The effects of milk fat globule membrane and its individual components on dough properties and bread quality

Abstract

The aim of this study was to investigate the effects of the addition of milk fat globule membrane (MFGM) and its individual components such as milk fat globule membrane protein (MFGMP) and milk fat globule membrane lipid (MFGML) on dough properties, microstructure, and the staling of wheat bread. The results showed that MFGM and its individual components' incorporation modified the rheological properties of dough. The addition of the MFGM, MFGMP and MFGML in bread significantly increased the specific volume of the samples compared with the control sample. In comparison with the control sample, bread prepared with the MFGM, MFGMP and MFGML effectively avoided moisture loss. The microstructure of bread slices was observed by scanning electric microscopy and exhibited the formation of liquid films with a lamellar structure between the gluten and starch in the samples prepared with the MFGM and MFGM components. Moreover, the addition of MFGM and MFGM components implied a decrease in both the hardness and chewiness of the crumb compared to the control sample after 5 days of storage. Crumbs with the addition of MFGM and MFGMP had the typical yellowish color of a bread loaf. The presence of the MFGM and MFGM components especially for the complete MFGM could significantly decrease the enthalpy of retrogradation of amylopectin. These results clearly indicated that the addition of MFGM and its individual components could effectively improve the quality and retard staling of wheat bread.

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Introduction

Bread, especially when prepared from wheat, is one of the most popular foods consumed globally. Technologies and various additives such as emulsifier, hydrocolloids, and enzymes have been developed to preserve the freshness of bread.^1–3 Staling strongly influences the quality of the bread by degrading the texture and taste of the product. The complicated process of bread staling is still not yet fully elucidated. An important factor affecting bread staling is water migration from the crumb to the crust. The water content of bread also determines recrystallization of starch polymers and therefore the texture of the bread.⁴ Another important factor inducing bread texture modifications is starch retrogradation that occurs during bread storage.^5–7

The milk fat globule membrane (MFGM) is a thin membrane surrounding the milk fat globule and can be directly separated from raw milk, butter serum, or buttermilk. It is mainly composed of milk fat globule membrane proteins (MFGMPs) and milk fat globule membrane lipids (MFGMLs).^8,9 The MFGMPs are mainly butyrophilin, xanthine oxidase, fatty acid-binding protein, and beta-glucuronidase inhibitor.^10,11 The MFGMLs are mainly neural lipids and polar lipids, neutral lipids including triglycerides, diglycerides, monoglycerides, esters and cholesterol, polar lipids including phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM).^12,13 Both MFGMPs and MFGMLs have beneficial functions on health such as inhibition of the growth of cancer cells,^8,14,15 and anti-bacterial¹⁶ or anti-adhesive^17,18 effects.

Emulsifiers are widely used to improve the quality and retard the staling of bakery products.¹ The natural emulsifier MFGM prevents the fat globules from coalescing and is considered as an efficient emulsifier because of its specific composition of proteins and lipids.⁸ Indeed, MFGM acting as an emulsifier prevents the lost of water contained in the bread crumb thanks to its high water-holding capacity. The inhibition of water migration can prevent bread from staling and hardening. Therefore, MFGM are used as improving agents of bread to increase the nutritional value of bread and improve the quality of bread.

The functional properties of MFGM and its individual components have attracted increasing attention in the exploration of different applications according to their amphiphilic nature.^19,20 Regarding the antistaling properties of MFGM and its components, it is not yet determine which of the components or a combination thereof, i.e. MFGMP, MFGML, or the complete MFGM, is responsible for the antistaling property. To clarify the participation of MFGM and its individual components in the antistaling of bread, this paper reports the evaluation of the antistaling properties of the different MFGM components and completes MFGM.

In contrast to other emulsifiers like sodium/calcium stearoyl lactylate and mono/diglycerides, which have been thoroughly studied,²¹ there are no data reporting the effect of MFGM and its individual components on the quality and staling of bread. The aim of the present work is to present the effect of MFGM and its individual components to improve the quality and reduce starch retrogradation of bakery bread.

Materials & methods

Materials

Commercial wheat flour (14.4% moisture content, 12.2% protein, 0.6% ash) for bread preparation and active dry yeast (Angel Co., Ltd., Hubei, China) was purchased from a local supermarket. The MFGM and its individual components were isolated from fresh bovine milk as described below.

Isolation of MFGM and its individual components

The complete MFGM, MFGML, and MFGMP were isolated from fresh bovine milk. The fresh bovine milk was fractionized using a 9NDS-50A cream separator (Qinghai Kangping, China). The cream was isolated and washed with phosphate buffer (PBS, 0.1 M, pH 6.8), and then centrifuged at 1500 × g for 10 min. The supernatant was discarded and the washing step was repeated three times. The washed cream was then used for the isolation of MFGM, MFGMP, and MFGML. (1) Separation of complete MFGM: the washed cream was allowed to crystallize for 4 h at 4 °C before churning in a mixer. The resulting butter and buttermilk were separated using a sieve. The pH of the buttermilk fraction was adjusted to 4.8 using hydrochloric acid (1 M) in order to precipitate the complete MFGM. The precipitated MFGM were centrifuged at 10 000 × g, and the MFGM pellet and supernatant were collected. The pH values of the MFGM pellet resuspended in water and of the supernatant were adjusted to 6.8 using sodium hydroxide (1 M). The isolated complete MFGM were then freeze dried; (2) fractionation of MFGMP: the separation of the MFGM proteins was based on the procedure reported by Lu et al.²² The washed milk cream was mixed with 0.4% SDS (1 : 1, v/v), then sonicated for 1 min and centrifuged at 1500 × g for 10 min. Subsequently, the MFGM proteins (bottom layer) were separated from the remaining fat and dialyzed with deionized water and then freeze-dried; (3) separation of MFGML: the remaining fat was separation by centrifuging at 6000 × g and then stirred at 100 rpm for 30 min, continue to centrifuged at 3000 × g for 15 min to get rid of butter and obtain the MFGML (bottom layer), the MFGML was concentrated by rotatory evaporator.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of MFGMPs

The MFGMPs were analyzed by SDS-PAGE as described by He et al.²³ The MFGMP samples were suspended in 0.5 mL of a reducing buffer (6% Tris-0.5 M, 10% glycerol, 5% β-mercaptoethanol, 2% SDS, and 0.05% bromophenol blue). Samples were heated in boiling water for 5 min and then centrifuged at 2500 × g for 30 min in order to remove the fat from the samples. The supernatants (10 μL) were loaded onto 12% SDS-polyacrylamide gels. Molecular mass markers ranging from 14.4 to 116 kDa (TransGen Biotech, Co., Ltd., China) and milk proteins migrated at 200 V. Protein bands were stained with a solution of Coomassie Brilliant Blue R-250. Gels were destained with a solution of methanol and glacial acetic acid at concentrations of 160 and 10 mL L⁻¹, respectively.

Composition analysis of the MFGM and its components

The analyses of the MFGM and its components were carried out using official AOAC methods, for the content determination of moisture (14.004), ash (14.006), and crude protein (47.021).²⁴ A nitrogen to protein conversion factor of 6.25 was used in the crude protein determination. The lipid content was calculated by subtracting the percentage of moisture, protein, and ash to the whole mass. All experiments were conducted as triplicates.

Bread preparation

The wheat bread was prepared from 100 g of wheat flour, 2% salt, 2% active dry yeast, and water was added in order to optimize the consistency of dough in a 500 Brabender unit. Then, the MFGM (2%), MFGML (2%), and MFGMP (2%) were introduced. All the ingredients were mixed and the dough was left to rest for 15 min. The resulting dough was proofed at 30 °C and 85% relative humidity for 50 min up to about three times the initial dough volume. After proofing, the subsequent baking was carried out at 230 °C for 30 min. Finally the breads were cooled to room temperature before sealing them in plastic bags. Each loaf was prepared in duplicate.

Dough rheology

Rheological measurements were conducted using a Kinexues rheometer (Malvern Instruments, England). Doughs prepared without yeast were allowed to rest for 5 min before measurement. Frequency sweep experiments were carried out between a frequency of 1.0–100 Hz, 0.5% strain was selected for tests. All measurements were conducted at 25 °C, using a 40 mm diameter parallel plate geometry, with 1 mm gap. Elastic (G′) and viscous (G′′) moduli were recorded for dough. Dough preparation was performed in triplicate. Experimental data were described by the power law model.²⁵

G′(ω) = K′ω^n′

G′′(ω) = K′′ω^n′′

where G′ is a storage modulus (Pa), G′′-loss modulus (Pa), ω-angular frequency (rad s⁻¹), and K′, K′′, n′, n′′-experimental constants.

Bread analysis

The bread volume was measured by the rapeseed replacement method,²⁶ and then the bread weight (g) was measured. The bread specific volume (cm³ g⁻¹) was determined by the ratio of the bread volume to its weight. Width/height ratio refers to the size of central slice.

Digital image analysis of the bread crumb was performed using the image processing software Image J (Image J, USA). Slices of 15 mm thick were scanned for each bread (M7650DNF Scanner, Lenovo, China). Each image was cropped to a square field of 30 × 30 mm in actual dimensions of the slice area and saved on the computer's hard disk. The saved images were converted to the gray scale by activation of the 8 bytes options with gray levels ranging from 0 to 255. The threshold routine was run using an automated fuzzy measure thresholding method that converted the images from gray level to black and white in order to differentiate the gas cells from the non-cells. The setting permitted to distinguish the radius of the cells from 50 to 50 000 μm. The mean cell area (mm²) and the cell area fraction (%) were then determined. All measurements were repeated with three replicates.

The color of the bread crumb was evaluated using a WSC-S colorimeter (Shanghai INESA Physico Optical Instrument, China) on a 15 mm thick slice of bread and at least eight L*, a*, b* values (L* = 100/0 white/black, ±a* = red/green, and ± b* = yellow/blue) were recorded.

The water content of the breads was determined at the center of loaf at different storage days and was measured according to the AACC method.²⁷ Each measurement was carried out in triplicate.

The crumb texture analysis was performed after 1, 3, and 5 days after baking on a TA-XTplus (Stable Micro Systems, England). A 25 mm thick slice was compressed with a P/50 probe up to 40% and the values of the pretest speed, test speed, post-test speed were all set to 2.00 mm s⁻¹ with a relaxation time of 5 s. The trigger force was 5.0 g and trigger mode was on the automatic mode. The resulting hardness (g), gumminess (g), and chewiness (g) were used as the indicators of textural changes during the storage and resulted from the average value of four replicates.

Thermal properties of the bread crumb were analyzed by a scanning calorimeter (DSC 204F1 Phoenix, PerkinElmer, USA). The calorimeter was calibrated according to the indium standard. The thermal transitions were undertaken for a control sample (without MFGM and its components) and for samples of bread crumb with MFGM (2%), MFGML (2%) and MFGMP (2%). After a post-baking period of 1, 3, and 5 days, the loafs were packed hermetically in an aluminum pan and heated in the calorimeter from 25 to 100 °C at rate of 10 °C min⁻¹. The empty aluminum pan was used as the reference. The enthalpy values were expressed as J g⁻¹ of dry matter.

Scanning electron microscopy (SEM) analysis

The crumb of the breads prepared from MFGM and its components was observed using a QUANTA 200 scanning electron microscope (FEI, America) at magnifications of 3000 × g. The bread samples were frozen at −20 °C and then freeze dried. The freeze-dried bread crumb samples having a size of approximately 10 × 10 × 3 mm were then sputter coated with gold using a SCD 005 sputter coater (BAL-TEC Machinery Ltd., Czech Republic), finally, the samples were transferred to the microscope operating at 10 kV.

Statistical analysis

The statistical difference between the mean values was analyzed by the one-factor analysis of the variance, and the least significant difference (LSD) at a significant level set at 0.05. The LSD values were calculated using Tukey's post hoc test. The calculations were performed using the Statistical Package for the Social Sciences v. 10.0 (SPSS, Chicago, IL, USA).

Results and discussion

Composition of MFGM and its components

The basic composition of the MFGM, MFGML and MFGMP is shown in Table 1. The complete MFGM was mainly composed of lipids (73.41%) and proteins (25.24%). The isolated sample of MFGML contained 94.47% of lipids. The MFGMP contained about 84% of proteins and 12% of lipids. The MFGMP were identified by SDS-PAGE (Fig. 1), the MFGMP were composed of mucin 1 (MUC), xanthine oxidase (XO), butyrophilin (BUT), and periodic acid-schiff 6 and 7 (PAS 6 and PAS 7). Casein and whey proteins were almost not found in the MFGMP sample. Gallier et al.²⁸ also reported the similar MFGM proteins composition in bovine milk.

Table 1 Composition of MFGM, MFGML and MFGMP^a

Sample	Moisture	Proteins	Lipids	Ash
a Under the detection limit.
MFGM	1.28 ± 0.25	25.24 ± 3.76	73.41 ± 2.12	0.07 ± 0.01
MFGML	5.18 ± 1.04	0.35 ± 0.07	94.47 ± 0.67	—
MFGMP	2.38 ± 0.20	84.35 ± 1.23	12.63 ± 0.45	0.64 ± 0.08

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Fig. 1 SDS-PAGE of samples. (1) The protein marker; (2) MFGMPs.

Rheological properties of dough

Analysis of rheological properties provides information about the effect of applied ingredients on rheological behavior of the dough. Fig. 2 presents the changes in storage modulus G′ and loss modulus G′′ for the control samples and samples with the addition of MFGM and its individual components. In all cases the values of G′ were higher than G′′ in all the frequency range studied, which indicates that elastic properties predominated viscous behavior. As shown, MFGM and its individual components incorporation affected the dough behavior during fermentation, the effect was different from one components to another, the MFGMP presents the highest G′ and G′′ values compared to control samples, followed by MFGML and MFGM, increase of moduli G′ and G′′ in comparison to control sample which was also confirmed by the values of K′ and K′′ (Table 2). Dough with the addition of MFGM and its individual components caused increase of moduli G′ and G′′ (Fig. 1) and a correspondence rise of K′ and K′′ (Table 2). On the other hand the application of MFGM and its individual components in dough formulation had no impact on the values of n′ and n′′ (Table 2). The higher values found in dynamic moduli (G′ and G′′) of doughs with MFGM and its individual components regarding control dough, clearly indicates that the application of MFGM and its individual components results in evident changes of dough rheological and introduces new interactions into the system. This result may be related to the emulsion properties of MFGM and its individual components. Sciarini et al.²⁹ reported that the presence of emulsifiers introduces new interactions into the system, and that their effect will also depend on the type of hydrophilic and hydrophobic interactions established. It has been reported that in wheat based systems, emulsifiers facilitate the interaction between lipids, proteins and starch,³⁰ possibly due to their amphiphilic nature, and that these interactions are responsible for dough reinforcement.

Fig. 2 Dynamic moduli (G′ and G′′) of doughs with MFGM and its individual components.

Table 2 Parameters of power-low-functions describing dependence of storage and loss moduli on angular frequency (mean value of two replications ± standard deviation)^a

	G′ = K′ω^n′			G′′ = K′′ω^n′′
	K′ × 10^−3 [Pa s^n′]	n′	r^2	K′′ × 10^−3 [Pa s^n′′]	n′′	r^2
a The values are the mean value obtained from four replicates ±standard deviation. Mean values marked with different letters in specific columns differ significantly from each other at a level of confidence set at 0.05.
Control	3.10 ± 0.68^a	0.20 ± 0.022	0.96	1.23 ± 0.240^a	0.22 ± 0.036	0.93
MFGM	3.95 ± 0.693^b	0.19 ± 0.004	0.99	1.47 ± 0.198^b	0.25 ± 0.016	0.95
MFGML	5.15 ± 1.379^b	0.20 ± 0.005	0.98	2.02 ± 0.552^b	0.26 ± 0.026	0.95
MFGMP	6.28 ± 1.047^b	0.18 ± 0.012	0.96	2.37 ± 0.318^b	0.27 ± 0.004	0.97

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Effect of the addition of MFGM and its components on the quality parameters of bread

The effect of the addition of MFGM and its components on the quality parameters of wheat bread is presented in Table 3. The control sample exhibited a lower specific volume than the other samples. The greatest increase was found for the bread prepared with the addition of complete MFGM (specific volume of 5.61 cm³ g⁻¹), followed by the MFGML prepared bread (specific volume of 5.47 cm³ g⁻¹), and by the complete MFGMP prepared bread (specific volume of 5.41 cm³ g⁻¹). In a previous study, it was shown that emulsifiers had the ability to form liquid films with a lamellar structure at the interphase between the gluten and starch of bread.³¹ The MFGM and its components, acting as emulsifiers, maybe also form liquid films with a lamellar structure. The liquid films with a lamellar structure could retain the carbon dioxide produced by yeast and therefore increase the specific volume of the bread loaf. The increase of the specific volume was mainly ascribed to the capacity of the dough to retain the carbon dioxide and to its suitable elasticity allowing expansion during baking.³² Moreover, other reports suggested that the crumb of bread mixed with different emulsifiers such as sodium/calcium stearoyl lactylate and mono/diglycerides could increase the specific volume of the crumb.¹ The ratio of the width to height of the crumb prepared with MFGM and its components showed no difference compared to the control crumb. As shown in Fig. 3, both the width and height values of the crumb prepared with the MFGM and its components were slightly higher than the control crumb. Consequently, the ratios of the width to height of the crumb of the bread prepared with the MFGM and MFGM components were similar to the control sample. The cell area fraction of the crumb was defined as the ratio of total cell area to the area of the analyzed image. It can be seen that the cell fraction of the crumb prepared with the addition of MFGML, MFGMP and complete MFGM was significantly higher than that of the control crumb. The results suggested that the crumb prepared with the addition of MFGML, MFGMP, and complete MFGM produced more gas cells than the control crumb. The mean cell area values of the crumb prepared with the addition of MFGML, MFGMP, and complete MFGM increased when more gas was produced in the bread. The results also showed that the addition of MFGM and its components produced more gas than in the control sample. This observation was consistent with the results obtained for the specific volumes, showing that a more important mean cell area induced an increase in the outside volume. These results further support that MFGM and its components, especially MFGM, improved the quality of bread.

Table 3 Effect of the addition of different MFGM and its components on selected parameters describing the freshness of the bread^a

	Control	MFGML	MFGMP	MFGM
a The values are the mean value obtained from four replicates ±standard deviation. Mean values marked with different letters in specific columns differ significantly from each other at a level of confidence set at 0.05.
Specific volume (cm^3 g^−1)	4.95 ± 0.17^a	5.47 ± 0.31^b	5.41 ± 0.27^b	5.61 ± 0.21^c
Width/height ratio	0.85 ± 0.04^a	0.93 ± 0.06^a	0.87 ± 0.05^a	0.95 ± 0.05^a
Cell area fraction (%)	39.55 ± 1.96^a	42.57 ± 2.11^b	42.00 ± 2.33^b	43.63 ± 2.70^b
Mean cell area (mm^2)	0.55 ± 0.02^a	0.66 ± 0.03^a	0.60 ± 0.04^a	0.76 ± 0.04^a

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Fig. 3 Photographs of bread slices from the control sample and bread loafs prepared with MFGM and its components.

Moisture content of bread

Moisture content of a product is closely related to the stability and quality of the food.³³ Moistness is a favorable sensory attribute for soft and tender baked-food products.³ The loss of moisture in bread results in the crumb hardening and starch recrystallization involved in bread staling.³⁴ In general, lower moisture lost corresponds to a slower firming rate of the baked product.

The crumb moisture contents of all samples during storage of 5 days are shown in Table 4. The moisture contents of all the samples decreased during the storage period but at different rates. The initial moisture content of all samples showed no significant difference. However, the moisture content of the control sample remarkably decreased from 39.83% to 27.37% after 5 days of storage. After 5 days of storage, the moisture contents of the crumb prepared with the complete MFGM, MFGML and MFGMP were significantly higher than the control crumb. Therefore, the bread prepared with the MFGM and its components retained better water in the crumb than the control sample. The results could be explained by the strong water-holding capacity of the MFGM and its components as emulsifiers due to their amphiphilic nature.

Table 4 Crumb moisture content (%) of the MFGM, MFGML and MFGMP-supplemented wheat breads and control (without MFGM and its components) during 5 days of storage (n = 3)^a

	0 day	1 day	3 days	5 days
a Values followed by different lower case letters in columns, and upper case letters in rows, indicate significant difference (p < 0.05).
Control	39.83 ± 0.56^aA	35.11 ± 1.15^aB	30.22 ± 1.30^aC	27.37 ± 1.13^aD
MFGM	39.90 ± 0.24^aA	38.43 ± 0.32^bA	34.65 ± 0.65^bB	31.20 ± 0.12^bC
MFGML	39.49 ± 0.45^aA	36.40 ± 0.93^aB	33.41 ± 0.95^bB	30.39 ± 0.89^bC
MFGMP	39.40 ± 0.72^aA	36.81 ± 0.32^aB	32.27 ± 0.62^abC	30.40 ± 0.83^bC

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Effect of the MFGM and its components on the bread microstructure

The bread microstructure was observed by SEM. The microstructures observed by SEM of the samples prepared with MFGM and its components are shown in Fig. 4. The control sample showed a discontinuous surface with a loosened structure of the connections among all components. The microstructure of the samples prepared with the addition of complete MFGM and MFGMP showed a more continuous and smooth surface than the control sample. The components of bread with the addition of complete MFGM and MFGMP could be assigned as described by Rojas et al.³⁵ Resistant networks were formed when continuous and tight structures were observed.³⁶ The samples prepared with MFGML also exhibited a more continuous and smooth surface than the control sample. Overall, the microstructure of the samples prepared with the MFGM and its components seemed to form a liquid film with a lamellar structure at the interface between the gluten and starch components. The film permitted to retain the gas produced by yeast, increasing the specific volume of bread, and at the same time retaining the water. Those observations indicated that the MFGM and its components could be interact closely with the dough constituents directly involved in the process of bread anti-staling.

Fig. 4 Cryo-SEM micrographs of crumb prepared with the different additions. (a) Control; (b) MFGM; (c) MFGMP; (d) MFGML.

Texture characteristics of bread

Selected texture parameters of bread crumb including hardness, gumminess, and chewiness at 1, 3, and 5 days of storage are summarized in Fig. 5. The hardness of the crumb increased when the days of storage increased, indicating that the staling of bread crumb occurred during the storage period. On the first day of the storage, the hardness of the crumb prepared with the MFGM and its components did not significantly change compared to the control sample. On the third day, only the hardness of the crumb of the sample prepared with complete MFGM was significantly lower than the control sample. However, the addition of the MFGM and its components caused a significant reduction of the hardness after storing for 5 days, especially for the sample prepared with complete MFGM. The results showed that the MFGM and its components could soften the crumb and inhibit the bread staling. The reduced hardness in bread samples containing MFGM and its components could be related to the high water-holding capacity. This functional property can prevent the lost of internal water of the crumb, resulting in a soft bread. Although there is no study reporting the effects of MFGM and its components on the texture properties of bread, emulsifiers such as distilled monoglycerides and sodium stearoyl lactylate are known to significantly reduce the hardness of bread.¹ Our results showed that a combination of proteins and lipids from MFGM afforded a better water-holding capacity than individual MFGM components. Gumminess refers to the steady-state energy required to break a sample during swallowing. The presence of only MFGM in bread could significantly lower the gumminess after storage 3 and 5 days. Chewiness refers to the steady-state energy required during swallowing of a solid sample. Low values of gumminess and chewiness reflect consumer-acceptable textural characteristics of bread. The evolution of the chewiness of bread prepared with the MFGM and its components was similar to the hardness. After a storage of 3 and 5 days, the chewiness of crumb prepared with complete MFGM significantly reduced, while the chewiness of the crumbs prepared with MFGML and MFGMP started to reduce after 5 days of storage. These results indicated that MFGM and its components, especially complete MFGM, had a positive impact on the texture properties of bread.

Fig. 5 Texture characteristics of control wheat bread and samples prepared with the MFGM and its components. Presented data are mean values of three replications. Error bars mean standard deviations. Mean values marked with the different letters for the same day differ significantly from each other at 0.05 level of confidence.

Bread crumb color

The crumb color of bread is an important attribute to consumers' preferences. The crumb color parameters of the samples prepared with the MFGM and its components are shown in Table 5. The MFGM, MFGML and MFGMP did not significantly modify the luminance L*, indicating that the addition of the different MFGM and its components did not impact the whiteness of the samples supporting the conclusions of Aguilar et al.³⁷ The samples with the addition of MFGML and MFGMP significantly increased the a* parameter, indicating that the concerned samples exhibited more red in their color. The addition of MFGM and MFGMP significantly increased the value of the parameter b*, pointing out a dominance of yellow as observed in typical bread crumbs.

Table 5 Color parameters of bread samples prepared with MFGM, MFGML and MFGMP^a

	L*	a*	b*
a Values were calculated as the mean value of four replicates ±standard deviation. Mean values marked with different letters differ significantly from each other at a level of confidence of 0.05.
Control	85.66 ± 0.37^a	1.92 ± 0.21^a	18.68 ± 0.54^a
MFGM	85.46 ± 0.84^a	1.94 ± 0.17^a	20.08 ± 0.72^b
MFGML	85.58 ± 0.64^a	2.11 ± 0.33^b	19.86 ± 0.43^a
MFGMP	84.20 ± 1.07^a	2.41 ± 0.28^b	20.68 ± 0.85^b

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Thermal properties of bread crumb

The enthalpy of retrograded amylopectin was measured by DSC for the control sample and the samples prepared with the MFGM and its components. All samples were stored at 4 °C for 1, 3, and 5 days. The values of the retrogradation enthalpy of the bread crumbs prepared with the MFGM and its components are shown in Fig. 6. The phase transition occurred at peak temperature ranging from 60 to 65 °C independently from the presence of the MFGM, MFGML and MFGMP or the storage time. The enthalpy of the retrograded amylopectin in all samples increased as with the storage time, indicating that the staling phenomenon occurred during the storage period for all the samples but at different rates. The enthalpy of the retrograded amylopectin in the control bread crumb presented a rapid increase during the 3 days of storage. However, the enthalpy of the retrograded amylopectin in bread prepared with the MFGM, MFGML and MFGMP showed a slower increase. The enthalpy of retrograded amylopectin in bread with the MFGM and its components was significantly lower than the control sample, especially for the case of complete MFGM. The values of the MFGM sample decreased by 36.67%, 34.92%, and 40.64% compared with the control experiment during 1, 3, and 5 days of storage, respectively. This observation could be explained by the presence in the complete MFGM of lipids and specific proteins such as xanthine oxidase and butyrophilin, considered as good emulsifiers retarding bread staling. Our experimental results support the theoretical consideration that the addition of emulsifier leads to a decrease in starch retrogradation.^38–42 It is worth noting that the addition of MFGML and MFGMP also showed retarding in the staling process, while the addition of MFGM exhibited a more beneficial effect than MFGML and MFGMP. This result suggests that the combination of the possible interactions between MFGML and MFGMP may result in a more effective retarding staling than the individual components.

Fig. 6 Enthalpy values of amylopectin retrogradation of the control sample and of the samples prepared with the MFGM and its components during storage. The presented data are mean values of three replications. The error bars mean standard deviations. Mean values marked with different letters differ significantly from each other at level of confidence of 0.05.

The low enthalpy value in the sample prepared with the MFGM and its components may be related to the low water contents. It had been reported that the moisture content in bread crumb is an important factor affecting the retrogradation of starch, illustrated by a positive correlation of the enthalpy values with the moisture control.^43–45 The results obtained for the hardening and the retrogradation of the amylopectin suggested that the MFGM and its components had the ability to delay the bread staling. These results also suggest that the addition of the MFGM and its components not only contributed to an improvement in the nutritional value of wheat bread, but also acted as an effective antistaling ingredient.

Conclusions

The contribution of the MFGM, MFGML and MFGMP could modified the rheological properties of dough, significantly increase the specific volume of bread and prevent the moisture lost in the crumb of the bread samples compared with the control sample. Addition of the MFGM and its components resulted in a significant decrease in hardness and chewiness of bread compared with the control sample. The addition of the MFGM and MFGM components also caused a decrease in the retrogradation of the amylopectin of the crumb. These results showed that the application of the MFGM and its components in bread not only improved the nutritional value of wheat bread, but also effectively prevented the bread from staling. The use of MFGM and its components, however, requires optimization of baking mixtures because of the significant differences in the water-holding capacity and emulsifying properties of the individual components of MFGM.

Acknowledgements

The authors acknowledge the financial support of the Fundamental Research Funds for the Central Universities (Grants No. HIT, NSRIF, and 2014094), and the China Postdoctoral Science Foundation (Grants No. 2012M520756 and 2014T70360).

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