The Production of Synbiotic Bread by Microencapsulation

Probiotics are de ned as live microorganisms that, when administered in adequate amounts, confer a health bene t to the consumer (1). In order to provide health bene ts of probiotic bacteria, they should be present at a minimum level of 106 CFU/g of food product or 107 CFU/g at point of delivery or be eaten in su cient amount to yield a daily intake of 108 CFU/g (2). Prebiotics are nondigestible substances that contribute to the well-being of their host by selectively stimulating the favourable growth or activity of a limited number of indigenous nonpathogenic bacteria (1,3). Fructooligosaccharides and inulin are among the most famous prebiotic compounds (4,5). Synbiotic foods are synergistic combinations of preand probiotics. The development of nondairy probiotic products is a challenge to the food industry in its e ort to utilize the abundant natural resources by producing high-quality functional products (6). Bread is a staple food in many countries and it constitutes a dominant portion of a standard diet, supplying a large fraction of the needs for energy, carbohydrates, proteins and micronutrients. In recent years, there is an increased interest in the role of food with health bene ts. The priority of the industry today is innovative approach in satisfying consumer needs. However, functional bread containing viable microorganisms has not been developed yet because of the high temperature during baking (7). A new approach to improve the probiotic survival is by physical protection by microencapsulation, which can help protect the bacterial cells ISSN 1330-9862 original scienti c paper


Introduction
Probiotics are de ned as live microorganisms that, when administered in adequate amounts, confer a health bene t to the consumer (1).In order to provide health bene ts of probiotic bacteria, they should be present at a minimum level of 10 6 CFU/g of food product or 10 7 CFU/g at point of delivery or be eaten in su cient amount to yield a daily intake of 10 8 CFU/g (2).Prebiotics are nondigestible substances that contribute to the well-being of their host by selectively stimulating the favourable growth or activity of a limited number of indigenous nonpathogenic bacteria (1,3).Fructooligosaccharides and inulin are among the most famous prebiotic compounds (4,5).Synbiotic foods are synergistic combinations of preand probiotics.
The development of nondairy probiotic products is a challenge to the food industry in its e ort to utilize the abundant natural resources by producing high-quality functional products (6).Bread is a staple food in many countries and it constitutes a dominant portion of a standard diet, supplying a large fraction of the needs for energy, carbohydrates, proteins and micronutrients.In recent years, there is an increased interest in the role of food with health bene ts.The priority of the industry today is innovative approach in satisfying consumer needs.However, functional bread containing viable microorganisms has not been developed yet because of the high temperature during baking (7).A new approach to improve the probiotic survival is by physical protection by microencapsulation, which can help protect the bacterial cells from the hostile conditions such as those present within gastrointestinal tract, thus potentially preventing cell loss (3).
Encapsulating lactobacilli in calcium alginate has been found to improve their heat tolerance and increase the survival by up to 80-95 % (8)(9)(10).Alginate is an approved food additive and the bene ts of its use as an encapsulating agent include: non-toxicity, formation of gentle matrices with calcium chloride to trap living microbial cells, simplicity and low cost (8,10).However, the use of alginate is limited due to its low stability in the presence of chelating agents and in acidic conditions below pH=2.0 (2,11,12).Combination of calcium alginate with prebiotics such as resistant starch improves both the viability of probiotics and structures of capsules (12,13).The coating of alginate beads and its e ectiveness in protecting probiotic bacteria has been extensively studied.Previous researchers have reported that coating alginate microcapsules with chitosan improves the stability of the alginate beads, increasing probiotic viability even further (2,8).Li le research has been carried out with an aim to incorporate probiotics into bakery products, due to destruction of live culture during heat treatment.The aim of this study is to obtain synbiotic bread, hence hamburger bun and white pan bread were selected.

Preparation of cell suspension
Pure freeze-dried Lactobacillus acidophilus LA-5 and L. casei 431 probiotic cultures were obtained from CHR--Hansen (Horsholm, Denmark) and were activated by inoculation in the MRS (de Man-Rogasa-Sharpe) broth at 37 °C for 24 h.The probiotic biomass was harvested in late log phase by centrifugation at 600×g for 10 min at 4 °C (3-18K; Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany), then washed twice in sterile 0.9 % saline solution under the same centrifugation conditions, and used in the microencapsulation process (3).

Encapsulation procedure
All glassware and solutions used in the protocols were sterilized at 121 °C for 15 min.Alginate beads were produced using a modi ed encapsulation method originally reported by Sheu and Marshall (10) and Sultana et al. (12).A 3 % alginate (batch number 71238; Sigma-Aldrich, London, UK) mixture in 100 mL of distilled water containing 2 % Hi-maize resistant starch (Hi-maize ® 260; Ingredion, London, UK) and cell suspension (0.1 %, by mass per volume) was prepared.The mixture was heated slightly (50 °C) until complete dissolution before cell suspension (0.1 %, by mass per volume) was dispersed into the solution.The mixture was added into 500 mL of corn oil containing 0.2 % Tween 80 and was stirred vigorously (at 19×g for 20 min) until full emulsi cation.Then the emulsion was broken by adding 500 mL of 0.1 M calcium chloride while stirring.The mixture was allowed to stand for 30 min to separate the prepared calcium alginate beads in the calcium chloride layer at the bo om of beaker.The oil layer was drained and beads in the calcium chloride solution were collected by low speed centrifuge at 350×g for 10 min and then washed with 0.9 % saline solution containing 5 % glycerol and stored at 4 °C (3,9,10,14).Low-molecular-mass chitosan (0.4 g; Sigma-Aldrich) was dissolved in 90 mL of distilled water acidi ed with 0.4 mL of glacial acetic acid to achieve a nal concentration of 4 g/L.The pH was then adjusted to between 5.7 and 6.0 by adding 1 M NaOH.The mixture was ltered through Whatman lter paper no. 4 and the volume adjusted to 100 mL before autoclaving at 121 °C for 15 min.Then a mass of 15 g of washed beads was immersed in 100 mL of chitosan solution for coating with gentle shaking at 1×g for 40 min on an orbital shaker (two-step method).The chitosan-coated beads were washed and kept in 0.1 g per 100 g of peptone solution at 4 °C for not more than 1 h (15), and then used on the same day.

Preparation of bread with encapsulated bacteria
Hamburger buns contained the following ingredients: sugar 3, salt 1, fresh yeast 5 and fat 3 g per 100 g of wheat our (extraction rate 72 %).Ingredients in white pan bread were: sugar 2, salt 1, fresh yeast 4 and fat 2 g per 100 g of wheat our (extraction rate 72 %).A mass of 1 g of microencapsulated bacteria was added per 100 g of nal product.Even distribution of the bacteria in the dough was obtained by mixing.Inulin was added so that 100 g of wheat our contained 5 g of HPX inulin (Beneo, Mannheim, Germany) to obtain prebiotic e ect per slice of bread equivalent to 0.7-1.2g of inulin.Studies have shown that the addition of inulin to bread generally results in smaller loaves with a harder crumb, darker colour and decreased overall acceptability.However, a forti cation with 5 % inulin appears to be acceptable (4).A er initial proo ng, dough was divided into 60-and 450-gram pieces for hamburger buns and white pan bread loaves, respectively.Dough pieces were rounded and shaped, then transferred to proo ng cabinet in trays and pans for 45 min at 37 °C with relative humidity of 85 %.

Baking conditions
Hamburger buns were baked for 15 min at 180 °C and white pan bread loaves were baked for 25 min also at 180 °C.Rotary oven was used for heating and the temperature of crumb centre was measured by thermocouple.

Enumeration of encapsulated probiotics
Bacterial counts were determined before and immediately a er microencapsulation, less than 24 h a er baking and during 4 days of storage at room temperature.Enumeration of probiotic bacteria was achieved as described by Haynes and Playne (16).All enumerating plates were incubated at 37 °C for 72 h under aerobic conditions.The average values of all results were expressed as colony-forming units per gram of sample (CFU/g) (3).To count the encapsulated bacteria, the entrapped bacteria were released from the beads according to the method of Sheu and Marshall (10).A mass of 10 g of bread was resuspended in 100 mL of phosphate bu er (0.1 M, pH=7.0),followed by blending in a stomacher for 10 min.Since chitosan-coated beads did not dissolve in phosphate bu er, they were suspended in citrate bu er (0.1 M, pH=6.2),blended in a stomacher for 1 min and then allowed to stand for 10 min to dissolve.The counts (CFU/g) were determined by plating on MRS agar (Merck, Darmstadt, Germany) as discussed above (3,17).

Size and morphology of microcapsules
In this study, the size of microcapsules was determined by particle size analyser (Mastersizer 2000, Malvern Instruments Ltd., Malvern, UK) with the standard deviation calculated from the cumulative distribution curve.Scanning electron microscopy (SEM) (LEO 440i; Oxford Instruments, Oxford, UK) was used to observe the surface and morphology of microcapsules.

Sensory analysis
Triangle test was performed on the rst and fourth day of storage at room temperature.Evaluation was carried out by ten expert panellists recruited among the employees of Sahar bread factory (Tehran, Iran) and Bread Research Centre (Tehran, Iran).The samples were assessed in a standardised tasting room equipped with individual booths along a wall that divided the room from the preparation area (18,19).The reference samples did not contain encapsulated bacteria and inulin.All samples were served in dishes with three-digit codes: ve judges tested two treatment samples and one reference sample, and the other ve judges received one treatment sample and two reference samples.The judges were asked to indicate and identify the odd sample regarding avour and texture.They were also asked to indicate the degree of di erence between the duplicate samples and the odd sample and nally the sample they preferred.The degree of di erence indicated by the ten judges, who correctly identi ed the odd samples, was scored on a four-point scale labelled 1 for slight, 2 for moderate, 3 for much and 4 for extreme (18,(20)(21)(22)(23)(24).

Statistical analysis
A complete randomised factorial design was used for all analyses and all results were expressed as mean values of triplicate trials.Factors selected were bread type, bacterial strain, coating type and storage time.Data analysis was carried out using Statistical Package for Social Sciences (SPSS) so ware v. 20 (SPSS Inc., Chicago, IL, USA).Signi cant di erences between the treatments were detected using least signi cant di erences at p<0.05.

Shape and size of calcium alginate microcapsules
Scanning electro n microscopy (SEM) showed that the beads were generally globular in shape and also showed that the starch granules were present in the alginate matrix and the cavities (Fig. 1).Previous studies indicated that microencapsulation of water-in-oil emulsions helps to avoid abnormal bead shape and that the selection of appropriate coating material determines the physical and chemical properties of the resulting microcapsules (12,25).Hi-maize resistant starch, which is a prebiotic, also acts as a synergist with alginate in gelling and may help in pro-viding additional protection to the entrapped bacterial cells.Studies have shown good compatibility between alginate and starch.The combination of calcium alginate with resistant starch produces beads with a good integrated structure that swells and absorbs water but does not gelatinize fully during heating (3,12,13,26,27).Since alginate gels have a porous structure, a lling material such as starch and a chitosan membrane coating can improve stability, maintaining the spherical shape, decreasing the shrinkage of the microcapsules and reducing bead permeability (28).
Addition of chitosan layer formed a smooth surface (Fig. 2), with Hi-maize resistant starch granules still visible on the surface (Fig. 3).SEM did not show any signicant di erences in capsule shapes between the calcium alginate-encapsulated and starch-encapsulated probiotic strains produced with and without chitosan coating.
The size distribution of calcium alginate and starch microcapsules was analysed with particle size analyser.Microcapsule diameter of monolayer alginate and starch beads containing L. acidophilus LA-5 ranged from 31.0 to 382.9 m with mean diameter of 216.6 m, while of those containing L. casei 431 ranged from 44.0 to 717.7 m with mean diameter of 352.8 m.These results indicated that beads containing L. casei 431 were bigger in size than beads containing L. acidophilus LA-5, while from a morphologic point of view, no di erence was observed.Therefore, our results showed that capsule size depends on the probiotic strain, which is in agreement with Chavárri et al. (2).
The size distribution of calcium alginate and starch microcapsules coated with chitosan was also analysed.Microcapsule diameter of chitosan-coated beads containing L. acidophilus LA-5 ranged from 78.0 to 574.2 m with mean diameter of 347.4 m, while of those containing L. casei 431 ranged from 93.04 to 895.7 m with mean diameter of 512 .6 m.Therefore, the mean diameter of double layer chitosan-coated beads was signi cantly (p<0.05)higher than of monolayer alginate and starch beads, which is in agreement with Mokarram et al. (29).Emulsion technique used in this experiment produces micrometer-sized beads rather than millimeter-sized ones produced by many

Survival of encapsulated bacteria in the bread
The initial cell count before and a er encapsulation was approx.10 11 CFU/g.The results show that there was no signi cant loss of viability of both strains during encapsulation and coating due to the gentle methods used, and 99.8 % of cells were successfully entrapped.This result implied that the encapsulation and coating methods had no e ect on cell viability, which is in agreement with Krasaekoopt et al. (17) and Mokarram et al. (29).
The survival of encapsulated probiotics was determined less than 24 h a er baking (Fig. 4a) and on day four of storage at room temperature (Fig. 4b).Longer storage time was avoided due to staling.Using alginate and starch beads with and without chitosan coating, viable microorganisms survived a er the baking process and both bread types met the standard criteria for probiotic products.Type of bread signi cantly a ected the probiotic survival, which was signi cantly higher in hamburger buns (p<0.05),probably due to shorter baking time than of white pan bread.The temperature of crumb centre was approx.93-94 °C.Temperatures above 45 °C are known to be critical for the survival of probiotics in free form.Elevated temperatures higher than 45-55 °C for longer time lead to a decrease in probiotic survival.It has been shown that temperature higher than 65 °C is fatal for all free probiotic bacteria.Ding and Shah (11) showed that time plays an important role in high temperatures.They observed that exposing encapsulated probiotics to the temperature mentioned above for an hour results in complete bacterial death and concluded that alginate started to disintegrate during this time and as a result, the protective layer surrounding the bacteria was destroyed.Our results indicated that survival of encapsulated probiotics was lower in white pan bread, which could be due to It was note d that four days of storage had no e ect on the viability of encapsulated bacteria (p>0.05).The goal of encapsulation is to create a microenvironment in which the bacteria will survive during processing and storage and be released at appropriate sites (e.g.small intestine) in the digestive tract (33).Ravula and Shah (34,35) reported that microencapsulation improved the counts of L. acidophilus compared to free cells in frozen fermented dairy desserts stored for 12 weeks.In frozen iced milk, 40 % more lactobacilli survived when they were entrapped in calcium alginate beads (10).In addition, it was demonstrated by Homayouni et al. (3) that encapsulated cells required longer time to decrease the viability for one log cycle.Therefore, microencapsulation of probiotic bacteria in beads can increase the viability of probiotics during storage.
A signi cant increase (p<0.05) in probiotic survival was observed when the protective outer layer of chitosan was used in addition to the rst layer of calcium alginate and Hi-maize resistant starch.According to Anal and Singh (36), the formation of a hydrogel barrier by the compacted sodium alginate layer retards the permeation of the gastric uid into the cells.Chandramouli et al. (37) and Iyer and Kailasapathy (38) have shown that only the microencapsulated probiotics were able to maintain via-bility in gastrointestinal conditions.Microencapsulation of probiotics in alginate beads had previously been tested to improve the viability of probiotic bacteria in simulated gastric conditions (2,8,(10)(11)(12)(13)27,29).Studies have shown that the survival of bacteria under di erent conditions is increased in calcium alginate-immobilized cell cultures, con rming that they are be er protected than the nonencapsulated ones (33).
Probiotic bacteria encapsulated with Hi-maize resistant starch also survived be er than the encapsulated bacteria without the prebiotic (2), and further coating with chitosan signi cantly enhanced their survival (38).Capsule membrane in microcapsules with alginate and starch allows su cient di usion of nutrients and metabolites to maintain the growth of encapsulated cells and their fermentation ability (8).Resistant starch is the starch that is not digested by pancreatic amylases in the small intestine and reaches the colon, where it can be fermented by human and animal gut micro ora.The fermentation of carbohydrates by anaerobic bacteria produces short-chain fa y acids and lowers the pH in the lumen.Resistant starch can be used to ensure the viability of probiotic populations from the food in the large intestine.It also provides an ideal surface for adherence of the probiotics to the starch granule during processing, storage and transit through the upper gastrointestinal tract (33).Studies have shown that the incorporation of Hi-maize starch improved the encapsulation of viable bacteria compared with the bacteria encapsulated without starch (12,13,33,38).It seems that speci c interactions occur during mixing of alginate and starch.Therefore, the precise ratio of the used materials is essential (39).Previous investigations have demonstrated that intermolecular interactions and good molecular compatibility take place between starch and alginate (26,28).This can be explained by strong interactions like hydrogen bonds and ionic interactions (26).
The survival rate of probiotic bacteria entrapped in alginate beads containing chitosan was higher than that of alginate beads without chitosan (40,41).Chitosan is a positively charged polyamine that forms a semipermeable membrane around a negatively charged polymer such as alginate.This membrane is not soluble in the presence of Ca 2+ -chelating or antigelling agents, and thus increases the stability of the gel, providing a barrier to cell release.Studies have reported that the probiotic organisms with chitosan coating had be er protection than the uncoated microcapsules and that their encapsulation in chitosan microspheres improved the survival in comparison with free cells (2,17,42,43).
L. casei 431 was more resistant to high temperatures than L. acidophilus LA-5 (p<0.05).Lactic acid bacteria (LAB) are the most important probiotic microorganisms typically associated with the human gastrointestinal tract (36).Their probiotic bene ts strongly depend on their ability to survive and multiply in the host.Therefore, in order to have bene cial e ects in the intestine of the host, the bacteria should be metabolically stable and active during and a er processing, and survive the passage through the upper digestive tract in large numbers (44).Overall, viability is essential for organisms targeted to proliferate within the human gut (36).The results of our

Sensory evaluation
Data from triangle test with replicates were analysed using the corresponding table for repeated triangle tests (20,(22)(23)(24)46).The results of sensory evaluation are shown in Tables 1 and 2. Sensory scores indicated that there were not any signi cant di erences in avour among the samples of bread and buns containing inulin.These results are in agreement with those of Morris and Morris (4), who concluded that bread containing 5 % inulin seems acceptable.In another study, Brasil et al. (47) evaluated the e ect of the addition of inulin on sensory, nutritional and physical parameters of white bread and, according to their results, a level of 6 % of inulin added to bread was considered to give good sensory quality.Our results showed that microcapsules had no signi cant e ect on the avour and texture of bread.Previous studies had shown that alginate and starch capsules are white and this could have been the reason why they did not impart a signi cant di erence in crumb colour (27).Alginate and starch beads in our study were also micrometer-sized, hence did not have an adverse e ect on bread texture (29)(30)(31)(32).

Conclusions
This study indicated that the production of synbiotic bread by using microencapsulation is possible and it can enhance the viability and thermal resistance of probiotic bacteria, and therefore signi cantly improve their surviv-al in bread and other bakery products.Using alginate and starch beads with and without chitosan coating, viable microorganisms survived a er baking and both types of bread met the standard criteria for probiotic products.A signi cant increase (p<0.05) in probiotic survival was observed when the protective outer layer of chitosan was used in addition to the rst layer of calcium alginate and Hi-maize resistant starch.L. casei 431 was more resistant to high temperature than L. acidophilus LA-5 (p<0.05), and our study showed that the survival of bacteria in unfavourable conditions was species-dependent.Type of bread signi cantly a ected the probiotic survival, which was signi cantly higher in hamburger bun (p<0.05),probably due to shorter baking time, than in white pan bread.Results showed that microcapsules had no signi cant effect on avour and texture of bread and adding 5 % of inulin as prebiotic was acceptable, leading to production of bread with similar characteristics to the common bread, but with additional health bene ts.Therefore, this work contributes to this area and its ndings can be applied by bakery industry to develop probiotic bread and cerealbased products.Further studies are needed to evaluate the survival of other probiotic strains using di erent microencapsulation techniques and other coating materials in cereal-based products.More investigations should be considered using consumer-and product-oriented tests to cover the in uence of sensorial factors on consumers' attitude towards the product.

Acknowl edgements
This research received no speci c grant from any funding agency in the public, commercial, or not-for-profit sectors.The authors declare that they have no con ict of interest.

Fig. 4 .
Fig. 4. The survival of encapsulated probiotics determined: a) less than 24 h a er baking, and b) on day 4 of storage at ambient temperature

Table 2 .
Sensory evaluation scores of hamburger buns with encapsulated probiotics

Table 1 .
Sensory evaluation scores of white pan bread with encapsulated probiotics