Wheat-based breads with slowly digestible starch properties by increasing the amylose content: an in vitro approach

2.1 Raw materials, recipes and baking conditions

The HAMS flour (Amylo-maize starch N-400; 65.0% amylose content according to the manufacturer) was obtained from Roquette Italia SpA (Sardigliano Alessandria, Italy). All other ingredients, including dry yeast (PaneAngeli, Italy) and salt with iodine (Salina di Margherita Savoia, Italy) were food grade. Three different wheat-based breads were prepared. For control bread (0-BR) the recipe was based on: 270 g soft NAWW flour (Barilla SpA, Parma, Italy; 25.0% amylose content according to the manufacturer), 2.7 g dry yeast, 2.1 g salt and 180 g water. Part of NAWW flour equivalent to 15% and 30% (on a total flour basis) was substituted with HAMS flour to formulate 15-BR and 30-BR experimental breads, respectively. The substitution levels were selected through preliminary baking trials, 30% HAMS being the maximum level where changes in bread texture characteristics were not noticeable. All ingredients were mixed and baked with a commercial bread maker machine (OW3101, Moulinex International, Italy) using the basic white bread program (program number 1; medium crust; total time 190 min). After the cycle was complete, products were cooled to room temperature and stored at –18°C in plastic bags. Prior to evaluation, breads were thawed for 24 h at 4 °C and then equilibrated to room temperature for 4 h. Three different batches of 0-BR, 15-BR and 30-BR were produced. Commercial wheat bread (soft white loaf; ingredients: wheat flour, water, olive oil, yeast, salt, sugar, malted barley flour) was used as reference.

2.2 Proximate composition of breads

For each bread, a representative portion was dried at 55 °C for 48 h in a forced-air oven, ground through a 1-mm screen using a laboratory mill (Retsch grinder model ZM1; Brinkman Instruments, Rexdale, ON, Canada) and analysed [15] for dry matter (DM; 930.15), ash (942.05), crude protein (976.05), crude lipid (954.02 without acid hydrolysis), total dietary fibre (Megazyme assay kit K-INTDF 02/15, which includes RS and non-digestible oligosaccharides [16]) and total starch contents (Megazyme assay kit K-TSTA 07/11). For each treatment, batches were analysed in triplicate.

2.3 Determination of starch digestive fractions

The RDS, SDS and RS levels were measured using the Englyst et al. [17] enzymatic protocol. Samples were pre-treated with a 0.05 M HCl solution containing pepsin (5 mg/ml; P-7000, Sigma-Aldrich^® Co., Milan, Italy) for 30 min at 37° C under gentle agitation. The pH was then adjusted to 5.2 by adding 20 ml of 0.1 M sodium acetate buffer prior to the addition of an enzyme mixture with an amylase activity of about 7000 U/ml given by pancreatin (about 7500 FIP-U/g; 7130, Merck KGaA, Darmstadt, Germany), amyloglucosidase (about 300 U/ml; A-7095, Sigma-Aldrich^® Co., Milan, Italy) and invertase (about 300 U/g; I-4504, Sigma-Aldrich^® Co., Milan, Italy). The glucose released from samples after 20 min and 120 min of incubation was measured colorimetrically using a glucose oxidase kit (GODPOD 4058, Giesse Diagnostic snc, Rome, Italy) and converted to RDS and SDS values [5]. The RS was measured as the starch that remained un-hydrolysed after 120 min of incubation [5]. A starch digestible index (SDI), which represents a measure of the rate of starch digestion, was calculated [5]. For each treatment, batches were analysed in triplicate.

2.4 In vitro starch digestion and calculations

The multi-enzymatic method proposed by Granfeldt et al. [18] was employed to evaluate the in vitro starch digestion of breads over time. This method has been shown to predict GI values with good accuracy for several cereal and legume foods. For each product, sample portions containing 1 g of available starch were tested. Healthy subjects participated in the chewing phases of the experiment to simulate the oral phase of the digestion [18]. The chewed materials were then carefully transferred into glass tubes containing 0.05 M phosphate buffer adjusted to pH 1.5 with HCl, and the mixture was incubated with pepsin (P-7000, Sigma-Aldrich^® Co., Milan, Italy) for 30 min at 37°C with gentle mixing. After neutralisation to pH 6.9 with NaOH, porcine pancreatic α-amylase (A-6255, Sigma-Aldrich^® Co., Milan, Italy) was added, the sample was brought to volume with phosphate buffer and then transferred to the dialysis tubing (13 cm strips, Spectra Por No. 2, width 45 mm, molecular weight cut-off 12,000–14,000) and incubated at 37 °C for 180 min in 800 ml of phosphate buffer. Every 30 min, aliquots from the dialysate were removed for analysis of reducing sugars by the 3,5-dinitrossalicylic acid method using a maltose standard curve [19]. The hydrolysis index (HI) was then derived from the ratio between the area under the hydrolysis curve (0–180 min) of breads 0-BR, 15-BR and 30-BR and the corresponding area of the reference sample chewed by the same person. From the HI obtained in vitro, the pGI was calculated using the formula pGI = 8.198+0.862 × HI [20].

To describe starch hydrolysis kinetics, a first-order exponential model (C_t = C _0 + C _∞–0 (1 –e^–kt) was applied, where C _t is the starch hydrolysed at time t (g/100 g dry starch), C ₀ is the starch solubilised in the buffer at 0 min (g/100 g dry starch), C _∞ is the equilibrium percentage of starch hydrolysed at infinite time (g/100 g dry starch), C _∞–0 is C _∞ minus C ₀ (g/100 g dry starch), k is the rate constant (min^–1) and t is the chosen time (min) [21]. For the purpose of data fitting, values were obtained by the Marquardt method using the PROC NLIN procedure of SAS 9.3 (SAS Inst. Inc., Cary, N.C., U.S.A).

2.5 Statistical analysis

Data were analysed as a completely randomised design using the MIXED procedure of SAS 9.3 (SAS Inst. Inc., Cary, N.C., USA) according to the model: Y ij = μ + α i + e ij where Y_ij is the dependent variable on the j th subject (bread batch) assigned to treatment i, μ is the overall mean, α _i is the fixed effect of HAMS substitution to NAWW flour (i = 0, 15 and 30%), and e _ij is the residual error. Orthogonal linear contrast was post-hoc carried out to study the linear response of fixed effect. Through the text, terms “increased” or “decreased” refer to the linear trend of treatments. Experimental unit was the bread batch. Significance was declared at p < 0.05.

3.1 Chemical composition and starch fractions of experimental breads

The chemical composition of experimental breads was influenced by HAMS addition (Table 1). Total starch content increased (p < 0.05) from 72.1 g/100 g DM to 73.6 and 74.5 g/100 g DM, whereas crude protein content decreased (p < 0.05) from 9.4 g/100 g DM to 7.3 and 6.6 g/100 g DM when the level of HAMS increased in the recipe. A similar pattern has been previously reported for cookies produced by the substitution of HA ingredients for a part of NA wheat flour [13]. The total dietary fibre content increased (p < 0.05) from 2.2 g/100 g DM to 3.6 and 5.2 g/100 g DM for control, 15-BR and 30-BR, respectively. Analogously, gluten-free breads prepared with a 20% replacement of NA maize starch with HAMS (on a total starch basis) showed a marked rise by 88% of total dietary fibre content with respect to control samples [22]. Greater amounts of dietary fibre in refined cereal products are favourable, aiming to raise the overall health profile of these types of foods [23].

Concerning starch fractions, both RS and SDS levels increased (p < 0.05) from 1.3 g/100 g DM to 4.4 and 6.1 g/100 g DM and from 5.8 g/100 g DM to 21.6 and 27.5 g/100 g DM, whereas the RDS level decreased (p < 0.05) from 64.9 g/100 g DM to 47.6 and 40.9 g/100 g DM for control, 15-BR and 30-BR, respectively. The conversion of RDS to SDS and RS fractions can improve the nutritional profile of foods aiming to formulate products with slowly digestion starch properties [24]. Similarly, Agama Acevedo et al. [25] obtained lower RDS and higher SDS contents in bakery products added with HA banana starch when compared to control samples. Present findings corroborate the hypothesis that the formation of RS in bakery products is strongly related to the amylose content [26]. Similarly, higher amounts of RS have been reported for arepa maize bread made from HA genotypes compared with corresponding products prepared with NA maize flours [27]. The hydration of native starch during heat treatment, commonly referred as gelatinisation, markedly increases starch susceptibility to amylolytic degradation. However, higher level of amylose can contribute to obstruct starch hydration, amylose molecules being more compact when compared to amylopectin [28]. This in turn may affect the degree of starch swelling during gelatinisation to such an extent that the overall accessibility of starch to hydrolysing enzymes can be significant lowered [29]. Consequently, the RS fraction in HA breads may correspond to amorphous amylose not gelatinised during normal bread-making conditions, as already indicated by Hoebler et al. [30]. In addition, on cooling after gelatinisation, part of the starch may undergo a re-association process called retrogradation. This process can produce firmly recrystallized structures which contribute to form RS, higher amounts of amylose being usually linked to a greater and much faster retrogradation tendency than amylopectin [31]. However, at the highest inclusion level of HAMS, the RS content of 30-BR was about 8% of the total starch content, lower than the minimum RS value (i.e., 14 %) proposed for baked goods [2]. Lastly, the SDI decreased (p < 0.05) from 90.1 to 63.3 and 52.2 with increasing HAMS levels in the recipe. Accordingly, Englyst et al. [5] reported a SDI value for white wheat bread of 90, whereas a SDI of 56 was obtained for maize RS products.

3.2 Predicted glycaemic index and in vitro starch digestion kinetics of experimental breads

The classification through a pGI is useful to predict the likely in vivo glycaemic response of a food of interest, this parameter being positively related to in vivo GI values for a wide number of food products [18]. As reported in Table 2, both HI and pGI decreased (p < 0.05) from 102 to 66 and from 96 to 65 when the HAMS increased in the composite. The pGI of the reference sample was similar to that measured for 0-BR (94 vs. 96, respectively). The lower pGI recorded for HAMS-enriched breads could be related to their respective SDS and RS contents, both fractions associated with decreased in vivo and in vitro glycaemic responses [27, 32]. Similarly, Hallström et al. [1] showed that wheat breads containing high amounts of RS (4.4 g/100 g DM) elicited the lowest glycaemic response when compared to other test products. In addition, present findings seem to confirm previous indications that the RS fraction may influence not only the amount of unavailable starch but also the digestibility of available starch itself, due to a possible encapsulation of gelatinised starch between RS layers [31].

Lastly, slower k values (p < 0.05) were calculated when the HAMS level increased in bread formulation, with values ranging from 0.176 min^–1 to 0.075 min^–1 and 0.041 min^–1 for 0-BR, 15-BR and 30-BR, respectively. Previous experiences revealed that the rate of in vitro starch hydrolysis corresponded well with the in vivo rate of starch uptake, the latter considered as one of the major determinants of the glycaemic response, faster starch digestion rates being usually associated with greater extent of starch digestion [9, 32]. The reduction in k values may be related to the variation in the proportion of amorphous starch material due to the presence of different HA levels in the composite. In addition, the possible formation of complexes between amylose and surrounding lipids and proteins during baking may be additional factors contributing to lower the rate of starch hydrolysis in products with elevated amylose content [31].

Taking together, present in vitro findings indicated that increase the amylose content through the substitution of NA with HA flours can enhance the overall slowly digestible starch properties of wheat-based breads, in agreement with current dietary indications [2]. Beyond the different amylose content of selected ingredients, structural characteristics of the starches used should be considered. Firstly, with respect to wheat starch, maize starch is characterised by larger size of starch granules [33]. A smaller surface area to volume ratio differentiates larger starch granules and thus a lower surface potentially subjected to enzyme hydrolysis [9]. Secondly, starch granules display different types of crystalline domains that resist enzyme hydrolysis to different degrees [14]. In particular, starch from cereal grains exhibits the A-type power diffraction pattern, which has an open structure that renders starches highly digestible, whereas tubers and HA starches give the so-called B-pattern, which is more resistant to digestion due to a close-packed arrangement [9].

3.3 Potential implications for human health

Current in vitro findings indicated that the use of flours with starch characterised by higher amylose content could favourably be used in wheat-based bread preparation aiming to increase slowly digestible starch properties. Foregoing results can therefore provide us the opportunity to discuss additional aspects in relation to human nutrition and health. In general, carbohydrate foods vary in their potential to provoke different postprandial glycaemic response in humans. This response has been quantified in several ways, by using both in vivo and in vitro approaches, including the GI assessment along with the quantification of nutritionally important starch fractions [5, 9]. Although there is an on-going debate, there is general consensus that the reduction of the dietary glycaemic response following the consumption of lower GI and higher SDS and RS content foods can favourably influence physiological parameters implicated as markers for conditions including overweight and obesity, diabetes mellitus and risk of coronary heart disease [2, 34]. For instance, in the long term the consumption of foods with low GI and high unavailable carbohydrate (UC) content improved blood glucose control in patients with type 1 diabetes [34, 35]. Similarly, there is evidence that lower GI and higher UC in diets can reduce the levels of glycated proteins for individuals with fasting blood glucose concentrations in excess of 5 mmol/l [34]. On the other hand, health benefits associated with SDS consumption are mainly related to stable glucose metabolism, diabetes management and improved satiety [6, 36]. Golay et al. [37] demonstrated that switching, at breakfast only, from standard cereals to foods containing SDS improved carbohydrate metabolism and reduced insulin requirement of insulin-treated type 2 diabetic individuals. A delayed return of hunger state has also been reported in human subjects after the consumption of SDS compared to RDS food sources [38]. Last, the replacement of digestible starch with RS has been proven to decrease post-prandial glycaemic and insulinaemic responses more than the addition of RS itself [2]. This starch fraction provides the colonic microbiota with a fermentable carbohydrate substrate, promoting higher proportion of butyric acid than other indigestible carbohydrates. Butyrate constitutes the major energy substrate for colonocytes and is usually associated with several benefits in relation to large intestine health [7, 8]. To promote aforementioned beneficial effects, the suggested RS intake level was set at about 20 g/person/day [39]. Considering that the average bread ingestion is about 150 g/person/day for European Countries [40], the consumption of experimental breads formulated with the highest HAMS inclusion (i.e., BR-30) could account for more than 50% of the suggested dietary RS intake.