THE IMPACT OF THE COOKED SAUSAGE ENRICHED WITH LACTULOSE AND FOOD FIBERS ON THE MORPHOFUNCTIONAL CONDITION OF THE MUCOUS MEMBRANE OF THE LARGE INTESTINE AND MICROBIOTA (MICROBIOCENOSIS) IN RATS

The researches on the development of medical and medical-preventive food products for people with violation of normal intestinal microflora are presented in the article. It was found that,  the introduction into the formulation of cooked sausage food beet  fibers based on sugar beet, hydrated in a ratio 1:5, in amount 10 %  to weight of mince and lactulose, synthesized from lactose, in  amount 640 mg/kg mince retains the traditional organoleptic  properties of the product. There were carried out comparative  morphometric, histochemical and bacterioscopic studies of boiled  sausage effect without additives and sausage enriched with food  fibers and lactulose on the morphofunctional condition of the mucous membrance of the colon (MMC) of rats. Was shown a significant  height  increase of epithelial surface of epithelium, an increase of frequency mitoses in the epithelium crypts of intestinal glands (from 0.6 ± 0.08 % to 1.1 ± 0.04 %), there is a tendency of increasing  content of goblet ekzokrinnye (from 21.3 ± 5.5 % to 32.4 ± 18.7  %), while the mucosal were intensively produced allopathically  mucus, which indicates the stimulation of sausage, enriched with  lactulose on the functional status of the surface epithelium and intestinal glands of the mucous membrane of the colon. Based on the studies results of the effect of food beet fibers and lactulose,  contained in the ration of rats in large and small intestine were fixed  on order greater amount of bifido- and lactobacteries in comparison  with the animals control group. Same time, it was found that in the  large intestine the number of lactobacilli were much higher in  animals receiving experimental sausage.

increase of epithelial surface of epithelium, an increase of frequency mitoses in the epithelium crypts of intestinal glands (from 0.6 ± 0.08 % to 1.1 ± 0.04 %), there is a tendency of increasing content of goblet ekzokrinnye (from 21.3 ± 5.5 % to 32.4 ± 18.7 %), while the mucosal were intensively produced allopathically mucus, which indicates the stimulation of sausage, enriched with lactulose on the functional status of the surface epithelium and intestinal glands of the mucous membrane of the colon.Based on the studies results of the effect of food beet fibers and lactulose, contained in the ration of rats in large and small intestine were fixed on order greater amount of bifido-and lactobacteries in comparison with the animals control group.Same time, it was found that in the large intestine the number of lactobacilli were much higher in animals receiving experimental sausage.

Introduction
Studies in our country and abroad have shown that, food is a source of natural food components, which have not only nutritional value, but also regulate numerous functions and reactions of the body.
It is known that, many diseases in humans are associated with the violation of normal intestinal microflora, which necessitates research on the development of therapeutic and preventive food, restoring it.
The most perspective direction of this problem solution is the creation of functional products, which include food fibers (FF) and other elements, belonging to the category of functional nutrition [1,2].The lack of food fibers in products brings to the decreasing of the human body resistance to the environment, the deterioration in the metabolism of carbohydrates in the gastrointestinal tract, the development of cancer, and the decreasing of the cardiovascular work and digestive systems [3].
There are necessary substances for stabilizing of the normal intestinal microflora, with bifidogenic action, which provide the microflora by a source of carbon and energy [4].As a bifidogenic material, in our country, lactulose received the greatest distribution [5].Lactulose was the first, artificially produced compound, introduced into wide practice as a bifidogenic factor.
Last years in the publications appeared the information about certain relationships in the intestine between plant fibers and microflora [6].It was shown, in performed researches, that food fiber and lactulose do not alter the organoleptics of products, but at the same time give them positive properties characteristic of these ingredients [7].
However there is no information about the influence of meat products containing food fibers and lactulose on the morphofunctional state of the intestinal mucosa in the available publications.
Taking into account these circumstances, there were performed researches conducted on the use of beet fibers and lactulose in the formulation of boiled sausage for restoring the beneficial microflora of the colon of experimental animals.
The aim of this work were comparative morphometric, histochemical and bacterioscopic researches of the boiled sausage effect without additives and sausage enriched with food fibers and lactulose on the morphofunctional state of the mucous membrane of the large intestine of rats.

Materials and methods
The researches were performed with a domestic concentrate of clarified food beet fibers on the base of sugar beet and lactulose syrup «Laktusan» synthesized from lactose special for the food industry of JSC «Felicata» (Moscow).
As an object of the study was chosen boiled sausage «Diabetic».Initially experimentally was fixed the number of injected hydrated 1: 5 beet fibers in the amount of 10 % to the mass of mince and lactulose in the amount of 640 mg/kg of mince in the formulation of sausages with aim to preserve the traditional organoleptic properties of the product [8].
Morphological and bacterioscopic researches were performed on laboratory rats of the Wistar line weighing 180 ± 10.0 g.
The animals were divided into groups (each group had 10 rats) depending on the nature of feeding: group 1-General food (GF) -intact control, group 2 -GF and sausage without additives in the ratio 1:1-control (K) and group 3 -GF and sausage in the ratio 1:1, containing lactulose and food fibers.There were included in GF wheat, sunflower seeds, millet, barley flakes, corn flakes, oatmeal, barley, flattened peas, peanuts, carob fruits, popcorn.The daily requirement for feed for adult rats is 30-32 g.The calculation of the minimum consumption of sausage for therapeutic effect for rats was 13 g of sausage per 100 g of body weight per day [9.10].
The animals received the indicated feed on the background of the usual water regime every day, during 21 day.24 hours after the last feeding, rats were removed from the experiment by the decapitation, distal portions of the colon were extracted, the biomaterial was immediately fixed in 10 % neutral formalin solution and poured into the wax by conventional method [11].
The research was performed on dewaxed slices 5-7 mcm thick, made on a rotary microtome painted with: alcyan blue, Erlich hematoxylin and eosin -for histological, histochemical and morphometric research; acridine orange -for bacterioscopic detection of campylobacters; basic fuchsine by ZIEHL-NEELSEN-for the detection of cryptosporidia [12,13].
The microstructural study of drugs and their photography was performed on a light microscope «Jenaval» (Germany) connected with computer system of image analysis «VedeTesE» using the software «Morpho -4,0» [11].
To obtain more objective morphological study of the mucous membrane of the colon (MMC) conducted the morphometric assessment of epithelial layer and lamina propria of the mucous membrane of the colon by 18 parameters conventional techniques in the PNYL laboratory of medical Cytology Russian medical Academy of postgraduate education of the Ministry of Health of the Russian Federation with the direct participation of the head of laboratory, MD, Professor E.G. Shcherbakova and MD I.T. Shcherbakov [14,15].
The results were processed by standard methods of variation statistics with the definition of average arithmetic (M), the deviations from the average arithmetic mean (m), significance coefficient of differences (t) with the definition of a credible interval p. the Differences of the indicators were considered to be significant with t ≥ 2 (p ≥ 0,05).

Results
Before starting the experiment were conducted the external examination of the animals.The safety of all animals was complete (100 %) during the whole period of the experiment.During the performing of the research chronic toxicity all feed mixtures were prepared «ex tempore».During the experiment were analyzed the behavioral reactions of rats, their appearance, and also the body weight and its average daily gain.During the whole experiment no differences in behavioral reactions were observed in control and experimental animals.
Histological studies of the material from the rats of the 1st group, which had been receiving GF, showed that the state of the colon did not differ from the physiological norm.The lumen of the colon had a scalloped shape.The thickness of the mucous membrane of the colon is given in (Table 1).
In 2 nd group of animals, receiving General Food and sausage in the ratio 1: 1, the lumen of the colon remained scalloped, similar to that in intact control.The thickness of the mucous membrane of the colon (MMC) is also not significantly changed.
In the superimpositions of mucus on the surface of epithelium of the mucous membrane of the colon (MMC) by acridine orange colouring were detected campylobacteria in the amount of 20-40 microbial cells in the field of vision.
Surface edematous epithelial cells had a cylindrical shape, there were no signs of dystrophy.Normochromic nuclei of these cells are basally located in the cytoplasm, acid glycosaminoglycans (GAG) poorly stained with alcyan by blue.Superficial epithelial cells (PE) presents a goblet ekzokrins (mucosal) that were in a state of moderate secretory activity, and the sour GAG in their cytoplasm stained very intensively.interepithelial lymphocytes (PE lymphocytes) were also found in the surface epithelium, the amount of which was 15.0 ± 2.3 %, and a certain amount (0.5 ± 0.96 %) of eosinophilic granulocytes.
Intestinal glands of the control group of animals had a tubular shape and closely adjoined to each other in the thickness of the mucous membrane of the colon; their length averaged 275 mkm.The lumen of almost all intesti-nal glands was filled allenopithecus secret, containing sulfoacids, related to glycosaminoglycan.
The columnar epithelial cells of the crypts of intestinal glands (epithelial cells of crypts -EC) had no degenerative changes.In the cytoplasm of these cells normochromic nucleus had a typical basal arrangement, acid glycosaminoglycans (GAG) poorly stained with alcyan by blue.Goblet ekzokrinn EK constituted 21.3 ± 5.5 % among the epithelial cells crypts and moderately secreted mucus, intensely painted by blue altianalis.The content of interepithelial lymphocytes in the epithelial formation of the intestinal glands (lymphocytes EK) was 12.3 ± 4.6 %.The frequency of mitosis in the epithelium of the intestinal glands (mitosis in the EC) was 0.6 ± 0.08 %.
The basal membrane of the superficial epithelium and intestinal glands was thin, with indistinct borders; acid glycosaminoglycans (GAG) poorly painted with alcyan by blue in it.In Table 1 shows the density of the cellular infiltrate in the lamina propria (stroma) of the animals mucosa of the colon of the control group was represented mainly by macrophages, lymphocytes, plasmocytoma also fibrocytes, fibroblasts and eosinophilic granulocytes.The blood vessels of the microvasculature lamina propria the mucous membrane of the colon (MMC) did not have any features, the nucleus of the endothelial cells were rounded, normochromic.
In mucus overlays on the superficial epithelium of the colon mucosa during the painting acridine by orange were detected campylobacters in the amount of 20-40 microbial cells in the field of vision.Cryptostegia was not detected.
In animals of the 2nd group, which received General Food and sausage without additives, the lumen of the colon remained scalloped, similar to that in intact control.The thickness of the mucous membrane of the colon (MMC) is also not significantly changed.
The main parameters reflecting the state of the superficial epithelium of the colon mucosa remained close to the adaptive norm -the shape and size of the superficial edematous epithelial cells, the location and nature of the staining of their nuclei and cytoplasm.Goblet ekzokrinn surface epithelial cells were moderately secreted allopathically mucilage, composed from acid glycosaminoglycans (GAG).Close to norm was also the percentage of interepithelial lymphocytes and eosinophilic granulocytes in the superficial epithelium (Table 1).
Intestinal glands had the usual location, there were no changes revealed in the occupancy of the lumen allenopithecus secret in compare with control.The depth of crypts also did not change significantly, although there was some tendency of this indicator decrease.There were no significant deviations in the state of columnar epithelial indicators of cells (the height of the cells, the absence of degenerative changes, basal location normochromic nucleus, weak staining of acid glycosaminoglycans (GAG) in the cytoplasm) and the percentage of goblet ekzokrinn EK, which were moderately secreted the allopathicall mucus.The frequency of mitosis significantly increased in germ zones of the epithelium of intestinal glands.
The basal membrane of the superficial and glandular epithelium in the animals mucous membrane of the colon of this group was thin and had clear contours.
In the lamina propria of the mucous membrane of the colon (MMC) rats of the second group is influenced by feeding a mixture of General Food and sausage, showed a significant increase of density of the cellular infiltrate in the control on 22.5 % because of increase of lymphocytes for 42.5 % and fibroblasts on 48,8 %.At the same time, there were no significant changes in the number of plasmocytes, macrophages and fibroblasts in the infiltration composition and there was only a slight tendency to the number of eosinophilic granulocytes increasing.The vessels of the microcirculatory bed of the own plate of the mucous membrane of the colon were moderately full-blooded, but not expanded.Wall vessels, as well as the nucleus of the endothelial cells did not have any specifics.
In mucus overlays on the superficial epithelium the number of campylobacteria increased to 60-80 cells in the field of vision and appeared single cryptosporidia (up to 10 cells in the field of vision).
In the main (3rd) group of rats, in whose feed sausage enriched with lactulose was introduced, morphometric parameters of surface and cryptal epithelium of the mucous membrane of the colon (MMC) in 78 % of cases were close to control and in 90 % of cases did not differ significantly from those in the 2nd group (Table 1).
The lumen of the colon of this animals group had normal scalloped shape.Surface edema epithelial cells of cylindrical shape had no signs of dystrophy and even increased in height on 40.8 % compared to the control.They normochromic nucleus had typical basal location in the cytoplasm, poorly painted altianalis by blue.The goblet ekzokrinn cells of the surface epithelium actively produced allopathically mucus, which stained very intensively.The percentage of lymphocytes PE did not differ significantly from the control group and group of animals, treated with sausage without lactulose in feed.
Tubular intestinal glands of the mucous membrane of the colon (MMC) rats of the 3rd group were closely located in the lamina propria of the mucous membrane of the colon (MMC), their lumen was filled allopathically glycosaminoglycans (GAG) secret containing.The depth of the crypts (252 ± 35 µm) and height kripalini epithelial cells (16.3 ± 1.6 µm) were at the physiological level, the content of the goblet ekzokrinn in the epithelium crypts was 32.4 ± 18.7 % and the mucosals were intensively produced allopathically mucus.The frequency of mitosis in the epithelium crypts was 1.1 ± 0.04 %.Eosinophilic granulocytes were not detected neither in the cryptal no in the surface of the mucous membrane of the colon (MMC epithelium.The basal membrane of the superficial and glandular epithelium was thin and had clear contours.
More significant changes under the influence of forage inclusion of the sausages in the food, enriched with lactulose, developed in the cellular infiltrate lamina propria of the mucosa of the colon.
In compare with the animals of the 2nd group, receiving GF and sausage without lactulose, there was a significant decrease in the density of cellular infiltration on 38.1 % and the approximation of this indicator to the adaptive norm.Significantly changed the ratio of cell infiltration of the lamina propria of the mucous membrane of the colon (MMC).The decrease in its density compared to the previous group was due to a decrease in the number of macrophages, fibroblasts and triple reduction in the number of eosinophilic granulocytes (Table 1).The number of lymphocytes in the cellular infiltration was 4855 ± 261 cl/mm 2 , and the number of plasma cells increased near in 1.5 times compare with the 1st and 2nd groups.
After application of the food, enriched with lactulose, contamination of the mucous membrane of the colon (MMC) with campylobacteria was minimal (10-20 microbial cells in the field of vision), and cryptosporidia was not detected [16].

Discussion
Performed researches have shown that in rats of the Wistar line, the introduction of sausage (animal feed) into the General food helps to increase the antigenic load on the body, as evidenced by the increase in the density of cellular infiltration of the stroma of the colon mucosa due to the content of lymphocytes and fibroblasts in it, as well as the increase in colon contamination by conditionally pathogenic microorganisms (campylobacteria) and protozoa (cryptosporidia).
In rats of the experimental group, which received sausage, enriched with lactulose, intergroup differences in comparison with control (adaptive norm) were reliable on 27.8 % of the considered morphometric parameters of a condition of a mucous membrane of a colon.Were observed a significant increase in the height of epithelial cells of the surface epithelium, increase in the frequency of mitoses in the epithelium of the crypts of intestinal glands (0.6 ± 0.08 % to 1.1 ± 0.04 %) and a trend to increasing of the content of goblet associacion (from 21.3 ± 5.5 % to 32.4 ± 18.7 %), while the mucosals were intensively produced allopathically mucus.This indicates the stimulating effect of sausage, enriched with lactulose, on the functional state of the superficial epithelium and intestinal glands of the colon mucosa.
Composed of lymphoplasmacytic infiltration of the lamina propria of the mucous membrane of the colon under the influence of lactulose has been a redistribution of cells, characterized by reliable reduction of the number of antigenpresenting cells -macrophages -in the context of increasing the number of lymphocytes and plasma cells in comparison with control.
Sausage, enriched with lactulose, had little effect on the morphofunctional state of the superficial epithelium and intestinal glands -when it was used, there was only a tendency of mitotic activity increase in the epithelium of crypts and there were no eosinophilic granulocytes in the superficial epithelium of the colon mucosa.The greatest differences between these groups affected the composition of the cellular infiltrate of the stroma of the mucous membrane of the colon (MMC).
Lactulose in the mixed (General food and animal) feed induced a significant decrease in the cellular density of stromal infiltration of the colon mucosa due to a significant decrease in the content of macrophages, fibroblasts and eosinophils in compare with those in the application of sausage without lactulose.The number of macrophages was in 2.6-2.67 times lower than in the 2nd group and in the control.Also was marked the tendency of the number of eosinophilic granulocytes decrease -in cellular infiltration they were revealed almost three times less than in the 2nd group.It can serve as an indicator of reduction of antigenic load, ever-present when used as General Food, and the introduction of the physiological for rats of animal origin products (sausage).
Reduction of antigenic load added to the feed of sausage, enriched with lactulose, also contributes the less contamination of the mucous membrane of the colon by campylobacteria and Cryptosporidium.Lactulose improves their elimination from the intestine due to the stimulating effect on the growth and reproduction of bifido and lactobacteria -the most important representatives of normal intestinal microflora responsible for providing colonization resistance of the large intestine mucosa and having antagonistic activity against many conditionally pathogenic microorganisms.
The number of plasma cells increased in cell infiltrate of the stroma of the mucous membrane of the colon of the animals of the 3rd group in 1.5 times compared with animal group that received a sausage without lactulose, as with the control.Such plasmocytic reaction against the background of reduced antigenic load indicates stimulation of cell factors of local immunity of the colon mucosa.
Based on the performed researches of influence of food beet fibres and lactulose, contained in the food of the rats in the large and small intestines are fixed to the high level of bifido and lactobacteria in comparison with the control group of animals.Same time was found, that in the large intestine the number of lactobacilli was much higher in animals receiving experimental sausage.

Conclusions
Based on the morphometric, histochemical and bacterioscopic studies, the influence of the General food with the addition of boiled sausage without additives and the content of beet fiber and lactulose on the morphological and functional state of the mucous membrane of the large intestine of rats showed that the inclusion of sausage in the General Food induces the restructuring of the cell infiltration of the own plate of the mucous membrane of the colon, this indicates the increase in antigenic load on the body of rats and promotes intestinal contamination with campylobacters and cryptosporides.
The data obtained in the course of experimental studies indicate that in the group of animals receiving General Food, including boiled sausage with dietary fibers and lactulose in the large and small intestine, more bifidobacteria was recorded, than in the groups of experienced rats receiving General Food diet and food with boiled sausage without additives.Same time was found that in the large intestine the number of lactobacteria in animals receiving experimental sausage was much higher in other groups.
Based on obtained datas can conclude that the food beet fibers and lactulose exert a corrective influence on the microbiocenosis of the large intestine of rats, which gives reason to recommend them for using in meat products with preventive orientation.