Microencapsulation of probiotic bacteria by sodium alginate and investigation in batter of probiotic wafer

The positive role of bacteria in human’s health introduced 1st time by a Russian scantiest named Metchnikov in the beginning of the 20th century. He believed that improving of intestine micro flora is possible by prescription of desirable bacteria (Piahoo et al, 2012).Regarding his theory in fermented dairy products, appropriate bacteria competes with undesirable pathogens. He also investigates the micro flora of intestine and found that aging process is due to some compounds produced by intestine bacteria. Probiotic bacteria define as live micro organism which play functional roles in human’s health (Najmeh and Shiva, 2011).

The importance using of probiotic bacteria is to improve lactose digestion, promoting calcium absorption, vitamin and protein synthesis, immune system, reduction of blood cholesterol, preventing of different kind of can- cers, decreasing allergic symptoms, adjustment of blood pressure, and treatment of cardiovascular disease and increasing of nutritional value (Piahoo et al, 2012; Marhamatizadeh et al, 2010; Aghajani et al, 2011 and Capelaa et al, 2007) of probiotic bacteria occur after their placement in different parts of body and their biological activity, hence this product classifies as functional food. It is thought that the sufficient bacteria must be present in order to carry probiotic affections on the body (Milan et al, 2011)

Probiotic bacteria inhibit the growth of pathogens through organic acid production and other anti microbial compounds (Moyano et al, 2008).

Today, clinical studies have reported the probiotic affections in curing of systemic diseases. The dominant of undesirable bacteria not only result in reduction of essential compounds production but increase the production of harmful compounds therefore the consumption of 100g of probiotic product including 106-108 live bacteria per gram of production can supply the considered optimized level (Taheri et al, 2010; Bengmark, 1998; Bergamini et al, 2005; Helland et al, 2004).

Mostly used probiotic products which have consumed have not exhibited side effects up to now (Khalkhali et al, 2009).Encapsulation of Probiotic cells is one of the novel methods of increasing shelf life of probiotics in order to transfer them through colon. The less amount of probiotic obtaining in difficult conditions such as bile acids has almost always have provoked scientist to find methods of improving this item.

Encapsulation may consider as one of the newest method in obtaining probiotic in conditions such as bile acids. Mostly, livability of probiotic cells in products like confectionary and their reproduction follow a gently rate due to the condition of low water activity in cereal products and confectionaries. Probiotics have injected to non dairy products such as sausages, chocolate, infant food, bread and mayonnaise (Mirzaei et al, 2013).

Encapsulation techniques are foodstuffs, enzymes, cells etc in especial capsules with the size of µm-mm. Encapsulation also uses as a method of flavor and taste covering which effects on organoleptic properties. Along with protecting affections of encapsulation, this method can consider as a promoter of flow properties and transferring of compounds. Gum, sugar protein, natural or modified poly saccharides are using in the capsule walls. Encapsulated compounds consider as inner core which can be in 3 liquid, gas and solid form, mostly liquid (Niazmand et al, 2006).From the viewpoint of microbiology, encapsulation defines as covering of a layer of hydrocolloid around microorganism cells and entrapping them to increase their shelf life. Overall the main goal of encapsulation is increasing the shelf life of probiotics along with the transforming possibility and safe releases of them in appropriate points of digesting system (Mirzaei et al, 2013). Alginates extracted from sea algae and create a hard structure in combination with calcium chloride. Calcium alginate is a non toxic compound with low cost in encapsulation and their gels are producing in presence of monovalent ions And chelating agent of calcium including phosphates, lactates and citrates results in destruction of alginate capsule (Khosravi Zanjani et al, 2013).

Alginates, non digestible compounds with no nutritional value, have a wide range of physiochemical properties. They are non toxic materials and find in soluble salt. Alginate with monovalent metal is in soluble salt form. While in the presence of double and triple valent ions like ca2+ create non soluble gel.

Alginate gel is non melt able and irreversible. These gels are formed from mixing of alginate and suitable salt (often calcium) or ion exchange with basic metals such as sodium form to liquid. The reason of forming this gel is binding of hydroxyl group with bonded side carboxyl group of and some extent fill the hole between parallel chains of G block. This arrangement calls egg box model (Alborzi, 2012).

Recent advances in encapsulation can be helpful in both industrial and non industrial goals about shelf life of probiotics in product and their maintenance in digestion systems and their affections on the body (Mirzaei et al, 2012).

Material and methodActivation and separation of probiotic bacteria

First, probiotic bacteria produced by E.S.M Co, Australia in powder form containing dominant variety named L. Casei carried to liquid lactose (Merck Co, Germany) in order to activating, the aforesaid materials incubated in incubator(produced by Nuve Co model, EN-055, Turkey) at 37°C for 48 hr. then to separate bacteria and reaching bacteria precipitation, tubes containing activated bacteria centrifuged with 6000rpm for 10 min, then its supernatant removed and the bacteria which is precipitated washed (Capelaa et al,2007; Kazemi Gorgi, 2012; P´erez Guerra et al, 2007; P´erez Guerra et al, 2000). By diluted water and centrifuged again then kept at 4°C (Capelaa et al, 2007; Kazemi Gorgi,. 2012; P´erez Guerra, 2007; P´erez Guerra et al, 2007).

Encapsulation using sodium alginate

All containers sterilized and autoclaved at 121°C for 15 min, then 3g of sodium alginate (Applichem, Germany) added to a beaker containing 100 ml distilled water and mixed with a magnet with 200rpm up to complete solution of sodium alginate in distilled water, then in order to uptake of sodium alginate, kept one night in refrigerator, besides 11.099g of sodium chloride dissolved in 100 ml of distilled water then 80 ml of it added to a flask 100ml and mixed with a magnet up to complete solution in distilled water Then 80 ml of it added to flask 100 ml and 10 ml of microbial suspension (containing at least 107-109 CFU/ml) added to it, distilled water added and the volume reached to 100 ml, mixed well for 5 min. in next stage 199 ml rapeseed oil poured in beaker 200ml, 1g of Tnue (emulsifier), then mixed with magnet for 5 min to produce a creamy color emulsion, rate adjusted at 750 rpm and 50 ml of mixture of alginate-bacteria carried to oil containing emulsifier by pipette, mixed 20 min.In next stage a solution of calcium chloride 0.1M poured in burette. Calcium chloride solution added gently to beaker containing alginate-bacteria mixture (mixture rate adjusted on 100 rpm) to create seeds, and then allow precipitating of seeds for 30 min, centrifuged with 350g for 15 min and kept at 4°C. (Capelaa et al, 2007; Alborzi, 2012; P´erez Guerra, 2007; P´erez Guerra et al,2007).

The number of initial encapsulated bacteria

0.1 g of microcapsules weighed and poured in 10 ml sodium citrate and allowed to separate in ambient temperature, then shook the mixture and diluted to dilution of 10^-7, cultured in MRS agar and incubated for 48 hr. Resulted colonies counted by colony counter model Funke Gerb, Switzerland and found that 2.3x10 5 FU/ml in 0.1 g of microcapsule (AOAC, 2002).

To each treatment, 3 g of encapsulated bacteria and to control 3 g non encapsulated bacteria added. To provide wafer cream (batter), date syrup concentrated and wafer produced as follows.

Preparation of batter

To provide batter, ingredients including oil 17%, sugar 47 %, inulin 15%, encapsulated probiotic bacteria with sodium alginate 0.5%, dry milk 1.5 %, water 17% and salt 2 % added to the mixer base on recipe. Sugar must used in powder from to mixed well and texture become soft. Mixing last to create a uniform cream finally treatment produced.

Microbial experiments The number of probiotic bacteria

Culture media of MRS agar was experimented base on National Iran Standard N.O. 365 with 3 replications.MRS agar (Merck Co, Germany) purchased and prepared base on its instruction present on the label,100g of prepared probiotic bacteria weighed and dissolved in 45 ml sodium citrate 2% and shook 20 min to completely separation of capsules, then required dilution provided so that 1 ml of probiotic bacteria solution added to 9 ml sterile peptone water and pour plated in 3 replications in MRS agar culture media, then cultures incubated at 37 ° C for 72 hr. resulted colonies counted by colony counter. The acceptable amount of probiotic in 100 g of foodstuffs is 10⁶ live cells (AOAC, 2002).

Conclusion and discussion

The results of microbial experiments have shown in table 1. Results exhibit more than 300 colonies found in MRS agar cultured in dilution of 10-1-10-2 and more. Re- sults also displayed that the number of colonies was less than 30 in dilution range of 10-6- 10-7. The number of colonies were 182.5, 79.5 and 36.5 in dilution of 10-3, 10-4 and 10-5 respectively.

On the other hand the number of probiotic bacteria in dilution of 10-3, 10-4 and 10-5 were 1.83×105, 7.95×105 and 3.65×105 respectively, besides no bacterial growth found after 6 weeks in MRS agar culture media. Comparing the results related to both non encapsulated and encapsulated bacteria, the prolongation of later revealed which was due to calcium alginate gel to stomach acid as protective factor. Encapsulation also causes to separate bacteria from outer surrounding medium and increasing livability of cells. These results are in agreement with Khosravi et al findings (2013).Therefore adding probiotic bacteria to batter can be performed by their encapsulation with sodium alginate. In another study the affection of simulated conditions of intestine investigated. Results revealed more obtaining of encapsulated bacteria (Milani et al, 2011).It must be noted that control cultured in MRS agar after 6 weeks, however no growth observed.

Conclusion

Results revealed that encapsulation of probiotic bacteria results in increasing of livability in comparison with non encapsulated bacteria in wafer batter. The remained number of encapsulated probioc bacteria after 6 weeks of storage at 4±1 ° C was in the permitted level for human’s health ,i.e. 10⁶ per g. this was due to resistance of calcium alginate to stomach acid. Sodium alginate is a resistant compound in presence of stomach acid.

Table 1. Results of microbial experiment in MRS agar after 6 weeks

Treatment concentration	10-1	10-2	10-3	10-4	10-5	10-6	10-7
Control	≥300	≥300	182.5	79.5	36.5	≤30	Not seen
25 % date juice	≥300	≥300	130	50	≤30	≤30	Not seen
50 % date juice	≥300	≥300	165	59	39.5	≤30	Not seen
75 % date juice	≥300	≥300	200	32	≤30	≤30	Not seen
100 % date juice	≥300	≥300	131	76	51	≤30	Not seen

And act as a protective cover and protect the en-                    Acknowledgement trapped bacteria via separation of them from outer medi- Authors would like to thank Mrs. Farzaneh Zeinolabedini, um and increasing livability of bacteria.                     Biology Graduated and all people who supports us in this study.