Biotechnological Production of PhenyllacticAcid and Biosurfactants from Trimming Vine ShootHydrolyzates by Microbial Coculture Fermentation
Noelia Rodrguez-Pazo & Jos Manuel Salgado &Sandra Corts-Diguez & Jos Manuel Domnguez
Received: 13 July 2012 /Accepted: 30 January 2013 /Published online: 16 February 2013# Springer Science+Business Media New York 2013
Abstract Coculture fermentations show advantages for producing food additives fromagroindustrial wastes, considering that different specified microbial strains are combinedto improve the consumption of mixed sugars obtained by hydrolysis. This technologydovetails with both the growing interest of consumers towards the use of natural foodadditives and with stricter legislations and concern in developed countries towards themanagement of wastes. The use of this technology allows valorization of both cellulosicand hemicellulosic fractions of trimming vine shoots for the production of lactic acid (LA),phenyllactic acid (PLA), and biosurfactants (BS). This work compares the study of thepotential of hemicellulosic and cellulosic fractions of trimming vine shoots as cheaper andrenewable carbon sources for PLA and BS production by independent or coculture fermen-tations. The highest LA and PLA concentrations, 43.0 g/L and 1.58 mM, respectively, wereobtained after 144 h during the fermentation of hemicellulosic sugars and simultaneoussaccharification and fermentation (SSF) carried out by cocultures of Lactobacillus planta-rum and Lactobacillus pentosus. Additionally, cell-bond BS decreased the surface tension(ST) in 17.2 U; meanwhile, cell-free supernatants (CFS) showed antimicrobial activityagainst Salmonella enterica and Listeria monocytogenes with inhibition halos of 12.10.6 mm and 11.50.9 mm, respectively.
Keywords Coculture .Trimmingvinewastes . Phenyllactic acid .Biosurfactants . Lactic acid .
Lactic acid bacteria
Appl Biochem Biotechnol (2013) 169:21752188DOI 10.1007/s12010-013-0126-1
N. Rodrguez-Pazo : J. M. Salgado : S. Corts-Diguez : J. M. DomnguezAgro-Food Biotechnology Laboratory, CITI-Research, Transfer and Innovation Centre,Parque Tecnolgico de Galicia, San Cibrao das Vias 32900 Ourense, Spain
N. Rodrguez-Pazo : J. M. Salgado : S. Corts-Diguez : J. M. Domnguez (*)Chemical Engineering Department, Sciences Faculty, University of Vigo (Ourense Campus),As Lagoas s/n, 32004 Ourense, Spaine-mail: email@example.com
Coculture fermentations have been defined by Bader et al.  as the combination ofincubation and metabolic activity of different specified microbial strains under asepticconditions. This technology offers advantages for production of food additives, pharma-ceuticals, fuels, antimicrobial substances, enzymes, and bulk and fine chemicals, since itmay result in increased yield and improved control of product quality. Additionally, itmakes possible the use of worthless waste materials, a critical factor in industrialbiotechnology because carbon source is often the greatest contributor to the cost ofmicrobial products .
Among these solid wastes, some authors have proposed the use of trimming vineshoots as a source of renewable sugars after a number of thermochemical and biochem-ical processing steps to convert the polymers to monomeric sugars to be furtherconverted by fermentative microorganisms into ethanol, xylitol, lactic acid (LA), andbiosurfactants (BS), among other substances . These studies were carried out evalu-ating independently the cellulosic and hemicellulosic fractions. Thus, in the first step,the hemicellulosic sugar hydrolyzates obtained from trimming vine were assayed toproduce LA and biosurfactants by Lactobacillus pentosus strains ; meanwhile, thesolid residue obtained after the delignification of trimming vines shoots was employedin a second step to produce LA by simultaneous saccharification and fermentation (SSF)by Lactobacillus rhamnosus .
Food and feed spoiling microorganisms cause great economic losses worldwide,being necessary to develop efficient biopreservatives. In this way, LA bacteria (LAB)can be applied at an industrial scale for the production of bioactive molecules such asorganic acids, phenolic compounds, fatty acids, hydrogen peroxide, diacetyl bacteriocins,and BS with antimicrobial activity against organisms and pathogens, thus offering apotential alternative to the use of synthetic preservatives . The coculture of LABallows the valorization of both cellulosic and hemicellulosic fractions of trimming vineshoots for the production of biopreservatives such as LA, with wide use in foodindustry, mainly as acidifying, coagulant, and preservative agent against several patho-genic bacteria, fungi, and yeast species ; phenyllactic acid (PLA), a novel antimi-crobial compound produced through the phenylalanine degradation metabolism that isactive against Gram-(+), Gram-() bacteria, yeast, and fungi ; and BS, amphi-philic compounds of microbial origin with a pronounced surface activity and withseveral advantages over chemical surfactants including lower toxicity, higher biodegrad-ability, and effectiveness at extreme temperatures or pH, being potential candidates formany commercial applications in the biomedical, petroleum, and food processing indus-tries [9, 1416].
The main purpose of this work was to develop a profitable technology for the use ofboth hemicellulosic and cellulosic fractions of trimming wastes by coculture of Lacto-bacillus plantarum and L. pentosus. Different bioactive compounds such as LA, PLA,and BS were analyzed in the exhausted culture media. This is, in our knowledge, thefirst report about the simultaneous use of both fractions, which can simplify theoperational conditions at an industrial scale considering that different sugars are metab-olized simultaneously in one bioreactor, thus making the overall process more compet-itive and controllable. The antimicrobial activity of cell-bond BS and cell-freesupernatants (CFS) was also assayed against the pathogenic bacteria Salmonella entericaand Listeria monocytogenes.
2176 Appl Biochem Biotechnol (2013) 169:21752188
Materials and Methods
DL-3-PLA (product code P7251) was purchased from Sigma-Aldrich (Milan, Italy), while DL-phenylalanine (product code 78040) was provided by Fluka (Sigma-Aldrich, Buchs, Switzer-land). Ultrapure water was produced using a Millipore Milli-Q System (Millipore, Bedford,MA, USA). Methanol high-performance liquid chromatographic (HPLC) grade and trifluoro-acetic acid (TFA, 99.8 %) were supplied by Panreac Qumica SAU (Barcelona, Spain). Formicacid 98 %v/v (product code 131030) and acetic acid (product code 122703.1611) from PanreacQumica SAU CULTIMED (Barcelona, Spain) as well as D(+) xylose (product code 95729),D(+) glucose (product code 49139), D() arabinose (product code A3131), 5-hydroxymethyl-2-furaldehyde (product code H40807), and 2-furaldehyde (product code 48070) were bought toSigma-Aldrich, (St. Louis, MO, USA) for HPLC analysis using H2SO4 (product code131058.1211) as mobile phase from Panreac Qumica SAU. Glycerol for maintenance ofstrains in cryovials and the ManRogosaSharpe (MRS) broth were obtained from PanreacQumica SAU.
Raw Material and Analysis
Trimming vine shoots were locally collected during the campaign of 2011. These wasteswere dried, milled to a particle size less than 1 mm, homogenized in a single lot to avoidcompositional differences, and stored until use. Aliquots from the homogenized lot weresubmitted to moisture determination and quantitative hydrolysis in a two-stage acid treat-ment according to Vzquez et al. . The first stage used sulfuric acid (72 % weight) at30 C during 1 h. The second step started after sulfuric acid dilution to 4 % weightfollowed by treatment at 121 C during another hour. The resulting solid was consid-ered as Klason lignin; meanwhile, hydrolyzates were analyzed by HPLC as describedbelow. This method allowed determination of glucose, xylose, arabinose, acetic acid,furfural, and hydroxymethylfurfural.
Acid Hydrolysis (Prehydrolysis) and Enzymatic Activity of Enzymes for SimultaneousSaccharification and Fermentation
Hydrolyzates of trimming wastes were obtained according to the conditions reported byBustos et al.  in an autoclave at 130 C with 3 % sulfuric acid solutions during15 min using a liquid/solid ratio of 8 g/g. Hydrolyzates were cooled, filtered through0.45-m membranes, and analyzed by HPLC, providing hemicellulosic hydrolyzates;meanwhile, the solid residue was treated with 8 % (w/w) NaOH solution at 130 C for120 min using a liquid/solid ratio of 10 g/g to a delignification treatment to obtain twoother phases: a solid residue with cellulosic sugars and a liquid phase with solublelignin. The cellulosic fraction obtained in this second step was employed for SSFexperiments, employing two commercial enzyme concentrates Celluclast and Novo-zym 188, with cellulase and -glucosidase activities, respectively. Both enzymes werekindly provided by Novozymes (Denmark). The cellulase activity of concentrates wasassayed according to previous reports  by the filter paper activity (FPA) test andexpressed as filter paper units (FPU) per milliliter; meanwhile, the -glucosidaseactivity was measured according to Paquot and Thonart .
Appl Biochem Biotechnol (2013) 169:21752188 2177
Microorganisms and Fermentation Conditions
L. pentosus CECT-4023 and L. plantarum CECT-221 used in this study for coculture and S.enterica subsp. enterica CECT-724 and L. monocytogenes CECT-934 employed to evaluatethe antimicrobial activity of cell-free extracts and BS obtained after fermentation processagainst pathogenic bacteria were obtained from the Spanish Collection of Type Cultures(Valencia, Spain) and maintained in cryovials at 80 C in MRS medium with 15 %v/vglycerol as cryoprotector. The MRS medium contains (per liter): 10 g peptone, 8 g beefextract, 4 g yeast extract, 20 g D-glucose, 2 g K2HPO4, 2 g diammonium hydrogen citrate,5 g CH3COONa, 0.2 g MgSO47H2O, 0.05 g MnSO42H2O, and 1 g Tween 80. The samemedium was employed for seed activation.
Inocula were prepared by inoculating one glycerol stock vial into a 250-mL Erlenmeyerflask containing 100 mL of activation medium, followed by growth in an incubator shaker(Optic Ivymen System, Comecta S.A., distributed by Scharlab, Madrid, Spain) at 31.5 Cand 100 rpm during 12 h. Two generations of activation cultures were required beforefermentation. Finally, cells were recovered and resuspended in the fresh broth medium forinoculation with a 5 % of the final culture volume. After inoculation, fermentations werecarried out in a 2-L Braun-Biostat fermenter (Braun, Melsungen AG, Melsungen, Germany)at 31.5 C, with pH automatically controlled to 6.2 with 5 N NaOH.
Hemicellulosic sugars supplemented with MRS nutrients (except glucose) and 0.6 g/L Phewere fermented by L. plantarum (Fermentation 1A) or applying coculture of L. plantarum andL. pentosus, adding the second microorganism after 20-h fermentation (Fermentation 1B) at100 rpm with a final working volume medium of 1,800 mL. SSF was conducted using 30 g/gliquid/solid ratio:30 g of distilled water per gram of cellulosic fraction by L. plantarum(Fermentation 2) or 30 g of hemicellulosic sugars (Fermentation 3) per gram of cellulosicfraction by coculture of L. plantarum and L. pentosus, supplemented, in both cases, with MRSnutrients (except glucose) and 0.6 g/L Phe and with a final working volume medium of1,500 mL. The agitation was fixed in 150 rpm to ensure complete mixing. Enzymes wereadded with a cellulose/substrate ratio of 28 FPU/g and cellobiase/cellulose ratio of 13 UI/FPU.
All media were sterilized at 101 C for 60 min in an autoclave (Trade Raypa SL, Terrassa,Barcelona, Spain) prior to the experiments and inoculated with each producer strain in apercentage of 5 % of the final culture volume. Samples were taken at given fermentation timesand centrifuged at 2,755g, 10 C for 15 min. Supernatants were filtered (0.22 m pore-sizemembrane, Millipore) and employed for HPLC analysis; meanwhile, cells were used for biomassdetermination and BS extraction. Culture filtrates were produced in triplicate for each experiment.
Glucose, xylose, arabinose, ethanol, and lactic, formic, and acetic acid concentrations duringfermentations were measured by a HPLC system (Agilent, model 1200, Palo Alto, CA,USA) equipped with a refractive index detector and an Aminex HPX-87H ion exclusioncolumn (Bio-Rad 3007.8 mm, 9 m particles) with a guard column, eluted with 0.003 Msulfuric acid at a flow rate of 0.6 mL/min at 50 C. PLA and Phe were measured by an HPLCsystem (Agilent, model 1200, Palo Alto, CA, USA) equipped with an Agilent Zorbax SB-AqC18 column (4.6150 mm, 5 m particles) with a guard column and using a UV detector.Linear gradient elution was used with methanol/0.05 % TFA (solvent A) and water/0.05 %TFA (solvent B) at 1 mL/min and A/B ratios of 10:90, 100:0, 100:0, and 10:90, with runtimes of 0, 20, 23, and 25 min, respectively. PLA and Phe were detected at 210 nm accordingto the procedure described by Valerio et al..
2178 Appl Biochem Biotechnol (2013) 169:21752188
Cell concentration in experiments was measured by dry cell weight. Cells of knownvolume of culture media were centrifuged at 2,755g, 10 C for 15 min and washed twicewith distilled water and centrifuged under the same conditions. The resulting pellets wereoven-dried at 105 C to constant weight.
Determination of Biosurfactants
The surface activity of BS produced by L. pentosus CECT-4023 and/or L. plantarum CECT-221 was determined by measuring the surface tension (ST) of samples using the Ring method employing a KRSS Tensiometer (Hamburg, Germany) equipped with a 1.9-cmDuNoyplatinum ring at room temperature. To obtain the samples for BS determination, cells wererecovered by centrifugation (10,000g, 15 min, 10 C) from fermentation media, washed twicein demineralized water, resuspended in phosphate buffer saline (PBS: 10 mMKH2PO4/K2HPO4 and 150 mM NaCl with pH adjusted to 7.0) using a fermentation mediumvolume/PBS volume ratio of 6, incubated for 2 h at room temperature, and centrifuged torecover the PBS extract free of biomass. The BS concentration [BS] was calculated using thecalibration curve reported by Portilla-Rivera et al. : [BS] (mg/L)=(ST (mN/m)76.984) /8.64658 that was obtained using surfactin at different concentrations below the critical micelleconcentration (CMC) with known values of ST.
The antimicrobial activity of the CFS and BS extracts obtained from all different fermentationswas determined by the well diffusion technique using tryptocase soy broth (TSB) with 20% (wv)agar as culture media, held at 45 C, and inoculated with 100 L of active culture of the targetorganism. About 25 mL of the seeded agar was poured into a sterile Petri dish and allowed tosolidify at room temperature. Wells were then cut in the solidified agar with a sterile metalcylinder and...