MYCOTOXINS AND

BREWING TECHNOLOGY

BYAPEH, DANIEL O.

MTECH/SSSE/2011/2892

SUPERVISED BYDR. MAKUN, HUSSAINI A.

DR.(MRS) MUHAMMAD, HADIZA L.DEPARTMENT OF BIOCHEMISTRY FEDERAL UNIVERSITY OF TECHNOLOGY,

MINNA

INTRODUCTIONBeer refers to beverages resulting from the germination and fermentation of starches mainly derived from cereal (Gutcho, 1976).

The third most popular drink overall after water and tea (en.wikipedia.org).

Africa is the third largest beer consuming continent (WHO, 2004).

Nigeria is among the first 30 top world consumers (FAO, 2003; WHO, 2004)

High demand for beer across various continents equals high risk of consumption of beer associated toxins (Karolína et al., 2012).

Mycotoxins are major cereal contaminants (Reviewed in Makun, et al., 2009; Chandrashekar et al., 2000).

Processed cereals in form of beer may be contaminated with mycotoxins.

MYCOTOXINSOver 300 fungi produced toxic compounds that

contaminate a wide variety of agricultural commodities (Nielson and Smedsgaard, 2003)

Produced as secondary metabolites (Pitt, 2000). At Pre-harvest, Post-harvest or During storage

(Reviewed in Kumar et al., 2008)Ingestion cause a range of toxic responses, from

acute to chronic health disorders (Roger, 1993)Affect trade Major producers Aspergillus species Penicillium species Fusarium species

FIGURE 1: STRUCTURAL REPRESENTATION OF SOME MYCOTOXINS

Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Aflatoxin G2 Aflatoxin

M1

FumonisinZearalenone Patulin Citrulin Ergot Alkaloid

HEALTH IMPACT OF MYCOTOXINS

Diseases resulting from mycotoxin exposure are called mycotoxicosis

They elicit short or long term impacts(Wayne, 2007) Inhibition of metabolic pathways Impairment of growth and development Immunosupression Carcinogenicity, mutagenicity and teratogenicity

T-2 toxin was responsible for the deaths of several Russians from 1942-1947 (Gao and Yoshizawa, 1997).

Aflatoxin intake relates to high incidence of liver cancer is S.A, Kenya etc. (Fabio, 1999); Aflatoxicosis

ECONOMIC IMPACT OF MYCOTOXINS Mycotoxin contamination affects up to 25% of global food and

feed (FAO, 2009). Crop losses and Reduced animal productivity

Costs from improving technologies for production, storage and transport of crops

Member states of the African Groundnut Council—The Gambia, Mali, Niger, Nigeria, Senegal, and Sudan—have calculated the annual cost of implementing a program to reduce aflatoxin contamination at US$7.5 million (Atanda, 2011).

The cost of analytical testing, especially as regulations become more stringent

EU regulation on aflatoxins cost Africa US$750 million each year in exports of cereals, dried fruit and nuts (World bank study; retrieved from http://www.mycotoxins.com)

BREWING TECHNOLOGY

Refers to the processes involved in beer production (Ertan Anli and Mert, 2010)

The most commonly used cereal grain is barley; others are wheat, maize (corn), sorghum and rice (Gutcho, 1976).

FIGURE 2: GENERAL CHART OF THE BREWING PROCESS

Malt

Hop

SyrupsWort

Malting (3 stages)

Mashing

Separation

Boiling

Cooling

Fermentation

MaturationYeast

Pre-treatment

Sugars

Grain (Barley, Sorghum, Wheat)

BEER

Adjunct (Rice, Wheat, Maize, Barley e.t.c)

Moisture contentMicroorganismsGibberellic Acid Hydrolytic Enzymes (Gutcho, 1976).

Ground malt + Adjuncts Amylases and proteases hydrolysis & extractionIncrease in particle size (Milling)

Wort is boiled sometimes with hops Boiling; Concentrate and sterilize the wort

Inactivate enzymes Coagulate and precipitate protein Caramelize the sugar slightly in

order to develop the color of the beer

Hops contribute antiseptic compoundsYeast is inoculated in WortYeast converts sugar into organic compoundsC6H12O6+2Pi+2ADP 2C2H5OH+2CO2+ 2ATP Glucose Ethanol + 2H2O Lactic acid bacteria is inoculated in WortResults in souring (Lerio, 1993)

MYCOTOXIN AND BREWING

Some mycotoxins seems to survive major beer production processes namely malting, mashing, boiling and fermentation into beer (Scott, 1996)

Mycotoxins in beer could either be developed or detoxified at various brewing processes (Mably et al., 2005)

FIGURE 3: AFLATOXIN AND BREWING

Malt

Hop

Wort

Malting

Mashing

Separation

Boiling

Cooling

Fermentation

Maturation

Yeast

Grain (Sorghum, Wheat)

BEER

Adjunct (Rice, Wheat, Maize, Barley e.t.c)

Steeping resulted in about 45% AFL lost (Yahl, 1971; Romer, 1984)

Adjunct is a source of AFL contamination (Karolína et al., 2012)

At 100°C and 250°C for 30min showed 10.4% and 99% reduction in AFB1 (Oluwafemi and Ikeowa, 2005)

For 72hrs showed 50% reduction of AFB1 (Yuan et al., 2008)Lactic acid fermentations lead to opening of the lactone ring (Nout 1994).

Sugars

Syrups

Pre-treatment

Upto 89% AFB1 was removed during Lager beer processing (Oluwafemi, 2004)

FIGURE 4: OCHRATOXIN AND BREWING

Malt

Hop

Wort

Malting

Mashing

Separation

Boiling

Cooling

Fermentation

MaturationYeast

Grain (Sorghum, Wheat)

BEER

Adjunct (Rice, Wheat, Maize, Barley e.t.c)

Almost completely lost ( Krogh et al., 1974).

40–89% was lost (Sylvie et al., 2011) Most probably due to proteolytic degradation (Ertan Mert, 2010).

Destroyed in the fermentation process (Kostecki et al., 1991; SCOOP, 2010)

Sugars

Syrups

Higher in the ‘‘non-alcoholic’’ beer (Tangni and Lanrondelle, 2003)Upto 70% of the OTA was degraded in the production steps beer(Nip et al., 1975)

Pre-treatment

FIGURE 5: FUMONISIN AND BREWING

Malt

Hop

Wort

Malting

Mashing

Separation

Boiling

Cooling

Fermentation

MaturationYeast

Grain (Sorghum, Wheat)

BEER

Adjunct (Rice, Wheat, Maize, Barley e.t.c)

Increase in Fusarium spp, and fumonisin

Brewing adjuncts e.g corn contributes fumonisin (Scott and Lawrence, 1995).

Heat-stable (Alberts et al., 1990)

Level change not significant (Scott et al., 1993).

Sugars

Syrups

Levels above maximum limit has been reported in severel countries (Drager, 1996; Mbugua and Gathumbi) e.g 150±24 ng/ml in Cameroonian beer (Roger, 2011). Gushing increases with fumonisin in beer (Casey,1996;).

Pre-treatment

FIGURE 6: DEOXYNIVALENOL (DON) AND BREWING

Malt

Hop

Wort

Malting

Mashing

Separation

Boiling

Cooling

Fermentation

MaturationYeast

Grain (Sorghum, Wheat)

BEER

Adjunct (Rice, Wheat, Maize, Barley e.t.c)

Increased by 18–114% of that present on the original barley in 5 day malts, (Scott, 1996; Schwarz et al., 2005, Lancova et al., 2008)

Stable after 7-9 days of alcoholic fermentation with S. cerevisiae (Scott, 1992)Detected at high levels in both the solid residue and the fermented liquid (Bennet and Richard 1996).

Sugars

Syrups

Pre-treatment

Trichothecenes are stable to brewing process; occurring in commercial beer in many countries (Shim et al., 1997; Molto et al., 2000; Baxter et al., 2006 ).After brewing, 80–93% of DON present on the malt grist was detected in the beer (Schwarz et al., 2005).

TABLE 2 : YEAST AND/OR YEAST CELL WALL MATERIAL AS POTENTIAL MYCOTOXIN DECONTAMINATING AGENTS

AGENT Mycotoxin bound

Level of Binding

Reference

S. cerevisiae AflatoxinB1 > 60% Shetty and Jespersen, 2006; Devegowda et al., 1996

Yeast from West African maize

AflatoxinB1 >60% Shetty and Jespersen, 2006

Cansida crusei AflatoxinB1 15- 39% Shetty and Jespersen, 2006

Mannan-oligosaccharides

Ochratoxins & ZearalenoneDON&FumonisinB1

High binding

Little binding

Raju & Devegowda, 2000; Devegowda et al., 1996

Modified mannan-oligosaccharides from the S. cerevisiae

AflatoxinB1 77% Devegowda et al., 1996

Mixture of 40% (w/w) sterilized yeast + 60% (w/w) residue of beer fermentation

Ochratoxin A High binding Grunkemeier, 1990

TABLE 3 : LACTIC ACID BACTERIA (LAB) AND/OR LAB MATERIAL AS POTENTIAL MYCOTOXIN DECONTAMINATING

AGENTSAGENT Mycotoxin bound Level of

BindingReference

Lactobacillus plantarum

Aflatoxin B1 40-59% Shetty and Jespersen, 2006

Paralactobacillus serangolensis

Aflatoxin B1 <15% Shetty and Jespersen, 2006

Lactobacillus rhamnosus

Aflatoxin B1 (more than B2, G1, G2)

80% El–Nezami et al., 1998a

Lactobacillus rhamnosus strains LGG and LC 705

Deoxynivalenol, 3-acetyldeoxynivalenol, nivalenol, fusarenon, diacetoxyscirpenol, T-2 toxin, and HT-2

Effective binding

El–Nezami, Chrevatidis, Auriola, Salminen, & Mykkanen, 2002

Lactic Acid Bacteria

Aflatoxins Inhibition of bio-synthesis

(Coallier-Ascah & Idziak, 1985; Thyagaraja & Hosono, 1994).

FIGURE 7: SACCHAROMYCES CEREVISIAE (YEAST) CELL WALL COMPONENTS

Retrieved from www.bioimages.net

FIGURE 8: LACTIC ACID BACTERIA CELL WALL COMPONENTS

Retrieved from www.bioimages.net

BIOCHEMICALMECHANISMS OF ACTION

OF SOME MYCOTOXINS

CONTAMINATING BEER

FIGURE 11: MECHANISM OF ACTION OF FUMONISIN

Dihydrosphingosine N-acyl transferase is one cellular target for fumonisin toxicity and carcinogenicity Inhibition of sphingolipid biosynthesis (Wang et al., 1991). Increases the ratio of sphingoid precursorsCell deregulationCell death (Riley et al., 1996)

TOXICOLOGY Suspected human carcinogen. Toxic to pigs and poultry. Cause equine eucoencepha-lomalacia (ELEM), a fatal disease of horses

FIGURE 12: MECHANISM OF ACTION OF DEOXYNIVALENOL

DON inhibits Translation by

1.Interaction with the peptidyl transferase centre on the 605 ribosomal subunit.

2.They also prevent release of the polypeptide, by suppressing hydrolysis of the peptidyl—tRNA at termination

TOXICOLOGY Feed refusalReduced weight gainDiarrheaVomiting

FIGURE 10: MECHANISM OF ACTION OF OCHRATOXIN

•Disruption Of Phenylalanine Metabolism•Reduced PEPCK at Translation level•Reduced Glyconeogenesis •Cell Death

Alanine and other glucogenic amino acids, Lactate

PREVENTION AND MANAGEMENT OF MYCOTOXIN CONTAMINATION IN

COMMODITIES

Implementation of GAPs from planting to harvesting

Proper storage conditionsBiotechnology; Resistant crop varieties (Hell et al.,

2011)Coordination between the different stakeholdersThe establishment of food safety measures needs

a legal basis, otherwise no enforcement is possible

Public awareness/ University curriculumHACCP and others

REGULATIONSEstablished in about 100 countries of which 15

are African.Agencies involved; WHO, FAO, WHO-JECFA,

EFSA, FSA, SON, ACADEMIC INSTITUTIONS Maximum allowable limits

4 µg/kg aflatoxinB1 for All foods (Nigeria standard)

0.5 µg/kg DON for grain (USA standard) 2-4 µg/kg FumunisinB1 for maize products

(EU standard) 5 µg/kg and 3 µg/kg Ochratoxin in cereals

and malt respectively (EU standard)

CONCLUSIONBrewing technology is a multi-phased process, each

phase contributes differently to the final quantity of different mycotoxins that may be found in beer.

Grain and/or adjuncts may be source of contamination by fungi and their mycotoxins.

Malting increases the risk of fungi and mycotoxin contamination but reduce ochratoxin levels.

Fermentums binds mycotoxins on their cell walls.Generally, DON and other tricothecenes are shown to

increase across the brewing process, fumonisin is relatively stable while aflatoxin and ochratoxin are generally observed to be reduced across the brewing process

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