Characterization of Cucumber Fermentation Spoilage Bacteria by Enrichment Culture and 16S rDNA Cloning Fred Breidt, Eduardo Medina, Doria Wafa, Ilenys P´erez-D´ıaz, Wendy Franco, Hsin-Yu Huang, Suzanne D. Johanningsmeier, and Jae Ho Kim Abstract: Commercial cucumber fermentations are typically carried out in 40000 L fermentation tanks. A secondary fermentation can occur after sugars are consumed that results in the formation of acetic, propionic, and butyric acids, concomitantly with the loss of lactic acid and an increase in pH. Spoilage fermentations can result in significant economic loss for industrial producers. The microbiota that result in spoilage remain incompletely defined. Previous studies have implicated yeasts, lactic acid bacteria, enterobacteriaceae, and Clostridia as having a role in spoilage fermentations. We report that Propionibacterium and Pectinatus isolates from cucumber fermentation spoilage converted lactic acid to propionic acid, increasing pH. The analysis of 16S rDNA cloning libraries confirmed and expanded the knowledge gained from previous studies using classical microbiological methods. Our data show that Gram-negative anaerobic bacteria supersede Gram-positive Fermincutes species after the pH rises from around 3.2 to pH 5, and propionic and butyric acids are produced. Characterization of the spoilage microbiota is an important first step in efforts to prevent cucumber fermentation spoilage. Keywords: pickled vegetables, Pectinatus, Propionibacteria, secondary cucumber fermentation, spoilage M: Food Microbiology Practical Application: An understanding of the microorganisms that cause commercial cucumber fermentation spoilage & Safety may aid in developing methods to prevent the spoilage from occurring. Introduction cucumbers fermented at 2.3% NaCl (Fleming and others 1989). Commercial cucumber fermentations are typically carried out In this fermentation tank, the initial lactic acid fermentation was in large 40000 L outdoor tanks (reviewed by Breidt and others completed within 2 wk, with 1.2% lactic acid formed (pH 3.6) 2007). The cucumbers are held under the surface of the brine and no detectable sugar remaining in the brine. The cucumbers by wooden headboards, and the brine surface is exposed to UV in the fermentation brine were microbiologically stable for more light from the sun, which helps to prevent yeast or mold growth. than 8 mo, with no change in pH or organic acid concentrations. Cucumber fermentations are typically done in a 6% NaCl brine, However, after 9 mo, a secondary fermentation occurred. Lactic and preservation is achieved by sugar utilization, pH reduction, acid was completely consumed, and acetic, propionic, and butyric and accumulation of lactic acid (typically, pH < 3.3 and approx- acids were produced. During this spoilage fermentation, the pH imately 1% lactic acid). However, spoilage-associated secondary rose above 4.6, resulting in a potential health hazard due to the fermentations have been observed in fermented cucumbers (Flem- possibility of Clostridium botulinum spore germination and growth. ing and others 1989; Kim and Breidt 2007; Johanningsmeier and C. butyricum was isolated from spoilage samples and the ability to McFeeters 2011; Franco and others 2012). convert lactic acid to butyric acid was demonstrated (Fleming and The first research report of spoilage in fermented cucumbers others 1989). However, the organisms responsible for production was from an anaerobic cucumber fermentation tank (4500 L), with of acetic and propionic acids were not identified. Spoilage fermentations have also been documented in con- ventional 6% NaCl cucumber fermentations (Franco and oth- MS 20121333 Submitted 9/27/2012, Accepted 1/2/2013. Authors Breidt, D´ıaz, and Johanningsmeier are with USDA-ARS, SAA Food Science Research Unit, 322 ers 2012). These fermentations were aerated during the primary Schaub Hall, Box 7624, North Carolina State Univ., Raleigh, NC 27695-7624, fermentation to prevent the buildup of CO2, which can cause de- U.S.A. Author Medina is with Inst. de la Grasa (Consejo Superior de Investigaciones structive gas pockets (bloating) that form within the fruit during Cientificas, CSIC) Ave. Padre Garcia Tejero 4, Seville 41042, Spain; and Dept. the primary fermentation. To prevent bloating, commercial fer- of Food, Bioprocessing and Nutrition Sciences, North Carolina State Univ., Raleigh, NC 27698-7624, U.S.A. Author Wafa is with Dept. of Food, Bioprocessing and mentation tanks are typically purged with compressed air using a Nutrition Sciences, North Carolina State Univ., Raleigh, NC 27698-7624, U.S.A. sparging system that circulates the brine (Humphries and Fleming and Dept. of Chemical Engineering and Bioprocess, Pontificia Univ. Catolica de Chile, 1988). The microbiota responsible for these spoilage fermenta- Santiago, Chile. Author Huang is with Natl. Taiwan Univ., Taipei, Taiwan, Republic tions were identified by classical microbiological methods. Isolates of China. Author Kim is with Dept. of Biology, Kyungsung Univ., Busan 608-736, from spoilage fermentations included: Pichia and Issatchenkia yeast South Korea. Direct inquiries to author Breidt (E-mail: [email protected]). species, as well as 3 Gram-positive bacteria: Lactobacillus buchneri, Mention of a trademark or proprietary product does not constitute a guarantee Clostridium sp., and Pediococcus ethanolidurans; and 1 Gram-negative or warranty of the product by the U. S. Dept. of Agriculture or North Carolina Agricultural Research Service, nor does it imply approval to the exclusion of bacterium Enterobacter cloacae (Franco and others 2012; Franco and other products that may be suitable. Perez-Diaz 2012). Previously, it has been shown that rumen and R Journal of Food Science C 2013 Institute of Food Technologists No claim to original US government works r M470 Journal of Food Science Vol. 78, Nr. 3, 2013 doi: 10.1111/1750-3841.12057 Further reproduction without permission is prohibited Cucumber fermentation spoilage . silage lactic acid bacteria can degrade lactic acid. Rumen mi- ratio of cucumbers and brine. The initial cover brine contained crobiota have been shown to convert lactic acid to propionic acid 60 mM calcium acetate (prior to equilibration with the cucum- (Baldwin and others 1962). Similarly, Lactobacillus buchneri has been bers). L. plantarum MU45 (malolactic decarbolylase mutant, USDA shown to convert lactic acid to acetic acid and 1,2-propanediol un- Food Fermentation Culture collection, Raleigh, N.C., U.S.A.) der acidic conditions in silage (Driehuis and others 1999; Holzer was grown for 15 h at 30 ˚C in De Man, Rogosa and Sharp and others 2003), fermented cucumbers (Johanningsmeier and (MRS) broth (BD Diagnostic Systems, Sparks, Md., U.S.A.). The others 2012), and microbiological media (Oude-Elferink and oth- cucumber fermentation was inoculated immediately after brin- ers 2001). Lindgren and others (1990)haveshownthatLactobacillus ing with 20 mL of the overnight culture, to give an approximate plantarum, in the absence of fermentable sugar, can metabolize L- initial inoculum of 1 × 106 CFU/mL in the brine. After 10 d lactic acid to acetic and formic acids. Other microbiota have been of fermentation in the pail sealed with a lid containing a gasket, implicated in the anaerobic conversion of lactic acid to other or- equal amounts of brine and cucumber pickles were removed and ganic acids. In olive fermentations, Propionibacterium species and blended into slurry and frozen in 350 mL (12 oz) jars at −20 ˚C. Clostridium species have been implicated as causative agents of Za- Prior to use, the slurry was thawed, NaCl was added to 2%, the patera spoilage, converting lactic acid to propionic and butyric pH was adjusted to 5.0 as indicated, and fermented slurry medium acids (Vaughn and Levin 1966; Fernandez and others 1995). (FSM) was autoclaved followed by refrigeration prior to use. A fer- Classical methods for microbial ecology may often identify only mented slurry broth (FSB) prepared by centrifugation at 10000 × a minority of the microbiota present in a given environment g in a Sorvall GSA rotor (Thermo Fisher Scientific Inc., Waltham, (Amann and others 1995; Hugenholtz and others 1998), indi- Mass., U.S.A.) for 10 min to remove particulate matter. The su- cating the need for culture-independent methods. One potential pernatant was then filtered through a 0.45 µm filter. Tryptic soy difficulty with nucleic-acid-based methods for the investigation agar (TSA) and MRS agar (BD Diagnostic) were used for isolating of the spoilage microbiota in fermented cucumbers is interfer- spoilage organisms. Plates were incubated anaerobically at 30 ˚C ence in sequencing or cloning due to the presence of cell-free for up to 4 d in an anaerobic chamber (Coy Laboratory Products, nucleic acids of Lactobacilli from the primary fermentation. Fol- Grass Lake, Mich., U.S.A.). lowing fermentation, viable Lactobacilli declineinnumbersbysev- eral orders of magnitude (Breidt and others 2007). It has been Biochemical analysis shown that propidium monoazide (PMA) pretreatment of sam- Spoilage samples were centrifuged at 13000 × g in a microcen- ples prior to DNA isolation can be used to selectively amplify trifuge to remove any residual particulate matter. The pH was de- DNA from living cells (Nocker and others 2006; Pan and Breidt termined with an IQ240 pH meter (IQ Scientific Instruments, San 2007). The objective of this study was to investigate the bacteria & Safety Diego, Calif., U.S.A.). High-performance liquid chromatography