Isolation and characterization of lactic acid bacteria from romanian ...

Romanian Biotechnological Letters Copyright © 2011 University of Bucharest

Vol. 16, No.6, 2011, Supplement Printed in Romania. All rights reserved SHORT COMMUNICATION

Isolation and characterization of lactic acid bacteria from romanian fermented vegetables Received for publication, October 7, 2011 Accepted, November 21, 2011 1

SILVIA SIMONA GROSU-TUDOR, 1*MEDANA ZAMFIR Institute of Biology Bucharest of Romanian Academy, 296 Splaiul Independentei, 060031 Bucharest, P.O. Box 56-53, ROMANIA * Corresponding author, phone: (021)2239072; fax: (021)2239071; e-mail: [email protected] 1

Abstract Fermentation was used since ancient times as an easy method of vegetables’ preservation, which also maintains and/or improves the nutritional and sensory properties of vegetables. The spontaneous fermentation process is mainly carried out by lactic acid bacteria (LAB). The aim of our study was to isolate and characterize the LAB involved in such spontaneous fermentations. Brine and vegetable samples were collected from 20 fermentations carried out at household level. They were analysed for acidification and plated on VRBG agar medium (for enumeration of enterobacteriaceae) and MRS agar medium (for enumeration and isolation of LAB). The cell morphology, growth at different temperatures, capsular polysaccharide production, mucoidness and ropyness of the colonies for the Gram-positive, catalase-negative isolates (139) were investigated. LAB were prevalent in all end-samples, as represented by their MRS counts, whereas enterobacterial counts were low in most cases, indicating good fermentation quality. All isolates grew well at 37ºC and 45ºC, in only 24h, while the growth at 10ºC was slower for some of them (72h). The cells are rod-shaped for most of the isolates, while the others are cocci and coccobacilli. All except five isolates were shown to produce capsular polysaccharide and 31 developed mucoid colonies on sucrose-based media.

Key words: lactic acid bacteria, fermented vegetables

Introduction Fermented vegetables represent a frequently used food in Romania, especially during the winter. The preservation is based on the property of lactic acid bacteria (LAB) to ferment sugars to lactic acid, causing an acidification and hence, a stabilization of the end product. The most common vegetables used are cucumbers, cabbage, and bell peppers (paprika). Green tomatoes, carrots, and cauliflower are used too, sometimes mixed with fruits (water melon, apples, or pears). During the spontaneous fermentation, LAB mostly found belong to Lactobacillus sp. (L. plantarum, L. brevis), Leuconostoc sp. (L. mesenteroides) and Pediococcus sp. (P. pentosaceus, P. cerevisiae) (Miambi & al. [1], Chao & al. [2], Nakayama & al. [3], Kim & al. [4]). Traditional preservation of vegetables and fruits by the process of lactic acid fermentation has numerous advantages beyond those of preservation. The proliferation of LAB in fermented vegetables enhances their digestibility and increases the vitamin levels and vitamin bioavailability. These beneficial organisms contribute to the organoleptic properties of the fermented product and to its preservation by in situ production of antimicrobial substances such as lactic and acetic acid, hydrogen peroxide, bacteriocins etc. (De Vuyst & Vandamme [5]). Their main end product, lactic acid, not only keeps vegetables and fruits in a state of perfect preservation but can also promote the growth of healthy microbiota throughout the intestine. Public awareness of effects of diet on gut health and a healthy gut microbiota is resulting in the consumption of products that mantain health in addition to 148

SILVIA SIMONA GROSU-TUDOR, MEDANA ZAMFIR

preventing disease. In Romania some fermented vegetables (especially sauerkraut) are sometimes used as “therapeutic agents”, especially for stomach disorders. Many LAB strains have the ability to produce exopolysaccharides (EPS), with an important role in the rheology and texture properties of fermented food products, and thus of interest for food applications as in situ produced, natural bio-thickeners (De Vuyst & Vaningelgem [6]). It has been suggested that some EPS produced by LAB have prebiotic activity (Ruijssenaars [7], Salazar [8]), contributing to the promotion of human gastrointestinal health. Traditionally fermented foods, including fermented vegetables, can be a rich source of new LAB strains, with interesting functional properties and with potential applications in food industry and health. In this context, the aim of our study was to isolate and characterize new LAB strains from various fermented vegetables, in a search for new EPS-producing strains.

Materials and methods Bacterial strains and media. Brine and vegetable samples were collected from 20 spontaneous vegetable fermentations (Table 1) carried out at a household level in the region of Valenii de Munte (Chiojdu) and Bucharest. For fermentations 19 and 20, nine additional samples were also collected during the 5 weeks of fermentation, at diferent time intervals. Samples were analysed for acidification (pH measurements) and plated on VRBG agar medium (for enumeration of enterobacteriaceae, Mossel & al. [9]) and MRS agar medium (for enumeration and isolation of LAB, de Man & al. [10]). Colonies were randomly picked up, purified and tested for catalase production and Gram-staining. Gram-positive, catalasenegative isolates were stored at -75°C in liquid MRS medium supplemented with 25% (vol/vol) of glycerol as cryoprotectant and used in our further experiments. Characterization of LAB isolated from Romanian fermented vegetables. To determine the mesophilic/thermophilic character, the strains isolated from Romanian fermented vegetables were incubated in MRS at different temperatures: 10°C, 37°C and 45°C. After 24– 72 h of incubation at these temperatures, the pH and the optical density at 600 nm was measured. If the growth of the strains occurs between 10°C and 37°C and there is no growth observed at temperatures higher than 45°C, they are mesophilic, while the strains showing a good growth at 45°C, but not able to grow at 10°C are thermophilic (G. Zarnea [11]). The cell morphology of the isolates was observed by microscopical examination of the Gramstained slides. In order to evaluate the capacity to produce exopolysaccharides, the isolates were cultivated on four different media: MRS with 20 g liter-1 of glucose, modified MRS (mMRS) with 50g liter-1 of sucrose instead of glucose, ST (Dave & Shah [12]) with 10 g liter-1 of lactose and modified ST (mST) with 50 g liter-1 of sucrose instead of lactose. After 24 h of incubation at 37°C, the mucoidness and ropyness of the colonies was observed. The mucoid colonies have a glistening and slimy appearance on agar plates, but are not able to produce strands when extended with a stik, whereas the ropy colonies form a long filament by this method (Dierksen & al. [13]). Capsular polysaccharide (CPS) formation was evaluated by the Chinese ink negative staining technique (Mozzi & al. [14]) after growing the strains on MRS with different carbon sources and at different temperatures (10°C, 37°C and 45°C). Ten microliters of a fresh culture was mixed with a drop of Chinese ink and spread on a slide in a thin film, coverred with a cover glass and examined.

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Isolation and characterization of lactic acid bacteria from romanian fermented vegetables

Results and discussions The final pH of the brines of all end-samples of the different spontaneous vegetable fermentations carried out was on average pH 3.6. LAB were prevalent in all end-samples, as represented by their MRS counts [ca. 109 (cfu/ml)], whereas enterobacterial counts were low in most cases, indicating good fermentation quality (data not shown). A total of 150 colonies were picked up from the MRS plates, purified and checked for catalase production and Gram staining. A number of 139 isolates were shown to be Grampositive and catalase-negative. The latter isolates were used for further characterization. All the isolates grew very well at 37°C, reaching an OD600nm of over 1.2 after 24 h of incubation. Generally, 37°C is considered the optimal temperature for most species of lactic acid bacteria (Zarnea, [11]). The isolates were able to grow well also at 10°C, 35 isolates reaching an OD600nm of over 0.5 after only 24 h of incubation. The other isolates needed 48 h (95 isolates) or even 72 h (9 isolates) of incubation to reach the same OD value. At 45°C, all isolates showed a good growth after 24 hours of incubation (OD600nm over 0.5) . The pH values of the bacterial cultures ranged between 3.0 and 5.0 for the isolates incubated at 37°C and 45°C, and around 5.0 for the isolates incubated at 10°C. Since all isolates could grow at all tested temperatures, we could not make, at this point, a distinction between their thermophilic/mesophilic character. The cell morphology of all 139 isolates was evaluated by microscopical observations using the immersion objective. The cells from most of the isolates (95) were rod-shaped, from other 32 were coccoid, from eleven were cocobacillar and from one isolate had a diplococcal shape (Table 1). Table 1. Overview of the spontaneous vegetables fermentations and characterization of the isolates Fermented product

150

Time of sampling

No. of isolates 4

Mucoid isolates (MRS suc) 0

CPS positive (MRS gluc) 4

4

0

4

Cell morphology

1. Cucumber and plums

8 weeks

2.Green tomato

4 weeks

3.Cucumber and beans

8 weeks

1

1

0

rods (2) cocobacilli (2) rods (3) cocci (1) rods (1)

4.Cucumber and carrots

6 weeks

0

0

0

-

5.Green tomato

8 weeks

6

0

4

rods (6)

6.Cucumber

12 weeks

0

0

0

-

7.Green tomato, beets and cabbage 8.Cucumber and cauliflower 9.Green tomato

8 weeks

1

0

0

cocobacilli (1)

6 weeks

6

1

2

4 weeks

4

2

3

10.Green tomato, apple, pear, and cucumber

2 weeks

9

2

9

11.Green tomato, apple, pear and cucumber

2 weeks

12

3

5

12.Green tomato, carrots and celery 13.Cabbage and beet

8 weeks

5

1

4

rods (4) cocobacilli (2) rods (3) cocobacilli (1) rods (2) cocci (4) cocobacilli (3) rods (7) cocobacilli (2) cocci (3) rods (5)

4 weeks

5

2

3

rods (5)

14.Cabbage

8 weeks

3

0

3

rods (3)

15.Green tomato, carrots and cauliflower

8 weeks

8

3

7

rods (8)


SILVIA SIMONA GROSU-TUDOR, MEDANA ZAMFIR 16.Cabbage

4 weeks

5

0

5

rods (5)

17.Pepper filled with cabbage 18.Cucumber

8 weeks

9

0

6

rods (9)

4 weeks

6

2

6

rods (6)

5 weeks

32

11

23

5 weeks

19

3

10

19.Green tomato, cauliflower, carrots, pepper and celery 20.Cauliflower

rods (16) cocci (15) diplococci (1) rods (10) cocci (9)

Most of the isolates (87) grew well in all four media used (MRS, mMRS, ST, and mST), but it was observed a preference for sucrose as a carbon source. Thirty-one strains developed mucoid colonies on MRS with sucrose (Table 1, Fig 1a) and less or no mucoid on media with glucose or lactose as a carbon source (data not shown). In some cases, due to the small size of the colonies, the mucoidness and ropyness phenotype could not be detected. a.

b.

Fig 1. Mucoidness (a) and ropynes (b) of the colonies developed on agar plates by the isolate 52 and isolate 371, respectively.

Results are in accordance with those obtained by Van Geel-Schutten & al. [15] who screened several Lactobacillus strains of different origins (fermented food, human dental plaque etc.) for mucoid phenotype in MRS medium supplemented with high concentrations (100 g liter-1) of different sugars: glucose, fructose, maltose, raffinose, sucrose, galactose or lactose. The MRS supplemeted with sucrose was the best medium for detecting the mucoide phenotype. The autors attributed the high percentage of positive isolates to the high content of sugar used in the medium. Also, Smitinont & al. [16] observed slimy colonies on agar media containing sucrose as a sole carbon source. None of the mucoid colonies showed a ropy phenotype. There were, however, two isolates (338 and 371) that developed ropy colonies on sucrose containing media (Fig 1b). Some LAB (e.g. Lactococcus lactis spp. cremoris Ropy352, Lactobacillus casei CG11) were described in the literature to express both ropy and mucoid phenotypes, depeding on the growth conditions (Dierksen & al. [13], Cerning & al. [17]). In Lactococcus lactis spp. cremoris Ropy352, it has been shown that this ability is due to the production of two exopolysaccharides with different chemical composition (Knoshaug & al. [18]). It was shown that ropy strains of LAB yield fermented milk products having smoother body, higher viscosity, and less syneresis than products made with nonropy strains (Bouzar & al. Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011)

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[19]) and are of particular interest in Scandinavian milk products such as viili and langmjolk. Ropy starter cultures also provide benefits for yogurt production (Broadbent & al. [20]). In Mexico, where the milk supply routinely faces shortages, yogurt can be made with less total milk solids if a ropy starter culture is used (Wacher-Rodante & al. [21]). In fermented vegetables, however, ropiness might be a disadvantage for the appearance of the final product. It was previously shown that depending on the culture medium and conditions, some strains can produce excessive ropiness and undesirable characteristics (Toba & al. [22]). The polysaccharide caspule formation was investigated for the 139 isolates, by microscopical observation after staining with Chinese ink. Ten out of the 139 isolates, randomly selected, were tested for capsule production on different media (MRS, mMRS, ST, and mST) and at different incubation temperatures (10°C, 37°C, and 45°C). Because no signifiant differences were observed in the presence/absence of the polysaccharide capsule in these different conditions, MRS medium was further used for the growth of tested isolates and incubation was performed at 37°C. Twenty six isolates showed a clear, large capsule (Fig 2), 72 showed a medium capsule, 36 showed a small capsule and for 5 of them no capsule was observed. CPS of different sizes was detected in all mucoid and ropy isolates. It is, however, difficult to make a precise correlation between the ropy/mucoid phenotype, CPS production and production of exopolysaccharaides (Van der Meulen & al. [23]).

Fig 2. Capsular polysaccharide sorrounding the cells of the isolate 326

Capsule formation can be found among both nonropy and ropy strains. Streptococcus thermophilus OR901, a strain isolated from commercial yogurt and able to form ropy strands from the cell mass, appeared encapsulated using light microscopy with Indian ink staining (Ariha & al. [24]). This was due to the production of two EPS with the same monosaccharide composition and branching linkage, but with different molecular mass. The EPS with the higher molar mass was assumed to affect physical (texture and viscosity) properties of yogurt and the EPS with lower molecular mass was thought to be located in the capsule, given that it was only released from the cell surface after sonication (Ariha & al. [24]). Thick-layered CPS have been shown to protect bacteria against phage infection (Kang & Cottrell, [25]). However, the involvement of CPS in the phage infection process of LAB is still unclear. The industrial interest in bacterial strains producing CPS has increased in recent years because the use of these strains for milk fermentation improves cheese moisture retention and cheesemelting ability (Broadbent & al. [26]). Thus, substantial efforts have been devoted to developing methods to optimize CPS biosynthesis. 152


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Conclusions This study was undertaken to isolate and characterize new lactic acid bacteria involved in the spontaneous Romanian vegetables fermentations. It was proven that fermented vegetables are a rich source of LAB, 139 isolates being obtained from 20 different fermented products. All isolates except five were shown to produce capsular polysaccharide and 31 developed mucoid clonies on sucrose-based media and some of them might be further selected as EPS-producing strains and tested for their potential applications in food industry (for the production of food with improved rheological properties) or health (prebiotics).

Acknowledgements The authors acknowledge their financial support of the Postdoctoral Research Project PD_33/2010 of the Romanian National Research Plan (PNII-RU). Part of this work was supported by the project no. RO1567-IBB05/2011 from the Institute of Biology Bucharest of Romanian Academy.

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