The Quality of Yoghurt Fortification Tuna Fishbone ( Thunnus sp.) Nanocalcium: Physicochemistry and Microstructure

Calcium is very important mineral for human body. Fortification of natural calcium source into yoghurt can be a way to provide calcium requirement. Fortification nanocalcium into yoghurt also can increase bioavailability of calcium. This research aimed to determine the effect of tuna fishbone nanocalcium fortification on total acidity, pH, water holding capacity, viscosity, syneresis, firmness, cohesiveness, particle size and particle distribution of yoghurt. The treatment used were 100 ml yoghurt + 0% nanocalcium (P0), 100 ml yoghurt + 0.12% nanocalcium (P1), 100 ml yoghurt + 0.24% nanocalcium (P2), and 100 ml yoghurt + 0.48% nanocalcium (P3). This research carried out 4 treatments and 3 repetitions. The results showed that the fortification had a significant effect (p < 0.05) on yoghurt's total acidity, pH, water holding capacity. The result of viscosity, firmness and cohesiveness showed the highly significant effect (p < 0.01) in yoghurt and the rest variable result like particle distribution, particle size and syneresis of yoghurt fortification showed no significant effect (p > 0.05). The average result P0; P1; P2; P3 of total acidity (%) was 1.395±0.085; 1.343±0.0471; 1.272±0.0364; 1.242±0.0469, pH was 4.38 ± 0.04; 4.39 ± 0.01; 4.40 ± 0.03; 4.45 ± 0.01, water holding capacity (%) was 71.17 ±1.90; 71.77±1.96; 74.56 ± 2.2, viscosity (%) was 84.473 ± 0.537; 90.317 ± 0.585; 92.873 ± 1.175; 95.073 ± 0.616, syneresis (%) was 28.140±6.97; 28.790 ±2,19; 27.253 ±4.72; 27.890 ± 4.04, firmness was 42.443 ± 1.093; 45.627 ± 0.994; 47.370 ± 1.295; 54.290 ± 2.509, cohesiveness was 12.679±0.244; -13.180±0.340; -13.913±0.270; -14.492±0.169, particle size (µm) was 9.820±1.950; 8.797±0.120; 8.470±1.869; 8.303±2.442, and particle distribution was 3.027 ±0.01; 2.781 ±0.16; 2.714 ±0.313; and 2.578 ±0.22. The conclusion of this research was fortification of tuna fishbone nanocalcium had effect to decrease total acidity, syneresis, cohesiveness, particle size and particle distribution of yoghurt. Also, the fortification can increase pH, water holding capacity, viscosity and firmness of yoghurt.


INTRODUCTION
Milk is a highly nutritious food due to its high content of essential nutrients such as protein and high-quality fats The main constituents of milk are water (87.9%), protein (3.5%), fat (3.5-4.2%),vitamins and minerals (0.85%) (Karseno et al., 2021).However, due to its high nutritional content and water content, milk is highly perishable as it can be used as a growth medium for microorganisms (Rohman and Maharani, 2020).Therefore, further processing is necessary to improve the quality and extend the shelf life of milk.One technique for processing milk is through the production of fermented milk, commonly known as yoghurt.
Yoghurt is a food that contains probiotics, which provide additional nutritional value.According to Yerlika et al. (2021), yoghurt is a rich probiotic fermented dairy product and have high digestible protein.Yoghurt also very impactfull to fulfill the important human nutrition because among fermented dairy product, yoghurt is rich by beneficial bacteria.Yoghurt is made by fermenting milk with lactic acid bacteria, such as Lactobacillus bulgaricus and Streptococcus thermophilus.
Yoghurt has a sour taste and thicker texture than milk.The sour taste in yoghurt is caused by the presence of lactic acid resulting from fermentation, while the thick texture is caused by the acidic environment in yoghurt, which causes milk protein or casein to clot or coagulate.Also, Yoghurt fermentation can prevent the growth of pathogenic microorganisms in the resulting product, increase its nutritional value compared to the raw material, and break down lactose in milk into smaller compounds and making it easier to adsorb the nutrition by the body (Widiastuti and Judiono, 2017).Simpler compounds, making it easier to digest.
Yoghurt also one of the good sources of minerals for consumption for human body.The consistency minerals of yoghurt contains mostly calcium, magnesium, potassium, sodium, and phosphorus minerals (Kirdar et al., 2017).A ccording to the US Department of Agriculture (2016), calcium is abundant in dairy products.Yoghurt is known to contain 100-180 mg of calcium per 100 g of yoghurt.Consuming adequate amounts of calcium is recommended due to its numerous benefits.The primary benefit of calcium is maintaining the health and density of bones and teeth.Calcium is known for its function in helping to maintain bone density, making bones stronger and less brittle.In addition to bone health, calcium also plays an important role in maintaining heart health (Rosa and Lolita, 2019).
One natural source of calcium is from tuna fish bones.The nano form of calcium in tuna fish bones has good bioavailability, which can improve calcium absorption to meet daily calcium intake requirements (Prinaldi et al., 2018).As of September 2017, the catch of tuna has reached 288 tons (Kementerian Kelautan dan Perikanan or KKP), 2018).Utilizing this source of calcium is highly suitable for increasing the economic value of tuna fish bone waste.According to Meiyasa and Tarigan (2020), tuna bone flour has a composition of 6.91% moisture content, 46.34% ash content, 12.57% fat content, 26.79% protein content, 7.38% carbohydrate content, and 41.61% calcium content.Calcium is known to reduce cholesterol and triglyceride levels in obese patients.The mechanism of calcium regulates intracellular metabolism, acting as The Quality of Yoghurt Fortification Tuna Fishbone a key regulator of adipocyte fat metabolism and triacylglycerol storage.However, the calcium content in yoghurt is considered insufficient to meet daily calcium intake needs, especially for those in the productive age range.According to the Cormick and Belizan (2019) recommendations for daily calcium intake, the average daily calcium requirement for individuals above 19 y age vary from 1000 mg to 1300 mg.In Asian society, the recommended daily intake of calcium is lower than that set by the WHO, at 868 mg/day.Unfortunately, the average calcium consumption in Indonesia is only 400 mg/day, which is far below the recommended amount.Therefore, fortification of calcium minerals in yoghurt is necessary to increase calcium intake.
To enhance the value of calcium in tuna fishbone, nanotechnology is applied through the implementation of science and engineering at the atomic scale.Nanocalcium is produced by utilizing nanotechnology, making it easier to absorb into the body due to its size, which reaches 500 x 10-19 (Anggraen et al., 2016).Nanokalsium has higher bioavailability compared to macro-sized calcium, resulting in lower excretion of nanocalcium through urine.The application of nanotechnology in food ingredients can enhance the taste, color, texture, and consistency of food products, as well as improve the absorption of nutrients and bioactive compounds.
Nanokalsium can be fortified in fresh cow's milk yoghurt.Fortification is the process of enriching nutrients by adding micronutrients to food.Calcium fortification is chosen for yoghurt because, in addition to having a high calcium content, adding calcium to yoghurt can increase the absorption of nutrients in the intestine.This is because calcium works with H + in yoghurt to increase the bioavailability of calcium.Ningrum et al. (2023) said that Nanotechnology can protect bioactive compounds from interacting with other material components and preserve their functional properties.Also, nanotechnology can enhance food stability during processing and storage, minimize impacts on the organoleptic properties of food, and improve the absorption and bioavailability of bioactive compounds.This study aims to determine the effect of fortification nanocalcium tuna fishbone on physicochemical yoghurt such as, total acidity, pH, water holding capacity, viscosity, syneresis, firmness, cohesiveness, particle size and particle distribution of yoghurt.

Research Location and Time
This research was conducted at the Animal Products Technology Laboratory, Faculty of Animal Husbandry, Brawijaya University; Food and Nutrition Research Laboratory, Faculty of Food Technology, Widya Mandala Catholic University, Surabaya; and the Pharmacy Laboratory, Maulana Malik Ibrahim State Islamic University, Malang.

Research Materials and Methods
This research used a laboratory experiment with a Completely Randomized Design with 4 treatments and 3 replications consisted of P0 = Yoghurt without the addition of tuna fish bone nanocalcium, P1 = Yoghurt with the addition of 0.12% tuna fish bone nanocalcium, P2 = Yoghurt with the addition of 0.24% tuna fish bones nanocalcium, P3 = Yoghurt with the addition of 0.48% tuna fish bone nanocalcium.
The materials used in this research were fresh cow's milk, yoghurt starter, tuna bones, lime, distilled water, 1.5 N NaOH, 1N HCl, Whatman No. 1 filter paper.

Research Procedure Tuna Fish Bone Nanocalcium Production
The first stage is the preparation of the raw material for tuna bone powder, which goes through several process stages including washing and cutting the bones, boiling for 12 h and adding lime juice.The preparation of nanocalcium refers to the method of Ratnawati et al. (2020) which has been modified where 700 g of tuna fish bones are baked in the oven for 24 h at a temperature of 80°C, then prepared and floured to 100 mesh, after which they are soaked in 1 N HCl (1 :2) for 72 h at room temperature, preparation with 1.5 N NaOH (1:1) until 2 precipitates are formed, and neutralized with distilled water until pH 7. Separate the filtrate using Whatman filter paper no. 1. Next, oven it for 24 h at a temperature of 60°C, re-oven for 3 h at a temperature of 105°C.Next, ashing was carried out in a furnace at a temperature of 600°C for 5 h followed by grinding with a mortal and pestle.

Production Yoghurt Fortified
The yoghurt incubation process begins with pasteurization of fresh milk for 15-30 min using a temperature of 90°C.Next, cool the milk until the temperature reaches 43°C and add tuna fish bone nanocalcium according to the treatment level.Add starter as much as 2% of the milk content.An incubation process was carried out for 12 h (Machadoo et al., 2017).
Meanwhile, testing the microstructure in the form of particle size of yoghurt using a Particle Size Analyzer (Sitanggang et al., 2019;Zhang et al., 2021).

Data Analysis
The data obtained was tabulated, then the values and standard deviations were calculated to determine the effect of tuna fish bone nanocalcium fortified yoghurt treatment on pH, viscosity, firmness and color.Calculations were carried out using analysis of variance (ANOVA) using a Completely Randomized Design (CRD).If there is a difference in influence then proceed with the Duncan Multiple Range Test (DMRT).

Results of Physicochemical Quality of Yoghurt with Different Treatments
Data from the analysis of variance shows that the addition of various levels of nano calcium concentration in tuna fish bones has an influence on pH, similarity, viscosity, syneresis, water holding capacity, firmness and cohesiveness, presented in Table 1.

pH
The analysis of variance shows that fortification with nano-calcium from tuna fish bones has a significant effect on the pH of yoghurt (p < 0.05), as shown in Table 1.The research results indicated that fortified yoghurt with nanocalcium shows a higher pH compared to the control treatment, where the highest pH value of 4.45 ± 0.01 was found in treatment P3 with the addition of 0.48% nano-calcium from tuna fish bones.
pH is an indicator of the hydrogen ion content in yoghurt, which is related to the total total acidity of the yoghurt itself.The decrease in total acidity and the increase in pH value are thought to be influenced by casein, which first binds to Ca 2+ ions rather than H + ions.This causes a decrease in the H + ion content in yoghurt with the highest nanocalcium concentration in this study.Muncan et al. (2021)), in his paper also told that in the form of suspended particles, casein micelles held together by colloidal calcium phosphate, which binds together numerous submicelles.The binding between micelles casein and calcium lowering possibility H + binding together with micelles casein in yoghurt.It lead to decrease the acidity and increase the pH value of yoghurt after the fortification.

Total Acidity
The analysis of variance shows that fortification with nano-calcium from tuna fish bones has a significant effect on the total total acidity of yoghurt (p < 0.05), as shown in Table 1.The research results indicated that yoghurt treated with fortification of nano-calcium from tuna fish bones shows lower total acidity compared to the control treatment.The total total acidity results showed a decrease in value with the addition of nanocalcium content to the yoghurt.
The total total acidity level in yoghurt can be an indicator of the activity and BAL content in the yoghurt.Calcium content is known to have a negative effect on the growth of lactic acid bacteria.Low BAL growth is suspected to be caused by the addition of nanocalcium in yoghurt.The addition of calcium in yoghurt can act as an antimicrobial agent for BAL.According to Al Mijan et al. (2014), yoghurt enriched with high levels of calcium carbonate eggshell powder has antimicrobial activity that can inhibit the growth of BAL.Nanocalcium also has a negative effect on bacterial growth.These antimicrobial components can reduce the growth of lactic acid bacteria.Nanoparticles of calcium carbonate can reduce damage to yoghurt caused by pathogenic bacteria.The mechanism by which calcium carbonate kills bacteria is as follows: calcium carbonate works in conjunction with natural antibacterials found in milk, typically lactoferrin and immunoglobulin.Firstly, calcium carbonate in its nano form, with its small size, can penetrate the surface of bacteria through the intermolecular layer.Furthermore, calcium carbonate increases the damage to bacterial cell walls and natural antibacterial agents in milk work to alter the permeability of cell membranes, causing the bacteria to die (Pan et al., 2018).Furthermore, the decrease in total acidity may also be attributed to the binding of calcium with casein.This binding reduces the total acid value in yoghurt (Muncan et al., 2021).

Viscosity
The analysis of variance shows that fortification of tuna fish bone nano-calcium has a highly significant effect on the viscosity of yoghurt (p < 0.01), as shown in Table 1.The research results indicated that yoghurt treated with fortification of tuna fish bone nanocalcium has a higher viscosity value compared to the control treatment.The average viscosity results of calciumfortified yoghurt with tuna bone nanocalcium in each treatment were P0: 84.473 ± 0.537, P1: 90.317 ± 0.585, P2: 92.873 ± 1.175, and P3 95.073 ± 0.616.
The viscosity results showed an increase that was consistent with the addition of nanocalcium from tuna fishbone treatment.This occurred due to the increase in the number of calcium cross-links contained in the yoghurt fortified with nanocalcium at different concentrations.Furthermore, it is believed that the increase in viscosity is due to calcium in nano form being able to disperse well in yoghurt, which increases the possibility of cross-linking between calcium and casein micelles.This also indicates that the addition of calcium to yoghurt can enhance its structure, making it stronger (Al-mijan et al. 2014).The high viscosity value in treatment P3 was also influenced by the pH of P3, which was close to the isoelectric point (pH 4.6).When the pH value of yoghurt approaches the isoelectric point, it causes the coagulation of casein, resulting in an increase in viscosity.When the pH decreased toward the isoelectric point (4.6) the colloidal calcium phosphate is completely dissolved, it caused the casein micelles agregate and form the gel molecule of yoghurt is created.Its also defined as the endpoint or the finish time of the yoghurt ferementation process (Mains et al., 2017).So, it caused the viscosity of the fortification yoghurt has increased.

Syneresis
The analysis of variance showed that the fortification of tuna fish bone nanocalcium had no significant effect on the yoghurt syneresis value (p > 0.05), as shown in Table 1.The research results indicated that the average value of yoghurt syneresis with the addition of tuna fish bone nanocalcium ranged from 27.14% to 28.79%, and the higher the concentration of added tuna fish bone nanocalcium in yoghurt will shows the lower of syneresis in yoghurt.
The pH of the yoghurt itself has a significant impact on the decrease in syneresis value.Yoghurt with a pH value close to the isoelectric point exhibits a weaker ability to bind water compared to pH values that are not close to the isoelectric point.Oktavia et al. (2016) explain that several factor play a role in the syneresis value of yoghurt is low product acidity (pH > 4.6) or high acidity product (pH < 4.0), temperature that used during incubation, and agitation process during production the yoghurt.The pH of yoghurt will influence the syneresis, which means when the pH is too low the syneresis of yoghurt will increase.This caused due to the decrease of water binding capacity power by micelles casein.Also, low pH lead the decrease of electrostatic interaction power between cell molecule (Malaka et al., 2020).Szajnar et al. (2017) concluded that the value of syneresis is influenced by water that interacts with calcium lactate molecules (lactate pentahydrate).The increase in syneresis value in yoghurt with added calcium chloride and D-gluconate is due to the degree of hydration of the calcium compound.The decrease in syneresis value is a result of the formation of a stronger and more abundant matrix that can trap more water.This is due to the increased number of hydroxyl groups that can form hydrogen bonds with water, resulting in a decrease in the amount of free and weakly bound water (Handani et al., 2016).

Water Holding Capacity
The analysis results show that fortification of tuna fish bone nanocalcium has a significant effect on the water binding capacity of yoghurt (p < 0.05), as shown in Table 1.The research findings indicated that the water holding capacity of yoghurt with added tuna fish bone nanocalcium has an average value ranging from 66.95 ± 2.86 to 74±2.21.The addition of increasing amounts of nanocalcium from tuna bone to yoghurt significantly increases its water holding capacity value.
The increase in water holding capacity is due to the hydrogen binding between the molecules of protein, which is reduced and enhances the hydrophilic properties of the protein.The reduction in hydrogen binding with the casein is caused by the molecules of casein that first bond with the nanocalcium of tuna bone.The addition of nanocalcium to yoghurt also affects the ability of mineral compounds to absorb water, resulting can increase the amount of water that can be bound by the curd (Josaphata et al., 2014).The water holding capacity of yoghurt is also influenced by pH value.In this study, the pH of the yoghurt ranged from 4.3 to 4.4, It is common for fermented products to experience the release of water molecules due to protein denaturation caused by pH (Kania et al., 2015).

Firmness
The analysis of variance indicated that fortification with nano-calcium from tuna fish bones has a significant effect on the firmness value of yoghurt (p < 0.01), as shown in Table 1.The research findings reveal that the firmness value of yoghurt with the addition of nano-calcium from tuna fish bones has an average value of P0: 42.443 ± 1.093; P1: 45.627 ± 0.994; P2: 47.370 ± 1.295; and P3: 54.290 ± 2.509.
The research findings indicated an increase in firmness values that correspond with the increase in nanocalcium concentration provided.The increase in firmness values in tuna bone-fortified yoghurt with various concentrations is influenced by the cross-linking of calcium networks in the yoghurt.Kaushik and Arora (2017), yoghurt enriched with calcium can cause a significant increase in tissue crosslinking.Additionally, calcium-enriched yoghurt can inhibit the growth of culture starters.As a result, the intensity of hardness will increase significantly with an increase in calcium content.The increase in firmness value in yoghurt fortified with nano-calcium from tuna bone is also influenced by the yoghurt's pH.If the pH of yoghurt approaches its isoelectric point, an increase in casein bonding will occur, resulting in an increase in the viscosity of fortified yoghurt.
This increase is due to the formation of casein micelle aggregation by acid and the interaction between casein micelles, resulting in the formation of a strong gel.According to Setianto et al. (2014), the texture of yoghurt is formed by the aggregation of casein micelles by acid and the interaction between casein micelles, resulting in the formation of a strong and smooth gel.The strength of the formed casein gel is determined by the strength of the bond between casein micelles.This bond is influenced by pH, calcium concentration, and temperature.A thick texture is supported by high viscosity results.

Cohesiveness
The analysis results show that fortification of tuna fish bone nanocalcium has a significant effect on the cohesiveness value of yoghurt (p < 0.01), as shown in Table 1.The research findings indicated that the cohesiveness value of yoghurt with the addition of nanocalcium from tuna fish bone has an average value ranging from -14.492 to -12.679.The texture of yoghurt is greatly influenced by its protein content.The more protein present in yoghurt, the thicker its texture will be.binding Protein with water the result showed in a smoother texture that appears uniform.Acid-coagulated proteins form a gel, resulting in a thicker yoghurt texture (Wardhani et al., 2015).In addition to the protein content, the total acidity level of yoghurt also affects its texture.According to Medeiros et al. (2015), the low pH and total acidity resulting from lactic acid fermentation induce significant structural changes that are responsible for the texture of yoghurt.
Higher total total acidity levels in yoghurt lead to increased thickness and texture.Higher cohesiveness values of yoghurt indicated stronger internal molecular bonds.In this study, the addition of nanocalcium from tuna bone resulted can decrease yoghurt cohesiveness.This is likely due to the electrostatic attraction between calcium and casein micelle proteins before acidification occurs as a result of BAL fermentation.The electrostatic bond between calcium ions and the negative charge of the casein micelle inhibits the effect of increased electrostatic attraction between protein molecules and acid, which ultimately causes coagulation.

Microstructure Quality Results of Yoghurt with Different Treatments
Data from the analysis of variance shows that the addition of various levels of nano calcium concentration in tuna fish bones has an influence on the microstructure including the yoghurt particle size and yoghurt particle distribution which is presented in Table 2.

Yoghurt Particle Size
The analysis of particle size in yoghurt fortified with nano-calcium from tuna bone showed that the addition of nano-calcium from tuna bone did not have a significant effect (p > 0.05) on the particle size of yoghurt in this study.The data on particle size of yoghurt fortified with nano-calcium from tuna bone showed smaller values compared to the control treatment (Table 2).The smallest particle size was observed in treatment P3 with the highest nanocalcium addition of 0.48%, and the average particle size in treatment P3 was 8.30.Particle size analysis of the yoghurt was conducted using the Particle Size Analyzer (PSA) method, which utilizes dynamic light scattering.PSA can be applied to measure the size and distribution of particles and molecules dispersed or dissolved in a solution, such as proteins, polymers, micelles, carbohydrates, nanoparticles, colloid dispersions, emulsions, and microemulsions (Trisnaeni, 2012).The particle size in yoghurt is greatly influenced by the size of protein particles, especially casein, which is contained in yoghurt.
The size of particles in yoghurt is influenced by the production process and the addition of ingredients (Kiani et al., 2008).The study found that particle size decreased with increasing concentration of nanocalcium.This is thought to be due to the interaction of Ca 2+ ions from tuna bone nanocalcium with the negative charge of the casein micelle.At neutral pH, the casein micelles carry a negative charge, allowing Ca 2+ ions to act as mineral bridges between them.The formation of mineral bridges between casein micelles is believed to increase their stability against pH changes when H+ ions from lactic acid fermentation by lactic acid bacteria are secreted by BAL cells.As a result, the size of casein micelle aggregates is smaller with the addition of nanotuna fish bone calcium.The smaller the particle size of the yoghurt, the better the quality of the yoghurt.This shows that the particles in yoghurt are in the smaller particle size range, causing the particle distribution to be more uniform and stable (You et al., 2014).The decrease in particle size in yoghurt is caused by the repulsive force between the particles.The higher the concentration of nano calcium in tuna fish bones makes the reciprocal repulsive force between particles greater, so that it is less likely that aggregates will form between particles.On the other hand, the lower the addition of nano calcium in tuna fish bones, the more easily the particles are attracted to each other and aggregate.

Distribution Particle
The analysis of the distribution of particles in yoghurt fortified with nanocalcium from tuna bone did not show a significant effect (p > 0.05) on the distribution of particles in this study.The average particle distribution was obtained from the treatment with added nano-calcium concentration in yoghurt, which was 2.578±0.22-3.027±0.01.
The addition of nanocalcium resulted in a decrease in particle distribution, but did not significantly affect the particle distribution of the yoghurt.Increasing the concentration of nanocalcium in yoghurt production showed that the particle distribution value of the resulting yoghurt decreased.A smaller particle distribution results showed more homogeneous yoghurt is, while a larger particle distribution results in a more heterogeneous yoghurt (Rasaie et al., 2014).
A small particle distribution indicates that the particles in the system are more concentrated in the smaller particle size range, resulting in a more uniform particle distribution (Liu et al., 2021).The stability of solution interactions can be evaluated by the zeta potential, which is the electrostatic repulsion force between particles.This causes the repulsive force between particles in the solution to increase, resulting in smaller-sized solutions that can reflect the degree of protein aggregation.

CONCLUSION
The conclusion of this study is the addition of nanocalcium derived from tuna fishbone causes an increase in pH value, viscosity, water holding capacity, and yoghurt firmness.Furthermore, the addition of nanocalcium from tuna fishbone leads to a decrease in total acid level, syneresis, cohesiveness, yoghurt particle size, and yoghurt particle distribution.The research obtained that the fortification of nanocalcium tuna fishbone can increase the quality of physicochemical yoghurt.

Table 1 .
Average pH, total acidity, viscosity, syneresis, water holding capacity, firmness, and cohesiveness in various treatments Different superscripts in the same column indicated a significant effect between treatments (p < 0.05).

Table 2 .
Average microstructure values of tuna fish bone calcium nano fortified yoghurt in various treatments