CN1692158A - Use of lactobacillus to produce exopolysaccharides in food and pharmaceutical compositions - Google Patents

Abstract

The present invention relates to a composition and a method of preparing or treating food products so as to improve their aroma, flavour, mildness, sweetness, consistency, texture, body, mouth feel, firmness, viscosity, gel fracture, wheying off, structure and/or organoleptic properties, nutrition and/or health benefits. In particular, the present invention provides a composition comprising a live micro-organism, an enzyme produced by said micro-organism and an exopolysaccharide (EPS) produced by the activity of said enzyme.

Description

Bacterium lacticum prepares the purposes of the exo polysaccharides in edible composition and the pharmaceutical composition

Technical field

The present invention relates to a kind of composition.

Foreword

At present, some useful microbial polysaccharides commercial be to prepare by fermentation process.These polysaccharide have special application in every field in for example foods/feeds industry, agrochemistry, Oil extraction, medical science and pharmacology, fine chemistry and makeup, isolation technique and the polymeric chemical.Useful microbial polymer is biodegradable, nontoxic, and is to generate from renewable resource by fermentation.Many microorganisms (bacterium, fungi, yeast, microalgae) in cell, extracellular (exo polysaccharides) or as the composition of its okioplast wall and generate this polymkeric substance.

Many lactic-acid-bacteriums (LAB) can produce exo polysaccharides (exopolysaccharide) (EPS) (Cerning, 1990, FEMS Microbiol.Rev.87:113-472).As used in this, term EPS comprises one type the exo polysaccharides or the exo polysaccharides of two or more types.

Sometimes, produce the LAB of EPS in foods prods such as grape wine (Llauberes etc., 1990, Carbohydr.Res.203:103-107) and hard cider (Whiting, 1975, the Bacterium lacticum in the drink and food, Academic Press, London, 69-86) the unusual haircuts of middle generation.But as a rule, the polysaccharide that LAB discharges in the extracellular brings benefit for the foods prods of various fermentations.In fact, lactic-acid-bacterium plays crucial effects in the processing of the milk preparation of fermentation such as yogourt, yogurt drink, cheese, fermenting cream, milk dessert etc.

From quality, chemical constitution, molecular weight, electric charge, have unprotected side chain and molecular rigidity aspect to consider, the multiple EPS that generated by Different L AB are in a large number arranged.EPS can be used as texture improving agent and stablizer, at first improves the viscosity and the elasticity of end product, secondly by the combination water Heshui and with other the composition of milk, for example protein and micelle interact and strengthen the rigidity of casein reticulation.As a result, EPS reduces synerisis effect (syneresis) (serum separation) and improves the stability of product.

The microflora of large intestine comprises hundreds of different bacterial species.Colony and metabolic activity thereof that this is complicated play an important role to the host.Although between the bacterium and and the host between ecological effects also lack complete understanding, testing data shows that some bacterium is useful to the host, and other bacteriums are deleterious owing to produce toxin and genotoxicity metabolite or invade mucous membrane.A kind of positive acting is the so-called probiotics of picked-up in the method for enteric microorganism system composition and metabolic activity thereof.Probiotics is defined as indigestible food ingredient, it produces beneficial effect by growth and/or the activity that optionally stimulates a kind of or a large amount of beneficial bacterias in the enteron aisle to the host, improve healthy state (Gibson and Roberfroid thereby promote, 1995, J.Nutr.125:1401-1412).It is generally acknowledged the bacterium of improving healthy state potentially be lactobacillus (Lactobacillus) and genus bifidobacterium (Orrhage and Nord, 2000, DrugsExp.Clin.Res.26:95-111).

Be reported that EPS may influence intestinal health.Can infer,, may increase the residence time of fermented milk in gi tract that is digested if improve the viscosity of the food that contains EPS, thereby instantaneous the settling down that little ecology is regulated bacterium is useful (German etc., 1999, Trends in Biotechnology, 17,492-499).By butterfat lactobacillus lactis butterfat subspecies (Lactobacillus lactis subsp cremoris) B40 and picogram Bacterium lacticum 0-1 (Lactobacillus sakei 0-1), the EPS that thermophilus streptococcus (S.thermophilus) SFi 20 generates is proved to be able to resist the Degradation (Ruijssenaars etc. of fecal microorganism, 2000, Current Microbiology, 40,194-199).

EPS is proved to be has antitumor, reducing cholesterol and immunoregulatory effect (Ode etc., 1983, Agricultural and Biological Chemistry, 47:1623-1625; Honoso etc., 1997, Bioscience.Biotechnology and Biochemistry, 61:312-316; Vincent etc., 2001, Gycobiology, 11:131-139).

EPS is proposed and is used as food ingredient.As an example, can be with reference to WO 94/12656, wherein described the method for preparing EPS from picogram Bacterium lacticum class bacterial strain, in the time of in being added to foods prods, described EPS has shearing-dilution (shear-thinning) characteristic and/or thickening and/or emulsification-stabilising characteristic.This method comprises with the bacterial isolates inoculation culture liquid of producing EPS; Under the conditions suitable that forms described EPS, cultivate described bacterium, and separate described EPS, EPS can be used as a kind of composition and is added in the foods prods then, and wherein said EPS brings shearing-dilution characteristic, thickening and/or emulsification-stabilising characteristic for the foods prods that contains described EPS.

US6,004,800 have described a kind of new butterfat Leuconostoc mesenteroides kind (Leuconostoc mesenteroides ssp.cremoris) that can produce Sucrose:glucan alpha1 (glucan sucrase), this kind of enzyme can be the polysaccharide dextran with sucrose inversion, then the polysaccharide dextran is used for setting up texture and improving taste to milk preparation.

WO 01/57234 describes a kind of natural butterfat lactobacillus lactis kind Ropy 352 isolates, and it contains the plasmid of 13 kinds of active genes of a kind of coding.Enzyme by these genes encodings makes this bacterium can produce charged exo polysaccharides (EPS 352), contain glucose, the 42%-46% semi-lactosi of 54%-58%, and molecular weight is 800,000 to 8,000, between 000.EPS 352 is added in milk or other liquid, in milk, produce the organoleptic feature of expectation, comprise making the milk retrogradation to have good and soft mouthfeel, have trickle sweet taste during " chewing " (" chewable-bite ").Should also be mentioned that and Ropy 352 or the independent EPS 352 that produces EPS can be added in any milks or the non-milk series products, comprise any liquid foodstuff, produce specific organoleptic feature.

Rawson ﹠amp; Marshall was at report in 1997, and haircuts bacterial strain (ropy strain) lactobacillus delbruckii Bulgaria's kind (Lactobacillus delbrueckii ssp.Bulgaricus) and lactobacillus bulgaricus (Lb.bulgaricus) can improve " viscosity " of the yoghourt production of stirring.The effect of secreted EPS though " viscosity " is proved to be, " flintiness " of the yoghourt production that is stirred and " elasticity " are done mutually and influence (the Rawson ﹠amp of the proteic matrix of yogourt by protein-protein; Marshall, 1997, Int.Journal of Food Science and Technology, 32,213-220).

Reports such as Broadbent, the thermophilus streptococcus lactic-acid-bacterium of the production EPS of involucrum rather than haircuts bacterial strain can be used to improve the moisture content of Mo Zeleile (Mozzarella) cheese and improve it and dissolve characteristic, and can not destroy whey viscosity (Broadbent etc., 2001, Int.Dairy, 11,433-439).

Reports such as Hess, the yogourt of planting the bacterial strain preparation with the lactobacillus delbruckii Bulgaria that produces exo polysaccharides has the low shear thinning of product for preparing than the bacterial strain by nonproductive EPS, this and shear-induced are coincide by the mechanism of the structural degradation of the yogourt of the bacterial strain preparation of producing EPS, the polymkeric substance relevant with the casein reticulated structure prevents the destroyed (Hess etc. of this reticulated structure part in this mechanism, 1997, J Dairy Sci, 80,252-263).

EP 0325872 has described the method for a kind of few dextran of preparation (oligodextran), be characterised in that when having the glucanotransferase that extraction obtains from the NRRL B-1299 bacterial strain of lactic-acid-bacterium Leuconostoc mesenteroides (Leuconostoc mesenteroides), sucrose and the saccharide acceptor and the glucose that are selected from the glucose of maltose, isomaltose, Isomaltotriose, methyl α-glucoside are in contact with one another.From the Leuconostoc mesenteroides culture of living, separate these oligomerization dextran then, and as the weighting material of sugar substitute or as food additive, human or animal's enteric microorganism system is had beneficial effect.

WO 01/90319 describe a kind of that produce by Lactobacillus rogosae (Lactobacillus reuteri), having can the catalysis oligosaccharides or the new albumen of the fructosyltransferaseactivity activity that generates of polysaccharide.The method that is used for producing described enzyme is included in nutrient solution and cultivates described Bacterium lacticum, and reclaim described enzyme or cell-free extract from described nutrient solution, and use contains the described Bacterium lacticum of the described enzyme that can produce oligosaccharides or polysaccharide as probiotics (probitic) or composite biological agent (sybiotic).

Similarly, described among the WO 01/90372 a kind of by new albumen Lactobacillus rogosae production, that have glucosyl transferase activity.The method that is used for producing described enzyme is included in nutrient solution and cultivates described Bacterium lacticum, and reclaim described enzyme or cell-free extract from described nutrient solution, and use contains the described Bacterium lacticum of the described enzyme that can produce oligosaccharides or polysaccharide as probiotics or composite biological agent.

EP 0881283 describes a kind of bacterial strain of new butterfat Leuconostoc mesenteroides kind, and it can generate a kind of enzyme (dextran-sucrase), and this endonuclease capable polymerization sucrose also forms dextran.This application has also been described and has been used for preparing the used method for compositions that comprises described enzyme and described dextran of foods and cosmetics industry.

Main aspect (Broad Aspect)

Aspect main, the present invention relates to a kind of composition that is used to eat.Said composition can be used as a kind of product that is used to eat or as the composition of the product that is used to eat.Especially, the present invention relates to a kind of composition, it comprises a kind of lactic-acid-bacterium (LAB) of work, by described lactic-acid-bacterium synthetic enzyme and the EPS that produced by described enzyme.Therefore, the present invention relates to the method for a kind of composition and a kind of preparation or processing food, with fragrance, local flavor, lightly seasoned (mildness), sugariness, denseness, texture, quality (body), mouthfeel, hardness, viscosity, gel crackle (gel fracture), removal whey (wheying off), the structure (structure) of improving food and/or characteristic, nutrition and/or the wholesome feature that influences sense organ.Especially, the invention provides a kind ofly comprise great-hearted microorganism, by the composition of the exo polysaccharides (EPS) of the enzyme of this microorganism preparation and the activity production by described enzyme.

So far, also nobody propose to use described composition as foods prods or as a kind of composition, the composition of for example a large amount of different foods prodss.

Other aspects of the present invention subsidiary claims and below description in illustrate.These aspects are introduced under the title of each chapters and sections.But, should be appreciated that the instruction that is produced by each chapters and sections is not to be limited in inevitably under the title of particular chapter.

Detailed aspect

An aspect the invention provides a kind of composition that is used to eat, wherein said composition comprise a kind of lactic-acid-bacterium (LAB) of work, by described lactic-acid-bacterium synthetic enzyme and the EPS that produces by described enzyme.

In yet another aspect, form the EPS product in position by cultivating lactic microorganisms and suitable enzyme substrates.Suitable enzyme substrates can be any glycan molecule, for example monose, disaccharides, trisaccharide or tetrose.As non-limitative example, monose comprises as sugar such as glucose, fructose or semi-lactosis, and disaccharides comprises as sugar such as maltose, lactose or sucrose.Other glycan molecules that can be used as suitable enzyme substrates comprise the galactoside glycan molecule, for example raffinose, stachyose (stacchyose) or verbascose.

Also have another aspect, a kind of composition is provided, wherein enzyme is glycosyltransferase (transglycosylase), for example fructosyl transferase or can polymerization sucrose and/or the Sucrose:glucan alpha1 of lactose and/or stachyose, raffinose or verbascose.

The present invention also provides the method for the product that a kind of preparation is used to eat, and for example a kind of food or food ingredient, this method comprise mixes the described product that is used to eat (consumption) with generation with composition of the present invention with another kind of composition.

The present invention also provides a kind of method that is used to prepare medicament production or pharmaceutical cpd, and this method comprises mixes composition of the present invention with another kind of composition.

In yet another aspect, the purposes of the composition of the EPS that the present invention relates to comprise lactic microorganisms, produces from the enzyme that can produce EPS of described lactic microorganisms with by described enzyme, with the fragrance that improves described foods prods of the present invention, local flavor, lightly seasoned, sugariness, denseness, texture, quality, mouthfeel, hardness, viscosity, gel crackle, remove whey, structure and/or influence the characteristic of sense organ, nutrition and/or wholesome feature, wherein said EPS is by the microorganisms producing of described work.

Also have another aspect, the present invention relates to the purposes according to composition of the present invention, wherein during the composition of product that is used to eat when conduct or medicament production, described lactic-acid-bacterium and EPS are as probiotics.

The invention still further relates to a kind of in-situ preparation method for compositions, comprise and in nutrient solution (being preferably edible nutrient solution), cultivate described lactic-acid-bacterium under certain condition, under this condition, generate enzyme and produce EPS or common EPS up to the appropriate amount that produces 2g-50g/l, perhaps up to generating the appropriate amount enzyme, the quantity of described Bacterium lacticum is generally every milliliter about 10 ³To about 10 ¹¹, be preferably about 10 ⁵To about 10 ¹¹Be more preferably about 10 ⁸To about 10 ¹¹Proper amt.

Another aspect, the invention provides a kind of screening and be suitable for method for compositions of the present invention, described method comprises the candidate set compound contacted with foods prods, and determines the improvement degree aspect texture, quality, mouthfeel, viscosity, structure and/or the organoleptics property at described foods prods; Wherein said composition comprises microorganism alive and by the enzyme of described microorganisms producing and the EPS that produced by described enzyme.

The composition that also has an aspect to relate to use a kind of EPS that forms by homopolysaccharide and different poly-polysaccharide molecule that comprises lactic microorganisms alive, produces of the present invention by described microorganism synthetic enzyme and by described enzyme.

Preferred aspect

One preferred aspect, be used for composition of the present invention and be used with spissated form.

Preferably, be used for composition of the present invention and be spray-dired, refrigerated, cryodesiccated and/or by resuspended or be concentrated, for example by membrane filtration or evaporation step.

Preferably, the microorganism that is used for work of the present invention is a lactic microorganisms, and it can be selected from lactic acid Coccus (Lactococcus), streptococcus (Streptococcus), Mycosphaerella (Pediococcus), enterococcus spp (Enterococcus), leuconostoc (Leuconostoc), Kano Pseudomonas (Carnobacterium), propionibacterium (Propionibacterium), genus bifidobacterium and lactobacillus.

One preferred aspect, the microorganism that is used for work of the present invention is from leuconostoc.

One preferred aspect, the microorganism that is used for work of the present invention is a Leuconostoc mesenteroides.

One preferred aspect, the Leuconostoc mesenteroides of living can generate a kind of enzyme, wherein said enzyme is a transglycosylase.

Another preferred aspect, transglycosylase is fructosyl transferase or glycosyltransferase.

One preferred aspect, endonuclease capable catalysis sucrose and/or lactose that Leuconostoc mesenteroides generates are EPS.

One preferred aspect, endonuclease capable catalysis raffinose, stachyose or verbascose that Leuconostoc mesenteroides generates are EPS.

One preferred aspect, the EPS of the enzyme production that is generated by described Leuconostoc mesenteroides comprises at least one long chain polymer (polysaccharide) and/or short chain polymer (oligosaccharides).

One preferred aspect, the polysaccharide of EPS or oligosaccharides composition comprise Polylevulosan and/or dextran.

One preferred aspect, the microorganism that is used for work of the present invention is from lactobacillus.

One preferred aspect, the microorganism that is used for work of the present invention is picogram lactobacillus species (Lactobacillus sakei spp.), plant lactobacillus kind (Lactobacilius plantarum spp.) or lactobacillus salivarius kind (Lactobacillus salivarius spp.).

One preferred aspect, the microorganism that is used for work of the present invention is from lactobacillus.

One preferred aspect, picogram lactobacillus species, plant lactobacillus or the lactobacillus salivarius kind of living can generate a kind of enzyme, wherein said enzyme is a Sucrose:glucan alpha1.

One preferred aspect, the endonuclease capable catalysis lactose that picogram lactobacillus species, plant lactobacillus or lactobacillus salivarius kind generate is EPS.

One preferred aspect, the endonuclease capable catalysis lactose, raffinose, stachyose or the verbascose that are generated by picogram lactobacillus species, plant lactobacillus or lactobacillus salivarius kind are EPS.

One preferred aspect, the EPS that is generated by picogram lactobacillus species, plant lactobacillus or lactobacillus salivarius kind is homotype EPS (homo-ESP).

One preferred aspect, the homotype EPS that is generated by described picogram lactobacillus species, plant lactobacillus or lactobacillus salivarius kind comprises at least one long chain polymer (polysaccharide) and/or short chain polymer (oligosaccharides).

Preferably, the invention provides a kind of composition that is used for fermented milk or its any other equivalent, wherein added yeast extract and magnesium ion, be used for producing and contain the EPS that forms structure (strutureforming) and/or the product that is used to eat of trophicity oligosaccharides and/or polysaccharide.

Preferably, the invention provides a kind of composition, it can be used to fermented milk or any other equivalent and production and contain the EPS that forms structure and/or the yogourt constituents of trophicity oligosaccharides and/or polysaccharide.

Preferably, the invention provides the composition that can be used as food ingredient, wherein the microorganism of the work of said composition, EPS and/or homotype EPS, oligosaccharides and/or polysaccharide component have beneficial effect for human consumer's gastrointestinal microorganisms system.

Another preferred aspect, the invention provides a kind of composition that can be used as food ingredient, wherein the EPS of said composition and/or homotype EPS and/or oligosaccharides composition and/or polysaccharide component have immunoregulation effect for human consumer's stomach and intestine immunity system.

Another preferred aspect, the invention provides a kind of composition that can be used as food ingredient, wherein when being used as the composition of the product that is used to eat or medicament production the EPS of said composition and/or homotype EPS and/or oligosaccharides and/or polysaccharide component as probiotics.

Another preferred aspect, the invention provides a kind of composition that can be used as food ingredient, wherein when being used as the composition of the product that is used to eat or medicament production, the EPS of said composition and/or homotype EPS and/or oligosaccharides and/or polysaccharide component can reduce the gas that is generated by the enteric microorganism system.That is to say that composition of the present invention can be used to reduce, limit or avoid gas to assemble, thereby reduces flatulence in enteron aisle.

Preferably, be used for the form that composition of the present invention is an enriched material, it comprises the very microorganism of the work of high density, and/or by the enzyme of described microorganisms producing and/or the EPS that produces by described enzyme.

Another preferred aspect, the invention provides a kind of composition that in nutrient solution, prepares, this nutrient solution has the pH scope that is conditioned, the degree that makes sucrose and/or lactose be converted into EPS can be conditioned.

Advantage

Some advantage of the present invention is introduced in following explanation.

The major advantage of composition of the present invention is the characteristic that it can improve the fragrance of the product that is used to eat that contains described composition, local flavor, lightly seasoned, sugariness, denseness, texture, quality, mouthfeel, hardness, viscosity, gel crackle, remove whey, structure and/or influence sense organ, nutrition.

Another advantage is that the microorganism of the work of composition of the present invention can produce and can utilize different sugar to produce the enzyme of EPS as substrate, and these sugar are as sucrose, lactose, raffinose, stachyose or verbascose.

Advantageously,, use composition of the present invention may be able to regulate the viscosity of whey concn and the product that is used to eat because lactose is present in whey and the milk natively, and the sugariness of this product of balance.That is to say,, may adopt the sugariness of the product that composition described here is used to eat by adjusting to generate product with suitable viscosity, retention ability and mouthfeel according to specific application.By reducing the lactose-content in the product, might obtain a kind ofly to be fit to not anti-lactose or to those products that people of lactose allergy is eaten.

Advantageously, composition of the present invention can be used to produce the complete product based on milk preparation that is used to eat.In other words, the product that is used to eat that comprises composition of the present invention can only be used milk preparation production, and does not add for example extra sucrose.As an example, the milk preparation that does not add sucrose and produce is good for human consumer's oral hygiene.

Another advantage of composition of the present invention is that lactic microorganisms can be converted into homopolysaccharide with lactose.Homopolysaccharide described herein has the height retention ability, and the viscosity that can be used to regulate the product that is used to eat.

For some application, composition of the present invention can cause the remarkable reduction of synerisis (serum separation), and improves the stability of product.

It is generally acknowledged that edible pulse family cereal for example bean or pea or soybean can cause producing gas in gi tract, causes discomfort.Advantageously, the invention provides a kind of composition, it can play a role and for example reduce by gastrointestinal microorganisms, reduces or suppress the generation of gas.

Composition of the present invention can also make up with other compositions of the product that is used to eat and realize described improvement.

The present invention comprises that also adopting composition of the present invention to combine to the human consumer as pharmaceutical cpd and other compositions brings medical science or physiological benefit.

The composition that a kind of (viable) microorganism that comprises the work that can produce a kind of enzyme and EPS be provided is wherein expressed and/or the polymerization level is conditioned also in protection scope of the present invention.Can by change microbial numbers alive, by changing leavening temperature and/or environment pH and/or expression and/or polymerization level by using acceptor molecule such as maltose to regulate the EPS in the composition.

Therefore, according to specific application, the present invention also provides a kind of composition that comprises microorganism alive, enzyme and EPS, and the expression of EPS and/or polymerization level have been conditioned to be fit to application-specific.

Microorganism

The microorganism that is used for suitable work of the present invention comprises bacterium, mould and/or yeast.

Term " microorganism that lives " is meant a kind of microorganism of g and D normally.It should also be understood that, unless point out especially in addition in the context, at this specification sheets, comprise that the microorganism of work mentioned in subsidiary claims or biocide are meant microorganism or one or more biocides that comprises that one or more are lived, or its mixture.

Microorganism can be a natural microbial or can be by microorganism transformed.Microorganism can also be the combination of multiple suitable microorganism.

One preferred aspect, the microorganism of work of the present invention can be selected from and comprise for example group that constitutes of the various bacterial strains of lactic acid Coccus, streptococcus, Mycosphaerella, enterococcus spp, leuconostoc, Kano Pseudomonas, propionibacterium, genus bifidobacterium and lactobacillus of lactic-acid-bacterium.

One preferred aspect, the microorganism that lives is the Leuconostoc mesenteroides kind.

Another preferred aspect, the microorganism that lives is picogram lactobacillus species, plant lactobacillus or lactobacillus salivarius kind.

The invention provides the lactic microorganisms of the work of production significant quantity and/or high-load glycosyltransferase (transglycosylase).Glycosyltransferase described here (transglycosylase) can be Transglucosylase or fructosyl transferase.

Advantageously, when this product is food, the microorganism that lives and/or in normal " sell-by date (sell-by) " or " keeping life " that provide this food to be used to sell by the dealer, should remain effective by the enzyme of described microorganisms producing and/or by the EPS of described enzyme production.Preferably, should prolong working lipe and surpass this time limit, up to begin to occur rotten normal quality warranty period end of addling when food.The time span of expectation will change along with different food with the normal storage life, one skilled in the art will recognize that the storage life will change along with kind, food volume, storage-temp, processing conditions, wrapping material and the packaging facilities of food.

EPS

EPS can be any suitable EPS, and it can obtain from lactic-acid-bacterium.

The EPS component of composition can comprise homopolysaccharide molecule and/or different poly-polysaccharide molecule.

As used in this, term " homopolysaccharide " or " homotype-EPS " are meant the polysaccharide molecule of the monose molecule that only contains a type, and wherein this monose molecule can be glucose or fructose.

As used in this, term " different poly-polysaccharide " is meant by two kinds of polysaccharide molecules that constitute in multiple monose, disaccharides, trisaccharide or the tetrose unit.As the example of indefiniteness, monose comprises as sugar such as glucose, fructose or semi-lactosis, and disaccharides comprises as sugar such as maltose, lactose or sucrose.

The content of EPS, and then the viscosity of nutrient solution can be conditioned.When term " adjusting " is used for EPS and/or viscosity, be meant that the polymerization level of various substrates can be conditioned.That is to say, can be raised or reduce.Therefore, the EPS of production amount can be conditioned, for example by changing the microbial numbers of living, the length of fermenting process, the maltose of the different content that exists in temperature and the nutrient solution.Preferably, the amount of regulating EPS by the pH that changes fermentation culture.The pH scope that EPS output raises preferably from pH5.7 to pH6.7, is more preferably at pH5.9 to pH6.5 in the scope from pH5.5 to pH6.9.The pH that the EPS amount reduces is in from 4.0 to 5.4 or from 7.0 to 8.0 scope.

Nutrient solution

The present invention also provides the nutrient solution of lactic microorganisms of the work of the enzyme that can catalysis generates EPS that comprises production significant quantity and/or increasing amount, improve the product that is used to eat fragrance, local flavor, lightly seasoned, sugariness, denseness, texture, quality, mouthfeel, hardness, viscosity, gel crackle, remove whey, structure and/or influence characteristic, nutrition and/or the wholesome feature of sense organ.

The invention provides the nutrient solution that comprises lactic microorganisms alive, this microorganism comprises significant quantity and/or high-load glycosyltransferase (transglycosylase) or fructosyl transferase.

In addition, the invention provides comprise generate significant quantity and/or high-load can polymerization sucrose and be translated into the nutrient solution of lactic microorganisms of the work of the glycosyltransferase (transglycosylase) of EPS or fructosyl transferase, EPS comprises at least one long-chain polysaccharide or a short chain oligosaccharides, and EPS can be used as the food component with optimized nutrition and health advantages.

Also have, the invention provides comprise generate significant quantity and/or high-load can the polymerization lactose and be translated into the nutrient solution of lactic microorganisms of the work of the glycosyltransferase (transglycosylase) of EPS or fructosyl transferase.The EPS that generates comprises at least one long-chain polysaccharide or a short chain oligosaccharides, and EPS can be used as the food ingredient with optimized nutrition and health advantages.Also be used to medical use with lactic acid as the EPS that enzyme substrates generates, for example as blood substitute.

In addition, the invention provides comprise generate significant quantity and/or high-load can polymeric maltose and be translated into the nutrient solution of lactic microorganisms of the work of the glycosyltransferase (transglycosylase) of EPS or fructosyl transferase.The EPS that generates comprises at least one long-chain polysaccharide or a short chain oligosaccharides, and EPS can be used as the food component with optimized nutrition and health advantages.

In addition, the invention provides the nutrient solution of the Leuconostoc mesenteroides that comprises the work that generates significant quantity and/or high-load glycosyltransferase (transglycosylase) that raffinose, stachyose or verbascose can be polymerized to EPS or fructosyl transferase.The EPS that generates comprises at least one long-chain polysaccharide or a short chain oligosaccharides, and EPS can be used as the food ingredient with optimized nutrition and health advantages.The invention provides the nutrient solution that comprises Leuconostoc mesenteroides alive, it generates significant quantity and/or the high-load glycosyltransferase (transglycosylase) or the fructosyl transferase that raffinose, stachyose or verbascose can be polymerized to EPS, the composition that also can be used as the product that is used to eat can reduce the gas generation and/or reduce flatulence.

In addition, the invention provides comprise can generate significant quantity and/or high-load can polymerization sucrose and be translated into the nutrient solution of great-hearted (viable) picogram lactobacillus species, plant lactobacillus kind or lactobacillus salivarius kind of the Sucrose:glucan alpha1 of EPS.The EPS that generates comprises at least one long-chain polysaccharide or a short chain oligosaccharides, and EPS can be used as the food ingredient with optimized nutrition and health advantages.Adopt sucrose as substrate, the EPS that generates by picogram lactobacillus species, plant lactobacillus or lactobacillus salivarius kind comprises the monomeric homotype exo polysaccharides of glucose molecule.

In addition, the invention provides comprise can generate significant quantity and/or high-load can the polymerization lactose and be translated into the nutrient solution of picogram lactobacillus species, plant lactobacillus or lactobacillus salivarius kind of work of the Sucrose:glucan alpha1 of EPS.The EPS that generates comprises at least one long-chain polysaccharide or a short chain oligosaccharides, and EPS can be used as the food ingredient with optimized nutrition and health advantages.Adopt lactose can also be used for medical use, as blood substitute as the EPS that substrate generates.

In addition, the invention provides comprise can generate significant quantity and/or high-load can the polymerization raffinose, stachyose or verbascose and they are polymerized to the nutrient solution of picogram lactobacillus species, plant lactobacillus kind or lactobacillus salivarius kind of work of the Sucrose:glucan alpha1 of EPS.The EPS that generates comprises at least one long-chain polysaccharide or a short chain oligosaccharides, and EPS can be used as the food ingredient with optimized nutrition and health advantages.Comprise the work that can generate significant quantity and/or high-load Sucrose:glucan alpha1 picogram lactobacillus species, plant lactobacillus kind or lactobacillus salivarius kind composition and can be used as the composition of the product that is used to eat by the EPS that raffinose, stachyose or verbascose generate as substrate, this product can reduce gas and produce and/or reduce flatulence.The product that is used for eating that contains the EPS that is generated by raffinose, stachyose or verbascose can be used to increase sanatory beneficial bacteria of gi tract.That is to say that composition of the present invention plays probiotics in gi tract.In addition, known flatulence is because multiple factor causes, for example abnormal fermentation or irregular fermentation pattern, this wherein can by as the probiotics of composition of the present invention be used for elimination.

When preparation composition of the present invention, can also adjust the ratio of sucrose/maltose, lactose/maltose or stachyose/maltose in the nutrient solution during the fermentation, so that obtain the polysaccharide of expectation or the molecular weight distribution of oligosaccharides.The viscosity that the ability of the molecular weight of control polysaccharide or oligosaccharides can also be used to regulate the product that comprises composition of the present invention.

As used in this, term " polysaccharide " is meant the carbohydrate molecule of being made up of the sugared unit more than 10 kinds, and wherein sugared unit can be monose, disaccharides, trisaccharide or tetrose.

In addition, be meant the carbohydrate molecule of being made up of the sugared unit below 10 kinds at this used term " oligosaccharides ", wherein sugared unit can be one or more monose, disaccharides, trisaccharide or tetrose.

Preferably, polysaccharide and/or oligosaccharides are made up of monose and/or disaccharides.As an example, monose includes, but are not limited to for example sugar of glucose and fructose etc., and disaccharides includes, but are not limited to for example sugar such as sucrose, lactose and maltose.

Therefore, in one aspect of the invention, this nutrient solution selectively adds any monose, disaccharides, trisaccharide or tetrose.At this, have one or more EPS by the composition of the lactic microorganisms production of living, wherein some (preferred most of) EPS is the homopolysaccharide molecule at least.

In yet another aspect, the nutrient solution that is used for method of the present invention selectively adds two or more monose, disaccharides, trisaccharide or tetrose.At this, have one or more EPS by the composition of the lactic microorganisms production of living, wherein some (preferred most of) EPS is different poly-polysaccharide molecule at least.

In yet another aspect, the nutrient solution that is used for method of the present invention selectively adds yeast extract and magnesium ion (Mg ²⁺), assist the growth of lactic microorganisms.

One preferred aspect, this nutrient solution is edible nutrient solution.Term " edible nutrient solution " is meant the grown cultures liquid of any support growth of lactobacillus, and the human consumer can buy and obtain, for example from animal, and the milk or the emulsion in plant or synthetic source as describing among the embodiment.In addition, according to specific purposes, said composition provides the purposes as edible nutrient solution, does not add any extra monose, disaccharides, trisaccharide or tetrose.Therefore, in one embodiment, the work of composition of the present invention the lactic microorganisms component produce can the polymerization lactose enzyme, lactose is present in milk preparation for example in milk or the whey natively.The lactic microorganisms component of the work in the composition of the present invention can production polymerization lactose ability make and can produce the product that is used to eat that this product has is low, significantly reduced lactose-content or be substantially free of lactose.It is edible that this product that is used to eat is suitable for not tolerating the human consumer of lactose or lactose allergy.The lactic microorganisms of living can the polymerization lactose ability make the milk preparation that can prepare lactose free.The product of this lactose free will reduce the deleterious effect to tooth.

When preparation composition of the present invention, can also regulate the pH of nutrient solution during the fermentation, to obtain the EPS of target content.The viscosity that the ability of control EPS content can be used to regulate the product that comprises composition of the present invention.

Therefore, in one aspect of the invention, can adjust the pH of nutrient solution, make and to control sucrose or lactose is converted into the EPS degree.At this, the pH that can adjust composition to be generating a certain amount of EPS, and then regulates the viscosity of nutrient solution.In addition, different pH condition may cause the viscosity change of composition by the equilibrium level that raises or reduce the EPS works.

Preparation of compositions

Can prepare the type of composition-referred in this by in-situ method.In this case, suitable microorganism or active glycosyltransferase (transglycosylase) are present in the edible nutrient solution, support that sucrose or lactose or other substrate conversion are EPS.The composition of processing produced in situ then provides the composition that is suitable for different application.As an example, composition can be frozen drying, spraying drying or any other freezing and storing method.Purpose is that the different methods of processing compositions can keep the vigor of lactic microorganisms.It is contemplated that, exist EPS may improve the survival ratio of the lactic microorganisms in the composition.Therefore, in process of cryopreservation, EPS can protect lactic microorganisms.

Selectively, the composition of composition can separately prepare, and each composition is combined formed composition then.As an example, can in commercial qualified nutrient solution, cultivate Bacterium lacticum and come in-situ preparing EPS by under the condition that generates EPS.The EPS that generates can separate from fermented liquid by any suitable method, and for example by with an organic solvent precipitating, EPS is insoluble in this organic solvent, perhaps has limited solubleness.Another kind of method of separating EPS is to take to remove moisture, for example by evaporation, membrane filtration or spraying drying.By producing EPS in commercial qualified nutrient solution, the product that is used to eat that contains described EPS does not need to be labeled as and contains additive.Used commercial qualified nutrient solution is meant and anyly can be used as a kind of nutrient solution of producing composition of the present invention.

In one embodiment of the invention, yeast extract and magnesium ion have been added in the commercial qualified nutrient solution.Be added to yeast extract in the nutrient solution in 0.01% to 1% scope of the cumulative volume of nutrient solution.Preferably, add yeast extract in the nutrient solution in 0.025% to 0.9% scope, be more preferably between 0.1% to 0.8%, even be more preferably between 0.25% and 0.7%.

In addition, composition of the present invention can be synthesized, and for example by recombinant microorganism, this microorganism also needs not to be lactic microorganisms.

Term " recombinant microorganism " is meant a kind of microorganism of recombinant nucleotide sequence that coding can be produced the enzyme of EPS of carrying, and this enzyme and this EPS can be used as the component of composition of the present invention.

And the lactic microorganisms composition of composition of the present invention can be separated to cultivate in commercial qualified nutrient solution, reaches the cell density that does not produce EPS or enzyme (producing the enzyme of described EPS).Importantly, keep the vigor of the culture of generation to realize, for example by spraying drying or lyophilize by the different methods that this area is known.

Method of the present invention can be used to the produced in situ composition, be included under the condition that generates enzyme and formation EPS, in commercial qualified nutrient solution, cultivate described lactic-acid-bacterium, this nutrient solution such as milky liquid nutrient solution, selectively added extra carbon source, for example disaccharides sucrose and/or maltose, they also are suitable enzyme substratess.During the fermentation, the ratio of disaccharides can be changed or adjust, with the molecular weight distribution of the polymkeric substance of realizing expectation.Therefore,, might regulate polymerization process artificially, and then adjust viscosity by changing the ratio of sucrose and/or maltose.Preferably, the product that obtains by described method need not carry out the intensive shearing treatment subsequently.Can be advantageously contain the culture of described composition or selectively various separated component is added in the product that contains the composition of suckling with this, for example water juice (dressing), oleomargarine, mayonnaise and smear sauce (spread), and in lower fat or fat free substitute, the yogurt drink etc.

Therefore, also in the scope that the present invention comprises, use maltose may be regulated the extent of polymerization of EPS to the ratio by changing maltose and sucrose or maltose and lactose as acceptor molecule.

Large-scale application

In a preferred embodiment of the invention, composition is used in large-scale the application.

Preferably, after cultivating lactic microorganisms, after cultivating lactic microorganisms, prepare composition with the amount of the total cell culture volume of the about 10%/L of 0.1%-.

Preferably, after cultivating lactic microorganisms, prepare composition with the amount of the total cell culture volume of the about 7.5%/L of 0.5%-.

Preferably, after cultivating lactic microorganisms, be produced aminoacid sequence with the amount of the total cell culture volume of the about 5%/L of 2.5%-.

Composition

Composition of the present invention can be used to improve fragrance, local flavor, sugariness, lightly seasoned, denseness, texture, quality, mouthfeel, hardness, viscosity, gel crackle, the structure of the product that is used to eat that contains described composition and/or influence the characteristic of sense organ, nutrition.In addition, composition of the present invention can also be realized described improvement with other combination of components of the product that is used to eat.

Though characteristic and nutrition-the present invention that first-selected composition of the present invention is used to improve fragrance, local flavor, lightly seasoned, sugariness, denseness, texture, quality, mouthfeel, hardness, viscosity, gel crackle, structure, the surface flatness of the product that is used to eat that contains described composition and/or influences sense organ comprise that also using composition of the present invention benefit medical science or physiological to be in conjunction with other components as pharmaceutical cpd passs the human consumer.

Combination with other component

Composition of the present invention can be used to and other combination of components.Therefore, the invention still further relates to various combinations.

Combination of the present invention comprises the component that composition of the present invention and other suitable animal or humans eat, and it can be benefit medical science or physiological passs the human consumer.

Other compositions of combination of the present invention comprise polyglucose, for example Litesse , and/or Star Dri 5.These other components selectively are added to comes the drying aid process and helps microbial survival in the composition.

Another example of other suitable ingredients comprises one or more: thickening material, jelling agent (gellingagent), emulsifying agent, tackiness agent, crystal modifier, sweeting agent (comprising artificial sweetner), VITAMIN, mineral substance, rheology modifiers, stablizer, antioxidant, dyestuff, enzyme, carrier (carrier), carrier (vehicle), vehicle, thinner, slipping agent, seasonings, coloring matter, suspension agent, decomposition agent (disintegrant), granulating tackiness agent (granulation binder) etc.These other components can be natural.These other components can be used method preparation chemistry and/or enzymatic.

As used in this, term " thickening material or jelling agent " is meant by slowing down or stoping the motion of drop, gas or the insoluble solids of particle, an insoluble liq to prevent isolating product.Multiviscosisty takes place when various hydrate molecules cause that viscosity raises, and separation slows down.Gelation takes place, thereby this three-dimensional netted thing is caught particle and is fixed them when hydrate molecule is connected to form three-dimensional netted thing.

Term " stablizer " is defined as preventing that product (for example food) in time and a kind of composition that changes or the combination of composition as used in this.

Term " emulsifying agent " is meant a kind of composition (for example food ingredient) that prevents separation of emulsions as used in this.Emulsion is two kinds of blended materials mutually, and a kind of form with drop exists, and is comprised by another kind of material.Emulsion can be formed by oil is water-soluble, and wherein drop or disperse phase are oil, and external phase is water; Perhaps water-soluble in oil, wherein water is that disperse phase and external phase are oil.Foam is that gas is water-soluble, and suspension to be solid be dissolved in liquid can be stablized by using emulsifying agent.Can ventilate in three-phase system, gas is caught by liquid oils, and emulsified then dose of stable aggegation grease crystallization fixed.Emulsifying agent has the polar group (hydrophilic) of affinity water molecules and attracts greasy non-polar group (lipophilic).They are absorbed on the interface of two kinds of materials, and the interfacial film of stable emulsion effect is provided.Hydrophilic/lipophilic the characteristic of emulsifying agent is subjected to the influence of molecular structure.These characteristics are determined by hydrophile/lipophile balance (HLB) value.The lipophilic tendency that low HLB value representation is higher is used for the emulsion that stabilize water is dissolved in oil.High HLB value is confirmed as hydrophilic emulsifying agent, is often used as the water-soluble emulsion of oil.These values are from simple system.Because contain other compositions of influential emulsification property in the food usually, the HLB value is not always selected the reliability index of emulsifying agent.

As used in this, term " tackiness agent " is meant a kind of composition that product is combined by physics or chemical reaction (for example food ingredient).(elation) in the process, for example moisture is absorbed, and produces adhesive effect in " activation ".But tackiness agent can absorb other liquid, and for example oil holds them in the product.In the present invention, tackiness agent is normally used in solid or the low moisture product, the product that for example bakes: cake (pastry), sugar are received son (doughnut), bread etc.

Term " crystal modifier " is meant a kind of grease or moisture crystalline composition (as food ingredient) of influencing as used in this.Owing to two reasons, the stable of ice crystal is very important.At first from separating on the viewpoint, stable directly related with product.Product is through many more freeze, and the ice crystal of formation is just big more.These macrocrystals can destroy the structure of product, comprise natural structure, as cell walls, perhaps pass through the structure that " activation " produces.Because water no longer is retained in the appropriate location, product in the back that thaws synerisis takes place or liquid oozes out.Secondly, when product is under the situation about being eaten when freezing, these big crystal can produce the mouthfeel as the undesirable gravel.

" carrier " (carrier) or " carrier " (vehicle) be meant the material that is suitable for mixing use, comprise any material known in the art, for example any liquid, gel, solvent, liquid diluent, solubilizing agent or the like, nontoxicity and not with composition in any component deleterious interaction takes place.

The example of acceptable carrier comprises on the trophology.For example water, salts solution, alcohol, siloxanes, cured, vaseline oil, vegetables oil, polyoxyethylene glycol, polypropylene glycol, liposome, sugar, gelatin, lactose, amylose starch, Magnesium Stearate, talcum powder, tensio-active agent, silicic acid, viscosity paraffin, volatile oil, fatty acid glycerine one acid esters and triglyceride, petroethral fatty acid ester, Walocel MT 20.000PV, polyvinylpyrrolidone etc.

The example of vehicle comprises one or more: the polyoxyethylene glycol of Microcrystalline Cellulose and other Mierocrystalline celluloses, lactose, Trisodium Citrate, lime carbonate, secondary calcium phosphate, glycine, starch, toffee (milk sugar) and macromolecule.

The example of decomposition agent comprises one or more: starch (being preferably corn, potato or tapioca (flour)), Explotab, cross-linked carboxymethyl cellulose sodium (croscarmellose sodium) and some composition silicate.

The example of granulating tackiness agent comprises one or more: polyvinylpyrrolidone, Vltra tears (HPMC), hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and Sudan Gum-arabic.

The example of slipping agent comprises one or more: Magnesium Stearate, stearic acid, Glyceryl Behenate and talcum powder.

The example of thinner comprises one or more: water, ethanol, propylene glycol and glycerine, and their combination.

Can also be simultaneously (for example when them be mixed together or even present) or order (for example presenting) by different paths by different paths use other components.

Preferably, when composition of the present invention mixed with any other component, lactic microorganisms still had vigor.

As used in this, term " is suitable for the component that the animal or human eats " and is meant a kind of quilt maybe can add in the composition of the present invention compound as additive to, it has nutritive value, has beneficial effect widely as the fiber substitute or to the human consumer.These compositions can be used to require in a large number gelation (gelling), veining (texturing), stablize, suspend, form film (film-forming) and structurizing (structuring), keep juice (retention of juiciness), and do not increase in the variant production of unnecessary viscosity.Preferably, this composition can prolong the stability of storage life and raising culture alive.

This composition can be a probiotics, for example alginate, xanthan gum, colloid (pectin), tracasol (locust bean gum) (LBG), chrysanthemum phenol (inulin), guar gum (guar gum), galactooligosaccharicomposition (GOS), fructo-oligosaccharides (fructo-oligosaccharide) (FOS), lactose sucrose, soy oligosaccharide, palatinose (palatinose), isomalto-oligosaccharides, glucose oligosaccharides (gluco-oligosacchride) and wood sugar oligosaccharides.

The composition that is used for the optimum quantity of combination of the present invention will depend on processed product and/or the method that product is contacted with composition and/or the specific end use of combination.Be used for composition work microorganism and/or should be sufficient by the enzyme of described microorganisms producing and/or by the content of the EPS of described enzyme production, it is effective, and contain fragrance, local flavor, lightly seasoned, sugariness, denseness, texture, quality, mouthfeel, viscosity, the structure of the food of described composition and/or influence the characteristic of sense organ in improvement, remain in nutrition and/or the wholesome feature fully effectively.The time span of validity will extend to the time that product is used at least.

Enriched material

Be used for the form that composition of the present invention can be an enriched material.Usually, these enriched materials comprise the very microorganism of the work of high density, and/or by the enzyme of described microorganisms producing and/or the EPS that produces by described enzyme.

The powder of enriched material form, particle and liquid composition can dilute with water or be resuspended in water or other suitable diluent in, for example suitable grown cultures liquid, for example milk or mineral substance or vegetables oil are to obtain operable composition.

The combination of the present invention of enriched material form can prepare according to the method that this area is known.

In one aspect of the invention, this product contacts with the composition of conc forms.Preferably, this product and spraying drying and/or resuspended composition contact.

Composition of the present invention carries out spraying drying or lyophilize by methods known in the art.

Adopting spray-dired method to prepare the particulate general method comprises solid material is dissolved in (for example microorganisms cultures is dissolved in the fermented liquid) in the appropriate solvent.In addition, this material can be suspended or is emulsified in and be formed suspension or emulsion in the non-solvent.Selectively add other compositions (as described above) or component for example biocide, stablizer, dyestuff and help the reagent of drying process in this stage.

Then, solution is sprayed and is formed superfine drop mist.Drop enters kiln rapidly, contacts with dry gas.Solvent evaporates from drop and enters dry gas and solidify drop, thereby forms particle.Separating particles and collection from dry gas then.

Product

Any product that can benefit from composition can be used to the present invention.Include, but are not limited to preserved fruit and milk-product and milk-product product, makeup and medicament production.

As an example, composition of the present invention can be used as nonalcoholic bevarage (soft drink), fruit juice or contain the beverage of whey-protein, health tea, cocoa drink, drinking milk and Bacterium lacticum beverage, yogourt and yogurt drink, cheese, ice-creams, ice cream and dessert, candy, biscuit, cake, snack, balance food (balanced food) and beverage, fruit is sandwich, care glaze, chocolate bread is sandwich, the cheese cake local flavor is sandwich, the fruit-like flavour cake is sandwich, cake and sugar are received sub-sugar-coat (cake and doughnut icing), instant-noodles double team heart cream, biscuit sandwich, promptly the bread of usefulness is sandwich, low in calories sandwich, the adult nutrition beverage, acidifying soy sauce/nectar, aseptic/pyrogenous chocolate drink, bar-shaped mixture (bar mix), drink powder, add the chocolate and chocolaty milk of calcium soy sauce/do not add, the composition that adds the coffee drinks of calcium.

Composition can also be used as a kind of composition in the foods prods, and for example American (American) cheese flavourings juice is used to rub with the hands broken and tears up caking inhibiter (the anti-caking agent for grated of cheese; Shredded cheese), potato slices dip in sauce (chip dip), cream cheese (cream cheese), do and mix whip top no resin acid cream (dry blended whip topping fat free sour cream), freeze/thaw milk system whip cream (dairy whipping cream), freeze/thaw is stablized natural cheddar cheese-(the stable whipped tipping low fat ﹠amp of whip top lower fat and less salt; Lite natural cheddar cheese), low-fat Switzerland yogourt, the frozen confection of inflation, the ice-creams of hard packing, easily mark, more economical and preserve the ice-creams of the hard packing that prolongs, low-fat ice cream: soft (soft serve), barbecue sauce (barbecue sauce), cheese dip in the rare cream (cottage cheese dressing) on sauce, the living cheese, the dried flavouring paste (dry mix Alfredo sauce) that mixes, blended cheese flavouring paste, do mixed tamato sauce etc.

In some aspects, preferably, the present invention can be used to yogourt production, Fa Jiao yogurt drink for example, and yogourt is drunk sour milk, cheese, fermenting cream, milk dessert etc.

The present invention also provides the method for a kind of food of preparation or food ingredient, and this method comprises and will mix with other food ingredients according to composition of the present invention.

Advantageously, the present invention relates to the product that contacts with composition of the present invention (selectively with other component/compositions), wherein said composition with the fragrance that can improve this product, local flavor, lightly seasoned, sugariness, denseness, texture, quality, mouthfeel, viscosity, gel crackle, remove whey, structure and/or influence the characteristic of sense organ, the amount of nutrition and/or wholesome feature is used.

Term " contact " is meant directly or indirectly composition of the present invention is applied to this product as used in this.The example of the application method that can be used comprises, but be not limited to, in comprising the material of composition, handle this product,, composition spray is immersed in the preparation of compositions thing to the surperficial of product or with product directly uses by composition is mixed with product.

When product of the present invention was food, composition of the present invention preferably mixed with this product.In addition, said composition can be included in the original composition of emulsion or food.In another kind selection, composition can be used as condiment, water juice (glaze), coloring agent mixture etc.

Use for some, importantly said composition can be added on the surface of the product that is applied/handles.Make said composition will be endowed one or more of following favorable characteristics: to improve fragrance, local flavor, lightly seasoned, denseness, texture, quality, mouthfeel, viscosity, structure, stability (serenity) and/or influence characteristic, nutrition and/or the wholesome feature of sense organ.

Composition of the present invention can be applied to the microorganism of the work of manipulated variable and/or by the enzyme of described microorganisms producing and/or by the EPS of described enzyme production intersperse, wrap by and/or pour into this product.The microorganism that lives and also can be used by the enzyme of described microorganisms producing with by the mixture of the EPS of described enzyme production can be respectively, simultaneously or in a sequence be used.

Food

Composition of the present invention can be used as-or preparation-a kind of food.At this, term " food " broadly uses, and comprises people's food and the food of animal (being feed).Aspect preferred, this food eats to the people.

Food can be solution form or solid-the depend on pattern of purposes and/or application and/or the pattern of using.

When as or prepare a kind of food for example during functional foodstuff (functional food), composition of the present invention can be used, in conjunction with one or more: acceptable carrier on the trophology, acceptable diluent on the trophology, acceptable vehicle on the trophology, acceptable adjuvant on the trophology; Activeconstituents on the trophology.

Preferably, composition is used to fermented milk or adds the milk of sucrose or have sucrose and/or the dairy medium of maltose (lactic media), wherein the nutrient solution of Sheng Chenging contains all components of composition, it is described Bacterium lacticum, and/or described enzyme and/or described EPS, can be used as a kind of composition and before yogourt fermentation or selectively, be added in the yogourt, for example with from about 0.1% to about 10% concentration with suitable concentration.Add composition and cause that viscosity raises, and generate the yogourt of mouthfeel firm (firm), the organoleptics property of stability decreases (serum separation) and improvement finished product.

Food ingredient

Composition of the present invention can be used as food ingredient.

Term " food ingredient " comprises the prescription that maybe can be added in functional foodstuff or the food as nutritional additive and/or fibre additive as used in this.This used term food ingredient also refer to be used for low content a large amount of different need gelation, veining, stable, suspend, form film and structurizing, reservation juice, improve mouthfeel and do not increase the prescription of the product of viscosity.

Food ingredient can be solution form or a kind of solid, depends on the pattern of its purposes and/or application and/or the pattern of using.

Foodstuff additive

Composition of the present invention can be-or be added to-foodstuff additive.

Functional foodstuff

Composition of the present invention can be-or be added to-functional foodstuff.

As used in this, term " functional foodstuff " is meant to provide and is not only nutritive validity and/or the sense of taste and satisfies, but also can present the food of other beneficial effect to the human consumer.

Therefore, functional foodstuff is a kind of bread and cheese with the component brought into wherein or composition (for example described here those), and these components or composition are given food specific function-for example, medical science or physiological benefit-and be not only trophism.

Though also there is not the legal definition of functional foodstuff, great majority approve all that to the interested group in this area functional foodstuff is to have the food that the quilt of specific health effect is sold.

Some functional foodstuff is nutritional drugs (nutraceutical).At this, term " nutritional drugs " is meant that a kind of trophism and/or sense of taste of can providing satisfies, but also treatment (perhaps other are useful) effect can be the food of passing the human consumer.Nutritional drugs has been crossed over traditional food and the line of delimitation between the medicine.

Investigation shows that the human consumer is placed on the attention of maximum on the demand of the functional foodstuff relevant with heart trouble.Preventing cancer is another aspect of trophology, and this also is very to attract the human consumer, but ironically, the human consumer feels that they are difficult in this respect and control.In fact, according to the statistics of the World Health Organization, at least 35% cases of cancer is relevant with diet.Other influence relevant demand with osteoporosis, intestinal health and obesity also is the key factor that the market development was bought and promoted to possible stimulatory function food.

Fibre additive

In yet another aspect, composition of the present invention can be used as or prepare fibre additive.

At first, in fact the key of food success be " fiber " or, subsequently, " wheat bran (bran) ".Although the result of study to the specific health benefit of fiber is inconsistent, in expert and human consumer, all consistent viewpoints are that most of people need more multifilament in its diet.Because its functional performance for example absorbs moisture, it is useful that fiber also is proved to be, and account for the major part of diet food.

For many years, fiber has many titles, comprises " coarse food grain (roughage) ", " fluffy goods (bulk) ", " wheat bran (bran) ", " fiber ", " plant residue (plant residue) ", " plantix " and " carbohydrate that is difficult to obtain ".Even today, determine that a kind of simple and clear and complete definition is not a simple thing for edible fiber, this is because food fiber is a kind of composite interstitial substance that has the various components of different definition to constitute at diverse discipline.

At this, when being used in the food, the term fiber is meant indigestible material equally.Especially, fiber is made up of Mierocrystalline cellulose, hemicellulose, colloid (pectin), viscose glue (gum), cement (mucilage) and xylogen.

Be not that every kind of fiber source all contains all these components.In fact, the possible combination results of absolute quantity be present in the physiological and functional effect of multiple difference in the different fibre compositions in a large number.As identical feature, be not various fiber sources are food fibers of 100%.

Canadian Harvest, Cambridge, the home sale of MN and marketing manager Diane Lardiere propose, " total food fiber (TDF) is defined as indigestible carbohydrate ", " Testa Tritici only is 40%TDF, but is considered to a kind of fibre composition ".

Therefore, composition of the present invention can add to-fibre additive.

Composition is used as a kind of additive and joins together in the food with the different traditional fibre source of describing in detail above, also in protection scope of the present invention.The recommended dose that the grownup absorbs fiber is between 20-35g/ days or the calorie that consumes of 10-13g/1000, for children, usually intake is based on their age, perhaps 0.5g fiber/kg body weight (perhaps 0.23g/ sterling body weight), on be limited to 35g fiber/sky.

The method that the daily fiber picked-up guaranteeing to realize to recommend (calorie that 20-35g/ days or 10-13g/1000 consume) is provided is also in protection scope of the present invention.Can dispensing tablet, pill, capsule, pearl, solution or suspension comes substitute food product or snack, particularly carry out in the process of the scheme that reduces body weight beginning.

From the viewpoint of health, importantly when with food food fiber tablet, pill, capsule, pearl, solution or suspension, will help to be reduced in the blood sugar increasing after having a meal and strengthen full sense.

Composition of the present invention is joined in the fibre drink also in the application's protection domain.Studies show that soluble fiber will help to keep digestive health, the diet of high in soluble fiber content (25g/ days at least) will help to keep normal cholesterol levels.

Probiotics

For some application, it is generally acknowledged that the lactic microorganisms of the work in the composition of the present invention can strengthen the effect of probiotics culture.At this, probiotics is:

" a kind of indigestible food ingredient, it acts on the host valuably by growth or the activity that optionally stimulates a kind of or limited amount bacterium in the large intestine.”(Am?Clin?Nutrit，2001；73：406S-409S.)。

According to nearest consistent the report (van Loo etc., 1999, Br.J.Nutr., 81:121-132), the definition of probiotics is quantity and/or the active increase of bifidus bacillus or lactic-acid-bacterium main in the gi tract.At this, known: bifidus bacillus has the bacterium that helps kill deleterious in a large number and foodborne illness, comprises E coli 0157 and E coli H88 that possibility is fatal; Bifidus bacillus can prevent that the child of tender age from suffering from diarrhoea; Lactobacillus rhamnosus GG helps to treat the diarrhoea relevant with microbiotic; And the case that Lactobacillus rhamnosus GG also is proved to be for treatment some traveler's diarrhea and rotavirus infection is effectively, rotavirus infection be the children's diarrhae in the worldwide most commonly encountered diseases because of.

Other little ecological regulated culture to add and also belongs to protection scope of the present invention in the composition of the present invention.

Term " little ecological culture of regulating " is defined as microorganism alive as used in this, and it advantageously acts on host organisms by the balance of improving enteric microorganism.As used in this term " probiotics " also comprise can stimulating immune system useful balance, the microorganism that reduces the work of the inflammatory reaction in the enteron aisle simultaneously.On this point, the purposes that the composition of the present invention that will contain described probiotics composition is used for anticancer therapy and Ammonium Glycyrrhizate and ulcerative colitis also is that the present invention considers.

Though do not have the bound of probiotics picked-up, it is generally acknowledged that the cell of at least 10,000 work/ml product can make microorganism be at war with in enteric microorganism flora (flora).

Except the probiotics effect of composition of the present invention, provide also within the scope of the invention as other probioticss that can be included in a compound in the combination with said composition.The probiotics component that comprises in the combination of composition of the present invention has the feature of slowing down the fermentation in the large intestine.This probiotics can produce positive acting to intestinal microflora, and particularly in the left side of colon, in specific intestinal cancer and ulcerative colitis, this zone of enteron aisle occurs especially easily unusually.

Composite biological agent

The present invention comprises that also to adopt probiotics and ecological regulator combined as composition and composition of the present invention, when being combined, and the formation composite biological agent.This purpose is to make up the hormesis of the beneficial bacteria of the effect of new beneficial bacteria and body itself.Because some have the collaborative trophism of intensive in these mixtures, the exploitation of this mixture and consumption have very big potentiality.

Therefore, can design composition of the present invention especially and contain different components, these components are brought symbiotics effect (symbiotics effect) to the human consumer.

Medicine

Composition of the present invention can be used as or be used to prepare medicine.At this, term " medicine " uses with broad sense, comprises medicine that is used for the people and the medicine that is used for animal (being that the animal doctor uses).Aspect preferred, medicine is to be used for human purposes and/or to be used for livestock industry.

Medicine can be used for the treatment of that purpose-it can be in fact curative anterethic or preventative.This medicine even can be used for diagnostic purpose.

When being used as or being used to prepare medicine, composition of the present invention can with one or more following associatings: pharmaceutically acceptable carrier, pharmaceutically acceptable thinner, pharmaceutically acceptable vehicle, pharmaceutically acceptable adjuvant, pharmacy activity component.

Medicine can be solution form or solid-depend on its purposes and/or application model and/or mode of administration.

Pharmaceutical cpd

Composition of the present invention can be used as pharmaceutical cpd (pharmaceutical ingredient).

At this, composition can be unique active ingredient or be one of a large amount of (being two or more) active ingredients at least.

Pharmaceutical cpd can be solution form or solid-depend on its purposes and/or application model and/or mode of administration.

Form

Composition of the present invention can any suitable form be used-and no matter be to exist separately or with other components or composition combination.Similarly, the combination that comprises composition of the present invention and other components and/or composition (being composition-as food ingredient, functional food ingredients or pharmaceutical cpd) can any suitable form be used.

Composition of the present invention can solid or one of liquid prepared product or boths' form be used.The example of solid prepared product includes but not limited to tablet, capsule, powder, particle and powder, and they can be moistening, spray-dired, freeze-dried or freeze dried.The example of liquid prepared product includes but not limited to the aqueous solution, organic or water-organic solution, suspension and milk sap.

The suitable example of form comprises one or more: tablet, pill, capsule, pearl, solution or suspension, it contains seasonings or tinting material, is used for immediately, time-delay, improved, that continue, application pulse or sustained release.

As an example, if composition of the present invention is a form with tablet to be used-for example can also contain one or more as functional ingredient-this tablet: comprise for example Microcrystalline Cellulose of vehicle, lactose, Trisodium Citrate, lime carbonate, calcium phosphate and glycine; Decomposition agent is starch (starch of preferred corn, potato or cassava) for example, Explotab, cross-linked carboxymethyl cellulose sodium and specific composition silicate; The granulating tackiness agent is polyvinylpyrrolidone for example, Vltra tears (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and Sudan Gum-arabic; Slipping agent is Magnesium Stearate for example, stearic acid, Glyceryl Behenate and talcum powder.

The example that is used to prepare acceptable carrier in the nutrition of this form comprises, water for example, salts solution, alcohol, siloxanes, cured, vaseline oil, vegetables oil, polyoxyethylene glycol, liposome, sugar, glycine, lactose, amylose starch, Magnesium Stearate, talcum powder, tensio-active agent, silicic acid, viscosity paraffin, volatile oil, fatty acid glycerine one acid esters and triglyceride, petroethral fatty acid ester, Walocel MT 20.000PV, polyvinylpyrrolidone etc.

The preferred vehicle that is used for this form comprises lactose, starch, Mierocrystalline cellulose, the polyoxyethylene glycol of toffee or macromolecule.

Suspension and/or vina (elixir) for the aqueous solution, composition of the present invention can with various sweeting agents or seasonings, coloring material or dyestuff, with emulsifying agent and/or suspension agent and with thinner for example water, propylene glycol and glycine and they combine combination.

This form can also comprise gelatine capsule; The fiber capsule, fiber tablet etc. or or even fibre drink.

Embodiment

Now the present invention is described by embodiment and with reference to subsidiary accompanying drawing.

The accompanying drawing summary

Accompanying drawing 1. is used to produce the evaluation of the optimum strain of exo polysaccharides (EPS).

The evaluation of the cell count of the bacterial strain of accompanying drawing 2. production EPS.

Accompanying drawing 3. adopts Leuconostoc mesenteroides 808 bacterial strains to produce the evaluation of the optimum temps of EPS.

The evaluation that EPS produces and viscosity raises when 4. pairs in accompanying drawing adopts Leuconostoc mesenteroides 808 bacterial strains.

The accompanying drawing 5A. evaluation that the EPS under different sucrose produces when adopting Leuconostoc mesenteroides 808 bacterial strains.

The accompanying drawing 5B. evaluation that the viscosity under different sucrose raises when adopting Leuconostoc mesenteroides 808 bacterial strains.

Accompanying drawing 6. is produced EPS by the Leuconostoc mesenteroides bacterial strain in the nutrient solution that contains different content maltose.

The viscosity change that accompanying drawing 7. is produced by Leuconostoc mesenteroides 808 bacterial strains in containing the grown cultures liquid of different content maltose.

Accompanying drawing 8. is produced EPS by picogram Bacterium lacticum 570 bacterial strains in having added the grown cultures liquid of sucrose/maltose and lactose.

Accompanying drawing 9. is produced EPS by plant lactobacillus 853 bacterial strains in having added the grown cultures liquid of sucrose/maltose and lactose.

Accompanying drawing 10. is produced EPS by lactobacillus salivarius kind 1502 bacterial strains in having added the grown cultures liquid of sucrose/maltose and lactose.

Accompanying drawing 11. maltose are to by Leuconostoc mesenteroides 808, picogram Bacterium lacticum 570, the influence of the EPS that plant lactobacillus 853 and lactobacillus salivarius 1502 are produced; By oligosaccharides product and the EPS product of lactose as enzyme substrates production.

Accompanying drawing 12. is produced EPS by Leuconostoc mesenteroides 808 in having added the grown cultures liquid of sucrose under condition of different pH.

Accompanying drawing 13. is produced EPS by picogram Bacterium lacticum 570 in having added the grown cultures liquid of sucrose under condition of different pH.

Accompanying drawing 14. is produced EPS by plant lactobacillus 853 in having added the grown cultures liquid of sucrose under condition of different pH.

Accompanying drawing 15. under condition of different pH, the viscosity of Leuconostoc mesenteroides 808 in having added the grown cultures liquid of sucrose.

Accompanying drawing 16. under condition of different pH, the viscosity of picogram Bacterium lacticum 570 in having added the grown cultures liquid of sucrose.

Accompanying drawing 17. under condition of different pH, the viscosity of plant lactobacillus 853 in having added the grown cultures liquid of sucrose.

Accompanying drawing 18. is by the structure of the EPS of NMR analysis.

Accompanying drawing 19. spraying dryings contain Leuconostoc mesenteroides 808 bacterial strains of EPS.

Method

Screening:

Can produce the EPS that is used for the object of the invention for definite which kind of Bacterium lacticum, a large amount of different strains are screened.Leuconostoc mesenteroides 808, the sample of plant lactobacillus 853 and lactobacillus salivarius kind 1502 have been deposited in Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH.Preserving number is respectively DMS 15792[" DC 808 "], DSM 15793[" DC 853 "] and DSM15794[" DC 1502 "].Preservation person [Danisco Niebull GmbH, Busch-JohannsenStr.1,25899 Niebull] the authorized application people in this application, relate to the biomaterial of this preservation, and without reserve and can not agree that this biomaterial by preservation is open to the public with going back on one's word.

Screening method comprises the steps:

Each strain microorganism is activated/is seeded in 10ml dairy medium or the 9% skimming milk nutrient solution (skim milk-media) (containing 0-2% sucrose), cultivates 8-20h to form pre-culture (pre-culture) at 25-42 ℃.

Subsequently, pre-culture is transferred on the microtiter plate.Contain 0.2-2ml dairy medium or 9% skimming milk nutrient solution in each hole of microtiter plate.Then, the pre-culture of inoculation 1-5% in containing each hole of selected grown cultures liquid, and at 25-42 ℃ of cultivation 8-20h.

By 96-replicator the culture in each hole is transferred to the surface of solid dairy substratum then, cultivate 8-20h again at 25-42 ℃.

After this incubation period, by naked eyes or under the 10X magnification, check bacterium colony by opticmicroscope in some cases, determine to have the bacterium colony of mucus vesicle (EPS).The bacterium colony that contains mucus vesicle (EPS) is confirmed as the positive and is used for further analysis.

Fermentation:

Prepare the culture that activation is spent the night at dairy medium or in based on the substratum (as indicated above) of milk.1% culture this overnight is transferred in the nutrient solution that contains 2% sucrose at least, and under fermentation conditions (25-42 ℃, 8-20h, pH5 8-7) cultivates.This pre-culture of inoculation 1-5% in the proving room then.

Emulsion growth substrate (lactic growth media) (lactic 100b) contains usually: 2% peptone (Merck, Germany), 0.5% yeast extract, 0.25% gelatin, 0.4%NaCl, 0.25% sodium-acetate, the 0.05%L-xitix, 0.05%Tween 80, at least 2% sucrose.

EPS measures

After pollutent for example separates the protein, precipitate to measure the amount of EPS by gravimetry by ice-cold (4 ℃) 96% ethanol that uses double volume.This precipitation is selectively improved settling step 4 ℃ of incubation a few hours, after this, deposits precipitated EPS by centrifugal and dry air or preferably freeze drying.

More detailed open:

The culture that 3ml is cultivated from proving room adds in 4 ℃ of 96% ice-cold ethanol of 6ml.

Then the mixture that obtains is mixed by rocking, then 4 ℃ of overnight storage.

4 ℃ be incubated overnight after, with 2500rpm centrifugation 15min.

Draw supernatant liquor, be deposited in 80 ℃ of dry 4-6h.

Viscosimetric analysis

After fermentation, adopt DV II viscometer (UT 401) to measure the viscosity of the lactic acid growth media of the maltose that has added various content according to manufacturer's introduction.

From Lb 570 purifying six poly-dextran

With 10,000rpm is centrifugal, and heat treated fermented liquid 10min removes cell.Use the EPS in the 96% ethanol sedimentation supernatant liquor of 2 times of volumes then, and with 12, the centrifugal 15min of 500rpm.Spend the night at 60 ℃ of dry EPS.Concentration with 5mg/ml is dissolved in EPS in the water again.

At 100 ℃, partial hydrolysis EPS 6h in 50mM TFA.Dry sample also is dissolved in the water with the concentration of 35mg/ml.The EPS that is not hydrolyzed precipitates with Virahol (1: 1 v/v).After with the centrifugal 10min of 10 000rpm, dry supernatant liquor is to the volume of half.By 0.45 μ m filter filtered sample, at Dionex ^TMSeparate on the PA1 post.The sample volume of 23 μ l is loaded on the post, with buffer A (0.1M NaOH) and buffer B (being dissolved in the 1M sodium-acetate among the 0.1M NaOH) wash-out, adopt following concentration gradient: 0-25min: with 5%B constant gradient wash-out (isocratic), 25-34min:5-8%B, 34-34.001min:8-100%B, 34.001-44: with 100%B constant gradient wash-out, 44-44.001; 100-5%B, 44.001-54: with 5%B constant gradient wash-out.In elution process, pass through Dionex ^TMDesalting plant CMD ^TMAutomatically crest is carried out desalination and collection.Analyze six poly-dextran by NMR.

Except in 500mM TFA, carrying out the partial hydrolysis, adopt the six poly-dextran of identical operations purifying from Ln808.Under the same conditions in the PA1 post again wash-out contain the crest of six poly-dextran to be further purified.

The NMR spectrophotometric analysis

With sample and 99.80%D ₂O exchange twice, and be dissolved in 99.99%D again ₂Among the O.From being dissolved in D ₂The NMR spectrum of record oligosaccharides in the solution of O.(operating frequency of 600.1MHz is used for BrukerAMX 600MHz spectrophotometer ¹The frequency of H NMR and 160.9MHz is used for ¹³C NMR) 300K record proton spectrum ( ¹H) and all two-dimension spectrums ( ¹H- ¹H COSY, ¹H- ¹H TOCSY, ROESY, ¹H-C HMQC and ¹H- ¹³C HMBC), in all tests, adopt the Bruker pulse protocol of standard.With respect to the chemical shift of the inherent acetone (δ that is in the news _H2.225; δ _C31.45).TOCSY is adopted the mixing time of 100ms.Along t ₂Obtain the increasing amount of 256t and the two-dimension spectrum of 2048 data points.For of the same race and xenogenesis correlation test (heterocorrelation experiment), for each t ₁Value needs 32 and 128 scanning spots respectively.Because carrying out Fourier transform (Fouriertransformation) before, at t ₁Carry out zero padding (Zero-filing) on the dimension, in two dimensions, use displacement square Bel or Gaussian function (shifted squared sine bell or Gaussian functions).

Glycan analysis

1. hydrolysis-2M TFA, 2h, 121 ℃;

2. reduction-Na (BH ₄);

3. acetylize-100 μ l pyridine and 100 μ l Ac ₂O, 30min, 85 ℃

4.GC analyze

Absolute configuration

1. methanolysis-oligosaccharides (about 0.2mg)+100 μ l 2M HCl/MeOH, 16h, 85 ℃;

2. butanols decomposes-100 μ l 2M HCl/BuOH, 16h, 85 ℃

3. acetylize-100 μ l pyridine and 100 μ l Ac ₂O, 30min, 85 ℃

4.GC analyze

Embodiment 1

A large amount of different strains are screened seek the lactic-acid-bacterium bacterial strain that to produce EPS.The proving room that contains 200ml emulsion growth medium (lactic 100b) with the pre-culture inoculation of spending the night.Cultivate culture 72h at 30 ℃, do not stir.When 24h and 72h, detect each bacterial strain and produce the ability of EPS.Result in the accompanying drawing 1 shows that butterfat Leuconostoc mesenteroides 808-bacterial strain generates the EPS of about 28g/l when 24h, generates the EPS (referring to accompanying drawing 1) of about 43g/l when 72h.

Further screening to different lactic microorganisms on sucrose agar shows, the picogram lactobacillus species, and plant lactobacillus or lactobacillus salivarius can both be produced EPS (referring to as follows).

Embodiment 2

In this test, produce the cell count of the cell of EPS and estimated.Therefore, the overnight culture of various lactic-acid-bacterium bacterial strains is used to the proving room that inoculation contains 200ml emulsion growth medium (lactic 100b).Cultivate various culture 72h at 30 ℃, do not stir.The result shows that when 48h that ferments and 72h, lactic microorganisms Leuconostoc mesenteroides 808 bacterial strains can be kept the cell count (referring to accompanying drawing 2) that is higher than any other detected bacterial strain.

Embodiment 3

Measure the temperature that Leuconostoc mesenteroides 808 bacterial strains produce best EPS level.The pre-culture that spends the night of Leuconostoc mesenteroides 808 is used to inoculate four proving rooms that contain 200ml emulsion growth medium (lactic 100b).After the inoculation, each proving room 22 ℃, 25 ℃, 30 ℃ and 35 ℃ of cultivations, does not stir respectively.PH value when mensuration begins to cultivate is 7.4, and the pH that measures when fermentation stage finishes is 4.1.The result who is shown in the accompanying drawing 3 shows that Leuconostoc mesenteroides 808 bacterial strains are at 25 ℃ of EPS that produce highest level.

Embodiment 4

Adopt Leuconostoc mesenteroides 808 bacterial strains to estimate EPS production and viscosity rising.The pre-culture that spends the night of Leuconostoc mesenteroides 808 is used to the proving room that inoculation contains 200ml emulsion growth medium (lactic 100b).Then, culture is cultivated 36h at 25 ℃, does not stir.PH value when mensuration begins to cultivate is 7.4, and the pH that measures when fermentation stage finishes is 4.1.The result of this test is presented in the accompanying drawing 4.

Embodiment 5

Adopt Leuconostoc mesenteroides 808 bacterial strains to measure because sucrose concentration changes the effect to EPS produces and viscosity raises.Inoculate five different proving rooms that contain 200ml emulsion growth medium (lactic 100b) with the pre-culture of spending the night of Leuconostoc mesenteroides 808, wherein replenished 2%, 4%, 6%, 8% and 10% sucrose concentration respectively.Five kinds of different cultures are cultivated 72h, do not stir.PH value when mensuration begins to cultivate is 7.4, and the pH that measures when fermentation stage finishes is 4.1.This test-results shows that because sucrose concentration changes, EPS output increases (referring to accompanying drawing 5A), and viscosity rising (referring to accompanying drawing 5B).

Embodiment 6

Some utilizes maltose as acceptor molecule to the endonuclease capable of producing the EPS response, and maltose can be controlled the chain length that the EPS glycopolymers constitutes conversely, and thereby control levels of viscosity.Adopt different Leuconostoc mesenteroides bacterial strains, and the picogram lactobacillus species, plant lactobacillus kind or lactobacillus salivarius kind are tested, to determine the influence of maltose to EPS chain length and viscosity.The pre-culture that spends the night of the different Leuconostoc mesenteroides bacterial strains of growing in the emulsion growth substrate of use 1% is inoculated the different fermentations chamber of containing 200ml emulsion growth substrate, has wherein added:

10% sucrose,

5% sucrose and 5% maltose (50/50 sucrose/maltose ratio)

7.5% sucrose and 2.5% maltose (75/25 sucrose/maltose ratio)

Cultivate culture 72h, do not stir, postvaccinal 24,48 and the time point of 72h on measure EPS output.The data that are presented in the accompanying drawing 6 show, have generated remarkable high-load EPS by Leuconostoc mesenteroides 808.These data also show, the content of the EPS that is produced by Leuconostoc mesenteroides 808 can be regulated by the maltose that add different amounts.Therefore, when 72h, Leuconostoc mesenteroides 808 bacterial strains can produce the EPS (accompanying drawing 6) of about 36g/L in the grown cultures liquid that adds 10% sucrose.The curve of accompanying drawing 6 also shows, when changing the content of the maltose that adds toward grown cultures liquid in, by the amount of the EPS of Leuconostoc mesenteroides 808 generations significantly descend (accompanying drawing 6).

The decline of EPS output very likely is the generation that maltose can be induced short polysaccharide chain.The polysaccharide chain contraction in length causes mobile the change greatly, and promptly viscosity reduces.Result in the accompanying drawing 7 shows, the viscosity that contains the grown cultures liquid of 10% sucrose surpasses 4000mPa, in containing the nutrient solution of sucrose/maltose ratio of 75/25, viscosity is reduced to 14mPa significantly, and further drops to 10mPa in the nutrient solution of sucrose/maltose ratio of 50/50.The result who is presented at accompanying drawing 6 and accompanying drawing 7 shows, produces short, low viscous chain with maltose during as acceptor molecule, causes EPS output to descend and the viscosity reduction.

Detect the influence (respectively referring to accompanying drawing 8, accompanying drawing 9 and accompanying drawing 10) that maltose generates sucrose to picogram lactobacillus species 570 bacterial strains, plant lactobacillus kind 853 bacterial strains or lactobacillus salivarius 1502 bacterial strains the ability of EPS.Cultivate culture 48h, do not stir, on the time point of postvaccinal 24h and 48h, measure EPS output.Be presented at accompanying drawing 8, the data in accompanying drawing 9 and the accompanying drawing 10 show that by picogram lactobacillus species 570 bacterial strains, plant lactobacillus kind 853 bacterial strains or lactobacillus salivarius 1502 generate high-load EPS from sucrose.These data show that also by picogram lactobacillus species 570 bacterial strains, the content of the EPS that plant lactobacillus kind 853 bacterial strains or lactobacillus salivarius 1502 generate can be regulated by the addition that changes maltose.Therefore, when 24h, picogram lactobacillus species 570 bacterial strains can generate the EPS (accompanying drawing 8) of about 27g/L in the grown cultures liquid that has added 10% sucrose.The curve of accompanying drawing 8 also shows, changes the amount of the maltose that adds in the growth media, significantly reduces (accompanying drawing 8) by the content of the EPS of picogram lactobacillus species 570 bacterial strains generation.

The inventor also detects Leuconostoc mesenteroides 808 bacterial strains, and picogram lactobacillus species 570 bacterial strains, plant lactobacillus kind 853 bacterial strains and lactobacillus salivarius 1502 bacterial strains utilize lactose to generate the ability of EPS as substrate.Result of the present invention shows that the enzyme that is generated by these lactic microorganisms can also be polymerized to lactose EPS (referring to accompanying drawing 11).Result in the accompanying drawing 11 shows that also when cultivating as enzyme substrates with lactose, detected lactic-acid-bacterium (lactic acid bacteria) can both generate the EPS of high density.Therefore, when postvaccinal 48h, Leuconostoc mesenteroides 808 bacterial strains generate the EPS of 50g/L, and picogram lactobacillus species 570 bacterial strains generate 20g/L EPS, and plant lactobacillus kind 853 bacterial strains generate 20g/L EPS, and lactobacillus salivarius 1502 bacterial strains generate 20g/L EPS.These results also prove, when adopting lactic acid as substrate, can regulate the growing amount of EPS by change the maltose amount in the reaction of joining.

Embodiment 7

Detect pH to by Leuconostoc mesenteroides 808 bacterial strains, picogram lactobacillus species 570 bacterial strains and plant lactobacillus kind 853 bacterial strains are produced the influence of EPS.The pre-culture that spends the night of any detected bacterial strain is used to the proving room that inoculation contains 200ml emulsion growth medium (lactic 100b).Cultivate culture 48h at 25 ℃ then, do not stir.At pH6.5, pH6.0 and pH5.8 detect the output of EPS.Result from these tests is presented at accompanying drawing 12, in accompanying drawing 13 and the accompanying drawing 14

It is when pH6.5 (referring to accompanying drawing 12) that Leuconostoc mesenteroides 808 bacterial strains generate production peak EPS.

It is when pH6.0 (referring to accompanying drawing 13) that picogram lactobacillus species 570 bacterial strains generate production peak EPS.

Embodiment 8

Also detected the influence of pH to viscosity.

It is when pH5.8 that Leuconostoc mesenteroides 808 bacterial strains produce high viscosity, and the MV minium viscosity that this bacterial strain produces is when pH6.0 (referring to accompanying drawing 15).

It is when pH6.5 that picogram lactobacillus species 570 bacterial strains produce high viscosity, and viscosity descends along with pH and reduces.Therefore, the MV minium viscosity of this bacterial strain generation is when pH5.8 (referring to accompanying drawing 16).

It is when pH6.0 (referring to accompanying drawing 17) that plant lactobacillus kind 853 bacterial strains produce high viscosity.

Embodiment 9

The structure that adopts nuclear magnetic resonance spectroscopy to generate derived from the EPS of oligomer by picogram lactobacillus species 570.The result is presented in the accompanying drawing 18, and proof EPS main chain is made up of the glycan molecule that comprises glucose.The EPS main chain is the dextran (dextran (Dextran)) that α-1-6 key connects.

Embodiment 10

After spraying drying, detect the activity of Leuconostoc mesenteroides 808 bacterial strains when having EPS.The pre-culture that spends the night of Leuconostoc mesenteroides 808 bacterial strains is used to inoculate the proving room that contains 20 liters of emulsion growth mediums that add 1% sucrose (lactic 100b).Cultivate culture 72h then, do not stir.Accompanying drawing 19 shown and has 5%Glucidex , when 10%Glucidex  and 10% skimming milk, before spraying drying and afterwards, the activity of Leuconostoc mesenteroides 808 bacterial strains.Glucidex  is the mixture of trophicity carbohydrate, by the production of control enzymically hydrolyse, is normally used for foodstuffs industry, especially for the fermentative production of milk.Glucidex  can be used as sweeting agent, but preferably improves technique effect, taste and the cost efficiency of composition of the present invention as the dense syrup of spray-dired sugar.

Patent application that each is mentioned in presents and patent, and in each above-mentioned patent application and patent, be included in and quote in each patent application and the patent examination process and every part of file (" application reference document ") of reference, and in each patent application and patent and in any application reference document, quote or any manufacturer of any product of mentioning introduces or products catalogue, all be incorporated herein by reference.In addition, the All Files of quoting in this article and in the file that this paper quotes, quoting or the All Files of reference, and quote in this article or any manufacturer of any product of mentioning introduces or products catalogue, all be incorporated herein by reference.

When not deviating from scope and spirit of the present invention, the various improvement and the variation of method and system described in the invention it will be apparent to those skilled in the art that.Though the present invention is described with specific preferred embodiment, should be appreciated that invention required for protection should be limited to these particular inadequately.In fact, be obvious for molecular biology or those skilled in the relevant art, the various modifications that are used to implement described pattern of the present invention should be in the protection domain of following claim.

Applicant or attorney docket PO 13723 WO

International application no

Explanation about microbial preservation

(PCT detailed rules and regulations 13 two)

A. following explanation relate to specification sheets the _ _ the 41__ page or leaf, the _ _ the capable described microorganism of 21__.
		B. the preservation item other be deposited in in the supplementary page
Germany microorganism and cell culture preservation center (the title von Mikroorganismen und Zellkulturen GmbH of Deutsche Sammlung depositary institution)
		Depositary institution address (comprising postcode and name of the country) Mascherode Weg 16 D-38124 Braunscheweig
Preservation date on July 23rd, 2003	Preserving number DSM 15792,15793,15794
		C. have supplementary page in supplementary notes (in case of necessity) this column
Our requirement, described by the sample of the microorganism of preservation, can only be according to relevant patent statute, for example, EPC rules 28 (4), and the corresponding laws and regulations of any another designated state, provide to give independently professional, and prerequisite be this kind behavior at designated state is feasible and law that also be designated state allows.
		D. this explanation is done (if explanation is not done for all designated states) for following designated state
		E. remark additionally (in case of necessity)
Following explanation will be subsequently provides (pointing out the classification that illustrates, for example: " numbering of preservation thing ") to international office

PCT/RO/134 shows (in July, 1998)

Applicant or attorney docket PO 13723 WO

International application no

Explanation about microbial preservation

(PCT detailed rules and regulations 13 two)

A. following explanation relate to specification sheets the _ _ the 41__ page or leaf, the _ _ the capable described microorganism of 21__.
		B. the preservation item other be deposited in in the supplementary page
Germany microorganism and cell culture preservation center (the title von Mikroorganismen und Zellkulturen GmbH of Deutsche Sammlung depositary institution)
		Depositary institution address (comprising postcode and name of the country) Mascherode Weg 16 D-38124 Braunscheweig
Preservation date on July 23rd, 2003	Preserving number DSM 15792,15793,15794
		C. have supplementary page in supplementary notes (in case of necessity) this column
Abide by EPC28 bar (1) (d), preservation person authorized application people relates to this preserved material in this application, and outspoken and public visit during this biomaterial of agreement with going back on one's word.
		D. this explanation is done (if explanation is not done for all designated states) for following designated state
		E. remark additionally (in case of necessity)
Following explanation will be subsequently provides (pointing out the classification that illustrates, for example: " numbering of preservation thing ") to international office

PCT/RO/134 shows (in July, 1998)

Budapest treaty is about the microbial preservation of the international endorsement that is used for patented procedure

The international office form

The addressee:

BAVARIAN?NORDIC?RESEARCH

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FRAUNHOFERSTRASSE?18B

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When being applicable to detailed rules and regulations 6.4 (d), the described date is the date that the qualification of international depositary institution is assert.

Form DSMZ.bp/4 (one page thus much)

Budapest treaty is about the microbial preservation of the international endorsement that is used for patented procedure

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Protect by the world that following one page is specified

INSTITUTE?GMBH

FRAUNHOFERSTRASSE 18B hides unit and provides the world according to detailed rules and regulations 10.2

D-82152?MARTINSRIED

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1 refers to initial preservation day, perhaps, when having carried out the conversion of new preservation or preservation, refers to the maximally related date (date of new preservation or the date of changeing preservation).

2 for the (ii) and (iii) situation of representative of those rules 10.2 (a), refers to nearest viability check.

3 usefulness are made fork and are represented to select.

Form DSMZ-BP/9 (only 1 page)

Budapest treaty is about the microbial preservation of the international endorsement that is used for patented procedure

The international office form

The addressee:

BAVARIAN?NORDIC?RESEARCH

INSTITUTE?GMBH

FRAUNHOFERSTRASSE?18B

D-82152?MARTINSRIED

Germany

When being applicable to detailed rules and regulations 6.4 (d), the described date is the date that the qualification of international depositary institution is assert.

Form DSMZ.bp/4 (one page thus much)

Budapest treaty is about the microbial preservation of the international endorsement that is used for patented procedure

The international office form

Addressee: survival proof

BAVARIAN?NORDIC?RESEAR

Protect by the world that following one page is specified

INSTITUTE?GMBH

FRAUNHOFERSTRASSE 18B hides unit and provides the world according to detailed rules and regulations 10.2

D-82152?MARTINSRIED

Germany

1 refers to initial preservation day, perhaps, when having carried out the conversion of new preservation or preservation, refers to the maximally related date (date of new preservation or the date of changeing preservation).

2 for the (ii) and (iii) situation of representative of those rules 10.2 (a), refers to nearest viability check.

3 usefulness are made fork and are represented to select.

Form DSMZ-BP/9 (only 1 page)

Budapest treaty is about the microbial preservation of the international endorsement that is used for patented procedure

The international office form

The addressee:

BAVARIAN?NORDIC?RESEARCH

INSTITUTE?GMBH

FRAUNHOFERSTRASSE?18B

D-82152?MARTINSRIED

Germany

When being applicable to detailed rules and regulations 6.4 (d), the described date is the date that the qualification of international depositary institution is assert.

Form DSMZ.bp/4 (one page thus much)

Budapest treaty is about the microbial preservation of the international endorsement that is used for patented procedure

The international office form

Addressee: survival proof

BAVARIAN?NORDIC?RESEAR

Protect by the world that following one page is specified

INSTITUTE?GMBH

FRAUNHOFERSTRASSE 18B hides unit and provides the world according to detailed rules and regulations 10.2

D-82152?MARTINSRIED

Germany

1 refers to initial preservation day, perhaps, when having carried out the conversion of new preservation or preservation, refers to the maximally related date (date of new preservation or the date of changeing preservation).

2 for the (ii) and (iii) situation of representative of those rules 10.2 (a), refers to nearest viability check.

3 usefulness are made fork and are represented to select.

Form DSMZ-BP/9 (only 1 page)

Claims (43)

1. composition that is used to eat, described composition comprises lactic microorganisms alive, by exo polysaccharides (EPS) product of described microorganism synthetic enzyme and described enzyme.

2. according to the composition of claim 1, wherein the EPS product is to come original position to generate by cultivate lactic microorganisms with suitable enzyme substrates.

3. according to the composition of claim 1 or 2, described enzyme is can polymerization sucrose and/or lactose and/or stachyose, and/or the glycosyltransferase of raffinose and/or verbascose, fructosyl transferase or Sucrose:glucan alpha1.

4. according to the composition of claim 3, wherein lactic microorganisms belongs to lactobacillus.

5. according to the composition of claim 4, the lactic-acid-bacterium of wherein said work is the picogram lactobacillus species, plant lactobacillus kind or lactobacillus salivarius kind.

6. according to the composition of claim 5, wherein said EPS is by by the picogram lactobacillus species that can produce Sucrose:glucan alpha1, and plant lactobacillus kind or the fermentation of lactobacillus salivarius kind obtain.

7. according to the composition of claim 6, wherein said EPS is homotype EPS.

8. according to the composition of claim 7, wherein said homotype EPS comprises at least a polysaccharide and/or a kind of oligosaccharide compositions.

9. according to the composition of claim 8, polysaccharide and/or the oligosaccharide compositions of wherein said EPS comprise glucose.

10. according to the composition of claim 3, wherein lactic microorganisms belongs to leuconos toc.

11. according to the composition of claim 10, the lactic microorganisms of wherein said work is a Leuconostoc mesenteroides.

12. according to the composition of claim 11, wherein said EPS is by being obtained by the Leuconostoc mesenteroides fermentation of expressing glycosyltransferase or fructosyl transferase.

13. according to the composition of claim 12, wherein the polysaccharide of EPS and oligosaccharide compositions comprise Polylevulosan or dextran.

14. according to the composition of claim 13, wherein oligosaccharides is fructo-oligosaccharides or glucose oligosaccharides.

15. according to the composition of aforementioned any one claim, wherein the amount of the EPS of Sheng Chenging can be conditioned.

16. according to the composition of claim 15, the lactic microorganisms quantity of the amount of wherein said EPS by living, fermenting process length, culture temperature, pH or acceptor molecule maltose are regulated.

17. according to the composition of aforementioned any one claim, wherein said EPS improves and contains the texture of described EPS as the food of composition, quality, mouthfeel, viscosity, structure and/or organoleptics property.

18. according to the composition of aforementioned any one claim, wherein said composition is used to the fermented milk and preparation contains the EPS that forms structure and/or the sour milk constituents of trophicity oligosaccharides.

19. according to the composition of aforementioned any one claim, wherein when when acting on a kind of composition of edible product or medicament production, said composition is as probiotics.

20. according to the composition of aforementioned any one claim, wherein when when acting on the composition of edible product or medicament production, the component of composition can reduce the gas that is produced by gastrointestinal microorganisms.

21. according to the composition of aforementioned any one claim, wherein said composition is a conc forms.

22. according to the composition of aforementioned any one claim, wherein said composition is cryodesiccated, and is spray-dired and/or resuspended.

23. a method for preparing the product that is used to eat, described method comprise composition is mixed with another kind of component to generate the described product that is used to eat; Wherein said composition is the composition according to any one of claim 1-16.

24. product that is used to eat that obtains by the method for claim 23.

25. a method that is used to prepare foods prods, described method comprise composition is mixed with another kind of component to generate described foods prods; Wherein said composition is the composition according to any one of claim 1-16.

26. foods prods that obtains by claim 25.

27. according to the product of claim 24 or 26, wherein said composition is added in the milk preparation, in the preferred sour milk.

28. according to the product of claim 27, wherein said composition is before the fermentation of described milk and/or be added in the sour milk afterwards.

29. according to the product of claim 27 or 28, wherein this product is a functional foodstuff.

Prepare described medicament production 30. a method that is used to prepare medicament production, this method comprise composition mixed with another kind of component, wherein said composition is the composition according to any one of claim 1-16.

31. a method that is used to prepare according to medicament production or a kind of pharmaceutical cpd of claim 30, this method comprises mixes composition of the present invention with another kind of component.

32. medicament production by the method preparation of claim 30 or 31.

33. comprise the container of composition, wherein said composition is the composition according to any one of claim 1-16.

34. comprise the container of composition, wherein said composition is the composition according to any one of claim 1-16, and has purposes that indicates the GI microbial balance of improvement behind edible this product and/or the label that gets the Green Light and use on the wherein said container.

35. the method for the enzyme producing EPS or define in any aforesaid claim comprises:

In commercial qualified nutrient solution, cultivate and be selected from the Leuconostoc mesenteroides kind that production can be synthesized the enzyme of EPS, the picogram lactobacillus species, the Bacterium lacticum of plant lactobacillus kind and lactobacillus salivarius kind and selectively separates EPS and/or enzyme.

36. a method that is used for preparing the EPS that defines as any aforesaid claim comprises:

Adding suitable enzyme substrates, sucrose for example, lactose, raffinose, stachyose or verbascose and be selected from the Leuconostoc mesenteroides kind that production can be synthesized the enzyme of EPS, picogram lactobacillus species, the Bacterium lacticum of plant lactobacillus kind and lactobacillus salivarius kind as cultivating in the commercial qualified nutrient solution of the acceptor molecule maltose of sole carbon source, selectively, separate EPS.

37. the EPS that the method by claim 36 is produced is used to regulate the purposes of the viscosity of the product that is used to eat.

38. one kind is used to prepare method for compositions, is included in the commercial qualified nutrient solution original position and cultivates described lactic microorganisms, reaches 10 up to the quantity of described lactic microorganisms ³To 10 ¹¹/ ml is preferably 10 ⁵To 10 ¹⁰/ ml and be more preferably 10 ⁹To 10 ¹⁰/ ml, wherein said lactic microorganisms production can be synthesized the enzyme of EPS.

39. in the purposes of preparation in the foods prods, wherein said composition improves the texture of this foods prods, quality, mouthfeel, viscosity, structure and/or organoleptics property according to any one the composition of claim 1-16.

40. an analytical procedure of screening composition, said composition is used for purposes described here substantially, and this analytical procedure comprises that the composition with the candidate adds in the foods prods, and definite foods prods is at texture, quality, mouthfeel, viscosity, the improvement degree of structure and/or organoleptics property aspect; Wherein said composition comprises lactic-acid-bacterium alive, from enzyme of described lactic-acid-bacterium and the EPS that is produced by described enzyme.

41. the composition conduct is the purposes of functional food ingredients substantially as described herein.

42. composition is as the purposes of the food ingredient of probiotics substantially as described herein and/or probiotics.

43. composition, foods prods, method, treatment process, purposes or analytical procedure substantially as described herein.

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