Acetolactate Decarboxylase-Deficient Spontaneous Mutants of Streptococcus Thermophilus Christophe Monnet, Georges Corrieu

Selection and properties of α-acetolactate decarboxylase-deficient spontaneous mutants of Streptococcus thermophilus Christophe Monnet, Georges Corrieu To cite this version: Christophe Monnet, Georges Corrieu. Selection and properties of α-acetolactate decarboxylase- deficient spontaneous mutants of Streptococcus thermophilus. Food Microbiology, Elsevier, 2007, 24 (6), pp.601-606. 10.1016/j.fm.2007.01.004. hal-01195414 HAL Id: hal-01195414 https://hal.archives-ouvertes.fr/hal-01195414 Submitted on 31 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. 1 Selection and properties of -acetolactate decarboxylase-deficient spontaneous mutants 2 of Streptococcus thermophilus 3 4 5 Christophe Monnet*, Georges Corrieu 6 7 Unité Mixte de Recherche Génie et Microbiologie des Procédés Alimentaires, Institut 8 National de la Recherche Agronomique, 78850 Thiverval-Grignon, France 9 10 11 12 13 Running title: alpha-acetolactate decarboxylase-deficient mutants 14 15 16 17 18 19 * Corresponding author. Mailing address: Unité Mixte de Recherche Génie et Microbiologie 20 des Procédés Alimentaires, Institut National de la Recherche Agronomique, 78850 Thiverval- 21 Grignon, France. Phone: +33 (0)1 30 81 54 91. Fax: +33 (0)1 30 81 55 97. E-mail: 22 [email protected] 23 24 Abstract 25 26 Many lactic acid bacteria produce diacetyl, which is a desirable aroma compound in 27 some fermented dairy products. Strains or mutants of Lactococcus lactis subsp. lactis biovar. 28 diacetylactis that are deficient in -acetolactate decarboxylase are used in some food 29 processes for their ability to produce large amounts of diacetyl. However, up until now, the 30 use of -acetolactate decarboxylase-deficient mutants of Streptococcus thermophilus for 31 increased diacetyl production has not been evaluated. The objective of the present study was 32 to devise a procedure for selecting spontaneous -acetolactate decarboxylase-deficient 33 mutants of Streptococcus thermophilus. We observed that in a chemically defined medium 34 containing -ketobutyrate plus leucine, or -ketobutyrate plus leucine plus isoleucine, the - 35 acetolactate decarboxylase-deficient mutant TIL865, obtained by directed mutagenesis, grew 36 faster than its parent strain. This property was used for selecting spontaneous -acetolactate 37 decarboxylase-deficient mutants on agar plates. The resulting mutants were able to grow in 38 milk, and their acidifying activity was slightly lower than that of the parent strain. Under 39 partial anaerobic or aerobic conditions, they produced approximately three times more 40 diacetyl than the parent strain. Such spontaneous mutants may be useful for increasing the 41 diacetyl content of fermented milks whose production involves Streptococcus thermophilus 42 strains. 43 44 2 45 1. Introduction 46 47 Diacetyl is a major flavor compound in many cultured dairy products. It is an end 48 product of citrate metabolism by certain lactic acid bacteria, such as Lactococcus lactis subsp. 49 lactis biovar. diacetylactis and Leuconostoc sp, which are used for the production of cultured 50 cream, cultured butter, buttermilk and fresh cheeses. Diacetyl is also a key aroma compound 51 in yogurt (Imhof et al., 1995; Ott et al., 1997), but in this case, it is mainly produced by 52 Streptococcus thermophilus, which is a lactic acid bacterium that is not able to use citrate. 53 54 Strains of Lactococcus lactis subsp. lactis biovar. diacetylactis that are deficient in - 55 acetolactate decarboxylase, the enzyme responsible for acetoin production, accumulate 56 significant amounts of –acetolactate in the culture medium. This results in higher diacetyl 57 production due to the spontaneous oxidative decarboxylation of -acetolactate (De Man, 58 1959). Such strains are widely used in butter-making processes (Veringa et al., 1976) and for 59 the production of aroma additives with a high -acetolactate (Kuiper et al., 1987) or diacetyl 60 (Jönsson et al., 1980) content. In Lactococcus lactis strains isolated from non-dairy 61 environments, -acetolactate decarboxylase is also involved in the regulation of leucine and 62 valine biosynthesis (Goupil-Feuillerat et al., 1997). Indeed, -acetolactate is a precursor of 63 these branched-chain amino acids, and when they are present in excess, the flux of - 64 acetolactate is diverted towards acetoin, due to an allosteric activation of the -acetolactate 65 decarboxylase (Phalip et al., 1994) and to a translational regulation of -acetolactate 66 decarboxylase synthesis (Goupil-Feuillerat et al., 2000). Goupil and co-workers (1996) took 67 advantage of the fact that this enzyme has a central role in the regulation of branched-chain 68 amino acid biosynthesis to select spontaneous -acetolactate decarboxylase-deficient mutants, 69 by using a defined medium containing leucine, but devoid of valine and isoleucine. 3 70 71 We recently showed that -acetolactate decarboxylase is also involved in the 72 regulation of leucine and valine biosynthesis by Streptococcus thermophilus (Monnet et al., 73 2003) (Fig. 1). The objective of the present study was to determine whether this property 74 could be used for devising a medium for the selection of spontaneous -acetolactate 75 decarboxylase-deficient mutants, and whether such mutants were able to produce large 76 amounts of diacetyl in milk. 77 78 2. Materials and Methods 79 80 2.1. Bacterial strains 81 82 Streptococcus thermophilus strains were routinely grown in M17 broth (Terzaghi and 83 Sandine, 1975). Strain CNRZ385 was obtained from UBLO (Unité Bactéries Lactiques et 84 Pathogènes Opportunistes, INRA, Jouy-en-Josas, France), and strain TIL865 is an - 85 acetolactate decarboxylase-deficient mutant selected from strain CNRZ385 by directed 86 mutagenesis (Monnet et al., 2003). 87 88 2.2. Growth in defined media 89 90 The effect of branched-chain amino acids and –ketobutyrate on the growth of 91 Streptococcus thermophilus was studied using a chemically defined medium (CDM), derived 92 from the medium described by Reiter and Oram (1962). It contained lactose (10 g/l), KH2PO4 93 (3 g/l), sodium acetate (1 g/l), ascorbic acid (0.5 g/l), adenine (5 mg/l), guanine (5 mg/l), 94 xanthine (5 mg/l), uracil (5 mg/l), pyridoxal hydrochloride (2 mg/l), niacin (1 mg/l), thiamine 4 95 hydrochloride (1 mg/l), riboflavin (1 mg/l), calcium pantothenate (1 mg/l), para-aminobenzoic 96 acid (10 µg/l), biotin (10 µg/l), folic acid (1 µg/l), vitamin B12 (1 µg/l), MgCl2.6H2O (200 97 mg/l), CaCl2 (50 mg/l), FeCl3.6H2O (5 mg/l), ZnSO4.7H2O (5 mg/l), CoCl2.6H2O (2.5 mg/l), 98 CuSO4.5H2O (2.5 mg/l), NiSO4.7H2O (10 mg/l), MnSO4.1H2O (10 mg/l), L-cysteine 99 hydrochloride (200 mg/l), L-alanine (150 mg/l), L-lysine hydrochloride (800 mg/l), L- 100 arginine (450 mg/l), L-proline (540 mg/l), L-methionine (70 mg/l), L-phenylalanine (100 101 mg/l), L-serine (200 mg/l), L-threonine (100 mg/l), L-tryptophane (100 mg/l), glycine (200 102 mg/l), L-hystidine hydrochloride.1H2O (550 mg/l), L-glutamate (3.2 g/l), L-glutamine (3.2 103 g/l), L-aspartate (300 mg/l) and L-tyrosine (100 mg/l). The medium was reconstituted using 104 concentrated stock solutions containing the nutrients. A mixture containing lactose, salts and 105 vitamins was prepared at a concentration double that in the CDM. After adjusting its pH to 106 6.8 using NaOH, the mixture was autoclaved for 15 min at 110°C. The amino acids were 107 prepared as a five times concentrated solution that was filter-sterilized (0.22 µm) after 108 adjustment of its pH to 6.8. Supplementation of CDM with L-leucine, L-isoleucine, L-valine 109 or sodium –ketobutyrate was done at a final concentration of 200, 100, 150 and 600 mg/l, 110 respectively. These compounds were prepared individually as 15 times concentrated solutions 111 that were filter-sterilized (0.22 µm) after adjusting their pH to 6.8. After two successive 112 cultures in CDM at 37°C, cells of Streptococcus thermophilus were recovered by 113 centrifugation for 10 min at 5,000 x g and 4°C, washed in a saline solution (9 g/l NaCl), and 114 resuspended in the same solution to reach an absorbance (575 nm) equivalent to 1.4. Cells 115 were then inoculated at 0.5% (v/v) in CDM. Cultures were performed at 37°C, under partial 116 anaerobic conditions (incubation without agitation, where the headspace volume represented 117 less than 2% of the culture volume). 118 5 119 Spontaneous mutants of Streptococcus thermophilus deficient in -acetolactate 120 decarboxylase were selected using an agar medium, which was prepared by supplementing 121 CDM with agar (15 g/l), L-leucine (200 mg/l) and sodium –ketobutyrate (600 mg/l). 122 123 2.3. Milk cultures 124 125 Cells were grown at 37°C in M17 broth and harvested at the end of exponential 126 growth phase. The density of the cell suspension in the broth was estimated by its absorbance 127 at 575 nm. Cells were washed with 0.9% NaCl and inoculated, at a concentration equivalent 128 to 0.02 absorbance unit, in reconstituted skim milk (100 g/l; Epi-Ingredients, Ancenis, France) 129 that had been heated for 10 min at 110°C. Static cultures (partial anaerobiosis) were grown in 130 250-ml conical flasks containing 200 ml of milk at 37°C. The pH electrodes, disinfected with 131 ethanol, were introduced through bores in the rubber stoppers.

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