Physicochemical and Microstructural Properties of Yoghurt Fortified with Anthocyanin Black Glutinous Rice ( Oryza sativa L. var. glutinosa) Extract

Anthocyanin has great potential as a natural additive to improve the quality of yoghurt drink and give more benefits for health. This research aimed to analyze the effect of adding anthocyanin black glutinous rice extract on the physicochemical and microstructural quality of yoghurt drink. The research material is skim milk, yoghurt starter, and anthocyanin extract of black glutinous rice. The method used in this study was a laboratory experiment with a Completely Randomized Design (CRD) with four treatments and three replications. The treatment given was the addition of anthocyanin extract from black glutinous rice with concentrations of 0% (P0), 10% (P1), 20% (P2), and 30% (P3). The variables observed were the total acid, pH, syneresis, viscosity, water holding capacity, particle size, and distribution. Data were analyzed by analysis of variance (ANOVA), and if there was a difference, the analysis was continued using Tukey's Test. The results showed that the addition of the anthocyanin extract of black glutinous rice had a significant effect (p < 0.05) on the total acid, viscosity, water holding capacity, particle size, and particle distribution and no significant effect (p > 0.05) on the pH and syneresis. The addition of anthocyanin extract black glutinous rice increases total acid, viscosity, water holding capacity and reduces pH, syneresis, particle size, and distribution. This study concludes that the addition of anthocyanin extract of black glutinous rice until 30% has a good effect on improving the physicochemical and microstructural quality of yoghurt drink.


INTRODUCTION
Dairy products are essential for human nutrition due to their high nutritional value and digestibility.Yoghurt is a fermented dairy product obtained through the fermentation of lactic acid bacteria (LAB).The longer the fermentation process, makes lower the pH of the yoghurt, resulting in a unique sour taste.In general, the composition of yoghurt drink contains carbohydrates, proteins, minerals, and essential vitamins (Gilbert and Turgeon, 2021).Additionally, yoghurt contains acetic acid, acetaldehyde, and other volatile compounds.Yoghurt is known to have nutraceutical, therapeutic, and probiotic effects, such as improving digestion, boosting the immune system, anticancer activities, and reducing blood cholesterol levels (Bankole et al., 2023).
Yoghurt is a food product prone to spoilage due to its composition and physicochemical properties.The decline in the quality of yoghurt is due to physical, chemical, and sensory changes, making it unsuitable for consumption, especially due to the unwanted contamination of yeast and mold (Zubairi et al., 2021).However, yoghurt drinks are also susceptible to physical damage that can reduce their quality, necessitating the fortification of other compounds that can improve their quality.Researchers have used natural additives to enhance the kinetic stability of yoghurt drinks.
Several studies to investigate the natural compound in order to improve the yoghurt quality, such as physicochemical properties, microstructure, and improve the number of viable bacteria (Anuyahong et al., 2020;Wijesekara et al., 2022).Black glutinous rice (Oryza sativa L. var.glutinosa) is one of thousands of rice varieties.This material has great potential as a source of carbohydrates, antioxidants, bioactive compounds, and fiber for health due to the phenolic compounds it contains, particularly anthocyanins (Zhu et al., 2022).Many found fortified yoghurt with anthocyanin from plants such as blackberries, blueberries, bilberries, grapes, strawberries, and black glutinous rice (Tian et al., 2019).However, anthocyanin is commonly used as a natural color food additive in dairy products such as ice cream and yoghurt (Morata et al., 2019).
Current studies have found that adding anthocyanins to yoghurt can support the growth of lactic acid bacteria during storage (Liu and Lv, 2018).Furthermore, Anuyahong et al. 2020 showed that fortifying anthocyanins from riceberry to yoghurt can reduce syneresis but not increase total acid.Additionally, anthocyanins can interact with milk protein and make binds through hydrophobic interactions, which can affect the physical properties of yoghurt, including texture, viscosity, water holding capacity, syneresis, and microstructure.This influence will also affect the level of total acid and total lactic acid bacteria (Rashwan et al., 2023).
Recent evidence reveals that anthocyanin has health benefits such as antioxidant, anti-hyperglycemia, antihyperlipidemia, anti-inflammatory, and gut microbiota improvement activities (Krga and Milenkovic, 2019).Those studies showed the potential of using anthocyanins compounds in yoghurt products.One potential source of anthocyanins that can be developed in Indonesia is colored rice.Currently, there are several types of rice that are rich in anthocyanins, one of which is black glutinous rice (Oryza sativa L var. glutinosa) as a local product.Based on the background above, adding black glutinous rice anthocyanin extract was expected to improve the quality of the yoghurt drink, particularly on its physicochemical properties (total acid, pH, syneresis, viscosity, water holding capacity) and microstructure (particle size and distribution).This study could be important to develop effective strategies to produce good quality, more nutritional, and healthy dairy products using different anthocyanin sources for fortification.

Research Method
The research method employed in this study was a laboratory experiment using a Completely Randomized Design (CRD) with four treatments and three replications.The treatments used were yoghurt drink without the addition of black glutinous rice anthocyanin extract (P0) and yoghurt drink with the addition of 10% (P1), 20% (P2), and 30% (P3) black glutinous rice anthocyanin extract (v/v).The variables observed in this research were physicochemical quality (total acid, pH, syneresis, viscosity, water holding capacity) and microstructure.

Black Glutinous Rice Anthocyanin Extract Preparation
The sample used in this study was black glutinous rice that was sorted, ground, and sifted using a 100-mesh sieve.A mixture of black glutinous rice and aquades in a 1:2 (w/v) ratio was heated to 45°C for 40 min with continuous stirring using a magnetic stirrer.The supernatant was then collected and filtered using Whatman No.1 filter paper and a vacuum pump.

Yoghurt Drink Production
Skim milk powder was dissolved in aquadest and mixed with the predetermined concentration of anthocyanin extract.It was then pasteurized at 72°C for 15 seconds while stirring gently.The milk temperature was then reduced to 43°C, and a 3% (v/v) yoghurt starter containing Lactobacillus bulgaricus and Streptococcus thermophillus bacteria were added.The milk was then incubated at room temperature (25-28 °C) for 24 h.The yoghurt was blended until smooth to become a yoghurt drink.

The Analysis of Physical and Chemical Qualities and Microstructure of Yoghurt Drink
The analysis was conducted using various methods such as the total acid test procedure by Mabrouk and Effat (2013), pH testing by Setianto et al. (2014), syneresis following the procedure by Jrad et al. (2024), viscosity testing by AOAC ( 2005), water holding capacity based on Ibrahim and Khalifa (2015), and microstructure testing for particle size and distribution using the PSA method by Zhang et al. (2021).

Statistical Analysis
The data obtained was tabulated using Microsoft Excel, and the mean and standard deviation were calculated.The data was analyzed using Analysis of Variance (ANOVA), and after obtaining significant differences, further testing was conducted using the test of Tukey's Test (p < 0.05).

Physicochemical Properties of Yoghurt Drink at Different Treatments
According to the analysis of variance, the addition of different concentrations of black glutinous rice anthocyanin extract has varying effects on the water holding capacity, pH, syneresis, viscosity, and acidity of yoghurt drinks.These effects are presented in Table 1.

Total Acid
Table 1 shows that total acid is significantly different (p < 0.05).Total acid yoghurt drink without fortification (P0) is 1.185%, and yoghurt drink with the addition of black glutinous rice anthocyanin extract has total acid ranges from 1.347% to 1.515%.This is in line with the standard SNI 2981: 2009, which states that the total acidity of yoghurt ranges from 0.5-2.0%.The higher the total acid, the more it affects the quality of yoghurt because it is related to the ability of lactic acid bacteria to produce lactic acid during fermentation.The addition of black glutinous rice anthocyanin extract makes a difference in the total acid of the yoghurt drink because the acidity of the black glutinous rice anthocyanin extract is lower than the total acid of the yoghurt.
The increase in the total acid of yoghurt is caused by the activity of bacteria to transform the lactose in milk into lactic acid during the process of fermentation.The addition of black glutinous rice anthocyanin extract before fermentation is believed to trigger an increase in the fermentation process, resulting in the production of lactic acid as a metabolite, causing the total acid content of the yoghurt to increase.Generally, black glutinous rice anthocyanin extract contains monosaccharides (fructose and glucose) and disaccharides (sucrose).The lactic acid bacteria use the high sugar content in the black glutinous rice anthocyanin extract as a source of energy and substrate to produce lactic acid during the fermentation process (Yulianto et al., 2022).In addition, lactic acid bacteria are generally more resistant to anthocyanin content compared to other microorganisms.According to Petruskevicius et al. (2023), black glutinous rice anthocyanin extract can inhibit the growth of gram-negative bacteria but is not active against gram-positive species.Based on this, the growth of lactic acid bacteria will not be disturbed and will increase because the Lactobacillus used is a gram-positive bacterium.This is supported by Liu and Lv (2018) opinion that the addition of anthocyanin extract can promote the growth of lactic acid bacteria during storage.Then, for gram-negative bacteria that die, they will produce acid, so the total acid will also increase.

pH
This study found that the treatment without black glutinous rice anthocyanin had the highest pH of 5.026, while the treatment with 30% black glutinous rice anthocyanin extract (P3) had the lowest pH of 4.293.The addition of black glutinous rice anthocyanin extract at various concentrations may decrease the pH of the yoghurt but is not significantly different (p > 0.05) when compared to the control yoghurt.The other studies were reported similar results of yoghurt fortified with anthocyanin from blueberry flower pulp and riceberry rice (Liu and Lv, 2019;Anuyahong et al., 2020).
The decreasing pH occurs by an increase in the number of H+ ions, which is caused by the presence of lactic acid bacteria in yoghurt and metabolizes lactose into lactic acid.According to Widagdha and Nisa (2015), the decrease in pH is one of the consequences of the fermentation process caused by the production of lactic acid from lactic acid bacteria.The pH of yoghurt is inversely proportional to the total acid, so the lower the pH, the higher the total acid (Fatmawati et al., 2013).

Syneresis
Based on the average value of syneresis, it was found that the treatment with a 30% addition of black glutinous rice anthocyanin extract (P3) had the lowest syneresis value compared to other treatments, which was 40.89%.Meanwhile, the highest syneresis value was obtained from the treatment without the addition of black glutinous rice anthocyanin extract (P0), which was 43.69%.
The syneresis value of the yoghurt drink decreased as the black glutinous rice anthocyanin extract concentration increased but was not significantly different (p > 0.05).However, syneresis occurs when the hydrogen bond between water molecules and protein molecules weakens, and the pores between the casein molecules loosen, allowing free water molecules to pass through.
The decrease in syneresis has an effect on improving the quality of the yoghurt drink.Syneresis is undesirable in yoghurt drink because there is a separation of the liquid phase in the gel which indicates a decrease in product quality (Aswal et al., 2012).

Viscosity
Based on the analysis of viscosity, it was found that the treatment with a 30% addition of black glutinous rice anthocyanin extract (P3) had the highest viscosity value compared to other treatments, which was 16.66 cps.In contrast, the lowest viscosity value was obtained from the yoghurt control (P0), which was 11 cps.Fermented products, such as yoghurt drinks, have a viscosity value between 8.28-13.00cps (Wanniatie et al., 2022).
This may be due to the acidic pH conditions, which can decrease the solubility of casein, resulting in hydrophobic interactions between casein micelles that form the structure and consistency of the yoghurt drink, causing it to become thicker and increasing its viscosity.The non-significant viscosity results are presumed to be due to the relatively similar coagulated milk protein, resulting in no significant difference in viscosity.This is in line with (Asaduzzaman et al., 2021) opinion that at pH approaching 4.6, the solubility of casein has disappeared, leading to hydrophobic interactions between casein micelles that form the main structure and consistency of the yoghurt.This process can affect the physical properties of yoghurt, including texture, viscosity, water-holding capacity, and syneresis.

Water Holding Capacity
Based on the average water-holding capacity values, it was found that the treatment with a 30% addition of black glutinous rice anthocyanin extract (P3) had the highest water-holding capacity value among the other treatments, which was 51.70%.In comparison, the lowest waterholding capacity value was found in treatment (P0), which was 47.89%.The higher water holding capacity value is important to improve the quality of the yoghurt drink as it is related to the ability of the drink to hold water.The increase in water holding capacity obtained is influenced by several factors, such as total acid and ingredients used in the yoghurt drink-making process, such as black glutinous rice anthocyanin.The water holding capacity of the yoghurt drink increases as the concentration of black glutinous rice anthocyanin extract increases.The addition of black glutinous rice anthocyanin extract can increase the waterholding capacity of yoghurt drink due to the interaction between protein, especially casein, and black glutinous rice anthocyanin.Additionally, skim milk, used as the base material for yoghurt production, has a relatively high protein content.Kania et al. (2015) stated that protein content is the component that has the most significant effect on water absorption.
Anthocyanin extract also causes a change in protein structure and makes the protein more hydrophilic (Cui et al., 2021).The interaction between protein and anthocyanin is in the form of hydrogen bonds formed between polyphenol hydroxyl groups and the main polypeptide chain of protein (Li et al., 2021).Non-covalent binding of the anthocyanin ligand can change the tertiary structure of the protein and make hydrophobic indigenous amino acids more hydrophilic (Liao et al., 2021).The level of protein hydrophobicity affects the ability of the protein to bind water.High levels of casein volume show a loose sponge-like structure with a large number of interstitials and hydrophilic groups on the surface of the casein micelle that is highly hydrated so that water will be bound by casein.The higher the concentration of added black glutinous rice anthocyanin extract, the higher the water-holding capacity of yoghurt, and vice versa.

Microstructure of Yoghurt Fortification Black Glutinous Rice Anthocyanin Extracts in Different Treatment
According to the analysis of variance, the addition of different concentrations of black glutinous rice anthocyanin extract has varying effects on the particle size and particle distribution.These effects are presented in Table 2.

Particle Size
Based on the data particle size of yoghurt, it was found that the treatment with a 30% concentration of added black glutinous rice anthocyanin extract (P3) had the smallest particle size among other treatments, which was 6.277 µm.On the other hand, the treatment without any addition of black glutinous rice anthocyanin extract (P0) had the largest particle size, which was 9.050 µm.With the increase in anthocyanin addition, the average particle size of yoghurt decreased from 9.050 µm to 6.277 µm.This indicates that the smaller particle size in yoghurt represents better quality.It also suggests that the particles in yoghurt were more concentrated in the range of smaller particle sizes, resulting in more uniform and stable particle distribution (You et al., 2014).
In the results of the particle size distribution of yoghurt with added black rice anthocyanin extract shown in Table 2, it can be seen that the concentration of 30% addition (P3) resulted in smaller particle sizes compared to the other treatments.Adding anthocyanin extract to yoghurt affects the repulsive forces between the anthocyanin extract particles and the protein in the yoghurt.The decrease in particle size in yoghurt is caused by the repulsive forces between the particles (Prajapati et al., 2016).The higher the concentration of black glutinous anthocyanin extract added, the greater the repulsive forces between particles, making it less likely for the particles to form aggregates. On the other hand, the lower the addition of anthocyanin extract, the easier it is for the particles to attract each other and form aggregates.

Particle Distribution
Based on the particle distribution results of yoghurt with the addition of black glutinous rice anthocyanin extract shown in Table 2, it can be observed that the concentration of 30% addition (P3) resulted in a smaller or narrower particle size distribution.A particle size distribution is considered narrow if the span value is ≤ 2.5.A narrow distribution indicates that the particle size is homogeneous, while a wide distribution indicates a heterogeneous particle size (Caetano et al., 2016).The particle distribution pattern is related to the particle size.A smaller particle size distribution indicates a more homogeneous particle size (Rasaie et al., 2014).The average span value for all four treatments was less than 2.5.This indicates that the particle size distribution of the resulting yoghurt is homogeneous.All treatments showed a consistent decrease in distribution values, indicating that increasing the addition of black glutinous rice anthocyanin extract will likely make the particle distribution more homogeneous.A homogeneous particle distribution indicates an increased ability to bind water.This is due to the more uniform and constant level of casein particles, which causes them to form protein-water bonds.The particle size distribution reflects the size and non-uniformity of particles in a solution.An increase in anthocyanin content is believed to increase intermolecular collisions, causing whey protein to aggregate (He et al., 2016).Interaction between anthocyanin and casein and whey proteins is thought to increase the zeta potential of the solution, resulting in an increase in repulsive forces among particles, causing the particle size in the solution to become smaller.Increased repulsion between larger particles will make the colloid system more stable (Mo et al., 2016).

CONCLUSION
From the research results, it can be concluded that adding up to 30% extract of anthocyanin from black glutinous rice to yoghurt drink affects the increase in total acid, water holding capacity, viscosity, as well as the decrease in pH, syneresis, particle size and distribution of the yoghurt drink.and rheological and nutraceutical benefits.Frontiers in Nutrition, 10 (1257439)

Table 1 .
The averaged value of total acid, pH, syneresis, viscosity, and water holding capacity of yoghurt at different treatments Different superscripts in the same column indicate a significant effect among the treatments (p < 0.05).

Table 2 .
The averaged value of particle size and distribution of yoghurt at different treatments Different superscripts in the same column indicate a significant effect among the treatments (p < 0.05).