crossmark

Hyperconcentrated Sweet Whey, a New Culture Medium That Enhances Propionibacterium freudenreichii Stress Tolerance Song Huang,a,b,c Houem Rabah,b,c Julien Jardin,b,c Valérie Briard-Bion,b,c Sandrine Parayre,b,c Marie-Bernadette Maillard,b,c Yves Le Loir,b,c Xiao Dong Chen,a,d Pierre Schuck,b,c Romain Jeantet,b,c Gwénaël Janb,c Suzhou Key Laboratory of Green Chemical Engineering, School of Chemical and Environmental Engineering, College of Chemistry, Chemical Engineering and Material Science, Soochow University, Jiangsu, Chinaa; INRA, UMR1253 STLO, Science et Technologie du Lait et de l’Œuf, Rennes, Franceb; Agrocampus Ouest, UMR1253 STLO, Rennes, Francec; Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, Chinad

ABSTRACT

Propionibacterium freudenreichii is used as a cheese-ripening starter and as a probiotic. Its reported physiological effects at the gut level, including modulation of bifidobacteria, colon epithelial cell proliferation and apoptosis, and intestinal inflammation, rely on active metabolism in situ. Survival and activity are thus key factors determining its efficacy, creating stress adaptation and tolerance bottlenecks for probiotic applications. Growth media and growth conditions determine tolerance acquisition. We investigated the possibility of using sweet whey, a dairy by-product, to sustain P. freudenreichii growth. It was used at different concentrations (dry matter) as a culture medium. Using hyperconcentrated sweet whey led to enhanced multistress tolerance acquisition, overexpression of key stress proteins, and accumulation of intracellular storage molecules and compatible solutes, as well as enhanced survival upon spray drying. A simplified process from growth to spray drying of propionibacteria was developed using sweet whey as a 2-in-1 medium to both culture P. freudenreichii and protect it from heat and osmotic injury without harvesting and washing steps. As spray drying is far cheaper and more energy efficient than freeze-drying, this work opens new perspectives for the sustainable development of new starter and probiotic preparations with enhanced robustness. IMPORTANCE

In this study, we demonstrate that sweet whey, a dairy industry by-product, not only allows the growth of probiotic dairy propionibacteria, but also triggers a multitolerance response through osmoadaptation and general stress response. We also show that propionibacteria accumulate compatible solutes under these culture conditions, which might account for the limited loss of viability after spray drying. This work opens new perspectives for more energy-efficient production of dairy starters and probiotics.

P

ropionibacterium freudenreichii, the main species of dairy propionibacteria, is consumed in large amounts both in traditional fermented foods and in probiotic supplements. Its promising probiotic properties include beneficial modulation of several gut parameters (1). Consumption of P. freudenreichii indeed leads to increased bifidobacterial intestinal populations in humans (2, 3). In an animal model of carcinogenesis, such consumption reduces proliferation while enhancing apoptotic depletion of colon cancer cells (4) in accordance with the proapoptotic effect of its major metabolites, the short-chain fatty acids (SCFAs) propionate and acetate (5–7). This cytotoxic effect, killing cancer but not healthy human colon epithelial cells (67), relies on survival and activity of P. freudenreichii in the ingested product and in the gut, key prerequisites for in situ release of SCFAs. Furthermore, selected strains of P. freudenreichii modulate gut inflammation through strain-specific surface layer proteins (8, 9), which induce the production of the immunomodulatory cytokine interleukin 10 (IL-10) by human immune cells and are expressed in fermented dairy products (10). This anti-inflammatory effect may be further enhanced by the ability of P. freudenreichii to release folic acid (11) and the menaquinone biosynthesis precursor 1,4-dihydroxy-2-naphthoic acid (DHNA) (12). Altogether, these promising studies indicate that the probiotic potential of P. freudenreichii relies on survival within the gut. Metabolic activity, leading to the release of SCFAs, has been demonstrated within the digestive tracts of rats (13) and humans (14), but it largely depends on the propionibacterial strain, its stress tolerance, and its physiological stage within the ingested product.

August 2016 Volume 82 Number 15

Stress adaptation is thus a key limiting factor of P. freudenreichii probiotic efficacy. The production of live and active starter and/or probiotic preparations, whether they are dried products or fermented food products, is a key issue mainly limited by the ability of the considered bacteria to adapt and survive stress. Dairy propionibacteria are subjected to various abiotic conditions during starter drying, whether it is freeze- or spray drying (15), and during Emmental cheese making (16) that constitute multistress processes, including variations in temperature, pH, water content, osmolarity, oxidation, and nutrient availability. Propionibacteria must cope with further stresses during transit through the digestive tract, including acidity and bile salts, which limit their probiotic efficacy.

Received 8 March 2016 Accepted 15 May 2016 Accepted manuscript posted online 27 May 2016 Citation Huang S, Rabah H, Jardin J, Briard-Bion V, Parayre S, Maillard M-B, Le Loir Y, Chen XD, Schuck P, Jeantet R, Jan G. 2016. Hyperconcentrated sweet whey, a new culture medium that enhances Propionibacterium freudenreichii stress tolerance. Appl Environ Microbiol 82:4641–4651. doi:10.1128/AEM.00748-16. Editor: D. W. Schaffner, Rutgers, The State University of New Jersey Address correspondence to Gwénaël Jan, [email protected], or Xiao Dong Chen, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.00748-16. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Applied and Environmental Microbiology

aem.asm.org

4641

Huang et al.

For several of these stresses, tolerance can be acquired by P. freudenreichii upon exposure to moderate sublethal doses of the stress, leading to tolerance for homologous lethal doses. This was demonstrated for acid stress (17), bile salt stress (18), and heat stress (19). Such adaptation relies on the enhanced expression of stress proteins involved in major processes of cellular injury remediation. Cross-protection was also shown between acid and heat (20), and technological stresses (heat, starvation, and osmolarity) may confer tolerance for digestive stresses (low pH and bile salts). Accordingly, growth media and growth conditions determine stress tolerance. Different formulations confer different tolerance levels in P. freudenreichii, among which fermented milk affords particularly high tolerance (21). When consumed as part of a fermented dairy product, P. freudenreichii exhibits higher survival and activity in vivo than the usual cultures, both in pigs (22, 23) and in humans (14). We thus investigated the molecular mechanisms responsible for tolerance induction upon growth within dairy products. As an example, growth within the Emmental cheese environment confers enhanced stress tolerance for acidic conditions, heat challenge, and the presence of bile salts compared to the usual laboratory culture medium (24). In this study, we investigated the possibility of using cheese whey to improve P. freudenreichii stress tolerance before drying. For industrial production, P. freudenreichii, like other probiotics, is currently grown using non-dairy complex culture media prior to harvesting, washing, and drying. P. freudenreichii, however, is known to grow in cheese, after the growth of lactic acid bacteria, using both lactose and lactate (produced by the lactic acid bacteria) as carbon sources. Since culture conditions are crucial for stress tolerance and survival under abiotic stresses encountered in technological processes and during digestion, we evaluated the possibility of growing P. freudenreichii on sweet whey (SW), a by-product of cheese manufacture close to the cheese aqueousphase composition. Furthermore, we investigated the impact of such culture on tolerance for key digestive and technological stresses. The dry matter of sweet whey was increased from 5 to 30%, leading to enhanced (i) biomass production after growth and (ii) viability after spray drying. MATERIALS AND METHODS Growth media and bacterial growth. The ITG P20 (also called CIRMBIA 129) strain of P. freudenreichii was kept and provided as a certified pure culture by the Centre International de Ressources MicrobiennesBactéries d’Intérêt Alimentaire (CIRM-BIA) (INRA, Rennes, France) International Biological Resource Center and routinely cultivated in yeast extract lactate (YEL) medium (25). In this study, the bacteria were cultivated in sweet whey medium containing (per liter) 50 g, 100 g, 200 g, or 300 g of spray-dried high-fluidity sweet whey powder (Lactalis Ingredients, Les Placis, Bourgbarre, France) supplemented with 5 g/liter of Casein Peptone Plus (Organotechnie, France), which was autoclaved (110°C; 30 min). P. freudenreichii was grown at 30°C without agitation under microaerophilic conditions as described previously (22) until stationary phase. This was achieved after 72 h of growth in 100-ml bottles (see Fig. S1 in the supplemental material). The medium was extremely turbid and rich in milk proteins, and bacterial growth was monitored by CFU counting on plates of YEL medium solidified with 10 g/liter agar (YEL agar) during growth in the autoclaved medium. Briefly, aliquots of cultures were subjected to serial 1/10 dilutions in isotonic peptone water (0.9% NaCl, 0.1% peptic digest of meat, adjusted to pH 7.0; Biokar Diagnostics, Beauvais, France), and dilutions were plated on YEL agar medium for CFU counting.

4642

aem.asm.org

Stress challenges. Acid, bile salts, and heat challenges were applied to stationary-phase cultures according to standardized procedures at pH 2 and 37°C for 60 min in YEL broth adjusted to pH 2.0 using HCl (17, 26) or in the presence of 1 g/liter of bile salts (an equimolar mixture of cholate and deoxycholate; Sigma Chemical, St. Louis, MO, USA) in YEL broth at 37°C for 60 min (18) or at 60°C for 10 min (20) prior to CFU counting on YEL agar to determine survival percentages. As a control, cultures were left untreated for the same time in order to determine the population corresponding to 100% survival. The viable and cultivable population of propionibacteria was determined by CFU counting on plates of YEL medium solidified with 10 g/liter agar for maximal recovery of both treated and untreated cultures. Percent survival was then determined by comparing stressed and unstressed cultures at the end of the same time period. Spray drying. Early-stationary-phase cultures (2 liters) of P. freudenreichii were enumerated on YEL agar plates as described above prior to spray drying on a pilot scale Mobile Minor spray dryer (GEA Niro, Denmark) with a maximal evaporation rate of 5 liters/h. A peristaltic pump (520S; Watson-Marlow, France) was equipped with a two-fluid nozzle with a diameter of 0.8 mm for feeding. The cultures were agitated gently for 10 min before spray drying. The spray-drying parameters were monitored using SD2P software (15). The inlet air temperature was fixed at 250°C, and the outlet air was controlled at 85°C and 0.2% relative humidity. After spray drying, the propionibacterial suspensions were reconstituted in sterile water to the original dry matter and slowly agitated for 15 min prior to enumeration on YEL agar plates as described above. Proteomic analysis. Two-dimensional electrophoresis was carried out as described previously (20). Briefly, propionibacteria were harvested by centrifugation, together with medium-insoluble materials; washed in 0.1 M Tris buffer, pH 8, containing 2% sodium citrate; centrifuged; washed twice in 0.1 M Tris buffer, pH 8, containing 0.001 M EDTA and then in phosphate-buffered saline (PBS); pelleted; frozen; and then resuspended in SDS lysis buffer prior to sonication and cell lysis using a Retsch MM301 mixer mill. The resulting SDS extracts were recovered by centrifugation (21,000 ⫻ g; 20°C; 20 min) and analyzed by one-dimensional SDS-PAGE (27) prior to protein precipitation using a two-dimensional (2D) cleanup kit (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Protein pellets, as well as immobilized pH gradient (IPG) dry strips, were rehydrated in destreaking solution (GE Healthcare Bio-Sciences AB) supplemented with IPG buffer, pH 4 or 7. For each two-dimensional gel, 300 ␮g of cellular proteins, whatever the treatment, were loaded onto the acid end of a linear-gradient IPG dry strip (pH 4 to 7) prior to isoelectric focusing on a MultiphorII up to 60,000 V · h according to a standardized procedure (17). The second dimension was SDS-PAGE (12.5% polyacrylamide; 16 by 16 by 0.1 cm; Protean II; Bio-Rad, Hercules, CA). The gels were stained using Bio-Safe Coomassie G-250 stain (Bio-Rad) prior to scanning on an ImageScanner III (GE Healthcare Bio-Sciences AB). Image analysis, gel matching, and quantification of the protein amounts in individual spots, calculated as average normalized volumes, were performed using Progenesis SameSpot software 3.1 (Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom). Molecular weights and isoelectric points were calculated with the software using molecular markers (LMW; GE Healthcare Bio-Sciences AB) and assuming a linear 4-to-7 pH gradient, as indicated by the supplier. Gel subgroups corresponding to growth in 5% SW and in 30% SW were compared to compute fold and P values of all spots using one-way analysis of variance (ANOVA) and principal-component analysis (PCA), as described previously (28). The spots of interest that had protein expression significantly upregulated in 30% SW compared to 5% SW (above a 1.2-fold change) were selected and spotted for further identification, as previously described (24). The fold changes are indicated in Table 1. Protein identification by NanoLC coupled on-line with tandem mass spectrometry. Gel pieces were washed with acetonitrile and ammonium bicarbonate solution and then dried under vacuum in a Speed-Vac concentrator (SVC100H-200; Savant; Thermo Fisher Scientific, Waltham, MA, USA). In-gel trypsin digestion was performed overnight at

Applied and Environmental Microbiology

August 2016 Volume 82 Number 15

Propionibacterium freudenreichii in Sweet Whey

TABLE 1 Proteins differentially expressed after growth of P. freudenreichii CIRM BIA 129 in isotonic 5% or hyperconcentrated 30% sweet whey as identified by NanoLC–MS-MS Log E valued

Cover %e

Masstheorf

pItheorg

No. of spectrah

Foldi

Chaperone ClpB 2 Methylmalonyl-CoAj mutase large subunit MutB Myoinositol catabolism IolD protein FeS assembly protein SufB Myoinositol catabolism IolA protein Aspartate ammonia-lyase Pyruvate kinase 1 Coenzyme A transferase Coenzyme A transferase Glycine hydroxymethyltransferase precursor Xaa-Pro aminopeptidase I

⫺524.4 ⫺426.4

75 75

94.2 80.1

5.33 5.21

220 160

1.5 1.5

⫺472.7 ⫺471.8 ⫺369.2 ⫺215.0 ⫺432.7 ⫺695.6 ⫺526.4 ⫺432.3

78 86 77 65 83 87 81 80

69.7 53.8 52.7 53.0 53.9 55.6 55.6 51.8

5.04 5.02 5.00 5.03 5.17 5.53 5.53 5.28

160 214 132 93 202 261 177 187

1.7 1.5 1.4 1.3 1.4 1.6 1.4 1.6

⫺302.5

74

54.4

5.32

127

1.7

ATP-binding protein OpuCA of osmoprotectant ABC transporter Dihydrolipoyl dehydrogenase Fumarate hydratase, class II Fumarate hydratase, class II 6-Phosphogluconate dehydrogenase, decarboxylating Citrate synthase Anaerobic glycerol-3-phosphate dehydrogenase subunit B Elongation factor Tu Tryptophan synthase beta chain (TrpB) Gamma-glutamyl phosphate reductase

⫺352.3

70

47.9

5.51

134

1.3

⫺395.4 ⫺611.8 ⫺636.2 ⫺398.4

75 78 90 84

49.5 51.1 51.0 52.5

5.08 5.34 5.34 5.25

179 261 311 191

1.4 1.6 1.4 1.5

⫺181.3 ⫺321.2

62 89

47.6 45.8

5.63 5.27

69 118

1.4 1.9

⫺344.6 ⫺250.1

68 72

43.6 44.5

5.05 5.47

138 72

1.4 1.4

⫺243.3

75

43.5

5.20

109

1.5

Putative isocitrate/isopropylmalate dehydrogenase Tryptophanyl-tRNA synthetase Acetate kinase Transaldolase 2 Transaldolase 2 Zinc-binding dehydrogenase Aldo-/ketoreductase Oxidoreductase Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase

⫺330.8

78

44.7

5.20

178

1.4

⫺227.9 ⫺600.6 ⫺300.4 ⫺295.4 ⫺522.9 ⫺301.6 ⫺274.2 ⫺424.3

77 89 78 79 83 80 89 85

40.7 42.4 39.3 39.3 37.1 36.9 38.7 36.0

5.45 5.50 5.10 5.10 5.52 5.36 5.57 5.46

113 303 150 173 201 140 142 306

1.2 1.5 1.7 2.8 1.2 1.7 1.6 1.2

⫺412.2

78

37.7

5.46

313

1.4

NADPH-quinone reductase Putative aldo-/ketoreductase (oxidoreductase) Putative aldo-/ketoreductase (oxidoreductase) Cysteine synthase 1 NADH-quinone oxidoreductase chain C Oxidoreductase Superoxide dismutase (Mn/Fe) SodM Superoxide dismutase (Mn/Fe) SodM Methylmalonyl-CoA epimerase

⫺171.3 ⫺233.0

74 81

33.6 31.1

5.59 5.48

64 135

1.9 1.4

⫺182.0

79

30.4

5.48

122

1.4

⫺175.6 ⫺227.1

87 80

31.6 28.6

5.11 5.36

62 138

1.8 1.8

⫺104.0 ⫺452.1

54 89

28.8 22.8

5.56 5.28

52 209

1.3 1.4

⫺533.7

93

22.8

5.28

319

1.6

⫺214.1

90

16.6

5.40

203

1.1

No.a

Massexpb

pIexpc

Locus tag

Description

1 2

93.2 82.5

5.71 5.55

PFCIRM129_06355 PFCIRM129_07240

3 4 4 5 6 7 8 9

72.1 56.8 56.8 55.5 55.2 54.6 54.2 53.9

5.30 5.28 5.28 5.38 5.45 6.07 6.18 5.68

PFCIRM129_10415 PFCIRM129_11225 PFCIRM129_10420 PFCIRM129_00340 PFCIRM129_11435 PFCIRM129_01440 PFCIRM129_01440 PFCIRM129_10235

10

53.5

5.56

PFCIRM129_11790

11

51.9

5.90

PFCIRM129_07855

12 13 14 15

49.7 49.1 48.6 48.6

5.33 5.63 5.74 5.54

PFCIRM129_09770 PFCIRM129_11895 PFCIRM129_11895 PFCIRM129_07025

16 17

47.2 44.5

6.15 5.50

PFCIRM129_05685 PFCIRM129_04835

18 19

44.5 44.4

5.34 6.05

PFCIRM129_08275 PFCIRM129_04290

20

43.6

5.63

PFCIRM129_06815

21

42.9

5.50

PFCIRM129_07640

22 23 24 25 26 27 28 29

42.5 42.2 40.6 39.9 39.5 39.1 38.6 37.9

5.70 6.06 5.23 5.32 6.05 5.78 6.11 5.52

PFCIRM129_02990 PFCIRM129_03745 PFCIRM129_10390 PFCIRM129_10390 PFCIRM129_07545 PFCIRM129_03400 PFCIRM129_05680 PFCIRM129_11300

30

37.6

5.66

PFCIRM129_11300

31 32

35.3 32.6

6.07 5.98

PFCIRM129_01130 PFCIRM129_01465

33

32.3

5.78

PFCIRM129_01465

34 35

31.2 30.1

5.45 5.61

PFCIRM129_07790 PFCIRM129_08515

36 37

28.3 24.5

5.91 5.41

PFCIRM129_10185 PFCIRM129_08020

38

24.0

5.57

PFCIRM129_08020

39

19.1

5.79

PFCIRM129_05095

(Continued on following page)

August 2016 Volume 82 Number 15

Applied and Environmental Microbiology

aem.asm.org

4643

Huang et al.

TABLE 1 (Continued) No.a

Massexpb

pIexpc

Locus tag

Description

40

18.0

4.71

PFCIRM129_02555

41

16.2

4.28

PFCIRM129_10610

Heat shock protein 20 1 (20-kDa chaperone 1) Phosphocarrier, HPr family

Log E valued

Cover %e

Masstheorf

pItheorg

No. of spectrah

Foldi

⫺139.6

77

17.3

4.73

119

1.4

⫺146.0

95

8.9

4.22

106

1.8

a

The numbers correspond to the spots in Fig. 2. b Massexp, molecular mass (kDa) evaluated from bidimensional electrophoresis migration. c pIexp, isoelectric point evaluated from bidimensional electrophoresis migration. d The E value is the probability that a given peptide score will be achieved by incorrect matches from a database search. Protein identifications were automatically validated when they showed at least two unique peptides with E values below 0.05, corresponding to a log(E value) of less than ⫺1.3. e Cover %, the percentage of the protein amino acid sequence covered by tandem mass spectrometry identification of the peptide. f Masstheor, protein molecular mass (kDa) automatically predicted from the corresponding gene on the Agmial annotation platform. g pItheor, isoelectric point automatically predicted from the corresponding gene on the Agmial annotation platform (http://genome.jouy.inra.fr/agmial/). h Number of MS-MS spectra with an individual E value of ⬍0.05 matched to a peptide sequence from the protein. i Fold change of protein expression in 30% SW compared to 5% SW as determined using Progenesis SameSpot software. j CoA, coenzyme A.

37°C and stopped with spectrophotometric-grade trifluoroacetic acid (TFA) (Sigma-Aldrich, Saint Quentin Fallavier, France) as described previously (24). The supernatants containing peptides were then vacuum dried in a Speed-Vac concentrator and stored at ⫺20°C until mass spectrometry (MS) analysis. Nanoscale liquid chromatography coupled online with tandem mass spectrometry (NanoLC–MS-MS) analysis was performed with an on-line liquid chromatography (MS-MS) setup using a Dionex U3000-RSLC NanoLC system fitted to a QStar XL (MDS SCIEX, Ontario, Canada) equipped with a nano-electrospray ion (ESI) source (Proxeon Biosystems A/S, Odense, Denmark). Samples were first concentrated on a PepMap 100 reverse-phase column (C18; 5-␮m particle size; 300-␮m inside diameter [i.d.] by 5-mm length) (Dionex, Amsterdam, The Netherlands). Peptides were separated on a reverse-phase PepMap 100 column (C18; 3-␮m particle size; 75-␮m i.d. by 150-mm length; Dionex) at 35°C, using solvent A (2% [vol/vol] acetonitrile, 0.08% [vol/vol] formic acid, and 0.01% [vol/vol] TFA in deionized water) and solvent B (95% [vol/vol] acetonitrile, 0.08% [vol/vol] formic acid, and 0.01% [vol/ vol] TFA in deionized water). A linear gradient from 10 to 40% solvent B in 17 min was applied for elution at a flow rate of 0.3 ␮l min⫺1. The eluted peptides were directly electrosprayed into the mass spectrometer operated in positive mode. A full continuous MS scan was carried, out followed by three data-dependent MS-MS scans. Spectra were collected in the selected mass range of 300 to 2,000 m/z for MS and 60 to 2,000 m/z for MS-MS spectra. Charged ions (2⫹ to 4⫹) were considered for the MS-MS analysis when the ion intensity was above 10 cps. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS-MS acquisition using Analyst QS 1.1 software. The instrument was calibrated by multipoint calibration using fragment ions that resulted from the collision-induced decomposition of a peptide from ␤-casein (␤-CN) (193 to 209). To identify peptides, all data (MS and MS-MS) were submitted to X! Tandem using the X! Tandem pipeline developed by Plateforme d’Analyze Protéomique de Paris Sud-Ouest (PAPPSO), INRA, Jouy-en-Josas, France (http://pappso.inra.fr). The search was performed against a database composed of (i) a homemade database containing all the predicted proteins of P. freudenreichii strain CIRM-BIA 129 used in this study and (ii) a portion of the UniProtKB database corresponding to P. freudenreichii. Database search parameters were specified as follows: trypsin cleavage was used, and the peptide mass tolerance was set to 0.2 Da for both MS and MS-MS. Oxidation of methionine and deamidation of asparagine and glutamine residues were included as variable modifications. Protein identifications were automatically validated when they showed at least two unique peptides with an E value below 0.05. Microscopy. Polyphosphate granule staining was done as recently described for lactobacilli (29) for the detection of polyphosphate granules using the procedure of Neisser. Bacterial cultures were smeared on microscope slides and heat fixed. The slides were covered for 1 min with a freshly prepared mixture of 1 volume of Neisser’s methylene blue solution (Fluka

4644

aem.asm.org

Analytical, France) and 2 volumes of 0.33% crystal violet solution in 10% ethanol. Excess dye was absorbed using blotting paper, and the slides were covered for 1 min with a 0.3% solution of chrysoidin G solution (SigmaAldrich, Saint Quentin Fallavier, France) prior to extensive rinsing with water. The slides were observed on an Olympus BX51 microscope at a ⫻1,000 magnification. In addition, polyphosphate granules were visualized by DAPI (4=,6=-diamidino-2-phenylindole) staining as previously described (30) and later modified (31) for cyanobacteria. Briefly, bacteria were washed in McIlvaine’s buffer (31), fixed in 4% formaldehyde, permeabilized in 0.3% Triton X-100, and stained in 20 ␮g/ml DAPI in the same buffer. The stained cultures were observed on an Olympus BX51 epifluorescence microscope equipped with a U-MWU2 fluorescence filter cube (excitation filter, 330 to 385 nm; emission filter, 480 to 800 nm) and an Olympus plan 100⫻/1.25 oil objective. For maximal resolution, bacteria were further imaged using a confocal Leica SP8 and a 63⫻/1.4 oil HC PL APO CS objective. Images were acquired using LAS-AF (Leica, Wetzlar, Germany) software. DAPI was excited with a 405-nm laser. Photomultiplier detector parameters were set to collect fluorescence between 412 and 450 nm and between 553 and 598 nm. Polyphosphate granules were further visualized by transmission electron microscopy as described previously for lactobacilli (29). Briefly, propionibacteria, grown in different media until stationary phase, were washed in PBS and in sodium cacodylate buffer (0.1 M, pH 7.3) prior to fixation with 2.5% glutaraldehyde for 3 h. The bacterial pellet was embedded in agarose prior to being cut into 1-mm pieces and fixed with 1% sodium tetroxide for 1 h. The agar pieces were rinsed with cacodylate buffer and dehydrated in ethanol prior to inclusion in Epon-AralditeDMP30 resin (polymerized at 60°C for 48 h). Thin sections (90 nm) were cut (Leica ultra microtome Ultracut E), collected on copper grids, and then stained with uranyl acetate. Samples were observed with a Jeol 1400 electron microscope (Jeol Co. Ltd., Tokyo, Japan), and images were digitally captured with a Gatan Orius camera (Digital Micrograph Software). Cellular glycan detection was done by using the periodic acid-Schiff (PAS) staining method, as previously described (32). The fixed bacterial smears on slides were stained with 1% periodic acid solution for 5 min and then transferred into absolute ethanol. The slides were rinsed with water, air dried, and subsequently stained with Schiff’s reagent (Sigma-Aldrich, France), consisting of 1% pararosaniline HCl and 4% sodium metabisulfite in 0.25 mol/liter hydrochloric acid. The slides were washed with flowing water for 5 min and then air dried before observation. For scanning electron microscopy (SEM) examination of the powder grains, powders were attached to SEM stubs using a two-sided carbon adhesive tape and then sputter coated with gold-palladium. Samples were analyzed at 5 kV at room temperature with a scanning electron microscope (JSM 7100F; Jeol Co. Ltd., Tokyo, Japan). Quantification of intracellular trehalose and glycogen. Trehalose and glycogen were assayed as described previously for Saccharomyces

Applied and Environmental Microbiology

August 2016 Volume 82 Number 15

Propionibacterium freudenreichii in Sweet Whey

cerevisiae (33) and adapted for propionibacteria (34). Briefly, propionibacteria were washed twice in PBS, resuspended in acetate buffer (40 mM, pH 5.2), heat inactivated for 5 min at 95°C, and disrupted using zirconium beads in a Retsch MM301 mixer mill prior to centrifugation of cellular debris. The resulting extract was divided into two parts, further digested using amyloglucosidase (from Aspergillus niger [no. 10115; Sigma-Aldrich]) or trehalase (from porcine kidney [no. T8778; Sigma-Aldrich]) for the hydrolysis of glycogen and trehalose at 57°C and 37°C, respectively. Samples were quickly frozen, and the resulting glucose was quantified using a glucose hexokinase assay kit (Sigma). The results were expressed as the concentration of generated glucose per unit weight of cellular wet matter. Statistical analysis. All experiments were independently repeated three times or performed with samples in triplicate (biological replicates). The results are presented as means ⫾ standard deviations. Significant differences (P ⬍ 0.05) between the mean values were determined by Tukey’s test. Statistical analysis was performed using R 3.2.1 with the Rcmdr package (R Development Core Team). The features observed with optical and electron microscopes were repeatedly observed in triplicate samples.

RESULTS AND DISCUSSION

Growth in hyperconcentrated sweet whey triggers a multitolerance response. Propionibacterium freudenreichii subsp. shermanii ITG P20, used in this study, is able to use various substrates as carbon and energy sources for growth. Carbohydrates, including sucrose, lactose, glucose, galactose, inositol, erythritol, adonitol, and esculine, may be used (35), as well as amino acids, glycerol, or its preferred carbon source, which is lactate (contained in the reference YEL growth medium). Within dairy products, it can thus utilize either lactose, the major milk carbohydrate, or lactate, resulting from lactic acid fermentation. Sweet whey and acid whey, which may contain either lactose or lactate or a mixture thereof, should thus sustain P. freudenreichii growth. As shown in Fig. S1 in the supplemental material, the use of sweet whey sustained P. freudenreichii growth. The final populations of propionibacteria were above 109 CFU/ml, whatever the growth medium, as determined by CFU counting. Hyperconcentrated sweet whey, 20 and 30% dry matter, allowed growth yields above that of the rich YEL reference medium, with a larger final population of P. freudenreichii, in accordance with the larger amount of substrates present in the medium. In contrast, growth was inhibited at 40% dry matter, suggesting inhibition at a concentration that was too elevated (data not shown). As a matter of fact, osmolarity increases with the concentration (see Fig. S1 in the supplemental material) to levels far above that of YEL medium, as hyperosmotic stress is a major stress for bacterial elongation and growth (36). P. freudenreichii was thus able to adapt to osmolality up to about 1,500 but not above 2,500 mosmol/kg. We then looked at the effect of such stress, undergone during growth, on tolerance for other stresses, imposed after growth. Growth in 20 and 30% dry matter sweet whey led to significant increases of stress tolerance in P. freudenreichii (Fig. 1). The most striking example was bile salts challenge survival, with 60.9% survival in 30% compared to 2.6% in 5% sweet whey. This result strongly suggests that the growth conditions imposed sublethal doses of stress responsible for stress adaptation; hyperosmotic stress is the main stimulus in hyperconcentrated sweet whey and is known to trigger bile salts tolerance in other bacteria (37, 38). The multitolerance response involves key stress proteins. To investigate the molecular mechanisms responsible for such adaptation, we performed a proteomic analysis in 5% versus 30% sweet

August 2016 Volume 82 Number 15

FIG 1 Hyperconcentrated sweet whey culture confers stress tolerance on P. freudenreichii. Propionibacteria were cultivated for 72 h in the indicated growth media until stationary phase. They were then subjected to heat (60°C for 10 min) (A), acid (pH 2.0 for 1 h) (B), or bile salts (1 g/liter for 1 h) (C) challenge as described in Materials and Methods. Viable propionibacteria were enumerated by plate counting in treated and control cultures. The results are expressed as percent survival. The error bars represent the standard deviations for triplicate experiments. Different letters above the columns indicate significant differences (P ⬍ 0.05).

whey culture media. As the media were turbid and rich in insoluble material, bacteria were centrifuged and washed using sodium citrate and EDTA prior to comparative two-dimensional electrophoresis. As shown in Fig. 2, at least 41 protein spots were enhanced in the pH range from 4 to 7 in 30% sweet whey. They were also enhanced in this hypertonic medium compared to the reference rich and isotonic YEL medium (see Fig. S2 in the supplemental material). The induced protein spots were then picked and subjected to in-gel trypsinolysis as previously described (20), and the resulting peptides were subjected to NanoLC–MS-MS as described in Materials and Methods. For each protein spot, the ma-

Applied and Environmental Microbiology

aem.asm.org

4645

Huang et al.

FIG 2 Hyperconcentrated sweet whey culture triggers overexpression of stress proteins. Propionibacteria were cultivated for 72 h in isotonic 5% (A) or hyperconcentrated 30% (B) sweet whey. The proteins accumulated in the cellular compartment were then analyzed by 2D electrophoresis (pH 4 to 7 isoelectric focusing and then SDS-12.5% PAGE) prior to differential analysis using the SameSpot software. The proteins induced in 30% sweet whey are indicated by arrows. Their identities, revealed by trypsinolysis and NanoLC–MS-MS, are shown in Table 1.

jor identified protein is presented in Table 1. Several of the identified proteins are known to participate in stress adaptation of P. freudenreichii and/or in other microorganisms. As an example, the ClpB 2 chaperone (spot no. 1 in Table 1), involved in acid stress adaptation in P. freudenreichii (20), is heat and salt inducible in Bifidobacterium breve (39). The protein synthesis elongation factor EF-Tu (spot no. 18) was also induced here. EF-Tu is known to alleviate the negative effect of heat stress by acting as a molecular chaperone (40). It is induced by acid stress in P. freudenreichii (20) and in Lactobacillus delbrueckii (41) and plays a role in osmotic adaptation in Escherichia coli (42). Citrate synthase (spot no. 16) is involved in stress tolerance, and its disruption leads to multistress susceptibility in Pseudomonas syringae (43), while its overexpression leads to aluminum tolerance in alfalfa (44), confirming a role in multistress tolerance response. We also reported the implication of cysteine synthase (no. 34), superoxide dismutase (no. 37), methylmalonyl-coenzyme A (CoA) mutase (no. 2), and aspartate ammonia-lyase (no. 5) in P. freudenreichii stress adaptation (20). The multitolerance response is linked with osmoadaptation. Our observations suggest that P. freudenreichii, in the 30% sweet whey culture medium, adapted via osmoadaptation mechanisms leading to intracellular accumulation of compatible solutes. Likewise, previous studies have evidenced that some strains of P. freudenreichii possess efficient osmoadaptation mechanisms using glycine betaine, dimethylsulfoniopropionate, and dimethylsulfonioacetate (45) and involving the synthesis of trehalose via the OtsA-OtsB pathway, induced by osmotic, acid, and oxidative stresses (46). Here, we observed the induction of the OpuCAcompatible solute transport system (no. 11 in Table 1) involved in

4646

aem.asm.org

glycine betaine uptake and osmoadaptation (47), which suggests adaptation of P. freudenreichii to hyperconcentrated sweet whey by increased transport of such compatible solute molecules. Compatible solutes, in addition to those transported by the OpuCA system, may also be synthesized de novo, as suggested here by the induction of serine hydroxymethyltransferase, also called glycine hydroxymethyltransferase (no. 9 in Table 1; EC 2.1.2.1), which is involved in accumulation of choline precursors and thus in osmoadaptation in the halotolerant cyanobacterium Aphanothece halophytica (48). Furthermore, induced proteins also comprised Xaa-Pro aminopeptidase (no. 10), which is involved in osmoprotection by proline-containing peptides in Bacillus subtilis (49), and gamma-glutamyl phosphate reductase (no. 20, encoded by proA), involved in osmoadaptive proline production in B. subtilis (50). They also comprised glycerol 3-phosphate dehydrogenase (no. 17), which is involved in osmoadaptation in yeasts, where glycerol plays a key role, while its mutational inactivation results in reduced salt tolerance (51). Altogether, these data strongly suggest that P. freudenreichii triggers an osmoadaptive response during growth in hyperconcentrated sweet whey. We also looked for accumulation of trehalose and glycogen, two carbohydrates known to participate in stress tolerance and in long-term survival in P. freudenreichii (34, 46). As shown in Fig. 3, both compounds were below the limit of detection when P. freudenreichii was grown in YEL medium. In contrast, both were accumulated in 30%, and to a lesser extent in 5%, sweet whey cultures. Such accumulation is known to confer enhanced osmotolerance and long-term survival under cheese-ripening conditions (34). Accordingly, periodic acid-Schiff staining of carbohy-

Applied and Environmental Microbiology

August 2016 Volume 82 Number 15

Propionibacterium freudenreichii in Sweet Whey

FIG 3 Hyperconcentrated sweet whey culture triggers intracellular accumulation of carbohydrates. (A to C) Propionibacteria were cultivated for 72 h in YEL medium (A) or in isotonic 5% (B) or hyperconcentrated 30% (C) sweet whey. Periodic acid-Schiff staining was applied to smears of the cultures. (D and E) Glycogen (D) and trehalose (E) were then enzymatically quantified in whole-cell extracts. The results are expressed as concentrations of generated glucose per unit weight of cellular wet matter. The error bars represent the standard deviations for the results of triplicate experiments. Different letters above the bars indicate significant differences (P ⬍ 0.05).

drates in smeared cultures of P. freudenreichii evidenced the presence of deeply stained round structures at both ends of the bacteria in 30% sweet whey that were not present in YEL cultures. Inorganic polyphosphate was also reported to play a key role in stress tolerance by acting as a protein-protecting chaperone and by regulating sigma factors involved in stress response in bacteria (52). Polyphosphate accumulation in the form of volutin granules has been evidenced in various Lactobacillus species (29), in Corynebacterium glutamicum (53), and in P. freudenreichii (54). The Neisser staining used by the above-mentioned authors was used here and revealed the characteristic intracellular dark-stained granules in P. freudenreichii (Fig. 4A to C). Granules were present in sweet whey cultures—more abundant in 30% than in 5% sweet whey—while they were not detected in YEL-cultured propionibacteria. In addition to Neisser staining, the fluorescent DAPI probe was also used to visualize polyphosphate (green) and DNA (blue). Green-light-emitting bacteria were evidenced in sweet whey cultures using epifluorescence microscopy, where emission wavelengths of 420 to 800 nm were seen together (Fig. 4D to F). This was further confirmed by confocal microscopy (Fig. 4G to I), where blue (412 to 450 nm) and green (553 to 598 nm) images were acquired and then merged. Furthermore, transmission electron microscopy (Fig. 4J to L) showed, in sweet whey cultures, circular and oval holes that had already been described when these granules were chipped or torn out during cutting of thin sections (29, 55, 56). Altogether, these data indicate that such granules are also accumulated in P. freudenreichii CIRM-BIA 129 under the conditions used in the present study. In lactobacilli, accumulation of intracellular polyphosphate in the form of polyphosphate granules depends on the species considered, the strain within the species, and the culture conditions (29). The presence of inorganic phosphate in the culture medium is a prerequisite for polyphosphate accumulation. In the sweet whey used here, inorganic phosphate accounts for 1.14% of the dry matter, so its concentration is

August 2016 Volume 82 Number 15

close to 6 mM and 36 mM for 5% and 30% sweet whey, respectively. This is close to the 37 mM KH2PO4 concentration in malic enzyme induction (MEI) medium (0.5% yeast extract, 0.5% tryptone, 0.4% KH2PO4, 0.5% KH2PO4, 0.02% MgSO4·7H2O, 0.005% MnSO4, 1 ml Tween 80 liter⫺1, 0.05% cysteine, 0.5% glucose), favoring polyphosphate accumulation in lactobacilli, which coincides with enhanced stress tolerance, including salt and low pH (29). Interestingly, abolishment of polyphosphate accumulation ability by mutational inactivation of the ppk gene leads to reduced stress tolerance (29). We propose that osmotically induced accumulation of polyphosphates, used as both energy storage molecules and compatible solutes by P. freudenreichii, participates in osmoinduced enhanced multitolerance. The multitolerance response protects from spray-drying stress. Dairy starters or probiotic products are mainly produced in a dried form, which is convenient to store, transport, and implement in food products (57, 58). Spray drying constitutes a promising alternative to freeze-drying to produce dried yet live bacterial powders because of its lower cost and higher production rate. Although spray drying of probiotic bacteria has been extensively investigated, the reports mainly focused on lactobacilli or bifidobacteria and rarely on propionibacteria. Schuck et al. reported for the first time the feasibility of spray drying Propionibacterium acidipropionici (15). The two P. acidipropionici strains were found to be robust during the pilot scale spray drying with a 130°C inlet temperature and 60°C outlet temperature (100% survival). In this work, P. freudenreichii cultures in 5%, 20%, and 30% sweet whey were subjected to a spray-drying trial with harsher, although usual, conditions of a 250°C inlet temperature and 85°C outlet temperature. It should be noted that at the beginning of drying, the droplets formed by spraying are submitted to a temperature close to the wetbulb temperature, which in this study was close to 52°C. The survival of bacteria after spray drying was increased around 5-fold as the sweet whey concentration increased from 5%

Applied and Environmental Microbiology

aem.asm.org

4647

Huang et al.

FIG 4 Hyperconcentrated sweet whey culture triggers intracellular accumulation of polyphosphate granules. Propionibacteria were cultivated for 72 h in YEL medium (A, D, G, and J) or in isotonic 5% (B, E, H, and K) or hyperconcentrated 30% (C, F, I, and L) sweet whey. (A to C) Polyphosphate metachromatic granules were evidenced by Neisser staining of fixed culture smears prior to microscopy observation at ⫻1,000 magnification. (D to I) Polyphosphate was visualized by DAPI staining prior to epifluorescence microscopy (D to F) or confocal microscopy (G to I) observation. Green fluorescence indicates cytosolic poly(P), and blue fluorescence indicates DNA. (J to L) Transmission electron micrographs of propionibacteria. Propionibacteria were fixed and stained, and ultrathin sections were observed using transmission electron microscopy. The positions that were occupied by granules appear as holes in the bacterial cytoplasm (examples are indicated by the arrows).

to 30%, with a maximum survival of 44.1% for the 30% sweet whey (Fig. 5A). It is known that variations in dry matter can change the drying behavior of particles during spray drying (59). Low concentrations of the drying medium result in low viscosity,

4648

aem.asm.org

less solid content within the droplets formed by spraying, and finer powder particles (Fig. 5B and C), which may potentially be exposed to longer retention times within the dryer (60). Moreover, the smaller the solid content within the particles, the less protective agent encap-

Applied and Environmental Microbiology

August 2016 Volume 82 Number 15

Propionibacterium freudenreichii in Sweet Whey

FIG 5 Hyperconcentrated sweet whey culture confers tolerance for spray drying. Propionibacteria were cultivated for 72 h in YEL medium or in isotonic 5% or 5% plus 25% (5% with 30% added before drying) or hyperconcentrated 20% or 30% (wt/wt) sweet whey and then spray dried at 250°C inlet temperature and 85°C outlet temperature. Viable propionibacteria were enumerated by plate counting in YEL agar before and after spray drying. For samples after spray drying, the powders were rehydrated with sterilized water to the same dry matter content as the culture before drying. (A) The results are expressed as percent survival. The error bars represent the standard deviations for the results of triplicate experiments. Different letters above the bars indicate significant differences (P ⬍ 0.05). (B and C) The powders dried from the 5% (B) and 30% (C) sweet whey powders were observed by scanning electron microscopy. Scale bars: 10 ␮m (upper panel of B and both panels of C) or 1 ␮m (lower panel of B).

sulates the bacteria during drying (61). Therefore, the use of hyperconcentrated sweet whey is advantageous for improving the viability of bacteria after spray drying, as P. freudenreichii bacteria were more exposed to heat stress in the 5% sweet whey cultures than in the 30% sweet whey cultures. Growing in hyperconcentrated medium is crucial, and addition of sweet whey just before drying does not completely afford such protection (Fig. 5A). Inactivation during spray drying or thermal convective drying is mainly caused by heat, osmotic, and oxidative stresses (62–64). Accordingly, adaptation to heat, osmotic, or oxidative stress has been used as a strategy to improve viability. For instance, a mod-

August 2016 Volume 82 Number 15

erate heat or salt pretreatment was reported to enhance Lactobacillus rhamnosus viability upon freeze-drying and air drying, together with enhanced expression of chaperones, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, enolase, and phosphoglycerate kinase (65). Similarly, Desmond et al. reported that the viability of the heat-adapted Lactobacillus paracasei NFBC 338 in reconstituted skim milk was enhanced 18-fold during spray drying, while salt-adapted cultures exhibited 16fold-greater viability than controls (66). We show here that growth in hyperconcentrated sweet whey not only renders drying easier, but also triggers the same kind of cross-protection with-

Applied and Environmental Microbiology

aem.asm.org

4649

Huang et al.

out additional treatment, causing multistress response with cross-protection against heat, osmotic, and oxidative stresses during drying, in accordance with previous reports of cross-protection triggered by osmoadaptation in other bacteria (38, 65). Conclusion. In conclusion, a new 2-in-1 culture and drying process was developed, using sweet whey as a culture and drying medium. It triggers different stress tolerance pathways, including overexpression of chaperones and accumulation of key compatible solutes. It leads to live and stable probiotics with induced multitolerance. Considering these adaptation processes, this work opens new avenues for the development of probiotic and starter preparations, ensuring enhanced efficacy in terms of survival during storage and stress tolerance. It will also lead to more sustainable production of probiotics, since it uses a low-cost by-product of the dairy industry. Furthermore, the energy cost of spray drying is far lower than that of the currently used freeze-drying.

11. 12.

13.

14.

15. 16.

ACKNOWLEDGMENTS Song Huang was the recipient of a Ph.D. grant from Soochow University and from Agrocampus Ouest. We thank Paulette Amet, Chantal Cauty, and Jessica Musset for their excellent technical support; Loïc Joanny from the Centre de Microscopie Électronique à Balayage et microAnalyse (CMEBA); and Stéphanie Dutertre from the Microscopy Rennes Imaging Center (BIOSIT, Université de Rennes 1) for expert assistance in experiments. We thank Eric Hotta and Oussama Roilles for useful advice.

17.

18.

19.

REFERENCES 1. Cousin FJ, Mater D, Foligné B, Jan G. 2011. Dairy propionibacteria as human probiotics: a review of recent evidence. Dairy Sci Technol 91:1–26. 2. Bouglé D, Roland N, Lebeurrier F, Arhan P. 1999. Effect of propionibacteria supplementation on fecal bifidobacteria and segmental colonic transit time in healthy human subjects. Scand J Gastroenterol 34:144 –148. http://dx.doi.org/10.1080/00365529950172998. 3. Hojo K, Yoda N, Tsuchita H, Ohtsu T, Seki K, Taketomo N, Murayama T, Lino H. 2002. Effect of ingested culture of Propionibacterium freudenreichii ET-3 on fecal microflora and stool frequency in healthy females. Biosci Microflora 21:115–120. http://dx.doi.org/10.12938/bifidus1996.21 .115. 4. Lan A, Bruneau A, Bensaada M, Philippe C, Bellaud P, Rabot S, Jan G. 2008. Increased induction of apoptosis by Propionibacterium freudenreichii TL133 in colonic mucosal crypts of human microbiota-associated rats treated with 1,2-dimethylhydrazine. Br J Nutr 100:1251–1259. http: //dx.doi.org/10.1017/S0007114508978284. 5. Jan G, Belzacq AS, Haouzi D, Rouault A, Metivier D, Kroemer G, Brenner C. 2002. Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria. Cell Death Differ 9:179 –188. http://dx.doi.org/10.1038/sj.cdd.4400935. 6. Lan A, Lagadic-Gossmann D, Lemaire C, Brenner C, Jan G. 2007. Acidic extracellular pH shifts colorectal cancer cell death from apoptosis to necrosis upon exposure to propionate and acetate, major end-products of the human probiotic propionibacteria. Apoptosis 12:573–591. http://dx .doi.org/10.1007/s10495-006-0010-3. 7. Cousin FJ, Jouan-Lanhouet S, Dimanche-Boitrel MT, Corcos L, Jan G. 2012. Milk fermented by Propionibacterium freudenreichii induces apoptosis of HGT-1 human gastric cancer cells. PLoS One 7:e31892. http://dx .doi.org/10.1371/journal.pone.0031892. 8. Foligné B, Deutsch SM, Breton J, Cousin FJ, Dewulf J, Samson M, Pot B, Jan G. 2010. Promising immunomodulatory effects of selected strains of dairy propionibacteria as evidenced in vitro and in vivo. Appl Environ Microbiol 76:8259 – 8264. http://dx.doi.org/10.1128/AEM.01976-10. 9. Foligné B, Breton J, Mater D, Jan G. 2013. Tracking the microbiome functionality: focus on Propionibacterium species. Gut 62:1227–1228. http://dx.doi.org/10.1136/gutjnl-2012-304393. 10. Plé C, Richoux R, Jardin J, Nurdin M, Briard-Bion V, Parayre S, Ferreira S, Pot B, Bouguen G, Deutsch SM, Falentin H, Foligne B, Jan G. 2015. Single-strain starter experimental cheese reveals antiinflamma-

4650

aem.asm.org

20.

21. 22.

23.

24.

25. 26. 27. 28.

29.

30.

tory effect of Propionibacterium freudenreichii in TNBS colitis model. J Funct Foods 18:575–585. http://dx.doi.org/10.1016/j.jff.2015.08.015. Rossi M, Amaretti A, Raimondi S. 2011. Folate production by probiotic bacteria. Nutrients 3:118 –134. http://dx.doi.org/10.3390/nu3010118. Mitsuyama K, Masuda J, Yamasaki H, Kuwaki K, Kitazaki S, Koga H, Uchida M, Sata M. 2007. Treatment of ulcerative colitis with milk whey culture with Propionibacterium freudenreichii. J Intest Microbiol 21:143– 147. Lan A, Bruneau A, Philippe C, Rochet V, Rouault A, Hervé C, Roland N, Rabot S, Jan G. 2007. Survival and metabolic activity of selected strains of Propionibacterium freudenreichii in the gastrointestinal tract of human microbiota-associated rats. Br J Nutr 97:714 –724. http://dx.doi.org/10 .1017/S0007114507433001. Hervé C, Fondrevez M, Cheron A, Barloy-Hubler F, Jan G. 2007. Transcarboxylase mRNA: a marker which evidences P. freudenreichii survival and metabolic activity during its transit in the human gut. Int J Food Microbiol 113:303–314. http://dx.doi.org/10.1016/j.ijfoodmicro.2006.08.013. Schuck P, Dolivet A, Méjean S, Hervé C, Jeantet R. 2013. Spray drying of dairy bacteria: new opportunities to improve the viability of bacteria powders. Int Dairy J 31:12–17. http://dx.doi.org/10.1016/j.idairyj.2012.01.006. Mocquot G. 1979. Reviews of the progress of dairy science: Swiss-type cheese. J Dairy Res 46:133–160. http://dx.doi.org/10.1017/S0022029900016940. Jan G, Leverrier P, Pichereau V, Boyaval P. 2001. Changes in protein synthesis and morphology during acid adaptation of Propionibacterium freudenreichii. Appl Environ Microbiol 67:2029 –2036. http://dx.doi.org /10.1128/AEM.67.5.2029-2036.2001. Leverrier P, Dimova D, Pichereau V, Auffray Y, Boyaval P, Jan G. 2003. Susceptibility and adaptive response to bile salts in Propionibacterium freudenreichii: physiological and proteomic analysis. Appl Environ Microbiol 69:3809 –3818. http://dx.doi.org/10.1128/AEM.69.7.3809-3818.2003. Anastasiou R, Leverrier P, Krestas I, Rouault A, Kalantzopoulos G, Boyaval P, Tsakalidou E, Jan G. 2006. Changes in protein synthesis during thermal adaptation of Propionibacterium freudenreichii subsp. shermanii. Int J Food Microbiol 108:301–314. Leverrier P, Vissers JP, Rouault A, Boyaval P, Jan G. 2004. Mass spectrometry proteomic analysis of stress adaptation reveals both common and distinct response pathways in Propionibacterium freudenreichii. Arch Microbiol 181:215–230. http://dx.doi.org/10.1007/s00203 -003-0646-0. Leverrier P, Fremont Y, Rouault A, Boyaval P, Jan G. 2005. In vitro tolerance to digestive stresses of propionibacteria: influence of food matrices. Food Microbiol 22:11–18. http://dx.doi.org/10.1016/j.fm.2004.05.003. Cousin FJ, Louesdon S, Maillard MB, Parayre S, Falentin H, Deutsch SM, Boudry G, Jan G. 2012. The first dairy product exclusively fermented by Propionibacterium freudenreichii: a new vector to study probiotic potentialities in vivo. Food Microbiol 32:135–146. http://dx.doi.org/10.1016 /j.fm.2012.05.003. Cousin FJ, Foligne B, Deutsch SM, Massart S, Parayre S, Le Loir Y, Boudry G, Jan G. 2012. Assessment of the probiotic potential of a dairy product fermented by Propionibacterium freudenreichii in piglets. J Agric Food Chem 60:7917–7927. http://dx.doi.org/10.1021/jf302245m. Gagnaire V, Jardin J, Rabah H, Briard-Bion V, Jan G. 2015. Emmental cheese environment enhances Propionibacterium freudenreichii stress tolerance. PLoS One 10:e0135780. http://dx.doi.org/10.1371/journal.pone .0135780. Malik AC, Reinbold GW, Vedamuthu ER. 1968. An evaluation of the taxonomy of Propionibacterium. Can J Microbiol 14:1185–1191. http://dx .doi.org/10.1139/m68-199. Jan G, Rouault A, Maubois JL. 2000. Acid stress susceptibility and acid adaptation of Propionibacterium freudenreichii subsp. shermanii. Lait 80: 325–336. http://dx.doi.org/10.1051/lait:2000128. Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680 – 685. http://dx.doi.org/10 .1038/227680a0. Morris JS, Clark BN, Wei W, Gutstein HB. 2010. Evaluating the performance of new approaches to spot quantification and differential expression in 2-dimensional gel electrophoresis studies. J Proteome Res 9:595– 604. http://dx.doi.org/10.1021/pr9005603. Alcantara C, Blasco A, Zuniga M, Monedero V. 2014. Accumulation of polyphosphate in Lactobacillus spp. and its involvement in stress resistance. Appl Environ Microbiol 80:1650 –1659. http://dx.doi.org/10.1128 /AEM.03997-13. Gunther S, Trutnau M, Kleinsteuber S, Hause G, Bley T, Roske I,

Applied and Environmental Microbiology

August 2016 Volume 82 Number 15

Propionibacterium freudenreichii in Sweet Whey

31.

32. 33. 34.

35.

36. 37.

38.

39.

40.

41.

42.

43. 44.

45. 46.

47.

48.

Harms H, Muller S. 2009. Dynamics of polyphosphate-accumulating bacteria in wastewater treatment plant microbial communities detected via DAPI (4=,6=-diamidino-2-phenylindole) and tetracycline labeling. Appl Environ Microbiol 75:2111–2121. http://dx.doi.org/10.1128/AEM .01540-08. Mukherjee C, Chowdhury R, Ray K. 2015. Phosphorus recycling from an unexplored source by polyphosphate accumulating microalgae and cyanobacteria: a step to phosphorus security in agriculture. Front Microbiol 6:1421. http://dx.doi.org/10.3389/fmicb.2015.01421. Moats WA. 1959. Application of periodic acid-Schiff type stains to bacteria in milk. J Bacteriol 78:589 –593. Parrou JL, Francois J. 1997. A simplified procedure for a rapid and reliable assay of both glycogen and trehalose in whole yeast cells. Anal Biochem 248:186 –188. http://dx.doi.org/10.1006/abio.1997.2138. Dalmasso M, Aubert J, Even S, Falentin H, Maillard MB, Parayre S, Loux V, Tanskanen J, Thierry A. 2012. Accumulation of intracellular glycogen and trehalose by Propionibacterium freudenreichii under conditions mimicking cheese ripening in the cold. Appl Environ Microbiol 78:6357– 6364. http://dx.doi.org/10.1128/AEM.00561-12. Loux V, Mariadassou M, Almeida S, Chiapello H, Hammani A, Buratti J, Gendrault A, Barbe V, Aury JM, Deutsch SM, Parayre S, Madec MN, Chuat V, Jan G, Peterlongo P, Azevedo V, Le Loir Y, Falentin H. 2015. Mutations and genomic islands can explain the strain dependency of sugar utilization in 21 strains of Propionibacterium freudenreichii. BMC Genomics 16:296. http://dx.doi.org/10.1186/s12864-015-1467-7. Empadinhas N, da Costa MS. 2008. Osmoadaptation mechanisms in prokaryotes: distribution of compatible solutes. Int Microbiol 11:151–161. Zhang Q, Feng Y, Deng L, Feng F, Wang L, Zhou Q, Luo Q. 2011. SigB plays a major role in Listeria monocytogenes tolerance to bile stress. Int J Food Microbiol 145:238 –243. http://dx.doi.org/10.1016/j.ijfoodmicro .2010.12.028. Flahaut S, Benachour A, Giard JC, Boutibonnes P, Auffray Y. 1996. Defense against lethal treatments and de novo protein synthesis induced by NaCl in Enterococcus faecalis ATCC 19433. Arch Microbiol 165:317– 324. http://dx.doi.org/10.1007/s002030050333. Ventura M, Kenny JG, Zhang Z, Fitzgerald GF, van Sinderen D. 2005. The clpB gene of Bifidobacterium breve UCC 2003: transcriptional analysis and first insights into stress induction. Microbiology 151:2861–2872. http: //dx.doi.org/10.1099/mic.0.28176-0. Fu J, Momcilovic I, Clemente TE, Nersesian N, Trick HN, Ristic Z. 2008. Heterologous expression of a plastid EF-Tu reduces protein thermal aggregation and enhances CO2 fixation in wheat (Triticum aestivum) following heat stress. Plant Mol Biol 68:277–288. http://dx.doi.org/10.1007 /s11103-008-9369-6. Zhai Z, Douillard FP, An H, Wang G, Guo X, Luo Y, Hao Y. 2014. Proteomic characterization of the acid tolerance response in Lactobacillus delbrueckii subsp. bulgaricus CAUH1 and functional identification of a novel acid stress-related transcriptional regulator Ldb0677. Environ Microbiol 16:1524 –1537. http://dx.doi.org/10.1111/1462-2920.12280. Berrier C, Garrigues A, Richarme G, Ghazi A. 2000. Elongation factor Tu and DnaK are transferred from the cytoplasm to the periplasm of Escherichia coli during osmotic downshock presumably via the mechanosensitive channel MscL. J Bacteriol 182:248 –251. http://dx.doi.org/10 .1128/JB.182.1.248-251.2000. Sengupta D, Sangu K, Shivaji S, Chattopadhyay MK. 2015. Tolerance of an Antarctic bacterium to multiple environmental stressors. Curr Microbiol 71:483– 489. http://dx.doi.org/10.1007/s00284-015-0874-y. Barone P, Rosellini D, Lafayette P, Bouton J, Veronesi F, Parrott W. 2008. Bacterial citrate synthase expression and soil aluminum tolerance in transgenic alfalfa. Plant Cell Rep 27:893–901. http://dx.doi.org/10.1007 /s00299-008-0517-x. Boyaval P, Deborde C, Corre C, Blanco C, Begue E. 1999. Stress and osmoprotection in propionibacteria. Lait 79:59 – 69. http://dx.doi.org/10 .1051/lait:199914. Cardoso FS, Castro RF, Borges N, Santos H. 2007. Biochemical and genetic characterization of the pathways for trehalose metabolism in Propionibacterium freudenreichii, and their role in stress response. Microbiology 153:270 –280. http://dx.doi.org/10.1099/mic.0.29262-0. Du Y, Shi WW, He YX, Yang YH, Zhou CZ, Chen Y. 2011. Structures of the substrate-binding protein provide insights into the multiple compatible solute binding specificities of the Bacillus subtilis ABC transporter OpuC. Biochem J 436:283–289. http://dx.doi.org/10.1042/BJ20102097. Waditee-Sirisattha R, Sittipol D, Tanaka Y, Takabe T. 2012. Overex-

August 2016 Volume 82 Number 15

49.

50.

51.

52. 53.

54. 55. 56.

57.

58. 59.

60. 61. 62.

63. 64. 65.

66. 67.

pression of serine hydroxymethyltransferase from halotolerant cyanobacterium in Escherichia coli results in increased accumulation of choline precursors and enhanced salinity tolerance. FEMS Microbiol Lett 333:46 – 53. http://dx.doi.org/10.1111/j.1574-6968.2012.02597.x. Zaprasis A, Brill J, Thuring M, Wunsche G, Heun M, Barzantny H, Hoffmann T, Bremer E. 2013. Osmoprotection of Bacillus subtilis through import and proteolysis of proline-containing peptides. Appl Environ Microbiol 79:576 –587. http://dx.doi.org/10.1128/AEM.01934-12. Brill J, Hoffmann T, Bleisteiner M, Bremer E. 2011. Osmotically controlled synthesis of the compatible solute proline is critical for cellular defense of Bacillus subtilis against high osmolarity. J Bacteriol 193:5335– 5346. http://dx.doi.org/10.1128/JB.05490-11. Yan H, Jia LH, Lin YP, Jiang N. 2008. Glycerol accumulation in the dimorphic yeast Saccharomycopsis fibuligera: cloning of two glycerol 3-phosphate dehydrogenase genes, one of which is markedly induced by osmotic stress. Yeast 25:609 – 621. http://dx.doi.org/10.1002/yea.1606. Gray MJ, Jakob U. 2015. Oxidative stress protection by polyphosphate— new roles for an old player. Curr Opin Microbiol 24:1– 6. http://dx.doi.org /10.1016/j.mib.2014.12.004. Klauth P, Pallerla SR, Vidaurre D, Ralfs C, Wendisch VF, Schoberth SM. 2006. Determination of soluble and granular inorganic polyphosphate in Corynebacterium glutamicum. Appl Microbiol Biotechnol 72: 1099 –1106. http://dx.doi.org/10.1007/s00253-006-0562-8. Clark JE, Beegen H, Wood HG. 1986. Isolation of intact chains of polyphosphate from “Propionibacterium shermanii” grown on glucose or lactate. J Bacteriol 168:1212–1219. Bode G, Mauch F, Ditschuneit H, Malfertheiner P. 1993. Identification of structures containing polyphosphate in Helicobacter pylori. J Gen Microbiol 139:3029 –3033. http://dx.doi.org/10.1099/00221287-139-12-3029. Auling G, Pilz F, Busse HJ, Karrasch S, Streichan M, Schon G. 1991. Analysis of the polyphosphate-accumulating microflora in phosphoruseliminating, anaerobic-aerobic activated sludge systems by using diaminopropane as a biomarker for rapid estimation of Acinetobacter spp. Appl Environ Microbiol 57:3585–3592. Ross RP, Desmond C, Fitzgerald GF, Stanton C. 2005. Overcoming the technological hurdles in the development of probiotic foods. J Appl Microbiol 98:1410 –1417. http://dx.doi.org/10.1111/j.1365-2672.2005 .02654.x. Anal AK, Singh H. 2007. Recent advances in microencapsulation of probiotics for industrial applications and targeted delivery. Trends Food Sci Technol 18:240 –251. http://dx.doi.org/10.1016/j.tifs.2007.01.004. Wu WD, Liu W, Gengenbach T, Woo MW, Selomulya C, Chen XD, Weeks M. 2014. Towards spray drying of high solids dairy liquids: effects of feed solid content on particle structure and functionality. J Food Eng 123:130 –135. http://dx.doi.org/10.1016/j.jfoodeng.2013.05.013. Jeantet R, Ducept F, Dolivet A, Mejean S, Schuck P. 2015. Residence time distribution: a tool to improve spray-drying control. Dairy Sci Technol 88:31– 43. Perdana J, Fox MB, Siwei C, Boom RM, Schutyser MAI. 2014. Interactions between formulation and spray drying conditions related to survival of Lactobacillus plantarum WCSF1. Food Res Int 56:9 –17. Simpson PJ, Stanton C, Fitzgerald GF, Ross RP. 2005. Intrinsic tolerance of Bifidobacterium species to heat and oxygen and survival following spray drying and storage. J Appl Microbiol 99:493–501. http://dx.doi.org/10 .1111/j.1365-2672.2005.02648.x. Huang S, Yang Y, Fu N, Qin Q, Zhang L, Chen XD. 2015. Calciumaggregated milk: a potential new option for improving the viability of lactic acid bacteria under heat stress. Food Bioproc Technol 7:3147–3155. Peighambardoust SH, Tafti AG, Hesari J. 2011. Application of spray drying for preservation of lactic acid starter cultures: a review. Trends Food Sci Technol 22:215–224. http://dx.doi.org/10.1016/j.tifs.2011.01.009. Prasad J, McJarrow P, Gopal P. 2003. Heat and osmotic stress responses of probiotic Lactobacillus rhamnosus HN001 (DR20) in relation to viability after drying. Appl Environ Microbiol 69:917–925. http://dx.doi.org/10 .1128/AEM.69.2.917-925.2003. Desmond C, Stanton C, Fitzgerald GF, Collins K, Ross RP. 2015. Environmental adaptation of probiotic lactobacilli towards improvement of performance during spray drying. Int Dairy J 11:801– 808. Cousin FJ, Jouan-Lanhouet S, Théret N, Brenner C, Jouan E, Le Moigne-Muller G, Dimanche-Boitrel MT, Jan G. 2016. The probiotic Propionibacterium freudenreichii as a new adjuvant for TRAIL-based therapy in colorectal cancer. Oncotarget 7:7161–7178. http://dx.doi.org/10 .18632/oncotarget.6881.

Applied and Environmental Microbiology

aem.asm.org

4651

Hyperconcentrated Sweet Whey, a New Culture Medium That Enhances Propionibacterium freudenreichii Stress Tolerance.

Propionibacterium freudenreichii is used as a cheese-ripening starter and as a probiotic. Its reported physiological effects at the gut level, includi...
2MB Sizes 0 Downloads 8 Views