. ._ __-__. _ \l ‘ r W 114 534 THS ‘QQ‘ ‘,’] N u‘ u (2‘: '7." . ' 3 '5': 4:44;»: “v-. ..~ 9? This is to certify that the -‘ a; thesis entitled '9 3 Factors Causing Olmdiness in V 1 $3.29 Pickle Brines. ‘ ‘L 3% presented by ; 35‘" Marvin H. Kragt . {5; 4- ' :'-‘{’$¥ I & a I .1 ;° has been accepted towards fulfillment of the requirements for C-l'b Ldegree in Bactzo 8: .H. :-. . 'LLA ‘ few . '3 Major professor pm May. 14, 1951 I 5" ' ' ' n ,' ‘H .‘ . . ,, . ' . h” ‘7‘ ‘. '. I I ’f ”a"; '_'.‘..“..T32 .' f‘" ’1 n‘..:" A; ‘..' fl, ‘ . ' =‘ f 1' a .7» ;L”€'3‘L)%‘$1" 3°” ’*"‘§“..‘53*1-’-71,-'7 ""‘ - ' , - ‘ ' I .4 '-(‘~.1";1..‘. (11“ ‘f h' as ’ ’k /' ‘;'( ‘ £Il,1l>;§ .;:.“ ,n ‘ ‘,I:‘Il:-":l!'..?’l.l'l ‘v': N .4". .. «114.1 ,C‘ ‘. ~ . s ' - - 7. ‘l I ."1 H "- lac. ‘ ' d' I -I. ii. .! ’ ‘- ..r. ‘1er . w vx'm ". ' ts'. C " t A- a EACIDRS CADSING’CLOUDINESS IN PICKLE BRINES By larvin Nelson Kragt A.IEESIS Submitted to the School of Graduate Studies of lichigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of EASIER OF SCIENCE Department of Bacteriology and Public Health 1951 THESIS ACKNOWLEDCMEN 1‘ no author wishes to express his sincere appreciation to Dr. F. W. Fabian, Professor of Bacteriology and Public Health, for his counsel and advise. as well as for his interest and sug- gestions in the preparation of this thesis. tfitiiitt$$ ensues *‘tttfi it“* fit a r, ,' w " r‘wt J‘ ‘ria :9‘; TIBLE OF'CONTENTS Page INWDUCTIONOOOO00......OOOOOOOOOOCOOOOOO0.000000000000000000000 1 LITERATURE REVIEW............................................... 2 EXPERIMENTAL PROCEDURE.......................................... 4 I. Bacteriological Studies................................. 4 II. Physical Studies........................................ 6 III. Chemical Studies........................................ 7 FACTORS PRODUCING CLOUDINESS IN PICKLE BM.................... 10 I. Bacteriological Tests................................... 10 11. Physical E1periments.................................... 12 III. Chemical E:periments.................................... 13 The effect of gum.emulsions......................... 12 The influence of whole spices and powdered spices... 13 The effect of water................................. 16 The effect of salt.................................. 18 The effect of acid.................................. 19 The effect of alum.................................. 20 The effect of pickles............................... 22 The effect of sugar................................. 23 SUMMARY......................................................... 25 CONCLUSIONS..................................................... 2? MELBOOOOOO0.00......OOOOOOOOOOOOOOOOO0.00000IOOOOIOOOOOIOOOOOO 28 LIWTU-RE CImOOOOOOOOO...0......OOOOOOOOOOOOOOOOOOOOOOO0.0... 42 IHEODUC TION The cloudiness in pickle brines is an important problem in the pickle industry. It is not only a problem of the pro- cessor but it has also become a problem to the consumer. Today with the shortage of tin, more and more pickles are being packed in glass. which allows the consumer to note any cloudiness in the brine. The general public has been educated to the fact that cloudiness in any food product usually indicates spoilage. Pickles that contain sediment or that are turbid usually influences a customer to buy the pickles that have the clear brine. An examina- tion of pickle products on the grocer shelves show that practically all fresh pasteurized or processed dill pickles show cloudiness or sediment ranging from slight to a heavy sediment. While other kinds of pickles have been observed in which this condition was noticable. it is not as prevalent as in the dill pickles. Since a clear brine is essential for consumer acceptance of pickle products, a study was made of the bacterial, chemical and physical factors causing turbidity in pickles. This study was confined mostly to fresh pasteurized and processed dill pickles. because the most sediment is found in them. LIIERLTURE KEVIN Etchells and Gorcsline (1940) observed turbidity in opened jars of fresh cucumber dill pickles. Bacterial counts proved negative gnd results indicated that the turbidity was due to soaking the slices in brine of 30° salometer for different lengths of time. Slices that had been previously soaked the shortest time had the greatest turbidity in the Jars, indicating that there hgd'becn less material removed from.the pickles before packing them.into the jar. ‘When this precipitate was tested with Millions Reagent for protein, results were positive indicating a protein precipitate. Krants (1928) studying vegetable gums and mineral oil emulsions noted that the range of greatest stability of either vegetable or numeral oil emulsions prepared with acacia was obtained when the pH of the outer phase was between pH 2 and pH 10. With gum tragacanth the range was from pH 1.9 and 2.3. The stable range of acacia emulsions was between pH 4.4 and 4.28, and‘with tragacanth the stable point was approximately pH 2.5. Bennett (1947) showed that the addition of electrolytes may act upon an emulsifying agent. If this happens, the electrolyte induces coagulation (salting out) or a change of the chemical nature of the emulsifying agent, thereby forming a precipitate. However, it is also possible that small quantities of electrolytes may stabil- ise emulsions. The presence of salts, especially calcium, magne- sium, aluminum, copper, iron, sine, bismuth, mercury, are likely to destroy emulsions. Cobb (1946) listed the factors which break down emulsions as: 1. Addition of electrolytes such as inorganic acids and salts. Adding electrolytes, after the emulsion was completed, was in many cases, less apt to result in breaking the emulsion. It must be remembered that there is no such a thing aerksrecipitationfa little bit of a colloid system - it is all or nothing. 2. Hardness of water - the softer the water the better, with distilled water representing the optimum. 3. Oxidation of even fairly inert material in finely divided form is rapid and destructive. Agitation should be carried out in a way to prevent inclusion of large quantities of air in the emlsion. Weyl (1877) stated that the chief protein found in mustard seed appears to be a globulin protein. Hones (1949) found vegetable guns to be composed of carbon, hydrogen and oxygen with small quantities of mineral matter and sometimes a little nitrogen. On hydrolysis with dilute mineral acids the guns form various sugars such as pentoscs, arabinosc, sylose, tragacanthose, and the heroes, galactose. Not all the gum is converted into sugars but usually about 20 per cent re- sists treatnent. Icodnan (1941) gives the general composition of mustard seed as containing considerable quantities of non-volatile oil, and protein material. This non-volatile oil is entirely lacking in ~4- pungency, being a tasteless oil somewhat like olive oil. IThe active constituent of mustard is the volatile oil which is not present in the mustard as such but is developed by an enzyme in an aqueous solution. The actual substance present is sinng or potassium myronate (K010 316 E32 09), a glucoside which by hydrolysis, through the agency of the enzymes also present, splits into glucose, potassium acid sulphate, and allyl iso- thiocyanate (63 H5 CNS). The latter is the pungent volatile mustard oil obtained from black mustard seeds. mmm PROCEDURE 1. Bacteriological studies Quart Jars of pasteurized dills were received which had a cloudy brine. these were Just a few of the many other jars of processed and pasteurized dill pickles, that had been cloudy when received at the laboratory for analysis. It was decided that the experimental work should first include a microbiological examination of all Jars. The various cloudy brines from the Jars were plated on three media to determine if the sediment was of bacterial origin. Tryptone glucose yeast extract agar was used to determine pepton- izers, acid bacteria, and inert bacteria. The composition of the asap (T.G.Y.E. )A used was as follows: Tryptone............ 10 grams Yeast extract....... 6 grams Beef extract. . . . . . . . 3 grams K2 HP04............. 1 gram Glucose............. 1 gram Agar................ 16 grams Distilled water. . . . .1000 ml . Brcm-cresol-purple. . 2 ml. of 1.6% solution One ml. of sterile skim milk and one ml. of sterile pickle brine were added as the plates were poured. It was noted that the sterile brine seemed to increase colony size of organisms. Potato dextrose agar (P.D.A.) was used to determine the pre- sence of yeasts. Its composition was as follows: Infusion from potato . . . 200 grams Dextrose............... 20 grams Agar................... 15 grams Distilled water. . . . . . . .1000 ml. The medium was acidified to a pH of 3. 5 with tartaric acid. lbmato juice agar (T.J.A.) was used to determine the lactic acid bacteria. Its composition was as follows: Tomato juice (400 ml). . 20 grams Peptene..... ........... 10 grams Peptonised milk. . . . . . . . 10 grams Agar................... 16 grams Difltilled water. o e o e e o .1000 lilo The T.G.Y.E. and T.J.A. plates were incubated for 48 hours at 35°C., and the P.D.A. plates were incubated at room tempera- ture for five days. A yeast extract was used as basal medium for the sugar re- actions of the bacteria. Its composition was as follows: Yeast extract...... 2.5 grams Peptone............ 5 grams ' Distilled water.... 1000 ml. Andrades indicator. 1 m1. In preparing the carbohydrate for the fermentation studies, the tubes with inserts were sterilised, the basic medium auto- claved, and then the carbon compounds were added in one percent concentration. Seven ml. was then pipetted into each tube and this was again autoclaved at 10 pounds pressure for 10 minutes. Bergey's Manual of Determinative Bacteriology, sixth edition was used to identin the organisms. (9). The percent salt was determined by titrating with standard silver nitrate solution using dichlcrcfluorescein indicator. Acidity, calculated as grains acetic acid was determined by using a 0.1666 N. NaOH solution and phenclphthalein indicator. the pH was determined by a Beckman pH meter. II. Physical studies Processed dill and fresh pasteurized dill brines were used in these experiments and standardised as shall be seen presently. The dispersing agents used for the essential oils consisted of a variety of vegetable gums, tweens and salt in the form of soluble oil of spice, 8.0.8. These agents containing the spices were that added to the two pickle brines in the ratio of 133000. The differ- ent combinations of brines and spices were placed in jars, sealed and pasteurized at 165° F. for 15 minutes. After one week the jars containing spices and brines were thoroughly mixed by shaking 25 times and examined for cloudiness by means of the photelcmeter. The cloudiness was measured by means of a Canoe-Sheard Smford photolometer Model No. 12335. The photolcmeter was standardized to 100 with distilled water, and the brine readings 'were determined on comparison with.distilled water. Tubular cells and adapters were used with the photelometer and a lantern red filter no. 2408. III. Chemical studies In order to standardize all the brines, the ingredients were added in a constant amount. 1. Vinegar - to give a dilution of 6.6 grain acid for fresh pasteurized dill brine, and eight grain acid for processed dill brine. 2. Salt - to give a 2.5 per cent dilution for fresh pasteurized dill brine and four per cent dilution for processed dill brine. 3. 4. 5. 8. -8- Alum - 0.2 per cent A12 (804); 18320, so that the final result would be 0.1 per cent A12 (3003. Spices - whole and powdered spices were added to give one ml. to each pint Jar. Spice oils - were added to give a l: 3000 dilution of spice oils in the brines. Vegetable guns in oil emulsions b. c. Mixed gun ‘ 0.1 part vegetable‘gun ‘ 1 part oil (dill, pepper, cloves) 2 parts water Resulting ratio of gun in brine - 1: 12,000 Gull arabic 1 part vegetable gum 4 parts oil 8 parts water Resulting ratio of gm in brine ' 1: 12.000 Gun tragacanth 3.7 parts gum 12.5 parts oil 59.2 parts water Resulting ratio of gun in brine - 1: 10,000 1' his is a mixed gun sold by llagnus, Haybee and Reynard, Inc. for making spice oil emulsions. d. Gun karaya 1 part gum 2 parts oil 40 parts water Resulting ratio of gun in brine II It 6000 e. Gun loccust bean 1 part gun 2 parts oil 40 parts water . ( Resulting ratio of gum in brine 3 1: 6000 7. Ratio of tweens andsalt base emlsifiers. a. Item 20‘ emulsifier 4 parts tween 20 1 part oil 4 parts water Resulting ratio of tseen in brine ' 1: 750 b. keen 80 emulsifier 3 parts tween 80 1 part oil 4 parts water Resulting ratio of tween in brine - 1: 1000 c. Salt base emlsifier 0.0.3. ' Resulting ratio of salt emulsifier in brine I ls 104 1- 0.0.8. spices made by Wm. J. Stange Co. For the chemical experimental work on cloudiness in brine, it was decided to make up brines and add various ingredients to determine if they caused cloudiness. Other chemical factors studied as possible causes of cloudi- ness were as follows: 1. Pickles in brine. Ihe pickles were in a brine of 16 per cent or‘ 60° salemeter. mesa pickles were desalted and placed in pint jars, after which a dill brine was added to cover them. he finished brine tested four per cent salt and eight grains acid. 2. Sugar in brine. A brine was made up to represent a standard sweet brine. Its composition was: two per cent salt, 22 grain acid and 35 Brix. Phote- 1ometer readings were observed to determine if sugar caused cloudiness. 3. Examination of comercial Jars. Photelometer read- ings were taken of sweet, processed and fresh pasteurized pickle brines. FACIORS TESTED I. Bacteriological Tests Eight quart jars of pasteurized dills were obtained which showed a very cloudy brine. Ihere were obtained frcn Michigan, -11- Georgia and Texas. Several additional Jars were added which con- tained a very slight precipitate to determine also if this pre- cipitate‘was of bacterial origin. 'lhen the brine'was examined bacteriologically these eight jars of pasteurized pickles appeared to contain largely gram positive rods ranging from.single to short and long chains. The brines were plated on the three media previously de- scribed, and microscopic examination of colonies isolated on the various media indicated relatively pure cultures of one organism. .A total of 12 colonies of the bacteria'were isolated and purified by repeated plating. These bacteria were further indentified'bfrgram.stains, cultural, morphorological, and bio- chemical characteristics. These tests placed them.in the family Lactobacteriaeeae. The two pickle jars containing a very slight precipitate showed a total count of 100 colonies per ml. Plates from these two Jars showed that these colonies were gram.positive, spore- genic, rod shaped bacteria. Since in these cases the total count did not indicate any great amount of sediment due to organisms, these organisms‘were not further indentified. Biochemical studies and other characteristics indicated that the 12 organisms could be divided into two types. Salt, acid and pH were determined on the various brines. The results of these experiments are found in tables 1, 2 and 3., h -12- Discussion of Results The he quart jars which showed only slight sedimmt gave only 100 organisms per ml. The other jars which were very cloudy showed large numbers of bacteria present which caused the cloudy brine. Chemical “12:13:“? otjfinvi’frious jars are-shown in table 1 showed that the eight jars which had cloudy brine had also in- creased acid, calculated as acetic acid which was undoubtly due to the lactic acid-producing organisms present in the brines. Biochemical studies and the morphological and cultural characteristics of the 12 organisms isolated indicated that they belonged to the genus, Lactobacillug and to two species plantarum and fermenti. ‘ Etchells and Jones (10) and Fabian and Orloff (11) have shown that proper pasteurization carried out at 166° F for 15 minutes has been adequate to kill bacteria, yeasts and molds which would ferment the pickles. The survival of lactobacilli in these jars showed that these dills were not properly pasteur- ized at the recommended time and temperature. This resulted in a fermentation of the pickles by organisms of the genus Lactobacillus. 11. Physical Experiment The effect of heat on gum emulsions and other oil emulsifiers. This experiment was set up to determine if heat caused cloudiness in brines. The brines tested had the same composition as those listed under 111 Chemical studies. The results are given in t‘blO 4e -13- Discussion of Results In this experiment a sediment oocured in all the gum emulsions in both the processed and the pasteurized brines. Although the vegetable gums acted as a stabilizer of the emul- sion, the electrolytic effect of the solution may have caused precipitation of the gums. Results in table 4 showed thggtggi brines heated at 165° F for 15 mdnutes resulted in increased cloudiness in the brine. This was doubtless due to the com- bined effect of heat on the vegetable emulsifier and the A electrolyte effect. Tests on the precipitate to determine if it ¥¥¥E carbo- hydrate in nature, by using the fillish test indicated that the precipitate was a carbohydrate. The results of salt base emulsifier (0.0.8.) and the tween emulsifier indicated that both served very well to disperse the oil into the brine, and what was more important neither one pro- duced a precipitate in the brine. III. Chemical Experiments Effect of Whole and Powdered Spices It has been known that the addition of whole and powdered spices causes increased sediment and sometimes cloudiness. There- fore, experiments wore set up to determine the factors involved. its—the Laboratory observations of the large Montana mustard seed, showed a gelatinous membrane being formed around the seeds -14- when they”were placed in both fresh pasteurized and processed dill brines. This membrane could be broken by continued jar- ing and shaking and resulted in a gelatinous precipitate in these brines. When the Montana mustard seeds were placed in ‘water and observed after a few days, the membrane extended its size to form a membrane of almost double the size observed in brines. Mustard seed placed in tap water and distilled water gave a gelatinous membrane. There was more formed in tap water than in distilled water. A.great variety of ground and whole spices were used, some of which were known to produce cloudiness in brines, such as powdered onion and garlic. These data are found in tables 5 and 6. Discussion of Results Results in table 5 indicate that powdered spices added to dill pickle brines, resulted in marked cloudiness as compared ‘with the flakes, buds and oils of the same spices. Dehydrated garlic flakes gave excellent results,'with a very slight re- duction in photelometer retdings. ' . In table 6 $833233?Exihis’omflia uuTbsidity W885 Microscopic examination of this sediment indicated that the sedi- ment was amorphorous. Mo oil globules were present in the sediment. Millionfia‘aewgeat for the protein but gave positive results. Ex- amination of the gelatinous precipitate obtained from soaking mustard seeds in tap and distilled water, showed the precipitate -15- to be amorphorous and it gave a positive Million's test'which indicated a protein. Assuming that this protein was globulin in nature, investi- gation of its globulin characteristics and comparison with the protein precipitated in brine solution, showed the two to be similiar. Globulin protein is soluble in dilute salt solutions and insoluble in pure water. Mustard seeds placed in tap water showed a great amount of cloudiness, while those placed in dis- tilled water did not show as much turbidity. There‘was not as much cloudiness in brine solution as there was in water. In this case the globudin protein precipitated in a dilute salt‘which may be considered a peptization process, and upon further increase of salt concentration, throughout a certain range, altered the surface forces in such a manner as to favor dispersion. Further increasing the salt concentration caused dehydration or the salting out of the protein micelles. There- fore, in the brine, it is possible that the salt concentration is in a peptization process. Furthermore tap water used had a pH 3 7, while the fresh pasteurized dill brine had a pH ' 4. This would result in the possibility that pH = 4 was not near the iso-olectric point of the protein fraction and, therefore, result- ed in less precipitation. However, results showed that the whole spices do cause cloudiness in brine, and that the brine serves chemdcally to in- crease this cloudiness depending cn the salt concentration and the pH. -15- Effect of Water Hardness i‘. Believing that 'thggggiofs w waters encounter- ed in many localities might result in cloudiness, tests were run using college water and distilled water. A typical analy- sis of Michigan State College water which is conditioned for laundry and power plant boilers is as follows. Total hardness as Ca 003 526 ppm Suspended matter 10 ppm Total dissolved solids 390 ppm Tbtal solids 400 ppm Tbtal alkalinity as Ca 003 310 ppm Carbon Dioxide V 25 ppm Oxygen 1° PPIn Iron. as Fe 1 1 ppm Silica 10 ppm Calcium 90 ppm Magnesium 24 ppm Chlorides 15 ppm Sulphates 15 ppm Bicarbonates 380 ppm pH 7.2 In addition the water containd Crenothrix, Leptothrix and other iron bacteria. In these experiments tests were run using tap water (M.S.C. water of the above composition) and distilled water. Photelometer -17- readings of both tap water and distilled water were 100, there- fore tests were made to determine differences in readings using acid alone, salt alone, and a regular processed brine. Various mustard seeds and gum emulsions were also used to determine ef- fect of the waters on them in the presence of acid salt and pro- cessed brine. Results are shown in table 7. Discussion of Results In table 7 the results indicated that water hardness contributed to the cloudiness in dill pickle brines. There was not as great varation in readings by the photelometer when using tap or distilled water. However, when acid or salt #3. added to tap or distilled water cloudiness oecured. Since the cloudiness was greater with tap water, this indicated that the high mineral content of the tap water was either salted out or the acid re- acted with the various anicns or cations present in the water which resulted in a cloudy brine. Salt when added to different kinds of mustard seeds in tap or "distilled water, at pH I 7, resulted in a salting out of the protein portion of the mustard seeds. Aeid when added to different kinds of mustard seeds also re- sulted in increased turbidity due possibly to the effect of the acid on various minerals present, or W that the protein of the various mustard seeds was at a pH near the iso- electric point. When acid, salt and whole spices were used together, the photolometer readings indicated that the various constituents apparently buffered each other and cloudiness was not as low as when either salt or acid'was used separately. The decreased cloudiness may have resulted because the pH was not near the iso- electric point or that the solubility of the protein was in the peptization process. Results of this experiment, however, indicated that the acid and salt precipitated out various minerals in tap water, and this resulted in higher cloudiness in pickle brines. Effect of Salt Salt as a constituent of brines should be of high purity. In this work granulated salt was used which was 03m grade comonly used in the pickle industry. Although the effect of salt on whole spices was recorded in table 7 this work showed the effect of salt on various other brine constituents. Salt brine of four per cent salt concentration was placed in pint jars, seal- ed and examined after one week. These results are shown in table 8e Discussion of Results The results in table 8, indicated that a salt brine of four per cent, has a cloudiness due to the action of the salt on the anions and cations present in tap water. Results also showed that -19- the salt'will "salt out" the protein fractions as well as pre- cipitate out a portion of the vegetable gum. Photelometer read- ings obtained from Brassica 33.139. and Brassica gigs}; are very low. This can be accounted for in that these mustard seeds are crushed; therefore, the brine contains many small particles of these seeds. Effect of Acid Vinegar as used in brines is commonly bought in.tank cars in concentrated form. It is diluted to the desired concentration depending on the type of pickle product being processed. In this work the acid was diluted to eight grains acid with tap water. various spices were also added to determine the effect of acid on them. The photolometer readings were observed after one week and are recorded in table 9. Discussion of Results In table 9, the results indicated that vinegar caused a slight turbidity in tap'water. This is due to the effect of the acid on the minerals present in the water. The increased cloudi- nose with acid and whole spices, may be due in part to the action of the acid on the water, or the cations and anions present caus- ing a precipitation'with the mustard protein. The effect of acid on the gum emulsions indicated that a portion of the gum.pre- cipitates out. -20- Effect of Acid and Salt In this experiment it was decided to determine if gum emulsions added to tap and distilled water would cause a cloudi- ness. The gun emulsions were added one part emulsions to 3000 parts water. The effect of acid and salt were also studied by making up a brine solution of eight grain acid and four per cent salt. Various ingredients were added to this brine to determine the combined effect. The results are shown in table 10. Discussion of Results These results showed that the acid and salt were not wholly responsible for increased turbidity, but that many factors may cause cloudiness. Results showed that the gum emulsions, when added to tap water and distilled water, were precipitated out. Distilled water gave only slightly higher results which would indicate that a certain amount of gum is precipitated as the oil is dispersed throughout the brine. It may also indicate that the vegetable gum which acts as an emulsion stabilizer was present, in excess. However, results as shown here and in table 6 do indicate that the acid and salt do cause protein precipitation which re- sults in increased cloudiness. Effect of Alum Alum is used extensively in the pickle industry to crisp pickles, especially processed and genuine dills. Alum is added -21- to the brine directly or may be added in the desalting of the salt stock pickles by allowing the pickles to soak in an alum solution. The amount of alum A12(SO4)3 18 H20, used was sufficient to give a final concentration of 0.1 per cent by weight in the brine. Photelometer readings were also made with alum and a buffered solution. The same concentration of alum was used as with brine solutions. Readings showed a large precipitation at pH 5.5 and up into the alkaline range with the greatest cloudi- ness at pH 6. The results using brines composed of various spices and gum emulsions with alum added are shown in table 11. Discussion of Results The experimental work as recorded in table 11 showed that alum is definitely precipitated in dill pickle brines and re- sulted in increased cloudiness. Further evidence was accumulated in work done on genuine dills. The genuine dills can be packaged in the brine in which they have fermented or they can be packaged in a new brine. The fermented brine was very cloudy due to the fermentation. Genuine dill pickleswwg'oepackaged in four when ways as follows; 1. Filtered the brine in a filter cell and placed this brine on the dills. 2. Placed non-filtered brine on the dill pickles. 5. Filtered the brine and then added alum. 4. Alum added to the brine unfiltered. -22- The results showed that the alum was precipitated after the genuine dills had been pasteurized, as shown in the following: 1. Brine was very clear, with no precipitate or sediment. 2. Brine was very cloudy, with precipitate on bottom.and on pickles. 3. Brine was fairly clear, however a floculant precipitate was observed on the bottom.of the jar. 4. Brine was very cloudy with precipitate on pickles and on the bottom.of the jar. The precipitate did not give a positive Mollish or Million's test and, therefore, it may be assumed that the alum itself pre- cipitated from, the genuine dill brine, upon pasteurizing. Effect of Pickles on Cloudiness A.study was made of the effect of the pickles themselves caus- ing the cloudiness. The pickles used were salt stock pickles in a brine of 16 per cent salt or 60° salometer. The pickles were desalted and placed in pint jars, after which a dill brine was added to cover them. The finished brine tested four per cent salt and eight grain acid. various spices were added to similate actual conditions of packaging. The results are given in table 12. Discussion of Results The results in table 12 showed that in this experiment the pickles did not cause extensive cloudiness of the brine. Un- -23- doubtdy any pickle which was not washed properly will show sedi- ment. lflcroscopic examination of this sediment did not show any epithelial cells to be the cause of turbidity. In other experimental work on the cloudiness of pickles in brine, various dill and sweet pickles from commercial companys 'were examined for turbidity by the photolometer. These results are given in table 13. Discussion of Results Examination of commercial packs by means of the photolometer showed.that the turbidity‘was evident in all dill and sweet pickles. The readings as given in table 13 were all acceptable from.the ' sales standpoint, however turbidity is evident in all the packs. It would seem.that photelometer readings below 80 would become objectionable. Effect of Sugar lhny examinations of high, normal and low brix sweet pickles, from.the grocers shelves, have shown little or no cloudiness, with no sediment. In this experiment the brine'was made up to have two per cent salt, 22 grain acid and a Brix of 35. This brine'was added to pint jars, sealed and observed after one week. The re- sults are shown in table 14. -24- Discussion of Results The results showed that sweet pickles also may have a cloudy brine. It Asa. probable that the sugar holds the various particles of sediment in suspension, as pectin does in jellies. Most sweet pickles are packed in small jars, and due to the smaller volume of brine, and to the better transmission of light, they do not appear cloudy. However, care should be used in the ingredients used such as emulsion gums, powdered spices, and the whole spices. -25- Summary Processed and fresh pasteurized dill pickle brines, simulating M commercial brines, were examined bacteriologically, physically and chemically to determine the various factors causing cloudiness in them. Samples of cloudy commercial pasteurized dills were examined bacteriologically and found to have undergone a lactic acid fermentation. Isolation and study of the predominant bacteria, showed them to be, Lactobaoillus and that they belonged to the species plantarum and fermenti. It is well known that proper pasteurization at 165° for 15 minutes is sufficient to kill all yeast and bacteria, except spore forming bacteria, which gradually die out. Vegetable gum emulsions were shown to cause turbidity in dill brines, due to the action of temperature, electrolytes and instability of gum emulsions in hard waters. Salt bases (0.0.8.) and Tween 20 and 80 which act as essential oil carriers were shown to cause little or no cloudiness in dill brines. The addition of whole spices to dill brines caused cloudiness. Montana mustard seed gave considerably more gelatenous precipitate than did the other two, Orientia and Superior seed, tested. -26- Tests made with tap water and hard water indicated that the various minerals present, as cations and anions in hard water, increased the cloudiness in the presence of acid and salt. Alum.caused cloudiness in dill pickle brines. This action may be due to increasing the electrolyte acitivity of the brine. Tests on sugar brines indicated that various sugar brines may also be cloudy as judged by the photolometer, but often the particles remain suspended and therefore are not seen in the small bottles. There are many factors which cause cloudiness. Chemically it may involve pH, electrolytes, iso-electric points, anions and cations, which acting together or separately may cause cloudiness. Physically heat causes cloudiness in certain brines by coagulation and precipitation of certain substances such as pro- teins. Bacteriologically cloudiness is due to the growth of certain acid tolerant bacteria. -2 7. Conclusion The factors responsible for cloudiness in pasteurized and processed dill brines may be listed as: 1. 2. 3. 4. 5. 6. 7. 8. Microorganisms in improperly pasteurized dills. Vegetable gum emulsions Whole spices. Powdered spices Alum Hard'water Vinegar - under certain conditions Salt - under certain conditions -28- Table l. - The chemical analysis of pasteurized dills containing a cloudy brine due to bacteria. Sample NO. la lb 2a 2b 3a 3b 4a 8‘ Ehotelometer reading 0 0 5 10 85 80 Bacteria Per mls 500,000 800,000 1,000,000 900,000 13'0993990 11'9999099 750,000 300,000 100 150 Per cent NC Clo 2.3 2.3 2.1 2.2 2.3 2.2 2.4 2.4 2.1 2.1 Grains acetic acid pH 12 13 13 13 12 13 11 12 3.45 3.4 3.5 3.5 3.6 3.9 3.9 -29- Table 2. - Cultural characteristics of 12 cultures of microaerophilic bacteria isolated from cloudy brine of pasteurized dills. Growth in nutrient broth Growth on nutrient agar mace Gm'th in TeGeYeEeA stabs Microscopic characteristics of the 12 colonies Type I +, no pedicle scant filiform, beaded Rods: size . 0.6 - 1 x 2.5-9 Microns short chains to rods no spores Type II 4, no pedicle scant filiform, beaded Rods: size 0.5- l x 3 - 18 Microns chains to rods no spores -30- Table 3. - Biochemical characteristics of 12 cultures of microaerophilic bacteria isolated from.cloudy brine of pasteurized dills. Fermentation of: Type I Type II . Type I Type II Arabinose . 3 - Maltose + + Zylose - + - hectose a - Rhamnose - - Raffinose + + Glucose 4 4 Starch - - Fructose + + Inulin - - Hannose + 4 Hannitol 4 - Galactose + + Sorbitol + - Sucrose - + Salicin + - Dulcitol 4 - Citrate utilization - - Starch hydrolysis - - Nitrate reduction - - Litmus:milk reaction acid - mm 59 pm #m 8 8 8 8 8 wnwcsou hepoaoaeuonm dado connoooam 8 8 8 8 3 0a 8 8 no woweoou aovcfioaevomm enaun vouwasepnam mocha .3532 3 .5 a 583 83.8 8 8 8 8. a 8 we a 8 mnacsoh Mafia vomuOOOhm mm mm mm mm 8 8 8 8 so wnfioseh acpoaoaovosm acvoaoacpomm eoaan connascvesm mocha ccvsco poo Hcavaoo, \s.‘ cm socks on mocha Aecaaaupoaouv codes Mo adoao seams no adeuo ocean unflaasc awo spasoswsuv_fism omen pasoooa sew emsaexissw ownsac_aow .lsw toads use: doe-Haas sou .osfian a“ mmoafieooae madness scoop mohauso Hfio heave cos umofiuaoao sow no peas mo pecan. Qua I .e sapna Table 5. - The effect of powdered spices on cloudiness in processed dill brine when used with various emulsifying gums. Photelometer readings of different gum.amulsicns Spice added added to pgocessed dill brine. locoust salt base Soluble 525313, bean tragacanth =£2E32_ 0.0.8. salt base brine only’ 88 90 91 91 94 98 synthetic garlic powder 76 . 83 84 81 95 92 synthetic onion . powder 84 88 88 89 92 . 95 oleoresin celery 58 64 59 68 70 72 ground mustard 32 49 33 52 ' 53 56 powdered onion 44 45 50 62 64 67 powdered onion . 45 58 42 60 57 68 powdered garlic 49 46 52 51 56 62 powdered garlic 4O 39 38 42 49 5? garlic flakes 87 89 91 91 93 96 garlic buds 85 88 90 89 91 94 garlic 011 88 89 91 90 94 98 Table 6. - The effect of whole spices on cloudiness of fresh pasteurized dill brine when used'with various emulsifying agents. Photelometer readings of different gum.emulsions when Spice added added to fresh pasteurized dill brine Control brine locoust mixed tween only karaya bean tragacanth gum 20 Brine only 96 92 91 90 85 99 Montana mustard 94 as 88 ‘ 35 7e 93 Oriental mustard 94 89 89 87 82 95 Superior mustard 94 90 89 88 82 94 mound Brassica Q: 73 61 58 64 65 71 smuml Brassica 3352 76 72 69 71 70 75 n1 spice 93 as 88 87 so 94 -O-.\0 -V..- -34- Table 7. - The effect of water hardness on cloudiness of processed dill brine when used with various ingredients. ' Photelometer reading Composition of brine distilled water tap water 8 grain acid, alone 100 98 «9 garlic flakes 99 96 4 Montana mustard seed 93 84 + Oriental mustard ....1 96 91 4 Superior mustard seed 95 90 e lixed gun emulsion .93 91 4 per cent salt, alone 99- 98 + Montana mustard seed 95 83 4 Oriental mustard seed 96 80 «7» Superior mustard seed 94 80 4» Mixed gm emlsion 91 88 main acid, 4 per out salt, alone 99 96 + lbntana mustard seed 95 94 “Oriental mustard seed 97 95 + Superior mustard seed 96 95 + lixed gm emulsion , 92 87 -35- Table 8. - 'lbe effect of salt in combination with various spices and a mixed gum in causing cloudiness in four per cent brine. Composition of brine Photelometer readini Salt 4 per cent, alone 98 Salt 4 per cent, alone 98 «v lbntana mustard seed 83 + Oriental mustard seed 80 + Superior mustard seed 80 e Montana mustard seed, mixed gum 71 6 Oriental mustard seed 7O 4» Superior mustard seed 72 + Brassica alba mustard seed 5? + Brassica n_i§_e_r_ mustard seed - 58 + Powdered garlic, mixed gum 51 + Powdered garlic, mixed gum 38 a Mixed gum emlsion 88 4 Mixed gun emulsion 87 v—~-. 'f -36- Table 9. - Elbe effect of vinegar in combination with whole spices and gum emulsion in causing cloudiness in eight grain acid brine. Photelometer Composition of brine ' reading Vinegar 8 grain, alone 98 Vinegar 8 grain, alone 99 hmntena mustard seed 84 +«Oriental mustard seed ' 91 + garlic flakes 96 i- gum emulsion 93 + gm emulsion 93 + lbntana mustard seed, gum emulsion 82 «6 Oriental mustard seed, gum emulsion 83 4» Superior mustard seed, gum emulsion 84 «r E. :_l_b_a_ mustard seed, gum emulsion 70 I _B_. 11535 mustard seed, gum emulsion 63 + powdered garlic, gum emulsion 54 + powdered garlic, gum emulsion 35 b. -3 7- Ihble 10. - the effect of acid and salt in combination with other brine constituents in causing cloudiness Composition of brine Photelometer reading. 4 per cent salt, 8 grain acid 98 9 gum arabic ‘ 91 + gum arabic 91 e Iontana mustard seed 94 + Oriental mustard seed 95 1' Superior mustard seed 95 tap water, gum arabic 90 ' distilled water, gum arabic 92 -3 8- Table 11. - Ihe effect of alum in combination with spices and gum in causing cloudy pickle brine. Photelometer readings with Composition of brine no alum alum 8 grain acid, 4 per cent salt 96 I 90 4» Montana mustard seed, gum emulsion 84 79 4- Oriental mustard seed, gum emulsion 86 80 e Superior mustard seed, gum emulsion 82 79 + Brassica 92:, gum emulsion 73 68 4- Brassica 33.5.9.9 gum emulsion ' 71 66 4- powdered garlic, gum emulsion 65 56 + powdered garlic, gum emulsion 62 56 4 gum emulsion . 89 81 + synthetic powdered garlic, gum arabic 95 83 + oleoresin celery, gum arabic 75 63 + synthetic powdered onion, gum arabic 93 85 e powdered onion, gum arabic 65 59' + powdered onion, gum arabic 66 46 + powdered garlic, gm arabic 57 53 4 powdered garlic, gum arabic 71 62 4 garlic flakes, gum arabic 93 83 4 gum arabic 97 86 c spthetic powdered garlic, salt base 93 83 + oleoresin celery, salt base 80 67 -.- .4.- -39- Table 12. - The effect of pickles and various ingredients as causing cloudiness in pickle brines. Composition of brine 4 per cent salt, 8 grain acid 4 pickles + pickles, gum emulsion + pickles, Montana mustard seed 4 pickles, Oriental mustard seed + pickles, alum, + pickles, alum, gum emulsion Photelometer reading, 95 ‘ 93 86 88 89 75 72 -40- Table 13. - Ibsts for cloudiness of samples of commercial dill and sweet pickles. 322‘ pack Photelometer reading_ Processed dill pickles 82 Processed dill pickles 85 Processed dill pickles 80 Processed dill pickles 85 Processed dill pickles 89 Processed dill pickles 78 Standard sweet pickles 85 Standard sanet pickles 87 Double sweet pickles 87 Double sweet pickles 89 Pasteurized dill pickles 86 Pasteurized dill pickles '85 Table 14. - The effect of sugar in combination with other ingredients as causing cloudy brines. Cogposition of brine Photelometer reading acid, salt, sugar 94 + gum emulsion ‘ 9O 4- gum, Montana mustard seed 88 + gum, Oriental mustard seed 88 4} gum, Superior mustard seed 87 + gum, _8_. £9. mustard seed ‘ 72 + gum, Pi.’ 9353: mustard seed 69 + gum, powdered onion 61 + gum, powdered garlic 55 .4 -42- LITERATURE CITED (1) Etchells, J. L. and H. E. Goresline, 1940. Methods of Exam- ining Fresh Cucumber Pickles. Fruit Producer Jour. .13, (111 353-335e (2) Krants, J. C. Jr. 1928, mulsions and the Effect of Hydrogen- Ion Concentration Upon heir Stability, 173-206. Ph. D. Dissertation, {hiversity of hryland. (3) Bennett, 3., 1947. Practical Enulsicns, 51-52. Chemical Publishing Co., Brooklyn, New York. (4) Cobb, R. n. x., 1946, Emulsion Technology, 26-27, Ind. Ed. Chemical Publishing Co., Brooklyn, New York. (5) Weyl,1‘., 1887, Zeitschrift fur Physikalische Chemie, _I_, 72. (6) Howes, F. N. 1949, Vegetable Guns and Resins, 8-9. Chromia Botonica Company, Waltham, Mass. (7) Woodman, A. G. 1941. Food Analysis. 4th. Ed., 416-417. McGraw - Hill Book Company, New York, New York. (8) Icrkman, C. 3., 1942. Manual of Pure Culture Study of Bacteria. 9th. Ed., 12, No. 4, VI 42-6. (9) Bergeys Manual of Determinative Bacteriology, 1948. 6th Ed. 11:. Williams and Wilkins Company, Baltimore, lid. (10) Etchells, J. L. and I. D. Jones, 1942. Pasteurisation of Pickle Products. Fruit Producer Journal, _2_1, (11), 330-332. (11) Fabian, F. W. and M. D. Orloff, 1950. Germicidal Detergents Are Not Needed For Washing Fresh Picked Cucumbers. Food Industries, _2_§_ 256-259. flail-«wit... a. ‘ .!n . ..u lhflllqls fr . uyfl. . ‘hij'u, EEEEEEE Mum mm 7138139917 u HHHHH WNW 12 1