434 Modern Food Microbiology content and more lipid saturation According to this investigator, cells cannot grow at temperatures below the solidification point of their lipids Marr and Ingraham33 showed a progressive increase in saturated fatty acids and a corresponding decrease in unsaturated fatty acids in E coli as the temperature of growth increased The general decrease in the proportion of unsaturated fatty acids as growth temperatures increase has been found to occur in a large variety of animals and plants Saturated fatty acids form stronger hydrophobic bonds than unsaturated Among the saturated fatty acids are branched-chain acids The preferential synthesis of branched heptadecanoic acid and the total elimination of unsaturated fatty acids by two thermophilic Bacillus spp have been demonstrated.60 Mesophilic bacteria display changes in their membrane lipids when grown significantly above or below their normal growth range When D57◦ C values of four heat-adapted strains of E coli (including E coli 0157:H7 and a rpoS mutant) were determined, the values were up to 3.9 minutes longer than controls for all strains.64 Palmitic acid (16:0) and cis-vaccenic acid (18:1 7c) increased in the membrane of two strains Total intracellular verotoxin decreased in the heat-adapted strain but extracellular toxin increased in the heat-adapted cells, apparently the result of increased membrane fluidity.64 Cellular Membranes The nature of cellular membranes affects thermophilic growth Brock11 reported that the molecular mechanism of thermophilism is more likely to be related to the function and stability of cellular membranes than to the properties of specific macromolecules This investigator pointed out that there is no evidence that organisms are killed by heat because of the inactivation of proteins or other macromolecules, a view that is widely held According to Brock, an analysis of thermal death curves of various microorganisms shows them to be a first-order processes compatible with an effect of heat on some large structure such as the cell membrane, as a single hole in the membrane could result in leakage of cell constituents and subsequent death Brock has also pointed out that thermal killing due to the inactivation of heat-sensitive enzymes, or heat-sensitive ribosomes, of which there are many copies in the cell, should not result in simple first-order kinetics The leakage of ultraviolet light-absorbing and other material from cells undergoing “cold shock” would tend to implicate the membrane in hightemperature death Because most animals die when body temperatures reach between 40◦ C and 45◦ C and most psychrophilic bacteria are killed at about this temperature range, the suggestion that lethal injury is due to the melting of lipid constituents of the cell or cell membrane is not only plausible, it has been supported by the findings of various investigators The unit cell membrane consists of layers of lipid surrounded by layers of protein and depends on the lipid layers for its biological functions The disruption of this structure would be expected to cause cell damage and perhaps death In view of the changes in cellular lipid saturation noted above, cellular membrane integrity appears to be critical to growth and survival at thermophilic temperatures Effect of Temperature Brock11 called attention to the fact that thermophiles apparently not grow as fast at their optimum temperatures as one would predict or is commonly believed Arrhenius plots of thermophile growth compared to E coli over a range of temperatures indicated that, overall, the mesophilic types were more efficient Brock noted that thermophile enzymes are inherently less efficient than mesophiles because of thermal stability; that is, the thermophiles have had to discard growth efficiency in order to survive at all Food Protection with High Temperatures 435 Genetics A significant discovery toward an understanding of the genetic bases of thermophilism was made by McDonald and Matney.36 These investigators effected the transformation of thermophilism in B subtilis by growing cells of a strain that could not grow above 50◦ C in the presence of DNA extracted from one that could grow at 55◦ C The more heat-sensitive strain was transformed at a frequency of 10−4 These authors noted that only 10–20% of the transformants retained the high-level streptomycin resistance of the recipient, which indicated that the genetic loci for streptomycin resistance and that for growth at 55◦ C were closely linked Although much has been learned about the basic mechanisms of thermophilism in microorganisms, the precise mechanisms underlying this high-temperature phenomenon remain a mystery The facultative thermophiles such as some B coagulans strains present a picture as puzzling as the obligate thermophiles The facultative thermophiles display both mesophilic and thermophilic types of metabolism In their studies of these types from the genus Bacillus, which grew well at both 37◦ C and 55◦ C, Bausum and Matney6 reported that the organisms appear to shift from mesophilism to thermophilism between 44◦ C and 52◦ C CANNED FOOD SPOILAGE Although the objective in the thermal canning of foods is the destruction of microorganisms, these products nevertheless undergo microbial spoilage under certain conditions The main reasons for this are underprocessing, inadequate cooling, contamination of the can resulting from leakage through seams, and preprocess spoilage Because some canned foods receive low-heat treatments, it is to be expected that a rather large number of different types of microorganisms may be found upon examining such foods As a guide to the type of spoilage that canned foods undergo, the following classification of canned foods based on acidity is helpful Low Acid (pH > 4.6) This category includes meat and marine products, milk, some vegetables (corn, lima beans), meat and vegetable mixtures, and so on These foods are spoiled by the thermophilic flat-sour group (Geobacillus stearothermophilus, B coagulans), sulfide spoilers (Clostridium nigrificans, C bifermentans), and/or gaseous spoilers (Thermoanaerobacterium thermosaccharolyticum) Mesophilic spoilers include putrefactive anaerobes (especially PA 3679 types) Spoilage and toxin production by proteolytic C botulinum strains may occur if they are present Medium-acid foods are those with a pH range of 5.3–4.6, whereas low-acid foods are those with pH ≥ 5.4 Acid (pH 3.7–4.0 to 4.6) In this category are fruits such as tomatoes, pears, and figs Thermophilic spoilers include B coagulans types Mesophiles include P polymyxa, P macerans (B betanigrificans), C pasteurianum, C butyricum, Thermoanaerobacterium thermosaccharolyticum, lactobacilli, and others 436 Modern Food Microbiology High Acid (pH < 4.0–3.7) This category includes fruits and fruit and vegetable products—grapefruit, rhubarb, sauerkraut, pickles, and so forth These foods are generally spoiled by non-sporeforming mesophiles—yeasts, molds, Alicyclobacillus spp., and/or lactic acid bacteria Alicyclobacillus spp can grow in and cause spoilage of apple and tomato juice and white grape juice.52 The fungus Byssochlamys can grow at pH as low as 2.0, and Neosartorya fischeri can grow as low as pH 3.0.9 Canned food spoilage organisms may be further characterized as follows: • Mesophilic organisms • – Putrefactive anaerobes – Butyric anaerobes – Aciduric flat sours – Lactobacilli – Yeasts – Molds Thermophilic organisms – Thermophilic anaerobes producing sulfide – Flat-four spores – Thermophilic anaerobes not producing sulfide The canned food spoilage manifestations of these organisms are presented in Table 17–15 With respect to the spoilage of high-acid and other canned foods by yeasts, molds, and bacteria, several of these have been repeatedly associated with certain foods The yeasts Torula lactis-condensi and T globosa cause blowing or gaseous spoilage of sweetened condensed milk, which is not heat processed The mold Aspergillus repens is associated with the formation of “buttons” on the surface of sweetened condensed milk Lactobacillus brevis (L lycopersici) causes a vigorous fermentation in tomato catsup, Worcestershire sauce, and similar products Leuconostoc mesenteroides has been found to cause gaseous spoilage of canned pineapples and ropiness in peaches The mold Byssochlamys fulva causes spoilage of bottled and canned fruits, and its actions cause disintegration of fruits as a result of pectin breakdown.5 Torula stellata has been found to cause the spoilage of canned bitter lemon, and to grow at a pH of 2.5.44 Frozen concentrated orange juice sometimes undergoes spoilage by yeasts and bacteria Hays and Reister23 investigated samples of this product spoiled by bacteria The orange juice was characterized as having a vinegary to buttermilk off-odor with an accompanying off-flavor From the spoiled product were isolated L plantarum var mobilis, L brevis, Leuconostoc mesenteroides, and Leuconostoc dextranicum The spoilage characteristics could be reproduced by inoculating the above isolates into fresh orange juice Minimum growth temperatures of spoilage thermophiles are of some importance in diagnosing the cause of spoiled canned foods B coagulans (B thermoacidurans) has been reported to grow only slowly at 25◦ C but grows well between 30◦ C and 55◦ C G stearothermophilus does not grow at 37◦ C, its optimum temperature being around 65◦ C with smooth variants showing a shorter generation time at this temperature than rough variants.17 T thermosaccharolyticum does not grow at 30◦ C but has been reported to grow at 37◦ C For reviews on the spoilage of acid and low-acid food products, see references 9, 38, and 52 Also of importance in diagnosing the cause of canned food spoilage is the appearance of the unopened can or container The ends of a can of food are normally flat or slightly concave When Food Protection with High Temperatures 437 Table 17–15 Spoilage Manifestations in Acid and Low-Acid Canned Foods Type of Organism B thermoacidurans (flat sour: tomato juice) Butyric anaerobes (tomatoes and tomato juice) Non-sporeformers (mostly lactics) Appearance and Manifestations of Can Acid products Can flat; little change in vacuum Can swells; may burst Can swells, usually bursts, but swelling may be arrested Flat sour Low-acid products Can flat; possible loss of vacuum on storage Thermophilic anaerobe Can swells; may burst Sulfide spoilage Can flat; H2 S gas absorbed by product Can swells; may burst Putrefactive anaerobe Aerobic sporeformers (odd types) Can flat; usually no swelling, except in cured meats when NO3 and sugar are present Condition of Product Slight pH change; off-odor and flavor Fermented; butyric odor Acid odor Appearance not usually altered; pH markedly lowered—sour; may have slightly abnormal odor; sometimes cloudy liquor Fermented, sour, cheesy, or butyric odor Usually blackened; “rotten egg” odor May be partially digested; pH slightly above normal; typical putrid odor Coagulated evaporated milk, black beets Source: From Schmitt.47 microorganisms grow and produce gases, the can goes through a series of changes that are visible from the outside The can is designated a flipper when one end is made convex by striking or heating the can A springer is a can with both ends bulged when one or both remain concave if pushed in or when one end is pushed in and the other pops out A soft swell refers to a can with both ends bulged that may be dented by pressing with the fingers A hard swell has both ends bulged, so that neither end can be dented by hand These events tend to develop successively and become of value in predicting the type of spoilage that might be in effect Flippers and springers may be incubated under wraps at a temperature appropriate to the pH and type of food in order to allow for further growth of any organisms that might be present These effects on cans not always represent microbial spoilage Soft swells often represent microbial spoilage, as hard swells In high-acid foods, however, hard swells are often hydrogen swell, which result from the release of hydrogen gas by the action of food acids on the iron of the can The other two most common gases in cans of spoiled foods are CO2 and H2 S, both of which are the result of the metabolic activities of microorganisms Hydrogen sulfide may be noted by its characteristic odor, whereas CO2 and hydrogen may be determined by the following test Construct an apparatus of glass or plastic tubing attached to a hollow punch fitted with a large rubber stopper Into a test tube filled with dilute KOH, insert the free end of this apparatus and invert it in a beaker filled with dilute KOH When an opening is made in one end of the can with the hollow 438 Modern Food Microbiology Table 17–16 Some Features of Canned Food Spoilage Resulting from Understerilization and Seam Leakage Feature Can Product appearance Odor PH Microscopic and cultural History Understerilization Flat or swelled; seams generally normal Sloppy or fermented Normal, sour, or putrid but generally consistent Usually fairly constant Pure cultures, sporeformers; growth at 98◦ F and/or 113◦ F; may be characteristic on special media, e.g., acid agar for tomato juice Spoilage usually confined to certain portions of pack In acid products, diagnosis may be less clearly defined Similar organisms may be involved in understerilization and leakage Leakage Swelled; may show defects Frothy fermentation; viscous Sour, fecal, generally varying from can to can Wide variation Mixed cultures, generally rods and cocci; growth only at usual temperatures Spoilage scattered Source: From Schmitt.47 punch, the gases will displace the dilute KOH inside the tube Before removing the open end from the beaker, close the tube by placing the thumb over the end To test for CO2 , shake the tube and look for a vacuum as evidenced by suction against the finger To test for hydrogen, repeat the test and apply a match near the top of the tube and then quickly remove the thumb A “pop” indicates the presence of hydrogen Both gases may be found in some cans of spoiled foods “Leakage-type” spoilage of canned foods is characterized by a biota of non-sporeforming organisms that would not survive the heat treatment normally given to heat-processed foods These organisms enter cans at the start of cooling through faulty seams, which generally result from can abuse The organisms that cause leakage-type spoilage can be found either on the cans or in the cooling water This problem is minimized if the cannery cooling water contains