Z. Lebensm. Unters.-Forsch. t62, 387--399 (1976)

Zeitschrif t fLir

Lebensmittel- Untersuchung

und-Forschung @ J. E. Bergmann-Verlag 1976

Sterilization of Dehydrated Potato Granules with Propy- lene Oxide

Leonard Steele and Dimitri Hadziyev Food Science Department, University of Alberta, Edmonton, Alberta, Canada T6G 2N2

Sterilisation von getrocknetem Kartoffelpulver mit Propylenoxyd

Zusammenfassung. Propylenoxyd wurde zur ,,Kaltsterilisation" yon Kartoffel- pulver mit 7% Feuchtigkeit und einen Chloridgehalt yon 0,044% verwendet. Bei einem optimalen Gehalt des Sterilisationsmittels yon 0,1% wurde die Behandlung w~ihrend 6 Tagen bei 22 ° C durchgeftihrt, was eine Reduktion des Keimgehaltes yon 3,4 x 10 5 auf weniger als 5 x 10 3 bewirkte. Der Glykolriick- stand war mit 29 ppm niedrig, ebenfalls das Chlorohydrin mit 12 ppm, wobei das 1-Chlor-2-propanol 94% der beiden Isomere ausmachte. Dies ergab einen Hinweis, dab im Pulver trotz des pH-Wertes 5,62 nicht protoniertes Propylen- oxyd als Acceptor nucleophiler/,)berg~inge dient. Aufgrund yon NMR-Unte r - suchungen wurde gefunden, dab die obengenannte Behandlung keine nach- weisbare Atherbildung der St~irke bewirkte, obwohl ihr Gehalt 83% des Trockengewichtes betr~igt und damit als potentieller Hauptreakt ionspartner anzusehen w~ire.

Summary. Results were provided for propylene oxide "cold sterilization" of dehydrated mashed potato granules that contained 7% moisture and 0.044% chloride. Treatment with an opt imum concentration of 0.1% w/w sterilant for 6 days at 22 ° C gave a bacterial count reduction from 3.4 x 105 to less than 5 x 10 3, a low glycol residue of 29 ppm, and 12 ppm of chlorohydrin, with the 1-chloro-2-propanol isomer constituting 94% of the total. This indicated that, in spite of the granule pH of 5.62, nucleophilic attack in dehydrated granules involves nonprotonated propylene oxide. Based on N M R analysis, evidence was given that such sterilization does not result in detectable etherification of starch, even though its content of 83% on a dry basis places it as the major potentially reactive constituent.

Introduction

The use of aliphatic epoxides in the food industry is receiving a great deal of attention for gaseous "cold sterilization" of a wide spectrum of organisms including insects, yeasts, moulds and bacteria. It is especially relevant when the foo d product has to be retained in its original state, free from the caramelization browning reaction, and the change in flavor and odor that often results from "heat sterilization".

388

In Canada the application of ethylene oxide on a commercial scale for the control of bacterial ring rot of raw potato during storage and transport was suggested as early as 1953 [1]. The same sterilant was proposed against the potato wart fungus [2]. Pappas and Hall [3], recommending the use of undiluted 1% ethylene oxide at room temperature, reported that 13 h were required to destroy the various metabolic type vegetative cells, compared with 25 h tO reduce the spore count to a negligible level. Their thermophilic count on a 1-g commercial sample of Idaho Potato Flour was 162 for fiat sour and 214 for total thermophilic spores. The counts dropped to zero after epoxide treatment. Recently, Griffith's Laboratories [4] revealed figures for reduction in moulds, yeasts and thermophilic bacteria when pure propylene oxide was used. The counts per gram in commercial potato flour of 3 x 103 for total bacteria, and 10 for yeasts and moulds decreased ten-fold after treatment, whilst the thermophile count of 320 dropped to zero. However, no test conditions were provided with these data.

A difficulty encountered with the use of ethylene oxide is its toxic hydrolytic products, ethylene and diethylene glycols, as well as chlorohydrin, all invariably found in treated foods. Thus, in commer- cially sterilized ground spice mixtures, Wesley et al. [5] found ethylene chlorohydrin up to 1030 ppm; with an average of 805. Simultaneously, they demonstrated that, in a nominally dry inert material in which glycol formation is avoided, all the sodium chloride present might react and account for the ease and extent of chlorohydrin formation.

The above facts discouraged the use of ethylene oxide and encouraged the use of propylene oxide, since its hydrolytic product, propylene glycol, is generally recognized as safe. Nevertheless, the use of propylene oxide has its own problems. In powdered and flaked foods, prolonged periods of treatment were needed to obtain a better than 90% reduction in bacterial count. In addition, it was not as affective as a sterilant for dried foods [6]. Lastly, toxic chlorohydrins occur in food in similar quanti- ties to those formed in ethylene oxide treatment [-5].

The present study attempts to clarify the propylene oxide sterilization of dehydrated mashed potato granules. It was of interest not only to establish the time and effectiveness of such treatment on total bacterial count, but to determine the reactivity of propylene oxide on the processed granules which contain a low 7% moisture and less than 0.05% sodium chloride. As well, the probability of etherification of starch, the major constituent of the granules, was investigated. Analytical methods were applied which proved to be suitable for analysis of the granules.

Material and Methods

Raw Potatoes: The potatoes used were variety Netted Gem (Russet Burbank) grown in Alberta. Proximate analysis gave the following percentages: protein 2.5 (N × 7.5); fat, oil 0.1; total carbohy- drates, 19.4, of which 17.5 was starch; crude fibre, 0.5; ash, 1.6. Vitamin contents after three months storage at 4 ° C were in mg/100g dry matter: ascorbic acid, 60; thiamine, 0.4; riboflavin, 0.09; niacin, 8.5. Alkali metals were the most abundant mineral constkuents with K, 1.3, and Na, 0.18%. The chloride content on a dry weight basis, amounting to 0.044%, was very low regardless of locality.

Dehydrated Mashed Potato Granules: They were prepared in our laboratory in a pilot plant scale stirred fluidized bed drier (modified Manesty Petrie 10 E, Manesty Machines Ltd., Liverpool, Eng- land) using a processing technique consisting of the following steps: peeling, steam cooking, mashing, freezing and thawing, pre-drying, granulation, drying, cooling, and sifting. Details of this technique were reported elsewhere [7, 8]. - - The potato granules exposed to propylene oxide treatment had the following characteristics: moisture content, 7%; broken cell count, 2%; total starch, 83.7%, with a 21.2% amylose content; free starch, expressed as Blue Value Index, 123; bulk density, 0.8 g/ml; granules of particle size less than 60 mesh, 83 %.

Propylene Oxide Treatment: Neat liquid propylene oxide doses in small open ampoules at 0 ° C were placed in air-tight jars containing 300 g of granules, and evaporated by exposing the jars to 4, 14, and 22 ° C for 1 to 7 days. The epoxide concentrations applied in percent (w/w) were 0.01, 0.1, 0.5, and 1.0, corresponding to concentrations in g/1 of 0.05, 0.5, 2.5, and 5. The treatment was terminated by subjecting the granules to alternating vacuum and a stream of filtered dry air for 30 min at room temperature.

Standard Plate Counts: Each potato granule sample of 11 g was suspended in 99 ml of sterile 0.25 M-K phosphate buffer, pH 7.2. From this primary dilution, decimal aliquots of 10 -2 to 10 4 were prepared and plated in duplicate on the commercially prepared plate count agar. Colonies on the plates were counted after incubation at 30 ° C for 3 days. Total counts per gram presented in the results section are the sum of total bacteria, moulds and yeasts.

389

Determination of Chlorohydrin: A 50g treated sample was mixed to a slurry with 250 ml of distilled and deionized water, and steam-distilled. Normally, 12 fractions, each of 50 ml, were collected and analyzed by GLC. - - The gas chromatography runs were performed on a Bendix Model 2500 chromatograph equipped with an FID detector and with "U"-shaped pyrex glass colums of 170 x 0.4 cm I.D., packed with 10% Carbowax 1500 on Chromosorb W, 60/80 mesh. The ca~-rier gas was N 2 with a flow rate of 30 ml/min. The runs were conducted isothermally at 105 ° C, with an injection-port temperature of 215 ° C. For an injection of 3 gl of aqueous distillate, the average suppression range was x 10 K, input attenuation x 10, and the recorder attenuation x 1. - - Separate calibrations were made for 1-chloro-2-propanol (I) and its isomer 2-chloro-1-propanol (II) (Eastman Organic Chemicals, Roch- ester, N.Y., USA) in the range of 0--5 ppm, 0--50 ppm, and 0--750 ppm. In the last concentration range, the input and recorder attenuations were adjusted to x 10 and x 20, respectively. Under these conditions of separation, the retention time was 3,5 min for I, and 4.5 min for II.

Chlorohydrin Coulometric Assay : A 50 ml aliquot of steam distillate was extracted with 5 x 10 ml of ~tfethyI ether. Then 7 gl of the extract was injected into a gas chromatograph. The emerging compounds were mixed with pure 02, burned in a pyrolyzing tube, and the corresponding oxidation products were eluted into a coulometric titration cell. - - A Pyrex glass column of 106 x 0.6 cm O.D. was packed with 15% of FFAP (Carbowax 20M treated with 2-nitroterephthalic acid) on Anakrom ABS, 130/140 mesh (Analabs, North Haven, Conn., USA). The column temperature was as before, whilst that of the injection port was raised to 220 ° C. Also, N 2 was retained as a carrier gas with flow rate of 75 ml/min. The pyrolyzing tube, of Quartz-Vycor type, was maintained at 820 ° C. The titration cell was a Dohrmann Model T-300 S, designed for halogens. It had a generator Ag anode, a generator Pt cathode, a Ag/AgC1 reference electrode, and a Ag sensor electrode. The chloride ions, resulting from pyrolysis of chlorohydrins, were then titrated with anodically generated Ag ions in an electrolyte of 70% aqueous acetic acid.

GLC-Mass Spectra Data: Analyses were done using a Varian Model 1200 gas chromatograph fitted with a conductivity detector and a 10% Carbowax 1500 column, attached to an AEI mass spectrometer 2, coupled with a DS-50 Data System and a Nova 2/10 24K computer.

Determination of Propylene Glycol: Under the separation conditions for chlorohydrin the first peak of the emerging doublet had a retention time of 16 min, whilst determination by replicate injections at a column temperature of 135--140° C reduced this time to only 5 min (see Fig. l).

Monitoring the Starch Etherification Degree: A Varian EM-360 nuclear-magnetic-resonance spec- trometer was used. The terminal methyl group of the hydroxypropyl substituent of the modified starch was measured. The group appears as a distinct doublet at about 1.5 ~. The area of this doublet was used as a basis of quantitation. - - Pure potato starch samples, isolated from tubers used for granule processing, unmodified or modified with propylene oxide treatment, were hydrolyzed with 10 ml of 10% HC1 at 100 ° C for 30 min [9]. In order to overcome their differing buffer capacity, potato granules (9.9% protein, 83% starch) were hydrolyzed with 17 mt of 20% HC1 for 50 min. Then the reaction mixture was allowed to cool, 2.5 ml of freshly prepared 10% acetic acid was added to serve as an internal standard, and the volume was made up to 30 ml with water. The unhydrolyzed protein was removed from this suspension by centrifugation at 10000 x g for 10 min. A 0.5-ml sample of the clear supernatant liquid was transferred to a standard 5-mm NMR tube. An illustration of integral mea- surements is given on Figure 2. The percentage hydroxypropyl group in the modified starch was calculated according to Stahl and McNaught [-9] by comparison of the methyl proton signals in the hydroxypropyl group in modified starch and in the standard acetic acid.

Results and Discussion

In t he a d d - b a c k p r o d u c t i o n of d e h y d r a t e d m a s h e d p o t a t o g r a n u l e s a d e q u a t e

w a s h i n g of t he r a w p o t a t o e s causes a r e d u c t i o n in t he n u m b e r of m i c r o - o r g a n -

i sms, as d o e s s t e a m peel ing. B l a n c h i n g usua l ly r e d u c e s t he c o u n t by 9 9 % , w h i l s t

s l ic ing h a s n o s ign i f i can t effect. T h e m a s h i n g s t ep fo l lows c o o l i n g a n d c o o k i n g .

T h e su r face of f r e sh ly m a s h e d g r a n u l e s is p r a c t i c a l l y f ree of m i c r o - o r g a n i s m s .

H o w e v e r , w h e n t h e m a s h is c o o l e d to 60 ° C, c o n t a m i n a t i o n o ccu r s f r o m m o u l d s ,

yeas t s a n d b a c t e r i a in t he air. T h e i r n u m b e r is i n c r e a s e d g r ea t l y d u r i n g c o n d i t i o n -

ing, a s t ep in w h i c h t h e d u r a t i o n a n d t e m p e r a t u r e r e g i m e s i m u l a t e a t e m p e r a t u r e -

390

2O

15

Ac~

10

ncei°ne ~ PROPYLENE I\ oL coL /

ppm = 3.38 ACM 2

A

~ / i I I

10 20 30 40 p p m , w/w

Fig. 1. Propylene glycol standard graph obtained by GLC. Acetone solutions (3 injection) gave a single peak, whilst aqueous solutions (9) gave a doublet, the total area (A) of which was used for the graph. For the effect of water on determination of low and high glycol concentrations, see Table 7

gradient incubator. The heat applied in the subsequent air-lift and fluidized bed drying steps causes a large reduction in the count. All yeasts and most bacteria are destroyed, but spores of bacteria and moulds usually survive, as do Vegetative cells of a few species of heat resistant bacteria. Thus, in a plant where the air is contaminated, equipment must be cleansed and sanitized to avoid a build-up of spores of thermophiles that occurs up to a count of 106, and exceeds the micro- biological standard of 5 x 103 or less. Similar contamination might occur in the freeze-thaw granule process where mashing and pre-drying are the critical steps affecting the final amount of bacteria.

"Cold sterilization" with propylene oxide (PO), 0.1% w/w, at 4 ° or 14°C showed that even 6 days of treatment could not reduce the original count of 3 .4x10 5 below 10 4. A satisfactory reduction was obtained only at 22°C

F--- %x102

of HOAc peak NMR

4001 GLYCOL I

~o T ~a; /

1 0 0

f 2 4 6 8 I0

propyJen e glycot %

MODIFIED ~ ~ S T A R C H

• / UNMODIFIED

/I S T A R C ~ _ . _ ~ _ _ _ j L ~ ~ ( b )

391

[ I

6 5 Fig. 2a and b

i I '

4 3 2 1 3

PROPYLENE GLYCOL (107oin water)

_t: I I I I 6 5 4 3

F i g . 2 c

, . .J

I I 2 1

Fig. 2a--c. Propylene glycol determination in starch hydrolysate by NMR. - - a Calibration graph. b Spectra of potato starch before and after propylene oxide etherification, c Spectrum of aqueous 10% propylene glycol

392

Table 1. The effect of temperature on propylene oxide treatment (with 0.1%, w/w) of mashed potato granules

4 ° C 14 ° C 22 ° C

2 4 6 2 4 6 2 4 6

days days days

Standard P la t eCoun t 5 .1x10 ~ 1.5×104 1.3×104 1 .1xl05 6 .8x10 ~ 2 .9x104 1.4x104 8.4x103 5.1x103

Chloropropanol, total (ppm) 7.4 7.3 11.8 3.0 7.7 8.1 11.4 11.7 12.1

1-chloro-2- propanol (ppm) 7.0 6.9 11.1 2.8 7.2 7.6 9.8 11.0 11.4

Table 2. Effects of duration of propylene oxide treatment on the bacterial count and chlorohydrin content of mashed potato granules (treatment with 0.1%, w/w, at 22 ° C; duration of propylene oxide treatment in days)

0 1 2 3 4 5 6 7

Standard P la t eCoun t 3 .4x10 s 3.4x104 1.4×104 9.9x103 8 .4x10 a 6.3x103 5 .1x10 a 8.5x102

Chloropropanol, total (ppm) 0 3.8 10.4 11.0 11.7 18.1 12.1 12.9

1-chloro-2- propanol (ppm) 0 3.4 9.8 9.5 11.0 16.6 11.4 11.8

(Table 1). At this temperature the optimum time appeared to be between 5 and 7 days, the latter reducing the count to 8.5 x 102 (Table 2). When PO concentra- tion was lowered to 0.01% w/w, even after 6 days about a three-fold higher count than the permitted standard of 5 x 103 was obtained. Permissible levels were only achieved with 0.1, 0.5, or 1.0% PO concentrations after 6 day treatment. However, as seen in Table 3, the levels of 0.5 and 1.0% did not substantially change the bacterial count after 2 or 4 day treatments, when compared to 0.1%, whilst after 6 days, the high levels of PO reduced the bacterial count from 5.1 to only 4.2 and 3.7 x 103, instead of a five- or ten-fold decrease, respectively, that might be antici- pated. However, the use of high concentrations of PO resulted in an abrupt nearly ten-fold rise in the content of toxic propylene chlorohydrin, a fact that endan- gered the whole PO sterilization approach. Thus, the final choice of PO treatment became dependent on the extent of chlorohydrin synthesis, and its accumulation in treated granules.

It was found that ethyl ether extraction of potato granules packed in a chro- matographic column results in a complete removal of chlorohydrin. However, lipids, carotenoids and flavouring constituents present in the extract interfere in the separation and quantitation by GLC. This interference was partly avoided when chlorohydrin was recovered by dry steam distillation, passing the steam through preheated granules in a flask with a perforated bottom. This approach

Tab

le 3

. E

ffec

ts o

f p

rop

yle

ne

oxid

e co

nce

ntr

atio

n o

n b

acte

rial

co

un

t an

d c

hlo

roh

yd

rin

co

nte

nt

of

trea

ted

mas

hed

po

tato

gra

nu

les

Ep

ox

ide

con

cen

trat

ion

in

% (

w/w

) af

ter

...

days

at

22 °

C

0.01

0.

1 0.

5 1.

0

2 4

6 2

4 6

2 4

6 2

4 6

Sta

nd

ard

P

late

Co

un

t 1

.8x

lO a

1.4

xlO

4

1.5

xlO

~

1.4

xlO

4

8.4

x1

03

5.

1×10

3 9

.3x

10

3

6.7

x1

03

4

.2x

10

3

7.4

x1

03

6.

0 x

lOa

3.7

×10

3

Ch

loro

pro

pan

ol

tota

l (p

pm

) 2.

1 4.

9 7.

2 10

.4

11.7

12

.1

73.7

93

.4

87.8

78

.3

96.2

12

3.5

1-ch

toro

-2-

pro

pan

ol

(ppm

) 2.

0 4.

6 6.

8 9.

8 11

.0

11.4

69

.3

87.8

10

6.8

73.5

90

.4

116.

1

Tab

le 5

. M

ass

spec

tra

dat

a

1-ch

loro

-2-p

ropa

nol

m/e

81

79

58

49

45

43

42

41

R

I a

1.3

5.2

2.5

8.6

100

39.8

9.

7 12

.4

%1;

m b

0.

4 16

0.

8 2.

6 30

.3

12.0

3.

0 3.

8

2-ch

loro

-l-pr

opan

ol

m/e

65

63

62

58

57

43

42

41

R

I 1.

6 2.

8 2.

7 24

.1

6.3

4.0

4.5

6.7

%~

'm b

0.

7 1,

2 1.

1 10

.2

2.6

1.7

1.9

2.8

Pro

pyle

ne o

xide

(Ref

. [-

14])

.

m/e

58

43

31

29

28

27

26

15

R

I 83

.9

43.8

35

,4

55.7

10

0.0

52.5

30

.6

15.1

39

38

37

14,8

6.

1 4.

6 4.

5 1.

9 1.

4

39

38

37

10.4

3.

5 3.

5 4.

4 1.

5 1

.5

32

12.6

3.

8

31

100 42,1

31

12.1

3.

7

30 4.6

1.9

29

36

11

29

55.9

23

.6

28

41

12.4

" R

elat

ive

inte

nsit

y b

Per

cent

of

the

tota

l io

n c

urr

ent

t~

394

isolated other volatiles, including some pyrazines. Ultimately, the method of adding water to the granules to form a slurry, followed by steam distillation, was adopted, since it provided distillates with the least number of interfering com- pounds. However, even though propylene chlorohydrin forms an azeotropic mix- ture with water of 46 to 54%, respectively, with a b.p. of 95.4 ° C, it was not readily recovered from the slurry. As shown in Table 4, the first 50-ml fraction of distillate gave 30% of the total chlorohydrin, whilst the recovery amounted to 98% only after the tenth fraction. An additional two fractions yielded a total of 99.6%. An attempt to collect only the first few fractions, and to calculate the chlorohydrin content by integration of the curve corresponding to an exponential equation, as presented in Figure 3, is not justified since the standard deviation and error in determination of these fractions are unacceptably high (Table 4).

Injection of the chlorohydrin distillate into the gas chromatograph gave two peaks. The first peak coincided with that of 1-chloro-2-propanol (I), and the second with with 2-chloro-l-propanol (II). Coulometric detection of both peaks gave chlorine contents matching those calculated from peaks obtained using a GLC equipped only with a flame ionization detector. The identities of both peaks were finally confirmed by mass spectra data.

As seen in Table 5, the presence in both GLC peaks of chlorine in a ratio of 1 to 3 was revealed by fragments at m/e 81 and 79 for I, and 65 and 63 for II. The chlorohydrin molecular ions (M ÷ 94) were not detectable. In spectrum I, the most abundant oxonium ion at m/e 45 appeared to be due to an e-cleavage of the carbon-carbon bond joining the hydroxyl and chlorine substituents. The next three most abundant fragments found at m/e 43, 29, and 28 were due to the ejection of a methyl radical from the epoxide furnished from I by removal of HC1, and to formyl and ethylene ion radicals.

0 m,,e 29

C I_CH~CH CH3_.J m/e58 I=.CH2= C= O H OH +. L H O = C H C H 3 ~ m,.'e 43

( I ) mle 45

The ethylene cation appeared to arise from a transannular cleavage of PO without a H transfer, a mechanism already reported for some terminal and non- terminal epoxides. Compound II had its most abundant fragment at m/e 31, followed by those at 29, 58, and 39:

-HCI ,,0,,

I CH2CHCH3 '

C H2 = O H ~ C H2-CI"I-CH 3"--J m,'e 58

OH CI / 1 + m,'e31 ÷. ~ r ,.cl.~

-H2° LcH: cHj (11) - HCI rote 39

H C - O

m/e 29

395

Table 4. Standard deviation and error in determination of chlorohydrin in distillates from granules treated with 0.1% (w/w) propylene oxide at 22 ° C for 2, 4, and 6 days

Fraction number Mean a Standard deviation b Standard error b (each 50 ml)

1 30.5 8.59 4.96 2 19.4 3.30 1.91 3 15.1 1.63 0.94 4 10.1 1.55 0.89 5 7.4 2.05 1.18 6 4.8 1.39 0.80 7 3.1 1.06 0.61 8 2.5 0.58 0.33 9 1.6 0.53 0.31

10 1.0 0.25 0.14 11 0.7 0.10 0.06 12 0.4 0.15 0.08

a Mean % in each fraction of the total amount of chlorohydrin isolated b n = 6

20A 2.26 12 v~1.3o

10 15.6 chlorohydrin total 2~ 1.93 120.7ppm ~ 1,11 p p m

8 SE /0 11.9

%4_ 1.23 ~0 .71

total ~-~ lO.6 0.92 6 '~ .53

4 ~ 4.5 0."/I

?-'g~ 39 " 3.0

2 4 6 8 10 12 14 16 1 8 fraction, 20ml

Fig. 3. The extent of chloropropanol recovery from steam disti l lation.--Ordinate-percentage of the total found in each fraction (in this case 20 ml fractions were collected rather than the usual 50 ml)

396

The stable oxonium ion, m/e 31, resulted again through the favoured path of a-cleavage. The fragmentation patterns coincided with neat chlorohydrins. Hence, their identity was considered proved, and, thus, GLC data for chlorohy- drin content were reported as the summation of both peaks.

As seen from Table 1, PO at 0.1% w/w brings about a total chlorohydrin content close to l0 ppm, a content which is not affected by temperature in the range of 4 ° to 22 ° C after 2, 4, or 6 day treatment. This suggested that the chloro- hydrin was built up during the initial days of treatment. Data from Table 2 support this assumption. At a temperature of 22 ° C, the chlorohydrin level after 1 day of treatment was 3.8 ppm, and after 2 days 10.4 ppm, whilest the increase in chloro- hydrin in the following days became negligible. Even after 7 days the content did not exceed 13 ppm, however, the effect of increased sterilization was maintained even beyond the 6 day treatment. The highest content of chlorohydrin was ob- tained by treating granules with 0.5 and 1.0% w/w PO, giving, after 2 days, contents of 74 and 78 ppm, respectively, with an average rise of 25% for addi- tional two day treatments (Table 3). Though these treatments resulted in much higher chlorohydrin contents, they did not substantially improve the decrease in bacterial count.

Regulations do not specifiy the allowable content of propylene chlorohydrin for many foods. Residual chlorohydrin in starch is limited to 5 ppm. Wesley et al. [5] reported the amount in canned soup to be 6.8 ppm when the ingredients were sterilized with gaseous PO. Contents of ethylene chlorohydrin reported for some foodstuffs are:flour, 260; spray-dried albumen, 310; ground air-dried green peas, 36; spices, from 490 to 1030 ppm [5]. So far, no data have been reported for potato granules, flakes or related products, but, compared to the above ethylene chlorohydrin contents, our findings for potato granules might be considered as low.

Evidence on the readiness of sodium chloride reaction with PO has been reported [5]. Therefore, based on the chloride content of 440 ppm on a dry weight basis in Alberta potatoes, the low average yields of chlorohydrin of only 0.4, 1.0, 8.5, and 9% for the corresponding treatments of 0.01, 0.1, 0.5, and 1.0% w/w PO indicate that chlorohydrin formation in granules was limited either by a lack in the granules of the buffering capacity necessary for synthesis, or by restricted availability of chlorides. As found by us, granules with sodium acid pyrophos- phate and sodium bisulfite as additives have a pH of 5.62 at which their buffering capacity is high. Hence, the second possibility seems valid.

Furthermore, based on chlorohydrin results, it has been proved that synthesis of 1-chloro-2-propanol occurs preferentially in the granules. The synthesis of its isomer, 2-chloro-l-propanol, averaged only 6% of the total chlorohydrin for all tre~ttments. This finding indicates that the dehydrated granules are not able to provide a proton to catalyze a protonated PO cleavage reaction. Instead, nucleo- philes in the granules are forced to attack the non-protonated epoxide, a reaction which proceeds with a low rate, and which results in addition at the least-hin- dered, terminal carbon, thus giving rise to 1-chloro-2-propanol in the reaction with chloride.

Table 6. The contents of propylene glycol found in mashed potato granules after propylene oxide treatment

397

Epoxide Days Yield (ppm) Percent treatment yield %, w/w Expected a Found

0.1 2 59.3 25 42.2 4 118.6 25 21.1 6 177,3 29 16.4

23 681,8 - - 1,312 b

0.5 2 296 107 36.1 4 593 118 19.9 6 886 110 12.4

23 3,410 - - 6,636 b

1.0 2 593 134 22.6 4 1,186 153 12.9 6 1,773 141 8.0

23 6,818 - - 13,123 b

a Theoretically expected values were based on the assumption that spontaneous ring opening of propylene oxide in water has a reaction rate K20 = 1.7 x 10 .5 min- 1, i.e., tl/; =22.3 days [101

Prolonged periods of PO treatment also brought about the PO hydrolytic product, propylene glycol. Glycol accumulation in the granules averaged 26, 112, and 142 ppm, respectively, for PO treatments of 0.1, 0.5, and 1.0% w/w/Table 6). These levels were attained after the second day, and changed only slightly after 4 and 6 day treatments. These results, compared to those expected stoichiometri- cally, were very low. This suggested that either water in granules is the reaction limiting factor, and/or that spontaneous ring opening, even in the presence of excess water, is limited by a slow reaction rate (K2o = 1.7 x 10 - 5) [10]. The find- ing that a PO increase from 0.1 to 0.5% was matched with a fivefold increase in glycol content, supports the latter suggestion. However, when PO was doubled to 1.0% from 0.5%, there was only a 1.3-fold increase in glycol content. This sug- gests that at high PO levels water availability becomes the reaction limiting factor.

A parallel study in our Department [11] demonstrated that the monolayer water capacity of dehydrated potato granules is 5.42%. Hence, this value sub- tracted from the granule moisture of 7% gives the actual amount of free water present. Although low in amount, it is still in a 50-, 10-, and 5-times excess than required for complete hydrolysis of PO at levels of 0.1, 0.5 or 1.0% w/w, respec- tively. The glycol yields of 42% or less, as found after two or more days of treatment, strongly suggest that only half of this free water is available for PO hydrolysis. Therefore, it appears that more than 50% of the "free" water in dehydrated granules is within the porous structure of the granules, and that the

398

Table 7. Solvent effect on GLC determination of propylene glycol using a flame ionization detector

Propylene Acetone Water glycol ppm Aa" SD b SE b Aw a SD SE

Aw-Aa % Change c

5.2 1.5 0.03 0.02 1.1 - - - 0 . 4 -26 .7 10.4 3.1 0.20 0.11 3.6 0.41 0.18 0.5 + 16.1 20.7 6.1 0.17 0.10 9.5 0.69 0.31 3.4 +55.7 31.0 9.3 0.44 0.25 14.8 0.47 0.18 5.6 +60.2 41.4 12.1 0.66 0.38 19.1 2.89 1.67 7.0 + 57.9

a Peak area (cm 2)

b SD, standard deviation; SE, standard error; Water, n = 3; Acetone, n = 5 c (Aw-Aa) x 100/Aa

pore size effectively limits PO penetration. Thus, the small increase in glycol yield with time of PO treatment merely reflects the gradual depletion of free water on the non-hindered outer surface of the granules.

Unlike the chlorohydrin, propylene glycol is on the list of safe and acceptable food additives. Nevertheless, tolerances of 300 ppm are reported as permitted for food ingredients, including starch, that are to be further processed into a final food form. Residual glycol of 110--210 ppm was reported for patent flour when the wheat was treated before milling with 1--1.5 g/1 PO below 28 ° C. At 48 ° C after 6 or 20 h treatments, 700--900 ppm were obtained. Residual glycol was 1000 ppm when the flour was treated, after milling, at 28 ° C. In order to produce flour with low glycol, it was recommended that any remaining PO be removed before milling by repeated cycles of aeration and evacuation [12].

For potato granules, flakes and related products no data on glycol residues have yet been reported.

Results of investigations into possible modification of potato granule starch by PO treatment are illustrated by NMR recordings presented in Figure 2. The starch granules isolated from raw potatoes after acid hydrolysis revealed the presence of hydroxyl absorption of water and hydrolyzed starch at 6.0 6 and, at 3.5 and 4.5 c5, the remaining hydrogen of the hydrolysate. The peak at 2.3 ~ corres- ponded to the methyl group of the acetic acid used as internal standard. When the hydrolysis was preceded with PO treatment of the same starch in the presence of an alkaline catalyst, a new doublet at 1.5 ~ appeared due to the methyl group of the hydroxypropyl substituent. Integration of this doublet permitted reliable quantitation down to 0.3%, though qualitative determination as low as 0.1% was possible. When the potato granules treated with 0.5 or 1.0% w/w of PO for 6 days were hydrolyzed, the doublet was absent. This finding indicated that even a prolonged period of granule sterilization does not result in detectable starch etherification. This contradicts findings obtained for PO treated patent flour [12] in which some reaction of epoxide with the starch was indic~tted by amylograph results. However, in flour the starch granules are free and are partially damaged, while in potato granules they are sterically hindered within intact potato cells. The cell damage of 2% for granules could provide an increased reactivity, but this

399

should be more than offset by intermolecular association of starch molecules caused by heat drying. Finally, the inert nature of starch in the granules is also indicated by the low Blue Value Index found for its free starch portion, i.e., 110-- 135 on a dry basis [7].

In conclusion, propylene oxide appears to be a slow but effective sterilant for dehydrated mashed potato granules. The requirement for a prolonged exposure of granules to the vapor does not adversely affect quality if the sterilant concen- tration is kept below 0.5% w/w. A level of 0.1% appears to be optimum. At this concentration a 6-day treatment brings about a bacterial count that is better than current standards. The content of toxic chlorohydrin is below 13 ppm, whilst that of nontoxic propylene glycol is only 30 ppm. Since these results are comparable to, or better than, those so far reported for many foods, propylene oxide may gain approval as a sterilant for dehydrated potato granules. However, as noted re- cently by Gammon and Kereluk [131 : "The burden of proof of its suitability is on the processor and not the Government Regulatory Agency".

Acknowledgements. The financial support from the National Research Council of Canada and the Alberta Potato Commission is gratefully acknowledged. Also, thanks are due to Mr. H. M. Mei for assistance in microbiological work.

References

1. MacLachlan, D.S., Monro, H.A.U., Racicot, H.N., King, J.E.: Can. J. Agr. Sci. 33, 132 (1953) 2. Monro, H.A.U., Olsen, O.A.: Can. J. Plant Sci. 50, 644 (1970) 3. Pappas, H. J., Hall, L. A.: Food Techn. 6, 456 (1952) 4. Griffith's Laboratories, Inc., Chicago, Ill., USA (1968) 5. Wesley, F, Rourke, B., Darbishire, O.: J. Food Sci. 30, 1037 (1965) 6. Bruch, C.W., Koesterer, M.G.: J. Food Sci. 26, 428 (1961) 7. Ooraikul, B., Packer, G. J.K., Hadziyev, D.: J. Food Sci. 39, 358 (1974) 8. Jadhav, S., Steele, L., Hadziyev, D.: Lebensm.-Wiss. -Technol. 8, 225 (1975) 9. Stahl, H., McNaught, R.P.: Cereal Chem: 47, 345 (1970)

10. Br~Snstedt, J.N., Kilpatrick, M., Kilpatrick, M.: J. Am. Chem. Soc. 51, 428 (1929) 11. Jericevic, D. : M.Sc. thesis, Food Sci. Dept., University of Alberta (1975) 12. Vojnovich, C., Pfeifer, V. F. : Cereal Sci. Today 12, 54 (1967) 13. Gammon, R, Kereluk, K.: In: Am. Inst. Chem. Eng. Symp. Ser., No. 132: Engineering of food

preservation and biochemical processes. 69, 91 (1973) 14. Stenhagen, E., Abrahamsson, S., McLafferty, F. W. : Atlas of Mass Spectral Data, New York: Inter-

sci. PuN. 1, 31 (1969)

Received, March 22, 1976