1H

qNMR of Alcoholic Cider - Analysis of Small Molecule and Residual Sugar Chemistry
John C. Edwards1,2

SpinMetrix

1) Process NMR Associates LLC, Danbury, CT USA. 2) SpinMetrix SRL, Montevideo, Uruguay

Cider Making Process

Abstract: 1H quantitative NMR (qNMR) has been utilized to assess the the small molecule and carbohydrate chemistry of a number of home-brewed and commercial alcoholic ciders. A quantitative chemistry distribution of the
products of the various fermentations that occur in cider making. Malolactic fermentation as well as fermentation by saccharomyces and wild yeasts occur in the cider making process which traditionally occurred without the
intentional addition of yeast by the manufacturer. The distribution of small molecules produced by the yeast and bacterial metabolomes at work in the process can yield information of the sensory perception of ciders produced in
different ways. An investigation of the residual sugar chemistry of commercial ciders gives some indication of the process of sweetening commercial cider products with sugar additions after fermentation is complete. These typical
commercial ciders are very different in chemistry distribution compared to very dry cider styles such as those found in the Basque region of Spain where fermentation is taken to the extreme resulting in complete conversion of
sugars to alcohol but also glycerols to 1,3 propandiol. Finally it was decided to determine how much quantitative chemistry information could be obtained from benchtop NMR systems operating in the 60 MHz range. These
benchtop NMR systems have a price and cost-of-ownership that would allow small laboratories of manufacturers to think about their use in QA and QC roles.

Racking

Filtration
Centrifugation

MaloLactic
Fermentation

Standard Material:
Maleic Acid (99.0%) Aldrich Lot#SLBC1970V - 10mg/100ml solution in D2O (99.9%D)

Extended Time
Esp. High Acidity

Experimental:
1H

qNMR experiments were carried out on a Varian Mercury MVX-300 equipped with a 5mm Varian ATB probe operating at a resonance
frequency of 299.67 MHz, and on a Varian Mercury 300 equipped with a 5mm Varian 4-Nucleus probe operating at 299.94. Experiments
were performed with a p/3 pulse with an 8 kHz spectral width collecting 64k over an 8 second acquisition time and with a 7 second
relaxation delay. For original cider samples 128 transients were co-averaged and for freeze dried samples 128 scans were co-averaged.
All data was post-processed using Mestrelab MNova software version 10.0.1-14719.

Sorting
Sweating
Washing

In the final data processing the maleic acid resonance at 6.4 ppm was normalized to 10 so that a direct calculation could be made of all
measured components on a mg/L basis

Back
Sweetening
Racking

Initial
Fermentation

Pasteurization
And
Packaging

saccharomyces

Sample Preparation:
Original sample analysis 175ml of cider was added to a 5mm NMR tube followed by 100ml of internal standard solution prepared such
that the concentration was 10mg per 100ml. Finally 475ml of D2O was added to the sample and the tube capped and agitated for 10
seconds.

Addition of SO2
Reduces wild fermentations

Chlorogenic
acid

Grind
To
Pomace

Freeze dried samples 1000ml of cider was placed in a 2 dram vial and freeze dried in a Virtis Benchtop K and the entire dried sample
was then dissolved in 650ml of D2O and 100ml of maleic acid internal standard solution (10mg/100ml) added and the sample agitated
before transfer to a 5mm NMR tube.
NMR Assignments:

Blending

The chemical components that can be identified and quantified in a number of different cider variants are shown in Figures 1-5. This
study centered on commercial hard ciders taken off the shelf at quality beverage centers. At this point in the study we have not
attempted to follow fermentation chemistry or product consistency. Rather we have focused on the finished product and what small
organic acid, fusel alcohol, tannin, and residual/back blended sugar chemistry can be readily quantified by a rapid screening NMR
analysis.
The following components were always obtained on the fresh cider sample as the freeze drying process would compromise the amount
present: ethanol, acetic acid, fusel alcohols (isobutanol and isopentanol (isoamyl alcohol)). The calculation utilized to quantify these
components was:

Pressing

1H

Component mg/L = 10 * ((Icomp / Ncomp) / 5) * (MWcomp / 116.1) * (1,000,000 / 175)

Where, wt of maleic acid = 10mg, Icomp = Integration of component resonance, Ncomp = number of protons integrated
IMA/NMA = 5, Mwcomp = molecular weight of component molecule, MW of Maleic Acid = 116.1 amu, the 1000000/175 factor rectifies
the volumetric component of the calculation to allow mg/L to be calculated.

Filtration
and Cooling

Figure1: Magners Cider assignments of the components quantified in the qNMR analysis spectrum was obtained
with water suppression on a 175ml aliquot of freshly degassed cider. This sample was the excpetion to the rule in the
experimental process in that 20mg of maleic acid was added to the sample. Sucrose, glucose and fructose are observed in
the sample and relatively high quinic acid indicating considerable tannin content.

The following components were always obtained on the freeze dried sample: lactic acid, acetic acid, succinic acid, malic acid, citrate,
fructose, glucose, sucrose, glycerol, quinic acid, chlorogenic acid. The calculation utilized to quantify these components was:
Component mg/L = 10 * ((Icomp / Ncomp) / 5) * (MWcomp / 116.1) * (1,000,000 / 1000)

Sugar Analysis of Commercial Ciders

Where, wt of maleic acid = 10mg, Icomp = Integration of component resonance, Ncomp = number of protons integrated

90000

IMA/NMA = 5, Mwcomp = molecular weight of component molecule, MW of Maleic Acid = 116.1 amu, , the 1000000/1000 factor rectifies
the volumetric component of the calculation to allow mg/L to be calculated.

80000
70000
60000
50000
40000
30000
20000
10000
0

Figure 2: Stella Artois Cidre assignments of the components quantified in the 1H qNMR analysis spectrum was obtained
with water suppression on a 175ml aliquot of freshly degassed cider. Spectrum shows low lactic content and only fructose
and glucose are observed. Tannin content id observed to be relatively low.
Sucrose mg/L

Glucose mg/L

Fructose mg/L

Small Organic Acid Analysis of Commecial Ciders

10000

Figure 3: 1H qNMR spectrum of freeze dried magners cider showing the improved signal-to-noise of the spectrum allowing more
accurate and higher sensitivity component measurements. Removal of the water resonance by pre-saturation is also seen to be more
effective.

8000

6000

4000

2000

Figure 4: 1H NMR spectrum of a dry cider with relatively high tannin content showing no residual sugar present in the cider.
High conversion of malic acid to lactic acid is seen with high lactic content and a low concentration of malic acid. This indicates
that malolactic fermentation occurred during the manufacturing process of this cider. The residual signal in the 3.5-3.8 ppm
region is due almost completely to glycerol (see Figure 5 below peaks in the 3.5-3.6 ppm region can be utilized to quantify
glycerol if identified in the spectrum).

Lactic Acid mg/L

Succinic Acid mg/L

Acetic Acid mg/L

Malic Acid mg/L

Tannin Analysis of Commercial Ciders

2500

Figure 6: 1H qNMR spectrum of cider allowed to ferment to ultimate completion yielding the highest possible ethanol content.
The malolactic fermentation continued and produced lactic acid and left no residual malic acid. The yeast then continued to
Ferment and turned the glycerol of the cider into 1,3-propandiol.

2000

1500

1000

500

Overlap

Figure 5: 1H NMR of glycerol compared to dry cider with no residual or back-blended sugars

Figure 7: Individual 1H NMR spectra of glucose, fructose and sucrose demonstrating the straightforward integration approach that can be used utilizing peaks
in unique regions of the spectrum. Overlap of sucrose resonances with the fructose peaks used for quantitation necessitate that the value of N (number of
Protons represented in a peak or series of peaks) be modified depending on whether sucrose is present in the cider.

Quinic Acid mg/L

60 MHz 1H NMR of Sugars and Hard Apple Ciders

Table I: Component Chemistry of Commercial Hard Ciders

Column1

Sample
Tim's Cider #1
Tims Cider #2
JK Scrumpy Famhouse Cider
Harpoon Craft Cider
Magners Cider
Stella Artois Cidre
Tim's Cider #3
Etienne Dupont Cidre Triple
Crispin Hard Apple Cider
Strongbow Gold Apple Cider
WoodChuck - Out on a Limb - Spitter
Splinter - Bourbon Barrel Aged Cider
Thatcher's Green Goblin - Oak Aged
English Cider
Magners Pear Cider
Etienne Dupont - Cidre Bouche Brut
de Normandie
Naked Flock Hard Cider
Doc's Draft Cider
Woodchuck Amber Cider
Blackthorn English Cider
Woodchuck 802 Hard Cider
ACE California Apple Cider
Angry Orchard Traditional Dry Cider
Woodchuck Gumption Hard Cider
Docs Draft Gold Rush Hard Cider

Chlorogenic Acid mg/L

Lactic Acid

Succinic Acid

Acetic Acid

Malic Acid

Ethanol

Ethanol

Label Ethanol

Sucrose

Glucose

Fructose

1,3-Propanediol

Glycerol

Quinic Acid

Chlorogenic Acid

Isobutanol

Isopentanol

Sorbate

Citrate

mg/L
5528
6770
2674
237
2838
296
4878
3156
1537
714

mg/L
494
494
706
378
523
145
465
1123
319
168

mg/L
1716
2504
1274
79
316
158
730
256
769
158

mg/L
0
0
4013
6072
5280
4752
0
993
2178
3708

mg/L
56256
64000
45766
34428
35487
34806
51627
84603
51082
42324

% v/v
7.1
8.1
5.8
4.4
4.5
4.4
6.5
10.7
6.5
5.4

%v/v
6.0
4.8
4.5
4.5
11.0
5.8
5.0

mg/L
0
0
403
0
33685
0
0
295
2181
1002

mg/L
0
0
10876
2514
24479
22883
6302
745
20754
51965

mg/L
0
0
52790
24676
15396
21251
8175
0
2566
31303

mg/L
3895
3671
0
0
0
0
0
0
0
0

mg/L
0
0
0
5892
0
0
4646
6799
0
4646

mg/L
0
0
0
348
1457
314
497
828
0
265

mg/L
0
0
0
397
946
275
580
1343
0
336

mg/L
97
146
55
61
49
24
49
73
49
24

mg/L
231
145
132
116
87
58
231
174
116
58

mg/L
0
0
0
0
0
0
0
0
0
0

mg/L
0
0
0
0
0
0
0
0
0
0

155

310

99

4158

42430

5.4

5.5

15975

10337

14877

7649

215

610

49

231

1314
72

457
239

99
138

1502
393

49070
37605

6.2
4.8

6.6
4.5

0
0

25982
29987

14799
28445

0
0

0
0

281
166

549
168

49
24

116
87

0
147

0
2896

3032
1247
3047
186
445
135
103
124
124
512

701
610
442
173
320
193
264
457
244
783

434
335
177
138
256
99
256
217
79
256

2310
6907
2368
4366
3916
3858
9876
5198
5359
6618

51702
59538
40267
37015
48738
36939
41522
43912
43322
59478

6.6
7.5
5.1
4.7
6.2
4.7
5.3
5.6
5.5
7.5

5.5
6.8
5.0
5.0
6.0
5.0
5.0
5.5
5.5
7.2

0
413
531
1710
5659
1710
0
26173
35428
0

652
18160
12572
48053
18346
31197
13783
10710
8847
838

15782
30212
24911
33209
10803
23189
86508
9254
15755
937

0
0
0
0
0
0
0
0
0
0

6176
0
0
0
0
0
0
0
0
4816

563
166
298
149
298
99
348
314
397
248

809
168
214
214
519
183
488
320
549
244

49
24
49
24
24
24
24
49
49
73

116
58
145
58
87
29
58
145
87
202

0
147
221
0
0
0
147
0
0
331

0
0
0
0
0
0
0
0
0
0

Figure 8: 1H NMR of sugars and example freeze dried ciders obtained on a 60 MHz NMR system demonstrating that sugar chemistry and organic acid chemistry can be observed. Also
60 MHz NMR of the fresh cider will also allow quantitation of the ethanol content.

Conclusions: 1H NMR can provide rapid broad range component analysis for a wide variety of commercial hard ciders providing targeted and
non-targeted analysis ranging from identification, qualitative comparisons, and fully quantitative component content on a mg/L or wt% basis.
Future Work:
1) Expand the number of flavor and aroma components quantifiable by 1H NMR pure component and spike analysis.
2) Utilize chemometrics to correlate quantitative results obtained at 300 MHz to data obtained on the same samples at 60 MHz.
3) Follow entire cider manufacturing process with the 1H qNMR analysis to provide quality control and process monitoring.
4) Fully automate the NMR processing, spectral and quantitative analysis and reporting.
5) Expand the analysis more thoroughly to mead, wine, spirits, fermented beverages, kombucha, vinegars, rice wine, fruit juices, distilled spirits and their precursor fermentations