FR2728588A1 - Detection of prodn. of diacetyl by bacterium - Google Patents

Abstract

Process for selecting bacterial strains deficient in alpha-acetolactate decarboxylase with a view to promoting the production of diacetyl, characterized in that the said bacterium is placed in the presence of hydroxylamine, and in that the dimethylglyoxime possibly formed is detected, and Lactococcus lactis subsp. lactis biovar. diacetylactis thus obtained. The invention also relates to the use of such a process for the production of a flavoring ferment which can be used in the food industry.

Description

METHOD FOR SELECTING BACTERIAL STRAINS
DIACETYL PRODUCERS
The present invention relates to a method for selecting bacterial strains deficient in CL-acetolactate decarboxylase, in order to promote the production of diacetyl.

 Diacetyl is a desirable aroma in certain food products such as butter, cream, fresh cheeses and some fermented milks.

In these products, the diacetyl concentration is most often less than 5 mg / l.

 In dairy products, diacetyl is mainly synthesized from citric acid, naturally present in milk at a concentration of about 1.7 g / l. The flavoring ferments used in the manufacturing processes generally contain strains of Lactococcus lactis subsp. lactis biovar.

diacetylactis or Leuconostoc spp., able to use citric acid. Citric acid gives rise to the formation of α-acetolactate, which is the precursor of diacetyl.

 Α-Acetolactate is an unstable compound that decarboxylates spontaneously to diacetyl or acetoin. Diacetyl is formed under oxidative conditions, for example in the presence of oxygen. Under nonoxidative conditions, acetoin is formed which does not possess the aromatic properties of diacetyl.

 Α-Acetolactate decarboxylase is an enzyme which decarboxylates α-acetolactate to acetoin to the detriment of diacetyl.

Several aroma production processes involve the synthesis of α-acetolactate by microorganisms. The α-acetolactate product can then be converted to diacetyl, for example by distillation in the presence of oxygen (JONSSON H. et al.,
Milchwissenschaft, 1980, 31, 8, 658-662). In the case of the manufacture of butter according to the "NIZO" process (developed by the Netherland Institute for Dairy
Research) the production of α-acetolactate by the ferment is due to the presence in said ferment of a strain of
Lactococcus lactis subsp. lactis biovar. diacetylactis naturally deficient in α-acetolactate decarboxylase.

In culture in milk, this strain produces high amounts of α-acetolactate, in contrast to strains possessing α-acetolactate decarboxylase (Hugenholtz J. et al., Appl Microbiol Biotechnol., 1992 38, 17-22).

 It has also been proposed to use α-acetolactate synthase, which is the enzyme responsible for the formation of α-acetolactate from pyruvate, in a strain deficient in α-acetolactate decarboxylase, to further increase the production of α-acetolactate. α-Acetolactate (European Patent Application No. 500,188, to the names of Unilever NV

and Unilever PLC).

 It would be interesting to have other mutants deficient in α-acetolactate decarboxylase, in particular mutants made from strains with interesting technological characteristics for the relevant manufacturing processes (dairy products) (phage resistance, acidifying activity, activity). proteolytic ...).

 Nucleic acid encoding Lactococcus lactis subsp. Α-acetolactate decarboxylase lactis was cloned and sequenced (French Patent Application 2,696,191 in the name of INRA). This French Patent Application 2,696,191 also describes processes for the preparation of lactic acid bacteria of the Lactococcus lactis subsp.

lactis in which the α-acetolactate decarboxylase has been inactivated by genetic engineering.

 However, genetic engineering techniques are relatively cumbersome to implement; the selection of the microorganisms obtained requires the use of a marker gene in addition, the use of recombinant microorganisms is the subject of restrictive regulations.

 Another method for obtaining <x-acetolactate decarboxylase-deficient mutants is to perform random mutagenesis from the microorganism strain whose mutants are desired to be obtained in this case, the main difficulty is to specifically identify the mutants possessing the mutant. the desired characteristics.

 The present invention aims to allow the detection of lactic acid bacteria deficient in β-acetolactate decarboxylase, and therefore the screening and selection of mutant bacteria. This method involves the detection, in the culture medium, of the diacetyl produced by the bacteria carrying the desired mutation.

 The subject of the present invention is a method for detecting the production of diacetyl by a bacterium, characterized in that the said bacterium is introduced in the presence of hydroxylamine, and in that the dimethylglyoxime formed by reaction of diacetyl is detected. optionally produced and hydroxylamine.

 The dimethylglyoxime formed can then be detected, for example, by formation of a red color complex in the presence of iron sulfate and ammonia.

 According to a preferred embodiment of the process according to the invention, it is a method of screening or selection, on agar medium, bacterial colonies producing diacetyl. However, it can be applied to non-agar culture screening, for example to the selection of microtiter plate clones.

 According to another preferred embodiment of the process according to the invention, the diacetyl production is detected after having subjected said bacterium to mutagenic treatment.

 This mutagenic treatment can be carried out by any means known in itself; it can be chemical mutagenesis, mutagenesis by irradiation, or mutagenesis by insertion or deletion of nucleic acid sequences.

 It may be a random mutagenesis treatment, but it may also be directed mutagenesis.

For example, to use the process according to the invention for the detection of lactic acid bacteria deficient in α-acetolactate decarboxylase; we operate as follows
The mutagenized cells are inoculated on petri dishes, so as to form colonies. The culture medium used allows the production of α-acetolactate by mutants deficient in α-acetolactate decarboxylase. Α-Acetolactate is partly converted by oxidative chemical decarboxylation to diacetyl due to the incubation of
Petriformed under aerobic conditions. Strains possessing α-acetolactate decarboxylase produce little diacetyl under these conditions and do not induce colony-like staining. Strains producing α-acetolactate are therefore readily demonstrated in this screen.

 Diacetyl reacts with hydroxylamine to form dimethylglyoxime. The dimethylglyoxime then forms a complex strongly colored red in the presence of iron sulfate and ammonia. As this series of chemical reactions is lethal for microorganisms, replicates of the Petri dishes are carried out prior to screening. The colonies revealed by the screening are then recovered on these replicas.

 The detection method according to the invention can also be used for screening and obtaining strains in which the α-acetolactate decarboxylase has been sufficiently attenuated, without having been totally inactivated, since the production of acetolactate by these strains is higher than in parental strains.

Among the selected mutant strains, mention may be made preferably of strain F2X2 deposited on December 20, under No. I-1510, with
National Collection of Culture of Microorganisms (CNCM) held by INSTITUT PASTEUR.

 In general, the method according to the invention can be used in any procedure for screening the production of α-acetolactate by microorganisms, since this screening is based on a specific detection of diacetyl.

The invention also relates to the use of the method for detecting the production of α-acetolactate for microorganisms other than Lactococcus lactis subsp. lactis biovar diacetylactis, for example for lactic acid bacteria of the genus Leuconostoc or
Lactobacillus. Strains of Lactococcus lactis subsp.

lactis biovar. diacetylactis are not the only strains capable of producing diacetyl. Stimulation of α-acetolactate and diacetyl production by inactivation of α-acetolactate decarboxylase is feasible for other bacterial species.

 Advantageously, said detection method makes it possible to select lactic acid bacteria deficient in α-acetolactate decarboxylase, suitable for use as a flavoring ferment.

 Such flavoring ferments selected in accordance with the present invention are preferably used in the preparation of fresh cheeses, fermented milks, matured cream and butter.

 Advantageously, said ferments comprise either leuconostocs or lactococci, selected in accordance with the invention, alone or as a mixture.

 In addition to the foregoing, the invention also comprises other arrangements, which will emerge from the description which follows, which refers to examples of implementation of the method that is the subject of the present invention.

 It should be understood, however, that these examples are given solely by way of illustration of the object of the invention, of which they in no way constitute a limitation.

EXAMPLE: OBTAINING LACTOCOCCUS STRAINS
LACTIS SUBSP. LACTIS BIOVAR. DIACETYLACTIS PRODUCING α-ACETOLACTATE.

1. MUTAGENESIS
A strain of Lactococcus lactis subsp. lactis biovar. diacetylactis is cultured at 300C in 5 ml of MRS broth (DE XAN JC et al J. Appl Bacteriol., 1960, 23, 130-135). The culture is stopped at the end of the exponential phase, then the cells are recovered by centrifugation, washed with 100 mM phosphate buffer (pH 7), and recovered with 0.5 ml of this same buffer.

A volume of 0.5 ml of a solution of N-methyl-N'-nitro
N-nitrosoguanidine (NTG) is added to the cell suspension; the mixture is then incubated for 1 h at 300 ° C. (the concentration of the NTG solution is adjusted so as to obtain 10% survival with the mutagenic treatment).

The cells are then recovered by centrifugation, washed with 5 ml of buffer and taken up in 5 ml of MURS broth. The cell suspension is then incubated for 1 h at 300 ° C. and then diluted in a solution of sodium chloride at 0.9%. This suspension is intended to seed the Petri dishes for screening.

2. INOCULATION OF PETRI BOXES
200 petri dishes are used. Their preparation is described below.

The following components are mixed in 950 ml of distilled water: 5 g of yeast extract, 10 g of polypeptone, 2 g of K2HPO4, 200 mg of MgSO4, 7H2O, 50 mg of MnSO4.4H2O, 15 g of trisodium citrate , 2H20 and 15 g of agar. The pH is adjusted to 6.5 and the agar is autoclaved for 15 minutes at 12 ° C. Before pouring the boxes of
To the medium, 500 ml of an autoclaved solution of glucose at 400 g / l are added for 950 ml of medium maintained at 500 ° C. Approximately 12 ml of agar per
Petri.

 The 200 Petri dishes are inoculated with the cell suspension from mutagenesis.

Inoculation is performed to obtain about 60 colonies per petri dish. The petri dishes are then incubated for 60 h at 300 ° C. in order to allow the colonies to develop.

3. SCREENING
Velvet replicas of petri dishes are made. These replicas allow the recovery of the colonies detected by the screening. The petri dishes are then covered with 6 ml of an overlay containing agar and hydroxylamine, maintained undercooled at 600 ° C (210 g / l NH 2, HCl / 50 g / l agar; , vol / vol, both solutions are mixed extemporaneously). After caking the agar overlay, the Petri dishes are incubated for 30 min at 750C to allow the formation of dimethyglyoxime from diacetyl and hydroxylamine.

 The Petri dishes are then removed from the oven and 8 ml of a solution of acetone phosphate (200 ml of acetone and 150 g of K 2 HPO 4 per liter of solution) are poured into each still-hot petri dish.

This operation is intended to fix the excess hydroxylamine and to allow a better subsequent development of the coloring.

After about 15 minutes, the acetone-phosphate is removed and 9 ml of an extemporaneous mixture of two solutions are added to each petri dish: (500 g / l sodium potassium tartrate / NH 4 OH, 3/1, vol / vol) / (50 g / l
FeSO4, H2O in 1% H2SO4; 8/1, vol / vol). The addition of this solution has the effect of forming a red colored complex from dimethylglyoxime. The red color peaks about 10 minutes after the addition of the reagent and persists for about 1 hour. To facilitate the screening, the reagents used are distributed in the boxes of
Petri with 1 using a vending machine. In addition, after incubation at 750C, all operations are performed under a chemical hood. Petri dishes are examined on a light table. Colonies surrounded by a red halo are spotted. These colonies are recovered from the replicas of the boxes of
Petriages performed before screening.

 Tables I and II and FIGS. 1 and 2 illustrate mutagenesis and characterization experiments on mutants obtained from three strains of Lactococcus lactis subsp. lactis biovar.

diacetylactis.

Table I illustrates the results of mutagenesis obtained with three strains of Lactococcus lactis lactis
Table II illustrates the measurements of the α-acetolactate decarboxylase activity of mutants and parental strains, as well as the production of α-acetolactate in milk.

 FIGS. 1 and 2 show the production kinetics of α-acetolactate and (diacetyl plus acetoin) in milk of a strain of Lactococcus lactis subsp. lactis biovar. diacetylactis (Figure 1), as well as a mutant obtained from this strain (Figure 2).

 Table I: Screening of aacetolactate production by colonies resulting from the mutagenesis of 3 strains of Lactococcus lactis subsp. lactis biovar.

diacetylactis.

<Tb>

Lactococcus <SEP> Concentration <SEP> Number <SEP> Number <SEP> of
<tb> lactis <SEP> subsp. <SEP> in <SEP> NTG <SEP> of <SEP> colonies <SEP> colonies
<tb> lactis <SEP> biovar <SEP> tested <SEP> positive
<tb> diacetylactis <SEP> (CLg / ml) <SEP>
<tb><SEP> CD <SEP> 300 <SEP> 5000 <SEP> 1
<tb><SEP> F2 <SEP> 200 <SEP> 14000 <SEP> 3
<tb><SEP> F5 <SEP> 20 <SEP> 12000 <SEP> 2
<Tb>
Strains of Lactococcus lactis subsp. lactis biovar. Diacetylactis used in the mutagenesis described in Table I (CD, F2 and F5) come from
André Gaillard Yoplait International Research Center (Ivry-sur-Seine, France).

 Strains CD, F2 and F5 are able to use lactose and citrate; only the CD and F2 strains moreover exhibit proteolysis characteristics.

Mutagenesis is performed according to point 1. above. NTG concentrations are adjusted to obtain 10% mutagenesis survival; this
Table I shows that strain F5 is much more sensitive to NTG than CD and F2 strains.

Table II: Characteristics of parental strains (CD, F2 and F5) and strains selected after mutagenesis (CDM1, F2M1, F2x2, F2X3, F5M1, F5M2).

<tb> Lactococcus <SEP> Activity <SEP> Production
<tb> lactis <SEP> subsp. <SEP> a-acetolactate <SEP> maximum
<tb> lactis <SEP> biovar. <SEP> decarboxylase <SEP> of a-acetolactate
<tb> diacetylactis <SEP> (U / mg) <SEP> (mM)
<tb><SEP> CD <SEP> 0.561 <SEP><0.10
<tb><SEP> CDM1 <SEP><0.002<SEP> 2.38
<tb><SEP> F2 <SEP> 0.628 <SEP><0.10
<tb><SEP> F2M1 <SEP><0.002<SEP> 2.28
<tb><SEP> F2x2 <SEP><0.002<SEP> 2.20
<tb><SEP> F2X3 <SEP><0.002<SEP> 2.30
<tb><SEP> F5 <SEP> 1,610 <SEP><0,10
<tb><SEP> F5M1 <SEP><0.002<SEP> 2.46
<tb><SEP> F5M2 <SEP><0.002<SEP> 2.12
<Tb>

 The frequency of appearance of positive colonies after mutagenesis as described above is of the order of 1 in 5000. The selected colonies were characterized at the enzymatic level, by measuring the α-acetolactate decarboxylase activity, from acellular extracts, with a concentration of 50 mM Da-acetolactate and in the presence of 40 mM leucine and for their ability to produce α-acetolactate; the 6 clones revealed by the screening no longer have α-acetolactate decarboxylase activity (Table II).

When inoculated into milk (at 107 cfu / ml, culture at 230C), they produce <x-acetolactate at an average concentration of 2.3 mM, whereas parental strains produce less than 0 , 1 mM (Table II).

The kinetics of a culture of the parental strain
Lactococcus lactis subsp. lactis biovar. diacetylactis F2 in milk (Figure 1) shows the following characteristics
The strain acidifies the milk to a pH below 4.5 due to the formation of lactic acid from lactose.

 - The citric acid of the milk is consumed by the strain F2.

 - No α-acetolactate production is detected.

 - Production is observed of the compounds from the decarboxylation of α-acetolactate (diacetyl plus acetoin, not dissociable in the assay used), until the depletion of citric acid. A more detailed study would show that the concentration of acetoin is much higher than that of diacetyl, due to the presence of α-acetolactate decarboxylase, which converts α-acetolactate to acetoin only.

The kinetics of a culture of the mutant F2X2 (Figure 2), isolated from Lactococcus lactis subsp. lactis biovar. diacetylactis F2 and selected by implementing the process of the invention, exhibits the following characteristics
- The strain F2M2 acidifies the milk and consumes the citric acid in a way similar to the parental strain.

 - There is a large production of aacetolactate until the exhaustion of citric acid. The production of this metabolic intermediate is due to the absence of the α-acetolactate decarboxylase activity in strain F2X2.

 Α-Acetolactate is then slowly degraded by chemical decarboxylation, not involving α-acetolactate decarboxylase, to acetoin and / or diacetyl, so that the sum diacetyl plus acetoin plus α-acetolactate remains almost constant. In the presence of oxygen, the formation of diacetyl from α-acetolactate is favored. In the opposite case, the α-acetolactate is converted mainly to acetone.

 As is apparent from the above, the invention is not limited to those of its modes of implementation, implementation and application which have just been described more explicitly; it encompasses all the variants that may come to the mind of the technician in the field, without departing from the scope or scope of the present invention.

Claims (12)

 1 - A method for detecting the production of diacetyl by a bacterium, characterized in that said bacteria are placed in the presence of hydroxylamine, and in that the dimethylglyoxime possibly formed is detected.  2 - Process according to claim 1, characterized in that it is used for the selection of bacteria producing diacetyl.  3 - Process according to claim 2, characterized in that it is carried out on a bacterial culture in agar medium, for screening bacterial colonies producing diacetyl.  4 - Process according to any one of claims 1 to 3, characterized in that one proceeds to the detection of diacetyl production after mutagenic treatment of said bacterium.  5 - Process according to claim 4, characterized in that said mutagenic treatment is a random mutagenesis treatment.  6 - Process according to any one of claims 1 to 5, characterized in that said bacterium is a lactic acid bacterium.  7 - Process according to claim 6, characterized in that said lactic acid bacterium is selected from the group consisting of Lactococcus, Leuconostoc or Lactobacillus.  8 - Process according to any one of claims 6 or 7, characterized in that said bacterium is of the type Lactococcus lactis subsp. lactis biovar. diacetylactis.  9 - Lactococcus lactis subsp. lactis biovar. National Microorganism Culture (CNCM) held by INSTITUT PASTEUR. diacetylactis obtained according to the process according to any one of claims 1 to 8, deposited on December 20, 1994 under the number I - 1510 from the Collection  10 - Use of a process for detecting diacetyl production according to any one of claims 1 to 8, for the selection of lactic acid bacteria deficient in α-acetolactate decarboxylase.  11 - Use of a process for detecting the production of diacetyl according to any one of claims 1 to 8, for the production of a flavoring ferment.  12 - Use of a process for detecting diacetyl production according to any one of claims 1 to 8 for the selection of bacteria suitable for use in the food industry.