IE912420A1

IE912420A1 - Process for the manufacture of microorganisms and¹microorganisms with improved cell productivity

Info

Publication number: IE912420A1
Application number: IE242091A
Authority: IE
Original assignee: Lacto Labo Sa
Priority date: 1990-07-11
Filing date: 1991-07-10
Publication date: 1992-01-15
Also published as: FR2664612B1; FR2664612A1; NO912655D0; EP0466560A1; CA2046719A1; FI913344A; PT98276A; NO912655L; FI913344A0

Abstract

The invention relates to a method for producing microorganisms which produce their own growth inhibitor, especially lactic bacteria, the aforementioned method enabling the specific cellular activity and the cellular productivity of the microorganisms to be improved and controlled. A subject of the invention consists of microorganisms having improved cellular productivity and improved specific cellular activity.

Description

The present invention relates to a process for the manufacture of microorganisms producing their own growth inhibitor, and to microorganisms whose specific cell activity and cell productivity are improved. The said microorganisms may be, in particular, lactic bacteria.

The manufacture of microorganisms is 10 conventionally carried out in a culture medium containing nutrient substrates diluted in water. This culture medium is first of all seeded with microorganisms, which thus develop in a virtually closed environment. This is known as batch culture.

Batch culture has the advantage of being simple, but low in efficiency. In fact, limiting factors exist, either in the nutrient substrates or in the inhibitors produced by the microorganisms whose accumulation in the fermentation medium reduces the rate of growth of the microorganisms and brings the fermentation to a halt. In parallel, the yield of biomass formed, in relation to the nutrient substrates introduced, especially in relation to the carbon and nitrogen sources, progressively decreases.

The Applicant Company has also been able to ascertain that microorganisms obtained by such a batch process have insufficient and uncontrollable specific cell activity and cell productivity, that is to say that, from one batch to another, the microorganisms obtained may exhibit a different specific cell activity and cell productivity. A specific cell activity means the expression of the capacity of a given quantity of microorganisms to perform the function assigned to them. The cell productivity is equal to the specific cell activity multiplied by the bacterial concentration and divided by the fermentation time. Thus, specific cell activity and cell productivity are characteristic of each type of microorganisms.

Specific cell activity and cell productivity can therefore be compared only between a microorganism of a certain type and a microorganism of the same type.

The determination of the specific cell activity and cell productivity of a microorganism can therefore be carried out only according to an established method which is specific for each type of microorganism.

Techniques which make it possible to determine the specific cell activity and cell productivity of a given microorganism are generally well known to people skilled in the art. However, in the case of a microorganism for which such methods have not yet been developed, it is well within the scope of a person skilled in the art to establish such a method. An example of determination of specific cell activity and cell productivity is given below insofar as lactic bacteria are concerned.

In order to overcome the disadvantages of batch culture, it has been proposed to prepare microorganisms continuously, that is to say by regular introduction of nutrient substrates in parallel with removal of the inhibitors from the fermentation medium The removal of inhibitors has been carried out by various means, for example by dialysis or by ultrafiltration.

European Patent 65,895 describes a process 10 for the manufacture of microorganisms with total recycling of the biomass, which employs removal of the growth inhibitors by ultrafiltration. However, such a process, while enabling high concentrations of microorganisms to be obtained, has disadvantages.

Thus, the increase in the biomass in the fermenter makes the process tricky to conduct, especially owing to the fact that the viscosity of the medium greatly increases .

The Applicant Company has also been able to ascertain that a process with total recycling of biomass does not make it possible to obtain microorganisms whose specific cell activity and cell productivity are improved or controllable.

In order to circumvent the disadvantages of the prior art, the Applicant Company has developed a process for the manufacture of microorganisms forming first subject of the present invention. This process makes it possible in particular to improve the yields of biomass in relation to the nutrient substrates and especially in relation to the carbon and nitrogen sources forming part of the culture medium employed. It also permits a simplified system management. Above all, however, it makes it possible to obtain microorganisms whose specific cell activity and cell productivity are not only improved but, above all, controlled, that is to say that it is possible to manufacture microorganisms whose specific cell activity and cell 10 productivity can be determined in advance and remain constant from one process of manufacture to another.

Such a process therefore enables the user of the microorganisms which are manufactured to provide for the necessary dosage of said microorganisms with a view to its subsequent applications. The process according to the invention can be applied in particular to the manufacture of lactic bacteria.

Another subject of the invention consists of microorganisms whose specific cell activity and cell productivity are improved. Such microorganisms may, in particular, be lactic bacteria.

The present invention relates, therefore, to a process for the manufacture of microorganisms producing their own growth inhibitor, according to which a culture medium is fermented using the said microorganisms so as to obtain a fermentation medium, at least part of the said inhibitor is removed from the fermentation medium by a physical separation process, and the retained material is recycled, the said process being characterised in that when the rate of growth of the said microorganisms reaches a value equal to the value of a rate of growth, measured in a steady state, corresponding to a specific cell activity of the microorganisms which has been predetermined, the fermentation medium is purged at a purge rate which is higher than or equal to the said rate of growth.

Other characteristics and advantages of the 10 invention are detailed below: The drawings attached hereto are briefly commented upon: Figure 1 is a diagrammatic view of a plant for the manufacture of lactic bacteria according to the process of the invention.

Figure 2 is a diagram showing the relationship between the specific cell activity of a Streptococcus cremoris strain in relation to its rate of growth.

- Figure 3 is a diagram showing the concentration of lactic acid as a function of measured conductivity.

Figure 4 shows the biomass yield of a Streptococcus cremoris strain in relation to the sugar in the culture medium, as a function of the purge rate.

The starting Streptococcus cremoris strain used for producing Figures 2 to 4 is a strain preserved in the strain collection of the Lacto Labo company under the code name Sc 301. This strain is marketed by Lacto Labo in the form of a leaven called SM 259.

The rate of growth which indicates the purge rate to be used within the scope of the present invention is therefore fixed in relation to a predetermined specific cell activity. The Applicant Company has, in fact, been able to ascertain that, surprisingly, the specific cell activity of the microorganisms is related to the rate of growth.

Figure 2 shows this relationship in the case of the said Streptococcus cremoris strain from Lacto Labo.

In parallel, the Applicant Company has been able to ascertain that by purging the fermentation medium according to the invention it is possible to impart to the microorganism a specific cell activity chosen by the manufacturer. Consequently, in order to manufacture microorganisms which have a high specific cell activity, it being possible, furthermore, for the latter to be controlled, it suffices to purge the fermentation medium at a purge rate which is higher than or equal to the purge rate corresponding to the predetermined and desired specific cell activity of the said microorganisms.

The steady state is obtained when the concentration of biomass, nutrient substrates and inhibiting products which are present in the fermentation medium is substantially constant.

The specific cell activity of a given type of microorganism is specific to it. Consequently, it is appropriate for each type of microorganism to determine the relationship which exists between its specific cell activity and its rate of growth measured in a steady state. This determination can be done by any means. However, a simple means consists in fermenting a culture medium with a microorganism in a chemostat, that is to say a fermenter of capacity V fed continuously with culture medium at a rate Q and from which the fermentation medium is drained continuously at the same rate Q. Under steady state conditions the rate Q is directly proportional to the rate of growth μ according to the formula μ - Q/V.

By varying the rate Q it is therefore possible to fix different rates of growth.

Each time the rate Q is varied, and after the steady state has been reached, microorganisms are sampled and their specific cell activity is measured.

Thus, in the case of each type of microorganisms, which it is intended to manufacture, the relationship between its rate of growth and its specific cell activity is obtained. Figure 2 shows the specific cell activity, expressed in g’1, of the said Streptococcus cremoris strain as a function of its rate of growth.

The determination of the specific cell activity of a given microorganism is carried out by a method which is specific to the said microorganism. By way of example, a method for determining the specific cell activity of lactic bacteria is given. In a first step this method consists in determining a certain volume Vo of fermentation medium in which the microorganism is manufactured; the said volume Vo is that needed to product 75° dornic in 10 ml of milk with a solids content of 10% during 15 hours at 21‘C. A degree dornic is defined as being equal to 0.1 g of lactic acid per litre of milk. The volume Vo corresponds to one unit of activity (UA).

The number of units of activity per litre of the fermentation medium is then determined.

It is then possible to determine the specific cell activity as being the number of units of activity per litre of fermentation medium divided by the bacterial concentration of the fermentation medium; the specific cell activity is expressed in UA/g.

When the fermentation medium is purged, it should be understood that part of the fermentation medium is drained out of the fermentation device, that is to say the nutrient substrates in aqueous solution forming the culture medium, as well as the microorganisms present therein. The purge rate, expressed in h1, is equal to the ratio of the flow at which the fermentation medium is purged to the volume of the fermentation medium.

Within the scope of the present invention it 5 suffices for the purge rate to be higher than or equal to the predetermined rate of growth of the microorganisms. However, the maximum value of the purge rate must preferably be lower than the theoretical maximum rate of growth μ max of the microorganisms which it is intended to manufacture, so as to avoid washing the device containing the fermentation medium. The purge rate is advantageously chosen at a value of between 10 and 80% of the theoretical maximum rate of growth, preferably between 15 and 50% of the theoretical maximum rate of growth of the microorganisms to be manufactured. The theoretical maximum rate of growth can be evaluated for each type of microorganism by a conventional method which is known to a person skilled in the art.

With some microorganisms cultured in a reactor and under special conditions it can happen that the theoretical maximum rate of growth cannot be reached. In this case the purge rate can be chosen at a value of between 15 and 80% of the experimental maximum rate of growth, more preferably between 20 and 50% of the experimental maximum rate of growth; the experimental maximum rate of growth being the maximum rate of growth which the said microorganisms can reach with the experimental conditions which are chosen. Such a maximum rate of growth can be defined by methods which are conventional for a person skilled in the art.

Advantageously, the fermentation medium 5 according to the invention is purged only when the predetermined rate of growth has been reached, the rate of growth being determined by continuous measurement of the concentration of inhibitors. This measurement can be carried out, for example, by the method described below in the particular case of lactic bacteria.

During the process of manufacture according to the invention the volume of the fermentation medium is generally kept constant by addition of culture medium. The pH of the fermentation medium is generally maintained at a value of between 5 and 8.

The pH can be maintained at a desired value by means of a base, in particular an inorganic base.

The inorganic base employed may be sodium hydroxide, potassium hydroxide, gaseous ammonia or an aqueous ammonia solution. Gaseous ammonia and ammonia in aqueous solution are preferred. Gaseous ammonia is very particularly preferred.

Before purging the fermentation medium according to the invention the culture medium may be advantageously fermented by the microorganisms to be manufactured batchwise. In this way, the rate of growth corresponding to that of the predetermined specific cell activity can be reached more rapidly.

* In addition, and optionally, after the culture medium has been fermented batchwise, at least part of the said growth inhibitor can already be separated by a physical separation process from the fermentation medium before the fermentation medium is purged. In the case where the inhibitor is excessively concentrated in the fermentation medium, this separation also makes it possible to reach the rate of growth corresponding to that of the predetermined cell activity more rapidly and with greater certainty.

Within the scope of the invention the said physical separation process may consist especially of an ultrafiltration, a phase separation, a centrifuging operation or a dialysis. However, ultrafiltration is preferred. It may be carried out by means of one or more ultrafiltration cells arranged in parallel. Whatever the separation process employed the retained material within the scope of the present invention means the recycled fraction of the fermentation medium.

The permeate means the fraction of the fermentation medium which is not recycled during the physical process of separation and which is free or virtually free from microorganisms or microbial debris.

The separation of the growth inhibitor is carried out at a dilution rate D.

When a physical separation process of this kind is used, the fermentation medium according to the invention may be purged continuously as soon as the . 12 concentration of the growth inhibitor in the said fermentation medium is substantially constant. Such a constant concentration is generally reached after 3 liquid residence times, more generally between 3 and 10 liquid residence times. The liquid residence time is expressed in hours and corresponds to the reciprocal of the dilution rate D.

The process according to the invention can be applied to any microorganism which produces its own growth inhibitor.

It is possible, in particular, to mention microorganisms whose growth inhibitor consists of a product such as propionic acid produced especially by propionibacterium, acetic acid obtained, for example, from acetobacters, or other organic acids such as lactic, citric, itaconic and butyric acids or alcohols such as ethanol.

The process according to the invention applies very particularly to the manufacture of lactic bacteria.

Lactic bacteria form a nontaxonomic group of Gram* bacteria which do not form spores and which can ferment water-soluble sugars to produce predominantly lactic acid as final product. These bacteria are employed especially in the dairy, cheesemaking and salting industries as a probiotic, that is to say as an additive for maintaining and enriching human or animal intestinal flora, or as an ensilage agent. These bacteria belong more particularly to the genera Lactobacillus, Streptococcus, Leuconostoc and Pediococcus. Lactic bacteria which belong to the genus Streptococcus are preferred. The taxonomy of these microorganisms has been described in BergeyManuel of Systematic Bacteriology (Sneath et al. 1986).

The process for the manufacture of lactic bacteria according to the invention can be carried out by means of a device such as that shown in Figure 1.

This device may, of course, be employed for fermentation of other microorganisms.

This device comprises a fermenter 1 containing the fermentation medium 2 and is connected: - to a storage vessel 3 of a carbon source in 15 aqueous solution, via a conduit 4 equipped with a feed pump 5 to a storage vessel 6 of a nitrogen source in aqueous solution, via a conduit 7 equipped with a feed pump 8 - to a storage vessel of ammonia (not shown) via a conduit 9 to a purge conduit 10, provided with a purge pump 11 and a valve 12 * to an ultrafiltration cell 18 via a conduit 14, equipped with a recirculation pump 15, a pressure gauge 16 and a valve 17.

The ultrafiltration cell has a downstream side 13 «ad 5 an upstream side 19.

The fermenter 1 also comprises: a level-measuring device 20 connected to the feed pump 5 and to the feed pump 8 by two electrical devices 21 and 22 respectively - a sample conduit 23 fitted with a valve 24, a device 25 for measuring the pH of the fermentation medium.

The upstream side 19 of the ultrafiltration cell 13 is connected to the fermenter via a conduit 26 equipped with a valve 27, a pressure gauge 28 and a heat exchanger 29. The downstream side 18 of the ultrafiltration cell 13 is provided with a conduit 30 equipped with a conductimetrie probe 31. After the conductimetric probe 31, the conduit 30 divides into two conduits 32 and 33.

Conduit 32 is equipped with a valve 34 and connected to the fermenter 1. Conduit 33 is equipped with a valve 35 and a pump 36.

The way in which the device which has just been described operates is as follows: At the beginning of an operation of manufacture of lactic bacteria, a volume of culture medium consisting partly of the source of carbon in aqueous solution originating from the storage vessel 3 and partly of the source of nitrogen in aqueous solution originating from the storage vessel 6 is introduced into the fermenter 1, followed by an inoculum of the lactic bacteria to be manufactured. In the present case the sources of carbon and the sources of nitrogen are contained in separate storage vessels, and this has the advantage, in particular, of facilitating the sterilisation of the said sources. However, it is possible to employ a single storage vessel containing both the source of carbon and the source of nitrogen in aqueous solution. Generally the procedure is such that the concentration of source of carbon in the fermenter is between 10 and 150 g/1, preferably between 35 and 80 g/1, and the concentration of the source of nitrogen is between 1 and 50 g/1 and preferably between 5 and 25 g/1.

Besides the source of carbon and the source of nitrogen, it is also possible to introduce inorganic salts which are necessary for the growth of certain microorganisms.

These salts may be introduced into the fermenter so that the concentration therein is between IE 0.001 and 1 g/1, preferably between 0.01 and 0.5 g/1. The inorganic salts may originate either from the storage vessel for the source of carbon 3 or from the storage vessel for the source of nitrogen 6.

As a source of carbon, there may be mentioned carbohydrates such as glucose, galactose, fructose, lactose, maltose, sucrose, starch and starch hydrolysates.

Organic and/or inorganic sources of nitrogen 10 may be employed as a source of nitrogen. Among the organic sources of nitrogen there may be mentioned especially proteins and protein hydrolysates such as peptones, tryptones, gelatin, casein and caseinates, animal and plant flours such as corn, soya bean, wheat and fish flours, corn step liquor, tomato pulp, yeast extracts. As an inorganic source of nitrogen there may be mentioned ammonium nitrate, alkali metal nitrates, ammonium chloride, mono- and diammonium carbonates, mono- and diammonium sulphates and mono- and diammonium phosphates.

As inorganic salts, there may be mentioned alkali or alkaline-earth metal sulphates and phosphates such as magnesium phosphate. The concentrations and the various compounds indicated above can be employed for the manufacture of lactic bacteria, but are also generally applicable to the manufacture of other bacteria or even of other microorganisms.

The carbon and nitrogen sources in aqueous solution are introduced into the fermenter 1 via the conduits 4 and 7 by the action of the feed pumps 5 and 8. The latter are connected to the level-measuring device 20 by electrical devices 21 and 22 respectively, so that the said pumps are actuated so as to keep constant the volume of the fermentation medium present in the fermenter 1.

The throughputs of the feed pumps 5 and 8 are adjusted so that the concentrations of nutrient substrates in the fermenter 1 remain constant.

The temperature of the fermentation medium is adjusted by means of a conventional device not shown in Figure 1. The said temperature may be between 25 and 32°C, preferably between 26 an 28°C.

The pressure in the fermenter is not critical, but is in general chosen conventionally, that is to say between 105 and 5x10s Pa.

Gaseous ammonia is introduced into the fermenter 1 by means of the conduit 9 and is bubbled into the fermentation medium. The gaseous ammonia allows the pH of the fermentation medium to be kept at a desired value. This value is generally between 6 and 8, preferably between 6.3 and 7.5.

The value of the pH is monitored by means of a pH-measuring device 25. This device may be optionally connected to the storage vessel of gaseous ammonia so as to control the delivery of gaseous ammonia to the fermentation medium automatically as a function of the requirements of the pH control.

In a first step it may be desired to cultivate the lactic bacteria batchwise. To do this, the valves 12 and 35 are closed, the valves 17, 27 and 34 being open.

After the batchwise culture it may be advantageous, in a second step, to remove at least part of the ammonium lactate formed, it being possible, in fact, for ammonium lactate to prevent the rate of growth from reaching the desired and predetermined value. To do this, the valves 12 and 34 are closed and the valves 17, 27 and 35 are open. The flowrate of the fermentation medium 2 leaving the fermenter is controlled by means of the pump 15.

The fermentation medium arrives at the ultrafiltration cell 13 on the upstream side 19. The retained material is recycled into the fermenter 1 by the conduit 26. The permeate, consisting chiefly of an aqueous solution of ammonium lactate, moves into the downstream side 18 and then into the conduit 30 and finally into the conduit 33, after which it is removed.

The pump 36 is adjusted so as to provide a dilution ratio D which is sufficient to permit an increase in the rate of growth up to said predetermined value. At this stage, the dilution rate D, expressed in h1, is equal to the flow rate of the permeate divided by the reaction volume.

When the rate of growth of the lactic bacteria reaches the desired value, it is possible to begin to purge the fermentation medium at a rate which is higher than or equal to the said rate of growth. To do this, valve 12 is opened and the throughput of the pump is adjusted. The purged fermentation medium leaves the conduit 10. On leaving this conduit, the said purged fermentation medium is treated so as to recover the microorganisms. Such a treatment may consist, for example, of deep freezing or freeze-drying.

Throughout the process of manufacture of the lactic bacteria such as just described, the rate of growth of the microorganisms can be evaluated continuously in a simple manner by determining the concentration of ammonium lactate in the permeate. In fact, the rate of growth μ is generally related to the concentration of ammonium lactate P by the following known formula: μ = μ max (1 - p)n Pl in which μ max is the maximum rate of growth of the lactic bacteria in the absence of any external inhibitor in the fermentation medium, Pl is the limiting concentration of the lactic acid, that is to say the maximum concentration of lactic acid at which the rate of growth of the lactic bacteria is zero for a given composition of the culture medium and n is a real number depending on the microbial strain employed. The values of μ max, Pl and n are determined by a person Λ 20 skilled in the art in a known manner for each type of microorganism.

The concentration of ammonium lactate P is known continuously by virtue of the conductimetric probe 31, which shows the value of this concentration in the permeate.

Although the use of such a conductimetric probe is advantageous, it is possible, of course, to measure the concentration of ammonium lactate by any other means which is suitable for this purpose. The measurement of the concentration of ammonium lactate by the said conductimetric probe relies on the measurement of the conductivity of ammonium lactate. It has been found, surprisingly, that, on the one hand, the concentration of ammonium lactate is proportional to the conductivity of the permeate and that, on the other hand, this measurement is not interfered with by other organic substances which are present in the permeate.

Figure 3 represents a curve showing the relationship between the concentration of ammonium lactate and the conductivity. Such a measurement can be carried out in the case of salts of organic acids produced by fermentation, other than ammonium lactate.

The microorganisms obtained according to the present invention exhibit an improved specific cell activity and cell productivity when compared with those of the same microorganisms obtained according to the processes of the prior art.

The process according to the invention also makes it possible to obtain microorganisms whose specific cell activity and cell productivity can be controlled. In fact, from one manufacture to another, it suffices to repeat the various parameters, essentially the purge rate, to manufacture microorganisms whose properties are very close or even identical.

Furthermore, it has been found that the fact 10 of purging the fermentation medium under the conditions of the invention makes it possible to increase the yield of biomass formed in relation to the source of carbon employed.

The present invention also relates to 15 microorganisms which can be obtained according to the process described above.

The said microorganisms may be, in particular, lactic bacteria, preferably lactic bacteria belonging to the genus Streptococcus.

The conditions of the preferred embodiments of the process making it possible to characterise the microorganisms, the second subject of the invention, are the same as those of the process described above, the first subject of the invention.

The microorganisms, in particular the lactic bacteria according to the invention, generally have a specific cell activity which is 20%, more generally from 50 to 100%, higher than the specific cell activity of microorganisms of the prior art and of the same type, but manufactured according to the processes of the prior art.

Finally, the invention relates to lactic 5 bacteria, preferably of the genus Streptococcus, whose cell productivity is higher than 13 UA/l/h and generally between 15 and 30 UA/l/h.

The following examples are intended to illustrate the present invention.

EXAMPLES The examples below were performed by starting with a Streptococcus cremoris strain from Lacto Labo whose code name is Sc 301 and which is marketed by this company in the form of a leaven called SM 259. At the outset, this strain manufactured by a conventional process has a maximum cell productivity of 13 UA/l/h.

The fermentations are performed in a device such as that shown in Figure 1.

The fermenter 1 has a working capacity of two litres. The ultrafiltration cell is made up of membranes whose membrane surface is equal to 0.1 m2. The pump 15 makes it possible to ensure the linear velocity of 5 m/s of the fermentation medium. The homogeneity of the fermentation medium is ensured by its recirculation. Three litres of culture medium in aqueous solution, comprising the following, are > introduced into the fermentation device of the fermenter 1; lactose : 68 g/1 yeast extract : 5 g/1 - tryptone : 7 g/1 MgSO4.7H2O j 0.2 g/1 The lactose and the MgSO,,.7H2O originate from the storage vessel for the source of carbon 3, the yeast extract and the tryptone originate from the storage vessel for the source of nitrogen 6.

During the fermentation, the pH is kept constant at 6.3, the temperature at 27eC and the pressure at 105 Pa.

The cell productivity is calculated by 15 multiplying the specific cell activity by the concentration of bacteria and by dividing by the fermentation time. The specific cell activity is determined according to the method described above.

The theoretical relationship which relates 20 the rate of growth of the said lactic bacteria to the concentration of ammonium lactate is the following: (0.9 being the theoretical maximum rate of growth) μ = 0.9 (1 - P/44) However, under the practical conditions of a 24 implementation which are described above it has been possible to determine that the experimental maximum rate of growth is not 0.9 but 0.45.

EXAMPLE 1 (not in accordance with the invention) Into the device are introduced 3 litres of culture medium, 1.5 litres of which originate from the storage vessel 3 and 1.5 litres from the storage vessel 6. The culture medium is seeded with 0.05 g of the said Streptococcus cremoris strain.

The fermentation is first of all carried out batchwise. Valves 12 and 35 are therefore closed and valves 17, 27 and 34 opened. The permeate is recycled completely towards the fermenter 1 after passing through the ultrafiltration cell 13 and the conductimetric probe 31. The conductimetric signal continuously shows the concentration of ammonium lactate present in the fermentation medium.

When the concentration of ammonium lactate reaches 30 g/1, the bacteria concentration is from 3 to 6 g/1. Valve 35 is opened, valve 34 is closed and the throughput of the pump 36 is adjusted so as to ensure a dilution rate D of 0.6 h'1. In parallel, culture medium originating from the storage vessels 3 and 6 is introduced into the fermenter in order to keep constant the volume of the fermentation medium. 17 hours after opening the valve 35, the bacteria concentration is 38 g/1. The concentration of ammonium lactate is 40 g/1 a and the rate of growth of the bacteria is 0.08 h'1.

Valve 12 is then opened and the purge pump 11 is adjusted so as to ensure a purge rate equal to 0.054 h1; 40 hours after the purge has been established, the bacteria concentration is 26 g/1 and that of ammonium lactate is 40.7 g/1. The cell productivity of the microorganisms thus manufactured is 11 UA/l/h.

EXAMPLE 2 (in accordance with the invention) The manufacture of Example 1 is continued but the lactate concentration is decreased to 22 g/1. To do this, the dilution rate D is adjusted to 1.15 h'1. The purge rate is then brought to 0.1 h1. The volume of the fermentation medium is still kept constant by introducing culture medium originating from the storage vessels 3 and 6.

The steady state maintained for 24 hours permits a bacterial concentration of 21.7 g/1 and a concentration of ammonium lactate of 25.5 g/1. The cell productivity of the microorganisms which is obtained is 20 UA/l/h.

EXAMPLE 3 (in accordance with the invention) The experiment of Example 2 is continued while the dilution rate D is adjusted to 0.60 h*1 and the purge rate to 0.19 h1. When the steady state is reached, the concentrations of bacteria and of ammonium * 26 lactate are 11.5 g/1 and 31 g/1.

The cell productivity is 26 UA/l/h.

EXAMPLE 4 (not in accordance with the invention) This example relates to the manufacture of lactic bacteria by a batch process .

The conditions for implementing this manufacture are the same as those described in the case of the batch culture conducted in Example 1. After 14 hours' fermentation, a bacterial concentration of 5 g/1 is obtained.

The cell productivity of the microorganisms thus manufactured is 13 UA/l/h.

EXAMPLE 5 (not in accordance with the invention) This example relates to the manufacture of lactic bacteria by a process with total recycling of the biomass, using a removal of the growth inhibitors by ultrafiltration. The conditions for implementing this manufacture are the same as those described in Example 1 before establishing the purge, except that the dilution rate D is 0.5 h'1.

After 90 hours' culture, the microorganisms are collected; their concentration in the reactor is 75 g/1 and their cell productivity is 10 UA/l/h.

Claims

1. Process for the manufacture of microorganisms producing their own growth inhibitor, according to which a culture medium is fermented using

2. Process for the manufacture of microorganisms producing their own growth inhibitor, according to which a culture medium is fermented using 20 the said microorganisms so as to obtain a fermentation medium, at least part of the said inhibitor is separated from the fermentation medium by a physical separation process and the retained material is recycled, the said process being characterised in that 25 the fermentation medium is purged at a purge rate which is lower than the theoretical maximum rate of growth of the said microorganisms, preferably the purge rate is 28 * between 10 and 80%, more preferably between 15 and 50% of the said theoretical maximum rate of growth.

3. Process for the manufacture of microorganisms producing their own growth inhibitor, 4. , characterised in that, before purging, the culture medium is fermented batchwise using the microorganisms to be manufactured.

4. Process according to claim 1, characterised in that the fermentation medium is purged only when the said predetermined rate of growth is reached. 20 5. Process according to one of claims 1 to

5. Genus Streptococcus. 5 preferred. 5 according to which a culture medium is fermented using the said microorganisms so as to obtain a fermentation medium, at least part of the said inhibitor is separated from the fermentation medium by a physical separation process and the retained material is 5 the said microorganisms so as to obtain a fermentation medium, at least part of the said inhibitor is separated from the fermentation medium by a physical separation process and the retained material is recycled, the said process being characterised in that

6. Process according to one of claims 1 to 25 5, characterised in that, before purging the fermentation medium, at least part of the said growth inhibitor is separated from the fermentation medium by a physical separation process. 29 »

7. Process according to one of claims 1 to 6, characterised in that the said physical separation process consists of an ultrafiltration, a centrifuging, a phase separation or a dialysis, ultrafiltration being

8. Process according to either of claims 6 and 7, characterised in that the fermentation medium is purged continuously starting when the concentration of growth inhibitor in the fermentation medium is 9. , characterised in that the said microorganisms are lactic bacteria.

9. Process according to any one of claims 1 to 8, characterised in that the pH of the fermentation medium is maintained at a value of between 5 and 8, preferably between 6 and 8. 15

10. Process according to one of claims 1 to 10 substantially constant. 10 recycled, the said process being characterised in that the fermentation medium is purged at a purge rate which is lower than the experimental maximum rate of growth of the said microorganisms, preferably the purge rate is between 15 and 80%, more preferably between 20 and 10 when the rate of growth of the said microorganisms reaches a value equal to the value of a rate of growth, measured in a steady state, corresponding to a specific cell activity of the microorganisms which has been predetermined, the fermentation medium is purged at a

11. Process according to claim 10, characterised in that the said lactic bacteria 20 belong to the genus Streptococcus.

12. Microorganisms capable of being obtained by the process of one of claims 1 to 11.

13. Microorganisms according to claim 12, characterised in that their specific cell activity is 25 20%, more generally from 50 to 100%, higher than the specific activity of microorganisms of the same type which are obtained by a process of the prior art.

14. Lactic bacteria characterised in that * 30 their cell productivity is higher than 13 UA/l/h and preferably between 15 and 30 UA/l/h.

15. Lactic bacteria according to claim 14, characterised in that the said bacteria belong to the 15 50% of the said experimental maximum rate of growth. 15 purge rate which is higher than or equal to the said rate of growth.

16. A process according to claim 1 or 2 for the manufacture of microorganisms producing their own growth inhibitor, substantially as hereinbefore described and exemplified.

17. A microorganism producing its own growth inhibitor, whenever manufactured by a process claimed in a preceding claim.

18. A lactic bacterium according to claim 14, substantially as hereinbefore described.