Introduction
Alcoholic beverage is any liquor containing more than 0.5% ethyl alcohol (ethanol) by volume/volume as the active agent. The manufacturing process of alcoholic beverages involves fermentation of cereal grains, fruit, vegetable, molasses or any other source of carbohydrates with the use of microorganisms depending on the type of beverage.
The global market of alcoholic beverage is forecasted to grow at Compound Annual Growth Rate (CAGR) of 7.30% from 2019 to 2026. The Indian market of alcoholic beverages is found to be growing at a CAGR of 8.8% in 2019, and it is expected to reach 16.8 billion litre of consumption by the year 2022. Owing to the enormous production and consumption of these alcoholic beverages, it becomes indispensable to maintain the final quality of the products.
Mycotoxins are produced during the storage of alcoholic beverages. These are secondary metabolites produced by certain filamentous fungi. The most critical mycotoxigenic fungi which will produce toxins in the alcoholic beverages during storage or transportation belong to the genera Aspergillus, Fusarium and Penicillium.
In 1993, the International Agency for Research on Cancer (IARC) classified aflatoxins as carcinogenic to humans Group 1, while ochratoxins, fumonisins and patulin were placed in a Group 2 as possible carcinogens. Trichothecenes and zearalenone were not classified as human carcinogens Group 3. Mycotoxins are suspected to be involved in Balkan endemic nephropathy and high frequency of urinary tract tumours. It also causes a high mortality rate due to neurotoxicity, genotoxicity, immune suppression and cytotoxicity.
Factors affecting the growth of mycotoxin in alcoholic beverages
The optimum temperature for the growth of mycotoxin during storage is 25- 30 degree with an incubation time of 14- 15 days. Other factors affecting the growth of mycotoxin in alcoholic beverages in production levels include meteorological condition like latitude, year of production, harvesting conditions and storage of crops used as raw materials, cultivations, use of pesticides, spoilage microorganism, production techniques like conditions of fermentation and type of maceration.
Table 1: Types of mycotoxins in alcoholic beverages
|
Mycotoxin
|
Types of alcoholic
beverages
|
|
Aflatoxin(AFB1,AFB2)
Ochratoxin
Fumanisins
Zearalenone
|
Beer, Craft beer
|
|
Ochratoxins
Patulins
|
Red wine, white wine, rose wines
|
The maximum permitted limit of mycotoxin in alcoholic beverages
The maximum limit for different mycotoxins (Aflatoxins 15 µg/kg, Ochratoxin 20 µg/kg, Patulin 50 µg/kg, Deoxynivalenol 1000 µg/kg) in alcoholic beverages were prescribed under FSSAI (Contaminants, Toxins and Residues) Regulations, 2011.
Detection techniques involved for mycotoxin
The detection of these types of mycotoxins becomes very important for maintaining the quality parameters of alcoholic beverages during export and import. The first step for any detection method is a sampling. Mycotoxins do not grow uniformly on a substrate so, homogenisation of the samples is a very critical point before analysis. The second step includes the extraction or purification step to remove the mycotoxins into the solvent that is suitable for cleaning up.
The common solvents used are Methanol: water 70% MeOH,Polyethylene glycol Acetronitrile:water:actetic acid(79:20:1), PBS. Recent days make use of QuEChERS,Super critical extraction columns.
The purified samples are then analysed by the different analytical method and Chromatographic techniques. Among the chromatographic techniques, the oldest method is the Thin Layer Chromatography (TLC) method. High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Liquid Chromatography (LC) are the other chromatographic methods which are combined with high-resolution mass spectroscopy using quadrupole or time of flight. The signals are then read through detectors, especially fluorescence detectors for these type of toxins.
Immunological methods include Enzyme-linked immunosorbent assay (ELISA) where the specific antibodies are used to capture the mycotoxin. It utilises the dual properties of antibody molecule being specific in reactivity and the ability to be conjugated to an active molecule such as enzymes. The antigen, which is a mycotoxin, in this case, is captured by the primary antibody, which is immobilised previously on a microtitre plate.
Further, the plate is treated with secondary antibody conjugated with an enzyme. This results in the trapping of antigen between two antibodies. The enzyme-linked with the secondary antibody gives colour in the presence of a substrate, and the intensity is proportional to the concentration of mycotoxin.
Biosensors are analytical device in which the biological recognition element such as enzyme, antibody, aptamers, nucleic acid and whole cells, is coupled with or near a transducer. A transducer is a device which senses the reaction between the analyte and the biological recognition element and converts it into a measurable signal. Biosensor provides an advantage over the conventional techniques in terms of rapid, selective and accurate analysis and minimum sample preparation.
Table 2: Comparison between different detection techniques
|
Method
|
Advanatage
|
Advantage
|
Disadvantage
|
|
Chromatographic
techniques
|
TLC
|
Low
cost, simple, rapid
|
Lack of
automation
|
|
|
GC
|
High
resolution, sensitivity, accuracy and precision, fast analysis of sample
|
Limited
to volatile sample, not suitable for thermally liable samples
|
|
|
HPLC
|
High
resolution, low limit of detection can be coupled with a multiple detection
automated system, specific
|
Expensive,
time-consuming, expensive equipment & clean-up procedure
|
|
|
LC-MS/MS
|
High
selectivity, high sensitivity, relatively easy sample clean-up,
multi-mycotoxin determination
|
Costly,
expensive, time-consuming, expensive equipment & clean-up procedure
|
|
Immunological
|
ELISA
|
Screening
method for different matrices, sensitive, specific, rapid, relatively low
cost & simple, low detection limit
|
Due to
the cross-reactivity with masked mycotoxins, ELISA results usually show an
overestimation of results, enzyme stability
|
|
Biological
|
Biosensor
|
Rapid,
sensitive, practical
|
Regeneration
of the receptor surface, specificity, sensitivity, reproducibility, stability
|
Methods for removal of mycotoxin in alcoholic industries
Mycotoxin are stable in different solvents, possesses resistance to acidity and high temperatures and difficult to remove. In industrial level charcoal, yeast strains (dead or alive), lactic acid bacteria has been widely used to remove the mycotoxins. The disadvantage which has been listed includes a reduction in polyphenol content, loss of colour and aroma of the alcoholic beverages, therefore directly affecting the efficacy of the system. Some mycotoxins like patulin and aflatoxin can be degraded at a higher temperature during the fermentation process and the distillation process up to a level of 30%.
Conclusion
As per the rising concern, FAO in 2019 announced that 25% of the food products worldwide contain mycotoxins which are not visible to the eye and are toxic. National or regional responsibility should be taken for food quality control, wine producers, importers and exporters to analyse the highest number of samples in the shortest time. Hence for controlling this issue, rapid and accurate monitoring tools or methods are necessary to ensure that the distributed products are safe and can be allowed to market.
(Dr Shanmugasundaram is Associate Professor [Food Engineering], Auddy and Nimbkar are PhD students at Indian Institute of Food Processing Technology [IIFPT], Thanjavur. They can be contacted at sasu@iifpt.edu.in )