Applications in Food Sciences

By Atta-ur-Rahman and M. Iqbal Choudhary

Applications of NMR Spectroscopy is a book series devoted to publishing the latest advances in the applications of nuclear magnetic resonance (NMR) spectroscopy in various fields of organic chemistry, biochemistry, health and agriculture.

The fourth volume of the series features several reviews focusing on NMR spectroscopic techniques in food sciences. Readers will find references on methods used to test food quality, food color analysis, the role of Tannins in wine taste as well as NMR studies on lipid oxidation and large protein complexes.

• Language: English
• Publisher: Bentham Science Publishers
• Release date: Apr 12, 2016
• ISBN: 9781681081434

Atta-ur-Rahman

Atta-ur-Rahman, Professor Emeritus, International Center for Chemical and Biological Sciences (H. E. J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research), University of Karachi, Pakistan, was the Pakistan Federal Minister for Science and Technology (2000-2002), Federal Minister of Education (2002), and Chairman of the Higher Education Commission with the status of a Federal Minister from 2002-2008. He is a Fellow of the Royal Society of London (FRS) and an UNESCO Science Laureate. He is a leading scientist with more than 1283 publications in several fields of organic chemistry.

Book preview

PREFACE

Volume 4 of the ebook series Applications of NMR Spectroscopy is mainly focussed on the use of NMR spectroscopy as a key method for food and beverage analysis and characterization. Food presents a complex mixture of many different compounds with different chemical structures, concentrations, solubilities, properties, and nutritional values. Any change in food composition can lead to a gross change in its quality, taste, and calorific value. The present book is based on six well-written reviews, each focussing on a unique set of applications of NMR spectroscopy in food analysis. In each of these articles, the optimum use of this powerful technique with reference to the field of food science is introduced in an easy to understand manner. The real strength of the book is its highly practical approach in describing both the concepts and applications of NMR spectroscopy for various purposes.

Review contributed by Melado-Herreros et al provides practical applications of several NMR techniques used in the multi-component analysis of food samples. Apart from introducing the concept of multi-component analysis with reference to food items, the authors have also explained the use of NMR techniques such as ¹H HR-MAS (High Resolution Magic Angle Spinning) for solid state analysis, MRI (Magnetic Resonance Imaging) and CSI (Chemical Shift Imaging) for physiological analysis of fruits and vegetables. The NMR technique called relaxometry mapping (relaxation time measurement) gives important information about water compartmentalization, structure and integrity.

Lipid oxidation/peroxidation is a key issue in the storage, and processing of edible oils and oil containing and oil-based food. This undesirable series of complex reaction leads to the development of off-flavour, odour, and degradation of the overall quality. It is therefore important to accurately and correctly measure the quantity and types of oxidized products. Hwang and Bakota have contributed an excellent review on the applications of various NMR techniques (¹H-NMR, ¹³C-NMR and ³¹P-NMR) for the analysis of the types, and extent of oxidative changes during the processing and manufacturing of oil-based food as well their storage. They demonstrate that NMR can be effectively used for determining the oxidative stability of lipids and oils, and their products.

Hermosin-Gutierrez et al review the various NMR techniques used for the study of the structures and dynamic properties of various classes of pyranoanthocyanins. Anthocyanins are plant-based pigments, which have the ability to protect against a myriad of human diseases. They frequently interact with other phytochemicals and give rise to new classes of compounds which are often difficult to decipher. Pyranooanthocyanins are however fairly stable compounds of complex structures. They occur as glycosides and exhibit complex structural variations in the flavone skeleton which often makes their structure determination quite challenging. NMR spectroscopy is especially suited for elucidating such structures. Pyranoanthocyanins also have a special significance in the color of foods and beverages, and their dynamic properties are important to be studied.

The article by Géan et al describes various key developments in NMR spectroscopy as a powerful tool for the study of the structures of tannins, their relationship with the taste of wine, as well as their health protective effects. Their anti-oxidant properties and their protective effect on membranes against lipid oxidation have been discussed.

Quantitative ¹H-NMR (qNMR) is an application of NMR spectroscopy for the determination of the concentrations of one or more chemical species in a solution with a very high level of precision. It is simple and rapid, yet an elegant technique in which the area of an NMR signal is directly proportional to its concentration and this response is the same for all molecules. Sugimoto et al have comprehensively reviewed the concept of qNMR methods, and their applications in complex food analysis, such as purity assessment, and quantification of mycotoxins, pesticides, preservatives, phytosterols, etc.

Kralicek and Ozawa have critically reviewed various NMR spectroscopic methods used in structure determination of large cell free proteins, and their complexes. The authors describe the various cell-free expression systems used for the rapid and cost effective production of target proteins with required isotope labeling.

At the end we would like to express our gratitude to all the contributors for their excellent contributions. The entire editorial team of Bentham Science Publishers, particularly Ms. Fariya Zulfiqar (Assistant Manager Publications), Mr. Shehzad Naqvi (Senior Manager Publications) and team leader Mr. Mahmood Alam (Director Publications) deserve our deep appreciation for compiling such an excellent volume which should prove to be of wide interest to the readers.

Atta-ur-Rahman, FRS

Kings College

University of Cambridge

Cambridge, UK

&

M. Iqbal Choudhary

H.E.J. Research Institute of Chemistry

International Center for Chemical and Biological Sciences

University of Karachi

Karachi

Pakistan

Application of NMR to Resolve Food Structure, Composition and Quality

Angela Melado-Herreros*, ¹, María E. Fernández-Valle², Pilar Barreiro¹

¹ LPF-TAGRALIA, Technical University of Madrid. ETSI Agronomos, Avda. Complutense s/n. 28040, Madrid, Spain

² CAI of NMR, Complutense University of Madrid. Avda. Juan XXIII, 1. 28040. Madrid, Spain.

Abstract

Food is a complex system formed by several chemical compounds and physical structures at different organization levels. For food analysis and characterization, it is not only important the study of the chemical composition, which will define the nutrient content, but also the physical distribution of the different compartments and structures that will define the physical properties of food products. Physical properties of food will define the palatability and texture of the food product and thus, the acceptance by the consumers. When talking about Nuclear Magnetic Resonance (NMR) spectroscopy we refer to several techniques that study the interaction of electromagnetic radiation with matter. Nuclear magnetic spectroscopy is the use of the NMR phenomenon to study physical, chemical and biological properties of matter, from the microscopic to the macroscopic. NMR spectroscopy is a very successful and multipurpose technique which is very suitable combined with chemometrics, for the analysis of food products [1]. In this chapter, we will review several NMR techniques that are related to both chemical and physical characterization. Such techniques are 1H High-Resolution Magic Angle Spin (1H HR-MAS), which provides a high resolution chemical spectrum without component extraction [2], relaxometry, which gives information about the water compartmentation, structure and integrity [3], magnetic resonance imaging (MRI) and chemical shift imaging (CSI), which is an efficient tool for the physiological analysis of fruit and vegetables [4]. The following chapter will address, first of all, what needs to be measured on food, as well as several NMR techniques that have been used for the analysis of food products.

These techniques are 1H High Resolution Magic Angle Spin (1H HR-MAS), MRI, 1D and 2D relaxometry, relaxometry mapping and chemical shift imaging. We further focus on the explanation of multicomponent analysis and finally offer some remarks about prospects in the field.

Keywords: Chemical properties, Chemometrics, Food structure, Macrostructure, Microstructure, Physical properties, Structure, Texture.

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* Corresponding author Angela Melado-Herreros: LPF-TAGRALIA, Technical University of Madrid. ETSI Agronomos, Avda. Complutense s/n. 28040, Madrid, Spain; Tel: [phone]; E-mail:[e-mail]

FOOD DIMENSIONS

To start with this chapter, we would like to offer an introduction of different food dimensions in order to understand the different parameters that affect the food. There are both chemical and physical characteristics that influence the nutrient composition, shelf life, texture, structure... and that are important to take into account when studying and/or designing a food product. These characteristics may be measured and controlled by means of nuclear magnetic resonance (NMR).

Composition

According to Skov et al. [5], from a physical, chemical and biological perspective, food matrices are complex multifactorial systems containing mixtures of heterogeneous classes of molecules (nutrients), and complex physical structures.

Nutrients

Nutrients are basically classified into macronutrients (fats, proteins and carbohydrates) and micronutrients (vitamins, minerals, phytochemical, zoochemicals, fungochemicals and bacteriochemical) [6]. Macronutrients provide energy to the body and are required for growth, metabolism and other functions. Macronutrients differ in the energy density, being highest for fat (above 12 kcal g-1) than for protein (9 kcal g-1), and last for carbohydrates (4 kcal g-1). Carbohydrates provide the glucose used by all cells as fuel, as well as deliver the fiber intake. Proteins are the unique source of nitrogen, basic for amino acids and tissue repair, and fat provides a mean for the absorption of fat soluble micronutrients [7].

Despite the generally accepted consideration of the role of macro- and micro-nutrients, the analysis of a limited number of compounds narrows the whole view of food. Bordoni et al. [8] pointed out that some foods contain more than 25,000 compounds with concentrations varying according to variety, breeding, season, and geographic origin, among the major factors. Human metabolome contains about 50,000 different detectable compounds with 20% circadian variations.

Throughout World War I and II, the main concerns in nutrition were the vitamin and mineral deficiencies, while in 1960’s the concern focused to the excess of nutrients (fat, cholesterol or sodium) or the imbalance in the intakes of fat and carbohydrates. In the 1980’s interest turned to fiber, vitamin A, C and E and selenium, and it was only in 1998 that the first recognition of functional food was set as those with proactive health path beyond the basic (adequate) nutritional functions. Also, functional foods are not considered as pills or capsules [9].

Functional foods can be classified into conventional (e.g., fruits and vegetables), modified (fortified, enriched or enhanced), medical (formulated by and to be used only under medical supervision, such as oral supplements in the form of phenyl ketonuria formulas free of phenylalanine, and diabetic, renal, and liver formulations), and special dietary (gluten-free, lactose-free…). It is expected that modifying foods through biotechnology for improving their nutritional value or health attributes will increase the number of new functional foods into the markets [9]. The above highlights the need for analytical methods that would allow a global analysis of food composition, as well as a specific assessment of selected nutrients.

Moreover, a recent discovery shows that individuals with different genotypes in a population may not benefit (or may even suffer) from increased level of nutrients in functional food [6]. It is estimated that human genome contains approximately 10 million of single nucleotide polymorphisms (SNP) which would lead to substantial differences in nutritional response among individuals. That is why there are large efforts involved toward linking nutrition science and genomics into a discipline called nutrigenomics.

Alternatively, food digestion may be manipulated to enhance nutrient absorption by changing digestion rate, and the site of absorption. The main methodologies for food digestion modification include food emulsification, gelation and encapsulation [7]. Fat emulsification (as a type of microstructure) improves digestion by increasing the area available for lipase attachment and activity. Protein gelation is a type of microstructure that decreases the kinetics of absorption of amino acids and also increases the satiety effect. The dietary carbohydrates are composed of simple sugars (monosaccharides and disaccharides) and complex carbohydrates (starch and fiber) the digestion and absorption of which are affected by structure, type and presence of fiber. More precisely, the physical state of the food affects the digestive motility, which is slower when there is a higher viscosity. The presence of mixtures of proteins and carbohydrates in the form of self-assembled structures also plays an important role in the digestion process. All of the above points extensively address the microstructure of food in order to align sensory, physical and nutritional values of foods.

Bio-availability

Cardoso et al. [10] highlight that not all the food components are directly available for the cells after food ingestion. The bioaccessible level is defined as the food component concentration that is released from the food matrix into the intestinal lumen after digestion, which may be different from the content found in the systemic circulation, the bioavailable content.

According to Cardoso et al. [10], the bioaccessibility factor is affected by the biochemical composition of the food matrix and by the synergies and antagonisms between components. On the other hand, the bioactivity stands for a set of phenomena that occur after the nutrient reaches the systemic circulation and finally to its last destination. In that sense, bioavailability is considered a combination of bioaccessibility and bioactivity.

There are two main approaches for assessing bioaccessibility: in vivo, and in vitro. The former includes balance studies and tissue analysis, while the latter makes use of static and dynamic digestion models or cell cultures. NMR foodomics can be used for metabolome profiling and fingerprinting on either of the two (tissues or cell cultures).

There is a large interest in incorporating micronutrients (vitamins, mineral and nutraceutical among others) into functional foods and beverages as to improve health and wellness through diet [11]. However, many micronutrients cannot be directly incorporated, due to low solubility, degradation susceptibility, off-flavor, adverse interaction with other food components, or variable bioavailability. The susceptibility of a micronutrient to degradation within a food is viewed as highly dependent on its molecular and physiochemical characteristics [11]. NMR looks like a promising technique for addressing accessibility and availability in this framework.

Food Matrix

Food is a complex system formed by several chemical compounds and physical structures at different levels of organization.

Physical properties of food are important since they define the distribution of the different compartments and structures, which are related to the palatability, shelf life and texture, among others.

There are several physical properties of food. In this chapter we will focus only on texture and structure, since we believe these to be the two important characteristics related to the product shelf life and the acceptance by the consumers, and much related to each other. In some cases, it is difficult to separate structure and texture, as one is a consequence of the other, and it is possible to use very similar NMR methods to characterize both.

Texture

Lawless and Heymann [12] defined food texture as all the rheological and structural (geometric and surface) attributes of the product perceptible by means of mechanical, tactile, and, where appropriate, visual and auditory receptors. According to the definition provided by Bourne [13], texture is the response of the tactile senses to physical stimuli that results from contact between some parts of the body and food. Food texture is related to the eating experience and it is associated not only to the consumer's acceptance of a product, but also to factors, such as readiness to harvest, shelf life, handling and processing. Thus, in the last decades, there has arisen the study of food texture involving aspects of food, materials and sensory sciences.

Food texture can be studied as a subjective (sensorial) or as an objective (instrumental) property. Sensory analysis usually involves time-consuming methods. In the recent years, a number of scientists are studying textural properties by the various instrumental methods [14]. The instrumental methods employ both destructive and non-destructive techniques. Destructive techniques include three-point bending test, single-edge notched bend test, compression and puncture test, stress relaxation test, Warner-Bratzler shear force test, a combination of mechanical and acoustic methods and imitative methods, among others. The non-destructive techniques are mechanical, ultrasound and optical techniques, among others [15, 16], as well as NMR techniques.

In fresh fruits there exist some internal problems affecting texture, such as mealiness, internal breakdown and internal browning in apples and pears, or wooliness in peaches. Both are negative textural attributes that combine the sensation of desegregated tissue with the loss of crispness and a lack of juiciness without variation of the total water content in tissues. Internal browning in apples (and pears) is characterized by softening and browning of tissues and development of cavities, which is only observable in the final stage of the commercial chain, and can cause important economic losses [17].

NMR techniques have provided good results for food texture assessment. Several NMR procedures can be applied to this, like MRI and NMR relaxometry, as further explained in this chapter.

Structure

Food is formed by different structures at several scales. According to their size, these structures can be classified as macrostructures (with the largest dimension above 100 µM) and microstructures (below 100 μm) [18], though more levels can be defined according to other sources.

To simplify the presentation, in this chapter we will only describe the general classification at the macrostructure and the microstructure level.

The Macrostructure Level

Food industry has experienced a huge development in the last century, thanks to the transfer of knowledge from other areas, such as chemical and mechanical engineering. This affected especially the macroscopic processing through the adaptation of several operations, and the design of processes to transform and preserve food.

To understand the macroscopic level, it should be stated that macrostructure comprehends numerous smaller structures (microstructures) that affect the macroscopic properties, like rheological behavior, textural and sensorial traits and transport properties, among others. According to Aguilera [18], macrostructure includes some plant cells, powder particles, bubbles and grains. To visualize and quantify such structures, imaging techniques have provided good results.

Therefore, it is difficult to talk about macrostructure without referring to microstructure insight. Biological materials appear to be continuous when viewed at the macroscopic scale and from predictive models based on macroscopic continuum physics [19-21] it is expected that these materials behave as a (non)linear (visco) elastic continuum. However, as stated by Mebatsion et al. [20], macroscopic properties of food depend on various features that cover a wide range of spatial scales, from nanoscopic to microscopic and to macroscopic (which is the actual geometry of the material). Mebatsion et al. [20] propose the construction of multiscale models, which are based on hierarchy of submodels that describe the material behavior at different spatial scales and also depending on the way by which the submodels are interconnected. Multiscale modeling involves challenging physical properties that are very difficult to solve at different scales. The solution would be to model at the coarser scale by including procedures to construct the equations at this scale and account for lower scales. On the other hand, equations can be solved for the fine scale. The up-scaling from smaller scales to a macroscale solution is known as homogenization. It is a collection of methods for extracting or constructing equations for the coarse scale (macroscale) behavior of material and systems, which incorporate many smaller (nano-, micro-, meso-) scales [20].

The Microstructure Level

Microstructure affects different properties of food, such as in-mouth sensation perceived by the consumers [22], texture [23], microbiological activity [24, 25] and the air and water distribution through the pores [25].

As stated before, microstructure affects food macrostructure. This fact can be taken into account when optimizing food technology and engineering processes in order to improve the macrostructural result, in terms of acceptance by the consumers, stability and better shelf-life of the food products. As an example, Zuñiga and Aquilera [26] studied the relation between microstructure and fracture and the importance of knowing the microstructure of gassed gels. The microstructure can affect several properties of foodstuffs, including the calorie density and satiety index, or aid in developing novel gastronomic qualities. In dried vegetables, thermal treatments have important role in microstructure and rheological properties, and could contribute to the manufacture of healthier processed foods, with lower artificial stabilizers [27].

NMR techniques are suitable to elucidate food structure. Nevertheless, it is important to define and take into account the resolution that each method can reach. Van As and van Duynhoven [28] reviewed several NMR methods used for multi-length scale architectures present in cereal plants and plant based food products. The choice of NMR technique could depend on the size of the sample to be analyzed. For example, for large particles with macrostructure, the most suitable technique is MRI. For microstructure elucidation, the most suitable NMR methods are relaxometry and diffusion. Thanks to these methods, the characterization of membrane permeability, cell walls, vacuoles and organelles on fresh food and plants is possible.

Stability and Processing

Currently, consumers demand food products having ‘fresh’ or ‘natural’ characteristics but with a long shelf-life, which has led to the application of several processing techniques that confer stability to those food products.

In general, adequate food stability is important to preserve the sensory characteristics. Food stability cannot be achieved using a single type of processing, but by means of multiple techniques that include heat, to reduce moisture content (aw) and antimicrobial chemicals, among others. Preservative factors also influence the sensory characteristics of the food product and can have a repercussion on flavor, texture or color [29]. The idea of combining several factors to preserve food has been developed by Leistner and Gorris [30], among others. They introduced the ‘hurdle’ concept, in which each factor is a hurdle that microorganisms have to overcome. Thus, the preservation of food by hurdle technology, is the use of a combination of several techniques for improving food stability, where the knowledge of temperature, aw, pH or chemical preservatives is used to design hurdles to control the proliferation of microorganisms [29].

In this chapter, we will introduce two transport phenomena that can be applied to food processing in order to confer stability to food products: water diffusion and thermal conductivity.

Water Diffusion

Water is a constituent of food, it is related to food stability, safety, quality and physical properties. State of water in a solution or a solid is expressed by the activity coefficient, which measures the chemical potential of water in the system. The water activity coefficient is defined by the following equation:

where p is the ratio of vapour pressure of water in food and p0 is the vapour pressure of pure water, at the same temperature and total pressure [31].

The state of water in food strongly depends on the structure of the food product. Water diffusion is a material transfer phenomenon, which is the main mechanism of moisture transport. Understanding water transport and behavior through a food product is crucial, since there is a need to optimize water diffusion processes, such as rehydration, and drying of food products. The macroscopic transport of water through the tissue in fruit and vegetables during drying can be controlled by the microscopic distribution of water and air on a cellular and subcellular distance scale and by the magnitude of membrane permeability barriers [32].

Drying of food is usually controlled by internal diffusion of water. It is considered as the main mechanism of moisture transport to the solid surface [33]. The average drying rate (DR) can be calculated by the expression [34]:

is the average moisture content and Δt is the time period.

Nevertheless, in most studies diffusion is accepted as the main mechanism of moisture transport to the solid surface [33], as assumed by Ramallo and Mascheroni [35]. These authors studied the effect of shrinkage on pineapple slices dehydration. They stated that if the sample is considered as a homogeneous solid with constant properties and the water movement in one dimension, the variation of the moisture content within the sample slice during drying can be considered as described by the Fick’s second law of diffusion:

where X is the moisture content (on a dry basis), x is the position inside the slice, t is the time and Deff is the effective moisture diffusivity. They built two different models: one without considering the thickness variation during drying, and another one considering it. The latter allowed a significant increase of the accuracy of simulation, with values of the mean percentage error of the estimation varying from 1.95-6.55%, whereas error values of the moisture estimation by application of the first model varied between 5.07-14.5%.

Diffusion coefficient was also determined during osmotic dehydration by Prociuncula et al. [36]. They modeled and quantified the mass transfer after the treatment by determining water loss (WL), mass loss (ML) and solids gain (SG) by means of:

where ms is the mass of dry solids at the end of treatment, ms0 is the initial mass of dry sample, m0 is the initial sample mass, mw0 is the mass of water in the non-treated sample, mw is the mass of water in the sample to the end of treatment and m is the sample mass after the treatment. Porciuncula et al. [36] concluded that model built by the diffusion equation with a moisture dependent diffusion coefficient is the best approach for predicting osmotic dehydration of banana.

Water diffusion (D) can be also determined by several methods, like optical and electron microscopy. Nevertheless, these methods have some limitations and suffer from artefacts introduced during sample preparation, and if an extensive image analysis is not performed, they only provide qualitative information. Other techniques, such as non-spatially resolved NMR techniques can provide this information, since they are non-invasive, do not introduce artefacts, and are quantitative. NMR diffusion techniques provide information about water diffusion through cell walls and membrane permeability coefficients. Biomembranes with different degrees of permeability separate different compartments containing water, with different diffusion properties.

NMR diffusion techniques can be applied to the study of fruit ripening, such as performed by Raffo et al. [37]. They measured the water self-diffusion coefficient by means of the standard PFG SE sequence. At a given amplitude G, and keeping fixed D, the time interval between the two gradient pulses of duration d, the amplitude of the NMR signal at fixed echo time is given by the following expression:

WhereA0 is the echo amplitude in absence of the pulsed gradients, k is given by (γ is the proton magnetogyric ratio). They determined D values by fitting the echo amplitudes measured at different G values.

2D NMR diffusion-relaxation correlation experiments, especially D-T2 , are very powerful in resolving water compartments with respect to their size and chemical composition [38]. This has been tested in several foods, such as cheese [39].

These methods are very useful also to separate fat/oil, as demonstrated in cream, cheese and yogurt [40] and for characterizing pure oil as a mixture of chemical species [41, 42].

Thermal Conductivity

Thermal conductivity is a transport phenomenon in food processing which involves momentum, heat and mass transfer. It needs thermo physical properties to solve heat transfer problems. According to [43], the rate of heat flow through a material by conduction can be predicted by Fourier's law as:

where Q is the rate of heat flow (J/s); A is the area of heat transfer normal to heat flow (m²); is the temperature gradient among the x- direction and k is the proportionality constant of thermal conductivity (W/m K).

Thermal