Characterization of Caulerpa racemosa yogurt processed using Lactobacillus bulgaricus and Streptococcus thermophilus

Lactic acid bacteria such as Lactobacillus bulgaricus and Streptococcus thermophilus in yogurt maintains the balance of the intestinal microflora by suppressing the growth of harmful bacteria. Dietary fibre and functional compounds in probiotic drinks also have a good effect on health. The modification of low - fat probiotic drinks is a value - added product and can be classified as a healthy drink. Caulerpa racemosa is a Chlorophyceae seaweed with high food fibre and functional compounds, including phenolic and chlorophyll as antioxidants. This study aimed to determine the effect of different lactic acid bacteria on the characteristics of the probiotic drink products by combining C. racemosa with low - fat cow ’ s milk. C. racemosa and low - fat cow ’ s milk with a ratio of 3:4 was fermented with Lactobacillus bulgaricus (A), Streptococcus thermophilus (B), and a combination of both (C). The products were analysed for protein and fat content, dietary fibre, total phenolic content, antioxidants activity, total lactic acid bacteria, total acid, pH, and with a sensory test for consumer preference. The addition of the different lactic acid bacteria had effects on the fat, antioxidants, dietary fibre, and total acid contents, while protein and total phenolic contents were not significantly different. The combination of L. bulgaricus and S. thermophilus produced a probiotic drink with the lowest amount of dietary fibre (1.27%) and total acid (0.64%), with antioxidant activity IC 50 of 183.57 ppm and total phenolic content of 0.11 mg GAE/g. The preference test showed that the panellists preferred yogurt fermented with L. bulgaricus and S. thermophilus for its aroma, texture, and colour.


Introduction
Caulerpa racemosa, also known as sea grape, is a green macroalga seaweed that, for human consumption, is usually served fresh or with salt (Sihono et al., 2018).C. racemosa contains bioactive components such as protein and polysaccharides, and secondary phenolic and flavonoid metabolites, namely 19.72% protein, 7.65% fat, and 11.51% fibre.It has a bright green colour (Bhuiyan et al., 2016;Sihono et al., 2018;Yap et al., 2019) and is known to have antioxidant properties.However, C. racemosa in food products is often either uncommon or sub-optimally used.Pires et al. (2013) found that polysaccharides contained in C. racemosa function as a catalyst for phospholipase enzymes and have pharmaceutical potential.As such, C. racemosa may be applied to the development of healthy foods.
Yogurt is a fermented drink known for its substantial health benefits, particularly in preventing digestive problems, boosting immunity, and increasing lactose tolerance for those who lactose intolerant.It is healthy because bacteria make it easily digestible, create a unique matrix that forms bioavailability and metabolic properties.It is satiating and good for weight control (Weerathilake et al., 2014;Rizzoli and Biver, 2017).The health benefits are what drive consumer demand, and the nutritional composition is what consumers consider first.As such, turnoffs for health-conscious consumers are fat and sugar contents, many consumers will prefer yogurt with low sugar and fat content (Miklavec et al., 2014).
Yogurt owes its healthy characteristics to bacteria that convert lactose into lactic acid, giving it a unique texture and flavour.Yogurt fermentation with lactic acid bacteria consists of glycolysis, proteolysis, and lipolysis and produces a sour taste and aroma.However, the fermentation and the formation of lactic acid are inherently linked to the quality of the bacteria (Obi et al., 2016).Lactobacillus bulgaricus and Streptococcus  (Corrieu and Beal, 2016;Chen et al., 2017).Agu et al. (2014), Rahman et al. (2016), andSarvari et al. (2014) have already studied the characteristics of yogurt made with combination of L. bulgaricus and S. thermophilus.However, research on yogurt made exclusively with these lactic acid bacteria individually is rarely conducted.Therefore, this is a novel study comparing yogurt made with either a combination of L. bulgaricus and S. thermophilus or individually.
Yogurt is generally produced with cow's milk as the main ingredient.C. racemosa is added to make it rich in antioxidants and dietary fibre, also giving it a green colour.The phenolic components and secondary metabolites in C. racemosa survive the heating and cooling process during production (Sihono et al., 2018;Nurjanah et al., 2019).Currently, C. racemosa is rarely used as a raw material besides biscuits (Kumar et al., 2017) and jelly candy (Tapotubun et al., 2018).It is not yet used in healthy drinks such as yogurt.It was assumed that the nutrient content in C. racemosa degrades during the fermentation process when different lactic acid bacteria are included.The goal of this study was to determine the characteristics of low-fat cow's milk made with C. racemosa and fermented with different types of lactic acid bacteria.

Materials
Ultra's low-fat cow's milk was obtained from local supermarkets in Semarang, Indonesia.Fresh C. racemosa was harvested in Jepara from the northern Central Java Sea, Indonesia.The commercial strains of lactic acid bacteria Lactobacillus bulgaricus FNCC 0041 and Streptococcus thermophilus FNCC 0040 were purchased from The Central of Food Research and Nutrition, Gadjah Mada University, Yogyakarta, Indonesia.

Microbial culture preparation
Lactobacillus bulgaricus and S. thermophilus were cultured by inoculating a loop of L. bulgaricus and S. thermophilus in 15 mL MRS agar media (Oxoid), followed by incubation at 37℃ for 72 hrs.The inoculum was made by transferring a loop of pure culture bacteria in a 9 mL milk solution incubated at 37℃ for 24 hrs.

Caulerpa racemosa preparation
Fresh C. racemosa was washed and cut into pieces, then chopped, mashed, and blended using a blender (Maspion, Indonesia) until homogenous.The ratio of C. racemosa to water was 1:9.

Yogurt preparation
Yogurt was produced according to Undugoda and Nilmini (2019) with some modifications.A mixture of approximately 30% C. racemosa, 40% low-fat milk, 5% sucrose, and 20% water was pasteurized using the HTST method at 72℃ for 15 mins.The mixture's temperature was then lowered and 5% L. bulgaricus (A), 5% S. thermophilus (B), and a mixture of 2.5% L. bulgaricus and 2.5% S. thermophilus (C) were added.Each treatment was then fermented for 6 hrs at 37℃.Finally, the yogurt was placed into glass bottles, covered with aluminium foil, and stored at 5℃.

Fat analysis
The analysis was carried out according to AOAC (1992).Briefly, a 1.5 mL of NH 4 OH was added to 10 g of the sample and subsequently mixed until becoming homogeneous to remove casein.Next, 10 mL of 95% alcohol was added and the mixture was shaken 15 times.The resulting mixture was added to 25 mL of ethyl ether, shaken for 1 min, then 25 mL of petroleum was added and shaken again for 1 min.Finally, the sample was centrifuged at 600 rpm for 30 secs to separate the ether and water phases (pink coloured) and then dried at ±100℃ for 30 mins in a vacuum oven at 70-75℃ for 7 mins until a constant weight was measured with the formula:

Protein analysis
The 2 g of the sample was poured into a Kjeldahl flask and 5 g of K 2 SO 4 , 200 mg of CuSO 4 , and 30 mL of concentrated H 2 SO 4 were added, homogenized, and distilled.The distillate was filled with a standard solution of 0.1 N HCl in 50 mL and three drops of 1% phenolphthalein indicator were added and titrated with a 0.1 N standard NaOH solution until a constant pink colour appeared (AOAC, 2006).The protein assay was done with the formula:

Dietary analysis
A 0.5 g sample was combined with 0.1 mL of an alpha-amylase enzyme and heated and stirred at 100℃ for 15 min.The sample was then cooled and 20 mL of distilled water and 5 mL of 1 N HCl were added.Next, eISSN: 2550-2166 © 2021 The Authors.Published by Rynnye Lyan Resources FULL PAPER 1 mL of 1% pepsin enzyme was added and the mixture was warmed in a water bath for 1 hr.After 1 hr, 5 mL of 1 N NaOH and 0.1 mL of a beta-amylase enzyme were added and again incubated in a water bath for 1 hr.Next, the sample was washed with ethanol and acetone, dried in an oven at 105℃ for 24 hrs, cooled in a desiccator, and weighed to determine the insoluble dietary fibre weight.The volume was adjusted to 100 mL of filtrate and 400 mL of 95% warm ethanol was added.The filtrate was allowed to settle for 1 hr after which it was filtered with ash-free filter paper and washed again with ethanol and acetone.The filtrate was then dried in an oven at 105℃ for 24 h and finally placed in a desiccator to weigh the dissolved dietary fibre.

Total phenolic content analysis
The total phenolic assay was performed using the Folin-Ciocalteau method and the gallic acid standard.The data obtained were expressed in milligrams of gallic acid equivalent to 1 g of dry extract (Ivanov and Dimitrova, 2019).

Antioxidant activity analysis
Antioxidant activity was analysed following the DPPH radical scavenging method and expressed as an IC 50 value.The sample was prepared in several concentrations.Each concentration consisted of 0.1 mL added with 5 mL of DPPH solution (0.00039 g in 1 L of methanol), followed by homogenization.The mixtures were then incubated at room temperature in a dark room for 30 mins.The colour of the mixture would change due to the reaction with the DPPH reagent.The samples were then spectrophotometrically measured at a 517 nm wavelength.The inhibition values obtained were then calculated as the IC 50 value (Srivastava et al., 2015).

Total lactic acid bacteria
The total lactic acid assay was performed using MRS agar with a pour plate method.The 1 mL sample was mixed with a 9 mL of sterile 0.85% NaCl solution and homogenized for 1 min, to make a dilution 10 -1 .Concurrently, similar sample dilution solutions (aliquots) were made for series 10 -2 -10 -7 .Samples of 1 mL were taken from each dilution series and placed onto sterile Petri dishes, followed by the addition of 10 mL of MRS agar warmed to 45℃.Homogenization was done by shifting the Petri dish to form a figure eight.After the media solidified, the plates were turned upside down and incubated at 37℃ for 48 hrs.The bacteria colonies were counted with a colony counter based on the appearance of clear zones with specific shapes (Daroonpunt et al., 2016).

pH analysis
The pH analysis was performed with a Mettler Toledo (Singapore) pH meter calibrated with a standard buffer solution of pH 4 and pH 7 for 15-30 mins.The electrodes were then rinsed with distilled water and dried.A sample of 5 mL was immersed in electrodes for the pH measurement.The electrodes were left until stable data were obtained and the results were recorded directly from the pH meter display (Ly et al., 2020).

Total acid analysis
A 10 mL sample was poured into an Erlenmeyer tube and three drops of 1% phenolphthalein were added.The sample was then titrated with a standardized 0.1 N NaOH solution, giving it a pink colour.The total acid was calculated with the formula:

The preference tests
The preference test was based on 30 semi-trained panellists (Ademosun et al., 2019) and based on their preferred colour, aroma, texture, and flavour of the yogurt.A five-point scale were used: 1 -dislike, 2moderate dislike, 3 -neutral, 4 -moderate like, 5 -like.

Statistical analysis
This study was conducted as a triplicate and analysed using ANOVA.If significant discrepancies were found, Tukey's HSD test would be carried out.Data were processed with SPSS 23.

Protein
All yogurts with C. racemosa had a protein content of 2.35-2.60%,as shown in Table 1, similar to the 2.35% protein content of commercial yogurt.The treatment of lactic acid bacteria on fermentation did not affect the protein content of the yogurt, which concurred with the findings reported by other researchers (Obi et al., 2016;Tenea and Suarez, 2020), who combined lactic acid bacteria L. bulgaricus and S. thermophilus with L. bulgaricus and L. fermentum.During the fermentation process, proteins may break into lactic acids and nonnitrogen proteins, making them undetectable by the ntotal protein method.It indicated that yogurt with C. racemosa had higher protein contents compared to yogurt moringa leaf fermented with L. bulgaricus (Rahmawati, 2017) In this phase, S. thermophilus uses more oxygen, thus supporting L. bulgaricus unable to grow at high oxygen conditions.In the transition phase, S. thermophilus slows due to the lack of free amino acids.In this phase, S. thermophilus cannot break down milk proteins to produce amino acids, so the role of L. bulgaricus is significant.L. bulgaricus can break down milk protein to produce amino acids that can support the growth of S. thermophilus in the second exponential phase (Sieuwert, 2016).The process leads to lower protein value in yogurt fermented with the combination of L. bulgaricus and S. thermophilus than the other treatments even though it is not statistically significant.

Fat
Table 1 shows that yogurt in this study contained fat ranging from 0.45 to 0.60%, which is lower than the 0.87% in commercial yogurt.The low-fat content in yogurts A, B, and C was due to the use of low-fat milk as the raw material.These results are in accordance with Singh et al. (2016) who reported that the fat content of low-fat yogurt with added fruit extracts ranges from 0.44 to 1.6%.Yogurts B and C contained less fat than yogurt A because lipolysis by lactic acid bacteria during the fermentation process converted fat into fatty acids, decreasing the fat content.The lactic acid bacteria strains used affected the outcome, especially S. thermophilus which plays a role in the long-chain fatty acid formation, and Lactobacillus requiring a longer time to degrade it (Widyastuti et al., 2014;Sieuwert, 2016;Chen et al., 2017).Thus, yogurt fermented with S. thermophilus contained less fat than with L. bulgaricus.

Dietary fibre
Table 1 shows the different lactic acid bacteria affect the dietary fibre content of yogurt.In this study, yogurt A had a higher dietary fibre content than yogurts B, C, and commercial yogurt.Lactobacillus bulgaricus is a superior starter culture for the fermentation of carbohydrates into dietary fibre than the other bacteria used in this study.Sorensen et al. (2016) demonstrated L. bulgaricus and S. thermophilus's inability to ferment sugars, mainly glucose and galactose.Its combination produces low glucose, and lactic acid bacteria alone can optimize their activity in fermenting lactose.During the fermentation process, pH decreased due to organic acid formation, mainly in the form of lactic and acetic acids.An increase in organic acid may lead to the activation of endogenous enzymes that play a role in the degradation process of biopolymer components.The fermentation process may also activate the β-glucosidase enzyme, which breaks down starch into glucose and dextrin.Finally, lactic acid bacteria may activate extracellular enzymes, such as amylase and pullulanase, which can break down starch into oligosaccharides and undigested starch components (Sari et al., 2017;Tsafrakidou et al., 2020).C. racemosa is a source of carbohydrate and contains 48.97% total carbohydrate and 11.51% fibre content (Bhuiyan et al., 2016).The low dietary fibre content of yogurt is presumably related to the combination of two starter cultures, such as S. thermophilus and L. bulgaricus.Tsafrakidou et al. (2020) reported that two Gram-positive thermophilic organisms did not properly ferment sugar into a simple compound.Dietary fibre is good for our health and especially beneficial to the digestive system.The consumption of dietary fibres is not linked to increased blood sugar levels, so it can be consumed without increasing the risk of contracting diabetes mellitus (Tsafrakidou et al., 2020).Furthermore, Beretta et al. (2018) noted that dietary fibre consumption reduces blood sugar levels in patients with diabetes mellitus type one.

Total phenolic content
Table 1 shows the total phenolic content in yogurt with added C. racemosa as a raw material ranged from 0.10 to 0.11 mg GAE/g, higher than the 0.04 mg GAE/g of commercial yogurt.Yap et al. (2019) explained that C. racemosa extracted with methanol has a total phenolic content of 10.33 mg GAE/g.According to Taneva and Zlatev (2020) and Adebo and Medina-Meza (2020), goji berries yogurt had high total phenolic contents.Lactic acid bacteria did not affect the total phenolic content, Nisa et al. (2019) also found that fermentation using different lactic acid bacteria did not affect the total phenolic content in fermented rice bran.FULL PAPER racemosa yogurt.The antioxidant activity of yogurt fermented with S. thermophilus was higher than yogurt fermented with L. bulgaricus and yogurt fermented with a combination of L. bulgaricus and S. thermophilus.Milk fermented with lactic acid bacteria had higher antioxidant activity than non-fermented milk.During the fermentation process, proteins, carbohydrates, and fats were broken down into amino acids, peptides, organic acids, and other compounds that are beneficial to our health.Amino acids and peptides have anti-hypertensive properties, increase antioxidant activity, and inhibit fat peroxidation (Gjorgievski et al., 2014).The high antioxidant activity in yogurt B was due to S. thermophilus more effectively degrading amino acids than L. bulgaricus (Sieuwerts, 2016).Antioxidant activity is proportional to the total phenol content in yogurt.Fermentation is known to increase the total phenol content due to the change in temperature, pH, fermentation time, and the presence of lactic acid bacteria which can break phenolic bonds and increase antioxidant activity (Tsafrakidou et al., 2020).This study's results were in line with Gjorgievski et al. (2014) who found that milk fermented with monocultural lactic acid bacteria strains produced higher antioxidant activity than milk fermented with multicultural lactic acid bacterial strains.

Total lactic acid bacteria and pH
C. racemosa yogurt with different strains of lactic acid bacteria had a significant effect on growth.Yogurt fermented with S. thermophilus contained more lactic acid bacteria than yogurt fermented with L. bulgaricus (Figure 1).However, combining S. thermophilus and L. bulgaricus produced a symbiotic mutualism where S. thermophilus was more active in the first phase of the fermentation process to degrade amino acids and trace elements than L. bulgaricus.Furthermore, S. thermophilus used more oxygen, which is beneficial for L. bulgaricus as it cannot withstand the presence of high amounts of oxygen (Sieuwerts, 2016).The growth of lactic acid bacteria is influenced by the fermentation duration (Taleghani et al., 2016) and the pH of yogurt.Optimal conditions for S. thermophilus range from 4 to 4.5 pH.Decreasing acidity would inhibit the growth of S. thermophilus (Chramostová et al., 2014).Yogurts B and C had 4.83 and 5.04 pH respectively, similar to Chramostová et al. (2014) who found that the total lactic acid bacteria ranging from 7 to 8 log CFU/mL had a pH of 4.26-4.32.The low pH of yogurt is caused by organic acids, mainly lactic acid, and acetic acid during the fermentation process (Chen et al., 2017).

Total acid
Fermentation by lactic acid bacteria converted carbohydrates into organic acids, which were summed up as the total acid (Figure 1).Lactic acid bacteria convert carbohydrates in milk, such as lactose, into lactic acid.Lactic acid production was affected by lactic acid bacteria strains.L. bulgaricus and S. thermophilus are known to produce lactic acid with different isomers, such as D(-) and L(+) lactic acid, through heterofermentative pathways.This process also generates acetic acid (Widyastuti et al., 2014;Gezginc et al., 2015;Chen et al., 2017).Table 2 shows that yogurt in this study had a total acid range of 0.64-0.71%.Yogurt with C. racemosa produced less acid than commercial yogurt (0.85%).Gezginc et al. (2015) explained the optimal conditions for L. bulgaricus and S. thermophilus to produce lactic acid through fermentation, such as an incubation temperature of 42℃.Incubation at 37℃ is optimal for both bacteria strains to produce acetaldehyde, which plays a role in giving yogurt its flavour.In this study, incubation and fermentation took place at 37℃, making the total acid content in yogurt with C. racemosa being lower than commercial yogurt to be expected.

The preference tests
The results of the preference test for C. racemosa yogurt are shown in Table 2.The different lactic acid bacteria played a role in the panelists' preferences for colour, aroma, texture, and flavour.Based on colour, the addition of C. racemosa was rated from neutral to moderate like.The green colour of C. racemosa yogurt is caused by a natural green pigment named chlorophyll (Sihono et al., 2018).In terms of aroma, the panellists rated the yogurt as moderate like.The aroma came from the formation of acetaldehyde during the breakdown of threonine during the fermentation process (Krisnaningsih and Yulianti, 2015).It was also related to the effectiveness of the combination of L. bulgaricus and S. thermophilus at degrading amino acids (Sieuwerts, thermophilus play a role in texture production.The texture comes from the total solids and protein content of the yogurt, which gives it firmness.Lactobacilli degrades amino acids in milk into glycine and histidine, which support the growth of streptococci.Combining the two bacteria converted milk sugar to lactic acid, giving it a pleasant flavour and consistency.Acid formation during the fermentation process caused the rearrangement of casein creating a more compact yogurt structure (Mohan et al., 2020).Yogurt has a strong sour taste related to the formation of lactic and acetic acid (Chen et al., 2017).Nowadays, consumers prefer a tasty and less sour taste, which the low pH resulting from L. bulgaricus provides.Combining L. bulgaricus and S. thermophilus produced a less sour taste.Sieuwert (2016) found that S. thermophilus plays a role in aroma formation, so the combination of L. bulgaricus and S. thermophilus produces yogurt with a mildly sour taste and a desirable aroma.

Conclusion
Yogurt with Caulerpa racemosa produced a probiotic drink with low fat, tasty, and had functional properties such as total phenolic content, antioxidant IC 50 and dietary fibre was better than commercial one.Yogurt made with combination L. bulgaricus and S. thermophilus (yogurt C) was the most preferred by panellists.
. Yogurt fermentation involves two exponential phases of bacterial growth separated by a transition phase.The first exponential phase indicates the growth of S. thermophilus at a neutral pH and requires eISSN: 2550-2166 © 2021 The Authors.Published by Rynnye Lyan Resources FULL PAPER more availability of free amino acids than L. bulgaricus.

Figure 1 .
Figure 1.Total lactic acid bacteria, pH and total acid of yogurt sample.Data with different superscripts are significantly different (P<0.05) for each parameter.

Table 1 .
Table 1 shows antioxidant activity expressed as an IC 50 value.Lactic acid bacteria affected the IC 50 of C. Chemical characteristics of yogurt Values are presented as mean±SD.Values with different superscripts in the same column are significantly different (P<0.05).

Table 2 .
Preference test of yogurt Values are presented as mean±SD.Values with different superscripts in the same column are significantly different (P<0.05).