Front-Face Fluorescence Spectroscopy (FFFS) is a powerful technique which works on the principle of Jablonski’s diagram which illustrates the electronic states of a molecule and transition between them. Fluorescence spectroscopy is a technique in which UV or visible light is absorbed by electrons in singlet ground state to move to the singlet excited state and returns back to the single ground state by emitting radiation (fluorescence) with lower energy (longer wavelength). Intensity of fluorescence is plotted as a function of emission wavelength. FFFS is used to measure more turbid or opaque samples, since the signal becomes more independent of penetration of light through the sample. It measures fluorescence emitted only from the sample surface, which reduces the influence of non-fluorescence disturbances. The angle between the sample and light beam can be changed. Thus milk and dairy products can be analyzed without any sample treatment prior to analysis. This is the most significant property of FFFS. The naturally occurring fluorophores in milk are the aromatic amino acids (Tryptophan, Tyrosine and Phenylalanine) and co-enzymes (Flavin adenosine dinucleotide and Nicotinamide adenosine dinucleotide). Trptophan shows the maximum fluorescence among others and it is considered as a biomarker molecule. FFFS is a rapid technique to evaluate quality of milk regarding maillard reaction products. This technique can reveal qualitative changes in the interaction of proteins at molecular level upon heating and acidic coagulation. FFFS technique saves time and gets the overall results quickly.

1. APPLICATIONS OF
FRONT FACE FLUORESCENCE
SPECTROSCOPY (FFFS)
IN MILK
ANUSHA KISHORE
CREDIT SEMINAR ON

2. PRESENTATION OUTLINE
 Introduction
 Basic principle
 Fluorescence properties of milk
 Applications in milk
 Applications in dairy processing
 Advantages
 Disadvantages
 Conclusions

3. INTRODUCTION
 Front Face Fluorescence Spectroscopy (FFFS) was developed by
Parker in 1968.
 It is a technique to reduce the scattering effect by changing the angle
of incidence onto the sample from 90° to 30/60° (Lakowicz, 1983).
 Used to characterize conformational changes that occur during
different manufacturing and storage conditions (Karoui, 2011).

4. BASIC PRINCIPLE
Fundamental principles- Jablonski diagram (Zude, 2008)
By absorbing light, the flurophore gets excited and undergoes vibrational
relaxation and reaches the lower level. When it reaches the lowest level of
excited state it undergoes fluorescence by emitting light (Ma, 2017).

5. Jablonski diagram- Fluorescence Spectroscopy Excitation and Emission spectra

6. BASIC SETUP

7. FLUORESCENCE PROPERTIES
OF MILK
Each fluorophore has a unique excitation and emission spectrum, which
can be used to identify compositional or structural changes of the
particular molecule (Shaikh & Odonnell, 2017).
.
Dairy products contain compounds that naturally or after chemical
modification emit fluorescence.
Fluorophores in milk- aromatic amino acids and coenzymes (Kulmyraez
et. al., 2005)

8. APPLICATIONS IN MILK

9. To determine the effect of heat
treatment in Dairy products
To determine the effect of storage on
Dairy products
Saif Shaikh and Colm Odonnell (2017)

10.  No physical or chemical
treatment is needed to provide
samples
 Deals with optically opaque
systems
 Non-destructive and rapid
method of measuring maillard
products
440 nm
343 nm
Normalized tryptophan emission Spectra (exc. 290nm) of Normal UHT2 milk
containing 0, 40 and 100% of Over-heated UHT milk
Asylbek Kulmyrzaev and Eric Dufour (2002)

11. 354
nm
515 nm
416
nm
429
nm
434
nm
517.5 nm
518 nm
363
nm
365
nm
320 nm
Normalized emission Spectra (exc. 360nm)
of advanced maillard reaction products in
NUHT1 (Normal UHT1), OUHT (over-
heated UHT) and Pasteurized milk
Normalized excitation Spectra (em. 440 nm)
of advanced maillard reaction products in
NUHT1 (Normal UHT1), OUHT (over-
heated UHT) and Pasteurized milk

12.  Qualitative changes in milk protein structures by
monitoring thermal and acidic denaturation of whole
milk proteins
 Ambient conditions (30oC)
 Heating conditions (Heating to 90oC and cooling down to
70oC)
 Acidification conditions (Addition of 1% citric acid at 70oC)
 Discriminate between cow milk, buffalo milk and mixed
milk under different conditions (raw milk, heated milk &
heated milk with coagulant).
Milk Type Proportion (% weight)
Buffalo
Milk (BM)
Cow Milk
(CM)
Mixed
Milk
(MM1)
Mixed
Milk
(MM2)
Mixed
Milk
(MM3)
BM 100 75 50 25 0
CM 0 25 50 75 100
Purba Chakraborty, Bhaswati Bhattacharya, Umashanker Shivhare and Santanu Basu (2020)

13. 345 nm
350 nm

14. Emission maxima and intensity values of the Trp residues in BM, MM and CM under different conditions

17.  Low-heat skim milk powder was
reconstituted to a total solids level
of 12 % (w/w) with distilled water
at ∼40 °C and stirred for 15 min at
150 rpm.
 The reconstituted milk was left to
rehydrate for 30 min at room
temperature, in the dark.
Noemf Ayala, Anna Zamora, Asmund Rinnan, Jordi Saldo and Manuel Castillo (2020)

18. Fluorescence spectra of tryptophan, corresponding to skim milk samples treated at different holding times and temperatures
of a) 70, b) 80, c) 90 and d)100 °C.

20. APPLICATIONS IN DAIRY
PROCESSING
A rapid method to quantify casein in fluid milk by front-face
fluorescence spectroscopy combined with chemometrics
Ma et. al., 2020
Utilization of front-face fluorescence spectroscopy for
monitoring lipid oxidation during Lebanese Qishta aging
Najiba et. al., 2020
Front-face fluorescence spectroscopy combined with
chemometrics to detect high proteinaceous matter in milk and
whey ultrafiltration permeate
Y. B. Ma and
J. K.
Amamcharla
2019
Development and validation of a front-face fluorescence
spectroscopy-based method to determine casein in raw milk
Ma et. al., 2019
Modeling of the changes in bovine milk caused by ultra-high
pressure homogenization using front-face fluorescence
spectroscopy
Liu et al., 2018
Quality Assurance of Model Infant Milk Formula Using a
Front-Face Fluorescence Process Analytical Tool
Heniman et.
al.,
2018

21. Application of front-face fluorescence spectroscopy as a tool
for monitoring changes in milk protein concentrate powders
during storage
K. S. Babu
and J. K.
Amamcharla
2018
Predicting lactulose concentration in heat-treated
reconstituted skim milk powder using front-face fluorescence
Ayala et. al., 2017
Monitoring of mild heat treatment of camel milk by front-
face fluorescence spectroscopy
Kamal, M. &
Karoui, R
2016
Use of Front-Face Fluorescence Spectroscopy to Differentiate
Sheep Milks from Different Genotypes and Feeding Systems
Hammami et.
al.,
2013
Utilisation of front-face fluorescence spectroscopy for the
determination of some selected chemical parameters in soft
cheeses
Karoui et. al., 2006
Utilization of Front-Face Fluorescence Spectroscopy for
Analysis of Process Cheese Functionality
Purna et. al., 2005

22. ADVANTAGES
High sensitivity (ng/ml)
• High selectivity (wavelength maximum)
Rapid and non-invasive analytical method
• Provides information on presence of fluorescent
molecules
Relatively inexpensive

23. DISADVANTAGES

24. CONCLUSION
The FFFS-based methods can be potentially implemented
into dairy foods production facilities and maintain product
qualities.
Fluorescence spectroscopy is able to determine several
properties (functional, composition, nutritional) without the
use of chemical reagents.
A sensitive and specific instrument was applied to characterize
protein leak occurrences in UF permeate.
None of the techniques can compete with FFFS technique in
consideration with the time needed to get the overall result.