Fermentation of Monascus Purpureus on Agri-by-Products to Make Colorful and Functional Bacterial Cellulose (Nata)

Chang-Chai Ng, Fuu Sheu, Chun-Ling Wang, and Yuan-Tay Shyu*
PLEASE PROVIDE DESIGNATION/POSITION OF THE OTHER 3 AUTHORS
*Professor and Chairman, Department of Horticulture
National Taiwan University
Taipei 106, Taiwan ROC, 2004-11-01

Abstract

Introduction

Nata, a white gelatinous bacterial cellulose, is produced by Acetobacter aceti ssp. xylinum through fermentation of fruit juices. Nata is considered one of the traditional holiday foods among Filipino people and is also popular in other Asian countries, including Indonesia, Japan, and Taiwan, due to its distinctly soft texture and high fiber content (Okiyama et al. 1992a, Roberfroid 1993). The biogenesis and fermentation for nata production have been well studied (Cannon et al. 1991, Banzon et al. 1990, Okiyama et al. 1992a, Embuscado et al. 1994). However, few studies have reported on the coloring of nata. The coloring of nata can improve its appearance and provide more variety of applications as a foodstuff. Due to its uniform 1?4?-glucan structure, nata is difficult to dye with only hydroxyl groups available.

Traditionally grown on steamed rice, bread, brans, and cereal meats, Monascus purpureus produces true red mycelium and pigments through solid culture, and has been used in the Orient for many centuries to color and flavor food and beverages (Johns and Stuart 1991, Chen and Johns 1993). The fermentative conditions influencing the pigment production have been extensively studied (Su 1978, Wong et al. 1982, Lin and Demain 1991, Chen and Johns 1993). Pigments from M. purpureus are very stable and suitable for use as a food additive (Fink-Gremmels et al. 1991, Fabre et al. 1993, Juzlova et al. 1996). Moreover, the Monascus fungi contain therapeutic metabolites such as monacolin k (mevinolin) (Juzlova et al. 1996), which is regarded as a healthy, nutritious food in Asia.

In the 1970s, monacolin k (mevinolin), an important metabolite of Monascus sp., was identified (Endo 1979) and shown to be able to inhibit the synthesis of cholesterol (Albert et al. 1980, Endo 1979, Endo et al. 1985, Endo et al. 1986). It also provides benefits to sufferers of cardiovascular disorders (Lin 1986). The critical reaction in the pathway of cholesterol synthesis is the formation of mevalonic acid from 3-hydroxy-3 methylglutaryl CoA (HMG-CoA) by HMG-CoA reductase. Monacolin k is structurally similar to HMG-CoA and plays a role as a competitive inhibitor, which competes with HMG-CoA and reduces the synthesis of cholesterol (Fears 1983). Since the discovery of this therapeutic function, the application of healthful Monascus sp. food has been broadly promoted in Asian countries (Juzlova et al. 1996).

The objective of this study was to develop a new foodstuff, the Monascus-nata complex, which which was prepared by fermenting with bacterial cellulose (nata). This study also sought to increase the availability of monacolin k in the Monascus-nata complex. The optimal culturing conditions for monacolin k production were investigated, and the stability of monacolin k in Monascus-nata complex, which is resistant to various processing treatments, was also studied. The structure of Monascus-nata complex was observed using scanning electron microscopy (SEM). The parameters affecting the fermentation and the coloration of M. purpureus were studied. The color stability of this complex was also examined and compared with pigment-dyed nata.

Materials and Methods

Materials and Chemicals

Bacterial cellulose (nata) in acetic acid was provided by the Cana Food Company (Tainan, Taiwan) and also can be easily cultured in the laboratory. The nata was cut into 2 x 2 x 1.5 cm pieces and then washed with running water for 4 h at room temperature to remove the acetic acid residue before fermentation.

Water-soluble Monascus pigment powder was obtained from the Zhejiang Chemicals Corp. (Hangzhou, China). Artificial red pigment Cochineal Red A (PONCEAU 4R) was obtained from E. Merck (Schuchardt, Germany).

Nata pieces were soaked in a solution containing 1% Cochineal Red A pigment or 1% Monascus pigment for 5 days. The pigment-dyed nata was used for the experiments of color stability.

Monascus ruber CCRC 31532 and Monascus purpureus Went CCRC3150 were obtained from the CCRC (Culture Collection and Research Center, Hshin Chu, Taiwan ROC) and Monascus pilosus NCHU M-35 was obtained from the National Chung Hsing University. The cultures were cultured on a potato dextrose agar (PDA) plate (Difco, USA) at 26oC for 3 days. Cultures of M. ruber CCRC 31532, M. purpureus and M. pilosus NCHU M-35 were used for inoculation.

Fermentation

Glucose-monosodium glutamate (MSG) medium described by Su (1978) was used with a glucose concentration of 50 g/L. The required maltose, rice powder, or sucrose (50 g/L) was substituted for glucose, whereas peptone, ammonium nitrate, or ammonium chloride (15 g/L) was substituted for MSG. The pH of the medium was adjusted using 1 M HCl or 1 M NaOH. Ten nata pieces were put into the medium (150 mL) in a 500 mL flask. After sterilization at 121oC for 20 min, each flask was inoculated with 2.0 mL of M. spp. in distilled water. Fermentation was carried out on a rotary shaker at 150 rpm at 30oC.

Structure Observation

The method of Okiyama et al. (1992b) was modified and used for the sample preparation. After 5 days of fermentation, the Monascus-nata complex was immersed in 5% glutaraldehyde overnight. The sample was dried using serially diluted ethanol (50%-100%) and anhydrous acetone, and then lyophilized using a SPI-DRYTM critical point dryer (Structure Probe Inc., West Chester, PA). The dried sample was removed from the specimen stubs, and was coated using a SC502 SEM coating system (Bio-Rad Laboratories Inc., Chicago, IL). A SEM-ABT-60 (Topcon Co., Tokyo, Japan) scanning electron microscope was used for observation at 15 kV.

Color Determination

A CIELAB colorimetry system, which was described in detail by Fabre et al. (1993), was used for color determination. Coloration was determined with a Color-PenTM handy color difference photometer (Dr. Bruno Lange GmbH, Berlin, Germany), which measured the spectrum of reflected light and converted it to a set of color coordinates (L, a, and b values). The C value, calculated from Equation 1, is a measure of the saturation or purity of the color. The percentage (%) of decolorization was calculated from the relationship of Equation 2. The measurement of each Monascus-nata complex sample was repeated 10 times.

C=(a2 + b2)1/2 Equation 1

Percent (%)=(Cafter treatment / Cbefore treatment)x100 Equation 2

Examination of Color Stability

The color resistance of Monascus-nata complex and of dyed nata to washing, heat, freezing, acidification, alkalization, and UV treatments were compared. For washing resistance evaluation, samples were washed under running tap water without any detergent at a constant flow rate of 10 L/h for 48 h. To examine the thermal stability of the color, the samples were autoclaved at 121oC for 1 h or frozen at -20oC for 5 days before analysis. For acidification and alkalization experiments, the samples were soaked in solutions at pH 2.5 or 12.5 for

5 days. To study the UV resistance, samples were exposed to ultraviolet light (366 nm) with a U60-110 Ultra-Violet lamp (Jepson Bolton & Co Ltd, Watford Herts, UK) for 36 h. The residual coloration on the surface of each sample was determined.

Statistical Analysis

Each treatment was carried out in triplicate (n=3), and all the experiments were repeated at least twice. Since the results of these experiments were similar, the result of only one experiment is shown in this paper. Significance of data was analyzed by one dimensional analysis of variance. Difference among the mean values was tested using the Least Significant Difference (LSD) multiple range test (Steel and Torrie 1980). Values were considered significant when p<0.05.

Extraction and HPLC Analysis

Six pieces of fermented Monascus-nata complex were collected and homogenized (POLYTRON®, Switzerland) with 100 mL deionized water. Later, 100 mL of CH2Cl2 was added for extraction. After centrifugation at 3000 x g for 8 min, the organic phase was collected for concentration (BÜCHI Rotavapor R-124 and BÜCHI 168 Vacuum/Distillation Controller, Switzerland) until it was dried. One mL of acetonitrile was added to dissolve monacolin k for HPLC analysis. HPLC (Intelligent HPLC System LC-800 series, JASCO, Japan), equipped with a system controller (model 801-SC), a pump (PU-980), a column (LichroCart RP18, 125 × 4 mm, 5 mm, Merck, Germany), a u.v. detector (870-UV, Jasco, Japan), a column oven (TU-100), a recorder (SISC ver. 2.1), and an autosampler (851-AS, JASCO, Japan), was used for the analysis (mobile phase acetonitrile: 0.1% phosphoric acid, 75:25 (v/v); a flow rate of 0.7 mL/min; an injection volume of 20 mL, and detection wavelength of 237 nm). A standard curve was obtained by using dilutions 10, 5, 2.5, and 1 ppm of monacolin k (Sigma, USA) in 100 mL acetonitrile.

Monacolin K Stability Test

The resistance of monacolin k in fermented Monascus-nata was tested by washing, heating, freezing, and incubating at various pH values. Samples were washed under running tap water at a constant flow rate of 10 L per hour for 60 h to determine washing resistance. In the heat resistance test, samples were cooked in boiled water for 15 min and autoclaved at 121oC for 15 min. In the freezing test, samples were frozen at -20oC for 24 h. Samples were soaked in solutions with pH values 3.0-7.0 for 48 h. The monacolin k content in the complexes was determined by HPLC over a particular period.

Results and Discussion

Production of Monascus-Nata Complex

Diced nata was fermented in Monascus-inoculated broth at 26oC for 6 days. After fermentation, Monascus mycelia penetrated the network of capillaries in nata, yielding the final Monascus-nata complex. The complex was uniformly and brightly colored.

Structure of Monascus-Nata Complex

For structural observation, M. purpureus was fermented on pieces of nata for 12 days at 30oC until the red color appeared. Figure 1 shows the SEM micrograph of nata, Monascus mycelium, and Monascus-nata complex. The capillaries of the nata network (ca. 0.5 mm-1mm) (Okiyama et al. 1992b) were wider than the diameter of Monascus mycelium (ca. 0.1 mm-0.5 mm). Therefore, the Monascus fungi could extend their mycelium through the capillaries of nata to be held in the network structure for further growth. After fermentation, a new structure composed mainly of mycelium network was formed. The original cellulose network of nata still filled the empty space between the mycelium (Fig. 1). However, the mycelium of M. purpureus could not grow well outside the piece of nata without the structural support of nata in a shaking environment. The porous network structure of nata was probably helpful for the initial growth of M. purpureus, and the original shape of nata was observed to limit the final shape of Monascus-nata complex.

According to the SEM micrography (Fig. 1), the permanent red of Monascus-nata complex was probably due to the red mycelium of M. purpureus. Water-soluble pigments produced extracellularly by M. purpureus (Lin et al. 1992) were not believed to be a major contributor to coloration of the complex. This was due to the much lighter color of the fermentation broth than that of the complex. A lack of bonding between the water-soluble pigment and the free hydroxyl groups of cellulose was a possible reason for this.

Fermentation Conditions for Coloration

Carbon source, nitrogen source, and pH have been shown to influence pigment production by Monascus purpureus (Su 1978, Wong et al. 1982, Lin and Demain 1991, Chen and Johns 1993). Therefore, the effect of these compounds on coloration of Monascus-nata complex was studied. When glucose, rice powder, maltose, or sucrose was used as a sole carbon source (5%), the most appealing and bright red color was observed with the rice powder medium after 12 days (Fig. 2). Fermentation using maltose and glucose as carbon sources yielded very dark liver color, while sucrose medium produced a light and uneven red, especially in a crosscut observation. For nitrogen source experiment, 1.5% MSG medium produced an appealing red appearance, while other nitrogen sources produced faint or foggy reds (Fig. 2). Additionally, initial pH value (3.5-10.5) and temperature (15-35oC) had no significant effect (p > 0.05) on coloration (data not shown). Minor inorganic elements (calcium, potassium, zinc, and manganese) did not show any obvious effect on coloration (data not shown). This result might be due to a crude carbon source. Rice powder that generally comprised 82% carbon, 17% water, 0.4% nitrogen, 0.3% phosphoric, and 0.3% potassium (Juliano 1986) was used as a major carbon source during fermentation.

Fig. 3 shows the progress of coloration on surface and center of the Monascus-nata complex. The red coloration in the center proceeded slower than on the surface. After 12 days of fermentation, red Monascus-nata pieces could be obtained with a fully red surface, and the central point appeared as ruby color. The results were consistent with previous research results (Wong et al. 1982, Lin and Demain 1991, Chen and Johns 1993). However, it must be noted that growth and pigment production are strain-specific among Monascus purpureus (Lin and Demain 1991). According to the results above, further experiments of color stability were carried out using a medium composed of 5% rice powder and 1.5% MSG for fermentation at 30oC for 12 days.

Color Stability

The color stability of Monascus-nata complex was examined and compared with Monascus pigment- and artificial pigment- (Cochineal Red A)-dyed nata pieces. The complex showed good resistance to washing decolorization (Fig. 4), 93.3% and 96.8% coloration were retained after 5 days of washing on the surface and the central point, respectively. In contrast, only 61.6% of coloration on Monascus pigment-dyed nata and 27.8% of coloration on artificial pigment-dyed nata were retained. These results also showed that the complex was only partially dyed by the extracellular pigments secreted by M. purpureus during fermentation. There was no appreciable color change of any sample after autoclaving, -20oC freezing, or pH 2.5 acidification (data not shown). However, at pH 12.5, the complex and Monascus pigment-dyed nata were significantly (p<0.05) decolorized by 9.6% and 7.2%, respectively. This result was in contrast to a study by Lin et al. (1992). In their report, pigments produced by M. purpureus were stable at pH 12.

Fading of Monascus-nata complex was observed under irradiation with 366 nm ultraviolet light for 36 h (Fig. 5). A 66.1% decolorization of the complex was greater than the nata dyed by water-soluble Monascus pigment. The UV decolorization of cell-bound Monascus pigment and the inferior UV resistance to the water-soluble Monascus pigment had been reported previously (Sweeny et al. 1981, Lin et al. 1992). Therefore, packaging of the complex is important to avoid the UV decolorization by sunlight.

Optimization of Culture Conditions

The effects of carbon sources, nitrogen sources, microelements, and pH on the production of monacolin k by M. pilosus NCHU M-35 and M. ruber CCRC 31532 were investigated. When glucose, sucrose, maltose, and lactose were utilized as alternative sole carbon sources (5%), the highest yield of monacolin k was produced with glucose as the carbon source (157 mg/L for M. pilosus NCHU M-35 and 148 mg/L for M. ruber CCRC 31532) (Fig. 6). Furthermore, ammonium chloride, ammonium phosphate, and peptone at 1.5% were similarly effective nitrogen sources, while MSG was poorly utilized. These results were consistent with those of Chen and Johns (1993). The lower utility of MSG in producing monacolin k on M. pilosus NCHU M-35 and M. ruber CCRC 31532 (77 mg/L and 93 mg/L, respectively) may be associated with its promotion of growth and the formation of pigments (Lin et al. 1992).

With respect to the microelements, KCl exhibited the highest production of monacolin k (160 mg/L for M. pilosus NCHU M-35 and 163 mg/L for M. ruber CCRC 31532), but ZnSO4 yielded the lowest production of monacolin k (127 mg/L for M. pilosus NCHU M-35 and 103 mg/L for M. ruberCCRC 31532). These results were similar to those of Bau and Wong (1979), who found that zinc ions diminished the growth, pigmentation, and antibacterial activity of Monascus. The addition of microelements did not markedly promote the production of monacolin k. Furthermore, the optimal pH for producing monacolin k ranged from 6.0 to 7.0 (monacolin k production >150 mg/L), whereas, a pH of lower than 5.0 yielded relatively low level of monacolin k. Therefore, the results indicate that M. pilosus NCHU M-35 yielded more monacolin k than did M. ruber CCRC 31532.

Most studies (Su 1978, Wong et al. 1981, Lin and Demain 1991, Lin et al. 1992, Chen and Johns 1993, Chen and Johns 1994) have focused on the production of pigments rather than monacolin k, so this study, in which a large amount of monacolin k is obtained, may be important for developing a functional Monascus-nata complex.

Stability of Monacolin K

The resistance of monacolin k toward processing treatments in the Monascus-nata complex was further investigated. After 60 h of washing with water, the monacolin k content fell from 160 mg/L to 119 mg/L, and from 147 mg/L to 109 mg/L for M-35-nata and 31532-nata complexes, respectively. These results indicate that the Monascus-nata complex exhibited washing resistance (Fig. 7). However, monacolin k was relatively thermally unstable. Treating M-35-nata complex with boiling water for 15 min reduced monacolin k to half of its original content (80 mg/L), while autoclave treatment removed almost all monacolin k (Fig. 7). Freezing storage reduced the content of monacolin k to 97 mg/L (Fig. 8). After soaking for 48 h in solutions with pH from 3.0 to 7.0, most of the original monacolin k content in the complex was retained, although at pH 3.0, a slight reduction in content was observed (Fig. 8).

The color of Monascus-nata complex was also relatively resistant to washing (Fig. 9). When washed, the complex retained the red pigment, with a slightly increased L value, and slightly decreased in its a value using Hunter Lab color spectrophotometer (data not shown). The results indicate that Monascus-nata lost relatively little color. When thermally treated using an autoclave, much of the red pigment in the complex was lost (Fig. 9). Freezing and treatments at various pH values caused shrinkage and yielded slightly dark red pigmentation.

The stability of monacolin k in Monascus-nata complex fermented by M. pilosus NCHU M-35 and M. ruber CCRC 31532 generally appeared to follow similar trends. These results show that fresh Monascus-nata complex possesses great potential for use in food processing, because it has more stable monacolin k and color, which are nevertheless reduced somewhat during thermal processing.

Conclusions

The production of a new Monascus-nata complex achieved the indelible coloration of nata. The coloration of this complex was influenced by the carbon and nitrogen sources during fermentation, but other parameters were not found to affect it significantly. In addition, the complex showed better color stability than Cochineal Red A or Monascus pigment-dyed nata in terms of resistance to washing, autoclaving, acidification, and alkalization, but inferior in UV resistance. The mycelium is regarded as the major contributor for color.

Monascus-nata complex, which combined the properties of nata and Monascus fungi, showed potential to be a new foodstuff as vegetarian meat or seafood replacement. Growth of Monascus mycelium did not impart a flavor on this new product and make it to be a good base as a taste-addable food. The color and texture of the complex were like those of liver, lean meat, or tuna meat (sashimi). The complex also provides high fiber content, limited calories, and healthful nutrients. Moreover, the waste broth of fermentation could be further used as a source of water-soluble pigment.

Acknowledgments

This paper is composed of research works previously cited in the theses of Mr. Chang-Chai Ng and Ms. Chun Ling Wang, and published in the J. Food Science and World J. Microbiol. Biotechnol.

References

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Index of Images

• 

  Figure 1 Sem Micrography of (a) Monascus-Nata Complex (1,500X); (B) Mycelium of Monascus Purpureus (1,700X); and (C) the Cellulose Network of Nata (9,800X)

• 

  Figure 2 Influences of (a) Different Carbon Sources and (B) Different Nitrogen Sources on the Coloration of Monascus-Nata Complex. for Carbon Source Experiments, MSG (15 G/L) Was Used As Nitrogen Source; for Nitrogen Source Experiments, Rice Powder Was Used As Carbon Source.

• 

  Figure 3 The Progress of Coloration of Surface and Center of the Monascus-Nata Complex.

• 

  Figure 4 Decoloration by Washing.

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  Figure 5 Decoloration by Uv Irradiation.

• 

  Figure 6 Monascus Pilosus Nchu M-35 Was Fermented on Nata and the Monacolin K Was Produced Using Various (a) Carbon Sources (Glu: Glucose; Suc: Sucrose; Mal: Maltose and Lac: Lactose); (B) Nitrogen Sources (a. C: Ammonium Chloride; a. P: Ammonium Phosphate); (C) Microelements and (D) PH Treatments of Medium Broth. Vertical Bars Represent S.D. (N 3).

• 

  Figure 7 Monacolin K Content in Monascus-Nata Complex after

• 

  Figure 8 (a) -20C Treatment for 48 H; (B) Treatment by Soaking in Solutions of Various PH Values for 48 H

• 

  Figure 9 Effect of Treatment of M-35-Nata Complex by (a) Washing for 60 H; (B) Heat Treatment; (C) Freezing; and (D) Solutions with Various Phvalues for 48 H

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