Preparation and Properties of Probiotic Chocolates Using Yoghurt Powder

Abstract

Milk chocolates were prepared by replacing skim milk powder in the formulation with yoghurt powder at 50% and 100% levels. The effect of incorporating yoghurt powder on quality of chocolates was studied. No significant changes were observed in fatty acid profile and hardness of the chocolates. Sour taste of chocolate with yoghurt powder due to its acidity was neutralized by adding calculated amount of sodium bicarbonate. Sensory analysis showed that probiotic chocolates were highly acceptable and similar to control chocolate. Microbiological studies of chocolates showed the presence of Lactobacillus species to the extent of 3.37 log·cfu/g, which were not present in the control sample prepared only with skim milk powder. Rheological studies showed that milk chocolate prepared using yoghurt powder at 50% showed no significant changes in yield value compared to that of control, but at 100% addition a considerable decrease in yield value was observed. Microstructural properties of chocolate with 50% addition of yoghurt powder showed smaller particles adhering to the cocoa and sugar crystals but at 100% addition of yoghurt powder, the cocoa particles were completely covered by smaller yoghurt powder matrix.

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Food and Nutrition Sciences, 2013, 4, 276-281
http://dx.doi.org/10.4236/fns.2013.43037 Published Online March 2013 (http://www.scirp.org/journal/fns)
Preparation and Properties of Probiotic Chocolates Using
Yoghurt Powder
Ramakrishna Chetana, Sunki Reddy Yella Reddy*, Pradeep Singh Negi
Human Resource Development CSIR—Central Food Technological Research Institute (Council of Scientific and Industrial Research)
Mysore, India.
Email: *syreddy52@gmail.com
Received January 2nd, 2013; revised February 7th, 2013; accepted February 15th, 2013
ABSTRACT
Milk chocolates were prepared by replacing skim milk powder in the formulation with yoghurt powder at 50% and
100% levels. The effect of incorporating yoghurt powder on quality of chocolates was studied. No significant changes
were observed in fatty acid profile and hardness of the chocolates. Sour taste of chocolate with yoghurt powder due to
its acidity was neutralized by adding calculated amount of sodium bicarbonate. Sensory analysis showed that probiotic
chocolates were highly acceptable and similar to control chocolate. Microbiological studies of chocolates showed the
presence of Lactobacillus species to the extent of 3.37 log·cfu/g, which were not present in the control sample prepared
only with skim milk powder. Rheological studies showed that milk chocolate prepared using yoghurt powder at 50%
showed no significant changes in yield value compared to that of control, but at 100% addition a considerable decrease
in yield value was observed. Microstructural properties of chocolate with 50% addition of yoghurt powder showed
smaller particles adhering to the cocoa and sugar crystals but at 100% addition of yoghurt powder, the cocoa particles
were completely covered by smaller yoghurt powder matrix.
Keywords: Probiotic Chocolates; SEM; Rheology; Yoghurt Powder
1. Introduction
Chocolate is a suspension of fine solid particles of sugar,
cocoa and milk powder in a continuous fat phase. Choco-
lates are solid at ambient (20˚C - 25˚C) and melt at body
temperature (37˚C) giving a smooth suspension of par-
ticulate solids with a pleasing cooling sensation in the
mouth [1]. The continuous phase influences the sensory
characteristics such as mouth feel or melt in mouth.
Despite high fat and sugar contents, chocolate con-
sumption makes a positive contribution to human nutria-
tion through provision of antioxidants, principally poly-
phenols including flavonoids such as epicatechin, cate-
chin and notably, the procyanidins [2]. Chocolates also
contain minerals, specifically potassium, magnesium,
copper and iron. Due to presence of cocoa, it is rich in
natural antioxidants having health benefits. Milk solids
added as spray-dried skimmed milk powder or full cream
milk powder contributes to flavour, texture and liquid
flow properties [3].
Numerous functional foods are consumed as part of a
normal diet and they provide consumers with well-docu-
mented and physiological benefits such as probiotic bac-
teria. Probiotics are live microorganisms and proliferate
in the human bowels that confer a health benefit by al-
tering the enteric microflora. The main sources of these
organisms are fermented dairy products, for example,
yoghurts. However, the functional dairy product must
contain a defined number of live probiotic bacteria (usu-
ally at least 106 cfu/g). Furthermore, their number at the
end of the shelf life is the most important criterion when
the health-promoting value of a given foodstuff is evalu-
ated [4-7].
Probiotic bacteria beneficially affect human health by
improving the gut micro biota balance and the defenses
against pathogens. Additional health benefits attributed
to probiotics are the stimulation of the immune system,
blood cholesterol reduction, vitamin synthesis, anti-car-
cinogenesis and anti-bacterial activities. Two other im-
portant criteria to determine the efficacy and the success
of the product containing probiotics are the acceptance of
the product by the consumers and the survival of probi-
otic microorganisms during its production [8]. Lactoba-
cilii, Bifido bacteria and several other lactic acid bacteria
are regarded as probiotics, as they do not induce mucosal
inflammation. The main sources of these organisms are
fermented dairy products for example yoghurt (curd). In
general, the food industry had applied the recommended
level of 106 cfu/gm at the time of consumption of lacto-
*Corresponding author.
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Preparation and Properties of Probiotic Chocolates Using Yoghurt Powder 277
bacillus acidiophilus, Bifidobacteria and other probiotic
bacteria [9]. Traditional yoghurt is produced from milk,
fermented by strains of Streptococcus thermophilus and
Lactobacillus delbrueckii subsp. bulgaricus [10].
Today, India is the largest producer of milk in the
world and the Indian dairy industry is witnessing rapid
changes. Yoghurt/curd is the most popular fermented dairy
product in India, prepared by the use of mixed mesophilic
cultures that ferment lactose to lactic acid. Products like
yoghurt are known more for their therapeutic significance
than nutritional value [11]. It has a limited keeping quality
of 1 - 2 days at ambient temperature and its quality is not
retained for more than 1 week under refrigerated conditions
[12]. Dried yoghurt powder has enhanced shelf life and it
can also be used as a base for formulation of health foods.
Nebesny et al. [13] have reported that chocolate formu-
lated with isomalt and enriched with viable cells of lactic
acid bacteria, introduced in the form of powdered yo-
ghurt, is not only a sucrose-free, low-calorie product but
additionally displays nutritional and dietetic attributes,
and can be regarded as a functional food additional spe-
cies in the fingerprints. Possemiers et al. [14] have re-
ported bacteria and chocolate to be a successful combina-
tion for probiotic delivery. They have showed that coating
of the probiotics in chocolate is an excellent solution to
protect them from environmental stress conditions and
for optimal delivery. The aims of the present study is to
incorporate dried probiotic powder for the preparation of
probiotic chocolate, and study the effects of replacing
skim milk powder with yoghurt powder on the rheologi-
cal and other quality parameters of milk chocolate.
2. Materials and Methods
2.1. Composition of Yogurt
Yoghurt was procured locally in bulk and used for the
studies. The yoghurt had protein content was 3.3%, fat
3.0%, minerals 0.7%, calcium 120 mg, vitamin A 35 ug
and carbohydrates (as Lactose) 3.9%.
2.2. Preparation of Yoghurt Powder
The yoghurt was dried in a freeze drier (Lyophilisation
Inc. USA, Model LT 5S): Freezing cycle : −26˚C for 2 h,
Drying cycle: −25˚C to +25˚C for 18 h and vacuum from
100,000 m·torr to 250 m·torr were used for a total period
of 20 h. The powder obtained was packed in polypro-
pylene covers and stored in refrigerator.
2.3. Composition of Yoghurt Powder
The prepared yoghurt powder had of: 3.0% - 3.5% mois-
ture, 35% protein, 1.5% - 2.0% fat, and total acidity of
5.8% as lactic acid.
2.4. Preparation of Chocolate
Milk chocolate was prepared using the formulation given
in Table 1. Skim milk powder was replaced at 50% and
100% level by yoghurt powder in milk chocolate formu-
lation and compared with control chocolate prepared
with skim milk alone. All the ingredients were mixed and
passed through a triple-roll chocolate refiner (Pascal,
England), three times, keeping the distance between the
rollers and number of passes constant for all the batches.
The mass was then conched by adding the remaining
cocoa butter and lecithin for 3 h at 50˚C - 55˚C. The
mass after conching was taken for viscosity measurement.
The mass was then tempered and moulded. The samples
were kept at refrigerated condition for further analyses.
2.5. Texture of Chocolate
The hardness of the chocolate was measured using a
Lloyd’s texture measuring system (model LR 5K, UK).
The samples of uniform dimension (4 × 8 × 1 cm·lbh),
were conditioned by keeping at 25˚C ± 2˚C for about 3 h
before measuring. Penetration was measured at 25˚C ±
2˚C using a probe of 3 mm dia at a speed of 50 mm/min
and 2 mm deflection using a 50 N load cell; the force
required to penetrate was measured as hardness (N) and
an average of 12 measurements was reported.
2.6. Fatty Acid Analysis
The fat from chocolate samples was extracted with chlo-
roform and converted to fatty acid methyl esters (FAME)
using KOH/Methanol (AOCS, 1993). FAME were ana-
lysed by GC (Varian GC 450, Hercudiesweg, The Neth-
erlands) with FID and using Supelco, SP-2340 (0.25 mm
× 30 m) capillary column, programmed from 50˚C to
200˚C at 5˚C/min and maintaining at 200˚C for 10 min;
injection temperature 230˚C, split ratio 1:20; detector
temperature 240˚C and nitrogen flow, 0.9 mL/min. The
fatty acids were identified by using authentic standards
and reported as relative percentage.
Table1. Viscosity of chocolates prepared with yoghurt powder.
Chocolate Casson Yield Value (Pa) τ0 Casson Plastic Viscosity (Pa S)

p R2
Control 40.23b 1.87a 0.99
50% Yoghurt Powder 41.02b 2.11a 0.99
100% Yoghurt Powder 28.65a 3.76b 0.99
a,bMean values (n = 6) bearing different superscripts in a column are significantly different (p ≤ 0.05).
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Preparation and Properties of Probiotic Chocolates Using Yoghurt Powder
278
2.7. Viscosity Measurement
The rheological behaviour of chocolate was measured at
40˚C using HAAKE Viscotester, VT550 (Haake, Karlsruhe,
Germany), using coaxial cylinder SV, from 0 to 100
shear rate in 3 min, and the curves of shear rate versus
shear stress and viscosity were recorded. The shear rate
and shear stress curves were subjected to various rheologi-
cal models to see the best fit and determine viscosity,
using Rheo Win (Haake, Karlsruhe, Germany) software.
All the measurements were made in triplicate and the
average was reported. The basic casson equation is used
to describe the flow behavior of chocolate. Casson yield
value and plastic viscosity were used to describe the flow
behavior of chocolate.
2.8. Sensory Evaluation
A trained panel was employed for carrying out sensory
evaluation of chocolate by following the method of
Quantitative Descriptive Analysis (QDA). The defini-
tions of the attributes were discussed and descriptors
were developed by asking the panelists to describe the
product with the suitable descriptive terms for develop-
ment of a score card, which consisted of each attribute on
a 15-cm line scale. Quantitative descriptive analysis
methods were adopted and the panelists were asked to
mark the intensity of each attribute [15]. The main de-
sirable sensory attributes of chocolate, such as gloss,
snap, melt-in-mouth, sweetness, chocolate flavour and
overall quality were assessed by a panel of 15 trained
judges. Calculated amount of sodium bicarbonate based
on their neutralization equivalent of the total acid present
in yoghurt powder was added to the chocolates to avoid
the acidic taste in chocolates. The judges were asked to
mark by drawing a vertical line on the scale for all qual-
ity attributes of the coded samples.
2.9. Scanning Electron Microscope (SEM)
The microstructure of chocolate samples was studied by
SEM using LEO Scanning electron model 435 VP (Leo
electronics system, Cambridge, UK). The chocolate was
defatted with hexane and placed on the sample holder
with the help of a double scotch tape and sputter coated
with gold (2 min, 2 m Bar) where it was observed at 15
kV.
2.10. Statistical Analysis
Data obtained was statistically analysed using Duncan’s
Multiple Range Test (DMRT) at significant level of P ≤
0.05 [16]. All the analyses were performed in triplicate
except for texture (n = 12). The mean values with stan-
dard deviations (SD) are reported.
2.11. Microbiological Analysis
Number of Lactobacilli was counted by pour plate method
using 1 ml of appropriate dilution of each sample in De
Man Rogosa-Sharpe agar (MRS agar, Hi media, Mum-
bai). After 2 days of incubation at 37˚C the colonies were
counted and the results expressed in logarithm of colo-
nies forming units per gram of product (log·cfu/g). The
representative colonies were tested for absence of cata-
lase to confirm the presence of LAB
3. Results and Discussion
3.1. Viscosity of Chocolates
The viscosity of chocolate plays an important role during
manufacture of chocolate. The solid ingredients, proc-
essing parameters and amount of fat influence viscosity
of chocolate mass. The rheological behavior of chocolate
followed non-Newtonian flow, as reported in literature,
due to presence of solids in molten fat and followed the
Casson model, which is the best fit as shown by the cor-
relation coefficient (Table 1). Casson model is recom-
mended by IOCCC [17] and has been used as an interna-
tionally accepted standard model for determination of
viscosity of chocolates. It is now accepted and applied as
an appropriate mathematical model for predicting flow
behavior and rheological analysis of different kinds of
chocolates [18,19].
Milk chocolate prepared using yoghurt powder at 50%
by weight of milk powder showed no significant changes
in yield value compared to that of control chocolate pre-
pared with milk powder. However, chocolate with 100%
addition of yoghurt powder showed considerable decrease
in yield value. Plastic viscosity of chocolate increased
with increase in addition of yoghurt powder from 50% to
100% (Table 1), which may be due to finer particles of
yoghurt powder compared to milk solids.
3.2. Texture of Chocolate
Only marginal differences in hardness were observed
among the chocolates, i.e., control and with added yo-
ghurt powder, ranging from 34 to 36 N.
3.3. Fatty Acid Composition
No significant differences in fatty acid composition were
observed among the chocolates prepared with yoghurt
powder as all samples contain cocoa butter and milk fat
as fat sources (Table 2). Short chain fatty acids are from
milk fat from milk powder or yoghurt powder. The prod-
uct contains about 30% of monounsaturated (oleic) fatty
acids and equal quantity of stearic acid, which is neutral
with respect to affecting serum cholesterol [20].
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Preparation and Properties of Probiotic Chocolates Using Yoghurt Powder 279
Table 2. Fatty acid composition (%) of fat in chocolates prepared with yoghurt powder.
Chocolate with
Fatty Acid Control 50% Yoghurt Powder 100% Yoghurt Powder
C4:0 2.50 2.45 2.50
C10:0 1.50 - -
C14:0 0.42 0.71 1.32
C16:0 24.63 25.18 25.87
C18:0 34.61 35.07 34.56
C18:1 32.2 32.32 32.82
C18:2 4.00 3.00 2.96
Mean values (n = 3).
3.4. Microstructural Properties of Probiotic
Chocolates
The outer topography of probiotic milk chocolates were
assessed by SEM (Figures 1(a)-(c)), in order to study the
effect of addition of yoghurt powder into chocolates.
Figure 1(a) shows coarse particles of cocoa mass sheared
during refining and conching process in the manufacture
of chocolate. A few sugar particles and skim milk pow-
der can also be seen along with the cocoa mass. Skim
milk powder particles are smaller than cocoa and sugar
particles and adhere to the larger and coarser cocoa parti-
cles.
In chocolates with 50% addition of yoghurt powder,
the smaller particles adhering to the cocoa and sugar crys-
tals are more in number (Figure 1(b)). In chocolate with
100% addition of yoghurt powder, the cocoa particles are
completely covered by smaller yoghurt powder matrix
(Figure 1(c)). Skim milk powder shows surface dents,
which can be attributed to the atomization conditions [21]
but yoghurt powder which is freeze dried has smaller
particle size compared to skim milk powder which forms
a cluster around the larger cocoa and sugar particles
without dents.
3.5. Microbiological Studies
Microbiological studies revealed that the chocolates pre-
pared with yoghurt powder showed the presence of lactic
acid bacteria to the extent of 3.37 log·cfu/g, which were
not present in the control sample prepared only with skim
milk powder (Table 3). There was no significant increase
in the viable LAB count in 100% substitution over 50%
as the cells were exposed (as seen in SEM) and probably
they were more susceptible to processing treatments. It is
well established that foods containing viable lactic acid
bacteria as probiotics, improve gut health.
3.6. Sensory Analysis
Sensory evaluation revealed that chocolate with 50%
addition of yoghurt powder was more acceptable than
0
2
4
6
8
10
12
14
Colour
Gloss
Snap
Melt in mouth
SweetnessSourness
choc f lavour
Off taste
OQ
Control 50% addition of yogh urt powder 100 % addition of yoghurt powder
Figure 1. Sensory evaluation of chocolates prepared with
yoghurt powder.
Table 3. Microbiological studies of chocolates prepared
with yoghurt powder.
Samples Lactobacillus sp. (log·cfu/g)
Yoghurt Powder 4.758 ± 0.11b
50% Yoghurt Powder 3.581 ± 0.23a
100% Yoghurt Powder 3.586 ± 0.24a
Control -Nil-
Mean values (n = 6) bearing different superscripts in a column are signifi-
cantly different (p ≤ 0.05).
that with 100% addition and both had slightly lower
overall quality when compared to control prepared only
with skim milk powder (Figure 2). No significant dif-
ferences were observed in attributes such as colour, gloss
and sweetness among all the three samples. Snap, melt in
mouth and chocolate flavour attributes decreased with
addition of yoghurt powder. Chocolates with added yo-
ghurt powder are sour in taste due to acid present in yo-
ghurt powder. However, chocolates with addition of re-
quired amount of sodium bicarbonate to neutralize the
acid did not show any sour taste. Similar results have
been reported by Nebesny et al. [13], where sensory
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Preparation and Properties of Probiotic Chocolates Using Yoghurt Powder
280
(a) Control chocolate
(b) Chocolate with 50% addition of yoghurt powder
(c) Chocolate with 100 addition of yoghurt powder
Figure 2. Scanning electron micrographs (SEM) of chocolates prepared with yoghurt powder (Mag. 4000×).
Copyright © 2013 SciRes. FNS

Preparation and Properties of Probiotic Chocolates Using Yoghurt Powder 281
attributes of sucrose-free yoghurt-containing dark and
milk chocolates and their yoghurt-free counterparts re-
vealed excellent quality. The sensory attributes of yo-
ghurt and isomalt containing dark chocolates received a
high average score which was only slightly lower than
that of analogous milk chocolates.
4. Conclusion
Milk chocolate with yoghurt powder was prepared re-
placing milk powder to make it probiotic. Marginal dif-
ferences in viscosity, texture and sensory evaluation of
chocolate with yoghurt were observed in comparison
with those of control milk chocolate. Chocolate with yo-
ghurt powder contain probiotic lactobacilli species and
thus making it probiotic. Chocolate is reported to contain
natural antioxidants and the nutritional quality of this
was further enhanced by making it probiotic. Thus, milk
chocolate with probiotic yoghurt powder was prepared
without affecting the desirable quality of conventional
chocolate.
REFERENCES
[1] S. T. Beckett, “Conching,” In: S. T. Beckett, Ed., In-
dustrial Chocolate Manufacture and Use, 3rd Edition,
Blackwell Science, Oxford, 1999, pp. 1-81.
[2] C. L. Hii, C. L. Law, S. Suzannah, S. Miswani and M.
Cloke, “Polyphenols in Cocoa (Theobroma cacao L.),”
Asian Journal of Food and Agro Industries, Vol. 2, No. 4,
2009, pp. 702-722.
[3] S. J. Haylock and T. M. Dodds, “Ingredients from Milk,”
In: S. T. Beckett, Ed., Industrial Chocolate Manufacture
and Use, 3rd Edition, Blackwell Science, Oxford, 1999,
pp. 1-77.
[4] B. German, E. J. Schiffrin, R. Reniero, B. Mollet, A.
Pfeifer and J. R. Neeser. “The Development of Functional
Foods: Lessons from the Gut,” Trends in Biotechnology,
Vol. 17, No. 12, 1999, pp. 492-499.
[5] M. A. F. Belem, “Application of Biotechnology in the
Product Development of Nutraceuticals in Canada,” Trends
Food Science and Technology, Vol. 10, No. 3, 1999, pp.
101-106. doi:10.1016/S0924-2244(99)00029-1
[6] N. M. Childs, “Nutraceutical Industry Trends,” Journal of
Nutraceutical, Functional and Medicinal Foods, Vol. 2,
No. 1, 1999, pp. 73-85. doi:10.1300/J133v02n01_07
[7] C. J. Dillard and J. B. German, “Phytochemicals: Nu-
traceuticals and Human Health,” Journal of Food Agri-
cultural Sciences, Vol. 80, No. 12, 2000, pp. 1744-1756.
doi:10.1002/1097-0010(20000915)80:12<1744::AID-JSF
A725>3.0.CO;2-W
[8] C. N. Heenan, M. C. Adams, R. W. Hosken and G. H.
Fleet, “Survival and Sensory Acceptability of Probiotic
Microorganisms in a Non-Fermented Frozen Vegetarian
Dessert,” LWT—Food Science & Technology, Vol. 37 No.
4, 2004, pp. 461-466. doi:10.1016/j.lwt.2003.11.001
[9] T. D. Boylston, C. G. Vinderola, H. B. Ghoddusi and J. A.
Reinheimer, “Incorporation of Bifidobacteria into Cheeses:
Challenges and Rewards,” International Dairy Journal,
Vol. 14, No. 5, 2004, pp. 375-387.
doi:10.1016/j.idairyj.2003.08.008
[10] V. Xanthopoulos, D. Petridis and N. Tzanetakis, “Char-
acterization and Classification of Streptococcus thermo-
philus and Lactobaccilus delbrueckii Sub Species Bulga-
ricus Strains Isolated from Traditional Greek Yoghurt,”
Journal of Food Science, Vol. 66, No. 5, 2001, pp. 747-
752. doi:10.1111/j.1365-2621.2001.tb04632.x
[11] R. P. Aneja, B. N. Mathur, R. C. Chandan and A. K.
Banerjee, “Technology of Indian Milk Products,” Dairy
India Year Book Publications, New Delhi, 2002.
[12] M. Kamruzzaman, M. N. Islam and M. M. Rahman,
“Shelf life of Different Types of Dahi at Room and Re-
frigeration Temperature,” Pakistan Journal of Nutrition,
Vol. 1, No. 6, 2002, pp. 263-266.
[13] E. Nebesny, D. Żyżelewicz, I. Motyl and Z. Libudzisz,
“Properties of Sucrose-Free Chocolates Enriched with
Viable Lactic Acid Bacteria,” European Food Research
and Technology, Vol. 220, No. 3-4, 2004, pp. 358-362.
doi:10.1007/s00217-004-1069-0
[14] S. Possemiers, M. Marzorati, W. Verstraete and T. Van
de Wiele, “Bacteria and Chocolate: A Successful Combi-
nation for Probiotic Delivery,” International Journal of
Food Microbiology, Vol. 141, No. 1-2, 2010, pp. 97-103.
doi:10.1016/j.ijfoodmicro.2010.03.008
[15] H. Stone and J. L. Sidel, “Sensory Evaluation Practices,”
3rd Edition, Academic Press/Elsevier, London/New York,
2004.
[16] B. D. Duncan, “Multiple Range and Multiple F-Test,”
Biometrics, Vol. 11, No. 1, 1955, pp. 1-42.
doi:10.2307/3001478
[17] IOCCC (International Office of Cocoa, Chocolate, and
Sugar Confectionery), “Viscosity of Cocoa and Chocolate
Products (Analytical Method: 46),” CABISCO, Brussels,
2000.
[18] S. Abbasi, “Food Texture and Viscosity: Concept and
Measurement,” Marzedanesh Publishers, Tehran, 2007.
(in Farsi)
[19] J. Chevalley, “Chocolate Flow Properties,” In: S. T. Be-
ckett, Ed., Industrial Chocolate Manufacture and Use,
3rd Edition, Blackwell Science, Oxford, 1999, pp. 182-
200.
[20] W. E. Connor, “Harbingers of Coronary Heart Disease:
Dietary Saturated Fatty Acids and Cholesterol. Is Chocolate
Benign Because of Its Stearic Acid Content?” American
Journal of Clinical Nutrition, Vol. 70, No. 6, 1999, pp.
951-952.
[21] E. Bylaite, P. R. Venskutonis and R. Mapdbieriene,
“Properties of Caraway (Carum carvi L.) Essential Oil
Encapsulated into Milk Protein-Based Matrices,” Euro-
pean Food Research Technology, 2001, Vol. 212, No. 6,
pp. 661-670. doi:10.1007/s002170100297
Copyright © 2013 SciRes. FNS