[Advances in Food Research] Advances in Food Research Volume 16 Volume 16 || Food Quality as Determined by Metabolic by-Products of Microorganisms

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    BY M. L. FIELDS AND BONNIE S. RICHMOND Department of Food Science and Nutrition University of Missouri, Columbia Missouri



    Department of Food Science and Nutrition, and School of Home Economics University of Missouri, Columbia, Missouri

    I. Introduction ........................ ............................... II. Definition and Criteria for Chemical Indicators ...........................

    III. Chemical Indicators of Quality for Foods with High Protein Content .... A. Background .............................................................. B. Dominant Spoilage Flora ............................................... C. Chemical Indicators of Microbial Spoilage .............................

    IV. Chemical Indicators of Quality for Foods with High Fat Content ........ A. Background ........................... ........................ B. Dominant Spoilage Flora ............................................... C. Chemical Indicators of Microbial Spoilage .............................

    Content ...................................................................... A. Background ................................................. B. Dominant Spoilage Flora ..................................

    V. Chemical Indicators of Quality for Foods with High Carbohydrate

    C. Chemical Indicators of Microbial Spoilage ............................. VI. Research Needs .................................... .....................

    References ...................................................................

    161 162 163 163 167 168 198 198 198 199

    203 203 204 206 218 2 19


    Food quality is largely the sum of the characteristics which register favorably or adversely on an individual's senses. These characteristics include freshness, flavor, odor, texture, tenderness, consistency, color, size and shape, degree of ripeness, and presence or absence of defects. Nutritive value, chemical residues, and disease-producing organisms are also a part of food quality, although these are not measured by the senses of smell, taste, sight, or touch.

    ' Formerly with Dept. of Horticulture, University of Missouri. 161


    When one visualizes a spectrum ranging from good to poor quality, the extremes are easily differentiated. This is not true for the central part of the spectrum, however, especially for products in which the manufacturing process tends t o mask differences, as with comminuted foods. When foods become more abundant, standards of quality are likely to be higher and more clearly defined. Quality which is acceptable in one society may not be in another.

    The consumer and many manufacturers depend upon the senses of smell, taste, sight, and touch for evaluating food quality. These organoleptic methods are qualitative and vary from individual to individual. The need for more adequate evaluation has focused atten- tion on chemical compounds which can be used for differentiation. Certain compounds arising from the metabolism of the dominant spoilage organisms answer this need and can be classified as chemical indicators of food quality.

    Chemical indicators may be the only means of evaluating quality in some foods, since processing methods, such as filtration, preclude the use of conventional methods such as plate counts. In some foods, indicator compounds may supplement microbiological methods of analyzing food quality, including plate counts, mold counts, rot fragment counts, and direct microscopic counts. The use of chemical indicators will assist the manufacturer in producing and maintaining high-quality foods.


    A chemical compound which indicates deterioration due to micro- organisms may be defined as a metabolic by-product which is produced by the dominant spoilage organisms as a result of their growth in the food. The dominant spoilage flora is that group of microorganisms which persists and brings about deterioration in the quality of a food under the usual handling and storage conditions. Since a specific spoilage situation may involve a mixed culture, measurement of multiple compounds may be superior to the use of a single metabolic by-product as an indicator of quality.

    Fields (1964b) proposed the following criteria for a chemical indicator of quality of foods: (1) The compound must be present a t low levels or absent in sound foods. (2) With increased spoilage, there must be an increase in the amount of the indicator. (3) The compound should make it possible to differentiate low-quality raw materials from


    poor processing conditions. (4) The indicator should be produced by the dominant spoilage flora. Farber (1952) stated that a spoilage index must be as reliable as organoleptic criteria and should indicate stages of spoilage which cannot be established definitely by organoleptic testing. To be useful as an index of quality for seafood and ground beef, the test for the compound must be rapid and the analysis must be simple (Novak et al., 1956; Rogers and McCleskey, 1961). Patterson (1945) emphasized that the compound should never yield a false positive test, and for this reason a companion test is desirable. It is true that some metabolic by-products of the dominant spoilage flora also might arise by autolysis, but the amount of the compound would be markedly lower than the levels associated with spoilage due to microorganisms.

    Table I summarizes the characteristics of potential and suggested chemical indicators of food quality. The suitability of the potential indicator may be determined, in part, by its physical properties. Compounds with low boiling points would be unsatisfactry where the use of heat in manufacturing or processing would volatilize them. Solubility of the compounds would affect the method of analyses. For example, acetic and formic acids can be removed from foods by steam distillation, while lactic acid must be extracted with ether or other solvents.

    Some of the chemical indexes may be very acceptable in certain foods but not in others. Acetylmethylcarbinol is a valuable constitutent of the flavor components in butter. In apple juice, however, the presence of large quantities indicates the use of unfit raw materials and/or poor sanitary conditions in the processing plant (Fields, 1962a). If the chemical compound has a useful function as a component in the food, it cannot be used as a chemical indicator of the quality of the food. One may compare the presence of a chemical indicator to the presence of some microorganisms in foods. Penicillia are very essential in producing certain cheeses but are detrimental to quality when they grow in citrus fruits and produce decay.



    Research on the quality of high-protein foods as indicated by meta- bolic by-products of microorganisms .has centered on seafoods, pro- bably because they are so highly perishable. Studies on the decomposi- tion of fish have been conducted by processors and by governmental


    Solubility ( s ) Boiling point Cold Alcohol

    Indicator Description ("C) water acid, etc.

    Acetic acid

    Acetylmethylcarbinol Liquid above 15OC with a pleasant

    Clear, colorless acid, liquid C,H,O, Pungent odor

    (AMC, acetoin, 3-

    butanone) Ammonia

    N H 3 Butyric acid

    Diacetyl (biacetyl,

    odor, a product of fermentation hydroxy-2-

    Colorless gas, pungent odor. Lower

    Colorless, limpid liquid. Rancid odor

    Yellowish green liquid. Quinone odor

    limit for human perception, 53 ppm


    C*H,O, 2, 3-butanedione)

    Dimethylamine Gas, strong arnmoniacal odor C,H,N

    Ethyl alcohol (ethanol) C,H,OH

    Formic acid Colorless, fuming liquid. Pungent CHZOZ penetrating odor. Dangerously

    Clear, colorless, flammable liquid


    needles Galacturonic acid White powder, forms monohydrate



    - 33.35








    very s







    s alcohol


    s alcohol; ether

    s alcohol; ether


    s alcohol; ether

    s alcohol; ether

    slightly s hot alcohol; insoluble ether

  • Histamine C,H&

    Hydrogen sulfide H,S


    Lactic acid C*H,N


    Oleic acid

    Palmitic acid

    Propionic acid

    Stearic acid

    Succinic acid



    c I sH34Oz C,tlH,,OZ




    C 3 H P


    Valeric acid C5HlOO2

    Yellow crystals (needles) from water, has medical uses, occurs as a result of putrefaction

    Colorless gas, flanlmable, offensive odor, sweetish taste, dangerously poisonous

    intense fecal odor Colorless to yellowish scales,

    Colorless to yellowish syrupy liquid

    Colorless or nearly so, odorless

    White, crystalline scales liquid

    Clear, colorless liquid. Pungent odor

    White leaflets

    Colorless crystals. Odorless. Acid

    Gas, pungent, fishy ammoniacal

    Essential amino acid for rats. Found



    in casein and other proteins Used as nutrient for humans White crystals

    Disagreeable odor and taste Clear, colorless liquid


















    slightly s alcohol

    s alcohol

    very s alcohol; ether s benzene

    s alcohol; ether glycerin; insoluble chlorine, petroleum ether


    slightly s in cold; very s in hot

    s alcohol; ether

    slightly s s

    S sp. s alcohol; ether

    very s

    slightly s s hot alcohol

    very s alcohol; slightly ether, benzene

    insoluble chlorine

    slightly s alcohol; ether



    Product Spoilage organism Reference

    Eggs Pseudomonas fluorescens Pseudomonas sp., Achromabacter sp.

    Proteus sp.

    Mucor, Thamnidium, Botrytis, Alternaria, Cladosporium, Penicilliwn, Sporotrichwn

    Alcaligenes. Flauobacterium, Paracolobactrum

    Fish, salt Serratia, Micrococcus, Bacillus, Achromobacter, Pseudomoms

    Flavobacterium, Achromobacter, Escherichia, Bacillus, Serratia

    Achromobacter , Micrococcus , Kurthia , Pseudomonns, Flavobacterium, Proteus

    Staphylococcus, Pseudomonns,

    Frazier, 1958 Frazier, 1958; Florian and

    Frazier, 1958; Florian and

    Frazier, 1958.

    Trussell, 1957.

    Trussell, 1957.

    Florian and Trussell, 1957.

    Frazier, 1958.

    Griffiths, 1937.

    Snow and Beard, 1939.

    Shellfish Flauobacterium, Pseudomonas, Bacillus, Frazier, 1958. Proteus, Achromobacter.

    agencies charged by law with regulation of the purity of our food supply.

    Because of the extreme perishability of some products, decomposed foods get into market channels occasionally. For example, the U. S. Department of Health, Education, and Welfare (HEW) (1966) publish- ed Notices of Judgment under the Federal Food, Drug, and Cosmetic Act which include seizures due to decomposed material in frozen eggs (FNJ 30337, 30338, 30340, 30341, 30342, 30343, 30345), frozen red snapper (FNJ 30351), frozen flounder fillets (FNJ 30354), and fro- zen salmon (FNJ 30356).

    The chemical composition of the food and the metabolic activities of the organisms growing in the food determine the compounds which can be used as indicators. Although the high-protein foods (8.4- 25.2y0) included in this discussion contain 0.8-14.5% fat and 0.0-1.770 carbohydrate, our concern here is with compounds derived from the protein. It must not be overlooked, however, that substances arising from components other than protein may have a potential as indexes of decomposition. Used to detect spoilage in the past have been ammonia nitrogen, reducing substances such as dextrose, acidity of the fat, and bacteriological examination (Macomber, 1927).


    The dominant spoilage floras for eggs and sea foods are listed in Table II. Molds cause spoilage in eggs, but to a lesser extent than do bacteria. Many of the bacteria which cause decomposition in fish may also be responsible for loss of quality in eggs. The dominant flora of fish is composed of species which grow in the sea. The organisms are mainly gram-negative rods present in the slime covering the surface of the fish. Since these bacteria are psychrophilic, spoilage may pro- ceed even under conditions of refrigeration or icing.

    According to Zobell (1946), marine bacteria are actively proteolytic, and they rapidly attack most kinds of proteinaceous materials. Nearly all of them liberate ammonia from peptones, but only a few produce indole from tryptophane. Zobell also stated that marine bacteria are weakly saccharolytic.




    Number of species

    Indicator Positive Negative

    Ammonia 60 0 Indole 2 58 Hydrogen sulfide 31 29 Hydrolyzed fat 13 47

    Achromobaeter. Flouobaeterium, Pseudomonos. Serratia, Microeoecw, Actinomyces, Vibrio, Bacterium,

    *Compiled from Zobell and Upham. 1944. and Bacillus.


    EIGHT GENERA'.^ Number of species

    Indicator Information

    Positive Negative not given

    Ammonia Indole Trimethylamine Hydrogen sulfide

    29 14 6


    7 107 101 28

    8 129 34 95

    aA chmmobacter . Flouobacterium , R e d o m o n a s , Sorcina , Serratia , A lcaligenes , Proteus, Kurthia , and bCompiled from Bergeys Manual of Determinative Bacteriology (Breed et al., 1957).



    If the breakdown is aerobic, the process is called decay, whereas a breakdown that is anaerobic is called putrefaction. When putrefaction occurs, various foul-smelling compounds are produced. These arise as a result of bacterial action on amino acids and include mercaptans, indole, hydrogen sulfide, ammonia, amines, and organic acids. If the protein is well aerated, digestion occurs and no ill-smelling compounds are formed.

    The enzyme systems of microorganisms causing spoilage of protein include proteinases, peptidases, deaminases, and decarboxylases. The amino acids freed by the action of peptidases and the breakdown pro- ducts of amino acids resulting from deaminase or decarboxylase activity have been suggested as chemical indicators for the quality of protein foods. Other suggested compounds include succinic, formic and acetic acids, ammonia, indole, and hydrogen sulfide. Tables 111 and IV list genera of microorganisms and the number of species of these genera which are known to produce ammonia, indole, trimethylamine, or hydrogen sulfide.


    I. Ammonia

    a. Biosynthesis of Ammonia. Ammonia is a metabolic by-product of several bacteria which hydrolyze proteins. In fish, one of the principal sources of ammonia is urea (Elliott, 1952). As described by Salle (1961), ammonia is produced in conjunction with other products of enzyme action:

    1. Production of a fatty acid and ammonia by deamination, de- carboxylation, and oxidation.

    R-CHNH,COOH + H,O + 2 0 --t R-COOH + H,O + CO, + NH:, 2. Production of an alcohol and ammonia by deamination and de-

    carboxylation. R-CHNH,COOH + H,O + R-CH,OH + CO, + NH:,

    3. Production of an acid and ammonia by reductive deamination.

    Data in Tables 111 and IV illustrate the potential occurrence of ammonia as a by-product of microbial metabolism. Of the eight gen- era associated with the spoilage of fish and eggs, 29 species produce ammonia, 7 do not, and the information on ammonia yield is not available for 107 species. However, Breed et al. (1957) listed eight species of Achromobacter which have proteolytic enzymes and liquefy gelatin but for which information on ammonia production is lacking (Table IV). All of the 60 species described by Zobell and Upham (1944) produced ammonia (Table III).



    6 . Ammonia as an Indicator of Quality of Seafood, Ground Beef, and Eggs. Ammonia and other volatile bases were found to be unreliable as indexes in determining the quality of frozen breaded shrimp because of variation among samples and in the batter used for breading the shrimp meat, and the dilution effect of the breading, which lowered the volatile bases even in highly decomposed products (Gagnon and Fellers, 1958). Ammonia and amines increased regularly during the decomposition of canned salmon, but Clough (1922) pointed out that it was probably formed during cooking, and therefore was of little value as an indicator of quality. Ammonia may contribute to spoilage odors more in nitrated fish than in fish containing no nitrate. Therefore, Vaisey (1956) felt that ammonia was unsuitable as an index for the quality of fish processed by nitrating.

    Because free ammonia is driven off by heat, its determination is of little value when applied to dried eggs. According to Macomber (1927), the evolution of ammonia varied so that the results were high or low depending upon the drying conditions rather than upon the quality of the eggs. However, ammonia has been used success- fully as an indicator of the quality of fresh and frozen crabmeat (Burnett, 1965), fresh eggs (Boyce, 1950), fresh haddock (Stansby and Lemon, 1933; Crooks and Ritchie, 1938), and shell and frozen eggs (Macomber, 1927).

    In Burnetts (1965) study on ammonia as an index of decomposition in crabmeat, the ammonia content was found to increase uniformly and rapidly with spoilage. As shown in Table V, ammonia could be detected before spoilage could be ascertained organoleptically.

    According to standards suggested by Crooks and Ritchie (1938) for haddock, flesh containing 35 mg or less of ammonia per 100 g was normally sound and of good quality. When the ammonia content in- creased to 45 mg, the freshness was questionable. At 60 mg of ammonia per 100 g there was a stale or fishy odor, and it became distinctly putrefactive as 75 mg was approached.

    Early research (Stansby and Lemon, 1933) indicated that evidence of bacterial decomposition and odor of haddock could be classified into greups according to the amounts of acid required to titrate suspensions of 5 g of fish in 100 ml of water to pH 6.0. When only 4 to 6 ml of 0.0165N HCI was required, there was little evidence of bacterial de- composition and the odor was predominantly fresh, with some evidence of sweetness. Fishy odors were noted when 8 to 10 ml of acid was required. Odors were described as stale or putrid when 14 to 17 ml of acid was used in adjusting the pH of the sample to 6.0. The increase in amounts of acid required to titrate the suspensions of fish of lower quality was attributed to the larger amounts of ammonia associated with decomposition by bacterial action.


    According to Reed (1925), lobster meat which was delayed between boiling and sterilization tended to have a higher content of ammonia than perfectly fresh meat. Also, when the meat with higher ammonia content was canned, more tin was dissolved from the tinplate. Thus, ammonia-forming bacteria may be partially responsible for the discoloration of canned lobster.

    Hughes (1959) demonstrated that fresh flesh of herring contained some ammonia which increased continuously through a test period of 10 to 100 hours when temperatures were 10-13OC. Also, Crooks and Ritchie (1938) showed that the ammonia content increased with the length of storage of fillets of fish at 4-5OC. Hughes (1959) evaluated fresh, spoiling, and cooked herring flesh, and found that both ammonia and trimethylamine were produced. He also indicated that cooking flesh in sealed glass tubes at 12OOC up to five hours resulted in the breakdown of trimethylamine oxide and the formation of ammonia along with tri-, di-, and monomethylamine.

    The relationships between bacterial count, pH, ammonia content, and methylene-blue reduction time were investigated by Rogers and McCleskey (1961). When ground beef was stored at 70C, little change occurred in ammonia content, reduction time, or pH, and the bacterial count remained low for the first three days. During the fourth and fifth days of storage, all criteria changed markedly. The first distinct odor of spoilage was evident after five days of storage. Ammonia increased from 0.15 mg/g of ground beef before storage to 0.21 mg at three days, 0.40 mg at five days, 0.88 mg at seven days, and 1.05 mg at ten days. Earlier research (Falk and McGuire, 1919) indicated that beef held a t low temperature underwent autolysis, with the formation of 1.0 mg ammonia nitrogen per gram of meat, before it was judged unacceptable as food. However, beef held at room temperature was unfit for food when it contained 0.3-0.4 mg ammonia nitrogen per gram of meat. Beef chilled immediately after slaughter and tested within 24 hours contained 0.03 and 0.10 mg ammonia nitrogen per gram.

    Ammonia was used for over twenty years in the Massachusetts Department of Public Health as a means of evaluating eggs (Boyce, 1950). The free ammonia in strictly fresh eggs varied within reason- ably narrow limits (1.0-1.8 mgl100 g egg), and the amount of ammonia increased in an orderly fashion as the protein of the yolk deteriorated.

    Macomber (1927) stated that for several years the Bureau of Chemistry used the determination of ammonia nitrogen, reducing sugars, and acidity of fat, along with bacteriological examination, in testing frozen egg products for decomposed eggs.

    c . Methods for Determining Ammonia in Foods. Bandemer and


    Schaible (1936) developed a microdetermination for ammonia nitrogen in eggs. A 5-cm glass ring was cemented to the bottom of a 10-cm Petri plate to give a vessel with an inner and an outer chamber. Standard hydrochloric acid was added to the inner chamber. Ammonia- free water, egg white or yolk, and potassium carbonate (to liberate the ammonia) were placed in the outer chamber and mixed. The apparatus, with a weighted cover, was incubated at 38OC for 1+ hours. At the end of this period, the excess acid was titrated with 0.0025 N NaOH, and the ammonia was calculated as milligrams of ammonia nit- rogen per 100 cc of egg.

    Tubis (1937) modified the Bandemer and Schaible method (1936) by changing the construction of the cell and the time of absorption. In Tubis modification, the inner chamber of the cell consisted of a small Petri dish held in place temporarily by grease, instead of the glass ring cemented to the floor of the Petri plate. This improved ease of cleaning, and the end point of the titration was easier to see. Tubis modification required more time than the Bandemer and Schaible method, but less than the AOAC (1935) aeration method. The aeration technique consisted of washing the egg sample into an aeration cylinder with ammonia-free water. Alcohol was added, and the mixture was allowed to stand before combining it with sodium fluoride, sodium carbonate, and kerosene. The ammonia was swept into standard sulfuric acid and titrated. Callaway (1939) compared two methods of recovery: the aeration method and the absorption procedures of Bandemer and Schaible (1936). On the whole, the absorption method was preferable to the aeration method because it gave slightly higher yields and required less apparatus and attention.

    In 1933, Stansby and Lemon developed a rapid test of buffer cap- acity which indicated the accumulation of metabolic by-products of bacteria and also autolytic breakdown. Those workers stated that since the buffer capacity of the inorganic constituents of fish was at a minimum at pH 6.0 and 4.2, titration of a solution made from ground flesh to pH 6.0 was a measure of ammonia and an indication of bacterial decomposition. Titration to pH 4.3 was suggested as a measure of pro- tein hydrolysis.

    Boyce (1950) described a modification of the original Folin procedure for the determination of free ammonia in urine, as adapted for ammonia in liquid eggs. Essentially, this was an aeration method. The blended eggs were combined with potassium oxalate, petroleum ether, and sodium carbonate in an aeration cylinder. The ammonia was then flushed from the sample and trapped in standard hydrochloric acid. The amount of ammonia was determined by Nesslerization and visual matching against standard ammonium sulfate solutions.


    More recently, gas chromatography was applied to study the production of ammonia in fresh, spoiling, and cooked herring flesh (Hughes, 1959). Gas chromatography made it possible to analyze mix- tures of volatile bases.

    A method based upon the color reaction between ammonia, thymol, and bromine was used successfully by Burnett (1965) to determine ammonia as an index of decomposition in fresh and frozen crabmeat (Table V). The color was extracted into n-butyl alcohol, and the net absorbance was determined by subtracting absorbance at 475 mp from that at 682 m,u. The method is very rapid, requires no special apparatus, and has a sensitivity of about 1.0 ppm ammonia. Good reproducibility and low results were obtained on fresh crabmeat.

    d. Disadvantages and Advantages of Indicator. The major limiting factor in using ammonia as an indicator of spoilage is its volatility. Since its boiling point is -33.35OC, it is best suited for fresh and



    Ammonia content Average Organoleptic class (pg NH:, Per 9 ) (,a NH:, per 9)

    0 Fresh 0 0 0 1.8

    14.4 9.8 8.0


    2 Slight but definite 75.6 decomposition 62.0

    66.7 77.3 70.4

    1 "Fishy" but still good

    3 Advanced decomposition 134.9 124.0 110.7 144.4 128.5

    4 Putrid 191.1 167.7 156.7 187.6 175.8



    %urnett, 1965.


    for frozen products. Thus, ammonia can be used as an index for quality in fresh eggs (Boyce, 1950) but not for dried eggs (Macomber, 1927). Likewise, it is appropriate as a measure of the decomposition of raw salmon but not canned (Clough, 1922).

    Variability among samples, as cited by Gagnon and Fellers (1958), is a distinct disadvantage in using ammonia as an indicator. Although Burnett (1965) illustrated an increase in ammonia associated with spoilage in fish, it may be seen that there was considerable variation in the amounts of ammonia within organoleptic classes (Table V). Crooks and Ritchie (1938) felt that the relationship between quality of haddock and amounts of ammonia was sufficiently reliable to propose it as a standard indicator. Although Rogers and McCleskey (1961) found that ammonia increased in ground beef stored at 70C from 0 to 10 days, they did not suggest that it be used as an indicator of quality for this product.

    2. Indole

    a. Biosynthesis of Indole. In 1903 it was proposed that tryptophane gives rise to indole and skatole (Hopkins and Cole, 1903). Krebs et al. (1942) suggested that tryptophane is converted to indole by oxidation of the indole ring with the formation of an o-aniline derivative such as kynurenine, followed by a breakdown of the side chain and the form- ation of o-aminophenylacetaldehyde, which in turn yields indole. It is possible that indole might be formed as a result of oxidation of o-amino- P-phenylethyl alcohol to an aldehyde which could yield indole spontan- eously rather than by action of Escherichia coli or other indole-forming organisms.

    The production of indole is used as a diagnostic characteristic in determinative bacteriology. Tables I11 and IV illustrate the potential occurrence of indole as a by-product of microbial metabolism. This compilation from Bergeys Manual of Determinative Bacteriology (Breed et al., 1957) indicates that, of eight genera, 14 species are producers of indole and 101 are nonproducers. Data were not available for 28 species (Table IV). Zobell and Upham (1944) also reported that only a few marine bacteria liberate detectable quantities of indole from tryptophane. They studied 60 species, of which only two formed indole (Table In). Clough (1922) stated that only 31% of 229 different cultures of bacteria isolated from raw or canned salmon gave positive indole tests.



    Cooked (pg/lOOg) Description

    Class 0 0.0 0.0

    Class 1 8.0 8.0

    Class 2 4.0

    Class 3 161.0 159.0

    Class 4 992.0 1080.0

    0.0 0.0

    10.8 11.2

    4.4 3.2

    178.0 168.0

    840.0 936.0

    Almost no odor, odor of fresh

    Strong, old, or fishy

    Faint but recognizable odor of decomposition; feverish red discoloration beginning at edge of segments

    Repugnant, deep-seated odor of decomposition; may be ammoniacal; may be described as tainted

    caught shrimp


    Deep-seated nauseating odor of putridity; feverish red color more pronounced than in 2 and 3

    Duggan and Strasburger (1946).

    b. Indole as a n Indicator of Quulity of Fish. Farber (1952) found indole to be of limited significance as an indicator of spoilage in such West Coast fish as tuna, mackerel, and sardines, because relatively few organisms formed the compound. Lartigue et al. (1960) evaluated indole tests for oyster quality. Indole concentrations showed no definite pat- tern during storage, and therefore were not recommended for assess- ment of oyster quality. On the basis of preliminary investigation, Gagnon and Fellers (1958) rejected the indole test because they felt it was not reliable enough for their study of biochemical methods of evaluating frozen breaded shrimp.

    Other investigators have found indole to be satisfactory as a chemical index of freshness. Indole was suggested as an indicator of decomposi- tion by Clough (1922) for salmon, Beacham (1946) for canned oysters and clams, Duggan and Strasburger (1946) for raw shrimp, and Hillig (1963) for raw and frozen shrimp. Tables VI, VII, VIII, and M illus- trate that large amounts of indole are associated with low organoleptic quality of shrimp and oysters.

    The results of different studies varied widely in the amounts of indole associated with good and with poor organoleptic quality. In





    Interval hours

    0 24 48

    72 120 144 288

    1 .o 1.5 1.8

    1.0 2.1 4.0 5.3

    Organoleptic quality

    Fresh Slightly stale but not decomposed Faint sweetish odor of decomposition; gaseous

    Offensive sour, yeasty odor Same as above but more pronounced Even more pronounced than above Same as above; almost completely liquefied


    %ng and Flynn (1945)

    work of King and Flynn (1945), shucked raw oysters were considered slightly stale when there was 1.5 ,ug indole per 100 g (Table VII), whereas 1.9 ,ug per 100 g was associated with the class of drained canned oyster meat categorized as good (Table Vm). Beacham (1946) described canned oysters which contained as much as 33.8 ,ug per 100 g as having an odor which was slightly off (Table IX). King and Flynn (1945) indicated that 34.0 pg per 100 g canned oyster was as- sociated with a slightly putrid odor (Table Vm). Unwashed oysters produced large quantities of indole (Beacham, 1946).



    Good Indole content oyster (%) (pd100 9) meats after canning

    100.0 1.9 Good

    of drained meats Odor of drained oyster

    97.2 12.7 Slight odor of decomposition 94.1 11.5 Same as above 93.6 8.4 Very slight odor of

    92.1 13.2 Slight odor of decomposition 90.3 11.5 Same as above 89.5 6.6 Same as above 76.6 34.0 Distinct odor of


    decomposition; slightly putrid

    King and Flynn (1945)



    Description of raw material

    1007 Sound oysters Odor which was slightly off ,

    only superficial in character not deep-seated or repugnant

    Noticeable and repugnant odor, regarded as unfit for food

    Same as above but more intense Revoltingly putrid

    Indole Odor of canned oysters (pd100 8)

    None 1.2-3.0 None 16.5-26.6 Slight 33.8

    Slight 53.0-87.2

    Strong 85.0-132.0 Strong 100.0-144.0

    Beacham (1946).

    Barry et al. (1956) stated that the amount of indole in canned decomposed shrimp decreased after storage periods of several months. In addition, odors associated with decomposition were less recognizable as storage was prolonged. In contrast, Duggan and Strasburger (1946) reported that no appreciable change occurred in the indole content of raw and cooked shrimp stored at commercial holding temperatures for extended periods.

    The effect of cooking on indole was investigated by Duggan and Strasburger (1946). No indole was formed in shrimp by cooking. Neither did indole concentration change appreciably during cooking. Shrimp cooked in brine previously used to cook decomposed shrimp acquired a part of the indole and odors of decomposition from the brine.

    The effect of chlortetracycline on extending the storage life of oysters was studied by Lartigue et al. (1960). Although the antibiotic limited bacterial growth and undesirable odors, it did not suppress the formation of indole. In both antibiotic-treated and control oysters, indole decreased during the first six days of storage but no consistent pattern was demonstrated thereafter. Organoleptic ratings indicated onset of spoilage on about the 18th day of storage. On the basis of these results, Lartigue did not recommend indole as a useful quality test for oysters .

    Two studies reported finding no indole in fresh material. Beacham (1946) detected no indole in canned oysters and clams prepared from some raw material known to be fresh. Duggan and Strasburger (1946) found that fresh shrimp did not contain indole. However, small amounts of indole are normally found in commercially packed shrimp. An average of 1.5 pg per 100 g was found in 27 random market samples with a range of values from 0.0 to 4.3 pg per g of cooked peeled


    shrimp all of which were judged as passable. Also, both Beacham (1946) and Duggan and Strasburger (1946) reported that as the amount of decomposed material increased, indole appeared in larger amounts. In 138 salmon, only small amounts of indole were found after 48 hours of storage. Therefore, Clough (1922) proposed that indole in amounts of 1.5 pup or more per 100 g canned salmon was indicative of a con- siderable degree of decomposition.

    Indole was investigated as a possible index for quality of butter by Clarke et al. (1937). Indole was present in far greater amounts in butter made from decomposed cream than in butter made from fresh cream, but this was not true in all cases.

    c. Methods for Determining Indole in Foods. A collaborative study by various laboratories showed that the percent recovery of indole from shrimp ranged from 86 to lOlY0 and averaged 96.37,. The samples were finely ground, mixed, frozen, and shipped under dry ice to the participating laboratories (Duggan, 1948b). Presumably because of these recoveries and data relating indole to spoilage, the method as given by Duggan (1948b) was the basis for the procedure included in the 9th edition of Methods of Analysis for the Official Agriculture Chemists (AOAC, 1960) for determining indole in shrimp, oysters, and crabmeat. For this method, 50 g of oyster meat and 25-50 g crabmeat or raw or cooked shrimp are blended in 80 ml of water for oysters and crabmeat or in 80 ml alcohol for shrimp. This slurry is steam distilled, and 350 ml of distillate is collected. The indole is extracted from the distillate with chloroform after dilute hydrochloric acid and sodium sulfate have been added. The color reagent (p-dimethylaminobenzal- dehyde in acetic acid with phosphoric acid and hydrochloric acid) is combined with the extract, and the mixture is allowed to separate into layers. The acid layer is diluted with acetic acid, and optical density is measured at 560 mp.

    d . Disaduantages and Advantages of Indicator. The potential occur- rence of indole is very low (Tables Ill and IV) . T o be useful as a chemical indicator, most of the flora involved in the spoilage should produce the compound. If not, the compound cannot be indicative of quality. That decomposition may take place without the formation of appreciable amounts of indole was reported by Duggan and Strasburger (1946). Also, Loeffler (1938), as cited by Snow and Beard (1939), demonstrated that processing may be responsible for various cleavages which cause false positive indole tests.

    Data in Table VI illustrate that there would be difficulty in inter- preting amounts of indole as a chemical indicator of quality. Both raw and cooked shrimp described as having almost no odor of fresh shrimp had no measurable indole, but that having a strong, old fishy odor


    contained 8.0 to 11.2 yg indole per 100 g shrimp. The next class, however, which had a red discoloration at the edge of se,ments, de- monstrated only a faint odor and contained only 3.2 to 4.4 p g indole per 100 g shrimp. Above this value, the amounts of indole increased tremendously (159.0 to 936 p g / l O O g shrimp) and offensive odors were apparent. Similar trends of increasing, then decreasing, followed by increasing amounts of indole with decreasing organoleptic quality were found by King and Flynn (1945) in raw oysters and in canned oysters (Tables VII and VIII). Beacham's study (1946) (Table M) showed an average increase in indole with decreasing organoleptic quality; however, the range of values of indole for each stage of quality was wide.

    3. Trimethylamine

    a. Biosynthesis of Trimethylamine. Trimethylamine (TMA) may be synthesized from creatine, betaine, choline, acetylcholine, and tri- methylamine oxide (TMAO). TMA and other amines arise by the pro- cess of decarboxylation of amino acids (Bramstedt, 1957). Much of the TMA apparently comes from TMAO (Beatty, 1938).

    Although Dyer (1952) found the amounts of TMAO to be similar in the same or related species from different parts of the world, Anderson and Fellers (1952) presented evidence of variation in TMAO content between marine and fresh water fish, among different species of marine fish, and among the same species of marine fish in different waters. TMAO occurs in both marine and fresh-water fish, but accord- ing to Anderson and Fellers (1952) the amount is small in fresh-water fish compared to that in marine fish. Dyer (1952) found no TMAO in fresh-water fish. Elasmobranch fish (dogfish, skate) generally contain 2-5 times as much TMAO as the marine teleosts (cod, haddock, pollock, flounder, mackerel, herring) (Elliott, 1952). Elasmobranch fish have 2-590 TMAO based on dry weight. The important food fishes of the cod and flounder families average 60-120 mg TMAO nitro- gen/100 g tissue (Dyer, 1952). No explanation has been found to account for the variation in amounts of TMA in different species of marine fish, nor among the same species of marine fish in different waters, but the feed of the fish may have some influence (Dyer, 1947).

    Triamineoxidease has been found in the cells of bacteria from widely different sources including spoiling fish muscle, well water, and surface-taint butter (Tarr, 1940). Several common bacteria were found to have TMAO-reducing enzyme systems which were less active when the temperature was reduced from 37' to OOC, but even at OC there was some reduction of TMAO. The enzyme systems were sensi- tive to acid conditions and were relatively inert below pH 6.0 (Castell


    and Snow, 1951). According to Elliott (1952) the optimum pH for tri- amineoxidease is 7.2 to 7.4. Neilands (1945) found that ammonium hydroxide and glycogen had very little effect on triamineoxidease; indole, skatole, and hydrogen sulfide caused partial inhibition; thioglycolate had no apparent influence; and cysteine showed definite inhibition.

    Wood and Baird (1943) tested bacteria in the family Entero- bacteriaceae for their ability to reduce TMAO to TMA. All the genera in the family were abIe to do so except some members of Shigella and Erwinia. None of the recognized Micrococcaceae examined by Baird and Wood (1944) reduced TMAO, but some unidentified Micrococci from fish did. In a study by Castell and Mapplebeck (1952) on the importance of Flavobacterium in fish spoilage, only 16% of the 245 cultures reduced TMAO, although 50% were proteolytic. Table IV illustrates the potential occurrence of TMA, compiled from data in Bergeys Manual (Breed et al., 1957). Of the eight genera associated with the spoilage of fish and eggs, six were listed as producers of TMA, and eight as nonproducers. The largest number of species, 129, were listed as neither producers nor nonproducers.

    b. Trimethylamine as an Indicator of Quality of Fish. Castell et al. (1961) reported that, for a given TMA level, haddock .had greater deterioration than cod. Also, there were variations in the amounts of TMA with seasons. Farber (1963) compared sensory judgments and TMA determinations on a number of white- and red-fleshed species of fish a t seven laboratories in seven countries. Correlations of TMA nitrogen tended to parallel those of the sensory judgments for white-fleshed fish, but were not related to stage of spoilage of herring.

    Tarr and Ney (1949) discussed tentative standards for TMA values (mg N/100 g flesh), which were as follows: fresh fish, 2 or below; fish beginning to spoil, 2-15; spoiled fish, 15-30. In data presented by Tarr and Ney two samples were very stale but had TMA values between 2.0 and 3.0. Therefore, there is doubt as to the suitability of the above standards.

    Dyer and Mounsey (1945) stated that to date TMA level provides the best indication of bacterial deterioration in sea fish of any of the various spoilage effects investigated. They found that large amounts of higher nonvolatile amines were formed in the advanced stages of deterioration of the muscle when TMA values were about 60-80 mg nitrogen or more per 100 g muscle. Fresh cod and haddock contained about 0.2 mg nitrogen per 100 g muscle as TMA. According to Hillig et al. (1959), significant increases occurred in TMA and other indexes in iced haddock and ocean perch until the fishreached


    a stage when it was no longer fit for consumption. At that stage there was an increase of severalfold in TMA.

    Dussault (1957) used TMA determinations in evaluating the quality of rosefish fillets. Samples having a flat taste ranged from 5.2 to 17.6 mg TMA per 100 g fish. The two samples showing excessive amounts of TMA were found slightly off to the taste but were not totally inedible. That investigator indicated that cold fillets with TMA values comparable to those reported for rosefish would have had greater evidence of loss of quality.

    According to Hillig et al., TMA and other compounds, including volatile acids and bases, correlated positively with organoleptic judgments for cod (1958), haddock (1959), ocean perch (1960), and flounder (1960). TMA was suggested as a chemical indicator for those fish (Hillig, 1963) and for shrimp (Campbell, 1962). Fieger and Friloux (1954), also working with shrimp, found that significant increases in bacterial plate counts preceded increases in TMA values, and amino nitrogen was negatively correlated with taste-panel eval- uation of flavor and quality. Bailey et al. (1956) stated that in most cases a TMA value of 1.5 mg/100 g shrimp tissue and a bacterial count of 10 X 10 per gram or higher on headless shell shrimp indic- ated unacceptable products.

    Tarr (1945) studied the relationship of relative humidity to water content in dried precooked fish and to the mold and bacterial and volatile base content of stored samples. TMA increased in samples held a t a relative humidity too low for growth of either molds or bacteria.

    Additives and preservatives have been found to influence the production of TMA in varied ways. Spinelli et al. (1964) studied the effect of irradiation on crabmeat with reference to chemical and sensory evaluations. TMA values showed only a fair degree of accuracy for assessing the quality of vacuum-packed irradiated crab- meat. In most cases values in excess of 0.9 mg% of TMA were associated with products which were borderline in acceptability. The TMA values were generally near 0.9 mg% when the bacterial popu- lation was about 1 X lo8 organisms per gram. Bacteria surviving the irradiation had reduced ability to form TMA.

    The effect of certain antibiotics on the production of TMA has been explored by several researchers. Castell and Greenough (1957) found that the antibiotics chlortetracycline, oxytetracycline, poly- cycline, and nisin in concentrations from 1 to 50 ppm did not retard the bacterial reduction of TMAO to TMA. Hillig et al. (1962b,c,d) made comparisons of organoleptic and chemical findings on the decomposition of perch, haddock, and cod treated with chhrtetra-


    cycline. Chemical indexes of decomposition continued to develop in some fish and fillets treated with chlortetracycline and graded as sat- isfactory from an organoleptic standpoint. The upper limits were set as 25 mg/100 g for perch, and 30 mg/100 g for haddock.

    Dyer (1949), in evaluating the effectiveness of low concentrations of nitrite in extending keeping time for fish, found that rapid re- duction of TMAO was inhibited by nitrite in concentrations up to 700 ppm. When nitrite was reduced to about 50 ppm, TMA was formed. The organoleptic quality of the fish usually remained accept- able up to the stage of rapid TMA production. Castell (1949) also stated that the concentrations of nitrite required to prevent or retard the formation of TMA at 3OC are much less than those required to inhibit bacterial growth on the fish under the same conditions. The formation of TMA was greatly retarded by 100 ppm. Nitrate-treated cod was reported by Vaisey (1956) to have less TMA than control samples. Therefore, it was considered unsuitable as an indicator of quality when this type of processing was used.

    c. Methods for Determining Trimethylamine in Foods. Dyer (1945) developed a colorimetric method for estimating spoilage of fish by determination of TMA as the picrate salt. In 1950, he suggested use of the Evelyn 400-mp filter rather than the 420 filter. Bethea and Hillig (1965) improved the accuracy of Dyers colorimetric procedure in determining TMA in frozen cod and haddock by using distillate rather than the extract for determination. They also found that it was possible to determine total volatile bases and TMA-N with only one weighed sample.

    The drip from frozen fish usually yields TMA values comparable to those of fillets. When fish are thawed under running water, how- ever, extracts may be lost, and chemical and organoleptic evaluations reflect this loss (Hillig et al., 1963).

    Hughes (1959) applied gas chromatography to the determination of TMAO in herring under various conditions.

    d. Disadvantages and Advantages of Indicator. There are conflicting views as to the reliability of TMA determination as an index of quality in fish. TMA values are probably useful in indicating whether spoil- age has occurred but are of considerably less value in yielding information on spoilage changes (Fieger and Friloux, 1954). The general opinion seems to be that TMA tests are not conclusive but would be useful in combination with other tests (Tarr, 1939; Bailey et al., 1956).

    Tarr and Ney (1949) indicated that TMA was not a very sensitive test for bacterial spoilage of fish, since the flesh of some fish con- tained substances which inhibit TMA formation. Also, the acidity


    of the samples and/or the types of bacteria influenced the amounts of TMA produced. Farber (1952) also found TMA determination of limited significance in evaluating West Coast fish such as tuna, mackerel, and sardines. Farber and Ferro (1956) stated that TMA values were not correlated significantly with organoleptic judgment in canned California anchovies, herring, mackerel, sardines, and tuna. Lartigue et al. (1960) did not recommend TMA determinations for assessment of oyster quality, since these tests showed no definite patterns during storage.

    In working with Pacific-coast fish, Tarr (1939) suggested that spoil- age in fish containing TMAO may occur without TMA formation. The ratio of TMA-forming to non-TMA-forming organisms varies in the natural flora. Of 30 microorganisms isolated from 7 samples in various stages of decomposition, he found only three which reduced TMAO to TMA. In contrast, Dyer and Mounsey (1945) found that, out of 3000 samples of Atlantic fish examined over a years time, there was only one case where TMA was low and decomposition was evident. Results of their study have shown that 10-40% of the organisms present in fish slime and spoiling fish reduced TMAO. In herring, the TMA values were not related to the progress of spoilage.

    The relationship between TMA and season of the year decreases its usefulness as an indicator of quality (Castell et al., 1961). Given levels of TMA are associated with lower quality for fish caught in the summer and late fall than for fish caught in the spring.

    In view of the differences in original TMA content and the factors which influence TMA, it seems reasonable to suggest that this indi- cator be used to follow the course of spoilage only if the original value of the fish in question is known. During spoilage, increases in TMA may be as great as 15-20 times the original value (Beatty and Gibbons, 1937).

    4 . Volatile Bases

    a. Biosynthesis of Volatile Bases. The biosynthesis of volatile bases and microorganisms known to produce them is discussed along with the information on ammonia and TMA .

    b. Volatile Bases as an Indicator of Quality of Fish. Volatile bases were found to be highly correlated with organoleptic judgment in cod (Hillig et al., 1958), haddock (Hillig et al., 1959), ocean perch (Hillig et al., - 1960a), flounder (Hillig et al., 1960b), pollock and whiting (Hillig et al., 1961). Campbell (1962) reported that volatile bases had promise of being useful in the analysis of frozen or fresh shrimp.

    Gagnon and Fellers (1958) studied biochemical, organoleptic, and bacteriological methods in relation to estimating degrees of fresh-


    ness in frozen breaded shrimp. They recommended that volatile bases be determined routinely as a quality-control measure on relatively homogeneous samples of shrimp. They stated that the only values having promise as an indicator of quality were those representing actual total nitrogen content of a sample plus the volatile bases.

    Chemical indexes of decomposition in perch and in haddock stored in natural ice and chlortetracycline ice were studied by Hillig et al. (1962b,c). The data indicated that the use of chlortetracycline ice and dip on perch and haddock permitted the biosynthesis of meta- bolic by-products in some fish and fillets graded as satisfactory from an organoleptic standpoint. The upper limits for volatile bases were set at 150 ml of 0.0liV per 100 g. Other limits were set also (Hillig et al., 1962a): volatile acid number expressed as ml 0.01 N/lOO g- perch, 30 ml and haddock, 40 ml, acetic acid expressed in mg/100 g-perch, 30 mg, and haddock, 40 mg; formic acid, any determinable amount; volatile amines expressed as ml 0.0 5 N KMn0,/100 g-350 ml for both perch and haddock; TMA expressed as mg/100 g-perch, 25 mg, and haddock, 230 mg.

    c. Methods for Determining Volatile Bases in Foods. In a spoilage- index study Stansby et al. (1944) compared methods of determining total volatile base and tertiary volatile base in fish flesh. Press juice, protein- free press juice, and suspension of ground fish in 60% ethanol were sampled.

    Microdiffusion, distillation, and aeration were used for obtaining volatile bases. The method of extracting the fish flesh with 60% ethanol and removing the volatile bases by distillation from the so- lution made alkaline with borax gave precise results. The procedure, which was a slight modification of the microdiffusion method of Beatty and Gibbons (1937), was most suitable for determining tertiary volatile bases.

    Fischbach (1945) proposed a new technique for study of decom- posed foods based on a low-temperature low-pressure system along with chemical and cold traps. With this type of apparatus, weakly dissociated volatile bases can be separated from strongly dissociated ones along with the neutral volatiles in one operation. Volatile acids and nonacidic material can also be separated.

    Tomiyama et al. (1956) suggested a vacuum distillation procedure for determination of volatile bases in fish flesh. This method had the advantages of being extremely rapid without sacrificing either accu- racy or precision.

    Mitchell and Honvitz (1941) measured volatile bases along with volatile acids and lipids and lipid phosphorus pentoxide in the de- tection of decomposition in eggs. They concluded that it is unlikely


    that any single chemical method can be developed to detect and evaluate decomposition in eggs. Their procedure for determining volatile bases involved the use of salt, acid, and alcohol to obtain a clear filtrate free of protein and lipid.

    Spinelli et al. (1964) studied irradiated crabmeat in relation to volatile-base production plus bacterial counts and TMA. Results indicated that total volatile-base values could be used with only a fair degree of accuracy to assess the quality of vacuum-packed irra- diated king crabmeat. In irradiated crab, bacterial counts approached 1 X los per gram before significant differences were indicated by sensory evaluations. In unirradiated crab, however, populations of slightly over 1 X lo6 per gram caused significant changes. Bacteria surviving irradiation were reduced in their ability to form volatile bases. Products were border-line in acceptability when values were in excess of 12.0 mg% of total volatile base. When total volatile bases were this high the bacterial population generally had reached about 1 x lo8 organisms per gram.

    d. Disadvantages and Advantages of Indicator. The disadvantages which apply to the individual indicators, ammonia and trimethylamine, apply also to the mixture called volatile bases. The fact that volatile bases are a mixture of compounds is a definite advantage, since not all spoilage organisms produce a single indicator. The multiple by- products may more nearly indicate the decomposition of a mixed culture.

    5 . Volatile Fatty Acids

    a. Biosynthesis of Volatile Fatty Acids. Acetic acid may be formed from aspartic acid, alanine, serine, glycine, cystine, threonine, and glutamic acid by the enzymes of microorganisms. Propionic acid may

    From fats upon hydrolysis by lipolytic microorganisms

    Volatile and nonvolatile acids & From carhhydrates via glycolysis and

    Krebs cycle by saccharolytic


    From prbteins by deamination of amino acids by

    proteolytic microorganisms

    FIG. 1. The possible origin of volatile and nonvolatile acids produced by micro- organisms in foods.


    be produced from alanine and serine. Butyric acid may be formed from threonine and glutamic acid (Salle, 1961). Volatile fatty acids may be synthesized also from fats and carbohydrates, as shown in Fig. 1.

    b. Volatile Fatty Acids as an Indicator of Quality of Fish. Volatile fatty acids have been used successfully as indicators of quality in several kinds of fish. Hillig (1939) and Clague (1942) studied volatile fatty acids as an approach to evaluating spoilage in canned fish roe and sardines. In both cases, fresh products were low in volatile fatty acids, and as quality decreased the amount of acids increased. Volatile fatty acids have a potential as an index of the quality of canned sardines. The determination is a measure of deteriorative changes occurring up to the time of canning the sardines. Both investigators mentioned that strong brines and sauces may inhibit the development of volatile fatty acids. Amano, as cited by Bramstedt (1957), showed that iso- valeric acid is one of the compounds contributing to the repulsive smell of fish.

    Volatile fatty acids have also been studied in relation to canned salmon and tuna. Hillig and Clark (1938) reported that when the freshest possible raw fish is used for canning, only small quantities of volatile fatty acids, chiefly formic and acetic, are present. With more spoilage, the quantity of volatile fatty acids was greater and higher members of the series appeared. Identification of all com- ponents was considered unnecessary, but it was stressed that accurate measurement of a definite proportion of total acids (VAN) and the amount of formic acid in this fraction was important.

    Hillig and co-workers (1950b) studied the individual volatile acids as indexes of decomposition in tuna. A small quantity of acetic acid was found in canned tuna from fresh fish. Amounts of both acetic and formic acids increased as quality decreased. When decomposition be- came considerable, propionic and butyric acids appeared. Thus, individual volatile acids in canned tuna were a good index of the stage of decomposition of the raw material used for the canned product. High holding temperatures accelerated the development of volatile fatty acids in sardines (Clague, 1942). In one series of determinations on raw sardines, Clague (1942) discovered no definite correlation between bacterial count and VAN. Under cannery conditions an appreciable increase in volatile acidity may be due to the holding of sardines in the open can after packing.

    VAN, along with formic and acetic acid, was found to be highly correlated with organoleptic judgments in studies of cod (Hillig et al., 1958), haddock (Hillig et al., 1959), flounder (Hillig et al., 1960b), pollock and whiting (Hillig et al., 1961), and ocean perch (Hillig et al.,


    1960a). Hillig et al. (1962a) summarized data on chemical indicators of the decomposition of cod, haddock, and perch. The principal vol- atile acids found were formic and acetic. When formic acid was present in measurable amounts, those workers concluded that decomposition had occurred without question. Indexes are given for the limits of decomposition. For perch, the upper limits were set at 30 ml for VAN (0.01 N/100 g), 30 mg for acetic acid (per 100 g), and any determin- able amount for formic acid (Hillig et al., 1962a). For haddock, the upper limits were set at 40 ml for VAN (0.01 N/100 g), 40 mg for acetic acid (per 100 g), and any determinable amount for formic acid (Hillig et al., 1962a).

    Campbell (1962) reported on the decomposition of shellfish. VAN, not formic acid alone, was considered to show promise as an indicator of the quality of frozen or fresh shrimp.

    VAN may have some diagnostic use in the detection of decom- position in eggs (Mitchell, 1940). High-quality yolks had little or no volatile acids, but yolks which had undergone decomposition had appreciable quantities. Mitchell and Horwitz (1941) used the Clark and Hillig (1938) steam distillation method in determining volatile acids in the detection of protein decomposition and/or lipid decom- position. The volatile acids were not identified in the above study. However, Clark and Hillig found formic, acetic, propionic, butyric, isobutyric, valeric, and isovaleric acids in decomposed egg yolk.

    c. Methods for Determining Volatile Fatty Acids in Foods. Ramsey and Patterson (1945) separated and identified the lower fatty acids (C, to C4) , formic, acetic, propionic, and butyric. In later work they separated C, to C,,, fatty acids (Ramsey and Patterson, 1948a), C , , to C,, fatty acids (Ramsey and Patterson, 1948b), and n-butyric and isobutyric (Ramsey, 1948). In a collaborative study of a partition chromato- graphic method for volatile fatty acids, Ramsey and Hess (1950) proposed a method that is accurate and suitable for application to studies on the decomposition of foods. Because of the small quantities of test material required, chromatographic separation is preferable to extraction and distillation of volatile acids.

    Van Dame (1957) recommended a method for the separation of all the volatile acids (formic, acetic, propionic, butyric, and valeric) and lactic and succinic acids on the same chromatographic column. The procedure was used with fresh and frozen eggs.

    Shelly et al. (1963) worked with gas chromatography and quanti- tative determinations of formic, acetic, propionic, and butyric acids in frozen whole egg and frozen fish fillets. Salwin (1965) later made a corroborative study on the quantitative determination of volatile fatty acids by gas chromatography and by column partition chromato-


    graphy. Gas chromatography was more sensitive for low concentrations of acids and was as accurate as partition chromatography.

    The advantages of gas chromatography included a single procedure for formic, acetic, propionic, and butyric acids, improved specificity and sensitivity, a permanent record of the analyses, and increased speed for some analyses.

    Schwartzman (1960) separated and identified sodium salts of C, to C, volatile fatty acids by paper chromatography. The sodium salts of acetic, propionic, butyric, and valeric acids were separated. On the basis of a preliminary investigation, this procedure was suggested as a screening test to detect decomposition as indicated by the presence of small amounts of volatile acids in food products.

    Young et al. (1965) improved earlier methods (Young and Schwartz- man, 1963; Schwartzman, 1960) for the separation and identification of sodium salts of acetic, propionic, butyric, and valeric acids on a silicic acid column. By paper chromatography and addition of acetone to the original solvent system (equal volumes of tertiary butanol, normal butanol, and concentrated sodium hydroxide), chromatograms could be developed in about 2$ hours with distinctly separated stable spots.

    Nakae and Elliott (1965), in a study of the selective formation of volatile fatty acids by degradation of amino acids by some lactic acid bacteria, investigated the kinds and amounts of volatile fatty acids produced from single amino acids, modified amino acid mixtures, and pyruvate. They found that the qualitative and quantitative balance of amino acids determined the volatile fatty acid patterns evolved from amino acid mixtures. Those investigators (1965) also determined that VFA produced by streptococci and lactobacilli from casein hydrolyzate were favored by: neutral or slightly alkaline pH; tem- peratures 32OC and 42"C, respectively; shaking; addition of sodium caseinate; but not addition of lactose or milk fat.

    Morton and Spencer (1926) separated formic acid from food pro- ducts such as ketchup, apple stock, and fruit juices, by distillation with excess xylene. The xylene did not interfere with determination of the amounts of formic acid by reduction of mercuric chloride. The mercurous chloride so obtained was either weighed or titrated in order to estimate the amounts of formic acid entering into the reaction. This procedure was simpler and required less time than the steam distillation method, and results were comparable.

    Distillation methods seem to predominate among the procedures for estimating volatile fatty acids. Dyer's method of 1917, also des- cribed in detail by Clark and Hillig (1938), .was based upon the principle of distillation constants. Design and size of the equipment


    influences distillation constants (Clark and Hillig, 1938). Distillation apparatus, constructed from the usual laboratory materials, was successfully used by Hillig and Knudsen (1942) in determining one to four acids. Formic acid was always present in four acid mixtures. The combination of five acids including formic, acetic, propionic, butyric, and isobutyric was never found by those investigators. By vacuum distillation, determination of volatile acids in fish flesh required only about ten minutes with satisfactory accuracy and precision (Tomiyama et al., 1956).

    Clague (1942) evaluated the Dyer distillation method as described by Clark and Hillig (1938), and the Loeffler method (1938). The Loeffler method differed from the Clark-Hillig method in that actual fish flesh instead of fish extract was placed in the distillation flask. Although the Loeffler method was much quicker and easier to use, the Clark-Hillig (originally Dyer) procedure was more reproducible.

    d. Disadvantages and Advantages of Indicator. Volatile acids, such as acetic, may be included as a natural ingredient of some foods. For example, vinegar is an essential ingredient in some canned food sauces. If vinegar is a normal constituent of a food, acetic acid cannot be used as a valid indicator of quality.

    The usefulness of the volatile acids may also be limited for foods which undergo processing procedures whereby the acids may be lost. However, Hillig (1956a) stated that losses during the canning of fish were not sufficient to allow decomposed fish to be acceptable when examined by chemical means. Neither did degradation by heat cause an increase in indexes such as acetic acid in the fish.

    According to Sigurdsson (1947), the determination of volatile acids or TMA as a quality index was more reliable for fish stored above 0C than for fish stored below 0C. A measurement of protein breakdown was necessary for fish stored below 0C. Even in fish stored above OOC, however, volatile acids or TMA alone did not give complete information on the degree of decomposition in the muscle.

    As a group, volatile acids are easily measured. If single acids are determined, however, the procedure is somewhat more complex. There is a greater probability that a mixed culture of microorganisms will synthesize several compounds within the group classified as volatile acids than a single compound such as indole.

    6. Volatile Reducing Substances

    a. Biosynthesis of Volatile Reducing Substances. The biosynthesis of acetylmethylcarbinol (AMC) and diacetyl is covered in the carbo- hydrate section, and hydrogen sulfide in this section. The group of compounds designated as volatile reducing substances (VRS) probably


    include several compounds in addition to AMC, diacetyl, and hydrogen sulfide.

    b. Volatile Reducing Substances as an Indicator of Quality of Fish and Meat. Since VRS are odorous compounds, decomposition can be judged organoleptically. Lang et al. (1944) made use of these volatile substances by measuring the VRS which were present. VRS values were determined in ground round steak stored at 37.5OC for as long as 50 hours. Microequivalents reduction per 5 ml press juice in- creased from 9.1 at zero hours to 10.4 at 19 hours, at which time the odor of the meat was still normal. After 26 hours, microequivalents reduction was 38.8 and the meat had a sour odor and appeared to be inedible (Lang et al., 1944).

    Farber (1952) found that VRS correlated better with organoleptic judgments of the condition of raw and canned fish (tuna, mackerel, and sardines) than total volatile nitrogen and trimethylamine nit- rogen (TMA-N), hydrogen sulfide, indole, oil acidity, and carbonyl compounds. Also, the levels of VRS in canned California anchovies, herring, mackerel, tuna, and sardines in brine and in tomato sauce correlated closely with organoleptic judgment, but the amounts of volatile bases and TMA did not (Farber and Ferro, 1956). It was concluded that the VRS determination was a more useful test than volatile bases and TMA for judging the quality of all kinds of fish and fish products and their diverse spoilage patterns under varying storage conditions (Farber and Lerke, 1958). More recent data further substantiated the negative correlation between sensory judgments and the quality of fish (Farber, 1963).

    Farber and Cederquist (1953) recommended VRS determination as a practical and accurate way of assessing the quality of fish pro- ducts, both in the sense of its wholesomeness and fitness for use and of attempting to establish grades, classes, or ratings.

    Moorjani and his associates (1958) reported on changes in VRS and bacterial count as indexes in the spoilage of fresh-water fish. At the time that definite spoilage set in, there was a corresponding rise in VRS values. VRS values greater than 20 were indicative of definite spoilage. Organoleptic evaluation, VRS, and bacterial count were interrelated.

    Although no data are available on the use of VRS as an analytical tool to detect decomposition in eggs, VRS would appear to be worthy of investigation. Data of Hillig et al. (1960~) indicated that the smell test is reliable as an indicator of the quality of eggs. Since VRS would be a part of the odorous material, it would seem that VRS might have potential as an indicator. The VRS test could conceivably detect decomposition below the threshold level detected by man.


    c. Methods for Determining Volatile Reducing Substances in Foods. Much of the work on VRS as an indicator of the quality of fish was done by Farber. Briefly, his method (Farber and Ferro, 1956) was as fol- lows: Air was circulated through the sample of fish (pressed juice) and through an alkaline permanganate reagent (0.02 N KMnO, in 1 N NaOH). After the aeration, sulfuric acid and potassium iodide in sodium carbonate were added. The liberated iodine was then titra- ted with sodium thiosulfate in a sodium carbonate-sodium borate solution with soluble starch used as an indicator. The reduction by the volatile constituents of the fish sample was calculated as micro- equivalents.

    d. Disadvantages and Advantages of Indicator. Determination of VRS requires rather elaborate equipment. This might be a handicap in certain laboratories.

    Since VRS include several compounds, there is a greater chance of metabolites from a mixed flora being present than would be true if a single compound were measured. Also, since the VRS test in- cludes several compounds, it would appear to be applicable to more species of fish than a specific test like ammonia or indole. Several studies demonstrated that spoilage in fish is accompanied by an in- crease in VRS whereas good quality is associated with low VRS values (Table X) .

    7. Lactic and Succinic Acids

    a. Biosynthesis of Lactic and Succinic Acids. Lactic and succinic acids may be produced from glucose (or other sugars) by bacteria. The mechanism of this conversion is covered under the carbohydrate sec- tion. Lactic acid may be formed also by hydrolytic deaminization



    Type of fish Researcher

    Tuna, mackerel, sardines Farber, 1952 Anchovies, herring, macherel, sardines (in brine or

    tomato sauce) Flat fish, rockfish, halibut, salmon, swordfish, white

    sea bass, corbina, whale, salted cod, herring, canned smoked yellowtail, canned kipper snacks, canned abalone, jack mackerel, and Pacific sardines in tomato sauce

    Farber and Ferro, 1956

    Farber and Lerke, 1958


    of alanine, while succinic acid may be formed by reductive deamin- ization of aspartic acid (Salle, 1961).

    b. Lactic and Succinic Acids, as Indicators of Quality of Milk, Eggs and Fish. The presence of various metabolic by-products of micro- organisms is considered objectionable in some foods whereas these same products in another food may be completely desirable. Such is in the case in the spoilage of milk, where lactic acid production in- dicates that the milk is spoiled even though the origination of lactic acid is highly desirable in the production of fermented milks and cheese. Since fresh milk contains no lactic acid, that which contains 0.03% lactic acid has been classified as decomposed (Hartmann and Hillig, 1933). Titratable acidity (expressed as lactic acid) is used as a screen- ing test for fresh milk and cream.

    Lactic and succinic acids have also been suggested as indicators of decomposition in eggs (Lepper el al., 1944). The lactic acid content of fresh eggs is very low or absent. As the bacteria grow, lactic acid is produced. Those workers suggested the following standards to in- dicate the presence of decomposed eggs; a) for liquid and frozen eggs, a microscopic count of over 5,000,000 per gram with a lactic acid content of 7 mg/100g; b) for dried eggs, a microscopic count of over 100,000,000 per gram with a lactic acid content of over 50 mg/ lOOg (on a dry basis).

    Succinic acid is not found in fresh shell eggs or in frozen or dried eggs made from acceptable shell eggs (Lepper and Hillig, 1948). The combination of lactic and succinic acids as an indicator is better than the individual acids. Succinic, lactic, acetic, and formic are produced by microorganisms present in eggs and increase with intensified micro- bial growth and breakdown of the protein, carbohydrate, and fat of the egg.

    Lepper et al. (1956) and Hillig et al. (1960~) studied the chemical composition of edible and inedible frozen eggs in authenic packs. They concluded that the smell test on frozen eggs was reliable if used by an experienced analyst. In addition, they suggested that succinic, formic, acetic, and lactic acids be measured and that bacteria counts be made.

    The usefulness of succinic acid as an indicator of decomposition varies with the type of fish, as shown in Table XI. Although succinic acid is formed when fish decompose, the data in this table illustrate that other indicators are more useful in some cases. The lactic acid content of haddock muscle (without struggle during catch) is 0.08%. Glycogen of fish disappears rapidly during the first three hours, and lactic acid accumulates to levels of 0.16 to 0.17% in 36-40 hours (Macpherson, 1932).



    Type of fish Correlation with raw material Reference

    Tuna Good Little tuna (Eulhynnus Good


    Hillig et al., 1950a. Hillig, 1954

    Cod Less than VAN," formic, acetic, Hillig et al., 1958 volatile bases, volatile amines and TMAb.

    Haddock Same as cod Hillig et al., 1959 Ocean perch Same as cod Hillig et al., 1960a

    "Volatile acid number bTrimethylamine.

    c. Methods for Determining Lactic and Succinic Acids in Foods. Both lactic and succinic acids may be extracted from foods by ether and may be titrated without separation and reported as nonvolatile acids. Lactic acid was separated in three hours (98% recovery) with a liquid extractor by Hartmann and Hillig (1933), the lactic acid was oxidized to oxalic acid with alkaline permanganate, and the oxalic acid was determined as calcium oxalate. A colorimetric method (ferric chloride) can be used to determine lactic acid in milk and milk products, but the lactic acid must first be separated from the food (Hillig, 1937). This method allows minute quantities (as low as 10 ppm) to be deter- mined in a day.

    An improved method (colorimetric) for the detection of lactic acid in dried nonfat milk was developed by Velasco and No11 (1957). After removal of the protein and separation and purification of the lactic acid (anion- and cation-exchange resins), the lactic acid was measured colorimetrically by using p-phenylphenol.

    d. Disadvantages and Advantages of Indicator. Succinic acid is pro- duced by microorganisms from amino acids and from carbohydrates. Since its boiling point is 235OC, it is stable during any heat pro- cessing that might be done. Succinic acid is low or absent from whole eggs of acceptable quality but increases with spoilage. Therefore, succinic acid meets one of the essential criteria for a good chemical indicator.

    Lactic and succinic acids have boiling points high enough to pre- vent loss from the product by heat processing. Lactic acid may be ex- tracted with ether along with succinic acid if both are present, but


    individual determinations must be made. A series of steps is required for measuring lactic or succinic acids. Neither can be measured directly. The more involved the chemical analysis, the less chance there is of the method being used in industry for quality-control pur- poses. A further restriction in the use of these metabolites is that their production may be limited to only part of the microbial Aora.

    8. Histamine

    a. Biosynthesis of Histamine. The enzymatic formation of histamine is given in the following formula:

    Production of histamine (decarboxylation of histidine)

    H H H H

    I I C-N

    I I C-N


    Histidine Histamine

    h. Histamine as an Indicator of Quality of Fish. Williams (1954) felt that histamine, because of its physical characteristics, could be used as an index of decomposition in the mackerel-like fishes when they are precooked and canned in normal factory practice. In fact, he found the histamine content of canned fish to be well correlated with its organoleptic properties. Only small quantities of histamine were present in high-quality canned fish, although small fish had greater histamine content than large ones.

    Histamine has been used successfully as one criterion for the relative freshness of fish, particularly marine fish rather than shellfish or fresh- water fish. Histamine was not detected by Hillig (1963) in samples of shrimp varying in organoleptic grade from good to decomposed.

    Biological and chemical evidence that the muscle tissue of marine fish contained histamine was shown by Geiger et al. (1944). In agree- ment with work of Williams (1954), they reported that the histamine content of fresh fish was very low but increased rapidly post mortem as a result of bacterial contamination. Geiger (1944) stated that


    changes in histamine content during the first 24 hours after the fish were caught were more marked than changes in odor, appearance, pH, free acids, or volatile acids. On the basis of experimental data, fish containing more than 10 mg of histamine-like substances per 100 g of tissue should not be regarded as fresh (Geiger, 1944).

    Histamine in fresh fish was present in the free form, but a small part of the histamine produced post mortem remained in peptide link- age within protein molecules. Proteolytic enzymes did not produce free histamine from protein which did not contain histamine groups (Geiger et al., 1944).

    Perhaps the most histamine determinations on a single kind of fish were done on tuna by Hillig. In 1954, he reported that progressive decomposition studies on little tuna demonstrated that histamine along with several other compounds could be used as indexes of de- composition of the raw material from which the canned product was prepared. In 1956(a) Hillig again reported on histamine formation as an index in decomposed tuna. However, he stated that more than one index is desirable in order to detect all types of decomposition that fish may undergo.

    Honeycombing has been accepted, both by the producing industry and by various examining laboratories, as definite evidence of decom- position since it occurs only when the raw fish is in an advanced stage of decomposition (Hillig, 195613). Williams (1954) found a relation between honeycombing and high levels of histamine in fish, regard- less of species.

    c. Methods for Determining Histamine in Foods. The original method for the determination of histamine was bioassay with a segment of guinea pig ileum. This method was based on measurement of the ileum response to histamine stimulation (Williams, 1954). To avoid some of the difficulties of this procedure, a colorimetric method for histamine was developed. Histamine was separated from the sample by column chromatography, a diazonium reagent was used for color develop- ment, and absorbence was read at 475 mp (Sager and Honvitz, 1957).

    A limited comparison of the biological and the chemical method was made by Williams (1957). He found that the histamine-like sub- stances measured by the biological method (calculated as histamine) approximated the histamine determined by the chemical method. The chemical method was simplified and the color was increased and stabilized by adding alcohol according to the method of Williams (1960). The cloudiness which developed after addition of the alcohol was removed by centrifuging.

    Discrepancies between the biological and chemical methods were eliminated by modifying the bioassay method so that the acid extracts


    were neutralized with sodium carbonate (Sager and Honvitz, 1957). When the extracts were neutralized in this manner, the pH was uni- formly about 7.5. Thus, alkaline stimulation of the guinea pig ileum was avoided, and results checked well with those obtained by the chemical method.

    d. Disadvantages and Advantages of Indicator. One disadvantage of using histamine as an indicator is that small fish appear to have greater histamine content than large fish. This would mean that the histamine content considered acceptable for small fish might be objec- tionable for large fish. In view of this, to give the best estimate of decomposition histamine should be used along with other indicators.

    An advantage of histamine as a chemical indicator is its stability. It also meets another criterion for a good index in that the histamine content of fresh fish is low and increases with bacterial action.

    9. Infrequently Used Indicators: Hydrogen Sulfide, Water-Insoluble Acids, Ethyl Alcohol, Tryptophane, and Acetylmethylcarbinol (Acetoin).

    a. Biosynthesis of Hydrogen Sulfide, Water-Insoluble Acids, Ethyl Alcohol, Tryptophane, and Acetylmethylcarbinol. The formation of hydrogen sulfide is used as a routine physiological test for the iden- tification of bacteria. According to Salle (1961), cysteine is dissimi- lated under aerobic conditions to hydrogen sulfide and other products. The biosynthesis of this compound would require that either cystine or cysteine be present. Cystine is converted to cysteine.

    Water-insoluble acids (WIA) are derived from the breakdown of triglycerides with the liberation of long-chain fatty acids. These acids have been found by Hillig (1947) to be mostly oleic and palmitic acids.

    Ethyl alcohol may be synthesized from amino acids like alanine, which is deamininated and decarboxylated. Carbon dioxide and ammonia are also produced. Ethyl alcohol may be formed from carbo- hydrate via glycolysis. Since ethyl alcohol is a product of carbohydrate fermentation, the major part of the discussion of this compound is included in the carbohydrate section,

    The biosynthesis of free tryptophane is accomplished by the hy- drolysis of proteins; the greater the enzymatic action the greater the production of tryptophane and the poorer the quality of the food. Free tryptophane has been used as an indicator of low quality in milk, cream, and butter (Duggan, 1948a).

    The formation of acetylmethylcarbinol (AMC) is described fully under the dissimilation of carbohydrate. However, amino acids (for example, alanine) can be converted into pyruvic acid, which is an intermediate compound in the synthesis of AMC.


    b. Hydrogen Sulfide as an Indicator of Quality of Meat, Fish, and Eggs. Hydrogen sulfide was found by Farber (1952) to be of limited signif- icance in his comparison of various methods for the determination of spoilage in fish such as tuna, California sardines, and Pacific mackerel. Snow and Beard (1939), however, found that 93% of nearly 2,000 fish samples produced hydrogen sulfide, so they thought it had possibili- ties as a quality indicator. Weaver (1927) used hydrogen sulfide produc- tion to detect spoilage in meat. Also, Kraft et al. (1956) found hydrogen sulfide in packaged meats and in broken-out shell eggs. Hydrogen sulfide probably originated from sulfur-containing amino acids (Bramstedt, 1957). Castell and Greenough (1957) showed that Chlortetracycline and oxytetracycline did not inhibit the reduction of cysteine to hydrogen sulfide by bacterial enzymes.

    c. Water-Insoluble Acids as a n Indicator of Quality of Eggs. The determination of WIA in relation to decomposition in eggs was studied by Hillig (1948a, 1950). Small amounts of WIA were found in edible dried eggs, but when liquid eggs were allowed to become sour before drying, the WIA in the dried product were increased several-fold. Commercial dried eggs of acceptable quality were found to contain WIA in the same concentration range as that found for edible dried eggs.

    d. Alcohol and Acetylmethylcarbinol as Indicators of Quality of Fish. In 1939, Holaday reported on ethyl alcohol as a measure of spoilage in canned fish. He found that good fish contained very small amounts of alcohol. As decomposition proceeded, the quantities became pro- gressively larger. Other researchers also found alcohol to increase with decomposition, but the increase was not significant. Correlations between alcohol content and organoleptic results were insignificant in experiments with haddock (Hillig. et al., 1959), cod (Hillig et al., 19581, ocean perch (Hillig et al., 1960a), and shrimp (Hillig, 1963).

    Groninger (1961) studied the formation of AMC in cod and other bottom-fish during refrigerated storage. He found acetaldehyde, butyraldehyde, and AMC in the spoiled flesh of sardines, mackerel, and flatfish, whereas he found only acetaldehyde in fresh tissue. The formation of AMC and volatile bases at about the same time in stored fish suggested that bacteria were responsible. The content of AMC increased from < 1 to 7-10 mg/100 g in 4-8 days of storage. The usefulness of AMC as a measure of quality appears limited, however, because AMC formation does not occur until just before the sample would be considered unacceptable.

    e. Tryptophane as a n Indicator of Quality of Milk, Cream, and Butter. Duggan (1948a) found negligible quantities of free tryptophane in normal sweet milk and cream. The amount of free tryptophane in milk and cream increased with age if the products were held under condi-


    tions conducive to bacterial and enzymatic activity. The amount of free tryptophane in butter depended on the tryptophane content of the original cream.

    f , Methods for Determination of Hydrogen Sulfide, WLA, Ethyl Alcohol, Tryptophane, and Acetylmethylcarbinol in Foods. The methods for deter- mining WIA, ethyl alcohol, and AMC are discussed in detail in other sections. Qualitative determinations for hydrogen sulfide can be made on such products as meats by placing filter paper soaked with lead acetate in packaged meats. If microorganisms which produce hydrogen sulfide are present, the color of the paper will change from white to black.

    Most methods for quantifying hydrogen sulfide in biological materials are based on the methylene blue reaction described by Sands et a1. (1949). An aliquot of distillate from the food is trapped in zinc acetate. Acid-diamine and ferric chloride are added, and color is developed and read at 745 mp.

    Free tryptophane is extracted from milk, cream, and butter with acetone. The acetone has two functions: coagulates the protein present to prevent the tryptophane held in the protein from being extracted, and extracts the free tryptophane into the acetone. The precipitated protein is removed by centrifuging. The acetone is removed by evapo- ration. The amount of tryptophane is determined colorimetrically by using p-dimethylaminobenzaldehyde in the presence of acetic, phosphoric, and hydrochloric acids (Duggan, 1948a, b). g. Disadvantages and Advantages of Indicators. The usefulness of

    tryptophane as an indicator for the quality of cream may be questioned since the level of this compound may vary in the original product. Free tryptophane, however, is present in sweet milk and cream in only a negligible quantity, and since bacterial activity causes an increase in free tryptophane, this compound may have potential as an indicator. More research is needed before its use is recommended.

    WIA, which are used frequently as indicators in fatty foods such as butter and cream, have been suggested as indicators for the spoilage of eggs. Hillig (1948a) found that the WIA increased when the eggs were sour prior to drying.

    Alcohols (mainly ethyl) have been used for detecting decomposi- tion in fish and eggs. Hillig (1958) found that alcohol could be used for processed foods as well as the raw material. Even though about one-half of the alcohol may be lost during steaming, the quantity remaining in decomposed fish is so large that this loss does not negate its use as an indicator.

    AMC has only limited use as an indicator in fish since it is not produced in any quantity until the fish is considered unacceptable.


    Hydrogen sulfide is produced by several bacteria (Tables m and IV) and therefore has a greater potential as an indicator than indole, which has had more attention.

    All of these compounds (hydrogen sulfide, WIA, ethyl alcohol, tryptophane, and AMC), when used by themselves as indicators, may not denote decomposed raw materials. For maximum advantage, several indicators should be used.



    This section emphasizes cream and butter as examples of fatty foods. The fat content of cream ranges from 18.0 to 40.0g/100 g, and butter contains 80.0 g/100 g, or more. Although certain fish (herring, Atlantic and mackerel, Atlantic) contain a rather high content of fat, the use of volatile acids as indicators of quality for fish is already included with the discussion of indexes for high-protein foods.

    Milk and cream are good growth media for microorganisms, and therefore spoil easily. If these foods are not handled properly, com- pounds resulting from decomposition occur in the finished product. Notices of Judgment under the Federal Food, Drug and Cosmetic Act provide numerous cases where butter has been seized by the govem- ment because it was made from decomposed cream (FNJ 23685, 1958; 24492, 1959; 25676, 1960; 26890, 1961.)

    Common methods of determining the quality of cream and butter, other than chemical tests, have been the mold count (the amount of mold filaments in cream), the bacterial plate count, and organoleptic tests. According to Hammer and Babel (1957), there is not necessarily a correlation between the mold content of butter and its organoleptic quality. In general, butter with a high mold count has a low quality score, but some butters with a high mold count may have a high quality score. Since molds are not the main cause of flavor deterioration in cream, no close correlation between organoleptic quality and mold count would be expected in butter. Chemical indicators of quality would assist the analyst in detecting decomposition in cream and butter.


    According to Hammer and Babel (1957), various species of molds grow on cream, with Geotrichum candidum the most important. This organism, according to Purko et al. (1952), is highly lipolytic and


    produces water-insoluble acids (WIA) from breakdown of the fat in the cream. Another lipolytic microorganism which produces rancidity is, according to Hammer, Pseudomonas fragi, which has been isolated repeatedly from rancid butter, thus indicating a causal relationship.

    Lipolytic microorganisms produce free fatty acids which may or may not be volatile. These acids, held to glycerol by an ester linkage, may be recovered from the food after hydrolysis of the fat by enzymes of the microorganisms. As the organisms grow, more and more of the acids are liberated and hydrolytic rancidity results.

    Mossell and Ingram (1955) listed the following genera as dominat- ing fatty foods when spoilage occurs during standard conditions of storage: Streptococcus, Lactobacillus, Microbacteriurn, Achromobacter, Pseudomonas, Flavo bacterium, and Bacillus.


    a. Biosynthesis of Volatile Fatty Acids. The biosynthesis of fatty acids from amino acids was discussed in the protein section. The biosynthesis of volatile fatty acids may occur in cream prior to, or after, it has been used in the manufacture of butter.

    Nakae and Elliott (1965) used three strains of lactic streptococci and two strains of lactobacilli to study the factors influencing the production of volatile fatty acids from caseine hydrolysate. They found that sodium caseinate added to the hydrolysate stimulated biosynthesis of volatile fatty acids whereas lactose or milk fat showed slight inhibitory effect. Hillig (1948b), however, postulated that the butyric acid in decomposed cream came from the breakdown of lactose, with lactic acid as an intermediate compound. Peters (1953), too, thought that butyric acid came from milk serum constituents, probably lactose.

    The degree of breakdown of animal and vegetable fat by pseudomo- nads depends more upon the particle size of the fat than on the type of fat (Goldman and Rayman, 1952). The composition of the growth media also influenced the degree of lipolysis. In a low-protein medium the main enzymatic activity was lipolytic, whereas in media contain- ing higher protein levels the degree of lipolysis was less. Goldman and Rayman (1952) found that Ps. fluorescens, Ps. oleovorans, and Ps. fragi were highly lipolytic.

    Air appears to have an important influence on the degradation of fats by microorganisms. Hydrolysis of glycerides into free fatty acids and glycerol by lipases is followed by'rapid oxidation of these compo-


    nents by oxidative enzymes of the microorganisms (Mukherjee, 1951). The contaminating organisms have an important influence upon the types of fatty acids formed. Bacteria synthesized less volatile acids and more long-chain fatty acids than did filamentous fungi. Contami- nating bacteria produced a greater proportion of butyric and caproic acids than the molds (Richards and El-Sadik, 1948).

    b. Volatile Fatty Acids as Indicators of Quality of Butter and Cream. To be an effective indicator of quality, the chemical compound must be absent or at low levels in good milk or cream. In studies of progress- ive decomposition made by Hillig (1948b), Hillig et al. (1949), and Hillig and North (1952), butyric acid was found in cream which was unfit for human consumption. Hillig (194813) held that the mere presence of butyric acid in butter indicated that decomposed cream was used in manufacture of the butter and that butyric acid is usually carried over into the butter. Peters (1953), however, found that all fresh samples of cream contained free butyric acid. Thus, the mere presence of this compound may not be as good an indication as Hillig suggested. On the other hand, Peters found that the quantity of butyric acid increased with incubation time in samples which did not contain formaldehyde to inhibit the spoilage organisms.

    A review of Hillig's data (1948b) indicates that butyric acid was not related closely to the Howard mold count, a common method of detecting the use of decomposed cream in the manufacture of butter. Out of 8 samples with mold counts greater than 50% (highest 88%), 5 had butyric acid contents of 3.0 mg/100 g, 1 had 4.0 mg/100 g, and 2 contained 23 and 28 mg/100 g. Other samples, all with the same mold count of 2276, had butyric acid contents of 9, 11, and 15 mg1'100 g. These data indicate that increased butyric acid content does not agree with increased mold count.

    In two experiments, Kester et al. (1953) showed that the presence of salt in butter retarded the highly lipolytic organism Ps. fragi. Ps. putrefaciens did not produce large quantities of butyric acid and was not influenced by the presence of salt. Synthesis of butyric acid by Ps. fluorescens was affected in one experiment and not in another. In one experiment in which it was used, A. lipolyticum was retarded in butyric acid production. Storage time, the presence of salt, and the kind of contaminating bacteria all influenced the amount of butyric acid released.

    c. Methods of Detecting Volatile Fatty Acids in Butter. The Association of Official Analytical Chemists (1960) induded methods for WIA and butyric acid and volatile acids. In the WIA-butyric acid procedure, the butyric acid is extracted with ether and separated on a silicic acid chromatographic column. The butyric acid, after elution from


    the column, is titrated with 0.01 N NaOH. For estimation of the total volatile acids, the sample is extracted with ether, diluted in water, acidified, and steam distilled. A known-volume standard quantity of the distillate is titrated with 0.01 N Ba(OH),.

    d. Disadvantages and Advantages of Volatile Acids as Indicators of Quality for Butter. Since butyric acid may be present in fresh cream, it appears that the mere presence of this acid in butter cannot be taken as conclusive evidence that decomposed cream was used in manufacture of the butter. Volatile acids alone are not adequate as an indicator, since some microorganisms produce more nonvolatile than volatile acids. The relationship between methods of estimating quality, such as the Howard mold count and bacterial methods, needs to be studied for a clear interpretation of what is and is not classified as decomposed. For example, do a low mold count and a high volatile acid content indicate decomposition? Although there is need for further investigation of chemical indicators for butter and cream, volatile acids in conjunction with WIA and the Howard mold count appear to be useful in the evaluation of butter.

    2. Water-Insoluble Acids

    a. Biosynthesis of Water-Insoluble Fatty Acids. Fat is hydrolyzed into its constituent fatty acids and glycerol (Hillig et al., 1949). Some of these fatty acids are long chains, such as palmitic and oleic, which are insoluble in water.

    Purko et al. (1952) studied 9 cultures of Geotrichum candidum and found that 8 cultures caused rapid and extensive hydrolysis of butter- fat in cream. The amount of WIA in sterilized cream increased in direct proportion to storage time, temperature, inoculum size, and surface of cream exposed to the air, whereas added lactic acid and glucose decreased the biosynthesis of WIA. Ps. fluorescem in cream produced higher WIA content than did S. lactis (Parmelee and Babel, 1953).

    b. Water-Insoluble Acids as Indicators of Quality of Cream and Butter. The fat in cream may be hydrolyzed by lipolytic microorganisms or milk lipase, with the liberation of fatty acids, including WIA. When such cream is churned, the butter has a higher content of WIA than when fat hydrolysis has not occurred in the cream. Regulatory officials have used WIA values in excess of 400 mg/100 g of cream or butter as an indication of decomposed cream (Nielsen, 1965).

    Season of the year influences the WIA content of butter. Butter produced from winter cream had the highest content, and fall cream the lowest. Freeman and Barkman (1953) gave no reason for these


    differences. The feeding of cows on dry feed in the winter might explain the high WIA content. Parmelee and Babel (1953) found WIA values greater than 400 mg per 100 g fat in butter from fresh milkof certain cows fed on dry feed. Hillig and Palmer (1952), however, presented data illustrating that feed had no effect on WIA content. Thus, further research is needed to clarify the influence of season and feed on WIA content of butter.

    Storage temperature and time can influence the WIA content of cream. Cream held at 4C prior to storage a t 25OC deteriorated rapidly (Hillig and North, 1952). Babel (1950) found, however, that there was no significant difference in WIA content between butters made from cream held at 55 or 75OF. He attributed this to the rapidity of spoilage by acid-producing bacteria, lowering the pH of the cream to a level which was inhibitory to lipases. He also observed that butter had a low WIA content when made from cream having a clean, acid flavor, and a high WIA value when putrefactive spoilage of the cream had occurred.

    In addition to the above factors, the apparent WIA content of cream and butt.er may be influenced by the amount of sodium hydroxide added to the fat-water-ether mixture during the neutralization pro- cedure in the WIA determination (Parmelee and Babel, 1953). With an increase in the amount of sodium hydroxide, WIA content increased. Hillig (1951), however, claimed that neutralization did not increase WIA content in cream or butter.

    Organoleptic grading of cream has been used as a means of detect- ing decomposition. Armstrong et al. (1951) described cream with a high WIA content as having an oily taste. Hillig and Ahlmann (1948) reported that cream judged to be decomposed contained higher quan- tities of WIA than cream classified satisfactory by organoleptic methods.

    c. Methods for Determining WTA in Cream or Butter. Hillig (1952) gave the following steps in determining WlA: ( 1) alkaline extraction of the fatty acids from an ether solution of the butter into the water- curd phase, (2) ether extraction of the fatty acids from the acidified water-curd phase, (3) alkaline extraction of the fatty acids from their ether solution into 50% alcohol solution, (4) separation of the WIA by precipitation and filtration, (5) solution of WIA in ether, evapora- tion of ether, drying and weighing the acids, (6) solution of dried acids in neutral alcohol or benzene, titration with standard alkali and cal- culation of mean molecular weight. For a detailed procedure, consult Methods of Analysis of the Association of Official Agricultural Chemists (1960).

    A rapid colorimetric method using alpha-napthylphthalein was


    described by Armstrong and Harper (1952) for measuring the hy- drolysis of fat. Although the method gives only an approximation, it can be performed in 10-15 minutes. One hundred determinations can be made by this method in the time required to do eight determi- nations by the Hillig (1947) procedure. The results of the colorimetric method agreed with those obtained by the Hillig procedures.

    Hillig (1953) also developed a rapid method for estimation of WIA in cream and butter. After the fat is isolated, freed of WIA, and dissolved in ether, the sample is titrated with standard sodium ethylate. Using a mean molecular weight of 270 for WIA, one can calculate the quantity of WIA in cream and butter. Total time required for the analyses is about 15 minutes.

    d. Disadvantages and Advantages of Indicator. Neither old cream nor high viable mold and yeast counts are consistently related to high WIA values. WIA content as an indicator of the quality of butter and cream is confused by the influence of the season. The amount of sodium hydroxide required for neutralization during the determination may also be an influencing factor. Naturally occurring milk lipases may give rise to WIA which would not come from contaminating micro- organisms. The usefulness of WIA as an indicator of quality depends upon clarification of these relationships by further research. High fatty acid content, regardless of i ts origin, indicates poor-quality cream.



    Research on chemical indicators has been less for fruit and vege- table products than for high-protein foods. The need for indicators is just as great for fruit and vegetable products as for other foodstuffs. Indexes would be especially useful for the comminuted foods such as sauces and purCes, in which faulty raw materials can be masked easily. As with the high-protein foods, if the processor is not extremely careful decomposed materials may contaminate the finished product. For example, frozen strawberries containing rotten or decomposed berries were seized by the federal government (U.S. Dept. Health, Education and Welfare) on several occasions (FNJ 23330, 1957; 23970, 1958; 25710, 1960; 26979, 1961). Frozen cherries were seized because they contained decomposed cherries while held for sale (FNJ 27374, 1961). Vegetable products were seized because they contained decom- posed materials: canned sweet potatoes (FNJ 24521, 1959; 26939,


    1961), canned mushrooms (FNJ 23708,1958; 23806,1958; 24728,1959; 24240, 1958; 24241, 1958; 25318, 1959; 25319, 1959), canned tomatoes (FNJ 25323, 1959), tomato paste (FNJ 24729, 1959), tomato puree (FNJ 24730, 1959), tomato ketchup (FNJ 24732, 1959), and tomato juice (FNJ 23813, 1958).


    The dominant spoilage floras for vegetables under standard con- ditions of storage consist of Achromobacter, Pseudomonas, Flavobacterium, Lactobacillus, and Bacillus. In addition to spoilage caused by these microorganisms, bacterial soft rot occurs on many vegetables because of the growth of Enuinia carotouora (Table XII). Rhizopus and Botrytis



    Vegetable Most frequent causes of spoilage after harvesting

    Artichokes Asparagus Beans, lima

    Beans, snap Beets Broccoli Brussel sprouts Cabbage


    ~ ~ ~ ~ _ _ _ _ ~~

    Gray moldb Bacterial soft rot' Gray mold, bacterial soft rot, watery soft rot: bacterial

    Sclerotinia, Rhizoctonia, bacterial blight, anthracnosef Bacterial soft rot, gray mold Bacterial soft rot Bacterial soft rot Bacterial soft rot, watery soft rot, Altemria leaf spot, black

    rot, gray mold Bacterial soft rot, watery soft rot, gray mold, Rhizopus,

    Fusariurn rot


    Cauliflower Celery Cucumbers Lettuce Bacterial soft rot Onions Peas Peppers Potatoes Bacterial soft rot Spinach Bacterial soft rot Sweet potatoes Tomatoes

    Bacterial soft rot, watery soft rot, Altemria rot Watery soft rot, bacterial soft rot Bacterial soft rot, watery soft rot

    Bacterial soft rot, gray mold, black rot Bacterial and watery soft rots Rhizopus rot, bacterial soft rot, gray mold, anthracnose

    Rhizopus, black and fusarium rots Phoma rot, late blight, bacterial soft rot, Rhizopus rot

    "Data fmm Agricultural Research Service. USDA, ARS-20-1. 1954. bBoohytis C h ,

    dSclerotinia sclerOtionun;

    Eminiu corotovom; 'Xanthomnus phasedi; Cdktohichwn lindemuthianum




    Fruit Most frequent causes of spoilage after harvesting

    Apples Apricots Cherries Grapefruit Grapes Lemons Limes Oranges Peaches Pears Plums, Prunes Pomegranates Strawberries

    Bull's eye rotb, blue rotr Brown rotd, Rhizopus rotr, gray mold' Rhizopus rot, green moldg, brown rot, gray mold Blue mold Gray mold, Rhizopus rot Blue mold, brown rot, alternaria roth Blue mold Blue mold Brown rot, Rhizopus rot Blue mold, gray mold Blue mold, Rhizopus rot Gray mold, blue mold Gray mold, Rhizopus rot

    'Data from Agricultural Research Service, USDA, ARS-ZCbl, 1954. *various organisms; 'Botrytis cinerea; Penicilliwn expcmsurn; Cladosporium;

    dSclerotinio sp.; h A l t e m ' a sp. ' Rhizopus nipricans;

    are involved frequently in post-harvest rots in both fruits and vege- tables (Tables XII and XIII) . Penicillium also causes considerable spoilage of fruits (Table Xm). In contrast to high-protein foods, vege- tables are spoiled by filamentous fungi, as well as by bacteria, and the dominant organisms spoiling whole fruits are the filamentous fungi (Table Xm) .

    In contrast to the spoilage of whole fruits, fruit juices, because of their pH, are dominated by yeast spoilage and later by bacteria and molds. Fruits and fruit juices are spoiled by the following genera under standard conditions of storage: Acetobacter, Lactobacillus, Saccharomyces, Torulopsis, Botrytis, Penicillium, Rhizopus, Candida, Zygosaccharomyces, and Hanseniasporu (Tomkins, 1936, 1951; Mrak et al., 1942; Mrak and McClung, 1940; Marshall and Walkley, 1952; Beneke et al., 1954).

    The.physiology of many of the filamentous fungi, which are involved in the spoilage of fruits and vegetables, has not been explored as much as that of bacteria. The taxonomy of the filamentous fungi is based on morphology rather than a mixture of morphology and


    physiology, as for bacteria. Research on the specific groups of fungi spoiling a particular food is fundamental to the development of a chemical index of quality.


    1. Acetylmethylcarbinol and Diacetyl.

    a. Biosynthesis of Acetylmethylcarbinol and Diacetyl. AMC is synthe- sized from acetaldehyde by some organisms and tissues (Gross and Werkman, 1947; Juni, 1952; Tomiyasu, 1937), while acetolactic acid is the intermediate in other cases (Dolin and Gunsalus, 1951; Juni, 1952; Watt and Krampitz, 1947). Diacetyl may arise in the fermenta- tion of sucrose via AMC (Suomalainen and Jannes, 1946).

    6 . Acetylmethylcarbinol and Diacetyl as Indicators. Since both AMC and diacetyl give positive tests with the Voges-Proskauer reagents, they may be referred to as Voges-Proskauer reactants.

    A survey of selected filamentous fungi for Voges-Proskauer reac- tants was conducted by Fields (1962b). Based on the AMC/dry-weight ratio, the test organisms fell into three classes: those that formed considerable AMC (0.01 10-0.0202), those that synthesized a small amount (0.0017-0.0035) and those that produced no AMC. Of the 16 organisms surveyed in that study, 13 were Voges-Proskauer-positive and 3 were negative.

    In a mixed culture, which occurs in foods, the type of flora instead of the level of contamination may influence the chemical indicator of quality. When Rhizopus nigricans and Oidium lac& were grown together in tomato serum, the amount of Voges-Proskauer reactants was 2.05 ppm. However, when R. nigricans was grown by itself the fungus yielded 28 ppm AMC. Although 0. lactis synthesized some AMC, it produced less than R. nigricans. Since 0. lactis dominated the fermentation, lower AMC values were found in the tomato-juice medium of the mixed culture (Fields, 1962b).

    Level of oxygen can also influence the amount of AMC or diacetyl produced. This was illustrated by data of Beisel et at. (1954) in which the anaerobic class of oranges were lower in Voges-Proskauer reactants than other classes of defective fruits. With increased aeration, more diacetyl was formed in fermenting apple juice, whereas AMC showed no consistent trend (Fields, 1964b). In Ledingham and Neishs work (1954), the relation among 2,3-butanediol, AMC, and diacetyl was indicated as follows:


    2,3-Butanediol AMC Diacetyl

    According to those workers, if the culture is aerated so that aerobic conditions exist, more AMC than 2,3-butanediol is synthesized. In such aerated cultures, diacetyl can be found in small amounts.

    In addition to the influence of oxygen on the levels of AMC, the amount may be influenced by the type of carbohydrate. As shown by Fields and Scott (1965), preformed mycelial mats of R. nigricans produced more AMC from glucose than from equivalent amounts of carbon in galactose, sucrose, maltose, or starch. Type of carbohydrate and environmental factors influenced the amount of AMC synthesized. Using preformed mycelial mats of R. nigricans and the sulfite-trapping method for estimating acetaldehyde, the pathway of the formation of AMC appeared to be via acetaldehyde. The mycelium of R. nigricans did not change AMC into diacetyl, but increased the amount of AMC in a solution of diacetyl in contact with preformed mycelial mats. However, the mycelial mats did not convert 2,3-butanediol to AMC. More work with various filamentous fungi is needed to elucidate the conversion of diacetyl to AMC and of AMC to 2,3-butanediol.

    Vitamins, pH, potassium chloride, magnesium sulfate, and potassium phosphate were studied to determine their influence on the biosynthesis of AMC by R. nigricans (Fields, 1964a). The mycelium was more efficient in synthesizing AMC at pH 3.0 and 4.0 than at 5.0 and 6.0. Thiamine stimulated the production of AMC, whereas potassium chloride, magnesium sulfate, and potassium phosphate significantly suppressed the production of AMC.

    Pure cultures of filamentous fungi, bacteria, and yeast grown in apple juice were surveyed by Fields (Table XIV). Of the organisms tested, only Mmor sp., Rhizopus nigricans, Botrytis cinerea, and Botryosphaeria ribis were negative for diacetyl. All other organisms were positive for both AMC and diacetyl. Most of the organisms produced Voges-Proskauer reactants; thus, these compounds fulfill one of the main requirements for an indicator of quality. Although diacetyl was produced in apple juice, data in Table XIV show that Penicillium expansum did not produce diacetyl in rotting apples. In


    fact, 71 rots out of 80 contained only AMC, not a combination of the two (Table XV) .

    c. Acetylmethylcarbinol and Diacetyl as Indicators of Quality of Apple Juice, Apple Jelly, and Orange Juice. In nine varieties of sound apples, AMC values ranged from zero to 1.0 ppm. The influence of rot on AMC values for apple juice is illustrated in Fig. 2. As the amount of rot increased, the AMC values also increased. Only apple juices with rot contents of 8% or more can be detected by taste tests (Fields, 1962a). Such rots would contain several metabolic by-products and would not resemble the buttermilk flavor of orange juice containing diacetyl (Hill and Wenzel, 1957; Beisel et al., 1954).

    Since yeasts form diacetyl, the presence of this compound can be used to indicate sanitary conditions within a processing plant. The use of diacetyl as an indicator of sanitation is possible because of its infrequent occurrence in rots.



    Type of inoculum Name of organism AMCb Diacetylb

    Filamentous fungus Filamentous fungus Filamentous fungus Filamentous fungus Filamentous fungus Filamentous fungus Filamentous fungus Yeastlike fungus Yeast, Y129 Yeast, Yl3129 Yeast, Yl31188 Yeast, ATCC4123

    Yeast, PCC253

    Yeast, PCC Yeast, isolates, 6, 23, 23a,

    Bacterial isolates 6, 8, 23, 37, 9

    29, 37, 43

    Mucor sp. Fusarium sp. Al temr ia sp. Rhiropus nigricans Botrytis cinerea Botryosphaeria ribis Penicillium expanswn Oidium lactis Saccharomyces sp. Saccharomyces sp. Saccharomyces sp. Saccharomyces cerevisiae var.

    Cryptococcus laurentii var.

    Saccharomyces logos Unknown, from rotting apple

    Unknown, from rotting apple



    - +' + + + + + - - - - -

    + + + + + + + + + + + + + + + + + + + + +' +'

    'Fields, 1964b. *Varied with age of culture.

    t indicates positive reaction, and - indicates negative reaction, by Voges-Proskauer teat.



    No. of Total No. of AMC samples Diacetyl

    Name of no. of samples with Type of inoculum fungus samples with AMC Range Meanb diacetyl Range Meanb

    Natural in: Jonathan, Red Delicious, Golden Delicious Mixed 60 54 0.0-100.9 10.9 4 1.2-7.7 3.5

    Inoculated in: Golden Delicious Botrytis

    cinerea 3 0 0.0 0.0 0 0.0 0.0 Mucor sp. 4 4 7.6-11.5 8.9 0 0.0 0.0 Penicilliwn

    expansum 6 6 0.9-10.8 5.1 0 0.0 0.0 Physalospora

    obtusa 3 3 0.5-3.0 2.5 0 0.0 0.0 Alternuria sp. 4 4 7.6-11.5 9.7 3 2.2-2.7 2.4

    Total rots examined 80 71 7

    Fields, 1964b. bMean value for 30 g of decayed tissue. Two samples contained no AMC.


    8 9 t r=0.849**

    0 I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 % Rot by Weight

    FIG. 2. The relation between acetylmethylcarbinol content and percent rot by weight (Fields, 1962a).

    Holck and Fields (1965a) analyzed 567 samples of apple juices stored at 0, 37, 72, and 91F for 242 days. Of these, 283 were tested for AMC, 195 for diacetyl, and 98 for ethyl alcohol. There was no signif- icant decrease in quantity of AMC or ethyl alcohol in apple juice in tin cans or glass bottles during the storage periods. The level of diacetyl, however, declined at a significant rate when it was added to apple juice in bottles and stored at 91 and 72'F for 219 days. Only AMC was tested for its stability during repeated thawing and freezing. No significant changes occurred in the quantities of AMC present in samples so treated.

    Both AMC and ethyl alcohol fulfill the criteria for a useful chemical index for determining the quality of apple juice. Since there was no decline in the quantities of AMC and ethyl alcohol during storage, these compounds reflect the conditions at the time of manufacturing. Although the level of diacetyl declined in glass containers in the study of Holck and Fields (1965a), the metabolite is still an effective indi- cator. As has been proposed (Fields, 1964b), the presence of diacetyl may be used as presumptive evidence of poor sanitation in a processing plant because it is produced by yeasts and usually is not found in tissue decomposed by filamentous fungi.

    AMC can also be used as an index of the quality of apple jelly. Holck and Fields (1965b) found no significant difference in AMC content between apple juice and apple jelly made from the same juice. Using AMC as an index, 19commercial samples were tested. Two jellies had AMC contents classified as unacceptable (4.05, 4.35 ppm), 3 were


    considered questionable (1.40, 1.65, 1.70 pprn), and 14 were accep- table (0.0 to 0.95 ppm).

    Diacetyl also has a potential for use as an indicator of the quality of orange juice. Hill et al. (1954) showed that, as processing time was lengthened, there was an increase in bacterial count and diacetyl content in pasteurized 20' Brix orange concentrate inoculated with Lactobacillus plantarum (Table XVI). Hill and Wenzel (1957) confirmed their earlier findings that an increase in plate counts corresponded with an increase in diacetyl when orange juice was concentrated in an evaporator. In 435 commercial samples of frozen concentrated orange juices collected from 24 Florida plants during the 1953-54 and 1954-55 seasons, amounts of diacetyl ranged from 0.1 to 3.3 ppm and 88% of the juices had values of 1.0 ppm or less. Five of these 435 samples were rated as poor in quality because of buttermilk-type off flavors. The diacetyl contents of these juices were 1.2, 2.6, 3.1,3.2, and 3.3 ppm.

    Voges-Proskauer reactants (AMC and diacetyl) have been found in juice from acceptable oranges (Beisel et al., 1954). The values ranged from 0.46 to 1.05 ppm, calculated as diacetyl. Defective fruit classified as splits, rots, and shriveled oranges contained Lactobacillus




    Processing time (hr)

    Off-flavor Diacatyl Plate count, orange, development content serum agar, pH 5.4

    Initial 5 6 7 8 9

    10 11 12 13 14

    none none none none none none slight slight slight slight


    0.6 102,000 0.0 0.0 0.0 840,000 1.6 1,205,000 1.0 1.5 3,060,000 3.2 3.4 6.2 7,500,000 8. I

    'Hill et d., 1954.


    and Leuconostoc whereas fruit with internal damage and fruit held under anaerobic conditions contained mainly yeast microflora, with a few bacterial isolates. The highest Voges-Proskauer reactants occurred in rots followed by splits. The lowest Voges-Proskauer values were obtained in oranges stored under anaerobic conditions, such as those held in the center of a load on a truck or fruit bin.

    Beisel et al. (1954) also isolated bacteria and yeast from various equipment in the processing plant and from defective fruit. Of 120 isolates, 67 were Lactobacillus, 13 were Leucomstoc, 1 was a Pediococcus, 20 were Acetobacter, and 6 were yeast cultures including the genera Candida, Kloeckera, and Torulopsis. Seven Voges-Proskauer-positive yeasts were isolated from frozen orange concentrates by Murdock (1964). Two of these were identified as Sacchuromyces carlsbergensis and S. cerevisiae. Yeasts were found to produce diacetyl on orange juice and orange juice concentrate, but they apparently destroyed the diacetyl after incubation of 2-3 days.

    d . Methods for Determination of AMC and Diacetyl. Analytical methods for the determination of AMC and diacetyl are based on the original observations of Voges-Proskauer for differentiation of Aerobacter aero- genes and Escherichia coli. A method which employs the Voges-Pros- kauer reaction and which has been used in the citrus industry was described by Hill and Wenzel (1957). The procedure requires 300 ml of the sample (orange juice or apple juice) for distillation. Colori- metric readings are made on the distillate by using a modification of the original Voges-Proskauer reagents. One reagent consists of 5 g of alpha naphthol dissolved in 95% ethyl alcohol. Creatin is dissolved in 40% sodium hvdroxide. When these reagents are mixed with either AMC or diacetyl, a red color develops. There is considerable variation in the method used by various plants even within the citrus industry, where it is used as a quality-control test of the sanitation of the pro- cessing (Murdock and Dennis, 1964).

    AMC and diacetyl can be measured in the same distillate by taking colorimetric readings at the end of 1 minute and after 10 minutes (Byer, 1954). AMC and diacetyl may also be determined separately by measuring the Voges-Proskauer reactants in the first 25-ml and the fourth 25-ml fractions of distillate. Most of the diacetyl is in the first fraction, whereas the distribution of AMC during distilla- tion is constant and independent of the concentration of the solution (Langlykke and Peterson, 1937). The amount of diacetyl in the first 25-ml fraction of distillate is concentrated 9.46 times, and the AMC 1.39 times (Fields, 1964b). Therefore, the factor 9.46 for diacetyl is divided into the value for the first 25-ml fraction to determine the diacetyl content in the original juice. The value of the fourth


    fraction is divided by 1.39 to determine the quantity in the original sample.

    Diacetyl may be determined independently from AMC by using hydroxylamine in a colorimetric reagent. Diacetyl, when treated with hydroxylamine, urea, and a phosphoric-sulfuric acid mixture, gives a yellow color, and AMC does not (White et al., 1946).

    e. Disadvantages and Advantages of Actylmethylcarbinol and Diacetyl as Indicators of Quality. One of the disadvantages of AMC as a quality indicator is that some rots were found to contain low amounts or no AMC. However, only 6 of 60 samples of mixed rots in apples were low in AMC (Fields, 1964b). It would be expected that the low boiling point of diacetyl (88OC.) would be a disadvantage. However, it did not appear to decrease its usefulness as an indicator of sanitation in plants processing apple juice. Ah4C content did not decrease with stor- age in either tin cans or glass bottles (Holck and Fields, 1965a). The quantity of diacetyl declined only in apple juice stored in glass bottles at 91 and 72'F (Holck and Fields, 1965a). Fields (1964b) found diacetyl in 17 of 31 commercial apple juice samples, indicating that it was not completely lost during processing. Quantitative methods for determin- ing AMC and diacetyl are rapid and effective ways of evaluating the quality of apple and orange juices.

    2. Ethyl Alcohol

    a. Biosynthesis of Ethyl Alcohol. Ethyl alcohol is a metabolic by- product of the growth of yeast, filamentous fungi, and bacteria. Classic- ally, however, ethyl alcohol is produced from some form of fermentable carbohydrate by yeast via the well known glycolysis pathway. The growth conditions determine the amount of alcohol formed.

    Mucors and Fusaria are generally associated with strong alcoholic fermentation (Foster, 1949). Members of the Aspergilli and Penicillia also produce volatile neutral compounds.

    6 . Ethyl Alcohol as an Indicator of Quality of Apple Juice. Hill and Fields (1966) evaluated ethyl alcohol as an indicator of the quality of apple juice. Ethyl alcohol in 80 ml of distillate from juice representing different varieties of sound apples ranged from 0.13 to 0.90 mg/ml. The mean for 39 samples representing four varieties was 0.47 mg/ml. With one exception, rots (72 samples) which were produced by pure and mixed cultures contained larger amounts of ethyl alcohol (0.0 to 24.8 mg/ml in 80 ml of distillate) than was found in distillate from juice of sound apples. When studied as still cultures, the levels of


    ethyl alcohol were considerably less (0.05 to 2.40 mg/ml in 80 ml of distillate) than those from naturally occurring rots.

    Table XVII shows the relation between percent rot by weight and the amount of ethyl alcohol (mg/ml) in 80 ml of distillate from apple juice prepared under pilot-plant conditions. Juices having larger amounts of rots were higher in ethyl alcohol than juices without rots.

    Yeasts formed considerable alcohol within 48 hours (Table XVm). Poor sanitation in the processing plant would contribute to yeast growth and would influence the levels of ethyl alcohol in the finished product.

    The alcohol values for sound apple juice (0.13 to 0.90 mg/ml in 80 ml of distillate) are lower than the quantities found in some com- mercial apple juices (0.25 to 4.13 mgl'ml in 80 ml of distillate). Hill and Fields (1966) suggested a standard for this metabolic by-product in determining the quality of apple juice. Their standard is as follows: ethyl alcohol values of 0.90 mg or less in 80 ml of distillate are con- sidered satisfactory, values greater than 0.90 mg/ml of ethyl alcohol in 80 ml of distillate are unsatisfactory. When this standard was applied



    Variety Percent Ethyl alcohol

    rot Type of rot content

    50% Jonathan 25% Red Delicious 25Yo Golden Delicious 0

    n L

    4 6 8

    10 12

    50y0 Jonathan 507, Golden Delicious 0

    4 8


    P. expansum 0.11 0.15 0.31 0.11 0.55 0.55 1.23

    Alternaria sp. 0.66 and 0.80

    P. expansum 0.76 0.90

    'Stored 2 years at OOF. *Hill and Fields, 1966.




    2 15



    Ethyl alcohol content

    Time (hr) Range



    0 6

    12 18 24 48 72 84

    0.20- 0.23 0.13- 0.15 0.15- 0.20 0.33- 0.45 0.55- 0.80

    18.00-23.50 47.50-50.00 67.50-67.50

    0.23 0.15 0.18 0.38 0.75

    21.00 50.00 67.50

    'Hill and Fields, 1966. em of three replicate SWPI~S.

    to the 8 commercial apple juices, 12 of 17 samples were graded as un- satisfactory. Using the AMC standard as suggested by Fields (1962a) and modified by Holck (1964), 11 of 17 samples were graded as unsatisfactory.

    c. Methods for Determining Ethyl Alcohol. Most methods for est- imating ethyl alcohol require distillation. To prevent the distillation of volatile acids during measurement of ethyl alcohol, it is necessary to adjust the pH of food to 8.0. If considerable protein is present, it should be precipitated with zinc hydroxide (Somogyi, 1930). All the simple alcohols pass into the distillate along with AMC. Corrections can be made for AMC. The ethyl alcohol in the sample can be oxidized with potassium dichromate. The potassium iodide is added. The amount of potassium iodide used can be determined by titrating with standardized sodium thiosulfate. This is not a specific, but a general, procedure, since the potassium dichromate will oxidize other materials also (Neish, 1957).

    Ethyl alcohol can also be determined by microdiffusion. This method was described by Conway (1947) and also by Winnick (1942).

    d. Disaduantages and Advantages of Ethyl Alcohol as an Indi- cator of Quality of Apple Juice. Probably the major disadvantage in the


    use of ethyl alcohol as an indicator is its low boiling point (78.5OC). Some of the alcohol could be lost if the food material were heat treated for a considerable time. In apple juice, however, extensive heat treat- ment vould damage the delicate flavor. Holck and Fields (1965a) found that there was no appreciable change in the ethyl alcohol content of apple juice samples preserved in glass bottles and tin cans during a 164-day storage period at 91, 72, and 37'F. In judging the quality of apple juice by using ethyl alcohol and AMC as indicators, 12 of 17 commercial apple juices were unsatisfactory according to the suggested alcohol standard, and 11 out of 17 were unsatisfactory with AMC the standard (Hill and Fields, 1966). About the same number of samples would be accepted or rejected with either the alcohol or the AMC index. The use of both tests would give a better indication of quality since the multiple metabolites would represent a greater part of the flora than would a single by-product.

    3. Nonvolatile and Volatile Acids

    a , Biosynthesis of Galacturonic, Succinic, Lactic, Acetic, and Formic Acids. In fruits and vegetables, galacturonic acid is a breakdown pro- duct of pectin. The biosynthesis of succinic acid is via amino acids and the tricarboxylic acid cycle. Lactic acid is synthesized from pyruvic acid by way of the Embden-Meyerhof-Parnas scheme of glycolysis. According to Fruton and Simmonds (1953), extracts of Escherichia coli convert pyruvic acid to acetic and formic acids.

    b: Nonvolatile and Volatile Acids as Indicators of Quality of Apple Juice, Strawberries, and Tomatoes. A considerable amount of rotten fruit can be used in the manufacture of fruit juices, jellies, and butters because its presence cannot be detected by odor or taste. Decomposi- tion of most fruits is brought about by the action of molds and yeast. Rot in fruits caused by bacterial action is rather rare. The mold count and the rot fragment count are used by food analysts but are of little value when applied to clear jellies and juices.

    Galacturonic acid liberated from pectin by enzyme hydrolysis would appear to have potential as an indicator of rot in certain apple products. After the removal of interfering substances it can be deter- mined in microgram quantities. Harris (1948) found that the enzyme, galacturonase, which is a member of the pectinase group and liberates galacturonic acid from yolygalacturonides, was present in tomatoes but absent in apples. Therefore, the increase in galacturonic acid in apples during rotting would be due to the action of galacturonase of microbial origin.

    Some galacturonic acid is present in ripe fruit, but the amount in


    rotten fruit is much greater (20 times or more). According to Harris (1948), amounts in juice from nine varieties of sound apples varied from 13 to 54 mg per ml of juice.

    In some cases commercial apple juice is clarified by enzyme prepara- tions and filtration prior to pasteurization. During enzyme treatment pectin is hydrolyzed to galacturonic acid. Obviously a method based upon measurement of galacturonic acid would be of no use on such a product. Harris (1948) suggested that such limitations would not render the method useless. It appears that it could be applied to such products as apple butter, jelly, and juice not clarified by enzyme.

    Galacturonic acid as an index of quality for strawberry juice was studied by Mills (1951, 1953). The galacturonic acid content of differ- ent varieties of sound fresh strawberries from two seasons were reason- ably uniform. Values ranged from 36 to 56 pglg juice, with an average of 42 ,ug/g juice.

    Succinic acid was considered as a chemical indicator of quality for tomato products and for spinach. However, in 1954, Van Dame con- cluded that the amounts of acetic, formic, succinic, or lactic acids found in tomato products were not indicative of the amount of rot due to molds, even though succinic acid is produced by Mucor, Rhizopus, Fusarium, Alternaria, and certain Aspergilli and Penicillia, all of which have been involved in the rotting of tomato fruits. Other spoilage organisms need to be investigated. Silverberg (1957) presented no data other than that related to improving the chemical method of recovery for succinic acid in spinach.

    Lactic acid is a metabolic by-product of both bacteria and fungi. Hillig and Ramsey (1945) studied the recovery of acetic and lactic acid from tomato to which was added various kinds of rots produced by microorganisms. The following types of rots were found to contain acetic, lactic, and formic acids: soft, black (Alternaria); Oospora; Anthracnose; black; and Mucor. The ranges in amounts of these acids were: 67-176 mg/100 g acetic; 60-509 mg/100 g lactic; and 1.8-518 mg/100 g formic. Good juice had the following content: 1.6-3.9 mg/ 100 g acetic, and 0.0-3.4 mg/lOO g lactic.

    c. Methods for Detecting Galacturonic, Lactic, Succinic, and Acetic Acids. Methods for lactic, succinic, and acetic acids are in the protein section. Winkler (1949; 1951; 1952; 1953) determined galacturonic acid in sound and rotten apples by the modified method of Deichmann and Dierker (1946), using naphthoresorcinol. The difference in galacturonic acid content


    juice, orange juice) and mixed acid solutions, recoveries were satis- factory. In 1952, Winkler reported the following disadvantages of the naphthoresorcinol method: instability of the reagent and lack of reproducibility in readings among different solutions of the reagent, particularly from different batches. A new carbozole method utilizing stable reagents and solutions was developed, but in 1953, Winkler reported that the results of this method were also disappointing. Winklers (1953) carbozole procedure for determination of galacturonic acid in fruits utilized a cation and anion column through which the juice was passed. Galacturonic acid was eluted from the anion column and measured by treating it with naphthoresorcinol reagent. Carbozole was also employed by Ashwell (1957) in determining uronic acids.

    Mills (1951) found that the naphthoresorcinol reaction was not specific for galacturonic acid but gave positive results for the general class of uronic acids as well as for pectic acid. However, galacturonic acid, or its poly combinations as pectin decomposition products, are all that would be expected to be present in fruit juice.

    d. Disadvantages and Advantages of Galacturonic, Lactic, Succinic, Formic, and Acetic Acids. The possibility of losing some or all of the volatile acids in processing some foods limits their potential as indi- cators. Of course, this depends upon the type of food and the method of processing. There also appear to be restrictions on the use of lactic and succinic acids as indicators. Preliminary studies have not shown a close relation between lactic and succinic acids and other quality- detecting methods such as mold counts. This lack of correlation may be due to the fact that yeasts, lactobacilli, and Acetobacter may also be involved in the spoilage.

    Both volatile and nonvolatile acids as a group are comparatively easy to determine. Therefore it seems that they would be suitable for use in combination with other possible indicators.


    Research to date on metabolic by-products of spoilage organisms as indicators of the quality of foods is little more than an introduction to the subject. Work on indicators of quality has been less for fruits and vegetables than for foods with high protein and fat content. Very little is known about the metabolic by-products of the organisms as they spoil various fruits and vegetables.

    Considerable research is needed on the physiology of the dominant flora for all foods. After the metabolic by-products of the organisms have been investigated, the levels of the potential indicators in sound


    fruits and vegetables and in various stages of decomposition need to be determined.

    Knowledge is essential on the influence of various processing methods upon the potential indicator. What is the influence of heat? What is the influence of vacuum concentration? What effects do container, storage temperature, and time have? At what level is the indicator found in the most carefully prepared product? What kind of standards can be recommended? How can industry be encouraged to use chemical indicators to raise the quality of the products?

    Chemical indicators of food quality must change as technology changes. For example, new acidulants are appearing on the market. Some of these, such as fumaric acid, may also be a metabolic by-product of microorganisms. If such substances are used in processing, this precludes their use as indicators of quality. The food analyst must be aware of trends in industry. If one indicator is invalidated, a new compound must be applied.

    Patterson (1945) explained that the delay in accepting chemical indicators has probably been related to the desire to find a universal indicator. The complexity of biological materials makes it unlikely that such a compound will be found. It may be possible, however, for a test to be developed which would detect the majority of undesirable samples.

    The complexity of the problem also has had an influence on the lack of progress in developing and using metabolic by-products of micro- organisms as indicators of food quality. Study of the biochemistry of the food product, that of the microorganisms, and the interactions involved in a spoilage situation requires either a team or multiple- discipline approach. The food scientist is uniquely suited to develop and promote the use of chemical indicators, because of his training in various disciplines.


    Appreciation is expressed to Drs. Bailey, Marshall, Ross, and Tweedy for their suggestions and assistance with this manuscript. This chapter was written when the authors were members of the Departments of Horticulture and School of Home Economics.


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