Stability and bioaccessibility of micronutrients and phytochemicals present in processed leek and Brussels sprouts during static in vitro digestion.

Vegetables are frequently processed before consumption. However, vegetable functionalization continues beyond ingestion as the human digestive tract exposes vegetable products to various conditions (e.g. elevated temperature, pH alterations, enzymes, electrolytes, mechanical disintegration) which can affect the stability of micronutrients and phytochemicals. Besides the extent to which these compounds withstand the challenges posed by digestive conditions, it is equally important to consider their accessibility for potential absorption by the body. Therefore, this study investigated the impact of static in vitro digestion on the stability (i.e. concentration) and bioaccessibility of vitamin C, vitamin K1, glucosinolates, S-alk(en)yl-l-cysteine sulfoxides (ACSOs) and carotenoids in Brussels sprouts (Brassica oleracea var. gemmifera) and leek (Allium ampeloprasum var. porrum). Water-soluble compounds, glucosinolates and ACSOs, remained stable during digestion while vitamin C decreased by >48%. However, all water-soluble compounds were completely bioaccessible. Lipid-soluble compounds were also stable during digestion but were only bioaccessible for 26-81%.

Impact of refrigerated storage on (bio)chemical conversions of health-related compounds in pretreated, pasteurized Brussels sprouts and leek.

Vegetable processing often consists of multiple processing steps. Research mostly focused on the impact of individual processing steps on individual health-related compounds. However, there is a need for more holistic approaches to understand the overall impact of the processing chain on the health potential of vegetables. Therefore, this work studied the impact of pretreatment (relatively intact versus pureed vegetable systems), pasteurization and subsequent refrigerated storage (kinetic evaluation) on multiple health-related compounds (vitamin C, vitamin K1, carotenoids, glucosinolates and S-alk(en)yl-L-cysteine sulfoxides (ACSOs)) in Brussels sprouts and leek. It could be shown that differences introduced by different types of pretreatment were not nullified during pasteurization and refrigerated storage. Clearly, enzymatic conversions controlled during pretreatment resulted in different health-related compound profiles still observable after pasteurization. Moreover, about -42% and -100% relative concentration differences of ACSOs and dehydroascorbic acid, respectively, were detected immediately after pasteurization, while glucosinolates concentrations decreased by about 47% during refrigerated storage. All other compounds were stable during pasteurization and refrigerated storage.

The volatile profile of pasteurized leek (Allium ampeloprasum var. porrum) and Brussels sprouts (Brassica oleracea var. gemmifera) (products), as a witness to (bio)chemical reactivity, influenced by pretreatment and successive refrigerated storage.

Processing can affect (bio)chemical conversions in vegetables and can act on their volatile properties accordingly. In this study, the integrated effect of pretreatment and pasteurization on the volatile profile of leek and Brussels sprouts and the change of this profile upon refrigerated storage were investigated. Pretreatments were specifically selected to steer biochemical reactivities to different extents. Volatile profiles were analyzed by headspace-solid phase microextraction-gas chromatography-mass spectrometry. For both vegetables, it was observed that different pretreatments prior to a pasteurization step led to diverse volatile profiles. The differences in volatile profiles observed in the different samples were presumably attributed to the different degrees of enzymatic conversions, further conversions of enzymatically formed products and thermally induced reactivities. Interestingly, the observed initial relative differences between volatile profiles of differently pretreated pasteurized samples were still observed after a refrigerated storage of 4 weeks at 4 °C. In conclusion, refrigerated storage only limitedly affected the resulting volatile profile.

The Volatile Profile of Brussels Sprouts (Brassica oleracea Var. gemmifera) as Affected by Pulsed Electric Fields in Comparison to Other Pretreatments, Selected to Steer (Bio)Chemical Reactions.

Pulsed electric fields (PEF) at low field strength is considered a non-thermal technique allowing membrane permeabilization in plant-based tissue, hence possibly impacting biochemical conversions and the concomitant volatile profile. Detailed studies on the impact of PEF at low field strength on biochemical conversions in plant-based matrices are scarce but urgently needed to provide the necessary scientific basis allowing to open a potential promising field of applications. As a first objective, the effect of PEF and other treatments that aim to steer biochemical conversions on the volatile profile of Brussels sprouts was compared in this study. As a second objective, the effect of varying PEF conditions on the volatile profile of Brussels sprouts was elucidated. Volatile fingerprinting was used to deduce whether and which (bio)chemical reactions had occurred. Surprisingly, PEF at 1.01 kV/cm and 2.7 kJ/kg prior to heating was assumed not to have caused significant membrane permeabilization since similar volatiles were observed in the case of only heating, as opposed to mixing. A PEF treatment with an electrical field strength of 3.00 kV/cm led to a significantly higher formation of certain enzymatic reaction products, being more pronounced when combined with an energy input of 27.7 kJ/kg, implying that these PEF conditions could induce substantial membrane permeabilization. The results of this study can be utilized to steer enzymatic conversions towards an intended volatile profile of Brussels sprouts by applying PEF.

The role of mechanical collapse by cryogenic ball milling on the effect of high-pressure homogenization on the microstructural and texturizing properties of partially pectin-depleted tomato cell wall material.

In the current study, the effect of different particle size reduction techniques, namely high-pressure homogenization (HPH) and cryogenic ball milling (CBM), on the microstructural and texturizing properties of the tomato acid-unextractable fraction (AcUF) in suspension was studied. Partial pectin depletion was performed by nitric acid pectin extraction on the alcohol-insoluble residue. In the absence of the aforementioned mechanical treatments, the partially pectin-depleted material, i.e., the AcUF, showed a cellular morphology and a high texturizing potential. By short-time CBM in dry-state, the AcUF was extensively fractured and clumped, resulting in a collapsed structure with negligible texturizing potential and low water binding capacity. In contrast, HPH could disrupt the cell wall network (destroying the cellular morphology) resulting in a continuum of interacting material having very similar texturizing potential and a slightly higher water binding capacity than the AcUF before HPH. Furthermore, the potential of HPH to (re)functionalize the collapsed cryogenic ball milled AcUF by its shear-induced disruption was shown. Indeed, the debris-like cell wall remnants could to some extent be reopened by HPH, which resulted in a partial recovery of the original texturizing potential and an improved water binding capacity. However, the potential of HPH at 20 MPa to revert the detrimental effect of CBM decreased with increasing CBM treatment time.

Production and molecular characterization of tailored citrus pectin-derived compounds.

In this study, tailored-made citrus pectin-derived compounds were produced through controlled enzymatic and/or chemical modifications of commercial citrus pectin with different degrees of methylesterification (DM) and similar average molecular weight (MW). In the first treatment, degradation of the citrus pectin (CP) materials by endo-polygalacturonase (EPG) yielded pectins with average Mw's (between 2 and 60 kDa). Separation and identification of the oligosaccharide fraction present in these samples, revealed the presence of non-methylesterified galacturonic acid oligomers with degree of polymerization (DP) 1-5. In the second treatment, exploiting the combined effect of EPG and pectin lyase, compounds with MW between 2 and 21 kDa, containing methylesterified and non-methylesterified polygalacturonans (DP 1-6), were generated. Finally, CP was sequentially modified by chemical saponification and the action of EPG. A sample of DM 11% and MW 2.7 kDa, containing POS (DP 1-5), was produced. Diverse pectin-derived compounds were successfully generated for further studies exploring their functionality.

Flavour stability of sterilised chickpeas stored in pouches.

The increasing need for sustainable food choices places a demand on developing palatable foods from lower impact production and with a suitable shelf-life. In this context, knowledge of the sensory properties of whole sterilised chickpeas is required to be able to make them more attractive to the consumers. The sensory quality of chickpeas is largely dependent on the aroma and flavour, which can be influenced by storage conditions. In this study, sterilised chickpeas in two different packaging materials with different oxygen permeabilities, stored up to 52 weeks at ambient temperature (20 °C) were investigated using both descriptive sensory profiling and gas chromatography - mass spectrometry - olfactometry analysis (GC-MS-O). During storage, intense, sulphury and meat-like aromas decreased in intensity, while with longer storage time hay-like, green-like and potato-like flavours and aromas became more apparent. A total of 40 odour-active volatiles were detected, of which some had properties reminiscent of the chickpea flavour profiles. However, no clear relationships could be established between these odorants and the sensory changes observed during storage by descriptive sensory analysis. It was concluded that, significant changes in the sensory attributes of sterilised chickpeas occurred during 52 weeks of storage, but that packaging material does not seem to largely influence the sensory attributes during storage.

Potential of Chickpea Flours with Different Microstructures as Multifunctional Ingredient in an Instant Soup Application.

Chickpea flours are an interesting multifunctional ingredient for different food products. This study investigated the potential of differently processed chickpea flours as alternative thickening agents in an instant soup recipe, replacing potato starch. Dry instant soup powders were compared on bulk density and powder flowability, whereas prepared liquid instant soups were studied in terms of rheological behaviour (as influenced by microstructure) and volatile composition. The chickpea-flour-containing soup powders possessed similar powder flowability to a reference powder but were easier to mix and will potentially result in reduced blockages during filling. For prepared liquid instant soups, similar viscosities were reached compared to the potato starch reference soup. Nevertheless, the chickpea-flour-containing soups showed higher shear thinning behaviour due to the presence of larger particles and the shear induced breakdown of particle clusters. Flavour compounds from the soup mix interacted with chickpea flour constituents, changing their headspace concentrations. Additionally, chickpea flours introduced new volatile compounds to the soups, such as ketones, aldehydes, alcohols, and sulphur compounds, which can possibly alter the aroma and flavour. It was concluded that chickpea flours showed excellent potential as alternative thickening ingredient in instant soups, improving the protein, mineral and vitamin content, and the powder flowability of the soups, although the flavour of the soups might be affected by the changes in volatile profiles between the soups.

Microstructural and Texturizing Properties of Partially Pectin-Depleted Cell Wall Material: The Role of Botanical Origin and High-Pressure Homogenization.

In the current study, the texturizing properties of partially pectin-depleted cell wall material (CWM) of apple, carrot, onion and pumpkin, and the potential of functionalization by high-pressure homogenization (HPH) were addressed. This partially pectin-depleted CWM was obtained as the unextractable fraction after acid pectin extraction (AcUF) on the alcohol-insoluble residue. Chemical analysis was performed to gain insight into the polysaccharide composition of the AcUF. The microstructural and functional properties of the AcUF in suspension were studied before HPH and after HPH at 20 and 80 MPa. Before HPH, even after the pectin extraction, the particles showed a cell-like morphology and occurred separately in the apple, onion and pumpkin AcUF and in a clustered manner in the carrot AcUF. The extent of disruption by the HPH treatments at 20 and 80 MPa was dependent on the botanical origin. Only for the onion and pumpkin AcUF, the water binding capacity was increased by HPH. Before HPH, the texturizing potential of the AcUFs was greatly varying between the different matrices. Whereas HPH improved the texturizing potential of the pumpkin AcUF, no effect and even a decrease was observed for the onion AcUF and the apple and carrot AcUF, respectively.

Modified Rhamnogalacturonan-Rich Apple Pectin-Derived Structures: The Relation between Their Structural Characteristics and Emulsifying and Emulsion-Stabilizing Properties.

In the context of the increasing interest in natural food ingredients, the emulsifying and emulsion-stabilizing properties of three rhamnogalacturonan-rich apple pectin-derived samples were assessed by evaluating a range of physicochemical properties. An apple pectin (AP74) was structurally modified by a ß-eliminative reaction to obtain a RG-I-rich pectin sample (AP-RG). Subsequent acid hydrolysis of AP-RG led to the generation of pectin material with partially removed side chains (in particular arabinose depleted) (AP-RG-hydrolyzed), thus exhibiting differences in rhamnose, arabinose, and galactose in comparison to AP-RG. All samples exhibited surface activity to some extent, especially under acidic conditions (pH 2.5). Furthermore, the viscosity of the samples was assessed in relation to their emulsion-stabilizing properties. In a stability study, it was observed that the non-degraded AP74 sample at pH 2.5 exhibited the best performance among all the apple pectin-derived samples evaluated. This emulsion presented relatively small oil droplets upon emulsion production and was less prone to creaming than the emulsions stabilized by the (lower molecular weight) RG-I-rich materials. The AP-RG and AP-RG-hydrolyzed samples presented a slightly better emulsion stability at pH 6.0 than at pH 2.5. Yet, neither pectin sample was considered having good emulsifying and emulsion-stabilizing properties, indicated by the presence of coalesced and flocculated oil droplets.

The Structure and Composition of Extracted Pectin and Residual Cell Wall Material from Processing Tomato: The Role of a Stepwise Approach versus High-Pressure Homogenization-Facilitated Acid Extraction.

In literature, different pectin extraction methods exist. In this study, two approaches starting from the alcohol-insoluble residue (AIR) of processing tomato are performed in a parallel way to facilitate the comparison of pectin yield and the compositional and structural properties of the extracted pectin and residual cell wall material obtained. On the one hand, pectin is extracted stepwise using hot water, chelating agents and low-alkaline conditions targeting fractionation of the pectin population. On the other hand, an industrially relevant single-step nitric acid pectin extraction (pH 1.6) is performed. In addition to these conventional solvent pectin extractions, the role of high-pressure homogenization (HPH) as a physically disruptive treatment to facilitate further pectin extraction from the partially pectin-depleted fraction obtained after acid extraction is addressed. The impact of HPH on the pectin cell wall polysaccharide interactions was shown as almost two thirds of the residual pectin were extractable during the subsequent extractions. For both extraction approaches, pectin obtained further in the sequence was characterized by a higher molecular mass and a higher amount of rhamnogalacturonan I domains. The estimated hemicellulose and cellulose content increased from 56 mol% for the AIR to almost 90 mol% for the final unextractable fractions of both methods.

Potential of 1H NMR fingerprinting and a model system approach to study non-enzymatic browning in shelf-stable orange juice during storage.

For the first time, a model system approach was combined with 1H NMR fingerprinting in studying non-enzymatic browning (NEB) of pasteurized shelf-stable orange juice during storage. Various NEB precursors were used individually or in combinations to formulate simple or complex model systems, respectively, in citric acid buffer. Based on orange juice composition, ascorbic acid, sugars (sucrose, glucose and fructose) and amino acids (proline, arginine, asparagine, aspartic acid, serine and glutamic acid) were selected as the precursors for the model systems. After pasteurization and during subsequent accelerated storage (42 °C, 16 weeks) the model systems displayed a three-phase browning development. The initial browning phase was mainly the result of ascorbic acid degradation especially in the presence of amino acids and sugars. In the later phases, the contribution of reactions of sugars and amino acids to browning became apparent. The application of 1H NMR fingerprinting on a simple model system containing ascorbic acid revealed that its degradation pathway to intermediates such as xylonic acid, acetic acid and erythrulose was responsible for the major changes during storage. When this model system was complexed by inclusion of sugars and amino acids, the hydrolysis of sucrose to glucose and fructose was identified as the main reaction leading to differences in the samples throughout storage. These three sugars dominated the NMR spectra of the samples, overshadowing several important compounds for NEB such as ascorbic acid and its degradation products. Other more advanced NMR experiments such as two-dimensional NMR analyses should be applied in future research to identify unknown compounds from NEB reactions.

Arabinoxylan, ß-glucan and pectin in barley and malt endosperm cell walls: a microstructure study using CLSM and cryo-SEM.

The architecture of endosperm cell walls in Hordeum vulgare (barley) differs remarkably from that of other grass species and is affected by germination or malting. Here, the cell wall microstructure is investigated using (bio)chemical analyses, cryogenic scanning electron microscopy (cryo-SEM) and confocal laser scanning microscopy (CLSM) as the main techniques. The relative proportions of ß-glucan, arabinoxylan and pectin in cell walls were 61, 34 and 5%, respectively. The average thickness of a single endosperm cell wall was 0.30 µm, as estimated by the cryo-SEM analysis of barley seeds, which was reduced to 0.16 µm after malting. After fluorescent staining, 3D confocal multiphoton microscopy (multiphoton CLSM) imaging revealed the complex cell wall architecture. The endosperm cell wall is composed of a structure in which arabinoxylan and pectin are colocalized on the outside, with ß-glucan depositions on the inside. During germination, arabinoxylan and ß-glucan are hydrolysed, but unlike ß-glucan, arabinoxylan remains present in defined cell walls in malt. Integrating the results, an enhanced model for the endosperm cell walls in barley is proposed.

Influence of pH and Composition on Nonenzymatic Browning of Shelf-Stable Orange Juice during Storage.

Nonenzymatic browning during storage of pasteurized shelf-stable orange juice causes a major color deterioration, which negatively affects consumer acceptance of the juice. This study, for the first time, investigated on a kinetic basis the effect of pH and suspected nonenzymatic browning reaction precursors such as ascorbic acid, fructose, and arginine on nonenzymatic browning during accelerated storage (42 °C) using an orange-juice-based model system. The results showed that lowering the pH of the model juice system from 3.8 to 1.5 significantly increased the rate of ascorbic acid degradation, the rate changes (increases and decreases) in different sugars, and the rates of furfural and 5-hydroxymethylfurfural formation. These changes coincided with a higher browning intensity, which became more pronounced toward the end of storage of the juice model system. Similarly, adding more ascorbic acid and fructose largely increased the formation of furfural and 5-hydroxymethylfurfural, respectively, and resulted in a higher browning intensity. In conclusion, lowering the pH of the orange juice or addition of ascorbic acid or fructose will enhance its browning during prolonged storage.

Changes in the Soluble and Insoluble Compounds of Shelf-Stable Orange Juice in Relation to Non-Enzymatic Browning during Storage.

For the first time in literature, this study revealed the participation of polymeric components of orange juice cloud and pulp (such as proteins, arabinogalactan proteins, or protein-pectin complexes) during nonenzymatic browning. In a quest to better understand the nonenzymatic browning of shelf-stable orange juice during storage, the juice was fractionated into different fractions depending on the solubility in water/ethanol and the obtained fractions were characterized. The results showed that brown compounds that were formed during storage of orange juice were distributed over water insoluble (pulp), ethanol insoluble (cloud), and ethanol soluble (serum) fractions. In the ethanol insoluble fraction, the brown compounds are hypothesized to be associated with proteins, arabinogalactan proteins, and/or protein-pectin complexes of this fraction without significantly changing their molecular weight distributions, monosaccharide compositions, and protein contents. The changes in the ethanol soluble fraction including ascorbic acid degradation, acid-catalyzed hydrolysis of sucrose, and formation of furfural and 5-hydroxymethylfurfural were highly correlated to the browning development of the juice during storage.

The potential of microalgae and their biopolymers as structuring ingredients in food: A review.

Microalgae are considered promising functional food ingredients due to their balanced composition, containing multiple nutritional and health-beneficial components. However, their functionality in food products is not limited to health aspects, since microalgae can also play a structuring role in food, for instance as a texturizing ingredient. Photoautotrophic microalgae are actually rich in structural biopolymers such as proteins, storage polysaccharides, and cell wall related polysaccharides, and their presence might possibly alter the rheological properties of the enriched food product. A first approach to benefit from these structural biopolymers consists of isolating the cell wall related polysaccharides for use as food hydrocolloids. The potential of extracted cell wall polysaccharides as food hydrocolloids has only been shown for a few microalgae species, mainly due to an enormous diversity in molecular structure and composition. Nevertheless, with intrinsic viscosities comparable or higher than those of commercial thickening agents, extracellular polysaccharides of red microalgae and cyanobacteria could be a promising source of novel food hydrocolloids. A more sustainable approach would be to incorporate the whole microalgal biomass into food products, to combine health benefits with potential structuring benefits, i.e. providing desired rheological properties of the enriched food product. If microalgal biomass would act as a thickening agent, this would actually reduce the need for additional texturizing ingredients. Even though only limitedly studied so far, food processing operations have been proven successful in establishing desired microstructural and rheological properties. In fact, the use of cell disruption techniques allows the release of intracellular compounds, which become available to create strong particle aggregates resulting in an improved viscosity and network structure. Food processing operations might not only be favorable in terms of rheological properties, but also for enhancing the bioaccessibility of several bioactive compounds. However, this research area is only very scarcely explored, and there is a demand for more standardized research studies to draw conclusions on the effect of processing on the nutritional quality of food products enriched with microalgae. Even though considered as promising food ingredients, some major scientific challenges have been pointed out throughout this review paper for the successful design of microalgal based food products.

Instability of common beans during storage causes hardening: The role of glass transition phenomena.

Long-term storage of common beans leads to loss of cooking quality and an ill-defined solution, appropriate storage, is recommended. Therefore, the polymer science theory of glasses that hypothesizes stability of a system below its glass transition temperature (Tg) was applied to determine bean stability during storage in relation to cooking behavior. Since composition influences Tg, powders of cotyledons and seed coats in addition to whole beans were equilibrated above different saturated salt solutions in order to generate materials with different moisture contents. A thermal mechanical compression test which measures compressibility changes in a system upon reaching its glass-rubber transition temperature region was conducted to obtain the Tg. A Tg-moisture relation was established, whose relevance was confirmed by storage and cooking experiments which showed development of hard-to-cook in beans stored above Tg but not below it. Therefore, this relation constitutes a stability map for storage of common beans.

Insight into the evolution of flavor compounds during cooking of common beans utilizing a headspace untargeted fingerprinting approach.

Beans age during storage leading to prolonged cooking times. Chemical reactions that occur during cooking lead to volatile production and flavor generation. Whereas few studies profiled the volatile fingerprint of either non-cooked beans or beans cooked for a specific time, this study explored the evolution of volatiles through headspace fingerprinting of beans cooked at 95 °C to different extents. The influence of aging of beans on this evolution was investigated. Cooking time clearly influenced the evolution of volatiles for both fresh (non-aged) and aged beans. Aged beans exhibited more discriminant compounds than fresh beans regardless of texture considerations due to differences in pre-history of the beans. Strecker aldehydes, sulphur compounds and furan compounds were identified as marker compounds and were linked to mainly lipid oxidation and Maillard reactions. In conclusion, both aging prior to cooking and the cooking process itself largely influence the evolution of volatile compounds during cooking.

Cotyledon pectin molecular interconversions explain pectin solubilization during cooking of common beans (Phaseolus vulgaris).

Dynamics of pectin extractability in cotyledons and seed coats were explored for mechanistic insight into pectin changes due to aging and cooking of beans. In addition, changes in mineral distribution during cooking were determined in order to investigate their retention in the matrix. Pre-soaked fresh and aged beans were cooked in demineralized water for different times and the cotyledons, seed coats and cooking water were lyophilized. From cotyledon and seed coat powders, alcohol insoluble residue (AIR) was extracted and sequentially fractionated into water-, chelator- and sodium carbonate-extractable pectin (WEP, CEP and NEP, respectively). Characterization of pectin in AIR and pectin fractions revealed inherent structural differences between cotyledon and seed coat pectin with the latter exhibiting a lower degree of methylesterification (DM) and being more linear. Due to aging, WEP decreased whilst NEP substantially increased and the CEP fraction and DM of pectin in AIR did not change significantly, suggesting a more crucial role of increased covalent bonding than cation-mediated crosslinking in aging-induced hardening of beans. During cooking, some NEP was converted into WEP and no pectin depolymerization was observed from molar mass distribution profiles. Pectin changes due to aging and cooking of beans were more pronounced in the cotyledon compared to the seed coat. Whilst Ca2+, Fe2+ and Zn2+ were largely retained in the bean matrix during cooking, Mg2+ was largely leached from cotyledons into the cooking water. In conclusion, aging-induced hardening of beans and softening during cooking were found to be premised on interconversion of pectin fractions in cotyledons.

Understanding the Relations Among the Storage, Soaking, and Cooking Behavior of Pulses: A Scientific Basis for Innovations in Sustainable Foods for the Future.

The world faces challenges that require sustainable solutions: food and nutrition insecurity; replacement of animal-based protein sources; and increasing demand for convenient, nutritious, and health-beneficial foods; as well as functional ingredients. The irrefutable potential of pulses as future sustainable food systems is undermined by the hardening phenomenon that develops upon their storage under adverse conditions of temperature and relative humidity. Occurrence of this phenomenon indicates storage instability. In this review, the application of a material science approach, in particular the glass transition temperature concept, is presented to explain phenomena of storage instability such as the occurrence of hardening and loss of viability under adverse storage conditions. In addition to storage (in)stability, application of this concept during processing of pulses is discussed. The state-of-the-art on how hardening occurs, that is, mechanistic insights, is provided, including a critical evaluation of some of the existing postulations using recent research findings. Moreover, the influence of hardening on the properties and processing of pulses is included. Prevention of hardening and curative actions for pulses affected by the hardening phenomenon are described in addition to the current trends on uses of pulses and pulse-derived products. Based on the knowledge progress presented in this review, suggestions for the future include: first, the need for innovation toward implementation of recommended solutions for the prevention of hardening; second, the optimization of the identified most effective and efficient curative action against hardening; and third, areas to focus on for elucidation of mechanisms of hardening, although existing analytical methods require advancement.