Maintaining a specific quality of the food and drinks today is a challenge for any manufacturer, because of today's modern methods by which impurities and fraud such as incorrect labeling of the product type or the type and origin of the ingredients. NMR spectroscopy is a reliable method to detect any deviation of the content to the informations on the Labelling.
In the beginning NMR spectroscopy was only used to solve the organic and inorganic structural problems. For example, reaction products can be determined, reaction mechanisms are elucidated or structural isomers are identified. In recent decades, however, through a lot of methodological and instrumental developments many new useful applications were added. Today the NMR spectroscopy finds an important application in the Metabolomic studies for example in biological fluids such as urine, plasma and blood. Together with standardized NMR spectra databases as reference for the identification of the metabolites that are contained in the sample. Benefits are the ease of use and low cost, for example a 1D spectrum is measured in a few minutes, this can be very well reproduced with minimal effort for sample preparation (typically only the addition of buffer).
Table of Contents
1. List of abbreviations
2. Introduction
3. Objective
4. Results and discussion
4.1 Identification of metabolites in the extracted apple juice
4.1.1 Identification of metabolites in the commercial apple juices
4.1.2 Investigation of the vitamine C content
4.1.3 Comparison of the relative metabolite concentration
4.1.4 Mixing of unripe fruits in apple juices
4.2 Identification of metabolites in the orange juices
4.3 Identification of metabolites in the multivitamine juices
4.4 Sugar content in commercial fruit juices
4.5 Changes in the fruit juice sample after three weeks
5. Investigation of Coca Cola
5.1 Comparison of spectra
5.2 Identification of metabolites
5.3 Differences between premium- and discountbrands
6. Experimental Section
6.1 Sample and standard preparation
6.1.1 Preparation of fruit juices
6.1.2 Preparation of phosphate buffer (pH 6 - 7 at 25°C)
6.1.3 Preparation of samples for NMR
6.1.4 1H NMR measurement at 400 MHz
6.2 Running an NMR Experiment using TOPSPIN as software
6.2.1 Insertion of sample tube into the spinner
6.2.2 Creating a new directory (edc)
6.2.3 Locking and shimming
6.2.4 Acquiring FID signal and modifying acquisition parameters
6.2.5 Processing the 1H NMR spectra with TOPSPIN
6.3 Working with Amix viewer
6.3.1 Displaying the spectra with Amix Viewer
6.3.2 How to show a series of spectra on top of selected spectrum
6.3.3 Matching spectra
6.3.4 Creating a new spectra base (Sbase)
6.3.5 Displaying spectra in overlaid mode
6.3.6 Displaying a series of spectra in horizontal- and vertical splitting
6.3.7 Showing a series of spectra on top of selected spectrum
6.3.8 Annotating peaks
7. Summary
8. References
1. List of abbreviations
Abbildung in dieser Leseprobe nicht enthalten
2. Introduction
Maintaining a specific quality of the food and drinks today is a challenge for any manufacturer, because of today's modern methods by which impurities and fraud such as incorrect labeling of the product type or the type and origin of the ingredients. NMR spectroscopy is a reliable method to detect any deviation of the content to the informations on the Labelling.
In the beginning NMR spectroscopy was only used to solve the organic and inorganic structural problems. For example, reaction products can be determined, reaction mechanisms are elucidated or structural isomers are identified. In recent decades, however, through a lot of methodological and instrumental developments many new useful applications were added. Today the NMR spectroscopy finds an important application in the Metabolomic studies for example in biological fluids such as urine, plasma and blood. Together with standardized NMR spectra databases as reference for the identification of the metabolites that are contained in the sample. Benefits are the ease of use and low cost, for example a 1D spectrum is measured in a few minutes, this can be very well reproduced with minimal effort for sample preparation (typically only the addition of buffer).
Figure 1 shows a very good reproducibility of the spectra of the fruit juices. It shows 50 spectra obtained under the same conditions. It can be seen that phase and baseline are perfectly set in the left part and the signal positions are absolutely stable as seen in the expanded signals of malic acid in apple juice. 1
Abbildung in dieser Leseprobe nicht enthalten
Figure 1: Reproducibility test on one apple juice 50 times injected with automatic preparation, measurement and processing. The expanded signals belong to the malic acid. 1
By comparing the measured spectra with the those of pure compounds in the database differences in the concentration of a particular metabolite, its availability or characteristic quality and authenticity issues, such as the addition of sugar can be discovered. Therefore, it is of great interest to the food chemistry in the classical juice evaluation method to determine the concentrations of the various constituents. Specific variations in the concentration of a particular compound or in the profile of a specific combination of compounds may show characteristic quality problems. One of the particular advantages of NMR spectroscopy against conventional methods of analysis is the possibility of simultaneous identification and quantification of a mixture of the Metabolte in the fruit juices. (Figure 3).
Table 1. An example of the metabolites contained in an orange juice. 2
Abbildung in dieser Leseprobe nicht enthalten
Figure 2: Structural formulas of some organic acids occurring in fruit juices.
Abbildung in dieser Leseprobe nicht enthalten
Figure 3: Structural formulas of some sugars and amino acids occurring in fruit juices.
Abbildung in dieser Leseprobe nicht enthalten
Figure 4: Peach puree screening by NMR at 400 MHz. The expanded region shows a 1D NOESY presat (water-suppressed) spectrum overlaid on a 2D spectrum. The J-resolved experiment provides easy identification of multiplets for citric acid (C) and malic acid (M), and the 1D spectrum provides quantitation. 1
In addition, further useful relationships between concentrations of certain metabolites can be calculated, for example, the ratio of glucose / fructose, or the ratio of sucrose to total sugar. This large amount of analytical results from one measurement enables the detection of fraud such as the addition of sugar, adding citric acid (for example, in apple juice), extraction of orange peel (Marker: phloretin). or the use of unripe fruit (e.g. high concentration of quinic acid in Apple Juice). 2
3. Objective
Using 1D 1H NMR spectroscopy, in this report we will investigate a series of fruit juices and Coca cola and try to identify the metabolites like sugars, amino acids and organic acids and compare their presence and concentrations, search for advertised vitamine ingredients, compare juices marketed as natural against the original one, distinguish between premium and discount brands, and recognize similarities between many brands. In addition we will try to determine the relative sugar content in the juices since a ctual fructose consumption levels are difficult to estimate because of the unlabeled quantity of fructose in fruit juices and since fructose consumption is hypothesized to be associated with risk for metabolic diseases. We also note that this analysis can be extended to other soft drinks and food products.
4. Results and Discussion
In this chapter a number of fruit juices will be analyzed on the basis of acquired 1H NMR data. Here, the metabolites contained will be identified and listed, besides the relative concentrations of metabolites in different fruit juices (apple,- orange- and multivitamin juices) will be compared.
Abbildung in dieser Leseprobe nicht enthalten
Figure 5: Pictures of some of the investigated apple and orange juices.
Abbildung in dieser Leseprobe nicht enthalten
Figure 6: The full 1H-NMR spectra (~4 minutes acquisition time) of the investigated natural apple juices (1- 11) which were obtained from different markets and produced by different companies showing very similar signals. The x-axis (expressed as ppm) corresponds to the chemical shifts of the proton resonances. The spectrum 8 was obtained from an apple juice which was directly extracted in lab for comparison.
Table 2: The corresponding brands and markets to the spectra in figure 6.
Abbildung in dieser Leseprobe nicht enthalten
Figure 7: The spectra (1-11) of apple juices in super imposed mode that shows that the spectra are congruent with the exception of the small signal at 2.56 ppm that belongs to (Rio d'oro Multivitamin, Aldi) since it also contains other component than the apple juice.
4.1 Identification of metabolites in the extracted apple juice
Prior to analyze the 1H NMR spectra of apple juices bought from different above mentioned markets, a spectrum of an apple juice from a ripe apple with high quality (Land of origin: Italy) will be presented, which has been extracted directly from an apple and, after centrifugation, its 1H NMR spectra were acquired in order to compare it with the ones from other commercial apple juices. The detected metabolites, and differences in the concentration will be compared.
The assignment of the peaks to the corresponding pure compounds in the following illustrated Spectra was done by matching them with reference spectra from the Spektra base "Human Metabolome data Base (HMDB) 3. Here, the raw “Free Induction Decay (FID)” files for spectral processing were downloaded and edited with the spectra editor MestReNova.
Abbildung in dieser Leseprobe nicht enthalten
Figure 8: The full 1H NMR spectrum of apple extract, land of origin Italy. Chemical shifts were determined at pH 6.1 in D2O, 298 K and expressed as relative values to that of TSP at 0 ppm.
Abbildung in dieser Leseprobe nicht enthalten
Figure 9: comparison of 1H NMR spectra of the metabolites Glucose, Sucrose and malic acid to that of apple extract. The comparison shows that the three metabolites are contained in the juice. The Reference spectra were obtained from the humen Metabolom Data Base (HMDB). 3
Abbildung in dieser Leseprobe nicht enthalten
Figure 10: Magnified section of 1H NMR spectrum (400 MHz) of apple extract, Land of origin Italy (Rewe) in the range of 0 - 3.1 ppm. Chemical shifts were determined at pH 6.1 in D2O, 298 K and expressed as relative values to that of TSP at 0 ppm.
Abbildung in dieser Leseprobe nicht enthalten
Figure 11: Magnified section of 1H NMR spectrum (400 MHz) of apple extract, Land of origin Italy (Rewe) in the range of 3.1 - 5.5 ppm. Chemical shifts were determined at pH 6.1 in D2O, 298 K and expressed as relative values to that of TSP at 0 ppm.
Abbildung in dieser Leseprobe nicht enthalten
Figure 12: Magnified section of 1H NMR spectrum (400 MHz) of apple extract, Land of origin Italy (Rewe) in the range of 5.4 - 8.6 ppm.
Table 3: Resonance Assignments with chemical shifts of metabolites identified in 400 MHz 1H NMR spectrum of apple extract.
Abbildung in dieser Leseprobe nicht enthalten
Abbreviations: d: doublet, dd: doublet of doublets, m: complex multiplet, s: singlet, t: triplet . Chemical shifts were determined at pH 6.1 in D2O and expressed as relative values to that of TSP at 0 ppm.
4.1.1 Identification of metabolites in the commercial apple juices
The three regions of the apple juice Rio d'oro as an example from the nine above mentioned apple juices could be identified corresponding to the amino acids region (0 - 3.1 ppm), the sugar region (3.2 - 5.5 ppm), and the phenolics region (5.4 - 8.6 ppm). Each major metabolite was identified after peak assignment using 1H NMR spectra from compounds associated with a comparison of data from HMDB 3 .
Abbildung in dieser Leseprobe nicht enthalten
Figure 13: The full 1H NMR spectrum of the apple juice branded Rio d'oro from Aldi market. Chemical shifts were determined at pH 6.1 in D2O, 298 K and expressed as relative values to that of TSP at 0 ppm.
Abbildung in dieser Leseprobe nicht enthalten
Figure 14: Magnified section of 1H NMR spectrum (400 MHz) of the apple juice (brand: Rio d'oro, Aldi) in the range of 0 - 3.1 ppm. Chemical shifts were determined at pH 6.1 in D2O, 298 K and expressed as relative values to that of TSP at 0 ppm.
Abbildung in dieser Leseprobe nicht enthalten
Figure 15: Magnified section of 1H NMR spectrum (400 MHz) of apple juice (brand: Rio d'oro, Aldi) in the range of 3.1 - 5.5 ppm. Chemical shifts were determined at pH 6.1 in D2O, 298 K and expressed as relative values to that of TSP at 0 ppm.
Abbildung in dieser Leseprobe nicht enthalten
Figure 16: Magnified section of 1H NMR spectrum (400 MHz) of orange juice (brand: Rio d'oro, Aldi) in the range of 5.4 - 8.6 ppm. Chemical shifts were determined at pH 6.1 in D2O, 298 K and expressed as relative values to that of TSP at 0 ppm.
Table 4: Resonance Assignments with chemical shifts and spin-spin coupling patterns of metabolites identified in 400 MHz 1H NMR spectra of commercial apple juices.
Abbildung in dieser Leseprobe nicht enthalten
Abbreviations: d: doublet, dd: doublet of doublets, m: complex multiplet, s: singlet, t: triplet, GABA: γ- Aminobutyric acid. Chemical shifts were determined at pH 6.1 in D2O and expressed as relative values to that of TSP at 0 ppm.
In the freshly extracted apple juice less metabolites could be detected in comparison to those in commercial juices. For example, ethanol could not be identified in the freshly extracted juice in lab. In all other juices the triplet typical for ethanol was seen at 1.2 ppm. This could be because of fermentation processes that happens despite the appropriate preservation conditions so that very small amounts of the glucose in the juice is reduced to ethanol. Besides γ-aminbutyric acid, formic acid, asparagine, alanine and tyrosine could not be detected in the freshly extracted apple juice, but in all other commercial ones. Those three missing acids and three amino acids could have been added as ingredients to the juice to improve the taste or the second possibility could be our extraction method in the lab, with which not all the components could be extracted out. Investigation of other apple varieties could answer this question more effectively.
Abbildung in dieser Leseprobe nicht enthalten
Figure 17: The full 1H NMR spectrum of apple juice (brand: hohes C, Rewe). Chemical shifts were determined at pH 6.1 in D2O, 298 K and expressed as relative values to that of TSP at 0 ppm.
4.1.2 Investigation of the vitamine C content
Many fruit juices or beverages packaging are labeled to contain nutritional ingredients such as vitamins. These labels do not give always the real amounts that are included in the product. This can be found out with NMR-spectroscopy as following.
Abbildung in dieser Leseprobe nicht enthalten
Figure 18: A picture of the bottle of apple juice branded "hohes C", which is characterized by higher content of vitamine C.
Abbildung in dieser Leseprobe nicht enthalten
Figure 19: Magnified section of 1H NMR spectrum (400 MHz) of the apple juice (brand: hohes C, Rewe) in the range of 0 - 3.1 ppm. Chemical shifts were determined at pH 6.1 in D2O, 298 K and expressed as relative values to that of TSP at 0 ppm.
Abbildung in dieser Leseprobe nicht enthalten
Figure 20: Magnified section of 1H NMR spectrum (400 MHz) of apple juice (brand: hohes C, Rewe) in the range of 3.1 - 5.5 ppm. Chemical shifts were determined at pH 6.1 in D2O, 298 K and expressed as relative values to that of TSP at 0 ppm.
Abbildung in dieser Leseprobe nicht enthalten
Figure 21: Magnified section of 1H NMR spectrum (400 MHz) of orange juice (brand: hohes C, Rewe) in the range of 5.4 - 8.6 ppm. Chemical shifts were determined at pH 6.1 in D2O, 298 K and expressed as relative values to that of TSP at 0 ppm.
Table 5: Resonance Assignments with chemical shifts and spin-spin coupling patterns of metabolites identified in 400 MHz 1H NMR nine spectrum of the apple juices branded: hohes C.
Abbildung in dieser Leseprobe nicht enthalten
Abbreviations: d: doublet, dd: doublet of doublets, m: complex multiplet, s: singlet, t: triplet. Chemical shifts were determined at pH 6.1 in D2O and expressed as relative values to that of TSP at 0 ppm.
Abbildung in dieser Leseprobe nicht enthalten
Figure 22: Structural formula of L-ascorbic acid (vitamine C) with two neighbouring chiral centers (C4, C5).
The numbering as in ref 4. Letters refer to the ABMX spin system.
Abbildung in dieser Leseprobe nicht enthalten
Figure 23: Comparison of the water suppressed (400 MHz, in D2O) proton spectra of apple juice „hohes C“ (dark red) and ascorbic acid (blue) 5 . Comparison reveals that the juice does not contains ascorbic acid.
The spectrum of ascorbic acid belonged to the Human Metabolome Database (HMDB) 3 to compare it with spectrum branded: hohes C (see figure 23).
The result of the comparison of the spectrum from the literature with the measured for the juice "high C" shows that the juice does not contain a particularly high or no vitamine C (ascorbic acid). The spectrum shows no signals that can be assigned to the vitamin C. The Spectrum may contain a very low concentration of vitamin C, which cannot be detected due to the low signal-to-noise ratio.
4.1.3 Comparison of the relative metabolite concentration
The relative concentrations of the metabolites in the apple juice in the region of amino acids (0 - 3 ppm) will be compared in order to visualize the difference and thus to make conclusions about the quality of the juice.
Abbildung in dieser Leseprobe nicht enthalten
Figure 24: The 1H-NMR spectra (~4 minutes acquisition time) of the investigated apple juices which were obtained from different markets and produced by different companies showing similar signals. The x-axis (expressed as ppm) corresponds to the chemical shifts of the proton resonances in the amino acid region (0 - 3.1 ppm).
The signal intensity is linearly proportional to the amount of each component (and the number of nuclei contributing to each signal). Remarkable is the relative concentrations of malic and aspartic acids in the extracted juice about one-third of the concentrations in the remaining commercial juices. This could be due to the industrial extraction method which is more effective or the reason for this could be the naturally lower content in this apple variety from Italy. Furthermore no ethanol (1.15 ppm) could be detected in the freshly extracted apple juice, but in all other commercial juices, since a small part of glucose is reduced to ethanol. The ethanol conc. is the lowest in the juices branded Hohes C, Albi and Bio. This indicates a better conservation conditions. The conc. of aspartic acid and asparagine (2.80 - 3.00 ppm) is just in Bio apple juice very low, otherwise it is almost the same in other juices. The conc. of the amino acid Threonine (1.33 ppm) is in the juices branded Ja and Hohes C remarkably high and is almost the twice compared to its content in other juices. As for the amino acid alanine (1.48 ppm), no difference in the concentration was observed between the ten analyzed spectra (see figure 24). Quinic acid signals (1.70 - 2.10 ppm) are missing in the extracted juice but visible with the same concentration in other spectra.
[...]
- Quote paper
- Sadik Mejid (Author), 2016, Usage of NMR Spectroscopy in Profiling Metabonomics, Munich, GRIN Verlag, https://www.hausarbeiten.de/document/475232
