Abstract: The research aimed to determine the Minimum Inhibitory Concentration (MIC) of L. plantarum supernatant in inhibiting the growth of Staphylococcus aureus ATCC 25923, determine the nutrients levels of cow’s milk dangke and sensory quality after the addition of cow’s milk fat 1 and 2% and determine the effect of the additional of L. plantarum supernatant against the growth of S. aureus ATCC 25923. Minimum inhibitory concentration is determined based on the lowest concentration of the supernatant that shown no growth in the media. This study used a Completely Randomized Design (CRD) with 2×2 factorial. Data of the growth of pathogenic bacteria was analyzed by ANOVA test and then continued by using Duncan test. The paired comparison test were used to determine sensory quality of cow’s milk dangke and then analyzed by Wilcoxon Signed Rank test. This experiment proved that L. plantarum upernatant could inhibit the growth of S. aureus TCC 25923 at VJA medium with MIC 10% but not shown inhibiting the growth of S. aureus ATCC 25923 in dangke medium. The addition of cow’s milk fat 1 and 2% into cow’s milk dangke able to increase the fat content dangke although not equal with buffalo’s milk dangke. The addition of cow’s milk fat 1 and 2% into cow’s milk dangke was able to improve the flavor of cow’s milk dangke to be equivalent with buffalo’s milk dangke. The addition of cow’s milk fat 2% into dangke could improve the aroma of cow’s milk dangke which equivalent to buffalo’s milk dangke.
INTRODUCTION
Dangke is a traditional food in Enrekang, South Sulawesi which made from buffalo’s milk. Dangke is made by heating the milk, clotting and furtherly packaged using banana leaf. Hatta et al. (2013) reported that dangke made from buffalo’s milk has more savory taste, smoother texture, softer and not sticky when ingested compared to cow’s milk. This could be due to higher fat content contained in buffalo’s milk. Buckle et al. (2007) reported that fat in buffalo’s milk is twice higher than cow’s milk.
Now, the market of dangke is not only in South Sulawesi but also marketed to Kalimantan, Jakarta, Papua, Malaysia and some other places where Enrekang society live in (Baba et al., 2012). One of obstacles faced in marketing dangke is the shelf-life which is relatively very short. Dangke contains of pH 6.4 which generally can be stored only for two days at room temperature and 5-7 days at refrigerator temperature (Hatta et al., 2013).
Contamination of dangke may occur due to the presence of pathogenic bacteria such as Staphylococcus aureus. Large amount of S. aureus is found in milk and its processing products including cheese (Nusrat et al., 2015). It was reported that 105-108 CFU mL–1 of S. aureus may produce enterotoxin (Kamal et al., 2013; Anunciacao et al., 1995). Enterotoxin can resistance for 30 min at 110°C. Enterotoxin causes intoxication with symptoms of nausea, vomiting, abdominal cramps and diarrhoea (Nusrat et al., 2015). According to Indonesian National Standard (Standar Nasional Indonesia) No. 7388 (SNI., 2009) regarding with cheese inspection, the maximum level of S. aureus is 1×102 colony per gram. Jayuska et al. (2014) reported that filtrate from Lactobacillus fermentation can inhibit the growth of pathogenic bacteria such as Streptococcus sp., S. aureus and Escherichia coli. The antibacterial capability of Lactobacillus filtrate doesn’t change even it had been stored for 6 months. The addition of lactic acid in cheese manufacturing process is able to increase the coagulant activity (Carvalho et al., 2007).
This research was designed as an effort to improve the sensory quality of dangke produced from cow’s milk with the addition of 1 and 2% cow’s milk fat so that it has flavor and texture be equivalent to buffalo’s milk dangke, to determine the nutrient of dangke after addition of cow’s milk fat, to determine the Minimum Inhibitory Concentration (MIC) of L. plantarum in inhibiting the growth of S. aureus ATCC 25923, and to study the influence of L. plantarum and cow’s milk fat added into dangke in inhibiting the growth of S. aureus ATCC 25923.
MATERIALS AND METHODS
This research was carried out at the Laboratory of Kesmavet, Faculty of Veterinary and Laboratory of Organoleptic, Faculty of Farms (FAPET) in Bogor Agricultural University (IPB). Staphylococcus aureus ATCC 25923 collected by Laboratory of Kesmavet, Department IPHK, FKH-IPB and L. plantarum collected by Department PAU UGM were selected as the tested pathogenic bacteria. Culture media and material used in this research were Nutrient Broth/NB, Mueller-Hinton Broth/MHB, de man Rogosa and Sharpe Broth/MRSB, Vogel Johnson Agar/VJA (Oxoid, Oxoid Ltd., Basingstoke, United Kingdom) and API 50 CHL carbohydrate fermentation strips (bioMérieux, Inc., Marcy l'Etoile, France). Other materials used were: Cow’s milk, buffalo’s milk, papaya’s latex, banana’s leaf and coconut’s shell as mold. Nine semi-skilled panelists were used for sensory quality test (Dzarnisa, 1999; Jinjarak et al., 2006).
Lactobacillus plantarum isolate was confirmed by Gram staining, catalase test and API 50 CHL test to obtain pure culture. An identified L. plantarum isolate was inoculated into 100 mL MRSB and incubated at 37°C for 24 h (Usmiati and Marwati, 2007). Cell separation was performed by centrifugation at 10,000 rpm (rotation per minute) for 15 min. Supernatant was sieved using filter membrane 0.2 μm and furtherly stored at 4°C as stock.
Determination of Minimum Inhibitory Concentration (MIC): Determination of MIC was conducted by dilution method (Sari et al., 2010). A total of 5 mL tested solution containing of NB, L. plantarum supernatant and 106 CFU mL–1 of pathogenic bacteria were placed into tube. Tested solution was made in several supernatant concentrations i.e., 0, 10, 20, 30, 40, 50, 60, 70, 80 and 90% and then incubated at 37°C for 24 h. Observation was performed in order to identify the bacteria growth which indicated by the presence of muddy condition at tested solution. About 0.1 mL was taken from each concentration of tested solution transferred into specific media for each bacteria type and incubated at 37°C for 24 h. The colony of bacteria was counted. The concentration with no growth of tested bacteria was defined as the Minimum Inhibitory Concentration (MIC) of L. plantarum supernatant.
Manufacturing of dangke: Cream was added into cow’s milk until milk fat content was 1 and 2% (g/v) of a total milk volume. Fat content in cream was determined by Kieferle and Charlotte method (Sudarwanto, 2012). Milk was then heated with small fire until the temperature reached ±70°C. Curd process was performed by adding papaya’s latex during milk heating process. After reaching boiling stage, milk was cooled for 30 min and during this cooling process L. plantarum was added into solution. The next stage was to sieve the curd and inserted it into coconut shell mold and pressed used spoon to separate curd from whey. Dangke was then packed with banana’s leaf.
Dangke proximate analysis: Proximate analysis was performed to determine the water content by gravimetric analysis, ash content by gravimetric analysis, fat content by hydrolysis-Soxhlet method, protein content by Kjeldahl micro (SNI., 1992; Geantaresa and Supriyanti, 2010), carbohydrate content (by difference) and pH (SNI., 1998; Rahayu et al., 2011).
Number of colonies of tested bacteria: Number of colonies was expressed as Colony Forming Unit (CFU) per gram or per milliliter or certain wide of sample (cm2) (Haryadi et al., 2013). Counting was carried out in accordance with the Standard Plate Count (SPC) method. Counting of S. aureus ATCC 25923 was conducted by pour plate method using VJA medium.
Sensory analysis: Sensory analysis to evaluate dangke quality including color, texture, aroma and flavor was conducted. Dangke produced from buffalo’s milk was used as comparison in this experiment. Sensory attributes were tested using paired comparison (Setyaningsih et al., 2010). Sensory test used 9 sensitive semi-skilled panelists (Dzarnisa, 1999; Jinjarak et al., 2006).
Data analysis: A Completely Randomized Design (CRD) with 2×2 factorials in time was applied in this experiment. The growth of pathogenic bacteria was analyzed by ANOVA test and followed by Duncan test if significance different occurred. Organoleptic data resulted from paired comparison test was analyzed by Wilcoxon Signed Rank.
RESULTS AND DISCUSSION
According to re-identification result on L. plantarum and S. aureus ATCC 25923, homogeny and pure colonies were found. Gram staining process against L. plantarum revealed that isolate was Gram-positive with a rod-shaped form, catalase-negative and could grow well at MRS agar medium. Identification using API test of 50 CHL system showed that the isolate was L. plantarum with validation of 99.9%. Identification against L. plantarum using API was also performed by Anas et al. (2014).
| Table 1: | Proximate analysis result of cow’s milk dangke with the addition of L. plantarum and cow’s milk fat 0, 1 and 2% |
| DSK: Buffalo’s milk dangke, DSSL0: Cow’s milk dangke, DSSL1: Cow’s milk dangke+cow’s milk fat 1%, DSSL2: Cow’s milk dangke+cow’s milk fat 2% | |
Lactobacillus plantarum isolate was known having good adaptability in high acidity environment. Acid condition was caused by accumulation of lactic acid produced by bacteria during fermentation in a homo-fermentation type. This acid product is furtherly used as the active compound to preserve food (Askari et al., 2012). Cisarova et al. (2009) reported that acid which produced by L. plantarum is not only able to inhibit Gram-negative bacteria such as clostridium and enterobacteriaceae but also inhibit Gram-positive bacteria such as S. aureus.
Minimum inhibitory concentration of L. plantarum supernatant: During observation for 24 h, the growth of S. aureus ATCC 25923 was only found at tube 1 (0%) with the presence of muddy. In VJA medium, the growth of S. aureus ATCC 25923 was found at petri dish containing of tube 1 (0% concentration of L. plantarum supernatant) which indicated by black color colonies surrounded by metallic light yellow zone. All petri dish containing of tube 2-10 (L. plantarum supernatant with various concentration) showed no growth of S. aureus ATCC 25923. Thus, it can be indicated that the minimum inhibitory concentration of S. aureus ATCC 25923 was 10%.
Nutrient content of cow’s milk dangke: The nutrient content of dangke measured in this experiment was: Water content, ash content, fat content, protein content, carbohydrate content and pH (Table 1).
Water and ash content: Water content is very important factor in determining cheese texture. The increasing water content will soften the texture. Dangke is one type of soft cheese as the water content is higher than 40% (Buckle et al., 2007). In this study, the water content of dangke is ±59%. The water content of dangke is influenced by the concentration of papaya’s latex. Diouf et al. (2012) stated that increasing concentration of papaya’s latex will reduce the water content of dangke. The concentration of papaya’s latex used in this research was 0.4% to avoid strong bitter taste when it is consumed (Yuniwati et al., 2008). Alkaloid carpaine in papaya’s latex will produce bitter taste in dangke.
The water content of buffalo milk dangke was the lowest among the four types dangke (45.65%). The results reported in accordance with Murtaza et al. (2014) which shows that the buffalo’s milk cheese has a lower water content than cow's milk cheese. The water content is influenced by the volume of solids dissolved in milk. The greater volume of dissolved solids in milk, the water content is getting smaller. Buffalo’s milk dangke has the greatest of total solids followed by cow's milk dangke plus milk fat 1%, cow's milk dangke plus cow’s milk fat 2% and last cow's milk dangke without adding cow’s milk fat. Solids constituent materials in milk include fats, proteins, carbohydrates and minerals. Buffalo milk contains a higher fat and protein than cow's milk (Murtaza et al., 2014; Salman et al., 2014). Therefore the forming curd from buffalo milk was more when clotting protein by papain.
Fat content: The highest fat content found in buffalo’s milk dangke (32.82%). This is caused by protein content of buffalo milk is higher than cow milk. Daulay (1991) stated that protein located in the outer layer of the fat globules membrane. The higher protein content in milk causes the milk fatmore bounded and retained in the curd. In addition, the size of fat globules and micellar casein buffalo milk is also greater than cow milk. Fat globule size of buffalo milk and cow milk respectively 5:05 and 3:55 μm and the casein micelles size of buffalo milk and cow's milk, respectively 190 and 180 μm (Hussain et al., 2012). This allows the fat out of buffalo milk curd is less than cow milk curd.
The addition of cow’s milk fat was confirmed could increase fat content of dangke. This is shown by the increasing of fat content dangke after addition of 1 and 2% cow’s milk fat. The lowest fat content found in cow’s milk dangke without adding milk fat (17:31%). Dangke with addition of fat 2% was confirmed to have lower fat content compared with dangke with addition of cow’s milk fat 1%. It is caused by fat particles out of the curd while heating. According to Abd El-Gawad and Ahmed (2011), fat content of cheese is influenced by the heating duration time and protein denaturation process. There is possibility that fat moves out from cheese during heating process at temperature above 80°C. Therefore, higher heating temperature or longer heating process will increase the possibility of fat moves out from curd and dissolved in whey.
Protein content: The highest protein content found in buffalo’s milk dangke (19.23%). This is caused by protein content of buffalo milk is higher than cow milk, so curd formed was more. Cow’s milk dangke with 2% cow’s milk fat has the lowest protein content (13.19%), while dangke without adding milk fat has 15.42% protein content. This revealed that addition of cow’s milk fat did not increase the protein content of dangke. Formed-curd is closely related with the coagulation process of milk protein to curd. This stage is the most difficult process to be controlled due to complex interaction which occurs among milk quality, heating temperature, milk volume, solid volume, pH, coagulant type, number and coagulation duration (Syah, 2012).
Carbohydrate content: The highest levels of carbohydrate found in cow’s milk dangke without addition of cow’s milk fat (4.68%), followed by cow’s milk dangke plus cow’s milk fat 2%, cow’s milk dangke with cow’s milk fat 1% and buffalo’s milk dangke (respectively 3.14, 0.62 and 0.53%). The results showed that overall buffalo’s milk dangke has a lower carbohydrate than cow’s milk dangke. The results reported in accordance with Barlowska et al. (2011) that the lactose content of milk cow (Bos taurus) higher than buffalo milk (Babalus bubalis), respectively 4.82 and 4.79%. But it is not in accordance with the results reported Murtaza et al. (2014) that the lactose content in buffalo’s milk cheese is higher than cow's milk cheese, (respectively 12.16 and 12.13%). Salman et al. (2014) and Hussain et al. (2012) reported that the lactose content of cow milk is only slightly higher than buffalo milk.
Lactose is a type of carbohydrate that provides a sense of sweet milk that is also called milk sugar. Most lactose left in the whey during curd formation. The lactose approximately 98% dissolved in whey and curd still contains 0.8-1.5% of lactose. Based on the results of measurements of the levels of carbohydrates in this study, it was found the addition of cow’s milk fat 1% and into dangke 2% did not show elevated levels of lactose. Dangke lactose content is related directly to the water content. If the water content of dangke is high, lactose content is high too.
pH: All types of dangke the range of pH close to neutral. The highest pH found in buffalo’s milk dangke (6.65). Although the processing of cow’s milk dangke added with L. plantarum supernatant (pH was 3.8) but the milk is able to maintain the pH conditions so there is no significant decline. That is caused by natural buffer compounds (phosphate, citric and protein) dissolved in milk.
Influence of addition of L. plantarum supernatant and cow’s milk fat (1 and 2%) on the colonies number of S. aureus ATCC 25923: Colonies number of S. aureus ATCC 25923 as resulted from counting (Table 2 and Fig. 1) was then analyzed with Anova test (alpha 5%).
| Fig. 1: | Interaction between the addition of L. plantarum supernatant with incubation time of dangke on number colonies of S. aureus ATCC 25923 (Log CFU) |
| Fig. 2: | Interaction between fat addition with dangke incubation time on number of colonies of S. aureus ATCC 25923 (Log CFU) |
| Table 2: | Interaction between addition of L. plantarum supernatant with incubation time on the number of S. aureus ATCC 25923 colonies (Log CFU) |
| A,B,C,D,EDifferent superscript at the same row indicates significant different at alpha 5% (Anova test) | |
| Table 3: | Interaction between fat content with incubation time on the number of S. aureus ATCC 25923 colonies (Log CFU) |
| A,B,C,D,EDifferent superscript at the same row indicates significant different at alpha 5% (Anova test) | |
The analysis result showed that significant factors which influence the number of bacteria colonies were: Addition of L. plantarum supernatant, incubation time (day), interaction between addition of L. plantarum supernatant with the time and addition of cow’s milk fat with the time. However, factors which not influence the number of bacteria colonies were: addition of cow’s milk fat (1 and 2%), interaction between addition of L. plantarum supernatant with addition of cow’s milk fat 1 and 2% and interaction between addition of L. plantarum supernatant and cow’s milk fat with the time.
The result of Duncan test on the effect of L. plantarum supernatant showed that addition of L. plantarum supernatant produced larger number of L. plantarum colonies. This indicated that addition of L. plantarum supernatant couldn’t inhibit the growth of S. aureus ATCC 25923 in dangke. Based on in vitro observation (stage 1), the addition of L. plantarum supernatant could inhibit the growth of S. aureus ATCC 25923 with concentration was 10% at NB medium. However, this observation hasn’t yet provided similar effect at dangke medium. This different inhibitory capability could be due to different pH of those two mediums. At medium NB, pH solution closed to pH supernatant i.e., 3.8. This acid condition is able to break down bacteria membrane and creates acid condition at cell environment leading to H+ ion moves from bacteria cell. The changes of pH of the bacteria cell leads to metabolic disorder and cell mortality. Immersion of dangke in L. plantarum supernatant caused small changes of pH from 6.5 to 6.1 (Table 1). Milk contain several compounds such as calcium, phosphate, citrate and protein are dissolved which role as natural buffer. This also had been reported by Carvalho et al. (2007) that soft cheese has pH 6.5 even with the addition of lactic acid during processing. Based on the observation, dangke with pH ±6 allowed S. aureus ATCC 25923 experienced optimum growth. Radovanovic and Katic (2009) reported that at skim milk medium, growth of L. plantarum only changes pH from 6.63 to 6.52. That increasing pH hasn’t yet inhibited the growth of S. aureus at skim milk. Fang and Liu (2002) also reported that antimicrobe capability of several lactic acid bacteria (L. acidophilus, L. bulgaricus, L. casei and Streptococcus thermophilus) against S. aureus decreases when the medium is near to neutral pH.
Based on the Duncan test, the interaction between the addition of cow’s milk fat and time on the growth of tested bacteria showed that addition of 1% cow’s milk fat supported higher growth of S. aureus ATCC 25923 at 2nd, 4th and 6th days compared to addition of fat 2% (Table 3 and Fig. 2). This indicated that dangke with fat 1% gave higher support on the growth of S. aureus ATCC 25923 compared to addition of cow’s milk fat 2%. Proximate test resulted that dangke with cow’s milk fat 1% had higher protein content than addition of cow’s milk fat 2%. Harris et al. (2002) reported that S. aureus can grow well in a medium with high nitrogen as organic nutrient source.
| Table 4: | Paired comparison test result |
| alpha 5% (Wilcoxon Signed Rank test), 103: Cow’s milk dangke+cow’s milk fat 1%, 234: Cow’s milk dangke+cow’s milk fat 2% | |
| Table 5: | Sensory paired comparison test score between cow’s milk dangke and buffalo’s milk dangke |
Dangke paired comparison test result
Color: The result of Wilcoxon Signed Rank on the color of cow’s milk dangke added with cow’s milk fat 1% (103) and 2% (234) showed no significant different (Table 4). The average value of dangke 103 and 234 was -1.1 (-1) and -0.9 (-1), respectively which showed that cow’s milk dangke was less white than buffalo milk dangke (Table 5).
White color on dangke is produced from light dispersion of fat, protein and mineral granules in milk. Milk casein evenly deflects the entire color of milk so it turns white. According to Hussain et al. (2012), casein content in buffalo’s milk is higher than cow’s milk i.e., 3.82 and 2.64%, respectively. Besides, cow’s milk has higher β-carotene compared to buffalo’s milk. Therefore, the color of cow’s milk is more yellowish.
Aroma: The result of Wilcoxon Signed Rank on the aroma of cow’s milk dangke added with cow’s milk fat 1% (103) and 2% (234) showed no significant different (Table 4). The average value of dangke 103 was -0.6 (-1). That number indicated that cow’s milk dangke added with cow’s milk fat 1% had less distinctive milk flavor than buffalo’s milk dangke. The average value of dangke 234 was -0.4 (0) (Table 5). This indicated that aroma of cow’s milk dangke added with cow’s milk fat 2% was not significantly different with buffalo’s milk dangke.
Milk aroma is influenced by volatile compound content. According to Friedrich and Acree (2015), buffalo milk contains of indole, nonanal and 1-oktentriol compound which make the aroma is stronger than cow’s milk. However, milk heating process changes the aroma profile. Volatile compound disappears after milk processed into cheese. Volatile compounds found in various types of cheese are 1-oktentrion, metional and 3-metilbutanal.
Texture: The result of Wilcoxon Signed Rank on the texture of cow’s milk dangke added with cow’s milk fat 1% (103) and 2% (234) showed no significant different (Table 4). The average value of dangke 103 and 234 was -0.8 (-1) and -0.6 (-1) (Table 5). This indicated that the texture of those two dangke was less chewy compared to buffalo’s milk dangke. Hussain et al. (2012) reported that micrograph detection finds that the curd texture from buffalo’s milk is thicker with denser porous compared to cow’s milk. This could be due to fat and protein content of buffalo’s milk is higher than cow’s milk leading to higher curd. Besides, the globule size of fat and micellar casein of buffalo’s milk is larger than cow’s milk i.e., 5.05 and 3.55 μm; 190 and 180 μm, respectively.
Flavor: The result of Wilcoxon Signed Rank on the flavor of cow’s milk dangke added with cow’s milk fat 1% (103) and 2% (234) showed no significant different (Table 4). The average value of dangke 103 was 0.2 (0) (Table 5). This indicated that the flavor of cow’s milk dangke was not significantly different with buffalo’s milk dangke. The average value of dangke 234 was 0.1 (0). This indicated that the flavor of cow’s milk dangke was not significantly different with buffalo’s milk dangke. Flavor of dangke is closely related with the nutrient content. Sweet flavor of dangke is produced from lactose and salty flavor is derived from chloride, citrate and other mineral salts (Buckle et al., 2007). A combination between sweet and salty flavor produces savory taste on dangke.
CONCLUSION
This experiment proved that L. plantarum supernatant could inhibit the growth of S. aureus ATCC 25923 at VJA medium with MIC 10% but couldn’t inhibit the growth of S. aureus ATCC 25923 at dangke medium. The addition of cow’s milk fat 1 and 2% into cow's milk dangke able to increase the fat content dangke although not equal with buffalo’s milk dangke.
The addition of cow’s milk fat 1 and 2% into cow’s milk dangke was able to improve the flavor of cow’s milk dangke to be equivalent with buffalo’s milk dangke. The addition of cow’s milk fat 2% into cow’s milk dangke could improve the aroma of cow’s milk dangke which equivalent to buffalo’s milk dangke. The addition of cow’s milk fat 1 and 2% into cow’s milk dangke couldn’t improve the quality of color and texture of cow’s milk dangke.