Chemical Properties of a Polysaccharide Purified From Solid-State Fermentation of Auricularia Auricular and its Biological Activity as a Hypolipidemic Agent Feng Zeng, Chao Zhao, Jie Pang, Zhanxi Lin, Yifan Huang, and Bin Liu
A water-soluble crude polysaccharide was extracted by hot water from Auricularia auricular mycelium grown under solid-state fermentation (SSF). The crude polysaccharide was purified by DEAE Sephadex A-50 and Sephadex G200 chromatography. Fourier transform infrared spectroscopy and nuclear magnetic resonance (1 H NMR) spectroscopy were used to investigate the structure of the purified A. auricular polysaccharide (AAP-I) and revealed that it is αglycosidically linked. After 14 and 28 days of AAP-I orally administered, the AAP-I significantly decreased the levels of total cholesterol, triglyceride, and low-density lipoprotein cholesterol in mice in which hyperlipidemia had been induced by a high fat diet (P < 0.05). The results revealed that AAP-I from SSF of A. auricular mycelium possesses potent hypolipidemic properties. The polysaccharide may be useful as a functional food additive and a hypolipidemic agent.
Abstract:
Keywords: Auricularia auricula, chemical properties, hypolipidemic, polysaccharide, solid-state fermentation
Introduction
H: Health, Nutrition & Food
ties, such as antitumor, anti-inflammatory, anticoagulant, hypocholesterolemic, hypoglycemic, and ameliorating properties, have reportedly been observed in A. auricula (Nguyen and others 2012; Li and others 2013). In addition, its ethanolic extracts have been proven to possess antioxidant and nitric oxide synthase activation properties (Acharya and others 2004). These medical applications could result in an increased commercial demand in the near future. Prior work has primarily focused on polysaccharides extracted from the fruiting body of macrofungi that were grown on a solid culture. Submerged culture is an alternative approach for producing a large amount of macrofungi polysaccharides, but SSF has many advantages. In recent years, researchers have shown an increasing interest in SSF as a potential alternative to submerged fermentation (Mitchell and Lonsane 1992) because it uses economical substrates (agricultural residues), requires fewer processing and down-streaming stages, requires less power, and generates less waste (Kumar 2003). Moreover, SSF has a higher product yield and offers better product stability. Previously, SSF was used primarily for the production of industrial enzymes; currently, it is also being exploited for the production of secondary metabolites (Suryanarayan 2003). A large number of studies have been focusing on the production of bioactive compounds with submerged culture (Kim and others 2002; Lee and others 2003; Lee and others 2004). However, few previous studies have been reported on bioactive polysaccharides produced by A. auricula mycelia grown in SSF. In this work, A. auricula mycelia were obtained by SSF. A water-soluble crude polysaccharide was extracted from A. auricular mycelia and further purified. The structure of the purified A. auricula polysacchaMS 20121760 Submitted 12/21/2012, Accepted 5/8/2013. Authors Zeng, Zhao, ride (AAP-I) was characterized, and its hypolipidemic activity was Pang, and Liu are with College of Food Science, Fujian Agriculture and Forestry Univ. investigated in vivo. Fuzhou, Fujian 350002, China. Authors Zeng, Lin, Huang, and Liu are with
Currently, hyperlipidemia, by triggering atherosclerosis, is the leading cause of cardiac illness and death. Elevated levels of plasma cholesterol are becoming a major risk factor for the development of hyperlipidemia and coronary heart disease (Peter and others 2013). It is desirable to maintain normal body functions by decreasing the elevated serum cholesterol to an appropriate level. However, the side effects of cholesterol-lowering drugs have become apparent over time. For instance, studies have reported that patients taking cholesterol-lowering drugs were unable to tolerate statin treatments due to musculoskeletal symptoms and other side effects (Rader 2001; Ballantyne and others 2003). The search for novel, natural cholesterol-lowering food components has increased in intensity. Since the technology of functional food emerged, an increasing number of functional foods have been developed from edible mushrooms (Man and others 2002; Journoud and Jones 2004). Traditionally, edible mushrooms have been produced in solid culture by using composts or lignocelluloses wastes, such as straws or wood, and it usually takes 4 to 6 months to culture their fruiting bodies (Solomons 1975). It is generally accepted that growing mushroom mycelia in a defined medium by solid-state fermentation (SSF) is a rapid method to obtain fungal biomass of consistent quality. Auricularia auricula, a precious macrofungus, has been used as a food and drug in China (Nguyen and others 2012). It has recently attracted interest due to its promising potential application in pharmaceutical industries (Wasser 2002). Immunomodulation activi-
Natl. Engineering Research Center of Juncao, Fuzhou, Fujian 350002, China. Author Huang is with College of Animal Science, Fujian Agriculture and Forestry Univ., Fuzhou, Fujian 350002, China. Direct inquiries to author Liu (E-mail: [email protected]). Authors Zeng and Zhao contributed equally to this study.
H1470
Journal of Food Science r Vol. 78, Nr. 9, 2013
Materials and Methods A. auricula 0020 was purchased from the Centre of Fungal Research (Fujian Agricultural and Forestry University, Fuzhou, China). It was maintained on potato dextrose agar (PDA) slants that R C 2013 Institute of Food Technologists
doi: 10.1111/1750-3841.12226 Further reproduction without permission is prohibited
Polysaccharide of Auricularia auricular . . .
Separation and purification of A. auricula polysaccharide The mycelia of A. auricula was dried at 70 ◦ C and ground to a particle diameter size to pass through a 60 mesh. The powders were extracted with hot water at 90 ◦ C for 120 min and filtered. The residue was collected and extracted twice more using the same procedure. The resulting suspension was then centrifuged at 3500 rpm for 15 min. The supernatant was concentrated by a rotary evaporator under reduced pressure. The concentrated solution was freeze-dried and precipitated with 3 volumes of absolute ethanol and maintained at 4 ◦ C for 12 h. The resulting precipitate was then separated by centrifugation, washed exhaustively with 95% alcohol, and dissolved in a minimum volume of demonized water; proteins were removed with polyamide. To the crude polysaccharide solution, hydrogen peroxide (H2 O2 ) was added dropwise until the final concentration of H2 O2 reached 5.0%; then, the solution was incubated in a water bath at 40 ◦ C for 1 h. The preparation was then dialyzed using cellulose sacks (JingHongKeDa Ltd., Beijing, China) in water flow dialysis for 48 h, followed by another 36 h distilled water dialysis. The preparation was vacuum concentrated and freeze-dried, yielding the crude A. auricula polysaccharide (CAAP). The CAAP was purified using anion-exchange chromatography. To this end, the CAAP obtained by freeze-drying was dissolved in distilled water and then centrifuged (4000 rpm, 10 min). The supernatant was then applied to a column (1.6 cm × 50 cm) of DEAE Sephadex A-50 (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) pre-equilibrated with distilled water. Fractions were collected in stepwise elution with increasing ionic strength of NaCl (0, 0.1, and 0.5 mol/L) at a flow rate of 0.5 mL/min. The main fraction from the eluted distilled water, which was quantified by the phenol–sulfuric acid method as described in the literature (Masuko and others 2005). The major fractions obtained were pooled, concentrated, and dialyzed against deionized water with dialysis tubing (molecular weight cutoff, 14 kDa). Next, the fraction was concentrated again and then lyophilized to give the polysaccharide-designated AAP-I. AAP-I was evaluated and then further purified by gel filtration chromatography on a Sephadex G-200 (Beijing Solarbio Science & Technology Co., Ltd.) column (2.5 cm × 60 cm). The sample was dissolved in a minimal volume of distilled water, added to the column, and then eluted with distilled water at a flow rate of 0.5 mL/min. The major fractions obtained were pooled, concentrated, and dialyzed against deionized water in a dialysis tube (molecular weight cutoff, 14 kDa). The resulting solution was concentrated and then lyophilized to give the pure A. auricula polysaccharide coded as AAP-I. The earlier methods were modified from previously reported methods (Zhang 2011).
Table 1–Serum TC levels of each group during the experimental period. Serum TC (mmol/L) of the time-course (wk) Treatment group
0
2
4
RD CED CED + 50AAP-I CED + 100AAP-I CED + 200AAP-I CED + simvastatin
1.99 ± 0.48b
2.73 ± 0.73b
2.35 ± 0.26c 4.83 ± 1.34a 3.71 ± 0.49b 3.73 ± 0.62b 3.28 ± 0.66b,c 4.76 ± 0.23a
4.49 ± 3.81a,b 4.46 ± 2.07a,b 5.07 ± 1.61a,b 4.33 ± 1.71a,b 5.12 ± 2.12a
4.38 ± 1.06a 3.14 ± 0.60b 2.34 ± 0.58b 2.54 ± 1.15b 2.33 ± 0.42b
The data are expressed as the mean ± SD (n = 10). Within a column, data with different superscript letters “a, b, c” are significantly different (P < 0.05) by DPS V7.05.
Fourier transform infrared (FT-IR) analysis of AAP-I IR spectra of AAP-I were recorded with an FT-IR spectrophotometer (Nicolet Instruments Corp., Madison, Wis., U.S.A.) in the range of 4000 to 400 cm−1 . The concentration of AAP-I in KBr was at a concentration of about 2.4%. The major peaks (intensity and wave number) were found using EZOMNIC version 6.0 software (Nicolet Instruments Corp.). The spectrum of the polysaccharide was calculated using the EZOMNIC software (Zhang 2011). 1
H NMR spectroscopy analysis of AAP-I Exchangeable protons were replaced with deuterium by suspending a sample in CD3 OD and lyophilizing 3 times. 1 H NMR spectra were recorded on a 600 MHz Bruker INOVA 600NB NMR spectrometer (Brucker, Rheinstetten, Germany). The relaxation delays for the NMR experiment were 1.0 s. The ionization potential was 70 eV, and the temperature of the ion source was 220 ◦ C. 1 H NMR experiments were conducted at 30 ◦ C in CD3 OD (Liu and others 2007). Treatment of hypolipidemic activity of AAP-I in vivo Sixty male Kunming mice weighing 18.0 to 20.0 g were purchased from Slaccas Laboratory Animal Center (Shanghai, China). All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals and conformed to the National Institutes of Health Guide for Care and Use of Laboratory Animals. Before commencement of the research, all mice were kept in stainless cages under controlled conditions (temperature 24.0 ± 0.5 ◦ C, humidity 55.0 ± 5.0%, and 12 h light/dark cycle). They were maintained according to the Guide for the Care and Use of Laboratory Animals and were acclimatized for a period of a week, whereas at the same time given free access to food and water. After acclimation, the mice were grouped according to a modified method from the literature (Luo 2009). The animals were randomly assigned into 6 groups (n = 10); 2 control and 4 treatment groups. During a 4-wk period, 2 control groups were fed either a regular diet (RD) or a cholesterol-enriched diet (CED), and 4 treatment groups were fed the CED, among which, 3 groups were provided AAP-I at 50.0, 100.0, and 200.0 mg/kg and 1 group was provided simvastatin 6.75 mg/kg day through orally administered. The CED was produced by supplementing 5.8% yolk powder, 0.5% cholesterol, 6.0% lard oil, and 0.2% bile salt (from pigs) with the RD. Every 2 wk, blood was collected from the orbital venous plexus for serum lipid analysis (Table 1–4). At weeks 2 and 4 of the experimental period, blood samples were taken from the orbital venous plexus of mice, after a 15-h fast, using a capillary tube without anesthesia. The blood samples were Vol. 78, Nr. 9, 2013 r Journal of Food Science H1471
H: Health, Nutrition & Food
were inoculated and incubated at 28 ◦ C for 12 days, then stored at 4 ◦ C for use. The seed culture’s medium contained the following components (in g/L): glucose 20.0, peptone 2.0, KH2 PO4 2.0, and MgSO4 ·7H2 O 1.0. The SSF medium consisted of the following components: 75.0% sugarcane bagasse and 25.0% bean dregs, with the moisture maintained at 55.0%. For preparation of the inoculum, 6 pieces (approximately 5 mm) of the mycelia of A. auricula were transferred individually into Erlenmeyer flasks containing 100 mL seed medium with the sterilized self-designed cutter. The flasks were then placed in a rotary shaker incubator at 160 rpm and 28 ◦ C for 5 days. The solid-state medium was placed in a mushroom bottle, sterilized, and inoculated with the inoculum. The SSF process was performed in biochemistry incubators at 28 ◦ C for 21 days, and the mycelia of A. auricula were obtained.
Polysaccharide of Auricularia auricular . . . Table 2–Serum TG levels of each group during the experimental period.
Results and Discussion
Purification of crude AAP A water-soluble crude polysaccharide was extracted by hot waTreatment group 0 2 4 ter from A. auricular mycelia cultured under SSF conditions and RD 1.17 ± 0.44b 1.47 ± 0.30a 0.99 ± 0.06a,b concentrated. The concentrated solution was precipitated, and CED 2.03 ± 0.22a,b 1.27 ± 0.54a,b 1.24 ± 0.10a after removal of the protein, the crude A. auricula polysacchaa,b a,b CED + 50AAP-I 1.70 ± 0.17 1.25 ± 0.23 1.00 ± 0.29a,b ride (CAAP) was obtained. The crude A. auricular polysaccharide CED + 100AAP-I 1.69 ± 0.12a,b 0.68 ± 0.34c 0.88 ± 0.13b (CAAP) was further separated by DEAE Sephadex A-50 column CED + 200AAP-I 1.96 ± 0.74a,b 0.85 ± 0.20b,c 0.96 ± 0.15a,b chromatography by stepwise elution with sodium chloride soa a,b,c a,b CED + simvastatin 3.01 ± 1.69 1.05 ± 0.10 1.22 ± 0.35 lutions (0, 0.1, and 0.5 mol/L) into 3 fractions, named AAP-I, The data are expressed as the mean ± SD (n = 10). Within a column, data with different AAP-II, and AAP-III (Figure 1A). The main peak (AAP-I) was superscript letters “a, b, c” are significantly different (P < 0.05) by DPS V7.05. further fractionated over a Sephadex G-200 column, and the main component was collected (Figure 1B). AAP-I was then further puTable 3–Serum LDL-C levels of each group during the experirified and analyzed. mental period. Serum TC (mmol/L) of the time-course (wk)
FT-IR spectroscopy analysis The FT-IR spectra of AAP-I show the characteristics of a polysaccharide. FT-IR spectra (Figure 2A) obtained from A. auRD 1.68 ± 0.66b 2.01 ± 0.20c 2.02 ± 0.38c ricular polysaccharide (AAP-I) display peaks at approximately 3500 a a a CED 4.69 ± 1.73 4.08 ± 1.04 5.22 ± 0.75 −1 CED + 50AAP-I 3.74 ± 0.48a,b 3.72 ± 0.53a,b 3.32 ± 0.47b and 500 cm in the carbohydrate region, characteristic of polysaca,b b b charides. In all of the spectra, the absorption between 1636 and CED + 100AAP-I 4.40 ± 2.58 3.09 ± 0.18 3.74 ± 0.36 −1 CED + 200AAP-I 4.20 ± 1.66a,b 3.02 ± 0.41b 3.78 ± 0.71b 579 cm can be attributed to bands of C–H, C–O, and O–H in CED + simvastatin 4.29 ± 1.93a,b 3.22 ± 0.32b 4.03 ± 0.66b the polysaccharide. In addition, this result is consistent with the The data are expressed as the mean ± SD (n = 10). Within a column, data with different previous research, which studied the chemical characterization of superscript letters “a, b, c” are significantly different (P < 0.05) by DPS V7.05. an A. auricula fruiting body polysaccharide, with the exception of the absorption between 1500 and 670 cm−1 (Wu and others Table 4–Serum HDL-C levels of each group during the experi- 2010), which demonstrates that there is a difference between the mental period. fruiting body polysaccharide and mycelia polysaccharide. Serum TC (mmol/L) of the time-course (wk)
Treatment group
Treatment group RD CED CED + 50AAP-I CED + 100AAP-I CED + 200AAP-I CED + simvastatin
0
2
4
Serum TC (mmol/L) of the time-course (wk) 1 H NMR spectroscopy analysis of AAP-1 0 2 4 1 H NMR spectroscopy was used to examine the structure of 1 a d b 1.55 ± 0.28 1.68 ± 0.21 1.83 ± 0.17 AAP-I. The 500 MHz H NMR spectrum of AAP-I, shown 2.57 ± 0.30a 3.30 ± 0.75a,b,c 3.09 ± 0.75a in Figure 2B, confirmed that the sugar residues are linked α2.17 ± 0.10a 3.47 ± 0.52a,b 3.32 ± 0.27a glycosidically (Perepelov and others 2000). Chemical shifts from 2.54 ± 0.35a 3.16 ± 0.28b,c 3.12 ± 0.28a 2.5 to 4.5 ppm were designated as the protons of carbons C-2 to 2.16 ± 0.46a 2.64 ± 0.23c 3.42 ± 0.66a 2.25 ± 1.00a 3.98 ± 0.48a 3.45 ± 0.46a C-6 on the glycodic ring (Chauveau and others 1996; Wu and
The data are expressed as the mean ± SD (n = 10). Within a column, data with different superscript letters “a, b, c” are significantly different (P < 0.05) by DPS V7.05.
H: Health, Nutrition & Food
placed in a plastic tube and incubated at 37 ◦ C for 15 min; then, the samples were centrifuged for 10 min at 3500 rpm. The serum samples were immediately analyzed. Total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) were analyzed by colorimetric method in an ultraviolet and visible light spectrophotometer (UNICO Ltd., Shanghai, China) using commercial kits from ZHONGSHENG (Beijing, China). Total triglyceride (TG) was measured as previously described (Chen and Cunnane 1992). The low-density lipoprotein cholesterol (LDL-C) concentration was calculated using the Friedewald formula (Santos and others 2012), where LDL-C = TC − (HDL-C + TG/5).
Statistical analyses All data in the tables that follow are presented as the mean ± SD (n = 10), and differences between groups were assessed by an analysis of variance using both directional and nondirectional hypothesis t-tests. Differences were considered to be statistically significant if P < 0.05. All statistical analyses used DPS for Windows, Version 7.05.
H1472 Journal of Food Science r Vol. 78, Nr. 9, 2013
others 2006).
The hypolipidemic activities of AAP-I in vivo To determine the anti-hyperlipidemia effect of AAP-I in vivo, mice fed a cholesterol-enriched diet were examined. For further investigation of the hypocholesterol effect of AAP-I, the effects of 3 orally administered dosages of 50, 100, and 200 mg/kg of AAPI were compared to a 6.75 mg/kg dosage of simvastatin. The medicine was given once everyday. After 1 wk of a cholesterolenriched diet, the content of mouse serum TC, TG, and LDLC increased significantly, compared with the RD group mice (Table 1–3) at week 0. As shown in Tables 1–4, the serum lipid profiles in each experimental group between weeks 2 and 4 was recorded. In general, serum cholesterol content remained stable throughout the study period in mice fed with RD and CED. In all 3 groups with treatments of orally administered AAP-I, serum TC contents were lowered significantly (P < 0.05) compared to that of the CED group (Table 1). At the end of week 2, 100 mg/kg dosage AAP-I infusion decreased serum TC by 46.6% compared with the CED group, which was similar to the simvastatin group; furthermore, at the end of week 4 in the 200 mg/kg dosage AAP-I infusion group, serum TC was decreased by 32.1%, compared with the CED group (P < 0.05). Similarly, there was a significant (P < 0.05) difference in serum TG of mice infused with
Polysaccharide of Auricularia auricular . . .
AAP-I (Table 2). At the end of week 2, 100 mg/kg dosage AAP-I infusion decreased serum TG by 46.4%, compared with the CED group, which was higher than the simvastatin group’s 17.0%; in addition, at the end of week 4 in the 100 mg/kg dosage AAPI infusion group, serum TG was decreased by 29.0%, compared with the CED group, whereas the simvastatin group decreased by 1.6%. This result demonstrates that the 100 AAP-I infusion is the optimum dosage for reducing serum TG. During the initial 2 wk of treatment, LDL-C content of all treatment groups was observed at similar levels (Table 3), with no significant difference between the RD and CED groups (P > 0.05). Nevertheless, at the end of weeks 2 and 4, the LDL-C content in the 200 mg/kg dosage AAP-I and 50 mg/kg dosage AAP-I groups were significantly lower than that of the CED group (P < 0.05). All treatment groups had significantly elevated HDL-C (P < 0.05) compared with the RD groups between weeks 0 and 4, but there was no significant difference among the AAP-I treatment groups (Table 4). In this study, AAP-I, a polysaccharide from a Chinese traditional edible mushroom, was fed via orally administered, and its potent pleiotropic preventive hyperlipidemic effect in mice was demonstrated. AAP-I has been formulated into bread as a health-promoting functional food (Fan and others 2006); however, knowledge about many aspects of this effective, nontoxic natural substance, grown with SSF, is lacking in the literature and in practical applications. Because the carbohydrate polymers in A. auricula are important nutraceutical ingredients and pharmaceutical components, this study explored this potent functional mushroom polysaccharide derived from SSF. Our study revealed the effect of purified A. auricula polysaccharide AAP-I on the serum lipid profile in hyperlipidemic mice (induced by a CED). The results indicated that AAP-I has a potent effect on lowering cholesterol as well as decreasing LDL-C.
During the 4-wk treatment period, AAP-I kept the serum TC, TG, and LDL-C contents at a stable lower level, showing a significant difference (P < 0.05) compared to the CED group. It was interesting to find that the 100 mg/kg dosage AAP-I group showed the lowest TC and TG contents; however, the 200 mg/kg dosage AAP-I group showed the lowest LDL-C levels among the AAP-I groups. Both groups showed a significant difference (P < 0.05) compared with the CED group. It may be suggested that 100 AAP-I was the optimal dose for adjusting the serum lipid profile. LDL-C is considered to be the “bad cholesterol” as it transfers cholesterol from the liver into circulation. These results suggest that AAP-I might be affecting the LDL-receptor’s upregulation or gene transcription, thereby facilitating the removal of cholesterol from circulation (Leanc¸a and others 2013). Compared with the CED group, the results showed no effect on HDL-C content in the AAP-I groups. This result was consistent with a previous study that studied the effect of a diet containing 2 edible mushroom materials (Nguyen and others 2012). There have also been many previous studies suggesting that factors of a functional food derived from flowers affect serum TC, with HDL-C being unchanged in animal studies (Zhang and others 2002), which has also been observed in humans (Schneider and others 2011). These studies indicate that the reduction of serum TC by AAP-I might be attributable to the fall of serum LDL-C; however, the 3 dosages of AAP-I had different effects on the levels of HDL-C. Further studies are needed to confirm the effects of these dosages on HDL-C. Although no obvious dose-dependent hypolipidemic effect was observed, 100 mg/kg was concluded to be as the optimal dosage for the serum lipid profile. AAP-I has a complicated mechanism for digestion and absorption; therefore, it could be difficult to establish the obvious dose-dependent manner in vivo. Moreover, the mechanisms of the hypocholesterol effect are Vol. 78, Nr. 9, 2013 r Journal of Food Science H1473
H: Health, Nutrition & Food
Figure 1–Purification of crude AAP. (A) The crude A. auricula polysaccharide was further separated by DEAE Sephadex A-50 column chromatography into 3 fractions, named AAP-I, AAP-I, and AAP-II. (B) The AAP-I was further separated by Sephadex G-200 column chromatography into one fraction.
Polysaccharide of Auricularia auricular . . . Figure 2–Structure analysis of the purified A. auricular polysaccharide (AAP-I). (A) FT-IR spectra obtained from AAP-I had peaks at approximately 3500 cm−1 and 500 cm−1 in the carbohydrate region. (B) 1 H NMR spectrum of AAP-I recorded in CD3 OD.
complicated; it is possible that synergistic or antagonistic interac- antagonistic interactions involved, meaning that AAP-I may affect the expression of certain genes. tions are involved.
Conclusions
H: Health, Nutrition & Food
Our results showed that the SSF is a rapid method to obtain fungal biomass of consistent quality, and the polysaccharide from the mycelia of A. auricula cultured by SSF possess hypolipidemic properties, the polysaccharide may be useful as a functional food additive and a hypolipidemic agent. In addition, we evaluated the preliminary chemical properties of the purified polysaccharide and revealed that it is α-glycosidically linked. Our research concluded that we could make use of food processing by-products bagasse and bean dregs to develop functional polysaccharide of fungi and enhance the value of these by-products. Moreover, from the in vivo study, AAP-I demonstrated hypolipidemic effects, however, the mechanisms of the hypocholesterol effect are complicated. The underlying mechanisms of the hypocholesterol effect need to be addressed in further investigations; perhaps there are synergistic or H1474 Journal of Food Science r Vol. 78, Nr. 9, 2013
Acknowledgments This study was funded by the Natural Science Foundation of China (No. 31071639), the school-enterprise cooperation major projects of Fujian, China (No. 2010N5005), and projects of science and technology of the Fujian Education Department, China (No. JB11038).
References Acharya K, Samui K, Rai M, Dutta B, Acharya R. 2004. Antioxidant and nitricoxide synthase activation properties of Auricularia auricula. Indian J Exp Biol 42:538–40. Ballantyne CM, Corsini A, Davidson MH, Holdaas H, Jacobson TA, Leitersdorf E, M¨arz W, Reckless JPD, Stein EA. 2003. Risk for myopathy with statin therapy in high-risk patients. Arch Intern Med 163:553–64. Chauveau C, Talaga P, Wieruszeski JM, Strecker G, Chavant L. 1996. A water-soluble β-Dglucan from Boletus erythropus. Phytochemistry 43:413–5. Chen ZY, Cunnane SC. 1992. Preferential retention of linoleic acid-enriched triacylglycerols in liver and serum during fasting. Am J Physiol 263:R233–9. Fan LS, Zhang SH, Yu L, Ma L. 2006. Evaluation of antioxidant property and quality of breads containing Auricularia auricula polysaccharide flour. Food Chem 101:1158–63.
Polysaccharide of Auricularia auricular . . . Perepelov AV, Babicka D, Shashkov AS, Senchenkova SN, Rozalski A, Knirel YA. 2000. Structure of the O-specific polysaccharide of the bacterium Proteus vulgaris O46. Carbohyd Res 328:229–34. Peter R, Bajwa H, Anthony S. 2013. Hyperlipidaemia and cardiovascular disease: newer antihyperglycaemic agents and cardiovascular disease. Curr Opin Lipidol 24(2):189–90. Rader DJ. 2001. A new feature on the cholesterol-lowering landscape. J Nat Med 7: 1282–4. Santos FA, Frota JT, Arruda BR, de Melo TS, da Silva A, de Castro Brito G, Chaves MH, Rao VS. 2012. Antihyperglycemic and hypolipidemic effects of α, β-amyrin, a triterpenoid mixture from Protium heptaphyllum in mice. Lipids Health Dis 11:98. Schneider I, Kressel G, Meyer A, Krings U, Berger RG, Hahn A. 2011. Lipid lowering effects of oyster mushroom (Pleurotus ostreatus) in humans. J Funct Foods 3(1):17–24 Solomons GL. 1975. Submerged culture production of mycelial biomass. London: Edward Arnold. p 249–64. Suryanarayan S. 2003. Current industrial practice in solid-state fermentations for secondary metabolite production: the Biocon India experience. Biochem Eng J 13:189–95. Wasser SP. 2002. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl Microbiol Biot 60:258–74. Wu Q, Tan Z, Liu H, Gao L, Wu S, Luo J, Zhang W, Zhao T, Yu J, Xu X. 2010. Chemical characterization of Auricularia auricula polysaccharides and its pharmacological effect on heart antioxidant enzyme activities and left ventricular function in aged mice. Int J Cast Met Res 46:284–8. Wu Y, Sun CR, Pan YJ. 2006. Studies on isolation and structural features of a polysaccharide from the mycelium of a Chinese edible fungus (Cordyceps sinensis). Carbohyd Polym 63:251–6. Zhang H, Wang ZY, Zhang Z, Wang X. 2011. Purified Auricularia auricular-judae polysaccharide (AAP I-a) prevents oxidative stress in an ageing mouse model. Carbohyd Polym 84:638–48. Zhang ZS, Ho WKK, Huang Y, James AE, Lam LW, Chen ZY. 2002. Hawthorn fruit is hypolipidemic in rabbits fed a high cholesterol diet. J Nutr 132:5–10.
H: Health, Nutrition & Food
Journoud M, Jones PJ. 2004. Red yeast rice: a new hypolipidemic drug. Life Sci 74:2675–83. Kim SW, Hwang HJ, Park JP, Cho YJ, Song CH, Yun JW. 2002. Mycelial growth and exobiopolymer production by submerged culture of various edible mushrooms under different media. Lett Appl Microbiol 34:56–61. Kumar D, Jain VK, Shanker G, Srivastava A. 2003. Citric acid production by solid state fermentation using sugarcane bagasse. Process Biochem 38:1731–8. Leanc¸a CC, Nunes VS, Nakandakare ER, de Faria EC, Quintao EC. 2013. Does plasma HDL-C concentration interact with whole-body cholesterol metabolism? Nutr Metab Cardiovasc Dis 23(4):279–84. Lee BC, Bae JT, Pyo HB, Choe TB, Kim SW, Hwang HJ, Yun JW. 2003. Biological activities of the polysaccharides produced from submerged culture of the edible Basidiomycete Grifola frondosa. Enzyme Microb Tech 32:574–81. Lee BC, Bae JT, Pyo HB, Choe TB, Kim SW, Hwang HJ, Yun JW. 2004. Submerged culture conditions for the production of mycelial biomass and exopolysaccharides by the edible Basidiomycete Grifola frondosa. Enzyme Microb Tech 35:369–76. Li C, Mao X, Xu B. 2013. Pulsed electric field extraction enhanced anti-coagulant effect of fungal polysaccharide from Jew’s ear (Auricularia auricula). Phytochem Anal 24(1):36–40. Liu CH, Lin QX, Gao Y, Ye L, Xing YY, Xi T. 2007. Characterization and antitumor activity of a polysaccharide from Strongylocentrotus nudus eggs. Carbohyd Polym 67:313–8. Luo GC. 2009. Evaluation of antioxidative and hypolipidemic properties of a novel functional diet formulation of Auricularia auricula and Hawthorn. Innov Food Sci Emerg 10:215–21. Man RY, Lynn EG, Cheung F, Tsang PS, Karmin O. 2002. Cholestin inhibits cholesterol synthesis and secretion in hepatic cells (HepG2). Mol Cell Biochem, 233:153–8. Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S, Lee YC. 2005. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem 339(1):69–72. Mitchell DA, Lonsane BK. 1992. Solid substrate cultivation. London: Elsevier Science Publishers. p 1–16. Nguyen TL, Chen J, Hu Y, Wang D, Fan Y, Wang J, Abula S, Zhang J, Qin T, Chen X, Chen X, Khakame SK, Dang BK. 2012. In vitro antiviral activity of sulfated Auricularia auricula polysaccharides. Carbohydr Polym 90(3):1254–8.
Vol. 78, Nr. 9, 2013 r Journal of Food Science H1475
A water-soluble crude polysaccharide was extracted by hot water from Auricularia auricular mycelium grown under solid-state fermentation (SSF). The crude polysaccharide was purified by DEAE Sephadex A-50 and Sephadex G200 chromatography. Fourier transform infrared spectroscopy and nuclear magnetic resonance (1 H NMR) spectroscopy were used to investigate the structure of the purified A. auricular polysaccharide (AAP-I) and revealed that it is αglycosidically linked. After 14 and 28 days of AAP-I orally administered, the AAP-I significantly decreased the levels of total cholesterol, triglyceride, and low-density lipoprotein cholesterol in mice in which hyperlipidemia had been induced by a high fat diet (P < 0.05). The results revealed that AAP-I from SSF of A. auricular mycelium possesses potent hypolipidemic properties. The polysaccharide may be useful as a functional food additive and a hypolipidemic agent.
Abstract:
Keywords: Auricularia auricula, chemical properties, hypolipidemic, polysaccharide, solid-state fermentation
Introduction
H: Health, Nutrition & Food
ties, such as antitumor, anti-inflammatory, anticoagulant, hypocholesterolemic, hypoglycemic, and ameliorating properties, have reportedly been observed in A. auricula (Nguyen and others 2012; Li and others 2013). In addition, its ethanolic extracts have been proven to possess antioxidant and nitric oxide synthase activation properties (Acharya and others 2004). These medical applications could result in an increased commercial demand in the near future. Prior work has primarily focused on polysaccharides extracted from the fruiting body of macrofungi that were grown on a solid culture. Submerged culture is an alternative approach for producing a large amount of macrofungi polysaccharides, but SSF has many advantages. In recent years, researchers have shown an increasing interest in SSF as a potential alternative to submerged fermentation (Mitchell and Lonsane 1992) because it uses economical substrates (agricultural residues), requires fewer processing and down-streaming stages, requires less power, and generates less waste (Kumar 2003). Moreover, SSF has a higher product yield and offers better product stability. Previously, SSF was used primarily for the production of industrial enzymes; currently, it is also being exploited for the production of secondary metabolites (Suryanarayan 2003). A large number of studies have been focusing on the production of bioactive compounds with submerged culture (Kim and others 2002; Lee and others 2003; Lee and others 2004). However, few previous studies have been reported on bioactive polysaccharides produced by A. auricula mycelia grown in SSF. In this work, A. auricula mycelia were obtained by SSF. A water-soluble crude polysaccharide was extracted from A. auricular mycelia and further purified. The structure of the purified A. auricula polysacchaMS 20121760 Submitted 12/21/2012, Accepted 5/8/2013. Authors Zeng, Zhao, ride (AAP-I) was characterized, and its hypolipidemic activity was Pang, and Liu are with College of Food Science, Fujian Agriculture and Forestry Univ. investigated in vivo. Fuzhou, Fujian 350002, China. Authors Zeng, Lin, Huang, and Liu are with
Currently, hyperlipidemia, by triggering atherosclerosis, is the leading cause of cardiac illness and death. Elevated levels of plasma cholesterol are becoming a major risk factor for the development of hyperlipidemia and coronary heart disease (Peter and others 2013). It is desirable to maintain normal body functions by decreasing the elevated serum cholesterol to an appropriate level. However, the side effects of cholesterol-lowering drugs have become apparent over time. For instance, studies have reported that patients taking cholesterol-lowering drugs were unable to tolerate statin treatments due to musculoskeletal symptoms and other side effects (Rader 2001; Ballantyne and others 2003). The search for novel, natural cholesterol-lowering food components has increased in intensity. Since the technology of functional food emerged, an increasing number of functional foods have been developed from edible mushrooms (Man and others 2002; Journoud and Jones 2004). Traditionally, edible mushrooms have been produced in solid culture by using composts or lignocelluloses wastes, such as straws or wood, and it usually takes 4 to 6 months to culture their fruiting bodies (Solomons 1975). It is generally accepted that growing mushroom mycelia in a defined medium by solid-state fermentation (SSF) is a rapid method to obtain fungal biomass of consistent quality. Auricularia auricula, a precious macrofungus, has been used as a food and drug in China (Nguyen and others 2012). It has recently attracted interest due to its promising potential application in pharmaceutical industries (Wasser 2002). Immunomodulation activi-
Natl. Engineering Research Center of Juncao, Fuzhou, Fujian 350002, China. Author Huang is with College of Animal Science, Fujian Agriculture and Forestry Univ., Fuzhou, Fujian 350002, China. Direct inquiries to author Liu (E-mail: [email protected]). Authors Zeng and Zhao contributed equally to this study.
H1470
Journal of Food Science r Vol. 78, Nr. 9, 2013
Materials and Methods A. auricula 0020 was purchased from the Centre of Fungal Research (Fujian Agricultural and Forestry University, Fuzhou, China). It was maintained on potato dextrose agar (PDA) slants that R C 2013 Institute of Food Technologists
doi: 10.1111/1750-3841.12226 Further reproduction without permission is prohibited
Polysaccharide of Auricularia auricular . . .
Separation and purification of A. auricula polysaccharide The mycelia of A. auricula was dried at 70 ◦ C and ground to a particle diameter size to pass through a 60 mesh. The powders were extracted with hot water at 90 ◦ C for 120 min and filtered. The residue was collected and extracted twice more using the same procedure. The resulting suspension was then centrifuged at 3500 rpm for 15 min. The supernatant was concentrated by a rotary evaporator under reduced pressure. The concentrated solution was freeze-dried and precipitated with 3 volumes of absolute ethanol and maintained at 4 ◦ C for 12 h. The resulting precipitate was then separated by centrifugation, washed exhaustively with 95% alcohol, and dissolved in a minimum volume of demonized water; proteins were removed with polyamide. To the crude polysaccharide solution, hydrogen peroxide (H2 O2 ) was added dropwise until the final concentration of H2 O2 reached 5.0%; then, the solution was incubated in a water bath at 40 ◦ C for 1 h. The preparation was then dialyzed using cellulose sacks (JingHongKeDa Ltd., Beijing, China) in water flow dialysis for 48 h, followed by another 36 h distilled water dialysis. The preparation was vacuum concentrated and freeze-dried, yielding the crude A. auricula polysaccharide (CAAP). The CAAP was purified using anion-exchange chromatography. To this end, the CAAP obtained by freeze-drying was dissolved in distilled water and then centrifuged (4000 rpm, 10 min). The supernatant was then applied to a column (1.6 cm × 50 cm) of DEAE Sephadex A-50 (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) pre-equilibrated with distilled water. Fractions were collected in stepwise elution with increasing ionic strength of NaCl (0, 0.1, and 0.5 mol/L) at a flow rate of 0.5 mL/min. The main fraction from the eluted distilled water, which was quantified by the phenol–sulfuric acid method as described in the literature (Masuko and others 2005). The major fractions obtained were pooled, concentrated, and dialyzed against deionized water with dialysis tubing (molecular weight cutoff, 14 kDa). Next, the fraction was concentrated again and then lyophilized to give the polysaccharide-designated AAP-I. AAP-I was evaluated and then further purified by gel filtration chromatography on a Sephadex G-200 (Beijing Solarbio Science & Technology Co., Ltd.) column (2.5 cm × 60 cm). The sample was dissolved in a minimal volume of distilled water, added to the column, and then eluted with distilled water at a flow rate of 0.5 mL/min. The major fractions obtained were pooled, concentrated, and dialyzed against deionized water in a dialysis tube (molecular weight cutoff, 14 kDa). The resulting solution was concentrated and then lyophilized to give the pure A. auricula polysaccharide coded as AAP-I. The earlier methods were modified from previously reported methods (Zhang 2011).
Table 1–Serum TC levels of each group during the experimental period. Serum TC (mmol/L) of the time-course (wk) Treatment group
0
2
4
RD CED CED + 50AAP-I CED + 100AAP-I CED + 200AAP-I CED + simvastatin
1.99 ± 0.48b
2.73 ± 0.73b
2.35 ± 0.26c 4.83 ± 1.34a 3.71 ± 0.49b 3.73 ± 0.62b 3.28 ± 0.66b,c 4.76 ± 0.23a
4.49 ± 3.81a,b 4.46 ± 2.07a,b 5.07 ± 1.61a,b 4.33 ± 1.71a,b 5.12 ± 2.12a
4.38 ± 1.06a 3.14 ± 0.60b 2.34 ± 0.58b 2.54 ± 1.15b 2.33 ± 0.42b
The data are expressed as the mean ± SD (n = 10). Within a column, data with different superscript letters “a, b, c” are significantly different (P < 0.05) by DPS V7.05.
Fourier transform infrared (FT-IR) analysis of AAP-I IR spectra of AAP-I were recorded with an FT-IR spectrophotometer (Nicolet Instruments Corp., Madison, Wis., U.S.A.) in the range of 4000 to 400 cm−1 . The concentration of AAP-I in KBr was at a concentration of about 2.4%. The major peaks (intensity and wave number) were found using EZOMNIC version 6.0 software (Nicolet Instruments Corp.). The spectrum of the polysaccharide was calculated using the EZOMNIC software (Zhang 2011). 1
H NMR spectroscopy analysis of AAP-I Exchangeable protons were replaced with deuterium by suspending a sample in CD3 OD and lyophilizing 3 times. 1 H NMR spectra were recorded on a 600 MHz Bruker INOVA 600NB NMR spectrometer (Brucker, Rheinstetten, Germany). The relaxation delays for the NMR experiment were 1.0 s. The ionization potential was 70 eV, and the temperature of the ion source was 220 ◦ C. 1 H NMR experiments were conducted at 30 ◦ C in CD3 OD (Liu and others 2007). Treatment of hypolipidemic activity of AAP-I in vivo Sixty male Kunming mice weighing 18.0 to 20.0 g were purchased from Slaccas Laboratory Animal Center (Shanghai, China). All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals and conformed to the National Institutes of Health Guide for Care and Use of Laboratory Animals. Before commencement of the research, all mice were kept in stainless cages under controlled conditions (temperature 24.0 ± 0.5 ◦ C, humidity 55.0 ± 5.0%, and 12 h light/dark cycle). They were maintained according to the Guide for the Care and Use of Laboratory Animals and were acclimatized for a period of a week, whereas at the same time given free access to food and water. After acclimation, the mice were grouped according to a modified method from the literature (Luo 2009). The animals were randomly assigned into 6 groups (n = 10); 2 control and 4 treatment groups. During a 4-wk period, 2 control groups were fed either a regular diet (RD) or a cholesterol-enriched diet (CED), and 4 treatment groups were fed the CED, among which, 3 groups were provided AAP-I at 50.0, 100.0, and 200.0 mg/kg and 1 group was provided simvastatin 6.75 mg/kg day through orally administered. The CED was produced by supplementing 5.8% yolk powder, 0.5% cholesterol, 6.0% lard oil, and 0.2% bile salt (from pigs) with the RD. Every 2 wk, blood was collected from the orbital venous plexus for serum lipid analysis (Table 1–4). At weeks 2 and 4 of the experimental period, blood samples were taken from the orbital venous plexus of mice, after a 15-h fast, using a capillary tube without anesthesia. The blood samples were Vol. 78, Nr. 9, 2013 r Journal of Food Science H1471
H: Health, Nutrition & Food
were inoculated and incubated at 28 ◦ C for 12 days, then stored at 4 ◦ C for use. The seed culture’s medium contained the following components (in g/L): glucose 20.0, peptone 2.0, KH2 PO4 2.0, and MgSO4 ·7H2 O 1.0. The SSF medium consisted of the following components: 75.0% sugarcane bagasse and 25.0% bean dregs, with the moisture maintained at 55.0%. For preparation of the inoculum, 6 pieces (approximately 5 mm) of the mycelia of A. auricula were transferred individually into Erlenmeyer flasks containing 100 mL seed medium with the sterilized self-designed cutter. The flasks were then placed in a rotary shaker incubator at 160 rpm and 28 ◦ C for 5 days. The solid-state medium was placed in a mushroom bottle, sterilized, and inoculated with the inoculum. The SSF process was performed in biochemistry incubators at 28 ◦ C for 21 days, and the mycelia of A. auricula were obtained.
Polysaccharide of Auricularia auricular . . . Table 2–Serum TG levels of each group during the experimental period.
Results and Discussion
Purification of crude AAP A water-soluble crude polysaccharide was extracted by hot waTreatment group 0 2 4 ter from A. auricular mycelia cultured under SSF conditions and RD 1.17 ± 0.44b 1.47 ± 0.30a 0.99 ± 0.06a,b concentrated. The concentrated solution was precipitated, and CED 2.03 ± 0.22a,b 1.27 ± 0.54a,b 1.24 ± 0.10a after removal of the protein, the crude A. auricula polysacchaa,b a,b CED + 50AAP-I 1.70 ± 0.17 1.25 ± 0.23 1.00 ± 0.29a,b ride (CAAP) was obtained. The crude A. auricular polysaccharide CED + 100AAP-I 1.69 ± 0.12a,b 0.68 ± 0.34c 0.88 ± 0.13b (CAAP) was further separated by DEAE Sephadex A-50 column CED + 200AAP-I 1.96 ± 0.74a,b 0.85 ± 0.20b,c 0.96 ± 0.15a,b chromatography by stepwise elution with sodium chloride soa a,b,c a,b CED + simvastatin 3.01 ± 1.69 1.05 ± 0.10 1.22 ± 0.35 lutions (0, 0.1, and 0.5 mol/L) into 3 fractions, named AAP-I, The data are expressed as the mean ± SD (n = 10). Within a column, data with different AAP-II, and AAP-III (Figure 1A). The main peak (AAP-I) was superscript letters “a, b, c” are significantly different (P < 0.05) by DPS V7.05. further fractionated over a Sephadex G-200 column, and the main component was collected (Figure 1B). AAP-I was then further puTable 3–Serum LDL-C levels of each group during the experirified and analyzed. mental period. Serum TC (mmol/L) of the time-course (wk)
FT-IR spectroscopy analysis The FT-IR spectra of AAP-I show the characteristics of a polysaccharide. FT-IR spectra (Figure 2A) obtained from A. auRD 1.68 ± 0.66b 2.01 ± 0.20c 2.02 ± 0.38c ricular polysaccharide (AAP-I) display peaks at approximately 3500 a a a CED 4.69 ± 1.73 4.08 ± 1.04 5.22 ± 0.75 −1 CED + 50AAP-I 3.74 ± 0.48a,b 3.72 ± 0.53a,b 3.32 ± 0.47b and 500 cm in the carbohydrate region, characteristic of polysaca,b b b charides. In all of the spectra, the absorption between 1636 and CED + 100AAP-I 4.40 ± 2.58 3.09 ± 0.18 3.74 ± 0.36 −1 CED + 200AAP-I 4.20 ± 1.66a,b 3.02 ± 0.41b 3.78 ± 0.71b 579 cm can be attributed to bands of C–H, C–O, and O–H in CED + simvastatin 4.29 ± 1.93a,b 3.22 ± 0.32b 4.03 ± 0.66b the polysaccharide. In addition, this result is consistent with the The data are expressed as the mean ± SD (n = 10). Within a column, data with different previous research, which studied the chemical characterization of superscript letters “a, b, c” are significantly different (P < 0.05) by DPS V7.05. an A. auricula fruiting body polysaccharide, with the exception of the absorption between 1500 and 670 cm−1 (Wu and others Table 4–Serum HDL-C levels of each group during the experi- 2010), which demonstrates that there is a difference between the mental period. fruiting body polysaccharide and mycelia polysaccharide. Serum TC (mmol/L) of the time-course (wk)
Treatment group
Treatment group RD CED CED + 50AAP-I CED + 100AAP-I CED + 200AAP-I CED + simvastatin
0
2
4
Serum TC (mmol/L) of the time-course (wk) 1 H NMR spectroscopy analysis of AAP-1 0 2 4 1 H NMR spectroscopy was used to examine the structure of 1 a d b 1.55 ± 0.28 1.68 ± 0.21 1.83 ± 0.17 AAP-I. The 500 MHz H NMR spectrum of AAP-I, shown 2.57 ± 0.30a 3.30 ± 0.75a,b,c 3.09 ± 0.75a in Figure 2B, confirmed that the sugar residues are linked α2.17 ± 0.10a 3.47 ± 0.52a,b 3.32 ± 0.27a glycosidically (Perepelov and others 2000). Chemical shifts from 2.54 ± 0.35a 3.16 ± 0.28b,c 3.12 ± 0.28a 2.5 to 4.5 ppm were designated as the protons of carbons C-2 to 2.16 ± 0.46a 2.64 ± 0.23c 3.42 ± 0.66a 2.25 ± 1.00a 3.98 ± 0.48a 3.45 ± 0.46a C-6 on the glycodic ring (Chauveau and others 1996; Wu and
The data are expressed as the mean ± SD (n = 10). Within a column, data with different superscript letters “a, b, c” are significantly different (P < 0.05) by DPS V7.05.
H: Health, Nutrition & Food
placed in a plastic tube and incubated at 37 ◦ C for 15 min; then, the samples were centrifuged for 10 min at 3500 rpm. The serum samples were immediately analyzed. Total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) were analyzed by colorimetric method in an ultraviolet and visible light spectrophotometer (UNICO Ltd., Shanghai, China) using commercial kits from ZHONGSHENG (Beijing, China). Total triglyceride (TG) was measured as previously described (Chen and Cunnane 1992). The low-density lipoprotein cholesterol (LDL-C) concentration was calculated using the Friedewald formula (Santos and others 2012), where LDL-C = TC − (HDL-C + TG/5).
Statistical analyses All data in the tables that follow are presented as the mean ± SD (n = 10), and differences between groups were assessed by an analysis of variance using both directional and nondirectional hypothesis t-tests. Differences were considered to be statistically significant if P < 0.05. All statistical analyses used DPS for Windows, Version 7.05.
H1472 Journal of Food Science r Vol. 78, Nr. 9, 2013
others 2006).
The hypolipidemic activities of AAP-I in vivo To determine the anti-hyperlipidemia effect of AAP-I in vivo, mice fed a cholesterol-enriched diet were examined. For further investigation of the hypocholesterol effect of AAP-I, the effects of 3 orally administered dosages of 50, 100, and 200 mg/kg of AAPI were compared to a 6.75 mg/kg dosage of simvastatin. The medicine was given once everyday. After 1 wk of a cholesterolenriched diet, the content of mouse serum TC, TG, and LDLC increased significantly, compared with the RD group mice (Table 1–3) at week 0. As shown in Tables 1–4, the serum lipid profiles in each experimental group between weeks 2 and 4 was recorded. In general, serum cholesterol content remained stable throughout the study period in mice fed with RD and CED. In all 3 groups with treatments of orally administered AAP-I, serum TC contents were lowered significantly (P < 0.05) compared to that of the CED group (Table 1). At the end of week 2, 100 mg/kg dosage AAP-I infusion decreased serum TC by 46.6% compared with the CED group, which was similar to the simvastatin group; furthermore, at the end of week 4 in the 200 mg/kg dosage AAP-I infusion group, serum TC was decreased by 32.1%, compared with the CED group (P < 0.05). Similarly, there was a significant (P < 0.05) difference in serum TG of mice infused with
Polysaccharide of Auricularia auricular . . .
AAP-I (Table 2). At the end of week 2, 100 mg/kg dosage AAP-I infusion decreased serum TG by 46.4%, compared with the CED group, which was higher than the simvastatin group’s 17.0%; in addition, at the end of week 4 in the 100 mg/kg dosage AAPI infusion group, serum TG was decreased by 29.0%, compared with the CED group, whereas the simvastatin group decreased by 1.6%. This result demonstrates that the 100 AAP-I infusion is the optimum dosage for reducing serum TG. During the initial 2 wk of treatment, LDL-C content of all treatment groups was observed at similar levels (Table 3), with no significant difference between the RD and CED groups (P > 0.05). Nevertheless, at the end of weeks 2 and 4, the LDL-C content in the 200 mg/kg dosage AAP-I and 50 mg/kg dosage AAP-I groups were significantly lower than that of the CED group (P < 0.05). All treatment groups had significantly elevated HDL-C (P < 0.05) compared with the RD groups between weeks 0 and 4, but there was no significant difference among the AAP-I treatment groups (Table 4). In this study, AAP-I, a polysaccharide from a Chinese traditional edible mushroom, was fed via orally administered, and its potent pleiotropic preventive hyperlipidemic effect in mice was demonstrated. AAP-I has been formulated into bread as a health-promoting functional food (Fan and others 2006); however, knowledge about many aspects of this effective, nontoxic natural substance, grown with SSF, is lacking in the literature and in practical applications. Because the carbohydrate polymers in A. auricula are important nutraceutical ingredients and pharmaceutical components, this study explored this potent functional mushroom polysaccharide derived from SSF. Our study revealed the effect of purified A. auricula polysaccharide AAP-I on the serum lipid profile in hyperlipidemic mice (induced by a CED). The results indicated that AAP-I has a potent effect on lowering cholesterol as well as decreasing LDL-C.
During the 4-wk treatment period, AAP-I kept the serum TC, TG, and LDL-C contents at a stable lower level, showing a significant difference (P < 0.05) compared to the CED group. It was interesting to find that the 100 mg/kg dosage AAP-I group showed the lowest TC and TG contents; however, the 200 mg/kg dosage AAP-I group showed the lowest LDL-C levels among the AAP-I groups. Both groups showed a significant difference (P < 0.05) compared with the CED group. It may be suggested that 100 AAP-I was the optimal dose for adjusting the serum lipid profile. LDL-C is considered to be the “bad cholesterol” as it transfers cholesterol from the liver into circulation. These results suggest that AAP-I might be affecting the LDL-receptor’s upregulation or gene transcription, thereby facilitating the removal of cholesterol from circulation (Leanc¸a and others 2013). Compared with the CED group, the results showed no effect on HDL-C content in the AAP-I groups. This result was consistent with a previous study that studied the effect of a diet containing 2 edible mushroom materials (Nguyen and others 2012). There have also been many previous studies suggesting that factors of a functional food derived from flowers affect serum TC, with HDL-C being unchanged in animal studies (Zhang and others 2002), which has also been observed in humans (Schneider and others 2011). These studies indicate that the reduction of serum TC by AAP-I might be attributable to the fall of serum LDL-C; however, the 3 dosages of AAP-I had different effects on the levels of HDL-C. Further studies are needed to confirm the effects of these dosages on HDL-C. Although no obvious dose-dependent hypolipidemic effect was observed, 100 mg/kg was concluded to be as the optimal dosage for the serum lipid profile. AAP-I has a complicated mechanism for digestion and absorption; therefore, it could be difficult to establish the obvious dose-dependent manner in vivo. Moreover, the mechanisms of the hypocholesterol effect are Vol. 78, Nr. 9, 2013 r Journal of Food Science H1473
H: Health, Nutrition & Food
Figure 1–Purification of crude AAP. (A) The crude A. auricula polysaccharide was further separated by DEAE Sephadex A-50 column chromatography into 3 fractions, named AAP-I, AAP-I, and AAP-II. (B) The AAP-I was further separated by Sephadex G-200 column chromatography into one fraction.
Polysaccharide of Auricularia auricular . . . Figure 2–Structure analysis of the purified A. auricular polysaccharide (AAP-I). (A) FT-IR spectra obtained from AAP-I had peaks at approximately 3500 cm−1 and 500 cm−1 in the carbohydrate region. (B) 1 H NMR spectrum of AAP-I recorded in CD3 OD.
complicated; it is possible that synergistic or antagonistic interac- antagonistic interactions involved, meaning that AAP-I may affect the expression of certain genes. tions are involved.
Conclusions
H: Health, Nutrition & Food
Our results showed that the SSF is a rapid method to obtain fungal biomass of consistent quality, and the polysaccharide from the mycelia of A. auricula cultured by SSF possess hypolipidemic properties, the polysaccharide may be useful as a functional food additive and a hypolipidemic agent. In addition, we evaluated the preliminary chemical properties of the purified polysaccharide and revealed that it is α-glycosidically linked. Our research concluded that we could make use of food processing by-products bagasse and bean dregs to develop functional polysaccharide of fungi and enhance the value of these by-products. Moreover, from the in vivo study, AAP-I demonstrated hypolipidemic effects, however, the mechanisms of the hypocholesterol effect are complicated. The underlying mechanisms of the hypocholesterol effect need to be addressed in further investigations; perhaps there are synergistic or H1474 Journal of Food Science r Vol. 78, Nr. 9, 2013
Acknowledgments This study was funded by the Natural Science Foundation of China (No. 31071639), the school-enterprise cooperation major projects of Fujian, China (No. 2010N5005), and projects of science and technology of the Fujian Education Department, China (No. JB11038).
References Acharya K, Samui K, Rai M, Dutta B, Acharya R. 2004. Antioxidant and nitricoxide synthase activation properties of Auricularia auricula. Indian J Exp Biol 42:538–40. Ballantyne CM, Corsini A, Davidson MH, Holdaas H, Jacobson TA, Leitersdorf E, M¨arz W, Reckless JPD, Stein EA. 2003. Risk for myopathy with statin therapy in high-risk patients. Arch Intern Med 163:553–64. Chauveau C, Talaga P, Wieruszeski JM, Strecker G, Chavant L. 1996. A water-soluble β-Dglucan from Boletus erythropus. Phytochemistry 43:413–5. Chen ZY, Cunnane SC. 1992. Preferential retention of linoleic acid-enriched triacylglycerols in liver and serum during fasting. Am J Physiol 263:R233–9. Fan LS, Zhang SH, Yu L, Ma L. 2006. Evaluation of antioxidant property and quality of breads containing Auricularia auricula polysaccharide flour. Food Chem 101:1158–63.
Polysaccharide of Auricularia auricular . . . Perepelov AV, Babicka D, Shashkov AS, Senchenkova SN, Rozalski A, Knirel YA. 2000. Structure of the O-specific polysaccharide of the bacterium Proteus vulgaris O46. Carbohyd Res 328:229–34. Peter R, Bajwa H, Anthony S. 2013. Hyperlipidaemia and cardiovascular disease: newer antihyperglycaemic agents and cardiovascular disease. Curr Opin Lipidol 24(2):189–90. Rader DJ. 2001. A new feature on the cholesterol-lowering landscape. J Nat Med 7: 1282–4. Santos FA, Frota JT, Arruda BR, de Melo TS, da Silva A, de Castro Brito G, Chaves MH, Rao VS. 2012. Antihyperglycemic and hypolipidemic effects of α, β-amyrin, a triterpenoid mixture from Protium heptaphyllum in mice. Lipids Health Dis 11:98. Schneider I, Kressel G, Meyer A, Krings U, Berger RG, Hahn A. 2011. Lipid lowering effects of oyster mushroom (Pleurotus ostreatus) in humans. J Funct Foods 3(1):17–24 Solomons GL. 1975. Submerged culture production of mycelial biomass. London: Edward Arnold. p 249–64. Suryanarayan S. 2003. Current industrial practice in solid-state fermentations for secondary metabolite production: the Biocon India experience. Biochem Eng J 13:189–95. Wasser SP. 2002. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl Microbiol Biot 60:258–74. Wu Q, Tan Z, Liu H, Gao L, Wu S, Luo J, Zhang W, Zhao T, Yu J, Xu X. 2010. Chemical characterization of Auricularia auricula polysaccharides and its pharmacological effect on heart antioxidant enzyme activities and left ventricular function in aged mice. Int J Cast Met Res 46:284–8. Wu Y, Sun CR, Pan YJ. 2006. Studies on isolation and structural features of a polysaccharide from the mycelium of a Chinese edible fungus (Cordyceps sinensis). Carbohyd Polym 63:251–6. Zhang H, Wang ZY, Zhang Z, Wang X. 2011. Purified Auricularia auricular-judae polysaccharide (AAP I-a) prevents oxidative stress in an ageing mouse model. Carbohyd Polym 84:638–48. Zhang ZS, Ho WKK, Huang Y, James AE, Lam LW, Chen ZY. 2002. Hawthorn fruit is hypolipidemic in rabbits fed a high cholesterol diet. J Nutr 132:5–10.
H: Health, Nutrition & Food
Journoud M, Jones PJ. 2004. Red yeast rice: a new hypolipidemic drug. Life Sci 74:2675–83. Kim SW, Hwang HJ, Park JP, Cho YJ, Song CH, Yun JW. 2002. Mycelial growth and exobiopolymer production by submerged culture of various edible mushrooms under different media. Lett Appl Microbiol 34:56–61. Kumar D, Jain VK, Shanker G, Srivastava A. 2003. Citric acid production by solid state fermentation using sugarcane bagasse. Process Biochem 38:1731–8. Leanc¸a CC, Nunes VS, Nakandakare ER, de Faria EC, Quintao EC. 2013. Does plasma HDL-C concentration interact with whole-body cholesterol metabolism? Nutr Metab Cardiovasc Dis 23(4):279–84. Lee BC, Bae JT, Pyo HB, Choe TB, Kim SW, Hwang HJ, Yun JW. 2003. Biological activities of the polysaccharides produced from submerged culture of the edible Basidiomycete Grifola frondosa. Enzyme Microb Tech 32:574–81. Lee BC, Bae JT, Pyo HB, Choe TB, Kim SW, Hwang HJ, Yun JW. 2004. Submerged culture conditions for the production of mycelial biomass and exopolysaccharides by the edible Basidiomycete Grifola frondosa. Enzyme Microb Tech 35:369–76. Li C, Mao X, Xu B. 2013. Pulsed electric field extraction enhanced anti-coagulant effect of fungal polysaccharide from Jew’s ear (Auricularia auricula). Phytochem Anal 24(1):36–40. Liu CH, Lin QX, Gao Y, Ye L, Xing YY, Xi T. 2007. Characterization and antitumor activity of a polysaccharide from Strongylocentrotus nudus eggs. Carbohyd Polym 67:313–8. Luo GC. 2009. Evaluation of antioxidative and hypolipidemic properties of a novel functional diet formulation of Auricularia auricula and Hawthorn. Innov Food Sci Emerg 10:215–21. Man RY, Lynn EG, Cheung F, Tsang PS, Karmin O. 2002. Cholestin inhibits cholesterol synthesis and secretion in hepatic cells (HepG2). Mol Cell Biochem, 233:153–8. Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S, Lee YC. 2005. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem 339(1):69–72. Mitchell DA, Lonsane BK. 1992. Solid substrate cultivation. London: Elsevier Science Publishers. p 1–16. Nguyen TL, Chen J, Hu Y, Wang D, Fan Y, Wang J, Abula S, Zhang J, Qin T, Chen X, Chen X, Khakame SK, Dang BK. 2012. In vitro antiviral activity of sulfated Auricularia auricula polysaccharides. Carbohydr Polym 90(3):1254–8.
Vol. 78, Nr. 9, 2013 r Journal of Food Science H1475
