FR2526808A1 - IMPROVED PROCESS FOR THE PREPARATION OF L-LYSINE - Google Patents

Abstract

THE SUBJECT OF THE PRESENT INVENTION IS A PROCESS FOR THE PREPARATION OF LYSINE INTENDED FOR FOOD USE. THIS PROCESS CONSISTS OF STARTING FROM WHEY OR LACTOSE JUICE WHICH IS SUBJECTED TO ENZYMATIC, ACIDIC OR CATALYTIC HYDROLYSIS IN THE PRESENCE OF A LYSINE-PRODUCING STRAIN THEN DRYING THE MIXTURE OBTAINED.

Description

AT

  IMPROVED PROCESS FOR THE PREPARATION OF L-LYSINE

  The subject of the present invention is an improved method for pre-

  L-lysine partion and relates more particularly to a process for obtaining a metabolizable mass containing L-lysine, this mass being intended to serve as a feed additive for animal feed.

  We know that it is necessary to supplement the food of animals

  sore by intake of protein when the amount of protein provided

  animals is too weak, which is particularly the case

  essentially based on, for example, cereal

  as we know, have a low protein content.

  Protein quality is in fact mainly defined by their

  in amino acids, particularly L-lysine, it is also possible to use

  L-lysine as an additive to improve the nutritional value of

  This L-lysine is, in general, placed on the market for animal feed in the form of

  a powder consisting of a mixture containing about 10% of lysine

  it has fillers and other metabolizable products, said powder

  being formed by mixing the various constituents.

  L-lysine used for this purpose has so far been essential

  prepared by chemical synthesis, protein hydrolysis or fermentation.

  The production of lysine by chemical synthesis is based mainly on the use of cyclic derivatives. For this purpose, we know production methods starting from caprolactam, dihydropyran or

  cyclohexane The disadvantage of these chemical methods

  time is to drive only a racemic mixture of D and L-lysine, and

  since D-lysine is not an assimilable food, it is necessary

  to carry out additional operations, either for the separation of the D and L isomers of lysine, or for the transformation of the L-isomer L isomer. This transformation is generally carried out by a racemase

  of Achromobacter obae followed by an aminohydrolase of Cryptococcus lauren

  tii and leads to the conversion of 99.8% of the racemic to L-lysine.

  This two-step method is complicated and expensive.

  The protein-lysine production by protein hyiirolyte has also not proved commercially viable, the yield being low and

the high cost price.

  In fact, these methods not chemical synthesis or protein hydrolysis proved too expensive, so we developed, the latter

  years, methods based on a fermentative process.

  Thus Davis (in the journal Nature, London 169, 534 (1952)

  evidence in E coli, auxotrophic, an accumulation of diaminopyridin

  (DAP) in a culture medium, whose conversion to lysine has been

  performed, with a yield of 100% by Acrobacter aerogens.

  Similarly, Haskins (USP 2902409) achieved a production yield of L-

  lysine 0.2 to 0.3 g / l using two strains of U Maydis In fact, U Mayais is the best wild excretory strain of L-lysine (1 g / l); however, better productions from the industrial point of view have been

  obtained by using auxotrophic mutants and regulatory mutants.

  Coryneform bacteria (article by Sano and Shiio in the review J Gen App Microbiol 13,349, (1967) In fact the fermentation is the

  industrial process currently the most economically profitable.

  All the processes thus described lead to lysines of high purity whose cost is relatively high. They involve, in fact, a large number of operations to achieve the purification and isolation of lysine and then to its combination with the various metabolizable fillers and products mentioned above for forming the food masses

  constituting the aforementioned food additive.

  The basic idea that led to the present invention was to obtain

  L-lysine using a process that does not require

  separation and isolation, nor the recombination

  the procedures implemented so far.

  From this idea, the applicants have sought a novel process for the preparation of lysine from a substrate which may itself constitute a food and giving rise to hydrolysis products which are

themselves foods.

  Applicants have thought of whey lactoserumetjuslactosé (ultraflltrat); however, in view of the fact that the strains capable of producing mutants of Escherichia coli develop on lactose, an attempt has been made to

  produce mutants of Escherichia coli that secrete L-lysine.

  It has indeed happened to produce lysine through the use of this mutant on lactose, but this method is not satisfactory because

  the amount of secreted L-lysine is too low.

  In fact, the research and developments that led to the pre-

  The present invention has made it possible to obtain a lysine-based additive

  tible to be used to supplement animal diets, not requiring purification during their preparation and containing

  in addition to the lysine of the constituents resulting from the hydrolysis or

  similarity of the starting mixture which are themselves foods.

  The subject of the present invention is a process for the preparation of

  sine intended for food uses, in particular for animal feed, which consists of whey or whey juice which is submitted to a

  enzymatic, acidic or catalytic hydrolysis in the presence of a

  lysine duct and then to dry the mixture obtained.

  It is recalled that whey is the remaining liquid fraction of milk

  after separation of the curd destined for the preparation of the cheese.

  The whey composition is a function of the milk itself and also of

the nature of the cheese made.

  The average composition of a whey is: 85% lactose, 5 to 6% protein,

at 10% of mineral salts.

  This process has the advantage that the rate of lysine formation is relatively high.

  that the lysine-producing strain consumes the glucose

  resulting from enzymatic hydrolysis, that is, from the reaction: lactose + glucose + galactose

  that it is easily reproducible and leads to

  tives. According to one embodiment of the invention, the enzymatic hydrolysis is carried out via enzymes, preferably of the β-galactosidase type.

  According to another embodiment of the invention, the strain produc-

  lysine is one of the strains belonging to the group Corynebac-

  glutamicum (= micrococcus glutamicus), Brevibacterium, Arthrobac-

ter sp, Corynebacterium lilium.

  The subject of the present invention is also a metabolizing mass

  containing lysine obtained by means of a process referred to above,

  lysine forming 5 to 30 of said mass, the remainder being composed of

  metabolizable products or products and preferably at least in part

  mass constituted by secretory residues of lysine.

  The present invention has the advantage of allowing the valorization

  whey, which is often dried but is valued

  irregular and weak, and which is often still rejected as waste.

  Other objects and advantages of the present invention will become apparent

  reading the following description and examples given as a non

  limiting. EXAMPLE 1 Example of production in stirred flask: the culture medium is prepared from a solution of lactoserum

  (10 U 9 / l) hydrolyzed by the action of o-galactosidase (concentration

  final concentration in glucose 45 g / l) to which is added:

(NH 4) 2 SO 4 13.4 9/1

  KH 2 PO 4 2.2 g / l Mg SO 4 0.8 g / 1 yeast extract 5 g / l thiamine 5 10 g / 1 biotin 5 10 g / 1

  After a sterilization treatment of 30 minutes at 1050 C, half

  Place of culture is inoculated with a strain of Corynebacterium.

  The flasks are incubated on a rotary shaker (200 rpm) at

  temperature of 30 C for 72 hours.

  After grinding, it is possible to obtain from 1 liter of culture medium, ie 103 g of dry powder containing: lysine 17.5% biomass 5.8% galactose 42.7% lactose 9.7% mineral salts 24.3 %

  Lysine is assayed according to the method of Chinard and Work (JL BIOL.

Chem 51,199 (1952).

  The strain of Corynebacterium used has the following characteristics: gram positive, aerobic, immobile, non-sporulating, resistant to ACS (0.5 g / 1), resistant to naledixic acid (30 μg) antibiotic acting on replication ,

  yellow pigmentation of clones, most often as they age.

Example 2

  Example of fermenter production:

  the same medium prepared from hydrolysed lactose juice (concentrated

  Glucose concentration of 50 g / l) is introduced into a 20 liter Biolafitte fermentor inoculated with the above-mentioned Corynebacterium strain. The following experimental conditions are used: temperature 30 CpH 6.8 Aeration 60 v / v / h

  and obtains 20 g / l of lysine in 48 hours.

  Under the same experimental conditions but with 80 g 9/1 of gli

  As a result, 28 g / l of lysine was obtained.

  In fact, 154 g / dry powder are obtained 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 cylinder

  as a recirculation of the medium, it is regulated in temperature, p H, con-

  dissolved oxygen center and equipped with a mechanical system against

  the foam This fermenter comprises a stirrer consisting of a

  only with slightly inclined pallets (angle of the order of 30) so that the culture medium is propelled from the periphery of the

  cylindrical tube to a circumaxial position.

Example 3

  Biolafitte fermenter production example:

  in a Biolafitte fermenter similar to that of the previous example.

  the following medium is used: (NH 4) 2 SO 04 20 g / l Fe S 4 32 10 / * FeS Oq 2 10 g / 1 * Mn 5 04 if 5 * @sts 3 oa 9 v * ea MS 04 ,,,, ,, o Biotin solution vitamin Bl yeast extract and fermentation is carried out on glucose

of it with time.

  The following results are obtained: final accumulation of lysine sugar consumes residual sugar biomass production -lysine yield / sugar -3 7.7 1 O g / 1

0.37 9/1

  ml 500 g 9/1 500 μg / kg / l at 50 9 g / l with addition 27.6 o O g / 1 gggi Example 4 Fermentation with demineralised hydrolized whey juice containing only 5% ash: dry matter 600 g / kg% hydrolized 90% nitrogenous material 23 g / kg Ashes 50 g / kg citrate 10 g / kg lactate 5 g / kg Using as medium the same medium as that indicated in Example 3, is obtained after fermentation to a powder having the following composition: final accumulation of lysine 19% lactose 5.5% galactose 49.5% residual glucose 10.3% biomass production (dry weight) 15.7%

  which corresponds to a yield on sugar consumed of 38.3%.

  Examples 5 and 6 A fermentation is carried out on powdered lactose powder to which glucose is added at the rate of 40 g / l and 62 g / l respectively

  and without any contribution in time. The same medium is used as in the Exem-

  The results are as follows: T lactose 8.8 g / l 14 g / l galactose 40 g / l 62.5 g / 1 glucose consumed 40 g / l 45.2 g / l residual glucose O 17.3 g / 1 biomass production (dry weight) 13 g / 1 16 g / 1 ratio (%): lysine / sugar consumed 28.75 24.3 total specific activity of lysine production 0.91 0 , 69 (biomass / 2 + lysine) produced yield = X 100 = 29.5% 42% glucose consumed Under these conditions: 40 g / 1 of glucose and without time contribution of it, we note:

  the end of bacterial growth and the end of the

  lysine are reached after 30 hours of culture.

  Bacterial growth is regular, that is, there is no

no break in slope.

  the phenomenon of alkalization appears in the first few hours

fermentations.

  In contrast, for 62 g / l glucose initially, the bacterial growth curve is biphasic (two growth phases).

  starts at two o'clock in the morning and ends at ten o'clock in the morning.

  twelfth begins at 10 pm and ends at 40 hours of culture The

  Glucose consumption is incomplete (residual glucose: 17.3 g / 1).

  In addition, glucose consumption becomes linear from the lag phase of the second growth phase. Examples 7 and 8 Examples of Biolafitte Fermentor Production on a Whey

GLUCOSE

  F 40% g / l 62% g / 1 final accumulation of lysine 11.8 g / 1 11 g / 1

252680.

  deprotein catalytic hydrolysis (thus demineralized) on the one hand, and enzymatic hydrolysis on the other hand, having the following compositions: total dry matter mineral matter nitrogenous material (total)% hydrolysis lactose glucose

galactose -

  p H The fermentation is carried out at 68

contribution of it in time.

  The following results are obtained: enzymatically catalyzed hydrolyzed whey example 7 example 8 g / 100 g traces 0.3 g / 100 g% 11.7 g / 100 g 24.0 g / 100 g 24.0 g / 100 g 100 g 7.5, 7 2.7, 8, 8 g / 100 g 9/100 g / 100 g% g / 100 g 9/100 g / 100 9

J I

  9/1 of glucose at the start and without Example 7 Example 8 catalytic route enzymatic

lysine produced 16 g / 11 g / 1.

  biomass produced (dry weight) 14 g / l 16.5 g / 1 lactose 34 g / l 20 g / 1 glucose consumed 68 g / 1 33 g / 1 residual glucose O 17.4 g / 1 galactose 68 g / 1 51 g / 1

9 2526808

  FIG. 1 represents an installation in which the process according to

  the invention is capable of being implemented.

  This plant comprises a conduit 1 for the arrival of the whey in a reactor 2 of acid hydrolysis or enzymatic hydrolysis, the mass thus treated being fed through line 3 into a fermentation tank 4. The mixture of the strain and the food base of the strain (glucose) is prepared in the chamber 5 and introduced via line 6 into said ba:

of fermentation 4.

  The fermented cup is brought by line 7 into a preconcentration zone.

  including several preconcentrators and hence the preconcentration mass

  obtained is conducted by line 9 in a zone of atomization 10 of which

  the lactoserum enriched in L-lysine is withdrawn in tank via line 11.

  The spray drying process can be replaced by any

other drying process.

  Of course, the present invention is not limited to modes of r'a.

  described and represented; it is capable of numerous variants accessible to those skilled in the art without departing from the V

of the invention.

1 '

Claims (9)

REVENDlCATIONS   1. Prxci for the preparation of lysine for food   airies, which consists of lactoserum or lactose juice which is subjected to enzymatic, acidic or catalytic hydrolysis in the presence of a lysine-producing strain and then to dry the mixture obtained.   2. Process according to claim 1, characterized in that the hydro-   Enzymatic enzyme is carried out via enzymes.   3. Method according to claim 1 or 2, characterized in that   the enzymatic hydrolysis is carried out via e-galac- tosidase.   4 Process according to claim 1, characterized in that   hyidrolysis is carried out catalytically on resins Ions.   5. Process according to any one of claims 1 to 4, characterized   in that the lysine-producing strain is one of the   belonging to the group Coryneb turium 'iutamicum (= micrococcus gluta-   wicus), Brevibacterium, Arthrobacter Sp, Corynebacterium lilium.   6. Metabolizable mass containing lysine obtained with the aid of   process according to any one of claims 1 to 5, characterized in that   that said lysine forms 5 to 30% of said mass, the remainder being constituted   loads or metabolizable products.   7. metabolizable mass according to claim 6, characterized in that the aforementioned rest is formed at least partly of biomass constituted   by the secretory residues of lysine.   Metabolizable mass according to any one of claims 1 to   7, characterized in that the whey is a demineralized whey.   The metabolizable mass of any one of claims 1 to   8, characterized in that the whey is an undemineralized whey,   the concentration of glucose in the fermentation medium being preferably the order of 40 g / l.