Synthesis of gamma-aminobutyric acid by lactic acid bacteria isolated from a variety of Italian cheeses

Abstract

The concentrations of gamma-aminobutyric acid (GABA) in 22 Italian cheese varieties that differ in several technological traits markedly varied from 0.26 to 391 mg kg(-1). Presumptive lactic acid bacteria were isolated from each cheese variety (total of 440 isolates) and screened for the capacity to synthesize GABA. Only 61 isolates showed this activity and were identified by partial sequencing of the 16S rRNA gene. Twelve species were found. Lactobacillus paracasei PF6, Lactobacillus delbrueckii subsp. bulgaricus PR1, Lactococcus lactis PU1, Lactobacillus plantarum C48, and Lactobacillus brevis PM17 were the best GABA-producing strains during fermentation of reconstituted skimmed milk. Except for L. plantarum C48, all these strains were isolated from cheeses with the highest concentrations of GABA. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48 by using primers based on two highly conserved regions of GAD. A PCR product of ca. 540 bp was found for all the strains. The amino acid sequences deduced from nucleotide sequence analysis showed 98, 99, 90, and 85% identity to GadB of L. plantarum WCFS1 for L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48, respectively. Except for L. lactis PU1, the three lactobacillus strains survived and synthesized GABA under simulated gastrointestinal conditions. The findings of this study provide a potential basis for exploiting selected cheese-related lactobacilli to develop health-promoting dairy products enriched in GABA.

FIG. 1.

Score plot of the first and second principal components after principal component analysis based on the concentrations of GABA, glutamic acid, and total FAAs of RSM started with lactic acid bacteria. Each fermented milk is indicated by the code of the lactic acid bacterium used for fermentation. The values are the averages of three batches of each milk fermentation.

FIG. 2.

Alignment of the internal deduced amino acid sequences of GAD of Lactobacillus paracasei PF6, Lactobacillus delbrueckii subsp. bulgaricus PR1, Lactococcus lactis PU1, and Lactobacillus plantarum C48 with other similar GAD sequences from Lactobacillus plantarum WCFS1 (accession number NP_786643.1), Lactobacillus brevis ATCC 367 (accession number YP_795941.1), Lactococcus lactis subsp. lactis Il1403 (accession number NP_267446.1), Listeria monocytogenes strain F6854 (accession number ZP_00234896.1), Clostridium perfringens strain 13 (accession number NP_562974.1), Enterococcus faecium DO (accession number ZP_00603789.1), Lactobacillus reuteri 100-23 (accession number ZP_01274543.1), and Escherichia coli CFT073 (accession number NP_753818.1). The deduced amino acid sequence was analyzed using ClustalW (1.81).

FIG. 3.

Survival of selected Lactobacillus paracasei PF6 (A), Lactobacillus delbrueckii subsp. bulgaricus PR1 (B), and Lactobacillus plantarum C48 (C) under gastric conditions (0 to 180 min) at pH 8.0 (▾), 3.0 (▪), and 2.0 (□) and at 2.0 with RSM added (11%, wt/vol) (▿) and with further intestinal digestion (180 to 360 min) at pH 8.0. The values were the averages of three replicates, and standard deviations are indicated by vertical bars.

FIG. 4.

Synthesis of GABA of Lactobacillus paracasei PF6 (⧫), Lactobacillus delbrueckii subsp. bulgaricus PR1 (▪), and Lactobacillus plantarum C48 (▴) in RSM after incubation under GI conditions and further incubation at 37°C for 24 h. The values were the averages of three replicates, and standard deviations are indicated by vertical bars.