Proteolytic Characterization of Trimmed Beef Fermented Sausages Inoculated by Indonesian Probiotics: Lactobacillus plantarum IIA-2C12 and Lactobacillus acidophilus IIA-2B4

Proteolysis is one of the most important biochemical changes affecting proteins during the ripening and preservation of fermented beef sausages. In this study, proteolytic activities of two Indonesian probiotics, Lactobacillus plantarum IIA-2C12 and Lactobacillus acidophilus IIA-2B4 used as starters in trimmed beef are investigated. L. plantarum IIA-2C12 and L. acidophilus displayed remarkable proteolytic activities against milk casein substrate, in which the activity of L. plantarum IIA-2C12 is higher than that of L. acidophilus IIA-2B4. Similar evidences were observed when proteolytic activities of both strains were visualized by using SDS-Page against meat sarcoplasmic proteins. The differences in the number of proteases encoded by the genomes of both starters might account for these differences. The activities of both strains were slightly reduced upon storage at room temperature for 28 days due to decreasing of the amount of substrate and or stability of proteases. In addition, we found also that the sausage inoculated by L. acidophilus IIA-2B4 tends to produce more aromatic amino acids than that of L. plantarum IIA-2C12. This might differently contribute to flavor (especially aroma) of both sausages. Altogether, this is, to our knowledge, first evidences for the proteolytic activity of L. acidophilus strain towards muscle proteins during sausage fermentation.


INTRODUCTION
Sausage is not only known as the oldest and most enduring form of processed meat, but also the most popular meat product in the world (Morgan and Tallard, 2006).In Indonesia, sausage is getting famous as one of meat product consumed to meet protein requirements.In 2012, the production of sausage in Indonesia is keeping increasing up to 30% with market value about USD 250 million.
Introduction fermentation in sausage manufacturing is aimed not only to increase shelf life of the sausages, but also taste and flavor development (Esmaeilzadeh et al., 2013).Fermented sausages are traditionally prepared from seasoned, raw meat that is stuffed in casings and allow fermentation process for ripening and maturation.Lactic acid bacteria (LAB) are commonly used as starters for the fermentation as it may improve safety and quality of the final product (Leroy et al., 2006).Beef commercial cuts are mainly used for fermented sausages, however non-commercial parts also promising to be used as raw material for the sausages.Trimmed beef, the non-commercial part obtained from remaining meat attached in the bone after deboning or cutting to have desired commercial parts, nutritionally is similar to beef commercial cuts (Mastuti, 2008).Trimmed beef can be easily obtained from slaughtering house and considered as waste product.Given these advantages of trimmed beef, however, the usage of trimmed beef remains limited.
Previously, two Indonesian probiotics, Lactobacillus plantarum IIA-2C12 and Lactobacillus plantarum IIA-2B4, were isolated from beef obtained from Indonesian cattle, Peranakan Ongole (Arief et al., 2015).As local probiotic, both lactobacilli strains are tought to display some advantages including adaptability to local environment so that the application of these strains does not need any additional modifications (Astawan et al., 2012).These bacteria had met requirements to be classified as probiotic (Arief et al., 2014).In addition, these bacteria also had displayed remarkable ability to prevent EPEC-causing diarrhea (Arief et al., 2010) and repair the hematology condition of diarrheal suspected rats (Astawan et al., 2011).
During fermentation, proteolysis is considerably the most important reaction (Candogan and Acton, 2001).It influenced both texture and flavor development that contribute to the overall quality of fermented sausages.The reaction involves fragmentation of protein by internal proteases in the raw material (indigenous proteases) and external proteases secreted out by bacteria used as starter (Fadda et al., 1999).It yields low molecular weight of protein (peptides) and their derivatives that contribute to formation of specific aroma, taste characteristic and meat tenderization.
Despite LAB is considered to exhibit weak proteolytic activity; however some of LAB have been reported to exhibit strong protease activity in fermented sausages (Nie et al., 2014).The proteolytic activities of LAB during fermentation is part of their attempts to obtain the energy from protein breakdown and also to utilize free amino acids for their growth.Unfortunately, comprehensive study on characterization of proteolytic activity of some LAB members in fermented sausages is limited.L. plantarum strains have been reported to exhibit strong proteolytic activity towards meat proteins (Fadda et al., 2002), however there is no report, to our knowledge, for the activity of L. acidophilus strains.
In this research, we use trimmed beef as raw material for the sausage fermentation, with the addition of two Indonesian probiotics, L. plantarum IIA-2C12 and L. acidophilus IIA-2B4 as starters.The objective of this study was to determine proteolysis activity of both strains towards muscle sarcoplasmic proteins.For the first time, we found that proteolytic activity of L. plantarum IIA-2C12 toward muscle sarcoplasmic proteins is significantly higher than that of L. acidophilus IIA-2B4.Some possibilities account for this difference are discussed in this study.

MATERIALS AND METHODS
Bacterial strains: LAB used in this study were Lactobacillus plantarum IIA-2C12 and Lactobacillus acidophilus IIA-2B4 isolated from Indonesian local beef, Peranakan Ongole breeds (Arief et al., 2015).To be used as a starter for sausage fermentation, the population of these strains should be more than 10 8 cfu/mL.To confirm, initial population of these strains were checked based on Arief et al. (2008).Briefly these strains were cultured in MRS broth (Man Ragosa Sharpe) at 37°C for 24 h.This culture then plated on MRS agar at 37°C for overnight.The population was determined based on number of colonies appear after overnight incubation.
Preparation of beef fermented sausage: Beef fermented sausage was prepared following the recipe of Arief et al. (2014).The sausage formulation consisted of 3 kg of meat with addition of 2.0% sugar, 2.0% common salt, 0.5% white pepper, 0.5% ginger, 0.5% nutmeg and 0.5% lime leaves.These materials were mixed in meat cutter and 2% of L. plantarum IIA-2C12 and L. acidophilus 2B4 (with population more than 10 8 cfu/mL) was added.The cocktail was then filled in 12.5 cm-diameter casing prior to 24 h conditioning to allow initial fermentation.Ripening was then performed through cold smoking at 27-30°C for 3 days, 4 h each.The sausages were prepared for two treatments and three replications each.
Total lactic acid bacteria analysis: Population of LAB on the sausages was determined according to AOAC (2005).Briefly, 25 g of sausages was aseptically transferred into 225 mL of buffered peptone water.The mixture was poured in MRS agar (Oxoid) and LAB colonies were calculated after 48 h incubation at 37°C.

pH and titratable acid measurement:
To measure pH values of the sausages, pH meter (Hanna Instrument, USA) was used.For this purpose, 50 g fermented beef sausages was as for titratable acid, 5 g of sausages were homogenized in 250 mL of distilled water.The initial pH was measured by pH meter (Hanna Instrument, USA) and titration was performed by drop-wise of 0.1 N NaOH until the solution reaches neutral pH.Titratable acid was then calculated based on the following formula: Extraction of sarcoplasmic proteins: Sarcoplasmic protein were extracted from the sausages based on Mauriello et al. (2002) with slight modifications.Briefly, 1 g of sausages were diluted 1:10 (w/v) with 20 mM phosphate buffer pH 6.5 and homogenized for 3 min followed by a centrifugation at 13 000 g for 20 min at 4°C.The supernatant (sarcoplasmic fraction) was sterilized by filtration 0.22 µm and stored at 4°C for further analysis.

Detection of proteolytic activity by agar plate method (Mauriello et al., 2002):
Proteolytic activity of the strains used for the fermentation was visually determined by agar plate method according to Mauriello et al. (2002).Sarcoplasmic extracts standardized at 5 mg protein/mL, were added at concentration of 1 mg/mL to an agar medium consisting of tryptone 0.5%, yeast extract 0.25%, glucose 0.1%, agar 1.5%, pH 6.9 and then poured into petri dishes.
After solidification of the medium, 70 µL of a 24 h culture broth of each strain were pipette in to wells (6 mm) borer in agar plate.Plates were incubated at 37°C

Analysis of proteolysis by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE):
To investigate proteolytic activity of both strains during the fermentation 12% SDS-PAGE was employed.For this purpose, sarcoplasmic proteins were extracted from the sausages as described by Mauriello et al. (2002).
Each sample was mixed with an equal volume of a sample buffer containing 2% of SDS, 5% of βmercaptoethanol, 10% of glycerol, 0.05% of bromophenol blue (as front marker), in 0.5% mmol l-1 Tris-HCl pH 6.8 and heated for 5 min at 90°C.Electrophoresis was carried out in a Mini Protean II (Biorad) at 220 V until bromophenol blue marker reached the bottom of the gel.Gels were subsequently stained for 1 h in a 0.1% (w/v) Brilliant Blue R in methanol: acetic acid: water (40: 10: 50) and then destined for 2 h.
Protein composition analysis: Amino acids composition were determined following the highperformance liquid chromatography.Sample preparation and extraction were performed according to Osthoff et al. (2002).HPLC analysis was performed by using liquid chromatography (Agilent Technologies).Five micro liters of samples were injected into the HPLC column and wait until complete separation of all amino acids, it takes run about 30 min.Work on stage OPA reagent addition until complete separation of amino acids is done automatically.The nitrogen content of total protein was determinated by the Kjeldahl method (AOAC, 2005).

Statistical analysis:
Factorial-based complete randomized design was used as experimental design.Data was then analyzed by Analysis of Variance (ANOVA) with Tukey test as post-hoc test (Steel and Torrie, 1995).

Survival of lactic acid bacteria in the sausages:
Population of LAB of the sausages 28 days room temperature storage is relatively stable (Fig. 1).Despite the population has tendency to increase until D14 storage and then slightly reduces up to D28, statistical analysis showed that these differences are not significant (p>0.05).In this study, we believe that the population of LAB are mostly dominated, if not all, by the strains used as fermentation starter: L. plantarum IIA-2C12 and L. acidophilus IIA-2B4.To be considered as a viable probiotic in food, the minimum LAB population is 10 8 cfu/mL (Arief et al., 2010).Figure 1 remarkably shown that the populations of LAB in both sausages during storage are higher than 10 9 cfu/mL indicating LAB in both sausages are acceptable to be considered as probiotic, in term of population.
Despite the population of LAB in both sausages are not significantly changed upon storage, population of LAB in L. plantarum IIA-2C12 fermented sausage was significantly higher than that of Lactobacillus acidophilus IIA-2B4.Johansson et al. (1993) reported that L. plantarum frequently predominant in lactic acid fermented foods.It might be due to adaptability of L. plantarum to many different conditions (Kleerebezem and Quadri, 2001).
The survival of LAB in both sausages can be monitored by the production of organic acids (mainly lactic acid) as final product of their fermentation activities.Figure 2 showed that pH and titratable acid of both sausages are not significantly different during 28 days storage at room temperature.This result is consistent with growth rate of LAB in the sausage (Fig. 1).Aro Aro et al. (2010) reported that high acidification rates usually accompanied by fast lactic acid bacteria growth rates in fermented beef sausages.pH and titratable acid are associated with the organic acid produced by LAB during fermentation and/or storage.Therefore, these parameters might indicate metabolism activity, especially in term of ability to produce lactic acid, of both strains used in the sausages.The pH values of both sausages are considerably low (less than 5) which provides uncomfortable environment for growing of pathogenic bacteria.Low pH is an important factor in the control of pathogenic and spoilage bacteria (Riebroy et al., 2008).Noteworthy, titratable acid of both sausages were higher than that of reported by Nie et al. (2014) that using L. plantarum ZY40 in grass carp sausages.It is plausible, as differences in the strain and raw materials used for sausage production might affect acidification rate.
Consistent with the population of LAB in plantarum-fermented sausages, pH values on this sausage also lower than that of acidohipllus-fermented sausages.The higher population of LAB in plantarumfermented sausage might promote production of more organic acid rather than in acidophilus-fermented sausage.In addition, L. plantarum IIA-2C12 had a better acidification capacity than fermented beef sausages with L. acidophilus IIA-2B4.
Proteolytic activity: By using agar plate method, L. plantarum IIA-2C12 and L. acidophilus IIA-2B4 demonstrated remarkable proteolytic activity towards casein as a substrate (Fig. 3).Proteolysis activity of both strains represents hydrolysis of casein which is indicated by a zone of clearing around the growing colony.Casein, milk, or gelatins are used to test the proteolysis activity of microorganism isolated from food (Mauriello et al., 2002).The clear zone formed by L. plantarum IIA-1C12 is significantly wider than that of L. acidophilus IIA-2B4 indicating that L. plantarum IIA-2C12 exhibited higher protease activity than L. acidophilus IIA-2B4 against casein.
Given the results of casein-containing agar plate and SDS-PAGE profiles, it is apparently L. plantarum IIA-2C12 exhibited higher proteolytic activity than L. acidophilus IIA-2B4.Strong proteolytic activity of L. plantarum IIA-2C12 towards sarcoplasmic proteins is in agreement with previous reports showing that L. plantarum strain, together with L. sake, L. casei, L. curvatus and L. pentsus, exhibited strong proteolytic activity towards muscle proteins (Castellano et al., 2012;Fadda et al., 1999;Sanz et al., 1999;Sriphochanart and Skolpap, 2010).The difference of proteolytic activities of L. plantarum IIA-2C12 and L. acidophilus IIA-2B4 is might be due to differences in the number of proteases enzyme encoded by their genomes.Mostly, among all strains of L. plantarum and L. plantarum that have been genomicaly sequenced and stored at gene bank, L. plantarum strains have more proteases (19-24 proteases) proteins compared to that of L. acidophilus (10)(11)(12)(13)(14)(15)(16)(17).
Amino acids such as aspartic acid, isoleucine and lysine significantly changes during room storage.Other amino acids like glutamic acid, serine, histidine, threonine, arginine, tyrosine, cystine, valine, methionine, phenylalanine, leucine, alanine and proline does not change significantly during storage on days 0, 7, 14, 21 and 28, respectively.Concentration of aspartic acid, isoleucine and lysine increased during storage.This is consistent with research Aro Aro et al. (2010) who also showed changes in aspartic acid and lysine in ripening up to 21 days.Nie et al. (2014) stated that the addition of L. plantarum ZY40 in fermented beef sausages effectively increase free amino acids in fermented fish sausage.The existence of L. plantarum ZY40 showed high level isoleucine and lysine.Nie et al. (2014) stated that the peptidase of L. plantarum ZY40 along with muscle aminopeptidase responsible for the increase of free amino acids from the N amino terminus acids of the protein muscle.
Table 2 shows that there is a change of amino acid composition in fermented beef sausages with Lactobacillus acidophilus IIA-2B4 in amino acid leucine, alanine and proline.Other amino acids did not change significantly during storage.Increasing the total of this amino acid showed a proteolytic activity of Lactobacillus acidophilus IIA-2B4.Proteolytic activity is breakdown of proteins into compounds such simpler amino acids by means of hydrolysis is carried out by microbial proteases (Poedjiadi, 1994).Hristova et al. (2006) found that Lactobacillus acidophilus has a good proteolytic activity with value 380 AU/mL.
Proteolysis during storage shows an increase of total amino acids composition.Proteolysis or degradation of protein into small peptides is combined action of endogenous cathepsins as well as microbial proteases and aminopeptidases (Latorre-Moratalla et al., 2014).On this case, the common raw meat origin has an identical endogenous enzyme activity.Therefore, the differences in the evolution of some amino acid among product would be partially atrributed to the distinct microbial enzymatic activity, such as Lactobacillus plantarum IIA-2C12 and Lactobacillus acidophilus IIA-2B4.

CONCLUSION
In the present study, two fermented beef sausages were prepared with Lactobacillus plantarum IIA-2C12 and Lactobacillus acidophilus IIA-2B4.Both strains displayed remarkable proteolytic activity toward casein and sarcoplasmic proteins as subsrrates.Proteolytic activity of L. plantarum IIA-2C12 is apparently higher than that of L. acidophilus IIA-2B4 which might be due to the differences number of proteases encoded by their genomes.In addition, the sausage inoculated by L. acidophilus IIA-2B4 produces more aromatic amino acid which might yield difference flavor formation compared to L. plantarum's sausage.

Fig. 2 :
Fig. 2: Changes of pH and titratable acid concent of the sausages with L. plantarum IIA-2C12 and L. acidophilus IIA-2B4 during storage

Table 1 :
Proteolysis activity of L. plantarum IIA-2C12 and L. acidophilus IIA-2B4 (mm) on fermented beef sausages during room storage Day of storage b -

:
Fig. 4: SDS-PAGE profile of sarcoplasmic proteins throughout the preservation of fermented beef sausages with L. plantarum IIA-2C12

Table 2 :
Amino acid composition (mg/100 g of samples) fermented beef sausages with L. plantarum IIA-2C12 during room storage