Utilization of shrimp shell as a substitute ingredient in mineral and protein enrichment of tempeh steak product

Indonesia has great potential for seafood products and has imported seafood products as one main imported product for the marine industry. The imported shrimp is deshelled, so the shell has a great potential to end waste and can be harmful if disposed to the environment. In this study, the shrimp shell will be utilized as a substitute in the production of tempeh steak to determine the effect of different concentrations of Shrimp Shell Flour (SSF) addition to the tempeh product. This study resulted that the addition of SSF added the ash (mineral) and protein content and lowered the water content along with the higher concentration of SSF added. The addition of SSF did not have an effect on the fat and carbohydrate content of the product produced. It can be concluded that the SSF can be utilized as a substitute to increase the ash (mineral) and protein content of tempeh steak products.


Introduction
Indonesia has great potential for seafood products.It is categorized as the main imported product in the marine ministry.Even in 2022, the marine export elevated to 10.66% [1].Besides imported, shrimp also process into several products.Shrimp processing has a great potential to end waste and can be harmful if disposed to the environment.Meanwhile, shrimp contains nutrition that can be used to improve food products like proteins and minerals [2].Not only the meat of the shrimp but also the waste shrimp including the shell and the tail contains a considerable amount of protein and ash [3].It gives the shrimp shell potential to be applied as a substitute for value-added human food ingredients.Therefore, it's critical to innovate with dietary items that are widely known and seen as acceptable by the general population, one of which is tempeh.
Tempeh also has gained a lot of attention in research and appears to be the most used keyword in the traditional Indonesian food field [4], especially for the last five years [5][6][7][8][9][10], which implies that it is worth making enhancing the tempeh-based product.Therefore, new tempeh-based products must be developed in order to get a formulation with a higher nutritional content that supports growth.In this case, the addition of shrimp shell flour can be applied.
However, to the best of our knowledge there has been no prior research has utilized this innovation, and the precise recipe for producing tempeh-based items with the addition of shrimp shells is unknown.This study, therefore, aimed to observe the effect of shrimp shell flour addition on the nutritional properties of tempeh steak, especially for the ash (mineral) and protein.

Materials
The tempeh used in this research was purchased fresh from the traditional market the same day for the production of tempeh steak.Shrimp shell (vaname (Litopenaeus vannamei)) was obtained from a shrimp export company in Makassar, distilled water was purchased from the chemical store (Sumber Rejeki), All chemical used were in the PA grade, including chloroform, selenium, H2SO4, distilled water, NaOH boric acid (H3BO3), bromocresol green, methyl red indicator, and 0.1 N HCl.

Flour shrimp shell production
Making Shrimp Shell Flour (SSF) is done by first cleaning the shrimp shells and then soaking them in a salt solution with a concentration of 20% for 30 minutes.Then rinsed and dried in an oven at 50ᵒC for 8.5 hours.Then ground and filtered to obtain SSF.

Tempeh steak production
Making tempeh steak was done by first prepared 250 grams of tempeh then steamed and added with different concentration of shrimp shell flour (0%, 5%, 10%, 15%, and 20%).Then added with 15 ml soy sauce, 6gram salt, 3gram flavouring and 25gram wheat flour.It is then mixed and moulded.

Parameters
2.4.1.Moisture content.Moisture content analysis following the [11] method using oven.The empty cup was dried in an oven for 30 minutes between 100 and 105⁰C.The sample is then weighed between 2 and 5 grams and placed in a cup whose weight is known.After that, place in an oven preheated to 100 to 105⁰C for 3 to 5 hours.The sample was then weighed after cooling in a desiccator for 15 minutes.The sample was then re-heated for 30 minutes, allowed to cool in a desiccator, and weighed once again.Up until a steady weight was achieved, the procedure was repeated.The water content is calculated using the formula: 2.4.2.Ash content.Ash content analysis following the [11] with modification.The porcelain dish is prepared first, then dried for 20 minutes in the oven.After cooling in a desiccator, the cup was weighed.
The sample was then weighed to a weight of up to 10 grams and put into a cup.The sample is then heated to between 600⁰C in a furnace until it turns to ash.After cooling in a desiccator, the final weight was measured.Ash then was calculated using the formula: First of all, the sample is weighed as much as 0.5 gram and then put into the Kjeldahl flask.Then 1 gram of selenium was added to each Kjeldahl flask which had been filled with samples, then 7 ml of H2SO4 solution was added.After that, the samples were destructed at 430⁰C for 1-2 hours until the solution became clear, then decomposed until the solution formed crystals.After cooling, the solution was added with 50 ml of distilled water and 7 ml of 40% NaOH and then distilled using a desilator at 100⁰C.Then the distillation results were collected in a 125 ml Erlenmeyer which had been filled with 10 ml of 2% boric acid (H3BO3) and added 2-4 drops of bromocresol greenmethyl red indicator.After the distillate volume reaches 50 ml and turns green, the distillation process is stopped.Then the distillate was titrated using 0.1 N HCl solution until it turned pink.The titrant volume is read and recorded.Protein content is calculated using the formula: Information: V1 = Volume sample titration V2 = Volume for blank titration fk = Conversion factor of protein (6.25) 2.4.5.Carbohydrate content.Carbohydrate measurement performed using by difference method following [11] that performed using following formula: Carbohydrate (%) = 100% − % (Protein+ Fat + sh + Water) (5)

Results and discussion
The steak tempeh made in this study was with the addition of shrimp shell flour that was expected to enrich the total mineral (ash) and protein along with the higher concentration of shrimp shell flour addition.The analysis performed in this research included water, ash, protein, fat, and carbohydrates content.

The effect of shrimp shell flour to the water content of Tempeh Steak
Water content of food is a total of water contains in food or food product.Water is very much correlated to the shelf life of food as it can be related to the growth of microorganisms.Besides, water also contribute to the texture of food or food product.In this study, it is expected that the water content of product will be lower along with the higher concentration of SSF since the flour is categorized as dry product which contains less water.The water content obtained in this study range between 48.275-55.349%.Statistical analysis shows that there is a significant effect (p<0.05) of the SSF concentration to the water content of tempeh steak product.The water content of tempeh steak with different concentration of SSF addition is presented in Figure 1. Figure 1 depicts that there is a tendency of lower water content as the higher concentration of SSF added.It was due to the characteristics of flour which is dry and by adding high concentration of SSF it will reduce the concentration of tempeh used and further will reduce the water content.All the water content found in this study were below 65% and it have met the quality standard set by Indonesian National Standard (SNI) for tempeh [14].

The effect of shrimp shell flour to the ash content of Tempeh Steak
Ash content is a total of ash contains in food.Ash content is represent the mineral content of food [15].In this study, it is expected that the ash content of product will be higher along with the higher concentration of SSFs since the SSF contains high amount of mineral.The ash content obtained in this study range between 1.65-5.35%.Statistical analysis shows that there was a significant effect (p<0.05) of the SSF concentration addition to the ash content of the tempeh steak product.The ash content of tempeh steak with different concentration of SSF addition is presented in Figure 2. Figure 2 depicts that the ash content (mineral) was increase along with the increase of the SSF concentration added.It is caused by the high mineral contained in SSF that contribute to the increase of the mineral content of product produced.As supported by Adeyeye and Aremu that shrimp shell contains high amount of mineral especially Ca [16].Liu et al., also reported that Litopenaeus vannamei shrimp shell and tail contains about 8.57 g/100g ash [3].

The effect of shrimp shell flour to the protein content of Tempeh Steak
Protein is one of nutrition that is very important to human body due to its effect on the growth of human.Also, protein contributes in the formation of cell due to the Nitrogen that builds the amino acids.Since shrimp shell was reported to contains protein in high amount, it is expected that the addition of SSF will increase the protein content of steak tempeh produced.
The protein content obtained in this study range between 17.67-21.79%.Statistical analysis shows that there was a significant effect (p<0.05) of the SSF concentration addition to the protein content of the tempeh steak product.The protein content of tempeh steak with different concentration of SSF addition is presented in Figure 3.In Figure 3, it can be seen that the increase concentration of SSF added to the product shows a tendency of product to increase the amount of protein contained.This due to the contribution of the protein contains in SSF added, as it has been reported that in shrimp shell and tail of Litopenaeus vannamei shrimps the protein is around 7.98 g/100g [3].Besides, all protein content in all product have met the standard from SNI which is a minimum of 16% [14].

The effect of shrimp shell flour to the fat content of Tempeh Steak
Fat is a nutrient that contribute to give high calory to food and food product.Since there is no report about the significance of fat content in SSF, in this study, the addition of SSF did not expect to have a significant effect on the tempeh steak produced.The fat content obtained in this study range between 0.035-0.375%.Statistical analysis shows that there was a significant effect (p<0.05) of the SSF concentration addition to the fat content of the tempeh steak product.The fat content of tempeh steak with different concentration of SSF addition is presented in Figure 4.In Figure 4 it can be seen that generally the fat content of product with different concentration of SSF was categorized very low with a value under 1% whereas the standard fat for tempeh was 8%.It is suspected that during the steam process of tempeh the water content increase and further lower the concentration of fat in tempeh.Besides, it was reported that the fat content in Litopenaeus vannamei shrimp shell and tail for vaname shrimp is only around 0.66g/100g which is very low, consequently it contributes to the low concentration of fat in product.

The effect of shrimp shell flour to the carbohydrate content of Tempeh Steak
Calculation of carbohydrate content was carried out using the by difference analysis method.Carbohydrate levels obtained in this study ranged from 22.56 to 25.30%.However, because there is no value that can be compared in SNI, the comparison is not made.Besides, statistically the addition of SSF in different concentration did not have a significant effect to the carbohydrates content of tempeh steak produced.
2)2.4.3.Fat content.Fat content was measured following Soxhlet method by[12]  with modification.The fat flask was dried and weighed until a constant weight was obtained (B), and the sample was weighed up to 3-5 grams (A) then wrapped in filter paper and put into a fat sleeve.The Soxhlet extraction apparatus is attached to the condenser with the paper containing the sample inside.The solvent chloroform is poured into a round bottom flask.Then the fat sleeve is inserted into the Soxhlet extractor and the Soxhlet circuit is properly installed.Then reflux for 3-4 hours.Furthermore, the flask fat was heated in an oven at 105 ⁰C for 2 hours.After drying, cooled in a desiccator for 30 minutes, the flask and its fat were weighed (C) to a constant weight.Fat weight can be determined by the formula: Fat content (%) = weight (g) B: Flask weight (g) C: Weight of flask plus fat (g) 2.4.4.Protein content.Protein measurement was performed using method by[13]  with modification.

Figure 1 .
Figure 1.The water content of Tempeh Steak product with the addition of Shrimp Shell Flour in variate concentrations.The different letter after the mean value indicated significant difference (p<0.05).

Figure 2 .
Figure 2. The ash content of Tempeh Steak product with the addition of Shrimp Shell Flour in variate concentrations.The different letter after the mean value indicated significant difference (p<0.05).

Figure 3 .
Figure 3.The protein content of Tempeh Steak product with the addition of Shrimp Shell Flour in variate concentrations.The different letter after the mean value indicated significant difference (p<0.05). 17

Figure 4 .
Figure 4.The fat content of Tempeh Steak product with the addition of Shrimp Shell Flour in variate concentrations.The different letter after the mean value indicated significant difference (p<0.05).

Figure 5 .
Figure 5.The carbohydrates content of Tempeh Steak product with the Addition of SSF in variate concentrations.