22 Accepted: 22/08/2023, Revised: 01/12/2023, Published: 31/01/2024 CHEMICAL AND ANTIOXIDANT CHARACTERISTICS OF FERMENTED YELLOW PUMPKIN FLOUR WITH VARIATION OF MICROORGANISMS AND FERMENTATION
TIME
Anisa Rachma Sari*, Aldila Sagitaning Putri, Devy Angga Gunantar
Department of Agricultural Technology, Faculty of Agricultural Technology, Universitas Semarang
Tlogosari Kulon Street, Pedurungan Subdistrict, Semarang City, 50198 Central Java Indonesia
*Corresponding Author, email : [email protected]
ABSTRACT
Pumpkin flour produced through fermentation can increase its nutritional value. This study aims to analyze the chemical characteristics and antioxidants of fermented pumpkin flour with variations in microorganisms and fermentation time, as well as the correlation between the two factors, and determine the best formulation. This study used a factorial group randomized design method with two factors, namely, variation of microorganism and fermentation time. The data obtained were analyzed using factorial ANOVA, followed by the Duncan Multiple Range Test (DMRT) test with a confidence level of 95% using SPSS 23. The chemical characteristics of fermented pumpkin flour showed an interaction between fermentation microorganisms and the length of fermentation time on the antioxidant activity of fermented pumpkin flour, but there was no interaction between fermentation microorganisms and the length of fermentation time on the moisture content, ash, protein, fat, and carbohydrates of fermented pumpkin flour. Fermentation by S. cerevisiae for 3 days had the best chemical characteristics and antioxidant activity of fermented pumpkin flour. Researchers hope that the application of fermentation in processing pumpkin into flour can increase the nutritional value, the economic value, and the value of health.
Keywords: Antioxidant activity, Proximate, Pumpkin flour
INTRODUCTION
Increasing the economic value of food is done by developing local food products (Budianto et al., 2019). Pumpkin, as one of the local food ingredients, is rich in nutrients such as high carbohydrates, dietary fiber, especially pectin, bioactive compounds β-carotene, several types of vitamins (A, B6, K, C, E, thiamine, and riboflavin), tocopherols, several types of minerals (K, P, Mg, Fe, and Se), ascorbic acid, phytosterols, selenium, lutein, zeaxanthin, and lenoleic acid, which act as antioxidants (Dhiman et al., 2009; Wahyono et al., 2018).
Pumpkin contains carotene as much as 6.10 mg/100g, total phenol content as much as 159.69 mg GAE/L, and antioxidant activity as much as 79.53 mg/L. β-carotene is identified as the pigment responsible for the yellowish color of pumpkin and acts as an antioxidant as well as a source of vitamin A (Dinu et al., 2016).
The lack of public awareness about optimizing the processing of nutrient-rich local food ingredients results in a lack of processed pumpkin products, so technology transfer is needed to support the improvement of pumpkin processing and utilization. Flour can be used as an alternative to pumpkin processing because of its several advantages, namely: it can be stored for a long period of time, easily processed into formulated food ingredients (bread products, cakes, instant noodles, pasta, and composite flour), natural coloring ingredients, and simplifies the packaging process (See et al., 2007). Pumpkin flour can be produced through fermentation
23 to increase the nutritional value of its protein (Yani & Akbar, 2018). Fermentation using L.
plantarum in making pumpkin flour produces energy-dense flour with reduced sugar and carotenoid content (Tedom et al., 2019). Fermentation by S. cerevisiae in the manufacture of tapioca flour showed increased starch solubility, protein content, amylose, Fe, Mg, and Ca, but decreased Zn content (Kustyawati et al., 2013).
During this time, pumpkin is only sold at a low price because it is limited to being processed into ready-to-eat foods with low shelf life even though it has the potential for high nutritional diversity. To increase economic value, it is necessary to apply pumpkin processing technology. Processing pumpkin into flour is expected to increase its economic value because it can be stored for a long time, easily processed into formulated food ingredients (bread products, cakes, instant noodles, pasta and composite flour), natural coloring ingredients, and facilitate the packaging process (See et al., 2007). The application of fermentation technology in making pumpkin flour can increase the nutritional value of its protein (Yani & Akbar, 2018).
Fermentation using L. plantarum in making pumpkin flour produces energy-dense flour with reduced sugar and carotenoid content (Tedom et al., 2019). Fermentation by S. cerevisiae in the manufacture of tapioca flour shows: increased starch solubility, protein content, amylose, Fe, Mg, Ca but decreased Zn content (Kustyawati et al., 2013).
Fermentation in the process of making pumpkin flour can affect β-carotene levels because β-carotene is labile to light, heat, and acidic pH (Nurrahman & Astuti, 2022). The combination of different types of microorganisms and fermentation time in making pumpkin flour is expected to improve the chemical characteristics and antioxidants in order to create flour that has health benefits and nutritional improvements for the creation of flour product diversification. The purpose of this study was to analyze the chemical and antioxidant characteristics of fermented pumpkin flour with variations of microorganisms and fermentation time, as well as the correlation between the two factors, and determine the best formulation.
METHODOLOGY
Materials
The materials used in the study were yellow pumpkin with a harvest age of 3 months and not damaged or rotten, L. plantarum isolate FNCC 0020 obtained from Fakultas Teknologi Pertanian Universitas Gadjah Mada, S. cerevisiae derived from fermipan, MRS agar, distilled water, petroleum ether, vitamin c and DPPH reagent. FRAP and FeCl36H2O solution.
Tools
The tools used in the study were erlenmeyer, petri dish, test tube, incubator, ose needle, digital balance, autoclave, colony counting chamber, food dehydrator, desiccator, porcelain ash cup, soxlet, UV-Vis spectrophotometer Genesys 10 S and cooking utensils.
Research Design
This research used an experimental method with factorial randomized group design with two factors, namely, variation of microorganisms and fermentation time. There were six treatment combinations, and each treatment was repeated four times. The total sample size of the study was 24 samples. The treatment factors used in this study are: M = variation of microorganisms consisting of M1 = fermentation without microorganisms; M2 = fermentation using Lactobacillus plantarum; and M3 = fermentation using Saccharomyces cerevisiae; and L = fermentation time consisting of L1 = 3 days and L2 = 6 days. The data obtained were analyzed for the effect and relationship between variables using factorial ANOVA (two-way ANOVA), followed by the Duncan Multiple Range Test (DMRT) with a significant level of α = 0.05 using SPSS 23.
Preparation of Pumpkin Ingredients (Tedom et al., 2019).
Pumpkins aged 95–120 days after planting were purchased from a vegetable shop in Tembalang, Semarang, in fresh and undamaged condition. The pumpkin was washed, then
24 peeled and cut into pieces with a thickness of 3–4 cm of flesh and seeds removed. Next, 400 g of pumpkin was weighed and put into a 500 mL Erlenmeyer, then pasteurized at 90 °C for 5 minutes to remove contaminating microorganisms.
Starter Preparation
Making starters by using the counting chamber method. Pasteurized pumpkin was put into a fermentation bottle, then L. plantarum and S. cerevisiae from baker's yeast were added without the addition of microorganisms (natural fermentation) into different bottles. For L.
plantarum, as much as 2% of the weight of pumpkin (Tandrianto et al., 2014), and for S.
cerevisiae, from baker's yeast, as much as 5 g : 1000 g of pumpkin (Yani & Akbar, 2018). Then, distilled water was added until the pumpkin was submerged (about 3:2 of the volume of pumpkin) (Tandrianto et al., 2014). Finally, incubate at 37 °C (L. plantarum) and 30 °C (S.
cerevisiae) and count the colonies periodically (minute 0, 30, 60, 90, 120, 150) until the cell density of each microorganism reaches 106 CFU/mL (Tandrianto et al., 2014).
Fermentation
Fermentation was carried out by inoculating 3% (v/v) of L. plantarum isolate inoculum (modification of Setiarto & Widhyastuti, 2016) into an Erlenmeyer containing pasteurized pumpkin, then homogenized and incubated at 30 °C for 3 and 6 days (modification (Tedom et al., 2019). The same method was applied to the S. cerevisiae isolate inoculum (3 g (w/v)). For natural fermentation, the inoculum solution was replaced with distilled water (control).
Flour Preparation
The fermentation results were filtered to separate the solids from the liquid. The solids were dried using a food dehydrator at 65 °C ± 2 °C for 24 hours and pulverized into flour with a particle diameter of 500 µm. (Pereira et al., 2020)
Methods
Chemical characteristics were analyzed in the form of proximate identification, namely:
water content, ash, protein, fat, carbohydrates, and antioxidant activity, which was carried out quantitatively.
Analytical Procedure
Gravimetric method AOAC 925.10 (AOAC, 2012) was used to analyze the moisture content of fermented pumpkin flour. The AOAC 923.03 method (AOAC, 2012) was used to analyze the ash content of fermented pumpkin flour. Soxhlet method The AOAC (AOAC, 2012) was used to analyze the fat content of fermented pumpkin flour. Kjeldahl method AOAC (AOAC, 2012) was used to analyze the protein content of fermented pumpkin flour. The by- difference method was used to analyze the carbohydrate content of fermented pumpkin flour (Siletty et al., 2022). Antioxidant activity testing is based on the ability of antioxidants to counteract DPPH radicals. The mechanism of deterrence is determined by the removal of DPPH radicals (Junita et al., 2017). Antioxidant activity testing uses the DPPH method. Test samples were mixed with ethanol (1 mg/mL) at several different ethanol concentrations. In addition, a DPPH solution was also prepared, which consisted of 1.3 g of DPPH mixed with ethanol up to 25 ml. A total of 0.5 mL of sample solution was pipetted to be put into a micropipette, and then 0.5 ml of DPPH solution was added. After that, the mixture was incubated at room temperature and in the dark for 30 minutes, and then the absorbance was measured with a spectrophotometer at a wavelength of 517 nm. Similarly, the blank or control.
The control used is vitamin C. Antioxidant activity is expressed as a percentage of inhibition (% inhibition) (Junita et al., 2017).
25 RESULTS AND DISCUSSION
The chemical characteristics and antioxidant activity of fermented pumpkin flour are shown in Table 1, while the analysis of the effect and relationship between variables is shown in the two ways anova analysis in Table 2.
Table 1. Chemical Characteristics of Fermented Pumpkin Flour Parameters
Testing
Without
Microorganisms (Control)
Lactobacillus plantarum 3 days
Lactobacillus plantarum 6 days
Saccharomyces cerevisiae 3 days
Saccharomyces cerevisiae 6 days Water (%) 13.20±0.892b 11.69±0.777a 11.16±0.421a 11.70±0.709a 10.23±0.637a
Ash (%) 9.44±0.401c 7.03±0.298a 4.63±0.156a 7.33±0.332b 6.43±0.672b Carbs (%) 72.45±0.769a 74.64±1.124b 72.89±0.777b 74.50±1.450ab 71.69±1.176ab Protein (%) 7.48±0.257a 7.91±0.282a 7.78±0.316a 8.08±0.878a 7.90±0.108a
Fat (%) 0.83±0.059a 0.86±0.033a 0.82±0.008a 1.00±0.022b 0.84±0.010b Antioxidant
(inhibition %)
12.69±0.326a 15.94±0.418b 16.01±0.469b 14.82±0.598b 15.90±0.150b Notes: The same superscript letters on one line indicate insignificant differences (sig > 0.05).
.
The moisture content of pumpkin flour in the control was 13.20%, which was higher than the moisture content of pumpkin flour fermented at different times by L. plantarum (11.69 and 11.16%) and S. cerevisiae (11.70 and 10.23%). These results show that the moisture content in all treatments exceeds the maximum limit of moisture content set by SNI 01-3727 in 1995, which is a maximum of 10% (Aini et al., 2016). The maximum moisture content for flour is < 14% to prevent mold growth (Tsaalitsati et al., 2016). The moisture content of fermented pumpkin flour is higher than the research of Aini et al. (2016), which showed that corn flour fermented by S. cerevisiae 80 hours and L. bulgaricus 80 hours was 8.7 and 8.9%, respectively. These results show that differences in fermentation substrates will produce different moisture content in flour, where corn substrates have a lower moisture content than pumpkin. When compared to the research of Tedom et al. (2019), yellow pumpkin flour fermented by L. plantarum for 70 hours showed a moisture content of 5.03%; this figure is much lower than the results of the research obtained.
Ash content shows the percentage of minerals contained in food ingredients (Fiqtinovri, 2020). The control treatment of pumpkin flour showed an ash content of 9.44%; this figure was higher than the ash content of pumpkin flour fermented at different times by L. plantarum (7.03 and 4.63%) and S. cerevisiae (7.33 and 6.43%). These results show that the ash content in all treatments is above the ash content threshold set by SNI 01-3727 in 1995, which is a maximum of 1.5% (Aini et al., 2016). When compared to the research of Tedom et al. (2019), the ash content of pumpkin flour fermented by L. plantarum for 70 hours was 7.39% higher than the results of the research obtained. The high ash content in fermented pumpkin flour is thought to be due to the high minerals found in pumpkin. Pumpkin is rich in dietary fiber and vitamins (especially pectin, β-carotene, tocopherols, thiamine, riboflavin, vit. B6, vit. K, and vit. C), as well as various minerals (potassium, phosphorus, magnesium, iron, and selenium) (Yanuwardana et al., 2013; Quintana et al., 2018; Millati et al., 2020). The fermentation process by L. plantarum and S. cerevisiae with different lengths of time was able to improve the quality of pumpkin flour compared to the control, as indicated by a significant decrease in ash content.
The ability of L. plantarum to reduce ash content was better than that of S. cerevisiae, but the longer the fermentation time, the decrease in ash content was not significant. The decrease in ash content is thought to be due to the soaking of the substrate in the fermentation process, which can dissolve vitamins B and C and some minerals present in pumpkin. This assumption
26 is in accordance with the opinion of Aini et al. (2010), which states that the decrease in mineral content occurs due to the leaching process due to substrate soaking during fermentation. Putri (2019) added that fermentation activities using Lactobacillus plantarum with a concentration of 5% caused the loss of some of the minerals contained in the raw materials due to the dissolution of minerals with fermentation bath water and waste before pressing.
In the control treatment, pumpkin flour showed a protein content of 7.48%; these results were lower than the protein content of pumpkin flour fermented at different times by L.
plantarum (7.91 and 7.78%) and S. cerevisiae (8.08 and 7.90%). These results show that the protein content in all treatments exceeds the standard protein content of SNI 01-3751 (BSN, 2006) which is at least 7% (Yanuwardana et al., 2013). The fermentation process by L.
plantarum or S. cerevisiae with different fermentation times did not significantly increase the protein content of pumpkin flour but was insignificant. When compared to research (Tedom et al., 2019), the protein content of pumpkin flour fermented by L. plantarum for 70 hours of 2.03%
is lower than the results of the research obtained. The high protein content in pumpkin flour during fermentation is thought to be due to the activity of microorganisms breaking down the peptide bonds in pumpkin. This is in accordance with the opinion of Triyani et al. (2013), who state that the decomposition of peptide bonds during fermentation produces simple peptide forms, and when the number of simple peptides increases, it increases the soluble protein content. Aini et al. (2016) added that LAB fermentation with different fermentation times will increase soluble protein because LAB will break down protein into amino acids. Another factor that causes the increase in protein content is the increase in protease activity produced by L.
plantarum, along with the length of fermentation time because the number is increasing (Tandrianto et al., 2014). The principle of the Kjeldahl method is the estimation of total nitrogen contained in food and the conversion of nitrogen presentation into protein, assuming that all nitrogen in food is protein. Determination of protein based on the amount of N shows crude protein because there are two types of protein, namely protein bound to N compounds and non-protein nitrogen (NPN) such as urea, nucleic acids, ammonia, nitrate, nitrite, amino acids, amides, purines, and pyrimidines (Tuankotta et al., 2015). L. plantarum and S. cerevisiae are proteolytic microorganisms that, during fermentation, are able to degrade proteins into dipeptides, and then dipeptides are degraded into NH3 (ammonia) and NH2 (amino acids that have nitrogen compounds) (Muthmainna et al., 2016).
Pumpkin flour in the control produced a fat content of 0.83%, which was lower than pumpkin flour fermented by L. plantarum (0.86 and 0.82%) and S. cerevisiae (1.00 and 0.84%) with different lengths of time. The fermentation process by S. cerevisiae with different lengths of time significantly increased the fat content compared to the other two treatments, while fermentation by L. plantarum did not occur. When compared to fermentation by L. plantarum on pumpkin substrate with different fermentation times, namely between 70 hours and 72 hours, it will produce pumpkin flour with a higher fat content (by 3.40%) at a fermentation time of 70 hours compared to a fermentation time of 72 hours (research results) (Tedom et al., 2019). Saskiawan & Nafi'ah (2014) stated that the fermentation process can increase the free fatty acid content and produce lipid compounds that are easily digested. However, along with the longer fermentation time, the fat content decreased (Siletty et al., 2022).
The results of the two-way ANOVA analysis in Table 2. above show as follows:
Separately, the first factor, namely fermentation microorganisms, had a significant effect (F count < 0.05) on the moisture, ash, fat, and antioxidant activity of fermented pumpkin flour, while it did not have a significant effect (F count > 0.05) on the protein and carbohydrate content of fermented pumpkin flour. The second factor, the length of fermentation time, had a significant effect (F count < 0.05) on the moisture, ash, protein, fat, and carbohydrate content of fermented pumpkin flour; however, it did not have a significant effect (F count > 0.05) on the antioxidant activity of fermented pumpkin flour. When combined, the first and second factors had a significant effect (F count < 0.05) on the ash, fat, carbohydrate, and antioxidant activity of fermented pumpkin flour but did not have a significant effect (F count > 0.05) on the moisture and protein content of fermented pumpkin flour. If the R squared value is close to 1, it indicates a strong correlation between the two factors. If the results of the plot diagram show parallel lines that approach each other but do not coincide, it means that there is no interaction between
27 the first factor, namely fermentation microorganisms, and the second factor, namely the length of fermentation time.
Tabel 2. Two Ways Anova Analysis Parameter Sig-F
Microorganism Fermentation
Sig-F Fermentation
Time
Sig-F Microorganism*
Fermentation Time
R Squared Plot Diagram
Water 0.00 0.02 0.19 0.76 Parallel lines
that are close to each other
Ash 0.00 0.00 0.00 0.93 Parallel lines
that are close to each other
Carb 0.06 0.00 0.11 0.04 Parallel lines
that are afr away to each
other
Protein 0.06 0.02 0.11 0.50 Parallel lines
that are afr away to each
other
Fat 0.00 0.00 0.01 0.82 Parallel lines
that are afr away to each
other
Antioxidant 0.00 0.30 0.02 0.93 Parallel lines
that intersect The relationship between the length of fermentation time and the significant effect of water content is that fermentation lasting more than 24 hours will increase the metabolic process of microorganisms so that the temperature in the material increases and the water produced during the fermentation process will evaporate. An increase in body temperature occurs because metabolism produces CO2 and heat energy. Another factor is the generation of heat due to the decomposition of organic compounds by the enzymatic activity of microorganisms (Putri, 2019). The length of fermentation time has a significant effect on protein content because changes in protein content (tending to decrease) are caused by L. plantarum degrading protein to meet nitrogen needs (Tedom et al., 2019).
Pumpkin flour (control) showed a carbohydrate content of 72.45%, which was lower than pumpkin flour fermented by L. plantarum (74.64 and 72.89%) and S. cerevisiae (74.50 and 71.69%) at different times. The fermentation process by L. plantarum with different lengths of time significantly increased the carbohydrate content compared to the control; however, fermentation between L. plantarum and S. cerevisiae did not significantly increase the carbohydrate content of pumpkin flour. When compared to the research of Tedom et al. (2019), the carbohydrate content of pumpkin flour fermented by L. plantarum for 70 hours was 78.25%
higher than the results of the research obtained. Carbohydrate levels are strongly influenced by other proximate levels; when other proximate levels increase, carbohydrate levels also increase (Siletty et al., 2022). Other proximate results (fat and protein) showed an increase by fermentation (referring to Table 1 above). The increase in carbohydrate content in fermented pumpkin flour is thought to be due to the formation of more monosaccharides resulting from the degradation of microorganism metabolism. This assumption is in accordance with the opinion of Yani & Akbar (2018), which explains that the activity of amylase and diastase enzymes in LAB metabolism causes the hydrolysis of starch into simple forms such as dextrin, maltose, and reducing sugar. To strengthen this assumption, it is necessary to test the total sugar content.
The antioxidant content of pumpkin flour in the control was 12.69%, which was lower than the antioxidant content of pumpkin flour fermented at different times by L. plantarum
28 (15.94 and 16.01%) and S. cerevisiae (14.82 and 15.90%). The fermentation process by L.
plantarum and S. cerevisiae with different lengths of time significantly increased antioxidant levels compared to the control; however, fermentation between L. plantarum and S. cerevisiae did not significantly increase the antioxidant levels of pumpkin flour. Wahyono et al. (2018) explained that drying at high temperatures will increase the formation of phenol compounds that are responsible for increasing antioxidant activity. Drying will have an increasing impact on several phenolic compounds, such as chlorogenic, syringic, p-hydrobenzoic, caffeic, gallic, p-coumaric, and sinapic acids. Another factor is the activation of other components that have antioxidant activity, such as maillard products, and the inactivation of oxidation and hydrolysis enzymes.
This is thought to be because the fermentation process causes the water content in the substrate to increase due to diffusion. The assumption is in accordance with the opinion of Mandasari et al. (2015), stating that the longer soaking causes the weakening of hydrogen bonds in starch due to starch OH-groups substituted by acetyl groups so that water becomes easier to penetrate into starch granules. The fermentation process by L. plantarum and S.
cerevisiae with different lengths of time was able to reduce the moisture content of pumpkin flour significantly compared to the control. The ability of L. plantarum in reducing water content is the same as S. cerevisiae, but the longer the fermentation time, the decrease in water content is not significant. This is due to the longer degradation activity by microorganisms on the fermentation substrate causing the reduction of water content in the substrate. The results obtained are in accordance with the opinion of Winarti et al. (2022), which states that the smaller the size makes the surface area larger and the longer the fermentation time results in the longer the microbes contact with the material and accelerate the degradation of compounds which results in damaged compound molecules so that water molecules easily evaporate
CONCLUSIONS
The chemical characteristics of fermented pumpkin flour showed an interaction between fermentation microorganisms and fermentation time on the antioxidant activity of fermented pumpkin flour, but there was no interaction between fermentation microorganisms and fermentation time on moisture, ash, protein, fat, or carbohydrate content of fermented pumpkin flour. Fermentation by S. cerevisiae for 3 days in flour making has the best ability to produce chemical properties and antioxidant activity.
ACKNOWLEDGMENTS
The author would like to thank the Institute of Research and Community Service of the University of Semarang for providing funds for the implementation of research with agreement number: 012/USM.H7.LPPM/L/2023.
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