Abstract

Cordyceps militaris and Lentinula edodes are known for their high extracellular proteolytic, amylolytic, and laccase activity, which is important for bakery production. This article describes the effect of enzymes obtained from mycelial biomass of C. militaris and L. edodes on such properties of flour mixes as amylolytic and proteolytic enzymic activity during baking, technological costs, and bread quality.
The research featured strains of C. militaris SRG4 and L. edodes 3790 fungi; mycelial biomass powder of these fungi on a sterile grain substrate (rice, wheat); experimental flour mixes of wheat bread and first-grade baking flour with mycelial biomass powder; bread made from the experimental flour mixes. The experimental part included standard methods used in the bakery industry.
The mycelial biomass of C. militaris and L. edodes fungi proved to be a promising baking additive because it possessed hydrolytic enzymes and was rich in protein substances (32.2 ± 1.5 and 26.4 ± 2.0%, respectively), specific mushroom polysaccharides (36.7 ± 0.8 and 52.2 ± 1.2%, respectively), and carotenoids (1600 ± 40 mcg/g of C. militaris biomass). The active amylases and proteinases in the mycelial biomass powder improved the sugar-forming ability and reduced the falling number. In addition, they raised the amount of washed gluten with a slight decrease in elasticity, as well as provided an acceptable structure and viscosity of the dough at 1–4% of mycelial biomass powder. This dose resulted in an optimal bread formulation of standard quality. A bigger amount caused the crumb to darken and increased its humidity while reducing the specific volume. The bread samples with C. militaris were too acid.
Mycelial biomass of C. militaris and L. edodes proved to be a good source of enzymes to be used with grain substrates in bakery production. Further research is needed to define the optimal dose and processing modes.

Keywords

Biotechnologies, ascomycetes, basidiomycetes, Cordyceps militaris, Lentinula edodes, enzyme activity, bread, dough, sugar-forming ability, gluten, quality

REFERENCES

1. Arshadi N, Nouri H, Moghimi H. Increasing the production of the bioactive compounds in medicinal mushrooms: An omics perspective. Microbial Cell Factories. 2023;22:11. https://doi.org/10.1186/s12934-022-02013-x
2. Turlo J. The biotechnology of higher fungi – current state and perspectives. Acta Universitatis Lodziensis. Folia Biologica et Oecologica. 2014;10:49–65. https://doi.org/10.2478/fobio-2014-0010
3. Mayolo-Deloisa K, González-González M, Rito-Palomares M. Laccases in food industry: Bioprocessing, potential industrial and biotechnological applications. Frontiers in Bioengineering and Biotechnology. 2020;8:222. https://doi.org/10.3389/fbioe.2020.00222
4. Berger RG, Ersoy F. Improved foods using enzymes from basidiomycetes. Processes. 2022;10(4):726. https://doi.org/10.3390/pr10040726
5. Gannochka E, Kolesnikov B, Salamahina A, Shamtsyan M. Technology of obtaining milk-clotting enzyme from fungal culture Funalia sp. for application in cheese production. 13th Baltic Conference on Food Science and Technology “FOOD. NUTRITION. WELL-BEING”; 2019; Jelgava. Jelgava: LLU, Faculty of Food Technology; 2019. p. 247–249. https://doi.org/10.22616/FoodBalt.2019.036
6. Krupodorova T, Ivanova T, Barshteyn V. Screening of extracellular enzymatic activity of macrofungi. Journal of Microbiology, Biotechnology and Food Sciences. 2014;3(4):315–318.
7. Kumar Chandrawanshi N, Koreti D, Kosre A, Kumar A. Proteolytic enzymes derived from a macro fungus and their industrial application. In: Haider S, Haider A, Catalá A, Surguchov A, editors. Hydrolases. IntechOpen; 2022. https://doi.org/10.5772/intechopen.102385
8. Wang Q, Cao R, Zhang Y, Qi P, Wang L, Fang S. Biosynthesis and regulation of terpenoids from basidiomycetes: Exploration of new research. AMB Express. 2021;11:150. https://doi.org/10.1186/s13568-021-01304-7
9. Minakov DV, Sevodina KV, Shadrintseva AI, Sevodin VP. Influence of vitamins on growth and development of mycelium of some basidiomycetes in liquid medium. Food Processing: Techniques and Technology. 2016;43(4):43–49. (In Russ.). https://elibrary.ru/XELEIT
10. Mata G, Salmones D, Pérez-Merlo R. Hydrolytic enzyme activities in shiitake mushroom (Lentinula edodes) strains cultivated on coffee pulp. Revista Argentina de Microbiología. 2016;48(3):191–195. https://doi.org/10.1016/j.ram.2016.05.008
11. Lin Q, Long L, Wu L, Zhang F, Wu S, Zhang W, et al. Evaluation of different agricultural wastes for the production of fruiting bodies and bioactive compounds by medicinal mushroom Cordyceps militaris. Journal of the Science of Food and Agriculture. 2016;97(10):3476–3480. https://doi.org/10.1002/jsfa.8097
12. Usacheva RV. Physiological and biochemical characteristics of some strains of cultivated fungus Lentinus edodes (Berk. Sing.): Cand. sci. biol. dis. Voronezh: K.D. Glinka Voronezh State Agrarian University; 2003. 126 p. (In Russ.).
13. Elkhateeb WA, El-Ghwas DE, Daba GM. Mushrooms as efficient enzymatic machinery. Journal of Biomedical Research. 2022;3(4):423–428. https://doi.org/10.37871/jbres1460
14. Li W, Chen W-C, Wang J-B, Feng J, Wu D, Zhang Z, et al. Effects of enzymatic reaction on the generation of key aroma volatiles in shiitake mushroom at different cultivation substrates. Food Science and Nutrition. 2021;9(4):2247–2256. https://doi.org/10.1002/fsn3.2198
15. Kobayashi N, Wada N, Yokoyama H, Tanaka Y, Suzuki T, Habu N, et al. Extracellular enzymes secreted in the mycelial block of Lentinula edodes during hyphal growth. AMB Express. 2023;13:36. https://doi.org/10.1186/s13568-023-01547-6
16. Baktemur G, Kara E, Yarar M, Yilmaz N, Ağcam E, Akyildiz A, et al. Yield, quality and enzyme activity of shiitake mushroom (Lentinula edodes) grown on different agricultural wastes. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 2022;50(1):12553. https://doi.org/10.15835/nbha50112553
17. Sousa MAC, Costa LMAS, Pereira TS, Zied DC, Rinker DL, Dias ES. Enzyme activity and biochemical changes during production of Lentinula edodes (Berk.) Pegler. Food Science and Technology. 2019;39(3):774–780. https://doi.org/10.1590/fst.38517
18. Semenova TA, Belozersky MA, Belakova GA, Borisov BA, Semenova SA, Dunaevsky YaE. Secreted proteinase of entomopathogenic fungus Cordyceps militaris. I. Development of cultivation medium and of purification protocol. Mycology and Phytopathology. 2010;44(6):535–541. (In Russ.). https://elibrary.ru/OITGBB
19. Semenova TA. Extracellular peptidases of fungi that form biotic connections with insects: Cand. sci. biol. dis. Moscow: Lomonosov Moscow State University; 2011. 130 p.
20. Shrestha B, Zhang W, Zhang Y, Liu X. The medicinal fungus Cordyceps militaris: Research and development. Mycological Progress. 2012;11:599–614. https://doi.org/10.1007/s11557-012-0825-y
21. Drozłowska-Sobieraj E. The use of enzymatic fungal activity in the food industry – Review. World Scientific News. 2019;116:222–229.
22. Frioui M, Gaceu L, Oprea O, Shamtsyan MM. The influence of fungal extract containing beta beta-glucans on the rheological characteristics of dough. Journal of International Academy of Refrigeration. 2018;(3):53–61. (In Russ.). https://doi.org/10.17586/1606-4313-2018-17-3-53-61
23. Chiozzi V, Eliopoulos C, Markou G, Arapoglou D, Agriopoulou S, El Enshasy HA, et al. Biotechnological addition of β-glucans from cereals, mushrooms and yeasts in foods and animal feed. Processes. 2021;9(11):1889. https://doi.org/10.3390/pr9111889
24. Nie Y, Zhang P, Deng C, Xu L, Yu M, Yang W, et al. Effects of Pleurotus eryngii (mushroom) powder and soluble polysaccharide addition on the rheological and microstructural properties of dough. Food Science and Nutrition. 2019;7(6):2113–2122. https://doi.org/10.1002/fsn3.1054
25. Kozubaeva LA, Kuzmina SS, Egorova EYu. Prospects for the use of dried Boletus edulis mushroom in the development of functional bakery products. News of KSTU named after I. Razzakov. 2021;60(4):189–195. (In Kyrgyz.). https://elibrary.ru/WSNCZL
26. Minakov DV, Kozubaeva LA, Kuzmina SS, Egorova EYu. Features of dough maturation and bread quality formation with Armillaria mellea mycelium biomass. Storage and Processing of Farm Products. 2022;(1):145–156. (In Russ.). https://doi.org/10.36107/spfp.2022.297
27. Yuan B, Zhao L, Yang W, McClements DJ, Hu Q. Enrichment of bread with nutraceutical-rich mushrooms: Impact of Auricularia auricula (mushroom) flour upon quality attributes of wheat dough and bread. Journal of Food Science. 2017;82(9):2041–2050. https://doi.org/10.1111/1750-3841.13812
28. Zhang Y, Ruan C, Cheng Z, Zhou Y, Liang J. Mixolab behavior, quality attributes and antioxidant capacity of breads incorporated with Agaricus bisporus. Journal of Food Science and Technology. 2019;56:3921–3929. https://doi.org/10.1007/s13197-019-03859-7
29. Nikolić NC, Krasić MS, Šimurina O, Cakić S, Mitrović J, Pešić M, et al. Regression analysis in examination the rheology properties of dough from wheat and Boletus edulis flour. Journal of Food Composition and Analysis. 2022;115:105022. https://doi.org/10.1016/j.jfca.2022.105022
30. Ulziijargal E, Yang J-H, Lin L-Y, Chen C-P, Mau J-L. Quality of bread supplemented with mushroom mycelia. Food Chemistry. 2013;138(1):70–76. https://doi.org/10.1016/j.foodchem.2012.10.051
31. Salehi F. Characterization of different mushrooms powder and its application in bakery products: A review. International Journal of Food Properties. 2019;22(1):1375–1385. https://doi.org/10.1080/10942912.2019.1650765
32. Vlaic RA, Mureșan CC, Muste S, Muresan V, Pop A. Boletus edulis mushroom flour-based wheat bread as innovative fortified bakery product. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Food Science and Technology. 2019;76(1):52–62. https://doi.org/10.15835/buasvmcn-fst:2018.0022
33. Sławinska A, Sołowiej BG, Radzki W, Fornal E. Wheat bread supplemented with Agaricus bisporus powder: Effect on bioactive substances content and technological quality. Foods. 2022;11(23):3786. https://doi.org/10.3390/foods11233786
34. Bentil JA. Biocatalytic potential of basidiomycetes: Relevance, challenges and research interventions in industrial processes. Scientific African. 2021;11:e00717. https://doi.org/10.1016/j.sciaf.2021.e00717
35. Chen C, Han Y, Li S, Wang R, Tao C. Nutritional, antioxidant, and quality characteristics of novel cookies enriched with mushroom (Cordyceps militaris) flour. CyTA – Journal of Food. 2021;19(1):137–145. https://doi.org/10.1080/19476337.2020.1864021
36. Jiaojiao Z, Fen W, Kuanbo L, Qing L, Ying Y, Caihong D. Heat and light stresses affect metabolite production in the fruit body of the medicinal mushroom Cordyceps militaris. Applied Microbiology and Biotechnology. 2018;102:4523–4533. https://doi.org/10.1007/s00253-018-8899-3
37. State standard bakery formulations. Moscow; 1998. 87 p. (In Russ.).
38. Chan JSL, Barseghyan GS, Asatiani MD, Wasser SP. Chemical composition and medicinal value of fruiting bodies and submerged cultured mycelia of caterpillar medicinal fungus Cordyceps militaris CBS-132098 (ascomycetes). International Journal of Medicinal Mushrooms. 2015;17(7):649–659. https://doi.org/10.1615/IntJMedMushrooms.v17.i7.50
39. Chaipoot S, Wiriyacharee P, Phongphisutthinant R, Buadoktoom S, Srisuwun A, Somjai C, et al. Changes in physicochemical characteristics and antioxidant activities of dried shiitake mushroom in dry-moist-heat aging process. Foods. 2023;12(14):2714. https://doi.org/10.3390/foods12142714
40. Wei X, Su Y, Hu H, Li X, Xu R, Liu Y. Quantification of aromatic amino acids in Cordyceps fungi by micellar electrokinetic capillary chromatography. Wuhan University Journal of Natural Sciences. 2019;24:245–250. https://doi.org/10.1007/s11859-019-1393-7
41. Turk A, Kim BS, Ko SM, Yeon SW, Ryu SH, Kim YG, et al. Optimization of cultivation and extraction conditions of pupae-Cordyceps for cordycepin production. Natural Product Sciences. 2021;27(3):187–192. https://doi.org/10.20307/nps.2021.27.3.187
42. Tao S-X, Xue D, Lu Z-H, Huang H-L. Effects of substrates on the production of fruiting bodies and the bioactive components by different Cordyceps militaris strains (ascomycetes). International Journal of Medicinal Mushrooms. 2020;22(1):55–63. https://doi.org/10.1615/IntJMedMushrooms.2019033257
43. Turk A, Abdelhamid MAA, Yeon SW, Ryu SH, Lee S, Ko SM, et al. Cordyceps mushroom with increased cordycepin content by the cultivation on edible insects. Frontiers in Microbiology. 2022;13:1017576. https://doi.org/10.3389/fmicb.2022.1017576
44. Turk A, Kim MH, Jeong SY, Kim BS, Woo S-I, Lee MK. Quality and composition of eggs laid by hens fed with Cordyceps militaris-supplemented feed. Journal of Mushrooms. 2022;20(4):254–257. https://doi.org/10.14480/JM.2022.20.4.254
45. Elkhateeb WA, Daba G. Review: The endless nutritional and pharmaceutical benefits of the Himalayan gold, Cordyceps; Current knowledge and prospective potentials. Asian Journal of Natural Product Biochemistry. 2020;18(2):74–81. https://doi.org/10.13057/biofar/f180204
46. Ashraf SA, Elkhalifa AEO, Siddiqui AJ, Patel M, Awadelkareem AM, Mejdi S, et al. Cordycepin for health and wellbeing: A potent bioactive metabolite of an entomopathogenic medicinal fungus Cordyceps with its nutraceutical and therapeutic potential. Molecules. 2020;25(12):2735. https://doi.org/10.3390/molecules25122735
47. Holbein S, Freimoser FM, Werner TP, Wengi A, Dichtl B. Cordycepin-hypersensitive growth links elevated polyphosphate levels to inhibition of poly(A) polymerase in Saccharomyces cerevisiae. Nucleic Acids Research. 2008;36(2):353–363. https://doi.org/10.1093/nar/gkm990
48. Yu C-X, Zhang Y-R, Ren Y-F, Zhao Y, Song X-X, Yang H-L, et al. Composition and contents of fatty acids and amino acids in the mycelia of Lentinula edodes. Food Science and Nutrition. 2023;11(7):4038–4046. https://doi.org/10.1002/fsn3.3392
49. Nallathamby N, Malek SNA, Vidyadaran S, Phan CW, Sabaratnam V. Lipids in an ethyl acetate fraction of caterpillar medicinal mushroom, Cordyceps militaris (ascomycetes), reduce nitric oxide production in BV2 cells via NRF2 and NF-κB pathways. International Journal of Medicinal Mushrooms. 2020;22(12):1215–1223. https://doi.org/10.1615/IntJMedMushrooms.2020037001
50. Lan L, Wang S, Duan, S, Zhou X, Li Y. Cordyceps militaris carotenoids protect human retinal endothelial cells against the oxidative injury and apoptosis resulting from H2O2. Evidence-Based Complementary and Alternative Medicine. 2022;2022:1259093. https://doi.org/10.1155/2022/1259093
51. Zheng Q, Wei T, Lin Y, Ye Z-W, Lin J-F, Guo L-Q, et al. Developing a novel two-stage process for carotenoid production by Cordyceps militaris (ascomycetes). International Journal of Medicinal Mushrooms. 2019;21(1):47–57. https://doi.org/10.1615/IntJMedMushrooms.2018029002
52. Yang Y, Bu N, Wang S, Zhang J, Wang Y, Dong C. Carotenoid production by caterpillar medicinal mushrooms, Cordyceps militaris (ascomycetes), under different culture conditions. International Journal of Medicinal Mushrooms. 2020;22(12):1191–1201. https://doi.org/10.1615/IntJMedMushrooms.2020036685
53. Zhao Y, Li S-L, Chen H-Y, Zou Y, Zheng Q-W, Guo L-Q, et al. Enhancement of carotenoid production and its regulation in edible mushroom Cordyceps militaris by abiotic stresses. Enzyme and Microbial Technology. 2021;148:109808. https://doi.org/10.1016/j.enzmictec.2021.109808
54. Lin P-J, Ye Z-W, Wei T, Wu J-Y, Zheng Q-W, Chen B-X, et al. Cross breeding of novel Cordyceps militaris strains with high contents of cordycepin and carotenoid by using MAT genes as selectable markers. Scientia Horticulturae. 2021;290:110492. https://doi.org/10.1016/j.scienta.2021.110492
55. Annepu SK, Sharma VP, Kumar S, Barh A. Cultivation techniques of shiitake (a medicinal mushroom with culinary delight). Chambaghat: ICAR-Directorate of Mushroom Research; 2019. 71 p.
56. Zhao P, Hou Y-C, Wang Z, Liao A-M, Pan L, Zhang J, et al. Effect of fermentation on structural properties and antioxidant activity of wheat gluten by Bacillus subtilis. Frontiers in Nutrition. 2023;10:1116982. https://doi.org/10.3389/fnut.2023.1116982
57. Clark AJ, Soni BK, Sharkey B, Acree T, Lavin E, Bailey HM, et al. Shiitake mycelium fermentation improves digestibility, nutritional value, flavor and functionality of plant proteins. LWT. 2022;156:113065. https://doi.org/10.1016/j.lwt.2021.113065
58. Turck D, Bohn T, Castenmiller J, de Henauw S, Hirsch-Ernst KI, Maciuk A, et al. Safety of pea and rice protein fermented by Shiitake (Lentinula edodes) mycelia as a Novel food pursuant to Regulation (EU) 2015/2283. EFSA Journal. 2022;20(4):e07205. https://doi.org/10.2903/j.efsa.2022.7205