Bioresource Technology 97 (2006) 1427–1430

Production of probiotic cabbage juice by lactic acid bacteria

Kyung Young Yoon, Edward E. Woodams, Yong D. Hang *

Department of Food Science and Technology, Cornell University, Geneva, NY 14456, United States

Received 29 March 2004; received in revised form 14 June 2005; accepted 30 June 2005Available online 24 August 2005

Abstract

Research was undertaken to determine the suitability of cabbage as a raw material for production of probiotic cabbage juice bylactic acid bacteria (Lactobacillus plantarum C3, Lactobacillus casei A4, and Lactobacillus delbrueckii D7). Cabbage juice was inoc-ulated with a 24-h-old lactic culture and incubated at 30 �C. Changes in pH, acidity, sugar content, and viable cell counts duringfermentation under controlled conditions were monitored. L. casei, L. delbrueckii, and L. plantarum grew well on cabbage juiceand reached nearly 10 · 108 CFU/mL after 48 h of fermentation at 30 �C. L. casei, however, produced a smaller amount of titratableacidity expressed as lactic acid than L. delbrueckii or L. plantarum. After 4 weeks of cold storage at 4 �C, the viable cell counts of L.

plantarum and L. delbrueckii were still 4.1 · 107 and 4.5 · 105 mL�1, respectively. L. casei did not survive the low pH and high acid-ity conditions in fermented cabbage juice and lost cell viability completely after 2 weeks of cold storage at 4 �C. Fermented cabbagejuice could serve as a healthy beverage for vegetarians and lactose-allergic consumers.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Cabbage juice; Probiotic; Lactic acid bacteria; Lactobacillus casei; Lactobacillus delbrueckii; Lactobacillus plantarum

1. Introduction

Probiotics are defined as live microbial feed supple-ment that beneficially affects the host by improving itsintestinal balance (Fuller, 1989). Most probiotic microor-ganisms are lactic acid bacteria such as Lactobacillus

plantarum, Lactobacillus casei, Lactobacillus acidophilus,and Streptococcus lactis (Sindhu and Khetarpaul, 2001).Research has shown that addition of probiotics to foodprovides several health benefits including reduction inthe level of serum cholesterol, improved gastrointestinalfunction, enhanced immune system, and lower risk of co-lon cancer (Berner and O�Donnell, 1998; Rafter, 2003;Saarela et al., 2002; McNaught and MacFie, 2001). Lac-tic acid bacteria are commercially used as starter culturesfor the manufacture of dairy-based probiotic foods

0960-8524/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2005.06.018

* Corresponding author. Tel.: +1 315 787 2265; fax: +1 325 7872284.

E-mail address: ydh1@cornell.edu (Y.D. Hang).

(Heenan et al., 2002). Traditionally, probiotics have beenadded to yogurt and other fermented dairy products, butlactose intolerance and the cholesterol content are twodrawbacks related to their consumption. In recent years,consumer demand for non-dairy-based probiotic prod-ucts has increased, and probiotics have been incorpo-rated into drinks as well as marketed as supplements inthe form of tablets, capsules, and freeze–dried prepara-tions (Shah, 2001). Fruits and vegetables are rich in func-tional food components such as minerals, vitamins,dietary fibers, and antioxidants (phytochemicals). Fur-thermore, fruits and vegetables do not contain any dairyallergens that might prevent usage by certain segments ofthe population (Luckow and Delahunty, 2004).

Cabbage is a cruciferous vegetable, which is rich inminerals, vitamin C, dietary fibers, and especially phyto-chemicals (Chu et al., 2002). The objective of this studywas to determine the suitability of cabbage as a rawmaterial for production of probiotic cabbage juice byprobiotic lactic acid bacteria.

Table 1Time course of lactic fermentation of cabbage juice by Lactobacillus

casei

Time(h)

pH Acidity(% lactic acid)

Sugar(mg/mL)

CFU/mL

0 5.0 ± 0.1a 0.11 ± 0.01d 45.6 ± 2.5a 3.0 ± 0.2 · 106a

24 3.7 ± 0.0b 0.38 ± 0.01c 41.7 ± 1.4b 6.3 ± 0.0 · 108b

48 3.4 ± 0.0c 0.6 ± 0.03b 39.5 ± 1.8bc 12 ± 0.0 · 108c

72 3.4 ± 0.1c 0.74 ± 0.02a 36.5 ± 1.9c 11 ± 0.1 · 108c

Means and standard deviations for n = 3. The experimental valueswithin rows that have no common superscript are significantly different(p < 0.05) according to Duncan�s multiple test range.

Table 2Time course of lactic fermentation of cabbage juice by Lactobacillus

plantarum

Time(h)

pH Acidity(% lactic acid)

Sugar(mg/mL)

CFU/mL

0 5.8 ± 0.0a 0.12 ± 0.0a 35.08 ± 0.09a 8.0 ± 6.26 · 105a

24 4.8 ± 0.2b 0.23 ± 0.06b 37.10 ± 0.39a 7.7 ± 3.41 · 108b

48 3.6 ± 0.0c 0.76 ± 0.03c 36.44 ± 3.77a 15.3 ± 0.92 · 108c

72 3.6 ± 0.0c 0.97 ± 0.03d 19.33 ± 1.04b 17.5 ± 7.05 · 108c

Means and standard deviations for n = 3. The experimental valueswithin rows that have no common superscript are significantly different(p < 0.05) according to Duncan�s multiple test range.

1428 K.Y. Yoon et al. / Bioresource Technology 97 (2006) 1427–1430

2. Methods

Cabbage (Brassica oleracea L. var. capitata L.) waspurchased from a local store and kept at 4 �C prior touse. Cabbage juice was obtained with a Loomis pressoperated at 2000 psi and sterilized for 15 min at 121 �C.

2.1. Probiotic lactic acid cultures

Lactobacillus casei A4, Lactobacillus debrueckii D7,and Lactobacillus plantarum C3 were obtained fromthe New York State Agricultural Experiment StationCulture Collection, Geneva, New York. The cultureswere grown at 30 �C for 24 h in MRS broth (dextrose20.0 g/L; meat peptone 10.0 g/L; beef extract 10.0 g/L;yeast extract 5.0 g/L; sodium acetate 5.0 g/L; disodiumphosphate 2.0 g/L; ammonium citrate 2.0 g/L; tween80 1.0 g/L; magnesium sulfate 0.1 g/L, manganese sul-fate 0.05 g/L).

2.2. Fermentation of probiotic cabbage juice

Fermentation experiments were conducted in testtubes (25 · 200 mm), each containing 40 mL of sterilecabbage juice. All samples were inoculated with a 24-hculture (>105 CFU/mL) and incubated at 30 �C for72 h. Samples were taken at 0, 24, 48, and 72 h for chem-ical and microbiological analyses.

2.3. Effect of cold storage on cell viability in probioticcabbage juice

After 72 h of fermentation at 30 �C, the fermentedsamples were stored at 4 �C for 4 weeks. Samples weretaken at weekly intervals, and the viability of probioticcultures in probiotic cabbage juice was determined andexpressed as colony forming units (CFU/mL).

2.4. Chemical and microbiological analyses

The pH of probiotic cabbage juice was measuredwith a pH meter. Total acidity, expressed as percent lac-tic acid, was determined by titrating with 0.02 N NaOHto pH 8.2. Sugar content was analyzed as glucose by thephenol sulfuric acid method of Dubios et al. (1956). Via-ble cell counts (CFU/mL) were determined by the stan-dard plate method with Lactobacilli MRS medium after48 h of incubation at 30 �C.

2.5. Statistical analysis

All experiments were carried out in triplicate, andeach sample was analyzed in duplicate. The results areexpressed as mean ± S.D. (standard deviation). TheSAS statistical computer package was used to analyzethe experimental data (SAS Institute, Cary, NC,

USA). The values within rows that have no commonsuperscript are significantly different (p < 0.05) accord-ing to Duncan�s multiple range test (SAS Institute, Cary,NC, USA). Any two means not marked by the samesuperscript (for example, a and b or b and c within rows)are significantly different (p < 0.05). Any two meansmarked by the same superscript (for example, a and aor b and b within rows) are not significantly different(p < 0.05).

3. Results and discussion

All the three species of lactic acid bacteria, L. casei,L. delbrueckii, and L. plantarum, were found capableof growing well on sterilized cabbage juice withoutnutrient supplementation. The time courses of lacticacid fermentation of cabbage juice by L. casei, L. plan-

tarum, and L. delbrueckii are presented in Tables 1–3,respectively. L. casei, L. plantarum and L. delbrueckii

grew rapidly on cabbage juice and reached nearly10 · 108 CFU/mL after 48 h of fermentation at 30 �C.Extending the fermentation beyond 48 h did not resultin a significant increase in the viable cell counts of lacticacid bacteria. Both L. plantarum and L. delbrueckii pro-duced significantly more titratable acidity expressed aslactic acid than L. casei. For example, L. plantarum

and L. delbrueckii produced nearly 1% titratable acidityexpressed as lactic acid after 72 h of fermentationat 30 �C. Under similar growth conditions, L. casei

Table 3Time course of lactic fermentation of cabbage juice by Lactobacillus

delbrueckii

Time(h)

pH Acidity(% lactic acid)

Sugar(mg/mL)

CFU/mL

0 5.8 ± 0.0a 0.12 ± 0.0a 35.08 ± 0.09a 4.3 ± 1.76 · 105a

24 4.5 ± 0.2b 0.30 ± 0.04b 36.32 ± 3.30a 7.6 ± 2.35 · 108b

48 3.7 ± 0.0c 0.74 ± 0.03c 29.62 ± 2.17b 15.4 ± 4.28 · 108c

72 3.6 ± 0.0c 0.95 ± 0.01d 28.45 ± 0.84b 11.0 ± 1.50 · 108ab

Means and standard deviations for n = 3. The experimental valueswithin rows that have no common superscript are significantly different(p < 0.05) according to Duncan�s multiple test range.

K.Y. Yoon et al. / Bioresource Technology 97 (2006) 1427–1430 1429

produced only 0.74% titratable acidity expressed as lac-tic acid. It is probable that L. casei requires some essen-tial growth nutrients which are deficient in cabbage juice(Pederson and Albury, 1969). Earlier studies havereported that an antibacterial substance is present incabbage (Pederson and Fisher, 1944; Dickermanand Liberman, 1952; Kyung and Fleming, 1994a). Thegrowth inhibitory substance of fresh cabbage was sug-gested to be carbohydrate in nature and of a low molec-ular weight (Dickerman and Liberman, 1952). Kyungand Fleming (1994b) reported that fresh juice of Cecilecultivar cabbage was inhibitory to the growth of lacticacid bacteria, and the inhibition was eliminated whenthe cabbage was heated (steamed 10 min) before juiceextraction.

The data in Table 4 illustrate the effect of cold storageon the viability of three species of lactic acid bacteria infermented cabbage juice. L. plantarum and L. delbrueckii

were capable of surviving in the fermented cabbage juiceat 4 �C for several weeks. For example, the viable cellcounts of L. plantarum and L. delbrueckii were still4.1 · 107 and 4.5 · 105 mL�1, respectively, after 4 weeksof storage at 4 �C. However, L. casei was unable to sur-vive the low pH and high acidity conditions in fer-mented cabbage juice at 4 �C and lost the cell viabilitycompletely after only 2 weeks of cold storage. For themaximum health benefits, the minimum number of pro-biotic organisms in a food product should be 106 CFU/g

Table 4Effect of cold storage on the viability of lactic cultures in fermentedcabbage juice

Time(weeks)

CFU/mL

Lactobacillus

casei

Lactobacillus

plantarum

Lactobacillus

delbrueckii

0 1.1 ± 0.01 · 109a 17.5 ± 7.05 · 108a 11.0 ± 1.50 · 108a

1 1.1 ± 5.3 · 106b 13.0 ± 3.10 · 108a 9.9 ± 1.72 · 108a

2 ND 10.4 ± 2.32 · 107b 15.9 ± 2.56 · 107b

3 ND 6.7 ± 0.61 · 107b 34.3 ± 8.52 · 105b

4 ND 4.1 ± 0.16 · 107b 4.5 ± 3.32 · 105b

ND: not detected.Means and standard deviations for n = 3. The experimental valueswithin rows that have no common superscript are significantly different(p < 0.05) according to Duncan�s multiple test range.

(Shah, 2001). Therefore, the viability of the lactic cul-tures is the most important factor during refrigeratedor frozen storage. The viability of probiotic organismsis dependent on the level of oxygen in products, oxygenpermeation of the package, fermentation time, and stor-age temperature (Shah, 2000). The viability of probioticbacteria is also affected by inhibitory substances suchas lactic acid produced during production and coldstorage. Other factors for loss of viability of probioticorganisms have been attributed to the decrease in pHof the medium and accumulation of organic acid as aresult of growth and fermentation (Hood and Zottola,1988; Shah and Jelen, 1990). In this study, we foundboth L. plantarum and L. delbrueckii could survive thehigh acidity and low pH in the fermented cabbage juice.

4. Conclusion

Three lactic acid bacteria, L. casei, L. plantarum, andL. delbrueckii were examined for their ability to utilizecabbage juice for cell synthesis and lactic acid productionwithout nutrient supplement. These lactic cultures grewwell in cabbage juice at 30 �C, and the viable cell countsreached nearly 10 · 108 CFU/mL after 48 h of fermenta-tion at 30 �C. Both L. plantarum and L. delbrueckii werecapable of surviving the low pH and high acidic condi-tions in fermented cabbage juice during cold storage at4 �C. In contrast, L. casei could not survive the low pHand high acidity in fermented cabbage juice, and lost cellviability completely after only 2 weeks of cold storage at4 �C. From the results of this study, it is concluded thatL. plantarum and L. delbrueckii could be used as probi-otic cultures for production of a healthy beverage fromcabbage for vegetarians or consumers who are allergicto lactose present in probiotic dairy products.

Acknowledgements

Dr. Kyung Young Yoon received a post-doctoral fel-lowship from Korea Science and Engineering Founda-tion (KOSEF). This work was supported in part byUSDA Regional Project NE-1008.

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