Use of hydrolysates from Atlantic cod (Gadus morhua L.) viscera as a complex nitrogen source for lactic acid bacteria

Authors

  • Stein Ivar Aspmo,

    1. Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003 1432 Ås, Norway
    2. Maritex AS Havnegt, 17 N-8400 Sortland, Norway
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  • Svein Jarle Horn,

    Corresponding author
    1. Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003 1432 Ås, Norway
    • Corresponding author. Tel.: +47 64947703; fax: +47 64947720., E-mail address: svein.horn@umb.no

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  • Vincent G.H. Eijsink

    1. Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003 1432 Ås, Norway
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  • Edited by W. Kneifel

Abstract

Hydrolysates of cod viscera were tested as an alternative to commonly used complex nitrogen sources (peptones and/or extracts) for the type strains of the lactic acid bacteria Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus sakei and Pediococcus pentosaceus. Comparative studies with MRS-like media containing different nitrogen sources showed that all the fish hydrolysates performed equally well or better than commercial extracts/peptones for all selected lactic acid bacteria.

1Introduction

Lactic acid bacteria (LAB) are important, fastidious food bacteria, which need rich growth media containing compounds such as amino acids, peptides, fatty acids, vitamins and nucleic acids. These compounds are usually provided in the form of a complex nitrogen source. For example, the standard laboratory medium for lactobacilli (MRS [1]), contains 22 g/L of a mixture consisting of a peptone (protein hydrolysate) and extracts from meat and yeast. Because of outbreaks of bovine spongiform encephalopathy (BSE) [2], and a growing demand for raw materials that are kosher approved and certified free of swine flu, there is a growing interest in alternative non-bovine/non-porcine complex nitrogen sources.

The use of fish materials as a source of nutrients for microorganisms was reported as early as 1949 [3]. Since then, several attempts to explore the use of fish peptones as a component of microbial growth substrates have been reported [4–11]. Recently, we have shown that hydrolysates of cod viscera, a hitherto scarcely explored, but abundant, raw material, perform well in standard growth media for several types of bacteria, including Lactobacillus sakei, the most fastidious of all LAB [12]. In the present study, we have further explored the potential of these new types of fish hydrolysates for growing LAB, by analyzing growth of type strains of several industrially relevant LAB on MRS-like media containing different complex nitrogen sources.

2Materials and methods

Sterile filtered (0.22 μm-Steritop™, Millipore, USA) fish viscera hydrolysates were produced by hydrolysis of cod viscera with endogenous enzymes at neutral pH (“Endo”), in some cases supplemented with Alcalase (Novozymes, Denmark; “Alc”) or papain (Biochem Europe, Belgium; “Pap”), as described by Aspmo et al. [13]. Special measures were taken to create non-oxidizing conditions (full reaction vessels, use of degassed water). The following commercial nitrogen sources were used: Bacto™ Tryptone (Beckton Dickinson, USA), Bacto™ Soytone (BD), Bacto™ Yeast Extract (Oxoid, UK), Lab lemco (Oxoid), L37 Bacteriological peptone (Oxoid), S490–70 salmon peptone (Seagarden, Norway) and Peptone F from cod silage (Maritex, Norway). Media were prepared according to the recipe for commercial MRS (as provided by Oxoid.com), but with only 5 g/L (instead of 22 g/L) of nitrogen containing components. This low concentration was selected after initial trial experiments had shown that a concentration of 5 g/L was sufficiently high to permit normal growth (i.e., in the same range as growth on standard MRS medium), while being sufficiently low to permit detection of differences in the growth performances of the nitrogen sources. The media contained (g/L): glucose, 20.0, dipotassium hydrogen phosphate, 2.0, sodium acetate · 3H2O, 5.0, triammonium citrate, 2.0, magnesium sulphate · 7H2O, 0.2, manganese sulphate · 4H2O, 0.05, complex nitrogen source, 5.0 and Tween 20, 1 ml/L. The pH was adjusted to 6.1 with 1 M NaOH for all media components (peptone, salt-mix and glucose solutions) before autoclaving.

Cultures (in triplicate) were started by inoculating 330 μl medium with 5 μl inoculum from a culture in late exponential growth phase, grown on standard MRS (Oxoid, UK). Growth curves were monitored by measuring the optical density (OD600) of the cultures every 15 min in a Bioscreen C apparatus (Labsystem, Helsinki, Finland). Before each measurement, the culture containing plates were shaken for 5 s at medium strength setting. The maximum cell density, maxOD600, was determined as the growth curves reached stationary phase. To determine maximum growth rate, μmax, growth curves were constructed by plotting ln(OD600) against incubation time. To determine the maximum specific growth rate, μmax=Δln(OD600)t, growth rates were determined for all sets of two consecutive measurements, i.e., between two time points (usually 15 min apart). The μmax for one growth experiment was defined as the average of the two highest of these growth rates.

Type strains were selected from the ATCC culture collection and grown at recommended temperatures. Lactococcus lactis ATCC 15346, Lactobacillus acidophilus ATCC 4356B, Lactobacillus helveticus ATCC 15009, Lactobacillus casei ATCC 334B and Pediococcus pentosaceus ATCC 33314 were grown at 37°C, while Lactobacillus sakei ATCC 15521 was grown at 30°C.

Total nitrogen and carbon contents were determined using a Dumas instrument (AOAC 990.03). For Dumas analysis tin cups with 80 μl samples were dried in an excicator over night, closed, and exposed to combustion in the Dumas instrument. Amino nitrogen was determined by triplicate measurements according to the method described by Nielsen et al. [14]. For the determination of the percentage of dry matter, samples were weighed before and after drying in air at 105°C for at least 16 h. Ash percentages were calculated by reweighing the samples subsequent to a 16 h incubation of the dried matter at 550°C.

3Results

Seven LAB were grown on ten MRS-like media only varying with respect to the complex nitrogen source, present at a concentration of 5 g/L (as opposed to 22 g/L in standard MRS). The chemical compositions of the 10 different nitrogen sources, three hydrolysates of cod viscera and seven commercial peptones are presented in Table 1. The nitrogen contents of the commercial media varied between 9.4% and 15.2%. The nitrogen contents of the cod viscera hydrolysates were between 11.5% and 12.8%. The commercial Peptone F from cod silage contained 13.2% nitrogen. The α-amino content varied from 1.5% for Soytone (BD) to 5.2% for Peptone F.

Table 1. Specifications for nitrogen sources –% of dry matter
NameTotal nitrogenAmino nitrogenAshSource
  1. aAs described in supplier's datasheet.

Bacto™ Soytonea9.41.512.0Soya
Bacto™ Tryptonea13.33.96.6Casein
Bacto™ Yeast Extracta11.43.713.1Yeast
Maritex Peptone F13.25.26.0Silage of cod viscera
Seagarden S490–7012.8N/D4.3Salmon
Oxoid L37 Bacteriological peptonea15.22.9N/DMeats
Oxoid Lab Lemcoa13.32.5N/DMeats
Endo12.83.16.0Cod viscera
Pap12.33.56.0Cod viscera
Alc11.54.36.0Cod viscera

An example of growth curves obtained is depicted in Fig. 1, whereas all results, in terms of maximum cell density (maxOD600) and specific growth rate (μmax) are depicted in Tables 2a and 2b, respectively. The results show that the peptones derived from cod viscera (The Endo, Pap, and Alc hydrolysates) outperform the commercial peptones/extracts for all LAB except Lb. casei. In the case of Lb. casei, only Alc outperformed the non-fish peptones, while the other fish peptones showed poor performance.

Figure 1.

Growth curves for Pediococcus pentosaceus ATCC 15521. Optical density was recorded every 15 min and every 10th point is shown as a symbol. All points are averages of triplicate measurements.

Table 2a. Growth data – maxOD600a
  Lc. lactis Lb. sakei Lb. acidophilus Lb. casei Lb. helveticus P. aciditactici
 ATCC15346ATCC15521ATCC4356BATCC334BATCC15009ATCC33314
  1. aThe highest value for each organism is highlighted in bold.

  2. bStandard Oxoid MRS contains in total 22 g/L nitrogen source/peptone while the other media contain 5 g/L.

Bacto™ Tryptone0.8750.6400.9620.1820.2750.953
Bacto™ Soytone1.1500.7300.9640.4630.2931.051
Bacto™ Yeast Extract1.3760.6841.3421.1100.4091.281
Maritex Peptone F1.4831.1511.5150.1530.5491.469
Endo1.6210.8771.4090.2020.4551.695
Pap1.6160.6941.6660.2240.4631.652
Alc 1.726 1.435 1.777 1.413 1.045 1.729
Seagarden S490-700.9660.8081.0990.6210.3890.968
Oxoid L37 Bacteriological peptone0.5150.4730.6410.2570.2760.783
Oxoid Lab Lemco1.0630.7091.0340.2660.2800.844
Oxoid MRSb1.7741.7061.8031.9131.8791.877
Table 2b. Growth data – specific growth rate, μmax,(h−1)a
  Lc. lactis Lb. sakei Lb. acidophilus Lb. casei Lb. helveticus P. aciditactici
 ATCC15346ATCC15521ATCC4356BATCC334BATCC15009ATCC33314
  1. aThe highest value for each organism is highlighted in bold.

  2. bStandard Oxoid MRS contains in total 22 g/L nitrogen source/peptone while all other media contain only 5 g/L.

Bacto™ Tryptone0.490.310.550.140.210.41
Bacto™ Soytone0.580.250.670.150.190.31
Bacto™ Yeast Extract0.760.180.750.310.190.43
Maritex F0.730.350.710.200.160.51
Endo0.810.210.710.18 0.23 0.56
Pap0.830.210.720.220.220.59
Alc 0.98 0.37 1.00 0.32 0.21 0.73
Seagarden S490–700.500.330.570.220.180.32
Oxoid L37 Bacteriological peptone0.410.250.640.210.190.33
Oxoid Lab Lemco0.630.280.690.220.170.40
Oxoid MRSb0.980.391.010.390.440.90

The Endo, Pap and Alc hydrolysates were produced from the same raw material, and have similar amino acid compositions [12]. Nevertheless, media containing these hydrolysates showed clear differences in growth performance. In all cases, Alc outperformed the other two hydrolysates (as well as the two commercial fish peptones), and in several cases the difference between Alc and the other two is huge, especially in terms of maxOD600 (e.g., for Lb. casei, Lb. sakei and Lb. helveticus). The choice of proteolytic enzyme thus clearly affects the growth performance that may be obtained when using peptones derived from the same raw material.

4Discussion

The superior performance of peptones obtained with Alcalase may be explained in several ways. One explanation is that Alcalase leads to optimal uptake of the available amino acid resources because this endoprotease's activity is most complementary to the bacteria's proteolytic and peptide-uptake systems [15]. We have previously shown that Alc, Pap and Endo have different peptide profiles, with Alc having a higher content of short peptides [12] and, accordingly, a higher content of α-amino groups (Table 1). Another possible explanation is that the Endo and Pap peptones contain peptides, which are inhibitory for some of the tested strains, and which are broken down (or never emerge) in hydrolysates obtained with Alcalase [16]. Hydrolysates of fish viscera produced under oxidizing conditions (S.I. Aspmo and S.J. Horn, unpublished observations) were inferior to the hydrolysates produced here, indicating that preservation of vitamins [17], antioxidants or unsaturated fatty acids [18] may also be of importance for peptone quality.

Direct measurements of optical density in a Bioscreen apparatus provide values which cannot be compared directly to values obtained when measuring in a spectrophotometer, mainly because the cultures cannot be diluted such as to yield accurate OD600 values (that is in the range below 1.0 [17–19]). Because of loss of linearity at OD600 values above 1, the real maxOD600 values are much higher than the values of 1.7–2.0 recorded in the Bioscreen apparatus [19]. However, Fig. 1 and the data presented in Tables 2a and 2b show that the Bioscreen experimental set-up permits clear discrimination between the various peptones, which was the purpose of the present study.

For several strains, the performance of the medium containing 5 g/L Alc was almost as good as the performance of commercial MRS, indicating that Alc and related hydrolysates are promising complex nitrogen sources for LAB. The use of fish viscera-derived peptones may reduce the need for meat-derived nitrogen sources in fermentation of LAB. The use of fish viscera-based peptones may facilitate kosher approval of LAB applications, may abolish BSE related problems, and generally allows the LAB to grow well on media containing reduced peptone concentrations.

Acknowledgements

This work was funded by the Norwegian Research Council, project no. 145616/130, MABIT (Marine Biotechnology in Tromsø, Norway), The Norwegian Raw Fish Association, and Maritex AS (Sortland, Norway). The authors are grateful to Linda Godager and Ingolf Nes at the Department of Chemistry, Biotechnology and Food Science, at the Norwegian University of Life Sciences, for providing lactic acid bacteria strains.

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