Impact of Using Organic Yeast in the Fermentation Process of Wine

The aim of this study was to find out what kind of “Bianca” wine could be produced when using organic yeast, what are the dynamics of the resulting alcoholic fermentation, and whether this method is suitable for industrial production as well. Due to the stricter rules and regulations, as well as the limited amount and selection of the permitted chemicals, resistant, also known as interspecific or innovative grape varieties, can be the ideal basic materials of alternative cultivation technologies. Well-designed analytical and organoleptic results have to provide the scientific background of resistant varieties, as these cultivars and their environmentally friendly cultivation techniques could be the raw materials of the future. The role of the yeast in wine production is crucial. We fermented wines from the “Bianca” juice samples three times where model chemical solutions were applied. In our research, we aimed to find out how organic yeast influenced the biogenic amine formation of three important compounds: histamine, tyramine, and serotonin. The main results of this study showed that all the problematic values (e.g., histamine) were under the critical limit (1 g/L), although the organic samples resulted in a significantly higher level than the control wines. The glycerin content correlated with the literature values, since it is well known that the glycerin-pyruvic acid transformation results in a 6–10 g/L concentration.


Introduction
Due to the recent trends in environmentally friendly and health conscious food consumption, organic products and organic wines produced in sustainable farming systems are gaining larger significance all over the world. Thus, organic yeast has also appeared among oenological products. Organic yeast is a selected yeast propagated and dried with biological methods. Due to the substrate used for the propagation, organic yeast originates from regulated organic production. Organic wine production requires special attention, not only in the vineyard, but in the whole winery. The process of drying is carried out carefully, and is regulated by the relevant EU rules for organic production [1] and the official NOP standards in the US. The packaging of organic yeast is also a certified organic material. According to the rules and standards, organic yeast production utilizes substrates deriving only from organic agriculture and GMO-free materials.
Several studies focus on the effects of organic yeasts, especially on the amount of biogenic amine, which is a beneficial, physiologically active compound in organic wines.
Yeast is an oval-shaped unicellular fungus with a eukaryotic structure, which is more developed than that of a prokaryotic organism.
body [10]. The effects of the biogenic amines studied in our recent publication are detailed below [9,[11][12][13][14]: • Methylamines: Methylamines are consumed via food, and consist of choline, carnitine, betaine, trimethylamine N-oxide (TMAO), and phosphatidylcholine [15]. Choline is an essential nutrient, which is present in higher amounts in egg yolk and liver. Together with betaine, it has several physiological functions. A high TMAO level of blood can prevent hyperammonemia, and it functions as osmo-protectants in case of osmotic stress [16]. TMAO reduces the neurotoxicity of glutamate, which is related to myocardial infarction and stroke [17]. In spite of these facts, it can be detected in higher levels in case of diabetes, Alzheimer's disease, and cardiovascular diseases. Its main function in the human body is the regulation of glucose transport in adipose cells, and supposedly the stimulation of the release of neurotransmitters through sodium channels of the nervous system. Neurotransmitters have a significant role in the transport mechanisms through the plasma membrane. • Ethylamine: Ethylamine is a component of mammalian, including human, urine. According to observations, it is excreted in higher amounts in patients with liver and kidney disease and with damage to the central nervous system [18]. It can be detected in plants, food products, animal waste, and even in tobacco smoke. In human physiology, it is connected to anesthetics, such as ketamine, which is a well-known NDMA-antagonist (N-methyl-d-asparaginate-antagonist), and recreational drugs, for example phencyclidine. NDMA is a neurotransmitter, which can substitute glutamate, while ethylamine is a possible precursor for ketamine [18]. • Histamine: Derives from histidine. The industry applies it scarcely: it can be a potential ingredient of pesticides and certain veterinary medicinal products. It is synthetized in tissues of living organisms, when allergens and antibodies meet, but it can be consumed via food as well. Histamine has both positive and negative properties regarding its effects on human physiology, depending on the way it enters the body. Its primary function lays in the allergic and inflammatory reactions. It participates in brain functions, and as a stimulus transmitter in the nervous system. It also has a significant role in indigestion (e.g., gastric acid production, intestinal motility, and mucosal functions), in the development of immune responses, or in the enhancements of the formation and metastases of tumors. Larger amount of histamine intake can cause an allergic reaction. Approximately 1% of the population has histamine intolerance (disorder in degradation and accumulation). Histamine poisoning is caused by consuming foods that are too high in histamine, for which histamine intolerance metabolic disorders are responsible, which can occur even after the intake of small amounts [19]. Symptoms include (migraine) headaches, flushing, runny or stuffy nose, arrhythmia, low blood pressure, digestive problems (such as diarrhea), or hives. Relief of symptoms can be achieved by a low-histamine diet, namely products with a lot of histamine should be avoided [20,21]. Larger amounts are contained in smoked foods, chocolate, and red wines. • Tyramine: Tyramine is formed from tyrosine. It is a side product of the fermentation and decomposition processes; it functions as a neurotransmitter in the human body, and is a metabolite of Escherichia coli. It can be found in large quantities in the basal ganglia and in the limbic system, so supposedly tyramine is connected to behavior and emotions [22]. It is a precursor in dopamine synthesis [23,24], which is a neurotransmitter, neuroendocrine-transmitter, and neurohormone. It is also called the reward hormone for its significance in the "reward mechanism" [25]. The following food products contain high amounts of tyramine: smoked, fermented or marinated fish and meat, fermented dairy products (tyramine derives from the Greek word "cheese"), fermented vegetables (for example sauerkraut), chocolate, various fruits and vegetables (banana, coconut, raisin, beans, etc.), and even yeast. Consuming large amounts of food with a high tyramine content can significantly raise blood pressure (even by 30 Hgmm or more). The tyramine concentration in wine (~25 µg/g) is considerably lower than in cheese (even 2000 µg/g). Tyramine can cause serious health problems with people taking certain antidepressants (e.g., monoamine oxidase inhibitors). • Serotonin: Serotonin is a monoamine neurotransmitter derived from L-tryptophane. It is a basic compound for the pharmaceutical industry, for example in antidepressant production. It has effects on the blood pressure, sleep and sexual behavior, state of arousal, and it also affects the manifestation of aggression. If a problem occurs in the serotonergic system, several diseases in the nervous system can be detected: depression, generalized anxiety disorder, obsessive-compulsive disorder, post-traumatic stress disorder, phobias, and epilepsy. Abnormally low serotonin levels lead to depression, which can result in suicide. High levels of serotonin have positive effects on health, such as improved perceptual ability and general well-being [26]. MDMA (3,4-methyl-enedioxy-methamphetamine) consumption comes with significant release of dopamine and serotonin [27]. During banana ripening, a large amount of serotonin is produced as well.

•
Putrescine: Putrescine is a cadaverine-related polyamine, which smells reminiscent of pepper. Industrial utilization: catalyst in resin technology, compound in medicines for hypertrophic scars after breast augmentation surgeries, and insect attractants. It promotes cell division in smaller doses, but in larger amounts it extends to toxic decomposition products. It affects reproduction (spermatogenesis), functions as a nerve protector, but also has significance in tumorigenesis. During the preparation of fatty foods on higher temperatures, putrescine can convert into N-nitroso pyrrolidine, which is a carcinogenic compound [28]. It is produced in both living and dead organisms. It is responsible not only for the unpleasant smell of carcasses but bad breath as well. Cheese (1560 mg/kg) and fermented vegetables (549 mg/kg) contain a high amount of it [29]. • Phenethylamine: Supposedly, phenethylamine (PEA) regulates the effectiveness of nerve cell connections (neuromodulator), and it is also a neurotransmitter [30]. It often can be found in food, especially in fermented products. Almonds and chocolate, and even tobacco contain high amounts of phenethylamine. Typically, people with schizophrenia have high levels of it in the urine, while children with attention-deficit hyperactivity disorder (ADHD) have low levels of PEA. There is no verified cure for ADHD [31], but a study states that PEA intake relieves symptoms almost instantly [32]. At the same time, it gets into the brain only in small doses, because monoamine oxidase-b converts it to phenylacetic acid. In certain cases, phenethylamine produced by the human body has similar effects as amphetamine [32,33]. • Cadaverine: Cadaverine is a foul-smelling ("death smell") diamine compound that is produced by the putrefaction of animal tissue, by the hydrolysis of proteins, although living organisms can emit it as well. Primarily, it was detected in bacteria; by now, it has been found in plant organs [34], for example in soy bean, chick peas, etc. It shows an elevated level in urine of persons with lysine degradation disorders. The pharmacological effects of cadaverine include low blood pressure, bradycardia (low heart rate), stiffness, or partial paralysis of the limbs [35]. Del Rio et al. [36] (2019) emphasized that sufficient information about toxicity is not available, but the NOAEL value (No Observed Adverse Effects Level) of laboratory experiments with rats proved a daily 180 mg/kg of body weight. Other authors measured the LOAEL (Lowest Observed Adverse Effect Level) values of cadaverine regarding cell cultures, which was proved to be 510.89 mg/kg. In case of putrescine LOAEL, it was 881.5 mg/kg. This value is significantly lower than in previous presentations (255.45 mg/kg for total biogenic amine and 2000 mg/kg for cadaverine) [37,38]. Cheese can contain cadaverine in large amounts (3170 mg/kg), but fish is also a significant source (1690 mg/kg).

Materials and Methods
The role of the yeast in wine production is crucial. In this study we wanted to find out what kind of "Bianca" wine can be produced when using organic yeast, what are the dynamics of the resulting alcoholic fermentation, and whether this method is suitable for industrial production as well.

Materials
We fermented wines from the "Bianca" juice samples three times where model chemical solutions were applied. The initial level of chaptalization was 20.6 MM • (sugar degree of the must), and the initial level of titratable acidity was 6.2 g/L. For the fermentation process, 2 L capacity glass carboys were used, and inoculation happened with 20 g/hL organic yeast. Wine nutrient salt was added in 15 g/hL doses in the middle and at the end of the fermentation process. The control sample was inoculated with the yeast Uvaferm CS2, at 20 g/hL, and the nutrient salt Uvavital was added mid-fermentation and at the end.

Chemical Analyses
There are multiple methods available to measure the level of biogenic amines. Due to the development of the analytical techniques, we have effective column chromatography methods (HPLC and IEC) and overpressure layer chromatography (OPLC) procedures at our disposal. Capillary electrophoresis methods are all suitable for defining the amines. The HPLC technique is suitable for processing the precise and high quantity sampling data.
We implemented practices used in everyday life to examine the acids in the wine: • titratable acid content-regulated by the Hungarian Standards Institution bearing the serial number MSZ 9472-86; • pH measurement using a combined glass electrode-MSZ-14849-79; • malic acid content-using the Boehringer Mannheim enzyme test and spectrophotometry; • citric acid content-using the Boehringer Mannheim enzyme test and spectrophotometry; • lactic acid content-using the Boehringer Mannheim enzyme test and spectrophotometry; • tartaric acid content-the vanadate ions of the reagent added to the wine combined with the tartrate ions in the wine turn the liquid into an orange-colored complex.
We measured the intensity of the color, which is proportional to the concentration level of the tartaric acid (MSZ-9489-78).
Examining the polyphenol content in wines: •

Statistical Analyses
The results were imported into and evaluated in the programs Microsoft Word for Mac and Excel for Mac. The results were evaluated by t-tests (α = 0.95).

Results
Results of the primer analysis are summarized in Table 1. The values show that there is a significant difference among the samples regarding alcohol and residual sugar concentration. In case of the control samples, a regular, normal fermentation is detectable, where the residual sugar content is only 2 g/L. Organic yeast showed partial effectiveness only, as 63.4 g/L of sugar remained in the wine. This was the reason for the significant differences in alcohol and residual sugar content. In wine making technology, the volatile acid content is an especially important factor. Our present study showed that all the problematic values were under the critical limit (1 g/L). The results of the biogenic amine content in "Bianca" wines, 2015-2017, are shown in Figure 1. The glycerin content correlated with the literary values, since its well known that the glycerin-pyruvic acid transformation results in a 6-10 g/L concentration.

Statistical Analyses
The results were imported into and evaluated in the programs Microsoft Word for Mac and Excel for Mac. The results were evaluated by t-tests (α = 0.95).

Results
Results of the primer analysis are summarized in Table 1. The values show that there is a significant difference among the samples regarding alcohol and residual sugar concentration. In case of the control samples, a regular, normal fermentation is detectable, where the residual sugar content is only 2 g/L. Organic yeast showed partial effectiveness only, as 63.4 g/L of sugar remained in the wine. This was the reason for the significant differences in alcohol and residual sugar content. In wine making technology, the volatile acid content is an especially important factor. Our present study showed that all the problematic values were under the critical limit (1 g/L). The results of the biogenic amine content in "Bianca" wines, 2015-2017, are shown in Figure 1. The glycerin content correlated with the literary values, since its well known that the glycerin-pyruvic acid transformation results in a 6-10 g/L concentration.  The histamine, tyramine, and serotonin concentration, due to their physiological effect, are presented in the first diagram.
In our study, the impact of organic yeast on biogenic amine formation regarding three important compounds (histamine, tyramine and serotonin) was examined. We found significant differences in the samples produced with organic yeast. In terms of physiological effect, the organic yeast produced serotonin in a higher concentration. We can conclude that the level was below the critical 5 mg/L, as suggested in a literature review [39], and it is in accordance with the health prescriptions concerning the three most important biogenic amines.
In the case of the histamine concentration, it can be stated that there is no relevant difference between the control and the organic samples at the 95% significance level. The histamine concentration is not allowed to exceed 5 mg/L in wines, and Figure 2 shows that the histamine levels in the two samples were well below that. The histamine, tyramine, and serotonin concentration, due to their physiological effect, are presented in the first diagram.
In our study, the impact of organic yeast on biogenic amine formation regarding three important compounds (histamine, tyramine and serotonin) was examined. We found significant differences in the samples produced with organic yeast. In terms of physiological effect, the organic yeast produced serotonin in a higher concentration. We can conclude that the level was below the critical 5 mg/L, as suggested in a literature review [39], and it is in accordance with the health prescriptions concerning the three most important biogenic amines.
In the case of the histamine concentration, it can be stated that there is no relevant difference between the control and the organic samples at the 95% significance level. The histamine concentration is not allowed to exceed 5 mg/L in wines, and Figure 2 shows that the histamine levels in the two samples were well below that. The tyramine concentration changed according to the relevant literature [40]. In neither sample were we able to detect a statistical difference. The serotonin content changed as expected, and based on the statistical test, we can state that there is a significant difference between the traditionally produced wine samples and those fermented with organic yeast (a = 0.05; s2 = 0.49; DF = 5).
In case of the other biogenic amines, there are relevant differences between the samples produced with organic and traditional yeast fermentation (Table 2.). The amount of amines, such as cadaverine, putrescine, and ethylamine, could be measured in significant quantities (a = 0.05; s2 = 0.22; DF = 5). At the same time, the methylamine level was higher in the control sample (6.5 mg/L). Based on our measurements, we could only prove significant differences between the samples in case of the methylamine concentration. In turn, 2-phenylethylamine could not be detected in any of the samples. We could not find significant differences between the samples regarding the biogenic amine composition, except for the methylamine concentration. Our results show that these yeast varieties can ensure the same quality as traditional yeast stocks. We consider it as a priority to analyze the so-called organic yeasts, as they can be the base of organic The tyramine concentration changed according to the relevant literature [40]. In neither sample were we able to detect a statistical difference. The serotonin content changed as expected, and based on the statistical test, we can state that there is a significant difference between the traditionally produced wine samples and those fermented with organic yeast (a = 0.05; s2 = 0.49; DF = 5).
In case of the other biogenic amines, there are relevant differences between the samples produced with organic and traditional yeast fermentation (Table 2.). The amount of amines, such as cadaverine, putrescine, and ethylamine, could be measured in significant quantities (a = 0.05; s2 = 0.22; DF = 5). At the same time, the methylamine level was higher in the control sample (6.5 mg/L). Based on our measurements, we could only prove significant differences between the samples in case of the methylamine concentration. In turn, 2phenylethylamine could not be detected in any of the samples. We could not find significant differences between the samples regarding the biogenic amine composition, except for the methylamine concentration. Our results show that these yeast varieties can ensure the same quality as traditional yeast stocks. We consider it as a priority to analyze the so-called organic yeasts, as they can be the base of organic grape growing and organic wine production. Our results showed that there was no significant difference to be described, not even at a very low confidence level (a = 0.2), in any of the three vintages.
Tyrosol is formed only through alcoholic fermentation, and the amount and formation of tyrosine is influenced by the yeast. The tyrosol level was high in the wines produced with organic yeast in all three vintages (Figure 3). grape growing and organic wine production. Our results showed that there was no significant difference to be described, not even at a very low confidence level (a = 0.2), in any of the three vintages.
Tyrosol is formed only through alcoholic fermentation, and the amount and formation of tyrosine is influenced by the yeast. The tyrosol level was high in the wines produced with organic yeast in all three vintages (Figure 3). The overall antioxidant capacity value developed evenly as it is formed from polyphenol, so there is no difference. As for epicatechin, in all three vintage years we measured lower values in the organic vines (Table 3).  In the samples produced with organic yeast, the catechin, leucoanthocyanin, and overall polyphenol concentration values supported the data in the relevant literature [41]. There was no significant difference between the polyphenol content in the samples of the control or the treated wines. The tyrosol concentration was higher in the organic samples. Regarding the simple phenol composition, there was no relevant difference between the samples. We can state that a considerable difference between the organic and the control samples was in the polyphenol and simple phenol composition and in the amount of tyrosol, which is the most important finding. The overall antioxidant capacity value developed evenly as it is formed from polyphenol, so there is no difference. As for epicatechin, in all three vintage years we measured lower values in the organic vines (Table 3).  In the samples produced with organic yeast, the catechin, leucoanthocyanin, and overall polyphenol concentration values supported the data in the relevant literature [41]. There was no significant difference between the polyphenol content in the samples of the control or the treated wines. The tyrosol concentration was higher in the organic samples. Regarding the simple phenol composition, there was no relevant difference between the samples. We can state that a considerable difference between the organic and the control samples was in the polyphenol and simple phenol composition and in the amount of tyrosol, which is the most important finding.
The results of the analysis turned out as expected. There was no considerable difference among the samples. We detected a great difference in polyphenol composition, which is a result of the interaction of the yeast with the polyphenol.
The biogenic amine content in the examined wines does not differ from that of the conventional wines.
The biogenic amine composition in the resistant varieties corresponds to the composition of the biogenic amines in the international varieties. The organic yeast also has an influence on the production of amines, but there is no clear distinction compared to the "traditional" types of yeast; thus, further examinations are necessary.
From the results of our research, we can conclude that organic yeasts cause significant differences in the glycerin concentration and in the residual sugar content of the samples. The volatile acid content was measured below the critical level of 1.0 g/L [42] in all cases.

Discussion
In this study, we aimed to find out what kind of "Bianca" wine can be produced when using organic yeast, what are the dynamics of the resulting alcoholic fermentation, and whether this method is suitable for industrial production as well. We fermented wines from the "Bianca" juice samples three times where model chemical solutions were applied.
Our present study showed that all the problematic values were under the critical limit (1 g/L), although the organic samples resulted in a significantly higher level than the conventional wines.
The glycerin content correlated with the literature values, since it is well known that the glycerin-pyruvic acid transformation results in a 6-10 g/L concentration.
In our research we aimed to find out how organic yeast influenced biogenic amine formation, regarding three important compounds: histamine, tyramine, and serotonin. We measured significant differences in the samples produced with organic yeast, where the values were higher.
We could not find significant differences in biogenic amine composition between the samples, except for the methylamine concentration. Our results showed that these yeast varieties could ensure the same quality as traditional yeast stocks.
There was no significant difference between the polyphenol content in the wines from the control or the treated items. The tyrosol concentration was higher in the organic samples. Regarding the simple phenol composition, there was no relevant difference among the samples. We can state that a considerable difference between the organic and control samples was in the polyphenol and simple phenol composition and in the amount of tyrosol; the latter is the most important finding.

Conclusions
Among the physiologically effective compounds, the biogenic amine composition was examined. The most important allergenic, histamine, was measured within the approved range (10 mg/L). The "Bianca" grape variety is perfectly suitable to produce organic wine regarding the biogenic amine content. In case of the other amines, we concluded that their values correlate with the values previously published in the relevant literature sources, and there is no significant difference either as a result of using organic yeast or the vine variety "Bianca" as a basic material. Regarding the polyphenol composition, we can conclude that it is suitable to produce high-quality "Bianca" organic wine. Following this project, we will examine the possibility of meeting the modern oenological technology requirements of a widespread grape variety "Bianca" in Hungary, in order to find what additional specifications we can define to further increase the market of this variety. Informed Consent Statement: "Not applicable" for studies not involving humans.