The effect of carboxymethyl cellulose (CMC) and banana puree ( Musa acuminata Colla ) addition on the characteristics and microstructure of goat milk yogurt

. Yogurt is one of the fermented products which may have increased functional value. However, yogurt made from goat milk has a weak gel strength and high level of syneresis which may reduce consumer acceptance. The addition of CMC and banana puree was expected to improve the characteristics of yogurt. The aim of this research was to study the effect of the addition of CMC (0.75; 1; and 1.25%) and banana puree (10 and 20%) on the characteristics of goat milk yogurt (viscosity, syneresis, total lactic acid bacteria, total acid, and pH). Further analysis was carried out using Scanning Electron Microscopy (SEM) to study the microstructure of goat milk yogurt in depth. The results showed that the addition of CMC had a significant effect on the viscosity, while the addition of banana puree had a significant effect not only on the viscosity, but also total acid and pH. There was an interaction between these two factors on the syneresis. The best treatment was obtained at the addition of 1% CMC and 20% banana puree with the viscosity of 7991 cP, syneresis of 6.23%, total lactic acid bacteria of 2.4 x 10 8 CFU/mL, total acid of 1.49% and pH of 4.49. The microstructure of best treatment goat milk yogurt had denser, more compact and smaller voids than those of control one.


Introduction
Goat milk production in Indonesia is quite abundant, reaching 370,708 tons in 2020 [1].Goat milk has many advantages over cow milk.These advantages include lower lactose content, higher digestibility, lower allergenicity, and contain some of oligosaccharides [2][3][4][5].However, goat milk also has a weakness such as having an unpleasant goaty flavor due to the high content of short chain fatty acids from goat milk (capric acid, caprylic acid, etc.) and branched chain fatty acids (4-ethyloctanoic acid and 4-methyloctanoic acid) [6].Goat milk can be processed into yoghurt to increase its health value due to the presence of Lactic Acid Bacteria (LAB).But the problem is goat milk yogurt generally has a less compact texture due to the low protein content of α s-1 casein and the intervention fat globules [7] which may reduce the consumer acceptance.
To overcome this problem, various approaches have been studied to improve the texture of goat milk yogurt such as the addition of stabilizers, the addition of milk solids content, the use of specific probiotics and starter cultures that produce exopolysaccharides, highpressure homogenization and the processing of milk with the enzyme transglutaminase, which make it possible to produce goat milk yogurt with acceptable consistency and rheological properties [8].However, the traditional ways such as increasing total solid content and adding various stabilizers remain the most commonly used techniques in modifying the texture of goat milk yogurt [9] CMC can be used to improve the characteristics of goat milk yogurt.CMC is one of hydrocolloid or stabilizer which is easy to find on the market, the production method is simple, efficient, does not require a lot of money and can be produced from waste [10,11].CMC is known to improve the texture of yogurt and reduce syneresis [12,13].
The addition of banana puree also may improve the texture and organoleptic value of goat milk yogurt.Banana production in Indonesia is very high, reaching 874,147 tons in 2021 [14].Banana contains inulin and fructo oligosaccharides (FOS) which can support the growth of LAB in yogurt [15].In addition, inulin is also a type of hydrocolloid that can improve the texture quality of yogurt [16].There are previous studies which state that banana can increase organoleptic values, total LAB, and yogurt viscosity [15,17].Therefore, banana was used in this study to improve the quality of goat milk yogurt, especially in unpleasant goaty flavor.Pisang mas were chosen because this type of banana has a fragrant aroma and sweet taste [18].
There are some previous researches on the addition of CMC and banana puree to improve the texture quality of yogurt, but the addition of CMC was utilized in cow milk yogurt [12,13] and banana puree was added without combination with hydrocolloid in goat milk yogurt [15,17].Based on these backgrounds, the aim of this research was to study the addition of CMC and banana puree on the characteristics of goat milk yogurt including viscosity, syneresis, total lactic acid bacteria, total acid, pH and also microstructure.Microstructural analysis carried on Scanning Electron Microscopy (SEM) to compare the structure of best treatment goat milk yogurt to those of control one which may be correlated to the texture characteristics.

Materials
The main ingredient in preparation of yogurt was Sapera goat milk obtained from goat milk farms in Bumiaji, Batu City.Other ingredients used were CMC (Wealthy), banana (pisang mas) with ripening index 7 obtained from traders in Sawojajar Market, Malang City, and commercial yogurt starter (Biokul Set Yogurt, Diamond).The materials used in the analysis include distilled water, NaOH (Merck, pa), filter paper (Whatman No. 1), phenolphthalein (PP) indicator (Merck, pa), MRSA media (Merck) and peptone (Merck).

Research design
The study was conducted using a factorial randomized block design (RBD Factorial) with two factors.The first factor was the concentration of CMC and the second factor was the concentration of banana puree.The CMC concentration factor consisted of 3 levels, i.e. 0.75; 1; and 1.25%.banana puree concentration factor consisted of 2 levels, i.e. 10 and 20%.Three repetitions were performed for each treatment to obtain 18 experimental units.The research data were analyzed using the Analysis of Variance (ANOVA) method at a 95% confidence interval.Factors that have a significant effect will be tested further using the LSD or DMRT method.The best treatment was determined by the Effectiveness Index Test [19].

Sample preparation
Banana puree was prepared by peeling and then steaming banana for 3 minutes.After that, the bananas were drained to reduce the water on the surface.Then, bananas were mashed using a blender to get the puree.
Yogurt starter was prepared by filtering 150 mL of goat milk and then the filtrate was pasteurized for 10 minutes at 90C.After that, the milk was transferred to a sterile glass jar then added with 6% (w/v) sugar and then homogenized.When the temperature of the milk reached 37-40C, 5% of commercial starter was inoculated.Furthermore, the milk fermented for 24 hours at 37C.After that, the back slopping starter was cooled in the refrigerator.
The preparation of goat milk yogurt was similar to the preparation of yogurt starter, but after the milk was transferred to a sterile glass jar and then added with 6% (w/v) sugar, 0.75; 1; and 1.25% CMC was added then homogenized.After that, 10 and 20% banana puree were added and then homogenized again.When the temperature of the milk reached 37-40C, a 5% back slopping starter was inoculated.Furthermore, the milk fermented for 24 hours at 37C.After that, the yogurt was cooled in the refrigerator to stop the fermentation process.

Determination of viscosity, syneresis, total lactic acid bacteria, total acid and pH
Determination of viscosity used the method described by Gad and Mohamad [12].Yogurt previously cooled in the refrigerator (24 hours; 4C) then kept in the room until it reached room temperature.A viscometer (Elcometer 2300 RV) along with spindle no.4 was prepared and the speed (rpm) of the spindle was set.The viscosity of yogurt was measured in each 30 seconds for 1.5 mins.The average viscosity (cP) was calculated.
Determination of syneresis followed the method described by Kenari and Razavi [20] with slight modifications.Filter paper Whatman No. 1 was prepared on the funnel and placed on the top of Erlenmeyer flask.Thirty grams of sample were placed on filter paper and kept in the refrigerator for 5 hours.The weight of fluid passed through the filter paper and fell into the Erlenmeyer was weighed.The calculation of syneresis was as follows: Total lactic acid bacteria were determined by the plate counting method [21], with slight modification.Peptone solution with dilution level from 10 -1 to 10 -5 was prepared and 1 mL of each peptone solution was pipetted and placed in sterile petri dish.Twelve to fifteen mL of MRSA (temperature ± 45C) was poured into each petri dish and the petri dish was shaken with the movement of forming the number '8' so that the media was homogenized with peptone then left until the substrate solidifies.The petri dish was put upside down into the incubator at 37C for 48 hours.The total colonies that appeared was calculated using the colony counter (Stuart) then the total lactic acid bacteria were obtained using the Standard Plate Count (SPC) formula.
Total acid used the acid-base titration method [22].A 1 g sample was weighed and placed in an Erlenmeyer flask and 9 mL of distilled water was added and mixed.Then 2-3 drops of 1% phenolphthalein indicator solution were added and mixed then titrated with NaOH standard solution (0.1 N).The volume of standard titration solution of NaOH consumed was recorded and used to calculate total acid by substituting the following equation: Total acid (lactic acid) =     0.09    × 100% (2) Where V is the volume of NaOH standard solution consumed during titration (mL), N is the normal concentration of NaOH standard solution (0.1 N), df is dilution factor, and m is mass of sample (g).The pH value was determined by a pH meter (Hanna instrument).

5 Microstructure analysis
Microstructure analysis was followed the procedure described by Pelaes Vital et al. [23] with slight modification.Yogurt samples were frozen in deep freezer (Nuve DF 490) at -80C and lyophilized for 24 h.Samples were mounted on aluminum stubs and coated with a gold layer (Quorum Q150 RS).Observations were made using a scanning electron microscope (SEM) (Fei Quanta FEG 650).

Viscosity
Results showed that increasing the concentration of CMC and banana puree leads to the higher viscosity of yogurt.The two factors had no interaction, so LSD test was performed.The results of the LSD test as the effect of CMC and banana puree concentrations on the viscosity parameters can be seen in Table 1 and Table 2, respectively.In this study the average viscosity was 7277.33 cP for commercial cow milk yogurt (with the addition of stabilizer and gelatin) and 4003.00 cP for control goat milk yogurt.
CMC can act as a stabilizer in yogurt which was indicated by an increase in viscosity [24].The increase in the viscosity of yogurt by CMC was due to the following mechanism.At a pH below its isoelectric point, the amino group of the protein (-NH 2 ) will form a positive charge (-NH 3 + ) through protonation with H 3 O + ions.The CMC group (-COO -) will form an electrostatic force with the proton group of the protein.Small molecules of CMC also form a protective layer on the surface of the caseins which can prevent the repulsive forces from the positive groups of the protein, so that casein precipitation increases with visible aggregates causing the texture of the yogurt to become thick [25].Meanwhile, banana puree increases viscosity by increasing total solids [15].Bananas contain inulin which can act as a hydrocolloid and interact further with casein micelles through hydrogen bonds and increase the total solids of yogurt which increases in viscosity [26][27][28].

Syneresis
Results showed that increasing the concentration of CMC and banana puree results in lower syneresis.The two factors have interactions so they were further tested using the DMRT.The result can be seen in Table 3. From this study, the average syneresis of commercial cow milk yogurt was 6.04% and for control goat milk yogurt was 22.75%.
The interaction that occurs between CMC and banana puree was related to the binding capacity of CMC to banana puree.According to Kumalasari et al. [29] who stated that there was an interaction between CMC and fruit puree in increasing product stability.CMC can bind fruit puree molecules to a certain concentration, preventing the puree from precipitating so that the product was more stable.Bananas contain quite high water (72%) and CMC can bind the water in these bananas [30].It is possible that this mechanism also occured in this study, where CMC can stabilize banana puree by binding the water in the puree, making it more stable and incorporating it into goat milk yogurt.
CMC and banana puree showed a synergistic effect in reducing yogurt syneresis by increasing water holding capacity.CMC act as a stabilizer that build a three-dimensional gel structure in yogurt to trapping water in this structure [13].Bananas contain dietary fiber which can bind water up to 5.8 mL/gram [31].Inulin and FOS are included in prebiotic which have the ability to reconstruct water, act as thickeners, and can form complexes with yogurt protein aggregates through hydrogen bonds [32].The addition of CMC and banana puree also increase the total yogurt solids [24,33].The high total solids of yogurt can result in decreased syneresis [34].

Total lactic acid bacteria
Total lactic acid bacteria in goat milk yogurt as the effect of the addition of CMC and banana puree are shown in Table 4.In this study, total lactic acid bacteria of commercial cow milk yogurt and control goat milk yogurt were 1.8 x 10 7 and 2.16 x 10 7 , respectively.From the Table 6, it can be concluded that yogurt with the addition of a higher concentration of banana puree produced a higher total LAB.Banana contains many sources of nutrients needed for the growth of lactic acid bacteria including various types of amino acids, the most dominant are lysine (1.16%), glutamic acid (1.1%), tryptophan (0.92%), and leucine (0.72%), as well as fatty acids including palmitic fatty acid (74.33-95.45mg/100g), linoleic acid (22.39-55.14mg/100g), and linolenic acid (23.53-42.22mg/100g) [35,36].In addition, the presence of prebiotic can also increase the growth of LAB.Banana is one of the fruits that contain lots of prebiotics in the form of dietary fiber such as oligosaccharides and inulin.Ripe bananas contain dietary fiber of 3.69 g/100g [37].According to Pongmalai & Devahastin [38], the content of oligosaccharides and inulin in bananas is strongly influenced by fruit ripeness.This can be affected by the higher amount of sucrose as banana maturity increases.Sucrose is the basic molecule of fructan so the higher sucrose in bananas can produce more fructan molecules (in this case the inulin and fructooligosaccharide types).The increase in total sucrose and 1-ketose also resulted in higher inulin content in bananas.Gallo et al. [39] presented that puree from ripe bananas has a higher sugar content than unripe bananas because fructans increase as bananas ripen.The amount of lactic acid in all yogurt samples complied with SNI 2981:2009 with a minimum of 10 7 CFU/mL.

Total acid
Results showed that increasing the concentration of banana puree causes the total acid of yogurt to be higher.Meanwhile, the CMC concentration factor did not significantly affect the acidity of yogurt.The results of total acid as the effect of banana puree and CMC addition are shown in Table 5 and Table 6.From this study, it was found that the total acid of commercial cow milk yogurt and control goat milk yogurt was 1.494%.
Table 5.Average total acid of yogurt as the effect of banana puree addition.

Concentration of banana puree (%)
Total Acid (%) 10 0.747 ± 0.00 b 20 1.494 ± 0.00 a Fruits that naturally have a low pH contain various types of organic acids, which will increase the total acid in yogurt [33].Banana puree can affect total acid of yogurt because of its prebiotic effect.Bananas contain the prebiotic inulin of 2.1% [26].Inulin content can accumulate more as banana maturity increases [38].Inulin can support the growth of lactic acid bacteria during the fermentation process.In addition, bananas also provide another source of energy for lactic acid bacteria in the form of amino acids, sugars, and several organic acids.The more lactic acid bacteria grow in yogurt, the more lactic acid is produced [40].Meanwhile, goat milk yogurt with the addition of 10% banana puree produced lower total acid than the control.This can be influenced by many factors, including the effect of CMC addition.Although the CMC concentration did not significantly affect the total acidity of goat milk yogurt.Research of [41] showed that the effect of CMC addition could inhibit lactic acid production even though these factors were not significantly different.CMC can act as an inhibitor in the production of lactic acid.The presence of a viscous gel that was formed makes the mobility of the reactants more difficult, so it can affect the metabolism of lactic acid bacteria which results in a decrease in the amount of lactic acid produced at the end of the fermentation [24].

pH
From this study it was found that the pH of commercial cow milk yogurt and control goat milk yogurt were 4.66 and 4.60, respectively.Increasing the concentration banana puree result in the pH of the yogurt to decrease so this factor was tested further.Meanwhile, the CMC concentration factor had no significant effect on the pH of yogurt.The results of pH of yogurt as the effect of banana puree and CMC addition are shown in Table 7 and Table 8.
The bananas used are ripe bananas which contain a lot of sugar in the form of sucrose.Although the main substrate utilized by lactic acid bacteria is lactose, but sucrose can also act as a source of energy for LAB in metabolism [42].Bananas also contain various nutrients such as fatty acids and amino acids which are a source of nutrition for lactic acid bacteria [35,36].The presence of inulin in bananas also contributes to increasing the activity of lactic acid bacteria so that more acid was produced and results in a decrease in the pH of the yogurt.Inulin in bananas acts as a prebiotic which provides nitrogen and carbon for the growth of lactic acid bacteria [16].Table 7.Average pH of yogurt as the effect of banana puree addition.The data of each parameter was further processed to obtain the best treatment from all goat milk yogurt samples.The method used for decision making was Effectiveness Index Test [19].The first step was to determine the order of priority of the parameters.In this study, the parameters prioritized were viscosity and syneresis then pH and total acid.Then, each parameter was assigned a score or weight based on its order.After that, the best reference value for each parameter was determined.The worst value of each parameter was also determined and the value was obtained from the inverse of the best value and then these two values were subtracted to get the difference.The determination of the best order, weight, and reference value can be seen in Table 9.The effectiveness value (EV) of each sample in each parameter was calculated by subtracting the worst value by the value in the sample divided by the difference.After obtaining the EV of each parameter, the Treatment Value (TV) is calculated by multiplying EV with the weight of the parameter.The best treatment was obtained at the sample with the highest TV.From the calculation of the Effectiveness Index Test method, the best treatment was obtained at goat milk yogurt with the addition of 1% CMC and 20% banana puree.The results of the best treatment order can be seen in Table 10.

Microstructure
From the previous parameter analysis, the best treatment was found in goat milk yogurt samples with the addition of 1% of CMC and 20% of banana puree.The best treatment was analysed for its microstructure by SEM at 500x and 2500x magnification.The microstructure of best treatment and control goat milk yogurt can be seen in Fig. 1.
In the control samples, it can be seen that there are a lot of voids with a large size and the network structure looks weak and less compact.Meanwhile, in the best treatment goat milk yogurt sample, the structure was denser, tighter, and had fewer voids than those in control sample.The addition of hydrocolloid leads to the reduced voids of the yogurt microstructure and the protein network become more compact.Hydrocolloids can intervene in the casein network, causing the yogurt gel to become denser [43].Packekrepapol et al [44] stated that the presence of a negative group of polysaccharides can bind to the positive group of casein in milk gel, resulting in a stronger electrostatic force.The same mechanism can occur in this study where CMC has a negative group (COO -) that can bind to a positive protein group (NH 3 + ) [13].Meanwhile, the reduced voids in the yogurt microstructure indicate that there are fewer liquid phases resulting in lower syneresis [43].

Conclusions
The best treatment for goat milk yogurt was obtained at the addition of 1% CMC and 20% banana puree with the viscosity of 7991 cP, syneresis of 6.23%, total lactic acid bacteria counted as 2.4 x 10 8 CFU/mL, total acid of 1.49%, and pH of 4.49.The results of the SEM analysis showed that the microstructure of best treatment yogurt had a tighter, more compact yogurt structure, and smaller voids than those of control one which may be correlated to the increase in gel firmness and lower syneresis due to the addition of CMC and banana puree.

Fig. 1 .
Fig. 1.Scanning Electron Microscopy of Control Goat Milk Yogurt (a, b) and Best Treatment Goat Milk Yogurt (c, d) and with Magnification at 500x (left) and 2500x (right).Arrow showed the void.

Table 1 .
Average viscosity of goat milk yogurt as the effect of CMC addition.

Table 2 .
Average viscosity of goat milk yogurt as the effect of banana puree addition.

Table 3 .
Syneresis of goat milk yogurt as the effect of CMC and banana puree addition

Table 4 .
Total Lactic Acid Bacteria of goat milk yogurt as the effect of CMC and banana puree addition.

Table 6 .
Average total acid of yogurt as the effect of CMC addition.

Table 8 .
Average pH of yogurt as the effect of CMC addition.

Tabel 9 .
The best reference value for each goat milk yogurt parameter.

Table 10 .
Best treatment order of goat milk yogurt.