Formation Mechanism of Bleaching Damage for a Biopolymer: Differences between Sodium Hypochlorite and Hydrogen Peroxide Bleaching Methods for Shellac.

Bleached shellac, a widely used material in food processing and products, was deeply affected in terms of structures and properties by the bleaching method. In the present study, a marked difference was observed between the damage performances of sodium hypochlorite-bleached shellac (SHBS) and hydrogen peroxide-bleached shellac (HPBS). The main bleaching damage reactions of sodium hypochlorite were the addition of double bonds to generate chlorine and the oxidation of hydroxyl to form aldehydes or ketones. In the case of hydrogen peroxide, degradation of shellac resin was caused by the hydrolysis of ester bonds and the oxidation of hydroxyl groups to form aldehydes and ketones, as well as carboxylic acids with deep oxidation. Based on the structural characterization of shellac resin, the bleaching damages were affected by the bleaching agent via the oxidizable groups, such as the unsaturated double bonds, hydroxyl and aldehyde groups in cyclic terpenes, and fatty acid chains. The differences could be attributed to the action of sodium hypochlorite on the hydroxyl group of aldehyde or ketone. Conversely, hydrogen peroxide bleaching oxidized the hydroxyl group and aldehyde group to carboxylic acid and initiated the hydrolysis reaction of the ester bond of the shellac resin, leading to the degradation of the resin. Thus, understanding the mechanism underlying the bleaching damage could provide a scientific basis for the subsequent targeted regulation of bleaching damage.

Introduction

Shellac is a mixture of natural resin, dye, and wax, secreted by lac insects Kerria lacca after the sap is oozed from host plants.[1,2] Because of its natural, green, nontoxic, and degradable characteristics and properties, such as proper film formation, adhesion, and barrier, shellac has applications in food,[3,4] medicine,[5−7] clean coating,[8,9] edible coating,[10,11] and functional materials.[12,13] Shellac resins are lactones and lactides formed from long-chain hydroxy fatty acids (such as aleuritic acid) and sesquiterpene acids (such as shelloic acid);[14,15] the structure of these resins is rich in functional groups, such as carboxyl, hydroxyl, aldehyde, and double bonds.[16,17] The deep purple-brown color of shellac caused by lac dyes is a limiting factor in the application of shellac resin in some fields. Thus, obtaining a light-colored resin while maintaining its original structure, properties, and safety is currently under intensive focus.[18]

Bleaching and decoloration are the two main methods for humans to obtain light-colored shellac resins,[19,20] with the former mainly adapted in China.[21] The traditional bleaching method is to dissolve the shellac in alkaline solution and then use the bleach to destroy the chromophores of the lac dyes.[22] Sodium hypochlorite is the most commonly used bleaching agent for shellac. However, when the bleaching agent attacks the chromophore group during the process, it also reacts with the carbon–carbon double bond in the shellac resin to form combined chlorine[23,24] that belongs to the category of organic chlorine and has limited application in the field of food and medicine due to its detrimental effects.[25] In addition, the combined chlorine could be easily removed during the storage of shellac, which catalyzes the polymerization of shellac resin and leads to a significant reduction in the thermal life of bleached shellac to only 6 months in storage.[26] Interestingly, cleanness and nonchlorination of shellac bleaching have become an inevitable requirement for the green and sustainable development of the bleached shellac industry.

Hydrogen peroxide decomposes to produce oxygen and water, which are nontoxic and safe. Thus, it is a typical sustainable green bleaching agent.[27] In a previous study, we used hydrogen peroxide as a bleaching agent to successfully achieve complete chlorine-free bleaching of shellac.[20,28] However, other issues were identified due to the damage of hydrogen peroxide bleaching, such as the decrease in softening point and the increase in the acid value of peroxide hydrogen bleaching glue.[28] This caused a significant decrease in the properties of heat resistance and film formation of bleached shellac. Nonetheless, in the medical field, controlling the variation range of physical and chemical indexes of shellac is the key to stabilizing product performance and distinguishing quality grades.[19]

Bleaching damage has become a scientific bottleneck restricting the application of bleached shellac and is also a major constraint for the replacement of sodium hypochlorite bleach with hydrogen peroxide bleach.[28] Currently, only a few research reports are available on shellac bleaching damage. Despite some relevant studies[18,20,22,23,26,28] with different perspectives and contents, there is no systematic description or conclusion on the mechanism of shellac bleaching damage yet. From the perspective of bleaching damage and by comparing the changes in the physical and chemical indicators and the molecular structure and monomer composition of bleached shellac with respect to sodium hypochlorite and hydrogen peroxide on shellac resin, we aimed to deduce the differences between the two bleaching methods. The parameters that were assessed included the damage mode, action site, and the reaction mechanism underlying the shellac resin. In addition, these findings would guide and promote the subsequent targeted development and comprehensive application of the regulatory method for the damage of bleaching shellac by hydrogen peroxide.

Results and Discussion

Bleaching Damage and Changes in Properties

As shown in Figure , the properties of SHBS and HPBS differed markedly. Compared to shellac resin, the obvious change in SHBS was the drastically shortened thermal life. Conversely, the thermal life of HPBS was greatly extended with a reduced softening point. Shellac is a rare animal-based natural resin with a low-molecular-weight polymer structure and high polymer performance; these properties were related to the specific molecular structure and intermolecular force.[29,30] Therefore, the different (Figure a, softening point) and completely opposite (Figure b, thermal life) performances exhibited by the two bleaching methods were attributed to the bleach damage mechanism of shellac resin. First, the two types of bleached shellac were solubilized in 95% ethanol and 0.2 mol/L sodium carbonate solution, respectively, to evaluate the particle size (Figure S1). As shown in Figure A, the particle size in the medium did not change significantly compared to shellac resin, which indicated the alcohol solubility of the two bleached shellac resins. However, in the sodium carbonate solution (Figure B), the particle size of the SHBS (3014.3 nm) changed slightly compared to that of shellac (3008.7 nm), while the particle size of the HPBS decreased to 144.8 nm, suggesting that the dispersion of HPBS in the alkaline solution was significantly better than that of shellac and SHBS.

Figure 1

Changes in softening point (a) and thermal life (b) of shellac, SHBS, and HPBS.

Figure 2

Median particle size of shellac, SHBS, and HPBS in (A) 10 wt % ethanol solution (B) 10 wt % sodium carbonate solution (0.2 mol/L).

To further elucidate the difference in the performance between the two bleached shellac resins, we tested the melting process of the samples (Figure a) and found that similar to the softening point, the melting temperature of SHBS was similar to that of the shellac resin. However, unlike the softening point test results, HPBS did not show a significant melting peak on the DSC curve. According to the study by Limmatvapirat et al.,[31] the disappearance of the melting peak of shellac was related to the degradation (or hydrolysis) of the resin. In addition, in the FTIR chart of bleached shellac, the main absorption characteristics of the two bleached shellac were consistent with those of natural shellac (Figure b). The stretching vibration absorption peak of the hydroxyl O–H bond was found at 3435–3424 cm–1; 2930 and 2859 cm–1 were the C–H stretching vibration absorption peaks of CH2 and CH3, respectively; 1735–1704 cm–1 was the vibration absorption peak of the carbonyl C=O bond in aldehyde, ketone, acid, and ester of shellac; 1633 cm–1 was the asymmetric stretching vibration peak of the C=C group;[32,33] and 1254 cm–1 was the C–O bond stretching vibration peak. However, the relative peak intensity was obviously different. For example, compared to natural shellac, the ratio of carbonyl and hydroxyl absorption intensities of the two bleached shellac samples increased significantly (Table S1), which indicated that carbonyl groups increased or hydroxy groups decreased in bleach. Thus, based on the altered characteristics obtained by DSC and FTIR experiments, did it mean that bleaching damage by hydrogen peroxide was related to the degradation of shellac resin during the bleaching process? To clarify this issue, we conducted a detailed test on the changes in the main functional groups of the two bleached shellac resins.

Figure 3

DSC (a) and FTIR (b) characteristics of shellac, SHBS, and HPBS.

Bleaching Damage Mode and Action Site

Figure shows the changes in the hydroxyl group, aldehyde group, carboxyl group, ester group, double bond number, and chlorine content in the two types of bleached shellac, which were represented by the hydroxyl value, aldehyde value, acid value, ester value, and iodine value, respectively. Compared to the shellac resin, SHBS bleaching damage showed a decrease in hydroxyl value (Figure a) and iodine value (Figure e), an increase in aldehyde value (Figure b), and unchanged acid value (Figure c) and ester value (Figure d), suggesting that sodium hypochlorite bleaching exerted a less degradation effect on shellac resin (breaking ester bond). The main damage reactions are summarized as the oxidation reaction in Scheme , including oxidation of primary hydroxyl and addition of double bond by sodium hypochlorite, which, in turn, led to an increase in the aldehyde value and the chlorine content of the bleached shellac, respectively. Moreover, the increased chlorine content of SHBS (Figure f) could be ascribed to the reduced thermal life of bleached shellac.[26] In the bleaching process of hydrogen peroxide (Scheme ), in addition to the increased aldehyde value and acid value by the oxidation reaction, the decreased ester value and increased hydroxyl value of the bleached shellac indicated an obvious degradation effect of hydrogen peroxide bleaching on shellac resin. In Figure d, the ester value of shellac was 2786 μmol/g, while that of HPBS decreased to 1454 μmol/g (a decrease of 1332 μmol/g), indicating that the damaging effect of hydrogen peroxide bleaching was exerted via the ester bond hydrolysis of the shellac resin. Due to the generation of carboxyl and hydroxyl groups after the hydrolysis of the ester bond, the acid value of HPBS increased from 1288 to 2959 μmol/g, an increase of 1671 μmol/g, which was significantly higher than the decrease in the ester value, suggesting that in addition to the hydrolysis of ester bonds, there were also other oxidized groups contributing to the increased acid value. This phenomenon could be explained by the altered hydroxyl value. In Figure d, the decrease in the ester value was found to reach 1332 μmol/g, while in Figure a, the increase in the hydroxyl value of hydrogen peroxide bleaching was only 116 μmol/g, which differed from the equimolar hydrolysis characteristics of the ester bond. Thus, it could be concluded that the hydrogen peroxide bleaching of shellac should be accompanied by the oxidation of the hydroxyl group, which, in turn, would result in a slight decrease in the hydroxyl value despite the markedly elevated acid value (due to hydrolysis of the ester bond). In addition, the bleaching process of hydrogen peroxide might also be accompanied by double-bond oxidation (Figure e); however, this oxidative damage was not responsible for the changes in HPBS performance.

Figure 4

Changes of functional groups of shellac, SHBS, and HPBS: (a) hydroxyl value, (b) aldehyde value, (c) acid value, (d) ester value, (e) iodine value, and (f) chlorine content.

Scheme 1

Schematic Graph of Damage Mechanisms and Action Sites for Sodium Hypochlorite- and Hydrogen Peroxide-Bleached Shellac

(a) Addition reaction of double bond, (b) hydroxyl oxidized as aldehyde by sodium hypochlorite; (c) hydroxyl oxidized as ketone by sodium hypochlorite; (d) hydroxyl oxidized as aldehyde/carboxyl by hydrogen peroxide, (e) hydroxyl oxidized as carbonyl by hydrogen peroxide, (f) double-bond oxidation by hydrogen peroxide.

The mutual conversion correlation among hydroxy, aldehyde, acid, and ester groups (Figure ) was quantified to evaluate the effect of damage mechanism of different bleaching methods on these four functional groups in the two bleaching processes. The derivation and calculation processes were as follows:

Figure 5

Transformational relationship among hydroxyl, aldehyde, carboxyl, and ester groups of bleached shellac in the bleaching process.

Since the hydrolysis of ester could increase the amount of hydroxyl group, and the hydroxyl groups continued to be oxidized to aldehydes and ketones, the amount of change in the hydroxyl value should be expressed as followswhere ΔH represents the variance of the hydroxy group of bleached shellac relative to the shellac resin, EH represents the amount of hydroxyl group generated by the hydrolysis of the ester, HA is the amount of aldehyde group formed by the oxidation of the hydroxyl group, and HC was the amount of ketone carbonyl group formed by the oxidation of the hydroxyl group.

The hydrolysis of the ester bond and the oxidation of the aldehyde group both increased the content of carboxyl groups and were designated as EC and AC, respectively; the change in the acid value could be expressed aswhere ΔC is the amount of carboxyl group change of bleached shellac relative to shellac resin, EC is the amount of carboxyl group generated by hydrolysis of the ester bond, and AC is the amount of carboxyl group generated by oxidation of the aldehyde group.

The change in the aldehyde group value was assessed from two aspects: oxidation of the hydroxyl group (increasing amount) and oxidation of aldehyde group to carboxylic acid (decreasing amount). Therefore, the change in the aldehyde value could be expressed as followswhere ΔA is the change in the carboxyl group of bleached shellac relative to shellac resin.

Since the hydrolysis of the ester bond produced equimolar amounts of carboxyl and hydroxyl groups, the amount of change in the hydroxyl and carboxyl groups caused by the hydrolysis of the ester bond could be expressed as followswhere ΔE is the altered amount of ester group of bleached shellac relative to shellac resin.

According to eqs –4, the calculation formulas for HC, HA, and AC were, respectively, as follows

The calculations of HC, HA, and AC are shown in Table .

Table 1

Transformational Amount of Hydroxyl, Aldehyde, Carboxyl, and Ester Groups of Bleached Shellac in the Bleaching Process

bleached shellac	ΔH	ΔA	ΔC	ΔE	HA	HC	AC	EH	EC
SHBS (μmol·g–1)	–269.8	254.0	21.80	–80.20	195.6	154.4	–58.40(0a)	80.20	80.20
HPBS (μmol·g–1)	115.5	51.20	1671	–1332	389.9	826.5	338.7	1332	1332

Due to the test error of different functional groups, when AC was negative, it was defined as zero.

According to the conversion equation shown in Figure and based on the changes in the hydroxyl value, aldehyde value, acid value, and ester value shown in Figure , the mutual conversion amount between the four functional groups was calculated (Table ). Thus, in the sodium hypochlorite bleaching process, only a small amount of resin ester bonds was hydrolyzed (EH or EC). The main reaction was the oxidation of hydroxyl groups to aldehyde groups (HA) and keto carbonyl groups (HC). In hydrogen peroxide bleaching, a large number of ester bonds were hydrolyzed to form hydroxyl and carboxyl groups. Simultaneously, large amounts of hydroxyl groups were further oxidized, mainly to keto carbonyl groups, and partially to aldehyde groups.

In summary, the damage mechanisms underlying sodium hypochlorite and hydrogen peroxide bleaching on shellac resin differed markedly. Supposedly, the damage of sodium hypochlorite bleaching on shellac was mainly affected by the oxidation reaction of the hydroxyl group and the double bond, with the former being the cause of the increased aldehyde value and the latter being the reason for the introduction of combined chlorine, which reduced the thermal life of SHBS; sodium hypochlorite bleaching did not cause significant degradation of shellac resin. On the other hand, hydrogen peroxide bleaching caused damage to the shellac resin through ester bond hydrolysis and hydroxyl oxidation. Moreover, ester bond hydrolysis caused a decrease in molecular weight and softening point and an increase in the acid value of bleached shellac, while the oxidation of primary hydroxyl groups resulted in elevated carboxyl groups (or aldehyde groups) and the oxidation of the secondary hydroxyl group raised the content of the ketone carbonyl group.

Characterization and Verification of the Damage Mechanism

Average Molecular Weight by Gel Permeation Chromatography (GPC)

Table shows the average-molecular-weight changes of the two bleached shellacs relative to the shellac resin. The similar molecular weights of SHBS and shellac indicated that sodium hypochlorite bleaching did not cause degradation of shellac resin. The number-average, weight-average, and Z-average molecular weights of HPBS were greatly reduced, proving that hydrogen peroxide bleaching caused significant degradation damage to shellac resin. This finding was well corroborated by the change in the number of ester functional groups (Figure d), confirming the degradation mechanism of hydrogen peroxide bleaching damage (Scheme ).

Table 2

Average Molecular Weights of Shellac, SHBS, and HPBS by the GPC Method

samples	Mn	Mw	Mz	D
shellac	1410 ± 14	3905 ± 35	2195 ± 7	2.76 ± 0
SHBS	1450 ± 28	4050 ± 240	2280 ± 156	2.77 ± 0.06
HPBS	996 ± 3	1580 ± 14	1195 ± 7	1.59 ± 0.01

13C-NMR

In the NMR carbon spectra of the two types of bleached shellac, the absorption characteristics of the carbon atoms related to bleaching damage altered significantly. First, the two bleached shellacs had a newly generated carbon peak at δ48.70 (Figure B,d′), which was attributed to the ortho carbon absorption peak of the carbonyl group due to the oxidation of the secondary hydroxyl group of the chain fatty acid (such as aleuritic acid) to the ketone group. In addition, the absorption peak of the carbonyl group at δ204.12 of the two bleached shellacs was enhanced (Figure A), which was the characteristic of the oxidation of hydroxyl groups to carbonyl groups and was consistent with the results of Table S1 in the FTIR test. Second, SHBS had a new absorption peak at δ51.88 (Figure B,c′), which was the absorption peak of the carbon–chlorine bond after the addition of sodium hypochlorite and a double bond. In addition, relative to the shellac resin, the absorption strengths of the primary hydroxyl groups of the chain end and the terpene acids (δ62.85, a; δ71.31, b) of the two bleached shellacs were reduced significantly. This phenomenon was consistent with the characteristic of the oxidation of primary hydroxyl groups of shellac resin by bleaching agents and also corroborated with the oxidation results of the hydroxyl value in Figure and Table .

Figure 6

13C-NMR of shellac, SHBS, and HPBS in the chemical shift ranges of 230–100 ppm (A) and 100–40 ppm (B).

Detection of the Monomer Composition

Shellac resin was a polymer of monomers formed by fatty acid chains (such as aleuritic acid) and cyclic terpene acids (such as shelloic acid). The bleaching damage referred to the changes in the structures or interaction forces of the shellac resin monomer and its constituent compounds. Therefore, the detection of the bleached shellac monomer composition was the key to confirm the above damage mechanism based on the composition of the compound and the molecular level of the shellac resin monomer; also, it was the core method to clarify the bleaching damage reaction mode and the action site. To this end, after saponification of the shellac resin, SHBS, and HPBS, the sample monomer compositions were detected by HPLC-QToF-MS/MS (Figure S2). According to the previous test results of shellac resin monomer composition,[34−36] the mass spectrum (Figure S3) and MS/MS spectrum (Figure S4) data were used to classify the types of compounds that made up the shellac resin monomer[37] (Table S2). To eliminate the detection error caused by the difference in ionization responsiveness of the monomer compounds of different structures in the shellac resin,[38] we employed the peak area normalization method to quantitatively analyze the composition of the shellac resin monomer compounds via HPLC-ELSD (Table ), and based on the retention time in Figure S2, the high content of terpene acid and fatty acid peaks were assigned (Figure ). Furthermore, the shellac resin monomer could be divided into two parts, cyclic terpene acid and chain fatty acid, with aleuritic acid (Ale) as the dividing threshold. As opposed to the shellac resin, the obvious changes in SHBS were the disappearance of the 16-H-9-A peak (the content drops to 0, Table ) and the decrease of chain component with double bonds, which was in accordance with the addition mechanism of double bonds by sodium hypochlorite (Scheme a); these findings were similar to the test results in Figure , showing reduced iodine value and increased chlorine content. However, in HPBS, the peaks of JA and 16-H-9-A vanished, and the peak intensities of JAI and SAI were significantly weakened. These phenomena were in agreement with the mechanism of oxidizing the hydroxyl groups and double bonds of shellac resin by hydrogen peroxide (Scheme d,f) that was confirmed by the test results in Figure , wherein the acid value and the aldehyde value increased and the iodine value decreased. This conversion relationship was further clarified by the relative percentage changes in terpene acid and the fatty acid of shellac resin before and after bleaching (Table ). In sodium hypochlorite bleaching, the relative percentage of LA, which had CHO as the R group in terpene, increased from 6.78 to 7.55%. This was in accordance with the characteristics of sodium hypochlorite bleaching that oxidized primary hydroxyl groups to aldehyde groups. Concurrently, the relative percentage of JA with CH2OH, similar to the R group, decreased from 7.75 to 2.57%. These were caused by the oxidation of CH2OH to CHO (Scheme b), while in hydrogen peroxide bleaching, this oxidation would be intense. The JA content was reduced from 7.75% to 0, and the relative content of SA was increased from 2.14 to 12.34%. Moreover, SA was obtained by oxidizing the R group of JA and CH2OH to COOH, which indicated that JA and SA could be converted by oxidation and that the R group of terpene was the oxidative damage site of hydrogen peroxide bleaching on the shellac resin. This finding was in line with the highly increased HPBS acid value.

Table 3

Relative Contents of Terpenic Acids and Fatty Acids Obtained from Saponified Shellac, SHBS, and HPBS

				R and R′ groups of terpenic acids		relative content/%
compounds and number		name	label	R	R′	shellac	SHBS	HPBS
terpenic acids	1	shellolic acid	SA	COOH	CH2OH	2.14 ± 0.02	1.60 ± 0.02	12.34 ± 0.05
	2	jaksholic acid	JA	CH2OH	CH2OH	7.75 ± 0.04	2.57 ± 0.01	0.00
	3	jaksholic acid isomer	JAI	CH2OH	CH2OH	7.51 ± 0.02	2.21 ± 0.01	0.66 ± 0.01
	4	shellolic acid isomer	SAI	COOH	CH2OH	15.52 ± 0.01	5.90 ± 0.05	0.47 ± 0.05
	5	laccilaksholic acid	LLA	CH2OH	CH3	3.70 ± 0.02	4.05 ± 0.08	9.00 ± 0.06
	6	laccijalaric acid, jalaric acid	LJA, LA	CHO	CH2OH	6.78 ± 0.04	7.55 ± 0.09	3.39 ± 0.09

Figure 7

Chromatogram of saponified shellac, SHBS, and HPBS by the HPLC-ELSD method.

Conclusions

Bleaching is a primary method for obtaining light-colored shellac resins, and revealing the mechanism of bleaching damage is the key to understanding the causes of performance changes of the bleached shellac. The present study focused on bleaching shellac by sodium hypochlorite and hydrogen peroxide and systematically evaluated the physicochemical properties, functional group content, resin structure, and monomer composition of bleached shellac from the perspective of bleaching damage. Thus, a significant difference was detected in the damage mechanism, action site, and bleach damage performance between sodium hypochlorite and hydrogen peroxide bleaching on shellac resin. The bleaching damage mechanism of sodium hypochlorite was the addition and oxidation reaction. The sites of action were unsaturated double bonds and hydroxyl groups in the cyclic terpenes and chain fatty acids of the shellac resin. The addition reaction of the double bond produced combined chlorine, which increased the chlorine content of SHBS and shortened the thermal life. The oxidation of hydroxyl groups to aldehydes or ketones greatly increased the aldehyde group value of bleached shellac. The mechanism of bleaching damage of hydrogen peroxide was the degradation and oxidation of shellac resin. The action sites were the ester and hydroxyl groups in the shellac resin, which affected hydrolysis and hydroxyl group oxidation reaction, respectively. The hydrolysis of the ester decreased the molecular weight of the bleached shellac, triggered the decrease of the softening point, and the acid value increased. Thus, hydrogen peroxide bleaching oxidized the hydroxyl groups not only to aldehydes or ketones but also to carboxylic acids.

Experimental Section

Materials

The sodium hypochlorite-bleached shellac (SHBS) and shellac were commercial products and purchased from Xilaike Biotechnology Co. Ltd. (Kunming, China). Hydrogen peroxide-bleached shellac (HPBS) was prepared in laboratory by our reported methods.[28] The other chemical agents were all of analytical grades and purchased from Aladdin Co. Ltd. (Shanghai, China).

Physicochemical Index Detection

The physicochemical indexes such as softening point and thermal life and the chemical indexes like acid value, iodine value, and chlorine content were evaluated according to the methods specified in the Chinese National Standard GB/T 8143-2008. The hydroxyl value was determined by the acetic anhydride method[39] based on the following formulawhere V1 is the volume of potassium hydroxide–ethanol solution used by the blank sample (L), V2 is the volume of potassium hydroxide–ethanol solution consumed by the sample (L), C1 is the concentration of potassium hydroxide–ethanol solution (mol·L–1), m1 is the sample mass (g), X1 is the acid value of the sample (mol·g–1), and X2 is the hydroxyl value of the sample (mol·g–1).

The aldehyde value was determined by the hydroxylamine hydrochloride method[40] using the formulawhere V3 is the volume of potassium hydroxide–ethanol solution consumed by the sample (L), V4 is the volume of potassium hydroxide–ethanol solution used by the hydroxylamine hydrochloride solution (blank, L), C2 is the concentration of potassium hydroxide–ethanol solution (mol·L–1), m2 is the sample mass (g), X1 is the acid value of the sample (mol·g–1), and X3 is the aldehyde value of the sample (mol·g–1). N,N-Dimethylformamide (DMF) was used as solvent of shellac or bleached shellac.

The ester value was determined by the saponification method,[41] which is widely used for oils. The calculation formula was as followswhere V5 is the volume (L) of HCL standard solution for titration of the blank, V6 is the volume (L) of HCL standard solution used for sample titration, C3 is the concentration of HCl standard solution (mol·L–1), m3 is the sample mass (g), X1 is the acid value of the sample (mol·g–1), and X4 is the saponification value (mol·g–1).

Characterization

Fourier transform infrared (FTIR) spectroscopy (Tenson 27, Bruker, Leipzig, Germany) and differential scanning calorimetry (DSC200F3, Netzsch, Germany) were employed to analyze the shellac and bleached shellac, respectively. The test conditions of DSC included a scanning range of 20–400 °C at a heating rate of 10 °C/min. Gel permeation chromatography (GPC, Agilent 1260 Infinity) was used to detect the average molecular weight and their distribution characteristics of shellac and bleached shellac. The calibration curve was calculated by polystyrene (Agilent, Mp = 162, 380, 580, 770, 970, 1370, 2170, 2930, 4910) as standard substance. The mobile phase and solvent were all tetrahydrofuran (THF, chromatographic grade), and the flow rate was 0.8 mL/min. Characterizations: The particle size distributions of the samples were measured using dynamic light scattering (DLS, Nanotrac wave, Microtrac, Montgomeryville, PA). The sodium hypochlorite- and hydrogen peroxide-bleached shellac were, respectively, dissolved in 95% ethanol and 0.2 mol/L sodium carbonate solution to 10% (w/v) concentration. Nuclear magnetic resonance (NMR) carbon spectroscopy was performed on a 500 MHz instrument (AVANCE III, Bruker, Germany). The chemical shifts relative to that of deuterated dimethyl sulfoxide (DMSO-d6, δc = 39.60) were recorded.

HPLC-TOF-MS/MS and HPLC-ELSD Analysis

Saponification of Shellac

The saponification process of shellac on the basis of the literature[42] and the main method are shown in Scheme . Shellac or bleached shellac (50 g) was put in a 250 mL round-bottom flask with a condenser, and the saponification process was implemented in 200 mL of a 25% (w/w) sodium hydroxide solution for 30 h at 90 °C. The saponified solution was salted out by a saturated sodium chloride solution to remove aleuritic acid. Diatomite was added into the salted out solution, and the filtrate was obtained after filtration. The filtrate was acidified by 18% HCl solution to pH ≈ 2.0, and the viscous deposition was obtained after washing, filtration, and drying. The product was dissolved in methanol for subsequent qualitative and quantitative analyses. The acidified filtrate was dried in an oven at 80 °C. The dried product was extracted by methanol and kept ready for qualitative and quantitative analyses.

Scheme 2

Schematic Diagram of the Saponification Process of Shellac and Bleached Shellac

HPLC-TOF-MS/MS Qualitative Analysis

Chromatographic separation was carried out on Waters Acquity HPLC (Waters). The temperature of column Zorbax 5 SB-C18 (250 mm × 4.6 mm, 5 μm) was 30 °C, the injection volume was 10 μL, and the mobile phase was acetonitrile (B) and 0.1% formic acid–water (D). A gradient elution program was utilized for the separation and determination, B (5–100%) 60 min, and the flow rate was 1.0 mL/min. Mass spectrometric analysis was performed on a Waters Micromass QuattroPremier triple quadrupole mass spectrometer (Waters) with the capillary voltage 3.5 kV, cone voltage 5 V, RF lens voltage 0.5 V, source temperature 120 °C, dissolution temperature 350 °C, airflow 600 L/h, cone airflow 50 L/h, and acquisition mode 4-channel selected ion recording (SIR) mode. MS and MS/MS spectra were analyzed by HPLC-MS MassLynx V4.1 software.

HPLC-ELSD Quantitative Analysis

Liquid chromatogram separation was performed on an Agilent 1260 HPLC system. The chromatographic separation condition was the same with the qualitative analysis method by HPLC-TOF-MS/MS. The optimum parameters of evaporative light scattering detector (ELSD) for evaporator temperature, drift tube temperature, and carrier gas (high-purity nitrogen) flow rate were determined as 70 °C, 60 °C, and 1.6 L/min, respectively. Aleuritic acid was chosen as standard to correct the difference of retention time between LC-MS and HPLC-ELSD.