Production of a Functional Frozen Yogurt Fortified with Bifidobacterium spp.

Abstract

Frozen dairy products have characteristics of both yogurt and ice cream and could be the persuasive carriers of probiotics. Functions of the frozen yogurt containing viable bifidobacterial cells are recognized and favored by the people of all ages. We developed a kind of yogurt supplemented by Bifidobacterium species. Firstly, five strains of Bifidobacterium spp. ( Bifidobacterium bifidum ATCC 11547, Bifidobacterium longum ATCC 11549, Bifidobacterium infantis ATCC 11551, Bifidobacterium adolescentis ATCC 11550, and Bifidobacterium breve ATCC 11548) were evaluated based on the feasibility criteria of probiotics, comprising acid production, bile tolerance, and adhesion to epithelial cells. Formerly, we combined the optimum strains with yogurt culture ( Lactobacillus delbrueckii subsp. bulgaricus EMCC 11102 and Streptococcus salivarius subsp. thermophilus EMCC 11044) for producing frozen yogurt. Finally, physiochemical properties and sensory evaluation of the frozen yogurt were investigated during storage of 60 days at −18°C. Results directed that Bifidobacterium adolescentis ATCC 11550 and Bifidobacterium infantis ATCC 11551 could be utilized with yogurt culture for producing frozen yogurt. Moreover, the frozen yogurt fermented by two bifidobacterial strains and yogurt culture gained the high evaluation in the physiochemical properties and sensory evaluation. In summary, our results revealed that there was no significant difference between frozen yogurt fermented by Bifidobacterium spp. and yogurt culture and that fermented by yogurt culture only.

Full text

Research Article
Production of a Functional Frozen Yogurt Fortified with
Bifidobacterium spp.
Amro Abdelazez,1,2 Zafarullah Muhammad,1Qiu-Xue Zhang,1Zong-Tao Zhu,1
Heba Abdelmotaal,3,4 Rokayya Sami,5,6 and Xiang-Chen Meng1
1Key Laboratory of Dairy Science of Ministry of Education, Northeast Agricultural University, Harbin 150030, China
2Department of Dairy Microbiology, Animal Production Research Institute, Agriculture Research Center,
Dokki, Giza 12618, Egypt
3Department of Microbiology, Soil, Water and Environment Research Institute, Agriculture Research Center, Giza 12619, Egypt
4Department of Microbiology and Biotechnology, College of Life Sciences, Northeast Agricultural University,
Harbin 150030, China
5Department of Nutrition and Food Science, Taif University, Taif, Al-Huwayah 888, Saudi Arabia
6Department of Food Science, Northeast Agricultural University, Harbin, Heilongjiang 150030, China
Correspondence should be addressed to Xiang-Chen Meng; xchmeng@hotmail.com
Received 11 November 2016; Accepted 5 January 2017; Published 11 June 2017
Academic Editor: Pratik Banerjee
Copyright ©  Amro Abdelazez et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Frozen dairy products have characteristics of both yogurt and ice cream and could be the persuasive carriers of probiotics. Functions
of the frozen yogurt containing viable bidobacterial cells are recognized and favored by the people of all ages. We developed
a kind of yogurt supplemented by Bidobacterium species. Firstly, ve strains of Bidobacterium spp. (Bidobacterium bidum
ATCC , Bidobacterium longum ATCC , Bidobacterium infantis ATCC , Bidobacterium adolescentis ATCC ,
and Bidobacterium breve ATCC ) were evaluated based on the feasibility criteria of probiotics, comprising acid production,
bile tolerance, and adhesion to epithelial cells. Formerly, we combined the optimum strains with yogurt culture (Lactobacillus
delbrueckii subsp. bulgaricus EMCC  and Streptococcus salivarius subsp. thermophilus EMCC ) for producing frozen
yogurt. Finally, physiochemical properties and sensory evaluation of the frozen yogurt were investigated during storage of  days
at −∘C. Results directed that Bidobacterium adolescentis ATCC  and Bidobacterium infantis ATCC  could be utilized
with yogurt culture for producing frozen yogurt. Moreover, the frozen yogurt fermented by two bidobacterial strains and yogurt
culture gained the high evaluation in the physiochemical properties and sensory evaluation. In summary, our results revealed that
there was no signicant dierence between frozen yogurt fermented by Bidobacterium spp. and yogurt culture and that fermented
by yogurt culture only.
1. Introduction
Diet plays an important role in preventing diseases and ensur-
ing health. Hence, the consumption of functional foods (i.e.,
benecial compounds or foods containing microorganisms)
which provide health benets with a reduction of coronary
heart disease, obesity risk, and diabetes has increased during
thelastdecade[].econceptofusingprobioticstoimprove
and maintain human health is not new at all. Probiotic
microorganisms are usually used as culture concentrates in
dried or deep-freeze forms to be added to food for industrial
or home uses []. In addition to the probiotic foods, there
are various health products and pharmaceutical preparations
containing probiotics on the market [].
Bidobacterium is an important group of probiotic cul-
tures and commonly used in fermented dairy products that
contributes a major part in the human intestinal micro-
biotainhealthyhumans.eyareconsideredtoprovide
Hindawi
BioMed Research International
Volume 2017, Article ID 6438528, 10 pages
https://doi.org/10.1155/2017/6438528

BioMed Research International
many benecial eects including improvement of lactose
digestibility, anticarcinogenic activity, reduction of serum
cholesterol level, synthesis of B vitamins, and facilitation in
calcium absorption []. Moreover, numerous studies with
dierent strains of Lactobacillus and Bidobacterium have
beenperformedinvitroandinvivo,inhumansandanimal
models to investigate their immunomodulatory properties
and probiotic potential to treat various infectious, allergic,
and inammatory conditions [, ]. Even though Bidobac-
terium strains have already been used in dairy products, they
have some inferior behavioral characteristics compared with
the traditional lactic acid bacteria (LAB) used in fermented
dairy products, hindering their possible applications [].
Vitally, they represent weaker growth and acid production
in cow milk and require long fermentation times, anaerobic
conditions, and low redox potential for their growth [].
ere are clear relationship between the food we eat and
our health. erefore, some reports have investigated ice
cream and yogurt as probiotic carrier. Hence, frozen yogurt
is a novel way of combining the characteristics of ice cream
with the therapeutic properties of yogurt that are considered
as a healthy alternative to ice cream for the people suering
from cardiovascular diseases and lactose intolerance [, –
]. e aim of study was to examine dierent factors
aecting survival and activity of ve species of bidobacteria,
study the viability of two chosen Bidobacterium species in
manufactured frozen yogurt under dierent conditions, and
investigate the eect of storage temperatures on their viability.
2. Materials
2.1. Additives. Skim milk powder, vanilla, and sugar were
purchased from local market. Stabilizer, emulsier, and Cre-
mondan SE  veg were provided by Danisco Ingredients,
Denmark.
2.2. Bacterial Strains. Freeze dried Lactobacillus delbrueckii
subsp. bulgaricus EMCC  and Streptococcus salivar-
ius subsp. thermophilus EMCC  and Bidobacterium
species including Bidobacterium bidum ATCC , Bi-
dobacterium longum ATCC , Bidobacterium infantis
ATCC , Bidobacterium adolescentis ATCC , and
Bidobacterium breve ATCC  were provided by Cairo
Microbiological Resources Center, Egypt.
3. Methods
3.1. Determination of Maximum Growth Rate and Maxi-
mumAcidicationofBidobacteriumspp.StrainsinMRSL.
Bidobacterium spp. were inoculated (% v/v) and grown
in MRSL (Man Rogosa Sharpe) broth (Oxoid, Basingstoke,
UK) supplemented with % (w/v) lactose (Win Lab, Gem-
ini House, Middlesex, Hab ET, UK) and .% (w/v) L-
cysteine-HCL (Merck, Germany) at ∘C under anaerobic
conditions (BBL Gas Pak, Becton Dickinson, Cockeysville
MA,USA).ebacterialgrowthwasmonitoredbymeasur-
ing the absorbance with a spectrophotometer (DU , Beck-
man Coulter,USA) at  nm. Moreover, pH was determined
by using pH meter (MP , Metler Toledo, Greifensee,
Switzerland). e maximum acidication rate was reported
according to [].
3.2. Bile Salts Tolerance of Bidobacterium spp. According to
[] Bidobacterium spp. strains were inoculated in MRSL
broth added to .% (w/v) oxgall powder (Merck, Germany)
and incubated at ∘C under anaerobic conditions for  hr.
Bacterial growth was monitored by measuring absorbance
with a spectrophotometer at  nm aer  hr. e obtained
absorbance values were plotted against the incubation time.
Strain inoculated in MRSL broth without oxgall powder was
taken as the control. Correlation between all the results of
Bidobacterium spp. resistance to bile salts was determined
by the principal component analysis (PCA) using XLSTAT
soware.
3.3. Calculation of Survival Rate in Bile Salts. e survival
rate was calculated by using the following formula reported
by []:
%Bilesurvival=log 𝑁1
log 𝑁0×100. ()
log 𝑁1is absorbance of culture in MRSL broth con-
taining .% bile salts.
log 𝑁0is absorbance of culture in MRSL broth with-
out bile salts.
3.4. Adhesion of Bidobacterium spp. to Intestinal Epithelial
Cells. According to [] for the adherence assay, ve Bi-
dobacterium spp. strains were tested for the adherence to
epithelial cells. Bidobacterium spp. strains were inoculated
in MRSL broth and incubated overnight at ∘C under anaer-
obic conditions. e cultures were adjusted overnight to .
×8CFU/mlandthenmlofBidobacterium spp. cultures
was removed and centrifuged at  ×gRPMformin.e
supernatant was discarded, and ml PBS (pH .) was added
and mixed using vortex. e crop scraping of epithelial cells
was prepared by scrapping o the epithelium from rabbit
duodenum with the edge of a microscope slide, washed by
phosphate buered saline, and suspended in buer (pH .).
Moreover, cell cultures were washed ve times with sterile
phosphate buered saline (PBS) (pH .). ereaer, .ml
of epithelial cell suspension was added to . ml of bacterial
cell suspension. e mixture was centrifuged at  ×gRPM
forminandthenincubatedat
∘C for  min. Finally,
binding between the bidobacterial cells and epithelial cells
was examined by gram stained phase contrast microscopy
(magnication fold, x). e adhered bidobacterial cells
were determined by counting adhering bidobacterial cells in
 randomly selected microscopic elds.
3.5. Manufacturing Procedure of Frozen Yogurt
3.5.1. Preparation of Yogurt. Experimental plain yogurt was
prepared by heating pasteurized whole milk at ∘Cfor

BioMed Research International 
minutes and subsequently cooled to ∘C en, it was divided
into ve separate containers:
Formula 1 (C) inoculated with % w/w starter yogurt
culture with no Bidobacterium spp.
Formula 2 (C + A) inoculated with % w/w starter yogurt
culture + % w/w of B. adolescentis.
Formula3(C+B)inoculated with % w/w starter yogurt
culture + % w/w of B. infantis.
Formula 4 (A + B) inoculated with % w/w B. adolescentis
+%w/wofB. infantis.
Formula5(C+A+B)inoculated with % w/w starter
yogurt culture + % w/w B. adolescentis +%w/wofB.
infantis.
e inoculated mixtures were incubated at ∘Cuntilthe
pH . was obtained.
3.6. Preparation of Frozen Yogurt. Five frozen yogurt blends,
each of three replicates, were prepared. All mixtures were
standardized to contain % fat, % milk solids not fat, %
sugar, .% stabilizer/emulsier, and .% vanilla. In each
treatment, mixed ingredients were homogenized together by
using the method described by [] with some modications
andthenheatedat
∘Cformin.Allmixeswerecooledat
∘C and then aged overnight at the same temperature. On the
other hand, prepared yogurt was added (% v/v) to ve ice
cream mixes prior to freezing. e freezing was performed in
a horizontal batch freezer (Taylor Co., USA) and hardened at
−∘C for  h before analyses.
3.7. Physicochemical Analyses. Frozen yogurt samples were
stored at − ±∘C for  days, and the physicochemical
analyseswereperformedat,,,andd.Titratableacid
(TA) and total solid (TS) were analyzed for all frozen yogurt
samples according to [], and pH was determined by pH
meter (MP , Metler Toledo, Greifensee, Switzerland).
3.8. Overrun and Meltdown Tests. e overrun was calculated
according to [].
Overrun =[(𝑊1 − 𝑊2)
𝑊2 ]×100, ()
where 𝑊1is weight of the mix and 𝑊2is weight of the same
volume of frozen yogurt. e meltdown test was conducted
in a chamber with controlled temperature (∘C). According
to the method described by []. Results were expressed as a
timeforcollectionofeachmlofliquid.
3.9. Hardness. Texture analysis was performed using Texture
Analyzer (TA.XT Plus Texture Analyzer, UK). e samples
were stored in  mm plastic containers at −∘Cuntil
analysis. Measurement was carried out by using a cylindrical
probe. Penetration depth at the geometrical center of the
sample was  mm and penetration speed was set at  mm/s.
e hardness was determined as the peak compression force
(g) during penetration [].
3.10. Enumeration of Viable Bidobacterium spp. in Frozen
Yo g ur t . e viable bidobacterial cell count in frozen yogurt
samples containing Bidobacterium spp. was determined and
expressed as colony forming units (CFU/mL) during storage
of , , , and  d at − ±∘C. Bidobacterial cell counts
were enumerated on MRSL agar using pour plate technique.
e plates were incubated anaerobically at ∘Cforhr.
Survival rates percentage of bidobacteria was calculated
according to [].
3.11. Sensory Appraisal. Organoleptic properties of frozen
yogurt were evaluated aer  days of storage according to
[], for avor ( points), body and texture ( points),
appearance ( points), melting quality ( points), and
total scores ( points) by  panelists of the experienced
sta members of the Dairy Science Department, Faculty of
Agriculture, Minia University, Egypt.
3.12. Statistical Analysis. All experiments and analyses were
performed in triplicate. e results were given as means ±the
standard error of mean (SEM) and analyzed by using Graph
Pad Prism  soware. Comparisons between groups were
performed by using one-way analysis of variance (ANOVA)
aer 𝑡-test. In addition, 𝑝 < 0.05 was considered signicant.
e PCA using XLSTAT soware determined the correlation
between all the experiments.
4. Results and Discussions
4.1. Growth Rate and pH of Bidobacterium spp. in MRSL at
37∘C. All the bidobacterial species showed a similar growth
prole when Bidobacterium spp. were incubated in MRSL at
∘C. e rst log phase was observed during the rst  to
 hr of growth and second log phase was started at  hr and
continued until  hr and aer that decline phase was started
(Figure (a)).
e kinetics growth of ve Bidobacterium spp. and pH
investigated that B.adolescentis,B.breve,and B. longum
grown well in lactose MRS and the rates of growth were
., ., and . at  hr, respectively, at log phase, while
resultsinFigure(b)haveshownthedecreaseofpHgradually
from . at zero time to ., ., and ., respectively, aer
 hr. However, growth of the B.adolescentis,B.breve,and
B. longum was ., ., and . at  hr, respectively,
whereas pH was ., ., and . at  hr, respectively.
On the contrary, growth of the B. infantis and B. bidum
was . and . at  hr of incubation and pH was .
and.,respectively.Meanwhile,thegrowthwas.and
. and pH . and ., respectively, at  hr. ese
results were in complete consensuses with [] that have
attributed this pattern of growth to the presence of two
dierent 𝛽-galactosidases. However, B. adolescentis showed
the highest growth rate, followed by B. breve and B. bidum.
Meanwhile, B. infantis and B. longum were the lowest at
 hr of incubation. Moreover, the dierences in growth rate
among species of Bidobacterium spp. correlated to dierent
levels of tolerance to aerobic conditions.
4.2. Resistance of Bidobacterium spp. to Bile Salts in MRSL
Incubated at 37∘C. Bile tolerance is one of the most crucial

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0
0.3
0.6
0.9
1.2
1.5
0 20 40 60 80 100 120
Time (h)
B. infantis
B. bidum
B. longum
B. adolescentis
B. breve
O.D/660 nm
(a)
2
4
6
pH
0 8 24 32 48 56 72 80 96
Time (h)
B. infantis
B. bidum
B. longum
B. adolescentis
B. breve
(b)
F : (a) Growth rate of dierent species of Bidobacterium spp. (b) pH of dierent species of Bidobacterium spp.
B. infantis
B. bidum
B. longum
B. adolescentis
B. breve
Observations (axes F1 and F2: 95.16%)
−2
−1
0
1
2
F2 (24.79%)
1023
−2−3−1−4
F1 (70.38%)
F : Plots of the 𝑥-loadings of Bidobacterium spp.
properties as it determines the ability of bacteria to survive in
the small intestine and play their functional role as probiotics.
A concentration of .% of bile salts closely appropriates the
bile level, which are found in the gastrointestinal tract [].
Common observations among this comparison of dierent
cultures for bile salts tolerance were shown in this study.
e highest and lowest resistance of ve Bidobacterium spp.
were observed in Figure . It was shown that B. infantis
and B. bidum were more resistant to bile salts than the
other three species that they reached O.D660 of . and .
athr,respectively.Onthecontrary,Badolescentishad a
dramatically decreased O.D660 of . at  hr according to
these results. Finally, we summarized that the growths of
Bidobacterium spp. were harmed by bile salts. Moreover,
these results were in convergence with [] who reported
the tolerance of Bidobacterium to bile or acid. erefore,
B. infantis had the highest survival rates followed by B.
bidum,B.breve,and B. longum, when exposed to bile salts
at concentrations ranging from zero to  g/L.
e result of the PCA was used to study the resistance of
Bidobacterium spp. to bile salts. Figures  and  presented
the plots of the scores and the correlation loadings, respec-
tively.escoreplotsofPCAillustratedthelargevariabilityof
the ve Bidobacterium spp. based on their resistance to bile
salts. e loadings are the coecients of the original variables
Without bile
at
Without bile With bile at
With bile at
Variables (axes F1 and F2: 95.16%)
−1
−0.75
−0.5
−0.25
0
0.25
0.5
0.75
1
F2 (24.79%)
0.50.250 0.75 1
−0.5−0.75 −0.25−1
F1 (70.38%)
24 h
zero h
zero h
at 24 h
F : Plots of the scores of Bidobacterium spp.
B. infantis
B. bidum
B. longum
B. adolescentis
B. breve
120
100
80
60
40
20
0
Survival rate (%)
Bidobacterium spp.
% survival rate at 0 h
% survival rate at 24 h
Bile salts tolerance of Bidobacterium spp.
F : Resistance percentage of Bidobacterium spp. to bile salts.
that dene each principal component. Inertia percentage and
correlated variables for axes  and  were displayed in Table .
Axis  explained .% of the total inertia. Axis  explained
.% of the inertia. Plots of the scores in Figure  indicated

BioMed Research International 
B. adolescentis Control
B. bidum B. longum
B. breve B. infantis
A = adhesion Bidobacterium, B = nonadhesion Bidobacterium
F : Adhesion of Bidobacterium spp. to intestinal epithelial cells.
T : Discriminate variables factors of principal components
analysis to study the resistance of Bidobacterium spp. to bile salts.
F F
Proper value . .
Variability (%) . .
Cumulative (%) . .
that the data cloud was mainly bidimensional with respect to
the explanatory variables. Figure  showed three clusters of
Bidobacterium spp. First cluster included the B. breve and B
adolescentis species. Second cluster included the B. bidum
and B. longum species. e third cluster (B. infantis species)
was individualized.
4.3.AdhesionofBidobacteriumspp.toIntestinalEpithelial
Cells. Major considerations in the choice of Bidobacterium
spp. to be used as dietary adjuncts are not only the capability
of survival and passing the harmful GI conditions, but also
being established within the digestive tract. Caco- cells
are human intestinal cell lines expressing morphologic and
physiologic characteristics of normal human enterocytes
[]. at has been exploited to select and assess probiotics
based on their adhesion properties.
erefore, the adhesion of Bidobacterium spp. to colum-
nar epithelial cells of the small intestine of rabbit was tested
asshowninFigure.Itappearedthattheabilityofadhesion
by B. adolescentis to Caco- cells was stronger than that of
other tested strains, but mainly with resistance to bile salts. In
contrary, B. infantis was less capable of adhering to epithelial
cells and acid production, but it was the best strain resistant
to bile salts.
According to data shown in Figures (a), (b), , and  B.
adolescentis have the highest percentage in survival rate at low
pHandstrongeradhesiontotheepithelialcells.Meanwhile,
B. infantis is best strain in resistance of bile salts. erefore,
we have chosen these strains to manufacture frozen yogurt.

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0.4
0.41
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0.44
0.45
0.46
C C + A C + B A + B C + A + B
Acidity (%)
Frozen yogurt mixes
0
15
30
60
(a)
5.55
5.6
5.65
5.7
5.75
5.8
5.85
5.9
C C + A C + B A + B C + A + B
pH
Frozen yogurt mixes
0
15
30
60
(b)
F : (a) Changes in titratable acidity of frozen yogurt during storage of , , , and  d at −∘C. (b) Changes in pH of frozen yogurt
during storage of , , , and  d at −∘C.
4.4. Physicochemical Characteristics of Frozen Yogurts during
60 Days of Storage at −18∘CAcidityandpH. ese studies
wereconductedtoseechangesinacidity,pH,andtotalsolids
of frozen yogurt made with yogurt culture and Bidobac-
terium spp. during  days of storage at −∘C.
Results indicate that there are similar changes of titratable
acidity and pH values development observed in dierent
frozen yogurt treated. Only slight changes were found in mix
(C+A+B),wheretheaciditywasincreasedtoreach.
at  days in the end of storage period. Furthermore, acidity
development and pH were steady for ve treatments of 
days of storage. No signicant dierences (p>.) in titrat-
able acidity and pH values were noted among dierent frozen
yogurt mixes during storage periods. ese results indicated
that the addition of Bidobacterium had no obvious changes.
Data in Figures (a) and (b) were in conformity with the
results obtained by [], who found that titratable acidity of
freshfrozenyogurtmadewithyogurtcultureorBidobac-
terium spp. culture was .. ese indicated that there were
no biochemical activities by yogurt culture during storage
of the product at −∘C. On the contrary, these results were
in disagreement with ndings by [] who reported that the
addition of the Bidobacterium spp.ledtolowerpH.
4.5. Total Solids. Total solids play an important role in the
qualityoffrozenyogurt.Resultsoffrozenyogurtsamples
made with yogurt culture and Bidobacterium spp. during
 days of storage at −∘C indicated that total solids in all
treatments made with yogurt culture and Bidobacterium
spp. were about . to .. ese results demonstrated
that there was no high signicance at p<., among frozen
yogurt samples during the storage periods. erefore, these
have close conformities with results obtained by [], who
found that total solids of frozen yogurt made with yogurt
culture and Bidobacterium spp.culturewithuptoweeksof
storage at −∘C did not signicantly changed. Moreover, []
reported that a slight increase in total solids was found in all
samples during storage period up to  days. ey attributed
an increase to the partial losses in free water during storage.
4.6. Changes in Rheological Properties of Frozen Yogurt Made
with Yogurt Culture and Bidobacterium spp. during 60
Days of Storage at −18∘C
4.6.1. Changes in Hardness (g) of Frozen Yogurt during 60 Days
of Storage at −18∘C. As seen in Table  hardness of frozen
yogurt made with yogurt culture only (C) was – while
hardness of frozen yogurt made with Bidobacterium spp. (C
+ A), (C + B), and (A + B) was , , and , respectively,
at  days of storage. On the contrary, hardness of mix (C
+ A + B) was the highest values; it was  at fresh samples
and ., , and  for , , and  days’ storage period,
respectively. ese obtained results were in agreement with
results obtained by [] who reported that no signicant (p
<.) dierences in hardness were found between frozen
yogurts samples. erefore, the addition of Bidobacterium
spp. did not aect the texture of the frozen yogurt.
4.6.2. Changes in Meltdown/Min of Frozen Yogurt during 60
Days of Storage at −18∘C. ResultsinTableshowedthatthe
meltdown of frozen yogurt made of yogurt culture (C) was in
therangefrom.to.minoffreshtodays’storage
at −∘C, while the time for collection was increased in mixed
yogurt culture + B. adolescentis (C + A) from . to . of
fresh to  days’ storage at −∘C. Moreover, frozen yogurt
made with yogurt culture + B. infantis (C+B)wasslightly
decreased from . to .. In addition, Bidobacterium
spp.culture(A+B)mixwasintherangefrom.to.
min of fresh to  days’ storage at −∘C. erefore, frozen
yogurt made with three combinations of cultures (C + A +
B)haddramaticallyincreasedfrom.tofromfresh
to  days of storage at −∘C. Finally, we summarized that
only slight changes were found in mix (C + A + B) which

BioMed Research International 
T : Changes in some rheological properties of frozen yogurt made with yogurt culture and Bidobacterium spp. culture during  days
of storage −∘C.
Treatment Storage time/days Hardness/g Meltdown/min % Overrun
C
±j. ±o
.
 . ±.hi . ±e
  ±h ±f
  ±f. ±d
C+A
 . ±g. ±p
.
  ±e ±h
  ±c ±j
  ±b. ±l
C+B
±i. ±g

  ±h. ±m
  ±e ±n
  ±d. ±l
A+B
±j. ±b
.
  ±hi  ±a
  ±g ±c
  ±e. ±c
C+A+B
 . ±f. ±q
.
 . ±.e ±k
  ±b. ±m
  ±a ±i
Values are the average of three individual samples each analyzed in duplicate ±standard deviation. Dierent lowercase superscript letters, respectively, indicate
signicant dierence (p<.) analyzed by Duncan’s multiple range test.
increasedinmeltdown/minoffrozenyogurt.Moreover,it
was clear that there was no signicant (p>.) dierence
in melting time and overrun values between dierent frozen
yogurt mixes. e melting behavior of the product coincided
with previous reports focusing on the melting behavior of ice
cream with and without probiotics []. ese ndings were
in close agreement with the ndings of [].
4.6.3. Changes in Overrun Percentage of Frozen Yogurt Made
with Dierent Bidobacterium spp. Overrun is one of the
most important quality parameters of frozen desserts, since it
aects the texture and consequently the price of the products.
Results in Table  showed that the overrun levels of the ve
studied frozen yogurt formulations were low (.%–.%)
andtheseresultswereincontrastto[],whoreportedthat
the addition of Bidobacterium spp. led to no high changes
in the overrun levels (p<.). We hypothesized that it
would lead to a poorer foaming capacity and decrease air
incorporation in samples with blending components.
4.6.4. Changes in the Viability of Bidobacterium spp. in
Frozen Yogurt during 60 Days of Storage at −18∘C. Results
in Figure  showed the revealed count of Bidobacterium
spp. at −∘C decreased with storage period. e count of
Bidobacterium spp.forfrozenyogurtmadewithyogurt
culture and B. adolescentis (C + A) was from . ×8to
. ×8CFU with decrease percent .% from fresh to
 days of storage period, while that of frozen yogurt made
with yogurt culture and B. infantis (C + B) was from . ×8
0
0.5
1
1.5
2
2.5
3
Fresh 15 days 30 days 60 days
CFU
Storage (d)
C + A
C + B
A + B
C + A + B
F : Changes in viability of Bidobacterium spp. in frozen
yogurt.
to . ×8CFU with decrease percent .% from fresh
to  days of storage. Moreover, frozen yogurt made with
Bidobacterium spp. culture of B. adolescentis +B. infantis (A
+B)hadcountof.×8to . ×8CFU with decrease
percent .% from fresh to  days of storage period.
Finally, the count of Bidobacterium spp. for frozen yogurt
mix made with yogurt culture + B. adolescentis +B. infantis

BioMed Research International
Flavor (45) Body and
texture (35)
Melting quality
(10)
Appearance (10) Total scores (100)
Parameters
C
A + B
C + B
C + A
C + A + B
0
10
20
30
40
50
60
70
80
90
100
Degrees
F : Sensory evaluation of frozen yogurt.
(C+A+B)wasfrom.×8to . ×8CFU with decrease
percent .% from fresh to  days of storage period. ese
data were in close agreement with data obtained by [, ]
whoreportedthatnosignicant(𝑝 > 0.05) dierence was
observed in the count of yogurt bacteria as well as B. bidum
count between dierent frozen yogurt mixes.
4.7. Sensory Evaluation of Frozen Yogurt aer 60 Days of
Storage at −18∘C. ResultsinFigurehaveshowntheeval-
uation scores of frozen yogurt made with yogurt culture
and Bidobacterium spp. aer  days of storage at −∘C.
It indicated that there were no high dierences between
samples in sensory evaluation. It appeared that frozen yogurt
made with yogurt culture + Bidobacterium adolescentis +
Bidobacterium infantis (C+A+B)gainedahighscore
of . In addition, samples made with yogurt culture +
Bidobacterium infantis (C+B)gainedscoreofintotal
as well.
ere are at least two important aspects that should
be highlighted while analyzing frozen yogurt. First, con-
sumersareusedtotheavorofdairyproductsproduced
with traditional yogurt bacteria, which would lead to lower
sensory scores to products that do not t into this cate-
gory. Secondly, Bidobacterium spp. are heterofermentative
organisms, which are able to produce several types of organic
acids (lactic, acetic, and formic acid) and ethanol [] which
can induce important avor modications. Considering the
potential benets provided by the probiotic microorganisms,
process adjustments could be implemented in order to
overcome any possible avor or aroma issues. In spite of
the slightly acidic avor of their samples, unfamiliar to our
consumers, all these samples were acceptable. e results in
Figure  closely agreed with results obtained by [], who
found that the overall acceptance of probiotic ice cream
depends on the preferred and accepted pH.
ResultsofthePCAwereusedtoanalyzephysicochemical
characteristics, some rheological properties, and sensory
C
C + A
C + B
A + B
C + A + B
Observations (axes F1 and F2: 76.35%)
−4
−2
0
2
4
6
F2 (21.27%)
6420810
−4−6−2−8
F1 (55.07%)
F : Plots of the 𝑥-loadings.
evaluation of frozen yogurts. Figure  presented the cor-
relation loadings. e scores plot of PCA illustrated the
large variability of ve mixes of frozen yogurt based on
dierent species of Bidobacterium spp. during  days of
storage at −∘C. Loadings were the coecients of the original
variables of each principal component. Inertia percentage
and correlated variables for axes  and  were displayed
in Table . Axis  explained .% of the total inertia.
Axis  explained .% of the inertia. With respect to the
explanatory variables, Figure  shows four clusters of mixes.
e rst cluster included the C + A and C + B, whereas the
second,third,andfourthclusterswereC,A+B,andC+A+
B, respectively, individualized.
5. Conclusion
Bidobacterium spp. can grow well and have ability to
withstand dierent conditions of acidity and bile. Moreover,
frozen yogurt can serve as an excellent vehicle for dietary
incorporation of probiotic bacteria. On the contrary, frozen

BioMed Research International 
T : Discriminate variable factors of principal components
analyses of analyzed physicochemical characteristics, some rheolog-
ical properties, and sensory evaluation.
F F
Proper value . .
Variability (%) . .
Cumulative (%) . .
storageoftheproductshaslittleeectsonthesurvivalofBi-
dobacterium spp., which are sucient to oer the suggested
therapeutic eects. Supplementation with Bidobacterium
spp. has been found to exert a little eect on avor or
compositional characteristics of frozen yogurt. Our previous
study indicated that there were no signicant dierence
changes (𝑝 > 0.05) during adding dierent Bidobacterium
spp. in the physiochemistry or sensory evaluation of frozen
yogurt.
Conflicts of Interest
e authors declare that there are no conicts of interest
regarding the publication of this study.
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