CN102747026A

CN102747026A - Use of compounds involved in biosynthesis of nucleic acids to increase yield of bacterial cultures

Info

Publication number: CN102747026A
Application number: CN2012102077386A
Authority: CN
Inventors: 罗格·温德尔·克林格拉姆; 尼尔斯·马丁·索罗森; 克里斯蒂·加里格斯; 马丁·B.·皮德森; 苏珊娜·格恩
Original assignee: HANSENS LAB
Current assignee: HANSENS LAB
Priority date: 2005-01-05
Filing date: 2006-01-05
Publication date: 2012-10-24
Also published as: DK1841855T3

Abstract

The invention relates to use of compounds involved in biosynthesis of nucleic acids to increase yield of bacterial cultures. Microbial starter cultures are provided. More specifically, a method for preparing a microbial starter culture wherein the microorganism is inoculated in a culture medium comprising at least one yield enhancing agent selected from the group consisting of a purine base, a pyrimidine base, a nucleoside, a nucleotide and a derivate thereof. Such microbial starter cultures are useful in the manufacturing of food, feed and pharmaceutical products.

Description

Compound at biosynthesizing nucleic acid with the application in the productive rate that increases bacterial cultures

The application is to be on January 5th, 2006 applying date, and denomination of invention is divided an application for the one Chinese patent application 200680007208.X's of " compound at biosynthesizing nucleic acid with the application in the productive rate that increases bacterial cultures ".

Technical field

The present invention relates to the mikrobe starter culture.More specifically; The invention provides the preparation method of milk-acid bacteria (LAB) starter culture; Wherein, Milk-acid bacteria is inoculated in the developing medium that contains at least a gain in yield agent and cultivates, and this gain in yield agent is to be selected from compound or the group that its one or more verivates are formed that is used for the biosynthesizing of nucleic acid by one or more.This starter culture can be used in the manufacturing of food, feed and pharmaceutical products.

Background technology

Microorganisms cultures is widely used in food, feed and medicine industry, in order to produce fermented product, comprises most milk preparations, for example cheese, sour milk and butter, and also comprise meat, bake, in wine or the vegetables product.In addition, microorganisms cultures also is used to produce protein, comprises enzyme and multiple useful compound.These microorganisms cultures are commonly referred to as starter culture, and quilt is produced and is distributed to the fermentation industry in industriallization breeding factory, and in milk products plant, wherein, starter culture is used to their production process.Lactic acid bacteria culture especially is widely used as starter culture.

Terminology used here " milk-acid bacteria " (LAB) is meant by Gram-positive, littlely has a liking for oxygen bacterium or anerobes, and it ferments to sugar, comprises lactic acid (for the main acid that produces), acetic acid and propionic acid in the acid that is produced.The most useful milk-acid bacteria is the milk-acid bacteria that belongs in lactococcus genus (Lactococcus species (spp.)), streptococcus (Streptococcus spp.), lactobacillus genus (Lactobacillus spp.), leuconos toc (Leuconostoc spp.), pediococcus acidilactici genus (Pediococcus spp.), brevibacterium sp (Brevibacterium spp.), faecalis (Enterococcus spp.) and the propionibacterium (Propionibacterium spp.) in the industry.In addition, belong to the bifidus bacillus (bifidobacteria) in the bacterium of lactic acid producing of strictly anaerobic flora, i.e. genus bifidobacterium, its normal coverlet is private to be made the food starter culture or is used in combination with milk-acid bacteria, is included in usually in the milk-acid bacteria group.Or even some staphylococcus (for example; Staphylococcus carnosus (S.carnosus), Staphylococcus equorum (S.equorum), Staphylococcus sciuri (S.sciuri), calf staphylococcus (S.vitulinus), staphylococcus xylosus (S.xylosus)) bacterium in belonging to also be called as LAB (Mogensen etc. (2002) Bulletin of the IDF No.377,10-19).

The production of LAB starter culture relates to LAB cell inoculation with proper amt in specific fermentation media, under suitable fermentation condition, breeds.In suitability for industrialized production, need do one's utmost to accomplish when fermenting process finishes, to obtain the cell of the breeding of high density.This is just to fermentation condition and the fermentation media generation high requirement of sustenticular cell growth with the high biological quality productive rate of acquisition expectation.

Liquid medium is at the optical density (OD) (OD of 600nm ₆₀₀) be a kind of exact method of estimating bacterial cell density in the culture samples.Term " high optical density (OD) condition " is meant that when fermenting process finished, the cell concn of breeding was up to OD ₆₀₀Be 10 or above fermentation.

In order to reduce production costs, industrialized fermentation uses complicated indefinite fermentation media to carry out usually.The staple of such medium can be yeast extract, W-Gum, whey proteins or other medium based on milk, and they all have complicated composition.For the fermentation of selecting, the medium that is to use the common chemical ingredients of processing by pure chemical substance to confirm.Pure chemical substance, for example specific energy source or carbon source also are added to usually and are used for specific purposes in the complex ferment medium.In either case, possibly be optimized as far as the compsn of the survival ability fermentation media of microorganism cells, but for the biological quality productive rate that obtains high mikrobe, just be not necessarily optimized.

The required compound of most cell growths is to require the expenditure of energy so that cells produce to be provided.This needs through expressing the gene of the corresponding biosynthetic enzyme of coding usually.The synthetic amino acid and the energy of needing of these enzymes.Grow owing to must synthesize more relatively enzyme, applied " protein burden " with regard to pair cell like this.Must obtain from other approach owing to form the required precursor of cellular constituent, this has brought extra burden to cell again.

More used compounds are found to be and are used for so-called cryoprotectant in biological nucleic acid is synthetic, and reduce the damaging influence of in freezing and the course of defrosting viability of viable cell being brought.WO00/39281 has described in the biosynthesizing of DNA, uses inosinate (IMP) and other compound to come the metabolic activity of the liquid starter culture in the stably stored.

Be known that LAB has complicated growth factor needs, and the compound that in the biosynthesizing of DNA and/or RNA, relates to has the growth that excites LAB in the medium of having confirmed chemical constitution.But some report demonstration, have produced short lag phase or higher initial growth speed although add these compounds, do not have or only improve productive rate (Klistrup (2005) FEMS Microbiol Rev.29,555-590 slightly; Nygaard (1951) J Bact 61,497-505; Weinman (1964) J Bact 87,263-269).Add relate to the compound in the DNA biosynthesizing in addition possibly suppress productive rate (Weinman (1964) J Bact 87,263-269).

As shown in

embodiment

3 and 4, adding the compound that relates in the DNA biosynthesizing not have increase to reach the productive rate of biological quality of the fermentation of high optical density (OD) condition yet.

Therefore, need provide a kind of new method to improve the productive rate of the biological quality of the fermentation of under high optical density (OD) condition, carrying out.

Summary of the invention

The problem that the present invention solves can be through the microorganisms cultures that provides a kind of method to prepare LAB, under high optical density (OD) condition, to improve the productive rate of biological quality.

Richardson is at 1936 (Biochem J.30,2186) report once, and uridylic is most important for staphylococcic anaerobic growth, when unimportant to identical organic aerobic growth.Therefore, the compound that in biological nucleic acid is synthesized, relates to adds in the complicated fermentation media, and makes us feeling very to be to find that the LAB starter culture might improve the productive rate of biological quality in aerobic cultivation or production in surprise.

Therefore, first aspect of the present invention relates at ventilation and high optical density (OD) condition bottom fermentation, the method for the productive rate of the lactic acid bacteria culture that acquisition increases, and this method may further comprise the steps:

I) in developing medium, cultivate milk-acid bacteria, and make that under certain condition fermentation proceeds to the optical density (OD) (OD that measures at 600nm ₆₀₀) be 10; Wherein, Said developing medium comprises at least a gain in yield agent, and it is selected from the group of being made up of purine bases, pyrimidine bases, nucleosides, Nucleotide and their verivate, and concentration is to guarantee that when fermentation ends developing medium contains the said at least a gain in yield agent of at least 1 μ M; And

Ii) collect said milk-acid bacteria with the acquisition lactic acid bacteria culture,

Wherein, compared with under the same conditions with similar medium in but contain each the gain in yield agent that is less than 1 μ M when the fermentation ends and come under the situation of culturing micro-organisms, the gain in yield agent has increased the productive rate of the milk-acid bacteria of collecting.

Preferably, said developing medium contains at least a gain in yield agent of at least 5 μ M, and for example, at least 10 μ M are like at least a gain in yield agent of at least 50 μ M.Special, said developing medium contains the reagent of the group that is selected from IMP, GMP, inosine and guanine of at least 5 μ M.

Preferably, said similar medium contains each the gain in yield agent that is less than 5 μ M, for example is less than 1 μ M, as is less than each gain in yield agent of 0.5 μ M.Special, said similar medium contains the reagent of the group that is selected from IMP, GMP, inosine and guanine that is less than 5 μ M.

Second aspect of the present invention relates to the starter culture that obtains according to first aspect present invention described here and its embodiment.

The third aspect of the invention relates to developing medium, and it comprises at least a gain in yield agent, and this gain in yield agent is selected from the group of being made up of purine bases, pyrimidine bases, nucleosides, Nucleotide and their verivate.

Fourth aspect of the present invention relates to the method for preparing food, feed goods, pharmaceutical products, milk flavor goods and cheese flavor goods; Said method comprises the mikrobe starter culture that in the parent material of food, feed or pharmaceutical products, adds according to the significant quantity of second aspect present invention and its embodiment, and the parent material of this inoculation is under the condition of microbe body maintenance metabolic activity.

The 5th aspect of the present invention relates to by the fourth aspect present invention of the description that Clicks here and food, feed or the pharmaceutical products of the fermentation of its embodiment acquisition.

Definition

Before the detailed embodiment of invention is done to describe, to used particular term further definition is provided here earlier.

Here, term " purine bases " is intended to comprise the base of encircling nitrogen that contains with purine core texture.Therefore, in this article, term " purine bases " means optional substituted purine.The example of concrete purine bases comprises VITAMIN B4, guanine, xanthine and xanthoglobulin.

Similarly, term " pyrimidine bases " is intended to comprise the base of encircling nitrogen that contains with pyrimidine core texture.Therefore, in this article, term " pyrimidine bases " means optional substituted pyrimidine.The example of concrete pyrimidine bases comprises cytosine(Cyt), thymus pyrimidine and uridylic.

Among this paper; Term " Nucleotide " means 2-deoxyribosyl (DNA) monomer or ribose (RNA) monomer; It is connected to purine bases through No. 1 carbon atom; On VITAMIN B4, guanine, xanthine or xanthoglobulin, or be connected to pyrimidine bases, on cytosine(Cyt), thymus pyrimidine or uridylic through No. 1 carbon atom.In addition, DNA or RNA monomer are connected on the phosphate through its No. 5 carbon atoms.The example of concrete Nucleotide comprises adenosine monophosphate (AMP), guanosine monophosphate (GMP), uridine list phosphoric acid (UMP), cytosine riboside monophosphate (CMP), xanthine list phosphoric acid (XMP), inosine list phosphoric acid (IMP), Desoxyadenosine list phosphoric acid (dAMP), pancreatic desoxyribonuclease list phosphoric acid (dGMP), thymidine 5'-monophosphate (dTMP), Deoxyribose cytidine list phosphoric acid (dCMP), deoxidation xanthine list phosphoric acid (dXMP) and Hypoxanthine deoxyriboside list phosphoric acid (dIMP).IMP is particularly preferred.

Terminology used here " nucleosides " means 2-deoxyribosyl (DNA) monomer or ribose (RNA) monomer; It is connected to purine bases through No. 1 carbon atom; On VITAMIN B4, guanine, xanthine or xanthoglobulin; Or be connected to pyrimidine bases through No. 1 carbon atom, on cytosine(Cyt), thymus pyrimidine or uridylic.Concrete nucleosides example comprises adenosine, guanosine, uridine, cytidine, inosine, Desoxyadenosine, pancreatic desoxyribonuclease, deoxythymidine, Deoxyribose cytidine and Hypoxanthine deoxyriboside.Inosine is particularly preferred.

Be appreciated that by above definition Nucleotide can be considered to comprise the nucleosides through No. 5 phosphate that carbon atom is connected of sugar unit to term " nucleosides " and " Nucleotide ".Therefore, Nucleotide described herein also can think " nucleosides "-5 '-single phosphoric acid.For example, t-inosinic acid (IMP) can refer to inosine-5 '-single phosphoric acid, deoxyinosine-5'-monophosphate (dIMP) (dIMP) can refer to Hypoxanthine deoxyriboside-5 '-single phosphoric acid etc.

Among this paper; Term " verivate " is when with term " Nucleotide " or " nucleosides " logotype; Mean those and the Nucleotide or nucleosides of modification taken place in its sugar (being 2-deoxyribosyl or ribose) unit; Or those contain the ring nitrogen base at it the Nucleotide or the nucleosides of modification have taken place, or those contain Nucleotide or the nucleosides that modification has all taken place the ring nitrogen base at its sugar unit and its.For example, ribodesose is unitary 2 '-H base or ribose is unitary 2 '-modification can take place in the OH base, for example, add 2 '-F base, 2 '-the O-methyl etc.Similarly, contain the base of encircling nitrogen and can contain one or more substituting groups on VITAMIN B4, guanine, xanthine, xanthoglobulin, cytosine(Cyt), thymus pyrimidine and the uridylic that do not appear at usually.Concrete example comprise 5-methylcytosine ( ^MeC), iso-cytosine, false iso-cytosine, 5-bromouracil, 5-proyl uridylic, 5-propine-6-Fluracil, 5-methylthiazol uridylic, adenine, 2-aminopurine, 2,6--diaminopurine, 7-propine-7-denitrification VITAMIN B4,7-propine-7-denitrification guanine and 2-chloro-adenine.

Term used herein " fermentation " is meant under aerobic or anaerobic condition the process of breeding or microorganisms cell.

Term used herein " starter culture " is meant the configuration thing that contains microorganism cells of the developing medium that is used to ferment.

In this article, term " productive rate " is meant the biological quality that in the fermentation of given volume, is produced.Productive rate can calculate through number of ways; Here, adopt two kinds of different methods to measure productive rate.1) is the biological quality (subtracting background) of the per unit of volume, when fermentation ends, passes through the optical density (OD) (OD that the 1cm light path is measured the 600nm of fermentation media ₆₀₀), perhaps 2) through at embodiment 2: have " souring activity " in the Pearce test of describing among the analytic process QAm-043 during fermentation ends measured its kg number for the F-DVS culture of 4.8-5.1.

Term " F-DVS " is meant the so-called freezing direct-throwing culture of in embodiment 1, describing.

Term " porphyrin compound " is meant ring-type tetrapyrrole verivate, and the extensibility of structure of these verivates is from porphyrin, is replaced the carbon atom at the place, summit that is positioned at pyrroles's core and is got by various functional groups.They also refer to the mixture of said verivate, wherein, form co-ordination bond by two in four nitrogen-atoms of atoms metal and porphyrin ring.This definition includes but not limited to: uroporphyrin, cp, protoporphyrin and porporino, and their salt and ester and with the mixture of atoms metal.Preferred especially porphyrin compound is a protoporphyrin IX and mixture, particularly protoheme and the protohemine of it and iron atom, and chlorophyllous verivate, like CHLOROPHYLLINE.

In this article; Words and phrases " milk-acid bacteria " (LAB) are meant by Gram-positive, katalase are negative, non-motion, little and have a liking for one group of bacterium that oxygen or anerobes are formed; They ferment to sugar, comprise in the acid that is produced that lactic acid is the main acid that produces, and comprise acetic acid, formic acid and propionic acid.The most useful milk-acid bacteria is the milk-acid bacteria that belongs in lactococcus genus (Lactococcus species (spp.)), streptococcus (Streptococcus spp.), lactobacillus genus (Lactobacillus spp.), leuconos toc (Leuconostoc spp.), pediococcus acidilactici genus (Pediococcus spp.), brevibacterium sp (Brevibacterium spp.), faecalis (Enterococcus spp.) and the propionibacterium (Propionibacterium spp.) in the industry.In addition, belong to the bifidus bacillus (bifidobacteria) in the lactic acid-producing bacteria of strictly anaerobic flora, i.e. genus bifidobacterium, normal coverlet is private to be made the food starter culture or is used in combination with milk-acid bacteria, is included in usually in the milk-acid bacteria group.Or even some staphylococcus (for example; Staphylococcus carnosus (S.carnosus), Staphylococcus equorum (S.equorum), Staphylococcus sciuri (S.sciuri), calf staphylococcus (S.vitulinus), staphylococcus xylosus (S.xylosus)) bacterium in belonging to also be called as LAB (Mogensen etc. (2002) Bulletin of the IDF No.377,10-19).

Usually the milk-acid bacteria that is used as LAB starter culture bacterial strain roughly is divided into has a liking for warm organism, and its optimum growth temperature is at about 30 ℃, and thermophilic organism, and its optimum growth temperature range is between about 40 to about 45 ℃.Typically belong to mesophilous organism and comprise lactococcus lactis ssp (Lactococcus lactis); Lactococcus lactis subsp (Lactococcus lactis subsp.cremoris); Leuconostoc mesenteroides sub species cremoris (Leuconostoc mesenteroides subsp.cremoris); Pediococcus pentosaceus (Pediococcus pentosaceus); Lactococcus lactis subsp.lactis di-acetyl lactic biological mutation (Lactococcus lactis subsp.lactis biovar.diacetylactis); The secondary cheese subspecies (Lactobacillus paracasei subsp.paracasei) of lactobacterium casei cheese subspecies (Lactobacillus casei subsp.casei) and lactobacillus paraceasi.The bacterial classification of thermophilic milk-acid bacteria comprise as, thermophilus streptococcus (Streptococcus thermophilus), faecium (Enterococcus faecium), lactobacillus delbruckii lactic acid subspecies (Lactobacillus delbrueckii subsp.lactis), lactobacterium helveticus (Lactobacillus helveticus), lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus) and Lactobacterium acidophilum (Lactobacillus acidophilus).

The concentration of the gain in yield agent in medium can owing to as therefore enter into the content of microorganism cells and, be necessary to indicate and will measure or the specific time point of the concentration of definite gain in yield agent along with the time changes.Therefore; The term " initial " of concentration that is used for describing the gain in yield agent of medium is meant the concentration of the gain in yield agent in the medium that in medium, adds before microorganism cells is cultivated, and perhaps is meant the concentration of the gain in yield agent in the medium that in medium, has just added after microorganism cells has been cultivated.

An important use according to starter culture of the present invention is as so-called probiotic bacterium.In this article, after term " probiotic bacterium " should be understood that those are ingested by the human or animal with the form of viable cell, the healthiness condition of improvement can be provided, for example suppress harmful microorganism in the gi tract through the enhancing immunity system or the nutrition that helps digest.An exemplary of the product that prebiotic effect is arranged like this is " a sweet sour milk ".

Below only embodiment of the present invention is described through embodiment.

Description of drawings

Fig. 1 shows the productive rate of the fermentation of under anaerobic carrying out 3 thermophilus streptococcuses (S.thermophilus); Accompanying drawing shows in fermentation container, the biological quality of measurement be non-spissated fermentation media sample OD600 to the time (hour) function.Solid triangle representes to have added 0.2%w/w IMP, and filled circles has represented to add the inosine of 0.2%w/w, and closed square representes not add the gain in yield agent.The inosine of 0.2%w/w is approximately the inosine of 7mM;

Fig. 2 in comprising the medium that is rich in mixture of a large amount of relatively purine, the multiple nuclear compound during the fermentation and the level of optical density (OD); In brief, lactococcus lactis ssp (bacterial strain CHCC2862) carries out grow aerobically in the complex media that comprises yeast extract and other mixture composition.The concentration (main shaft) and the OD600 (secondary axes) that represent with μ M mapped to the time.Abbreviation: G, guanine; A, VITAMIN B4; Hx, xanthoglobulin; X, xanthine; IR, inosine; GR, guanosine; GdR, pancreatic desoxyribonuclease; AR, adenosine;

The level of the purine compound in the fermenting process of Fig. 3 in the medium of the inosine that has added 2g/L; In brief, except in every liter of medium, having added the inosine of 2g, lactococcus lactis ssp (bacterial strain CHCC2862) carries out grow aerobically in the complex media identical with Fig. 2.Concentration (main shaft) and OD600 * 100 (secondary axes) so that μ M representes were mapped to the time.Notice that the level of inosine is to be represented by secondary axes.Abbreviation: G, guanine; A, VITAMIN B4; Hx, xanthoglobulin; X, xanthine; IR, inosine; GR, guanosine; GdR, pancreatic desoxyribonuclease; AR, adenosine, and IR, inosine (attention) with different axle expression inosines.

Embodiment

Scheme of dealing with problems according to the present invention provides the method for the microorganisms cultures of preparation LAB; With the productive rate that obtains under high optical density (OD) condition, to increase; This method is under aerobic conditions; Fermenting culture in the medium that contains at least a gain in yield agent, this gain in yield agent are selected from by one or more and relate to the biosynthetic compound of nucleic acid or the group of being made up of one or more verivates of any such compound.

Be not limited to any theory, can think that the increase of productive rate is relevant with the situation that at least a gain in yield agent is present in the fermentation media always in the process of whole fermentation.Observe when the gain in yield agent in the medium is exhausted, caused synthetic again (the seeing embodiment 6) of plurality of enzymes system, and can think the synthetic again reduction that has caused productive rate of this power consumption.

The situation that this at least a gain in yield agent is present in the process of whole fermentation in the fermentation media always can be cultivated LAB in the medium through the initial gain in yield agent that contains capacity, thereby guarantees that at least a such reagent remaines in the medium in the process of whole fermentation all the time.Such medium for example can be originally to contain 1mM at least; Preferred 3mM at least; The more preferably developing medium of at least a gain in yield agent of 3mM at least, these gain in yield agent are selected from by one or more and relate to the biosynthetic compound of nucleic acid or the group of being made up of one or more verivates of any such compound.

Such medium can be through using the configuration of assigning to of those one-tenth that particularly are rich in the gain in yield agent, and these gain in yield agent are selected from compound or the group of being made up of one or more verivates of any such compound that is related to the biology of nucleic acid by one or more.A kind of such composition can be a yeast extract, particularly so-called " enrichment " or " enhanced " yeast extract preparations, and it is rich in purine and/or pyrimidine especially.

The preparation that is rich in the gain in yield agent except use comes the collocating medium, also can in the medium prescription of standard, add pure preparation.For example, developing medium can be the complex ferment medium, in every liter of medium, adds 0.2g at least, is preferably 0.8g at least, more preferably at least a gain in yield agent of 2g at least.

Also can add said at least a gain in yield agent, thereby guarantee in the process of whole fermentation, to have at least a kind of gain in yield agent to remain in the medium through one or many in fermentation.

The present invention is particularly useful under the high optical density (OD) condition that many industry member adopt.In the embodiment of selection of the present invention, the characteristic of said high optical density (OD) condition is when fermentation ends, OD ₆₀₀Be higher than 15, preferably be higher than 20, more preferably be higher than 30, further preferably be higher than 40, most preferably be higher than 50.

In preferred embodiment; At least 1.2 times of the gain in yield of the mikrobe that obtains in the wherein said first aspect present invention method are preferably at least 1.3 times of increases, more preferably increase at least 1.4 times; Further be preferably at least 1.5 times of increases, most preferably be at least 1.6 times of increases.

According to the present invention, mikrobe is under aerobic conditions, to ferment.Preferably, microorganisms cultures ferments in ventilation and nutrient media, contains or added at least a porphyrin compound in the medium.In preferred embodiment; LAB cultivates in the nutrient media that contains porphyrin under aeration condition; This has description in WO00/0542; Wherein, said medium further contains at least a gain in yield agent, and it is selected from by one or more and relates to the biosynthetic compound of nucleic acid or the group of being made up of one or more verivates of any such compound.WO00/0542 is annex as a reference.Those skilled in the art can ventilate with the skill of knowing, for example, and through shaking or the stir culture medium, or through in developing medium, feeding the gaseous mixture like air that contains aerobic.

In preferred embodiment, said gain in yield agent is to be selected from by purine bases, pyrimidine bases, nucleosides, Nucleotide, and the group of their verivate composition.

Said gain in yield agent can be purine bases, preferably is selected from the group of being made up of VITAMIN B4, guanine, xanthine and xanthoglobulin.

Said gain in yield agent can be pyrimidine bases, preferably is selected from the group of being made up of cytosine(Cyt), thymus pyrimidine and uridylic.

Said gain in yield agent can be a nucleosides, and preferably, wherein said nucleosides is to be selected from the group of being made up of adenosine, guanosine, uridine, cytidine, inosine, Desoxyadenosine, pancreatic desoxyribonuclease, deoxythymidine, Deoxyribose cytidine and Hypoxanthine deoxyriboside.

In preferred embodiment, said nucleosides is to be selected from the group of being made up of adenosine, guanosine, uridine, cytidine, thymidine and inosine.Most preferably, wherein said nucleosides is an inosine.

Said gain in yield agent can be a Nucleotide; Preferably, said Nucleotide is to be selected from the group of being made up of adenosine monophosphate (AMP), guanosine monophosphate (GMP), uridine list phosphoric acid (UMP), cytosine riboside monophosphate (CMP), xanthine list phosphoric acid (XMP), inosine list phosphoric acid (IMP), Desoxyadenosine list phosphoric acid (dAMP), pancreatic desoxyribonuclease list phosphoric acid (dGMP), thymidine 5'-monophosphate (dTMP), Deoxyribose cytidine list phosphoric acid (dCMP), deoxidation xanthine list phosphoric acid (dXMP) and Hypoxanthine deoxyriboside list phosphoric acid (dIMP).

In preferred embodiment, said Nucleotide is to be selected from the group of being made up of AMP, GMP, UMP, CMP, XMP and IMP.Most preferred, said Nucleotide is IMP.

One preferred embodiment is, said developing medium comprises at least two kinds of gain in yield agent, preferably is selected from the group of being made up of purine bases, pyrimidine bases, nucleosides, Nucleotide and their verivate.

Preferably, said developing medium comprises at least two kinds of gain in yield agent, is selected from the group of being made up of nucleosides and Nucleotide.Most preferably, wherein said nucleosides is an inosine, and said Nucleotide is IMP.

One preferred embodiment is, wherein said developing medium is initiated with each the gain in yield agent that comprises 1-70mM.

Preferred, wherein said developing medium is initiated with each the gain in yield agent that comprises 1-60mM, and for example 1.3-60mM like 1.5-50mM, is preferably 2-40mM, like 2.5-30mM, and for example 3-20mM, more preferably 3-15mM, for example 4-10mM, 7mM according to appointment.

It is shocking,, can obtain sometimes that concentration is enough to be used in producing F-DVS and the LAB culture that need not to concentrate culture through method of the present invention.But even adopt present method, but most of culture need pass through concentrated and obtain commercial starter culture.Such culture can preferably be collected and concentrate through centrifugal or ultra-filtration.

One preferred embodiment in, the cultivation of mikrobe is under the industrial correlated condition of high optical density (OD) condition, to carry out.

Therefore, one preferred embodiment is that the high optical density (OD) (OD) of culture reaches OD in the 1cm of 600nm light path medium ₆₀₀=10-OD ₆₀₀=200, OD more preferably ₆₀₀=15-OD ₆₀₀=100, further OD preferably ₆₀₀=20-OD ₆₀₀=90, OD most preferably ₆₀₀=25-OD ₆₀₀=80.

In addition, preferred embodiment in, culturing process is to contain 5L-100,000L, preferred 300L-20 carries out in the big formula fermentation container of the developing medium of 000L.

One preferred embodiment in, culturing process comprises controlled temperature and/or pH.

Preferably; Culture comprises one or more organism; Be selected from by genus bifidobacterium (Bifidobacterium spp.), brevibacterium sp (Brevibacterium spp.), propionibacterium (Propionibacterium spp.), lactococcus genus (Lactococcus spp.) (comprising Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis), lactococcus lactis subsp (Lactococcus lactis subsp.cremoris)), lactobacillus genus (Lactobacillus spp.) (comprising Lactobacterium acidophilum (Lactobacillus acidophilus)), streptococcus (Streptococcus spp.), enterococcus spp (Enterococcus spp.), pediococcus acidilactici and belong to (Pediococcus spp.), leuconos toc (Leuconostoc spp.) and wine Coccus (Oenococcus spp.).

Culture can comprise one or more optimum growth temperatures at about 30 ℃ warm organism of having a liking for, and preferably one or more are selected from the warm organism of having a liking in the group of being made up of the secondary cheese subspecies of lactococcus lactis ssp (Lactococcus lactis), lactococcus lactis subsp (Lactococcus lactis subsp.cremoris), leuconostoc mesenteroides sub species cremoris (Leuconostoc mesenteroides subsp.cremoris), Pediococcus pentosaceus (Pediococcus pentosaceus), Lactococcus lactis subsp.lactis di-acetyl lactic biological mutation (Lactococcus lactis subsp.lactis biovar.diacetylactis), lactobacterium casei cheese subspecies (Lactobacillus casei subsp.casei) and secondary lactobacillus johnsonii (Lactobacillus paracasei subsp.paracasei).

Culture can comprise one or more optimum growth temperatures and arrive about 45 ℃ thermophilic organism at about 40 ℃, and preferably one or more are selected from the thermophilic organism in the group of being made up of thermophilus streptococcus (Streptococcus thermophilus), faecium (Enterococcus faecium), lactobacillus delbruckii lactic acid subspecies (Lactobacillus delbrueckii subsp.lactis), lactobacterium helveticus (Lactobacillus helveticus), lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus) and Lactobacterium acidophilum (Lactobacillus acidophilus).

Preferred culture is the LAB-culture, and it comprises one or more and is selected from the organism in the group of being made up of lactococcus genus (Lactococcus spp.), streptococcus (Streptococcus spp.), enterococcus spp (Enterococcus spp.), lactobacillus genus (Lactobacillus spp.), leuconos toc (Leuconostoc spp.), pediococcus acidilactici genus (Pediococcus spp.) and genus bifidobacterium (Bifidobacterium spp.).

Culture can be the LD-culture, and it comprises one or more and is selected from the organism in the group of being made up of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis), lactococcus lactis subsp (Lactococcus lactis subsp.cremoris), Lactococcus lactis subsp.lactis di-acetyl lactic biological mutation (Lactococcus lactis subsp.lactis biovar.diacetylactis) and leuconostoc mesenteroides sub species cremoris (Leuconostoc mesenteroides subsp.cremoris).

Culture can be the O-culture, and it comprises one or more and is selected from the organism in the group of being made up of Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) and lactococcus lactis subsp (Lactococcus lactis subsp.cremoris).

In preferred embodiment, contain lactococcus lactis ssp (Lactococcus lactis) in the culture.

Commercial starter culture usually can the refrigerated culture form supply with.At low temperatures, in such refrigerated culture, most of Metabolic activity of cell stops usually, but cell can be kept its vigor in the time of an elongated segment in this suspension-s.

Because spissated frozen cultures can directly be cultivated in producing container, thereby commercial very attractive.Through using so spissated frozen cultures, the final user has eliminated the middle fermentation step of wasting time and energy, and in this step, starter culture is through amplification, and the final user has also further reduced the danger of strong pollution.Spissated nutrient solution like this can be called as the DVS-direct-throwing ^TMCulture.

Also can prepare FD-DVS ^TM, spissated freeze dried direct-throwing ^TMCulture replaces spissated frozen cultures.Such culture has extra advantage, and their advantage is can be freezing and transport.

Therefore, one preferred embodiment in, the method that is used to prepare the microorganisms cultures of gain in yield is described below and further comprises:

Iii) the mikrobe of freezing said collection is to obtain the refrigerated microorganism cells.

Said method can further comprise:

Iv) from said refrigerated cell, make water sublimed to obtain freeze dried cell.

In other words, wherein collected microorganisms cultures is converted into freeze dried cell culture.

This method can further comprise:

V) pack the cell that obtains in iii) or iv) by above-mentioned steps.

Usually observe and freezingly can produce damaging influence with the survival ability that thaws to the cell of living.These influences generally are described to cell dehydration and in refrigerating process, in cytosol, form ice crystal.

Yet, can use some cryoprotectants to guarantee under controlled and the form that causes minimum injury, to carry out freezing, for example guarantee in refrigerating process, not take place in the cytosol ice crystalization or ice crystalization is suppressed to minimum.

Preferably, in the mikrobe of collecting, add at least a cryoprotectant.

Preferably, cryoprotectant is to be selected from the group of being made up of one or more one or more verivates that relate to biosynthetic compound or any such compound of nucleic acid.The example that preferably is suitable for joining the preferred cryoprotectant in the mikrobe of collection corresponds essentially to preferred gain in yield agent described here.In the mikrobe of collecting, add cryoprotectant and in the previous patent application PCT/DK2004/000477 that proposes, description was arranged.The preferred cryoprotectant of in PCT/DK2004/000477, describing also is the preferred cryoprotectant among the present invention.The full content of PCT/DK2004/000477 is incorporated into as a reference at this.

Other embodiment of the present invention is the method for the microorganisms cultures of preparation gain in yield described here, and further comprises through spraying drying, vacuum-drying, dry air or any next dry mikrobe of collecting of drying means that is applicable to the dry bacterial culture.

The starter culture of preferred second aspect of the present invention is that the form with the starter culture enriched material provides, and for example contains at least 10 ⁸The starter culture organism of CFU.

The third aspect of the invention relates to a kind of developing medium, and it comprises at least a gain in yield agent, is selected from the group of being made up of purine bases, pyrimidine bases, nucleosides, Nucleotide and their verivate.Although a large amount of during the fermentation gain in yield agent is consumed, still have in the supernatant that remaines in culture of q.s medium (seeing embodiment 5 or 6) to guarantee that spissated culture can be confirmed to be present method and draws.

The food article of preferred fourth aspect of the present invention is to be selected from the group of being made up of milk preparation, vegetables product, meat product, beverage, fruit juice, wine and baked goods.

Preferred milk preparation is to be selected from the group of being made up of the sweet milk and the liquid cultured-milk product of cheese, sour milk, butter and inoculation.

One interesting aspect, the invention provides the method that obtains gain in yield by the compound of microorganisms, said method comprises following steps:

I) culturing micro-organisms in the developing medium of the gain in yield agent in containing the group that at least a one or more verivates that are selected from the biosynthetic compound that related to nucleic acid by one or more or any such compound form; And

Iii) obtain said compound by microorganisms,

Wherein, with in same media when there not being the situation of culturing micro-organisms under measurable gain in yield agent condition to compare, the gain in yield agent has caused the increase by the productive rate of the compound of microorganisms.

Compound by described microorganisms includes but not limited to: enzyme, protein, metabolite, glycolipid class, microbiotic, bacteriocin, amino acid, monosodium glutamate, volatile matter.These compounds can pass through recombinant DNA technology or other produced in conventional processes.

The present invention further explains through following non-limiting example and accompanying drawing.

Embodiment

Embodiment 1: the productive rate that in three kinds of dissimilar developing mediums, ferments

For the effect that in medium enrichment and that optimize, adds the compound of extra purine-containing is described, compared the culture of three types industrial scale.

The cultivation of ventilating in the three types all culture bacterial strain nutrient medias; In this medium; Have at least a kind of porphyrin compound to exist; Or as being added the International Patent Application WO 00/05342 (EMIL method), and, in all cases; This culture is so-called " O-culture ", and it comprises Lactococcus lactis subsp.lactis (Lactococcus lactis subsp.lactis) and lactococcus lactis subsp (Lactococcus lactis subsp.cremoris).The O-culture be normally used for making atresia cheese (Cheddar, Cheshire, Feta).Concrete culture can obtain through the commercial channel, and commodity are called R 604, by Hansen A/S, and Hoersholm, Denmark (catalog number (Cat.No.): 200113) provide.

Three kinds of media are shown in Table 1.

^*BD-5-ex3 is the developing medium that contains porphyrin according to the optimization of WO 00/05342 and WO 01/52668.

^*Standard yeast extract and new yeast extract are two kinds can pass through the yeast extract that the commercial channel obtains.

^{* *}IMP is: inosine-5 '-single phosphoric acid (IMP) (Alsiano A/S, Birkeroed, DK).

glycosides is: inosine (Alsiano A/S; Birkeroed, DK).

Culturing process is in 550L or 10, and under 30 ℃, the above-mentioned culture as inoculum of use 0.5% (w/w) carries out in the industrial fermentation groove of 000L.Fermentation is to carry out under aerobic conditions by such described in the WO 00/05342.Fermented product is acidificable to be 6.2 to pH.Then, through controllably adding 13.4N NH ₄0H and pH is maintained 6.2.

When the consumption that does not have further alkali is detected, cool off corresponding culture to about 10 ℃.

After the cooling, the bacterium in the developing medium is concentrated to 6-18 doubly through centrifugal, in liquid nitrogen, is frozen into bead under 1 normal atmosphere then, processes so-called freezing direct-throwing culture (F-DVS).The F-DVS bead is stored in-50 ℃ and treats further analysis.

Confirm the productive rate of fermentation through two kinds of different modes, 1) through optical density (OD) (OD to measure at 600nm ₆₀₀) biological quality of acquisition of expression, perhaps 2) through at embodiment 2: have kg number that " souring activity " is the F-DVS culture of 4.8-5.1 in the Pearce test of describing among the analytic process QAm-043.

The result is shown in the following table 2.

Table 2. is with OD ₆₀₀The fermentation production rate of measuring

^*According to the Pearce test, the souring activity of F-DVS is 4.8-5.1

Conclusion:

Can find out from these results, in developing medium, before aerobic fermentation begins, add the toughener of forming by 0.2%w/wIMP and 0.2%w/w inosine, can increase productive rate.

Embodiment 2: analytic process QAm-043, souring activity-" sequencing temperature distribution " Chr.Hansen A/S (Denmark).

Use

Present method is used to confirming according to the souring activity of Pearce test.The Pearce test is adopted by IDF standard (international milk-product standard).

Principle

Acidifying is to carry out according to the temperature distribution that reflects warm change process, and this is that culture is used as when producing dairy products and can runs into usually.

For the Pearce test, this is the cheese production temperature when producing cheddar cheese-(Cheddar).PH measured in the set time

For those cultures that in analytic process, does not add rennet, can adopt successive pH to measure.

Analytical parameters

Analytical parameters is according to the specificity of product, and it provides in LIMS.

The definition of temperature distribution (for the product that does not use the Pearce test).

Use the control standard.

The type that pH measures.

Inoculation per-cent to sample and control standard.

Diluted milk: 206.9g cold (4 ℃) LAB-milk (that is, and the reconstruction skimmed milk (RSM) of UHT-sterilization, it contains 9.5% (w/w) solid matter, and 99 ℃ of heating 30 minutes).

Active milk: the sterilization full-fat milk of 200g cold (4 ℃), lipid content 3.5%.

Rennet:

border accurate 190 is diluted with water to 1: 40.

Device and reagent

PH appearance eks.

PHM92 that the semicontinuous pH of pH appearance/be used for measures.

PH electrode radiometer

Buffer reagent: pH 7.00 ± 0.01 and pH 4.01 ± 0.01.

Water-bath, band constant temperature program is in ℃ heating down of temperature distribution ± 0.2 of prediction.

TP.

Scale is accurate to 0.01g (two-decimal at least).

Timing register.

Magnetic stirrer.

Magnet.

Beaker, 50ml.

Little plastic cup.

Swivel arrangement.

Process

Analyze and prepare

All bottles are unified lot number, promptly produce on the same day.

Water-bath is adjusted to the starting temperature of the temperature distribution that will use earlier.

The bottle that is used to dilute (=the first weighs) and be used for activity (second weighs) and be placed on 4 ℃ before use.

Before calibration pH appearance, the damping fluid of pH 4.01 and pH 7.00 was placed on earlier in the water-bath of specified temp ± 0.2 ℃ 30 minutes at least.

The preparation of sample before analyzing.

Frozen cultures:

Freezing sample/reference standard is prepended in the foam box that is having dry ice weighing first, and is stored in wherein, up to all completion of weighing.

The refrigerated culture thaws earlier before use:

For frozen prods, when whole box by the time spent, according to current indication product is thawed.

After the thawing, the sample before using can be 4 ℃ of keep 30 minutes the longest.

Freeze dried culture:

Freeze dried sample and reference standard place under the room temperature to be analyzed at least in 15 minutes again.

If sample will be tested in next day once more, can be+8 ℃ of storages.

Seeded process

The direct weight of weighing product/reference standard in milk.

The actual amount of inoculum (weighing for the first time) is got 2 significant digits at least.

The careful product that overturns refrigerated and thaw 4 times then, lets bottle stand about 50 seconds.

To freeze dried product, must use swivel arrangement.Must dissolve fully with normal speed driving 5 minutes or up to product.This can be through staying bottle desk the preceding paragraph time, observes a bottle end then and check that solution controls.

Attention:

If work together words easily, before weighing for the second time, the cold bottle of weighing first can at room temperature be placed maximum 15 minutes.

Weigh for the second time:

Before carrying out weighing the second time, the upset dilution bottle.

The actual amount of inoculum (weighing for the second time) is got 2 significant digits at least.

Overturn active bottle, and carry out seeded process repeatedly by sample/control standard.

Those from the identical first time the weigh active bottle graft that begins to inoculate and inoculate.

The rennet that before or after weighing for the second time, in each bottle, adds 2ml.In these bottles of upset, rennet is also stirred.

Then, as described in 6, simultaneously bottle is inoculated.

At last, 2 nonvaccinated feeding bottles are placed in the water-bath; One is used to measure bath temperature, and another is used to measure the pH of blank milk.

Cultivate

Attention: when the more water-bath of needs, the control standard must be cultivated in identical water-bath with corresponding sample.

All active bottles are cultivated in the water-bath that is preheating to the predetermined start temperature all at one time.

Temperature distribution begins to launch in water-bath putting into bottle.

After this, cultivate temperature through the constant temperature time variable control, this program is corresponding to later specific temperature distribution.For the Pearce test, see table 3.

Horizontal plane in the water-bath should exceed 2cm at least than the surface of milk.

The temperature program(me) (IDF after) of table 3. in the Pearce spectrum

Time, minute	Temperature, ℃	Error
				0	31.0	±0.2℃
50	31.0	±0.2℃
			54	31.7	±0.5℃
58	32.2	±0.5℃
			62	32.8	±0.5℃
66	33.3	±0.5℃
			70	33.9	±0.5℃
73	34.4	±0.5℃
			76	35.0	±0.5℃
79	35.6	±0.5℃
			82	36.1	±0.5℃
85	36.7	±0.5℃
			87.5	37.2	±0.5℃
90	37.8	±0.2℃
			360	37.8	±0.2℃

The calibration of pH electrode

According to current indication, starting temperature is calibrated about electrode calibration and maintenance.

The measurement of pH

After the cultivation, bottle fully shakes and measures pH.

The measurement of pH is poured in the beaker of 50ml in bottle or with sample, under magnet stirs, carries out.

The pH value is got 2 significant digits.

Can write down note possible in the measurement.

Measuring process lasts till till all measured mistake of milk of all sample/reference standard and not cultivation.

PH in the final buffer liquid is through measuring and writing down.

The measurement of successive pH

, temperature distribution gathers the pH value of moment when beginning.After cultivating completion, be recorded in the pH observed value in the following two kinds of damping fluids of starting temperature.

Embodiment 3: the productive rate of the lactococcus lactis ssp fermentation of carrying out under standard anaerobic height-OD condition

For the effect that in medium enrichment and that optimize, adds the compound of extra purine-containing is described, the fermentation of having compared two kinds of dissimilar industrial scale has added the inosine of 0.3%w/w in a kind of, does not have in the another kind.

Culture:

R 604 cultures that can obtain through the commercial channel are used in this test, and (Denmark (catalog number: 200113)) carries out for Chr.Hansen A/S, Hoersholm.

Fermentation media:

Fermented product is cultivated in having the medium of following compsn: casein hydrolysate (Oxoid, Basingstoke, UK, product code name L41), 30g/l; Primatone RL (Quest, Naarden, The Netherlands, product code name 5X59051), 30g/l; Soya peptone (Oxoid, Basingstoke, UK, product code name L44), 30g/l; Yeast extract (Oxoid, Basingstoke, UK, product code name L21), 15g/l; MgSO4,1.5g/l; Sodium ascorbate, 3g/l; With lactose 50g/l.

(143 ℃ 8 seconds) are handled and sterilized to medium with UHT-.The pH of the medium of accomplishing is 6.5.

The fermentation condition of culture:

Cultivation be in the industrial fermentation groove of 550L under 30 ℃, use the above-mentioned culture of 1% (w/w) to carry out as inoculum.Under high OD condition, fermentation is anaerobic basically.It is 6.0 that culture allows to be acidified to pH.Then, through controllably adding 13.4N NH ₄OH and pH is maintained 6.0.When the consumption that does not have further alkali is detected, cool off corresponding culture to about 10 ℃.

After the cooling, the bacterium in the developing medium is concentrated to 6-18 doubly through centrifugal, in liquid nitrogen, is frozen into bead under 1 normal atmosphere then, processes so-called freezing throw type leaven culture (F-DVS).The F-DVS bead is stored in-50 ℃ and treats further analysis.

Confirm the productive rate of fermentation through two kinds of different modes:

1) through optical density (OD) (OD to measure at 600nm ₆₀₀) biological quality of acquisition of expression, perhaps

2) the kg number through F-DVS culture in every 100L fermentation media, wherein culture is through at embodiment 2: have " souring activity " in the Pearce test of describing among the analytic process QAm-043 is 4.8-5.1.

The result is shown in the following table 4.

Table 4.

Inosine is: and inosine (Alsiano A/S, Birkeroed, DK).

^§See embodiment 2

Conclusion:

Can find out from these results, in developing medium, before pressing aerobe fermentation to begin, add the toughener of forming by the 0.3%w/w inosine, not cause the increase of productive rate.

Embodiment 4: the productive rate of the streptococcus thermophilus fermentation that carries out under standard anaerobic height-OD condition

This test is the effect that adds the compound of extra purine-containing for research to the medium enrichment that is used for anaerobically fermenting under the high OD-condition and that optimize.In current test; The culture that has prepared three kinds of thermophilus streptococcuses; A kind of is the inosine that in medium, has added 0.2%w/w, and another kind is the IMP that in medium, has added 0.2%w/w, and last a kind of be the compound that in medium, does not add extra purine-containing.

Culture:

The culture CHQ-18 of the thermophilus streptococcus that can obtain through the commercial channel is used in this test, and (Chr.Hansen A/S, Hoersholm Denmark) carry out.

Fermentation media:

Fermented product is in the medium based on the enrichment of component, BioSpringer enzyme extract 207, Arla skim-milk (Milex 240) and the lactose of complex media, to cultivate.

(143 ℃ 8 seconds) are handled and sterilized to medium through UHT-.The pH value of the medium of accomplishing is 6.0.

The fermentation condition of culture:

Culturing process be in the stirring formula fermenter of 3L under 40 ℃, use the above-mentioned culture of 0.1% (w/w) to carry out as inoculum.PH is through adding 13.4N NH ₄OH and maintain 6.0.Guarantee anaerobic condition through feeding nitrogen (1.51/min) from headspace.Agitation condition is 300rpm.

Optical density (OD) (the OD of the productive rate of fermentation through measuring at 600nm with the not concentrating sample of gathering ₆₀₀) biological quality of acquisition of expression calculates.

The result of these 3 kinds of fermentations is shown in down among Fig. 1.

Conclusion:

Can find out from these results, add the toughener of forming by 0.2%w/w inosine or 0.2%w/w IMP, not cause the increase of productive rate.

Embodiment 5: adopt chemical analysis method to detect the existence of unnecessary nuclear compound in the fermentation

Though milk-acid bacteria is anauxotrophic as far as purine and pyrimidine usually, but still can synthesize these compounds, thereby cell can utilize obtainable external purine and pyrimidine source in fermentation.Concrete, all conventional purine nuclear compounds all can be just by the completely consumed (see figure 2) when about OD 15.What is interesting is that although purine compound is exhausted, the increase of biological quality still can continue until about OD 45.Similarly (data not shown goes out) also appears in the result in pyrimidine compound.This shows, fermented liquid (being acellular fermented product) is not purine-containing and pyrimidine compound producing when finishing.

On the other hand, in growth medium, added excessive inosine, this compound, and/or examine base xanthoglobulin (owing to the hydrolysis of inosine produces) accordingly and will when growth ending, appear at (see figure 3) in the fermented liquid.This excessive nuclear compound can detect in fermented liquid easily.

In order to produce like F-DVS etc., cell needs to concentrate times in fermented product.Although the cell levels among the F-DVS is that several times in the fermented product are high, in F-DVS, still there is very a large amount of fermented liquids.This pure fermented liquid can obtain through further concentrating cells.The method that is used for separate fermentation liquid can be through defrosting F-DVS, uses strainer then or in centrifugal, uses the higher g-power than production F-DVS.

Can test the existence whether nuclear compound is arranged with a spot of obtainable pure liquid in fermented product, thereby learn whether this compound is joined in the fermentation media by excessive.This detection method is traditional HPLC (as seeing http://www.laubscherlabs.com/Presentation/YMC%20ODS-AQ.pdf); Wherein, nuclear base and nucleosides (cytosine(Cyt), cytidine, uridylic, Deoxyribose cytidine, guanine, VITAMIN B4, xanthoglobulin, guanosine, xanthine, thymus pyrimidine, inosine, guanosine, Hypoxanthine deoxyriboside, pancreatic desoxyribonuclease, xanthosine-, thymidine, adenosine and Desoxyadenosine) commonly used can easily be detected.Other available method can be used for surveying corresponding Nucleotide.The existence of any of these compound will clearly show to ferment and carry out according to the present invention in fermented liquid.

Embodiment 6: use proteoplast to learn the existence that (proteomics) surveys excessive nuclear compound in the fermented product

In process of growth, what cell needed purine and pyrimidine nucleotide continues to flow to synthesize RNA and DNA.These Nucleotide can be supplied with by the dried rhizome of rehmannia outside medium (recovery) carried out, or carry out again synthetic from simpler compound.Synthetic for again, specific synthetic gene again must be expressed.On the contrary, when having exogenous origin, these genes do not need to be expressed.

In purine synthetic again, approximately relate to 10 gene prods.Found in the past to receive about 35 times adjusting, and this depends on existence/do not exist external purine source (Nilsson and Kilstrup 1998) with the relevant purDEK operon that synthesizes again at the purine of lactococcus lactis ssp.And, also find the some purine existence of synthetic proteins again/do not exist and depend on whether external purine source (Gitton etc. 2005) is arranged on the 2D protein gel.

In medium, have/do not exist the concrete grammar of external purine in order to establish detection, we are inoculating lactic acid lactococcus spp lactic acid subspecies CHCC2862 in containing the SA medium of confirming of 1% glucose.Do not contain nuclear compound (Jensen and Hammer 1993) in this medium.Culture is set in 30 ℃, is having and do not having overnight under the situation of 0.2% inosine.Then, the cell that is exponential growth is inoculated in fresh medium, and its OD600 is about 0.1.When OD is 0.8 (exponential growth), and at quiescent phase, collecting cell, and produce the 2D protein gel.

Usually, under pH scope 4-7, on gel, can detect about 3-400 protein spots.When OD 0.8, when not having inosine, be less than 10 points and from culture, obtain, when when inosine is arranged, there is not (or very faint) point from culture, to obtain.These some appearance/absent variable patterns show that corresponding proteins matter does not only just exist when having external purine source.Only appear at by 4 points the strongest on the gel that produces in the apueinic culture and carried out digestion and mass spectrum affirmation in the gel.These protein are confirmed to be: purH (difunctional purine biosynthesizing protein); PurM (Phosphoribosyl aminooimidazole synthetic enzyme (phosphoribosylaminoimidazole synthase)), yphF (Phosphoribosyl formylglycinamidine synthetic enzyme (phosphoribosylformylglycinamidine synthase PurS) and fhs (Tetrahydrofolic formylase (formyltetrahydrofolate synthetase)).These three kinds of gene purH, the gene product of purM and yphF (purS) is all directly relevant with the biosynthesizing again of purine, and wherein, fhs relates to the unitary formation of carbon atom, and it is used for the synthetic again of purine.For the cell that is in quiescent phase, the situation among this and the F-DVS is similar, has also obtained similar nothing/have pattern.Generally speaking, this is illustrated in and exists excessive nuclear compound can use proteoplast to detect in the medium.

Although present embodiment has been showed purine how to survey gain in yield, also can carry out similarly the existence of excessive pyrimidine.Therefore, proteoplast is learned the evidence that can be used for providing powerful and is proved whether fermentation is to carry out according to according to the invention.

Material and method

(Fey etc. 1998 in the standard method of announcing before the method for below describing is based on; Vido etc. 2004; Gitton etc. 2005).

The preparation of acellular extract.Cell is through centrifugal and at ice-cold 10mM Tris-HCl, and pH 7, washes twice in the multitudinous sugar of 0.25M and collects.Cell is transferred to the Eppendorf pipe of 2mL, wherein contains the 1.0g granulated glass sphere (0.25-0.50mm) of having an appointment, and in mixing tank, shakes 6 minutes twice.Then, 10, centrifugal extract is 5 minutes under the 000rpm, and the supernatant of 250-300 μ L is transferred in the new Eppendorf pipe.Supernatant is once more 15, under the 000rpm centrifugal 5 minutes, except the 10-20 μ 1 of test tube bottom, all transfers in another new Eppendorf pipe.From the stoste of 1M, adding DTT is 10mM to ultimate density, and with the lysate refrigerated storage at-20 ℃.

2D gel electrophoresis (isoelectronic focusing and gel electrophoresis).To each gel,, and be suspended to once more in the damping fluid (8M urea, 50mMDTT, 4% CHAPS, 0.2% carrier ampholyte) of the hydration again of 190 μ 1 through the protein of chloroform/methanol deposition 75-300 μ g.One dimension is on the 11cm IPG band (Bio-Rad) of pH 4-7 and pH 4.7-5.9, there being activity to move 12 hours under the hydration again, then 11cm is with in the enterprising column criterion program of Protean IEF groove (Bio-Rad).To the band of pH3.9-5.1, again protein is loaded with cup-shaped after the hydration again at band.Behind the IEF electrophoresis, band perhaps directly is ready for use on the electrophoresis of two dimension perhaps-20 ℃ of refrigerated storage.

Before two-dimentional PAGE, handle band with the SDS damping fluid, under DTT 15 minutes for the first time, under excessive IAA 15 minutes for the second time.Then, sticking through agarose envelope, band is attached to the polyacrylamide gel (the Criterion Tris-HCl of Bio-Rad) of 10-20% and 12.5%, two dimension goes up under 200V at flux electrophoresis chamber (Criterion Dodeca Cell) and moves 1 hour.Gel dyes in BioSafe Coomassie, and goes up scanning at densitometer GS-800 (Bio-Rad).

Proteinic affirmation.The proteinic affirmation of institute's reconnaissance is reached through digestion in the gel with the analysis that mass spectrum carries out peptide spectrum and aminoacids content.The data that produce further be used in public's DB retrieval have similar performance protein (Alphalyse A/S, Odense, Denmark).

Reference

Fey，S.J.，A.Nawrocki，M.R.Larsen，A.Gorg，P.?Roepstorff，G.?N.Skews，R.Williams，and?P.?M.Larsen.1997.Proteome?analysis?of?Saccharomyces?cerevisiae：a?methodological?outline.Electrophoresis?18：1361-1372.

Gitton，C.，M.Meyrand，J.Wang，C.Caron，A.Trubuil，A.Guillot，and?M.Y.?Mistou.2005.Proteomic?signature?of?Lactococcus?lactis?NCDO763?cultivated?in?milk.Appl?Environ?Microbiol?71：7152-7163.

Jensen，P.?R.and?Hammer，K.1993.Minimal?requirements?for?exponential?growth?of?Lactococcus?lactis.Appl.and?Env.Microbiol.59：4363-4366.

Nilsson?D.and?Kilstrup?M.1998.Cloning?and?expression?of?the?Lactococcus?lactis?purDEK?genes，required?for?growth?in?milk.Appl?Environ?Microbiol.64：4321-4327.

Vido，K.，D.Le?Bars，M.Y.Mistou，P.?Anglade，A.Gruss，and?P.?Gaudu.2004.Proteome?analyses?of?heme-dependent?respiration?in?Lactococcus?lactis：involvement?of?the?proteolytic?system.J?Bacteriol?186：1648-1657.

Claims

1. ventilate and high optical density (OD) condition bottom fermentation, obtain the method for the lactic acid bacteria culture of increase productive rate, this method may further comprise the steps:

I) in developing medium, under certain condition, cultivate milk-acid bacteria, make fermentation proceed to the optical density (OD) (OD that measures at 600nm ₆₀₀) be 10; Wherein said developing medium comprises at least a porphyrin compound and at least a gain in yield agent; Said gain in yield agent is selected from the group of being made up of purine bases, pyrimidine bases, nucleosides, Nucleotide and their verivate; Its concentration is to guarantee that when fermentation ends developing medium contains the said at least a gain in yield agent of at least 1 μ M, wherein said verivate be selected from 5-methylcytosine ( ^MeC), iso-cytosine, false iso-cytosine, 5-bromouracil, 5-proyl uridylic, 5-propine-6-Fluracil, 5-methylthiazol uridylic, adenine, 2-aminopurine, 2,6-diaminopurine, 7-propine-7-denitrification VITAMIN B4,7-propine-7-denitrification guanine and 2-chloro-adenine; And

Wherein, and come culturing micro-organisms to compare under the same conditions with in the similar medium that contains each the gain in yield agent that is less than 1 μ M, the gain in yield agent has increased the productive rate of the milk-acid bacteria of collecting when fermentation ends.

2. according to the method for claim 1; Wherein, Said developing medium contains at least a gain in yield agent of 1mM at least at first; It is selected from compound or the group of being made up of one or more verivates of any such compound that is used for the biosynthesizing of nucleic acid by one or more, wherein said verivate be selected from 5-methylcytosine ( ^MeC), iso-cytosine, false iso-cytosine, 5-bromouracil, 5-proyl uridylic, 5-propine-6-Fluracil, 5-methylthiazol uridylic, adenine, 2-aminopurine, 2,6-diaminopurine, 7-propine-7-denitrification VITAMIN B4,7-propine-7-denitrification guanine and 2-chloro-adenine.

3. according to the method for aforementioned any claim, wherein, said developing medium is the mixture fermentation media, in every liter of this medium, adds at least a gain in yield agent of 0.2g at least.

4. according to the process of claim 1 wherein, said at least a gain in yield agent adds during the fermentation.

5. according to the process of claim 1 wherein, the characteristic of the condition of said high optical density (OD) is OD when fermentation ends ₆₀₀Be higher than 15.

6. according to the process of claim 1 wherein at least 1.2 times of said gain in yield.

7. according to the process of claim 1 wherein, said gain in yield agent is purine bases, and these purine bases are selected from the group of being made up of VITAMIN B4, guanine, xanthine and xanthoglobulin.

8. according to the process of claim 1 wherein, said gain in yield agent is pyrimidine bases.

9. according to Claim 8 method, wherein, said pyrimidine bases are to be selected from the group of being made up of cytosine(Cyt), thymus pyrimidine and uridylic.

10. according to the process of claim 1 wherein, said gain in yield agent is a nucleosides.

11. according to the method for claim 10, wherein, said nucleosides is to be selected from the group of being made up of adenosine, guanosine, uridine, cytidine, inosine, Desoxyadenosine, pancreatic desoxyribonuclease, deoxythymidine, Deoxyribose cytidine and Hypoxanthine deoxyriboside.

12. according to the method for claim 11, wherein, said nucleosides is to be selected from the group of being made up of adenosine, guanosine, uridine, cytidine and inosine.

13. according to the method for claim 12, wherein, said nucleosides is an inosine.

14. according to the process of claim 1 wherein, said gain in yield agent is a Nucleotide.

15. method according to claim 14; Wherein, said Nucleotide is to be selected from the group of being made up of adenosine monophosphate (AMP), guanosine monophosphate (GMP), uridine list phosphoric acid (UMP), cytosine riboside monophosphate (CMP), xanthine list phosphoric acid (XMP), inosine list phosphoric acid (IMP), Desoxyadenosine list phosphoric acid (dAMP), pancreatic desoxyribonuclease list phosphoric acid (dGMP), deoxythymidine list phosphoric acid (dTMP), Deoxyribose cytidine list phosphoric acid (dCMP), deoxidation xanthine list phosphoric acid (dXMP) and Hypoxanthine deoxyriboside list phosphoric acid (dIMP).

16. according to the method for claim 15, wherein, said Nucleotide is to be selected from the group of being made up of AMP, GMP, UMP, CMP, XMP and IMP.

17. according to the method for claim 16, wherein, said Nucleotide is IMP.

18. according to the process of claim 1 wherein, said developing medium comprises at least two kinds of gain in yield agent, is selected from the group of being made up of purine bases, pyrimidine bases, nucleosides, Nucleotide and their verivate, wherein said verivate be selected from 5-methylcytosine ( ^MeC), iso-cytosine, false iso-cytosine, 5-bromouracil, 5-proyl uridylic, 5-propine-6-Fluracil, 5-methylthiazol uridylic, adenine, 2-aminopurine, 2,6-diaminopurine, 7-propine-7-denitrification VITAMIN B4,7-propine-7-denitrification guanine and 2-chloro-adenine.

19. according to the method for claim 18, wherein, said developing medium comprises at least two kinds of gain in yield agent, is selected from the group of being made up of nucleosides and Nucleotide.

20. according to the method for claim 19, wherein, said nucleosides is an inosine, said Nucleotide is IMP.

21. according to the process of claim 1 wherein initial each the gain in yield agent that comprises 1-70mM of said developing medium.

22. according to the method for claim 21, wherein, initial each the gain in yield agent that comprises 1-60mM of said developing medium.

23. according to the method for claim 21, wherein, initial each the gain in yield agent that comprises 1.3-60mM of said developing medium.

24. according to the method for claim 21, wherein, initial each the gain in yield agent that comprises 1.5-50mM of said developing medium.

25. according to the method for claim 21, wherein, initial each the gain in yield agent that comprises 2-40mM of said developing medium.

26. according to the method for claim 21, wherein, initial each the gain in yield agent that comprises 2.5-30mM of said developing medium.

27. according to the method for claim 21, wherein, initial each the gain in yield agent that comprises 3-20mM of said developing medium.

28. according to the method for claim 21, wherein, initial each the gain in yield agent that comprises 3-15mM of said developing medium.

29. according to the method for claim 21, wherein, initial each the gain in yield agent that comprises 4-10mM of said developing medium.

30. according to the method for claim 21, wherein, initial each the gain in yield agent that comprises 7mM of said developing medium.

31. according to the process of claim 1 wherein the OD of developing medium ₆₀₀Reaching an optical density (OD) is OD ₆₀₀=10-OD ₆₀₀=200.

32. according to the process of claim 1 wherein the OD of developing medium ₆₀₀Reaching an optical density (OD) is OD ₆₀₀=15-OD ₆₀₀=100.

33. according to the process of claim 1 wherein the OD of developing medium ₆₀₀Reaching an optical density (OD) is OD ₆₀₀=20-OD ₆₀₀=80.

34. according to the process of claim 1 wherein, culturing process is to contain 5L-100, carries out in the big formula fermentation container of the developing medium of 000L.

35. according to the process of claim 1 wherein, culturing process is to contain 300L-20, carries out in the big formula fermentation container of the developing medium of 000L.

36. according to the process of claim 1 wherein, culturing process comprises controlled temperature and/or pH.

37. method according to claim 1; Wherein, Culture comprises one or more organism, is selected from the group of being made up of genus bifidobacterium, brevibacterium sp, propionibacterium, the lactococcus genus that comprises Lactococcus lactis subsp.lactis and lactococcus lactis subsp, the lactobacillus genus that comprises Lactobacterium acidophilum, streptococcus, enterococcus spp, pediococcus acidilactici genus, leuconos toc and wine Coccus.

38. according to the process of claim 1 wherein, culture comprises one or more and has optimum growth temperature at the warm organism of 30 ℃ have a liking for.

39. method according to claim 1; Wherein, culture comprises one or more and is selected from the warm organism of having a liking in the group of being made up of lactococcus lactis ssp, lactococcus lactis subsp, leuconostoc mesenteroides sub species cremoris, Pediococcus pentosaceus, the mutation of Lactococcus lactis subsp.lactis di-acetyl lactic biological, lactobacterium casei cheese subspecies and the secondary cheese subspecies of lactobacillus paraceasi.

40. according to the process of claim 1 wherein, culture comprises one or more and has optimum growth temperature at 40 ℃ to 45 ℃ thermophilic organism.

41. method according to claim 1; Wherein, culture comprises one or more and is selected from the thermophilic organism in the group of being made up of thermophilus streptococcus, faecium, lactobacillus delbruckii lactic acid subspecies, lactobacterium helveticus, lactobacillus delbruockii subspecies bulgaricus and lactobacillus acidophilus.

42. method according to claim 1; Wherein, Culture is the LD-culture, and it comprises one or more and is selected from the organism in the group of being made up of Lactococcus lactis subsp.lactis, lactococcus lactis subsp, the lactic biological mutation of Lactococcus lactis subsp.lactis di-acetyl and leuconostoc mesenteroides sub species cremoris.

43. according to the process of claim 1 wherein, culture is the O-culture, it comprises one or more and is selected from the organism in the group of being made up of Lactococcus lactis subsp.lactis and lactococcus lactis subsp.

44. according to the process of claim 1 wherein, culture comprises lactococcus lactis ssp.

45. according to the method for claim 1, said method further comprises:

46. according to the method for claim 45, said method further comprises:

47. according to the method for claim 45 or 46, said method further comprises:

V) pack the cell that obtains in iii) or iv) by above-mentioned steps.

48., wherein, in the mikrobe of collecting, add at least a cryoprotectant according to method any among the claim 44-46.

49., wherein, in the mikrobe of collecting, add at least a cryoprotectant according to the method for claim 47.

50. a developing medium, it comprises at least a gain in yield agent and at least a porphyrin compound, and said gain in yield agent is selected from the group of being made up of purine bases, pyrimidine bases, nucleosides, Nucleotide and their verivate.

51. according to the developing medium of claim 50, wherein, said medium such as claim 1 definition.