Development and characterization of a new active and intelligent packaging system based on soluble soybean polysaccharide-Malva sylvestris extract

Abstract

This work aimed to develop an active and intelligent film based on soluble soybean polysaccharide (SSPS)/Malva sylvestris extract (MSE) to extend the shelf life of foodstuff and detect indirectly the food spoilage. The influence of MSE content on physico-mechanical properties, biological activity, and pH sensitivity of the SSPS-based films was examined. When the MSE concentration increased from 0 to 6% (w/w), water solubility and water vapor permeability of the films decreased (p ˂ 0.05). Clear antioxidant and antibacterial capacities were observed for SSPS films incorporated with different concentrations of MSE. SSPS/MSE films could sense the pH variation in a pH range of 7 to 8. SSPS/MSE film was used to detect the spoilage of shrimp and showed a pH-sensitive highly distinctive color variation from grey to dark green as the shrimp’s quality altered. Overall, SSPS/MSE film can be introduced as a promising candidate for application as active and intelligent packaging.

Introduction

With ever-increasing public awareness of the quality, safety, and freshness of the foods, the development of eco-friendly active, and intelligent packaging has attracted the attention of scientists in recent years. Active packaging helps to secure food safety and increase the shelf life of foods, while intelligent packaging is a system that provides quality information over the storage of food products (Pereira de Abreu et al. 2012).

Malva sylvestris flower extract (MSE) is a natural colorant that has been utilized for its emollient and laxative properties since ancient times (DellaGreca et al. 2009). The antioxidant and antibacterial activities of Malva sylvestris extract have been documented in various studies (Beghdad et al. 2014; Benso et al. 2016; Mohajer et al. 2016). MSE comprises a high content of anthocyanin, which is a kind of functional colorant and has an extensive range of physiological functions (Zhen-Yu 2005). The color of anthocyanins is pH-dependent. This property has a vital role in its application in intelligent film formulations. Considering the high amount of anthocyanins in MSE, it is a new interesting candidate for detecting the pH alterations and enhancing the biological activity of bio-based films.

Different biopolymers such as proteins, polysaccharides, and lipids are commonly used to fabricate eco-friendly multifunctional packaging systems. Among them, polysaccharides have received considerable attention due to their good film-forming properties, mechanical strength and high biodegradability (Rhim et al. 2013). Soluble soybean polysaccharide (SSPS) is extracted from the cell-wall material of the cotyledon of soybeans. Various studies have been conducted on the development of active and intelligent SSPS-based films. The literature review demonstrated that the SSPS based films incorporated with different types of antimicrobial agents could effectively inhibit the growth of bacteria and extend the shelf life of foodstuffs (Salarbashi et al. 2016, 2017; Salarbashi et al. 2018a, 2018b; Salarbashi et al. 2013). A literature review also demonstrated that two recent papers have been written on the pH sensitivity of SSPS films incorporated with curcumin (Salarbashi et al. 2021; Salarbashi et al. 2020). However, in the present study, for the first time, a new pH-responsive film based on SSPS/MSE was developed, and then its physico-mechanical properties were evaluated. Finally, the antibacterial and antioxidant activities, pH sensitivity, and its ability to detect the quality of shrimp were examined.

Materials and methods

Materials

Soluble soybean polysaccharide (SSPS) was provided by Fuji Oil Company (Osaka, Japan). Malva sylvestris flower was supplied from a local herbal store in Tehran, Iran. All the chemicals used in this work were obtained from Merck Co. (New Jersey, USA). Microbial cultures were purchased from Himedia (Mumbai, India).

Methods

Extraction of Malva sylvestris anthocyanins

The extraction of Malva sylvestris anthocyanins was carried out as described by Almasian et al. (2020) with a slight adjustment. Briefly, 700 g dried flowers of M. sylvestris were lyophilized and extracted by ethanol/distilled water with a volume ratio of 80/20 at 23 ± 2 °C using maceration technique (72 h for three times by 6 L solvent). The resulting extracts were combined and filtered using a paper filter and dried under vacuum at 40 °C.

Determination of anthocyanin and total phenol contents

For determination of anthocyanin content, the pH-differential method was employed (Muanda et al. 2011). Two buffer systems including potassium chloride and sodium acetate buffers were used. Total anthocyanin content was reported as mg of Cyaniding-3-glucoside Equivalents (CgE). The total phenol content of the samples was measured using the Folin-Ciocalteu method, reported by Singleton, Orthofer, and Lamuela-Raventós (1999).

Fabrication of pH-responsive films

SSPS/MSE films were prepared using casting method as reported by Salarbashi et al. (2021) with some modifications. SSPS powder (2.4 g) was dispersed in distilled water (40 mL) under magnetic stirring (500 rpm) at ambient temperature and then kept at 4 ºC overnight for hydration. Afterward, glycerol (35 wt%) as plasticizer was added to SSPS dispersion under mechanical agitation (700 rpm). Different contents of MSE (0, 2, 4, and 6 w/w%) were mixed with the dispersions. The resultant film-forming dispersions were degassed, cast on Petri-dishes, and dried using a temperature-controlled chamber at 25 °C for 20 h. Finally, the films were peeled and stored at zip-kips until further experiments.

Thickness, water solubility (WS), and water vapor permeability (WVP)

The film thickness was determined by a hand-held digital micrometer (Tester Sangyo Co, Tokyo, Japan) at 5 arbitrary positions of each film. The water solubility of the films was measured by drying at 105 ºC using an oven-drier until they reached a constant weight. The resulting samples were weighted before (W₀) and after immersing in distilled water (W₁):

$$WS\left(\%\right)=\frac{W_0-W_1}{W_0}$$ 1

WVP was measured gravimetrically as described by ASTM E96-95 standard method (Astm 2004). For this purpose, the films were cut and mounted horizontally on the top of the cups containing calcium anhydride. Then, the cups were kept in a desiccator containing saturated sodium chloride solution (NaCl − 75% RH) at 25 °C and weighed at a pre-determined time interval for 4 days. The following equation was used to determine WVP:

$$WVP=\frac{\Delta m}{A\Delta t}\frac{X}{\Delta p}$$ 2

in which, Δm/Δt, X, and A show the weight of moisture gain per unit of time (g/s), the thickness of films (mm), and the film’s surface area (m²).

Mechanical properties

The mechanical characteristics of the neat and MSE-loaded SSPS films such as tensile strength (TS) and elongation at break (EB) were determined by an M350-10CT Machine (Testometric Co., Ltd., England) based on the standard method of ASTM D 882 − 95 (ASTM 1995). The instrument was operated at a crosshead speed of 10 mm min^− 1 at 25 °C. Following equations were used to calculate TS and EB:

$$TS=\frac{F_{\max }}{A_{\min }}$$ 3

$$EB\left(\%\right)=\frac{L_{\max }}{L_0}\times 100$$ 4

where, F_max, A_min, L_max, and L0 indicate the maximum load, the cross-section area, the extension at rupture point and the original length of the specimen, respectively.

Antibacterial activity

The antibacterial activity of neat SSPS and MSE (6%)-loaded SSPS dispersions were tested as to their inhibitory effect against two foodborne pathogenic bacteria Staphylococcus aureus PTCC 1112 (ATCC 6538p) and Escherichia coli (ATCC 25,922). The inhibition zone of the samples was determined according to Salarbashi et al. (2018c).

Antioxidant activity

Antioxidant capacity of neat and MSE-loaded films was determined by measuring the free radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay as described by Roy and Rhim (2019). The antioxidant capacity of MSE and ascorbic acid in different concentrations were also examined.

pH-sensitive property

The pH-responsive color changes of the films were investigated as reported by Qin et al. (2019). The films (2 cm × 2 cm) were dipped in various buffer solutions with different pH levels for 1 min and the color variation of the films was determined by a digital camera (Canon 6D, Japan).

Application of the films as a measure of shrimp’s freshness

The SSPS/6%MSE films were cut and placed on the inner surface of the petri dish with 4 g shrimps. The dishes were covered with Parafilm and placed in an incubator at 25 °C with 50% RH. The of total volatile basic nitrogen (TVBN) content of the shrimps were measured at pre-determined time interval using stream distillation technique (Cai et al. 2011).

Statistical analysis

One-way analysis of variance (ANOVA) was carried out and a significant difference was determined by Duncan multiple tests using SSPS software (p value < 0.05). All the experiments were repeated three times.

Results and discussion

Physical properties of fabricated films

WVP is a determinant characteristic of packaging films that has a considerable influence on food safety and shelf-life of foodstuff. The effect of MSE concentration on WS and WVP of the SSPS films are given in Table 1. When the MSE concentration increased from 0 to 6%, the values of WS and WVP decreased from 66.12 to 54.35% and 6.93 to 5.07 (g.m^− 1 s^− 1 Pa^− 1), respectively (p ˂ 0.05). The reason for that has been attributed that the H-bonding interactions between hydroxyl groups of SSPS and MSE that reduced the availability of OH groups, and as a results decrease the WS and WVP of the films.

Table 1.

The physico-mechanical properties of SSPS/MSE films as a function of MSE concentration*

MSE conc (%)	WS (%)	WVP (g.m− 1.s− 1.Pa− 1)	TS (MPa)	EB (%)	Thickness (mm)
0	66.12 ± 2.12a	6.93 ± 0.452a	14.22 ± 1.14c	32.12 ± 1.30a	0.140 ± 0.006a
2	63.27 ± 1.60ab	5.70 ± 0.313b	16.24 ± 0.41b	27.35 ± 0.89b	0.141 ± 0.002a
4	61.71 ± 0.91b	5.63 ± 0.217b	17.31 ± 0.26a	24.39 ± 0.73c	0.137 ± 0.003a
6	54.35 ± 1.11c	5.07 ± 0.031c	16.23 ± 0.77b	26.02 ± 1.21bc	0.142 ± 0.004a

*a, b, c, and d, Different letters in the same column indicate significant differences at 5%

Likewise, Qin et al. (2019) revealed that incorporation of the anthocyanin extracted from Lycium ruthenicum Murr into cassava starch-based films led to decrease of water vapor permeability. The authors demonstrated that this effect is due to the hydrogen bonding between cassava starch and anthocyanin. Other study also indicated that WVP of chitosan/ propolis extract films decreased significantly when the extract concentration increased (p ˂ 0.05). In conclusion, MSE can be used as a promising filler to reduce WVP of packaging systems and thereby extend the shelf life of food products.

Mechanical properties

The tensile strength (TS) and elongation at break (EB) are commonly used to investigate the mechanical performance of packaging films. The mechanical properties of neat and SSPS/MSE films are summarized in Table 1. In can be observed that with increasing MSE concentration from 0 to 4 (%), TS showed an increasing trend (p˂0.05), but beyond this point, TS decreased The increasing effect has been attributed to the formation H-bonding between OH groups in SSPS and MSE that resulted in stronger interfacial adhesion between them (Wang et al. 2019). On the other hand, the decrease of TS at high MSE concentration has been related to the formation of agglomerates that collapse the compactness of SSPS network (Zhai et al. 2017). On the other hand, as the MSE concentration increased from 0 to 4 (%), EB decreased. This observation is due to the formation of H-bands between SSPS and MSE that limited the stretchability of SSPS/MSE films (Prietto et al. 2017). Similar results have been reported that by Qin et al. (2019), who showed that incorporation of the anthocyanin extracted from Lycium ruthenicum Murr into cassava starch-based films up to a certain point, led to an increase of TS strength. Conversely, Mei et al. (2020) reported that with an increase of incorporating anthocyanin-rich torch ginger extract into the sago starch-based films, TS decreased while EB increased. This might be associated with the increase in the plasticity of the starch-based film matrix. Therefore, it can be found that the effect of anthocyanin addition on the mechanical properties of polymeric films is dependent on the nature anthocyanin’s nature.

Antibacterial activity

The antibacterial capacity of MSE has been well proven in the literature (Dowek et al. 2020). The inhibitory effect of neat and MSE-loaded SSPS films against both gram-negative (Escherichia coli (ATCC 25,922)) and gram-positive (Staphylococcus aureus PTCC 1112 (ATCC 6538p)) bacteria are presented in Table 2. The neat film did not show any antibacterial capacity, while SSPS films incorporated MSE exhibited clear antibacterial activity against tested bacteria. This effect is related to the presence of high content of phenolic compounds in MSE including 4-hydroxybenzoic acid, 4-methoxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, 4-hydroxycinnamic acid, ferulic acid, methyl 2- hydroxydihydrocinnamate, scopoletin, N-trans-feruloyl tyramine, sesquiterpene, (3R,7E)-3-hydroxy-5,7-megastigmadien9-one and s (10E,15Z)-9,12,13- trihydroxyoctadeca-10,15-dienoic acid (DellaGreca et al. 2009). Phenolic compounds interact with membranal proteins of bacterial cells and consequently inhibit bacterial growth (Aliabbasi et al. 2021; Haslam et al. 1988). Similarly, Liu et al. (2021) revealed that addition of anthocyanins extracted from purple potato or roselle to chitosan/polyvinyl alcohol/nano-ZnO films could weakly improve the antibacterial capacity of films.

Table 2.

Inhibition zone of SSPS, MSE and SSPS/MSE film *

	Zone inhibition (mm)
	Organism
	S. aureus PTCC 1112 (ATCC 6538p)	E. coli ATCC 25,922
SSPS	–	–
MSE	7.2 ± 0.3b	5.3 ± 0.2b
SSPS/MSE film	9.4 ± 0.6a	7.0 ± 0.3a

*a and b, Different letters in the same column indicate significant differences at 5%

As presented in Table 2, the inhibitory effect of SSPS/MSE films against gram-positive bacteria was higher than gram-negative bacteria. Similar results have been reported in previous studies (Liu et al. 2021; Sun et al. 2020). This observation may be attributed to the distinct structure of cell wall between Gram-negative and Gram-positive bacteria. Gram-negative bacteria contain a double membrane envelope that acts as an obstacle against accessing antibacterial agents to their targets (Gao et al. 2020). Overall, SSPS/MSE-based film can be introduced as a promising active packaging system to delay or prevent bacterial growth.

Antioxidant capacity

Since free radicals can result in rancidity, off-flavor and discoloration of packed foods, radical scavenging ability is an important characteristic need for active packaging (Liu et al. 2019). The inhibitory radical activity of MSE, neat SSPS film and SSPS/MSE films were 17, 11 and 43%, respectively, showing all the tested samples could effectively inhibit free radicals. The antiradical activity of neat SSPS film indicated that SSPS had an electron-donating ability. Likewise, several studies have been reported that polysaccharides interact with free radicals, and produce products with high stability (Dokht et al. 2018; Salehi et al. 2019). The incorporation of MSE into SSPS film’s formulation significantly improved the antioxidant activity of the films, exhibiting a synergistic effect between MSE and SSPS. The improved antioxidant capacity of SSPS/MSE films is related to the presence of anthocyanins that could inhibit free radicals. Total phenolic compound and anthocyanin contents for M. sylvestris extract were 7.44 mg PyE/g DW and 0.80 mg Cg/g, respectively. These values were higher than those reported by Almasian et al. (2020). This variability could be related to the different growing conditions of the plant.

pH-responsive properties

pH is commonly used to monitor the bacterial growth. The photos and color parameters of SSPS/MSE films at different pHs were evaluated and the results are presented in Fig. 1A–C. The films were highly pH-responsive and exhibited clear color alterations in the pH range of 7–8. Thus, SSPS/MSE films could effectively use to sense the quality of foods over storage. The color change of anthocyanin in contact with the media with different pHs has been related to the chemical transformation of the anthocyanin structure. Such alteration of color properties of anthocyanin extracted from different resources have been documented in previous studies (Mei et al. 2020; Roy and Rhim 2021; Sani et al. 2021). The color of anthocyanins is depending on the pH of the solution. This is because of the molecular structure of anthocyanins having an ionic nature (Khoo et al. 2017). In acidic condition, some of the anthocyanins appear red. Anthocyanins have a purple hue in neutral pH while the color changes to blue and green in an increasing pH condition.

Fig. 1.

The color parameters of SSPS/MSE films at different pHs

The color attributes of the films such as lightness (L*) and redness (a*) as a function of pH are shown in Fig. 1B, C. The value of L* was 53.2 at pH 7 and reduced to 32.4 when pH increased to 8. L* is a color parameter that shows the degree of lightness. Hence, the increase in pH led to a decrease in the film’s lightness. With increasing the medium’s pH from 7 to 8, a* showed a decrease from 4.3 to − 26.1, revealing the reduction of the intensity of redness. In conclusion, SSPS/MSE films are ideal to detect the pH increase arisen from both acidic and basic spoilages.

Application of SSPS/MSE films for sensing the spoilage of shrimp

Anthocyanin is a pH-sensitive compound that can use to monitor the quality of food products. Many kinds of factors such as microbial spoilage and subsequent accumulation of total volatile base nitrogen (TVBN) can lead to an increase in pH of seafood. The pH change in shrimp with microbial growth was investigated at room temperature and the correlation between TVBN content of shrimps and color properties of the films were evaluated during the storage period. Based on Fig. 2, the initial pH value of the shrimps was 6.92 ± 0.05 and then the pH sharply elevated to 7.95 ± 0.03 as the time of storage increased from 0 to 24 h. The TVB-N content at 18 h was 31.22 mg/100 g, which was more than the safe consumption threshold of 30 mg/100 g for marine fish and shrimp (GB 2733−2015).

Fig. 2.

TVBN content of shrimps and color properties of the films

The color parameters of the films as a function of storage time were also determined and the results are shown in Fig. 2. When the TVBN content increased, the a* and L* values of the films showed a decreasing trend. Pearson correlation test exhibited that there was a strong correlation between TVBN content of shrimp and b* values of the films over storage time (Pearson correlation = 0.973). Accordingly, SSPS/MSE films can be used as a promising intelligent packaging system to detect shrimp spoilage.

Conclusion

In the present work, a novel film based on soluble soybean polysaccharide (SSPS)-Malva sylvestris extract (MSE) with good mechanical characteristics, antibacterial and antioxidant activities, and intelligent response property was developed and applied for monitoring the freshness of shrimp. The films indicated a visual color variations in various pH because of the presence of anthocyanin in MSE. Considering highly pH sensitivity and good functional properties, SSPS/MSE films can be introduced as a promising intelligent packaging system to monitor the freshness of a wide range of food products like pork and shrimp.

Abbreviations

MSE

Malva sylvestris flower extract

SSPS

Soluble soybean polysaccharide

ASTM

American Society for Testing and Materials

WVP

Water vapor permeability

TS

Tensile strength

EB

Elongation at break

TVBN

Total volatile basic nitrogen

ANOVA

One-way analysis of variance

Authors’ contributions

Authors MJ and PT critically reviewed previous data and prepared the first draft of the paper. Author SS participated in the study design and critically reviewed the manuscript draft.

Funding

Not Applicable.

Data Availability

Data available on request due to privacy/ethical restrictions.

Code Availability

Not Applicable.

Declarations

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Authors declare that they are free of any conflicts of interest.