Effect of Degree of Konjac Glucomannan Enzymatic Hydrolysis on the Physicochemical Characteristic of Gluten and Dough.

Influences of different enzymatic hydrolysis degrees of konjac glucomannan (KGM) with various addition proportions on the structural characteristic of gluten protein and dough properties were evaluated. Results revealed that addition of KGM decreases the free sulfhydryl and freezable water content of dough, and KGM with different enzymatic hydrolysis degrees had more beneficial effects on strengthening the gluten structure by the raised molecular weight of gluten proteins and the increased disulfide bonds and β-sheet content, especially KGM with 15 min enzymatic hydrolysis treatment (KGM II). Besides, microstructure observation and thermal analysis results illustrated that addition of KGM promotes gluten cross-linking and improves the thermal stability of the gluten network structure. Dough possessed better elasticity, as well as tensile and texture properties with the addition of KGM than the control sample. And the KGM with 15 min enzymatic hydrolysis showed the most positive effect on dough quality than others, and 2.0% addition proportion is the most acceptable.

Introduction

Dough is a complex mixture of wheat flour and many other ingredients. There are different processing methods to produce different traditional flour products like bread, biscuit, noodles, steamed bread, and many other forms. Assessment index of dough quality mainly includes appearance, texture, cooking, and rheology properties, among which the rheology properties play an important role in determining dough quality.[1,2] Numerous researchers have proved that gluten protein quality is an important influencing factor of wheat processing suitability and has a positive correlation with dough rheology properties.[3,4] Therefore, dough quality can be evaluated by the gluten protein content of flour, which has obvious effects on viscoelasticity and the rheological properties and serves as a support skeleton in dough.

Addition of various food additives not only affects the texture and enhances the volume of final products but also decreases the risk of physical damage induced by different food processing approaches. Addition of 2.0% trehalose improves the specific volume and granular structure of baking products made from frozen dough and hydroxypropyl methylcellulose reduces the mobility of water and restrains the formation and growth rate of ice crystals to make the microstructure and network structure of gluten more stable during the freezing process.[5,6] In has been proved that pectin and fruit polyphenols could affect the interactions between water and gluten proteins during dough formation and bread baking, which resulted in change in cross-linking of the gel structure and texture properties of bread.[7]

Konjac glucomannan (KGM) is a typical high polymer dietary fiber, which was extracted from the roots and tubers of Amorphophallus konjac. It consists of d-mannosyl and d-glucosyl β-1,4-linked at a molar ratio of 1.6:1.0, while β-1,3-mannosyl units connect to the other side chain.[8] The molecular weight (Mw) of native KGM ranges from 1.0 × 105 to 2.0 × 106 Da and is influenced by species and producing areas. It was widely used as gelatine, thickening agent, and water-retaining agent in the food and pharmaceuticals industry.[9,10] KGM can be decomposed into oligosaccharides with different degrees of degradation via physical and chemical treatment and enzymatic hydrolysis.[11−14] Recently, enzymatic hydrolysis has been commonly used in KGM degradation and KGM skeletons have been hydrolyzed into monosaccharides by β-mannanase, which catalyzes β-d-1,4-mannopyranosyl into mannobiose and mannotriose.[15−17] The degradation products with various Mw values have excellent dispensability, specific physicochemical properties, and antioxidant activities.[18−20] In addition, degraded KGM can serve as a kind of additive to promote synergistic effects when combined with protein to form different rheologies of dough.[21−23] Understanding the enzymatic hydrolysis of KGM will be beneficial to broaden its development scope in the food industry.

In this study, a microrheology analyzer (Rheolaser MASTER) was employed to analyze the change of the viscoelastic properties of dough with different enzymatic hydrolysis degrees of KGM addition. This approach is built in accordance with the measurement of the mean-square displacement (MSD) of particles by laser light scattering and gives a deep understanding on the elastic and viscosity index (moldflow viscosity index), storage modulus (G′), and loss modulus (G″). Based on the diffusing-wave spectroscopy and theory of Brownian motion, decorrelation curve quantifies particle motion, which was obtained and characterized by the viscoelasticity of soft materials.[24,25] Based on this result, we can have a better understanding about dough viscoelastic properties.

It has been proved that KGM could improve the external and internal appearances of loaf. However, there are few studies about the influence of different enzymatic hydrolysis degrees of KGM on dough quality. The objective of this study was to evaluate the effect of different enzymatic hydrolysis degrees of KGM addition on the structural characteristic of gluten proteins and dough properties; the viscoelasticity of dough was detected by a microrheology analyzer and provides information about variation on the texture and tensile properties. The purpose is to focus on the interaction mechanisms between different enzymatic hydrolysis degrees with the gluten protein and dough quality. The consequence may stimulate the progress of KGM with different enzymatic hydrolysis degrees and lay the theoretical foundation for the application of KGM in flour products.

Results and Discussion

Effect of Degree of Konjac Glucomannan Enzymatic Hydrolysis on the Molecular Weight of Gluten Proteins

Molecular weight distribution of gluten proteins with different enzymatic hydrolysis degrees of KGM addition was measured by high-performance liquid chromatography (HPLC), and the corresponding chromatogram is shown in Figure S1. Protein fractions were divided into four fractions with elution time ranges of 5.15–8.46 min (peak 1), 8.67–15.91 min (peak 2), 15.77–18.23 min (peak 3), and 18.17–18.26 min (peak 4). Relative percentage and Mw values of individual fractions were calculated, and are shown in Table .

Table 1

Effect of Different Proportions of KGM Added on the Molecular Weight Changes of Gluten Proteina

		protein molecular weight
	proportion (%)	Mw (Da)	peak 1 area (%)	peak 2 area (%)	peak 3 area (%)	peak 4 area (%)
KGM	0	1.98 × 105	23.35 ± 0.27f	48.59 ± 0.37f	18.52 ± 0.11a	9.54 ± 0.03a
	0.5	2.10 × 105	25.23 ± 0.09e	49.56 ± 0.11e	18.12 ± 0.13b	7.09 ± 0.04b
	1.0	2.16 × 105	26.13 ± 0.11d	50.73 ± 0.12cd	17.62 ± 0.31c	5.52 ± 0.04c
	1.5	2.18 × 105	27.62 ± 0.08c	51.08 ± 0.45bc	17.22 ± 0.04d	4.08 ± 0.04d
	2.0	2.20 × 105	29.13 ± 0.16ab	51.20 ± 0.22b	17.03 ± 0.16de	2.64 ± 0.03e
	2.5	2.25 × 105	29.24 ± 0.14a	51.83 ± 0.31a	16.63 ± 0.03f	2.30 ± 0.06f
KGM I	0	1.98 × 105	23.35 ± 0.27f	48.59 ± 0.37f	18.52 ± 0.11a	9.54 ± 0.03a
	0.5	2.11 × 105	25.42 ± 0.15e	51.65 ± 0.21e	15.26 ± 0.12b	7.67 ± 0.02b
	1.0	2.17 × 105	26.85 ± 0.12d	52.32 ± 0.31d	14.11 ± 0.11c	6.72 ± 0.06c
	1.5	2.23 × 105	27.07 ± 0.11c	54.12 ± 0.20bc	12.74 ± 0.13d	6.07 ± 0.11d
	2.0	2.25 × 105	28.23 ± 0.05a	55.02 ± 0.11a	11.08 ± 0.15f	5.67 ± 0.05f
	2.5	2.29 × 105	27.48 ± 0.02b	54.36 ± 0.15b	12.30 ± 0.21e	5.86 ± 0.04e
KGM II	0	1.98 × 105	23.35 ± 0.27f	48.59 ± 0.37f	18.52 ± 0.11a	9.54 ± 0.03a
	0.5	2.15 × 105	25.80 ± 0.21d	57.83 ± 0.35e	12.36 ± 0.12b	5.11 ± 0.09b
	1.0	2.20 × 105	26.91 ± 0.22bc	58.16 ± 0.21cd	11.58 ± 0.09c	4.01 ± 0.11d
	1.5	2.24 × 105	27.17 ± 0.25ab	59.78 ± 0.26ab	11.01 ± 0.11e	3.35 ± 0.10e
	2.0	2.28 × 105	27.20 ± 0.12a	60.06 ± 0.39a	10.73 ± 0.06f	2.05 ± 0.08f
	2.5	2.35 × 105	25.72 ± 0.16e	58.23 ± 0.22c	11.56 ± 0.05cd	4.49 ± 0.11c
KGM III	0	1.98 × 105	23.35 ± 0.27f	48.59 ± 0.37f	18.52 ± 0.11a	9.54 ± 0.03a
	0.5	2.01 × 105	23.58 ± 0.15cde	48.83 ± 0.25e	18.35 ± 0.11b	9.24 ± 0.06bc
	1.0	2.06 × 105	23.60 ± 0.21cd	49.19 ± 0.23d	18.03 ± 0.12c	9.18 ± 0.05bcd
	1.5	2.10 × 105	23.96 ± 0.11c	49.41 ± 0.31bc	17.97 ± 0.14cd	8.66 ± 0.01e
	2.0	2.12 × 105	24.38 ± 0.06a	50.90 ± 0.22a	17.62 ± 0.21e	7.10 ± 0.12f
	2.5	2.15 × 105	24.16 ± 0.09b	49.43 ± 0.26b	17.15 ± 0.19ef	9.26 ± 0.15b

All experiments were in triplicate and data were expressed as mean ± standard deviation (n = 3); means in the same row of the same parameter with different letters indicate a significant difference (P < 0.05).

As shown in Table , with increased proportion of KGM addition with different enzymatic hydrolysis degrees, Mw of gluten proteins showed a continuous increase in each group. With increase in the additional proportion of KGM I, peak 1 and peak 2 areas increased and peak 3 and peak 4 areas decreased, resulting in the increment of Mw. This indicated that with the existence of KGM, gluten proteins with low molecular weight polymerized and formed into large polymers and shifted into peak 1 and peak 2. KGM has been reported to have high cohesiveness and it can interact with smaller subunits and bind to gluten proteins.[9] The samples with KGM addition also showed a continuous increase of Mw, which was in accordance with the result of elution time for different peaks (Figure S1) that peak 1 and peak 2 were left-shifted. Partly because KGM has better water absorption ability than gluten protein, the effect of protein structure is related to the development of aggregates with ample space in dough.

For group with KGM II addition, Mw increased significantly compared to other samples because KGM molecules without enzymatic hydrolysis would tend to self-aggregate or form larger aggregates with gluten proteins. This behavior resulted in the molecular chain entangling more easily and large glutenins not dissolving in the extract solution. In addition, some researchers have proved that glutenin as a kind of high-molecular-weight protein plays a decisive role during wheat application processing, leading to strong elongation resistance of gluten. An increasing number of experiments proved that high-molecular-weight subunit of wheat glutenin has a remarkable influence on gluten quality and the baking quality of dough. HPLC result agreed with the next result of free sulfhydryl content, and both can be explained similarly.

Effects of Degree of Konjac Glucomannan Enzymatic Hydrolysis on the Free Sulfhydryl Content of Gluten Proteins

Disulfide bonds and hydrogen-bond interaction are critical elements to decide the gluten characteristics and influence the dough properties. Glutenin polymers were formed by the interaction between high- and low-molecular-weight subunits through disulfide bonds in the gluten network structure.[26,27] Studies show that noncovalent hydrophobic and hydrogen-bond interactions promote gliadin cross-links with glutenin polymer. This particular three-dimensional network structure affords the dough with specific rheological properties.[28] Therefore, the effect of different enzymatic hydrolysis degrees of KGM addition in the exchange of free sulfhydryl was investigated in this study, as shown in Figure . The higher free sulfhydryl values (7.81 μmol/L) of control group than other groups shows that the gluten network of control group samples is highly unstable. While for KGM groups, as the KGM amount increased, the free sulfhydryl content markedly decreased, which suggested that addition of KGM could increase the stability of the gluten network.

Figure 1

Effect of enzymatic hydrolysis degree of konjac glucomannan added on free sulfhydryl content of gluten protein.

With different enzymatic hydrolysis degrees of KGM added, the sulfhydryl content decreased to a different extent for every group. Compared to samples without KGM addition, the sulfhydryl content decreased from 7.81 to 5.13 μmol/L with the proportion of 2.0% KGM I added and the sulfhydryl transformed into disulfide bonds, besides the same variation of sulfhydryl content occurred with KGM II and KGM III added. It is interesting that KGM II addition results in more significant changes of sulfhydryl content than KGM I and KGM III, i.e., decrease from 7.81 to 4.92 μmol/L. After KGM I, KGM II, and KGM III added up to 2.0%, the free sulfhydryl content dropped by 2.69, 2.89, and 2.85%, respectively. When the additional proportion reached 2.5%, the sulfhydryl content in increased in comparison to the KGM group, which demonstrated that KGM I, KGM II, and KGM III addition restricts the formation of disulfide bond. The result is possibly ascribed to the synergistic effects of KGM. This protective effect was relevant to the KGM substitution levels, and changes in the free sulfhydryl content showed that different enzymatic hydrolysis degrees of KGM had a better impact on preventing protein denaturation, especially KGM II addition.

Effects of Degree of Konjac Glucomannan Enzymatic Hydrolysis on the Secondary Structure of Gluten Proteins

Fourier transform infrared (FTIR) spectroscopy can quickly detect the secondary structure of protein. In general, amide I band (1600–1700 cm–1) was used to characterize the secondary structure of gluten proteins, and the characteristic absorption peaks of the amide I band are illustrated in Figure S2, which were classified into α-helix (1650–1660 cm–1), β-sheet (1610–1640 cm–1), β-turn (1660–1670 cm–1), and random coil (1640–1650 cm–1); the content of these four regions are shown in Figure .[26] The results illustrated that the secondary structure of gluten protein in all groups was mainly β-sheet and random coil accounts for a small fraction. As proportion of KGM increased to 2.0%, the β-sheet content increased from 39.12 to 40.45%, while with KGM I, KGM II, and KGM III added, β-sheets increased from 39.12 to 44.01, 45.99, and 40.02% respectively. Meanwhile, some reports have verified that β-sheet was the main structure of gluten proteins and the formation of β-sheet at the cost of β-turn.[29] So, β-turn decreased from 39.25 to 37.5, 34.98, 34.24, and 39.01%, respectively. The higher β-sheet content and lower β-turn manifested that gluten structure became more stable and elastic protein polymerization increased in each group. Interestingly, KGM II addition has significant influence on the secondary structure than KGM. This was because KGM would tend to self-aggregate or form larger aggregates with gluten proteins and attributed to the molecular chain entanglements more easily.[30] And then with enzymatic hydrolysis occurred, more hydrophilic groups appear and have excellent surface activity to bind with gluten protein;[31] thus, 2.0% addition was the acceptable proportion. Besides, the higher α-helix content corresponds to a more ordered structure and β-sheet is referred to as the most stable protein conformation.

Figure 2

Effect of enzymatic hydrolysis degree of konjac glucomannan added on the secondary structure changes of gluten protein. The secondary structure contents included α-helix content (A), β-sheet content (B), β-turn content (C), and random coil content (D).

Effects of Degree of Konjac Glucomannan Enzymatic Hydrolysis on the State of Water in Dough

Lower-field nuclear magnetic resonance (LF-NMR) was used to study water migration and state in dough. A typical T2 relaxation time distribution curve is shown in Figure S3, which shows three peaks of T21, T22, and T23, representing bound water, immobilized water, and free water, respectively.[5]Figure shows the proportion of every peak of T2 distribution with different enzymatic hydrolysis degrees of KGM addition.

Figure 3

Effect of enzymatic hydrolysis degree of konjac glucomannan added on the water fluidity of dough. The T2 relaxation time distribution includes T21 (A), T22 (B), and T23 (C).

Addition of KGM resulted in a decrease in freezable water. According to previous studies, a more tight structure of the KGM-gluten compound could be expected, which can bind more water tightly. After different enzymatic hydrolysis degrees of KGM addition, freezable water content of the KGM I, KGM II, and KGM III groups decreased by 4.65, 5.88, and 1.50%, respectively, with addition proportion of up to 2.0%. It is evident that free water in the KGM I and KGM II groups was lower than that in the control group, which indicates that addition of KGM I and KGM II tended to lower the content of freezable water. This demonstrated that KGM could inhibit freezable water flow. Besides, introduction of the hydrophilic groups of KGM increased with the occurrence of enzymatic hydrolysis and the substitution of oligo mannose groups is in conformity with both substituents degree and position, thereby making hydrogen bonds more complex.[31,32] Several hydrophilic groups exist in KGM molecule, which bind the water in a certain space and reduce the water mobility and the ability of water vapor to escape. The unit of water molecules with the polar and nonpolar amino acids of the gluten proteins was protected and the bound water content increased. Although high molecular weight makes KGM have good gelatinization and thickening properties, it affects the solubility of KGM in water and limits the application scope of KGM. Compared to KGM and different enzymatic hydrolysis degrees of KGM addition, the augment of T21 and T22 and the decrease of T23 in dough system indicated that the addition of KGM increases the ability of gluten networks to bind water. But T23 decreased with different enzymatic hydrolysis degrees of KGM addition proportion up to 2.0%, while increased for 2.5% KGM addition, which could be attributed to the high capacity of water holding, while with KGM added, T21 and T22 increased and T23 decreased with KGM addition proportion up to 2.5%. In conclusion, if the optimum addition proportion was different, then the water state is significantly different from each other. So, addition of KGM II exhibited better advantageous effects on inhibiting the formation of free water. A change of state of water is the basis of the transformation of gluten protein characteristics and dough properties.

Effects of Degree of Konjac Glucomannan Enzymatic Hydrolysis on the Elasticity of Dough

Dough with different enzymatic hydrolysis degrees of KGM addition affects the water state and disulfide bond changes, which could modify the rheological properties of dough. The dynamic rheology results are expressed as elasticity index (EI) and describe the elastic properties of dough samples. The parameter EI is proportional to storage modulus (G′) and describes the process of change of sample elasticity with time. The gradual reduction of platform area height corresponds to the reduction of the lattice and represents the further formation of a network structure. And reduction in lattice size corresponds to the increase of product elasticity, which means that the network structure is built more tightly.[33]Figure shows the elasticity index (EI) values for KGM I, KGM II, and KGM III groups affected by different enzymatic hydrolysis degrees of KGM addition with various proportions. Compared to other samples, dough with 2.0% KGM II addition has the best elasticity index. This phenomenon was due to the more even distribution of KGM II than KGM I and KGM III in dough network, and it will form a larger gel network after absorbing water. As a result, it works more effectively with gluten and reduces the probability of forming large aggregates. So it has less damage to the gluten network continuity and preserves the higher elasticity of dough. Dough with 2.0% KGM III addition exhibited lower elasticity than samples with 2.0% KGM II addition because the degree of enzymatic hydrolysis increases with extension of enzymatic hydrolysis time and exposure to more hydrophilic groups, which may form larger aggregates through hydration and destroy the continuity of gluten network structure. For KGM I, the molecular weight is relatively higher than for KGM II; thus, the capacity of water holding became lower, which causes the gluten protein cross-linking with a small amount, but is not stable. This result is in accordance with the analysis of water state in dough.

Figure 4

Effect of enzymatic hydrolysis degree of konjac glucomannan added on the elasticity index of dough. Dough with different proportions of KGM I (A), KGM II (B), and KGM III (C) added.

Effects of Degree of Konjac Glucomannan Enzymatic Hydrolysis on Microstructure of Dough

Scanning electron microscopy (SEM) was used to observe the dough microstructure. From Figure , we can see that dough microstructure has a continuous network and starch granules were inserted in the gluten network. Figure A shows that dough with 2.0% KGM present numerous aggregated starch particles implanted in continuous gluten network, indicating that KGM tended to gelatinize during dough production slightly. Micrographs of samples with proportions of KGM II at 2.0% (Figure C) were similar to the control group. In the complex system of gluten-KGM, KGM joint connection with gluten network and discrete starch granules could observe clearness. Figure C shows that starch granules are enclosed in a tight gluten network, demonstrating that a higher amount of KGM addition binding relationship between protein and KGM affects the textural and rheological properties of dough. Li et al.[34] applied polymer science technology to study gluten properties, indicating that the relationship between gluten and starch influences the behavior of dough system. With 2.0% KGM II filling into the gluten network (Figure C), a stronger stable connection between starch, protein, and KGM is formed. Gluten network was marginally influenced with KGM III substitution at 2.0%, which could see the exposure of starch granules to gluten, losing regular structure in other parts of Figure D. Besides, Figure B,C shows that the starch undergoes deformation, implying the competition between KGM and other components in dough seriously.

Figure 5

Scanning electron micrographs of dough with different enzymatic hydrolysis degrees of KGM added. Dough sample with 2.0% KGM (A), 2.0% KGM I (B), 2.0% KGM II (C), and 2.0% KGM III (D).

Effects of Degree of Konjac Glucomannan Enzymatic Hydrolysis on the Textural and Tensile Properties of Dough

The textural properties can effectively evaluate the dough quality that takes consumers’ feeling into consideration. Textural parameters, including hardness, springiness, cohesiveness, and chewiness, are shown in Figure . Dough made with KGM II obtained higher values than control group. The total trend is the continuous increase in hardness, springiness, cohesiveness, and chewiness as the proportion was up to 2.0%.

Figure 6

Effect of enzymatic hydrolysis degree of konjac glucomannan added on the textural properties of dough. Textural properties include hardness (A), springiness (B), cohesiveness (C), and chewiness (D).

However, the 2.5% dough sample with KGM addition has comparatively higher values of hardness, whereas the opposite effect of KGM was evident at 2.5% KGM on springiness. The lower acceptability of the samples with KGM I might be due to its unsatisfactory textural property to cater to Chinese consumers’ chewing preference. This finding might infer that KGM addition amount up to 2.5% for dough could effectively improve dough textural properties. The increased hardness of the dough samples may be explained by the relatively low water distribution inside the dough network. Considering the values of springiness and cohesiveness, a significant increase was found with KGM II added, supporting the extraordinary influence of the protein network formation. Chewiness also increase with KGM II addition, but not significantly. Therefore, KGM II addition was not affected significantly by the water competition due to the molecular weight compared to the other samples. Besides, dough treated with KGM III exhibited a weaker influence on dough textural properties than KGM I.

As shown in Figure , dough treated with a series of KGM (KGM, KGM I, KGM II, and KGM III) exhibited increased extensibility, extension area, and maximum resistance. The large extensibility of dough with KGM addition could form more developed protein network by the KGM and potential better water-holding capacity. However, the strength of the effect remarkably depends on the special KGM molecular weight.[35] In fact, addition of KGM II increased the parameters of extensibility, extension area, and maximum resistance. It also partially promotes a more stable linkage between proteins and the KGM II molecules. Moreover, addition of the KGM II in dough resulted in samples having reduced probability of the chain slide when a stress was applied and easy breaking apart owing to the enhanced chain–chain interactions between the KGM II and the gluten protein molecules. The same tendency is observed for the addition of KGM I and KGM III. In contrast, KGM I and KGM III influenced the parameters to a lesser extent, with an increase of extensibility and extension area, respectively, while KGM III addition up to 2.5% significantly decreased chewiness. Therefore, dough with KGM II addition exhibited the most acceptable tensile properties of dough.

Figure 7

Effect of enzymatic hydrolysis degree of konjac glucomannan added on the tensile properties of dough. Tensile properties included extensibility (Ek) (A) and extension area (Ak) (B), which were estimated by calculating Rkmax × Ek and maximum resistance (Rkmax) (C).

Effects of Degree of Konjac Glucomannan Enzymatic Hydrolysis on the Thermal Properties of Gluten Proteins

To investigate the effects of different enzymatic hydrolysis degrees of KGM addition to the gluten protein thermal properties, differential scanning calorimetry (DSC) was carried out. Tp and ΔH are the key parameters acquired from the DSC test that are useful to analyze the protein thermal properties, and are presented in Table . As vital parameter, Tp increased as the gluten protein molecules aggregation.[36] Dough with KGM II addition had significantly higher values of Tp and ΔH than other samples. Moreover, Tp became higher as the additional proportion increased. However, dough samples with KGM III addition displayed the lowest Tp and ΔH values than the others. These evident changes demonstrate a limitation of the network structure formation when KGM was added, due to the given enzymatic hydrolysis degree. The interaction between gluten protein and the KGM would be weakened due to the strong self-aggregation of the KGM molecules. Thus, KGM molecules may appear to self-aggregate to form larger aggregates with gluten proteins. This phenomenon has been attributed to the molecular chain entanglements more easily. In addition, the ΔH values required for melting the gluten depend on the extent of network structure aggregation and the internal available water.[37] Therefore, KGM II molecules could interact easily with the other components of the dough. Finally, addition of KGM II provided a more coherent microstructure of the dough or a better water distribution in dough network capable of undergoing thermal resistance. Then, these changes of Tp and ΔH values in different groups agreed with the results of T2 distribution, and bound water content was higher in dough with KGM II addition than in other groups, thus preventing water fluidity.

Table 2

Denaturation Peak Temperature (Tp) and Enthalpy of Denaturation (ΔH) of Gluten Protein with Different Proportions of KGM Addeda

		proportion (%)
	thermal parameter	0	0.5	1.0	1.5	2.0	2.5
KGM	Tp (°C)	54.74 ± 1.58e	62.70 ± 2.26d	67.30 ± 1.30bc	67.43 ± 2.13bc	69.54 ± 1.16b	72.75 ± 1.25a
	ΔH (J/g)	114.45 ± 3.56f	123.49 ± 3.87e	129.07 ± 1.33d	153.92 ± 4.21bc	156.22 ± 1.64b	160.32 ± 2.35a
KGM I	Tp (°C)	56.74 ± 1.38f	63.12 ± 2.75de	68.54 ± 1.25cd	70.12 ± 1.22bc	72.09 ± 2.11b	70.28 ± 1.25a
	ΔH (J/g)	116.89 ± 4.56ef	126.58 ± 6.10e	135.26 ± 4.87d	157.36 ± 3.56bc	162.34 ± 2.54ab	160.87 ± 6.32a
KGM II	Tp (°C)	58.26 ± 1.08	64.28 ± 1.24de	70.25 ± 1.11cd	72.14 ± 2.15bc	74.87 ± 2.06b	73.05 ± 2.22a
	ΔH (J/g)	120.37 ± 4.11ef	126.87 ± 6.57e	137.78 ± 5.04d	162.87 ± 6.53c	168.94 ± 4.44b	164.24 ± 5.26a
KGM III	Tp (°C)	53.01 ± 1.05e	55.24 ± 1.87cd	62.35 ± 4.10cd	66.13 ± 2.14bc	67.74 ± 1.98b	67.09 ± 2.58a
	ΔH (J/g)	116.52 ± 4.12f	124.59 ± 6.12de	131.74 ± 4.57cd	155.64 ± 4.82c	158.25 ± 4.44b	155.92 ± 5.89a

All experiments were in conducted triplicate and data are expressed as mean ± standard deviation (n = 3); means in the same row of the same parameter with different letters indicate a significant difference (P < 0.05).

Conclusions

The effects of different enzymatic hydrolysis degrees of KGM addition on the water distribution, elasticity, microstructure, and textural and tensile properties of dough were evaluated in the present study. Results revealed that different enzymatic hydrolysis degrees of KGM addition promoted gluten cross-linking through decreased free sulfhydryl and freezable water. KGM with 15 min enzymatic hydrolysis treatment showed a significant increase in bound water and decrease in free water. Besides, thermal tests demonstrated that the thermal stability of gluten protein improved with KGM addition and the microstructure became denser. In addition, dough possessed the most acceptable elasticity and tensile and texture properties with 2.0% KGM II addition. Therefore, these results will provide the theoretical basis for KGM to widen its application in the development of food industry.

Materials and Methods

Materials

Wheat flour with 14.38% protein, 72.19% starch, 0.45% ash, and 12.83% moisture (dry basis) was supplied by Wudeli Flour Group Co., Ltd. (Henan Province, China). The protein, starch, ash, and moisture content analyses of wheat flour were performed according to AOAC International.[38] Konjac glucomannan (KGM) with ≥98% purity was obtained from Bomei Biotechnology Co., Ltd. (Hefei Province, China), and β-mannanase was purchased from Solarbio Biotech Co., Ltd. (Beijing, China). The activity of the β-mannanase unit was 60 000 U/g. All other reagents and chemicals were of analytical purity, and deionized distilled water was used for all experiments.

Konjac Glucomannan Solution Preparation

Different enzymatic hydrolysis degrees of KGM were determined according to previous study.[31] KGM (1.00 g) was dissolved with 100 mL of citrate buffer solution (0.1 mol/L) and then added with 0.1 mg of β-mannanase to start the experiment. The solution pH of the reaction mixture was set as 4.8 for time ranges of 5 min (KGM I), 15 min (KGM II), and 30 min (KGM III), and the water bath temperature was kept steady at 40 °C. The reaction was stopped by boiling after the set time, and then the samples were centrifuged at 4000 rpm for 10 min and filtered through a 0.22 μm poly(vinylidene difluoride) (PVDF) membrane filter. High-performance liquid chromatography (HPLC) was performed with a differential refraction detector (Shimadzu RID-10A, Kyoto, Japan) according to the study by Yang et al.[39] with some modifications. A 20 μL solution of KGM with enzymatic hydrolysis treatment was injected into a gel chromatography column (OHpak SB-805 HQ, Kyoto, Japan), and ultrapure water was used as moving phase with a flow rate of 0.8 mL/min at 25 °C. The molecular weights (Mw) of KGM with different enzymatic hydrolysis treatment times are listed in Table .

Table 3

Molecular Weight (Mw) of KGM with Different Enzymatic Hydrolysis Time Treatment

name of KGM	enzymatic hydrolysis time (min)	molecular weight (Mw)
KGM	0	1.9306 × 106
KGM I	5	1.5974 × 106
KGM II	15	1.2769 × 106
KGM III	30	0.7912 × 106

Dough Sample Preparation

Wheat flour (50 g), KGM I, KGM II, KGM III (0.0, 0.5, 1.0, 1.5, 2.0, and 2.5% of wheat flour dry basis) solution, and deionized distilled water were mixed and knead for 5 min. The sample with KGM addition was set as the control group. The total amount of deionized distilled water added was 55% of wheat flour dry basis. Dough was kneaded for 10 min with all ingredients. Then, the dough was covered with food-grade plastic film, fermented for about 30 min, and then washed by 2.0% sodium chloride solution and lyophilized for further analyses.

Gluten Molecular Weight Analysis

Gluten molecular weight was detected by high-performance liquid chromatography (HPLC) on a Shimadzu LC-20AT system equipped with an RF-20A UV–vis detector (Shimadzu, Japan) according to the method of Ceresino.[40] Gluten protein was extracted from the wheat flour with 1 mL of acetic acid (500 mM) solution. Then, the suspension was centrifuged for 10 min at 5000 rpm and filtered through a 0.22 μm PVDF membrane filter. The filtrate (20 μL) was injected into the size exclusion column (BioSep-SEC-S4000, Phenomenex, 300 × 7.8 mm2, Torrance), and the samples were eluted with acetic acid solution (500 mM) at a flow rate of 0.8 mL/min and detected at 280 nm.

Free Sulfhydryl Content Analysis of Gluten Proteins

Free sulfhydryl content of gluten proteins was analyzed according to the method described by Zhou et al.[41] Freeze-dried gluten proteins (5 mg) were dissolved in 5 mL of urea solution (8 moL/L, 1.04% Tris, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.69% Gly, 1.5% sodium dodecyl sulfate (SDS), 8 M urea, pH 8.0) and centrifuged at 3000 rpm for 10 min. The supernate (1 mL) was then mixed with 2 mL of Tris–Gly solution (1.04% Tris, 1 mM EDTA, 0.69% Gly, 1.5% SDS) and 200 μL of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) reagent (4 mg/mL), and then the mixture was shaken for 30 min at 25 °C. Each supernatant was measured at 412 nm. For measurement of the total sulfhydryl equivalent groups (SHeq), 2 mL of Tris–Gly solution and 0.02 mL of β-mercaptoethanol were continuously added to 1 mL of supernatant after centrifugation and shaken for 1 h at 25 °C. Then, 10 mL of 12% trichloroacetic acid was added to precipitate gluten protein. The sediment was washed by 12% trichloroacetic acid and centrifuged at 3000 rpm for 10 min three times. The gluten proteins sediment was redissolved in 10 mL of Tris–Gly solution and 0.04 mL of DTNB reagent. The absorbance measurement was also recorded at 412 nm. The SHF and SHeq values were calculated by the following equationwhere 73.53 = 106/(1.36 × 104) and 1.36 × 104 are DTNB molar absorption coefficients, A412 is the detection absorbance of the sample at 412 nm, D is the dilution factor of the sample, and C is the sample concentration (mg/mL). In addition, the disulfide bonds (SS) content was calculated from SHF and SHeq. Besides, the disulfide bond (SS) content was calculated based on SHF and SHeq determination

Secondary Structure Analysis

The secondary structure of gluten protein was studied using Fourier transform infrared (FTIR) spectroscopy according to the method of Nawrocka et al.[42] FTIR spectra were recorded in the wavelength range of 4000–400 cm–1 using a Nicolet IS50 FTIR spectrometer (Thermo Nicolet Corp.). Gluten protein sample (1 mg) was mixed with 150 mg of KBr powder and compressed at a force of 5 kN for 30 s. FTIR spectra were recorded with 64 scans. The secondary structure contents of the samples were analyzed using OMNIC software package and Peakfit software (Origin, version 9.1).

Differential Scanning Calorimetry Analysis

The thermal changes of gluten protein were analyzed using a DSC system (DSC-60 Plus, Shimadzu, Co., Japan) according to Wang et al.[43] Gluten protein was freeze-dried and ground through 120-mesh sieve. Gluten samples (3.0–5.0 mg) were tableted into an aluminum pan (S201-52943, Shimadzu, Japan) and then heated to 250 °C with constant nitrogen purging at a constant rate of 5 °C/min. A sealed empty aluminum pan was used as a reference. The enthalpy change (ΔH) and peak temperature (Tp) of gluten protein with addition of different amounts of KGM were obtained using TA-60 Analysis software (version 2.21, Shimadzu Co., Japan).

Low-Frequency Nuclear Magnetic Resonance Analysis

Water fluidity of dough was detected by low-frequency nuclear magnetic resonance (LF-NMR) to analyze T2 according to the report of Ding et al.[44] NMR probes (10 mm diameter) filled with 2.0–3.0 g of dough samples were covered with a layer of plastic film. The T2 relaxation curves were obtained using a Carr–Purcell–Meiboom–Gill pulse sequence. Resonance frequency was set as 22 Hz; magnetic strength, 0.5 T; coil diameter, 60 mm; and magnetic temperature, 32 °C.

Microrheology

In each experiment, 2 g of dough was loaded into a 4 mL glass bottle seamless, and the sealed glass cell was loaded into the chamber (Rheolaser Lab, Formulaction, France) and maintained at 25 °C for 30 min prior to measurements. For this experiment, a coherent laser beam (650 nm) was applied to the sample. The mean-squared displacement (MSD) of the tracer particles was calculated using decorrelation functions. Three key parameters were obtained from the MSD curves.[24] Elasticity index (EI) is derived from the MSD curve of low decorrelation time using the following equationwhere MSD is the mean height value of the curve at low decorrelation times (<0.1 s) and EI is in direct proportion to storage modulus (G′). Besides, the slope of the MSD curve platform represents the ratio of the product’s solid and liquid properties; the smaller the slope, the slower the particle moves, which means the dough properties are closer to solid state.

Microstructure of Dough

The dough microstructure was obtained from scanning electron microscopy (SEM) based on the method of Li et al.[45] Freeze-dried dough samples were coated with gold particles using metal spraying. Pictures were taken by an SU-1510 scanning electron microscope (Hitachi Co., Ltd., Japan) with a 5 kV acceleration voltage with 2000× magnification.

Texture and Tensile Tests

The texture tests of dough were performed as previously described with minor modification.[45] The dough sample was prepared in the shape of 30 × 30 × 30 mm3, and texture analyses were determined using a texture analyzer equipped with a P/100 aluminum plate, using 1.0 mm/s testing speed, 40% pressure, 5.0 s testing time, and 5.0 nm distance. The hardness, springiness, cohesiveness, and chewiness were recorded.

The tensile test was performed on a texture analyzer equipped with a Kieffer extensibility rig. Strips of dough (⌀1 mm × 50 mm) were mounted on the tensile grips, using 5.0 mm/s pretest speed, 2.0 mm/s test speed, 5.0 mm/s post-test speed, 5.0 s test time, 0.05 N trigger force, and 75.0 mm distance. The Rmaxk (maximum resistance), Ek (extensibility), and Ak (extension area) were estimated by calculating Rmaxk × Ek.

Statistical Analysis

Statistical data analysis for three independent replicates was carried out and the data were expressed by mean ± standard deviation. Results were calculated and graphs were obtained using the Origin software (version 9.1), followed by the one-way analysis of variance at the significance level of 0.05. SPSS was used to determine differences among mean values at significance levels of 0.01 and 0.05 for the correlations between rheological, textural characteristic, and structure parameters.