Tucker (1930) studied the proteolytic activity of a Clostridium putrificum isolate from sour ham. Proteolysis was inhibited by 4.0% sodium chloride (salt), 44,000 µg/g of sodium nitrate, or 12,000 µg/g of sodium nitrite in a pork infusion broth buffered to pH 7.6 and held at 37°C. He concluded that, of the three, only salt was used in sufficient concentration in commercial hams to prevent proteolytic activity at 37°C. The same results were obtained with Clostridium putrefaciens at 23°C. He suggested that nitrite, not nitrate, is responsible for retarding proteolysis by C. putrificum. Tanner and Evans (1933), using Clostridium botulinum, C. putrificum, and Clostridium sporo- genes, found that sodium nitrate up to 22,000 µg/g did not inhibit the 12 strains tested in either pork infusion or egg-meat media. At levels between 22,000 and 44,000 µg/g, irregular inhibition occurred among seven botulinal cultures. At 44,000 µg/g, 6 of the 7 cultures were inhibited. The authors concluded that nitrate at concentrations as low as 22,000 µg/g cannot be relied on to inhibit putrefactive anaerobes if other conditions are favorable for growth.
Tanner and Evans (1934) reported that 5900 µg/g of sodium nitrite inhibited 9 of 12 strains of clostridia in nutrient broth and all 12 strains in dextrose broth. Levels of 3100 µg/g in pork infusion and 3900 µg/g in egg-meat, the highest levels tested, were not inhibitory to any of the strains,
although the nitrite was added to the media (pH 7.4) before autoclaving. The authors concluded that reliance cannot be placed on nitrite alone to prevent spoilage by clostridia. From tests with various blends of salt, nitrite, and nitrate in peptic digest broth (pH 7.4), raw pork, and cooked pork, Evans and Tanner (1934) concluded that “the most effective component in curing mixtures is sodium chloride” and “the sodium nitrite present apparently produced no effect on the organisms.” Botulinal toxin was produced in the cooked meat and a few samples of raw meat. Repression of toxin formation in the raw meat was a result of other bacteria that caused a decline in pH to inhibitory levels (pH 4.49 to 5.3).
It is curious that Tanner and Evans (1933), citing earlier work by MacNeal and Kerr, state the following:
[P]otassium nitrate in neutral or alkaline solutions exerted no special restrictive effect on bacterial activity. Under these conditions, it was used as a food. In acid solutions, however, the results were quite different. A marked inhibition was noticed. They said that this effect was incomparably greater than that of salt and was best ascribed to the production of small amounts of nitric acid, and of nitrous acid also, in mixtures containing reducing substances. Potassium nitrate was therefore considered to be particularly effective in restricting acid fermentation of organic substances that are already slightly acid. They further believed that the claim of meat packers that small amounts of nitrate in the pickle produced better preservation of the meat, was borne out by their results. It seemed that nitrate was especially valuable in preventing high degrees of acidity or souring of meat. MacNeal and Kerr stated that the effect of saltpeter was probably due to the oxidizing action of the nitrate ion in the presence of hydrogen ion.
It is surprising that the fundamental importance of pH, so clearly stated and available to researchers at that time, was not pursued.
Perishable (i.e., insufficiently heated to be stable at room temperature) canned, chopped, spiced ham and luncheon meats containing nitrate, nitrite, and sugar swelled in 1 to 90 days during temperature abuse at 37°C. Examining 1000 cans representing all manufacturers at the time, the sole cause of the spoilage was found to be nitrate-reducing Bacillus species (Jensen et al., 1934). A medium was developed with nitrate, sugar, and cured meat to detect the gas-producing bacteria in raw materials and the plant environment. Nitrite could not replace nitrate. To obtain gas produc- tion in the medium, both nitrate and cured meat had to be present. The reason for this specific combination of ingredients remains unknown. Perhaps the cured meat provides a source of heme, as the more recent results suggest (Jacobs et al., 1964).
The opinions expressed by Jensen et al. in 1934 laid the foundation for a new concept. They stated that if nitrate were omitted from the product, the usual fermentative carbon dioxide swells caused by Bacillus species would be prevented and conditions for the growth of clostridia and other anaerobes would prevail. Their bold statements that a mixed nitrate–nitrite cure favors most species of aerobes but a nitrite cure “always inhibits fermentation and aids in putrefaction” were to have long-term effects within the meat industry and federal government.
Brooks et al. (1940) described the contemporary method of making bacon in the United Kingdom. After the slaughtering process, pork sides were held at ambient temperature overnight and then moved into refrigeration for 24 hours to achieve 5.6°C in the meat. The sides were then immersed in a tank and cured using a pickle solution containing potassium nitrate. Several inter- esting questions were raised. Does nitrate serve any function other than as a precursor for nitrite? Does nitrate appreciably retard or inhibit the growth of putrefactive anaerobes? What is the mech- anism behind nitrite depletion, and what factors influence depletion? Would the more rapid chilling process as practiced in North America adversely affect the quality of English-style bacon? Can bacon acceptable to the English trade be produced by the use of rapidly chilled meat and with nitrite in place of nitrate? Not all the questions could be answered, but the research led to the following general conclusions. Satisfactory bacon can be produced with the use of nitrite. The characteristic cured flavor of bacon is primarily the result of the action of nitrite. The conversion
Nitrite 173
of nitrate to nitrite in commercial bacon curing brines is mainly the result of the growth of micrococci. The presence of nitrate or microbial action during the curing process is not essential for bacon flavor. Rapid chilling of the meat is not detrimental.
Tarr and coworkers published a series of reports on the possible use of nitrite for preservation of fish. Tarr and Sutherland (1940) concluded that “as far as can be ascertained from the literature, little or nothing is known regarding the possible bacteriostatic (or bactericidal) action of nitrites in meat pickles or in cured meats themselves.” Their tests with fish muscle showed that nitrite delayed spoilage. Subsequent tests (Tarr, 1941) demonstrated the importance of pH to the efficacy of nitrite. At pH 7.01, little or no inhibition was observed. At pH 5.7 and 6.0, complete or strong microbial inhibition occurred.
The effect of pH was more firmly documented in a series of tests in nutrient broth inoculated with a variety of aerobes. Later tests involving C. botulinum and C. sporogenes yielded similar results (Tarr, 1942). Investigations on the mode of action led Tarr to conclude that nitrite is not inhibitory by sole virtue of its toxicity toward aerobic respiratory catalysts (Tarr, 1941) and that the mode of action was still unknown (Tarr, 1942).
While Tarr was demonstrating that nitrite has strong antimicrobial properties, Jensen and Hess (1941) advocated the virtues of nitrate and perpetuated the belief that the sole function of nitrite was for cure color development. They stated that the literature shows “unmistakenly that nitrate in the cure exerts a definite inhibitory effect upon bacteria.” They postulated the anticlostridial mech- anisms to consist of partial conversion of nitrate to hydroxylamine, which inactivates catalase and allows the accumulation of hydrogen peroxide, which destroys anaerobes.
Jensen and Hess stated that nitrite reacts with protein during heating and is destroyed, thus leaving the meat in much the same state as freshly cooked uncured meat. Others have shared this concern (Scott, 1955). Even if nitrite had been uniformly accepted as a critical factor in microbial protection, workers at the time would have had difficulty applying the information to commercial practice. How could reliance be placed on an unstable preservative that disappears during processing and storing? This question is still valid.
The purpose of Jensen and Hess’ work in 1941 was to determine the specific value of nitrate as a bacteriostat and as an inhibitor of bacterial spore germination and toxigenicity and its effect on the anaerobic flora of cured meats. Two tests were reported with perishable canned ham. In one, nitrite alone inhibited anaerobic putrefaction during 30 days of abuse at 37.2°C. In the other, fewer cans became putrid in the product with both nitrate and nitrite than in the product with nitrite only. Despite these results, the authors concluded that nitrate is beneficial for preventing the growth of putrefactive anaerobes in abused perishable canned ham. In both tests some cans with nitrate swelled because of the growth of Bacillus species, thus serving as a desirable indicator of temperature abuse. Using the spiced ham medium they reported earlier, Jensen and Hess found that heat (93°C for 4 hours) combined with 5000 µg/g, but not 2000 µg/g, of sodium nitrate, prevented growth of C. sporogenes during 4 weeks of incubation at 37.2°C. The authors suggested that the combination of heat, nitrate, nitrite, and salt caused destruction of anaerobic spores at much lower temperatures. Yesair and Cameron (1942) pursued this idea but concluded that curing salts do not assist in thermal destruction but inhibit outgrowth. Stumbo et al. (1945a) reached the same conclusion from tests in meat processed at 116°C. They also reported (Stumbo et al., 1945b) that nitrite appreciably delayed germination, although salt was the stronger inhibitor. Nitrate, alone or in combination with other ingredients, did not appreciably influence spoilage in tubes processed from F0 = 1 to 16 and
held for a year at 28°C.
Jensen et al. (1949) also examined the combined effect of heating and curing salts. Greatly increased inhibition occurred in tubes of pork heated in the range of 50°C to 65°C for 30 minutes. Higher temperatures, longer heating times, or repetitive heating did not increase this effect. Within the levels normally added to canned ham, increasing salt and nitrite caused increased inhibition. However, increasing nitrate did not increase the inhibition of C. sporogenes 3679.
Vinton et al. (1947) found the heat resistance of spore crops was not altered by adding nitrite and nitrate to the meat on which the spores were grown, but the condition of the meat was a factor. Spores produced in raw, pasteurized, and sterilized meat were of low, intermediate, and high heat resistance, respectively.