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Vol. 29, 2011, No. 6: 616–623 Czech J. Food Sci.

Preparation and Characterisation of Food-Grade Chitosanfrom Housefly Larvae

A -J ZHANG 1,2 , Q -L QIN1 , H ZHANG 1 , H -T WANG 1 , X LI 1 ,

L MIAO1 and Y -J WU 1

1State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute

of Zoology, Chinese Academy of Sciences, Beijing, P.R. China; 2Graduate University of Chinese

Academy of Sciences, Beijing, P.R. China

Abstract

Z A.-J., Q Q.-L ., Z H. , W H.- ., L X., M L., W Y.-J. (2011): Preparation and char-acterisation of food-grade chitosan from housefly larvae . Czech J. Food Sci., 29 : 616–623.

he preparation and characterisation of food-grade chitosan from housefly larvae are reported. A refinement procedurewas developed to remove larval mouth hooks from the primary chitosan product, which greatly improved the qualityof the final product and simplified the production procedures. Different factors affecting chitosan preparation werestudied and an orthogonal experiment was designed to determine optimal preparation conditions. When prepared un-der optimal reaction conditions, the end product was snow-white in colour, had a high deacetylation percentage, good viscosity, and a low ash content. he end product was characterised by Fourier transform infrared spectral analysis ,X-ray diffraction analysis, thermo-gravimetric analysis, and differential scanning calorimetry. Its physical and chemi-cal properties and sanitary index were determined and compared to the relevant Chinese standards. he results showthat the chitosan we produced under optimal conditions meets the Chinese Fishery rade Standard SC/ 3403-2004 forfood-grade chitosan.

Keywords : Musca domestica ; commercial applications; chitin; insect; derivates

Abbreviations

DDA – degree of deacetylation; PSM – prortion of the solution to material; RSM – ratio of the solution (ml) to mate-rial consider changing PSM to RSM, GA – thermo-gravimetric analysis; DSC – differential scanning calorimetry;F IR – Fourier transform infrared; XRD – X-ray diffraction

he housefly Musca domestica (Diptera: Mus-cidea) is commonly regarded as an importantsanitary pest insect. However, due to its shortlife cycle, high fecundity, and efficient digestionof organic waste, it has also become a source ofprotein, chitin and chitosan (O et al. 2001;J et al. 2007; H et al. 2008). Housefly larvaecould be used as a high protein livestock feed and

could thereby improve human nutrition by increas-ing the protein content of meat , milk, butter, andeggs (B 1991). he simplicity and low costof rearing housefly larvae on a commercial scalehas made this activity very popular in China. hemost feasible and easiest commercial utilisation ofhousefly larvae is to raise them on poultry manureand other organic wastes and then feed them fresh

Supported by the Knowledge Innovation Program of the Chinese Academy of Sciences , Grants No. KSCX2-YW-G-040and No. KSCX2-YW-N-081.


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to poultry or other livestock. In addition to their value as a livestock feed, other valuable mater ials,such as protein or peptides, chitosan, phospholipid,and antibiotics have been extracted from houseflylarvae (B & P 1970; I et al. 1998;H et al. 2007; A et al. 2008). Our preliminaryobservations indicate that dried housefly larvaecontain approximately 55% protein, 9.1% crudechitin, and 8.8% fat (unpublished data). We weremotivated to develop better techniques for rearingand utilising housefly larvae because we had previ-ously reared lepidopteran insects on an artificialdiet, much of which was wasted. Housefly larvaeprovide a potential means of converting this wasteinto useful biological products. A series of attemptswere carried out to isolate and characterise pro-

tein, chitin, fat, and other materials from houseflylarvae reared on the above-mentioned diet. hispaper describes the results of the experimentsundertaken to characterise chitosan extractedfrom the cuticle of these larvae.

Chitin (β-(1,4)-2-acetamido-2-deoxy- -glucose)is the second most abundant natural carbohydratepolymer. Chitin an d its derivative chitosan arethought to have more than two hundred uses inareas as diverse as agriculture, biomedicine, cos-metics, food, textiles, as well as the potential for

use as chelating agent and in refining industrialeffluents (B et al. 2000; R K 2000;R 2006; A et al. 2009). Currently, themost available source of chitin is the exoskeletonsof crustaceans, particularly shrimps and crabs(M & B 1978; A et al. 1993).However, the relatively high calcium, wax, andpigment contents of shrimp and crab exoskeletonscause the extraction of chitosan from them to berelatively expensive. Compared to shrimps or crabs(R et al. 2008; Y et al. 2009), the cuticle ofthe housefly larvae is much easier to extract chitinfrom because it contains smaller amounts of crudeprotein, crude fat, and ash (our unpublished data).However, no one has yet developed a method toobtain high-grade chitosan from housefly lar valcuticle. In this paper, we describe the results ofthe experiments aimed at optimising the extrac-tion of chitosan from housefly larvae.

MATERIAL AND METHODS

Chitin extraction . Four-day old housefly larvaewere isolated from waste feed, rinsed and boiled

for 10–15 min, and subsequently macerated in ahome juicer. he resultant granules of crude cu -ticle were strained from the mixture with a meshsieve, extensively washed, and freeze-dried. Proteinand lipid in the dry cuticle were removed by thetreatment with 1 mol/l NaOH solution (HengyeZhongyuan Chemical Co., Beijing, China) at 100°Cfor 3 hours. Crude chitin was obtained by rinsingthe mixture with water until reaching neutral pHfiltered with mesh sieve to remove water and thenfreeze-dried.

Chitosan preparation . Several single-factorexperiments were conducted to evaluate variousreaction conditions for the deacetylation of chitin.

he factors were the granularity of the raw chi-tin, NaOH concentration, reaction temperature,

proportion of solution to material (PSM), reactiontime, and steeping time. Four grams of raw chitinwere used in each experiment. he experimentswere designed so that when one factor was testedat different levels, a ll the other factors were fixed.Other experimental constants included the diam-eter of the raw chitin granules (ca. 0.085 mm),NaOH concentration (50% w/v), reaction tem-perature (125°C), PSM (30 ml/g), reaction time(4 h), and steeping time (0.5 day). Based on thesesingle-factor experiments, an orthogonal experi-

mental design was applied to identify the optimaldeacetylation conditions ( able 1). he data fromthe orthogonal experiment were analysed by generallinear model analysis of variance with Duncan’smultiple range test using SPSS software version16.0 (SPSS Inc., Chicago, USA).

Removal of impurities from raw chitosan . oavoid the negative impact of the impurities onchitosan quality, a refinement procedure was developed for their removal from the deace tylatedproduct. After deacetylation, chitosan was filteredand rinsed until neutral pH was achieved. Chitosanwas then dissolved in acetic acid solution (1%) andthe impurities were removed by filtration throughnylon net with a mesh diameter of 0.050 mm. he

able 1. Orthogonal experimental combination of differentfactors at various levels

ParametersLevels

1 2 3

Proportion of solutionto material (ml/g) 22.5 25.0 27.5

Reaction temperature (°C) 130 125 120

Reaction time (h) 4 6 8


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pellucid filtrate was neutralised and precipitatedwith NaOH solution until reaching neutral pH.

he precipitate was rinsed several times and thenfreeze-dried after which the yield, i.e. the amountof pure chitosan (% m/m) obtained from crudechitin, was calculated.

Determination of the degree of deacetylation,viscosity and ash content . he degree of deacetyla-tion (DDA) was measured by the acid-base titrationmethod (D & R 1983) with modi-fications. In brief, chitosan (0.1 g) was dissolvedin 30 ml HCl aqueous solution (0.1 mol/l) at roomtemperature with 5–6 drops of methyl orangeadded. he red chitosan solution was titrated with0.1 mol/l NaOH solution until it turned orange.

he DDA was calculated by the formula:

DDA (%) =(C 1V 1 – C 2V 2)

× 0.016 M × 0.0994

where:C 1 – concentration of standard HCl aqueous solution

(mol/l)C 2 – standard NaOH solution (mol/l)V 1 – volume of the standard HCl aqueous solution used

to dissolve chitosan (ml)V 2 – volume of standard NaOH solution consumed

during titration (ml) M – weight of chitosan (g)

he number 0.016 (g) is the equivalent weightof NH 2 group in 1 ml of standard 1 mol/l HClaqueous solution, and 0.0994 is the proportionof NH 2 group by weight in chitosan.

One gram of the end product was dissolved in1% acetic acid solution (100 ml) and its viscositywas measured by means of NDJ-1 rotation vis-cometer (Hengping Instrument Co., Shanghai,China) at 25°C. he ash content was determinedgravimetrically after the incineration of the sam-ple (0.5–1.0 g) in a muff le furnace at 500°C for atleast 4 hours.

Chitosan FTIR analysis . Fourier transform in-frared (F IR) spectrum of the end product sampleswas measured in KBr pellets in the transmissionmode in the range of 400–4000 cm –1 using a BrukerEquinox 55 spectrophotometer (Bruker Optik,Ettlingen, Germany).

X-ray diffraction analysis . he X-ray diffraction(XRD) experiment was performed in the rangeof 3–60° (2θ) using a PANalytical X’Pert PRO X-

ray diffractometer (PANalytical Co., Almelo, theNetherlands).

Thermal analysis . hermo-gravimetric analy-sis ( GA) and differential scanning calorimetry(DSC) were carried out using Netzsch S A 499Cthermal analyser (NE ZSCH-Gerätebau GmbH.Selb, Germany) at a heating rate of 10°C/min undernitrogen atmosphere.

Determination of physical and chemical prop-erties and sanitary indices . Some physical andchemical properties of the chitosan prepared underthe determined optimal processing conditions weretested, including the amounts of water, ash, andheavy metals, such as lead (Pb) and arsenic (As), theDDA and viscosity, and bacteriological detections.Lead (Pb) content was determined by graphitefurnace atomic absorption spectrometry accord-ing to the method of O et al . (2005) with

slight modifications. Arsenic (As) was measuredby hydride generation atomic fluorescence spec-trometry according to the method of E -H etal . (2007). Aerobic bacterial count was determinedusing the method described by J et al . (1977)and coliforms were enumerated by the method ofF et al . (1994). Pathogens, including Sta-

phylococcus aureus and Salmonella , were screenedby the methods of B et al . (1986) and Jet al . (1995). he data were compared with thoseof Chinese Fishery rade Standard SC/ 3403-2004

(Ministry of Agriculture of the People’s Republicof China 2005).

RESULTS AND DISCUSSION

Chitosan preparation

he effects of variation of different factors on theDDA, viscosity, yield, and ash content of the endproduct are shown in Figures 1 and 2. As shownin Figure 1, DDA generally increased with thegranularity of the raw chitin, NaOH concentra-tion, reaction temperature, PSM, reaction time,and steeping time, while viscosity was negativelycorrelated with these factors. NaOH concentra-tions of below 50% resulted in significantly reduced yie lds (Figure 2B). None of the factors examinedhad a significant effect on ash content (Figure 2).

hese results suggest that optimal conditions forthe preparation of chitosan from housefly larvaewere granularity of the raw chitin particles of about0.85 mm, steeping time ≤ 1 day, reaction time of

4–8 h, reaction temperature of 120–130°C, andPSM of 20–30 ml/g.


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Different factors had different effects on thedeacetylation reaction. Using exoskeleton particlesthat were too ne resulted in a great decrease of the yield and viscosity of the end product, while thosethat were too coarse reduced DDA (Figures 1A and2A). Although NaOH concentrations below 50%reduced the yield by inhibiting the deacetylation reac-tion (Figure 2B), excessive NaOH reduced viscosity(Figure 1B). A low DDA and yield also occurred athigh ash content at 120°C while high temperaturescaused a sharp decrease in viscosity and yield (Fig-

ures 1C and 2C). Peak yields were obtained whenPSM was 25 ml/g or 30 ml/g (Figure 2D). AlthoughDDA increased with PSM, viscosity generally de-creased (Figure 1D). A PSM of 15 ml/g seemedunsuitable because of inadequate soaking of theraw chitin resulting in a low DDA, viscosity, and ahigh ash content in the end product (Figures 1D and2D). Prolongation of the reaction or steeping timeimproved DDA (Figures 1E and 1F), but excessivereaction or steeping time decreased the yield and viscosi ty (Figures 1E, 1F, 2E and 2F).

79

80

81

82

83

84

2.0 0.85 0.60 0.425 0.30

Chitin sample granularity (mm)

D D A ( % )

240

270

300

330

360

(

m P

a . s )

DDA visc osity

70

74

78

82

86

90

50 55 60

NaOH concentration (%)

D D A ( % )

120

180

240

300

360

( m P a . s )

D.D. viscosity

Figure 1. Effect of different factors on the degree of deacetylation (DDA) and viscosity of chitosan prepared fromhousey larvae. Te DDA ( □ ) and viscosity ( ▲ ) were measured by the modied acid-base titration method and NDJ-1 viscosimetry, respectively, under different conditions, including chitin sample granularity (A), NaOH concentration(B), reaction temperature (C), proportion of solution to material (D), reaction time (E) and steeping time (F)

(A) (B)

0

20

40

60

80

100

120 125 130 135 140

Reaction temperature (°C)

D D A ( % )

0

150

300

450

600

750

(

m P a

. s )

50

58

66

74

82

90

15 20 25 30 35 40

Proportion of solution to material (ml/g)

D D A ( % )

150

210

270

330

390

450

(

m P a • s )

65

70

75

80

85

90

2 4 6 8 10 12Reaction time (h)

D D A ( % )

0

80

160

240

320

400

(

m P a . s

)

76

78

80

82

84

86

0.5 1.0 1.5 2.0 2.5 3.0

Steeping time (day)

D D A ( % )

290

305

320

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350

(

m P a

. s )

(C) (D)

(E) (F)


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Optimal preparation conditions for chitosan

DDA, yield, viscosity, and ash content are thefour main parameters influencing the prepara-tion efficiency and chitosan quality. Our single-factor experiments (Figures 1 and 2) indicatedthat DDA and yield were the two key parametersthat should be considered in the orthogonal ex-

periment. Figure 3 shows the results of the or-thogonal experiment on the effects of various

levels of different factors on DDA and yield. PSMhad no significant effect on DDA or yield. hereaction temperature and reaction time were thekey factors affecting the DDA, while the yieldwas significantly affected only by the reactiontemperature. aking into account the produc-tion costs, these results suggest that optimalconditions for chitosan preparation are PSM of22.5 ml/g, a reaction temperature of 125°C, anda reaction time of 6 hours.

56

60

64

68

72

76

2.0 0.85 0.60 0.425 0.30

Chitin sample granularity (mm )

Y i e l d ( % )

0

0.1

0.2

0.3

0.4

0.5

A s h c o n

t e n

t ( % )

yield ash content

0

20

40

60

80

100

40 45 50 55 60

NaOH concentration (%)

Y

i e l d ( % )

0

0.1

0.2

0.3

0.4

0.5

A s h c o n

t e n

t ( % )

yield ash content

40

48

56

64

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80

120 125 130 135 140

Reaction temperature (°C)

Y i e l d ( % )

0

0.1

0.2

0.3

0.4

0.5

A s h c o n

t e n

t ( % )

40

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15 20 25 30 35 40

Proportion of solution to material (ml/g)

Y i e l d ( % )

0

0.1

0.2

0.3

0.4

0.5

A s h

c o n

t e n

t ( % )

50

55

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2 4 6 8 10 12

Reaction time (h)

Y i e l d

( % )

0.00

0.10

0.20

0.30

0.40

0.50

A s h c o n

t e n

t ( % )

60

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0.5 1.0 1.5 2.0 2.5 3.0

Steeping time (days)

Y i e l d ( % )

0

0.1

0.2

0.3

0.4

0.5

A s h c o n

t e n

t ( % )

Figure 2. Effect of different factors on the yield and ash content of chitosan obtained from housey larvae. Te yield(□ ) and ash content ( ▲ ) were determined by standardised methods under different conditions, including chitin samplegranularity (A), NaOH concentration (B), reaction temperature (C), proportion of solution to material (D), reactiontime (E) and steeping time (F)

(A) (B)

(C) (D)

(E) (F)


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Received for publication March 26, 2010Accepted after corrections December 30, 2010

Corresponding author:

Dr. Q -L Q and Dr. Y -J W , Chinese Academy of Sciences, Institute of Zoology, State Key Laboratoryof Integrated Management of Pest Insects and Rodents, Beijing 100101, P.R. China

tel.: + 86 10 648 070 56 (Q.-L. Qin); + 86 10 648 072 51 (Y.-J. Wu), e-mail: [email protected] (Q.-L. Qin); [email protected] (Y.-J. Wu)

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