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] DE.VELOPMENTOF RANCIDITY IN WArnUTS J L. Carl Greve, Rebecca Damell and John M. Labavitch ABSTRACT

'Ibis year's research was divided into ~ categories; a) ExperiIrents to J further understand the biochem:::ial (chemical) mechanisms of rancidity in and b) Work related to industry practices which might affect the ] probability of rancidification in walnuts. . ExperiIrents were done to verify the existence of a enzyne in kernel tissue in agreezrent with last year's proposed mJdel for j rancidity developrent in walnuts. Additional experiIrents verified that certain experiIrental varieties of walnuts contain oil of different Irolecular carp:>sition than that of ] existing ccmrercial varieties.

The possibility of antioxident potential in the walnut pellicle was ] examined. These experiIrents showed pellicle to have considerable antioxident activity and the possibility of varietal differences is nCM being examined.

] Additionally, experirrents designed to test the use fullness of low oxygen atmJsphere in walnut storage were run. The results of the experiIrents were canplicated by control materials which have failed to go rancid at ] the time of this writing.

A collaboration has been established with Dr. Clyde T. Young (Professor, ] North Carolina State Uni versi ty) in order to test for walnut production of volatile materials which are indicators of rancidification by specialized gas chranatographic techniques. '!he pw:pose of these experiIrents was the early detection of rancidity in walnuts and the 1 further elucidation of the exact mechanism of the rancidification process.

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The overall objectives of this project are (as stated last year) the following:

1) Elucidate the IreChanism of rancidification in walnuts and define the m::>lecular entities involved; 2) Develop diagnostic tests for the identification of incipient rancidity in walnuts; 3) Examine existing walnut varieties with the purpose of providing plant breeders the infonnation necessary to produce a walnut less susceptible to rancidity.

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] The objectives of this year's work done in order to canplete or fill voids in last year's results were the following: ] l) Verify last year's results with respect to the unusual oil canposition of sane experimental varieties; 2) Examine the possibility of the existence of a lipoxygenase enzyne in walnut kernel tissue; ] 3) Test varietal differences with respect to rancidification

potential; -. 4) Test low oxygen at:m::>spheres as potential handling -procedures for ] preventing rancidification; 5) The developrent of special gas chranatographic procedures for testing the onset of rancidity and to deteDnine the IreChanism of

] rancidification.

PR(XE)URES AND RESULTS

I Part of the process of understanding rancidity in walnuts involved the generation of a model of the chemistry involved in their rancidification. Last year we proposed such a IOOdel and then proceeded to show that at least sane of the canponents we proposed were present in walnut kernel tissue. That IOOdel is shown below*.

'mG-PUFA

Lipase 1 FFA + Lipoxygenase, Rene Iron or Inorganic Iron, and 02 1 Md~yoos, Upo~d~, etc.

1 Rancid Products

We verified-.- the existence of a and showed a correlation between its inducenent and the onset of rancidity last year. We also showed a positive requirement for 02 in the rancidification process. However, we were unable to find ~ipoxygenase activity in walnut tissue. Upoxygenase enzynes are difficult to isolate for several technical

*TAG-PUFA (Polyunsaturated Triacylglycerols), PUFA (Polyunsaturated Fatty Acids), FFA (Free Fatty Acids).

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1 reasons, not the least of which is the fact that the products they generate tend to poison the enzyrre the m:rnent they are produced. However, we have now deroc>nstrated a lipoxygenase activity in walnuts l.. (Serr) and have thus validated our m:Xlel. We believe, however, that this is probably only one mechanism through whichrancidity develops in walnuts.

J MJre important to the prevention of rancidity in walnuts was the deteD11ination that at least two experi1rental walnut varieties in Dr. Gale McGranahan's seedling block possess nuts with unusual oil J catq?Ositions as canpared to existing carmercial varieties. Nuts fran numbered varieties 77-12 and 76-21 were again tested this year for the degree of unsaturation of their oils (Degree of unsaturation = [C 18: 2] ] + [C 18:3] /total oil = DUS). The tendency of any oil, or oil nut, to go rancid is dependent on its DUS. The nore polyunsaturated oil present the greater the tendency for rancidification. C 18: 2 and C 18: 3 are the predaninant polyunsaturated fatty acids in walnut oil. The average DUS ] fran canrercial cultivars fran the UCDvarietal block was 86% + 5.6%*. Last year, seedlings 77-12 and 76-21 had DUS values of 49 -and 68, respectively. This year they had DUS of 61 and 72, respectively. 1 Whether this increase in DUS represents a trend because of maturation or was envirorurentally induced can only be guessed at present. This will be evaluated by arulUal analyses. 1 It was suggested by industry representatives that a nore ccmplete evaluation of the existing ccmrercial cultivars be carried rot this year with respect to oil canposition. Last year we examined 6 varieties; ) Hartley, Ashley, Howard, Vina, Eureka and Chandler. This year we examined 19 varieties (see footnote) and found that all had essentially the same oil canposition. . I An important finding which developed out of our research regarding the lipoxygenase was that there are naturally ocurring in both stored and genninating walnuts. These antioxidants are located in the I pellicle of stored walnuts and can be found in the fleshy tissue of genninating nuts. It is believed that the antioxidants are phenolic in nature and efforts are underway to deteDnine whether there are varietal I differences in concentrations or types of these canpounds.

A set of experi1rents to deteDnine the effect of low oxygen content in the storage atIrosphere on the rancidification process in walnuts has given us mixed results. Experiments where half kernels were stored either in air or a atIrosphere containing approximately 1.0% 0 were carried out. '!he nut halves were stored in a static environment o~ 50°C and 100% relative humidity to (preStm1ably) accelerate the onset of

*Cultivars examined in 1986 were; Payne, Franquette, Mayette, Early, Erhardt, Eureka, Pedro, Idaho, Waterloo Vina, Ashley, Amigo, Howard, Chandler, SUnlund, Chico, Tehema, Serr, and Westside.

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J rancidity. Nuts fran Chandler and Hartley varieties were examined. Nut -. meats were examined organoleptically every fourth day for a four-week I period. No lots which were stored in the low oxygen atIrospheres went .I rancid during the four week storage period. However, only half of the control lots (four 50 gram lots of each variety) were found to be rancid after four weeks of storage in air. This inability to initiate ] rancidity in stored walnuts under nmrerous conditions has been a difficulty with this project fran the beginning. This fact and other data have led us to believe that there may be handling factors which are important to the inducement of rancidity in walnuts. - In palm nuts 1 mechanical injury is an important ParaIreter in the initiation of rancidity in the whole fruits. Because of this we intend""t.o examine the role of bruising in the onset of rancidity in walnuts. Data fran these } experinents should be available by the time of the Walnut Research Conference.

I Dr. Clyde Young (North Carolina State University) has had considerable success in correlating the onset of rancidity (and other flavor defects) in with the em:mation of trace aIOOunts of volatile chemicals fran the nuts. Our collaboration with Dr. Young should enable us to evaluate stored nut neats for incipient rancidity with sane degree of certainty. However, the need for special aparatus in order to perfonn these procedures may limit the application of this analysis. Data fran these exper:iIrents will hopefully be available by early 1987. OONCLUSIONS

After tYIO years of research it appears that there are several factors that contribute to rancidity in walnuts. It is probably that there are a mnnber of nechanisms that lead to "Off Flavors" in walnut nut neats. In other oil nuts (peanuts) the canpounds responsible for these "Off Flavors" are different dependent on the chemical mechanism which has been induced. The situation may be the same in walnuts.

Certainly a major factor in rancidification of walnuts is the degree of unsaturation of walnut oil. The finding that certain seedlings (77-12 and 76-21) produce nuts containing oil with a reduced DUS is a Il'Ost pranising observation for the long tenn solution to ~ancidity. The finding that both lipase and lipoxygenase activities are present in walnut kernel tissues may be important to future breeding programs. Certai.n+Y& screening programs could be established to test for reduced levels of these activities in walnuts thus, potentially, leading to a reduced probability of rancidity.

At present perhaps the Il'Ost interesting observations we have made with respect to the short tenn solution for rancidification of walnuts in storage relates to our finding that in our hands walnuts are not particularly susceptible to rancidity. This leads us to believe that while it is certainly true that walnuts can be made rancid there must be sane set of factors related to industrial practices that potentiate the tendency for walnuts to go "bad". Our observations that walnuts contain significant aIOOunts of natural antioxidents suggest that these fruits

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J should be resistent to oxidative processes in general. Because of this we believe that research in the area of mechanical injury may be very rewarding. We will direct considerable research effort in this J direction.

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