Brines containing NaCl and/or TPP were incorporated into the selected muscles (10% w/w) with an automatic multi-needle injector (36 needles, Fricor, Buenos Aires, Argentina) to give the final concentrations (g/100 g injected muscle) of 0.70 NaCl; 0.25 TPP; 0.70 NaCl + 0.25 TPP, and 1.20 NaCl + 0.25 TPP. Non injected (NI) muscles were used as control. Injected and NI muscles were vacuum packaged (Cryovac CN510, Sealed Air Co., Buenos Aires, Argentina) and submitted to continuous tumbling at 5.0 rpm, 8 h, 2-4 °C (Lance Industries tumbler, model LT-15, Allenton, USA) in order to improve brine sorption and protein extraction. Two slices (2.0 cm wide) of each muscle were frozen at -80 °C until thermal analysis and the rest of the muscles were kept at 2-4 °C until protein extraction.

### *<IP_ADDRESS>. Myofibrillar and sarcoplasmic proteins extraction and myofibrils isolation*

Myofibrillar proteins extraction was conducted as mentioned in **<IP_ADDRESS>.** After centrifugation, the supernatant containing sarcoplasmic proteins was separated and stored at -80 °C until electrophoretic analysis. The precipitate was suspended in SDS buffer and filtered through a home-made nylon strainer to remove connective tissue. An aliquot of the filtrate, which included proteins of the myofibrillar system, was subjected to SDS-PAGE analysis. Protein concentration of both sarcoplasmic and myofibrillar extracts was measured according to Lowry et al. (1951).

Myofibrils isolation from minced meat of the different saline treatments was conducted according to Culler et al. (1978), and as detailed in Pighin et al. (2008). The filtered myofibrils collected were used for DSC analysis. Protein concentration of isolated myofibrils and of whole muscles was determined by a macro-Kjeldahl method (Foss Tecator equipment, Sweden), with a conversion factor of N = 6.25.

### *<IP_ADDRESS>. SDS-PAGE analysis and thermal analysis*

Electrophoretic profile of protein extracts was monitored by discontinuous SDS-PAGE (Laemmli, 1970), carried out under the same conditions previously described in **<IP_ADDRESS>.**

A Perkin-Elmer Pyris-1 differential scanning calorimeter (DSC) was employed to study the thermal properties of proteins of the whole muscle and of the isolated myofibrils. For

temperature and heat flow calibration, Indium was used as standard (melting point 156.6 °C, enthalpy 28.46 J/g). Five to fifteen milligrams of each sample, accurately weighed, were placed into an aluminum pan, which was hermetically sealed and equilibrated for 2 min at the initial scanning temperature. During the study, a heating rate of 10 °C /min was applied over a 20-90 °C temperature range. All samples were re-scanned (submission to a second thermal analysis) to check for the irreversibility of the denaturation process. Endotherm (mW vs. temperature), denaturation enthalpy (DH) and endothermic peak (Td) were obtained using the software Pyris-7 (Perkin-Elmer). The total enthalpy change (DHT) was estimated by the algebraic sum of the individual denaturation enthalpies.

Electrophoresis as a Useful Tool in Studying the Quality of Meat Products 123

approximation because of the absence of suitable HMW standards for SDS-PAGE. Nevertheless, the MW found for nebulin is consistent with previous published data

reporting a value of 600– 900 kDa for this protein (Locker & Wild, 1986; Wang, 1982).

**Figure 1.** Myofibrillar protein isolation and SDS-PAGE (3–12%) analysis of protein fractions Lane 1 = HMW standard. Lane 2 = supernatant. Lane 3 = precipitate. (Pighin & Gonzalez, 2008)

**Figure 2.** Western blot analysis of myofibrillar protein extract Lane 1 = HMW standard.

It is important to remark that the use of gradients is usually recommended when SDS-PAGE analysis is applied to high-MW proteins, in order to improve separation and resolution of bands. The addition of glycerol has been reported to facilitate gradient formation and also to improve resolution of the SDS-PAGE technique (Sobieszek, 1994). However, Chen et al.

Lane 2 = myofibrillar protein extract. (Pighin & Gonzalez, 2008)

### *<IP_ADDRESS>. Statistical analysis*

The experiments described were conducted in duplicate. The study of saline incorporation included 5 treatments (NI; 0.70% NaCl; 0.25% TPP; 0.70% NaCl + 0.25% TPP; 1.20% NaCl + 0.25% TPP). A set of five muscles were assigned at random to each treatment. SDS-PAGE analysis and DSC measurements were performed in all muscles included in the treatments. Analysis of variance (ANOVA) for DSC measurements was conducted using the statistical package SAS (version 8, SAS Institute Inc., 2004, Cary, NC). Duncan´s Multiple Range Test (with a significance of a = 0.05) was used to determine differences among treatments.

### **4. Results and discussion**

### **4.1. Isolation of titin, nebulin, MHC and actin**

### *4.1.1. Separation of myofibrillar proteins*

Figure 1 shows the electrophoretic pattern of supernatant and precipitate fractions obtained after the extraction of myofibrillar proteins. It can be seen (lane 2) that most of the soluble proteins (28–70 kDa) remained in the supernatant fraction. Instead, proteins of the precipitate (lane 3) have a wider MW range (25–2,500 kDa). The precipitate included almost exclusively myofibrillar proteins, yielding 64 g of extracted protein per kilogram of muscular tissue. It can also be observed that the most intense band is MHC (MW: 220 kDa, Rf: 0.47), which represents approximately 35 % of total myofibrillar proteins extracted. Actin (MW: 43 kDa) is the second most intense (20 %) having one of the largest Rf (0.92). Titin band represents approximately 4 % of the proteins extracted. It has the lowest Rf (0.06) in the SDS-PAGE analysis with an estimated MW of 2,500 kDa. A similar MW for titin was found by Kellermayer & Grama (2002) and Kurzban & Wang (1988). A comparable electrophoretic pattern of myofibrillar proteins has been reported previously by Chen et al. (2005).

Nebulin could not be precisely identified on the present SDS-PAGE due to the presence of a set of several proteins in the range of 600–800 kDa. Therefore, the use of monoclonal antibodies appeared to be a useful tool to corroborate nebulin localization in the gel. Figure 2 shows the Western blot analysis of protein extract (precipitate fraction). Only one band was detected by the commercial kit against nebulin, with an Rf of 0.20 and an estimated MW of 710 kDa. It is important to denote that the MW of titin and nebulin were calculated in approximation because of the absence of suitable HMW standards for SDS-PAGE. Nevertheless, the MW found for nebulin is consistent with previous published data reporting a value of 600– 900 kDa for this protein (Locker & Wild, 1986; Wang, 1982).

122 Electrophoresis

*<IP_ADDRESS>. Statistical analysis*

**4. Results and discussion**

by Chen et al. (2005).

**4.1. Isolation of titin, nebulin, MHC and actin**

*4.1.1. Separation of myofibrillar proteins*

temperature and heat flow calibration, Indium was used as standard (melting point 156.6 °C, enthalpy 28.46 J/g). Five to fifteen milligrams of each sample, accurately weighed, were placed into an aluminum pan, which was hermetically sealed and equilibrated for 2 min at the initial scanning temperature. During the study, a heating rate of 10 °C /min was applied over a 20-90 °C temperature range. All samples were re-scanned (submission to a second thermal analysis) to check for the irreversibility of the denaturation process. Endotherm (mW vs. temperature), denaturation enthalpy (DH) and endothermic peak (Td) were obtained using the software Pyris-7 (Perkin-Elmer). The total enthalpy change (DHT) was

The experiments described were conducted in duplicate. The study of saline incorporation included 5 treatments (NI; 0.70% NaCl; 0.25% TPP; 0.70% NaCl + 0.25% TPP; 1.20% NaCl + 0.25% TPP). A set of five muscles were assigned at random to each treatment. SDS-PAGE analysis and DSC measurements were performed in all muscles included in the treatments. Analysis of variance (ANOVA) for DSC measurements was conducted using the statistical package SAS (version 8, SAS Institute Inc., 2004, Cary, NC). Duncan´s Multiple Range Test

Figure 1 shows the electrophoretic pattern of supernatant and precipitate fractions obtained after the extraction of myofibrillar proteins. It can be seen (lane 2) that most of the soluble proteins (28–70 kDa) remained in the supernatant fraction. Instead, proteins of the precipitate (lane 3) have a wider MW range (25–2,500 kDa). The precipitate included almost exclusively myofibrillar proteins, yielding 64 g of extracted protein per kilogram of muscular tissue. It can also be observed that the most intense band is MHC (MW: 220 kDa, Rf: 0.47), which represents approximately 35 % of total myofibrillar proteins extracted. Actin (MW: 43 kDa) is the second most intense (20 %) having one of the largest Rf (0.92). Titin band represents approximately 4 % of the proteins extracted. It has the lowest Rf (0.06) in the SDS-PAGE analysis with an estimated MW of 2,500 kDa. A similar MW for titin was found by Kellermayer & Grama (2002) and Kurzban & Wang (1988). A comparable electrophoretic pattern of myofibrillar proteins has been reported previously

Nebulin could not be precisely identified on the present SDS-PAGE due to the presence of a set of several proteins in the range of 600–800 kDa. Therefore, the use of monoclonal antibodies appeared to be a useful tool to corroborate nebulin localization in the gel. Figure 2 shows the Western blot analysis of protein extract (precipitate fraction). Only one band was detected by the commercial kit against nebulin, with an Rf of 0.20 and an estimated MW of 710 kDa. It is important to denote that the MW of titin and nebulin were calculated in

(with a significance of a = 0.05) was used to determine differences among treatments.

estimated by the algebraic sum of the individual denaturation enthalpies.

**Figure 1.** Myofibrillar protein isolation and SDS-PAGE (3–12%) analysis of protein fractions Lane 1 = HMW standard. Lane 2 = supernatant. Lane 3 = precipitate. (Pighin & Gonzalez, 2008)

**Figure 2.** Western blot analysis of myofibrillar protein extract Lane 1 = HMW standard. Lane 2 = myofibrillar protein extract. (Pighin & Gonzalez, 2008)

It is important to remark that the use of gradients is usually recommended when SDS-PAGE analysis is applied to high-MW proteins, in order to improve separation and resolution of bands. The addition of glycerol has been reported to facilitate gradient formation and also to improve resolution of the SDS-PAGE technique (Sobieszek, 1994). However, Chen et al.

(2005) did not find a great improvement of the gel system with the use of this reagent. Present research supports the first statement, given the fact that the addition of glycerol both to the resolving and the stacking gel solutions has successfully improved band resolution (comparison data not shown).

Electrophoresis as a Useful Tool in Studying the Quality of Meat Products 125

higher in the saline ranges of 40–60 g/L and 60–100 g/L. Protein profiles of the ranges 0–20 g/L and 20–40 g/L were similar, except for the amount of the titin band (2,500 kDa), which is about four times higher in the second saline range. A saturation range of 40–60 g/L showed that the amount of titin and nebulin extracted is the highest. A higher saturation range (60–

100 g/L) did not include the giant proteins in the precipitated fraction.

**Figure 3.** SDS-PAGE analysis of the different ammonium sulfate precipitates

saline range of 40–60 g/L, having 3.6 and 4.0 times increment, respectively.

brackets mean times increment. (Pighin & Gonzalez, 2008)

electroelution.

Lane 1 = HMW standard. Lane 2 = range 0–20 g/L. Lane 3 = range 20–40 g/L. Lane 4 = range 40–60 g/L. Lane 5 = range 60–100 g/L. T, titin; N, nebulin; MHC, myosin heavy chain; A, actin. \*Numbers between

If this protein profile (Fig. 3) is densitometrically compared to the myofibrillar protein extract (Fig. 1), it becomes evident that titin and nebulin have been greatly enriched in the

Even though the analysis of the gel in Fig. 3 indicates that the saline ranges of 20–40 g/L and 40–60 g/L were appropriate for isolation and enrichment of titin, and 40–60 g/L was so for the enrichment of nebulin, the unified range of 20–60 g/L appeared to be an interesting option to increase the amount of these proteins in subsequent experiments. Instead, the saline range of 60–100 g/L was suggested to isolate actin and MHC. Once the saline precipitation ranges were selected, SDS-PAGE was chosen to continue the purification protocol. For this purpose, each ammonium sulfate precipitate was resuspended and electrophoretically developed in an SDS-PAGE gel (3–12%). After the identification of the proteins on the gel by means of its Rf´s, they were first excised, and then submitted to

Figure 4 shows the SDS-PAGE of each electroeluted protein (lanes 1–4) and the Western blot analysis of nebulin (lane 5). It can be seen that MHC (lane 2) was obtained in an almost pure state. Actin and titin (lanes 3 and 4) were obtained enriched but not in a pure state. The reason for this result is that actin and titin concentrations obtained after the salt precipitation

### *4.1.2. Enrichment of titin, nebulin, MHC and actin*

The isolation of myofibrillar proteins requires time-consuming procedures that usually separate them individually. Thus, myosin isolation generally involves the removal of sarcoplasmic proteins by means of washings with diluted phosphate buffer and subsequent protein extraction with specific solutions containing elevated amounts of KCl (Dudziak & Foegeding, 1988; Hermanson et al., 1986). Actin isolation requires a prior separation of Gactin by breaking intermolecular linkages of F-actin. One of the most commonly used agents is acetone, which can break the linkages without denaturing the protein (Syrovy, 1984). After that, a second isolation stage involves several steps including washing, filtration, centrifugation, polymerization/depolymerization cycles, dialysis and centrifugation (Pardee & Spudich, 1982; Spudich & Watt, 1971). Gel filtration chromatography can also be employed for further actin purification (Kuroda, 1982). More recently, Perez-Juan et al. (2007) have succeeded in the isolation of actomyosin and actin in an interesting attempt to amalgamate myofibrillar protein isolations in a single extraction process.

High-molecular weight proteins (i.e. titin and nebulin) extraction specifically involves the addition of protease inhibitors to avoid degradation during processing. The isolation methodology initiates with myofibrils extraction and posterior protein–SDS solubilization, followed by the precipitation of titin/nebulin–SDS complex by salt fractionation. For further purification, the use of gel filtration is recommended because of the great size of these proteins (Wang, 1982). A more exhaustive extraction of titin was proposed by Pospiech et al. (2002), which demanded several centrifugation steps followed by suspension/ homogenization of the sediments in specific solutions, dialysis, and hydroxyapatite and ionexchange chromatography. Previous description shows the requirement of time and effort to achieve the isolation of one or another myofibrillar protein. For that reason, the present research was focused on looking for a more simple methodological strategy.

Salting out fractionation is believed to be a useful step for protein isolation because the procedure is inexpensive and relatively easy to apply. Sodium chloride had been proposed for the precipitation of the giant proteins, titin and nebulin (Wang, 1982; Wang & Wright, 1988). However, in the present research, the use of ammonium sulfate as precipitating agent produced a proper isolation of these two proteins and also of myosin and actin. In order to select the appropriate saturation range, different ammonium sulfate ranges were assayed: 0– 20, 20–40, 40–60 and 60–100 g/L. After each centrifugation step, the protein profile of the precipitated fractions was monitored by SDS-PAGE. The different saturation ranges showed the following protein pattern (Fig. 3): the band corresponding to MHC (MW: 220 kDa) was the most noticeable one, showing similar concentrations in all saline ranges (see numbers between brackets). Actin (MW: 43 kDa) was also detected in all fractions, being its intensity higher in the saline ranges of 40–60 g/L and 60–100 g/L. Protein profiles of the ranges 0–20 g/L and 20–40 g/L were similar, except for the amount of the titin band (2,500 kDa), which is about four times higher in the second saline range. A saturation range of 40–60 g/L showed that the amount of titin and nebulin extracted is the highest. A higher saturation range (60– 100 g/L) did not include the giant proteins in the precipitated fraction.

124 Electrophoresis

(comparison data not shown).

*4.1.2. Enrichment of titin, nebulin, MHC and actin*

(2005) did not find a great improvement of the gel system with the use of this reagent. Present research supports the first statement, given the fact that the addition of glycerol both to the resolving and the stacking gel solutions has successfully improved band resolution

The isolation of myofibrillar proteins requires time-consuming procedures that usually separate them individually. Thus, myosin isolation generally involves the removal of sarcoplasmic proteins by means of washings with diluted phosphate buffer and subsequent protein extraction with specific solutions containing elevated amounts of KCl (Dudziak & Foegeding, 1988; Hermanson et al., 1986). Actin isolation requires a prior separation of Gactin by breaking intermolecular linkages of F-actin. One of the most commonly used agents is acetone, which can break the linkages without denaturing the protein (Syrovy, 1984). After that, a second isolation stage involves several steps including washing, filtration, centrifugation, polymerization/depolymerization cycles, dialysis and centrifugation (Pardee & Spudich, 1982; Spudich & Watt, 1971). Gel filtration chromatography can also be employed for further actin purification (Kuroda, 1982). More recently, Perez-Juan et al. (2007) have succeeded in the isolation of actomyosin and actin in an interesting attempt to

High-molecular weight proteins (i.e. titin and nebulin) extraction specifically involves the addition of protease inhibitors to avoid degradation during processing. The isolation methodology initiates with myofibrils extraction and posterior protein–SDS solubilization, followed by the precipitation of titin/nebulin–SDS complex by salt fractionation. For further purification, the use of gel filtration is recommended because of the great size of these proteins (Wang, 1982). A more exhaustive extraction of titin was proposed by Pospiech et al. (2002), which demanded several centrifugation steps followed by suspension/ homogenization of the sediments in specific solutions, dialysis, and hydroxyapatite and ionexchange chromatography. Previous description shows the requirement of time and effort to achieve the isolation of one or another myofibrillar protein. For that reason, the present

Salting out fractionation is believed to be a useful step for protein isolation because the procedure is inexpensive and relatively easy to apply. Sodium chloride had been proposed for the precipitation of the giant proteins, titin and nebulin (Wang, 1982; Wang & Wright, 1988). However, in the present research, the use of ammonium sulfate as precipitating agent produced a proper isolation of these two proteins and also of myosin and actin. In order to select the appropriate saturation range, different ammonium sulfate ranges were assayed: 0– 20, 20–40, 40–60 and 60–100 g/L. After each centrifugation step, the protein profile of the precipitated fractions was monitored by SDS-PAGE. The different saturation ranges showed the following protein pattern (Fig. 3): the band corresponding to MHC (MW: 220 kDa) was the most noticeable one, showing similar concentrations in all saline ranges (see numbers between brackets). Actin (MW: 43 kDa) was also detected in all fractions, being its intensity

amalgamate myofibrillar protein isolations in a single extraction process.

research was focused on looking for a more simple methodological strategy.

**Figure 3.** SDS-PAGE analysis of the different ammonium sulfate precipitates Lane 1 = HMW standard. Lane 2 = range 0–20 g/L. Lane 3 = range 20–40 g/L. Lane 4 = range 40–60 g/L. Lane 5 = range 60–100 g/L. T, titin; N, nebulin; MHC, myosin heavy chain; A, actin. \*Numbers between brackets mean times increment. (Pighin & Gonzalez, 2008)

If this protein profile (Fig. 3) is densitometrically compared to the myofibrillar protein extract (Fig. 1), it becomes evident that titin and nebulin have been greatly enriched in the saline range of 40–60 g/L, having 3.6 and 4.0 times increment, respectively.

Even though the analysis of the gel in Fig. 3 indicates that the saline ranges of 20–40 g/L and 40–60 g/L were appropriate for isolation and enrichment of titin, and 40–60 g/L was so for the enrichment of nebulin, the unified range of 20–60 g/L appeared to be an interesting option to increase the amount of these proteins in subsequent experiments. Instead, the saline range of 60–100 g/L was suggested to isolate actin and MHC. Once the saline precipitation ranges were selected, SDS-PAGE was chosen to continue the purification protocol. For this purpose, each ammonium sulfate precipitate was resuspended and electrophoretically developed in an SDS-PAGE gel (3–12%). After the identification of the proteins on the gel by means of its Rf´s, they were first excised, and then submitted to electroelution.

Figure 4 shows the SDS-PAGE of each electroeluted protein (lanes 1–4) and the Western blot analysis of nebulin (lane 5). It can be seen that MHC (lane 2) was obtained in an almost pure state. Actin and titin (lanes 3 and 4) were obtained enriched but not in a pure state. The reason for this result is that actin and titin concentrations obtained after the salt precipitation and the electrophoresis steps were low and made difficult the removal of a thinner band from the electrophoretic gel to improve their isolation by electroelution. Nebulin band stained with Coomassie Brilliant Blue dye- was not accompanied by any other protein band (data not shown). However, the band was vaguely stained and it required the identification and confirmation by the previously mentioned immunoblotting technique (lane 5).

Electrophoresis as a Useful Tool in Studying the Quality of Meat Products 127

consequence of the application of NaCl into muscles. Hence, an attenuated intensity of any of the bands in Fig. 6 was expected. It is probable that the increment of 70 kDa or the appearance

**Figure 5.** SDS-PAGE analysis of sarcoplasmic proteins obtained from muscles treated with NaCl and/or TPP

**Figure 6.** SDS-PAGE analysis of myofibrillar proteins extracted from muscles treated with NaCl and/or TPP

Gel B: Lane 1, HMW-SDS; Lane 2, NI; Lane 3, 0.70 % NaCl + 0.25 % TPP; Lane 4, 1.20 % NaCl + 0.25 % TPP.

Gel B: Lane 1, HMW-SDS; Lane 2, NI; Lane 3, 0.70 % NaCl + 0.25 % TPP; Lane 4, 1.20 % NaCl + 0.25 % TPP.

Gel A: Lane 1, HMW-SDS; Lane 2, NI; Lane 3, 0.70 % NaCl; Lane 4, 0.25 % TPP.

Gel A: Lane 1, HMW-SDS; Lane 2, NI; Lane 3, 0.70 % NaCl; Lane 4, 0.25 % TPP.

(Pighin et al., 2008)

(Pighin et al., 2008)

of 65 kDa band found in Fig. 5 is a result of the degradation of myofibrillar proteins.

**Figure 4.** SDS-PAGE and Western blot analysis of the electroeluated proteins Lane 1 = HMW standard. Lane 2 = myosin heavy chain extract (SDS-PAGE analysis). Lane 3 = actin (A) extract (SDS-PAGE analysis). Lane 4 = titin (T) extract (SDS-PAGE analysis). Lane 5 = nebulin (N) extract (Western blot analysis). (Pighin & Gonzalez, 2008)

### **4.2. Study of protein contribution to the effect of saline addition to meat**

### *4.2.1. Electrophoretic analysis of proteins extracts*

The electrophoretic analysis of sarcoplasmic proteins extracts belonging to saline treated muscles are shown in Fig. 5. As can be seen, proteins bands ranged between 30 and 75 kDa. It is worth noting that the intensity of the band with an estimated MW of 70 kDa was increased about 2.5 times in samples of muscles containing NaCl alone or combined with TPP (see arrows, lanes 3-A, 3-B and 4-B) with respect to NI. Even more, an additional band with an estimated MW of 65 kDa was revealed in these saline treated muscles (see dashed arrows, lanes 3-A, 3-B and 4-B). On the contrary, the presence of TPP alone did not modify the protein pattern found in NI muscles (lanes 2-A and 2-B). These findings suggest that the presence of NaCl collaborates in the solubilization of myofibrillar proteins, releasing them to the soluble fraction.

Myofibrillar proteins were separated by SDS-PAGE into a broad range of molecular weight (Fig. 6). Bands corresponding to MHC (MW 220 kDa, see arrow) and actin (MW 46 kDa, see dashed arrow) were the most noticeable ones. No differences among treatments were verified by means of densitometric analysis. This result does not agree with the one previously shown in Fig. 5, which had suggested an augmented solubilization of myofibrillar proteins as a consequence of the application of NaCl into muscles. Hence, an attenuated intensity of any of the bands in Fig. 6 was expected. It is probable that the increment of 70 kDa or the appearance of 65 kDa band found in Fig. 5 is a result of the degradation of myofibrillar proteins.

126 Electrophoresis

and the electrophoresis steps were low and made difficult the removal of a thinner band from the electrophoretic gel to improve their isolation by electroelution. Nebulin band stained with Coomassie Brilliant Blue dye- was not accompanied by any other protein band (data not shown). However, the band was vaguely stained and it required the identification

and confirmation by the previously mentioned immunoblotting technique (lane 5).

**Figure 4.** SDS-PAGE and Western blot analysis of the electroeluated proteins

*4.2.1. Electrophoretic analysis of proteins extracts*

extract (Western blot analysis). (Pighin & Gonzalez, 2008)

Lane 1 = HMW standard. Lane 2 = myosin heavy chain extract (SDS-PAGE analysis). Lane 3 = actin (A) extract (SDS-PAGE analysis). Lane 4 = titin (T) extract (SDS-PAGE analysis). Lane 5 = nebulin (N)

The electrophoretic analysis of sarcoplasmic proteins extracts belonging to saline treated muscles are shown in Fig. 5. As can be seen, proteins bands ranged between 30 and 75 kDa. It is worth noting that the intensity of the band with an estimated MW of 70 kDa was increased about 2.5 times in samples of muscles containing NaCl alone or combined with TPP (see arrows, lanes 3-A, 3-B and 4-B) with respect to NI. Even more, an additional band with an estimated MW of 65 kDa was revealed in these saline treated muscles (see dashed arrows, lanes 3-A, 3-B and 4-B). On the contrary, the presence of TPP alone did not modify the protein pattern found in NI muscles (lanes 2-A and 2-B). These findings suggest that the presence of NaCl collaborates in the solubilization of myofibrillar proteins, releasing them to the soluble fraction.

Myofibrillar proteins were separated by SDS-PAGE into a broad range of molecular weight (Fig. 6). Bands corresponding to MHC (MW 220 kDa, see arrow) and actin (MW 46 kDa, see dashed arrow) were the most noticeable ones. No differences among treatments were verified by means of densitometric analysis. This result does not agree with the one previously shown in Fig. 5, which had suggested an augmented solubilization of myofibrillar proteins as a

**4.2. Study of protein contribution to the effect of saline addition to meat**

**Figure 5.** SDS-PAGE analysis of sarcoplasmic proteins obtained from muscles treated with NaCl and/or TPP Gel A: Lane 1, HMW-SDS; Lane 2, NI; Lane 3, 0.70 % NaCl; Lane 4, 0.25 % TPP. Gel B: Lane 1, HMW-SDS; Lane 2, NI; Lane 3, 0.70 % NaCl + 0.25 % TPP; Lane 4, 1.20 % NaCl + 0.25 % TPP. (Pighin et al., 2008)

**Figure 6.** SDS-PAGE analysis of myofibrillar proteins extracted from muscles treated with NaCl and/or TPP Gel A: Lane 1, HMW-SDS; Lane 2, NI; Lane 3, 0.70 % NaCl; Lane 4, 0.25 % TPP. Gel B: Lane 1, HMW-SDS; Lane 2, NI; Lane 3, 0.70 % NaCl + 0.25 % TPP; Lane 4, 1.20 % NaCl + 0.25 % TPP. (Pighin et al., 2008)

### *4.2.2. Thermal analysis of whole muscle*

Thermal analysis of NI whole muscles (Fig. 7) showed three characteristic endothermic peaks. Published data allowed to relate them to myosin and its subunits denaturation (I; 57.0 °C), sarcoplasmic proteins and collagen denaturation (II; 67.4 °C) and actin denaturation (III; 80.3 °C) (Graiver et al., 2006; Kijowski & Mast, 1988; Stabursvik & Martens, 1980). Enthalpies involved in each thermal transition were 1.32, 0.65 y 1.74 J/g protein, respectively. Re-scanned samples did not show any thermal response, corroborating the irreversibility of the transitions. The calculated DHT of NI muscles was 3.71 J/g protein. It can be seen in Table 1 that NaCl treatment (0.70 %) significantly reduced myosin DH and actin Td. Only slight decreases were found in myosin Td and actin DH. These results make evident that actin was destabilized by NaCl, becoming the more susceptible protein to thermal denaturation (decreased Td). Instead, the effect of NaCl upon myosin could be seen on the DH, which reduction could be related to the decrease of hydrogen bonds and/or to the increment of protein aggregation (exothermic event) exerted by the salt. Total enthalpy (DHT) for NaCl-treated muscles (calculated by adding DH of individual values related to transitions I, II, and III) decreased from 3.71 J/g protein (NI muscle) to 3.04 J/g protein, demonstrating system instability.

Electrophoresis as a Useful Tool in Studying the Quality of Meat Products 129

The described findings reveal an important effect of the salt in decreasing the thermal stability of proteins. In order to support this fact, it was proposed that addition of neutral salt causes anions to compete with water molecules for specific sites of proteins, altering its hydration properties, and consequently requiring lower denaturation energies (Von Hippel & Wong, 1965). Treatment with TTP (0.25 %) significantly increased sarcoplasmic proteins/collagen Td. Also, Td of myosin and DH of actin were slightly increased. Whole muscle treated with 0.25 % TPP increased the DHT from 3.71 (NI muscles) to 4.28 J/g protein. These findings show an important stabilizing effect exerted by TPP, probably by altering hydrophobic interactions rather than affecting pH or ionic strength (Trout & Schmidt, 1986). With regard to the effect of phosphates on meat, Kijowski & Mast (1988) studied the effect of different phosphates (sodium pyrophosphate or sodium tripolyphosphate) on chicken breast muscles and its isolated myofibrils. They stated that low levels of sodium pyrophosphate or sodium tripolyphosphate (0.25 % or 0.50 %) caused the maximum stabilization (DHT) of chicken muscle and isolated myofibrils by means of myosin stabilization (increased DH). These results agree with present ones, even they were

When both additives were used together (0.70 % NaCl + 0.25 % TPP), Td corresponding to sarcoplasmic proteins/collagen was significantly increased in comparison to NI muscles, suggesting stabilization of the related proteins. This stabilization went along with a significant decreased of the DH, probably associated to an increment of the exothermic protein aggregation. On the contrary, actin Td showed a significant reduction in comparison to NI. Destabilization of actin in the presence of NaCl and phosphates was also described by Kijowski & Mast (1988) in chicken breast muscles treated with 2 % NaCl and 0.25/0.50 % pyrophosphate. When the effect of this combination of salts is compared to the one previously described, 0.70 % NaCl effect, it can be speculated that two effects appear to coexist: the same stabilizing effect of TPP on sarcoplasmic proteins/collagen and the destabilizing effect of NaCl on actin. Even though, the last effect is smaller in magnitude than the one produced by NaCl alone. It is important to denote that DHT of the muscle treated with the combination of salts (0.70 % NaCl + 0.25 % TPP) was slightly higher than the NI one (3.83 vs. 3.71 J/g protein, respectively). Comparing the DHT obtained individually for NaCl and TPP treatments, it can be seen that the effect of the addition of both salts is contrary to the effect of NaCl alone and in the same direction as TPP. Hence, the final increase could be assigned to the increase (even non significant) of myosin DH induced by TPP. Even more, when NaCl was raised to 1.20%, a maximal drop in actin stability was achieved (higher reduction of Td and DH). This event was accompanied by a maximal drop in DHT (from 3.71 to 2.75 J/g protein). It becomes evident that the increased amount of NaCl in the brine produced a destabilizing effect similar to the one found for NaCl alone, which

Regarding this issue, Findlay & Barbut (1992) had stated that the addition of NaCl (0.5-2.0 %) reduced myosin Td and conversely TPP (0.2-0.6 %) increased its stability. Additionally, DHT varied depending on salts concentration. At low NaCl levels (less than 1.00 %) the effect of TTP on the increment of DHT was predominant. Instead, as NaCl

also turned to be predominant to expenses of the effect of TPP.

obtained in beef muscles.

**Figure 7.** Thermal analysis of NI whole muscles (Pighin et al., 2008)

**Table 1.** Endothermic peaks (Td) and denaturation enthalpies (DH) of whole muscles treated with NaCl and/or TPP

a-c Means within a column having different letters are significantly different (p < 0.05). (Pighin et al., 2008)

The described findings reveal an important effect of the salt in decreasing the thermal stability of proteins. In order to support this fact, it was proposed that addition of neutral salt causes anions to compete with water molecules for specific sites of proteins, altering its hydration properties, and consequently requiring lower denaturation energies (Von Hippel & Wong, 1965). Treatment with TTP (0.25 %) significantly increased sarcoplasmic proteins/collagen Td. Also, Td of myosin and DH of actin were slightly increased. Whole muscle treated with 0.25 % TPP increased the DHT from 3.71 (NI muscles) to 4.28 J/g protein. These findings show an important stabilizing effect exerted by TPP, probably by altering hydrophobic interactions rather than affecting pH or ionic strength (Trout & Schmidt, 1986). With regard to the effect of phosphates on meat, Kijowski & Mast (1988) studied the effect of different phosphates (sodium pyrophosphate or sodium tripolyphosphate) on chicken breast muscles and its isolated myofibrils. They stated that low levels of sodium pyrophosphate or sodium tripolyphosphate (0.25 % or 0.50 %) caused the maximum stabilization (DHT) of chicken muscle and isolated myofibrils by means of myosin stabilization (increased DH). These results agree with present ones, even they were obtained in beef muscles.

128 Electrophoresis

*4.2.2. Thermal analysis of whole muscle*

**Figure 7.** Thermal analysis of NI whole muscles

(Pighin et al., 2008)

NaCl and/or TPP

(Pighin et al., 2008)

Thermal analysis of NI whole muscles (Fig. 7) showed three characteristic endothermic peaks. Published data allowed to relate them to myosin and its subunits denaturation (I; 57.0 °C), sarcoplasmic proteins and collagen denaturation (II; 67.4 °C) and actin denaturation (III; 80.3 °C) (Graiver et al., 2006; Kijowski & Mast, 1988; Stabursvik & Martens, 1980). Enthalpies involved in each thermal transition were 1.32, 0.65 y 1.74 J/g protein, respectively. Re-scanned samples did not show any thermal response, corroborating the irreversibility of the transitions. The calculated DHT of NI muscles was 3.71 J/g protein. It can be seen in Table 1 that NaCl treatment (0.70 %) significantly reduced myosin DH and actin Td. Only slight decreases were found in myosin Td and actin DH. These results make evident that actin was destabilized by NaCl, becoming the more susceptible protein to thermal denaturation (decreased Td). Instead, the effect of NaCl upon myosin could be seen on the DH, which reduction could be related to the decrease of hydrogen bonds and/or to the increment of protein aggregation (exothermic event) exerted by the salt. Total enthalpy (DHT) for NaCl-treated muscles (calculated by adding DH of individual values related to transitions I, II, and III) decreased from 3.71 J/g

protein (NI muscle) to 3.04 J/g protein, demonstrating system instability.

**Table 1.** Endothermic peaks (Td) and denaturation enthalpies (DH) of whole muscles treated with

a-c Means within a column having different letters are significantly different (p < 0.05).

When both additives were used together (0.70 % NaCl + 0.25 % TPP), Td corresponding to sarcoplasmic proteins/collagen was significantly increased in comparison to NI muscles, suggesting stabilization of the related proteins. This stabilization went along with a significant decreased of the DH, probably associated to an increment of the exothermic protein aggregation. On the contrary, actin Td showed a significant reduction in comparison to NI. Destabilization of actin in the presence of NaCl and phosphates was also described by Kijowski & Mast (1988) in chicken breast muscles treated with 2 % NaCl and 0.25/0.50 % pyrophosphate. When the effect of this combination of salts is compared to the one previously described, 0.70 % NaCl effect, it can be speculated that two effects appear to coexist: the same stabilizing effect of TPP on sarcoplasmic proteins/collagen and the destabilizing effect of NaCl on actin. Even though, the last effect is smaller in magnitude than the one produced by NaCl alone. It is important to denote that DHT of the muscle treated with the combination of salts (0.70 % NaCl + 0.25 % TPP) was slightly higher than the NI one (3.83 vs. 3.71 J/g protein, respectively). Comparing the DHT obtained individually for NaCl and TPP treatments, it can be seen that the effect of the addition of both salts is contrary to the effect of NaCl alone and in the same direction as TPP. Hence, the final increase could be assigned to the increase (even non significant) of myosin DH induced by TPP. Even more, when NaCl was raised to 1.20%, a maximal drop in actin stability was achieved (higher reduction of Td and DH). This event was accompanied by a maximal drop in DHT (from 3.71 to 2.75 J/g protein). It becomes evident that the increased amount of NaCl in the brine produced a destabilizing effect similar to the one found for NaCl alone, which also turned to be predominant to expenses of the effect of TPP.

Regarding this issue, Findlay & Barbut (1992) had stated that the addition of NaCl (0.5-2.0 %) reduced myosin Td and conversely TPP (0.2-0.6 %) increased its stability. Additionally, DHT varied depending on salts concentration. At low NaCl levels (less than 1.00 %) the effect of TTP on the increment of DHT was predominant. Instead, as NaCl increased the phosphate effect was minimized. In accordance with such observations, present results indicate that NaCl exerts a destabilizing effect in the presence of TPP (0.25 %) that becomes stronger as NaCl level raises.

Electrophoresis as a Useful Tool in Studying the Quality of Meat Products 131

decreased, being the myosin DH reduced in almost 2.60 J/g protein with respect to NI muscles (NI: 3.87 vs. 1.27 J/g protein; p < 0.05). These findings confirm the destabilizing effect of NaCl previously described for actin in the whole muscle. Even more, this effect seems to be stronger in the myofibril fraction since myosin transition was also affected. Further increments of NaCl (1.20 %) -in the presence of TPP- produced similar effects on both proteins, with the exception of an intensified effect on actin DH. It is important to remark that the effect of both additives together was definitively higher than the effect of any of the additives individually. Offer & Knight (1998) proposed that polyphosphates -at the concentrations used in the industry- may assist NaCl in causing depolymerisation of thick filaments and dissociation of actomyosin, facilitating in consequence the myosin extraction. The action of NaCl as a mild structure-breaker aided by the presence of TPP

**Table 2.** Endothermic peaks (Td) and denaturation enthalpies (DH) of myofibrils isolated from muscles

As it was mentioned, the physicochemical mechanisms involved in water binding and retention in the protein matrix of cooked meat were not completely elucidated. These results showed that the combined presence of NaCl (0.70 % or 1.20 %) and TPP (0.25 %) changes the heat susceptibility of muscle proteins (especially actin) so that they denature and coagulate at a lower cooking temperature. These modifications suggest the occurrence of protein conformational changes due to the salts, probably by altering hydrophobic and electrostatic interactions that stabilize the protein structure (Franks & Eagland, 1975). Evidently, these changes collaborate in the binding and posterior retention of water inside the tissue by any of the mechanisms proposed for the action of NaCl and polyphosphates (Offer & Knight, 1998). Increased solubilization of proteins found in whole muscles treated with NaCl (alone or in combination with TPP) could be related to the removal of transverse myofibrillar proteins which may act as structural constrains to myosin extraction. Also, some other low molecular weight myofibrillar proteins could be removed. The softening of the structure together with conformational modifications of myofibrillar proteins would allow entrapping water inside the tissue. This event would be the

could be the reason of the significant reduction of protein stability found.

a-d Means within a column having different letters are significantly different (p < 0.05).

reason of the increased WHC of *Semitendinosus* muscle found by Vaudagna et al. (2008).

The isolation protocol previously described in this chapter has been demonstrated to be a successful methodology for individually isolating four of the main myofibrillar proteins.

treated with NaCl and/or TPP

(Pighin et al., 2008)

**5. Conclusion**

### *4.2.3. Thermal analysis of isolated myofibrils*

Thermal analysis of isolated myofibrils from NI muscle showed only two endothermic peaks (Fig. 8), corresponding to denaturation enthalpies of myosin and its subunits (I: 58.1 °C) and actin (II: 73.5 °C). Data obtained from Table 2 showed a significant decrease of myosin and actin DH in myofibrils isolated from 0.70 % NaCl-treated muscles and washed in the same brine solution. No changes in Td of these proteins were found. The destabilizing effect of NaCl was in agreement with data obtained from the whole muscle. TPP (0.25 %) treatment produced a significant increase of both, myosin and actin Td. In contrast, DH of myosin was strongly reduced (p < 0.05) by the treatment. These findings indicate that the salt is capable of stabilizing actin and myosin structures. However, the smaller energy required to denature myosin (lower DH) suggests a small amount of protein available in the myofibril fraction. Supporting this comment, it had been proposed that phosphates can induce myosin solubilization by promoting both, thick filament depolymerisation and actomyosin dissociation (Granicher & Portzehl, 1964; Xiong, 2005). Even though, an increased protein aggregation (exothermic event) taking place immediately after myosin denaturation, might not be discarded. In the case of whole muscle treated with TPP, myosin and actin Td did not suffer any change and their DH were only slightly increased. Therefore, it can be affirmed that the effect of TPP on the whole muscle and myofibrils is almost alike.

**Figure 8.** Thermal analysis of myofibrils isolated from NI muscles (Pighin et al., 2008)

When NaCl and TPP were used together (0.70 % NaCl + 0.25 % TPP) both, myosin and actin Td of isolated myofibrils significantly decreased (p < 0.05). Enthalpy of the proteins was also decreased, being the myosin DH reduced in almost 2.60 J/g protein with respect to NI muscles (NI: 3.87 vs. 1.27 J/g protein; p < 0.05). These findings confirm the destabilizing effect of NaCl previously described for actin in the whole muscle. Even more, this effect seems to be stronger in the myofibril fraction since myosin transition was also affected. Further increments of NaCl (1.20 %) -in the presence of TPP- produced similar effects on both proteins, with the exception of an intensified effect on actin DH. It is important to remark that the effect of both additives together was definitively higher than the effect of any of the additives individually. Offer & Knight (1998) proposed that polyphosphates -at the concentrations used in the industry- may assist NaCl in causing depolymerisation of thick filaments and dissociation of actomyosin, facilitating in consequence the myosin extraction. The action of NaCl as a mild structure-breaker aided by the presence of TPP could be the reason of the significant reduction of protein stability found.

**Table 2.** Endothermic peaks (Td) and denaturation enthalpies (DH) of myofibrils isolated from muscles treated with NaCl and/or TPP

a-d Means within a column having different letters are significantly different (p < 0.05). (Pighin et al., 2008)

As it was mentioned, the physicochemical mechanisms involved in water binding and retention in the protein matrix of cooked meat were not completely elucidated. These results showed that the combined presence of NaCl (0.70 % or 1.20 %) and TPP (0.25 %) changes the heat susceptibility of muscle proteins (especially actin) so that they denature and coagulate at a lower cooking temperature. These modifications suggest the occurrence of protein conformational changes due to the salts, probably by altering hydrophobic and electrostatic interactions that stabilize the protein structure (Franks & Eagland, 1975). Evidently, these changes collaborate in the binding and posterior retention of water inside the tissue by any of the mechanisms proposed for the action of NaCl and polyphosphates (Offer & Knight, 1998). Increased solubilization of proteins found in whole muscles treated with NaCl (alone or in combination with TPP) could be related to the removal of transverse myofibrillar proteins which may act as structural constrains to myosin extraction. Also, some other low molecular weight myofibrillar proteins could be removed. The softening of the structure together with conformational modifications of myofibrillar proteins would allow entrapping water inside the tissue. This event would be the reason of the increased WHC of *Semitendinosus* muscle found by Vaudagna et al. (2008).

### **5. Conclusion**

130 Electrophoresis

that becomes stronger as NaCl level raises.

*4.2.3. Thermal analysis of isolated myofibrils*

myofibrils is almost alike.

(Pighin et al., 2008)

**Figure 8.** Thermal analysis of myofibrils isolated from NI muscles

When NaCl and TPP were used together (0.70 % NaCl + 0.25 % TPP) both, myosin and actin Td of isolated myofibrils significantly decreased (p < 0.05). Enthalpy of the proteins was also

increased the phosphate effect was minimized. In accordance with such observations, present results indicate that NaCl exerts a destabilizing effect in the presence of TPP (0.25 %)

Thermal analysis of isolated myofibrils from NI muscle showed only two endothermic peaks (Fig. 8), corresponding to denaturation enthalpies of myosin and its subunits (I: 58.1 °C) and actin (II: 73.5 °C). Data obtained from Table 2 showed a significant decrease of myosin and actin DH in myofibrils isolated from 0.70 % NaCl-treated muscles and washed in the same brine solution. No changes in Td of these proteins were found. The destabilizing effect of NaCl was in agreement with data obtained from the whole muscle. TPP (0.25 %) treatment produced a significant increase of both, myosin and actin Td. In contrast, DH of myosin was strongly reduced (p < 0.05) by the treatment. These findings indicate that the salt is capable of stabilizing actin and myosin structures. However, the smaller energy required to denature myosin (lower DH) suggests a small amount of protein available in the myofibril fraction. Supporting this comment, it had been proposed that phosphates can induce myosin solubilization by promoting both, thick filament depolymerisation and actomyosin dissociation (Granicher & Portzehl, 1964; Xiong, 2005). Even though, an increased protein aggregation (exothermic event) taking place immediately after myosin denaturation, might not be discarded. In the case of whole muscle treated with TPP, myosin and actin Td did not suffer any change and their DH were only slightly increased. Therefore, it can be affirmed that the effect of TPP on the whole muscle and

> The isolation protocol previously described in this chapter has been demonstrated to be a successful methodology for individually isolating four of the main myofibrillar proteins.

Despite the proteins were not isolated in a complete pure state, this approach seems to be useful for using them for further analysis. The estimation of proteins' Rf by means of an immunological method looks like an interesting tool to complement the identification of proteins in unclear electrophoretic patterns.

Electrophoresis as a Useful Tool in Studying the Quality of Meat Products 133

Culler, R.D.; Parrish, F.C. Jr.; Smith, G.C. & Cross, H.R. (1978). Relationship of myofibril fragmentation index to certain chemical, physical and sensory characteristics of bovine longissimus muscle. *Journal of Food Science*, Vol.43, pp. 1177-1182. ISSN: 1750-3841. Church, I.J. & Parsons, A.L. (2000). The sensory quality of chicken and potato products prepared using cook-chill and sous vide methods. *International Journal of Food Science*

Chen, S.P.; Sheu, J.R.; Lai, C.Y.; Lin, T.Y.; Hsiao, G. & Fong, T.H. (2005). Detection of myofibrillar proteins using a step gradient minigel with an ambiguous interface.

Clark, K.A.; Mcelhinny, A.S.; Beckerle, M.C. & Gregorio, C.C. (2002). Striated muscle cytoarchitecture: An intricate web of form and function. *Annual Review of Cell and*

Dudziak, J.A. & Foegeding, E.A. (1988). Isolation of actomyosin and myosin from post-rigor turkey breast ant thigh. *Journal of Food Science,* Vol.53(S), pp. 1287– 1290. ISSN: 1750-

Findlay, C.J. & Barbut, S. (1992). A response surface investigation of the effects of sodium chloride and tripolyphosphate on the thermal properties of beef muscle. *Meat Science*,

Franks, F. & Eagland, D. (1975). The role of solvent interactions in protein conformation. *CRC Critical Reviews in Biochemistry*, Vol.3 No.2, pp. 165–219. ISSN: 0045-6411. García-Segovia, P.; Andrés-Bello, A.; & Martínez-Monzó , J. (2007). Effect of cooking method on mechanical properties, color and structure of beef muscle (*M. pectoralis*). *Journal of*

Graiver, N.; Pinotti, A.; Califano, A. & Zaritzki, N. (2006). Diffusion of sodium chloride in

Granicher, D. & Portzehl, H. (1964). The influence of magnesium and calcium pyrophosphate chelates, of free magnesium ions, free calcium ions, and free pyrophosphate ions on the dissociation of actomyosin in solution. *Biochimica et*

Hermanson, A.M.; Harbitz, O. & Langton, M. (1986). Formation of two types of gels from bovine myosin. *Journal of the Science of Food and Agriculture,* Vol.37, pp. 69–84. ISSN:

Ho, C.Y., Stromer, M.H. & Robson, R.M. (1994). Identification of the 30-kDa polypeptide in post mortem skeletal muscle as a degradation product of troponin-T. *Biochimie* Vol.76,

Houmeida, A.; Holat, J.; Tskhovrebova, L. & Trinick, J. (1995). Studies of the interaction between titin and myosin*. The Journal of Cell Biology,* Vol.131, pp. 1471–1481. ISSN: 0021-

Karcher, R.E. & Landers, J.P. (2006). Electrophoresis, In *TIETZ Textbook of Clinical Chemistry and Molecular Diagnostics*. Carl A. Burtis, Ph.D. Edward R. Ashwood, M.D. David E. Bruns, M.D., Eds. Fourth Edition. pp. 102-111. Elsevier Inc. ISBN-13: 978-0-7216-0189-2.

pork tissue. *Journal of Food Engineering*, Vol.77, pp. 910–918. ISSN: 0260-8774.

*and Technology,* Vol*.*35, pp. 361–368. ISSN: 0950-5423.

Vol.31, pp. 155–164. ISSN: 0309-1740.

3841.

1097-0010.

9525.

pp. 369–375. ISSN: 0300-9084.

ISBN-I0: 0-7216-0189-8. Washington, USA.

*Analytical Biochemistry,* Vol.338, pp. 270–277. ISSN: 0003-2697.

*Developmental Biolology.* Vol.18, pp. 637–706. ISSN: 1081-0706.

*Food Engineering*, Vol.80 No.3, pp. 813–821. ISSN: 0260-8774.

*Biophysica Acta*, Vol.86, pp. 567–578. ISSN: 0304-4165.

The thermal behavior of the myofibrillar proteins is quite involved in the thermal behavior of whole muscle. NaCl incorporation into meat leads to an important protein destabilizing effect upon myofibrillar proteins, even at relative low doses when compared to industrial usage. The use of NaCl in combination with TPP produces a destabilizing global effect, suggesting that TPP may assist NaCl in acting as structure-breaker. The increase of WHC of *Semitendinosus* muscles injected with saline additives and submitted to *sous vide* cooking would be mainly associated to the conformational modifications of myofibrillar proteins and to the weakening of myofibrillar structures due to protein removal.

Taken together, the use of gel electrophoresis demonstrated to be a useful tool not only in isolating major muscle proteins but also in contributing to study the effect of saline additives in meat, in order to improve sensorial properties. Its combination with advanced technologies like DSC represents an interesting issue to look forward in the meat science approach.

### **Author details**

Dario G. Pighin *Institute of Food Technology, National Institute of Agricultural Technology – INTA, Argentina*

### **Acknowledgement**

Special thanks are given to the Instituto Nacional de Tecnología Agropecuaria (INTA), Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET) and Universidad de Morón (UM) for their financial support in the studies carried out and compiled in the present chapter.

### **6. References**

Culler, R.D.; Parrish, F.C. Jr.; Smith, G.C. & Cross, H.R. (1978). Relationship of myofibril fragmentation index to certain chemical, physical and sensory characteristics of bovine longissimus muscle. *Journal of Food Science*, Vol.43, pp. 1177-1182. ISSN: 1750-3841.

132 Electrophoresis

approach.

**Author details**

**Acknowledgement**

compiled in the present chapter.

*Science*, Vol.72, No.4, pp. 704–712, ISSN: 0309-1740.

http://www.fao.org/docrep/T0562E/T0562E00.htm.

Vol.76, pp 2415–2434. ISSN: 0021-8812.

Dario G. Pighin

**6. References**

proteins in unclear electrophoretic patterns.

to the weakening of myofibrillar structures due to protein removal.

Despite the proteins were not isolated in a complete pure state, this approach seems to be useful for using them for further analysis. The estimation of proteins' Rf by means of an immunological method looks like an interesting tool to complement the identification of

The thermal behavior of the myofibrillar proteins is quite involved in the thermal behavior of whole muscle. NaCl incorporation into meat leads to an important protein destabilizing effect upon myofibrillar proteins, even at relative low doses when compared to industrial usage. The use of NaCl in combination with TPP produces a destabilizing global effect, suggesting that TPP may assist NaCl in acting as structure-breaker. The increase of WHC of *Semitendinosus* muscles injected with saline additives and submitted to *sous vide* cooking would be mainly associated to the conformational modifications of myofibrillar proteins and

Taken together, the use of gel electrophoresis demonstrated to be a useful tool not only in isolating major muscle proteins but also in contributing to study the effect of saline additives in meat, in order to improve sensorial properties. Its combination with advanced technologies like DSC represents an interesting issue to look forward in the meat science

*Institute of Food Technology, National Institute of Agricultural Technology – INTA, Argentina*

Special thanks are given to the Instituto Nacional de Tecnología Agropecuaria (INTA), Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET) and Universidad de Morón (UM) for their financial support in the studies carried out and

Baublits, R.T.; Pohlman, F.W. Jr.; Brown, A.H.; Yancey, E.J. & Johnson, Z.B. (2006). Impact of muscle type and sodium chloride concentration on the quality, sensory and instrumental colour characteristics of solution enhanced whole-muscle beef. *Meat*

Bender, A. (1992). *Meat and meat products in human nutrition in developing countries*, FAO,

Boehm, M.L.; Kendall, T.L.; Thompson, V.F. & Goll, D.E. (1998). Changes in the calpains and calpastatin during postmortem storage of bovine muscle. *Journal of Animal Science*,

Kellermayer, M.S.Z. & Grama, L. (2002). Stretching and visualizing titin molecules: Combining structure, dynamics and mechanics. *Journal of Muscle Research and Cell Motility,* Vol.23, pp. 499–511. ISSN: 0142-4319.

Electrophoresis as a Useful Tool in Studying the Quality of Meat Products 135

Pighin, D.G.; Sancho, A.M. & Gonzalez, C.B. (2008). Effect of salt addition on the thermal behavior of proteins of bovine meat from Argentina. *Meat Science*, Vol.79, pp. 549-556.

Pospiech, E.; Greaser, M.L.; Mikolajczack, B.; Chiang, W. & Krzymdzinska, M. (2002). Thermal properties of titin from porcine and bovine muscles. *Meat Science,* Vol.62, pp.

SENASA (National Service of Animal Health) Decreto #4238/68 (1969). *Despostadero*. In Reglamento de Inspección de Productos, Subproductos y Derivados de Origen Animal

Sobieszek, A. (1994). Gradient polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate: A practical approach to muscle contractile and regulatory proteins.

Spudich, J.A. & Watt, S. (1971). The regulation of rabbit skeletal muscle contraction*. The Journal of Biological Chemistry,* Vol.246 No.15, pp. 4866–4871. ISSN: 0021-9258. Stabursvik, E. & Martens, H. (1980). Thermal denaturation of proteins in post rigor muscle tissue as studied by differential scanning calorimetry. *Journal of the Science of Food and*

Syrovy, I. (1984). Separation of muscle proteins. *Journal of Chromatography A,* Vol.300, pp.

Szerman, N.; Gonzalez, C.B.; Sancho, A.M.; Grigioni, G.; Carduza, F. and Vaudagna, S.R. (2012). Effect of the addition of conventional additives and whey proteins concentrates on technological parameters, physicochemical properties, microstructure and sensory attributes of *sous vide* cooked beef muscles. *Meat Science*, Vol.90, pp. 701–710. ISSN:

Trout, G.R. & Schmidt, G. (1986). Effect on the functional properties of restructural beef rolls: The role of pH, ionic strength, and phosphate type. *Journal of Food Science*, Vol.51,

Vaudagna, S. R.; Sánchez, G.; Neira, M. S.; Insani, E. M.; Picallo, A. B.; Gallinger, M. M. & Lasta, J.A. (2002). *Sous vide* cooked beef muscles: Effects of low temperature - long time treatments on their quality characteristics and storage stability of product. *International*

Vaudagna, S.R.; Pazos, A.A.; Guidi, S.M.; Sanchez, G.; Carp, D.J. & Gonzalez, C.B. (2008). Effects of salt addition on *sous vide* cooked whole beef muscles from Argentina. *Meat*

Von Hippel, P.H. & Wong, K.Y. (1965). On the conformational stability of globular proteins. The effects of various electrolytes and nonelectrolytes on the thermal ribonuclease transition*. The Journal of Biological Chemistry*, Vol.240 No.10, pp. 3909–3923. ISSN: 0021-

Wang, K. (1982). Purification of titin and nebulin. *Methods in Enzymology,* Vol.85, pp. 264–

Wang, K. (1996). Titin/Connectin and nebulin: Giant proteins rulers of muscle structure and

function. *Advances in Biophysics,* Vol.33, pp. 123–134. ISSN: 0065-227X.

*Journal of Food Science and Technology*, Vol.37, pp. 425-441. ISSN: 0950-5423.

ISSN: 0309-1740.

187–192. ISSN: 0309-1740.

225– 247. ISSN: 0021-9673.

pp. 1416–1423. ISSN: 1750-3841.

0309-1740.

9258.

274. ISSN: 0076-6879.

(G. Say, ed.) pp. 115–116, Buenos Aires, Argentina.

*Electrophoresis,* Vol.15, pp. 1014–1020. ISSN: 0173-0835.

*Agriculture*, Vol.31, pp. 1034–1042. ISSN: 1097-0010.

*Science*, Vol.79 No.3, pp. 470–482. ISSN: 0309-1740.

Pighin, D.G.; Sancho, A.M. & Gonzalez, C.B. (2008). Effect of salt addition on the thermal behavior of proteins of bovine meat from Argentina. *Meat Science*, Vol.79, pp. 549-556. ISSN: 0309-1740.

134 Electrophoresis

ISSN: 1750-3841.

0021-9258.

0756.

1872. ISSN: 1756-2651.

Kellermayer, M.S.Z. & Grama, L. (2002). Stretching and visualizing titin molecules: Combining structure, dynamics and mechanics. *Journal of Muscle Research and Cell*

Kijowski, J.M. & Mast, M.G. (1988). Effect of sodium chloride and phosphates on the thermal properties of chicken meat proteins. *Journal of Food Science*, Vol.53 No.2, pp. 367–370.

Kuroda, M. (1982). Direct extraction of G-actin from the myosin-removed myofibrils under the conditions of low ionic strength. The *Journal of Biochemistry (Tokyo)*, Vol.92, pp. 1863–

Kurzban, G.P. & Wang, K. (1988). Giant polypeptides of skeletal muscle titin: Sedimentation equilibrium in guanidine hydrochloride. *Biochemical and Biophysical Research*

Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of

Linke, W.A.; Kulke, M.; Li, H.; Fujita-Becker, S.; Neagoe, C.; Manstein, D.I.; Gautel, M. & Fernandez, J.M. (2002). PEVK domain of titin: An entropic spring with actin-binding

Lowry, O.H.; Rosebrough, M.J.; Farr, M.L. & Randall, R.L. (1951). Protein measurement with the Folin phenol reagent. *The Journal of Biological Chemistry*, Vol.193, pp. 265–275. ISSN:

Offer, G. & Knight, P. (1998). The structural basis of water-holding in meat. Part I: General principles and water uptake in meat processing. In R. Lawrie (Ed.), *Developments in Meat Science 4,* pp. 63-171, ISBN-13:<PHONE_NUMBER>860, London, Elsevier Applied Science. Pan, K.M. & Damodaran, S. (1994). Isolation and characterization of titin T1 from bovine

Pardee, J.D. & Spudich, J.A. (1982). Purification of muscle actin. *Methods in Enzymology,*

Perez-Juan, M.; Flores, M. & Toldrá, F. (2007). Simultaneous process to isolate actomyosin and actin from post-rigor porcine skeletal muscle. *Food Chemistry,* Vol.101 No.3, pp.

Pietrasik, Z. & Shand, P.J. (2004). Effect of blade tenderization and tumbling time on the processing characteristics and tenderness of injected cooked roast beef. *Meat Science*,

Pietrasik, Z. & Shand, P.J. (2005). Effects of mechanical treatments and moisture enhancement on the processing characteristics and tenderness of beef semimembranosus roast. *Meat Science*, Vol.71 No.3, pp. 498–505. ISSN: 0309-1740. Pighin, D.G. & Gonzalez, C.B. (2008). Enrichment of myofibrillar proteinsfrom beef muscle by a simple sequential method. *Journal of Muscle Foods*, Vol.19, pp. 362-373. ISSN: 1046-

cardiac muscle. *Biochemistry,* Vol.33, pp. 8255–8261. ISSN: 0006-2960.

properties. *Journal of Structural Biology,* Vol.137, pp. 194–205. ISSN: 1047-8477. Locker, R.H. & Wild, D.J.C. (1986). Acomparative study of high molecular weight proteins in various types of muscle across the animal kingdom*. The Journal of Biochemistry*

*Motility,* Vol.23, pp. 499–511. ISSN: 0142-4319.

*Communications,* Vol.150, pp. 1155–1161. ISSN: 0006-291X.

*(Tokyo),* Vol.99, pp. 1473–1484. ISSN: 1756-2651.

Vol.85, pp. 164–179. ISSN: 0076-6879.

Vol.66 No.4, pp. 871–879. ISSN: 0309-1740.

1005–1011. ISSN: 0308-8146.

Bacteriophage T4. *Nature,* Vol.227, pp. 680–685. ISSN: 0028-0836.

Wang, K. & Wright, J. (1988). Architecture of the sarcomere matrix of skeletal muscle: Immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line. *The Journal of Cell Biology,* Vol.107, pp. 2199– 2212. ISSN: 0021-9525.

**Chapter 8**

© 2012 González-Arenzana et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Application of the Different Electrophoresis**

**of Lactic Acid Bacteria in Wines**

Lucía González-Arenzana, Rosa López, Pilar Santamaría and Isabel López-Alfaro

http://dx.doi.org/10.5772/45759

**1. Introduction**

Additional information is available at the end of the chapter

complex microbiota involved in them is necessary.

**Techniques to the Detection and Identification**

The microorganisms play an essential role in winemaking since a mixed culture of numerous microorganisms including fungal, yeast, and bacteria species are involved in this process and are the responsible for the final quality of the wine (Bisson et al., 1993). Therefore, in order to control the fermentation processes knowing and understanding the

Yeast are able to convert sugar from grapes into ethanol and many other changes that lead to wine. Lactic acid bacteria (LAB) that are often present on the surface of the grapes and can represent significant populations in musts (Lonvaud-Funel, 1999) play dual roles in wine fermentations: as wine spoilage agents and as the main effectors of malolactic fermentation (MLF). Numerous studies have been conducted on the LAB that occur on grapes, grape musts and wines and it is generally agreed that a succession of species happens during the different stages of winemaking and conservation of wines (Ribéreau-Gayon et al., 2006). Most bacterial species present in wine fermentations have been identified by traditional microbiological techniques involving cultivation. However, as it was observed with microbial ecology studies of other environments, cultivation-dependent methods often exhibit biases resulting in an incomplete representation of the true present bacterial diversity (Amann et al., 1995; Hugenholtz et al., 1998). Applications of cultureindependent molecular techniques to monitor the microbial successions of various food and beverage fermentations have revealed microbial constituents and microbial interactions not

Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis to separate bacterial 16S ribosomal DNA (rDNA) amplicons are common

and reproduction in any medium, provided the original work is properly cited.

properly cited.

witnessed by previous plating analyses (Giraffa & Neviani, 2001).

## **Application of the Different Electrophoresis Techniques to the Detection and Identification of Lactic Acid Bacteria in Wines**

Lucía González-Arenzana, Rosa López, Pilar Santamaría and Isabel López-Alfaro

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/45759

### **1. Introduction**

136 Electrophoresis

2212. ISSN: 0021-9525.

Wang, K. & Wright, J. (1988). Architecture of the sarcomere matrix of skeletal muscle: Immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line. *The Journal of Cell Biology,* Vol.107, pp. 2199–

Wang, S.M. & Greaser, M.L. (1985). Immunocytochemical studies using a monoclonal antibody to bovine cardiac titin on intact and extracted myofibrils. *Journal of Muscle*

Warris, P.D. (2000) The Chemical Composition and Structure of Meat, In *Meat Science. An Introductory Text*. pp. 37-67. CABI Publishing, ISBN: 0-85199-424-5, Oxfordshire, UK. Xiong, Y.L. (2005). Role of myofibrillar proteins in water-binding in brine-enhance meats.

*Research and Cell Motility,* Vol.6, pp. 293–312. ISSN: 0142-4319.

*Food Research International*, Vol.38, pp. 281–287. ISSN: 0963-9969.

The microorganisms play an essential role in winemaking since a mixed culture of numerous microorganisms including fungal, yeast, and bacteria species are involved in this process and are the responsible for the final quality of the wine (Bisson et al., 1993). Therefore, in order to control the fermentation processes knowing and understanding the complex microbiota involved in them is necessary.

Yeast are able to convert sugar from grapes into ethanol and many other changes that lead to wine. Lactic acid bacteria (LAB) that are often present on the surface of the grapes and can represent significant populations in musts (Lonvaud-Funel, 1999) play dual roles in wine fermentations: as wine spoilage agents and as the main effectors of malolactic fermentation (MLF). Numerous studies have been conducted on the LAB that occur on grapes, grape musts and wines and it is generally agreed that a succession of species happens during the different stages of winemaking and conservation of wines (Ribéreau-Gayon et al., 2006). Most bacterial species present in wine fermentations have been identified by traditional microbiological techniques involving cultivation. However, as it was observed with microbial ecology studies of other environments, cultivation-dependent methods often exhibit biases resulting in an incomplete representation of the true present bacterial diversity (Amann et al., 1995; Hugenholtz et al., 1998). Applications of cultureindependent molecular techniques to monitor the microbial successions of various food and beverage fermentations have revealed microbial constituents and microbial interactions not witnessed by previous plating analyses (Giraffa & Neviani, 2001).

Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis to separate bacterial 16S ribosomal DNA (rDNA) amplicons are common

© 2012 González-Arenzana et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

culture independent methods employed to characterize microbial communities from specific environmental niches (Lopez et al., 2003; Muyzer & Smalla, 1998). These approaches are attractive since they enable to detect individual species as well as to get overall profiling of community structure changes with time.

Application of the Different Electrophoresis Techniques to the Detection and Identification of Lactic Acid Bacteria in Wines 139

drawn off into tanks and were allowed to undergo spontaneous MLF with the endogenous microbiota. The sampled winery had never used commercial starters for MLF. One fermentation tank was sampled in each vintage. Wine samples were collected aseptically for chemical and microbiological analysis at different times: must (stage 1), tumultuous AF (density around 1,025; stage 2), at final AF (< 2 g/L glucose + fructose; stage 3), initial MLF (consumption of 10% of the initial malic acid; stage 4), tumultuous MLF (consumption of 60% of the initial malic acid; stage 5) and at final MLF (L-malic acid concentration < 0.5 g/L;

Sixteen commercial starter cultures employed to induce MLF derived from six different companies were analyzed. These commercial cultures were selected between the most

Alcohol degree, pH, total acidity, volatile acidity, reducing sugars, free and total sulphur dioxide and L-malic and L-lactic acid content were measured according to the European

Must or wine samples were diluted in sterile saline (0.9% NaCl) solution and plated on modified MRS agar (Scharlau Chemie S.A., Barcelona, Spain) plates supplemented with tomato juice (10% v/v), fructose (6 g/L), cysteine-HCl (0.5 g/L), D,L-malic acid (5 g/L) and pymaricine (50 mg/L) (Acofarma, S. Coop., Terrassa, Spain). Samples were incubated at 30 ºC under strict anaerobic conditions (Gas Pak System, Oxoid Ltd., Basingstoke, England) for at least ten days, and viable counts were reported as the number of CFU/mL. Fifteen colonies from each wine sample were selected for reisolation and identification. Isolates

Every commercial lyophilized starter culture was hydrated in saline solution (0.9% NaCl) and then 100 μL aliquot from the appropriate dilution was plated at the surface of modified MRS agar without pymaricine. Because of their low viability in laboratory conditions (Maicas et al., 1999a; Maicas et al., 1999b) glycine (40 mM) and ethanol (10% v/v) were added to this medium. The plates were incubated for at least 10 days at 30 ºC under anaerobic atmosphere (Gas Pak System, Oxoid Ltd.) and five colonies were isolated from

Species identification was carried out by previously recommended methods, which included bacteria morphology, Gram staining, and catalase (Holt et al., 1994). *Oenococcus*

**2.2. Commercial** *Oenococcus oeni* **starter samples**

**2.3. Chemical analysis of the musts and wines**

were stored in 20% sterile skim milk (Difco) at –20 ºC.

Community Official Methods (European Community, 1990).

stage 6).

each one.

*2.4.2. Species identification*

frequently used in Spain.

**2.4. Culture dependent methods**

*2.4.1. Bacterial enumeration and isolation*

Otherwise, ecology, interactions and development of the different bacterial strains during alcoholic fermentation (AF) and MLF are still a field of active research. Efficient and precise methods of strain identification and discrimination have been developed during the last years, either to prepare well-defined starters of biotechnological interest in winemaking, or to quickly assess the presence of certain strains in a wine, or to gain insight of such a complex ecosystem as wine.

Pulsed field gel electrophoresis (PFGE) has proved to be an useful tool for the identification of a wide LAB strains variety and especially for species belonging to the genus *Lactobacillus* (Charteris et al., 1997). Several restriction enzymes have been used for obtaining profiles of *Oenococcus oeni* strains: *Not*I (Kelly et al., 1993; Prevost et al., 1995; Sato et al., 2001), *Apa*I and *Sfi*I (Daniel et al., 1993; Kelly et al., 1993; Lopez et al., 2007). Other used methods for LAB strain identification include ribotyping or restriction fragment length polymorphism (RFLP) of genes encoding rRNAs (Charteris et al., 1997; Zavaleta et al., 1997), random amplified polymorphic DNA (RAPD-PCR) analysis with arbitrary primers (Cocconcelli et al., 1997; Lopez et al., 2008; Spano et al., 2002), rep-polymerase chain reaction (rep-PCR) analysis (Parry et al., 2002) and multilocus sequence typing (MLST) system (de las Rivas et al., 2004; de las Rivas et al., 2006). RAPD-PCR and PFGE of macrorestriction fragments are the most frequently used (Guerrini et al., 2003; Tenreiro et al., 1994; Viti et al., 1996; Zapparoli et al., 2000; Zavaleta et al., 1997), and more recently Ruiz et al. (Ruiz et al., 2008) have obtained a better discrimination in the study of bacterial diversity by the combination of results from those two techniques.

For all these reasons, the aims of this work were: (a) applying the DGGE, PFGE and RAPD-PCR techniques to the analysis of the LAB species diversity and the intraspecific diversity of *Oenococcus oeni* in a winery of La Rioja region during three consecutive vintages, (b) getting a better knowledge of bacterial ecology throughout both AF and MLF in wine elaborated with Tempranillo, the classic red grape variety of Spain and native of Appellation of Origin Rioja, (c) evaluating the occurrence of genotypes from commercial *Oenococcus oeni* strains between the autochthonous *Oenococcus oeni* strains isolated from non inoculated fermenting wines, and (d) contributing to the maintenance of the LAB biodiversity in Rioja red wines.

### **2. Methodology**

### **2.1. Wine production and wine samples**

Traditional red wine fermentations from c.v. Tempranillo local grapes of 2006, 2007 and 2008 vintages at one winery of the Spanish northern region of Rioja were studied. Winemaking practices were the typical of this wine-producing area: AFs were conducted in the presence of grape skins, seeds and stalks, after the addition of sulphur dioxide and until the residual reducing sugar content was under 2 g/L. At this final point of AF, wines were drawn off into tanks and were allowed to undergo spontaneous MLF with the endogenous microbiota. The sampled winery had never used commercial starters for MLF. One fermentation tank was sampled in each vintage. Wine samples were collected aseptically for chemical and microbiological analysis at different times: must (stage 1), tumultuous AF (density around 1,025; stage 2), at final AF (< 2 g/L glucose + fructose; stage 3), initial MLF (consumption of 10% of the initial malic acid; stage 4), tumultuous MLF (consumption of 60% of the initial malic acid; stage 5) and at final MLF (L-malic acid concentration < 0.5 g/L; stage 6).

### **2.2. Commercial** *Oenococcus oeni* **starter samples**

Sixteen commercial starter cultures employed to induce MLF derived from six different companies were analyzed. These commercial cultures were selected between the most frequently used in Spain.

### **2.3. Chemical analysis of the musts and wines**

Alcohol degree, pH, total acidity, volatile acidity, reducing sugars, free and total sulphur dioxide and L-malic and L-lactic acid content were measured according to the European Community Official Methods (European Community, 1990).

### **2.4. Culture dependent methods**

138 Electrophoresis

community structure changes with time.

complex ecosystem as wine.

**2. Methodology**

**2.1. Wine production and wine samples**

culture independent methods employed to characterize microbial communities from specific environmental niches (Lopez et al., 2003; Muyzer & Smalla, 1998). These approaches are attractive since they enable to detect individual species as well as to get overall profiling of

Otherwise, ecology, interactions and development of the different bacterial strains during alcoholic fermentation (AF) and MLF are still a field of active research. Efficient and precise methods of strain identification and discrimination have been developed during the last years, either to prepare well-defined starters of biotechnological interest in winemaking, or to quickly assess the presence of certain strains in a wine, or to gain insight of such a

Pulsed field gel electrophoresis (PFGE) has proved to be an useful tool for the identification of a wide LAB strains variety and especially for species belonging to the genus *Lactobacillus* (Charteris et al., 1997). Several restriction enzymes have been used for obtaining profiles of *Oenococcus oeni* strains: *Not*I (Kelly et al., 1993; Prevost et al., 1995; Sato et al., 2001), *Apa*I and *Sfi*I (Daniel et al., 1993; Kelly et al., 1993; Lopez et al., 2007). Other used methods for LAB strain identification include ribotyping or restriction fragment length polymorphism (RFLP) of genes encoding rRNAs (Charteris et al., 1997; Zavaleta et al., 1997), random amplified polymorphic DNA (RAPD-PCR) analysis with arbitrary primers (Cocconcelli et al., 1997; Lopez et al., 2008; Spano et al., 2002), rep-polymerase chain reaction (rep-PCR) analysis (Parry et al., 2002) and multilocus sequence typing (MLST) system (de las Rivas et al., 2004; de las Rivas et al., 2006). RAPD-PCR and PFGE of macrorestriction fragments are the most frequently used (Guerrini et al., 2003; Tenreiro et al., 1994; Viti et al., 1996; Zapparoli et al., 2000; Zavaleta et al., 1997), and more recently Ruiz et al. (Ruiz et al., 2008) have obtained a better discrimination in the study of

For all these reasons, the aims of this work were: (a) applying the DGGE, PFGE and RAPD-PCR techniques to the analysis of the LAB species diversity and the intraspecific diversity of *Oenococcus oeni* in a winery of La Rioja region during three consecutive vintages, (b) getting a better knowledge of bacterial ecology throughout both AF and MLF in wine elaborated with Tempranillo, the classic red grape variety of Spain and native of Appellation of Origin Rioja, (c) evaluating the occurrence of genotypes from commercial *Oenococcus oeni* strains between the autochthonous *Oenococcus oeni* strains isolated from non inoculated fermenting wines, and (d) contributing to the maintenance of the LAB biodiversity in Rioja red wines.

Traditional red wine fermentations from c.v. Tempranillo local grapes of 2006, 2007 and 2008 vintages at one winery of the Spanish northern region of Rioja were studied. Winemaking practices were the typical of this wine-producing area: AFs were conducted in the presence of grape skins, seeds and stalks, after the addition of sulphur dioxide and until the residual reducing sugar content was under 2 g/L. At this final point of AF, wines were

bacterial diversity by the combination of results from those two techniques.

### *2.4.1. Bacterial enumeration and isolation*

Must or wine samples were diluted in sterile saline (0.9% NaCl) solution and plated on modified MRS agar (Scharlau Chemie S.A., Barcelona, Spain) plates supplemented with tomato juice (10% v/v), fructose (6 g/L), cysteine-HCl (0.5 g/L), D,L-malic acid (5 g/L) and pymaricine (50 mg/L) (Acofarma, S. Coop., Terrassa, Spain). Samples were incubated at 30 ºC under strict anaerobic conditions (Gas Pak System, Oxoid Ltd., Basingstoke, England) for at least ten days, and viable counts were reported as the number of CFU/mL. Fifteen colonies from each wine sample were selected for reisolation and identification. Isolates were stored in 20% sterile skim milk (Difco) at –20 ºC.

Every commercial lyophilized starter culture was hydrated in saline solution (0.9% NaCl) and then 100 μL aliquot from the appropriate dilution was plated at the surface of modified MRS agar without pymaricine. Because of their low viability in laboratory conditions (Maicas et al., 1999a; Maicas et al., 1999b) glycine (40 mM) and ethanol (10% v/v) were added to this medium. The plates were incubated for at least 10 days at 30 ºC under anaerobic atmosphere (Gas Pak System, Oxoid Ltd.) and five colonies were isolated from each one.

### *2.4.2. Species identification*

Species identification was carried out by previously recommended methods, which included bacteria morphology, Gram staining, and catalase (Holt et al., 1994). *Oenococcus*

*oeni*, *Lactobacillus plantarum* and *Lactobacillus brevis* species were confirmed by the speciesspecific PCR method (Beneduce et al., 2004; Zapparoli et al., 1998). In case of the identification of other species, PCR amplification of partial 16S rRNA genes was performed with WLAB1 and WLAB2 as previously described (Lopez et al., 2007). PCR products were sequenced by Macrogen Inc. (Seoul, South Korea) and sequences were used for comparison to the data in GenBank using the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990).

Application of the Different Electrophoresis Techniques to the Detection and Identification of Lactic Acid Bacteria in Wines 141

zirconium hydroxide (7 g/L) were added to the pellet to facilitate pelleting of the bacteria in wine (Lucore et al., 2000). After horizontal shaking during 10 min at room temperature, the suspension was again centrifuged (10 min, 500xg, 7 ºC) and finally DNA was purified from the cell pellet by using a PowerSoil® DNA isolation kit (MO BIO Laboratories, Inc.,

PCR was performed using an Applied Biosystem, GeneAmp® PCR System 2700 thermocycler at a final volume of 50 μL. To amplify the region V4 to V5 of 16S rDNA gene, primers WLAB1 and WLAB2GC were used as López et al. described (2003). Moreover, primers *rpo*B1, *rpo*B1o, and *rpo*B2GC were employed to amplify the region of the *rpoB* gene as it was described by Renouf et al. (2006) with the next modifications: 0.5 μM of each primer, 1

The separation of the respective PCR products was performed with the D-CODETM universal mutation detection system (Bio-Rad, Hercules, Calif.). PCR products obtained from WLAB1- WLAB2GC primers were run on 8% (wt/V) polyacrilamide gels in a running buffer containing 2 M Tris base, 1 M Glacial acetic acid and 50 mM EDTA pH 8 (TAE), and a denaturing gradient from 35 to 55% of urea and formamide. The electrophoresis was performed at 20 V for 10 min, and 80 V for 18 h at a constant temperature of 60 °C. PCR products generated with the *rpo*B1, *rpo*B1o, and *rpo*B2GC primers were separated with 8% polyacrylamide gels containing a 32 to 50% urea-formamide gradient. Electrophoresis was performed for 10 min at 20 V, and 16 h at 60 V at a constant temperature of 60 °C. The DGGE gels were stained in ethidium bromide after the electrophoresis and then were visualized with UV transiluminattion (GelDoc, Bio-Rad). Blocks of polyacrylamide gels which contained selected DGGE bands were excised and later incubated overnight in 20 μL of sterile and pure water at 4 ºC to make DNA bands diffuse to the liquid. One microliter of

PCR products were sequenced by Macrogen Inc. (Seoul, South Korea). The quality and characteristics of the obtained sequences were analyzed with the software InfoQuestTM FP 5.10, only those ones considered as appropriate were used for comparison to the GenBank database with the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990). After this preliminary study, our sequences and their homologous ones (obtained from the Nucleotide Database: http://www.ncbi.nlm.nih.gov/nuccore) were assembled and submitted to phylogenetic and evolutionary analysis with MEGA version 4.0.2 (Tamura et al., 2007). The Neighbor-Joining analysis (Saitou & Nei, 1987) allowed to get information about the relations between the gotten sequences and the reliability of the identifications provided by the Nucleotide Database. The bootstrap test was based on 1000 replicates (Felsenstein, 1985).

mM dNTP mix and 0.5 μL of *PfuUltra* II Fusion HS DNA Polymerase (Stratagene).

Carlsbad, CA USA) as per the manufacturer's instructions.

this solution was used to reamplify the PCR product.

*2.5.4. DNA sequencing and phylogenetic analysis*

*2.5.2. PCR conditions*

*2.5.3. PCR-DGGE analysis*

### *2.4.3. Oenococcus oeni typification by PFGE*

PFGE was carried out according to the method described by Birren et al. (1993) with some modifications (Lopez et al., 2007) for agarose block preparation. Because of the difficulty of typing commercial strains first of all these cells underwent to fifteen minutes of ultrasounds, moreover a higher quantity of lysozime (100 μL/block) was added and incubated for 2 h. Macrorestriction analysis was performed with two endonucleases: *Sfi*I, following the method reported by López et al. (2007), and *Apa*I by the method reported by Larisika (2008) with the following modifications for optimal separation of fragments: 1.2% (w/v) agarose gels were submitted to 24 h with a pulse ramping between 0.5 and 20 s at 14 ºC and 6 V/cm in a CHEF DRII apparatus (Bio-Rad).

### *2.4.4. Oenococcus oeni typification by RAPD-PCR*

RAPD-PCR was carried out following the procedure described by Ruiz et al. (2010b) with some modifications: MgCl 100 mM, dNTP 50 mM and primer M13 100 mM. RAPD-PCR reaction was developed in a total volume of 50 μL and it was carried out with a Perkin Elmer, GeneAmp PCR System 2400 thermocycler. 20 μL of amplified products were resolved by electrophoresis in a 1.4% agarose gel in 0.5x TBE (45 mM Tris base, 89 mM, boric acid, 2.5 mM EDTA pH 8) for 3 h at 70 V.

### *2.4.5. Numerical analysis of PFGE and RAPD-PCR images*

The conversion, normalization and further processing of images were carried out by InfoQuestTM FP software version 5.10 (Bio-Rad, USA). Comparison of the obtained PFGE patterns was performed with Pearson's product-moment correlation coefficient and the Unweighted Pair Group Method using Arithmetic averages (UPGMA). Comparison of the pulse types from the PFGE and RAPD was made by composite data set comparison with average molecular analysis by Unweighted Pair Group Method using Arithmetic averages (UPGMA) (Ruiz et al., 2008).

### **2.5. Culture independent methods**

### *2.5.1. Direct DNA extraction from wines samples*

A volume of 10 mL of each must or wine sample was centrifuged (30 min, 10000xg, 4 ºC). The supernatant was discarded and 1.2 mL of saline solution (NaCl 0.9%) and 2.4 mL of zirconium hydroxide (7 g/L) were added to the pellet to facilitate pelleting of the bacteria in wine (Lucore et al., 2000). After horizontal shaking during 10 min at room temperature, the suspension was again centrifuged (10 min, 500xg, 7 ºC) and finally DNA was purified from the cell pellet by using a PowerSoil® DNA isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA USA) as per the manufacturer's instructions.

### *2.5.2. PCR conditions*

140 Electrophoresis

al., 1990).

*2.4.3. Oenococcus oeni typification by PFGE*

in a CHEF DRII apparatus (Bio-Rad).

acid, 2.5 mM EDTA pH 8) for 3 h at 70 V.

(UPGMA) (Ruiz et al., 2008).

**2.5. Culture independent methods**

*2.5.1. Direct DNA extraction from wines samples*

*2.4.4. Oenococcus oeni typification by RAPD-PCR*

*2.4.5. Numerical analysis of PFGE and RAPD-PCR images*

*oeni*, *Lactobacillus plantarum* and *Lactobacillus brevis* species were confirmed by the speciesspecific PCR method (Beneduce et al., 2004; Zapparoli et al., 1998). In case of the identification of other species, PCR amplification of partial 16S rRNA genes was performed with WLAB1 and WLAB2 as previously described (Lopez et al., 2007). PCR products were sequenced by Macrogen Inc. (Seoul, South Korea) and sequences were used for comparison to the data in GenBank using the Basic Local Alignment Search Tool (BLAST) (Altschul et

PFGE was carried out according to the method described by Birren et al. (1993) with some modifications (Lopez et al., 2007) for agarose block preparation. Because of the difficulty of typing commercial strains first of all these cells underwent to fifteen minutes of ultrasounds, moreover a higher quantity of lysozime (100 μL/block) was added and incubated for 2 h. Macrorestriction analysis was performed with two endonucleases: *Sfi*I, following the method reported by López et al. (2007), and *Apa*I by the method reported by Larisika (2008) with the following modifications for optimal separation of fragments: 1.2% (w/v) agarose gels were submitted to 24 h with a pulse ramping between 0.5 and 20 s at 14 ºC and 6 V/cm

RAPD-PCR was carried out following the procedure described by Ruiz et al. (2010b) with some modifications: MgCl 100 mM, dNTP 50 mM and primer M13 100 mM. RAPD-PCR reaction was developed in a total volume of 50 μL and it was carried out with a Perkin Elmer, GeneAmp PCR System 2400 thermocycler. 20 μL of amplified products were resolved by electrophoresis in a 1.4% agarose gel in 0.5x TBE (45 mM Tris base, 89 mM, boric

The conversion, normalization and further processing of images were carried out by InfoQuestTM FP software version 5.10 (Bio-Rad, USA). Comparison of the obtained PFGE patterns was performed with Pearson's product-moment correlation coefficient and the Unweighted Pair Group Method using Arithmetic averages (UPGMA). Comparison of the pulse types from the PFGE and RAPD was made by composite data set comparison with average molecular analysis by Unweighted Pair Group Method using Arithmetic averages

A volume of 10 mL of each must or wine sample was centrifuged (30 min, 10000xg, 4 ºC). The supernatant was discarded and 1.2 mL of saline solution (NaCl 0.9%) and 2.4 mL of PCR was performed using an Applied Biosystem, GeneAmp® PCR System 2700 thermocycler at a final volume of 50 μL. To amplify the region V4 to V5 of 16S rDNA gene, primers WLAB1 and WLAB2GC were used as López et al. described (2003). Moreover, primers *rpo*B1, *rpo*B1o, and *rpo*B2GC were employed to amplify the region of the *rpoB* gene as it was described by Renouf et al. (2006) with the next modifications: 0.5 μM of each primer, 1 mM dNTP mix and 0.5 μL of *PfuUltra* II Fusion HS DNA Polymerase (Stratagene).

### *2.5.3. PCR-DGGE analysis*

The separation of the respective PCR products was performed with the D-CODETM universal mutation detection system (Bio-Rad, Hercules, Calif.). PCR products obtained from WLAB1- WLAB2GC primers were run on 8% (wt/V) polyacrilamide gels in a running buffer containing 2 M Tris base, 1 M Glacial acetic acid and 50 mM EDTA pH 8 (TAE), and a denaturing gradient from 35 to 55% of urea and formamide. The electrophoresis was performed at 20 V for 10 min, and 80 V for 18 h at a constant temperature of 60 °C. PCR products generated with the *rpo*B1, *rpo*B1o, and *rpo*B2GC primers were separated with 8% polyacrylamide gels containing a 32 to 50% urea-formamide gradient. Electrophoresis was performed for 10 min at 20 V, and 16 h at 60 V at a constant temperature of 60 °C. The DGGE gels were stained in ethidium bromide after the electrophoresis and then were visualized with UV transiluminattion (GelDoc, Bio-Rad). Blocks of polyacrylamide gels which contained selected DGGE bands were excised and later incubated overnight in 20 μL of sterile and pure water at 4 ºC to make DNA bands diffuse to the liquid. One microliter of this solution was used to reamplify the PCR product.

### *2.5.4. DNA sequencing and phylogenetic analysis*

PCR products were sequenced by Macrogen Inc. (Seoul, South Korea). The quality and characteristics of the obtained sequences were analyzed with the software InfoQuestTM FP 5.10, only those ones considered as appropriate were used for comparison to the GenBank database with the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990). After this preliminary study, our sequences and their homologous ones (obtained from the Nucleotide Database: http://www.ncbi.nlm.nih.gov/nuccore) were assembled and submitted to phylogenetic and evolutionary analysis with MEGA version 4.0.2 (Tamura et al., 2007). The Neighbor-Joining analysis (Saitou & Nei, 1987) allowed to get information about the relations between the gotten sequences and the reliability of the identifications provided by the Nucleotide Database. The bootstrap test was based on 1000 replicates (Felsenstein, 1985).

The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2007) that allowed to calculate the equivalent units to the base substitutions per site.

Application of the Different Electrophoresis Techniques to the Detection and Identification of Lactic Acid Bacteria in Wines 143

fermentation could also influence, as it has been reported by other authors (du Plessis et al.,

Figure 1 shows the number of isolates of the viable LAB species identified at each stage and year of vinification. A total of 251 LAB isolates were recovered and identified as belonging to eight different species. The greatest diversity of LAB species was detected during the AF. *Oenococcus oeni* was present in all studied stages of the fermentation process except in 2007. It was isolated in must and tumultuous AF in 2006 and 2008 vintages, and it was the only species isolated at MLF in the three years, being therefore the predominant species, followed by *Lactobacillus plantarum, Leuconostoc mesenteroides* and *Lactobacillus mali*. The other non-*Oenococcus oeni* species appeared at stages 1-2 in variable rates in the three vintages. A similar distribution of species has been also reported by other authors (Fugelsang & Edwards, 2007; Ruiz et al., 2010) and they also concluded that *Oenococcus oeni* was the main responsible species for MLF. The diversity of species found at each year was different, being the number of species isolated in 2007 almost double that in 2006 and 2008, and missing *Oenococcus oeni* until the end of the 2007 AF, a fact that could also have influence in the MLF

**Figure 1.** Number of isolates of the viable LAB species identified at each stage of vinification (1, must; 2, tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF) and in each vintage.

123456<PHONE_NUMBER>56 **Stage 2006 vintage Stage 2007 vintage Stage 2008 vintage**

*O.oeni L. plantarum L. mali L. brevis L. coryniformis L. uvarum Ln. mesenteroides P. parvulus*

PCR-DGGE analysis of the sampled wine fermentations in the three studied vintages using primers WLAB1/2 (16S rDNA-based primer sets) and primers *rpo*B1/1o/2 (β subunit of the

*3.2.2. Culture independent microbiological analysis and comparison with culture*

2004; Lonvaud-Funel, 1999; Reguant et al., 2005a; Reguant et al., 2005b).

**3.2. Species identification**

duration, as it was indicated above.

*dependent method*

**Nº of isolates of each LAB species**

*3.2.1. Culture dependent microbiological analysis*

### **3. Results and discussion**

### **3.1. Oenological parameters of wine samples and fermentation development**

Results for analytical composition of wines during three vintages are displayed in Table 1. Data were within the usual range of Tempranillo wines from this Spanish region (González-Arenzana et al., 2012b). After completion AF, alcohol content ranged between 13.0% and 14.0%, pH was between 3.32 and 3.64 and free SO2 level was between 4.24 and 18.1 mg/L. During MLF a decrease in total acidity and a subsequent increase in pH were observed. In addition, an increase in volatile acidity was noted as it was expected. The wine from 2006 vintage showed less restrictive parameters for microbial growth, so it presented higher pH and the lowest values of alcohol content and SO2.

**Table 1.** Analytical composition of wines at final AF (stage 3) and final MLF (stage 6) at each vintage.

AF completion lasted for six, sixteen and eleven days in 2006, 2007 and 2008 vintages, respectively. Viable LAB counts during AF were in the range of 102 - 103 CFU/mL, increasing to 107 – 108 CFU/mL during MLF, similar to spontaneous MLF results reported by other authors (European Community, 1990; Lopez et al., 2008). The development of the MLF was related to the viable population of LAB and there was a relation between bacterial population and decrease in L-malic acid (data not shown). Important differences in MLF duration were observed between vintages and MLF completion lasted for 21, 239 and 136 days in 2006, 2007 and 2008 vintages, respectively. Different temperatures at each vintage (wine temperature below 12 ºC after AF in 2007) and the lack of temperature control in the winery were the determinant factors in these differences, but factors such as pH, composition of the wine and the interaction with other microorganisms implicated in the fermentation could also influence, as it has been reported by other authors (du Plessis et al., 2004; Lonvaud-Funel, 1999; Reguant et al., 2005a; Reguant et al., 2005b).

### **3.2. Species identification**

142 Electrophoresis

substitutions per site.

**3. Results and discussion**

and the lowest values of alcohol content and SO2.

The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2007) that allowed to calculate the equivalent units to the base

**3.1. Oenological parameters of wine samples and fermentation development**

Results for analytical composition of wines during three vintages are displayed in Table 1. Data were within the usual range of Tempranillo wines from this Spanish region (González-Arenzana et al., 2012b). After completion AF, alcohol content ranged between 13.0% and 14.0%, pH was between 3.32 and 3.64 and free SO2 level was between 4.24 and 18.1 mg/L. During MLF a decrease in total acidity and a subsequent increase in pH were observed. In addition, an increase in volatile acidity was noted as it was expected. The wine from 2006 vintage showed less restrictive parameters for microbial growth, so it presented higher pH

Year 2006 2007 2008

Stage 3 6 3 6 3 6 Alcohol content (% v/v) 13.0 - 13.8 - 14.0 pH 3.64 3.83 3.41 3.57 3.32 3.50 Total acidity (g/L tartaric acid) 7.98 5.91 7.63 6.71 9.00 7.20 Volatile acidity (g/L acetic acid) 0.25 0.46 0.37 0.49 0.26 0.37 Total SO2 (mg/L) 28.4 - 38.1 - 31.6 - Free SO2 (mg/L) 4.24 - 18.1 - 13.2 - L-malic acid (g/L) 2.60 0.04 1.48 0.16 2.61 0.21 L-lactic acid (g/L) - 1.81 - 1.10 - 1.72

**Table 1.** Analytical composition of wines at final AF (stage 3) and final MLF (stage 6) at each vintage.

AF completion lasted for six, sixteen and eleven days in 2006, 2007 and 2008 vintages, respectively. Viable LAB counts during AF were in the range of 102 - 103 CFU/mL, increasing to 107 – 108 CFU/mL during MLF, similar to spontaneous MLF results reported by other authors (European Community, 1990; Lopez et al., 2008). The development of the MLF was related to the viable population of LAB and there was a relation between bacterial population and decrease in L-malic acid (data not shown). Important differences in MLF duration were observed between vintages and MLF completion lasted for 21, 239 and 136 days in 2006, 2007 and 2008 vintages, respectively. Different temperatures at each vintage (wine temperature below 12 ºC after AF in 2007) and the lack of temperature control in the winery were the determinant factors in these differences, but factors such as pH, composition of the wine and the interaction with other microorganisms implicated in the

### *3.2.1. Culture dependent microbiological analysis*

Figure 1 shows the number of isolates of the viable LAB species identified at each stage and year of vinification. A total of 251 LAB isolates were recovered and identified as belonging to eight different species. The greatest diversity of LAB species was detected during the AF. *Oenococcus oeni* was present in all studied stages of the fermentation process except in 2007. It was isolated in must and tumultuous AF in 2006 and 2008 vintages, and it was the only species isolated at MLF in the three years, being therefore the predominant species, followed by *Lactobacillus plantarum, Leuconostoc mesenteroides* and *Lactobacillus mali*. The other non-*Oenococcus oeni* species appeared at stages 1-2 in variable rates in the three vintages. A similar distribution of species has been also reported by other authors (Fugelsang & Edwards, 2007; Ruiz et al., 2010) and they also concluded that *Oenococcus oeni* was the main responsible species for MLF. The diversity of species found at each year was different, being the number of species isolated in 2007 almost double that in 2006 and 2008, and missing *Oenococcus oeni* until the end of the 2007 AF, a fact that could also have influence in the MLF duration, as it was indicated above.

*O.oeni L. plantarum L. mali L. brevis L. coryniformis L. uvarum Ln. mesenteroides P. parvulus*

**Figure 1.** Number of isolates of the viable LAB species identified at each stage of vinification (1, must; 2, tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF) and in each vintage.

### *3.2.2. Culture independent microbiological analysis and comparison with culture dependent method*

PCR-DGGE analysis of the sampled wine fermentations in the three studied vintages using primers WLAB1/2 (16S rDNA-based primer sets) and primers *rpo*B1/1o/2 (β subunit of the RNA polymerase-based primer sets) (Figure 2a and 2b, respectively) revealed different species and a profile of bacterial community structure changes during AF and MLF.

Application of the Different Electrophoresis Techniques to the Detection and Identification of Lactic Acid Bacteria in Wines 145

(a)

**Figure 2.** DGGE gels of the sampled wines at each vintage and stage of vinification (1, must; 2, tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF). Letters indicate bands excised for each gene, 16S rRNA (a) and *rpo*B (b).

The sequences obtained from the DNA excised DGGE-bands of each sample and their homologous ones from Nucleotide Database (Altschul et al., 1990) constituted a tree for each studied gene (Figure 3). Figure 3a shows a tree based on 16S rDNA gene composed by four ramifications or branches belonging to the genus *Oenococcus*, *Lactobacillus*, *Weissella* and *Leuconostoc*; and Figure 3b shows a tree based on *rpo*B gene composed by three branches belonging to *Oenococcus*, *Leuconostoc* and *Lactobacillus.*

Application of the Different Electrophoresis Techniques to the Detection and Identification of Lactic Acid Bacteria in Wines 145

144 Electrophoresis

(a)

a b c d

(b)

a

a

b

a

a a

b b

b

RNA polymerase-based primer sets) (Figure 2a and 2b, respectively) revealed different

 2006 2007 2008 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

species and a profile of bacterial community structure changes during AF and MLF.

**Figure 2.** DGGE gels of the sampled wines at each vintage and stage of vinification (1, must; 2, tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF). Letters indicate bands

a

2006 2007 2008

a a

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

a b

a a

a

b

a

a b b a b c

a

c

a

a a

a a a

The sequences obtained from the DNA excised DGGE-bands of each sample and their homologous ones from Nucleotide Database (Altschul et al., 1990) constituted a tree for each studied gene (Figure 3). Figure 3a shows a tree based on 16S rDNA gene composed by four ramifications or branches belonging to the genus *Oenococcus*, *Lactobacillus*, *Weissella* and *Leuconostoc*; and Figure 3b shows a tree based on *rpo*B gene composed by three branches

a a a

excised for each gene, 16S rRNA (a) and *rpo*B (b).

a

a b

belonging to *Oenococcus*, *Leuconostoc* and *Lactobacillus.*

 LAB species detected with culture independent techniques (16S rDNA/PCR-DGGE and *rpo*B/PCR-DGGE) and culture dependent methods at each stage of vinification (1, must; 2, tumultuous

Identification of the *Oenococcus oeni* strains of this study was successfully achieved by PFGE of DNA digested with *Sfi*I and each strain presented a characteristic PFGE pattern. Digestions with *Apa*I enzyme were not more discriminating than *Sfi*I restriction (data not

AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF) during three vintages.

and their influence in MLF duration.

Detected LAB species

\*

*UB*; uncultured bacterium w; detected with 16S rDNA/DGGE b; detected with *rpo*B/DGGE c; detected in plate after culture

◊; LAB species detected with all the employed methods

**3.3. Strain typing of** *Oenococcus oeni*

*3.3.1. PFGE analysis of the strains of this study*

**Figure 3.** Phylogenetic trees generated from the method of 16S rDNA (a) and *rpo*B (b) sequences of recovered bands were inferred using the Maximun Parsimony method (Felsenstein, 1985). Numbers over the branches are bootstrap values (1000 repetitions). *Escherichia coli* and *Listeria inocua* were used as outgroups. Scale bar represents (calculated) distance. Reference strains are closely related to the sequenced bands and accession number of each gene is indicated. Band number isolated from fermenting wines are indicated by the sampled stage of vinification (1, must; 2, tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF) the letter of the position in the gel and the isolation year.

The species identification at each stage of vinification in the three studied years with culture independent techniques (16S rDNA/PCR-DGGE and *rpo*B/PCR-DGGE) and culture dependent method (plating on modified MRS) are recovered in Table 2.

A total of fourteen different LAB species were identified in the three studied vintages by traditional and culture independent methods. PCR-DGGE analysis allowed to identify nine species in comparison to the eight ones detected by culture in plate of the sampled wine. Thus, *Fructobacillus ficulneus*, *Fructobacillus tropaeoli*, *Lactobacillus buchneri*, *Leuconostoc pseudomesenteroides* and *Weissella* were not detected in the employed culture medium, while *Lactobacillus coryniformis*, *Lactobacillus brevis*, *Lactobacillus mali*, *Lactobacillus uvarum* and *Pediococcus parvulus* were not detected by PCR-DGGE. Therefore, results obtained by both methods were complementary and demonstrated the importance of using a combined analytical approach to explore microbial communities as other authors have concluded in different ecological niches (Iacumin et al., 2009). In relation with DGGE analysis, PCRamplified bands from 16S rDNA gene gave better results than the *rpo*B bands amplification, with eight and three identified species, respectively. Nevertheless, the results obtained with the two genes were again complementary so *Lactobacillus buchneri* was only detected by *rpo*B/PCR-DGGE.

Results about diversity of LAB species found at each year and stage of vinification were very similar to those described above for culture dependent method. The greatest diversity was detected again during AF, opposite to MLF were only two species were present, *Oenococcus oeni* and *Leuconostoc mesenteroides*. The presence of *Leuconostoc mesenteroides* in

2007 at stage 4 confirmed again the possible competition of other LAB with *Oenococcus oeni* and their influence in MLF duration.

\* stage of vinification (1, must; 2, tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF) -; not detected *UB*; uncultured bacterium w; detected with 16S rDNA/DGGE b; detected with *rpo*B/DGGE c; detected in plate after culture ◊; LAB species detected with all the employed methods

**Table 2.** LAB species detected with culture independent techniques (16S rDNA/PCR-DGGE and *rpo*B/PCR-DGGE) and culture dependent methods at each stage of vinification (1, must; 2, tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF) during three vintages.

### **3.3. Strain typing of** *Oenococcus oeni*

146 Electrophoresis

(b)

*rpo*B/PCR-DGGE.

**Figure 3.** Phylogenetic trees generated from the method of 16S rDNA (a) and *rpo*B (b) sequences of recovered bands were inferred using the Maximun Parsimony method (Felsenstein, 1985). Numbers over the branches are bootstrap values (1000 repetitions). *Escherichia coli* and *Listeria inocua* were used as outgroups. Scale bar represents (calculated) distance. Reference strains are closely related to the sequenced bands and accession number of each gene is indicated. Band number isolated from fermenting wines are indicated by the sampled stage of vinification (1, must; 2, tumultuous AF; 3, final AF; 4, initial MLF; 5,

The species identification at each stage of vinification in the three studied years with culture independent techniques (16S rDNA/PCR-DGGE and *rpo*B/PCR-DGGE) and culture

A total of fourteen different LAB species were identified in the three studied vintages by traditional and culture independent methods. PCR-DGGE analysis allowed to identify nine species in comparison to the eight ones detected by culture in plate of the sampled wine. Thus, *Fructobacillus ficulneus*, *Fructobacillus tropaeoli*, *Lactobacillus buchneri*, *Leuconostoc pseudomesenteroides* and *Weissella* were not detected in the employed culture medium, while *Lactobacillus coryniformis*, *Lactobacillus brevis*, *Lactobacillus mali*, *Lactobacillus uvarum* and *Pediococcus parvulus* were not detected by PCR-DGGE. Therefore, results obtained by both methods were complementary and demonstrated the importance of using a combined analytical approach to explore microbial communities as other authors have concluded in different ecological niches (Iacumin et al., 2009). In relation with DGGE analysis, PCRamplified bands from 16S rDNA gene gave better results than the *rpo*B bands amplification, with eight and three identified species, respectively. Nevertheless, the results obtained with the two genes were again complementary so *Lactobacillus buchneri* was only detected by

Results about diversity of LAB species found at each year and stage of vinification were very similar to those described above for culture dependent method. The greatest diversity was detected again during AF, opposite to MLF were only two species were present, *Oenococcus oeni* and *Leuconostoc mesenteroides*. The presence of *Leuconostoc mesenteroides* in

tumultuous MLF; 6, final MLF) the letter of the position in the gel and the isolation year.

dependent method (plating on modified MRS) are recovered in Table 2.

### *3.3.1. PFGE analysis of the strains of this study*

Identification of the *Oenococcus oeni* strains of this study was successfully achieved by PFGE of DNA digested with *Sfi*I and each strain presented a characteristic PFGE pattern. Digestions with *Apa*I enzyme were not more discriminating than *Sfi*I restriction (data not shown). Cluster analysis and visual inspection of the PFGE patterns from the 187 *Oenococcus oeni* isolates recovered from wine fermentations in the three years gave a total number of 37 distinct genotypes (Figure 4). Twenty-four of them were detected in 2006, five in 2007 and fifteen in 2008 vintages (Figure 5). The lower clonal diversity found in 2007 vintage would be related with the low temperatures during MLF (below 12 ºC) what could make few genotypes be able to get adapted to the difficult conditions.

Application of the Different Electrophoresis Techniques to the Detection and Identification of Lactic Acid Bacteria in Wines 149

<sup>36</sup>

Comparing coincident genotypes for the three vintages, it was observed that between the genotypes isolated in 2006 vintage two were found in 2007 (genotypes 18 and 20) and four in 2008 (genotypes 3, 13, 17 and 18). Moreover, two genotypes isolated in 2007 (genotypes 18 and 25) were also detected in 2008 vintage. Only one genotype (18) was identified in the three studied years. The frequency of participation of each genotype varied from year to year, thus dominant genotypes one year were minority or not present at other one which suggested the adaptation of *Oenococcus oeni* strains to the winery conditions every year. Similar results were reported by other authors in studies about bacteria and yeast populations (Gutierrez et al., 1999; Reguant et al., 2005a; Ruiz et al., 2010; Santamaría,

Interestingly, no genotype was isolated in all fermentation stages so fourteen genotypes appeared only at AF (stages 1-3), six were present at all MLF stages (4, 5 and 6) and three of them were also detected at the end of AF. Most fermentation stages showed mixed *Oenococcus oeni* strains populations, which confirmed that several *Oenococcus oeni* strains occurred in a single spontaneous MLF (Lopez et al., 2007; Renouf et al., 2009; Ruiz et al., 2010). The number of different genotypes identified at each stage ranged from 0 to 5, and from 3 to 9, at stages 1-3 and 4-6, respectively. Genotypes 9 and 18 in 2006 vintage, 20 and 26 in 2007, and 13 in 2008 were the predominant ones during MLF. Three out of these genotypes (13, 18 and 20) could be considered as interesting *Oenococcus oeni* strains for the selection of new malolactic starter cultures as individual or mixed strains, because in addition to be dominant in most of the MLF stages at each vintage, they were isolated at

**Figure 5.** Frequency of appearance (%) of *Oenococcus oeni* genotypes at each stage (1, must; 2, tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF) and vintage. Genotypes without color only appeared once, genotypes with grey color appeared only in one year at more than

<sup>26</sup>

123456 3456 123456 **Stage 2006 vintage Stage 2007 vintage Stage 2008 vintage**

25

one stage and genotypes with color appeared in more than one vintage.

2009).

more than one year in quality wines.

<sup>3</sup> <sup>3</sup>

7

<sup>4</sup>

0%

20%

40%

60%

**Appearance (%)**

80%

100%

**Figure 4.** UPGMA dendrogram based on the *Sfi*I PFGE patterns of the 37 *Oenococcus oeni* genotypes. Figure from González-Arenzana et al. (2012a).

Comparing coincident genotypes for the three vintages, it was observed that between the genotypes isolated in 2006 vintage two were found in 2007 (genotypes 18 and 20) and four in 2008 (genotypes 3, 13, 17 and 18). Moreover, two genotypes isolated in 2007 (genotypes 18 and 25) were also detected in 2008 vintage. Only one genotype (18) was identified in the three studied years. The frequency of participation of each genotype varied from year to year, thus dominant genotypes one year were minority or not present at other one which suggested the adaptation of *Oenococcus oeni* strains to the winery conditions every year. Similar results were reported by other authors in studies about bacteria and yeast populations (Gutierrez et al., 1999; Reguant et al., 2005a; Ruiz et al., 2010; Santamaría, 2009).

148 Electrophoresis

shown). Cluster analysis and visual inspection of the PFGE patterns from the 187 *Oenococcus oeni* isolates recovered from wine fermentations in the three years gave a total number of 37 distinct genotypes (Figure 4). Twenty-four of them were detected in 2006, five in 2007 and fifteen in 2008 vintages (Figure 5). The lower clonal diversity found in 2007 vintage would be related with the low temperatures during MLF (below 12 ºC) what could make few

**Figure 4.** UPGMA dendrogram based on the *Sfi*I PFGE patterns of the 37 *Oenococcus oeni* genotypes.

Figure from González-Arenzana et al. (2012a).

genotypes be able to get adapted to the difficult conditions.

% Similarity

Interestingly, no genotype was isolated in all fermentation stages so fourteen genotypes appeared only at AF (stages 1-3), six were present at all MLF stages (4, 5 and 6) and three of them were also detected at the end of AF. Most fermentation stages showed mixed *Oenococcus oeni* strains populations, which confirmed that several *Oenococcus oeni* strains occurred in a single spontaneous MLF (Lopez et al., 2007; Renouf et al., 2009; Ruiz et al., 2010). The number of different genotypes identified at each stage ranged from 0 to 5, and from 3 to 9, at stages 1-3 and 4-6, respectively. Genotypes 9 and 18 in 2006 vintage, 20 and 26 in 2007, and 13 in 2008 were the predominant ones during MLF. Three out of these genotypes (13, 18 and 20) could be considered as interesting *Oenococcus oeni* strains for the selection of new malolactic starter cultures as individual or mixed strains, because in addition to be dominant in most of the MLF stages at each vintage, they were isolated at more than one year in quality wines.

**Figure 5.** Frequency of appearance (%) of *Oenococcus oeni* genotypes at each stage (1, must; 2, tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF) and vintage. Genotypes without color only appeared once, genotypes with grey color appeared only in one year at more than one stage and genotypes with color appeared in more than one vintage.

### *3.3.2. Comparison of the PFGE and RAPD-PCR profiles from the wine fermentation strains and commercial strains*

The thirty-seven genotypes of the indigenous O*enococcus oeni* strains from wine fermentations and fourteen PFGE and RAPD-PCR patterns obtained of strains from several commercial cultures were submitted to comparison by bioinformatics and visual analysis (data not shown). Figure 6 shows that genotypes I, II, III and IV from commercial starter cultures resulted indistinguishable from four indigenous genotypes (23, 13, 21 and 18, respectively), despite commercial malolactic cultures had never been employed in the sampled cellar.

Application of the Different Electrophoresis Techniques to the Detection and Identification of Lactic Acid Bacteria in Wines 151

a future selection process regardless of their oenological properties as malolactic starter

This study has been a contribution to a better description of the LAB ecology along the

The study about the microbial diversity of viable LAB populations showed that the species diversity was higher at the AF stage where eight different species were identified. *Oenococcus oeni* was detected during AF in variable proportions and it became the majority

This work allowed to increase the endogenous strain collection of LAB isolated from fermenting wines of the Appellation of Origin Rioja what meant a contribution to the preservation of biodiversity and wine peculiarity of this region and a starting point for

The analysis of the total LAB populations by culture independent techniques (PCR-DGGE) showed that the species diversity detected along the winemaking process was higher than the one found by the study of viable LAB, identifying up to nine different LAB species. The LAB species variability was also higher at the previous stages to the MLF. Once spontaneous MLF started this variability was greatly reduced, with *Oenococcus oeni* and

The results obtained with culture dependent and independent techniques were complementary so in studies conducted in microbial ecology they both should be used to

PFGE has shown to be a suitable method for strain differentiation, for monitoring individual strains and determining which strains actually survive and carry out MLF. The results of *Oenococcus oeni* typification indicated the high diversity of indigenous *Oenococcus oeni* strains responsible for MLF of the wines of this study and the complexity of the ecology involved in a fermentating wine. The frequency of participation of each genotype varied from year to year, thus dominant genotypes one year were minority or not present at other one, which suggested the adaptation of *Oenococcus oeni* strains to the winery conditions

Several genotypes could be considered as interesting *Oenococcus oeni* strains for the selection of new malolactic starter cultures as individual or mixed strains because, in addition to be isolated at more than one year in quality wines, they were dominant in most of the MLF stages at each vintage. The comparison of the patterns from commercial cultures and the genotypes from indigenous *Oenococcus oeni* strains showed four indistinguishable genotypes. The presence of these four genotypes for one to three years, and in some cases with a high frequency of appearance, demonstrated the significance of this study in order to

cultures.

**4. Conclusion**

future research.

every year.

process of Tempranillo wines winemaking.

*Leuconostoc mesenteroides* as the only detected species.

achieve a broader view of the studied ecosystem.

exclude these genotypes from a future selection process.

species during spontaneous MLF.

**Figure 6.** Consensus dendrogram obtained by combination *Sfi*I-PFGE, *Apa*I-PFGE and M13 RAPD-PCR patterns corresponding to the commercial strains (I to IV) and their respective indistinguishable genotypes from indigenous *Oenococcus oeni* strains from this winery.

These all four autochthonous genotypes were detected in 2006 vintage, year which showed the greatest strain diversity, two of them occurred in 2008 and one in 2007, with an important frequency of appearance in some cases (Table 3).

**Table 3.** *Oenococcus oeni* genotypes indistinguishable to commercial patterns; isolation stage (1, must; 2, tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF) and frequency (%) of their appearance (frequency of appearance (%) = nº of isolates that presented a specific PFGE pattern × 100/total nº of isolates per vintage) at each vintage.

Therefore, despite two of these strains had been previously considered as interesting for the selection of new malolactic starter cultures, the possible identical strain identification with already marketed strains suggested reject these two indigenous *Oenococcus oeni* isolates from a future selection process regardless of their oenological properties as malolactic starter cultures.

### **4. Conclusion**

150 Electrophoresis

sampled cellar.

*strains and commercial strains*

*3.3.2. Comparison of the PFGE and RAPD-PCR profiles from the wine fermentation*

The thirty-seven genotypes of the indigenous O*enococcus oeni* strains from wine fermentations and fourteen PFGE and RAPD-PCR patterns obtained of strains from several commercial cultures were submitted to comparison by bioinformatics and visual analysis (data not shown). Figure 6 shows that genotypes I, II, III and IV from commercial starter cultures resulted indistinguishable from four indigenous genotypes (23, 13, 21 and 18, respectively), despite commercial malolactic cultures had never been employed in the

**Figure 6.** Consensus dendrogram obtained by combination *Sfi*I-PFGE, *Apa*I-PFGE and M13 RAPD-PCR patterns corresponding to the commercial strains (I to IV) and their respective indistinguishable

These all four autochthonous genotypes were detected in 2006 vintage, year which showed the greatest strain diversity, two of them occurred in 2008 and one in 2007, with an

Genotype Isolation stage Frequency of appearance at each vintage (%)

13 4-5-6 2 - 45 18 3-4-5-6 15 14 3 21 6 2 - - 23 6 2 - - **Table 3.** *Oenococcus oeni* genotypes indistinguishable to commercial patterns; isolation stage (1, must; 2, tumultuous AF; 3, final AF; 4, initial MLF; 5, tumultuous MLF; 6, final MLF) and frequency (%) of their appearance (frequency of appearance (%) = nº of isolates that presented a specific PFGE pattern ×

Therefore, despite two of these strains had been previously considered as interesting for the selection of new malolactic starter cultures, the possible identical strain identification with already marketed strains suggested reject these two indigenous *Oenococcus oeni* isolates from

2006 2007 2008

genotypes from indigenous *Oenococcus oeni* strains from this winery.

important frequency of appearance in some cases (Table 3).

100/total nº of isolates per vintage) at each vintage.

This study has been a contribution to a better description of the LAB ecology along the process of Tempranillo wines winemaking.

The study about the microbial diversity of viable LAB populations showed that the species diversity was higher at the AF stage where eight different species were identified. *Oenococcus oeni* was detected during AF in variable proportions and it became the majority species during spontaneous MLF.

This work allowed to increase the endogenous strain collection of LAB isolated from fermenting wines of the Appellation of Origin Rioja what meant a contribution to the preservation of biodiversity and wine peculiarity of this region and a starting point for future research.

The analysis of the total LAB populations by culture independent techniques (PCR-DGGE) showed that the species diversity detected along the winemaking process was higher than the one found by the study of viable LAB, identifying up to nine different LAB species. The LAB species variability was also higher at the previous stages to the MLF. Once spontaneous MLF started this variability was greatly reduced, with *Oenococcus oeni* and *Leuconostoc mesenteroides* as the only detected species.