459

Journal ofFood Protection, Vol. 44, No.6, Pages 459-470 (June 1981) Copyright©, International Association of Milk, Food, and Environmental Sanitarians Detoxification of Rapeseed Products

P. N. MAHESHWARil, D. W. STANLEY! andJ. I. GRAY2•

Department ofFood Science, University of Guelph, Guelph, OntarioNI G2Wl, Canada, and Department ofFood Science and Human Nutrition, Michigan State University, East Lansing, Michigan 48824

(Received for publication June 19, 1980)

ABSTRACT oil as it relates to the fatty acid composition that becomes The full potential of rapeseed products has not yet been the primary concern as it determines the value and utility Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/6/459/1654352/0362-028x-44_6_459.pdf by guest on 02 October 2021 realized because of the presence of certain toxic compounds. of the vegetable oil. There are wide variations in the fatty This paper reviews development of low erucic acid rapeseed, acid composition of the world's major edible oils and the extensive experimental scrutiny to which this oil has (fable 1). Rapeseed and mustard oils produced from been subjected. The significance of the presence of gluocosino­ conventional varieties differ from most other vegetable lates as well as their decomposition products (isothiocyanates oils by containing significant amounts of long-chain and oxazolidinethiones) in rapeseed meal is also discussed. monoenoic fatty acids with 20 and 22 carbon atoms. Various methods for removing these toxic constitutents from Indeed, the rapeseed and mustard oils produced in the the meal are reviewed. Far East still contain as much as SO% erucic acid and 8-lOo/o eicosenoic acid in their total fatty acid composition. Because of this unique fatty acid composi­ Rapeseed is an important oilseed crop and ranks fifth tion, rapeseed oil has been used in several industrial in the world production of oilseeds. India is the world's applications as well as in the manufacture of margarines, largest single producer of the crop (55), while Canada is shortenings and salad and cooking oils (42). the biggest exporter and supplier of the crop to the world The unique composition of rapeseed oil intrigued market (18). This crop has been cultivated for at least nutritionists to study its dietetic properties. Consequent­ 3500-4000 years in India, and today it is the second major ly, some Canadian nutritional investigations carried out oilseed crop (preceded only by peanuts), supplying about in the early 1950's by Carroll and his colleagues (34-36) 20% of the total edible oil supply in the country. In suggested that oils high in erucic acid, if fed to laboratory Canada, rapeseed was first grown commercially in 1942 animals as a large proportion of the diet, can result in as a wartime measure to supply oil for lubrication of retarded growth and poor animal performance. It was marine engines. Extraction of oil for edible purposes first the result of these early studies that led to a Canadian occurred in 1956. Since then production has expanded plant breeding program to either reduce or alter the rapidly so that rapeseed now represents a very important erucic acid levels in the rapeseed oil. crop for Canadian farmers, and accounts for more than Genetic investigations ascertained that the fatty acid SOo/oofthe oilseed crops grown. composition of the seed oil was determined by the Unfortunately, the full potential of rapeseed products genotype make-up of the developing embryo and not by has not been realized because of the presence of toxic the maternal plant (46), and that the erucic acid content compounds. This paper reviews progress in development ofthe seed oil was controlled by genes (39,64). By 1963, of erucic acid-free rapeseed oil as well as the problems this breeding program in Canada had produced strains associated with rapeseed meal. The various techniques in summer rape and summer turnip rape which involved in detoxification of this meal will also be contained less than 1 o/o erucic acid in their oil (40,129). reviewed. Commercial testing and industrial evaluation of the pro­ cessing quality and storage stability of the oil from these COMMERCIAL PROCESSING OF RAPESEED new strains of rapeseed was completed by 1966, and was Rapeseed oil found to be highly satisfactory. However, exploitation of The commercial processing of rapeseed in modern oil the low erucic rapeseed in Canada was very much limited mills yields approximately 40% oil and SO% protein-rich due to an unfavorable pricing situation for its oil on the meal. Oil quantity and quality are usually of the greatest world ma.rket. In the late 1960s and early 1970s, several interest to the oilseed processor since oil prices are reports of nutritional investigations from Canadian and normally several times higher than that of the by-product European laboratories were published which showed that meal. With the fixed quantity of oil, it is the quality of the the high erucic acid rapeseed oil was poorly used by young laboratory animals. When used as a major source 1 University of Guelph. of energy in the diet. it resulted in reduced growth and 2 Michir;an State University. undesirable physiological and histopathological changes

JOURNAL UFFOO!J PROJECTION. VOL.44,JUNE 19H1 460 MAHESHWARI, STANLEY AND GRAY

in the heart (cardiac lesions) and the skeletal muscles in biosynthesis of eicosenoic and erucic acids was confirmed the rat (2), ducks (1,3,133,147), guinea pigs (147) and by Downey and Craig (44). While low erucic acid (less monkeys (62). The nutritional properties of rapeseed oil than 1 o/o) rapeseed strains are being cultivated in Canada have been thoroughly reviewed by VIes (146) and Slinger and some European countries, goals have been set up for (122). These reports clearly established that a high level future quality improvement by breeding and include of erucic acid in the oil is undesirable from a nutritional advances such as palmitic acid up to 10o/o, linoleic acid point of view and abruptly changed the status of low above 40o/o and linolenic acid below 3o/o. Altering the erucic rapeseed in Canada and subsequently in Europe. levels of linoleic and linolenic acids appears feasible The changeover to low erucic acid varieties was (107), but it will probably require a few years to initiated in Canada in 1971 (42) and was soon followed by incorporate this characteristic into commercial varieties Sweden (10), Germany (50,68), France (89), Poland and (122). other European countries. The Canadian conversion was Low erucic acid rapeseed oil has also been subjected to essentially completed in 1974. The European conversion extensive experimental scrutiny. Rocquelin and Cluzan has been hampered by sharply increasing erucic acid (1 08) found that rapeseed oil from low erucic acid levels during seed and commercial multiplications varieties with 1-2 o/o levels of erucic acid in the oil also Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/6/459/1654352/0362-028x-44_6_459.pdf by guest on 02 October 2021 (perhaps as a result of volunteer high erucic plants resulted in cardiac lesions in rats when included in the originating from seed of old varieties lying dormant in the diet. These findings have been subsequently confirmed fields) but is expected to be completed shortly. by several groups of workers (4,23, 78, 79,109,111). It has Conversion to low erucic acid rapeseed and mustard also been shown that early lipidosis in the heart is strains in Asia has not yet been initiated. directly related to the concentration of erucic acid in Genetic isolation of rapeseed plants with less than 1 o/o dietary oils (5,21,22, 79,86), and the erucic acid erucic acid in their seed gave simultaneous selection for accumulation in cardiac lipids is proportional to the low eicosenoic acid and a corresponding increase in oleic concentration of this acid in the diet (79). Since the acid (fable 1). These observations suggested that oleic incidence of myocardial lesions on feeding rapeseed oil to acid was the precursor of eicosenoic and erucic acids in weanling male rats for about 16 weeks was observed their biosynthesis and that addition of an acetate group irrespective of the level of erucic acid, it has been to the carboxylic end of oleic acid would result in suggested that additional factors such as unsaponifiable eicosenoic acid. Addition of a second acetate group components or a fatty acid imbalance may be involved would give rise to erucic acid. This pathway for (78, 79,109, 111). However, Abdellatif and VIes (5)

TABLE 1. Percent fatty acid composition of rapeseed oils and some selected vegetable oils. a

Rapeseed oil Indian mustard b Low erucic before 1969 Commer- B. camp· Sun- Saf- Corn Fatty acid Canada Europe B.juncea cial oil B. napus estosis Soybean Peanut flower flower USA 1. Palmitic 3.5 3.5 2.1 1.7- 2.1 3.8 3.2 15.0 9.2 5.9 7.2 11.5 (16:0) 2. Stearic 1.4 1.2 1.2 1.1- 1.2 1.5 1.5 4.0 3.1 5.2 1.9 2.2 (18:0) 3. Arachidic 1.4 (20:0) 4. Behenic 2.6 (22:0) 5. Lignoceric 1.8 (24:0) 6. Oleic 30.5 12.0 11.2 11.2-16.2 62.6 60.2 24.0 57.2 15.9 12.4 26.6 (18: 1) 7. Linoleic 21.5 15.0 15.9 11.2-16.3 19.7 21.3 49.0 23.4 72.5 78.5 58.7 (18:2) 8. Linolenic 6.6 8.7 10.0 4.9-10.0 8.9 10.9 8.0 0.8 (18:3) 9. Eicosenoic 10.0 8.0 6.8 6.5- 8.2 1.4 1.3 1.4 (20: 1) 10. Erucic 25.5 49.5 48.5 48.5-52.7 0.4 0.5 (22:1) 11. Tetracosenoic 1.1 (24: 1) a From Downey (42). bFrom Srivastava eta!. (126). t =Trace amounts.

JOURNAL OF FOOD PROTECTION. VOL. 44, JUNE 1981 DETOXIFICATION OF RAPESEED PRODUCTS 461

ascribed the cardiopathogenicity of rapeseed oil solely to characterized not only by low content of erucic acid but the erucic acid. To establish if the long-term lesions also by incidental elimination, through breeding, of observed in male rats were caused by the triglycerides or Factor X. by some non-triglyceride components of rapeseed oil, In a study on the intake and digestibility of high and Kramer and co-workers (77) fractionated Brassica low erucic acid rapeseed oils in a mixed Canadian diet campestris var. Span (a low erucic rapeseed oil) by (142), it was shown that foods baked and fried with molecular distillation and adsorption chromatography rapeseed oil as a sole source of added fat were accepted and carried out cardiotoxicity studies with various equally well and that there was no difference in the fractions by long-term feeding trials. From these studies, intake of fat by the subjects, digestibility or absorption of Kramer et al. (78) concluded that the cardiopathogenic ingested fat and erucic acid. Metabolic studies on rats by properties appear to be associated with the triglycerides Kienle et al. (73) and Hsu and Kummerow (67) suggest of the oil and not with the non-triglyceride minor that erucic acid is oxidized at lower rates than other components such as brassicasterol, present in the fully long-chain unsaturated fatty acids. It appears that oleic

refined oil. Beare-Roger et al. (23) reported that the acid is the major form of use of erucic acid after its Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/6/459/1654352/0362-028x-44_6_459.pdf by guest on 02 October 2021 refining of rapeseed oil did not appear to change its conversion, which is effected greatly and at a faster rate nutritional properties, but partial hydrogenation of high in the liver than in the heart (1 03). or low erucic acid rapeseed oils reduced the incidence of Thus there is no direct evidence that the presence of a cardiac lesions. Hydrogenation resulted in a marked large amount of erucic acid in the diet constitutes a reduction in the linolenic acid content of the oils, human health hazard, but on the basis of animal whereas the erucic acid content was only slightly experiments, it was considered prudent by various affected. They postulated the presence of a pathogenic countries to limit the erucic acid intake of their citizens factor, other than erucic acid, in rapeseed oils which is through issuing guidelines to the industry. Such removed or destroyed by hydrogenation. Rocquelin eta!. guidelines recommended that the erucic acid content (110) have also reported that hydrogenated rapeseed oil should not exceed a certain percentage of fatty acids does not cause as much accumulation of myocardial present in a fatty food. In Canada, the maximum lipids as non-hydrogenated rapeseed oil. Similar results suggested level is 5% and this is now a gazetted have also been obtained by Slinger (122) and Beare­ regulation (42,122). Rogers and Nera (20). Rapeseed meal McCutcheon et al. (87), on the basis of an experiment Rapeseed meal after oil extraction is an excellent involving 10 different oils or oil blends, including source of protein and contains well above 40% protein rapeseed oils, simulated rapeseed-type oils and modified (fable 2). Rapeseed proteins have a very well-balanced rapeseed-type oils, have postulated that linolenic acid essential amino acid composition which compares plays a role in the etiology of cardiac necrosis observed favorably with soy proteins and the FAO pattern of when rats are fed diets containing low erucic acid essential amino acid requirements for human adults. rapeseed oils. Since hydrogenation markedly reduces the They are especially rich in lysine and methionine, which levels of linoleic and linolenic acids in rapeseed oil and is are limiting amino essential amino acids in most cereals accompanied by a marked reduction in severity of heart and other vegetable proteins (50,51). The potential value lesions, the hypothesis of McCutcheon et a!. (87) has a of rapeseed proteins as one of the best oilseed proteins sound analytical basis. Observations of Slinger (122) lend for supplementation of the human diet is well support to this hypothesis. documented (33,50.51,58, 71,81,82, 123). In addition, VIes (146) and Biedermann et a!. (31) have reviewed rapeseed protein products having satisfactory functional the nutritional properties of rapeseed oil and have properties are acceptable for use in various food discussed the toxic effects of erucic acid and a formulations for human consumption (98). pathogenic agent designated as Factor X, present in low The prevailing malnutrition in heavily populated erucic acid rapeseed oils but not yet isolated and nations, and the growing shortage of protein throughout identified. VIes (146) stated that low erucic acid rapeseed the world due to rapidly increasing populations have oils in the nutrition of man and animals are as created an obvious and critical need to fmd and use more satisfactory as other vegetable oils and concluded that sources of protein. Consequently, there has been the cardiac lesions induced in male weanling rats fed considerable interest in using rapeseed meal as a source rapeseed oils were due to erucic acid and not to other factors. He also did not find any statistically significant TABLE 2. Protein and oil content ofBrassica oilseeds (88). differences between the cardiotoxicity of rats fed Percent (dry basis) Brassica napus var. Tower (a Canadian double low Oil Protein (N x 6.25) variety of rapeseed) oil containing 1.4o/o erucic acid and ______W_hole seed Defatted meal_ sunflower oil, while there was a much higher incidence Brassica oilseeds with conventional rapeseed oil containing 45% erucic B. napus 44.7 23.8 43.1 acid. Biedermann et al. (31) on the other hand, point out B. campestn's 42.7 23.8 41.5 that some recently developed rape cultivars are B. 37.2 27.5 43.8

JOURNAL OF FOOD PROTECTION, VOL.44.JUNE 1981 462 MAHESHWARI, STANLEY AND GRAY of food protein, not only because of its high content and quality of protein, but also for reasons of its low cost and substantial availability. However, use of rapeseed meal, GLUCOSINOLATE UNSTABLE GLUCOSE BISULFATE as a protein source in livestock rations and human diets, ORGANIC is severely limited due to the presence of high levels of AGLUCONE sulfur-containing glucosinolates in the seed and the defatted meal (9,143). There are certain other undesir­ able constituents present in the seed such as phytates (70), polyphenols (75), odoriferous and bitter principles R- s-c;; /!~N R-C;:N + S (9.65, 112), indigestible carbohydrates (mainly cellulose) in appreciable amounts (up to 3 %) in dehulled seed (96) THIOCYANATE ISOTHIOCYANATE NITRILE SULFUR and unidentified antinutritional factors in recently Figure 1. Enzymatic hydrolysis ofglucosinolates (143). developed low glucosinolate varieties (72,125). Bronowski in Poland and Erglu in Germany, are Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/6/459/1654352/0362-028x-44_6_459.pdf by guest on 02 October 2021 GLUCOSINOLATES IN RAPESEED relatively low in their glucosinolate content (Table 4). Members ofthe genus Brassica are goitrogenic. Goiter However, rapeseed cultivars virtually free from glucosin­ -enlargement and improper functioning of the thyroid olates are not yet available. gland due to deficiency of iodine - is consistently The intact glucosinolates are apparently free from produced if Brassica seeds are fed to experimental toxicity, but on hydrolysis by an endogenous enzyme, animals (143). The glucosinolates have been identified as myrosinase (thioglucoside glucohydrolase (E.C.3.2.3.1), the source of goitrogens in rapeseed and are a deterrent present in the seed and unheated meal, these compounds to greater use of this oilseed crop. The basic structure for yield undesirable and potentially toxic products such as glucosinolates, which was revised in 1956 and firmly isothiocyanates, thiocyanate, nitrites and oxazolidine­ established in 1957 by Ettlinger and Lundeen (54), is thiones (24,119.122.124,143,151). These hydrolytic pro­ shown in Fig. 1. ducts of glucosinolate hydrolysis, when ingested by The rapeseed species are not identical with respect to non-ruminants, produce various manifestations of their glucosinolate contents. Brassica juncea (brown or toxicity ranging from depressed growth and loss in oriental mustard) just like other mustard seeds (e.g. weight, loss of reproductive potential, enlarged thyroids Brassica hirta or white/yellow mustard and Brassica and kidneys to death in rats and other species of nigra or black mustard) is characterized by the presence experimental animals (12,43.45,125.126). Cattle and of only one glucosinolate (sinigrin) in contrast to the other ruminants are not so adversely affected by the complex situation in B. campestris and B. napus which goitrogenic factors of the rapeseed meal as the toxic contain several glucosinolates. The major glucosinolates oxazolidinethiones are partly destroyed in the rumen of in the latter species are gluconapin and progoitrin, and the animal (113). However, Rutkowski (113) maintains to some extent glucobrassicanapin. These glucosinolates that the rapeseed meal is less palatable and less readily differ from one another in terms of their organic radical, digested than many of the other high-protein oilseed R, as indicated in Table 3. The total glucosinolate meals, and that its taste and actual feeding needs are the content of dry defatted seed meals of some common sole factors limiting the dosage of rapeseed meal in the cultivars of rapeseed from Canada, Europe, and the feeding of ruminants. Consequently, a small portion of Indian subcontinent are high in their glucosinolate the rapeseed meal produced is used as animal feed after content, whereas newly bred Canadian and European it is diluted with other meals, while the major portion is cultivars, such as Tower and Candle in Canada, used as a fertilizer. No rapeseed preparation is currently

TABLE 3. Glucosinolates in rapeseed (144).

Oilseed Glucosinolate Organic radical 'R'

1. B. napus (i) Gluconapin 3-butenyl- CH CH CH 2 -CH2 - (ii) Progoitrin (R}-2 hydroxy-3-butenyl- CH2 = CH- CHOH CH2 (iii) Glucobrassicanapin 4-pentenyl- CH2 =CH (CH2)l- (iv) Giuconasturtiin 2-phenylethyl- C6H 5 CH2 -CH2- (v) Glucoiberin 3-methyl sulfmyl propyl CH 3 -SO - (CH2)l

(vi) Sinalbin p-hydroxybenzyl- p-OH-C6H,.- CHz-

2. B. campestris (i) Gluconapin 3-butenyl- CH2 =CH CH2 -CHz- (ii) Progoitrin (R) -2 hydroxy-3-butenyl CH 2 = CH- CHOH CH2 (iii) Glucobrassicanapin 4-pentenyl- CH2 = CH - (CH 2)3 \!v) Glucoalyssin 4-methylsulfinyl-butyl- CH3 so (CH2)4- (v) Glucoraphanin 5-methylsulfinyl-pentyl- CH3 SO- (CH2)5

3. B.juncea (i) Sinigrin Allyl- CH2 = CH-CH2

JOURNAL OF FOOD PROTECTION. VOL. 44, JUNE 1981 DETOXIFICATION OF RAPESEED PRODUCTS 463

TABLE 4. Glucosinolate contents of dry defatted seed meals _,s-c6H110 5 ofsome selected cultivars CH2 : CH-CHOH-CH2-c '-N-O-S020-K+ PROGOITRIN Total glucosino­ THIOGLUCOSIDE H~ GLUCOHYDROLASE late content E.C. 3.2.3.1 Rapeseed species and cultivars (mg/g) I B. napus CeH1~6 + kH2zcH-CHOH-CH2-C,:Ns-_ J + KHS04 GLUCOSE L ,, aBronowski, (97) 0.94 BISULFATE bErglu, (90) 0.85 UNSTABLE AGLUCONE bLesira, (90) 16.70 CTarget, (126) 9.50 CTower, (126) 1.29 B. campestris /J ~ ccandle, (43) 1.10 THIOCYANATE ISOTHIOCYANATE NITRILE ddichotoma (Pusa kalyan), (126) 9.70 + Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/6/459/1654352/0362-028x-44_6_459.pdf by guest on 02 October 2021 lCYCLIZATION s dT-g, Toria, (126) 17.80 SULFUR dT-42, Yellow Sarson, (126) 6.06 B.juncea dT-63, Brown mustard, (126) 15.10 a Polish cultivar. bGerman cultivar. ccanadian cultivar. GOITRIN (S- 5- VINYLOXAZOLIDINE-2-THIONE) dlndian cultivar. Figure 2. The enzymatic hydrolysis of progoitrin to goitrin (115,143). used in food for human consumption. The glucosinolates in rapeseed and other crucifers are rapeseed proteins and meal. associated with the enzyme, myrosinase, which hydro­ lyzes them to glucose, bisulfate and an unstable organic Myrosinase enzyme system in rapeseed aglucone when wet, unheated material is crushed. The The myrosinase enzyme system has been widely organic aglucone portion of the molecule may undergo studied because of the multiplicity of the breakdown an intramolecular rearrangement to form an isothio­ products of glucosinolates (52.53,57.59.60,66,83,85,93, cyanate. Without such a rearrangement, the aglucone 99-102,105,116-118,134-138,1 45,149}. From these inves­ may form a nitrile and sulfur or a thiocyanate (Fig. 1). tigations, it has been demonstrated that myrosinase is a Thiocyanates, isothiocyanates and nitrites block the group of isoenzymes or isozymes and exists in multiple capture of iodine by the thyroid gland and set free the molecular forms. The physical and chemical properties iodine accumulated in it (113), thus acting as goitrogens of each molecular form of the enzyme are distinctly only when the iodine content in the diet is low (1 15). different from one another (99). The enzyme is a However, the potent antithyroid compound is goitrin glycoprotein with a molecular weight of about 135,000 (5-vinyloxazolidine-2-thione) which arises from the and consists of two polypeptide chains. cleavage of its precursor, progoitrin, and is formed There is a multiplicity of pathways for the enzymatic through cyclization of the postulated unstable isothio­ breakdown of glucosinolates (Fig. 1 and 2), the various cyanate containing a hydroxyl group (Fig. 2). The products identified being formed under different but thyroid-inhibiting effect of goitrins or oxazolidinethiones appropriate conditions. The enzyme is brought into close is due to the inhibition of organic binding of iodine, and contact with the glucosinolates when the rapeseed is this type of goiter is not alleviated by increasing iodine in crushed during the extraction of the oil. Under favorable the thyroids (1 15). conditions of temperature (40-70 C) and moisture Van Etten (143) has stated that most of the content (13o/o and above), enzymatic hydrolysis of glucosinolates have been characterized in part through glucosinolates occurs before the oil is extracted. Such a the isothiocyanate formed by hydrolysis because its hydrolysis has been reported to be extremely fast (92), formation appears to be favored over the nitrile and 90o/o hydrolysis being obtained in 1 min at 15.5o/o thiocyanate. The allyl isothiocyanate from sinigrin is moisture and 55 C. If the hydrolysis of glucosinolates easily recognized because of its pungency, a characteris­ occurs during crushing before the oil is extracted, the tic of mustard oils which has long been recognized and sulfur-containing hydrolytic products - isothiocyanates sought after in condiments to impart a desirable culinary and oxazolidinethiones - enter the oil (38,95). Daun and t1avor to the preparation. This is the reason why B. Hougen (37) reported that industrially processed crude Juncea species of rapeseed is used more as a condiment rapeseed oils contained 10-57 ppm of sulfur which could than as an oilseed. However, the products derived from never be completely removed by conventional refining glucosinolate hydrolysis are potentially toxic and are also procedures of degumming, alkali refming and bleaching. responsible for undesirable t1avors associated with Consequently, a substantial amount (3-5 ppm) of sulfur

JOURNAL OF FOOD PROTECTION. VOL. 44, JUNE 1981 464 MAHESHWARI. STANLEYANDGRAY is always present in refined rapeseed oils. This sulfur is Recently, Mukherjee et al. (90) have proposed a objectionable in refined oil as it has been implicated as a method of autolysis of ground rapeseed by the action of catalyst poison, causing difficulties during the hydrogen­ heat (45 C), moisture (15-40o/o) and myrosinase (4-12 h) ation of the oil (14,31) and also as a cause of certain for the decomposition of glucosinolates, followed by unpleasant odors in heated rapeseed oils. It is, therefore, defatting with hexane, for the production of low important to prevent sulfur contamination of the oil glucosinolate rapeseed meals. However, this method has during extraction, and this can be achieved by effectively three serious disadvantages, namely, the organic aglu­ inactivating the enzyme myrosinase by proper heat cones formed from the glucosinolates are extracted in the treatment and keeping the glucosinolates intact. oil, the free fatty acid content of the oil is increased due However, if the cooking temperatures employed for the to the action of lipase present in the seed, and the purpose exceed 110 C, chemical breakdown of glucosino­ defatted meal is not completely free from oxazolidinethi­ lates may occur, with possible formation of oil-soluble ones. Vaccarino and co-workers have reported detoxifi­ sulfur-containing products (95). cation procedures for ground as well as whole rapeseeds, involving precooking to hydrolyze the glucosinolates, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/6/459/1654352/0362-028x-44_6_459.pdf by guest on 02 October 2021 followed by acetone extraction of the ground seed (I 39) DETOXIFICATION OF RAPESEED and boiling water and salt solution extractions of the Much research has been directed towards detoxific­ whole seed (140,141) for production of the detoxified ation of rapeseed and its meal. The detoxification rapeseed flour. procedures investigated and reported in the literature protein quality or heavy contamination of the oil with can be grouped into the following five categories: (a) sulfur-containing hydrolytic products of glucosinolates potentiation ofthe enzyme and removal of the hydrolytic or appreciable contents of oxazolidinethiones in the products; (b) inactivation of the enzyme myrosinase; (c) residual meal, none of these methods have gained removal of glucosinolates and their hydrolytic products commercial acceptance. from rapeseed and its meal; (d) destruction of Inactivation ofmyrosinase glucosinolates and their hydrolytic products in rapeseed Inactivation of myrosinase has offered a more feasible and its meal and (e) breeding out glucosinolates from solution to the problems in processing and utilization of rapeseed. rapeseed. The intact glucosinolates are neither extracted Potentiation of the enzyme and removal of hydrolytic during oil extraction nor cause toxicity on ingestion, products provided they remain unhydrolyzed in the digestive Goering (61) developed a process in which ground system. rapeseed was moistened with cold water to potentiate Myrosinase starts inactivating at temperatures above the enzyme or the rapeseed meal was slurried with 6 to 8 70 C, if the moisture content in the rapeseed is 6 to 10%. parts of water. Myrosinase was added, if necessary, and Reynolds and Youngs (1 06) demonstrated that cooking the digestion carried out at 45-55 C. The liberated the crushed rapeseed without addition of water at volatile oils of mustard, isothiocyanates, were removed by temperatures greater than 80 C destroyed the myrosinase steam stripping. However, the oxazolidethiones and activity and produced oil of a satisfactory quality if the other non-volatile products of the enzymatic hydrolysis temperature in the cooking did not exceed 105 C. remained in the meal. There was also a risk of impairing Appelquist and Josefsson (11) found in laboratory studies the quality of ground rapeseeds. In a similar process (91) that the myrosinase of seed with 8% moisture content developed to produce oil, meal and allyl isothiocyanate was completely inactivated by heating at 90 C for 15 min. from B.juncea, the residual meal contained only 0.004% Today in Canada and Europe, the modern procedures of allyl isothiocyanate, but its nitrogen solubility index was extracting rapeseed oil include a heat treatment which lowered to 3.4. causes inactivation of the glucosinolate-splitting enzymes A process for preparation of bland protein meal from (71). It is, however, obvious that in a commercial dehulled defatted mustard (Indian rapeseed) was operation, some hydrolytic enzyme reaction still may developed at the Central Food Technological Research occur during the interval required to raise the Institute, Mysore, India and presented at the FAO/ temperature from ambient to deactivation level, even WHO/UNICEF Protein Advisory Group Meeting held in though this interval is kept as short as possible. One Geneva, 1969. In this process, the dehulled defatted seed might also speculate that another way to avoid the in the form of flour was mixed with water (1:3 w/v) and unwanted reaction would be to remove all possible the pH was adjusted to 8.2. A paste of ground, cold moisture from the seed before crushing. Unfortunately, defatted mustard seed was added as enzyme source and this is only a partial solution since a certain level of the glucosinolate hydrolysis was carried out at room moisture is as necessary as elevated temperatures for temperature for 4-5 h. The allyl isothiocyanate was inactivation of the enzyme. With very low moisture, the removed by steam distillation. The aqueous slurry was hydrolysis is certainly minimized; however, myrosinase cooled and its pH was adjusted to 5.0. The solid matter survives to cause problems at some later stage, which was collected by centrifugation, was washed with particularly in meal when adequate moisture levels for water to remove a bitter compound and dried. hydrolysis may occur again.

JOURNAL OF FOOD PROTECTION. VOL. 44. JUNE 1981 DETOXIFICATION OF RAPESEED PRODUCTS 465

Residual myrosinase activity in rapeseed meals has Therefore, removal of glucosinolates, as well as their been destroyed by treating the meal with hot water at hydrolytic products, from rapeseed and its meal has been 90 C (28) and steam autoclaving for 15 min at 1.2 kg/cm2 attempted by several researchers in Canada and Europe. pressure (25) or at 248 C (28). The method of steam Sosulski et al. (124) reported that five to six 1-h autoclaving (25) does not have any apparent effect on the diffusion extractions of the intact seeds, after inacti­ glucosinolate content of the meal while that of Belzile et vating the seed myrosinase by boiling in water for 3 min, al. (28) produced dried meals devoid of glucosinolates with 0.01 N NaOH at 60 C were sufficient to remove most and their hydrolytic products. of the seed glucosinolates. Bhatty and Sosulski (30) used Eapen et al. (47) reported extensive studies on ethanolic NaOH to effectively extract rapeseed glucosi­ inactivation of myrosinase using dry as well as wet heat nolates as well as inhibiting the myrosinase activity. A treatments, and simultaneously studied the effect of more detailed study on aqueous and ethanolic extrac­ these treatments on the quality of the oil produced. Dry tions of glucosinolates from intact and crushed rapeseed heat treatments of cracked seeds of B. napus var. Tanka and rapeseed meal was reported by Kozlowska et a!. (76). in a jacketed pan maintained at approximately 104.5 C The various extraction procedures employed included: and with continuous stirring for 30 min (achieving a final (a) aqueous extraction of rapeseed meal; (b) enzyme Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/6/459/1654352/0362-028x-44_6_459.pdf by guest on 02 October 2021 seed temperature of approximately 83 C) and in a hot air inactivation of the seed, followed by wet grinding and oven maintained at 104.5 C, with seeds kept in a thin aqueous extraction; (c) diffusion extraction of intact layer (achieving a final seed temperature of 88.3 C for seeds in O.Ql N NaOH; (d) diffusion extraction of 30 min) were found unsatisfactory from the dual enzyme-inactivated seeds with 0.01 N NaOH and (e) standpoint of myrosinase inactivation and oil color. diffusion extraction of whole seeds against 50% (v /v) Steam blanching treatments of seeds for periods varying ethanolic NaOH adjusted to pH 12. All treatments were from 5 to 30 min were all effective in completely effective in detoxifying low and intermediate glucosino­ inactivating the enzyme but resulted in darkening the oil. late samples, but two processes in which boiling water Immersion of seeds in boiling water was found to be treatment was given for 3 min to inactivate the enzyme effective in inactivating the enzyme (47). One minute and failed to fully detoxify the high glucosinolate samples. 1.5 min treatments for B. campestris and B. napus seeds, The other effects of these extraction processes are respectively (depending upon the size of the seeds), were summarized in Table 5. required to completely destroy the enzyme. This treatment also produced a light colored oil. However, if Using the approach of enzyme inactivation by treatment time exceeded the time necessary to inactivate boiling water treatment and aqueous extraction of the enzyme, the oil color darkened. glucosinolates from the slurry obtained by grinding of Eapen et al. (47) demonstrated that microwave-heating these seeds followed by air classification of the dried of whole seeds at their original moisture content of 5.2 defatted rapeseed meal, Eapen et al. (48) worked out a and 6.0o/ofor 3 min inactivated the enzyme but produced process for preparing the rapeseed flour for human oil with a darker color and meal with a burnt aroma. The consumption. This process was further improved (131) to effectiveness of a short microwave treatment in reduce losses of soluble solids during the aqueous inactivating myrosinase has also been reported in studies extraction process. However, the losses of solids could by Armstrong (13) who suggested that the material not be reduced below 17% and the solids lost contained should be preconditioned to a certain moisture level approximately 20% protein, 23% fat, 2% fiber, 8% before microwave treatment for the effective transmis­ mineral matter and 47% nitrogen-free extract. Subse­ sion of microwave energy and the rapid inactivation of quent modifications employed finer screens and counter­ the enzyme. current equipment for extraction (49). A similar principle Most of these enzyme inactivation studies have has been observed in the Tropical Products Institute, invariably neglected to investigate the deleterious effects London, England (TPI) process for aqueous fractiona­ of these procedures on the nutritional and functional tion of rapeseed (84), in which coarsely ground seeds are properties of the meal proteins and the correlation suspended in water and stirred, then left in a vat to between the extent of reduction in enzyme activity and separate into two fractions. The lower fraction sedi­ the quality ofthe resulting rapeseed meal. menting at the bottom collects all the dark brown seedcoats while the aqueous and most of the upper Removal qf glucosinolates and their hydrolytic products fraction contains the major portion of the seed kernels. from rapeseed and its meal The kernel particles are recovered by acidification to pH Dry heat treatment at 80-90 C for 15-30 min in com­ 3.5 and filtering. This process removes 93-94% of mercial practice to inactivate myrosinase leaves the glucosinolates present initially in rapeseed. glucosinolates intact in the meal. If this meal is used for Ballester et a!. (16) carried out steaming, simple and feed and food purposes, the compounds may be double water extractions of presscake meal of rapeseed hydrolyzed to toxic isothiocyanates and oxazolidine­ (B. napus) and reported that the double water extraction thiones by the myrosinase enzyme reintroduced by other procedure gave better results for removal of toxic factors, dietary ingredients or common microflora of the but did not eliminate them completely. In 1973, these gastrointestinal tract of man or other animals (63,94). workers (17) reported that a 2·h continuous water

JOURNAL OF FOOD PROTECTION, VOL. 44,1UNE 1981 466 MAHESHWARI, STANLEY AND GRAY extraction procedure with stirring (Li:xiviation process) SOo/o of the isothiocyanates and oxazolidinethione completely removed the isothiocyanates and reduced contents of meal, but resulted in about one-third oxazolidinethiones by 97o/o. The detoxified meal obtained decrease in the amount of soluble proteins. He also by this process contains only 0.02o/o oxazolidinethiones maintained that the more drastic the heat treatment and gives a PER and growth rates similar to casein diets applied, the greater will be the destruction of goitrogens. in rats even at a 20o/o protein level in the diet (1 5). The However, the solubility of proteins will also be decreased weights of liver, thyroid, heart, spleen, adrenals and proportionately. kidneys were normal in rats fed this detoxified meal and Youngs and Perlin (150) reported the ferrous-catalysed no histopathological changes were detected in the liver. decomposition of sinigrin and related glucosinolates. However, the thyroid exhibited changes similar to, but of Sallans et al. (114), Bell et al. (26) and Kirk et al. (74) lesser severity than those in animals fed crude rapeseed have demonstrated the salts of iron, copper and nickel to meal. It has been, therefore, suggested by Ballester and be the most active decomposers of glucosinolates. his co-workers that there may be other toxic compounds Although the lysine content of the meal proteins has been present in the rapeseed meal that are not extracted by reported to remain unaffected (73), the decomposition Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/6/459/1654352/0362-028x-44_6_459.pdf by guest on 02 October 2021 lixiviation. products, chiefly nitriles and the toxic and nonvolatile Some investigators have employed cold or warm water 1-cyano-2-hydroxy-3-butene remained in the meal (114). (8.28.56) or buffers (27) to extract glucosinolates and Szewczuk et al. (130) attempted to destroy glucosino­ their hydrolytic products from crushed rapeseed or its lates in rapeseed meal at elevated temperatures with defatted meal. A process developed by Alfa Laval AB weak solutions of sulfuric acid, but apart from (Sweden) for manufacture of rapeseed protein concen­ removing the glucosinolates this method caused a trate uses countercurrent water extraction for removal of considerable destruction of lysine. Recently, Kroliczek et glucosinolates (69). Ultrafiltration techniques have also al. (80) treated whole rapeseed with sulfuric acid and been suggested by Bockelman et al. (32). reported that the treatment reduced the levels of Several workers have reported that glucosinolates from glucosinolates and toxic isothiocyanates and oxazolidine­ full fat ground rapeseed (6, 90) as well as its defatted meal thiones, but did not affect the basic composition of the (104.112) could be effectively extracted with aqueous treated rapeseed varieties. The treatment, however, mixtures of methanol, ethanol or acetone, without adversely affected the quality of the oil. Anderson et al. extracting much of the meal solids. While the loss of (7) reported that treating rapeseed flours with hydrogen meal solids to the extent of 5-10o/o can be tolerated, fat peroxide effectively reduced the levels of glucosinolates, loss of 5 o/o or more, as reported by Mukkerjee et al. (90) but the production of oxidized sulfur amino acids, for extraction of ground rapeseed using 70o/o aqueous especially methionine sulfone, reduced considerably the acetone, is quite substantial for the industry to tolerate. nutritive value of proteins. Microbial fermentation techniques have also been Destruction of glucosinolates and their hydrolytic employed to destroy the glucosinolates in rapeseed meal. products in rapeseed and its meal Staron (127.128) has reported biological destruction of Processes involving destruction of glucosinolates and glucosinolates through the action of the fungus their hydrolytic products in rapeseed and its meal by Geotrichum candidum on rapeseed presscake. The heat, chemicals and microorganisms have been reported. action of the fungus also results in more soluble rape Rutkowski (112) reported that temperatures of 120 C and proteins. above in industrial toasting trials destroyed more than Methods of destroying glucosinolates are not entirely

TABLE 5. Effects ofaqueous and ethanolic extractions ofglucosinolates from rapeseed (76). Nitrogen Losses in extract(% Total seed solids) Sulfur content solubility Yield (o/o) Total in oil (%of total Treatment solids Nitrol!en Oil (ppm) Oil Meal nitrogen) Control 0.9 40.8 54.7 65.6 Aqueous extraction 17.9 1.2 37.8 60.8 Enzyme inactivation, 25.0 1.2 6.7 5.8 33.9 36.8 64.2 aqueous extraction Diffusion extraction 10.9 0.3 0.1 12.0 40.7 46.4 55.8 in O.ot N NaOH Enzyme inactivation, 12.0 0.3 0.1 2.0 40.6 45.3 39.5 diffusion extraction in 0,01 NNaOH Diffusion extraction against 11.5 0.3 0.2 0.0 40.6 46.6 22.5 SOo/oethanolic NaOH

JOURNAL OF FOOD PROTECTION. VOL. 44. JUNE 1981 DETOXIFICATION OF RAPESEED PRODUCTS 467

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Downey, R. K.1965. Rapeseed botany, production and utilization and K. Darlington. 1975. Influence of rapeseed meal on the to the market. Chern. Ind. (London), May 1, p. 401. odor and tlavor of eggs from different breeds of chickens. Can. 43. Downey, R. K. 1978. Results of chemical analysis of Candle Inst. Food Sci. Techno!. J. 8:51-54. CZY3-1821 rapeseed and its products commercially grown and 66. Henderson, H. M., and T. J. McEwen. 1972. Effect of ascorbic acid processed in 1976. Personal communication. on the thioglucosidases from different crucifers. Phytochemistry 44. Downey, R. K., and B. M. Craig. 1964. Genetic control of fatty 11:3127-3133. acid biosynthesis in rapeseed (/Jrassica napus L.). J. Am. Oil 67. Hsu, C. M. L., and F. A. Kummerow. 1977. Influence of Chern. Soc. 41:475-478. elaidate and erucate on heart mitochondria. Upids 12:486-494. 45. Downey. R. K., B. M. Craig, and C. G. Youngs. 1%9. Breeding 68. Hudalle, B. 1977. Rapeseed research and utilization. J. Am. 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