Shelf-Life Determination of “Hiwan Tahu” Using Accelerated Shelf-Life Testing (ASLT) with Arrhenius Model

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I. INTRODUCTION
Tofu meatballs are traditional food from Ungaran made from tofu filled with meat.CV.Maju Jaya Indonesia is one of the medium food industries in Ungaran, which can produce tofu meatballs up to ±15,000 pieces per day.The production of tofu meatballs in this company has implemented quality control through a sorting process to separate tofu that is qualified and doesn't qualify for physical quality standards.This quality control produces by-products of tofu meatballs in the form of tofu that doesn't qualify for physical quality standards, for example, torn, having a size or thickness that is different from the standard, having a dull and uneven color, and having a texture that is too hard or too soft [1].These byproducts can be used as filling for "hiwan tahu" to increase their economic value.
"Hiwan" is a popular food among Chinese people in Indonesia."Hiwan" is a fish meatball product with pork filling, so it is haram consumed by Muslims.It is necessary to develop a halal version of "hiwan" to expand consumers."Hiwan tahu" is the halal version of "hiwan"."Hiwan tahu" is made from chicken meat, tapioca flour as the outer part, and tofu as the filling material."Hiwan tahu" is easily damaged during storage because it contains high water content, high protein, and a neutral pH suitable for bacterial growth.Therefore, shelf-life testing is essential to ensure the quality and safety of "hiwan tahu" until it reaches consumers.Industries use two methods to determine shelf life: the conventional method and the acceleration method.The conventional method is carried out by periodically observing product quality until the product is damaged at the end of its shelf life.The acceleration method is carried out by observing the quality of the product, which is accelerated by storing it in extreme conditions [2].Using the ASLT method, the product's shelf life can be predicted by extrapolating the equation under normal storage conditions.The acceleration method can be done quickly and accurately [3].This method calculates the shelf life of "hiwan tahu".
The acceleration method has several approaches, including a semi-empirical approach using the Arrhenius model and the critical water content approach using the Labuza model [4].The Arrhenius method is usually used for products sensitive to temperature changes, such as "hiwan tahu" [5].Shelf-life testing of "hiwan tahu" in this study involved parameters of Total Plate Count (TPC), pH, water content, and water activity ( ).During product storage at extreme temperatures, an increase in microbial growth can occur which also causes an increase in pH, moisture content, and of "hiwan tahu".

II. MATERIALS AND METHOD
A. The Process of Making "Hiwan Tahu" "Hiwan Tahu" was made using a predetermined formulation summarized in Table 1.Making "hiwan tahu" begins with the chicken breast being ground in a chopper and the mustard greens being mashed in a blender.Seasonings such as garlic, candlenut, pepper, salt, and Monosodium Glutamate (MSG) were mashed.Tapioca flour, eggs, mustard greens, and ground spices were mixed with the chicken meat.The dough was mixed until evenly distributed.The byproduct of tofu meatballs was cut into small squares weighing ± 6 g.Next, the dough was formed into a round shape, and then the middle of the dough was filled with tofu.After that, "hiwan tahu" was boiled in boiling water for five minutes, drained, and cooled.

B. Product Storage and Observation of Quality Changes
"Hiwan tahu" were packed in vacuum packaging and stored at three different storage temperatures, including 5℃, 27℃, and 40℃.Furthermore, the initial characteristics and quality degradation of "hiwan tahu" during storage were determined by testing the total plate count (TPC) [6], pH, water content, and water activity ( ) [7].Observations were carried out every 3 hours until the 12 th hour for storage at 40℃, every 24 hours until the 96 th hour at 27℃, and every 48 hours until the 192 nd hour for storage at 5℃.The pH was determined [8] with a pH meter that had previously been calibrated using a standard buffer solution of pH 4 and pH 7. Furthermore, 5 g of samples were mashed and dissolved into 45 mL of distilled water, and then the suspension was stirred until homogeneous.The electrode was dipped into the suspension, and the pH value can be seen on the monitor screen.

C. Shelf-Life Determination of "Hiwan Tahu"
Determination of the shelf life of "hiwan tahu" using the ASLT method with the Arrhenius model begins with the data obtained from observation during storage are plotted with storage time to obtain a regression equation for zero order graphic, while a regression equation for the first order graphic is obtained by change the data into ln and then plotted with storage time.In the graphic, three regression equations y = a + bx will appear to represent quality changes in each of the three storage temperatures, where y is the change in product quality, x is the storage time (hours), a is the initial quality value of "hiwan tahu", and b obtained from the slope is the rate of change in quality (k value).The reaction order is determined by comparing the R 2 value of each equation.The reaction order chosen is the reaction order that has the largest R 2 value or is closest to 1.The next step is making the graphic correlation between the ln k value and 1/T (K).The quality degradation constant (k value) obtained from the regression equation on the reaction order graph is changed to ln k for each storage temperature.The ln k value is then plotted with 1/T, where T is the storage temperature in Kelvin units [9].The equation obtained from this graphic is equal to this equation: The equation above comes from this Arrhenius equation: where k is quality degradation constant, k0 is pre-exponential constant, Ea is activation energy, T is absolute temperature (K), and R is gas constant (1986 cal/mol K) The next step is determining the critical parameters based on the lowest activation energy.The slope in the graphic correlation of ln k value and 1/T is equivalent to the value (Ea/R), so the activation energy can be obtained by substituting the R-value (ideal gas constant = 1.986 cal/mol K) into the equation.The equation on the graphic correlation of the ln k value with 1/T can also be used to find the k value.The k value is the Arrhenius constant which indicates the quality degradation rate at each storage temperature.The k value is obtained by substituting the storage temperature into the equation.The k value obtained will be used in calculating the shelf life of "hiwan tahu" using the following formula: for 0 order reaction rate: for 1 st order reaction rate: where ts is shelf life, $ is initial quality value of the product, $ % is critical value, and k is quality degradation constant.

A. Initial Characteristics of "Hiwan Tahu"
Based on the observations, "hiwan tahu" has a fresh light green color, a springy, elastic, and not mushy texture, a distinctive chicken aroma, and a savory chicken meat taste.The initial characteristics of "hiwan tahu" are summarized in Table 2.The initial Total Plate Count (TPC) value of "hiwan tahu" was 7.4×10 3 CFU/g.The initial value of TPC still qualified the maximum TPC value for meatballs which is 1×10 5 CFU/g.An increase in total microbes indicates a decrease in the microbiological quality of processed meat products, such as "hiwan tahu" during storage.The initial pH of "hiwan tahu" is 5.31."Hiwan tahu" is classified as a chicken meat product.The pH of chicken meat is 5.8 [10].High pH will accelerate the growth of microbes' growth, resulting in meat products deteriorating more quickly.The initial water content of "hiwan tahu" is 67.3116%.The initial water content still qualified the maximum water content limit for meatballs which is 70%.High water content can accelerate the deterioration of meat products [11].The initial water activity ( ) of "hiwan tahu" is 0.740."Hiwan tahu" is classified as a processed meat product.Meat products twithan around 0.91 tend to have a long shelf life because the pathogens can't grow [12].The high of will accelerate microbial growth in chicken meat products [13].

B. Total Plate Count (TPC) Changes During Storage
The results of the Total Plate Count (TPC) analysis of "hiwan tahu" during storage are summarized in Figure 1.The TPC value of "hiwan tahu" increases during storage.The total microbes of "hiwan tahu" at the beginning of storage is 7.4×10 3 CFU/g, while the total microbes at the end of storage at 5℃ (192 nd hour), 27℃ (96 th hour), and 40℃ (12 th hour) are 2.4×10 6 CFU/g, 1.3×10 6 CFU/g, and 2×10 5 CFU/g.The maximum limit for the TPC value of meatballs is 1×10 5 CFU/g [14].The TPC value of "hiwan tahu" at the beginning of storage still qualified for SNI standard, but the TPC value at the end no longer qualifies for the requirement.The reaction rate of changes in TPC at each storage temperature can be seen from the graph's k value (slope).The k values at 5℃, 27℃, and 40℃ are 14.311, 14.326, and 14.340.The higher the k value (slope), the higher the rate of change in the TPC value, so the increase in TPC at a storage temperature of 40℃ occurs faster than at 5℃ and 27℃.

C. The pH Changes During Storage
The results of the pH analysis of "hiwan tahu" during storage are summarized in Figure 2. The pH of "hiwan tahu" increased during storage.The pH of "hiwan tahu" at the beginning of storage is 5.31 while pH at the end of storage at 5℃ (192 nd hour), 27℃ (96 th hour), and 40℃ (12 th hour) are 6.02, 6.30, and 6.42."Hiwan tahu" is made from chicken meat which has a pH standard of 5.8.A high pH value (6.1-7.2) causes the structure of chicken meat to become closed and facilitates microbial growth [17].The reaction rate of changes in pH at each temperature can be seen from the k value (slope) on the graph.The k value at 5℃, 27℃, and 40℃ are 0.0041, 0.0085, and 0.078.The higher the k value (slope), the higher the rate of change in pH, so that the increase in pH at a storage temperature of 40℃ occurs faster than at 5℃ and 27℃.Storage temperature can affect pH because it is closely related to microbial growth, affecting the pH of meat products.Spoilage bacteria generally grow optimally at temperatures of 35℃-47℃ and pH 6.5-7.5 [18].Besides temperature, storage time also affects the pH of "hiwan tahu".The longer the storage, the higher the pH of meat products increases due to the accumulation of metabolite compounds resulting from microbial activity through protein deamination reactions [19].

D. Water Content Changes During Storage
The results of the water content analysis of "hiwan tahu" during storage are summarized in Figure 3-the water content of "hiwan tahu" increases during storage.The water content of "hiwan tahu" at the beginning of storage is 67.3116%, while the water content at the end of storage at 5℃ (192 nd hour), 27℃ (96th hour), and 40℃ (12th hour) are 70.34%;70.28%; and 70.04%.The maximum water content for meatballs is 70% [14], so the water content of "hiwan tahu" at the beginning of storage still qualified for the SNI requirement, but at the end of storage, it doesn't qualify for the requirement.The reaction rate of changes in water content at each storage temperature can be seen from the k value (slope) on the graph.The k value at 5℃, 27℃, and 40℃ are 0.0137, 0.0296, and 0.2168.The higher the k value (slope), the higher the reaction rate of changes in water content, so the increase in water content at a storage temperature of 40℃ occurs faster than at 5℃ and 27℃.Storage temperature can affect the water content.The water content of "hiwan tahu" increases at higher temperature storage.During the storage of meatballs, microbes carry out metabolism by breaking down glucose into water and CO2 as well as releasing energy [20].The higher number of microbes, the more water is produced from metabolism which can contribute to the water content of the product.

E. Water Activity (aw) Changes During Storage
The results of the water activity ( ) analysis of "hiwan tahu" during storage are summarized in Figure 4. Water activity ( ) of "hiwan tahu" increases during storage.Water activity ( ) of "hiwan tahu" at the beginning of storage is 0.740; while aw at the end of storage at 5℃ (192 nd hour), 27℃ (96 th hour), and 40℃ (12 th hour) are 0.7515, 0.758, and 0.7885.Microbes generally can grow in food products with a minimum value of 0.60 [21].The rate change of at each storage temperature can be seen from the k value (slope).The k value at 5℃, 27℃, and 40℃ are 0.00006, 0.0002, and 0.0039.The higher the k value (slope), the higher the reaction rate of changes in the value so that the increase in value at a storage temperature of 40℃ occurs faster than at 5℃ and 27℃.
Storage temperature can affect water activity ( ) because it is related to the growth and activity of microbes.The higher tthe temperature and the longer the torage of "hiwan tahu", the higher the rate of increase in the water activity ( ).Storage at higher temperatures causes microbes to grow rapidly in a shorter time.During storage, the degradation of material molecules by microbes occurs by releasing bound water to form free water [22].

F. Determination of Reaction Order
The Arrhenius model can the rate of food product deterioration accelerated under extreme storage temperature conditions.The rate of quality degradation in food products generally follows the zero-order or first-order reaction.Zeroorder reactions occur when the quality degradation follows a linear line, while first-order responses occur when the quality degradation follows an exponential line [23].The reaction order of each quality parameter must be determined to determine the degradation of "hiwan tahu" quality during storage based on these parameters, following a zero or firstorder reaction.Determining the correct reaction order will result in better accuracy in calculating the shelf life of "hiwan tahu".The determination of the reaction order of each parameter is summarized in Table 3.The reaction order is determined based on the highest R 2 value.The higher of R 2 value, the more accurate the equation [24].If the value of zero-order R 2 is higher than that of first order R 2 , then the rate of quality degradation will follow zeroorder.If the value of first order R 2 is higher than that of zeroorder R 2 , then the rate of quality degradation will follow the first-order.Based on Table 3, the deterioration reaction of "hiwan tahu" based on the TPC parameter has an R 2 value of first order that is higher than the R 2 value of zero-order.Hence, the rate of change in the TPC value follows the first order.In contrast to the pH, water content, and parameters which have R 2 values of zero-order that are higher than the value of R 2 of the first order so the rate of change in pH, water content, and follow zero-order.

G. Determination of Critical Parameter
After determining the reaction order, the next step is plotting the ln k value with 1/T (Kelvin).The k value (slope) or the quality degradation rate constant obtained from the regression equation on the reaction order graph is changed to the ln k and plotted with 1/T (Kelvin) to form a linear regression equation graph which is equivalent to the Arrhenius equation ln k = ln k0 -Ea/RT so that the activation energy (Ea) of each parameter can be calculated to select one critical parameter [25].The results of plotting the ln k and 1/T for each parameter are summarized in Table 4.

H. Determination of Shelf-Life
The next step after determining the critical parameter is calculating the k value or the degradation rate at each storage temperature based on the selected critical parameter.The Arrhenius equation in the correlation graph between the ln k value and 1/T (Kelvin) can be used to find the k value.The k value is obtained by substituting the storage temperature into the equation.The k value can be obtained through the calculation below: The k value that has been obtained is then used to calculate the shelf life of "hiwan tahu" using the formula t = "#( )

!
for the first order reaction rate, where N0 is the initial quality value while Nt is the critical quality limit, and k is the degradation rate constant at temperature T (K).Shelf life of "hiwan tahu" at storage temperature T(K): The higher the storage temperature, the shorter the "hiwan tahu" shelf life.The shelf life of "hiwan tahu" based on ASLT calculations according to the Total Plate Count (TPC) parameter at freezer temperature (−18℃), refrigerator temperature (5℃), and room temperature (27℃) are 68.54days, 16.27 days, and 5.05 days.The shelf life of "hiwan tahu" in this study is similar to that of other processed meat products such as meatballs.Based on the research [26], the shelf-life of chicken meatballs stored at freezer temperature (-20℃) is 2 months.Other research [27] states that beef meatballs have a shelf life of 9 days if stored at ±5℃ while chicken meatballs with edible coating control treatment at ±5℃ temperature storage are no longer suitable for consumption on the 10 th day because TPC value is 5.92 logs CFU/mL and exceeds the SNI standard, which is 5 logs CFU/mL [28].When stored at room temperature (25℃), the shelf-life of vacuum-packed beef meatballs is 4.81 days [29].
Processed meat products such as "hiwan tahu" are easily damaged and have a short shelf life because they contain relatively high-water content, high nutrients, especially protein, and a pH close to neutral (5.5 -6.5) which supports microbial growth [30].Spoilage bacteria can decompose proteins, carbohydrates, and fats which causes off-flavors, a sticky or slimy texture, and discoloration of meat.The breakdown of protein into peptides or amino acids causes offodors and off-flavors in the meat products due to the formation of ammonia, hydrogen sulfide, methyl sulfide, amines, and other volatile compounds [31]."Hiwan tahu" is easily damaged, especially if stored at high temperatures.The higher temperature of storage, the faster the rate of change in the "hiwan tahu" quality.Therefore, the recommended storage to extend the shelf life of "hiwan tahu" is frozen storage at -18℃ in the freezer.During frozen storage, some of the product's water content undergoes crystallization, inhibiting water mobility so that water activity decreases and microbial growth can be inhibited due to dehydration [32].
Dehydration effectively inhibits microbial growth and can also affect the texture, flavor, and nutritional content of the meat.Additionally, proper hygiene, storage conditions, and packaging are critical to maintaining the quality and safety of dehydrated meat products.Following food safety regulations and guidelines is important to ensure that the dehydration process meets industry food safety standards [33].

IV. CONCLUSION
Based on the research, it can be concluded that the quality of "hiwan tahu" stored at three different storage temperatures decreased during storage.During storage, the Total Plate Count (TPC), pH, water content, and value of "hiwan tahu" decreased.The higher the temperature of storage, the faster the degradation rate.The critical parameter is the Total Plate Count (TPC) because it has the lowest activation energy.The shelf life of "hiwan tahu" stored at −18℃, 5℃, and 27℃ was 68.54 days, 16.27 days, and 5.05 days.

Fig. 1
Fig. 1 Changes in Total Plate Count (TPC) during storage

Fig. 2
Fig. 2 Changes in pH during storage

Fig. 3
Fig. 3 Changes in water content during storage

Fig. 4
Fig. 4 Changes in water activity during storage

TABLE II INITIAL
CHARACTERISTICS OF "HIWAN TAHU"

TABLE IV ARRHENIUS
EQUATION AND ACTIVATION ENERGY