6.1 Methods of lactose hydrolysis

The enzymatic hydrolysis of lactose is the most common method of lactose hydrolysis (Figure 3) and can be achieved in several ways. The common industrial conversion of lactose to ethanol uses an ethanol producing microbe, K. marxianus which enzymatically hydrolyzes lactose [8, 9]. Whey or whey permeate is cooled to the microbe's optimum fermentation temperature and then inoculated. Hydrolysis of lactose is achieved intracellularly via β-galactosidase and the organism subsequently metabolizes the constituents to produce ethanol [18]. It should be noted that the traditional brewing and distilling yeast used to produce ethanol, S. cerevisiae does not express the genes necessary to produce β-galactosidase, as an alternative a β-galactosidase producing yeast, K. marxianus is used. Genetic engineering of S. cerevisiae to produce β-galactosidase has been explored on an experimental scale

Figure 3. Enzymatic hydrolysis of lactose.

for bioethanol production, but to the authors' knowledge this is not being used in beverage production [19–21].

A common method of lactose hydrolysis in dairy product production is the addition of lactase, an exogenous enzyme belonging to the β-galactosidases family [22–24]. The addition of this enzyme requires no additional processing equipment and lactase is widely available. Using lactase to hydrolyze lactose allows for the use of microbes, which do not produce β-galactosidase, to be used in the fermentation. This approach has been explored and documented on an experimental scale for bioethanol production [25, 26].

Other methods of hydrolysis of lactose include the use of immobilized enzyme systems, membrane reactor processes used to recover enzymes/cells and acid hydrolysis [24, 27]. Immobilized enzyme and membrane reactor systems could help reduce cost because both are enzyme conservation processes, but they require additional processing technology and are not widely implemented commercially. Acid hydrolysis requires the use of ultrafiltration because the whey permeate stream must be free of protein. The process involves the acidification and short heat treatment ranging from approximately 100–150°C. This treatment causes a brown discoloration in serum which requires color removal and purification steps [24, 27]. The color removal process would not be necessary during ethanol production. While these technologies and processes are currently not used in the commercial conversion of whey to ethanol, some have been explored to increase production efficiency [26, 28–30].

### 6.2 Fermentation after lactose hydrolysis

Alcoholic fermentation is a form of anaerobic energy production commonly used by plants, yeast and other microbes [31]. This metabolic pathway has been exploited by humans for food and beverage production for several millennia. During industrial production of ethanol from whey, an ethanol-fermenting strain of K. marxianus is used to convert lactose into ethanol. This strain of K. marxianus is used because it can intracellularly hydrolyze lactose and efficiently produce ethanol.

Alcoholic fermentation has two distinct phases. The first phase is glycolysis which converts glucose to pyruvate. The glycolytic pathway is common to nearly all

Figure 4. Alcoholic fermentation after lactose hydrolysis.

Whey to Vodka DOI: http://dx.doi.org/10.5772/intechopen.81679

cells and generates adenosine triphosphate (ATP) which is used for intracellular energy transfer. Galactose is enzymatically converted to glucose 6-phosphate, an intermediate product of glycolysis (Figure 4). The conversion of galactose to glucose 6-phosphate is a four step process; however, the cellular energetic cost is the same as the phosphorylation of glucose. The outcome of this glycolysis process is net production of 4 ATP, the conversion of glucose and galactose to 4 pyruvate molecules and the reduction of NAD<sup>+</sup> to NADH.

The second phase of alcoholic fermentation converts pyruvate into ethanol to regenerate NAD<sup>+</sup> used during glycolysis. Pyruvate is decarboxylated enzymatically which results in the production of CO2 and the formation of acetaldehyde. The reduction of acetaldehyde to ethanol is catalyzed by alcohol dehydrogenase and NAD<sup>+</sup> is replenished in the process [31]. Ethanol is then passively diffused from the cell into the fermentation substrate.
