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The impact of the soluble protein fraction and kernel hardness on wheat flour starch digestibility

Wheat is the staple crop for 35% of the world’s population, providing a major source of calories for much of the world’s population. Starch is the main source of energy in wheat flour, but the digestibility of wheat starch varies greatly between different flours and wheat products. This has relevance from a health perspective because wheat starch products that are rapidly digested and elicit large post-prandial glucose peaks are associated with a host of cardiac and metabolic disorders. In this study, we investigate the impact of protein on starch digestion in three commercially sourced flours with different grain hardness. Grain hardness impacted on flour particle size, but not significantly on starch digestion. A soluble extract of wheat proteins was found to dramatically reduce starch digestion, even following gastric proteolysis. Proteomic analysis revealed that this soluble extract was enriched in proteinaceous α-amylase inhibitors which were partially degraded during gastric proteolysis. Therefore, we conclude that the soluble proteins of wheat flour have a significant contribution towards retarding starch digestion, even following gastric digestion.


Introduction
Starch is one of the most abundant sources of polysaccharides widely distributed in seeds, roots and tubers of plants, which supports about half of the carbohydrate caloric intake for much of the population of the planet (Jobling, 2004;Whistler & Daniel, 2000). Amongst starchy staple crops, one of the most widely consumed is bread wheat (Triticum aestivum), and understanding the factors which impact wheat starch digestion has the potential to yield nutritional benefit (Shewry, Hazard, Lovegrove, & Uauy, 2020). Starchy foods vary greatly in their digestibility in the gastrointestinal tract, and starchy foods which are rapidly digested can result in large peaks in post-prandial blood glucose concentration (Brouns et al., 2008). Regular consumption of foods which produce large excursions in post-prandial glycaemia, including wheat based products such as white bread, are has been associated with health problems such as insulin resistance, hyperinsulinemia, obesity, cardiovascular diseases and type 2 Diabetes (Borneo & León, 2012;Lanzerstorfer et al., 2018). Thus, reducing the digestion rate of starch in wheat based foods is a potential route to reduce post-prandial glycaemic responses (Brouns et al., 2008;Eelderink, Schepers, Preston, Vonk, Oudhuis, & Priebe, 2012).
Foods made from bread wheat are generally made from the milled endosperm of the wheat grain (although whole wheat and other products may include other parts of the grain). The resulting flour is mainly starch (80-85%), but also contains other components which can impact on the digestibility of the starch, including protein, which comprises 10-15% of bread wheat flour. The main proteins in bread wheat are the gluten proteins, glutenin and gliadin, which are storage proteins that comprise ~80% of the protein in bread flour (Juhász, Békés, & Wrigley, 2015). These proteins form the largely insoluble gluten network which give the typical properties of many wheat based foods such as bread. Bhattarai, Dhital, and Gidley (2016)  However, the relative contribution of these different protein families to starch digestion in wheat based foods is not fully understood. A further challenge is that to impact significantly on starch digestion in the small intestine, wheat proteins must escape proteolysis in the stomach. L. Li, Fan, and Zhao (2022) investigated proteinaceous α -AIs extracted from different plants by analysing their inhibition activities following treatment with different temperature, pH and digestive proteinases.
Inhibition activities of the extracts were decreased under certain conditions, especially after treating with pepsin. Therefore, the effectiveness of different protein components to reduce starch digestion will be further impacted by gastric digestion.
In this study we aimed to elucidate the impact of protein fractions within wheat flour on the digestibility of starch. Using commercially available flours with different grain hardness and protein contents we determined the impact of gastric protein digestibility on starch digestibility using an in vitro model system. Proteomic analysis was used to identify the soluble proteins present in the digestion mixture before and after gastric digestion. We demonstrate that, while grain hardness and insoluble proteins had a relatively minor impact on starch digestibility, the soluble albumin fraction containing high levels of proteinaceous α -AI had a significant impact on starch digestibility, even following extensive gastric digestion. The role of wheat proteinaceous α -AI should be considered a significant factor impacting wheat starch digestibility in future studies.

Starch extraction from wheat flours
Pre-milled wheat flours were suspended in water at a ratio of 1 to 12, then the pH of slurry was adjusted to 8.5 with 0.1 M NaOH. The slurry was mixed for 5 minutes and then centrifuged at 3000 rpm for 10 minutes. The viscous top layer was removed, and the rest of the precipitate was re-suspended into deionised water. After mixing, the resuspended slurry was centrifuged again to remove top layers. The washing process was repeated two more times, then all the supernatant was removed. The resulting white precipitate was then suspended in deionised water and sequentially filtered through a 300 μ m sieve and a 53 μ m sieve. Starch extracted from wheat flours was collected after air drying.

Starch content analysis for wheat flours
Total starch contents (TTS) of each of the wheat flours was analysed using the total starch assay kit (K-TSTA

Digestion of control wheat starch with addition of wheat flour extracts
Each of the wheat flours was weighted based on 100 mg starch and transferred in to 15 ml conical tubes followed by the addition of 1 ml of deionised water and 2 ml of 0.05 M HCL. The tubes were mixed and then incubated in a 37 o C water bath for 30 minutes. After incubation, 2 ml of 0.05 M NaOH was added into each tube for neutralising pH. PBS (5 ml) was then added to establish 10 ml of solution. The tubes were vortexed for 10 seconds, then centrifuged at 3000 rpm for 10 minutes. The supernatant (9 ml) from each tube was then respectively transferred new tubes containing pre-weighed 100 mg control wheat starch and 1 ml of PBS (the control group was prepared by adding 1 ml of water, 2 ml of 0.05 M HCL, 2 ml of 0.05 M NaOH and 5 ml of PBS into 100 mg control wheat starch). The continue steps for starch digestion with α -amylase were the same as method described in the section 2.4.1. Analysed samples were all prepared in duplicates.

Analysis of reducing sugar released from starch by using PAHBAH method
Reducing sugar in the digesta samples collected in 2.

Analysis of relative crystallinity for all wheat flours by using X-ray diffraction (XRD)
All three different wheat flours were analysed with the Rigaku Smartlab SE X-ray diffraction system (Cu X-ray tube, Rigaku, Tokyo, Japan) operating at 40 kV -50

Scattering (SLS)
Each wheat flour was suspended into deionised water to establish a 10% (w/v) slurry and their particle size distribution was measured with a Beckman Coulter LS 13-320 (Beckman Coulter, Brea, California, US). The pre-set model "Wheat flour in water" was applied for analysis. Each sample was examined in triplicate.

Characterisation of proteinaceous
α -amylase inhibitors using Mass

Spectrometry and Proteomics
Each wheat flour was weighed into a 15 mL conical tube on a 100 mg starch basis.
Samples were then treated with pepsin by adding 1 ml deionised water and 2 ml pepsin solution (described on section 2.4). A pepsin free control group was also prepared as section 2.4 described. After incubation, the pH of the solution was neutralised by adding 2 ml of 0.05 M NaOH. Pepsin treated samples and controls were centrifuged at 3000 rpm for 10 minutes and then 4 ml supernatant was collected as flour extract for proteomic analysis.
Proteins were precipitated from the flour extract with acetone (Nickerson & Doucette, 2020 were set as variable modifications and carbamido-methylation (CAM) of cysteine as fixed modification. The Mascot search results were imported into Scaffold 4.11.0.
Statistical analysis was carried out by using Scaffold 5.0.1. Statistical significance of differences between the control group and gastric group for each wheat flour were assessed by Fisher's Exact Test (no specific correction method applied) with the significant level of P-value < 0.05.

Inclusion of a gastric digestion step increase the digestibility of starch in wheat flour
Starch digestion was carried out for 3 different commercial wheat flours and wheat starch. There were two groups analysed, the gastric group which was treated with 5 mg/ml pepsin solution to mimic the gastric phase, and the control group which was not treated with pepsin. As Figure 1 shows, all 3 types of wheat flours treated with pepsin (Green bars) had significantly higher digestion rates of starch comparing with control group (Blue bars). There was 30.4%, 37.9% and 28.1% starch digested respectively for the BKG, Titan and HG with a gastric digestion stage, but only 8.0%, 10.9% and 5.9% of starch (respectively) were digested for the control group of each flour. Wheat starch showed overall higher digestibility than any of the flour samples, with 44.0% starch digestion for the control and 43.5% for sample with gastric digestion sample. These findings are in agreement with the study of Zou et al. (2015) using durum wheat, who reported increases in starch digestibility for pasta and semolina with pepsin treatment prior to hydrolysis by α -amylase. Pepsin is known as a non-specific proteinase capable of hydrolysing a broad range proteins (Nelson, Lehninger, & Cox, 2008). The differences in digestibility of starch with and without gastric digestion was not observed for the purified wheat starch. Therefore, the results implied that the digestibility of starch in wheat flours was reduced due to the presence of protein. to different digestibility of starch, relative crystallinity, particle size distribution and thermal behaviour was examined for all 3 different wheat flours.

Analysis of particle size distribution, gelatinisation of starch and relative crystallinity for wheat flours
Static light scattering (SLS) was used to analyse particle size distributions for each of the wheat flours. Particle distribution and Mean and Median values are presented in Figure 2. BKG and Titan showed minor differences in size distribution. starch digestion for raw durum wheat flours milled into different particle size and found that starch digested slower in raw durum wheat flours with larger particle sizes.
However, the differences in particle size observed in this study are smaller than in the referenced literature, so may not contribute a significant impact. Wheat flours were analysed using XRD to investigate the level of starch crystallinity.
As presented in The thermal behaviour and gelatinisation of the different wheat flours were investigated using DSC. The gelatinisation enthalpy and temperature is indicative of the relative amount and stability of the crystallites present in the starch (Warren, Gidley, & Flanagan, 2016). The results shown in Table 1 illustrate that the onset and peak temperatures and enthalpy of starch gelatinisation did not differ significantly between the wheat flours, except the gelatinisation enthalpy for BKG which was slightly lower (4.43 J/g) than other samples (4.49 J/g for Tian and 4.93 J/g for HG).
Taken together, the results of the XRD and thermal analysis indicate that there were only minor differences in starch crystallinity between the different flours.

Aqueous wheat flours extracts are capable of reducing the digestion of purified wheat starch
To further investigate differences in starch digestion between the wheat flours, native starch was extracted from each flour and its digestibility was evaluated. As   As shown in Figure 4, there were significant reductions in the abundances of all of these amylase inhibitors as a result of gastric proteolysis for all three of the wheat flours tested. Therefore, this data indicates that the reduction in abundance of proteinaceous α -AI inhibitors may explain larger differences observed between wheat flour digestion with and without a gastric phase, and as indicated in Figure 1, the incomplete proteolysis of these inhibitors (which were still identified by proteomics following the gastric phase) may impact wheat flour digestion and explain differences in digestibility between wheat flours from different sources with different protein contents.
In addition, there were also significant reductions in some glutenin and gliadin proteins. Wheat gluten had been extensively studied and indicated as not just a physical barrier to digestive enzymes but an effective inhibitor that binds to However, in this aqueous extract, the glutenin and gliadin proteins were found at a much lower abundance than the α -AI's, so may be playing a lesser role.

Conclusion
In this study, the role of wheat proteins and grain hardness in controlling starch digestibility was explored. Using wheat flours with different hardness and protein content, we demonstrated that wheat flour was significantly more slowly digested than purified starch, and that this effect was significantly reduced by gastric digestion and proteolysis. While grain hardness impacted on flour particle size, it did not have a large effect on starch digestibility. We then identified that an aqueous extract of wheat   a  p  i  d  a  n  d  q  u  a  n  t  i  t  a  t  i  v  e  p  r  o  t  e  i  n  p  r  e  c  i  p  i  t  a  t  i  o  n  f  o  r  p  r  o  t  e  o  m  e  a  n  a  l  y  s  i  s  b  y  m  a  s  s  s  p  e  c  t  r  o  m  e  t  r  y  .   J  o  u  r  n  a  l  o  f  p  r  o  t  e  o  m  e  r  e  s  e  a  r  c  h  ,  1  9   (  5  )  ,  2  0  3  5  -2  0  4  2  .  S  a  l  e  s  ,  P  .  M  .  ,  S  o  u  z  a  ,  P  .  M  .  ,  S  i  m  e  o  n  i  ,  L  .  A  .  ,  M  a  g  a  l  h  ã  e  s  ,  P  .  O  .  ,  &  S  i  l  v  e  i  r  a  ,  D  .  (  2  0  1  2  )  .  α  -A  m  y  l  a  s  e  I  n  h  i  b  i  t  o  r  s  :  A  R  e  v  i  e  w  o  f  R  a  w  M  a  t  e  r  i  a  l  a  n  d  I  s  o  l  a  t  e  d  C  o  m  p  o  u  n  d  s  f  r  o  m  P  l  a  n  t  S  o  u  r  c