World

Journal

01 Microbiology

and Biotechnology

Fermentation Lactobacillus mesenteroides

9, 176-l 79

of brined turnip roots using plantarum and Leuconostoc starter cultures

M.I. Yamani The controlled fermentation of turnip slices using Lactobacillus plantarum or Leuconostoc mesenferoides as starter cultures led to earlier acid production and earlier and more pronounced inhibition of Enterobacteriaceae than with uninoculated (natural) fermentation. Unlike the natural fermentation, the controlled fermentations did not show a yeast secondary fermentation and also had a better colour. Due to its ability to produce higher amounts of acid, the use of Lad plantarum is more desirable than of Leuc. mesenteroides. Key

Controlled

words;

vegetable

fermentation,

Lactobacillus

Fermentation of vegetables is an important means of preservation because of its low energy requirements and the favourable sensory properties it imparts to the product (Fleming 1982; Daeschel et al. 1987). Most vegetables can be fermented naturally when kept in brine for a sufficient time at an appropriate temperature (Pederson 1979; Vaughn 1985). However, this type of fermentation causes many problems. Softening, off-colour and off-flavour are probably the most important spoilage problems and are common to all naturally fermented vegetables. These undesirable changes are brought about by the activity of some of the microbial flora and/or indigenous enzymes during the fermentation process (Pederson 1979; Fleming 1984; Vaughn 1985). Controlled fermentation procedures, including acidification and buffering (Etchells et al. x973), the use of pure lactic acid bacteria cultures (Etchells et al. 1964; Fleming & McFeeters 1981) and the use of anaerobic tanks for fermentation (Humphries & Fleming 1986) have been used to overcome these problems. The turnip (Brussica rupu) is an important crop in many countries. Like beets and carrots, it is grown both for its roots and for its foliage (Thompson & Kelly 1957). The roots are tuberous and usually globular, white fleshed, with a reddish purple crown (Tindall 1983). In many countries, turnips are fermented. In the Orient, they are either fermented separately, in brine or by dry

phnfarum,

salting, or as part of various vegetable blends, with carrots, cauliflowers and/or beets (Pederson 1979). In many Middle Eastern countries, fermented turnip roots are next to cucumbers in importance as pickles. Turnip pickles are served as savouries or appetizers. Information about the fermentation of turnips is sparse in comparison with that of other vegetables. Miyao & Aoki (1979) and Aoki & Miyao (1979) examined turnip pickling brine during fermentation for changes in the microflora and contents of sugars and acids during storage at 5’ to 20°C. They reported that the shelf life of the fermented turnips was < 10 days at 20°C, 10 days at 10°C and 100 days at 5°C. This study investigates the use of pure cultures of Lactobacillus plantarum or Leuconostoc mesenferoides as starters in the fermentation of brined turnip roots and describes their effect on some of the characteristics of the fermented turnips.

Materials

@ 1993 Rapid

176

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of Oxford

Ltd

World @mai of Mmobiology and Bv&chnology, Vol 9. 1993

and Methods

The turnips used in this study were purchased from a local market in Amman, Jordan. The roots were 6 to i’cm in diameter, free from mechanical damage and generally of good quality. They were carefully selected for uniformity of size and shape. Brining,

M.I. Yamani is with the Department of Nutrition and Food Technology, Faculty of Agriculture, University of Jordan, Amman, Jordan; fax: (962)(6) 832318.

Leuconosfoc mesenteroides, turnip fermentation.

Inoculation

Procedures

and Fermentation

Turnips were hand washed and each root was cut into eight pieces, which were packed into 2-l glass jars. To prevent the pieces from rising, semi-rigid thin plastic sheets were placed over them. A cover

Controlled brine containing 6% (w/v) NaCl was added to the jars. The final pack-out ratio was about 55 :45 (w/w), tumips:brine. Nine jars were filled in this way. The inoculation of lactic acid bacteria cultures was done I day after brining the turnips, to allow the salt to diffuse into and fermentable nutrients to diffuse out of the turnip pieces (Fleming et al. 1973). Before inoculation, the nine jars were divided into three groups of three. The first group was inoculated (5 ml/jar) with a culture of Lactobacihs plantawn, the second with a

fermentation

of turnips

I”

98-

Leuconostoc mesenferoides culture, while the third group, which served as a control, was not inoculated. All jars were kept at room temperature

(25 f

2°C).

Culture Preparation The Lacfobacih plantamm and Letlconostoc mesenteroides cultures used were isolated previously from fermented cucumber brine.

They were identified and maintained according to the methods described by Kandler & Weiss (1986) for the Lactobacilh and by

10.8

6-

Gravie (1986) for the Leuconostoc. Each species was grown separately in 50 ml cucumber juice broth for 24 h at 32°C (Fleming & Etchells 1967) before use as inocula. Sampling

and Enumerafion

Procedures

From each jar, a 10 ml brine sample was taken periodically during the fermentation. For analysis, a composite sample from the three jars forming a group was used. Ten-fold dilutions were prepared using 0.1% peptone water. The pour plate technique was used for the enumeration of microorganisms. Total lactic acid bacteria (LAB) were counted using MRS agar medium (Oxoid) (de Man et al. 1960) after the plates had been incubated at 32°C for 72 h (Fleming et al. 1984). Enterobacteriaceae were counted using Violet Red Bile salts agar (Oxoid) with I% (w/v) glucose, after the plates had been incubated at 32°C for 24 h (Fleming et al. 1984). Yeasts were counted using standard methods agar containing 100 mg/l each of chloramphenicol and chlortetracycline.HCl, after 5 days at 25°C (Koburger & Marth 1984). Duplicate plates were used for each

count. Chemical Analysis pH and titrable acidity were determined described by Fleming et al. (1984).

according

to procedures

Sensory Evaluation The procedure for sensory evaluation was as that described by Fleming et al. (1978) for sliced, fermented cucumber. A taste panel of five trained individuals evaluated the turnips on day 24 of fermentation. The panel was asked to note any differences in flavour and appearance between samples of the naturally fermented turnips and those of the controlled fermentations, without knowing the identity of the samples.

Results Natural

Fermentation

of Turnips

Figure I shows the changes in the microbial flora, pH and acidity of the naturally fermented turnips. A rapid increase in the count of LAB up to [email protected] was noticed; these bacteria predominated throughout the fermentation. An increase in acidity and a decrease in pH, which correlated with the LAB counts, were also observed. A transient increase in the Enterobacteriaceae count was noted, followed by a steep

4

8

12

Fermentation

16 20 time

24

(days)

Figure 1. Microbiological changes and acid brine of naturally fermented turnips. O-Lactic V-Enterobacteriaceae; O-yeasts; A-pH;

production in the acid bacteria; A-acidity.

decline; by the tenth day Enterobacteriaceae were not detectable. The yeast count decreased and reached undetectable levels by day 4. However, a gradual increase in the yeast count was noticed starting on day 12 of the fermentation. Naturally fermented turnips showed a bleached appearance. No off-flavour was noticed. Controlled

Fermentation

of Turnips

The addition of LAB resulted in a [email protected] increase in initial levels compared with the natural fermentation (Figure 2). In the first 5 days, higher LAB counts (Figure 2), higher acidities and lower pH values (Figure 3) were noticed in brines to which LAB were added, whereas higher Enterobacteriaceae counts were noticed in naturally fermented turnips (Figure 2). The drop in the Enterobacteriaceae counts was more rapid in the brines of the controlled fermentation. In the first week of fermentation the highest LAB counts and the most rapid decline in Enterobacteriaceae were observed in the brine to which lucf. plunturum was added (Figure 2). This was also associated with a more rapid drop in pH and an increase in acidity (Figure 3). In the second half of the fermentation, the acidity of the natural fermentation brine was higher than that of the controlled fermentations and the pH was slightly lower (Figure 3).

M.I. Yamani

10 9

8

4

8 Fermentation

12

16 time

20

24

(days)

4

8

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Fermentation

16 20

24

time (days)

Flgure 2. Growth of lactic acid bacteria (LAB), Enterobacteriaceae, and yeasts during the natural fermentation (m-LAB; V-Enterobacteriaceae; (J-yeasts) and controlled fermentations of brined turnips using a LactobacNus plantarum starter culture (@-LAB; 0-Enterobacteriaceae; O-yeasts) or a Leuconostoc mesenferoides starter culture (A-LAB; xEnterobacteriaceae; A-yeasts).

Figure 3. Changes in the pH and acidity % (as lactic acid) during the natural fermentation (O-pH; m-acidity) and controlled fermentations of brined turnips using a Lactobacihs plantarum starter culture (O-pH; O-acidity) or a Leuconostoc mesenteroides starter culture (A-pH; A-acidity).

Yeast counts were relatively low in all brines in the first few days of fermentation (Figure z), being lowest in the naturally fermented turnips. By day 4 of fermentation, yeasts were not detected in any of the brines. In contrast to the naturally fermented turnips, no increase in yeast counts was noticed in the brines of the controlled fermentation. Turnips of the controlled fermentation had a uniform pinkish appearance and no off-flavour was noticed.

at < 10°C. A mixed flora of Gram-negative and Gram-positive bacteria was detected during the initial stages of fermentation, after which Lacf. planfartrm predominated, with a concurrent increase in the yeast count. The speed at which desirable LAB are established and the manner in which undesirable microorganisms (e.g. Enterobacteriaceae) are excluded during the initiation stage are among the most important factors which determine the quality of naturally fermented vegetables (Fleming 1984; Daeschel et al. 1987). In this study, rapid establishment of LAB and exclusion of Enterobacteriaceae were achieved by an early drop in the pH which resulted from the growth of the LAB cultures added. It was more pronounced when Lacf. planfarum was added. This was accomplished without acidification or buffering of the brines, as practised in some vegetable fermentations (Etchells et al. 1973). Whether this is a constant phenomenon, which could be applied to other vegetables, needs further investigation. A gradual increase in yeast numbers in the brines to which no LAB were added (Figure 1) is expected at the secondary stage of fermentation of naturally fermented vegetables (Etchells 1941; Fleming 1982). This may account for the increase in acidity noticed. The disappearance of the yeasts

Discussion Natural fermentation of brined vegetables involves a dynamic microbial succession, categorized into four stages: initiation, primary fermentation, secondary fermentation and post-fermentation (Fleming 1983). It seems that the first three stages were exhibited in the natural fermentation of turnips described in this study (Figure I). This pattern of fermentation is similar to the natural fermentation of cucumbers (Etchells & Jones 1943). Miyao & Aoki (1979) described the microbial and chemical changes observed during the fermentation of turnips, packed tightly in jars containing 3% (w/v) NaCl and pressed for about one week

178

World ]oumal

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Mimbiology

and Biotechnology. Vol 9. 1993

Confrolled

from the brines to which LAB were added (Figure 2) is worth further study. The rapid and early drop in the pH and the early increase in the acidity (Figure 3), and/or the production of inhibitory substance(s), e.g. bacteriocins (Klaenhammer 1988), by the added LAB, might be responsible for this. A major problem in the natural fermentation of turnips is the bleached appearance of the product, which is not acceptable to the consumer. To overcome this, producers tend to add natural or artificial colouring agents to the turnips at the time of brining. The turnips produced by the controlled fermentation were pink in colour, with a more pronounced colour in the turnips to which Lat. plunfarum was added. In contrast, the naturally fermented turnips had the typical bleached appearance. It seems that the pigments present in the crowns of the turnips had diffused into the brine and consequently into the flesh of the turnip pieces during fermentation. The destruction of these pigments, by the non-lactic flora, in the turnips of the natural fermentation may be responsible for their bleached appearance. It is concluded from this study that the use of the two lactic acid bacteria cultures as described may be more advantageous than natural fermentation of turnips. The use of Lact. planfurum is more favoured than that of Leuc. mesenferoides because of the former’s ability to produce higher amounts of acid and its earlier inhibition of the non-lactic flora represented by the Enterobacteriaceae.

Acknowledgements The author gratefully acknowledges from the Scientific Research Council Jordan.

the financial support at the University of

References Aoki, M. & Miyao, S. 1979 Quality and behaviour of microorganisms in pickles. III. Shelf-life of fermented turnips. Journal of the Japanese Society of Food Science and Technology 26, 447-449. Daeschel, M.A., Anderson, R.E. & Fleming, H.P. 1987 Microbial ecology of fermenting plant materials. FEMS Microbiology Reviews 46, 357-367. de Man, J.C., Rogosa, M. & Sharpe, M.E. 1960 A medium for the cultivation of lactobacilli. ]ournal of Applied Bacteriology 23, 130-135. Etchells, J.L. 1941 Incidence of yeasts in cucumber fermentations. Food Research 6, 95-104. Etchells, J.L., Bell, T.A., Fleming, H.P., Kelling, R.E. & Thompson, R.L. 1973 Suggested procedure for the controlled fermentation of commercially brined pickling cucumbers-the use of starter culture and reduction of carbon dioxide accumulation. Pickle Pak Science 3, 4-14.

femenfufion

of turnips

Etchells, J.L., Costilow, R.N., Anderson, T.E. & Bell, T.A. 1964 Pure culture fermentation of brined cucumbers. Applied Microbiology 12, 523-535. Etchells, J.L. & Jones, I.D. 1943 Bacteriological changes in cucumber fermentation. Food Industries 15, 54-56. Fleming, H.P. 1982 Fermented vegetables. In Economic Microbiology. Vol. 7: Fermented Foods, ed Rose, A.H. pp. 227-258. London: Academic Press. Fleming, H.P. 1984 Developments in cucumber fermentation. ]ourna[ of Chemical Technology and Biotechnology 34B, 241-252. Fleming, H.P. & Etchells, J.L. 1967 Occurrence of an inhibitor of lactic acid bacteria in green olives. Applied Microbiology 15, 1178-1184. Fleming, H.P. & McFeeters, R.F. 1981 Use of microbial cultures: vegetable products. Food Technology 35, 84-88. Fleming, H.P., McFeeters, R.F., Etchells, J.L. & Bell, T.A. 1984 Pickled vegetables. In Compendium of Methods for the Microbiological Examination of Foods, 2nd edn, ed Speck, M.L. pp. 663-681. Washington: DC: American Public Health Association. Fleming, H.P., Thompson, R.L., Bell, T.A. & Hontz, L.H. 1978 Controlled fermentation of sliced cucumbers. journal of Food Science 43, 888-891. Fleming, H.P., Thompson, R.L., Etchells, J.L., Kelling, R.E. & Bell, T.A. 1973 Bloater formation in brined cucumbers fermented by Lactobacillw plantarum. Journal of Food Science 38, 49~503. Gravie, E.I. 1986 Genus Leuconostoc. In Bergey’s Manual of Systematic Bacteriology, Vol. 2, ed Sneath, P.H.A. pp. 1071-1075. Baltimore: Williams & Wilkins. Humphries, E.G. & Fleming, H.P. 1986 Closed-top fermentation tanks for cucumbers. Transactions of the American Society of Agricultural Engineers 2, 45-48. Kandler, 0. & Weiss, N. 1986 Genus Lactobacillus. In Bergey’s Manual of Systematic Bacteriology, Vol. 2, ed Sneath, P.H.A. pp. 1209-1235. Baltimore: Williams & Wilkins. Klaenhammer, T.R. 1988 Bacteriocins of lactic acid bacteria. Biochemie 70, 337-349. Koburger, J.A. & Marth, E.H. 1984 Yeasts and molds. In Compendium of Methods for the Microbiological Examination of Foods, 2nd edn, ed Speck, M.L. pp. 197-202. Washington DC: American Public Health Association. Miyao, S. & Aoki, M. 1979 Quality and behaviour of microorganisms in pickles. II. Lactic acid fermentation of turnips. Journal of the japanese Society of Food Science and Technology 26, 444-446. Pederson, C.S. 1979 Microbiology of Food Fermentations, 2nd edn. Westport, CT: Avi Publishing. Tindall, H.D. 1983 Vegetables in the Tropics. London: Macmillan Education. Thompson, H.C. & Kelly, W.C. 1957 Vegetable Crops, 5th edn. New York: McGraw-Hill. Vaughn, R.H. 1985 The microbiology of vegetable fermentations. In Microbiology of Femented Foods, Vol. 2, ed Wood, B.J.B. pp. 49-109. London: Elsevier.

(Received

in revised

form

7 August

1992;

accepted

14 August

1992)

World ]ournal of Microbiology and Biotechnology, Vof 9. 1993

179

Fermentation of brined turnip roots using Lactobacillus plantarum and Leuconostoc mesenteroides starter cultures.

The controlled fermentation of turnip slices using Lactobacillus plantarum or Leuconostoc mesenteroides as starter cultures led to earlier acid produc...
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