Ultrasound in the deproteinization process for chitin and chitosan production.

Recently, chitin and chitosan are widely investigated for food preservation and active packaging applications. Chemical, as well as biological methods, are usually adopted for the production of these biopolymers. In this study, modification to a chemical method of chitin synthesis from shrimp shells has been proposed through the application of high-frequency ultrasound. The impact of sonication time on the deproteinization step of chitin and chitosan preparation was examined. The chemical identities of chitin and chitosan were verified using infrared spectroscopy. The influence of ultrasound on the deacetylation degree, molecular weight and particle size of the biopolymer products was analysed. The microscopic characteristics, crystallinity and the colour characteristics of the as-obtained biopolymers were investigated. Application of ultrasound for the production of biopolymers reduced the protein content as well as the particle size of chitin. Chitosan of high deacetylation degree and medium molecular weight was produced through ultrasound assistance. Finally, the as-derived chitosan was applied for beef preservation. High values of luminosity, chromatid and chrome were noted for the beef samples preserved using chitosan films, which were obtained by employing biopolymer subjected to sonication for 15, 25 and 40 min. Notably; these characteristics were maintained even after ten days of packaging. The molecular weight of these samples are 73.61 KDa, 86.82 KDa and 55.66 KDa, while the deacetylation degree are 80.60%, 92.86% and 94.03%, respectively; in the same order, the particle size of chitosan are 35.70 μm, 25.51 μm and 20.10 μm.

Introduction

Among the biopolymers, chitosan has become very attractive in the last few decades. It is well known for its wide range of applicability in the fields of cosmetics, environment, food and health. Chitosan is usually obtained from its chitin precursor through the deacetylation reaction. Meanwhile, chitin is a polysaccharide, which is widely distributed in nature, available from renewable resources, and being the second largest natural polymer (following cellulose). It is found in the crustaceańs skeleton, insect́s cuticle, the cell wall of fungi, and annelids. Consequently, its rate of replacement in the biosphere is almost double as that of cellulose. In general, chitin is a non-reactive and insoluble biopolymer, resembling the characteristics of cellulose in solubility and chemical stability. Crustaceans are the main source of chitin. The production of chitosan generally involves three steps, namely, demineralization, deproteinization and deacetylation. Sometimes, an additional depigmentation step is also employed. The first step of synthesis of biopolymer requires treatment with strong acids such as HCl, while the second and third steps require reacting with strong bases such as NaOH and KOH.

A range of applications arising from chitosan motivated investigations on the new routes of obtaining it or its synthesis as well as intense optimization of the current processes. The main efforts have been addressed on the deacetylation reaction, where chitin is converted into chitosan. Different concentrations of NaOH or KOH and a range of relatively high temperatures, together with a longer treatment time are some of the strategies studied for the conventional production of biopolymers [1]. Microwave and ultrasound-assisted [2], [3] deacetylation reactions have also been proposed for the synthesis of chitosan, especially the former, was developed with non-isothermal and isothermal experiments to derive the biopolymer with controlled characteristics [4]. However, the major disadvantage of all these methods is the generation of sludge owing to the employed chemicals, including those generated in the demineralization and deproteinization reactions. Reducing the amount of residues as well as reaction time without affecting the properties of chitosan would be desirable, mainly when the process is implemented industrially.

In the food industry, ultrasound or sonication is employed in different ways and for different purposes. The isolation of several cephalosporins from milk was successfully performed with the application of ultrasound [5]. The inclusion of heat, pressure and their combination, along with ultrasound, has shown efficacy in the inactivation or killing of the microorganisms [6]. Application of ultrasound in food technology including processing, preservation and extraction as well as inactivation of enzymes has been reported to be responsible for the deterioration of fruit and juices [7], [8]. Hence, including ultrasound to a wide range of food processes improve overall process efficiency giving rise to maximum reproducibility, higher purity of the final product, reduction in the processing time and lowering of the operational cost. Also, usage of ultrasound in the deproteinization step for the synthesis of chitosan could eliminate the post wastewater treatment and eventually reduce the time and energy associated with the synthesis of biopolymer. In this study, the optimization of obtaining chitosan from shrimp skeletons through ultrasound application is addressed, specifically on the deproteinization step. The employment of ultrasound in this step without utilizing any chemicals is beneficial in the production process. The impact of ultrasonication time on the resultant products was considered. Chitin was deacetylated to obtain chitosan, which was further used in the preparation of active packaging film to be applied in the preservation of beef.

The present work is focused on eliciting the effect of ultrasound in the production of chitin. Studies on protein content, crystallinity as well as morphology and particle size were carried out to support the relevance of ultrasound application. After obtaining chitosan, its functionality was studied in the preservation of the bright red colour of beef samples. This property was measured through the luminosity, the chromatid and the chrome. Infrared spectroscopy, molecular weight and deacetylation degree complemented characterization of the obtained samples.

Experimental details

Sample collection and preparation

Raw shrimp shells were obtained from a local seafood restaurant and cleaned thoroughly. They were washed with distilled water and dried in a conventional heater at 90 °C for 3 h. After that, the shells were pulverized effectively to a particle size in the range of 44–53 μm. Each 1 kg of shrimp generates 0.40 kg of skeletons; the size of the former is approximately 15 cm.

Isolation of chitin

The demineralization of the sample was performed using 0.6 M HCl with the ratio of dried shells to an acid solution of 1:11 (w/v) for 3 h at 30 °C and stirring at 300 rpm. The demineralized sample was then washed until its pH reached neutral and then dried at 90 °C for 3 h. The deproteinization was carried out using a high-frequency ultrasonic bath. The demineralized sample was sonicated in deionized water for 10, 15, 20, 25, 30, 35, and 40 min. For comparison, chitin deproteinization using NaOH was used as a control [9]. The final chitin product was washed with distilled water until it became neutral and then dried at 90 °C for 3 h.

Determination of the protein content

The remnant of proteins in the samples was determined by measuring the total nitrogen using the micro-Kjeldahl method [10]. The protein content was determined using Eq. (1).

Where N is the normality of HCl, V is the sample’s HCl volume (mL) - control’s HCl volume (mL), 14.01 is the atomic weight of nitrogen, m is the sample mass (g), and F is 6.25 (the assigned value to proteins).

Deacetylation of chitin

To convert the as-obtained chitin into chitosan, the following alkali treatment was considered: NaOH (50%) was added to chitin in the proportion of 1:4 (w/v), and the system was constantly stirred at 700 rpm. It was initially heated and maintained first at 70 °C for 2 h, followed by heating at 115 °C for another 2 h. A condensing system was used to avoid the evaporation loss of NaOH and to enhance the contact between NaOH and chitin. Following this, the obtained deacetylated product was filtered, washed until neutral and finally dried to obtain chitosan. The temperatures were maintained constant during this deacetylation.

Characterization using infrared spectroscopy

The identification of chitin and chitosan was made using infrared spectroscopy. A Perkin Elmer spectrophotometer with a fast Fourier transform and ATR system was employed for this purpose. The scan was carried out in the range of 4000–650 cm−1 employing eight scans with a resolution of 2 cm−1. Higher scan rates and resolution did not lead to any significant difference in the obtained results. Humidity (0%) and a temperature of 26 °C were maintained while obtaining the spectra to ensure proper determination of the –OH groups. Before analysis, the samples were dried in a heater at 105 °C for 4 h, and about 0.05 g of the sample was subjected to analysis.

Determination of the deacetylation degree of chitosan

The deacetylation degree (DD) was determined by acquiring the infrared spectrum of chitosan, as reported earlier [11]. The absorption bands at 1320 cm−1 (amine III) and 1420 cm−1 (–CH2) were taken as the base. Accordingly, DD was determined using the following Eq. (2) [12].

Where, AD is the acetylation degree (%), DD = 100-AD, A is the area under the curve of the absorption band at 1320 cm−1, and A is the area under the curve of the absorption band at 1420 cm−1.

Determination of the molecular weight of chitosan

The molecular weight was determined from the viscosity measurements. An Ubbelohde capillary viscometer was introduced into a thermostatic bath. Solutions of chitosan of different concentrations (0.08, 0.12, 0.16, 0.2 and 0.24 g mL−1) were prepared in a buffer solution of acetic acid (0.30 M)/sodium acetate (0.20 M) at 25 °C. The flow times for the pure solvent and the solutions of chitosan were recorded to calculate the relative viscosity. The intrinsic viscosity was determined using the following Mark-Houwink-Kunh-Sakurada (MHKS) equation (Eq. (3)).

Where [η] is the intrinsic viscosity, K and a are the empirical constants which are dependent on the solvent-polymer system at a given temperature. For the given chitosan solution system, the values of the constants were taken as K = 7.4 x10-4 cm3 g−1 and a = 0.86, as reported by Rinaudo [13].

X-ray diffraction

The X-ray diffraction patterns of chitin and chitosan were determined using a Bruker D8 Discover diffractometer with CuKα radiation of wavelength (λ) 1.54 Å and Linux Elle detector at the operational conditions of 40 kV and 40 mA. The continuous scanning was performed using 0.2 g of sample, a step size of 0.2° and a step time of 3 s in the range of 5-70°.

Scanning electron microscopy

The microscopic characteristics of chitin and chitosan were observed through a JEOL JSM 6610 LV scanning electron microscope to understand the morphology of the as-obtained biopolymers. The samples were sputtered with gold before the measurements to enhance their conductive nature.

Chitosan functionality in the preservation of beef

Steak and ground beef purchased from a local supermarket were used to study the functionality of chitosan. Portions of 1 g of the meat were covered with a chitosan membrane of 10 cm diameter. Membranes were prepared from chitosan which was previously obtained in an aqueous solution of acetic acid (1 M) with glycerol as an additive. About 0.2 g of chitosan was dissolved in 10 mL of 1 M acetic acid solution. Then, 0.4 mL of comestible glycerin was added with continuous stirring until a gel-like mixture was obtained. This was then deposited in a Petri dish and dried at 40 °C for 12 h to obtain the final membrane [14]. All food samples were stored at 4 °C for the entire study period of 10 days. The control samples for this analysis correspond to those without any coverage. The luminosity of the samples was measured with a HunterLab colorimeter for every 24 h. With this parameter, the chroma (Cab*), the tune (hab*) and the colour difference (ΔE) for the meat samples were obtained using the following Eqs. (4)-(6), respectively [15].

Where a* represents the red and green colours, b* the yellow and blue colours and L* the luminosity.

Results and discussion

Chitin and chitosan samples

Chitin and chitosan samples were obtained in a powder form. The colour of chitosan was measured with a HunterLab colorimeter, and the whiteness index (WI) of the polymer was estimated through Eq. (7) [16].

Where L* is the luminosity, a* measures the red and green colours and b* measures the yellow and blue colours. The WI of the obtained chitosan samples subjected to different sonication times is reported in Table 1. For 20 min of sonication, it reached a higher value. With an increase in sonication time from 25 to 35 min, a decrease in WI was observed. This could be due to the oxidation of pigments in the skeletons, namely, astaxanthin which intensifies the colour of chitosan [17]. At 40 min, this index again augmented due to an important transformation of the molecule, which is discussed in the following section.

Table 1

Whiteness index (WI), deacetylation degree (DD), molecular weight (MW) and classification according to the MW of chitosan samples.

Sonication time (min)	WI	DD (%)	MW	Classification
10	40.974	73.314	1.15	Low
15	41.697	80.604	73.61	Medium
20	43.307	90.965	11.8	Low
25	39.478	92.860	86.82	Medium
30	35.918	100.000	9.68	Low
35	35.784	93.134	60.51	Medium
40	36.542	94.034	55.66	Medium

Infrared spectroscopy analysis

Fig. 1a shows the infrared spectra of the chitin samples. The spectrum of chitin obtained through the chemical method is also included. The characteristic absorption bands of chitin could be observed in all the samples. The –OH vibration is reflected in the region of 3431–3484 cm−1, whereas NH2 and NH amides are in the region of 1621–1624 cm−1 and 1533–1545 cm−1, respectively. Concerning the chitin fingerprint, the C-O-C band stretching could be identified in the region of 1146–1156 cm−1 and C-O at 1073–1014 cm−1. Additionally, the deformation of C–H is found in the region of 883–897 cm−1. Similar absorption bands were also reported for chitin obtained through various methods [18], [19].

Fig. 1

(a) FTIR spectra of chitin extracted from shrimp shells using ultrasound in the deproteinization at different times of sonication. (b) FTIR spectra of chitosan obtained from the deacetylation of chitin using ultrasound in the deproteinization process at different times of sonication.

Besides the fingerprint of chitin, the depth in the absorption bands of the amines (NH and N) was larger in the chitin sample obtained using longer sonication time, attributed to changes in the acetylation [20]. This indicates that the yield in the extraction of proteins is higher when the sonication time was increased, resulting in the formation of purer chitin [21]. This was also confirmed through the absence of bands in the range of 1650–1700 cm−1 corresponding to the C = O groups of the terminal COOH, which is the characteristic of proteins. It could be observed from Fig. 1a that the chitin structure obtained from the chemical method matches the structure of chitin obtained through ultrasound. Moreover, the existence of a few absorption bands in the range of 1700–1758 cm−1 indicate that some proteins are not eliminated. Deproteinization with chemical method requires 24 h whereas for the ultrasound technique, it is less than 40 min notably without incorporating any chemical reagents.

The main derivative of chitin is chitosan. Fig. 1b depicts the infrared spectra of all the chitosan samples obtained from their respective chitin precursors. Each of them contains the characteristic absorption bands and match with the standard chitosan sample (procured from Sigma) and those reported in the literature [22]. The stretching of –OH and –NH could be found in the regions of 3432–3439 cm−1 and 3255–3306 cm−1, respectively. The asymmetric stretching of CH2 and the symmetric stretching of CH are in the range of 2914–2927 cm−1 and 2847–2875 cm−1, respectively. The reflex peaks of NH2 for the amides could be observed in the region of 1621–1653 cm−1 whereas for NH in the region of 1541–1558 cm−1. Also, the absence of absorption bands around 1540 cm−1 indicates the absence of proteins, as reported in the literature [23]. The fingerprint bands of the asymmetric stretching of C-O-C and the symmetric stretching of C-O along with CH3 are in the regions of 1148–1154 cm−1 and 1061–1072 cm−1, respectively, confirming the presence of chitosan. In the chitosan sample obtained completely through the chemical method, an absorption band could be observed at 1544 cm−1, confirming that the proteins were not eliminated even in the deacetylation step. This elucidates the need for more aggressive condition in the deproteinization process of the chemical method, especially with the inclusion of more chemicals and longer treatment times.

The characteristic absorption bands (1541–1558 and 1306–1312 cm−1) of the residual N-acetyl group show decreased intensity with an increase in sonication time. In contrast, the absorption bands of the NH2 groups (1613–1653 cm−1) and CH2 (1415–1421 cm−1) exhibit a higher intensity, suggesting higher deacetylation degree with an increase in the sonication time.

X-ray diffraction analysis

The crystalline structures of all the obtained chitin samples are similar (Fig. 2a) and demonstrate crystallinity of short-range as well as amorphous phase. The diffraction peaks observed around 9° and 19° are the characteristics of chitin molecule [24]. The peak intensities of the samples increased with an increase in the sonication time. This confirms that more crystalline chitin is formed with fewer impurities for longer sonication time during the preparation of biopolymer [25]. The strong reflections in the diffractograms of the chitin samples correspond to α- chitin [22]. With an increase in the sonication time, the less intense peak moved to a higher 2θ, indicating a good degree of deacetylation in the resultant chitin samples [26]. The pattern obtained with the chemical method exhibits an additional peak around 26° corresponding to the residual CaCO3 [27].

Fig. 2

(a) X-ray pattern of chitin obtained from shrimp shells using ultrasound in the deproteinization at different times of sonication. (b) X-ray pattern of chitosan obtained from chitin using ultrasound in the deproteinization at different times of sonication.

Considering chitosan, two main characteristic diffraction peaks could be observed for the samples obtained using sonication times from 15 to 35 min (Fig. 2b). The first (less intense) peak could be observed around 10.2° while the second (more intense) peak is in the range of 19.8–20.1° [28]. The diffractogram of all the as-prepared chitosan samples matches with the characteristic peaks of standard chitosan. Similar to chitin, chitosan contains short-range crystalline regions and amorphous zones. The crystallinity increased with an increase in the sonication time. Hence, in the preparation of biopolymers, ultrasound enhances the crystallinity of the resultant chitin and chitosan [29]. These findings agree with the results of earlier work [30]. Ultrasound degraded chitosan samples showed increased crystalline index as a result of an increase in the ultrasound exposure. For 10 min of sonication, there is no crystallinity as the deproteinization is not efficient, and deacetylation is not initiated. The peak around 32° in the samples at 30, 35 and 40 min of sonication is associated with the residual NaOH, which does not eliminate during the deacetylation. It is also observed that the peak at 10.2° tends to disappear, a change associated with a transformation to β- chitosan [31].

Analysis of the protein content of chitin

The content of nitrogen associated with proteins is shown in Fig. 3. It could be observed that the nitrogen content decreases as the sonication time increased. This indicates that the vibration effects of ultrasonic waves produce cavitation in the samples [21]. In general, cavitation produces shear stress that induces the breakage of nitrogen bonds and other molecular/intramolecular bonds, degrading the protein-chitin matrix. This favours the leaching of proteins and consequently, the deproteinization process [31]. The non-linear trend in the variations of protein highlights that the extraction of proteins was carried out without control on the quotient solid/solvent [19], [21]. The lower protein content of 1.96% was attained for 30 min of sonication, which is ~ 69% lower as compared to 10 min of sonication. This is consistent with that of the results obtained from IR and XRD. However, for 35 min of sonication, an increase in the protein content could be observed. Generally, an increase in the solvent increases the propagation of ultrasound leading to cavitation, which in turn enhances the extraction of protein. When water is used as a solvent, the excess solvent produces free radicals under prolonged sonication owing to induced dissociation of water, which reduces the extraction efficiency [32]. The obtained results suggest a good alternative for obtaining chitin with an efficient deproteinization. Moreover, the utilisation of reactants, as in the chemical method, is avoided in this technique along with an advantage of shorter treatment time. The average time in the biotechnological methods is also often longer, even compared to the chemical method. Thus, the method proposed in this study may be considered to be a cheap, eco-friendly and rapid approach for the synthesis of chitin and chitosan. As an example, silver nanoparticles were successfully synthesized using ultrasound [33].

Fig. 3

Total nitrogen content in the chitin samples. Ultrasound was used in the deproteinization. This parameter has the value of 5.66 in the chitin obtained using the chemical method.

Analysis of the deacetylation degree of chitosan

The deacetylation degree of the resultant chitosan increased with an increase in the sonication time (Table 1). A lower DD value of 73% was obtained for 10 min, while the highest DD value of 100% was attained for 30 min of sonication. Higher values of DD match with the lower values of protein in the chitin samples. Lower protein content facilitates the accessibility of reactants, thereby improving the deacetylation reaction [22], [31]. As it could be seen, for 30 min of sonication, 100% of DD was obtained, corresponding to the lower protein content. This value for DD indicates that all the acetamide groups could be transformed into amine groups, demonstrating the complete conversion of chitin into chitosan [34]. This confers different properties to the polymer chain. Few authors have found that the sonication process decreased the DD of chitosan [35]. Acetic acid solutions of different concentrations were used in this study, which could facilitate the breakdown of glycoside bonds. Also, water utilised permits the propagation of ultrasound without affecting the polymer chain. Moreover, the recovery of the sample is easier when water is used as a solvent.

Molecular weight analysis of chitosan

This parameter was estimated using capillary viscometry. The classification of chitosan as low and medium molecular weight was taken from the earlier report [36]. The as-obtained chitosan samples possess both medium and low molecular weights. It is evident that the deproteinization process affects this parameter. Low molecular weight product with low protein content is obtained for 30 min of sonication. According to the DD analysis, this sample corresponds to chitin, instead of chitosan. In the case of the polymer obtained for 20 min of sonication, molecular weight with high DD was obtained (Table 1), corresponding to chitin with a high protein content. All other product samples recorded a medium molecular weight. The synthesis of chitosan with high MW from chitin with low protein content has been reported [37]. Also, the production of chitosan with high MW from chitin with high protein content has been reported [38]. In this study, both these trends have been observed, and this provides further research scope for molecular weight analysis in the ultrasound-assisted chitosan production technique.

Scanning electron microscopy analysis

Fig. 4 shows the representative SEM images of both chitin (Fig. 4a-g) and chitosan (Fig. 4h-n) particles. A characteristic flake shape could be observed for the obtained biopolymer samples. Initially, with sonication (Fig. 4a,h) the particles appear compact and with multiple layers. As time increased, the surface of the polymers erodes. This could be attributed to the cutting force, the shock waves, as well as due to turbulence caused by ultrasound during the synthesis of the polymer. In some of the chitin particles, the erosion manifests in micro-scale levels (Fig. 4e), which is associated with the superposition effects of ultrasound standing waves [39]. A significant level of porosity could also be noted (Fig. 4d,j), which makes the particles to be brittle. For 35 min of sonication (Fig. 4m), a more compact particle is observed, which is also associated with more crystallinity, as identified through XRD studies. It is observed that the chitosan particles are highly eroded due to the effects of solvent (NaOH) during the deacetylation process.

Fig. 4

SEM images for different sonication times of chitin (a) 10 min, (b) 15 min, (c) 20 min, (d) 25 min, (e) 30 min, (f) 35 min, (g) 40 min; and chitosan (h) 10 min, (i) 15 min, (j) 20 min, (k) 25 min, (l) 30 min, (m) 35 min, (n) 40 min particles . As the sonication time increases more fragmentation is observed.

The particle sizes of various as-prepared biopolymer samples could be obtained from the SEM images. Due to their irregular form, a similar circular shape is assumed, and the observed diameter is reported. Fig. 5a shows the particle size of chitin as a function of sonication time. The particle size decreased rapidly from 10 to 30 min (about 35 μm) of sonication followed by a slight decrease in the size from 30 min to 40 min (only 3 μm) of sonication. Ultrasound produces a fragmentation effect which causes the breakage of the particle due to the shock waves between the particles originating from cavitation [39]. A similar effect has also been observed already [40]. Thus, it could be observed that the sonication time had an inverse effect on the particle size. A similar trend in the variation of particle size could be observed for the chitosan product also (Fig. 5b). The smallest particle size of chitosan was estimated as 20 μm. Employing ultrasound to reduce the particle size is a very practical approach and has been effectively used to synthesize nanoparticles [39].

Fig. 5

Particle size as a function of sonication time a) chitin and b) chitosan.

Biopolymer yield

The product yield in a process is a very important parameter and is obtained using Eq. (8).

Where RR is the quantity (g) of the final product, and RT is the initial sample quantity (g).

In this investigation, with the development of novel ultrasound method for deproteinization, the chitin yield is in the range of 35–71% (Table 2), which is higher than the yield obtained from the chemical method (13.12–17.36%) [41]. The utilisation of ultrasound in the deproteinization process eliminates the need for an alkaline base as well as the washing of the samples, thereby enhancing the product yield. The yield of chitosan from chitin varies in the range of 3–23%. It could be observed that the yield decreased in the first 20 min of sonication; after that, a significant increase was reached. Exposure to ultrasound for longer times caused fragmentation of the particles, resulting in dissociation and decomposition of chitin and proteins. This leads to the formation of small molecules which are lost in the washing step considerably [41], [42]. A similar trend of decreased product yield with longer sonication times has been reported [30].

Table 2

Yield of chitin and chitosan using ultrasound in the deproteinization step.

Sonication time (min)	Skeletons (g)	Chitin (g)	Chitosan (g)	Chitin yield (%)	Chitosan yield (%)
10	10	3.592	1.534	35.916	15.341
15	10	3.918	1.043	39.175	10.427
20	10	2.277	0.356	22.765	3.563
25	10	5.342	0.652	53.42	6.524
30	10	7.154	1.107	71.538	11.074
35	17	9.094	2.203	53.494	12.959
40	11	6.321	2.571	57.464	23.373

Functionality of chitosan in the preservation of beef

According to the official Mexican standard NOM-093-SSA1-1994, the sensory characteristics of the beef to be used are bright red, firm texture and characteristic smell. The beef colour is an indicator that determines the life of anaquel. Chitosan has been used successfully in the food preservation, both in the film [43] and powder [44] forms; particularly, in storage, it has shown remarkable efficiency [11], [45]. In this study, the as-obtained chitosan was used to examine the conservation of the bright red colour in the beef samples. In the beginning, the beef had a characteristic bright red colour, and the control turned dark red at the end. This property was measured through the luminosity (L*), the chromatid (a*) and the chrome (Cab*).

A higher value (18.472) of luminosity was obtained in the sample covered with the membrane prepared using chitosan derived using 20 min of sonication. Higher values of this parameter indicate higher bright red in the beef [46]. The L* values obtained for the beef samples preserved using the chitosan films of 15 and 25 min presented similar performance; the control sample, as well as other membranes, reached a luminosity below 13 at the end of this study (Fig. 6a). When the membrane at 25 min with the same preservation period, the ground beef maintained the luminosity as 16.504. The lower values were obtained with the membranes of 35 and 40 min as well as the control (11.023) (Fig. 6b).

Fig. 6

Luminosity as a function refrigeration time a) beef and b) ground samples covered with chitosan membranes.

Fig. 7a exhibits variations in the chromatid as a function of time. The membrane made from chitosan derived for 35 min of sonication showed higher values of a*. This indicates that the beef preserved in this film maintained higher intense red at the end of the period. Consequently, the chitosan membrane prevented the oxidation of beef, enlarging its life of anaquel; this property is associated with the chelating capacity of chitosan. The control sample was a little less intense red whose chromatid showed a value of 5.446. Ground beef maintained better values of a* (4.299) with the membrane at 25 min of sonication (Fig. 7b). All the membranes presented good performance concerning this parameter. The control sample at the end of 10 days diminished this indicator to 3.404. It is worthwhile to mention that the membrane with 40 min of sonication reached the highest value of a* (8.168) after 4 days which is twice as that of the control at the same time. This behaviour was maintained for 7 days. In general, the life of beef and ground beef in anaquel is 3 days. The study on beef preservation using the as-obtained chitosan-based membrane films extended the shelf-life of the food sample to 7 days. This would consequently enhance the sales potential and reduce wastage losses.

Fig. 7

Chromatid as a function of refrigeration time a) beef and b) ground beef samples covered with chitosan. The experiment was developed for 10 days.

In the beef, an ideal colour is obtained for higher values of Cab*. According to this indicator, the chitosan membrane with better performance corresponds to 15 min of sonication. Membranes with 20 and 25 min of sonication performed better than the control, which signifies that these are also useful to maintain acceptable colour in the beef (Fig. 8a). In the case of ground beef, all membranes have better performance compared with the control (Fig. 8b). Membranes of 20 and 25 min of sonication are more recommended for the refrigeration storage of beef. Globally, 15, 20 and 25 min of sonication in the deproteinization process gives rise to chitosan membranes with higher antioxidant activity, reducing the oxidative deterioration of beef. Moreover, it has also been observed that the membrane is absorbed by the surface of beef, disabling its detachment without affecting the quality of the sample. These membranes were done with chitosan of medium molecular weight, and the obtained results agree well with the earlier results where chitosan with high viscosity presented higher antioxidant effect [15], [47]. It has been demonstrated that chitosan films with high, medium and low molecular weight have better preservation effect than the commercial plastic packaging films [48]. The former assures us in the adequate beef status for human consumption and also increases customer choice. Additionally, the methodology for preparing this smart packaging may be easily translated for its fabrication, as proposed for other chitosan-based films [49].

Fig. 8

Chroma as a function of refrigeration time a) beef and b) ground beef samples covered with chitosan. The experiment was developed for 10 days.

Conclusions

The effect of ultrasound in the deproteinization to obtain chitin and chitosan from shrimp skeletons was investigated. The obtained results show that with an increase in the sonication time, deproteinization is more efficient, as confirmed from the low residual nitrogen content in the chitin samples. The increased crystallinity of the obtained biopolymer products from higher sonication times confirmed the effectiveness of ultrasound in this process. The particle sizes of both chitin and chitosan products were reduced due to ultrasound. The morphological analysis revealed that the surface of biopolymers was eroded due to ultrasound effects. Higher sonication times resulted in chitosan product with high deacetylation degree and medium and low molecular weights. Also, the pigments of the skeletons were oxidized, resulting in the formation of chitosan with lower whiteness index. The functionality of chitosan was investigated through beef preservation study for 10 days of analysis. The fresh red colour of the beef was maintained, owing to the potent antioxidant activity of the as-prepared chitosan. Specifically, the films made from chitosan after subjecting to ultrasound for 15, 20 and 25 min led to molecular weights of 73.61 KDa, 86.82 KDa and 55.66 KDa, deacetylation degrees of 80.60, 92.86 and 94.03% and particle sizes of 35.70, 25.51 and 20.10 µm, respectively, which are better than the results obtained from control. Finally, the obtained results suggest a good alternative for obtaining chitin with an efficient deproteinization. The utilisation of reactants, as in the chemical method, is avoided in this technique along with an advantage of shorter treatment time. The methodology for preparing this smart packaging may be easily translated for its fabrication, which would consequently enhance the sales potential and reduce wastage losses.

CRediT authorship contribution statement

D. Vallejo-Domínguez: Data curation, Investigation, Methodology. E. Rubio-Rosas: Formal analysis, Methodology. E. Aguila-Almanza: Data curation, Investigation, Methodology. H. Hernández-Cocoletzi: Conceptualization, Funding acquisition, Supervision, Writing - original draft. M.E. Ramos-Cassellis: Conceptualization, Funding acquisition, Supervision, Writing - original draft, Methodology, Validation. M.L. Luna-Guevara: Methodology, Validation. K. Rambabu: . Sivakumar Manickam: Conceptualization, Funding acquisition, Supervision, Writing - original draft. Heli Siti Halimatul Munawaroh: Funding acquisition, Project administration, Supervision. Pau Loke Show: Funding acquisition, Project administration, Supervision.

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.