Targeted Inhibition of Enzymatic Browning in Wheat Pastry Dough

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Targeted Inhibition of Enzymatic Browning in Wheat Pastry Dough Linda Brütsch,† Seline Rugiero,† Stéphanie Spoerry Serrano,† Christian Städeli,‡ Erich J. Windhab,† Peter Fischer,*,† and Simon Kuster† †

Institute of Food Nutrition and Health, ETH Zürich, 8092 Zürich, Switzerland Jowa AG, 8604 Volketswil, Switzerland

J. Agric. Food Chem. Downloaded from pubs.acs.org by UNIV OF TEXAS AT EL PASO on 11/07/18. For personal use only.

‡

ABSTRACT: Enzymatic browning primarily affects fruits and vegetables but also occurs in wheat-based food. Herein, the browning behavior in wheat pastry dough was investigated aiming toward a targeted inhibitory treatment without influencing the pastry dough properties such as workability or taste. Dough discoloration is attributed to several subsequent enzyme− substrate reactions, which can selectively be inhibited by food additives. In most cases, an effective and lasting inhibition is only guaranteed by compounds acting upon multiple inhibition pathways. Despite their effectiveness, the unlimited use of commercial inhibitors is nondesirable due to necessary labeling, thus sustainable and natural inhibitors usually occurring as conventional food ingredients are of interest. It is shown that white wine combined with lemon juice revealed itself as an ideal combination for prevention of enzymatic browning in pastry dough. KEYWORDS: enzymatic browning, wheat dough, targeted inhibition, shelf life, consumer acceptance

1. INTRODUCTION Appearance, flavor, texture, and nutritional value are the main attributes considered by the majority of consumers when selecting food. Consequently, reactions and processes that negatively affect one of these attributes are undesirable and need to be avoided as best as possible. One process is enzymatic browning, which is regarded as one of the leading natural phenomena occurring during food processing and storage influencing the quality of many products and deteriorating customers’ acceptance.1−4 In certain foods including dried fruits, tea, tobacco, and cocoa it improves or contributes to the desired organoleptic properties being essential for color and taste development. However, in most cases enzymatic browning is negatively influencing the foods quality or its perception. The prevention remains an ongoing challenge driven by the quality loss of e.g. sensory and nutritional aspects causing reduced consumer acceptance and thus tremendous economic losses. The existing knowledge on the structure of polyphenol oxidase (PPO), its mechanism of action, and chemistry of enzymatic browning reaction in food provides information on inhibiting the process to minimize the discoloration.3−10 Enzymatic browning is generally associated with the reaction catalyzed by PPO, and as enzymatic browning is widely observed in plant products, it is not surprising that they have been extensively studied and that the enzyme was isolated from numerous fruits and vegetables.6−9,11−15 Browning in food products is generally related to loss of cell integrity caused by mechanical or thermal processes. This results in the disruption of membranes, cell walls, and other cellular structures enabling interaction of enzyme and its substrate. In the presence of oxygen, PPO is then capable of catalyzing melanin formation, which is the colored metabolic end product of enzymatic browning. Under the trivial name PPO, two kinds of enzyme are classified based on their substrate specificity. The first one consists of two similar enzymes named catechol oxidase (EC © XXXX American Chemical Society

1.10.3.1) and monophenol monooxygenase (EC 1.14.18.1) and the second class named laccase (EC 1.10.3.2), which is not considered further. In the presence of oxygen, both monophenol monooxygenase and catechol oxidase catalyze two distinct reactions shown in Figure 1. The oxygen dependent hydroxylation of monophenols into o-diphenols and subsequently the oxidation of the o-diphenols into o-quinones and water. The highly reactive o-quinones take part in various other enzymatic and spontaneous nonenzymatic reactions (as indicated with double arrows in Figure 1) forming intensely colored polymerization products.6 However, also autooxidation might play a major role in discoloration of dough. As enzymatic browning can only take place if all the essential reaction components are present, inhibitory approaches aim to eliminate one or more of them. Possible target points are the (i) elimination or chemical modification of the two substrates, i.e. polyphenols and oxygen, (ii) the inactivation or suppression of the enzymatic catalytic ability, or (iii) the addition of compounds acting on the reaction products to suppress enzymatic and nonenzymatic polymerization reactions.5,7,15−18 Based on the different target points and underlying inhibition mechanisms, all antibrowning agents can be divided into six main categories to control enzymatic browning: (i) substrate analogous, (ii) reducing agents, (iii) complexing agents, (iv) acidulants, (v) quinone couplers, and (vi) chelators.16,19 As shown in Figure 1, acidulants and chelators are targeted toward PPO, substrate analogous, reducing and complexing agents toward the substrates, oxygen and polyphenols and the quinone couplers and reducing agents toward the reaction products. Additionally, other enzymes Received: August 17, 2018 Revised: October 18, 2018 Accepted: October 19, 2018

A

DOI: 10.1021/acs.jafc.8b04477 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Schematic representation of known possibilities to chemically inhibit enzymatic browning reactions. General accepted enzymatic browning reaction starting with tyrosinase activity from a para-phenolic compound to a 3,4-polyphenol followed by mainly enzymatic PPO activities to yield the corresponding ortho-quinone derivative. Colored compounds are marked with an asterisk, whereas the color gradient indicated the increase in discoloration with progression of the reaction. The inhibition of the reaction can be targeted toward the substrates: Oxygen or polyphenols (bold i.e. substrate analogous), the reaction products (bold and italic i.e. quinone couplers), or the enzyme (gray i.e. acidulant). The polyphenol oxidase PPO is represented by its active center containing two copper ions bond to six histidine residues. Adapted from Nicolas et al.20

Table 1. Overview of the Selected Anti-Browning Agents To Investigate Their Inhibitory Effect Targeted toward Decreasing Enzymatic Browning in Pastry: The Anti-Browning Agents, Suppliers, and Modes of Action Are Listed below Antibrowning agent

Supplier

Citric acid (monohydrate) L-Ascorbic acid N-Acetyl L-cysteine Thiourea Ferulic acid trans-Cinnamic acid Cinnamaldehyde Kojic acid Chitosan Ethylenediamine tetraacetic acid L-Cysteine Sodium acetate L-Phenylalanine L-Lysine White Grape juice (G) (polyphenols, malic and tartaric acid) Lemon juice (L) (ascorbic and citric acid) White Wine (W) (polyphenols, malic and tartaric acis, alcohol)

Sigma-Aldrich Sigma-Aldrich Merck Suchard OHG Merck Suchard OHG Merck Suchard OHG Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Molekula ACROS SAFC Sigma-Aldrich Fluka Analytical Merck KGaA Merck KGaA JaMaDu - Coop Freshly squeezed Swiss Fendant − Coop

modifying either the reaction products or PPO itself can thus be used to prevent enzymatic browning.16,20 The consequences of enzymatic browning reaction are mainly characterized for fruits and vegetables although the same phenomenon is observable in wheat-based products such as pastry dough.21−23 All wheat-based products acquire their share of PPO and substrate with the wheat flour, which contains aleuron and bran cells. PPO and its substrates are highly concentrated in the aleuron, a single-cell layer between bran and endosperm. Contamination of the flour and acquisition of PPO results during milling. Due to the multistage milling process and grain morphology a complete separation of bran and endosperm cannot be ensured while staying economically viable. Especially with increasing extraction rate, i.e. using a higher percentage of the endosperm, the amount of bran and aleuron increases.15 Additionally, the mechanical impact during milling disrupting the cell structure might also lead to enzyme and substrate damage.20,24−27 Enzymatic browning is initiated during dough formation and remains a major challenge for fresh-pastry goods as

Mode of action Acidulant, chelator Reducing agent, acidulant Acidulant, Reducing agent, chelator Quinone coupler Substrate analogous Substrate analogous Substrate analogous Tyrosinase inhibitor Quinone coupler and weak chelator Chelator Quinone coupler and chelator Substrate analogous Chelator and quinone coupler Acidulant, reducing agent, chelator, substrate analogous Acidulant, reducing agent, chelator Acidulant, reducing agent, chelator, substrate analogous

deterioration influences the optical dough properties and thus decreases consumer acceptance. To overcome the browning and produce high quality wheat-based dough and batters, commonly extra white flours are utilized. These flours consist of only the innermost endosperm but also leave for lower quality flours to be used elsewhere. Herein, the goal was to replace the extra white flour (type 380) with standard white flour (type 550), which is prone to undergo enzymatic browning during storage. To minimize the discoloration during a four-week shelf life of the fresh pastry dough different inhibitory treatments were elucidated. The inhibiting additives are selected based on their reported impact on product flavor, taste, color, and texture, inhibitory potential, innocuousness, cost, and legal admission.14,28 In a first step, the extent of enzymatic browning was assessed in flour-water mixtures by quantitative color measurements as well as optical investigation. Second, the inhibitory potential of a selection of antibrowning agents was tested in pastry dough during a shelflife of 4 weeks. Finally, a set of natural alternatives providing B


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Journal of Agricultural and Food Chemistry Table 2. Overview on the Different Dough Samples and Their Composition Sample Reference 1 Reference 2 Low inhibitor conc. High inhibitor conc. Low natural inhibitor conc. Low natural inhibitor conc. High natural inhibitor conc.

Flour 280 280 280 280 280 280 280

g g g g g g g

flour flour flour flour flour flour flour

type type type type type type type

Salt 550 380 550 550 550 550 550

4 4 4 4 4 4

Citric acid

g g g g g g

Inhibitor

Natural Inhibitor

0.5 g 0.5 g 1g 5g 1 gdm 5 gdm 10 gdm

Ice water 80 80 80 80 74 50 20

mL mL mL mL mL mL mL

Margarine 155 155 155 155 155 155 155

g g g g g g g

Figure 2. Color change in (A) flour−water mixtures and (B) pastry dough due to enzymatic browning. The discoloration was assessed colorimetrically and optically. In (A) the colometrically measured discoloration over a period of 24 h using the CIE L*a*b* color space is presented (measured in triplicates for both flour types). Flour type 380 (black symbols) did not demonstrate optically determinable changes going in line with constant brightness and redness. While flour type 550 (gray symbols) showed a distinct discoloration resulting from the enzymatic browning. The enzymatic browning resulted in a decreasing brightness and increasing redness of the flour−water mixtures. In (B) the pastry dough’s made from flour type 380 and 550 after two months of storage represent the discoloration and the appearance of shell particles in flour type 550. antibrowning agents make them highly potential enzymatic browning inhibitory. 2.3. Investigation of the Flour Browning Discoloration. To assess the extent of discoloration independently of the effect of fatty acids or other ingredients added during dough formation, a flour− water mixture was studied. Flour type 380 or type 550 was mixed with water to achieve a moisture content of 40%wb. The mixture was filled into Petri dishes forming a 2 mm thick layer mimicking the final dough. The appearance of the flour−water mixtures was judged optically and the color change was determined colorimetrically in triplicate (CR300 Chroma Meter, Konica Minolta Sensing Singapore Pte Ltd.) using the CIE L*a*b* color space over a period of 24 h until leveling out. 2.4. Pastry Dough Preparation and Inhibitory Treatment. The browning behavior and inhibitory effectiveness of the antibrowning agents was analyzed in pastry dough samples. Flour type 380 and type 550 served as references illustrating the targeted and occurring browning, respectively. The inhibitory potential of the browning agents was solely studied in samples made from flour type 550. All pastry dough samples were prepared according to the following recipe: 280 g flour and 4 g salt were mixed with a KitchenAid KSM175 (Whirlpool Corporation, USA) on level 1 for 1 min at room temperature. For the reference samples 0.5 g of citric acid were dissolved in 80 mL of ice water and added to the flour mixture. Ice water was used to control the temperature during dough formation, i.e. counteract the temperature increase due to the mixing process and thus prevent sticky dough. To assess the effectiveness of inhibitors in pastry dough made from flour type 550, citric acid was replaced by 1 or 5 g of the selected additives. For the natural

the same inhibitory potential is proposed for the envisaging natural products.

2. MATERIAL AND METHODS 2.1. Flour Types. Flour type 550 IPS (standard white flour) was obtained from Meyerhans Mühlen AG (Weinfelden, Switzerland). Flour type 380 S (extra white flour) was purchased from Group Minoteries, Bruggmühle Goldach (Goldach, Switzerland). Wheat (Triticum aestivum L.) flour type 550 consists of inner, peripheral endosperm and parts of the aleuron layer using an extraction rate of 72%. For flour type 380 only the innermost endosperm is used, resulting in an extraction rate of approximately 30%. The different extraction rates employed give rise to flour types with compositional and nutritional differences. This in turn affects their dough forming and viscoelastic properties. From an economical point of view, flour type 550 is preferably utilized for pastry dough production but shows enzymatic browning during processing and storage. Therefore, suitable inhibitory treatments of the browning need to be found allowing for its industrial applicability. 2.2. Food Additives. The inhibitory potential of a variety of food additives listed in Table 1 was elucidated in dough aiming at improving the pastry quality. The additives were selected and classified according their inhibitory effect (Figure 1). Despite of their effectiveness the necessity of labeling them induced the search for natural alternatives. Thus, lemon juice, grape juice, and white wine were investigated as potential natural alternatives to chemical antibrowning agents. Their richness in acids, polyphenols, and other C


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Journal of Agricultural and Food Chemistry inhibitors (lemon juice, grape juice, and white wine) three different concentrations were tested: 1, 5, and 10 g dry matter corresponding to 6, 30, and 60 g of liquid, respectively. The liquid addition was compensated by reducing the amount of ice water added. After 1 min of mixing on level 1, 155 g of baking margarine (Mifa AG, Frenkendorf, Switzerland) was added and kneaded on level 3 until a smooth, viscoelastic dough was formed. The dough was flattened to a thickness of 3 mm. Thereof three A5 sized rectangles were cut, wrapped in plastic bags, and put into storage at 8 °C for 4 weeks. An overview on the different pastry dough samples and their composition is given in Table 2. 2.5. Optical Pastry Dough Evaluation. Browning behavior and inhibitory potential were determined based on the induced changes in optical pastry dough properties. The appearance of the dough was examined by (i) optical inspection including counting of aleuron shell particles and (ii) quantitative color measurements using the CIE L*a*b* color space. In combination, both methods provide an objective pastry dough assessment as well as an indication if differences are perceivable and acceptable by the consumer. The optical inspection consisted of counting and classifying the amount of shell particles on the dough surface and judging the dough color. Shell particles visible from a viewing distance of 30 cm and diameter equal to 1 mm or larger were considered in the counting. Samples with 0 to 1 visible particle per cm2 are regarded as ideal. Samples with 1 to 5 particles per cm2 dough were in the acceptable range. Exceeding 5 particles per cm2 gave rise to nontolerable dough. The particle counting and optical judgment was carried out every 2 days for the first 2 weeks of storage and every 3 days for the remaining 2 weeks. All particles on the A5 sizes dough samples were counted followed by calculating the number of particles per cm2. Colorimetric measurements were performed four times for the freshly prepared dough, i.e. directly after production and after 1, 2, and 4 weeks of storage. All experiments have been conducted in triplicates on different days and different batch order. Reference dough made from flour type 380 and type 550 were prepared with every pastry dough series for direct comparison and evaluation of the differences between the different batches. The individual samples were examined in six different spots. The average of the values with corresponding standard deviation was calculated.

increasing from 11.1 to 22.3 (Figure 2A, black symbols). The samples additionally demonstrated a lower initial brightness and increased redness. The brightness L* decreased from 81.3 to 64.4, while the redness a* increased from −1.7 to 1.4 during the 24 h storage. Consequently, enzymatic browning induced alterations affected the brightness and redness of the dough. The induced alterations in yellowness may derive from different discoloration reactions including oxidation being independent of the flour type. The discrepancy in initial dough color and browning behavior between the two flour types originated from constituents such as polyphenols and polyphenol oxidase, which are present in larger quantities in flour type 550 than in type 380. This in turn derived from the differing milling extraction rates producing flours with compositional variations. With increasing extraction rate the amount of aleuron, acquisition of polyphenols and polyphenol oxidase generally rises. Their presence in larger quantities gives rise to more pronounced and accelerated browning during storage. Pastry dough made from flour type 380 and type 550 demonstrated comparable changes in optical properties as the flour−water mixture (Figure 2B). The browning behavior of mixtures and pastry dough solely differed in reaction speed, which was considerably longer for the later one. The gluten network in dough and additional ingredients may have hindered the enzymatic browning reaction resulting in a retarded progression. For dough made from flour type 550 first changes became apparent a few days after preparation. It took up to 4 weeks to reach the full extent of discoloration. During storage a decrease in brightness L* values from 79.8 to 69.1, slight increase in redness (a* = 1.5), and increased amount of shell particles (≫10/cm2) was recorded. The increase in b* value (yellowness) derived from nonenzymatically induced reactions occurring in both sample types. Pastry dough from flour type 380 demonstrated only slight alterations over storage time. A decrease in L* value was measured after 4 weeks storage from 84.4 to 81.8, which cannot be perceived by the human eye. Similar discoloration was observed by Jukanti et al.,29 Fuerst et al.,30 and Kruger et al.,31 who investigated the behavior in wheat noodles. They reported a decrease in brightness L* and increase in yellowness b* during storage. Discoloration of durum wheat (Triticum durum) products might lead to altered yellowness while hard wheat (Triticum aestivum) products demonstrate changes in redness. 3.2. Chemical Inhibition of the Enzymatic Browning. Production of pastry dough with standard flour type 550 makes prevention of the occurring discoloration indispensable. Inhibition or retardation of the browning is achieved by physical (packaging, thermal treatment) and chemical (pH reduction, addition of inhibitors) methods. In this work, chemical inhibitors were investigated as they do not require changes in the pastry dough making process in contrast to physical methods: (i) Lemon juice and citric acid were added in different concentrations lowering the pH to elucidate the effect of acidulation on the browning reaction and the pH sensitivity of the enzyme, (ii) a wide range of chemicals and (iii) natural ingredients (Section 3.3) targeting toward the enzyme, its substrates (oxygen or polyphenols), their reaction products, or combinations thereof were examined (Table 1). 3.2.1. Inhibition by pH Reduction. Acidification by lemon juice and citric acid is commonly used in affected foods such as apples and offered a viable solution to decrease PPO activity by pH reduction. To ensure inhibition of enzymatic browning, i.e.

3. RESULTS AND DISCUSSION The inhibitory potential of several additives to suppress the enzymatic browning and to improve the optical pastry dough properties was investigated in flour−water mixtures (Section 3.1) and dough under simulated shelf-life conditions for 4 weeks (Section 3.2). Industrially applicable and clean label solution using natural ingredients such as lemon juice, grape juice, and white wine are discussed in Section 3.3. 3.1. Characterization of the Discoloration in Flour− Water Mixtures. The color change of the flour−water mixtures over 24 h was investigated by optical and colorimetric means intending to characterize the induced alterations (Figure 2). The flour−water mixtures prepared from flour type 380 did not demonstrate distinct, optically perceivable alterations. Also, no increase in amount of shell particles on the pastry dough surface was observed. In contrast, the sample made from flour type 550 indicated two specific changes. The base color turned reddish-brown and the amount of shell particles increased from 0−1 to over 10 particles per cm2 on the sample surface. The colorimetric evaluation presented in Figure 2A confirmed the findings of the optical evaluation. For the flour water mixture made of flour type 380, the brightness L* (84.1 ± 0.9) and redness a* (−2.7 ± 0.1) value remained constant over 24 h (Figure 2A, gray symbols). The b* value (yellowness) increased from 10.6 after 1 h to 22.8 after 12 h and remained constant thereafter. Progression of the b* value for the mixtures of flour type 550 followed the same trend D


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Figure 3. (A) Average color changes of dough samples after 4 weeks of storage. Low values of brightness L* indicated darker, more colored dough samples, while increasing Hue a* reflected more reddish appearance of the dough samples. The color change of the reference samples made from flour type 380 (light gray circles, dough image in Figure 3B) and 550 (black circles, freshly made dough image in Figure 3D, aged dough image in Figure 3F) as well as the inhibitory potential of selected antibrowning agents is presented (dough images 3C, 3E, and 3F). A decrease in L* and increase in a* value was observed for all investigated samples. The amount of shell particles on the dough is indicated by the circle size. With increasing circle size higher amounts of shell particles were counted. The tolerance range, in which the color of the dough is acetated by the consumer, is marked with the red square in the upper left corner. Samples lying in the tolerance range demonstrate a remarkable improvement of the optical properties of the dough compared to the sample made from flour type 550 without additives.

Figure 4. Overview illustrating the improvement of the optical properties of pastry dough made from flour type 550 (A) treated with antibrowning agents. Antibrowning agents improved the amount of shell particles (B), the dough color (D), or both parameters (C). Chemicals reported in bold demonstrated a significant improvement of the indicated property, whereas the ones in regular only had a slight beneficial effect. All pictures were taken of dough samples that were stored for 4 weeks at 8 °C.

pH interfered with Maillard and caramelization reactions during baking. Below a pH of 6.5 the baking time increased with decreasing pH. At pH 4 solely the edges of the pastry dough turned brown after more than 25 min of baking and no puffing was observed. Acidification proved itself as potent inhibitory treatment for enzymatic browning in dough but applicability is restricted due to the impairment of taste, smell, and baking behavior.

to render the enzyme inactive during the storage time of 4 weeks, a pH value of 4 or lower is required.19,32 A pH between 4 and 5.5 retarded the browning, enabling storage without quality decrease for 2 weeks. Values larger than pH 5.5 lead to a comparable browning as the untreated standard pastry dough made from flour type 550. Exceeding pH 7 accelerated the discoloration. Consequently, a satisfying inhibition was only achieved in very acidic dough affecting the organoleptic properties including taste and smell. Moreover, lowering the E


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Journal of Agricultural and Food Chemistry 3.2.2. Inhibitory Effect of Antibrowning Agents in Dough. Aiming at decreasing browning during storage, antibrowning agents with differing inhibitory mechanisms were elucidated. The inhibitory potential of the compounds was evaluated based on color alterations (L* and a* value) and the amount of shell particles as summarized in Figure 3. The effect is studied by comparing the treated doughs to those made from flour type 550. Pastry dough made from flour type 550 demonstrated the most pronounced discoloration (Figure 3A, black circles and Figure 3D (freshly made dough) and Figure 3F (dough after 4 weeks of storage)) with decrease in brightness L* and increase in redness a*. Pastry dough made from flour type 380 demonstrated the targeted optical properties (Figure 3A, light gray circles and Figure 3B (freshly made and aged dough)). The inhibitory effect on the amount of shell particles was best illustrated for citric acid (Figure 3A, blue circles). With addition of 5 or 1 g of citric acid the amount of shell particles was decreased to 0−1 and 3−4 from over 5 per cm.2 The particles visible on the dough surface are about 1 mm in diameter and the color did not intensified during storage or turned reddish. Addition of thiourea and DTPA (Figure 3A, red and dark gray circles) among others did not influence the base color of the initial dough when freshly prepared but reduced the discoloration during storage resulting in brighter dough. Thiourea and DTPA were not capable of impacting the amount of shell particles on the dough surface. Out of all chemicals investigated only few including N-acetyl-Lcysteine were capable of beneficially influencing both quality aspects. N-Acetyl L-cysteine was effective in decreasing the amount of shell particles to 0−1 or 2−4 per cm2 if 5 or 1 g was added (Figure 3A, green and dark green circles and Figure 3C). It further improved the initial dough color. Nevertheless, it remained darker than the reference pastry dough made from flour type 380. Moreover, no significant decrease in brightness was measured in samples treated with N-acetyl-L-cysteine during storage. Due to their inhibitory potential presented in Figure 3 the antibrowning agents were classified into three groups according to their inhibition mechanism as illustrated in Figure 4, with Figure 4A showing the target dough color. The first group (Figure 4B) with citric acid demonstrating a positive effect on the amount of shell particles consisted of kojic acid, sinapic acid, ferulic acid, and trans cinnamic acid. This group includes many acidulants, which shift the PPO activity outside its natural active pH range and thus reduces significantly its activity. However, in the concentration added the pH reduction was nonsufficient. Hence, the reported inhibitory effect must originate mainly from other inhibitory pathways. Citric acid inhibited the reaction due to its additional chelating properties capable of sequestering the copper atom at the active center of the enzyme. This disabled or decreased the catalytic activity of the enzyme and reduced browning around the shell particles.15 Kojic acid successfully inhibited the enzyme due to its similarity with the common reaction products. It irreversibly bound to the active center decreasing the catalytic ability. The inhibition of cinnamic, sinapic, and ferulic acid originated from their ability to act as substrate analogous. They were oxidized instead of the polyphenols hindering the formation of colored reaction products. The second group of added ingredients contains N-acetyl-L-cysteine, L-cysteine, ascorbic acid, EDTA, and chitosan (Figure 4C). The multipathway inhibition of the antibrowning agents improves both the amount of shell

particles and the dough color. Attacking on different stages along the reaction mechanism allowed for a long-lasting inhibition and should therefore be favored. In case of N-acetylL-cysteine the inhibition potential derived from the ability to act as reducing agent, quinone coupler, and PPO inhibitor through complex formation.15 Ascorbic acid functioned as acidulant and reducing agent while L-cystein acted as quinone coupler and chelator. Chitin is capable of chelating the copper atom and acting as quinone coupler. The third group of antibrowning agents (Figure 4D) included thiourea, sodium acetate, cinnamaldehyde, L-phenylalanin, and L-lysine beneficially influencing the dough color. This group consisted of quinone couplers, reducing agents, and substrate analogous. Quinone couplers and reducing agents both interact with the colored quinones. Quinone couplers form colorless reaction products when interacting with the quinones. Reducing agents reverse the progressing discoloration by reducing the formed quinones to less intensely colored compounds. Moreover, they are capable of reacting with the oxygen decreasing the amount available for the polyphenol oxidase. Substrate analogous hindered or decreased the formation of colored reaction products binding to the enzyme instead of the polyphenols. Consequently, they did not improve the initial dough color but positively affected the dough during storage. The results indicated that the action of reducing agents and quinone couplers was responsible for an improved pastry dough color. The decrease in shell particles was linked to modification of the catalytic ability of the enzyme. Pairing of different chemicals or employing inhibitors targeting several reaction pathways hence provided the most potent inhibition of enzymatic browning in pastry dough. Even though the optical dough properties were improved by addition of the investigated antibrowning agents no treatment was sufficient to reach the brightness of the referenced pastry dough manufactured with flour type 380 (Figure 3A and Figure 3B for fresh and aged dough). Achieving a sufficient and enduring inhibition would require the addition of higher antibrowning agents’ concentration, which is nondesirable from a labeling point. 3.3. Natural Inhibition of the Enzymatic Browning. Taking the results of the previous section into account, the inhibitory potential of natural inhibitors was investigated aiming toward lasting, natural dough. Lemon juice, grape juice, and white wine were selected as potent inhibitors based on their composition. Freshly pressed lemon juice was expected to affect the browning similarly as a combination of the chemically produced citric and ascorbic acid. Pastry dough samples based on flour type 550 containing 5 g of lemon juice showed brighter initial base color (see Figure 3A, orange circles, Figure 3G for dough image). The discoloration during shelf life is shown in Figure 5 (pink line). On the initial dough no shell particles were observed, but during storage the L* value decreased and the amount of visible shell particles increased. The improved initial optical properties of the freshly prepared pastry dough with lemon juice in comparison to reference dough made from flour type 550 (Figure 5, black line) most likely derived from the inhibitory potential of citric and ascorbic acid acting as chelator and reducing agent. The color alteration at later stages during storage was attributable to depletion of the inhibitors. Grape juice was used due to its polyphenol and acid content, but it demonstrated a low inhibitory potential independent of concentration. The weak inhibition potential is shown by the decrease in brightness L* F


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pastry dough with wine and lemon juice showed values of 82.0, −1.1, and 15.4 (freshly prepared) and 80.3, 0.7, and 14.4 (shelf life 4 weeks) as also depicted in Figure 3A (light gray and orange circle) and Figure 3G. The most distinct color differences were found for a* values, indicating a greenish tone for flour type 380 and reddish for the wine and lemon juice treated dough. Moreover, the count of shell particles was reduced; that is, no particles were visible in the freshly prepared dough, and no increase in size, color intensity, or number occurred during storage. In summary, addition of white wine and lemon juice enabled browning inhibition and yielded dough samples with improved optical properties such as color and reduced amount of discolored shell particles. The alcohol content in combination with the lower pH value is seen as the main reason for the determined prolongation. Addition of natural ingredients such as a combination of lemon juice and white wine to pastry dough based on flour type 550 was capable of inhibiting enzymatic browning during storage. This work aimed at replacing extra white flour type 380 with standard white flour type 550 without impacting the pastry dough quality. The replacement allows for increased milling extraction rate so that flour type 550 instead of flour type 380 can be used for pastry doughs. However, utilization of flour type 550 resulted in enzymatic browning of pastry dough during shelf-life. An investigation of the enzymatic browning pathways, transient browning behavior and inhibitory possibilities was carried out for dough to decrease the deterioration thereof during storage. Discoloration of the dough can be attributed to the darkening of the dough base color and an increase in size, amount, and color intensity of aleuron shell particles. This darkening is observed in a decrease of brightness L* and increase in the a* value. Antibrowning agents were added targeting toward the individual inhibition possibilities of the enzymatic browning and were generally able to increase the brightness of the dough or decrease the amount and intensity of the shell particles. However, to reach a lasting and extensive inhibition, high amounts of inhibitors are required, which increases the number of food additives and moves away from a natural product. Therefore, natural food additives were selected based on the trials with the individual chemicals and tested for their inhibitory potential. Especially acidulants, chelators, quinone couplers, and reducing agents have been proven as viable inhibitors. All of them are found in considerable concentrations in grape juice, lemon juice, and white wine. Lemon juice demonstrated the highest inhibitory potential while white wine revealed itself as ideal additive for prevention of enzymatic browning and mold formation. In summary, a combination of lemon juice and white wine yield pastry dough (flour type 550) with optical properties comparable to those made form flour type 380.

Figure 5. Changes in brightness L* of dough samples over a storage time of 4 weeks. Reference samples 380 and 550 are presented in gray and black, respectively. Dough based on flour type 550 treated with grape juice (blue), with lime juice (purple), and treated with white wine (red) show similar browning as the untreated 550 dough (black). Dough based on flour type 550 with addition of white wine and lemon juice (green) showed minimum discoloration similar to the untreated 380 dough (gray). The average color of the measured dough showed an RGB plot transformed by Adobe RGB1998.icc from the CIE L*a*b* color space. All data points were measured in real triplicate, and the error bars indicate standard deviation.

value (79.0 to 71.0) in Figure 5 (blue line) at a concentration of 5 g. Compared to the reference dough made from flour type 550, the pastry dough treated with grape juice is slightly brighter and less reddish after 4 weeks of storage. The amount of shell particles remained unaffected by grape juice giving similar counts as for the reference flour type 550. White wine contains alcohol in addition to the polyphenols and acid present in lemon and grape juice. Already concentrations as low as 1 g of white wine improved the optical dough properties when freshly prepared (Figure 5, red). During storage L* decreased following the progression of lemon and grape juice. Increasing concentrations of wine delayed the decrease in brightness and increase in redness. The amount of shell particles followed a similar trend, i.e. increase in number and size with storage time and lower concentrations of wine added. The more pronounced inhibitory potential of lemon juice compared to grape juice or white wine is linked to their stronger chelating effect: Citric acid and ascorbic acid tented to bind stronger to the active center than tartaric or malic acid present in grape juice and wine. For improvement of the browning inhibition the natural ingredients were combined aiming at interfering at several reaction points of the browning (see Figure 1). Lemon juice and white wine were paired because of their whitening effect and the ability to reduce the amount of browning shell particles. Addition of 1 g dry mass each was not sufficient in achieving lasting inhibition. Increasing the concentration to 5 g dry mass of both components reduced the pH to 5.5. The pastry dough exhibited a similar brightness as the reference dough made from flour type 380 (see Figure 5, gray line). An initial but small decay in brightness L* (82.0 to 80.0) was monitored in the first week of storage and then remained constant during the remaining storage time following the progression of flour type 380. Dough samples made with flour type 380 and the one treated with wine and lemon juice were comparable in terms of color after 4 weeks of storage. The reference pastry dough (flour type 380, Figure 5, gray line) showed L*, a*, and b* values of 83.8, −2.3, and 16.6 while the

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AUTHOR INFORMATION

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*E-mail: peter.fi[email protected]. ORCID

Peter Fischer: 0000-0002-2992-5037 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported and funded by Jowa AG and Swiss Food Research. We acknowledge the assistance of Horst G


Article


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Adelmann (ETH Zürich) and the analytical laboratory of Jowa AG for their assistance.

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Targeted Inhibition of Enzymatic Browning in Wheat Pastry Dough

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