DEVELOPMENT OF RANCIDITY IN WALNUTS

L. Carl Greve and John M. Labavitch

ABSTRACT

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

The fatty acid compositions of oils from six commercial and two experi-mental walnut varieties were determined. The implications of thesestudies 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 themolecular entities involved:

2. Develop diagonostic test(s) which would enable industrialiststo detect incipient rancidity~

3. Examine existing walnut varieties with the purpose of providingplant breeders with information which might lead to the productionof 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 rancidificationprocess in walnuts. Drawing on existing literature dealing withrancidity in other oil seeds, we proposed the following:

TAG-PUPA

..J,..LipaseFFA + Glycerol

1Lipoxygenase, Heme protein or Inorganic iron+ 0

Aldehyde co~pounds, Lipid hydroperoxidesConcomitant decrease in FA concentrations

R~Kcid products (Volatile aldehydes, etc.)

Where TAG-PUFA are triacyl glycerols (TAG) containing polyunsaturatedfatty acids (PUFA), and lipase is an enzyme capable of hydrolyzingthe ester link between the glycerol backbone and fatty acid therebygenerating free fatty acids (FFA). Lipoxygenase, heme protein andinorganic iron represent catalysts, specific (lipoxygenase) and non-specific (heme protein and iron) which are capable of oxidizing thePUFA to lipohydroperoxides which can then spontaneously decomposeto 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 torancidification.

Nuts used in these studies were harvested by ourselves in order toreduce the impact of different cultural procedures. The nuts wereall acquired from the same region (Gridley, CA) at the appropriateharvest dates. Nuts were dried using (1) ambient air or (2) hotair in a commercial dryer until they were 4% with respect to moisturecontent. The nuts were then transferred to storage in a nitrogenatmosphere 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 thevalidi ty of our model we carefully cracked nuts from each of thesix test varieties so that we had whole or half nuts. These nutmeats were further divided so we had only quarter nuts as experimentaltissue. These pieces were then stored in either a nitrogen atmosphereor in air at 38°C with a RH of 100%. At zero time and weekly intervalsthere after the nuts were evaluated for the following:

1) Total oil composition (cold pressed oil collected; TAG hydrolyzedand fatty acid methyl esters generated in a sulfuric acid, methanoland benzene mixture; methyl esters examined by gas chromatography(GC) ;

2) Lipase activity (extracts prepared from kernels; assays performedat pH 702 using a synthetic substrate; fatty acids generatedwere assayed by GC, as in 3, below;

3) FFA concentration (cold pressed oil collected; fatty acid methylesters generated in diazomethane - no hydrolysis; assayed byGC) ;

4) Rancidity (organoleptic).

The zero time oil compositions of the six varieties are shown inTable 10 The degree of polyunsaturation of an oil can be used asan index of its susceptibility to rancidification." This parameteris 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 willsee that there is some variation in degree of unsaturation but thatthey 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 resultsare 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 twovarieties as representative of the two groups we found. Hartleyis the representative of the stable varieties (Hartley, Ashley, Vinaand Eureka) and Howard is representative of the unstable group (Howardand Chandler). Table 2 shows what occurs to the FFA concentrationsin Hartley and Howard during a 3-week storage period in a nitrogenatmosphere. What is apparent is the clear increase in FFA in theHoward; this is not so apparent in the Hartley. Table 3 shows thesame parameter in a normal (oxygen-containing) atmosphere. Herewe see less of a FFA increase in the Howard.

Figure I depicts the lipase activities of each of the varieties duringthe same storage period. What is obvious is that there is activityonly in the Chandler and Howard varieties. Note also that at zerotime there is no measurable lipase and that it only becomes apparentafter elevating the storage temperature. Table 4 shows there islittle change in the total fatty acids in Howard and Hartley oilsif the kernels were stored in nitrogen. However, if we look at Table5 we see what occurs with respect to total FA composition in air.Here there are great differences in the oil fatty acid compositionbetween Hartley and Howard, especially in the PUFA.

How does all this' fit into the proposed model? We have shown thatin Howard there is an increase in FFA during storage. In the absenceof molecular oxygen the FFA tend to accumulate. In the presence

of 02 they don't accumulate; they are apparently metabolized furtherand ultimately the fatty acid composition of the oil is dramaticallychanged. In the varieties where there is no assayable lipase noneof these changes occur. There is, thus, a direct correlation betweenthe 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 onthe fourth week showed the Howards to be "rancid" and by the sixthweek the Chandlers had "off flavors". During this period the otherfour varieties remained sound.

Gas chromatographic analysis of methylated derivatives of the rancidHoward oil showed numerous additional compounds which we presumeto be decomposition products involved in "off-flavor" development.We are currently studying these. Because it is highly probably thatthe molecular species that make walnut oil so susceptible to rancidifi-cation are the PUFA any variety we could find with a smaller proportionof these entities, in its oil would probably be less likely to gorancid during storage. Initial evaluations of current cultivarshowever indicate that most of these now in production have highlyunsaturated oils (Table I). This is probably because the breedingprograms which have led to our current varieties utilized similargenetic stocks.

There is, however, an ongoing variety evaluation of new stocks onour campus under the direction of Dr. Gale McGranahan. We have shownpreviously (Figure 2) that during nut development the oil is the

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last component "loadedII into the nut. Additionally, the degree ofunsaturation of this oil (Figure 3) continually rises during thisperiod. 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 evaluationblock and we examined two of these. The results are shown in Table6. What we see is that the- PUFA of the numbered varieties 77-12and 76-21 are significantly reduced relative to Hartley as are theirdegrees of unsaturation. The degree of unsaturation for almond oilis 27% - almonds have little problem with rancidity. Other oil crops(oil palm, soy bean, etc.) have had their oil composition markedlychanged by breeding programs and it is now obvious that there arewalnut varieties which possess less unsaturated oils. It is probablethat a wider search (international) would locate varieties with oilcompositions having even less unsaturated fatty acids.

CONCLUSIONS

If we examine the overall objectives of this project, we can evaluateour first year results.

1. Elucidate the mechanism of rancidity in walnuts and define themolecular entities involved.

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

2. Developed a diagnostic test for incipient rancidity.

In all cases we have detected a rise in FFA concentrations of pressedwalnut oil 3 to 5 weeks before rancidity can be detected organolep-tically. We have developed a new gas chromatographic assay for FFAwhich 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 giventhe degree of inbreeding of our current cuItivars, we think lookingat varieties on a more international scope would provide us witha broader range of parameters for oil improvement.

4. Please note that our experiment, which indicates a high tendencyfor 'Chandler' and 'Howard' to go rancid examined only quarteredkernels. We have not yet examined shell and pellicle factors thatmay play a role in rancidification. Our inshell nuts proved to bevery stable in cold storage and at elevated temperatures.

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TABLE 1. Percentage distribution of the major fatty acids in coldpressed walnut oil.

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DEGREE OFFATTY 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 84VIN 2.4 .6 7.0 72.0 17.6 89ASH 4.4 .6 5.7 69.0 20.6 89CHA 2.3 .7 6.1 66.2 25.0 91HOW 3.2 .7 6.6 72.0 18.5 90EUR 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 kernelsof Hartley and Howard walnuts stored at 38°C in a nitrogenatmosphere.

WEEKS OF STORAGEHART 0 1 2 3

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

HOW 0 1 2 3

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

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

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

WEEKS OF STORAGEHART 0 1 2 3

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

HOW 0 1 2 3

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

WEEKS OF STORAGE

? HART 0 1 2 3..

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

HOW 0 1 2 3

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

* Note that total % of fatty acids identified in3-week stored Howards is much less than 100%.

Other materials are accumulating in oil.

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

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

WEEKS IN STORAGEHART 0 1 2 3

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

HOW 0 1 2 3

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

FATTY ACID TYPE DEGREE OFC: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

Figure 1.

LIPASE

HARoto

ASHoto

>-!::>.i=u 0« ~-

oto

o~

2 3 WEEK

EUR

VIN

CHA

2 3

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

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Figure 2.

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Accumulation of oil in walnut kernels over a season of

development. Both curves are for Hartley - onerepresents 1979 and the other 1980.

Figure 3.

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M II 1.1 A S 0

Degree of un saturation in oil being accumulated in walnut

kernels during developm~nt (refer to Figure 2).

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