Chlorine in Food Plant Sanitation

Chlorine in Food Plant Sanitation

BY WALTER A. MERCER

AND

IRA I. SOMERS

Western Research Laboratory, National Canners Association, Berkeley, Calijornia Page I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 11. Historical Review. 130 111. The Germicidal Ac ne . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 1. Mechanism of Microbial Death from Chlorine.. . . . . . . . . . . . . . . . . . . . . 133 134 2. The Germicidal Agent in Chlorine Solutions. . . . . . . . . . . . . . . . . . . . . . . 3. The Pattern of Bacterial Death from Chlorine.. . . . . . . . . . . . . . . . . . . . 136 4. Evaluation of the Germicidal Activity of Chlorine Solutions. . . . . . . . . 140 IV. The Application of Chlorine in Food Plant Sanitation.. . . . . . . . . . . . . . . . . . 141 1. Definition of Chlorination Terms.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 . a. Available Chlorine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 b. Chlorine Dosage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 c. Chlorine Demand.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 d. Total Residual Chlorine.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 e. Free Available Chlorine.. . . . . . . . . . . . . . . . . . . . 143 f. Combined Availab e..... . . . . . . . . . . . . . . . . . 143 g. Marginal Chlorina . . . . . . . . . . . . . . . . . 143 h. Break-Point Chlor .................................. 143 2. Chlorine Compounds Commonly Used.. . . . . . . . . . . . . . . . . . . . . . . . . . . 145 . . . . . . . . 145 c.

Chloramine Compounds. . . .

a. In-Plant Chlorination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 b. Chlorination of Can-Cooling Waters.. ........................ 158 c. Chlorination of Waters Reused for Purposes Other V. The Effects of Food Plant Chlorination.. . . . . . . . . . . . . . . . . . . 1. Effect of Chlorination on Plant Sanitation. . . . . . . . . . . . . 2. Effect of Chlorination on Containers and Equipment. . . . 3. Effect of Chlorination on Quality of Foods., . . . . . . . . . . . VI. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

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I. INTRODUCTION Chlorine compounds, correctly used in food plant sanitation, have proved to be safe and dependable germicides. The addition of chlorine to waters used for washing and conveying raw food products, for cleansing food-handling equipment, and for cooling heat-sterilized cans of food has been of inestimable esthetic and practical value in food processing. The ability of chlorine in water solution, even in trace amounts, to destroy microorganisms has made possible longer periods of continuous plant operation under conditions which have enabled the food processor to prepare and package foods which meet the highest standards of sanitation.

11. HISTORICAL REVIEW Chlorine compounds were used long before the element itself was discovered in 1774 by Scheele, who named it ‘ I dephlogisticated muriatic acid.” In 1785, Berthollet, because of the method used in preparing chlorine, considered it a compound of hydrochloric acid and oxygen. He called it “ oxygenized muriatic acid.” Sir Humphry Davy, in 1810, proved that chlorine was an element and gave it the name by which it is known today. The value of chlorine in preventing disease was recognized before the germ theory of disease was established and before the cause of fermentation and decay was known. I n 1846, Semmelweis, using chloride of lime, succeeded in eradicating puerperal fever from his medical clinic. Koch, in 1881, made the first investigation of the bactericidal properties of hypochlorites. Five years later, the American Public Health Association issued a report favorable to the use of hypochlorite solutions as disinfectants. Chlorine compounds began to be widely used as disinfectants during World War I when Dakin’s Solution (Dakin, 1915) was introduced for the irrigation-disinfection of wounds. The irritating action of this hypochlorite solution on living tissue caused Dakin and his associates (1916, 1917) to search for other chlorine-bearing compounds which would be effective as germicides yet would not harm tissue. p-Toluene sulfonchloramide, now known as chloramine-T, was their final choice. As early as 1850, chlorinated lime was used for treating water, and in 1854 a report by the first Royal Sewage Commission of Great Britain referred t o the use of chlorinated lime as a deodorant in London sewage (Phelps, 1909). It is probable, however, that Traube (1894) first focused attention on the disinfecting properties of chlorine compounds when added to water supplies. The first attempt in North America t o purify water by chlorination was that by Johnson (1911). He reported very satisfactory results when 1.5 parts per million chlorine was added to the

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effluent from the Bubbly Creek Filter Plant in Chicago. These results led to the first permanent chlorination installation in this country, when, in 1908, hypochlorites were added to the Boonton, New Jersey, water supply of Jersey City. From this time, the use of hypochlorites for water purification rapidly increased and by 1911 it was estimated that 800,000,000 gal. of water per day were being chlorinated. I n 1913, Darnall completed development of equipment for chlorinating water supplies by the introduction of gaseous chlorine. Today unchlorinated municipal water is rarely found. Among food processors the dairy industry was the forerunner in utilizing the germicidal and deodorant properties of chlorine. I n 1912, Whittaker and Mohler referred to the use of calcium hypochlorite as a sanitizer for milk bottles. Subsequently, many others studied and reported on the uses of chlorine compounds in cleansing dairy farm and milk plant equipment. Prucha (1927) and Loveless (1934) were among the early workers to compare and report on the effectiveness of various methods of applying chlorine solutions to milk-handling equipment, Johns (1930, 1934), Myers (1930), and others studied the comparative germicidal efficiency of the different chlorine products available for use in dairy sanitation. I n 1939, the United States Milk Ordinance and Code recommended chlorine as one of the agents for the bactericidal treatment of milk equipment between each usage. The use of chlorine in the canning, freezing, and dehydrating of foods began with its addition to the water used for washing and rinsing equipment during routine cleaning periods. I n 1931 (Scott, 1937) the canning industry began to experiment with the addition of chlorine to water used for cooling heat-sterilized cans. Can spoilage caused by aspiration through apparently normal seams of minute amounts of contaminated cooling water was an annoying and costly problem. The results of experimental chlorination (Merrill et al., 1938) of can-cooling waters soon demonstrated that this “leaker ” spoilage could be drastically reduced by chlorine. Development of the principles of “ break-point ” chlorination (Griffin, 1946) indicated the possibility of more extensive use of chlorine in food plants. I n 1946, Hall and Blundell reported on the beneficial results obtained by chlorination beyond the “break-point ” of the general water supply for a pea cannery and a vegetable freezing plant. This method of chlorination became known as “in-plant chlorination.” It provided a continuous application of germicidal chlorine to the food preparation equipment during its operation. Critical surveys (Zuch and Somers, 1946; Vaughn and Stadtman, 1946) of the results obtained showed that the use of chlorinated water sprays a t selected points on the preparation

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lines greatly reduced or prevented the accumulation of microbial slimes. Washing and conveying the raw product in chlorinated water resulted in much lower bacterial counts in the finished product. Odors of fermentation were avoided and the time required for satisfactory plant cleaning shortened. Generally, the food processing industry approached with considerable caution the widespread use of in-plant chlorination (Cameron, 1939). Additional information was needed on (1) the possibility of chlorine causing off-flavors in the product, ( 2 ) the effect of chlorine on metal equipment, and (3) the concentrations and the costs of chlorine required to maintain satisfactory sanitary conditions. In 1946 and 1947 several in-plant chlorination installations were made. Geographically, these installations were widespread and in plants handling a considerable variety of fruits and vegetables. The reported results from the use of chlorine in these plants served as a basis for a preliminary evaluation of in-plant chlorination. Conclusions regarding the advantages of this method of chlorination were set forth in a conference in December 1946 (Ritchell, 1947) a t which various food processors, the National Canners Association, and a manufacturer of chlorinating equipment were represented. The conclusions reached by this conference are virtually unchanged today. Essentially they were as follows: 1. The use of chlorine prevents or greatly reduces the accumulation of microbial slimes on all equipment surfaces which are continuously or frequently washed with chlorinated water. Odors due to fermentation and decay are prevented. 9. Use of chlorinated water permits longer hours of operation by reducing the time required for cleanup. 3. Total bacteria counts on the finished product are reduced if the raw product is washed in chlorinated water and conveyed over preparation lines bathed with chlorinated water. Q. No apparent corrosion of metal equipment occurs from continuous contact with water having a chlorine content normally used in food plant operation. 6. Chlorine must not be applied indiscriminately. The following precautions should be taken: (a) It must be determined that the flavor of the product will not be adversely affected by chlorine; (b) fruit canning sirups should not be made with chlorinated water; (c) compounds containing phenol or related chemicals should not be present in a plant using chlorinated water; (d) frequent tests should be made of the chlorine concentration in the water; (e) standard industrial safety measures should be observed in the use of chlorine.

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These conclusions formed on the basis of preliminary studies and observations were confirmed by a succession of studies carried out under varied conditions of food plant operation (Harris, 1946; Brownlee et al., 1947; Scarlett and Martin, 1948; Haynes and Mundt, 1948; Stanley, 1948; Mercer, 1951; Filice, 1953). I n 1951, Somers reported that in the United States there were 230 known installations of in-plant chlorination. Of these, 165 were in fruit and vegetable canning or freezing plants and 57 were in fish processing plants. Today it is estimated th a t only a small percentage of the canning plants in the United States do not use chlorinated water a t some point in their operations. 111. THE GERMICIDAL ACTIVITYOF CHLORINE The most remarkable characteristic of chlorine in aqueous solution is its ability even in trace amounts to exert rapid germicidal action. T h e mechanism of this action is not thoroughly understood despite the long and wide use of chlorine in sanitation. Much of the confusion in this regard resulted from the difficulties experienced by early workers in identifying and measuring the germicidally active chlorine in their working solutions. 1. Mechanism of Microbial Death from Chlorine

Many early workers were of the opinion that the killing action of chlorine was due t o oxidative reactions involving nascent oxygen which was assumed t o be liberated in a union of chlorine with the hydrogen of water. The nascent oxygen, in turn, was supposed t o combine with unsaturated components of the cell protoplasm. Well-founded objections t o this theory have been brought forth. Oxygen from sources other than chlorine does not kill bacteria as readily as does the amount of chlorine theoretically necessary to yield an equivalent amount of nascent oxygen. Chlorine is also known to have germicidal activity under conditions which exclude direct oxidation of bacterial protoplasm. Experimental proof is lacking also for other hypotheses advanced to explain the bactericidal action of chlorine. These include suggestions tha t bacterial proteins are precipitated by chlorine; th a t cell membranes are altered by chlorine t o allow diffusion of cell contents; and th a t cell membranes are mechanically disrupted by chlorine. More acceptable theories conceive of the direct chemical combination of chlorine with the protoplasm of the bacterial cell. Chlorine would replace one or more hydrogen atoms in amino groups t o produce chloramines which would be toxic t o the cell and eventually cause its death. Rudolph and Levine (1941) concluded on the basis of results from their studies with hypochlorite th at two phases exist in the death of the

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bacterial cell: (1) the penetration of the active principle into the cell, and ( 2 ) the chemical union of this principle with the protoplasm which is directly responsible for the death of the cell. The majority of investigators now apparently believe that bacterial death by chlorine is a poisoning process in which a form of chlorine combines chemically with the protoplasm of the bacterial cell to produce toxic organic complexes. If penetration of undissociated chloramines or perhaps intracellular chlorination of nitrogen compounds occurs, it is assumed to be followed by dissociation of the chloramine to produce more specific inhibiting reactions. More recent theories are concerned principally with the mode of the chemical combination. The small amounts of chlorine needed to cause effective killing of cells in a bacterial suspension has led to recent theories that chlorine must attack certain oxidizable radicals in essential enzymes. Because of the marked bactericidal efficiency of chlorine in water in concentrations of 0.2 to 2.0 p.p.m., Green and Stumpf (1946) and Knox et al. (1948) considered chlorine a ‘‘biologically active trace substance.” They found experimentally that a precise parallelism existed between the effect of chlorine on bacterial growth and its effect on the rate of glucose oxidation by certain bacteria. This correlation of the inhibition of glucose oxidation with cell death was considered sufficiently general to suggest that chlorine acts by specifically inhibiting one or more of the enzymes required in glucose oxidation (Knox et al., 1948). The effect of chlorine on cells not dependent on glucose for life would be explained if chlorine interrupts in other essential metabolic systems. The mechanism of this inhibition is thought to involve oxidation of the sulfhydryl groups (-SH) of essential enzymes. Attempts to reverse the effects of chlorine by the addition of cysteine or glutathione have been unsuccessful (Ingols et al., 1953). After the oxidative change in the essential enzyme the bacterial cell cannot be helped to restore the functional sulfhydryl group. Additional support for the suggestion that chlorine exerts its bactericidal action by enzyme inhibition is found in the work of Douglas and Johnson (1938), who showed that sulfhydryl radicals are oxidized to sulfonyl compounds by chlorine gas. Furthermore, Black and Goodson (1952) have reported that hypochlorous acid will oxidize sulfide to sulfate a t concentrations and pH values normal in water chlorination. 2. The Germicidal Agent in Chlorine Solutions

Considerable discussion in the early literature was concerned with the identity of the germicidal agent in chlorine solutions. The undissociated

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molecules of both hypochlorites and chloramines and the hypochlorite ion (OC1-) have been suspected of being the toxic agent. With reference t o hypochlorite solutions, Andrews and Orton (1904) were the first to suggest that hypochlorous acid (HOC1) was the active agent. Later workers have concurred in this opinion (Charlton and Levine, 1937; Rudolph and Levine, 1941; Marks et al., 1945; Knox et al., 1948; and others). With regard to chloramines, opinions differ as to the identity of the active agent. Leech (1923) and Johns (1934) were among those who believed that chloramines hydrolyze to give hypochlorous acid and that this was the germicidal agent. Holwerda (1928, 1930) considered that chloramines could undergo the following hydrolysis to yield hypochlorous acid : R-NHzCl

+ 2Hz0

-+

R-NHIOH

+ HOCl

However, his methods of analysis could not detect the presence of hypochlorous acid in a chloramine solution containing 10 p.p.m. available chlorine. I n comparative studies with hypochlorites, monochloramine, and chloramine-T Charlton and Levine (1937) found that death rate curves for spores exposed to monochloramine and chloramine-T approximated the straight-line pattern of a monomolecular reaction. This was in contrast to the curved-line patterns found with hypochlorite solutions in which they believed hypochlorous acid t o be the active agent. They interpreted this evidence to indicate that in (‘solutions of chloramine with an =N :C1 linkage the germicidal action is not due to hypochlorous acid.” They suggested instead that in disinfection with chloramine solutions death of the bacteria was due to the direct chlorinating action of positive chlorine atoms or to the undissociated chloramine molecule. Marks et al. (1945) suggested that the bactericidal activity of chloramine compounds ‘(may be due in whole or in part to the hypochlorous acid formed in accordance with the hydrolysis and ionization equilibria.” From the results of their studies with various N-chloro compounds they concluded that these compounds effect their killing action by two different mechanisms: 1. Action by the HOCl formed by the hydrolysis of the chloramine. 2. Direct action by the undissociated molecule of the chloramine. The action of HOCl formed by hydrolysis they considered to be the most reactive germicidal mechanism. The concentration of HOCl in the unionized form was dependent on the pH of the solution. At pH levels where HOCl remained substantially ionized bactericidal activity was decreased. When the ratio of nitrogen to chlorine was in excess of that

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necessary t o form the N-chloro compound the rate of sterilization depended on whatever germicidal properties the N-chloro molecule possessed. For purposes of chlorination in food plant sanitation it can be assumed that the time required by any type of chlorine solution to kill exposed bacteria is a n inverse function of the concentration of undissociated hypochlorous acid. Reference to Fig. 1 will show the relationship between

-

a

TEST ORGANISM B. MACERANS SOURCE OF CHLOFUNE CHLORINE GAS CONC. OF AVAILABLE CHLORINE 1 4 . 5 PPM

-

-

p H OF BUFFERED WATERS

FIG.1. Relationship between the concentration of free chlorine (HOC1) a t varied pH levels and the rate of killing for bacterial spores. (From Mercer, 1953.)

available chlorine residuals, the calculated concentrations of undissociated HOC1, and survivor curves for spores of B. macerans. 3. The Pattern of Bacterial Death from Chlorine

It has long been known th at cells of a bacterial population exposed to a lethal agent do not all die a t the same time. Early workers concluded from their studies that the death of bacteria in contact with certain chemicals was a n orderly process which required time for completion. Numerous workers have since studied the cause of cell death in bactericidal processes with the hope that such studies would explain the nature of the reaction between the lethal agent and the cell, and would explain the shape of the time curve of the process. Considerable controversy still exists as to whether the process is essentially chemical or physical in nature, or is controlled by complex biological laws. Adherents to the chemical theory of bacterial death have

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interpreted their data to show that the destruction of bacteria is an orderly process of the nature of a monomolecular chemical reaction where only one substance undergoes change. Chick (1908), among others, early reached the conclusion that the rate of death of bacteria showed a logarithmic order. The data upon the logarithmic order of death of microorganisms have been reviewed and discussed by Rahn (1945). Many data questioning the logarithmic order of bacterial death have been reviewed by Knaysi (1930a, 1930b), Buchanan and Fulmer (1930), and others. Falk and Winslow (1926) cite experimental evidence to indicate that close analysis of survivor curves will show more or less marked deviations from the logarithmic rate of death. They suggest, however, that when the death rate cannot be described by a unimolecular equation the process may sometimes be described by a multimolecular reaction. Recently, a nonlogarithmic order of death of heated spores of food spoilage organisms has been reported by Andersen et al. (1949) and Reed, Bohrer, and Cameron (1951). Reynolds and Lichtenstein (1952) have presented data that are inconsistent with the assumption that thermal death rate curves for bacterial spores are exponential. They found that survivor curves exhibited an initial phase of accelerated rate of death and a declining rate of death in the vicinity of complete spore destruction. The shape of the curves was sigmoid in character, indicating that the spore population was made up of individuals with varying resistance. In the case of disinfection, McCulloch (1945) recognized the existence of an initial “period of lag” in the survivor curve and a final retardation of the death rate a t t,he end of the exposure period. He believed, however, that during most of the exposure period the order of death was essentially logarithmic, and recommended that the logarithmic rate be used to calculate the velocity of disinfection phenomena. Bacterial death from chlorine is an apparent exception to the logarithmic order found in sterilization processes with other bactericidal agents. Charltnn and Levine (1937) observed thah survivor curves for spores of Bacillus matiens exposed to hypochlorite solutions showed marked deviation from the straight-line relationshir, between the logarithms of numbers of survivors and time of exposure. Later, Rudolph and Levine (1941) in their studies with hypochlorite solutions again found that bacterial spores exposed to chlorine did not die a t a uniform rate. Rahn (1945), in stating that evidence was “overwhelmingly in favor of logarithmic order of death in bacteria” took note of the experiments by Charlton and Levine (1937) and Rudolph and Levine (1941) and considered their results to be remarkable exceptions which could not be disregarded.

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WALTER A. M E R C E R AND IRA I. SOMERS

The typical survivor curve for bacterial spores exposed to chlorine is S-shaped, as shown in Fig. 2. A definite initial lag is shown even when the exposure time is very short. Following the initial lag there is a middle phase of increasing death rate. In most survivor curves a final phase of

9

DEATH R A T E C U R V E FOR SPORES EXPOSED T O CHLORINE IN WATER

100

DEATH RATE C U R V E FOR SPORES H E A T E D IN PHOSPHATE

0

5

BUFFER

10

15

20

25

30

T I M E IN MINUTES

FIQ.2. Comparison of death rate curves for bacterial spores to show the logarithmic order of death when exposed to moist heat and the nonlogarithmic order when exposed to chlorine in water. (From Mercer, 1953.)

retarded death rate for the last surviving spores is apparent. If the velocity coefficient of the death rate K is calculated from the formula for logarithmic death

where a and b are the survivors at times T Iand Tz,respectively, it will

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be found that the values of K progressively increase instead of being constant as would be found in true monomolecular reactions. Explanation of the nonlogarithmic death rate for bacteria exposed to chlorine can be based only on assumption. Charlton and Levine (1937) and Rudolph and Levine (1941) suggested that the lag phase of the survivor curve was the time required for the bactericidal form of the A B C D E

-

WATER - DISTILLED LAURYL SODIUM S U L F A T E - CHLORINE AND ALKYL ARYL SULFONATE CHLORlNE ONLY -- CHLORINE A N D LAURYL SODIUM S U L F A T E

I

200 000

-

TEST ORGANISM B . COAGULANS SPORES CONC. OF AVAILABLE CHLORINE 6 . 5 PPM CONC. O F COMPOUNDS ADDED - 0.10 PERCENT

-

TIME IN MINUTES

FIG.3. Effect on the germicidal activity of chlorine when it is combined with compounds having wetting and detergency properties. (From Mercer, 1955.)

chlorine to penetrate the cell. The length of the lag phase and the rate of death thereafter would depend on the concentration of germicidal chlorine. Rahn (1945) theorized that survivor curves of the type obtained in chlorine disinfection could result only if “a fairly large number of molecules of the cell surface must be destroyed to produce an injury from which the cell cannot recover.” However, it seems reasonably apparent that the important bactericidal action of chlorine is not on the cell surface, but on intracellular protoplasm. Death of the bacterial cell, at least within a time period practical for large-scale chlorination purposes, must depend on penetration of bactericidal chlorine into the cell. Treatment which allows more rapid cell penetration shortens the time required to complete the death process. Fisher (1942) found that the addition of surface-active agents in dilu-

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tions of 1:1000, whether anionic, neutral, or cationic, enhanced the bactericidal activity of mercury bichloride, mercury oxycyanide, chloramine-T, argyrol, and potassium permanganate. MacGregor and Elliker (1953) observed that addition of inorganic chelating agents to quaternary ammonium compounds increased germicidal effectiveness by removal of interfering cations from the cell surface. Preliminary studies (Mercer, 1953) have indicated that addition to chlorine solutions of certain compounds having wetting and detergency properties decreases the time required to kill bacterial spores. The shortened lag in survivor curve E shown in Fig. 3 is evidence that cell penetration was more rapid when lauryl sodium sulfate was added. Alkyl aryl sulfonate, however, caused a decrease in germicidal activity by neutralizing part of the chlorine.

4. Evaluation of the Germicidal Activity of Chlorine Solutions The fact that bacterial cells exposed to chlorine do not die in a predictable manner means that methods based on a logarithmic rate of death cannot be used for evaluating and comparing the germicidal efficiency of the various chlorine compounds. Standard methods such as the “phenol coefficient” test now used for evaluating disinfectants and antiseptics are dependent on a constant concentration of the test compound. The very reactive nature of chlorine means that in the presence of organic or inorganic matter the concentration of active chlorine in a given solution will not be constant throughout the test period. The temperature of the solution may vary the concentration of active chlorine since the solubility of elemental chlorine in water is governed by the temperature. Other factors such as the effect of p H on the activity of chlorine solutions cannot be adequately accounted for in the usual standard methods of testing. Numerous methods have been used for ascertaining the germicidal activity of chlorine compounds. Johns (1934) developed his Glass Slide Method in an effort to simulate the conditions under which these compounds would be used in sanitizing farm dairy utensils and dairy plant equipment. One advantage of this method is that it determines the ability of the test compound to destroy bacteria in a moist film of dilute milk. The organisms surviving after a definite contact period are counted. From the results of the test it can be determined how much time would be required for each germicide to kill 99.9999% of the organisms present in the milk film. The survivor-curve procedure is a t present the most practical method of evaluating chlorine compounds. The vagaries of the death rate for bacteria exposed to chlorine are of no consequence if the test criterion

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used is the “killing time” which represents the time required to kill a definite percentage of the cells exposed. This type of testing procedure enabled Charlton and Levine (1937), Rudolph and Levine (1941), and others to study the effect of concentration, pH, and temperature on the germicidal activity of chlorine compounds. The technique of the tests for evaluating chlorine compounds on the basis of their germicidal activity has been discussed in detail by Charlton and Levine (1937). Essentially it consists of suspending a known number of bacterial cells in water to which a known amount of available chlorine has been added. At various time intervals, a portion of the water is removed, the chlorine is neutralized, and the number of cells still living in a measured amount of the water are counted. To be counted as living, the cell must be able to produce a colony in a suitable culture medium within a short period of time, usually 48 hours. From the results a survivor curve can be drawn which shows the rate and extent of the bactericidal activity.

IV. THE APPLICATION OF CHLORINE IN FOOD PLANTSANITATION Many factors must be considered in the successful practice of chlorination. The results obtained are definitely related to the degree of knowledge employed in the application of chlorine as a germicide. When chlorination of a water supply fails to give the results desired, the failure is often due to conditions, either chemical or physical, which have limited or completely destroyed the germicidal activity of the chlorine.

I . Definition of Chlorination Terms A number of specific terms are used in the literature t o identify various aspects of chlorination. These terms are found also in manuals describing laboratory procedures used to control large-scale applications of chlorine. a. Available Chlorine. The germicidal strength of commercial hypochlorite and chloramine preparations is ordinarily expressed in terms of “per cent available chlorine.” This expression is not a reliable indication of the germicidal efficiency of these compounds. A statement of the “per cent available chlorine ” of hypochlorite, for example, accounts for all the chlorine which was used to make the compound, including that reacted to form chloride which does not influence the germicidal power of the solution. The confusion arose from early methods of measuring the oxidizing power of acidified chlorinated lime. The gas evolved was measured and expressed as “per cent chlorine available.” When hypochlorite is acidified with HC1 two chlorine atoms are

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released in oxidation-reduction reactions, for each hypochlorite ion (OC1-) present. The following equations will illustrate this.

+

+

+

+

+

1. NaOCl 2KI 2HC1- 1 2 NaCl 2KC1 H20 2. Ca(OC1)z 4KI 4HC1-+ 212 CaC12 4KC1 2Hz0 3. Clz 2KI --t Iz 2KC1

+

+

+

+

+

+

+

The number of iodine atoms released in each equation is equal to the number of chlorine atoms present in the reaction, yet the number released in equations 1 and 2 is twice the number of atoms available in the hypochlorite molecule. However, the term “ available chlorine ” is universally used to denote “parts per million available chlorine’’ on the basis that 1 ml. of 0.1 N sodium thiosulfate solution is equivalent to 0.003546 g. available chlorine. b. Chlorine Dosage. The amount of chlorine compound added to a water is often called the “chlorine dosage.” This value is given in parts per million available chlorine and is not a measure of the resulting concentration of germicidally active chlorine in the solution. e. Chlorine Demand. The “chlorine demand” of a water is the difference between the amount of chlorine dosage and the amount of residual chlorine remaining a t the end of a specified contact period. The chlorine demand for any given water varies with the amount of chlorine added, time of contact, water temperature, and the concentration and type of impurities in the water. When inorganic impurities, such as hydrogen sulfide or nitrites, are present, part of the reactive HOCl combines with the impurities. The reactions between HOCl and inorganic impurities are rapid and stoichiometric. I n the usual water, this loss of active chlorine ranges from 0.25 to 1.0 p.p.m. Loss of chlorine to inorganic impurities does not increase with longer contact. The rapidity of these reactions which satisfy the chlorine demand does not allow for bactericidal action from the chlorine lost. This action must come from chlorine added in excess of the chlorine demand. If organic impurities, such as fruit and vegetable solids, are present HOCl reacts with amine-type nitrogenous compounds to form monoand dichloramines. The organic chlorine demand is satisfied relatively slowly and is not stoichiometric. The chlorine loss increases with time, with increased temperature, and with increased concentration of chlorine. The chlorine in the chloramines formed has a considerably reduced germicidal activity. Other reactions between chlorine and organic materials may completely destroy the germicidal activity of the chlorine ions entering the reaction. Milroy (1916) has pointed out that in the case of

CHLORINE IN FOOD PLANT SANITATION

143

aromatic or heterocyclic amino acids, such as tyrosine and tryptophan, partial chlorination of the ring takes place in addition to oxidation. The active chlorine reduced in these reactions is no longer germicidal. d. Total Residual Chlorine. When a given amount of chlorine is added to water the amount remaining after the chlorine demand has been satisfied is termed the “total residual chlorine” without regard to the type of residual. e. Free Available Chlorine. That portion of the total chlorine residual remaining in the water which reacts chemically and biologically as hypochlorous acid (HOC1) or as hypochlorite ion (OC1-) is the “free chlorine residual.” f. Combined Available ChEorine. That portion of the total chlorine residual which is combined with ammonia-nitrogen or other amine-type nitrogen is known as “combined available chlorine.” Free available and combined available chlorine may exist in water a t the same time. At pH 8.4 or above, the combined available chlorine is present as monochloramine. At pH 4.5 and below, dichloramines are largely formed. Between these pH levels a mixture of the two can be found. g. Marginal Chlorination. The addition of small amounts of chlorine to water without regard to the type of residual produced, or to the persistence of the residual, is termed “marginal chlorination.” This type of chlorination is used most often to render water potable with respect to pathogenic bacteria. Marginal chlorination is of little value for in-plant chlorination purposes. The chlorine residual is low and persists for only a short time, and is not of the type necessary to destroy odors and tastes in the water. The germicidal efficiency of such a method is, therefore, low and unpredictable. h. Break-Point Chlorination. When small amounts of chlorine are added intermittently to water under controlled conditions, the first chlorine is used up in satisfying the chlorine demand of the water. At the same time, chlorine combines loosely with nitrogenous matter present to form chloramines or other chloro-nitrogen compounds. As additional chlorine is added a free chlorine residual appears. This free chlorine residual gradually increases until it reaches a concentration, determined by the physical and chemical nature of the water, a t which an oxidation reaction occurs between the free chlorine and the chloronitrogen compounds. The free chlorine residual is decreased by the amount necessary to oxidize the chloro-nitrogen compounds. Further additions of chlorine beyond this point will result in a second rise in a free chlorine concentration which persists and increases almost in direct proportion to the rate of chlorine application. That point after the first rise in concentration a t which the free residual reaches its lowest

144

WALTER A. MERCER AND IRA I. SOMERS

-

8.E

4.

-2 -

d

8,

3

1.8-

3: 0

9E w

a 6 2 I w

+BREAK-POINT

7 . t

9

u

?i

2-1

7.4

I

I

1

2

I 3

I 4

I 5

I

6

-I

8.0-

d

w

7.8-

!ji El

w

7.L

E

0 X

% 7 . e

CHLORINE DEMAND

I

6 TOTAL CHLORINE ADDED (PPM)

FIG.5. Break-point curve for tap water containing 0.5 ml. of tomato juice per liter. (From Mercer, 1955.)

CHLORINE I N FOOD PLANT SANITATION

145

level is known as the “break-point.” If curves be drawn (Figs. 4 and 5 ) t o show the rise and fall in free residual concentration, this point will be indicated by a “dip” following a “hump” which represents the first rise in free residual. Waters in which the chlorine addition does not reach the break-point may retain a high concentration of the chloro-nitrogen compounds. T he presence of chlorine in certain of these compounds greatly intensifies their unpleasant odor and taste. Beyond the breakpoint, however, these odors and tastes are practically eliminated and the chlorine residual is most efficient as a germicide. All waters, however, may not exhibit the break-point effect, and in those which do many modifications of the typical break-point curve may be found. Surface waters which originate from such sources as melting snow and which are almost pure in respect t o the absence of chemical and organic matter, will show no break-point, or one so slight that it is difficult t o demonstrate. The break-point on a n individual water can be determined only by tests on th at water. 2. Chlorine Compounds Commonly Used

Chlorine, as the element, is a greenish yellow gas which, at 0°C. and 1 atm. of pressure, is about two and one-half times as heavy as air. T h e characteristic odor is highly irritating and penetrating. As a dry gas, chlorine is not unusually reactive, but in the presence of moisture, it is one of the most reactive elements known. It attacks many kinds of metals and has a great affinity for most organic materials. Chlorine replaces bromine and iodine from their salts. I n the presence of sunlight or arclight chlorine combines with hydrogen with explosive violence. Chlorine is never found free in nature, but chlorine-bearing compounds are very abundant. The most important natural source is sodium chloride. Potassium chloride and carnallite, which is a mixture of potassium and magnesium chloride, are other natural sources. Generally, the chlorine used for food plant sanitation is a n aqueous solution containing active chlorine which comes from one of three commercial sources: ( I ) liquid elemental chlorine, (2) hypochlorites, or (3) organic chloramine compounds. a. Liquid Chlorine. The most important commercial preparation is liquid chlorine. It is obtained principally b y the electrolysis of sodium chloride. The chlorine gas produced in this process is compressed and cooled t o a liquid which is shipped in steel cylinders equipped with special release valves. Upon release the liquid flashes back t o the gaseous form. Chlorine gas is generally considered the best source for in-plant chlorination where large volumes of water are t o be chlorinated to 5 t o 7 p.p.m. Since chlorine gas is a pure substance, no extraneous materials

146

WALTER A. MERCER AND IRA I. SOMERS

are added. It is the most inexpensive source on the basis of pounds of available chlorine. The main objection to use of chlorine gas is the initial cost of the equipment needed to feed the gas into the water. However, when compared with the cost of chlorine from other sources, the lower cost of the gas will off set the more expensive chlorinating equipment. b. Hypochlorites. The hypochlorites are salts of hypochlorous acid formed by reacting gaseous chlorine with an alkali, an alkaline earth hydroxide, or an alkali carbonate. Calcium hypochlorite is a powder prepared by passing chlorine gas into milk of lime. The chlorination reaction may be represented as follows: 2C12

+ 2Ca(OH)2 = Ca(OCl)z + CaCL + H 2 0

Chloride of lime (30% to 35% available chlorine) was largely used in the early days of chlorination. I n recent years high-test calcium hypochlorite containing 60% to 70% available chlorine has been developed. Sodium hypochlorite is liquid in form. The commercial solutions are prepared by reacting gaseous chlorine with caustic soda, according to the following equation: 2NaOH

+ Cl2 = NaOCl + HzO

Sodium hypochlorite solutions are available in two ranges of strength, 2 % to 6 % available chlorine for household bleaches and disinfectants, and 10% to 18% for industrial uses. These solutions are used without dilution for chlorinating water supplies but as a 5 % solution for other uses as a sanitizer. As a source of chlorine for sanitizing purposes, hypochlorites have some disadvantages. One is that on storage they slowly lose chlorine. Moisture, heat, and light increase the rate of loss. Packaging in sealed containers which exclude light, and storage a t cool temperatures retard the rate of chlorine loss. Attempts have been made to stabilize hypochlorite preparations. Mixtures of sodium and calcium hypochlorite are more stable than either compound alone. These mixtures are more moderately alkaline than calcium hypochlorite solutions of the same strength. Other hypochlorite preparations contain trisodium phosphate or sodium carbonate as stabilizing agents. However, addition of stabilizers involves some sacrifice of germicidal activity. As McCulloch (1945) points out, no hypochlorite can be both highly stable and rapidly germicidal. If a higher degree of stability is desirable, then the content of available chlorine as hypochlorous acid must be reduced. Any reduction in the content of available chlorine as hypochlorous acid means a corresponding reduction in germicidal efIiciency.

CHLORINE I N FOOD PLANT SANITATION

147

Hypochlorites are the second choice for in-plant chlorination for the following reasons: i. They are not pure chemicals; chlorides and hydroxides are added with the chlorine. These unnecessary chemicals may have a n adverse effect on the product. 2. If hypochlorites are added to hard waters, deposition of salts on cans and equipment will be increased. 3. The amount of chlorine added as hypochlorite is more difficult to control . 4. On the basis of available chlorine content hypochlorites are more expensive than chlorine gas. Hypochlorites are superior as a source of chlorine when only small amounts are needed, such as in can-cooling systems, for hot applications during cleanup, and for dripping chlorine solution on belts and other equipment to prevent slime growth. c. Chloramine Compounds. Before World War I only gaseous chlorine and hypochlorites were commercially available. The germicidal properties of chloramines had been known since 1910, when Rideal observed the use of chlorine-ammonia in sewage chlorination. Chloramine-T (Dakin et al., 1916) was the first commercially produced chloramine compound. It was formed by reacting p-toluene sulfonamide with sodium hypochlorite. The chlorine in sodium p-toluene sulfonchloramine has the 1inkage:NCl as shown in the following formula: (CH3CsHaS02N:C1)-Na+. Chloramine-T is a white crystalline powder freely soluble in water. At room temperature saturated aqueous solutions contain about 15 % available chlorine. Dichlorodimethyl hydantoin has recently been introduced as a sanitizer for use in food industries. It is a white powder of low density, soluble in water up to concentrations of 1000 p.p.m. available chlorine. Aqueous solutions are slightly acidic in contrast t o the alkalinity of hypochlorites and chloramine-T. Chloramines should not be considered as a source of chlorine for chlorination of in-plant water. Their germicidal action is slow. However, the slow release of chlorine is a n advantage in uses where a long contact time is possible. 3. Conditions Aflecting the Germicidal Activity of Chlorinated Waters

Whether a chlorine product be one of the hypochlorites, a chloramine, or gaseous chlorine, its power as a germicide will depend on the concentration of undissociated hypochlorous acid which it makes available in water solution. The weakness of HOCl as a n acid will favor hydrolysis of the (OC1)- ion. Its strength as an oxidizing agent will cause its loss

148

WALTER A. MERCER AND IRA I. SOMERS

through chemical reactions. Thus, the germicidal activity of a given chlorine solution will depend on the influence exerted by conditions in the water which is chlorinated. a. Efect of p H . The pH of any chlorine solution has a profound influence on the rate a t which exposed bacteria will die. Rideal and Evans (1921) were among the first to indicate the influence of pH by demonstrating that the addition of alkali to hypochlorite solutions caused a marked decrease in oxidizing power. Johns (1934) was able to show that the bactericidal activity of hypochlorite solutions was dependent on the amount of hypochlorous acid formed and that both the concentration of the parent chlorine product and the pH were deciding factors in the activity of the acid. Charlton and Levine (1937) and Rudolph and Levine (1941) concluded that pH was the most important single factor influencing the germicidal activity of hypochlorite solutions. The influence of hydrogen ion concentration on the formation of hypochlorous acid from gaseous chlorine, hypochlorite, and chloramine is represented by the following equations: H+ + HzO OHHOCl + H+ + C1H+ 2. NaOCl + HzO HOCl + Naf + OH6HH+ 3. NHzCl + Hz0 F - HOCl + NHI+ + OHOH-

1. Clz

F==

__

The pH also influences the hydrolysis of HOCl formed in the above reactions. OC1-

Hf + HzO OHHOCl + OH~

=

f

The hydrogen ion concentration determines the fraction of the HOCl concentration present as the undissociated molecule or as hypochlorite ion. As the hydrogen ion concentration decreases the ionization of HOCl increases. Holwerda (1928), using the ionization constant of 3.7 X 10-8, calculated the per cent undissociated HOCl which would be present in hypochlorite solution a t different pH levels. This is shown in Table I. Figure 6 shows the results of studies (Mercer, 1953) to determine the effect of pH change on the activity of solutions prepared from sodium hypochlorite. I n Fig. 7 a comparison is made of the time required for four chlorine compounds to kill 99% of 3. macerans spores exposed to solutions having a pH range of 6.0 to 8.0. With equal concentrations of available chlorine and the same pH, no significant difference in sporicidal

CHLORINE IN FOOD PLANT SANITATION

149

activity was found between chlorine gas and the hypochlorites. The chloramine required approximately 10 times as long a t each p H level to be equally effective as a sporicide. b. Efect of Concentration. It might be assumed that the germicidal activity of chlorine solutions will increase as the concentration of chlorine compound in solution is increased. However, this assumption is true only in the case of buffered solutions where conditions of pH, temperature, and organic content are held constant. Under conditions where only the concentration of chlorine is varied, a plot of the logarithms of the killing TABLE I Calculated Percentages of Hypochlorous Acid Present in Hypochlorite Solutions at Various pH Levelso

pH level 4.0 5.0 6.0 7.0 8.0 9.0 10.0 a

Per cent hypochlorite present as undissociated HOCl ca. 100.0

99.6 95.8 69.7 18.7 2.2 0.2

After Holwerda (1928).

time against the logarithm of the concentration of chlorine will approximate a straight line. Mallmann and Schalm (1932) reported killing times of 5, 10, and 15 minutes for concentrations of 2.0, 1.2, and 0.6 p.p.m., respectively, of available chlorine in solutions at pH 9. Reducing the chlorine concentration to 0.3 p.p.m. did not cause death in 30 min. Analysis of these results indicated that doubling the concentration reduced the killing time about 33 % for the lower concentrations of chlorine. With higher chlorine content, doubling the concentration reduced the killing time about 50%. It was assumed that in solutions of low concentration a greater portion of the germicidal chlorine is neutralized in side reactions. When the concentration of available chlorine is increased in unbuffered solutions the effect on germicidal activity is dependent on two interrelated factors, (1) the shift of p H toward acidity or alkalinity, and (2) the influence of the pH shift on the concentration of HOC1. I n the case of chlorine gas, increasing the concentration of available chlorine in unbuffered solution will increase the acidity and thereby

150

Q

i

! w

0

8

E

0 r4

w

-

FIG. 6. Survivor curves for B . macerans spores exposed in buffered waters at various pH levels to sodium hypochlorite in a concentration of 15 p.p.m. available chlorine. (From Mercer, 1953.)

I I

1

6.0 6.5

pH

=

7.0 7.5

8.0

6.0

6.5 pH

7.0 7.5

8.0

6.0 6.5 pH

7.0 7.5-

8.0

PH

-7 . 0 1 7.5

47.0

-

- '

6.5

CHLORlNE GAS

13.0

20.5

8 . 5 MlNUTES

- 1

6.0

9 . 5 MINUTES

10.3

9.2

12.8

SODIUM HYPOCHLORITE

20.5

42.0

8 . 5 MINUTES

11.0

14. 3

CALCIUM HY POCHLORlT E

19.0

48.0

105 MINUTES

ORGANIC CHLORAMINE

115

190 250

FIQ.7. Comparison of time required to kill 99% of 3.macerans spores exposed to different chlorine compounds present in concentration of 15 p.p.m. available chlorine in solutions at varied pH levels. (From Mercer, 1953.)

CHLORINE I N FOOD PLANT SANITATION

151

increase the germicidal activity. The source of the added hydrogen ion is shown in the following equation: Cln

+ Ha0 + H + + C1- + HOCl

The case of acidic chloramines is essentially that of gaseous chlorine. When the source of the increased concentration of available chlorine is hypochlorite or an alkaline chloramine, the added hydroxyl ion will increase the alkalinity of the solution and thus decrease the germicidal activity. The effect of dilution of calcium hypochlorite solutions was studied by Rudolph and Levine (1941), who found that a solution containing 25 p.p.m. available chlorine had a killing time of 35.5 minutes. At 100 p.p.m. the killing time increased to 57.0 minutes, while a t 1000 p.p.m. the killing time was 95.5 minutes. The pH for these solutions was 9.35, 9.75, and 11.1, respectively. The equations for sodium hypochlorite in water will show the source of the added hydroxyl ion. NaOCl

+ H20

--f

Na+

+ OH- + HOCl

c. Efect of Temperature. Very few comprehensive studies have been reported of the effect of temperature on the bactericidal efficiency of chlorine compounds, although temperature has been shown to be a very important factor. Charlton and Levine (1937) using chloramine-T solutions containing 2000 p.p.m. available chlorine with an initial pH of 6.0 a t 55°C. (131°F.) found that each 10°C. rise in temperature gave a reduction of 82% in the killing time. With solutions having an initial pH of 8.7 the reduction in killing time was 71%. The effect of temperature on the bactericidal efficiency of calcium hypochlorite solutions of pH 10 was observed by Rudolph and Levine (1941) a t 20", 25", and 50°C. (68", 77", and 122°F.) I n general, the killing time was reduced by 60% to 65% for each 10°C. rise in temperature. Weber and Levine (1944) concluded that a drop of 10°C. required a twofold increase in exposure time with chlorine and a three- to fourfold increase for chloramine to achieve equivalent bactericidal action. Ames and Smith (1944) found that the ability of chlorine to destroy E. coli in the presence of 0.25% organic nitrogen was markedly affected by the temperature a t which it was used. A given dosage of chlorine required nine times as long to sterilize a t 8°C. (45°F.) as a t 40°C. (104°F.). I n food plant sanitation, the feasibility of elevating the temperature of chlorine solutions in order to take advantage of increased germicidal activity depends on the type of chlorine compound from which the solution is prepared and the purpose for which the solution is to be used. I n the case of solutions prepared by dissolving gaseous chlorine in water, the solubility of chlorine gas in relation to temperature should

152

WALTER A. MERCER A N D IRA I. 8 0 M E R S

be considered. The solubilities of chlorine in water at different temperatures are listed in Table 11. Unless the water in which the chlorine gas is dissolved also contains organic nitrogen to allow chloramine formation, elevating the temperature of the solution will cause considerable depletion of chlorine content. Ordinarily, chlorinated waters prepared by dissolving gaseous chlorine are used in large-scale operations such as product washing or can cooling where the contact time between the bacterial cell and the chlorine is TABLE I1 Solubility of Chlorine Gas in Water at Various Temperatures" Temperature

a

"C.

OF.

Maximum per cent chlorine dissolved

0 10 20 30 40 50 60 70 80 90 100

32 50 68 86 104 122 140 158 176 194 212

1.46 0.98 0.76 0.56 0.45 0.39 0.32 0.27 0.22 0.12 0.00

Hodgman, Handbook of Chemistry and Phuaica, 1947.

sufficiently long to allow adequate germicidal action at natural water temperature. Chloramine solutions are more stable a t elevated temperatures than solutions of dissolved gaseous chlorine. The feasibility of using these solutions a t moderately elevated temperatures would depend on the convenience of heating for the intended use. Hypochlorite solutions apparently lose little available chlorine a t moderately elevated temperatures. Hadfield (1954) states that he found no appreciable loss of available chlorine from sodium hypochlorite solutions held at 55°C. (131OF.) for 180 minutes. He concluded that the use of hypochlorite solutions as sanitizing agents for food handling and processing equipment need not be restricted to tap water temperature, but could advantageously be used a t higher temperatures. d. Efect of Organic Matter. It has long been recognized that the presence of certain types of organic matter in chlorine solutions causes a marked reduction in germicidal activity. In the chlorination of waters

153

CHLORINE I N FOOD PLANT SANITATION

used in food processing this fact has not always been fully appreciated. Especially has this been true in the chlorination of recirculated cancooling waters and waters used for fluming raw products. UnIess adjustments are made in the chlorine dosage to allow for the organic chlorine demand of these waters, serious bacteriological problems may result. The reaction of chlorine with organic material may be of the nature of adsorption or true chemical combination depending on the type of organic material involved. Table I11 shows the effect on chlorine concentration of some organic materials common to food plant processing waters.

TABLE III* Effect of Organic Matter on Concentration of Free Available Chlorine0 in Water Concentrationbof free available chlorine Time after adding Clz (min.)

No organic matter

5 % starch (1.0 ml./l.)

40 % cane sugar (1.0 ml,/l.)

(0.5 m1.b.)

(1.0 ml./l.)

1 3 6 9 12

5.00 4.95 4.95 4.92 4.90

4.98 4.96 4.95 4.92 4.90

4.75 4.60 4.40 4.30 4.25

4.20 3.70 3.45 3.20 3.00

3.45 2.65 1.95 1.60 1.50

Canned tomato juice

~~

*Mercer (1963). Source of chlorine was stock aohtion prepared from gaseoua ohlorine. b Free available c h h h measured by amperometrio titration.

From the results given in Table I11 it can be seen that the presence of organic compounds such as starch and cane sugar in chlorine solutions cause no significant reduction in available chlorine. Guiteras and Schmelkes (1934) found that chlorine loss t o nonnitrogenous substrates was negligible. Using a sodium hypochlorite solution with a concentration of 200 p.p.m. available chlorine they determined the chlorine demand of various sugars and alcohols. Included were the monosaccharides ; glucose and levulose; the disaccharides; maltose, lactose, and sucrose; the trisaccharide; raffinose; and the alcohol, mannitol. Of this group only levulose took up 69 p.p.m. of the original 200 p.p.m. sodium hypochlorite. Chlorine in the presence of such organic materials as sugars and starches retains the major portion of its germicidal activity. This is shown in Fig. 8, where survivor curves are drawn for yeast cells suspended in buffered chlorine solutions containing starch, cane sugar, tomato juice, and tomato serum. The property of sugar solutions to adsorb and retain chlorine in active forms is of special significance in fruit canning where the fruits are packed

154

WALTER A. MERCER AND IRA I. SOMERS

in sugar sirup. Even low concentrations of chlorine (1.0 t o 2.0 p.p.m.) in the water used to make the sirup dilutions have been known t o cause off-flavors in the finished product. The nature of the organic material, that is, whether in solution or suspended in the chlorinated water, is a factor in the time required by 2.0.

1. 5 10

el

-

u1.0

-

TEST ORGANISM YEAST CELLS AVAlLABLE CHLORINE ADDED 4 . 2 5 PPM CONC. OF ORGANIC MATTER 1 . 0 M L / LITER

0

E $

-

20.3

!2 u

W ao.0

u

4. -0.3

0.5

1.0

1. 5

2.0

2.5

3.0

3.5

TIME IN MINUTES

FIG.8. Effect of organic matter on the germicidal activity of chlorine solutions. (From Mercer, 1955.)

the chlorine t o kill bacteria. Suspended particulate matter, both organic and inorganic, will prolong the life of bacteria exposed to the solution. This protective action is mechanical in nature. The chlorination of water which contains turbidity of inorganic nature over 2 mg. per liter has given poor results because of the protective action of the suspended matter. Table I V gives the results of experiments in which test organisms were suspended in two chlorine solutions having equal concentrations of free available chlorine, one chlorine solution containing whole tomato juice and the second a clear tomato serum. The organic chlorine demand of each preparation was the same. The killing time for Streptococcus lactis

155

CHLORINE I N FOOD PLANT SANITATION

cells was two and one-half and for the yeast cells four times longer in the solution containing suspended tomato solids. TABLEIV Comparison of the Effect of Suspended and Soluble Organic Matter on the Germicidal Action of Chlorine *

Test organism Yeast (non-spore-forming) Streptococcus lactis

Amount and type of organic matter added to watera

Total* residual (p.p.m.1

99.9 % of cells

Time to kill

0 . 5 ml. tomato juice 0 . 5 ml. tomato serum 0 . 5 ml. tomato juice 0 . 5 ml. tomato serum

3.90 3.90 3.14 3.17

4 .O 1.5 2.5 1 .o

(min.)

*Mercer (1953). Water buffered at pH 7. Chlorine dosage = 5.00 p.p.m. in all oaae8.

5

The pH of the chlorine solution is also a factor in the amount of chlorine lost to organic matter in solution. I n general, as the pH becomes more alkaline, the concentration of total available chlorine becomes less.

2

I

\

>-

FREE RESIDUAL CHLORINE

1

c

.

a

-

WITH ORGANIC MATTER

6.0

6.5

7.0

7.5

8.0

0

8.5

pH O F BUFFERED WATERS

FIG.9. Effect of organic matter (tomato juice, 0.5 ml. per liter) on chlorine residuals. (Chloramine compound added sufficient to give 5.5 p.p.m. available chlorine in solutions a t various pH levels.) (From Mercer, 1953.)

This is shown in Fig. 9, where curves for total available chlorine and HOCl concentrations are plotted against change in pH. Some discussion in the literature has indicated that nitrogenous

156

WALTER A. MERCER AND IRA I. SOMERS

organic materials consume less chlorine from chloramine solutions than from solutions prepared from gaseous chlorine or hypochlorites. Guiteras and Schmelkes (1935) observed that all the various organic substrates which they tested consumed more available chlorine from sodium hypochlorite than from chloramine-T, and still less from axochloramide. However, the chlorine solutions used in these experiments contained 200 p.p.m. available chlorine. The hypochlorite solution of this strength would be much more alkaline than the chloramine solutions, and for this reason would consume more available chlorine. Langheld (1909) pointed out that sodium hypochlorite, because of its alkalinity, would react with a-amino acids to form chloramine acids which more or less readily break down t o give aldehydes, ketones, ammonia, carbonic acid, and sodium chloride. I n addition, Milroy (1916) observed th a t hypochlorites would be greatly reduced in efficiency by oxidation reactions with such amino acids as tyrosine and trypotophan where chlorine is substituted into the ring. The results of experiments with the chloramine, dichlorodimethyl hydantoin, have shown th at this compound was a s greatly affected b y heterogeneous organic matter as hypochlorite or gaseous chlorine if the strength and pH of the solutions were the same. Figure 9 shows th a t the concentrations of total available chlorine and HOCl were considerably reduced,by the addition of tomato juice to the solution. e. Bacterial Resistance to Chlorine. I n food plant chlorination the high resistance of certain bacterial forms to killing by chlorine must be taken into consideration. This factor becomes appreciable only when the bacterial cells are in the spore stage. Tonney et al. (1930) found bacterial spores t o be 10 to 1100 times more resistant to chlorine than vegetative forms. As a group, the aerobic spore-formers were found to be more resistant than the anaerobes. Phillips (1952), in discussing the relative resistance of spore and vegetative forms of bacteria, pointed out that spores are much more resistant t o chlorine and quaternaries than t o alkylating agents, such as ethylene oxide, methyl bromide, and formaldehyde. The latter group have the notable ability of attacking the cell at multiple points. Four or more reactive groups found in proteins are susceptible to attack. Phillips (1952) agreed with others (Green and Stumpf, 1946; Knox et al., 1948) t ha t the point of attack for chlorine is probably the -SH groups of essential enzymes. Spore formation could involve changes in molecular configuration which would give protection from attack on the -SH groups. I n this case the killing action of chlorine would be greatly slowed down. Williams and Reed (1942) and others have shown th a t the resistance

157

CHLORINE IN FOOD PLANT SANITATION

of bacterial spores to killing by heat increases, within certain limits, with increases in incubation temperature a t which the spores are formed. An interesting correlation exists between optimum growth temperature of spore-forming bacteria and resistance of the spores to killing by moist heat or chlorine. Increased resistance to moist heat is accompanied by

,,,r

I

\ -\

-

OPT. GROWTH RANGE 105 TO 130.F HEAT RESlSTANCE 20 MIN. AT 2 5 0 . F

-

I* 1 0 0 0 L

8 . COAGULANS

2-

- 0 -. 7 100MIN.T OATI2O.F250.F

OPT. GROWTH RANGE

3-

HEAT RESISTANCE

F-

M

2 v)

loo

8d z -

-

OPT. GROWTH RANGE 95 TO 105.F HEAT RESISTANCE 0 . 2 2 MIN. AT 2 5 0 . F

-

I0

I

40

I

80

I

I20

I

16 0

I

200

I

240

TIME IN MINUTES

FIG.10. Survivor curves for spores of three bacterial species. (Exposed to 15 p.p.m. available chlorine to show relationship between chlorine resistance, growth temperature range, and heat resistance.) (From Mercer, 1955.)

increased resistance to chlorine. This correlation for four food-spoilage bacteria is shown in Fig. 10. The usual chlorination procedures in food plant operations are designed t o destroy only the vegetative forms of bacteria. The concentration of chlorine in processing water which would be required t o kill all types of spores within a practical period of time would not be feasible. For example, the water used for cooling cans of food after the heat sterilization process and th at used for product washing has a chlorine content of 5 to 7 p.p.m. This low chlorine concentration and the shortness of the usual contact time could not destroy large numbers of resistant, spores. The objective in the chlorination of food-processing waters is to

158

WALTER A. MERCER AND IRA I . SOMERS

apply chlorine continuously in a concentration sufficiently high to kill all vegetative forms within the contact time allowed, and by this means to prevent the development of spore forms which, if present in the product, might survive the sterilization process and cause spoilage of the food.

4. Large Scale Applications of Chlorine The extensive use of chlorine in food plants as a sanitary control procedure requires accurate and controlled methods of application. a. In-Plant Chlorination. The addition of chlorine to the entire incoming water supply is known as in-plant chlorination. Automatic solution injectors are used to continuously maintain in the water a free available chlorine residual of 5 to 7 p.p.m. During cleanup periods the rate of chlorine addition is increased to obtain residuals of 15 to 20 p.p.m. Various types of chlorinators are used to inject chlorine into the water. Gaseous chlorine is added by means of equipment which dissolves the gas in water and then injects the strong chlorine solution into the water supply line. The rate of addition may be manually controlled or may be automatically proportioned to the rate of water flow. Hypochlorites are usually added by means of chemical pumps which inject the hypochlorite solution directly into the water supply line. b. Chlorination of Can-Cooling Waters. After heat-sterilization, cans of food are usually cooled with water either in the retort, in a cooling canal, by spraying, or by combinations of these methods. During the cooling process, an internal vacuum is developed in the cans. Often an apparently normal can seam will allow the aspiration of minute amount,s of cooling water. If the water is contaminated, bacteria may be taken into the can and its contents spoiled. A rapid build-up of bacteria can occur in cooling water held in tanks or recirculated over cooling towers. The continuous addition of chlorine to these waters will prevent a bacterial build-up and maintain a water of good bacteriological quality. Merrill et al. (1938) demonstrated a reduction from 9000 bacteria per milliliter in one cannery cooling water and 100,000 per milliliter in a second cooling water to less than 25 per milliliter by the addition of chlorine to give a free residual of 1 p.p.m. Such reductions in cooling water contamination result in marked reductions in can spoilage rates. I n one of the earliest experiments on cooling water chlorination, spoilage in No. 10 cans of cream-style corn was reduced from 7.80 cans per 1000 cans cooled in unchlorinated water to 1.37 per 1000 for cans cooled in chlorinated water (Scott, 1937). A reduction of can spoilage from 11 per 1000 for cans cooled in unchlorinated water to 0.33 can per 1000 after chlorination of the same cooling water was reported by Merrill et al. (1938).

CHLORINE IN FOOD PLANT SANITATION

159

The method of chlorinating cooling water will depend on how the cooling water is applied. When the water is used only once, then discarded, the concentration of chlorine maintained by an in-plant chlorinator is adequate. However, if the water is recirculated for can cooling or is held in a cooling canal, more chlorine is required than the amount added in the fresh make-up water, unless the volume of make-up water is unusually large.

FIG.11. General plan for counterflow reuse of flume water in a pea cannery involving successive rechlorination of the water. (From Mercer, 1953.)

Addition of chlorine to cooling water in canals or recirculation systems is usually by means of chemical solution feed pumps. Hypochlorite solutions are best suited for this. c. Chlorination of Waters Reused for Purposes Other than Cooling. When an inadequate supply of water makes it necessary to reuse water in food preparation departments, the counterflow method should be employed wherever possible. This provides for successive uses of the water in reverse order to the flow of the product over the lines. Fresh water is used for the final washing or fluming of the product before canning. This water is collected and second, third, and fourth uses made of the same water in intermediate stages of product preparation. The last use may be for pumping or washing the raw product as it enters the preparation lines, after which the water is discarded or used for fluming waste. I n Fig. 11 is shown the general plan of a typical counterflow system for reusing flume water in a pea cannery. I n the counterflow reuse system, the water is collected in a separate

160

WALTER A. MERCER AND IRA I. SOMERS

tank after each use where it is rechlorinated before the next reuse. A gaseous chlorinator other than the in-plant chlorinator is used for this purpose. It is thought that successive rechlorination with hypochlorite would result in the accumulation in the water of chemicals other than chlorine which might affect the quality of the product. Sufficient chlorine is added to the reused water before each use to give a trace of free available chlorine at the end of the next use. The organic

Log of bacteria count ml.of reused water

per

Date of sampling

FIG.12. Effect of the rate of production on chlorine residua18 and the numbers of bacteria per milliliter of reused water. (FromMercer, 1951.)

chlorine demand of reused water is ordinarily high. This results in a high concentration of combined available chlorine having a lower but significant germicidal effect. In Fig. 12 is shown the effect of the rate of production in a pea cannery, as measured by the number of cases packed per hour, the gallons of water used per case, chlorine residual, and the numbers of bacteria per milliliter of the reused flume water.

V. THE EFFECTS OF FOOD PLANTCHLORINATION The increasingly more widespread use of chlorine in food plant sanitation has demonstrated that general use of chlorination has been found to be an efficient, economical, and effective means of improving plant sanitation. At the same time, experience in chlorination has shown that it cannot be used as a panacea for sanitation problems. In-plant chlorination does not redeem a raw product of poor quality nor does it minimize the importance of proper sanitary practices in the plant.

CHLORINE IN FOOD PLANT SANITATION

161

1. Efect of Chlorination on Plant Sanitation

Addition of chlorine to the usual food plant processing water results in a marked reduction in the number of microorganisms present in the water, on equipment surfaces, and in the product. Scarlett and Martin (1948) reported th at the improvement in sanitation resulting from in-plant chlorination allowed a n average reduction of 50% in time required for plant cleanup. Stanley (1948) wrote th a t chlorination improved cleanliness, reduced overhead costs, and lowered the accident rate from falls on floors slippery with bacterial slime. Haynes and Mundt (1948) found th at use of in-plant chlorination reduced the bacterial count on frozen beans by 35% to 56% and at the same time eliminated slime from equipment surfaces and markedly reduced the time needed for plant cleanup. Goresline et al. (1951) reported on the use of chlorine in a poultry eviscerating plant. With 10 p.p.m. chlorine added to the water used for the final wash of the carcass, a 78% reduction in bacterial contamination was achieved. Addition of 20 p.p.m. gave a reduction of 90%. The chlorine cost for a plant dressing 20,000 birds a day was approximately $2.00. Filice (1953) concluded after a season of experimental chlorination in a fruit cannery th at chlorine was a useful tool in plant sanitation. Chlorination gave a cleaner plant, lessened the chances of can spoilage, and reduced t he cost and time required t o keep the plant clean. 2. E$ect of Chlorination on Containers and Equipment

Chlorine in high concentrations is corrosive to the common metals, as shown in Table V. However, under usual food plant conditions and with the concentrations recommended (5 t o 7 p.p.m.) for in-plant chlorination, chlorine does not noticeably corrode either cans or equipment. Somers (1951) after surveying a number of plants using in-plant chlorination reported th at there were no indications of corrosion attributable t o chlorination where recommended levels of chlorine were used during operating periods and no more than 10 t o 25 p.p.m. during cleanup. Some plant operators stated that less corrosion occurred when chlorine was used, since this prevented slime deposits underneath which serious corrosion could occur. The role of microorganisms in the corrosion of iron and steel has been discussed by Thomas (1941). The more extensive use of chlorine in food plant sanitation during the last few years has not caused any reported instances of corrosion to food-handling equipment. However, the possibility of corrosion of cans cooled in chlorinated water should not be overlooked. If the cooling

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WALTER A. MERCER AND IRA I. SOMERS

water contains sulfates or chlorides the addition of chlorine increases the tendency toward can corrosion. This may be counteracted by the addition of a corrosion inhibitor such as sodium chromate. TABLEV Effect of Chlorine on Metal and Other Surfaces* Effect of chlorine solutions ~

Material Glass, earthenware, silver,a tantalum, most precious metals, bitumastics (tar), hard rubber Soft gum rubber, fabrics, concrete Wood Iron, steel, stainless steel, copper, brass, aluminum, tin

~

5 p.p.m.

100 p.p.m.

1000 p.p.m.

None None None

None None .None

None Disintegrates

None

-

Corrodesb ~~

Corrodes ~

*National Canners Assoc. A Laboratory Manual for the Canning Industry, 1954. Protection of silver is due to formation of silver chloride and if this is removed by abrasion corrosion will result. b Corrosion occurs if application is continuous. A periodic application of a few minutes contact may have very little effect. The lower t h e p H the more corrosion will result. a

3. E$ect of Chlorination on Quality of Foods

Accumulated experiences with the chlorination of water used in the preparation of fruits and vegetables indicate that chlorine levels sufficient to give satisfactory plant sanitation can be used in washing and conveying the product without damage to its quality. However, chlorine must be excluded from the sirups used in the canning of fruits. As already discussed, chlorine does not undergo a true chemical reaction with sugars. When present in a sugar sirup it retains the characteristic taste and odor of chlorine. This may cause an off-flavor in fruits to which the sirup is added. The effects of chlorine on 29 fruits and vegetables are shown in Table VI. Except in the case of Rome Beauty apples, no off-flavors resulted when chlorine in a concentration of 50 p.p.m. was present in all processing waters except that used in making sirups and brines. When 5 p.p.m. was added directly to the canned food or to the brine or sirup used in packing the foods, off-flavors resulted in apples, figs, clingstone peaches, pears, strawberries, vegetable juice cocktail, and yams. It was concluded on the basis of these results that off-flavors from chlorine would not occur in the products tested if the process water contained no more than the recommended dosage of 4 to 5 p.p.m. and if unchlorinated water was used for making the sirup.

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CHLORINE IN FOOD PLANT SANITATION

TABLEVI Effect of Chlorine Treatment on Flavor of Canned Foodso Lowest concentration which produced off-flavor when 2, 5, 10, and 50 p.p.m. of chlorine were added

Product Applesauce, Rome Beauty* Applesauce, Gravensteinb Apricots, halves unpeeled Apricots, whole peeled Asparagus, all green Beans, green cut Beans, green limas Beans, with pork (recanned)b Beets, red sliced Carrots, sliced Carrots, purBedb Cherries, Royal Anne Corn Figs, whole Kadota Grapefruit juice (recanned)b Orange juice (recanned)b Peaches, clingstone halves Peaches, Elberta halves Peas Pears Pineapple juice (recanned)6 Potatoes, sweet, solid packb Pumpkin, solid packb Prunes, Italian Spinach Strawberries, whole Tomato juiceb Vegetable juice cocktail (recannedlb Yams, sirup pack a

b

Somers, 1951. Chlorine added directly t o the product.

Partial treatment chlorination of all water except brines & sirups

Complete treatment chlorination of all water including brines & sirupa

Chlorine (p.p.m.) 10 (None at 50) (None a t 50) (None at 50)

Chlorine (p.p.m.)

50 50 50 50

(None a t 50) (None at 50) (None at 50) 50

-

(None at 50) (None at 50)

50

(None at 50) (None at 50) (None at 50) 50

(None a t 50) -

~~

5 10 50 50 50 10 10 50

10

10 50 50

(None with 15) 5 50 50

5 10 (None with 10) 2 to 5 10 50 50

10 10 5 to 10 10 5 5

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WALTER A. MERCER AND IRA I. SOMERS

T A B LVIIa ~

Effect of Chlorine on pH and Ascorbic Acid of Canned Foodsb

Product Apricot Apricot Apricot Apricot Apricot

halves halves halves halves halves

Asparagus, Asparagus, Asparagus, Asparagus, Asparagus,

all green all green all green all green all green

Clingstone peaches Clingstone peaches Clingstone peaches Clingstone peaches Clingstone peaches Strawberries Strawberries Strawberries Strawberries Strawberries Tomato juice Tomato juice Tomato juice Tomato juice Tomato juice

Degrees Brix

Chlorine concentration (p.p.m.Y

PH

Ascorbic acid (mg./100 g.)

22.2 19.2 19 .o 19.2 20.2

0.0 2.0 5 .O 10.0 50 .O

3.86 3.88 3.88 3.88 3.86

9.1 9.1 9.6 9.4 8.9

-

-

0 .o 2 .o 5 .O 10 .o 50 .O

5.56 5.63 5.64 5.62 5.57

8.9 7.6 7.1 7.5 8.8

21.6 21.6 22.8 20.8 20.6

0 .o 2 .o 5 .O 10 .o 50 .O

4.04 3.97 4.06 3.93 4.09

5.7 5.7 5.8 6.2 5.8

-

0 .o 2 .o 5.0 10 .o 50 .O

3.73 3.60 3.63 3.70 3.63

26.8 25.1 24.3 27.8 25 . O

0 .o 2.0 5 .O 10 .o 50.0

4.50 4.48 4.51 4.50 4.51

27.8 27.2 27.5 26.8 25.2

-

-

-

Somers (1951). Testa made on samples 3 days after packing. * Chlorine waB added t o all water used. including brines and sirup.

a

The use of chlorinated water for the making of brines for canning vegetables is considered t o be optional. Brine-packed vegetables are not sensitive t o off-flavors from chlorine. However, canned vegetables intended for use in low-sodium diets should not be packed in water containing chlorine. The pH and ascorbic acid content of five products canned with varying amounts of chlorine are given in Table VII. The p H of the foods was

CHLORINE IN FOOD PLANT SANITATION

165

not noticeably affected by the addition of chlorine. T h e ascorbic acid content of tomato juice was slightly reduced when 50 p.p.m. chlorine was added. High concentrations of chlorine may cause discoloration of the fruit or vegetable t o which it is added. Somers (1951) reported that with apples and pears the addition of 50 p.p.m. to the sirup or pureed product produced a slight darkening. Lower concentrations of chlorine did not cause the discoloration. Such vegetables as green peas show slight fading of color if they are exposed to high concentrations of chlorine. Green peas exposed to 50 p.p.m. chlorine in water showed no apparent bleaching after 1 minute but were slightly faded after 3 minutes. Similar tests with asparagus showed no color changes from chlorine concentrations u p t o 60 p.p.m. (Mercer, 1955). Chlorine in combination with other chemicals sometimes present in food-processing plants may cause off-flavors and odors in the finished product. Chlorine in reaction with cresols, phenols, or phenol-like compounds, will produce chlorophenol, which, when present in minute concentrations in foods, imparts a strong unpleasant taste and odor. Such materials a s marking inks, paints, fly sprays, wood preservatives, hand lotions, and boiler feed water compounds, may produce chlorophenol when brought in contact with chlorinated water.

VI. SUMMARY The use of chlorine in food plant sanitation began with the addition of chlorine compounds to waters used for washing and rinsing equipment during routine cleaning periods. The canning industry extended the use of chlorine by adding it to waters used for cooling heat-sterilized cans. This prevented or reduced can spoilage due to aspiration of bacterial contaminants during the cooling process. Freezers, dehydrators, and canners of foods found that dripping chlorine solutions onto conveyor belts and other equipment surfaces improved sanitation by inhibiting the growth of slime-producing bacteria. The development of “break-point ” chlorination led to “in-plant ” chlorination which allowed the continuous addition of germicidal concentrations of chlorine to all processing waters. This more extensive use of chlorine inhibited microbial growth on equipment, reduced bacteria counts on the finished product, and permitted longer periods of plant operation by reducing the time required for plant cleaning. The remarkable germicidal efficiency of chlorine in water is attributed to its ability t o attack and inactivate enzymes essential for life of the microbial cell. Hypochlorous acid (HOC1) formed by chlorine compounds in solution is shown to be the germicidal agent. For purposes of food plant

166

WALTER A. MERCER AND IRA I. SOMERS

chlorination the time required by a solution of gaseous chlorine, hypochlorite, or chloramine to kill exposed bacteria is a n inverse function of the concentration of undissociated HOCl formed in the solution in accordance with hydrolysis and ionization equilibria. The successful chlorination of food-processing waters requires a n understanding of the chemical and physical conditions which influence the germicidal activity of chlorine solutions. Included in these conditions are the p H of the solution, the concentration of available chlorine, the concentration of organic material, and the temperature of the solution. Of these conditions the p H has the most pronounced influence. The hydrogen ion concentration determines the fraction of the hypochlorous acid which is present as the undissociated molecule (HOC1) and as hypochlorite ion (OC1-). As the hydrogen ion concentration decreases the ionization of HOCl increases to give hypochlorite ion which has no practical germicidal properties. With a given chlorine solution its germicidal efficiency increases as the p H becomes more acid. The extensive use of chlorine as a sanitizing agent in food plants requires accurate and controlled methods of application. Automatic chlorine solution injectors are widely used to maintain a chlorine residual of 5 t o 7 p.p.m. in processing and can-cooling waters. During plant cleaning the rate of addition may be increased to 15 to 20 p.p.m. T h e continuous addition of chlorine solution to recirculated processing and can-cooling waters has made possible considerable reductions in water consumption without undesirable effects on food quality or plant sanitation. The application of chlorine in food plants must not be indiscriminate. Off-flavor may result from excessive use of chlorine in the processing of certain sensitive fruits. Canning sirups must be free of chlorine. When used as recommended, chlorination has not been detrimental t o food quality, and corrosion of metal equipment due to chlorine has not been appreciable. Experience has now demonstrated th a t chlorination is a n effective and economical aid in maintaining good plant sanitation.

REFERENCES Ames, A. M., and Smith, W. W. 1944. The temperature coefficient of the bactericidal action of chlorine. J . Bacleriol. 47, 445. Andersen, E. E., Esselen, W. B., and Fellers, C. R. 1949. Effect of acids, salt, sugar, and other food ingredients on thermal resistance of B . Thermoacidurans. Food Research 14,499. Andrews, F. W., and Orton, K. J. P. 1904. A study of the disinfectant action of hypochlorous acid with remarks on its practical application. Zentr. Bakteriol. Abt. Orig. 36, 645,811. Black, A. P., and Goodson, J. B., Jr. 1952. The oxidation of sulfides by chlorine in dilute aqueous solutions. J. Am. Water Works Assoc. 44, 309.

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BrownIee, D. S., Guse, U. C., and Murdock, D. I. 1947. In-plant chlorination of cannery water supply. Food Packer 28 (l), 52. Buchanan, R. E., and Fulmer, E. I. 1930. “Physiology and Biochemistry of Bacteria,” Vol. 11. Williams & Wilkins, Baltimore. Cameron, E. J. 1939. Advantages and disadvantages of chlorination of cooling water. Canning Trade 61 (28), 58. Charlton, D., and Levine, M. 1937. Germicidal properties of chlorine compounds. Iowa Eng. Expt. Sta. Iowa State Coll. Bull. 132. Chick, H. 1908. An investigation on the laws of disinfection. J . Hyg. 8,92. Dakin, H. D. 1915. The antiseptic action of hypochlorites. Brit. Med. J . ii, 809-810. Dakin, H. D., Cohen, J. B., and Kenyon, J. 1916. Studies in antiseptics. 11. On chloramine, its preparation, properties, and use. Brit. Med. J . i, 160. Dakin, H. D., Cohen, J. B., Daufresne, M., and Kenyon, J. 1917. The antiseptic action of substances of the chloramine group. Proc. Roy. Soe. B89, 232. Darnall, C. R. 1913. Apparatus for purifying water by chlorine gas. U. S. Patent 1,060,669. Douglas, I., and Johnson, T. B. 1938. The interaction of chlorine with different types of organic sulfur compounds. J . Am. Chem. Soc. 60, 1486. Falk, I. S., and Window, C. E. A. 1926. A contribution to the dynamics of the toxicity a,nd the theory of disinfection. J . Bacteriol. 9, 1. Filice, B. A. 1953. Chlorination as applied to sanitation in a fruit cannery. Western Canner and Packer 46 (4), 25, 55. Fisher, C. V. 1942. Influence of wetting agents on various antiseptics. Am. J. Public Health 32, 389. Goresline, H. E., Howe, M. A., Baush, E. R., and Gunderson, M. F. 1951. In-plant chlorination does a 3-way job. U.S. Egg and Poultry Mag. 67 (4), 12. Green, D. E., and Stumpf, P. K. 1946. The mode of action of chlorine. J. Am. Water Works Assoc. 38 ( l l ) , 1301. Grifin, A. A. 1946. Break-point chlorination practices. “Technical Publication No. 213.” Wallace and Tiernan, Newark, N. J. Guiteras, A. F., and Schmelkes, F. C. 1934. The comparative action of sodium hypochlorite, chloramine-T, and Azochloramid on organic substrates. J . Biol. Chem. 107, 235. Hadfield, W. A. 1954. Chlorine and chlorine compounds. “Antiseptics, Disinfectants, Fungicides, and Sterilization.” Lea & Febiger, Philadelphia. Hall, J. E., and Blundell, C. C. 1946. The use of break-point chlorination and sterilized water in canning and freezing plants. National Canners Assoc. Information Letter No. 1073. Harris, J. J. 1946. Chlorination in the food plant. Canner, Part I, 103 (9), 18, 48; Part 11, 103 (lo), 14, 26. Haynes, R., and Mundt, J. 0. 1948. These chlorine sprays improve processed green beans. Food Inds. 20, 71. Holwerda, K. 1928. On the control and degree of reliability of the chlorination process of drinking water, in connection with the chloramine procedure and the chlorination of ammoniacal water. Mededeel. Dienst Volksgenzondheid Ned. Indie 17, Part 2, 251. (Seen in abstract.) Holwerda, K. 1930. Concerning the control and the degree of reliability of the chlorination process of purifying drinking water, especially in relation to the use of chloramine for this purpose and the chlorine process as applied to water containing ammonia in solution. Mededeel. Dienst Volksgenzondheid Ned. Indie 19, Part 2, 326. After Charlton and Levine, 1937.

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Ingols, R. S., Wyckoff, H. A., Kethley, T. W., Hodgden, H. W., Fincher, E. L., Hildebrand, J. C., and Mandel, J. E. 1953. Bactericidal studies of chlorine. Znd. Eng. Chem. 46, 996. Johns, C. K. 1930. The speed of germicidal action of chlorine compounds upon bacteria commonly occurring in milk. Sci. Agr. 10, 553. Johns, C. K. 1934. Germicidal potency of chlorine compounds. I. Hypochlorites. Sci. Agr. 14, 585. Johnson, G.A. 1911.Hypochlorite treatment of public water supplies, its adaptability and limitations. Am. J . Publzc Health 1, 562. Knaysi, G. 1930a.Disinfection; do bacteria die logarithmically? J. Infectious Diseases 47, 322. Knaysi, G. 1930b.Disinfection; some properties of frequency curves and their use in studies of disinfection. J. Infectious Diseases 47, 328. Knox, vc‘. E., Stumpf, P. K., Green, D. E., and Auerbach, V. H. 1948. The inhibition of sulfhydryl enzymes as the basis of the bactericidal action of chlorine. J . Bacteriol. 66, 451. Koch, R. 1881. Ueber disinfection. Mztt. Kaiser. Gesundheit. 1,234. (After Chick, H., 1908. J . Hyg. 8,92.) Langheld, K. 1909. Degradation of a-amino acids to aliphatic aldehydes b y means of sodium hypochlorite. Ber. dent. chem. Ges. 42, 2360. Leech, P. N. 1923. Examination of American-made chloramine-T, dichloramine-T, halasone and preparations. J . Am. Pharm. Assoc. 12, 592. Loveless, W. G. 1934. Chlorine products as germicides on dairy farms. Vermont Agr. Expt. Sta. Bull. No. 369. McCulloch, E. C. 1945. “Disinfection and Sterilization.” Lea & Febiger, Philadelphia. MacGregor, D. R., and Elliker, P. R. 1953. Effect of chelating agents on the bactericidal activity of quaternary ammonium compounds. Proc. 69rd General Meeting, SOC.Am. Bacteriol. p. 13. Mallmann, W. L., and Schalm, 0. 1932. The influence of the (OH) ion on the germicidal action of chlorine in dilute solution. Mich. State COX Eng. Expt. Sta. Bull. No. 44. Marks, H. C., Wyss, O., and Strandskov, F. B. 1945. Studies on the mode of action of compounds containing available chlorine. J. Bacteriol. 49,299. Mercer, W. A. 1951. Chlorination studies on reused water in pea canneries. MS. Prepared for Annual Meeting of Northern California Section of Institute of Food Technologists. Mercer, W. A. 1953. Conditions which affect the germicidal activity of chlorine com7th Ann. Conf. Food Znd. Sanitarians. pounds. PTOC. Mercer, W. A. 1955. Unpublished data. National Canners Assoc. Western Research Laboratory. Merrill, C. M., Cameron, E. J., and Williams, C. C. 1938. Chlorination studies. Canner 86 (121,58. MiIroy, T. H. 1916. Action of hypochlorites and allied substances on proteins, and their behavior on injection. Biochem. J. 10, 453. Myers, R. P. 1930. What chemicals are best adapted for the sterilization of dairy Znst. Assoc. Milk Dealers 23, 75. equipment? I. Milk Bottles. PTOC. Phelps, E. B. 1909. The disinfection of sewage and sewage effluents. U. S. Geol. Survey Water Supply Paper No. 229. Phillips, C. R. 1952. Symposium on the biology of bacterial spores. Part IX. Relative resistance of bacterial spores and vegetative bacteria to disinfectants. Bacteriol. Revs. 16, 135.

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Public Health Service. 1939. Milk Ordinance and Code, Public Health Bull. 220. Prucha, M. J. 1927. Chemical sterilization in the dairy industry. Proc. Znst. Assoc. Dairy and Milk Inspectors 16, 319. Rahn, 0. 1945. Injury and death of bacteria by chemical agents. Biodynamica Monograph No. 3. Reed, J. M., Bohrer, C. W., Cameron, E. J. 1951. Spore destruction rate studies on organisms of significance in the processing of canned foods. Food Research 14, 499. Reynolds, H., and Lichtenstein, H. 1952. Symposium on the biology of bacterial spores. Part VIII. Evaluation of heat resistance data for bacterial spores. Bacteriol. Revs. 16, 126. Rideal, S. 1910. The influence of ammonia and organic nitrogenous compounds on chlorine disinfection. J. Roy. Sanzt. Znst. 31, 33. (After Charlton and Levine, 1937.) Rideal, S., and Evans, U. R. 1921. The effect of alkalinity on the use of hypochlorites. J . Soc. Chem. Znd. 40, 64. Ritchell, E. C. 1947. Chlorination of cannery water supply. National Canners Assoc. Information Letter No. 1200. Rudolph, A. S., and Levine, M. 1941. Factors affecting the germicidal efficiency of hypochlorite solutions. Iowa Eng. Expt. Sta. Iowa State Coll. Bull. No. 160. Scarlett, W. J., and Martin, R. D. 1948. Value of chlorination in frozen food processing. Canner 106 (13), 12, 30. Scott, G . C. 1937. Cooling tank contamination. Canning Age 18 (5),190. Semmelweis, I. P. 1861. Die aetiologie der begriff und die prophylaxis des Kindbettfiebers. Pest. Vienna, and Leipzig, Hartleberr. After Reddish, G. 1954. “Antiseptics, Disinfectants, Fungicides, and Sterilization,” p. 17. Lea & Febiger, Philadelphia. Somers, I. I. 1951. Studies on in-plant chlorination. Food Technol. 6 (2), 46. Stanley, M. R. 1948. Benefits of in-plant chlorination at Puyallup. Western Canner and Packer 40(6), 70. Thomas, A. H. 1941. Role of bacteria in the corrosion of iron and steel. Proc. 2nd Ann. Water Conf. Eng. SOC.Western Penn. Reprinted by Wallace and Tiernan, Technical Publication No. 245. Tonney, F. O., Greer, F. E., and Liebig, G. F., Jr. 1930. The minimal “chlorine death points” of bacteria. 11. Vegetative forms. 111. Spore-bearing organisms. Am. J. Public Health 20, 503. Traube, M. 1894. Einfaches verfahren wasser in grossen menger keirnfrei zu machen. 2.Hyg. 16, 149. After Charlton and Levine, 1937. Vaughn, R. H., and Stadtman, T. C. 1946. Sanitation in the processing plant and its relation to the microbial quality of the finished product. Food Freezing July, 1946. Weber, G. R., and Levine, M. 1944. Factors affecting germicidal efficiency of chlorine and chloramine. Am J. Public Health 34, 719. Whittaker, H. A., and Mohler, B. M. 1912. The sterilization of milk bottles with calcium hypochlorite. Am. J . Public Health 2, 282. Williams, 0. B., and Reed, J. M. 1942. The significance of the incubation temperature of recovery cultures in determining spore resistance to heat. J . Infectious Diseases 71, 225. Zuch, T. L., and Somers, I. I. 1946. Unpublished data. National Canners Assoc. Western Research Laboratory .