Impact of whey protein hydrolysates on the formation of 2,5-dimethylpyrazine in baked food products

Food Research International 132 (2020) 109089

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Impact of whey protein hydrolysates on the formation of 2,5dimethylpyrazine in baked food products

T

Gustavo Luis Leonardo Scalonea,c, Angelos Gerasimos Ioannidisa,b, Prabin Lamichhanea, ⁎ Frank Devliegherea,b, Norbert De Kimpec, Keith Cadwalladerd, Bruno De Meulenaera, a

Department of Food Technology, Safety and Health, nutriFOODchem group, member of Food2Know, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium b Department of Food Technology, Safety and Health, Food Microbiology and Food Preservation Research Unit, Member of Food2Know, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium c Department of Green Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium d Department of Food Science and Human Nutrition, University of Illinois, 1302 W. Pennsylvania Avenue, Urbana, IL 61801, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: Peptides Whey protein Hydrolysates Maillard reaction Pyrazines HS-SPME-GC/MS GPLC

Peptides have been reported to serve as precursors in the generation of alkylpyrazines, key aroma compounds in heated foods. Most previous studies, concerned with the generation of pyrazines via the Maillard reaction, were conducted using model systems of varying complexities. However, the formation of pyrazines in real food systems has received less attention. The aim of this study was to investigate the impact of adding protein hydrolysates as precursors for the generation of alkylpyrazines in baked food products such as bread and cookies. Two whey protein hydrolysates, obtained using either trypsin or proteinase from Aspergillus melleus, were used in the presented study. 2,5-Dimethylpyrazine was produced in both food systems. Therefore, its formation was quantitatively monitored using a stable isotope dilution assay. Additionally, sensory evaluation was performed. Results demonstrated that the addition of the protein hydrolysates were effective in promoting the generation of 2,5-dimethylpyrazine and other aroma compounds in two well-known food products.

1. Introduction The acceptability of a food product is determined by a combination of various attributes, including appearance, flavor, texture, composition and nutritional value. Flavor perception is the integrated response to the aroma and taste of a product. While there are only five universally recognized basic tastes, such as sweet, sour, bitter, salty and umami, the number of possible food aromas is abundant. Pleasant aromas associated with a food are important since they positively impact product acceptability and can stimulate the appetite of a person. On the other hand, unpleasant aromas tend to decrease food acceptance. Therefore, it is obvious that the flavor quality of a food product can be altered by changing its aroma profile without modifying its taste. Various aroma substances are the result of biosynthethic pathways (Schwab, Davidovich-Rikanati, and Lewinsohn (2008)). The reactions that occur between the different ingredients of a food product during cooking, baking, thermal preservation processes and storage are however also of great importance in the formation of aroma, taste and color. Apart from lipid oxidation (Papastergiadis, Mubiru, Van Langenhove, & De

⁎

Meulenaer, 2012), the Maillard reaction possesses a remarkable prominence and has been the subject of numerous publications (Lane & Nursten, 1983; Ledl & Schleicher, 1990; Lee, Chung, & Kim, 2012; Maga & Sizer, 1973; Mottram, 1994, 2009; Parker, 2013; Tan & Yu, 2012). The Maillard reaction involves not only one reaction, but instead a cascade of complex and interdependent reactions that are known to generate more than 2500 different flavor compounds (Adams & De Kimpe, 2006; Jousse, Jongen, Agterof, Russell, & Braat, 2002). Among the many volatile compounds that can be formed as a result of the Maillard reaction, alkylpyrazines are of particular importance. These nitrogen-containing heterocyclic compounds are important Maillard reaction products known to contribute to the roasted, nutty, meaty, earthy, popcorn-like aroma notes of many roasted and baked goods (Maga, 1992), and in non-thermally treated food, like Chinese Baijiu, soybean and cocoa bean-based fermented foods (Fan, Xu, & Zhang, 2007; Zhang, Cao, Tong, & Xu, 2019). Additionally, many alkylpyrazines possess low odor detection thresholds, thus even at low levels certain pyrazines can contribute significantly to the overall aroma of a food product (Cerny, 2008). Additionally, for cocoa beans, the

Corresponding author. E-mail address: [email protected] (B. De Meulenaer).

https://doi.org/10.1016/j.foodres.2020.109089 Received 10 July 2019; Received in revised form 3 February 2020; Accepted 9 February 2020 Available online 14 February 2020 0963-9969/ © 2020 Elsevier Ltd. All rights reserved.

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considering the formation of flavor compounds in food products. Taylor et al. (Taylor, Sivasundaram, Moreau, Channell, & Hill, 2010), described how some physical properties of food matrices, such as the mobility of the reactants, the temperature gradient in the matrix and/or phase changes caused by heating, can affect the course of the Maillard reaction. Further, the organoleptic properties of food are highly related to the property to flavor release. Food matrixes, such as proteins, fats and carbohydrates also have an important effect on the flavor release because of a strong interaction between flavor compounds and the food matrix (Ma et al., 2019). Nevertheless, the use of food systems instead of models have evolved interestingly (Ewert, Granvogl, & Schieberle, 2011; Fiore et al., 2012; Gökmen et al., 2011; Vaclavik, Capuano, Gökmen, & Hajslova, 2015). Other aspects that play an important role in the formation of volatiles in food products are the multiple interactions between the different ingredients. The aroma of most foods that are thermally processed originates in part from the Maillard reaction, and in part from lipid autoxidation products (Negroni, D'Agostina, & Arnoldi, 2001). Nevertheless, there is evidence that the products of both reactions can interact to form compounds in the intermediate and final stages of the Maillard reaction (Whitfield, 1992; Zamora & Hidalgo, 2005). Several authors have studied the generation of flavor compounds in model systems (Cerny, 2008; Low, Parker, & Mottram, 2007; Madruga, Elmore, Oruna-Concha, Balagiannis, & Mottram, 2010; Zheng, Chung, & Kim, 2015). Recently, Liu et al. investigated the effect of a pretreatment on cocoa beans to control the generation of pyrazines via modification of the pH of the Maillard reaction, and later on, the same authors investigated the use of sucrose, fructose and glucose as precursors to enhance the formation of Maillard volatiles. Moreover, the authors of these investigations achieved interesting economic perspectives, as they could make Robusta coffee more similar to Arabica coffee, the latter being less available and more expensive in the global market (Liu, Yang, Yang, et al., 2019; Liu, Yang, Linforth, Fisk, & Yang, 2019). However, besides the aforementioned research, to the best of our knowledge, the generation of pyrazines in actual food products as a result of the addition of specific flavor precursors for the Maillard reaction has not been sufficiently studied. This study aimed to evaluate the effect of peptides as precursors of alkylpyrazines during the Maillard reaction in real food systems and processing conditions. It therefore evaluated the potential of the addition of peptides to serve as aroma precursors in baked or roasted products to generate characteristic roasted aromas without the necessity of adding artificial flavors. Therefore, the formation of a relevant alkylpyrazine was quantitatively monitored using a stable isotope dilution assay. Additionally, sensory evaluation was performed

concentration of tetramethylpyrazine, has been proposed as a marker of proper roasting parameters such as time and temperature (Starowicz & Zieliński, 2019). Further, due to the flavor importance of many alkylpyrazines, their generations have been studied via other procedures in an effort to produce and use them as flavor additives, implying the economic potential of these volatiles. Zhang et al. have studied the generation of 2,5-dimethylpyrazine and 2,3,5-trimethylpyrazine by microbial mechanisms using Bacillus subtilis (Zhang et al., 2019), while Liu et al. investigated direct selective synthesis of pyrazines using a byproduct-promoted strategy (Liu et al., 2018). Moreover, Gupta et al. have investigated the formation of pyrazines among other flavor volatiles in ash gourd with the purpose of using it as food additive (Gupta, Chikkala, & Kundu, 2019). The Maillard reaction is influenced by temperature, time, pH, aw and the nature and proportion of the reactants. Furthermore, the impact and generation of flavor precursors has been studied as well. Flavor precursors are compounds which are naturally present or intentionally added to a formula to obtain a desired specific flavor. Methionine, for example, leads to the production of a “vegetable character” (Baines, 2009). Further, In the case of yeast extracts, the yeast cells can produce a wide variety of extracts enriched in nucleic acids, amino acids and some other Maillard precursors. This product is characterized as food ingredient and is recognized as safe. When processed in food systems, it undergoes a series of thermally induced reactions at elevated temperatures, generating a wide variety of volatile compounds responsible for the roast like, meaty and caramel like odor (Raza, Begum, Song, Li, & Li, 2019). The use of other flavor enhancers, such as nucleotides, glutamate and amino acids, and the use of soy sauce, are being investigated as part of several strategies to reduce sodium in bread without compromising its sensory profile and consumer acceptability (Sinesio et al., 2019). Protein hydrolysates and peptides in general have been recognized as important flavor enhancers and precursors of the Maillard reaction (Arihara, Zhou, & Ohata, 2017; Baines, 2009; Gao, Yan, Yang, Lu, & Jin, 2014; Hashiba, 1975; Janek, Niewienda, Wöstemeyer, & Voigt, 2016; Jiang & Brodkorb, 2012; Scalone, Lamichhane, Cucu, De Kimpe, & De Meulenaer, 2019; Scalone, Textoris-Taube, et al., 2019; SchlichtherleCerny & Amado, 2002; Van Lancker, Adams, & De Kimpe, 2011; Voigt, Janek, Textoris-Taube, Niewienda, & Wöstemeyer, 2016; Voigt, Textoris-Taube, & Wöstemeyer, 2018). As protein hydrolysates contain a large number of peptides, it is reasonable that Maillard reaction products derived from protein hydrolysates can exhibit a wide variety of aroma (Fu, Zhang, Soladoye, & Aluko, 2019). Moreover, their use in Maillard model systems was effective in generating alkylpyrazines among other volatiles (Adams & De Kimpe, 2006; Adams, Polizzi, van Boekel, & De Kimpe, 2008; Scalone, Cucu, De Kimpe, & De Meulenaer, 2015; Scalone, Lamichhane, et al., 2019; Scalone, Textoris-Taube, et al., 2019; Van Lancker, Adams, & De Kimpe, 2010; Van Lancker et al., 2011; Wang, Zhang, Wang, Wang, & Liu, 2019; Zhang, Zhao, Yang, & Liu, 2017). A large number of animal and plant proteins can be a source for the production of protein hydrolysates, these hydrolysates can be produced through chemical hydrolysis, enzymatic hydrolysis and microbial fermentation of proteins (Fu et al., 2019). Further, addition of protein hydrolysates to food systems can exert an impact on the quality of products thereof, including flavor properties (Lafarga, Gallagher, Aluko, Auty, & Hayes, 2016; Wouters, Rombouts, Fierens, Brijs, & Delcour, 2016). As demonstrated in the literature, the factors that affect the Maillard reaction have been extensively studied. However, most of these studies have been done using model systems. While such insight is invaluable, it does not immediately allow one to predict what will happen in a real food, as for example according to Mottram et al. model systems never deliver all the sensory characteristics of cooked foods (Mottram, 2009). The weak point in the current modelling approaches is that the translation from simplified model systems to real food systems is problematic as several parameters need to be taken into account when

2. Materials and methods 2.1. Chemicals D-(+)-glucose (99.5%), trypsin from porcine pancreas and proteinase from Aspergillus melleus, were purchased from Sigma–Aldrich (Bornem, Belgium). 2,5-Dimethylpyrazine (99%), was purchased from Acros Organics, Thermo – Fisher Scientific (Erembodegem, Belgium). Whey protein isolate LACPRODAN DI-9224 was donated from Arla foods (Aarhus, Denmark). Deuterated standard, consisting of a mixture of 2-[2H3]-methyl-3-methylpyrazine (~83%) and 2-[2H3]-methyl-6methylpyrazine (~17%), was synthetized (Fang & Cadwallader, 2013). Wheat flour [(15.5% moisture, 10.5% protein), Delhaize, Brussels, Belgium], eggs (chicken), sugar (sucrose), dried baker’s yeast (Bruggeman, Gent, Belgium), salt, butter, sodium bicarbonate and parchment paper were purchased in a local store. 2.2. Hydrolysis of whey protein Whey protein isolate (84% protein) was dissolved in potassium 2

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phosphate buffer 0.1 M (pH 7.8) at a concentration of 5.0 mg/ml, heated at 95 °C for 5.0 min and then cooled down to room temperature. The whey protein solution was divided into two aliquots. To the first aliquot trypsin was added at a ratio of 1:20 (w:w), and to the second one, proteinase from Aspergillus melleus was added at a ratio of 1:20 (w:w) according to the literature (Abubakar, Saito, Kitazawa, Kawai, & Itoh, 1998). Both mixtures were incubated at 37 °C for 20 h in a water bath and then heated at 95 °C for 5.0 min to inactivate the enzymes. Final hydrolysates were frozen and freeze-dried for later use.

was added and mixing continued for one minute at high speed, followed by addition of 114.5 g of wheat flour (15.5% moisture, 10.5% protein content) with an additional mixing at low speed for two minutes. The resulting dough was rolled out to a thickness of 1 cm and then cut into several cookies using a 5.5 cm round cookie cutter. Finally, the cookies were placed on a baking sheet previously covered with parchment paper and then baked at 205 °C for 10 min in an oven (Memmert, Fisher Scientific, Erembodegem, Belgium.) (Fig. 1). The percentages of the different ingredients in the cookie samples are detailed in Table 2.

2.3. Characterization of whey hydrolysates

2.6. Preparation of bread samples

The free amino acid content of both hydrolysates was analyzed and quantified by HPLC coupled with a fluorescent detector, as described by Mestdagh et al. (Mestdagh, Kerkaert, Cucu, & De Meulenaer, 2011). The protein hydrolysate was further characterized by gel-permeation chromatography coupled with UV detection as described in an earlier work (Scalone et al., 2015).

The bread was prepared using a modification of a recipe from a traditional cook book (Ons Kookboek; KVLV vzw, 2013, Wijgmaal, 2013), except the milk content was replaced by 10.0 g of whey protein and/or hydrolyzed whey protein and water. Initially, 20.0 g of dry yeast powder and 10.0 g of whey protein isolate and / or hydrolysate (Table 1) were mixed with 50 ml of water. The resulting suspension was then manually mixed with 50 g of wheat flour (15.5% moisture, 10.5% protein content) and then left to rest for 15 min. Afterwards, 150 g of wheat flour, one egg, 15.0 g of sucrose and 5.0 g of glucose were added and the mixture kneaded manually until it reached an appropriate consistency. Then the resulting mixture was kneaded intensively for seven minutes. Subsequently, 40 g of soft butter was added and the mixture was kneaded automatically until the butter was fully incorporated. Salt (4.0 g) was added and the mixture was kneaded for an additional five minutes. The resulting dough was then left to rest at room temperature (≈20 °C) under a plastic wrap for a total of 45 min. During this period the dough was flattened, stretched and folded every 15 min. Finally, after 45 min, the dough was flattened and shaped into a firm ball on a baking sheet, left to rest for 15 min (Fig. 2A) and then baked in an oven (Memmert) at 200 °C for 25 min (Fig. 2B). The percentages of the different ingredients used in the preparation of the bread samples are detailed in Table 2.

2.4. Preparation of food matrices Two food products, cookies and bread were selected for the present research work, as they represent different formulations and manufacturing processes. Whey protein isolate and hydrolyzed whey protein were added as ingredients in quantities which correspond to 9.40 g for the cookie dough and 10 g for the bread dough, respectively. The quantities of whey protein and hydrolyzed whey protein were added in different proportions ranging from 0 to 100% over the total amount present in the food formulation. Formulations used for preparation of the cookies and bread are provided in Table 1. 2.5. Preparation of cookie samples The cookies were prepared following the AACC method 10-50D (American Association of Cereal Chemists, 2000). Additionally, the whey protein and/or hydrolyzed whey protein (9.40 g) were added in quantities comprising 4% of the original recipe (Table 2) in different ratio’s (Table 1). Soft butter (32 g), 60 g of sucrose, 6.0 g of glucose, 1.0 g of salt, 1.25 g of sodium bicarbonate and 9.40 g of whey protein and / or hydrolyzed whey protein in different proportions (Table 1) were combined in a bowl of about 3 l and mixed for three minutes at low speed using an automatic kitchen mixer (Kenwood, UK). Then, water (21.5 g)

2.7. HS-GC-MS analysis 2.7.1. Selection of the volatile compounds to monitor Several publications suggest that peptides are important precursors of alkylpyrazines (Lee et al., 2012; Scalone et al., 2015; Van Lancker et al., 2010, 2011; Van Lancker, Adams, & De Kimpe, 2012; Yang, Wang, & Song, 2012). Therefore, two preliminary experiments were conducted in order to determine which alkylpyrazines among other volatiles were most abundant in the cookies samples C-TH3 and CAMH3 (Table 1). Volatiles were measured by solid phase micro extraction coupled with gas chromatography and mass spectrometry (HSSPME-GC/MS) using the method described by Scalone et al. (Scalone et al., 2015). Moreover, for this particular analysis, pyrazines and other volatiles were identified by comparison of their mass spectrum with mass spectral libraries (Nist 98, Nist 08, Wiley 6th and HPCH2205) and by comparison with authentic samples. As shown in Table 3, 2,5-dimethylpyrazine was present in both samples, while 2-ethyl-3-methylpyrazine was present at only trace levels in the cookie prepared with the hydrolysate from A. melleus (C-AMH3). Other alkylpyrazines were not detected in any of the samples. Such results are not surprising, and despite of the fact of having a relatively high odor threshold, 2,5-dimethylpyrazine has been found in several, past and current foods and model systems of variating complexity whenever there is generation of alkylpyrazines (Koehler, Mason, & Odell, 1971; Koehler & Odell, 1970; Lee et al., 2012; Maga, 1992; Maga & Sizer, 1973; Rizzi, 1972; Scalone et al., 2015; Scalone, Lamichhane, et al., 2019; Van Lancker et al., 2010, 2012; Vandecan, De Schutter, Saison, & Delvaux, 2007; Zou, Liu, Song, & Liu, 2018). Moreover, it is considered an important pyrazine from a mechanistic point of view, while some authors even analyze its formation to assess working conditions in the food industry (Pasqualone, Paradiso, Summo, Caponio, & Gomes, 2014). Therefore,

Table 1 Quantities of whey protein and whey protein hydrolysate present in cookies and bread.

Food product Cookies

Bread

Name of the sample

Whey protein isolate content (g)

Tryptic hydrolysate content (g)

A. melleus protease hydrolysate (g)

C C-TH1 C-TH2 C-TH3 C-AMH1 C-AMH2 C-AMH3 B B-TH1 B-TH2 B-TH3 B-AMH1 B-AMH2 B-AMH3

9.40 7.05 4.70 0.00 7.05 4.70 0.00 10.0 7.50 5.00 0.00 7.50 5.00 0.00

0.00 2.35 4.70 9.40 0.00 0.00 0.00 0.00 2.50 5.00 10.0 2.50 5.00 0.00

0.00 0.00 0.00 0.00 2.35 4.70 9.40 0.00 0.00 0.00 0.00 2.50 5.00 10.0

3

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Table 2 Bread and cookie sample formulations. Cookies formulation

Bread formulation

Ingredients

Quantities (g)

Percentage (%)

Ingredients

Quantities (g)

Percentage (%)

Butter Sucrose Glucose Salt Sodium bicarbonate Water Wheat flour

32 60 6 0.5 0.62 21.5 114.5

13.1 25.52 2.55 0.21 0.26 9.14 48.70

Whey protein / hydrolysate Total

9.40 235.12

4 100

Yeast powder Water Wheat flour Eggs Sucrose Glucose Butter Salt Whey protein / hydrolysate Total

20 50 200 50 15 5 40 4 10 394

5.08 12.69 50.76 12.69 3.81 1.27 10.15 1.02 2.54 100

since the main objective was to investigate the formation of alkylpyrazines as a consequence of peptide addition to food matrices, and since it is well-known and widely spread in several foods and model systems, 2,5-dimethylpyrazine was selected to estimate the overall impact of the whey hydrolysate addition in the Maillard reaction in the selected food samples.

2.7.2. Preparation of the samples Samples were ground separately in an automatic mixer (Kenwood, UK) to assure sample homogeneity. For each of the obtained powders, 1.0 g was put in a 22-mL glass vial (Gerstel, Mulheim, Germany). Then, 0.5 µl of the deuterated standard, consisting of a mixture of 2-[2H3]methyl-3-methylpyrazine (~83%) and 2-[2H3]-methyl-6-methylpyrazine (~17%) was added as a 0.05 mg/ml ethyl acetate solution using a 10 µl Hamilton syringe (Hamilton Company, Reno Nevada USA), and the vial was capped immediately [silicone/polytetrafluoroethylene, PTFE face, hardness 55° (Durometer: shore A), 1.5 mm, magnetic;

Fig. 1. Cookies obtained following the procedure detailed in “Preparation of cookie samples”

Fig. 2. A. Bread samples before baking B. Bread samples after baking. C. Detail of the crumb of a bread sample D. Detail of the crumb of a bread containing Asp. melleus hydrolysate (sample B-AMH3). 4

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Table 3 Overall identified volatiles in two different cookie formulations. Compounda

Butanal a 2-Methylbutanal 3-Methylbutanal Pentanal a Pyridine b Methional a

b a

2,5-Dimethylpyrazine

a

2-Ethyl-3-methylpyrazine a Limonene a Phenylacetaldehyde a 2-Nonanone a 3,5-Dihydroxy-6-methyl-2,3-dihydropyran-4-one 2-Undecanone b 2-Tridecanone b 4-Heptyltetrahydropyran-2-one b a b c

b

c

Cookies with tryptic hydrolysate (C-TH3)

Cookies with Asp. melleus hydrolysate (C-AMH3)

Odor description

GC/MS peak area × 106 N/D N/D N/D N/D 94.93 N/D

N/D N/D N/D N/D 48.85 N/D

6.94 5.16 16.91 39.69 118.35 0.29

N/D

1.40

4.81

N/D 2.16 N/D N/D N/D 1.16 0.91 N/D

N/D 2.27 N/D 1.99 10.54 2.50 1.36 0.81

0.85 2.05 0.46 3.58 42.32 3.51 2.45 1.89

Cocoa, green, fermented Musty cocoa nutty, fermented Fruity, rancid, pungent Fermented, bready, fruity, nutty Non-described Musty tomato, potato, mold ripened cheeses, onion Nutty, musty, earthy, roasted, cocoa powder Nutty, musty, earthy, bread Herbal, peppery Grass, sweet, floral, clover, cocoa Waxy, soapy, cheese, grass, Not described Waxy, fruity, fatty Fatty, waxy, dairy, milky Fatty, dairy, buttery, fruity, nutty

Cookies with whey protein isolate (C)

Identification confirmed by comparison of GC retention time and mass spectrum with authentic compound. Identification based in the best spectra match available in spectra libraries (Nist 98, Nist 08, Wiley 6th and HPCH2205). Odor descriptors obtained from http://www.thegoodscentscompany.com/.

selected for statistical analysis. The Tukey correction was applied to control the family-wise error rate at 5% for all multiple pairwise comparisons. In the sensory analysis, Friedman’s test and post hoc analysis were done using a significance level of p < 0.05.

Gerstel, Germany]. 2.7.3. GC-MS conditions SPME vials containing the food samples were incubated at 70 °C for 15 min in order to release the flavor compounds to the headspace of the vial. Then the headspace volatiles were sampled using a 2.5-mL HDtype gas tight syringe (CTC Analytics, Agilent technologies, Zwingen, Switzerland) using a CombiPAL multipurpose sampler (CTC Analytics, Agilent technologies). Further, the headspace technique was used instead of SPME as in the Section 2.7.1. This due to the fact that the solvent in which the internal standard was diluted would saturate the absorption capacity of the fiber, affecting then the detection of 2,5dimethylpyrazine. GC-MS analyses of the HS extracts were done with an Agilent 7890A GC Plus apparatus coupled to a quadrupole mass spectrometer 5975 MSD (Agilent Technologies, Diegem, Belgium) equipped with a DB-624 capillary column (60 m length × 250 µm i.d; 1.4 µm film thickness) (Agilent Technologies). Working conditions were the following: transfer line to MSD 280 °C, carrier gas (He) 1.0 ml/min; ionization: EI 70 eV; acquisition parameters: SIM low resolution, monitored ions (108 m/z for 2,5-dimethylpyrazine and 111 m/z for 2-[2H3]methyl-3-methylpyrazine and 2-[2H3]-methyl-6-methylpyrazine; oven temperature started at 35 °C, was held for 1 min, then programmed from 35 to 120 °C at 15 °C /min, then from 120 to 130 °C at 2 °C /min and held for 2 min, then from 130 to 150 °C at 2 °C /min and from 150 to 250 °C at 20 °C /min and held 5 min. 2,5-Dimethylpyrazine was identified by comparison of its mass spectrum and retention time of that of the authentic compound. The generation of 2,5-dimethylpyrazine was followed by quantification using an external calibration curve to determine the response factor between the authentic compound (2,5-dimethylpyrazine) and a mixture of 2-[2H3]-methyl-3-methylpyrazine (~83%) and 2-[2H3]-methyl-6-methylpyrazine (~17%) as internal standards, calibration points ranged from 0 to 100 ng.

2.9. Sensory analysis Sensory analysis was conducted in order to identify the products with the highest preference in terms of aroma and appearance among the cookies and bread samples. The tests used, followed the general guidelines described by Meilgaard et al. (Meilgaard, Carr, & Civille, 2006). Furthermore, a preference-ranking test was conducted examining two attributes, the overall appearance and the aroma (Lawless & Heymann, 1999). Additionally, Friedman’s test was used to reveal if significant differences exist among the samples, this is, if the calculated test value exceeds the upper-α critical value of a chi-square random variable for (t-1) degrees of freedom (Meilgaard et al., 2006). Hence, in our case a significant difference would be present for each attribute examined among the samples, if the chi-square is greater than χ2(3) = 7.81 for p = 0.05. Furthermore, two samples are characterized to be significantly different (α = 0.05) if their rank sums difference was greater than LSRD = 16.4 from (Eq. (1)).

LSRD = ta/2, ∞

JK (J + 1) 6

(1)

Sensory evaluations of the bread and the cookies were done in the sensory laboratory facilities of the Faculty of Bioscience Engineering at Ghent University. A group of 21 panelists characterized as frequent consumers of bread and cookies was recruited and briefly instructed prior to the analysis. The sensory test was carried out in two consecutive days, one for each product. During both days, three groups of seven people entered the sensory evaluation lab and four different samples were presented at room temperature (20–22 °C) in random order and labeled with randomly-generated three-digit codes. The bread and cookie samples were one day old when evaluated. Panelists were asked at first to evaluate the degree of preference with respect to the appearance of the samples, then these samples were shuffled the panelists evaluated the preference for aroma under red light to prevent the visual appearance of the samples from affecting the judgment of the panelists. For each sensory attribute a different set of

2.8. Statistical analysis Statistical analyses were done using SPSS Statistics version 22. The data points obtained from the HS-GC-MS analysis of 2,5-dimethylpyrazine were normally distributed (Kolmogorov-Smirnov test: (p < 0.050) for all standardized residuals) and represent mean values of three independent determinations. Therefore, one-way ANOVA was 5

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fact that the tryptic hydrolysate contained small amounts of free amino acids and medium chain molecular weight peptides, while, on the other hand, the A. melleus hydrolysate had a considerable higher amount of free amino acids and an abundance of small peptides. Additionally, the use of both whey protein hydrolysates generated statistically similar quantities of 2,5-dimethylpyrazine for cookies if 2.35 g of hydrolysate, the lowest amount evaluated, was added (samples C-TH1 and CAMH1). Similarly, for the bread samples at the lowest amount evaluated of 2.50 g hydrolysate, statistically similar amounts the pyrazine were produced for both hydrolysates (B-TH1 and B-AMH1). However, the use of hydrolysate from A. melleus resulted in an enhanced generation of 2,5-dimethylpyrazine in both the cookie and bread samples at higher concentrations. It is also interesting to note that, in general, more 2,5-dimethylpyrazine was found in bread samples than in cookie samples. This is most likely related to the addition of yeast during the bread manufacturing. Munch et al. reported that yeast contains a high content of free ornithine (Munch, Hofmann, & Schieberle, 1997; Munch & Schieberle, 1998). The Strecker degradation of this amino acid generates 4-amino-butanal, which further cyclizes to form 1-pyrroline. 1Pyrroline is a precursor of 2-acetyl-1-pyrroline and 2-acetyltetrahydropyridine, two key odorants of bread (Adams & De Kimpe, 2006). However, as a result of the Strecker degradation, ornithine generates αaminoketones as well. These α-aminoketones can condense to form pyrazines. Further, the bread formulation included eggs to a percentage of 12.69%. Previously, Umano et al. identified 2,5-dimethylpyrazine in cooked egg whites (Umano, Hagi, Shoji, & Shibamoto, 1990). Additionally, bread samples were heated longer than the cookie samples. During the baking process, the bread crust is exposed to a higher temperature than the crumb. As the bread matrix dehydrates, it concentrates some water-soluble solutes in the outer regions of the product (Taylor et al., 2010). Since the presence of pyrazines in the bread crust was reported previously (Schieberle & Grosch, 1991, 1994), it is likely that the combination of the factors mentioned above influenced the formation of 2,5-dimethylpyrazine. Moreover, the interplay between lipid oxidation products and Maillard reaction products could be taken into account as a possible explanation of pyrazine formation. Negroni et al. reported that different oils can promote the formation of pyrazines in Maillard model systems depending on their degree of unsaturation (Negroni et al., 2001). Butter, an ingredient used in the preparation of both the bread and cookies, contains a moderate level of unsaturated fatty acids (Table S1). Nevertheless, bread and cookie samples contained butter in similar percentages (Table 2).

samples was introduced. An example of the sensory analysis survey can be found in Appendix 1 of the supporting information. The score sheets required from the panelists to rank four samples in the order of preference from 1 to 4, with 1 being least preferred and 4 being most preferred. In addition, the panelists were asked to comment on their choices. The rank sums were used to determine which samples were the most preferred and Friedman’s test (p < 0.05) was used to determine whether significant differences were present among the samples (Meilgaard et al., 2006). 3. Results and discussion 3.1. Characterization of the hydrolysates – peptides and amino acids Two types of whey protein hydrolysates were selected to be used in the formulation of bread and cookies. One hydrolysate was obtained from the digestion of whey protein with trypsin while the other was obtained using proteinase from Aspergillus melleus. These hydrolysates were previously characterized in terms of their free amino acid composition and their approximate peptide profile. Both hydrolysates were significantly different with respect to their peptidic profile and free amino acid composition (Scalone, Lamichhane, et al., 2019). Briefly, the total amount of free amino acids amounted 1.53 and 23.65 mg/ 100 mg for the hydrolysate obtained by trypsin and the proteinase from A. melleus respectively. The dominant amino acids in the trypsin hydrolysate was lysine (0.99 mg/100 mg), while leucine was dominant (4.48 mg/100 mg) in the other. In the hydrolysate obtained using the proteinase from A. melleus, the following amino acids were present at a concentration > 1.0 mg/100 mg (in order of abundance): serine, alanine, phenylalanine, tyrosine, threonine, valine, iso-leucine, lysine and leucine (Scalone, Lamichhane, et al., 2019). Further, both hydrolysates were significantly different with respect to their peptide profile. The hydrolysate obtained using the proteinase from A. melleus contained a high amount of peptides with molecular weights below 1355 Da, while in the tryptic hydrolysate the majority of the peptides had a molecular weight between 1355 and 8000 Da. Hence, it was expected that the flavor formation resulting from the addition of these hydrolysates to the bread and cookies would also differ. 3.2. Formation of 2,5-dimethylpyrazine As is generally known, the production of different flavors in food can determine their acceptability. Recently, Pacynsky et al. expressed that gluten-free bread did not contain some bread key-odorants, like 2acetyl-1-pyrroline and pyrazines, and, therefore, it is less appealing to potential consumers (Adams & De Kimpe, 2006; Pacynski, Wojtasiak, & Mildner-Szkudlarz, 2015). Furthermore, the authors reported that the use of different amino acids and sugars improved the aroma of glutenfree bread due to an enhanced formation of the mentioned key-odorants (Adams & De Kimpe, 2009; Pacynski et al., 2015). However, the use of peptides was not considered by these researchers. Both amino acids and peptides are effective precursors for the formation of pyrazines in Maillard model systems. Recent results imply that the role of peptides in the generation of pyrazines was underestimated (Scalone et al., 2015; Scalone, Lamichhane, et al., 2019; Van Lancker et al., 2010, 2012). The results of the present investigation showed that the addition of whey protein hydrolysates was effective in generating 2,5-dimethylpyrazine in the selected real foods. For all cases, the use of whey protein hydrolysates resulted in an enhanced production of 2,5-dimethylpyrazine with respect to the control samples (C and B) (Table 4). Further, for both food products, the samples containing tryptic hydrolysate exhibited a moderate rise in the detected amounts of 2,5-dimethylpyrazine. This increase was proportional to the amount of tryptic hydrolysate used in the preparation of the samples. However, in samples containing the A. melleus hydrolysate, this trend was considerably more pronounced. This could be due to the

3.3. Appearance and aroma preference With respect to appearance and aroma attributes, panelists had no preference (p ≤ 0.05) for breads made with or without tryptic whey protein hydrolysate. In agreement with these results, panelists commented that it was difficult to spot visual differences among the samples. For the breads made with A. melleus hydrolysates, both appearance and aroma attributes were preferred for the control samples over those containing the highest level of hydrolysate (Table 5). This can be attributed to the dark, burnt appearance and an irregular crumb of the bread samples containing high quantities of the hydrolysate from A. melleus (Fig. 2D). Furthermore, the panelists reported that the characteristic smell of the same samples was undesirable. In particular, some panelists commented that the odor of sample B-AMH3 was quite intense and described it as a grilled cheese-like. Therefore, it was clear that the trypsin hydrolysate had a less pronounced impact on the aroma of the breads compared to the hydrolysate obtained with proteinase from A. melleus. In contrast with the bread samples, the addition of tryptic whey protein hydrolysate affected the preference for both the appearance and aroma of the cookie samples. The same patterns were observed for both the appearance and aroma of the cookies, where the control sample C 6

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Table 4 Concentrations (ng/g) * of 2,5-dimethylpyrazine detected in bread and cookie samples. Cookies

C

C-TH1

C-TH2

C-TH3

C-AMH1

C-AMH2

C-AMH3

Amount of whey protein hydrolysate 2,5-Dimethylpyrazine Bread Amount of whey protein hydrolysate 2,5-Dimethylpyrazine

0g 11.78 ± 0.76 a B 0g 17.29 ± 0.88 a

2.35 g 12.02 ± 0.38 a B-TH1 2.5 g 24.01 ± 1.28b

4.7 g 13.30 ± 1.10 ab B-TH2 5g 28.44 ± 2.26c

9.4 g 14.66 ± 0.28b B-TH3 10 g 33.80 ± 2.41 d

2.35 g 13.26 ± 0.15 ab B-AMH1 2.5 g 22.21 ± 0.68 ab

4.7 g 22.99 ± 0.91c B-AMH2 5g 29.47 ± 1.60 dc

9.4 g 41.99 ± 1.73 d B-AMH3 10 g 46.25 ± 2.85 e

* Values represent mean values of three independent determinations. Values in the same row followed by different letters are significantly different (p < 0.05). TH (tryptic hydrolysate), AMH (Aspergillus melleus hydrolysate).

its concentration. As can be observed in Table 4, the production of 2,5dimethylpyrazine had a considerable upward tendency for both products. At the same time, the sensory panel described the appearance of the samples C-TH3 and C-AMH3 as burnt compared to the control samples, which is obviously due to the increased production of melanoidins due to the Maillard reaction. However, this observation was not so clear for bread samples (Table 5), probably because the majority of the melanoidins are formed in the bread crust, while no clear differences could be observed in the crumb. Further, the addition of the A. melleus hydrolysate generated flavor compounds that the sensory panel considered as undesirable. These aromas were described as “grilled cheese” and “pungent” flavors.

and samples C-TH1 and C-TH2 were preferred over sample C-TH3 which contained the highest content of the hydrolysate (Table 6). Moreover, for sample C-TH1, the sensory panel reported roasted and malty flavors which are typically associated with the generation of pyrazines like 2,5-dimethylpyrazine. Therefore, it is remarkable that the addition of 2.35 g of tryptic hydrolysate (1% of the food composition) resulted in an acceptable product for a considerable number of the panelists. Nevertheless, the results revealed that the sensory impact of the addition of hydrolysate on appearance and aroma was subtle, except when used at the highest level (C-TH3) making clear that the use of tryptic hydrolysate in higher amounts is not recommendable. Similar results were observed for cookies containing the A. melleus hydrolysate. The appearance of control sample C and AMH1 and CAMH2 were preferred over sample C-AMH3 which contained the highest level of the hydrolysate (Table 6). A slightly different trend was observed for aroma, where the aroma of the control sample C was significantly preferred over samples C-AMH2 and C-AMH3, while no significant difference was observed between the samples C and CAMH1. Panelists noted that sample C-AMH3 had a prominent burnt off odor, which explains its low preference ranking. Considering the overall quantities of whey protein hydrolysates used in the formulation of both food products, it is remarkable that a small addition of whey protein hydrolysate could have such a large impact on the generation of 2,5-dimethylpyrazine among other volatiles for both tested products. The results previously presented, indicate that for both products, the use of hydrolyzed whey protein was effective in generating flavor compounds that were not present in the respective control samples (Table 4). However, in both cookie and bread samples, the production of 2,5-dimethylpyrazine in samples containing the A. melleus hydrolysate, was accompanied by a production of several other volatiles that were perceived as undesirable by the sensory panel. Moreover, the appearance of both tested products resulted as undesirable as well when this particular hydrolysate was used. The use of the A. melleus hydrolysate, produced remarkable differences depending on

3.4. Proposed explanation of the undesirable aromas formed in samples containing A. melleus hydrolysate The production of undesirable aromas in the tested food products can be explained as a direct consequence of the addition of the whey protein hydrolysate from A. melleus. Firstly, due to Maillard reactions, the high concentration of free amino acids in the hydrolysate (23.653 mg/100 mg hydrolysate) could generate a variety of Strecker aldehydes, like 2-methylpropanal and 3-methylbutanal, formed through the Strecker degradation of valine and leucine, respectively, which are present in significant quantities in this hydrolysate (Table S2). As can be seen in Table 3, butanal, 2-methylbutanal and 3-methylbutanal were detected by GC-MS full scan mode in the cookie samples containing whey protein hydrolysate from A. melleus. Although 2-methylpropanal was not detected, most likely due to its overlapping with other highly volatile compounds, it is probable that it was present as well due to the considerable quantities of valine in this hydrolysate. Methional was also detected in the cookie samples containing whey protein hydrolysate from A. melleus. This compound is generated from the degradation of methionine, which is present in the free amino acid

Table 5 Results of preference ranking for appearance and aroma attributes of bread samples. Bread samples containing tryptic hydrolysate Appearance Sample § B B-TH1

B-TH2

B-TH3

Aroma B

B-TH1

B-TH2

B-TH3

Rank Sum Mode Min value Max value

40b 2 1 4

57 a 3 1 4

51 a 1 1 4

49 a 2 1 4

52 a 2 1 4

58 a 4 1 4

B-AMH2 43b 2 1 4

B-AMH3 44b 1 1 4

Aroma B 71 a 4 1 4

B-AMH1 59 ac 3 1 4

B-AMH2 48 bc 2 1 4

B-AMH3 32b 1 1 4

63 a 4 1 4

50 ab 1 1 4

Bread samples containing A. melleus hydrolysate Appearance Sample § B B-AMH1 Rank Sum 65 a 58 ab Mode 4 3 Min value 1 1 Max value 4 4

§ Sample abbreviations correspond to those in Table 1. Values in the same row followed by different letters are significantly different in ranked preference. Highest rank sum score corresponds to the highest preference. The minimum difference in rank sums required for two samples to differ was calculated to be LSRD = 16.4 for α = 0.05.

7

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Table 6 Results of preference ranking for appearance and aroma attributes of cookie samples. Cookie samples containing tryptic hydrolysate Appearance Sample

§

Rank Sum Mode Min value Max value

Aroma C

C-TH1

C-TH2

C-TH3

C

C-TH1

C-TH2

C-TH3

64 a 4 1 4

67 a 4 1 4

52 a 3 1 3

27b 1 1 3

68 a 4 2 4

59 a 4 1 4

52 a 2 1 4

31b 1 1 4

C-AMH2 55 a 3 1 4

C-AMH3 27 b 1 1 4

Aroma C 70 a 4 1 4

C-AMH1 60 ab 3 1 4

C-AMH2 52b 2 2 4

C-AMH3 28c 1 1 4

Cookie samples containing A. melleus hydrolysate Appearance Sample C C-AMH1 Rank Sum 69 a 59 a Mode 4 2 Min value 1 2 Max value 4 4 §

Sample abbreviations correspond to those in Table 1.Values in the same row followed by different letters are significantly different in ranked preference. Highest rank sum score corresponds to the highest preference The minimum difference in rank sums for two samples to be significantly different was calculated, LSRD = 16.4 for α = 0.05.

4. Conclusions

composition of the hydrolysate (1.103 mg/100 mg hydrolysate), (Table S2). Methional has been reported to have a strong cabbage, potato and sulfur aroma (Pripis-Nicolau, de Revel, Bertrand, & Maujean, 2000). Moreover, it has an extremely low odor threshold value, capable to contribute significantly to the overall aroma at the part per billion level or lower (Cerny, 2008; Mottram, 2009). Other volatiles that were detected in the preliminary scan could contribute as well to the cheeselike flavors described by the panelist (Table 3), like for example 2nonanone. On the other hand, it has been shown recently that the perception of mixtures of odorants, even if each is correctly identified alone, is not just a simple sum of the precepts of the individual components. Furthermore, for mixtures containing more than four components, the odorants were found to lose their individuality and produce a new odor (Dunkel et al., 2014). Despite of the fact that it is difficult to identify individual odorants in a mixture, humans can easily discriminate mixtures from each other (Dunkel et al., 2014). Nevertheless, it must be noticed that the primary objective of this investigation was to evaluate the formation of 2,5-dimethylpyrazine as a consequence of the use of protein hydrolysates in food products. Regarding the use of whey protein hydrolysate resulting from tryptic digestion, the production of Maillard volatiles and melanoidins was less pronounced. As shown in Tables 5 and 6, the sensory panel had more difficulty differentiating samples containing different concentrations of the tryptic hydrolysate. In agreement, the results given in Table 4 show that the trend in production of 2,5-dimethylpyrazine was less pronounced. However, the main difference between the use of these two hydrolysates is the fact that the sensory panel did not describe the samples containing tryptic hydrolyzed whey as unpleasant. Thus, the expected production of pyrazines was not accompanied by the same generation of amino acid specific volatiles which were present in the samples containing proteolytic hydrolysate (Table 3). As commented before, the tryptic hydrolysate contained much lower quantities of free amino acids (1.527 mg/100 mg hydrolysate) compared to the A. melleus hydrolysate (23.653 mg/100 mg hydrolysate), (Table S2). Therefore, the production of Maillard volatiles was mainly due to the peptides present in the hydrolysate. Similar results were obtained in model systems containing hydrolyzed whey protein and glucose. Since peptides cannot follow the Strecker degradation mechanism, the formation of pyrazines from peptidic sources did not generate Strecker aldehydes, which are known to have strong flavor properties. Hence, this explains why the sensory panel only described typical roasted or baked flavors, which are associated with pyrazines in samples containing tryptic hydrolyzed whey.

The use of whey protein hydrolysates can clearly promote the formation of volatiles in food products even when they are added in small quantities. On the other hand, it is clear as well, that the use of high amounts of hydrolysate results in negative effects both in appearance and aroma. The peptide and amino acid composition of the hydrolysates also plays an important role; it is clear that high concentration of amino acids not only promote the formation of 2,5-dimethylpyrazines but also the formation of other volatile compounds resulting in a complex way of promoting specific flavors. Moreover, as the tryptic hydrolysate contained small quantities of amino acids, therefore, the peptides contained in this hydrolysate were appropriate flavor precursors, which generated 2,5-dimethylpyrazine without an excessive formation of undesirable volatiles. A remarkable aspect of this study is that it was conducted under commercial conditions. It can be concluded that whey protein hydrolysates, or other protein hydrolysates added to a food formulation may increase its roasted or malty character during different thermal processes common in the manufacturing of baked food products, increasing then the validity of the obtained results. Moreover, this opens the possibility of using whey protein hydrolysates as flavor precursors to promote flavor properties and thus acceptance of products like glutenfree bread or meat substitutes, foods for which moderate amounts of pyrazines could be desirable. CRediT authorship contribution statement Gustavo Luis Leonardo Scalone: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization, Project administration. Angelos Gerasimos Ioannidis: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Visualization. Prabin Lamichhane: Investigation, Visualization. Frank Devlieghere: Supervision. Norbert De Kimpe: Writing - review & editing, Supervision. Keith Cadwallader: Resources, Writing - review & editing. Bruno De Meulenaer: Conceptualization, Methodology, Validation, Resources, Writing - review & editing, Supervision, Project administration. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to 8

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influence the work reported in this paper.

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