P. Fernández de Palencia

Isolation and characterization of an intracellular esterase from Lactobacillus casei subsp. casei IFPL731

An intracellular esterase from Lactobacillus casei subsp. casei IFPL731 was purified 1000‐fold by ion exchange chromatography and gel filtration chromatography. The relative molecular mass of the native enzyme was 105 kDa, while the subunit molecular mass was estimated to be 38 kDa. The esterase hydrolysed tributyrin and had a preference for esters of short‐chain fatty acids (butyrate, caproate and caprylate), while it did not hydrolyse palmitate and sterate esters. The apparent Michaelis‐Menten constant of the enzyme on p‐nitrophenyl butyrate was 0·3 mmol l−1 while on p‐nitrophenyl caprylate, it was 0·04 mmol l−1. The esterase was active over a broad range of pH and temperature values, and retained about 50% of maximal activity at pH 5·0 and 12 °C. Activity was strongly inhibited by 5 mmol l−1 phenylmethylsulphonyl fluoride, β‐mercaptoethanol and N‐ethylmaleimide, and was stimulated by Zn2+ at 1 mmol l−1.

Evaluation of yogurt and various beverages as carriers of lactic acid bacteria producing 2-branched (1,3)-β-d-glucan

Probiotic cultures are increasingly being incorporated into a wide variety of food products. Although lactobacilli and bifidobacteria are the most frequently used, other lactic acid bacteria (LAB) have been reported to be potential probiotics. Of these, the cider isolates Pediococccus parvulus (strains 2.6 and CUPV22) and Lactobacillus suebicus CUPV221 produce a 2-branched (1,3)-β-d-glucan exopolysaccharide that decreases serum cholesterol levels and affects the activation of human macrophages. For this reason, these 3 strains were incorporated into yogurt, orange juice, and 2 juice-milk beverages to evaluate the effect of the food matrix on the resistance of these strains to simulated gastrointestinal tract conditions. Our results showed that incorporation of the LAB did not significantly affect the physical and rheological properties of the food matrices tested. When incorporated in yogurt, LAB strains population decreased by 2 to 3 log orders of magnitude during the shelf life of the product (28 d). However, no significant decrease was observed in the juice and juice-milk beverages during the same storage period, except for Lb. suebicus, whose viability decreased by 3 log orders of magnitude. When strains were subjected to gastrointestinal tract conditions, a decrease in the survival was observed at the lower pH (1.8). However, incorporation of these LAB strains into orange juice increases their resistance to lower pH conditions, thus improving survival to gastrointestinal stress. Moreover, a protective effect was observed for P. parvulus CUPV22 and 2.6 to gastric stress in juice-milk beverages and to gastrointestinal stress in yogurt. Lactobacillus suebicus CUPV221 did not survive when incorporated into yogurt and juice-milk beverage.

Discrepancies between the phenotypic and genotypic characterization of Lactococcus lactis cheese isolates

Aims:  The use of randomly amplified polymorphic DNA (RAPD)‐PCR fingerprinting and plasmid profiles to determine at the strain level, the similarity of Lactococcus lactis isolates obtained during sampling of traditional cheeses and to verify its correspondence to the selected phenotypic characteristics.

Specificity of the bound and free forms of the cell‐envelope proteinase of Lactobacillus casei subsp. casei IFPL 731 towards the αs1‐casein‐(1–23)‐fragment

P. FERNÁNDEZ DE PALENCIA, c. PELÁEZ AND M.C. MARTÍN‐HERNÁNDEZ. 1997. The specificity of the bound and free forms of Lactobacillus casei subsp. casei IFPL 731 proteinase towards the αs1‐casein‐(1–23)‐fragment has been studied. The use of the chelant agent EDTA for the extraction of the proteinase affects its specificity compared to either the use of lysozyme and mutanolysin or the whole‐cell proteinase form. This gives a different pattern of the αs1‐casein‐fragment hydrolysis, as observed by HPLC. The effect of different chemical agents on the specific activity of the proteinase also varies depending on the method used to release the proteinase.

Isolation and characterization of proteinase- and aminopeptidase-deficient mutants of Lactobacillus casei subsp. casei IFPL 731.

Proteinase‐deficient (Prt−) and aminopeptidase‐deficient (Amp−) variants of Lactobacillus casei subsp. casei IFPL 731 were isolated and characterized. The Prt− mutant was isolated from strains that developed poorly on glucose milk agar. The Amp− mutant was isolated on the basis of its inability to hydrolyse L‐leucine‐β‐naphtylamide. The Prt− variant developed poorly, while in milk the Amp− variant grew at about the same rate as the parental strain. The characterization of aminopeptidase activity in more detail showed that at least two enzymes are involved The results of the present study suggest that the proteolytic system of Lactobacillus casei is subjected to a regulatory system.