¹Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

²Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

Corresponding author Email: L.kh52834@yahoo.com

Article Publishing History

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. sinensis are glossy green with serrated edges. It has a small white flowers with yellow stamens (Moore et al., 2003; Pastore and Fratellone, 2006; Goenka et al., 2013). C. sinensis is full from flavonoids with high antimutagenic, antioxidant, anticarcinogenic activities and can prevent from development of cardiovascular and neurodegenerative diseases (Moore et al., 2003; Pastore and Fratellone, 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 benefits to humans’ or animals’ bodies, usually the maintenance and development of the microbial balance of the intestine 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. sinensis 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 (Bifidobacterium and Lactobacillus) is widely used in dairy manufactures (Hekmat and McMahon, 1992; Nagpal 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 bifidobacteria are normal inhabitants of the human intestine and numerous health benefits have been reported for them. Several health benefits are attributed to these groups of probiotics, including anti-carcinogenic, anti-infection, serum cholesterol reduction, nutritional and antimutagenic effects, stimulation of immune system, and alleviation of lactose intolerance symptoms (Hekmat and McMahon, 1992; Nagpal et al., 2012; Dehkordi et al., 2014). Several investigations 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 during 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 functional foods. Prebiotics are the foods of probiotic bacteria. In the other hand, prebiotics are distinct as non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth of one or a limited number of bacterial species in the colon, such as bifidobacteria and lactobacilli. Prebiotic components have several types including herbs, fruits, sweet vegetables, 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 present 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 symbiotic 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 identified by the experts of the botany 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 mechanical grinder. Five-hundred grams of the dried plants were mixed with 2 liters distilled water. Contents of the previous stage were then filtered using a 0.22 μm membrane. Then, additional 1 liter of the distilled water was added to the non-filtrated contents and shaken for 48 hrs. Contents were filtered in a same condition and finally filtered 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 incubated 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 inoculated to the MRS broth media and incubated anaerobically 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 concentration 10 times higher than optical density of 1. An overall 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 1.2×10⁹ and 1×10⁹ 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 procedure, 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 fluid milk), 11% milk solid not-fat (MSNF, Mashhad, Iran), 15% sucrose, 0.1% vanilla (Polar Bear Brand, China), 0.15% emulsifier (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 homemade 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 polyethylene 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 prepared. Then, 100 microliters of each diluent were taken and superficially 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 differences between groups of the investigation. In all tests, P <0.05 was considered as a significant 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 symbiotic 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 significant 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. Significant statistical differences were seen between the percent of free and encapsulated L. casei strains (P <0.05).

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	15	30	45	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. caseiwith 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 significant 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 significant difference about 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 significant differences were found for the 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. Significant statistical differences were seen between the percent of free and encapsulated B. lactis strains (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	15	30	45	60	75	90
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 significant 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 significant difference about P <0.05.

Microencapsulation is a novel method to protect from probiotic bacteria like lactobacillus and bifidobacterium species and also increase their survival in hard conditions and also the maintenance period. In keeping with the hard conditions of acidic foods, protection of probiotic bacteria from the acidic conditions of stomach is another important reason for using from microencapsulation 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 first prevalence report of the application 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.

Several investigations have been conducted in this field. Akin et al. (2007) (Akin et al., 2007) showed that ice-cream is a good dairy-based food product for transmission of probiotic bacteria to customers, while Kailasapathy and Sultana (2003) (Kailasapathy and Sultana, 2003) mentioned that decrease in the numbers of probiotic 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 transmission of probiotics to human. We found that the survival percent of free and encapsulated L. casei and B. lastics probiotics in ice-creams were 62.20% and 69.10% and 26.70% and 40%, respectively which was acceptable. Reduction in the numbers of probiotic bacteria is neither a surprising finding neither in ice-cream and nor in any other types of dairy products. It a common finding 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 probiotics in ice-creams. Shah and Ravula (2000) (Shah and Ravula, 2000) represented that the microencapsulation procedure is a good and functional procedure to increase the viability of probiotic bacteria in frozen desserts which was similar to our findings. 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 probiotics 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. lactisstrains in compare to the L. casei is the fact that bifidobacteria 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 bifidobacteria.

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 protection 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 bacteria through 90 days maintenance was decreased from 10⁷ to 10⁶ log CFU/g, while in the control group was decreased to 10⁵ 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 microencapsulation 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 significant practical provision. This work was supported by the University of Tehran.

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