Correction of Bifidobacterium spp . and Lactobacillus spp . Populations in Mice with Experimental Intestinal Dysbiosis after Therapy with Enterosorbent-Immobilized Probiotics Stored at – 80 and – 196 ° C

The research revealed the recovery of the Bifidobacterium spp. and Lactobacillus spp. cenobiont populations in mucin of mice with experimental intestinal dysbiosis after treatment with free and immobilized on enterosorbents probiotics, stored for a year at a temperature of –80, –196°C. Experimental intestinal dysbiosis was simulated by Ampicillin and Metronidazole oral administration. Mice with induced dysbiosis were treated with free cells of S. boulardii, B. bifidum, L. bulgaricus probiotics, the mixtures of free cells with enterosorbents as well as the complexes of probiotic cells immobilized on enterosorbents. The enterosorbents based on activated carbon Sorbex and SCMS-1 were used. Immobilized probiotics have been found to provide a more rapid and complete recovery of cenobiont populations. Low-temperature storage for a year (observation period) and temperature storage regimen did not affect the therapeutic properties of free and immobilized cells of probiotics. The mechanisms of action of immobilized probiotics are under discussion.

Various groups of human population suffer from dysbiosis, a clinical and laboratory syndrome characterized by a changed qualitative and/or quantitative composition of the normal intestinal flora, metabolic and immune disorders [14,17].To treat and prevent an intestinal dysbiosis and even intestinal infections one uses probiotic preparations: probiotics, prebiotics, synbiotics, metabiotics [4,6,8,31].Probiotics are live microorganisms positively affecting physiological, biochemical and immune responses of a host body after natural intake due to stabilizing and optimizing the functions of the gut microbiota [6,13,14,17].There are large amounts of probiotic products produced worldwide possessing the clinical efficacy proved in the randomized multicenter double-blind studies [1,13,14,31].However, the existing products do not properly meet some clinical requirements and in this connection there is a need in developing medications of new types, in particular, probiotics, immobilized in polysaccharide hydrogel carriers [7,16] and on enterosorbents [28].Modern pharmaceutical products being the probioticts immobilized on sorbents (Baktistatin; Kraft, Bifidumbacterin forte, Ecopolis, Probifor, Partner, Ecoflor, Vector-Bialgam, Russia) are produced by freeze-drying and stored in a dry form [5].The studies on creating the liquid forms of probiotic immobilized on sorbents and their storage are single [25].
We have obtained the experimental products of probiotics immobilized on enterosorbents SCMS-1 (Spherical Carbon-Mineral Sorbents) and Sorbex [30].It has been shown that immobilization on enterosorbents and storage at -80 and -196°C did not alter the initial biological properties of probiotics and, during the correction of the experimental dysbiosis in immune suppressed mice, the products provided faster recovery of microbiota and eradication of intestinal microflora translocated to internal organs if compared with treatment with free cells and the mixtures of free cells and enterosorbents [2,3].To the date there are no comprehensive studies of the effect of immobilized on enterosorbents probiotics stored at different temperatures rendered on the recovery of populations of cenobionts and colonic mucosa mucin of the animals with chemotherapeutic dysbiosis without immune suppression.

Materials and methods
The research objects were Saccharomyces cerevisiae, Saccharomyces boulardii, bacteria Bifidobacterium bifidum, LVA-3 strain (B.bifidum), Lactobacillus delbrueckii subspecies bulgaricus (L.bulgaricus).S. boulardii yeasts were isolated from Enterol commercial drug (Biocodex, France).B. bifidum bacteria were obtained from the All-Russian Collection of Industrial Microorganisms of Research Institute for Genetics (Russian Federation), L. bulgaricus bacteria were from the collection of Saint-Petersburg Branch of the Research Institute of the Bakery Industry (Russia).
Free and immobilized on enterosorbents microbial cells were frozen in 5% sucrose solution.Suspensions of free cells and the carrier-cells complexes were placed into Corning cryovials (Corning Incorporated, USA) with 1.8 ml handling volume.The part of the samples was cooled in a programmable freezer Cryoson (Germany) down to -40°C with the rate of 1 deg/min, followed by the immersion into liquid nitrogen.These cryopreservation conditions ensured a high preservation rate of the carrier-cells complexes [2].The second part of the samples was placed into low-temperature chamber Jouan VX380 (France) with temperature of (-80 ± 4)°C.The samples were stored for a year at -196 and (-80 ± 4)°C, and were thawed in a water bath at 30°C.
The experiments were carried out in accordance with the General Principles of Experiments in Animals approved by the 5 th National Congress in Bioethics (Kiev, 2013) and consistent with the statements of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 1986).
Experimental dysbiosis was simulated in white 7-8-month-old outbred laboratory mice of 18-20 g weight.Dysbiosis in animals was initiated by oral administration for 3 days of Ampicillin (Kyivmedpreparat, Ukraine) and Metronidazole (Unique Pharmaceutical Laboratories, India) [9] dissolved in a distilled water проблемы криобиологии и криомедицины problems of cryobiology and cryomedicine том/volume 25, №/issue 3, 2015 by 5 and 2 mg of pure substance, respectively.There have been performed three series of experiments in which the animals were administered with the enterosorbents and preparations of B. bifidum cells (1 st series), L. bulgaricus (2 nd series), S. boulardii (3 rd series).
The groups of animals are specified in Tables 1-4.
Each of control and experimental groups consisted of 10 animals.Groups were formed from the same batch of animals randomly.The microbiological status of all mice before experiments was defined as improvedconventional (category 2).The diet basis was a standard feed mixture.Maintaining, nutrition and care of the animals were in accordance to the special requirements and under the similar conditions (temperature, humidity, light, diet) [32].The animals were ether premedicated for anesthesia when perished.
In 48 hrs after the last administration of medications (observation day 1) the mice were also orally (with enteral feeding tube) administered within 12 days (once a day) the preparations of probiotics and enterosorbents (free cells, a mixture of free cells with enterosorbents, and immobilized probiotics).Daily doses of probiotics and enterosorbents were calculated by weight of the animals in accordance with the application instructions of the products.For free S. boulardii cells they were 1.6×10 4 , for B. bifidum the number was 2.5×10 6 and 2.5×10 5 CFU for L. bulgaricus.The daily dose of SCMS-1 was 0.02 g, and 0.01 for Sorbex.Taking into account the established previously survival of the complexes of immobilized cells after low temperature storage their therapeutic doses were twice increased.
To days 5, 12 of a dysbiosis therapy (observation days 5, 12), and in 5, 10 days after the last treatment with probiotics (observation days 17,22) the colon mucin samples were collected for microscopic and microbiological examinations [6,11].To do this, different parts of the colon were dissected in two adjacent fragments of 5 cm length.Chyme was removed by washing the intestine fragments with 4°C Ringer's solution (pH 7.0-7.4),then the fragments were twisted by plastic rods with mucosa outside, then placed into the vessels with chilled Ringer's solution (5 ml) and shaken for 10 min, afterwards the removed with a spatula mucin was placed into the solution.
For statistical analysis of the results the Student t-test and SPSS 17.0 (IBM, USA) software were used.The threshold of statistical error was set at 5%.

Results and discussion
To day 3 after the Ampicillin and Metronidazole treatment the animals got the symptoms of intestinal dysbiosis: polyfekalia, diarrhea and constipation interchange, altered stool consistency, appetite and weight loss by (3.5 ± 0.5) g.In 48 hrs after administration of medications the colon mucin had number of lg CFU/g of Bifidobacterium spp.and Lactobacillus spp.bacteria decreased from 7.4 down to 2.0 and from 8.1 down to 2.3, respectively (Tables 1 and 2).
During 17 days the content of Bifidobacterium spp.and Lactobacillus spp.bacteria in parietal mucin of the animals non-treated with probiotics and enterosorbents was not changed.To observation day 22 the content of these bacteria in mucin was significantly increased, but it was still lower than in the control.All the animals of these groups had the mentioned above clinical manifestations of gastrointestinal tract digestive functions disorders during the entire period of observation.
In the animals of groups 1-3, treated with native or stored at -80, -196°C free B. bifidum cells, the total number of Bifidobacterium spp.bacteria in mucin was significantly increased to day 12 of therapy as compared with the values after administration of chemical drugs (see Table 1).To day 5 after the treatment (observation day 17) the total number of Bifidobacterium spp.bacteria decreased if compared to day 12 of B. bifidum receiving.To day 10 after the end of therapy course (observation day 22) the total number of Bifidobacterium spp.bacteria increased again and exceeded the value corresponding to day 12 of the treatment.For all the terms of observation (during therapy and 10 days after its finishing) the total number of Bifidobacterium spp.bacteria in mucin was lower than before the dysbiosis induction.
In the animals of groups 4-9 the therapy was performed using the mixtures of enterosorbents SCMS-1, Sorbex with free native or stored at -80 and -196°C B. bifidum cells (see Table 1).In the animals of these groups, as well as in those of groups 1-3, a significant increase of the total number of Bifidobacterium spp.bacteria in mucin was observed to days 5-12 of treatment.The number of bifidobacteria in mucin within 10 days after the therapy stop did not change and was significantly lower than before the dysbiosis induction.
Для статистического анализа полученных результатов использовали t-тест Стьюдента и пакет программ «SPSS 17.0» («IBM», США).Порог статистической погрешности был установлен на уровне 5%.Table 1).To day 5 of therapy, the total number of Bifidobacterium spp.bacteria in mucin was significantly increased.To day 12 of therapy and during the next 10 days (observation days [17][18][19][20][21][22], the total number of Bifidobacterium spp.bacteria in mucin did not differ from the one prior to the dysbiosis induction.In all the groups of experimental animals we found no differences in recovery of the total amount of Bifidobacterium spp. in mucin after administration of free and immobilized B. bifidum cells stored or not stored at low temperatures.There were no differences in therapeutic effect of B.bifidum products stored at either -80 and -196°C as well.

Результаты и обсуждение
After treatment of intestinal dysbiosis with free and immobilized L. bulgaricus bacteria the results similar to the ones when using B.bifidum were obtained.The total amount of Lactobacillus spp.bacteria in mucin of the animals treated with free cells and the mixtures of free cells with enterosorbents significantly increased to day 12 of administration (see Table 2: groups 1-3 and 4-9).In 5 days after the treatment the index declined and 10 days later it again increased to the values close to the ones prior to the dysbiosis induction.In the groups of animals treated with immobilized L. bulgaricus cells, the total amount of Lactobacillus spp.bacteria in mucin started rising to observation day 5 (Table 2: groups 10-15), and to day 22 it restored to the control values.
In mucin of the animals treated with L. bulgaricus products stored and not stored at low temperatures the amount of Lactobacillus spp bacteria did not differ like in previous experiments.There were no differences in therapeutic effect of L.bulgaricus stored at -80 and -196°C as well.
In the third set of experiments we examined the persistence of S.boulardii yeast in the colon mucin and recovery of Bifidobacterium spp.and Lactobacillus spp.bacteria populations after introduction of free and immobilized S. boulardii cells.Saccharomyces spp.cells were absent in mucin of the animals before dysbiosis induction and 48 hrs later administering the chemical drugs.To days 5 and 12 of treatment (observation day 5 and 12) the equal amounts of Saccharomyces spp.cells were isolated from the mucin of the animals treated with S.boulardii cells and mixtures of free cells with enterosorbents, i. e. from 3.5 to 4.4 lg CFU/g (Table 3: Groups 1-9).In 5 days after the treatment (observation day 17) only single Saccharomyces spp.cells were identified in mucin of the animals of these groups, and 10 days later (observation day 22) the yeast cells were absent in mucin.During the whole period of treatment with the enterosorbent-immobilized S .boulardiicells and 5 days later the equal amounts of yeast cells were isolated from the mucin, which were significantly higher ные выше клинические проявления нарушений пищеварительных функций ЖКТ.
In addition to the testing the persistence of yeast cells in mucin the groups of the S. boulardii treated animals were assessed for the presence of Bifidobacterium spp.and Lactobacillus spp.bacteria.Before the dysbiosis induction the animals of these groups had the number of lg CFU/g for bifidobacteria of 7.6 ± 0.5, for lactobacilli this was 8.0 ± 0.4, and 48 hrs later the administration of chemodrugs the amounts were 2.1 ± 0.4 and 2.4 ± 0.5, respectively.In mucin of the animals of groups 1-3 (Table 4, 5) to day 12 day of therapy the number of lg CFU/g for bifidobacteria was 3.3, for lactobacilli this was 4.2.In 10 days after the therapy termination, when the yeast cells were already absent in mucin, the amount of bifidobacteria was increased up to 4.3 lg CFU/g and the one of lactobacilli enhanced up to 4.8 lg CFU/g.Similar results were observed in the groups 4-9, where the animals were treated with the mixture of S. boulardii cells and enterosorbents.In groups 10-15 of the animals treated with preparations of S. boulardii cells immobilized on enterosorbents, the number of lg CFU/g of bifidobacteria and lactobacilli were higher than in groups 1-9.In 10 days after treatment, the number of yeast cells in mucin of mice of groups 10-15 decreased and the number of bifidobacteria and lactobacilli increased if compared to the last day of therapy.
Storage of free and immobilized S. boulardii cells throughout the year at -80 and -196°C did not affect their ability of recovering the Bifidobacterium spp.and Lactobacillus spp.cenobiont populations.
The increase of the numbers of bifido-and lactobacteria in mucin after receiving the non-stored and stored at low temperatures free or immobilized probiotics correlated with a decrease in clinical manifestations of disorders in gastrointestinal tract digestive functions.In the groups of mice treated with immobilized probiotics no clinical manifestations were found to observation day 22.In 30% of the animals treated with a mixture of free cells and enterosorbents the polyfecalia was kept, thereat the animals treated with free probiotic cells had just some clinical manifestations less pronounced than in the animals non-treated with probiotics and enterosorbents after the dysbiosis induction.
Note: c -statistically significant changes with data of animals, which received free cells preparations (p < 0.05).
Параллельно с исследованиями по восстановлению популяций ценобионтов Bifidobacterium spp.и Lactobacillus spp.было проведено сравнительное микроскопическое изучение муцина толстой кишки животных всех групп.При микроскопическом анализе препаратов муцина мышей после индукции дисбиоза наблюдали характерную картину микро-simultaneously with studies on the recovery of Bifidobacterium spp.and Lactobacillus spp.populations.Microscopic analysis of the mucin preparations of mice after dysbiosis induction revealed a characteristic microbiota of wall layer of the colon, i. e. presence of single gram-positive and gram-negative bacterial cells of different structures and a small amount of mucus in the vision field (Figure 1).Mucin microscopic preparations did not differ in case of treatments with cells stored and non-stored at -80 and -196°C, so thereafter the data for the cells stored at -80°C are provided.
The colon mucin in mice of groups 1-15 to day 12 of treatment (observation day 22) had increased number of cells, similar in morphology to those of Bifidobacterium spp.and Lactobacillus spp.(Fig. 2-4).Some preparations contained the particles of enterosorbents.
The yeast cells, bifidobacteria and lactobacilli were observed in the animals treated with free cells of the S. boulardii probiotic (groups 1-3) and a mixtures of yeast cells with enterosorbents (groups 4-9) to day 12 after treatment and in 5 days after finishing the course.In 5 days after treatment the number of yeast cells decreased.In 10 days, the yeast cells in mucin were absent (Fig. 5).
Throughout the observation period, mucin of animals of the groups of 10-15 treated with immobilized S. boulardii cells contained yeast cells, enterosorbent granules without cells and the ones with immobilized yeast cells, as well as the bifidobacteria and lactobacilli.The number of yeast cells in mucin in 10 days after the therapy end was insignificant.
The research results suggest a more pronounced therapeutic effect of enterosorbent-immobilized probiotics if compared to free cells.Studying internal and external mechanisms of formation and preservation of intestinal natural bacterial homeostasis revealed that aggregation of microbial cells into conglomerates (microcolonies) of different size contributed to a successful transfer of eubiotics from an external environment to a recipient and subsequent formation of intestinal microbiocenosis [25].
The conglomerates of microbial cells are formed due to co-adhesion (immobilization of cells on carriers which do not originate from living tissues).In biological aspect the colonies originated from conglomerates, unlike the clones generated from single free cells, are characterized by heterogenic population and higher probability of preserving the viability as a part of the conglomerate when exposed to damaging physical and chemical factors [11,26].Developed experimental products of enterosorbent-immobilized probiotics are also conglomerates with an amount of cells on their surface capable of reproduction in the local site of biotope.After adhesion of the carrier-cells complexes Таблица 4. Содержание Bifidobacterium spp.в пристеночном муцине толстой кишки мышей с химиотерапевтическим дисбиозом кишечника после введения свободных и иммобилизованных S. boulardii и энтеросорбентов, lg КОЕ/г, x ± Sx Table 4. Content of Bifidobacterium spp. in parietal mucin colon of mice with chemotherapeutical intestinal dysbiosis after injection of free and immobilized S. boulardii cells and enterosorbents, lg CFU/g, x ± Sx Примечание: b -статистически значимые отличия по сравнению с данными на 12-е сутки терапии (р < 0,05).
Note: b -statistically significant changes if compared with data on 12 th day of treatment (p < 0.05).
S. cerevisiae yeasts are not referred to microbiota of homoiothermal animals and are not able to adhere to intestinal mucosa [12,13,27], but their clinical efficacy during treatment of several pathologies in adults, as well as acute gastroenterites and prevention of antibiotic-associated diarrhea in children is described  in several studies and meta-analyzes [10,12,21].Application of S. cerevisiae is recommended by the European Society of Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) and the European Society for Paediatric Infectious Diseases [27].
This study demonstrates that the animals with experimental dysbiosis treated with free S.boulardii cells, mixtures of free cells with enterosorbents and the ones immobilized on enterosorbents had an increased amount of bifidobacteria and lactobacilli in mucin (see Table 4).The concentration of bifidobacteria or lactobacilli to observation day 22 was close to the values prior to the dysbiosis formation.The carrier-cells complexes served also as as a depot of 'transient' probiotic, leaving the intestine in a certain time after introduction.The complexes adhered to the mucous membrane mucin by the carrier surface.Due to its specific properties [19] the microcolony of yeast cells immobilized on a carrier, suppressed opportunistic pathogenic and pathogenic microflora, provided the conditions for populating mucosa with bifidobacteria or lactobacilli and their subsequent reproduction.Under the influence of chyme advancing through the intestine lumen there was a desorption of yeast cells from the carriers and their following elimination.This was evidenced by a reduced concentration of yeast cells 10 days later the therapy end by 2.8-3.1 lg CFU/g in mucin of the animals of groups 10-15.
Storage of the products at temperatures of -80 and -196°C did not affect the colonization properties of bifidobacteria, lactobacilli and the ability of the carriersenterosorbents to adhere to intestinal mucosa.Unlike the preparations of free cells of probiotics and particles or granules of enterosorbents the carrier-cells complexes are a novel medical prodcut providing the benefits when administered into a digestive tract.These studies can be used when developing new classes of probiotic commercial products.

Conclusions
The preparations of probiotics immobilized on enterosorbents provide a rapid and complete recovery of Bifidobacterium spp.and Lactobacillus spp.cenobiont populations in animals with experimental intestinal dysbiosis compared to the preparations of free cells and the mixtures of free cells with enterosorbents.A significant therapeutic effects of enterosorbent-immobilized probiotics is due to the fact that they are conglomerates representing a carrier with a reproductive dose of cells on its surface.The carriercells complexes adhere to mucosal mucin and the microcolonies of probiotics formed in these loci provide the conditions which are necessary for the recovery of cenobiont populations.
Storage at -80, -196°C for one year did not affect the colonizing properties of B. bifidum, L. bulgaricus