Rice Bran and American Ginseng Residue as Media for Black Truffle Solid-State Fermentation

Abstract

American ginseng (Panax quinquefolium L.) residue from the extraction industry can be dried and mixed with rice bran as media for black truffle solid-state fermentation to enhance reuse and bioactive functions. Different ratios of rice bran (R) and American ginseng residue (G) mixtures were used as solid-state media for 5 weeks of black truffle fermentation, and then their bio-component contents and whitening effects were analyzed. Finally, four drying methods—hot air drying (HA), microwave drying (MW), hot air-assisted radio frequency (HARF) drying, and radio frequency vacuum (RFV) drying—were assessed to optimize drying efficiency for fermented medium. The results showed that using a 3:1 ratio of rice bran and American ginseng residue as the medium increased the crude polysaccharide and flavonoid contents by approximately threefold and enhanced the ginsenoside Rg3 content about twelvefold. Additionally, the 100 µg/mL ethanol extract of the fermented product inhibited 70% of tyrosinase activity and reduced the melanin area on zebrafish embryos by 42.74%. In the drying study, RFV drying R2G1 required only 13 min without exceeding 70 °C, demonstrating superior drying efficiency, temperature control, and low energy consumption. Overall, this study demonstrates the potential of black truffle fermentation of solid-state media from rice bran and American ginseng residue mixtures for whitening applications and highlights RFV drying as an efficient method for by-products.

1. Introduction

Ginseng (Panax spp.) is a perennial herb of the Araliaceae family, known for its ginsenosides, which provide whitening, immune regulation, and cardiovascular protection. The most common types include American ginseng (P. quinquefolius), Korean ginseng (P. ginseng), and Chinese ginseng (P. notoginseng) [1]. Typically, American ginseng residues are by-products from extraction processes [2], and they are often discarded or underutilized. Fungal bioconversion can modify the ginsenoside composition of the residues, enhancing the value and utilization of this by-product [3]. Fungal glycosidases hydrolyze the glycosyl groups of ginsenosides from American ginseng residue during fermentation to convert Rb1 to bioactive Rg3 [4,5,6].

To ensure the safety of American ginseng residue during storage, it must be dried first to reduce water activity and prevent microbial growth and spoilage [7]. Microwave (MW) and radio frequency (RF) heating are electromagnetic heating technologies that cause dipole rotation or ionic migration of water molecules within food, enabling rapid overall heating and overcoming the heat transfer limitations of conventional hot air drying [8]. In addition, hot air or vacuum can remove evaporated water vapor from food in RF drying systems. The vacuum system can decrease the water evaporation temperature, preventing prolonged high-temperature exposure from degrading nutrients [9]. Nevertheless, samples with high moisture content tend to exhibit elevated dielectric loss factors, which may cause excessive energy absorption during RF drying. This can trigger equipment shutdowns and complicate the drying process [10].

Rice bran is another agricultural by-product rich in nutrients and commonly incorporated in fungal cultivation media. Rice bran was incorporated into American ginseng residues in the present study to lower their initial moisture levels. Furthermore, RF treatment has the added benefit of deactivating lipase enzymes in rice bran, thereby minimizing lipid oxidation and prolonging shelf life [11]. Additionally, incorporating rice bran into the medium supports the proliferation of fungal mycelia and enhances the synthesis of bioactive metabolites [12].

Truffles are edible fungi of the genus Tuber, highly valued for their distinctive aroma. They are also rich in bioactive compounds such as polysaccharides, sterols, polyphenols, flavonoids, and triterpenes [13], exhibiting excellent effects in whitening [14], anti-tumor activity [15], and immune modulation [16]. However, harvesting wild truffles is challenging due to their low yields. Even with semi-artificial cultivation, it takes 4 to 12 years to harvest them [17]. Therefore, truffle solid-state fermentation offers a cost-effective and high-quality alternative.

Most truffle solid-state fermentation studies have used edible grains as the fermented medium for truffles to obtain fermented products rich in bioactive compounds [18,19,20], and adding methionine into the medium can enhance the sulfur-containing characteristic aroma compounds in fermented products [21]. Consequently, utilizing rice bran mixed with American ginseng residues as media for truffle solid-state fermentation presents a promising strategy to increase the production of bioactive compounds. To determine the functional potential of the truffle-fermented products, it is important to investigate their skin-whitening properties, focusing on tyrosinase inhibition activity and verification through an in vivo zebrafish embryo model.

Tyrosinase is the key enzyme responsible for melanin deposition [22,23]. Zebrafish embryos can absorb tyrosinase inhibitors through osmosis to reduce melanin synthesis [24]. Moreover, since zebrafish embryos lack feeding ability within 120 h post-fertilization (hpf), they comply with the cosmetic research regulations of Taiwan’s Ministry of Health and Welfare and the EU Directive 2010/63/EU [25]. Therefore, zebrafish embryos serve as an animal welfare-compliant model organism for evaluating the whitening effects of test samples [26]. When using 200 μg/mL water and ethanol extracts from Poria cocos solid-state fermented rice bran and brown rice product for in vitro tyrosinase inhibition, P. cocos fermented rice bran product had better inhibition [12].

Although the whitening effects of American ginseng have been extensively studied [27,28], research on the fungal fermentation of American ginseng residue is limited. The whitening effect of truffle fruiting bodies has been mentioned in only a few patents [14]. The objective of this study was to use different ratios of rice bran and dried American ginseng residue as media for black truffle solid-state fermentation and analyze the bioactive compound contents and whitening effects on zebrafish embryos.

2. Materials and Methods

2.1. Materials

American ginseng residue (approximately 85% moisture content) was collected from the ultrasonic extraction processes of ACEXTRACT Biotechnology Co., Ltd. (Yilan, Taiwan) and dried at 45 °C using hot air to reduce moisture before use. Rice bran (variety: Indica rice bran of Taichung No. 10) initially containing 14% moisture was obtained from Minfeng Farm in Jiaoxi Township (Yilan, Taiwan) and bleached by a 5 kW HARF for 2 min to inactivate lipase [29]. Fertilized wild-type AB zebrafish embryos, 6 h post-fertilization, were purchased from the Taiwan Zebrafish Center Branch (TZCNHRI, Miaoli, Taiwan).

2.2. Strain Preservation and Pre-Activation

The black truffle strain (Tuber melanosporum) was obtained from Professor Hong-Dao Hu’s laboratory at National Taiwan University. It was cultivated on potato dextrose agar (PDA) plates in a 25 °C incubator (LM-600R, Yihder Co., Ltd., New Taipei, Taiwan). Two pieces of 1 cm² black truffle were taken and placed in the potato dextrose broth (PDB) flasks. They were cultivated at 25 °C and 150 rpm for 7 days.

2.3. Petri-Dish Preparation and Black Truffle Cultivation

A 30 g petri dish (Asahi Glass Co., Ltd., Amagasaki-shi, Hyōgo-ken, Japan) containing different mixtures of rice bran and American ginseng residue at ratios of 4:0, 3:1, 2:2, 1:3, and 0:4, with 40% moisture, was sterilized in a 121 °C autoclave (Tommy SS-325, Tokyo, Japan). Then, 10 mL of black truffle preactivated solution was added to the petri dish and cultivated at 25 °C for 5 weeks.

2.4. Bioactive Compounds Analysis of Black Truffle Fermented Products

2.4.1. Microwave Extraction

The microwave extraction system (Yen Hua Biotech Co., Ltd., Tainan, Taiwan) was self-assembled following an optimized protocol based on previously established methods for white truffles [20]. Briefly, a 2.5 g sample of fine powder (<60 mesh) was mixed with 50 mL of water or 95% ethanol and subjected to microwave-assisted extraction at 300 W for 5 min [30]. The water extract was used for crude polysaccharide analysis, while the ethanol extract was used for the analysis of other components.

2.4.2. Crude Polysaccharide Analysis

Crude polysaccharide analysis was modified from the sugars and related substances determination of Dubois et al. [31]. In short, the water extract was mixed with ethanol to obtain alcohol-insoluble solids. These solids were re-dissolved in water, then reacted with 5% phenol solution and concentrated sulfuric acid for 2 min. Finally, the absorbance was measured at 488 nm to determine the crude polysaccharide content.

2.4.3. Crude Triterpenoid Analysis

Crude triterpenoid analysis was performed using a modified method from Sun et al. [32]. The ethanol extract (0.1 mL) was evaporated to dryness, and then reacted with 0.4 mL of 5% vanillin-acetic acid and 1 mL perchloric acid at 60 °C for 15 min. Subsequently, 5 mL of acetic acid was added and reacted for 15 min. The absorbance was subsequently recorded at 548 nm, and the crude triterpene content was calculated from the linear regression equation of the oleanolic acid standard curve.

2.4.4. Total Phenol Analysis

Total phenol analysis was performed using the analytical method [33]. In short, 0.1 mL ethanol extract was mixed with 1 mL of Folin–Ciocalteu’s phenol reagent and 0.8 mL 7.5% sodium carbonate, then incubated in the dark for 30 min at room temperature. The absorbance was subsequently measured at 765 nm, and the total phenolic content was determined from the linear regression equation of the gallic acid standard curve.

2.4.5. Total Flavonoid Analysis

Total flavonoid analysis was performed from the analytical method [33]. A total of 1 mL ethanol extract was reacted with 1 mL 2% AlCl₃·6H₂O methanol solution in the dark for 10 min. After measuring the absorbance at 430 nm, the total flavonoid content was determined from the linear regression equation of the quercetin standard curve.

2.4.6. Ginsenoside Rg3 Analysis

The method of Hsu et al. [6] was modified slightly to conduct this analysis. First, a 2 g sample was extracted using ultrasound with 40 mL of 80% methanol in a 50 °C water bath (DC-600H, Macro Fortunate Co., Ltd., New Taipei, Taiwan) for 1 h, followed by centrifugation at 6000 rpm for 15 min. The supernatant was filtered through a 0.2 μm membrane, and 10 μL of the filtrate was injected into the HPLC system (Waters^TM, Milford, MA, USA). The separation column employed was an Ascentis^R C18 (25 cm × 4.6 mm, 5 µm, Supelco, St. Louis, MO, USA). The results were then applied to the standard curve to determine the ginsenoside Rg3 content in the sample.

2.5. Whitening Experiment

2.5.1. Tyrosinase Inhibitory Activity

This process was performed using a modified version of the method of Masuda et al. (2005) [34]. The tyrosinase solution (TYR) was prepared at a concentration of 250 units/mL using 0.1 M phosphate-buffered saline (PBS, pH 6.8) and stored at −18 °C. Then, 30 mg L-tyrosine was dissolved in 100 mL of PBS to prepare a 300 mg/L L-tyrosine solution. The following mixtures were added to a 96-well (model 90015-2NB, Alpha Plus Scientific Corp., Taoyuan, Taiwan) microplate: (A) 120 µL PBS + 40 µL TYR, (B) 160 µL PBS, (C) 80 µL PBS + 40 µL TYR + 40 µL of sample, and (D) 120 µL PBS + 40 µL of sample. The mixtures were incubated at room temperature (25 °C) for 10 min. Then, 40 µL of the L-tyrosine solution was added to each well and incubated for another 10 min. The absorbance was measured at 475 nm, and the tyrosinase inhibition rate was calculated using Equation (1).

$$\mathrm{Tyrosinase} \mathrm{inhibition} (\%)={\displaystyle \frac{(\left(\mathrm{A}–\mathrm{B}\right)-\left(\mathrm{C}–\mathrm{D}\right))}{(\mathrm{A}–\mathrm{B})}}\times 100\%$$

(1)

2.5.2. Image Analysis of Zebrafish Embryos

Image analysis was performed using ImageJ image analysis software (version 1.54f, 2023). In the HSB color model, a color thresholding approach was applied to select melanin patches on the zebrafish body (excluding the eyes) with brightness values ranging from 0 to 150. This technique enabled the quantification of changes in melanin-covered areas on the zebrafish surface based on the captured images.

2.6. Different Drying Methods for Rice Bran and American Ginseng Residue Medium Treatment

2.6.1. Hot Air-Assisted Radio Frequency Drying (HARF)

A 40.68 MHz, 5 kW radio frequency (RF) heating system (EDC-10D, YH-DA Biotech Co., Ltd., Yilan, Taiwan) was used, with 100 °C forced hot air introduced from the right side at an airflow velocity of 1.7 m/s. A 500 g sample was placed in a perforated No. 3 plastic basket (26 cm in diameter, 8.5 cm in height) and dried under a 10 cm electrode gap until the moisture content decreased to below 10%. The surface temperature was recorded by an infrared thermometer (Testo 104-IR, Hot Instruments Co., Ltd., New Taipei, Taiwan) during drying.

2.6.2. Radio Frequency Vacuum Drying (RFV)

A 40.68 MHz, 5 kW RF heating system (EDC-10D, YH-DA Biotech Co., Ltd., Yilan, Taiwan) with a small glass vacuum chamber (outer diameter: 18 cm, inner diameter: 14 cm, height: 16 cm) was used. The chamber contained 0.5 kg of sample at 150 mmHg absolute pressure and a 17 cm electrode gap. The sample was dried until its moisture content decreased to below 10%.

2.6.3. Microwave Drying (MW) and Hot Air Drying (HA)

The microwave drying system (CF-003, TWLT Enterprise Co., Ltd., Taoyuan, Taiwan) was set at 1 kW with a temperature limit of 100 °C. The hot air drying system was set at 45 °C. In both drying methods, a 500 g sample was dried until its moisture content decreased to below 10%.

2.6.4. Calculation of Energy Consumption and Greenhouse Gas Emissions During Drying

We utilized a clamp meter to measure the power of equipment, recording the current in amperes (A) by AC clamp (3280-10, Hioki Taiwan Co., Ltd., Taipei, Taiwan) [30]. The energy consumption was calculated using Equations (2) and (3):

Three-phase electrical power:

$$\mathrm{HARF} \mathrm{and} \mathrm{RFV} \mathrm{energy} \mathrm{consumption} \left(\mathrm{kWh}\right)={\displaystyle \frac{220\times \sqrt{3}\times \mathrm{A}\times \mathrm{t}}{1000}}$$

(2)

Single-phase electrical power:

$$\mathrm{MW} \mathrm{and} \mathrm{HA} \mathrm{energy} \mathrm{consumption} \left(\mathrm{kWh}\right)={\displaystyle \frac{220\times \mathrm{A}\times \mathrm{t}}{1000}}$$

(3)

Greenhouse gas emissions were calculated based on the carbon dioxide emission factors provided by the Environmental Protection Administration, Executive Yuan, R.O.C. (Taiwan) in 2022 [35], using energy consumption to estimate greenhouse gas emissions [36].

$$\begin{array}{l}\mathrm{Greenhouse} \mathrm{gas} \mathrm{emissions} \left({\mathrm{kgCO}}_2\mathrm{e}\right)\\ =\mathrm{electricity} \mathrm{emission} \mathrm{coefficient} \left({\displaystyle \raisebox{1ex}{$0.495{\mathrm{kgCO}}_2\mathrm{e}$}\!\left/ \!\raisebox{-1ex}{$\mathrm{kWh}$}\right.}\right)\times \mathrm{energy} \mathrm{consumption} \left(\mathrm{kWh}\right)\end{array}$$

(4)

2.7. Statistical Analysis

The experimental results were presented as mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) was performed and subsequently subjected to Duncan’s multiple range tests of treatment means using Statistical Analysis System (SAS 9.4, SAS Institute, Cary, NC, USA). The significance level was set at 0.05.

3. Results and Discussion

3.1. Cultivation Conditions and Bioactive Components of Black Truffle

Rice bran is an agricultural byproduct from rice milling. It is often added to fungal medium as a carbon source to promote the growth of fungal fruiting bodies and mycelium [37]. American ginseng residue is primarily composed of fiber [38], and it has a high level of ginsenoside Rb1 [39], which can be bio-transformed into the highly bioactive ginsenoside Rg3 [4]. Therefore, rice bran and American ginseng residue were mixed in different ratios and used for black truffle petri dish fermentation (Figure 1). The media with rice bran and ginseng residue ratios of 2:2, 3:1, and 4:0 started to develop black truffle mycelia in the first week of fermentation. A higher proportion of rice bran accelerated mycelial growth, and the 3:1 and 4:0 ratios covered the Petri dish by the fifth week.

The addition of rice bran in the medium helps provide essential nutrients for mycelial growth, leading to improved mycelial growth with a higher proportion of rice bran. Given the objective of increasing the utilization of American ginseng residue in the fermented medium while ensuring optimal black truffle growth, the 3:1 ratio of rice bran to American ginseng residue, which yielded the best fermentation results, was selected as the medium for black truffle solid-state fermentation.

The bioactive compounds in the media and fermented product were analyzed (

Table 1. The bioactive compounds and antioxidant activity of rice bran (R), ginseng residue (G), mixture media (R3G1), and black truffle 5 weeks solid-state fermented products (FR3G1).

Extracts	Polysaccharides (mg DE/g DW)	Triterpenoids (mg OE/g DW)	Polyphenols (mg GE/g DW)	Flavonoids (μg QE/g DW)	Ginsenoside (mg Rg3/g DW)
R	15.45 ± 1.03 d	32.10 ± 3.96 a	4.91 ± 0.01 b	52.33 ± 0.32 d	ND
G	80.29 ± 0.79 b	30.80 ± 2.91 a	2.55 ± 0.08 d	203.66 ± 3.21 b	2.84 ± 0.28 b
R3G1	38.96 ± 0.55 c	31.63 ± 3.68 a	4.52 ± 0.08 c	101.53 ± 4.25 c	0.33 ± 0.02 c
FR3G1	114.97 ± 1.36 a	32.02 ± 3.95 a	5.16 ± 0.07 a	272.84 ± 6.49 a	4.04 ± 0.12 a

R: rice bran, G: American ginseng residue, R3G1: the mixture of rice bran and American ginseng residue in a ratio of 3:1, FR3G1: black truffle solid-state fermented the media of 3:1 rice bran and American ginseng residue product. DE: dextrose equivalent, OE: oleanolic acid equivalent, GE: gallic acid equivalent, QE: quercetin equivalent. Scavenging DPPH free radicals of 20 mg/mL ascorbic acid and BHA are 94.47% and 95.27%, respectively. ^a–d Means with different superscript letters in the column were significantly different (p < 0.05).

). The results showed that, compared to the mixture of rice bran and American ginseng residue at a 3:1 ratio (R3G1), the black truffle solid-state fermented product (FR3G1) exhibited a significant increase in crude polysaccharides, total polyphenols, and flavonoid content. Notably, crude polysaccharides increased by approximately threefold, representing the most substantial enhancement. Previous studies have consistently reported that the bioactive compound content in truffle mycelium is higher than that in its fruiting body and fermented medium [20,40].

The ginsenoside Rg3 content was only 0.33 mg/g DW in R3G1, whereas the fermented product FR3G1 increased the ginsenoside Rg3 content to 4.04 mg/g DW, representing approximately a twelvefold increase. This enhancement may be attributed to the ability of T. melanosporum to produce glycosidases, which facilitate the deglycosylation of ginsenoside Rb1, the major ginsenoside in American ginseng residue, during fermentation, thereby increasing the Rg3 content [1]. In previous studies, whether using Ganoderma lucidum liquid fermentation or Hericium erinaceus solid-state fermentation, the ginsenoside Rg3 content in American ginseng residue could only be increased by approximately threefold [6,41]. This may be related to the choice of fermentation method and fungal strain. Therefore, black truffle solid-state fermentation can bio-transform a higher amount of ginsenoside Rg3, making it more advantageous as a functional food ingredient compared to previously fermented American ginseng residue products.

3.2. Whitening Effect of Black Truffle Solid-State Fermented Product

The ginsenosides in the American ginseng ethanol extract are related to its whitening effects [42]. Therefore, the ethanol extracts of the medium and black truffle fermented products were used for the whitening experiment, with kojic acid (K) as the positive control in the study. Tyrosinase is a key enzyme in melanin synthesis within biological systems. When a sample inhibits tyrosinase activity, it indicates potential whitening effects [23]. Under treatment with ethanol extracts at the same concentration, the black truffle solid-state fermented product (FR3G1) exhibited a greater ability to inhibit tyrosinase activity compared to the medium (R3G1). When the concentration of the ethanol extract increased, the inhibition of tyrosinase activity was further enhanced and was significantly higher than that of the kojic acid treatment (Figure 2).

It was essential to avoid excessive ethanol extract concentrations, which could cause zebrafish embryo mortality. A total of 100 µg/mL of the FR3G1 ethanol extract was able to inhibit 70% of tyrosinase activity. The zebrafish embryos treated with the 100 µg/mL ethanol extract showed no visible abnormalities, indicating no significant toxicity. Moreover, the relative melanin area in zebrafish embryos treated with FR3G1 was 57.26%, demonstrating a superior depigmentation effect compared to the medium-treated group (

Table 2. Depigmenting effect of 100 µg/mL ethanol extracts from rice bran (R), American ginseng residue (G), their 3:1 mixture (R3G1), and black truffle fermented product (FR3G1) on melanogenesis of zebrafish embryos treated at 78 hpf (72 h treatment). Kojic acid (K) was a positive control.

Sample	Relative Area (%)	Picture
Control	100.00 ± 0.00 a
K	64.34 ± 2.78 c,d
R	81.17 ± 9.38 b
G	71.61 ± 4.32 c
R3G1	78.98 ± 3.82 b
FR3G1	57.26 ± 6.08 d

Data are expressed as mean ± S.D. (n = 6). ^a–d Means labeled with different letters indicate significant differences among treatments (p < 0.05).

).

This effect may be attributed to the biotransformation of ginsenoside composition in American ginseng residue by black truffle solid-state fermentation, which not only increased the content of the bioactive ginsenoside Rg3 but also enhanced the whitening effect of its ethanol extract on zebrafish embryos. In this study, image analysis was used to quantify the melanin area on the zebrafish body surface, excluding melanin accumulation in the eyes to eliminate its influence on the total melanin measurement. This approach provided more practical research results, further confirming the excellent whitening effect of FR3G1.

Previous studies have investigated the whitening effects of various ginseng extracts. The ethanol extracts of ginseng leaves and roots at 1000 µg/mL could only inhibit 22.8% and 37.7% of tyrosinase activity, respectively [43]. Additionally, 120 ppm of methanol extract from ginseng fruit reduced melanin deposition in melanocytes by only 23.9% [28]. Moreover, ginsenoside CY must be extracted and isolated from fresh American ginseng before it can reduce melanin content in zebrafish embryos by 20% at 20 µM [44]. In contrast, this study demonstrates that a crude extract derived from black truffle-fermented American ginseng residue can reduce 42.74% of the melanin area on zebrafish embryos. It can be effectively reutilized and holds potential as a whitening cosmetic ingredient.

3.3. Scale-Up Production of the Medium for Black Truffle Solid-State Fermentation

The medium conditions, bioactive compound contents, and whitening effects of black truffle fermented product have been well characterized in the above studies. To enhance the mass production potential of this fermentation method, this study first processed wet American ginseng residue using different drying methods. By mixing rice bran and wet American ginseng residue in a 1:2 ratio before drying, a final 3:1 mixture of rice bran and dried American ginseng residue was obtained for further black truffle solid-state fermentation.

In the heating curves of different drying methods (Figure 3), the surface temperature rapidly increased to 82 °C and 64.4 °C within the first 2 min of heating under HARF and RFV drying, respectively. After 2 min, the moisture absorbed latent heat for evaporation, slowing the temperature increase. Under MW drying, the sample took 10 min to reach a stable temperature of 51.6 °C, indicating a slower heating rate but a lower drying temperature. In contrast, the sample temperature ranged between 30.6 °C and 38.6 °C in HA drying, showing the slowest heating rate and the lowest drying temperature, which hindered efficient heat transfer for moisture evaporation.

The drying trends of HARF, RFV, and MW samples were more linear in the drying curves (Figure 4). The R² values of their linear regression curves were 0.988, 0.988, and 0.986, respectively (

Table 3. Effect of different drying methods on the drying rate, time, energy consumption (E), and total greenhouse gas emissions (GHG) for the mixture of 1:2 rice bran and wet American ginseng residue.

Methods	Drying Curve	R2	Rate (g/min)	Time (min)	Energy (kWh/kg)	GHG (kg CO2e/kg)
HARF	W = −29.380 t + 508.5	0.988	29.38	9.56	2.27	1.12
RFV	W = −22.325 t + 513.0	0.988	20.97	13.03	3.80	1.88
MW	W = −3.778 t + 499.9	0.986	3.79	75.53	2.18	1.08
HA	W = −0.904 t + 457.8	0.903	0.90	260.63	15.72	7.78

HARF is hot air-assisted 5 kW radio frequency drying; RFV is 5 kW radio frequency vacuum drying; MW is 1 kW microwave drying; HA is hot air drying.

), indicating that their drying kinetics were more consistent with a zero-order reaction. In contrast, HA exhibited a more curved trend, with the lowest R² value of 0.903. The lowest R² value of the HA drying curve suggested the occurrence of a heat transfer barrier in the final stage of HA drying, which affected the absorption of thermal energy by moisture and its phase transition into water vapor. Therefore, the HA drying time required for the mixture to reach a 10% moisture content was extended to 260.63 min, which was significantly longer than the drying times of HARF, RFV, and MW at only 9.56 min, 13.03 min, and 75.53 min, respectively (Table 3).

Because RF and MW can oscillate water molecules within the sample, enabling rapid and uniform heating while reducing heat transfer removes barriers and shortens the drying time [8]. Additionally, the energy consumption required to dry the samples using HARF, RFV, MW, and CA was 2.27, 3.80, 2.18, and 15.72 kWh/kg, respectively. However, RFV drying consumes more electricity than the hot air system due to the vacuum pump, resulting in total energy consumption of 1.53 and 1.62 kWh/kg higher than HARF and MW, respectively. Considering these factors, RFV drying can be an energy-efficient and low-carbon alternative for rapidly drying a 1:2 mixture of rice bran and wet American ginseng residue at low temperatures. The dried rice bran and American ginseng residue mixture can be used as a solid-state fermentation medium for black truffle cultivation, facilitating the large-scale production of black truffle fermented products.

4. Conclusions

A solid-state fermentation medium composed of rice bran and American ginseng residue at a 3:1 ratio and 40% moisture content effectively promoted black truffle mycelial growth and enhanced the production of bioactive compounds, including crude polysaccharides, triterpenes, polyphenols, flavonoids, and ginsenoside Rg3. The ethanol extract of the fermented product exhibited significant tyrosinase inhibition and suppressed melanin synthesis in zebrafish embryos, demonstrating notable whitening potential. Additionally, radio frequency vacuum (RFV) drying efficiently reduced the moisture content of the medium within 13 min at 70 °C. This study demonstrated the feasibility of valorizing agricultural by-products through fungal fermentation and RFV drying, offering a novel, eco-friendly, and sustainable strategy for generating high-value functional ingredients for cosmetic applications.