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",isbn:"978-1-83969-452-3",printIsbn:"978-1-83969-451-6",pdfIsbn:"978-1-83969-453-0",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,hash:"a6e1a11c05ff8853c529750ddfac6c11",bookSignature:"Dr. René Mauricio Barría",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10734.jpg",keywords:"Neonatal Intensive Unit, Neonatal Diagnostic Techniques, Neonatal Nurses, Neonatologists, Newborn Diseases, Premature Diseases, Breast Feeding, Kangaroo-Mother Care Method, Neonatal Survival, Limit of Viability, Minimal Handling, Neonatal Stress",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"February 5th 2021",dateEndSecondStepPublish:"March 5th 2021",dateEndThirdStepPublish:"May 4th 2021",dateEndFourthStepPublish:"July 23rd 2021",dateEndFifthStepPublish:"September 21st 2021",remainingDaysToSecondStep:"4 days",secondStepPassed:!1,currentStepOfPublishingProcess:2,editedByType:null,kuFlag:!1,biosketch:"The principal investigator and academic expert in epidemiological methods and evidence-based health with an emphasis on children's health. His research interests lie in the areas of Maternal-Child Health, Neonatal Care, and Environmental Health. From 2010 until 2017 he was Director of the Evidence-Based Health Office and currently serves as Director of the Nursing Institute at the Universidad Austral de Chile.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"88861",title:"Dr.",name:"R. Mauricio",middleName:null,surname:"Barría",slug:"r.-mauricio-barria",fullName:"R. Mauricio Barría",profilePictureURL:"https://mts.intechopen.com/storage/users/88861/images/system/88861.jpg",biography:"R. Mauricio Barría, DrPH, is a Principal Investigator and Associate Professor at the Faculty of Medicine at Universidad Austral de Chile. He was trained as an epidemiologist and received his MSc in Clinical Epidemiology from Universidad de la Frontera in Temuco, Chile, and his DrPH from Universidad de Chile in Santiago, Chile. His research interests lie in the areas of Maternal-Child Health, Neonatal Care and Environmental Health. He is skilled in epidemiological studies designs with special interest in cohort studies and clinical trials. Since 2010 until 2017 he was Director of the Evidence-Based Health Office and currently serves as Director of the Nursing Institute at the Universidad Austral de Chile. He has published several articles related to the care and health of the newborn and is a reviewer of several international journals.",institutionString:"Austral University of Chile",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"4",totalChapterViews:"0",totalEditedBooks:"4",institution:{name:"Austral University of Chile",institutionURL:null,country:{name:"Chile"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"16",title:"Medicine",slug:"medicine"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"345821",firstName:"Darko",lastName:"Hrvojic",middleName:null,title:"Mr.",imageUrl:"//cdnintech.com/web/frontend/www/assets/author.svg",email:"darko@intechopen.com",biography:null}},relatedBooks:[{type:"book",id:"6550",title:"Cohort Studies in Health Sciences",subtitle:null,isOpenForSubmission:!1,hash:"01df5aba4fff1a84b37a2fdafa809660",slug:"cohort-studies-in-health-sciences",bookSignature:"R. 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This substance is widely applied as antioxidant and antibacterial in many processed foods, being the most preservative used in the wine industry. In wines, SO2 prevents undesirable sensory properties and the spoilage of wines produced by chemical or microbiological agents. However, in recent times, it has been shown that the intake of SO2 implicates a wide range of adverse health consequences, such as allergic reactions and cumulative harmful effects [1]. Therefore, negative perceptions toward sulfites have been induced, and a significant increase on the demand of wines with low content of SO2 has been displayed by consumers in the last years [2]. For this reason, reducing the amount of SO2 in wines is a decisive strategy for the wine industry and one of the current topics on the oenological science.
In wines, SO2 is composed by total SO2, bound SO2, free SO2, and molecular SO2. Proper adjustment of the SO2 dosage is difficult because it depends on the equilibrium between its free and bound forms. The active form is molecular SO2, which depends on the concentration of free SO2 and the pH [3]. This active form has the antimicrobial and antioxidant properties. In terms of antimicrobial, an insufficient addition of SO2 will not ensure the wine protection, increasing the risk of yeast and bacteria proliferation. In terms of antioxidant, an inadequate dosage will allow an excessive oxidation of aromas and flavors, compromising the quality of wines [4]. Contrary, excessive dosages in wines may cause organoleptic alterations and also health reactions in consumers. Taking this into account, the International Organization of Vine and Wine (OIV) has progressively reduced the maximum limits of the total SO2 in wines, which is nowadays 150 mg/L for red wines and 200 mg/L for white wines, with some exceptions depending on the sugar content (Regulation (EC) No 607/2009).
Today, there is not a commercial product or recipe able to replace the widespread SO2 actions. Consequently, diverse technological strategies should be considered by winemakers in each stage of the winemaking process, according to the type of wine to be produced and the winery capabilities. From our point of view, these strategies should be addressed from three joint perspectives; microbiological strategies, physical technologies, and chemical treatments. In this sense, the Wine Technology Centre (VITEC) has been working in this research field since 2012. Our studies have been focused in red and white wines, especially regarding Tempranillo and Albariño grape varieties.
From a microbiological point of view, many factors should be taken into account to reduce the quantity of SO2 in wines. First, it should be considered that an endogenous content of SO2 is naturally produced by yeasts during alcoholic fermentation. Second, grape juice composition, yeast nutrition, and fermentation management may strongly influence the ability of yeasts to produce sulfites. Finally, microbiological stability of the SO2-free wines remains uncertain yet.
As mentioned above, the European Union regulates the levels of total sulfites in wines following the Regulation (EC) 607/2009. Therefore, wines must be labeled with the indication “contains sulfites,” when the total content of SO2 is over 10 mg/L, either exogenous or endogenous. Most organisms produce sulfites as a normal intermediate during digestion or synthesis of the sulfur-containing amino acids, such as methionine and cysteine [5]. Sulfites are minor by-products of yeast fermentation, and therefore, they are natural wine constituents. The ability of yeasts to form SO2 has been reported in different types of wines and geographical areas, and it was known long time ago and investigated intensively over the years [6, 7].
One of the most important factors to elaborate SO2-free wines is the choice of the suitable yeast strains used for the development of the alcoholic fermentation. During winemaking process, sulfur (naturally available as sulfate in grape juice) is used by yeasts in the synthesis of amino acids. In particular, Saccharomyces cerevisiae produces sulfite as an intermediate product during the assimilatory reduction of sulfate to sulfide, via adenosine-5′-phosphosulfate [6, 8]. The available sulfide (S2−) can be used in the synthesis of amino acids, as well as being excreted as hydrogen sulfide (H2S). Eventually, the sulfur amino acid biosynthesis (SAAB) pathway plays a crucial role in the active transport of sulfate (SO42−) into the cell, as well as in the reduction and production of SO2 and in the resistance of yeasts against this additive [9]. Yeast strains differ in their capacity to form SO2, estimating a total average content ranged from 0 to 115 mg/L [10, 11, 12, 13, 14]. Most strains of S. cerevisiae produce between 10 and 30 mg/L of total SO2. However, some of them may produce less than 10 mg/L, which were commonly called “low sulfite-forming strains” [6]. On the opposite side, “high sulfite-forming strains” are able to produce more than 100 mg/L. These classifications according to their ability to form SO2 during the alcoholic fermentation have been reported by several authors over the time [6, 7, 12, 14].
In the last years, the use of yeast strains with a low capacity to produce SO2 has been one of the most used strategies to reduce the amount of SO2 in wines [15]. Several studies have compared the amount of SO2 produced during alcoholic fermentation by different commercial and indigenous yeast strains. In 1985, Suzzi et al. [13] investigated the biological sulfite role in the stabilization of white wines by comparing 1700 strains of Saccharomyces isolated from spontaneous fermentations. The majority of them produced less than 10 mg/L of total SO2, around 350 produced between 10 and 20 mg/L, 52 strains produced between 20 and 40 mg/L, and just two strains produced more than 40 mg/L. More recently, an experiment carried out at industrial scale by Werner et al. [14] showed two distinguishable groups of yeasts, among 22 commercial strains. The first one produced under 10 mg/L of total SO2, and the second one produced between 10 and 20 mg/L. Significant differences among yeasts strains in production of SO2 (free and bound-SO2) were also described by Wells and Osborne [7]. In this case, values ranged from 25 to 60 mg/L of bound-SO2 were observed. In 2015, Miranda-Castilleja et al. [11] studied the production of total SO2 of 52 indigenous species of Saccharomyces from Querétaro (Mexico), and the obtained results ranged from 37 to 115 mg/L. More recently, VITEC has investigated the natural production of SO2 of 21 selected yeast strains (commercial and indigenous). Fermentations were conducted using Muscat grape juice at 18 and 25°C. These results showed a total SO2 production lesser than 10 mg/L in all cases. The results in agreement with other works which also showed diverse yeast strains are able to produce small amounts of total SO2 (<1.4 mg/L) [16, 17]. Thus, several commercial and indigenous yeast strains have proved to be able to produce SO2-free wines. However, other considerations should be taking into account, such as the organoleptic properties and microbial stability of this type of wines.
The formation of SO2 by yeasts is influenced by a complex interaction of genetic, physiochemical, and metabolic factors. H2S is one of the most undesirable metabolites derived from the alcoholic fermentations due to its unpleasant smell and taste. It should be noted that the biosynthesis and the production of H2S and SO2 are linked [18, 19]. As occurs in the case of SO2, the formation of H2S varies widely depend on the yeast strains [20, 21]. The release of H2S by yeast during the fermentation is a long-standing problem that has been extensively studied in comparison to the SO2 production. There has been an ever-growing interest in wine yeasts with low production in H2S. The selection of suitable strains has so far been the principal way of limiting excessive H2S formation. Other engineering strategies have been used for limiting its production, which generally consisted of overexpression or inactivation of some genes involved in the sulfate reduction pathway [22, 23, 24].
Both sulfites and hydrogen sulfides are produced during the biosynthesis of the sulfur containing amino acids, methionine, and cysteine, starting from sulfate assimilation. Given the metabolic link between H2S and SO2, such kind of biotechnological and engineering strategies firstly applied to reduce H2S production could also be applied to decrease SO2 formation by yeasts. Nonetheless, few works have been aimed to obtain both low SO2 and low H2S production. Three strains with low SO2 production (SO2 < 10 mg/L) and with reduced H2S production were selected by De Vero et al [25]. These authors proposed a strategy that combines sexual recombination and specific selective pressure to generate nongenetically-modified S. cerevisiae with desired oenological characteristics. More recently, new insight into the regulation of sulfur metabolism in wine yeasts by the identification of variants of MET2 and SKP2 genes within SAAB has been reported to modulate the production of sulfites and sulfides [26]. These results provide novel targets for the improvement of wine yeast strains orientated to produce SO2-free wines. This knowledge on the sulfate pathway provides a chance to successfully apply engineering strategies to select “low sulfite-forming” yeast strains. However, as we previously highlighted, the production of sulfites by yeast during fermentation not only depend on metabolic factors but also on the environment, including nutrients and fermentation management, among others. Hence, grape juices composition is an imperative factor that should be considered in order to elaborate this type of wines. The insoluble solids contained in the grape juice also appeared to have an effect on the SO2 content, and wines with the higher insoluble solids obtained lower values of SO2 [27]. In contrast, results obtained in our experimental cellar showed that grapes with higher content of soluble solids produced higher content of total SO2 (Figure 1). The biplot of the principal component analysis (PCA) shows that the amount of SO2 produced during the alcoholic fermentation is mainly favored by a high amount of sugars and a low quantity of nitrogen. Furthermore, musts fermented at low temperatures (18°C), and a low titratable acidity may contribute on the production of SO2.
Biplot performed by 74 wines produced from Tempranillo and Albariño musts.
In addition, the supplementation of musts with amino acids can significantly affect SO2 and H2S production depending on the amount added, the time of addition, and the nitrogen concentration [26, 28]. Individual amino acids such as methionine, cysteine, asparagine, and arginine have been shown to influence sulfite formation [18, 28]. Higher the concentration of methionine and cysteine in the grape must, lower the formation of SO2 [6]. Under ammonia limitations, the addition of nonsulfur amino acids tended to increase the formation of SO2 (but inhibits the formation of H2S). The addition of cysteine seems to increase the H2S content but inhibits the sulfite formation, and the addition of methionine inhibits both SO2 and H2S formation [28]. More recently, it was stated that methionine repressed the cysteine-induced increase in the H2S production but had no effect on the formation of SO2. Both compounds were produced in greater quantities by yeast when grown in the presence of increasing concentrations of cysteine [18]. It has been reported that yeasts produce higher concentrations of SO2 under higher yeast assimilable nitrogen (YAN) quantities [7, 29]. The supplementation on nitrogen using ammonium salts (sulfate or phosphate) allows higher growth rates and biomass yielding and also the stimulation of the fermentative activity [30, 31]. The addition of diammonium phosphate (DAP) significantly decreases H2S production and improves the kinetics of fermentation and aroma profile of wine [32]. In the last 5 years, VITEC has been studying the effect of ammonium sulfate and DAP addition on the amount of SO2 produced by yeast along of the alcoholic fermentation. Results obtained showed that the addition of the N-sources slightly increases the total content of SO2 in wines. The addition of ammonium sulfates and DAP using low sulfite-forming strains to ferment musts showed no significant differences. In the case of musts fermented by “high sulfite-forming” strains, the addition of DAP significantly increased the total content of SO2 [33].
Other important consideration to elaborate SO2-free wines is the management of the alcoholic fermentation. In this sense, it has been stated that temperature has several effects on biochemical and physiological properties in yeast cells. Some changes in the sulfur assimilation pathway by S. cerevisiae depending on temperature may occur [34]. Our results are in agreement with other authors, who reported that at low temperature, the SO2 production increases [26]. SO2 and H2S production is also affected by pH (acidic pH facilitate SO2 uptake) and concentration of some minerals (copper and zinc) and vitamins, such as pantothenate or thiamine [9, 26, 35]. Thiamine is a vitamin used as a co-enzyme in the alcoholic fermentation pathway. It stimulates yeast growth, speeds up fermentation, and reduces production of SO2 binding compounds. Thiamine supplementation allows the transformation of pyruvic acid to acetaldehyde and limits the accumulation of ketonic compounds on wine being considered a factor to reduce the SO2 amount on wines [36]. A deficiency in thiamine may reduce yeast growth, slow fermentation, and promote the accumulation of pyruvic acid and acetaldehyde, the components responsible of wine oxidation. The effect of major SO2 binding compounds (acetaldehyde, pyruvic, and α-ketoglutarate) on the production of SO2 by different yeasts strains is still poorly understood, and more studies should be performed to better understand their role on the SO2 production [7]. In this way, the results obtained in VITEC are in agreement with the results obtained by Comuzzo and Zironi [33, 36], who showed that the addition of DAP + thiamine reduced the production of α-ketoglutarate.
From a physical point of view, different technologies have been used to ensure the wine microbiological stability and to prevent oxidations [37]. The main advantage of using physical methods is the nonaddition of chemical substances that may affect human health. By these technologies, the preservation of the organoleptic properties of wines and the antimicrobial effect should be produced at the same time. Pulsed electric fields (PEF), ultraviolet radiation (UV), high hydrostatic pressure (HHP), and flash-pasteurization lead an antimicrobial result, while the use of ultrasounds (US) or inert gases does not share this property [38, 39, 40, 41]. The PEF consists in the application of short electric pulses of high intensity between two electrodes, producing electroporation of the cell membranes increasing their permeability. It has been shown that this technique is effective to inactivate both bacteria and yeasts [42]. Thus, PEF may be applied to eliminate undesirable microorganisms at different winemaking stages, for example, before bottling. It has been stated that the treatments with PEF also reduces the activity of enzymes, such as polyphenol oxidases and peroxidases, increases the extraction of phenolic compounds and affects the aromas of white wines [42, 43]. VITEC has evaluated the antimicrobial effect of PEF, HHP, US, and EMR (electromagnetic radiation). Figure 2 shows the obtained results after the quantification of viable yeasts and acetic acid bacteria (AAB) in Petri dishes culture. The PEF conditions were electric field 35 kV/cm, voltage 23 kV, pulse rate 0.65 kHz, pulse duration 2.5 μS, initial conductivity 5.04 mS/cm, flow 25 l/h, and initial temperature 20.8°C. The PEF 1 and PEF 2 differed on the final temperature of the treatment which was 23 and 31°C, respectively. Worthy results of PEF as antimicrobial technique were obtained, although high colony-forming units of yeast were observed in the case of PEF 1.
Evaluation of different physical treatments in Tempranillo and Albariño wines (at the end of the alcoholic fermentation) by the quantification of viable yeasts and acetic acid bacteria in Petri dishes culture (cfu, colony forming units).
The use of high hydrostatic pressures (HHP) was evaluated in our studies at different pressures (from 400 to 600 MPa) and times (1, 3, 5, and 10 min). HHP results showed that the inhibition of microorganism by this methodology depends not only on the time and pressure applied but also on the variety and the type of microorganisms (Figure 2). Tempranillo and Albariño yeast growth were inhibited by all pressures and times applied. However, in the case of acetic acid bacteria, the HHP treatment was very efficient for Tempranillo but not for Albariño wines. Even so, low levels of viable AAB (102 cfu/100 mL) were found. According to Bartowsky et al. [44], AAB populations from either spoiled or unspoiled wines ranged between 102 and 103 cfu/mL. According to the literature, pressures above 700 MPa may inhibit the polyphenol oxidase, although lower values of pressure are enough to inactivate yeasts and bacteria [45]. In our experiments, HHP results as a very effective technique against yeast and lactic acid bacteria and a lesser extent against AAB. At the studied conditions, HPP and PEF showed a noteworthy preservation of the organoleptic properties of wines (data not shown), according to other authors [45, 46, 47].
Other techniques, such as ultrasounds (US) and EMR, were also evaluated. The EMR is one of the most recent physical technologies evaluated in wines, which has shown a good potential in food processing, such as fruits, vegetables, and juices. This technique allows increasing the wine temperature for a short time period without any external heating source. EMR allows achieving the reduction of microorganisms with low effect on the organoleptic properties of wines, when compared with other heating techniques, such as flash pasteurization. However, recently studies have shown that the application of lower power microwave exposures may increase the growth of Bretttanomyces cells [48]. In agreement, Figure 2 shows an increase on AAB after the treatment with EMR in both cases. The application of US at different conditions considering time of application (from 1 to 3 min) and wavelengths (12, 43 and 75 μm) inhibited the yeasts growth but not the bacteria population (Figure 2). The effectiveness of US resulted lower than HHP, at least at the experimental conditions studied. As occurred with EMR treatment, an increase on the colony-forming units was observed after the treatment with US. Ultraviolet radiation reduces the population of wine microorganisms, but different resistances to the radiation have been stated depending on species. It appears to be an effective method against Brettanomyces, Saccharomyces, Acetobacter, Lactobacillus, and Pediococcus [46]. Furthermore, it has been described that phenolic compounds can absorb UV radiation and is therefore less effective in red wines. This technique seems to be more effective in white wines at the end of fermentation, when wines present low turbidity. In order to increase the total polyphenol, it could be also applied at maceration stage [38, 49].
In general, all the physical treatments assessed clearly affect the viability of lactic acid bacteria in Tempranillo and Albariño varieties. In both cases, only viable lactic acid bacteria were detected in the control (data not shown). The employed treatments reduced the viability of yeasts and lactic and acetic acid bacteria. However, in this study, both US and EMR were not effective enough to reduce the population of viable acetic acid bacteria. According to the results, AAB were more resistant to the treatments than lactic acid bacteria (LAB). Regarding techniques, a higher antimicrobial effect of HHP and EMR was observed in comparison to the other methodologies employed. Besides, some wines produced by US and EMR showed oxidation characteristics. As occurred in the antimicrobial assays, the optimization of methods and experimental conditions is an imperative action to avoid adverse effects on the sensory quality of wines. It should be noted that some of these physical techniques are commonly used in food industry, but their implementation on the wine sector is so far to be available for a daily work routine, mainly due to economic and technique questions.
The oxidation is one of the main processes that affect SO2-free wines. Apart from the mentioned technologies and despite of its antimicrobial effect is limited, the use of inert gases is more and more applied throughout the winemaking process. The oxygen control by the management of the inert gases during the winemaking process must be considered because they have an important impact on the organoleptic properties. Caps are the ultimate physical barrier to preserve wines during storage, and so their oxygen permeability should be considered. The long-term protection is one of the most concerns for wineries in bottled wines with reduced SO2 content [50]. The assays carried out in VITEC using argon and carbon dioxide showed valuable sensory results (Figure 3). The SO2-free red wines produced by the use of Ar and CO2 showed higher significant color intensity, tannic intensity, and dryness. Greater aroma intensity and mouthfeel were also found, although values did not show significant differences. In general, Tempranillo-bottled SO2-free wines obtained higher global punctuations than wines with SO2 addition.
Comparison of the sensory evaluation of Tempranillo wines elaborated using argon (Ar), carbon dioxide (CO2) and sulfur dioxide (SO2). * Significant differences by HSD Tukey test (p < 0.05).
The oxygen control during all the production process of this type of wines is an imperative engagement. It is important to take into account that wines without sulfite addition are exposed to physicochemical and microbiological alterations. Considering the techniques available in any winery, to avoid microbiological alterations, sterilizing filtration may be an alternative. However, this technique could reduce the sensorial quality of the wine because it is a very oxidative process. To ensure a correct conservation of the SO2-free wines, the amount of oxygen incorporated into wine should be controlled, especially at bottling, where concentrations from 0.2 to 4 mg/L may be incorporated, depending on conditions [51]. The amount of oxygen incorporated at bottling is the sum of the dissolved oxygen and the headspace oxygen, which is called TPO (total packaged oxygen). By our experience, between 0.5 and 1.5 mg/L of dissolved O2 is usually incorporated at this process. Moreover, the oxygen in the headspace changes depending on the type of closure. In submerged caps, the headspace height is commonly 1–2 cm, and the normal values of dissolved oxygen ranged from 0.5 mg/L (with the use of inert gases) to 2 mg/L (without inertization). In the case of screw caps, the headspace height is higher, about 4 to 6 cm, and the oxygen values ranged from 2 to 6 mg/L. In summary, in submerged caps, values of TPO around 1 or 2 mg/L could be optimum, but values over 3 mg/L are not suitable. In screw caps, TPO values around 2.5 mg/L are optimum, but values over 7 mg/L are not suitable. The type of caps employed not only changes the amount of oxygen incorporated at bottling but also is the ultimate barrier physic to protect wines during the storage period. Thus, a correct cap should be selected depending on the type of wine, and also its permeability to oxygen should be measured to estimate the optimum storage period. The measure of the oxygen transmission rate (OTR) helps to carried out these purposes. Figure 4 shows “high” and “low” oxygen permeability of different types of caps measured in VITEC by the MOCON® equipment. The OTR measurement corresponds to two natural corks stoppers. As can be seen in the figure, the cork stopper represented in green reached the stability of the oxygen permeability at 24 h, while the stopper represented in red did not reach this stability until the third day. Moreover, once reached the stability, the values of OTR were 4 times higher for “red” stopper than for “green”. It can be also observed a great decrease in the case of the “red” stopper, likely due to higher content of oxygen inside of the cork and therefore higher porosity.
Representative oxygen transmission rate (OTR) of caps with different oxygen permeability.
The addition of chemical substances to wines is the most used alternative to reduce the SO2 addition in wines. Over the years, the addition of several chemical substances has been allowed by the OIV with different purposes. Accordingly, new antioxidant and antimicrobial additives have been evaluated as possible alternatives to the use of the SO2 [37, 52]. Particularly, the addition of dry yeasts enriched in glutathione, chitosan, and dimethyl dicarbonate, and different hydrolyzed and condensed tannins were evaluated by our research group. The most relevant results and some considerations related to these practices are summarized below.
In the last years, the potential application of glutathione (GSH) has increased the attention of many winemakers and researchers. The addition of reduced glutathione to grape juices or wines is allowed by OIV up to 20 mg/L (OIV OENO 445/2015). The use of GSH in the wine production was reviewed in 2013 by several authors [36, 53]. Following studies also demonstrated that the combination of SO2 and GSH involves a notable protective effect in wines [54]. Recent studies have shown that the addition of glutathione-rich dry inactivated yeast to grape juices modifies the white wine aroma influencing the concentrations of some volatile compounds and precursors with some benefits on its preservation [55, 56, 57]. The GSH amount of wine changes depending on the winemaking period. Hence, this compound decreases after wine aging and storage; at pressing could increase its content up to 20 times [58].
Chitosan is a natural polymer formed by deacetylation of chitin, which has a wide range of applications in different field research, such as agriculture, food, and pharmaceutical industry, among others [59]. The use of this polysaccharide in oenology was approved in 2009 by the OIV to fining musts (OIV-OENO 336A-2009). Moreover, it also used as antimicrobial and antioxidant. Chitosan allows the growth of Saccharomyces strains but is an antimicrobial against Brettanomyces, acetic, and lactic acid bacteria [60, 61, 62, 63]. Commonly, it is used to preserve wine from oxidation and also as fining agent for white wine protein stabilization [64, 65]. Figure 5 shows the potential of chitosan as antimicrobial. In this case, a significant decrease on yeasts, LAB, and AAB after the addition of 10 g/hL of chitosan to Tempranillo wines (after alcoholic fermentation) was observed. This effectiveness was greater for yeasts, decreasing up to 1 × 104 cfu/100 mL.
Viable yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) quantified in Petri dishes culture (cfu; colony-forming units) from Tempranillo wines before and after a treatment with chitosan (10 g/hL). *Significant differences by HSD Tukey test (p < 0.05).
Dimethyl dicarbonate (DMDC) was also accepted by European Union to be used in wine with a maximum limit amount of 200 mg/L (Regulation (EC) No 643/2006). DMDC is an organic chemical compound, which acts inhibiting the growth of microorganisms [9, 66]. When it is added to wines, it is quickly transformed to methanol and produces certain content on methyl and alkyl carbonates as products reaction by polyphenols or organic acids. These products are usually found at a low concentration, and so the quality of wine, flavors and aromas, should not be affected [67]. DMDC seems to be more effective against yeasts than against bacteria, although its activity depends on several factors, such as the pH [66, 67, 68]. In this sense, Figure 6 shows the results obtained by the addition of DMDC to Albariño musts. The above-mentioned antimicrobial effect can be observed in yeast, LAB, and AAB. However and as occurred with chitosan, DMDC treatment was clearly more effective in yeasts than in bacteria.
Viable yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) quantified in Petri dishes culture (cfu, colony-forming units) from Albariño musts treated with dimethyl dicarbonate (DMDC = 20 g/hL). *Significant differences by HSD Tukey test (p < 0.05).
The addition of oenological tannins to wine is an accepted practice by the OIV (OENO 12/2002 and revisions OENO 5/2008, OENO 6/2008, OENO 352/2009, and OENO 554/2015), which mainly aims the color stabilization and the improvement of the wine mouthfeel and flavor. Quite a few studies have evaluated the influence of the tannin addition on the chemical and sensory properties of wines. However, the results obtained are not as promising as expected. In 2005, Bautista-Ortiz et al. [69] did not observe any improvement on the chromatic and sensory properties of wines treated with different oenological tannins. Harbertson and co-workers [70] observed that some additions may be unjustified and have limited or negative impacts on the wine quality. A wide range of commercial tannins exists on the market; nonetheless, a lack of information about the composition and origin of the product is a common pattern. This fact could lead to technological problems according to the expected final wine [71]. The antioxidant properties of tannins, with related health beneficial effects, and their benefits when added to wines are also well known [72]. Both characteristics make tannins a very attractive alternative to the use of SO2 in wine. Some studies showed hopeful results when mixed with antimicrobials, such as lysozyme [17, 73]. The studies carried out in VITEC have recently shown that the addition of tannins mixed with glutathione may be an effective alternative to the use of SO2 [74]. Figure 7 shows the sensory analysis of Tempranillo wines with addition of grape seed tannins (ST), grape skin tannins (SKT), oak tannins (OAK), and tara tannins (GAL). In general, the sensory profiles of wines produced with the addition of different tannins were similar (and even better) than wines elaborated by addition of SO2. Significant higher color intensity was observed between control and treated wines. Treated wines also obtained significant dryness and tannic intensity. Astringency and mouthfeel reached higher values but not significant. Lower persistence and higher aroma intensity can also be observed. Low differences between treatments were found, which may be due not only to the different quantity of tannins added but also to their qualitative profile. Recent studies performed by other authors have confirmed the importance of the anthocyanin/tannin ratio on the wine oxidation process and especially on the acetaldehyde formation. Wines with higher tannin addition showed lower production of acetaldehyde [75].
Sensory profile of Tempranillo wines elaborated by different enological tannin additions to grape juices. SO2: Wine control. ST: Grape seed tannins (40 g/hL), SKT: Grape skin tannins (30 g/hL), GAL: Tara tannin (20 g/hL), OAK: oak tannins (30 g/hL). *Significant differences by HSD Tukey test (p < 0.05).
Other chemical substances, such as ascorbic acid and lysozyme, may also be able alternatives to SO2. Ascorbic acid has the ability to scavenge molecular oxygen before the oxidation of phenolic compounds occurs. It is a highly efficient antioxidant in combination with sulfur dioxide; nonetheless, a pro-oxidation effect may occur when the content of SO2 and ascorbic acid is low [76]. The reaction between ascorbic acid and oxygen results in dehydroascorbic acid and hydrogen peroxide, which would be removed by sulfites. Under certain conditions, ascorbic acid both accelerates oxygen removal and reduces the O2:SO2 molar reaction ratio [4]. In wines, it is generally employed in winemaking stages with high oxygen dissolution, such as grape crushing, after racking or just before bottling. The addition of ascorbic acid in white wines improves color and flavor retention during bottling aging [77]. Certain carbonyl compounds, such as furfural, acetaldehyde, glyoxal, and diacetyl, formed from the oxidation of ascorbic acid may involve the formation of brown pigments by reacting with phenolic compounds. Higher browning was observed in catechin model solutions containing ascorbic acid than in model solutions containing sulfite [78]. These oxidation products of ascorbic acid bind to SO2 reducing in some extent the ratio between free and total SO2 content [76]. The mixture of ascorbic acid together with SO2 seems to be a better antioxidant combination than the use of SO2 alone, avoiding the oxidation of wine and preserving the aroma profile. In white wines, ascorbic acid provides considerable protection against oxidation under conditions of low oxygen [79]. However, it should be highlighted that the impact of the addition of ascorbic acid to wine composition and sensory characters is far to be clarified [36, 77].
Lysozyme belongs to glycoside hydrolases, which is a type of enzyme that catalyzes the hydrolysis of bonds between N-acetyl muramic acid and N-acetyl-D-glucosamine residues in peptidoglycans, and it is found in the cell walls of bacteria, especially in Gram-positive bacteria. These enzymes are therefore destructive to many bacteria like lactic acid bacteria (LAB). In winemaking, indigenous LAB, such as Lactobacillus brevis, Oenococcus oeni, Lactobacillus kunkeei, Pediococcus parvulus and Pediococcus damnosus, can be completely inhibited by lysozyme, being this efficacy strongly affected by winemaking and dosage [80, 81]. The addition of lysozyme did not have any negative effect on yeast growth and sugar reduction and may prevent the increase of volatile acidity during the stuck/sluggish of the alcoholic fermentation [17, 81]. This substance had little or no effect on the content of alcohol, titratable acidity, and pH value and did not cause important changes on the sensory characteristics of wines. Nonetheless, it may produce esters in certain wines, contributing to their complexity [73, 82]. Lysozyme may involve changes on yeast nitrogen consumption and the amino nitrogen metabolism, although it does not appear to have an effect on the formation of biogenic amines [16]. The addition of lysozyme may produce a color loss associate with the formation of precipitates in red wines and may induce protein haze in white wines [82]. Lysozyme does not possess an antioxidant activity and therefore does not prevent the wine oxidation. Hence, it becomes necessary the addition of antioxidants, such as proanthocyanidins, in combination with lysozyme to replace the SO2 actions [16, 73]. A critical point of lysozyme is the safety of wines treated with this additive, since it is an egg allergen (allergen Gal d 4 according to the International Allergen Code) that remains in bottled wine. The OIV issued limitation of 500 mg/L [83], and this quantity is removed by an efficient fining treatment using, for example, bentonite or metatartaric acid [84].
The use of yeast strains with a low capacity to produce SO2, during the alcoholic fermentation is essential to reduce the final amount of SO2 in wines. Both commercial and indigenous yeasts strains can be used with this purpose. However, factors as grape juice composition, the management of the fermentation, and musts supplementation will be decisive. Different physical technologies and methodologies can be used to elaborate this type of wines. The replacement of the antioxidant and antimicrobial action of the SO2 is a complex mission. However, the combination of different physical techniques together with a good management of inert gases to control oxygen appears to be a suitable practice to achieve this purpose. In addition, some chemical treatments will help to complete the effects caused by these practices. In general, chemical treatments should be combined at different wine production stages to complete their respective actions. The combination of chemical additions even with SO2 may help to reduce its use during the winemaking. It should be noted that still today, there is a lack on the knowledge of the microbiological stability of SO2-free wines during the aging period. Therefore, more research is needed to better understand the effect of the low concentration of SO2 in wines as well as the use of new additives, especially regarding the wine stability after storage and the effects on the human health.
In summary, multidisciplinary approaches should be considered to elaborate high-quality SO2-free wines. The combination of microbiological strategies, physical methods, and chemical treatments becomes indispensable to achieve this ambitious purpose. Several yeast strains are able to generate low quantities of SO2 during alcoholic fermentations (<10 mg/L), and several physical and chemical treatments have shown their antioxidant and antimicrobial effect. Therefore, reducing the SO2 amount in wine production may be achieved. Nonetheless, more research should be done to adapt winemaking procedures according to the particular working conditions and the desired product of each winery.
Thanks are due to the Spanish MICINN for their financial support of VINNO_SO2 Project (Ref. IPT-2012-0967-060000). The authors also thank AGROVIN S.A. for supplying the yeast strains, Bodegas RODA S.A. (Haro, La Rioja, Spain) and Adegas Valmiñor S.L. (O Rosal, Pontevedra) for supplying the grape samples. We also thank Programa de Desenvolupament Rural de Catalunya 2014–2020 (N° expdte. 56 30032 2017 2A).
Four hundred years back, Paracelsus stated that, “All substances are poisons; there is none which is not a poison.” If the right dose is taken, it could become a remedy, otherwise poisonous [1, 2]. The therapeutic index or ratio, i.e., LD50/ED50, tells whether the chemical is safe or not.
Poisons are generally found in cases of homicides, suicides, or accidents. They have a significant role to play as the silent weapon to destroy life mysteriously and secretively.
Every poison has almost similar action on the victim’s body. In many cases, they either stop the transfer of O2 to the tissues or create an obstacle in the respiratory system by inhibition of enzymes which are associated with the process. In this, the myoneural junction and the ganglions and synapses are the sites of action. In some cases of insecticidal poisoning, hyperexcitement of voluntary and involuntary muscles can cause death. There are four categories of action of poisons—(i) local action, (ii) remote action, (iii) local and remote actions, and (iv) general action.
Local action: Local action means direct action on the affected site of the body. Examples include irritation and inflammation in strong mineral acids and alkalis, congestion and inflammation by irritants, the effect on motor and sensory nerves, etc.
Remote action: Remote action affects the person due to absorption of that poison into the system of that person. For example, alcohol is absorbed in the system and then it affects the person.
Local and remote actions: Some poisons can affect both local and remote organs. Thus, they not only affect the area with contact to the poison but also cause toxic effect after absorption into the system, for example, oxalic acid.
General action: General action means the absorbed poison affects more than one system of the body, for example, mercury, arsenic, etc.
Toxicity of a poison depends upon its inherent properties such as physiochemical as well as pharmacological properties.
The action of poisons mainly depends upon the following factors discussed below:
Forms of poison: There are three forms of poison:
Physical form: Gaseous/volatile/vaporous forms of poisons act faster than liquid poisons as they are quickly absorbed. Similarly, liquid poisons act faster than solid poisons.
Gaseous or volatile > liquid > solid.
For solid poisons, powdered poisons act quickly than the lumps. For example, there are certain seeds that escape the gastrointestinal tract as they are solid, but when crushed, they can be fatal.
For solids: powdered > lumps
Chemical form: Few substances like mercury or arsenic are not poisonous as they are insoluble and cannot be absorbed when they are in combination with other substances like mercuric chloride, arsenic oxide, etc.
In other cases, the action is vice versa. For example, there are some substances that become inert in combination with silver nitrate and hydrochloric acid and are deadly and poisonous when present in pure forms.
Mechanical combination: The effect of poisons is significantly altered when they are combined with inert substances.
Quantity: Large doses of toxin cause much lethal effect. But this statement is not always true. For example, sometimes when a toxin is taken in very large amount, the body produces a mechanism against it such as vomiting, and thus the intensity of the toxin is reduced.
Concentration: The absorption speed of poison is dependent on concentration; thus poison of higher concentration is fatal. However, there are still some exceptions. For example, a dilute oxalic acid is less corrosive, but the absorption rate is high and so it is more dangerous.
Methods of administration: It has a unique role in the process of absorption. It is fastest through inhalation and then through injection as compared to the oral mode.
Condition of the body: Different persons react differently when exposed to a poison. It is because the condition of our body is also responsible for the increase or decrease of the effect of a poison on the body:
Age: Children and older people are more affected than an adult by the same quantity of toxin.
Sleep: The body functions are slower during sleep; thus toxin circulation in the body is also slower.
Health: Healthy persons can tolerate a toxin better than a weak or ill person.
Dosage: The effect of the poison depends upon its dosage. It is said that the dose determines whether a substance is a poison or remedy. A substance is usually considered a poison after a certain fixed quantity. Although this quantity is not fixed for all people, it is considered according to the average effect on the population. There are two considerable effects of poison on the body of a person; these are the subtle long-term chronic toxicity and immediate fatality.
Some poisons are lethal in microquantities, while others can affect in large doses. The significance of a dose can be understood by taking an example of a metal essential in the food, for example, iron, copper, manganese, zinc, etc.; if its dose is higher than the body requires, it can be lethal.
Effective dose (ED): The effective dose is the quantity of a substance at which it shows its effect in the population. In most cases, ED50 is measured as a dose which induces a response in half of the targeted population.
Lethal dose: The lethal dose (LD) 50 is the amount of drug which is expected to cause death of 50% population.
Hypersensitivity: It is basically the type of reaction initiated by the body against any other substances. Sometimes, it could be related to allergy. There is an assumption that hypersensitivity does not depend on wrong doses. Every person who is hypersensitive to a particular substance has a dose related that defines the quantity required to cause hypersensitivity to that person. The allergic response is actually a toxic response and can be sometimes fatal.
Idiosyncrasy: It is defined as a reaction produced by the body to a chemical genetically. It is a type of person that affects only those people who are genetically sensitized to that particular chemical or substance but will show no effect on others. In such cases, the person experiences discomfort for several hours or if the dose is high can be fatal also. For example- peanut allergy in some people.
Tolerance: It is the capability of a person to not produce any effect against a chemical that usually causes reaction to normal persons. It is a state of reduced or no reaction to a chemical. There are basically two types of mechanism that induces tolerance. First is when the toxin reaches the effective site, its quantity is very less. This is called dispositional tolerance. The second is because the tissues show reduced response to the toxin.
Tolerance can also be achieved if a drug is taken in a small quantity on a regular basis. This can be explained by taking the example of alcohol. When any human consume alcohol for the first time, he/she will show an effect even when the quantity is small, but eventually the effect will decrease and the person can tolerate a large amount also.
Individual susceptibility: It is defined as the different kinds of responses produced by different individuals to a particular harmful compound. It can be due to occupational or environmental factors and exposures. It is determined by complex genetic factors. Its effect depends upon the intensity of exposure. There is a gene uniqueness that varies from person to person; thus the same amount of exposure can show no effect in one individual, cause illness to other individual, and also could be fatal to someone as well.
The route of administration is the path through which a drug, toxin, or poison is taken or administered into the body of a person which is distinguished by the location where any drug is applied. It is mostly classified on the basis of its target:
Topical—which has a local effect
Enteral—which has a wide effect, i.e., affect the whole system
Parental—which follows a systemic action
Poisons are given or taken so that death can occur at once by shock due to stoppage of body’s vital systems. Drug addicts take drugs through inhalation or injection.
Route of administration plays a very important role in determination of death by poison as time in which death occurs are fastest in inhaled poisons, relatively slow in injected and lastly when ingested orally.
Some important features that are considered during the administration of poisons and can make a poison fatal are:
Rate of dissolution of the poison that depends upon the physical form of the poison, i.e., gaseous, vapors, liquid, solid, etc.
The surface area affected at the site of administration of the poison
The circulation rate of blood in that route
The solubility of the poison, i.e., lipid soluble or water soluble
The concentration of the poison
The time required by the poison to be absorbed completely from the site of administration
Routes of administration can be classified into two categories:
Enteral routes/gastrointestinal routes.
Parenteral routes.
Enteral routes: When the drug is administered through the gastrointestinal tract, it is defined as an enteral route. It has both oral and rectal routes. It also includes sublingual and sublabial routes. It is comparatively a slower mode of action for absorption of drugs:
Oral route: Generally absorption takes place in the tongue and the gums of the oral passage. The pH of the buccal cavity and mouth ranges from 4 to 5. Sublingual and supralingual routes have a significant role in absorption. The sublingual absorption is faster as the toxin is transformed directly to the heart, but it takes more time.
Rectal route: Administration of drugs can be done through anus which directly absorbed in bloodstream through membrane of mucous. This administration can cause the burning of tissues or bleeding in rectum as the area is very sensitive.
Parental route: It includes all the other routes that does not involve the gastrointestinal tract. It has a systemic effect on the body. It has the following categories of administration:
Intradermal: Here, the administration of drugs takes place from surface of skin. This type of poisoning is mostly found in chronic poisoning cases.
Intravenous: It is one of the fastest modes of drug administration as the injection is directly taken and the drug is transferred directly into the veins and thus is directly circulated into the blood quickly. Immediate death might be caused by this type of drug.
Intraosseous: It involves an administration of a drug directly into the bone marrow. This mode is actually used for administration of drugs for medical purposes.
Intra-arterial: It involves an administration of a drug into the artery directly through injection. It is a fast mode of administration.
Intramuscular: In this mode, the drug or poison is administered into the muscle of the thigh, upper arm, or buttock. The time required in this mode is greater than other parental modes.
Subcutaneous: In this mode, the drug is injected into the layer beneath the skin, i.e., the subcutaneous layer. The drug then goes to the small blood vessels and then to the bloodstream. This mode is used for mostly those protein drugs that would be destroyed if administered through the gastrointestinal tract.
Inhalation: In this mode, the nose is the primary path. Because of the presence of mucous membrane, the nasal aperture is very absorptive. The microparticles of poisons are easily absorbed and transported quickly to the lungs. From the lungs, they are circulated into the blood.
Poisons are classified into two ways:
Based on their action on the body.
Based on their physical and chemical properties [1].
Classification based upon the effect of poison on the body:
Corrosive: The poisons burn the tissues or organs when they come in contact with them, e.g.:
Strong acids such as H2SO4, HNO3, HCL, etc.
Strong alkalis such as hydroxides of Na, K, NH4, etc.
Irritants: The poisons irritate the tissues or organs when they come in contact with them [3]:
Inorganic:
Nonmetallic phosphorous, chlorine, bromine, iodine, etc.
Metallic salts of arsenic, antimony, mercury, copper, lead, zinc, etc.
Organic:
Vegetable—castor oil, madar, croton oil, etc.
Animals—snake venom, cantharides, insect bites, etc.
Mechanical—glass powder, needles, diamond dust, hair, etc.
Neurotics: Poisons affect the nervous system and the brain [3]:
Cerebral:
Narcotic—opium and its alkaloids
Inebriant (depressant)—alcohol, ether, chloroform, and chloral hydrate
Spinal:
Excitant (stimulants)—nux vomica and strychnine
Depressant—gelsemium
Cardiorespiratory:
Cardiac—aconite, digitalis, oleander, and hydrocyanic acid (HCN)
Asphyxiants—carbon monoxide, carbon dioxide, and hydrogen sulfide
Miscellaneous: A number of chemicals having diverse actions on their body are included in this group [4]:
Animal poisons
Curare (an arrow poison)
Poisonous food articles
Industrial poisons—methyl isocyanate (MIC)
Fuels—petroleum and kerosene
Insecticides—endrin, dichlorodiphenyltrichloroethane (DDT), and
naphthalene
Radioactive substances
Classification of poisons based upon their properties:
Inorganic poisons
Metallic poisons:
Arsenic: It has been the most known and exclusively used throughout
the ages to poison men and animals [1].
It is a white tasteless powder and a pinch of the poisons can kill two adult persons.
Arsenic for homicidal purposes is mixed with various food articles, e.g., cooked food, milk, tea, liquors, or medicines.
Arsenic in a metal form is not poisonous; its oxides are highly poisonous. It is extensively used in insecticides, etc. [5].
Mercury: Chloride and nitrites of mercury are highly poisonous. They
are used in chemical industry and as fungicides.
Lead: Most of its compounds are poisonous. This is a slow poison,
e.g., Sindoor adulterated with red lead oxide.
Copper: Its salts are used in electroplating; copper sulfate is a poison.
Thallium: Thallium salt is used as rat poison [6].
Antimony: Its effect is like that of arsenic.
Nonmetallic poisons:
Cyanides: Cyanides of potassium and sodium are extremely
poisonous, even in small quantities. They react with the acid of
gastric juices in the stomach to form hydrocyanic acid, which
paralyzes the respiratory center in the brain resulting in death due to
respiratory failure [4].
Yellow phosphorus: In olden days it was used in match industry and
several times proved highly poisonous.
Iodine: Only elemental iodine in high quantity is poisonous.
Strong acids and alkalis: These are highly poisonous with corrosive
effects, e.g., sulfuric acid, nitric acid, sodium, potassium
hydroxides, etc.
Gases: Phosphine gas kills rats when used on the rat holes and is
poisonous for infants. MIC killed over 2000 persons and invalidated
several others in a gas leak tragedy in Bhopal in 1984. Some other
poisonous gases are HCN, carbon monoxide, hydrogen sulfide,
arsine, etc. [3].
Organic poisons
Volatile poisons:
Ethyl alcohol: It is poisonous if taken in excess.
Other alcohols: Methyl alcohol and isopropyl alcohol are poisonous.
Methanol, used in polish and chemical industries, is used in illicit
liquor, and its intake causes paralysis, blindness, and death [3].
Phenol: Phenol or carbolic acid could be poisonous. It is mostly used
as a disinfectant [6].
Miscellaneous substances: Various industrial chemicals like
chlorinated hydrocarbons, benzene, chloral hydrate, etc. are
poisonous. In several cases of poisoning, chloral hydrate could be
used in illicit liquors.
Nonvolatile substances:
Alkaloids: Several narcotics and vegetable poisons contain alkaloids,
e.g., strychnine, morphine, cocaine, nicotine, etc.
Barbiturates: These drugs are synthetic and induce sleep [1].
Glycosides: These drugs can cause cardiac arrest and could be fatal
such as aconite, oleander digitalis, etc.
Insecticides and pesticides
Poisoning: It is known as the injurious effect caused by the action of a poison or a detrimental chemical substance. It leads to the development of adverse reaction toward the harmful chemicals or drugs. It is basically differentiated in three categories: suicidal, homicidal, and accidental. Cattle poisoning is the poisoning related to animals. Accidental poisoning is caused by negligence and carelessness. Homicidal poisoning includes the killing of a person due to the poison. Suicidal poisoning refers to the use of toxic chemicals in order to kill oneself.
Corrosive poisoning: It is caused by poisons such as acids and alkalis. They produce a corrosive action on the human body by causing ulcers and acute inflammation.
Metallic poisoning: Metals such as arsenic, mercury, lead, etc., when ingested, cause a deleterious effect. This is known as metallic poisoning.
Plant poison: The study of plant poisons is known as phytotoxicology. Plant poisons, or phytotoxins, comprise a vast range of biologically active chemical substances, such as alkaloids, polypeptides, amines, glycosides, oxalates, resins, toxalbumins, etc.
An alcohol is a drink that contains ethanol. Ethanol is made by fermentation of grains, fruits, and some resources of sugar. Chemically, it is a group of compounds whose saturated carbon chain has a “-OH” group. Alcohol is also a depressant, and in low dose, it can reduce tension, cause euphoria, and improve sociability, but in high dose it can cause stupor, drunkenness, and even death. Regular alcohol intake can cause cancer, alcoholism, dependency, etc. 33% of the total people in the world consumes alcohol. Drinks containing alcohol are broadly classified into three classes, i.e., beer, spirit, and wine, whose alcohol content varies between 3% and 50%. When diluted, alcohol has nearly sweet taste, but when concentrated it gives a burning sensation. 90% of the absorbed alcohol is metabolized by the liver and broken down into less toxic metabolites. Alcohol acts on the central nervous system (CNS) as a depressant on the cells of the cerebral cortex. Its adverse effects like a decrease in cognitive and psychomotive skills are well documented. Alcohol percentage (ABV) differs from one brand to another, for example, beers contain 5%, wines contain typically 13.5%, fortified wines contain 15–22%, spirits contain 30–40%, fruit juice contains less than 0.1%, and cider/wine coolers contain 4–8% ABV [1].
The goal of blood alcohol test is to check the concentration of alcohol in the body. This test result is known as blood alcohol concentration (BAC) which indicates alcohol % in the blood. It is directly proportional to the alcohol in the body, and alcohol hinders with people’s decision, control on them and other characteristics [3]. This test can tell the presence of alcohol in blood for 12 hours [4]. Blood quickly absorbs alcohol and is measured within minutes of consuming alcoholic drink. The highest level of BAC result can be reached within an hour of consuming alcohol. Intake of food can vary the result. Liver breaks down almost 90% of alcohol and rest are given out from exhalation and urine [5].
In case of deaths due to alcoholic intoxication, the viscera is collected and preserved in saturated saline. Preservation of sample is very important as if wrongly preserved it can ruin the examination. Generally, urine and blood are taken as samples.
A sterile needle must be cleaned up by the swab of a nonalcoholic disinfectant like aqueous mercuric chloride and aqueous benzalkonium chloride (Zephiran) before the suspect’s skin is punctured with it. The use of an alcoholic disinfectant either may give false-positive results or may contribute to falsely high alcohol contents of blood. About 5–10 ml of the sample (blood) is taken in a test tube; an anticoagulant such as potassium oxide and EDTA and a preservative such as NaF are added and stored in the refrigerator at 40°C. The anticoagulant will prevent blood from clotting, and the preservative will inhibit the presence of microorganisms. The urine sample is also collected in the usual manner and preserved with 30 mg of phenyl mercuric nitrate for every 10 ml of urine [6].
Ethyl alcohol is isolated from biological materials by acid distillation. Viscera, vomit, stomach contents, and other materials should be analyzed separately. About 50–100 g of the viscera is taken and is finally minced by thin gruel and adding water (3–5 times) and sulfuric acid. It is passed to steam distillation which is generally heating it on the water bath. The condenser and the receiving flask should be well cooled with ice especially in the hot season, the outlet of the condenser being dipped in little water or NaOH solution. Some pieces of pumice stone are stored in the flask to avoid bumping. It is better to collect the distillate in 4–5 fractions, out of which the first one should not exceed 20 ml and the remaining fractions should be 50 ml each. The distillate contains alcohol and other volatile acids, etc. [6].
There are some tests which show the presence of ethyl alcohol in the exhibits.
Also known as triiodomethane reaction, it is used in the detection of CH3CH (OH) which is present in alcohol. There are mainly two types of different mixtures used in this reaction which are mainly chemically equivalent. A pale yellow precipitate occurs if the result is positive [6].
In the above structure, “R” can be hydrogen or alkyl group or any other hydrocarbon group. In case when R denotes hydrogen, then the compound we have the possibility to find is primary alcohol ethanol. Ethanol is the only alcohol that gives an iodoform reaction. In case R is any hydrocarbon group, then it gives secondary alcohol groups. Tertiary alcohol is not able to contain R group because of the absence of hydrogen atom [7].
In 1 ml of distillate, a few drops of 10% NaOH are added dropwise till the solution becomes brown and warmed for a few minutes. A few drops of iodoform solution are added to change the color to yellow. The mixture has to be again heated on low flame/water bath; a yellow-colored precipitate is formed on standing. The precipitate has to be observed under a microscope. Characteristic hexagonal crystals of iodoform are seen which usually shows the presence of ethanol, acetaldehyde, isopropanol which on standing for long time breaks into flower like structure. This test initially involves oxidation followed by substitution and hydrolysis [6].
Add 1 gm of molybdic acid in 25 ml of a concentrated sulfuric acid which has the reagent. Mix 2 ml of this reagent when hot and with 2 ml of distillate. At the junction of both liquids, a ring will be formed which is deep blue in color. On shaking, the whole mixture will become deep blue which is due to ethyl alcohol. This test is very sensitive and it gives a negative result with acetone, acetaldehyde, and dilute solution of methyl alcohol. Only the strong solution of methyl alcohol gives a light blue color after several minutes [6].
Mix two drops of benzoyl chloride with 2 ml of the distillate. Add 10% of sodium hydroxide drop by drop till the solution becomes alkaline. By providing heat the irritating smell of benzoyl chloride will be replaced by sweet fruity odor of ethyl benzoate. Methyl alcohol gives this test also but not the iodoform test [6].
In case of drunkenness, alcohol detection in the body is very important. Observing behavioral abnormalities of the suspect is the best method, but analyzing the breath, blood, and urine is the only way of confirming it. The analysis of breath alcohol can be performed on the spot with the help of breath-analyzer instruments like Alco-Sensor, Breathalyzer, etc. However, the alcohol content of the blood could be determined by using the modified version of the Kozelka and Hine/Cavett method [6].
In recent years, several methods in determining the alcohol in body fluids are described. Kent-Jones and Taylor reported the results of an investigation into the merits of two methods—the micro Cavett and that of Kozelka and Hine. The micro Cavett method is more accurate, but it suffered from serious inconsistencies in reproducibility, but the Kozelka and Hine method is less accurate and more time-consuming but gives good reproducibility.
Nickolls modified the micro Cavett method which appears to give a more accurate result in comparison with the unmodified method. The simplicity of this procedure increases its use for routine work in laboratory [8].
The principle behind this method is the oxidation of alcohol, which is easy with acetic acid in the presence of oxidizing agents such as sulfuric acid and potassium dichromate. Reduction of each mL of N/20 potassium dichromate solution takes place that is equivalent to 0.575 mg of alcohol [6].
This formula is used to estimate the amount in which alcohol is present in the body.
a. For blood analysis
Here, a = Total amount of alcohol absorbed in the body; p = Weight of the person; c = Concentration of alcohol in the blood; r = Constant which is 0.5 in women and 0.68 in men
b. In urine analysis.
Here, a = Total alcohol content present in the body; p = Total weight of the person; q = Alcohol concentration in the urine; r = Constant, namely, 0.68 for men and 0.5 in women [6].
There are several methods in determining ethanol in the blood, urine, and serum. One of the most important methods is gas chromatography (GC). The sample is injected in a heating chamber, and due to its high temperature, alcohol converts in vapors which are carried by inert carrier gas such as nitrogen through the column which is packed by an adsorbent material. Separation of different types of components depends on their different affinity, i.e., partition coefficient toward adsorbent phase which is stationary and later detected as shown in the figure below. A chromatogram so obtained helps in qualitative as well as quantitative analysis [6].
Various components of gas chromatography are [9]:
Carrier gas
Flow regulator
Injector
Column
Stationary phase
Oven
Detectors
Display device
The area covered by the peak represents the amount and position of a particular type of compound [6].
Operating conditions [10]:
Column: Porapak polymer bead 80–100 mesh or its equivalent, which can separate or resolve the ethanol.
Column temperature: 1600°C.
Carrier gas: Nitrogen.
Rate of gas flow: 50 ml/minute.
Detector: Flame ionization detector.
Alternative operating conditions:
Column: 0.3% Carbowax 20 M on 80–100 mesh Carbopak C, 2 m × 2 mm ID or its equivalent.
Column temperature: 350°C for 2 minutes and then programmed at 50°C per minute to 1750°C and hold for at least 8 minutes.
Carrier gas: Nitrogen at 30 ml/minute [6].
The purpose of this chapter is to discuss the mode of action and function of poisons once they reached in the human body. The impacts of poisons are severe and even cause death if not treated properly.
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