Characteristics of GABA (Gamma Amino Butyric Acid), antioxidant and sensory quality of modified Tempeh

ABSTRACT GABA (Gamma Amino Butyric Acid) is a non-protein amino acid that is beneficial to health. Tempeh is a fermentation product with GABA and antioxidant components that contribute to its health-promoting properties. This study aimed to determine the effect of the type of raw material and coloring method on the characteristics of the functional content of modified Tempeh. The research utilized a Completely Randomized Design with raw material types (sorghum, adlay, soybeans, and mung beans) and drying methods (oven drying and freeze-drying) as treatments. The results showed that the type of raw material and drying method had significant effect on the levels of GABA, flavonoids, total phenolic content, and the antioxidant activity of the modified Tempeh. The sorghum tempeh dried with a freeze dryer exhibits the highest GABA content at 1421 mg/100 grams. From a statistical perspective, the antioxidant capacity generated in fresh green bean and soybean tempeh is categorized as moderate. Modified Tempeh made from soybean had sensory acceptance and it did not significantly different from the control.


Introduction
Local food products have the potential to be developed into functional foods that have added value and can contribute to public health to prevent and help treat symptoms caused by the outbreak of the pandemic caused by the COVID-19 virus.Based on Global Market Forecast shows an increase in functional food that was increasing between 2017-2022, where for food and beverages it is 7.9, and for supplement products is 7.6 (CAGR %) [1] .This increase should have been utilized in Indonesia because of its functional food research and development in the COVID-19 Pandemic Era.
The prevalence of ethnic cuisine has exhibited a steady ascent in recent years. [2]One of the prospective fermented products from local food sources is Tempeh.Fermented soybeans (tempeh) hold significant promise as a nutritional food product for human consumption. [3]Tempeh is recognized as a functional food due to its composition of certain ingredients that positively impact health.Despite existing research generally addressing tempeh production by blending soybeans with various ingredients, there is a gap in research focusing on the characteristics of tempeh produced from a blend of soybeans and corn during the processing phase. [4]An alternative for raw materials in tempeh production includes sorghum, millet, and mung beans, aside from the widely used soybeans.Tempeh modification technology involves not only utilizing various raw material sources but also employing different main and additional starters to enhance the functional value of the product.One potential aspect of tempeh is its content of Gamma Amino Butyric Acid (GABA) and other functional nutrients.
GABA commonly referred Gamma-aminobutyric acid is a four-carbon non-proteinaceous amino acid that is ubiquitously distributed in the natural environment. [5]In recent years, GABA has garnered significant interest among researchers owing to its multifaceted physiological implications across humans, animals, microorganisms, and plants. [6]GABA assumes a crucial role in information transmission, neuronal development regulation, and enhancement of sleep and mood. [7]Moreover, various investigations have elucidated that GABA manifests several noteworthy health advantages, including its anti-hypertensive, anti-diabetic, and anti-inflammatory properties. [1]everal cereal products contain varying concentrations of GABA as well.Some fermented products generally contain higher concentrations of GABA than non-fermented products.The microbial fermentation enrichment principle centers on the elevated activity of glutamate decarboxylase (GAD) in microorganisms, enabling them to catalyze the conversion of glutamate into gammaaminobutyric acid (GABA) as part of the branched metabolism of GABA. [6]Lactic acid bacteria (LAB) represent the microbial strain exhibiting the greatest potential for GABA enrichment.Among these LAB, particularly Lactobacillus and Lactococcus, demonstrate substantial GABA titers and possess elevated endogenous glutamate decarboxylase (GAD) activity. [5]More recently, Lactiplantibacillus plantarum KB1253 was identified as the most prolific GABA producer among 74 LAB strains, yielding 41.0 ± 1.1 mM GABA in tomato juice under optimized fermentation conditions (pH 4.0, 20°Bx). [8]everal studies using microbes for fermentation to produce GABA include L plantarum [9] ; L. brevis [10] ; L. bulgaricus [11] ; L. rhamnosus [12] ; L. fermentum [13] ; H. adolescents [14] ; and B. bifidum. [15]The utilization of lactic acid bacteria in the fermentation process holds significant promise for enhancing the GABA content and functional qualities of fermented foods.Research using several types of microbes for fermentation can also produce different qualities and quantities of GABA of modified products.
GABA is found in many fermented products with varying levels.The concentration of GABA in fermented products depends on the type of raw material and the microbes contained therein.Some literature shows levels of high or low concentrations of GABA.GABA is found in both fermented raw food products and processed products.That is related to the processing technology implemented that affect the concentration of GABA contained therein.This potential also should be analyzed for the bioactive components contained therein, related to their potential for health.Other active components, macro, and micro components as well as the antioxidant activity contained therein.The objective of this research to modify types of raw materials and processing methods to improve the characteristics of GABA content, antioxidant, and sensory quality in the produced tempeh.

Materials and equipments
This research was conducted at the Laboratory of the Center for Agricultural Postharvest Research and Development, Cimanggu-Bogor, Indonesia.The materials used include soybeans comercial, green beans commercial, sorghum (Bioghuma 1), adlay (Sumedang District Variety), monosodium glutamate (Sasa commercially merk), Tempeh yeast (Raprima Ragi Tempe), lactic acid bacteria culture (Mixture of S. thermophilus, L. bulgaricus, L. casei, B. longum from Gadjah Mada University), GABA standard Sigma and chemicals Merck for analysis.Furthermore, the equipment used in this study was a soybean crusher, a boiling pot, a fermentation rack, an oven dryer Maxindo, and a freeze dryer Buchi Lyoxapor L-200.The analysis equipment includes Chroma meter CR 410 Konica Minolta.INC, centrifuge, and spectrophotometer UV-Vis Agilent Technologies Cary 60.

Process production of modified tempeh
This research consisted of two stages, namely the process of making modified Tempeh, each of which used four different types of raw materials i.e sorghum, adlay, soybeans, and mung bean.The technology employed in the production process of fresh tempeh is based on the method utilized by Syukri et al. [4] modification.Subsequently, the obtained Tempeh underwent drying using two methods: the oven tray dryer and freeze dryer.The process of making modified Tempeh products was carried out in the following way: Soybeans are boiled, then soaked for 12-24 hours.The soaked soybeans were then drained and crushed using a soybean crusher.Then do the washing and separation of the epidermis using water.The cleaned soybeans were drained and combined with Tempeh yeast, monosodium glutamate, and a mixture of Lactic Acid Bacteria (LAB).The resulting mixture was left to age for a day in a dark room to condition the yeast and warm the soybean blend.Once the initial spore formation occurred, the fermentation process was optimized in an open space until achieved Tempeh with an optimal maturity level.This optimization process was guided by findings from previous studies on the production of modified Tempeh, with GABA levels and functional components identified in commercial products serving as reference points.
Furthermore, an analysis of the levels of Gamma Amino Butyric Acid (GABA), flavonoids, total phenols, and antioxidant activity was carried out in fresh and dried Tempeh.Modified Tempeh in fresh form was analyzed for color and sensory quality on the best product compared to the control.An analysis of the amino acid profile of modified Tempeh products was also carried out for the best results. [16]) Preparation of the γ-aminobutyric acid test sample according to the preparation method by Zhang et al.. [16] 1 mg of sample was dissolved in 10 mL of distilled water in a 15 mL centrifuge tube.Samples were extracted for 60 minutes using a shaker with a speed of 200 rpm.The sample extract mixture was then separated using centrifugation at 3,000 rpm for 60 minutes to obtain the extract supernatant.The supernatant was then filtered using a nylon membrane with a pore size of 0.45 μm until a clear supernatant was obtained.

Analysis of γ-aminobutyric acid levels (Kitaoka and Nakano
Analysis of γ-aminobutyric acid levels in samples was based on the omega amino acid testing procedure. [17]The test was started by taking 0.5 mL of sample extract supernatant, followed by the successive addition of 0.2 mL of 0.2 M borate buffer and 1.0 mL of 6% phenolic reagent.The solution was mixed homogeneously and cooled in ice water, before adding 0.4 mL of 9% sodium hypochlorite reagent.The tube containing the mixture was vortexed, then placed in boiling water for 10 minutes.After heating, the tubes were cooled rapidly by immersion in ice water for 20 minutes.The analysis was carried out by measuring the absorbance using a spectrophotometer at a wavelength of 645 nm.Determination of sample concentration based on comparison with a standard blank reagents without samples.GABA standards were used as a comparison with graded concentrations of 40, 80, 120, 160, and 200 ppm.Based on the results of plotting the standard curve, the resulting equation is Y = 0.00011 × + 0.0787.With an R 2 value of 0.9495.

Polyphenolic analysis
In this study, the reaction mixture consisted of 2 mg sample in 25 ml of 80% methanol, shaken until homogeneous and allowed to stand for 1 day as sample solution.Dissolve 0.5 ml sample solution in 10 ml 80% methanol, shake well, then pipette the mixture 0.5 ml +0.5 ml 10% AlCl3 and vortex for 1 minute.Furthermore, the measurement of polyphenol levels was carried out spectrophotometrically with a length of 432 nm.The calibration curve uses 100 ppm gallic acid and the results are expressed in extracts (%).Linear regression from absorbance data and standard solution concentration: where x is sample concentration (mg/L), y is absorbance of the sample, b: is intercept of the standard curve, and a is the standard slope of the standard curve.The polyphenol content is calculated using the following formula: where, Abs is absorbance of the sample, bis calibration curve intercept, a is slope of V of the standard curve, Vs is volume (mL), fp is dilution factor, and b is weight of the extract used (g).

Total flavonoid analysis
The process of analyzing flavonoids was carried out using the Marinova et al [18] method approach.The aluminum chloride colorimetric method was carried out to determine the total flavonoid content of the samples.For assay, quercetin was used to create a standard calibration curve.A stock solution of quercetin was prepared by dissolving 5.0 mg of quercetin in 1.0 mL of methanol.Quercetin standard solutions were prepared by serial dilution using methanol (5-200 μg/mL).A 0.6 mL amount of diluted standard quercetin solution or extract was mixed separately with 0.6 mL of 2% aluminum chloride.After mixing until homogeneous, the solution was incubated for 60 minutes at room temperature.Measurement of the absorbance of the reaction mixture was measured using a blank at a wavelength of 430 nm with a UV-Vis spectrophotometer.The concentration of total flavonoid content was calculated from the calibration plot and expressed as mg quercetin equivalent (QE)/g dry plant material.All analyses were performed in triplicate.

DPPH radical scavenging analysis
The tempeh sample (0.5 mg) was added with 10 ml of methanol in a test tube.The mixture was then extracted by ultrasonic for 30 minutes, then centrifuged and the supernatant was taken.The supernatant 0.2-0.5 ml added 1 ml of methanol and shaken until evenly distributed.The mixture was added 1 ml of DPPH and let stand for 90 minutes, then measured with a spectrophotometer with a wavelength of 517 nm.For IC 50 standard use of ascorbic acid or catechins was carried out.

Colour analysis
Color analysis used the Minolta Chromameter on the parameters Lightness (L), redness (a), and yellowness (b).Color measurement is done with Chroma Meter CR 300 (Konica Minolta, Japan) with calculations as Lin et al [19] and Selimovic et al . [20]The calibration process was carried out using black and white standards before the instrument was used for measurement.Sample is placed in a sample holder and covers the entire Croma Meter lens.The analysis was carried out by identifying the values of L, a and b.The values of L (white/black), a (red/green) and b (yellow/blue).The degree of whiteness of the values L, a and b is calculated according to the equation formula as shown below.

Sensory analysis
Sensory analysis was carried out using 30 general panelists.Parameters observed included: color, aroma, texture, and general acceptance of raw Tempeh and there were additional taste parameters for cooked Tempeh.The level of preference is analyzed with hedonic parameters by using 5 levels sequentially from number 1 to 5, namely: dislike, somewhat dislikes, neutral, rather like, and like.
Panelists were asked to observe according to the senses used.The panelists then fill out the assessment form, and the next step is to collect data.The data obtained were then compiled and analyzed based on the level of preference and analyzed the differences based on the existing treatment with commercial samples.

Data analysis
The data obtained were tested statistically using the Analysis of Variance method at α = 0.05% and Duncan's further test using SPSS 2.1 software to determine differences between samples and treatments.

GABA content
GABA is one of the important parameters analyzed in the development of this modified Tempeh product.GABA is a non-protein amino acid that is reported to have various health benefits.The average GABA content of the modified Tempeh products can be seen in Table 2. From Table 2 it can be seen that the raw materials and the drying method have significantly different effects on the GABA levels of the resulting Tempeh.Soybeans produced the highest levels of GABA followed by sorghum.Previous research reported GABA levels in soybean Tempeh at 0 hours of fermentation of 2.7 mg/ 100 g to 21.4 mg/100 g dry basis after 24 hours of fermentation. [3]Anaerobic fermentation with R. microsporus var.oligosporus IFO 32,002 was reported to yield 1740 mg/100 g dry basis. [21,22]ABA formation is contingent upon the precursor amino acid glutamate's abundance. [3]Anaerobic conditions and the use of lactic acid bacteria strains are reported to enhance GABA production during soybean Tempeh fermentation. [21]Co-fermentation with different bacteria strains may also stimulate GABA production due to strain competition or environmental stress. [22]oreover, the choice of drying method significantly impacts the GABA levels.Freeze-drying was found to be markedly increased GABA levels, while oven tray drying reduced them.Previous studies on freeze-drying have reported higher carotenoid and total phenol levels in quinine powder compared to oven drying, vacuum oven drying, or spray drying. [23]The use of lower temperatures in the freezedrying method is advantageous for preserving heat-sensitive active components.

Total flavonoid content
Flavonoids and total phenols serve as parameters closely tied to the antioxidant activity of a product, contributing to its functional characteristics.The mean flavonoid levels in modified Tempeh are presented in Table 1.In fresh Tempeh, the highest flavonoid content was in soybean Tempeh (ST-Soybean) and the lowest was in adlay Tempeh (ST-Adlay).Furthermore, the drying process using a freeze dryer can increase the levels of flavonoids from modified Tempeh.Analysis of variance results indicated that both the type of raw material and the drying method had significantly different effects.
Flavonoids are polyphenolic components that have biological activities that are beneficial to health.Flavonoids are bioactive components that are widely found in fruits and vegetables but are also important active components in several types of cereals and nuts. [24]For instance, the flavonoid content in soybeans is reported to be 4.62 mg QE/100 g, [25] whereas in this study, the total flavonoid level in soy Tempeh was 65.72 ± 0.32 mg QE/g.Furthermore, the level of flavonoids in green beans is 12.79 mg QE/100 g. [25] In this study, modified soybean Tempeh had a flavonoid content of 24.68 ± 0.01 mg QE/g.Then the total level of flavonoids from adlay seeds was 209.3 micrograms RE/g dry weight and increased to 647.4 after germination with pre-treatment of immersion in citric acid. [26]In this study, the level of flavonoids was 12.32 ± 0.04 mg GAE/g.The fermentation processes can contribute to increased flavonoid levels in cereals.The activity of enzymes such as alpha-amylase, xylanase, and alpha-glucosidase has been correlated with heightened flavonoid levels during cereal fermentation. [24]Additionally, the use of a freeze dryer in the drying process resulted in higher total flavonoid content compared to drying using an oven tray dryer (Table 2).Previous studies on quinine powder manufacturing reported higher flavonoid levels in grape pulp dried by the freeze-drying method than those dried by the oven method.Freeze-drying methods in plant drying are known to retain bioactive components, including antioxidants. [27]

Polyphenolic content
The polyphenol of the modified Tempeh ranged from 58.23 ± 21.18 to 180.51 ± 0.28 mg GAE/g extract (Table 2).The analysis of variance showed that raw materials and drying methods had significantly different effects.Polyphenol in modified fresh Tempeh was highest in soybean raw materials and lowest in adlay.Additionally, the use of a freeze dryer in the drying process appeared to slightly increase the total phenolic content of the resulting modified Tempeh.
In this study, oven adlay Tempeh had a higher polyphenolic content, 180.51± (mg GAE/g extract).Previous research stated that the polyphenolic content of Tempeh from four soybean varieties ranged from 13.96 to 16.26 mg GAE/g extract). [28]The modification of the Tempeh-making process by introducing lactic acid bacteria and using monosodium glutamate was reported to enhance the total phenol content of the resulting soybean Tempeh.
While the polyphenolic content of sorghum was 1069.2 micrograms GAE/g DW, which increased to 2790.8 after germination with citric acid pre-treatment. [29]The modified Tempeh fermentation process, involving Tempeh yeast, LAB, and the addition of monosodium glutamate, was found to increase the total phenol content of the resulting Tempeh.

Antioxidant activity
The analysis of antioxidant activity in fresh and dried tempeh was conducted using the DPPH method, as antioxidants are a desired functional property in food products known for their healthmaintaining benefits.The antioxidant levels of the modified tempeh, in both fresh and dried forms, ranged from 128 ± 1 to 973 ± 2 ppm (see Table 2).It was observed that the type of raw material and the drying method exerted significantly distinct effects on the antioxidant activity of the modified tempeh.The lowest antioxidant activity was found in fresh tempeh made from green beans value of 128 ± 1 ppm, and did not show a significant difference statistically compared to the use of soybeans value of 132 ± 12 ppm.The classification of antioxidant activity is determined based on the IC 50 value, where activity is considered very strong if IC 50 is less than 50 ppm, strong if IC 50 is 50-100 ppm, moderate if IC 50 is 101-150 ppm, and weak if IC 50 is 150-200 ppm. [30]According to this categorization, the produced tempeh falls under the category of having moderate antioxidant activity.
The modification of the drying process, whether using an oven or a freeze dryer, generally decreases the antioxidant capacity, as evidenced by the increase in the IC50 value of antioxidant activity measured in ppm units.The drying process has the potential to compromise the sample's ability to counteract free radicals.Tempeh underwent multiple heating treatments during the drying process, specifically steaming and oven drying (drying cabinet) at 50°C for 10 hours. [31]This drying procedure has an impact on the antioxidant activity and may diminish the samples' capacity to scavenge radicals. [32,33]he analyzed antioxidant components in tempeh are quite diverse, in addition to the components investigated in this study, namely total flavonoids and polyphenols.Antioxidants can be derived from various compounds in products, including isoflavones. [34]Isoflavones are recognized for their ability to shield cells from the detrimental effects of free radicals. [35]In fermented soy products like natto, miso, soy sauce, or fermented soymilk, isoflavone glucosides undergo hydrolysis by microorganism αglucosidases, converting them into isoflavone aglycone form.Wang et al. [35] documented a decrease in total glucosides from 49.68 μg/mL to 14.49 μg/mL, while total aglycones increased from 21.91 μg/mL to 59.01 μg/mL after the fermentation of soybean milk by LAB and bifidobacteria. [36]Antioxidant activity also stems from lactic acid produced by LAB during the fermentation process. [37]The antioxidant potential of fermented products is not limited to flavonoid compounds and total phenols but also includes contributions from lactic acid, bioactive peptides, amino acids, and other substances.

Amino acid profile
Freeze-dryer-modified Tempeh had the highest GABA content.Furthermore, the characterization of the amino acid profile of the Tempeh can be seen in Table 3.Each type of modified Tempeh had a significantly different amino acid profile.Soy-modified Tempeh has the highest content of the amino acid L-glutamic acid, followed by L-aspartic acid, L-leucine, and L-arginine.Previous research reported that the dominant amino acid content in Tempeh and Tempeh flour from soybeans are glutamic acid, lysine, and leucine.Lysine and leucine are essential amino acids in soy Tempeh. [38]urthermore, it was also reported by Cahyani et al. [39] in water-soluble soybean Tempeh flour, the major amino acid fractions were glutamic acid, aspartic acid, arginine, and leucine.
The amino acids of dominant modified sorghum Tempeh were L-glutamic acid, L-leucine, L-alanine, L-proline, and L-phenylalanine.According to Osman et al., [40] the dominant amino acid content of sorghum protein are leucine, alanine, proline, and phenylalanine.Compared with the raw material, this modified Tempeh contains higher L-glutamic acid.This is due to the use of monosodium glutamate as a GABA precursor.
In the modified mung bean Tempeh, the amino acid L-histidine had the highest levels, followed by L-glutamic acid, followed by L-leucine, and L-phenylalanine.According to Elobuike et al., [41] the dominant amino acids in mung bean flour are glutamic acid, aspartic acid, leucine, and arginine.According to Mubarok et al., [42] the main amino acids in green beans are glutamic acid, aspartic acid, leucine, and arginine.
The amino acids of adlay modified Tempeh were glutamic acid, L-leucine, L-alanine, and L-proline.The dominant amino acids in adlay are glutamic acid, aspartic acid, glycine, and alanine.The variety may affect the amino acid composition of adlay and its processed products.In this modified Tempeh, glutamic acid became an amino acid with the highest average level compared to other types of amino acids, except for the modified mung bean Tempeh.This happens because of the addition of monosodium glutamate during the process of making Tempeh.Glutamic acid is a non-essential amino acid but is an amino acid that gives the product an umami taste.
The soybean-modified Tempeh as raw material has higher essential amino acids compared to green beans, sorghum, and adlay.Furthermore, Tempeh made from peanuts, namely soybeans and green beans, has a higher content of essential amino acids than modified Tempeh made from cereals, namely sorghum, and adlay.The levels of essential amino acids in Tempeh made from adlay are higher than in sorghum.Amino acids are the building blocks of protein.Protein is a polypeptide composed of chains of amino acids.Foodstuffs with high protein content have higher amino acids as their constituent components.Nuts have a higher protein content than cereals.

Colour
Color is an important parameter to assess food product characteristics.Product color analysis is carried out using the chromameter value parameters, namely the value of L, a, b, C and the calculation result is the value of WI (Whiteness Index).The results of the color analysis of the samples and their drying results are as shown in Table 4 below.
The use of raw materials affects the color values of both L, a, b, and C as well as WI of fresh and dried Tempeh.Sorghum Tempeh, which has a white raw material, produces a higher WI value compared to other raw materials.Selimovic et al. [20] linked the effect of L, a, and b values on the capacity of total phenol values and the capacity of antioxidant activity.

Sensory characteristic
Sensory analysis of modified Tempeh with the highest GABA had done to determine the panelists' acceptance of the product.Selected raw and cooked modified Tempeh samples from soy and sorghum Tempeh were analyzed compared to control Tempeh.Control Tempeh made from soybean that come from home industry of Tempeh.The results of the sensory analysis are shown in Table 5.
Based on the results of the sensory analysis, GABA soybean Tempeh was not significantly different from the control soybean Tempeh that was usually processed by Tempeh producers (control), both for parameters of color, aroma, texture, and general acceptance.The criteria for sorghum Tempeh as a whole quite like it, while for GABA soybean Tempeh and the control it produces like criteria for all parameters including color, aroma, texture, and general acceptance.
The results of the sensory analysis showed that the GABA soybean Tempeh and the control soybean Tempeh were not significantly different compared to the ripe GABA sorghum Tempeh for the parameters of color, aroma, taste, texture, and general acceptance.This is possible because sorghum Tempeh has an optimal fermentation time that is different from soy Tempeh.Physically, it could be seen that ripe and unripe sorghum Tempeh had not yet formed perfect filaments, as is the case with soy Tempeh.

Conclusion
Tempeh modification technology with the treatment using a mixture of 4 microbes and monosodium glutamate increases GABA levels.Tempeh can be processed using raw materials from vegetable sources not only from soybean, but also use raw materials from mung beans, sorghum, and adlay.One of the techniques to extend the shelf life of Tempeh is using the drying methods, namely an oven drying or freeze-drying.The highest GABA content is found in sorghum tempeh dried using a freeze dryer, amounting to 1420.59 ± 0.00 mg/100 grams.Statistically, the antioxidant capacity produced in fresh green bean and soybean tempeh falls within the moderate category.Modified soybean Tempeh with the addition of a mixture of microbes and monosodium glutamate was still well received by the panelists in the hedonic test.In the next stage, it is urgently needed for the process of extracting GABA components and analyzing their functional value capacity in vitro for health.

Table 1 .
Raw Material and Drying Methods in Modified Tempeh Process Production.

Table 2 .
Results of Analysis of GABA (Gamma Amino Butyric Acid), Flavonoid, Polyphenolic and Antioxidant Activity Levels from Modified Tempeh.

Table 3 .
Amino Acid Profile Analysis from Modified Tempeh.

Table 4 .
Color Results Analysis of Modified Tempeh.Numbers followed by different letters in the same column are significantly different at the 95% level.

Table 5 .
Results of Sensory Analysis of Fresh and Dried Tempeh.Numbers followed by different letters in the same column are significantly different at the 95% level.