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Research: 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 is a phe- , 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 to mainly originate from cell walls of unpleasant in terms of mouthfeel. Ropy plants, while 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 - Physico-chemical analysis bushes and can develop in improperly handled or stored maple sap (Morin et Each sample was analyzed for its al., 1993). (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 and cultured Dilution and plating of sap and on LB Miller 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 . 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. 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 (, and ) 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, , , arabi- to characterize the microbial profile of nose and 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 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. than saps. mesenteroides and isolate N belonged to Enterobacteriaceae family. They already Phylogenetic tree of bacterial have been reported for EPS produc- isolates tion and enhancing the viscosity of the Analysis of 16s rDNA sequences medium in which they were inoculated of the 18 isolates through BLASTn re- (Beech & Carr, 1977; Korkeala, Suortti vealed that 15 bacterial isolates were and Mäkelä, 1988; Anderson and Rog- Gammaproteobacteria with nine iso- ers, 1963). lates belonging to the genus Pseudo- Influence of exopolysaccharides monas and six isolates belonging to the producing bacteria inoculated in family of Enterobacteriaceae (Figure 1). maple sap concentrate Pseudomonas are psychrophilic micro- organisms and are usually present in To test growth conditions of the sap throughout the season. Pseudomo- selected bacterial isolates in maple nas genus is generally associated with syrup concentrate and properties of good quality sap or concentrate (Laga- corresponding syrup, isolates A, 2 and cé et al., 2004, Filteau et al., 2012). Bacte- N were inoculated at 106 CFU/ml in rial isolates A and 2 were identified as 8°Brix sap concentrate and incubated at Leuconostoc mesenteroides. 4°C, 15°C and an alternation of 23°C for

Table 1: Estimated economic impacts of ropy maple syrups production, Quebec (2008-2017) 12 Maple Syrup Digest Figure 1: Neighbor-joining phylogenetic tree based on partial 16S rRNA gene of 18 bacterial isolates retrieved from sap and concentrate samples of producers having ropy maple syrup issues. eight hours and 4°C for 16 hours, over The filtration step was therefore not three days for unaerated static fermen- fully effective to remove all microor- tations (Figure 2). ganisms initially found in the sap con- centrate. However, this contamination Higher viscosity was noticed at 15°C didn’t influence pH or viscosity values for Leuconostoc (isolates A and 2) com- at 23°C after three days. pared to higher temperature (23°C) after three days of incubation. At 15°C Properties of corresponding syrup and 23°C, the pH of the fermenting con- Following inoculation and fermenta- centrate dropped rapidly to about 5.7 tion with isolates A, 2 and N, concen- by three days of fermentation (except trates were evaporated into syrups at for isolate 2 at 15°C) whereas at 4°C, lab-scale. All syrups corresponding to the pH did not vary. The pH decrease concentrate fermentation at 4°C and is suspected to result from metabolic controls are similar regarding °Brix, microbial activity and corresponding string length and viscosity (Figure 3). organic acids production. This sharp Indeed, average viscosity of a syrup decrease is also correlated with bacte- without viscosity defect range from rial proliferation during fermentation. 120 to 160 cP. However, in two cases Indeed, all bacterial isolates counts in- (Leuconostoc incubated at 15°C) evapo- creased from 6 to about 8 log CFU/ml ration was interrupted due to the ex- at all growth conditions except for Leu- treme viscosity of the boiling solution conostoc 2 at 4°C. Control showed a mi- making the evaporation very difficult crobial contamination reaching 5.00 log forcing us to stop the evaporation. Fur- CFU/ml after three days of incubation. Ropy:continued on page 15

December 2018 13 Figure 2: Aerobic mesophilic bacteria count profiles (A), pH (B) and viscosity (C) of ster- ile concentrate inoculated at 106 UFC/ml with three different bacterial isolates, incubated at three different temperatures over three days. Ropy: continued from page 13 4 and 6 cm long when incubated at 15 thermore, syrup corresponding to fer- and 23°C respectively, making it con- mentation with Leuconostoc A at 23°C form to current regulations and mar- was too viscous to show a measure by ketable. the viscometer in spite of its complete Those results suggest that factors evaporation to 66°B. Fermentation with promoting ropy slime formation in L. mesenteroides isolates produced ropy syrup are mainly uncontrolled fermen- syrup with strings up to 30 cm long at tation of Leuconostoc species at higher 15°C and 23°C. Only Enterobacteriaceae N gave syrups with shorter strings with Ropy: continued on page 16

December 2018 15 Figure 3: Degree Brix Profiles (A), string length (B) and viscosity (C) of maple syrup pro- duced after inoculating sterile maple sap concentrate at 106 UFC/ml with three different bacterial isolates followed by incubation at three different temperatures over three days.

Ropy: continued from page 15 more, a slight increase of sap viscosity temperature (15 to 23°C) in concen- can contribute to the production of a trate of 8°B. The slime production of L. highly ropy syrup. Various species in mesenteroides is well known (Bamforth, Enterobacteriaceae family are also EPS 2008; Strausbaugh & Gillen, 2008). producers such as Enterobacter agglom- They are ubiquitous and can produce erans which was reported to secrete EPS ropy slime with or without sucrose in concentrated maple sap or Aerobacter and they usually need small concentra- aerogenes when fermented in diluted tions of glucose as source maple syrup resulted in a production (Korkeala, Suortti and Mäkelä, 1988; of highly ropy syrup that could stretch Giglio & McCleskey, 1953). Further- up to 10 feet long (Morin et al., 1993,

16 Maple Syrup Digest Figure 4: Monosaccharide composition of purified polysaccharides from 3 ropy maple syrup samples.

Fabian & Buskirk, 1935). Storage of sap microorganisms. or concentrate at a lower temperature The analysis of monomeric such as 4°C can be a good way to pre- after of PS mix purified from vent the growth of slimy bacteria and each ropy maple syrup sample showed the production of ropy syrup. that glucose was present in each sam- Monosaccharide composition of ple, with the highest percentage ob- ropy maple syrups served in sample #2 with 98.2%, while it represented 60.0% and 32.5% in sample The (PS) compo- #1 and #3 respectively (Figure 4). This sition of three ropy maple syrups suggests that polysaccharides of glu- samples retrieved from producers cose such as dextrans are largely pres- was carried out and each sample con- ent in ropy syrup. The latter had been tained several PS of different molecular reported to be synthetized by weights (results not shown). Sample #1 bacteria (LAB) such as L. mesenteroides showed seven chromatographic peaks in sucrose-based medium and in maple with molecular weights ranging from syrup (Han et al., 2014; Roberts, 1982; <1000 Da up to more than 800000 Da, Storz, Darvill and Albersheim, 1986). suggesting that seven different PS were Dextran was possibly the main PS pres- present in this syrup sample. Sample ent in sample #2. Other potential PS #2 and #3 showed four and eight chro- present in sample #1 could potentially matographic peaks respectively. This be and arabinogalactans due analysis showed that a large variety of to the presence of galactose and arabi- PS were present in ropy syrup and each of them could be produced by different Ropy: continued on page 18

December 2018 17 Ropy: continued from page 17 in the food industry as texture modifi- nose. Arabinogalactan was previously ers or thickeners such as dextran. Such reported in maple syrup (Adams & an approach could possibly allow the Bishop, 1960; Lamport, 1977) and Dar- valorization of this type of non-compli- vill et al. (1980) explained that PS isolat- ant maple syrup. ed from primary cell walls of plants are similar to the composition of arabino- present in maple syrup. This ACKNOWLEDGEMENTS leads us to suspect that some (presum- We thank the maple advisors of the ably a low portion) PS slimes isolated Ministère de l’agriculture, des pêcheries et from ropy syrup can be originated from de l’alimentation du Québec for their help the maple tree. Sample #3 could include on sample collection and the Fédération arabinoglucans, rhamnoglucans, and de producteurs acéricoles du Québec for dextrans due to the presence of glucose, providing industry statistics. rhamnose and . REFERENCES CONCLUSION Adams, G. A., & Bishop, C. T. (1960). Slimy PS isolated from maple syrup Constitution of an arabinogalactan may be composed of various chemi- from maple sap. Canadian Journal of cal structures. Some are EPS produced Chemistry, 38(12), 2380-2386. by various bacteria naturally found in the vicinity of the sugarbush such as Anderson, E. S., & Rogers, A. H. (1963). L. mesenteroides or Enterobacteriaceae, Slime polysaccharides of the Entero- others could possibly derive from the bacteriaceae. Nature, 198(4881), 714. tree cells. Further chemical analysis Bamforth, C. W. (2008). Food, fermenta- on each PS purified would permit to tion and micro-organisms. John Wiley confirm their identification and char- & Sons. acterization of slime produced by L. mesenteroides and Enterobacteriaceae iso- Beech, F. W., & Carr, J. G. (1977). Al- lates and could provide information on coholic beverages. London: AH Rose, the specific production of EPS by each 185. isolate. Otherwise, the probability to Britten, M., & Morin, A. (1995). Func- produce a ropy maple syrup increases tional characterization of the exo- with the increase of temperature and polysaccharide from Enterobacter storage time. A proper handling of sap agglomerans grown on low-grade and concentrate is essential, especially maple sap. LWT-Food Science and when temperature rises as the sugar Technology, 28(3), 264-271. season progresses. Controlled storage temperature and cleanliness of sap col- Conseil des Productions Végétales du lection and storage equipment will help Québec. (1984). Le sirop d’érable prevent ropy syrup production. filant. Gouvernement du Québec - Ministère de l’Agriculture, des Our research hypothesis was that Pêcheries et de l’Alimentation ; 1984 the production of EPS by bacteria in the ; AGDEX 300/80. (Érablière). sap was responsible for the highly vis- cous texture of the syrup. Some EPS are Darvill, A. L. A. N., McNeil, M., Al- known and used in other applications bersheim, P., & Delmer, D. P. (1980).

18 Maple Syrup Digest The primary cell walls of flowering (rDNA) restriction analysis and plants. The of plants, 1, rDNA sequencing. Applied and envi- 91-162. ronmental microbiology, 70(4), 2052- 2060. Edson, H. A., Jones, C. H., and Carpen- ter, C. W. (1912). Micro-organisms of Lamport, D. T. A. (1977). Recent Advanc- Maple Sap. Vermont Agricultural Ex- es in Phytochemistry. by FA Loewus periment Station. Bull. 167. and VC Runeckles, Plenum Press, New York, 79-115. Fabian, F. W., & Buskirk, H. H. (1935). Aerobacter aerogenes as a cause of Morin, A., Moresoli, C., Rodrigue, N., ropiness in maple sirup. Industrial & Dumont, J., Racine, M., & Poitras, Engineering Chemistry, 27(3), 349-350. E. (1993). Effect of carbon, nitrogen, and agitation on exopolysaccharide Filteau, M., Lagacé, L., LaPointe, G., & production by Enterobacter agglomer- Roy, D. (2012). Maple sap predomi- ans grown on low-grade maple sap. nant microbial contaminants are cor- Enzyme and microbial technology, 15(6), related with the physicochemical and 500-507. sensorial properties of maple syrup. International journal of food microbiol- Quebec, Règlement des producteurs ogy, 154(1-2), 30-36. acéricoles sur les normes de qualité et le classement, chapitre M-35.1, r. Giglio, D. M., & McCleskey, C. S. (1953). 18, a.17. The fermentation of sucrose by Leuco- nostoc mesenteroides. Journal of bacteri- Storz, G., Darvill, A. G., & Albersheim, ology, 65(1), 75. P. (1986). Characterization of poly- saccharides isolated from maple syr- Han, J., Hang, F., Guo, B., Liu, Z., You, up. Phytochemistry, 25(2), 437-441. C., & Wu, Z. (2014). Dextran synthe- sized by Leuconostoc mesenteroides Strausbaugh, C. A., & Gillen, A. M. BD1710 in tomato juice supplement- (2008). Bacteria and yeast associated ed with sucrose. Carbohydrate poly- with root rot at harvest in mers, 112, 556-562. the Intermountain West. Plant disease, 92(3), 357-363. Holmes, D. S., & Quigley, M. (1981). A rapid boiling method for the prepara- Sun, J., Ma, H., Seeram, N.P., Rowley, tion of bacterial plasmids. Analytical D.C. 2016. Detection of , a pre- biochemistry, 114(1), 193-197. biotic polysaccharide, in maple syr- up. J. Agric. Food. Chem, 64, 7142-7147. Korkeala, H., Suortti, T., & Mäkelä, P. (1988). Ropy slime formation in van Geel-Schutten, G. H., Flesch, F., Ten vacuum-packed cooked meat prod- Brink, B., Smith, M. R., & Dijkhuizen, ucts caused by homofermentative L. (1998). Screening and characteriza- lactobacilli and a Leuconostoc species. tion of Lactobacillus strains produc- International Journal of Food Microbiol- ing large amounts of exopolysac- ogy, 7(4), 339-347. charides. Applied Microbiology and Biotechnology, 50(6), 697-703. Lagacé, L., Pitre, M., Jacques, M., & Roy, D. (2004). Identification of the bacterial community of maple sap by using amplified ribosomal DNA

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