Characteristics and mechanism of <i>Leuconostoc citreum</i> as a novel bioflocculant for starch granules in starch production

Xuan Wang; Pamela Owusu Osei; Lei Rao; Xiaomeng Wu; Xiaojun Liao; Xuan Wang; Pamela Owusu Osei; Lei Rao; Xiaomeng Wu; Xiaojun Liao

doi:10.48130/FIA-2023-0030

Figures (6) Tables (3)

Figure 1.
FT-IR spectra of (a) untreated starch & Leuc. citreum SJ-57 adhered starch and (b) untreated Leuc. citreum SJ-57 & starch-adhered Leuc. citreum SJ-57. (c) The effect of different metal ion concentrations and types on Leuc. citreum SJ-57's FR, (d) the zeta potential of starch, and (e) the zeta potential of bacteria. (f) Effects of chemical treatments on Leuc. citreum SJ-57's FR: The floc were formed in 0.01 mol/L KCl (I), 0.01 mol/L KCl and 0.01 mol/L HCl (II), 0.01 mol/L KCl and 5 mol/L urea (III), 0.01 mol/L CaCl₂ (IV), and 0.01 mol/L CaCl₂ and 0.01 mol/L EDTA (V). Statistical analysis of differences between FRs with cells after various treatments performed using t-test. * represents p < 0.05.
Figure 2.
Total long-range interaction energy (Tot) and the individual partial interaction profiles between (a) Leuc. citreum SJ-57 and starch, (b) Leuc. citreum SJ-57 and Leuc. citreum SJ-57, (c) starch and starch, in 0.01 mol/L KCl solution at pH = 7 calculated by DLVO theory. FT-IR spectra of (d) untreated Leuc. SJ-57 and (e) starch in 0−10 mmol/L KCl solution. (f) Changes in the equivalent diameter of strains and starch in 0−10 mmol/L KCl solution. LW, EL, and AB denote van der Waals, electrostatic interactions, and Lewis acid/base interactions, respectively.
Figure 3.
Effects of (a) thermal treatment and (b) enzymatic treatments of Leuc. citreum SJ-57 on their FR. Statistical analysis of differences in FRs between cells after various treatments and the control group was performed using t-test. * represents p < 0.05.
Figure 4.
(a) Effect of extraction reagents on the starch-flocculating activity of Leuc. citreum SJ-57. Different letters represent significant differences (p < 0.05). (b) SDS–PAGE analysis of whole cell surface proteins (lane 1), unbound starch of cell surface proteins (lane 2), and starch-binding surface proteins (lane 3). The molecular masses (in kDa) of the standard proteins (lane M) are indicated on the left. (c) Sedimentation of starch emulsions after resting for 5 min. Control: natural sedimentation of starch emulsions. Cell surface proteins: sedimentation of starch mixed with surface proteins. SJ-57: sedimentation of starch mixed with Leuc. citreum SJ-57.
Figure 5.
(a) Changes in FR as a function of time when Leuc. citreum SJ-57 cells were washed with NaCl. (b) The protein and EPS content changes in the wash solution as a function of time when Leuc. citreum SJ-57 cells were washed with NaCl. TEM pictures of Leuc. citreum SJ-57 (c) before and (d) after washing with NaCl for 6 h.
Figure 6.
Proposed starch flocculation mechanism of Leuc. citreum SJ-57.

Surface tension parameters (mJ/m²)
	γ^LW	γ⁺	γ⁻	γ^Tot
H₂O	21.8	25.5	25.5	72.8
C₃H₈O₃	34.0	3.9	57.4	64.0
CH₂I₂	50.8	0.0	0.0	50.8
Note: γ^LW represents the LW component of the surface tension. γ⁺ and γ^- are the electron-acceptor and electron-donor parameters of the surface tension, respectively.

Table 1.

Surface tension parameters of probe liquids used for contact angle measurements for Leuc. citreum SJ-57 and starch granules.

	Contact angles (degree)			Surface tension parameters (mJ/m²)			ΔG_iwi (mJ/m²)
	H₂O	C₃H₈O₃	CH₂I₂	γ^LW	γ⁺	γ⁻	ΔG_iwi (mJ/m²)
Leuc. citreum SJ-57	53.5 ± 4.3	49.2 ± 2.0	24.5 ± 2.9	46.3	0.3	21.9	−15.8
Starch	26.8 ± 2.8	31.3 ± 7.5	18.5 ± 2.7	48.2	41.6	0.6	13.7
The contact angles are presented as the means ± standard error. Parameters γ^LW, γ^+, and γ⁻ are the surface tension components. Hydrophobicity was evaluated by ΔG_iwi, which was calculated using the surface tension parameters according to Eqn (2).

Table 2.

Contact angles of water, glycoside, and diiodomethane on Leuc. citreum SJ-57 and starch granules, along with the derived surface tension parameters and hydrophobicity of the surfaces.

Band	Protein	Accession number	Number of matches	Score	Isoelectric point	Molecular weight (KD)
1	GW (glycine-tryptophan)) dipeptide domain-containing	WP_146992371.1	154	3546	5.26	230
2	GW (glycine-tryptophan)) dipeptide domain-containing	WP_146992371.1	867	22986	5.26	230
3	GW (glycine-tryptophan)) dipeptide domain-containing	WP_146992371.1	761	18792	5.26	230
4	molecular chaperone DnaK	WP_040177072.1	281	6808	4.60	65
5	Chaperonin GroEL	WP_004901547.1	1085	28391	4.60	57
6	Elongation factor Tu	WP_004900335.1	297	6485	4.82	43
7	Lysozyme M1	WP_004901157.1	974	26689	5.08	36
8	Lysozyme M1	WP_004901157.1	110	2473	5.08	36

Table 3.

Candidate starch-binding proteins extracted from the surface of Leuc. citreum SJ-57.