*3.3.1. A look at yoghurt texture*

The transformation of milk into yoghurt is called acid gelation. This gelation is a phenomenon that results in a remarkable change in the physical state of the system which changes from a liquid to a system with the characteristics of a solid. Several phases in the formation of a gel can be distinguished:


The slow acidification of milk is due to bacteria that metabolize lactose and produce lactic acid. While casein micelles are stable at normal milk pH and room temperature, this supramolecular structure becomes unstable and leads to the formation of a gel with the slow progressive acidification of milk.


Rheology is used to characterize the texture of yoghurt that specifically targets the mechanical properties. The rheological characterization of a product involves the application of a shear stress and measurement of the deformation, or application of a deformation (compression, stretching or shear) and measurement of its ability to withstand this distortion. Yoghurt can be defined as a viscoelastic fluid. It therefore has both the viscous properties of a liquid and the elastic properties of a solid.

### *3.3.2. Effect of Eh on a model acid skim milk gel*

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

the milk.

**3.3. Texture** 

*3.3.1. A look at yoghurt texture* 

held together;

of the liquid phase.

progressive acidification of milk.

formation of a gel can be distinguished:

gel, with dominant elastic rheological behaviour;

potential ζ and begin to form groups of micelles.

between the solution and the acid gel.

are relationships between them that give yoghurt its distinctive flavour.

Some authors have also demonstrated that the association of *S. thermophilus* and *Lb. bulgaricus* affects the production of volatile compounds involved in flavour development in yoghurt [15]. *S. thermophilus* produces more acetaldehyde, acetoin and diacetyl than *Lb. bulgaricus*, contrary to the rest of the bibliography concerning acetaldehyde [9, 16, 17]. Quantities of these molecules and other carbonyl compounds are not crucial per se for yoghurt flavour, but there

Finally, Ebel *et al.* [18] showed that during the manufacture and storage of a fermented dairy product, the populations of *Lb. bulgaricus* and *S. thermophilus* are the same whatever the Eh of

The transformation of milk into yoghurt is called acid gelation. This gelation is a phenomenon that results in a remarkable change in the physical state of the system which changes from a liquid to a system with the characteristics of a solid. Several phases in the




The slow acidification of milk is due to bacteria that metabolize lactose and produce lactic acid. While casein micelles are stable at normal milk pH and room temperature, this supramolecular structure becomes unstable and leads to the formation of a gel with the slow




Rheology is used to characterize the texture of yoghurt that specifically targets the mechanical properties. The rheological characterization of a product involves the

due to reduced electrostatic repulsion, leading to micelle aggregation.

It has been shown that dairy products are affected by Eh [4, 19]. Delbeau *et al.* [19] showed that the use of gas to change the Eh of milk can modify the sensory properties of a fermented dairy product. However, we do not know if these modifications are due to the impact of Eh on physicochemical phenomena, lactic acid bacteria, or both. For this purpose, Martin *et al.* [20] wanted to determine to what extent chemical phenomena affect acid milk gelation under different Eh conditions. Glucono-δ-lactone (GDL) was used to acidify milk to avoid variations caused by microorganisms sensitive to Eh.

Martin *et al.* [20] studied the effects of Eh on model acidified skim milk gels obtained using GDL and prepared under different gaseous conditions. The milk prepared in air is an oxidizing medium; nitrogen, which is a neutral gas, can be used to remove oxygen from milk - even so the milk Eh remains oxidizing in these conditions - and hydrogen leads to a reducing Eh (below 0). Martin *et al.* [20] focused on the effect of gas bubbling on gel structure through viscoelastic properties and measurement of whey separation (Table 1).


a-c: different letters indicate that groups were significantly different at an α risk of 5% (ANOVA test). Values in the same column should be compared.

Reprinted from Journal of Dairy Science, Vol 92, Martin F, Cayot N, Marin A, Journaux L, Cayot P, Gervais P, Cachon R, Effect of oxidoreduction potential and of gas bubbling on rheological properties and microstructure of acid skim milk gels acidified with glucono-δ-lactone, Pages No. 5898-5906, Copyright (2009), with permission from Elsevier.

**Table 1.** Characteristics of gel structure depending on the different Eh conditions (milk acidified using GDL):

• Apparent viscosity η at 500 1/s of GDL-gel at pH 4.6 and 4 °C. Measurements were carried out 24 hours after addition of GDL.

• Evolution of average whey separation (WS) over 28 days in GDL-gels.

Values are means from triplicate experiments (mean value standard deviation).

The apparent viscosity of each gel was characterized at pH 4.6, 4 °C, 24 hours after addition of GDL under the different Eh conditions (Table 1). For GDL-gels, apparent viscosity ranged from 0.032 to 0.039 Pa.s. GDL-gels produced in air had the highest apparent viscosity, whereas values obtained with air and N2 – H2 bubbling were similar and significantly lower than those obtained with N2 bubbling. So, for GDL-gels, the viscosity was affected by bubbling. Martin *et al.* [20] showed that the type of gas used for bubbling has a significant influence but no clear trend can be deduced from these results in terms of the influence of an oxidizing or reducing environment.

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 83

At t=0 of yoghurt) At t=3.5

hours

20 171a ± 2 63.60b ±

11 139b ± 5 25.29c

CEPS

3.72

0.74

 ± 10 62.70b ± 0.75

 ± 0.40

(mg/L) η (Pa.s) WS (g/100g

0.046a ±

0.046a ±

0.035b ±

0.021c ±

0.00 1.98a ± 0.54

0.00 1.76a ± 0.31

0.00 1.03ab ± 0.27

0.01 0.59b ± 0.12

pH Eh7 (mV)

0.0 4.6a ± 0.0 425a ±

0.0 4.6a ± 0.0 285b ±

0.0 4.6a ± 0.0 -345c

hours At t=0 At t=3.5

0.0 4.6a ± 0.0 435a ± 3 241a ± 8 15.22a ±

± 4 -309c

a, b, c: different letters indicate that groups were significantly different at an α risk of 5% (ANOVA test). Values in the

Reprinted from Journal of Food Res. Int., Vol 431, Martin F, Cayot N, Vergoignan C, Journaux L, Gervais P, Cachon R, Impact of oxidoreduction potential and of gas bubbling on rheological properties of non-fat yoghurt, Pages No. 218-

**Table 2.** Characteristics of gel structure depending on the different Eh conditions (milk acidified using

• Apparent viscosity η at 500 1/s of yoghurt at pH 4.6 and 4 °C. Measurements were made 24 hours

The apparent viscosity of each yoghurt was characterized at pH 4.6 and 4 °C, 24 hours after addition of bacteria under the different Eh conditions (Table 2). The apparent viscosity ranged from 0.021 to 0.046 Pa.s. Yoghurts produced in air and with air bubbling had the highest apparent viscosity. The apparent viscosity of yoghurts made with N2 bubbling was lower (0.035 Pa.s) than other oxidizing conditions (0.046 Pa.s), and values obtained with N2 – H2 bubbling were the lowest (0.021 Pa.s). Apparent viscosity is clearly affected by the gas

Apparent viscosity depends on the solid fraction in the gel as well as the relationships between the different solid elements. In yoghurt, solid particles include milk proteins, lactic acid bacteria and their EPS. Indeed, the gel of yoghurts produced under N2 – H2 conditions is weaker despite greater EPS production [23]. It is a common assumption that EPS produced by bacteria contribute to the rheological properties of yoghurt [31-33] but, as reported by Hassan *et al.* [34], van Marle [35] and Martin *et al.* [23], no correlation between

Whey separation of yoghurts produced under different Eh conditions over 28 days of storage was then studied [23] (Table 2). Concerning GDL-gels, whey separation of yoghurts occurred from the very first day of storage and the volume of whey separation was relatively constant over the 28 days of storage. Whey separation ranged from 0.59 to

• Concentrations of exopolysaccharides (CEPS) in yoghurts after one day of storage.

type. A reducing environment reduces the apparent viscosity of yoghurt.

the viscosity of yoghurt and EPS concentrations was found.

• Evolution of average whey separation over 28 days (WS) in yoghurts.

Gaseous conditions applied to milk

Air 6.80a ±

Air bubbling 6.80a ±

N2 bubbling 6.81a ±

same column should be compared.

after addition of starter culture.

6.81a ±

223, Copyright (2010), with permission from Elsevier.

Values are means from triplicate experiments.

N2 – H2 bubbling

lactic starters):

The gel structure was then observed during storage for up to 28 days. The mean whey separation values of GDL-gels produced under different Eh conditions are presented in Table 1. For each gaseous condition, the authors observed that whey separation occurred from the very first day of storage and the volume of whey separation was relatively constant during the 28 days of storage [20]. Whey separation ranged from 0.59 to 4.74 g / 100 g of GDL-gels. The highest whey separation was obtained with air but this value was lower than values reported in the literature: 18.48% of GDL-gels in the work by Lucey *et al*. [21] and 10% in a study by Fiszman *et al.* [22]. One explanation for is that in the study by Lucey *et al.* [21] the method used to measure whey separation was to remove the gels from their flasks and thus whey separation could have been over-estimated. Whey separation obtained with gas bubbling was lower (1.26 g / 100 g with air bubbling, 1.93 g / 100 g with N2 bubbling and 0.59 g / 100 g with N2 - H2 bubbling). The lowest whey separation was observed with GDL-gels made under N2 – H2. Adjusting the Eh of milk to reducing conditions (under N2 – H2) could be a possible way of significantly decreasing the phenomenon of whey separation.

### *3.3.3. Effect of Eh on a non-fat yoghurt*

In a second step, the authors proposed studying the extent to which lactic acid bacteria affect acid milk gelation under different Eh conditions [23]. Indeed, oxygen modifies the growth capacity of bacteria and the formation of end products. So, Eh may contribute to the quality of fermented products [2, 24, 25]. Martin *et al.* [23] wanted to determine the effects of Eh on yoghurts made under various gaseous conditions. In this study they focused on exopolysaccharide production and gel structure (Table 2). The same gaseous conditions as in the study on the effect of Eh on model acid skim milk gels were chosen.

*Lb. bulgaricus* and *S. thermophilus* produce exopolysaccharides (EPS) which can contribute to improving the texture and viscosity of fermented dairy products [26]. In standard yoghurts (produced in air) the concentration of EPS was 63.60 mg.L-1, in accordance with the literature (50 to 350 mg.L-1) [27, 28]. The concentration was lower in yoghurts produced with air bubbling (15.22 mg.L-1) than in yoghurts produced with N2 bubbling, which was lower than those made with N2 – H2 bubbling. The EPS concentration of yoghurts made in Air and with N2 – H2 bubbling were similar. In reducing Eh conditions, lactic acid bacteria produced the same amount of EPS as in ambient air. This result has already been observed in the literature. Indeed, *Lactobacillus sake* 0-1 was reported to have optimal EPS production in anaerobic conditions [29], while higher EPS yields were correlated with a lower oxygen tension [30].


a, b, c: different letters indicate that groups were significantly different at an α risk of 5% (ANOVA test). Values in the same column should be compared.

Reprinted from Journal of Food Res. Int., Vol 431, Martin F, Cayot N, Vergoignan C, Journaux L, Gervais P, Cachon R, Impact of oxidoreduction potential and of gas bubbling on rheological properties of non-fat yoghurt, Pages No. 218- 223, Copyright (2010), with permission from Elsevier.

**Table 2.** Characteristics of gel structure depending on the different Eh conditions (milk acidified using lactic starters):


Values are means from triplicate experiments.

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

oxidizing or reducing environment.

*3.3.3. Effect of Eh on a non-fat yoghurt* 

tension [30].

The apparent viscosity of each gel was characterized at pH 4.6, 4 °C, 24 hours after addition of GDL under the different Eh conditions (Table 1). For GDL-gels, apparent viscosity ranged from 0.032 to 0.039 Pa.s. GDL-gels produced in air had the highest apparent viscosity, whereas values obtained with air and N2 – H2 bubbling were similar and significantly lower than those obtained with N2 bubbling. So, for GDL-gels, the viscosity was affected by bubbling. Martin *et al.* [20] showed that the type of gas used for bubbling has a significant influence but no clear trend can be deduced from these results in terms of the influence of an

The gel structure was then observed during storage for up to 28 days. The mean whey separation values of GDL-gels produced under different Eh conditions are presented in Table 1. For each gaseous condition, the authors observed that whey separation occurred from the very first day of storage and the volume of whey separation was relatively constant during the 28 days of storage [20]. Whey separation ranged from 0.59 to 4.74 g / 100 g of GDL-gels. The highest whey separation was obtained with air but this value was lower than values reported in the literature: 18.48% of GDL-gels in the work by Lucey *et al*. [21] and 10% in a study by Fiszman *et al.* [22]. One explanation for is that in the study by Lucey *et al.* [21] the method used to measure whey separation was to remove the gels from their flasks and thus whey separation could have been over-estimated. Whey separation obtained with gas bubbling was lower (1.26 g / 100 g with air bubbling, 1.93 g / 100 g with N2 bubbling and 0.59 g / 100 g with N2 - H2 bubbling). The lowest whey separation was observed with GDL-gels made under N2 – H2. Adjusting the Eh of milk to reducing conditions (under N2 – H2) could be a possible way of significantly decreasing the phenomenon of whey separation.

In a second step, the authors proposed studying the extent to which lactic acid bacteria affect acid milk gelation under different Eh conditions [23]. Indeed, oxygen modifies the growth capacity of bacteria and the formation of end products. So, Eh may contribute to the quality of fermented products [2, 24, 25]. Martin *et al.* [23] wanted to determine the effects of Eh on yoghurts made under various gaseous conditions. In this study they focused on exopolysaccharide production and gel structure (Table 2). The same gaseous conditions as in

*Lb. bulgaricus* and *S. thermophilus* produce exopolysaccharides (EPS) which can contribute to improving the texture and viscosity of fermented dairy products [26]. In standard yoghurts (produced in air) the concentration of EPS was 63.60 mg.L-1, in accordance with the literature (50 to 350 mg.L-1) [27, 28]. The concentration was lower in yoghurts produced with air bubbling (15.22 mg.L-1) than in yoghurts produced with N2 bubbling, which was lower than those made with N2 – H2 bubbling. The EPS concentration of yoghurts made in Air and with N2 – H2 bubbling were similar. In reducing Eh conditions, lactic acid bacteria produced the same amount of EPS as in ambient air. This result has already been observed in the literature. Indeed, *Lactobacillus sake* 0-1 was reported to have optimal EPS production in anaerobic conditions [29], while higher EPS yields were correlated with a lower oxygen

the study on the effect of Eh on model acid skim milk gels were chosen.

The apparent viscosity of each yoghurt was characterized at pH 4.6 and 4 °C, 24 hours after addition of bacteria under the different Eh conditions (Table 2). The apparent viscosity ranged from 0.021 to 0.046 Pa.s. Yoghurts produced in air and with air bubbling had the highest apparent viscosity. The apparent viscosity of yoghurts made with N2 bubbling was lower (0.035 Pa.s) than other oxidizing conditions (0.046 Pa.s), and values obtained with N2 – H2 bubbling were the lowest (0.021 Pa.s). Apparent viscosity is clearly affected by the gas type. A reducing environment reduces the apparent viscosity of yoghurt.

Apparent viscosity depends on the solid fraction in the gel as well as the relationships between the different solid elements. In yoghurt, solid particles include milk proteins, lactic acid bacteria and their EPS. Indeed, the gel of yoghurts produced under N2 – H2 conditions is weaker despite greater EPS production [23]. It is a common assumption that EPS produced by bacteria contribute to the rheological properties of yoghurt [31-33] but, as reported by Hassan *et al.* [34], van Marle [35] and Martin *et al.* [23], no correlation between the viscosity of yoghurt and EPS concentrations was found.

Whey separation of yoghurts produced under different Eh conditions over 28 days of storage was then studied [23] (Table 2). Concerning GDL-gels, whey separation of yoghurts occurred from the very first day of storage and the volume of whey separation was relatively constant over the 28 days of storage. Whey separation ranged from 0.59 to

1.98 g/100 g of yoghurt. The highest whey separation was obtained with air and air bubbling and these values are in accordance with the literature [22]. Whey separation obtained with N2 bubbling (1.03 g / 100 g) and N2 - H2 bubbling (0.59 g / 100 g) was lower. So, the more reducing the environment, the lower the whey separation. Adjusting the Eh of milk to reducing conditions (under N2 – H2) could be a possible way of significantly decreasing the phenomenon of whey separation.

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 85

diketones [40, 41]. Moreover, during storage at 4 °C, the concentration of the two diketones

Agitating a mixed culture of *Lactococcus* and *Leuconostoc* promotes diacetyl production by allowing oxidative decarboxylation of 2-acetolactate [42, 43]. In unstirred cultures, the redox potential of the medium decreases rapidly at the start of fermentation. Only acetoin and 2 acetolactate are produced. The authors also showed that controlled oxygenation of the *Lactococcus lactis* ssp. *lactis* culture medium favoured diacetyl production by increasing the

Neijssel *et al.* [40] showed that the distribution of carbon flux from pyruvate depended on the NADH / NAD+ ratio, intracellular redox potential or the concentration of metabolites and particularly that of pyruvate. Finally, the authors suggested adding air or oxygen to

References [36] and [37] are the only articles that mention dimethyl sulphide as a compound having a significant impact on the flavour of yoghurt. The metabolic pathways involved in the synthesis of sulphur compounds are not well-known in yoghurt. However, the literature

In general, the majority of sulphur aromatic compounds come from methionine [46]. Methanethiol is easily oxidized to dimethyl disulphide and dimethyl trisulphide [47]. The appearance of these compounds is the direct result of the methanethiol content and is modulated by the low redox potential in Cheddar. Dimethyl sulphide is produced by a metabolic pathway that does not involve methanethiol, but that is different to that of

Studies have also shown that when the redox potential decreases, methanethiol and hydrogen sulphide concentrations increase [45]. Moreover, the cheeses to which reducing compounds (dithiothreitol or glutathione) were added contained higher amounts of sulphur compounds and had better qualitative and quantitative flavour performances [45]. It therefore seems that a reducing environment is essential for the production of aroma compounds by bacteria. If a cheese is exposed to air, the redox increases and this leads to the

Studies on aroma biosynthesis by LAB usually take into account environmental factors such as pH and temperature. However, the Eh of the medium has not yet been considered, although it is supposed to affect bacterial metabolism [49, 50]. Martin *et al.* [51] determined to what extent Eh can affect the metabolic pathways involved in the production of aroma compounds in *Lb. bulgaricus* and *S. thermophilus.* Four aroma compounds (acetaldehyde, dimethyl sulphide, diacetyl and pentane-2,3-dione) were chosen as metabolic tracers of lactic acid bacteria metabolism. The same gaseous conditions as in the study of the effect of Eh on model acid skim milk gels and non-fat yoghurt were chosen. The amounts of each of the four aroma compounds extracted using a headspace solid-phase micro-extraction

increases slightly [41] due to the basal metabolic activity of the bacteria.

milk in order to increase the amount of diacetyl in cheese [45].

mentions these synthetic pathways in the development of cheese flavour.

dimethyl sulphide and trimethyl disulphide from methionine [48].

oxidation of sulphur compounds, resulting in lower quality aromatics.

*3.4.2. Impact on aroma biosynthesis by lactic acid bacteria* 

activity of diacetyl synthase [44].
