PRODUCT DEVELOPMENT OF LOW SUGAR READY-TO-DRINK

Soymilk was processed into a nutritious jelly drink product with low sugar level to overcome the problems of current comercial jelly drink. The optimum concentration of iotacarrageenan (0.1-0.3%) was determined based on sensory evaluation and texture analysis. The preferable sweetness level (2.5-7.5%) was determined based on sensory evaluation. The optimum combination of stevia and sucrose (g/l, 0.211:45, 0.27:45, 0.579:45, 0:75) was determined based on sensory evaluation. The effect of the heating time (30-60 min) and the effect of storage temperature (4 and 25 C) were evaluated by using product characteristic and microbial activity. Results presented in this work suggest that iota-carrageenan was more suitable to be used to produce RTD soy jelly drink than kappa-carrageenan, with 2% addition as the optimum concentration. The combination of 4.5% sucrose and 0.0579% stevia could produce less sugar RTD soy jelly drink with good sensory acceptance. Standard heating process (30 minutes) could hold the microbial activity below the maximum limit up to 21 day at 4 C storage condition, but only has 6 hours at 25 C storage condition. The additional heating time could hold up to about 11 hours. The heating process could increase the texture value of product, and the texture characteristic product would be changing during storage at 4 C.


INTRODUCTION
Jelly drink is a ready to drink beverage that was firstly created as a snack to delay hunger that contains water, sweeteners, carrageenan (that creates jelly texture), artificial flavoring, sweetener, and coloring that create an end product with some unique characteristics such as chewy and sippable by straw (Agustin and Putri, 2014;Nuraeni et al., 2019). However, this product is lacking nutritional value.
People nowadays are getting busier with their work and in need of something practical yet nutritious to temporarily replace their meal with something that is able to delay hunger without disturbing their work time. People also tend to be concerned about the sugar level contained in a product (Sardarodiyan and Hakimzadeh, 2016). By developing the common jelly drink to be more nutritious and less sugar content but still practical, it will surely overcome those problems.
Soy milk extracted from soya bean or Glycine max L. is known to be good for human's general nutrition as the protein source (Shurtleff and Aoyagi, 2016). Many studies have reported the beneficial impacts of the addition of soya bean's derivatives to other food products, such as reducing the risk of coronary heart disease and treatment for iron deficiency. The nutritional content that has the role on the beneficial of soymilk are iron, protein, and amino acids (Baba et al., 2005;Hugelshofer, 1986;Setyaningrum et al., 2017). Regarding that nutritional content, soymilk could be processed into a nutritious jelly drink product.
The combination of sugar with stevia has a potential to produce soy milk jelly drink products with less sugar content but still high in sensory acceptance (Gasmalla et al., 2014;Saniah and Samsiah, 2012;Takaichi and Hatai, 1998). Formulation the proper combination ratio of sugar with stevia is important to be known to achieve that condition.
To make a jelly drink product, carrageenan can be used because of its ability as a gel-forming agent (Kaya et al., 2015). Jelly drink texture is different from normal jelly products because it should be firm enough to form a jelly but soft enough to be sipped using straw with a little amount of syneresis presence (Hartati and Djauhari, 2017). It is important to know the optimum concentration of carrageenan addition to produce soy milk jelly drink products with good texture characteristics.
Microbes tend to grow rapidly in soybean-based products, and soy milk jelly drink has a similar problem. Heating treatment could kill bacteria and prolong the shelf-life of a soy milk product (Kohli et al., 2017). Heating process also takes place in the production of soy milk jelly drink to activate the gelling properties of carrageenan, and producing jelly texture. The observation of the effect of the heating process to the texture characteristic, microbial number, and stability during storage of RTD soy jelly drink product is important.
In this context, the objectives of this work were to determine the optimum concentration of carrageenan addition, the optimum combination ratio of sugar with stevia, and to evaluate the effect of heating process to the texture characteristic, microbial activity, and stability during storage. The parameters used were sippable and microscopic observations. The optimum concentration of iota carrageenan was obtained from analysis of sensory evaluation and texture analysis parameters. The preferable sweetness level of RTD soy jelly drink was determined from analysis of sensory evaluation parameter. The optimum combination of stevia and sucrose to produce less sugar-RTD soy jelly drink was obtained from the analysis of sensory evaluation parameters.

Soy milk production
The selected soybeans were soaked in NaHCO3 solution (0.5% w/w) for 8 h minimum in room temperature. The soaked soybeans were drained and washed using clean water. The soybeans skins were separated from its kernel. The soybeans kernels were collected and blanched in boiling water for 30 min. One hundred g of blanched soybeans were grinded with 600 g of hot water (80-90 °C) for 2 min at high speed. The grinded soy milk slurry was filtered through a strainer and clean kitchen towel (Nirmagustina and Rani, 2013).

Jelly drink production and preliminary analytical stage
Soy milk, carrageenan, sugar, and stevia were mixed and heated at 90 °C for 30 min. This process also aimed to be the pasteurization process of the product. Specifically to evaluate the effect of the heating process, 2 additional heating times were conducted (15 and 30 min). After the product was heated, the product was poured into cups (for analysis) or packed and sealed into pouches directly after to prevent microbial contamination. The products were chilled in the refrigerator for 4-6 h minimum to let the gelling process complete. At the preliminary analytical stage, the production used two types of carrageenan (iota-carrageenan and kappa-carrageenan), in four concentrations of carrageenan addition ( 0.3, 0.4, 0.5, 0.6 % w/v), and used 5% of sugar concentration. Products from all combinations were tested for sippable test and microscopic observation to determine the carrageenan type and concentration used for further analytical stages.

Sippable test
The products from two types of carrageenan in preliminary analytical stages were tested by using the straw of commercial jelly drink. The samples were sucked through the straw to check whether the samples were able to be sipped or not.

Microscopic observation
The product from two types of carrageenan were observed by using stereo microscope with objective lens magnification: 4.5X, and ocular lens magnification: 16X. The analysis was used to determine the suitable carrageenan type to be used for RTD soy jelly drink production.

Texture analysis
Three different iota-carrageenan concentrations (0.1%, 0.2%, 0.3%) on day of production (day 0 at 4 °C) were measured by Penetrometer and statistically analyzed with ANOVA. The result was used to determine the Optimum Carrageenan Concentration.
Texture of chosen carrageenan concentration was analyzed by Penetrometer. The analysis was done after the heating process, and during 3 weeks storage, at 4 °C and 25 °C of storage condition. Then the result was statistically analyzed with nonparametric analysis. The analysis was done to evaluate the effect of the heating process to the texture characteristic, microbial activity, and stability during storage.

Sensory evaluation
The hedonic test was used to determine the optimum concentration of carrageenan addition (sample: 0.1%, 0.2%, 0.3%), evaluation of preferred sweetness level of RTD soy jelly drink (sample: 2.5%, 5%, 7.5%), and the optimum combination ratio of sugar with stevia (stevia:sucrose, g/l, 0.211:45, 0.27:45, 0.579:45, 0:75). The panelists involved in this work were 30 students and laboratory staff from Swiss German University Faculty of Life Science and Technology that were familiar with commercialized jelly drink products. Sensory evaluations using Hedonic tests were statistically analyzed with Friedmann's test and Wilcoxon test with 0.05 as the probability level based on overall likeness of the product using 9points hedonic test where 1 represents "like extremely" and 9 represents "dislike extremely".

PH and brix value
The pH analysis was done by Digital pH meter and Brix value analysis was done by Refractometer. The analysis was applied to evaluate the stability of the product during 3 weeks storage time, at 4 °C and 25 °C of storage condition.

Microbial activity analysis (SNI 2897:2008)
The microbial activity that was represented by Total Plate Count (TPC) Analysis, and compared to Indonesia National Standard of TPC parameter for jelly drink (104 colony/g). Media used for Total Plate Count Analysis was Plate Count Agar using the Pour Plate method. 25 g of sample was diluted to 225 ml 0.1% Buffered Peptone Water (BPW) and homogenized. Then 1 ml of the solution was diluted into 9 ml of BPW as dilution factor 10-1. The diluted sample was again diluted until dilution factor 10-5 was obtained. 1 ml sample from each dilution was poured into a sterilized petri dish. About 12-15 ml of sterilized Plate Count Agar (PCA) was poured into each petri dish and 8figured motion was done to homogenize the sample.
After the agar was set, the petri dish was turned upside down, sealed with Parafilm M and incubated in 371°C for 48 h. Colonies of microbes from range 25-250 were counted as colony form units (CFU)/ml, and converted to colony/g. The microbial analysis was applied to evaluate the effect of heating process and storage.

Preliminary analytical stage
The result shown on Table 1, that the texture characteristic of kappa-carrageenan product at all concentrations caused the product could not be sipped. For the iota-carrageenan product, 0.3% of iota-carrageenan concentration was the only one that sippable. It means the maximum concentration of iota-carrageenan used for the further analysis was 0.3%. Jelly drink production from other raw materials, such as Averrhoa blimbi L. (Agustin and Putri, 2014) and soursop fruit (Gani et al.) could produce good texture characteristics by using both kappa or iota-carrageenan. Regarding its molecular structure in the normal condition, gel characteristic of iota-carrageenan is less-rigid texture than kappa-carrageenan (Median-Torres, 2003). Mineral content in the food added with carrageenan could give effect to the gel characteristic of carrageenan.
The potassium ions in soy milk would interact with kappa-carrageenan and produce firmer texture that was hard to sipped. The calcium ions in soymilk could interact with iota-carrageenan and produce soft elastic gel that is sippable (Popescu et al., 2007). appearance from microscopic observation can be seen on Figure 1. Table 2 shows that the increase of Iota-carrageenan Concentration could increase the texture value of products exponentially (R2 = 0.9922). The texture value would be highly increasing at the iotacarrageenan concentration more than 13%, which led to lower sippability of product. The data also shows that the maximum texture value for the sippable product is 15.00 ± 0.37 N. Iotacarrageenan tends to set after the heating process at 40 -70 °C, the gel-strength ability of the carrageenan increases as the heating process takes place (Thomas, 1997).

Determination of the optimum iota-carrageenan concentration
The sensory hedonic test was observed only based on the texture profile of the product. Table 2 also shows that 0.2 % of iota-carrageenan is highly preferred by the consumers as jelly drink products much higher than 0.1 and 0.3 % carrageenan concentrations. The product with 0.3 % iotacarrageenan was too firm and hard to be sipped, though still sippable. The product with 0.1 % iotacarrageenan was too thin in texture, and it was not firm enough to be categorized as a jelly drink.
It can be concluded that RTD soy jelly drink product with texture value 7.0 ± 0.35 N was highly accepted by the consumers because of its characteristic that was solid enough yet still be able to be sipped using a straw. Those texture characteristics could be achieved by the addition of 0.2 % iota-carrageenan.

The optimum combination ratio of sugar with stevia
According to BPOM regulation (BPOM No. 13:2016 -"Pengawasan Klaim Pada Label dan Iklan Pangan Olahan"), a product can be claimed as "Less Sugar Product" if the product consists of less than 2.5 % sugar in liquid form or less than 5 % in solid form. While the jelly drink is a semisolid form product, it should contain less than 5 % of sugar to be claimed as a less sugar product (BPOM, 2016).  Table 3 shows that the concentration of sucrose 5.0 and 7.5 % gave the preferable sweet taste in the same level, but 2.0 % sucrose gave an un-preferable sweet taste. Each concentration of sucrose also gave different texture to the product as answered by 30 out of 30 panelists stated that higher concentration of sucrose gave firmer texture. Since the sweetness profile of 5.0 and 7.5 % of sucrose were similar, all panelists preferred to consume the product with 7.5% sucrose. Therefore, 7.5 % sucrose concentration was chosen as the most desired RTD soy jelly drink sweetness profile in this stage, and chosen as the sweetness level target on the optimization of combination ratio of sugar with stevia. Since 5 % is the limitation of less sugar product, 4.5 % of sucrose was used as the maximum level of sucrose concentration in sweetener combination with stevia. The three combinations of stevia and sucrose (g/l) respectively were 0.211:45 (Gasmalla, Yang and Hua, 2014), 0.579:45 (Saniah and Samsiah, 2012), 0.27:45 (Takaichi and Hatai, 1998), where all of them were categorized a lesssugar product. Table 4 shows that the combination ratio stevia:sucrose 0.579:45 (g/l) and standard had the similar taste and overall acceptance, even though the aroma and texture were different. This result also shows that 3% of sucrose in the product could be substituted by 0.0579% of stevia addition. Since the amount of sugar used in the final product is less than 5%, the product could be claimed as less sugar product. Based on data, the desired composition to RTD soy jelly drink was 0.2 % iota-carrageenan with combination of 4.5 % sucrose and 0.0579 % stevia. Table 4. Hedonic evaluation of soy jelly drink using several combination ratio of stevia and sucrose

Sensory attribute evaluated
Hedonic score in soy jellydrink that is using stevia and sucrose with combination ratio (g/l)  Table 5 shows that the standard heating time (30 min) was able to kill the microbial activity in the products. The maximum of microbial contamination based on SNI about "Jelly Agar" products is 104 colony/g (Standar Nasional Indonesia, 1994). From the result, it also could be concluded that heating time on 30 min created an edible and safe-to-consume soy milk jelly drink product.   Figure 2 shows that even though the heating process could produce RTD soy jelly drink with no microbes appearing on microbial activity tests, the microbes would be growing rapidly if the product was stored at 25 °C. The shelf life was only 6 h. The additional 15 min of heating time could prolong the shelf life up to about 11 h, but the more heating time addition (30 min) could not give an additional impact to the shelf life.A study showed preservation by high-pressure processing treatment was more effective to kill aerobic and anaerobic bacteria in soy milk product (Kohli et al., 2017). It might be caused by the insufficient heat temperature treated to the product so the spores of microbes from the original sample or the packaging itself tend to grow rapidly at 25 °C storage temperature. Another method of the production process should be considered to get a longer shelf life of RTD soy jelly drink products that are able to be stored at 25 °C. Table 6 shows that the increase of heating time could increase the texture value of the product, while the result of pH and Brix value were not significantly changed. The strength of carrageenan gel is increasing as the increase of temperature, and will be decreasing when the temperature of heating  (Tziboula and Horne, 2000). The heat will alter the networks chain of gel, and at the certain heating condition the gel strength will be lost due to the loss of networks chain of gel (Ainsworth and Blanshard, 1879) . In this work, the temperature used for production was 90 o C, and it was still under 100 o C as the maximum temperature of carrageenan heat treatment. This heating condition still gave the positive impact to the gel strength of the product. The increase of texture value has a potential to change the sensory acceptance and sippability of the product. Unfortunately, this work has no data regarding the sensory evaluation of products on different heating times.

60
12.1 ± 0.65 c 7.1 ± 0.121 c 3.5 ± 0.00 <900 Figure 3 A and B show that the texture characteristics of products with standard heating processes during storage at 4 °C were changing. Based on data, the texture of products was still under the maximum acceptability limit (15.0 N) until day 21, even though the texture was becoming harder. This changing characteristic was caused by the condition that water inside gel formcarrageenan was moved out from the solid matrix, producing a harder gel form and higher syneresis value of product.

CONCLUSION
Results presented in this work suggest that iotacarrageenan was more suitable to be used to produce RTD soy jelly drink than kappacarrageenan, with 2 % addition as the optimum concentration. The combination of 4.5 % sucrose and 0.0579 % stevia could produce less sugar RTD soy jelly drink with good sensory acceptance. Standard heating process could hold the microbial activity below the maximum limit up to 21 day at 4 °C storage condition, but only has 6 h at 25 °C A B J. Functional Food & Nutraceutical 2020:2(1), pp.44-52 51 storage condition. Additional heating time could hold the microbial activity below the maximum limit only up to about 11 h at 25 °C storage condition. The heating process could increase the texture value of the product, and the texture characteristic product would be changing during storage at 4 °C.