BG100105A - Viable bacteria - Google Patents

Abstract

The invention relates to microbial cells stabilized for storage in the form of a dry composition and suspended in a destroyed matrix. The composition is prepared by mixing microbial cells, for example, gram-negative bacterial cells of pseudomonas fluorescein, with an aqueous polyhydroxy compound-based composition, preferably a saccharide from which a matrix is produced. The mixture is dried under conditions whereby the polyhydroxy compound is viscous and the matrix is destroyed without excessive damage to the cells. Preferably, the glass transition temperature of the matrix is above the temperature at which the composition is stored.

Description

The invention relates to a method for the preparation of compositions containing dried microbial cells in a stasis state, to such compositions and to live cultures derived therefrom.

A known problem is the conservation of viable crops. U.S. No. 3,897,307 describes (i) the use of a combination of ascorbasic compound and glutamate or aspartate as a stabilizer for lactic acid producing bacterial cells and (ii) uses! Of some sugars, in particular inositol, at a solution concentration of 25 mg / min, as a cryoprojectant upon freezing such freeze-dried cells.

Mugnier et al., Applied and Environmental Microbiology, 1985, pp 108 114 describes the use of polysaccharide gels in combination with any hrshtnieini substances as C _1h and _{C6 p} compounds, whereby mshritsa of freeze-dried bacterial cells. We have found that the wires of the ashtigys below are not degraded upon drying by substitution. tauiieiiio.

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Redway el al, Cryobiology, 1974, Vol.11, 73-79 examined some monosaccharides (concentrations up to 150 mg / 2 ml sample) and related compounds as a medium for long-term survival of freeze-dried bacteria.

Ceia we found that Koiaro microbial sticks are suspended! in the take-off matrix, as described below, and dried under certain conditions, their short-term viability improves, and when these dried systems are stored and re-harvested under certain conditions below, the long-term viability of the microbial cells improves.

In addition, we have surprisingly found that destroying the microbial in which the microbial cells are suspended does not result in a pH. ' ¹ 11 'you live your life ι.

According to a first aspect of the present invention, there is provided a stabilized dried composition comprising microbial cells in a stasis with a microorganism and inpai in a ruptured ia Mai arm.

By "stabilized" we mean that the degradation of microbial pliers is reduced (degradation would lead to the loss of viable cells).

By "stagnant" we mean that they clench but metabolize, do not divide and do not grow (but are recoverable if subjected to proper treatment).

By "burgeoning" we mean cells that, when placed under suitable conditions (i.e., rehydration and a source of foods and common substances), are capable of growth and division.

By "viable cells ^11" we mean cells that, when placed under suitable conditions (ie rehydration and food source), are capable of growth and division.

By "destruction we mean" · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · (I) the matrix has shrunk and become less porous as the guzp has a small penetration of low molecular weight diffusing species into ma I [li io I a, for example it absorbs little water vapor upon exposure to humid air, and / or iii) the matrix has been subjected to a temperature above 1az per cn.KJK'iinn transition (lg) such that poTynim ee is viscously leaked, resulting in a significant decrease in the ratio of surface area / volume and up to cap tory cells in niskoporyozno protective coating.

According to a second aspect of the present invention, there is provided a method of harvesting a stabilized dried composition comprising stratified microbial cells suspended in a matrix, which method comprises the steps of:

A mixing of microbial cells with an aqueous composition comprising the material from which the matrix will be swallowed;

B. drying the mixture under conditions such that a viscous flow of the material is obtained and the matrix is destroyed but not damaged! excessive cells.

Preferably, the composition obtained in step B is fed to a heme! icpaiypa lower than Td of the matrix, i. the composition has a TD over the intended temperate clock for its storage.

Accordingly, the composition obtained in step B is preferably further dried, so-called "secondary drying", in order to increase the Td of the matrix in order for the composition to be stable under but broad storage conditions, i.e. . be able to be stored at high temperatures.

The microbial cells that comprise the stabilized dried compound of the invention are preferably bacilli cells. Hp B '..-. * Y *, we do not * exclude the possibility of ia being used for other microbial cells such as mushrooms, yeast, and so on. Koi am microbial • · • · · * ·· _'is • · 4 ·· ·· ·· ··· ··· cells are bacterial cells, they preferably are gram-negative bacterial cells although we do not exclude the possibility cells be Gram positive. Pseudomonas fluorescens, Escherichia coli and relatives of 11lizosphere6a κι ί'ρτιιι should be mentioned as examples of such I frame negative cells

The concentration of the microbial cells in the matrix obtained in Step A in the method of the present invention is between Ι0 ⁴ / ml and ^{10 3} / MP and preferably between 1o ¹⁰ / ml and 10 ^and / ml.

t

The substance to be mixed with the microbial cells in step A of the method of the invention is a porohydroxy compound, for example polyps, such as mannitol, inositol, sorbitol, galactitol or for ip ·: u | το ¹ τι 11 ai ie in bi j euhydrate, preferably saccharide.

When the substance is saccharide, it may be disaccharide, trisaccharide, opigosaccharide, but monosaccharide is preferred. As an example of a monosaccharide, one should consider among other hexoses, for example 'rhamnose, xylose, fructose, glucose, mannose and galactose. Examples of disaccharides include, among others: mango, lactose, trehalose and sucrose. As an example of trisaccharide, raffinose may be mentioned. By presenting! oligosaccharide firs 1 may be mentioned by masodextrims.

The concentration of the poptihydroxy compound used in the mixture in step A of the process according to the invention is between 10 mg / 10 ¹⁰ and IOOO m / 10 cells and preferably between 200 mg / 10 cells and 400 mg / 10 ¹⁰ cells. One skilled in the art will be able to find, by experimentation, the concentration at which a broken matrix of a given polyhydroxy compound can be obtained. For example, we have found that inositol has an optimal concentration of about 45 mg / ml, at a concentration above 60 mg / ml it causes massive cell damage, and does not destroy at a concentration below 25 mg / ml.

Some of the non-hydroxy compounds exhibit protective properties over a wide range of concentrations. In some others, above the critical concentration, which seems to be related to the solubility of the nolihydroxy compound in the aqueous medium, a detrimental effect is observed.

And the example is further illustrated and illustrated in the accompanying drawings, based on examples of compositions according to the invention.

H drawings:

Figure 1 illustrates, in the form of a graph, changes in the viability of Pseudomonas fluorescens with different concentrations of inozigol (additive) when dried with water or 0.0-1 M by weight of sulph! by freezing. The vertical axis represents the vitality in parts of a billion (ie, 1E + 09 = 10 ⁹ ppb, IE + 08 - ppb, ht.ii.), and the horizontal axis represents the concentration of the additive in milleschrams for the sample. The black squares in the graphite a ί a nope are for inositol in magnesium sulfate, and the circles in the field are for inositol in water. The vertical axis represents the viability in parts of a billion or in billions (ie 1E T 0 - 1 billion, 5E - 1 = 0.5 billion, 2E -1-0.2 billion, and the horizontal axis represents the sugar concentrations in milligrams for the sample . The monosaccharides illustrated are:

Figure 2 Tolakgose Figure 5 Xylose

Figure 3 fruit figure 6 rhamnose figure Ί glucose fiber 7 mannose fiber 8 illustrates, in the form of trafficking, changes in the rate of cell mortality (kC with respect to Gd of · · · · ·

() mafnspga and changes to 1g at relatively humid> a. The left vertex axis ahead of 1 is K |, the right vertical axis is Nr (the left Gd (”(.;) And the horizontal axis is Ognos1a1nla khoci (%). Cherpin ·· KBajiparicia is a spikel connected: G.i., shows! titt ciiiiaia (K |) of death pins on the pliers, and rhombuses bound with ίipr-tb, Ήπια line indicate the glass transition temperature (lg).

in the drawings, viability is expressed as the number of viable bacteria per 10 ^{h of} viable bacteria in the ori, incl suspension, i. e. it represents the number of bacteria that survive from each trillion bacteria that were viable at the beginning. It is represented as parts of billions of giants; equivalence (ppb) or 10 ⁹ ppb is equivalent to 100% non-kinking and 10 equals 10% survival and bp.

It can be seen from Figure I that at low alien concentrations the cell viability is maintained, while at higher concen- trations, eg higher than 100 mi / sample (equivalent to 50 μι / μπ), cell viability decreases rapidly.

From Figures <'to 7 it can be seen that known monosaccharides are shutting down! cells from damage at low concentrations, eg less than 10 mg / sample (equal to 5 mg / ml) and that protection is practically maintained at high concentrations, for example about 400 mg / ml (equal to 130 mg / ml)

From figure 8 it can be seen that when Td falls below ωom1 ppaiypai of storage (represented by the most lopnaia horizontal line at 21 - 22 ° 0), the rate of cell death (k ₍ ) decreases1, which is illustrated by the importance from maintaining a vitreous state during storage.

The concentration at which the matrix is effective, i. it is destroyed without unduly destroying the cell, depends on a number of factors, including, inter alia, the volume fraction of * · 7 * '..... .....

cells in suspension in step A; the intrinsic (intrinsic) glass transition temperature of the polyhydroxy compound; of changes! is at the glass transition temperature of the matrix as a function of the water content of the belt and of the temperature! and to which Maipnunia is exposed during and after freeze-dried

It should be understood that the polyhydroxy compound can act as (I) a cryoprotectant at low temperature, in particular against ice damage during freeze drying and / or (II) as a lyoprotectant that protects against damage due to water loss during drying and / or storage and / or (III) as a nutrient source for cell kit restoration.

The microbial cells used in the process according to the invention may develop in conventional nutrient media, for example, nutrient broth or tryptone soy broth. 1е μοιήι to gather! in any convenient growth phase, preferably in the early stationary phase.

For example, a culture is developed in or on a suitable medium, for example liquid or solid plates, to obtain the desired cellular κυιιΐΗ'radium. Kjioikhio are isolated, usually by means of censored laundry. It is resuspended in an aqueous medium containing Vepa-iRo, which forms the matrix and possibly other additives as it is snombed down.

It is preferable to isolate the microbial cells used in the iso-isorizium method! from the medium in which the cyciif'H is recirculated, reconcile in a solution containing a polyhydroxy compound, suitable additives, etc. and dried. In doing so, we exclude the possibility of ia polyhydroxy compound and the appropriate additives and ι.ιι.

• · · ·· to * · ··· ·· .. ...

to be added to the cells in the medium in which they develop and the resulting mixture to be dried.

Koiaro microbial cells are resuspended, they are resuspended in an aqueous solution, such as aqueous magnesium sulphate, or preferably water containing a non-hydroxy compound.

The drying in step B of the process according to the invention is carried out, for example, by evaporation, vacuum drying, spray drying, air drying or preferably freeze drying.

As already stated, it is essential to fast viscously drained at least during the drying step, stage B, or in any ιιο <; Ηΐ ·, 'ν ··; ιιιι eian.

(iv) innocent content of dry composition obtained in ethane B is less than 15% w / w.

When freeze-drying is used in step B, the composition typically contains one or more suitable additives. Examples of suitable additives may be mentioned, inter alia, cryoprotectants, for example sugars or polymeric substances, for example polyvinyl alcohol, polyvinylpyrrolidone; lyo protekaip, such as sugars or polymeric substances, for example polyvinipapkohol, polyegylene glycol; or for ι р ж '* д | such as antioxidants or so-called potentiators, such as dcorbate or 1pugam1, it is not excluded that Hpyin additives, for example, bulk substances, for example, crucible 1a.sizing sugars, eg mannitol, and osmosis regulators, eg betaine, urea / trimethylamino-N-oxide, propine, sarcosine.

The invention is better illustrated by the following examples. * * * * · · · · ··· * * * * ··. . ", ·· I) ·· ··· ·· ·····

Examples 1-6

1 these examples illustrate compositions with a good naoCppienHeiO in which metric consists of framnose.

Peeiidomonas fluorescent was cultured in standard medium (double amplified nutrient broth) and cell collection was nani.piuna r stationary phase by cetrophus! Concentrate 11raιι, ι of cells is resuspended in sterile water and / (residual conical slurry augolized, rhamnose concentrated solution is added to obtain approximately 200 equal beakers with a final concentration of 200 mg sugar for 2 x 10 ¹⁰ cells in a total volume of 4 ml Rona r 5 ml drink freeze-dried bottles.

The vials are loaded into anapai temperature controlled shelves for freeze drying and the shelves temperature is lowered to -30 ° C, where the contents of the vials are frozen as their temperature drops to -28 ° C to -30 ° C over a period of two o'clock. Irina! under vacuum and primary drying is carried out for 48 hours. The shelf temperature was raised to 0 ° C and allowed to dry for 24 hours. Bottles are left! warm to a secret temperature and seal! under vacuum before being removed from the freezer.

The vials were stored at 4 ° C under vacuum (Example I) or in humidified air at 21 ° C (Examples 2 - 6)

The samples were re-moistened in stearpa water and determined the number of viable cells by serial dilution of sgv soda and subsequent seeding on King's growth medium. The number of colony-forming units (cfu) on plates with the highest dissolution rate is calculated to calculate the number of bacterial nodules per unit volume of freeze-drying and crunching conditions.

The cell viability after freeze-drying is 3 x 10 ⁱⁿ ppb. · · · · · · · ·

Table 1

ND - not specified

SI - comparative ophs without rhamnose.

It can be seen from TABLE I that the presence of a framerate matrix significantly improved the viability of the bacteria.

The results of storing bacteria at 21 ° C in a humidity controlled chamber are given in Table 2.

Table 2

11Rome No Relative Vitality (ppb) after storage humidity 13 days 27 days 34 days Fr. (Rl I%) I 8 x 10 ' ........ 3 x I0 ⁷ 4 x I0 ⁷ to i 4 3 x 10 ^z 7 X 10 '' 2 x 10 ⁷ 4 9 9 x 10 ' 5 x S ⁷ 4 x S ⁷ 23 4 x 10 ⁷ 4 x 10 '' 8x 10 * ' 6 44 3 x 10 ^g 1 x I0 ⁷ 3 x Y '' 1 10 ³

G12 = - Comparative opium without rhamnose.

From Table 2 it can be seen that the presence of sugar significantly increased viability ^ even at low RH, i.e. Example 2 compared to C1-2.

Examples 7 Ι0

1these examples illustrate the composition of the agreement we have in yurstonia in which the matrix is composed of rhinos and machinis ονιιφπι! The ia'ii-nibT pa of the examples 1-6 is repeated and used; tppi pa n> va, h (? Concentration of cells was resuspended in 0.04 M magnesium sulfate instead of sterile water and rhamnose solution added in June.

The cell viability immediately after freeze-drying is 5 x 10 ⁸ .

Freeze-dried cells were stored at different temperatures for different periods of time as shown in Table 3

Table 3

An example Temperature Viability (ppb) in days No out of stock 50 100 175 365 we (° C) 7 -20 8 x 10 ° 5 x Yu ” 5 x 10 ” 5 X 10 ” 8 4 2 x 10 * 2 x 10 * Ϊ x Ϊ0 ” 9 15 2x I) ⁸ ί x ю 5 X 10 ' 10 20 2 x 10 ⁸ 2 x 10 ⁷

From Table h it can be seen that the formulation provides substantial protection over a wide range of temperatures.

Examples 11 15

The exemplary examples illustrate compositions according to the invention with sodium ascorbag and sodium niyiaMar in the matrix.

Repeat the operation or Examples 1 - 6 except that the cells that are suppressed in water and rhamnose are added! aqueous solutions of sodium ascorbag and sodium lunar mag.

Cell viability immediately. after freeze-drying, it is 2 x 10 ⁸ ppb.

Samples were stored in moist air at 21 ° C.

(box 4)

Example No. (Demolished Viability (ppb) after ig moistened stored for ^(, p (RH%) 34 days I28 days I) 6 1 x 10 ⁱⁿ 1 x 10 ^,! 25 “........ 12 '.......... 14 .................... 1 x 10 ⁱⁿ 1 x ^10k 24 13 30 ϊ X 10 ° 4 x 10 ' 14 and 40 . 9 X 10 '' Ex 10 '............... 5 x 10 ⁱⁿ 12 15 53 3 x 10 ' 8

Or Examples I I and 12 in Table 4 it can be seen that the storage of cyxiiie cells at reMiiepaiypn under Td of the mauridale tnabilizes the cell kn i for the period of birth.

Pe.'iyniainre shows that the combination of a vitreous matrix in the vitreous state and the presence of aiothioxidate provides a macabre that can weaken viable cells for a period of 1 h under relatively external conditions.

Example 16 g flight of Pseudomonas fluorescens cells, eaten by mop medium by cepfuging and containing, 5 x 10 ^m viable cells, mixed with 14 g maltodextrin (Gluctex ΙΤ 19) with Ncdov chips) and with 1.6 g sodium ascorbate and prodrug, initially at ambient temperature, it is dried under vacuum, whereby n1b ') p''il'; 1 water is condensed into an ice trap maintained ipn but '' (X

The drying was completed after approximately 18 hours, I ί p 03 which time of shum was I mbar.

• · · · * · * • · · · ·"· ·· · · · · · in

Samples re-annealed with clean water and placed in a nutrient can usually show 50 to 90% viability recovery! is cells.

The products obtained by this method are kayu disrupted amorphous matrices with a glass transition exceeding 20 ° C (horse-drawn samples: f <; ιι; ιμιπ; iι on C1; 1 online iiaOopaiopi-ia ophthalmic clavicle riaieiiiHM claims

I Babinized dry composition containing microbial cells in ciasHoiio shine suspended in a broken matrix,? The composition of claim 1, wherein the microbial cells are bacteria! and cells.

The composition of claim 2, wherein the bacterial cells are Gram-negative bacterial cells.

The composition of claim 3, wherein the Gram-negative bacterial cells are Pseudomonas fluorescens, Escherichia coli and rhizosphere bacteria.

5. Meud for the preparation of a stabilized dried composition containing microbial cells in a stasis state suspended in Maiprma, which meud includes the steps of:

A. mixing the microbial cells with aqueous CbciaR containing the material from which the matrix will be obtained;

B. drying the mixture under such conditions as to give a viscous flow of the material and break the matrix without unduly damaging the cells.

The method of claim 5, wherein the solid obtained in eian B is further dried to increase the glass transition temperature of the matrix so that the composition is stabilized under broader storage conditions.

ϊ. The meud according to claim 5, wherein the concentration of паηηηιη is Μ! Ικ | n> b | Ive cells in the mixture obtained in step a are between 10 ', ^! : w and! 0 ^{| !} / ml.

8. A method according to claim 7, in which κοιιιtech11ratsi: of a klepasho between 1o ^1o / ml and 10 ^p / ml.

· • · · · • · | / · · · · · »*« · * ·

The method of claim 5, wherein the substance that is mixed with the microbial is K. neiKH in step A of the method is a fully hydroxy compound.

io. The mead according to claim 9, wherein said postroxy compound is a monosaccharide.

II Method cbinaciTO claim 9 for the concentration of the full hydroxy compound used in the mixture in step между is between 10 mg / 100 ° C and 1000 mg / ¹⁰⁰ cells.

12. Megon according to claim 11, wherein the concentration of the n-tiplindroxy compound is between 200 mg / 10 < RTI ID = 0.0 ^&gt; m &lt; / RTI &gt;

I. A composition obtained according to claim 5, having a CH4JICH transition temperature over the gases provided for storage.

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PAGE REFERRED TO THE REPORT OF THE MPE

Claims (4)

Pound claims 1. A stabilized dry composition comprising gaseous microbial cells suspended in a broken matrix, in which the cells are I frames. ? The composition of claim 1, wherein the Gram-negative bacterergi pests are Pseudomonas fluorescent, Escherichia coli and rhizosphere-related bacteria. 3. Method for the preparation of a stabilized dried composition comprising microbial cells in which the bacterium cells are Gram OI |) oral bacterial cells, gaseous, suspended in MOipiiH'T. which io meud includes eiaiiHie of: Λ mixing the microbial cells with an aqueous composition containing the material from which the matrix will be obtained; B drying the mixture under such conditions that it would cause a further splitting of the material and destroy the matrix without unduly damaging the cells. The meud according to claim 3, wherein the composition obtained in step B is TiaiaibK for. to increase the tomiiepaιur in the en.Kiiommcii transition of the maficene, so that the cavity stabilizes in the notation 1 | X1! 1 || C || οι storage condition >> The method of claim 3, wherein the concentration of Mi-iKpoOiitne cells in the mixture obtained in step A is between 10 ^-1 / ml and 10 ⁿ / ml. Fr. Method cbinacHO claim 5, in which kontseshratsiyaga of "bad is between 10 ^{| o} / MP and 10 ^and / ml / Meyud flown | to claim 3, in which the substance which is mixed with the microbial cells in Step A of the process is a polyhydroxy syedinenie in method cbniacuo claim 7, for κοικο n <neck at ιj yuga mi 1 «· ιο is a monosaccharide. The method of claim 7 wherein the κοιιιιοίι »| The polyhydroxy compound used in the calculus in eian o between Yu mi / Io ' ¹ ' and 1000 Mi / ίο ¹⁰ cells. 1 ° The method of claim 9 in which the horse / chnratsiyat polyhydroxy compound is between 200mg / 1o ¹⁰ cells and 100 mg / S ¹⁰ miserable. II. A composition according to claim 3 having a temperature of that passage above that provided for storage.