World Journal of Microbiology and Biotechnology

, 26:15

Solid-state fermentation of cornmeal with the ascomycete Morchella esculenta for degrading starch and upgrading nutritional value

Authors

  • Gui-Ping Zhang
    • Institute of Loess PlateauShanxi University
    • Department of Biology & ChemistryChangzhi College
  • Feng Zhang
    • Institute of Loess PlateauShanxi University
  • Wen-Ming Ru
    • Department of Biology & ChemistryChangzhi College
    • School of Life Science and TechnologyShanxi University
Original Paper

DOI: 10.1007/s11274-009-0135-y

Cite this article as:
Zhang, G., Zhang, F., Ru, W. et al. World J Microbiol Biotechnol (2010) 26: 15. doi:10.1007/s11274-009-0135-y

Abstract

The ability of the ascomycete Morchella esculenta to degrade starch and upgrade nutritional value of cornmeal during solid-state fermentation (SSF) was studied. On the basal medium, α-amylase activity of M. esculenta reached its maximum value of 215 U g−1 of culture on day 20 after inoculation. Supplementation of glucose, yeast extract to the basal medium caused a significant increase in either the degradation rate of starch or the mycelial biomass as compared with control (P < 0.01). Through orthogonal experiments, the theoretical optimum culture medium for SSF of this fungus was the following: 100 g cornmeal, ground to 30-mesh powder, moistened with 67 ml of nutrient salt solution supplemented with 3 g yeast extract and 10 g glucose per liter. Under the optimum culture condition, the degradation rate of starch reached its maximum values of 74.8%; the starch content of the fermented product decreased from 64.5 to 23.5%.

Keywords

CornmealMorchella esculentaMycelial biomassSolid-state fermentationStarch

Introduction

Mushrooms have been used as food and food-flavouring material in soups and sauces for centuries, due to their unique and subtle flavor. Recently mushrooms also have become attractive as functional foods and as a source of physiologically beneficial medicine (Mau et al. 2004).

Morels (Morchella spp.) are some of the most desirable, edible mushrooms known (Royse and May 1990). Currently, morels are highly valued in China, partially due to their rareness and difficulty in cultivation. In addition to dried morels, alternative or substitute mushroom products are mycelia of Morchella spp., mainly prepared from submerged culture. Morchella spp. mycelia cultivated by submerged culture have a similar smell and stiffness to the morel mushroom, which suggested cultured mycelia may be able to be utilized in processed foods. In the USA Morchella mycelia grown in submerged culture has been produced with the trade name “morel mushroom flavoring” (Litchfield 1955).

There are numerous studies dealing with the spore germination, culture, cytology, morphology, anatomy and physiology of morels (Amir et al. 1993). Morel mycelium has the same nutritive content and aroma as its ascocarps (Gilbert 1960). The morphological, cytological and nutritional characteristics of the morel vegetative mycelium are well known (Hervey et al. 1978). In various studies (Janardhanan et al. 1977), Morchella mycelia have been grown on agar media with a variety of undefined substrates such as vegetable wastes, sulfite liquor, peat hydrolysates, citrus wastes, cheese whey, pumpkin and carob bean. Güler et al. (1996) reported that black olive juice, beet molasses, potato, pumpkin and carob are used as substrates in submerged culture mediums. Nowadays, more research is carried out in relation to submerged culture. However, studies on solid-state fermentation (SSF) of Morchella spp. on amylaceous substrates, such as cornmeal and potato meal, have not been reported.

Solid-state fermentation is now being used for enzyme production and upgrading the values of existing foods, especially oriental foods (Pandey et al. 2000). SSF is a process whereby an insoluble substrate is fermented with sufficient moisture but without free water. SSF, unlike that of slurry state, requires no complex fermentation controls and has many advantages over submerged liquid fermentation (SLF). SSF requires less energy input than SLF, making its potential application of interest (Pandey et al. 2000). Filamentous fungi seem to be more suitable for SSF than single-celled microorganisms such as bacteria and yeasts. During previous studies (Han 2003), we found that corn starch could be degraded by the basidiomycete Hericium erinaceum, and nutritional value of cornmeal was upgraded strikingly. Encouraged by these results, we envisaged a new approach to develop a more efficient and dependable method for the culture of Morchella spp. We have focused on the SSF of cornmeal by Morchella esculenta. The aim of this research was to study the ability of the ascomycete M. esculenta to degrade starch and upgrade nutritional value of cornmeal during SSF. The present report described a series of experiments on the SSF of M. esculenta in cornmeal medium and analyzed the amylase activity, mycelial biomass and main nutritional component differences between the fermented product and nonfermented control.

Materials and methods

Organisms

Morchella esculenta ACCC51589 was obtained from China Agricultural Microbiological Culture Collection Center, and routinely maintained on potato dextrose agar (PDA) slants.

Chemicals

Casamino acid and peptone were purchased from Sigma (St Louis, MO, USA). Ergosterol (Sigma) was recrystallized from ethanol. All other chemicals were reagent grade. Methanol was of HPLC grade.

Preparation of medium

The basal medium for SSF consisted of 100 g of dry cornmeal, which were ground to 30-mesh powder, moistened with 67 ml of nutrient salt solution which contained the following per liter of distilled water (g): KH2PO4 1, MgSO4·7H2O 0.5, FeCl3 0.05, CaCl2·2H2O 0.1. The moisture content at this stage was about 40% (w/w). The initial pH of the salt solution was adjusted to 6.5. The saccharides and nitrogen sources being added to the basal medium are described later.

Solid-state fermentation

The solid medium containing 100 g of dry substrate was dispensed into each Erlenmeyer flask of 250 ml capacity. The flasks were sealed with cotton plugs to facilitate air transfer, autoclaved twice for 30 min each at 121°C. After sterilization, each flask was surface inoculated with five discs of mycelium and agar, 6 mm in diameter, from agar plate cultures of the organisms on PDA medium. Three replicate were prepared for each treatment, and an uninoculated flask served as control. All of the flasks were incubated at 25°C in the dark. The entire contents of flasks were harvested at 10, 15, 20 and 25 days for the assay of α-amylase activity. Then the samples were dried to constant weight at 70°C and used for the assay of main nutritional components and ergosterol.

Enzyme extraction and assay

One gram of the culture of SSF from each flask was mixed with 8 ml of deionized water and acid-washed sand in an ice-cold mortar. The homogenate was centrifuged at 2,000 g for 5 min. The supernatant was assayed for α-amylase activity within 12 h of preparation. Amylase (α-1, 4-glucan-4-glucanohydrolase, EC 3.2.1.1) was assayed with the substrate 1% soluble starch in 20 mmol l−1 phosphate buffer (pH 6.9). A mixture of 0.5 ml of extract and 0.5 ml of 20 mmol l−1 phosphate buffer (pH 6.9) containing 1% soluble starch was incubated at 25°C for 3 min. The reducing sugars released therein were then determined by the method of Bernfeld (1955) with reference to a standard curve of maltose. One α-amylase unit (U) was defined as the amount of enzyme producing 1 μmol of maltose per min at 25°C on soluble starch.

Main nutritional components assay

The crude protein content of the samples was calculated from the nitrogen content (as N × 6.25) as determined by the micro-Kjedahl method. The crude fat content was estimated gravimetrically after continuous ether extraction of the dried samples in a Soxhlet apparatus. The reducing sugar content was determined by the Folin capacity method (Huang 1989). The starch content was determined with the multi-starch plant sample assay method described by Huang (1989). The starch degradation rate was calculated as follows:
$$ {\text{Starch degradation rate }}\left( \% \right) = {\frac{{{\text{Starch}}\,{\text{content}}\,{\text{of}}\,{\text{control }} - {\text{Starch}}\,{\text{content}}\,{\text{of}}\,{\text{fermented}}\,{\text{sample}}}}{{{\text{Starch}}\,{\text{content}}\,{\text{of}}\,{\text{control}}}}} \times 100 $$

Measure of ergosterol

Ergosterol is a membrane component of most fungi and ergosterol levels are commonly used to estimate fungal biomass on various substrates (Charcosset and Chauvet 2001). In present the study, the ergosterol content in fermented products was measured by the method of Matcham et al. (1985) with some modifications. The fermented samples were extracted twice with ethanol (2 ml ethanol per gram of sample). The mixture was steeped for 1 h, then, the supernatant was decanted. Following clarification by centrifugation for 10 min at 2,000 g, the two washings were combined. The extracts were evaporated to dryness under oxygen free nitrogen prior to saponification in a solution of 1 mol l−1 KOH in 95% (v/v) ethanol for 1 h at 70°C. After cooling, the mixtures were diluted with two volumes of water and the non-saponifiable fraction containing ergosterol extracted with three washings of petroleum ether. The washings were combined and dried over sodium sulfate prior to assay.

Ergosterol was separated by reverse-phase HPLC (Hitachi, Tokyo, Japan) using a pre-packed LiChrospher 100 RP-18 column (4 × 250 mm, 5 μm particle size) of Merck (Darmstadt, Germany). The samples were eluted with 97:3 methanol/water (v/v) with a flow rate of 0.5 ml min−1 and monitored at 282 nm in a Hitachi L-4000 UV detector. Fifty microliters of ergosterol was injected into the HPLC system. The analysis was calibrated against a standard curve obtained from ergosterol solutions of 0–60 μg ml−1.

Experimentation and analysis

All experiments were replicated in three flasks and the data are presented as the mean and standard error of three independent experiments. Duncan’s multiple range test (Du 1985) was used to determine the significant differences among mean values at 1% level of confidence.

Results

Effect of fermentation time

The α-amylase activity, starch content and ergosterol yield of every sample were determined after 10, 15, 20 and 25 days. The results (Table 1) showed that the α-amylase activity of M. esculenta reached its maximum value of 215 U g−1 of culture on day 20 after inoculation. When plotting the degradation rate of starch versus the fermentation time, a positive linear relation was found. On day 25 after inoculation, this fungus gave the highest degradation rate of starch (62.3%).
Table 1

Effect of fermentation time on starch degradation and mycelial biomass of Morchella esculenta

Fermentation time (days)

α-Amylase activity (U g−1 of culture)

Degradation rate of starch (%)

Ergosterol yielda (μg flask−1)

10

169 ± 7 Ab

40.4 ± 4.1 A

320 ± 25 A

15

187 ± 8 B

50.8 ± 4.1 B

350 ± 25 A

20

215 ± 10 C

58.2 ± 4.5 C

410 ± 30 B

25

180 ± 8 B

62.3 ± 4.5 C

430 ± 30 B

aThe ergosterol yield was used to estimate the mycelial biomass of M. esculenta during solid-state fermentation

bSuperscript letters within a column indicate that values followed by the same letter did not differ significantly (P < 0.01) in Duncan’s multiple range test

Considering that the degradation rate of starch was affected by both amylase activity and mycelial biomass, we tested the effect of fermentation time on ergosterol yield (as an indicator of mycelial biomass) in every sample (Table 1). The results showed that the ergosterol yield per flask had also a positive correlation with the fermentation time. So on day 25 after inoculation, M. esculenta gave the highest ergosterol yield (430 μg flask−1).

Effect of supplementary nitrogen sources

Due to the nitrogen deficiency in cornmeal, addition of nitrogen to the basal medium was required for increasing the mycelial biomass and enhancing the degradation rate of starch. We examined the effect of supplementary nitrogen sources on degradation rate of starch and ergosterol yield (Table 2). In this experiment, the basal medium served as control. The supplementation of nitrogen sources (2 g l−1) was conducted through salt solution. Among four kinds of nitrogen sources, yeast extract gave the highest degradation rate of starch and ergosterol yield respectively, followed by KNO3.
Table 2

Effect of supplementary nitrogen sources on mycelial biomass and starch degradation by Morchella esculenta

Adjuvant

Degradation rate of starch (%)

Ergosterol yielda (μg flask−1)

KNO3

70.1 ± 4.7 Ab

500 ± 33 A

NH4NO3

69.3 ± 4.6 A

480 ± 32 AB

(NH4)2SO4

67.5 ± 4.4 AB

450 ± 31 B

Yeast extract

71.1 ± 4.7 A

510 ± 34 A

Controlc

62.3 ± 4.5 B

430 ± 30 B

aThe ergosterol yield was used to estimate the mycelial biomass of M. esculenta during solid-state fermentation

bSuperscript letters within a column indicate that values followed by the same letter did not differ significantly (P < 0.01) in Duncan’s multiple range test

cControl means without supplementation of nitrogen sources to basal medium

Effect of supplementary saccharides

In cornmeal as amylaceous substrate, starch is the main carbon source that can be utilized by the strain. Generally, compared to polysaccharides, glucose and maltose are easier utilized by various fungi. We examined the effect of selected saccharides on degradation rate of starch and ergosterol yield (Table 3). The supplementation of saccharides were conducted through salt solution and remained the concentration of 5 g l−1 of salt solution. The results showed that supplementation of three types of saccharides (i.e. glucose, sucrose and maltose) to the basal medium caused a significant increase in both the degradation rate of starch and the ergosterol yield as compared with control (P < 0.01). Glucose gave the highest degradation rate of starch and ergosterol yield, respectively.
Table 3

Effect of supplementary saccharide on mycelial biomass and starch degradation by Morchella esculenta

Adjuvant

Degradation rate of starch (%)

Ergosterol yielda (μg flask−1)

Glucose

70.5 ± 4.8 Ab

515 ± 33 A

Fructose

60.8 ± 3.8 B

445 ± 29 B

Sucrose

69.5 ± 4.6 A

500 ± 34 A

Maltose

68.3 ± 4.7 A

510 ± 34 A

Lactose

61.6 ± 3.7 B

450 ± 30 B

Controlc

62.3 ± 4.5 B

430 ± 30 B

aThe ergosterol yield was used to estimate the mycelial biomass of M. esculenta during solid-state fermentation

bSuperscript letters within a column indicate that values followed by the same letter did not differ significantly (P < 0.01) in Duncan’s multiple range test

cControl means without supplementation of saccharides to basal medium

Identification of optimum SSF condition

Through orthogonal experiments (Table 4), the theoretical optimum culture medium for SSF of M. esculenta was the following: 100 g cornmeal, ground to 30-mesh powder, moistened with 67 ml of nutrient salt solution supplemented with 3 g yeast extract and 10 g glucose per liter. Under the optimum culture condition, the degradation rate of starch by M. esculenta reached its maximum values of 74.8%. The result of orthogonal experiment also showed that the single factor effect of either fermentation time or yeast extract on the degradation rate of starch was significant (P < 0.05) (Table 5), the interaction of several factors achieved a better degree of starch degradation than any individual factor.
Table 4

Result of orthogonal experiment to assess optimal conditions for solid-state fermentation by Morchella esculenta

Experimental no

Fermentation time (days)

Yeast extract (g l−1)

Glucose (g l−1)

Degradation rate of starch (%)

1

25

1

5

71.2 ± 4.5

2

25

2

7.5

73.4 ± 4.5

3

25

3

10

74.8 ± 4.8

4

20

1

7.5

68.5 ± 4.2

5

20

2

10

69.5 ± 4.3

6

20

3

5

70.6 ± 4.4

7

15

1

10

65.4 ± 4.1

8

15

2

5

67.7 ± 4.2

9

15

3

7.5

69.1 ± 4.2

Table 5

Variance analysis of orthogonal experiment

Variation source

df

Sum of squares

F value

F0.05

F0.01

Fermentation time

2

50.39

111.16**

19

99

Yeast extract

2

14.87

32.8*

  

Glucose

2

0.45

0.9854

  

Error

2

0.451

   

Sum

8

66.16

   

Effect of SSF on the main nutritional components content

Under the condition of orthogonal experiment 3, the main nutritional components content of the fermented product and the control were summarized in Table 6. The starch content of the nonfermented control reached 64.5%; the reducing sugar content was only 4.4%. However, the starch content of the fermented product decreased significantly (P < 0.001) from 64.5 to 23.5%; while the reducing sugar content increased significantly (P < 0.001) from 4.4 to 21.7%. SSF also produced a significant (P < 0.01) increase from 11.2 to 16.9% in protein content. After SSF, the crude fat content decreased significantly (P < 0.01).
Table 6

Main nutritional components of fermented product of Morchella esculenta and a nonfermented control on a dry basis

Component (%)

Nonfermented

Fermented

P (t-test)

Protein

11.2 ± 0.6

16.9 ± 0.7

<0.01

Crude fat

10.7 ± 0.5

8.6 ± 0.3

<0.01

Starch

64.5 ± 1.5

23.5 ± 0.8

<0.001

Reducing sugar

4.4 ± 0.2

21.7 ± 0.8

<0.001

Discussion

Fermentation has historically been one of the most common ways to enhance the nutritional properties and palatability of agricultural and dairy products. After SSF of M. esculenta, the starch content of cornmeal was reduced greatly, and quite a proportion of the starch was converted into dextrin and reducing sugar. The digesting and absorbing ratio of cornmeal was strikingly increased. This also demonstrated that M. esculenta was able to produce high-active α-amylase during SSF on cornmeal. High α-amylase activity was attributed to the close contact of hyphae of M. esculenta with the substrate in SSF, which resulted in a high degradation rate of starch. Yang et al. (1992) discussed the importance of α-amylase in SLF of Candida tropicalis. However, in their research, the α-amylase was not produced by C. tropicalis during SLF, but commercial amylase was added into the medium at the same time as inoculation with the yeast.

The nutritional values and taste components of Morchella spp. mycelia have been studied (Weng 2003). In addition to their nutritional value, M. esculenta mycelia are of high gastronomic value (Phillips 1991). In this experiment, the ergosterol in the fermented product was not only as an indicator of mycelial biomass but also a physiologically active substance (Lin et al. 1991). Certainly, the possibility that the fermented product contains some physiologically active substances besides the ergosterol cannot be ruled out. This implied that cornmeal could be processed into many special functional foods containing some bioactive substances after SSF by M. esculenta. Functional foods as a marketing term was initiated in the 1980s and is used to describe foods fortified with ingredients capable of producing health benefits. This concept is becoming increasingly popular with consumers because of a heightened awareness of the link between health, nutrition, and diet. Food manufacturers are enthusiastic about developing such products because the added ingredients give increased value to food (Hilliam 1998). In this case, all bioactive components present in the fermented product should be assayed. The importance of further studies on SSF of M. esculenta in cornmeal or other cereals can readily be seen. The SSF of M. esculenta is still far from being thoroughly studied.

Acknowledgments

Support for this research by the Shanxi Province Science Foundation (No. 2008012010-2) is gratefully acknowledged.

Copyright information

© Springer Science+Business Media B.V. 2009