Ultrasound assisted fabrication of the yeast protein-chitooligosaccharide-betanin composite for stabilization of betanin

Graphical abstract

Highlights

• •The ultrasound-induced fabrication of the YCB by was studied.
• •Ultrasound improved the surface hydrophobicity and solubility of yeast protein.
• •Ultrasound facilitated binding of betanin and oligosaccharide to the yeast protein.
• •YCB exhibits a high color stability of betanin and controls its sustained release.
• •This work offers a scheme to stabilize colorant by an ultrasound-assisted method.

Abstract

Betanin, a water-soluble colorant, is sensitive to light and temperature and is easily faded and inactivated. This study investigated the formation of yeast protein-chitooligosaccharide-betanin complex (YCB) induced by ultrasound treatment, and evaluated its protective effect on the colorant betanin. Ultrasound (200–600 W) increased the surface hydrophobicity and solubility of yeast protein, and influenced the protein’s secondary structure by decreasing the α-helix content and increasing the contents of β-sheet and random coil. The ultrasound treatment (200 W, 15 min) facilitated binding of chitooligosaccharide and betanin to the protein, with the binding numbers of 4.26 ± 0.51 and 0.61 ± 0.06, and the binding constant of (2.73 ± 0.25) × 10⁵ M⁻¹ and (3.92 ± 0.10) × 10⁴ M⁻¹, respectively. YCB could remain the typical color of betanin, and led to a smaller and disordered granule morphology. Moreover, YCB exhibited enhanced thermal-, light-, and metal irons (ferric and copper ions) -stabilities of betanin, protected the betanin against color fading, and realized a controlled release in simulated gastrointestinal tract. This study extends the potential application of the fungal proteins for stabilizing bioactive molecules.

1. Introduction

Betalains are a kind of naturally water-soluble pigments predominantly found in beetroot, amaranth, and cactus pear [1]. Betanin is an important component of betalains, and has a characteristic red–purple color [2]. It can be applied as a natural colorant in food formulation, such as yogurt, gummy candy, and ice cream [3]. Betanin demonstrates a free radical-scavenging ability due to the phenolic and amine groups [4]. It also has many pharmacological activities such as anticarcinogenic, anti-diabetes properties, and anti-inflammatory [5]. Thanks to its nutraceutical potential, dietary, and biological activities, betanin has received much attention for laboratory and industrialization in food industry. Nevertheless, the instability of betanin, exacerbated by high temperatures, oxygen, and light, leads to its gradual discoloration to light yellow and a notable reduction in its biological activity, which will limit its food applications [6], [7]. Therefore, how to protect the stability and activity is crucial for its applications and popularization of betanin.

Utilizing the protein- and polysaccharide- based nanoparticles to bind, deliver, and stabilize the pigment may be practicable during its food storage and processing. Yeast proteins are a kind of novel fungal proteins, and have an overall and balanced amino acid composition. The contents of protein account for 40 % to 60 % of the dry weights of the yeast cell [8], which is considered as high quality protein sources. Yeast protein shows functional characteristics such as good solubility, high water holding ability, and emulsifying property, which facilitate their use as protein supplements in food and nutritional applications [9], [10]. To the best of our knowledge, there are few reports on utilizing the yeast protein as a carrier of bioactive compounds. Specifically, the non-covalent conjugation of polysaccharide to protein, including the electrostatic interaction, hydrophobic force, hydrogen bonds, may strengthen the functions of the proteins and provide an opportunity to stabilize bioactive compounds [11]. Chitooligosaccharide, a naturally cationic amino oligosaccharide, has a high antibacterial property and has a good biocompatibility [12], [13]. Many attentions have been received owing to the non-toxic feature and the cationic conjugation role of chitooligosaccharide to protein. Inspired by these foundations, the effect of a two-layer material of yeast protein and chitooligosaccharide system on the color stability of structural and the interaction mechanisms of yeast protein-chitooligosaccharide-betalain composite are valuable and intriguing research questions.

Ultrasonic treatment is an important food processing technology, and it affects protein spatial structures by disrupting hydrogen bonds and hydrophobic interactions through cavitation and sound influx. This leads to the alterations in protein conformation, such as the destruction of tertiary and secondary structures, protein aggregation, or dissociation, depending on the ultrasonic wave intensity [14]. Some hidden hydrophobic groups become exposed, resulting in increased surface hydrophobicity of the proteins [15]. Up to the present, many studies have indicated that the application of ultrasonic processing is capable of influencing protein structures and enhancing their functional properties [16], [17], nevertheless, whether the ultrasonic processing could influence the yeast protein-saccharide-pigment interactions systems and the effects of ultrasonic processing the stabilization of betalain in a steady state are unclear.

In this study, the stability of the food colorant-betalain was studied in the form of protein-chitooligosaccharide-betalain composite system induced by ultrasound assistance. The objectives included: 1) evaluating the impact of ultrasound treatment on the surface properties of yeast protein and the binding dynamics between betalain, chitooligosaccharide, and yeast protein; 2) examining the structure, morphology, and size distribution of the resulting composite system; and 3) assessing the stability of betanin, the primary component of betalain, under various conditions such as thermal, light, and ferric ion exposure. This paper provides a theoretical guideline for the stabilization of the instable colorant, and will be beneficial for extension of the utilization of yeast protein as a carrier system to deliver bioactive substances.

2. Materials and methods

2.1. Materials

Yeast protein was supplied by Hubei Provincial Key Laboratory of Yeast Function, Angel Yeast Co., Ltd., China, with a protein content of 75 % (w/w). The concentration was detected by the Lowry method. Betanin (molecular weight of 550.475) and other chemicals were from Shanghai Macklin Biochemical Co., Ltd (China). Chitooligosaccharide (deacetylation degree of 97 %) was purchased from Zhejiang Golden-Shell Biochemical Co. Ltd. (Hangzhou, China).

2.2. Preparation of yeast protein-chitooligosaccharide-betanin complex

The yeast protein (100 mg) dissolved in phosphate buffer (0.2 mol/L, pH 7.0) was stirring for 10 min, followed by centrifugation at 6000 r/min for 15 min to remove insoluble substances. The chitooligosaccharide with different concentrations (1:1–11:1, chitooligosaccharide/protein), and the betanin with different concentrations (0.1:1–1.2:1, betanin /protein) were mixed with the yeast protein (0.5 mg/mL), respectively, followed by treatment by ultrasonic processing (200, 400, and 600 W, 15 min) in the dark at 4 °C for 60 min. The mixtures were dialyzed (5 kDa) by phosphate buffer (0.2 mol/L, pH 7.0) for 1 h with twice buffer changes to remove the unbound chitooligosaccharide and betanin molecules to prepare the yeast protein-chitooligosaccharide-betanin complex (YCB), protein-chitooligosaccharide complex (YC), and the yeast protein-betanin complex (YB), respectively. The YCB complex was also prepared without ultrasonic processing. The applying amount of chitooligosaccharide and betanin for all the samples were the same.

2.3. Fluorescence fitting

The fluorescence change of yeast protein induced by chitooligosaccharide and betanin were evaluated by a Cary Eclipse spectrophotometer (Agilent Technologies, USA). Different concentration of chitooligosaccharide or betanin were added to yeast protein sample (0.5 mg/mL, 1 mL), respectively. The mass ratio of chitooligosaccharide to protein was 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 11:1, and the mass ratio of betanin to protein was 0.1:1 to 1.0:1 with a gradient increase of 0.1:1. The excitation and emission slit widths were 5 nm and 10 nm. The excitation wavelength was set at 290 nm, and emission spectra were collected from 300 to 500 nm. The binding constant K and the binding number n stoichiometric were fitted by the formulation (1) [18].

$$I=I_0-\left(\frac{I_0-I_{\infty }}{2n{\left[P\right]}_0}\right)\times \left[\frac{1}{K}+{\left[C\right]}_0+n{\left[P\right]}_0-\sqrt{{\left(\frac{1}{K}+{\left[C\right]}_0+n{\left[P\right]}_0\right)}^2-4n{\left[P\right]}_0{\left[C\right]}_0}\right]$$ (1)

Where [P]₀ represents the concentration of protein, [C]₀ represents the concentrations of chitooligosaccharide or betanin, I₀ and I_∞ are the fluorescence intensity without the attachment of chitooligosaccharide or betanin and with chitooligosaccharide or betanin as the binding sites were saturated, respectively.

2.4. Fluorescence analysis of the yeast protein-chitooligosaccharide-betanin complex

The interactions of yeast protein to chitooligosaccharide or betanin were studied with a spectrophotometer (Agilent Technologies, USA). The formations of the yeast protein-chitooligosaccharide-betanin complex, protein-chitooligosaccharide complex, and the yeast protein-betanin complex were performed by respectively blending the protein with chitooligosaccharide or betanin for an incubation of 5 min (25 °C). The excitation wavelength was 290 nm, and emission spectra were corrected from 300 to 500 nm. The excitation slit width was 5 nm, and the emission slit width was 10 nm.

2.5. Analysis of hydrophobicity

The 1-anilino naphthalene-8-sulfonic acid (ANS) method was used determine the surface hydrophobicity of the samples treated by the ultrasonic power [19]. The ultrasonic power was selected as 200, 400, and 600 W for 15 min. The protein samples (5 mL) with gradient concentrations were mixed with ANS (20 μM) and reacted for 15 min in dark condition. The fluorescence spectrum was detected by a spectrophotometer (Agilent Technologies, USA) with the excitation/emission wavelength of 390 and 470 nm. The surface hydrophobicity (So) was calculated as initial slope of fluorescence intensity with protein concentration.

2.6. Solubility analysis

The yeast protein was treated with ultrasonic processing with a power of 200, 400, and 600 W for 15 min. The yeast protein samples were then adjusted to pH (2 to 11) with 1 M NaOH or HCl, and were centrifuged for 20 min at 8000 rpm. The protein contents in the supernatants were analyzed by a Bradford method. The solubility index of the yeast protein was figured up by the formula (2) [20]:

$$\mathit{So}lubilityindex\left(\%\right)=\mathit{the}contentinthe\sup erna\tan ts/\mathit{the}totalcontent\times 100$$ (2)

2.7. Secondary structure analysis

The circular dichroism (CD) spectra of the samples were measured by using a MOS-450 spectrometer at 25 °C. These samples included the yeast protein, yeast protein-chitooligosaccharide-betanin complex, protein-chitooligosaccharide complex, and the yeast protein-betanin complex (0.5 mg/mL). The path length was 1 cm and the scan ranges were from 190 to 260 nm. The proportion of the secondary structures was analysed by the program CDNN.

2.8. SEM analysis

The liquid samples of the yeast protein, protein-chitooligosaccharide complex, and the yeast protein-betanin complex, and the yeast protein-chitooligosaccharide-betanin complex (pH 7.0, 0.5 mg/mL) were diverted to the carbon-coated grid and were respectively stained with 2 % uranyl acetate for 5 min. Then, the excess liquid was absorbed with filter paper before being examined by Scanning electron microscopy (SEM).

2.9. DLS analysis

The dynamic light scattering (DLS) experiment was conducted by a device from Malvern, UK to analyze the diameter of yeast protein, protein-chitooligosaccharide complex, and the yeast protein-betanin complex, and the yeast protein-chitooligosaccharide-betanin complex (pH 7.0, 0.5 mg/mL). Each of the samples was placed for 2 h before measurements to guarantee the reaction was finished. All experimental data were analyzed by OmniSIZE software.

2.10. Stability of betanin

The thermal stabilities of betanin in the form of FB, YCB, and a free state were performed from 30 to 80 °C to imitate the storage processes (30 °C), machining processes (60 °C), and pasteurization processes (80 °C), respectively. Each sample (1 mL) covered with silver paper was set in a water bath at 30, 60, and 80 °C for 2 h. During the heating process, the absorption of the sample was detected at 535 nm every 30 min by an 8430 ultraviolet spectrophotometer (Agilent Technologies, USA) to calculate the betanin content with a calibration curve [3]. The retention ratio after treatment of betanin in different samples was counted referring to formula (3):

$$\text{Re}tentionratios\left(\%\right)=\text{Re}sidualbe\tan in/\mathit{The}bindingbe\tan in\times 100\%$$ (3)

The influence of light on the stability of betanin in the form of YB, CFB, and a free state was assayed at UV radiation and natural light. Each sample (1 mL) was placed under a UV lamp with ultraviolet light at 254 nm or natural light for 6 h. Especially, the treated samples were assessed to quantify the remaining betanin according to measuring the absorption at 535 nm every 1.5 h [21]. The retention ratio after treatment of betanin in different samples was counted referring to formula (3).

The effect of Fe³⁺ and Cu²⁺on the stability of betanin in the form of YB, YCB, and a free state was evaluated by recording the changes of the absorbance at 535 nm. A certain amount of FeCl₃ and CuCl₂ solution was respectively added to each sample (0.5 mg/mL, 1 mL) so that the concentration of Fe³⁺ and Cu²⁺ in the sample was 0.05, 0.1, 0.15, and 0.2 mM, respectively. The absorbance at 535 nm was detected after standing the mixtures for 10 min kept in dark place. The retention ratio betanin in different groups were counted referring to formula (3).

2.11. Color analysis

The colorimeter (CS-810, Beijing, China) was used to determine the color changes of the betanin, YB, and YVB after the thermal, light, and metal ions treatments. The procedure was conducted according to the method as described in section 2.10. A standard calibration whiteboard was applied to calibrate the colorimeter. The ΔE was fitted according to formula (4) [22],

$$\mathrm{\Delta }E=\sqrt{{\left(L_n-L_m\right)}^2+{\left(a_n-a_m\right)}^2+{\left(b_n-b_m\right)}^2}$$ (4)

Where L represents light/dark; a expresses the red/green and b expresses yellow/blue, respectively, and n and m represent the final treatment time and the initial time, respectively.

2.12. The simulated gastrointestinal analysis

The yeast protein-betanin complex and the yeast protein-chitooligosaccharide-betanin complex were subjected to the gastrointestinal digestion referring to reported work [23]. All the samples (5 mL) were respectively subjected to the simulated gastric fluid (pH 2.0) with pepsin (2 %) for stirring for 2 h at 37 °C. The liquid (0.5 mL) was sampled each 40 min to be centrifuged at 10000 rpm (15 min) and was quantified to determine the betanin content. For the intestinal digestions, the sample was conducted to the simulated intestinal fluid (pH 7.5) with pancreatin (0.4 %) for stirring for 2 h at 37 °C to simulate digestion. The mixture (0.5 mL) was collected each 40 min to be centrifuged at 10000 rpm (15 min) and was quantified to determine the betanin content.

2.13. Statistic analyses

All the obtained data were analyzed by a SPSS software (11.0) based on one-way analysis by Duncan’s multiple range tests. A confidence of 95 % was used to analyze the statistical significance.

3. Results and discussion

3.1. Effect of ultrasound on the hydrophobicity and solubility of yeast protein

Yeast protein is a novel alternative fungal protein, the influence of ultrasonic processing on the structure and function of the yeast protein and the form of YCB complex has been studied in this work. The molar weight and the purity of the yeast protein were checked by a SDS PAGE method, and the molar weight was distributed in the range of 31.0 to 66.2 Kda with multiple subunits (Fig. S1, Supporting Information). Firstly, to study the surface properties of the yeast protein, the surface hydrophobicity and the solubility were evaluated. The hydrophobic interaction is a key force in the formation and stabilization of the spatial structure of protein. The variations of the surface hydrophobicity can reflect the folding and shielding of the amino acids on protein, and can indicate the changes of the surface properties. The effect of different ultrasonic powers (200–600 W) on the surface hydrophobicity of the yeast protein was evaluated by using the fluorescent probe of ANS (Fig. 1a). The ultrasound treatments could remarkably increase the So of yeast protein, and the ultrasonic powers from 0 to 200 W and 400 W significantly enhanced the So by 12.8 % and 14.9 % (p < 0.05). However, continuing increase of the ultrasonic power to 600 W had little effect on the So. These results suggested that the ultrasound treatment caused an increase in surface hydrophobicity of the yeast protein. This finding was consistent with the report about the increased surface hydrophobicity of the pea protein isolates at 300 W, 600 W, and 900 W [15]. The potent cavitation effects engendered by ultrasonication have the capacity to diminish intermolecular associations within protein molecules, subsequently leading to the exposition of internal hydrophobic residues to the ambient environment [24]. By comparison, Kang et al. [25] discerned that as the ultrasonic power intensified, the So of chickpea protein ascended to a certain apex before experiencing a subsequent recession. Overzealous ultrasonication could engender anomalous denaturation of proteins, culminating in protein agglomeration and the obfuscation of hydrophobic residues [26]. We inferred that some hydrophobic aromatic amino acids may be exposed and some hydrophilic groups may become folded due to the ultrasound treatment deposing on the amino acid of yeast proteins.

Fig. 1.

(a) Surface hydrophobicity index and (b) the solubility of the yeast protein with different treatment of ultrasonic powers (200, 400, and 600 W). CD spectra (c) (200–260 nm) and (d) (285–300 nm) of the yeast protein with different treatment of ultrasonic powers (200, 400, and 600 W).

Solubility serves as a crucial indicator of protein hydration and represents a key physicochemical characteristic that influences protein functionality and determines its applications [27]. As shown in the Fig. 1b, different ultrasonic treatments exhibit varying effects on protein solubility, and the ultrasonic treatment significantly enhanced the solubility of yeast protein, reaching its peak at an ultrasonic power of 200 W and duration of 15 min. This improvement in solubility may be attributed to conformational and structural changes in proteins caused by ultrasonication, which in turn exposed hydrophilic domains within the protein to water [28]. Moreover, the augmentation of surface area, concomitant with the diminution of particle dimensions and exposure of hydrophilic moieties, results in a partial unfolding of protein structures. This phenomenon amplifies the intermolecular interactions between protein molecules and the surrounding aqueous medium, thereby enhancing solubility. In congruence with this observation, Tang et al. [29] reported that when subjected to ultrasonication at varying amplitudes (20–100 %) for a span of 15 min, the water-soluble proteins derived from Moringa seeds manifested a notable increment in solubility. Similarly, Martínez-Velasco et al. [30] discerned a significant amplification in the solubility of fava bean isolate proteins post-ultrasonic treatment. However, as the ultrasonic power continued to increase, protein solubility decreased, possibly due to protein reaggregation under excessively high ultrasonic power, leading to a reduced solubility. Compared to other treatment methods, ultrasonic treatment acts directly on proteins. The ultrasonic waves generate instantaneous high pressure, resulting in shear, cavitation, turbulence, surface electrostatics, and thermodynamic effects, which endows the protein surface with more negative charges, thereby enhancing hydration [31].

3.2. Secondary structure analysis of yeast protein induced by ultrasound

The CD spectroscopy ranging from 200 to 260 nm was collected to gain more information about the structure of yeast protein. Fig. 1c showed that the ellipticity curves of yeast protein samples with different ultrasonic power treatment (200 W, 400 W, and 600 W). The ultrasonic treatment on ferritin led to a decrease in the α-helix content and an increase in the contents of β-sheet and random coil (Table 1). For example, the α-helix content of the protein with ultrasonic treatment at 200 W decreased by 4.10 %, and corresponding, 2.55 % of β-sheet and 1.35 % of the random coil content were increased. However, the improvement of the ultrasonic power to 400 W and 600 W did not significantly decrease the α-helix content (p > 0.05), while the group with 400 W treatment remarkably increased the β-sheet content relative to that at 200 W treatment (p < 0.05).

Table 1.

Secondary structure content of yeast protein after treatment with different ultrasonic powers.

Samples	α-helix	β-sheet	random coil
Yeast protein	17.23 ± 0.08 %a	52.35 ± 0.13 %c	30.42 ± 0.11 %c
Yeast protein (200 W, 15 min)	13.13 ± 0.07 %b	54.90 ± 0.12 %b	31.97 ± 0.08 %b
Yeast protein (400 W, 15 min)	12.75 ± 0.11 %b	56.01 ± 0.11 %a	31.24 ± 0.06 %b
Yeast protein (600 W, 15 min)	10.14 ± 0.11 %c	55.72 ± 0.09 %a	33.14 ± 0.05 %a

Results are expressed as mean value ± SD (n = 3). Different letters after the analysis data in the same row indicate significant analysis results (p < 0.05).

Moreover, the CD spectroscopy from 285 to 300 nm that predominantly represent the tryptophan residues were analysed [32]. The ultrasonic treatment (200 W, 400 W, and 600 W) remarkably decreased the spectroscopy intensity of 290 nm (Fig. 1d), and the yeast protein after 400 W and 600 W treatments showed higher value of the ellipticity than that after 200 W treatment. In addition, the relative broad spectroscopy of the yeast protein changed to a steeper form after ultrasonic treatment. These findings indicated that the ultrasonic treatment led to a perturbational effect on the environment of tryptophan residues of the yeast proteins. We inferred that the ultrasonic treatment-induced cavitation and sound influx effects affected the spatial structures of proteins by disrupting the weak interactions such as hydrogen bond and hydrophobic interaction [14], which was evidenced by the decreased content of the α-helix structure and increased proportion of the β-sheet (Table 1). This may lead to alterations in the conformation and the property of the yeast proteins and in turn influence its interaction with other compounds such as chitooligosaccharide and betanin as discussed in the following sections.

3.3. Effect of ultrasound on the binding of chitooligosaccharide and betanin to yeast protein

Regarding the ultrasound-induced structure and property changes of the yeast protein, the interaction between the yeast protein with chitooligosaccharide or betanin were studied to explore the interaction mechanism of the protein-saccharide-betanin three-layer systems and to clarify the effect of the yeast protein- and chitooligosaccharide-based carrier on the color stability of the betanin. The tryptophan residue on the protein usually causes the endogenous fluorescence of protein, and the change in the intensity and shift of tryptophan fluorescence is deemed as an important symbol of the structure change of yeast proteins.

Firstly, the interactions of yeast protein with chitooligosaccharide were studied by fluorescence spectroscopy. As shown in Fig. 2a, the fluorescence intensities of yeast protein reduced regularly following the gradient increases of the mass ratio of chitooligosaccharide (0:1 to 11:1). The interaction stoichiometry between yeast protein and chitooligosaccharide was further studied to obtain the stoichiometric number-n and the constant-K, which contribute to predicting the binding number and the binding affinity [33]. The data were fitted into formula (2) on the basis of the emission peaks of yeast protein after chitooligosaccharide treatment with an increasing gradient (Fig. 2b and Table 2). The result showed n was 3.12 ± 0.12, indicating that one mass concentration of yeast protein can be combined with approximately 3.1 mass ratio of chitooligosaccharide molecules. The binding constant-K valued (1.24 ± 0.16) × 10⁵ M⁻¹, which corresponds to the binding constant between chitosan and ferritin reported in the previous article [34], suggesting that the chitooligosaccharide binding to yeast protein includes electrostatic interaction. Contrastively, the n and K for the interactions between the yeast protein and chitooligosaccharide assisted by the ultrasonic treatment (200 W, 15 min) were 4.26 ± 0.51 and (2.73 ± 0.25) × 10⁵ M⁻¹ (Fig. 2c, d, and Table 2), respectively, which was significantly higher than that without ultrasonic treatment (p < 0.05). We deduced that the interaction between the yeast protein and chitooligosaccharide can be strengthened due to the structural change of the surface and the unfolding of protein (Fig. 1c and d); the changes in the hydrophobicity and the distribution of the amino acids on the protein may facilitate the binding of the cationic chitooligosaccharide molecules [31].

Fig. 2.

(a) Fluorescence spectrum of yeast protein treating with different amounts of chitooligosaccharide (0–11 EGCG/PC, mass ratios) at 25 °C. (b) The fitting curve of the intensity based on (a) at 330 nm. (c) Fluorescence spectrum of yeast protein treating with different amounts of chitooligosaccharide (0–11 EGCG/PC, mass ratios) and ultrasonic power (200 W) at 25 °C. (d) The fitting curve of the intensity based on (a) at 330 nm.

Table 2.

Binding stoichiometry of yeast protein with chitooligosaccharide and betanin.

Samples	Chitooligosaccharide/yeast protein		Betanin/yeast protein
	n	K (M−1)	n	K (M−1)
Without ultrasound	3.12 ± 0.12	(1.24 ± 0.16) × 105	0.46 ± 0.02	(3.13 ± 0.23) × 104
Ultrasound (200 W, 15 min)	4.26 ± 0.51	(2.73 ± 0.25) × 105	0.61 ± 0.06	(3.92 ± 0.10) × 104
Ultrasound (400 W, 15 min)	4.72 ± 0.26	(2.50 ± 0.12) × 105	0.59 ± 0.03	(3.78 ± 0.12) × 104
Ultrasound (600 W, 15 min)	–	–	0.23 ± 0.02	(3.18 ± 0.18) × 104

The binding of yeast protein with the betanin was also investigated (Fig. 2e-h), and the n and K was compared (Table 2) to study the influence of the ultrasonic treatment and the binding of chitooligosaccharide molecules. It was showed that n was 0.51 ± 0.02 for the interaction between yeast protein and the betanin without ultrasonic treatment, indicating that one mass concentration of yeast protein can be combined with about 0.5 mass ratios of betanin molecules. The K was (3.13 ± 0.23) × 10⁴ M⁻¹, suggesting that the binding of betanin molecules to yeast protein mainly includes weak bonds, such as hydrophobic forces and van der Waals forces. After ultrasonic treatment (200 W, 15 min); however, these values were all significantly improved to 0.61 ± 0.06 and (3.92 ± 0.10) × 10⁴ M⁻¹ (Table 2). Continuing increase of the ultrasonic power (400 W and 600 W, 15 min) did not guarantee the improvement of the binding number. For example, the ultrasonic treatment at 400 W and 15 min resulted in a binding number n of 0.62 ± 0.05, while the ultrasonic treatment at 600 W and 15 min even decrease to a binding number n of 0.23 ± 0.02, indicating a high ultrasonic power played a negative role in the binding of betanin to the yeast protein. The increase of the surface hydrophobicity and the decrease of the solubility (400 W and 600 W) were inferred as important reasons for the decrease of binding number (Fig. 1a and b). Higher ultrasonic power at 400 W and 600 W could cause conformational and structural changes in proteins, such as the reduction of helical structure and increase of the sheet structure (Fig. 1c and d), which may in turn limit the binding of betanin to the protein [28].

3.4. Analysis of the loading amount of betanin

The effect of ultrasound and chitooligosaccharide attachment on the actual loading amount of betanin to yeast protein was evaluated. A loading ratio of 22.56 % (betanin/yeast protein, mass ratio) was obtained when the ultrasonic power was applied at 200 W for 15 min (Table S1), which was remarkably higher than that for the ultrasound-untreated group (p < 0.05). This result suggested that the ultrasonic treatment can increase the loading of the betanin. However, further increase of the ultrasonic power to 400 W and 600 W led to a decrease in the loading ratio; even so, the loading ratios of betanin (19.15 ± 1.29 % and 17.19 ± 2.01 %) were significantly higher than the untreated group (15.32 ± 1.02 %) (p < 0.05). The reduced protein solubility with the increase of ultrasonic power was inferred as an important reason. These findings were consistent to the fluorescence fitting data that the relatively high ultrasonic power was detrimental to the betanin binding (Fig. 2).

On the other hand, the influence of chitooligosaccharide on the loading amount of betanin to yeast protein was investigated. The bindings of chitooligosaccharide to the protein with a 1:1 and 3:1 mass ratio could significantly improve loading ratio of betanin, with values of 18.56 ± 1.29 % and 21.66 ± 1.56 %, respectively, these values were significantly higher than that for the yeast protein without chitooligosaccharide binding (p < 0.05). The binding of chitooligosaccharide to yeast protein with a 5:1 mass ratio, however, resulted in a remarkable decrease in the loading ratio of betanin (12.57 ± 1.22 %). A high amount of the attachment of the chitooligosaccharide may change the structure of yeast protein, cover the binding site of the betanin, and in turn hinder the loading of betanin. This trend was also appropriate for the ultrasonic power treatment (200 W) with the ratios of 1:1 and 3:1 (chitooligosaccharide/yeast protein) could facilitate the loading of the betanin molecules (Table S1). In addition, the ultrasonic treatment (200 W and 400 W) had little effect on the loading ratio of betanin for the 5:1 group (chitooligosaccharide/yeast protein), while the ultrasonic treatment (600 W) led to a significant decrease in the loading ratio (12.57 ± 1.22 %). We inferred that the binding of the betanin to the yeast protein would be hindered due to the changes in the hydrophobic property and the second structure upon a high power of the ultrasound. These findings were consistent to the fluorescence fitting data that the relatively high ultrasonic power (400 W and 600 W) was detrimental to the betanin binding (Table 2). Based on these findings, we selected the ultrasound treatment (200 W, 15 min) with a binding ratio of 3:1 (chitooligosaccharide/yeast protein) for the formation of YCB complex for subsequent discussions.

3.5. UV−vis spectrum and dissolved state observation

UV–Vis spectrophotometry was analyzed to study the formation of the yeast protein-chitooligosaccharide-betanin complex. Results showed that the yeast protein and betanin displayed their typical absorption peaks (Fig. 3a). The yeast protein presented an absorption peak at 280 nm, attributable to the absorbance properties of tryptophan and tyrosine residues inherent within its molecular structure. Conversely, betanin unveiled its apical peak at 530 nm. The YB also showed a maximum absorption peak at 530 nm, which is the characteristic absorption of the betanin. It’s noteworthy that the absorbance peak of YB surpassed that of the solitary yeast protein, hinting at potential protein conformational transitions. In juxtaposition, the hallmark absorption peak for betanin within the YCB complex was discerned at 539 nm, evidencing a pronounced red-shift of 9 nm. Conventionally, the red shift of the absorption indicates an exposure of the chromophore to the hydrophilic environments influenced by certain structural modification [35]. We deduced that the change of the structure of the yeast protein after the sound influx effects of the ultrasonic treatment and the attachment of the chitooligosaccharide influenced the binding of the betanin and facilitate the red shift of YCB.

Fig. 3.

(a) UV–vis absorption of yeast protein, betanin, YB, and YCB with the same protein content (0.5 mg/mL) and betanin content (0.3 mg/mL). (b) The solution state of yeast protein, betanin, YB, and YCB with the same protein content (0.5 mg/mL) and betanin content (0.3 mg/mL), which orders from left to the right.

The aqueous solution of aforementioned samples in solution was also observed to ascertain their dissolved state. The yeast protein had a transparent soluble state, and the betanin showed its red color in a transparent state (Fig. 3b). Specifically, with a same loading amount of the betanin (26.26 ± 1.12 %), the YCB and YB both showed the typically red color, suggesting that the ultrasonic treatment did not influence dissolved state of the protein and sustained the color of the betanin as a colorant. The maintenance of the naturally typical red color is crucial to its usage in food formulation, such as ice creams, beverage, yogurt, and candies that need red colors.

3.6. Morphology and particle size analysis

To investigate the effect of binding of chitooligosaccharide and betanin on the micromorphology of yeast protein, the TEM images of various samples - namely, yeast protein, ultrasound-treated yeast protein, YB, and YCB - were compared, as depicted in Fig. 4. The yeast protein exhibited a porous and networked flake-like morphology (Fig. 4a), a structural characteristic that potentially renders it highly suitable for the transport of bioactive substances [36]. The ultrasound-treated yeast protein displayed relatively smaller granules (Fig. 4b), indicating that the ultrasound led to the dispersion of block shapes. The unordered hydrophobic interactions may be reorganized during the ultrasonic treatment, facilitating the aggregated state of the protein to a smaller size. Both YB and YCB morphologies appeared in a granulated form, and even showed a more irregular and aggregated status (Fig. 4c and d), which suggested that the binding of chitooligosaccharide and betanin could change the morphology of yeast protein to be dispersed and disordered.

Fig. 4.

(a) SEM image of yeast protein. (b) SEM image of yeast protein with ultrasonic power (200 W) treatment. (c) SEM image of YB (The mass ratio of the yeast protein to betanin was 1:0.6). (d) SEM image of YCB (The mass ratio of the yeast protein to chitooligosaccharide to betanin was 1:3:0.6). (e) DLS of yeast protein. (f) DLS of yeast protein with ultrasonic power (200 W) treatment. (g) DLS of YB (The mass ratio of the yeast protein to betanin was 1:0.6). (h) DLS of YCB (The mass ratio of the yeast protein to chitooligosaccharide to betanin was 1:3:0.6).

DLS technology was subsequently employed to analyze the R_H of the particles in solution (Fig. 4e-h). The R_H value of yeast protein was in 478.6 nm, the ultrasound treatment led to the decrease of the main R_H of CFB to 408.3 nm, indicating that the ultrasound would reduce the diameter of the hydrated layer of the yeast protein. The formation of the YB generated the main R_H to 380.2 nm (Fig. 4g), a similar size distribution to that of ultrasound-treated yeast protein. In addition, a small proportion with a R_H of 199.3 nm was emerged (Fig. 4g), indicating that the binding of the betanin triggered the dispersion of the yeast protein aggregates. Similar results were also found in the size distribution of the YCB (Fig. 4h), indicating that the chitooligosaccharide binding maintained the majority of the typical size of ferritin at about 389 nm. These findings proved that the ultrasound-induced formation of the three-layer yeast protein-chitooligosaccharide-betanin complex could decrease the aggregation extent of the yeast protein, which may be useful for the preparation of the liquid form foods with a relatively stable and dispersed state.

3.7. Stability analysis of betanin

3.7.1. Thermal stability

Betanin is generally chemically unstable upon exposure to heat, light, and metal ions, leading to low bioavailability and a short half-life. Thermal treatment is a widely used food processing to disinfect and induce chemical reaction of ingredients. The stability of betanin was investigated in different samples, including betanin, YB, and YCB, in the temperature range from 30 °C, 55 °C, to 80 °C (Fig. 5). The results showed that as treatment time increased, the retention rate of betanin declined (Fig. 5), a phenomenon attributed to the decarboxylation reactions from heating that produce decarboxylated betanin [37]. Specifically, betanin demonstrated a higher retention in YB and YCB groups than that in the free betanin group for 30 °C treatment after 1 h, and the retention ratios gradually decreased with the increase in time (Fig. 5a), indicating that the yeast protein and the chitooligosaccharide were effective in improving the stability of betanin. Interestingly, a relatively higher retention rate of betanin in YCB at 55 °C for 3 h was observed, with a value of 58.7 %, a value significantly higher than that of YB (52.5 %) and free betanin (41.6 %) (p < 0.05) (Fig. 5b). By contrast, a higher temperature treatment (80 °C) even led to a rapid reduction in the retention ratio of betanin for all these three samples with no significant differences (p > 0.05) (Fig. 5c). Jiang et al. [38] have posited that alterations in pigment solution hues may not solely be attributed to pigment degradation but could also stem from the formation of pigment complexes. The yeast protein and the chitooligosaccharide in the form of YCB could protect betanin against its degradation and revealed a synergistic effect in protecting the betanin below 60 °C.

Fig. 5.

The retention ratio of the betanin in the free betanin, YB, and YCB after different treatments. (a) Thermal treatment at 30 °C. (b) Thermal treatment at 55 °C. (c) Thermal treatment at 80 °C. (d) Natural light treatment. (e) Natural light treatment (254 nm). (f) The metal lions (Cu²⁺ and Fe³⁺) treatment.

3.7.2. Light stability

The influence of natural light and UV radiation treatment on stability of betanin was studied to assess the defensive functions of the yeast proteins and the binding with chitooligosaccharide. It was found that the free betanin and the betanin in YB and YCB all underwent varying degrees of degradation after natural light and UV radiation treatment (Fig. 5d and e). The retention ratio of free betanin was 71.1 %, which was significantly lower than that of the YB with a value of 76.8 % after 6 h under natural light treatment, while the retention of YCB reached 81.0 % under a same treatment (Fig. 5d). The retention ratios of these three samples reduced more evidently under UV irradiation. Consistent with natural light exposure, betanin in YB and YCB remained significantly higher retention ratios (34.0 % and 39.5 %) than that in the free betanin (22.3 %) (p < 0.05) (Fig. 5e). These finding suggested that the photostability of betanin based on the yeast proteins and chitooligosaccharide could bear UV radiation and natural light treatments to a larger extent relative to the free betanin. This enhanced resilience is likely attributable to the attachment of yeast protein or chitosan oligosaccharide, which imparts a protective barrier against the light sources, thereby safeguarding the betanin.

3.7.3. Ion iron stability

Metal irons such as Cu²⁺ and Fe³⁺ have great destructive effect on the color of pigment. To investigate the roles of yeast protein and chitooligosaccharide in the stability of betanin, the retention ratio of the betanin in YB and YCB were evaluated (Fig. 5f). Notably, the retention rate of betanin in YB (52.2 %) was significantly higher than that in the untreated betanin (45.2 %) (p < 0.05), and the retention rate of betanin in YCB (57.1 %) was significantly higher than that in YB and betanin with the same Fe³⁺ loading level (0.3 mM) (p < 0.05). As for the Cu²⁺ treatment, the retention ratio of free betanin decreased by 32.9 %, which was significantly higher than that in YB and YCB with the decreased retention rates of 24.3 % and 22.8 % (p < 0.05), respectively. This phenomenon proved that the formation of YCB three-layer complex was crucial in protecting the betanin against the metal irons.

The protective effects of the yeast protein-chitooligosaccharide loading carrier can be attributed to several factors. One reason was that the yeast protein showed a shielded effect on the betanin. This impact was particularly evident in especial below 55 °C, a higher temperature higher than that may influence the structure of the yeast protein and thus destabilize the betanin. The weak interactions such as hydrogen bonds and van der Waals forces between the betanin and the protein would be helpful for the stabilization of bioactive molecules upon high temperature treatments [39]. Notably, the ultrasound treatment was effective in the loading of betanin to the protein, which revealed a pronounced effect in stabilizing the betanin. This deduction is supported by the significantly higher retention ratio of betanin compared to the untreated sample. Another important reason was inferred that the chitooligosaccharide could bind to the proteins and influence the interaction between the yeast protein and betanin (Fig. 2 and Table 2). This binding force can be strengthened resulted as a result of the chitooligosaccharide, and consequently, the ultrasound-assisted formation of YCB strengthened the binding among these three compounds and showed superiority in the stabilization of betanin in food processing.

3.8. Color difference analysis

Fig. 6a-f showed the images of the free betanin, YB, and YCB samples after different thermal-, light-, and metal ion-exposing treatments as shown in Section 2.10. The red color of the YB and YCB was obviously darker than that of the free betanin after the 30 and 55 °C incubation for 3 h (Fig. 6a and b). The 80 °C treatment, however, led to a dramatic fading of the typical color of the betanin in these three samples (Fig. 6c). Light exposure intensified the discoloration of betanin in YB and YCB (Fig. 6d and e), and YCB exhibited a deeper red relative to the YB after UV light treatment (Fig. 6e), suggesting that chitooligosaccharide binding decelerates betanin degradation upon light exposure. As for the metal ion treatment, a clear fading of the betanin was evident after 20 min incubation of Cu²⁺, while the YB and YCB remained a deeper red color (Fig. 6f-1). The Fe³⁺ had a more adverse effect on the free betanin state, and the YCB could effectively improve the color stability of the betanin (Fig. 6f-2).

Fig. 6.

The dissolved state (a-f) and the ΔE (g-l) of yeast protein, YB, and YCB after different treatments. (a) Thermal treatment at 30 °C. (b) Thermal treatment at 55 °C. (c) Thermal treatment at 80 °C. (d) Natural light treatment. (e) Natural light treatment (254 nm). (f1-2) The metal lions (Cu²⁺ and Fe³⁺) treatment. (g) Thermal treatment at 30 °C. (h) Thermal treatment at 55 °C. (i) Thermal treatment at 80 °C. (j) Natural light treatment. (k) Natural light treatment (254 nm). (l) The metal lions (Cu²⁺ and Fe³⁺) treatment.

The color changes were further observed by a colorimeter to analyze the color differences of different samples. ΔE value is an important parameter to indicate the overall color differences of the sample, and it was calculated by the formula (4). The ΔE values were compared for these three groups including the YB, YCB, and the free betanin. Usually, the higher the ΔE value, the greater the color it can change. In agreement with the image in Fig. 6a and Fig. 5b, the ΔE values for YB and YCB were significantly lower than that for the free betanin after 30 and 55 °C treatments (Fig. 6g and h), indicating that the yeast protein and chitooligosaccharide played a protective role for the betanin color upon a relative lower temperature. These three samples had similar ΔE values after 80 °C treatment, which was consistent with the state as shown in Fig. 6c. The ΔE for the samples after the natural and UV light treatments exhibited a similar trend to that after the thermal treatment at 30 and 55 °C (Fig. 6i and j). Specifically, the YCB had a lowest ΔE value after UV light exposure among these groups, indicating that the binding of chitooligosaccharide would weaken the easily fading property of the betanin. Furthermore, Fe³⁺ induced fading in betanin was substantially mitigated in YCB, as evidenced by its minimal ΔE value (Fig. 6k). Compared with the ΔE for the free betanin induced by the Cu²⁺, the YB, and YCB had a higher and similar ΔE (Fig. 6l), showed that the binding of chitooligosaccharide had little effect on the protective role in the betanin exposing to the Cu²⁺. Betanin as the natural pigment shows a characteristic red–purple color [2], its color stability is crucial for its utilization as natural colorant in food applications. The yeast protein and the chitooligosaccharide show superiority in protecting the color of the pigment thanks to their natural source and easy accesses, making them potentially suitable as novel base materials to extend the applications of betanin.

3.9. Analysis the betanin stability in the simulated gastrointestinal tract

The betanin stability in the gastrointestinal tract serves as an index to evaluate the functions of the yeast protein-chitooligosaccharide based particle to deliver pigments. The instability of the carrier material may cause the enzymic hydrolysis and the leak of the compounds in gastrointestinal tracts. To investigate the effects of yeast protein and chitooligosaccharide on the release of betanin, the retention ratio of betanin in the simulated fluids was studied. Different samples were conducted for pepsin digestion at pH 2.0 and were exerted to intestinal trypsin (pH 7.5), respectively. The YB showed a rapid release of betanin (Table 3), with a retention ratio of 45.6 % in simulated gastric tract during incubation of 2 h. In comparison, the release of betanin in YCB was more sustained by 10.2 % after 2 h of pepsin digestion, suggesting that the attachment of chitooligosaccharide was effective in slowing the release of betanin primarily due to the protective role of chitooligosaccharide against the hydrolysis by pepsin. The retention ratios of betanin in the simulated intestinal tract under different incubation time showed a similar trend to that in the simulated gastric digestion. The YCB could decrease the release ratio of betanin by 8.9 % after 2 h of intestinal digestion. It was deduced that the interaction between chitooligosaccharide and yeast protein potentially shields the enzymatic interaction sites on the protein, thereby diminishing its exposure, curbing protein hydrolysis, and consequently moderating the release rate of betanin in YCB [40]. The non-covalent interactions such as hydrogen bonds and van der Waals forces are likely involved in the binding of betanin to yeast protein, similar to the bindings observed in rice protein and soy isolate protein with betanin [36]. These interactions are key to betanin’s stability in the composite. Moreover, we consider the alterations in the secondary and tertiary structures of the yeast protein, induced by the conjugation with chitooligosaccharide and betanin (Fig. 1c, 1d, 2, and 3a), as significant factors. These structural modifications likely impact the folding of amino acids, further restricting protein hydrolysis and the subsequent release of betanin [41]. Such modulations are pivotal for betanin’s effectiveness, as its rapid exposure can be detrimentally impacted by the intricate pH and enzyme matrix within the gastrointestinal environment. The triple layer particles in the form of YCB sustained the release of bioactive pigment and increased the contact to the Intestinal cells, which will be beneficial for the bioavailability improvement of the betanin in in gastrointestinal environments.

Table 3.

Retention ratios of betanin in simulated gastric/intestinal tracts over 2 h of incubation.

Samples	Simulated gastric tract			Simulated intestinal tract
	40 min	80 min	120 min	40 min	80 min	120 min
YB YCB	73.2 ± 3.1 %b 80.1 ± 2.1 %a	61.2 ± 1.5 %b 68.0 ± 2.0 %a	45.6 ± 1.2 %b 55.8 ± 2.3 %a	38.2 ± 1.5 %b 45.7 ± 3.3 %a	28.1 ± 3.0 %b 36.2 ± 2.8 %a	11.2 ± 2.3 %b 20.1 ± 1.9 %a

Values in the row with different superscript letters (a and b) were significantly different (p < 0.05).

4. Conclusion

In this study, the novel fungus protein-yeast protein was applied to interact with the chitooligosaccharide and betanin induced by ultrasound treatment and to protect the color stability. The interaction mechanisms among the protein, chitooligosaccharide, and betanin assisted by ultrasound were evaluated and the role of the ultrasonic technique was highlighted. The ultrasound treatment influenced the binding, structure, and functional properties of the yeast protein-chitooligosaccharide-betanin complexes. Spectroscopic analyses, including circular dichroism and fluorescence spectroscopy, corroborated alterations in the secondary and tertiary structures of the yeast protein. Furthermore, scanning electron microscopy revealed that the complexes, YCB, crafted with the assistance of ultrasound, exhibit a porous and networked flake-like protein structure, which notably enhanced the thermal, photic, and metal ion stability of betanin. The triple layer particles also protected betanin against the color fading, and realized a controlled releasing of betanin in YCB. This work clarified the ultrasound assisted interaction mechanisms of protein-oligosaccharide-pigment molecules, and provides a guide for the application of the fungal protein source for stabilization of the thermal- and light-sensitive pigment. This work is also meaningful for the development of pigment combined with yeast proteins in food formulations, such as yogurt, ice creams, and beverage. In addition, the design of yeast protein-based food systems can offer a reference for the utilization of this new fungal protein resource.

CRediT authorship contribution statement

Rui Yang: Writing – review & editing, Writing – original draft. Jiangnan Hu: Writing – review & editing, Writing – original draft. Jiaqi Ding: Writing – review & editing. Runxuan Chen: Writing – review & editing. Demei Meng: Writing – review & editing. Ku Li: Writing – review & editing. Hui Guo: Writing – review & editing. Hai Chen: Writing – review & editing. Yuyu Zhang: Writing – review & editing, Supervision, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: