Heat and osmotic stress responses of probiotic Lactobacillus rhamnosus HN001 (DR20) in relation to viability after drying

Abstract

The viability of lactic acid bacteria in frozen, freeze-dried, and air-dried forms is of significant commercial interest to both the dairy and food industries. In this study we observed that when prestressed with either heat (50 degrees C) or salt (0.6 M NaCl), Lactobacillus rhamnosus HN001 (also known as DR20) showed significant (P < 0.05) improvement in viability compared with the nonstressed control culture after storage at 30 degrees C in the dried form. To investigate the mechanisms underlying this stress-related viability improvement in L. rhamnosus HN001, we analyzed protein synthesis in cultures subjected to different growth stages and stress conditions, using two-dimensional gel electrophoresis and N-terminal sequencing. Several proteins were up- or down-regulated after either heat or osmotic shock treatments. Eleven proteins were positively identified, including the classical heat shock proteins GroEL and DnaK and the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, enolase, phosphoglycerate kinase, and triose phosphate isomerase, as well as tagatose 1,6-diphosphate aldolase of the tagatose pathway. The phosphocarrier protein HPr (histidine-containing proteins) was up-regulated in cultures after the log phase irrespective of the stress treatments used. The relative synthesis of an ABC transport-related protein was also up-regulated after shock treatments. Carbohydrate analysis of cytoplasmic contents showed higher levels (20 +/- 3 microg/mg of protein) in cell extracts (CFEs) derived from osmotically stressed cells than in the unstressed control (15 +/- 3 microg/mg of protein). Liquid chromatography of these crude carbohydrate extracts showed significantly different profiles. Electrospray mass spectrometry analysis of CFEs revealed, in addition to normal mono-, di-, tri-, and tetrasaccharides, the presence of saccharides modified with glycerol.

FIG. 1.

Effect of heat shock on the growth of L. rhamnosus HN001 cultures. The cultures were grown in MRS broth at 37°C to an OD₆₁₀ of 0.4 to 0.5 and subjected to heat shock at 37°C (•), 45°C (○), 50°C (▾), or 55°C (▿) for 30 min. Further growth was monitored at 37°C.

FIG. 2.

Effect of osmotic shock on the growth of L. rhamnosus HN001 cultures. The cultures were grown in MRS broth at 37°C to an OD₆₁₀ of 0.7 to 0.8 before being harvested and resuspended in MRS broth containing no NaCl (•) or 0.3 M (○), 0.4 M(▾), 0.5 M (▿), 0.6 M (▪), or 0.7 M (□) NaCl and then further incubated at 37°C.

FIG. 3.

Storage stability of L. rhamnosus HN001 in dried form at 30°C. Cultures were grown in MRS broth, stressed by using either heat or salt, and then harvested and dried as described in Materials and Methods. The powders were stored (30°C) under ambient atmospheric conditions away from sunlight in plastic containers. •, nonstressed; ▿, heat shocked at log phase; ▪, heat shocked at stationary phase; ⋄, osmotically shocked.

FIG. 4.

Protein synthesis in an unstressed (control) mid-log-phase L. rhamnosus HN001 culture. An autoradiogram of a 2D PAGE gel of [³⁵S]methionine- and [³⁵S]cysteine-pulse-labeled proteins derived from strain HN001 grown to mid-log phase at 37°C and labeled for 30 min is shown. The proteins were assigned unique spot numbers. GAP, glyceraldehyde-3-phosphate dehydrogenase; LDH, lactate dehydrogenase; TBPA, tagatose 1,6-diphosphate aldolase; PGK, phosphoglycerate kinase; TPI, triose phosphate isomerase.

FIG. 5.

Protein synthesis in a heat-shocked mid-log-phase L. rhamnosus HN001 culture. An Autoradiogram of a 2D PAGE gel of [³⁵S]methionine- and [³⁵S]cysteine-pulse-labeled proteins derived from strain HN001 grown to mid-log phase at 37°C, shock treated at 50°C, and labeled for 30 min is shown. The proteins on the gel were identified with spot numbers. For abbreviations, see the legend to Fig. 4.

FIG. 6.

Protein synthesis in an osmotically shocked mid-log-phase L. rhamnosus HN001 culture. An autoradiogram of a 2D-PAGE gel of [³⁵S]methionine- and [³⁵S]cysteine-pulse-labeled proteins of strain HN001 grown to mid-log phase, harvested, resuspended in preheated (37°C) MRS broth (with 0.6 M NaCl), and labeled for 30 min is shown. The proteins on the gel were given unique spot numbers. For abbreviations, see the legend to Fig. 4.

FIG. 7.

Differential regulation of L. rhamnosus HN001 protein synthesis during different stages of growth and stress treatments. After 2D gel electrophoresis and blotting followed by autoradiography, the autoradiograms were scanned by using a Fluor-S Multimager System (Bio-Rad). The gels were compared by using the Z3 2D-PAGE Analysis System (Compugen Inc.). The relative absorbance units (relative protein synthesis) corresponding to protein spots in different gels were used to determine the changes in protein synthesis. (A) Heat shock proteins; (B) glycolysis-related enzymes; (C) other proteins. For each enzyme the bars are as follow, from left to right: nonstressed log-phase cultures, cultures heat shocked after log-phase growth, cultures osmotically stressed after log-phase growth, nonstressed stationary-phase cultures, and cultures heat shocked after stationary phase growth. For abbreviations of the enzymes, see Fig. 4 (GAPDH, glyceraldehyde-3-phosphate dehydrogenase).

FIG.8.

(a) Carbohydrate profiles of CFEs derived from control (dashed line) and osmotically stressed (solid line) L. rhamnosus HN001 by using a Dionex PA1 column. The HPLC system used was a Dionex DX500 system with electrochemical detection. The gradient was 18 mM NaOH for 40 min, followed by a linear rise to 200 mM NaOH over 15 min with the elution held at 200 mM for a further 5 min. Peaks 1 and 2, monosaccharides; peaks 3, 4, and 5, disaccharides; peaks 6 through 12, tri- and tetrasaccharides. (b) Electrospray mass spectrometry of methylated control and osmotically stressed L. rhamnosus HN001 CFEs. Normal di-, tri-, and tetrasaccharides have mass/charge ratios (m/z M + Na) of 477, 681, and 885, respectively, when methylated. When the oligosaccharides are modified with glycerol, the methylated m/z values shift to 565, 769, and 973, respectively.