977

Journal ofFood Protection Vol. 41, No.l2. Pages 977·979 (December, 1978) Copyright © 1978, International Association of Milk, Food, and Environmental Sanitarians Microbiology of the Soda Cracker Process

I. Isolation and Identification of Microflora

T. FRANK SUGIHARA

Western Regional Research Center

Science and Education Administration, U.S. Department ofAgriculture Downloaded from http://meridian.allenpress.com/jfp/article-pdf/41/12/977/1649777/0362-028x-41_12_977.pdf by guest on 24 September 2021 800 Buchanan Street, Berkeley, California 94710

(Received forpublicationApril7, 1978)

ABSTRACT from 3 to 6 C. For isolation and enumeration of the microorganisms, ll g of sample was aseptically blended for 90 sec, at a reduced speed with A survey of the soda cracker manufacturing process revealed that starter cultures were not used and that fermentation for the process 99 ml of sterile 0.1% aqueous peptone in a sterilized 8-oz. Osterizer 1 relied primarily on chance contamination. Over 200 isolates from jar • After further serial dilution, also in sterile 0.1 '7o peptone, samples sponge and samples, obtained from two commercial plants, were were plated on selected agar media by the spread plate technique and screened. Microorganisms responsible for fermentation of commercial incubated for 2 days at 30 C. Preliminary counts were made using soda cracker sponge and dough were isolated and identified. Besides tomato juice agar (Difco Laboratories, Detroit, Michigan), APT agar Bakers' (), which is pati of the standard (Baltimore Biological Laboratories, Cockeysville, Maryland), Mycophil agar (BBL), Wort agar (BBL), Malt agar (Difco), PCA agar (Difco), formulation, three species of lactic bacteria were found to have prominent roles in the fermentation process. Lactobacillus plantarum L-agar (BBL), MRS agar (Difco) and SDB agar (5). MRS agar and was found to be the dominant species. Two other species found in tomato juice agar were found to be the only suitable media. MRS agar significant numbers were Lactobacillus delbrueckii and Lactobacillus was selected for visual ease in colony counting and reproducibility of the leichmannii. media. Samples from the two commercial plants were received once a month, for six consecutive months, to study the possibility of changing microflora. Soda crackers have been made since 1840 and their I dentijication ofmicroorganisms manufacture has grown into a giant industry which now Single colonies from spread plates were picked and carried on MRS includes crackers of various forms and flavor. It is slants. Several hundred isolates were collected for screening. The difficult to comprehend that the soda cracker industry were eliminated from the screening process since the standard formula still relies on chance contamination for fermentation of includes 5 x 10" Bakers' yeasts (Saccharomyces cerevisiae) per gram of sponge. Contaminating yeasts would be few, if any. sponge and dough. A few microbiological and chemical The MRS medium, in a modified form (6), was used for biochemical studies have been published, notably by Micka (7,8,9). studies. It was made up as follows: peptone, 10.0 g; yeast extract, 5.0 g; Chemical and biochemical changes during fermentation Tween-SO, 1.0 g; ammonium citrate, 2.0 g; sodium acetate, 5.0 g; of sponge and dough have been studied but the magnesium sulfate, 0.1 g; manganese sulfate, 0.05 g; and disodium microorganisms responsible for the process have never phosphate, 2.0 g per liter of distilled water. The pH of the basal medium was adjusted to 6.5 with HCI. Bromthymol blue (0.1 gil) was been isolated or identified (1,3,4,12,13,14). added to give a dense green color. Carbohydrate solutions (6%) were Following our successful studies on the lactic made and then sterilized by passing through a 0.20-micron filter fermentation of San Francisco French membrane. Fermentations were conducted in 20 mm x 125 mm screw (5,10) and the subsequent development of pure culture capped culture tubes with a 10 mm x 44 mm inverted culture tube technology (JJ), these techniques have been directed insert for gas collection. Culture tubes, with inserts and 8 ml of basal media, were sterilized at 15 psi steam for 15 min. After cooling, 4 ml of toward the complex lactic fermentations in soda cracker sterile carbohydrate solution was added. sponge and dough. It was the goal of these studies to Inoculum was prepared by suspending the growth of 24-h-old MRS develop pure culture fermentation to eliminate batch slant in 5 ml of sterile distilled water. Tubes were inoculated with 0.1 ml variations and quality control problems that have of the suspension and incubated at 30 C. continuously plagued the industry. Metabolic data were recorded at 24 and 48 h. Bergey's Manual of Determinative Bacteriology (2) was used for determining species of MATERIALS A:SD METHODS lactic bacteria isolates. Isolation of bacteria Sponge and dough samples from two commercial soda cracker plants RESULTS AND DISCUSSION were studied. Samples packed in insulated boxes, with frozen Soda cracker sponge samples had total counts of 4 to refrigerant (canned gel), were air·freighted and received for analysis within 24 h of shipment. Temperature of the samples, on arrival, varied 49 x 107 per gram (yeast counts were 1 to 15 x 107 and bacteria counts 1 to 41 x 107). Soda cracker dough 'Reference to a company or product name does not" imply approval or samples had total counts of 9 to 90 x 101 per gram (yeast recommendation of the product by the U.S. Department ofAgriculture to the exclusion of others that may be suitable. counts were 1 to 12 x 107 and bacteria counts 9 to 80 x 978 SUGIHARA

TABLE I. Microbial counts' ofsoda cracker sponge and (commercia[).

Souree

A 49 8 41 44 9 35 A 13 10 3 90 10 80 A 7 6 1 60 5 55 A 5 1 4 10 1 9 A 15 1 14 9 1 8 B 23 5 18 12 2 10 B 28 2 26 20 9 11 B 40 15 25 60 12 48 B 4 2 2 26 4 22 B 30 2 28 30 5 25 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/41/12/977/1649777/0362-028x-41_12_977.pdf by guest on 24 September 2021

counts for all figures are 1 x 107•

7 TABLE 3. Species distribution of lactic bacteria found in soda 10 ). Table 1 summarizes the microbial counts over a period of 5 months. The data show a 10-fold variation in counts. However, this spread did not have any noticeable effect on quality and production of soda crackers. The 47 16 yeast and lactic bacteria ratio and numbers were quite Lactobacillus leichmannii 13 similar to those found in our studies on San Francisco Lactobacillus brevis 5 sour dough French bread fermentation (5,10). Lactobacillus casei 4 Lactobacillus fermentum 3 A total of 183 bacterial isolates were examined during Other lactic bacteria 12 this study. They were all found to be gram-positive, catalase-negative and non-motile rods. Carbohydrate Further research will be conducted to develop reactions, shown in Table 2, when compared with stabilized concentrated cultures of L. plantarum. L. descriptive data in Bergey's Manual, indicated that the delbrueckii and L. leichmannii for possible use in pure dominant species found was Lactobacillus plantarum. culture fermentation of soda cracker sponge and dough. This was true for both of the two commercial soda cracker sponge and dough systems studied. Two other species found in most samples, in substantial numbers, REFERENCES were Lactobacillus delbrueckii and Lactobacillus leich­ mannii. Lactic bacteria found in smaller numbers were 1. Bohn, R. T., and R. J. Martz. 1926. A rapid method for the Lactobacillus brevis, Lactobacillus casei and Lacto­ colorimetric determination of hydrogen-ion concentration of bacillus fermentum. Table 3 shows the distribution of crackers. Cereal Chern. 3:183-187. bacteria found in the studies. 2. Buchanan, R. E., and N. E. Gibbons. 1974. Bergey's manual of determinative bacteriology, 8th ed. The Williams and Wilkins Co., TABLE 2. Carbohydrate reactions Baltimore. 3. Johnson, A. H .. and C. H. Bailey. 1924. A physico-chemical study of cracker dough fermentation. Cereal Chern. 1:327- 410. Carbohydrate Isolate No.9 Isolate No. 24 Isolate No. 160 4. Johnson, A. H. 1925. Identification and estimation of the organic + acids produced during bread dough and cracker dough + fermentation. Cereal Chern. 2:345-364. Cellobiose + + 5. Kline. L.. and T. F. Sugihara. 1971. Microorganisms of the San Fructose + + + Francisco sour dough bread process. II. Isolation and characteri­ Galacto>e + ± zation of undescribed bacterial species responsible for the souring Glucose (acid) + + + activity. Appl. Microbiol. 21:459-465. Glucose (gas) Lactose + + 6. Man, J. C. de, M. Rogosa, and M. E. Sharpe. 1960. A medium for Maltose + + + the cultivation oflactobacilli. J. Appl. Bacteriol. 23:130-135. Mannitol + 7. Micka, J. 1941. Soda requirements of cracker . Cereal Chern. Man nose + + 18:483-497. Melezitose + 8. Micka. J. 1948. Lactic fermentation and trace iron effect on cracker Melibiose + quality. Food Ind. 20(1):102-107, 234. Ratlinose + 9. Micka. I. 1955. Bacterial aspects of soda cracker fermentation. Rhamnose Cereal Chern. 32:125-131. Rhibose + 10. Sugihara, T. F., L. Kline. and M. W. Miller.197l. Microorganisms Salicin + + Sorbitol + of the San Francisco sour dough bread process. I. Yeasts responsible for the leavening action. Appl. Microbiol. 21:456-458. Sucrose + + + Trehalose + + 11. Sugihara. T. F .. and L. Kline. 1975. Further studies on a growth + medium for Lactobacillus sanjrancisco. J. Milk Food Techno!. + 38:667-672. 12. Triebold, H. 0 .. and C. H. Bailey. 1932. A chemical study of rancidity. I. Autoxidation of shortenings and crackers. Cereal L. delbrueckii. Chern. 9:50-64. MICRO FLORA OF CRACKER DOUGH 979

13. Triebold, H. 0., and C. H. Bailey. 1932. A chemical study of 14. Vaufel, E. A., and W. Hanks. 1944. pH variations in soda crackers rancidity. II. Factors influencing the keeping quality of shortenings and their control with diammonium phosphate. Cereal Chern. and crackers. Cereal Chern. 9:91-103. 21:16-27.

Report of the Committee on development of two APHA texts recently Subcommittee on Laboratory Methods for the Applied laboratory Methods published: Compendium of Methods for Examination of Foods the Microbiological Examination of International Association of The members of this subcommittee have Foods and Quality Assurance Practices been reviewing microbiological methods pub­ Milk. Food. and Environmental for Health Laboratories. lished in AOAC and APHA laboratory Sanitarians. 1976-1978. As of January 1, 1978, the Chairpersons for manuals to determine projects worthy of three Subcommittees of this Committee had consideration for subcommittee comparative This committee, during the past 2 years, has started on their new assignments. Mr. Charles and collaborative studies. provided in-depth assistance and consultation Huhtanen had transferred to Chairperson of Downloaded from http://meridian.allenpress.com/jfp/article-pdf/41/12/977/1649777/0362-028x-41_12_977.pdf by guest on 24 September 2021 These include: Reports of microbial growth to the Association and others in the following the Subcommittee Laboratory on Methods for on acidit1ed media tor enumeration of yeast ways: the Examination of Foods, Dr. Michael Wehr and/or mold contaminants has resulted in I. Conducted collaborative and/or compara­ accepted the position of Chairperson of the publications specifying the need of one or tive studies on new or modified methods. Subcommittee on Laboratory Methods for the more antibiotics in media to suppress which have been published in the Associa­ Examination of Milk and Milk Products, and bacterial growth. and the influence of water tion journal. Most of these studies have Mr. Ernest Shipe, Jr. became Chairperson of activity (Awl on microbial growth, suggesting provided necessary information on meth­ the Subcommittee on Methods for the the need for preenrichment media prior to ods to Chapter Chairpersons and the Examination of Water and other Environ­ examination and detection of pathogens and Intersociety Council of the American mental Samples. food spoilage microorganisms. The Chairper­ Public Health Association (APHA) in the A. Richard Brazis, Ph.D. son has authored a chapter on poten­ development of the 14th edition of Chairperson, Applied Laboratory tially pathogenic microorganisms and para­ Standard Methods for the Examination of Method Committee sites of meat which will be published in a Dairy Products (SMEDP). handbook of The American Meat Science 2. Provided consultation on abnormal milk Association in 1979. testing procedures to The National Mesti­ Subcommittee on Laboratory Methods for C. H. Huhtanen, Chairperson tis Council (NMC) and the NMC Research Examination of Milk and Milk Products Committee. Members: Harold Bengsch, J. J. Jezeski, This subcommittee has continued to be very 3. Provided consultation on microbiological Ph.D., W. S. LaGrange, Ph.D., R. T. active in the affairs of the Association. methods and abnormal milk screening Marshall, Ph.D., Don Pursch, E. L. Sing, Changes in administrative responsibilities of tests to the National Conference of Inter­ Ph.D., H. Michael Wehr, Ph.D. state Milk Shippers (NCJMS) Laboratory the two former Co-chairpersons forced their request for reassignment of a new Subcom­ and Abnormal Milk Committees. Subcommittee on Laboratory Methods for the mittee Chairperson in 1977-78; Dr. Mike 4. Provided assistance and consultation on Examination of Water and Other Environ­ Wehr of the State Department of Agriculture Abnormal Milk Confirmation Procedures mental Samples to the responsible AOAC Associate Refe­ in Oregon has accepted Chairpersonship of ree and his committee. this subcommittee provided he also does not This subcommittee has had difficulty the past two 5. Continued to stress the importance of receive new responsibilitcs. which unfor­ getting off the ground during variation of analyst data because criteria tunately for the Association appear to be years. There are critical questions which should be resolved concerning presumptive have not been developed for certification headed his way. The activities of this test media for coliforms in water supplies, of microbiological media, reagents, Subcommittee for 1977 were shown in the influence of transit time on recovery of materials. and instrumentation in all 1977 report of the Committee, which has not coliforms, on-site methods for rapid enumera­ laboratory disciplines. been published. The changes in leadership in tion of coliforms in rural water supplies. 6. Provided liaison to the Association Farm this subcommittee have resulted in no action on new studies. variability of coliform growth in different lots Methods Committee and to laboratory of brilliant green bile lactose broth, steriliza­ committees of Regional Associations of H. Michael Wehr, Ph.D., Chairperson tion of media containing carbohydrate. impor­ Food and Drug Oflicials. Offered the Members: William Arledge, Guenther Blank­ tance of magnesium sulfate in dilution water, assistance of this committee to Regional enagel, Ph.D .. Edwin H. Connell, Earl W. chemical quality of laboratory water and last, Food and Drug Otlicials Associations. Cook, Ph.D .. Vernon R. Cupps, Roy Ginn, but not least, sanitary significance of entero­ 7. Assisted in the development of Drinking Clair Gothard, James J. Jezeski, Ph.D .. Oliver Water Laboratory Certification Regula­ W. Kaufmann, Ph.D., Wesley N. Kelly, Will­ bacteriaceae in water supplies. tions of the Environmental Protection iamS. LaGrange, Ph.D., H. E. Randolph, Ph. Ernest L. Shipe, Jr., Chairperson Agency. D., James A. Rolloff, Edmond Sing. Ph.D., Members: Berry Gay, Jr., Arnold Salinger, 8. Participated as Chapter members in the Maurice Weber, Ph.D., Kenneth W. Whaley Kenneth Whaley