Long-term fungal inhibitory activity of water-soluble extracts of Phaseolus vulgaris cv. Pinto and sourdough lactic acid bacteria during bread storage

Abstract

The antifungal activity of proteinaceous compounds from different food matrices was investigated. In initial experiments, water-soluble extracts of wheat sourdoughs, cheeses, and vegetables were screened by agar diffusion assays with Penicillium roqueforti DPPMAF1 as the indicator fungus. Water-soluble extracts of sourdough fermented with Lactobacillus brevis AM7 and Phaseolus vulgaris cv. Pinto were selected for further study. The crude water-soluble extracts of L. brevis AM7 sourdough and P. vulgaris cv. Pinto had a MIC of 40 mg of peptide/ml and 30.9 mg of protein/ml, respectively. MICs were markedly lower when chemically synthesized peptides or partially purified protein fractions were used. The water-soluble extract of P. vulgaris cv. Pinto showed inhibition toward a large number of fungal species isolated from bakeries. Phaseolin alpha-type precursor, phaseolin, and erythroagglutinating phytohemagglutinin precursor were identified in the water-soluble extract of P. vulgaris cv. Pinto by nano liquid chromatography-electrospray ionization-tandem mass spectrometry. When the antifungal activity was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all three proteins were inhibitory. A mixture of eight peptides was identified from the water-soluble extract of sourdough L. brevis AM7, and five of these exhibited inhibitory activity. Bread was made at the pilot plant scale by sourdough fermentation with L. brevis AM7 and addition of the water-soluble extract (27%, vol/wt; 5 mg of protein/ml) of P. vulgaris cv. Pinto. Slices of bread packed in polyethylene bags did not show contamination by fungi until at least 21 days of storage at room temperature, a level of protection comparable to that afforded by 0.3% (wt/wt) calcium propionate.

FIG. 1.

Dry biomass (milligrams) of P. roqueforti DPPMAF1 incubated in WFH for 48 h at 25°C under different conditions. Treatments were as follows: 1, WFH alone (control); 2, WFH with 0.3% (wt/vol) calcium propionate added; 3, WFH with 30% (vol/vol, 1.71 mg of peptide/ml) water-soluble extract of sourdough L. brevis AM7 added; 4, WFH with 27% (vol/vol, 5 mg of protein/ml) water-soluble extract of P. vulgaris cv. Pinto added; 5, WFH with a mixture of 30% (vol/vol, 1.71 mg of peptide/ml) water-soluble extract of sourdough L. brevis AM7 and 27% (vol/vol, 5 mg of protein/ml) water-soluble extract of P. vulgaris cv. Pinto added. Data are the mean of three independent experiments, and the values marked with different letters are statistically significantly different (P ≤ 0.001).

FIG. 2.

SDS-PAGE of the water-soluble extract of P. vulgaris cv. Pinto. Lanes: 1, protein standard (Bio-Rad, Hercules, CA); 2, crude water-soluble extract; 3, polypeptides after FPLC purification. Identified polypeptides are indicated. Mr, molecular mass.

FIG. 3.

Slices of different breads stored at room temperature for 21 days without fungal spore inoculum. Bread 1, control; bread 2, with 0.3% (wt/wt) calcium propionate added; bread 3, fermented with sourdough L. brevis AM7; bread 4, with 27% (vol/wt, 5 mg of protein/ml) P. vulgaris cv. Pinto water-soluble extract added; bread 5, fermented with sourdough L. brevis AM7 with 27% (vol/wt, 5 mg of protein/ml) P. vulgaris cv. Pinto water-soluble extract added. The formulas used for bread making are shown in Table 1.