Identification of Microbial Spoilage in Maple Syrup Samples Kathryn M. Hopkins*1, Beth L. Calder, Ph. D2, Wyatt Marshall3, Vitoon Lim3, Rafael Garcia3, and Seanna L. Annis3, Ph.D.

Introduction and Project Goals

A number of maple syrup samples containing floating masses or surface mold have been identified. Formerly, consumers were advised to skim the mold, reboil and consume. Examination of the samples has been conducted to determine if the floating masses (“mother”) in syrup are fungal in origin, if they are a food safety hazard to consumers and if Brix levels are related to microbial growth.

Contaminated syrup samples were collected and transported to Dr. Seanna Annis’ laboratory at the University of Maine. Floating mass samples were sampled under aseptic conditions and samples were streaked onto Sabouraud Agar plates. Plates were incubated at 20ºC and individual colonies were isolated by transfer onto new plates of the same agar. Colonies were observed under a microscope and identified to genus. Syrup samples were also analyzed in the Department of Food Science & Human Nutrition for pH, and Brix (% soluble solids) levels with and without centrifugation at 10,000 g for 10 min.

DNA was extracted from each isolate using 5x107 spores and the MoBio PowerPlant extraction kit (MoBio) with 0.5mm zirconia silica beads. DNA was tested for quality and then the internal transcribed spacer regions in the ribosomal RNA gene cassette was amplified using the polymerase chain reaction with universal fungal primers ITS1 and ITS4.

The effect of temperature on survival of spores in

maple syrup was tested on one isolate of Aspergillus and one isolate of Pencillium.

1University of Maine Cooperative Extension, Extension Educator, Somerset County, 7 County Drive, Skowhegan, ME 04976-4209 2University of Maine Cooperative Extension, Food Science Specialist, Dept. of Food Science & Human Nutrition, 5735 Hitchner Hall, Orono, ME 04469-5735 3University of Maine School of Biology and Ecology, Mycologist, 5735 Hitchner Hall, Orono, ME 04469-5735

Funding for parts of this project was provided by the North American Maple Syrup Council

Strains of Organisms Found

Figure 2. Penicillium Figure 3. Aspergillus

Abstract An increasing number of maple syrup samples containing floating masses or surface mold have arrived at the University of Maine Cooperative Extension. These samples originated from New Hampshire, Maine, Minnesota, Rhode Island, Vermont in the USA and New Brunswick and Quebec in Canada. Conventional practices have been to discard obvious mold growths, reboil and consume the syrup. This practice may be risky, especially with the increasing number of food borne illness outbreaks with other food products. To determine if floating masses or “mother” from contaminated syrup samples are fungal in origin and to determine the Brix and pH ranges, maple syrup samples have been analyzed. Fungal species have been detected in samples, which include Penicillium, Wallemia, Aspergillus, and yeasts. The pH levels ranged from 3.8 to 7.32. Brix values were mostly above 66 degrees Brix. Research indicates these fungal species are slow growing in syrup, but can persist in processed maple syrup at levels above 66 Brix, which was not previously thought to be possible. Future research should continue fungal identification of contaminated syrup samples, conduct DNA sequencing to identify if these fungal species are ones known to produce mycotoxins, and determine if mycotoxins can be produced in contaminated maple syrup.

•  Twenty-nine samples have been analyzed from 2009 to 2011 •  pH levels ranged from 3.8 to 7.32 (Mean: 6.47) •  Brix (% Soluble Solids) centrifuged ranged from 630 to 680 (Mean: 65.80) •  12 of 29 samples had Brix levels <660, but there were no differences in the type or abundance of isolates and the Brix level. •  Wallemia isolates were only found in syrup samples with pH above 6.9. There was no difference in pH preference for the other fungi. •  Preliminary experiments found spores of the Aspergillus isolate survived 75°C and were killed at 82°C. The spores of the Penicillium isolate survived 65°C but not 70°C. Temperature sensitivity of other isolates will be tested.

Results

•  Fungi can persist in maple syrup at Brix levels >660 despite boiling and a sugar concentration thought to prevent fungal growth. •  Fungi can produce mycotoxins that are safety risks such as aflatoxins in grains and patulin in apple cider. •  Wallemia, Aspergillus & Penicillium have the potential to produce mycotoxins and it is not known if the isolated species do produce toxins in maple syrup. •  Further research (DNA sequencing) needs to be conducted to determine food safety implications.

Our results indicate that skimming off mold and re-boiling syrup should no longer be recommended. Fungi

isolated from our samples are from genuses that can produce mycotoxins. The best advice at this time is:

When in doubt, throw it out!

Methods

Summary and Food Safety Implications

Data Discussion

Table 1. Data Summary

Figure 1. Syrup samples with floating masses

Figure 4. Wallemia

Fungus Range of pH# of Samples found

% of Samples found (29 samples processed to date)

Wallemia 6.98 -7.3 6 20.7Pink Yeast 6.18-6.32 2 6.9White yeast 5.02-6.49 5 17.2Penicillum 3.8-7.3 8 27.6Aspergillus 5.02-7.32 13 44.8Cladosporium 5.02 1 3.4Unknowns 3.8-6.54 3 10.3