Acetaldehyde Production by Strains Used As Probiotics in Fermented Milk

JourTUll of Food Protection, Vol. 57, No.5, Pages 436-440 Copyright©, International Association of Milk, Food and Environmental Sanitarians Acetaldehyde Production by Strains Used as Probiotics in Fermented Milk SILVIA GONZALEZ, VILMA MORATA DE AMBROSINI, MARiA MANCA DE NADRA, AiDA PESCE DE RUIZ HOLGADO and G. OLIVER* Universidad Nacional de Tucumdn, Centro de Referencia para Lactobaci/os (CERELA), Chacabuco 145, 4000 Tucumdn, Argentina Downloaded from http://meridian.allenpress.com/jfp/article-pdf/57/5/436/1664941/0362-028x-57_5_436.pdf by guest on 02 October 2021 (Received July 23, 1993/Accepted January 10, 1994) ABSTRACT flavor varies widely among dairy products. In cheese, cultured butter and buttermilk only relatively small amounts Lactic acid bacteria have diverse shunts for the metabolism are required for the development of a balanced flavor, in of acetaldehyde, which is involved in the metabolism of carbohydrates, proteins and nucleic acids. In Lactobacillus acidophilus fermented milk intermediate amounts are needed, and in and Lactobacillus casei, strains isolated from feces of healthy yogurt relatively large amounts are required to give the children, acetaldehyde can be formed from different sources. characteristic flavor (24). Phosphotransacetilase, acetate kinase, aldehyde dehydrogenase In our laboratory two strains of lactobacilli (L. casei and 2-deoxiriboaldolase activities were found in both strains. and L. acidophilus) were isolated from feces of healthy u-Carboxilase and threonine aldolase activities only occurred in children. The strains were able to survive in gastric juice L. acidophilus. In contrast, phosphoketolase activity was present and bile and, moreover, they showed an inhibitory effect on L. casei, in but absent in the other strain studied. The accumula- the growth of enteropathogens (1,7) producing a fermented tion of acetaldehyde in the growth medium is possible because the milk with outstanding properties for the treatment of infan- enzymes specific activities to form it are higher than those able to convert it to ethanol. tile diarrhea. The incidence of diarrhea in children receiv- ing fermented milk was smaller than in the control: 17% Key Words: Lactobacillus acidophilus, Lactobacillus casei, acet- versus 59%, respectively (6). aldehyde, probiotics, fermented milk In this paper we examine the production of characteristic flavor compound (acetaldehyde) by lactobacilli strains Children consume dairy products (yogurt, fermented used in fermented milk. The enzymes involved in the milk) for its flavor more than anything else. Large-scale acetaldehyde metabolism from crude cell-free extract and production has often led to rather bland products and one of dialyzed cell-free extract were determined. the major preoccupations of the industry is to find some way to improve the flavor characteristics of their products MATERIALS AND METHODS within this production system. Flavor is the most important Microorganisms quality of foods and beverages for determining consumer Lactobacillus casei and L. acidophilus were isolated from acceptability. Flavor production can be described as a feces of healthy children and characterized in our laboratory by a series of reactions related to lactic acid metabolism, lipoly- biochemical test, according to the method described by Kandler sis, oxidation and proteolysis, which can be followed by and Weiss (11). reactions between the products formed in each pathway. In these reactions, microorganisms play a significant role Cultured medium and growth conditions through their enzymatic system. The basal medium (Laptg) for the growth of lactobacilli was Acetaldehyde, a product of the metabolism of microor- described by Raibaud et al. (18) with the following composition: I% yeast extract, 1.5% peptone, 1% tryptone, I% glucose and ganisms used in the manufacture of cultured dairy products, O.l% Tween 80. The pH was adjusted to 6.8 with 0.2 N sodium has attracted considerable interest because of its association hydroxide (NaOH) and then the medium was sterilized by auto- with the development of desirable flavor. claving for 20 min at 118°C. The cells were grown in the Lactic acid bacteria form varying amounts of acetalde- appropriate medium in precultures of 10 ml, 100 ml and a final hyde and ethanol during growth and they contain enzymes culture of 1,000 ml. After being cultured for 12 h at 37°C, the which catalyze the formation of acetaldehyde from carbo- cells were harvested by centrifugation at 8,000 g for 15 min at hydrate, protein or nucleic acid sources. The accumulation 4°C in a Measuring and Scientific Equipment LTD (MSE) refrig- of acetaldehyde in the growth medium depends on whether erated centrifuge. the organism has enzymes which convert this compound to Cell-free extract other metabolites, principally ethanol. The amount of acet- The cells harvested by centrifugation were washed in 0.2 M aldehyde required for the development of a characteristic Tris-HCI buffer pH 7. They were suspended at 30% wt/vol in the JOURNAL OF FOOD PROTECTION, VOL. 57, MAY 1994 same buffer and ruptured using a French press. Cell debris were Alcohol dehydrogenase. Activity was measured by the rate of removed by centrifugation at 20,000 g for 20 min at 4°C. One- oxidation or reduction of pyridine nucleotides at 340 nm in the half of the supernatant solution was stored at 4°C for assays in the presence of acetaldehyde or ethanol. The reaction mixture (final crude form; the remaining solution was dialized against 0.02 M volume 2.5 ml) for the reduction of acetaldehyde contained: 200 ; Tris-hydrochloric acid (Tris-HCI) buffer pH 7.0 for 12 h at 4°C. Ilmoles Na phosphate buffer, pH 6.5; O.4llmoles NAD(P)H2 and Protein was determined by the method of Lowry et al. (16) using 35 Ilmoles acetaldehyde. The reaction mixture (total volume serum albumin as a standard. 3.5 ml) for the reverse reaction contained 200 Ilmoles NazHP04- NaOH buffer, pH 9.3; O.4llmoles NAD(P); and 6 Ilmoles ethanol. Assays of enzymatic activities The addition of 0.2 ml of a suitable bacterial cell-free extract Enzymes associated with the metabolism of carbohydrates (crude or dialized) started the reaction. include: Phosphoketolase. Activity was measured by the method of Enzyme associated with the metabolism of amino acids Horecker (10). The reaction mixture, total volume 1.0 ml, con- Threonine aldolase activity was assayed by the rate of forma- tained: 0.55 ml, 0.1 M sodium phosphate buffer, pH 6.5; 10 tion of acetaldehyde semicarbazone in Conway microdiffusion Ilmoles magnesium chloride; 30 Ilmoles glutathione; 0.5 Ilmoles units. The reaction mixture (total volume 3.0 ml) in the outer well thiamine pyrophosphate (cocarboxylase); 5 Ilmoles D-xylulose 5- contained: 140 Ilmoles sodium phosphate buffer, pH 6.5; 125 P; and 0.1 ml (crude or dialyzed) cell-free extract. The acetyl Ilmoles D-L-threonine; 100 nmoles pyridoxal-5-phosphate; and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/57/5/436/1664941/0362-028x-57_5_436.pdf by guest on 02 October 2021 phosphate formed was estimated by the method of Lipman and 0.5 ml (crude or dialyzed) cell-free extract. The inner well of the Tuttle (15). Conway unit contained 1.2 ml of 6.7 mM semicarbazide-HCI in Pyruvate decarboxylase (a-carboxylase). Activity was esti- 0.2 M sodium phosphate buffer pH 7.0. The incubation was mated by the rate of formation of acetaldehyde semicarbazone in stopped after 30 min at 40°C by the addition of 0.25 mlof 12% Conway micro-diffusion units. The reaction mixture in the outer trichloroacetic acid into the outer well. The acetaldehyde produc- chamber, total volume 2.0 ml, contained 75 Ilmoles sodium tion was determined spectrophotometrically at 224 nm according phosphate buffer, pH 6.5; 5 Ilmoles magnesium sulfate; 0.5 mg to Burbridge et al. (4). thiamine pyrophosphate; 20 Ilmoles sodium pyruvate and 0.5 ml of (crude or dialyzed) cell-free extract. Then 1.2 ml of 6.7 mM Enzyme associated with the metabolism of nucleic acids semicarbazide HCI in 0.2 M sodium phosphate buffer pH 7 was Deoxyriboaldolase was assayed within the breakdown of poured into the center chamber. Incubation was stopped after 30 2-deoxyribose-5-phosphate, forming acetaldehyde and glyceralde- min at 40°C by the addition of 0.25 ml of 12% trichloroacetic acid hyde-3-phosphate. The reaction mixture was added to the outer to the outer well. The acetaldehyde production was determined well of a Conway microdiffusion unit and the acetaldehyde spectrophotometrically at 224 nm, according to Burbridge et al. formed was absorbed into semicarbazide in the center well deter- (4). mined by the method of Burbridge et al. (4). This mixture (total Phosphotransacetylase. Activity was estimated by the method volume of 2 m!) contained: 3 Ilmoles 2-deoxy-D-ribose-5-phos- of Stadtman (23). The reaction mixture, total volume 1.0 ml, phate; 50 Ilmoles sodium phosphate buffer, pH 6.5; and 0.25 ml contained: 0.4 ml distilled water; 10 Ilmoles tris-HCI buffer, pH (crude or dialized) cell-free extract. The incubation was stopped 8.2; 61lmoles acetyl phosphate; 16 nanomoles (nmoles) coenzyme after 30 min at 40°C by the addition of 0.25 ml of 12% trichlo- A (CoA); 10 Ilmoles cysteine HCI; and 0.1 ml (crude or dialyzed) roacetic acid into the outer well. cell- free extract. All enzyme assays were performed in triplicate. Specific After incubation for 5 min at 30°C, 0.1 ml of 0.5 M activities were defined in the tables. potassium arsenate pH 8 was added, and the incubation was continued for IS min. The residual acetyl phosphate was esti- RESULTS mated by the method of Lipman and Tuttle (15). Acetate kinase. Activity was determined by measuring the The main reactions involving acetaldehyde studied in adenine nucleotide-dependent utilization of acetyl phosphate.

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