Effects of tilapia bone gelatin addition in broccoli jelly candy towards the protein, fat, fiber, and vitamin C content

This study aims to evaluate the effect of tilapia bone gelatin addition to the formulation of broccoli jelly candy and determine the best formulation from its protein, fat, fiber, and vitamin C content. The results show that tilapia bone gelatin addition to broccoli jelly candy significantly increases protein content from 8.17 - 12.19%, due to the gelatin composition that mostly consists of protein. This addition also increases the vitamin C content from 7.26 - 15.18 mg/g, due to gelatin’s ability to improve the thermal stability of ascorbic acid. However, the addition of gelatin does not affect the fat and fiber content. The best formulation based on protein content is Formulation 6 (13g of gelatin), meanwhile, formulation 4 (11g of gelatin) has the best formulation for vitamin C content.


Introduction
Indonesia is one of the countries that has a problem of malnutrition in children who dislike vegetables.93.6% of these children are preschool-aged children [1].Vegetables are essential foods that must always be consumed at meals.Goal 3 of UNSDGs, "Ensure healthy lives and promote well-being for all at all ages," emphasizes the importance of addressing health and well-being issues, particularly in children.This goal aligns with the critical need for vegetable consumption, especially during the age of 3-6 years, which is considered a golden period for growth and development in children.Unfortunately, children often dislike vegetables due to their bitter taste, texture, and unique odor.Moreover, this aversion can be influenced by the lack of provision of vegetables by parents in the children's daily meals and the lack of parental knowledge of the importance of vegetable consumption [2].
Jelly candy is a food product that can be processed from various colors, raw materials, and flavors.The main ingredient of jelly candy is sugar which is used in the form of sucrose or glucose syrup and combined with gelling agents, acids, and aromas [3].Jelly candy is widely consumed, especially by children, because of its soft and elastic texture and comes in various of fruit flavors [4].Jelly candy is a snack children favor because of its sweet taste and chewy texture.Children need sugar as energy for the brain during growth and development, so jelly candy can be an easy alternative for children to consume [5].In the manufacture of jelly candy, it usually uses gelling agents that have a reversible reaction or if the gel is heated it will form a liquid and when cooled it will form a gel again [6].Gelatin is one of the food additives that is often used in the jelly candy-making industry.The use of gelatin in the food industry is as an emulsifier, stabilizer, foaming agent encapsulant, and film-forming material [7].Fishbone gelatin is one type of gelatin that contains a high source of protein.Tilapia bone gelatin was chosen because tilapia bones is still not widely utilized [8].Gelatin is obtained by partial thermal hydrolysis of collagen and contains essential and high hydrocolloids.Gelatin which is widely used is derived from pigskin, cowhide, and cow bone.Because there are some problems such as allergies and halal issues.Therefore, gelatin from tilapia bones is an excellent alternative to collagen as a source of gelatin [9].The broccoli jelly candy mixture uses broccoli as the main ingredient and uses gelatin derived from tilapia bones.This gelatin can be derived from the extraction of tilapia fish bones.Gelatin derived from fish bones has a unique character because it has a reasonably high collagen content [10].Broccoli jelly candy is an innovation to overcome children's dislike of vegetables.Jelly candy made from vegetables has a higher nutrition content than jelly candy on the market [11].
Broccoli (Brassica oleracea L. italica) can be said to be a superfood because broccoli is one type of vegetable plant that comes from the cabbage family and contains various kinds of nutrients such as carbohydrates, protein, and minerals as well as multiple types of vitamins that have beneficial effects for the health of the body [12].The most extensive nutrient content in broccoli is vitamin C (89.2 mg/100 g) [13].Other nutrients that are also included in broccoli are vitamin K, iron, and potassium [14].Broccoli also contains calcium similar to milk and is a good source of P, Ca, Mn, Fe, Mg, Se, and Zn.In addition, broccoli is also a rich source of fiber [15].This vegetable from the cabbage family contains the most essential antioxidant compound called sulforaphane.In addition, broccoli also contains betacarotene, quercetin, and glutathione [16].
Until now, there have not been many studies related to making broccoli jelly candy using tilapia bone gelatin and the effect of gelatin addition on various concentrations in terms of protein, fat, fiber, and vitamin C content.It makes this research necessary to do.It is hoped that through this research, the formulation and the nutritional content of broccoli jelly candy will be one way to fulfill children's vegetable consumption needs and contribute to the fulfillment of Goal 3 of the UN Sustainable Development Goals (SDGs), which aims to ensure healthy lives and promote well-being for all at all ages.

Materials
This research used broccoli obtained from the market, the gelatin obtained from the commercial online marketplace Tokopedia.Chemical reagents used for analysis such as ethanol, H2SO4, NaOH, n-hexane, HCl, selenium mixture reagents, iodine, etc. were obtained from Merck (PT Merck Tbk.Merck Groups Companies in Indonesia) and from Smart Lab (PT Smart Lab in Indonesia).

Preparation of broccoli juice
Broccoli (100g) is washed with water and soaked in salt water for 10 minutes.Broccoli is blanched at 100 o C for 2-3 minutes.Broccoli is removed, drained, and cut. 100 mL of water is added and pureed with a blender until smooth.Broccoli juice is filtered using a filter cloth [17].

Making of broccoli jelly candy
Tilapia bone gelatin weighed as much as 8, 9, 10, 11, 12, and 13 grams and was dissolved with broccoli juice as much as 25 mL.Gelatin that has dissolved completely is added with 33 grams of sugar, 32 grams of high fructose syrup, and 2 grams of citric acid.Gelatin was cooked at 80-90 o C for 5 minutes.The solution that has been formed is poured into the mold and cooled at room temperature for 1 hour.Broccoli jelly candy was put into the refrigerator for 12 hours.The hardened broccoli jelly candy was placed at room temperature for 1 hour and removed from the mold [18].

Chemical analysis
The chemical analysis was carried out using the AOAC 2005 method.Analysis of protein was carried out using Kjeldhal method.Analysis of fat was carried out using Soxhlet method.Analysis of fiber was carried out using gravimetric method.Analysis of vitamin C was carried out using titrimetic methods.

Data analysis
Data analysis for broccoli jelly candy protein, fat, fiber, and vitamin C content was carried out using one-way ANOVA with Statistical Package for the Social Science (SPSS) software version 25.0.The resulting data will be presented as mean or average.One-way ANOVA was used to assess the differences of each formulation in broccoli jelly candy.Duncan's test was used as part of the Posthoc test to identify significantly different mean values, if the p value <0.05 then the results obtained are significantly different [19].Based on the data shown above with the Post-hoc Duncan test on the protein content of the broccoli jelly candy formulation, the results obtained are significantly different, with a value of 0.011 (p<0.05).F2, F3, and F4 were not significantly different from F1. F5 was significantly different from F1, but not significantly different from different from F2, F3, and F4.The highest protein content was found in F6 with gelatin addition treatment of 13 grams, which was significantly different from F1 with a protein content 8.17% and F3 with a protein content of 9.67%, but not significantly different from F2, F4, and F5.This is because the more gelatin is used in the making of broccoli jelly candy, the greater the protein contained in the jelly candy.According to [20], gelatin made from tilapia bone waste contains 30.6% of protein.

Protein content
Protein levels in each formulation used in the test have significantly different values.The increase in protein content of broccoli jelly candy follows the increase in the amount of bone gelatin used in each formulation because the higher of tilapia bone gelatin used in each formulation can affect the increase in protein content of broccoli jelly candy.This is in accordance with the statement of [21] which says that the more of the amount of gelatin is added to each jelly candy formulation, the higher the protein content.

Fat content
Notes: Treatments that have the same number of notations indicate not significant difference by Duncan test (p>0.05)

Figure 2. Fat Content of broccoli jelly candy in each formulation
Based on the image data above with the Post-hoc Duncan test on broccoli jelly candy, it was found that the fat content of broccoli jelly candy from F1 to F6 did not change, with a p-value of 0.957 (p>0.05), which means that the fat content of broccoli jelly candy is not significantly different or has no significant effect on broccoli jelly candy.
The fat content of broccoli jelly candy is not significantly different because the most significant content in gelatin is protein and the amount of fat in gelatin can be classified as minor or none because in the manufacture of gelatin from tilapia bones, a degreasing process is carried out for fat removal.The method of making tilapia bone gelatin using acids and bases is one method to remove the fat content contained in tilapia bones [22].The fat content of broccoli jelly candy is in accordance with the fat content of tilapia bone gelatin which ranges from 0.64% -2.073%.The low fat content is due to the use of NaOH which can remove the remaining fat in fish bones [23].

Fiber content
Notes: Treatments that have the same number of notations indicate not significant difference by Duncan test (p>0.05)

Figure 3. Fiber content of broccoli jelly candy in each formulation
Based on the data shown above with the Post-hoc Duncan test on the fiber content of broccoli jelly candy in various formulations of adding tilapia bone gelatin with a p-value of 0.373 (p>0.05) was found.This indicates there was no significant difference between the different formulations.Tilapia bone gelatin does not give a significant difference effect because gelatin does not contain fiber and the largest content of gelatin itself is protein.The amount of fiber content contained in broccoli jelly candy can be influenced by the fiber content of broccoli.According to [24], broccoli contains 10.27 grams/100 grams of fiber and the amount of broccoli juice that used in broccoli jelly candy is not different.
Tilapia bones are a source of sodium, phosphorus, and calcium minerals.Tilapia bone gelatin does not contain fiber because this gelatin comes from bone, obtained from animal food.Gelatin is a protein extracted from collagen found in animal connective tissue [25].F1 is not significantly different from F3, F4, F5, and F6.F2 is significantly different from F3, F4, F5, and F6.Based on the results of statistical tests, vitamin C in broccoli jelly candy has a p-value of 0.001 (p<0.05),meaning that the levels of vitamin C contained in broccoli jelly candy are significantly different.The changes in the vitamin C content of broccoli jelly candy can be influenced by several factors, such as the heating of broccoli and gelatin, the cutting process of broccoli, and storing broccoli jelly candy.The increase in vitamin C content in formulations 3 to 6 in the broccoli jelly candy is possible due to an increase in the amount of tilapia bone gelatin.This in accordance with the research of [26], which states that the higher the concentration of gelatin in a product, it will affect the higher the vitamin C contained in it.The high level of vitamin C in gelatin treatment is caused by the withdrawal of more colloidal particles, with the withdrawal of colloidal particles causing less free oxygen that can cause oxidation reactions to broccoli jelly candy.In the research of [27], gelatin can bind the components of vitamin C, so that the vitamin C is not easily diffused.This interaction will increase the thermal stability of vitamin C in jelly candy processing by using heat.The amount of vitamin C that diffused is inversely proportional to the viscosity of broccoli jelly candy caused by the higher concentration of gelatin that is used.

Conclusion
The formulation of broccoli jelly candy with the addition of tilapia bone gelatin does not have a significant effect on fiber content and fat content, but the formulation of broccoli jelly candy with the addition of tilapia bone gelatin has a significant effect on the vitamin C content and protein content.The increase in protein content of broccoli jelly candy is because of the gelatin largest component, which is protein.Vitamin C levels in broccoli jelly candy are increased, possibly due to the use of tilapia bone gelatin, which causes the interaction of colloidal particles that bind free oxygen, reduce oxidation reactions and increase thermal stability.The best formulation based on protein content is Formulation 6 (13g of gelatin), meanwhile, formulation 4 (11g of gelatin) has the best formulation for vitamin C content.

Figure 1 .
Figure 1.Protein content in of broccoli jelly candy in each formulation Notes: Treatments that have different number of notations indicate the significant difference by Duncan test (p<0.05)

Figure 4 .
Figure 4. Vitamin C content of broccoli jelly candy in each formulation

Table 1 .
Composition of Broccoli Jelly Candy Formulations