Characterization of Fresh Cheese with Addition of Probiotics and Prebiotics

Abstract

Sensorial and microbiological characteristics of a Brazilian fresh cheese samples with Bifidobacterium animalis subps.
lactis as well as samples with this probiotic and polydextrose, a prebiotic ingredient, were evaluated. The addition of this
microorganism was studied as: (1) lyophilized probiotic added to cheese curd and (2) by using milk previously fermented by this
probiotic to produce the cheese. Cheese samples were microbiologically characterized after 0, 7, 14, 21 and 28 days of storage at a
temperature of 4 °C. The microbiological analyses conducted were quantification of total lactic acid bacteria, mesophilic
microorganisms, Bif. animalis subps. lactis, coliforms at 30 °C and 45 °C. Affective sensory test was conducted for two different
cheese samples (with probiotic and with probiotic and prebiotic) as well as for control one week after manufacturing date. Cheese
samples provided acceptable results for coliform counts at 30 °C and 45 °C in compliance with legislation. The cheese samples
produced using milk fermented by probiotic showed counts of 107-108 CFU/g after 28 days of storage, which assures functional
property for this product to be claimed.

Full text

Feb. 2013, Vol. 7, No. 2, pp. 189-195
Journal of Life Sciences, ISSN 1934-7391, USA
Characterization of Fresh Cheese with Addition of
Probiotics and Prebiotics
Natália Chinellato Azambuja1, Patrícia Blumer Zacarchenco1, Luciana Francisco Fleuri2, Juliana Cunha Andrade3,
Izildinha Moreno1, Ariene Gimenes Fernandes Van Dender1.and Darlila Aparecida Gallina1
1. Food Technology Institute—Dairy Technology Center (TECNOLAT-ITAL), Jardim Chapadão 13070-178, Campinas, São Paulo
State, Brazil
2. Chemitry and Biochemistry Department, Institute of Biosciences, São Paulo State University “Júlio de Mesquita Filho” UNESP,
Botucatu 18618-000, São Paulo State, Brazil
3. Food Technology Institute—Meat Technology Center (CTC-ITAL), Jardim Chapadão 13070-178, Campinas, São Paulo State,
Brazil
Received: August 29, 2012 / Accepted: December 3, 2012 / Published: February 28, 2013.
Abstract: Sensorial and microbiological characteristics of a Brazilian fresh cheese samples with Bifidobacterium animalis subps.
lactis as well as samples with this probiotic and polydextrose, a prebiotic ingredient, were evaluated. The addition of this
microorganism was studied as: (1) lyophilized probiotic added to cheese curd and (2) by using milk previously fermented by this
probiotic to produce the cheese. Cheese samples were microbiologically characterized after 0, 7, 14, 21 and 28 days of storage at a
temperature of 4 °C. The microbiological analyses conducted were quantification of total lactic acid bacteria, mesophilic
microorganisms, Bif. animalis subps. lactis, coliforms at 30 °C and 45 °C. Affective sensory test was conducted for two different
cheese samples (with probiotic and with probiotic and prebiotic) as well as for control one week after manufacturing date. Cheese
samples provided acceptable results for coliform counts at 30 °C and 45 °C in compliance with legislation. The cheese samples
produced using milk fermented by probiotic showed counts of 107-108 CFU/g after 28 days of storage, which assures functional
property for this product to be claimed.
Key words: Probiotic, prebiotic, fresh cheese.
1. Introduction
The consumers’ interest for food products with
benefits for health has produced an increase in search of
“functional” food, stimulating innovation and
development of new products [1]. In Brazil, a large
consumption of milk products is observed. Cheese
production in our country was 801,440 tons in 2010.
Among fresh cheese types, 68% of production is
represented by Minas-type fresh cheese [2]. Minas-type
fresh cheese is one of the most important dairy products
in Brazil, being considered as a truly national cheese. It
Corresponding author: Natália Chinellato Aambuja,
research fields: dairy science and technology. E-mail:
natalia.ferreira@fca.unicamp.br.
is widely accepted in internal market and its simplified
production technology makes this product very
attractive for many industries [3].
Probiotic foods belong to functional food group.
One of current definitions for probiotic is “live
microbial food supplements which beneficially affects
the host animal by improving its intestinal microbial
balance” [4]. In order to obtain health benefits a daily
dose of 108-1010 CFU of probiotic should be ingested,
which represents consuming 100 g of food with at
least 106 CFU/g [5]. Most known bacteria with
probiotic function in organism are those from
Bifidobacterium and Lactobacillus genera [4]. There
are technological advantages of using the probiotic

Characterization of Fresh Cheese with Addition of Probiotics and Prebiotics
190
species investigated in this paper such as good
feasibility, compatibility with other lactic cultures,
and tolerance to oxygen [6, 7]. Haschke et al. [8]
emphasized, among functional qualities of
Bifidobacterium animalis subps. lactis (BB12), the
excellent survival during intestinal transit as well as
adhesion to enterocytes, which are essential conditions
so a culture can be considered as probiotic.
Prebiotic is a term used to define food ingredients
that are not hydrolysable or absorbed in the small
intestine [8, 9] and which benefit the host by
selectively stimulating bacterial growth and/or activity
in the intestine favoring the growth of probiotics,
mainly for lactobacilli and bifidobacteria. These
substances are essentially composed by carbohydrate
of different sizes from disaccharides and
oligosaccharides to large polysaccharides.
Currently the prebiotics mostly used in studies and
in commercial products are inulin, FOS
(fructooligosaccharides) and polydextrose. Craig et al.
[10] described polydextrose as an oligosaccharide
formed by glucose polymers, defined as dietary fiber
and belonging to the same class as inulin, both of low
energy value.
According to Kolida, Gibson [11, 12], symbiotics
are mixtures of probiotics and prebiotics, implying
that the efficacy of each component will be
established for a symbiotic formulation.
The purpose of this paper was evaluated two ways
of incorporation of the probiotic BB12 to fresh cheese
in order to improve industrial technology in cheese
manufacture. By including the probiotic and, also, the
prebiotic, the authors can offer to the consumers a
fresh cheese containing health benefits instead of its
intrinsic nutritional value. The centesimal composition,
as well as physical, chemical and sensory properties
was evaluated.
2. Material and Methods
2.1 Materials
Cheese elaboration: Milk used in process was
low-fat, homogenized and pasteurized type milk. Two
processing types were conducted, in which three
cheese formulations were prepared with 3 kg per
treatment. Steps for cheese obtainment are detailed
below based on control cheese flowchart (Fig. 1), as
per the technique recommended by Furtado and
Lourenço-Neto [13].
In Q1 (probiotic cheese) treatment, lyophilized
probiotic culture Bifidobacterium animalis subps.
Lactis. (BB12—Christian Hansen) was added into
Minas-type fresh cheese curd. These cheese samples
from Q1 treatment were obtained by the same method
Milk heated in water bath in a vat previously sterilized at 35 °C

Cheese dough formation (Coagulation)  Addition of: lactic acid 30 mL, calcium chloride 6 mL, and
rennet 11 mL diluted in sterile water 200 mL

Dough cutting with lyres

Serum removal/Clot separation

Weighting
  Dough salting (solution at 10%)
Shaping (Put clot into molds)

Turning

Package
Fig. 1 Flowchart of control cheese (with no addition of probiotic and prebiotic)

Characterization of Fresh Cheese with Addition of Probiotics and Prebiotics
191
as control cheese (Fig. 1). After curd weighting,
lyophilized culture was added at a previously
calculated concentration in order to obtained 108 CFU/g
in final product. This concentration was determined by
quantification of the viable cells in the lyophilized
culture: BB12 culture was previously dispersed in
0.01% peptonated water, then it was plated in MRS
(Man Rogosa Sharp) agar modified as described by
Antunes et al. [7] for probiotic count. In Q2 treatment,
probiotic culture was inoculated in milk and incubated
overnight at probiotic and prebiotic cheese 35-37 ºC
so milk fermentation could occur reaching pH of
5.7-5.8 before cheese samples were obtained by acid
coagulation conducted in previous treatments, control
and Q1. Also, in treatment Q2, polydextrose prebiotic
(Danisco) was added at an amount of 60 g for 1.2 kg
cheese dough. Then, prebiotic was diluted into 300
mL of cheese whey and added before final product
salting. It was produced two batches of the three
cheeses.
2.2 Methods
2.2.1 Microbiological Analyses
Microbiological analyses were conducted in
duplicate according with the methodology described
in Michael & Frank [14] during 28 days of storage on
the following time points: days 0, 7, 14, 21 and 28.
Analyses conducted were coliform count at 30 °C and
45 °C by the MPN (most probable number) method,
total mesophilic microorganisms count in PCA (plate
count agar) and lactic acid bacteria count in MRS agar.
These analyses were performed in milk, raw material
and control cheese. All three treatments, control
cheese, Q1 and Q2, were prepared at the same time.
Therefore samples for total count, coliform at 30 °C
and 45 °C and thermotolerant coliforms (45 °C) count
and lactic acid bacteria count were obtained from
control cheese.
2.2.2 Quantification of BB12 in Cheese Samples
According with modification made by Antunes et al.
[7], the quantification of BB12 was conducted in Man
Rogosa Sharp Agar supplemented by 10% HCL
L-cysteine, 10% lithium chloride, and aniline blue dye.
Incubation was made under anaerobic conditions
using one anaerobiosis jar with gas generator for
anaerobiosis (Anaerogen, Oxoid/Interlab) for 72 h at a
temperature of 45 °C ± 1 °C.
2.2.3 Sensory Analysis
This analysis was conducted in accordance with
legal proceedings on enrollment in Research Ethics
Committee under protocol number 350/2011.
Sensory evaluation of control, Q1 and Q2 cheese
samples was performed after one week of
manufacturing. Affective test was applied as
described by Meilgaard et al. [15].
The test was conducted with 33 male and female
panelists of 21 to 50 years old, not previously trained,
with the purpose of evaluating the acceptance of
products obtained on a hedonic scale (nine
corresponding to “I liked very much” and one
corresponding to “I disliked very much”). Each
panelist was given two cubes (2 cm) of each
Minas-type fresh cheese treatment (control,
Minas-type cheese with addition of lyophilized
probiotic Q1 and Minas-type cheese with addition of
probiotic and prebiotic Q2) in a 50 mL disposable
glass coded with a 3-digit number. Together with the
samples, each panelist received a glass of water (200
mL) at room temperature and one cracker type biscuit
to be used between samples evaluations. Analyses
have occurred in separate cabins illuminated by white
light. The panelist was requested to evaluate each
coded sample, comparing it with the others in order to
indicate the degree of difference between the samples
in the following attributes: overall, odor, texture, and
taste. At the end of evaluation, the panelist was
requested to organize the samples as the one he/she
liked the least (1) liked (2) and liked the most (3) by
rank test with the purpose of verifying if treatments
were different between each other.
Results were evaluated by ANOVA (Analysis of
Variance) and Tukey’s test at a confidence level of

Characterization of Fresh Cheese with Addition of Probiotics and Prebiotics
192
95% (P  0.05) to check if there is any difference
between Minas-type fresh cheese treatments.
2.2.4 Physical-Chemical Analysis
After processing, control cheese, Q1 and Q2 were
characterized for the following physical and chemical
parameters: pH by a digital potentiometer B375
(Micronal), total dry matter by gravimetric
methodology, ash content by drying in mufla oven at
550 °C, titratable acidity in lactic acid, total nitrogen
by Kjeldahl method; all analyses have followed the
methodology described by physical-chemical analysis
manual of Instituto Adolfo Lutz [16]. Nitrogen values
were multiplied by 6.38 factor to obtain equivalent
values for protein, carbohydrate and humidity by
method of difference and sodium chloride determined
by Volhard method as described by Pereira [17]. All
physical-chemical analyses were conducted in
triplicate.
3. Results and Discussion
3.1 Microbiological Analysis
Microbiological analyses conducted for low-fat,
homogenized, and pasteurized type A milk used as
raw material for cheese preparation allowed to verify
their compliance to Brazilian legislation [17], with
counts for total mesophilic microorganisms and acid
lactic bacteria of < 10 CFU/mL and coliform at 30 °C
and 45 °C of < 0.3 NMP/mL. Table 1 shows values
regarding microbiological analyses conducted to
characterize cheese samples obtained after processing.
As per the results shown in Table 1, cheese samples
had counts within minimum hygienic sanitary
standards established in the Brazilian legislation [18].
These microbiological conditions were achieved due
to characteristics of milk used, which are described
above. Cheese samples were elaborated according
with good manufacturing practices, with raw material
playing an important role on cheese quality since it
had a good hygienic sanitary quality.
Table 2 shows Bif. animalis subps. lactis. counts in
Q1 and Q2 cheese samples during storage time.
Q1 and Q2 cheese samples with probiotic had
counts around 107-108 CFU/g, except for one Q1
batch. These are encouraging results since, as
mentioned by Antunes et al. [7], bifidobacteria
are fastidious microorganisms with low survival under
Table 1 Microbiological characterization of control cheese elaborated in processing 1 and processing 2 (repetition) during storage
period.
Storage
Time
Total mesophilic microorganisms’
count (CFU/g) Acid lactic bacteria count
(CFU/g)
Coliform count at 30-35ºC
(NMP/g) Coliform count at 45ºC
(NMP/g)
Proc. 1 Proc. 2 Proc. 1 Proc. 2 Proc. 1 Proc. 2 Proc. 1 Proc. 2
0 8.10 × 102 1.09 × 103 < 10 < 10 43 < 3.0 < 3.0 < 3.0
7 2.50 × 105 7.00 × 102 1.85× 105 < 10 23 9.2 < 3.0 < 3.0
14 1.06 × 106 1.10 × 103 2.22× 105 < 10 23 43 < 3.0 < 3.0
21 1.95 × 107 4.30 × 105 1.00× 106 1.23× 103 > 1,100 23 < 3.0 < 3.0
28 4.50 × 109 7.00 × 106 2.27× 107 3.30× 104 > 1,100 > 1,100 < 3.0 < 3.0
CFU/g = Colony-Forming Unit per gram of cheese analyzed.
Table 2 BB12 probiotic culture count in MRS Agar modified in probiotic (Q1) and probiotic with prebiotic (Q2) cheese samples.
Storage Time Probiotic cheese (Q1) (CFU/g) Probiotic and prebiotic cheese (Q2) (CFU/g)
Proc. 1 Proc. 2 Proc. 1 Proc. 2
0 1.40 × 108 2.20 × 108 9.50 × 107 4.10 × 109
7 1.95 × 107 3.00 × 108 9.50 × 106 9.20 × 108
14 2.60 × 108 2.02 × 107 1.20 × 107 1.49 × 109
21 6.50 × 108 6.40 × 106 6.30 × 107 1.18 × 109
28 6.30 × 108 1.70 × 105 3.30 × 107 9.50 × 108
CFU/g = Colony-Forming Unit per gram of cheese analyzed.

Characterization of Fresh Cheese with Addition of Probiotics and Prebiotics
193
certain conditions, therefore BB12 culture was chosen
for this research, since it is industrially used as
probiotic in yogurts, fermented milk, cheese,
beverages, sauce, diet supplements, infant formula and
cereal for over 10 years. BB12 strain used in this
study showed appropriate populations resisting to
barriers such as oxygen during processing
incorporation.
Minas-type fresh cheese samples of this study that
had probiotic and prebiotic (Q2) showed count values
around 107-108 CFU/g after 28 days of storage at 4 °C.
Cheese samples with prebiotic and probiotic Q2 were
obtained from milk fermented by BB12, which
enables to reduce the lyophilized culture amount used
by the industries. Investigations such as the one
conducted by Buriti [20] evaluated the use of
lyophilized probiotic added in symbiotic cream fresh
cheese only.
Capela et al. [21] have verified that by adding 2%
of fiber such as inulin, corn resistant starch and
oligofructose, viability of Lactobacillus acidophilus,
Lactobacillus casei, Bifidobacterium longum and
Lactobacillus rhamnosus GG strains was increased in
yogurt in comparison with control yogurt with no
addition of fiber during 4 weeks of storage at 4 °C.
Buriti et al. [22] have studied probiotic fresh cream
cheese samples added or not with prebiotic and have
not found differences in probiotic survival during
storage between cheese samples with and without
prebiotic.
Considering the requirements of the Brazilian
legislation for functional foods [22], cheese samples
with probiotic have shown a potential to functional
property to be claimed.
3.2 Sensory Analysis
Most panelists (54.6%) who have evaluated the
products were within age group of 21 to 30 years old.
The frequency of consumption for this type of product
may be considered as high: 63.3% consume it on a
weekly and biweekly basis.
Table 3 shows results for sensory analysis,
conducted on 7 days of storage for different treatments
of Minas-type fresh cheese, as follows: (1) control, (2)
Minas-type fresh cheese with addition of probiotic
(Q1), and (3) Minas-type fresh cheese with addition of
probiotic and prebiotic (Q2).
By analyzing the results described in the table
above, samples were not significantly different (P >
0.05) in overall, odor and flavor attributes. These
results allow to infer that the probiotic BB12 to the
cheeses will not interfere in their acceptance by
consumer in market.
On the other hand, as expected for cheese with
addition of prebiotic (fiber) (Q2) the authors observed
that panelists noticed cheese consistency was different
in comparison with Q1 and control; however, even
with this statistical difference such cheese had a
higher score for texture attribute than other cheese
samples.
When compared with studies performed by other
researchers, sensory changes were irrelevant among
different samples evaluated. In a study conducted by
Buriti [20], the use of probiotic cultures has not
caused sensory changes in fresh cream cheese samples
Table 3 Sensory evaluation results.
Attributes Treatments
Control Q1 Q2
Overall 5.67
a ± 2.16 5.91 a ± 2.01 6.48 a ± 2.00
Odor 6.06
a ± 1.80 6.36 a ± 1.67 6.58 a ± 1.89
Texture 5.03
b
± 2.38 5.12
b
± 2.29 6.64 a ± 1.80
Flavor 5.64
a ± 1.88 5.67 a ± 1.87 5.70 a ± 2.39
Overwritten letters in the same line means there is no significant difference between samples evaluated (p ≤ 0.05). Scale ranging
from 9—I liked very much to 1—I disliked very much.

Characterization of Fresh Cheese with Addition of Probiotics and Prebiotics
194
Table 4 Physical-chemical characterization of control, probiotic (Q1) and probiotic added with prebiotic (Q2) after processing
cheese samples
Analyses Treatments
Control Q1 Q2
pH 6.47 5.32 6.32
Acidity 0.103 ± 0.000 0.512 ± 0.000 0.102 ± 0.000
Ashes (g/100g) 3.856 ± 0.029 3.118 ± 0.064 3.636 ± 0.076
Total protein (g/100g) 26.726 ± 0.849 25.508 ± 0.150 26.430 ± 0.493
Total dry stratum (g/100g) 35.398 ± 0.494 35.807 ± 0.733 35.634 ± 0.529
Salt (g/100g) 1.288 ± 0.014 1.128 ± 0.015 1.153 ± 0.045
Humidity (%) 64.602±0.494 64.193 ± 0.733 64.367 ± 0.529
after 7 days of storage at a temperature of 4 °C.
According with results obtained after performing the
sensorial test, no significant difference (P > 0.05) was
observed among samples evaluated in comparison
with control sample. However, most panelists rated
the samples evaluated with the attribute “I liked”.
3.3 Physical-Chemical Analysis
Table 4 shows the results for physical-chemical
characterization of cheese samples produced in this
study.
Contents of humidity, ashes, total protein, dry
matter, salt and pH for the three types of cheese
produced in this study did not show any significant
difference even with the variations among cheese
samples (addition of probiotic in Q1 and addition of
probiotic and prebiotic in Q2). In accordance with
Brazilian Law [23], Minas-type fresh cheese is a
product of very high humidity content and may show
values above 55%. Cheese samples produced in this
study fall within this characteristic. By studying
symbiotic cream cheese with the addition of probiotic
and prebiotic at various amounts, Alves [25] observed
that cheese formulations have not had significant
differences for physical-chemical characteristics as fat,
ashes, and protein, in comparison with control
treatment.
4. Conclusion
The cheeses with prebiotic and probiotic Q2 were
obtained from milk fermented by the probiotic BB12,
which enables to reduce the lyophilized culture
amount used by the industries. This finding was
reinforced by the sensorial scores of the cheeses Q2
and Q1 (containing lyophilized probiotic) that were
statistically equal. The probiotic used in this study was
technologically appropriate for application in
Minas-type fresh cheese, as it showed appropriate
populations resisting to barriers such as oxygen during
processing incorporation. The addition of probiotic
culture and prebiotic studied will not interfere in the
consumer acceptance of the cheese. Consequently, the
authors have verified that this study was conducted as
expected in order to offer to the market a value-added
product with benefits for consumer health, since
cheese is highly consumed in the country.
Acknowledgments
The authors’ special thanks to ITAL (Instituto de
Tecnologia de Alimentos), which has provided labs
and pilot plants for conduction of this study and
especially to the department TECNOLAT (Centro de
Tecnologia de Laticínios) and CTC (Centro de
Tecnologia de Carnes).
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