Wheat Germ Lipoxygenase: Its Effect on Dough Rheology, Microstructure, and Bread Making Quality

Abstract

Extracted lipoxygenase from defatted wheat germ of commercial bread wheat along with raw and defatted germ were used to study their effect on rheological characteristics of wheat flour dough and bread making quality. The addition of 500 U and 1000 U of lipoxygenase increased the water absorption from 59.5 to 62.3 and 66.7%, respectively. The dough stability increased to 10.5 min, whereas mixing tolerance index values marginally decreased, and the addition of raw or defatted germ did not affect the mixing profile of the wheat flour dough. Breads with lipoxygenase were softer with a lower firmness value of 546 g when compared to the control (594 g) and had brighter crumb as seen in the reduction in ΔE values from 22.34 (control) to 19.04. The addition of gluten along with the lipoxygenase showed a synergistic effect. The specific volume of breads increased to 3.95 cc/g and the firmness values decreased to 538 g. Scanning electron micrographs at different stages of bread processing have shown improvement in the gluten network.

Keywords:

• Wheat germ
• Lipoxygenase (LOX)
• Farinograph characteristics
• Alveograph characteristics
• Bread
• SEM

INTRODUCTION

Enzymes play an important role in the food industry, such as in the production of cheese, alcohol, and sweeteners; clarification of beer, wines, and fruit juices; and meat tenderization, and most importantly in the production of bakery products. The number of enzymes is being explored in the bakery industry of various classes, such as amylases, proteases, and oxido-reductases, to improve the dough handling properties and quality of bakery products. Very recently, gluco-oxidases and lipoxygenase (LOX) have been exploited for beneficial effects. Oxidoreductase is a group that produces hydrogen peroxide (H₂O₂) and includes enzymes like lipoxygenase, peroxidases, glucose oxidase, etc. Lipoxygenase catalyzes peroxidation of unsaturated fatty acids, and shows high specificity for cis-cis methylene interrupted dienoic fatty acids. The H₂O₂ produced promotes the formation of di-sulfide linkages in gluten protein.[1] Soybean is the richest source of lipoxygenase and contains about 40 times as much as in wheat.[2] Lipoxygenase of wheat is mainly concentrated in the scutellum and embryo with very little in endosperm. The lipoxygenase activity in wheat in different streams varies with a wide range from 0.24–4.3 U/g. Activity is higher in tail end streams since they contain a small amount of bran and germ.[3] Lipoxygenase has a number of functions in the bakery industry. Apart from its bleaching action,[4] it promotes cleavage of disulfide bonds and initiates new cross linking between free SH groups of gluten protein.[5,6] It also increases the amount of free lipids in dough,[7] which in turn increases the mixing tolerance and relaxation time of dough resulting in enhanced loaf volume.[6,8–11 11] Lipoxygenase isozymes (L1, L2, and L3) were studied for rheological and bread making characteristics and it was observed that lipoxygenase L1 acted on the free unsaturated fatty acids, whereas L2 and L3 acted on bound unsaturated fatty acids.[11] Lipoxygenase showed improvement in rheological, baking, and nutritional properties of wheat flour dough.[12–15 15 Wheat germ LOX activity has been measured but utilization of the same has not been explored in either dough rheology or bread making quality. Hence, an attempt has been made to extract LOX from wheat germ and wheat bran. The objective of this study was to study the effect of partially purified lipoxygenase from either wheat germ or bran at different levels on the macroscopic properties (dough rheology and bread quality) and on the molecular structure.

MATERIALS AND METHODS

Commercial wheat flour (11.13% moisture, 0.55% ash, 10.46% gluten, 24 ml sedimentation value, and 373 s falling number) and compressed yeast (Tower brand, Mumbai, India), sugar powder, and vegetable fat (Hindustan Unilever Ltd., Bangalore, India) were procured from a local market for the study. Wheat germ and coarse bran (from a commercial local roller flour mill of 20T capacity) and gluten powder (Burn Philips India Ltd., Pune, India) were used for the study.

Lipoxygenase Extraction and Estimation

Lipoxygenase extraction was carried out as described by Shiiba et al.[6] Defatted wheat germ (100 g) was stirred with 600 ml of 50 mM acetate buffer (pH 5.5) and kept overnight in a stirrer at 4°C. The enzyme was precipitated using 40% ammonium sulfate and dialyzed against 10 mM phosphate buffer for 48 h and centrifuged. The supernatant was considered as the partially purified lipoxygenase extract (lipoxygenase).

Linoleic acid, the lipoxygenase substrate, was prepared in an atmosphere of nitrogen by dissolving 100 μl of pure linoleic acid in a mixture of 0.12 ml Tween 20, 2.5 ml of 50 mM acetate buffer (pH 7.0), and 0.32 ml of 1.0 M sodium hydroxide and diluted to 50 ml with 50 mM acetate buffer. The reaction mixture consisted of 2.9 ml of 50 mM acetate buffer (pH 5.5), 90 μl stock substrate, and 10 μl of lipoxygenase. Activity was assayed spectrophotometrically at 234 nm. A unit of lipoxygenase activity is defined as the amount of enzyme that produced a change of one unit of absorbance at 234 nm per minute. Enzyme activity was expressed in terms of hydroperoxide formed (μmol/min) using an extinction coefficient value of 2.5 × 10⁴ M⁻¹ cm⁻¹.

Rheological Characteristics

Blends were prepared using raw and defatted raw germ at 1 and 2% by replacing wheat flour. Dough properties for wheat flour-germ blends and with 500 U and 1000 U of LOX per 100 g of wheat flour were determined using a farinograph.[16]

Extensible properties for the same were determined using an alveograph according to standard AACC methods.[16]

Bread Making Characteristics

Breads were prepared in triplicate using a straight dough method according to the standard ‘Remix’ baking test with slight modification.[17] Fat at a 1% level was included in the formulation. Doughs were mixed in a planetary mixer. Ingredients other than flour (100 g, 14.0% moisture base), were fresh yeast (2.0%), sugar (2.5%), and salt (1.0%). Mixing time was 4 min and water absorption was based on farinograph water absorption. Fermentation was for 90 min with a knock back, molded after 25 min, final proof was 55 min, and baking was 35 min at 220°C. Breads were prepared from wheat flour-germ blends and with lipoxygenase extract at different levels, cooled to room temperature, then packed in polypropylene bags till further analyses.

Evaluation of Breads

Evaluation of breads was carried out after storing overnight for weight and loaf volume (rapeseed displacement method).[18] Objective measurement of texture (crumb firmness) was carried out in a texture analyzer (TAHDi, Stable Micro Systems, Godalming, UK) by the standard AACC method.[16] Crumb firmness was measured by compressing a 25-mm-thick bread slice by 25% using a 10 kg load cell and a plunger of 36 mm diameter at 100 mm/min crosshead speed. Objective evaluation of the color of breadcrumb was measured using a UV-visible recording spectrophotometer (Model UV 2100, Shimadzu Corporation, Kyoto, Japan) with a reflectance attachment of illuminant G. The crumb color of the bread in terms of total color difference value (ΔE) was measured against a standard white board made of barium sulphate (100% whiteness). The bread slice was placed in the sample holder and the reflectance was auto recorded for wavelengths of 360 to 800 nm. The ΔE for the bread slice in comparison with the standard barium sulphate was spectrophotometrically recorded.

Sensory Evaluation

Six panelists who were briefed about the characteristics beforehand carried out evaluation of bread for crust and crumb characteristics on a seven-point hedonic scale by assigning different scores for various parameters. Samples were presented as whole bread for appearance, half-bread for crumb color, texture by hand feel, and 2-cm slices for mouth feel and eating quality by serving in a randomized order. The data was subjected to statistical analysis using analysis of variance (ANOVA) followed by Duncan's Multiple Range Test at significance level of p < 0.05.[19]

Scanning Electron Microscopy (SEM)

Samples for SEM were prepared according to the method described by Sidhu et al.,[20] with slight modifications. A representative portion of freshly mixed dough, dough after first and second fermentations were cut using a sharp scissors with minimum distortion, frozen at –20°C, and freeze dried using a Heto freeze dryer (model DW3, Allerød Denmark). Breadcrumb of the same sample was freeze-dried. The freeze-dried pieces of dough at different stages were mounted on the specimen holder using double-sided scotch tape and were exposed to gold sputtering (2 min, 2 mbar). The samples were then subjected to a SEM examination using a Leo Scanning Electron Microscope (model 435 VP, Leo Electronic System, Cambridge, UK). Micrographs of appropriate magnifications were selected for presentation of results.

RESULTS AND DISCUSSION

Lipoxygenase Activity

Extraction of lipoxygenase was separately carried out for raw and defatted germ along with wheat bran. Lipoxygenase activity was found to be higher in defatted wheat germ (6.361 × 10⁻³ U) when compared to raw germ (4.392 × 10⁻³ U) and wheat bran (1.231 × 10⁻³ U) at pH 5.5 and 4°C. Hence, defatted wheat germ was used for extraction of lipoxygenase.

Table 1  Effect of germ and lipoxygenase on the rheological characteristics of wheat flour dough

Sample	FWA (%)	DDT (min)	DS (min)	MTI (BU)	P (mm H2O)	L (mm)	P/L	W (×10^−4) (J)
Control	59.5d	5.0b	8.0c	30c	102b	55a	1.84e	197a
1% raw germ	60.1c	5.0b	8.0c	30c	98c	39c	2.59b	143c
2% raw germ	60.2c	4.0d	7.5d	35b	84e	35d	2.36c	108d
1% defatted germ	60.0c	4.5c	7.0e	40a	101b	41b	2.45bc	158b
2% defatted germ	60.8c	4.5c	7.0e	40a	90d	41b	2.19d	133c
500 U lipoxygenase	62.3b	6.0a	9.0b	25d	114a	40bc	2.82a	184a
1000 U lipoxygenase	66.7a	5.0b	10.5a	20e	90d	35d	2.55b	132c
SEM (±)	1.02	0.14	0.54	1.14	3.51	1.23	0.28	2.16
Values for a particular column followed by different letters differ significantly (p < 0.05).
SEM: standard error of mean at 14 degrees of freedom; FWA: Farinograph water absorption; DDT: dough development time; DS: dough stability; MTI: mixing tolerance index; BU: Brabender unit; P: maximum over pressure (mm H2O); L: abscissa at rupture (mm); P/L:curve configuration ratio; W: energy of dough (10^−4 J).

Farinograph Characteristics

The farinograph characteristics for wheat flour-germ blends and with different levels of lipoxygenase are summarized in Table 1. The data show that there was a marginal increase in farinograph water absorption on increase in addition of either raw or defatted germ from 0 to 2%. Upon addition of 500 U of lipoxygenase, the water absorption increased to 62.3% and on addition of 1000 U, it further increased to 66.7%. There was no change in dough development time on addition of lipoxygenase, whereas, with raw and defatted germ, there was marginal reduction in dough development time. Dough stability gradually reduced from 8 min (control) to 7 min on germ addition, whereas on addition of lipoxygenase, the dough stability increased to 10.5 min. These results indicated that incorporation of either raw or defatted wheat germ had a marginal adverse effect on the dough properties indicating weakening effect on the rheological characteristic of the dough as reported earlier.[21] However, the addition of lipoxygenase at different levels improved the dough characteristics indicating that LOX had an oxidative effect on the dough properties. Lipoxygenase is liable to promote cleavage of disulphide bonds and initiates new cross linking between free SH groups of gluten protein, thereby increasing the number of SS groups due to the oxidative effect of the enzyme.[5,6] Lipoxygenase extracted from soy protein improved the rheological characteristics of wheat flour.[12,13,15] The result also agrees with the result obtained by Bonet et al.,[22] wherein glucoprotein oxidation was used to study the effect on rheological characteristics.

Alveograph Characteristics

Effect of germ and LOX on the alveograph characteristics of wheat flour dough is presented in Table 1. An addition of 1% level of raw germ or defatted germ did not show changes in the maximum over pressure (P), a measure of dough elasticity, whereas at 2% level there was a deteriorating effect on the tensile force, which is evident in the decrease in maximum over pressure (P) value. Addition of 500 U lipoxygenase increased the ‘P’ value indicating that lipoxygenase might have functioned as an oxidizing agent resulting in formation of three-dimensional elastic network of protein, starch, and lipids. On the other hand, addition of raw germ, defatted germ, or lipoxygenase did not increase the L values (abscissa at rupture). The values for configuration ratio varied from 1.84 to 2.82. Maximum pressure ‘P’ values and values of abscissa at rupture ‘L’ decreased on addition of either raw or defatted germ causing a reduction in energy values of dough ‘W’, a measure of alveogram area. An addition of 500 U of lipoxygenase, on the other hand, did not show a reduction in deformation energy of dough. Gujral and Rosell[1] attributed the strengthening effect of glucose oxidase on the wheat flour dough to the formation of additional protein crosslinks via disulphide.

Bread Making Quality

The addition of both raw and defatted wheat germ marginally decreased the specific volume of bread from 3.69 to 3.43 and 3.30 cc/g with increase in raw and defatted germ (Table 2). On the addition of 500 U and 1000 U of LOX, the specific volume increased to 3.99 and 3.86 cc/g, respectively. Similar results were obtained at lower levels of enzyme units as reported by Shiiba et al.[6]

Table 2  Physical and sensory characteristics of breads

				Crust		Crumb
Sample	Specific volume* (cc/g)	Texture** (g force)	Color* (ΔE)	Shape (7)	Symmetry (7)	Color (7)	Grain (7)	Texture (7)	Overall quality (7)
Control	3.69 ± 0.05bc	594 ± 2.3e	22.34 ± 0.1c	6.0a	6.0a	5.5b	5.5c	5.5bc	6.0b
1% raw germ	3.59 ± 0.03c	646 ± 5.2d	28.06 ± 0.3a	6.0a	5.5ab	5.0c	5.5c	5.0c	5.5c
2% raw germ	3.43 ± 0.02cd	669 ± 2.9c	28.70 ± 0.5a	6.0a	5.0b	4.5d	4.5	4.5d	4.5e
1% defatted germ	3.39 ± 0.05d	1041 ± 5.3b	27.47 ± 0.3b	6.0a	5.5ab	5.0c	5.0d	5.0c	5.0d
2% defatted germ	3.30 ± 0.05e	1118 ± 7.6a	27.16 ± 0.4b	6.0a	5.0b	4.0d	4.5e	4.0e	4.0
500 U LOX	3.97 ± 0.03a	546 ± 4.3g	19.04 ± 0.2d	6.0a	6.0a	6.5a	6.0b	6.5a	6.0b
1000 U LOX	3.86 ± 0.01b	560 ± 5.1f	20.15 ± 0.3d	6.0a	6.0a	5.5b	6.5a	6.0ab	6.0b
2% gluten	4.02 ± 0.02a	562 ± 3.1f	23.74 ± 0.2c	6.0a	6.0a	5.5b	6.0b	6.0ab	6.0b
2% gluten + 500 U LOX	3.95 ± 0.05a	538 ± 4.3g	22.97 ± 0.1c	6.0a	6.0a	6.0a	6.5a	6.5a	6.5a
SEM (±)	0.05	3.58	0.12	0.02	0.033	0.12	0.19	0.18	0.24
df	32	48	32	48	48	48	48	48	48
Values are means ± standard deviation (*n = 3, **n = 6). Values for a particular column followed by different letters differ significantly (p < 0.05); SEM: standard error of mean; df: degrees of freedom.

The compression force for control bread was 594 g and increased to 669 g on addition of raw germ, and further increased when defatted germ was added (1041–1118 g). At 500 U level of lipoxygenase, the force required was 546 g, whereas at 1000 U level, it decreased to 560 g. Lower firmness value on addition of lipoxygenase extract either at 500 or 1000 U level, when compared to control, indicated improvement in baking performance, which may be attributed to the oxidizing effect of lipoxygenase during the bread making process. It was reported that soy flour lipoxygenase converted fatty acid to H₂O₂, which in turn reacted with the flour component.[11] Hence, there was a strengthening effect and development of dough. Also, specific soy lipoxygenase isoenzymes (L2/L3) increased the bread loaf volume and reduced the crumb firmness.

Lipoxygenase is also known for its bleaching effect[6,23,24] and, hence, the color of bread crumb samples was analyzed. The ΔE value, which indicates the total color difference between the sample and standard white (barium sulphate), was 22.34 for control bread and reduced to 19.04 and 20.15 on the addition of 500 and 1000 U of enzyme (Table 2). The reduction in ΔE values, on addition of lipoxygenase depicts that lipoxygenase, at 500 U, has greater bleaching effect. The bleaching action observed upon the addition of lipoxygenase and the isozyme, which is mostly responsible, is supposed to be L3.[6,22] On the addition of 1 and 2% of raw and defatted germ, the ΔE increased, indicating that crumb became slightly darker than the control. The probable reason for this may be due to other pigments present in germ has overshadowed the effect of enzyme, which is present in very less quantities. The sensory evaluation data presented in Table 2 indicate that the crust color was similar to control for all the breads, the shape and symmetry scores for breads containing LOX, was higher. Breads were brighter and softer with LOX as also seen in the lower compression values.

Under similar conditions of processing, on the addition of gluten alone and along with 500 U of lipoxygenase, the bread volume increased to 545 and 560 cc, respectively, as compared to the control, which had a volume of 500 cc. Similarly, specific volume also increased to 4.02. The bread obtained was much softer and the compression force required was 562 and 538 g force on addition of gluten and gluten along with 500 U lipoxygenase. The bleaching effect was more on addition of lipoxygenase alone than on addition of gluten and lipoxygenase in combination with gluten. This can be seen by reduction in ΔE values when only lipoxygenase was added (Table 2). Gluten addition has contributed to higher bread volume when added alone and in combination with lipoxygenase with similar softness. This also shows that lipoxygenase has no significant role on gluten with respect to expansion during baking. Sensory analysis showed no significant difference for crust characteristics. Gluten when added in combination with LOX showed higher scores for crumb color, texture and overall quality scores indicating synergistic effect on the bread quality.

Scanning Electron Microscopic (SEM) Studies

SEM is a powerful tool for structural observation of either dough or bread. It also provides useful information about the gluten strands and starch granules interaction[25] or in relation to other protein mixtures.[26] Figure 1 shows the SEM pictures of the fresh bread dough samples. The starch granules are seen dispersed in the developed gluten matrix. The dough surface has become more uniform in case of addition of lipoxygenase with or without gluten. The starch granules are seen at the early stage of dough development. Hence, the dough, which has been optimally mixed, has been interpreted as a three dimensional sponge like structure. After first fermentation, the dough is quite normal in the gluten matrix covering most of the starch granules. There are small and large starch granules densely distributed throughout the formed protein matrix (Fig. 2).

Figure 1 Scanning electron micrograph of bread dough just after mixing: (a) control; (b) 500 U lipoxygenase; (c) 2% gluten; (d) 2% gluten + 500 U lipoxygenase; (e) 1000 U lipoxygenase.

Figure 2 Scanning electron micrograph of bread dough after fermentation: (a) control; (b) 500 U lipoxygenase; (c) 2% gluten; (d) 2% gluten + 500 U lipoxygenase; (e) 1000 U lipoxygenase.

However, after second fermentation (proofing) the large starch granules look like more swollen and more evenly dispersed in the protein matrix (Fig. 3). In case of addition of lipoxygenase or gluten or in combination showed a more uniform gluten matrix when compared to control. There is uniform matrix in experimental samples, when compared to control, indicating better gas retention property. Addition of lipoxygenase or gluten has resulted in a membrane-like structure of optimally developed gluten with good gas retention property. The starch granules are more swollen and more evenly dispersed within the protein matrix than in the first fermentation.

Figure 3 Scanning electron micrograph of bread dough after proofing: (a) control; (b) 500 U lipoxygenase; (c) 2% gluten; (d) 2% gluten + 500 U lipoxygenase; (e) 1000 U lipoxygenase.

The bread microstructure showed a strong connection among all the components resulting into a complex structure with numerous cavities (Fig. 4). Some starch granules are seen on the surface of the crumb. Gelatinisation has covered the surface of starchy granules in various processes of distortion and degradation appeared in the protein matrix. Addition of lipoxygenase, at 500 U levels, resulted in a smooth surface with fewer cavities. The gas cells in the sample containing 500 U lipoxygenase showed a more continuous and smooth surface with a thicker appearance than the control bread. An addition of 1000 U of lipoxygenase has further formed a thick sheet and the components are not at all revealed. This shows that the lipoxygenase has become linked with other dough constituents.

Figure 4 Scanning electron micrograph of bread: (a) control; (b) 500 U lipoxygenase; (c) 1000 U lipoxygenase.

CONCLUSION

Lipoxygenase activity was found to be higher at pH 5.5 and at 4°C in defatted germ. The addition of lipoxygenase improved the dough characteristics due to the oxidizing effect of the enzyme on gluten. Breads prepared with the enzyme had greater loaf volume and softer texture. The crumb was brighter due to the bleaching action of the enzyme. Sensory analysis showed that breads with lipoxygenase had higher overall acceptability when compared to breads with either raw or defatted germ. The addition of gluten along with lipoxygenase showed synergistic action contributing to greater volume and softer crumb. Electron micrograph studies confirmed a better network of gluten embedding starch granules than in control.

ACKNOWLEDGMENTS

The authors are thankful to Mr. Anbalagan, CIFS, CFTRI, Mysore, for his help in carrying out scanning electron microscopy.