Characteristic of Lamb Sausages Fermented by Indonesian Meat-Derived Probiotic, Lactobacillus Plantarum IIA-2C12 and Lactobacillus Acidophilus IIA-2B4

Probiotic is a group of microorganism, mainly from lactic acid bacteria (LAB), widely used to increase functionality of various foodstuffs, including lamb which was limited by its goaty odor and short life issue. This study aimed to evaluate the characteristic of lamb sausages fermented by either Lactobacillus plantarum IIA-2C12 or L. acidophilus IIA-2B4 isolated from local cattle in Indonesia, and stored for 21 days at low temperature (4oC). Fermented lamb sausages were made with the addition of L. plantarum IIA-2C12 and L. acidophilus IIA-2B4 with three replications. The result showed that pH value, protein, and cholesterol contents of the sausages with addition of L. acidophilus IIA-2B4 were higher (P<0.05) than that of L. plantarum IIA-2C12. Meanwhile, the sausage fermented with L. plantarum IIA-2C12 had higher titratable acid (TA) value, texture, and the content of fat, carbohydrate, tyrosine, lysine, myristoleic (C14:1), pentadecanoic (C15:0), heneicosanoic (C21:0) and cis-11-eicosatrienoic (C20:1) as compared to that of L. acidophilus 2C12-2B4. Final population of LAB in the sausage fermented by L. plantarum IIA-2C12 was also higher than that of L. acidophilus IIA-2B4, yet both can be categorized as a probiotic. The differences between characteristics of the physicochemical traits and microbiological quality of the sausage fermentation associated with the addition of L. plantarum IIA-2C12 or L. acidophilus IIA-2B4. The 21 days of storage at cold temperatures with probiotics addition could extend shelf life and maintain quality of fermented sausage.


INTRODUCTION
The use of probiotics for various foodstuffs in Indonesia remarkably increased due to the need of functional foods having high nutritional values and health benefits. Probiotics refer to living microorganisms when administered in adequate amounts results in health benefits, which are mostly belong to lactic acid bacteria (LAB) (Al-Sheraji et al., 2013). LAB is usually used as a starter in a controlled fermentation for probiotics purpose. Fermented lamb sausages with the addition of starter culture are considered as manufactured through controlled fermentation. Fermentation aimed to extend the shelf life and to give a distinctive flavor on the characteristics product (Esmaeilzadeh et al., 2013).
Fermented sausages are possibly manufactured through spontaneous fermentation process as shown in Urutan, a pork-based fermented sausage from Bali (Darmayanti et al., 2014). The traditional fermented sausages made spontaneously using natural starter of raw materials during the maturation process for the purpose of adding flavor (Leroy et al., 2006). The probiotic L.
plantarum IIA-2C12 can be used as starter culture of fermented lamb sausage. The physicochemical, microbiology, and sensory properties of fermented lamb sausages with the addition of L. plantarum IIA-2C12 (population 8 log cfu/g) were better than that of control (without probiotic) (Arief et al., 2014).
The probiotic strains used in sausage commonly belong to Lactobacilli and Bifidobacteria that are also present in the intestines (De Vuyst et al., 2008). Bacteria are considered as probiotics when they are able to survive in the digestive tract, which implies their resistances toward acidic conditions and the presence of bile acids (Sunny-Roberts & Knorr, 2008).
Previously, L. plantarum IIA-2C12 and L. plantarum IIA-2B4, isolated from meat of Peranakan Ongole cattle collected from traditional markets proved to be a probiotic LAB (Arief et al., 2015a). Low temperature storage is widely used to prevent pathogenic contamination risk on fermented lamb sausages. Lindqvist & Lindblad (2009) stated that the population of pathogenic bacteria of the fermented sausages decreased during storage at low temperature (refrigerator) for 20 d. Storage at low temperature might increase the length of the adaptation phase of microbial growth that might affect the shelf life of fermented lamb sausages. This study aimed to evaluate the characteristic of lamb sausages fermented by either L. plantarum IIA-2C12 or L. acidophilus IIA-2B4 isolated from local cattle in Indonesia, and stored for 21 d at low temperature (4 o C).

Bacterial Strains
LAB used in this study were L. plantarum IIA-2C12 and L. acidophilus IIA-2B4 isolated from meat of Peranakan Ongole cattle from traditional markets in Indonesia (Arief et al., 2015a). Culture of L. plantarum IIA-2C12 and L. acidophilus IIA-2B4 were growth on media Man Rogoso Sharpe Agar (MRSA) (Oxoid, UK) to determine the initial population. According to Arief et al. (2008), the population of the strains used to ferment lamb sausage were over than 10 8 cfu/mL.

Preparation of Fermented Lamb Sausage
Fermented lamb sausage was prepared by following the recipe of Arief et al. (2014a). As much as 1000 g of lamb meat was ground first and then inserted sequentially with 2% salt, 2% starter culture of 10 8 cfu/mL L. plantarum IIA-2C12 or L. acidophilus IIA-2B4, 0.5% spices nutmeg, 2% sugar, 0.5% ginger, 0.5% black pepper, and 0.5% white pepper. The dough was incorporated into the sausage casing with diameter of 12.5 cm, and then conditioning for 24 h at room temperature (27°C). After that, cool smoking was conducted for 5 h for 2 d with a temperature of 28-30°C. Fermented sausages were made with the addition of L. plantarum IIA-2C12 and L.
acidophilus IIA-2B4 with 3 replications. Fermented sausages were packed with polyethylene and stored at refrigerator with a temperature of 4-8°C. Physicochemical and microbiological analysis were carried out at 0, 7, 14, and 21 h according to the research of Afiyah et al. (2015).

Physicochemical Analysis
The moisture, protein, fat, and ash contents of the sausages were analyzed using proximate analysis (AOAC, 2005). Carbohydrate content was calculated by difference from the proximate analysis. The pH value was analyzed using a hanna instrument pH meter (USA). Water activity (a w ) was determined using an a w meter (Novasino). The measurement was conducted according to manufacturer's manual (Ravyts et al., 2010). Total lactic acid was measured by titratable acidity (Afiyah et al., 2015). Texture was determined using a texture analyzer Steven-LFRA as kg/cm 2 .

Microbiological Analysis
Population of LAB, E. coli, and Salmonella spp on fermented lamb sausages was determined according to AOAC (2005). Fermented lamb sausage was weighed as much as 25 g and put into 225 mL of buffered peptone water (BPW) and homogenized. LAB was grown on media MRSA (Oxoid, UK) and incubated at 37°C for 24-48 h. Meanwhile, the total population of Salmonella spp and E. coli pathogenic bacteria were determined using the Xylose Lysine Deoxycholate Agar (Oxoid, UK) and Eosin Methylen blue Agar (Oxoid, UK), respectively, and incubated at 37 o C for 24-48 h.

Amino Acids Composition
The amino acid composition was determined using the method of High-Performance Liquid Chromatography (HPLC) Agilent Technologies. A total of 3 mg fermented lamb sausage samples were hydrolyzed with acid following the method of Osthoff et al. (2002). A total of 10 mL sample was added with 25 mL of reagent ortoftalaldehida (OPA). A total of 5 mL was injected into the HPLC column and waited until separation of all the amino acids was complete. The time required was approximately 25 min. The concentration of amino acids in samples of fermented sausage can be calculated.

Fatty Acids Composition
The sausages fatty acid composition was analysed according to the AOAC (2005). The fatty acids were methylated by transesterification. The injector and detector temperatures were set at 250°C and 300°C. Helium was used as the carrier gas with a flow rate of 1.0 mL/min. The results were expressed as mg FFA/g of extracted lipid. All the tests were performed in 175 triplicates.

Analysis of Cholesterol
Cholesterol analysis was conducted by using a method by Lieberman-Buchards method (Kleiner & Dotti, 1962). This method is an analysis of the concentration of cholesterol chemically. A total of 0.1 g of sample was put into the centrifuge tube and added 8 mL alcohol hexane : ether alcohol (3:1) and then centrifuged for 10 min at a speed of 3000 rpm. The remaining residue was dissolved using chloroform as it was poured in a tube scale up volume 5 mL, and added with 2 mL of acetic anhydride and 0.2 mL of concentrated H 2 SO 4 . The tube was whirled by using vortex and left in the dark for 15 min. The resulting color was a bluish green color that was read at absorbance with 420 nm wavelength.

Statistical Analysis
Data were expressed as means with standard of error of at least 3 independent experiments. Data on the physicochemical and microbiological properties were statistically analyzed using analysis of variance (ANOVA) under completely randomized design based on Steel & Torrie (1995). Post hoc analysis was performed by Duncan test. Data of physicochemical and microbiological properties during storage were analyzed by using factorial completely randomized design.

RESULTS
The physicochemical properties of fermented sausages were shown in Table 1. Addition of L. plantarum IIA-2C12 or L. acidophilus IIA-2B4 had shown to significantly (P<0.05) contribute to pH values of the sausages. The content (percentage) of titratable acid of fermented sausage with the addition of L. plantarum IIA-2C12 was shown to be higher than that of L. acidophilus IIA-2B4 (P<0.05). This might explain the significant differences (P<0.05) on the effect of addition of probiotics in the sausage textures found in this study, in which the addition of L. plantarum IIA-2C12 produces a texture that was harder than L. acidophilus IIA-2B4.
The addition of L. plantarum IIA-2C12 produced higher ash content of the products than that of L. acidophilus IIA-2B4 (P<0.05) ( Table 1). The protein content with the addition of L. acidophilus IIA-2B4 was significantly (P<0.05) higher than that of L. plantarum IIA-2C12. The carbohydrate content was significantly higher (P<0.05) with the addition of L. plantarum IIA-2C12 than that of L. acidophilus IIA-2B4. The cholesterol value on the product with the addition of L. plantarum IIA-2C12 or L. acidophilus IIA-2B4 showed significantly (P<0.05) lower than that of lamb before processing at 162.67 mg/100g.
Further, Table 1 also displayed microbiological quality of fermented sausage. The population of LAB in fermented sausages had significant difference (P<0.05). Tyrosine and lysine showed significant larger (P<0.05) in the fermented sausage products with the addition of L. plantarum IIA-2C12 than that with L. acidophilus IIA-Note: Means in the same row with different superscripts differ significantly (P<0.05); cfu/g= colony forming unit per gram; na: not analyzed.

Variables
Without probiotic With probiotic Arief et al. (2014) L  Table 2). The fatty acids of myristoleic, pentadecanoic, heneicosanoic, and cis-11-eicosenoic on fermented sausage by the addition of L. plantarum IIA-2C12 were higher than L. acidophilus IIA-2B4 (P<0.05) (Table 3). Microbiological properties of fermented lamb sausages during cold storage can be seen in Table 4. The population of LAB in fermented sausages until the 21 st day after storaging was 8.95 log cfu/g for fermented sausage with the addition of L. plantarum IIA-2C12 and 8.81 log cfu/g for fermented sausage with the addition of L.
acidophilus IIA-2B4 (P<0.05). Physicochemical properties of fermented lamb sausages during cold storage can be seen in Table 4.

DISCUSSION
As expected, the addition of probiotics used in this study significantly reduces pH values (Table 1). This is due to production of lactic acid as end product of fermentation by L. plantarum IIA-2C12 and L. acidophilus IIA-2B4 added in the sausages. The pH values of fermented sausage with the addition of L. plantarum IIA-2C12 and L. acidophilus IIA-2B4 are in the range of pH optimum for BAL to growth (3.5-4.5) and produce antibacterial compounds against pathogenic bacteria. Indeed, Riebroy et al. (2008) stated that low pH value was an important factor in the control of bacteria.
This might be due to the ability of L. plantarum IIA-2C12 to produce acid compound is relatively higher than that of L. acidophillus IIA-2B4. Nevertheless, Afiyah et al. (2015) suggested that meat-based substrate (in this study is lamb) was apparently more favorable for biochemical activities of L. plantarum IIA-2C12. Noteworthy, titratable acid of fermented sausage with the addition of L. plantarum IIA-2C12 was higher from that of lamb sausage with no addition of probiotic culture as reported by Arief et al. (2014a). This is plausible since the more LAB presence in the sausage, more acid compounds are produced. This also indicated that the addition of probiotic cultures can be used for specific purposes.
Further, pH value, as well as lactic acid content, was assumed to have some effect on the product texture as proposed by Seo et al. (2015). This is because of the dependency of protein structure changes (denaturation and gelation) on the pH value. Riebory et al. (2008) clearly showed that apart from water loss, denaturation and gelation of protein in the food systems were indeed related to the texture as it was mainly dealing with shear force or gel strengh.
LAB were known to produce lactic acid associated with the increasing of ash content (Nie et al., 2014). Digestibility of the product with the presence of LAB was mostly found to have higher digestibility as the indegenous proteins were digested into smaller fragments (peptide) by starter LAB (Liu et al., 2011). The differences between the sausages with addition of L.
plantarum IIA-2C12 and L. acidophillus IIA-2B4 reflected the differences on the proteolytic system including activity and specificity.
LAB in both fermented sausages utilized carbohydrates as a source of glucose in fermentation process. The addition of skim milk in fermentation sausage was degraded lactose to glucose and galactose which had Note: Means in the same row with different superscripts differ significantly (P<0.05); lamb meat: not analyzed statistically.

Variables
Lamb meat With probiotic Mean L. plantarum IIA-2C12 L. acidophilus IIA-2B4 Amino acid (w/w) % benefit for brain growth (Zhang et al., 2010). It is interesting that the probiotics used in this study displayed the ability to lower cholesterol concentration. Lye et al. (2010) and Wang et al. (2014) reported that only some of LAB had ability to do so. Tsai et al. (2014) summarized the possible mechanism by which probiotic, including LAB, reduced the cholesterol. Firstly, probiotc may utilise bile-salt hydrolase (BSH) to deconjugate bile salt. Secondly, probiotics may assimilate cholesterol into bacterial cell membrane. Thirdly, probiotics might produce short-chain fatty acids during the growth period. Lastly, cholesterol might be converted into coprostanol. The population of LAB on fermented sausages was 9.24 log cfu/g with the addition of L. plantarum IIA-2C12 and 9.18 log cfu/g with the addition of L. acidophilus IIA-2B4. Total population of LAB in this study met the requirement for being considered as probiotics according to Arief et al. (2014) stated that the population of the strains used to ferment lamb sausage were more than 10 8 cfu/mL. The LAB population of both sausages were higher than LAB population reported by Arief et al. (2008) using L. plantarum 1B1 in fermented lamb sausages. The results of this study indicated that in the production of lamb sausages, the cultures of L. plantarum IIA-2C12 and L. acidophilus IIA-2B4 were considered as better starters to increase the population of LAB. Nie et al. (2014) stated that LAB was the main microorganisms that played a role in the sausage fermentation process.
The population of E. coli in fermented sausage was 0.33 log cfu/g with the addition of L. plantarum IIA-2C12 or L. acidophilus IIA-2B4. According to the MLA (2003), population of E. coli that was acceptable was 3.6 log cfu/g. Salmonella spp was not found in fermented sausages on both products with the addition of L. plantarum IIA-2C12 or L. acidophilus IIA-2B4. L. plantarum produces antimicrobial subtances, called bacteriocin plantaricin, that can inhibit and kill pathogenic bacteria, such as S. aureus (Arief et al., 2015b) and E. coli (Kia et al., 2016;Arief et al., 2013). According to the MLA (2003) and Arief et al. (2014b), with the absence of Salmonella spp, the product was considered to be safe for consumption. Accordingly, fermented lamb sausages in this study can be consumed safety because appropriate with MLA (2003).
Amino acids were formed due to proteolysis activity during fermentation process by LAB. Glutamic is an amino acid component which is dominant on both sausages with the addition of bacteria L. plantarum IIA-2C12 and L. acidophilus IIA-2B4. Xu et al. (2008) suggested that amino acid contributed to the taste of fermentation products, such as glutamic. Protease activity in the meat or the addition of LAB in fermented sausage led to an increase in free amino acids (Candogan et al., 2009). Free amino acids also could affect the flavor of fermented sausages (Nie et al., 2014).
Tyrosine and lysine showed in the fermented sausage products with the addition of L. plantarum IIA-2C12 was larger than that of L. acidophilus IIA-2B4. The higher proteolysis activity of culture yielded, the higher amount of free amino acids released (Aro et al., 2010). et al. (2014) assumed that the indigenous activity of proteolytic enzymes in the raw material used in this study were similar. Hence, the differences in the profiles of some amino acids attributed to the distinct microbial proteolytic activities, including L. plantarum IIA-2C12 and L. acidophilus IIA-2B4. The dynamic of amino acid composition in the product is related to the metabolism process covering both catabolism and anabolism. For instance, the decreasing of some amino acid contents found in this study might be due to a high rate of anabolism in which amino acids were used by LAB for growing. This assumption is supported by Marathe & Ghosh (2009) that stated that microorganisms such as lactic acid bacteria utilized amino acids and vitamins for growth.

Latorre-Moratalla
The sausage with the addition of L. plantarum IIA-2C12 had greater fatty acid component as compared with fermented sausage with the addition of L.
acidophilus IIA-2B4. This difference might be due to the discrepancy on lipolytic activity of probiotics during fermentation. Stearic fatty acid is the dominant component in fermented sausages by the addition of L. acidophilus IIA-2B4 and oleic fatty acid is the dominant component in fermented sausages by the addition of L. plantarum IIA-2C12.
The addition of L. plantarum IIA-2C12 or L. acidophilus IIA-2B4 in lamb meat could detect the content of arachidonic fatty acids. Dhanapal et al. (2012) stated that the content of arachidonic fatty acid increased following the cooking process. Zhang et al. (2010) stated that the oxidation of fat was occurred on fermented lamb sausages and this situation was anticipated by using some seasoning that contained antioxidants to slow down the oxidation of fat. Total population of LAB at the end of storage period is sufficient for probiotic LAB candidates to provide beneficial effects to the body. Parvez et al. (2006) found that probiotics LAB could prevent and had a therapeutic effect against diarrhea, protected against inflammation, and improved the immune system.
The addition of L. plantarum IIA-2C12 or L. acidophilus IIA-2B4 to sausage on storage day-0 found 0.33 log cfu/g of E. coli; while on day-7, 14, and 21, E. coli was not detected. Arief et al. (2015a) stated that L. plantarum IIA-2C12 and L. acidophilus IIA-2B4 had antimicrobial activity and good co-aggregation against pathogenic bacteria E. coli. According to Astawan et al. (2011), the use of culture of L. plantarum 2C12 and L. acidophilus 2B4 could cause a decrease in the number of E. coli populations.
Salmonella spp bacteria was not found in fermented sausages on both of products with the addition of L. plantarum IIA-2C12 or L. acidophilus IIA-2B4 during storage.
De Vuyst et al. (2008) suggested that fermented meat products with certain bacteria could inhibit the growth of pathogenic bacteria. The pH value is associated with the production of lactic acid by LAB. Low pH values generally related to organic acids produced by LAB (Nie et al., 2014).
According to De Vuyst et al. (2008), fermented meat products with certain bacteria could inhibit the growth of pathogenic bacteria, indicated by the decrease of pH due to acidity and low a w . The percentage of lactic acid on both the sausage with the addition of L. plantarum IIA-2C12 or L. acidophilus IIA-2B4 increased in during storage. Arief et al. (2010) stated that the increase of TA was caused by increase of LAB activity that produce lactic acid.

CONCLUSION
Population of L. plantarum IIA-2C12 and L. acidophilus IIA-2B4, isolated from local cattle in Indonesia, in the sausage could be categorized as a probiotic. The differences between the characteristics of physicochemical traits and microbiological quality of the sausage fermentation associated with the addition of L. plantarum IIA-2C12 or L. acidophilus IIA-2B4. The 21 days of storage at cold temperatures with probiotics addition could extend the shelf life and maintain quality of fermented sausage.