Growth of Lactobacillus plantarum in media containing hydrolysates of fish viscera

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1 Journal of Applied Microbiology 2005, 99, doi: /j x Growth of Lactobacillus plantarum in media containing hydrolysates of fish viscera S.J. Horn 1, S.I. Aspmo 1,2 and V.G.H. Eijsink 1 1 Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, A s, Norway, and 2 Maritex AS, Sortland, Norway 2005/0042: received 17 January 2005, revised 26 May 2005 and accepted 27 May 2005 ABSTRACT S.J. H O R N, S. I. A S P M O A N D V. G. H. E I J S I N K Aims: To compare growth of Lactobacillus plantarum on media containing hydrolysates (peptones) from cod viscera with growth on commercial media. Methods and Results: Growth of Lact. plantarum on various fish peptones and commercial peptones/extracts was evaluated using both a Bioscreen apparatus (microtiter plates, no ph control) and fermentors (with ph control). Generally, the performance of the fish peptones was good and only beaten by the performance of yeast extract. Replacement of the 22 g l )1 complex nitrogen source in standard MRS medium with only 5 g l )1 fish peptone reduced the biomass yield with only 10%, whereas replacement with a mixture of 2Æ5 gl )1 fish peptone and 2Æ5 gl )1 yeast extract increased the biomass yield by 10%. Conclusions: Peptones derived from cod viscera support excellent growth of Lact. plantarum. Significance and Impact of the Study: We show that peptones derived from cod viscera are promising constituents of growth media for fastidious food bacteria such as lactobacilli. Media containing these peptones show excellent performance while problems associated with the use of meat-derived peptones (BSE, kosher status) or plant-derived peptones (genetically modified organisms) are avoided. Keywords: cod, fish hydrolysate, growth medium, lactic acid, Lactobacillus, NC8. INTRODUCTION Lactic acid bacteria (LAB) are used in the production of numerous fermented food products, where they contribute to important qualities such as taste, texture and shelf life (Stiles and Holzapfel 1997; Carr et al. 2002). Some LAB are thought to have probiotic effects, e.g. through their interactions with the human gut and its microflora (Fioramonti et al. 2003). The LAB genus Lactobacillus comprises fastidious bacteria with numerous growth requirements. Lactobacilli thus need rich media containing compounds such as amino acids, peptides, vitamins and nucleic acids. One of the most widespread Lactobacillus species used in Correspondence to: S.J. Horn, Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, 1432 Ås, Norway ( svein.horn@umb.no). food technology is Lactobacillus plantarum, which has a homofermentative metabolism, a high acid tolerance and is, like most LAB, a Generally Regarded As Safe (GRAS) organism. Experiments with defined media have shown that Lact. plantarum NC8 needs arginine, leucine, isoleucine, tyrosine, valine and pantothenic acid for growth (Møretrø et al. 1998). The most common medium for growth of lactobacilli in lab scale is MRS broth (de Man et al. 1960), which contains 22 g l )1 of a complex nitrogen source consisting of a peptone (protein hydrolysate) and extracts (from meat and yeast). The costs of both MRS medium and defined media are prohibitive for use in commercial applications (Zhang and Greasham 1999). Another disadvantage of MRS concerns the presence of constituents of bovine origin, which should be avoided in food production because of risks related to the occurrence of bovine spongiform encephalopathy (BSE; Wells et al. 1987). ª 2005 The Society for Applied Microbiology

2 L. PLANTARUM GROWTH ON FISH VISCERA 1083 Generally, an ideal medium for a food bacterium should not only be cheap and free of constituents of bovine origin, but also free of constituents of porcine origin (ensuring the ÔkosherÕ status) and free of plant materials (e.g. hydrolysates of soya proteins) for which the absence of genetically modified constituents cannot be guaranteed. This makes fish-derived peptones/extracts an interesting alternative for commonly used peptones/extracts, such as those present in MRS. Annually tons of cod (Gadus Morhua L.) are fished off the coast of Norway (Anonymous 2003). Viscera constitute up to 17% of the wet weight of the cod and represent an important fish processing waste, which, due to environmental legislation, no longer can be discarded into marine waters by land-based processing plants. This waste is a rich source of proteins and vitamins and is thus a promising source for ingredients in microbial growth media. Several microorganisms have been tested for growth on media containing fish materials (Krishnaswamy and Lahiry 1962; Green et al. 1977; Clausen et al. 1985; Gildberg et al. 1989; Vecht-Lifshitz et al. 1990; de la Broise et al. 1998; Dufosse et al. 2001; Poernomo and Buckle 2002; Vazquez et al. 2004). However, to our knowledge, the use of fish viscera in growth media has rarely been explored, despite the fact that viscera are a rich source of amino acids and other growth factors such as vitamins (Waterman 1968). We have previously described methods for effective solubilization of cod viscera, using different treatments with proteolytic enzymes (Aspmo et al. 2005). We have now evaluated the potential of several of the resulting hydrolysates (hereafter referred to as peptones) as components in a growth media for Lact. plantarum. Bacterial growth was evaluated both in a microtiter plate format without ph control (Bioscreen) and in fermentors with ph control. The performance of the fish peptone-based media was compared to media based on commercial peptones, including MRS. MATERIALS AND METHODS Microorganisms and media All growth experiments were done with Lact. plantarum NC8 (Aukrust and Blom 1992). The stock culture of this bacterium was stored at )80 C in MRS broth with 15% glycerol. All media were based on the MRS medium recipe (Oxoid) and contained (g l )1 ): glucose, 20.0, dipotassium hydrogen phosphate, 2Æ0, sodium acetate Æ3H 2 O, 5Æ0, triammonium citrate, 2Æ0, magnesium sulfate Æ7H 2 O, 0Æ2, manganese sulfate Æ4H 2 O, 0Æ05, complex nitrogen source (referred to as peptone or extract), 5Æ0 and TritonX100, 1 ml l )1 (note that the amount of complex nitrogen source in standard MRS is 22 g l )1 ). The media components were dissolved in distilled water, adjusted to ph 5Æ5 or6æ1 and sterilized by filtration (0Æ22 lm). The following peptones/extracts were tested: Bacto TM Tryptone from casein (Beckton Dickinson (BD), CA, USA), Bacto TM Soytone from soybeans (BD), Yeast Extract from yeast (Oxoid, Hampshire, UK), Lablemco meat extract (Oxoid), (NH 4 ) 2 SO 4 (Merck, Darmstadt, Germany) and Maritex Peptone F TM from fish silage (Maritex, Sortland, Norway). In addition three hydrolysates from cod (Gadus morhua L.) viscera (Aspmo et al. 2005) were tested. These hydrolysates were prepared by enzymatic hydrolysis for 24 h at 55 C with either endogenous enzymes only (Endo), or with endogenous enzymes supplemented with Papain (Pap) or Alcalase (Alc). The standard MRSmedium used for preculture growth and in reference cultures was obtained from Oxoid (CM0359; a home-made MRS variant containing only 5 g l )1 of the standard complex nitrogen source is referred to as ÔMRS5Õ). Culture conditions For the Bioscreen experiments, 300 ll medium (ph 6Æ1) in microtitre wells was inoculated with 5 ll inoculum (from an overnight culture). Growth curves (in triplicate) were recorded at 30 C by measuring the optical density (OD 600 ) of the cultures every 15 min in a Bioscreen C (Labsystem, Helsinki, Finland). The maximum cell density, OD max,was determined as the growth curves reached stationary phase. The maximum specific growth rate, l max, was determined by calculating the slope of the exponential growth phase (l max ¼ Dln(OD 600 )/Dt). Fermentations were carried out in 3 l fermentors (Applicon) using a 1 l working volume. The ph was automatically kept at ph 5Æ5 by controlled addition of 5 mol l )1 NaOH and the temperature was maintained at 30 C. The fermentation medium was initially flushed for 30 min with N 2 and then inoculated with 10 ml of an overnight preculture with a cell density of about 0Æ7 gl )1. Growth was monitored by taking regular samples and measuring the biomass concentration (g l )1 ). The l max was calculated using at least three samples taken in the exponential growth phase. All precultures were grown in MRS medium at 30 C for 20 h. Analytical methods Organic acids and glucose were analysed using a slightly modified variant of the method described by Marsili et al. (1981). One gram of sample (wet weight) was added to 0Æ2 ml0æ5 mol l )1 H 2 SO 4,2Æ5 ml deionized water and 8 ml acetonitrile (Rathburn Chemicals, Borders, UK), followed by mixing for 30 min. After centrifugation and filtration (0Æ2 lm), the samples were analysed using a 300 7Æ8 mm Aminex 87H HPLC column (Bio-Rad Labs., Richmond, CA, USA), held at 30 C and connected to a Perkin-Elmer

3 1084 S.J. HORN ET AL. HPLC (Perkin-Elmer, Norwark, CT, USA). The mobile phase was 10 mmol l )1 H 2 SO 4 and the flow rate was 0Æ4 ml min )1. Glucose and organic acids were identified according to their retention times. The system was externally calibrated using mixed standard solutions in deionized water, which had been treated in exactly the same way as the samples. Standard deviations for the values reported below were typically below 1% of the value. For the determination of cell dry weight, cells from 25 ml culture were collected by centrifugation (3500 g, 10 min), washed with distilled water, heated for 10 h at 105 C and weighed. Comparison of measurements of dry cell weight and OD 600 revealed a linear relationship, dry weight (g l )1 ) ¼ OD 600 0Æ260, with a correlation factor r 2 of 0Æ994 (OD 600 ¼ 0 8Æ3). Amino acids were determined using the Pico-tag method (Cohen et al. 1989). The analyses were carried out by, The National Institute of Nutrition and Seafood Research (NIFES) in Bergen, Norway. Total nitrogen contents and carbon/nitrogen (C/N) ratios were determined using a Dumas instrument (AOAC ). Ash contents were determined after heating samples at 540 C for 16 h. The details of these analyses are described elsewhere (Aspmo et al. 2005). RESULTS Some characteristics of the peptones/extracts used in this study are presented in Tables 1 and 2. Note that all peptones, except Bactosoytone, have rather similar contents of total nitrogen and total amino acids. Bactosoytone has a clearly lower content of nitrogen and amino acids than the other peptones/extracts. Table 1 Chemical specifications of nitrogen sources Name Total nitrogen (%) Ash (%) C/N (g g )1 ) Source Bactosoytone* 9Æ4 12Æ0 4Æ40 Soya Tryptone* 13Æ3 6Æ6 3Æ37 Casein YE* 11Æ4 13Æ1 3Æ86 Yeast Maritex 13Æ2 6Æ0 3Æ27 Silage of fish viscera (NH 4 ) 2 SO 4 15Æ1 N/D Lab Lemco* 13Æ3 N/D 3Æ15 Meat Endo 12Æ8 6Æ0 3Æ17 Fish viscera Pap 12Æ3 6Æ0 3Æ18 Fish viscera Alc 11Æ5 6Æ0 3Æ40 Fish viscera YE, yeast extract; Pap, Papain; Alc, Alcalase; Endo, endogenous; C/N, carbon/nitrogen. *As described in suppliersõ datasheets. Calculated. Bioscreen Figure 1 shows the growth of Lact. plantarum NC8 on standard MRS medium (containing 22 g l )1 of a complex nitrogen source) and on nine MRS-like media containing only 5 g l )1 of different nitrogen sources of varying complexity, namely three different peptones from cod viscera (Aspmo et al. 2005), five commercial peptones/ extracts, and ammonium sulfate. Growth on MRS yielded both the highest maximum OD (1Æ81) and the highest maximum growth rate (l max ¼ 0Æ60; see Table 3). Despite the fact that the other media contained only 5 g l )1 of the nitrogen source, several of these media yielded reasonable growth rates and maximum OD values which were only 10 20% lower than the rates obtained with MRS (Fig. 1, Table 3). The four fish-based media were among the bestperforming ones. The Alc, Pap and Endo peptones were made by hydrolyzing cod viscera with different enzymes, as described in Materials and methods and in Aspmo et al. (2005). Fig. 1 and Table 3 show that growth media based on the Endo and Pap peptones gave similar growth curves, while the Alcbased medium performed better that the other two, in terms of both growth rate and yield. Two of the commercial complex nitrogen sources, Maritex fish peptone and yeast extract, performed approximately equally well as Pap and Endo. The Maritex peptone is also produced from cod viscera, but through a different manufacturing process (acidification with formic acid and subsequent hydrolysis with endogenous pepsin). The other commercial peptones/ extracts performed less well, and no significant growth was observed in the ammonium sulfate medium. Fermentors The media that best supported growth in the Bioscreen experiments were also tested in fermentors with ph control. For all media, the growth rates (l max ) obtained in the fermentor set-up were higher than the rates obtained in the Bioscreen set-up (Table 3). MRS medium yielded the highest growth rate with a l max of 0Æ71. However, the highest biomass concentration (2Æ76 g l )1 ) was reached in a medium (MY) containing 2Æ5 gl )1 Maritex fish peptone and 2Æ5 gl )1 yeast extract. The MY medium also yielded a high growth rate (l max ¼ 0Æ62), only surpassed by the growth rate obtained with MRS. The three media containing peptones derived from hydrolysis of cod viscera at neutral ph (Alc, Endo, Pap) yielded very similar growth curves (Fig. 2) and performed better than the medium based on the commercial fish peptone (ÔMaritexÕ). Of the commercial peptones/extracts, yeast extract resulted in a slightly higher biomass production than the fish peptones, whereas growth on Bactosoytone and Bactotryptone was clearly inferior.

4 L. PLANTARUM GROWTH ON FISH VISCERA 1085 Table 2 Amino acid composition of selected nitrogen sources (mg g )1 ) Amino acid Bactotryptone* Bactosoytone* Yeast extract* Maritex Pap Alc Endo Rsd (%) Alanine Æ5 Arginine Æ0 Aspartate Æ0 Cysteine N/D N/D N/D N/D N/D N/D N/D Glutamate Æ0 Glycine Æ5 Histidin Æ0 Hydroxyproline N/D N/D N/D Isoleucine Æ0 Leucine Æ0 Lysine Æ5 Methionine Æ0 Phenylalanine Æ0 Proline Æ5 Serine Æ0 Taurine N/D N/D N/D Threonine Æ0 Tryptophan N/D N/D N/D N/D N/D 5 5 3Æ0 Tyrosine Æ0 Valine Æ0 Sum Essential amino acids for Lactobacillus plantarum NC8 are shown in bold. Rsd, relative standard deviation for the values in the Maritex, Pap, Alc and Endo peptones (percentage of the average value); N/D, not determined. *As described in suppliersõ datasheets. Table 3 Growth data from Bioscreen and fermentation experiments 1 6 Bioscreen Fermentation* O.D. (600 nm) Time (h) Fig. 1 Growth in batch cultures of Lact. plantarum at 30 C, as recorded automatically in a Bioscreen apparatus as optical density (OD) at 600 nm. The growth media are named after their complex nitrogen source, except MRS (see text) and MRS5 (see text). The concentration of complex nitrogen source was 5 g l )1 in all media except MRS, in which this concentration was 22 g l )1.( ) MRS; (s) Maritex; (4) Bactosoytone; (,) Bactotryptone; (() yeast extract; ( ) ammonium sulfate; (j) Lab Lemco; (black line) Papain; (dark grey line), Alcalase and (light grey line), Endogenous Medium l max OD max l max (g l )1 ) Max biomass MRS Æ81 0Æ71 2Æ52 MRS5 N/D N/D 0Æ51 1Æ64 Maritex 0Æ40 1Æ51 0Æ50 2Æ13 Endo 0Æ35 1Æ40 0Æ57 2Æ29 Pap 0Æ39 1Æ48 0Æ55 2Æ29 Alc 0Æ41 1Æ62 0Æ55 2Æ23 YE 0Æ44 1Æ37 0Æ57 2Æ45 YE + Maritex (MY) N/D N/D 0Æ62 2Æ76 Bactotryptone 0Æ28 1Æ26 0Æ48 0Æ96 Bactosoytone 0Æ31 1Æ00 0Æ43 1Æ51 Lab Lemco 0Æ40 0Æ95 N/D N/D AmSO4 0Æ07 0Æ07 N/D N/D OD, optical density; YE, yeast extract; Pap, Papain; Alc, Alcalase; Endo, endogenous. *Standard deviations were <7 and <3% of the value, for maximum biomass and l max, respectively. The growth media are named after their complex nitrogen source, except MRS (see text) and MRS5 (see text). The concentration of complex nitrogen source was 5 g l )1 in all media except MRS, in which this concentration was 22 g l )1.

5 1086 S.J. HORN ET AL. Biomass concentration (g l 1 ) Time (h) Fig. 2 Growth in batch cultures of Lact. plantarum, grown at ph 5Æ5 and 30 C in a fermentor with ph control. ( ) MRS; (s) Maritex; (4) Bactosoytone; (,) Bactotryptone; (() yeast extract; (d) Maritex + yeast extract; (+) MRS with 5 g l )1 of the complex nitrogen source (MRS5); (black line) Papain; (dark grey line) Alcalase and (grey line), Endogeneous MRS, despite being a very rich medium, yielded only 10% more biomass than the best of the fish peptone-based media, whereas the MY medium outperformed MRS by about 10%. To enable further comparisons with MRS, a modified MRS medium (MRS5) containing only 5 g l )1 of the complex nitrogen source (2Æ27 g l )1 Bactotryptone, 1Æ82 g l )1 Lab Lemco and 0Æ91 g l )1 yeast extract) was tested. While still yielding a reasonable l max of 0Æ51, biomass production in MRS5 was much lower than for most of the other media (Table 1). Figure 3 shows data from the growth of Lact. plantarum in MY-medium. Glucose was consumed and lactate formed Concentration (g l 1 ) Time (h) Fig. 3 Growth of Lactobacillus plantarum in Maritex-Yeast Extract (MY) medium in a fermentor with ph control (ph 5Æ5, 30 C). (d) biomass concentration; (s) lactate concentration; (() glucose concentration; (line) cumulative consumption of base Biomass concentration (g l 1 ) mol l 1 NaOH (ml) with a total yield of 0Æ84 g lactate per g glucose, while 38Æ8 ml 5 mol l )1 NaOH (0Æ19 mol NaOH) was consumed. As expected, the total amount of lactate produced (17Æ0 gl )1, equalling 0Æ19 mol) was identical to the amount of consumed NaOH. The halt in growth coincided with the exhaustion of glucose and the stop in base consumption. The other fermentations showed similar curves (results not shown). However, in the case of Bactosoytone, Bactotryptone and MRS5 the halt in growth occurred before all glucose was consumed. Figure 4 shows base consumption in all cultures. For the best-performing media, the curves in Fig. 4 are similar to the corresponding growth curves shown in Fig. 2 and the total amounts of consumed base correlate with the measured maximum biomass levels (Table 3). For example, the MY medium shows both the highest total base consumption and the highest level of produced biomass. Some deviations are observed though. The fermentations on the MRS and Maritex media both resulted in a total consumption of 37 ml base, despite the fact that the total amount of biomass produced in the slower growing Maritex culture was considerably lower than in the MRS culture. Deviations between base-consumption curves and growth curves are also observed for the cultures where growth stopped before all glucose was consumed (Bactosoytone, Bactotryptone and MRS5 cultures); in these cultures base-consumption (Fig. 4) continued after growth (Fig. 2) had stopped. Glucose was still present in the last sample taken (26 h) from the Bactotryptone fermentation. Thus the consump- Consumption of 5 mol l 1 NaOH (ml) Time (h) Fig. 4 Consumption of base in batch cultures of Lactobacillus plantarum grown at ph 5Æ5 and 30 C in a fermentor with ph control. ( ) MRS; (s) Maritex; (4) Bactosoytone; (,) Bactotryptone; ((), yeast extract; (d) Maritex + yeast extract; (+) MRS with 5 g l )1 of the complex nitrogen source (MRS5); (black line) Papain; dark grey line, Alcalase; (grey line) Endogenous

6 L. PLANTARUM GROWTH ON FISH VISCERA 1087 tion of 25 ml base (Fig. 4) does not represent the end point in the Bactotryptone fermentation. To investigate effects of different peptone/sugar ratios, some further fermentations were conducted with variants of the Alc medium. Use of 2 g l )1 instead of 5 g l )1 Alc peptone yielded a biomass concentration of 1Æ48 g l )1, and consumption of 32Æ9 ml base. The halt in growth occurred before all the glucose was consumed and before base consumption stopped (not shown). A medium containing 22 g l )1 Alc, i.e. the same concentration of complex nitrogen source as in MRS, yielded a biomass concentration of 3Æ71 (1Æ5 times higher than MRS) and led to consumption of 39Æ3 ml of base. A medium with increased glucose concentration (30 g l )1 ) and the standard 5 g l )1 Alc concentration yielded a biomass concentration of 2Æ60 g l )1 and led to consumption of 52 ml of base. As in the medium containing 2gl )1 Alc and 20 g l )1 glucose the halt in growth occurred before all the glucose was consumed and before base consumption stopped (not shown). To further study the potential of using byproducts from the food industry for LAB fermentation, the glucose (20 g l )1 ) in the medium based on the Maritex peptone was substituted with whey permeate resulting in a medium containing 23 g l )1 lactose. Growth in the resulting medium showed a rather long lag phase, but all lactose was consumed within 20 h, using a total of 40 ml 5 mol l )1 NaOH and reaching a maximum growth rate and biomass of 0Æ42 and 2Æ50 g l )1, respectively (results not shown). Thus, the yield of Lact. plantarum with this medium was close to the yields obtained with MRS or with the best of the media containing fish peptones and glucose. This preliminary result indicates that combination of waste materials from the fish and dairy industry may yield good media for growing LAB. DISCUSSION The general picture emerging from the growth experiments with Lact. plantarum NC8 described above is that media containing only 5 g l )1 of several of the tested peptones, including the fish-based peptones, result in growth rates and biomass yields which are only slightly lower than those obtained with the much richer MRS medium. The MY medium, containing 5 g l )1 of a mixture of two of the tested peptones, out-competed MRS in terms of biomass production. The three fish peptones which were produced at neutral ph (Endo, Pap and Alc) showed very similar performances in the fermentation experiments, while Alc performed better than the other two in the Bioscreen experiments lacking ph control. Table 2 shows that Alc, Pap and Endo have very similar amino acid compositions (as illustrated by the low standard deviations in the column labeled ÔRsdÕ). We have previously shown that the peptide profiles of Alc, Pap and Endo are somewhat different, Alc having the highest content of short peptides (Aspmo et al. 2005). Accordingly, Alc was found to have a higher alpha amino/total nitrogen ratio than Pap and Endo (Horn and Aspmo, unpublished results). One of the effects of such a higher degree of hydrolysis is that the Alc medium has a better buffer capacity than the Endo and Pap media, which may be an advantage in the Bioscreen experiments without ph-control. The Maritex peptone is also made from fish viscera and has a similar amino acid composition as Endo, Pap and Alc (Table 2). The fact that the Maritex peptone performed less well than the other three fish peptones in the fermentor experiments, may be due to the presence of low concentrations (0Æ19 g l )1 ) of formic acid, which may be inhibitory for Lact. plantarum NC8 (Woolford 1975). It is intriguing that media containing only 5 g l )1 of one of the fish peptones or yeast extract showed biomass yields that were only slightly (10%) lower than the yields obtained with MRS medium (containing 22 g l )1 complex nitrogen sources), both in the Bioscreen set-up and in the fermentor. This indicates that the nitrogen sources in MRS are in excess and are not incorporated into biomass. The MRS medium was originally composed to sustain growth of lactobacilli that did not grow well in tomato juice medium (de Man et al. 1960). Thus, this medium was designed to support growth of the most fastidious LAB and may therefore contain an excess of nutrients when used for less nutrient-demanding species such as some lactobacilli. It has been shown that essential growth factors for Lact. plantarum NC8 are arginine, leucine, isoleucine, tyrosine, valine and pantothenic acid (Møretrø et al. 1998). Table 2 shows that all the essential amino acids are found in the fish peptones, in amounts which seem balanced and which are comparable with those in e.g. yeast extract (which performs slightly better than the fish peptones). Apparently, the fish peptones also contain sufficient amounts of pantothenic acid (vitamin B2). Møretrø et al. (1998) also showed that nicotinic acid, biotin, folic acid, pyridoxal, adenine and uracil stimulate growth of Lact. plantarum NC8, while not being essential. It is conceivable that these components are most abundant in the rich MRS medium, which may explain that MRS yielded the highest l max values in both Bioscreen and fermentor experiments. It should be noted that the internal organs, i.e. viscera, of cod have been shown to contain much higher concentrations of vitamins than, e.g. cod muscle (Waterman 1968). Thus the fish peptones used in this work are probably better for growth of LAB than peptones produced from fish muscle or whole fish. In the fermentor experiments, MY medium yielded a higher biomass than any of the other tested media with 5 g l )1 of a complex nitrogen source. This result indicates synergetic effects between yeast extract and the fish peptone, possibly caused by complementarity in the amino acid compositions

7 1088 S.J. HORN ET AL. (Table 2). For example, the Maritex peptone contains 60% more of the essential amino acid arginine than yeast extract, while yeast extract contains 42% more of the essential amino acid isoleucine than the Maritex peptone. Yeast extract is known to be a rich source of vitamins, which may contribute to its suitability for growing LAB in general, and which may complement possible deficiencies in the fish peptones. Media with Bactotryptone, Bactosoytone, Lab Lemco or ammonium sulfate as nitrogen sources did not perform well in the Bioscreen experiments. Two of these, Bactotryptone and Bactosoytone, were tested in the fermentor were they also showed inferior performance. The bad performance of the ammonium sulfate medium was to be expected for a bacterium that has specific amino acid and vitamin requirements. The bad performance of the other three-nitorgen sources is less straightforward to explain and may be due to suboptimalities in amino acid composition, peptide length and composition and/or vitamin content. Kwon et al. (2000) reported that Bactosoytone contains too low levels of vitamins to support efficient growth of Lactobacillus rhamnosus. The content of pantothenic acid in Bactosoytone was found to be only 5% of the content in yeast extract. In addition to lack of vitamins, the relatively low level of amino acids, particularly the essential amino acids isoleucine, leucine and valine (Table 2), may explain the inferior performance of the Bactosoytone medium. Since the amino acid content and composition of Bactotryptone are similar to those in yeast extract (Table 2), it is conceivable that the bad performance of the Bactotryptone medium is due to low levels of pantothenic acid or other essential growth factors. The experiments with different glucose/alc ratios demonstrated that the combination 20 g l )1 glucose and 5 g l )1 peptone used as a standard in this study yields a wellbalanced medium, in which the peptone concentration is kept to a minimum, while being sufficiently high (at least for the better peptones) to support utilization of all available glucose. Higher glucose/alc (20/2 or 30/5 g l )1 ) ratios led to a halt in growth before all the glucose was consumed. Interestingly, increase of the amount of Alc to 22 g l )1 in a medium containing the standard 20 g l )1 glucose led to a 67% increase in biomass concentration. Thus, the same amount of glucose led to significantly more biomass in a medium with 22 g l )1 Alc than in standard MRS with 22 g l )1 of the complex nitrogen source. This is consistent with the general notion that during LAB fermentation, sugar is only a catabolic substrate leading to metabolic end products and energy, while biomass is formed from anabolic precursors such as amino acids (Novak and Loubiere 2000). Some glucose may be utilized in anabolic reactions (Novak and Loubiere 2000), but under conditions of a low glucose/ peptone ratios, and hence an excess of anabolic precursors, most of the glucose is presumably used as a catabolic substrate. The results thus indicate that Alc has more, and/ or a better composition, of such anabolic precursors than the complex nitrogen source used in MRS. The current study shows that media containing fish peptones provide a good alternative to MRS for growing Lact. plantarum and other LAB with similar nutritional requirements. The fish peptones are promising ingredients of industrial media for Lact. plantarum and other food bacteria, not only because they support growth very well, but also because fish are not associated with meat-related diseases such as BSE nor with plant materials that may contain components derived from genetically modified organisms (e.g. soya). Our own preliminary data indicate that good media may be developed, by combining fish peptones with lactose-containing waste from the dairy industry, such as whey permeate. Thus, further development of media for LAB on the basis of the results described above may eventually lead to better utilization of important waste streams from both fisheries and the dairy industry. ACKNOWLEDGEMENTS This work was supported by the Norwegian Research Council, project No /130, MABIT (Marine Biotechnology in Tromsø, Norway), and The Norwegian Raw Fish Association. 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