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J. Agric. Food Chem. 1981, 29, 296-302
Detoxification of Jojoba Meal by Lactobacilli Anthony J. Verbiscar,* Thomas F. Banigan, Charles W. Weber, B. L. Reid, R. Spencer Swingle, John E. Trei, and Edward A. Nelson
Selected strains of Lactobacillus acidophilus and Lactobacillus bulgaricus were found to grow well on jojoba seed meal and reduce the levels of simmondsin and other cyano toxicants. After standing for 21 days at 26 "C on a 30% moist jojoba meal, L. acidophilus 629 lowered total toxicant levels 9598%. Ammonia used in the process facilitated the detoxification. The Lactobacilli apparently modify the cyano groups of the toxicants during their growth, thereby detoxifying the meal. This is the first time that Lactobacilli of any species or strain have been reported to act on cyano groups, indicating the possible presence of a nitrilase in this food grade microorganism. In addition to rendering jojoba meal nontoxic to mice, poultry, sheep and cattle, the Lactobacillus treatment increases palatability of deoiled jojoba meal, which is otherwise poorly accepted in animal rations. The treatment of jojoba meal with a Lactobacillus resembles an ensilage process.
Jojoba meal is the high protein material remaining after jojoba seeds containing some hulls are deoiled. Jojoba seeds contain simmondsin, a cyanomethylenecyclohexyl glucoside that is toxic to rats (Booth et al., 1974) and some animals. This principal toxicant is present at levels as high as 6% in deoiled jojoba meals. In addition, simmondsin 2'-ferulate and two other structurally related cyano glucosides are present in the meal at high levels (Elliger et al., 1974; Verbiscar and Banigan, 1978). We have investigated a number of solvent extraction, heat, and chemical methods to detoxify jojoba for use as a livestock feed ingredient (Verbiscar et al., 1980). Microbial detoxification methods and feeding studies of the treated meals in animal diets are reported here. There were several factors in our rationale for using microorganisms to detoxify jojoba meal. We reasoned that the glucoside toxicants could be a source of carbon and energy to support microorganism growth. That is, because of the glucose moiety of the toxicants, these could be utilized by the microorganism as a nutrient. This would result in an increased protein level in the treated meal. We did not want the microorganism to merely cleave glucose from the CN-containing aglycons, which could be inherently toxic or rearrange to toxic phenylacetonitriles (Elliger et al., 1973). Microbial modification of the CN groups of the toxicants was also necessary. The use of enzymes to detoxify food and feed plant materials have been reviewed recently (Liener, 1977), but no microbial methods were mentioned. Many plants including their edible parts contain cyano compounds that are toxic to animals and humans (Montgomery, 1965; Conn, 1973). Nitrilase enzymes that hydrate CN groups have been identified in a number of plants, (Thimann and Mahadevan, 1964), fungi, and bacteria (Jallageas et al., 1980). It was apparent that microorganisms already in the food chain would be most desirable for animal feeding, although not necessarily for CN group modification. Accordingly, we screened 15 microorganisms for their ability to grow in jojoba meal and modify the toxicants. These included one Saccharomyces cerevisiae, nine strains of LactobaAnver Bioscience Design, Inc., Sierra Madre, California 91024 (A.J.V. and T.F.B.), College of Agriculture, University of Arizona, Tucson, Arizona 95721 (C.W.W., B.L.R., and R.S.S.), and School of Agriculture, California Polytechnic University, Pomona, California 91708 (J.E.T. and E.A.N.). 0021-8561/81/1429-0296$01.25/0
cillus acidophilus, and five strains of Lactobacillus bulgaricus. The S. cereuisiae was Fleischmann's Active Dry Yeast obtained in a grocery store. L. bulgaricus Alta was an unflavored yogurt obtained from a local dairy. L. acidophilus Knudson was a sweet acidophilus milk obtained from another dairy. The other 12 Lactobacilli strains were provided through the courtesy of Dr. C. W. Hesseltine, Culture Collection, U.S.Department of Agriculture, Peoria, IL. Growth of these latter bacteria in soybean milk has been reported (Wang et al., 1974). Advantages in using the lactic acid bacteria are that these micoorganisms are notably nontoxic themselves and can be the basis for an ensilage process. The Lactobacilli can be adapted to use in a silo, and such a process can conceivably be used by small jojoba oil processors. However, there are no prior examples demonstrating nitrilase activity in Lactobacilli. Our purpose was to reduce toxicity of deoiled jojoba meal for use as a livestock feed. The detoxification process needed to be practical and provide a meal that was acceptable as a feed ingredient in poultry, sheep, or cattle diets. An improvement in palatability was also necessary because deoiled meal is somewhat bitter for humans and has low acceptability in animal diets. A successful process would conserve this half of the annual jojoba oilseed harvest, a crop being cultivated and developed in the southwestern United States, in Mexico, and in other gemiarid lands. EXPERIMENTAL SECTION
Solutions. Medium 2 is 10% solution of Carnation Instant Non-Fat Dry Milk in distilled water. Solution X is solution of 0.1 g of MgS04.7H20,0.2 g of K2HPO4.3Hz0, 0.4 g of Difco yeast extract, 1.2 g of Sheffield NZ amine, and 20 mL of distilled water. Inoculum Preparation. Inocula were prepared by suspending lyophilized pellets of sterile bovine serum containing the dormant bacteria as obtained from the Culture Collection,U.S. Department of Agriculture, Peoria, IL, in 10 mL of autoclaved medium Z. The culture tubes were incubated at 37 "C until curdling occurred, usually in 1-3 days. A 1-mL transfer of this culture was made to a second 10 mL of medium Z which was incubated to the curdling point and then stored at 8 "C until used in a detoxification experiment. Cultures of the Lactobacilli were routinely maintained in this manner. Culture Protocol for Screening Microorganisms. A 125-mL Erlenmeyer flask with a cotton plug closure was charged with 2.0 g of deoiled jojoba meal, 18 mL of distilled 0 1981 American Chemical Society
J. Agric. Food Chem., Vol. 29, No. 2, 1981
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Table I. Microorganism Screening organism con trolb L. acidophilusC B-629 B-1833 B-1868 B-1910 B-1911 B-1912 B-2178 B-2092 Knudsend L. bulgaricusC B-548 B-734 B-1909 B-1918 Ahe S. cerevisiaef
toxicants as % meala simmondsin sim. 2'-ferulate
culture conditions PH
4.9
2.3
6.2
1.1 1.5 2.5 2.8 0.58 2.1 3.8 3.7 2.1
0.25 1.3 1.3 0.39 0.33 0.90 1.5 1.6 1.0
6.8 7.6 7.4 6.9 7.0 7.1 5.5 5.6 6.2
growth
heavy heavy light light heavy light light light heavy
odor
musty sour fresh sour fresh fresh fresh fresh sour
4.1 1.3 4.6 light fresh 1.9 1.5 4.1 medium sour 2.8 1.5 4.7 heavy fresh 3.5 1.1 7.0 light fresh 3.5 1.5 4.4 light fresh 3.7 1.5 5.9 heavy yeasty Meal suspended in a After 10 days, submerged cultures were assayed by high-performance LC. sim. = simmondsin. medium and autoclaved. All B strains were supplied by the Northern Regional Research Center, U.S.Department of Agriculture. Obtained from Knudsen Creamery Co. e Obtained from Alta-Dena Dairy. f Fleischmann's dry yeast powder.
water, and 1.0 mL of solution X and then autoclaved at 121 "C for 15 min. The resulting suspensions were inoculated with 2 mL of the prepared bacterial inoculum and placed on a rotary shaker. For the S. cerevisiae experiment, a small amount of dry yeast powder was added directly to the jojoba meal suspension. The cultures were shaken at 200 rpm for 30 min at 2-day intervals. After 10 days the flasks were removed, the contents were inspected, the pH was measured, and the contents were assayed for toxicants (Table I). Analytical Sample Preparation. Screening Study. Each of the 10-day-old cultures and the control were diluted with 25 mL of acetone, mixed well, and vacuum filtered, washing the insoluble residue twice with (2:l) acetone-water. The combined filtrates were concentrated to near dryness on a rotary vacuum evaporator and then transferred to a 10-mL volumetric flask by using acetone. A 5-mL aliquot of this solution was concentrated to dryness on the rotary evaporator, and the residue was redissolved in 1 mL of methanol. The methanol solution was diluted with 3 mL of ethyl acetate and passed through a column containing 0.5 g of Merck silica gel G. The column was washed with 10 mL of (73) ethyl acetate-ethanol, the eluates were concentrated, and the residue was redissolved in methanol to a volume of 10 mL for assay. Treated Meals. Microbially treated meals were dried in an oven at 75 OC to a moisture level of
