Probiotic composition based on the enterococcus strain and used as a treatment means and method for the production thereof



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32. EP1478246 - 04.09.2003
DIETETIC AND/OR PHARMACEUTICAL COMPOSITIONS FOR HUMAN AND/OR ANIMAL USE BASED ON PROBIOTIC MICROBIAL PREPARATIONS

URL EPO = http://v3.espacenet.com/textdoc?F=3&CY=ep&LG=en&IDX=EP1478246


Inventor(s): BIANCHI SALVADORI BRUNA (IT); PIROVANO FRANCO (IT); ROTTIGNI CLAUDIO (IT)
Applicant(s): CT SPERIMENTALE DEL LATTE S P (IT); BIANCHI SALVADORI BRUNA (IT); PIROVANO FRANCO (IT); ROTTIGNI CLAUDIO (IT)
IP Class 4 Digits: A61K; C12N; A23L
IP Class: C12N1/20; A61K35/74; A23L1/03; A61K35/72; A23L1/30; C12N1/16
E Class: A61K35/74; A23K1/00C2B; A23K1/18K; A23L1/03M; A23L1/30M; A23K1/18L2; A23C9/123D; A23K1/18V; A61K35/66; A61K35/72
Application Number: WO2003EP01867 (20030224)
Priority Number: IT2002MI00399 (20020228)
Family: EP1478246
Equivalent: AU2003212265; ITMI20020399
Cited Document(s): WO02060276; WO9306208; US5716615; RU2178975; EP1177794; WO9962348
Abstract:

THE PRESENT INVENTION RELATES TO DIETETIC AND/OR PHARMACEUTICAL COMPOSITIONS FOR HUMAN AND/OR ANIMAL USE, AND GENERAL FOODSTUFFS, BASED ON MICROBIAL CULTURES CONSISTING OF AUTOCHTHONOUS AND ALLOCHTHONOUS SPECIES WITH RESPECT TO HUMAN BEINGS AND ANIMALS, SELECTED FROM SPECIES OF LACTIC BACTERIA, PROPIONIBACTERIA, YEASTS AND/OR MOLDS. THEY HAVE AN EQUILIBRATING ACTION OF THE INTESTINAL FLORA OF THE HOST HUMAN BEING OR ANIMAL, AS WELL AS HAVING VARIOUS BENEFICIAL/PROBIOTIC EFFECTS TOWARDS THE HOST ORGANISM.Description:

DIETETIC AND/OR PHARMACEUTICAL COMPOSITIONS FOR HUMAN AND/OR ANIMAL USE BASED ON PROBIOTIC MICROBIAL PREPARA- TIONS

The present invention relates to dietetic and/or pharmaceutical compositions for human and/or animal use based on probiotic microbial preparations.


In particular, the present invention relates to die- tetic and/or pharmaceutical compositions for human and/or animal use and foodstuffs in general, based on microbial cultures consisting of autochthonous and allochthonous species with respect to human beings and animals, se- lected from the lactic bacterial species Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei subsp. casei, Lactobacillus casei subsp. rhamnosus, Lac- tobacillus zeae, Lactobacillus salivarius, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus reuteri, Lactobacillus amylovorus, Lactobacillus crispatus, Lacto- bacillus curvatus, Lactobacillus delbrueckii subsp. del- brueckii, Lactobacillus delbrueckii and all its subspe-

cies, Lactobacillus gasseri, Lactobacillus johnsonii, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, optionally associated with Streptococ- cus thermophilus ; Lactobacillus fermentum, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Leuconostoc spp. ; Entercoccus faecium, Pediococcus pentosaceus, Pediococcus acidilac- tici ; Bifidobacteria such as Bifidobacterium Longum, Bi- fidobacterium Breve, Bifidobacterium bifidum, Bifidobac- terium infants, Bifidobacterium lactis ; and/or propioni- bacterial species, yeast species and/or mold species ; the above species being live and vital and/or devitalized, and said species being present in microbial cultures in a dried concentrated form with a concentration ranging from 106ufc/g to 101l ufc/g.


The above-mentioned probiotic micro-organisms shall be indicated hereafter with the term"pr micro- organisms".
The compositions, object of the present invention, have an equilibrating action of the intestinal flora of the host (human being and animal), in addition to produc- ing various beneficial/probiotic effects towards the or- ganism which vary according to the destination target, such as children, adults, elderly people, expectant women, persons with various kinds of deficiencies or gas-

tro-intestinal disturbances (dismicrobisms) or with acute or chronic diseases, for example of the vaginal or uro- logical type.


These components, with a reciprocal association, act according to a biological and synergic succession on an intestinal level.
It is also known that bacteriophages and bacteria form a more or less indissoluble pair to the extent that it can be declared that there are no micro-organisms without bacteria. The development of bacteriophages is generally considered as being a production problem, but it can also be observed in intestinal flora after the in- gestion of microbial preparations which exert a probiotic effect. In this case, the problem must be solved in the product formulation phase so as to preserve the positive effects of the composition.
It has been found that the most effective solution consists in the differentiation of the species and avail- ability, for each of these, of numerous strains with a different sensitivity to bacteriophages so that all the species are always present in the intestines.
If, on the contrary, the product only consists of a single strain, it is not possible to guarantee the pres- ence of all the species in the case of lysis of this strain and the beneficial effects obtainable with their

ingestion will be lost.


The positive effects of a bacterial culture consist- ing of a single strain of a certain species are conse- quently rather limited as it can be easily attacked by its specific bacteriophages.
Another factor which can inhibit the development of bacteriophages is the administration of preparations for oral bacterium therapy for quite a limited period of time in order to reduce the possibility of the development of bacteriophages.
When preparations for oral bacterium-therapy are ad- ministered for very prolonged periods of time to a large number of individuals, as is the case for example in hos- pitals, the risk of phagic infection is extremely high.
The problem can be solved by substituting the strains used with other strains definitely resistant to those specific bacteriophages.
Bacteriophages are an extremely important biological reality, even more widely diffused than micro-organisms themselves. Cases of diarrhea not due to the action of pathogenous germs can be correlated to the phagic attack of some normal constituents of intestinal flora. It should be pointed out that phages are harmless for human beings even though they may interfere with the intestinal flora.

The continuous administration of bacteria of the same strain, with a probiotic action, leads to the devel- opment of specific bacteriophages which destroy said bac- terial strain, thus annulling its probiotic prophylaxis.


A knowledge of bacteriophages can be of help in the preparation and use of cultures adopted in oral bacte- rium-therapy.
In order to obtain a effective protection with re- spect to intestinal disturbances, the problem of bacte- riophages has been considered and solved with the compo- sition according to the present invention.
An object of the present invention therefore also relates to a composition which comprises different strains of the same species having a different sensitiv- ity to bacteriophages (lysogeny and lysotypy) but with the same biological and probiotic properties.
The composition according to the present invention can also comprise at least one of the following compo- nents: other micro-organisms, enzymes, mineral salts, vi- tamins, prebiotics, natural fibres, phyto-derivatives, antioxidants, fermented milk, paps, feeds.
The live and vital and/or devitalized yeasts of the compositions, object of the present invention, are yeasts with a low fermentative capacity for probiotic use, rich in essential amino acids. In particular, the yeast can be

Saccharomyces cerevisiae or Saccharomyces boulardii.


In the composition according to the present inven- tion, at least one of the pr micro-organisms is prefera- bly present in a concentration lower than 109 ufc/g.
In particular, a dietetic and/or pharmaceutical com- position according to the present invention comprises Streptococcus thermophilus, Bifidobacteria such as Bifi- dobacterium longum, Bifidobacterium breve, Bifidobacte- rium infantis, Bifidobacterium lactis, Bifidobacterium bifidum, Lactobacillus acidophilus in a concentration ranging from 109 to 1011 ufc/g, Lactobacillus plantarum, Lactobacillus casei subsp. casei, Lactobacillus del- brueckii subsp. bulgaricus, Enterococcus faecium in a concentration ranging from 106 to 109 ufc/g.
In particular, S. thermophilus and L. delbrueckii subsp. bulgaricus are developed in symbiosis (or proto- cooperation).
The natural enzymes used essentially consist of a mixture made up of ss-glucanase and xylanase produced by micro-organisms of the Thricoderma type.
The mixture comprising these natural enzymes is par- ticularly useful for optimizing the digestive catalase of hydrosoluble polysaccharides (NSP). This mixture develops a synergic action on the various NSP contained in wheat, barley, oats, rye and triticum, and is defined by its

specific efficacy on the various substrates, the analyti- cal characteristics of the single components, the product properties studied for commercial use in association with a pool of pr micro-organisms in human and animal nutri- tion.


The natural fibres, possibly present in the composi- tion according to the present invention, are selected from fibres of acacia, oats, apples, inulin, psyllium, microcrystalline cellulose (which act as prebiotics).
The composition according to the present invention comprising pr micro-organisms and prebiotics, such as natural fibres, is of particular interest.
The fibres represent a nutritional substrate for the pr micro-organisms. The metabolic activity of pr micro- organisms on the fibres (prebiotic) makes them easier to digest and causes the free release of substances useful for the nutrition of the organism (human or animal), of the pr micro-organisms and autochthonous intestinal flora.
The phyto-derivatives are preferably selected from extracts from Eleuterococcus and green tea.
The antioxidants are preferably natural antioxidants and can be selected from oleuropein (from extravirgin ol- ive oil), lycopene (from tomatoes), bioflavonoids (from citrus fruit), phenol components (from red grapes).

The vitamins and mineral salts are selected from vi- tamin A, B1, B2, B6, B12, niacin, C, D3, E, folic acid, calcium, phosphorous, magnesium, iron, zinc.


A further object of the present invention therefore relates to the use of said dietetic and/or pharmaceutical compositions as integrators and/or dietetic-therapeutic products for human and/or animal nutrition.
In particular, an object of the present invention also relates to the use of said dietetic and/or pharma- ceutical compositions for preparing integrators, die- tetic-therapeutic food products, food, drinks and/or feeds for human and/or animal nutrition.
The food can consist of milk, cheese, paps, homoge- nized products (based on meat, milk, cheese, fruit, vege- tables), dietetic food products destined for diabetics such as jams, chocolate, sweeteners other than sucrose, or animal feeds.
The milk can be fermented or non-fermented milk, with the direct inoculum of suitable pr micro-organisms in a concentrated dried form.
The pr micro-organisms in a concentrated dried form inoculated into milk are suitable for the processing of 1000 or more liters of milk without any intermediate pas- sage with the presence on the finished product of the probiotics identified.

Therapeutic dietetic cheese can be obtained by the addition of suitable pr micro-organisms in a concentrated dried form in a certain processing phase of the cheese in order to guarantee, in a certain gram-weight of cheese, the supply of the dose of pr micro-organisms necessary for the organism.


The drinks can be instantaneous drinks or water con- taining the compositions according to the present inven- tion.
The present patent application also relates to inte- grators, food, dietetic-therapeutic food products, drinks and/or feeds for human and/or animal nutrition, charac- terized in that they contain a dietetic and/or pharmaceu- tical composition according to the present invention.
The use of the compositions according to the present invention for human nutrition has an equilibrating action on the intestinal flora of the host (human being and ani- mal), in addition to producing various benefi- cial/probiotic effects towards the organism which depends on the destination target (children, adults, elderly peo- ple, expectant women, people with various kinds of defi- ciencies or gastro-intestinal disturbances i. e. dismicro- bisms, or with acute or chronic diseases, for example of the vaginal or urological type). This use also regulates intestinal functioning, improves constipation, hemor-

rhoids and intestinal irritation; it reduces the absorp- tion of sugar and cholesterol.


The use of the compositions according to the present invention in zootechnics allows growth promotion, the control of enteric pathologies of a bacterial origin, an improvement in the digestive efficiency (I. C. A. ) of ani- mals for breeding, the food conversion index and an im- provement in the quality of the end-product (meat or eggs, in the case of fowl), thus solving problems linked to antibiotic residues.
In addition to this, the use of the compositions ac- cording to the present invention results in a better con- sistency of feces, a reduction in enteric pathologies of a bacterial origin (colibacillosis), an improvement in the consistency of the egg-shells produced, an improve- ment in the egg-laying itself, a reduction in therapeutic treatment and an improved immunitary response.
In particular, as far as human nutrition is con- cerned, the compositions according to the present inven- tion, containing suitable live and vital and/or devital- ized pr micro-organisms in concentrated dried form, can be used as a dietetic and/or therapeutic product in pow- der form as tablets, capsules or sachets both for pharma- ceutical use and as food integrators, optionally combined with natural principles.

Pr micro-organisms can, in fact, interact with nu- tritional principles of a natural origin such as fibres of acacia, oats, apples, Psyllium-which already have health-giving properties and whose reduced food contribu- tion can be correlated with various pathologies, such as constipation, obeseness, cardiovascular diseases, diabe- tes.


These substances, also classified as prebiotics, in- teract with pr micro-organisms (such as lactic bacteria and bifidobacteria) as they act as a substrate on which the pr micro-organisms themselves develop thus creating a synergic effect. The symbiotic combination between pr mi- cro-organisms and prebiotic fibres, such as inulin, fa- vours intestinal functioning, improves constipation, hem- orrhoids and intestinal irritation; it reduces the ab- sorption of sugar and cholesterol.
Another extremely interesting combination is that between pr micro-organisms and antioxidants of a natural origin, such as: bioflavonoids, anthocyanins, lycopene, orthophenols, extracts from citrus fruit, red grapes, tomatoes, olive oil, respectively. These antioxidants are not only effective in fighting AOR (Active Oxygen Radi- cals), but they are also capable of protecting the integ- rity and functionality of microflora on an intestinal level, guaranteeing a greater beneficial effect on human

beings.
Pr micro-organisms can also be"coupled"with vita- mins and minerals, especially in cases where a lack of these is likely. Deficiencies in specific trace elements are now associated with chronic health problems: typical examples are fluorine and dental decay, chromium and tol- erance to glucose, copper and hypercholesterolemia, zinc and the immunitary system.


The association of vitamins and minerals with pr mi- cro-organisms improves the absorption, i. e. bio- availability, of nutritional principles, a factor which is often neglected in evaluating the composition of inte- grators and/or the necessity for their use. A second im- portant aspect is linked to the action synergy, for exam- ple between zinc and pr micro-organisms which are both implied in the modulation of the immunitary function.
This synergy also develops between various species of vegetable substances, phyto-derivatives (extracts from Eleuterococcus and green tea), known for their tonic, adaptogenous, antistress, immuno-potentializing activity, but at the same time a hypocholesterolemizing, antioxi- dant activity, for controlling glycemia, modulating the ecosanoid cascade, with safe health effects.
The combination with pr micro-organisms favours the effect of phyto-estrogens as they increase the bio-

availability and absorption of these vegetable princi- ples, in addition to normalizing the equilibrium of in- testinal flora altered in situations of stress.


Pr micro-organisms in association can also be used for enriching dietetic food products destined for chil- dren and adults, for example in homogenized products (based on meat, milk, cheese, fruit, vegetables, etc. ) or in products destined for diabetics (jams, chocolate, sweeteners other than sucrose), etc.
The pr micro-organisms can be mixed with the above food preparations, with fermented milk and with food products in general, in different proportions.
The addition to food of pr micro-organisms in con- centrated dried form is justified in that these products have equilibrating properties of intestinal flora and stimulate the immunological properties and natural de- fence system of the organism in addition to those against tumours.
In animal nutrition, on the other hand, the composi- tions according to the present invention, containing live and vital and/or devitalized pr micro-organisms, in a dried concentrated form, can be used for the following purposes.
The biological processes linked to microbial life present with respect to the enteron are extremely impor-

tant for animals, as they can influence both the diges- tive processes and also the absorption processes of the nutritional principles. In this respect, two main types of micro-organisms can be distinguished on an intestinal level: fermentation micro-organisms which produce, start- ing from glucosides, various short-chain fatty acids and putrefaction micro-organisms which degrade the amino ac- ids producing biogenic amines. Under normal physiological conditions, the putrefaction processes are controlled by fermentative processes, whereas when there is an enteric pathology, bacterial strains with a high putrefactive ac- tion, above all Escherichia coli, are predominant.


In this particular context, resort is made to the use of selected cultures of pr micro-organisms which form the typical and most effective antagonist of putrefactive flora. The antagonist action is in fact carried out by means of a"barrier effect", exerted with adhesion to the intestinal epithelium, and an acidifying activity which makes the enteric environment unsuitable for the develop- ment of pathogenous flora. Suitable specifically combined pr micro-organisms allow the development of a beneficial microflora to be activated on an intestinal level. This is an innovative technology which is extremely effective and completely without side-effects, for controlling en- teritis of a bacterial origin and for improving the di-

gestion of breeding animals, the food conversion index and problems linked to residues in meat or eggs (in the case of fowl). In particular, the following selection criteria are adopted for selecting the strains which form these cultures: adhesion capacity to the epithelium of the enteron; -resistance to gastric acidity ; development and production rate of lactic acid at the level of the enteron; - processing of the enzymes useful for food degrada- tion.


100,000, 000 quintals of feed form the basic datum for evaluating the potential market of selected cultures of pr micro-organisms used as growth promoters and as ac- tive principles for the prophylaxis of enteric diseases.
In both cases, the advantages in terms of quality im- provement (in particular of meat, reduction in the use of antibiotics and consequently the elimination of residues) which products based on pr micro-organisms offer, are im- portant.
In addition to a greater control of enteric patholo- gies of a bacterial origin and an improvement in the di- gestive efficiency (I. C. A. ), the application of lactic bacteria shows: - an improved consistency of feces ;

- a reduction in enteric pathologies of a bacterial origin (colibacillosis); - an improvement in the consistency of the egg-shells produced; an improvement in the laying; a reduction in therapeutic treatment; an improvement in the immunitary response.


In particular, technology development in animal nu- trition is increasingly emphasizing the problem of the use of antibiotics for auxinic purposes in feeds.
The pros and cons, advantages and disadvantages, use and abuse of these molecules have often been the object of discussion between scientists, which frequently in- volves the public. In England, the"Swam Institute"pub- lished a report in 1969 in which the use of antibiotics for zootechnical productions was connected to the risk of allergic or toxic reactions, and to the danger of the formation of antibiotic-resistant microbial strains, with cases of a wide diffusion of resistance to said antibiot- ics from animals to human beings. When antibiotics are absorbed by animals, in fact, they can be found in the form of residues in meat.
There is also the problem relating to possible in- tolerance on the part of those working in feed producers and of zootechnical operators, who can come into contact

with the various active principles.


The identification and use of natural auxinic fac- tors which do not produce negative effects such as those mentioned above, is therefore of great interest.
The use of gastrointestinal flora"modulators", such as yeasts, lactic bacteria and bifidobacteria, is becom- ing more and more well known in the preparation of food destined for animal nutrition of the concentrated type or in the form of fodder.
Limitations in the use of these products derive from the current legislations in force which only allow the use of some species of pr micro-organisms (lactic bacte- ria and yeasts), at very low concentrations, which often jeopardize the efficacy in zootechnical applications.
Detailed studies in the selection and preparation of new species of pr micro-organisms suitable for use in various zootechnical species, are consequently extremely important.
Another differentiation to be taken into considera- tion as a selection criterion of these products is the different action mechanism according to the animal spe- cies, distinguishing between monogastric species and those with a polygastric digestive system.
The formulations of the composition according to the present invention are polyvalent and therefore play an

important role in the preventive treatment of gastric and intestinal diseases.


These preparations contain not only pathogen agent antagonists, but also compounds which produce biologi- cally active substances for regulating the metabolic pro- cesses in animals and raising their resistance to infec- tions.
Said compositions consist of a mixture of pr micro- organisms (for example, lactic bacteria, propionibacteria and yeasts) in a concentrated and dried form.
Lactic bacteria are in fact antagonists of many pathogenous varieties and are vitamin producers, raising the resistance of the organism to illnesses. Propionibac- teria (which form part of the intestinal microflora of ruminants) produce propionic acid (important for the regular growth of calves), acetic acid (essential for the synthesis of milk fat, during lactation) and B12 vita- mins.
For yeasts, biomasses have been selected and pro- duced, which due to their biochemical characteristics, increase their probiotic properties and are functional for zootechnical use, also identifying the specificity for the different animal species and various productions.
This solves the present situation whereby yeasts destined for zootechnical use consist, in the best of cases, of

surpluses of yeast production for bread-making, with the selection and production of yeasts which therefore have: - a high adaptability under the conditions present in the digestive system of animals ; - a high content of particular cellular elements (amino acids, vitamins, etc.).


Yeasts favour a series of important biochemical reactions in the rumen, which improve the fermentative activity, allowing the contemporaneous presence, in milk cows, of the three volatile fatty acids precursors of milk compo- nents (fat, casein and lactose), in the highest possible concentrations.
In the zootechnical field, in particular as far as cattle breeding is concerned, it has been demonstrated that autochthonous microflora affects the animal's health as it can participate in the digestion and metabolism of the nutritive substances, supplying energy, amino acids and sugar which could otherwise not be available to the host.
An appropriate equilibrium between the autochthonous micro-organisms normally present in the intestines or ru- men, ensures, among other things, the mutual beneficial relationship between host and microflora and consequently the health of the animal.
As a result, the treatment of animals with the poly-

valent compositions according to the present invention, consisting of a mixture of pr micro-organisms (for exam- ple lactic bacteria, propionibacteria and yeasts), cre- ates not only an antagonistic action against pathogenous agents, but also the production of biologically active substances which regulate the metabolic processes of ani- mals and raise their resistance to infections.


To actuate similar treatment in intensive breeding it is necessary however to have cultures which are highly concentrated in cells and are dried, to facilitate trans- portation and use.
In conclusion, the use of the compositions according to the present invention containing pr micro-organisms in a concentrated and dried form to be used as food integra- tors for cattle leads to: - an improvement in the state of health of animals with a consequent increase in meat yield; - a reduction in the use of antibiotics in feeding breed- ing cattle, with indirect effects for consumption due to a greater hygienic reliability of the meat; - positive ecological consequences on the waste water disposal of farms together with the advantages already mentioned and deriving from the concentrated and dried form (multiple and simple use and easy transportation).
According to research effected on fowl species

(hens, broilers, turkeys) it has been found that the ad- ministration of specific cultures of pr micro-organisms, is extremely effective from the very first day of the animal's life.


The development of a totally beneficial intestinal flora and consequently the start of the productive cycle under optimal functioning conditions of the digestive system, has, in fact, been observed.
The use of pr micro-organisms in animal breeding is extremely practical as it can be effected both via the food (feed) and also via the drinking water, care being taken not to subject the pr micro-organisms to tempera- tures higher than 65-70 C, so as not to jeopardize their vitality.
The pr micro-organisms selected for the compositions object of the present patent application have different nutritional demands and require the application of a wide variety of fermentative parameters (growth temperatures, pH conditions, fermentation times, drying curves, etc.) for which a general production scheme is provided below (Scheme 1)

SCHEME 1-Process diagram


Pure strains trom Raw materials strain library (Warehouse) (Microbiology laboratory) weigning ot meaium ingredients

Vegetative phase (Preparation department)

Preparation of pre-inoculum (Microbiology laboratory)

Preparation, dissolution and pasteurization or sterilization of the cultural medium (Production dept.) s L v

Primary fermentation Final fermentation

Incubation of pre-inoculum Incubation of inoculum

200/400 liters 5, 000/10, 000 liters (Production dept.) Production dept.)

Concentration by centntugation or utira-nttration or micro-filtration, etc..
(Production dept.) 1

Homogenization with cryo-protective products

Raw materials (Production dept.) l (Warehouse)

IF mxng-------unng (Production dept.) (Spraying, freeze drying, zeolite, etc..) (Production dept.)

IF

Microbial culture in dried concentrated form


For illustrative purposes only, the manufacturing process is described below of one of the components of the mixtures of pr micro-organisms such as the species Streptococcus thermophilus.
The manufacturing process comprises a vegetative phase in a flask, starting from collection strains (in a laboratory), a production phase including primary and fi- nal fermentation and the actual production phase (concen- tration, homogenization, drying).
Vegetative phase Preparation of the pre-inoculum

Different strains of the same species can be used for the manufacturing of the species S. thermophilus.


For the preparation of the pre-inoculum (vegetative phase) the contents of the various phials containing the collection strains (1 per strain) are dissolved sepa- rately with 1.5 ml of a solution of peptone and sterile salt. The suspensions contained in the phials are mixed and inoculated in equal quantities into 10 ml test-tubes of sterile skimmed milk or dried milk re-formed at 10% in water. They are incubated at 37 C until the milk coagu- lates or until the logarithmic growth phase of the cul- ture thus obtained (first culture).
The first culture is transferred to a 250 ml flask of sterile skimmed milk. It is incubated at 42 C until

coagulation (second culture) and, if necessary, it can be conserved at 5 C for 3 days.


The contents of the second culture are transferred (in a ratio of 2% = 20 ml/1) into a flask containing 4 1 (or 8 1 in relation to the expected fermentation volume) of a substrate of re-formed sterile skimmed milk in a ra- tio of 10% and containing 1% of yeast extract and incu- bated at 37 C for about 2 hours (until coagulation). The flask is then left to cool in a refrigerator (4 C) until use. The phase contrast microscopic morphological con- trol, the growth and acidification curve control and the control of the contaminating germs and acidity developed are then contemporaneously effected on the third culture (laboratory preinoculum). ist production phase Primary fermentation

This phase is carried out in a 200/400 litre fermen- tor using 200 1 (or 400 1 in relation to the expected fermentation volume) of dried skimmed milk re-formed at 8% (containing 0. 1% of Antifoam additive), as culture me- dium. The components of the medium were previously weighed and dissolved, using an automatic plant. Pas- teurization of the culture medium is then effected, under slow stirring, at 90 C for 30 minutes. The medium is cooled to 42-44 C and is sterilely inoculated with the


contents of the flask (preinoculum, 4 or 81) of the vege- tative phase. The fermentation is continued for 2-3 hours, after which rapid cooling is initiated to 4-8 C with icy water.


The culture is maintained at 5 C until the end of the quality control.
Final fermentation Preparation of the culture medium

Culture medium 5000 1 (or 10000 1).


A 5000 1 fermentor is used (in the case of 10000 1 the quantities are doubled), previously washed and flushed with a CIP cycle. 4400 liters of water are charged, and the fermentor is heated to 60 C.
The following products are weighed: -Lactose 80 Kg -Yeast extract 25 Kg -Dextrose 150 Kg - Ammonium sulfate 15 Kg - Monobasic potassium phosphate 7.5 Kg - Manganese sulfate 0.5 Kg - Antifoam additive 0.5 Kg

The components of the medium are dissolved using an automatic plant. Pasteurization is then effected, under slow stirring, at 80 C for 30 minutes.


Inoculation and incubation
The culture medium mass is cooled to 40-42 C ; it is sterilely inoculated with 200 liters of the primary fer- mentation culture and is thermostat-regulated ; it is kept under stirring and incubated for 3-4 hours approximately, controlling the pH which is continuously adjusted with NaOH at 30% to maintain a value of 6.1-6. 2. The regula- tion is effected continuously, under stirring.
Various samplings are taken for the different con- trols and rapid cooling to 5 C with icy water, is initi- ated. The operations are carried out so as to complete the fermentation within the same day, enabling the micro- biological purity control (e. g. absence of coliforms) to be effected before the operations of the following day.
2nd production phase Concentration of the cellular mass (e. g. by centrifuga- tion) Preparation of the centrifuges

The Westphalia centrifuges (or ALFA LAVAL) are washed and flushed using a CIP plant with NaOH and nitric acid, washed with hot water and pre-cooled (hollow cav- ity) with icy water to 5 C.


The culture to be concentrated is directly trans- ferred, by means of a lobe pump, to the centrifuges, us- ing a closed circuit plant, flushed in CIP, maintaining a flow-rate of 1000 1/hour (2500 1/hour for ALFA LAVAL).

The first three discharges of concentrate are eliminated and the whole of the remaining quantity is directly transferred, still in line, to the homogenizer.


Homogenization of the concentrate with cryoprotective products

The concentrate collected is added with a cryopro- tective product (in a ratio of 16% with respect to the weight of the concentrate collected) and homogenized. The cryoprotective product is thus composed: (dose per 300 Kg of concentrate) : - Dried skimmed milk 4.0 Kg - F. U. Lactose 12.8 Kg - Sucrose 4.0 Kg - Yeast extract 3. 2 Kg - Water 24.0 1 (the cryoprotective product is previously prepared and pasteurized at 80 C), the pH is corrected to 6.3-6. 5 with NaOH at 30%, samplings are taken for the controls and it is transferred to a vacuum freeze drier.


At the end of the operations, the centrifuge is im- mediately flushed with nitric acid-soda in CIP, and is rinsed, first with rinsing water and then with hot water.
The homogenizer is immediately flushed with nitric acid- soda and water at 90 C.
Drying (e. g. by vacuum freeze drying)
All the vacuum freeze drying accessories must be flushed, before use, in compliance with the specific pro- cedures whereas the chambers and fixed parts are decon- taminated with vapour and/or a solution of sodium hypo- chlorite or another suitable decontaminant.
The concentrate is directly distributed by means of a pump to the vacuum freeze drying basins, stratifying about 1.5 cm per basin (also in relation to the actual quantity of material obtained). The vacuum freeze drying process is activated according to the automatic program of the lyostat; the vacuum freeze drying is considered as being terminated (normally after about 36-40 hours) when all the temperature registration curves of the product are stable for at least 8 hours and the chamber isolation test does not reveal vacuum drops higher than 10% (less than 100 microbar).
The basins are extracted from the lyostat, the prod- uct is immediately discharged and the lyophilized product is collected in double polyethylene bags. The product is ginned on an oscillating blade granulator previously washed and equipped with an inox grid of 2.77 x 1 mm and collected in double polyethylene bags or in steel drums.
Each drum or bag is sampled for control and immediately placed in a quarantine cell. At the end of the drying process, the yield % to dried product obtained is calcu-

lated, with respect to the damp concentrate.


Final mixing (standardization and stabilization)

The product destined for the use listed above can consist of the mixture of several pr micro-organisms in a predefined cellular ratio. This predefinition obviously cannot be actuated in a hypothetical fermentation in a mixed culture but can only be effected from an appropri- ate mixing of the single species which takes into account the single cellular numerical charges of the single com- ponents.


Through said mixing, (in addition to the necessary "titre"standardization according to specifications) the result of a stabilization of the cellular numerical charge is reached, together with its ratios between the various species. For this purpose, suitable inert dilu- ents are used (such as F. U. Lactose or, alternatively and for use in lactose-free specialties, maize starch, Si02, Mg Stearate).
The mixing is effected using a 750/1000 1 biconic mixer, previously washed and flushed with a suitable de- contaminating agent, by means of a validated mixing cy- cle. The diluent can be added in two or three successive aliquots, in relation to the relative quantities.
At the end of the mixing, the product is discharged into double polyethylene bags, and immediately sampled

for specific quality controls; the bags are immediately thermo-sealed and placed, in adequate packaging, in the quarantine area of the refrigerator at +2/+8 C"Awaiting Analysis".


Some typical schemes are provided below for the preparation of mixtures standardized and stabilized with a predetermined titre. As they are raw materials to be dosed at a minimum vital cellular charge, (UFC-Unit Forming Colonies), the quantities naturally vary slightly from lot to lot, whereas the diluent must, necessarily, be calculated each time according to the a. n. (as neces- sary) scheme.
Scheme a) (total microbial charge not less than 300 bil- lion cells/g) 200 Kg Lot

Species (Minimum) UFC Quantity S. thermophilus 4. 6x10l6 Bifidobacteria 2. 1x1016 L. acidophilus 4. 4x1014 L. delbrueckii subsp. bulgaricus 0. 68xlO" L. plantarum 0. 50x1014 L. casei 0. 50x1014 E. faecium 0. 068x1014 (minimum 50 g) F. U. Lactose a. n. 200 Kg Scheme b) (total microbial charge not less than 100 bil-


lion cells/g) 200 Kg Lot

Species (Minimum) UFC Quantity S. thermophilus 1. 54x10l6 Bifidobacteria 0. 7x1016 L. acidophilus 1. 47x1014 L. delbrueckii subsp. bulgaricus 0. 234x1014 L. planta-rum 0. 174x1014 L. casei 0. 174x10" E. faecium 0. 023x10l4 (minimum 50 g) F. U. Lactose a. n. 200 Kg Scheme c) (total microbial charge not less than 70 bil- lion cells/g-Lactose free) 200 Kg Lot Species (Minimum) UFC Quantity S. thermophilus 1. 07x1016 Bifidobacteria 4. 9x1015 L. acidophilus 1. 03x10l4 L. delbrueckii subsp. bulgaricus 0. 16x1014 L. plantarum 0.11x1014 L. casei 0. 11x10" E. faecium 0. 016x1014 (minimum 50 g) SiO2 2.0 Kg Mg Stearate 1.0 Kg F. U. Lactose a. n. 200 Kg
The quality control of the formulation after mixing is effected as described below.
Control of the differential microbial charge in the mixture.
Peptone-salt diluent: 1 g of peptone and 8.5 g of NaCl are dissolved in 1000 ml of water; the mixture is stirred carefully, heated and, if necessary, the pH is adjusted so as to obtain, after sterilization in an auto- clave, a value of 7.0 at 25 C.
Method: 10 g approximately, carefully weighed, of dried concentrated culture are diluted to 100 ml (dilu- tion 1: 10) with the diluent solution.
The mixture is stirred carefully and homogenized in a Stomacher homogenizer for 1.5 minutes and the cells are left to revitalize in a thermostat at 37 C for 20 min- utes. 10 ml of the first dilution (10-1) are transferred to an empty sterile bottle, diluted with 90 ml of diluent (10-2) and stirred carefully. The decimal dilutions are repeated in succession to 10-9 ; the dilutions thus ob- tained will be used in triplicate form.
Preparation of the plates for the counting of the pr micro-organisms: Three replicates per dilution to be ex- amined are distributed on an adequate number of 90 mm Petri plates, together with 14 ml of HHD commercial broth, containing 20 g/1 of agar and 1 g/1 of Tween 80.

The mixture is left to cool and dried under a horizontal flow hood. 0.1 ml of the pre-selected dilution is planted at the centre of each plate and is distributed by palet- ting/rotation with a 70-75 mm glass spatula until the complete absorption of the planting. The plates are incu- bated in anaerobiosis with a Gas-Pack system for 72 hours at 37 C

The counting is effected (referring to the different dilutions) by distinguishing the various species on the basis of the different morphologies, colouring of the colony and by means of stereoscopic microscopic control.
When there are doubts, a part of the colony in question is removed with a needle and a microscopic preparation is transferred to a sample slide with a drop of sterile wa- ter and is covered with a covering slide. It is observed in phase contrast with a 40x lens and a lOx eyepiece (for Bifidobacteria a 100x immersion lens is used).
Examples of compositions based on probiotic microbial preparations for human and/or animal use.
The examples are provided for illustrative purposes only and in no way limit the scope of the present inven- tion.
EXAMPLE 1

Homogenized products destined for children

1 g of live and vital L. bulgaricus and S. thermo-

philus (in concentrations of 1 to 5x109 ufc/g), in symbi- otic association, in a dried concentrated form are mixed with 50-100 grams of homogenized product.


EXAMPLE 2

Food integrators based on live and vital lactic bac- teria and bifidobacteria.


These integrators contain the following live and vi- tal pr micro-organisms: St. thermophilus # 27% equal to 540x106 ufc/g Bifidobacteria # 20% 400x106 ufc/g Lc. lactis, Lc. cremoris, Leuconostoc sp ~5. 5 % equal to 110x106 ufc/g L. casei ~ 8% 100X106 ufc/g L. helveticus-5. 5% 110x106 ufc/g L. plantarum ~ 37% 740X106 ufc/g

Total 100% Total 2000x106 ufc/g and also contain, according to the use and for illustra- tive purposes, the following compounds: A) natural fibres (fibres of acacia, oats, apples, inu- lin, psyllium, microcrystalline cellulose) for regulating the intestines and weight control.


The integrator thus obtained is particularly suit- able for hypocaloric diets, constipation, hemorrhoidal disturbances, for the prevention of obesity, varicose veins, intestinal irritation;

B) natural antioxidants for free radical defence, and anti-aging effect.


The antioxidants comprise oleuropein (from extravir- gin olive oil), lycopene (from tomatoes), bioflavonoids (from citrus fruits), phenol components (from red grapes). The integrator thus obtained is suitable for people subject to oxidative stress deriving from unbal- anced nutrition, a disorderly life-style, cigarette smoke and pollution, it is also suitable for the prevention of degenerative and cardiovascular diseases and against de- terioration of the cellular functions, including aging; C) vitamins and mineral salts for the re-equilibrium of the intestinal flora with a supply of nutritional sub- stances.
The nutritional substances comprise vitamins A, Bl, B2, B6, B12, niacin, C, D3, E, folic acid, calcium, phos- phorous, magnesium, iron, zinc.
This combination is suitable for people following hypocaloric diets and do-it-yourself dieters, those who carry out moderate or intense sporting activities, eld- erly people and inappetent children, adolescents in the development phase, expectant women and during breast- feeding, smokers, after the administration of antibiotics and as a preventive treatment for osteoporosis; D) vitamin C, Siberian ginseng (Eleuterococcus), ginger

and green tea for increasing resistance to stress, and controlling intestinal problems correlated therewith. The integrator thus obtained is suitable for people who travel widely, live in situations of any kind of stress (family, work, studies and life-stile), managers and for improving physical efficiency and protection against in- fective diseases.


EXAMPLE 3

Fermented milk prepared with a mixture of lactic bacteria and bifidobacteria.


A 20-30 g sachet of dried concentrated product is thus composed: S. thermophilus + L. delbrueckii substp. bulgaricus grown in symbiosis 50x109 ufc/g L. acidophilus 10x109 ufc/g L. casei 50x109 ufc/g L. plantarum 10x109 ufc/g Bifidobacteria 50x109 ufc/g

This composition is inoculated directly into 1000 liters of milk previously pasteurized and cooled to a temperature of 44 C. It is left to incubate at this tem- perature for the time necessary for the development of the micro-organisms inoculated (7-8 h) and at the end of the process, a fermented milk is obtained with high pro- biotic qualities, having a pH of 4.1-4. 3 and containing


per ml: 1000-2000 x 106 ufc S. thermophilus 100-200 x 106 ufc L. delbrueckii subsp. bulgaricus 10-100 x 106 ufc L. acidophilus 10-100 x 106 ufc L. casei 10-100 x 106 ufc L. plantarum 50-100 x 106 ufc bifidobacteria.


EXAMPLE 4

Food integrators for broilers based on lactic bacte- ria.


These integrators contain the following lactic bac- teria: L. acidophilus 1Ox109 ufc/g L. plantarum 50x109 ufc/g S. thermophilus/L. bulgaricus 2. 5x109 ufc/g L. salivarius 1x109 ufc/g Excipient: lactose

100 g of product are administered to drinking water and this quantity of product is sufficient for 10,000 subjects.


EXAMPLE 5

Feed for broilers based on lactic bacteria as in Ex- ample 4 with the addition of exogenous enzymes.


1 g of exogenous enzymes/kg of feed is used.
This mixture allows the hydrolysis of specific glu-

cosides both of the non-starchy type and also those par- tially indigestible. It develops a synergic effect with the enzymes in the catabolism of polysaccharides of wheat, barley, oats, rye and triticum and contemporane- ously integrates the intestinal flora of the animals.


The administration to broilers of both preparations allows an improvement in the retention of nitrogen, growth and ICA; a reduction in cholesterol in the blood and cecum coliforms.
EXAMPLE 6

Food integrator for hens based on lactic bacteria and bifidobacteria.


This integrator contains the following combination of pr micro-organisms ; L. acidophilus 10x109 ufc/g L. plantarum 50x109 ufc/g L. casei 5x109 ufc/g S. thermophilus/L. bulgaricus 2. 5x109 ufc/g L. salivarius 1x109 ufc/g Bifidobacterium bifidum 50x109 ufc/g Excipient: lactose

100 g of product are administered to drinking water and this quantity of product is sufficient for 10,000 subjects.


EXAMPLE 7
Feed for hens based on lactic bacteria and bifido- bacteria as in Example 3 with the addition of exogenous enzymes.
1 g of exogenous enzymes/kg of feed is used.
Unlike what is specified in Example 5, this mixture contains pr micro-organisms conditioned to the catabolism of polysaccharide monomers and oligomers, in particular arabinose and xylose, particularly studied for developing a synergic effect with the exogenous enzymes in the com- plete catabolism of polysaccharides of wheat, barley, oats, rye and triticum.
The administration of the preparations of Example 6 and 7 to hens stimulates the animal's appetite and allows an improvement in the phytasic activity in the digestive tract, in the P retention of N and Ca, the ICA laying rate, the egg/hen mass, the specific weight of the eggs and thickness of the shells; a lowering of the pH in the goiter and intestines and cholesterol in the yoke.
EXAMPLE 8

Preparation of an antibiotic-free integrated feed as a substitution of milk destined for calves.


- lactic bacteria : 0.5 % L. plantarum 2x109 ufc/g E. faecium 2x109 ufc/g S. thermophilus/L. bulgaricus 10x109 ufc/g

Excipient: lactose - dried whey 85 % - coconut oil 8 % - fat 4 % - cereal flour 2 % - mineral and vitamin integrator 0.5 %

The substitutive integrated feed has a pH equal to 5.0 after re-formation in water at 10%.
EXAMPLE 9

Complementary feed for milk cows/meat cattle based on a mixture of pr micro-organisms.


The complementary feed comprises the following pr micro-organisms: L. plantarum 10x109 ufc/g S. thermophilus/L. bulgaricus 50x109 ufc/g Propionibacteria 10x109 ufc/g Saccharomyces cerevisiae 10x109 ufc/g L. casei 10x109 ufc/g L. helveticus 10x10 9 ufc/g.
Excipients: cereals and their by-products to obtain a charge of 2x109 ufc/g.
30-100 grams/head/day are administered.. The prepara- tion increases the ingestion of dry substances, it con- tributes to reducing. post-birth hygiene problems, exerts a detoxifying and hepato-protective action ;- it increases

the production of milk with a high fat and protein titre; it stimulates rumenal fermentation and favours fibre di- gestion.


The concentration of live yeasts, total anaerobic bacteria and celluloselytic bacteria in rumenal liquid (ufc loglO/ml) gave the following results:

Control Preparation (15g/head/day) - Yeasts 5.40 6.87 - Total anaerobic bacteria 8.98 10. 35 - Celluloselytic bacteria 7.28 8.82 EXAMPLE 10

Food integrator for piglets with a mixture of lactic bacteria.
The following ingredients are introduced and mixed in a double-chamber (or V-shaped) mixer having the appro- priate capacity: - Live and vital lactic bacteria in a dried concentrated form (100x109 ufc/g)

L. acidophilus 10x109 ufc/g

L. plantarum 50x109 ufc/g

S. thermophilus/L. bulgaricus 30x109 ufc/g

E. faecium 10x109 ufc/g - A carrier consisting of lactose or whey in powder or any other form, in a quantity sufficient for obtaining a

microbial charge of 2 billion cells per gram of integra- tor product.


The product thus obtained is introduced, in the de- sired quantities, into a horizontal mixer and mixed with the products forming the various feeds in the following proportions: - dried skimmed milk 10% - dried whey 5% - soybean flour 10% - maize flour 20% - fish meal 5% barley flour 5% - oat flour 2. 5% - barley flakes 20% - oat flakes 5% - bran 15% - mineral salts 2% - vitamin mixture 1% - integrators with lactic bacteria 0. 5%Claims:

WHAT WE CLAIM IS 1. Dietetic and/or pharmaceutical compositions for hu- man and/or animal use based on microbial cultures con- sisting of autochthonous and allochthonous species, with respect to human beings and animals, selected from the lactic bacterial species Lactobacillus acidophilus, Lac- tobacillus plantarum, Lactobacillus casei subsp. casei, Lactobacillus casei subsp. rhamnosus, Lactobacillus zeae, Lactobacillus salivarius, Lactobacillus lactis, Lactoba- cillus helveticus, Lactobacillus reuteri, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus curva- tus, Lactobacillus delbrueckii subsp. delbrueckii, Lacto- bacillus delbrueckii and all its subspecies, Lactobacil- lus gasseri, Lactobacillus johnsonii, Streptococcus ther- mophilus, Lactobacillus delbrueckii subsp. bulgaricus, optionally associated with Streptococcus thermophilus ; Lactobacillus fermentum, Lactobacillus brevis, Lactococ- cus lactis subsp. lactis, Lactococcus lactis subsp. cre- moris and Leuconostoc spp.; Entercoccus faecium, Pedio- coccus pentosaceus, Pediococcus acidilactici ; Bifidobac- teria such as Bifidobacterium Longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis ; and/or propionibacterial species, yeast species and/or mold species; the above species be- ing live and vital and/or devitalized, and said species


being present in microbial cultures in a dried concen- trated form with a concentration ranging from 106 ufc/g to lOll ufc/g.


2. The compositions according to claim 1, characterized in that they comprise different strains of the same spe- cies with a different sensitivity to bacteriophages (ly- sogeny and lysotypy) and with the same biological and probiotic properties.
3. The compositions according to claim 1, characterized in that they also comprise at lest one of the following components: other micro-organisms, enzymes, mineral salts, vitamins, prebiotics, natural fibres, phyto- derivatives, antioxidants, fermented milk, paps, feeds.
4. The compositions according to claim 1, characterized in that they comprise prebiotics, such as natural fibres.
5. The compositions according to claim 1, characterized in that at least one of the pr micro-organisms is present in a concentration lower than 109 ufc/g.
6. The compositions according to claim 1, characterized in that they comprise Streptococcus thermophilus, Bifido- bacteria such as Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infants, Bifidobacterium lactis, Bifidobacterium bifidum, Lactobacillus acidophilus in a concentration ranging from 109 to 1011 ufc/g, Lactobacil- lus plantarum,. Lactobacillus casei subsp. casei, Lactoba-

cillus delbrueckii subsp. bulgaricus, Enterococcus fae- cium in a concentration ranging from 106 to 109 ufc/g.


7. The compositions according to claim 1, characterized in that the live and vital and/or devitalized yeasts are yeasts with a low fermentative capacity for probiotic use, rich in essential amino acids.
8. The compositions according to claim 7, characterized in that the yeast is Saccharomyces cerevisiae or Saccha- romyces boulardii.
9. The compositions according to claim 3, characterized in that the natural enzymes consist of a mixture made up of ss-glucanase and xylanase produced by micro-organisms of the Thricoderma type.
10. The compositions according to claim 3, characterized in that the natural fibres are selected from fibres of acacia, oats, apples, inulin, psyllium, microcrystalline cellulose.
11. The compositions according to claim 3, characterized in that the antioxidants are natural antioxidants.
12. The compositions according to claim 11, character- ized in that the natural antioxidants are selected from oleuropein (from extravirgin olive oil), lycopene (from tomatoes), bioflavonoids (from citrus fruits), phenol components (from red grapes).
13. The compositions according to claim 3, characterized

in that the vitamins and mineral salts are selected from vitamin A, B1, B2, B6, B12, niacin, C, D3, E, folic acid, calcium, phosphorous, magnesium, iron, zinc.


14. The compositions according to claim 3, characterized in that the phyto-derivatives are selected from those ex- tracted from Eleuterococcus and green tea.
15. The compositions according to claim 1, characterized by the presence of S. thermophilus and L. delbrueckii subsp. bulgaricus developed in symbiosis (or proto- cooperation).
16. The use of the compositions according to any of the claims from 1 to 15, as integrators, foodstuffs and/or dietetic-therapeutic products for human and/or animal nu- trition.
17. The use of the compositions according to any of the claims from 1 to 15, for preparing integrators, food- stuffs and/or dietetic-therapeutic products, drinks and/or feeds for human and/or animal nutrition.
18. The use according to claim 17, wherein the food products are milk, cheese, paps, homogenized products (based on meat, milk, cheese, fruit, vegetables), die- tetic food products destined for diabetics such as jams, chocolate, sweeteners other than sucrose, or animal feeds.
19. The use according to claim 17, wherein the milk is a

fermented or non-fermented milk, with the direct inoculum of pr micro-organisms in a dried concentrated form.


20. The use according to claim 17, wherein the cheese is a therapeutic dietetic cheese obtained by the addition of pr micro-organisms in a dried concentrate form in a cer- tain processing phase of the cheese..
21. The use according to claim 17, wherein the drinks can be instantaneous drinks or water containing the com- positions according to any of the claims from 1 to 15.
22. Integrators, foodstuffs, and/or dietetic-therapeutic products, drinks and/or feeds for human and/or animal nu- trition characterized in that they contain a composition according to any of the claims from 1 to 15.
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