THE EFFECTS OF PHYSICAL AND CHEMICAL PRE-BRINING TREATMENTS ON THE QUALITY OF SALT-STOCK PICKLES By Paul James Ward A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1982 ABSTRACT THE EFFECTS OF PHYSICAL AND CHEMICAL PRE-BRINING TREATMENTS ON THE QUALITY OF SALT-STOCK PICKLES By Paul James Ward The effect of treating cucumber wash water with chlorine dioxide was evaluated to determine if the storage life of the fresh fruit could be lengthened. Under certain conditions, chlorine dioxide treated cucumbers remained free of visible mold one day longer than controls. Chlorine dioxide was found to be effective in reducing microbial populations in cucumber wash water and was found to be more efficient than chlorine or Anthium Dioxcide . No appreciable effect upon the initiation of natural fermentation was noted. The effects of piercing fresh cucumbers, vacuum impreg- nation of brine and the use of sorbate in cucumber brines were also studied. Vacuum treatment resulted in increased initial brine penetration, rapid cure color development and increased bloater formation in the absence of sorbate. Piercing treatments increased initial brine penetration but were insufficient to significantly effect the finished salt- stbck quality. Sorbate reduced the incidence of salt- stock defects in all treatments. To My Wife Paula ii ACKNOWLEDGEMENTS I would like to express my deepest appreciation for my wife, Paula. Her constant love and encouragement throughout the writing of this thesis and the course of my graduate studies at Michigan State University were indispensible. I would also like to thank my family and friends for their encouragement and the "listening ear" I often requested of them. Finally, I would like to thank my major professor, Dr. Mark Uebersax, for serving as my advisor and for his valuable input into this project. Also, many thanks to my committee members, Dr. Ralph Costilow, Dr. Pericles Markakis and Sterling Thompson for their participation in my M.S. program. TABLE OF CONTENTS LIST OF TABLES. . . . . . . . . . . . . . . . , . LIST OF FIGURES . . . . . . . . . . . . . . . . . INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . The Natural Fermentation Process. . Bloater Formation and Control . . Enzymatic Softening of Salt- Stock Pickles . Influence of Postharvest Handling and Storage on Salt- Stock Quality. . . . . . . . . . . Chlorine Dioxide. MATERIALS AND METHODS . Experimental Design of Research Conducted Source of Fresh Cucumbers . . . . . . . . Chlorine Dioxide Studies. . . . . . . . . Effect of c102, C12, Anthium Dioxcide09 and Blanching on the Microbial Popu- lations of Blended Cucumbers. . . . Effect f C102, Clz and Anthium Diox- cide on the M1crobial Populations in Cucumber Wash Water. . . Effect of C102 on Natural Fermentations of Salt Stock Pickles . . . . . Cucumber Piercing and Vacuum Studies. Preliminary Salt Penetration Study. Five Gallon Pail, Vacuum X Pierce X Sorbate, Fermentation Study . Analytical Procedures Preparation and Measurement of Water Saniti- zers. . Polygalacturonase Activity Assay. . Cucumber and Salt Stock Pickle Texture Evalua- tion. . . . . Microbiological Media and Methods Brining and Fermentation Procedures . . Visual Evaluation of Salt Stock Pickles iv 23 24 25 27 27 30 30 32 34 35 36 Chloride Ion Concentration of Salt Stock Pickles. . . . . . . . . . . . . Statistical Analysis . . . . . . . . . . RESULTS AND DISCUSSION . . . . . . . . . . Chlorine Dioxide Studies . . . . . . . . . . . . . Effect of c102, c12, Anthium Dioxcide® and Blanching on Microbial Populations of Fresh Cucumbers. Effect of moi, Cl .and.Anthium.Dioxcide®.on . Microbial Popula ions in Cucumber Hash Hater. . . . . Effect of c10° on Natural Fermentations of Salt- Stock ickles . . . . . . . . Cucumber Piercing and Vacuum Studies . . . . . . Preliminary Salt Penetration Study . . . Five Gallon Pail, Vacuum X Pierce X Sorbate, Fermentation Study . . . . . . . . . . . CONCLUSIONS. . . . . . . . . Chlorine Dioxide Studies . . Cucumber Piercing and Vacuum Studies APPENDICES . LITERATURE CITED . Page 36 37 38 38 38 44 47 56 60 67 67 69 71 75 Table TO LIST OF TABLES Mean values for microbial populations of green- stock cuc mbers treated with C102, C12, Anthium Dioxcide or blanched for 48 hours at 70°C for 5 min. and then held at 20°C and 95% R.H. . Analysis of variance of microbial populations of green-stock cucumbe 5 treated with C102, Clz, Anthium Dioxcide or blanched at 70°C for 5 min. and then held at 20°C and 95% R.H. for 48 hours. . . . . . . . . . . . . . . . . Mean values for polygalacturonase activity and texture of green-stock cucumfifrs treated with ClOz, Clz, Anthium Dioxcide or blanched at 70°C for 5 min. and then held at 20°C and 95% R.H. for 48 hours . . . . . . . . . . . . . . Analysis of variance of polygalacturonase activity and texture of green-stock cucumgfrs treated with C10 , Clz, Anthium Dioxcide or blanched at 7 0C for 5 min. and then held at 20°C and 95% R.H. for 48 hr. . . . Effect of Cl02, Clz and Anthium Dioxcide® on microbial populations in cucumber wash water I. Effect of C102, Clz and Anthium Dioxcide®on microbial populations in cucumber wash water 11. . . . . . . . . . Microbial populations in cucumber wash water treated with C102 Firmness of salt-stock and PG activity of brine from cucumber fermentations following pre- brining treatment of green-stock with C102. Mean values for microbial populations of green- stock cucumbers treated with Cl02 and held at l0 and 20°C and 75% R.H. for up to 6 days Mean values for NaCl concentration of green- stock and PG activity of brine after vacuum and piercing treatments . . . . . . . . . . vi Page 40 41 42 43 45 48 50 54 55 57 Table Page ll Analysis of variance of polygalacturonase activity in brine and NaCl concentration of green-stock immediately and 24 hours following piercing and vacuum treatments of fresh cucum- bers brined in 1 gallon jars with a 450$ cover brine. . . . . . . . . . . . . . . . . . . . . . 58 l2 Mean values for defects and texture of vacuum, pierced and sorbate treated salt-stock . . . . . 62 vii LIST OF FIGURES Figure l Piercing variables for the preliminary salt penetration study. . . . . . 2 Effect of C102, Clz and Anthium Dioxcide® on microbial populations in cucumber wash water . 3 Total acidity and pH of cucumber fermentations following treatment of green-stock with l00 ppm Cl02 for 15 min. versus control. . . . . . 4 Total acidity and pH of cucumber fermentations following treatment of green-stock with 2.5 ppm C102 for 15 min. versus control. . . . . . 5 Main effects of vacuum and piercing treatments on the NaCl concentration of green-stock cucum- bers . . . . . . . . . . . . . . . . 6 Main effects of vacuum and piercing treatments on the defect classification of salt-stock . 7 Main effects of sorbate treatment on_the defect classification of salt-stock and the main effects of piercing, vacuum and sorbate treat- ments on the texture of salt-stock viii Page 29 46 49 53 59 64 65 INTRODUCTION Michigan is a leading state in the growing and manu- facture of pickling cucumbers and pickle products, and thus, this commodity makes up an important part of the State's economy. Fresh cucumbers are either l) processed at the time of harvest as "Fresh Pak" products, by pasteuri- zation in individual containers, or 2) cured in bulk, via the natural fermentation process. Salt-stock defects and fresh product deterioration contribute greatly to product devaluation and economic loss for the pickling industry. The length and condition of storage of fresh pickling cucumbers has a direct effect on the subsequent quality of the finished product. As the trend towards transporting fresh cucumbers from out-state growing areas to lengthen the packing season becomes ever more prominent, the importance of proper post harvest handling and storage becomes more critical. Brining techniques are also continually being improved in an effort to insure the consistent development of good quality salt-stock once the fruit are in brine. Large populations of microorganisms build up quickly in hydrocooling, wash and flume waters that come in contact with cucumbers. Microbial populations of such water may significantly increase the load of undesirable organisms on the green-stock cucumbers and consequently decrease their storage life. Molds are of particular importance because they remain active in cucumber brines and produce pectino- lytic enzymes capable of causing softening spoilage. The use of chlorine as a germicidal agent in such water is not practical due to the pH and the high content or organic matter of the water. Chlorine dioxide has been used effectively to treat vegetable processing waters when chlorinehas been ineffective. However, chlorine dioxide has not been evaluated for use in cucumber processing waters. The chlorine dioxide studies were initiated to possibly answer questions specifically related to the Pickle packing industry. These questionable areas include the efficiency of chlorine dioxide in maintaining low microbial populations in cucumber processing water and on the fresh fruit. Whether an increase in the storage life of fresh cucumbers will be realized from the use of chlorine dioxide. And whether the use of chlorine dioxide has any effects upon the initiation of natural fermenta- tions. Despite much research in the area of cucumber fermen- tations, salt-stock defects such as softening and bloating still occur. The Cucumber Piercing and Vacuum Studies were initiated to evaluate the effect of such prebrining treatments on the quality of the finished salt-stock pickles. The piercing and vacuum treatments were intended to increase initial brine penetration into the fresh fruit and to allow gases entrapped within the cucumber tissues to escape more efficiently. Sorbate is known to reduce bloater formation and sof- tening in cucumber fermentations due to its selective anti- microbial action against yeasts and molds. A sorbate treatment was added to the cucumber Piercing and Vacuum Studies to observe the interaction between sorbate and the piercing and vacuum treatments. LITERATURE REVIEW The Natural Fermentation Process The brining and subsequent fermentation of cucumbers is an ancient method of preservation for this perishable commo- dity. Cucumbers are placed in a vessel and covered with a 5-lO% salt brine. Next, the cucumbers are "headed down" by means of a false head which keeps the bouyant cucumbers beneath the surface of the brine. The brine draws water and other soluble cellular components out of the cucumber tissues via osmotic pressure. These cellular fluids dilute the cover brine, so additional salt must be added after 24-48 hr. to equilibrate the brine at the desired salt concentration. A comprehensive review of proper commercial brining techniques- is provided by Etchells andMoore (l97l). The fermentation that ensues is dependent upon the naturally occurring microflora associated with the surface of the cucumbers and the soil. Of this complex heterogeneous mixture of yeasts, molds and bacteria, only the non-gas producing lactic acid forming bacteria are desirable organ- isms. These bacteria, which include Lactobacillus plantarum, Lactobacillus brevis, and Pediococcus cerevisiae, make up approximately .02% of the total initial microbial population (Lingle, 1975). A proper initial salt concentration favors the growth of the lactic acid forming bacteria and inhibits the growth of many competing organisms. The lactics utilize the sugars and other nutrients drawn from the cucumbers by the action of the brine and produce lactic acid as a by-product of metabo- lism. As the fermentation proceeds, lactic acid accumulates and the pH drops to a point where the lactic acid formers themselves are inhibited. At this point, the total acidity should range from .6 to 1.2 percent when calculated as lactic acid (Etchells, Fabian & Jones, 1945). Also, there should be little or no remaining sugar present in the brine at the completion of the fermentation as these sugars can be utilized by salt and acid tolerant yeast and reduce the quality of the salt-stock. Jones gt 31. (1940) describe the course of natural fermentations in terms of the microbiologi- cal and chemical changes which occur. The total fermentation period may last from two to six weeks, depending upon the temperature, brining techniques and the variability of the naturally occurring microflora (Etchells, Fabian and Jones, 1945). Following the completion of fermentation, the brine strength is gradually increased to 60-800 salometer(s). The salt-stock can be held under these conditions for up to a year and are available for desalting and finishing when needed. Although the preservation of cucumbers via natural fermentation appears to be a straightforward process, exten- sive research has proved it to be highly complex. This is evidenced by the incidence of salt-stock defects such as bloater formation and softening. Over the past forty five years, extensive investigations have been conducted on cucumber fermentations in an attempt to learn more about these spoilage problems. This research has helped the pickle packing industry make numerous advances toward understanding and solving brining problems. The results include manufac- ture of higher quality products in an economical fashion. Bloater Formation and Control Formation of bloaters during the fermentation of the larger sizes of pickling cucumbers constitutes a major source of product devaluation. The bloater is characterized by the presence of gas pockets within the cured cucumber tissues. Severe bloaters, termed mflloon bloaters, are characterized by a large hollow cavity within the cucumber, with the carpel tissues compressed toward the perimeter of the fruit. The Bloater Chart of Etchells gt 11. (1974), pictures examples of various types and degrees of severity of typical bloated cucumbers. 'These cucumbers are unsuitable for many high quality pickle products such as spears, slices or whole dills and are usually diverted for use in less valuable products such as relish. Etchells _£._l- (1968) describe the mechanism of bloater formation in the following manner. C02 formed in the brine during active fermentation accumulates until it becomes supersaturated. When this brine diffuses into the cucumbers, C02 is released from solution and accumulates in areas of structural weakness. As C02 accumulates around the carpel tissues, they are gradually pressed towards the side of the fruit which may become distended due to the excessive gas pressure. Fleming gt al. (1973a) revised this theory some- . what by showing that CO2 need not be supersaturated in the brine to cause bloating. There are several variables that contribute to bloater formation. One would easily conclude from Etchells gt El- (1968) theory of bloater formation that the presence and amount of C02 in the brine is a critical factor. Veldhuis and Etchells (1939) studied the composition of the gas evolved from cucumber fermentations and found that it consisted mainly of C02, with H2 occasionally present. Furthermore, the gases found inside of bloaters from any given vat were found to be similar in composition to the gas evolved from that vat. Bloater formation was not observed in microbiologically inactivated brines (Fleming gt 31., 1973b). And finally, Fleming, Thompson and Monroe (1978) induced bloater formation by artificial carbonation of cucumber brines. Brining techniques have an influence on the amount of C02 produced by a given fermentation and therefore on the inci- dence of bloater formation. Veldhuis and Etchells (1939) found that if the initial salt concentration was high, a gaseous fermentation ensued. Fleming gt_al, (1977) reported that a greater amount of C02 was evolved from fermentations as brining temperatures increased. And Jones gt 31. (1940) reported that addition of sugar to brines increased bloater formation. Proper brining techniques, adjusted to the climate and fruit size, are necessary to control excess 002 production and to promote a healthy lactic fermentation. Yeast populations in cucumber fermentation have been shown to produce large amounts of C02 (Etchells gt 11., 1968). Yeasts are an endemic portion of the normal microbial populations of cucumber fermentations. Etchells and Bell (1950b), isolated and identified numerous species of surface and subsurface yeast from commercial fermentations. As mentioned previously, salt and acid tolerant yeasts may ferment any residual sugars after the completion of lactic fermentation. Costilow (1957) found that bloater formation could be reduced by the addition of sorbate to cucumber brines. Sorbate is known to selectively inhibit the growth of yeasts, molds and some species of bacteria. Bacteria of the genus Enterobacter were also reported to produce C02 as well as H2 in cucumber fermentations by Etchells gt ai. (1945). These organisms were thought to account for the H2 evolved from some fermentations as well as the H2 found inside of some bloaters. Etchells gt 31. (1964) developed a pure culture fermen- tation process whereby populations of all naturally occurring microbes were drastically reduced by acidification of the cover brine with acetic acid. This was followed by raising the pH and buffering it at about pH 4.7 with sodium acetate. Next, the brine was inoculated with a starter culture of lactic acid bacteria. This process reduced the initial popu- lations of yeasts, molds and all other unwanted organisms and insured a large population of lactics. Buffering the brine allowed the lactics to completely utilize all of the sugars present in the brine by holding the pH at a level tolerable for them. However, this procedure did not successfully eliminate salt-stock defects such as bloating. Further studies showed that lactics common to pickle brines were also capable of producing C02 in quan- tities sufficient to induce bloater formation (Etchells _t‘_l., 1968). Removing C02 from cucumber fermentations by purging proved to be the most significant innovation of the 1970's in terms of reducing bloater formation. Purging is accom- plished by bubbling N2, air, combusted air, or inert gases through the brine. These gases all have much lower solubility in brine than does C02. Therefore, the bubbles remain intact 10 and as they pass through the brine, molecules of C02 absorb into these bubbles. All the gases are liberated from the brine as the bubbles reach the surface (Fleming gt 11., 1975). Fleming _t__l, (1975) found that N2 was the most satis- factory purging gas. N2 was found to efficiently remove C02 from the fermentations studied without effecting the lactic fermentation. The reduction in bloater formation was sub- stantial. The drawbacks of purging with N2 were the expense and a delay in cure color development. None the less, Etchells, Bell and Fleming (1973), incorporated N2 purging into their suggested procedure for pure culture fermentations as an important step in inhibiting bloater formation. Due to the expense of N2 purging, interest was generated for defining the maximum amount of C02 tolerable in cucumber brines before bloater formation was induced, Etchells gt gt. (1973 ). A satisfactory level has yet to be defined due to the variable solubility of C02 in brines of different strengths and temperatures. Also, the minimum amount of C02 necessary to cause bloating has not been determined. Etchells, Bell and Fleming (1973) suggested a value of not more than 20 mg C02 per 100 m1 of brine. Fleming gt _1. (1978) suggested that C02 should not exceed 50% saturation. And Costilow gt gt. (1981) reported that values as high as 90 mg per 100 ml or 75% saturation have not resulted in excessive bloater formation in purged fermentations. 11 At first, air was deemed unsuitable for purging due to softening, off flavors and discoloration of the salt stock (Fleming _t__l,, l975). Softening was attributed to the growth of molds which was supported by the oxygen present in the air (Costilow gt gt., 1980). Off flavors and discolora- tion of the salt-stock were considered to be the results of oxidation when air bubbles became entrapped beneath the. cucumbers and remained in contact with the fruit for some period of time (Fleming _t gl., 1975). The disadvantages of purging with air were overcome by several modifications in purging apparatus and purging schedules. Costilow gt_gt. (1977) developed a device called a sidearm purger. This device draws brine from the bottom of the vat, purges the brine and expels it at the surface of the vat all through an L shaped tube. The sidearm purger circu- lates the brine as it purges and the purging gas does not come in contact with the cucumbers, thus eliminating oxida- tion problems. Next, Costilow gt gt. (1981) recommended intermittent purging to allow dissolved oxygen to dissipate from the brine. It was surmised that molds deprived of oxygen for even short periods of time would be rendered nonviable. Finally, Costilow gt gt. (1981) recommended that the flow rate should not exceed 20 ft3/hr when air was used as the purging gas. Extensive studies of commercial fermen- tations purged with N2 and air with the above modifications concluded that there were no significant differences between 12 N2 and air purged stock, Costilow and Uebersax (1981). These advances made the replacement of costly N2 with air as a purging gas practical and further increased the economic benefits derived from purging. Enzymatic Softening of Salt Stock Pickles The enzymatic softening of fermented cucumbers represents a second type of spoilage commonly encountered by pickle briners. Like bloaters, soft pickles are unacceptable for many pickle products and at best they may be incorporated into low quality products such as relish. Fermented cucumbers are composed mainly of cellulose and pectic substances and the hydrolysis of these substances has been implicated as the main cause of softening (Etchells gt gl., 1958a). Pectin is considered to be the main component responsible for the characteristic crispiness of pickles. Pectin is a component of cell walls and also functions as a cementing substance that binds cell walls together in plant tissues. It is a complex polymer consisting of 1,4 alpha linked galacturonic acid units. The carboxyl groups may be free, esterified with methyl groups or in a salt form. In plant tissues pectin is located within the middle lamella or may be bound to cell wall constituents (Fenema gt gt., 1976). It is known that many microorganisms can produce enzymes capable of hydrolyzing pectic substances. Fruit tissues also produce active pectinolytic enzymes and these account 13 in part for the natural softening of fruit upon ripening. These enzymes can be placed in two main classifications: 1) pectinmethylesterases, which are enzymes capable of catalyzing the deesterification of the methyl groups attached to the pectin molecule and 2) polygalacturonases (PG), which are enzymes capable of hydrolyzing pectin into galacturonic acid sub—units. Although the deesterification of pectin alone does not cause softening, some degree of deesterifica- tion must take place before complete hydrolysis of the molecule can occur. Bell, Etchells and Jones (1950) found polygalacturonase activity in cucumber brines and showed a correlation between this enzyme activity and softening. Furthermore, they con- cluded that the enzyme had a pH optimum of about 4.0 and was active in cucumber brines with a pH range of 3.2 to 3.9, salt content of 10-20% and brine acidity of .2-1.2% lactic acid. Aerobes (Fabian and Johnson, 1938), yeasts (Etchells and Bell, 1950a), and molds (Etchells, Bell and Jones, 1955a) were investigated as possible sources of this specific polygalacturonase since all are known to produce pectino- lytic enzymes under certain condition. However, only molds were found to produce a polygalacturonase that was active in cucumber fermentations and met the specifications listed previously., Etchells ££.£l- (1958) observed pectinolytic activity from species of Penicillium, Ascochyta, Fusarium, Cladosporium, Alternaria and others isolated from commercial 14 brines. These organisms are part of the normal flora of the soil and the surface of the cucumber fruit. Their popula- tions on any lot of cucumbers vary with harvest conditions, time of year, area, and length and conditions of post harvest storage (Etchells _t _t., 1973). Cucumber blossoms were found to harbor large populations of molds. Etchells, Bell and Jones (1955) observed that cucumbers brined with a large number of attached blossoms resulted in a high incidence of soft stock. They also showed that softening could be induced by the addition of cucumber blossoms to cucumber fermentations. Thus, molds were indicated as the causitive agents in the softening of brined cucumbers. As mentioned earlier, fruit tissues may also be a source of pectinolytic enzymes. Bell (1951) measured pectinolytic activity in cucumber fruits and plant parts. He found that staminate and pollinated pistillate flowers were strongly positive for pectinolytic enzyme activity. Seeds and ripe fruit were also positive. Unpollinated flowers, unripe fruit and stems were weakly positive to negative. Bell and Etchells (1950) observed that pectin esterase diffused out of cucumber fruit when placed in 13.2% salt brine. Hamilton and Johnston (1961) also studied polygalacturonase like enzymes produced by molds in commercial brines. They did not find pectinesterase production by molds and concluded that both pectinesterase produced by the cucumber, plus the 15 polygalacturonase produced by molds were responsible for the enzymatic softening of brined cucumbers.. Management of softening problems in cucumber brines has been focused upon removing or inactivating enzyme activity in brines. Etchells, Bell and Jones (1955b) suggested that attached blossoms be mechanically removed before brining, or perhaps development of new cucumber varieties which have less tendency to retain the blossoms. Etchells gt gt. (1955b). also suggested that the brines of small sized cucumbers, containing a majority of the softening enzyme, be drained off and replaced with a fresh brine after the first 24-36 hr. This procedure has been used with some degree of success, especially in southern brining areas. However, the disposal problems that spent brine presents to the industry at this time somewhat limits the practicality of this pro- cedure. For years, home recipes for brining cucumbers have included the addition of grape leaves to the fermentation. Etchells, Bell and Williams (1958) used a crude extract of grape leaves to inhibit pectinase activity in cucumber fer- mentations. The extract did not disturb the fermentation process nor add any off flavors. Also, it was noted that the degree of inhibition corresponded to the amount of grape leaf extract added and that pectinolytic activity returned when the inhibitor was removed. Thus, the grape leaf extract was thought to contain a competitive inhibitor of 16 pectinolytic enzymes. Bell, Etchells and Singleton (1965) noted similar inhibition effects from Sericea extracts. However, the addition of plant extracts in the large quan- tities required for commercial fermentations is impractical. And at this point in time, there is no commercially available purified enzyme inhibitor for cucumber fermentations. Chavana (1976) studied the thermal inactivation of pectinase in cucumber brines. Pectinolytic enzyme produced by Penicillium janthinellum was found to be the most stable in terms of resistance to thermal denaturation. Heat stability of this enzyme was found to be affected by pH and salt concentration. However, it was concluded that a pas- teurization treatment of 175°F for 30 seconds would be sufficient to inactivate this enzyme. This procedure is also somewhat impractical for commercial brining of cucum- bers. However, heat inactivation of pectinolytic enzymes may have some value in the reuse of brines and in the pas- teurization of fresh pack pickles. Bell and Etchells (1961) studied the effect of salt on pectinase activity. They found that the enzyme activity decreased as salt concentration increased. Briners have felt for many years that low salt concentrations in brines increased the incidence of softening and these findings lend some credence to such thought. The addition of calcium chloride to cucumber brines has also been shown to enhance the texture of the cured salt 17 stock (Buescher, Hudson and Adams, 1979). Calcium and other divalent ions are known to form cross linkages between adjacent pectin molecules. Cross linked pectin molecules are thought to be more resistant to hydrolysis by polygalac- turonase enzymes. Calcium chloride or calcium citrate are commonly added to commercial fermentations to enhance the texture of salt-stock. Influence of Post Harvest Handling and Storgge on Salt-Stock Quality Post harvest handling and storage of cucumbers has a profound effect upon the quality of the finished salt-stock. It has been said that even the finest briner can not make a decent pickle from an old moldy cucumber. Indeed this is so; the best pickles are manufactured from fresh, undamaged fruit of good size and variety. In order to increase the brining season many pickle packers are transporting green stock from out state areas. In Michigan, pickle packers commonly buy early season cucum- bers from as far away as Texas. This means increased time from field to brine. Lee, Uebersax and Herner (1982) reported that increasing holding times and temperatures of fresh cucumbers prior to brining increased weight loss and respiration and decreased firmness. Also, bloater formation and softening were shown to increase dramatically in salt- stock as prebrining holding time and temperatures increased. 18 These changes are probably attributable to senescence of the fruit followed by microbial invasion. Rough handling of fresh cucumbers may also contribute to quality loss in salt-stock. Marshall, Cargill and Levin (1972) reported that repeated drops of several feet may bruise internal cucumber tissues and render them more susceptible to bloater formation. Limiting the number of transfers made, reducing drop heights and the use of 3-4 inches of foam or 3-4 feet of cushion brine in bins and brining tanks were suggested to reduce such damage. Abrasions, cuts and scrapes on fresh cucumbers can also be detrimental. These types of injuries can increase respiration rates and provide an avenue for invasion by microorganisms. Sarig gt gt. (1975) suggested that rough areas, exposed bolts and welds be eliminated from conveyor belts used for sorting and washing operations. Mechanically harvested fruit have been shown to have higher respiration rates (Garte and Weichmann, 1974). This is due to the abrasions and scrapes the fruit sometimes endure as they come in contact with machinery. Broken or smashed cucumbers are also more common among machine harvested fruit. To cope with these problems, mechanical cucumber harvesters are being refined and new varieties of cucumbers are being developed specifically for mechanical harvesting (Van Ee gt gt., 1981; Baker gt gt., 1973). l9 Chlorine Dioxide Chlorine dioxide is a germicidal compound similar in action to chlorine. A gas at room temperature, it is slightly soluble in water, to .29%, and will impart a yellowish tint and a prominent chlorine odor to an aqueous solution. Bernarde ££.11- (1965) found chlorine dioxide to be an effective rapid acting germicidal agent against bacteria, viruses and spores. In sewage effluent, 2 ppm chlorine dioxide drastically reduced microbial populations within one minute. Also, the effectiveness of chlorine dioxide was noted to increase with time and temperature (White, 1972). In organic free water (low demand) at low pH the germicidal power of chlorine dioxide was noted to be com- parable to that of chlorine. However, under alkaline conditions or in the presence of a large amount of organic matter, chlorine dioxide was found to be far superior (Bernarde t al., 1965). Unlike chlorine, in aqueous solution, chlorine dioxide is a true dissolved gas. Therefore, it is not directly affected by pH, as is chlorine, which is dependent upon the presence of hypo- chlorous ions for its most effective germicidal power (White, 1972). Also, chlorine dioxide is a more selective oxidizing agent. It enters into far fewer side reactions than does chlorine which will combine readily with many 20 organic compounds via addition and substitution reactions (Ward, 1975). Therefore, chlorine dioxide can be used more efficiently in water high in pH or organic matter. And although chlorine dioxide is more costly than chlorine, it may be used cost effectively under certain conditions. Due to some of the unique properties of chlorine dioxide mentioned above, its use has been found to be very advantageous for certain applications. In the city of Niagara Falls, the presence of phenolic wastes in river water resulted in off tastes and odors in municipal water supplies following chlorination. Chlorine reacts with phenolic compounds to form chlorophenols that are respon- sible for objectionable taste and odors in such water. Chlorine dioxide, which completely oxidizes phenols to odorless compounds, has been used since 1945 by the city of Niagara Falls to eliminate such problems in the municipal water supply (Ward, 1977). Vegetable processors have also found use for chlorine dioxide in plant waters. Welch and Folinazzo (1959) inves- tigated the use of chlorine dioxide for the control of microbial populations in recycled water at a vegetable canning operation. They found that five to ten ppm chlorine dioxide maintained acceptable levels of bacteria in plant waters and on finished product. Chlorine was found to be less effective under the conditions studied. 01in Water Services (1978) provides similar case histories 21 where chlorine dioxide was used effectively to control bacterial populations and odor problems in tomato flume water and potato processing waters. In both cases the use of simple chlorination was insufficient. Also, Baran gt gt. (1973) reported that microbial counts on whole turkeys were further reduced when five to seven ppm chlorine dioxide was added to chilling waters following processing in chlorinated water as opposed to when chlorine was used alone. In the range of concentrations used in the studies above, no off flavors, odors or bleaching of the ' products were noted. As a gas, chlorine dioxide is unstable and explosive at concentrations above 10-15%. Also, as noted earlier chlorine dioxide is not highly soluble in water and con- centrated solutions are not available. Therefore, develop- ment of appropriate technology was necessary before chlorine dioxide could be made available for widespread use. Some companies have developed on-site generation systems where chlorine dioxide is liberated from a precur- sor solution of NaC102 by mixing with water containing free residual chlorine. This residual chlorine can be provided by dissolving chlorine gas into the water supply or by liberating it from NaOCl with acidification (Olin Water Services, 1978). Other companies have developed so called stabilized chlorine dioxide solutions. Anthium Dioxcide® 22 (International Dioxcide, Clark, N.J.) is one such solution manufactured by passing C102 gas through sodium carbonate peroxide solutions. The process is completed upon removal of the peroxides. Free chlorine dioxide is said to be released from this product when the pH is reduced or in the presence of free residual chlorine. MATERIALS AND METHODS Experimental Design of Research Conducted Source of Fresh Cucumber All of the cucumbers used in these studies were donated by Green Bay Foods Inc., and were obtained from the plant in Eaton Rapids, Michigan. Size 18 (7/8 to 1 1/16" dia.) cucumbers were used throughout the C102 studies and size 38 (1 1/2 to 2" dia.) cucumbers were used exclusively in the Cucumber Piercing and Vacuum studies. Chlorine Dioxide Studies Effect of C102,_C12L Anthium Dioxcide® and Blanching on the Microbial Populations of Blended Cucumbers This study was designed to study the effects of C102, C12, and Anthium Dioxcide® on the microbial populations of cucumbers. Size 18 cucumbers, carefully selected to be free from visible disease or mechanical damage, were used in this study. Prior to treatment, they were rinsed to remove adhering soil. The treatments were applied by washing a sample size of one hundred fruit per treatment in 10L of water containing the following com- pounds for 15 min: C102, C12 and Anthium 0ioxcide® in 23 24 concentrations of 2.5, 25, and 150 ppm each. A control treatment consisted of washing the cucumbers in pure water and a positive control consisted of blanching the cucumbers for five minutes at 70°C. Following the treatment, the cucumbers were stored at 20°C and 95% relative humidity. A subsample of 10 cucumbers was withdrawn at one, twenty- four and forty-eight hours after treatment. These cucum- bers were then homogenized with water in a 1:5 ratio. Serial dilutions were prepared from these homogenates and used to estimate microbial populations. In this and sub- sequent studies the following media were used: (LBS) agar with .05% bromocresol green (pH 5.6) for lactics, Yeast Nitrogen Base agar (YNBA) acidified with tartaric acid for yeasts and molds and Plate Count agar (PCA) for an esti- mated total count. Three replications with duplicate plates within each replication were counted to estimate the microbial populations within each treatment. Addi- tional analysis included measuring polygalacturonase activity and texture at one, twenty-four and forty-eight hours. Each treatment was carefully observed at the times of analysis for the appearance of visible mold growth. Effect of C102, C12 and Anthium Dioxcide® on the Microbial Populations in Cucumber Wash Water These two studies were designed to study the effect of C102, C12 and Anthium Dioxcide® 0" the microbial populations in cucumber wash water under two conditions: 25 i.e., high and low demand environments. In the first experiment, the cucumbers were carefully selected to be free from mechanical damage and rinsed to remove any adhering soil prior to treatment. The treatments consisted of washing twenty cucumbers each in one liter volumes of water containing 2.5, 25 and 150 ppm of C102, Cl2 or Anthium Dioxcide ® for fifteen minutes. The cucumbers were removed and the water samples from each treatment were I evaluated for microbial populations and compared to a control. In the second study in this section, a high demand environment was generated by soaking one bushel of cucum- bers in ten gallons of water for four hours. These cucum- bers included broken and damaged fruit and adhering soil was not removed. Following this soaking procedure, the cucumbers were removed and the water was divided into ten lots. 2.5, 25 and 150 ppm of C102, C12 and Anthium Diox- cide ®were added to the different water samples. Then, after fifteen minutes, the microbial populations of these water samples were enumerated. Effect of C102 on Natural Fermentations of Salt Stock Pickles These two studies were initiated to determine whether or not the use of C102 in cucumber wash waters had any effect upon the natural fermentation process. In the first study, clean undamaged lB cucumbers were washed for 26 fifteen minutes in clean water containing 1 to 100 ppm C102. C102 concentration was measured before and after each treatment. Also, following the treatments the micro- bial populations of the water samples were estimated. Treated cucumbers were brined in 1 gallon jars with a 45° salometer cover brine. Dry salt was added the following day to equilibrate the brines at 25° salometer. Total- acidity and pH were monitored on these fermentations on a daily basis for one week. In the second study cucumber fermentations were monitored over a longer period of time. Also, an attempt was made to simulate wet tank or commercial cucumber wash water. This was done by washing ten bushels of cucumbers in twenty gallons of water. Continuous washing of cucum- bers in this comparatively small volume of water generated a source of water that represented a high demand environ- ment for c162. This water was used for the following four treatments: 1) cucumbers were washed in this water for fifteen minutes and then dipped in clean water containing 2.5 ppm C102. This treatment was designed to simulate a commercial situation whereby the fresh cucumbers would be sprayed with a solution containing C102 following wet grading: 2) C102 was added to the high demand water until a residual of approximately 2.5 ppm C102 was achieved. This required approximately 25 ppm C102 initially. Then the cucumbers were washed in this water for fifteen 27 minutes. This treatment was designed to simulate the effect of adding C102 directly to cucumber wash water or wet tanks. Obviously, in a continuous system, C102 would have to be constantly replaced as it is used up; 3) cucum- bers were washed in the water, then rinsed with clean tap water. This treatment simulates common commercial prac- tices at this time; 4) cucumbers were washed in the water and used directly without a final rinse of any kind. A sample of fifty cucumbers from each treatment was held at 20°C and 10°C at 75% relative humidity and observed daily for the appearance of visible mold. Subsamples of ten cucumbers from each treatment of the stored fruit were taken daily and used for enumeration of mold populations. In addition two five gallon pails of cucumbers from each treatment were brined immediately following the treatments. These fermentations were monitored for six weeks by measuring pH and total acidity every other day. Each fermentation was also evaluated for polygalacturonase activity. Following the fermentation, the stock was evaluated for texture using the Instrom universal testing machine. Cucumber Piercing and Vacuum Studies Preliminary Salt Penetration Study A 2X2X2X3X2 factorial design Was employed. The variables included two depths and three frequencies of piercing, two diameters of piercing implements, a vacuum 28 treatment and two replications. A total of fifty two, one gallon jars were used to evaluate the effect of piercing and vacuum treatments on brine penetration as measured by NaCl concentration of the stock at zero and twenty four hours. Piercing variables included depth and frequency of pierces as well as size, or diameter of the piercing imple- ment. Two depths were used to evaluate whether salt penetration was enhanced by piercing into the carpel space or if piercing just through the skin would be sufficient. These two depths are referred to as radius and 1/2 radius, respectively. Three frequencies were incorporated into this study. These include eight, sixteen or thirty two punctures per 3B cucumber. Also, needles of two diameters were used as piercing implements to evaluate the effect of hole size on brine penetration. Piercing variables are illustrated in Figure 1. All piercing was done by hand so that the piercing angle was perpendicular to the cucumber surface and did not tear the skin upon withdrawal. Vacuum treatment consisted of submerging the green stock in a 45° salometer brine and pulling a vacuum equivalent to 25 inches of Hg for ten minutes. A sample of two cucumbers from each jar was removed after the initial vacuum treatment and again after twenty four hours in brine. These samples were towel dried and stored frozen for later evaluation of NaCl concentration. 29 2 SIZES: Small Needle = .4 mm dio. Large Needle : .7mm dio. 2 DEPTHS‘ : l/Z RADIUS 3RADIUS 3 FREQUENCIES8 O O O O 4 PUNCTURES/ SIDE @0629 2 SIDES 4 SIDES 8 SIDES (8 holes) (I6 holes) (32 holes) Figure 1. Piercing variables for preliminary salt pene- tration study. 30 In addition, after twenty four hours, a twenty m1 sample of each brine was withdrawn and evaluated for polygalac- turonase activity. All treatments were prepared in duplicate. Five Gallon Pail, Vacuum X Pierce X Sorbatg, Fermentation Stud A 2X2X2X2 factorial design was employed. Sixteen, five gallon pails of 3B cucumbers were brine following vacuum, piercing and sorbate treatments. Again, vacuum treatment consisted of submerging the green stock in a 450 salometer brine and pulling a vacuum equivalent to twenty five inches of Hg for ten minutes. The piercing treatment consisted of piercing each treated cucumber sixteen times, to a depth of one half the radius with a .4 mm diameter needle. Sorbate treatment consisted of adding .1% sorbate to the brine. Following a six week fermentation period, the salt stock was evaluated for texture, defect classification and general appearance. Analytical Procedures Preparation and Measurement of Water Sanitizers Working solutions of chlorine were prepared from com- mercial solutions of hypochlorite by dilution with deionized water. Initial chlorine concentrations, in ppm, were calculated on the basis that the stock solutions contained 5.25% available chlorine as stated on the label. 31 Working solutions of Anthium Dioxcide® were prepared from a concentrated solution of stabilized chlorine dioxide supplied by International Dioxcide, Inc., Clark, N.J. The stabilized chlorine dioxide was activated by lowering the pH to 4J1with acetic acid per label directions. Initial concentrations of Anthium Dioxcide® were calculated on the basis that the stock solution contained 50,000 ppm- chlorine dioxide as stated on the label. Chlorine dioxide was prepared fresh daily by combining equal parts of chilled dilute solutions of hypochlorite, sodium chlorite and HCl, see Appendix I for formulation. This stock solution, containing 300-500 ppm chlorine dioxide, was diluted with deionized water to the desired working concentrations. A spectrophotometric method (Wheeler _t _t., 1977) was used to determine the concentration of C102 in water samples. The procedure is as follows: add 2.0 m1 of a 3.3 x 10'4M standard chlorophenol red solution to a 125 m1 Erlenmeyer flask; add 1 m1 of a pH 7.0 buffer and swirl to mix. The solution should immediately turn purple. Add 50 ml of a water sample containing up to .1 mg C102 to the flask, mix and measure the absorbance at 575 nm. Absor- bance at 575 nm was transformed to ppm C102 using a standard curve where an absorbance reading of .70 equals zero C102 and .1 absorbance is approximately 2.5 ppm C102. 32 For samples containing greater than 2.5 ppm C102 dilu- tions were prepared. For example, a 50 ml sample containing 4.0 ppm would be diluted to 100 ml and a subsample of 50 ml of this solution would be used to make a measurement. All samples requiring such a dilution to measure initial con- centrations of C102 were diluted in a similar manner to measure residual concentrations of C102 after treatments. Samples containing background interference such as suspended dirt were filtered through Whatman No. 1 paper prior to analysis. The chlorophenol red solution was prepared by dissol- ving .1436 g of chlorophenol red in 100 ml of .01 N NaOH and diluting this solution to one liter with distilled deionized water. After standing overnight the solution was filtered through a .45-um Millipore filter. The chloro- phenol red solution was standardized by titrating 10 ml of .01 N potassium dichromate in 25 ml sulfuric acid and 15 ml deionized water to a pink end point. Polygalacturonase Activity Assay Polygalacturonase (PG) activity of cucumbers and brines was measured using the procedure described by Bell gt gt. (1955), with modifications by Costilow E£.il- (1981). Brine samples had to be dialyzed to remove salt which reacts with the pectate solution to form a gel. Dialysis was accomplished by filling a twenty ml sample of brine 33 into a length of cellophane tubing and securing both ends. These tubes were then suspended in a container of tap water which was replenished with fresh water at a flow rate of about one gallon per five minutes. After three hours in tap water the samples were resuspended in a large volume of deionized distilled water for an additional hour. Samples were then transferred to sterile test tubes con- taining about five drops of toluene as a preservative. These samples could be capped and refrigerated for analysis at a later date or used immediately. For testing green stock the samples were first homogenized with water in a 1:5 ratio, filtered and used directly. To perform the assay, 4 ml of a 1.2% sodium polypectate solution (see Appendix II for formulation) were added to a viscosity pipette, which was suspended in a 32°C water bath, allowing ten minutes for the solution to come up to temperature. 1 ml of the dialyzed brine or fresh filtered cucumber homogenate was added and the two solutions were mixed by gently blowing air through the viscometer. The solution was drawn into the upper bulb of the viscometer and the time required for the solution to flow from the upper etch mark to the lower etch mark was measured with a stopwatch. A few drops of toluene were placed into the viscometer to prevent contamination. The viscometer was tightly stoppered to prevent evaporation of the toluene. The viscometer was incubated at 32°C and the flow time 34 remeasured after 20 and 44 hours. The following formula was used to estimate polygalacturonase activity: % Loss in Viscosity after 20 hr. initial flow time - 20 hr. flow time x 100 initial fTow‘tTme - flow time of H20 in the viscometer Bell t 1. (1955) provide a table that relates % loss in viscosity to activity units of polygalacturonase. Cucumber and Salt-Stock Pickle Texture Evaluation Two means of evaluating the texture of fresh and salt- stock cucumbers were employed. A Magness-Taylor fruit pressure tester (FPT) with a 7/16“ diameter tip was used according to the procedure described by Bell gt gt. (1955). Firmness was recorded as the force in lbs. required to puncture the wall of the cucumber. In all cases, 10 cucum- bers from each sample were measured to evaluate the firm- ness of the lot. The Instron Universal Testing Machine (Model TTBM, Instron Corp., Canton, Massachusetts) with a 7/16" diameter probe was the second method of measuring the texture of cucumbers in these studies. As with the Magness-Taylor (FPT), firmness was measured as the force in Kg required to puncture the wall of the cucumber. 35 Microbiological Media and Methods Lacto-Bacillus Selective Media (LBS) with .05% bromo- cresol green, acidified to pH 5.6 i .05 was used to enu- merate lactic acid forming bacteria (Costilow, Etchells & Anderson, 1964). Yeast and mold colonies were enumerated on Yeast Nitrogen Base Agar (YNBA) acidified with 10% tartaric acid. Total counts were estimated using plate count agar (PCA). See appendix II for formulation of media. Microbial populations of cucumbers were estimated by homogenizing 10 cucumbers per sample in a 1:5 ratio with water. Serial dilutions were prepared from the homogenate and plated on the various media in duplicate. To estimate the microbial populations of water samples, serial dilutions were prepared and plated in duplicate directly from the sample. Brining and Fermentation Procedures All cucumbers brined in five gallon pails followed the procedures described by Costilow gt gt. (1982) for pilot scale fermentations. The fresh cucumbers are washed and covered with a forty five degree salometer brine with .05% acetic acid to enhance C02 solubility in the brine. Dry salt was added as needed to equilibrate the brine at 25° salometer. The pails were fitted with purging apparatus and purged with nitrogen gas on the following schedule: 36 immediately following brining for twenty minutes and thereafter fifteen minutes each morning and afternoon for approximately two weeks. Measurement of total acidity and pH were used to moni- tor fermentations. Total acidity was estimated as percent lactic acid using .1N NaOH as a titrant and phenylphtha- line as an indicator. A standard pH meter was used to. measure pH. These measurements were taken daily. Visual Evaluation of Salt-Stock Pickles Following a six week fermentation period, salt stock from the 5 gallon pail, Pierce X Vacuum X Sorbate study were evaluated in the following manner. Thirty cucumbers were removed from each pail, sliced lengthwise, and grouped according to the following defect classifications: No Damage, Honeycomb, Lens, Balloon,om Soft Center. The "Bloater Chart" of Etchells gt gt. (1974) was used to help determine these classifications. These groupings were then rearranged by three judges until a consensus was reached. Pierced stock was also observed for evidence of visible puncture marks. Chlorine Ion Concentration of Salt Stock Chloride ion concentration of pierced and/or vacuum treated salt stock was measured using an Orion Micropro- cessor Ionanalyzer 901 (Orion Research, Cambridge, Mass.), equipped with a selective ion electrode specific for chloride ions (Cl'). 37 Two cucumbers from each sample to be tested were removed from the brine, the surface towel dried and then homogenized in a Waring blender with 1% nitric acid in a 1:5 ratio. The nitric acid served to extract the Cl' from the cucumber tissues. The C1' ion concentration was related directly to the NaCl concentration by calibrating the instrument with stan- dard NaCl solutions (.1 to 10% NaCl), diluted with nitric acid in a 1:5 ratio.. When the instrument is calibrated in this manner, the % NaCl of the sample is presented on the instrument read out (personal communication with Orion technical representative). Statistical Analysis The CDC 6500 computer, located at Michigan State Uni- versity, was used in conjunction with the "Statistical Package for the Social Sciences" (SPSS), (Nie gt gt., 1975) to assist with the statistical analysis of the data herein. Subprogram ANOVA was used to determine analysis of variance. Mean squares were reported after rounding and those with significant F tests were indicated with sig- nificant probability levels of P S .05 (*), P S .01 (**) and P S .001 (***). Mean values were separated by L.S.D. and those which were not significantly different at P f .05 were indicated with like letters. RESULTS AND DISCUSSION Chlorine Dioxide Studies Effect of C102, C12, Anthium Dioxcide® and Blanching on Microbial Populations of Blended Cucumbers . Since the cucumbers used in this experiment were all commercial run and of undetermined post-harvest age, the initial microbial populations measured varied widely. Therefore, microbial populations were analyzed as percent reduction as compared to controls within each lot of cucum- bers used. As a positive control, blanching for five minutes at 70°C reduced the mold, yeast and lactics popu- lations by greater than 100 fold. Although there were significant differences between some of the treatment means, none of the other treatments applied caused more than a ten fold reduction in microbial populations. In terms of microbial growth patterns, reductions of such magnitude may not be important. Under the storage conditions employed in this study, 95% R.H. and 20°C, which are ideal for mold growth, it is not surprising then that the time before the appearance of visible mold growth and a corresponding decline in texture of the fresh fruit were not suppressed. It is probable that the rough, porous cuticle of the cucumber, at the 38 39 microscopic level, is capable of harboring vast numbers of microorganisms and protecting them from such water treat- ments as were applied in this experiment. Trends noted in the data (Table 1) were first, that population reductions generally increased gradually as concentrations of C102, C12 and Anthium Dioxcide® were increased. Aside from blanching, the largest reductions occurred when using 150 ppm of either C102, Cl2 or Anthium Dioxcide® in the water treatment. Such concen- trations are far too high for commercial application. In the commercially applicable range from 2.5 to 25 ppm, generally less than a one fold reduction in microbial populations of molds, yeasts and lactics was observed. At similar concentrations the different water treatment compounds had comparable effects. The second trend noted, was that the population reduc- tions decreased as the time following the treatment increased. Very little differences were noted between treatment means 48 hours following the treatments as compared to 24 hr estimates (Tables 1 and 2). Polygalacturonase activity was not significantly different between treatments or control (Tables 3 and 4). The PG activity observed was probably due to a combination of enzymes of microbial origin and from the fruit itself. As noted earlier there was a gradual decline in texture over time. 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Although the blanching treatment successfully eliminated molds, yeasts and lactics, the blanched fruit are extremely vulnerable to bacterial decay, especially from spore formers. Effect of C102, C12 and Anthium Dioxcide® on Microbial Populations in Cucumber Wash Water In the first study, where cucumbers were sorted and rinsed prior to use, the initial microbial populations of the wash water were approximately 104 organisms per ml for the total count with nearly 103 molds and yeasts per ml. The water was practically free from soil and other visible debris. These conditions constituted a low demand environ- ment. Results showed that C102 concentrations of 2.5 ppm were adequate to reduce total microbial populations by ten fold and nearly eliminate yeast and mold populations. Similar results with C12 and Anthium Dioxcide ® required initial concentrations of 25 ppm each (Table 5, Figure 2). In the second study, where cucumbers were not sorted or rinsed prior to use, initial microbial populations were approximately 108 organisms per ml as a total count and 4 just over 10 molds and yeasts per ml. There was also considerable soil and other organic material present in 45 Table 5. Effect of C102, C1 and Anthium Dioxcide® on microbial populati6ns in cucumber wash water. Microbial Populations (organisms/m1) Treatment Total Count Yeasts and Molds Initial Population I X 104 8 x 102 Chlorine Dioxide (ppm) 0102, 2.5 2 x 102 . 1x 101 25 3 x 101 O x 101 250 2 x 101 0 x 101 Chlorine (ppm 012 2.5 1 x104 5 x 102 25,0 1x 103 1x 101 250 2 x 101 0 x 101 Anthium Dioxcide R (ppm) 2.5 1x 104 7 x 102 25.0 5 x 103 1x 101 250,0 3 x 101 0 x 100 1Values represent means; n=6. High Demand Low Demand 46 ANTHIUM OIOXCIDE 2509mm lllllIIlllllllllllllllllllllllIIII 2" TOTAL CT 7 fl YEAST. MOLD |||||||||||||||||||IIIIIIIIIII CIO2 2 Sum Cl2 250ppm Ill||||||||||||||l|||||||I“ IIII|III|||||III|||||ll|l|l||||||l||I|||||llm1| . Q> :3 “b “g 'b o CONTROL 106' e h 9 o lw/SWSINVDUO ANTHIUM DlOXCIDE 25ppm - Ill"llllllllllllllllllllllllIllllll OTAL CT. 0 .l O 2 g..." m < ILI >. Illlll NV CE 25mm IIIIII||||l|||IIIIIIIIIIIIIIIIIIII C102 2.5ppm CONTROL \\\\\\ lllllllllllllllmlflflmmfllfl 1 (D Q' N O 2 e 2 IW/SWSIN‘VSUO =5) Figure 2. Effect of C102, C12 and Anthium DioxcideC) on cucumber wash water.(n 47 these water samples, all of which increase the demand for the water treatment compounds. Under these conditions, C102 concentration of 25 ppm was required to achieve an effective residual capable of significantly reducing microbial popu- lations. In contrast, an initial concentration of 250 ppm of C12 and Anthium Dioxcide® were necessary to get similar reductions in microbial populations (Table 6, Figure 3). These studies indicate that the demand for C102 varies depending upon the microbial and organic content of the environment, as is true with the other water treatment com- pounds. C102 was also found to be up to ten times more efficient than either Cl2 or Anthium Dioxcide® under the conditions in the two studies. This data suggests that C102 may be used successfully in low concentrations to control microbial p0pulations of water coming in contact with fresh cucumbers. Effect of C102 on Natural Fermentations The first study in this section examined the effects of washing cucumbers in water containing 0-100 ppm C102 on the microbial populations of the wash water and the effect of the treatment on the subsequent brining and fermentation of the fruit. Table 7 shows the initial and residual levels of C102 and the corresponding microbial populations of the water samples. The initial microbial count was relatively low, 48 Table 6. Effect of C10 , C1 and Anthium Dioxcide® on microbial popalatin in cucumber wash water. Microbial Populations (organisms/ml) Treatment Total Count Yeasts and Molds Initial Population >108 4 x 104 0102, 2.5 ppm 1 x 107 6 x 103 25.0 5 x 102 1 x 101 250.0 6 x 101 0 x 101 012, 2.5 ppm 1 x 108 2 x 104 25.0 4 x 107 3 x 104 250.0 2 x 103 1 x 101 Anthium Dioxcide ® 2.5 ppm 1 x 108 4 x 104 25.0 3 x 107 2 x 104 250.0 5 x 103 1 x 101 1Values represent mean; n=6. 49 l l ------ Control C102 100 ppm I 0 Total Acidity Figure 3. Total acidity and pH of cucumber fermentations following treatment of green-stock with 100 ppm C102 for 15 min. versus control. (n=4) 50 Table 7. Microbial populations1 treated with C102. in cucumber wash water Chlorine Dioxide (ppm) Organisms per m1 Initial Residual Total Mold Yeasts Lactics Concentration Count 0.0 0.0 10,000 70 1,000 250 1.0 0.0 5,000 10 110 0 3.5 1.0 3,000 10 O O 6.0 2.0 500 O 0 0 10.0 4.0 250 0 0 0 25.0 20.0 180 0 0 0 50.0 44.0 70 0 0 0 100.0 96.0 100- 0 0 0 1Values represent means, n=4. 51 approximately 104 organisms per m1, and there was little observable soil or organic matter in the water samples following the treatments. Under these conditions, low initial concentrations of C102 were sufficient to obtain an effective residual capable of greatly reducing microbial populations in the water samples. The optimum efficiency was obtained when a residual of 2 ppm C102 was reached. Under the conditions in this study, an initial concentration of 6 ppm C102 was required to achieve the desired residual. Figure 3 shows a plot of titratable acidity and pH for the control and the 100 ppm C102 wash water treatment fermentations. There was no significant difference between the initiation and course of fermentation between the two treatments. Similar results were observed for the other levels of C102 used in this study. It was concluded from this data that concentrations of C102 as high as 100 ppm in cucumber wash water does not inhibit subsequent natural fermentation of the cucumbers. The second study in this section monitored the fermen- tations of ClO2 treated and untreated stock over a six week period. In addition, subsamples of treated and untreated stock were held at 10 and 20°C and 75% R.H. These subsam- ples were observed for the appearance of visible mold growth and the yeast and mold populations of each were measured intermittently. 52 The fermentations of all four treatments proceeded normally in terms of developed acidity and corresponding drop in pH. Figure 4 is a plot of total acidity and pH for the C102 treated and untreated cucumbers which are essentially the same. Once again, this supports earlier findings that C102 in cucumber wash water does not inhibit the natural fermentation process. Moderate polygalacturonase activity was found in the brine of one of the replicates of treatment four (untreated) and this corresponded to slightly softer stock. Otherwise there were no significant differences of texture among salt-stock from the four treatments (Table 8). Stock from treatments three and four (no C102) held at 20°C and 75% relative humidity showed profuse mold growth after two days of storage. Stock from treatments one and two (C102 treated) did not show visible mold growth until the third day of storage. These results were consistent for both replicates in each of the treatments. However, at 10°C and 75% relative humidity, stored fruit from all four treatments showed signs of visible mold growth on the sixth day. Also, there were no significant differences between the enumerated mold populations of the four treat- ments under any conditions in this study (Table 9). This may be due to the difficulty of accurately measuring the populations of molds with standard plating methods. 53 x pH No C10 0102 13 Wash Water Total Acidity Figure 4. Total acidity and pH of cucumber fermentations following treatment of green-stock with 2.5 ppm C102 for 15 min. versus control. (n=4). 54 Table 8. Firmness of salt-stock and PG activity of brine from cucumber fermentations following pre- brining treatment of green-stock with C102. Treatment1 Firmness2 PG Activity3 1 12.0+2.0 12.0% 2 12.2+1.4 12.8% 3 11.8+2.0 9.0% 4 ll.O+2.6 15.0% 1Pre-brining treatments are as follows: 1) 2.5 ppm residual C102 in wash water 2) 2.5 ppm C102 rinse following washing 3) Tap water rinse following washing 4) Washed then brined directly 2Firmness is in Kg, measured by Instrom. n=60. 3Measured as % loss in viscosity after 44 hr: n=4. 55 wees: memes “cemegeeg meewe> .mepesem mg» :e ewes epnwmw> we eecegemeee . m": age maeecweeea e.e_ am.mm N.eN m.a ee.wm m.NN e o.N we.es o.mN O.“ e.mp m.m_ e.m e.mp o.eF m N.m e.mp e.PF e.m ae.mm e.m_ 0.x m.e m.Nw N.e e.ew e.__ N m.w e.m 0.0 m.e m.w m.m e.w m.N e.m e.m e._ e.« _ 0.x P.F m.~ «.e N._ m.m e.m _.. m.N s.e ~._ m.m e mewuemA ewe: mumee> mewuuee ewe: mumem> mewueee ewe: mumee> mewpeee ewe: mpmee> aaa m.N .Newe Nope ez . Sea m.N .Nope Nose az mop x seem\msmwcemgo 11 Awhmmv .=.m emu .eeeN .=.m em“ .eco. eewewaz .msee e as a: cow .=.m emw .eoeN eee e, ea ewe; eee Ne_e new; eeuemgu mgenszeee xeeumuceegm we meewueweeee _ewnegews Lew meepe> cam: .m mwnew 56 Cucumber Piercing and Vacuum Studies Preliminary Salt Penetration Study Results of the preliminary salt penetration study indicates that both piercing and vacuum treatment of fresh stock significantly enhanced the initial penetration of the cover brine as measured by NaCl concentration. Vacuum treatment was superior in that NaCl concentrations in this stock were initially higher and remained significantly higher than in non-vacuum treated stock after twenty four hours in the cover brine. Polygalacturonase activity was very sporadic, occurring in only two samples, and apparently did not correlate to any of the treatments involved in the study (Tables 10 and 11). The main effects of the piercing variables are as follows. First, the depth of piercing is not significant. Piercing just through the skin (1/2 radius), or all the way into the caprel space (radius) gave nearly identical results in terms of NaCl concentration. Secondly, increas- ing the frequency of piercing from four through thirty two holes per cucumber increased the NaCl concentration in a stepwise fashion. However, these increases were not great enough to be considered significant. Thirdly, the diameter of the piercing implement appeared to be the most critical factor for enhancement of brine penetration. The large size needle (.7 mm) increased NaCl concentration significantly Table 10. Mean values1 57 for NaCl concentration of gree a and stock ang PG activity of brine after vacuum piercing treatments. Piercing Variables Vacuum No Vacuum Size Depth Freq. o gait 24 Hr pgT 24 Hr P64 (Needle (z) Salt Acti- Salt Acti- dia) (x) vity (%) vity 2 Sides .14de 4.1” 3.3” 3.3Jk 6.4” 1/2 Rad 4 Sides .24abc 4.2a” 4.8” 3.413 7.6b 8 Sides .20bc“e 3.9de 3.1” 3.9de 0.9a” Small ~- .4 mm 2 Sides .15°°e 3.4U 5.5” 3.3jk 6.5” Radius 4 Sides .14de 3.5”‘ 21.9a 3.8ef 3.9” 8 Sides .20dee 4.1”c 3.0” 4.0Cd 9.2” 2 Sides .14de 2.91 3.3” 3.2k 4.5” 1/2 Rad 4 Sides .28ab 4.3a 4.2” 3.7fg 3.5” 8 Sides .23ade 3.9de 5.0” 3.3jk 4.0” Large 2 Sides .24°°C 4.1bc 5.2b 3.6gh 5.0b .7 mm .. Radius 4 Sides .20dee 3.8ef 4.3” 3.41J 9.3” 8 Sides .30a 3.413 5.4” 3.3Jk 8.3” Control .12e 3.9de 6.1” 4.1”c 3.2” 1n=2, like letters within columns indicate no significant differences by LSD at P>.05. 2Vacuum = 25" Hg/lO min. in 40°S brine. 3Piercin depths I cies (2, 4 and 8 sides X 4 punctures per side). 4PG Activity = % loss in viscosity of pectin solution after 44 hr at 30°C. = 2 sizes of needles (.4 mm and .7 mm dia.), 2 7/16"=radius and 7/32"=1/2 radius) and 3 frequen- 58 Table 11. Analysis of variance of PG activity in brine and NaCl concentration immediately and twenty four hours following piercing and vacuum treatments of fresh cucumbers brined in 1 gallon jars with a 4505 cover brine. Mean Squares1 Source of Variation NaCl NaCl PG df at 0 Hr. at 24 Hr. Activity Main Effects 5 .097*** .373 29.70 Vacuum l .466*** .656* 2180 Size 1 .011* .212 33.33 Depth 1 .000 .086 51.25 Frequency 2 .004 .455 30.54 Two-Way 9 .003 .135 24.40 Vacuum X Size 1 .011* .000 6.75 Vacuum X Depth 1 .000 .089 28.83 Vacuum X Frequency 2 .004 .078 48.13 Size X Depth 1 .001 .027 .12 Size X Frequency 2 .001 .179 16.55 Depth X Frequency 2 .004 .292 27.27 Three-Way 7 .002 .396* 43.03 Vac X Size X Depth 1 .001 .566 100.92 Vac X Size X Freq 2 .001 .075 68.04 Vac X Depth X Freq 2 .004 .176 16.54 Size X Depth X Freq 2 .002 .853** 15.57 Four-Way Vac X Sixe X Depth X 2 .002 .147 35.35 Freq Explained 23 .023*** .267 32.18 Residual 24 .002 .152 34.25 Total 47 .012 .208 33.23 1Significant F tests indicated by *** (ps.001), ** (ps.01), * (ps.05). 59 o m-Omm q- 03-0““) N q,_.oum C(O-Dm Q ¢.05. 2Vacuum = 25" Hg/lO min. in 4005 brine. 3Pierce = 1/2 radius depth, cucumber. .4 mm dia. needle, 16 pierces/ 63 .Ame.mav s .Ape.mav 44 .A_ee.mav «as so easeeweew mamas a eeaewwwemwm_ m«.w mN.mN o«.«mm mm.Nw Pm.N«p Nm.owm mp Pepe» No.w Fm.m_ mo.oom mm.Pm mm.om Fm.«NN m Peeewmea «xsmw.«F Nm.«m 4mm.mom.P Nm.om x—m.w«N xxmm.mom.~ N eeewepexm mN. mm. om.w—— ««.«« mo. mm.OmN _ eueaLem x ee> x eecewe aezieegnw w«SNN.«m mN.om om.wmm oo.mNN xmo.«m« mN.oom F maesgem x s==ee> xpm.m me. om.wmm mw.~wp mo.«m mm. 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