HUT67883A - Process for manipulating the intestinal structure and enzymes in animals - Google Patents

Abstract

A method of decreasing the reduction in digestive enzymes and altering mucosal structure in the intestine of animals is described wherein a probiotic is applied to the animals to achieve such results where the animal may benefit from such a change. Further, a method for maintaining such a benefit after weaning is demonstrated comprising in administering the probiotic to the animal prior to weaning.

Description

Attempts to influence the intestinal flora of animals have increased over the years. Properly influencing the intestinal microflora of animals has been found to have a positive effect on the growth rate of the animal. One possible route of action on microflora is the use of antimicrobial agents, and while the use of such agents in animal feed has been well documented, the effects of changes in the microbial population in the small and large intestine have not been understood.

One of the antimicrobial compounds used contains dietary copper, which has been shown to be effective in improving the growth rate of pigs (Braude, R., 1967, Animal Production 3, 69-75). Antibiotics have also been used to eliminate the pathogenic flora of the digestive tract. However, the agricultural industry has attempted to avoid the use of antibiotics because their ultimate impact on humans is not sufficiently clear. There is widespread public opinion that antibiotic additives for livestock are undesirable, and efforts have thus turned to other devices that have a beneficial effect on the intestinal microflora.

As both consumers and manufacturers were concerned about the side effects of using antibiotics as therapeutic agents, more attention was paid to the use of probiotics in this role. Probiotics are generally defined as a live microbial nutritional supplement that has a beneficial effect on the host by improving the intestinal microbial balance [Fuller, R., J. Appl. Bacteriol. 66: 365-378 (1989)].

The big problem with probiotics is that there is no information on how these substances work. The beneficial effects on growth are influenced by the great variety of formulations, their interaction with other growth promoting substances in the diet and the poor determination of the organisms tested. Therefore, while there is evidence that these formulations are effective, the magnitude of the effect on the animal's growth and the mechanism by which they act are unclear. Understanding this process would provide a rational basis for determining the level and frequency of dose required to optimize the beneficial effect. With such knowledge, changes in the need for and response to probiotic treatment resulting from changes in the gut microbial balance due to growth and development of the gastrointestinal tract and physiological and environmental stress in the animal would be foreseeable.

Many animals experience the phenomenon that occurs after weaning and appears in weight loss and increased susceptibility to infections. This phenomenon has often been studied in pigs, since significant losses in livestock following weaning are not uncommon. This lag phase of piglet growth after weaning has become a phenomenon recognized in early-weaning production systems [Lecce, J.G., Armstrong, W.D., Crawford, P.C. and Duchame, G.A.; J. Anim. Sci. 48: 10071014 (1989)].

It was observed that the intestinal structure after weaning

changed. Cross-sectional examination of the gastrointestinal tract shows that all regions of the gastrointestinal tract are structurally similar and that there are muscle layers beneath the outer serum. The innermost layer of the tract has three components: the muscular mucosa, which consists of two layers of smooth muscle, a thick layer of connective tissue, and a mucous membrane. The mucous membrane, the luminal surface of the tract, consists of columnar epithelial cells, excluding the esophagus. Throughout the small intestine, there are depressions which extend into the connective tissue (lamina propria), which form the so-called Liberkhun lacunae. The surface of the small intestine is also increased by the folds of the mucosa and the submucosa (submucosa), and by the mucosal protrusions called bohol. The pleural epithelial cells are continuously located on the lacunar epithelial cells. Separation has been found to have a significant effect on this structure and on the function of the small intestine epithelium [Miller, B.G., James, P.S., Smith, N.W. and Bourne, F. J.; J. Agric. Sci. Camb. 107: 579-589 (1986)]. After weaning, the height of the fluff decreases and the complexity of the fluff structure increases. The decrease in the height of the bohol after weaning is accompanied by an increase in the depth of the lacuna, since the increase in lacuna cell production causes the migration of young enterocyte cells into the bohol, which can therefore be present on the surface for a shorter period. This limits the digestive and absorption capacity of epithelial tissue. A decrease in the digestive and absorption capacity of the intestinal tissue may cause the piglets to stop growing after weaning and weaning. In addition, poor bowel function is associated with a change in the composition of the gastrointestinal microflora, in particular an increase in enteropathogenic strains of Escherichia coli, which can cause acute intestinal infection after weaning.

Therefore, there is a clear need for a better understanding of the effects of probiotics in animals in order to better use and apply such probiotics. In addition, it is necessary to overcome the negative effects on the intestinal tract of animals, especially pigs and other slaughter animals, after weaning.

The present invention relates to the use of probiotics for the propagation of gastrointestinal digestive enzymes, to the improvement of the intestinal epithelial structure to improve digestive and adsorption capacity, and to reduce the negative effects that occur in the intestinal tract after weaning and reduce the growth of animals.

Thus, it is an object of the present invention to provide a solution which results in a beneficial change in the digestive enzymes of animals.

It is a further object of the present invention to alter the mucosal structure of the gastrointestinal tract of an animal, to the extent that it is beneficial to the animal.

Another object of the invention is to modify the intestinal tract and its chemical components in order to reduce the negative effect on the intestinal tract of animals.

It is a further object of the present invention to provide a method of improving the health of an animal after weaning.

Other objects of the invention will become apparent from the following description.

The present invention relates to a method of altering the digestive enzymes of an animal which has a beneficial effect on the animal by administering to the animal a probiotic strain of Lactobacillus and Streptococcus. The present invention relates to a method of altering the mucosal structure of the intestinal tract of an animal using such probiotic strains which has a beneficial effect on the animal. The present invention relates to a method for improving the health of an animal after weaning comprising administering to the animal such strains.

The following is a brief description of the figures.

Figure 1 is a graphical representation of alkaline phosphatase activity in control piglets treated with tylosin and Probios at 7, 17, 42 and 80 days.

Figure 2 shows the sucrase activity of pigs treated with tylosin and Probios for 7, 17, 42 and 80 days.

Figure 3 is a graph showing the lactase activity of pigs treated with tylosin and Probios at 7, 17, 42 and 80 days.

Figure 4 is a graph showing the tripeptidase activity of pigs treated with tylosin and Probios at 7, 17, 42 and 80 days.

Figure 5 is a graph showing the dipeptidase activity of pigs treated with tylosin and Probios at 7, 17, 42 and 80 days.

Figure 6 with 8, 20, 28 and 35 days of control and Probios ♦ ·

- The alkaline phosphatase activity of 1 treated piglet is plotted.

Figure 7 is a graph showing the lactase activity of pigs treated with Probios at 8, 20, 28 and 35 days.

Figure 8 is a graph showing the tripeptidase activity of pigs treated with Probios at 8, 20, 28 and 35 days.

Figure 9 is a graph showing the dipeptidase activity of pigs treated with Probios at 8, 20, 28 and 35 days.

The following is a detailed description of the invention.

The mode of action of probiotics in animals, especially how they affect the animal's intestinal tract, is not fully understood. As already indicated, a better understanding of this function could greatly assist in understanding how and with which strains to administer probiotics to an animal.

Unexpectedly, it has been found, and the invention is based on this recognition, that probiotics alter the digestive enzymes that are involved in the better digestion of nutrients. It has also been found that the mucosal structure of the intestinal epithelium is altered in the intestinal tract of animals by the appearance of mature cells with more nutrient-digestive capacity than younger cells. It has also been found that when animals are given such probiotics prior to weaning, these probiotics multiply the enzymes and mucosal structures associated with improved nutrient digestion and reduce the negative effects after weaning.

The digestive enzymes studied here include sucrose • · «

- 8 (sucrose α-D-glucohydrolase), lactase (α-D-galactoside galactohydrolase), dipeptidase (substrate L-leucylglycine) and tripeptidase (substrate L-leucylglycylglycine). These enzymes are active in the final stages of digestion before the nutrient is absorbed into the body. Of these, carbohydrates are located in the brush border of the enterocyte cell and dipeptidase is located in the cytosol. Tripeptidase activity binds primarily, though not exclusively, to the brush border region of the cell. Within the scope of these enzymes studied, it was found that the probiotic used had a significant effect on all enzymes except tripeptidase. The study described below shows that when piglets to which Probios is given at day 17 of birth are compared to control groups, the enzymes in the control group are found to be lower compared to the probios receiving group or to that control. to which antibiotics were administered. Furthermore, the decrease in enzyme activity from pre-weaning to post-weaning was significantly reduced by the use of Probios.

In addition, probiotic administration also had an effect on intestinal morphology. The ratio of bohol height and bohol height to lacuna depth from pre-weaning to post-weaning does not decrease significantly when probiotics are administered. Administration of the probiotic to piglets influenced pre-weaning changes in mucosal structure, typically by preventing the reduction of the bohol height and the extension of the lacuna to the lungs. sunny day.

While not wishing to be hypothesized, it is believed that this enhancement of enzyme activity is a consequence of changes in epithelial cell turnover resulting in the presence of a greater number and / or functionally more mature enterocytes on the boholy. Postnatal (postnatal) changes in the piglet's nest structure may be influenced by the composition of the endogenous microbial population. It is believed that the administration of the probiotic will affect the balance of the microorganism and the bacterial metabolism pattern in the gastrointestinal tract, resulting in changes in small intestine histology and enzyme activity, as observed in Example 1.

Pre-weaning probiotic administration also has a beneficial effect on the animal's growth rate. The growth rate of the animals shown in Example 1, compared to the control group, increased from day 26 onwards. In the antibiotic-treated control, growth growth only begins at day 36 when compared to the normal control.

The probiotics of the present invention may be diverse, with the requirement that they have a favorable effect on enzyme activity and gut structure. The first composition shown is a variant of 12 strains wherein Lactobacillus acidophilus (3 strains); There are L. casei (1 strain), L. plantarium (4 strains) and Streptococcus faecium (2 strains). The five strains are composed of L. acidophilus, L. casei, two L. plantarum strains and Streptococcus faecium. These strains in Germany are found in the Deutsche Sammlung von Mikroorganismen Und Zell * ··· ············································

Kulturen GmbH has been deposited with the American Type Culture Collection and will be deposited with the public after the grant of the patent.

The form of probiotic administration was not found to be critical. The livestock is typically fed in the form of granules mixed into the feed. The granules are commonly used in the event of stress, transportation or nutritional changes. A gel composition may also be used to administer the probiotic and may be used to replace the addition of the granules to the diet. The gel is typically administered at birth, at 7-10 days of age, and at weaning or other times when the piglet is under stress. Because deleterious enzyme reduction and gut changes following weaning can be influenced by the probiotic, its administration prior to weaning is most effective. Such gel formulations are known to contain other components and their administration is not critical as long as they do not interfere with their increased enzymatic activity or altered gut structure. Such additives include, for example, yeast, vegetable oils, silica, titanium dioxide, vitamins, dyes and preservatives.

The amount of lactic acid bacteria present in the composition should be sufficient to induce the desired enzymatic and intestinal changes. The effective amount employed is about 10 ⁷ live germs / g formulation.

The invention is further illustrated by the following non-limiting examples.

Example 1

Growth rate is measured from birth to 80 days of age and compared with growth of non-growth-promoting diet (negative control), pig industry antibiotic, growth-promoting diet (positive control) and probiotic-fed diet. Microbiological and gastrointestinal developmental studies are performed at 7, 17, 42 and 80 days of age as follows.

Probiotic preparations

The probiotic formulations administered during the assay are Probios ^R (Pioneer Hi-Bred International, Inc., Johnston, Iowa, USA) formulations containing 12 bacterial strains, including dried Lactobacilus plantarum, Streptococcus faecium, Lactobacillus caseit and Lactobacillus acidophilus, and products. The formulations used in the study are of two types:

Probios oral gel for pigs

Composition: dried fermentation products and selected strains of lactic acid bacteria (7 Lactobacillus sp. And 5 Streptococcus sp.), Together with: yeast culture, vegetable oil, sucrose, silica, titanium dioxide, vitamins A, D, E and B, F , D and C yellow No. 6 Lacquer ”, Polysorbate 80, TBHQ and ethoxyquin preservatives. Total lactic acid bacteria count is 10 ⁷ live cells / gram.

Recommended dose: newborn piglets -lg at birth or at first treatment. An additional 1 g was added at 7-10 days of age.

At weaning / starter piglets - 2 g at weaning, feed12 ···· · ♦♦ · · · · · · · · · · · · · · · · · · · · · · · · · ·

Probios granules for pigs

Composition: dried fermentation products and selected strains of lactic acid bacteria (7 Lactobacillus sp. And 5 Streptococcus sp.), Together with: yeast culture, calcium carbonate, corn cobs, vegetable oil, TBHQ and ethoxyquin preservatives. Total lactic acid bacteria = 10 ⁷ live cells / g.

Recommended dose: For weaning and starter pigs (up to 18 kg body weight) 1 kg granule / tonne creep feed in case of environmental change, transport, feed change. For growth piglets (18-45 kg), the granules are added to complete feed at a rate of 500 g / ton during environmental changes, transport or nutritional changes. Adult pigs (45 kg to market weight) are fed granules at 250 g / ton complete feed for environmental, transport and nutritional changes.

Sows are given 10 g / person / day granules for 5 days prior to parturition, and 500 g granules / ton are added to the complete feed during lactation.

Use of tylosin in a positive control diet

Tylosin is a macrolide antibiotic isolated from Streptomyces fradiae (Brander, G.L., Chemical Forensic Health Control Taylor and Francis Ltd. London (1986)) and is active against Gram-positive organisms. Tylosin is used extensively at the sub-therapeutic level to promote growth in pigs and poultry. In our studies, tylosin is used as a positive control in feeds such as Tylamix Premix at 100 g / kg (product license: No · · · · · ···

0006/4055. Elanco Products Ltd., Basingstoke, England) contains 100 g tylosin phosphate equivalent to 100 g tylosin base per kg feed.

The study includes three treatments: (1) a basic diet that does not contain a growth promoter and contains nutritional grade copper; (2) basic nutrition and antibiotic tylosin growth promoter (Elanco Products Ltd., Basingstoke, Hants), a macrolide antibiotic isolated from tylosin Streptomyces fradiae, active against Gram-positive organisms (Brander, supra, 40 mg per kg dose); (3) basic nutrition and Probios ^B probiotic formulation (Pioneer Hi-Bred International Ltd., Johnston, Iowa, USA) administered as an oral gel at 1g 24 hours after birth and again at 1g at 7 days of age, and 2 g at separation. The probiotic is used in the form of granules in both the starter feed and the subsequent continuous feed at a rate of 1 kg / tonne. Both forms of probiotic contain 10 ⁷ colony forming units / g of a mixture of the following strains: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei and Streptococcus faecium.

Experimental animals

The studies were performed on the offspring of large cross breeds (Large White / Landrace / Duroc) and, due to lack of space in the litter house, were performed in three phases with two treatments in each phase. In each phase, all four sows were calved within 48 hours and there was a 2-week interval between phases. Sows by date of calving ···· · · · ···

- 14 were mated and each litter was split into two litters, with the sex ratio being equal for each litter. Thus, each sow received a group of piglets, half of them own piglets, the other half being raised as nurses. The smallest animals in the litter were also involved in the cross-dressing. The animals were selected and graded three times (i.e., the study was performed in three phases). For the treatments in a given phase, the four groups were randomly assigned to each sow pair for each treatment. While each group was enclosed in a separate pen in the shed, after weaning, the animals were enclosed in each treatment group, with two groups in one treatment per treatment. In each case, precautions were taken to avoid cross-contamination between treatment groups.

Nutrition and nutrition

The creep feed and the follow-on feed were designed to contain 253 and 200 g of crude protein per kg (protein = nitrogen x 6.25), respectively, with a lysine content of 0.133 and 0, 11 g / kg. The nutritional formulations and their corresponding chemical analysis results are shown in Table 1. Both foods contain nutritional grade copper (20-70 mg / kg). Reproductive feeding is provided from the age of 7 days onwards and the piglets are weaned from the housing at 21 days of age and transferred to a flat-deck housing. Each piglet is weighed weekly.

Table 1

Ingredients (g / kg fresh weight) and nutrients for starter feed and continuous feed

starter Flap feed Wheat 320 250 Barley 250 470 Dried skimmed milk 210 10 soy flour 150 150 fish meal 55 100 Vitamin - mineral mixture 15 20 Dry matter (g / kg) 921.4 915.9 Ash (g / kg bw) 52.26 59.92 Oil (g / kg bw) 20.68 18.68 Crude protein (nitrogen x 6.25: g / kg bw) 245.00 233.00 Total energy (MJ / kg bw) 20.24 20.24 Copper (mg / kg bw) 20.90 68.30

Receiving tissue sample

At days 7, 17, 42, and 80, a pig is randomly selected from each group for tissue sampling.

At these times, an attempt is made to select animals in a balanced manner in terms of sex and natural / nurse mother. Accordingly, 4 piglets per treatment were sampled for each of the four ages. In order to obtain intact mucosal tissue, the animals are captured and then exposed to an open mask using Halothane (4%) - oxygen -

- Anesthetized with 16 nitric oxide (gas) mixtures. The animals were opened and then sacrificed by injection of sodium barbitone via the portal vein. The gastrointestinal tract is immediately removed and the small intestine is freed from the mesentery (mesenterium). Mucosal tissue samples were isolated from 50 mm long sections of the gut along the small intestine at positions 0.05, 0.1, 0.3, 0.6 and 0.8. The intestinal sections are opened lengthwise, rinsed with ice-cold saline (9 g sodium chloride / liter), watered dry and placed on chilled porcelain tiles. The mucous membrane is scraped from the muscle layer by microscope plate, wrapped in stanol foil, and frozen by rapid freezing in liquid nitrogen and stored at -70 ° C.

The enzyme assays are described below.

Each assay was performed with about 1 g of mucosa homogenate from frozen tissue, homogenized with 12 mL of a Teflon-glass homogenizer with 15 mL of 50 mM mannitol and 2 mM Tris hydrochloride, pH 7.1. The homogenate is made up to 25 ml with the same buffer and stored in portions at -70 ° C until assay.

The sucrose and lactase assays were analyzed by Dahlqvist and Arne, Analyt. Biochem. 7, 18-25 (1964)] for dipeptidase (substrate L-leucylglycine) and tripeptidase (substrate L-leucylglycylglycine) Kim. Y.S .; Analyt. Biochem. 63: 110-117 (1975), which are incorporated herein by reference. In these assays, the hydrolysis of the di- or tripeptidase to release amino acids is bound by the oxidation of leucine by L-amino acid oxidase. · ·· ·· ···· «·«

17 to give hydrogen peroxide. Oxidation of o-dianisidine with hydrogen peroxide following the action of peroxidase gives a specific endpoint for the reaction, which is observed at 530 nm absorption. When using the leucylglycine substrate and the leucylglycylglycine substrate, the two hydrolases can be distinguished. The final product of the tripeptidase assay, glycylglycine dipeptidase, is not reactive in the assay system when hydrolyzed by activity. Each assay is set up to obtain linear proportions of the homogenate volume and substrate concentration over time.

Examination by scanning electron microscope (scanning electron microscope, S.E.M.)

Changes in bowel morphology with age in sections S.E.M. below. Small sections (3 mm x 3 mm) of 4% neutral buffered formaldehyde tissue were removed and the samples were dehydrated in a series of ethanol solutions (25, 50, 70, 90, 95 and 100% by volume) for 30 minutes each. The tissue sections are then processed by the Department of Plant Biology (University of Newcastle upon Tyne). Each section was placed in 50% amyl acetate, 50% ethanol for 15 minutes, and then 100% amyl acetate. Finally, the tissue sections were dried to a critical point (to avoid distortion) and S.E.M. (Jeol Si S.E.M., Jeol (UK), Ltd., London) A splutter-coated carbon and gold spray was prepared.

Light microscopic examination

Quantitative examination of intestinal morphology

... ..., • • «« «« · · · · · · · «« «

- 18 tissue sections under light microscopy. Originally fixed with 3% glutaraldehyde, the tissues were placed in smaller sections (1 mm x 2 mm) under a Vickers M69 autopsy microscope and stored overnight in phosphate buffer, pH 7.4. The tissue sections are then processed by Medical School, Newcastle upon Tyne. Intestinal sections were examined at 50x magnification with a Vickers V80 stereomicroscope and accurately plotted using a projection camera attached to the microscope. A scale (0-50 μιη) is drawn (x50) from an object micrometer, copied onto an acetate plate and used to determine the exact size of the tissue section by superimposing the image from the imaging camera. Measured values include bohol height (the distance from the apex of the bohol to the collapse of the bohol / lacuna), and lacuna depth (the distance from the bohol / lacuna collapse to the muscle mucosa). An average of 6 measurements per section were made and the bohol height to lacuna depth ratio was calculated.

Statistical analysis of the data was performed by the GLM Statistical Package [Baker, R.J. and Nelder, J.A., GLM System Release 3 Numerical Algorithms Group, Oxford (1978)], which allows sequential matching of all factors that may affect the variable under investigation. The baseline analysis included all age groups studied using pooled intestinal data and was designed to examine whether there was any significant effect of age and treatment or inhibitory factors, phase, gender and maternal . The latter factor has been included so that they can be found in the · *.... <<<<<<<· · ·.

- 19 identify the effects that may be due to the fact that certain piglets were reared by their mother, not by their natural mother. In addition, the interaction of all these factors was fitted and the residual squares sum was used to calculate the error against which all null hypotheses were tested. As a result of this initial analysis, we determined that the factors of gender and maternity had no significant effect on any of the enzyme activities, and limited the analysis to phase, treatment and age, and treatment x age interactions. The phase was retained in the analysis because it was found to be significant in many cases, although this may have been related to the unbalanced nature of the experimental design (not all treatments included all phases), we attribute this effect to the former rather than to biological origin. The analysis includes a breakdown of the effect of age on the pre-weaning and post-weaning stages (7 + 17 days and 42 + 80 days), and the effect of treatment on control tylosin + probios (C v T + P) and tylosin Compared to Probios (T v. P). A summary of the analysis of the variance table is shown in Table 2.

Analyzed data for each of four piglets at each age were expanded to include five intestinal sites. In the analysis, factors such as phase, treatment, intestinal site and treatment x intestine were fitted, and the effect of treatment was then divided to evaluate the analysis of control versus tylosin + probios and tylosin versus probios. Intestinal sites were re-analyzed to determine proximal (0.05, 0.10, and 0.30) sites versus distal (0.60 and 0.80) sites. The analysis of the variance table is outlined in Table 3.

Table 2

Sources of Variation and Degrees of Freedom in Analysis of Aggregated Enzyme Activity Data by 7, 17, 42, and 80 Day Piglets

Sources of variation Degrees of freedom Sum 47 Phase 2 C v. T + P 1 T v. P 1 Pre or Post 1 Other age effects 2 (pre or post) x (C v. T + P) 1 (Pre or Post) x (T or P) 1 Other treatments x age effects 4 the rest 34

Treatment: C, Base Nutrition: T, Base Nutrition + Tylosin (40 mg / kg);

P, basic power + Probios; age: Pre, before weaning (7 days + days); post, weaning (42 days + 80 days).

• · «

Table 3

Sources of variation and degrees of freedom in Ί, 17, 42, and 80-day-old piglets when analyzing the effect of treatment and bowel site on enzyme activity

Sources of variation Degrees of freedom Sum 59 Phase 2 C v. T + P 1 T v. P 1 Prox v. Ornament. 1 Other space effects 3 (Prox v. Trim) x (C v. T + P) 1 (Prox v. Decor) x (T v. P) 1 Other treatment x location effects 6 the rest 43

Treatment: C, Basic Nutrition: T, Basic Nutrition + Tylosin (40 mg / kg);

P, basic power + Probios; location, Prox, proximal sites (0.05,

0.10, 0.30); ornament, distal sites (0.60, 0.80).

Below we summarize our results.

Activity of mucosal enzymes

In our Figures, the mean values ± SEM are expressed as International Units / g wet mucosal mass units of alkaline phosphatase activity (Figure 1), sucrase activity (Figure 2), lactase activity (Figure 3), tripeptidase activity (Figure 4). ) and dipeptidase activity (Figure 5) at the five intestinal sites of the pig at 7, 17, 42 and 80 days of age.

- 22 treatments.

Table 4 summarizes the effects of treatment and age on each of the studied mucosal parameters at each intestinal site and on each piglet treated at 7, 17, 42, and 80 days of age.

Table 4

Summarizing the effects of treatment, age, and treatment x age on small intestine enzymatic activity in piglets, summarized along intestinal sites

Enzyme active treatment Treatment Age Treatment x Age

saccharose NS Pre <Post * * * * [Pre V. Post]: OT + P * [EC 3.2.1.48) [Pre V. Post]: T <P * lactase NS Pre <* Position " [Pre V. Post]: C <T + P ** (EC 3.2.1.23) [Pre V. Post]: T> P * dipeptidase NS Pre <Pos zt * * * [Pre V. Post]: C <T + P ** (EC 3.14.13.11) tripeptidases NS NS NS

(EC 3.4.11.4)

Treatment: C, basic; T, base feed + tylosin (40 mg / kg); P, basic power + Probios; age: Pre, activity in weaning piglets (7 and 17 days); Post, activity after weaning (42 and 80 days old); NS - not significant.

* P <0.05; ** P <0.01; *** P <0.001.

The activity of alkaline phosphatase, lactase and dipeptidase was significantly higher in pre-weaned piglets than after weaning. In contrast, · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

- 23 saccharase activity increased from pre-weaning to post-weaning. No significant change in tripeptidase activity was observed before and after weaning. At certain ages, a significant effect was observed in piglets when treated with Probios and the antibiotic for all enzymes except dipeptidase.

7 days old:

At this age, alkaline phosphatase showed a significant interaction between treatment and site, the increase in activity from proximal to distal small intestine was significantly lower in control pigs than in treated pigs (p <0.05 at IE / gMWW and p <0.01). specific activity). Other enzymes do not show a treatment effect at this age. The activity of alkaline phosphatase and tri- and dipeptidase enzymes in the proximal tissues was significantly lower than in the distal intestine (p <0.001 for all enzymes except tripeptidase specific activity, for which p <0.05). Conversely, sucrase activity was significantly higher in proximal tissue (p <0.001). The site had no significant effect on lactase activity.

17 days old:

Activity of each enzyme tested was significantly lower in control pigs at all sites than in treated animals except for dipeptidase (p <0.001 for sucrase IE / gMWW and lactase activity; p <0.01 for alkaline phosphatase IE / gMWW and sucrase specific activity; p < 0.05 alkaline phosphatase for specific activity and tripeptidase activity). Activity of all enzymes studied, except tri- and dipeptidases, in the form of IE / gmWW · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· ·· ·· · ♦ · ····

- 24 significantly greater in tylosin-treated piglets than in Probios-treated pigs (p <0.001 for lactase activity; p <0.01 for alkaline phosphatase activity; p <0.05 for sucrase activity and dipeptidase specific activity).

Regardless of treatment effect, sucrase and lactase activities were significantly higher in proximal intestinal tissues than in distal intestine (p <0.001 IE / gMWW for both enzymes and p <0.001 for both enzymes). In contrast, all other enzymes tested had significantly lower activity in the proximal intestine compared to distal (p <0.001 for all activities except tripeptidase, IE / gMWW, for which p <0.05).

The magnitude of change in activity between the proximal and distal regions is significantly lower in control pigs than in treated animals for sucrase, tripeptidase, and dipeptidase (p <0.001 for tripeptidase specific activity; p <0.01 for sucrase IE / gMWW, tripeptidase IE / g) p <0.05 for sucrase specific activity and dipeptidase IE / gMWW). Sucarase activity increased between the proximal and distal small intestines in control animals, while decreased in the tylosin and Probios treated groups. In contrast, tripeptidase activity decreased between these two regions in control animals, whereas it increased in tylosin and Probios treated animals. The increase in tylosin treated animals was greater than in Probios treated animals (p <0.05). Dipeptidase activity increased in the control and treated piglets between the proximal and distal small intestines, the specific activity of this enzyme + · · · · · · · · · · · · · · · · · · 4 * · ν ··· ····

- 25 increase in tylosin-treated animals (p <0.01) compared to probios-treated animals. There was no significant interaction between site and treatment for alkaline phosphatase or lactase activities.

sunny age

At this age, the activity of tylosin-treated animals was significantly lower than that of Probios-treated animals (p <0.001 for sucrase ΙΕ / gMWW; p <0.01 for sucrose specific activity; p <0.05 for alkaline phosphatase ΙΕ / gMWW). At each of these sites, treatment had no significant effect on alkaline phosphatase specific activity, tri- or dipeptidase activity at this site. Alkaline phosphatase ΙΕ / gMWW and saccharase activity did not differ significantly between control and treated piglets. Lactase activities were significantly (p <0.05) higher in control pigs than in treated animals.

The site was found to have no effect on alkaline phosphatase activity or tripeptidase ΙΕ / gMWW. Sucarase activity and tripeptidase specific activity were significantly lower in proximal tissue than in distal bowel (p <0.001). Conversely, proximal activity was significantly higher than distal for lactase (p <0.001) and for dipeptidase ΙΕ / gMWW (p <0.05). Dipeptidase specific activity is not significantly different between the proximal and distal regions. The degree of increase in tripeptidase specific activity between the proximal and distal intestines does not differ significantly in control piglets compared to treated animals. However, this increase does not occur in animals treated with tylosin.

- 26 significantly (p <0.05) lower than in the Probios treated group.

sunny day:

Sucarase and dipeptidase activity were significantly lower at the proximal sites studied than at the distal sites for sucrase activity (p <0.001); IE / gMWW for dipeptidase (p <0.01) and dipeptidase for specific activity (p <0.05). Conversely, lactase activity was significantly higher in proximal tissue than in distal gut (p <0.001). No significant difference was found in the level of alkaline phosphatase or tripeptidase activity of proximal and distal small intestine.

Except for dipeptidase, the activity of all enzymes tested was found to be statistically lower in 17-day-old control pigs at all sites than in the treated groups, and higher in tylosin-treated animals than in Probios-treated animals (although not tripeptidase activity when wet mucosa). basis). The specific activity of dipeptidase in 17-day-old piglets is higher with tylosin than in Probios-treated animals, but the activity is similar in the control and Probios-treated groups. Although statistically, treatment had no effect on dipeptidase (IE / gMWW) activity, it was higher in tylosin-treated pigs than in Probios-treated pigs, and the lowest values were found in control piglets. Whether antibiotic or probiotic was added to the animal feed, this affected the pre- and post-weaning changes in enzyme activity.

• · *

- 27 Effects of treatment and age on the gut structure

Table 5 shows the mean intestinal height and lacuna depth for 7 treatments for each treatment.

Pigs at 17, 42 and 80 days of age and 0.60 at the sites of the bohol (± SE) and the bohol height to lacuna depth ratio.

Table 5

Disease Treatment Boholy height (/ Un) Lacuna depth (/ Un) Pile / lacuna average gold NEITHER average NEITHER átlaq NEITHER 0.10 small intestine space C 793 (4) 33 296 (4) 55 2.95 (4) 0.43 7 days T 676 (4) 82 263 (4) 40 2.66 (4) 0.32 P 664 (3) 114 304 (3) 108 3.10 (3) 1.37 C 582 (3) 43 510 (3) 116 1.31 (3) 0.30 17 days T 666 (3) 91 302 (3) 32 2.18 (3) 0.11 P 709 (4) 99 217 (4) 15 3.25 (4) 0.54 c 397 (4) 38 400 (4) 30 1.10 (4) 0.11 42 days T 331 (3) 15 310 (3) 47 1.13 (3) 0.32 P 396 (3) 35 307 (3) 71 1.55 (3) 0.53 c 413 (1) - 363 (1) - 1.14 (1) - 80 days T 422 (3) 23 365 (3) 15 1.17 (3) 0.14 P 455 (3) 21 395 (3) 26 1.15 (3) 0.03

Table 5 (continued)

Disease Treatment Boholy height (Gm) Lacuna depth (/ Un) Boholy / lacuna ratio average NEITHER average NEITHER average NEITHER 0.60 small bowel space C 844 (3) 87 306 (3) 59 3.07 (3) 0.74 7 days T 794 2) 84 345 (2) 42 2.31 (2) 0.05 P 914 (4) 129 312 (4) 68 3.31 (4) 0.72 C 535 (4) 82 265 (4) 36 2.13 (4) 0.39 17 days T 746 (4) 108 307 (4) 46 2.52 (4) 0.30 P 583 (4) 85 251 (4) 29 2.85 (4) 0.70 C 504 (3) 58 325 (3) 11 1.57 (3) 0.26 42 days T 385 (3) 72 497 (3) 119 0.82 (3) 0.15 P 275 (1) - 404 (1) - 0.68 (1) - C 343 (2) 18 410 (2) 10 0.75 (2) 0.13 80 days T 406 (3) 30 366 (3) 24 1.27 (3) 0.18 P 531 (2) 4 315 (2) 20 1.72 (2) 0.24

Treatments: C - control; T - tylosin; P - Probios.

Initial analysis of bohol height: lacuna depth showed a significant interaction between the treatment and the mother (natural or nurse), which was therefore included as a blocking factor in further analyzes. The treatment had a significant effect on bohol height and lacuna depth at different early ages of the animal.

The administration of tylosin or Probios to piglets during the study prevented a decrease in the height of the cochlea and an increase in lacuna depth at the 0.10 gut site in animals aged 7 to 17 days compared to control animals. At 0.60 sites, only antibiotic-treated piglets showed a clear decrease in cohol height between 7 and 17 days of age. All animals had a decrease in cohol height between the ages of 17 and 42 at 0.10. At site 0.60, there was no significant change in bohol height or lacuna depth in control piglets between 17 and 42 days of age, however, a decrease or increase in these parameters was evident in both tylosin and Probios treated animals.

The administration of probiotics and antibiotics to piglets has altered the pattern of epithelial changes prior to weaning, particularly in the proximal intestine. These changes reflect the theory that the turnover rate of epithelial cells in these treated animals is slower, resulting in an increased population of functionally mature enterocytes. Among the potential benefits attributable to such changes in bowel function are improved digestion as a result of treatment and increased growth rate after weaning. Probiotics also have the advantage of eliminating consumer concerns about antibiotic treatment.

Example 2

In this example, it is facing and separating

Further examples of the effect of feeding probiotic animals to 30 animals are provided.

The methods used are essentially the same as those described in Example 1, except that the probiotic preparation has only four Lactobacillus sp. and a Streptococcus sp. contains strains.

The starter and parent (continuous) feeds are slightly different from those described in Example 1. No antibiotic-treated group is used, only control treatment or Probios treatment is given. The nutrient components are shown in Table 6.

Table 6

Components of starter and parent nutrition

Starter feed Growth feed (% fresh weight) (% fresh weight)

Complan 25.0 Barley 48.0 Wheat 24.0 Wheat 25.0 Barley 24.0 soy flour 15.0 soy flour 15.0 fish meal 10.0 fish meal 10.5 Vitamin / mineral Vitamin / mineral material mixture 2.0 material mixture 1.5 Calculated nutrient quantity fresh by weight: Protein 23 40 20.40 Daily energy (MJ / kg) 15 , 20 13.10 Avis lysine (%) 1 39 1.07 Cu (mg / kg) 30 40 36.50 Oil (%) 7 81 1.91

Fiber (%) 2.73 3.84 Salt (%) 0.57 0.45 Methionine and Cysteine (%) 0.86 0.69 Threonine (%) 0.72 0.54 Ca (%) 1.19 1.13 P (%) 0.77 0.71

There were four phases in the study. All piglets were exposed to the normal unit system, weaned at 21 days of age, except animals born in the first and second stages, which were weaned on day 28. Samples were taken at the age of 8 days, when the pig had become accustomed to the sow and had not received starter feed; At the age of 20 days, when the pig has already received a starter feed but has not been weaned; At the age of 28 days, when the pig has been weaned for a week but not fully accustomed to rearing; At the age of 35, when the pig has recovered from the weaning stress and has become accustomed to rearing.

The results are presented below.

The five probiotic strains resulted in greater recovery of the two peptidase enzymes tested. The activity of all other enzymes was reduced from day 20 to day 28 in the weaned animals at day 21, and by day 35 activity was restored to a higher level in both dipeptidase enzymes in the treated animals. In weaned animals at 28 days of age, the decrease in enzyme activity after weaning was not as high as in weaned animals at 21 days of age. Significant treatment for treatment as shown in Example 1.

The effect of 32 was not repeated here, but the above reversal effect occurred, and a significant increase in dipeptidase activity was observed in the Example 2 assay on day 8. In control animals, weight loss after weaning was not seen in Probios treated animals.

From the table it can be seen that from 8 days to 20 days, the animals treated with Probios show a smaller decrease in the height of the cohol than the control piglets. Between 20 and 28 days of age, the decrease in cohol height and consequently the cohol height to lacuna depth ratio is greater in Probios treated pigs than in control animals.

At site 0.6, the effect of treatment was evident at 28 and 35 days. This observation was performed by determining the lacuna depth per pig in each treatment. Probiotic treatment does not have a significant effect on the changes in the structural parameters studied between the pre-weaning and post-weaning periods for the five aforementioned strains; Between the ages of 20 and 20 days, it appears to increase compared to control piglets, although lacuna elongation is not unequivocally affected by treatment between the two ages. Conversely, between 20 and 28 days of age, the control piglet height loss in control pigs is not evident in Probios treated animals.

- 33 Table 7

Treatment Disease (Sun) Boholy height (Gm) Lacuna depth (Gm) Boholy / lacuna average gold NEITHER average NEITHER average NEITHER 0.10 small intestine space control 8 636 (5) 9 117 (5) 10 5.88 (5) 1.38 20 384 (8) 22 186 (8) 13 2.18 (8) 0.26 28 294 (6) 11 194 (6) 13 1.55 (6) 0.09 * 28 386 (2) 37 198 (2) 6 1.94 (2) 0.13 35 302 (4) 35 213 (4) 23 1.48 (4) 0.22 * 35 330 (2) 44 314 (2) 30 1.04 (2) 0.04 Trial os 8 555 (9) 80 129 (9) 12 4.52 (9) 0.71 20 446 (8) 46 199 (8) 19 2.28 (8) 0.13 28 242 (5) 25 223 (5) 28 1.15 (5) 0.14 * 28 319 (1) - 171 (1) - 1.87 (1) - 35 337 (3) 22 194 (3) 24 1.78 (3) 0.11 * 35 350 (2) 27 263 (2) 1 1.23 (2) 0.03

0.60 Small bowel space

control

8 753 (8) 98 99 (8) 5 7.75 (8) 1.05 20 519 (7) 3 160 (7) 3 3.75 (7) 0.38 28 288 (4) 19 166 (4) 22 1.81 (4) 0.19 - 28 447 (2) 3 248 (2) 16 1.82 (2) 0.10 35 153 (1) - 162 (1) - 0.94 (1) - * 35 342 (3) 8 271 (3) 26 1.29 (3) 0.09

···· ··· «• ·

Table 7 (continued)

Treatment Disease (Sun) Boholy height (/ Un) Lacuna depth (/ Un) Boholy / lacuna ratio average NEITHER átlaq NEITHER average NEITHER Probios 8 781 (6) 91 108 (6) 9 7.73 (6) 1.21 20 381 (8) 3 179 (8) 12 2.18 (8) 0.17 28 369 (4) 30 245 (4) 36 1.60 (4) 0.19 28 411 (3) 53 223 (3) 17 1.94 (3) 0.42 35 288 (1) - 223 (1) - 1.29 (1) - * 35 329 (3) 36 266 (3) 15 1.22 (3) 0.07

weaned piglets

The decrease in calf height at the 0.10 gut site between day 20 and day 28 is not evident in animals that were weaned at 28 days. In piglets weaned at 21 days of age, the high heel height loss of 0.06 at 20 to 28 days of age does not occur in piglets weaned at 28 days.

In the first example study, the improvement in weight gain was not evident in the Probios and Day 36 groups treated with Probios and Day 36, and it is possible that animals in the latter study had a greater correlation with the treatment regimen if the measurements to be continued. It is noteworthy that, in this study, the control group exhibited weaning-related weight loss, which was not evident in Probios treated animals, and the probiotic treated group at day 34 ···· ····

- He had 35 weights. In both animal experiments conducted in this study, we observed that treatment had an effect on recovering from a reduction in growth rate and enzyme activity during weaning.

Accordingly, our studies show that Probios is correlated with its effect on facilitating adverse changes in gut structure and enzyme reduction following separation.

The use of Probios involves increased activity of digestive enzymes, including alkaline phosphatase, sucrase, lactase and tripeptidase. The effect varies across the bowel sites.

The decrease in bohol height to lacuna depth ratio also shows the effect of probios administration. Typically, post-weaning changes in enzyme activity development and in bohol height and concomitant lacuna depth are reduced by the use of probiotics.

Thus, it can be seen that at least all the objects of the invention are accomplished.

Claims (30)

CLAIMS 1. A method of increasing the growth of domestic animals and / or achieving higher meat yields comprising administering to the animals a novel probiotic composition for increasing the activity of their digestive enzymes. The method of claim 1, wherein the probiotic composition comprises Lactobacillus and Streptococcus. The method of claim 2, wherein the composition comprises strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantorium and Streptococcus faecium. 4. The method of claim 3, wherein the probiotic composition comprises Lactobacillus strain,,,,, ATCC and,,, ATCC. Contains Streptococcus strain. 5. The method of claim 3, wherein the probiotic composition comprises Lactobacillus strain ATCC and Streptococcus strain ATCC. 6. The method of claim 1, wherein the activity of sucrase, lactase, alkaline phosphatase, dipeptidase and tripeptidase, or combinations thereof, is increased. 7. A method of increasing the growth of a pet and / or achieving a higher yield of meat, comprising administering to the animal a probiotic composition to provide the animal with a «♦. - Change the structure of intestinal mucosa. 8. The method of claim 7, wherein the probiotic composition comprises Lactobacillus and Streptococcus strains. 9. The method of claim 8, wherein the composition comprises strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantorium and Streptococcus faecium. 10. The method of claim 9, wherein the probiotic composition comprises Lactobacillus strain,,,,, ATCC and,,, ATCC. Contains Streptococcus strain. 11. The method of claim 9, wherein the probiotic composition comprises Lactobacillus strain ATCC and Streptococcus strain ATCC. 12. The method of claim 7, wherein the ratio of bohol height to lacuna depth is increased. 13. The method of claim 7, wherein after separation, a reduction in the proportion of bohol height to lacuna depth is achieved. 14. The method of claim 13, wherein the probiotic is administered to the animal prior to weaning. 15. A method of increasing the growth of a pet and / or achieving a higher meat yield, comprising administering to the animal a probiotic composition of the gut structure of the animal and of the animal. - after 38 weaning to change the enzymatic processing of the digested nutrient in the animal. 16. The method of claim 15 wherein the probiotic composition comprises Lactobacillus and Streptococcus. The method of claim 16, wherein the composition comprises strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantorium and Streptococcus faecium. 18. The method of claim 17, wherein the probiotic composition is a // t / / Lactobas ATCC No. cillus and t f f , ATCC No. Streptococcus strain contain. 19. The method of claim 17, wherein the probiotic composition is a Lactobacillus strain ATCC No. Contains Streptococcus strain ATCC. 20. The method of claim 15, wherein the probiotic is administered prior to weaning. 21. The method of claim 16, wherein the pig is administered a probiotic. 22. The method of claim 21, wherein the probiotic comprises a lactic acid bacterium and is administered at a dose of about 10 ⁷ live lactic acid bacteria per gram of composition. 23. The method of claim 22, wherein the probiotic composition is administered in the form of a gel. 24. The method of claim 23, wherein X that the probiotic composition is administered to a newborn animal at about 7 to 10 days of age, at weaning or during stress. 25. The method of claim 22, wherein the probiotic composition is administered in the form of granules in the medium. 26. The method of claim 25, wherein the probiotic composition is administered to the animal during stress, transport, or nutritional changes. 27. A method for increasing the growth of livestock and / or achieving a higher meat yield, comprising administering to the animal a probiotic composition comprising Lactobacillus and Streptococcus before weaning and altering the gut structure and gut chemical composition of the animal after weaning. 28. The method of claim 27, wherein the probiotic composition comprises Lactobacillus strain ATCC and Lactobacillus strain ATCC. Contains Streptococcus strain. 29. The method of claim 27, wherein the probiotic composition is a Lactobacillus strain ATCC No. Contains Streptococcus strain ATCC. 30. The method of claim 27, wherein the animal is stimulated with digestive enzymes and separates the ratio of bohol height to lacuna depth.