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Effect of liquid fermentation on bread fortified with Lycium ruthenicum: A quality attribute and in vitro digestibility study
Food Chemistry 299 (2019) 125131
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Effect of liquid fermentation on bread fortified with Lycium ruthenicum: A quality attribute and in vitro digestibility study
T
Hua Wanga,b, Xiaoyan Xiaa, Hongzhu Yub, Xiaohong Zhaob, Xue Zhongb, Qian Lib, , ⁎ ⁎ Jintian Tangb, , Yuqing Zhaoa, ⁎
a b
School of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang 110016, China Key Laboratory of Particle & Radiation Imaging, Ministry of Education, Department of Engineering Physics, Tsinghua University, Beijing 100084, China
ARTICLE INFO
ABSTRACT
Keywords: Lycium ruthenicum Yeast Liquid fermentation Polyphenols Bread Quality attributes Digestibility
The aim of this study was to research the effect of yeast fermentation on the polyphenol content of Lycium ruthenicum (LR) to guide the production of bread with a lower digestibility. Liquid fermentation from 0 to 2 h significantly increased the phenolic, flavonoid and anthocyanin content of LR, while further liquid fermentation from 2 to 16 h had little additional effect. The addition of LR without prior liquid fermentation impaired the bread quality, while a prior liquid fermentation for at least 2 h improved the bread quality. The bread digestibility was decreased after adding LR, and it was further reduced with the extended liquid fermentation time. The results of this study indicate that adding LR with prior liquid fermentation to the dough during bread making could result in a higher quality and a lower digestibility.
1. Introduction Bread is a staple food that is popular throughout the world, particularly in western countries. However, bread is also a high-carbohydrate food with a large quantity of rapidly digestible starch and has a high glycaemic index (GI) (Shumoy, Van Bockstaele, Devecioglu, & Raes, 2018). A prolonged high-carbohydrate diet for long time may increase the risk of chronic diseases. It has been reported that the excessive consumption of bread, particularly white bread, could increase the risk of being overweight and obese, and of related diseases such as type 2 diabetes mellitus (T2DM) (Bautista-Castano & Serra-Majem, 2012; de la Fuente-Arrillaga et al., 2014; Loria-Kohen et al., 2012). Polyphenol-rich diets have been shown to help decrease the risk of T2DM (Xiao, Guo, Sun, & Zhao, 2017). Additionally, digestive enzyme inhibitors from polyphenol-rich plants are found to be promising approaches to help maintain a low GI diet, especially for starch-rich foods. Therefore, the use of polyphenol-rich additives in bread has become an increasingly popular way to produce bread with a low GI and is a potential way to prevent T2DM. Sui, Zhang, and Zhou (2016) found that the fortification of anthocyanin-rich black rice extract powder into bread could inhibit the activity of α-amylase and cause a lower digestion rate. Similarly, Goh et al. (2015) found that the addition of green tea extract could significantly reduce the glycaemic potential of bread via inhibition of α-amylase. Lycium ruthenicum (LR), which is a polyphenol-rich plant
⁎
mainly distributed in the Qinghai and Xinjiang Provinces in China, has the potential of inhibiting digestive enzymes. Hence, the addition of LR in bread may result in a lower digestion rate, thus creating a low GI food. While the polyphenol content in bread can paly an important role in improving bread digestibility, the presence of polyphenols can also impaired the quality of bread (Shumoy et al., 2018; Sui et al., 2016). In addition, a large amount of polyphenol-rich additives in bread will dramatically increase its cost. Therefore, development of a cost-effective method that can increase polyphenol content while maintaining the quality of bread is essential. One potential method would be linked to the fermentation process. Through a series of complex reactions, yeast fermentation makes the dough fluffy and elastic and endows the bread with a unique colour, aroma and taste. At the same time, the enzyme systems of yeast may lead to formation of numerous biocatalysts and biotransformations of polyphenol-rich additives during the process of fermentation, which may increase polyphenol content and influence the digestion rate. Bei, Chen, Liu, Zhang, and Wu (2018) found that microbial fermentation could improve phenolic compositions and the bioactivity of inhibiting α-amylase and α-glucosidases. Moreover, the mass reproduction of yeast during the fermentation and the production of metabolites may influence the quality of bread. Therefore, we hypothesized that the yeast fermentation process would have a positive effect on LR and result in the desired changes in the digestibility while maintaining the quality of bread.
Corresponding authors. E-mail addresses: [email protected] (Q. Li), [email protected] (J. Tang), [email protected] (Y. Zhao).
https://doi.org/10.1016/j.foodchem.2019.125131 Received 12 February 2019; Received in revised form 2 July 2019; Accepted 3 July 2019 Available online 04 July 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
Food Chemistry 299 (2019) 125131
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2. Materials and methods
spectrophotometer (Shimadzu, Jiangsu, China) at a wavelength of 760 nm. The total phenolic content was expressed as mg gallic acid equivalents per 1 g of dry weight (mg GAE/g DW) from a calibration curve.
2.1. Materials and reagents Freeze-dried Lycium ruthenicum was purchased from Qinghai Geermu Juxin Shuifeng Agricultural Development Co., Ltd., China. Bread flour containing 13.5% protein and 12.2% water (Golden Statue, Jiangsu, China), instant dry yeast (Saccharomyces cerevisiae, S.I. Lesaffre, China), eggs, fine salt, shortening, blended bread improver and sugar were purchased from a local supermarket. Pepsin (from porcine gastric mucosa, product number E022), α-amylase (from porcine pancreas, product number S31302-50ku), and α-glucosidases (from yeast, product number RM1035). Sodium carbonate (Na2CO3), sodium nitrite (NaNO2), aluminium nitrate (Al(NO)3), sodium hydroxide (NaOH), dibasic sodium phosphate (Na2HPO4), potassium chloride (KCl), hydrogen chloride (HCl) and sodium acetate trihydrate (CH3COONa) were purchased from Beijing Chemical Works (Beijing, China). Folin-Ciocalteu reagent was obtained from Sigma-Aldrich (St. Louis, MO, USA). Rutin and Gallic acid standards were provided by Macklin (Shanghai, China).
2.4. Total flavonoid content (TFC) determination The total flavonoid content of the extracts was determined using the method of Park et al. (2008) with minor modifications. Briefly, samples dissolved in 70% ethanol (5 mg/mL, 1 mL) were added into test tubes containing 0.4 mL of NaNO2 (5%, w/v). After 6 min, 0.4 mL of Al(NO)3 (10%, w/v) was added and gently mixed. After an additional 6 min, 4 mL of NaOH (4%, w/v) and 4.2 mL of water were added. Then, the absorbance was measured at 506 nm using a spectrophotometer. The total flavonoid content was calculated using a calibration curve and expressed as mg rutin equivalents per 1 g of dry weight (mg RE/g DW). 2.5. Total anthocyanin content (TAC) determination The total anthocyanin content of the extracts was measured using the spectrophotometric pH differential method (Chamorro-Cevallos, 2016) with minor modifications. Briefly, samples dissolved in 70% ethanol (1.25 mg/mL, 0.2 mL) were added into two types of test tubes containing either 3.8 mL of KCl solution (0.025 M, pH 1.0) or 3.8 mL of CH3COONa solution (0.4 M, pH 4.5), the pH being adjusted with HCl. The absorbance of both tubes was measured at 515 (maximum absorption wavelength) and 700 nm using a spectrophotometer. The results were calculated using Eqs. (1), (2) and (3) and were expressed as mg cyanidin-3-O-glucoside equivalents per 1 g of dry weight (mg CGE/ g DW).
2.2. Liquid fermentation and phytochemical extraction of Lycium ruthenicum Approximately 50 g of freeze-dried LR were crushed into powder using a herb pulveriser (Dingli, Zhejiang, China), and 5 g of freeze-dried LR powder was added to 10 groups respectively with a mark of 0, 1, 2, 3, 4, 5, 6, 7, 8, and 16 h. Subsequently, 1 g of instant dry yeast and 10 mL water (35 °C) were added to each portion. The dose of freezedried LR and instant dry yeast corresponded to the bread production. After the three parts were mixed well, the mixtures were placed in an incubator (Kewei, Beijing, China) at 28 °C. Then, 90 mL of ethanol (77.8%) was poured into every group to stop fermentation and prepare for extraction at times of 0, 1, 2, 3, 4, 5, 6, 7, 8, and 16 h. Although fermentation takes a long time to complete, we chose a shorter fermentation time for the consideration of actual production. Every group was extracted using an ultrasonic extraction apparatus (Shinva, Shandong, China) for 30 min at 50 °C with constant stirring. The crude extracts were then filtered through a Büchner funnel with two layers of filter paper. Then, a rotary evaporation apparatus (Shinva, Shandong, China) was used to remove the solvent under vacuum at 50 °C, and the residues were further lyophilized for investigation. A control group (marked C) was extracted under the same conditions without the addition of the instant dry yeast.
A= (A
515
A
Anthocyanin content
TAC
700)pH 4.5
(1)
mg A·MW·DF·103 = L ·L
(2)
700)pH 1.0
(A
515
A
mg CGE V·Anthocyanin content = 100g DM m
(3)
2.6. The preparation of bread As the dough was not suitable for a long fermentation time, we adopted a new kind of method called prior liquid fermentation to obtain a bread with a lower digestibility. Approximately 5 g of LR powder subjected to 0, 0.5, 1, 2 and 8 h of liquid fermentation (referring to the method described in Section 2.2) was added to 100 g of bread flour. The choice of the liquid fermentation time was based on the outcome of TPC, TFC and TAC and the actual production time. Then, bread flour with fermented LR was mixed with 4 g sugar, 3 g shortening, 10 g egg, 1.2 g salt, 0.5 g blended bread improver and 40 mL of water at a slow speed for 1 min followed by an intense mixing for 7 min in a mixer (Jinben, Guangzhou, China). After mixing, the dough was relaxed for 30 min at 28 °C and divided into 50 g each with a molder. Subsequently, the different dough pieces were proofed at 38 °C and 85% relative
2.3. Total phenolic content (TPC) determination The total phenolic content of the extracts was determined according to the previously reported Folin-Ciocalteu method (Mokrani & Madani, 2016) with minor modifications. Briefly, samples dissolved in 70% ethanol (0.5 mg/mL, 0.5 mL) were added into test tubes, and 2.5 mL of Folin-Ciocalteu reagent (10%) and 2 mL of Na2CO3 (7.5%, w/v) were subsequently added. The tubes were mixed well and placed in darkness at room temperature for 2 h. The absorbance was then detected by a Table 1 Fermentation time of different kinds of bread with and without LR. Sample
Liquid fermentation
Relaxing time (h)
Proofing time (h)
Fermentation time in dough (h)
Total fermentation time (h)
Control (0%)* 0 h (5%) 0.5 h (5%) 1 h (5%) 2 h (5%) 8 h (5%)
0 0 0.5 1 2 8
0.5 0.5 0.5 0.5 0.5 0.5
1 1 0.5 0.5 0.5 0.5
1.5 1.5 1 1 1 1
1.5 1.5 1.5 2 3 9
* Control contained no LR. Other samples contained 5% LR with liquid fermentation times as indicated. 2
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et al. (1997) with minor modifications. The samples (0.1 g) marked C, 0 h, 0.5 h, 1 h, 2 h and 8 h were placed in 3.8 mL of phosphate buffer containing 0.075 mM of CaCl2, which was previously adjusted to a pH of 1.5 using HCl (12 M). Subsequently, 0.8 mg of pepsin (10,000 units/ mg) was added to the mixture to start the hydrolysis. The mixture was incubated in a magnetic stirring thermostatic water bath at 37 °C for 1 h. Then, 1 mL of Na2HPO4 solution (0.4 M) was poured into the mixture to stop the enzyme reaction and achieve a pH of 6.8 for the following enzymatic reaction. The small intestine digestion phase was initiated by adding 0.1 mL of pancreatic α-amylase solution (30 units/ mL) and 0.1 mL of α-glucosidases solution (165 units/mL) in the mixture at 37 °C with constant stirring. Although the starch digestion of the small intestine phase recommended in the literature (Reshmi, Sudha, & Shashirekha, 2017) is normally evaluated using a combination of pancreatic α-amylase and fungal amyloglucosidase, the reaction conditions, the hydrolytic mechanism and influence in physiologic responses of fungal amyloglucosidase and small intestinal α-glucosidases are quite different according to a recently published review (Lin, Lee, & Chang, 2016). Therefore, a combination of α-amylase and α-glucosidases was used to more reasonably simulate the intestine phase. The dosage of α-glucosidases corresponded to the dosage of amyloglucosidase from a previous study (Reshmi et al., 2017). Aliquots of 0.1 mL were withdrawn at 30, 60, 90 and 120 min and placed into boiling water with constant shaking for 5 min to inactivate the enzyme reaction. After cooling to room temperature, the samples were kept in a refrigerator at 4 °C until the end of the incubation time. The released glucose was measured by a glucose oxidase–peroxidase (GODPOD) kit. The absorbance was detected by a spectrophotometer at a wavelength of 505 nm and the reagent blank was subtracted.
humidity for 0.5 h and 1 h, respectively (the fermentation time is shown in Table 1). Finally, the dough pieces were baked at 200 °C for 8 min in an oven (Sinmag, Guangzhou, China). 2.7. The evaluation of the quality of the breads The weight of the bread was determined, and the volume of the loaves was measured by the rapeseed displacement method (Sudha & Leelavathi, 2008). The measurement of the texture characteristics (hardness, cohesiveness, springiness and chewiness) of the bread crumb with and without LR were evaluated by a texture analyser (FTC, Virginia, USA). The force required to compress 50% of the bread crumb was recorded using an aluminium cylindrical probe with a 75 mm diameter at a speed of 0.5 mm/s. 2.8. Assessment of dough rheological properties assessed by Mixolab The effects of LR with or without liquid fermentation on the dough rheological properties were assessed by Mixolab (Chopin, Paris, France) using the Chopin+ protocol according to ICC No.173. Briefly, dough weight was kept constant at 75 g and the LR with or without liquid fermentation was also added into the Mixolab bowl as extra additive. The dough rheological properties were evaluated by recording the realtime torque variation of two kneading knives under the following gradient heating program: 0–8 min, 30 °C; 8–23 min, 30–90 °C; 23–30 min, 90 °C; 30–40 min, 90–50 °C; 40–45 min, 50 °C. The following Mixolab curve indicators represent different dough properties: C1 (Nm), initial maximum torque during mixing used for evaluating the water absorption (when reaching a torque of 1.10 ± 0.05 Nm); C2 (Nm), minimum torque during mixing and initial heating used for determining protein weakening; C3 (Nm), maximum value (peak) of torque during heating stage; C4 (Nm), stability of hot starch paste; C5 (Nm), the torque after dough coiling at 50 °C for 5 min used for determining the amount of retrogradation.
2.11. Statistical analysis All experiments were replicated in triplicate, and the results are presented as the mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) or t-test at p < 0.05 was performed to identify the significance using SPSS software version 17.0 (SPSS Inc., IL, USA).
2.9. Total starch (TS) The total starch was measured using the method of Goni, GarciaAlonso, and Saura-Calixto (1997) with some modifications. Briefly, samples (0.1 g) marked C, 0 h, 0.5 h, 1 h, 2 h and 8 h were dispersed in 4.88 mL of 0.4 M Sodium acetate buffer (pH 4.75) at 37 °C. After 15 min, 120 μL of amyloglucosidase solution (3300 units/mL) was added to this suspension and incubated for 45 min at 60 °C in a magnetic stirring thermostatic water bath (Hengyan, Zhengzhou, China). The starch was measured as glucose by a glucose oxidase–peroxidase (GODPOD) kit (Robio, Shanghai, China). The factor for conversion from glucose to starch was 0.9.
3. Results and discussion 3.1. Total phenolic, flavonoid and anthocyanin content The TPC, TFC and TAC with different fermentation times are shown in Fig. 1 (A), (B) and (C), respectively. The fermentation process increased TPC, TFC and TAC of LR, which was similar to results reported by Bei, Liu, Wang, Chen, and Wu (2017). The addition of yeast (0 h group) compared with the control group had no significant effect on TPC (from 49.89 to 48.23 mg GAE/g) and TAC (from 25.19 to 25.25 mg CGE/g) but had a significant effect on TFC (from 57.91 to 53.91 mg RE/ g). The increase of fermentation time from 0 to 2 h increased TPC from 48.23 to 95.55 mg GAE/g, TFC from 53.91 to 89.07 mg RE/g and TAC from 25.25 to 38.02 mg CGE/g, each of which were significant
2.10. In vitro digestibility study The in vitro digestibility study was based on the method of Goni
Fig. 1. The total phenolic (A), flavonoid (B) and anthocyanin (C) content of LR with different fermentation times. The different letters indicate significant differences (p < 0.05). 3
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(p < 0.05). However, a further increase of fermentation time from 2 to 8 h could not increase any of the contents significantly except TFC which increased by 5.88 mg RE/g (from 89.07 to 94.95 mg RE/g) from 2 to 3 h. Yeast contains vitamin B, minerals (selenium, chromium, zinc and copper), soluble protein and other components, which is the reason why the addition of yeast can slightly change the TPC and TAC of the extract. For the significant decrease of TFC, some insoluble ingredients may be produced via a chemical or physical reaction between flavonoids and yeast such as by flavonoid-binding protein. During the first 2 h of fermentation, the yeast multiplies rapidly utilizing the carbohydrate of LR. Simultaneously, the occurrence of enzymatic reactions significantly increases the content of phenolics, flavonoids and anthocyanins. Yeast is capable of an in situ catabolic conversion and affects the metabolism of some phenolic compounds (Adeboye, Bettiga, Aldaeus, Larsson, & Olsson, 2015). In the natural form, phenolic acid and flavonoids generally occur as conjugated (glycosides) and bound forms (Nardini, 2004). This type of glycosidic bond can be broken down by the yeast glycosidases, which may be one of the main reasons for the enhancement in the content (Rodríguez, Lopes, Valles, Giraudo, & Caballero, 2007). Additionally, the decomposition of plant cell walls by the enzymes produced by the yeast may liberate phenolic and flavonoid compounds (Cai et al., 2012). In addition, the synthesis of phenolic and flavonoid compounds by yeast may also occur through the polyketide pathway, which may also cause an increase of the components (Bond, Tang, & Li, 2016; Hertweck, 2009; Schumann & Hertweck, 2006). Overall, decomposition and synthesis reactions of enzymes commonly change the contents of total phenolics, flavonoids and anthocyanins. The fact that the extension of the duration of the fermentation process is not associated with further increases in TPC, TFC, and TAC is likely due to the inactivation of enzymatic reactions caused by unsuitable conditions or to the lack of remaining reactive substrate.
to 8 h slightly, though not significantly (p > 0.05), decreased the bread volume (to 4.87 cm3/g), which was probably due to a decline of yeast activity towards the end. In general, the addition of LR would decrease bread volume and thus impair the quality of bread, while the impaired quality of bread was improved with the increase of liquid fermentation time. 3.3. Texture analysis The various quality attributes of bread crumbs with different liquid fermentation times is presented in Table 2. The hardness of the bread crumbs was significantly (p < 0.05) related to the addition of LR. Previous reports indicated that the presence of polyphenols increases the hardness of bread in a dose-dependent manner (Sui et al., 2016; Wang, Zhou, & Isabelle, 2007). Similarly, the increase of the polyphenol content during the first hour of fermentation, as shown in Fig. 1, leads to an augmentation of the hardness from 13.95 to 15.29 N in spite of a lack of significant differences (p > 0.05). However, an opposite effect was found that further liquid fermentation from 1 to 2 h decreased the hardness of the bread crumb with increased polyphenol content. Moreover, a further decrease of hardness was found as the liquid fermentation time increased. A similar trend was also found for chewiness. These results indicate that liquid fermentation may affect the hardness and the chewiness of bread crumb due to the influence of yeast reproduction and its following metabolites. For springiness and cohesiveness, no significant difference (p > 0.05) was found between the bread crumb with and without LR except for the 8 h group and the C group. The trend of hardness and chewiness from 0 to 8 h shows that polyphenol content and liquid fermentation time commonly influenced quality attributes of the bread crumb. The addition of LR has a negative effect on the quality of bread which may be due to the polyphenols yielding less SS bonds and therefore weakening the bread matrix structure (Sui et al., 2016). However, the negative impact caused by the increase of polyphenol content on the quality of bread can be alleviated by the extension of the liquid fermentation time. A recently published study investigating the effects of yeast metabolites on bread dough fermentation showed that yeast metabolites played an important role on the gas holding capacity and dough fermentation performance (Rezaei et al., 2014). With increasing liquid fermentation time, the amount of yeast increased followed by an increase of metabolite production, such as organic acids, exopolysaccharides and enzymes. This might affect relaxing and proofing as well as baking and therefore change the quality of the bread crumb (Arendt, Ryan, & Dal Bello, 2007).
3.2. Specific volume analysis The specific volume of the bread shown in Table 2 is derived from dividing the bread volume (cm3) by its weight (g). A significant difference (p < 0.05) was found between the control bread (5.32 cm3/g) and 0 h bread (4.62 cm3/g), which indicates that the addition of 5 g dried LR per 100 g flour impaired the quality of bread. This result was in agreement with the results of Reshmi et al. (2017), Sui et al. (2016) who found that the addition of polyphenols reduced the specific bread volume and thus impaired the quality of bread. There was no significant difference (p > 0.05) between 0 h bread (4.62 cm3/g) and 0.5 h (4.24 cm3/g) bread despite the existence of the 0.5 h proofing time discrepancy. Remarkably, we found that the increase of the prior liquid fermentation time from 0.5 to 2 h led to an increase of the specific volume of the bread (from 4.24 cm3/g to 5.02 cm3/g), which has not been reported previously. The activity and amount of yeast play essential roles in bread volume (Gao, Tay, Koh, & Zhou, 2017). Hence, it is understandable that increasing the extent of prior liquid fermentation can enlarge bread volume as a result of the substantial reproduction of yeast. However, a further increase of the prior liquid fermentation time
3.4. The effects of LR and liquid fermentation on the water absorption and dough rheological properties The water absorption and dough rheological properties are closely related to the quality of bread. To further determine the reason why the addition of LR and liquid fermentation influenced the quality of bread, the water absorption and dough rheological properties were assessed by
Table 2 Specific volume and texture analysis of bread with LR and with different liquid fermentation times. Specific volume (cm3/g)
Sample *
Control (0%) 0 h (5%) 0.5 h (5%) 1 h (5%) 2 h (5%) 8 h (5%)
5.32 4.62 4.24 4.58 5.02 4.87
± ± ± ± ± ±
a
0.37 0.09b 0.38b 0.33b 0.03ab 0.10ab
Hardness (N)
Cohesiveness
b
8.97 ± 1.04 13.95 ± 1.53a 14.82 ± 0.51a 15.29 ± 0.73a 14.80 ± 0.78a 12.92 ± 1.78a
0.47 0.41 0.39 0.41 0.40 0.40
± ± ± ± ± ±
a
0.02 0.01a 0.04a 0.01a 0.01a 0.01a
Springiness 0.81 0.79 0.80 0.76 0.75 0.74
± ± ± ± ± ±
Chewiness (N) a
0.02 0.06ab 0.11ab 0.03ab 0.03ab 0.02b
99.30 ± 12.12b 123.28 ± 17.29ab 126.12 ± 12.91a 129.56 ± 3.36a 121.12 ± 2.36ab 104.17 ± 13.28b
* Control contained no LR. Other samples contained 5% LR with liquid fermentation times as indicated. The values are the means ± standard deviation (n = 3); values with different letters in the same column indicate significant statistical differences (p < 0.05). 4
Food Chemistry 299 (2019) 125131
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Fig. 3. Rate of starch hydrolysis of bread fortified with LR with and without liquid fermentation.
demonstrated a similar digestion profile in which the starch hydrolysis markedly increased in the first 30 min and then gradually increased from 30 to 90 min until it reached a plateau between 90 and 120 min. Nevertheless, a significant lower rate of starch hydrolysis was observed in the bread to which unfermented LR was added (0 h group) during the first 60 min especially during the first 30 min compared with wheat bread (C group), which indicated that the addition of LR reduced starch digestibility of bread. In addition, the rate of starch hydrolysis was found to further significantly decrease after the liquid fermentation from 0 to 2 h in a time-dependent manner. The extension of liquid fermentation time from 2 to 8 h did not noticeably influence the rate of starch hydrolysis any further. These results suggest that the digestibility of bread with LR added to it could be further effectively reduced using the method of liquid fermentation. The polyphenol content plays a crucial role in bread digestibility (Shumoy et al., 2018; Sui et al., 2016). On the one hand, the polyphenols may limit the extent and velocity of starch gelatinization and as a result reduce the susceptibility to enzymatic attack, owing to the interaction between phenolic and amylose via hydrogen bonding (Chai, Wang, & Zhang, 2013). On the other hand, the polyphenols may inhibit the activity of α-amylase and α-glucosidase, as has been reported previously (Bei et al., 2018). As shown in Fig. 4, the increased polyphenol content (the sum of TPC, TFC and TAC) via liquid fermentation dramatically reduced the bread digestibility in the first 60 min, particularly in the first 30 min. Brand-Miller et al. (Brand-Miller, Stockmann, Atkinson, Petocz, & Denyer, 2009), who assessed the in vivo glycaemic response of over one thousand foods, found that the GI had a weak correlation with the glucose concentration after 120 min of digestion and a strong correlation with the glucose concentration after 30 min and 60 min of digestion, as well as with the actual peak digestion. Consequently, liquid fermentation is an effective method to reduced bread digestibility by increasing polyphenol content from LR, which also makes it possible to obtain equal digestibility with a smaller amount of polyphenol-rich additives, thus increasing the cost-effectiveness.
Fig. 2. Mixolab curves of flour fortified with LR with and without liquid fermentation.
Mixolab. The flour required a water absorption of 65% to reach a torque of 1.10 ± 0.05 Nm. Addition of LR to the flour (5% w/w) was found increase the water absorption to 68%. The water absorption plays an essential role in specific volume and textural properties. Consequently, insufficient flour hydration was one of reasons that the quality of bread decreased after adding LR. However, there were no difference found on water absorption of flour added LR with and without fermentation, in spite of the existing difference on bread quality. As for dough rheological properties, the addition of LR was found to mainly enhance the protein weakening and the amount of retrogradation (Fig. 2) which would impair the quality of bread. On the one hand, the increased protein weakening might lead to a weakening of the bread matrix structure (Sui et al., 2016). On the other hand, the increase of retrogradation could speed up the aging velocity of bread and thus enhance the hardness and chewiness (Zhang, Li, Yang, Jin, & Xu, 2018). Meanwhile, it was found that the negative impact brought by LR was improved with the extension of liquid fermentation from 2 to 8 h. During the fermentation, mass reproduction of yeast and the production of metabolites as well as the changes of some compounds in LR may influence the protein weakening and retrogradation. In general, the quality of bread was reduced by the addition of LR due to the increase of water absorption, protein weakening and retrogradation. However, with the extension of liquid fermentation time from 2 to 8 h, the quality of bread was improved by obtaining dough rheological properties closer to that of flour without LR. 3.5. In vitro digestibility analysis As shown in Fig. 3, the starch hydrolysis rate was calculated as the percentage of total glucose released from bread compared to that released at 30, 60, 90 and 120 min. In general, all of the bread samples
Fig. 4. The relationship between polyphenol content and rate of starch hydrolysis at 30 (A), 60 (B), 90 (C) and 120 (D) min. 5
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4. Conclusion
Chamorro-Cevallos, G. (2016). Methods for Extraction, Isolation and Purification of Cphycocyanin: 50 years of research in review. International Journal of Food and Nutritional Science, 3(3), 1–10. de la Fuente-Arrillaga, C., Martinez-Gonzalez, M. A., Zazpe, I., Vazquez-Ruiz, Z., BenitoCorchon, S., & Bes-Rastrollo, M. (2014). Glycemic load, glycemic index, bread and incidence of overweight/obesity in a Mediterranean cohort: The SUN project. BMC Public Health, 14, 1091. Gao, J., Tay, S. L., Koh, A. H. S., & Zhou, W. (2017). Dough and bread made from highand low-protein flours by vacuum mixing: Part 2. Yeast activity, dough proofing and bread quality. Journal of Cereal Science, 77, 275–283. Goh, R., Gao, J., Ananingsih, V. K., Ranawana, V., Henry, C. J., & Zhou, W. (2015). Green tea catechins reduced the glycaemic potential of bread: An in vitro digestibility study. Food Chemistry, 180, 203–210. Goni, I., Garcia-Alonso, A., & Saura-Calixto, F. (1997). A starch hydrolysis procedure to estimate glycemic index. Nutrition Research, 17(3), 427–437. Hertweck, C. (2009). The biosynthetic logic of polyketide diversity. Angewandte Chemie (International ed. in English), 48(26), 4688–4716. Lin, A. H.-M., Lee, B.-H., & Chang, W.-J. (2016). Small intestine mucosal α-glucosidase: A missing feature of in vitro starch digestibility. Food Hydrocolloids, 53, 163–171. Loria-Kohen, V., Gomez-Candela, C., Fernandez-Fernandez, C., Perez-Torres, A., GarciaPuig, J., & Bermejo, L. M. (2012). Evaluation of the usefulness of a low-calorie diet with or without bread in the treatment of overweight/obesity. Clinical Nutrition, 31(4), 455–461. Mokrani, A., & Madani, K. (2016). Effect of solvent, time and temperature on the extraction of phenolic compounds and antioxidant capacity of peach (Prunus persica L.) fruit. Separation and Purification Technology, 162, 68–76. Nardini, M. (2004). Determination of free and bound phenolic acids in beer. Food Chemistry, 84(1), 137–143. Park, Y.-S., Jung, S.-T., Kang, S.-G., Heo, B. G., Arancibia-Avila, P., Toledo, F., ... Gorinstein, S. (2008). Antioxidants and proteins in ethylene-treated kiwifruits. Food Chemistry, 107(2), 640–648. Reshmi, S. K., Sudha, M. L., & Shashirekha, M. N. (2017). Starch digestibility and predicted glycemic index in the bread fortified with pomelo (Citrus maxima) fruit segments. Food Chemistry, 237, 957–965. Rezaei, M. N., Dornez, E., Jacobs, P., Parsi, A., Verstrepen, K. J., & Courtin, C. M. (2014). Harvesting yeast (Saccharomyces cerevisiae) at different physiological phases significantly affects its functionality in bread dough fermentation. Food Microbiology, 39, 108–115. Rodríguez, M. E., Lopes, C., Valles, S., Giraudo, M. R., & Caballero, A. (2007). Selection and preliminary characterization of β-glycosidases producer Patagonian wild yeasts. Enyzme and Microbial Technology, 41(6–7), 812–820. Schumann, J., & Hertweck, C. (2006). Advances in cloning, functional analysis and heterologous expression of fungal polyketide synthase genes. Journal of Biotechnology, 124(4), 690–703. Shumoy, H., Van Bockstaele, F., Devecioglu, D., & Raes, K. (2018). Effect of sourdough addition and storage time on in vitro starch digestibility and estimated glycemic index of tef bread. Food Chemistry, 264, 34–40. Sudha, M. L., & Leelavathi, K. (2008). Influence of micronutrients on rheological characteristics and bread-making quality of flour. International Journal of Food Science and Nutrition, 59(2), 105–115. Sui, X., Zhang, Y., & Zhou, W. (2016). Bread fortified with anthocyanin-rich extract from black rice as nutraceutical sources: Its quality attributes and in vitro digestibility. Food Chemistry, 196, 910–916. Wang, R., Zhou, W., & Isabelle, M. (2007). Comparison study of the effect of green tea extract (GTE) on the quality of bread by instrumental analysis and sensory evaluation. Food Research International, 40(4), 470–479. Xiao, T., Guo, Z., Sun, B., & Zhao, Y. (2017). Identification of anthocyanins from four kinds of berries and their inhibition activity to alpha-glycosidase and protein tyrosine phosphatase 1B by HPLC-FT-ICR MS/MS. Journal of Agriculture and Food Chemistry, 65(30), 6211–6221. Zhang, Y., Li, D., Yang, N., Jin, Z., & Xu, X. (2018). Comparison of dextran molecular weight on wheat bread quality and their performance in dough rheology and starch retrogradation. LWT, 98, 39–45.
In conclusion, the addition of LR significantly decreases the digestibility of bread, but at the same time causes a decline in the quality of bread. However, using the method of prior liquid fermentation counteracts the negative impact brought about by the addition of LR by decreasing the protein weakening and retrogradation of dough added LR. Additionally, the liquid fermentation process further effectively reduces the digestibility of bread fortified with LR by increasing polyphenol content. These results indicate that a suitable liquid fermentation of polyphenol-rich additives for bread making might help to attain a lower digestibility of bread, while maintaining its quality. This makes it possible to use a smaller amount of polyphenol-rich additives to produce bread with greater function and quality, which would greatly reduce its cost. Declaration of Competing Interest None. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125131. References Adeboye, P. T., Bettiga, M., Aldaeus, F., Larsson, P. T., & Olsson, L. (2015). Catabolism of coniferyl aldehyde, ferulic acid and p-coumaric acid by Saccharomyces cerevisiae yields less toxic products. Microbial Cell Factories, 14, 149. Arendt, E. K., Ryan, L. A., & Dal Bello, F. (2007). Impact of sourdough on the texture of bread. Food Microbiology, 24(2), 165–174. Bautista-Castano, I., & Serra-Majem, L. (2012). Relationship between bread consumption, body weight, and abdominal fat distribution: Evidence from epidemiological studies. Nutrition Reviews, 70(4), 218–233. Bei, Q., Chen, G., Liu, Y., Zhang, Y., & Wu, Z. (2018). Improving phenolic compositions and bioactivity of oats by enzymatic hydrolysis and microbial fermentation. Journal of Functional Foods, 47, 512–520. Bei, Q., Liu, Y., Wang, L., Chen, G., & Wu, Z. (2017). Improving free, conjugated, and bound phenolic fractions in fermented oats (Avena sativa L.) with Monascus anka and their antioxidant activity. Journal of Functional Foods, 32, 185–194. Bond, C., Tang, Y., & Li, L. (2016). Saccharomyces cerevisiae as a tool for mining, studying and engineering fungal polyketide synthases. Fungal Genetics and Biology, 89, 52–61. Brand-Miller, J. C., Stockmann, K., Atkinson, F., Petocz, P., & Denyer, G. (2009). Glycemic index, postprandial glycemia, and the shape of the curve in healthy subjects: Analysis of a database of more than 1,000 foods. American Journal of Clinical Nutrition, 89(1), 97–105. Cai, S., Wang, O., Wu, W., Zhu, S., Zhou, F., Ji, B., ... Cheng, Q. (2012). Comparative study of the effects of solid-state fermentation with three filamentous fungi on the total phenolics content (TPC), flavonoids, and antioxidant activities of subfractions from oats (Avena sativa L.). Journal of Agriculture and Food Chemistry, 60(1), 507–513. Chai, Y., Wang, M., & Zhang, G. (2013). Interaction between amylose and tea polyphenols modulates the postprandial glycemic response to high-amylose maize starch. Journal of Agriculture and Food Chemistry, 61(36), 8608–8615.
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Effect of liquid fermentation on bread fortified with Lycium ruthenicum: A quality attribute and in vitro digestibility study
T
Hua Wanga,b, Xiaoyan Xiaa, Hongzhu Yub, Xiaohong Zhaob, Xue Zhongb, Qian Lib, , ⁎ ⁎ Jintian Tangb, , Yuqing Zhaoa, ⁎
a b
School of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang 110016, China Key Laboratory of Particle & Radiation Imaging, Ministry of Education, Department of Engineering Physics, Tsinghua University, Beijing 100084, China
ARTICLE INFO
ABSTRACT
Keywords: Lycium ruthenicum Yeast Liquid fermentation Polyphenols Bread Quality attributes Digestibility
The aim of this study was to research the effect of yeast fermentation on the polyphenol content of Lycium ruthenicum (LR) to guide the production of bread with a lower digestibility. Liquid fermentation from 0 to 2 h significantly increased the phenolic, flavonoid and anthocyanin content of LR, while further liquid fermentation from 2 to 16 h had little additional effect. The addition of LR without prior liquid fermentation impaired the bread quality, while a prior liquid fermentation for at least 2 h improved the bread quality. The bread digestibility was decreased after adding LR, and it was further reduced with the extended liquid fermentation time. The results of this study indicate that adding LR with prior liquid fermentation to the dough during bread making could result in a higher quality and a lower digestibility.
1. Introduction Bread is a staple food that is popular throughout the world, particularly in western countries. However, bread is also a high-carbohydrate food with a large quantity of rapidly digestible starch and has a high glycaemic index (GI) (Shumoy, Van Bockstaele, Devecioglu, & Raes, 2018). A prolonged high-carbohydrate diet for long time may increase the risk of chronic diseases. It has been reported that the excessive consumption of bread, particularly white bread, could increase the risk of being overweight and obese, and of related diseases such as type 2 diabetes mellitus (T2DM) (Bautista-Castano & Serra-Majem, 2012; de la Fuente-Arrillaga et al., 2014; Loria-Kohen et al., 2012). Polyphenol-rich diets have been shown to help decrease the risk of T2DM (Xiao, Guo, Sun, & Zhao, 2017). Additionally, digestive enzyme inhibitors from polyphenol-rich plants are found to be promising approaches to help maintain a low GI diet, especially for starch-rich foods. Therefore, the use of polyphenol-rich additives in bread has become an increasingly popular way to produce bread with a low GI and is a potential way to prevent T2DM. Sui, Zhang, and Zhou (2016) found that the fortification of anthocyanin-rich black rice extract powder into bread could inhibit the activity of α-amylase and cause a lower digestion rate. Similarly, Goh et al. (2015) found that the addition of green tea extract could significantly reduce the glycaemic potential of bread via inhibition of α-amylase. Lycium ruthenicum (LR), which is a polyphenol-rich plant
⁎
mainly distributed in the Qinghai and Xinjiang Provinces in China, has the potential of inhibiting digestive enzymes. Hence, the addition of LR in bread may result in a lower digestion rate, thus creating a low GI food. While the polyphenol content in bread can paly an important role in improving bread digestibility, the presence of polyphenols can also impaired the quality of bread (Shumoy et al., 2018; Sui et al., 2016). In addition, a large amount of polyphenol-rich additives in bread will dramatically increase its cost. Therefore, development of a cost-effective method that can increase polyphenol content while maintaining the quality of bread is essential. One potential method would be linked to the fermentation process. Through a series of complex reactions, yeast fermentation makes the dough fluffy and elastic and endows the bread with a unique colour, aroma and taste. At the same time, the enzyme systems of yeast may lead to formation of numerous biocatalysts and biotransformations of polyphenol-rich additives during the process of fermentation, which may increase polyphenol content and influence the digestion rate. Bei, Chen, Liu, Zhang, and Wu (2018) found that microbial fermentation could improve phenolic compositions and the bioactivity of inhibiting α-amylase and α-glucosidases. Moreover, the mass reproduction of yeast during the fermentation and the production of metabolites may influence the quality of bread. Therefore, we hypothesized that the yeast fermentation process would have a positive effect on LR and result in the desired changes in the digestibility while maintaining the quality of bread.
Corresponding authors. E-mail addresses: [email protected] (Q. Li), [email protected] (J. Tang), [email protected] (Y. Zhao).
https://doi.org/10.1016/j.foodchem.2019.125131 Received 12 February 2019; Received in revised form 2 July 2019; Accepted 3 July 2019 Available online 04 July 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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2. Materials and methods
spectrophotometer (Shimadzu, Jiangsu, China) at a wavelength of 760 nm. The total phenolic content was expressed as mg gallic acid equivalents per 1 g of dry weight (mg GAE/g DW) from a calibration curve.
2.1. Materials and reagents Freeze-dried Lycium ruthenicum was purchased from Qinghai Geermu Juxin Shuifeng Agricultural Development Co., Ltd., China. Bread flour containing 13.5% protein and 12.2% water (Golden Statue, Jiangsu, China), instant dry yeast (Saccharomyces cerevisiae, S.I. Lesaffre, China), eggs, fine salt, shortening, blended bread improver and sugar were purchased from a local supermarket. Pepsin (from porcine gastric mucosa, product number E022), α-amylase (from porcine pancreas, product number S31302-50ku), and α-glucosidases (from yeast, product number RM1035). Sodium carbonate (Na2CO3), sodium nitrite (NaNO2), aluminium nitrate (Al(NO)3), sodium hydroxide (NaOH), dibasic sodium phosphate (Na2HPO4), potassium chloride (KCl), hydrogen chloride (HCl) and sodium acetate trihydrate (CH3COONa) were purchased from Beijing Chemical Works (Beijing, China). Folin-Ciocalteu reagent was obtained from Sigma-Aldrich (St. Louis, MO, USA). Rutin and Gallic acid standards were provided by Macklin (Shanghai, China).
2.4. Total flavonoid content (TFC) determination The total flavonoid content of the extracts was determined using the method of Park et al. (2008) with minor modifications. Briefly, samples dissolved in 70% ethanol (5 mg/mL, 1 mL) were added into test tubes containing 0.4 mL of NaNO2 (5%, w/v). After 6 min, 0.4 mL of Al(NO)3 (10%, w/v) was added and gently mixed. After an additional 6 min, 4 mL of NaOH (4%, w/v) and 4.2 mL of water were added. Then, the absorbance was measured at 506 nm using a spectrophotometer. The total flavonoid content was calculated using a calibration curve and expressed as mg rutin equivalents per 1 g of dry weight (mg RE/g DW). 2.5. Total anthocyanin content (TAC) determination The total anthocyanin content of the extracts was measured using the spectrophotometric pH differential method (Chamorro-Cevallos, 2016) with minor modifications. Briefly, samples dissolved in 70% ethanol (1.25 mg/mL, 0.2 mL) were added into two types of test tubes containing either 3.8 mL of KCl solution (0.025 M, pH 1.0) or 3.8 mL of CH3COONa solution (0.4 M, pH 4.5), the pH being adjusted with HCl. The absorbance of both tubes was measured at 515 (maximum absorption wavelength) and 700 nm using a spectrophotometer. The results were calculated using Eqs. (1), (2) and (3) and were expressed as mg cyanidin-3-O-glucoside equivalents per 1 g of dry weight (mg CGE/ g DW).
2.2. Liquid fermentation and phytochemical extraction of Lycium ruthenicum Approximately 50 g of freeze-dried LR were crushed into powder using a herb pulveriser (Dingli, Zhejiang, China), and 5 g of freeze-dried LR powder was added to 10 groups respectively with a mark of 0, 1, 2, 3, 4, 5, 6, 7, 8, and 16 h. Subsequently, 1 g of instant dry yeast and 10 mL water (35 °C) were added to each portion. The dose of freezedried LR and instant dry yeast corresponded to the bread production. After the three parts were mixed well, the mixtures were placed in an incubator (Kewei, Beijing, China) at 28 °C. Then, 90 mL of ethanol (77.8%) was poured into every group to stop fermentation and prepare for extraction at times of 0, 1, 2, 3, 4, 5, 6, 7, 8, and 16 h. Although fermentation takes a long time to complete, we chose a shorter fermentation time for the consideration of actual production. Every group was extracted using an ultrasonic extraction apparatus (Shinva, Shandong, China) for 30 min at 50 °C with constant stirring. The crude extracts were then filtered through a Büchner funnel with two layers of filter paper. Then, a rotary evaporation apparatus (Shinva, Shandong, China) was used to remove the solvent under vacuum at 50 °C, and the residues were further lyophilized for investigation. A control group (marked C) was extracted under the same conditions without the addition of the instant dry yeast.
A= (A
515
A
Anthocyanin content
TAC
700)pH 4.5
(1)
mg A·MW·DF·103 = L ·L
(2)
700)pH 1.0
(A
515
A
mg CGE V·Anthocyanin content = 100g DM m
(3)
2.6. The preparation of bread As the dough was not suitable for a long fermentation time, we adopted a new kind of method called prior liquid fermentation to obtain a bread with a lower digestibility. Approximately 5 g of LR powder subjected to 0, 0.5, 1, 2 and 8 h of liquid fermentation (referring to the method described in Section 2.2) was added to 100 g of bread flour. The choice of the liquid fermentation time was based on the outcome of TPC, TFC and TAC and the actual production time. Then, bread flour with fermented LR was mixed with 4 g sugar, 3 g shortening, 10 g egg, 1.2 g salt, 0.5 g blended bread improver and 40 mL of water at a slow speed for 1 min followed by an intense mixing for 7 min in a mixer (Jinben, Guangzhou, China). After mixing, the dough was relaxed for 30 min at 28 °C and divided into 50 g each with a molder. Subsequently, the different dough pieces were proofed at 38 °C and 85% relative
2.3. Total phenolic content (TPC) determination The total phenolic content of the extracts was determined according to the previously reported Folin-Ciocalteu method (Mokrani & Madani, 2016) with minor modifications. Briefly, samples dissolved in 70% ethanol (0.5 mg/mL, 0.5 mL) were added into test tubes, and 2.5 mL of Folin-Ciocalteu reagent (10%) and 2 mL of Na2CO3 (7.5%, w/v) were subsequently added. The tubes were mixed well and placed in darkness at room temperature for 2 h. The absorbance was then detected by a Table 1 Fermentation time of different kinds of bread with and without LR. Sample
Liquid fermentation
Relaxing time (h)
Proofing time (h)
Fermentation time in dough (h)
Total fermentation time (h)
Control (0%)* 0 h (5%) 0.5 h (5%) 1 h (5%) 2 h (5%) 8 h (5%)
0 0 0.5 1 2 8
0.5 0.5 0.5 0.5 0.5 0.5
1 1 0.5 0.5 0.5 0.5
1.5 1.5 1 1 1 1
1.5 1.5 1.5 2 3 9
* Control contained no LR. Other samples contained 5% LR with liquid fermentation times as indicated. 2
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et al. (1997) with minor modifications. The samples (0.1 g) marked C, 0 h, 0.5 h, 1 h, 2 h and 8 h were placed in 3.8 mL of phosphate buffer containing 0.075 mM of CaCl2, which was previously adjusted to a pH of 1.5 using HCl (12 M). Subsequently, 0.8 mg of pepsin (10,000 units/ mg) was added to the mixture to start the hydrolysis. The mixture was incubated in a magnetic stirring thermostatic water bath at 37 °C for 1 h. Then, 1 mL of Na2HPO4 solution (0.4 M) was poured into the mixture to stop the enzyme reaction and achieve a pH of 6.8 for the following enzymatic reaction. The small intestine digestion phase was initiated by adding 0.1 mL of pancreatic α-amylase solution (30 units/ mL) and 0.1 mL of α-glucosidases solution (165 units/mL) in the mixture at 37 °C with constant stirring. Although the starch digestion of the small intestine phase recommended in the literature (Reshmi, Sudha, & Shashirekha, 2017) is normally evaluated using a combination of pancreatic α-amylase and fungal amyloglucosidase, the reaction conditions, the hydrolytic mechanism and influence in physiologic responses of fungal amyloglucosidase and small intestinal α-glucosidases are quite different according to a recently published review (Lin, Lee, & Chang, 2016). Therefore, a combination of α-amylase and α-glucosidases was used to more reasonably simulate the intestine phase. The dosage of α-glucosidases corresponded to the dosage of amyloglucosidase from a previous study (Reshmi et al., 2017). Aliquots of 0.1 mL were withdrawn at 30, 60, 90 and 120 min and placed into boiling water with constant shaking for 5 min to inactivate the enzyme reaction. After cooling to room temperature, the samples were kept in a refrigerator at 4 °C until the end of the incubation time. The released glucose was measured by a glucose oxidase–peroxidase (GODPOD) kit. The absorbance was detected by a spectrophotometer at a wavelength of 505 nm and the reagent blank was subtracted.
humidity for 0.5 h and 1 h, respectively (the fermentation time is shown in Table 1). Finally, the dough pieces were baked at 200 °C for 8 min in an oven (Sinmag, Guangzhou, China). 2.7. The evaluation of the quality of the breads The weight of the bread was determined, and the volume of the loaves was measured by the rapeseed displacement method (Sudha & Leelavathi, 2008). The measurement of the texture characteristics (hardness, cohesiveness, springiness and chewiness) of the bread crumb with and without LR were evaluated by a texture analyser (FTC, Virginia, USA). The force required to compress 50% of the bread crumb was recorded using an aluminium cylindrical probe with a 75 mm diameter at a speed of 0.5 mm/s. 2.8. Assessment of dough rheological properties assessed by Mixolab The effects of LR with or without liquid fermentation on the dough rheological properties were assessed by Mixolab (Chopin, Paris, France) using the Chopin+ protocol according to ICC No.173. Briefly, dough weight was kept constant at 75 g and the LR with or without liquid fermentation was also added into the Mixolab bowl as extra additive. The dough rheological properties were evaluated by recording the realtime torque variation of two kneading knives under the following gradient heating program: 0–8 min, 30 °C; 8–23 min, 30–90 °C; 23–30 min, 90 °C; 30–40 min, 90–50 °C; 40–45 min, 50 °C. The following Mixolab curve indicators represent different dough properties: C1 (Nm), initial maximum torque during mixing used for evaluating the water absorption (when reaching a torque of 1.10 ± 0.05 Nm); C2 (Nm), minimum torque during mixing and initial heating used for determining protein weakening; C3 (Nm), maximum value (peak) of torque during heating stage; C4 (Nm), stability of hot starch paste; C5 (Nm), the torque after dough coiling at 50 °C for 5 min used for determining the amount of retrogradation.
2.11. Statistical analysis All experiments were replicated in triplicate, and the results are presented as the mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) or t-test at p < 0.05 was performed to identify the significance using SPSS software version 17.0 (SPSS Inc., IL, USA).
2.9. Total starch (TS) The total starch was measured using the method of Goni, GarciaAlonso, and Saura-Calixto (1997) with some modifications. Briefly, samples (0.1 g) marked C, 0 h, 0.5 h, 1 h, 2 h and 8 h were dispersed in 4.88 mL of 0.4 M Sodium acetate buffer (pH 4.75) at 37 °C. After 15 min, 120 μL of amyloglucosidase solution (3300 units/mL) was added to this suspension and incubated for 45 min at 60 °C in a magnetic stirring thermostatic water bath (Hengyan, Zhengzhou, China). The starch was measured as glucose by a glucose oxidase–peroxidase (GODPOD) kit (Robio, Shanghai, China). The factor for conversion from glucose to starch was 0.9.
3. Results and discussion 3.1. Total phenolic, flavonoid and anthocyanin content The TPC, TFC and TAC with different fermentation times are shown in Fig. 1 (A), (B) and (C), respectively. The fermentation process increased TPC, TFC and TAC of LR, which was similar to results reported by Bei, Liu, Wang, Chen, and Wu (2017). The addition of yeast (0 h group) compared with the control group had no significant effect on TPC (from 49.89 to 48.23 mg GAE/g) and TAC (from 25.19 to 25.25 mg CGE/g) but had a significant effect on TFC (from 57.91 to 53.91 mg RE/ g). The increase of fermentation time from 0 to 2 h increased TPC from 48.23 to 95.55 mg GAE/g, TFC from 53.91 to 89.07 mg RE/g and TAC from 25.25 to 38.02 mg CGE/g, each of which were significant
2.10. In vitro digestibility study The in vitro digestibility study was based on the method of Goni
Fig. 1. The total phenolic (A), flavonoid (B) and anthocyanin (C) content of LR with different fermentation times. The different letters indicate significant differences (p < 0.05). 3
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(p < 0.05). However, a further increase of fermentation time from 2 to 8 h could not increase any of the contents significantly except TFC which increased by 5.88 mg RE/g (from 89.07 to 94.95 mg RE/g) from 2 to 3 h. Yeast contains vitamin B, minerals (selenium, chromium, zinc and copper), soluble protein and other components, which is the reason why the addition of yeast can slightly change the TPC and TAC of the extract. For the significant decrease of TFC, some insoluble ingredients may be produced via a chemical or physical reaction between flavonoids and yeast such as by flavonoid-binding protein. During the first 2 h of fermentation, the yeast multiplies rapidly utilizing the carbohydrate of LR. Simultaneously, the occurrence of enzymatic reactions significantly increases the content of phenolics, flavonoids and anthocyanins. Yeast is capable of an in situ catabolic conversion and affects the metabolism of some phenolic compounds (Adeboye, Bettiga, Aldaeus, Larsson, & Olsson, 2015). In the natural form, phenolic acid and flavonoids generally occur as conjugated (glycosides) and bound forms (Nardini, 2004). This type of glycosidic bond can be broken down by the yeast glycosidases, which may be one of the main reasons for the enhancement in the content (Rodríguez, Lopes, Valles, Giraudo, & Caballero, 2007). Additionally, the decomposition of plant cell walls by the enzymes produced by the yeast may liberate phenolic and flavonoid compounds (Cai et al., 2012). In addition, the synthesis of phenolic and flavonoid compounds by yeast may also occur through the polyketide pathway, which may also cause an increase of the components (Bond, Tang, & Li, 2016; Hertweck, 2009; Schumann & Hertweck, 2006). Overall, decomposition and synthesis reactions of enzymes commonly change the contents of total phenolics, flavonoids and anthocyanins. The fact that the extension of the duration of the fermentation process is not associated with further increases in TPC, TFC, and TAC is likely due to the inactivation of enzymatic reactions caused by unsuitable conditions or to the lack of remaining reactive substrate.
to 8 h slightly, though not significantly (p > 0.05), decreased the bread volume (to 4.87 cm3/g), which was probably due to a decline of yeast activity towards the end. In general, the addition of LR would decrease bread volume and thus impair the quality of bread, while the impaired quality of bread was improved with the increase of liquid fermentation time. 3.3. Texture analysis The various quality attributes of bread crumbs with different liquid fermentation times is presented in Table 2. The hardness of the bread crumbs was significantly (p < 0.05) related to the addition of LR. Previous reports indicated that the presence of polyphenols increases the hardness of bread in a dose-dependent manner (Sui et al., 2016; Wang, Zhou, & Isabelle, 2007). Similarly, the increase of the polyphenol content during the first hour of fermentation, as shown in Fig. 1, leads to an augmentation of the hardness from 13.95 to 15.29 N in spite of a lack of significant differences (p > 0.05). However, an opposite effect was found that further liquid fermentation from 1 to 2 h decreased the hardness of the bread crumb with increased polyphenol content. Moreover, a further decrease of hardness was found as the liquid fermentation time increased. A similar trend was also found for chewiness. These results indicate that liquid fermentation may affect the hardness and the chewiness of bread crumb due to the influence of yeast reproduction and its following metabolites. For springiness and cohesiveness, no significant difference (p > 0.05) was found between the bread crumb with and without LR except for the 8 h group and the C group. The trend of hardness and chewiness from 0 to 8 h shows that polyphenol content and liquid fermentation time commonly influenced quality attributes of the bread crumb. The addition of LR has a negative effect on the quality of bread which may be due to the polyphenols yielding less SS bonds and therefore weakening the bread matrix structure (Sui et al., 2016). However, the negative impact caused by the increase of polyphenol content on the quality of bread can be alleviated by the extension of the liquid fermentation time. A recently published study investigating the effects of yeast metabolites on bread dough fermentation showed that yeast metabolites played an important role on the gas holding capacity and dough fermentation performance (Rezaei et al., 2014). With increasing liquid fermentation time, the amount of yeast increased followed by an increase of metabolite production, such as organic acids, exopolysaccharides and enzymes. This might affect relaxing and proofing as well as baking and therefore change the quality of the bread crumb (Arendt, Ryan, & Dal Bello, 2007).
3.2. Specific volume analysis The specific volume of the bread shown in Table 2 is derived from dividing the bread volume (cm3) by its weight (g). A significant difference (p < 0.05) was found between the control bread (5.32 cm3/g) and 0 h bread (4.62 cm3/g), which indicates that the addition of 5 g dried LR per 100 g flour impaired the quality of bread. This result was in agreement with the results of Reshmi et al. (2017), Sui et al. (2016) who found that the addition of polyphenols reduced the specific bread volume and thus impaired the quality of bread. There was no significant difference (p > 0.05) between 0 h bread (4.62 cm3/g) and 0.5 h (4.24 cm3/g) bread despite the existence of the 0.5 h proofing time discrepancy. Remarkably, we found that the increase of the prior liquid fermentation time from 0.5 to 2 h led to an increase of the specific volume of the bread (from 4.24 cm3/g to 5.02 cm3/g), which has not been reported previously. The activity and amount of yeast play essential roles in bread volume (Gao, Tay, Koh, & Zhou, 2017). Hence, it is understandable that increasing the extent of prior liquid fermentation can enlarge bread volume as a result of the substantial reproduction of yeast. However, a further increase of the prior liquid fermentation time
3.4. The effects of LR and liquid fermentation on the water absorption and dough rheological properties The water absorption and dough rheological properties are closely related to the quality of bread. To further determine the reason why the addition of LR and liquid fermentation influenced the quality of bread, the water absorption and dough rheological properties were assessed by
Table 2 Specific volume and texture analysis of bread with LR and with different liquid fermentation times. Specific volume (cm3/g)
Sample *
Control (0%) 0 h (5%) 0.5 h (5%) 1 h (5%) 2 h (5%) 8 h (5%)
5.32 4.62 4.24 4.58 5.02 4.87
± ± ± ± ± ±
a
0.37 0.09b 0.38b 0.33b 0.03ab 0.10ab
Hardness (N)
Cohesiveness
b
8.97 ± 1.04 13.95 ± 1.53a 14.82 ± 0.51a 15.29 ± 0.73a 14.80 ± 0.78a 12.92 ± 1.78a
0.47 0.41 0.39 0.41 0.40 0.40
± ± ± ± ± ±
a
0.02 0.01a 0.04a 0.01a 0.01a 0.01a
Springiness 0.81 0.79 0.80 0.76 0.75 0.74
± ± ± ± ± ±
Chewiness (N) a
0.02 0.06ab 0.11ab 0.03ab 0.03ab 0.02b
99.30 ± 12.12b 123.28 ± 17.29ab 126.12 ± 12.91a 129.56 ± 3.36a 121.12 ± 2.36ab 104.17 ± 13.28b
* Control contained no LR. Other samples contained 5% LR with liquid fermentation times as indicated. The values are the means ± standard deviation (n = 3); values with different letters in the same column indicate significant statistical differences (p < 0.05). 4
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Fig. 3. Rate of starch hydrolysis of bread fortified with LR with and without liquid fermentation.
demonstrated a similar digestion profile in which the starch hydrolysis markedly increased in the first 30 min and then gradually increased from 30 to 90 min until it reached a plateau between 90 and 120 min. Nevertheless, a significant lower rate of starch hydrolysis was observed in the bread to which unfermented LR was added (0 h group) during the first 60 min especially during the first 30 min compared with wheat bread (C group), which indicated that the addition of LR reduced starch digestibility of bread. In addition, the rate of starch hydrolysis was found to further significantly decrease after the liquid fermentation from 0 to 2 h in a time-dependent manner. The extension of liquid fermentation time from 2 to 8 h did not noticeably influence the rate of starch hydrolysis any further. These results suggest that the digestibility of bread with LR added to it could be further effectively reduced using the method of liquid fermentation. The polyphenol content plays a crucial role in bread digestibility (Shumoy et al., 2018; Sui et al., 2016). On the one hand, the polyphenols may limit the extent and velocity of starch gelatinization and as a result reduce the susceptibility to enzymatic attack, owing to the interaction between phenolic and amylose via hydrogen bonding (Chai, Wang, & Zhang, 2013). On the other hand, the polyphenols may inhibit the activity of α-amylase and α-glucosidase, as has been reported previously (Bei et al., 2018). As shown in Fig. 4, the increased polyphenol content (the sum of TPC, TFC and TAC) via liquid fermentation dramatically reduced the bread digestibility in the first 60 min, particularly in the first 30 min. Brand-Miller et al. (Brand-Miller, Stockmann, Atkinson, Petocz, & Denyer, 2009), who assessed the in vivo glycaemic response of over one thousand foods, found that the GI had a weak correlation with the glucose concentration after 120 min of digestion and a strong correlation with the glucose concentration after 30 min and 60 min of digestion, as well as with the actual peak digestion. Consequently, liquid fermentation is an effective method to reduced bread digestibility by increasing polyphenol content from LR, which also makes it possible to obtain equal digestibility with a smaller amount of polyphenol-rich additives, thus increasing the cost-effectiveness.
Fig. 2. Mixolab curves of flour fortified with LR with and without liquid fermentation.
Mixolab. The flour required a water absorption of 65% to reach a torque of 1.10 ± 0.05 Nm. Addition of LR to the flour (5% w/w) was found increase the water absorption to 68%. The water absorption plays an essential role in specific volume and textural properties. Consequently, insufficient flour hydration was one of reasons that the quality of bread decreased after adding LR. However, there were no difference found on water absorption of flour added LR with and without fermentation, in spite of the existing difference on bread quality. As for dough rheological properties, the addition of LR was found to mainly enhance the protein weakening and the amount of retrogradation (Fig. 2) which would impair the quality of bread. On the one hand, the increased protein weakening might lead to a weakening of the bread matrix structure (Sui et al., 2016). On the other hand, the increase of retrogradation could speed up the aging velocity of bread and thus enhance the hardness and chewiness (Zhang, Li, Yang, Jin, & Xu, 2018). Meanwhile, it was found that the negative impact brought by LR was improved with the extension of liquid fermentation from 2 to 8 h. During the fermentation, mass reproduction of yeast and the production of metabolites as well as the changes of some compounds in LR may influence the protein weakening and retrogradation. In general, the quality of bread was reduced by the addition of LR due to the increase of water absorption, protein weakening and retrogradation. However, with the extension of liquid fermentation time from 2 to 8 h, the quality of bread was improved by obtaining dough rheological properties closer to that of flour without LR. 3.5. In vitro digestibility analysis As shown in Fig. 3, the starch hydrolysis rate was calculated as the percentage of total glucose released from bread compared to that released at 30, 60, 90 and 120 min. In general, all of the bread samples
Fig. 4. The relationship between polyphenol content and rate of starch hydrolysis at 30 (A), 60 (B), 90 (C) and 120 (D) min. 5
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4. Conclusion
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In conclusion, the addition of LR significantly decreases the digestibility of bread, but at the same time causes a decline in the quality of bread. However, using the method of prior liquid fermentation counteracts the negative impact brought about by the addition of LR by decreasing the protein weakening and retrogradation of dough added LR. Additionally, the liquid fermentation process further effectively reduces the digestibility of bread fortified with LR by increasing polyphenol content. These results indicate that a suitable liquid fermentation of polyphenol-rich additives for bread making might help to attain a lower digestibility of bread, while maintaining its quality. This makes it possible to use a smaller amount of polyphenol-rich additives to produce bread with greater function and quality, which would greatly reduce its cost. Declaration of Competing Interest None. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125131. References Adeboye, P. T., Bettiga, M., Aldaeus, F., Larsson, P. T., & Olsson, L. (2015). Catabolism of coniferyl aldehyde, ferulic acid and p-coumaric acid by Saccharomyces cerevisiae yields less toxic products. Microbial Cell Factories, 14, 149. Arendt, E. K., Ryan, L. A., & Dal Bello, F. (2007). Impact of sourdough on the texture of bread. Food Microbiology, 24(2), 165–174. Bautista-Castano, I., & Serra-Majem, L. (2012). Relationship between bread consumption, body weight, and abdominal fat distribution: Evidence from epidemiological studies. Nutrition Reviews, 70(4), 218–233. Bei, Q., Chen, G., Liu, Y., Zhang, Y., & Wu, Z. (2018). Improving phenolic compositions and bioactivity of oats by enzymatic hydrolysis and microbial fermentation. Journal of Functional Foods, 47, 512–520. Bei, Q., Liu, Y., Wang, L., Chen, G., & Wu, Z. (2017). Improving free, conjugated, and bound phenolic fractions in fermented oats (Avena sativa L.) with Monascus anka and their antioxidant activity. Journal of Functional Foods, 32, 185–194. Bond, C., Tang, Y., & Li, L. (2016). Saccharomyces cerevisiae as a tool for mining, studying and engineering fungal polyketide synthases. Fungal Genetics and Biology, 89, 52–61. Brand-Miller, J. C., Stockmann, K., Atkinson, F., Petocz, P., & Denyer, G. (2009). Glycemic index, postprandial glycemia, and the shape of the curve in healthy subjects: Analysis of a database of more than 1,000 foods. American Journal of Clinical Nutrition, 89(1), 97–105. Cai, S., Wang, O., Wu, W., Zhu, S., Zhou, F., Ji, B., ... Cheng, Q. (2012). Comparative study of the effects of solid-state fermentation with three filamentous fungi on the total phenolics content (TPC), flavonoids, and antioxidant activities of subfractions from oats (Avena sativa L.). Journal of Agriculture and Food Chemistry, 60(1), 507–513. Chai, Y., Wang, M., & Zhang, G. (2013). Interaction between amylose and tea polyphenols modulates the postprandial glycemic response to high-amylose maize starch. Journal of Agriculture and Food Chemistry, 61(36), 8608–8615.
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