WO2018029595A1 - Préparation probiotique, son procédé de préparation et son utilisation. - Google Patents

Préparation probiotique, son procédé de préparation et son utilisation. Download PDF

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WO2018029595A1
WO2018029595A1 PCT/IB2017/054824 IB2017054824W WO2018029595A1 WO 2018029595 A1 WO2018029595 A1 WO 2018029595A1 IB 2017054824 W IB2017054824 W IB 2017054824W WO 2018029595 A1 WO2018029595 A1 WO 2018029595A1
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Prior art keywords
alginite
grains
probiotic
preparation according
probiotic preparation
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PCT/IB2017/054824
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English (en)
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Eva STYKOVÁ
Radomíra NEMCOVÁ
Igor VALOCKÝ
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Univerzita Veterinárskeho Lekárstva A Farmácie V Košiciach
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Publication of WO2018029595A1 publication Critical patent/WO2018029595A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/733—Alginic acid; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/10—Fertilisers containing plant vitamins or hormones

Definitions

  • Probiotic preparation method of its preparation and use of probiotic preparation
  • the technology concerns a new carrier of the probiotic microorganisms, mainly for veterinary or human consumption, pharmaceutics, food industry or cosmetics.
  • the invention discloses a method of preparation of the probiotic preparation, where the beneficial living microorganisms are tied to new application form of the carrier.
  • the specific use of the probiotic preparation in the veterinary and human medicine is also new.
  • probiotics Positive effect of the probiotics is known. Pure cultures of living microorganisms are applied in forms of capsules, tablets, syrups and so one. In case of peroral use it is important that probiotic cultures are tied to the carrier which allows its transfer between the stomach and small intestine.
  • the live cultures have application, especially concerning the short expiration time of the preparation (usually 21 days) and specific conditions for its storage (mainly due to low temperatures). Lyophilization often (depending on the conditions and process of the lyophilization) significantly lowers the amount of present live bacteria, which in the end means that the resulting preparation does not have to have desired probiotic features.
  • Published patent file CZ 303986 B6 discloses a preparation which contains one- species or multiple-species probiotic culture and a carrier where the probiotic culture is in a preparation in form of a biofilm adjacent to the carrier.
  • the basic features of the biofilms related to the solid surfaces are know and described (Costerton et al., 1985). Sterile milk or whey or synthetic substances with casein or casein hydrolyzate or peptone are used as a cultivation medium. Microcrystalline cellulose or starch is used as carrier. Effective creation of biofilm is not achieved with this carrier in case of multiple species of lactobacilli, for example strain Lactobacillus reuteri.
  • Publication US 2015/0202136 A1 discloses dermatologically acceptable carrier of the probiotics in case of application of large group of probiotics on a skin. This publication does not address peroral application.
  • Publication US 2015/0150295 A1 discloses production of a porous powder which serves as a carrier of probiotic microorganisms. The directed process of the transformation of the liquid concentrate to the foam and its drying allows controlling various resulting features of the product.
  • File US 2014/0010918 A1 discloses a method of production of micro-capsule carrier with various types of matrix for carrying probiotic cultures.
  • a natural carrier of the probiotic cultures is desired and not known, which would be broadly usable, easily available, ensuring symbiosis with the probiotic cultures, and allows their gradual and long-term release.
  • a probiotic preparation which includes living microorganisms forming a biofilm on a solid surface which is at least partially saturated by a substrate according to this invention which essence lies in the fact that the carrier is an alginite, preferably an alginite in form of grains with size up to 3 mm, especially preferably up to 1 ,3 mm.
  • Alginite forms a basic skeleton, solid porous matrix for solid state fermentation (SSF) in order to produce bacterial biofilm.
  • Alginite is an organic-mineral rock of clay character, occurring in volcano craters by deposition of dead algae parts and inorganic material with a high content of humus, mineral nutrients and trace elements. It also contains humic acids. Alginite is entirely ecological material with unique structure and excellent composition on exclusively natural basis; it contains no unnatural chemicals or artificial additives and it has naturally low content of the salts and heavy metals. Alginite has been hitherto known as a fertilizer, or earth fertilizer for use in agriculture. The effects of alginite in this field are known; alginite increases fertility and helps the plants to endure the droughts without much harm. The use of alginite in its basic natural form for pharmaceutical, medicinal or food purposes are not known.
  • the proposed invention discloses that the alginite can be preferably used as a carrier of the probiotic cultures, advantageously for lactobacillus strains.
  • Alginite grains are covered with biofilm from respective probiotic microorganism, where most of the bacterial cells in the biofilm is in dorminant, resting state with minimal metabolism.
  • the proposed invention provides crucial advantage in the fact that microorganisms, for example lactobacilli are stabilized in a form of the biofilm, where they are present in the dorminant state. In this state they show increased resistance against various types of stress and do not require specific conditions for storing.
  • the present microorganisms are resistant to environment for which they are not designed (esophagus, stomach and so on), whereby they are activated in the desired environment (lower gastrointestinal tract), where they absorb necessary nutrients and begin to reproduce. Subsequently the biofilm is disintegrated and gastrointestinal tract recolonized, which causes a release of the substances in the alginite itself.
  • the comparison of results from the tests shows higher amount of surviving microorganisms (lactobacilli in particular in the tests) as in case of application of live cultures, which is crucial advantage.
  • the alginite has high natural humidity and high porosity. These features contribute to good conditions for fermentation with creation of bacterial biofilms.
  • the surface of the alginite grains creates excellent conditions for production of the stable biofilm.
  • alginite Since alginite is mined in the ground, it is apt that the grains of the alginite of the necessary fraction are sterilized in advance. In practice the mined alginite is cleaned from the little rocks, it is milled and sterilized. The alginite grains are subsequently saturated by the cultivation medium and the microorganisms are then cultivated in the alginite grains.
  • the alginite is saturated by a suitable substrate, for example lactose or glucose or their combination. It is preferable that the alginite is connected with lactobacillus strains, for example Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus buchneri. Lactobacillus reuteri has positive effects on living microorganism; this strain has been identified directly in the digestive tract of animals which significantly predetermines its potential use. The positive effects on people - especially newborns and children - have been found out. It has also positive effects on various types of digestive tract diseases and oral health.
  • lactobacillus strains for example Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus buchneri. Lactobacillus reuteri has positive effects on living microorganism; this strain has been identified directly in the digestive tract of animals which significantly predetermines its potential use. The positive effects on people - especially newborns and children - have been found out. It
  • alginite with lactobacillus strains leads to stabilization of the beneficial microorganisms cultivated in the alginite grains.
  • the bond to alginite carrier saturated by suitable cultivation medium stabilizes microorganisms during their storage and after their application.
  • the probiotic organisms forming a biofilm on the alginite grains are live for a long time and capable of reproduction in microenvironments of humans and animals. Probiotic microorganisms are released from the biofilm for long time.
  • the experiment has been designed to test the colonization capacity of the strain Lactobacillus reuteri L2/6, stabilized in biofilm on alginite in a digestive tract of the model animals in predefined conditions - BALB/c germ-free mice.
  • the first group of animals - LR2/6A where the Lactobacillus reuteri L2/6 has been applied, was compared with second group of animals - LR 2/6, where the strain has been applied without alginite.
  • Lactobacillus reuteri L2/6 has been applied according to this invention in a form of a biofilm on sterile alginite grains (0,05 % of the dry matter 72 hours fermented grains of alginite into sterile feed).
  • the second group of animals LR 2/6 received a strain without alginite as a night culture of planktonic cells of the strain applied to the feed.
  • the experiment lasted for three subsequent periods: period of adaptation (4 days), period of application of additives in the feed (21 days) and period after the application of additives ended (5 days).
  • the samples of droppings (feces) for microbiological examination during the period of application of additives were collected on 5 th , 10 th , 15 th and 20 th day since the beginning of the application individually from each animal.
  • the last collection of the droppings was done after the application of additives ended - on 25 th day.
  • the numbers of strain Lactobacillus reuteri L2/6 were significantly higher in group LR2/6A (p ⁇ 0,001) as compared to LR 2/6.
  • the numbers of strain Lactobacillus reuteri L2/6 in LR2/6A were constant until the end of application (8, 11 ⁇ 0,48 - 8,25 ⁇ 0,21 log/g of droppings).
  • the numbers of strain Lactobacillus reuteri L2/6 had slightly increasing tendency from 6,38 ⁇ 0,43 to 6,75 ⁇ 0.21 log/g of droppings, but these values were lower by approximately 1 ,2 - 1 ,7 log as compared to LR 2/6A group.
  • the number of strain Lactobacillus reuteri L2/6 in LR2/6A dropped to log 4,42 ⁇ 0, 1 1/g.
  • the presence of the tested strain in the droppings was approximately log 1 ⁇ 0,22/g on 5 th after the end of application.
  • the probiotic preparation in form of 0,05 % of 72 hours fermented mixture with amount approximately 3x10 4 KTZ/g was mixed into the feed of the mice.
  • the detection of the applied strain in droppings 5 days after last feed of the mice with Lactobacillus reuteri L2/6 proves a colonization of the intestine of the animals by this strain and its ability to reproduce in this environment.
  • probiotic preparation according to this invention on the reparation of the microbial settlement of the intestines in the conventional mice after the treatment by antibiotics (ATB) has been tested on following experiment.
  • Probiotic strain Lactobacillus reuteri L2/6 has been stabilized in alginite pursuant to abovementioned description.
  • the intestinal dysbiosis has been caused in mice by daily application of ATB amikacin in amount 5 mg/mouse during 7 days. Amikacin causes reversible lowering of the level of lactobacilli in colon.
  • the experiment was directed for monitoring of the effects of the strain Lactobacillus reuteri L2/6 stabilized in alginite on numbers of chosen microflora of jejunum, ileum and caecum, and droppings, and concentration of organic acids in conventional mice (BALB/c).
  • the animals were divided into three groups: group 2/6A - strain Lactobacillus reuteri L2/6 R
  • the microbiological analysis of the intestinal tract of the mice after 14 day application of the additives has show the effects of the strain Lactobacillus reuteri L2/6 on the numbers of chosen intestinal bacteria in the digestive tract of mice.
  • the numbers of bacteria of Lactobacillus reuteri species were also positively affected (p ⁇ 0,001) in a group where Lactobacillus reuteri L2/6 R
  • the total bacteria were more significantly affected in group 2/6A in frontal parts of the intestine - in jejunum and ileum (p ⁇ 0,01 ; p ⁇ 0,01).
  • F in group 2/6A set by the cultivation method after 14 days of application of the strain, were following: jejunum - 6,55 ⁇ 0,23 log/g; ileum - 7,02 ⁇ 0,25 log/g; caecum - 7,20 ⁇ 0,26 log/g; droppings - 7,26 ⁇ 0,38 log/g.
  • droppings - 7,26 ⁇ 0,38 log/g The most significant production of the organic acids (p ⁇ 0,05) in droppings of group 2/6A compared to other groups have been detected on 14 th day of application of the additives, namely in cases of acetoacetic acid and acetic acid.
  • the protective effect of the strain Lactobacillus reuteri L2/6 stabilized on the alginite has been tested by the experiment during which the infection has been incited in case of model animals (mice) by strain Salmonella Typhimurium CCM 7205.
  • the experiment was directed on testing of the effect of the strain Lactobacillus reuteri L2/6 stabilized in the alginite on the inhibition of Salmonella Typhimurium CCM 7205 and on morphological, biochemical and immunological parameters in case of model animals (mice).
  • the animals have been divided into four groups: group LAB - strain Lactobacillus reuteri L2/6 as biofilm stabilized in alginite (0,05 % of dry matter 72 hours fermented grains of alginite into sterile feed); group PK - positive control (alginite without lactobacillus strain); group NK1 - negative control (without additives); and group NK2 - negative control (alginite without lactobacillus strain).
  • group LAB - strain Lactobacillus reuteri L2/6 as biofilm stabilized in alginite (0,05 % of dry matter 72 hours fermented grains of alginite into sterile feed
  • group PK - positive control alginite without lactobacillus strain
  • group NK1 - negative control without additives
  • group NK2 - negative control alginite without lactobacillus strain
  • liver parenchyma Histological analysis of liver parenchyma in mice after 14 days of application of additives have shown the effects of strain Lactobacillus reuteri L2/6 on melioration of post- infectional harm to liver parenchyma of mice.
  • the presence of an inflammatory infiltrate has been detected to be significantly lower (p ⁇ 0,05), forming 1 ,67 ⁇ 0,09 % of liver parenchyma, as compared to PK group (without application of probiotic strain) with percentual presence of 5,70 ⁇ 1 ,23% of inflammatory infiltrate in liver parenchyma.
  • the liver of the mice from experimental groups inoculated with Salmonella Typhimurium bacteria had a typical image of the inflammatory response presented by the infiltration of the inflammatory cells of the inborn or received element of the immunity, mainly by increased number of Kupffer cells in the interstitial space and diffusion of these cells from the lumen of the veins and arteries.
  • the perceived repleted inflammatory bearings in PK group confirmed the breach of integrity of the parenchyma.
  • the intensity of the inflammatory bearings was significantly lower; the bearings had character of specified granulation bearing and, as opposed to PK group, no unlimited diffuse inflammation throughout parenchyma of the liver was detected.
  • a necrosis of the liver cells occurred, which has happened by the violation of their integrity and structure.
  • the overall effect on the mineral (Ca, P) metabolism is important, too, which has been perceived in all groups with application of alginite.
  • Significantly higher level of Ca (p ⁇ 0,05) has been perceived in LAB group in comparison with group which received pure feed without alginite (NK1).
  • the highest concentration P has been detected in group of mice with application of alginite itself, where level P has been significantly higher (p ⁇ 0,05; p ⁇ 0,01 ; p ⁇ 0,001) as compared to other groups (LAB, NK1 a PK).
  • Higher concentration P has been perceived in LAB group (p ⁇ 0,05), too, in comparison with infected group PK.
  • CD4+CD25+ lymphocytes were not significantly affected. Lactobacilli have statistically significantly (p ⁇ 0,001) increased the presence of B-lymphocytes in peripheral blood as compared to other groups. In spleen as well as in peripheral blood the significantly higher CD4:CD8 has been perceived, as well as significantly higher presence of CD4+CD8+ as well as NK cells in LAB group. As opposed to peripheral blood, the presence of CD4+ as well as CD8+ lymphocytes has been statistically significantly lower in all experimental groups as compared to NK1 group. In mesenteric lymph nodes a higher CD4:CD8 ratio has been detected - similarly as in spleen; the highest presence of CD4+CD8+ as well as NK cells was in LAB group. The highest presence of CD4+ was detected in LAB and PK groups as compare to NK1 and NK2 group.
  • strains have been isolated and identified from the skin lesions: Streptococcus dysgalactiae, Staphylococcus warneri, Staphylococcus epidermidis, Corynebacterium glutamicum, Streptococcus porcinus and Bacillus spp.
  • the strains demonstrated high hemolytic activity. The results after examination of presence of scabies and Dermatophilus congolensis were negative. Before the beginning of the experiment in March the fur has been cut on the limbs.
  • the strain Lactobacillus buchneri 5/K has been chosen on the basis of earlier in vitro selection.
  • nodules hyperkeratose, hyperplastic to granulomatous to hypergranulomatous prominent formations
  • bleeding-prone nodules dry crackling rhagades (cracks in the horny skin)
  • bleeding rhagades wet lesions
  • dry peeling lesions without exudation dry lesions which are wet after scraping
  • hyperkeratosis thickening of the stratum corneum often associated with the presence of abnormally high amounts of keratin
  • lichenification the result of constant scratching and friction.
  • the outer layer of the skin succumbs to hypertrophy (thickening of the skin and highlighting of the skin signs, which gives the skin a leather appearance similar to the bark), seborrhoea (excessive sebum production by sebaceous glands and its accumulation on the skin surface).
  • the horses did not limp; the temperature of the lesions corresponded to the environment temperature.
  • strain Lactobacillus reuteri L2/6 stabilized in alginite in the host organism leads to induction of its reproduction, which is manifested by higher presence of the strain in droppings of the animals and by longer life-time of the strain in the digestive tract.
  • the application of strain Lactobacillus reuteri L2/6 stabilized in alginite had positive effect on reparation of settlement of the intestine of the convention mice by lactobacilli after ATB treatment.
  • Multicolor FISH analysis of the intestinal tract of the conventional mice after 14 day application of the strain Lactobacillus reuteri L2/6 stabilized in alginite has proven its ability to colonize jejunum, ileum a caecum of the mice at the presence of competitive microflora, and to modulate the monitored intestinal microflora by the parallel increase of the production of the organic acids. This effect has been manifested by significant increase of the number of representatives of Lactobacillus and Bifidobacterium genera, and by significant decrease of the number of representatives of Clostridium, Bacteroides and Enterobacteriaceae genera.
  • the applied strain stimulated the population caecal lactobacilli belonging to the species Lactobacillus Reuteri. Lactobacillus reuteri L2/6 reduced the invasion of salmonelli into liver and spleen, which was manifested by more moderate negative effect on the liver parenchyma of the mice.
  • strain Lactobacillus reuteri L2/6 significantly stimulated immunity response of the cells, which has been manifested by significant increase in CD4:CD8 ratio of lymphocytes, higher presence of young or - eventually - memory T-lymphocytes, NK cells and B lymphocytes, whereby this higher presence was both in peripheral blood as well as in spleen and in mesenteric lymph nodes.
  • Alginite itself has positively affected the absorption abilities of phagocytes. Immunostimulation effect has been confirmed by significantly increased gene expression for pro-inflammatory cytokines TNF-a, IL-1 b, and MCP-1 , too.
  • Alginite has positively affected chosen parameters of mineral metabolism, especially Ca and P.
  • strain Lactobacillus buchneri 5/K stabilized in alginite had positive effect on elimination of presence of acute skin lesions in distal parts of limbs of horses.
  • Deficiencies in the prior state of the art are significantly remedied by a method of preparation of probiotic preparation, where the inoculated cultivation solution containing living probiotic microorganisms is applied onto solid carrier, then the fermentation takes place at temperature of 40 °C and the carrier is at least partially covered by biofilm from probiotic microorganisms according to this invention which essence lies in the fact that the alginite is crumbled into grains with fraction up to 3 mm; the alginite grains are sterilized and saturated with substrate which contains lactose and/or glucose. Subsequently, the inoculated cultivation solution is applied to alginite grains and during the fermentation a biofilm is produced from probiotic microorganisms.
  • Gamma rays can be advantageously used for sterilization of the alginite grains, which achieves the effective sterilization more quickly than it happens with use of common autoclave.
  • Figure 1 is a graph which depicts the saturation of alginite grains by the glucose substrate at various fraction of alginite.
  • Horizontal axis depicts 10%, 25% and 40% of glucose.
  • Vertical axis depicts saturation in percents, whereby the value of saturation is given as a mean of three measured values.
  • Figure 2 is a graph which depicts the saturation of alginite grains by the lactose substrate at various fraction of alginite.
  • Horizontal axis depicts 10%, 25% and 40% of lactose.
  • Vertical axis depicts saturation in percents, whereby the value of saturation is given as a mean of three measured values.
  • Alginite is milled to grains with fraction on the level of 1-1 ,3 mm. Milled and sieved grains are sterilized by gamma rays. Subsequently, in this example, they are saturated by 40% lactose. At this concentration, the alginite grains were bound with ca. 7% of lactose.
  • Lyophilized strain of Lactobacillus reuteri L2/6 (10 10 KTZ/g) has been resuspended in sterile cultivation medium (peptone, yeast autolysate, casein hydrolyzate, humic acid) into absorbance value 0,9 - 1 (OD 6 2o), which corresponds roughly to 1 x 10 9 KTZ/ml.
  • sterile cultivation medium peptone, yeast autolysate, casein hydrolyzate, humic acid
  • Resulting probiotic preparation has been used with positive effect for recolonization of biocoenosis of gastro-intestinal tract of animals and also for recolonization of biocoenosis of skin.
  • the alginite grains with biofilm were used as an additive to feed mixtures for animals. It is probable that similar positive effects will occur in human use, too.
  • various alginite grains with biofilm were mixed in water into form of emulsion which is applied directly onto affected skin.
  • Alginite grains with biofilm produced by previous example form a basis for production of capsules and tablets.
  • Substrates formed by glucose or lactose had concentration: 10 - 25 - 40%. Tested samples with 1g of alginite have been deposited in vessels followingly:
  • Saturated alginite is cleaned by sterile water during centrifugation for 30 minutes; in this example the rotations are 4500/min.
  • the sediment is frozen and lyophilized for 24 hours.
  • the drying by infrared radiation took place at temperature of 105 °C.
  • the weight difference against the original non-saturated alginite is counted.
  • the results are display on figures 1 and 2.
  • Probiotic preparation stabilized in alginite is in this example used for recolonization of biocoenosis of skin.
  • the probiotic preparation is applied onto the affected skin of the limbs of animals, for example horses. Preventive application on healthy skin is also successfully tested.
  • Probiotic preparation according to this invention is in this example used for recolonization of biocoenosis of gastro-intestinal tract.
  • the preparation is applied to feed mixtures, or directly consumed by animals.
  • Strain Lactobacillus reuteri L2/6 has shown high ability to reproduce and form biofilm on alginite grains in conditions of solid substrate fermentation. At 72 hour cultivation a decrease of cultivable colonies forming germs or nuclei between desorbed cells from biofilm has been detected. Most bacterial cells in the biofilm were in dorminant (resting) state with minimal metabolism. Solid substrate cultivation of lactobacilli with subsequent drying of the matter led to production of significantly higher number of non-cultivable but live cells, which looked like coccobacilli with all features of rest forms: stress resistance, loss of ability to grow on live media. Industrial applicability
  • Probiotic preparation is stabilized in alginite by means of production of biofilm in alginite grains, which has been repeatedly proven as effective application form of beneficial microorganisms designed for harmonization (recolonization) of micro-environments of animals and humans.

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  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Animal Husbandry (AREA)
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Abstract

L'alginite est utilisée comme support de culture probiotique; l'alginite est désagrégée en grains avec une fraction allant jusqu'à 3 mm, de préférence jusqu'à 1,3 mm. Les grains d'alginite sont stérilisés et saturés avec un substrat qui contient du lactose et/ou du glucose. L'évacuation et l'action ultérieure de la pression de l'atmosphère extérieure peuvent être de préférence utilisées; le substrat pénètre à l'intérieur des grains d'alginite. Il est préférable d'utiliser une fraction de 1-1,3 mm et une saturation de 40 % de lactose. Ensuite, une solution de culture inoculée est appliquée sur les grains d'alginite et, pendant la fermentation, un biofilm provenant de micro-organismes probiotiques est produit, principalement à partir du genre Lactobacillus. La Fermentation a lieu à 40 °C; la matière obtenue est séchée. Le produit final est utilisé comme additif dans des mélanges d'aliments pour animaux, ou après dissolution dans l'eau en tant qu'émulsion applicable sur la peau affectée d'animaux, ou d'êtres humains, ou en tant que produit intermédiaire pour la préparation de produits finaux sous forme de capsules, de comprimés, d'émulsions à des fins humaines, vétérinaires ou cosmétiques.
PCT/IB2017/054824 2016-08-07 2017-08-07 Préparation probiotique, son procédé de préparation et son utilisation. WO2018029595A1 (fr)

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SKPP50050-2016 2016-08-07
SK50050-2016A SK288782B6 (sk) 2016-08-07 2016-08-07 Probiotický prípravok, spôsob jeho prípravy a použitie probiotického prípravku

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113812631A (zh) * 2020-06-18 2021-12-21 陈信行 腐植酸益生菌复方及其制程

Citations (1)

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Publication number Priority date Publication date Assignee Title
CZ303986B6 (cs) * 2011-10-07 2013-07-31 Rysávka@Petr Prípravek obsahující probiotickou kulturu, zpusob jeho výroby a pouzití

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CZ303986B6 (cs) * 2011-10-07 2013-07-31 Rysávka@Petr Prípravek obsahující probiotickou kulturu, zpusob jeho výroby a pouzití

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I KUBASOVÁ ET AL: "ALGINITE AS SUITABLE COMPOUND FOR COMBINATION WITH LACTOBACILLUS FERMENTUM CCM 7421 IN DOGS", 21 June 2016 (2016-06-21), XP055423439, Retrieved from the Internet <URL:http://www.probiotic-conference.net/files/ALGINITE AS SUITABLE COMPOUND FOR COMBINATION WITH LACTOBACILLUS FERMENTUM CCM 7421 IN DOGS web.pdf> [retrieved on 20171109] *
K. HLUBENOVÁ ET AL: "The Efect of Probiotic Lactobacilli and Alginite on the Cellular Immune Response in Salmonella Infected Mice", FOLIA VETERINARIA, 27 June 2017 (2017-06-27), pages 61 - 66, XP055423446, Retrieved from the Internet <URL:https://www.degruyter.com/downloadpdf/j/fv.2017.61.issue-2/fv-2017-0020/fv-2017-0020.pdf> [retrieved on 20171109], DOI: 10.1515/fv-2017-0020 *
STROMPFOVÁ VIOLA ET AL: "Health benefits observed after probioticLactobacillus fermentumCCM 7421 application in dogs", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER, DE, vol. 101, no. 16, 18 July 2017 (2017-07-18), pages 6309 - 6319, XP036290403, ISSN: 0175-7598, [retrieved on 20170718], DOI: 10.1007/S00253-017-8425-Z *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113812631A (zh) * 2020-06-18 2021-12-21 陈信行 腐植酸益生菌复方及其制程

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