SUGAR
The effect of sugar was tested on both the mold and the yeast by adding it to tomato bouillon. It was supposed that a low percentage of sugar like the salt would plasmolyze the cells, and in this way check growth, but it seemed to have no effect until the amount was increased to 25 grams per 100 cc of bouillon. In this solution growth appeared as soon as with the weaker solutions, but there was a smaller amount. In the 25 to 40 gram solutions there was less development as the amount of sugar increased. In the 70 and 75 gram solutions growth was delayed one day in its appearance. In the 80, 85, and 90 gram solutions growth was delayed two days, the colonies growing submerged at first, but after a time forming on the surface. The mycelium remained very thin, but a thick layer of spores formed. From this point on the amounts were increased by 10 grams up to 200. The development became slower and less successively until 170 grams were added. In this case a small colony appeared on the surface in seven days, but seemed to grow less after that. The solutions were held, and in time crystals separated from the thick sirups. After two months dry-looking colonies developed along the edges, forming a ring, and some formed on the surface, these occurring also in the flasks containing 170, 180, 190, and 200 grams of sugar per 100 cc. The colonies were a dull greenish drab in spots, the remainder being white.
For the yeast the 80-gram solution of sugar was the strongest in which any development took place.
SPICES
Experiments to determine the value of the spices as antiseptics were made, using water infusions, acetic-acid extracts, and oil extracts.
WATER INFUSIONS
In making the water infusions 20 grams of the whole spices, with 200 cc of water, were boiled for forty-five minutes. This is approximately the length of time that the spices are cooked in the ketchup in the factory. The liquid was then filtered and from 0.1 to 5 cc of the filtrate was used in 10 cc of tomato bouillon. The same organisms were used as in the former experiments.
The tests showed that cinnamon and cloves were the strongest antiseptically. These checked growth when used in small amounts, but it required 3 cc of the cinnamon and 1 cc of the cloves to inhibit the growth of the mold. Mustard, paprika, and cayenne pepper checked growth also, but 5 cc, the highest strength used, did not inhibit growth. The ginger, mace, and black pepper had no apparent effect in the quantities used.
The effect of the spices on the development of the yeast was somewhat different from their effect on Penicillium. The cinnamon showed the strongest action, 3 cc being effective, whereas 5 cc of the cloves was required. The cayenne pepper came next in effectiveness, and after that the black pepper. The ginger, mace, and mustard solutions had no effect in the strengths used.
The remainder of the spice infusions were kept in glass-stoppered bottles in the laboratory, and in a few weeks’ time there was a coating of mold formed over the surface of the mace, the mustard, and the black and cayenne peppers. The paprika had small, stunted colonies dotting the surface.
At the time that these experiments were made a quantity of the ground spices were placed in large petri dishes and water was added to make a heavy paste. One set of these was inoculated with the mold, and another set with the yeast, and all were kept in a warm place. No development of either organism appeared on the cinnamon, cloves, or mustard; on the others a growth first showed in three days. On a normal medium growth appears in twenty-four hours. On the mace, paprika, and cayenne pepper the Penicillium and yeast with which the pastes were inoculated were overgrown in a few days with black mold (Rhizopus nigricans).
ACETIC-ACID EXTRACTS
In the manufacture of ketchup acetic-acid extracts of the spices are sometimes used instead of the whole spices, on account of their supposed antiseptic properties as well as their greater strength and convenience in handling. One minim of the standard acetic-acid extracts is equal in strength to 1 grain of the whole spices. The acid extracts obtained included allspice, celery, cloves, coriander, garlic, and black pepper.
In the tests 0.1, 0.2, 0.3, 0.4, 0.5, and 1 cc, respectively, of the extract was added to 10 cc of tomato bouillon. One set was inoculated with the mold and another set with the yeast. In the case of the mold, no growth occurred with the allspice and cloves; the celery checked the growth materially, there being no indication of mold until the sixth day. Normally a fairly strong growth occurs in twenty-four hours. In the solution containing 0.3 cc there was only one small colony in thirteen days, and no further development. In the solution containing the coriander, the growth in the 0.5 cc solution did not appear for three days, the 1 cc solution showing no growth. The garlic had practically the same effect as the coriander, while the black pepper was stronger, no growth appearing in the solution containing 0.5 cc.
The yeast was slightly stronger in resisting the effect of the extracts. No growth appeared with the allspice and cloves; 0.5 cc of the celery and 1 cc of the coriander were required to inhibit growth, and the garlic and black pepper gave similar results, a weak development occurring in the solutions containing 1 cc.
OIL EXTRACTS
Oil extracts of the spices were tested in the same manner as the water infusions and the acetic-acid extracts. The oils were so strong that in order to handle them easily they were mixed with equal volumes of alcohol, except that the mace, which was in the form of a paste, was mixed with two-thirds its volume of alcohol. To 10 cc of tomato bouillon were added 0.1, 0.2, 0.3, 0.4, and 0.5 cc, respectively, of the oils of cinnamon, cloves, mace, mustard, and black pepper.
In the case of the mold, there was no development in the solutions containing cinnamon, cloves, and mustard; in those containing mace and black pepper the development was slower than the normal, that in the black pepper being more pronounced. On the yeast the effect was similar, no development occurring in the cinnamon, cloves, and mustard, and a retarded development taking place in the mace and black pepper, that in the black pepper being the more pronounced.
The experiments show that some of the spices, notably allspice, cinnamon, and cloves have decided antiseptic value, but that the peppers are not as valuable as is generally supposed.
The oil extracts have been advocated for use in ketchup instead of the whole spices, but in quantities which would be useful antiseptically their use would be objectionable, for when present in approximately the same proportions as are the whole-spice infusions, the flavor is too strong and masks the more delicate flavor of the tomato. The acetic-acid extracts are more effective than are the water infusions, and they are not objectionable in the ketchup.
VINEGAR AND ACETIC ACID
An experiment was made to determine the antiseptic value of vinegar and acetic acid. Commercial 50-grain distilled vinegar was used. It was found that when 30 per cent of this vinegar was added to the tomato bouillon the development of mold was checked and the extent to which it was checked increased with the increased amounts of vinegar. The development in the solution containing 30 per cent of the vinegar was two days later than the normal in starting, while the solution containing 100 per cent was eleven days delayed and showed but little growth.
An 80 per cent solution of glacial acetic acid was used. One-half of 1 per cent added to the tomato bouillon checked growth to the same extent as 30 per cent of vinegar, and no development occurred when the quantity was increased to 2 per cent.
Experiments were then made in which vinegar was added to the ketchup in proportions varying from 1 part in 32 to 1 part in 8, with the result of greatly delaying the appearance of the mold as the proportion increased. With the increase in vinegar it was necessary to add sugar and slightly more spices to overcome the pungency of the acid and thus insure good flavor. The addition of the vinegar to the pulp had the effect of arresting the action of the oxidase and thus the bright color was maintained.
The usual custom in factory practice is to add the vinegar near the close of the cooking process otherwise a considerable portion of the acid will be driven off. This practice was followed in the experimental work, but it has since been found that continued heating in the presence of the acid has some effect upon sterilization, and therefore the increased amount of vinegar is effective not only because of the additional acid present, but also because the heating in the after process is thereby rendered more efficacious.
This line of experiments gives promise of practical results in producing a ketchup which will not only keep while in the bottle, but will also keep longer after it is opened. Each manufacturer must work out the quantities that could be used with his formula and still retain the character of his goods.
OIL
In ketchup manufacturing it is customary, if an agitator is not used, to put a small amount of fat in the kettle to check the ebullition during the reduction of the pulp. The amount used in this manner is not sufficient, however, to be apparent in the ketchup. Brannt[4 - Brannt, W. L., A Practical Treatise on the Manufacture of Vinegar, 1900, p. 455.] states that in some factories, where the trimmings are allowed to accumulate for the season, they are given liberal doses of oils and condiments when cooked, in order to disguise their defects, so that the product can be placed on the market as “fresh tomato catchup.” That the use of oils is increasing is evident from the comparison of the ketchup of the past season with that of former years.
When oil is used in ketchup, it is easily detected under the microscope, as it appears in the form of shining, yellow globules which blacken gradually when treated with osmic acid. Besides this, the oil comes to the surface of the ketchup, where it can be seen readily, and if considerable oil has been used a distinct layer is formed. When the ketchup has been made for some time, the oil changes so that the ketchup has a peculiar “greasy” odor, or the oil may be so changed as to give a decidedly rancid smell to the ketchup. Oil usually causes a deterioration in flavor and odor, though some of the ketchups to which it has been added do not spoil readily. Olive oil, cottonseed oil, and oleomargarine are used. That the oil is not considered one of the regular known ingredients of the ketchup is shown by the failure to declare its presence on the label.
To test the antiseptic value of oils in ketchup, experiments were made, using olive oil, cottonseed oil, and oleomargarine in the proportions of 1 part of oil to 1,000, 750, and 500 parts of ketchup, respectively. The ketchup was made in small quantities, 2 gallons for each experiment. After bottling, all except the check bottles were inoculated with Penicillium and kept at kitchen temperature. All spoiled, and neither the quantity nor kind of oil used had any marked effect in preventing spoilage. That the oils affected the development of the mold was evident. The mold developed first at the junction of the ketchup with the bottle forming a ring which spread gradually over the surface developing a somewhat heavy mycelium. This remained white longer than usual, spores forming very gradually, as indicated by the change in color from white to a delicate blue. At the end of three weeks only spots of color appeared on the surface and these were still blue, though in ordinary development the blue color changes to green in two or three days.
Another test was made, using olive oil only, and in the proportions of 1 part of oil to 500, 400, and 300 parts, respectively, of the ketchup. Reduction was made in a steam-jacketed kettle, the oil being added when the ebullition of the ketchup was the strongest, after which the boiling was continued for fifteen minutes. The ketchup was bottled, unsterilized bottles being used, then covered loosely with the metal caps.
The time required for the ketchup to spoil was longer than in the first set, but there was not sufficient difference nor enough uniformity in the time to indicate that the use of oil in ketchup is desirable, even if the change of flavor and odor be not taken into consideration. The average number of days before spoilage for those containing 1 part of oil to 500 parts of ketchup, was thirteen and two-thirds days; one has not yet spoiled (a period of forty-five days), while the first bottle spoiled in four days. Those having 1 part of oil to 400 parts of ketchup had an average life of nine and three-fourths days, the minimum being three days, and the maximum twenty-six days. Those having 1 part to 300 parts of ketchup on an average did not spoil for six and three-fourths days, the minimum being four days, and the maximum eleven days.
The failure of some of the bottles to spoil, though similar in every known respect to those which did spoil, is a feature peculiar to ketchup and is familiar to manufacturers who make careful tests before putting their product on the market. For this reason a rather large number of bottles should be used in a test in order that the results may be approximately accurate and represent general conditions.
STUDY OF PENICILLIUM IN KETCHUP
Penicillium is a plant which is distributed widely and apparently is able to grow wherever organic matter is found, though flourishing best when the material contains acid. It causes loss in canneries, breweries, distilleries, etc., the only use made of it being in the manufacture of Roquefort cheese, the immature cheese being inoculated with the conidia for the effect the mold produces in the maturing process.
DEVELOPMENT
In developing, the mold forms a white felt-like mass, covering the medium on which it is growing; then as development proceeds, it changes to bluish-green, and finally to a darker, duller color. The change in color is accompanied by a change in structure, the surface becoming powdery in appearance, a slight current of air being sufficient to dislodge a cloud of fine dust. This fine dust is formed of small, spherical bodies, the spores or conidia (from the Greek meaning dust). These need no resting period, but are able to develop at once. When the conidia lodge on a moist substance they swell to a much greater size and then send out a tube from some part of their surface. The tube lengthens and septa form, dividing the tube into sections, or cells. At the same time branches are sent out, which again form other branches. The original conidium sends out a second branch shortly after the first one, and usually from the opposite side, and may even send out a third one. The formation of the septa and the subbranching goes on in all, so that in a short time the branches mat together and form a felt-like cover.
REPRODUCTION
After a shorter or longer period of development, dependent on the conditions, branches are sent perpendicularly from the substratum, and into the air. These branches cease their growth in length, sending out branches near the tip, which take the same general direction as the original branch. Each of these subbranches is called a sterigma (from the Greek word meaning support). In vigorous development the sterigmata may form secondary branches, the whole forming a tassel-like arrangement. The tip of a sterigma enlarges, a septum forms around the enlargement, cutting it off from the sterigma, and forming a conidium. The sterigma develops to the original length and another conidium is formed, the operation being repeated many times, thus forming a chain of spores. As the other sterigmata are also forming conidia in the same manner, a series of these chains is formed close together. After the cessation of conidial development, the filament below the sterigmata is disorganized, setting free the conidia. The filament and head together are called the conidiophore (Greek, dust-bearer).
Penicillium forms spores sexually, but the conditions for their formation are unknown. Brefeld obtained them by growing the mold on damp bread placed between two glass plates, and excluding the air. Lindner obtained carpospores on a wort gelatin culture in a petri dish, from which the air was excluded. The writer has tried various methods for obtaining carpospores, but so far without success. Moist chambers were used with various media, excluding the air. The development of the mold is seemingly dependent on the amount of air in the chamber at the time of sealing. After the air is exhausted, the conidiophores assume fantastic forms, developing only one or a few sterigmata, and on these one or few conidia. In other cases the conidiophores are fascicled, in no cases, however, forming the conidia as luxuriantly as when air is supplied. The hyphæ become clear, much vacuolated, and develop more septa, and some of the cells become much enlarged. An enlarged cell will often contain two or three septa, thus forming cells that are not larger than disks. In cultures from which the air was excluded from the start, no development took place. In test-tube cultures sealed with paraffin after twenty-four hours, the mold developed on the surface of the gelatin, forming a felted white mass, but no conidia nor carpospores were formed.
GROWTH IN KETCHUP
The form of Penicillium which was used in the experiments was isolated from ketchup in which it grew luxuriantly. When conidia are first formed on the ketchup, they are a delicate blue in color; they then become bluish green, then green, and finally olive. The development of the color of mold growing on ketchup is practically the same as when grown in wort, tomato bouillon, pea bouillon, or gelatin made with these solutions as a basis. In ketchup containing sodium benzoate, the blue color appearing first remains for a long time, and in old cultures the mold is a dull drab, not olive, as in normal development.
In ordinary ketchup made without a preservative, the mold forms a heavy, wrinkled mycelium, showing a large development of conidia. In the bottles of ketchup, the mold pushes down into the ketchup, becoming entirely submerged, a clear liquid covering the mold and separating it from the ketchup. This occurred in more than one hundred bottles. No secondary mycelium formed on the surface of the liquid, a method of development which frequently occurs in ordinary media when a mass of mold is submerged.
An exception to this was shown in ketchup which had developed the mold in the laboratory. The bottles were then put in the refrigerator for two weeks. During this time scarcely any development took place; but after they were again placed in the laboratory, the mycelium pushed down into the ketchup and a new, very thin mycelium developed on the surface. The filaments when seen under the microscope were swollen, had irregular outlines, and a comparatively smaller number of septa, and were filled with a coarsely granular protoplasm. The ends were blunt and misshapen and the sterigmata were irregular, tending more toward a fasciculated arrangement, and forming fewer conidia. The filaments from the vinegar and acetic acid media had the same appearance as those developed on ketchup, but had a smoother outline.
TEMPERATURE TESTS
The limits for the germination of Penicillium, as given by W. J. Sykes,[5 - Principles and Practice of Brewing, 1907, p. 284.] are 2° to 43 °C. (35° to 110° F.), and the most favorable temperature 22° to 26 °C. (72° to 79° F.). This author states also that according to Pasteur the dry spores retained their vitality at 108 °C. (226° F.), but that they were soon killed when immersed in boiling water. Klöcker[6 - Ibid., p. 281.] quotes Pasteur as saying that the conidia are killed if exposed to a temperature of 127° to 132 °C. for half an hour, but that they retain life at 119° to 121 °C.
A series of tests was made to determine the thermal death point of the moist and dry conidia of the Penicillium used in the experiments, a young, vigorous development on ketchup being used. The flasks were kept under observation for a month after the tests were made, as in many cases a development does not occur in the usual time. The high temperatures applied for longer periods of time were tried first, but both temperature and time were reduced as results from the series were obtained. Only the conditions obtaining in the final tests are given in the table. It was found that the Penicillium used did not have the high resistance supposed.
The tests were made in small flat-bottomed 10-cc flasks, tomato bouillon being used for the tests on moist conidia. The bouillon was used so as to have the conidia in a nutritive medium after the test was made, without transferring. The time for those at 100 °C. was estimated from the time of ebullition. At the end of the specified time, the flasks were cooled promptly under running water. As the flat bottoms gave comparatively large surface, the heating and the cooling could be effected in a short time. For the tests below 100 °C. a vessel of water was heated to the desired temperature, and the flasks were immersed in it and shaken constantly. The dry conidia were placed in test tubes which were immersed in boiling water for the desired time and cooled under running water, after which 10 cc of sterilized tomato bouillon was added. After determining the death point in this manner and finding it to be much lower than had been supposed, it was decided to make the test again, but using ketchup as the medium. Ten grams of ketchup were sterilized, then inoculated from a vigorous growth of mold, and tested with a set in which the tomato bouillon was used. For those below 100 °C. the two flasks which were to receive the same temperature were held in the vessel of water at the same time, so that as nearly as possible the treatment would be identical. The following results were obtained:
Thermal death point of moist and dry conidia of Penicillium.
The moist heat was very effective in destroying the vitality of the conidia of Penicillium, the death point being 27 °C. higher than the maximum temperature for germination as given by Sykes. The heating was more effective in destroying germs when applied to bouillon than to ketchup, no development taking place for any temperature above 65 °C., even when applied for a short time.