Biotechnological

Communication

Biosci. Biotech. Res. Comm. 10(1): 72-77 (2017)

Effects of

Camellia sinensis

on survival of

encapsulated

Lactobacillus casei

and

Bi dobacterium lactis

in ice-cream

Negin Noori

, Leila Khaji

, and Hassan Gandomi

Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, University of Tehran,

Tehran, Iran

Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

ABSTRACT

Camellia sinensis is considered as one of the most common beverages with high ant oxidative effects al-around the

world. High levels of selenium caused it to be a good prebiotic for enhancement of the survival of probiotic bacteria.

The present investigation was done to study the effects of C. sinesis extract on the survival of encapsulated Bi do-

bacterium lactis and Lactobacillus casei in ice-cream. Aerial parts of the C. sinesis were dried and their extract were

puri ed using the distilled water. B. latis and L. casei strains and also extract of C. sinesis were encapsulated using

the chitosan-alginate procedure. Encapsulated materials were added to the content of ice-cream in the  nal stage of

procedure at the homemade ice-cream machine. L. casei and B. lactis strains were decreased through maintenance

period. Numbers of free and encapsulated strains of L. casei and B. lactis during 90 days of maintenance had a range

of 7.9 to 30.7 and 21.7 to 38.7 CFU/g and 65.6 to 6.6 and 48.6 to 15.6 CFU/g. Reduction in the numbers of probiotics

was entirely lower in encapsulated bacteria. Percent of the survival of probiotics in encapsulated groups was entirely

higher than control (P <0.05). Application of chitosan-alginate based microencapsulation along with the using from

C. sinensis extract is a good way to produce symbiotic ice-cream with high numbers of L. casei and B. lactis probiotic

bacteria.

KEY WORDS:

CAMELLIA SINENSIS

, ENCAPSULATION,

LACTOBACILLUS CASEI

BIFIDOBACTERIUM LACTIS

, ICE-CREAM

ARTICLE INFORMATION:

*Corresponding Author: L.kh52834@yahoo.com

Received 12

Jan, 2017

Accepted after revision 12

March, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF : 4.006

reserved.

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

Noori, Khaji, and Gandomi

INTRODUCTION

Green tea which is derived from the aerial parts of the

Camellia sinensis (C. sinensis) is considered as one of

the most common beverages al-around the world. It is

a part of the evergreen family. Leaves of the C. sinen-

sis are glossy green with serrated edges. It has a small

white  owers with yellow stamens (Moore et al., 2003;

Pastore and Fratellone, 2006; Goenka et al., 2013). C.

sinensis is full from  avonoids with high antimutagenic,

antioxidant, anticarcinogenic activities and can prevent

from development of cardiovascular and neurodegen-

erative diseases (Moore et al., 2003; Pastore andFratel-

lone, 2006; Goenka et al., 2013). This plant can easily

growth at the zone of North of Iran and especially Gilan

and Mazandaran province, Iran.

Probiotics can be distinct as live microorganisms

(bacteria and yeasts) that can bring health bene ts to

humans’ or animals’ bodies, usually the maintenance

and development of the microbial balance of the intes-

tine environment and inhibition form the substitution

of dangerous pathogenic strains into the intestinal tract

(Hekmat and McMahon, 1992; Nagpal et al., 2012;

Dehkordi et al., 2014). It is essential for most of these

live cultures to survive during their shelf life prior being

consumed. Therefore, probiotic producing companies

have been tried to increase the survival of probiotic

bacteria using plant extracts, phenolic compounds and

anti-oxidative substances (Hekmat and McMahon, 1992;

Nagpal et al., 2012; Dehkordi et al., 2014). Presence of

various chemical components in the leaves of C. sin-

ensis caused it to be used as a good prebiotic agent for

production of symbiotic products (Sourabh et al., 2014).

Combination of lactic acid starters with probiotic

(Bi dobacterium and Lactobacillus) is widely used in

dairy manufactures (Hekmat and McMahon, 1992; Nag-

pal et al., 2012; Dehkordi et al., 2014). Two of the most

important species of these bacteria are B. lactis and L.

casei. L. casei and bi dobacteria are normal inhabitants

of the human intestine and numerous health bene ts

have been reported for them. Several health bene ts are

attributed to these groups of probiotics, including anti-

carcinogenic, anti-infection, serum cholesterol reduc-

tion, nutritional and antimutagenic effects, stimulation

of immune system, and alleviation of lactose intoler-

ance symptoms (Hekmat and McMahon, 1992; Nagpal

et al., 2012; Dehkordi et al., 2014). Several investiga-

tions revealed that application of B. lactis and L. casei

together can cause higher amount of decrease in the

acidic pH of dairy products which will cause higher

survival of both bacteria in hard conditions and dur-

ing maintenance of foods (Hekmat and McMahon, 1992;

Nagpal et al., 2012; Dehkordi et al., 2014). Alienation of

prebiotic components is one of the most common ways

to increase the survival of probiotic bacteria in func-

tional foods. Prebiotics are the foods of probiotic bac-

teria. In the other hand, prebiotics are distinct as non-

digestible food ingredients that bene cially affect the

host by selectively stimulating the growth of one or a

limited number of bacterial species in the colon, such

asbi dobacteriaandlactobacilli. Prebiotic components

have several types including herbs, fruits, sweet vegeta-

bles, fructooligosaccharides and inulin (Patel and Goyal,

2012).

Functional components of the C. sinensis besides its

high frequency in Iran caused us to carried out the pre-

sent study in order to investigate the effects of the C.

sinensis extract on the survival of L. casei and B. lactis

bacteria encapsulated with alginate chitosan in symbi-

otic ice-cream during 90 days maintenance in freeze

conditions.

MATERIAL AND METHODS

Plants

From July to August 2015, aerial parts of the C. sinensis

L. were collected from the farms of Gilan province, north

of Iran. Plants were identi ed by the experts of the bot-

any of the Faculty of Pharmacology, Tehran University

of Medical Sciences, Tehran, Iran. Plants were recorded

by the herbarium number of L481-8732.

Preparation and extraction of plant’s extract

Plant material was dried in shade for ten days. Then,

Plant material was ground to a powder in a mechani-

cal grinder. Five-hundred grams of the dried plants were

mixed with 2 liters distilled water. Contents of the previ-

ous stage were then  ltered using a 0.22 μm membrane.

Then, additional 1 liter of the distilled water was added

to the non- ltrated contents and shaken for 48 hrs. Con-

tents were  ltered in a same condition and  nally  l-

tered extracts were dried on a freeze dryer device for 48

hrs. Residual materials were kept at 4 °C.

Preparation of probiotic bacteria

Freeze dried Lactobaciliu casei (Lc-01) and L. casei (Bb-

12) (CHR, Hansen, Denmark) was used in this study. L.

casei strains were transferred to the Man Rogosa and

Sharpe broth media (MRS Broth, Sigma, Uk) and incu-

bated aerobically at 37 °C for 24 hrs. Both bacteria were

also cultured on slant MRS agar media and kept at

refrigerator.

Bacterial inoculation

One-loop of the MRSA agar media was taken and inoc-

ulated to the MRS broth media and incubated anaero-

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECTS OF

CAMELLIA SINENSIS

ON PROBIOTICS OF ICE-CREAM 73

Noori, Khaji, and Gandomi

bically at 37 °C for 48 hrs. bacterial cultures were then

centrifuged in a 4000 rpm at 4 °C for 10 min. After

deposits of bacteria, the supernatant was evacuated and

2 mL of sterile saline was added to the tube. All tubes

were then vortexed. Operations of centrifugation and

washing were done 2 times. Spectrophotometer device

was set at 600 nm and a cuvette containing sterile saline

was used for its calibration. Appropriate amounts of the

prepared bacterial suspension were added to a cuvette

and its optic absorption was set on the 1 number. The

bacterial suspensions were then prepared at a concentra-

tion 10 times higher than optical density of 1. An over-

all of 0.5 ml of the prepared bacterial suspension were

taken, and transmitted to a tube contained 4.5 ml of

the 0.1% peptone water media (Merck, Germany). Then,

the serial dilutions were prepared. Totally, from the 10

-5

and 10

-6

dilutions were cultured on the MRS agar media.

Colonies were enumerated after 48 hrs of incubation on

37 °C. Numbers of the L. casei and B. lactis were 1.2×10

and 1×10

CFU/ml.

Bacterial microencapsulation

Microencapsulation procedure was done according to

the method described previously by Ahmadi et al. (2014)

(Ahmadi et al., 2014) and Allan-Wojtas et al. (2008)

(Allan-Wojtas et al., 2008). In the encapsulation pro-

cedure, bacterial suspension was added to 5 ml of 0.1%

peptone water media and then 40 ml of sterile Sodium

Alginate and 1% of the C. sinensis extract were added

to the mixture. In the other hand, encapsulated bacteria

were mixed with the extract of C. sinensis.

Preparation of ice-cream

Formulation of ice-cream was consisted of 10% fat (cream

and  uid milk), 11% milk solid not-fat (MSNF, Mash-

had, Iran), 15% sucrose, 0.1% vanilla (Polar Bear Brand,

China), 0.15% emulsi er (Puratus, Belgium), and 0.35%

stabilizer. Milk and cream (fat) samples were mixed and

heated until the temperature of mixture reached to 40

°C. Then, sucrose (270 g), vanilla (0.9 g) and stabilizer

(10 g) were added to the mixture and heated at 80 °C

for 20 min. Then, mixture was transferred to the home-

made ice-cream machine (Elegant BL 1380) and 1% of

encapsulated and also free (control group) bacteria were

added to them and process was continuing for 20 min.

Samples of ice-creams were then packaged in the 50 g

plastic containers and kept at -18 °C.

Enumeration and study the survival of bacteria

in ice-cream

Two-hundred milliliters of the 0.1 molar sodium citrate

were added to the 50 grams of ice-creams in the poly-

ethylene bags. Polyethylene bags were then settled on

the bag mixer. After 10 min remaining at the laboratory,

the bag mixer device was used 3 times for their well

mixing. Then, 0.5 ml of the contents of the bag mixture

was added to the tubes containing 4.5 ml of 0.1 peptone

water. After well vortex of tubes, 10

-5

dilutions were pre-

pared. Then, 100 microliters of each diluent were taken

and super cially cultured on the MRS agar media. Plates

were then incubated on the aerobic (for L. casei) and

anaerobic (for B. lactis) conditions at 37 °C for 48 hrs.

Finally, bacterial colonies were encountered.

Statistical analysis

All statistical analyses were carried out using the SPSS

statistical software (version 20, USA). One-way ANOVA

and Tukey tests were used to study an any statistical dif-

ferences between groups of the investigation. In all tests,

P <0.05 was considered as a signi cant level.

RESULTS AND DISCUSSION

The present investigation was done to study the effects

of the C. sinensis extract on the survival of L. casei and

B. lactis bacteria encapsulated with alginate chitosan

in symbiotic ice-cream during 90 days maintenance in

freeze conditions. Table 1 represents the results of the

enumeration of free and encapsulated L. casei in sym-

biotic ice-cream during 90 days. Results showed that

numbers of free strains of L. casei had a range of 7.9

to 30.7 CFU/g. We found that the numbers of L. casei

strains were decreased through maintenance period in

symbiotic ice-cream. In the encapsulated L. casei strains

with C. sinensis extract, reduction in the numbers of

bacteria through the maintenance period had a slower

tilt. Ranges of encapsulated L. casei strains were 21.7

to 38.7 CFU/g. Statistically signi cant differences were

found for the numbers of bacteria of both groups in the

days of 0, 30, 45, 75 and 90 of maintenance. Percent of

the survival of the free and encapsulated L. casei strains

during 90 days maintenance period is shown in table

2. Signi cant statistical differences were seen between

the percent of free and encapsulated L. casei strains (P

<0.05).

Table 3 represents the results of the enumeration of

free and encapsulated B. lactis in symbiotic ice-cream

during 90 days. Results showed that numbers of free

strains of B. lactis had a range of 65.6 to 6.6 CFU/g.

We found that the numbers of B. lactis strains were

decreased through maintenance period in symbiotic

ice-cream. In the encapsulated B. lactis strains with C.

sinensis extract, reduction in the numbers of bacteria

through the maintenance period had a slower tilt. Ranges

of encapsulated B. lactis strains were 48.6 to 15.6 CFU/g.

Statistically signi cant differences were found for the

74 EFFECTS OF

CAMELLIA SINENSIS

ON PROBIOTICS OF ICE-CREAM BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Noori, Khaji, and Gandomi

Table 1. Average log of the numbers of free and encapsulated L. casei in symbiotic ice-cream during 90 days of

maintenance.

Types of bacteria

Numbers of bacteria (Log CFU/g) in various days

0 153045 60 75 90

Free L. casei 30.7±0.46a* 26.7±0.61a 25.7±0.72b 22.7±0.58b 21.7±0.73a 15.7±0.62b 7.9±0.57b

Encapsulated L. casei

with C. sinensis

38.7±0.4b 30.7±0.33a 33.7±0.71a 30.7±0.53a 27.7±0.29a 26.7±0.64a 21.7±0.36a

*Dissimilar leather in each column represent the signi cant difference about P <0.05.

Table 2. Percent of the survival of free and encapsulated L. casei in symbiotic ice-

cream during 90 days of maintenance.

Types of bacteria

Survival (%) in various days

15 30 45 60 75 90

Free L. casei 91.40a* 90.40a 86.60a 83a 70.90a 62.20a

Encapsulated L. casei with

C. sinensis

90.20a 83.80b 84.30a 78.60b 77.40a 69.10a

*Dissimilar leather in each column represent the signi cant difference about P <0.05.

Table 3. Average log of the numbers of free and encapsulated B. lactis in symbiotic ice-cream during 90 days of

maintenance.

Types of bacteria

Numbers of bacteria (Log CFU/g) in various days

0 153045 60 7590

Free B. lactis 65.6±0.33a 45.6±0.54a 34.6±0.81a 18.6±0.19a 11.6±0.22b 8.6±0.51b 6.6±0.25b

Encapsulated B. lactis with

C. sinensis

48.6±0.28b 30.6±0.39b 30.6±0.64a 18.6±0.44a 17.6±0.36a 17.6±0.75a 15.6±0.61a

*Dissimilar leather in each column represent the signi cant difference about P <0.05.

Table 4. Percent of the survival of free and encapsulated B. lactis in

symbiotic ice-cream during 90 days of maintenance.

Types of bacteria

Survival (%) in various days

15 30 45 60 75 90

Free B. lactis 66.70a 50a 33.90b 28.90b 28.30b 26.70

Encapsulated B. lactis with

C. sinensis

76.30a 66.70a 50a 49.20a 50.80a 40

*Dissimilar leather in each column represent the signi cant difference about P <0.05.

numbers of bacteria of both groups in the days of 0, 15,

60, 75 and 90 of maintenance. Table 4 shows the percent

of survival for the free and encapsulated B. lactis strains

during 90 days of maintenance. Signi cant statistical

differences were seen between the percent of free and

encapsulated B. lactis strains (P <0.05).

Microencapsulation is a novel method to protect from

probiotic bacteria like lactobacillus and bi dobacterium

species and also increase their survival in hard condi-

tions and also the maintenance period. In keeping with

the hard conditions of acidic foods, protection of pro-

biotic bacteria from the acidic conditions of stomach is

another important reason for using from microencapsu-

lation procedure.

To date, several methods have been applied to protect

from the probiotic microorganisms and increase their

survival in food science. As far as we know, the present

investigation is the  rst prevalence report of the appli-

cation of the extract of C. sinensis on the survival of

encapsulated L. casei and B. lactis on the symbiotic ice-

cream during the 90 days of maintenance. Our results

represented that application of C. sinensis can protect

from the numbers of the L. casei and B. lactis during the

period of maintenance.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECTS OF

CAMELLIA SINENSIS

ON PROBIOTICS OF ICE-CREAM 75

Noori, Khaji, and Gandomi

Several investigations have been conducted in this

 eld. Akin et al. (2007) (Akin et al., 2007) showed that

ice-cream is a good dairy-based food product for trans-

mission of probiotic bacteria to customers, while Kaila-

sapathy and Sultana (2003) (Kailasapathy and Sultana,

2003) mentioned that decrease in the numbers of probi-

otic bacteria during the freezing stages in ice-creams is

an inhibitory factor for survival of bacteria. Similar to

the results of the Akin et al. (2007) (Akin et al., 2007), we

found that ice-cream has a high capacity for transmis-

sion of probiotics to human. We found that the survival

percent of free and encapsulated L. casei and B. last-

ics probiotics in ice-creams were 62.20% and 69.10%

and 26.70% and 40%, respectively which was accept-

able. Reduction in the numbers of probiotic bacteria is

neither a surprising  nding neither in ice-cream and nor

in any other types of dairy products. It a common  nd-

ing in all researches and even in all factory’s products.

An important matter is the fact that C. sinensis extract

can increase the viability of L. casei and B. lastics pro-

biotics in ice-creams. Shah and Ravula (2000) (Shah

and Ravula, 2000) represented that the microencapsula-

tion procedure is a good and functional procedure to

increase the viability of probiotic bacteria in frozen des-

serts which was similar to our  ndings. Krasaekoopt et

al. (2004) (Krasaekoopt et al., 2004) approved that the

microencapsulation using the alginate and chitosan is

the best practical method to increase the viability of pro-

biotics in dairy products which support the basic idea of

our study.

Survival of probiotics in ice-cream has been impacted

in unappropriated environmental conditions such as

presence of oxygen, freezing procedure and its bad

effects due to the creation of ice crystals, mechanical

stress of the production procedures and its storage at low

temperature. The main reason for the higher decrease in

the numbers of B. lactis strains in compare to the L.

casei is the fact that bi dobacteria are mainly anaerobic

and aeration process which is necessary for formation of

ice-creams can enter high amounts of oxygen into the

ice-cream which is resulted in the destruction of bi do-

bacteria.

Picot and Lacroix (2003) (Picot and Lacroix, 2003)

reported that the extract of C. sinensis has a high degree

of selenium which can facilitate and improve the growth

of L. casei and B. lactis bacteria in functional foods.

They also stated that high antioxidant and antiradical

effects of the C. sinensis extract cause its strong pro-

tection on probiotic bacteria. Another study which was

conducted by Molan et al. (2009) (Molan et al., 2009)

showed that the survival of B. lactis and L. acidophilus

probiotics in ice-cream was increased using 1% inulin

powder. They showed that the numbers of probiotic bac-

teria through 90 days maintenance was decreased from

to 10

log CFU/g, while in the control group was

decreased to 10

log CFU/g.

CONCLUSION

This research project showed that application of the C.

sinensis extract can improve the survival of L. casei

and B. lactis bacteria in ice-cream. We recommended

the application of chitosan-alginate microencapsula-

tion procedure along with the application of C. sinensis

extract to production of symbiotic ice-cream with high

numbers of L. casei and B. lactis probiotic bacteria even

after 90 days of maintenance in freeze temperature.

ACKNOWLEDGEMENTS

Authors want to thank Prof. Afshin Akhondzadeh Basti

and Prof. Ali Misaghi at the Department of Food Safety,

University of Tehran, Tehran, Iran for their signi cant

practical provision. This work was supported by the Uni-

versity of Tehran.

REFERENCES

Ahmadi, A.,Milani, E.,Madadlou, A.,Mortazavi, S.A.,Mokar-

ram, R.R. andSalarbashi, D. (2014). Synbiotic yogurt-ice cream

produced via incorporation of microencapsulated lactobacillus

acidophilus (la-5) and fructo oligosaccharide. Journal of Food

Science and Technology, Vol. 51, No. 8, pp. 1568-1574.

Akin, M.B., Akin, M.S. and Kirmaci, Z. (2007). Effects of inulin

and sugar levels on the viability of yogurt and probiotic bacte-

ria and the physical and sensory characteristics in probiotic ice

cream. Food Chemistry, Vol. 104, No. 1, pp. 53–59.

Allan-Wojtas, P., Truelstrup, H.L. and Paulson, A.T. (2008).

Microstructural studies of probiotic bacteria-loaded alginate

microcapsules using standard electron microscopy techniques

and anhydrous  xation. LWT-Food Science and Technology,

Vol. 41, No. 1, pp. 101–108.

Dehkordi, F.S., Yazdani, F., Mozafari, J. and Valizadeh, Y.

(2014). Virulence factors, serogroups and antimicrobial resist-

ance properties of Escherichia coli strains in fermented dairy

products. BMC Research Notes, Vol. 7, pp. 217.

Goenka, P., Sarawgi, A., Karun, V., Nigam, A.G., Dutta, S.

andMarwah, N. (2013). Camellia sinensis (Tea): Implications

and role in preventing dental decay. Pharmacognosy Review,

Vol. 7, No. 14, pp. 152-6.

Hekmat, S.and McMahon, D.J. (1992). Survival of Lactobacil-

lus acidophilus and Bi dobacterium bi dum in ice cream for

use as a probiotic food. Journal of Dairy Science, Vol. 75, No.

6, pp. 1415-22.

Kailasapathy, K. and Sultana, k. (2003). Survival and b-D-

galactosidase activity of encapsulated and free Lactobacillus

acidophilus and Bi dobacterium lactis in ice cream. Australian

Journal of Dairy Technology, Vol. 58, No. 3, pp. 223–227.

76 EFFECTS OF

CAMELLIA SINENSIS

ON PROBIOTICS OF ICE-CREAM BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Noori, Khaji, and Gandomi

Krasaekoopt, W., Bhandari, B. and Deeth, H. (2003). Evaluation

of encapsulation techniques of probiotics for yoghurt. Interna-

tional Dairy Journal, Vol. 13, No. 1, pp. 3-13.

Molan, A.L., Flanagan, J., Wei, W. and Moughan, P.J. (2009).

Selenium-containing green tea has higher antioxidant and

prebiotic activities than regular green tea. Food Chemistry, Vol.

114, No. 3, pp. 82–87.

Moore, R.J.,Jackson, K.G. andMinihane, A.M. (2009). Green

tea (Camellia sinensis) catechins and vascular function. British

Journal of Nutrition, Vol. 102, No. 12, pp. 1790-802.

Nagpal, R., Kumar, A., Kumar, M., Behare, P.V., Jain, S.

and Yadav, H. (2012). Probiotics, their health bene ts and

applications for developing healthier foods: a review. FEMS

Microbiology Letters, Vol. 334, No. 1, pp. 1-15.

Pastore, R.L. andFratellone, P. (2006). Potential health bene ts

of green tea (Camellia sinensis): a narrative review. Explore

(NY), Vol. 2, No. 6, pp. 531-9.

Patel, S. and Goyal, A. (2012). The current trends and future

perspectives of prebiotics research: a review. 3 Biotechnology,

Vol. 2, No. 2, pp. 115–125.

Picot, A. and Lacroix, C. (2003). Effects of microniza-

tion on viability and thermotolerance of probiotic freeze-

dried cultures. International Dairy Journal, Vol. 13, No. 6,

pp. 78-84.

Shah, N.P. and Ravula, R.R. (2000). Microencapsulation of pro-

biotic bacteria and their survival in frozen fermented dairy

desserts. Australian Journal of Dairy Technology, Vol. 55, No.

3, pp. 139–144.

Sourabh, A,Kanwar, S.S,Sud, R.G,Ghabru, A. andSharma,

O.P. (2014). In uence of phenolic compounds of Kangra

tea, Camellia sinensis (L) O Kuntze] on bacterial patho-

gens and indigenous bacterial probiotics of Western Hima-

layas. Brazilian Journal of Microbiology, Vol. 44, No. 3,

pp. 709-15.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECTS OF

CAMELLIA SINENSIS

ON PROBIOTICS OF ICE-CREAM 77