The objectives of this study were to inactivate the enzymes α-amylase, lipase, protease, and peroxidase in flour with supercritical carbon dioxide (scCO2), and to optimize the enzymatic treatment conditions. Enzyme inactivation is important, due to the undesirability of certain flour enzymes that cause adverse reactions during storage as unpleasant rancidity of flour, and, at the same time, reduce the shelf life of flour. Therefore, crude enzymes and flour were initially exposed to scCO2 to determine the effect on specific enzyme activity under appropriate conditions. The activity of the unwanted enzymes lipase and peroxidase decreased under optimal process conditions of scCO2 exposure, lipase by 30%, and peroxidase by 12%, respectively. It was discovered that the inactivation of enzymes in wheat flour occurred, where, at the same time, this sustainable method allows the regulation of enzyme activity in the baking process. Afterwards, the effect of scCO2 on the physicochemical properties of flour, morphological changes on starch granules, and content of total lipids was studied. In scCO2-treated white wheat flour, the fat content decreased by 46.15 ± 0.5%, the grain structure was not damaged, and the bread as the final product had a lower specific surface volume. Therefore, this could be a promising technology for flour pretreatment, potentially impacting the prolonging of its shelf-life.
The numerous enzymes present in the bran and germ fractions of a wheat kernel initiate many chemical changes that affect the compositional and functional properties of whole wheat flour. In general, wheat flour is composed mainly of macronutrients such as starch, water, proteins, and non-starch polysaccharides, such as lipids and ashes and also contains several technologically important enzymes, mainly amylases, proteases, lipoxygenase, polyphenol oxidase, and peroxidase. In addition, there are the three most important groups of enzymes with regard to the process of baking: Enzymes that hydrolyse carbohydrates, such as cellulase, amylases, and pentosanases, enzymes that hydrolyse proteins, such as proteases, and enzymes that affect fats and oils, mainly lipase and lipoxygenases. Although the enzymes are inactive during the storage of grain and flour, they become active when water is added, and, thus, play a significant role in determining the functional attributes of flour.
One of the challenges faced by the food industry is the stability of whole wheat flour during storage. The development of hydrolytic and oxidative rancidity during storage decreases the sensory acceptability, as well as the compositional and functional properties of flour. For these reasons, maintaining the quality of whole wheat flour to be used in product development and formulations is a challenge to milling companies and the food industry. The accumulation of free fatty acids in flour during storage is related positively to the initial lipase activity of flour. Reducing/inactivating lipase activity would be one of the methods to reduce the accumulation of free fatty acids. Another class of enzymes, peroxidases which are oxidoreductase enzymes, use hydrogen peroxide or organic hydroperoxides as oxidants. Peroxidase also plays a significant role in carotenoid bleaching during dough mixing, and may be responsible for the undesirable browning of flour.
Previous studies have used different thermal processing methods, including steaming, microwave heating, and passing through infrared and gamma radiation, to decrease enzyme activity. The disadvantage of these methods is the use of high temperatures, which, besides high energy consumption, also affect the quality of the food product. Conventional heat treatment applications cause nutritional loss and undesired changes in sensorial qualities. Moreover, an excessively high concentration of protease in wheat flour can result in a total breakdown of the gluten protein structure, and a small amount of enzyme α-amylase is necessary to break down starch, which then provides an adequate supply of fermentable sugars. This is often supplied by the addition of malt or commercial preparations of fungal or bacterial α-amylase. However, if an excess enzyme is present, starch is broken down to dextrins and simple sugars, resulting in a sticky, hard-to-handle dough that produces bread with a wet, sticky crumb.
This paper is focused on the enzyme activities and compositional properties of different flour types for different applications. Optimization of the protein extraction process was performed, in order to describe the differences in the protein concentrations in various flour types. Furthermore, another aim of this work was to observe the effects of scCO2 treatment on the compositional and functional properties of whole wheat flour, as CO2 is a thermodynamically stable molecule with low reactivity. In principle, the technology of scCO2 treatment, as a green, waterless, more energy-efficient, cleaner, and safer production method, has attracted considerable interest in different industries in recent years. ScCO2 is used commonly, and has become the solvent of choice for the food, cosmetic and pharmaceutical industries as a result of its non-toxicity and moderate critical properties (critical pressure: 7.38 MPa, critical temperature: 304.35 K). It is well known that, in scCO2, the most widely used supercritical fluid, enzyme activity may be tuned with different pressure/temperature/time combinations, and, at certain conditions, it may be reduced. This could be attributed to the formation of carbonic acid in the presence of water and carbamates by reaction with amine groups on the enzyme. Under certain pressure and temperature conditions, a change in the protein structure can happen with an increase in the β-sheet structure that leads to the burial of the catalytic center. Subsequently, scCO2 treatment of flour may reduce enzymatic activities efficiently without thermal treatment, ensuring the same quality of flour after this process is finished. Due to the undesirable effects of some enzymes, a series of experiments were performed to achieve the inactivation of these enzymes in wheat flour.
Certainly, an important parameter is scCO2’s solubility, which is needed for understanding the fermentation of bread dough modeling. Fan et al. researched CO2 solubility in aqueous dough during baking, and found that CO2 solubility is responsible for the early oven rise of the dough, since vapor makes a significant contribution to the later expansion. Therefore, the solubilities of CO2 were also measured in different flour types.
In addition white wheat flour was exposed to scCO2, with the aim to inactivate the enzymes in the flour. Thus, after exposure of the flour to scCO2, the proteins were extracted, and the residual activities of the enzymes α-amylase, lipase, protease, and peroxidase were determined in the supernatant. Moreover, a comparison was conducted between the enzyme activity in unexposed flour to the residual enzyme activity in scCO2 treated flour and the activities of free enzymes treated in scCO2. To understand the mechanism of enzyme inactivation using scCO2 it is essential to know the interactions between biological molecules like amino acids, proteins, and CO2. Amino acids are the structural constituents of the proteins, containing amine (NH2) and carboxyl (COOH) functional groups with a side chain (R group—aliphatic, acyclic, aromatic) specific to each amino acid. Moreover, an aqueous solution of amino acids and amino acid salts is advantageous in capturing CO2 where dipole-quadrupole interactions become significant in the investigation of such complicated thermodynamic systems (water + amino acid + carbon dioxide). The fundamental chemical modifications in the amino acids exposed to SCCO2 are oxidation, sulfonation, hydroxylation, ring-opening, and amidation, wherein the inactivation of enzymes is mostly attributable to the type of amino acid in the active site. The knowledge gained from this study may also help promote the application of a sustainable approach of using scCO2 in the food industry and food science.
2. Materials and Methods
White wheat flour type 500, wheat flour type 850, rye flour type 1250, wholegrain rye flour, graham flour, and wholegrain spelt flour were kindly supplied by the bakery Hlebček, d.o.o. (Pragersko, Slovenia). Experiments were conducted using carbon dioxide (99.5% purity) produced by Messer, Ruše. Ethanol (96%), phosphoric acid (≥85%), sodium chloride (≥99.5%), Coomassie-Brilliant Blue G250 (1.15444.0025) and acetonitrile (99.9%) were supplied from Merck, while the bovine serum albumin (≥96%), sodium acetate (≥99.0%), acetic acid (GR for analysis), p-nitrophenyl butyrate (≥98%) were supplied from Sigma. The enzymes α-amylase (~30 U/mg) from Aspergillus oryzae and protease (≥0.6 U/mg) from Aspergillus saitoi were obtained from Sigma, while the lipase (~200 U/g) from Aspergillus niger was obtained from BioChemics. Peroxidase from horseradish (232-668-6) was purchased from BBI Enzymes (UK). The petroleum ether (32247) used for lipid extraction was supplied by Honeywell, Riedel-de Haen. All other chemicals used in the laboratory were of analytical grade.
2.1. Protein Extraction Process
The extraction of proteins was performed in 0.1 M acetate buffer pH 5.3 with 5 g of wheat flour in an Erlenmeyer flask, followed by incubation at room temperature for a specific time with horizontal shaking at 300 rpm. Afterwards, centrifugation at 8000 rpm was performed for five minutes. To optimize the extraction process, three different combinations of centrifugation and filtration were performed, to obtain the highest value of protein concentration. The first sample was, thus, filtered through a filter with a pore size of 0.45 µm. For the second sample, additional centrifugation was performed under the same conditions (8000 rpm, five minutes). In the third sample, the supernatant was decanted after the first centrifugation. For all three combinations, the supernatant was separated and considered as a crude protein extract. The protein concentration was determined in the supernatant according to the Bradford method. Each sample was assayed twice, and three individual absorbance measurements per extracted sample were recorded within an experimental error of about 0.03%.
2.2. scCO2 Medium Treatment
An application of scCO2 technology was implemented to determine the effects of scCO2 on enzymes’ activities. First, the native enzymes were exposed to scCO2 under different conditions in a high-pressure batch reactor, and then white wheat flour was exposed to the scCO2 medium under the same conditions. A high-pressure batch reactor with a volume of 60 mL with a CO2 gas supply was used in the experimental research. When the temperature in the high-pressure batch reactor reached 35 °C, the pressure was raised to the desired value of 200 and 300 bar with cooled CO2. Afterwards, the system was maintained at a constant temperature and pressure for a preestablished exposure time. After the scCO2 medium treatment, the reactor was depressurized rapidly (0.37 ± 0.08 bar/min). Later, enzymes were extracted from the scCO2 treated wheat flour, where the supernatant was used to determine the enzyme activity and protein concentration. Residual enzyme activities in the treated wheat flour were correlated to the enzyme activities in untreated wheat flour, taken as 100%. The flour samples were subjected to the same conditions of scCO2 treatment for three times to ensure repeatability, whereby the percentage error was ±2.0%.
2.3. Flour Physicochemical Analyses
2.3.1. Moisture Content
The moisture was determined from the weight loss of a sample dried by heating to 130 °C. Moisture determination in the flour samples was repeated three times, to obtain reliable results within an experimental error of about 0.5%.
2.3.2. Particle Size Analysis
Previous studies have shown that the particle size distribution of flours from all wheat types could be measured more precisely by laser diffraction than by sieve analysis. Therefore, the particle size distribution of flour samples was measured by a laser diffraction particle size analyzer (Analysette 22, Fritsch, Schlieren, Switzerland), using the dry analysis method within the selected size range from 0.3 to 300 µm. The sample was placed into the sample hopper manually and then aspirated into the apparatus. The particles cross the laser beam, and the particle size distribution was measured in this way. The distribution was reported both as a Gaussian curve and arithmetic mean (µm). Subsequently, the flour particle size distributions were determined in triplicate measurements with an experimental error of 0.5%.
2.3.3. Environmental Scanning Electron Microscope
A Quanta FEI 200 3D, Oregon, United States microscope was used in environmental mode to observe the morphological changes in the starch granules in the flour that occurred during the scCO2 high-pressure treatment ESEM. The pressure chamber was 60 Pa, and the accelerating voltage for imaging was 10 kV.
2.3.4. Determination of CO2 Solubility in Flour with Magnetic Suspension Balance (MSB)
The phase equilibria behavior was studied by determination of the CO2’s solubility and the volumetric expansion of flour saturated with CO2. The solubility of CO2 is the total amount of carbonate species that can be dissolved in the solution. The solubilities of CO2 in different flour types were measured by a gravimetric method involving magnetic suspension balance (MSB, RUBOTHERM, Berlin, Germany) at pressures ranging from 1 to 350 bar (Figure 1). The applied MSB is designed for a maximum operating pressure of 350 bar and an operating temperature of 562 K. The temperature of the cell was kept constant using a Lauda P5 thermostat (Lauda, Königshofen, Germany). The measuring cell of the MSB has a sapphire window, which allows observation of the sample and estimation of volume modifications during the sorption measurements. The software MessPro (SL-2500-00216, Ver.11.05) recorded the conditions (mass, temperature, pressure). The duration of the measurement at each pressure was approximately 120 min, as equilibrium was reached after this time. To define the volume of the sample, a photo of the sample was taken with a digital camera and analyzed using ImageJ 1.52 software (Freeware).
2.3.5. Fourier Transform Infrared Spectroscopy
Fourier transform infrared spectroscopy (FTIR, IRAffinity-1s, Shimadzu, Slovenia) using an ATR accessory was performed for the untreated flour and flour samples after scCO2 medium treatment with rapid and slow expansion. FTIR analysis was conducted to observe the changes in functional groups qualitatively, and further evaluate the modifications in the components of starch granules due to the impact of the scCO2 process.
2.3.6. Determination of the Total Lipids in the Flour
Lipids are a group of substances that are soluble in organic solvents. In general, Soxhlet extraction is one of the methods used most commonly for the determination of total lipids in dried foods. Therefore, a Soxhlet apparatus was used to extract the lipids from the untreated and scCO2 treated flour samples into petroleum ether. A total of 10 g of flour sample was used to extract the lipids with 180 mL of petroleum ether, boiling at 60 °C for 5 h. In the Soxhlet extraction, the solvent was accumulated in the extraction chamber (the flour sample was held in a filter paper thimble) for 5–10 min and then returned to the boiling flask. This method provides a soaking effect for the sample. The solvent in the round bottom flask with the dissolved lipids was removed after a certain period of time, and the lipids were isolated by evaporating the organic solvent using a rotavapor apparatus at 40 °C, which is used for the recovery of lipids from the solution to near dryness. The mass of the extracted lipids obtained was determined by calculating the extraction yield.
Moreover, the percent of lipid content in the sample was calculated using Equation (1),
Where mextract is the mass of lipids weighed after evaporating the petroleum ether, and mflour sample is the mass of flour used for lipid extraction.
2.4. Monitoring of Enzyme Activity
The activity of a specific enzyme was determined based on enzymatic activity assays using a UV-spectrophotometer (Varian, Cary 50 Probe, Agilent Technologies, Santa Clara, CA, USA) at different wavelengths.
The α-amylase activity was determined based on the increase in the amount of glucose in the solution by using the DNS reagent (3,5-dinitrosalicylic acid). The absorbance was measured with a UV-Vis spectrophotometer at a wavelength of 540 nm.
The activity of lipase was determined based on P-nitrophenol (pNP) reaction, which was monitored at 400 nm for 5 min. One unit of lipase activity released 1 nanomole of p-nitrophenol per minute at pH 7.2 and 37 °C using p-nitrophenyl butyrate (PNPB) as the substrate.
The protease activity was measured using casein as a substrate. The procedure for the determination of the protease activity was described previously in research.
The peroxidase activity was measured using phenol, 4-aminoantipyrine (4-AAP), and hydrogen peroxide as substrates. The method for peroxidase activity determination and a detailed calculation procedure of peroxidase activity has been published in our previous research.
The residual activities of different enzymes were calculated using the following Equation (2).
All experiments were carried out in triplicate, and the mean values express the average ± standard deviation.
R version 4.1.1 and Rstudio, version 1.4.1717 were used to perform the statistical analysis and evaluate the differences between the residual enzyme activity within enzyme types in native form and in scCO2-treated flour after the performance of a two-way analysis of variance (ANOVA). The data were distributed normally and were presented with mean and standard deviation. A post hoc Tuckey honest significant difference test was performed to define the inactivation of enzymes in different groups.
2.5. Baking Test
The baking test was developed to establish the suitability of the flour for making bread, and to determine the difference between untreated and scCO2-treated white wheat flour in the bread making process. A bread-making machine, an SBB 850 F2, 850 W (SilverCrest, Lidl, Slovenia) was used for dough baking, according to the recipe from the bakery Hlebček d.o.o, Pragersko, Slovenia. The dough was prepared from 540 g of white wheat flour with 1.8% yeast, 2% salt, 58% water, and 2.5% of professional dough improver, which contains wheat flour, wheat gluten, sugar, wheat malt flour, flour treatment agent (ascorbic acid) and enzymes (α-amylase, hemicellulase). In general, bakeries add dough improver to improve the baking functionality in their bread production. For this purpose, a dough improver was added in the same quantity in baking bread with both types of flour, untreated and scCO2-treated white wheat flour. The ingredients were first preheated for 10 min, and the dough was mixed for 12 min and fermented for 20 min, whereas stirring and fermenting were then repeated for 7 min and 35 min. The bread loaf was baked for 60 min at 220 °C. The dough baking test was repeated with untreated white wheat flour and scCO2-treated white wheat flour under the same conditions and bread-making program. The loaves of bread were weighed after cooling, and their volume (cm3) was determined by the water displacement method. The specific volume (cm3/g) was calculated as loaf volume/bread weight. Values obtained were the mean of three replicates. A sensory analysis was carried out, involving evaluation of the sensory attributes of appearance, aroma, taste, and texture. Ten non-trained testers (8 females and 2 males, ranging in age between 26 and 58) made a hedonic evaluation of the acceptability of bread baked from untreated and scCO2-treated white wheat flour. In the test, participants were asked about their preferences concerning the differences in the experimental bread. The average height of the baked bread was measured, and determined using the ImageJ program.
Source: MDPI AG (Multidisipliner Digital Publishing Institute)