**2. Redox potential**

Eh, like pH, is a parameter of the state of biological media which indicates the capacity to either gain or lose electrons. During oxidation, electrons are transferred from an electron donor to an electron acceptor, which is reduced. Electrochemical measurement of Eh is not new but has attracted little attention as a parameter for controlling fermentation processes due to the sensitivity of its measurement. However, Eh is already indirectly taken into account in industry through oxygen, of which the inhibitory effect on lactic acid bacteria is well-known. Indeed, oxygen modifies the growth capacity of microorganisms and the formation of end products, and so may contribute to the quality of fermented products [1, 2].

### **2.1. Definition of Eh**

**Oxidation** is a reaction in which a molecule, atom or ion, loses electrons.

**Reduction** is a reaction in which a molecule, atom or ion, gains electrons.

An **oxidant** (also known as an oxidizing agent, oxidizer or oxidiser) can be defined as a substance that removes electrons from another reactant in a redox reaction.

A **reductant** (also known as a reducing agent or reducer) can be defined as a substance that donates an electron to another species in a redox reaction.

In the same way pH defines acid-base characteristics of a solution, Eh defines the reducing and oxidizing characteristics.

Presented below is the reduction half-reaction of an oxidant (Ox) to its corresponding reduced species (Red):

$$\text{Ox} + \text{ne}^- \leftrightarrow \text{Red} \tag{1}$$

The Nernst equation gives the relationship between the redox potential and the activities of the oxidised and reduced species:

$$\mathbf{E}\_{\mathrm{h}} = \mathbf{E}\_{\mathrm{h}}^{0} + 2.3 \times \left(\frac{\mathrm{RT}}{\mathrm{nF}}\right) \times \log\left(\frac{\left[\mathrm{Ox}\right]}{\left[\mathrm{Red}\right]}\right) \tag{2}$$

where:

74 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

environment surrounding the microorganism.

be advantageously exploited in industry.

**2. Redox potential** 

**2.1. Definition of Eh** 

[1, 2].

acid bacteria applications in the food industry.

adaptation of lactic acid bacteria to extracellular redox depends on their ability to positively or negatively interfere with oxidants (electron acceptors) or reducing molecules (electron donors). Carbon and electron flow management by the cell will thus be highly dependent on

Potentially, all biochemical reactions in the cell, and therefore the enzymatic activity, may be influenced by the redox state of the environment. Dissolved oxygen is an oxidizer and can reach concentrations of 8 mg.L-1 of medium (equilibrium with air). Despite the strict anaerobic metabolism of some lactic acid bacteria, the majority are aerotolerant and can react with dissolved oxygen at varying levels. Lactic acid bacteria provided with NADH oxidase can reduce oxygen to water (reduction reaction coupled with the re-oxidation of NADH). This process influences both the intracellular and extracellular redox environment, and will result in a change in the metabolism, cellular physiology and physico-chemical

Changes in the extracellular environment can be monitored by measuring the redox potential (Eh). This parameter plays a key role in the quality of fermented dairy products, but is still rarely taken into consideration or is completely ignored during the manufacturing process. The reasons for this lack of interest can be attributed to difficulties associated with its measurement and control. Over the past ten years, several studies advocate the monitoring and control of Eh in fermented products using lactic acid bacteria selected for their reducing ability, redox molecules, or heat treatment. In terms of food applications, the variation in Eh must involve compounds that do not alter the product characteristics. So, modifying the Eh using gas, which enables the product characteristics to be maintained, may

The aim of this chapter is to present the latest knowledge concerning the adaptation of lactic acid bacteria to their redox environment, and the interest of modifying Eh using gas for lactic

Eh, like pH, is a parameter of the state of biological media which indicates the capacity to either gain or lose electrons. During oxidation, electrons are transferred from an electron donor to an electron acceptor, which is reduced. Electrochemical measurement of Eh is not new but has attracted little attention as a parameter for controlling fermentation processes due to the sensitivity of its measurement. However, Eh is already indirectly taken into account in industry through oxygen, of which the inhibitory effect on lactic acid bacteria is well-known. Indeed, oxygen modifies the growth capacity of microorganisms and the formation of end products, and so may contribute to the quality of fermented products

**Oxidation** is a reaction in which a molecule, atom or ion, loses electrons.

the ability of the microorganisms to interact with the redox environment.

Eh = redox potential (mV) (in relation to a normal hydrogen electrode).

0 *<sup>h</sup> E* = standard redox potential (mV) (in relation to a normal hydrogen electrode) at pH 0

F = Faraday constant (96500 C.mol-1)

n = number of electrons exchanged

R = gas constant (8.31 J.mol-1.K-1)

T = temperature in K

$$2.3 \times \frac{\text{RT}}{\text{F}} = 59 \text{ mV (at } 25 \text{ } ^\circ \text{C)}$$

However, chemical reactions in aqueous media involve protons, and the following halfreaction:

$$\text{Ox} + \text{mH}^+ + \text{ne}^- \leftrightarrow \text{Red} + \text{H}\_2\text{O} \tag{3}$$

From Equation (2) it can be written:

$$\mathbf{E}\_{\mathrm{h}} = \mathbf{E}\_{\mathrm{h}}^{0} - 2.3 \times \left(\frac{\mathrm{mRT}}{\mathrm{nF}}\right) \times \mathrm{pH} + 2.3 \times \left(\frac{\mathrm{RT}}{\mathrm{nF}}\right) \times \log\left(\frac{\left[\mathrm{Ox}\right]}{\left[\mathrm{Red}\right]}\right) \tag{4}$$

m = number of protons involved in the reaction

Equation (4) is used to determine 0' <sup>h</sup> E defined as the standard redox potential at pH 7, which is closer to biochemical and biological processes (Figure 1).

Redox Potential: Monitoring and Role in Development of Aroma Compounds, Rheological Properties and Survival of Oxygen Sensitive Strains During the Manufacture of Fermented Dairy Products 77

Ag / AgCl KCl 3M <sup>r</sup> E 207 0.8 (25 T) (6)

Calomel Saturated KCl E 244 0.7 (25 T) <sup>r</sup> (7)

hmr EE E (5)

h7 h E E α x (7 ) (8)

reference electrode. The reference system is the standard hydrogen electrode, but in practice two other references are used: the calomel electrode and the silver / silver chloride (Ag/AgCl) electrode. The redox potential is expressed in volts or millivolts. Redox values should always be expressed in relation to the hydrogen electrode. Consequently, potential measurements (Em) using other references must be adjusted according to the reference

For example, Er of the Ag/AgCl electrode is equal to 207 mV at 25 °C [3]. According to data from Galster [3], we propose the following equations linking Er and temperature for the two

Before use, the redox electrodes must be polished with ne aluminium powder to restore the platinum surface, and controlled in tap water. Three measurements in tap water should be compared and need to be within the condence interval around their mean value

Equation (4) shows the dependence of Eh on pH. It is possible to overcome pH dependency

To calculate Eh7 in biological media, the Nernst factor (α) must be determined experimentally by measuring Eh variation as a function of pH using an acid or a base. This value may vary according to the nature of the oxido-reducing molecules in the media. For

Gas applications in the food industry are numerous: modified atmosphere packaging (MAP), beverage distribution, cooling, freezing or carbonation. The advantage of using gases such as hydrogen (H2), nitrogen (N2) or carbon dioxide (CO2) to modify Eh is that they are not directly toxic to microorganisms. There are no safety issues for the product with these gases and they can be used sequentially. Finally, their use is authorized at European

(calculated at 20 mV, 95% condence level) to ensure correct measurement [4].

potential of the hydrogen electrode (Er):

by applying the Leistner and Mirna equation [5]:

= Nernst Eh–pH correlation factor (mV/pH unit).

example, the Nernst factor is 40 mV/pH unit in milk [6].

Eh7 = redox potential (mV) at pH 7 Eh = redox potential (mV) at pH

**2.3. Use of gas to modify Eh** 

reference electrodes:

where:

= pH of medium

**Figure 1.** Standard reduction potential 0' <sup>h</sup> E (mV) of some important half-reactions involved in biological processes at 25 °C and pH 7.

### **2.2. Measurement of Eh**

The first technique for measuring Eh is based on the use of coloured indicators (redox indicators), which are mostly indophenols or indigo derivatives with a reversible structure between oxidized (coloured) and reduced (colourless) state. However, the use of coloured indicators for measuring Eh, including biological media or food, is limited. Indeed, these molecules behave as electron donors and acceptors; they affect and can change the equilibrium. These compounds can also catalyse or inhibit biological reactions and may be toxic to microorganisms. Furthermore, in some cases it is difficult to appreciate a significant colour change and some Eh indicators also change colour with the pH of the medium. For these reasons, redox indicators are rarely used. They are more often used as indicators of redox thresholds, especially in the manufacture of strictly anaerobic culture media (resazurin) where maintaining a minimum level of reduction is essential for the growth of anaerobic microorganisms. Resazurin is also used to evaluate the reducing activity of starter cultures, for sterility testing and for the detection of microorganisms in dairy milk.

The second method commonly used in microbiology is a potentiometric technique which, contrary to redox indicators, is a direct method. The principle consists in measuring a potential difference determined between an inert electrode (usually made of platinum or gold) in contact with a redox couple in solution and a reference electrode. Electron exchange with the reduced and oxidised species takes place at the inert electrode. The inert electrode is made of stainless metals with a high enough standard potential to be electrochemically stable. These metals act as electron conductors between the measuring medium and the reference electrode. The reference system is the standard hydrogen electrode, but in practice two other references are used: the calomel electrode and the silver / silver chloride (Ag/AgCl) electrode. The redox potential is expressed in volts or millivolts. Redox values should always be expressed in relation to the hydrogen electrode. Consequently, potential measurements (Em) using other references must be adjusted according to the reference potential of the hydrogen electrode (Er):

$$\mathbf{E\_h = E\_m + E\_r} \tag{5}$$

For example, Er of the Ag/AgCl electrode is equal to 207 mV at 25 °C [3]. According to data from Galster [3], we propose the following equations linking Er and temperature for the two reference electrodes:

Ag / AgCl KCl 3M <sup>r</sup> E 207 0.8 (25 T) (6)

$$\text{Calculel} \left( \text{Saturated KCl} \right) \qquad \qquad \to\_r = 244 + 0.7 \times \left( 25 - \text{T} \right) \tag{7}$$

Before use, the redox electrodes must be polished with ne aluminium powder to restore the platinum surface, and controlled in tap water. Three measurements in tap water should be compared and need to be within the condence interval around their mean value (calculated at 20 mV, 95% condence level) to ensure correct measurement [4].

Equation (4) shows the dependence of Eh on pH. It is possible to overcome pH dependency by applying the Leistner and Mirna equation [5]:

$$\mathbf{E}\_{\mathbf{h}\mathcal{T}} = \mathbf{E}\_{\mathbf{h}\mathfrak{B}} - \alpha \ge (\mathcal{T} - \mathfrak{B}) \tag{8}$$

where:

76 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

**Figure 1.** Standard reduction potential 0'

+600

+400

biological processes at 25 °C and pH 7.

Fe2+/Fe3+

**2.2. Measurement of Eh** 

Eh

0'

(mV)

+800

O2/H2O

<sup>h</sup> E (mV) of some important half-reactions involved in

0

Dehydroascorbate/Ascorbate


Pyruvate/Lactate


GSSG/GSH (G=Glutathione)

NAD+/NADH

CSSC/CSH (C=Cystein)

H+/H2

The first technique for measuring Eh is based on the use of coloured indicators (redox indicators), which are mostly indophenols or indigo derivatives with a reversible structure between oxidized (coloured) and reduced (colourless) state. However, the use of coloured indicators for measuring Eh, including biological media or food, is limited. Indeed, these molecules behave as electron donors and acceptors; they affect and can change the equilibrium. These compounds can also catalyse or inhibit biological reactions and may be toxic to microorganisms. Furthermore, in some cases it is difficult to appreciate a significant colour change and some Eh indicators also change colour with the pH of the medium. For these reasons, redox indicators are rarely used. They are more often used as indicators of redox thresholds, especially in the manufacture of strictly anaerobic culture media (resazurin) where maintaining a minimum level of reduction is essential for the growth of anaerobic microorganisms. Resazurin is also used to evaluate the reducing activity of starter

+200

cultures, for sterility testing and for the detection of microorganisms in dairy milk.

The second method commonly used in microbiology is a potentiometric technique which, contrary to redox indicators, is a direct method. The principle consists in measuring a potential difference determined between an inert electrode (usually made of platinum or gold) in contact with a redox couple in solution and a reference electrode. Electron exchange with the reduced and oxidised species takes place at the inert electrode. The inert electrode is made of stainless metals with a high enough standard potential to be electrochemically stable. These metals act as electron conductors between the measuring medium and the Eh7 = redox potential (mV) at pH 7 Eh = redox potential (mV) at pH = pH of medium = Nernst Eh–pH correlation factor (mV/pH unit).

To calculate Eh7 in biological media, the Nernst factor (α) must be determined experimentally by measuring Eh variation as a function of pH using an acid or a base. This value may vary according to the nature of the oxido-reducing molecules in the media. For example, the Nernst factor is 40 mV/pH unit in milk [6].

### **2.3. Use of gas to modify Eh**

Gas applications in the food industry are numerous: modified atmosphere packaging (MAP), beverage distribution, cooling, freezing or carbonation. The advantage of using gases such as hydrogen (H2), nitrogen (N2) or carbon dioxide (CO2) to modify Eh is that they are not directly toxic to microorganisms. There are no safety issues for the product with these gases and they can be used sequentially. Finally, their use is authorized at European

level. Of the gases used in the food industry, in this chapter we will focus more particularly on nitrogen and hydrogen.

Redox Potential: Monitoring and Role in Development of Aroma Compounds, Rheological Properties and Survival of Oxygen Sensitive Strains During the Manufacture of Fermented Dairy Products 79

lactic acid bacteria for acidification, proteins act on the texture and minerals help stabilize the gel. These components vary in cow's milk according to race, diet, stage of lactation of the animal and season, which is why, during yoghurt manufacture, it is necessary to standardize the milk fat and protein content to meet the nutritional and organoleptic

characteristics of the product and obtain consistent quality throughout the year. Homogenization: homogenization has two main effects on milk fat and proteins. The fragmentation of the fat globules prevents the separation of the lipid phase and the rest of the mixture, thus preventing the cream rising to the top during fermentation.

 Heat treatment: This eliminates most of the microbial flora originally present in the milk, including pathogenic or spoilage flora. It denatures the whey proteins, improves the consistency and viscosity of fermented milks and prevents whey separation. The

Heat treatment of milk also has a positive effect on enzyme activity by providing a supportive environment. The environment becomes reductive through the elimination of a high proportion of oxygen. This medium is more conducive to fermentation that

Cooling: After heat treatment, the mixture must be cooled to temperatures approaching

 Fermentation: "Yoghurt" refers to a product fermented by *Streptococcus thermophilus* (*S. thermophilus*) and *Lactobacillus delbrueckii* ssp. *bulgaricus* (*Lb. bulgaricus*). In general, milk is fermented at 40-45 °C, the optimum growth temperature, with an incubation time of 2 and a half hours. However, a longer incubation period of 16-18 hours can be used at a

**3.2. Yoghurt strains: Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus** 

The association of *S. thermophilus* and *Lb. bulgaricus* is called proto-cooperation. Each species produces one or more substances, initially absent from the culture medium, that stimulate the growth of the other species [10]. During the symbiosis observed in yoghurt, the growth phases of these two bacterial species are staggered. Initially, growth of *S. thermophilus* is observed which is then slowed by the inhibitory effect of the lactic acid produced; the

*S. thermophilus* is a strain that often shows little proteolytic activity, due to general low activity or absence of a wall protease. Its growth is limited because the peptides and amino acids initially present in milk are insufficient to cover its needs. In contrast, *Lb. bulgaricus* membrane protease degrades milk caseins releasing small peptides and amino acids which

The cooperation between these two strains also involves the production by *S. thermophilus* of pyruvic acid, formic acid, and carbon dioxide (CO2 obtained from the decarboxylation of milk urea by urease) which stimulates the growth of *Lb. bulgaricus* [9, 13]. However, formic acid is released late in fermentation and in small quantities. The two bacterial species also

Homogenization also stabilizes the proteins.

takes place under anaerobic conditions.

growth rate of *Lb. bulgaricus* then increases [11].

can be used by *S. thermophilus* intracellular peptidases [12].

consume the formic acid resulting from the heat treatment of milk [14].

43 °C for inoculation and incubation of the starter culture.

temperature of 30 °C, or until the desired acidity is attained [9].

risk of syneresis is reduced.

**thermophilus** 

Nitrogen (N2) is odourless, colourless, tasteless, non-toxic, and non-flammable. It is used to extend the life of packaged products (authorized additive E941). It is used to expel oxygen from the packaging before it is closed, which prevents oxidative phenomena involving pigmentation, flavours and fatty acids. It is also used for rapid freezing and refrigeration of food during transport.

Hydrogen (H2) has major potential in food as it is colourless, odourless and has no known toxic effects. It is already used in the food industry for the hydrogenation of liquid oils and their transformation into solid products such as margarine or peanut butter. Hydrogen is a powerful reducer in solution, even at very low concentrations. It has been used to demonstrate the effect of Eh on the heat-resistance of bacteria [7]. Hydrogen is a special reducing agent: it imposes an Eh value on the medium associated with the introduction of the H+/H2 couple ( 0' *<sup>h</sup> E* = -414 mV). This Eh value is highly dependent on the concentration of this couple that mainly influences the stability of the Eh imposed.

With the prospect of food use, hydrogen has the advantage over chemical reducing agents of not changing the product formulation, and therefore not altering the taste. Its industrial use has been rarely seen in this context because of its low flammability limit of 4% in air at 20 °C [8], this is why N2-H2 (96%-4%) is preferred to pure hydrogen. Its use in food technology is authorised at the European level (E949).
