**1. Introduction**

Modern functional food products are available on the market, ranging from isolated nutrients, dietary supplements, and specific products to processed or engineered foods. Peptides from foodstuff are candidates for functional food ingredients due to their beneficial health aspects such as immune-boosting, anti-oxidative stress, hypolipidemic and tumor suppressing activity [1,2]. One of the above-mentioned beneficial aspects is the hypolipidemic activity on adipocytes, affecting lipid storage, directly associated with obesity, a contemporary health problem. Obesity is caused by excessive triacylglycerol (TAG)

**Citation:** Krobthong, S.;

Yingchutrakul, Y.; Visessanguan, W.; Mahatnirunkul, T.; Samutrtai, P.; Chaichana, C.; Papan, P.; Choowongkomon, K. Study of the Lipolysis Effect of Nanoliposome-Encapsulated *Ganoderma lucidum* Protein Hydrolysates on Adipocyte Cells Using Proteomics Approach. *Foods* **2021**, *10*, 2157. https:// doi.org/10.3390/foods10092157

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Academic Editor: Antonello Santini

Received: 22 July 2021 Accepted: 10 September 2021 Published: 12 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

accumulation in the adipocytes. Increasing TAG breakdown or hypolipidemic activity might contribute to reducing body fat and triglyceride (TG) levels. Several natural-sourced peptides could be combined with foodstuffs, and their effective delivery could display beneficial aspects [3].

Most edible mushrooms such as *Volvariella volvacea*, *Lentinula edodes*, and *Ganoderma lucidum* are beneficial for health. They have been generally consumed as basic food, as they provide plenty of dietary nutrients including fibers, minerals, and vitamins. They are also excellent sources of proteins [4]. Beyond their role as foodstuffs, edible mushrooms feature in certain types of holistic or alternative medicine. *G. lucidum*, locally known as Lingzhi, is defined as a medicinal mushroom for the prevention of various diseases, as well as for recuperation and health improvement. Ganoderma species are generally found all over the world. Lingzhi exhibits the prevailing features of being an excellent nutrient source of proteins, lipids, and carbohydrates [5]. Lingzhi has been consumed widely in East-Asia as a traditional remedy for centuries [6]. Many of its pharmacological effects have been widely reported such as immune modulation enhancement, soothing the nerves, inflammatory response reduction, cancer growth suppression, cell-aging deceleration, oxidative stress reduction, and anti-aging and lipid accumulation suppressive effects [7–9].

Foodstuff-derived protein hydrolysates contain a high level of functional peptides. The dominant features of the protein hydrolysates are their lower molecular weight and relative lack of high-order structure, as well as the increased number of functionally ionizable and exposed hydrophobic groups compared to those of intact proteins. These features denote that their surface interactions, water-solubility, host-receptors, and biological activities might be different from those of proteins. This includes the transduction triggering capability of various signaling pathways, leading to the activation or deactivation of regulators and biological activities above their generic nutritional value [10]. However, the major obstacle in introducing peptides into functional food ingredients is their functional stability during commercial processing and under human physiological conditions [11]. Therefore, functional peptides might partially or completely lose their activity before reaching the target cells or organs [12]. Hence, choosing a delivery system that is highly compatible with human physiological conditions would alleviate this problem.

Liposome encapsulation is a well-known compatibility delivery approach for foodstuff hydrolysates. The advantage of encapsulation within small particles is the stability and bioactivity enhancement of the protein hydrolysates [13]. This approach is suitable for protein and peptide delivery as their molecules possess various polar and non-polar regions similar to their liposome properties [14]. Several studies revealed the potential of liposomal encapsulation of peptides. For example, a pharmacological study of ghrelin, the appetite-stimulating peptide hormone, indicated increasing ghrelin stability and circulation period in the blood [15]. Both the pharmaceutical and cosmetic industries generally use liposome-based carriers to store and deliver functional proteins and peptides for specific purposes [16,17]. Although the use of liposomal encapsulation can be observed in a small number of products in the food industry market, liposomal encapsulation would be a promising approach as its safety and efficiency are proven by the pharmaceutical and food industries.

In this study, we established liposome carriers for protein hydrolysates to enhance the biological activities and stability of the latter. In addition, we also investigated the lipolysis-stimulating activity of the encapsulated Lingzhi protein hydrolysates on 3T3-L1 adipocyte cells. A possible signaling pathway for the encapsulated hydrolysates on the stimulation of lipid breakdown was also investigated using quantitative proteomic analysis. Finally, the possible beneficial mechanisms of the nano-liposomal hydrolysates are clarified and their value as a functional food additive supported.

#### **2. Materials and Methods**

#### *2.1. Ganoderma Lucidum Hydrolysate Preparation*

Dried Lingzhi (200 g) was powdered using an ultra-centrifugal mill (Retsch Co., Haan, Germany) equipped with a sieve (diameter = 1 mm3) at 8000 rounds per minute (rpm). The powdered mushroom was heated using a modified Pressurized Hot Water Extraction method [18]. Briefly, the Lingzhi was mixed with deionized water at a ratio of 1:2 ( *w*/*v*) and incubated at 121 ◦C, 15 psi for 20 min. The extracted Lingzhi was left to cool down and hydrolyzed with pepsin as the first independent factor (X1) at 0.25%, 0.5%, and 1% in 0.1% of HCl for digestion times of 3, 6, and 9 h as the second independent factor (X2) at a constant temperature of 37 ◦C. Next, the crude was filtrated with a 0.22-μm nylon membrane and fractionated through Vivaspin-20 (GE Healthcare Co., Amersham, UK), with a molecular weight cut-off of 3 kDa. Peptides of <3 kDa were subjected to Solid-Phase Extraction (SPE) (Waters Co., Milford, MA, USA). An amount of 3 mg of small peptides was loaded on an equilibrated SPE column (Sep-Pak C18) and eluted using acetonitrile: water (1:1, *v*/*v*). The supernatant was dried using a freeze-drying machine.

#### *2.2. Lingzhi Protein Hydrolysate Optimization by Response Surface Methodology (RSM)*

The two independent variable factors used in this study were the digestion time (X1) and the enzyme concentration (X2). The experimental outputs were the degree of hydrolysis (DH) (Y1) and the product yield (Y2). The DH determination was performed according to the method of Nielsen et al. [19] and the product yield was calculated as a percentage of the proteins found in the hydrolysates divided by the raw protein content. While calculating the optimal condition of an independent factor, the values of the other independent factors were fixed. An experimental design was set with 11 conditions, including 9 experimental conditions and 2 central points. The correlation of the independent factors and experimental outputs was used to generate RSM by the following equation:

$$y = \beta\_0 + \varepsilon + \sum\_{i=1}^{k} \beta\_i \mathbf{x}\_i + \sum\_{i=1}^{k} \sum\_{j=1}^{k} \beta\_{ij} \mathbf{x}\_i \mathbf{x}\_j + \sum\_{i=1}^{k} \beta\_{ii} \mathbf{x}\_{ij}^2 \tag{1}$$

where *y* is the experimental output; *β*0 is constant intercept value; *β<sup>i</sup>*, *βii*, and *βij* are the linear, quadratic, and interaction coefficients, respectively; and *xi* and *xj* are the independent variable factors. Three-dimensional response surface plots were drawn to illustrate the correlation between the levels of the process variable factors and the outcome results.

#### *2.3. Nano-Liposome Carrier Preparation and Characterization*

Soybean lecithin (Sigma Aldrich Co., St. Louis, MO, USA) and cholesterol (Sigma Aldrich Co., St. Louis, MO, USA) (8:1, *w*/*w*) were dissolved in 10 mL of diethyl ether in a 50-mL round bottom flask for 5 min. Once the lipids were thoroughly mixed in diethyl ether, the solvent was removed to yield a lecithin-cholesterol film layer by rotary evaporation (Buchi Co., Flawil, Switzerland) at 100 rpm under reduced pressure. The hydration of the lecithin-cholesterol film layer was accomplished by adding 10 mL of Lingzhi extract and agitating on an orbital shaker at 220 rpm for 6 h at 28 ◦C to obtain a vesicular white suspension. The vesicular suspension was forced through a membrane filter with a defined pore size of 200 nm by an extruder (GE Healthcare Co., Amersham, UK). After day 7, the loading efficiency of the loaded nanoliposome was determined by a protein-based spectrophotometric analysis. We mixed 100 μL samples of loaded liposomes with 1% Triton X-100 (Sigma Aldrich Co.) and sonicated for 10 min (10 s-interval) to disassemble the liposomes and release the extract. Afterward, the protein content of the clearance solution was assessed by Lowry protein assay using Bovine Serum albumin (Sigma Aldrich Co.) as a reference. The loading efficiency was calculated using the following equation:

Extracted loading Efficiency ( *w*/*w*) (%) = (protein extracted of which encapsulated in

liposomes (mg) ÷ protein content of extracted Lingzhi (mg)) × 100 (2)

The hydrodynamic diameter of the liposomal formulations in deionized water was measured by dynamic light scattering (DLS) using ZetaSizer Nano-ZS (Malvern Instruments, Worcestershire WR, UK), in which the zeta potential was also examined (*n* = 3).

#### *2.4. Effect of Loaded Nanoliposomes on 3T3-L1 Adipocyte Cells*

Cell cytotoxicity of the loaded liposome and unloaded liposome control was evaluated through an MTT assay. Human fibroblasts (American Type Culture Collection., Manassas, VA, USA)) and 3T3-L1 mouse differentiated adipocyte cells (induced by an adipogenic cocktail containing 2.5 mM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 10 g/mL insulin for 8 days) were tested for cytotoxicity at various concentrations (104.68, 52.34, 26.17, 13.09, 6.54, 3.27, 1.64, 0.82, 0.41, and 0.20 μg/mL) of loaded liposomes and unloaded liposome as control for 24 h. Next, we measured the optical absorbance at 570 nm using a microplate reader and transformed the results into cell survival rate percentage [20].

The lipolytic effect of the loaded nanoliposome was used to quantify glycerol, a byproduct of lipolysis (EnzyChrom ™ Glycerol Assay Kit, BioAssay Systems, Hayward, CA., USA) in cell culture supernatant after 24 h of treatment with the loaded nanoliposome. To determine the intracellular TG content, the differentiated 3T3-L1 cells were treated with the loaded nanoliposomes, as described previously, for 24 h. The cells were washed twice with PBS and fixed with 4% paraformaldehyde for 1 hour at room temperature. Next, the cells were washed once with PBS and isopropanol 60% (*v*/*v*), then they were allowed to dry. Next, the cells were stained with 0.5% (*v*/*v*) Oil Red O (ORO) (Sigma Aldrich Co.) in an isopropanol solution of 60% for 1 hour. After staining, the unstained dye was removed by rinsing with distilled water. The stained lipid droplets were observed under a stereomicroscope. The stained oil droplets indicating lipid accumulation were solubilized by absolute isopropanol for 15 min and their absorbance was measured at 510 nm using a microplate reader (Multiskan Go, Thermo Scientific, Waltham, MA, USA).

#### *2.5. Proteomic Analysis and Data Processing*

To investigate the adipocyte protein expression profiles after the exposure to the loaded liposomes, the cells were lysed by a lysis buffer solution (10 mM HEPES-NaOH pH 8.0 and 0.5% Triton X-100) supplemented with a protease inhibitor cocktail (Thermo Scientific Co.). The supernatant was collected by centrifugation, followed by ice-cold acetone precipitation (1:5 *v*/*v*). After precipitation, the protein pellet was reconstituted in 0.2% RapidGest SF (Waters Co.) in 10 mM of Ammonium bicarbonate (Sigma Aldrich Co.). The total protein (50 μg) was subjected to gel-free based digestion. Next, sulfhydryl bond reduction was performed using 5 mM DTT (Sigma Aldrich Co.) in 10 mM ammonium bicarbonate at 72 ◦C for 1 h and sulfhydryl alkylation using IAA (Sigma Aldrich Co.) at room temperature for 30 min in the dark. The solution was cleaned up using a Desalting Zebra-spin column (Thermo Scientific Co.). The flow-through solution was enzymatically digested by Trypsin (Promega Co., Madison, WI, USA) at a ratio of 1:50 (enzyme: protein) and incubated at 37 ◦C for 3 h. The digested solution was dried and reconstituted in 0.1% formic acid before being subjected to tandem-mass spectroscopy using a nanoLC-system coupled with high resolution 6600 TripleTOFTM (AB-Sciex, Concord, ON, Canada). The LC conditions were as follows: mobile phase A and B were used, with mobile phase A being composed of 0.1% formic acid in water and mobile phase B comprising 95% acetonitrile with 0.1% formic acid. The LC-method parameters comprised a 135-min long process for a single injection. The analytical column was maintained at 55 ◦C. Using the datadependent acquisition mode of mass spectroscopy, the MS scans over a mass range of 400–1600 *<sup>m</sup>*/*<sup>z</sup>*, selecting the top 20 most abundant peptide ions with charge state in the range of 2–5 (positive mode) for fragmentation. The dynamic exclusion duration was set at 15 s. The raw MS-spectra resulting (.wiff) file was extracted and annotated with protein sequences using the Paragon ™ Algorithm by ProteinPilot ™ Software [21]. The *Mus musculus* protein database, retrieved from UniProtKB (16,477 sequences) and used in Paragon ™, was assembled in FASTA format and downloaded in May 2021. We set a

detected protein threshold of (Unused ProtScore (Conf)) ≥ 0.05 with 1% false discovery rate (FDR) with ≥10 peptides/protein. The protein and peptide comparisons exhibiting >20% coefficient of variation (C.V.) between the replicates were rejected. Both library and SWATH-MS data were imported into SWATHTM processing microapp in PeakView® software. The normalization of the relative protein abundances was performed using the R package, NormalyzerDE [22], in which Quantile-normalization was applied to expression data analysis, after adding 1 to all expression values to avoid errors upon log transformation.
