[advances in food research] advances in food research volume 3 volume 3 || control of microorganisms...

Control of Microorganisms Causing Spoilage in Fruit and Vegetable Products

BY MATHILDE VON SCHELHORN

Institut fur Lebeitsmitteltechnologie. Munich. Germany

Translated by A . L . Gardner

CONTENTS Page

I . Introduction . . . . . . . . . . . . . . . . . . . . . . 431 I1 . Sterilization of Fruit and Vegetable Products by Heat Treatments . . 433

1 . Fundamental Research on the Destruction by Heat of Microorganisms 433

a . Temperature-Time Relationship for Destruction of Microorganisms 435

b . Influence of pH on Heat Destruction Values . . . . . . . . . 437 c . Basic data for thermal death t iTe curves for the organisms in

question . . . . . . . . . . . . . . . . . . . . . 437 2 . Practical Applications of Heat Sterilization of Fruit and Vegetable

Products . . . . . . . . . . . . . . . . . . . . . . 440 a . Recent Developments in Sterilizing in Cans . . . . . . . . . 440 b . High-Temperature Short-Time Sterilization . . . . . . . . . 441

I11 . Exclusion of Oxygen as a Supplement to Heat Sterilization . . . . . 443 I V . The Inhibitory Effect of Carbon Dioxide on Germination . . . . . . 444

445 VI . Sterilization of Fruit and Vegetable Products by Electronics, Ultra-

sonics, etc . . . . . . . . . . . . . . . . . . . . . . . 44d . 446

1 . Behavior of Microorganisms at Low Temperatures . . . . . . . 446

That Can Cause Spoilage in Fruit and Vegetables . . . . . . 446

Occurring in Fruit and Vegetable Products . . . . . . . . . . Occurriug in Fruits and Vegetables . . . . . . . . . . . .

V . Sterilization of Juices by Filtration . . . . . . . . . . . . . .

V I I . Preservation of Fruit and Veget. able Products at Low Temperatures .

a . Temperature Limits of Activity for Bacteria, Yeasts, and Moulds

b . Influence of the Freezing Temperature and Rate of Freezing on the Death of Microorganisms . . . . . . . . . . . . . . . by Freezing . . . . . . . . . . . . . . . . . . . .

to Minimize Microbiological Activity . . . . . . . . . . . . a . Treatment before Freezing . . . . . . . . . . . . . . b . Defrosting . . . . . . . . . . . . . . . . . . . .

c . Influence of the Substratum on the Destruction of Microorganisms

2 . Treatment of Fruit and vegetable Products before and af ter Freezing

V I I I . Reduction in Microbial Spoilage by Removal of Water . . . . . . . 1 . Limits within Which Organisms Can Live in Highly Concentrated

Fruit and Vegetable Products . . . . . . . . . . . . . . . 2 . Possibilities of Reducing Microbial Spoilage in Concentrated Foods .

447

448

449 449 4.50 4.50

450 453

a . Preservation by Increasing the Concentration . . . . . . . . 453

429

430 MATKILDE VON SCHELHORN

b . Preservation of Highly Concentrated Fruit and Vegetable Products

c . Gas Treatment of Dried Fruit with Ethylene Oxide and Propylene Oxide . . . . . . . . . . . . . . . . . . . . . .

d . Protection of Dried Fruit and Vegetable Products against Water

by Heat Sterilization . . . . . . . . . . . . . . . . .

Absorption by Suitable Packing . . . . . . . . . . . . . IX . Preservation of Vegetables by Means of Lactic Acid Fermentation . . . X . Preservation of Fruit and Vegetable Products by the Addition of Chemi-

cals . . . . . . . . . . . . . . . . . . . . . . . . . 1 . Present State of Knowledge Regarding the Preservation of Fruit and

a . Acetic Acid . . . . . . . . . . . . . . . . . . . . b . Benzoie Acid . . . . . . . . . . . . . . . . . . . . c Derivatives of Benzoie Acid d . Combination of Benzoic Acid and I t s Derivatives with Other Pre-

servatives . . . . . . . . . . . . . . . . . . . . . e . Vanillie Acid Esters . . . . . . . . . . . . . . . . .

Vegetable Products by Various Chemical Preservatives . . . . . .

. . . . . . . . . . . . . . .

f . Sulfurous Acid . . . . . . . . . . . . . . . . . . . g . Other Proposals for Preservatives

ticular Groups of Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . .

2 . The Problem of the Efficiency of Individual Preservatives against Par-

3 . Comparison of Dosages Required with Different Preservatives . . . XI . Fungicidal Dips and Wrapping? for Fruit and Storage in Fungicidal

Gases . . . . . . . . . . . . . . . . . . . . . . . . 1 . Dip Treatments . . . . . . . . . . . . . . . . . . .

a . Sodium Tetraborate . . . . . . . . . . . . . . . . . b . Sodium Bicarbonate . . . . . . . . . . . . . . . . . c . Sodium Silicate . . . . . . . . . . . . . . . . . . . d . Sodium Orthophenylphenate (Dowicide A) . . . . . . . . . e . Na-chloro-2-phenylphenate (Dowicide C) . . . . . . . . . .

. . . . . . . a . Iodine . . . . . . . . . . . . . . . . . . . . . . b . Diphenyl . . . . . . . . . . . . . . . . . . . . . e . o-Phenyl-phenol d . Other Proposals and General Conclusions Regarding Fungicidal

Wrappings for Fruit . . . . . . . . . . . . . . . . . 3 . Fungicidal Gases for Treatment of Stored Fruit . . . . . . .

a . Ammonia . . . . . . . . . . . . . . . . . . . . . b . Sulfur Dioxide . . . . . . . . . . . . . . . . . . . c . Nitrogen Trichloride . . . . . . . . . . . . . . . . . d . Ozone . . . . . . . . . . . . . . . . . . . . . . e . Ethylene Oxide and Propylene Oxide . . . . . . . . . . .

XI1 . Storage of Fruit and Vegetables in Controlled Atmospheres without the Addition of Fungicides . . . . . . . . . . . . . . . . . . 1 . Composition of Atmosphere in Storerooms for Fruit and Vegetables

in Relation to Microbiological Spoilage . . . . . . . . . . . 2 . Erec t of Storage Temperature . . . . . . . . . . . . . . 3 . Relative Air Humidity in the Storeroom . . . . . . . . . . . 4 . Combination of Gas Storage with Other Processes . . . . . . .

. 2-Aminopyridine . . . . . . . . . . . . . . . . . . 2 . Impregnants for Fungicidal Wrappings for Fruit

455

457

457 45 7

458

458 458 458 459

459 460 460 462

462 462

463 463 463 464 464 464 464 464 464 464 465

465 465 465 465 465 465 466

466 467 468 468 469 469

CONTROL OF MICROORGANISMS CAUSING SPOILAQE 43 1

XIII. The Significance of Low Initial Infection and Clean Handling on the Life

XIV. Conclusions . . . . . . . . . . . . . . . . . . . . . . 471 References . . . . . . . . . . . . . . . . . . . . . . 472

of Fruit and Vegetable Products . . . . . . . . . . . . . . 470

I. INTRODUCTION

Spoilage of fruit and vegetable products can be caused by a wide variety of microorganisms including bacteria, moulds, and yeasts. The types of microorganisms that can damage any given product and which should therefore be destroyed or suppressed depend on the chemical composition of the product, primarily its acidity. Acid foods with a pH value less than 4.5, including most fruits, are attacked chiefly by yeasts and moulds. Among the bacteria, only acetic and lactic acid bacteria can tolerate high degrees of acidity. Spore-forming bacteria can propagate only in media with a p H value greater than 4.5 ; hence it is mainly the less acid vegetables that they threaten. B a d u s t h e m + acidurms which propagates as a spore-forming bacillus in the relatively acidic tomato juice is an exception.

pH value

IC acid bacteria utyric acid bacteria

Acid-insensitive Coli aerogenes types

Acid-sensitive Coli aerogenes types

Acid -insensitive spore-forming aerobes

Acid-insensitive putrefactive bacteria Acid-sensitive putrefoctive bacteria

c Moulds and film yeasts on surface of acid nutrients

FIO. 1. Limits of growth of microorganisms as affected by pH (after Rusch- mann, 1939).

Figure 1 illustrates the ranges of survival conditions of the various groups of microorganisms in relation to pH. Table I shows pH values of the pressed juices of a number of fruits and vegetables (taken mostly from Friese, 1941). From the writer’s own experience it can be said that these values are merely examples and not standards. The values in the figure, however, give an idea of which microorganisms can normally attack various fruit and vegetables in their natural state, i.e., when nothing has been added to the latter.

432 MATHILDE VON SCHELHORN

TABLE I

pH Values o f Some Vegetables and Fruits in Their Natural Condition

Vegetables PH t Tomatoes 4.5-4.9 Gherkins 5.8 Salsify 5.8 Carrots (short) 5.9 Asparagus 5.9 Red cabbage 5.9 Beans 6.0 Spinach 6.0 White cabbage 6.2 Savoy cabbage 6.3 Carrots 6.3 Peas 0.5 Brussel sprouts 6.6 Cauliflower 6.7 Kohlrabi 6.9 Sweet corn 7.3

* Friese, 1941. t Determinations made on the pressed juice.

Fruits Lemons Oranges Cranberries Red currants Plums Apples Black currants Blackberries Gooseberries Cherries Pears Strawberries Peaches Mandarins Bananas

PH t 2.4 2.8 2.8 3.0

3.24.6 3.3 3.3 3.3 3.3

3.3-4.7 3.4-4.7 3.54.2

3.6 4.6 5.2

Basically. the same principles of control are often employed for the microorganisms causing spoilage of fruit and vegetable products as are used for those affecting other foodstuffs. Much of the scientific knowledge gained, for example, with regard to the preservation of milk and meat products can be and is applied in the preservation of fruits and vegetables. Only the degree of the effects is often different, according to whether (as a result of the different substratum) the one group of microorganisms or the other constitutes a danger and must be suppressed; or whether the special properties of the substratum and the requirements of the consumer necessitate particular methods of treatment. The greatest resistance of microorganisms to methods of control is not associated with any single characteristic of them. Bacteria, for example, are difficult to control because of resistance to heat, whereas certain yeasts offer a problem because of their resistance to high osmotic values.

The object of this chapter is to discuss the scientific basis of the protection of fruit and vegetable products against microbiological spoilage. This includes a range of different problems, some closely interlinked, others involving widely diverse fields of natural science. All these methods (and their scientific bases) have in common the fact that they contribute to the protection of fruit and vegetable products against microscopic spoilage and hence to their preservation for human consump-

CONTROL OF MICROORQANISMS CAUSINQ SPOILAQE 433

tion. The enormous range of material is limited herein to fruit and vegetable products and to the avoidance of spoilage in these products. The scope of the paper is further restricted by omitting any discussion of the various microorganisms in foods that may affect human health but are not directly responsible for food spoilage.

Certain phases of the field to be discussed are already treated in the publications listed below :

The Canned Food Reference Manual. W. V. Cruess. Commercial Fruit and Vegetable Products, 3rd Edition. McGraw-Hill

Book Company, New York, 1948. E. C. McCulloch. Disinfection and Sterilization. Lea and Febinger, Philadelphia,

1945. Morris B. Jacobs. In-

terscience Publishers, New York, 1944. T. N. Morris. Chapman and Hall,

Ltd., London, 1946. F. W. Tanner. Garrard Press, Urbana,

Illinois, 1944. D. K. Tressler, M. A. Joslyn, and G. L. Marsh. Avi

Publ. Co., New York, 1939.

American Can Co., Maywood, Ill., 1947.

The Chemistry and Technology of Food and Food Products.

Principles of Fruit Preservation, 2nd Edition.

The Microbiology of Foods, 2nd Edition.

Fruit and Vegetable Juices.

These and other references that are quoted later in this paper have to a great extent already brought together the latest scientific developments on the various problems with which they deal so that it is possible in a number of places to give only a brief statement here and to devote more attention to points that have hitherto received less attention.

11. STERILIZATION OF F R U I T A N D VEGETABLE PRODUCTS

BY HEAT TREATMENTS

1. Pundamenta l Research on the Destruction b y Heat of Microorganisms Occwring in Pr td and Vegetable

Products

The fundamental investigations of Bigelow and Esty (1920), Esty and Meyer (1922), and Ball (1923), in particular, resulted in the development of the basic conceptions for the modern calculation of heat sterilization.

The various formulas, both as regards content and history, have been collected and discussed many times in other works, e.g., in “The Canned Food Reference Manual.” The treatment here will therefore be restricted to a short summary of those parts necessary to an understanding of the data that are given later, referring especially to fruit and vege- t abl cs .

The normal rate of destruction of bacteria by heat may be mathe-

434 MATHILDE VON SCEELHORN

matically described by the same formula as is used to describe the rate of a monomolecular reaction, namely

in which Co is the concentration (bacteria per unit volume) a t the be- ginning of the reaction, when time is zero, and C is the concentration after time t has elapsed (Viljoen, 1926; Ball, 1943; Rahn, 1945; Stumbo, 1948a, 1949a).

With the more highly organized forms of life the conditions become more complex and cannot be expressed by means of simple mathematical equations. A characteristic example of this among the microorganisms is provided by the moulds. Figure 2 shows rate of destruction curves

Minutes

Fro. 2. Typical destruction rate curves for various organisms (Williams el al.,

1. Penicillium sp. Ascospores at 81" C. (177.8" F.). 2. Putrefactive Anaerobe No. 3679 at 115" C. (239O F.). 3. Escherichia coli at 51.7' C. (125" F.). 4. Penicillium sp. Sclerotia at 90.5" C. (195O F,).

1r4 l ) .

for bacteria and also for ascospores and sclerotia of moulds (Williams et al., 1941). The sagging ascospore curve is indicative of a decreasing death rate. According to Rahn (1932) a curve of this type is charac- terestic of a system containing both sensitive and resistant cells. Upon exposure to heat the sensitive cells are killed off early, after which the rate is largely determined by the more resistant cells. The bulging

CONTROL OF MICROORGANISMS CAUSING SPOILAQE 435

sclerotia curve, indicative of an increasing death rate, is similar to the curves for higher organisms (Rahn, 1945). According to his interpreta- tion, such a curve pictures the death rate of organisms when destruction is the result of the reaction involving more than one molecule per cell.

The time for which a given temperature must be employed in order to kill a given number of microorganisms depends on the type of organism and on the nature of the medium containing them, e.g., on the pH and concentration. The temperature-time values that achieve equal effects can be shown graphically as a thermal death time curve. With logarithmic coordinates the "curve" is generally a straight line. With higher temperatures, other conditions being equal, the time required for killing the organisms is decreased. The number of degrees by which the sterilizing temperature must be raised in order to reduce the sterilizing time by one-tenth is designated as z. z indicates the slope of the thermal death time curve and is specific for the individual organism. Clostrid- ium botulinum in neutral phosphate buffer has a value for z of 10" C. (18' F.).

So far as the preservation of fruit and vegetable products is concerned, the data concerning the temperature conditions required for killing the relevant microorganisms are as yet rather incomplete. This is particularly so with regard to the' organisms occurring in acid substrates, i.e., true yeasts, film yeasts, moulds, and nonspore-forming bacteria. Further investigations in this field would be desirable.

a. Temperature-Time Relationship for Destruction of Microorganisms Occwring in Fruits and Vegetables. True yeasts and related organisms have relatively little resistance to heat. Table I1 gives some collected data on the relation between temperature and time on the destruction of fermentation yeasts. The table, which is based mainly on the experimental results obtained by Cruess and his co-workers, is taken from Tressler, Joslyn, and Marsh (1939). The juices to which the figures relate had a pH of between 3 and 4.

Useful conclusions on the destruction of yeasts by heat were also obtained by the work of Lund (1946). These investigations were, it is true, carried out on beer, but corresponding values have been obtained with fruit juices having a pH of 4. According to Lund's results species of Seccharmyces, Hansewla, Mycoderma, and Torula were killed within 15 minutes when heated in bottom fermentation beer a t 56' C. (132.8' F.).

The nonspore-forming bacteria that affect fruit and vegetable products are also relatively nonresistant to heat. Bigelow (1921) observed that most of the nonspore-forming bacteria found in raw fruits succumb in a few minutes to temperatures of about 60' C. (140" F.) in fruit


TABLE I1

Relationship between Temperature and Time for Destruction of Wine Yeast (After Tressler, Joslyn and Marsh, 1939)

Temperature Time for complete "C. O F . destruction (minutes) Medium

57.5 135.5 10 Grape juice * 56.5 133.5 20 1 6 l i

55.5 131.5 40 l t ' 1

54.7 130.5 60 i l 11

53.8 128.8 120 l i 11

62.0 143.6 59.0 138.2 56.0 132.8

1 5

15

62.8 145.0 2 57.0 134.6 10 55.9 132.6 20 54.8 130.6 40 54.2 129.5 60 53.3 127.9 120

* Aref and Cruess (1934). t Tracy (1932). f. Cruess, Aref, and Irish (1933).

Grape juice t ii L i

( 1 I (

Apple juice t 11 ( 1

I 1 1 1

6 1 i I

<i i l

11 11

syrups or in media comparable with fruit syrups. According to Lund (1946)) Acetobacter and Pediococcus, as well as Lactobacillus pasteur- {anus, were also killed within 15 minutes by heating to 56" C. (132.8" F.) ; while certain strains of the more heat-resistant Lactobacillus lind- l ter i withstood 20 minutes a t 58" C. (136.4' F.), but not a t 60" C. (140" F.).

Certain moulds are very heat-resistant. Tanner (1944) mentions Byssochlumys fulwa as being especially heat-resistant [based on the reports of Olliver and Smith (1933), and Olliver and Rendle (1934)]. The ascospores of this mould withstood 86"-88" C. (186.8'-190.4" F.) for 30 minutes in a number of fruit syrups. According to Williams et d. (1941) the sclerotia of a heat-resistant Penkillium withstood 93.3" C. (200" F.) for 9 minutes. Ruyle, Pearce, and Hays (1946) noted that the sclerotia of probably the same organism withstood 85" C. (185" F.) for several hours, and 87.8" C. (190" F.) f o r 1 hour.

Among the spore-forming bacteria there are definitely heat-resistant types. Fur the products with which we are dealing, they play a part only so far as the slightly acid vegetables are concerned. The standard forms introduced for determining the thermal processes of such substrata are Clostridium botulinum (Esty and Meyer, 1922) and Clos-

CONTROL OF MICROORGANISMS CAUSING SPOILAGE 437

tridium sporogenes 3679 (Williams, 1940). For various data on the heat-resistance of these forms reference should be made to ' ' The Canned Food Reference Manual."

In relatively acid media is found the spore-forming Bacillus thermoacidurans, the producer of flat-souring in tomatoes. Sognefest and Jackson (1947) state that the spores of this heat-resistant bacillus were killed in 0.73 to 1 minute a t 121" C. (249.8" F.) ; a t the boiling point they require 140 minutes. For the earlier literature on the destruction of Bacillus thermoacidurans by heat, reference may be made to Sognefest and Jackson (1947).

The question of the influence of the pH on the heat destruction of microorganisms is of decisive importance, particularly in the sterilization of f rui t products. It is therefore the more to be regretted that really methodical research on this problem is rarely undertaken, espccially in regard to yeasts and moulds.

Levine and Fellers (1940) compared the temperatures at which, with 10 minutes heating, various types of spore-forming and nonspore-forming organisms, Xaccharomyces ccrevkiae and Aspergillus niger, were killed by heat, in media a t various p H values. Unfortunately the selected p H values used (4.5 to 6.8) were too high, so that with Saccharomyces cerevisiae and Aspergillus niger no difference was observed between the tests a t the lower and higher p H values-unlike the tests made with bacteria. The reduction in lethal temperature was more marked with Bacillus mesentericus and Bacillus cereus than with the nonspore-forming organisms. The corresponding values are shown in Table 111.

Etchells and Jones (1943) investigated conditions during the pasteurization of pickled gherkins. They found that the strains of microorganisms used for the tests ( 1 strain of an acid-forming bacterium and 2 of yeasts) showed, with a treatment of 15 minutes a t 49" C. (120" F.) and 54.5" C. (130" F . ) , a definite correlation between the number of surviving acid-forming bacteria and yeasts and the acid contents of the liquors used. Results a t 60" C. (140" F.) and 65.5" C. (150" F.) indicated that the organisms added in inocula were killed in the most acid liquor and that some survived i n the two less acid liquors.

Investigations have more frequently been made on the influence of the pH on the heat destruction of spore-forming bacteria (Bigelow and Esty, 1920; Esty and Meyer, 1922; Sognefest e t al., 1948). I n all these investigations the results showed the common feature that in a n acid medium the test bacterial spores were more readily destroyed by heat.

c. Basic Data f o r Thermal Death Time Curves for the Organisms in Question. Very few data are available that can serve as a basis for the

b. Ififluence of p H on Heat Destruction Values.


TABLE 111

Edect of pH on the Thermal Death Point of Microorganisms

Lethal temperature t Organism

Salmonella aertrycke

Staphylococcus aureus

Phytomonas phaseoli

Bacillus cereus

Bacillus mesentericus

Saccharomyces cerevisiae

Aspergillus niger

PH 6.6 5.0 6.6 5.5 6.6 5.7 6.6 5.5 6.6 5.5 6.8 4.5 6.8 5.0 4.5

"C. O F .

55 131 50 122 65 149 60 140 55 131 50 122

100 212 60 140

100 212 60 140 60 140 60 140 60 140 60 140 60 140

* After Levine snd Fellers, 1940. t Exposure period 10 minutes.

construction of thermal death time curves for nonsporing bacteria, moulds, and yeasts, particularly in fruit and vegetable products. For the yeasts the values of z are 3.4-5' C. (6-9' F.) (Table 11).

Williams and his co-workers (1941) found thermal death time curves for the facultative anaerobic mould in blueberry juice to have a slope value ( z ) of about 5.85' C. (10.5' F.).

Spiegelberg (1940) reported data on thermal death times for acid- forming, nonsporulating bacteria in pineapple juice. According to these data the most resistant of the types investigated were killed within 1 minute a t 70.5' C. (158.9' F.), and within 10 minutes a t 64.5' C. (148.1" F.). A less resistant type was killed in 1 minute a t 60.5' C. (140.9' F.) and in 10 minutes a t 52.5' C. (126.5' F.). The value of z for the first organism is 6" C. (10.8' F. ) and for the second, 8' C. (14.4' F.). Figure 3 shows the thermal death time curves constructed by Williams and co-workers for heat-resistant ascospores and sclerotia of Penicillium sp. Figure 4 shows the curves given by Spiegelberg for acid-forming nonsporulating bacteria.

All these values of z are lower than the standard value z = 10' C. (18' F.) that was set up for CEostridium bdulinum in neutral phosphate. This means that, with fruit and tomato juices, the time required to achieve a given sterilizing effect decreases more rapidly with increase in temperature than is the case with foodstuffs in the neutral pH range.


I n general, i t is assumed (e.g., Ball, 1943) that the less heat-resistant microorganisms have smaller z values ; however, there does not appear to be any close correlation between heat resistance and a high z value. The strongly heat-resistant ascospores and sclerotia of the extremely heat- resistant moulds of the penicillia (used by Williams and co-workers in

1 . 1 . 1 . 1 . 1 . 1 . 1

1 96 92 88 04 80 76 72 Temperature, 'C.

FIG. 3. Thermal death time curves for sclerotia and ascospores of a heat-resistant mould of the type Penicillium (Williams et al., 1941) .

Sclerotia curve 1. z = 10.3. Ascospores curve 2. z = 10.6.

their investigations) have lower z values than the less heat-resistant nonsporulating bacteria that were employed by Spiegelberg. I n the latter case the lower values and steeper thermal death time curves were found for more heat-resistant types than fo r less heat-resistant types.

Even these few examples serve to show how little is known about the thermal death time curves of microorganisms other than the Clostrkikz, and how necessary i t is, to carry out systematic investigat,ions of the specific values for the members of the various groups of organisms, such as the fermentive yeasts, yeast-like organisms, moulds, nonspore-forming and spore-forming bacteria. It must also be assumed that the substrate has, in this respect, different effects on the various individual groups of organisms ; these relationships also need investigation.


Temperature, ‘C.

FIQ. 4. Thermal death time curves for single strains of nonspore-forming bacteria (after Spiegelberg, 1940).

A, B, C, 1, and 240 are individual strains of bacteria.

2. Practical Applications of Heat Sterilization of Fruit and Vegetable Products

The thermal death time curves for the microorganisms, under the con. ditions set by the product, form the basis for calculating the sterilization process that is to be used in practice. This basis must allow for the destruction of the most heat-resistant type of microorganism that occurs in the product in question. I n calculating the temperature-time values that must be employed, if greatest accuracy is to be attained, in the actual process for sterilization it is necessary to take into account the time-temperature relationships throughout the container of food (Stumbo, 1949a,b). In modern practice there is a general trend toward retaining better quality in the product by reducing the heating time- without, of course, any sacrifice of microbiological safety. This is possible by increasing the temperature in accordance with the relations that exist between sterilizing temperature and the corresponding necessary time.

a. Recent Developments in Sterilizing in Cans. It is not possible in this review to cover details of the various systems for the mathematical or graphical determination of the sterilization process. Reference must again be made to “The Canned Food Reference Manual,’’ and to the latest published works of Stumbo (1948a,b; 1949a,b) and Ball (1949) on questions relating to the region within the can in which the majority of survivors are found after heat treatment. Temperature distribution within the can is dependent on the size, shape, and makeup of the con-

CONTROL OF MICROORQANISMS CAUSINQ SPOILAGE 441

tainer, consistency and filling density of the contents, position of the can in the autoclave, i.e., whether standing o r lying, and on whether it is stationary or in motion during the sterilizing process (Roberts and Sognefest, 1947 ; Wilbur, 1949). The extreme instances of dependence of temperature distribution on the consistency and filling density of the contents are found in the cases of heat transfer by pure conduction and transfer by pure convection. The first case would include products of thick consistency, e.g., spinach, and sliced fruit ; the second would include fruit juices and perhaps brine-packed peas. Uniform temperature distribution is achieved most quickly as a result of convection, and most slowly by pure conduction, especially when there is no liquid to act as a conducting medium between solid substances, e.g., dried fruits, in which case the intervening air is a poor conductor of heat. Heat transfer takes place very quickly when the vacuum-pack process is used (Roberts and Sognefest, 1947) if the cans are in motion.

This process was developed mainly in the laboratories of the American Can Company during the last 15 years (Ball, 1936, 1943). The scientific basis for the modern high-short sterilization process depends (apart from the investigations by Bigelow, Esty and Meyer, Ball, and others) on investigations on the relationship between temperature and the acceleration of thermochemical processes.

The advantages of this process may be summarized as follows (Cour- tis, 1943) : The thermochemical processes are accelerated, so that all the "cooking effects, " including loss of aroma, caramelization, and destruction of the thiamin, are increased only two- or threefold by an increase of 10" C. (18" F.) in processing temperature. On the other hand, the time required for killing a given number of spores of the culture of Clostridium botdinum used as a test organism is reduced to one-tenth the original time when the temperature is increased 10" C. (18" F.). Thus, by increasing the temperature 30" C. (54" F.) i t is possible to achieve the same microbiological effect with only 2.7% of the cooking effect. Theoretically, the high-temperature short-time process can result in a smaller risk to the quality of the product and at the same time give a far higher safety from bacteria than with the older process. For example, when the temperature is raised 30" C. (54" F.), the heating time employed is three times longer than is necessary according to the thermal death time curves and yet quality destruction is only 8.1% of that occurring when the lower temperature for a minimum time period is used.

A good example of the progress represented by the introduction of this process is the possibility of destroying the heat-resistant spores of Bacillus thermoacidurans in tomato juice (Sognefest and Jackson, 1947).

71. High-Temperature Short-Time Sterilization.


The process has been a commonly used method in the United States since 1937 for the preservation of apple juice (Pedersen and Tressler, 1938 ; Marshall and Kremer, 1938 ; Marshall, 1947).

In order to achieve satisfactory results with the process it was necessary to solve the fairly difficult problem of sterile filling and avoidance of contamination a t this point. Sognefest and Jackson (1947) employ the following procedure: The tomato juice is heated rapidly to 122"- 138' C. (250"-280" F.) in heating coils, then held at this temperature for 0.7 minute. This process kills the heat-resistant Bacillus thermoacidurans. The juice is then cooled quickly to below the boiling point and is filled into cans at 88" C. (190" F.). This temperature is maintained for 3 minutes before cooling the cans with water. The 3 minutes' treatment at 88' C. (190' F.) is not sufficient to kill Bacillus thermoacidurans and is used only to destroy any other less resistant bacterial or mould spores that may enter the product during the filling process.

In the most up-to-date process, [the Pilot Unit process developed and patented by Martin (1949) ] , the product is enclosed and protected from outside air during its passage through the heating and cooling system to the filling apparatus. The process (Figure 5) includes the following stages : (1) sterilization of the product by flash heating in a tubular type heat-exchange system j (2) sterilization of the containers and covers with superheated steam; ( 3 ) aseptic filling of the cold sterile product into

Can

' .-.- ~ _ _ _ _ _ _.-..---.---

H Holding Tube I

I I I

Heating Cooling Section Section

ma. 5. Layout of high-short pasteurization (Martin, 1949).

CONTROL OF MICROORQANISMS CAUSINQ SPOILAGE 443

the sterile containers; (4) application of sterile covers to the filled containers and sealing in an atmosphere of either saturated or superheated steam.

For a long time, difficulty was experienced in constructing thermal death time curves for temperatures above 116" C. (240" F.), and such curves were necessary for high-short sterilization. Stumbo (1949b), however, designed a n apparatus by means of which these values could be determined u p to 131" C. (270" F.). Thus it is now possible to obtain thermal death time values in this higher temperature range,

111. EXCLUSION OF OXYGEN AS A SUPPLEMENT TO HEAT STERILIZATION

While the less heat-resistant yeasts are still able to cause fermentation when oxygen is excluded, the more heat-resistant moulds need oxygen for their development. Cruess (1933) stated explicitly that the investigations of his co-workers showed that i t was essential to kill off the yeasts by heat, while the moulds could be held in check by the exclusion of oxygen. I n 1938, Pedersen and Tressler pointed out that with the technics then available it was in practice impossible to sterilize apple juice to such an extent that all mould spores were killed without causing an adverse effect on flavor. They therefore recommended tha t the cans be filled completely and hence air be excluded to such a n extent that germination of mould spores cannot occur.

I n this connection, it is interesting to consider the amount of oxygen that will permit the development of moulds in canned foods, and how complete the removal of oxygen must be for protection against mould growth.

Williams e t al. (1941) report the occurrence of two strains of a heat- resistant Penicillium in cans of blueberries that had been considered sufficiently evacuated. According to these investigators, growth of ascospores was still possible in a vacuum of 28.5 in. (725 mm.) of mercury, and of sclerotia in a vacuum of 26 in. (660 mm.). Ruyle et al. (1946), who also investigated the behavior of these moulds, found that growth occurred even when the headspace was filled with an atmosphere consisting of 0.5% oxygen and 99.5% nitrogen (an oxygen partial pressure of 18 mm. of mercury).

I n 1949 Miller and Golding investigated the minimum amounts of oxygen necessary for the development of moulds. It was found tha t the inhibiting effect of reduced oxygen was in proportion to its solubility and not directly in proportion to the composition of the gas above the medium or mycelium. NO inhibition of growth of the culture investigated was noted until the dissolved oxygen was below 0.9 volume of


oxygen (at normal temperature and pressure) per 1,000 volumes of water. Complete inhibition of growth of any culture occurred in 7 days until the dissolved oxygen content was below 0.08 volume. The different' moulds tested varied considerably in respect to their oxygen requirements. Aspergillus niger, for instance, was inhibited significantly at approximately 0.56 volume of oxygen per 1,000 volumes of water and no germination occurred in 7 days at 0.01 volume of oxygen per 1,000 volumes of water.

The investigations of Miller and Golding (1949) which were carried out with organisms useful or injurious to milk products should be repeated with moulds that commonly develop on fruit and vegetable products.

IV. THE INHIBITORY EFFECT OF CARBON DIOXIDE ON

GERMINATION

The effects of carbon dioxide in inhibiting germination have long been known and are frequently used to advantage in the preservation of foodstuffs. For the effects of COz on moulds, reference is made to the work of Brown (1922) and Section XI1 of this paper which is concerned with gas storage of fruit. Ruyle e t d. (1946) found that the development of Penicillium spores was inhibited in atmospheres consisting of 10% oxygen and 90% carbon dioxide, and of 10% oxygen, 45% nitrogen, and 45% of carbon dioxide.

At this point it is worth noting the inhibitory effect of high concentrations of carbon dioxide on yeasts and the applications made of this fact in practice. The effect of carbon dioxide on fermentation yeasts was recognized by Delbruck as early as 1887. A process by which it was possible to preserve fruit juices with carbonic acid in suitable pressure containers was developed by the Swiss worker, Bohi in 1912. Bohi charged the juice coming from the presses with about 1.5% by weight of carbon dioxide. After sedimentation, clearing by filtration (some- times it was left cloudy), and the addition of further carbon dioxide, the juice was supplied to the consumer in pressure bottles. This procedure, however, had several weaknesses. The main problem was due to the fact that the carbon dioxide acted as an inhibitor and not a sterilizing agent. The process attained its present wide application only after the aforementioned COz treatment was combined with the sterile filtration process described in the next section. As a result of the increase in basic biological data (Schmitthenner, 1949), and physical data (Gatjen, 1937; Osterwalder and Jenny, 1939; Jenny, 1940), the storage of juice saturated with carbon dioxide has undergone many variations at the

CONTROL O F MICROORGANISMS CAUSING SPOILAQE 445

hands of technicians and manufacturers. Bohi 's fundamental idea, however, has been retained.

The essential feature of the carbon dioxide process is the complete inhibition of yeast growth by dissolving in the juice 1.5% by weight of carbon dioxide [7.7 atm. a t 15" C. (59' F.)]. Alcohol formation is not completely inhibited by the carbon dioxide. However, since it is dependent on the number of yeast cells present in the juice, it is possible to hold down the alcohol production by reducing the number of yeast cells to a minimum before storage. This is achieved by clarification accomplished through fermentation, fining, centrifuging, or filtration. After this, further inhibition of growth is effected by saturation with carbon dioxide. This is accomplished by use of an impregnating pump or a pressure separator and then storage in high-pressure tanks in which the pressure is maintained to preserve the COZ charge. After removal of the carbon dioxide, the juice is freed from any remaining fermentation agents by sterile filtration and is then put aseptically into sterilized bottles.

The Rueff and the Frank processes are based on the same principles.

V. STERILIZATION OF JUICES BY FILTRATION

The number of microorganisms removed from juices by filtration varies according to the fineness of the filter. This fact induced Schmitt- henner (1923) to construct an efficient and reliable asbestos-cellulose sterilization filter with pores so fine that the smallest microorganisms were retained. Sterile filter pads, such as the patented ones, produced by the Seitz factory a t Bad Kreuznach, may be used in any required number in a layer filter. The filter performance depends on the number of these layers employed. Because of the fineness of the pores only juices completely free of sediment and perfectly clear can be filtered. Unclear juices deposit so much residue on the filter layers that they soon become clogged. I n America, where cloudy juices are preferred, this process would not find wide usage. Marshall (1937) pointed out that this process does not cause enzyme destruction, hence the treated juice is unstable and likely to form objectionable precipitates, unless pre- heated.

In Europe, on the other hand, the process of filtration is widely used in connection with the Seitz-Bohi process described in the previous section.


VI. STERILIZATION O F F R U I T AND VEGETABLE PRODUCTS

BY ELECTRONICS, ULTRASONICS, ETC.

The use of various areas of the electromagnetic spectrum for the sterilization of foods is covered thoroughly by Proctor and Goldblatt in this volume in the chapter entitled " Electromagnetic Radiation Funda- mentals and Their Application in Food Technology." Therefore a discussion of this means of preservation is omitted from this particular review.

VII. PRESERVATION OF FRUIT AND VEGETABLE PRODUCTS AT Low TEMPERATURES

1. Behavior of Microorganisms at Low Temperatures a. Temperature Limits of Activi ty for Ba,cteria, Yeasts, and Moulds

That Can Cause Spoilage in Fruit and Vegetables. It has been shown by Haines (1931), that many organisms have the ability to grow at -1" to 0" C. (30"-32' F . ) , a smaller number a t -3.9" C. (25" F.) and a few forms at temperatures as low as -6.6" C. (20" F.). Thorough investigations have been made by Berry and his co-workers during the past fifteen years on the growth of microorganisms at subfreezing temperatures. Berry and Magoon (1934) reported that Pseudomoms fEuo- rescens and species of Lactobacillus, Torula, Monilia, and Penicillium grow at -4' C. (24.8" F.). Growth of Cladosporium and Xporotrichum species was observed at -6.6" C. (20" F.) and some bacteria a t -3.9" C . (25" F.). All these organisms were isolated from both fruits and vegetables. Diehl e t al. (1934) stated that certain moulds grow slowly at about -6.6" C . (20" F.). It is clear that moulds are particularly resistant to low temperatures (Berry, 1934a) ; they are able to grow even at -6.6" C. (20" F.). Russian investigators have also come to the conclusion that moulds can develop a t temperatures lower than any other type of microorganism (Tschistjakow and Botscharowa, 1938 ; Panassenko and Tatarenko, 1940). It has not yet been established which type of mould is the most resistant to low temperatures. Since the problems of cold resistance are closely connected with osmophilic char- acters it would be worth while investigating whether or not osmophilic cultures of Aspergillus glaucus are very resistant to low temperatures. Smart (1935) found that fermentation yeasts develop slowly on agar at -8.89" C. (16" F.). In the case of yeasts as for A. glauaus it would be interesting to investigate the relationship between resistance to low temperatures and adaptability to high concentrations of sugar. An investigation of species of Zygwucchnromyces would be also of particular interest.

CONTROL OF MICROORGANISMS CAUSINQ SPOILAGE 447

The experiments made so far show that growth and multiplication of microorganisms are possible at temperatures of -9.5" C. (15' F.). At appreciably lower temperatures there is a very considerable reduction in the count, but not complete sterilization. According t o Magoon (1932) individual microorganisms can even survive temperatures of - 252" C. (-421.6' F.) for several hours.

Tanner and Williamson (1928) record that certain types of yeast were still alive after freezing a t - 15' C. (5' F.) for 160 weeks. Accord- ing to Berry (1937b) some parts of the bacterial population of vegetables were still alive after 4 years of storage at -18' C. (-0.4" F.) ; Smart (1935) records that numerous species of bacteria and several species of moulds and yeasts survived three years' storage at -9.5" C. (15" F.).

Berry and Diehl (1934) found that in frozen apple juice stored a t temperatures of between -7" and -21' C. (19.4" and 5.8" F.) the count was reduced by 90 to 96% after one month's storage. From then onwards it decreased much more slowly, although after 10 months' storage the apple juice had not decreased to zero. According to ljochhead and Jones (1936) the reduction during the first few weeks is particularly marked with vegetables, while with fruit the reduction takes place more gradu- ally. I n considering this fact it must be remembered that we are dealing with populations of organisms. The less resistant types are killed off during the first few weeks, whereas the more resistant organisms remain alive for months or even years. It would appear that, with pure lines of single-celled microorganisms, death by cold, like death by heat, follows the law of monomolecular chemical reactions (Tanner and Williamson, 1928 ; Stille, 1943).

b. Influence of the Freezing Temperature and Rate of Freezing on the Death of Microorganisms. I n numerous investigations i t has been observed that a greater number of organisms are killed a t a few degrees below 0" C. (32" F.) than when the temperature is much lower. A number of such findings are set out in Table IV.

Gasbelein (1940) attempted to explain this phenomenon on the basis of coarse ice crystals a t -4" C. (24.8" F.) and their tendency to cause a maximum disruption of the cells at this temperature. Another possibility suggested by the investigations of Haines (1938) is the occurrence of a more pronounced flocculation of proteins in microorganisms at temperatures slightly below 0' C. (32" F.) than at lower temperatures.

A question closely related to that just discussed is whether freezing is rapid or slow. Luyet and Gehenio (1940) stated that, during rapid freezing, the water in the cells is not changed to .ice crystals, but to a glasslike amorphous mass which results in less injury to the cells than

Not all microorganisms are so resistant to cold.


TABLE IV

Mortality of Microorganisms at High and Low Freezing Temperatures

Substratum

Low Temperature

High Organisms destroyed temperature

Duration of ~ . - destroyed __

storage "C. ( O F . ) TO 00. ( O F . ) "lu Strawberries in 4 sucrose solution * months -10 (14) 89 -20 (-4) 60

Blueberries in 5070 9 sugar syrup t months - 6.7 (20) 99.9 -17.8 (0) 60

Blackberries in 5070 24 sugar syrup t weeks -10 (14) 99 -20 (-4.0) 75

* Berry, 1933s. t Smart, 1939. $ McFarlane, 1940b.

when freezing a t lower rates. Injury to cells by ice crystals during the process of slower freezing would result in lower bacterial counts. Van Eseltine e t al. (1948) came t o the conclusion, in respect of vegetables, that freezing very rapidly in liquid air does not materially change the bacterial count of the product. This does not mean, however, that slower freezing produces a greater reduction in the number of organisms than rapid freezing at lower temperatures. One impoftant factor is the possibility of organisms multiplying for a period of time before a sufficiently low temperature to destroy or inhibit them is reached. Further- more, experiments such as those of Hess (1934) (quoted by Heiss, 1939), show that there is a considerably greater reduction in living salt water bacteria when freezing is rapid rather than slow. An explanation for this is that with rapid freezing the bacteria are enclosed between ice crystals, while with slow freezing they are found mainly in the unfrozen residual solution. A similar situation would be expected to occur when substrata rich in sugar are frozen.

c. Influence of the Xubstratum on the Destructiolz of Microorganisms b y Freezing. McFarlane (1941, 1942) investigated the factors favoring the survival of yeasts a t low temperatures. He found that 30 to 50% sucrose solutions retarded the destruction of the cells a t subfreezing temperatures. The observations made by McFarlane are in agreement with the modern views on the cell physiology of yeasts (discussed more fully in the section on osmophilic yeasts). The greater the concentration of the nutrient medium, the greater is the concentration in the yeast cell and the smaller is the amount of water in the cell freezing at a given temperature. McFarlane 's experiments appeared to show that very high concentrations of sugar seemed to favor the destruction of yeast cells at low temperatures. This can probably be explained by the use,

CONTROL O F MICROORGANISMS CAUSING SPOILAGE 449

in these experiments, of yeasts that were not particularly osmophilic and that had a limited tolerance t o sugar.

I n McFarlane's experiments (1942) a reaction of p l I 6.5 was found to be more favorable than a reaction of p H 5 for hastening the destruction of Xaccharomyces. I n both Xaccharomyces and Escherichia coli the mortality of frozen cells was greater a t pH 3.6 to 3.7 than at higher pH values. Stille (1943) also stated that in his tests with yeasts more cells were killed by cold a t low pH values. These and similar findings must be contrasted with the fact that a t an optimum pH the microorganisms can readily tolerate extremely high osmotic values.

I n any case it would be valuable to have further studies of the cell physiology problems relating to this field,

2. T r e a t m e n t of Fruit a n d Vegetable Products before and af ter Freezirtg t o Minimize Microbio logkd A c t i v i t y

Berry and his co-workers, Haines, and others have concluded that since freezing does not sterilize the product, the latter should have a very low bacterial count before freezing. Berry (1946), for instance, remarked on the importance of getting fruit into the freezer as soon as possible after harvesting. The maximum degree of cleanliness is important in all preparatory steps. Pedersen (1947) discussed a number of possible sources of bacterial contamination of vegetables prior to freezing.

Obold and Hutchings (1947) indicated that fruits and vegetables, after packing and before freezing, should not be stored for longer than 24 hours a t 4.5" C. (40" F.), 5 hours a t 10' C. (50" F.), or 2 hours a t 26.6' C. (80" F.). I n the case of berries, which are frozen in large containers, there is danger of fermentation occurring in the innermost parts of the mass before the entire mass freezes. Ireland (1941) recommended that the fruit be precooled before barrelling for freezing and that the barrels be rolled frequently when in the freezer.

Vegetables are, of course, blanched before freezing, and this kills a large proportion of the microorganisms occurring on them. According to Diehl e t al. (1936) as many as 99% of the organisms on peas may be destroyed by blanching.

Care must be taken, however, that during the precooling or handling prior to freezing reinfection does not occur. This could develop very rapidly in the open tissues produced by blanching. According to Proctor (1948) and Proctor and Nickerson (1948) this danger can be avoided entirely by employing simultaneous blanching and sterilization by radar irradiation at 3,000 megacycles for a period of 30 seconds or

a. Treatment before Freezing.


less. The vegetables then can be frozen in the same containers immediately after irradiation.

The open and shattered cells of thawed fruits and vegetables permit rapid growth of microorganisms. It is therefore essential that frozen fruit and vegetables be thawed as quickly as possible and only immediately before being used. Here again electronic heating offers new possibilities of application (Sherman, 1946 ; Cathcart and Parker, 1946 ; Bartholomew, 1948).

b . Defrosting.

VIII. REDUCTION IN MICROBIAL SPOILAGE BY

REMOVAL OF WATER Control of microorganisms in foodstuffs by decreasing thd r moisture

content may be achieved by the following processes: (1) drying fruits and vegetables, (2) condensing juices and syrups, with o r without addition of sugar, (3) production of jams and fruit jellies with a high sugar content. For a recent summary of the technical details of these processes reference may be made to Cruess (1948). Here we shall discuss specifically the microbiological problems relating to these processes.

1. Limits witlain Which Organisms Can Live in Highly Concentrated Fruit and Vegetable Products

Tanner (1944) has given a number of references (mainly from earlier literature) on this subject. These provide most of the data and argu- ments considered here.

Richter (1912) introduced the term “osmophilic” for microorganisys that are adapted to substrates extremely low in moisture content. Only foodstuffs that cannot be attacked by these osmophilic organisms are free from moulding or fermentation.

A measure of the concentration of a substrate in relation to the possible development on it, of microbial colonies, is its osmotic value (Walter, 1931), or vapor pressure which, of course, is related to it. Vapor pressure is generally measured and expressed as the relative humidity of the air with which the substrate is in equilibrium.

I n order to ascertain the extent of water removal providing certain protection against microbial deterioration, i t is necessary to obtain a clear picture of the limits of activity of microorganisms with respect t o the osmotic value of their substrates.

Bacteria are in general not well adapted to live in substrates of high osmotic values. An example of an osmophilic bacterium, Bacillus sac- ch,arolyticus n.sp., was described by Nepomnayachyaya and Liberman (1938). This form can propagate in jams with 50 to 60% sugar content,


which, according to the present authors' calculation, corresponds to about 85% relative humidity.

Detailed studies have been made of the limiting concentrations for moulds, and on the growth of moulds in relation to moisture content (Heintzeler, 1939). The mould best adapted to low moisture condition has been recognized as being Aspergillus glaucus and A. repens (Klebs, 1928). Stille (1942) gave a relative humidity of 70% as the lower limit for spore germination of Aspergillus glaucus, with 74% as the limit for spore formation ; in 1948 he found the limit for spore formation under optimum temperature conditions of 30" C. (86" F.) to be 73% relative humidity. The present writer's own investigations have demonstrated the relationship between the limiting concentration for Aspergillus glaucus and the pH value of the substrate. Aspergillus glaucus be- longs to the basophilic (neutrophilic) moulds (Delitzsch, 1944). The writer's investigations showed that with strongly acid substrates, e.g., syrups from acid berries o r fruits, the limit for the formation of macro- scopically visible moulds was higher than with neutral or slightly acid substrates (in low concentrations), Samples of syrup (73% relative humidity equilibrium r.h.) a t pH 7 were inoculated with spores of Aspergillus glaucus; these were covered with a visible mould growth within two months. Fruit syrups of pH 5 under the same conditions and temperature [30° C. (86" F.)] developed a covering of mould within 9 months. These investigations also showed that the absolute limit for mould growth on slightly acid to neutral concentrates occurred when the substratum was in equilibrium with air a t 72 to 73% relative humidity. With acid fruit concentrates of p H 3, on the other hand, no mould growth was observed a t concentrations less than the equivalent of 77 to 78% relative humidity. Jams, fruit jellies, and dried fruits are therefore the better protected against mould growth the lower the p H value of the product.

The group of microorganisms that can tolerate the lowest moisture content in foodstuffs are the osmophilic yeasts. Particularly detailed investigations have been made of these yeasts by Lochhead and co-workers (Lochhead and Farrel, 1931 ; Lochhead, 1934 ; Lochhead, 1942) in Can- ada and by Kromer, Krumbholz, and co-workers (Kromer and Krumb- holz, 1931, 1932; Krumbholz, 1931a,b, 1936, 1939) in Germany. These investigators found that the highest osmotic values and lowest moisture contents could be tolerated better by yeasts of the genus Zygosaccharo- myces (which are definitely small-celled yeasts) than by any other microorganism living in foodstuffs. Lochhead's investigations were carried out mainly on fermenting honey, but the results obtained are applicable to jams and other products. Kromer and Krumbholz (1931, 1932) dealt


with the osmophilic yeasts in the course of investigations on dry fruit pressing in wine production.

Curl e t al. (1946) and also Ingram (1949) studied fermentation in concentrated orange juice of 65" Brix by osmophilic yeasts. According to Mrak (1940) and Mrak et al. (1942), osmophilic yeasts are the cause of the souring of dried dates. I n these investigations, Zygosaccharo- rnyces again showed themselves to be particularly osmophilic. Accord- ing to Fellers and Clague (1942) souring of dates by osmophilic yeasts occurs when the water content of the dates is higher than 25%. It is rather striking and, in the present author's opinion, worth further study, that Fellers and Clague (1942) found cultures of Torula (Tom- lopsis ?) and Willia (Hamsenela?) but none of Zygosaccharomyces. Mrak and Stadtman (1946) gave further references to the literature on the souring of dates and other alterations occurring in dried fruit by osmophilic yeasts.

Detailed investigations have been made relative to the concentration which inhibits the propagation and life of extremely osmophilic yeasts such as Zygosaccharomyces. Kromer and Krumbholz (1931) stated that the growth of certain types of yeasts was prevented only by 90% of sugar in the substrate (90 g. of sugar in 100 ml. of HzO, which equals in per cent weight about 68% of sugar and a relative humidity of 78%). According to the present author's own investigations (Schelhorn, 1950) the limit for an extremely osmophilic yeast, namely Zygosacchuromyces barkeri, under optimum conditions of about pH 5, occurs a t a sugar concentration in equilibrium with about 61.5% relative humidity. This corresponds, for example, to pear concentrate containing 82% solids, Frui t syrup will ferment up to such a concentration, although at a very slow rate. Fermentation in highly concentrated substrates is helped by the presence of other conditions that favor the development of yeasts, for instance higher temperatures and optimum pH. The pregent author's experiments agreed with those of Hjorth-Hansen (1939) in giving an optimum pH value in the region of 4 t o 5 for Saccharomyces cerevisiae and Saccharomyces ellipsoideus. Thus slightly acid jams and jellies ferment most readily, whereas definitely acid or neutral products ferment less easily.

It was observed that osmophilic yeasts in substrates too concentrated for propagation did not die immediately but remained alive for weeks or even months. This also applies to spore-forming bacteria and moulds. Thus it is readily understandable that living microorganisms have been found on dried fruit and vegetables (for fruits see Fellers, 1930; Clague and Fellers, 1933; for fruit and vegetables, see Jones, 1943).

In this connection salt tolerance is also of interest. Joslyn and Cruess


(1929) made a detailed study of yeasts in pickle brine. A number of types were able to propagate even with a salt content of 19 to 20%. In connection with these observations i t is interesting to note that an NaCl solution that is saturated at 20" C., i.e., a 26.4% solution, is in equilibrium with air a t a relative humidity of 75% (Obermiller, 1924). Hence, the occurrence of yeasts in strong salt pickle brine agrees with the investigations on the limits under which osmophilic yeasts can occur in substances that are very rich in sugar. I n these investigations the salt tolerance of the strains investigated also depended on pH, as may be seen in Figure 6. The individual strains (curves 1, 10, 13, and 14 in

pH value

FIQ. 6. Effect of pH 011 salt tolerance of film-forming yeasts (Joslyn and Cruess, 1929).

the figure) obviously behaved differently. In the investigations of Joslyn and Cruess (1929) , however, the interval from p H 3.2 to p H 5 is too wide; it would be better to have a determination on the optimum pI-1 range between pH 4 and 5, since it is in this pH range that the greatest salt tolerance is to be expected.

2. Possibilities of Reducing Microbial Spoilage in Concentrated Poods

a. Preservation by Increasing the Concentration. Figure 7 shows the present author's own data on the relation between dry matter content and relative humidity for apple juice concentrate a t 20" C. (68" F). These sorption isotherms were formulated in accordance with the method


described by Kaess (1949). Similar curves for other fruit juice eon- centrates correspond almost exactly to this curve. Mould growth has been found to occur, under optimum conditions, down to 72 to 73% relative humidity, and for yeasts down to 61% ; thus i t is apparent that complete preservation of fruit concentrates is practically impossible. This agrees with the finding of Heid (1943) that citrus concentrates with more than 20% moisture content will not keep.

Relative humidity, %

FIQ. 7. Sorption isotherms for apple concentrate at 20" C. (68" I?.) (Schelhorn, 1950b).

The keeping quality of fruit juices like those of jellies and jams is enhanced by an increase in dry matter content. Thus, for a long time it has been an established practice to increase concentration by adding sugar. According to Tarkow e t al. (1942) the addition of glucose is more effective, pound for pound, in preservation than is sucrose because the former is in greater molecular concentration. The amount of glucose that can be added for sweetening fruit juice, however, is limited, since its solubility is comparatively low. Mixtures of glucose and sucrose such as a combination of invert sugar and sucrose, or fructose and sucrose, are more soluble. Hence Heiss and Schachinger (1949) have proposed the use of a mixture of several sugars for sweetening fruit concentrates, in order to obtain higher concentrations and far greater protection against microbial spoilage without the risk of crystallization. I n practice, problems relating to the amount of sugar that can be taken up by fruit concentrates are important with respect to concentrates obtained by use of the freezing process. It is known that this process is par-

CONTROL OF MICROORGANISMS CAUSINQ SPOILAQE 455

ticularly suitable for concentrating fruit juices with the least loss of aroma. At present, however, it is not possible with this process to achieve a higher concentration than about 60% of solids. Such products are not safe microbiologically, but by addition of sugar their keeping properties can be increased without sterilization or the addition of preservatives.

With properly cooked jams and jellies, the danger of fermentation by osmophilic yeasts is not great, since these organisms are killed during heating and later contaminants grow very slowly, if a t all. Frui t jellies, which according to Grover (1947) are in equilibrium with air a t a relative humidity of 59 to 76% and have an average p H value of 3, are not subject to attack by moulds. Jams are in equilibrium with air a t a relative humidity of 75 to 8270, and therefore come in the range within which mould growth is possible. If the jars are not sterilized again after being filled, there is a danger that when the jars are closed before the jam is cooled the upper layer will be covered by condensed water and will provide a particularly favorable condition for mould growth. Thus in the German jam industry it is common for the filled jars and cans to be allowed to cool while still open ; in this way a surface skin is formed, which on account of its high osmotic value does not permit the development of mould. As an additional precaution a cover paper impregnated with a fungicide may be used to seal the jar.

b. Preservation of Highly Concentrated Frui t and Vegetable Products by Heat Xteriliza.tiow I n the sterilization of highly concentrated products it must be remembered that the resistance of microorganisms to temperature increases with rising osmotic pressure in the substratum [Owen, 1914; Rahn, 1928 (cited by Tanner, 1944) ; Krumbholz, 19361. The high osmotic value of the substrate gives a certain protection against low temperatures for the remaining microorganisms, just as it does against heat. I n the case of yeasts both of these effects are connected with the cytorrhysis, that is to say, with the fact that the cells become reduced in size with a rising concentration of the surrounding medium, and the osmotic value in the interior of the cell is increased (Beetlestone, 1930; Windisch and Enders, 1946). The conditions with other types of microorganisms require closer investigation in relation to cell physi-

The rate of heat penetration into the substrate being sterilized decreases with increasing viscosity of the medium (Irish et al., 1928). In view of the protective effect of higher concentrations on the microorganisms to be killed, it may be concluded that higher time-temperature values are necessary for sterilizing concentrates than for the plain juice. On the other hand, the sterilization of concentrated juices may be easier

ology.

456 MATHILDE VON GCEIELHORN

in view of the fact that the osmophilic Aspergillus types are less resistant to heat than the Penicillium types, while the latter are less able to tolerate high concentrations of sugar. Planned investigations on this point have not been carried out.

Experiments on “pasteurization” (perhaps better called sterilization) have been carried out by Fellers (1930), and also by Clague and Fellers (1933). Working with dried dates, they found that the lower the moisture content of the fruit the slower the heat penetration. They gave data regarding the time-temperature value necessary to achieve sterilization for different moisture contents of the dates, but gave no exact relationship between the water content of the fruit, temperature, and sterilization time.

FIQ. 8. 1949).

CONTROL O F MICROORQANISMS CAUSING SPOILAQE 457

c . Gas Treatment of Dried Fruit with Ethylene Oxide and Propylene Oxide. This method of destroying organisms on fruit will be discussed in Section X I ( 3 ) .

d. Protection of Dried Fruit and Vegetable Products agluinst Water Absorptirm. by Suitable Packing. When the surrounding air has a relative humidity greater than the equilibrium value for the product, highly concentrated foodstuffs draw moisture from it and may become liable to spoilage by microorganisms.

Figure 8 shows a series of absorption isotherms for dried vegetables. The curves were constructed by Heiss (1949). The present author has drawn in the ordinate a t a relative air humidity of 72.5%, which according to our investigations marks the limit for the possibility of mould growth on these vegetable products. Analogous curves could and should be provided for dried fruit, particular attention being paid to the limit for the propagation of osmophilic yeasts, which occurs a t 61% relative air humidity.

I n general, permissible limit for the moisture content of dried vegetables from the standpoint of microbial growth is considerably higher than that which is permissible if changes are to be minimized. I n certain dried vegetables the texture suffers a t the low moisture content. I n such cases recourse may be made to cold storage (which delays the growth of microorganisms as well as chemical changes) or to gas treatment with ethylene oxide in the container.

Since in many areas the atmosphere is of sufficiently high humidity to enable dried fruits and vegetables to absorb moisture rapidly, protective packaging must be used. The scientific basis for packing foodstuffs sensitive to moisture has been investigated by Heiss (1949).

IX. PRESERVATION OF VEGETABLES BY MEANS OF LACTIC ACID FERMENTATION

Although the use of lactic acid fermentation is one of the oldest methods for the preservation of vegetables, it is only in recent years that a scientific knowledge has been gained of the processes involved, thus making it qossible to develop proper controls. It has been found that several types of microorganisms (particularly lactobacilli and fermenting yeasts) are responsible for certain of the changes (at least aroma) that take place in vegetables preserved by this means. These organisms must act in the right succession in order to obtain a usable product. For the fermentation to follow the desired course, optimum conditions of temperature and composition (particularly sugar and salt) are essential. It is only by providing these optimum conditions for the fermentation process that failures such as butyric acid fermentation and excessive


yeast growth can be avoided. The addition of chemical preservatives is not desired. Innoculation with pure cultures of lactic acid bacteria such as the so-called starters are regarded of doubtful value by Pederson and Kelly (1938) and Pederson (1949). The exclusion of air plays a great part in the success of the fermentation, since this enables keeping moulds under control. The film-forming yeasts have a high oxygen demand and are often found on the surface of vats containing pickles. As with other processes, clean raw materials and containers play an important role. Fabian and Faville (1949), for example, describe a case of pickle spoilage caused by an infection of Oospora lactis resulting from the use of old barrels.

Comprehensive reviews on the question of lactic acid fermentation of vegetables and of the associated problems are available in the technical literature. It is therefore appropriate to keep the present review of this subject brief and to refer the reader to the contributions of Cruess (1948) and Fabian (see Jacobs, 1944). Reference should also be made to the work of Etchells et d . (1947) and to the collection “Abstracts of Papers Presented at the Technical School for Pickle and Kraut Pack- ers, ” Michigan State College, 1949.

X. PRESERVATION OF FRUIT AND VEQETABLE PRODUCTS BY THE ADDITION OF CHEMICALS

In Volume I of ‘‘Advances in Food Research,” 0. Wyss (1948) gave a thorough review of microbial inhibition by food preservatives. Here, therefore, it is necessary to review only the latest knowledge and applications of chemicals used for preserving frui t and vegetable products and to make a few general suggestions regarding the effectiveness of certain preservatives.

1. Present State of Knowledge Regarding the Preserva- tion of Fruit and Vegetable Products by Various

Chemicd Preservatives

a. Acetic Acid. Levine and Fellers (1940), and Morse, Fellers, and Levine (1948) investigated the preservative effect of acetic acid. Their results confirmed the findings of earlier authors that the lethal effect of acetic acid is not due to the hydrogen ion activity alone, but also seems to be a function of the undissociated acetic acid molecule. In comparisons with lactic acid, hydrochloric acid, and citric acid, the inhibitive or lethal effect of acetic acid on Salmonella aertrycke, Sac- charomyces cerevisiae, and Aspergillw niger was, even at high p H values, as great as those of the other acids.

b. Benzoic Acid. Benzoic acid and its salts still comprise one of the

CONTROL O F MICROORQANISMS CAUSING SPOILAQE 459

most widely used group of chemical agents for preserving fruit and vegetable products. I n many countries, including the United States and Great Britain, the only preservatives officially approved for such pur- poses are benzoic acid, sodium benzoate, and sulfur dioxide. Since benzoic acid is effective only in the unionized form (Cruess and Richert, 1929; Cruess et al., 1931; Cruess and Irish, 1932; Sabalitschka and Marx, 1947), it is particularly suitable for use with acid types of fruit.

The methyl, ethyl, and propyl esters of paraoxybenzoic acid were introduced as preservatives by Sabalitschka about 1926. Serger (1929a,b, 1935) recommends these esters as suit-able for use with fruit products. The esters axe stated to be less injurious to health than benzoic acid (Sabalitschka, 1939). Further information on this question is given by Heim and Poe (1948), and Bleyer e t ul. (1933). The latter investigators were concerned with the effects of preservative agents on the digestive enzymes.

As far as the paraoxybenzoic esters are concerned, Liese (1933) claimed that they are the most suitable preservatives for foodstuffs and are superior to benzoic acid. The present writer’s own investigations confirm the observation that in certain cases (particularly for neutral o r slightly acid foods) these esters have a greater preservative effect than benzoic acid.

The pure esters are difficultly soluble in water but their solubility is increased by adding alkaline metals in the parachydrosy group (Saba- litschka, 1939). Recently calcium, instead of sodium, was introduced into this group. For further information regarding the composition and trade names of the various preparations based on these esters, reference may be made to a publication by Jacobs (1944). I n Germany, use is also made of the parachlorbenzoic acid and its sodium salts. Their use is permitted as preservatives in fruit products without the necessity of declaration on the label, provided they are employed in small quantities. According to the manufacturer, the effect.iveness of parachlorbenzoic acid is even more dependent on strongly acid media than is benzoic acid.

d. Combination of Benzoic Acid and Its Derivatives with Other Pre- servatives. The question as to whether and how far it is desirable to combine the use of benzoic acid and its derivatives with other preservatives is one on which there is disagreement. A large number of such combinations have been put on the market under a wide range of fancy names and are a t present in use. Those who use these combinations claim that a number of preservatives used together will reinforce one another and so effect a saving in the quantity required. Such a reduction in the quantity of benzoic acid used as a preservative for fruit and vegetable products is certainly desirable, since all these preservatives, in

c . Derivatives of Befizoic Acid.


spite of statements to the contrary, are undesirable so far as human health is concerned. The guiding principle in estimating the value of these combinations of preservatives must therefore be to the effect, that a combination is preferred only when the sum of the value of the function (Quantity of preservative) x (Injury to health) is smaller than the value of the same function for each of the materials used alone (Schelhorn, 1950a). From the discussions in the literature on benzoic acid and from the present writer’s own investigations, i t may be said that, from this point of view, there is a great deal in favor of the commercially produced combinations of benzoic acid and benzoate. These combinations may include substances which are in themselves not preservatives but which lower the p H value, eg., combinations of benzoic acid and benzoate with citric acid or tartaric acid. Such additions often result in a saving of benzoic acid. The use of formic acid along with benzoic acid o r benzoate also makes possible a saving in benzoic acid. It is often claimed that combinations of benzoic acid with esters of paraoxybenzoic acid and their alkali compounds are to be recommended as effecting a saving in the total amount of preservatives used. The present writer’s own investigations show that this is a very complicated problem requiring a thorough investigation.

e. Vmillic Acid Esters. Sabalitschka and Dietz (1931) found that the vanillic acid esters were good inhibitors of glucose fermentation. Pearl and McCoy (1945) tested their effects on Aspergillus niger, Aero- bacter aerogenes, and Bacillus mycoides and came to the conclusion that vanillic acid esters can be classed as good preservatives for fruit juices and that they should be considered for legal use in foods. According to Evans and Curran (1948) the isobutyl ester is particularly effective. A drawback is the noticeable flavor of the product. f. Sulfurcncs Acid. Sulfurous acid is, like benzoic acid, one of the

most commonly used preservatives for fruit pulps and similar products. Like benzoic acid and its salts, sulfurous acid and its salts axe effective

only in acid media, a point which was observed as long ago as 1914, by the Swiss workers Miiller-Thurgau and Osterwalder. Cruess, Richert, and Irish (1931) hinted that the antiseptic properties might be con- fined to undissociated H&03 ions or to molecular SOz and be absent in SO; ions.

The range of application and effectiveness of sulfurous acid is affected by still another factor-that it forms reaction products with the alde- hydes and sugars present in fruit juices. For this reason it seems scarcely possible, by adding sulfurous acid, to prevent further fermentation of a juice in which fermentation has already begun, since the added acid might be rendered ineffective by the formation of reaction products


with the acetaldehydes resulting from the initial fermentation. This latter observation was made as early as 1914 by Swiss workers, i.e., Miiller- Thurgau and Osterwalder (1914). Rosenfeld (according to Feigenbaum and Israelashvili, 1949) describes in a British patent a method of desulfur- iiig fruit juices preserved with gaseous sulfur dioxide by use of acetalde- hyde, pelargonic aldehyde, citral, and capric aldehyde. Downer (1943), investigating the preservative qualities of free and bound sulfur dioxide, found that 55% combined with the reducing sugar of the concentrated juice. According to Vas and Ingram (1949) the greater part of the added sulfurous acid for the preservation of fruit concentrate becomes ineff ective, i.e., to the extent that SO: is formed. Only the relatively small proportion of undissociated, free sulfur dioxide is microbiologically effective. It is, however, possible to increase this effective proportion by acidifying the products with acids (which alone are not preservatives), such as hydrochloric acid, tartaric acid, and citric acid. (See Figure 9.) According to Vas and Ingram, lowering the pH value also delays the formation of reaction products between the sulfur dioxide and the sugar. "The combination of sulfur dioxide with sugar, etc. is greatly delayed by increasing the acidity. A decrease of only 0.3 pH unit roughly halves

A3 a B2 5 10 15

r l L I r I L

Days at 25.C

FIQ. 9. Growth curve of yeasts, at 25" C. (86" F.) in the presence of varioi amounts of SO, and at different pH values (Vaa and Ingram, 1949).

pH 3.40 A 0 mg. SO,/1. pH 2.88 B, 0 mg. SO,/]. A l , 90 B1, 90 A2, 270 B2, 270 A3, 560 B3, 560


the rate of combination.’’ On the other hand Feigenbaum and Israelash- vili (1949) came to a different conclusion, which they set out in a series of tables : “The percentage of sulfur dioxide combined with glucose increases with decreasing pH.” It is clear that the question of the effect of the pH on the formation of reaction products between sugar and sulfur dioxide needs further investigation.

These come and go in large numbers, but experience has shown that only a few of such preparations prove to be useful. Hence we will refrain here from describing all the preparations, since the critical evaluation of them would require long years of research. Mention may, however, be made of vitamin Kg (Prat t e t al., 1948), since this is completely noninjurious to human health.

g . Other Proposals for Preservatives.

2. The Problem of the Efficacy of Individual Preservatives against Particular Groups of Microorganisms

I n numerous references scattered throughout the literature it is re- oorted that commonly used preservatives such as sulfurous acid, benzoic acid and its derivatives, and formic acid are particularly effective against certain groups of microorganisms (bacteria, moulds, fermenta- tive and other types of yeasts), but are less effective against other groups of agents causing microbiological spoilage of fruit and vegetable products. These various references will not, however, be quoted here, since the results reported conflict so much and it appears that the observations reported were not always the results of careful investigations. A thorough investigation, on a comparative basis, of these commonly used preservatives is yet to be made. This might be a promising field for investigation ; however, the development of physical methods for preservation and the continuous development of new preservatives would seem to make such a proposed study rather superfluous.

3. Comparison of Dosages Required with Different Preservatives

It is a fundamental fact that the best preservative is not the one which will preserve foodstuffs, when present in the smallest quantities, for consideration must be given to possible injury to health. Thus Sabalitschka (1939) states that chlorobenzoic acid is in fact a stronger preservative than benzoic acid, but that its toxicity to humans is also greater. The best chemical preservative is the one in which the quantity required gives the least injury to health. This problem, however, can only be solved after really detailed investigations have been made of the comparative effects of the individual preservatives on human health.


A good comparison of the effectiveness of the individual preservatives in common use for the preservation of fruit and vegetable products is given in Table 5, which is taken from the latest proposals for legal requirements for preservatives in Western Germany. In this table it may be assumed that the maximum legal permissible quantity of the individual preparations would be that which is just sufficient to provide the preservative effect.

TABLE v Permissible Quantities of Chemical Preservatives in the West German Federation

Maximum permissible amount of preservative (mg. per 100 9 . )

Foodstuff

Liquor for gherkins, beets, and tomatoes

Grated horseradish

Fruit juices and fruit pulps, in so far as they are to be used for f urtlier processing

Paraoxy- Benzoic benzoic acid or esters or sodium their sodium

benzoate compounds

200 90

125 75

150 90

Formic Sulfurous acid acid 150

(mixed with - 50 of

benzoic acid)

250 125

XI. FUNGICIDAL DIPS AND WRAPPINGS FOR FRUIT AND

STORAGE IN FUNGICIDAL GASES

Scientific investigations that can be considered more than mere empiri- cal searches for suitable preparations, are scarcely to be found at all. Reference must therefore be made again to Wyss (1948) in Volume I of “Advances in Food R.esearch.” Discussion here will be restricted to a short treatment of the individual means suggested for fruit and vegetables.

1 . Dip Treatments

a. Sodium Tetraborate. Robson (1935) stated that a satisfactory treatment for citrus fruits involves immersion for several minutes in a hot 5% solution of sodium tetraborate. This treatment is said to be excellent against green mould attack (Penicillium digdatum) but less satisfactory against blue mould (PeniciZlium i tdicum) .


The effect of borates is increased by raising the pH of the dip (Tom- kins, 1937). A 27. borate solution to which 1% of caustic soda is added is twice as effective as a 5740 borax solution (Fidler and Tomkins, 1938; Furlong and Tomkins, 1931, cited by Furlong, 1948). Ryall and God- frey (1948) recommend a combination of dipping in a 5% sodium tetraborate solution and treatment with nitrogen trichloride. The gas was used in concentrations of 0.03 to 0.04% for 2 to 4 periods of approximately 4 hours each.

b . Sodium Bicarbonate. According to Robson (1935) a 3% solution of sodium bicarbonate is eff ective, particularly against blue mould.

c. Sodium Silicate. A 2740 solution of this substance also was found to be effective by Robson (1935).

d. Sodium Orthophemylphenate (Dowicide A ) . Miller, Winston, and Meckstroth (1944) found, during investigations of a series of substances for surface treatment of citrus fruits, Na-orthophenylphenate (Dowicide A) to be one of the most promising. Its use as a disinfectant, however, was limited because it tended to cause rind injury. This difficulty was overcome by adding formaldehyde to the solution [ (1 past of formaldehyde solution to 5 parts of Dowicide A when lemons were treated at 43.5' C. (110" F.), and 1 : 1.5 when oranges were treated at 38' C. (100" F. ) 1. Recommended concentrations of Na-orthophenylphenate ranged from 1.25 to 2.0%.

The following may be reported from an English work by English, Wright, and Smith (1948). As the result of an investigation of about 60 compounds for control of pear and apple storage rots (particularly blue mould, gray mould, and lenticell rots) it was found that 0.4% Na-chloro-2-phenylphenate in the final rinse water o r 1.2% in Na-silicate wash solution were effective. It was suggested that use in the silicate solution is preferable, since with this procedure there is less likelihood of causing injury.

Winston, Meckstroth, and Roberts (1947) recommend the use of 2-amjnopyridine as a 5% solution or incorporated in a wax emulsion for control of spoilage in oranges.

e. Na-chloro-2-phenylphenate (Dowicide C).

f. 2-aminopyridine.

2. Impregnants for Fungicidal Wrappings for Fruit

a. Zodine. Tomkins (1934, 1936) tested numerous substances for their suitability as impregnants for wrapping papers for fruit. Among the chemicals investigated the only one, besides diphenyl, to prove satisfactory was iodine. A more comprehensive review of the literature and developments for these impregnants for fruit wrappers has been made by Manley (1948). According to this writer iodine impregnants have proved particularly successful, especially for the transport of f rui t

CONTROL OF MICROORQANISMS CAUSING SPOILAQE 465

overseas. Oranges, tomatoes, grapes, peaches, cherries, passion fruit, and melons are benefited by iodine impregnated wraps. Losses due to the moulds are reduced 50-80%. Application is in the form of iodine- impregnated paper, wood shavings, cork dust, or cotton wool. The success with iodine suggested the organic compounds and derivatives containing iodine as suitable mould inhibitors. Iodoform and numerous complex chemicals mere tested but with little success, for they usually have strong odors, are much more volatile, more costly, and necessitate the use of expensive solvents for impregnation.

This substance was found by Tomkins (1934, 1936) to be equal to iodine as a fungicide for fruit wrappings and was introduced by him for that purpose.

c. o-Phenyl-phenol. This substance is also a suitable impregnant for fruit wrappings and mas also introduced by Tomkins (1937). The amount of this impregnant in the paper, however, must be limited or the fruit will be damaged.

d . Other Proposals and General Conclusions Regarding Fungicidal Wrappings for Fruit. Manley (1948) listed a number of other preservatives which have been tested in fungicidal wrappings for fruit. I n his opinion, however, iodine is superior for most fruits. Tomkins (1945) concluded with respect to these impregnants : “None of the effective substances has been found to be ideal in every respect. Color, odor and possible conflict with regulations concerning preservatives in food have constituted problems. ”

b . Diphenyl.

3. Pungicidal Gases for Treatment of Stored Fruit

a. Ammonia. Tomkins and Trout (1931) recommend the use of ammonia for gas treatment of citrus fruits for the control of green rot (Penicillium digitatum). On the other hand Robson (1935) considered the effects of ammonia treatment of citrus fruits inadvisable because of the development of undesirable skin blemishes at certain concentrations (higher than those necessary to kill spores) and the possibility of affecting flavor of the juice. Tomkins (1933) obtained good results when tomatoes were treated with ammonia, but similar tests with grapes were unsuccessful.

b. Sulfur Dioxide. Pryor (1949) reported that gas treatment with sulfur dioxide was particularly successful with grapes.

c. Nitrogen Trichloride. Robson (1935) considered this gas to be promising for gas treatment of citrus fruits. Ryall and Godfrey (1948) recommended the use of nitrogen trichloride in conjunction with a sodium tetraborate wash.

Ac- d . Ozone. It is difficult to kill moulds completely with ozone.


cording to Heise (1917) (cited by Heiss, 1939) the mycelium of Penic3- lium glaucum and Mucw stolonifer are severely damaged and in part destroyed by ozone at concentrations ranging from 2.7 to 27 mg./m.3. On the other hand the spores of this fungus were resistant even to high ozone concentrations. In the experiments of Heiling and Scupin (1935) ozone treatment provided a marked inhibition, but not absolute suppres- sion, of Penicdliuna glaucum. According to Kaess (1940) ozone is very strongly absorbed by foods treated and therefore microorganisms situ- ated immediately beneath the surface are killed and hence the foodstuffs are protected. The use of stronger doses of ozone is not possible because of physiological damage to the fruit. This is dependent to some extent on the type of fruit. Hence the experiments on the preservation of stored fruit by use of ozone have often not been entirely satisfactory. A number of references to this effect are quoted from the literature by Heiss (1939). It is stated that ozone treatment for stored fruit gave only moderately successful results. According to Schomer and McColloch (1948) ozone used in apple storage in concentrations up to three parts per million in the atmosphere did not control decay of apples and did not reduce infection of inoculated wounds. It did, however, retard the rate of mould spreading in the inoculated areas. Reports by Ewe11 (1938) that strawberries and grapes could be preserved fresh twice as long at an ozone concentration of 3 mg./m.3 as they could without ozone are considered by Heiss (1939) to be open to doubt. I n any event, only a surface mould growth can be controlled by ozone treatment, and the effect on decay progressing from the interior outwards must be regarded as unsatisfactory.

These substances are used for st6rilization of dried fruits (Mrak, 1941; Whelton et al., 1946; Mrak and Stadtman, 1946). These writers.deny any injury to health: “If one assumes that the ethylene oxide present in packages of dried fruits is converted to ethylene glycol it would be necessary to ingest 1,000 pounds of treated fruit to obtain the amount reported lethal.” Luh (1949) describes the use of epoxides for prolonging the keeping time of fresh fruit. It appears, however, that an investigation would be desirable to establish whether or not the latter application introduces too much glycol into the human system.

e. Ethylene Oxide and Propylene Oxide.

XII. STORAGE OF FRUIT AND VEQETABLES IN CONTROLLED ATMOSPHERES WITHOUT THE ADDITION OF FUNQICIDES

The storage of fruit and vegetables in atmospheres of specific compo- sitions has grown during the last twenty years into a science of its own.


The leading part in this development has been played by the Low Tem- perature Research Station a t Cambridge, England.

By far the greatest number of the investigations have been directed to the physiological behavior of the stored fruit and vegetables. The microbiological problems have been often touched on only incidentally. The present review, on the other hand, aims a t bringing the microbiological aspects of the problems into the foreground. For technical and physiological details not given here, references may be made to the comprehensive review by Heiss (1939).

1. Composition of Atmosphere in Storerooms for Fruit and Vegetables in Relation to Microbiological Spoilage

Kidd and West (1936, 1949) carried out a comprehensive series of experiments on the storage of apples and pears in atmospheres of specific composition. The essential nature of these experiments may be summarized as follows. The transpiration and ripening processes in stored fruit can be considerably retarded by maintaining the oxygen content of the storage atmosphere low and the COZ content high. Since ripening fruit is in general less liable to attack by agents of decay than overripe fruit, the microbiological spoilage of fruit is limited by retarding the ripening process. Numerous experiments (Brown, 1922 ; see also Sec- tions I11 and IV) have shown that oxygen deficiency hinders the development of moulds; carbon dioxide in high concentrations restrains the growth of numerous microorganisms. Unfortunately the work of Kidd and West and others shows that it was impossible in storerooms for apples and pears, to keep the oxygen content of the air sufficiently low and the carbon dioxide sufficiently high as to achieve both a noticeable reduction in the growth of moulds and avoidance of abnormal ripening. In the absence of oxygen, it was found that the respiration of the stored fruit and vegetables became intercellular, and breakdown products such as alcohol and aldehyde were thereby formed. Thus, if stored in atmospheres too low in oxygen or too high in carbon dioxide the fruit was reduced in quality as a result of physiological disturbances. Hence i t was found necessary to retain a certain proportion of oxygen in the atmosphere and not to raise the carbon dioxide content to an excessively high level. It was found that the proportion of oxygen and, in fact, the constitution of the storage atmosphere as a whole, had to be varied with the varieties of apples and pears used. For certain of the varieties the optimum oxygen content of the air was 2.5%, while for others it was much higher (up to 21%). The optimum carbon dioxide content ex- ceeded 10% only in a few cases. Similar observations with respect to composition of atmosphere and microbial control on different varieties


of apples and pears have been made by American, Canadian, Australian, and German investigators (Allen and McKimmon, 1935 ; Smock and van Doren, 1938, 1941 ; Fisher, 1942 ; Plagge, 1943 ; Kaess, 1943 ; Huelin and Tindale, 1947).

There has not been very much planned quantitative investigation of the oxygen and carbon dioxide contents of atmospheres that permit, restrict or inhibit the growth of individual types of mould. Brown (1922) found that inhibition of Botrytis cinerea did not occur until the oxygen content of the atmosphere was less than 1%. From the investigations referred to in Sections I11 and IV, it is certain, however, that many moulds are capable of growth at very low oxygen tensions and that high concentrations of carbon dioxide are necessary to obtain any appre- ciable control of mould growth. Thus it may be considered an established fact that, under the conditions existing during gas storage of apples and pears, the control of mould growth is mainly a secondary effect resulting from delayed ripening of the fruit.

The prospect of successfully storing other fruits and vegetables in gas depend mainly on the physiological characteristics of these products and whether or not they can tolerate storage in atmospheres low in oxygen or high in carbon dioxide. Emblick (1938) stored fruits and vegetables in nitrogen atmospheres low in oxygen content. He found that practically all the types of fruits and vegetables tested suffered marked deterioration in quality when the oxygen content of the atmosphere was reduced sufficiently to reduce appreciably mould growth, The only reasonably satisfactory results with storage a t low-oxygen atmospheres were obtained with egg plums and blackberries.

A high concentration of carbon dioxide can give good results when the purpose is to secure a very short period of protection, for example, during rail transport of berry fruits to the place of storage. Kaloyereas (1949) recommended the use of atmospheres high in carbon dioxide (conveniently provided by dry ice) for the transportation of peaches. This provided good protection against microbial spoilage without im- pairment of quality.

2. Effect of Xtorage Temperature

It has already been mentioned that moulds can grow even at temperatures below O* C. (32' F.). Among the most resistant to cold are Fusi- clladium dendriticum (which causes storage deterioration in apples) and Gloeosporiurn album and fructigenum, and Botrytis cinerea (Krumb- holz, 1939). Investigations on the most favorable storage temperature for particular fruits and vegetables have shown that because of the physiological nature of these products it is impossible to keep the tem-

CONTROL OF MICROORQANISMS CAUSING SPOILAQE 469

perature so low that this factor alone will make the development of moulds impossible ; unless freezing storage, which causes marked changes in structure of the tissues, is employed (Heiss, 1939). Many fruits undergo undesirable physiological changes when the storage temperature is too low. Furthermore, aroma development in many fruits is inhibited in cold storage. In any case the temperature of the storeroom, when direct freezing is not being used, should he lowered only to such a point that (allowing for local variations and temporary fluctuations in temperature) the cell juices of the stored fruit or vegetables will a t no time be below their freezing temperature.

3. Relative Air Humidity in the Storeroom

In choosing the desirable relative humidity for storage Kidd and West again distinguish between requiremenhs to inhibit microorganisms and those for preservation of the fruit in the best possible condition. For microbiological inhibition the relative humidity must be kept as low as possible. Immediately surrounding fruits and vegetables the vapor pressure approaches saturation, a condition which f avors rapid growth of moulds. On the other hand, if the vapor pressure in the storeroom is kept too low, the stored goods soon begin to shrivel. Thus i t is necessary to make a compromise and, by employing lower humidity, to achieve a reduction of losses from fungal rotting a t the cost of increased loss of weight (Heiss, 1939). The optimum values for different fruits and vegetables lie between 80 and 95% relative humidity. Detailed data concerning these relationships are given in a review by Heiss (1939). A recent and inexpensive method of keeping air humidity constant in storerooms is the Sapak-Leicester process (Faure and Chouard, 1948 ; Leicester, 1949). Heiss (1940) investigated the humidity conditions in the interior of boxes of apples. I n normal conditions the relative humidity was not constant in a cross section of the box. On the other hand, a t a low relative humidity in the room, the humidity was lowest in the upper layers and highest in the center of the box. The humidity gradient depends on the permeability to air of the container, the air velocity in the room, the closeness of stacking, and resistance to air movement of the stored fruit.

4. Combination of Gas Storage with Other Processes

As mentioned above, as a result of limits set by the nature of the products to be stored and because of the great adaptability and resistance of moulds, neither ordinary cold storage nor gas storage alone are sufficient to control fungal decay. Better results were achieved by a combination of both processes, i.e., combined gas and cold storage.

470 YATHILDE VON SCHELHORN

Within certain limits improvement can be effected by supplementary processes such as ultraviolet irradiation, ozone treatment, and treatment with fungicidal gases. Finally it must be remembered that for successful storage only perfect fruit and vegetables should be used and that certain pre-harvest measures must be taken against conditions that may be caused by microbiological agents during storage. I n any case the problem of protecting fresh frui t and vegetables with certainty against microbiological losses during storage cannot be considered as solved. It remains to be seen to what extent new methods, e.g., high-velocity elec- tron streams, will prove themselves in practice.

XIII . THE SIGNIFICANCE OF Low INITIAL INFECTION AND

CLEAN HANDLINQ ON THE LIFE OF FRUIT AND

VEGETABLE PRODUCTS

In the previous discussions it has been mentioned several times that the effectiveness of the various methods of preventing microbial spoilage and the effort necessary to achieve this depend on the amount of original infection present in the product.

On the one hand, there is the destruction of microorganisms by heat, radiant energy, low temperatures, and by use of certain poisons. The extent to which these are used vary according to the extent of infection. The lower the initial infection, the sooner will be the desired reduction in count achieved.

On the other hand, when for certain reasons complete destruction of the microorganisms cannot be achieved, the survivors will multiply and cause spoilage. This is more rapid when the initial number of organisms is greater or the more they have increased during handling of the products.

It was for this reason that in the discussion of the control of microbiological spoilage by freezing, great significance was attached to a low initial infection.

I n fruit juice concentrates rich in sugar, fermentation is slower when fewer osmophilic yeasts are present initially.

I n preservation by the use of a lactic acid fermentation it is essential that the materials employed should be, in so fa r as possible, free from undesirable microorganisms ; otherwise these will suppress the lactic acid bacteria.

With respect to chemical preservatives, it was pointed out in the discussion on sulfurous acid that substrates in which fermentation has already started cannot be protected by the addition of this preservative. Opinion is still divided in respect to other preservatives. Liese (1933) carried out experiments to determine the extent to which the preserva-


tive effects of benzoic acid, sodium benzoate, p-chlorbenzoic acid, and p-oxybenzoic acid esters were dependent on the number of microorganisms. He observed that: “ I f in one test, the samples were inoculated heavily, and in another series the samples were so inoculated as to give at the most ten germs to each sample, it was found that in general no difference could be observed in the concentration required to deal with the weaker inoculation against that required to deal with the stronger. l 1

This observation may be accepted within certain limits but must not be generalized too widely.

Ae Stumbo (1947) stated, it is essential in carrying out the preservation process that the number of microorganisms present be small and that the control measures taken should attack the organisms in their lag phase. The latter requirement applies both to physical and to chemical methods of preservation (Tamiya et d., 1947).

The necessity for sanitary handling of the raw products from the fields until they reach the factory is, from the scientific point of view, a factor that comes under this heading. I n this respect, those organisms which get on to the raw materials in particles of dust are not as danger- ous as infection from contaminated plant parts. Contamina.tion of apparatus in the factory with residues of the product may also be very troublesome. The microorganisms depositing on foods in particles of dust, etc., are in a mature condition and hence their lag phase until reproduction starts anew. ‘This might require quite a period of time; on the other hand, the organisms living in contaminated parts of the equipment are in optimum condition of growth and are therefore ready to continue propagation immediately.

Although numerous references dealing with cleanliness in food processing have been given, it is pertinent that a few more recent ones be given here: Vaughn and Stadtman (1946), Berry (1947), Vaughn et al. (1948), Oilespy (1948), Cruess (1948), Kraska (1949).

XIV. CONCLUSIONS

In 1933 Galloway published a paper in which he covered in broad out- line similar points of view to those underlying the present paper. A number of ideas and discoveries are given in this present paper that were not available to GaIIoway (1933). I f these are considered, one obtains an idea of the progress of research and technical development that has been made during the last decade and a half. Nevertheless the above review will certainly give the impression that there is a great deal of need for further research. Sterilization by electrical methods is still in the experimental stage, and its future use in the control of microbial spoilage in fruit and vegetable products cannot be foreseen


with certainty. So far as chemical methods of preservation of foodstuffs are concerned, the progress made during the last 15 years is not so great and so satisfactory as in the field of preservation by physical methods. There is, in particular, a special need for preseryatives suitable for use in slightly acid t o neutral products.

Chemical preservatives can be considered fully satisfactory only when they are fully effective against injurious microorganisms, but have no ill effect on human health. The latter point includes effects on the necessary intestinal microflora as well as digestive enzymes. The problem of preservation of fresh, untreated fruit and vegetables has not been solved by the use of such procedures as storage in special atmospheres and fungicidal washes and wraps. In many cases there is a recognizable trend toward combined processes, i.e., a process using supplementary treatment in support of a treatment which does not otherwise produce the desired effect with the necessary degree of certainty o r without involving qualitative damage. A general example of this is provided by the many methods for reducing the initial degree of infection such as the use of combined storage under gas a t low temperatures.

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