hexane in a Soxhlet apparatus for16hr. General properties such as the iodine and saponification values of theoils extracted with supercritical CO2, as well as those obtained by extractionwith the CM-mixture or hexane, are analyzed. To study theeffects of addition of wheat germ on empirical rheological properties of breaddough, processed wheat germ and raw wheat germ will be added to the flourseparately at different ratios in the bowl of a with capacity of 50 g. Thecontrol was 50 g wheat flour of 14% moisture content. Addition of wheat germ tothe flour increased the dry weight of the samples, which could affect the waterabsorption and empirical rheological properties of the dough. Therefore,samples made with wheat flour having weights equal to the samples containinggerm were also prepared and tested with the Farinograph.

Water is then added tothe mixture until the dough is formed with a consistency of 500BU. Theempirical rheological properties of the dough, including dough arrival andstability times, mixing tolerance, and softening after 12 min were determinedfrom the obtained Farinogram.4.      ConclusionWheat germaddition increases water absorption and development time but decreasedstability after over-kneading, dough tenacity, extensibility, and doughstrength. The addition of extruded wheat dough improved stability and decreasedextensibility and strength.

Bread made from dough with added wheat germpresented decreased volume, cohesiveness, and elasticity and increasedfirmness. However, extrusion increased the volume of breads with added wheatgerm and improver and decreased firmness. All breads obtained positiveacceptability scores in sensory analysis, although wheat germ addition slightlydecreased texture, appearance, and overall acceptability scores of breads. Germextrusion therefore improves dough rheology and bread quality and constitutes asuitable treatment to stabilize wheat germ in bread dough.