An intelligent film based on cassia gum containing bromothymol blue

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Food Safety and Toxicology

An intelligent film based on cassia gum containing bromothymol blueanchored cellulose fibers for real-time detection of meat freshness Lele Cao, Guohou Sun, Cijian Zhang, Wenbo Liu, Jian Li, and Lijuan Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06493 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 6, 2019

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Journal of Agricultural and Food Chemistry

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An intelligent film based on cassia gum containing bromothymol blue-anchored

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cellulose fibers for real-time detection of meat freshness

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Lele Cao, †,‡ Guohou Sun, †,‡ Cijian Zhang, † Wenbo Liu, † Jian Li, †,‡ and Lijuan Wang*,†,‡

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†

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Northeast Forestry University, 26th Hexing Road, Xiangfang District, Harbin 150040, P. R. China

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‡

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Road, Xiangfang District, Harbin 150040, P. R. China

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*Corresponding author. Tel.: 86-451-82191693

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Email address: [email protected]

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Key Laboratory of Bio-based Materials Science and Technology of Ministry of Education,

Research Center of Wood Bionic Intelligent Science, Northeast Forestry University, 51th Hexing

Graphical abstract

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Synopsis: Intelligent film based on cassia gum prepared by incorporating cellulose fibers anchored

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bromothymol blue for monitoring meat freshness.

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Abstract 1

ACS Paragon Plus Environment


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To prepare intelligent cellulose fiber (ICF), cellulose fibers were modified by grafting

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hydroxypropyltriethylamine groups, to which bromothymol blue (BTB) was anchored. The ICFs

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were incorporated into cassia gum (CG) to prepare a pH-sensitive intelligent film. The Fourier

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transform-infrared results indicated that BTB has been introduced in the CG-ICF5 film. Scanning

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electronic microscopy indicated that the addition of ICF can loosen the structure of the film. The

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incorporation of ICF decreased the light transmittance and water vapor permeability, while did not

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significantly affect the thermal stability. The mechanical properties weakened with 3% ICF addition

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and improved with 5% ICF addition. The release experiment indicated that 46.784% and 8.297% of

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BTB released from the CG-ICF5 film under oscillating to 50% and 95% alcohol/water solution,

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respectively. The response of the intelligent films to triethylamine in environments with different

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relative humidities was investigated. A visible color change occurred in the triethylamine

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environment within 20 min. Pork and chicken spoilage experiments were performed to study the

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application of the intelligent film in monitoring meat freshness during spoilage. Obvious color

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changes appeared, demonstrating that the intelligent film has potential for use in real-time indication

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of meat spoilage.

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Keywords: Cellulose fiber; Bromothymol blue; Intelligent film; Cassia gum; Triethylamine response;

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Meat freshness

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Introduction

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Fresh meat is rich in nutrients and is a component of many foods. However, the shelf life of fresh

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meat is very short, owing to the growth and reproduction of microorganisms and decomposition from

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its own enzymes. As people's living standards and food safety awareness improve, consumers are

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becoming increasingly worried about the freshness of purchased meat. Slight spoilage in meat

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products cannot be detected via the naked eye or nose. Another traditional method of detection of 2


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spoilage in meat products is based on chemical analysis, called destructive testing, which requires

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expensive inspection instruments and professional operators.1 However, for most consumers, such

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detection is not feasible or accessible. Therefore, a visible real-time monitoring technology for food

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freshness, which is low-cost, rapid and non-destructive, is urgently needed.2 In recent years,

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intelligent packaging films and indicators have attracted researchers’ attention for real-time detection

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of the freshness of foods.3-9 During spoilage, protein-rich foods (such as fish, pork and chicken) can

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release organic amines, termed total volatile basic nitrogen compounds (TVB-N), consisting of

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dimethylamine (C2H7N), trimethylamine (C3H9N) and ammonia (NH3), as a result of microbial

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degradation. The TVB-N content is a key indicator of the freshness of protein-rich foods.10, 11 Because

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the pH is increased by organic amines,12 intelligent films13,

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potentially be applied for real-time monitoring of the freshness of protein-rich foods. Some synthetic

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dyes are highly sensitive to pH changes and have been used in indicators16, 17, 18 and packaging films.

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Atchareeya Nopwinyuwong19 has prepared an indicator based on dye by coating a solution of

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bromothymol blue (BTB) and methyl red on a linear low-density polyethylene nylon film, and has

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used this film for monitoring the freshness of Thai golden drop pastries; the results demonstrated that

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the freshness could be monitored according to a color change. A bromocresol green-based sensor has

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been investigated by Alexis Pacquit.20 That sensor was made by coating a mixture of bromocresol

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green and cellulose acetate on optically clear polyethylene terephthalate discs, and it has been applied

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to real-time monitoring of the freshness of fish via an obvious color change. However, the inedibility

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and the potential toxicity of synthetic dyes have limited their widespread use as food monitoring

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indicators. Consequently, natural pigments in vegetables and fruits for use as indicators have attracted

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the attention of researchers. Yanina S. Musso21 has prepared a gelatin-based smart film with added

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curcumin. The films are yellow in an acid liquid and red in a basic liquid. Ma22 has reported a pH3


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sensitive to pH change could


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sensing tara gum film made by incorporating grape skin extractive, which has been used for indicating

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milk spoilage. However, natural pigments are not sensitive to small pH changes because the color

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changes are not obvious with a pH variation of 2. Therefore, pH-sensitive synthetic dyes are necessary

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for the preparation of highly pH-sensitive materials, but leakage of the dyes from the matrix must be

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prevented to ensure food safety.

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BTB has been widely used in indicators, owing to its high sensitivity to pH.23, 24, 25 Cellulose fiber

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(CF) is a renewable, non-toxic and naturally abundant material26, 27, 28 that can serve as a carrier to

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which to anchor synthetic dyes via grafting of active groups. No previous studies have reported

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anchoring BTB onto cellulose fibers in alkaline solution to prepare pH-sensitive fibers. Cassia gum

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(CG) is a representative galactomannan found in the endosperm of CatsiatoraLinn, and CG solution

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is weakly acidic. In our previous work, flexible CG films were prepared by the addition of glycerol

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and sorbitol as plasticizers.29

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In this study, to prepare intelligent cellulose fiber (ICF), cellulose fibers were modified by grafting

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hydroxypropyltriethylamine groups to which BTB was firmly anchored. ICFs were incorporated into

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the CG to prepare a pH-sensitive intelligent film. Characterization of the film through techniques

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including Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and

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thermogravimetric analysis (TGA). The light transmittance performance, water vapor permeability

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and mechanical properties of the films were also investigated. The relationship between film color

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change and the TVB-N was investigated by using triethylamine as a simulant at different relative

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humidity (RHs). The films were applied to monitor the freshness of pork and chicken during spoilage.

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Materials and methods

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Materials

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Cellulose fiber (CF) was supplied by Henfeng Paper Co., Ltd. (Mudanjiang, China). Bromothymol 4


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blue (BTB) was purchased from Shandong Jiaying Chemical Co., Ltd. (Jinan, China). Cassia gum

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(CG, food grade) was purchased from Anli Fine Chemical Co., Ltd. (Henan, China). Calcium chloride,

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lithium chloride, potassium acetate, magnesium chloride and sodium chloride anhydrous were

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supplied by Zhiyuan Chemical Reagent Co., Ltd. (Tianjin, China). All other chemicals including

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sodium hydroxide, epoxy chloropropane, triethylamine (C6H15N), hydrochloric acid, ethyl alcohol

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and glycerol were of analytical grade and purchased from Yongda Chemical Reagent Co., Ltd.

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(Tianjin, China).

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Intelligent cellulose fiber (ICF) preparation

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Intelligent cellulose fiber (ICF) was prepared according to our previous work30 with a little

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modification. CF was ground with a multi-functional crusher and powders of 80-120 mesh were

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selected for use. 5 g of CF powder and NaOH solution (150 mL, 20% w/w) were stirred at ~500 rpm

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and 25 oC for 3 h. After filtration, 150 mL of NaOH solution (10% w/w) and epichlorhydrin (70 mL)

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were added to the mixture and stirred at ~500 rpm and 65 oC for 7.5 h. Subsequently, the solution

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were removed from the mixture by filtration, and then triethylamine/ethyl alcohol solution (80 mL,

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35% v/v) was added under stirring of ~500 rpm and 75 oC for 4.5 h. The separated product (QCF)

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through filtration was washed by ethyl alcohol to remove the rest triethylamine. Then, 0.1 M NaOH,

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0.1 M HCl and distilled water were used in turn to wash the product until the pH of the filter liquor

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reached 7. The QCF was dried at 70 oC in a vacuum oven. 2 g of QCF and BTB/NaOH solution (200

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mL, 1,000 mg/L) were stirred at ~200 rpm and 50 oC for 4 h. Then, an aspirator filter pump was used

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to separate the mixture and the solid was washed with distilled water until the filter liquor colorless

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to obtain the intelligent cellulose fiber (ICF). The ICFs were dried at 60 oC overnight.

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Preparation of films

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CG was stirred in distilled water at 45 oC for 30 min to gain a solution of 0.6 wt% that was fully 5



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pre-dispersed in 10 mL of absolute ethyl alcohol. ICFs (0%, 1%, 3%, 5% [w/w], based on CG weight)

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and 45% (w/w, based on CG weight) glycerol were added to the CG solution and stirred at 500 rpm

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and 45 oC for 15 min. Then, the film-forming solution was cast into a mold made of plexiglass, and

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dried at 65 oC for ~30 h. The resulting films were marked as CG-ICF0, CG-ICF1, CG-ICF3 and CG-

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ICF5 based on the amount of the ICF.

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Characterization

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A D/MAX-2500 diffractometer (Rigaku, Tokyo, Japan) was used to record the XRD patterns of

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the films with and without ICF. The measurements were operated at 1200 W with a voltage of 40 kV

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and an electricity of 30 mA.

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The infrared spectra of the samples with and without ICF were conducted in the range of 4000-750 cm-1 by using a Nicolet is50 spectrometer (ThermoFisher, USA) with a resolution of 4 cm-1.

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A JSM-7500F (Japan) scanning electron microscope was used to observe the upper surface and

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cross-section of the films. The samples were put in liquid nitrogen for 30 s to obtain the cross-section,

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and then pasted the cross-section samples on the stage. A thin layer of gold was sputtered by using a

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JEC-3000FC ion sputtering apparatus before observation.

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Property measurements

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Color

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Films color was measured with a portable colorimeter (Xrite2600d, MI, 101, USA), including L

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(lightness), a (soft pink-green) and b (tulip yellow-blue). Six measurements were conducted for each

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sample. The total color difference (∆E) was calculated by using the eq 1.

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∆E =

(𝐿 ― 𝐿𝑠)2 + (𝑎 ― 𝑎𝑠)2 + (𝑏 ― 𝑏𝑠)2

(1)

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where Ls, as and bs are color parameters of the white standard plate and taken of 99.417, -0.077 and -

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0.110, respectively. L, a and b are color parameters of the film samples. 6


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Violet-visible absorption spectra

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The transmittance of films were tested by using an ultraviolet-visible (UV-2600, Shimadzu, Kyoto,

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Japan) and conducted from 200 to 800 nm with air as the reference. Films were cut into rectangles (4

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cm × 2 cm) uniformly.

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Water vapor permeability (WVP)

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The water vapor permeability (WVP) of film samples were conducted according to the gravimetric

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method31 with a slight modification. Film samples were uniformly cut into round sheets with diameter

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of 5 cm, and then, sealed onto the mouth of the weighing bottles containing ~23 g of calcium chloride

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anhydrous as an environment with zero relative humidity (0% RH) after recording the thickness.

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These weighing bottles were stored in a dryer equipped with silica-gel desiccant over night to remove

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the moisture. Then, the weighting bottles were transferred in another dryer containing saturated NaCl

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solution (as an environment with 75% RH) after recording the initial weight of them. The driving

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power of the water vapor infiltration process is resulting from the differentials of 75% RH and 0%

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RH and taken of 1753.55 Pa. Weight variations (∆m, g) were obtained by weighting the weight bottles

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periodically to calculated the quality of moisture that was permeated to the desiccant through the film.

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WVP was achieved within the stabilization stage of the time versus weight and acquired by using the

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eq 2.

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WVP = (∆m × d)/(∆t ×A × ∆P)

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where d (mm) and A (m2) presents the thickness and area of the film, respectively. ∆t (s) denotes the

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time intervals and ∆P (Pa) is the vapor pressure difference of the two side of the film of 1753.55 Pa.

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(2)

Mechanical properties

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The thicknesses of films were tested by using a micrometer (ID-C112XBS, Mitutoyo Corp., Tokyo,

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Japan).Tensile strength (TS)32 and elongation at break (EB) of films were obtained by using an auto 7



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tensile tester (XLW-PC, PARAM, Jinan, China) at 300 mm/min of cross-head speed. Films were

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stored at 53% RH (Mg(NO3)2 saturated solution) and 25 oC for 12 h before determination.

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Thermogravimetric analysis (TGA)

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A TA instruments TGA Q500 (TA Instruments, USA) was employed to determine the TGA of the

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film samples. And the measurements were conducted from 30 oC to 600 oC with a heating rate of 20

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oC/min.

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Leakage-resistance of BTB

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In order to investigate the leakage of BTB in different food, 50% (v/v) and 95% (v/v) alcohol/water

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solution were used to simulate the alcoholic food and fatty food, respectively. 200 mg of CG-ICF5

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film was oscillated in 50 mL of 50% and 95% alcohol/water solution at 30 oC, respectively. 3 mL of

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samples was withdrawn and analyzed for BTB release until it reached the equilibrium. A

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spectrophotometer (UV-2600) was employed to measure the absorbance of BTB released in 50%

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alcohol/water solution at 425 nm and 95% alcohol/water solution at 423 nm, respectively. The

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standard curves of BTB in 50% and 95% alcohol/water solution were also obtained.

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However, in actual application, the film will not directly contact the food being tested. A sample

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operation was done to simulate the practical use to investigate the BTB leakage from the film. NaOH

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solution (0.1 M) was dropped on a piece of filter paper, then, the CG-ICF5 film (3 cm × 4 cm) that

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was paste on the wet part of the filter paper. After 5 min, the film was removed. Subsequently, the

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color of the wet filter paper was observed before and after drying.

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Response to triethylamine

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Transparent containers with lids were used to investigate the responses of films containing various

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amounts of ICF to triethylamine at different RHs. The film samples were cut into ribbons of 15 mm

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× 30 mm, which were placed on the inner surface of the lid and conditioned at 23% RH (saturated 8


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CH3COOK solution), 33% RH (saturated MgCl2 solution), 53% RH (saturated Mg(NO3)2 solution)

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and 75% RH (saturated NaCl solution) for 10 h after recording of the color. Subsequently, 5 mL of

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triethylamine was rapidly injected into the container, as shown in Figure 1. The color of the film

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samples was recorded with a portable colorimeter without taking out from the containers at various

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response times.

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Figure 1. Schematic of the C6H15N response test.

Monitoring freshness of pork and chicken

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After the skin, fat and bone were removed, 25 g of fresh pork or chicken cut into dices (1 cm3) was

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placed in a plastic petri dishes whose internal surface of the lid was pasted with the films of 15 mm

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× 20 mm. The same experiment was repeated for four times. Then, the plastic petri dishes samples

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were conditioned at 20 oC. The color of films, pH and TVB-N of pork or chicken were recorded at

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various response times. The pH of the pork and chicken samples was measured according to

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GB5009.237-2016, and the standards were as follows: pH 5.8~6.2 (fresh meat); pH 6.3~6.6 (sub-

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fresh meat); and pH> 6.7 (metamorphic meat). Pork or chicken dices was ground in a blender, then

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20 g of minced pork or chicken and 100 mL of distilled water were shocked for 30 min in a shaking

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table. The pH of the filtrate was measured with a pH meter after filtration, and the average of three

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measurements was recorded for each sample. The TVB-N was obtained according to GB2707-2016 9



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by using a Kjeltec distillation unit33 and the following standards: ≤ 15 mg/100 g (fresh meat) and >

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15 mg/100 g (metamorphic meat). The plastic petri dishes samples, preparation of the steeping liquor

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of the pork (or chicken) samples, and the pH and TVB-N measurement procedures are shown in

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Figure 2A–2D.

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Figure 2. Plastic petri dishes samples equipped with films and pork or chicken (A), preparation steeping liquor of

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pork and chicken (B), pH (C) and TVB-N (D) measurement process.

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Statistical analysis

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Data were handled by Excel 2010 and SPSS and reported as the average ± standard deviation.

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Duncan’s multiple range tests were used to determine the difference among the average values and

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conducted with significance of P < 0.05.

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Results and discussion

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Analysis of the films

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Figure 3A shows the XRD patterns of ICF and films with and without ICF. Characteristic peak of 10


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ICF appeared at 20.32°, indicating that the ICF showed a typical structure of cellulose II. Peaks at

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11.08°, 16.68° and 19.88° are the characteristic peaks of CG. The patterns of films with and without

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ICF are very similar, and the intensity around 20.30° gradually increased as the ICF addition

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increasing.

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Figure 3. XRD patterns of ICF and films with and without ICF (A), FTIR spectra of CG-ICF0 and CG-ICF5 film (B), SEM photographs of film surfaces (C)-(a), (b), (c) and (d), film cross-section (D)-(a), (b), (c) and (d).

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The FTIR spectra of the CG-ICF0 and CG-ICF5 films are shown in Figure 3B. The spectrum of

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the CG-ICF0 film showed a band at ~3280 cm−1 that resulted from the O–H stretching vibration. The

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band from 2990 to 2870 cm−1 represents the C–H vibration34. The bands at ~863, ~1020, and ~1152

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cm−1 correspond to the C–O–C stretching of the glucosidic bonds and O–H vibrations due to the

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existence of pyranose35. The band around 1605 cm-1 and 1242 cm-1 are the C=O and C–C stretching

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vibration, respectively. The spectrum of CG-ICF5 was similar to that of the CG-ICF0 except the 11



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difference as follows: O–H stretching vibration bands enhanced and broadened, the intensity of the

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peaks for C–H, C–O–C and C–C increased, and a new band at 1726 cm-1 attributed to C=O vibrations

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appeared. All of the changes illustrates that the BTB has been introduced in the film of CG-ICF5.

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The SEM images of the upper surfaces and cross-sections of film samples are displayed in Figure

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3C and Figure 3D. The surface of CG-ICF0 (Figure 3C-a) was smooth and continious without voids.

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The ICF incorporated in the CG film became increasingly visible as the amount of ICF increased.

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Meanwhile, the surfaces remained continious and without ICF exposed on the surface. The cross-

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section of CG-ICF0 was continious and regular. With the amount of ICF inceresing, the cross-section

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of CG films was still continious, and the freeze-fractured cross sections of ICF became increasingly

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clear, as shown in Figure 3D-c and Figure 3C-d, thus indicating a looser film sturcture.

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Simultaneously, no voids were observed in the cross-sections of films with and without ICF, thus

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indicating that the films were compact.

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Color and light transmittance of the films

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The color parameters of film samples with and without ICF, including L, a, b and ∆E are shown in

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Table 1. Although the brightness of the film decreased as the amount of ICF increased, a high

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brightness (high L) was maintained. The values of a changed from negative to positive and not

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significantly. The values of b increased significantly from −4.700 to 54.360 with increasing ICF, thus

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demonstrating that the film became incrementally yellow. Values of ∆E also markedly increased, thus

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indicating that the films with different amounts of ICF could be distinguished with the naked eye36.

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The UV-vis spectra of film samples with and without ICF are shown in Figure 4A. The light

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transmission of the films showed no significant differences at 280 nm (decrease from 3.685% to

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1.403%) with increasing ICF. Meanwhile, the light transmission exhibited marked changes at 600 nm

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and decreased rapidly from 70.437% to 39.705% with the amount of ICF increased from 0% to 5%. 12


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The results showed that the films became increasingly opaque with increase of ICF, indicating that

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the presence of ICF destroyed the ordered structure of the films. However, the films with ICF retained

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favorable optical properties, and a plant (Plectranthus hadiensis var. tomentosus) covered with films

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containing ICF was visible, as shown in Figure 4B. The results of light transmittance indicated that

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the addition of ICF improved the UV barrier property.

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Table 1. Color Parameters of Films with and without ICFa Film

L

a

b

∆E

CG-ICF0

87.625±0.204d

−0.238±0.035c

−4.700±0.219a

12.658±0.123a

CG-ICF1

86.740±0.285b

−1.510±0.079a

13.250±0.202c

18.473±0.329b

CG-ICF3

79.052±0.457c

0.680±0.324b

52.757±1.730b

57.796±1.720c

CG-ICF5

75.632±0.668a

2.110±0.089c

54.360±2.571d

58.712±2.407d

aDifferent

Photos

letters in the same column indicate significant differences (P < 0.05).

249 250 251

Figure 4. UV–vis spectra of the films (A) and films covered plant (B).

WVP and mechanical properties

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The water vapor permeability and mechanical properties of the films with and without ICF are

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exhibited in Table 2. The values of WVP increased from 2.16 g m−1 s−1 Pa−1×10−10 to 2.74 g m−1 s−1

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Pa−1×10−10 as the ICF addition increased from 0 to 5%, which could be due to the hydrophilic nature 13



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of ICF that with abundant hydroxyl groups. This tendency may be explained by structural

256

modifications of the CG network which becomes less dense resulted from the dispersion of a small

257

amount of ICF. It must be noted that the reorganization of the CG network could increase the free

258

volume and segmental motions, resulting water molecules to diffuse more easily and giving a higher

259

WVP37. The values of TS initially decreased followed by an increase as the ICF increased, whereas

260

the EBs declined. These results may have occurred because the interaction between CG chains was

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weakened by the dispersion of the small amount of ICF, and the interaction between ICF and CG

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chains compensated for that between CG chains as the amount of ICF increased up to 5%.

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Table 2. WVP and Mechanical of Films with and without ICFb Film

WVP (g m-1 s-1 Pa-1×10-10)

TS (MPa)

EB (%)

CG-ICF0

2.16±0.09d

18.53±0.4d

36.53±0.25d

CG-ICF1

2.21±0.26a

13.18±1.62a

28.56±6.93a

CG-ICF3

2.53±0.21b

13.57±2.15a

27.35±4.18a

CG-ICF5

2.74±0.14c

17.18±2.71b

22.80±2.69a

bDifferent

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Thermogravimetric analysis

265

Thermogravimetric analysis (TGA) was performed to determine the thermal stability of the films

266

with various amounts of ICF; the results are shown in Figure 5. The corresponding temperatures of

267

CG-ICF0, CG-ICF1, CG-ICF3 and CG-ICF5 film were 290.77 oC, 295.24 oC, 296.41 oC and 296.80

268

oC

269

(DTG, Figure 5B) showed three weight loss stages as follows: the first one occurred at ~100 oC and

270

contributed to evaporation of residual moisture; the second one was observed at ~200 oC and resulted

271

from a loss of glycerol38; the third one occurred at ~300 oC and was due to the degradation and

at a weight loss of 50% (Figure 5A), respectively. The results of derivative thermogravimetry

14


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decomposition of CG29. These results indicated that the addition of ICF did not obviously affect the

273

thermal stability of the films.

274 275 276 277 278 279 280

Figure 5. TGA (A) and DTG (B) of the films.

Leakage-resistance of BTB

281

The amount of BTB incorporated in 0.5 g of CG-ICF5 film is 0.633 mg30. The BTB concentration

282

and release rate over time were shown in Figure 6. The equilibrium concentration of BTB is 6.178

283

mg/L and the maximum release rate is 46.784% in 50% alcohol/water solution which was colorless

284

at initial (Figure 6A-a) and then changed to pale yellow (Figure 6A-b) while the release reached

285

equilibrium. In 95% alcohol/water solution (Figure 6B), the equilibrium BTB concentration is 1.051

286

mg/L and the maximum release rate is 8.297%. The color of the solution was still colorless (Figure

287

6B-b) when the release reached equilibrium. The results show that a portion of BTB released from

288

the CG-ICF5 film into fatty food simulants much lower than that in alcoholic food simulants,

289

indicating that the film are more suitable for fatty foods.

290

The CG-ICF5 film immediately changed to blue-green when 0.1 M NaOH solution was added. The

291

filter paper under the film remained colorless after a few minutes, as shown in Figure 6C, thus

292

indicating that the BTB on the ICF was not leakage during the actual application.

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295 296 297 298 299 300 301 302 303 304 305

Figure 6. BTB concentration and release rate over time in alcoholic food simulants (A) and fatty simulants (B), leakage test of BTB during actual use (C).

306 307

Figure 7. The values of a (A) and b (B) changes of CG-ICF5 film exposed to triethylamine at different relative

308

humidities over time, the corresponding photos of CG-ICF5 film at different relative humidities for 150 min (C),

309

chemical change of ICF (D) and schematic of the film’s color change.

310 16


Page 17 of 25

311


Response to triethylamine

312

The responses to triethylamine of the film containing 5% ICF was measured by exposing the CG-

313

ICF5 film in environments with various RH levels (23%, 33%, 53%, and 75% RH) and 5 mL of

314

triethylamine. The color parameters of films, including L, a and b, were tested to analyze the color

315

changes over time. The values of a were positive and decreased significantly in a triethylamine-

316

containing environment over time and reached equilibrium at around 120 min (Figure 7A), thus

317

indicating that the films’ color gradually changed to green. The b values decreased rapidly with the

318

extension of response time under different RHs, and the range was higher with increasing RH (Figure

319

7B). The color of the CG-ICF5 film changed from yellow to blue (b changed from positive to negative)

320

within 50, 80, and 120 min in an environment of 75%, 53%, and 33% RH, respectively (Figure 7C).

321

The slower volatilization of triethylamine resulted in a low concentration of triethylamine in the

322

container, and the high RH provided more water molecules to penetrate the film and promote the

323

contact of C16H15N and H2O, thus forming a weak-base environment on the surface of the CG-ICF5

324

film. Therefore, the OH− can react with the ICF (Figure 7D), and the structural change resulted in the

325

color change (Figure 7E). More OH− formed in higher RH and expedited the color change of the CG-

326

ICF5 film.

327

Monitoring freshness of pork and chicken

328

The films with different amounts of ICF were applied to monitor the freshness of meat. The photos

329

of films with meat are shown in Figure 8 (A-pork) and Figure 8 (B-chicken), and the corresponding

330

color parameters of films and TVB-N levels over time are in Table 3. It is a quite spectacular contrast

331

that the color of CG-ICF3 and CG-ICF5 clearly changed from yellow to blue-green at 36 h for pork

332

(Figure 8A-a and Figure 8A-d). It is equally true that the CG-ICF3 color changed to pale green at 24

333

h for chicken (Figure 8B-e and Figure 8B-g), and then changed to green-blue at 36 h (Figure 8B-h), 17



Page 18 of 25

334

the CG-ICF5 color changed to blue-green at 36 h (Figure 8B-e and Figure 8B-h). Correspondingly,

335

the TVB-N of pork increased from 2.808 mg/100 g to 12.270 mg/100 g, and increased from 4.089

336

mg/100 g to 13.971 mg/100 g (CG-ICF3 started to change color) for chicken. Meanwhile, the increase

337

of TVB-N indicated that the content of organic amines (dimethylamine, trimethylamine and ammonia)

338

was increasing. Therefore, the color of the CG films changed. The a values of films changed from

339

positive to negative, indicating that the color of the films tends to change to green. And the b values

340

of films decreased, showing that the films gradually changed to blue. Furthermore, the color of CG-

341

ICF3 film and pH values of pork and chicken over time were exhibited in Figure 8C and Figure 8D,

342

respectively. The sensitivity of CG films incorporated with ICFs is much higher than that of the others.

343

Kuswandi’s group studied a package sticker sensor based on curcumin for detection of volatile amines,

344

and the sensor changed from orange to reddish orange with a 25 mg/100 g of TVB-N.33 Wang et al

345

prepared a k-carrageenan and curcumin based film for freshness monitoring, and the film changed

346

from yellow to red while the TVB-N of pork changed from 4.91 to 31.11 mg/100 g, and the TVB-N

347

of shrimp increased from 7.15 to 41.53 mg/100 g.39 Therefore, it can be inferred that the CG film

348

incorporated ICFs can be applied as an effective detector for monitoring the freshness of animal based

349

protein-rich foods.

350

Table 3. Films’ Color Parameters, TVB-N of Pork and Chicken Over Timec

Meat

CG-ICF1

Tim

CG-ICF3

CG-ICF5

TVB-N

e (h)

a

b

a

b

a

b

(mg/100 g)

0

0.32±0.08b

20.15±0.20c

4.48±0.32b

67.71±0.73d

2.15±0.09c

50.84±0.57b

2.808±0.13a

12

0.32±0.12c

18.19±0.16d

4.08±1.03a

65.87±0.41b

1.56±0.92a

50.23±0.64c

6.517±0.21a

24

0.28±0.06a

15.85±0.57b

2.81±0.96d

64.15±0.94a

1.24±0.09b

49.55±0.30d

11.229±0.54c

36

-3.43±0.3d

13.68±1.50a

-15.79±0.28c

45.58±0.32c

-16.38±0.1d

41.83±0.16a

12.270±0.34b

Pork

18


Page 19 of 25


0

0.32±0.08c

20.15±0.20b

2.81±0.32a

67.71±0.73d

2.15±0.09b

50.84±0.57a

4.089±0.19a

12

0.19±0.06b

14.19±0.17c

2.35±0.10c

61.82±0.95a

1.97±0.51c

49.55±0.23c

11.804±0.43a

24

-2.11±0.2d

15.88±0.51a

-10.89±0.02b

40.63±0.02c

-6.87±0.32d

46.43±0.52d

13.971±0.84c

36

-5.80±0.5a

16.06±0.37d

-15.87±0.48d

29.53±0.52b

-15.94±0.3a

42.25±0.47b

16.056±0.57b

Chicken

cDifferent


351 352 353 354 355 356 357 358 359 CG-ICF1

CG-ICF3

CG-ICF5

360 361 362 363 364 365

Figure 8. The photos of films with pork (A) and chicken (B) over time, the color parameter of CG-ICF3 and pork

366

pH changes (C), the color parameter of CG-ICF3 and chicken pH changes (D).

367

Conclusion 19



Page 20 of 25

368

An intelligent cassia gum film was prepared, which contained quaternized cellulose fibers as a

369

carrier loading BTB. The FTIR results indicated that the BTB has been introduced in the CG film.

370

The SEM results indicated that the ICFs were wrapped by the CG, and the film remained compact.

371

The addition of ICF decreased the light transmittance from 70.437% to 39.705% at 600 nm while did

372

not significantly affect the thermal stability of the films. Meanwhile, the water vapor permeability of

373

the intelligent films was slightly decreased, and the mechanical properties showed no obvious

374

changes. The release experiment indicated that 46.784% and 8.297% of BTB released from the CG-

375

ICF5 film under oscillating to 50% and 95% alcohol/water solution, respectively. The intelligent films

376

had a sensitive in-situ response to triethylamine, with a highly visible color change from pale yellow

377

to blue-green. The light yellow films changed to blue as the meat (pork or chicken) transitioned from

378

a fresh to a sub-fresh state, thus demonstrating that the intelligent films can accurately indicate the

379

freshness of meat products. This study offers a promising intelligent film for use in monitoring the

380

freshness of animal based protein-rich foods and for preventing consumption of slightly spoiled foods.

381

In further experiments, we will study how to keep the dye from releasing at all to insurance the safety

382

of the tested food.

383 384

Acknowledgement This work was supported by the Fundamental Funds for Central Universities (2572018AB14) and

385

the National Natural Science Foundation of China (31770618).

386

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505 25


An intelligent film based on cassia gum containing bromothymol blue

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