NMR spectroscopy applications in food analysis A. Spyros NMR Laboratory, Dept. of Chemistry, Univ. of Crete

Spectroscopy Solutions eConference, May 20, 2014

“Food” and “NMR or MRI” articles in Scopus (title, keywords or abstract)

Why has NMR become so successful in food analysis ? • NMR spectrometers are now more accessible to food scientists • Sophisticated NMR hardware and user-friendly software has been developed • 2D NMR spectroscopy has allowed easy metabolite identification • Ease of use, minimal sample preparation • Quantitative chemical composition information • Multivariate statistical analysis tools has enabled food metabolomics and chemometrics • Public awareness on food safety and quality issues

NMR spectroscopy in food analysis • • • • • •

Fats and oils Beverages Fruits and vegetables Dairy products Meat products Whole foods

• Chemical characterization • Quality control • Authentication • Adulteration • Genetic modification • Protected denomination of origin

NMR methodology scheme for food analysis metabolomics

Solid state NMR

Metabolomics

L. Mannina et al., Progr. NMR Spectrosc. 66, 1 (2012)

1H

NMR spectrum of beer

Rich compositional information (metabolites)

• • • • •

Brewing site Type (ale, lager) Barley or wheat malt Production date Storage effects

J. E. Rodrigues and A. M. Gil, Magn. Reson. Chem., 2011, 49, S37

Multivariate statistical analysis modelsMetabolomics One metabolite – one line Many spectra, many metabolites ? Too much information ! To visualize differences between samples we need the data to be broken down to a few components or factors

Metabolomics variants • Targeted vs untargeted metabolomics Concentrations of known metabolites vs pure spectra (buckets)

• Unsupervised (PCA, HCA) vs supervised models (DA, PLS, OPLS-DA, NN, etc.) The model does not know sample class vs the model knows the classification already

Oils and beverages analysis by NMR spectroscopy • • • • • • • • • • •

Wine Beer Spirits Coffee Tea Cocoa Fruit juices Vinegar Honey Olive oil Vegetable oils

Dr Markus Link’s presentation later today E. F. Boffo et al, LWT - Food Science and Technology, 2009, 42, 1455.

NMR-based metabolomics on teas and herbal teas

A. Spyros, P. Dais. “NMR Spectroscopy in Food Analysis”, RSC, Cambridge UK, 2013.

Tea metabolomics

A. Ohno et al, J. Agric. Food Chem., 2011, 59, 5181.

Cultivar discrimination of wine : local Cretan wine cultivars aged in barrels

A. Spyros et al, manuscript in preparation, 2014.

Red wine aging in barrels

A. Spyros et al, manuscript in preparation, 2014.

Quality control of spirits - Cretan raki Public safety issues (mainly distributed hand in hand, unbottled and without traceability information)

3-Me-butanol

2-Me-propanol Propanol

MeOH Ethyl acetate Acetaldehyde

Phenylethanol Ethyl lactate

Alcohols Acetate

A. Spyros, unpublished results, 2014.

Controlled compounds in raki Acetaldehyde

Methanol

Acetaldehyde (g/100 Lt AA)

Methanol (g/100 Lt AA) Official limit = 1000

Official limit = 500

350

24 22

300

20 18

250

16 200

14 12

150

10 8

100

6 50

4 2

0 1

3 2

5 4

10

8 6

9

12 11

16

14 13

15

18 17

20 19

22 21

MeOH

0

Ace 1

3

5

8

10

12

14

16

18

20

22

So far no sample was found over the official limits

A. Spyros, unpublished results, 2014.

NMR spectroscopy in olive oil analysis • • • •

Quality Control of olive oil (composition, age) Geographical and varietal classification Year of production variations Detection of adulteration with vegetable oils and low quality olive oils

Quality control in virgin olive oils Diglycerides analysis by NMR • Virgin oils contain 1-3% total DGs • Fresh virgin oils contain mainly 1,2-DGs D=1,2-DGs/DGs > 0.9 D is further reduced during storage, thus it is a good index of aging

A. Spyros, A. Filippidis, P. Dais, J.Agric. Food. Chem., 52, 157 (2004)

Classification of 13 types of vegetable oils

30 25 20 15

Root 2

10

virgin olive soybean hazelnut corn sesame groundnut sunflower almond safflower palm coconut walnut rapeseed

5 0 -5 -10 -15 -20 -40

-20

0

20

40

60

80

Root 1

G. Vigli, A. Filippidis, A. Spyros, P. Dais, J.Agric. Food. Chem., 51, 5715 (2003)

Adulteration of olive oil with vegetable oils 25 virgin olive

20 15

hazelnut

mhazelnut

sunflower

msun

soybean

msoybean

corn

mcorn

Root 2

10 5 0 -5 -10 -15 -30

-20

-10

0

Root 1

10

20

30

Adulteration of virgin olive oil with low quality olive oils

Lampante

Extra virgin

Refined

G. Fragkaki, A. Spyros, G. Siragakis, A. Salivaras, P. Dais, J.Agric. Food. Chem., 53, 2810 (2005)

NMR analysis of solid and semisolid foods • Solids foods, due to immobility, provide NMR spectra with broad lines • Rotation of the sample at the magic angle (θ=54.7o) leading to narrow lines, just like liquid NMR • High Resolution Magic Angle Spinning (HR-MAS) is suitable for semisolid foods • Cross-Polarization Magic Angle Spinning (CP-MAS) is suitable for rigid foods, like grains.

Solid state NMR spectroscopy Normal magnet

Special probe

MAS rotors

•20-200 mg of sample • Suitable pulse sequences allow 1D and 2D NMR spectral acquisition

High resolution MAS NMR in food science • Fruits and vegetables (sweet pepper, mango pulp, apple, tomato), nuts and grains (wheat, rice, almonds, barley beans, green coffee beans) • Cheeses (Parmigiano reggiano, emmentaler), meat (dried beef, salmon) Citric acid Ala

1 day 1H

HR-MAS study of ripening of mango juice pulp 5

15

19 Gil et al., J. Agric. Food Chem. 2000, 48, 1524-1536

1H

HR-MAS NMR analysis of triterpenoids in olive leaves

ppm

24 26

16 11

27

20

29 23

30

30 40

18

19b

19a 9

50

5

60

70

3

80

90 3.2

3.0

2.8

2.6

2.4

2.2

2.0

1.8

1.6

1.4

1.2

1.0

0.8

0.6

ppm

E. Manolopoulou et al., Magnetic Resonance in Food Science: Food for Thought, Duynhoven, J. (Ed), RSC, Cambridge UK, 2013, pp. 164-170.

NMR-based metabolomics on vegetables

Vegetable processing: soya beans thawing MR imaging 4 min

24 min

44 min

64 min

Oil M. Koizumi et al, Magnetic Resonance Imaging 2006, 24, 1111

NMR-based metabolomics on fruits

A. Spyros, P. Dais. “NMR Spectroscopy in Food Analysis”, RSC, Cambridge UK, 2013.

Post-harvest ripening of fruits by MRI mature green

red ripe

Distribution of sugars and lycopene in tomato during ripening

Sugars

Lycopene

Y. C. Cheng, T. T. Wang, J. H. Chen and T. T. Lin, Postharvest Biol. Tec., 2011, 62, 17.

Quality evaluation of fruits by MRI On-line seed detection in mandarins during sorting

photos

MR images

Motion-corrected MR images

N. Hernández-Sánchez, P. Barreiro and J. Ruiz-Cabello, Biosystems Engineering, 2006, 95, 529.

Meat analysis by HR-MAS NMR NMR spectra of a sample of Longissimus dorsi muscle of a Friesian Holstein bull

M. Ritota et al. Meat Science 2012 92,754.

NMR-based metabolomics on meat

Discrimination of beef sirloin from Australia, Korea, New Zealand and the United States.

Y. Jung et al, J. Agric. Food Chem., 2010, 58, 10458.

NMR-based metabolomics of dairy products

Discrimination between milk of different animal origin

sheep

cow

R. Lamanna, A. Braca, E. Di Paolo and G. Imparato, Magn. Reson. Chem., 2011, 49, S22

Whole foods: Rye bread storage Solid state Cross Polarization MAS 13C NMR spectroscopy

A. Mihhalevski et al. Journal of Agricultural and Food Chemistry 2012, 60, 8492

Whole foods: Sausage fermentation Distribution of 1H T2 relaxation times during fermentation

S.M. Møller, A. Gunvig, and H.C. Bertram Meat Science 2010, 86, 462

Whole foods: Rice cooking

MRI images obtained at different stages of rice (cooking time indicated in minutes) White indicates high water concentration

M. Kasai, A. Lewis, F. Marica, S. Ayabe, K. Hatae and C. A. Fyfe, Food Res. Int., 2005, 38, 403.

Many thanks to the organizers for their kind invitation, and Thank you for your attention! [email protected] www.chemistry.uoc.gr/aspyros