Fermentation of Philippine Vegetable Blends1

C. A. ORILLO, E. C. SISON, M. LUIS,2 AND C. S. PEDERSONI College ofAgriculture, University ofthe Philippines College, Laguna, Philippines Received for publication 10 October 1968

Seven blends of Philippine vegetables, two of which contained soybeans and one mongo bean sprouts, were prepared for fermentation and study of microbiological and chemical changes. The fermentations were typical lactic acid bacterial fermentations, initiated by Leuconostoc mesenteroides and continued by Lactobacillus brevis, Pediococcus cerevisiae, and L. plantarum. The combination of high acidity and low pH resembled other vegetable fermentations, such as sauerkraut. The procedure offers a method of preserving surplus vegetables, and, in addition, a method for in- corporating and preserving the high-protein-containing soybeans.

Preservation of vegetables by fermentation major vegetable blends that could be made in the probably originated in Asia with methods that Far East. have been in use for centuries. Yen tsai, paw tsay, MATERIALS AND METHODS kimchi, sajur asin, mostasa, and nukamiso pickles The vegetables used were purchased on the open are among the better known preserved foods of market. The vegetables of higher sugar content, such the Far East. In recent years, the Filipinos have as cabbage and singkamas, were included inthe blends. become increasingly aware of the value of vege- The edible roots of singkamas, a yam bean grown in tables to their diets. Although recently cucumber the Philippines, resemble small white turnips in ap- pickling has become an important industry, the pearance, but are white, crisp, and sweet. The blends not contained the percentages of trimmed vegetables Filipinos have utilized fermentation as a given in Table 1. The vegetables were washed, peeled method of preserving other vegetables. They are when necessary, cleaned, sliced or shredded, and then accustomed to eating sweet-sour foods, such as mixed with 1.75 to 2.25% salt. The mixtures were "Atchara," a vegetable blend sweetened with packed into jars of 1 to 3-gal-capacity. A bag-like sugar and acidified with vinegar. The increase in plastic cover was placed on the surface of the vege- vegetable production, particularly in the moun- table, and sufficient water was placed in the bag to tain province, has resulted in seasonal overpro- contact the entire surface and to bring the vegetable duction which could be alleviated by preserving brine to a height level with the surface. vegetables by fermentation for later use. During fermentation, samples of brine were re- Palo and Lapuz isolated moved at regular intervals for determination of pH, (2) strains of bacteria total acidity, and salt content. With four blends, from fermenting mustard leaves, "mostasa,) samples ofbrine were plated for estimation ofnumbers which were presumably strains of Leuconostoc ofbacteria, isolation ofcolonies, and study ofthe flora. mesenteroides. Kim and Whang (1) reported that Plates were incubated for 2 days at room temperature, this species played an important role in the fer- which usually approximated 30 C. Colonies were mentation of kimchi. In view of the importance of counted, and usually 25 colonies were isolated from L. mesenteroides, Lactobacillus plantarum, and each plating. Later the cultures isolated were identified other species in the production of sauerkraut of by the method of Pederson and Albury (3). Repre- superior quality (4) and the necessity for establish- sentative cultures were commonly studied further to ing optimal environmental conditions for growth assure the validity of identification. of these species, it seemed desirable to determine RESULTS AND DISCUSSION whether they also played a major role in all of the With the uniform and comparatively high tem- I Approved by the Director of the New York State Agricul- peratures of the Philippines, fermentations were tural Experiment Station for publication as Journal Paper initiated rapidly and continued at a rapid rate. No. 1630. Acidities (as lactic) were attained as follows: no. 2 College of Home Economics, University of the Philippines, 1, 1.34% in 7 days; no. II, 1.72% in 11 days; no. Diliman. ' Visiting Professor, 1 July, 1965 to 31 August, 1967 from New III, 1.60% in 8 days; no. IV, 1.75% with a pH York State Agricultural Experiment Station, Cornell University, of 3.70 in 12 days; no. V, 1.83% with a pH of Geneva, N. Y. 14456. 3.78 in 7 days; no. VI, 1.75% with a pH of 3.70 10 VOL. 17, 1969 FERMENTATION OF PHILIPPINE VEGETABLE BLENDS 11 TABLE 1. Approximate percentages of vegetables used in each blend

Asparau Mustard Soybean Mongo Blend no. Cabbage Singkamas Radishes Carrot Pepper beans green cooked banu

I 48 25 25 II 36 19 24 5 8 6 III 32 31 35 IV 36 27 10 15 10 V 32 21 10 4 31 VI 39 21 11 9 18 VII 38 20 11 9 20

TABLE 2. Microbiological and chemical changes during the fermentation of vegetable blend with soybean, blend no. V Estimated no. of each species of bacteria X 106 Total Total-- - -_ __ Time Temp Salt acid pH count Lacto,- PeIc Laco- X 106 Aerobic Leuconostoc bacillus occus bacillus Sp mcsenteroides brevis cercisca~e plaxbarum hr C % 3 30 0 5.90 7 6.3 0.7 10 2.77 0.17 5.10 470 470 21 30 2.23 0.49 4.64 3,700 3,700 27 32 2.34 0.65 4.50 2,700 2,600 100 34 33 2.25 0.77 4.30 1,200 864 46 144 144 45 31 2.49 0.99 4.15 1,630 68 204 340 1,020 51 31 2.23 1.14 4.09 2,100 84 336 336 1,343 60 2.40 1.24 3.94 1,540 124 62 1,354 68 2.55 1.32 3.94 1,660 330 1,330 76 31 2.23 1.41 3.90 2,500 900 1,600 94 31 2.27 1.50 3.86 1,320 50 1,270 119 30 2.30 1.51 3.86 1,340 60 1,280 143 30 2.34 1.63 3.75 570 250 320 165 30 2.23 1.83 3.78 64 6 58 213 2.30 1.85 3.80 171 64 107 285 29 2.34 1.86 3.78 90 35 55 in 5 days; and no. VII, 1.74% with a pH of 353. to reduce the acidity to about 1.2%. They in 7 days. were then heated to 165 F (74 C), packed into Blends no. I and III resembled sauerkraut in heated glass jars at this temperature, sealed, and both appearance and flavor. The varying shades cooled. of green of mustard leaves, beans, and peppers, Microbiological and chemical changes. The combined with the white of the other vegetables, microbiological and chemical changes that oc- cabbage, singkamas, and radish, in blend no. II curred during fermentation were quite similar to presented the least attractive appearance of all those that occur in the sauerkraut fermentation blends and also had the least appealing flavor. (3). The detailed microbiological and chemical In contrast, blend no. IV was most attractive in changes that occurred in blend no. V (Table 2) color and flavor. The red and green colors of the show the rapid rise in acidity with a corresponding peppers and with the carrot-red hue of the carrots reduction of pH. L. mesenteroides was entirely blended well with the white vegetables. These responsible for the early stages of this fermenta- vegetables were included also in blends V, VI, tion as it was in the other blends studied. A maxi- and VII. Although the soybeans in blends V and mal count of 3,700 X 106 was attained in 21 hr. VI were very noticeable, the mongo bean sprouts The count dropped to 2,700 X 106 during the in blend VII were barely discernible. next 6 hr, and a marked change in flora occurred After fermentation was completed, the blends during the next 7 hr, when an acidity of 0.77% were diluted with one-half their volume of water was attained. This change in flora occurred at a 12 ORILLO ET AL. APPL. MICROBIOL.

TABLE 3. Fermentation of vegetable blend containing soybean, blend no. VI Estimated no. of each species of bacteria X 106 Time pH Total acid Salt Total plate - ______-actobacillu_ count X 106 Leuconosloc Lactobacillus Laclobacillus- mesenteroides brevis plantarum hr 0 1.69 0.135 9 0.1 2.49 7.20 7.2 21 0.59 2.09 1,410 1,410 27 4.50 0.76 1.90 900 900 34 4.30 0.80 2.02 800 544 96 160 45 4.10 0.98 1.90 610 80 50 48& 52 3.92 1.13 1.90 1,020 43 43 934 58 3.85 1.18 1.84 700 28 672 68 3.79 1.44 1.90 1,000 1,000 73 3.78 1.70 1.90 82 3.72 1.75 1.90 260 260 97 3.69 1.75 1.90 470 470 116 3.70 1.75 1.90 224 224 164 3.73 1.78 1.90 121 121 212 3.75 1.80 1.90 15 15 lower acidity than that which occurs in sauerkraut table was not attained rapidly, and the soybean fermentation. Lactobacillus plantarum became the protein apparently remained within the bean. predominant florum. L. brevis and Pediococcus Therefore, the buffering effect of the fermenting cereviseae played minor roles; the acid-tolerant brine was not affected greatly. A pH below 4.00- L. brevis persisted in spite of the high acidity was attained at an acidity level of 1.0 to 1.2%. attained. The blend of carrots and red peppers contrasted In one of the vegetable fermentations without well to the white cabbages, singkamas, and soybeans, e.g., blend no. IV, and the second radishes, providing an attractive and acceptable fermentation with soybeans, blend no. VI (Table appearance. In general, the Americans who were 3), P. cereviseae was never isolated at any stage; accustomed to eating sauerkraut found the flavor Lactobacillus brevis played a less important role of the blends acceptable. The processed blends than in blend V. In blend no. IV, strains of strep- were too sour for the majority of the Filipinos; tococci and micrococci were isolated from the however, when they were sweetened with 10 to second plating when the acidity was low, e.g., 15% sugar or blended with crushed pineapple, 0.26%, but they were entirely absent thereafter. the products were relished by them. They com- The soybeans were added to vegetable blends pared them to their "Atchara." The soybeans no. V and IV to increase the protein content. acquired a somewhat chewy, peanut-like flavor. Protein analysis of the heat-processed sample of There may be a chemical change responsible for blend no. IV yielded 8.80% protein (N X 6.25) development of this character. The mongo beans calculated on the dry basis. The soybean sample, in blend VII were barely discernible. blend no. V, yielded 34.44% protein on the dry Technically, the low oxidation-reduction po- basis. Although this may seem abnormally high tential attained in lactic acid fermentation systems when only 32% by weight of soybean had been is effectual in retaining the light color and bright added, soybeans even after cooking contain a appearance of the vegetables. Low pH or high high percentage of total solids, whereas the other acidity delays enzyme action and inhibits growth vegetables are low in total solids. Blend no. VI, in of other microorganisms. which only 18% soybeans were added, contained Preservation of mixed vegetables, such as those 23.0% protein. seasonally available, can be readily adapted to Because of the high-protein content of blend V, small-scale operations using almost any blend of it was assumed that the buffer content of the vegetables as is done in other Asian countries. brine may have increased to such an extent that After fermentation, the vegetable blends may insufficient acid would be produced to lower the be preserved by heating the blend to 165 F (74 C), pH for effective preservation. Actually equilib- filling into containers, and hermetically sealing rium between the soybean and the other vege- the containers at a temperature of 165 F. VOL. 17, 1969 FERMENTATION OF PHILIPPINE VEGETABLE BLENDS 13

LITERATURE CITED the synthesis of the gum and its production in coconut water. Philippine J. Sci. 83:327-357. 1. Kim, Ho-Sik, and Kyn-Chan Whang. 1959. Microbiological 3. Pederson, C. S., and M. N. Albury. 1954. The influence of salt and temperature on the microflora of sauerkraut fermenta- studies on kimches. Part I. Isolation and identification of tion. Food Technol. 8(1):1-5. anaerobic bacteria. Bull. Sci. Res. Inst. (Seoul, Korea) 4:56. 4. Pederson, C. S., and M. N. Albury. 1969. The sauerkraut fer- 2. Palo, M. A., and M. M. Lapuz, 1955. On a new gum-forming mentation. N.Y. State Agr. Expt. Sta. (Geneva, N.Y.) streptococcus, with studies on the optimal conditions for Bull. 824.