α-Amylase (Thermostable) (Bacillus sp.)

Background and objectives: The importance of selectively measuring available and unavailable carbohydrates in the human diet has been recognized for over 100 years. The levels of available carbohydrates in diets can be directly linked to major diseases of the Western world, namely Type II diabetes and obesity. Methodology for measurement of total carbohydrates by difference was introduced in the 1880s, and this forms the basis of carbohydrate determination in the United States. In the United Kingdom, a method to directly measure available carbohydrates was introduced in the 1920s to assist diabetic patients with food selection. The aim of the current work was to develop simple, specific, and reliable methods for available carbohydrates and digestible starch (and resistant starch). The major component of available carbohydrates in most foods is digestible starch. Findings: Simple methods for the measurement of rapidly digested starch, slowly digested starch, total digestible starch, resistant starch, and available carbohydrates have been developed, and the digestibility of phosphate cross‐linked starch has been studied in detail. The resistant starch procedure developed is an update of current procedures and incorporates incubation conditions with pancreatic α‐amylase (PAA) and amyloglucosidase (AMG) that parallel those used AOAC Method 2017.16 for total dietary fiber. Available carbohydrates are measured as glucose, fructose, and galactose, following complete and selective hydrolysis of digestible starch, maltodextrins, maltose, sucrose, and lactose to glucose, fructose, and galactose. Sucrose is hydrolyzed with a specific sucrase enzyme that has no action on fructo‐oligosaccharides (FOS). Conclusions: The currently described “available carbohydrates” method together with the total dietary fiber method (AOAC Method 2017.16) allows the measurement of all carbohydrates in food products, including digestible starch. Significance and novelty: This paper describes a simple and specific method for measurement of available carbohydrates in cereal, food, and feed products. This is the first method that provides the correct measurement of digestible starch and sucrose in the presence of FOS. Such methodology is essential for accurate labeling of food products, allowing consumers to make informed decisions in food selection.

Grape pomace powder (GPP), a by-product from the winemaking process, was used to substitute flour for wheat bread fortification within 0, 5, and 10 g/100 g. Rheological properties of control and fortified doughs, along with physicochemical and nutritional characteristics, antioxidant activity, and the sensory analysis of the obtained bread were considered. The GPP addition influenced the doughs’ rheological properties by generating more tenacious and less extensible products. Concerning bread, pH values and volume of fortified products decreased as the GPP inclusion level increased in the recipe. Total phenolic compounds and the antioxidant capacity of bread samples, evaluated by FRAP (ferric reducing ability of plasma) and ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) assays, increased with GPP addition. Moreover, the GPP inclusion level raised the total dietary fiber content of bread. Regarding sensory evaluation, GPP fortification had a major impact on the acidity, the global flavor, the astringency, and the wine smell of bread samples without affecting the overall bread acceptability. The current results suggest that GPP could be an attractive ingredient used to obtain fortified bread, as it is a source of fiber and polyphenols with potentially positive effects on human health.

This study investigated the physicochemical characteristics of protease-treated wheat starch (PT-WST) to understand the role of starch granule-associated proteins (SGAPs) and the potential capability of PT-WST to provide a nutrient delivery system (NDS). Protease treatment was conducted at 4°C and 37°C (PT04 and PT37), respectively. A model delivery system was assessed with PT37 granules infiltrated by λ-carrageenan (λC) under variations of molecular size (λC hydrolysates produced from 0, 2.5, 100, and 500 mM HCl solution), agitation time, and temperature. Protein-specific (3-(4-carboxybenzyl)quioline-2-carboxaldehyde) or non-reactive (methanolic merbromin) fluorescent dye staining revealed that removal of SGAPs on surfaces and channels were more effective for PT37 than for PT04. Consistent amylose content, swelling, and gelatinization temperature before and after protease treatment suggested minimal impact on the starch structure. PT37 presented higher solubility and pasting viscosity than PT04. This resulted from excessive SGAP removal, which enhanced entrapment capacity. λC molecular size and agitation temperature showed a negative correlation with the content of λC entrapped within PT37, and this content depended on the interplay between the agitation time and λC molecular size. As λC molecular size decreased, the λC distribution became uniform throughout the granules, which confirmed the potential of PT-WST as a carrier for NDS.

In the present study, BSG was hydrolysed using cellulolytic enzymes and used as a growth medium supplement for cultivation of the thermophilic bacterium, Bacillus stearothermophilus in the production of α-amylase. A central composite design involving five parameters and four levels viz. starch, peptone, KCl, and MgSO₄ along with BSG hydrolysate was used to derive the optimal media composition. The fermentation was conducted using shake flasks for 36 h at a temperature of 50°C and pH 7.0 at 220 rpm. Optimization trials revealed that maximal amylase production (198.09 U/ml) occurred with a medium composition of starch (0.2% w/v), peptone (0.2% w/v), KCl·4 H₂O (0.02% w/v), MgSO₄·7 H₂O (0.01% w/v) and hydrolysate (0.22% v/v). A 1.3-fold increase in amylase activity was obtained following novel media composition. All the factors considered in the study were found to be significant. The enzyme was purified by three step purification strategy, characterised and tested for anti-biofilm activity.