Yeast Activation Methods Used in Fermentation Industries

Abstract

The reasons why it is practically impossible to maintain optimal conditions for the development of cultural yeast populations under production conditions are briefly substantiated. A simplified classification of yeast activation methods is given: chemical, physical, and combined. In each of the mentioned groups, the varieties of the proposed technological methods and the modes of their implementation are considered. Experimental data obtained in recent years on the influence of the sound in the audible range (20–20,000 Hz) and light in the visible range on the development of Saccharomyces cerevisiae yeast used in brewing are presented. An attempt made to compare the effectiveness of various ways to improve technological indicators: the increase in the total titer of cells, the percentage of nonviable cells, the accumulation of ethyl alcohol.

1. Introduction

A wide range of productions, including food production, are based on the growth of microbial populations, such as yeast, among others. A technologist in a production of this nature should be seeking to culture a yeast population as intensively as possible. The aim is to obtain biomass or metabolism products within the shortest possible period of time with the maximum possible utilization of nutrients within the cultivation medium.

The most logical approach to address this issue would be to maintain ideal conditions for microorganisms, specifically for cultivating yeast of a particular genus and species [1]. However, achieving this objective is often unfeasible for various reasons. One clear example is the production of bottom-fermentated beer. In traditional brewing techniques, brewers cultivate a yeast population at the stage of fermentation under highly unfavorable conditions for this yeast type [2]. The process begins with a high osmotic pressure and ends with a relatively high ethanol concentration. The temperature remains too low, and the physiological condition of the inoculated yeast is suboptimal, especially in the case of pure-culture yeast or late-generations seed yeast. Furthermore, despite the technologist’s effort, the composition of the growth medium can quite often be unbalanced, with low-molecular nitrogenous compounds typically being a limiting factor. In contemporary conditions, the growth medium may also contain abiotic substances such as heavy metals, pesticides, and radionuclides that have entered the process flow from the raw materials, although their content in the latter is restricted and monitored. These factors, either individually or collectively, create suboptimal conditions for yeast population growth. Consequently, the activation of the process through additional processing methods becomes desirable or even necessary. Considerable attention has been directed toward the development of such methods. This chapter aims to provide a concise overview of these techniques (addition of activators of various natures; removal of undesirable components from a nutrient medium; physical processing of inoculum yeast, wort, or fermentation medium; or a combination of these methods) and, if possible, to compare their efficacy. It should be noted that we have intentionally excluded from the discussion any approach that relies based on enhancing the characteristics of yeast through genetic manipulation [3].

A comprehensive classification system [4, 5] for methods aimed at enhancing yeast activity has been proposed. This classification is based on various criteria, including teleological, genetic, and technological aspects. Additionally, it was suggested distinguishing activating agents based on their chemical properties, composition, origin, production stage, and intended final objectives. Nevertheless, we believe that even a complex classification fails to take into account every difference. Therefore, we find it more reasonable to categorize all yeast activation methods, whether proposed or implemented on an industrial scale, into three main groups:

• Chemical methods: These involve the addition of specific agents and substances to the food medium, or conversely, the removal of unwanted components from the food medium.

• Physical methods: These rely on the utilization of specialized equipment, which does not form a part of the traditional hardware circuit typically used for the given process.

• Combination of the above (combined methods).

The information presented in this chapter is organized in accordance with this subdivision.

2. Chemical methods of activation

Agents employed as chemical yeast activators can be categorized based on their intended functions. These categories encompass the following:

Deficient (limiting) compounds: These agents are employed to supplement the medium with essential compounds that may be lacking for yeast growth.

Biostimulators promoting yeast activity.

Extraction of unwanted components: Specific agents are used to remove undesirable components from the medium that could hinder yeast development.

Stressors.

Antibacterial agents: These agents are introduced to create an environment conductive to the growth of the main yeast culture.

Biopolymer degraders: Certain agents are employed to break down biopolymers found in raw and semifinished products.

Adjustments to the medium composition can be made at various stages - from the preparation of the nutrient medium to the stage of cultivation of the yeast population. These additions or agents may vary in terms of their chemical nature, falling into categories such as organic, nonorganic, or mixed (complex). They can be obtained through chemical synthesis, microbiological processes, or have a natural origin. The proposed classification system enables a targeted approach in selecting agents capable of altering yeast culture metabolic activity by adjusting the composition of the culture media [4]. Below, we provide examples of agents belonging to the discussed groups.

In order to fulfill the yeast’s requirements for microelements and vitamins, various specialized preparations are currently in widespread use (“yeast feedings”, “yeast nutrition”). These preparations came in both single-component and multicomponent forms, containing amino acids, vitamins, and minerals: zinc sulfate, “Istex,” “Eastfield,” “Hi Vit,” “Eastfood,” “Alkoten,” “Rhodium Zumesit,” and others [6]. The utilization of these compounds accelerates the start of wort fermentation, prevents fermentation from slowing down and halting, reduces fermentation duration, fosters thorough fermentation of wort sugars, increases yeast growth and resistance to autolysis, and, in some instances, assists in reducing diacetyl content in beer. Typically, these feedings are added to the wort before fermentation. However, it is important to note that the composition of these feedings includes inorganic substances as mineral components (such as diammonium phosphate, manganese and zinc sulfates, and potassium metabisulfite), which are foreign to the food product and considered undesirable from a hygiene standpoint.

A study of the chemical composition of sweet potatoes revealed the presence of a complex of biologically active substances. In order to increase their yield, the use of enzyme preparations became necessary. A biologically active additive, derived from sweet potatoes, was employed as a fermentation activator, exhibiting a positive impact on brewer’s yeast and beer quality [7]. These dietary supplements were added to the wort during the fermentation stage, at concentration of 1% and 2%. Fermentation was carried out at temperatures ranging from 5 to 8° C. The research findings indicate that the use of dietary supplements in beer production positively influences several parameters of young beer, including the degree of fermentation (increased by 5.8%) and ethanol content (elevated by 3.6%). Furthermore, it was observed that the experimental sample, utilizing the sweet potato preparation, outperformed the control sample by 44% in terms of yeast biomass growth. The experiment results demonstrated that the introduction of the sweet potato preparation into the wort accelerates the fermentation process by 2–3 days compared to the control sample. Moreover, the use of this biologically active sweet potato additive during fermentation led to a more significant reduction of diacetyl, with the control and experimental beer samples having diacetyl content of 0.14 and 0.09 mg/dm³, respectively.

The organs of the Far Eastern wild plant Aralia mandshurica were chosen as a biostimulant for Saccharomyces cerevisiae yeast 34/70 [8]. Alcohol-containing extracts of aralia (derived from roots, branches, or leaves) were introduced after the main fermentation, before the start of 16 days post-fermentation period. This increased the physiological activity of the yeast, as assessed by the number of physiologically active yeast cells containing glycogen. In the control sample, there was a gradual decline in the number of physiologically active cells containing glycogen (from 47 to 15%). This decline is explaining by the natural, modest reduction in the functional (fermentation) activity of yeast during the beer fermentation process. In the experimental samples with Aralia extracts, the initial number of glycogen-containing cells was lower compared to that in the control sample, averaging around 33%. According to the authors, this can be attributed to a certain “shock” experienced by microorganisms due to the ethanol content in Aralia extracts. However, in the days following fermentation, the number of viable cells in all experimental samples increased by the 8th to 10th day, surpassing the control sample by an average of 10% by the end of the experiment. The most significant impact in terms of the number of glycogen-containing microorganisms was observed in the experiment using an extract from Aralia leaves.

The study examined the results of using S. cerevisiae yeast strains 11 and 8a (M) in the production of kvass—a traditional low-alcohol fermentation beverage in the Russian Federation. The authors propose methods to enhance the accumulation of physiologically active yeast biomass using succinic acid and yeast autolysate obtained through their developed technology [9].

The impact of succinic acid at concentrations ranging from 0.01% to 0.2% on the metabolism of the pure culture of alcohol yeast S. cerevisiae (strain XII) is demonstrated. Changes were established in the consumption of dry substances, yeast biomass accumulation, active and titrated acidity values, CO₂ production, concentration of dry substances, and yeast cells titer during alcohol yeast reproduction. After 24 hours of cultivation with the introduction of 0.1% succinic acid, the number of yeast cells increased by 14%, budding cells by 71%, and cells with glycogen by 11.5% compared to the control sample [10].

The results [11] of applying an amino acid-vitamin activator (AVA), a natural stimulant obtained from residual brewer’s yeast cells of the first-generation strain Saflager S-189, were also studied. AVA serves as a source of amino acids, vitamins, and other essential compounds required for cell development. The addition of the activator to a 12% wort in quantities of 0.2 and 0.5% stimulated the growth and accumulation of reserve nutrients by yeast cells. After 5 days of fermentation, the increase in cell count in the control sample was 2.46 million/cm³, whereas in samples with 0.2% AVA, it reached 4.6 million/cm³, and with 0.5%, it was 1.9 million/cm³. After 7 days of fermentation, the percentage of budding cells in the aforementioned samples was 52%, 74%, and 57%, respectively. Additionally, the proportion of cells containing glycogen was 62%, 83%, and 70%, respectively. However, it was observed that an excessive amount of AVA in a dosage of 1.0% somewhat suppressed yeast growth and activity. During testing in a small-scale brewery, the activator was found to be more effective as the generation number of seed yeast increased. When fermenting 12% wort with seed yeast of the third generation, the experimental sample with 0.2% AVA achieved a visible extract of 3.8% and a visible degree of fermentation of 68.3% after 6 days. In contrast, the control sample reached similar values of 4.3% and 64.1%, respectively, but only after 7 days. Similar results were obtained when assessing the effectiveness of using the AVA activator in combination with a preparation derived from the microalgae Spirulina platensis [12].

A method has been developed to increase the viability of brewing yeast using exclusively preparations derived from Spirulina platensis algae. Yeast activated with these preparations exhibit an increased reproduction rate. They also demonstrate efficient flocculation, resulting in the formation of a dense precipitate by the conclusion of the main fermentation. The use of this preparation enables yeast to maintain their activity over an extended period. The incorporation of this preparation into the wort is recommended not only as a routine practice but also as a means of providing “first aid” to reactivate yeast that may have been weakened during technological processes or have been reused multiple times. This approach also serves to stabilize their fermentation characteristics [13].

The impact of baking yeast hydrolysate on fermentation and the physiological state of Saccharomyces cerevisiae yeast strains 34, 776, 8Am was investigated [14]. In order to assess the influence of hydrolysate on the extent of wort fermentation, it was added in quantities of 50, 100, and 150 mg per 100 cm³ of wort. The fermentation process employed sterile hopped wort with varying concentrations of dry substances, specifically 8%, 10%, 11%, 12%, and 14%. Fermentation was conducted within a temperature range of 7 to 9° C for a duration of 7 days, with daily measurements of the visible extract. The experiment revealed that the hydrolysate did not significantly impact the extent of wort fermentation when the initial dry substances concentration was 8% and 10%. However, in wort with an initial dry matter concentration of 11%, 12%, and 14% and containing 100–150 mg per 100 cm³ of hydrolysate, a more profound fermentation occurred, resulting in the degree of attenuation enhancement of 20–30%. This allowed for a reduction in the fermentation duration by 1 to 1.5 day. Beer produced with the inclusion of hydrolysate exhibited deeper attenuation, higher alcohol content, lower diacetyl levels, and superior organoleptic characteristics compared to the control sample.

Yeast extracts also exhibit effectiveness in fermentation high-gravity beer wort with dry substances concentration ranging from 16.2% to 20.3%. The addition of 1% of the extract, on average, reduced the fermentation duration by 1.5 days. Furthermore, it was found to stimulate yeast flocculation ability, fermentation activity, and the number of budding cells [15].

Yeast extracts have proven to be efficient in activating Saccharomyces cerevisiae yeast, commonly used in alcohol production [16, 17]. As a result, they contribute to the intensification of alcohol wort fermentation [16].

Complex yeast feeding (CYF) was developed, and its impact on the wort fermentation process and beer quality using dry yeast of the Saflager strain W-34/70 was evaluated. CYF can be added to yeast or to the wort before yeast introduction. CYF consists of a mixture of coarsely ground natural zeolite-containing tuffs and yeast. The use of CYF was found to significantly increase the activity of yeast cell enzymes compared to the control sample: α-glucosidase increased by 1.7–2.7 times, zymase by 1.6–2.4 times, and invertase by 9–30%. The positive impact of CYF on the physiological state of the yeast culture was observed even at a dosage of 0.05 g per 100 cm³ and further improved with higher doses. The number of budding cells increases by 1.5–2.0 times compared to the control sample, and cells containing glycogen increased by 1.6–1.7 times. Additionally, there was a reduction in the concentration of dead cells, ranging from 8 to 30%. In order to study the wort fermentation process, yeast was added to the wort at a rate of 20 million cells/cm³. The fermentation was conducted at a temperature of 12° C for 7–8 days, followed by secondary fermentation lasting 30 days at a temperature of 2–3° C. The following samples were compared: experiment sample 1—fermentation of wort with yeast pre-activated using CYF at a dose of 0.1 g/100 cm³; experiment sample 2—fermentation of wort into which CYF was previously introduced at a rate of 0.05 g/100 cm³, with reactivated yeast without prior treatment; control sample - wort fermented with yeast starter without prior activation and without CYF application. In the experiment samples, the processes of yeast propagation and consumption of extract and amino nitrogen were notably more vigorous. In the experiment samples of young beer, the concentration of yeast cells in the suspended state by the end of fermentation was significantly lower than in the control sample. This is expected to have a positive impact on the beer clarification process during secondary fermentation and filtration and potentially enhance the colloidal stability of the finished drink. In the samples initially enriched with the yeast feeding, several favorable outcomes were observed. These include a higher degree of attenuation and increased volume fraction of alcohol, a reduced content of polyphenolic compounds, and a lower presence of high-molecular-weight proteins. In addition, in the samples where CYF was applied, the beer exhibited a harmonious and full flavor profile, accompanied by a gentle hop bitterness [18].

Many small beer production enterprises employ active dry brewing yeast for pitching the wort, but in most cases, the viability of the yeast is reduced. Therefore, yeast activation is necessary before fermentation. The effectiveness of increasing the activity of brewing dry yeast [19] Saflager W-34/70 was established through the use of preparations of natural organic and inorganic origin. Yeast activation involved the utilization of antler-containing raw materials in the form of a dry preparation as a source of various biologically active organic substances. The antler-containing preparation composes lipids, nitrogenous compounds, calcium, phosphorus, and other components. The lipid complex includes phospholipids; mono-, di-, and triglycerides; sterols; fatty acids; and sterol esters. The medium for processing the yeast culture, as well as for fermentation, was industrial hopped beer wort with an extra activity of 12%. Biochemical parameters of the culture were assessed both in the initial yeast and during its activation, focusing on the activity of α-glucosidase (maltase), invertase (β-fructofuronosidase), and the zymase complex. The results indicate that adding the antler preparation to the yeast starter during its preparation for wort fermentation leads to a significant increase in the activity of the yeast cell enzymes under investigation when compared to the control sample: α-glucosidase (maltase) by 2.0–6.8 times, zymase by 1.5–2.5 times, and invertase by 4.5–6.2 times. The introduction of the antler-containing preparation, either separately or in combination with CYF, during both yeast preparation for fermentation and directly into the wort before yeast pitching, ensures high yeast viability and high biochemical and physiological activity of the culture. The effectiveness of these feedings is further enhanced when applied under aeration conditions during the dry yeast preparation stage for wort fermentation. Recommended doses of the antlers-containing preparation (% by volume of the medium) are (0.10–0.75) × 10⁻³, and CYF should be in the range of 0.05–0.075.

The study presents the results demonstrating the positive impact of multifunctional components of geothermal water in the yeast culture medium on ethanol biosynthesis [20]. It was observed that an increase in carbohydrate content and the fermentation duration in a nutrient medium with geothermal water resulted in a 20% increase in alcohol synthesis compared to the previously established technology. Morphophysiological parameters of the cells further validate the active state of the experimental yeast.

The positive effects of silicon on the carbohydrate and nitrogen metabolism of Saccharomyces yeast were revealed. Silicon dioxide was sourced from natural quartz sand found in the Sarykum dune and industrial quartz glass. This led to an increase in the biological activity of the yeast population impacting growth, vital activity, generative activity, and adaptive variability in the shape and size of cells [21].

The research also explored the potential for activating dry brewing yeast using an energy exchange regulator: a mixture of organic acids from the Krebs cycle, including succinic, malic, fumaric, citric, and oxalic acid in a 1:1:1:1:1 ratio, at concentrations ranging from 10⁻⁸ to 10⁻¹⁰ mol/dm³). The influence of various concentrations of this acid mixture was evaluated, demonstrating a positive effect on yeast cell enzymes activity and the physiological state of the yeast culture due to increased cell membranes permeability. The use of yeast activated in the acids solution at a concentration of 1x10⁻¹⁰ mol/dm³ positively impacted the beer wort fermentation process, as evidenced by production tests. This resulted in a reduction of the fermentation process duration by 1 day and improved physiological parameters of the yeast culture compared to the control sample without special treatment. Additionally, the quality indicators and organoleptic characteristics of the beer met the standard requirements [22].

The utilization of milk whey as an activator of brewer’s yeast was investigated [23]. The efficacy of yeast treatment through exposure in a medium consisting of whey, as well as a mixture of whey and beer wort, at a temperature of 4–6° C, was also defined. During the exposure, the number of budding cells was shown to increase twofold, cells with glycogen by 1.3 times, the zymase activity of yeast by over 2-fold, and maltase activity by 21–42.4%. This led to a reduction in the main fermentation duration by 1 day. At the same time, in experimental beer samples, higher degree of attenuation and alcohol content were achieved, with reduced diacetyl formation. An additional effect is achieved through a joint rotary-pulsation process, as described in the section on combined processing methods.

Furthermore, the optimization of the nutrient medium composition for yeast cultivation can be achieved not only by introducing components with a positive impact but also by eliminating those with a negative influence [24]. This task can be addressed, in part, by employing sorbents.

A proposal is made to employed oyster mushroom mycelium (Pleurotos ostreatus), a food additive, for the removal of undesirable components from the wort, thereby optimizing the fermentation process. The sorbent was added to the 12% beer wort before commencing the primary fermentation stage. After 7 days of fermentation, the alcohol concentration in the experimental samples, compared to the control sample, exhibited a significant increase of 145.0% and 152.95% when using biosorbent dosage of 0.5 and 0.3 wt. %/wort volume, respectively [25].

Biosorbent “OD-2,” derived from initiated autolysis of sedimentary brewer’s yeast, when added to the wort before pitching at dosages ranging from 0.1% to 0.5% by weight/vol., led to a significant increase in the concentration of ethanol in young beer by 17–46% compared to the control sample [26].

While baking may not be directly related to the fermentation industry, one of the crucial stages in bread production involves fermenting dough using Saccharomyces cerevisiae yeast. Therefore, the findings of studies on the activation of baking yeast with fruit and berry extracts are of interest.

Biologically active components and acids found in fruit and berry extracts can either inhibit or activate Saccharomyces cerevisiae yeast. The impact of various berry extracts on the activity of S. cerevisiae baking yeast has been investigated. These experiments were conducted using “Extra” baking yeast and dry extracts of raspberry, chokeberry, sea buckthorn, and rosehip. Raspberry extract (3–4%) suppressed the growth and propagation of yeast. After 1 hour of exposure, the number of yeast cells decreased by 1.5–2 times, when compared to the control sample. Sea buckthorn extract had a stimulating effect: the growth rate of yeast cells increased up to 40% when compared to the control sample. Chokeberry and rosehip extracts had minimal effect on yeast cell growth rate. However, 2–3% chokeberry extract enhanced dough fermentation. The extracts of fruit and berries naturally increased the dough’s acidity, which influenced the growth rate of yeast cells. Sea buckthorn extracts significantly elevated the acidity (up to 4.24 pH units), resembling acid stress, leading to increased yeast cell growth rate of (1.53 × 10⁶–1.55 × 10⁶ compared to 1.10 × 10 ⁶ in 1 cm³ of the control sample). On the other hand, samples with raspberry extract exhibited the slowest growth rate, as raspberry extract is known for its pronounced fungistatic effect, resulting in a 1.5–2 times decrease in the number of yeast cells after an hour of fermentation [27].

A method [28] has been proposed for the preliminary activation of baking yeast to enhance their resistance to acid stress. It was observed that yeast treated with solutions of hydrogen peroxide in concentrations ranging from 0.5 to 3.0 mM retained higher fermentation activity when subjected to stressful conditions (such as a 2% lactic acid solution) compared to the control sample.

The effectiveness of several chemical activation methods was compared by increasing in the concentration of ethyl alcohol and decreasing in the content of the apparent extract (Figure 1).

Figure 1.

Increase in ethyl alcohol content and decrease in apparent extract (%) in test samples of finished beer compared to similar values in control samples.

3. Physical activation methods

Activation methods in this group are primarily based on direct or indirect wave/field effects, with the exception of the use of mechanical vibrations (rotary pulsation action). This will be discussed below when examining the combined yeast processing method [23].

Based on the analysis of the literature, it can be concluded that the greatest interest is in the study of the effectiveness of ultrasonic treatment. Examples of such an approach are provided at the beginning of this section.

3.1 Ultrasound and audible range sound processing

Ultrasonic treatment is widely employed for the destruction or inactivation of treated objects, including yeast cells [29]. However, altering the parameters of such treatment can yield the opposite effect and activate yeast cell population, as confirmed by the data presented below.

The same physical and mechanical effects utilized in sonochemistry, such as strong shear forces, particle fragmentation, and increased mass and heat transfer, are also applied in the food industry. Powerful ultrasound is employed to influence the development of living cells, aiming to boost sterilization efficiency, impact enzyme activity [30], and enhance the overall quality of food products [31].

Fermentation processes involve enzymes produced by microorganisms’ cells to carry out chemical transformations. Ultrasound can be employed in such processes for monitoring the course of fermentation or influencing its. High-frequency ultrasound (>2 mHz) is well-known as a tool for measuring changes in chemical composition during the fermentation process, offering real-time insights into the reaction’s progress. Low-frequency ultrasound (20–50 kHz) can affect the fermentation process by improving mass transfer and cell permeability. This leads to increased process efficiency and productivity. Additionally, this type of ultrasound can be used to eliminate microorganisms that may disrupt the fermentation process [32].

The activation method of Safale T-58 brewer’s top fermentation yeast Saccharomyces cerevisiae was examined, employing ultrasound at an oscillation frequency of 44 kHz. Ultrasonic treatment significantly intensified the fermentation process and improved the quality of final products. The yeast suspension was placed in an ultrasound machine and processed at the oscillation frequency of 44 kHz. During the exposure, the temperature was measured every minute from the 1st to 20th, as well as at the 25th, 30th, 35th minutes. The survival of yeast cells was recorded by defining the percentage of dead cells. It was found that under the influence of ultrasound, the medium was heated by 1° C for 1 min, and by the 40th minute of treatment, the temperature of the medium reached 57° C. The yeast treated with ultrasound served as an inoculum at the stage of the fermentation of beer wort. The fermentation was carried out at a temperature of 22° C. The fermentation activity increased by 36% in the sample after treating seed yeast with ultrasound for 2 min. The remaining samples differed slightly from the control sample during the first 72 hours of fermentation and lost their fermentation activity after 80 hours of fermentation [33].

In their later work, the same group of authors [34] researched the activating and disintegrating effect of ultrasound at a frequency of 44 kHz and intensity of 1.0 W/cm² on brewer’s yeast. It was found that a 10-minute ultrasonic treatment of yeast is sufficient to achieve a stimulating effect. Further ultrasound treatment is impractical, since the percentage of dead cells in the yeast suspension exceeds the permissible level (more than 10%). The experiment showed that two-minute ultrasound treatment improved the physiological activity of the seed yeast. The beer obtained using this technique had a higher quality.

The effect of ultrasound on the results of fermentation of beer wort from Korean six-row barley was also studied. Beer samples were processed in an ultrasonic bath for 4 days during the primary fermentation. The ultrasound frequency was 40 kHz, and the input power was 120, 160 and 200 watts. The ultrasound treatment was carried out for 2, 6, and 12 hours for each input power. The physicochemical and organoleptic properties were measured, and the quality of the beer was evaluated. Ultrasound with a power of 160 W increased the yield of ethanol by 13.18% [35].

The study examined the impact of ultrasound at a frequency of 22 kHz and an intensity of 1.0 W/cm² on the outcomes of water–heat treatment of wort derived from winter triticale grain [36]. It was established that ultrasonic exposure results in increased activity of grain α-amylases enzymes. The wort obtained was used for cultivating seed yeast with simultaneous ultrasound treatment. It was also observed that such treatment leads to an augmentation of biomass and intensification of the seed yeast growth process. The processed wort was then fermented with activated yeast to produce alcohol with a yield of 67.3 dal/t of conditional starch and a reduced content of toxic impurities.

The potential for increasing ethanol yield in alcohol production through the treatment of Saccharomyces cerevisiae yeast using low-intensity ultrasonic irradiation at fixed frequencies of 20, 23, 25, 25, 28, 33, and 40 kHz was considered [37]. Under optimal ultrasonic treatment conditions (ultrasound frequency - 28 kHz, power density - 180 W/l, processing time - 24 h), the maximum ethanol yield increased by 34.87% compared to the control sample. Transcriptome sequencing revealed that ultrasound treatment regulated the expression of genes involved in pyruvate metabolism, glycolysis, the pentose phosphate pathway, and glucose transport. Quantitative polymerase chain reaction in real time also confirmed changes in gene expression. Metabolomics showed that ultrasound treatment increased the intracellular glucose and nicotinamide adenine dinucleotide (NADH) content, crucial metabolites for ethanol synthesis. Additionally, ultrasound treatment reduced the levels of acetate and its derivatives, leading to a decrease in the reverse consumption of pyruvate, thereby facilitating ethanol synthesis. These alterations in gene expression and metabolite content are likely the primary reasons for the increase in ethanol yield in Saccharomyces cerevisiae after ultrasound irradiation [37].

The study investigated the impact of ultrasonic treatment with different operating modes and different frequencies on the accumulation of cells and metabolites of Saccharomyces cerevisiae yeast. The results demonstrated that in situ ultrasound treatment can enhance the controlled parameters, and the effect of ultrasound with a fixed frequency was greater than that of wide-frequency ultrasound. When exposed to ultrasound with a fixed frequency, the concentration of metabolites in the culture medium increased, whereas it decreased after treatment with wide-frequency ultrasound. Conversely, when the ultrasound frequency exceeded 33 kHz, the accumulation of S. cerevisiae biomass weakened, accompanied by an increase in the proportion of nonviable cells in the culture medium. At a frequency of 23 kHz and a fermentation duration of 48 hours, the ethanol content increased by 19.33%, as well the content of β-phenylethanol and other volatile metabolites, such as esters [38].

Another study examined the effect of ultrasound treatment on the growth of S. cerevisiae yeast, utilizing ultrasonic frequencies of 20, 25, 45, and 130 kHz, with processing duration ranging from of 2 to 30 min. The kinetics of yeast biomass accumulation following ultrasonic treatment was assessed using modeling techniques and scanning electron microscopy (SEM). It was revealed that ultrasound at frequencies of 45 and 130 kHz had no adverse impact on the lag phase and growth rate of yeast populations, unlike the 20 kHz frequency. Ultrasound treatment at 20 kHz resulted in a significant reduction in yeast concentration (by 1.3 log CFU/cm³) and caused significant damage to the external structures of S. cerevisiae cells [39].

The effect of low-intensity ultrasound, varying in frequencies, processing time, and ultrasound power, on Saccharomyces cerevisiae in different growth phases was assessed by measuring biomass growth. Additionally, the permeability of cell membranes and the ethanol tolerance of ultrasound-treated Saccharomyces cerevisiae cells were studied. It was discovered that under optimal processing conditions during cultivation (ultrasound frequency: 28 kHz, power: 140 W/dm³, treatment duration: 1 hour), yeast biomass increased by 127.03%. Ultrasound exposure improved the permeability of cell membranes, resulting in increased extracellular protein, nucleic acid, and fructose-1.6-diphosphate content. Furthermore, ultrasound treatment could increase damage to yeast cells when exposed to high concentration of alcohol. However, this effect did not significantly impact yeast tolerance to ethanol [40].

It was also observed that the fermentation process could be activated due to the indirect impact of ultrasound: it does not process the yeast biomass at one stage or another, but the fermentation medium before pitching with yeast.

Consequently, the influence of ultrasound and thermal pretreatment on ethanol yield from cassava chips was investigated. Cassava suspensions were treated with ultrasound for 10 and 30 seconds at amplitudes of 80, 160, and 320 microns/s, corresponding to low, medium, and high power levels, respectively. Processed and untreated (control) ultrasound samples were then subjected to simultaneous liquefaction-saccharification and fermentation. Based on the efficiency of converting cassava starch into ethanol, it was concluded that higher ethanol yields are directly related to the duration of ultrasound treatment, rather than to its power level. The ethanol yield from the ultrasound-treated sample was 2.7 times higher than that from the control sample. The fermentation rate was also significantly higher, with the fermentation duration being shortened by nearly 24 hours for samples treated with ultrasound to achieve the same ethanol yield as in control samples. Thus, ultrasound pretreatment increased both the total ethanol yield and the fermentation rate. Compared to the heat-treated samples, the ethanol yield in ultrasound-treated samples was almost 29% higher. The combined heat and ultrasound treatment did not significantly affect the overall ethanol yield from cassava chips. Ultrasound was also preferred over preheat treatment due to its lower energy requirements [41].

Similar findings were reported in a study [42] where pretreatment with ultrasound during the liquefaction stage was tested in the subsequent combined saccharification/fermentation of corn flour using Saccharomyces cerevisiae var. ellipsoideus for bioethanol production through a periodic method. Preliminary ultrasonic treatment (at a frequency of 40 kHz) was conducted for various durations at different temperatures before adding a liquefying enzyme. Based on glucose concentration, the optimal duration of ultrasound treatment was determined to be 5 minutes at a treatment temperature of 60° C. This resulted in an increase in glucose concentration by 6.82% compared to the untreated control sample. Additionally, the kinetics of combined saccharification/fermentation was evaluated, and the impact of ultrasound pretreatment on increasing ethanol yield was studied. Ultrasound pretreatment led to an 11.15% increase in ethanol concentration (compared to the control sample) after 32 hours of saccharification/fermentation. The study found that ultrasound’s effect, driven by cavitation and acoustic flow, stimulated the breakdown of starch granules and the release of glucose, thereby accelerating starch hydrolysis.

Furthermore, research conducted at our university has focused on the acoustic effects on raw materials and intermediates in brewing production over several years. Saccharomyces cerevisiae yeast from various strains was subjected to audible sound exposure at fixed frequencies. It has been established that sound processing could either enhance or impair the technological characteristics of brewer’s yeast [43]. This depends primarily on the processing parameters and the frequency of the sound. The following parameters were applied: sound frequency of 2765 Hz, duration of exposure - 30 min, oscillation amplitude - 100%, source power - 2 W, and distance from the source to the treated object - 5 cm. Such preliminary acoustic treatment of dry seeded yeast that were applied to the fermentation of the model nutrient medium (5% sucrose solution) led to a utilization rate increase in nutrient compared to the control sample by 66%, along with a 5% reduction in the proportion of nonviable cells [44].

3.2 Exposure to pulsed electromagnetic field

There is an opinion [45] that a pulsed electromagnetic field (PEMF) has a positive impact on yeast metabolism, cell biomass growth, and ethanol production, especially with low-frequency electromagnetic waves, which are known to have biological effects. An electromagnetic pulsed generator was used to investigate the influence of electromagnetic pulsed fields on yeast cell reproduction and ethanol accumulation in a fermenting medium, with a focus on intensifying the technological process. The fermentation of wort was carried out in a two-stage mode with treatment using a pulsed electromagnetic field (frequency - 4 Hz, power - 1 μT). The study employed beer wort with a density of 15–35% (15% unmalted raw material) and Saccharomyces cerevisiae top fermenting yeast. A positive effect of the pulsed electromagnetic field was shown on yeast cell proliferation and the fermentation process. This approach also revealed the potential for accelerating alcoholic fermentation and increasing ethanol yield by 15%. The physicochemical evaluation of fermented materials showed a significant increase in foaming and foam resistance. The fermented wort underwent distillation; gas chromatography analysis of the distillate indicated variations in the content of trace impurities, such as higher alcohols (n-butyl, isobutyl, and isoamyl). The proposed technology has the potential to optimize the fermentation process, adjust the quality of volatile components, and improve the sensory characteristics of fermented materials and distillates for alcoholic beverage production (beer, whiskey wort, brandy, alcoholic beverages, etc.).

The impact of extremely low-frequency magnetic fields on ethanol accumulation by Saccharomyces cerevisiae yeast during the fermentation of sugarcane molasses in a periodic fermentation process was explored. The cell suspension from the fermenter was directed through two magnetic field generators in recirculation mode. The recirculation rate and magnetic field strength varied within the range of 0.6–1.4 m•s⁻¹ and 5–20 μT, respectively. The best results were achieved when treated with a magnetic field of 20 μT at a recirculation rate of 0.9–1.2 m• s⁻¹, resulting in a yield of ethanol approximately 17% higher than in the control sample. Moreover, the fermentation duration was reduced by 2 hours [46].

3.3 Electron-ion processing (EIP)

The potential for activating brewer’s yeast through EIP to enhance membrane permeability and increase nutrient and oxygen availability for cells has been confirmed. These activated cells were found to maintain their viability for 3–5 cycles after EIP, attributed to the activation of the permease system. A correlation was established between the EIP modes and glycogen content of yeast cells. When using yeast that has undergone EIP, the main fermentation cycle of beer can be shortened by 15–40%. These activated cells facilitate vigorous wort fermentation without the need for additional fermentation activators. Implementing the proposed EIP method for brewer’s yeast activation prior to its introduction into the fermentation apparatus results in beer with a fermentation degree exceeding 80%, enhancing the stability of the final product. EIP-treated yeast can be utilized for 10–11 generations, extending their operational lifespan by 1.5 times compared to untreated yeast. This also reduces the cost associated with maintaining a pure yeast culture. Production tests confirmed the effectiveness of EIP, showing that processing low-quality yeast led to a 10–60% increase in viability and a 3–8% boost in the final fermentation degree while maintaining high beer quality standards. The beer produced using EIP-treated yeast met all standards and exhibited superior physicochemical and organoleptic properties compared to beer samples produced using traditional methods [47, 48].

Glushhenko [49] provides a description of the equipment used for electron-ion processing of yeast. The impact of electron-ion treatment on yeast cells viability was investigated, revealing a reduction in nonviable yeast cells numbers The most pronounced effect was observed when yeast was processed in a “yeast + wort” medium. The efficiency of electron-ion processing is contingent on the intensity of the nonuniform electric field and the exposure time. Implementing electron-ion processing results in a significant enhancement of the yeast’s physiological state.

3.4 Processing with visible light and laser radiation in the infrared range

Another approach for yeast-saccharomycete activation is based on exposure to visible spectrum light wavelengths.

The earliest publication we found on this subject dates back to 1984 [50]. This doctoral dissertation research demonstrates that exposure to shortwave optical radiation (410–520 nm) with an intensity of 0.12–0.20 W/m² leads to a substantial activation of metabolite transformations within the glycolytic system. Visible light exposure triggers the decarboxylation of pyruvate, facilitating the generation of a substrate for the operation of the di- and tricarboxylic acid cycle, thus alleviating substrate deficiencies in the Krebs cycle. In addition, it increases the activity of alcohol dehydrogenase, the final enzyme of alcoholic fermentation, and some respiratory enzymes. It also enhances the activity of isocitrate dehydrogenase, succinate dehydrogenase, aldehyde dehydrogenase, and the pyruvate dehydrogenase complex. Research also revealed that exposure to 410 nm light wavelength increases the rate of protein biosynthesis by 1.6 times compared to the control sample. Furthermore, an optimal parameter for applying optical radiation to influence the technological characteristics of beer seeding yeast was established. Exposure to 410 nm light at an intensity of 0.12–0.20 W/m² for 6 hours prior to inoculation into the wort was found to boost yeast fermentation activity by 20–25%. This lead to a reduction of 1 day in the main fermentation duration without compromising the quality of the final product. Additionally, beer produced with photoactivated yeast displayed enhanced resistance to colloidal turbidity, attributed to a significant reduction in the concentration of high-molecular-weight protein substances compared to the control sample. Regrettably, in our view, this research, although promising, did not receive further attention at that time and was only resumed three decades later.

Thus Kobelev and Bagaeva [51], Saccharomyces cerevisiae baker’s yeast was used. The initial yeast was grown on a solid nutrient Reader medium until the beginning of the stationary phase of population growth (72 hours). Then, they were transferred to a liquid medium, where the cells were exposed to light of various spectra. The experiments were carried out in three light-isolated blocks: 1st - white light (the source of illumination is fluorescent lamps LDS–40); 2nd – blue light (the source is fluorescent lamps LG-40, the transmission area is 420–460 nm); and 3rd – red light (the source is fluorescent lamps LC–40, the transmission area is 620–640 nm). The exposure time was 6 hours. The control was a yeast culture grown in the dark. The research results indicated a notable enhancement in yeast biomass when exposed to both blue and red light. Specifically, the biomass increase under blue light exposure was found to be 10% higher compared to the control sample, while under red light, this increase amounted to 5%. The rise in yeast biomass under white light conditions was consistent with the growth observed in the dark. This suggests that the effect of blue light is associated with the direct absorption of blue light quanta by components of the mitochondrial energy system, leading to an increased synthesis of high-energy compounds. Red light also affects yeast cells, but unlike blue light, its impact appears to influence a broader spectrum of cellular systems associated with overall metabolism.

For several years, our university has also been engaged in researching the effects of monochromatic light exposure within the visible spectrum, as well as near-ultraviolet and infrared spectra, on brewer’s yeast. Previously, a positive impact of such treatment on top-fermentation yeast [52] was demonstrated. This research is currently ongoing, utilizing bottom fermentation Saccharomyces cerevisiae Saflager S-189 yeast. Light with a certain wavelength is applied to dry yeast for 60 minutes, after which seeding is performed. Cultivation is carried out for 3 days at 28° C in a model nutrient medium, which is a sterilized 5% sucrose solution. The controlled parameters and their values (expressed as a percentage relative to the control samples) after 3 days of cultivation are presented in Figure 2.

Figure 2.

The effect of fixed wavelength light treatment on the indicators of bottom fermenting brewer’s yeast population.

Based on this data, it can be concluded that light with such wavelengths has a multidirectional effect on the state of the yeast population. The processing effect is less pronounced than most of the activation methods described earlier. Nevertheless, light treatment with a wavelength of 650 nm provided a higher degree of utilization of nutrients, a smaller proportion of nonviable cells, and a significantly lower increase in biomass than in the control sample. This can be an advantage in the brewing industry, as it helps in reducing the quantity surplus yeast, which, in compliance with environmental regulations, must be disposed of in an environmentally responsible manner. We have decided to continue the research, in order to consider the possibility of optimizing the processing parameters.

The effectiveness of low-intensity laser radiation (LILR) in the infrared range on brewer’s yeast of top fermentation was evaluated as a means of intensification of fermentation processes [53]. A treatment duration equal to 2 minutes was chosen as rational. The impact of radiation with pulse frequencies was tested at 300, 600, 760, 1200, 1500, and 3000 Hz. The object of treatment was Saccharomyces cerevisiae top fermentation dry yeast. It was found that when processing experimental samples with LILR at a frequency of 600 Hz, the number of nonviable yeast decreases by 75% and when processing at a frequency of 300 and 3000 Hz, by 48% compared to the control sample. In all samples, with the exception of treatment at a frequency of 3000 Hz, an increase in the number of cells with glycogen was observed. The greatest extent was observed in the case of treatment at frequencies of 300 and 600 Hz—by 42%—compared to the control sample. The maximum accumulation of biomass was revealed in the experimental sample after treatment at 600 Hz. In the same sample, on the 7th day of fermentation of 12% beer wort, the visible extract was 4.6%, while in the control sample only on the 9th day, it was 5.8%. This proves the possibility of reducing the duration of the main fermentation stage by 2 days. In the given sample, the amount of diacetyl and 2-butanol (0.6 and 0.2 mg/dm³) was lower than in the control sample (0.9 and 0.4 mg/dm³). The beer obtained with the use of top fermentation yeast treated with low frequencies (300, 600, 760 Hz) has a mild full taste and floral aroma. At the same time, it was found that processing by LILR at frequencies of 1500 and 3000 Hz led to a deterioration in most of the indicators monitored.

3.5 Other physical methods of yeast activation

The effect of a magnetic field on alcoholic fermentation using Saccharomyces cerevisiae yeast strain DAUFPE-1012 was studied. The yeast culture was exposed to a constant magnetic field of 220 microTesla generated by NdFeB (Neodymium-Iron-Boron) rod magnets for 24 hours at a temperature of - 23 +/−1°C. The magnets were placed diametrically opposite (N to S) on a cylindrical tubular reactor. Biomass growth in the culture medium of the reactor (yeast extract + glucose of 2%) was monitored over 24 hours by measuring changes in optical density, which correlated with the dry mass of cells. Ethanol and glucose concentrations were measured every 2 hours. As a result of this treatment, the biomass (g/dm³) increased by 2.5 times, and the ethanol concentration increased by 3.4 times compared to the control sample. Glucose consumption was higher in the magnetized cultures, which was in line with increased ethanol production [54].

However, an opposite viewpoint was also published, suggesting that constant or variable magnetic fields have no influence on cellular processes in Saccharomyces cerevisiae yeast [55].

The impact of electric current on yeast characteristics has also been studied. When a direct current of 10 mA or alternating current of 100 mA was applied to the culture medium, a significant increase in the rate of cell growth and accumulation of ethyl alcohol was observed. The content of higher alcohols, esters, and organic acids in culture media treated with direct and alternating current differed from that in the untreated sample. Several compounds, such as acetaldehyde and acetic acid, were formed from ethanol as a result of the electrode reaction [56].

Pulsating electromagnetic-induced currents (PEMIC, PEMF, PMF), simple alternating currents, and direct currents have been explored for their effects on cells, tissues, and organisms, stimulating membrane permeability and various metabolic processes [57].

4. Combined processing methods

A method for activating brewer’s yeast was investigated [5] and patented [58]. This method provides for the treatment of a suspension of yeast cells with an activation medium (whey, beer wort, or a mixture of whey and beer wort) in a ratio of 1:0.5 through acoustic exposure to low-frequency vibrations (20–2·10⁴ Hz) in a rotary pulsating machine for 2 minutes. The rotor speed and the inter-cylinder clearance were 2000 min⁻¹ (209.33 c⁻¹) and 0.2–0.3 mm, respectively. The processing parameters were selected to avoid an excessive number of dead cells, which should be kept under 10%. This processing method significantly improved the quality of brewer’s yeast. Compared to the control sample, the total concentration of cells increased by 3.9–8.1%, the percentage of dead cells decreased by 1.7–2.3 times, and the number of budding cells increased almost twofold. After 7 days of fermentation of 11% wort in the control sample, the actual extract was 4.9%, while in the experimental samples, similar concentrations of dry substances (4.6–4.5) were achieved after 5–6 days of the main fermentation. The volume fraction of ethyl alcohol in the finished beer of the control sample was 4.58%, whereas in the experimental variants, it ranged from 5.27 to 5.37% of the volume, with the actual extract at 3.5% and 2.3–2.4%, respectively.

A patent [59] was obtained for a yeast activation method designed for the alcohol industry. In this method, a suspension of alcoholic yeast in the production grain wort is subjected to an acoustic field with an oscillation frequency of 22 kHz and an oscillation intensity of 1.0 W/cm² for 3.5–4.5 minutes at a temperature of 28–34°C. A fraction of 5–10% of the total volume of the processed yeast suspension is then selected and undergoes a secondary treatment with an acoustic field at an oscillation frequency of 22 kHz and an oscillation intensity of 1.0 W/cm² for 35–45 minutes at a temperature of 48–52°C. This secondary treatment is aimed at destroying yeast shells and obtaining yeast extract. The resulting extract is subsequently mixed with the suspension of processed yeast. This process leads to a significant increase in yeast biomass by 2–3 times, faster start of wort fermentation at the first day of fermentation, and a reduced fermentation duration by 10–12 hours.

5. Comparing the effectiveness of various methods of activation of Saccharomyces cerevisiae yeast

From the information provided, it is evident that various methods are employed for different types of yeast, and their effectiveness is evaluated using diverse criteria. Nevertheless, we have chosen to conduct a comparative analysis of the effectiveness of selected methods by consolidating the data from in the literature into a single table (Table 1).

Activation method	The field of yeast use	Increasing the degree of fermentation, %	Increase in ethanol concentration, %	Increase in cell titer/yeast biomass growth, %	Shortening the duration of the stage	Increase in the proportion of budding cells, %	Increase in the proportion of cells with glycogen, %	Reduction of the proportion of non-viable cells	A source
Chemical
Sweet potato supplement	Brewing	5.8	3.6 in green beer	44	Lagering - for 2–3 days				7
Succinic acid	Alcohol production			14*		71*	11.5*		10
Amino acid-vitamin activator 0.2%	Brewing	6.6		87	The main fermentation - for 1 day	42	33.9		11
Complex yeast feeding	Brewing					50–100	60–70	8–30	18
Milk whey	Brewing				The main fermentation - for 1 day	100	30		23
Physical
Ultrasound 23 kHz	Alcohol production		19.3						38
Sound at frequency of 2765 Hz	Brewing	66						5	44
Light with a wavelength of 410 nm	Brewing	20–25			The main fermentation - for 1 day				50
Low-intensity laser radiation 600 Hz	Brewing				The main fermentation - for 2 days		42	75	53
Magnetic field			240	150					54
Combined
Rotary pulsation treatment + whey	Brewing		15–17	3.9–8.1	The main fermentation - for 1-2 days	100		70–130	58
Ultrasound + yeast extract	Alcohol production			100–200	Fermentation of alcoholic wort - for 10–12 hours				59

Table 1.

The effectiveness of various methods of activation of Saccharomyces cerevisiae yeast (change in the value of the indicator compared to the control sample).

*

After 24 hours of cultivation.

6. Conclusion

It can be seen that the proposed methods for improving the technological characteristics of Saccharomyces cerevisiae yeast are very diverse and aim at improving various properties of the cells of the population. At the same time, different authors recommend various parameters of the same type of exposure, in order to ensure an activating effect. Separate groups of researchers express sometimes opposing opinions about the effect of one and the same type of treatment. In our perspective, this suggests that the efficacy of treatment depends not only on the nature and methods of exposure but also on a large number of other variables, including the initial characteristics of yeast cells, yeast strain, the environmental conditions in which yeast develops within the culture medium, and various other factors. Therefore, it is advisable to continue the research and obtain more detailed information about the effectiveness of yeast activation methods. A number of the approaches herein discussed seem promising when applied on an industrial scale in the aims of intensifying the various technological stages in fermentation industries, ranging from yeast generation to wort fermentation.

Conflict of interest

The authors declare no conflict of interest.