3.1 Color analysis
For all food products, including yogurt, color is one of the visual attributes which has a part in influencing consumers’ preferences. Data of each yogurt sample’s L* (lightness), a* (redness), b* (yellowness), c (chroma), and H (hue) are shown in Table 2. Treatments of angkak forms had a significant difference on L*, a*, b*, and h value of yogurt, particularly on the a* value. MFDS has a bright red color from the pigments produced by Monascus purpureus during fermentation e.g. the yellow pigments: monascin and ankaflavin, orange pigments: rubropunctatin and monascorubrin, and the red pigments: rubropunctamine and monascorubramine (Feng, Shao, & Chen, 2012). These added pigments would give a significant effect to the yogurt color in comparison to the control. Yogurt with added MFDS powder also produced the highest a* values and the lowest H values throughout storage, while yogurt with added water MFDS extract produced the second highest a* values. Overall, yogurt with added MFDS powder produced a more red color compared to other yogurt samples.
Table 2
Changes in lightness (L), redness (a*), yellowness (b*), chroma (c), and hue (H) of yogurt samples during storage (4⁰C)
Color parameter | Storage time (day) | Treatment |
Control | Powder | Water | Ethanol |
L | Before fermentation | 89.32 ± 0.93abcd | 88.12 ± 0.53a | 88.10 ± 2.14a | 89.60 ± 1.11abcd |
0 | 89.32 ± 1.63abcd | 89.45 ± 0.93abcd | 90.63 ± 0.38cde | 89.93 ± 1.94bcde |
7 | 90.68 ± 0.77de | 88.65 ± 0.89ab | 89.03 ± 1.53abc | 91.35 ± 0.94e |
14 | 90.00 ± 1.18bcde | 88.55 ± 1.28ab | 90.40 ± 0.57cde | 90.88 ± 0.53de |
a* | Before fermentation | -0.33 ± 0.54a | 2.38 ± 0.10fg | 1.25 ± 0.16e | -0.03 ± 0.22ab |
0 | 0.17 ± 0.23abc | 2.08 ± 0.34f | 1.30 ± 0.70e | 0.18 ± 0.12abc |
7 | 0.73 ± 0.12d | 2.25 ± 0.05fg | 1.52 ± 0.17e | 0.53 ± 0.26cd |
14 | 0.12 ± 0.24abc | 2.63 ± 0.17g | 1.33 ± 0.26e | 0.38 ± 0.50bcd |
b* | Before fermentation | 11.13 ± 0.62bA | 9.68 ± 0.39aA | 10.22 ± 0.59abA | 10.70 ± 0.98ab |
0 | 11.98 ± 0.39bB | 10.90 ± 0.74aB | 10.90 ± 1.27aAB | 11.18 ± 0.93a |
7 | 12.08 ± 0.39B | 11.62 ± 0.82B | 11.82 ± 0.63B | 11.45 ± 0.89 |
14 | 11.58 ± 0.15AB | 11.45 ± 1.15B | 11.53 ± 0.90AB | 11.43 ± 0.72 |
c | Before fermentation | 11.15 ± 0.60A | 9.98 ± 0.39A | 10.32 ± 0.59 | 10.70 ± 0.98 |
0 | 11.98 ± 0.39BC | 11.87 ± 1.82B | 11.30 ± 1.84 | 11.25 ± 1.02 |
7 | 12.08 ± 0.39C | 11.82 ± 0.82B | 11.92 ± 0.63 | 11.45 ± 0.89 |
14 | 11.58 ± 0.15AB | 11.75 ± 1.15AB | 11.63 ± 0.90 | 11.43 ± 0.72 |
H | Before fermentation | 87.48 ± 2.27de | 76.18 ± 0.18a | 83.03 ± 0.90c | 90.27 ± 1.23f |
0 | 88.75 ± 0.79def | 78.70 ± 1.86b | 83.37 ± 3.14c | 88.88 ± 0.76ef |
7 | 86.52 ± 0.68d | 78.98 ± 0.87b | 82.67 ± 1.15c | 87.42 ± 1.09de |
14 | 89.43 ± 1.18ef | 77.00 ± 0.64ab | 83.32 ± 1.74c | 88.20 ± 2.36def |
a, b, c, d, e, f Mean values with different letters are significantly different (p < 0.05). A, B Mean values in the same column with different letters are significantly different (p < 0.05). |
Table 2 placement
The extraction process of MFDS could decrease the color intensity of the water and ethanol MFDS extracts by losing some of the pigments during extraction. Some pigments may have been degraded from the high temperature of extraction and pasteurization. Most of Monascus pigments are unstable outside of 30–60⁰C (Feng, Shao, & Chen, 2012). Also, the pigments may have not been fully extracted from the MFDS powder due to the solvent used. Srianta, Ristiarini, & Nugerahani (2019) found that MFDS extracted using ethanol:water solvent with the ratio of 7:3 produced higher red pigment content compared to extraction using 10:0 ratio solvent. Meanwhile, MFDS powder was added straight into the yogurt mixture, thus most of the pigments were able to be incorporated into the yogurt without any further degradation from the extraction process.
Yogurt with added ethanol extract was less red in comparison to the yogurt with added water extract. The six main Monascus pigments are ethanol-soluble, but reactions with –COOH or NH3 groups of amino acids would produce water-soluble derivative pigments (Feng, Shao, & Chen, 2012). Srianta, Hendrawan, Kusumawati, & Blanc (2012) found more water-soluble yellow, orange, and red pigments in MFDS, which might be due to the 3.4% protein content in durian seeds being a source of NH3. More pigments were extracted with distilled water, resulting to a yogurt with a more red color than using the ethanol extract.
3.2 Total phenolic content
Total phenolic content of all yogurt samples are shown in Fig. 1. Yogurt with added MFDS powder reached the highest total phenolic content (TPC) throughout 14 days of storage, with 1.54 ± 0.24 mg GAE/g on day-0, 2.37 ± 0.42 mg GAE/g on day-7, and 2.21 ± 0.46 mg/GAE g on day-14. Yogurt with added MFDS ethanol extract reached the second highest TPC (1.35 ± 0.21, 1.53 ± 0.17, and 1.89 ± 0.25 mg GAE/g on day-0, day-7, and day-14 respectively), while addition of MFDS water extract did not affect TPC in comparison to the control. MFDS water extract and ethanol extract were reported to have TPC as high as 3.58 mg GAE/g and 3.61 mg GAE/g respectively (Srianta, Nugerahani, Kusumawati, Suryatanijaya, & Subianto, 2014). Durian seeds by themselves contain phenolics and exhibit antioxidant activity (Subianto, Srianta, & Kusumawati, 2013). According to Ramli, Sukri, Azelee, & Bhuyar (2021), freeze-dried and rotary evaporated durian seeds had the TPC of 4.46 mg GAE/mL and 29.52 mg GAE/mL respectively. Monascus sp. fermentation is able to further increase TPC of substrates and their bioavailability by releasing enzymes including amylases, cellulases, esterases, tannases, and glucosidases to break down cell walls and facilitate phenolics extraction (Suraiya et al., 2018; Zhihao et al., 2022). These enzymes can also break bonds between phenolic compounds and other groups such as polysaccharides, lipids, and amines (Cheng, Lee, Palaniyandi, Suh, & Yang, 2015; Wang, Luo, Wu, & Wu, 2018). Monascus sp. can also produce micromolecular phenolic compounds by itself (Bei, Liu, Wang, Chen, & Wu, 2017).
Addition of MFDS powder in yogurt resulted in the highest TPC of all the samples, which is due to less processing steps of MFDS before being incorporated into the yogurt. A larger amount of phenolic compounds in the MFDS powder could have been preserved and thus able to be detected during the TPC analysis. Ethanol and water extracts of MFDS underwent a few more heat processing steps and would have lost more phenolics due to degradation. Based on the TPC of the yogurt samples, it is observed that phenolics in MFDS were more ethanol-soluble rather than water-soluble. Abd Razak, Abd Rashid, Jamaludin, Sharifudin, & Long (2015) also found similar results where methanol extracts of rice bran fermented with Rhizopus oligosporus and Monascus purpureus had higher amounts of TPC compared to the water extracts.
TPC of yogurt with added MFDS powder and ethanol extract experienced an increase throughout 14 days of storage. Addition of MFDS powder increased TPC of yogurt from 1.54 ± 0.24 to 2.21 ± 0.46 mg/GAE g during 14 days of storage, while MFDS ethanol extract increased the TPC from 1.35 ± 0.21 to 1.89 ± 0.25 mg GAE/g. Aside from the phenolic compounds present in MFDS, degradation of milk proteins during fermentation by LAB could release phenolic amino acids and non-phenolic compounds which could have interfered during TPC analysis (Shori, 2013; Baba, Najarian, Shori, Lit, & Keng, 2014). Similar results were found by Szoltysik et al. (2020), in which the phenolic compounds (anthocyanins, favan-3-ols, ellagitannins, and flavonols) in yogurt with added Rosa spinosissima extract experienced a slight elevation after 14 days of storage. Yogurt with added Azadirachta indica also experienced an increase of TPC from 38.5 ± 7.2 µg GAE/mL on day 14 to 74.9 ± 6.2 µg GAE/mL by day 28 (Shori & Baba, 2013).
3.3 Antioxidant activity
Antioxidant activity of yogurt samples were determined based on their DPPH scavenging capacities and are shown in Fig. 2. On day-0, yogurt with added MFDS water extract reached the highest antioxidant activity (0.0210 ± 0.0035 mg GAE/g) while the control had the lowest activity (0.0165 ± 0.0043 mg GAE/g). Through day-7 and day-14, yogurt with added MFDS powder produced the highest antioxidant activity (0.0118 and 0.0125 ± 0.0032 mg GAE/g respectively), while the control reached the lowest values with 0.0093 ± 0.0003 mg GAE/g on day-7 and 0.113 ± 0.0014 mg GAE/g on day-14. Addition of any form of MFDS increased the antioxidant activity of yogurt, which was due to the bioactive compounds in MFDS such as Monascus pigments and phenolics. Tan, Xing, Chen, & Tian (2018) found that water-soluble yellow Monascus pigments exhibited DPPH and ABTS+ radical scavenging activity, reaching up to 86.33% inhibition from 1 mg/mL sample and 99.95% from 0.25 mg/mL respectively. Srianta et al. (2017) reported that the Monascus pigments monapilol B and rubropunctamine had a high correlation with the DPPH radical scavenging activity of Monascus-fermented products. Phenolics from Monascus-fermented oats were also reported to have high correlation with DPPH and ABTS+ radical scavenging activity (Bei et al., 2017). Other metabolites such as GABA, monacolin K, and dihydromonacolin MV also contribute to DPPH radical scavenging (Srianta, Nugerahani, Kusumawati, Suryatanijaya, & Subianto, 2014).
After fermentation, the antioxidant activity of all the yogurt samples were significantly higher than the initial yogurt mixture. The increase of antioxidant activity of yogurt mixture after fermentation was due to LAB activity. Using the thiobarbituric acid method, Kim et al. (2005) found that Lactobacillus bulgaricus and Lactobacillus acidophilus exhibit antioxidant activity (81.30% and 65.32% respectively). They are able to demonstrate radical scavenging activity, inhibitory activity towards lipid peroxidation, show strong reducing power, and produce proteolytic enzymes which help release antioxidative milk peptides (Virtanen, Pihlanto, Akkanen, & Korhonen, 2007; Zhang et al., 2011; Aloglu & Oner, 2011). Gjorgievski, Yomovska, Dimitrovska, Makarijoski, & Shariati (2014) found that yogurt fermented with Streptococcus thermophilus and L. bulgaricus mix culture and L. acidophilus monoculture also exhibited antioxidant activity by DPPH radical scavenging (52.44% and 63.99% respectively).
The antioxidant activity of the yogurt samples then significantly decreased by day-7 and stayed stagnant between day-7 and day-14. This data has a negative correlation with the TPC of yogurt samples throughout storage time, in which yogurt with added MFDS powder and ethanol extract had increasing TPC up until day-14. This suggest that the phenolics detected in yogurt with added MFDS did not have DPPH radical scavenging properties. Bei, Liu, Wang, Chen, & Wu (2017) found that the bound phenolic fraction of fermented oats with Monascus anka, mainly ferulic acid, showed greater ABTS+ scavenging activity than DPPH radical scavenging activity. Abd Razak, Abd Rashid, Jamaluddin, Sharifudin, & Long (2015) reported that TPC and radical-scavenging activity of rice bran fermented with R. oligosporus and M. purpureus were poorly correlated for the water and methanol extracts, which might be due to many factors such as the concentration and chemical structures of the phenolics detected. This result suggests that other antioxidant compounds in MFDS might have stronger DPPH scavenging activity than the phenolics detected i.e. pigments, monacolins, and GABA (Srianta, Nugerahani, Kusumawati, Suryatanijaya, & Subianto, 2014; Suraiya et al., 2018). These compunds would have degraded during storage, thereby decreasing the antioxidant activity of yogurt samples. Similar results have been observed by Chouchouli et al. (2013), in which the antiradical capacities of grape seed-fortified full fat yogurt decreased during 32 days of storage time (from 1487.4 ± 38.2–1567 ± 52.8 mg TE/100 g on day 1 to 234.8 ± 20.6-440.5 ± 40.3 mg TE/100 g by day 32).
3.4 pH and titratable acidity
Changes in yogurt pH added with different MFDS forms are shown in Table 3. pH of unfermented yogurt mixture decreased significantly after fermentation due to the formation of acids by LAB. pH data from yogurt with added MFDS powder experienced the biggest pH reduction during storage time from 4.492 ± 0.093 on day-0 to 4.175 ± 0.087 by day-14. Yogurt with added water extract reached the lowest pH on day-0 and day-7, while MFDS powder yogurt has the lowest pH by day-14. These results were in line with the titratable acidity data shown in Fig. 3. The majority of acids found in yogurt are lactic acids, produced by LAB by converting lactose in milk (Chen et al., 2017). The total lactic acid percentage of all yogurt mixture samples increased significantly after fermentation and increased gradually throughout storage time, which is in line with the pH data. The highest total acid percentage was reached by MFDS powder yogurt by day-14. Overall, addition of MFDS forms to yogurt only gave a slight effect to the pH and titratable acidity in comparison to the control. Darwish, Darwish, & Ismail (2017) and Melani, Nurliyani, & Indratiningsih (2021) found that the addition of angkak did not give a significant effect towards the pH value and acidity of yogurt and goat milk kefir respectively. Pyo & Song (2009) found that the addition of Monascus-fermented soybean decreased the pH and increased the acidity of yogurt, while Jeon, Lee, Choi, & Kwon (2011) found that pH of yogurt increased after the addition of Monascus-fermented Chinese yam. Different substrates of Monascus-fermentation will produce different amount and types of compounds which could affect the characteristics of each Monascus-fermented product.
Table 3
Changes in pH, syneresis percentage, and total plate count of yogurt samples during storage (4⁰C)
Parameter | Treatment | Storage time (day) |
Before fermentation | 0 | 7 | 14 |
pH | Control | 6.397 ± 0.048c | 4.468 ± 0.068bAB | 4.291 ± 0.096aAB | 4.289 ± 0.130aAB |
Powder | 6.415 ± 0.034c | 4.492 ± 0.093bB | 4.275 ± 0.111aAB | 4.175 ± 0.087aA |
Water | 6.396 ± 0.032c | 4.353 ± 0.028bA | 4.215 ± 0.045aA | 4.249 ± 0.097aAB |
Ethanol | 6.389 ± 0.033b | 4.473 ± 0.105aAB | 4.390 ± 0.089aB | 4.373 ± 0.087aB |
Syneresis (%) | Control | - | 2.22 ± 0.63A | 2.42 ± 0.60A | 2.56 ± 0.68A |
Powder | - | 3.29 ± 0.43aB | 3.10 ± 0.27aA | 5.24 ± 0.51bB |
Water | - | 2.83 ± 0.82AB | 2.67 ± 0.50A | 3.60 ± 0.60AB |
Ethanol | - | 2.59 ± 0.46A | 2.68 ± 0.60A | 2.92 ± 0.69A |
Total plate count (CFU/mL) | Control | 2.65 x 107 a | 6.27 x 109 b | 3.36 x 1010 bc | 1.96 x 1011 c |
Powder | 3.05 x 106 | 6.22 x 109 | 2.04 x 1010 | 1.90 x 107 |
Water | 1.34 x 106 a | 7.82 x 1010 b | 4.84 x 1010 b | 1.16 x 109 ab |
Ethanol | 4.00 x 106 a | 1.55 x 1010 bc | 1.12 x 1011 c | 1.22 x 109 b |
a, b, c, Mean values in the same row with different letters are significantly different (p < 0.05). A, B Mean values in the same column with different letters are significantly different (p < 0.05). |
Table 3 placement
3.5 Syneresis
As a commercial product, syneresis percentage is best kept at a minimum for yogurt to suit consumer preferences. Table 3 shows the effect of different MFDS forms addition towards syneresis percentage in yogurt. Addition of MFDS forms gave a significant effect in the syneresis percentage of yogurt samples, although only yogurt with added MFDS powder experienced an increase of syneresis percentage by day-14. Syneresis percentages of yogurt with added MFDS powder (3.29 ± 0.43–5.24 ± 0.51%) throughout storage are higher compared to other yogurt samples. Syneresis takes place due to weakening of yogurt gel structure thus losing the ability to entrap water (Sah, Vasiljevic, McKechnie, & Donkor, 2016). Higher syneresis percentage found in yogurt with added MFDS powder might be related to its TPC. Yogurt with MFDS powder reached the highest TPC out of all the samples (1.54 ± 0.24–2.21 ± 0.46 mg/GAE g) and high amount of phenolic compounds have been proven to effect yogurt syneresis by other studies. Increased rates of syneresis were detected in yogurts with added ingredients containing phenolic compounds such as blueberry juice, green tea powder, Ferulago angulata extract, and grape pomace (Marchiani et al., 2015; Jeong et al., 2018; Dimitrellou, Solomakou, Kokkinomagoulos, & Kandylis, 2020; Keshavarzi, Sharifan, & Ardakani, 2020). Excess polyphenol concentrations could reduce the gel matrix that confines the yogurt serum by decreasing the volume of each individual protein-phenolic cage and preventing gel matrix formation (Jeong et al., 2018). Increasing the number of particle-particle junctions in the gel structure could lead to the shrinkage of the network and dismissing interstitial liquid (Dönmez, Mogol, & Gökmen, 2017).
3.6 Total plate count
Changes in total plate count of yogurt with added MFDS are shown in Table 3. Addition of MFDS forms to yogurt did not give a significant difference towards the total plate count in comparison to the control. For all the yogurt samples, the total plate count yogurt mixture increased after fermentation. However, total plate count of yogurt with added MFDS did not increase significantly from day-0 to day-7. By day-14, the total plate count of all samples except the control experienced a decrease. Yogurt with added MFDS powder was not significantly affected during storage time even though it produced the same pattern of data as the other samples. Addition of MFDS powder to yogurt resulted in the lowest total plate count reached, from 6.22 x 109 on day-0 to 1.90 x 107 on day-14). Yogurt without any added MFDS experienced an increase of total plate count by day-14, reaching 1.96 x 1011 from 6.27 x 109 on day-0.
Monascus fermentation produces various compounds, among them are pigments and phenolics which have antibacterial characteristics. These compounds may have inhibited the growth of LAB during storage. Pigments are the major bioactive compounds found in Monascus-fermented products along with monacolins (Zhu et al., 2019), with each group of pigments having different antibacterial mechanisms. Kim, Jung, Kim, & Shin (2006) found that amino acid derivatives of red Monascus pigments are able to suppress growth of Gram positive bacteria, which indicates that the red pigment derivatives from MFDS could have inhibited LAB growth. Hydrophobicity of bacteria cell surfaces increases when the pigments are adsorbed into the cell, which leads to cell aggregation into pellets. Pellet formation of cells results in limited transfer of oxygen and nutrients into bacteria cells. Orange pigments has a good affinity with liposomes, resulting in an interaction with the phospholipid of bacteria cytoplasmic membranes. The interaction disrupts the bacteria membrane and causing cellular leakage. Orange pigments can also stimulate pellet formation on cells (Zhao, Li, Yang, & Cui, 2016). On the other hand, phenolics can disrupt cytoplasmic membrane and cause lysis, and interact with enzymes, substrates, and metal ions which prevents bacteria metabolism (Vaquero, Alberto, & Nadra, 2007; Oulahal & Degraeve, 2021; Melani, Nurliyani, & Indratiningsih, 2021).
MFDS water and ethanol extracts underwent further processing, starting with the extraction of compounds with various polarities using purely polar or non-polar solvent and then be treated to sterilization. These caused the amount of compounds in the extracts to be less than the MFDS powder. This would explain why the addition of MFDS powder suppressed the growth of LAB in yogurt the most. Melani, Nurliyani, & Indratiningsih (2021) found that the total plate count of angkak-supplemented kefir also decreased during storage time. Pyo & Song (2009), however, found that Monascus-fermented soybeans had an increase of viable LAB due to the free and essential amino acids produced from Monascus fermentation being a source of nutrients. According to the Indonesian national standard for yogurt, the minimal amount of starter bacteria in yogurt is 107 log/g thus MFDS-supplemented yogurt is in accordance to the standard.
3.7 Sensory evaluation
According to Fig. 4, the acceptance score of all yogurt samples were in the range of 2.56 (dislike-neutral) to 4.06 (like-most like). Addition of MFDS powder had a significant difference in the panelist acceptance score for color and taste, while addition of any form of MFDS did not give a significant effect towards the consistency acceptance score. Storage time did not give a significant effect towards the panelist acceptance scores, which meant the quality of all yogurt samples remained stable after seven days of storage. Yogurt with added MFDS powder obtained the lowest acceptance score for color with the scores of 3.28–3.34) and taste (2.56–2.81) on day-0 and 7 respectively. The highest score for each parameter was reached by yogurt with added ethanol extract, with the score of 3.97-4.00 for color, 3.87–3.84 for taste, and 3.53–3.97 for consistency. The results show that addition of MFDS, particularly the ethanol extract, on yogurt was well liked by panelists.
Color analysis results from Table 2 shows that yogurt with added MFDS powder produced a more red-colored yogurt in comparison to the other samples. Without information of any flavor or added ingredient in yogurt, panelist could have an expectation of a white color regularly found in plain yogurt. A difference in the color of yogurt with added MFDS powder in comparison to other samples might affect panelists’ acceptance scores. MFDS powder was not incorporated evenly in the yogurt, which lead to the accumulation of powder at the bottom of the yogurt cup. This overall appearance might also affect the scores. Panelists have also commented that yogurt with added MFDS powder had a bitter taste. The bitterness might be due to the presence of phenolics in the yogurt (Li & Duan, 2018). According to the total phenolic content data from Fig. 2, yogurt with added MFDS powder was found to have the highest TPC out of all the samples. Reginio, Hurtada, & Dizon (2016) also reported that bitterness and astringency were detected by panelists from Monascus biopigment beverage.