WO1993000012A1 - Manipulation de la structure intestinale et d' enzymes digestives chez les animaux - Google Patents

Manipulation de la structure intestinale et d' enzymes digestives chez les animaux Download PDF

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Publication number
WO1993000012A1
WO1993000012A1 PCT/US1992/005534 US9205534W WO9300012A1 WO 1993000012 A1 WO1993000012 A1 WO 1993000012A1 US 9205534 W US9205534 W US 9205534W WO 9300012 A1 WO9300012 A1 WO 9300012A1
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WIPO (PCT)
Prior art keywords
probiotic
streptococcus
weaning
animal
atcc number
Prior art date
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PCT/US1992/005534
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English (en)
Inventor
David Parker
K. Georgina Collington
Original Assignee
Pioneer Hi-Bred Internatinal, Inc.
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Filing date
Publication date
Application filed by Pioneer Hi-Bred Internatinal, Inc. filed Critical Pioneer Hi-Bred Internatinal, Inc.
Priority to JP5501255A priority Critical patent/JPH06508522A/ja
Publication of WO1993000012A1 publication Critical patent/WO1993000012A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis

Definitions

  • One antimicrobial compound which has been used includes dietary copper which has been shown to be effective in improving growth rates in pigs. Braude, R. , "Copper is a Growth Stimulant in Pigs.”, Animal Production 3:69-75 (1967). Antibiotics have also been used in order to eliminate . pathogenic flora within the digestive tract.
  • the agriculture industry has attempted to move away from use of antibiotics since their ultimate effect upon humans is not well established. A widely spreading perception of the public is that antibiotic additives in livestock are undesirable and efforts have turned away from such use and towards other me_-.ns of favorably effecting the intestinal microflora.
  • a probiotic is generally defined as a live microbial feed supplement which beneficially effects the host by improving its intestinal microbial balance. See, Fuller, R.. "Probiotics in Man and Animals.”, J_ ⁇ Appl. Bacteriol. 66:365- 378 (1989).
  • a major problem with probiotics is the lack of information on how such substances function. Beneficial affect upon growth is influenced by a wide variety of preparations used, their interaction with other growth- promoting substances in the diet and poor definition of the organisms tested. Therefore, while there is data to suggest that such preparations can be effective, the extent t which they influence animal growth in production and the mechanisms of their actions are still unclear. An understanding of this process would provide a rational basis for the level and frequency of dosing required to optimize beneficial effects. With such knowledge changes in requirement and response to probiotic treatment might be anticipated with the stage of growth and development of the gastrointestinal tract and in animals subject to physiologica or environmental stress which alter the microbial balance of the gut.
  • the intestinal structure has been observed to change pos weaning. Transverse sectioning of the gastrointestinal tract shows that all regions of the tract are structurally similar and beneath the outer serous coating are layers of musculature.
  • the innermost layer of the tract has three components: the muscularis mucosae which consists of two layers of smooth muscle, a thick layer of connective tissue, and a mucous membrane.
  • the mucous membrane, luminal surface of the tract is composed of columnar epithelial cells and al but the oesophagus.
  • the small intestine has depressions throughout which project down into the connective tissue (lamina basement) creating what is termed the crypts of Liberkhun.
  • This invention relates to application of probiotics to increase digestive enzymes in the intestinal tract, to improve the intestinal epithelial structure for increased digestive and absorptive ability, and to reducing negative effects which occur in the intestinal tract post-weaning which can cause decrease in growth of animals.
  • a further object of this invention is to provide for a method of altering the mucosal structure of the intestinal tract in animals, where such animals are benefited by such change.
  • a still further object of this invention is to cause changes to the intestinal tract and the chemical components o the same in order to decrease negative effect on the intestinal tract of animals.
  • a still further object of the invention is to provide fo a method of improving animal health post-weaning.
  • the invention relates to a method to alter digestive enzymes in animals where the animal can benefit from such alteration comprising administering to the animals probiotic strains of Lactobacillus and Streptococcus.
  • the invention also relates to a method of altering mucosal structure in the intestinal tract of animals where the animal can benefit from such alteration through the application of such probiotic strains.
  • the invention provides for a method of improving animal health post-weaning by applying such strains to the animal.
  • Fig. 1 is a graphic depiction showing alkaline phosphatase activity in control, tylosin, and Probios-treated pigs at 7,17,42 and 80 days of age.
  • Fig. 2 is a graphic depiction showing sucrase activity i control, tylosin, and Probios-treated pigs at 7,17,42 and 80 days of age.
  • Fig. 3 is a graphic depiction showing lactase activity i control, tylosin, and Probios-treated pigs at 7,17,42 and 80 days of age.
  • Fig. 4 is a graphic depiction showing tripeptidase activity in control, tylosin, and Probios-treated pigs at 7,17,42 and 80 days of age.
  • Fig. 5 is a graphic depiction showing dipeptidase activity in control, tylosin, and Probios-treated pigs at 7,17,42 and 80 days of age.
  • Fig. 6 is a graphic depiction showing alkaline phosphatase activity in control and Probios-treated pigs at 8,20,28 and 35 days of age.
  • Fig. 7 is a graphic depiction showing lactase activity i control and Probios-treated pigs at 8,20,28 and 35 days of age.
  • Fig. 8 is a graphic depiction showing tripeptidase activity in control and Probios-treated pigs at 8,20,28 and 35 days of age.
  • Fig. 9 is a graphic depiction showing dipetidase activity in.control and Probios-treated pigs at 8,20,28 and 35 days of age.
  • probiotics alter digestive enzymes, which enzymes are associated with improved capacity to digest nutrients.
  • the inventors have also discovered that the mucosal structure of the intestinal epithelial surface in the intestinal tract of animals is changed in a manner which exposes more mature cells associated with better capacity to digest nutrients than younger cells. Where such probiotics are applied to an animal prior to weaning, it has further been found that the probiotic will increase such enzymes and mucosal structure associated with improved nutrient digestion and can decrease negative post-weaning effects.
  • sucrase sucrosefo -D-glucohydrolase
  • lactase f -D-galactoside galactohydrolase
  • dipeptidase substrate L-leucylglycine
  • tripeptidase substrate L-leucylglycylglycine
  • morphology of the intestine was affected when the probiotic was applied.
  • the villus height and ratio of villus height to crypt depth was not as greatly reduced pre- to post-weaning when the probiotic was applied.
  • the probiotic administration to pigs influenced the pre-weaning changes in mucosal structure typically by preventing the reduction of villus height and crypt elongation between Day 7 and 17 of age.
  • the probiotics of this invention can take a variety of forms as long as they improve enzyme activity and structure of the intestine.
  • the first preparation here is a 12 strain version including Lactobacillus acidophilus (3 strains); L. casei (one strain), L ⁇ plantarium (four strains) and Streptococcus faeciu (2 strains).
  • the five strain version includes _ ⁇ acidophilus, L. c. ei, two strains of ⁇ plantarum and Streptococcus faecium. These are deposited in Germany at the Deutsche Sammlong von Mikroorganismen Und ZellWriten G bh. and will be deposited with the American Type Culture Collection to be made available to the public upon allowance of any claims.
  • the form of administration of the probiotic has not been found to be critical.
  • livestock it typically is administered in the form of granules included in feed.
  • the granules are ordinarily applied at times of stress, arrival or change in ration of feed.
  • a gel composition may be used to administer the probiotic and may supplement the addition of granules to feed.
  • a gel is typically administered at birth, 7 to 10 days of age and at weaning or other times of stress. Since the detrimental enzyme reduction and intestinal structure changes occurring post weaning can be impacted by the probiotic, dosing prior to weaning is most effective. It is known to include other components in such gel compositions and the additions are not critical as long as they do not interfere with the increased enzyme activity or changed intestinal structure. Additions such as those to improve delivery include yeast, vegetable oil, silicon dioxide, titanium dioxide, vitamins, coloring agents and preservatives.
  • the amount of lactic acid bacteria included must be sufficient to cause the desired enzymatic and structural changes to the intestine.
  • the effective amount used in these experiments include about 10 7 viable counts per gram of composition.
  • the probiotic preparations administered during this study were Probios ® brand (Pioneer Hi-Bred International, Inc., Johnston, Iowa, U.S.A.) which contained twelve bacterial strains, including dried Lactobacillus plantarum. Streptococcus faecium, Lactobacillus casei and Lactobacillus acidophilus and their fermentation products.
  • the preparations used in this experiment were of two types: Probios Oral Gel for Swine
  • Composition Dried fermentation products and selected strains of lactic acid bacteria (7 Lactobacillus spp. and 5 Streptococcus spp. ) together with: yeast culture, vegetable oil, sucrose, silicon dioxide, titanium dioxide, vitamins A,D,E and B,F,D and C yellow No. 6 Lake, Polysorbate 80, TBHQ and ethoxyquin preservatives.
  • Total lactic acid bacteria 10 7 viable counts/g.
  • Composition Dried fermentation products and selected strains of lactic acid bacteria (7 Lactobacillus spp. and 5 Streptococcus spp. ) together with yeast culture, calcium carbonate, corn cob fractions, vegetable oil, TBHQ and ethoxyquin preservatives.
  • Total lactic acid bacteria 10 7 viable counts/g.
  • Tylosin is a acrolide antibiotic, isolated from Streptomyces fradiae (Brander, G.L. "Chemicals for Animal Health Control” Taylor and Francis Ltd. London (1986)) and active against Gram- positive organisms. At subtherapeutic levels tylosin is used extensively as a growth-promoter in pigs and poultry. In the present study tylosin was included in 'positive control' diets as Tylamix Premix 100 g/kg (Product License No 0006/4055. Elanco Products Ltd., Basingstoke, England.) each kilogram of which contains tylosin phosphate equivalent to 100 g of tylosin base. TREATMENTS
  • the trial consisted of three treatments: (1) basal diet containing no added growth promoter and with copper at nutritional levels; (2) basal diet and antibiotic growth promoter Tylosin (Elanco Products Ltd, Basingstoke, Hants), a macrolide antibiotic isolated from Streptomyces fradiae and active against Gram positive organisms (Brander, Supra, at 40 mg/kg; (3) basal diet and probiotic preparation Probios ® (Pioneer Hi-Bred International Ltd, Johnston, Iowa, USA) administered as an oral gel 24 hours after birth (1 g), again at 7 days of age (1 g) and at weaning (2 g).
  • the probiotic was included as granules in both the creep feed and follow-on diet at 1 kg/ton.
  • Both forms of the probiotic contained 10 7 colony-forming units/g and comprised a mixture of multiple strains of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei and Streptococcus faecium.
  • the treatments represented in a particular phase were randomly allocated to the four cohorts such that within each pair of sows one cohort was allocated to each treatment. While in the farrowing house all cohorts were penned individually, and following weaning animals were penned in treatment groups with the two cohorts from each treatment in each pen. At all time precautions were taken to avoid cross-contamination between treatment groups. DIETS AND FEEDING
  • the creep feed and the follow-on ration were formulated to provide respectively 253 and 200 g crude protein/kg (nitrogen x 6.25) with lysine contents of 0.133 and 0.11 g/kg.
  • the diet formulations and relevant chemical analyses are shown in Table 1. Both diets contained copper at nutritional levels (20-70 mg/kg). Litters were offered creep feed from 7 days of age and piglets were weaned from the farrowing house to flat- deck housing at 21 days of age. All pigs were weighed at weekly intervals.
  • Sucrase and lactase were assayed by the method of Dahlqvist, Arne, "Method for assay of intestinal disaccharidases” Analyt. Biochem. 7:18-25 (1964), and dipeptidase (substrate L-leucylglycine) and tripeptidase (substrate L-leucylglyclglycine) by that of Nicholson, J.T. and Kim, Y.S., "A one-step L-aminoacidoxidase assay for intestinal peptide hydrolase activity" Analyt. Biochem. 63:110-117 (1975) incorporated herein by reference.
  • tissue sections were viewed under the light microscope. From tissues fixed initially in 3% glutaraldehyde smaller sections (1 mm x 2 mm) were taken under a Vickers M69 dissecting microscope and stored overnight in phosphate buffer (pH 7.4). Tissue sections were then processed by the Department of Anatomy, Medical School, Newcastle upon Tyne. Intestinal sections were viewed (x50) using a Vickers V80 stereoscopic microscope and accurately drawn using a camera lucida attachment. A scale (0-50 urn) was drawn (x50) from a stage micrometer, copied onto an acetate sheet and used to overlay the camera lucida drawing to allow precise measurement of the tissue section.
  • villus height (distance from villus tip to villus/crypt junction) and crypt depth (distance from villus/crypt junction to muscularis mucosa). On average 6 measurements of each parameter were made per section, and the ratio of villus height:crypt depth calculate .
  • the data were analyzed using the GLM statistical package (Baker, R.J. and Nelder, J.A. , "GLM System Release” 3 Numerical Algorithms Group, Oxford (1978)) which allows for the sequential fitting of all factors which may affect the variable being analysed.
  • the initial analysis was carried ou across all ages studied with the summed data for all sites within the intestine and was designed to test whether there were any significant effects of age and treatment or the 'blocking' factors phase, sex and mothering. The latter factor was included to identify effects that might have arise due to the fact that some pigs were reared on a foster rather than a natural mother.
  • C basal diet: T, basal diet + tylosin (40 mg/kg): P, basal diet + Probios; age: Pre, pre-weaning (7 d + 17 d): Post, post-weaning (42 d + 80 d) .
  • C basal diet: T, basal diet + tylosin (40 mg/kg): P, basal diet + Probios: site, Prox, proximal sites (0.05, 0.10, 0.30): Dist. distal sites (0.60, 0.80).
  • Table 4 summarizes the affects of treatment and age on each mucosal parameter studied for all intestinal sites and pigs on each treatment at 7,17,42 and 80 days of age.
  • C basal diet: T, basal diet + tylosin (40 mg/kg): P, basal diet + Probios: age: Pre, activity in pre-weaned pig (7 and 17 d of age): Post, activity in post-weaned pigs (42 and 80 d of age): NS, not significant.
  • alkaline phosphatase activity showed significant interactions between treatment and site, the increase in activity from proximal to distal small intestine being significantly lower in control pigs compared with treated animals (p ⁇ 0.05 for IU/gMWW and p ⁇ 0.01 for specific activity) .
  • Other enzymes at this age did not show treatment effect.
  • the activities of alkaline phosphatase, tri- and dipeptides enzymes in proximal tissue were significantly lower than that in the distal intestine (p ⁇ 0.001 for all except tripeptidase specific activity for which p ⁇ 0.05).
  • sucrase was significantly greater in the proximal tissue (p ⁇ 0.001). There were no significant effects of site on lactase activity. 17 DAYS OF AGE:
  • sucrase and lactase activities were significantly higher in tissue from the proximal intestine than .. the distal gut (p ⁇ 0.001 for IU/gMWW of both enzymes and p ⁇ 0.01 for specific activity of both enzymes).
  • the activities of all the other enzymes studied were significantly lower in the proximal intestine compared to the distal (p.0.001 for all activities except tripeptidase IU/gMWW for which p ⁇ 0.05).
  • the magnitudes of change in activity between proximal an distal regions was significantly lower in control pigs compared with treated animals for sucrase, tripeptidase and dipeptidase (p.0.001 for tripeptidase specific activity; p ⁇ 0.01 for sucrase IU/gMWW, tripeptidase IU/gMWW and dipeptidase specific activity; p ⁇ 0.05 for sucrase specific activity and dipeptidase IU/gMWW).
  • Sucrase activity increase between the proximal and distal small intestine in control animals while decreasing in the tylosin- and Probios-treated groups.
  • tripeptidase activity decreased between these two regions in control animals while increasing in tylosin- and Probios-treated animals.
  • Tylosin or Probios administration to pigs during the present experiment was associated with prevention of reduction in villus height and the increase in crypt depth at the 0.10 intestinal site seen between 7 and 17 days of age in control animals.
  • At the 0.60 site only antibiotic-treated pigs showed no obvious loss in villus height between 7 and 17 days of age. All animals had reduced villus height from 17 to 42 days of age at the 0.10 site.
  • At the 0.60 site there were no marked changes in villus height or crypt depth in control pigs between 17 and 42 days of age, however, reductions and increases respectively in these parameters were apparent in both tylosin- and Probios-treated animals.
  • the five strain probiotic has the effect of greater recovery of both peptidase enzymes studied.
  • the activities o all enzymes fell between 20 and 28 days in the animals weaned at 21 days and by Day 35 there was evidence of a recovery in activity of both dipeptidase enzymes which was greater in treated animals.
  • the fall in enzymes after weaning in the animals weaned at 28 days was not as great as in 21 day weaned animals.
  • the significant effect of treatment seen in the first experiment was not repeated here, however, there was the recovery affect noted with treatment and a significant increase in dipeptidase was seen in the second experiment at Day 8. There was a reduction in weight seen with control animals past weaning not apparent in Probios-treated animals.
  • the table shows that between 8 and 20 days of age, compared with controlled pigs, there was a smaller reduction in villus height in Probios-treated animals. Between 20 and 28 days of age, the loss in villus neight and therefore the change in villus height:crypt depth ratio was greater in Probios treated pigs than control animals.
  • Probios has been associated with increased activity of digestive enzymes which can include alkaline phosphatase, sucrase, lactase and tripeptidase. The effect varies across intestinal sites.

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Abstract

Procédé consistant à faire décroître la réduction d'enzymes digestives et à modifier la structure muqueuse de l'intestin d'animaux. Pour ce faire, on administre auxdits animaux une substance probiotique leur permettant de bénéficier des effets favorables de ce changement. En outre, un procédé permettant de maintenir ces effets après le sevrage consiste à administrer à l'animal la substance probiotique avant le sevrage.
PCT/US1992/005534 1991-06-28 1992-06-28 Manipulation de la structure intestinale et d' enzymes digestives chez les animaux WO1993000012A1 (fr)

Priority Applications (1)

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JP5501255A JPH06508522A (ja) 1991-06-28 1992-06-28 動物における腸の構造および酵素の操作

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US72439891A 1991-06-28 1991-06-28
US724,398 1991-06-28

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EP (1) EP0563337A4 (fr)
JP (1) JPH06508522A (fr)
AU (1) AU2299392A (fr)
CA (1) CA2093131A1 (fr)
HU (1) HUT67883A (fr)
WO (1) WO1993000012A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5784994A (en) * 1994-07-04 1998-07-28 Van Der Lely; Cornelis Apparatus and method for automatically milking animals
WO2000054788A1 (fr) * 1999-03-15 2000-09-21 Italmed S.N.C. Di Galli G. E Pacini G. Composition pharmaceutique a usage medical et veterinaire utilisee pour regenerer la flore intestinale en cas de diarrhee ou de syndrome dyspeptique

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5340555B2 (ja) * 2007-04-09 2013-11-13 コンビ株式会社 絨毛伸張剤

Citations (2)

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Publication number Priority date Publication date Assignee Title
US4910024A (en) * 1988-07-05 1990-03-20 Micro Chemical, Inc. Method and apparatus for administering live bacteria as feed additives to livestock and poultry
US4999193A (en) * 1987-06-16 1991-03-12 Etablissements Guyomarc'h S.A. Feed additive for animals, feeds containing such an additive and method for improving the growth of animals

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US4999193A (en) * 1987-06-16 1991-03-12 Etablissements Guyomarc'h S.A. Feed additive for animals, feeds containing such an additive and method for improving the growth of animals
US4910024A (en) * 1988-07-05 1990-03-20 Micro Chemical, Inc. Method and apparatus for administering live bacteria as feed additives to livestock and poultry

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
British Journal of Nutrition, Volume 27, issued 1972, R.C. SIDDONS et al., "The influence of the intestinal microflora of disaccharidase activities in the chick", pages 101-112, see entire document. *
British Journal of Nutrition, Volume 64, issued 1990, G.K. COLLINGTON et al., "The influence of inclusion of either an antibiotic or a probiotic in the diet on the development of digestive enzyme activity in the pig", pages 59-70, see entire document. *
Clinical and Experimental Gnotobiotics, Zbl. Bakt, Supplement 7, issued 1979, J. SZABO, "Protein, Carbohydrate, and Fat Degrading Enzymes in the Intestine of Germfree and Conventional Piglets", pages 125-128, see entire document. *
Journal of Animal Science, Volume 67 (Supplement 2), issued 1989, M.A. GIESEMANN et al., "Effect of a probiotic, Streptococcus faecium M74, on growing-finishing pig performance", pages 127-128, see entire document. *
Journal of Chem Technol Biotech, Volume 51, issued 1991, S. MINNEY, "Probitics- The Success Story", pages 557-559, see entire document. *
See also references of EP0563337A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5784994A (en) * 1994-07-04 1998-07-28 Van Der Lely; Cornelis Apparatus and method for automatically milking animals
WO2000054788A1 (fr) * 1999-03-15 2000-09-21 Italmed S.N.C. Di Galli G. E Pacini G. Composition pharmaceutique a usage medical et veterinaire utilisee pour regenerer la flore intestinale en cas de diarrhee ou de syndrome dyspeptique

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HU9300875D0 (en) 1993-06-28
EP0563337A4 (en) 1993-10-13
JPH06508522A (ja) 1994-09-29
AU2299392A (en) 1993-01-25
HUT67883A (en) 1995-05-29
EP0563337A1 (fr) 1993-10-06
CA2093131A1 (fr) 1992-12-29

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