Selection of Autochthonous Strains as Starter Cultures for Fermented Fish Products Barbara Speranza, Angela Racioppo, Antonio Bevilacqua, Luciano Beneduce, Milena Sinigaglia, and Maria Rosaria Corbo
This paper was the 1st research focusing on the design of a halophilic lactic starter for the production of fermented fish products using a quantitative approach, based on the evaluation of the growth index and acidification score, as well as on the use of a multivariate approach to select the most promising strains. Fifty-nine strains were randomly selected from salted fish and phenotypically characterized through Gram staining, catalase activity, glucose metabolism, H2 S and indole production, nitrate reduction, citrate utilization, and hydrolysis of arginine, esculin, casein, gelatin, starch, Tween 80, and urea. Then the Gram positive isolates (44 out of 59) were studied for their growth at different temperatures (10, 25, 40, and 55 °C), salt (0%, 20%, and 30%), pHs (4.5 and 9.5), and acidification score in lab medium. Data were modelled through growth index and used as input to run a preliminary cluster analysis and a principal component analysis. Three promising strains were selected, identified as members of the genus Pediococcus and used for the validation at laboratory level through the assessment of their performances in the production of a fermented fish sauce. The results were really promising as their use not only reduced the fermentation time (2 d) but also improved the microbiological quality of the final product. This paper represents a 1st report on the use of a simple step-by-step methodology to select promising halophilic strains for the optimization of a starter for fish-fermented products.
Abstract:
Practical Application: The isolation, targeted selection, characterization of halophilic bacteria, or strongly halotolerant microorganisms, as well as their appropriate use, could be of great interest to standardize the quality of fermented fish products. The use of autochthonous cultures to produce a fish-type product is attracting increasing interest, as it could increase processing rates and product consistency, improve the sensory characteristics and microbiological quality, and shorten the fermentation time.
Introduction
ones, so representing the most important of all fermented fish products (Adams 2009). In recent years, using pure bacterial cultures to produce a fishtype product is attracting increasing interest. The use of starters in food fermentations has become a mean to increase processing rates and product consistency improving the sensory characteristics and microbiological quality and shortening the fermentation time (Visessanguan and others 2006). As generally observed for meat and dairy fermented products, various researchers have stated that the use of lactic acid bacteria (LAB) could significantly improve also the quality of fish products accelerating the formation of lactic acid and significantly inhibiting the growth of spoilage bacteria and pathogens (Kose and Hall 2011). LAB are found to be as the dominant microorganisms in many fermented fish products where their primary role is to ferment available carbohydrates and thereby cause a decrease in pH. The combination of low pH (below 4.5) and organic acids (mainly lactic acid) is the main preservation factor in this kind of products. In addition, the use of salt and spices (such as garlic, pepper, or ginger) can contribute to additional safety to these products. Salt concentration may range from 1% to 20% (w/w), thus the fermentation by halophilic LAB could be an advantageous solution. To avoid spontaneous fermentations that are not easily controllable, it would be appropriate to select halophilic bacterial starters, in order to optimize and control the MS 20141557 Submitted 9/17/2014, Accepted 10/22/2014. Authors are with evolution of biochemical processes that occur during the proDept. of the Science of Agriculture, Food and Environment (SAFE), Univ. of Foggia, Via Napoli 25, 71122, Foggia, Italy. Direct inquiries to author Corbo (E-mail: duction cycle of the products concerned. The isolation, targeted selection, characterization of halophilic bacteria or strongly [email protected]). tolerant microorganisms, as well as their appropriate use, could
Fish fermentation is one of the most common methods of seafood preservation. It has many benefits and could be used as a low-cost convenient technique for the preservation of fish muscle, improving the organoleptic qualities of fish and increasing the nutritional value and/or digestibility of the raw material. Nowadays, fermented fish products are largely confined to east and south-east Asia, although some products are being to be produced elsewhere. Most of the fermented fish products are still produced on a cottage industry or domestic scale, so there are numerous variants of some common themes and a host of local names used to describe them. Essentially, fermented fish products can be divided into 2 main categories: (1) fish/salt products including fish pastes and sauces that contain relatively high levels of salt, typically in the range 15% to 25% and are used mainly as a condiment; (2) fish/salt/carbohydrate products ranging from those that resemble the fish sauces and pastes in which an extensive autolysis has occurred, to products analogous to other lactic fermented foods, where the bacterial production of lactic acid is a major feature (Kose and Hall 2011). In economic terms, the fish sauces are produced on the largest scale being exported from oriental countries to European and North American
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12721 Further reproduction without permission is prohibited
Vol. 80, Nr. 1, 2015 r Journal of Food Science M151
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Keywords: fermented fish, growth index, quantitative approach, starter selection
% Identity
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Halotolerant bacteria collection This study focused on 59 isolates of bacteria recovered from salted anchovies (Engraulis encrasicholus) purchased from 3 different local markets (Foggia, Italy). The samples were stored at 3 different temperatures (4, 15, and 30 °C) and bacterial isolations carried out after 0, 3, 6, and 9 d of storage. Fish samples (25 g) were aseptically transferred in a sterile plastic bag (Seward, London, England) and homogenized for 1 min in a stomacher Lab-blender 400 (Seward) using 225 mL of sterile saline solution (9 g/L NaCl). Decimal dilutions of the samples were plated on JCM377 medium incubated at 37 °C for 1 wk. The composition of the medium was the following: 100 g/L NaCl, 5 g/L amino acids of the casein (Amicase, Sigma Aldrich, Milan), 5 g/L yeast extract (Oxoid, Milan, Italy), 1 g/L glutamic acid (Sigma Aldrich), 2 g/L KCl, 3 g/L trisodium citrate, 20 g/L MgSO4 ·H2 O, 36 mg/L FeCl2 ·4H2 O, 0.36 mg/L MnCl2 ·4H2 O, 20 g/L agar (pH 7.5; unless otherwise specified, the salts were of C. Erba, Milan, Italy). From each plate, 5 to 10 colonies were randomly selected and stored at 4 °C on JCM377 agar slants; the isolates were labeled with a numeric code ranging from 1 to 59. Phenotyping Initial typing was based upon Gram staining and microscope examination, followed by evaluation of catalase activity, glucose metabolism (homo or hetero-fermentative metabolism), H2 S and indole production, nitrate reduction, and citrate utilization; all these tests were performed as described by Barrow and Feltham (1993). Hydrolysis of arginine, esculin, casein, gelatin, starch, Tween 80, and urea were also determined (Namwong and others 2005). Growth assays These assays were conducted in aliquots of JCM377, inoculated to 5 log cfu/mL with each strain separately, adjusted to different amounts of NaCl (0%, 20%, and 30%), pHs (4.5 or 9.5) through HCl or NaOH 1.0N or incubated at different temperatures (10, 25, 40, and 55 °C). Microbial growth was evaluated every 24 h (for 7 d) by reading absorbance at 600 nm through a UV-Vis Spectrophotometer; M152 Journal of Food Science r Vol. 80, Nr. 1, 2015
Table 2–Sequence comparison with reference 16S ribosomal RNA sequences in GenBank database.
Materials and Methods
KJ997937 KJ997939 KJ997938 1489 1481 1476
Strain sequence length
be of great interest to standardize the quality of fermented fish products. Thus, the main goal of this paper was the selection of promising halophilic autochthonous strains as potential starter cultures for fermented fish products using a step-by-step procedure. The research was divided in different phases: (1) evaluation of some simple traits: growth at different temperature, with salt added and at various pH, acidification of a laboratory medium; (2) selection of the most promising strains through a multivariate approach and molecular identification; and (3) validation of the selected strains through the assessment of their performances in the production of a fermented fish sauce.
99 99 99
1.5
NR_042058.1 NR_042057.1 NR_042057.1
pH decrease (pH)
75%
Reference accession no.
Growth index
Reference sequence
0 1 2
Strain
Codes
Pediococcus pentosaceus strain DSM 20336 16S ribosomal RNA gene, complete sequence Pediococcus acidilactici strain DSM 20284 16S ribosomal RNA gene, complete sequence Pediococcus acidilactici strain DSM 20284 16S ribosomal RNA gene, complete sequence
Table 1–Qualitative codes for the multivariate analyses.
41 17 51
Strain separation accession no.
Halophilic starters for fish products . . .
Halophilic starters for fish products . . . Table 3–Descriptive indices for GI (growth index) after 48 h in JCM377 added with NaCl, adjusted to pH 4.5 or 9.5 or incubated at 10, 25, 40, and 55 °C or for acidification (pH) performed by the isolates in the same medium after 24, 48, and 120 h. Growth index after 48 h
Arithmetic mean Median Minimum Maximum 1 quartiles 3 quartiles Standard deviation Coefficient of variation (CV, %)
Acidification
NaCl 0%
NaCl 20%
NaCl 30%
10 °C
25 °C
40 °C
55 °C
pH 4.5
pH 9.5
24 h
48 h
120 h
55.25 26.60 4.29 210.35 12.59 89.01 61.44 111
49.82 55.78 7.30 78.41 26.42 70.40 23.45 47
16.17 7.10 0.00 45.99 2.17 33.94 15.86 98
10.26 1.87 0.00 75.81 0.00 4.54 20.15 196
74.36 74.83 26.70 157.51 49.89 90.22 27.21 37
79.45 70.76 34.22 223.60 58.17 87.68 35.26 44
3.13 2.54 0.00 20.85 0.00 4.32 3.79 121
15.80 6.87 1.33 124.09 4.71 12.83 23.57 149
15.17 12.31 0.00 77.34 2.05 21.62 16.22 107
1.05 1.17 0.52 1.40 0.78 1.29 0.29 28
1.50 1.62 0.81 1.80 1.37 1.70 0.29 19
1.67 1.70 1.38 1.91 1.55 1.77 0.14 8
The assays were performed on the Gram positive isolates (n = 44).
GI =
Abss × 100 Absc
Aliquots of JCM377 containing glucose (10 g/L) were individually inoculated with each strain to 5 log cfu/mL and incubated at 37 °C; the pH of the medium was evaluated after 24, 48, and 120 h through a pH-meter Crison (Crison Instruments, Barcelona,
Figure 1–Frequency histogram of Growth Index in JCM377 incubated at 10, 25, 40, and 55 °C. GI range was divided into 10 homogenous classes; the numbers on the bars indicate the isolates within each class. Vol. 80, Nr. 1, 2015 r Journal of Food Science M153
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samples of not-modified JCM377 (10% NaCl–pH 7.5), inoculated where Abss is the absorbance of the samples at different temas reported above and incubated at 37 °C, were used as controls. peratures, pH, or added with salt, whereas Absc is the absorbance The experiments were performed over 2 different batches; for of the control. each batch the experiments were repeated twice. For each time point, data were modeled as growth index, as reported by Blaszyk and Holley (1998) and Bevilacqua and others (2010): Acidification in lab medium
Halophilic starters for fish products . . .
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Spain). The experiments were performed in duplicate over 2 dif- 30 °C for 48 h under anaerobiosis for LAB. The pH was evaluated on homogenized samples with a pH meter (Crison, Barcelona, ferent batches. Spain). Selection of suitable strains and identification of isolates The analyses were performed twice over 2 different batches; Data of growth assays, as well as the results of acidification, were the results were analyzed through one-way analysis of variused for the evaluation of the descriptive statistical indices (arith- ance (ANOVA) and Tukey’s test as the post hoc comparison test metic mean, median, standard deviation, coefficient of variation, (P < 0.05). 1 and 3 quartiles) and to build frequency histograms, reporting on the x-axis growth index or pH decrease. Thereafter, they were converted into qualitative codes, as reported in Table 1, and used to run a cluster analysis and divide the strains in some homogeneous group, through the approach of dissimilarity. Then, the variables were used to carry out a principal component analysis to choose the most promising strains for the validation in a fermented fish sauce. The statistical analyses were performed through the software Statistica for Windows (Statsoft, Tulsa, Okla., U.S.A.). Selected strains for validation experiments (strains 17, 41, and 51) were identified by sequencing the 16SrDNA. Briefly, DNA of each strain was extracted with Ultraclean Microbial DNA Isolation kit (MoBio, Carlsbad, Calif., U.S.A.) and amplified in a PCR reaction with BSF8/BSR1510 primers targeting 16SrDNA genes (Eden and others 1991). PCR program was: initial denaturation at 95 °C for 5 min; 30 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 45 s, and extension at 72 °C for 1 min. PCR products were sequenced by Primm Biotech (Milan, Italy) with either the forward and the reverse primer in separate reactions. The sequences obtained were assembled into a consensus sequence using Mega 6.06 software (Tamura and others 2013). Assembled sequences were compared with the sequences available in the GenBank database using the Basic Local Alignment Search Tool version 2.2.27 (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences are freely available with the following accession nr: KJ997937, KJ997938, and KJ997939 (Table 2).
Validation at laboratory level: production of fish sauce The fish sauces were prepared using gilthead sea bream fillets (Sparus aurata) kindly furnished from Lepore Mare (Fasano, Brindisi, Italy). The fish was thawed in running tap water and then gutted, eviscerated, deboned, and finally passed through a strainer (Meat Strainer Mod. MMG22, Electrolux, Rimini, Italy) to remove the scale, pin bones, debris, and connective tissues. The resulting samples were homogenized with equal volume of 5% NaCl solution and mixed with 4% sucrose and 1% glucose. Then, appropriate amounts of 3 selected strains (17, 41, and 51) were inoculated to a final level of 106 cfu/g of the ground mince. A control sample was also prepared with the same ingredients, but not inoculated. After mixing through a sterile glass rod, samples were packaged in commercially available nylon/polyethylene bags (thickness 95 μm, Tecnovac, San Paolo D’Argon, Bergamo, Italy) and left to ferment at 37 °C for 5 d. During the fermentation, the changes in pH, aerobic microflora (APC), Pseudomonadaceae (PSE), Enterobacteriaceae (E), and LAB populations were measured. For microbiological analyses, 25 g of each sample were aseptically placed in a stomacher bag, diluted with saline solution, and homogenized in a Stomacher LabBlender 400 (Pbi Intl., Milan, Italy). Decimal dilutions were carried out using the same diluent. The media and conditions used were: Plate Count Agar incubated at 30 °C for 24 to 48 h for APC; Pseudomonas Agar Base with CFC selective supplement incubated at 25 °C for 48 h for Pseudomonas spp.; Violet Red Bile Glucose Agar, incubated at 37 °C Figure 2–Frequency histogram of Growth Index in JCM377, without or with for 24 h for Enterobacteriaceae; de Man Rogosa Sharp incubated at 20% to 30% NaCl. M154 Journal of Food Science r Vol. 80, Nr. 1, 2015
Halophilic starters for fish products . . .
Phenotyping During the sampling operations, 59 isolates were collected: these microorganisms were mainly Gram positive (44 isolates), nonspore-forming, catalase-negative, rods or cocci shaped, able to decrease the pH of a lab medium, and not producing CO2 from glucose and citrate (data not shown). Most of the isolates showed a typical profile comprising the production of ammonium from arginine (93.22% of the studied population), the hydrolysis of esculin (62.71%), gelatin and urea (ca. 80%), the reduction of nitrate, and the synthesis of indole, whereas casein, starch, and Tween 80 were never hydrolyzed and H2 S never produced (data not shown). Growth assays and acidification performances The assays concerning the acidification of the lab medium and the growth with salt added or as a function of the pH and temper-
ature were performed only on the Gram positive isolates (44 out of 59). The results (decrease of pH and growth index values, GI) were used to calculate some descriptive indices (arithmetic mean, median, quartiles, maximum and the minimum of the distribution, standard deviation, and the coefficient of variation) able to give an imprinting of the kind of statistical distribution (symmetrical or not) and the existence of a possible common trend for each trait (Table 3). A 1st common trend for some data is the difference between the arithmetic mean and the median, thus they suggest the existence of an asymmetric distribution, mainly characterized by a negative skewness (arithmetic mean > median) (GI without salt, with 30% of NaCl, at 10 °C, at pH 4.5 and 9.5); the mean and the median were closer for the other traits, however the high values of the coefficient of variation suggested also for this data an extreme variability and the impossibility to use any kind of distribution. From a biological point of view, the existence of a large variability in the indices pointed out a strong difference among the different isolates, with some targets completely inhibited and
Figure 3–Frequency histogram of Growth Index in JCM377 adjusted to pH 4.5 or 9.5.
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Results
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other growing or moderately growing throughout the different assays. Figure 1 shows the distribution for GI as a function of the temperature; at 10 °C most of the isolates (35) were strongly inhibited, as they showed a GI < 7.58% and only some targets were able to experience a moderate growth, with a GI included in the range 25% to 75% (6 isolates), as reported previously by Bevilacqua and others (2009, 2010). The targets with the highest GI (68.23% to 75.81%) were the isolates 4 and 8 (Figure 1A). As expected many targets were able to grow both at 25 °C (figure 1B) and 40 °C (figure 1C). Namely at 25 °C the strains could be divided into 3 groups: those showing a GI in the range 26.70% to 79.02% (moderate inhibition according to the guidelines of Bevilacqua and others 2009; 24 isolates); a 2nd group characterized by a GI of 79.02% to 118.27% (growth similar to the reference temperature, 37 °C; 18 isolates) and 2 targets with GI > 131%, thus suggesting that they were more adapted to 25 °C than in the control (isolates 2 and 32). A similar approach could be used to divide the isolates in 3 classes following their growth at 40 °C: the 1st group comprising 23 isolates showing a moderate inhibition at this temperature (GI < 72%), the 2nd group including the isolates with a growth similar to the reference temperature (17 isolates, GI of 72.09% to 128.9%), and 4 strains with a moderate or a strong thermophilic trend (GI > 128%) (isolates 2, 4, 32, and 38). Finally at 55 °C, the isolates were strongly inhibited as they experienced a GI < 20.9% (Figure 1D). Figure 2 reports growth profile in the lab medium added with salt; for this assay, a concentration of salt of 10% (basal composition of JCM377) was used as the reference. The results from the experiment with 0% NaCl (Figure 2A) pointed out the preference of many isolates toward the reference concentration, as 18 targets were strongly inhibited in the broth without the salt (GI < 25%). However, 10 isolates showed a GI > 127%, thus suggesting that they did not possess an halophilic trend. An increase of salt to 20% (Figure 2B) affected strongly the growth of the targets, being 7 isolates completely inhibited (GI < 21.52%) and the remaining ones experiencing a moderate-to-strong inhibition with a GI in the range 21% to 78%. As expected, the inhibition was stronger in the broth with 30% salt added (Figure 2C). pH affected significantly the growth of the isolates both under acidic and alkaline conditions; namely at pH 4.5, 37 isolates experienced a GI < 25% (complete inhibition), while for 50 targets the inhibition was moderate-to-strong (GI in the range 25.87% to 50.43%), and only 2 isolates (3 and 19) showed a growth similar to the control (Figure 3A). Under alkaline conditions, 10 isolates were partly inhibited (GI in the range 23.20% to 54.14%) and only 1 out of 44 showed a moderate growth (GI of 69.61% to 77.35%, isolate 26), while the remaining 33 isolates were completely inhibited (GI < 23%). The last assay for the technological characterization was the acidification of the basal medium after 24, 48, and 120 h (Figure 4). The results from this experiment were interesting and promising, as after 24 h twenty-seven isolates decreased the pH of the medium by more than 1 and twenty-four microorganisms experienced a pH of 1.6 to 1.8 after 48 h. As expected, the acidification increased over time (Figure 4B and 4C).
4 groups, labeled as I, II, III and IV, including respectively 20, 3, 8 and 13 isolates. A focus on the technological traits revealed that the group I was characterized by a common trend for the following traits: optimal growth at 37 °C or with 10% of salt (conditions generally used during fermentation of fish products). Thereafter, a principal component analysis (PCA) was run (Figure 6); for this kind of analysis we used only the variables with some differences
Selection of suitable strains and identification of isolates The results from the technological characterization were used as input variables to run a preliminary cluster analysis and divide the strains in some homogeneous groups. Cluster analysis is reported Figure 4–Frequency histogram for the acidification experienced by the in the Figure 5; the multivariate approach divided the strains into targets in JCM377 after 24, 48, and 120 h. M156 Journal of Food Science r Vol. 80, Nr. 1, 2015
among the different isolates (GI at 25 and 40 °C or with 20% salt added, acidification of the lab medium after 48 h). PCA divided the isolates into 2 classes: the left and the right regions of the factorial space; the isolates on the left side showed interesting traits in terms of acidification, growth with 20% NaCl added, and at 40 °C. Moreover, the strains placed below the xaxis experienced some interesting values for GI at 25 °C (room temperature). The selection of the most promising strains based on a multivariate approach is a kind of risk/benefit analysis, as none of the isolates showed all the technological traits at their optimal values. Thus, the selection was based on the choice of the most important characteristics for the production of fermented fish products: acidification, growth at 37 to 40 °C, and resistance to salt. As a result of this approach, 3 isolates on the left side were chosen; moreover, 2 out of 3 were selected also for their ability to grow and perform a fermentation at lower temperatures: following this approach some promising candidates were the isolates 17, 41, and 51. They were respectively identified as Pediococcus acidilactici (17 and 51) and Pediococcus pentosaceus (isolate 41).
Production of fish sauce After the selection, the research was focused on the evaluation of acidification performances of the promising isolates throughout the fermentation of a fish sauce. Changes in pH of not-inoculated (control) and inoculated fish mix are shown in Figure 7. The initial pH of fish sauce was 6.50. As the fermentation proceeded, all samples inoculated with the target strains exhibited lower pH than the control and attained a threshold level of 4.50 within 2 d. However, fermentation was stopped after 3 d in the control sample, as a strong spoiling event (off-odors) was found. Microbiological data of fish sauce are shown in Table 4. In the control sample, LAB count was low (3.3 log cfu/g) immediately after sauce preparation, while the inoculum of the starter cultures increased LAB counts to 6.6 to 6.8 log cfu/g. In the inoculated samples, LAB prevailed over the autochthonous microbiota and attained after 5 d a level of ca. 9 log cfu/g, thus they inhibited and/or controlled the growth of Pseudomonadaceae and Enterobacteriaceae (ca. 3 log cfu/g in the inoculated samples and 8 log cfu/g in the control after 3 d).
Discussion It has been reported that diverse and complex microbial communities may be responsible for seafood fermentation (Roh and others 2010) including LAB, which are found to be the dominant microorganisms; their primary role is to ferment the available carbohydrates and cause a decrease of pH. Generally, pH should be below 4.5 in order to inhibit pathogenic and spoilage bacteria; the
combination of low pH and organic acids (mainly lactic acid) is the main preservation factor to which the effects of salt and spices (such as garlic, pepper, or ginger) may add. The salt concentration often range from 1% to 10% (w/w) and this is likely to have a pronounced influence on the microbial growth and the rate of fermentation, and thereby on the sensory quality and safety of the product; therefore, the use of autochthonous halophilic bacteria or strongly halotolerant microorganisms with desirable properties, isolated from salted fish, could be a valid tool to get reproducible and improved quality of this kind of products. The selection of LAB as suitable starters for fermented fish production, such as for other fermented foods, is a complex process, involving the evaluation of some technological performances and desired metabolic traits, as well the identification and the attribution to a species included in the qualified presumption of safety (QPS) list. Some studies performed in the past have primarily focused on culture-dependent approaches to obtain an understanding of microbial communities and their function in fermenting seafood (Paludan-Muller and others 2002; Kim and others 2009; Guan and others 2011). However, the most of them focused on the characterization of the autochthonous microflora based on some molecular approaches (both dependent and independent techniques) and on the evaluation of some desired technological and metabolic traits, without a real selection of the most promising strain for a future optimization of a starter cultures. Therefore, this paper was focused on a quite different topic, that is, the evaluation of some simple technological traits through a quantitative approach (growth index) and their use as input for the selection of promising strains through a multivariate approach, as proposed elsewhere for different starter cultures by Rodriguez-Gomez and others (2012), Bevilacqua and others (2013), and Speranza and others (2015). Which variables and which traits? The choice relied upon the flow-sheet for the production of fermented fish sauces or pastes; even if different flow charts for several fish/salt fermented products are used, generally the fish substrate is mixed with salt and carbohydrates and the product is packed into containers and left to ferment at various temperatures (from 10 to 45 °C) for a period ranging from 1 wk to several months. Some fish sauces require a shorter fermentation time (2 to 5 d): Som Fug or Plaa-som, for example, are prepared mixing minced fish with ground steamed rice (2 to 15 g/100 g) and salt (2.5 to 5 g/100 g), stuffing this mixture into plastic bags, followed by natural fermentation for 2 to 5 d at 30 °C (Adams 2009; Kose and Hall 2011). Thus, acidification and growth at low and relatively high temperatures, resistance to salt, and growth under acidic conditions can be considered as
Figure 5–Statistical groups of the targets as revealed by the cluster analysis. Vol. 80, Nr. 1, 2015 r Journal of Food Science M157
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Halophilic starters for fish products . . .
Halophilic starters for fish products . . . the necessary characteristics for a starter to be used in fermented fish products. In this research, temperature profile of the strains isolated from salted fish was studied in a wide range, due to the strong technological impact of this variable. The use of a quantitative approach (that is, the evaluation of the growth index) resulted in a kind of printing of the population, with some additional details. A strain could grow, but its growth could be partially inhibited or stimulated and this information could be recovered by the value of GI, as proposed elsewhere (Bevilacqua and others 2009, 2010; Speranza and others 2015); namely, a severe growth inhibition was observed at 10 and 55 °C, whereas the recovery of GIs ranging around 80%, highlighted that the studied strains had a preference for temperatures of 25 and 40 °C. This trait is a positive characteristic because most of fermented fish products are produced at these temperatures (Adams 2009; Kose and Hall 2011).
The resistance to salt is absolutely the most important trait required for starters to be used in fish fermentation: even with 200 g/L of salt, the obtained results were promising with most isolates (11 strains) showing a growth similar to that recovered in the control and a strong halophilic trend. Another important property is the growth at prohibitive pHs and the acidifying ability; almost 8 isolates showed good performances with a good growth under stressful conditions and a pH decrease >2, thus suggesting their suitability as potential starter cultures for halophilic fermentation. Therefore, after the technological characterization, the following step was the selection of the most promising strains; the difficulty of this phase is the management of a large amount of data. A possible approach is the simplification of the input through a multivariate technique; in this research, this approach consisted of 2 different analyses: a cluster analysis, run as a screening tool, and a principal component analysis, used as a selection tool. For the selection of
Figure 6–Principal component analysis run for the targets of the group I (cluster analysis): (A) variable projection and (B) case projection. 25 and 40 °C, growth index at 25 and 40 °C; NaCl 20%, growth in the lab medium added with NaCl 20%; acidification, decrease of pH after 48 h.
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Halophilic starters for fish products . . . Table 4–Changes in viable counts of aerobic bacteria (APC), lactic acid bacteria (LAB), Pseudomonadaceae (PSE) and Enterobacteriaceae (E) of fish sauces inoculated with the selected strains (17, 41, and 51) during the fermentation at 37 °C. Bacteria (Log CFI/g) APC d 0 1 2 3 5 PSE 0 1 2 3 5
LAB
Control
17
41
51
Control
17
41
51
4.20 ± 0.40aA 6.43 ± 0.49aB 7.35 ± 0.20aB 8.04 ± 0.24aB,C ND E
6.28 ± 0.10bA 8.03 ± 0.19bB 8.58 ± 0.60bB 8.67 ± 0.03aB 8.75 ± 0.30a,bB
6.20 ± 0.17bA 8.23 ± 0.01bB 8.50 ± 0.30bB 8.17 ± 0.63aB 8.41 ± 0.02aB
6.00 ± 0.13bA 8.18 ± 0.21bB 8.32 ± 0.38bB 8.65 ± 0.53aB 8.80 ± 0.02bB
3.30 ± 0.18aA 5.35 ± 0.52aB 6.28 ± 0.20aC 7.32 ± 0.13aD ND
6.84 ± 0.21bA 7.94 ± 0.14bB,C 8.68 ± 0.21bC 8.60 ± 0.13bC 9.01 ± 0.22aC
6.64 ± 0.25bA 7.99 ± 0.34bB 8.38 ± 0.65bB 8.50 ± 0.12bB 8.91 ± 0.24aB
6.60 ± 0.28bA 8.19 ± 0.35bB 8.68 ± 0.25bB 9.04 ± 0.19bB 8.85 ± 0.34aB
4.88 ± 0.23aA 6.03 ± 0.15aB 7.58 ± 0.14aC 8.27 ± 0.53aC ND
4.83 ± 0.16aA 4.93 ± 0.15bA 3.58 ± 0.71bA 3.67 ± 0.33bA 3.61 ± 0.32aA
4.67 ± 0.48aA 4.12 ± 0.15bA 3.94 ± 0.02bA 3.14 ± 0.93bA 3.18 ± 0.55aA
4.41 ± 0.25aA 4.11 ± 0.60bA 3.38 ± 0.17bA 3.24 ± 0.03bA 3.40 ± 0.25aA
3.20 ± 0.03aA 5.20 ± 0.19aB 7.08 ± 0.10aC 8.17 ± 0.53D ND
3.23 ± 0.06aA 3.35 ± 0.04bA 3.09 ± 0.11bA 2.65 ± 0.56bA 2.64 ± 0.33bA
3.20 ± 0.21aA 3.90 ± 0.13bA 3.25 ± 0.15bA 3.29 ± 0.50bA 3.33 ± 0.10bA
3.25 ± 0.10aA 3.26 ± 0.18bA 3.43 ± 0.25bA 3.23 ± 0.06bA 2.93 ± 0.40bA
the most suitable strains, the technological traits were set as follows: (a) acidification, pH after 48 h > 1.50 (that is, acidification score higher that the mean value of the normal distribution of the data); (b) growth at 25 and 40 °C >75% (growth similar to the control, as reported by Bevilacqua and others 2009); (c) growth index with 20% NaCl added >75%. These values were used for the selection of the most promising strains, namely strain 41 as target resistant to high concentrations of salt and strains 17 and 51 for their ability to grow and perform a fermentation at lower temperatures. These strains were identified as members of the genus Pediococcus. The microflora of salted and fermented fish is quite variable and relies upon different factors, like the raw material, the flow-sheet, the time of sampling, etc. Some authors in the past performed similar researches and found that the bacterial flora of these products is dominated by Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus fermentum, and P. pentosaceus (Paludan-Muller and others 2002; Riebroy and others 2008). In 2007, Namwong and others isolated Halobacterium salinarum and Halococcus thailandensis from fish sauce samples in Thailand. These bacteria were able to grow optimally at 20% to 25% NaCl, like our strains. Other researchers isolated moderately halophilic microrganisms with an optimal growth at 3% to 15% NaCl, that is, Tetragenococcus halophilus, Tetragenococcus muriaticus, Halobacillus thailandensis, and Lentibacillus juripiscarius (Thongsanit and others 2002; Namwong and others 2005). Mod-
erately halophilic microorganisms are widely distributed in many kinds of fermented foods: for example, strains of Jeotgalibacillus alimentarius were found in jeotgal in Korea (Yoon and others 2001), L. juripiscarius and Lentibacillus halophilus, T. halophilus, and T. muriaticus in Thai fish sauce (Thongsanit and others 2002; Namwong and others 2005; Tanasupawat and others 2006), Bacillus vietnamensis in Vietnamese fishsauce (Noguchi and others 2004), Salinicoccus siamensis, Lentibacillus kapialis, and Oceanobacillus kapialis in Thai fermented shrimp paste (Pakdeeto and others 2007; Namwong and others 2009), and Piscibacillus salipiscarius, Salinivibrio siamensis, and Gracilibacillus thailandensis in salt-fermented fish (pla-ra) in Thailand (Tanasupawat and others 2007; Chamroensaksri and others 2009, 2010). As a final step of this study, a lab-validation was conducted evaluating the performance of the 3 selected strains during the production of a fermented fish sauce. As expected, the use of the proposed strains reduced the fermentation time (2 d) but also improved the microbiological quality of the final product. Regardless the strains used, no significant differences were observed among the samples: both P. acidilactici (17 and 51) and P. pentosaceus (41), in fact, showed similar results. In 2008, Riebroy and others investigated the effect of inoculation of different LAB on the fermentation and quality of Som-fug from bigeye snapper: Somfug inoculated with P. acidilactici at 104 cfu/g showed a greater
Figure 7–Changes in pH of not-inoculated (control) or inoculated fish sauces during the fermentation at 37 °C.
pH
6,5
4,5
2,5 0 control
1 17
2 41
3 51
4
5
6
Days
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ND, not determined. The letters indicate for each group (APC, LAB, PSE, or E) the differences among the different samples (control 17, 41, and 51) (lowercase letters) or throughout the fermentation for each sample (uppercase letters) (one-way ANOVA and Tukey’s test, P < 0.05). Control, non-inoculated sample.
Halophilic starters for fish products . . . acceptability than those inoculated with Lb. plantarum and P. pentosaceus at either 104 or 106 cfu/g and the control (without inoculum). Based on desired pH (4.5), the fermentation was completed within 36 h for P. acidilactici, as observed in this study.
Conclusions This paper was the 1st research focusing on the design of a lactic starter for the production of fermented fish products. Another key factor of the manuscript was the use of a quantitative approach, based on the evaluation of the growth index and acidification score, as well as on the use of a multivariate approach to select the most promising 3 strains with the best technological performances to be used as potential starters for fish fermentation.
Acknowledgments This work was supported by the Italian Ministry of the Univ. and Research (M.I.U.R.) by the research project PON01_01962: “Technologies for Nutraceutic Seafood with Extended Shelf Life” (Tecnologie per la valorizzazione e l’estensione di shelf life di trasformati ittici ad elevata valenza salutistica).
References
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Adams M. 2009. Fermented fish. In: Rhea F, editor. Microbiology handbook fish and seafood. Surrey: Leatherhead Publishing. p 123–40. Barrow GI, Feltham RKA. 1993. Cowan and Steel’s manual for the identification of medical bacteria. 3rd ed. Cambridge: Cambridge University Press. Bevilacqua A, Perricone M, Cannarsi M, Corbo MR, Sinigaglia M. 2009. Technological and spoiling characteristics of the yeast microflora isolated from Bella di Cerignola table olives. Int J Food Sci Tech 44:2198–207. Bevilacqua A, Altieri C, Corbo MR, Sinigaglia M, Ouoba LII. 2010. Characterization of lactic acid bacteria isolated from Italian Bella di Cerignola table olives: selection of potential multifunctional starter cultures. J Food Sci 75:M536–544. Bevilacqua A, Beneduce L, Sinigaglia M, Corbo MR. 2013. Selection of yeasts as starter cultures for table olives. J Food Sci 75:M742–751. Blaszyk M, Holley RA. 1998. Interaction of monolaurin, eugenol and sodium citrate on growth of common meat spoilage and pathogenic organisms. Int J Food Microbiol 39:175–83. Chamroensaksri N, Tanasupawat S, Akaracharunya A, Visessanguan W, Kudo T, Itoh T. 2009. Salinivibrio siamensis sp. nov., from fermented fish (pla-ra) in Thailand. Int J Syst Evol Micr 59:880–85. Chamroensaksri N, Tanasupawat S, Akaracharunya A, Visessanguan W, Kudo T, Itoh T. 2010. Gracilibacillus thailandensis sp. nov., from fermented fish (pla-ra) in Thailand. Int J Syst Evol Micr 60:944–48. Eden PA, Schmidt TM, Blakemore RP, Pace NR. 1991. Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reaction-amplified 16s rRNA-specific DNA. Int J Syst Bacteriol 41:324–25.
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Guan L, Cho CH, Lee JH. 2011. Analysis of the cultivable bacterial community in jeotgal, a Korean salted and fermented seafood and identification of its dominant bacteria. Food Microbiol 28:101–13. Kim MS, Park EJ, Jung MJ, Roh SH, Bae JW. 2009. Analysis of prokaryote communities in Korean traditional fermented food, jeotgal, using culture-dependent method and isolation of a novel strain. Korean J Microbiol 45:26–31. Kose S, Hall GM. 2011. Sustainability of fermented fish-products. In: Hall GM, editor. Fish processing- Sustainability and new opportunities. Surrey: Leatherhead Publishing. p 138–66. Namwong S, Tanasupawat S, Smitinont T, Visessanguan W, Kudo T, Itoh T. 2005. Isolation of Lentibacillussalicampi strains and Lentibacillus juripiscarius sp. nov., from fish sauce in Thailand. Int J Syst Evol Micr 55:315–20. Namwong S, Tanasupawat S, Visessanguan W, Kudo T, Itoh T. 2007. Halococcus thailandesis sp. nov. from fish sauce in Thailand. Int J Syst Evol Micr 57:2199–203. Namwong S, Tanasupawat S, Lee KC, Lee JS. 2009. Oceanobacillus kapialis sp. nov., from fermented shrimp paste in Thailand. Int J Syst Evol Micr 59:2254–59. Noguchi H, Uchino M, Shida O, Takano K, Nakamura LK, Komagata K. 2004. Bacillus vietnamensis sp. nov., a moderately halotolerant, aerobic, endospore-forming bacterium isolated from Vietnamese fish sauce. Int J Syst Evol Micr 54:2117–20. Pakdeeto A, Tanasupawat S, Thawai C, Moonmangmee S, Kudo T, Itoh T. 2007. Lentibacillus kapialis sp. nov., from fermented shrimp paste in Thailand. Int J Syst Evol Micr 57:364– 69. Paludan-M¨uller C, Madsen M, Sophanodora P, Gram L, Moller PL. 2002. Fermentation and microflora of plaa-som, a Thai fermented fish product prepared with different salt concentrations. Int J Food Microbiol 73:61–70. Riebroy S, Benjakul S, Visessanguan W. 2008. Properties and acceptability of som-fug, a thai fermented fish mince, inoculated with lactic acid bacteria starters. Food Sci Technol-LEB 41:569–80. Rodr´ıguez-G´omez F, Romero-Gil V, Bautista-Gallego J, Garrido-Fern´andez A, Arroyo-L´opez FN. 2012. Multivariate analysis to discriminate yeast strains with technological applications in table olive processing. World J Microb Biot 28:1761–70. Roh SW, Kim KH, Nam YD, Chang HW, Park EJ, Bae JW. 2010. Investigation of archeal and bacterial diversity in fermented seafood using barcoded pyrosequencing. Int J Syst Evol Micr 4:1–16. Speranza B, Bevilacqua A, Corbo MR, Altieri C, Sinigaglia M. 2015. Selection of autochthonous strains as promising starter cultures for Fior di Latte, a traditional cheese of southern Italy. J Sci Food Agr 95:88–97. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol 30:2725–29. Tanasupawat S, Pakdeeto A, Namwong S, Thawai C, Kudo T, Itoh T. 2006. Lentibacillus halophilus sp. nov., from fish sauce in Thailand. Int J Syst Evol Micr 56:1859–63. Tanasupawat S, Namwong S, Kudo T, Itoh T. 2007. Piscibacillus salipiscarius gen. nov., sp. nov., a moderately halophilic bacterium from fermented fish (pla-ra) in Thailand. Int J Syst Evol Micr 57:1413–17. Thongsanit J, Tanasupawat S, Keeratipibul S, Jatikavanich S. 2002. Characterization and identification of Tetragenococcus halophilus and Tetragenococcus muriaticus strains from fish sauce (nam-pla). Jap J Lactic Acid Bact 13:46–52. Visessanguan W, Benjakul S, Smitinont T, Kittikun C, Thepkasikul P, Panya A. 2006. Changes in microbiological, biochemical and physico-chemical properties of Nham inoculated with different inoculum levels of Lactobacillus curvatus. Food Sci Technol 39:814–26. Yoon JH, Weiss N, Lee KC, Lee IS, Kang KH, Park YH. 2001. Jeotgalibacillus alimentarius gen. nov., sp. nov., a novel bacterium isolated from jeotgal with Lysine in the cell wall, and reclassification of Bacillus marinus R¨uger 1983 as Marinibacillus marinus gen. nov., comb. nov. Int J Syst Evol Micr 51:2087–93.
This paper was the 1st research focusing on the design of a halophilic lactic starter for the production of fermented fish products using a quantitative approach, based on the evaluation of the growth index and acidification score, as well as on the use of a multivariate approach to select the most promising strains. Fifty-nine strains were randomly selected from salted fish and phenotypically characterized through Gram staining, catalase activity, glucose metabolism, H2 S and indole production, nitrate reduction, citrate utilization, and hydrolysis of arginine, esculin, casein, gelatin, starch, Tween 80, and urea. Then the Gram positive isolates (44 out of 59) were studied for their growth at different temperatures (10, 25, 40, and 55 °C), salt (0%, 20%, and 30%), pHs (4.5 and 9.5), and acidification score in lab medium. Data were modelled through growth index and used as input to run a preliminary cluster analysis and a principal component analysis. Three promising strains were selected, identified as members of the genus Pediococcus and used for the validation at laboratory level through the assessment of their performances in the production of a fermented fish sauce. The results were really promising as their use not only reduced the fermentation time (2 d) but also improved the microbiological quality of the final product. This paper represents a 1st report on the use of a simple step-by-step methodology to select promising halophilic strains for the optimization of a starter for fish-fermented products.
Abstract:
Practical Application: The isolation, targeted selection, characterization of halophilic bacteria, or strongly halotolerant microorganisms, as well as their appropriate use, could be of great interest to standardize the quality of fermented fish products. The use of autochthonous cultures to produce a fish-type product is attracting increasing interest, as it could increase processing rates and product consistency, improve the sensory characteristics and microbiological quality, and shorten the fermentation time.
Introduction
ones, so representing the most important of all fermented fish products (Adams 2009). In recent years, using pure bacterial cultures to produce a fishtype product is attracting increasing interest. The use of starters in food fermentations has become a mean to increase processing rates and product consistency improving the sensory characteristics and microbiological quality and shortening the fermentation time (Visessanguan and others 2006). As generally observed for meat and dairy fermented products, various researchers have stated that the use of lactic acid bacteria (LAB) could significantly improve also the quality of fish products accelerating the formation of lactic acid and significantly inhibiting the growth of spoilage bacteria and pathogens (Kose and Hall 2011). LAB are found to be as the dominant microorganisms in many fermented fish products where their primary role is to ferment available carbohydrates and thereby cause a decrease in pH. The combination of low pH (below 4.5) and organic acids (mainly lactic acid) is the main preservation factor in this kind of products. In addition, the use of salt and spices (such as garlic, pepper, or ginger) can contribute to additional safety to these products. Salt concentration may range from 1% to 20% (w/w), thus the fermentation by halophilic LAB could be an advantageous solution. To avoid spontaneous fermentations that are not easily controllable, it would be appropriate to select halophilic bacterial starters, in order to optimize and control the MS 20141557 Submitted 9/17/2014, Accepted 10/22/2014. Authors are with evolution of biochemical processes that occur during the proDept. of the Science of Agriculture, Food and Environment (SAFE), Univ. of Foggia, Via Napoli 25, 71122, Foggia, Italy. Direct inquiries to author Corbo (E-mail: duction cycle of the products concerned. The isolation, targeted selection, characterization of halophilic bacteria or strongly [email protected]). tolerant microorganisms, as well as their appropriate use, could
Fish fermentation is one of the most common methods of seafood preservation. It has many benefits and could be used as a low-cost convenient technique for the preservation of fish muscle, improving the organoleptic qualities of fish and increasing the nutritional value and/or digestibility of the raw material. Nowadays, fermented fish products are largely confined to east and south-east Asia, although some products are being to be produced elsewhere. Most of the fermented fish products are still produced on a cottage industry or domestic scale, so there are numerous variants of some common themes and a host of local names used to describe them. Essentially, fermented fish products can be divided into 2 main categories: (1) fish/salt products including fish pastes and sauces that contain relatively high levels of salt, typically in the range 15% to 25% and are used mainly as a condiment; (2) fish/salt/carbohydrate products ranging from those that resemble the fish sauces and pastes in which an extensive autolysis has occurred, to products analogous to other lactic fermented foods, where the bacterial production of lactic acid is a major feature (Kose and Hall 2011). In economic terms, the fish sauces are produced on the largest scale being exported from oriental countries to European and North American
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12721 Further reproduction without permission is prohibited
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Keywords: fermented fish, growth index, quantitative approach, starter selection
% Identity
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Halotolerant bacteria collection This study focused on 59 isolates of bacteria recovered from salted anchovies (Engraulis encrasicholus) purchased from 3 different local markets (Foggia, Italy). The samples were stored at 3 different temperatures (4, 15, and 30 °C) and bacterial isolations carried out after 0, 3, 6, and 9 d of storage. Fish samples (25 g) were aseptically transferred in a sterile plastic bag (Seward, London, England) and homogenized for 1 min in a stomacher Lab-blender 400 (Seward) using 225 mL of sterile saline solution (9 g/L NaCl). Decimal dilutions of the samples were plated on JCM377 medium incubated at 37 °C for 1 wk. The composition of the medium was the following: 100 g/L NaCl, 5 g/L amino acids of the casein (Amicase, Sigma Aldrich, Milan), 5 g/L yeast extract (Oxoid, Milan, Italy), 1 g/L glutamic acid (Sigma Aldrich), 2 g/L KCl, 3 g/L trisodium citrate, 20 g/L MgSO4 ·H2 O, 36 mg/L FeCl2 ·4H2 O, 0.36 mg/L MnCl2 ·4H2 O, 20 g/L agar (pH 7.5; unless otherwise specified, the salts were of C. Erba, Milan, Italy). From each plate, 5 to 10 colonies were randomly selected and stored at 4 °C on JCM377 agar slants; the isolates were labeled with a numeric code ranging from 1 to 59. Phenotyping Initial typing was based upon Gram staining and microscope examination, followed by evaluation of catalase activity, glucose metabolism (homo or hetero-fermentative metabolism), H2 S and indole production, nitrate reduction, and citrate utilization; all these tests were performed as described by Barrow and Feltham (1993). Hydrolysis of arginine, esculin, casein, gelatin, starch, Tween 80, and urea were also determined (Namwong and others 2005). Growth assays These assays were conducted in aliquots of JCM377, inoculated to 5 log cfu/mL with each strain separately, adjusted to different amounts of NaCl (0%, 20%, and 30%), pHs (4.5 or 9.5) through HCl or NaOH 1.0N or incubated at different temperatures (10, 25, 40, and 55 °C). Microbial growth was evaluated every 24 h (for 7 d) by reading absorbance at 600 nm through a UV-Vis Spectrophotometer; M152 Journal of Food Science r Vol. 80, Nr. 1, 2015
Table 2–Sequence comparison with reference 16S ribosomal RNA sequences in GenBank database.
Materials and Methods
KJ997937 KJ997939 KJ997938 1489 1481 1476
Strain sequence length
be of great interest to standardize the quality of fermented fish products. Thus, the main goal of this paper was the selection of promising halophilic autochthonous strains as potential starter cultures for fermented fish products using a step-by-step procedure. The research was divided in different phases: (1) evaluation of some simple traits: growth at different temperature, with salt added and at various pH, acidification of a laboratory medium; (2) selection of the most promising strains through a multivariate approach and molecular identification; and (3) validation of the selected strains through the assessment of their performances in the production of a fermented fish sauce.
99 99 99
1.5
NR_042058.1 NR_042057.1 NR_042057.1
pH decrease (pH)
75%
Reference accession no.
Growth index
Reference sequence
0 1 2
Strain
Codes
Pediococcus pentosaceus strain DSM 20336 16S ribosomal RNA gene, complete sequence Pediococcus acidilactici strain DSM 20284 16S ribosomal RNA gene, complete sequence Pediococcus acidilactici strain DSM 20284 16S ribosomal RNA gene, complete sequence
Table 1–Qualitative codes for the multivariate analyses.
41 17 51
Strain separation accession no.
Halophilic starters for fish products . . .
Halophilic starters for fish products . . . Table 3–Descriptive indices for GI (growth index) after 48 h in JCM377 added with NaCl, adjusted to pH 4.5 or 9.5 or incubated at 10, 25, 40, and 55 °C or for acidification (pH) performed by the isolates in the same medium after 24, 48, and 120 h. Growth index after 48 h
Arithmetic mean Median Minimum Maximum 1 quartiles 3 quartiles Standard deviation Coefficient of variation (CV, %)
Acidification
NaCl 0%
NaCl 20%
NaCl 30%
10 °C
25 °C
40 °C
55 °C
pH 4.5
pH 9.5
24 h
48 h
120 h
55.25 26.60 4.29 210.35 12.59 89.01 61.44 111
49.82 55.78 7.30 78.41 26.42 70.40 23.45 47
16.17 7.10 0.00 45.99 2.17 33.94 15.86 98
10.26 1.87 0.00 75.81 0.00 4.54 20.15 196
74.36 74.83 26.70 157.51 49.89 90.22 27.21 37
79.45 70.76 34.22 223.60 58.17 87.68 35.26 44
3.13 2.54 0.00 20.85 0.00 4.32 3.79 121
15.80 6.87 1.33 124.09 4.71 12.83 23.57 149
15.17 12.31 0.00 77.34 2.05 21.62 16.22 107
1.05 1.17 0.52 1.40 0.78 1.29 0.29 28
1.50 1.62 0.81 1.80 1.37 1.70 0.29 19
1.67 1.70 1.38 1.91 1.55 1.77 0.14 8
The assays were performed on the Gram positive isolates (n = 44).
GI =
Abss × 100 Absc
Aliquots of JCM377 containing glucose (10 g/L) were individually inoculated with each strain to 5 log cfu/mL and incubated at 37 °C; the pH of the medium was evaluated after 24, 48, and 120 h through a pH-meter Crison (Crison Instruments, Barcelona,
Figure 1–Frequency histogram of Growth Index in JCM377 incubated at 10, 25, 40, and 55 °C. GI range was divided into 10 homogenous classes; the numbers on the bars indicate the isolates within each class. Vol. 80, Nr. 1, 2015 r Journal of Food Science M153
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samples of not-modified JCM377 (10% NaCl–pH 7.5), inoculated where Abss is the absorbance of the samples at different temas reported above and incubated at 37 °C, were used as controls. peratures, pH, or added with salt, whereas Absc is the absorbance The experiments were performed over 2 different batches; for of the control. each batch the experiments were repeated twice. For each time point, data were modeled as growth index, as reported by Blaszyk and Holley (1998) and Bevilacqua and others (2010): Acidification in lab medium
Halophilic starters for fish products . . .
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Spain). The experiments were performed in duplicate over 2 dif- 30 °C for 48 h under anaerobiosis for LAB. The pH was evaluated on homogenized samples with a pH meter (Crison, Barcelona, ferent batches. Spain). Selection of suitable strains and identification of isolates The analyses were performed twice over 2 different batches; Data of growth assays, as well as the results of acidification, were the results were analyzed through one-way analysis of variused for the evaluation of the descriptive statistical indices (arith- ance (ANOVA) and Tukey’s test as the post hoc comparison test metic mean, median, standard deviation, coefficient of variation, (P < 0.05). 1 and 3 quartiles) and to build frequency histograms, reporting on the x-axis growth index or pH decrease. Thereafter, they were converted into qualitative codes, as reported in Table 1, and used to run a cluster analysis and divide the strains in some homogeneous group, through the approach of dissimilarity. Then, the variables were used to carry out a principal component analysis to choose the most promising strains for the validation in a fermented fish sauce. The statistical analyses were performed through the software Statistica for Windows (Statsoft, Tulsa, Okla., U.S.A.). Selected strains for validation experiments (strains 17, 41, and 51) were identified by sequencing the 16SrDNA. Briefly, DNA of each strain was extracted with Ultraclean Microbial DNA Isolation kit (MoBio, Carlsbad, Calif., U.S.A.) and amplified in a PCR reaction with BSF8/BSR1510 primers targeting 16SrDNA genes (Eden and others 1991). PCR program was: initial denaturation at 95 °C for 5 min; 30 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 45 s, and extension at 72 °C for 1 min. PCR products were sequenced by Primm Biotech (Milan, Italy) with either the forward and the reverse primer in separate reactions. The sequences obtained were assembled into a consensus sequence using Mega 6.06 software (Tamura and others 2013). Assembled sequences were compared with the sequences available in the GenBank database using the Basic Local Alignment Search Tool version 2.2.27 (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences are freely available with the following accession nr: KJ997937, KJ997938, and KJ997939 (Table 2).
Validation at laboratory level: production of fish sauce The fish sauces were prepared using gilthead sea bream fillets (Sparus aurata) kindly furnished from Lepore Mare (Fasano, Brindisi, Italy). The fish was thawed in running tap water and then gutted, eviscerated, deboned, and finally passed through a strainer (Meat Strainer Mod. MMG22, Electrolux, Rimini, Italy) to remove the scale, pin bones, debris, and connective tissues. The resulting samples were homogenized with equal volume of 5% NaCl solution and mixed with 4% sucrose and 1% glucose. Then, appropriate amounts of 3 selected strains (17, 41, and 51) were inoculated to a final level of 106 cfu/g of the ground mince. A control sample was also prepared with the same ingredients, but not inoculated. After mixing through a sterile glass rod, samples were packaged in commercially available nylon/polyethylene bags (thickness 95 μm, Tecnovac, San Paolo D’Argon, Bergamo, Italy) and left to ferment at 37 °C for 5 d. During the fermentation, the changes in pH, aerobic microflora (APC), Pseudomonadaceae (PSE), Enterobacteriaceae (E), and LAB populations were measured. For microbiological analyses, 25 g of each sample were aseptically placed in a stomacher bag, diluted with saline solution, and homogenized in a Stomacher LabBlender 400 (Pbi Intl., Milan, Italy). Decimal dilutions were carried out using the same diluent. The media and conditions used were: Plate Count Agar incubated at 30 °C for 24 to 48 h for APC; Pseudomonas Agar Base with CFC selective supplement incubated at 25 °C for 48 h for Pseudomonas spp.; Violet Red Bile Glucose Agar, incubated at 37 °C Figure 2–Frequency histogram of Growth Index in JCM377, without or with for 24 h for Enterobacteriaceae; de Man Rogosa Sharp incubated at 20% to 30% NaCl. M154 Journal of Food Science r Vol. 80, Nr. 1, 2015
Halophilic starters for fish products . . .
Phenotyping During the sampling operations, 59 isolates were collected: these microorganisms were mainly Gram positive (44 isolates), nonspore-forming, catalase-negative, rods or cocci shaped, able to decrease the pH of a lab medium, and not producing CO2 from glucose and citrate (data not shown). Most of the isolates showed a typical profile comprising the production of ammonium from arginine (93.22% of the studied population), the hydrolysis of esculin (62.71%), gelatin and urea (ca. 80%), the reduction of nitrate, and the synthesis of indole, whereas casein, starch, and Tween 80 were never hydrolyzed and H2 S never produced (data not shown). Growth assays and acidification performances The assays concerning the acidification of the lab medium and the growth with salt added or as a function of the pH and temper-
ature were performed only on the Gram positive isolates (44 out of 59). The results (decrease of pH and growth index values, GI) were used to calculate some descriptive indices (arithmetic mean, median, quartiles, maximum and the minimum of the distribution, standard deviation, and the coefficient of variation) able to give an imprinting of the kind of statistical distribution (symmetrical or not) and the existence of a possible common trend for each trait (Table 3). A 1st common trend for some data is the difference between the arithmetic mean and the median, thus they suggest the existence of an asymmetric distribution, mainly characterized by a negative skewness (arithmetic mean > median) (GI without salt, with 30% of NaCl, at 10 °C, at pH 4.5 and 9.5); the mean and the median were closer for the other traits, however the high values of the coefficient of variation suggested also for this data an extreme variability and the impossibility to use any kind of distribution. From a biological point of view, the existence of a large variability in the indices pointed out a strong difference among the different isolates, with some targets completely inhibited and
Figure 3–Frequency histogram of Growth Index in JCM377 adjusted to pH 4.5 or 9.5.
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Results
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other growing or moderately growing throughout the different assays. Figure 1 shows the distribution for GI as a function of the temperature; at 10 °C most of the isolates (35) were strongly inhibited, as they showed a GI < 7.58% and only some targets were able to experience a moderate growth, with a GI included in the range 25% to 75% (6 isolates), as reported previously by Bevilacqua and others (2009, 2010). The targets with the highest GI (68.23% to 75.81%) were the isolates 4 and 8 (Figure 1A). As expected many targets were able to grow both at 25 °C (figure 1B) and 40 °C (figure 1C). Namely at 25 °C the strains could be divided into 3 groups: those showing a GI in the range 26.70% to 79.02% (moderate inhibition according to the guidelines of Bevilacqua and others 2009; 24 isolates); a 2nd group characterized by a GI of 79.02% to 118.27% (growth similar to the reference temperature, 37 °C; 18 isolates) and 2 targets with GI > 131%, thus suggesting that they were more adapted to 25 °C than in the control (isolates 2 and 32). A similar approach could be used to divide the isolates in 3 classes following their growth at 40 °C: the 1st group comprising 23 isolates showing a moderate inhibition at this temperature (GI < 72%), the 2nd group including the isolates with a growth similar to the reference temperature (17 isolates, GI of 72.09% to 128.9%), and 4 strains with a moderate or a strong thermophilic trend (GI > 128%) (isolates 2, 4, 32, and 38). Finally at 55 °C, the isolates were strongly inhibited as they experienced a GI < 20.9% (Figure 1D). Figure 2 reports growth profile in the lab medium added with salt; for this assay, a concentration of salt of 10% (basal composition of JCM377) was used as the reference. The results from the experiment with 0% NaCl (Figure 2A) pointed out the preference of many isolates toward the reference concentration, as 18 targets were strongly inhibited in the broth without the salt (GI < 25%). However, 10 isolates showed a GI > 127%, thus suggesting that they did not possess an halophilic trend. An increase of salt to 20% (Figure 2B) affected strongly the growth of the targets, being 7 isolates completely inhibited (GI < 21.52%) and the remaining ones experiencing a moderate-to-strong inhibition with a GI in the range 21% to 78%. As expected, the inhibition was stronger in the broth with 30% salt added (Figure 2C). pH affected significantly the growth of the isolates both under acidic and alkaline conditions; namely at pH 4.5, 37 isolates experienced a GI < 25% (complete inhibition), while for 50 targets the inhibition was moderate-to-strong (GI in the range 25.87% to 50.43%), and only 2 isolates (3 and 19) showed a growth similar to the control (Figure 3A). Under alkaline conditions, 10 isolates were partly inhibited (GI in the range 23.20% to 54.14%) and only 1 out of 44 showed a moderate growth (GI of 69.61% to 77.35%, isolate 26), while the remaining 33 isolates were completely inhibited (GI < 23%). The last assay for the technological characterization was the acidification of the basal medium after 24, 48, and 120 h (Figure 4). The results from this experiment were interesting and promising, as after 24 h twenty-seven isolates decreased the pH of the medium by more than 1 and twenty-four microorganisms experienced a pH of 1.6 to 1.8 after 48 h. As expected, the acidification increased over time (Figure 4B and 4C).
4 groups, labeled as I, II, III and IV, including respectively 20, 3, 8 and 13 isolates. A focus on the technological traits revealed that the group I was characterized by a common trend for the following traits: optimal growth at 37 °C or with 10% of salt (conditions generally used during fermentation of fish products). Thereafter, a principal component analysis (PCA) was run (Figure 6); for this kind of analysis we used only the variables with some differences
Selection of suitable strains and identification of isolates The results from the technological characterization were used as input variables to run a preliminary cluster analysis and divide the strains in some homogeneous groups. Cluster analysis is reported Figure 4–Frequency histogram for the acidification experienced by the in the Figure 5; the multivariate approach divided the strains into targets in JCM377 after 24, 48, and 120 h. M156 Journal of Food Science r Vol. 80, Nr. 1, 2015
among the different isolates (GI at 25 and 40 °C or with 20% salt added, acidification of the lab medium after 48 h). PCA divided the isolates into 2 classes: the left and the right regions of the factorial space; the isolates on the left side showed interesting traits in terms of acidification, growth with 20% NaCl added, and at 40 °C. Moreover, the strains placed below the xaxis experienced some interesting values for GI at 25 °C (room temperature). The selection of the most promising strains based on a multivariate approach is a kind of risk/benefit analysis, as none of the isolates showed all the technological traits at their optimal values. Thus, the selection was based on the choice of the most important characteristics for the production of fermented fish products: acidification, growth at 37 to 40 °C, and resistance to salt. As a result of this approach, 3 isolates on the left side were chosen; moreover, 2 out of 3 were selected also for their ability to grow and perform a fermentation at lower temperatures: following this approach some promising candidates were the isolates 17, 41, and 51. They were respectively identified as Pediococcus acidilactici (17 and 51) and Pediococcus pentosaceus (isolate 41).
Production of fish sauce After the selection, the research was focused on the evaluation of acidification performances of the promising isolates throughout the fermentation of a fish sauce. Changes in pH of not-inoculated (control) and inoculated fish mix are shown in Figure 7. The initial pH of fish sauce was 6.50. As the fermentation proceeded, all samples inoculated with the target strains exhibited lower pH than the control and attained a threshold level of 4.50 within 2 d. However, fermentation was stopped after 3 d in the control sample, as a strong spoiling event (off-odors) was found. Microbiological data of fish sauce are shown in Table 4. In the control sample, LAB count was low (3.3 log cfu/g) immediately after sauce preparation, while the inoculum of the starter cultures increased LAB counts to 6.6 to 6.8 log cfu/g. In the inoculated samples, LAB prevailed over the autochthonous microbiota and attained after 5 d a level of ca. 9 log cfu/g, thus they inhibited and/or controlled the growth of Pseudomonadaceae and Enterobacteriaceae (ca. 3 log cfu/g in the inoculated samples and 8 log cfu/g in the control after 3 d).
Discussion It has been reported that diverse and complex microbial communities may be responsible for seafood fermentation (Roh and others 2010) including LAB, which are found to be the dominant microorganisms; their primary role is to ferment the available carbohydrates and cause a decrease of pH. Generally, pH should be below 4.5 in order to inhibit pathogenic and spoilage bacteria; the
combination of low pH and organic acids (mainly lactic acid) is the main preservation factor to which the effects of salt and spices (such as garlic, pepper, or ginger) may add. The salt concentration often range from 1% to 10% (w/w) and this is likely to have a pronounced influence on the microbial growth and the rate of fermentation, and thereby on the sensory quality and safety of the product; therefore, the use of autochthonous halophilic bacteria or strongly halotolerant microorganisms with desirable properties, isolated from salted fish, could be a valid tool to get reproducible and improved quality of this kind of products. The selection of LAB as suitable starters for fermented fish production, such as for other fermented foods, is a complex process, involving the evaluation of some technological performances and desired metabolic traits, as well the identification and the attribution to a species included in the qualified presumption of safety (QPS) list. Some studies performed in the past have primarily focused on culture-dependent approaches to obtain an understanding of microbial communities and their function in fermenting seafood (Paludan-Muller and others 2002; Kim and others 2009; Guan and others 2011). However, the most of them focused on the characterization of the autochthonous microflora based on some molecular approaches (both dependent and independent techniques) and on the evaluation of some desired technological and metabolic traits, without a real selection of the most promising strain for a future optimization of a starter cultures. Therefore, this paper was focused on a quite different topic, that is, the evaluation of some simple technological traits through a quantitative approach (growth index) and their use as input for the selection of promising strains through a multivariate approach, as proposed elsewhere for different starter cultures by Rodriguez-Gomez and others (2012), Bevilacqua and others (2013), and Speranza and others (2015). Which variables and which traits? The choice relied upon the flow-sheet for the production of fermented fish sauces or pastes; even if different flow charts for several fish/salt fermented products are used, generally the fish substrate is mixed with salt and carbohydrates and the product is packed into containers and left to ferment at various temperatures (from 10 to 45 °C) for a period ranging from 1 wk to several months. Some fish sauces require a shorter fermentation time (2 to 5 d): Som Fug or Plaa-som, for example, are prepared mixing minced fish with ground steamed rice (2 to 15 g/100 g) and salt (2.5 to 5 g/100 g), stuffing this mixture into plastic bags, followed by natural fermentation for 2 to 5 d at 30 °C (Adams 2009; Kose and Hall 2011). Thus, acidification and growth at low and relatively high temperatures, resistance to salt, and growth under acidic conditions can be considered as
Figure 5–Statistical groups of the targets as revealed by the cluster analysis. Vol. 80, Nr. 1, 2015 r Journal of Food Science M157
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Halophilic starters for fish products . . .
Halophilic starters for fish products . . . the necessary characteristics for a starter to be used in fermented fish products. In this research, temperature profile of the strains isolated from salted fish was studied in a wide range, due to the strong technological impact of this variable. The use of a quantitative approach (that is, the evaluation of the growth index) resulted in a kind of printing of the population, with some additional details. A strain could grow, but its growth could be partially inhibited or stimulated and this information could be recovered by the value of GI, as proposed elsewhere (Bevilacqua and others 2009, 2010; Speranza and others 2015); namely, a severe growth inhibition was observed at 10 and 55 °C, whereas the recovery of GIs ranging around 80%, highlighted that the studied strains had a preference for temperatures of 25 and 40 °C. This trait is a positive characteristic because most of fermented fish products are produced at these temperatures (Adams 2009; Kose and Hall 2011).
The resistance to salt is absolutely the most important trait required for starters to be used in fish fermentation: even with 200 g/L of salt, the obtained results were promising with most isolates (11 strains) showing a growth similar to that recovered in the control and a strong halophilic trend. Another important property is the growth at prohibitive pHs and the acidifying ability; almost 8 isolates showed good performances with a good growth under stressful conditions and a pH decrease >2, thus suggesting their suitability as potential starter cultures for halophilic fermentation. Therefore, after the technological characterization, the following step was the selection of the most promising strains; the difficulty of this phase is the management of a large amount of data. A possible approach is the simplification of the input through a multivariate technique; in this research, this approach consisted of 2 different analyses: a cluster analysis, run as a screening tool, and a principal component analysis, used as a selection tool. For the selection of
Figure 6–Principal component analysis run for the targets of the group I (cluster analysis): (A) variable projection and (B) case projection. 25 and 40 °C, growth index at 25 and 40 °C; NaCl 20%, growth in the lab medium added with NaCl 20%; acidification, decrease of pH after 48 h.
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Halophilic starters for fish products . . . Table 4–Changes in viable counts of aerobic bacteria (APC), lactic acid bacteria (LAB), Pseudomonadaceae (PSE) and Enterobacteriaceae (E) of fish sauces inoculated with the selected strains (17, 41, and 51) during the fermentation at 37 °C. Bacteria (Log CFI/g) APC d 0 1 2 3 5 PSE 0 1 2 3 5
LAB
Control
17
41
51
Control
17
41
51
4.20 ± 0.40aA 6.43 ± 0.49aB 7.35 ± 0.20aB 8.04 ± 0.24aB,C ND E
6.28 ± 0.10bA 8.03 ± 0.19bB 8.58 ± 0.60bB 8.67 ± 0.03aB 8.75 ± 0.30a,bB
6.20 ± 0.17bA 8.23 ± 0.01bB 8.50 ± 0.30bB 8.17 ± 0.63aB 8.41 ± 0.02aB
6.00 ± 0.13bA 8.18 ± 0.21bB 8.32 ± 0.38bB 8.65 ± 0.53aB 8.80 ± 0.02bB
3.30 ± 0.18aA 5.35 ± 0.52aB 6.28 ± 0.20aC 7.32 ± 0.13aD ND
6.84 ± 0.21bA 7.94 ± 0.14bB,C 8.68 ± 0.21bC 8.60 ± 0.13bC 9.01 ± 0.22aC
6.64 ± 0.25bA 7.99 ± 0.34bB 8.38 ± 0.65bB 8.50 ± 0.12bB 8.91 ± 0.24aB
6.60 ± 0.28bA 8.19 ± 0.35bB 8.68 ± 0.25bB 9.04 ± 0.19bB 8.85 ± 0.34aB
4.88 ± 0.23aA 6.03 ± 0.15aB 7.58 ± 0.14aC 8.27 ± 0.53aC ND
4.83 ± 0.16aA 4.93 ± 0.15bA 3.58 ± 0.71bA 3.67 ± 0.33bA 3.61 ± 0.32aA
4.67 ± 0.48aA 4.12 ± 0.15bA 3.94 ± 0.02bA 3.14 ± 0.93bA 3.18 ± 0.55aA
4.41 ± 0.25aA 4.11 ± 0.60bA 3.38 ± 0.17bA 3.24 ± 0.03bA 3.40 ± 0.25aA
3.20 ± 0.03aA 5.20 ± 0.19aB 7.08 ± 0.10aC 8.17 ± 0.53D ND
3.23 ± 0.06aA 3.35 ± 0.04bA 3.09 ± 0.11bA 2.65 ± 0.56bA 2.64 ± 0.33bA
3.20 ± 0.21aA 3.90 ± 0.13bA 3.25 ± 0.15bA 3.29 ± 0.50bA 3.33 ± 0.10bA
3.25 ± 0.10aA 3.26 ± 0.18bA 3.43 ± 0.25bA 3.23 ± 0.06bA 2.93 ± 0.40bA
the most suitable strains, the technological traits were set as follows: (a) acidification, pH after 48 h > 1.50 (that is, acidification score higher that the mean value of the normal distribution of the data); (b) growth at 25 and 40 °C >75% (growth similar to the control, as reported by Bevilacqua and others 2009); (c) growth index with 20% NaCl added >75%. These values were used for the selection of the most promising strains, namely strain 41 as target resistant to high concentrations of salt and strains 17 and 51 for their ability to grow and perform a fermentation at lower temperatures. These strains were identified as members of the genus Pediococcus. The microflora of salted and fermented fish is quite variable and relies upon different factors, like the raw material, the flow-sheet, the time of sampling, etc. Some authors in the past performed similar researches and found that the bacterial flora of these products is dominated by Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus fermentum, and P. pentosaceus (Paludan-Muller and others 2002; Riebroy and others 2008). In 2007, Namwong and others isolated Halobacterium salinarum and Halococcus thailandensis from fish sauce samples in Thailand. These bacteria were able to grow optimally at 20% to 25% NaCl, like our strains. Other researchers isolated moderately halophilic microrganisms with an optimal growth at 3% to 15% NaCl, that is, Tetragenococcus halophilus, Tetragenococcus muriaticus, Halobacillus thailandensis, and Lentibacillus juripiscarius (Thongsanit and others 2002; Namwong and others 2005). Mod-
erately halophilic microorganisms are widely distributed in many kinds of fermented foods: for example, strains of Jeotgalibacillus alimentarius were found in jeotgal in Korea (Yoon and others 2001), L. juripiscarius and Lentibacillus halophilus, T. halophilus, and T. muriaticus in Thai fish sauce (Thongsanit and others 2002; Namwong and others 2005; Tanasupawat and others 2006), Bacillus vietnamensis in Vietnamese fishsauce (Noguchi and others 2004), Salinicoccus siamensis, Lentibacillus kapialis, and Oceanobacillus kapialis in Thai fermented shrimp paste (Pakdeeto and others 2007; Namwong and others 2009), and Piscibacillus salipiscarius, Salinivibrio siamensis, and Gracilibacillus thailandensis in salt-fermented fish (pla-ra) in Thailand (Tanasupawat and others 2007; Chamroensaksri and others 2009, 2010). As a final step of this study, a lab-validation was conducted evaluating the performance of the 3 selected strains during the production of a fermented fish sauce. As expected, the use of the proposed strains reduced the fermentation time (2 d) but also improved the microbiological quality of the final product. Regardless the strains used, no significant differences were observed among the samples: both P. acidilactici (17 and 51) and P. pentosaceus (41), in fact, showed similar results. In 2008, Riebroy and others investigated the effect of inoculation of different LAB on the fermentation and quality of Som-fug from bigeye snapper: Somfug inoculated with P. acidilactici at 104 cfu/g showed a greater
Figure 7–Changes in pH of not-inoculated (control) or inoculated fish sauces during the fermentation at 37 °C.
pH
6,5
4,5
2,5 0 control
1 17
2 41
3 51
4
5
6
Days
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ND, not determined. The letters indicate for each group (APC, LAB, PSE, or E) the differences among the different samples (control 17, 41, and 51) (lowercase letters) or throughout the fermentation for each sample (uppercase letters) (one-way ANOVA and Tukey’s test, P < 0.05). Control, non-inoculated sample.
Halophilic starters for fish products . . . acceptability than those inoculated with Lb. plantarum and P. pentosaceus at either 104 or 106 cfu/g and the control (without inoculum). Based on desired pH (4.5), the fermentation was completed within 36 h for P. acidilactici, as observed in this study.
Conclusions This paper was the 1st research focusing on the design of a lactic starter for the production of fermented fish products. Another key factor of the manuscript was the use of a quantitative approach, based on the evaluation of the growth index and acidification score, as well as on the use of a multivariate approach to select the most promising 3 strains with the best technological performances to be used as potential starters for fish fermentation.
Acknowledgments This work was supported by the Italian Ministry of the Univ. and Research (M.I.U.R.) by the research project PON01_01962: “Technologies for Nutraceutic Seafood with Extended Shelf Life” (Tecnologie per la valorizzazione e l’estensione di shelf life di trasformati ittici ad elevata valenza salutistica).
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