GB2120240A - Lysine from lactose by fermentation - Google Patents

Abstract

A process for the preparation of lysine destined for alimentary use consists in starting from lactoserum or lactose containing juice, subjecting it to an enzymatic, acid or catalytic hydrolysis in the presence of a lysine- producing microorganism and drying the mixture thus obtained. The hydrolysis is preferably effected by an enzymatic route, e.g. with beta -galactosidase, or by a catalytic route with an ion exchange resin. The lysine producing microorganism may be Corynebacterium glutamicum, Brevibacterium Arthrobacter sp. or Corynebacterium lilium.

Description

SPECIFICATION Improved process for the preparation of L-lysine The present invention concerns an improved process for the preparation of L-lysine and relates more particularly to a process for the obtention of a metabolisable mass containing L-lysine, this mass being destined to serve as an alimentary additive for animal foodstuffs.

It is known that it is necessary to complement animal foodstuff with respect to proteins when the quantity of the proteins supplied to the animals is too low, which is especially the case of a foodstuff essentially based, for example, on cereal grains that, as is well known, have a low protein content.

The quality of the proteins is, in fact, above all, defined by their aminated acid content and especially their L-lysine content; therefor L-lysine is currently used as an additive in order to improve the alimentary value of certain foodstuffs destined to be fed to animals. This L-lysine is generally available on the animal foodstuff market in the form of a powder constituted by a mixture containing 10% lysine associated to fillers or additives and other metabolisable products, the said powder being formed by mixing the various constituents.

The L-lysine used for this purpose has up until now been essentially prepared by chemical synthesis, proteic hydrolysis or fermentation.

The production of lysine by chemical synthesis is substantially based on the use of cyclic derivatives.

Therefore, with this purpose, production methods are known which start from caprolactam, dihydropyrane, or also cyclohexane. The drawback of these chemical processes, however, is that they only lead to a racemic mixture of D- and L-lysine, and as D-lysine does not constitute an assimilable aliment, it is necessary to effect supplementary operations, either the separation of D- and L-isomers of the lysine, or the transformation of D-isomer into L-isomer. This transformation is generally carried out by an Achromobacter obae racemase followed by an aminohydrolase of Cryptococcus laurentii and leads to the conversion of 99.8% of the racemic into L-lysine.

This method, comprising two stages, is complicated and expensive.

Neither has the production of L-lysine by hydrolysis of proteins shown itself to be industrially economical, the yield being low and the cost price high.

In fact, these methods by chemical synthesis or proteic hydrolysis have proved to be too expensive, and during recent years methods based on a fermentative process have been developed.

Thus DAVIS (in the journal Nature, London, 169,534(1952) has has shown that auxotrophic E. coli exhibit an accumulation of diaminopimelic acid (DPA), in a culture medium, the conversion of which acid into lysine has been carried out with a yield of 100% by aerogenic Acrobacter.

Furthermore, HASKINS (USP 2.902.409) has obtained a production yield of L-lysine from 0.2 to 0.3 g/l by using two strains of U. Maydis.

In fact, U.Maydis is the best wild L-lysine excreting strain (Ig/l); however the best productions from the industrial point of view have been obtained by using auxotrophic mutants and regulation mutants of Coryneforme bacteria (article of SANO and SHIIO in the journal J. Gen. app. Microbiol. 13,349(1967). In fact, fermentation is currently the most economically viable process.

All the processes thus described lead to extremely pure lysines, the cost price of which is relatively high.

They imply, in fact, a large number of operations in order to achieve the purification and isolation of the lysine, then its combination with the various fillers and metabolisable products mentioned herein-above in order to form alimentary masses constituting the foodstuff additive mentioned herein-before.

The fudamental idea that has led to the present invention was to obtain L-lysine by using a process that requires neither separation and isolation operations, nor recombination operations as necessitated by the processes carried out up to now.

Starting from this idea, it was endeavoured to develop a novel process for the preparation of lysine from a substrate able to constitute in itself an aliment and giving rise to hydrolysis products that are themselves aliments.

It has been decided to start from lactoserum and lactosed containing juice (ultrafiltrate); however, taking into consideration that the strains liable to produce Escherischia Coli develop on lactose, an attempt was made to produce Escherischia Coli mutants that secrete L-lysine.

Production of lysine was achieved due to the use of this mutant on lactose, but this method is not satisfactory since the quantity of the secreted L-lysine is too small.

In fact, the research and development that led to the present invention has allowed obtention of a lysine-based additive, liable to be used to complement animal alimentary diets, that does not require purification during its preparation and that contains beside lysine, the constituents resulting from the hydrolysis or the assimilation of the original mixture that are themselves aliments.

The present invention concerns a process for the preparation of lysine destined for alimentary uses, especially for animal foodstuffs, that consists in starting from lactoserum or lactose-containing juice, subjecting it to an enzymatic, acid or catalytic hydrolysis in the presence of a lysine-producing strain then drying the mixture thus obtained.

It will be recalled that lactoserum is the liquid fraction of milk that exists after separation of the curds destined for the preparation of cheese.

The composition of lactoserum depends on the milk itself and equally on the nature of the cheese produced.

- 80 à 85% lactose; - 5 à 6% proteins; - 15 à 10% mineral salts.

This process has the advantage: - that the speed of formation of lysine is relatively high due to the fact that the lysine-producing strain consumes the glucose resulting from the enzymatic hydrolysis, i.e. dfrom the reaction: lactose # glucose + galactose - that it is easily reproducible and leads to constant yields.

According to one embodiment of the invention, the enzymatic hydrolysis is carried out through the intermediary of enzymes, preferably of the ss-galactosidase type.

According to another embodiment of the invention, the strain producing the lysine is one of the strains of the group comprising Coryne-bacterium glutamicum (= micrococcus glutami cus), Brevibacterium, Arthrobacter sp., Corynebacterium lilium group.

The present invention also concerns a metabolisable mass containing lysine obtained by using the above-mentioned process, the said lysine forming 5 to 30% of the said mass, the remainder being constituted by fillers or metabolisable products and preferably at least partly by a biomass constituted by the lysine-secreting residues.

The present invention has the advantage of allowing the valorisation of lactoserum, a product often dried but the valorisation of which is irregular and weak, and which is often furthermore discarded in the form of waste.

Other objects and advantages of the present invention will appear from the following description and examples given by way of non-limitative example, and from the appended drawing.

EXAMPLE 1 Production in stirred flask: - the culture medium is prepared from a solution of hydrolysed lactoserum (100 g/l) by the action of ss-galactosidase (final concentration of glucose 45 g/l) to which is added: .(NH4)2SO4 .................................... 13.4 g/l .KH2PO4 ....................................... 2.2 g/l .MgSO4 ........................................ 0.8 g/l yeast extract ........................................ 5 g/l thiamine ............................................. 5.10-4 g/l biotine .............................................. 5.10-4 g/l After a sterilization during 30 minutes at 105 C, the culture medium inoculated with a strain of Corynebacterium. The flasks are incubated on a rotative stirrer (200 rpm) at a temprature of 30 C during 72 hours.

After possible grinding 103 g of dry powder is obtained from 1 liter of culture, said powder having the following composition: .lysine 17.5% .biomass 5.8% .galactose ...................................42.7 % .lactose ........................................ 9.7 % .mineral salts ........................................ 24.3 % The lysine content is determined according to the method of CHINARD and WORK (JL BIOL. Chem. 51,199 (1952).

The Corynebacterium strain used has the following characteristics: -gram positive, - aerobic; - immobile; - does not sporulate; - ECA resistant (0,5 g/l); - resistant to naledixic acid (30 g), an antibiotic acting on the replication; - yellow pigmentation of the spirits, most frequently while aging.

EXAMPLE 2 Example of production in a fermenter: -the same medium prepared from hydrolysed lactose-containing juice (glucose concentration 50 g/l) is introduced into a Biolafitte fermenter of 20 liters in oculated with the Corynebacterium strain mentioned herein-above. The experimental operating conditions are as follows: .temperature ........................................ 30 C .pH 6.8 aeration 60 vivlh 20 g/l of lysine is obtained in 48 hours.

Underthe same experimental conditions, but using 80 g/l glucose, 28 g/l lysine is obtained.

In fact, one obtains 154 g/l of dry powder containing: .L-lysine ............................................... 18.2% biomass ................................................. 9.7% galactose ............................................... 45.5% lactose .............................................. 13.0% mineral salts ........................................ 13.6 % The Biolafitte fermenter used is equipped with a director cylinder allowing a recirculation of the medium and its temperature, pH, dissolved oxygen concentration is regulated. it is further equipped with a mechanical anti-frothing system.This fermenter comprises a stirrer consisting of a disc with slightly tilted blades (angle of about 30 ) so that the culture medium is propelled from the peripheral of the cylindrical tube towards a circumaxial zone.

EXAMPLE 3 Example of production in Biolafitte fermenter: - in a Biolafitte fermenter identical to that used in the previous example, the following medium is used: .(NH4)2SO4 ......................................................... 20 g/l .Fe SO4 ........................................ 2.10-3 g/l .Mn SO4 ...................................... 7.7.10-3 g/l .Mg SO4 ........................................ 0.37 g/l .solution .......................................... 50 ml .biotine ............................................ 500 g/l .vitamine B1 ........................................ 500 g/l .yeast extract ...................................... 5 g/l and the fermentation is carried out on glucose at 50 g/l its contribution being made with time.

The following results are obtained: final accumulation of lysine ........................................ 27.6 g/l .sugar consumed .............................................. 0 g .residual sugar ............................................... 0 g .production of biomass ........................................ 10 g .lysine/sugar yield ........................................... 34 % EXAMPLE 4 Fermentation with demineralized, hydrolized lactose-containing juice, only containing 5% ash:: . dry matter ........................................ 600g/kg . % hydrolysed ...........................................90% .nitrogenated matter ........................................ 23 g/kg .ash ......................................................... 50 g/kg .citrate ..................................................... 10 g/kg .lactate ..................................................... 5 g/kg Using as the medium the same as that indicated in EXAMPLE 3 is obtained after fermentation a powder having the following composition:: .final accumulation of lysine ................................... 19 % .lactose ................................................... 5.5% .galactose ................................................................. 49.5 % .residual glucose .......................................................... 10.3 % .production of biomass (dry weight) ........................................ 15.7 % that corresponds to a yield related to consumed sugar of 38,3 %.

EXAMPLES 5 and 6 A fermentation is carried out on lactose-containing juice, in powder, to which is added 40 g/l and 62 g/l glucose respectively, without gradual admixture. The same medium as that of EXAMPLE 3 is used, except the glucose content. The following results are obtained: GLUCOSE 40% g/l 62% g/l - final accumulation of lysine .................... 11.8 g/l 11 g/l lactose ......................................................... 8.8 g/l 14 g/l -galactose ...................................................... 4040 9/l 62.5 g/l - consumed glucose .............................................. 40 g/l 45.2 g/l - residual glucose .............................................. 0 17.3 g/l -productionofbiomass(dryweight) ................................. 13 g/l 16 g/l - ratio (%): lysine/sugar consumed .................................... 28.75 24.3 - overall specific activity of lysine production ........................................ 0.91 0.69 (biomass/2 + lysine) produced -yield = x100= 29.5 % 42 % glucose consume Under these conditions: 40 g/l of glucose and without further gradual glucose admixture, the following is noted: - the end of bacteria growth as well as that of the lysine roduction occur after 30 hours of culture; - the bacteria growth is regular, i.e. there is no break in the curve; - the alkalinisation appears at the first hours of fermentation.

On the other hand, for 62 g/l glucose originally used the curve of bacteria growth is diphasic (two phases of growth). The first begins at 2 hours of culture and finishes at 22 hours and the second starts at 22 hours and finishes at 40 hours of culture. The consumption of glucose is incomplete (residual glucose:17,3 g/l). It is noted, furthermore, that the glucose consumption becomes linear as from the latent phase of the second phase of growth.

EXAMPLES 7 and 8 - examples of production in Biolafitte fermenter on a deproteinised lactoserum hydrolysed by catalytic route (thus demineralized), on the one hand, and hydrolysed by enzymatic route, on the other hand, having the following compositions: lactoserum hydrolyzed by catalytic by enzymatic route route Example 7 Example 8 total dry matter 60 g/100 g 60 g/100 g mineral matter ........................................ traces 5.7 g/100 g nitrogenated matter (total ............................ 0.3 g/100 g 2.7 g/100 g %hydrolysed ........................................... 80 % 80% lactose ........................................ 11.7 g/100 g 10 g/100 g glucose ........................................ 24.0 9/100 g 20.8 9/100 g galactose ........................................ 24.0 9/100 g 20.8 g/100 g pH ........................................ 7.5 6 Fermentation is carried out with 68 g/l of the glucose initially used and without glucose admixture with time.

The following results are obtained: Example 7 Example 8 catalytic enzymatic route route lysine produced 16 g/l 11 g/l biomass produced (dry weight) .................. 14 g/l 16.5 g/l lactose ........................................ 34 g/l 20 g/l consumed glucose ................................. 68 g/l 33 g/l residual glucose ................................. 0 17.4 g/l galactose ........................................ 68 g/l 51 g/l Figure 1 represents an installation in which the process according to the invention can be operated.

This installation comprises a conduit 1 for the admission of the lactoserum in a reactor 2 for the acid hydrolysis or enzymatic hydrolysis, the mass thus treated being lead by a conduit 3 into a fermentation tank 4. The mixture of the strain and the alimentary base of the strain (glucose) is prepared in an enclosure 5 and introduced by conduit 6 into the said fermentation tank 4.

The fermented mass is brought by conduit 7 into a preconcentration zone comprising several preconcentrators and from there the preconcentrated mass obtained is carried by conduit 9 into an atomisation zone 10 from which is withdrawn in a vat the lactoserum enriched with L-lysine by conduit 11.

The process of drying by atomisation can be replaced by any other drying process.

Of course, the present invention is in no way limited to the embodiments described and represented herein; it to the adapted to numerous variants available to the man skilled in the art without departing from the scope and spirit of said invention.

Claims (10)

1. Process for preparing lysine, destined for alimentary uses, that consists in starting from lacteroserum or lactose - containing juice, submitting it to an enzymatic, acid or catalytic hydrolysis in the presence of a lysine - producing strain and drying the mixture thus obtained.

2. Process according to claim 1, wherein the enzymatic hydrolysis is operated by the intermediary of enzymes.

3. Process according to claims 1 or 2, wherein the enzymatic hydrolysis is operated through the intermediary of p-galactosidase.

4. Process according to claim 1, wherein the hydrolysis is operated by catalytic route on ion exchanger resins.

5. Process according to any one of claims 1 to 4, wherein the lysine - producing strain is one of the strains belonging to the group of Corynebacterium glutamicum (= micrococcus glutamicus), Brevibacterium, Arthrobacter sp., Corynebacterium lilium.

6. Metabolisable mass containing lysine obtained by using the process according to one of claims 1 to 5, wherein the said lysine forms 5 to 30% of the said mass, the remainder being constituted of metabolisable loads or products.

7. Metabolisable mass according to claim 6, wherein the above-mentioned remainder is formed at least in part of the biomass constituted by lysine-secreting residues.

8. Metabolisable mass according to one of claims 1 to 7, wherein the lactoserum is demineralized lactoserum.

9. Metabolisable mass according to one of claims 1 to 8, wherein the lactoserum is a non demineralized lactoserum.

10. Any novel feature or combination of features described herein.