The functional food market keeps holding the lead- ing positions  around the world  as consumers  tend  to choose products that taste better and provide addition- al health benefits. Most consumers would like to pre- vent some diseases and cure the ones they already have. Therefore, they buy products with bioactive supplements that are able to support their health physiologically. It has been scientifically proven that non-microbial and mi- crobial functional products have a therapeutic effect and

can be used in preventive medicine. However, these bio- logically active ingredients are prone to rapid degrada- tion during food processing, storage, and gastrointestinal transit. One of the best ways to prevent the degradation of these non-microbial and microbial bioactive compo- nents is to encapsulate them.

Recently, the popularity  of  functional  foodstuffs on the global food market has increased significantly. The turnover of the global functional food market will reach several hundred billion dollars in the nearest fu-

Copyright © 2019, Voblikova et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.

ture. In addition to the positive effect they exert on hu- man health, functional foods correspond to and satisfy all basic nutritional needs. Functional food products with probiotics and prebiotics have gained significant mar- ket share worldwide, especially in Europe, Asia (Japan), Australia, and, more recently, in the United States.

Despite all their fundamental differences, probiotic and prebiotic approaches to functional foods are equally beneficial for gastrointestinal tract (GIT). As a result, a symbiotic approach, i.e. a combination of probiotic and prebiotic approaches, is becoming more and more pop- ular. Therefore, a number of symbiotic products are cur- rently being developed for functional food markets.

Low survival rate of potential probiotics during stor- age and intestinal passage may limit the positive qual- ities of food products. Microencapsulation helps reduce the adverse effects on the viability of probiotics in GIT, as well as during food or nutraceutical processing, stor- age, and consumption. Microencapsulation separates and protects probiotic cells from the environment before their release.

There are various methods of gel microencapsulation that involve polymers: extrusion method, emulsification method, spray drying technology, etc. The main advan- tage of microencapsulation is in the controlled release of bacteria.

Microencapsulation is the process of enclosing sub- stances in microcapsules, i.e. a material or a mixture of materials covered, or encapsulated, by another material or system. The coated material is called active, or base, material. It can be solid, liquid, or gaseous. The coating material is called shell, wall material, carrier, or encap- sulating agent. Microparticles have a multicomponent structure with a diameter of 1–1,000 micrometers [1]. As a rule, microspheres are described as a matrix system where the active ingredient is dispersed/dissolved in a carrier matrix. Microcapsules have, at least, one discrete domain of the active agent, sometimes more (reservoir system) [2]. As a result, every microcapsule consists of a layer of an encapsulating agent that isolates and protects the active substance from any negative impact. Micro- capsules can have a regular (spherical, tubular, or oval) or irregular shape [3].

An analysis of scientific resources resulted in the fol- lowing list of substances used for microencapsulation of probiotics in food industry: sodium alginate, pectin, chitosan, carrageenan, gelatin, xanthan-gelatin mixture, and cellulose acetyl phthalate. All these substances help mitigate the process of immobilisation, thus, preserving the biological properties of substances and cell integri- ty. The most common encapsulating material is sodium alginate: it is simple, biocompatible, non-toxic, and cost effective. Alginate is a polysaccharide extracted from algae. It consists of β-d-mannuronic and α-l-guluronic acids. Different amounts and sequential distribution of β-d-mannuronic and α-l-guluronic acids in the chain can affect the functional properties of alginate as an auxilia- ry material [17].

If a polymer base is chosen as a shell, it results in the

formation of microcapsules of various sizes, as well as in

a good packing degree, molecular weight, structure, and shape, which guarantees targeted delivery of viable pro- biotics into the GIT as a part of food matrix.

When microencapsulating probiotics, one should take into account the chemical nature of coating materi- als. The use of microencapsulation techniques increases the viability of probiotics, both within food products and during their passage through the GIT. However, coating materials behave differently, and, therefore, their ability to protect living microorganisms and deliver biologically active substances also varies. In addition, the effective- ness of material depends not only on its encapsulating properties and strength, but also on its low cost, avail- ability, and biocompatibility [18].

Microcapsules are currently applied in food [4], tex- tile, pharmaceutical [5, 6], cosmetic, and agrochemical [7] industries. This method allows the producers to im- prove and/or modify the characteristics and properties of the active material, as well as its protection, stabilisa- tion and slow release.

Microencapsulation can modify the colour, shape, volume, pressure sensitivity, heat sensitivity, and photo- sensitivity of the encapsulated substance [8]. In addition, microencapsulation:

• protects the base material from ultraviolet rays, mois- ture, and oxygen;
• increases the shelf life of the volatile compound;
• reduces the rate of evaporation or transfer of active material from the core to the medium;
• prevents chemical reaction; reduces the problems of

fine powders’agglomeration;

• improves the processing properties of sticky materials;
• controls the release of substances; and
• reduces toxicity.

Thus, a research in the following spheres seems very promising: immobilizing methods of bifidobacterial cells and their use in the development of enforced dairy prod- ucts from goat or sheep milk. Microencapsulation of bi- fidobacteria is important since it allows one to preserve the useful properties of bifidobacteria in foodstuffs. In addition, it helps to protect the viable cells from gastric juice, bile, and other external conditions.

The research objective  was to provide  a scientific basis for choosing a natural polymer as a method of im- mobilisation of bifidobacteria; to evaluate their physical and chemical characteristics; to study the process of mi- croencapsulation of probiotics with prebiotics; to study the morphological features of microparticles, formed by natural biodegradable polymer (sodium alginate), using optical and electron microscopy.

STUDY OBJECTS AND METHODS

The research featured extrusion technique in alginate gel microencapsulation of bifidobacteria. Thus, there were two study subjects: microcapsules with bifido- bacteria and resistant starch (Hi-maize) as a part of the capsular structure and microcapsules without starch. De- hydrated and hydrated microcapsules were assessed for

average diameter, size, and general morphology charac- teristics.

To obtain a ≥ 109 liquid bifidobacteria concentrate, we used a serial passage. For the first passage, we prepared a culture medium. A sublimated Bifi obacterium bifi um 791 was inoculated into the culture medium and incu- bated at 37–38°C to obtain microbacterial mass with the bifidobacteria content of 108 CFU/ml. During the second passage, 10% of the cultured Bifi obacterium bifi um 791 was inoculated into the second culture medium at 37–38°C. During the third passage, 10% of the cultured Bifi obacterium bifi um 791 was inoculated into the third culture medium at 37–38°C and incubated for 6 hours. Next, it was centrifuged at 4°C for 20 min at 5,000 rpm. The resulting bacterial concentrate contained  at  least 1010 CFU/ml and was used to obtain microcapsules.

The microcapsules were obtained using extrusion technique. Two kinds of solution were prepared: the first solution contained 1% of sodium alginate and 1% of pre- biotic Hi-maize, the second – 1% of sodium alginate. After the polymers dispersed completely, Bifidobacte- rium bifi  um 791 was added to the solutions. The mul-

ticomponent composition was sprayed from 30 cm into

mended in Procedural Guidelines 4.2.999-00**.

To study the protective properties of microcap- sules, we used solutions with the following pH gradi- ents: pH 2.0 – gastric environment model  (exposure time = 30–120 min); pH 4.5 – duodenum environment model (15–60 min); pH 6.8 – jejunum environment (60–

120 min); pH 7.2 – ileum environment (60–120 min); and pH 5.8 – large intestine environment (18 hours). The temperature was 37 ± 1°C. The sample vials were in- termittently stirred with circular motions. At each time point, we tested the probiotic survivability using the ti- tration method of tenfold serial dilutions from 10–9 to 10–1 CFU/ml in two parallel rows of test tubes. The cell cultures were thermostated at 37 ± 1°C for 72 hours.

If no significant differences were registered in the number of CFU/ml of control and test samples, it was concluded that the concentration of the substance had no effect on the in vitro GIT model. If the CFU/ml in the test samples decreased significantly (up to one logarith- mic order) in comparison with the control sample, the ef- fect was declared inhibitory.

The general morphology of the microcapsules was

using an airbrush (model EW 110) with a

determined with the help of a scanning electron micro-

0.3 mm nozzle that was attached to an air compressor (model Jas – 1203). The resulting particles were stirred

scope  (SEM)  (MIRA3  TESCAN).  The  microcapsules

were placed on the substrate of the mechanical micro-

solution to ensure complete gela-

scope stage using a double-sided gold-flashed tape. The

tion. After that they were removed from the solution.

To define the specific activity of the encapsulated probiotics, we used Procedural Guidelines 1.2.2566-09*. The acidic gastric environment was modeled by add-  ing two components into the sterile physiological solu- tion. The first component was 0.5 mg/ml of acidin-pepsin manufactured  by  RUP  Belmedpreparaty  (Minsk,  Be- larus), registration number LS-001355. Each tablet con- tained 50 mg of proteolytic pepsin enzyme and 200 mg of acidin. The second component was a 0.1 mol solution of HCl (pH ≤ 2.0), which corresponds to the average gastric juice acidity [State Pharmacopoeia CCC ed. XI]. The small intestine environment was modeled by ad- ding 2.5 mg/ml of panzinorm forte 20,000 produced by OOO KRKA-RUS (Istra, Moscow region, Russia), regis- tration number P No. 014602/01. The pH level was adjust-

ed by sterile 0.1 mol solutions of NaOH and HCl.

As a rule, survival studies of probiotic microor- ganisms are  performed  on a  model  in vitro, simulat- ing the process of digestion in the human body. During the first stage, microorganisms are incubated at 37 ± 1°C first in an acidic model environment with acidin-pepsin (pH 2.0) and then in an alkaline model with panzinorm

accelerating direction of the microscope was 5 kV. The

diameters of the microcapsules were determined using the ImageJ software (NIH, USA). The average diameter was calculated by measuring 100 microcapsules.

Fifty white mice of the SHK line of both sexes were se- lected to assess the safety level of the microcapsules. The animals were provided by the Research Centre for Bio- medical Technologies of the Russian Federal Medical-Bi- ological  Agency  (certifi ate  number  05815,  11.05.2017

–  19.10.2017;  veterinary  certificate   250  №  0860445,

11.05.2017). The mice had a 21-day quarantine.

The dermonecrotic properties (irritant effect on mucous tissues) were studied on rabbits of Soviet Chinchilla breed. The animals were provided by the Federal State Unitary Enterprise, Experimental produc- tion farm (Manihino branch) (veterinary certificate 250

№0819660,  11.22.2016).  The  rabbits  were  quarantined

for 30 days.

Three series of experiments were conducted to esti- mate the levels of safety, acute toxicity, chronic toxicity, and dermonecrotic properties.

Sample  groups  were  formed  according  to  body

weight and age factor. The animals were kept in a vivari-

forte 20,000 (pH 6.8–5.8). The incubation time equals                                                                      

the period it takes mixed food to pass through the GIT. After that, the number of surviving microorganisms is assessed according to colony forming units of bifidobac- teria (CFU/ml) in tenfold limiting dilutions, as recom-

* Procedural Guidelines 1.2.2566-09. Security assessment of nanoma- terials based on in vitro and in vivo model systems. Moscow: Federal Center of Hygiene and Epidemiology Publ., 2010. 71 p.; General phar- macopoeia monograph 2.2.1.0005.15 Solubility. Moscow: Ministry of Health of the Russian Federation Publ., 2015. 4 p.

** Procedural Guidelines 4.2.999-00. Bifidobacteria count in fermented milk products. Moscow: Federal Center of Hygiene and Epidemiol- ogy Publ., 2001. 18 p.; Procedural Guidelines 2.2602-10 System of pre-registration preclinical study of drug safety. Selection, verification, and storage of master seed strains used in the probiotics production. Moscow: Federal Center of Hygiene and Epidemiology Publ., 2010. 61 p.; State Standard R 4.1.1672-03 Guidelines for quality and safety control of dietary supplements. Moscow: Federal Center of Hygiene and Epidemiology Publ., 2004. 240 p.; General Pharmacopeia Article 1.7.2.0009.15 Determination of the specific activity of probiotics. Mos- cow: Ministry of Health of the Russian Federation Publ., 2015. 24 p.

um according to Sanitary Rules 2.2.1.3218-14***.

The analysis of safety and acute toxicity of microcap- sules was conducted according to Procedural Guidelines 4.2.2602-10****. As a result, three experimental groups of five animals were formed. According to the key selec- tive factors, each laboratory rodent had to be clinically healthy, well-fed, active and mobile, with a good pel- age, normally coloured mucous membranes, and formed stool. The average animal weight was 20 ± 5 g.

A paste of microcapsules with concentration of bi- fidobacteria 1×108  CFU of microbial cells per 1 g was diluted with saline C080812 (expiration date September 2017). The paste was prepared in three doses. The ratio of the paste and the saline in the first dose was 1:5 while the amount of microbial  cells  was  2×107  CFU/ml.  In the second dose, the ratio was 1:10, and the amount of cells equalled 1×107 CFU/ml. As for the third dose, the ratio was 1:15, and the amount of microbial cells was 6.7×106 CFU/ml. The solutions were administered orally using a 1 ml feeding tube. Four hours before the manip- ulations, the animals had stopped taking food and water. Feeding was resumed two hours after the procedure. As a control measure, physiological saline C080812 (expi- ration date: September 2017) was orally administered to the control group. The control group of five mice was used simultaneously in parallel experiments in safety, acute toxicity, and chronic toxicity. All the animals be- longed to the same lot.

The dermonecrotic properties of the microcapsules were tested on rabbits of Soviet Chinchilla breed.

In vivo dermal irritation tests were performed on the anterior segment of the eyes of three rabbits. The an- imals weighed 1,500–2,500 g and were kept in a stan- dard vivarium at 22 ± 2°C with a 12-hour synchronised change of the light period (Sanitary Rules 2.2.1.3218- 14)*****. The animals were grouped according to body weight and age factor. During the whole research peri- od, the animals received briquetted feed produced by OOO Laboratorkorm.

Animal testing was performed according to State Standard ISO 10993-10-2011******.

The bifidobacteria microcapsules were  tested  for the chronic toxicity according to Procedural Guidelines 4.2.2602-10****. Three groups of six animals were formed. All animals received specialized briquetted feed produced by OOO Laboratorkorm and water.

Group no. 4 was experimental. These animals re- ceived an experimental preparation, i.e. microcapsules composed of natural biodegradable polymers containing bifidobacteria.

*** Sanitary  Rules  2.2.1.3218-14. Procedural  Guidelines  4.2.999-00. Bifidobacteria count in fermented milk products. Moscow: Federal Center of Hygiene and Epidemiology Publ., 2001. 18 p..

**** Procedural Guidelines 4.2.2602-10. System of pre-registration preclinical study of drug safety. Selection, verification, and storage of production strains used in probiotics production. Federal Center of Hy- giene and Epidemiology Publ., 2010. 60 p.

***** Sanitary Rules 2.2.1.3218-14. Sanitary and epidemiological re- quirements for the organization, equipment, and maintenance of exper- imental biological clinics (vivaria). Moscow: Official Publ., 2009. 7 p.

****** State Standard ISO 10993-10-2011. Medical devices. Biologi- cal evaluation of medical devices. Part 10. Tests for irritation and de- layed-type hypersensitivity. Moscow: Standartinform Publ., 2014. 42 p.

Group no. 5 was control group. Animals received an alternative preparation, i.e. Bifidobakterin produced by ZAO Ecopolis (Kirov, Russia), series 792 (release date: March 2017; expiration date: April 2019).

Physiological saline C 080812 (expiration date: Sep- tember 2017) was orally administered to the animals of the control group. To analyse the chronic toxicity, the preparations were administered orally once a day for 14 days to six animals in the experimental groups. The amount was equivalent to that proposed for humans. The dose was calculated according to the number of bifido- bacteria in Bifidobacterin as stated in the product label. The dose of bifidobacteria in the microcapsules was identical to that in Bifidobacterin. For a 14 g mouse the concentration was 2.14×105 CFU. The animal behaviour and state of health were registered during the 14 days of administration and 7 days after the trial. Death rate of animals, state of hair, activity, colour of mucous mem- branes, body weight, and bowel movements were daily recorded.

RESULTS AND DISCUSSION

There are a lot of modern methods of microen- capsulation, and this number continues to increase as companies keep patenting more and more innovative microencapsulation technologies [10, 11]. The methods make it possible to encapsulate active material. How- ever, the final choice of microencapsulation method de- pends on the type of encapsulated material, the release characteristics of the encapsulated compound, applica- tion, and regulatory requirements, which can affect the final characteristics and properties of the microparticles. The whole spectre of microencapsulation methods can be divided into three main categories: chemical process- es, which included interfacial and in situ polymerization methods; physicochemical processes, which involved coacervation (phase separation) and evaporation/emulsi- fied solvent extraction; and physicomechanical process- es, which involved air suspension method, spray drying, spraying, spray cooling, and fluidized bed coating.

Table 1 shows various methods of microencapsula- tion. The data presented in the table characterize the ef- fectiveness of each encapsulation process.

Although complex and simple coacervation are the most effective methods, they are more costly. Spray drying and extrusion are second best according to the efficiency rating. Spray cooling and molecular incorpo- ration are the least effective encapsulation techniques.

Table 2 demonstrates some of the most important and common methods of microencapsulation, the size

Table 1. Efficiency characteristics of encapsulation methods

Microencapsulation method              Maximum load, % Simple coacervation  < 60

Complex coacervation                      70–90

Molecular inclusion                          5–10

Spray drying                                     < 40

Spray cooling                                   10–20

Extrusion                                          16–40

Table 2. Size of particles obtained by various methods of microencapsulation; advantages and disadvantages of each method

Microencapsula- tion method

Particle size  Advantages                                                  Disadvantages                                                   Scientific

resources

Simple coacerva- tion

Complex coacer- vation

20–200          high encapsulating efficiency;

effective particle size control

5–200            dissolving capacity of the active com- pound for further processing;

product oxidation

expensive method; particle aggregation; complex scaling;

evaporation of volatile substances expensive method;

particle aggregation;

complex scaling;

evaporation of volatile substances

[12, 13]

Spray drying             1–50              cheap;

easy scaling technique

uniform particles;

low level of microcapsule loading, further processing is required

[12, 13]

Spray cooling           20–200          suitable for water soluble substances          high engineering costs                                  [18]

Film coating              > 100             low operating costs;

high thermal efficiency;

full temperature control

long process                                                  [14]

Emulsification              0.1–100         small drops;

limited particle size distribution;  suitable for biodegradable and non-bio- degradable polymer microparticles and a wide range of liquid and solid materials

low efficiency of encapsulation;

expensive method

[12, 13]

Interfacial polym- erization

0.5–1,000      easy scaling technique;

high encapsulating efficiency

difficult to control;

possibility of non-biodegradable and / or non-biocompatible monomers’ formation

[13, 15,

16]

Extrusion                    from 150 to 2,000 mi-

crometers

easy scaling technique                                  formation of rather large particles                 [15, 16]

of particles obtained by various methods of microencap- sulation, as well as the advantages and disadvantages of every method.

According to Table 2, there are several advantageous techniques for immobilisation of bifidobacteria. Howev- er, extrusion proves to be the most acceptable variant, given the limitations described.

Polysaccharides are the most widely used materials for various encapsulation techniques. They are followed by proteins and lipids.

The following types of carriers were selected to ob- tain bifidobacteria microcapsules, based on the cost pa- rameter, as well as on safety and technology indexes.

Alginate gels are quite suitable for encapsulation of eukaryotic and prokaryotic cells. Microencapsulation us- ing alginate gel was evaluated as a possible method for improving the viability of probiotics during the low pH exposure and storage.

Table 3. Physicochemical parameters of biopolymers

Table 3 presents some physicochemical parameters of natural non-toxic biodegradable biopolymers.

Thus, the analysis of the physicochemical properties presented in Table 3 allows one to conclude that all these samples of biological polymers can be used for the im- mobilisation of bifidobacteria.

As for the molecular aspect, the alginate creates a particularly strong molecular structure in the pres- ence of Ca2+. As a result, one can obtain cold-pre- pared, thermoreversible, and freeze-thaw resistant microcapsules.

Probiotics are living microorganisms that help con- sumers to improve their health. However, such kinds of microorganisms lose their viability and stability rather

9

6

Bio-

polymer sample

Opti- mum pH range

Gelation conditions

Score

Duration of disso- lution, max, s

Mois- ture content, max %

Ash content, max, %

3

0

0               1,800            3,600            7,200

Potas- sium alginate Sodium alginate

4.7–6.3   Exposure

to gelling ions

1. 5. –6.5   Exposure

to gelling ions

720           10.0         23.0–

25.0

720           10.0         18.0–

22.0

Incubation time in the model environment, s

Test sample (protected by a microcapsule) Control sample (unprotected by a microcapsule)

Fig. 1. Survival rate of bifidobacteria in the stomach model,

pH 1.2.

900

1,800

3,600

log 10 of a cell concentartion, CFU/g

log 10 of a cell concentartion, CFU/g

6                                                                                                         4

3                                                                                                         2

Incubation time in the model environment, s

Test sample (protected by a microcapsule) Control sample (unprotected by a microcapsule)

0

0                            3,600                          7,200

Incubation time in the model environment, s

Test sample (protected by a microcapsule) Control sample (unprotected by a microcapsule)

Fig. 2. Survival rate of bifidobacteria in the duodenum envi- ronment model, pH 4.5.

Fig. 4. Survival rate of bifidobacteria in the ileum environ- ment model, pH 7.2.

4

2

0

Incubation time in the model environment, s

Test sample (protected by a microcapsule) Control sample (unprotected by a microcapsule)

Fig. 3. Survival rate of bifidobacteria in the jejunum environ- ment model, pH 6.8.

easily due to various physical and physiological condi- tions and factors.

The selected immobilisation method has a great ef-

fect on the viability of associated probiotic bacteria. It proved to be an effective method of probiotic viability improvement. Figs. 1–4 show some results of probiotic survivability in vitro in a GIT model.

confirms the protective effect of the biodegradable natural polymer on bifidobacteria during their  passage  through the in vitro gastric model. However, because the total plate count fell down to 4.0 lg CFU/g in acidity gradients (in vi- tro GIT model), the titers of the initial microencapsulated biomass had to be increased up to > 109 CFU/g. Probiot- ics must have a 106–107 level of living microorganisms per 1 gram of product when administered orally to maintain vi- ability when passing through the GIT.

Alginate immobilisation of bifidobacteria protects them from aggressive external factors. To increase the stability of bifidobacteria, resistant starch (Hi-maize) was introduced into the composition of the biodegrad- able microcapsules. If prebiotics  are  introduced  into the walls of probiotic microcapsules, it provides an im- proved protection for active microorganisms.

1. 1. 1. (b)

Fig. 5. Optical microscopy of alginate microparticles + Hi-maize. (a) and alginate microparticles: 1 – sodium alginate inside the particle; 2 – the microorganism inside the particle (200 ×); 3 – the prebiotic Hi-maize (200 ×); (b) Alginate microparticles: 1 – sodium alginate inside the particle; 2 – the microorganism inside the particle (200 ×).

(a)                                                                (b)                                                                          (c)

Fig. 6. Morphology and microstructure of lyophilized microparticles (alginate + Hi-Maize), obtained using scanning electron microscopy. (a) Surface of the microparticles with microorganisms (1 – the microorganism inside the particle); (b) microparticle (alginate + Hi-Maize); (c) particle distribution.

Some researchers also reported of a higher bacteri- al survival rate in alginate microcapsules containing prebiotics in a GIT model (fructo-oligosaccharides, ga- lacto-oligosaccharides/inulin, fructo-oligosaccharides, monosaccharides, respectively) compared with alginate microcapsules without prebiotics [19, 20].

Some studies suggest that alginate-based microcap- sules may provide a limited protection for probiotics due to its specific properties. For example, microcapsules ob- tained by extrusion using alginate as the main carrier and biopolymer are not stable in an acidic medium. More- over, the microspheres obtained on the basis of alginate are characterized by a porous structure and provide an easy diffusion of acid into and out of the microspheres. These disadvantages can be effectively eliminated: algi- nate can be combined with other polymers or structurally modified using various additives [21].

Our method of obtaining microcapsules based on biodegradable non-toxic polymers of natural origin al- lows one to obtain microcapsules with a closed surface and specific sizes.

The optical microscopy of wet microparticles was performed using a microscope and a digital-still camera. The morphology of the lyophilized microparticles was evaluated using a scanning electron microscope. Micro- capsules were mounted on aluminum plugs using a dou- ble-sided adhesive tape and then sprayed with gold.

The shape  of  the  wet  microparticles  was  close  to spherical, and the core material was distributed through- out the matrix (Fig. 5). The optical micrographs show that alginate particles and microorganisms were found inside the microparticle. Thus, microencapsulation of Bi-

fi obacterium bifi um 791 was effective for both treat- ments.

Scanning electron microscopy (Fig. 6) revealed that the morphology of freeze-dried microparticles had  a high agglomeration of particles and a variety of particle size distribution for both treatments.

A sharp dehydration of lyophilized polysaccharide gels results in a porous matrix. In the process of lyo- philization, all microcapsules were exposed to low tem- peratures. This led to the formation of ice crystals and the sublimation of ice under reduced pressure, resulting in a porous, dry product. The microparticles containing resistant Hi-Maize starch were more agglomerated if compared to the alginate microparticles.

The use of Hi-maize resistant starch in the process of microencapsulation did not significantly affect the diam- eters of wet microparticles.

As a result, the lyophilized microparticles had an average diameter of 150 and 97 micrometers for the ma- trix of microparticles alginate + Hi-maize and the algi- nate matrix, respectively. The structural changes caused by the process of freeze-drying increase the pore size, which results in a quick and full rehydration. Thus, freeze-dried microparticles swell quickly after being im- mersed in water and get larger than those wet micropar- ticles that have not been lyophilized.

Nontoxicity and harmlessness of the new compo- nents, as well as the benefits to the human health, are the key factors in using additives in food dairy products.

For this purpose, acute toxicity, chronic toxicity, and dermonecrotic properties were tested in animals. The tests on the safety level and acute toxicity proved that

Table 4. Clinical score of animals (groups 1, 2, and 3) during the test on safety level and acute toxicity

Groups           Amount,

The number

Group mass be-

Reaction         Observation time,

Results            Group mass

microcapsules made of natural biodegradable polymers with bifidobacteria were harmless to white mice of the SHK line of both sexes.

The mice were observed for 7 days. The experiment lasted 14 days. All animals survived the tests.

All the animals looked healthy and active, had a good appetite and a nice white thick tight pelage. The abdominal zone was not enlarged. The urinary frequency and urine colour corresponded to the physiological norm. The colour of the mucous membranes and the bowel habits remained the same during the entire time of the experiment. The be- haviour of the test animals did not differ from that of the control group. Table 4 demonstrates the results of weighing. On day 14 after the administration of the preparations,  the animals were euthanized with chloroform, and further

morphological studies of the internal organs followed.

A macroscopic examination did not register any ef- fect of the preparations on the state of the internal or- gans of mice; no differences were found between the control and experimental groups.

The location of the internal organs was proper. Free fluid in the pleural and abdominal cavities was not de- tected. The lumen of the trachea and bronchi was unob- structed; the mucous membrane was moist and slimy. The spleen was elongated, not enlarged, with a dense texture and smooth surface. The liver was not enlarged, had a proper shape, with a dense homogeneous smooth and slimy texture without inclusions.

When administered orally, the dose of LD50 was not determined since the administered doses caused no clin- ical signs of poisoning (dose limitation was due to the

possibility of administering a concentrated preparation through a probe).

When determining the chronic toxicity of micro- capsules, the biodegradable microcapsules did not pro- duce any chronic toxicity effect on the white mice of the SHK-line of both sexes when administered orally.

When determining the dermonecrotic properties of microcapsules, the assessment of the local irritant ef- fect on the ocular mucosa was carried out on rabbits: 1–2 drops (0.1 ml ≤ 100 mg) of suspended microcap- sules were introduced into the conjunctival sac of the left eye in diluted form. The ratio of the paste and the sa- line was 1:5 while the amount of bacterial cells equalled 2×107 CFU/g. Five minutes after application, the eyes were rinsed with distilled water. The ocular mucosa was inspected 1 hour after the introduction of the preparation and on the next day. The right eye of the animal served as a control sample. Observation of animals continued for 14 days. 24 hours after the preparations were applied to the rabbits’ eyes, the following results were obtained: hyperemia – 0 points, swelling – 0 points, accumulation of serous secretions in the canthi – 0–1 points; cornea damages were not observed in any animal. The total score was 0–1. The results of irritation of the conjuncti- va were assessed according to a 5-point scale, as recom- mended by Mikhailov [22].