Agricultural and Food Science in Finland 37 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 9 (2000): 37–48. Use of inoculated lactic acid bacteria in fermenting sour cabbage Esko Petäjä, Päivi Myllyniemi, Pasi Petäjä Department of Food Technology, Meat Section, PO Box 27, FIN-00014 University of Helsinki, Finland, e-mail: esko.petaja@helsinki.fi Velimatti Ollilainen, Vieno Piironen Department of Applied Chemistry and Microbiology, Food Chemistry, PO Box 27, FIN-00014 University of Helsinki, Finland Fermentation of sour vegetables has to date occurred through the use of lactic acid bacteria (LAB) naturally present in vegetables. The present article deals with preliminary studies on the effects of some LAB inocula (Lactobacillus alimentarius or Pediococcus pentosaceus) on fermenting sour cab- bage. The effect of LAB on yeast growth, a problem in sour vegetables, was also studied through the use of dual yeast and LAB inocula. The pH of cabbage juice decreased to levels under pH 4 during the first 10 days of fermentation, which is near the final values, pediococci decreasing the pH to the lowest values. The LAB count in inoculated cabbages increased by 0.5–2.0 log cfu (colony forming unit) /g during the first 10 days of fermentation and thereafter decreased. Pediococci formed predominant part of microbial flora almost in all experimental batches. In cabbage challenged with yeasts, yeast counts rose only when the pH was < 3.5. Yeasts appeared almost regularly also in cabbages inoculated only with LAB. Pediococci fermented cabbage effectively decreasing the pH to lower levels than lactobacilli or natural LAB. However, too strong a decrease in pH may result in a decrease of LAB count which may subsequently lead to yeast growth. The yeast problem could not be solved with the LAB inocula used in our study. Key words: fermented foods, sauerkraut, vegetables Introduction Souring is a proven method for preparing vege- tables as food in many areas of the world. Acid- ification can be carried out through the use of organic acids or by fermentation, which is the © Agricultural and Food Science in Finland Manuscript received June 1999 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 9 (2000): 37–48. original method for preparing sour vegetables. Natural lactic acid bacterial (LAB) flora ferment the sugars in vegetables to lactic acid and small amounts of other organic acids. Salting vegeta- bles before fermentation and acid production by LAB aids LAB in growing over other bacteria and becoming predominant microbes of the flo- mailto:esko.petaja@helsinki.fi 38 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Petäjä, E. et al. Some lactic acid bacteria inocula in fermenting of sour cabbage ra. The fermentation temperature should be 20– 24°C, since higher temperatures often permit growth of undesirable microbes (ICMFS 1980). In the first phase of natural cabbage fermen- tation leuconostocs are the main type of LAB and are later replaced by lactobacilli as the pre- dominant microbes of the microbial flora (Fra- zier 1958, Vaughn 1985, Buckenhüskes 1997). Buckenhüskes (1993) used Leuconostoc me- senteroides as a starter inoculum in sour cabbage fermentation. During storage leuconostocs were overgrown by Lactobacillus plantarum. Also pediococci have been shown to replace leucon- ostocs at later stage of fermentation (ICMFS 1980, Vaughn 1985). Delclos (1992) reports that mixed starter culture composed of Leucon- ostoc mesenteroides and Lactobacillus pentosus gave the closest resemblance to the product obtained following a natural commercial fermen- tation. Fermentation of sour vegetables has to date occurred through the use of LAB naturally present in vegetables, whereas in many foods, especially in milk and meat products inocula- tion of specific LAB strains has been used to improve quality and reproducibility. The present paper deals with the preliminary studies in which the effects of different artificial inocula on fer- menting sour cabbage are examined. The effect of LAB on yeast growth, a problem in sour veg- etables, was also studied through the use of dual yeast and LAB inocula. Material and methods Preparation of sour cabbage The sour cabbage was prepared using 12 kg cabbage, 2 kg carrots, 0.7 kg onions, 0.2 kg salt and 0.1 kg garlic. Cabbage (the race: rinda) orig- inated from the same batch. Cabbage was sliced with a cutter (Seydelman Rasant 40; Seydelman Gmbh, Germany) into strips 5 mm in width. Carrots and onions were sliced with a vegetable cutter (Hälde RG 7, Metos, Finland) into strips 5 mm in diameter, and garlic was cut with a knife into small pieces. The raw materials were mixed and placed in 15-kg plastic pot by layers, add- ing salt after each layer. An additional 1.5 l of water with bacterial inoculum were added after the last 2 layers. The experimental batches were closed tightly, kept at 22°C for 7 days and there- after at 6–7°C for 5 months. The sour cabbage was long ripened type; the product was ready 3 months after preparation. The experimental batches were sampled by taking sour cabbage juice through the velvets near the bottom of plas- tic pot. Samples were taken immediately after preparation and after 10 and 21 days of fermen- tation and 2, 3 and 5 months of storage. Lactic acid bacteria inoculated The commercial starter Lactobacillus alimenta- rius (Flora Carn FM, Chr. Hansen A/S, Hörs- holm, Denmark) as a rapid acid producer and Pediococcus pentosaceus strain POHK (pork- kanahapankaali) as a strong acid producer were selected for use as inocula. L. alimentar- ius was isolated from a commercial preparation and P. pentosaceus strain POHK from sour car- rot strips (Petäjä and Puolanne 1997) on APT (all-purpose agar containing Tween) agar / pH 5.6 (BBL 10916). The strain POHK was identi- fied as belonging to the species Pediococcus pen- tosaceus. Both strains were stored in APT agar at 4°C. Both LAB strains were inoculated into the vegetable mixture as APT broth culture grown for 2 days at 30°C. APT broth culture (400 ml) was mixed with 1.5 l water, and this mixture added to the mixture of vegetable strips (15 kg). The APT broth culture count was about 8.7 log cfu (colony forming unit) /ml, and the aim count in the vegetable strip mixture was 7 log cfu/g. Yeasts The yeast strain used was isolated from good quality sour cabbage containing yeast as contam- 39 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 9 (2000): 37–48. inant. The strain was isolated on Rose-Bengal agar (Labm lab36 and X085) at 25°C and main- tained on Rose-Bengal agar at 4°C. The yeast strain was identified as belonging to the species Candida sake and it proved facultatively fermen- tative. The yeast strain was inoculated into the vegetable mixture as a nutrient broth (composi- tion: 5 g peptone, 3 g meat extract and 1 g glu- cose in 1 l water) culture grown for 2 days at 30°C; 10 ml nutrient broth culture was mixed with 1.5 l water which was added to the vegeta- ble mixture. The nutrient broth culture count was 6.8 log cfu/ml; the aim count in the vegetable mixture was 3 log cfu/g. Grouping of sour cabbages The purpose was to examine how L. alimentar- ius and P. pentosaceus strain POHK ferment sour cabbage and the effect of these bacteria on the growth of yeast in sour cabbage. The following groups of cabbage were prepared according to the microbial inocula: 1 Control, no inoculation (Control in tables) 2 L. alimentarius (400 ml) (Lactob. in tables) 3 L. alimentarius (200 ml) + P. pentosaceus strain POHK (200 ml) (Lactob. + Ped. in ta- bles) 4 L. alimentarius (200 ml) + C. sake yeast strain (10 ml) (Lactob. + yeast in tables) 5 P. pentosaceus strain POHK (400 ml) (Ped. in tables) 6 P. pentosaceus strain POHK (400 ml) + C. sake yeast strain (10 ml) (Ped. + yeast in ta- bles) Lactobacilli and pediococci were added as an APT broth culture, yeast as a nutrient broth cul- ture. Three experimental series of each group of sour cabbage were prepared; the microbiologi- cal results of each are presented separately be- cause in series III the pH of 4 cabbage groups (3–6) was much lower than the pH levels of the corresponding groups in the other series and because then the predominating bacterial group of different experimental series can be enclosed behind the counts. The pH value and titrated acid The pH value was measured directly from the sour cabbage juice by an Ingold 104043041 elec- trode (Ingold Messentechnik AG, Utdorf, Swit- zerland). Acid titrations were conducted with 0.1-N NaOH on the filtrates (20 ml) obtained from sour vegetable juice by filtering through filter paper (Whatman 4 Cat No 1004090, 90 mm ∅), and the results were calculated as percent- ages (wt/vol) of sour cabbage juice. The pH val- ues and titrated acid were determined from the fresh cabbage juice (0 days), and from the sour juice 10 and 21 days and 2, 3 and 5 months after preparation. Redox potential and water activity value The redox potential of the sample cabbage juices was measured using an Inlab 501 electrode (Mettler Toledo, Utdorf, Switzerland) directly from juices 10 and 21 days and 2, 3 and 5 months after preparation. The a w values (water activity values) were measured with a Luft -a w measurer (Luft, G. Luft Mess und Regelteknik Gmbh, Germany) after 10 and 21 days of fermentation. Sugars The levels of sucrose, D-glucose and D-fructose were determined using a UV method based on the measurement of the stoichiometrically formed NADPH (cat. no. 716260, Boehringer- Mannheim, Germany). The sample solution was clarified using Carrez reagent precipitation ac- cording to the instruction leaflet. UV spectro- photometric measurements were performed with a Lambda Bio UV/VIS spectrometer (Perkin- Elmer, USA). A recovery value of 94.1% (n = 3) for D-glucose was obtained. 40 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Petäjä, E. et al. Some lactic acid bacteria inocula in fermenting of sour cabbage Organic acids Acetic acid (99.8%; Riedel de Haen, nr. 33209, Germany) and DL-lactic acid (∼90%; Fluka, nr. 69785, Germany) were used as calibrants. The contents of lactic acid and acetic acid were de- termined using Waters liquid chromatography equipment including a chromatography pump, autosampler, air-bath column temperature mod- ule and UV detector. Chromatographic data were collected and processed with a Millennium Chro- matography Manager software package (Waters, USA). The organic acid fraction was purified with disposable strong anion-exchange (SAX) solid- phase extraction (SPE) cartridges prior to liquid chromatographic determination. Organic acids were separated using a reversed-phase column (Spherisorb S50DS2, 250 x 4 mm; PhaseSepa- rations, UK). The mobile phase, a 50 mmol po- tassium phosphate buffer, pH 2.4, was isocrati- cally pumped at a flow rate of 1 ml/min, and the column temperature was set at 30°C. Relative retention values (retention factor, k) for lactic acid and acetic acid were 1.3 and 1.4, respec- tively. Acetic acid and lactic acid were detected at a wavelength of 214 nm and quantitated using an external standard method. The relative standard deviation of repeated DL-lactic acid and acetic acid measurements derived from an in-house control sample (n = 5) were 11.0% and 24.8%, respectively. Peak identification was based on the retention time in the chromatogram and spik- ing of the sample extract with standard com- pound. Recovery values were calculated accord- ing to AOAC (1990). Recovery values of an add- ed standard (n = 3) were 70.6% and 72.5% for lactic acid and acetic acid, respectively. Microbiological determinations Each experimental series was examined micro- biologically after preparation (0 days) and after 4, 10 and 21 days of fermentation and 2, 3 and 5 months of storage. The following determinations were performed: total count of aerobically grow- ing bacteria (APT agar / pH 7.0, BBL10918, 4 d a y s a t 3 0 ° C ) , L A B ( A P T a g a r / p H 5 . 6 , BBL10918, 4 days at 30°C), staphylococci (Baird-Parker agar, Labm lab 85 and X085, 2 days at 37°C), pseudomonads (GSP, glutamate- starch-phenolred, agar, Kielwein 1969, 4 days at 25°C) and yeast and moulds (Rose-Bengal agar, Labm lab 36 and X009, 2–4 days at 30°C). The type of predominating LAB was con- firmed by examining microscopically three col- onies of predominating colony type/types from APT agar / pH 5.6. The predominating LAB type/ types are presented in the Table 3 behind the LAB counts. Sensory evaluations The total palatability of 1-, 2-, 4- and 5-month- old cabbage was evaluated by comparing them to a 3-month-old commercial product, using a paired test. The evaluation was performed by a panel of 5–7 persons familiar with sensory eval- uation of foodstuffs. Statistical methods The results of pH, titrated acid and sugar deter- minations were subjected to statistical tests (mul- tivariate analysis/StatGraphics win 32). Results and discussion The pH value and titrated acid The mean pH value of cabbage juice measured immediately after preparation ranged from 5.84 to 6.20 (Table 1). The pH decreased during the first 10 days of fermentation to values below pH 4, which is near the final values. This pH level is comparable to those announced for sour veg- etables in the literature (ICMSF 1980). P. pen- 41 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 9 (2000): 37–48. tosaceus strain POHK decreased the pH to 3.5 which is significantly (P<0.05) lower than fer- mentations using L. alimentarius, which did not decrease pH more than did wild LAB in control cabbage groups. This is in agreement with the fact that pediococci are active at lower pH val- ues than lactobacilli (ICMSF 1980). In experimental series III the pH values de- creased to levels under pH 3.5 in the cabbage groups containing pediococci as inoculum, which confirms the strong acid-producing capac- ity of pediococci. At low pH the yeasts grew in series III. These results are sporadical, but indi- cate that pediococci as fermentation starter LAB may lead to growth of yeasts when they are no more competitive at low pH. Frazier (1958) observed that the acid percent- ages of sour cabbages rose from 0.7% (wt/vol) to 2.0% during fermentation achieved with nat- ural LAB flora. The range of titrated acid ob- tained in experimental sour cabbages immedi- ately after preparation (0 days) was 0.4–0.9%. During the 10-day fermentation the acid content increased in inoculated cabbage groups to over 1.2%, with the highest amounts in those groups inoculated with P. pentosaceus strain POHK with or without yeast inoculum (range 1.4–1.7%). The content of titrated acid increased slowly there- Table 1. The pH value of experimental sour cabbage groups (1–6) during fermentation (0, 10 and 21 days) and storage (2, 3 and 5 months). The number of experimental series = 3. Cabbage group 0 days 10 days 21 days 2 months 3 months 5 months 1. Control x 6.18ab 3.87a 3.90a 3.97a 3.94a 3.62a s 0.10 0.07 0.05 0.06 0.05 0.20 2. Lactob. x 5.84a 3.86a 3.89a 3.97a 3.88a 3.58a s 0.10 0.07 0.05 0.06 0.05 0.20 3. Lactob. + Ped. x 6.03ab 3.62b 3.57b 3.61b 3.60b 3.32b s 0.10 0.07 0.05 0.06 0.05 0.20 4. Lactob. + yeast x 5.98ab 3.55b 3.50b 3.63b 3.56b 3.29b s 0.10 0.07 0.05 0.06 0.05 0.20 5. Ped. x 6.02ab 3.48b 3.54b 3.61b 3.57b 3.28b s 0.10 0.07 0.05 0.06 0.05 0.20 6. Ped. + yeast x 6.20b 3.51b 3.55b 3.64b 3.57b 3.18b s 0.10 0.09 0.06 0.07 0.06 0.24 x = mean s = standard deviation of mean Means within the vertical line not followed by the same small letter are significally different (P<0.05). If there are no letters after the means listed there are no differences among them. 1. Control, no inoculation 2. L. alimentarius 3. L. alimentarius + P. pentosaceus strain POHK (from sour carrot strips) 4. L. alimentarius + C. sake yeast strain 5. P. pentosaceus strain POHK 6. P. pentosaceus strain POHK + C. sake yeast strain 42 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Petäjä, E. et al. Some lactic acid bacteria inocula in fermenting of sour cabbage after with the highest amounts appearing in POHK groups, (range 2.2–2.5%) 5 months after preparation. In noninoculated cabbage groups the content of titrated acid rose, but remained near 1% during the fermentation and storage periods. The titrated acid content of noninoculated cab- bages and cabbages inoculated with L. alimen- tarius were significantly lower than cabbages with predominating pediococci but only 5 months after preparation. Organic acids The content of liquid-phase lactic acid and ace- tic acid immediately after preparation of sour cabbage batches ranged from 0.03 to 0.05 % (wt/ vol) (Fig. 1). Most acid formation occurred dur- ing the first 10-day period; the amount of lactic acid was 0.4–0.8% after the 10-day fermenta- tion period and was usually maintained at that level (Fig. 1a). Using P. pentosaceus POHK Fig. 1. Content (%; wt/vol) of lac- tic acid (a) and acetic acid (b) in experimental sour cabbage groups (1–6) during fermentation (0, 10 and 21 days) and storage (2 and 3 months). The number of experi- mental series = 3. 1 Control, no inoculation 2 L. alimentarius 3 L. alimentarius + P. pentosaceus strain POHK (from sour carrot strips) 4 L. alimentarius + C. sake yeast strain 5 P. pentosaceus strain POHK 6 P. pentosaceus strain POHK + C. sake yeast strain a) b) 43 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 9 (2000): 37–48. starter inoculation the formation of lactic acid was continued for a longer period, and the acid level attained values of 0.7–1.1% during 3 months of fermentation and storage. The lower acidity level was obtained with L. alimentarius inoculation, although lactobacilli were replaced by pediococci during fermentation. A rise in ace- tic acid content was observed during the first 10- day fermentation period, the contents increas- ing only slightly thereafter (Fig. 1b). The differ- ences in overall acetic acid contents formed by various inoculation combinations were smaller than those of lactic acid. Redox potential and a w value The redox potential of experimental sour cab- bages ranged from +50 to +250 mV after prepa- ration. The values decreased during fermenta- tion and storage, but remained distinctly posi- tive 5 months after preparation. The a w value of experimental sour cabbages ranged from 0.975 to 0.985 after 10 and 21 days of fermentation. Sugars The liquid phase of sour cabbage before ferment- ing (0 days) contained 0.6% (wt/vol) sucrose, 0.6% glucose and 0.4% fructose (Table 2). Ras- tas et al. (1989) observed the following contents of these sugars in cabbage: glucose no informa- tion, 0.1% saccharose and 0.4% fructose. Frazi- er (1958) reported higher sugar contents for cab- bage, ranging from 2.9% to 6.4%. During the 10-day fermentation period the content of su- crose rose to 1–2%, decreasing thereafter to counts that were mostly under 0.9% 2 months after preparation. In our study differences (P<0.05) among cabbage groups were not ob- served. Glucose contents rose during fermentation due to the splitting of sucrose; the highest val- ues (about 1%) were obtained after 21 days of fermentation (Table 2). During storage the glu- cose contents did not decrease noticeably. The differences (P<0.05) among cabbage groups were not observed. The fructose contents rose to their highest levels (1–2%) during 10 days of fermentation, but decreased during the remaining period of fermentation and storage (Table 2). The differ- ences (P<0.05) among cabbage groups were not observed. The contents of all 3 sugars increased during fermentation, due to gradual removal of the sug- ars from the cabbage to the liquid phase. Accord- ing to Buckenhüskes (1993) sugars should be removed during fermentation to prevent yeast growth in sour cabbage. Lactic acid bacteria The LAB count corresponded to the total count of bacteria in all samples. The LAB counts ranged from 6.2 to 7.4 log cfu/g after inocula- tion, increasing by 0.0–1.9 log cfu/g during the first 10 days of fermentation (Table 3). During this time the pH decreased to levels under pH 4. During the next 11 days the LAB counts de- creased by 0.2–3.8 log cfu/g in 17 batches out of 18; this decrease continued mostly during the following 4 months. The counts varied from <2 to 6.5 log cfu/g in experimental products 5 months after preparation, suggesting that LAB may disappear completely from the product dur- ing storage. Lactobacilli and pediococci formed the pre- dominant part of the microbial flora in sour cab- bages prepared without bacterial inocula or with L. alimentarius inocula (Table 3). The predomi- nant type varied among experimental series. When L. alimentarius was used with P. pen- tosaceus strain POHK or C. sake yeast as inocu- la pediococci already displaced lactobacilli dur- ing the first 10 days of fermentation, which agrees with earlier information on the develop- ment of bacterial flora in fermented vegetables 44 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Petäjä, E. et al. Some lactic acid bacteria inocula in fermenting of sour cabbage (ICMSF 1980). When P. pentosaceus POHK alone was used as an inoculum, it remained the predominant form of LAB. In experimental series III, yeasts appeared as the predominant microbes after 2 months of fer- mentation in all inoculated experimental groups, which can be explained by the low pH values (<3.5) encountered in this series. This would indicate that at low pH even pediococci do not survive and that yeasts begin to dominate the microbial flora. Yeasts In the sour cabbage groups inoculated with C. sake yeast strain, the yeast counts did not rise during fermentation and storage (Table 4). On the other hand, in cabbage groups prepared with- out yeast inocula, yeasts appeared almost regu- larly. In experimental series III, yeasts formed the predominant microbial group in inoculated cabbages 2 months after preparation. On the ba- sis of these observations, it can be concluded that Table 2. Content (%) of sucrose, glucose and fructose in experimental sour cabbage groups (1–6) during fermentation lasting 10 and 21 days (d) and storage lasting 2 and 5 months (m). Contents (%) at the beginning of fermentation (0 days) were as follows: sucrose 0.6, glucose 0.6 and fructose 0.4. The number of experimental series = 3. Sucrose Glucose Fructose Cabbage group 10 d 21 d 2 m 5 m 10 d 21 d 2 m 5 m 10 d 21 d 2 m 5 m 1. Control x 1.6 1.0 0.2 0.8 1.1 1.1 0.4 0.8 1.9 1.1 0.4 0.8 s 0.1 0.7 0.0 0.1 0.4 0.2 0.4 0.6 0.1 0.8 0.4 0.9 2. Lactob. x 0.9 0.9 0.3 0.1 0.9 1.1 0.7 0.9 1.9 1.3 0.4 0.5 s 0.4 0.2 0.0 0.1 1.0 0.3 0.1 0.7 0.1 0.4 0.2 0.4 3. Lactob. + Ped. x 1.3 0.9 0.9* 0.1 1.0 0.9 0.6* 0.7 1.6 1.6 0.6* 1.0 s 0.4 0.2 0.1 0.3 0.4 0.8 0.6 0.5 0.7 4. Lactob. + yeast x 1.4 0.9 0.1 0.2 1.0 0.8 0.7 1.1 1.6 1.4 0.4 0.9 s 0.2 0.3 0.0 0.1 0.1 0.6 0.9 0.1 0.6 0.2 0.4 0.5 5. Ped. x 1.3 0.6 0.5 0.4 0.9 1.6 0.9 0.9 1.6 0.6 0.7 0.8 s 0.6 0.2 0.1 0.3 0.5 0.8 0.6 0.6 0.6 0.7 0.1 0.7 6. Ped. + yeast x 1.8 1.9* 0.6 1.4 1.9* 1.0 1.4 0.8* 1.1 s 0.8 0.5 0.2 0.7 0.7 0.1 * only one value x = mean s = standard deviation of mean Means within the vertical line not followed by the same small letter are significally different (P< 0.05). If there are no letters after the means listed there are no differences among them. 1. Control, no inoculation 2. L. alimentarius 3. L. alimentarius + P. pentosaceus strain POHK (from sour carrot strips) 4. L. alimentarius + C. sake yeast strain 5. P. pentosaceus strain POHK 6. P. pentosaceus strain POHK + C. sake yeast strain 45 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 9 (2000): 37–48. yeasts may appear in sour vegetables at least when ripened and stored 2 months or more. They may even form the predominant part of the mi- crobial flora. Buckenhüskes (1993) suggested that all sugars should be removed during fermen- Table 3. Lactic acid bacterial counts (log cfu/g) of experimental sour cabbage groups (1–6) on APT agar (pH 5.6) during fermentation (0, 10 and 21 days) and storage (2, 3 and 5 months). The counts of different experimental series (I–III) are presented separately. The pH values after 10 days of fermentation are also presented. Cabbage 0 days 10 days pH 10 21 days 2 months 3 months 5 months group days 1. Control I 7.9 s 3.86 6.3 s 6.2 l 5.2 l, p 4.3 l II 8.6 l, (p) 3.80 8.2 p 6.5 p, (l) 6.7 l, (p) 6.8 p, (l) III 5.2 l (p) 5.6 l 3.96 5.1 p, (l) 4.6 p 5.1 p 6.5 p 2. Lactob. I 7.3 l 8.4 p, (l) 3.74 5.8 l 5.5 l 4.4 l, (p) 4.6 p II 6.7 l 8.6 p, (l) 3.90 6.4 p 8.2 p, (y) 7.8 p, (y) 7.4 p III 6.4 l < 5 3.92 4.1 l 6.3 l, (y) 3.0 y 7.5 y 3. Lactob. + Ped. I 6.5 p, 7.0 l 8.0 p 3.65 7.8 p 6.9 p 4.0 p < 2 II 6.8 p, 6.9 l 7.9 p 3.74 7.3 p 5.4 p 3.8 p 6.5 p III 7.5 l 7.6 p 3.46 4.6 y 7.3 y 6.6 y 6.9 y 4. Lactob. + yeast I 7.0 l 7.3 p 3.46 6.1 p 5.8 p 3.9 p 4.0 p, (y) II 6.4 l 6.9 p 3.76 7.4 p 5.9 p 4.3 3.6 p, (y) III 7.4 l 7.3 p 3.44 5.5 p, (y) 5.5 y 6.0 y 6.6 y 5. Ped. I 6.8 p 8.5 p, (l) 3.39 6.4 p, (l) 4.0 p 5.9 p 2.3 l II 7.2 p 8.4 p, (l) 3.57 6.2 p 6.5 p, (y) 6.5 p, (y) 4.5 y, (p) III 6.2 p 7.6 p 3.47 4.0 p 2.3 p, (y) 2.5 l 4.6 y 6. Ped. + yeast I II 7.2 p 7.5 p 3.53 6.8 p 4.5 p, (y) 4.5 p, (y) < 2 III 6.2 p 8.0 p 3.50 4.2 6.3 5.0 y 6.7 y s = streptococci p = pediococci l = lactobacilli y = yeast ( ) = secondary microbial group 1. Control, no inoculation 2. L. alimentarius 3. L. alimentarius + P. pentosaceus strain POHK (from sour carrot strips) 4. L. alimentarius + C. sake yeast strain 5. P. pentosaceus strain POHK 6. P. pentosaceus strain POHK + C. sake yeast strain tation to prevent yeast growth; however, those amounts of sugars present in the sour cabbages after fermentation in this investigation did not appear to enhance the growth of yeasts in the present study. 46 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Petäjä, E. et al. Some lactic acid bacteria inocula in fermenting of sour cabbage Table 4. Yeast counts (log cfu/ g) of experimental sour cabbage groups (1–6) on Rose-Bengal agar during fermentation (0, 10 and 21 days) and storage (2, 3 and 5 months). The counts of different experimental series (I–III) are presented separately. The pH values after 10 days of fermentation are also presented. Cabbage 0 days 10 days pH 10 21 days 2 months 3 months 5 months group days 1. Control I 3.2 3.86 < 2 < 2 < 2 2.6 II < 2 < 2 3.80 2.0 < 2 < 2 < 2 III 3.0 3.6 3.96 < 2 < 2 < 2 3.7 2. Lactob. I 5.3 3.74 < 2 < 2 < 2 < 2 II 2.3 4.3 3.90 5.2 5.6 5.0 5.7 III 2.5 3.7 3.92 2.8 < 2 < 2 7.0 3. Lactob. + Ped. I 3.7 3.2 3.65 < 2 < 2 < 2 < 2 II < 2 < 2 3.74 < 2 2.3 < 2 < 2 III 4.3 3.6 3.46 < 2 6.0 5.9 6.8 4. Lactob. + yeast I 3.7 2.8 3.3 < 2 < 2 3.4 II 3.7 2.0 3.76 3.0 2.3 < 2 2.9 III 4.0 3.9 3.44 3.6 5.5 4.7 4.5 5. Ped. I 3.5 5.6 3.39 < 2 < 2 < 2 < 2 II 5.2 3.57 4.8 3.9 5.0 6.4 III 3.9 3.8 3.47 < 2 < 2 2.0 4.5 6. Ped. + yeast I II 3.7 < 2 3.53 < 2 2.0 < 2 < 2 III 4.2 4.0 3.50 4.2 6.0 4.6 6.3 1. Control, no inoculation 2. L. alimentarius 3. L. alimentarius + P. pentosaceus strain POHK (from sour carrot strips) 4. L. alimentarius + C. sake yeast strain 5. P. pentosaceus strain POHK 6. P. pentosaceus strain POHK + C. sake yeast strain Staphylococci and pseudomonads The staphylococcal and pseudomonad counts decreased to <2 log cfu/g during the first 10 days of fermentation. Sensory evaluation Commercial sour cabbage (3 months old) proved significantly better than 1-, 2- and 4-month-old experimental cabbages when the paired test fig- ures of different cabbage groups were summed and the sums were compared using paired tests (Table 5). Five-month-old cabbages were not significantly worse than the commercial prod- uct when compared in the manner described. The results can be summarized as follows The counts of inoculated LAB were on the level of 7.0 log cfu/g increasing by 0.0–1.9 log cfu/g during the first 10 days of fermentation. The pH 47 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Vol. 9 (2000): 37–48. decreased during that time to levels under 4, which is near the final values. Pediococci formed predominant part of microbial flora almost in all experimental batches. Pediococci fermented cab- bage effectively, decreasing the pH to lower lev- els than lactobacilli or natural LAB flora of con- trol group. The counts of LAB, also pediococci, decreased after 10 days of fermentation. The low pH and decreasing of LAB counts enhanced the growth of yeasts in some cases. Table 5. Sensory evaluation of total palatability of experimental sour cabbages by comparing cabbages of different ages to reference cabbage (3-month-old commercial product). The figures are the numbers of better evaluations of paired comparisons. Cabbage group 1 m – 3 m 2 m – 3 m 4 m – 3 m 5 m – 3 m 1. Control 0 7 2 5 1 4 3 2 2. Lactob. 1 6 2 5 1 4 0 5 3. Lactob. + Ped. 2 5 2 5 1 4 1 4 4. Lactob. + yeast 2 5 2 5 1 4 2 3 5. Ped. 1 6 2 5 1 4 2 3 6. Ped. + yeast 1 6 1 6 1 4 3 2 Total 7 35 * 11 31 * 6 24 * 11 19 * significantly better in paired comparison m = months 1. Control, no inoculation 2. L. alimentarius 3. L. alimentarius + P. pentosaceus strain POHK (from sour carrot strips) 4. L. alimentarius + C. sake yeast strain 5. P. pentosaceus strain POHK 6. P. pentosaceus strain POHK + C. sake yeast strain References AOAC 1990. Official Methods of Analysis. 15th ed. As- sociation of Official Analytical Chemists, Inc., Virgin- ia, USA. Buckenhüskes, H.J. 1993. Selection criteria for lactic acid bacteria to be used as starters for various food com- modities. In: FEMS. Microbiology Reviews 12. Else- vier. p. 253–271. – 1997. Fermented vegetables. In: Doyle, M.P. et al. (eds.). Food Microbiology, Fundamentals and Fron- tiers. ASM Press. Washington D.C. p. 595–610. Delclos, M. 1992. Vegetable preservation by a mixed acid fermentation. Dissertation thesis. Univ. Surrey. UK. Frazier, W.C. 1958. Food Microbiology. McGraw-Hill Book Company, Inc. New York. p. 166. ICMSF 1980. Microbial Ecology of Foods. vol. 2. Aca- demic Press. London. p. 521. Kielwein, G. 1969. Ein Nährböden zur selektiven Züch- tung von Pseudomonaden und Aeromonaden. Archiv für Lebensmittelhygienie 20: 131. Petäjä, E. & Puolanne, E. 1997. Use of two Pediococcus strains isolated from sour vegetables as starters in dry sausage. Proceedings of the 43rd International Congress of Meat Science and Technology. Auckland, New Zeeland. p. 448–449. Rastas, M., Seppänen, R., Knuts, L.-R., Karvetti, R.-L. & Varo, P. (eds.). 1989. Nutrient Composition of Foods. Publications of the Social Insurance Institution, Fin- land. Helsinki. 1989. p. 104. Vaughn, R.H. 1985. The microbiology of vegetable fer- mentations. In: Wood, B.J.B. (ed.). Microbiology of Fermented Foods. Vol. 1. Elsevier Applied Science Publishers. London. p. 49–108. 48 A G R I C U L T U R A L A N D F O O D S C I E N C E I N F I N L A N D Petäjä, E. et al. Some lactic acid bacteria inocula in fermenting of sour cabbage SELOSTUS Maitohappobakteerien hyödyntäminen hapankaalin fermentoinnissa Esko Petäjä, Päivi Myllyniemi, Pasi Petäjä, Velimatti Ollilainen ja Vieno Piironen Helsingin yliopisto Hapanvihannesten fermentointi on tapahtunut perin- teisesti vihannesten omien maitohappobakteerien (MHB) avulla. Monissa muissa hapatetuissa elintar- vikkeissa fermentointi tapahtuu valmisteeseen lisät- tyjen MHB:n avulla. Tämä artikkeli käsittelee tutki- musta, jossa selvitettiin hapankaalin fermentointia tiettyjen MHB-siirrostuksien (Lactobacillus alimen- tarius ja Pediococcus pentosaceus) avulla. Koska hii- vojen esiintyminen hapanvihanneksissa on ollut on- gelma, tutkittiin myös MHB-siirrostuksien vaikutus- ta hiivojen kasvun estäjänä. Kaalimehun pH laski kymmenen ensimmäisen fermentointipäivän aikana alle neljän, eli lähes lopul- liselle pH-tasolle. Inokuloidut pediokokit laskivat pH-arvoa eniten. Myös korkeimmat titratun hapon pitoisuudet, 2,2–2,5 % (paino/tilavuus) viisi kuukaut- ta valmistuksen jälkeen, esiintyivät pediokokeilla in- okuloiduissa kaaleissa. Sokerien määrä lisääntyi kaa- limehussa fermentoinnin aikana johtuen niiden asteit- taisesta siirtymisestä kaalisolukosta nesteosaan. In- okuloitujen MHB:n lukumäärä kaalimehussa kasvoi 0,0–1,9 log pmy (pesäkkeen muodostava yksikkö)/g kymmenen ensimmäisen fermentointivuorokauden aikana laskien sen jälkeen. Pediokokit olivat valta- organismeja lähes kaikissa kaalierissä. Hiivoilla in- okuloitujen kaalien hiivapitoisuus nousi vain kun pH- arvo oli alle 3,5. Hiivoja esiintyi usein myös kaali- erissä, joita ei oltu inokuloitu hiivoilla. Tutkimus osoitti, että hapankaalia voidaan val- mistaa fermentoimalla kaali valituilla maitohappo- bakteereilla. Pediokokit fermentoivat kaalia tehok- kaasti laskien pH-arvon alemmaksi kuin laktobasil- lit tai kontrolliryhmän MHB-floora. Seurauksena voi kuitenkin olla liian voimakas haponmuodostus ja pH- arvon lasku, jotka johtavat maitohappobakteerimää- rän vähenemiseen. Tämä edistää hiivojen kasvua. Hiivojen esiintymisongelmaa ei siten voitu ratkaista pelkällä maitohappobakteerilisäyksellä. Title Introduction Material and methods Results and discussion References SELOSTUS