Growth of Pediococcus acidilactici on sugar cane blackstrap molasses

Abstracts

Pediococcus acidilactici (IL01) cresceu em caldo MRS (Man, Rogosa and Sharpe) modificado por adição de 2,0% (MRS-2), 3,0% (MRS-3), 4,0% (MRS-4) and 5,0% (MRS-5) de melaço de cana de açúcar, em substituição à glicose. A maior produção de ácido ocorreu em caldo MRS-5 com pH constante 5,0. A produção de biomassa foi mais acentuada em caldo MRS-5 com pH inicial de 6,5, embora a produtividade tenha sido maior em caldo MRS-2 (28,16%). Em caldo MRS-2 e em pH inicial de 6,5 durante uma fermentação de 20 horas, a velocidade de crescimento (dx/dt) foi maior entre a 8ª e 16ª hora (0,290 g celulas/L.h) enquanto a velocidade específica de crescimento µ foi 0,175 (h-1) para este período, diferente de 0,441 (h-1) obtido no período compreendido entre a 4ª e 12ª hora. O crescimento em caldo MRS foi 5,08% (2,95 g/l) maior que em caldo MRS-2 (2,80 g/l). Os dados obtidos mostraram que P. acidilactici cresceu bem em melaço como principal fonte de carbono e que é possível substituir a glicose do MRS por esta fonte de carbono, com o objetivo de obter um meio de crescimento mais econômico para eventuais produções em grande escala.

Pediococcus acidilactici; "starter"; salame

GROWTH OF PEDIOCOCCUS ACIDILACTICI ON SUGAR CANE BLACKSTRAP MOLASSES

Ernani S. Sant’Anna^* and Regina Coeli O. Torres

Departamento de Ciência e Tecnologia de Alimentos, Universidade Federal de Santa Catarina, Florianópolis, SC, Brasil.

Approved: July 23, 1998

ABSTRACT

Pediococcus acidilactici (IL01) has grown in MRS (Man, Rogosa and Sharpe) broth modified by substitution of glucose by 2.0% (MRS-2), 3.0% (MRS-3), 4.0% (MRS-4) and 5.0% (MRS-5) sugar cane blackstrap molasses. The highest acid production was obtained in MRS-5 broth maintained at a constant pH of 5.0. The highest biomass production was obtained when P. acidilactici was grown in MRS-5 broth at initial pH 6.5, while productivity was higher in MRS-2 broth (28.16%). When the MRS-2 broth was utilized at initial pH 6.5 for a 20-hour fermentation period, the highest growth rate (dx/dt) was found in a period of 8 to 16 hours (0.290 g cells/L.h), while the specific growth rate (µ) was 0.175 (h^-1) for that period, differently from the 0.441 (h^-1) obtained for the period comprising the 4th to the 12th hour. The growth in MRS broth was 5.08% (2.95 g/l) higher than in MRS-2 broth (2.80 g/l). The data obtained have shown that P. acidilactici has had a significant growth in molasses as the main carbon source, and that it is possible to substitute MRS glucose by this carbon source with the purpose of obtaining a more economical growth medium for the potential large scale productions.

Key words: Pediococcus acidilactici, starter, salami

In the fermentation phase, the lactic acid bacteria produce mainly lactic acid that contributes to color formation (nitrosomyoglobin). During ripening, several changes occur which emphasize the degradation of protein and fat. Fermentation can occur naturally by indigenous microorganisms or through starter cultures which are intentionally added (18, 20). When the starter culture is used, there is a better control of the process and many other advantages, such as a decrease in fermentation time and damage (2). However, very good quality fermented meat products can be produced without starter cultures.

Starter cultures with lactic acid bacteria affect the quality of fermented sausage in many ways. The formation of lactic acid from an added carbohydrate source is of major importance. The resulting decrease in pH causes the coagulation of meat proteins, the acid taste, improves hygienic stability and reddening (9).

Chemical acidulants such as gluconodelta lactone (GDL) have been used to accelerate the reduction in pH, but products manufactured with GDL do not have a traditional dry-sausage flavor. In contrast, the use of defined microorganisms has provided an acceptable means through which dry-sausage fermentation could be accelerated (4).

Starter cultures should be GRAS (generally recognized as safe)-approved. Laws vary from country to country, but only a few countries demand official permits for the use of specific starter cultures (7). Brazilian laws require that only GRAS microorganisms be used.

Bacterial starter cultures are very important for converting food into new products and for exerting preservative actions on the food. Their influence with regard to exerting preservative actions involves antagonistic action towards other types of microorganisms (8). This aspect of starter culture activity has aroused an interest in applying starter cultures to foods that traditionally do not undergo fermentation in order to enhance their preservation (8, 16).

These starters are usually obtained from synthetic media which are equal or similar to MRS broth. In addition, the cells are separated (centrifuged), washed in isotonic solutions and freeze-dried. These operations are very expensive. The cells produced will be used with the substrate (growing medium of molasses). The starter in this stage (with a known number of cells) will be added to the formulation of cured meats as the main purpose of a future study.

Cane molasses are by-products of the sugar industry. There are different kinds of cane molasses depending on the process utilized to obtain sugar. Among them, blackstrap molasse is considered the one which is no longer used to recover sugar, and is normally used for industrial fermentation. The overall composition of the various molasses differs according to specific geographical areas of production. In Brazil, molasses are the cheapest carbon sources in addition to being a good substrate which is rich in fermentable sugars and minerals.

The industrialization of meat products is changing with respect to productivity, quality and volume of products and Brazil must keep up with this trend. Meanwhile, the meat industry needs to import starters from Europe, the USA and most recently from Argentina if it wants to keep up with the latest trend.

The aim of this study was therefore to investigate the growth characteristics of Pediococcus acidilactici in sugar cane blackstrap molasses and to prepare the material to be used in salami manufacturing, in compliance with the requirements prescribed by the Brazilian laws.

Pediococcus acidilactici

The MRS broth was modified by the addition of 2.0% (MRS-2), 3.0% (MRS-3), 4.0% (MRS-4) and 5% (MRS-5) of sugar cane blackstrap molasses (w/v) to replace the glucose contained in the MRS broth formulation. The microorganism was conditioned to growth for 2 weeks in each broth. After that, it was maintained on MRS-5 broth agar slants at 4ºC to be used in all experiments.

The blackstrap molasses was diluted in 1:10 and centrifuged at 10,000 rpm per 15 min, after freezing at -40ºC for utilization in all experiments. The full composition of the molasses was not determined, but only the total reducing sugar, that which was 54.1% w/w (17).

The experiment was carried out in a shaker (New Brunswick) using Erlenmeyer flasks of 250 ml (180 rpm and 35ºC) with 50 ml of broth and inoculum levels of 3 x 10

³

The acid production was determined by measuring the volume of a solution of NaOH 2.5 M, delivered from a 50 ml graduated reservoir, added to maintain at constant pH of 5.0, 6.0 or 6.5 (± 0.2) during growth. In this case, the experiment was carried out in fermentation flasks under magnetic agitation in 500 ml of the MRS-2, MRS-3, MRS-4 and MRS-5 broth. This control was carried out at intervals of 60, 30 or 15 min depending upon the fermentation period.

Bacterial growth was estimated by turbidimetry with absorbance at 650 nm with reference to a standard curve of absorbance versus dry weight. Counting of Pediococcus acidilactici were determined in 1.0 ml of sample diluted in 9.0 ml of sterile water from which 0.1 ml was spread on agar medium. Colony forming units were enumerated on MRS agar (30ºC for 48 hours).

The productivity was calculated as yielded biomass/consumed sugar x 100.

MicrococcusLactobacillus Pediococcus

Lactic acid starter cultures are added to meat for reliable and consistent acid production in the preparation of fermented sausages. Acids produced by Pediococcus acidilatici lower the pH and contribute to the preservation of sausage products (15). This is an important aspect of the lactic acid bacteria, and the acid production during the phase of growth is a very good indicative of quality for meat fermentation. Fig. 1 shows the acid produced by P. acidilactici in a 20-hour growth period. Pediococcus acidilactici showed the highest acid production at a constant pH of 5.0 and in MRS-5 broth. The optimum growth pH to P. acidilatici is 6.5 (3). In this pH the acid production was lower than at pH 5.0. In order to determine acid at pH 6.5, 13.3 ml of a solution of NaOH 2.5 M was utilized to titrate 500 ml of the MRS-5 broth. At pH 5.0, 23.4 ml of the alkali for the same volume of broth was utilized. In MRS-2, MRS-3 and MRS-4 broth, the acid production was lower, maybe due to substrate limitation (carbohydrates). The growth of P. acidilactici is showed in Fig. 2 and can be compared with that in Fig. 1. The biomass production (dry weight) was higher when the initial pH was 6.5 in MRS-5. Meanwhile, similar biomass production was achieved in MRS-4. Hence when 5.0% molasses (MRS-5 broth) was utilized, the acid production was higher at a constant pH of 5.0 while the biomass production was higher at initial pH 6.5. We can say that the substrate was utilized for acid production at a lower pH (5.0) and for biomass production at a near optimum growth pH (6.5).

Figure 1
- Acid production of Pediococcus acidilactici in MRS broth (without glucose) with of 2.0% (MRS-2), 3.0% (MRS-3), 4.0% (MRS-4) and 5.0% (MRS-5) of sugar cane molasses, at constant pH of 5.0; 6.0 and 6.5.

Figure 2
- Growth of Pediococcus acidilactici in MRS broth (without glucose) with of 2.0% (MRS-2), 3.0% (MRS-3), 4.0% (MRS-4) and 5.0% (MRS-5) of sugar cane molasses, at initial pH of 5.0; 6.0 and 6.5.

The maximum biomass produced was 3.80 g/l at initial pH 6.5 after a 16-hour growth period in MRS-5 broth. As the total reducing sugar of the blackstrap molasses utilized in this experiment was 54.1% (w/w), the total reducing sugar was 1.082; 1.623; 2.164 and 2.705% corresponding to 2.0% (MRS-2); 3.0% (MRS-3); 4.0% (MRS-4) and 5.0% (MRS-5) molasses addition, respectively. Thus, fig. 3 shows the maximum biomass produced (3.80 g/l) with the addition of 5% molasses (MRS-5) but it was not the best result. Table 1 shows the carbohydrates consumed and the productivity on different molasses amounts after a 20-hour fermentation. The productivity was 28.16; 21.20; 19.31 and 16.31% corresponding to 2.0% (MRS-2); 3.0% (MRS-3); 4.0% (MRS-4) and 5.0% (MRS-5) molasses amounts, respectively.

Figure 3
- Growth of Pediococcus acidilactici in MRS broth (without glucose) with of 2.0% (MRS-2), 3.0% (MRS-3), 4.0% (MRS-4) and 5.0% (MRS-5) of sugar cane molasses, at initial pH 6.5.

Table 1 - Productivity of Pediococcus acidilactici in MRS broth (without glucose) with of 2.0% (MRS-2), 3.0% (MRS-3), 4.0% (MRS-4) and 5.0% (MRS-5) of sugar cane molasses, after 20 hours of fermentation at initial pH 6.5.

Productivity = biomass yielded / consumed sugar x 100

Several authors have reported that the growing of cells is better on low carbohydrates concentrations (14). In this experiment the same results were found, and the highest productivity of 28.16% was reached with 2.0% (MRS-2) molasses amounts. The data in Table 1 indicate that all experiments showed non-fermented sugars after a 20-hour fermentation, and the highest amounts in the highest molasses additions. As illustration, Table 2 shows the growth of P. acidilactici in 2.0% molasses amounts (MRS-2) and MRS broth at initial pH 6.5 during a 20-hour fermentation. For MRS-2 the highest growth rate verified (dx/dt) occurred in an 8 to 16 hour period (0.290 g cells/l.h) when the specific growth rate µ was 0.175 (h

^-1-1

Table 2 - Growth of P. acidilactici in MRS broth and in MRS-2 broth (without glucose) with 2.0% of sugar cane molasses, at initial pH 6.5 for a 20-hour fermentation period.

dx/dt= calculation of derivates using numerical differentiation.

(-) null or negative values.

Fig. 4 shows the viable cells of Pediococcus acidilactici in MRS-2, MRS-3, MRS-4 and MRS-5, for a 20-hour period. The maximum production was 12 Log

₁₀

Figure 4
- Viable cells of Pediococcus acidilactici in MRS broth (without glucose) with of 2.0% (MRS-2), 3.0% (MRS-3), 4.0% (MRS-4) and 5.0% (MRS-5) of sugar cane molasses, at initial pH 6.5.

Villavicencio (19) studied the growth of Pediococcus pentosaceus in sugar cane molasses and reported that 5.0% of molasses (24.25 g/l total reducing sugar) was a feasible substrate when improved with meat extract, yeast extract, peptone and minerals. In this case, the maximum specific growth rate µ was 0.391 (h

^-1

Comparing the growth in MRS broth and in MRS-2 broth, there is not a significant difference. The cells yield was higher in the MRS broth (2.95 g/l or 5.08% higher) in a 20-hour period. Other results, such as dx/dt; µ; rs and fermented sugar showed small differences.

The data obtained show that P. acidilactici grew in sugar cane blackstrap molasses and that it is possible to replace the MRS glucose source.

RESUMO

Crescimento de Pediococcus acidilactici em substrato de melaço de cana-de-açúcar

Pediococcus acidilactici (IL01) cresceu em caldo MRS (Man, Rogosa and Sharpe) modificado por adição de 2,0% (MRS-2), 3,0% (MRS-3), 4,0% (MRS-4) and 5,0% (MRS-5) de melaço de cana de açúcar, em substituição à glicose. A maior produção de ácido ocorreu em caldo MRS-5 com pH constante 5,0. A produção de biomassa foi mais acentuada em caldo MRS-5 com pH inicial de 6,5, embora a produtividade tenha sido maior em caldo MRS-2 (28,16%). Em caldo MRS-2 e em pH inicial de 6,5 durante uma fermentação de 20 horas, a velocidade de crescimento (dx/dt) foi maior entre a 8ª e 16ª hora (0,290 g celulas/L.h) enquanto a velocidade específica de crescimento µ foi 0,175 (h

^-1-1P. acidilactici

Palavras-chave: Pediococcus acidilactici, "starter", salame

Food technology

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J. Food Science

• ¹
  Bacus, J. N.; Brown, W. L. Use of microbial cultures: Meat products. Food technology, Jan: 74-77, 1981.
• ⁴
  Civentry, J.; Hickey, M.W. Growth characteristics of meat starter cultures. Meat Science, 30: 41-48, 1991.
• ⁵
  Deibel, R.H.; Niven, C.F. Pediococcus cerevisae: a starter culture for summer sausage. Bacteriol. Proc, 14: 1957.
• ⁶
  Everson, C.W.; Danner, W.E.; Hammes, P.A. Bacterial starter cultures in sausage products. J. Agr. Food Chem., 18: 570-571, 1970.
• ⁷
  Jessen, B. Fermented meats 1995, p.131-159.
• ⁸
  Gilliland, S.E. Bacterial starter cultures for foods CRC Press, Boca Raton, 1977, 185p.
• ⁹
  Hammes, W.P.; Bantleon, A.; Min, S. Lactic acid bacteria in meat fermentation. FEMS Microbiology Reviews, 87: 165-174, 1990.
• ¹⁰
  Marchesini, B.; Bruttin, A.; Romailler, N.; Moreton, R.S. Microbial events during commercial meat fermentations. J. Appl. Bacteriol, 73: 203-209, 1992.
• ¹¹
  Niinivara, F.P. Uber den einflurs van bakterienreinkulturen auf die reifung und umrotung der rohwurst. Acta agralia Fenica, 84: 1-128, 1955.
• ¹²
  Nurmi, E. Effect of bacterial inoculations on characteristics and microbial flora of dry sausage. Acta Agralia Fennca, 108: 1-77, 1996.
• ¹³
  Pederson, C.S. Microbiology of food fermentations 2. ed. AVI, Wesport, 1979, 384p.
• ¹⁴
  Salminen, S.; von Wright, A. Lactic acid bacteria. Marcel Dekker, New York, 1993.
• ¹⁵
  Smith, J.L.; Palumbo, S.A. Microorganisms as food additives. J. Food Prot, 44: 936-955, 1981.
• ¹⁶
  Smith, J.L.; Palumbo, S.A. Use of starter cultures in meats. J. Food Prot, 46: 997-1006, 1983.
• ¹⁷
  Somogy, M. A new reagent for the determination of sugars. J. Biol. Chem, 160: 6168, 1945.
• ¹⁸
  Vignolo, G.M.; Holgado, A.P.R.; Oliver, G. Use of bacterial cultures in the ripening of fermented sausages. J. Food Prot, 52: 787-791, 1989.
• ¹⁹
  Villavicencio, A.R.N. Avaliação da utilização do melaço da cana-de-açúcar, como substrato para o crescimento de Pediococcus pentosaceus. Florianópolis, 1996, 88p. (Mestrado. Departamento de Ciência e Tecnologia de Alimentos. UFSC).
• ²⁰
  Wu, W.H.; Rule, D.C.; Busboom, J.R.; Field, R.A.; Ray, B. Starter culture and time/temperature of storage influences on quality of fermented mutton sausage. J. Food Science, 56: 916-919, 1991.

Publication Dates

• Publication in this collection
  26 Feb 1999
• Date of issue
  Sept 1998

History

• Received
  20 May 1997
• Reviewed
  20 Oct 1997
• Accepted
  23 July 1998