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Solid-state fermentation of cornmeal with the basidiomycete Hericium erinaceum for degrading starch and upgrading nutritional value
International Journal of Food Microbiology 80 (2003) 61 – 66 www.elsevier.com/locate/ijfoodmicro
Solid-state fermentation of cornmeal with the basidiomycete Hericium erinaceum for degrading starch and upgrading nutritional value Jianrong Han* Department of Life Science, Shanxi University, Taiyuan 030006, People’s Republic of China Received 8 June 2001; received in revised form 4 February 2002; accepted 5 March 2002
Abstract The ability of the basidiomycete Hericium erinaceum to degrade starch and upgrade nutritional value of cornmeal during solid-state fermentation was studied. On the basal medium which consisted of cornmeal and salt solution, H. erinaceum produced a strong a-amylase on the 15th day after inoculation, which resulted in a 52% degradation of the starch. By supplementation with 5 – 15 g soybean meal per 100 g cornmeal the a-amylase activity and degradation rate of starch was raised significantly ( P < 0.01). Prolongation of fermentation time from 15 to 30 days did not increase significantly the degradation rate of starch, though the a-amylase activity reached its maximum value of 179 U/g on the 20th day after inoculation. Under conditions close to the theoretical optimum fermentation conditions, that was after 25 days at 25 jC in the medium with added 15 g soybean meal per 100 g cornmeal, the starch content in the product decreased from 63% to 22% ( P < 0.001) and protein content increased from 12% to 17% ( P < 0.01). In the protein in the product, the lysine content was increased from 36 to 56 mg/ g and tryptophan from 9 to 13 mg/g. Using egg protein as a standard, an evaluation on the protein quality of the fermented product showed that it was superior to that of the nonfermented control and to other cereals, was close to that of soybean and chicken, but was inferior to that of milk and red meats. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Cornmeal; Solid-state fermentation; Hericium erinaceum; Starch; Protein
1. Introduction Hericium erinaceum is well known as a traditional and valuable mushroom in China. Recently, it has been paid much attention regarding some health effects when used as a home remedy. It has been reported that a preparation from either the fruitbodies or the mycelium of H. erinaceum worked effectively to cure gastric *
Fax: +86-351-7011981. E-mail address: [email protected] (J. Han).
ulcer (Yang, 1988). The lysine and tryptophan contents of the protein are very high (Ma and Chen, 1994). A biscuit product supplemented with the fruitbodies of H. erinaceum has been used in prevention or cure of nutritional anemia of preschool children (Liu et al., 1992). Consequently, H. erinaceum is cultivated widely in China, and mycelium is being produced by submerged fermentation. However, studies on solidstate fermentation (SSF) of H. erinaceum on amylaceous substrates, such as cornmeal and potato meal, have not been reported.
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 2 ) 0 0 1 2 2 - 8
62
J. Han / International Journal of Food Microbiology 80 (2003) 61–66
Cornmeal shows a nutritional deficiency because of lack of lysine and tryptophan in the protein (Li, 1987). Methods have been reported to improve the nutritional value of cornmeal including liquid state fermentation (LSF) of Bacillus spp. or yeasts (Fields et al., 1988; Yang et al., 1992), but there have been no reports about the use of SSF of edible or medicinal basidiomycetes to improve the nutritional value of cornmeal. Because H. erinaceum has the special metabolic ability to synthesize lysine and tryptophan, SSF on a solid medium containing cornmeal may be able to reduce the shortage of lysine and trytophan in cornmeal protein. We describe here a series of experiments on the SSF of H. erinaceum in cornmeal medium and analyze the a-amylase activity and main nutritional component differences between the fermented product and nonfermented control. By means of the fuzzy discernment method (Wu and Chen, 1985), the nutritional value of the fermented product was analyzed and evaluated.
cornmeal. Then the solid medium was dispensed into Erlenmeyer flasks (250 ml capacity). After sterilization, each flask was inoculated with five discs of mycelium and agar, 6 mm in diameter, agar plate cultures of the fungus on complete medium (Raper and Miles, 1958). Three replicates were prepared for each treatment, and an uninoculated flask served as control. All of the flasks were incubated at 25jC in the dark. The entire contents of flasks were harvested at 15, 20, 25, 30 days for the assay of a-amylase activity. Then the samples were dried to constant weight at 70jC and used for the assay of the main nutritional components. 2.3. Enzyme extraction and assay
The strain H 18 of Hericium erinaceum was obtained from the Edible Fungi Research Institute, Shanghai Academy of Agricultural Science, China and routinely maintained on the complete medium agar slant as outlined previously by Raper and Miles (1958).
One gram of the culture of SSF from each flask was mixed with 8 ml of deionized water and acidwashed sand in an ice-cold mortar. The homogenate was centrifuged at 2000g for 5 min. The supernatant was assayed for a-amylase activity within 12 h of preparation. Amylase (a-1, 4-glucan-4-glucanohydrolase, EC 3.2.1.1) was assayed with the substrate 1% soluble starch in 20 mM phosphate buffer (pH 6.9). A mixture of 0.5 ml of extract and 0.5 ml of 20 mM phosphate buffer (pH 6.9) containing 1% soluble starch was incubated at 25jC 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 aamylase unit (U) was defined as the amount of enzyme producing 1 Amol of maltose per min at 25jC on soluble starch.
2.2. Solid-state fermentation
2.4. Main nutritional components assay
The basal medium for SSF consisted of 100 g of dry cornmeal, which were ground to 20-mesh powder, moistened with 67 ml of nutrient salt solution which contained the following per liter of distilled water (g): KH 2 PO 4 1, MgSO 4 7H 2 O 1, FeSO 4 7H 2 O 0.5, CaCl2H 2O 0.1 (a modified formula previously described by Jia et al., 1997). The moisture content at this stage was about 40% (w/w). The initial pH of the salt solution was adjusted to 6.5. The dry soybean meal added to the basal medium as a supplementary nitrogen source. Supplements of soybean meal were added at the rate of 5, 10 and 15 g per 100 g
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). An amino acid analyser (Hitachi Model 83550) was used for amino acid and tryptophan determination by fluorescence analysis (Huang, 1989). The starch content was determined with the multi-starch plant sample assay method described by Huang
2. Materials and methods 2.1. Fungus
J. Han / International Journal of Food Microbiology 80 (2003) 61–66 Table 1 Effect of supplementary soybean meal on starch degradation by H. erinaceum Soybean meal quality (g/100 g cornmeal)
a-amylase activity (U/g of culture)a
Degradation rate of starch (%)b
0 5 10 15
44.0Ac 79.5B 121.8C 152.3D
52.3A 56.4A 59.5AB 61.7B
a
One a-amylase unit (U) is defined as the amount of enzyme producing 1 Amol of maltose per min at 25jC on soluble starch. b Degradation rate of starch=(starch content of fermented sample minus the starch content of control)/(starch content of control) 100. c Means in the same column followed by the same letter are not significantly different at the P < 0.01 level according to Duncan’s multiple range test.
(1989). The starch degradation rate was calculated as follows: Starch degradation rate ð%Þ ¼ ðstarch content of fermented sample minus the starch content of the controlÞ=starch content of control 100
Duncan’s multiple range test (Ray, 1985) was used on the isolation treatment means to test for significant differences at the 1% level of confidence. 2.5. Evaluation on the nutritional value of protein Ten kinds of foods including the fermented product and control were evaluated for the nutritional value of the protein using egg protein as the standard, by means of fuzzy discernment (Wu and Chen, 1985). If the evaluated object is Ui and the egg protein is a, then out of the Lan’s distance method (Wu and
Table 2 Effect of fermentation time on starch degradation by H. erinaceum
63
Table 3 Result of orthogonal experiment to assess optimal conditions for solid state fermentation by H. erinaceum Experimental Soybean Fermentation Moisture Degradation Protein no. meal time content rate of content (g)a (days) (%) starch (%) (%) 1 2 3 4 5 6 7 8 9
15 15 15 10 10 10 5 5 5 a
15 20 25 15 20 25 15 20 25
35 37.5 40 37.5 40 35 40 35 37.5
62.1 65.2 66.5 59.6 60.6 61.4 57.0 59.0 60.3
15.0 16.4 16.8 13.8 14.2 14.9 12.6 12.7 13.4
Supplementary quantity of soybean meal in100 g cornmeal.
Chen, 1985), the close degree is defined between the object Ui and the sample plate a, that is 8 X Aak Uik A lða; Ui Þ ¼ 1 C ð1Þ ak þ Uik k¼1 where Uik is the essential amino acid content of the evaluated protein and C is a properly selected constant (here C = 0.09) (Zhang et al., 1996), ak is the essential amino acid content of the egg protein (mg/g protein)(CAPM, 1992), and where a1, a2,. . . a8 are, respectively, Val (54.19), Leu (81.09), Ile (48.76), Thr (44.73), Phe (48.22), Trp (17.21), Met (28.14) and Lys (65.89). According to Eq. (1), the close degree of each evaluated object was calculated out, and the bigger the close degree, the higher the nutritional value.
3. Results 3.1. Effect of supplementary soybean meal Due to the nitrogen deficiency in cornmeal, addition of nitrogen to the basal medium was required for SSF of Table 4 Main nutritional components of fermented product of H. erinaceum and a nonfermented control on a dry basis (X F S, n = 3)
Fermentation time (days)
a-amylase activity (U/g of culture)
Degradation rate of starch (%)
Component (g/100 g)
Nonfermented
Fermented
P
15 20 25 30
152.3B 178.6C 164.2BC 130.9A
61.7A 64.6A 65.7A 66.4A
Protein Crude fat Starch Reducing sugar
11.6 F 0.6 10.6 F 0.5 62.8 F 1.3 4.0 F 0.2
16.8 F 0.7 9.7 F 0.4 21.9 F 0.6 19.4 F 0.6
< 0.01 < 0.01 < 0.001 < 0.001
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J. Han / International Journal of Food Microbiology 80 (2003) 61–66
Table 5 Amino acids composition of the protein (mg/g protein) produced by solid state fermentation by H. erinaceum Amino acids
Nonfermented
Fermented
Val Leu Ile Thr Phe Trp Met Lys Tyr Cys Asp Pro Ser Glu Gly Ala Arg His EAAa Totalb E/Tc
45.90 110.80 42.35 32.12 50.51 9.05 18.35 35.96 31.06 18.98 77.80 53.76 35.30 162.23 30.69 63.70 50.29 26.53 346.04 895.38 0.3864
56.12 106.72 46.60 42.71 42.35 13.25 16.53 56.12 34.30 18.07 73.41 50.49 32.19 157.42 29.52 57.60 52.05 23.96 380.40 909.41 0.4293
a b c
Essential amino acids. Total quantity of amino acids. Ratio of essential amino acids in total amino acids.
H. erinaceum. The results (Table 1) showed that during SSF of 15 days the a-amylase activity of H. erinaceum had a positive correlation with the supplementary quantity of soybean meal (r = 0.9983). The higher the supplementary quantity was, the higher the a-amylase activity. Consequently, the starch degradation rate was increased. When 15 g soybean meal was added to 100 g cornmeal, the degradation rate of starch in the fermentative product increased from 52% to 62% ( P < 0.01). 3.2. Effect of fermentation time The a-amylase activity and starch content of every sample was determined after 15, 20, 25 and 30 days.
The results (Table 2) showed that prolongation of fermentation time from 15 to 30 days did not increase significantly the degradation rate of starch, though the a-amylase activity reached its maximum value of 179 U/g on the 20th day after inoculation. After the 20th day, the amylase activity decreased, but the degradation rate of starch continued to increase. 3.3. Identification of optimum SSF condition Through orthogonal experiments (Table 3), the theoretical optimum SSF condition was the following: 100 g cornmeal supplemented with 15 g soybean meal, a fermentation time of 25 days and a moisture content of 37.5%. Under the condition of orthogonal experiment 3 that was similar to the theoretical optimum SSF condition, the degradation rate of starch and the protein content in the product reached its maximum values of 67% and 17%, respectively (Table 3). The results indicated that the interaction of several factors achieved a better degree of starch degradation than any individual factor.
3.4. Effects of SSF on the main nutritional components content Under the condition of orthogonal experiment 3, the main nutritional components content of the fermentative product and the control are summarized in Table 4. The starch content of the nonfermented control reached 63%; the reducing sugar content was only 4%. However, the starch content of the fermented product decreased significantly ( P < 0.001) from 63% to 22%; while the reducing sugar content increased significantly ( P < 0.001) from 4% to 19%. SSF also produced a significant ( P < 0.01) increase from 12% to 17% in protein content. After SSF, the crude fat content decreased significantly ( P < 0.01).
Table 6 Comparison between the essential amino acid content of a fermented sample and the FAO/WHO reference pattern (mg/g protein) b
FAO/WHO Nonfermented Fermented a b
Ile
Leu
Lys
Met + Cys
Phe + Tyr
Thr
Trp
Val
Amino acid scoresa
40 42.35 46.60
70 110.80 106.72
55 35.96 56.12
35 38.33 34.60
60 80.57 76.65
40 32.12 42.71
10 9.05 13.25
50 45.90 56.12
65.4 98.8
The scores of first limiting amino acid. From Yang et al. (1992).
J. Han / International Journal of Food Microbiology 80 (2003) 61–66
65
Table 7 The essential amino acid contents (mg/g protein) and close degree of the appraised food protein
ValUi1 LeuUi2 IleUi3 ThrUi4 PheUi5 TrpUi6 MetUi7 LysUi8 CDe a b c d e
NFa
Fb
Wheat Flourc,d
Milletc
Soybeanc
Milkc
Lean porkc
Beefc
Chickenc
Hairtailc
U1
U2
U3
U4
U5
U6
U7
U8
U9
U10
45.90 110.80 42.35 32.12 50.51 9.05 18.35 35.96 0.8843
56.12 106.72 46.60 42.71 42.35 13.25 16.53 56.12 0.9339
47.16 70.46 36.97 28.35 47.16 12.39 12.84 25.69 0.8644
53.66 129.57 43.55 36.34 54.84 19.78 32.37 19.57 0.8968
49.31 80.55 52.95 40.99 52.67 13.00 10.99 63.92 0.9305
46.21 84.48 39.66 34.83 38.97 13.10 22.41 71.38 0.9350
51.71 83.46 45.41 45.61 43.80 13.17 20.68 74.93 0.9567
49.15 80.15 44.62 45.88 41.06 11.01 24.47 87.09 0.9442
43.75 70.80 42.05 38.70 37.70 11.70 23.30 73.70 0.9295
45.76 74.18 42.17 40.60 38.70 11.68 22.07 80.05 0.9308
Nonfermented control. Fermented product. The data are from CAPM (1992). Standard grade. Close degree.
3.5. Effects on constitution of amino acids in the protein Under the condition of orthogonal experiment 3, the quantity of the essential amino acids in the product increased from 346 to 380 mg per g protein and the ratio of essential amino acids to the total amino acids increased from 0.3864 to 0.4293 (Table 5). Among the essential amino acids, the contents of lysine and tryptophan increased significantly ( P < 0.05), the former from 36 to 56 mg per g protein, the latter from 9 to 13 mg per g protein. According to the standard essential amino acid balance model of FAO/WHO (Yang et al., 1992), the essential amino acids of proteins were analyzed by a chemical score method. The results are shown in Table 6. In the nonfermented control, the first limiting amino acid was lysine and its amino acid score was only 65.4. After SSF, only sulphur-containing amino acids were lower than the standard pattern, the other essential amino acids were higher than the standard pattern. This showed that after SSF of H. erinaceum, the shortage of lysine and trytophan in cornmeal protein was made up for and the protein quality had been improved greatly. Table 7 showed the close degree of the protein of 10 kinds of foods evaluated and the standard protein. The nutritional value of the 10 kinds of foods protein were placed in turn according to their close degree: lean pork > beef>milk>the fermented product>hair-
tail>soybean>chicken>millet>the nonfermented control>wheat flour. This showed that among the 10 kinds of foods, the nutritional value of the fermented product was superior to that of nonfermented control and other cereals, was close to that of soybean and chicken, but was inferior to that of milk and red meats.
4. Discussion After SSF of H. erinaceum, the starch content of corn meal was reduced greatly, and quite a proportion of the starch was converted into dextrin and reducing sugar. The digesting and absorbing ratio of corn meal was strikingly increased. This also demonstrated that H. erinaceum was able to produce high-active amylase during SSF on corn meal. High a-amylase activity was attributed to the close contact of hyphae of H. erinaceum with the substrate in SSF, which resulted in a high degradation rate of starch. Yang et al. (1992) had discussed the importance of a-amylase in LSF of Candida tropicalis. However, in their research, the a-amylase was not produced by C. tropicalis during LSF, but commercial amylase was added into the medium at the same time as inoculation with the yeast. In contrast, SSF is a process whereby an insoluble substrate is fermented with sufficient moisture but without free water. It requires no complex fermentation controls and has many advantages over LSF
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J. Han / International Journal of Food Microbiology 80 (2003) 61–66
(Chahal, 1983). First, SSF requires less energy input than LSF, making its application of potential interest. For example, the method is now being used for enzyme production and upgrading the values of existing foods, especially oriental foods (Chahal, 1983). Second, the products of SSF remain in the product, so increasing the nutritional state of corn meal, the substrate used here. This implied that corn meal or other cereals food could be processed into many kinds of special function food after SSF by some medical fungi. The importance of further studies on SSF of H. erinaceum in cornmeal or other cereals can readily be seen. Acknowledgements Support for this research by the Chinese National Science Fund (grants no. 30070021) and the Shanxi Province Science Foundation (no. 20001082) is gratefully acknowledged. References Bernfeld, P., 1955. Alpha-amylase. In: Colowick, S., Kaplan, N.O. (Eds.), Methods in Enzymology vol. 1. Academic Press, New York, pp. 149 – 152. CAPM (Chinese Academy of Preventive Medicine), 1992. Tables of Food Composition People Hygiene Press, Beijing.
Chahal, D.S., 1983. Protein production and growth characteristics of Chaetomium cellulolyticum during solid state fermentation of corn stover. Mycologia 75, 597 – 603. Fields, M.L., Shoshan, A.A., Poosiri, Y., 1988. Mixed culture fermentation to improve nutritional value of corn meal. Journal of Food Protection 51, 866 – 868. Huang, W.K., 1989. Examination and Analysis of Food. China Light Industry Press, Beijing. Jia, X.L., Yu, Y.Z., Liu, Z.H., 1997. A study on the optimum vegetative growth conditions of Hericium erinaceum. Acta Edulis Fungi 4, 33 – 39. Li, J.R., 1987. Food Chemistry. China Light Industry Press, Beijing. Liu, X.W., Lu, Y.H., Xu, X.Q., 1992. A study on preparation of sandwich biscuits with hedgehog fungus and therapeutic approach for nutritional anemia of preschool children. Acta Nutrimenta Sinica 14, 109 – 111. Ma, G.R., Chen, M.C., 1994. Studies on the nutrient composition of common edible mushrooms. Acta Nutrimenta Sinica 16, 344 – 347. Raper, J.R., Miles, P.G., 1958. The genetics of Schizophyllum commune. Genetics 43, 530 – 546. Ray, A.A., 1985. SAS Users Guide: Statistics SAS, Cary, NC. Wu, W.M., Chen, Y.Y., 1985. Applied Fuzzy Set Methods. Beijing Teachers University Press, Beijing. Yang, X.M., 1988. The Cultivation of Edible Mushrooms in China. Agriculture Press, Beijing. Yang, J.X., Tao, S.X., Song, T.S., 1992. Improvement of nutritional value in enzymatically hydrolyzed corn meal by Candida tropicalis. Acta Nutrimenta Sinica 14, 182 – 185. Zhang, Y.F., Zhang, F.Y., Bai, Z.M., 1996. Appraisal on the protein quality of Pleurotus ostreatus growing in vinegar waste. Acta Nutrimenta Sinica 18, 155 – 158.
Solid-state fermentation of cornmeal with the basidiomycete Hericium erinaceum for degrading starch and upgrading nutritional value Jianrong Han* Department of Life Science, Shanxi University, Taiyuan 030006, People’s Republic of China Received 8 June 2001; received in revised form 4 February 2002; accepted 5 March 2002
Abstract The ability of the basidiomycete Hericium erinaceum to degrade starch and upgrade nutritional value of cornmeal during solid-state fermentation was studied. On the basal medium which consisted of cornmeal and salt solution, H. erinaceum produced a strong a-amylase on the 15th day after inoculation, which resulted in a 52% degradation of the starch. By supplementation with 5 – 15 g soybean meal per 100 g cornmeal the a-amylase activity and degradation rate of starch was raised significantly ( P < 0.01). Prolongation of fermentation time from 15 to 30 days did not increase significantly the degradation rate of starch, though the a-amylase activity reached its maximum value of 179 U/g on the 20th day after inoculation. Under conditions close to the theoretical optimum fermentation conditions, that was after 25 days at 25 jC in the medium with added 15 g soybean meal per 100 g cornmeal, the starch content in the product decreased from 63% to 22% ( P < 0.001) and protein content increased from 12% to 17% ( P < 0.01). In the protein in the product, the lysine content was increased from 36 to 56 mg/ g and tryptophan from 9 to 13 mg/g. Using egg protein as a standard, an evaluation on the protein quality of the fermented product showed that it was superior to that of the nonfermented control and to other cereals, was close to that of soybean and chicken, but was inferior to that of milk and red meats. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Cornmeal; Solid-state fermentation; Hericium erinaceum; Starch; Protein
1. Introduction Hericium erinaceum is well known as a traditional and valuable mushroom in China. Recently, it has been paid much attention regarding some health effects when used as a home remedy. It has been reported that a preparation from either the fruitbodies or the mycelium of H. erinaceum worked effectively to cure gastric *
Fax: +86-351-7011981. E-mail address: [email protected] (J. Han).
ulcer (Yang, 1988). The lysine and tryptophan contents of the protein are very high (Ma and Chen, 1994). A biscuit product supplemented with the fruitbodies of H. erinaceum has been used in prevention or cure of nutritional anemia of preschool children (Liu et al., 1992). Consequently, H. erinaceum is cultivated widely in China, and mycelium is being produced by submerged fermentation. However, studies on solidstate fermentation (SSF) of H. erinaceum on amylaceous substrates, such as cornmeal and potato meal, have not been reported.
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 2 ) 0 0 1 2 2 - 8
62
J. Han / International Journal of Food Microbiology 80 (2003) 61–66
Cornmeal shows a nutritional deficiency because of lack of lysine and tryptophan in the protein (Li, 1987). Methods have been reported to improve the nutritional value of cornmeal including liquid state fermentation (LSF) of Bacillus spp. or yeasts (Fields et al., 1988; Yang et al., 1992), but there have been no reports about the use of SSF of edible or medicinal basidiomycetes to improve the nutritional value of cornmeal. Because H. erinaceum has the special metabolic ability to synthesize lysine and tryptophan, SSF on a solid medium containing cornmeal may be able to reduce the shortage of lysine and trytophan in cornmeal protein. We describe here a series of experiments on the SSF of H. erinaceum in cornmeal medium and analyze the a-amylase activity and main nutritional component differences between the fermented product and nonfermented control. By means of the fuzzy discernment method (Wu and Chen, 1985), the nutritional value of the fermented product was analyzed and evaluated.
cornmeal. Then the solid medium was dispensed into Erlenmeyer flasks (250 ml capacity). After sterilization, each flask was inoculated with five discs of mycelium and agar, 6 mm in diameter, agar plate cultures of the fungus on complete medium (Raper and Miles, 1958). Three replicates were prepared for each treatment, and an uninoculated flask served as control. All of the flasks were incubated at 25jC in the dark. The entire contents of flasks were harvested at 15, 20, 25, 30 days for the assay of a-amylase activity. Then the samples were dried to constant weight at 70jC and used for the assay of the main nutritional components. 2.3. Enzyme extraction and assay
The strain H 18 of Hericium erinaceum was obtained from the Edible Fungi Research Institute, Shanghai Academy of Agricultural Science, China and routinely maintained on the complete medium agar slant as outlined previously by Raper and Miles (1958).
One gram of the culture of SSF from each flask was mixed with 8 ml of deionized water and acidwashed sand in an ice-cold mortar. The homogenate was centrifuged at 2000g for 5 min. The supernatant was assayed for a-amylase activity within 12 h of preparation. Amylase (a-1, 4-glucan-4-glucanohydrolase, EC 3.2.1.1) was assayed with the substrate 1% soluble starch in 20 mM phosphate buffer (pH 6.9). A mixture of 0.5 ml of extract and 0.5 ml of 20 mM phosphate buffer (pH 6.9) containing 1% soluble starch was incubated at 25jC 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 aamylase unit (U) was defined as the amount of enzyme producing 1 Amol of maltose per min at 25jC on soluble starch.
2.2. Solid-state fermentation
2.4. Main nutritional components assay
The basal medium for SSF consisted of 100 g of dry cornmeal, which were ground to 20-mesh powder, moistened with 67 ml of nutrient salt solution which contained the following per liter of distilled water (g): KH 2 PO 4 1, MgSO 4 7H 2 O 1, FeSO 4 7H 2 O 0.5, CaCl2H 2O 0.1 (a modified formula previously described by Jia et al., 1997). The moisture content at this stage was about 40% (w/w). The initial pH of the salt solution was adjusted to 6.5. The dry soybean meal added to the basal medium as a supplementary nitrogen source. Supplements of soybean meal were added at the rate of 5, 10 and 15 g per 100 g
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). An amino acid analyser (Hitachi Model 83550) was used for amino acid and tryptophan determination by fluorescence analysis (Huang, 1989). The starch content was determined with the multi-starch plant sample assay method described by Huang
2. Materials and methods 2.1. Fungus
J. Han / International Journal of Food Microbiology 80 (2003) 61–66 Table 1 Effect of supplementary soybean meal on starch degradation by H. erinaceum Soybean meal quality (g/100 g cornmeal)
a-amylase activity (U/g of culture)a
Degradation rate of starch (%)b
0 5 10 15
44.0Ac 79.5B 121.8C 152.3D
52.3A 56.4A 59.5AB 61.7B
a
One a-amylase unit (U) is defined as the amount of enzyme producing 1 Amol of maltose per min at 25jC on soluble starch. b Degradation rate of starch=(starch content of fermented sample minus the starch content of control)/(starch content of control) 100. c Means in the same column followed by the same letter are not significantly different at the P < 0.01 level according to Duncan’s multiple range test.
(1989). The starch degradation rate was calculated as follows: Starch degradation rate ð%Þ ¼ ðstarch content of fermented sample minus the starch content of the controlÞ=starch content of control 100
Duncan’s multiple range test (Ray, 1985) was used on the isolation treatment means to test for significant differences at the 1% level of confidence. 2.5. Evaluation on the nutritional value of protein Ten kinds of foods including the fermented product and control were evaluated for the nutritional value of the protein using egg protein as the standard, by means of fuzzy discernment (Wu and Chen, 1985). If the evaluated object is Ui and the egg protein is a, then out of the Lan’s distance method (Wu and
Table 2 Effect of fermentation time on starch degradation by H. erinaceum
63
Table 3 Result of orthogonal experiment to assess optimal conditions for solid state fermentation by H. erinaceum Experimental Soybean Fermentation Moisture Degradation Protein no. meal time content rate of content (g)a (days) (%) starch (%) (%) 1 2 3 4 5 6 7 8 9
15 15 15 10 10 10 5 5 5 a
15 20 25 15 20 25 15 20 25
35 37.5 40 37.5 40 35 40 35 37.5
62.1 65.2 66.5 59.6 60.6 61.4 57.0 59.0 60.3
15.0 16.4 16.8 13.8 14.2 14.9 12.6 12.7 13.4
Supplementary quantity of soybean meal in100 g cornmeal.
Chen, 1985), the close degree is defined between the object Ui and the sample plate a, that is 8 X Aak Uik A lða; Ui Þ ¼ 1 C ð1Þ ak þ Uik k¼1 where Uik is the essential amino acid content of the evaluated protein and C is a properly selected constant (here C = 0.09) (Zhang et al., 1996), ak is the essential amino acid content of the egg protein (mg/g protein)(CAPM, 1992), and where a1, a2,. . . a8 are, respectively, Val (54.19), Leu (81.09), Ile (48.76), Thr (44.73), Phe (48.22), Trp (17.21), Met (28.14) and Lys (65.89). According to Eq. (1), the close degree of each evaluated object was calculated out, and the bigger the close degree, the higher the nutritional value.
3. Results 3.1. Effect of supplementary soybean meal Due to the nitrogen deficiency in cornmeal, addition of nitrogen to the basal medium was required for SSF of Table 4 Main nutritional components of fermented product of H. erinaceum and a nonfermented control on a dry basis (X F S, n = 3)
Fermentation time (days)
a-amylase activity (U/g of culture)
Degradation rate of starch (%)
Component (g/100 g)
Nonfermented
Fermented
P
15 20 25 30
152.3B 178.6C 164.2BC 130.9A
61.7A 64.6A 65.7A 66.4A
Protein Crude fat Starch Reducing sugar
11.6 F 0.6 10.6 F 0.5 62.8 F 1.3 4.0 F 0.2
16.8 F 0.7 9.7 F 0.4 21.9 F 0.6 19.4 F 0.6
< 0.01 < 0.01 < 0.001 < 0.001
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J. Han / International Journal of Food Microbiology 80 (2003) 61–66
Table 5 Amino acids composition of the protein (mg/g protein) produced by solid state fermentation by H. erinaceum Amino acids
Nonfermented
Fermented
Val Leu Ile Thr Phe Trp Met Lys Tyr Cys Asp Pro Ser Glu Gly Ala Arg His EAAa Totalb E/Tc
45.90 110.80 42.35 32.12 50.51 9.05 18.35 35.96 31.06 18.98 77.80 53.76 35.30 162.23 30.69 63.70 50.29 26.53 346.04 895.38 0.3864
56.12 106.72 46.60 42.71 42.35 13.25 16.53 56.12 34.30 18.07 73.41 50.49 32.19 157.42 29.52 57.60 52.05 23.96 380.40 909.41 0.4293
a b c
Essential amino acids. Total quantity of amino acids. Ratio of essential amino acids in total amino acids.
H. erinaceum. The results (Table 1) showed that during SSF of 15 days the a-amylase activity of H. erinaceum had a positive correlation with the supplementary quantity of soybean meal (r = 0.9983). The higher the supplementary quantity was, the higher the a-amylase activity. Consequently, the starch degradation rate was increased. When 15 g soybean meal was added to 100 g cornmeal, the degradation rate of starch in the fermentative product increased from 52% to 62% ( P < 0.01). 3.2. Effect of fermentation time The a-amylase activity and starch content of every sample was determined after 15, 20, 25 and 30 days.
The results (Table 2) showed that prolongation of fermentation time from 15 to 30 days did not increase significantly the degradation rate of starch, though the a-amylase activity reached its maximum value of 179 U/g on the 20th day after inoculation. After the 20th day, the amylase activity decreased, but the degradation rate of starch continued to increase. 3.3. Identification of optimum SSF condition Through orthogonal experiments (Table 3), the theoretical optimum SSF condition was the following: 100 g cornmeal supplemented with 15 g soybean meal, a fermentation time of 25 days and a moisture content of 37.5%. Under the condition of orthogonal experiment 3 that was similar to the theoretical optimum SSF condition, the degradation rate of starch and the protein content in the product reached its maximum values of 67% and 17%, respectively (Table 3). The results indicated that the interaction of several factors achieved a better degree of starch degradation than any individual factor.
3.4. Effects of SSF on the main nutritional components content Under the condition of orthogonal experiment 3, the main nutritional components content of the fermentative product and the control are summarized in Table 4. The starch content of the nonfermented control reached 63%; the reducing sugar content was only 4%. However, the starch content of the fermented product decreased significantly ( P < 0.001) from 63% to 22%; while the reducing sugar content increased significantly ( P < 0.001) from 4% to 19%. SSF also produced a significant ( P < 0.01) increase from 12% to 17% in protein content. After SSF, the crude fat content decreased significantly ( P < 0.01).
Table 6 Comparison between the essential amino acid content of a fermented sample and the FAO/WHO reference pattern (mg/g protein) b
FAO/WHO Nonfermented Fermented a b
Ile
Leu
Lys
Met + Cys
Phe + Tyr
Thr
Trp
Val
Amino acid scoresa
40 42.35 46.60
70 110.80 106.72
55 35.96 56.12
35 38.33 34.60
60 80.57 76.65
40 32.12 42.71
10 9.05 13.25
50 45.90 56.12
65.4 98.8
The scores of first limiting amino acid. From Yang et al. (1992).
J. Han / International Journal of Food Microbiology 80 (2003) 61–66
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Table 7 The essential amino acid contents (mg/g protein) and close degree of the appraised food protein
ValUi1 LeuUi2 IleUi3 ThrUi4 PheUi5 TrpUi6 MetUi7 LysUi8 CDe a b c d e
NFa
Fb
Wheat Flourc,d
Milletc
Soybeanc
Milkc
Lean porkc
Beefc
Chickenc
Hairtailc
U1
U2
U3
U4
U5
U6
U7
U8
U9
U10
45.90 110.80 42.35 32.12 50.51 9.05 18.35 35.96 0.8843
56.12 106.72 46.60 42.71 42.35 13.25 16.53 56.12 0.9339
47.16 70.46 36.97 28.35 47.16 12.39 12.84 25.69 0.8644
53.66 129.57 43.55 36.34 54.84 19.78 32.37 19.57 0.8968
49.31 80.55 52.95 40.99 52.67 13.00 10.99 63.92 0.9305
46.21 84.48 39.66 34.83 38.97 13.10 22.41 71.38 0.9350
51.71 83.46 45.41 45.61 43.80 13.17 20.68 74.93 0.9567
49.15 80.15 44.62 45.88 41.06 11.01 24.47 87.09 0.9442
43.75 70.80 42.05 38.70 37.70 11.70 23.30 73.70 0.9295
45.76 74.18 42.17 40.60 38.70 11.68 22.07 80.05 0.9308
Nonfermented control. Fermented product. The data are from CAPM (1992). Standard grade. Close degree.
3.5. Effects on constitution of amino acids in the protein Under the condition of orthogonal experiment 3, the quantity of the essential amino acids in the product increased from 346 to 380 mg per g protein and the ratio of essential amino acids to the total amino acids increased from 0.3864 to 0.4293 (Table 5). Among the essential amino acids, the contents of lysine and tryptophan increased significantly ( P < 0.05), the former from 36 to 56 mg per g protein, the latter from 9 to 13 mg per g protein. According to the standard essential amino acid balance model of FAO/WHO (Yang et al., 1992), the essential amino acids of proteins were analyzed by a chemical score method. The results are shown in Table 6. In the nonfermented control, the first limiting amino acid was lysine and its amino acid score was only 65.4. After SSF, only sulphur-containing amino acids were lower than the standard pattern, the other essential amino acids were higher than the standard pattern. This showed that after SSF of H. erinaceum, the shortage of lysine and trytophan in cornmeal protein was made up for and the protein quality had been improved greatly. Table 7 showed the close degree of the protein of 10 kinds of foods evaluated and the standard protein. The nutritional value of the 10 kinds of foods protein were placed in turn according to their close degree: lean pork > beef>milk>the fermented product>hair-
tail>soybean>chicken>millet>the nonfermented control>wheat flour. This showed that among the 10 kinds of foods, the nutritional value of the fermented product was superior to that of nonfermented control and other cereals, was close to that of soybean and chicken, but was inferior to that of milk and red meats.
4. Discussion After SSF of H. erinaceum, the starch content of corn meal was reduced greatly, and quite a proportion of the starch was converted into dextrin and reducing sugar. The digesting and absorbing ratio of corn meal was strikingly increased. This also demonstrated that H. erinaceum was able to produce high-active amylase during SSF on corn meal. High a-amylase activity was attributed to the close contact of hyphae of H. erinaceum with the substrate in SSF, which resulted in a high degradation rate of starch. Yang et al. (1992) had discussed the importance of a-amylase in LSF of Candida tropicalis. However, in their research, the a-amylase was not produced by C. tropicalis during LSF, but commercial amylase was added into the medium at the same time as inoculation with the yeast. In contrast, SSF is a process whereby an insoluble substrate is fermented with sufficient moisture but without free water. It requires no complex fermentation controls and has many advantages over LSF
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(Chahal, 1983). First, SSF requires less energy input than LSF, making its application of potential interest. For example, the method is now being used for enzyme production and upgrading the values of existing foods, especially oriental foods (Chahal, 1983). Second, the products of SSF remain in the product, so increasing the nutritional state of corn meal, the substrate used here. This implied that corn meal or other cereals food could be processed into many kinds of special function food after SSF by some medical fungi. The importance of further studies on SSF of H. erinaceum in cornmeal or other cereals can readily be seen. Acknowledgements Support for this research by the Chinese National Science Fund (grants no. 30070021) and the Shanxi Province Science Foundation (no. 20001082) is gratefully acknowledged. References Bernfeld, P., 1955. Alpha-amylase. In: Colowick, S., Kaplan, N.O. (Eds.), Methods in Enzymology vol. 1. Academic Press, New York, pp. 149 – 152. CAPM (Chinese Academy of Preventive Medicine), 1992. Tables of Food Composition People Hygiene Press, Beijing.
Chahal, D.S., 1983. Protein production and growth characteristics of Chaetomium cellulolyticum during solid state fermentation of corn stover. Mycologia 75, 597 – 603. Fields, M.L., Shoshan, A.A., Poosiri, Y., 1988. Mixed culture fermentation to improve nutritional value of corn meal. Journal of Food Protection 51, 866 – 868. Huang, W.K., 1989. Examination and Analysis of Food. China Light Industry Press, Beijing. Jia, X.L., Yu, Y.Z., Liu, Z.H., 1997. A study on the optimum vegetative growth conditions of Hericium erinaceum. Acta Edulis Fungi 4, 33 – 39. Li, J.R., 1987. Food Chemistry. China Light Industry Press, Beijing. Liu, X.W., Lu, Y.H., Xu, X.Q., 1992. A study on preparation of sandwich biscuits with hedgehog fungus and therapeutic approach for nutritional anemia of preschool children. Acta Nutrimenta Sinica 14, 109 – 111. Ma, G.R., Chen, M.C., 1994. Studies on the nutrient composition of common edible mushrooms. Acta Nutrimenta Sinica 16, 344 – 347. Raper, J.R., Miles, P.G., 1958. The genetics of Schizophyllum commune. Genetics 43, 530 – 546. Ray, A.A., 1985. SAS Users Guide: Statistics SAS, Cary, NC. Wu, W.M., Chen, Y.Y., 1985. Applied Fuzzy Set Methods. Beijing Teachers University Press, Beijing. Yang, X.M., 1988. The Cultivation of Edible Mushrooms in China. Agriculture Press, Beijing. Yang, J.X., Tao, S.X., Song, T.S., 1992. Improvement of nutritional value in enzymatically hydrolyzed corn meal by Candida tropicalis. Acta Nutrimenta Sinica 14, 182 – 185. Zhang, Y.F., Zhang, F.Y., Bai, Z.M., 1996. Appraisal on the protein quality of Pleurotus ostreatus growing in vinegar waste. Acta Nutrimenta Sinica 18, 155 – 158.