Maple sap predominant microbial contaminants are correlated with the physicochemical and sensorial properties of maple syrup

Marie Filteau a, Luc Lagacé b, Gisèle LaPointe a, Denis Roy a,
a Institut des nutraceutiques et aliments fonctionnels (INAF), Département des Sciences des aliments et de nutrition, Université Laval, QC, Canada G1V 0A6
b Centre de recherche, de développement et de transfert technologique acéricole inc. (ACER) St-Norbert d'Arthabaska, QC, Canada G0P 1B0

Source : International Journal of Food Microbiology 154 (2012) 30–36
www.elsevier.com/locate/ijfoodmicro avec mots clés: Microbial ecology, Quantitative PCR, Multiple factor analysis, Pseudomonas fluorescens, Mrakia
 

Abstract
Maple sap processing and microbial contamination are significant aspects that affect maple syrup quality. In this study, two sample sets from 2005 and 2008 were used to assess the maple syrup quality variation and its relationship to microbial populations, with respect to processing, production site and harvesting period. The abundance of maple sap predominant bacteria (Pseudomonas fluorescens group and two subgroups, Rahnella spp., Janthinobacterium spp., Leuconostoc mesenteroides) and yeast (Mrakia spp., Mrakiella spp.,Guehomyces pullulans) was assessed by quantitative PCR. Maple syrup properties were analyzed by physicochemical and sensorial methods. Results indicate that P. fluorescens, Mrakia spp., Mrakiella spp. G. pullulans and Rahnella spp. are stable contaminants of maple sap, as they were found for every production site throughout the flow period. Multiple factor analysis reports a link between the relative abundance of P. fluorescens group and Mrakia spp. in maple sap with maple and vanilla odor as well as flavor of maple syrup. This evidence supports the contribution of these microorganisms or a consortium of predominant microbial contaminants to the characteristic properties of maple syrup.

Évaluation de l’alcool isopropylique pour l’assainissement du système de collecte de la sève d’érable

712 Ko

RÉSUMÉ
Des essais ont été réalisés en 2009 et 2010 dans 6 érablières afin d’évaluer l’efficacité de l’alcool isopropylique à 70% utilisée par aspiration pour l’assainissement du système de collecte de la sève d’érable comparativement à l’hypochlorite de sodium (600 ppm) utilisé par refoulement. Les résultats obtenus par ATP bioluminescence ont montré une diminution significative du niveau de contamination des surfaces internes des réseaux de collecte après une période d’attente de quelques mois suite au traitement à l’alcool isopropylique, comme le spécifie la procédure proposée. Cette diminution a été observée dans tous les systèmes évalués et pour tous les types de surfaces mesurées (collecteurs, latéraux, chute et chalumeaux). L’efficacité du traitement à l’hypochlorite de sodium par contre a seulement été observée après une courte période suivant le traitement (2 jours). Après une période d’attente de quelques mois, les systèmes traités à l’hypochlorite de sodium avaient des niveaux de contamination presque comparables à ceux avant les traitements contrairement à ce qui a été observé pour l’alcool isopropylique. La méthode d’assainissement à l’alcool isopropylique 70% est donc un moyen efficace de gestion de la salubrité des systèmes de collecte de la sève d’érable. La poursuite des travaux précisera si cette pratique offre des avantages sur la qualité des produits acéricoles et sur la productivité à l’entaille des systèmes de collecte. Étant donné que ce produit est inflammable, il doit donc être utilisé et entreposé selon les normes de sécurité prescrites.

ABSTRACT
Tests were performed in 2009 and 2010 in 6 maple sugarbushes to evaluate the performance of isopropyl alcohol 70% used with vacuum for the maple sap collection system sanitation in comparison with the method using a bleach solution at 600 ppm pushed with air in the tubing system. Results obtained by ATP bioluminescence showed a significant decrease in internal surface contamination of the sap collection system using isopropyl alcohol and after a few months following the treatment as it is suggested by the procedure. The decrease in contamination was observed for all systems evaluated and all types of surfaces tested (main lines, lateral lines, drop lines and spouts). The performance of the bleach solution however was only observed following a short period of time (2 days) after the treatment. After a longer period (few months), systems treated with bleach solutions had levels of contamination almost comparable to the ones before treatment in opposition to what was found for isopropyl alcohol. Sanitation with isopropyl alcohol 70% is therefore an efficient way to maintain the sap collection system clean. Further work will be pursued in order to evaluate if this practice has an impact on the quality of maple products and on sap yield. As a reminder, isopropyl alcohol is a flammable product that has to be used and stored according to proper safety standards applicable in your jurisdiction.

Correlation of maple sap composition with bacterial and fungal communities determined by multiplex automated ribosomal intergenic spacer analysis (MARISA)
Marie Filteau a, Luc Lagacé b, Gisèle LaPointe a, Denis Roy a,*  Source : Food Microbiology

a Institut des nutraceutiques et aliments fonctionnels (INAF), Département des Sciences des aliments et de nutrition, Université Laval, 2440, Boul. Hochelaga, QC G1 V 0A6, Canada
b Centre de recherche, de développement et de transfert technologique acéricole inc. (ACER), St-Norbert d’Arthabaska, QC G0P 1B0, Canada

ABSTRACT
During collection, maple sap is contaminated by bacteria and fungi that subsequently colonize the tubing system. The bacterial microbiota has been more characterized than the fungal microbiota, but the impact of both components on maple sap quality remains unclear. This study focused on identifying bacterial and fungal members of maple sap and correlating microbiota composition with maple sap properties. A multiplex automated ribosomal intergenic spacer analysis (MARISA) method was developed to presumptively identify bacterial and fungal members of maple sap samples collected from 19 production sites during the tapping period. Results indicate that the fungal community of maple sap is mainly composed of yeast related to Mrakia sp., Mrakiella sp., Guehomyces pullulans, Cryptococcus victoriae and Williopsis saturnus. Mrakia, Mrakiella and Guehomyces peaks were identified in samples of all production sites and can be considered dominant and stable members of the fungal microbiota of maple sap. A multivariate analysis based on MARISA profiles and maple sap chemical composition data showed correlations between Candida sake, Janthinobacterium lividum, Williopsis sp., Leuconostoc mesenteroides, Mrakia sp., Rhodococcus sp., Pseudomonas tolaasii, G. pullulans and maple sap composition at different flow periods. This study provides new insights on the relationship between microbial community and maple sap quality.

Effet de matériaux de récolte neufs et d’un chalumeau anti-retour sur la contamination de l’entaille et le rendement en coulée de la sève d’érable

323Ko

RÉSUMÉ
Ce projet réalisé pour CDL visait à évaluer la performance de différents systèmes sur le rendement en coulée et la contamination microbienne de la sève d’érable à l’entaille. Un 1er dispositif étudiait la performance sous vide intermittent d’un système équipé de chalumeaux neufs et chutes neuves comparativement à un système installé en parallèle équipé de chalumeaux usagés et de chutes usagées et dont les entailles étaient appariées sur les mêmes arbres (200). Les résultats obtenus ont montré un rendement supérieur (17%) pour le système muni de matériaux neufs surtout attribuable à la fin de saison. La charge microbienne à l’entaille en fin de saison était faible pour les 2 systèmes mais tout de même moindre pour le système équipé de matériaux neufs. Un 2ème dispositif évaluait sous vide intermittent, la performance d’un système équipé de chalumeaux neufs et de valves anti-retour neuves comparativement à un système équipé de chalumeaux neufs sans valves anti-retour installé en parallèle et dont les entailles étaient appariées sur les mêmes arbres (200). Dans les conditions particulières de cette étude, les résultats ont montré des rendements comparables pour les 2 systèmes contrairement à ce qui a été avancé par les études antérieures sur les valves anti-retour. La contamination microbienne à l’entaille faible pour les 2 systèmes mais tout de même moindre pour le système avec valve anti-retour n’a pas donné de gain notable sur le rendement. Ces résultats suggèrent que la contamination microbienne de l’entaille ne serait pas le seul facteur limitant du rendement en coulée de la sève d’érable et que les chalumeaux à valve anti-retour ne seraient pas efficaces en toutes circonstances.

ABSTRACT
This research sponsored by CDL evaluated the performance of different systems related to maple sap yield and microbial contamination of tapholes. A first experiment made under vacuum (stopped at night) compared a system equipped with new spouts and droplines to another equivalent system in parallel equipped with old spouts and droplines (3 years old with 2 years in storage). Both systems had new main and lateral lines. Tapholes of these two systems were made pair wise on the same trees (200) that had never been tapped prior to the experiment. Results showed an increased sap yield of 17% for the system equipped with new material. This increase in sap yield was mostly observed during the last part of the sap flow season. Although microbial contamination of tapholes was relatively low for both systems at the end of the season, it was significantly lower for the system equipped with new material. A second experiment made under vacuum (stopped at night) compared a system equipped with new check valve adaptors to another equivalent system in parallel equipped with new spouts of the same diameter. Again, both systems had new main and lateral lines and tapholes of these two systems were made pair wise on the same trees (200) that had never been tapped prior to the experiment. In the specific conditions of this experiment, results showed that there was no significant difference in sap yield between systems, which is in contradiction with already published data on check valve adaptors. A relatively low microbial contamination of tapholes was found at the end of the season for both systems. Although the system equipped with check valve adaptors did not show any increase in microbial contamination of tapholes at the end of the season, it did not seemed to be enough to observe a positive effect on sap yield. These results suggest that microbial contamination of tapholes would not be the only limiting factor to increase sap yield and that check valve adaptors would not necessarily improve sap yield in all circumstances.

Détermination d’une méthodologie de lavage de la tubulure

543 Ko

Deux méthodes de lavage de la tubulure ont été comparées soit le lavage à l’eau et le lavage à l’hypochlorite de sodium. Ces lavages ont été effectués à l’érablière expérimentale à 50% et à 100% de la coulée. Les résultats ont permis d’observer l’évolution de la contamination durant toute la saison et ce en fonction de la nature des contaminants (bactéries totales, Pseudomonas, levures et moisissures). La flore contaminante était essentiellement constituée de bactéries du genre Pseudomonas. L’effet d’une coulée forte sur la charge microbienne est observé de façon évidente avec les suivis des populations. Cependant, la charge initiale de contamination étant élevée (environ 1x10⁷ UFC/ml), il a été difficile d’observer l’effet des méthodes de lavages proposées. Une légère baisse des populations a été observée après les traitements à l’eau et à l’hypochlorite de sodium sans toutefois déceler de différence entre les deux types de traitements. La présence de biofilm pourrait expliquer la reprise rapide de la contamination dans la tubulure. La formation du biofilm à la surface de la tubulure a été observée en microscopie électronique à balayage. Les photos au microscope électronique n’ont pas démontré que le biofilm s’installait de façon préférentielle sur la surface de la tubulure (bas versus haut du tube). Les effets des traitements de lavage n’ont également pas été démontrés grâce à la microscopie électronique. La poursuite des travaux sera consacrée à l’étude de différents produits de lavage et sur différents procédés pouvant limiter l’accumulation du biofilm à la surface de la tubulure.

Rapid Prediction of Maple Syrup Grade and Sensory Quality by Estimation of Microbial Quality of Maple Sap Using ATP Bioluminescence
(Source : IFT Institute of Food Technologists)

ATP bioluminescence was evaluated as a method for assessing the level of microbial contamination in sap and predicting maple syrup characteristics. This approach provided results that were strongly correlated with the standard plate count and took less time than the modified resazurin technique. ATP bioluminescence measurement of sap proved to be reliable for predicting physicochemical and sensory characteristics as indicated by the color and flavor of maple syrup. Most of the syrups made from saps with higher ATP bioluminescence values were darker in color and presented off-flavors. Based on these results, ATP bioluminescence could be used to improve sanitary practices associated with collecting and storing maple sap.

Identification of the Bacterial Community of Maple Sap by Using Amplified Ribosomal DNA (rDNA) Restriction Analysis and rDNA Sequencing
(Source : American Society for Microbiology )
 

The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons. Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified by 16S rRNA gene fragment sequencing. Results showed a wide variety of organisms, with 22 different genera encountered. Pseudomonas and Ralstonia, of the - and ß-Proteobacteria, respectively, were the most frequently encountered genera. Gram-positive bacteria were also observed, and Staphylococcus, Plantibacter, and Bacillus were the most highly represented genera. The sampling period corresponding to 50% of the cumulative sap flow percentage presented the greatest bacterial diversity according to its Shannon diversity index value (1.1). -Proteobacteria were found to be dominant almost from the beginning of the season to the end. These results are providing interesting insights on maple sap microflora that will be useful for further investigation related to microbial contamination and quality of maple products and also for guiding new strategies on taphole contamination control.

The bacterial microflora of maple sap and biofilms in collection system tubing were studied through the use of bacterial counts, scanning electron microscopy (SEM) of surfaces and the analysis of 16S rRNA gene by denaturing gradient gel electrophoresis (DGGE). Samples were taken at five times during the 2002 and 2003 seasons in order to follow the changes in the microflora of this complex ecosystem. Bacterial counts showed the growth of bacterial populations as the season advanced. These populations were mainly composed of psychrotrophic bacteria and Pseudomonas spp. SEM results confirmed the suspected presence of biofilms on the inner surfaces of tubing samples. Bacterial colonization and biofilm formation progressively increased during the season for both lateral and main line surfaces, and biofilms were mainly composed of rod shape bacteria. The bacterial microflora profiles obtained for sap and corresponding biofilm by DGGE showed up to 12 major bands. The Shannon-Weaver index of diversity (H) calculated from DGGE bands were statistically higher for sap samples compared to biofilm. The diversity index was relatively stable or increasing for lateral line sap and biofilm samples during the season while the diversity index for sap and biofilm samples of the main line showed a decreasing profile as the season progressed. Sequence analysis of major DGGE bands revealed the predominance of bacteria from the genera Pseudomonas, Rahnella and another, unidentified genus. The results describe the composition of sap collection system microflora as well as the formation of biofilms and will be useful for further studies on factors affecting maple product quality.

Biofilm formation and Biocides sensitivity of Pseudomonas marginalis isolated from a Maple Sap Collection System
L.Lagacé, M. Jacques, A.A. Mafu and D. Roy
(Journal of Food Protection, Vol. 69, No 10, 2006, Pages 2411-2416)

The susceptibility of planktonic and biofilm cells of Pseudomonas marginalis toward four commonly used biocides at different temperatures (15 and 30°C) and biofilm growth times (24 and 48 h) was assessed. Using the MBEC biofilm device, biofilm production in maple sap was shown to be highly reproducible for each set of conditions tested. Biofilm formation was influenced by growth temperature and time. A temperature of 15°C and incubation time of 24 h yielded fewer CFU per peg and showed fewer adhered cells and typical biofilm structures, based on scanning electron microscopy observations as compared with other conditions. Minimal biofilm eradication concentration values for P. marginalis were significantly greater (P < 0.001) than were MBCs for planktonic cells and for every biocide tested, with the exception of minimal biofilm eradication concentration values for peracetic acid at 15°C and 24 h. Sodium hypochlorite and peracetic acid sanitizers were able to eliminate P. marginalis biofilms at lower concentrations as compared with hydrogen peroxide- and quaternary ammonium-based sanitizers (P < 0.001). According to the results obtained, sodium hypochlorite and peracetic acid sanitizers would be more appropriate for maple sap collection system sanitation.

Évaluation des principaux facteurs associés à la détérioration microbienne du sirop d’érable en vrac
Luc Lagacé, Ph.D., Centre de recherche, de développement et de transfert technologique acéricole Inc. 3600 boul. Casavant Ouest, St-Hyacinthe, QC, Canada. J2S 8E3.

Résumé

L’importance de différentes variables sur la conservation du sirop d’érable en barils a été étudiée dans le cadre de ce projet. Premièrement, une comparaison de 4 types de barils et de 2 techniques d’échantillonnage (standard et Morneau) a été effectuée à partir de 9 lots de sirop et en utilisant des conditions rigoureusement contrôlées de mise en contenant. Les résultats obtenus ont démontré que la couleur du sirop a été influencée par le type de barils durant la période d’entreposage de 24 mois.  Les barils Barinox et Vulcan ont permis une meilleure conservation de la couleur du sirop comparativement aux barils Morneau et CDL (sac laminé d’aluminium). Les conditions contrôlées de mise en contenant ont permis d’obtenir un faible taux (2%) de détérioration microbienne du sirop durant l’entreposage. Cependant, l’échantillonnage à l’aide de la technique standard a fait augmenter considérablement ce taux (16,6%) notamment pour les barils Morneau et CDL.  L’échantillonnage à l’aide de l’échantillonneur Morneau a permis un taux réduit de détérioration de 3,7%.  Deuxièmement, une analyse a été effectuée de la population de 53206 barils reçus des producteurs après la récolte et entreposés environ pendant 6 mois avant d’être reclassés à l’automne 2004 en vue d’être pasteurisés. Cette analyse a permis d’évaluer l’importance de différentes variables sur l’apparition de la détérioration microbienne du sirop à l’entreposage. Les résultats révèlent notamment que les pratiques de mise en contenant sont considérablement importantes pour la conservation du sirop. Une concentration en solides solubles inférieure à 66°Brix ainsi qu’un taux de remplissage inférieur à 90% sont parmi les variables qui sont les plus fortement corrélées à la détérioration microbienne. De plus, une certaine catégorie de sirops venant d’entreprises de plus petite taille semblent plus fortement associés à la détérioration microbienne du sirop à l’entreposage. Finalement, le baril de plastique Morneau s’est révélé plus susceptible aux pratiques défavorables à la conservation du sirop faisant en sorte qu’il est associé à une proportion importante de barils présentant une détérioration microbienne (43%).  Ces résultats révèlent l’importance de contrôler les pratiques de mise en contenant à l’érablière ainsi que l’échantillonnage et l’entreposage afin de prévenir la dégradation de la qualité du sirop en vrac et d’éviter des pertes monétaires importantes pour l’industrie acéricole.

Mots clés : sirop, érable, barils, entreposage, conservation,  fermentation, saveur

Abstract

The importance of different variables on the stability during storage of bulk maple syrup was investigated in this project. First, 4 types of 32-gallon drums and 2 sampling techniques (standard and Morneau) were evaluated for 9 different lots of maple syrup produced in rigorously controlled packaging conditions. Results obtained demonstrate that the colour of maple syrup was influenced by the type of drums over the storage period of 24 months. Metal drums from Barinox and Vulcan showed a slightly better colour stability of maple syrup compared to Morneau and CDL (aluminium barrier bag) plastic drums. Under controlled packaging conditions, the rate of microbial spoilage of maple syrup during storage was low (2%). But after sampling of containers using the standard method, the rate of microbial spoilage considerably increased (16.6%) especially for the Morneau and CDL containers. Sampling with the Morneau device
permitted to control the microbial spoilage rate to 3.7%.  Second, an analysis was performed on the population of 53206 maple syrup drums that were received from Quebec producers following the 2004 season and kept in storage for approximately 6 months before being re-inspected and
pasteurized for long period storage. From this analysis, some variables showed to be highly related to microbial spoilage during the 6 month storage period. Results showed that the packaging operations performed by the maple syrup producers are critical to maple syrup stability during storage. A total solid concentration lower than 66°Brix and a percentage of drum filling lower than 90% were among the variables that most strongly correlated with microbial spoilage. Furthermore, a category of syrup coming from smaller sugarbushes was also highly related to microbial spoilage during storage. Finally, a certain type of drum (Morneau plastic
drum) showed to be highly susceptible to improper packaging operations and was related to a high proportion of syrups (43%) that had microbial spoilage.  These results reveal the importance of appropriate packaging operations at the sugarhouse and of good sampling practices for inspection to prevent microbial spoilage of bulk maple syrup during storage and avoid important economical loss for the maple syrup industry.

 Key words: maple, syrup, drums, storage, conservation, fermentation, flavour
 

Keywords: Maple sap contamination, Community PCR fingerprint method, Isolate typing,16S rRNA gene clone librairy, Community membership, Community structure, Operational taxonomic units, Pseudomonas, Rahnella