Medical

Communication

Biosci. Biotech. Res. Comm. 10(1): 117-122 (2017)

Effects of inulin and fat percentage on the viability of

Bi dobacterium lactis

Bb12 in chocolate milk

Hadi Pourjafar

, Valiollah Ayareh

, Guity Karim

, Fereshteh Ansari

* and Hamid Kaboosi

Department of Public Health, Maragheh University of Medical Sciences, Maragheh, Iran

Islamic Azad University, Science and Research-Ayatollah Amoli Branch, Amol, Iran

Department of Food Hygiene, Tehran University, Tehran, Iran

Iranian evidence based center of excellence, Tabriz University of Medical Sciences, Iran

ABSTRACT

Bi dobacterium genus has several bene cial effectsas a probiotic bacterium. It has been demonstrated that Inulin

and fat percentage of the probiotic product may modify the viability of this bacterium during storage time. The aim

of this study was  nding the best formulation of the chocolate milk regarding bacterial viability and sensory prop-

erties of the  nal product. Four types of probiotic chocolate milk, with and without inulin in 1% fat samples, with

and without inulin in 2% fat samples, were manufactured in duplicate. The count of probiotic cells during 12 days

of storage in the refrigerator was assessed. In all samples, the viable cells of Bi dobacterium lactis increased except

1% fat samples without inulin in which bacterial count decreased (not signi cantly). The rate of change in the bacte-

rial count was signi cantly p<0.05 higher in the 2% fat chocolate milk both in with and without inulin samples (0.6

and 0.8) compared with 1% fat samples (0.5 and-0.4). Inulin inoculation had some effects on biochemical and orga-

noleptic changes. After 12 days storage, the acceptability of 2% fat milk with probiotic and inuline was the highest.

The results illustrated that the most signi cant Bi dobacterium lactis viability was in 2% fat chocolate milk. Adding

inulin improved the viability of bacteria in 1% fat milk; however, ithad not any signi cant effects on the viability

of probiotic bacteria in 2% fat milk. In conclusion, 2% fat chocolate milk with inuline could be recommended to be

the best group.

KEY WORDS: BIFIDOBACTERIUM LACTIS, CHOCOLATE MILK, INULIN, VIABILITY

117

ARTICLE INFORMATION:

*Corresponding Author: fereshtehansari66@gmail.com

Received 27

Feb, 2017

Accepted after revision 29

May, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

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Online Contents Available at: http//www.bbrc.in/

118 EFFECTS OF INULIN AND FAT PERCENTAGE ON THE VIABILITY OF

BIFIDOBACTERIUM LACTIS

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Hadi Pourjafar et al.

INTRODUCTION

Nowadays the acceptability of probiotic products is

increasing across the world, especiallyin Europe, USA, and

Japan. More than 90% of probiotic food products contain

Lactobacilli and Bi dobacteria (Gueimonde et al. 2004,

Hemalatha et al. 2017, Zoumpopoulou et al. 2017).

Bi dobacterium genus is one of the most important

genera of probiotic bacteria. It was isolated for the  rst

time from breastfeed newborns’ feces by Tisser in 1906.

This bacterium helps food digestion by secretion enzymes

like beta-galactosidase and protease. Suppressing the

intestinal infective bacteria, producing some vitamins,

anti-cancer and anti-mutation properties, immune sys-

tem stimulation, reducing blood serum cholesterol, and

increasing calcium absorption are some of its other bene -

cial effects. Bi dobacterium lactis is the most usable Bi -

dobacteria in probiotic dairy products (dos Santos Cruxen

et al. 2017, Gomes and Malcata 1999, Monteagudo-Mera

et al. 2016, Novak and Katz 2006, Poutsiaka et al. 2017).

Inulins are a group of fructooligosaccharides mainly

extracted from chicory root. Inulin is non-digestible;

therefore, passes throughout the small intestine and

isfermented in the colon. Inulin may be able to feed and

stimulate the growth and propagation of Bi dobacteria

species and other probiotics in the colon (Guarino et al.

2017, Lightowler et al. 2017, Lopes et al. 2017, Valdés-

Varela et al. 2017, Vandeputte et al. 2017).

Dairy products provide an important portion of the

essential nutrientsin every age group. These days unfer-

mented and avoured probiotic milks are drawing the

attention of researchers and industrialists due to their-

sensory properties, increasing consumption, and nutri-

tional value. Therefore, by using chocolate milk as a

carrier for probiotics, we can improve the healthiness

level of society (Ashurst 2016, Aune et al. 2015, Henry

et al. 2015, Oliveira et al. 2017).

The objective of this study was  nding the best com-

position of the probiotic bacteria (Bi dobacteria) and

prebiotic (inulin) regarding bacterial viability and sen-

sory properties of the chocolate milk as the  nal product.

Chocolate milk can be industrially produced with differ-

ent percentages of fat. In this study, we used 1% and 2%

fat containing chocolate milk to examine the effects of

increased fat on the  nal product. To our knowledge, it

is the  rst time that the effects of inulin and fat percent-

age are concurrently studied in probiotic chocolate milk.

MATERIAL AND METHODS

Sample production method and its volume

The sterile chocolate milk was prepared. And then, com-

mon microbial control tests (total count and Enterobac-

teriaceae) were conducted based on Iran National Stand-

ard. The Bi dobacteriumx lactis Bb12 was obtained from

a Dutch company (DSM) asa direct vat set (DVS). They

were cultured in MRS broth in anaerobic condition.

Then, they were incubated at 37°C for 18 hours.

According to the manufacturer’s instruction, 0.1% of

the bacteria was added to the normal salineand incubated

at 37°C for 18 h to reach the logarithmic growth. Then,

they were added to sterile milk with 1 and 2 percent

fat. Both of the milk percentages were divided into two

parts. A 0.5% sterile inulin (Belgium Co.) was added into

one part of each sample. Another part of each sample

was left without inulin. Two other samples were selected

as controls. Each of the samples (6 samples) was divided

into 8 bottles of 100 ml capacity (total, 48 bottles). A

two- liter containing bottle of each sample was stored in

the 5°C refrigeratorto taste panel test. The bottles with a

dilution of 10-3 to 10-7were cultured in anaerobic MRS

agar (MEREK, Germany) and type A gas pack (MEREK,

Germany), then incubated at 37 °C for 96 h. At the end

of 96 hthe colony counting was conducted using the

colony counter (Klindt-Toldam et al. 2016, Mirzaei et al.

2012, Mirzaei et al. 2011, Pourjafar et al. 2016).

Biochemical indices

Physicochemical properties (pH, acidity, and viscosity)

were measured; and, organoleptice valuation was con-

ducted at each time of culturing. Evaluation of pH: The

samples’ pH was measured during refrigerated storage

using the pH-meter. The device was calibrated using

standard buffers (pH4, pH7). Aciditymeasurements: In

order to measure titrable acidity 10 ml of sample and

10 ml distilled water was poured into Erlenmeyer  asks

and 0.1 normal salinewas titratedat the presence of phe-

nolphthalein. The rate of the index was measured using

Dornic degree as follows a formula: Titrable acidity

(Dornicdegree) = usedsaline volume (ml) ×9: Viscosity

measurement The viscosity of the samples was meas-

ured using refractometer (Zherber Co.) during refriger-

ated storage. Sensory evaluation: chocolate milk sam-

ples were examined by 15 experts on the days 4 and 12.

They used scoring methodto evaluate sensory properties

of the chocolate milk, such as  avor, smell, sweetness,

and viscosity based on the score 10. Total results were

evaluated based on 20 scores (Marsanasco et al. 2015,

Mirzaei et al. 2012).

Statistical analysis

Bacterial count was measured in days 0, 4, 8 and 12 in

duplicate. The rate of changes in bacterial count from

the  rst day to twelfth day was compared across groups

using ANOVA test and post hoc Bonferroni test. Further,

SPSS-IBM 21 was used for statistical analysis. P<0.05

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECTS OF INULIN AND FAT PERCENTAGE ON THE VIABILITY OF

BIFIDOBACTERIUM LACTIS

119

Hadi Pourjafar et al.

was considered as signi cance level. Graphpad prism

ver. 6 used for drawing the graphs.

RESULTS AND DISCUSSION

In all samples, bacterial count increased after 12 days

except 1% fat samples without inulin in which bacterial

count decreased. The rate of increase the bacterial count

was higher in 2% fat milk (both with and without inu-

lin samples) compared with 1% fat milk. The change in

bacterial count was signi cantly different between 1%

fat milk with and without inulin samples, but we did not

observe any signi cant differences between 2% fat milk

with and without inulin in this case (See table1). Varia-

tions in counts of B. lactis during 12 days storage period

in the refrigerator (cfu ml-1) are shown in Fig. 1.

The viscosity of 2% fat milk was higher than 1%

fat milk. In samples containing inulin, the viscosity

was higher than inulin free samples with comparable

fat percentages. Viscosity did not change during stor-

age period. There has been just a small increase in the

viscosity of samples containing inulin with both 1%

and 2% fat. Acidity was similar in all study groups and

stayed constant during the research period. pH was

higher in 2% fat samples and samples containing inulin

had higher pH in comparison with control and probiotic

without inulin samples with the similar fat percentage.

pH reduced slightly during 12 days in all samples. In

2% fat samples containing inulin pH reduction was the

highest (See table 2).

The 1%-fat chocolate milk with and without inulin

sample was examined and compared to the control sam-

ple (probiotic-free) by 15 experts on the days 4 and 12

during refrigerated storage. The most and least accept-

able groups after 4 days storage were 1% fat probiotic

milk with inulin and 1% fat probiotic without inulin.

After 12 days milks with 2% fat were generally more

acceptable than 1% fat. Samples containing inulin and

probiotic and at the second stage samples with probiotic

and without inulin were more acceptable than control

groups with similar fat percentage (See table 3).

There are many studies that con rm the role of inu-

lin in improving sensory quality, enhancing the probi-

otic viability, asa prebiotic, fat replacer and texturizerin

some potentially synbiotic dairy products; for instance

FIGURE 1. Survival of B. lactis (BL) cells in chocolate milk during 12 days storage in the refrigerator (Duplicate

bacterial count).

120 EFFECTS OF INULIN AND FAT PERCENTAGE ON THE VIABILITY OF

BIFIDOBACTERIUM LACTIS

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Hadi Pourjafar et al.

Table 2. The results of the biochemical characteristics of 1% and 2% fat milk (without probiotic, with probiotic,

with both probiotic and inulin) during 12 days storage in the refrigerator.

1% Fat 2% Fat

Storage day Control Probiotic Probiotic & Inulin Control Probiotic Probiotic & Inulin

Viscosity 0 17.1 17.1 17.2 16.1 16.1 16.2

4 17.1 17.1 17.15 16.1 16.1 16.2

8 17 17.1 17.3 16.15 16.1 16.25

12 16.9 17.1 17.3 16.15 16.1 16.35

Acidity 0 15 15 15 15 15 15

4151515 151515

8151515 151515

12 15 15 15 15 15 15

pH 0 6.6 6.6 6.75 6.7 6.7 6.75

4 6.62 6.6 6.62 6.7 6.69 6.73

8 6.59 6.6 6.63 6.7 6.71 6.73

12 6.59 6.58 6.62 6.68 6.68 6.72

Table 1. The Mean ± standard deviation of log 10 of duplicate probiotic count in 1% and 2% fat

milk (without probiotic, with probiotic, with both probiotic and inulin) during 12 days storage in the

refrigerator in 104 dilution (n/group. day=4).

1% Fat 2% Fat

Storage day Probiotic

Probiotic & Inulin

Probiotic

Probiotic & Inulin

Bacterial count 0 6.4±0.1 6.4±0.1

6.0±0.1 6.1±0.1

4 6.4±0.0 7.2±0.0

5.3±0.8 5.4±0.5

8 7.0±0.2 7.0±0.1

6.6±0.1 6.6±0.2

12 6.0±0.4 6.9±0.2

6.9±0.4 6.7±0.1

The result of two way ANOVA test showed that there was a signi cant difference between the bacterial count

in different examination days (P=0.004) and different groups (P<0.001). There was also a signi cant difference

between the change of bacterial count during storage time in different groups (P=002). The groups shown by

different lowercase letters have statistical difference. Statistical differences between days in each group are

shown by different uppercase letters.

Table 3. The mean and standard deviation of the organoleptic acceptability of 1% and 2% fat milk (without probiotic,

with probiotic, with both probiotic and inulin) after 4 and 12 days storage in the refrigerator (examined by 15

experts).

1% Fat 2% Fat

Properties Control Probiotic Probiotic & Inulin Control Probiotic Probiotic & Inulin

Day 4 Flavor (from 10) 5.7 5.3 6.6 6.5 6.5 6.7

Smell (from 10) 5.2 4.9 6 6 6.1 6.4

Sweetness (from 10) 6.1 5.8 6.9 4.5 5.5 5.5

Viscosity (from 10) 5.4 5.9 6.5 6.36 6.72 6.45

Total result (from 20) 14.6±0.7 14.1±0.6 15.6±0.5 14.4±0.5 15.2±0.7 14.9±0.6

Day 12 Flavor (from 10) 5.3 5.9 6 5.8 6.45 7.09

Smell (from 10) 5.1 5.9 5.6 6.1 6.2 6.2

Sweetness (from 10) 6.1 6.2 7.4 6 7.1 6.4

Viscosity (from 10) 6 5.7 6.1 6.09 6.2 6.2

Total result (from 20) 13.0±0.7 13.5±0.8 14.5±0.4 14.1±0.7 14.3±0.7 15.4±0.5

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECTS OF INULIN AND FAT PERCENTAGE ON THE VIABILITY OF

BIFIDOBACTERIUM LACTIS

121

Hadi Pourjafar et al.

ice-cream, yogurt, cheese and other fermented dairy

beverages (Cardarelli et al. 2008, de Souza et al. 2017,

Guggisberg et al. 2009, Karimi et al. 2015, Kolida et al.

2002, Valero-Cases and Frutos 2017). de Souza et al.

(2017) studiedthe effect of inulin in different propor-

tions up to 3%, on the survivability and resistance to

simulated gastrointestinal conditions of Bi dobacterium

animalis Bb 12 added in probiotic and synbiotic mar-

garine. In this study, Inulin enhanced Bb 12 survival

signi cantly (P<0.05) (de Souza et al. 2017). Valero-

Cases and Frutos (2017) investigated the prebiotic effect

of diverse concentrations of inulin (0, 1 and 2%) on the

growth and viability of Lactobacillus plantarum CECT

220 in mixed carrot and orange juices. Throughout stor-

age time, the inulin improved the survivability of Lacto-

bacillus plantarum and the monosaccharide concentra-

tion remained upper regarding the juice without inulin

(40% lower). At 30 days, the fermented juices with 2%

inulin following in vitro digestion presented the maxi-

mum viability of Lactobacillus plantarum (Valero-Cases

and Frutos 2017).

To the best of our knowledge, present study is the  rst

work that shows the effects of inulin and fat percentage

concurrently on the viability of Bi dobacterium lactis.

According to the results of this investigation, the most

important status for the viability of Bi dobacterium lac-

tis was in 2% fat chocolate milk. We conclude that the

viability of this probiotic bacterium is higher in samples

with higher fat content, and adding inulin may increase

the viability of bacteria in low fat milk (1%) but not in

high fat milk (2%). Inulin inoculation had some effects on

biochemical and organoleptic changes. In sensory exami-

nation, all groups were more favorable than the control

sample (without the probiotic bacterium and inulin). It is

suggested that chocolate milk with 2% fat and inulin be

as a more effective probiotic dairy product.

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