Experimental Evolution Reveals a Novel Ene Reductase That Detoxifies α,ß-Unsaturated Aldehydes in Listeria monocytogenes.

The plant essential oil component trans-cinnamaldehyde (t-CIN) exhibits antibacterial activity against a broad range of foodborne pathogenic bacteria, including L. monocytogenes, but its mode of action is not fully understood. In this study, several independent mutants of L. monocytogenes with increased t-CIN tolerance were obtained via experimental evolution. Whole-genome sequencing (WGS) analysis revealed single-nucleotide-variation mutations in the yhfK gene, encoding an oxidoreductase of the short-chain dehydrogenases/reductases superfamily, in each mutant. The deletion of yhfK conferred increased sensitivity to t-CIN and several other α,ß-unsaturated aldehydes, including trans-2-hexenal, citral, and 4-hydroxy-2-nonenal. The t-CIN tolerance of the deletion mutant was restored via genetic complementation with yhfK. Based on a gas chromatography-mass spectrometry (GC-MS) analysis of the culture supernatants, it is proposed that YhfK is an ene reductase that converts t-CIN to 3-phenylpropanal by reducing the C=C double bond of the α,ß-unsaturated aldehyde moiety. YhfK homologs are widely distributed in Bacteria, and the deletion of the corresponding homolog in Bacillus subtilis also caused increased sensitivity to t-CIN and trans-2-hexenal, suggesting that this protein may have a conserved function to protect bacteria against toxic α,ß-unsaturated aldehydes in their environments. IMPORTANCE While bacterial resistance against clinically used antibiotics has been well studied, less is known about resistance against other antimicrobials, such as natural compounds that could replace traditional food preservatives. In this work, we report that the food pathogen Listeria monocytogenes can rapidly develop an elevated tolerance against t-cinnamaldehyde, a natural antimicrobial from cinnamon, by single base pair changes in the yhfK gene. The enzyme encoded by this gene is an oxidoreductase, but its substrates and precise role were hitherto unknown. We demonstrate that the enzyme reduces the double bond in t-cinnamaldehyde and thereby abolishes its antibacterial activity. Furthermore, the mutations linked to t-CIN tolerance increased bacterial sensitivity to a related compound, suggesting that they modify the substrate specificity of the enzyme. Since the family of oxidoreductases to which YhfK belongs is of great interest in the mediation of stereospecific reactions in biocatalysis, our work may also have unanticipated application potential in this field.

Study of the Fermentation Characteristics of Non-Conventional Yeast Strains in Sweet Dough.

Despite the diverse functions of yeast, only a relatively homogenous group of Saccharomyces cerevisiae yeasts is used in the baking industry. Much of the potential of the natural diversity of yeasts has not been explored, and the sensory complexity of fermented baked foods is limited. While research on non-conventional yeast strains in bread making is increasing, it is minimal for sweet fermented bakery products. In this study, the fermentation characteristics of 23 yeasts from the bakery, beer, wine, and spirits industries were investigated in sweet dough (14% added sucrose w/w dm flour). Significant differences in invertase activity, sugar consumption (0.78-5.25% w/w dm flour), and metabolite (0.33-3.01% CO2; 0.20-1.26% ethanol; 0.17-0.80% glycerol; 0.09-0.29% organic acids) and volatile compound production were observed. A strong positive correlation (R2 = 0.76, p < 0.001) between sugar consumption and metabolite production was measured. Several non-conventional yeast strains produced more positive aroma compounds and fewer off-flavors than the reference baker's yeast. This study shows the potential of non-conventional yeast strains in sweet dough.

Application of multivariate data analysis for food quality investigations: An example-based review.

These days, large multivariate data sets are common in the food research area. This is not surprising as food quality, which is important for consumers, and its changes are the result of a complex interplay of multiple compounds and reactions. In order to comprehensively extract information from these data sets, proper data analysis tools should be applied. The application of multivariate data analysis (MVDA) is therefore highly recommended. However, at present the use of MVDA for food quality investigations is not yet fully explored. This paper focusses on a number of MVDA methods (PCA (Principal Component Analysis), PLS (Partial Least Squares Regression), PARAFAC (Parallel Factor Analysis) and ASCA (ANOVA Simultaneous Component Analysis)) useful for food quality investigations. The terminology, main steps and the theoretical basis of each method will be explained. As this is an example-based review, each method was applied on the same experimental data set to give the reader an idea about each selected MVDA method and to make a comparison between the outcomes. Numerous MVDA methods are available in literature. Which method to select depends on the data set and objective. PCA should be the first choice for data exploration of two-dimensional data. For predictive purposes, PLS is the most appropriate method. Given an underlying experimental design, ASCA takes into account both the relation between the different variables and the design factors. In case of a multi-way data set, PARAFAC can be used for data exploration. While these methods have already proven their value in research, there is a need to further explore their potential to investigate the complex interplay of compounds and reactions contributing to food quality. With this work we would like to encourage food scientists with no or limited knowledge of MVDA to get some first insights into the selected methods.

Antinutrient to mineral molar ratios of raw common beans and their rapid prediction using near-infrared spectroscopy.

The presence of antinutrients in common beans negatively affects mineral bioavailability. Therefore, this study aimed to predict the antinutrient to mineral molar ratios (proxy-indicators of in vitro mineral bioavailability) of a wide range of raw bean types, using near-infrared (NIR) spectroscopy. Iron, zinc, phytate and tannin concentrations and, antinutrient to mineral molar ratios were determined. Next, model calibration using NIR spectra from milled beans was performed. This entailed wavelength selection, pre-processing and partial least squares regression. Bean type had a significant effect on tannin content. The average values of phytate to iron (Phy:Fe), phytate to zinc (Phy:Zn), tannins to iron (Tan:Fe) and phytate and tannins to iron (Phy + Tan:Fe) MRs were 27.6, 61.7, 16.0 and 43.6, respectively. With determination coefficients for test set prediction above 75%, the PLS-R models for Phy:Zn, Tan:Fe and Phy + Tan:Fe molar ratios are useful for screening purposes.

Reaction pathways and factors influencing nonenzymatic browning in shelf-stable fruit juices during storage.

The occurrence of nonenzymaticbrowning in fruit juices during storage is a major quality defect. It negatively affects consumer acceptance and consumption behavior and determines the shelf-life of these products. Although nonenzymatic browning of fruit juices has been the subject of research for a long time, the exact mechanism of the nonenzymatic browning reactions is not yet completely understood. This review paper aims to give an overview of the compounds and reactions playing a key role in nonenzymatic browning during the storage of fruit juices. The chemistry of the plausible reactions and their relative importance will be discussed. To better understand nonenzymatic browning, factors affecting these reactions will be reviewed and several strategies and methods to evaluate color changes and browning will be discussed. Nonenzymatic browning involves three main reactions: ascorbic acid degradation, acid-catalyzed sugar degradation, and Maillard-associated reactions. The most important NEB pathway depends on the matrix. Nonenzymatic browning is affected by many factors, such as the juice composition, the pH, the oxygen availability (packaging material), and the storage conditions. Nonenzymatic browning can thus be considered as a complex problem. To characterize color changes and browning and obtain insight into the browning mechanism of fruit juices, food scientists applied several approaches and strategies. These included the use of model systems with/without the addition of labeled compound and real systems as well as advanced analytical methods.

The Impact of Drying and Rehydration on the Structural Properties and Quality Attributes of Pre-Cooked Dried Beans.

Fresh common beans can be made 'instant' to produce fast-cooking beans by first soaking and cooking the beans before drying to create a shelf-stable product that can be rehydrated at the time of use. This study investigated the interplay between the drying process (air, vacuum and freeze drying), the microstructure and functional attributes of rehydrated pre-cooked beans. The microscopic study revealed that the three different drying techniques resulted in distinctly different microstructures, with the freeze drying process resulting in highly porous materials, while the air- and vacuum-dried samples underwent shrinkage. Additionally, the rehydration behavior (modeled using empirical and diffusion models) demonstrates that the high rehydration rate of freeze-dried beans is due to capillarity, while rehydration, in the case of air- and vacuum-dried beans, is primarily diffusion-controlled. Irrespective of the drying technique, the high rehydration capacity supports little to no structural collapse or damage to the cell walls. The color and texture of the rehydrated beans did not differ greatly from those of freshly cooked beans. The total peak area of the volatiles of rehydrated beans was significantly reduced by the drying process, but volatiles characteristic of the cooked bean aroma were retained. This new understanding is beneficial in tailoring the functional properties of pre-cooked dry convenient beans requiring short preparation times.

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.

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.

Insight into non-enzymatic browning of shelf-stable orange juice during storage: A fractionation and kinetic approach.

BACKGROUND: Non-enzymatic browning (NEB) is the main quality defect in shelf-stable orange juice and other fruit juices during storage. Previous studies on NEB focused solely on the soluble fraction of orange juice, regardless of the fact that both soluble and insoluble fractions turn brown during extended storage. Clear evidence of the relative contribution of both fractions to NEB is currently lacking in the literature. This study investigated the contribution of the soluble and insoluble fractions of orange juice, which were obtained by centrifugation and ethanol precipitation, to NEB during storage. Changes in different NEB-related attributes, such as ascorbic acid (AA) degradation, and the browning index (BI), were quantified and kinetically modeled. RESULTS: Evaluation of color during storage showed that the orange juice and the soluble compound-containing fractions turned brown whereas the insoluble fractions did not. The soluble compound-containing fractions showed exactly the same browning behavior with storage as the plain orange juice. Based on the kinetic parameters obtained, the degradation of AA, the hydrolysis of sucrose, the increase in the glucose and fructose content, and the formation of furfural and 5-hydroxymethylfurfural during storage were similar for the plain orange juice and the soluble compound-containing fractions. CONCLUSION: This work provided evidence that the soluble fraction of orange juice plays the major role in NEB, unlike the insoluble fraction, which seems to make no contribution. Results from this work also demonstrate the potential use of the soluble fraction as an orange-juice-based model system of reduced complexity that can be used for the further investigation of NEB processes. © 2020 Society of Chemical Industry.

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.

Combining untargeted, targeted and sensory data to investigate the impact of storage on food volatiles: A case study on strawberry juice.

An integrated science-based approach, combining analytical and sensorial data and different data analysis methods, proved successful to study the impact of storage time, storage temperature and oxygen availability on strawberry juice volatiles and allowed to get a multi-perspective view on these changes. An untargeted GC-MS approach showed that the volatile fraction of shelf-stable strawberry juice clearly changed during ambient storage and that oxygen availability (linked to the type of bottle) had a limited effect. To gain further insight, several characteristic aroma compounds were quantified during storage at ambient (20 °C) and accelerated (28-42 °C) temperatures, kinetic parameters were estimated and odour activity values were calculated. The kinetic parameters showed that all characteristic aroma compounds changed significantly during storage at all temperatures and that the rate of change in some compounds was accelerated by storage at higher temperatures. The observed changes in strawberry juice volatiles caused sensorial differences between non-stored and 20 °C stored samples as shown by the sensory evaluations and odour activity values.

The potential of kiwifruit puree as a clean label ingredient to stabilize high pressure pasteurized cloudy apple juice during storage.

In the fruit juice industry, high pressure (HP) processing has become a commercial success. However, enzymatic browning, cloud loss, and flavor changes during storage remain challenges. The aim of this study is to combine kiwifruit puree and HP pasteurization (600 MPa/3 min) to stabilize cloudy apple juice during storage at 4 °C. A wide range of targeted and untargeted quality characteristics was evaluated using a multivariate approach. Due to high ascorbic acid content and high viscosity, kiwifruit puree allowed to prevent enzymatic browning and phase separation of an apple-kiwifruit mixed juice. Besides, no clear changes in organic acids, viscosity, and particle size distribution were detected in mixed juice during storage. Sucrose of apple and mixed juices decreased with glucose and fructose increasing during storage. The volatile changes of both juices behaved similar, mainly esters being degraded. Sensory evaluation demonstrated consumer preferred the aroma of mixed juice compared to apple juice.

Shelf-life dating of shelf-stable strawberry juice based on survival analysis of consumer acceptance information.

BACKGROUND: Accurate shelf-life dating of food products is crucial for consumers and industries. Therefore, in this study we applied a science-based approach for shelf-life assessment, including accelerated shelf-life testing (ASLT), acceptability testing and the screening of analytical attributes for fast shelf-life predictions. Shelf-stable strawberry juice was selected as a case study. RESULTS: Ambient storage (20 °C) had no effect on the aroma-based acceptance of strawberry juice. The colour-based acceptability decreased during storage under ambient and accelerated (28-42 °C) conditions. The application of survival analysis showed that the colour-based shelf-life was reached in the early stages of storage (≤11 weeks) and that the shelf-life was shortened at higher temperatures. None of the selected attributes (a* and ΔE* value, anthocyanin and ascorbic acid content) is an ideal analytical marker for shelf-life predictions in the investigated temperature range (20-42 °C). Nevertheless, an overall analytical cut-off value over the whole temperature range can be selected. CONCLUSIONS: Colour changes of strawberry juice during storage are shelf-life limiting. Combining ASLT with acceptability testing allowed to gain faster insight into the change in colour-based acceptability and to perform shelf-life predictions relying on scientific data. An analytical marker is a convenient tool for shelf-life predictions in the context of ASLT. © 2017 Society of Chemical Industry.

Role of structural barriers in the in vitro bioaccessibility of anthocyanins in comparison with carotenoids.

Although natural structural barriers are factors limiting nutrient bioaccessibility, their specific role in anthocyanin bioaccessibility is still unknown. To better understand how natural barriers govern bioactive compound bioaccessibility, an experimental approach comparing anthocyanins and carotenoids was designed, using a single plant matrix. Initial results revealed increased anthocyanin bioaccessibility in masticated black carrot. To explain this observation, samples with increasing levels of bioencapsulation (free-compound, homogenized-puree, puree) were examined. While carotenoid bioaccessibility was inversely proportional to the level of bioencapsulation, barrier disruption did not increase anthocyanin bioaccessibility. This means that mechanical processing is of particular importance in the case of carotenoid bioaccessibility. While micelle incorporation is the limiting factor for carotenoid bioaccessibility, anthocyanin degradation under alkaline conditions in the gastrointestinal tract dominates. In the absence of structural barriers, anthocyanin bioaccessibility is greater than that of carotenoids.

Relative importance and interactions of furan precursors in sterilised, vegetable-based food systems.

Mitigation strategies aimed at an intervention in the reaction pathways for furan formation (e.g., by adjusting precursor concentrations) might offer an additional route for furan reduction in sterilised, vegetable-based foods, without adverse effects on other food safety or quality attributes. As a first step towards product reformulation, the aim of the present study was to determine the relative importance and interactions of possible furan precursors in these types of foods. Based on an I-optimal experimental design, potato purée (naturally low in furan precursors) was spiked with known amounts of sugars, ascorbic acid, olive oil and ß-carotene, and subjected to a thermal sterilisation. Significant correlations were observed between furan concentrations after thermal treatment and starting concentrations of ascorbic acid and monosaccharides (i.e., fructose and glucose). Ascorbic acid had a clear furan-reducing effect as an antioxidant by protecting (polyunsaturated) fatty acids against oxidative degradation. Fructose and glucose were the main precursors, which can most probably be attributed to their high, but realistic, concentrations in the product. The contributions of fatty acids and ß-carotene were strongly dependent on redox interactions with other food constituents. In the same potato purées, only low concentrations (0-2 ng g(-1) purée) of 2-methylfuran were detected, indicating that the direct importance of the spiked food constituents as a precursor for methylfuran formation was rather small. Based on the results of this study, reducing the amount of monosaccharides or adjusting the redox conditions of the matrix are suggested as two possible approaches for furan mitigation on the product side.

Furan formation during storage and reheating of sterilised vegetable purées.

To this day, research for furan mitigation has mostly targeted the levels of food production and handling of prepared foods by the consumer. However, part of the furan concentrations found in commercially available food products might originate from chemical deterioration reactions during storage. A range of individual vegetable purées was stored at two different temperatures to investigate the effects of storage on the furan concentrations of shelf-stable, vegetable-based foods. After 5 months of storage at 35°C (temperature-abuse conditions), a general increase in furan concentrations was observed. The furan formation during storage could be reduced by storing the vegetable purées at a refrigerated temperature of 4°C, at which the furan concentrations remained approximately constant for at least 5 months. Following storage, the vegetable purées were briefly reheated to 90°C to simulate the effect of the final preparation step before consumption. Contrary to storage, furan concentrations decreased as a result of evaporative losses. Both refrigerated storage and the reheating step prior to consumption showed the potential of mitigation measures for furan formation in vegetable-based foods (e.g. canned vegetables, ready-to-eat soups, sauces or baby foods). Next to furan, the vegetable purées were analysed for 2- and 3-methylfuran. Tomato was very susceptible to the formation of both alkylated derivatives of furan, as opposed to the other vegetables in this study. Methylfuran concentrations rapidly decreased during storage, which was contrary to the results observed for furan.