Acidic and saline stresses modify microflora composition of table olives produced by spontaneous fermentation.

(1)

ACIDIC

AND

SALINE

STRESSES

MODIFY

MICROFLORA COMPOSITION OF TABLE OLIVES

PRODUCED BY SPONTANEOUS FERMENTATION

Andrea CARIDI, Alessandra DE BRUNO, Amalia PISCOPO,

Angela ZAPPIA, Marco POIANA

Department of AGRARIA, “Mediterranea” University of Reggio Calabria, Loc. Feo di Vito, 89124

Reggio Calabria, Italy

Introduction

Table olives have a great diffusion in the Mediterranean countries, with a worldwide production which currently exceeds 2.5 million tons/year. The main components of the table olive microflora are lactic acid bacteria (LAB) and yeasts. Their growth is affected by both environmental and technological stresses. Among others, nutrient depletion [1], salt concentration and low pH levels [2] can induce a shift from homo- to hetero-fermentative metabolism of LAB [3]. When LAB grow in a high salt medium, the structural properties of the cell wall change [4]. Several LAB produce exopolysaccharides as a resistance mechanism to phenolic stress [5]. On the other hand, under stressing conditions yeasts synthesize a number of bioactive antioxidant compounds [6,7]. In order to improve the industrial production process, we tested the effect of acidic (by lactic acid addition) and saline (by sodium chloride addition) stresses on the autochthonous LAB and yeasts during table olive production.

Materials and methods

The trials were carried out in 25-liter containers using olive of the cultivars

Carolea, Geracese, and Nocellara messinese. No starter culture - neither

of LAB neither of yeasts - was added; consequently, all the fermentations are spontaneous. Four different stressing conditions were tested:

• addition of sodium chloride until 8% (w/v);

• addition of sodium chloride until 8% (w/v) and addition of lactic acid to reach pH 4.30;

• addition of sodium chloride until 5% (w/v); after 20 days further addition of 3% of sodium chloride: in total sodium chloride 8%;

• addition of sodium chloride until 5% (w/v) and addition of lactic acid to reach pH 4.30; after 20 days further addition of 3% of sodium chloride: in total sodium chloride 8%.

After 7, 15, and 30 days the following counts were carried out on olives: • LAB in MRS agar under anaerobic incubation at 32 C for two days; • Aerobic microflora in STPA (Standard Plate Count Agar) at 26 C for two days.

The best representative strains of LAB and yeasts were isolated and at present they are under identification and technological characterization.

Results and Discussion

The information obtained allow us to better understand the complex microbial population dynamic in a stressing environment like the table olive fermentation. Phenolic compounds, low nutrient content, low pH, high sodium chloride content: each constitutes an agent able to induce an anti-stress reaction in the microflora. Data can be used to improve the quality and safety of this fermented vegetable, by promoting the growth of desirable microbial species and inhibiting spoilage and mycotoxin producer ones. Resistance to stressing conditions is a trait useful to select starter cultures of LAB and/or yeasts.

For the Carolea cultivar (Table 1) the addition of 8% (w/v) of sodium chloride is the condition that greatly reduces both LAB and yeast content after 7 and 30 days. For the Geracese cultivar (Table 2) the addition of 8% (w/v) of sodium chloride and the addition of lactic acid to reach pH 4.30 is the condition that greatly reduces both LAB and yeast content after 7, 15, and 30 days. For the Nocellara messinese cultivar (Table 3) the same condition greatly reduces both LAB and yeast content after 7 and 15 days.

These preliminary results clearly underline that there are significant differences among the three cultivars and the four stressing conditions. In addition, the observed differences persist until at least 30 days of fermentation.

This research was supported by POR CALABRIA FESR 2007/2013 - 1.1.1.2 NUOVE TECNOLOGIE PER LA

VALORIZZAZIONE DELLA FILIERA DELLE

CONSERVE: CONSERVO

References

1. Ruiz-Barba J.L., Jiménez-Díaz R. - Vitamin and amino acid requirements of Lactobacillus plantarum strains isolated from green olive fermentations. Journal of Applied Bacteriology 76, 350-355, 1994.

2. Bobillo M., Marshall V.M. - Effect of acidic pH and salt on acid end-products by Lactobacillus plantarum in aerated, glucose-limited continuous culture. Journal of Applied Bacteriology 73, 67-70, 1992.

3. Panagou E.Z., Schillinger U., Franz C.M.A.P., Nychas G.-J.E. - Microbiological and biochemical profile of cv.

Conservolea naturally black olives during controlled

fermentation with selected strains of lactic acid bacteria. Food Microbiology 25, 348-358, 2008.

4. Piuri M., Sanchez-Rivas C., Ruzal S.M. - Cell wall modifications during osmotic stress in Lactobacillus

casei. Journal of Applied Microbiology 98, 84-95, 2005.

5. Manca de Nadra M.C., Strasser de Saad A.M. -Polysaccharide production by Pediococcus pentosaceous from wine. International Journal of Food Microbiology 27, 101-106, 1995.

6.Cruz J.M., Domínguez J.M., Domínguez H., Parajó J.C. - Solvent extraction of hemicellulosic wood hydrolysates: a procedure useful for obtaining both detoxified fermentation media and polyphenols with antioxidant activity. Food Chemistry 67(2), 147-153, 1999.

7. Martínez-Montañés F., Pascual-Ahuir A., Proft M. – Repression of ergosterol biosynthesis is essential for stress resistance and is mediated by the Hog1 MAP kinase and the Mot3 and Rox1 transcription factors. Molecular Microbiology 79(4), 1008-1023, 2011.

Cultivar Stress 7 days 15 days 30 days 7 days 15 days 30 days

1 317,000a _13,800,000a _875,000a _970,000a _6,050,000a _1,470,000a

2 1,040,000a _10,500,000a _1,250,000a _1,010,000a _13,100,000a _1,790,000a

3 10,400,000b _46,400,000b _11,300,000b _12,600,000b _53,000,000b _12,200,000b

4 6,680,000ab _28,000,000ab _10,900,000b _11,100,000b _33,100,000ab _10,600,000b

Table 1. Evolution of lactic acid bacteria and yeast content in olives of the cultivar Carolea until 30 days of spontaneous

fermentation under four different stressing conditions.

Legend. 1=addition of sodium chloride until 8% (w/v); 2=addition of sodium chloride until 8% (w/v) and addition of lactic

acid to reach pH 4.30; 3=addition of sodium chloride until 5% (w/v); after 20 days further addition of 3% of sodium chloride: in total sodium chloride 8%; 4=addition of sodium chloride until 5% (w/v) and addition of lactic acid to reach pH 4.30; after 20 days further addition of 3% of sodium chloride: in total sodium chloride 8%; superscript letters indicate

the homogeneous groups (Fisher’s Least Significant Differences, level of confidence of 95%) in the same column.

Lactic acid bacteria Yeasts

Carolea

Microorganisms

1 355,000a _6,450,000ab _940,000a _565,000ab _8,950,000ab _1,060,000ab

2 25,000a _545,000a _495,000a _240,000a _1,270,000a _400,000a

3 2,730,000ab 15,600,000b 6,500,000b 7,810,000c 8,040,000ab 3,860,000c

4 4,720,000b _17,700,000b _2,900,000a _6,700,000bc _21,400,000b _2,690,000bc

Table 2. Evolution of lactic acid bacteria and yeast content in olives of the cultivar Geracese until 30 days of

spontaneous fermentation under four different stressing conditions.

Microorganisms Lactic acid bacteria Yeasts

Geracese

1 139,000a _141,000a _1,180,000b _228,000a _655,000a _435,000ab

2 18,500a _66,000a _910,000ab _182,000a _410,000a _880,000b

3 1,760,000b _7,000,000a _1,150,000b _2,660,000b _7,500,000a _865,000b

4 45,200a _195,000a _45,000a _415,000a _425,000a _60,000a

Table 3. Evolution of lactic acid bacteria and yeast content in olives of the cultivar Nocellara messinese until 30 days of

spontaneous fermentation under four different stressing conditions.

Microorganisms Lactic acid bacteria Yeasts

Nocellara messinese