Eri Silkworm a Source of Edible Oil With a High-libre

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Research ArticleReceived: 20 May 2011 Revised: 29 November 2011 Accepted: 30 November 2011 Published onlinein Wiley OnlineLibrary: 30 January 2012

(wileyonlinelibrary.com) DOI 10.1002/jsfa.5572

Eri silkworm: a source of edible oil with a high

content of

-linolenic acid and of signicantnutritional valueThingnganing Longvah, Korra Manghtya and Syed S Y H Qadri

Abstract

BACKGROUND: The study was undertaken to provide value addition to spent eri silkworm as an alternative source of edible oilfor the food and feed industry by carrying out a short-term nutritional and toxicological evaluation of eri silkworm pupae oilusing Wistar NIN rats.

RESULTS: Growthperformanceof ratsfed either sunoweroil (Control)orerisilkwormpupae oil (Experimental)wascomparable.Histopathological examination of the various tissues showed no signs of toxicity even after feeding the eri silkworm oil for18 weeks. Serum cholesterol and triglyceride was signicantly reduced ( P < 0 . 05) while high-density lipoprotein cholesterolwas signicantly increased ( P < 0 . 05) which is attributed to the high -linolenic acid content of eri silkworm oil.

CONCLUSION: The study showed that eri silkworm pupae oil is safe and nutritionally equivalent to commonly usedvegetable oils. Eri silkworm pupae can be harvested to provide a cost effective alternative edible oil that can be used tonutritional advantage in the food and feed industry. Therefore eri silkworm and its host plants offer an excellent exampleof multiple product crops and of sustainable agricultural practice with excellent opportunity for economic and nutritionalbenets.c 2012 Society of Chemical Industry

Keywords: eri silkworm ( Samia ricinii ) oil; fatty acid composition; nutritional; toxicological evaluation

INTRODUCTIONErisilkworm(Samiaricinii )isoneoftheimportantnon-mulberrysilk produced primarily in north-east India. Eri silkworm is polyfagusand grows on a wide range of plants such as castor ( Ricinuscommunis ), tapioca ( Manihot utilizsima ), kissaru (Heteropanax fragrans ), payam ( Evodia axinifolia), barpat ( Ailanthus grandis ),papaya ( Carica papaya ), jatropha ( Jatrophacurcus ) and barkesseru( Ailenthus excels).1 Eri silk is a staple bre, darker and heavier thanother silks. Eri silk blends well with cotton, wool, jute or mulberrysilk to create a new range of fabrics which is in great demand. Therefore, eri culturewas introducedin many statesoutsidenorth-east India, wherever the host plants are being cultivated on largetracts of land, in order to not only increase eri silk productionbut also to improve the economic status of the marginal farmersgrowing the host plant.

The population in north-east India uses a variety of insects asfood, one of which is the eri silkworm ( Samia ricinii ). The spent erisilkworm pre-pupae and pupae are considered a delicacy and isalways in great demand in local markets. 2 Nutrient compositionand protein quality evaluation of eri silkworm pre-pupae andpupae has shown that it is an excellent source of good qualityprotein. 3 Someinsectsarealso known to secrete toxic metabolitesor sequester toxic chemicals from plant foods. 4 7 However,the long history of human use suggests that insects harvestedfor human consumption may not pose any signicant healthproblem.

In 20092010, India imported 10.1 million tons of edible oil asagainst the domestic production of 8.2 million tons. The demandfor imported oil remains strong due to growing consumptionand the constraints on supply. 8 Therefore, the government of India is exploring alternative sources of edible oil to augment theincreasing shortage in order to meet the growing demand andsave precious foreign exchange.

Silk production in India reached 19600 metric tons in2009 2010. Pupae, which accounts for 60% of the cocoonweight, is discarded as waste material. Silkworms such as Samiaricinii, Bombyx mori and Antheraea pernyi have been reportedto contain good amounts of fat. 3,9,10 With the introductionof eri culture outside north-east India the production of erisilk has increased substantially, with a concomitant increasein the production of eri pre-pupae and pupae. Harvesting oilfrom spent silkworm could help augment the acute shortagefor edible oil in the food and feed industry in the country. Therefore the study was undertaken to provide value additionto spent eri silkworm pre-pupae and pupae by carrying out short-term nutritional and toxicological evaluation of the eri silkwormpupae oil.

Correspondenceto: ThingnganingLongvah,NationalInstituteof Nutrition,Ja-maiOsmaniaPO,Hyderabad 500007,AP, India.E-mail:[email protected]

National Institute of Nutrition, Jamai Osmania PO, Hyderabad 500 007, AP,India

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Eri silkworm as a source of linolenic acid www.soci.org

MATERIALS AND METHODSSample preparationAll the samples of eri silkworm pre-pupae and pupae requiredfor the study was supplied by the Central Silk Board, Governmentof India, Ministry of Textiles, Bangalore, through its local ofcein Hyderabad. The eri silkworm pre-pupae and pupae samplesgrown on either castor or tapioca were washed separately withdeionised water and spread on lter paper sheets. Samples of eri silkworm pre-pupae and pupae grown on either castor ortapioca were taken separately for fatty acid analysis. The rest of the samples were homogenised and dried overnight in a hot airoven at 6070 C. Dried samples of eri silkworm pre-pupae andpupae grown on either castor or tapioca were extracted withn-hexane using a Soxhlet apparatus to separate the oil which waspooled and used for the nutritional and toxicological evaluationin vivo.

Fatty acid analysisLipids were extracted from the eri silkworm pre-pupaeand pupaeaccording to the method of Bligh and Dyer 11 and methylatedas described by Loweinstein etal .12 The fatty acid compositionof the samples was determined by gas chromatography ona Shimadzu 2010 gas chromatograph equipped with ameionisation detector (Shimadzu, Kyoto, Japan). Separation wascarried out in a Phenomenex BPX-70 fused silica column (30 m 0.32 mm). The temperature of the injection and detection portswas maintained at 230 C. The initial temperature of 140 C wasincreased at the rate of 4 C min 1 and maintained at 226 C for20 min. Individual fatty acids were identied and quantied bycomparing the retention time and peak area with authentic fattyacid methyl ester standards.

Nutritional and toxicological evaluation of eri silkworm oil The animal experiment was carried out after the experimentalprotocol was approved by the institutional animal ethics com-mittee. Weanling Wistar NIN rats (2123 days old) were dividedinto two groups of 24 animals each (12 males and 12 females)and fed the diet containing either 10% sunower oil (Control)or eri silkworm oil (Experimental). The fatty acid composition of the sunower and eri silkworm oil used in the experiment isgiven in Table 1. The diets consisting of 633 g kg 1 corn starch,200 g kg 1 casein protein, 40 g kg 1 mineral mixture, 10 g kg 1

vitamin mixture, 15 g kg 1 cellulose, 2 g kg 1 choline chloride,10 mg kg 1 DL-methionine and either 10% sunower oil (Con-trol) or eri silkworm pupae oil (Experimental) was fed to theanimals ad libitum for 18 weeks as described earlier. 13 Towardsthe end of 18 weeks, animals were transferred to metabolic

cages and faeces were collected for 3 days. The diet and fae-ces of the animals were analysed for nitrogen by the AOAC 14

Kjeldahl method (984.13), calcium by using a Varian Techtron100 Atomic absorption spectrophotometer (Varian, Amsterdam,Netherlands) after dry ashing the sample and phosphorus by theFiske and Subbarow method as described in AOAC 14 method(931.01). The apparent retention of these nutrients was calcu-lated from the dietary intake and faecal excretion. At the end of the 6, 12 and 18 weeks, blood was drawn from the retro-orbitalsinus of the animals for the estimation of serum cholesterol,triglycerides, alkaline phosphatase, creatinine and urea usingstandard kits procured from Biosystems SA (Costa Brava 30,Barcelona, Spain).Liver sampleswere analysedfor total cholesterolcontent.

Table 1. Fatty acid prole of eri silkworm pupae oiland sunower oilused in the diet

Fatty acid Eri silkworm pupae oil Sunower oil

16 : 0 Palmitic acid 26.98 5.616 : 1 Palmitoleic acid 1.82 18 : 0 Stearic acid 4.73 2.2

18 : 1 Oleic acid 15.89 25.118 : 2 Linoleic acid 5.49 66.218 :3 -Linolenic acid 44.73 20 : 0 Arachidic acid 0.924 : 0 Lignoceric 0.4 Total saturates 31.71 9.1Monounsaturates 17.71 25.1Polyunsaturates 50.23 66.2

Results are the means of duplicate analyses and are given as the % of the total fatty acids.

Organ weights and histopathology

The animals were sacriced and liver, spleen, kidneys, lungs, heart,brain, testes and ovaries were carefully dissected out. Individualorgan weights of each animal in the sunower and eri silkwormpupae oilgroupswereweighedand recordedimmediately.Tissuesof liver, spleen, kidneys, lungs, heart, brain, pancreas, trachea,thymus,adrenal,ovaries,testes,sternum, stomach,small intestine,large intestine, sciatic nerves and abdominal aorta were xed,processed, embedded andsectioned by theconventionalmethod.Five-micrometre sections stained in Mayers haematoxylin&eosinwere examined under light microscopy for any changes in thetissues due to the consumption of eri silkworm pupae oil.

Statistical analysis

The data from animal experimentation was tested for statisticalsignicancebyANOVAusingtheSPSS11.0package(SPSS,Chicago,IL, USA).

RESULTS AND DISCUSSIONFatty acid composition of eri silkworm The fatty acid composition of eri silkworm pre-pupae and pupaegrown on either castor or topiaca wascomparable forall fatty acidsexcept -linolenic acid which was higher in in eri silkworm grownon castor (Table 2). Developmental stages of the insect appear tohave a bearing on the fatty acid content as stearic acid increasedfrom 3.91 to 5.61% as the worm transformed from the pre-pupae

stage tothe pupae stagewith concomitantdecreasein thelinoleicacid content. Palmitic acid and -linolenic acid were the majorfatty acids comprising 6972% of the total fatty acids, which issimilar to that reported earlier for eri pupae fat by Shanker etal .15

Total saturated fatty acids of 32% in eri silk worm pupae oil in thepresent investigation was comparable to 33.2% in spent silkworm(Bombyx mori ) pupae oil reported by Rao. 9 The total unsaturatedfatty acids of eri silkworm pupae oil (6769%) and spent silk worm pupae oil (66.8%) 9 were also comparable but vegetable oilslike perilla,16 with substantial -linolenic acid content had higherlevels of total unsaturated fatty acids (87.3%).

The -linolenic acid content in eri silkworm pre-pupae andpupae oil reported in the present study (4145%) was similar tothat reportedearlierfor erisilkwormby Shanker etal .,15 buthigher

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www.soci.org T Longvah, K Manghtya, SSYH Qadri

Table 2. Comparison of the fatty acid composition of eri silk worm pre-pupae and pupae oil with other silkworms and vegetable oils

Pre-pupae oil Pupae oil

Fatty acid Castor Tapioca Castor Tapioca Bombyx mori a Perilla oilb

16 : 0 Palmitic acid 26.45 27.52 26.75 27.18 26.2 8.916 : 1 Palmitoleic acid 1.90 1.83 1.76 1.79

18 : 0 Stearic acid 3.91 3.94 5.61 5.45 7.0 3.818;1 Oleic acid 16.57 18.32 16.19 18.46 36.9 12.918 : 2 Linoleic acid 5.62 6.10 4.93 5.29 4.2 17.61 8 : 3 -Linolenic acid 45.26 41.52 44.74 41.38 25.7 56.8 Total saturates 30.36 31.46 32.36 32.63 33.2 12.7 Total unsaturates 69.35 67.77 67.62 66.89 66.8 87.3Monounsaturates 18.47 20.15 17.95 20.22 36.9 12.9Polyunsaturates 50.88 47.62 49.67 46.67 29.9 74.4

Values are means of duplicate analyses and are expressed as a percentage of the total fatty acids.a From Rao.9b From Longvah and Deosthale. 16

than 25.5%reported forspent silk wormpupaeoil9

or17 33.4%inBombyx mori L.17 In the present study both the eri pre-pupae andpupae on castor leaves showed higher -linolenic acid contentcompared to tapioca leaves which is contrary to the ndingsof Shanker etal .15 The higher -linolenic acid content in the eripupae on tapioca has been attributed by Shanker etal .15 to thehigher content of -linolenic acid in tapioca (45%) compared tocastor(40%). However, on a dryweight basis crude fatcontent hasbeen reported to be 38 g kg 1 in cassava leaves 18 and 148 g kg 1

in castor leaves 19 which can offset the small differences in their -linolenic acid content. Further, the high palimitic acid and lowoleic acid content in both castor andtapioca leaves is notreectedin the eri pupae.

Studieshavealsoshownthatthe -linolenicaciddecreasedfrom

8.7%in the dietto 3.3%in the larvaeof Trichoplusiani eventhoughit is known that -linolenic is required for wing development. 20

Biotin21 and choline 22 has also been shown to inuence fatty acidsynthesis/accumulation. Further the fatty acid prole including -linolenic acid of Bombyx mori L. has been shown to vary greatlyduring the developmental stages of the larvae. 23 Therefore theremay be other factors involved in the synthesis/accumulation of -linolenic acid in the silkworm which cannotbe just explainedbythe -linolenic acid content in the diet of silkworms as this acid isfound in thelipidof insects andinsect larvaewithdifferent feedinghabits. In the insect order Lepidoptera, to which the silkwormsbelong, -linolenic acid content has been reported to be as lowas 0.6% in Heliothis virescens to as high as 51% in Hyalophoraceropia .24 However, among the silkworm species it appears thateri silkworm has the highest content of -linolenic acid. In the

lightof the importance of n-3 polyunsaturated fatty acid in healthanddisease thehighcontent of -linolenicacid in silkwormpupaeassumes signicance.

Growth performance of rats fed 10% sunower oil or erisilkworm oil for18 weeks Table 3 shows the food intake, gain in body weights and drymatter digestibility of the animals fed either sunower (Control)or eri silkworm pupae oil (Experimental) oil diets. No statisticalsignicance could be observed for dry matter digestibility or gainin body weights between the sunower or eri silkworm pupae oilgroups which shows that eri silkworm pupae oil supports growthandweightgainsimilarto edible oilslike sunoweroil. Erisilkwormpupae oil in thediet didnot affect theretention of nitrogen (58%),phosphorus (72%) and calcium (70%) adversely and the retentionswere comparable to those of sunower oil fed animals.

Organ weights Table 4 shows the organ weightsof animals which were fed eitherthe eri silkworm pupae oil or sunower oil diet for 18 weeks.No apparent differences were observed in the organ weights of these animals thus indicating no gross abnormality due to theconsumption of eri silkworm pupae oil.

Histopathological studiesHistopathologyof theorgansrevealedno signsof toxicity(Table 5). The lesions of periportal inammatory cell in ltrates and focalareas of necrosis in the livers have been observed in one animal

Table 3. Growth performance of rats fed 10% sunower oil or eri silkworm oil for 18 weeks

Sunower oil Eri silkworm pupae oil

Parameter Males Females Males Females

Food intake (g) 1652 68 . 74 1280 68 . 61 1559 76 . 17 1282 89 . 12Gain in body weights (g) 353 24 . 19 195 17 . 50 299 27 . 16 179 22 . 36Dry matter digestibility (%) 95 . 6 0 . 87 95 . 9 1 . 00 95 . 0 0 . 94 95 . 3 1 . 00

Values are mean SD (n = 12 males and 12 females in each group).

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Table 4. Organ weights (g) of control and experimental animals atthe end of 18 weeks


Organ Males Females Males Females

Liver 12. 07 1 . 438 7 . 30 0 . 168 11 . 83 1 . 258 7 . 69 0 . 437Spleen 0 . 89 0 . 264 0 . 53 0 . 105 0 . 72 0 . 103 0 . 57 0 . 166Kidney 2 . 94 0 . 336 1 . 86 0 . 231 2 . 97 0 . 427 1 . 93 0 . 257Lungs 1 . 89 0 . 284 1 . 66 0 . 885 1 . 58 0 . 186 1 . 27 0 . 186Heart 1 . 15 0 . 151 0 . 75 0 . 067 1 . 02 0 . 129 0 . 76 0 . 116Brain 1 . 50 0 . 191 1 . 42 0 . 164 1 . 36 0 . 215 1 . 36 0 . 156 Testis 5 . 62 0 . 918 5 . 13 0 . 619 Ovaries 3 . 60 1 . 322 2 . 94 0 . 840

Values are mean SD (n = 12 males and 12 females in each group).

each of both the sunower and perilla oil fed groups respectively. Therefore, the above lesions in the liversare incidentaland notdueto the test material. The lung lesions of peribronchial lymphoid

aggregates present may be due to latent infection. These lesionsare normally present as background pathology in the lungs of many strains of rats and hence not attributed to the treatmenteffect.Thelymphoidcellcollectionsinthesubmucosaareroutinely

found in the rodent species and not attributed to the treatmenteffect.Allotherorganswerenormalanddidnotshowanychanges. Therefore, it can be concluded that no signs of toxicity wereobserved when the animals were fed with the eri-silkworm pupaeoil even at 10% level in the feed.

Biochemical parameters Table 6 shows the biochemical parameters measured at threetime points (6, 12 and 18 weeks) of the study period in theplasma of the sunower oil and eri silkworm oil fed animals. The creatinine levels showed a gradual increase with increasein body weights of the animals. However, the creatinine levelswere comparable between the control and experimental groups. The serum creatinine, alkaline phosphatase and urea levels of theeri silkworm pupae oil and sunower oil fed animals were alsocomparable indicating no functional changedue to thefeedingof eri silkworm pupae oil.

Serum triglyceride levels lowered signicantly ( P < 0. 05) inboth the male and female groups fed eri silkworm pupae oil at18 weeksas compared to the animals fed sunower oil. This effectcan be attributed to the high -linolenic acid content in the diet

which is absent in sunower oil diet. There are many reports toshow that -linolenic acid in the diet of animals can signicantlyreduce serum triglyceride levels. 25 High levels of triglyceridesin the blood stream have been linked to atherosclerosis and

Table 5. Histopathology ndings in various organs of rats fed 10% sunower oil (Control) or eri silkworm oil (Experimental) after 18 weeks(sex-pooled data)

S. no. Organ Code Diagnosis Sunower oil a Silkworm pupae oil a

1 Liver 1 Normal 95.8 (23) 95.8 (23)3 Periportal inammatory cell inltrates 4.16 (1) 05 Focal areas of necrosis 0 4.16 (1)

2 Spleen 1 Normal 95.8 (23) 95.8 (23)99 Not available 4.16 (1) 4.16 (1)

3 Kidney 1 Normal 100 (24) 100 (24)4 Lungs 1 Normal 45.83 (11) 66.66 (16)

2 Peribronchial lymphoid aggregates 54.17 (13) 33.33 (8)5 Heart 1 Normal 100 (24) 100 (24)6 Brain 1 Normal 100 (24) 100 (24)7 Pancreas 1 Normal 100 (24) 100 (24)8 Trachea 1 Normal 95.8 (23) 100 (24)

2 Tracheitis 4.16 (1) 09 Thymus 1 Normal 91.66 (22) 87.5 (21)

99 Not available 8.33 (2) 12.5 (3)10 Adrenals 1 Normal 100 (24) 100 (24)11 Ovaries 1 Normal 100 (12) 100 (12)

12 Testes 1 Normal 100 (12) 100 (12)13 Sternum 1 Normal 100 (24) 100 (24)14 Stomach 1 Normal 95.8 (23) 95.8 (23)

2 Lymphoid cell collections in submucosa 4.16 (1) 4.16 (1)15 S. intestines 1 Normal 87.5 (21) 87.5 (21)

2 Lymphoid cell collections in submucosa 12.5 (3) 12.5 (3)16 L. intestines 1 Normal 66.66 (16) 41.66 (10)

2 Lymphoid cell collections in submucosa 4.16 (1) 4.16 (1)99 Not available 29.16 (7) 54.16 (13)

17 Sciatic nerve 1 Normal 100 (24) 100 (24)18 Abdominal aorta 1 Normal 100 (24) 100 (24)

Number of animals: 12 male and 12 female in each group.a Results are given as the % of animals, with the number of animals in parentheses.

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Table 6. Serum biochemical parameters in rats fed 10% sunower oil (Control) or eri silkworm oil (Experimental)


Parameter Period (weeks) Males Females Males Females

Creatinine (mg dL 1) 6 0. 82 0 . 161 0 . 79 0 . 158 0 . 73 0 . 178 0 . 73 0 . 19612 0 . 98 0 . 176 0 . 96 0 . 126 0 . 91 0 . 203 0 . 99 0 . 128

18 1 . 03 0 . 176 1 . 02 0 . 181 0 . 96 0 . 181 1 . 11 0 . 170Urea (mg dL 1) 6 30. 7 6 . 25 31 . 5 3 . 82 27 . 6 4 . 56 29 . 7 5 . 43

12 30 . 1 2 . 92 38 . 7 3. 53 31 . 9 5 . 22 38 . 1 5 . 8518 28 . 1 4 . 14 30 . 6 7. 06 26 . 0 4 . 04 25 . 2 4 . 64

Alkaline phosphatase (U L 1 ) 6 78. 7 10 . 45 63 . 2 12 . 59 86 . 9 10 . 80 71 . 6 19 . 6112 82 . 0 13 . 34 63 . 7 16 . 38 81 . 6 6 . 73 77 . 4 10 . 6418 91 . 1 17 . 95 81 . 3 11 . 72 91 . 4 14 . 99 87 . 7 6 . 04

Triglyceride (mg dL 1) 6 78. 5 14 . 62 65 . 7 4 . 59 78 . 6 14 . 06 71 . 3 7 . 7012 80 . 8 11 . 20 66 . 7 6. 15 63 . 3 6 . 92 55 . 8 7 . 4818 86 . 2 10 . 34a 69 . 6 5 . 33a 63 . 2 4 . 37b 53 . 7 6 . 42b

Total cholesterol (mg dL 1) 6 127 11 . 15 103 19 . 22 114 19 . 52 104 15 . 7512 136 13 . 63a 105 16 . 04 109 6 . 45b 103 3 . 8018 169 12 . 78a 128 15 . 13a 107 7 . 38b 99 3 . 60b

HDL cholesterol (mg dL 1) 6 18. 1 3 . 91 15 . 6 2 . 35 16 . 9 2 . 93 15 . 7 2 . 35

12 12 . 6 6 . 07a 12 . 1 7 . 55a 17 . 1 1 . 99b 17 . 7 1 . 50b18 10 . 3 2 . 09a 11 . 0 2 . 07a 22 . 7 3 . 01b 20 . 2 3 . 10b

Values are mean SD (n = 12 males and 12 females in each group).a,b Different superscripts are signicant at P < 0 . 05.

increased risk of heart disease and stroke. Therefore, the reducedserum triglyceride levels in eri silkworm pupae oil fed rats isof signicance even though it was achieved after prolongedfeeding.

Serum cholesterol level in both the male and female animalsfed eri silkworm pupae oil was signicantly ( P < 0. 05) lower at18 weeksas compared to the malesand females fed sunower oil. The liver cholesterol levels were comparable between the controlfed sunower oil (230 7 . 8 mg 100 g 1) and the experimentalanimals fed the eri silkworm pupae oil (215 8 . 2 mg 100 g 1). This shows that the decrease in serum cholesterol level does notleadto hepaticcholesterol accumulation. Thehigh -linolenicacidoferilsilkwormpupaeoilmightinhibitcholesterolsynthesisormaystimulate cholesterolcatabolism to bileacids and to neutral sterolsexcreted in faeces or may stimulate the excretion of cholesteroland its metabolites as dermal lipids. 26 Similar lowering of serumcholesterol level due to feeding of oil rich in -linolenic acid hasalso been observed by feeding perilla oil 13 or -linolenic acid richdiet. 27

High-density lipoprotein cholesterol increased signicantly

(P

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Eri Silkworm a Source of Edible Oil With a High-libre