DEVELOPMENT OF RANCIDITY IN

L. Carl Greve and John M. Labavitch

ABSTRACT

The rancidification of walnuts was promoted by storing shelled kernels at 38°C for 4 weeks. Of six varieties tested, only 'Howard' and 'Chandler' kernels went rancid during this period. Their deterioration was preceded by the appearance of (an oil digesting enzyme) in kernels and an accumulation of free fatty acids in the oil. The implications and limitations of these observations are discussed.

The compositions of oils from six commercial and two experi- mental varieties were determined. The implications of these studies for "genetic" reductions in rancidity potential are discussed.

OBJECTIVES

The objectives of this year's research efforts were as follows:

1. Elucidate the mechanism of rancidity in walnuts and define the molecular entities involved: 2. Develop diagonostic test(s) which would enable industrialists to detect incipient rancidity~ 3. Examine existing walnut varieties with the purpose of providing plant breeders with information which might lead to the production of a walnut which would be more stable with respect to rancidity. PROCEDURE AND RESULTS

In order to evaluate the previously mentioned objectives it was neces- sary for us to generate an appropriate model of the rancidification process in walnuts. Drawing on existing literature dealing with rancidity in other oil seeds, we proposed the following: TAG-PUPA ..J,..Lipase FFA +

Lipoxygenase, Heme protein or Inorganic iron 1 + 0 co~pounds, Lipid hydroperoxides Concomitant decrease in FA concentrations

R~Kcid products (Volatile , etc.)

Where TAG-PUFA are triacyl (TAG) containing polyunsaturated fatty acids (PUFA), and lipase is an enzyme capable of hydrolyzing the ester link between the glycerol backbone and fatty acid thereby generating free fatty acids (FFA). , heme protein and inorganic iron represent catalysts, specific (lipoxygenase) and non- specific (heme protein and iron) which are capable of oxidizing the PUFA to lipohydroperoxides which can then spontaneously decompose to generate rancid products.

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-- --- If rancidification in walnuts procedes as proposed in this model, the following should be true:

a 0 Before the appearance of rancidity there should be an increase' (or appearance) of lipase followed by an increase in FFA concentra- tions in the walnut oil. b 0 CuI tivars with little or no PUFA should not be susceptible to rancidification.

Nuts used in these studies were harvested by ourselves in order to reduce the impact of different cultural procedures. The nuts were all acquired from the same region (Gridley, CA) at the appropriate harvest dates. Nuts were dried using (1) ambient air or (2) hot air in a commercial dryer until they were 4% with respect to moisture content. The nuts were then transferred to storage in a atmosphere at 2°C with a relative humidity (RH) of 55%.

The varieties we chose to evaluate were Hartley, Ashley, Howard, Chandler, vina and Eureka 0 In an experiment designed to test the validi ty of our model we carefully cracked nuts from each of the six test varieties so that we had whole or half nuts. These nut meats were further divided so we had only quarter nuts as experimental tissue. These pieces were then stored in either a nitrogen atmosphere or in air at 38°C with a RH of 100%. At zero time and weekly intervals there after the nuts were evaluated for the following:

1) Total oil composition (cold pressed oil collected; TAG hydrolyzed and fatty acid methyl esters generated in a sulfuric acid, methanol and benzene mixture; methyl esters examined by gas chromatography (GC) ; 2) Lipase activity (extracts prepared from kernels; assays performed at pH 702 using a synthetic substrate; fatty acids generated were assayed by GC, as in 3, below; 3) FFA concentration (cold pressed oil collected; fatty acid methyl esters generated in diazomethane - no hydrolysis; assayed by GC) ; 4) Rancidity (organoleptic). The zero time oil compositions of the six varieties are shown in Table 10 The degree of polyunsaturation of an oil can be used as an index of its susceptibility to rancidification." This parameter is defined as follows:

Degree of Unsaturation = [C:18:2] = [C:l~:3] TC)t-al"" ei'I . j If we look at the normal oil composition of our six cultures we will see that there is some variation in degree of unsaturation but that they are all highly unsaturated (values ranging between 84-91%). This can be compared to the degree of unsaturation of almond oil (27%) - almonds have little problem with rancidity. These results are not really surprising in as much as all the varieties we examined, except for Eureka, have very similar genetjc backgrounds. During storage, however, there were significant differences in the

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------manner in which these varieties behaved. We present data from two varieties as representative of the two groups we found. Hartley is the representative of the stable varieties (Hartley, Ashley, Vina and Eureka) and Howard is representative of the unstable group (Howard and Chandler). Table 2 shows what occurs to the FFA concentrations in Hartley and Howard during a 3-week storage period in a nitrogen atmosphere. What is apparent is the clear increase in FFA in the Howard; this is not so apparent in the Hartley. Table 3 shows the same parameter in a normal (oxygen-containing) atmosphere. Here we see less of a FFA increase in the Howard.

Figure I depicts the lipase activities of each of the varieties during the same storage period. What is obvious is that there is activity only in the Chandler and Howard varieties. Note also that at zero time there is no measurable lipase and that it only becomes apparent after elevating the storage temperature. Table 4 shows there is little change in the total fatty acids in Howard and Hartley oils if the kernels were stored in nitrogen. However, if we look at Table 5 we see what occurs with respect to total FA composition in air. Here there are great differences in the oil fatty acid composition between Hartley and Howard, especially in the PUFA.

How does all this' fit into the proposed model? We have shown that in Howard there is an increase in FFA during storage. In the absence of molecular oxygen the FFA tend to accumulate. In the presence of 02 they don't accumulate; they are apparently metabolized further and ultimately the fatty acid composition of the oil is dramatically changed. In the varieties where there is no assayable lipase none of these changes occur. There is, thus, a direct correlation between the induction of the lipase and the appearance of FFA in the walnut. Secondarily, it appears that °2 is needed for further decomposition (rancidification) of the oil.

At the end of the third week of storage, all nut meats in this experi- ment were sound by organoleptic evaluation. However, testing on the fourth week showed the Howards to be "rancid" and by the sixth week the Chandlers had "off flavors". During this period the other four varieties remained sound.

Gas chromatographic analysis of methylated derivatives of the rancid Howard oil showed numerous additional compounds which we presume to be decomposition products involved in "off-flavor" development. We are currently studying these. Because it is highly probably that the molecular species that make walnut oil so susceptible to rancidifi- cation are the PUFA any variety we could find with a smaller proportion of these entities, in its oil would probably be less likely to go rancid during storage. Initial evaluations of current cultivars however indicate that most of these now in production have highly unsaturated oils (Table I). This is probably because the breeding programs which have led to our current varieties utilized similar genetic stocks. There is, however, an ongoing variety evaluation of new stocks on our campus under the direction of Dr. Gale McGranahan. We have shown previously (Figure 2) that during nut development the oil is the

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--- last component "loadedII into the nut. Additionally, the degree of unsaturation of this oil (Figure 3) continually rises during this period. Therefore, if there were varieties with shorter "leaf out" to "harvest" periods they might produce oils that were less unsatur- ated. Dr. McGranahan has several such varieties in her evaluation block and we examined two of these. The results are shown in Table 6. What we see is that the- PUFA of the numbered varieties 77-12 and 76-21 are significantly reduced relative to Hartley as are their degrees of unsaturation. The degree of unsaturation for almond oil is 27% - almonds have little problem with rancidity. Other oil crops (oil palm, soy bean, etc.) have had their oil composition markedly changed by breeding programs and it is now obvious that there are walnut varieties which possess less unsaturated oils. It is probable that a wider search (international) would locate varieties with oil compositions having even less unsaturated fatty acids. CONCLUSIONS

If we examine the overall objectives of this project, we can evaluate our first year results. 1. Elucidate the mechanism of rancidity in walnuts and define the molecular entities involved.

With respect to this point, we have developed a strong correlation between the appearance of lipase activity and the involvement of PUFA in rancidification of walnuts. We have not yet isolated an active lipbxygenase but that work is currently underway. We think, however, that a more likely target for breeding to reduce rancidity problems will be the enzyme systems responsible for the synthesis of PUFA in oil.

2. Developed a diagnostic test for incipient rancidity.

In all cases we have detected a rise in FFA concentrations of pressed walnut oil 3 to 5 weeks before rancidity can be detected organolep- tically. We have developed a new gas chromatographic assay for FFA which is extremely simple and' very quick and reliable. If this correlation continues to hold, this point will have been accomplished.

3. Examine the existing walnut varieties, etc.

This work has been started with some initial success. Though given the degree of inbreeding of our current cuItivars, we think looking at varieties on a more international scope would provide us with a broader range of parameters for oil improvement.

4. Please note that our experiment, which indicates a high tendency for 'Chandler' and 'Howard' to go rancid examined only quartered kernels. We have not yet examined shell and pellicle factors that may play a role in rancidification. Our inshell nuts proved to be very stable in cold storage and at elevated temperatures.

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DEGREE OF FATTY ACID TYPE UNSATURATION* C:16 C:18 C:18:1 C:18:2 C:18:3

HAR 3.2 .8 11.1 67.3 17.4 84 VIN 2.4 .6 7.0 72.0 17.6 89 ASH 4.4 .6 5.7 69.0 20.6 89 CHA 2.3 .7 6.1 66.2 25.0 91 HOW 3.2 .7 6.6 72.0 18.5 90 EUR 3.6 .8 7.7 71.0 16.3 87

4t-,," * defined as: [C:18,2) + [C:18:3J x 100 Total Oil

TABLE 2. FFA concentrations (Jlg/50Jll oil) in oil pressed from kernels of Hartley and Howard walnuts stored at 38°C in a nitrogen atmosphere.

WEEKS OF STORAGE HART 0 1 2 3

C:16 0 0 0 0 C:18 0 0 0 0 C:18:1 0 0 2 5 C:18:2 4 2 1 42 C:18:3 2 2 2 44

HOW 0 1 2 3

C:16 0 0 16 27 C:18 0 2 8 11 C:18:1 0 5 26 50 C:18:2 6 45 121 608 C:18:3 5 12 54 170

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;..- TABLE 3. As in Table 2 - kernels stored in air.

WEEKS OF STORAGE HART 0 1 2 3

C:16 0 0 0 0 C:18 0 0 0 0 C:18:1 0 2 2 0 C:18:2 4 6 7 44 C:18:3 1 0 4 14

HOW 0 1 2 3

C:16 0 0 12 23 C:18 0 2 25 45 C:18:1 6 16 28 33 C:18:2 9 45 67 66 C:18:3 5 12 22 13

TABLE 4. Total fatty acid composition (as % of oil weight) in cold pressed oil collected from Hartley and Howard kernels stored at 38°C in nitrogen.

WEEKS OF STORAGE HART .. ? 0 1 2 3

C:16 3.1 3.2 3.1 3.4 C:18 .8 .9 1.2 1.5 C:18:1 11.1 10.8 11.5 9.5 C:18:2 67.3 66.5 67.0 66.0 C:18:3 17.4 18.5 18.0 18.0

HOW 0 1 2 3

C:16 3.2 3.6 3.7 4.1 C:18 .7 .9 1.0 1.0 C:18:1 6.6 6.5 6.0 5.8 C:18:2 72.0 74.0 72.0 68.0 C:18:3 18.3 18.6 17.5 16.6

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-- --- TABLE 5. As in Table 4 - kernels stored in air.

WEEKS IN STORAGE HART 0 1 2 3

C:16 3.2 3.3 3.1 3.3 C:18 .8 .6 .9 1.0 C:18:1 11.1 12.1 9.9 10.8 C:18:2 67.3 67.0 66.8 66.2 C:18:3 17.4 17.5 17.0 17.8

HOW 0 1 2 3

C:16 3.2 3.2 4.1 5.8 C:18 .7 .6 2.2 3.2 C:18:1 6.6 6.4 4.2 3.2 C:18:2 72.0 65.0 61.0 58.5 C:18:3 18.5 15.5 12.2 10.2

* Note that total % of fatty acids identified in 3-week stored Howards is much less than 100%. Other materials are accumulating in oil.

TABLE 6. Total fatty acid composition (% of oil weight) in cold pressed oils from Hartley kernels and kernels of 2 numbered varieties.

FATTY ACID TYPE DEGREE OF C:18. C:18 C:18:1 C:18:2 C:18:3 UNSATURATION

HARTLEY 3.2 .8 11.1 67.3 17.4 84

77-12 14.1 3.8 30.6 35.6 13.1 49

76-21 1.7 0 29.4 61.5 7.2 68

* Degree of un saturation for almond oils is 27

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--- -- LIPASE

HAR EUR o to

ASH VIN o to >- !:: >. i= u 0 « ~-

CHA o to

o ~

3 2 3 WEEK 2

Figure 1. Lipase activities in extracts of walnut kernels stored in air at 38°C. Samples were taken weekly.

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40ror / /"79 % 30

20

10 --- M J J A s o

Figure 2. Accumulation of oil in walnut kernels over a season of development. Both curves are for Hartley - one o represents 1979 and the other 1980. (J)

r-01 , 0 0 X

0 .. Ij)

N<.{ 001-T.

o

M II 1.1 A S 0 Figure 3. Degree of un saturation in oil being accumulated in walnut kernels during developm~nt (refer to Figure 2).

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