Aquaphotomics-Exploring Water Molecular Systems in Nature.

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Aquaphotomics Monitoring of Lettuce Freshness during Cold Storage.

Fresh-cut leafy vegetables are one of the most perishable products because they readily deteriorate in quality even during cold storage and have a relatively short shelf life. Since these products are in high demand, methods for rigorous quality control and estimation of freshness that are rapid and non-destructive would be highly desirable. The objective of the present research was to develop a rapid, non-destructive near-infrared spectroscopy (NIRS)-based method for the evaluation of changes during cold storage of lettuce using an aquaphotomics approach to monitor the water molecular structure in lettuce leaves. The reference measurements showed that after 6 days of dark, cold storage, the weight and water activity of lettuce leaves decreased and ß-carotene decreased, while chlorophylls slightly increased. Aquaphotomics characterization showed large differences in the lettuce leaves' spectra depending on their growth zone. Difference spectra, principal component analysis (PCA) and linear discriminant analysis (LDA) confirmed the differences in the inner and outer leaves and revealed that spectra change as a function of storage time. Partial least squares regression (PLSR) allowed the prediction of the time spent in storage with a coefficient of determination of R2 = 0.80 and standard error of RMSE = 0.77 days for inner, and R2 = 0.86 and RMSE = 0.66 days for outer leaves, respectively. The following water absorbance bands were found to provide the most information in the spectra: 1348, 1360, 1373, 1385, 1391, 1410, 1416, 1422, 1441, 1447, 1453, 1466, 1472, 1490, 1503, 1515, 1521, 1534 and 1571 nm. They were further used as water matrix coordinates (WAMACs) to define the water spectral patterns (WASPs) of lettuce leaves. The WASPs of leaves served to succinctly describe the state of lettuces during storage. The changes in WASPs during storage reveled moisture loss, damage to cell walls and expulsion of intracellular water, as well as loss of free and weakly hydrogen-bonded water, all leading to a loss of juiciness. The WASPs also showed that damage stimulated the defense mechanisms and production of vitamin C. The leaves at the end of the storage period were characterized by water strongly bound to collapsed structural elements of leaf tissues, mainly cellulose, leading to a loss of firmness that was more pronounced in the outer leaves. All of this information was reflected in the changes of absorbance in the identified WAMACs, showing that the water molecular structure of lettuce leaves accurately reflects the state of the lettuce during storage and that WASPs can be used as a multidimensional biomarker to monitor changes during storage.

Adaptive Spectral Model for abnormality detection based on physiological status monitoring of dairy cows.

This research study developed milk spectral data-driven approach, called Adaptive Spectral Model for Abnormality Detection - ASMAD, for detection of physiological abnormalities of individual dairy cows. The algorithm is based on the logic that milk spectra of each individual cow is highly animal-specific, which means it could be used as a respective individual marker for identification. When the algorithm fails to recognize the milk spectra as coming from a certain animal, instead of being treated as a mistake, this outcome is accepted as a deviation of the respective individual marker, and a potential indicator of abnormal physiological state. For the purpose of ASMAD development, near infrared spectra of milk of seven dairy cows have been collected daily during 1-year period. The abnormality detection model is built using supervised recognition method Soft Independent Modeling of Class Analogies - SIMCA, and optimized with respect to spectral pre-processing, choice of the wavelength region and size of the time-window when developing the adaptive model. The sensitivity and specificity of ASMAD were dependent on the animal, and in the ranges 40.00-64.29% and 87.23-98.86%, respectively. Considering significant level of day-to-day spectral variation and multitude of physiological and environmental factors influence on milk constituents and spectra, these results represent a significant potential for creating a health-status monitoring and detection of abnormal physiological states in dairy animals. The adaptive modeling based on the time series of spectral data collected from the individual organism utilized in this work for monitoring physiological status and abnormality detection in dairy cows, has a good potential to be used for similar purposes in other animals and humans.

Aquaphotomics monitoring of strawberry fruit during cold storage - A comparison of two cooling systems.

The objective of this study was to use aquaphotomics and near-infrared (NIR) spectroscopy to follow the changes in strawberries during cold storage in the refrigerator with an electric field generator (supercooling fridge, SCF) and without it (control fridge, CF). The NIR spectra of strawberries stored in these refrigerators were collected over the course of 15 days using a portable mini spectrometer and their weight was measured daily. The spectral data in the region of the first overtone of water (1,300-1,600 nm) were analyzed using aquaphotomics multivariate analysis. The results showed a decrease in weight loss of strawberries, but the loss of weight was significantly lower in SCF, compared to the CF. The reduction of weight loss due to exposure to an electric field was comparable to the use of coatings. The aquaphotomics analysis showed that the NIR spectra adequately captured changes in the fruit over the storage period, and that it is possible to predict how long the fruit spent in storage, regardless of the storage type. During aquaphotomics analysis, 19 water absorbance bands were found to be consistently repeating and to have importance for the description of changes in strawberries during cold storage. These bands defined the water spectral pattern (WASP), multidimensional biomarker that was used for the description of the state and dynamics of water in strawberries during time spent in storage. Comparison of WASPs of strawberries in CF and SCF showed that exposure to an electric field leads to a delay in ripening by around 3 days. This was evidenced by the increased amount of structural, strongly bound water and vapor-like trapped water in the strawberries stored in SCF. This particular state of water in strawberries stored in SCF was related to the hardening of the strawberry skin and prevention of moisture loss, in agreement with the results of significantly decreased weight loss.

Aquaphotomic Study of Effects of Different Mixing Waters on the Properties of Cement Mortar.

The mixing water used for cement concrete has a significant effect on the physical properties of the material after hardening; however, other than the upper limit for the mixed impurities, not enough consideration has been given to the functions and characteristics of water at the molecular level. In this study, we investigated the effect of four different types of water (two spring-, mineral waters, tap water and distilled water) on the drying shrinkage of the hardened cement by comparing the material properties of the concrete specimens and analyzing the molecular structure of the water and cement mortar using aquaphotomics. The near infrared (NIR) spectra of waters used for mixing were acquired in the transmittance mode using a high-precision, high-accuracy benchtop spectrometer in the range of 400-2500 nm, with the 0.5 nm step. The NIR spectra of cement paste and mortar were measured in 6.2 nm increments in the wavelength range of 950 nm to 1650 nm using a portable spectrometer. The measurements of cement paste and mortar were performed on Day 0 (immediately after mixing, cement paste), 1 day, 3 days, 7 days, and 28 days after mixing (cement mortar). The spectral data were analyzed according to the aquaphotomics' multivariate analysis protocol, which involved exploration of raw and preprocessed spectra, exploratory analysis, discriminating analysis and aquagrams. The results of the aquaphotomics' analysis were interpreted together with the results of thermal and drying shrinkage measurements. Together, the findings clearly demonstrated that the thermal and drying shrinkage properties of the hardened cement material differed depending on the water used. Better mechanical properties were found to be a result of using mineral waters for cement mixing despite minute differences in the chemical content. In addition, the aquaphotomic characterization of the molecular structure of waters and cement mortar during the initial hydration reaction demonstrated the possibility to predict the characteristics of hardened cement at a very early stage. This provided the rationale to propose a novel evaluation method based on aquaphotomics for non-invasive evaluation and monitoring of cement mortar.

Pilot Aquaphotomic Study of the Effects of Audible Sound on Water Molecular Structure.

Sound affects the medium it propagates through and studies on biological systems have shown various properties arising from this phenomenon. As a compressible media and a "collective mirror", water is influenced by all internal and external influences, changing its molecular structure accordingly. The water molecular structure and its changes can be observed as a whole by measuring its electromagnetic (EMG) spectrum. Using near-infrared spectroscopy and aquaphotomics, this pilot study aimed to better describe and understand the sound-water interaction. Results on purified and mineral waters reported similar effects from the applied 432 Hz and 440 Hz frequency sound, where significant reduction in spectral variations and increased stability in water were shown after the sound perturbation. In general, the sound rearranged the initial water molecular conformations, changing the samples' properties by increasing strongly bound, ice-like water and decreasing small water clusters and solvation shells. Even though there was only 8 Hz difference in applied sound frequencies, the change of absorbance at water absorbance bands was specific for each frequency and also water-type-dependent. This also means that sound could be effectively used as a perturbation tool together with spectroscopy to identify the type of bio, or aqueous, samples being tested, as well as to identify and even change water functionality.

Aquaphotomics for monitoring of groundwater using short-wavelength near-infrared spectroscopy.

Water spectrum of any aqueous system contains information about OH covalent and hydrogen bonds that are highly influenced by the environment and the rest of the molecules in the system. When aquaphotomics is used to analyze the water near infrared (NIR) spectra, the information about the water molecular structure can be obtained as a function of internal and external factors. The objective of this research is to apply aquaphotomics analysis to evaluate different groundwaters by using their NIR unique spectral pattern, robust to external influences of temperature and humidity, that can potentially be used for water type identification and screening practice. Two groundwaters obtained at different depths and their mixture, differing in mineral content and molecular structure were monitored on a daily basis using portable visible/NIR (vis/NIR) spectrometer during three consecutive years. The spectra were pre-processed by smoothing and multiplicative scatter correction (MSC) to remove noise and baseline effects. Results showed that NIR spectral patterns of groundwater samples were affected by changes in environmental factors - temperature, humidity, time and others. The water absorbance bands which are highly influenced by humidity and temperature in short wavelength NIR region were identified. Their avoidance resulted in obtaining consistent spectral patterns during the entire monitoring period, unique for each groundwater, that can be used as its fingerprint and monitored over time. Consistency and uniqueness of the spectral pattern for each groundwater provide a potential to use the deviation of spectral pattern as an indicator of changes in the water. These results confirm that vis/NIR spectral pattern can be used as an integrative marker of water status, stable over time, providing the basis for an efficient cost-effective method for monitoring of water functionality.

Identification of Stingless Bee Honey Adulteration Using Visible-Near Infrared Spectroscopy Combined with Aquaphotomics.

Honey is a natural product that is considered globally one of the most widely important foods. Various studies on authenticity detection of honey have been fulfilled using visible and near-infrared (Vis-NIR) spectroscopy techniques. However, there are limited studies on stingless bee honey (SBH) despite the increase of market demand for this food product. The objective of this work was to present the potential of Vis-NIR absorbance spectroscopy for profiling, classifying, and quantifying the adulterated SBH. The SBH sample was mixed with various percentages (10−90%) of adulterants, including distilled water, apple cider vinegar, and high fructose syrup. The results showed that the region at 400−1100 nm that is related to the color and water properties of the samples was effective to discriminate and quantify the adulterated SBH. By applying the principal component analysis (PCA) on adulterants and honey samples, the PCA score plot revealed the classification of the adulterants and adulterated SBHs. A partial least squares regression (PLSR) model was developed to quantify the contamination level in the SBH samples. The general PLSR model with the highest coefficient of determination and lowest root means square error of cross-validation (RCV2=0.96 and RMSECV=5.88 %) was acquired. The aquaphotomics analysis of adulteration in SBH with the three adulterants utilizing the short-wavelength NIR region (800−1100 nm) was presented. The structural changes of SBH due to adulteration were described in terms of the changes in the water molecular matrix, and the aquagrams were used to visualize the results. It was revealed that the integration of NIR spectroscopy with aquaphotomics could be used to detect the water molecular structures in the adulterated SBH.

Aquaphotomics Research of Cold Stress in Soybean Cultivars with Different Stress Tolerance Ability: Early Detection of Cold Stress Response.

The development of non-destructive methods for early detection of cold stress of plants and the identification of cold-tolerant cultivars is highly needed in crop breeding programs. Current methods are either destructive, time-consuming or imprecise. In this study, soybean leaves' spectra were acquired in the near infrared (NIR) range (588-1025 nm) from five cultivars genetically engineered to have different levels of cold stress tolerance. The spectra were acquired at the optimal growing temperature 27 °C and when the temperature was decreased to 22 °C. In this paper, we report the results of the aquaphotomics analysis performed with the objective of understanding the role of the water molecular system in the early cold stress response of all cultivars. The raw spectra and the results of Principal Component Analysis, Soft Independent Modeling of Class Analogies and aquagrams showed consistent evidence of huge differences in the NIR spectral profiles of all cultivars under normal and mild cold stress conditions. The SIMCA discrimination between the plants before and after stress was achieved with 100% accuracy. The interpretation of spectral patterns before and after cold stress revealed major changes in the water molecular structure of the soybean leaves, altered carbohydrate and oxidative metabolism. Specific water molecular structures in the leaves of soybean cultivars were found to be highly sensitive to the temperature, showing their crucial role in the cold stress response. The results also indicated the existence of differences in the cold stress response of different cultivars, which will be a topic of further research.

Analysing the water spectral pattern by near-infrared spectroscopy and chemometrics as a dynamic multidimensional biomarker in preservation: rice germ storage monitoring.

Water activity is an important phenomenon not yet explained in terms of water molecular structure. This paper aims to find the relationship between the water activity and water molecular structure of the rice germ, based on its spectral pattern which can be measured using non-destructive technology. Aquaphotomics near-infrared spectroscopy was used to study rice germ stored at different levels of water activity and atmosphere. The findings show that state of the rice germ is governed by the water activity upon storage, which is defined by the structure of water within germ matrix. The structure of water can be described solely by the absorbance spectral pattern at the following absorbance bands: proton hydrates, hydration shells and water vapor (1364, 1375 and 1382 nm), trapped water (1392 nm), free water (1410 nm), hydration water (1425 nm), adsorbed water (1455 nm), non-bonded hydroxyl (1436 nm) and bound water (1520 nm).

Water Spectral Patterns Reveals Similarities and Differences in Rice Germination and Induced Degenerated Callus Development.

In vivo monitoring of rice (Oryza sativa L.) seed germination and seedling growth under general conditions in closed Petri dishes containing agar base medium at room temperature (temperature = 24.5 ± 1 °C, relative humidity = 76 ± 7% (average ± standard deviation)), and induced degenerated callus formation with plant growth regulator, were performed using short-wavelength near-infrared spectroscopy and aquaphotomics over A period of 26 days. The results of spectral analysis suggest changes in water absorbances due to the production of common metabolites, as well as increases in biomass and the sizes of the samples. Quantitative models built to predict the day of the development provided better accuracy for rice seedlings growth compared to callus formation. Eight common water bands were identified as presenting prominent changes in the absorbance pattern. The water matrix of only rice seedlings showed three developmental stages: firstly expressing a predominantly weakly hydrogen-bonded state, then a more strongly hydrogen-bonded state, and then, again, a weakly hydrogen-bonded state at the end. In rice callus induction and proliferation, no similar change in water absorbance pattern was observed. The presented findings indicate the potential of aquaphotomics for the in vivo detection of degeneration in cell development.

Relating Near-Infrared Light Path-Length Modifications to the Water Content of Scattering Media in Near-Infrared Spectroscopy: Toward a New Bouguer-Beer-Lambert Law.

In near-infrared spectroscopy (NIRS), the linear relationship between absorbance and an absorbing compound concentration has been strictly defined by the Bouguer-Beer-Lambert law only for the case of transmission measurements of nonscattering media. However, various quantitative calibrations have been successfully built both on reflectance measurements and for scattering media. Although the lack of linearity for scattering media has been observed experimentally, the sound multivariate statistics and signal processing involved in chemometrics have allowed us to overcome this problem in most cases. However, in the case of samples with varying water content, important modifications of scattering levels still make calibrations difficult to build due to nonlinearities. Moreover, even when calibration procedures are successfully developed, many preprocessing methods used do not guarantee correct spectroscopic assignments (in the sense of a pure chemical absorbance). In particular, this may prevent correct modeling and interpretation of the structure of water. In this study, dynamic near-infrared spectra acquired during a drying process allow the study of the physical effects of water content variations, with a focus on the first overtone OH absorbance region. A model sample consisting of aluminum pellets mixed with water allowed us to study this specifically, without any other absorbing interaction terms related to the dry mass-absorbing constituents. A new formulation of the Bouguer-Beer-Lambert law is proposed, by expressing path length as a power function of water content. Through this new formulation, it is shown that a better and simpler prediction model of water content may be developed, with more precise and accurate identification of water absorbance bands.

Author Correction: Water molecular structure underpins extreme desiccation tolerance of the resurrection plant Haberlea rhodopensis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Novel Tool for Visualization of Water Molecular Structure and Its Changes, Expressed on the Scale of Temperature Influence.

Aquaphotomics utilizes water-light interaction for in-depth exploration of water, its structure and role in aqueous and biologic systems. The aquagram, a major analytical tool of aquaphotomics, allows comparison of water molecular structures of different samples by comparing their respective absorbance spectral patterns. Temperature is the strongest perturbation of water changing almost all water species. To better interpret and understand spectral patterns, the objective of this work was to develop a novel, temperature-scaled aquagram that provides standardized information about changes in water molecular structure caused by solutes, with its effects translated to those which would have been caused by respective temperature changes. NIR spectra of Milli-Q water in the temperature range of 20-70 °C and aqueous solutions of potassium chloride in concentration range of 1 to 1000 mM were recorded to demonstrate the applicability of the proposed novel tool. The obtained results presented the influence of salt on the water molecular structure expressed as the equivalent effect of temperature in degrees of Celsius. The temperature-based aquagrams showed the well-known structure breaking and structure making effects of salts on water spectral pattern, for the first time presented in the terms of temperature influence on pure water. This new method enables comparison of spectral patterns providing a universal tool for evaluation of various bio-aqueous systems which can provide better insight into the system's functionality.

Real-Time Monitoring of Yogurt Fermentation Process by Aquaphotomics Near-Infrared Spectroscopy.

Automated quality control could have a substantial economic impact on the dairy industry. At present, monitoring of yogurt production is performed by sampling for microbiological and physicochemical measurements. In this study, Near-Infrared Spectroscopy (NIRS) is proposed for non-invasive automated control of yogurt production and better understanding of lactic acid bacteria (LAB) fermentation. UHT (ultra-high temperature) sterilized milk was inoculated with Bulgarian yogurt and placed into a quartz cuvette (1 mm pathlength) and test-tubes. Yogurt absorbance spectra (830-2500 nm) were acquired every 15 min, and pH, in the respective test-tubes, was measured every 30 min, during 8 h of fermentation. Spectral data showed substantial baseline and slope changes with acidification. These variations corresponded to respective features of the microbiological growth curve showing water structural changes, protein denaturation, and coagulation of milk. Moving Window Principal Component Analysis (MWPCA) was applied in the spectral range of 954-1880 nm to detect absorbance bands where most variations in the loading curves were caused by LAB fermentation. Characteristic wavelength regions related to the observed physical and multiple chemical changes were identified. The results proved that NIRS is a valuable tool for real-time monitoring and better understanding of the yogurt fermentation process.

Aquaphotomics approach for monitoring different steps of purification process in water treatment systems.

Currently, the water quality assessment is based on determination of a range of physico-chemical, and microbiological parameters, which is costly, time-consuming and requires multiple tests. Aquaphotomics strategy for water monitoring is based on the principle that the spectra of water are sensitive to all the chemical and physical perturbations, and as such reflect the state of water. In this study, aquaphotomics approach was applied on the near infrared spectra of water samples which underwent different filtration treatments performed by a commercially available water treatment system intended for the household use. The applicability of this approach was demonstrated using difference spectra, multivariate classification analysis and by visualizing the water spectral pattern in aquagrams. The results consistently showed that water samples produced by different filtration treatments have distinctive spectral patterns which characterize the state of water molecular network, and can be used for identification of the respective treatment, tracking efficiency of the treatment and monitoring the performance of water treatment systems.

Aquaphotomics-From Innovative Knowledge to Integrative Platform in Science and Technology.

Aquaphotomics is a young scientific discipline based on innovative knowledge of water molecular network, which as an intrinsic part of every aqueous system is being shaped by all of its components and the properties of the environment. With a high capacity for hydrogen bonding, water molecules are extremely sensitive to any changes the system undergoes. In highly aqueous systems-especially biological-water is the most abundant molecule. Minute changes in system elements or surroundings affect multitude of water molecules, causing rearrangements of water molecular network. Using light of various frequencies as a probe, the specifics of water structure can be extracted from the water spectrum, indirectly providing information about all the internal and external elements influencing the system. The water spectral pattern hence becomes an integrative descriptor of the system state. Aquaphotomics and the new knowledge of water originated from the field of near infrared spectroscopy. This technique resulted in significant findings about water structure-function relationships in various systems contributing to a better understanding of basic life phenomena. From this foundation, aquaphotomics started integration with other disciplines into systematized science from which a variety of applications ensued. This review will present the basics of this emerging science and its technological potential.

Water molecular structure underpins extreme desiccation tolerance of the resurrection plant Haberlea rhodopensis.

Haberlea rhodopensis is a resurrection plant with an extremely high desiccation tolerance. Even after long periods of almost full desiccation, its physiological functions are recovered shortly upon re-watering. In order to identify physiological strategies which contribute to its remarkable drought stress tolerance we used near infrared spectroscopy to investigate the state of water in the leaves of this plant and compared it to its relative, non-resurrection plant species Deinostigma eberhardtii. Here we show, using a novel aquaphotomics spectral analysis, that H. rhodopensis performs a dynamic regulation of water molecular structure during dehydration directed at drastic decrease of free water molecules, increase of water molecules with 4 hydrogen bonds, and a massive accumulation of water dimers in the full desiccation stage. Our findings suggest that changes in water structure mirror the changes in major metabolites and antioxidants which together constitute a robust defense system underlying the desiccation tolerance of the resurrection plant, while the water dimer may hold special importance for the "drying without dying" ability.

Essentials of Aquaphotomics and Its Chemometrics Approaches.

Aquaphotomics is a novel scientific discipline involving the study of water and aqueous systems. Using light-water interaction, it aims to extract information about the structure of water, composed of many different water molecular conformations using their absorbance bands. In aquaphotomics analysis, specific water structures (presented as water absorbance patterns) are related to their resulting functions in the aqueous systems studied, thereby building an aquaphotome-a database of water absorbance bands and patterns correlating specific water structures to their specific functions. Light-water interaction spectroscopic methods produce complex multidimensional spectral data, which require data processing and analysis to extract hidden information about the structure of water presented by its absorbance bands. The process of extracting information from water spectra in aquaphotomics requires a field-specific approach. It starts with an appropriate experimental design and execution to ensure high-quality spectral signals, followed by a multitude of spectral analysis, preprocessing and chemometrics methods to remove unwanted influences and extract water absorbance spectral pattern related to the perturbation of interest through the identification of activated water absorbance bands found among the common, consistently repeating and highly influential variables in all analytical models. The objective of this paper is to introduce the field of aquaphotomics and describe aquaphotomics multivariate analysis methodology developed during the last decade. Through a worked-out example of analysis of potassium chloride solutions supported by similar approaches from the existing aquaphotomics literature, the provided instruction should give enough information about aquaphotomics analysis i.e. to design and perform the experiment and data analysis as well as to represent water absorbance spectral pattern using various forms of aquagrams-specifically designed aquaphotomics graphs. The explained methodology is derived from analysis of near infrared spectral data of aqueous systems and will offer a useful and new tool for extracting data from informationally rich water spectra in any region. It is the hope of the authors that with this new tool at the disposal of scientists and chemometricians, pharmaceutical and biomedical spectroscopy will substantially progress beyond its state-of-the-art applications.