Application Note 1: Monitoring
Fermentation by Benchtop NMR
Fermentation is the alteration or production of products with the help of microorganisms.
It can be used to alter or preserve foods, for example in bread, yoghurt, vinegar or wine.
Fermentation can be spontaneous, like milk turning sour in the fridge, or controlled byadding specific microorganisms. Examples of controlled fermentation are the productionof cheese or wine. In industrial processes, it is important to monitor the fermentation
progress as uncontrolled fermentation through contamination can spoil entire batches ofa product.
In this note, we show how the Spinsolve benchtop NMR spectrometer can be used tomonitor the conversion of sugar into ethanol through fermentation without any sample
processing. The different outcomes of controlled and wild fermentation are demonstrated.

FERMENTATION PROCESSThe fermentation happens in several stages. In afirst step, the dimer sucrose (C6H12OH) is broken
into two glucose molecules:
C12H22O11 + H2O
2 C6H12O6
The next step is the conversion of glucose intoethanol and carbon dioxide:C6H12O6
2 C2H5OH + 2 CO2The production of carbon dioxide createsthe characteristic bubbles observed duringfermentation. Under the presence of oxygen, acetic
acid bacteria, acetobacter, convert ethanol into
acetic acid:C2H5OH + O2 CH3COOH + H2O

Sample preparation and measurement
Two samples from a batch of fresh, untreated apple
juice were transferred into three litre plastic bottles.
One of these samples was inoculated with abrewer’s yeast for the controlled fermentation. The
other sample was left without any further treatment
to undergo wild fermentation. Both bottles werekept at room temperature.
An example spectrum of the wild fermentation
sample is shown in Figure 1.

Figure 1

Figure 1: NMR spectrum apple juice after undergoing wild fermentation.

Figure 1: NMR spectrum apple juice after undergoing wild fermentation.

NMR spectra of fresh and fermented juice samples

The NMR spectra of the completely fermented samples (the juice now converted to cider) are
shown in Figure 2 along with the spectrum of the fresh juice. All sample spectra show a large

NMR SPECTRA OF FRESH AND FERMENTED JUICE SAMPLESThe NMR spectra of the completely fermentedsamples (the juice now converted to cider) are
shown in Figure 2 along with the spectrum of thefresh juice. All sample spectra show a large peak at
4.7 ppm, which is due to the water.

A vertical expansion reveals a number of smaller
peaks between 1 and 4 ppm. These peaks are
assigned in Figure 2.

Figure 232

32

Figure 2: NMR spectra of fresh apple juice and apple cider from wild andcontrolled fermentation after 20 days. The juice and cider spectra aredominatedby theprominent
peak,
but wildtheandverticalexpansionafter
by 20a days. TheFigure
2: NMR spectraof freshapple juicewaterand apple
cider fromcontrolled fermentation
factor50spectra
reveals
the presenceof smaller
peaks,
which
all bejuice
and cider
are dominatedby the prominent
water peak,
but theverticalcan
expansionby aassigned.
factor 50 reveals
In order to aid peak assignment, the spectrum of a 99% ethanol/water
the presence of smaller peaks, which can all be assigned. In order to aid peak assignment, the spectrum of a 99%
mixture is also shown.
ethanol/water mixture is also shown.

Measurement of alcohol content and conversion rate

The CH3 peaks at 1.2 and 2.0 ppm are well resolved and can be integrated for a quantitative
measurement of ethanol and acetic acid content. A plot of the measured volumeMEASUREMENT OF ALCOHOL CONTENT AND CONVERSION RATEThe CH3 peaks at 1.2 and 2.0 ppm are wellresolved and can be integrated for a quantitative
measurement of ethanol and acetic acid content.

A plot of the measured volume concentrations isshown in Figure 3. Characteristic for the controlled
fermentation is the higher conversion rate and theabsence of secondary fermentation.

Figure 3Concentration (% v/v )12Ethanol CH3 controlled
30.086 % v/v per hour

10Ethanol CH3 wild
30.023 % v/v per hour
Acetic acid wild

80.007 % v/v per hour
Ethanol to acid conversion
Asymptotic level

6

Figure 3: Volumeconcentrations of
ethanol and acetic
acid obtained fromthe CH3 peak integralof the NMR spectra.
The lines show theregression of thedata points in thelinear region.

4
200100200

Time (hours)

300

400

Figure 3: Volume concentrations of ethanol and acetic acid obtained fromthe CH3 peak integral of the NMR spectra. The lines show the regression ofConclusion
the data
points in the linear region.

The Spinsolve benchtop NMR spectrometer has been used to monitor the fermentation process of
untreated apple juice. Clear differences between controlled and wild fermentation were detected.
The samples were taken straight out of the fermentation vessel without any further processing orConclusions
purification. This shows that the Spinsolve benchtop NMR spectrometer can be readily used as an
The Spinsolve
benchtop
NMR spectrometerhas been used to monitor the fermentation
on-line processmonitoringtool.

process of untreated apple juice. Clear differences between controlled and wild fermentation
were detected. The samples were taken straight out of the fermentation vessel without any
further processing or purification. This shows that the Spinsolve benchtop NMR spectrometer
CONTACT INFORMATION
can beFor
readily
as an on-line
processsales@magritek.com
monitoring tool.
furtherused
information,please contact:
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