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JOURNAL OFFOOD COMPOSITION

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Journal of Food Composition and Analysis 19 (2006) 850–853

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Short Communication

Proximate, amino acid and mineral composition of pupae of thesilkworm Antheraea pernyi in China

Jun Zhou�, Dingxian Han

Department of Chemistry, Huazhong University of Science and Technology, Wuhan 430074, PR China

Received 12 September 2005; received in revised form 10 February 2006; accepted 20 April 2006

Abstract

The chemical composition and the nutritional quality of protein of pupae of the silkworm Antheraea pernyi were investigated.

Investigations showed that the pupal powder contained 7.6% moisture, 71.9% crude protein, 20.1% fat and 4.0% ash on a dry matter

basis. The mineral analysis indicated high K content with a low Na/K ratio and low heavy metal content. The pupal protein contained 18

known amino acids, including all of the essential amino acids and sulphur-containing amino acids. Compared with the amino acid profile

recommended by FAO/WHO, the pupal protein was of high quality due to its high content of essential amino acids. The results of the

present study provide some technical information and suggestions for the food industry for more effective utilization of pupae of the

silkworm A. pernyi.

r 2006 Elsevier Inc. All rights reserved.

Keywords: Silkworm pupae; Antheraea pernyi; Amino acids; Mineral; Proximate composition; Nutritive value

1. Introduction

Silkworms are well known as an efficient large-scaleproducer of silk threads. Among the various species ofsilkworms, the mulberry silkworm (Bombyx mori L.) andthe non-mulberry silkworm (Antheraea pernyi) are ofcommon use in sericulture. The mulberry silkwormB. mori L., reared on mulberry leaves, belongs to familyBombycidae while the non-mulberry silkworm A. pernyi,reared on oak leaves, belongs to family Saturniid (Mishraet al., 2003). However, it is not well known amongconsumers that silkworm pupae are an interesting by-product obtained after the extraction procedure of silkthreads. The pupae of mulberry and non-mulberry silk-worms were traditionally accepted and used as fertilizer,animal feed and edible insects in some countries, such asJapan, Korea, India and Thailand (Pereira et al., 2003;Mishra et al., 2003; Zhu, 2004). In China, humanconsumption of silkworm pupae has been practised sincethe very earliest times. In recent years, silkworm pupae

e front matter r 2006 Elsevier Inc. All rights reserved.

a.2006.04.008

ing author. Tel.: +8627 87543532; fax: +86 27 87543632.

ess: hustzhj@163.com (J. Zhou).

have been approved as a new food resource by the Ministryof Health of the People Republic of China (Zhu, 2004).While pupae of the silkworm A. pernyi are commonly

eaten, there is little available information on their nutritivevalue. Published works include: Rao (1994) on thechemical composition of silkworm (B. mori L.) pupae;Pereira et al. (2003) on proximate composition and fattyacid profile of B. mori L. chrysalis toast; Mishra et al.(2003) on proximate composition of non-mulberry andmulberry silkworm pupae in India. Therefore, based on theinterest of some countries in using the product in animalfeeds or for human nutrition, additional information onthe nutritional composition of pupae of the silkworm A.

pernyi is necessary. The present study was designed toevaluate the proximate, amino acid and mineral composi-tion of pupae of the silkworm A. pernyi.

2. Materials and methods

2.1. Sample preparation

Pupae of the silkworm A. pernyi were purchased fromthe villages in Luoyang (Henan Province, China) and

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Table 1

Proximate composition of pupae of the silkworm Antheraea pernyi

(g/100 g dry weight)b

Component Composition (mean7S.D.a)

Moisture 7.670.51

Protein 71.971.2

Fat 20.170.3

Ash 4.070.04

aS.D., standard deviation, n ¼ 3.

bThe values are on a dry-matter basis. The moisture content is 77.0% on

a fresh-weight basis, so data may readily be converted to fresh basis if

needed, using this tabulated data.

J. Zhou, D. Han / Journal of Food Composition and Analysis 19 (2006) 850–853 851

visually identified by Associate Professor Dingxian Han(Department of Chemistry, Huazhong University ofScience and Technology, Wuhan, China) who was certifiedin determining the pupae. The characteristics of the pupaeconformed to the standard criteria. After the sample waswashed with distilled water and ground in a meat grinder, itwas centrifuged at 3000 rev/min for 20min to get rid ofdeposits (mainly pupa shell). The liquid centrifugate wasspray-dried and ground into fine powder.

2.2. Proximate composition

Moisture, ash and fat contents were assayed by theAssociation of the Official Analytical Chemists (AOAC,1984) Methods 14004, 14009 and 14006, respectively.Nitrogen was determined using the Kjeldahl method(Matissek et al., 1989). The quantity of protein wascalculated as 6.25�N (method 7015, AOAC, 1984).

2.3. Amino acid analysis

The PICO TAG method with modification was em-ployed for determining the amino acid profile of the sample(Bidlingmeyer et al., 1987; Deng et al., 2004). The aminoacids were determined from standard curves based on peakarea measurements. Five-point standard curves were madefor all analysed amino acids using reference materials. Inaddition, intraday variability (R.S.D.) and spike recoverystudies were performed. Thirty milligrams of silkwormpupae powder were hydrolysed in evacuated sealedampoules with 6M hydrochloric acid at 110 1C for 24 h.Ten millilitres of internal standard (a-amino butyl acid)were added to the mixture. After derivatisation, 100mLPICO TAG diluent were added and mixed; 100mL sampleswere then injected into the HPLC and analysed withWaters PICO TAG amino acid analyser. Tryptophancontent was then determined using the basic hydrolysismethod (Deng et al., 2004). Silkworm pupae powder(weight equivalent to 1�2mg tryptophan) was treated with4.2 M sodium hydroxide (100mL) and 0.3mL thioglycerinand placed in the oven at 110 1C for 24 h. After cooling, thepH was adjusted to 4.5, and the mixture was made up to acertain volume. Tryptophan content was determined bycolorimetric analysis at 440 nm under the conditions of pH5.0�5.5, column temperature 55 1C, reactor temperature100 1C, reaction time 10�15min.

2.4. Mineral content

Mineral content was analysed with a Perkin-Elmer(optima) 3000 DV analyser with induction coupled plasmaatomic emission spectroscopy (ICPAES; Eknayake et al.,1999). Arsenic was measured as inorganic arsenic. Thesample (2 g) was digested with 20mL concentrated nitricacid (BDH-Aristar) until a transparent solution wasobtained. The instrument was calibrated with knownstandards and samples analysed at corresponding wave-

lengths. Five-point standard curves were made for allanalysed minerals using reference materials. Linear regres-sion analysis of the standard curves indicated they werelinear with correlation coefficients in the range of0.997–0.999. Selenium was determined as hydride using ahydride generator (VGA-76). The samples digested inHNO3 were mixed with concentrated HCl and heated to70�90 1C for 10min and cooled before injection intohydride generator followed by NaBH4.

3. Results and discussion

The proximate composition of pupae of the silkworm A.

pernyi is given in Table 1. The values obtained formoisture, crude protein and ash content from the presentstudy are 7.6%, 71.9% and 4.0% respectively, in agreementwith the values reported by Pu and Chen (Pu and Chen,2002; namely, 8.5%, 70.6% and 3.9%, respectively). Thecrude protein content is high on a dry matter basis, whichmakes pupae of the silkworm A. pernyi a good supplementto protein food. The crude fat content is 20.1%, and mostof it is constituted by unsaturated fatty acids, which haveimportant physiological functions according to previousstudies (Lei and Song, 1998; Rao, 1994).The mineral analysis (Table 2) indicates high concentra-

tion of potassium (34.0mg/g). Furthermore, Na/K ratio isvery low (0.08), which is interesting from the point of viewof nutrition, since the intake of sodium chloride and dietswith a high Na/K ratio have been related to the incidenceof hypertension. Most of the trace elements present inpupae of the silkworm A. pernyi are necessary traceelements (Cu, Zn, Fe, Se), which have important physio-logical functions (Liu, 1996). The selenium level is belowthe toxic limit (Ministry of Health, PR China, andNational Standardization Management Committee PR,China, 2005), but the lead and arsenic levels are above themaximum levels for lead and arsenic (0.5mg/kg) estab-lished for a range of foods in Chinese Standards (Ministryof Health, PR China, and National StandardizationManagement Committee, PR China, 2005). However,considering that silkworm pupae are consumed in a freshstate, and that silkworm pupae contain 77.0% moisture on

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Table 3

Amino acid composition of pupae of the silkworm Antheraea pernyi (mg/g

crude protein)

Amino acid Silkworm (Antheraea

pernyi) pupae

Silkworm (Bombyx

mori L.) pupae

Egg

Aspartic acid 64.1 109 89.2

Threonine 46.4 54 44.7

Serine 46.4 47 67.2

Glutamic acid 127.4 149 121.3

Glycine 44.2 46 30.2

Alanine 62.6 55 50.3

Cysteine 1.5 14 19.0

Valine 66.3 56 54.2

Methionine 14.7 46 28.1

Isoleucine 79.5 57 48.8

Leucine 32.4 83 81.1

Tyrosine 20.6 54 38.1

Phenylalanine 81.0 51 48.2

Lysine 45.4 75 65.9

Histidine 29.4 25 20.9

Arginine 41.2 68 57.0

Proline 122.2 40 33.8

Tryptophan 40.5 9 17.2

Total 965.8 1038 915.2

Table 4

Essential amino acid composition of pupae of the silkworm Antheraea

pernyi compared with the FAO/WHO pattern (mg/g crude protein)

Amino acid Silkworm

(Antheraea

pernyi) pupae

FAO/WHO

pattern

Amino acid

scores

Isoleucine 79.5 40 1.99

Leucine 32.4 70 0.46

Lysine 45.4 55 0.82

Methionine+cysteine 16.2 35 0.46

Phenylalanine+tyrosine 101.6 60 1.69

Threonine 46.4 40 1.16

Tryptophan 40.5 10 4.05

Valine 66.3 50 1.33

Table 2

Mineral composition of pupae of the silkworm Antheraea pernyib

Mineral Composition (mean7S.D.a)

Potassium (mg/g) 34.070.6

Sodium (mg/g) 2.8170.07

Calcium (mg/g) 0.6370.01

Magnesium (mg/g) 1.5470.03

Phosphorus (mg/g) 2.7270.05

Iron (mg/g) 40.071.3

Selenium (mg/g) 0.1170.02

Copper (mg/g) 7.2770.34

Zinc (mg/g) 35.771.8

Lead (mg/g) 1.1370.04

Arsenic (mg/g) 1.3070.03

aS.D., standard deviation, n ¼ 3.

bThe values are on a dry-matter basis. The moisture content is 77.0% on

a fresh-weight basis, so data may readily be converted to fresh basis if

needed, using this tabulated data.

J. Zhou, D. Han / Journal of Food Composition and Analysis 19 (2006) 850–853852

a fresh-weight basis (Zhou and Yang, 1993), the lead andarsenic levels will be lower than 0.5mg/kg. The ash contentis somewhat lower than the sum of the contents ofminerals, presumably as a result of analytical variability.The difference in mineral contents from those reported inthe literature (Pu and Chen, 2002) possibly results fromgeographical place of harvest, seasonal, annual andenvironmental factors, type of processing, and so on.

Validation of the amino acid analysis method showedthe standard curves for all analysed amino acids were linearwith correlation coefficients in the range of 0.998–0.999.The intraday variability (R.S.D.) was less than 2.0% for allanalysed amino acids (n ¼ 6). Recoveries ranged from97.6% to 101.5% for various amino acids (n ¼ 6). Theamino acid composition of pupae of the silkworm A. pernyi

is shown in Table 3. The results showed that pupae of thesilkworm A. pernyi contained eighteen known amino acids,including all of the essential amino acids (EAAs) andsulphur-containing amino acids. The percentage of EAAsin total amino acids was 42%, and the ratio of EAAs tonon-EAAs was 0.7, which approximated to the referencevalues of 40% and 0.6 recommended by FAO/WHO(1973). In addition, the percentages of savoury amino acids(asparagines and glutamic acid) and sweet amino acids(glycine and alanine) to total amino acids were as high as19.8% and 11.0%, respectively. While it is known thatcystine can substitute for part of the requirement formethionine, FAO/WHO/UNU (1985) does not give anyindication of the proportion of total sulphur-containingamino acids which can be met by cystine. For the rat, chickand pig, the proportion is about 50% (FAO/WHO, 1991).Most animal proteins are low in cystine; in contrast, manyvegetable proteins contain substantially more cystine thanmethionine. Thus, for animal protein diets or mixed dietscontaining animal protein, cystine is unlikely to contributemore than 50% of the total sulphur-containing amino acids(FAO/WHO, 1991; Adeyeye and Afolabi, 2004). This pointis amplified by our results with the value of 9.2%.

In addition, we compared the amino acid composition ofpupae of the silkworm A. pernyi with those of pupae of thesilkworm B. mori L., and with egg (Rao, 1994; Wang,1991). The EAA content of pupae of the silkwormA. pernyi (406.2mg/g protein) was lower than that of pupaeof the silkworm B. mori L. (431mg/g protein) and higherthan that of egg (388.2mg/g protein). However, the pupalprotein of the silkworm A. pernyi had higher contents ofisoleucine, phenylalanine, tryptophan and valine than thatof the silkworm B. mori L. and the whole egg protein.Threonine content was higher in the pupal protein of thesilkworm A. pernyi than in the whole egg protein.The EAA profile of the pupal protein of the silkworm A.

pernyi (Table 4) revealed that the levels of EAAs werecomparable with those of the FAO/WHO amino acidreference pattern (FAO/WHO, 1973) established for hu-mans. The EAA contents of the silkworm pupal protein

ARTICLE IN PRESSJ. Zhou, D. Han / Journal of Food Composition and Analysis 19 (2006) 850–853 853

(428.3mg/g protein, including cysteine and tyrosine) werehigher than those of the FAO/WHO pattern (360mg/gprotein). According to the amino acid scores, the firstlimiting amino acids of the pupal protein were leucine andmethionine+cysteine, and the second was lysine, whereasthe contents of other EAAs were higher than the referencevalues recommended by FAO/WHO.

The preliminary results presented in this paper indicatethat pupae of the silkworm A. pernyi have a great potentialfor narrowing the amino acid deficits that are prevalent inthe diets in many developing countries. Silkworm pupaecould serve as an alternative dietary supplement of proteinand amino acids, or as animal feed. In China, more than100,000 tonnes of silkworm pupae are produced annually(Wei and Xu, 1999). Therefore, in order to fully utilize thisnutritional resource in abating nutritional deficiencies,intensive efforts towards the popularization of pupae ofthe silkworm A. pernyi are being encouraged. Furthermore,detailed analysis of pupae of the silkworm A. pernyi forother nutrients, anti-nutrients and secondary metaboliteswith medicinal potential should be undertaken. The resultsfrom the present analysis might strengthen the industrialuse of this by-product as a supplement of protein, aminoacids and trace minerals in foods.

Acknowledgement

The authors would like to thank Food Quality Inspec-tion and Testing Center of Agricultural Ministry (Wuhan,PR China) for their technical assistance.

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