Wheat Milling by-Products: an Alternative to Produce Amylolytic Enzymes by Mushrooms Strains

Paludo, Luana Cristina; Salles, Patrícia Maria Santos; França, Jessica Steinwandt de; Strobel, Christian Scapulatempo; Soccol, Carlos Ricardo; Spier, Michele Rigon

doi:10.1590/1678-4324-2023210514

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Article - Food/Feed Science and Technology • Braz. arch. biol. technol. 66 • 2023 • https://doi.org/10.1590/1678-4324-2023210514 copy

Wheat Milling by-Products: an Alternative to Produce Amylolytic Enzymes by Mushrooms Strains

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

The goal of this study was to evaluate the α-amylases production by basidiomycetes in submerged cultivation (SmC), using wheat milling by-products as substrate. Enzymatic activity was evaluated to select the best strain, the ideal concentration, the by-product and the influence of supplementation in the culture medium. The best producer was chosen among three strains, Ganoderma applanatum (MR-56), Schizophyllum commune (MR-01), and Ganoderma stipitatum (MR-72). All of them were cultivated under the same conditions. MR-56 was selected for an initial screening activity of 5.10±0.31 U/mL in 192h. The Ganoderma applanatum strain was cultured in a medium with various starch concentrations to determine which concentration resulted in the highest α-amylase activity. After 120h of cultivation, activity in the medium containing 4.5% (w/v) had increased from 0.40±0.03 U/mL to 20.70±0.9 U/mL. Subsequently, the wheat milling by-products [Glue Flour (GF), Clean-Out Flour (CF4) Flour, and Low-grade Flour (LGF)] were evaluated in aqueous suspension with and without nutritional supplementation. It was shown that GF and LGF are potential starch sources which do not require nutritional supplementation, exhibiting increases in α-amylase activity of 31.79 U/mL and 30.98 U/mL, respectively. This is the first report involving the application of Ganoderma applanatum in SmC for α-amylase production using synthetic and wheat milling by-products. The results also demonstrated that wheat milling by-products may also be used without supplementation for cell growth and enzyme production, contributing to the development of more sustainable forms of enzyme production through the innovative use of edible organisms with medicinal properties.

Keywords:
α-amylases; Submerged cultivation; Grain by-products; Flour; Basidiomycetes.

HIGHLIGHTS

• One of the first works on the amylase production by Ganoderma applanatum;

• Production of α-amylase in the wheat milling by-product without supplementation;

• This strain showed potential to produce amylase in several wheat milling by-products;

• Amylase production costs 42-fold less with by-product than synthetic medium;

GRAPHICAL ABSTRACT

Tests	Wheat milling by-products	Description	Characteristics of the medium
1	Soluble starch medium (control)	4.5% (w/v) starch	Soluble corn starch P.A.
2	GF	The flour used in the adhesive manufacturing industry	13.24% moisture, 0.85% ashes, 15.18% proteins, 78.98% starch, 35.14 mg/100g Ca²⁺, 149.14 mg/100g P, 43.33 mg/100g Mg²⁺, 182.24 mg/100g K⁺ and 27.42 mg/100 g Na⁺ [²³23 Frantz SC, Paludo LC, Stutz H, Spier MR. Production of amylases from Coprinus comatus under submerged culture using wheat-milling by-products: Optimization, kinetic parameters, partial purification and characterization. Biocatal Agric Biotech. 2019 Jan;17(2019):82-92.]
3	CF4	“Clean-out” Flour (a residual flour of silos, pipes, and floor)	13.48% moisture, 0.92% ashes, 14.57% proteins, 81.07% starch, 50.75 mg/100g Ca²⁺, 156.79 mg/100g P, 46.83 mg/100 g Mg²⁺, 219.28 mg/100g K⁺ and 252.84 mg/100 g Na⁺ [²³23 Frantz SC, Paludo LC, Stutz H, Spier MR. Production of amylases from Coprinus comatus under submerged culture using wheat-milling by-products: Optimization, kinetic parameters, partial purification and characterization. Biocatal Agric Biotech. 2019 Jan;17(2019):82-92.]
4	LGF	The by-product of ground wheat flour	12.98% moisture, 5.02% ashes, 15.80% proteins, 43.95% starch, 122.00 mg/100g Ca²⁺, 379.50 mg/100g P, 153.60 mg/100g Mg²⁺, 727.39 mg/100g K⁺ and 31.88 k mg/100g Na⁺ [²³23 Frantz SC, Paludo LC, Stutz H, Spier MR. Production of amylases from Coprinus comatus under submerged culture using wheat-milling by-products: Optimization, kinetic parameters, partial purification and characterization. Biocatal Agric Biotech. 2019 Jan;17(2019):82-92.]
5	GF + salts	The flour used in the adhesive manufacturing industry	GF + (KH₂PO₄ (2.0) mg/mL, (NH₄)₂SO₄ (1.4) mg/mL, CaCl₂ (0.3) g/L, MgSO₄.7H₂O (0.3) mg/mL and urea (0.3) mg/mL)
6	CF4 + salts	“Clean-out” Flour (a residual flour of silos, pipes, and floor)	CF4 + (KH₂PO₄ (2.0) mg/mL, (NH₄)₂SO₄ (1.4) mg/mL, CaCl₂ (0.3) g/L, MgSO₄.7H₂O (0.3) mg/mL and urea (0.3) mg/mL)
7	LGF+ salts	The by-product of ground wheat milling	LGF + (KH₂PO₄ (2.0) mg/mL, (NH₄)₂SO₄ (1.4) mg/mL, CaCl₂ (0.3) g/L, MgSO₄.7H₂O (0.3) mg/mL and urea (0.3) mg/mL)

Basidiomycete	Diameter Ø (cm)				Growth increased	Growth^ Growth sign (+, ++, +++), the + sign represents the lowest growth of the colonies; the ++ signal represents intermediate growth; +++ represents the highest growth.	Relative growth^* ***** Relative growth (%) is the diameter of the growth of a specific fungus to growth of the highest growing fungus (MR-56).
Basidiomycete	0 day	1^st day	4^th day	5^th day	∆Ø		%
MR-01	0.5	0.7	2.8	4.2	3.7	+	56.9
MR-72	0.5	0.7	3.0	5.2	4.7	++	72.3
MR-56	0.5	0.8	4.6	7.0	6.5	+++	100.0

Strain	72 h			144 h								192 h
Strain	A	P		A		P		A		P	A		P
MR-56	0.72±0.18^b	0.010	1.67±0.28^a		0.012		3.30±0.34^a		0.020			5.10±0.31^a		0.027
MR-01	0.94±0.11^a	0.013	1.60±0.34^a		0.011		2.00±0.41^b		0.012			2.80±0.42^b		0.015
MR-72	0.60±0.09^c	0.008	0.79±0.1^b		0.005		0.94±0.21^c		0.005			1.10±0.13^c		0.006

Cultivation time (h)	Starch 4.5%		GF		CF4		LGF
Cultivation time (h)	A	P	A	P	A	P	A	P
0	2.27±0.32^b	0.00	2.22±0.02^b	0.00	1.79±0.23^c	0.00	2.38±0.15^a	0.00
24	2.25±0.18^b	0.09	2.39±0.22^b	0.10	2.03±0.17^c	0.08	2.85±0.02^a	0.12
72	5.18±0.23_b	0.07	3.99±0.35^d	0.05	4.43±0.33^c	0.06	5.49±0.11^a	0.07
120	18.72±0.14^b	0.16	24.89±0.07^a	0.21	6.28±0.01^d	0.05	17.98±0.38^c	0.15
168	24.83±0.10^b	0.15	25.61±0.43^a	0.15	13.09±0.03^d	0.08	17.98±0.43^c	0.11
192	24.93±0.08^a	0.13	24.17±0.28^b	0.12	12.96±0.21^d	0.07	16.68±0.36^c	0.08

Conditions	α-amylase Activity (U/mL)
	Control (Starch 4.5%)		GF		CF4		LGF
	0 h	120 h	0 h	120 h	0 h	120 h	0 h	120 h
Without supplementation	0.89^F	9.58^f	1.11^E	32.90±0.31^b	2.79^A	8.46±0.05^g	2.32^B	33.3±0.13^a
With supplementation	0.89^F	9.58^f	1.38^D	27.66±0.21^c	1.94^C	22.83±0.18^d	1.90^C	17.97±0.45^e

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[1] Editor-in-Chief: Paulo Vitor Farago