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Chemical and Microbial Characterization of Ropy Maple Sap

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Chemical and Microbial Characterization of Ropy Maple Sap Research: Syrup production Chemical and microbial characterization of ropy maple sap and syrup Luc Lagacé, Mariane Camara, Simon Leclerc, Carmen Charron, Mustapha Sadiki Centre de recherche, de développement et de transfert technologique acéricole Inc. (Centre ACER) opiness of maple syrup is a phe- dextrans, arabinogalactans and rham- nomenon that can occur several nogalacturonans (Sun et al., 2016; Storz, Rtimes in the season. The altera- Darvill and Albersheim, 1986; Adams & tion known as “ropiness” is character- Bishop, 1960) were previously reported ized by a viscous, thick, slimy/jelly-like in maple syrup. Arabinogalactans and texture which, although not noticeably rhamnogalacturonans were suspected altering the taste, renders the product to mainly originate from cell walls of unpleasant in terms of mouthfeel. Ropy plants, while dextran was presumed maple syrup is unsaleable according to to result from bacterial contamination Quebec’s current regulation, causing it of sap (Storz, Darvill and Albersheim, to be discarded and leading to a sub- 1986). stantial loss for the industry (Quebec, The aim of this study was to estimate M-35.1, r. 18, a.17). Year after year, this the economic impact of production of type of defective syrup is produced to ropy maple syrup in the region of Que- varying extent. A syrup is graded ropy bec, to more deeply identify and char- when the length of the string is equal acterize bacteria associated to this type or above 10 cm (http://www.centreacer. of quality defect, and to study the com- qc.ca/Service/document-formulaire). position of PS found in stringy maple It is automatically graded as improper syrup. and must be destroyed. Ropy maple syrup is generally MATERIAL AND METHODS caused by fermentation of bacteria Sampling present in sap (Fabian and Buskirk, A total of 25 samples were obtained 1935). These bacteria possess the ability in 2011, including 15 ropy maple syr- to produce exopolysaccharides (EPS) in ups, six concentrates and four saps, maple sap resulting in a stringy maple from several producers in different syrup after concentration. Several bac- regions of Québec. It should be noted teria were found to contribute to the that sampling sap corresponding to development of stringiness in concen- ropy maple syrup was not always pos- trated maple sap such as Aerobacter sible since ropiness cannot always be aerogenes, Bacillus ,aceris or Enterobacter predicted from sap or concentrate. agglomerans (Fabian and Buskirk, 1935; Samples were then stored at -20°C until Edson and Jones, 1912; Britten and further analysis. Morin, 1995). These bacteria are usu- ally found in the environment of sugar- Physico-chemical analysis bushes and can develop in improperly handled or stored maple sap (Morin et Each sample was analyzed for its al., 1993). Polysaccharides (PS) such as Ropy continued on page 10 December 2018 9 Ropy: continued from page 9 Germany) and carried out in a TGradi- soluble solids (°Brix), pH and viscos- ent PCR thermocycler (Biometra, Goet- ity. Light transmittance (at 560 nm) was tingen, Germany) according to a pub- measured in syrup samples. Ropiness lished protocol (Lagacé et al., 2004). was measured in each syrup sample by Cloning and plasmidic DNA measuring the string’s length by dip- extraction ping a spatula into the ropy syrup. Amplified PCR products were ligat- Microbial counts and culture ed into the pCR2.1-TOPO plasmid, in- isolation serted in Escherichia coli and cultured Dilution and plating of sap and on LB Miller agar for 24 hours at 37°C. concentrate samples were performed Prior to sequencing, plasmids were to provide total bacterial mesophilic extracted according to the method de- counts in aerobic and anaerobic condi- scribed by Holmes and Quigley (1981) tions, and total bacterial psychrophilic and digested with EcoRI enzyme. DNA aerobic counts. Specific growth media fragments were then sequenced and were prepared to obtain counts of mi- partial sequences were obtained. Con- croorganisms of the genus Pseudomo- sensus sequences of 1500 bp were re- nas and total yeast and mold counts. trieved with InfoQuestTMFP software The viable cell counts were expressed and identified by comparison to data- in terms of log of colony forming unit base of DNA sequences with BLASTn per millimeter (log CFU/ml). Eighteen (NCBI). different colonies with distinctive mor- Identification of microorganisms phologies were purified with three sub- causing stringy maple syrup cultures in their corresponding growth medium before storage at -80°C in Each of the 18 bacterial isolates were tryptic soy broth (Difco, NJ, USA) until inoculated in an 8°Brix maple sap con- DNA extraction. centrate previously filtered through a membrane (0.22 µm) to remove micro- Total DNA extraction bial biomass. Identification of microor- The DNA of bacterial isolates was ganisms responsible for stringy maple extracted with the Nucleospin® Tis- syrup was performed with inoculation sue extraction kit (Marcherey-Nagel, of each isolate at 106 CFU/ml in sterile Düren, Germany). Concentrations of 8°Brix concentrate, incubated at 15°C purified DNA were measured using for two days followed by 4°C for four ND-1000 spectrophotometer (Nano- days. Resulting fermented media were drop Technologies, USA). evaluated for ropy properties and cor- responding bacterial isolates were se- PCR Amplification lected for growth conditions and asso- Amplified 16S rRNA gene was ob- ciated syrup characteristics evaluation. tained from each isolates by PCR, Fermentation of concentrated maple by using the universal primers F27 sap by slimy bacterial isolates and (5’-AGAGTTTGATCMTGGCTCAG-3’) syrup production and R1492 (5’-GGYTACCTTGTTAC- GACTT-3’) (Invitrogen, Carlsbad, CA). Fermentations were done by inocu- PCR amplification was achieved with lating selected bacterial isolates at 106 Taq PCR Core kit (QIAgen, Hilden, CFU/ml in an 8°Brix maple sap con- 10 Maple Syrup Digest centrate previously filtered through a RESULTS AND DISCUSSION membrane (0.22 µm). Three incubation conditions were selected: 4°C, 15°C Economic impact and alternating incubations of 23°C for Ropy maple syrup volumes may eight hours followed by 4°C during 16 vary from year to year. For the last ten hours for three days. Syrups were sub- years, a maximum was reached in 2014 sequently produced and physicochemi- with 358,607 lbs. of bulk ropy syrups cal analysis were performed. produced (Table 1). It is important to mention that the amount presented is Polysaccharides purification and underestimated since producers tend characterization to destroy ropy maple syrups them- Three out of the 15 ropy syrup sam- selves when they are detected after ples were selected for polysaccharides evaporation. However, in 2014, more characterization based on the difference than $1 million (CAD) was lost without between total soluble solids measured including those barrels discarded by by the refractometer and total sugar producers. In total, an estimated more (sucrose, glucose and fructose) content than $5.5 million was lost in production quantified by high-performance liquid of ropy maple syrup since 2008. chromatography (HPLC). The greater Over the last ten years, the highest the difference, the more polysaccha- proportion of ropy syrups has been rides were suspected to be present in classified as dark color syrup, repre- syrup. senting 39.77% of total ropy maple Polysaccharides of each syrup sam- syrups (Results not shown). This is ples were purified using HPLC (Waters, followed by amber and medium syr- Milford, MA, USA). Molecular weights ups representing 25.20% and 17.57% of polysaccharides were estimated by respectively, while extra-light and HPLC with a TSK-GEL 4000PWXL col- light ropy syrups represented less than umn (Waters, Milford, MA, USA) heat- 10%. Therefore, the darker the syrup, ed at 40°C and using ultrapure water as the higher the probability of ropiness the eluent at a flow rate of 0.5 ml/min. occurrence. Ropy syrup production is an important issue that needs to be ad- Polysaccharides were then hydro- dressed. lyzed by adding trifluoroacetic acid (TFA) 2N and monomeric saccharides Microbial counts were identified by GC Agilent 6890 Sap and concentrate samples re- equipped with an MS 5973 as detec- trieved from sugarbushes producing tor. Reference solutions of glucose, ropy syrups were diluted and plated fructose, mannose, rhamnose, arabi- to characterize the microbial profile of nose and galactose were as well used each sap and concentrate samples (Re- to confirm the peak identification. Re- sults not shown). Aerobic plate count sults were presented as area percentage ranged from 6.12 to 8.49 log CFU/ out of total areas of identified peaks to ml. For most samples, psychrotrophic demonstrate relative monosaccharide counts were higher than Pseudomonas proportions. Peaks were identified us- counts and varied between 6.14-8.41 ing the NIST database (2008) as well log CFU/ml and 5.77-7.63 log CFU/ml as reference solutions of monosaccha- rides. Ropy continued on page 12 December 2018 11 Ropy: continued from page 11 Identification of exopolysaccharides respectively. Other psychrotrophic spe- producing isolates cies are therefore suspected to be pres- The ability of the isolates to produce ent in sap/concentrate samples. Anaer- ropy slime was tested by inoculation obic plate counts were lower or equal at 106 CFU/ml in filtered 8°B concen- to aerobic plate counts and ranged trate (0.22 µm) and incubation at 15°C from 5.05 to 8.56 log CFU/ml. Yeast and for two days followed by 4°C for four mold counts ranged from 2.22 to 5.05 days. The viscosity of resulting concen- log CFU/ml. trates was monitored. Three isolates Globally, samples showed a high (A, 2 and N) were able to enhance the total bacterial load, with concentrates viscosity of the concentrate. Isolates A containing higher aerobic plate counts and 2 were previously identified as L.
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