UNIVERSITÀ CATTOLICA DEL SACRO CUORE Sede di Piacenza Scuola di Dottorato per il Sistema Agro-alimentare Doctoral School on the Agro-Food System Cycle XXV S.S.D.: AGR/15 Optimization of oenological practices to reduce biological contaminants in wine Coordinator: Ch.mo Prof. Romeo Astorri _______________________________________ Candidate: Alessandro Moncalvo Matriculation n. : 3810664 Tutor: Prof. ssa Angela Silva Co-tutor: Dott.ssa Maria Daria Fumi Academic Year 2011/2012 1 PhD in Food Science, Technology and Biotechnology Those who dream by day are cognizant of many things which escape those who dream only by night Edgar Allan Poe Il faut être toujours ivre. Mais de quoi? De vin, de poésie ou de vertu, à votre guise. Mais enivrez-vous. Charles Baudelaire 2 Contents Preface and aim of PhD thesis 6 Introduction 8 Chapter 1 – Ochratoxin A 9 1.1 Chemical characters of ochratoxins 9 1.2 Toxicity of ochratoxin A 9 1.3 Ochratoxin A occurence in food and wine 10 1.4 Ochratoxin A production in the vineyard 11 1.5 Ochratoxin A prevention in vineyard 12 1.6 The fate of ochratoxin A during winemaking 13 1.7 Removal of ochratoxin A in wine 14 1.7.1 Adsorbent material 15 1.7.2 Biological methods \ 17 1.7.2.1 Enzymes 17 1.7.2.2 Yeast 17 1.7.2.3 Lactic acid bacteria 20 1.8 References 22 Chapter 2 – Biogenic amines 30 2.1 Chemical characteristics of biogenic amines 30 2.2 Toxicological aspects 30 2.3 Biogenic amines presence in food and beverages 31 2.4 Biogenic amines production in winemaking 34 2.4.1 Yeast 35 2.4.2 Lactic acid bacteria 36 2.4.3 Factors affecting biogenic amines formation in winemaking 38 3 2.5 Prevention and reduction of biogenic amines in wine 41 2.6 References 43 PhD thesis project 50 Development of the research project 51 Chapter 3 – Ochratoxin A reduction by Lactobacillus plantarum 52 3.1 Introduction 52 3.2 Materials and methods 54 3.2.1 Standards, reagents and biochemicals 54 3.2.2 Bacterial origin and growth conditions 54 3.2.3 Ochratoxin A reduction assay in wine 55 3.2.4 Ochratoxin A degradation by B. linens 55 3.2.5 Ochratoxin A removal assay in YNB 55 3.2.6 HPLC analysis 56 3.2.7 Statistical analysis 58 3.3 Results 59 3.3.1 Ochratoxin A reduction preliminary assay in wine 59 3.3.2 Ochratoxin A degradation by B. linens 59 3.3.3 Ochratoxin A removal assay by L. plantarum in YNB 62 3.4 Discussion 66 3.5 References 68 Chapter 4 – Bacteria strains screening able to perform the malolactic fermentation without produce biogenic amines 72 4.1 Introduction 72 4.2 Materials and methods 73 4.2.1 Microorganisms, growth conditions and malic acid degradation tests 73 4.2.2 DNA extraction 74 4.2.3 PCR conditions 75 4 4.3 Results 76 4.3.1 Bacteria growth and malic acid degradation trials 76 4.3.2 PCR detection of L. casei and L. plantarum 78 4.3.3 PCR detection of hdc, tdc and odc genes 79 4.4 Discussion 79 4.5 References 81 Chapter 5 – Biogenic amines in wine related to Lactobacillus plantarum inoculation time 84 5.1 Introduction 84 5.2 Materials and methods 86 5.2.1 Grape cultivar 86 5.2.2 Yeast and bacteria strains 86 5.2.3 Standards and reagents 86 5.2.4 Chemical analysis of must and wine 86 5.2.5 Biogenic amines samples preparation and analysis 86 5.2.6 Winemaking trials 87 5.2.7 Statistical analysis 87 5.3 Results and discussion 88 5.3.1 Oenological parameters 88 5.3.2 Biogenic amines evolution 91 5.4 Conclusion 95 5.5 References 96 Conclusions 100 Chapter 6 – Conclusions and future perspectives 101 References 105 Acknowledgements 107 5 Preface and aim of PhD thesis Wine is an alcoholic fermentation made from fermented juice of grape with appropriate processing. During the processes, the grapes, the must and the wine are susceptible to various safety and quality hazards. The quality hazards are usually related to products appearance, acceptability, taste, flavour, colour and components. As far as safety, physical, chemical and microbiological hazards can affect consumer health. Hazards introduced to the wine may come from the environment, the processes equipment and the processing operations and treatments in the winery. Quality and safety are significant variables for a product as the wine; the quality is important for the consumer acceptability, while the assurance of the safety is obligatory for protection of human health. Safety of the wine product may only be assured by taking care of the relative hazards throughout the whole winemaking process. Normally the wine is considered a safe product, the low pH, the ethanol and the presence of SO 2 ensure a microbiological stability to the product during winemaking and the ageing. The possible risks may be due to the presence of chemical residues derived from pesticides used in vineyard, and biological metabolites products by microorganisms in the vineyard or during winemaking. Two of the major biological metabolites present in wine are the ochratoxin and the biogenic amines. The first of these contaminants was studied in recent decades because of its toxicity in humans, although its presence is not frequent in wines. The biogenic amines are present in every types of wine in different concentration, and some of them, in high concentrations, can cause allergenic reactions in humans. The research managed in this PhD is focused on the use of the selected starter for malolactic fermentation (MLF) to reduce these contaminants at guarantee of wine quality and safety assurance for the consumers. The objectives regard three different aspects. In the first part, it was investigated the biological methods to reduce ochratoxin A in wine during winemaking; in particular the study is focused to use a Lactobacillus plantarum strain as malolactic starter. Previous studies have described different methods to reduce ochratoxin in wine; in particular, adsorbent material (carcoal, bentonite, and yeast cell wall) and enzymes (proteases). The ochratoxin A is usually present in must and wine in particular regions of Europe as South Italy, Greece and Spain. These wines have different characteristics respect to the wines original of colder regions; a relevant condition that affects the MLF is the high pH values. The L. plantarum used in 6 this study was already tested in winemaking trials that have demonstrated the ability of this strain to carry out MLF especially in wine with high pH levels. A problem related to the high pH is the formation of biogenic amines as histamine, tyramine and putrescine. Some bacteria are able to produce these compounds in winemaking, in particular during MLF, or can be already present in the grapes before crushing. The second part of this work has the objective to investigate the presence of Lactobacillus spp., isolated from must and wine, able to produce the amines, using molecular techniques as polymerase chain reaction (PCR) to detect the genes that encode for the enzymes responsible of the synthesis of these compounds. This study is important to identify the real risk related to malolactic bacteria normally present in wine. In the last part of this PhD a malolactic starter, unable to produce biogenic amines, is tested to investigate the ability to perform MLF in relationship with inoculation time to shorter overall process of winemaking and to have a final product that maintains the varietal characteristics and has a good quality. Furthermore, the trend of biogenic amines already present in must was investigated to assess the relationship between the L. plantarum and the amines naturally present on the grapes that are affected by vintage, cultivar and other variables not related with the lactic acid bacteria. 7 Introduction 8 Chapter 1- Ochratoxin A 1.1 Chemical characters of ochratoxins The ochratoxins are a group of mycotoxins that contain a dihydro-isocumarin moiety linked to L-β- phenylalanine by an amide bond. Ochratoxin A (OTA), (R) N-[(5-chloro-3,4-dihydro-8-hydroxy-3- methyl-1-oxo-1H-2-benzo-pyran-7-yl) carbonyl]-L-phenylalanine, and its ethyl ester (ochratoxin C) are the most toxic compounds. The isocoumarin moiety is known as ochratoxin α (OTα), and is commonly reported to be less toxic than OTA. Strong acidic conditions cause hydrolysis of the amide bond, and strong bases open the lactone ring, reformed by acidification (Pohland et al., 1992). OTA can be converted into OTα and L-β-phenylalanine by heating under reflux for 48 h in 6 M hydrochloric acid (van der Merwe et al., 1965) or by hydrolysis with carboxypeptidase A (Pitout, 1969). In fact the amide bond mimics a peptide bond and is therefore susceptible to the action of hydrolytic proteases. 1.2 Toxicity of ochratoxin A OTA is a well-known nephrotoxin and it is classified by International Agency for Research on Cancer (IARC) as possible human carcinogen (group 2B) (IARC, 2003). A tolerable weekly intake of 120 ng/kg of body weight (bw) for humans was fixed on the basis of the lowest observed adverse effect level of 8 µg/kg (bw) per day for early markers of renal toxicity in pigs, the most sensitive animal species (EFSA, 2006). Long-term exposure to OTA has been implicated in Balkan Endemic Nephropathy and associated with urinary tract tumours because of rather high OTA levels detected in food samples and in blood or urine from affected persons (EFSA, 2006). The toxicity of OTA involves several mechanisms. OTA inhibits protein synthesis by competing with the phenylalanine amino acylation reaction catalysed by Phe-tRNA synthase (Creppy et al., 1984). This involves the inhibition of protein as well as DNA and RNA synthesis. OTA also disrupts hepatic microsomal calcium homeostasis by impairing the endoplasmic reticulum membrane via lipid peroxidation (Omar and Rahimtula, 1991). Based on animal studies (Kumagai, 1988), OTA is easily absorbed through the gastrointestinal tract mainly in the duodenum and jejunum. There are no studies on skin or inhalational absorption of 9 OTA. When absorbed, OTA has a high binding affinity for plasma protein. OTA was found in decreasing order of concentrations in kidney, liver, fat, and muscle tissues (Krogh et al., 1974); excretion is mainly via renal elimination (Chang and Chu, 1977). The half-life of OTA in humans is about 35 days; blood concentration is considered to represent a convenient biomarker of exposure and has been used in epidemiological studies. The human blood levels of OTA, in geographical areas with relatively high dietary exposure, are at least two order of magnitude below the mean concentration of OTA in the blood of rats that is known to cause nephrotoxicity and kidney tumours with long-term treatment (Mally et al., 2007). There is a positive correlation between plasma levels of OTA and the consumption of cereals, processed meat, animal fat, mutton/lamb meat, wine, beer, and jam/honey, Di Giuseppe et al. (2011) found a strong positive association between OTA intake, cardiovascular risk, and C-reactive protein. Recently, it was detected the presence of OTα in human plasma and urine suggesting the OTA- detoxification process in humans; in the same study the differences of OTα levels, especially in urine, between males and females (Muñoz et al., 2010a) were revealed. Another study investigated the presence of OTA and OTα in human milk; OTA was detected with an average concentration of 106±45 ng/L, while levels of OTα were 40±30 ng/L, but increased upon enzymatic hydrolysis with β-glucuronidase/sulfatase, indicating that the majority of OTα in milk is excreted as glucuronide or sulphate (Muñoz et al., 2010b). These new data provide further insight into the fate of OTA in the human organism, documenting partial detoxification of OTA to OTα and conjugation of this metabolite. 1.3 Ochratoxin A occurrence in food and wine OTA has been found in many food and raw materials such as cereals, coffee, cocoa and other crops in warmer and tropical regions. In a study on coffee OTA was detected in almost half of the samples analyzed (299 positive samples out of 633) and high OTA values (>10 ng/g) were very rare (Stegen et al., 1997). In the cocoa Bonvehí (2004) reported that the average of OTA was 11 µg/kg (range of 2.9-23.1 µg/kg) (100% positive samples) for the roasted cocoa shells and 2.60 µg/kg in raw cocoa shells. Ariño et al. (2007) investigated the effect of the sorting, washing and peeling of fresh liquorice on the concentration of OTA and the transfer of OTA from dry liquorice roots to derived products; in 10