Time Course Evaluation of Biochemical Contents and Biocatalytic Activities of Jiaosu from Fruit Wastes During One-Year Natural Fermentation

Abstract

Jiaosu is a multifunctional solution derived from the fermentation of a mixture of fruit or vegetable wastes, sugar, and water for a typical period of three months. The present study evaluated the changes in pH, proteins, phenolics, carbohydrates, alcohols, and organic acids (oxalic, tartaric, malic, lactic, acetic, citric, and succinic) as well as amylase, protease, and lipase activities of different groups of jiaosu throughout one year of natural fermentation. Three jiaosu groups, each with different types of fruit peels, were prepared: orange–papaya–watermelon (OPW), grapefruit–mango–pineapple (GMP), and durian–jackfruit–passion fruit (DJP). A total of 18 jiaosu samples (days 0, 7, 14, 21, 28, 42, 56, 70, 84, 120, 150, 180, 210, 240, 270, 300, 330, and 360) were analyzed for each group. Using repeated measures multivariate analysis of variance (MANOVA) over the one-year fermentation period, the pH, the concentrations of proteins, phenolics, carbohydrates, alcohols, and lactic acid, and the amylase, protease, and lipase activities were significantly different (p < 0.05) between all three groups of jiaosu. Notably, GMP showed the highest total protein and phenolic concentrations and the lowest specific protease activity (p < 0.05) among the jiaosu groups. Meanwhile, DJP exhibited higher specific lipase activity and lactic acid concentration, but lower total alcohol concentration (p < 0.05) compared to OPW and GMP. The results indicated that the biochemical contents and enzyme activities of jiaosu were influenced by fermentation duration and the types of fruit peels used for the fermentation.

1. Introduction

Wet markets, supermarkets, restaurants, and food industries produce high amounts of decomposable food waste such as fruit peels and vegetable dregs. In Malaysia, out of 17,000 tons of food waste generated daily, 76% are unavoidable food waste [1]. The management of unavoidable food waste via combustion is a less efficient method due to the high moisture content in the waste and it may also cause air pollution. Food waste can be decomposed in landfills, but this approach generates about 12% of the global methane emissions that contribute to the depletion of the Earth’s ozone layer [2]. Hence, the reduction in unavoidable food waste should be given much attention as the amount is comparatively higher than avoidable food waste and thus, more detrimental to the environment.

One of the approaches to reducing unavoidable food waste is the recovery of beneficial organic compounds from the waste or the transformation of the waste into functional bio-products through fermentation [3]. Dr. Rosukon Poompanvong, the founder of the Organic Agriculture Association of Thailand, introduced an approach to convert fruit and vegetable wastes into a multipurpose liquid called garbage enzyme [4]. It is produced by the fermentation of a mixture of fruit or vegetable wastes, sugar (brown or white sugar, jaggery, or molasses), and water at room temperature for a typical period of three months. The end product of the fermentation is a liquid that contains various components such as proteins or enzymes, organic acids, phytochemicals, minerals, and alcohols, as well as microorganisms. The liquid is also known as eco-enzyme, bio-enzyme, or jiaosu. However, the term jiaosu, which means elements of fermentation, more accurately represents the content of the liquid than just merely enzymes.

Due to the presence of various bioactive components such as proteins, phenolic compounds, organic acids, enzymes, and alcohols in jiaosu, it has been widely deployed as a natural fertilizer, pesticide, hand sanitizer, disinfectant, or as an organic treatment in waste management [5,6]. A 10% jujube jiaosu significantly enhanced the plant height, root length, fresh and dry weights, and leaf area of bok choi (Brassica chinensis) [7]. This jiaosu was also found to have inhibitory activity against Botrytis cinerea, a common agricultural pathogen, due to the synergistic effect of organic acids and beneficial microorganisms [8]. Jiaosu prepared from orange and watermelon peels could remediate oil-contaminated soils due to its high amylase, catalase, lipase, and protease activities [9]. Our previous study using jiaosu derived from different combinations of fruit peels, i.e., orange–papaya–watermelon, grapefruit–mango–pineapple, and durian–jackfruit–passion fruit demonstrated substantial antibacterial and antifungal activities against human pathogens. These jiaosu samples also possessed killing effects on the larvae of Aedes aegypti and Aedes albopictus, which are the vectors of mosquito-borne diseases such as dengue fever and chikungunya [10], indicating the potential use of these jiaosu as cleaning agents and biopesticides.

As most of the studies on jiaosu employed three months as the fermentation period and focused on the applications or uses, there is a lack of exploration of the biochemical changes during the fermentation period of three months and beyond. Therefore, this research aimed to measure the biochemical contents and biocatalytic activities of jiaosu throughout the one-year fermentation process. The biochemical contents included proteins, carbohydrates, phenolics, organic acids, and alcohols while the biocatalytic activities comprised amylase, protease, and lipase of jiaosu prepared from three different combinations of fruit peels, orange–papaya–watermelon, grapefruit–mango–pineapple, and durian–jackfruit–passion fruit.

2. Materials and Methods

2.1. Materials

The following chemicals and bioreagents were used: phenol crystals, potassium permanganate, and sodium acetate trihydrate from Bendosen (Damansara, Selangor, Malaysia), gallic acid and tyrosine from Bio Basic (Markham, ON, Canada), sodium dodecyl sulfate, trichloroacetic acid, Triton X-100 from Fisher Scientific (Waltham, MA, USA), bovine serum albumin from HiMedia (Thane, Maharashtra, India), casein from bovine milk, Folin–Ciocalteu’s phenol reagent, sodium dihydrogen phosphate, sodium hydrogen phosphate, and sodium sulfite from Merck (Rahway, NJ, USA), brown sugar from MSM Prai (Perai, Penang, Malaysia), Bradford reagent and hexadecyltrimethylammonium bromide from Nacalai Tesque (Kyoto, Japan), disodium tetraborate, glacial acetic acid, and maltose from R&M Chemicals (Semenyih, Selangor, Malaysia), 3,5-dinitrosalicylic acid, 4-nitrophenol, 4-nitrophenyl palmitate, acetic acid, citric, acid, D-glucose, ethanol, lactic acid, lipase from porcine pancreas (type II), malic acid, oxalic acid, protease from bovine pancreas (type I), succinic acid, tartaric acid, and α-amylase from porcine pancreas (type VI-B) from Sigma-Aldrich (St. Louis, MO, USA) and soluble starch from Systerm (Shah Alam, Selangor, Malaysia).

2.2. Collection of Fruit Waste

Nine types of fruit peels were selected based on their availability during this study. The fruit peels were sourced from households, fresh fruit sellers, and fruit juice shops located in the towns of Ipoh, Batu Gajah, and Tanjung Tualang of Perak state. The fruit peels were allocated into three groups with three types of fruit peels in each group: orange–papaya–watermelon (OPW), grapefruit–mango–pineapple (GMP), and durian–jackfruit–passion fruit (DJP).

2.3. Jiaosu Preparation and Sampling

The jiaosu for each group of fruit peels was prepared by mixing 6 kg of three different fruit peels (2 kg of each fruit peel) with 2 kg of brown sugar and 20 L of deionized water based on the ratio of 3:1:10 (w/w/w) [10]. Each group was prepared in three 30 L high-density polyethylene plastic barrels that were stored at room temperature protected from sunlight and subjected to natural fermentation for one year. Samples were taken before fermentation (day 0), weekly during the first month of fermentation (day 7, 14, 21, and 28), biweekly during the second and third months of fermentation (day 42, 56, 70, and 84), and monthly from the fourth month until the twelfth month (Figure S1). The jiaosu sample collected at each time point was centrifuged at 10,000 rpm for 15 min and the resulting supernatant was used for analysis in triplicates. Prior to the centrifugation, the pH value of the sample was measured using a calibrated pH meter.

2.4. Proteins and Biocatalytic Characterization

2.4.1. Total Protein Concentration

The total protein concentration was measured using a linearized Bradford protein assay [11] with bovine serum albumin (1.25–25 µg/mL) as a standard for the calibration curve. The standard solutions and samples of 100 µL were each added with an equal volume of Bradford reagent in a 96-well microplate. The microplate was incubated in the dark at room temperature for 5 min. The negative control was 200 µL of deionized water. After incubation, the absorbance was measured at 450 nm and 590 nm using a spectrophotometric microplate reader (FLUOstar^® Omega, BMG Labtech, Mornington, VIC, Australia). The calibration curve was plotted using the absorbance ratio (590 nm/450 nm) vs. bovine serum albumin concentration.

2.4.2. Amylase Activity

The amylase activity was determined using the 3,5-dinitrosalicylic acid (DNSA) method [12]. Maltose was used as the standard to construct a calibration curve ranging from 100 to 1000 µg/mL. In separate test tubes, 250 µL of the sample and deionized water (negative control) were each added with 2.5 mL of the substrate 1% starch solution. The mixtures were incubated at 37 °C for 10 min. After that, 1 mL of DNSA reagent was added to the mixtures and incubated at 95 °C for 10 min. After cooling at room temperature, the mixtures were transferred to a 96-well microplate, and the absorbance was measured at 570 nm using the microplate reader. The specific amylase activity was calculated based on the concentration of maltose formed and expressed as µmol/min/µg of protein.

2.4.3. Protease Activity

The protease activity was quantified using a non-specific protease assay that employed casein as the substrate [13]. An enzyme diluent was prepared by mixing 10 mM sodium acetate trihydrate, 5 mM glacial acetic acid, and 5 mM calcium acetate hydrate and adjusted to a pH of 7.5. Then, 0.5 mL of the sample was added with 0.5 mL of the enzyme diluent and 5 mL of 0.65% casein in a test tube. The mixture was incubated at 37 °C for 5 min. Later, 0.5 mL of the enzyme diluent was added to the mixture and incubated at 37 °C for 10 min. Another 0.5 mL of the enzyme diluent and 5 mL of 0.11 M trichloroacetic acid were added to the mixture and incubated for 30 min. The mixture was then filtered using a 0.45 µm nylon syringe filter. The filtered sample and standard solutions were added with 5 mL of 0.5 M sodium carbonate and 1 mL of 0.5 M Folin–Ciocalteu’s phenol reagent. The solutions were transferred to a 96-well microplate and the absorbance was read at 660 nm using the microplate reader. For negative control, the sample was replaced with deionized water. The amount of tyrosine released from the casein was determined using the tyrosine standard curve (20–100 µg/mL). The specific protease activity was expressed as µmol/min/µg of protein.

2.4.4. Lipase Activity

The lipase activity was measured using a spectrophotometric assay in which 4-nitrophenol (5–50 µg/mL) was used as the standard and 4-nitrophenyl palmitate (4-NPP) as the substrate [14]. Four solutions were prepared before the assay: A (sodium dodecyl sulfate with Triton X-100), B (Triton X-100 and 4-NPP), C (solution A added with Tris-HCl buffer (pH 7)), and D (solution B added with Tris-HCl buffer). For the generation of a calibration curve, 100 µL of solution C was added into a 96-well microplate and incubated in the microplate reader at 30 °C for 5 min, followed by the addition of 100 µL of the standards, and the absorbance was recorded at 410 nm. The negative control was 100 µL of deionized water. For the enzymatic assay, 100 µL of solution D was added into the 96-well microplate and pre-incubated in the microplate reader at 30 °C for 5 min. After that, 100 µL of the samples was added into the wells and the absorbance was measured at 410 nm every 5 min for a reaction time of 1 h. The highest sample absorbance was used to determine the 4-nitrophenol concentration from the calibration curve and the specific lipase activity expressed in µmol/min/µg of protein was calculated.

2.5. Total Phenolic Concentration

The total phenolic concentration was quantified based on the Folin–Ciocalteu method [15] with modifications. Gallic acid (15.6–750 µg/mL) was used to construct a calibration curve while deionized water was used as a negative control. After treating the samples, standards, and negative control with 50% Folin–Ciocalteu’s phenol reagent, followed by 700 mM sodium carbonate, the microplate was incubated in the dark at room temperature for 90 min. The absorbance value was then read at 765 nm using the microplate reader. The total phenolic concentration of each sample was expressed as mg gallic acid equivalent (GAE)/mL.

2.6. Total Carbohydrate Concentration

The total carbohydrate concentration was analyzed using the phenol-sulfuric acid method [16] with slight modifications. Briefly, 100 µL of glucose standard or samples were mixed with 200 µL of 96% sulfuric acid, followed by the addition of 80 µL of 5% phenol solution. The mixture was incubated at 90 °C for 5 min. After cooling at room temperature for 5 min, the absorbance value at 490 nm was recorded using the microplate reader. A plot of the absorbance value against the glucose standard concentration (200–1000 µg/mL) was used to determine the total carbohydrate concentration of jiaosu samples.

2.7. Total Alcohol Concentration

The total alcohol concentration was determined using a spectrophotometric method with ethanol and D-glucose as the standards [17]. The jiaosu samples were pre-treated with 20% trichloroacetic acid solution and centrifuged at 10,000 rpm for 5 min. The supernatant was filtered with 0.22 µm nylon syringe filters and added with 20% hexadecyltrimethylammonium bromide. The mixture was then incubated at 65 °C for 10 min and centrifuged again at 10,000 rpm for 10 min. The resulting supernatant was used for 3,5-dinitrosalicylic acid (DNSA) assay and potassium permanganate assay.

For the DNSA assay, D-glucose (0.16–2.50 mg/mL) was used as the standard for the calibration curve. Each pre-treated sample (20 µL) was added with 180 µL of deionized water, followed by 600 µL of DNSA reagent and incubated at 95 °C for 5 min. After cooling at room temperature, the mixture was transferred into a 96-well microplate, and the absorbance at 550 nm was taken using the microplate reader. As for the potassium permanganate assay, ethanol (0.039–0.625 µL/mL) and D-glucose (0.016–0.250 mg/mL) were used as the standards to construct two calibration curves separately. The pre-treated sample (2 µL) was added with 198 µL of deionized water, followed by 200 µL of potassium permanganate solution, and incubated at 40 °C for 90 min. After cooling at room temperature, the mixture was then transferred into a 96-well microplate for absorbance measurement at 526 nm. After subtracting the portion of absorbance increase contributed by reducing sugars from the DNSA assay, the remaining absorbance decrease was used to calculate the total alcohol concentration in percentage (% v/v) for all jiaosu samples.

2.8. Organic Acid Content

The organic acid content was quantified using high-performance liquid chromatography (HPLC) coupled with an ultraviolet detector (Model 1100, Agilent Technologies Inc., Santa Clara, CA, USA) set to 210 nm. A C18 column (150 mm × 4.6 mm × 5 µm; Purospher^® STAR RP-18, Merck KGaA, Darmstadt, Germany) was used as the stationary phase for separation of organic acids at 30 °C. The mobile phase was 20 mM sodium dihydrogen phosphate (pH of 2.7) running at a flow rate of 1.0 mL/min [18]. The standard solutions of tartaric, malic, lactic, acetic, citric, and succinic acids (all 100–1000 µg/mL) and oxalic acid (20–100 µg/mL) were prepared using the mobile phase and filtered using 0.22 µm nylon syringe filters before use. The samples were filtered using 0.22 µm nylon syringe filters and diluted with an equal volume of the mobile phase before injection. The injection volume was 20 µL. The organic acid concentrations in the jiaosu samples were interpolated from the plots of peak areas versus standard concentrations.

2.9. Data Analysis

All data were presented in mean ± standard deviation of three replicates. Two sets of data representing the first three months of fermentation (day 0, 28, 56, and 84) and from the third month to one year (day 84, 6th month, 9th month, and 12th month) were examined for statistical significance (p < 0.05) by repeated measures multivariate analysis of variance (MANOVA) using JMP statistical software version 16.2 (JMP Statistical Discovery LLC, Cary, NC, USA). Wilk’s Lambda test was used as the multivariate test and the Greenhouse–Geisser Epsilon (G-G) estimates of sphericity were applied when Mauchly’s test indicated that the assumption of sphericity had been violated (p < 0.05).

3. Results

3.1. pH Values

The pH values of OPW, GMP, and DJP at day 0 were 5.19 ± 0.11, 4.29 ± 0.06, and 5.01 ± 0.09, respectively. Within one week, the pH values dropped sharply to 3.36 ± 0.06, 3.05 ± 0.01, and 3.43 ± 0.03, correspondingly (Figure 1). After that, the pH values of jiaosu samples remained relatively stable from day 14 to the 12th month; 2.60 ± 0.02–3.37 ± 0.09 for OPW, 2.82 ± 0.04–3.39 ± 0.06 for GMP, and 2.61 ± 0.01–3.39 ± 0.05 for DJP. The pH values of GMP were significantly lower compared to that of OPW and DJP throughout the one year of fermentation (p < 0.05).

3.2. Proteins and Biocatalytic Characterization

3.2.1. Total Protein Concentration

The total protein concentrations of OPW, GMP, and DJP before fermentation (day 0) were 5.00 ± 0.47, 3.13 ± 0.38, and 1.20 ± 0.12 µg/mL, respectively. The fermentation process led to an increase in the protein concentrations, with the highest concentrations of 6.24 ± 0.40 µg/mL at 10th month for OPW, 21.45 ± 1.38 µg/mL at 7th month for GMP, and 4.77 ± 0.29 µg/mL at 5th month for DJP (Figure 2). At the end of one year of fermentation, the protein concentrations of OPW, GMP, and DJP were lower (2.98 ± 0.36 µg/mL), tripled (9.57 ± 0.48 µg/mL), and slightly higher (1.72 ± 0.20 µg/mL), respectively, than the ones before fermentation. The total protein concentrations of GMP were the highest among the jiaosu groups throughout the one year of fermentation (p < 0.05).

3.2.2. Amylase Activity

The jiaosu samples showed different specific amylase activities before fermentation (day 0) with OPW having the highest activity (2507.97 ± 15.32 µmol/min/µg of protein), followed by GMP (1209.24 ± 25.45 µmol/min/µg of protein) and DJP (237.31 ± 10.47 µmol/min/µg of protein). Within a week, the specific amylase activity of OPW increased by 2.3 fold to 5725.36 ± 73.84 µmol/min/µg of protein whereas the specific activity decreased by more than two fold for GMP and DJP, as shown in Figure 3a. From day 14 until 12th month, the specific amylase activities, in µmol/min/µg of protein, varied between 8.06 ± 0.58–110.64 ± 13.15 for OPW, 11.79 ± 0.70–174.37 ± 2.11 for GMP, and 32.02 ± 1.00–173.80 ± 20.46 for DJP. The specific amylase activities were significantly different (p < 0.05) between the three groups of jiaosu throughout the one year of fermentation.

3.2.3. Protease Activity

Before fermentation, OPW had the lowest specific protease activity (3.70 ± 0.19 µmol/min/µg of protein), followed by GMP (4.26 ± 0.23 µmol/min/µg of protein) and DJP (10.29 ± 0.44 µmol/min/µg of protein). After one week of fermentation, the specific protease activity of OPW increased to 9.42 ± 0.29 µmol/min/µg of protein and decreased thereafter until the lowest specific activity of 2.45 ± 0.11 µmol/min/µg of protein observed at the 10th month (Figure 3b). In contrast, the specific protease activities of GMP and DJP decreased after fermentation. The specific activity varied between 0.71 ± 0.04 and 3.73 ± 0.05 µmol/min/µg of protein for GMP and between 3.41 ± 0.09 and 6.97 ± 0.21 µmol/min/µg of protein for DJP. The specific protease activities of GMP were the lowest among the jiaosu groups throughout the one year of fermentation (p < 0.05).

3.2.4. Lipase Activity

Jiaosu OPW had the lowest specific lipase activity among the jiaosu samples at day 0 with 1.89 ± 0.96 µmol/min/µg of protein and the specific activity increased gradually until the highest level of 7.47 ± 3.02 µmol/min/µg of protein at the 12th month of fermentation (Figure 3c). The specific lipase activity of GMP varied between 0.75 ± 0.09 µmol/min/µg of protein at the 4th month and 5.49 ± 1.16 µmol/min/µg of protein at day 0. As for DJP, it had the highest specific lipase activity (17.86 ± 3.48 µmol/min/µg of protein) among the jiaosu samples tested before fermentation. It is noticed that there was a surge in the specific activity for this jiaosu sample during the 4th month to 7th month of fermentation compared to the other two jiaosu samples (Figure 3c). The specific lipase activity of DJP was significantly higher compared to OPW and GMP throughout the one year of fermentation (p < 0.05).

3.3. Total Phenolic Concentration

The total phenolic concentrations of all jiaosu samples before fermentation were low, with 0.60 ± 0.04, 0.84 ± 0.02, and 0.17 ± 0.00 mg GAE/mL for OPW, GMP, and DJP, respectively. The concentrations started to increase after day 7 in all jiaosu samples and remained relatively stable throughout the one-year fermentation period (Figure 4). The concentrations varied between 1.85 ± 0.05 and 3.48 ± 0.19 mg GAE/mL for OPW, 3.66 ± 0.05 and 5.90 ± 0.06 mg GAE/mL for GMP, and 1.47 ± 0.05 and 3.12 ± 0.11 mg GAE/mL for DJP. Jiaosu GMP contained the highest concentrations of phenolics, followed by OPW and DJP (p < 0.05) throughout the one year of fermentation.

3.4. Total Carbohydrate Concentration

All jiaosu samples contained high total carbohydrate concentrations before fermentation, with 104.98 ± 0.71, 105.31 ± 2.46, and 52.58 ± 1.43 mg/mL for OPW, GMP, and DJP, respectively. The carbohydrates were used extensively during the first month of fermentation, in which the concentrations at day 28 decreased to 10.76 ± 0.16 mg/mL (89.7% reduction) for OPW, 14.34 ± 0.77 mg/mL (86.4% reduction) for GMP, and 8.20 ± 0.37 mg/mL (84.4% reduction) for DJP (Figure 5). After one year of fermentation, the total carbohydrate concentrations of the jiaosu samples ranged from 2.42 ± 0.04 to 3.09 ± 0.16 mg/mL. The total carbohydrate concentrations were significantly different between all three jiaosu groups throughout the one year of fermentation (p < 0.05).

3.5. Total Alcohol Concentration

The alcohol concentration in jiaosu was only detectable from day 7 onward for GMP (0.93 ± 0.37% v/v) and day 14 onward for OPW (0.90 ± 0.24% v/v) and DJP (0.52 ± 0.24% v/v). The total alcohol concentration peaked at the 4th month of fermentation for OPW (8.91 ± 0.29% v/v) and day 84 for GMP (10.43 ± 0.37% v/v) and DJP (4.43 ± 0.41% v/v), as shown in Figure 6. After that, the alcohol concentrations decreased gradually to a range of 0.22 ± 0.19 to 0.63 ± 0.31% v/v at the end of one year of fermentation. Comparatively, DJP had the lowest total alcohol concentrations as compared to OPW and GMP (p < 0.05) throughout the one year of fermentation.

3.6. Organic Acid Content

Oxalic acid was the least abundant organic acid in all jiaosu samples either before or after fermentation (Figure 7a). At day 0, the oxalic acid concentrations in OPW, GMP, and DJP were 44.52 ± 0.33, 12.88 ± 0.38, and 5.85 ± 0.31 µg/mL, respectively. After one year of fermentation, the concentration decreased to 15.55 ± 0.33 and 7.09 ± 0.24 µg/mL for OPW and GMP, respectively, but increased to 18.46 ± 0.24 µg/mL for DJP. However, the oxalic acid concentrations were not significantly different between all three jiaosu groups throughout the one year of fermentation (p > 0.05).

Before fermentation (day 0), the jiaosu samples had different levels of tartaric acid with OPW having a much higher concentration (2100.63 ± 11.02 µg/mL) compared to GMP (189.83 ± 3.08 µg/mL) and DJP (38.11 ± 2.20 µg/mL). The tartaric acid in OPW decreased by 88.2% to 246.88 ± 5.42 µg/mL after three weeks of fermentation and the concentrations varied between 49.81 ± 1.00 µg/mL and 280.38 ± 6.47 µg/mL after that (Figure 7b). For GMP, the tartaric acid concentration slowly increased to the highest concentration of 652.02 ± 2.31 µg/mL in the 6th month and then decreased to 97.77 ± 2.27 µg/mL in the 12th month. For DJP, the concentration increased after fermentation to a maximum of 606.44 ± 1.09 µg/mL on day 56 and slowly decreased to 334.18 ± 1.87 µg/mL on the 12th month. Statistically, there was no significant difference in tartaric acid concentration between the three jiaosu groups (p > 0.05).

For malic acid, the concentrations of OPW, GMP, and DJP before fermentation were 125.83 ± 4.75, 159.63 ± 2.51, and 67.36 ± 1.93 µg/mL, respectively. The concentrations increased after fermentation to the highest levels of 266.81 ± 7.42 (day 70), 456.52 ± 5.40 (4th month), and 367.29 ± 9.59 (4th month) µg/mL, respectively. After that, the concentrations decreased and by the end of one year of fermentation, the concentrations remained were 150.56 ± 2.19, 202.81 ± 0.60, and 167.23 ± 2.50 µg/mL, respectively (Figure 7c). Similar to tartaric acid, there was no significant difference in malic acid concentration between the three jiaosu groups (p > 0.05).

The lactic acid concentrations of all jiaosu samples were low before fermentation, ranging from 67.30 ± 1.10 to 133.41 ± 2.26 µg/mL. However, the lactic acid concentrations increased significantly after fermentation. For OPW, the concentration increased to the highest level of 4648.66 ± 28.29 µg/mL on the 4th month and then declined to 953.10 ± 26.93 µg/mL on the 12th month (Figure 7d). Similarly, the concentration for GMP increased to a maximum of 588.46 ± 7.13 µg/mL at day 56 and then decreased to 269.54 ± 2.52 µg/mL on the 12th month. In contrast, the concentration for DJP increased drastically to 5737.62 ± 125.73 µg/mL after one week of fermentation and steadily to the highest level of 8216.75 ± 30.99 µg/mL after one year of fermentation (Figure 7d). Hence, DJP had the highest concentrations of lactic acid, followed by OPW and GMP over the one year of fermentation (p < 0.05).

Similarly to lactic acid, the acetic acid concentrations of all jiaosu samples at day 0 were low, ranging from 61.97 ± 6.74 to 171.84 ± 8.62 µg/mL. Fermentation significantly increased the concentrations of acetic acid in all the samples. For OPW, the concentration increased drastically to 9986.40 ± 86.51 µg/mL on day 28 and then decreased gradually to 785.69 ± 6.78 µg/mL on the 12th month (Figure 7e). For GMP, the concentration also increased sharply to a maximum of 7856.30 ± 29.81 µg/mL at day 70 and then decreased to 3514.53 ± 28.25 µg/mL on the 12th month. For DJP, the concentration increased and maintained relatively stable in the range of 2395.83 ± 18.28 µg/mL (12th month) to 3281.04 ± 36.29 µg/mL (4th month). However, the acetic acid concentrations were not significantly different between the three jiaosu groups throughout the one year of fermentation (p > 0.05). The results also implied that acetic acid was the most abundant organic acid for OPW and GMP.

The citric acid concentrations of all jiaosu samples were low before fermentation (32.49 ± 2.24–52.67 ± 1.13 µg/mL) and increased after fermentation. The highest concentrations were recorded at day 70 with 558.22 ± 4.84, 712.93 ± 13.78, and 1062.38 ± 7.53 µg/mL for OPW, GMP, and DJP, respectively (Figure 7f). After one year of fermentation, the concentrations declined to 184.90 ± 6.31, 253.52 ± 10.43, and 465.18 ± 17.78 µg/mL, correspondingly. There was no significant difference in the citric acid concentrations between the three jiaosu groups throughout the one year of fermentation (p > 0.05).

The concentration of succinic acid in jiaosu samples was relatively higher than other organic acids before fermentation; 566.54 ± 7.27 µg/mL for OPW, 584.63 ± 12.03 µg/mL for GMP, and 433.61 ± 12.60 µg/mL for DJP. The concentration increased slowly after fermentation but the time to achieve the highest concentration for each jiaosu sample was different: the 11th month for OPW with 2398.50 ± 18.84 µg/mL, day 56 for GMP with 3066.33 ± 40.92 µg/mL, and day 84 for DJP with 2318.25 ± 23.22 µg/mL, as shown in Figure 7g. Nevertheless, the succinic acid concentrations were not significantly different between the three jiaosu groups throughout the one year of fermentation (p > 0.05).

4. Discussion

This study showed that all jiaosu products are acidic, regardless of the types of fruit peels used. The reduction in the pH after one week of fermentation can be attributed to the microorganism-induced conversion of the bulk of sugars in the fermentation mixtures to acids, ethanol, and carbon dioxide and the presence of organic acids naturally found in the fruit waste that could have been drawn out into the fermented solutions by microbial activities [5].

The increase in total protein concentration for all jiaosu groups after fermentation corresponded to the decrease in total carbohydrate concentration (Figure 5). Microorganisms utilize carbohydrates as the major carbon and energy sources and metabolize them to produce carbon dioxide as a by-product which in turn causes the build-up of nitrogen that aids in the production of proteins [19,20]. The decrease in total protein concentrations at the latter stage of fermentation could be due to the utilization of proteins as the alternative source of energy for microbial growth [21]. The high total protein concentration in GMP compared to OPW and DJP (Figure 2) suggested that the microbial profile involved in the fermentation for GMP is likely different from the microbial profiles for the other two jiaosu samples. Besides that, the total protein concentrations of all jiaosu samples in this study were lower compared to the jiaosu produced using papaya, banana, sapodilla, and pomegranate fruit peels, which recorded a concentration of 4.23 mg/mL [22]. This also suggests that the types of fruit peel used may affect the total protein concentration in jiaosu. However, further study is needed to verify these suggestions.

The difference in specific amylase activity among the jiaosu samples before fermentation was indicative of the presence of this enzyme in the fruit peel wastes. Comparing the specific amylase activities (237.31–2507.97 µmol/min/µg of protein) to the total carbohydrate concentrations (52.58–105.31 mg/mL) at day 0 in the three jiaosu samples leads us to postulate that the amylases present in the samples may have different biocatalytic capacities with the highest capacity being the one present in OPW. The high amylase activities at the first few weeks of fermentation (Figure 3a) corresponded well to the drastic decrease in total carbohydrate concentrations of the jiaosu samples, particularly for OPW and GMP (Figure 5), as amylase is the enzyme responsible for converting carbohydrates to oligosaccharides and glucose [21]. The variations in amylase activity in jiaosu samples also imply the involvement of microorganisms during the fermentation period. For example, Garg et al. [23] conducted a fermentation of mango stone waste using Lactobacillus plantarum, and the amylase activity increased from 0.0573 U/mL/min on day 7 to 0.4424 U/mL/min on day 13 and then decreased to 0.3626 U/mL/min at day 28. Chin et al. [24] evaluated the total carbohydrate concentration of mixed fruits (pomelo, watermelon, and melon) fermented for three months. The fermentations that were supplemented with Saccharomyces cerevisiae and yogurt (lactic acid bacteria) resulted in lower carbohydrate concentrations, 4.30 and 8.80 mg/mL, respectively, compared to the natural fermentation (13.10 mg/mL), indicating the utilization of carbohydrates by microorganisms during fermentation.

The specific protease and lipase activities varied between the jiaosu samples before fermentation as well as after fermentation, suggesting the types of fruit peels and microorganisms play important roles in the fermentation. Chin et al. [24] compared the protease activity between orange peels and pineapple peels after three months of fermentation and found that fermented orange peels showed higher protease activity (0.129 U/mL) than fermented pineapple peels (0.046 U/mL). Selvakumar and Sivashanmugam [25] compared the lipase activity of pomegranate, orange, and pineapple waste at two different ratios, 35:20:35 (w/w/w) and 10:40:40 (w/w/w) and found that the combination with a higher portion of pomegranate exhibited higher lipase activity (9.7 U/mL) than the other combination (6.8 U/mL). The surge in lipase activity in DJP between the 4th month and 7th month (Figure 3c) most likely signifies the presence of lipase-producing fungi or bacteria during that period. Microbial isolation or metagenomic study could shed some light on the identity of these exoenzyme-producing microorganisms. Nevertheless, the results of biocatalytic activities from this study reaffirmed the reports from other studies that jiaosu contained amylase, protease, and lipase [9,22].

The increase in total phenolic concentration in all jiaosu samples after fermentation could be caused by the metabolic activities of microorganisms. During fermentation, microorganisms secreted enzymes to break down the structural integrity of the cell walls of the fruit peels which liberated the bound phenolics and hence, increased the total phenolic concentration [26]. Rusdianasari et al. [27] reported an increase in the total phenolic concentration from 184.6 mg/L to 762.2 mg/L after three months of fermentation using fruit peel waste. The peels of some fruits such as grapefruit, lemon, orange, and pomegranate have been shown to have higher total phenolic content compared to their pulps or peeled fruits [28,29]. The relatively stable concentrations of phenolics in all jiaosu samples throughout the one year of fermentation suggest that the phenolics originated from the fruit peels rather than as the end products of microbial metabolism. It is also of interest to look into the polyphenol profile of these combinations and their changes during the entire fermentation period using more advanced techniques such as ultra-performance liquid chromatography–mass spectrometry.

Under anaerobic conditions, glucose is broken down into pyruvic acid by glycolysis, which is then converted to acetaldehyde by pyruvate decarboxylase. Acetaldehyde is then catalyzed to ethanol and carbon dioxide by the action of alcohol dehydrogenase [6]. In the present study, alcohol was detected in the jiaosu samples only after one to two weeks of fermentation and increased to the highest levels between day 84 to 4 months of fermentation (Figure 6). This shows that the presence of alcohol in jiaosu samples during fermentation resulted from the enzymatic activities of microorganisms. Singh et al. [30] reported that the maximum ethanol production from banana peels fermented using Saccharomyces cerevisiae could be achieved in a shorter period by increasing the fungal cell concentrations. The decrease in total alcohol concentration after the peaks may be due to the hydrogenation of alcohol to form acetaldehyde which is then converted by aldehyde dehydrogenase to form acetic acid [31]. In this study, the highest alcohol level achieved by each jiaosu group was different, with 10.43% v/v for GMP, 8.91% v/v for OPW, and 4.43% v/v for DJP. In contrast, jiaosu produced from a three-month fermentation of papaya, banana, sapodilla, and pomegranate peels contained 0.18 mL/mL (=18% v/v) of alcohol [22]. These results strongly suggest that the alcohol content could be influenced by the types of fruit peels used and the microorganisms involved in the fermentation.

In this study, seven types of organic acids, i.e., oxalic, tartaric, malic, lactic, acetic, citric, and succinic acids were detected in all three jiaosu samples before fermentation (day 0), suggesting these organic acids originated from the fruit peels. This is supported by the fact that some of these organic acids have been reported from the peels of oranges, papaya, and jackfruit [32,33,34]. The concentrations of these seven organic acids were found to be increased in all three jiaosu samples after fermentation (except for tartaric acid in OPW), signifying the involvement of microorganisms in producing organic acids for jiaosu samples during fermentation. The three most abundant organic acids in the jiaosu samples, in descending order, were acetic, lactic, and succinic acids for OPW, acetic, succinic, and citric acids for GMP, and lactic, acetic, and succinic acids for DJP (Figure 7).

Acetic acid is a major product of jiaosu fermentation. It is usually produced from the oxidation of alcohol, which is generated from glycolysis of glucose, by bacteria such as Acetobacter and Gluconobacter [35]. The concentrations of acetic acid obtained in this study were comparable to the value (78.14 g/L) obtained for jiaosu fermented using a mixture of tomato, cauliflower, pineapple, orange, and mango peels for 90 days [36].

The high and sustained lactic acid concentration in DJP (Figure 7d) suggested that the population of lactic acid-producing microorganisms was more abundant in DJP than in the other two jiaosu groups. Bacteria such as Lactobacillus casei and Lactobacillus delbrueckii could use orange and mango peels as the substrate for the production of lactic acid [37]. In a study using pre-treated corn stover as a substrate, mixed cultures of Lactobacillus rhamnosus and Lactobacillus brevis produced a lactic acid concentration of 0.70 g/g, which was about 18.6% and 29.6% higher than that by single cultures of Lactobacillus rhamnosus and Lactobacillus brevis, respectively [38]. Similarly, the filamentous fungus Rhizopus oryzae has been used to produce lactic acid from pineapple waste [39]. Microbial profiling or the metagenomic study of jiaosu during fermentation is essential for us to have a better understanding of the microorganisms involved in lactic acid production.

Microorganisms can produce succinic, citric, and malic acids from their metabolic activities via the tricarboxylic acid cycle. This cycle involves the conversion of carbohydrates, proteins, and fats into carbon dioxide and water for microbial growth [40]. Woo et al. [41] presented the application of the bacterium Actinobacillus succinogenes in fermenting the hydrolysates of durian husk for succinic acid production. Zhang et al. [42] used the yeast Saccharomyces cerevisiae to ferment apple juice and the concentration of succinic acid produced increased from 19.21 mg/L to 290.53 mg/L in 18 days. Production of citric acid through the fermentation of orange peels or pineapple waste was achieved by using the filamentous fungus Aspergillus niger [43,44]. In apple juice inoculated with S. cerevisiae, the citric acid concentration increased from 116.52 mg/L at day 0 to 512.89 mg/L at day 18 [42]. The decrease in citric acid concentration after the peak at day 70 for all jiaosu samples may be due to the conversion of citric acid to lactic acid by microorganisms. A comparison between natural fermentation and Lactobacillus plantarum-supplemented fermentation of fig pulp revealed that the addition of the bacterium significantly reduced citric acid concentration but increased malic acid concentration [45]. The production of malic acid during fermentation is due to the tricarboxylic acid cycle in which malic acid can be reduced from oxaloacetic acid via malate dehydrogenase or rearranged from fumaric acid via fumarase [46]. Conversely, malic acid can be broken down by microorganisms to form lactic acid during malolactic fermentation [47].

Oxalic acid was the least abundant organic acid in all jiaosu samples. The increase or decrease in oxalic acid during fermentation can be attributed to the metabolic activity of microorganisms. Fu et al. [48] showed that the oxalic acid concentration of blueberry wine declined sharply from 110.08 mg/L to 26.17 mg/L after 4 weeks of yeast fermentation. Another study reported that the oxalic acid content of naturally fermented Huyou (Citrus aurantium) peels was 0.15 mg/g, whereas Huyou peels fermented with 5.18% lactic acid bacteria had an increased oxalic acid content of 0.18 mg/g [49].

At day 0, the tartaric acid concentration in OPW was 11.1 fold and 55.1 fold higher than that in GMP and DJP, respectively (Figure 7b). This could be due to the abundant tartaric acid in the peels of papaya [34] and possibly citrus fruits such as orange [50]. The reduction in tartaric acid concentration could be due to the conversion of tartaric acid into oxaloacetic acid and then into acetic acid, lactic acid, and carbon dioxide by tartaric acid dehydratase produced by bacteria during fermentation [51]. Yao et al. [45] reported a tartaric acid concentration of 122.00 µg/mL for fig pulp before fermentation, and the concentration increased to 340.10 µg/mL after natural fermentation and 1921.79 µg/mL after fermentation with Lactobacillus plantarum. The decrease or increase in tartaric acid after fermentation again reflected the importance of microorganisms in the fermentation process.

5. Conclusions

The present study showed that all jiaosu solutions were acidic. Fermentation caused significant increases in total protein concentration, total phenolic concentration, and total alcohol concentration but decreases in total carbohydrate concentration and specific amylase activity for all three jiaosu samples. The concentrations of oxalic, tartaric, malic, lactic, acetic, citric, and succinic acids for all jiaosu samples were found to be increased after fermentation, except for the tartaric acid concentration in OPW which was decreased after fermentation. Among the jiaosu samples, GMP had a significantly higher total protein concentration and total phenolic concentration, but lower specific protease activity and pH values than the other two groups. On the other hand, DJP showed significantly higher specific lipase activity and lactic acid concentration, but lower total alcohol concentration compared to the other two samples. However, the concentrations of oxalic, tartaric, malic, acetic, citric, and succinic acids were not markedly different between OPW, GMP, and DJP. This study provided a useful evaluation of how the duration of fermentation and the types of fruit wastes could influence the end-product quality of jiaosu. The results from this study could serve as a guide for the more efficient preparation of jiaosu based on the effective level of bioactive compounds for their specific applications in various fields. For example, GMP could be used as a natural fertilizer due to its high phenolic and protein concentrations while DJP could be used as a detergent as it contains high lipase activity. Future research on the microbial population could provide a clearer understanding of the biochemical content levels and biocatalytic activities of jiaosu during natural fermentation.