Microbiological Changes and Biodiversity of Cultivable Indigenous Bacteria in Sanbao Larger Yellow Croaker (Pseudosciaena crocea), a Chinese Salted and Fermented Seafood He Zhang, Yan Li, Kunhua Xu, Jiajia Wu, and Zhiyuan Dai
Sanbao large yellow croaker is a kind of Chinese salted and fermented seafood processed by adding 50% of salt and fermenting at high temperature [around 30 °C] over 20 d. To get a comprehensive understanding of cultivable microorganism’s diversity present in its fermentation, the chemical and microbial properties of this product, were detected initially, followed by identification of bacteria recovered from different fermentation periods using PCR-RFLP and 16S rRNA gene sequencing analysis. In total, 105 indigenous isolates were recovered with 3 different medium and majority of the isolates picked up from both MRS and MSA medium were finally clustered into the genus of Staphylococcus (S.). Among the 90 Staphylococci, S. xylosus, S. saprophyticus, and S. nepalensis were the most prevailing cultivable species recovered throughout the whole production process (70 isolates, 77.8%), while 5 other species, namely, S. aureus, S. vitulinus, S. sciuri, S. equorum, and S. succinus formed a minor fraction (20 isolates, 22.2%) of the Staphylococcus communities. Lactic acid bacteria, Pseudomonas, Proteus, and Bacillus constituted trivial populations in the initial period of the fermentation and then gave the way to the Staphylococcus immediately. High salt concentration used during the processing is like to have a pronounced influence on the microbial populations involved. Data obtained in this work could be referred in control and optimization of the fermentation process and selecting suitable strains for aquatic product fermentation. Abstract:
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Keywords: diversity, fermentation, lactic acid bacteria, Sanbao large yellow croaker, Staphylococcus
Introduction Traditional spontaneous fermented fish products have long been consumed in Southeast Asian countries and Northeastern regions of Asia, such as Thailand, Korea, Japan, and China (Kopermsub and Yunchalard 2010; Zeng and others 2013). In China, there are more than 100 kinds of fermented fish products manufactured with different processing techniques mainly produced in Zhejiang, Jiangsu, Hubei, Fujian, and Shandong provinces (Xu 2012). In Zhejiang Province, the traditional fermented fish products are always processed with high-salt content according to local preferences and the “Sanbao” (in Chinese) technique is one of the popular traditional fermentation processing methods in this district. During the Sanbao processing, salts are added 3 times on the 0 (fresh fish), 1st , and 5th fermenting days with the salt to fish ratio of 10%, 20%, and 20%, respectively. After 20 d fermentation, the off-odor could become a part of the desirable odor profile in the fermented fish products while the products are free of fishy odor and taste. Previous studies have indicated the microorganisms play a critical role in fish fermentation and the species involved in the fish fermentation is of great variety (Guan and others 2011). Normally, in the low-salt processed fish products, the Gram-negative bacteria are inhibited while the Gram-positive bacteria, particularly
MS 20141515 Submitted 9/9/2014, Accepted 12/4/2014. Authors are with College of Food Science and Biotechnology, Zhejiang Gongshang Univ. , Hangzhou, Zhejiang, 310035, P. R. China and with Inst. of Aquatic Products Processing Zhejiang Gongshang Univ., Hangzhou, Zhejiang Province, 310035, P. R. , China. Direct inquiries to author Jiajia Wu (E-mail: [email protected]). Authors He Zhang and Yan Li contributed equally to this work.
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lactic acid bacteria (LAB), become predominant microorganisms (Paludan-M¨uller and others 2002; Hwanhlem and others 2011; Dai and others 2013). The beneficial effect of LABs involved in the fermentation of those products have been associated with their ability to initiate a fast pH drop by producing lactic acid in the first fermenting days, which contributes to a safe product by preventing the growth of spoilage and pathogenic organisms (Leroy and others 2006). Meanwhile, in the high-salt processed fish product, Staphylococcus constitute the highest number (Guan and others 2011). Generally, gram positive and catalase positive cocci, especially the coagulase-negative Staphylococci, participate in the development of flavor of many high-salt fermented fish and meat products mainly through amino and fatty acid degradation (Hammes and Hertel 1998; Talon and Leroy 2006). All of these desirable proteolytic and lipolytic reactions induced by both microorganisms and endogenous from fish body influence both texture and flavor development through the formation of several low-molecular weight compounds, mainly peptides, amino acids, aldehydes, organic acids, amines, and fatty acids, which are known as important flavor compounds or precursors of flavor compounds (Leroy and others 2006). To date, the fermented fish products are mainly manufactured with the traditional spontaneous fermentation process which relies exclusively on indigenous microorganisms that occur naturally on or in fresh fish or producing environment and thus, it is hard to ensure the consistency of the final products. Moreover, growth of the spoilage and (opportunistic) pathogenic bacteria widely existed in the environment, such as Enterobacteriaceae, Enterovocci, and Staphyloccus aureus (Marty and others 2012), also threaten the safety of the product. Sometimes, activities of the bacterial decarboxylation reactions from precursor amino acids within certain R C 2015 Institute of Food Technologists
doi: 10.1111/1750-3841.12818 Further reproduction without permission is prohibited
Microbiological changes and biodiversity. . . .
Materials and Methods Preparation of sanbao LYC LYCs (weight around 230 to 270 g) were purchased from Yingzhou Aquatic Products Company in Zhoushan city, Zhejiang province and transported to the laboratory on ice within 4 h. Initially, noneviscerated LYCs were cleaned with tap water and smeared with salt (10% of the fish weight) outside of the body. After incubating at 30 °C for 1 d, a partial of salt (8% of the fish weight) was stuffed into the fish belly through the anus with a chopstick and another portion of salt (8% of the fish weight) was coated on the fish body evenly. Later, the fish were placed in a barrel with a layer of salt (4% of the fish weight) at the bottom and all the fish belly was kept upward. The prepared fish layer were subjected to a pressure (40% of the fish weight) and incubated at 30 °C for 4 d. Finally, the treated fish were coated with salt (20% of the fish weight) again and kept with pressure (15% of the fish weight) at 30 °C for another 15 d.
Enumeration and isolation of microorganisms Ten grams of the collected sample were cut into small species and then added into a 90 mL sterile saline solution (0.85% NaCl, w/v) and mixed gently. Aliquots of 100 µL serial decimal dilutions were spread on selective media in duplicate. Aerobic mesophilic bacteria, LAB, Enterobacteriacea, and Staphylococcus were enumerated using Plate Count Agar (PCA), de Man Rogosa and Sharp (MRS) agar, Violet Red Bile Glucose Agar (VRBGA), and Mannitol Salt Agar (MSA, Beijing Land Bridge, Beijing, China), respectively, and all of the plates were incubated at 30 °C for 48 h. Extra 5% of NaCl was added to each of the commercial medium applied in this study. Plates obtained 30 to 300 colonies forming units (CFU) per gram were selected for enumeration. Ten colonies were picked up from the suitable diluted plate based on different morphologies and then purified 2 times. Gram-staining and catalase production test were performed with the purified cultures. For long time preservation, the purified cultures were stored at –80 °C with 30% glycerol. Genomic DNA extraction and gene amplification Genomic DNA was extracted with bacteria genomic DNA kit (Beijing Zoman Biotechnology Co. LTD., Beijing, China) according to the instruction manual. Amplification of the 16S rDNA was accomplished with primers 27F (5 -AGA GTT TGA TCC TGG CTC AG-3 ) and 1492r (5 -GGC TAC CTT GTT ACG ACT T-3 ) according to the protocol described before (Dai and others 2013) using a T100TM Thermal Cycler (Bio-rad laboratories, U.S.A.). Amplicon of partial ropB gene fragments of Staphylococcus were generated using primer pairs of 2643F (5 -CAA TTC ATG GAC CAA GC-3 ) and 3241R (5 -GCD ACD TGD TCC ATA CCT GT-3 ) described previously (Drancourt and Raoult 2002) and the PCR reaction was carried out following the protocols reported by Marty and others (2012). All the PCR products were resolved by electrophoresis in 1% (w/v) agarose gels and the R ChemiDocTM XRS+ gels were visualized with Molecular Image (Bio-rad laboratories, U.S.A.).
Restriction analyses of PCR amplicons Restriction of the amplified fragment Staphylococci isolates were carried out by digestion of the amplicons with Mbo II first. Then the enzyme combination of Hae III and Hha I, or TspR I and Taq I were selected for the further digestion according to the initial results. Enzymes Hae III, Msp I, and Afa I were chosen for the restriction analysis of presumptive Enterobacteriaceae isolates. All the tetrameric restriction endonuclease enzymes used in this study were purchased from Thermo Fisher Scientific Co. (U.S.A.). The restriction patterns were examined using 2% (w/v) agarose gel with the100 bp DNA ladder (Takara biotechnology (Dalian) Co. Sampling and physicochemical characterization Ltd., China). Image analysis of restriction patterns were performed Samples were collected at predetermined days (0, 1st, 5th, 10th, with Image LabTM Software (Bio-Rad Laboratories, U.S.A.). 15th, 20th of the fermenting day) for the measurement of following physical properties: pH value was detected using pH-meter 16S rRNA gene sequencing and molecular identification The amplicons of the 16S rDNA were initially amplified as (Ultra Basic Benchtop, Denver Instrument, U.S.A.) with 10 g samples homogenized in 90 mL sterilized water; salt content was described above and purified with the Gel Mini Purification determined according to the AOAC (1997) procedures and mois- Kit (Zomanbio, China) and then submitted for sequencing ture content was measured by moisture meter (MB45, Ohaus by Nanjing Jinsirui Biotechnology Co. Ltd. The sequencInstrument, N.Y., U.S.A.); histamine content of the fresh fish ing data were analyzed and determined through basic local and the final product were analyzed by high-performance liquid alignment search tool searching in NCBI database (http:// chromatography system following the method proposed by Ben- blast.ncbi.nlm.nih.gov/Blast.cgi) and the assigned GenBank Gigirey and others (1999) with the 5 µm, 150 × 4.6 mm Sunfire Accession number is KJ958195-KJ958217. Determined 16S C18 column (Waters, Milford, U.S.A.). Histamine standard pur- rRNA gene sequences were aligned with the sequences of the type strains obtained from the database and then the phyletic chased from Sigma Chemical Co. was used as positive control. Vol. 80, Nr. 4, 2015 r Journal of Food Science M777
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LABs (Lactobacillus, Pediococcus, and Streptococcus) and Enterobacteriaceae (Klebsiella, Escherichia, Proteus, Pseudomonas, Shigella) could lead to hazardous amounts of biogenic amine formation in fermented meat and fish products ( Ten Brink and others 1990; Anastasio and others 2010). In this case, the use of the starter cultures would be an optimum approach for the control and optimization of the fermentation process (G¨otz 1990). It has been known that the microbial diversity of the spontaneous fermented food products is fundamental for searching starter cultures providing novel properties and the species identification of the fermenting microorganisms would enable a more predictable fermentation process, giving the product greater consistency in quality and decrease in hygienic risks (Kopermsub and Yunchalard 2010; Marty and others 2012). Consequently, investigation of the microbial diversity, technological traits, and hygienic quality of the fermenting microorganisms in spontaneous fermented fish product may provide precious data for the starter selection of the corresponding products. This study was designed to evaluate the diversity of the cultivable indigenous microorganisms involved in the traditional spontaneous fermented Sanbao large yellow croakers (LYC, Pseudosciaena crocea) production, which could provide the basic microbial background knowledge and data for starter culture collection. The Polymerase Chain Reaction-restriction fragment length polymorphism (PCR-RFLP) methods combined with sequencing approach were mainly used as a fast and powerful approach for species identification of the isolates picked up from different fermenting periods during the Sanbao LYC production.
Microbiological changes and biodiversity. . . .
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tree was constructed by the neighbor-joining method using the 2.74 ± 0.05 and 2.06 ± 0.08 log CFU/g. This similar variation trend predicted that the Staphylococci were likely to be the MEGA 4.1 software. dominant bacterial population at the end of the fermenting phase. Among all the tested microorganisms, presumptive EnterobacteResults and Discussion riaceae were most sensitive ones to the salt concentration as their Physicochemical characters along the fermentation counts decreased immediately and rapidly after the salt adding process (Figure 1). The Enterobacteriaceae were progressively eliminated The fermentation was carried out following traditional Sanbao with initial population of 3.8 ± 0.05 log CFU/g and could not be LYC processing technique. Six samples were collected from dif- detected until the 10th fermenting day. As the salt added for the 1st ferent fermenting periods for the physicochemical character tests. time, growths of the natural microbial flora in the fresh fish were The pH value of the fish increased steeply from 6.82 to 7.82 after inhibited obviously with the relatively high salt concentration. Afthe first salt adding step and then decreased gradually to 6.80 at the ter the second salt adding step, the less halophilic and salt-tolerant end of the fermentation. The initial rise in pH was possibly due to microorganisms are eliminated, giving way to the more halophilic the formation of nitrogenous basic compounds as a result of en- and salt-tolerant ones already present in the fish (Anihouvi and zyme and microbial proteolytic activity, while the gradual increase others 2007). in fermenting microbial population may lead to an accumulation of the organic acid and correspond to the following decrease of Identification of Staphylococcus isolates the pH value. In total, 42 presumptive LABs and 52 presumptive StaphyloAs the salt content of the Sanbao LYC increased steadily from cocci were picked up from MRS and MSA agars, respectively, and 0.4% to 35.65% (w/v), the moisture content descended rapidly from 77.58% to 50.93% until the 10th fermenting day as the all of these isolates were gram positive and catalase positive. In this salt was added 3 times. Then, the variation of the salt content case, we tried to amplify the partial rpoB gene of all the isolates and moisture content tended toward stable and stopped finally at gotten from both MRS and MSA medium and 90 of amplicons 36.20% and 46.51%, respectively. This high level of NaCl is likely were obtained. Later, 2-step RFLP analysis was carried out for to have a pronounced influence on the microbial growth and the clustering those isolates. The initial RFLP analysis with the rerate of fermentation, and thereby on the sensory quality and safety striction enzyme Mbo II resulted in 2 clusters, Type H and Type T (Table 1). Isolates grouped into Type H were digested with Hae of the product (Paludan-M¨uller and others 2002). Although there is no histamine detected in the fresh LYC sam- III and Hha I further, while isolates in Type T were distinguished ple, it is noteworthy that the histamine content of the final product by combination of TspR I and Taq I instead. Finally, all of the 90 even reached to 91.67 mg/kg which has exceeded the guidance isolates were classified into 13 different RFLP clusters with fraglevel of 50 mg/kg established by U.S. Food and Drug Administra- ment size ranged from 104 to 591 bp (Table 1). At least 1 isolate tion ( U.S. FDA, 2001) and was also near the limit for human con- from each RFLP profile type was selected for 16S rRNA gene sumption of 100 mg/kg in some fishery products prescribed by the sequencing, which confirmed identity of the isolates on species European Union [Commission Regulation (EC) No 1441/2007]. level (Table 1), and most of the sequences were perfectly identical As far as we knew, the consumption of foods with high levels of to the data in the Gene Bank (99% to 100%). Staphylococcus was histamine can cause allergy-like symptoms in sensitive humans and confirmed as the predominant genus recovered from both MRS the histamine contamination in fish and fish products may cause and MSA agar, which is similar to that occurring in isolates picked food poisoning (Rabie and others 2009; Joint FAO/WHO Expert up from the Plasom (Paludan-M¨uller and others 2002). FurtherMeeing 2012). Therefore, the result obtained in our study sug- more, the Staphylococcus isolates from the Sanbao LYC displayed a gests careful monitoring of histamine contents in these products is pressing. The high value of histamine in Sanbao LYC might be attributed to the fact that the viscera and gills remained in the fish body during the whole fermentation process, which provide more opportunity for the growth of histamine-forming bacteria that usually inhabit in the intestinal tract, viscera, and gills.
Growth of microbial population during Sanbao LYC fermentation The microbial population variation during the Sanbao LYC fermentation was revealed in Figure 1. The data of the initial microbial account in the Sanbao LYC sample indicated that the fresh fish was a good habitat for the microorganisms. With the salt added for the first time, all of the bacterial populations investigated in this study experienced a rapid decrease. Thereafter, the salt added for the 2nd time at the end of the 1st fermenting day, and except for the Enterobacteriaceae, the numbers of the aerobic mesophilic count, LAB, and Staphylococcus counts all rebound to relatively high levels of 5.26 ± 0.06, 4.93 ± 0.10, and 5.10 ± 0.01 log CFU/g, respectively, till the 5th fermenting day. After the 3rd salt adding process, both of the aerobic mesophilic count and Staphylococcus counts decreased gradually in a similar manner and kept at a moderate level at the end of the fermentation, about M778 Journal of Food Science r Vol. 80, Nr. 4, 2015
Figure 1–Growth of the bacteria species during fermentation of Sanbao Large Yellow Croaker. ∗ Total aerobic bacteria counts (open squares), lactic acid bacteria (open circles), Staphylococcus (open triangles), Enterobacteriaceae (open inverted triangles) were counted on PCA, MRS agar, MSA agar, and VRBGA, respectively.
Microbiological changes and biodiversity. . . . Table 1–Species assessment of the Staphylococcus isolates based on the ropB gene RFLP and 16S rDNA sequencing. Restriction profile Cluster B Isolate code
Ampa
Cluster A
SX4, SX8, S5–4, S5–10, L10–8, L10–10 SX2, SX3, SX6, S1–2, S1–4, S1–10, L5–10 SX5, SX7, S5–1, S15–5, LX5,L5–2 S5–5, S10–1, S10–3, S10–4, S10–8 to S10–10, S15–10, S20–1, L1–3, L1–9, L1–10, L5–1, L5–5, L10–1, L10–3 to L10–5, L15–1 S20–9, S20–10, S20–4, S20–8, L10–6, LX6 S5–7, S5–8, S10–6 S5–2, S5–3, S10–7, S15–2, S15–9, S20–7, S20–2, S20–3, L5–4, L10–7, L15–2, L15–4 LX9, L1–2, L1–6, L1–7, L5–3, L5–6, L5–8, L10–2, L20–1 S1–8, S1–9, S5–6, S5–9, S10–5, S15–7, S15–8, S20–5, S20–6, L1–4, L1–8 LX10, L1–1, L5–7, L15–7 S1–3, S1–7, L1–5 S15–1, L15–6 S15–4, S15–6
600 600 600 600
Enzyme a
Enzyme b
Species
T T T T
551 547 300–245 551
315–236 321–289 310–220 279–217–116
S. vitulinus S. aureus S. xylosus S. xylosus
600 600 600
T T T
551 551 366–164
551 292–138 268–116–104
S. xylosus S. saprophyticus S. saprophyticus
600 600
T T
589 319–262
326–140–135 585
S. saprophyticus S. nepalensis
600 600 600 600
T T T H
410–190 273–226 428–198 410–221
315–226 510 570 591
S. nepalensis S. sciuri S. succinus S. equorum
a
great diversity with 8 species identified including S. vitulinus, S. aureus, S. xylosus, S. saoriohyticus, S. nepalensis, S. sciuri, S. succinus, and S. equorum. Moreover, genetic diversity within the species of S. xylosus, S. saprophyticus, and S. nepalensis were also observed as they all possessed varied RFLP profile patterns. The relationship of different Staphylococus species was reflected by the phyletic tree constructed on 16S rRNA gene (Figure 2). Many studies have concluded that coagulase-negative Staphylococci are frequently recovered from food-related sample and especially in fermented products (Cheese or dry sausage; Blaiotta and others 2004; Mounier and others 2005; Mart´ın and others 2006; Coton and others 2010; Irlinger 2008). The species of S. xylosus, S. succinus, S. equorum, and S. saprophyticus are 4 main
Staphylococcus frequently picked up from the fermented sausages and cheese products (Coton and others 2010). In this work, S. xylosus, S. saprophyticus, and S. nepalensis were the most prevailing cultivable species recovered throughout the whole production process (Table 2) with 70 isolate taken part of 77.8% of the Staphylococcal populations. This was partially consistent with studies on traditionally spontaneously fermented sausage showing S. xylosus is frequently dominant in them, followed by S. saprophyticus (Ravyts and others 2012; Janssens and others 2014). The species S. xylosus are eligible as starter cultures for fermented meat products and has been used widely as commercial starter, and characterized by rounded aroma and less acidic taste (Mauriello and others 2004). S. sarprophyticus is often isolated in large numbers from southern European fermented salamis and especially dominant in some of the traditional Greek salami (Samelis and others 1998; Mauriello and others 2004). Although, S. sarprophyticus exhibited the unexcellent ability to reduce nitrate, the first criterion in the selection of strains to be used as starter cultures for sausage manufacture, it processed high values of SOD and catalase activity which could help to prevent off-flavors produced by lipid oxidation during sausage ripening. Therefore, it was suggested to be selected and validated as starter culture in some of the fermented food productions (Samelis and others 1998). However, special careful consideration should be taken before it applied in food industry since this species is deemed to be potentially pathogenic and frequently associated with clinical cases (Hammes and Hertel 1998; Marty and others 2012). S. nepalensis (15 strains, 16%) was the 3rd largest bacterial population recovered from the Sanbao LYC. Although this species was not often reported in the food-associated product, S. nepalensis strains picked up from Japanese fish sauce has been proved to obtain the ability to improve fish sauce odor and is likely to improve the unpleasant odor of fermented seafoods (Fukami and others 2004a,b). Five other species, namely, S. aureus, S. vitulinus, S. sciuri, S. Figure 2–A phylogenetic tree based on 16S rDNA sequences constructed by equorum, and S. succinus formed a minor fraction of the staphylothe neighborhood-joining method. ∗ The type strains M. caseolyticus ATCC coccal communities of the Sanbao LYC biota (Table 2). S. aureus is 13548T was used as outgroup. Numerals at the nodes indicate bootstrap the best known staphylococcal pathogen which produces a great values (%) derived from 1000 replications. Vol. 80, Nr. 4, 2015 r Journal of Food Science M779
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ropB gene amplified product size in bp; restriction fragment under 100 bp are not considered. The isolates with bold mark were subjected to sequencing. PCR-RFLP was based on ropB gene sequence with Mbo II as restriction enzyme first. Cluster A with H type profile was digested by Hae III(a) and Hha I (b) further. Isolates with T type profile were then digested with TspR I (a) and Taq I (b). (Cluster T: 320–280 bp; Cluster H: 330–220 bp).
Microbiological changes and biodiversity. . . . Table 2–Distribution of different isolated Staphylococcus species during the fermentation process. Samples Species S. xylosus S. saprophyticus S.nepalensis S. aureus S. vitulinus S. sciuri S. equorum S. succinus Total isolates
Isolates Total No.
Percentage (%)
0d
1d
5d
10 d
15 d
20 d
31 24 15 7 6 3 2 2 90
34.4% 26.7% 16.7% 7.8% 6.7% 3.3% 2.2% 2.2%
4 1 1 3 2 0 0 0 11
3 3 5 3 0 3 0 1 17
5 8 3 1 2 0 0 0 19
11 4 1 0 2 0 0 0 19
3 4 3 0 0 0 2 1 13
5 4 2 0 0 0 0 0 11
Table 3–Species assessment of the VRBDA isolates based on RFLP analysis and sequencing of the 16S rRNA gene. Restriction profile Isolate code EX7,EX8,EX9, E1–2, E1–4, E1–8 E1–1 E1–6,E1–10 EX4 E1–3
Ampa
Hae III
Msp I
Rsa I
Species
1500 1500 1500 1500 1500
690–240–173–114 286–262–149–108 286–262–149–108 1124–296 678–276–208–157–120
585–520–152–142 611–384–195–141 526–463–218–113 549–484–116–105 540–443–320–140
641–334–200–118–106 494–438–122 487–434–402 486–397–121 475–387–336–128
Ps. putida Pr. vulgaris Pr. penneri Ps. fulva B. subtills
a
16S rRNA gene amplified product size in bp; restriction fragment under 100 bp are not considered. The isolates with bold mark were subjected to sequencing.
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number of toxins causing foodborne intoxications (Akineden and others 2008). Fortunately, less S. aureus isolates was just detected in the fresh fish and initial fermenting samples and no S. aureus was found after the 5th fermenting day. S. vitulinus, which were deemed as host specific in the fresh LYC, have been also recovered from several kinds of fermented sausages (Coton and others 2010). Generally, S. succinus and S. equorum play important roles in the ripening process of certain fermented dry sausages production (Ravyts and others 2012). Corbi`ere Morot-Bizo and others (2006) have indicated that those 2 species represented 82% of Staphylococcal populations in French naturally fermented sausages, and considerable proportion among the other fermented cheese, meat sausage, as well as fermented fish products (Mauriello and others 2004; Guan and others 2011). It has been predicted that gram positive and catalase positive cocci, especially the coagulase-negative Staphylococci, participate in the development of flavor of many fermented meat products mainly through amino and fatty acid degradation (Talon and Leroy 2006). Berdagu´e and others (1993) suggested that Staphylococcus spp., rather than LAB, could have a predominant effect on sausage flavor development. In this case, considerable high percentage in microbial population and good diverse among the species in the Staphylocuccus investigated in this work could be of great importance to the taste and flavor of the final product.
Identification of LAB and presumptive Enterobacteriacea isolates LABs have been considered as the predominant species in various fermented food products including many fermented fish products (Franc¸oise 2010; Hwanhlem and others 2011), however, in this study a marginal presence of LAB was detected. Four LAB isolates picked up from fresh fish were identified as Macrococcus (M.) caseolyticus and the other one was Carnobacterium divergens through direct 16S rDNA sequencing. The remarkably high NaCl concentration applied for the Sanbao LYC processing may cause a broad inhibition to the nonhalophilic bacteria including LAB, which was consistent with previous studies showing that salt M780 Journal of Food Science r Vol. 80, Nr. 4, 2015
concentration up to 7% results in the inhibition of LAB (Horner 1997). Accordingly, low amount of LAB population could be the reason why no stronger variation in pH was observed during the Sanbao LYC fermentation. Species in Enterobacteriacea family are frequently detected on fresh or processed seafood and responsible for the decomposition of fish (Yin and others 2002). In the work present herein, Enterobacteriacea constituted a small population in the initial period of the Sanbao LYC fermentation. Eleven isolates recovered from the VRBDA medium were finally classified into 3 genus and 5 species, namely, Pseudomonas (Ps.) putida, Ps. fulva, Proteus (Pr.) penneri, Pr. Vulgaris, and Bacillus (B.) subtilis (Table 3). Only 2 Pr. Penneri and 1 Pr. vulgaris belong to Enterobacteriacea family. Note that 55% of the VRBDA isolates belong to the genus Pseudomonas, which has been considered as the most prolific microorganisms during the storage of fish regardless of its origin (Gennari and others 1999). The predominant species Ps. putida was also frequently isolated from fresh and fermented samples and usually involved in spoilage of fish, meat, and milk for producing H2 S and/or biogenic amine.
Conclusion The Sanbao LYC was processed following the traditional Sanbao technique with high concentration of NaCl, which may have a pronounced influence on the microbial populations involved as well as the flavor of the final products. Although, seldom microorganisms could survive under the high osmotic pressure environment, a good species diversity among the predominant bacteria Staphylococus recovered from the Sanbao LYC was still observed. A thorough knowledge of microbiological diversity and variations during the fermentation of the fish product should be helpful for the development of indigenous starters, which is very promising because it enables products to be produced with both high sanitary and sensory qualities. In addition, the major problem related to this product is the considerable high histamine content. The causing of the histamine accumulation in this product should be investigated carefully in future studies. Data obtained in this work could be referred in control and optimization of the fermentation
process and selecting suitable strains for aquatic product fermentation. Further study is required to obtain a better understanding of the metabolic (technological features) characters of the dominant species involved in the fermentation.
Acknowledgment This research work was financially supported by the Zhejiang Provincial Public Program (2014C32048) and National Key Technology R&D Program (2012BAD29B06).
Author Contributions H. Zhang and Y. Li designed the study and interpreted the results. J.J. Wu and Z.Y. Dai collected test data and drafted the manuscript. K.H. Xu attended the bench work.
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G¨otz F. 1990. Staphylococcus carnosus: a new host organism for gene cloning and protein production. J Appl Bacteriol 69:49–53. Guan L, Cho KL, 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. Hammes WP, Hertel C. 1998. New developments in meat starter cultures. Meat Sci 36:155–68. Horner WFA. 1997. Preservation of fish by curing (drying, salting and smoking). In: Hall GM, editor. Fish processing technology. 2nd ed. London: Blackie Academic & Professional (Chapman & Hall)). p 32–72. Hwanhlem N, Buradaleng S, Wattanachant S, Benjakul S, Tani A, Maneerat S. 2011. Isolation and screening of lactic acid bacteria from Thai traditional fermented fish (Plasom) and production of Plasom from selected strains. Food Control 22:401–7. Irlinger F. 2008. Safety assessment of dairy microorganisms: coagulase-negative staphylococci. Int J Food Microbiol 126:302–10. Janssens M, Van der Mijnsbrugge A, S´anchez Mainar M, Balzarini T, De Vuyst L, Leroy F. 2014. The use of nucleosides and arginine as alternative energy sources by coagulase-negative staphylococci in view of meat fermentation. Food Microbiol 39:53–60. Joint FAO/WHO Expert. 2012. Meeting on the Public Health Risks of Histamine and other Biogenic Amines from Fish and Fishery Products. Rome: FAO headquarters. p 26–8. Kopermsub P, Yunchalard S. 2010. Identification of lactic acid bacteria associated with the production of plaa-som, a traditional fermented fish product of Thailand. Int J Food Microbiol 138:200–4. Leroy F, Verluyten J, De Vuyst L. 2006. Functional meat starter cultures for improved sausage fermentation. Int J Food Microbiol 106:270–85. Mart´ın B, Garriga M, Hugas M, Bover-Cid S, Veciana-Nogu´es MT, Aymericha T. 2006. Molecular, technological and safety characterization of Gram-positive catalase-positive cocci from slightly fermented sausages. Int J Food Microbiol 107:148–58. Marty E, Buchs J, Eugster-Meier E, Lacroix C, Meile L. 2012. Identification of Staphylococci and dominant lactic acid bacteria in spontaneously fermented Swiss meat products using PCR-RFLP. Food Microbiol 29:157–66. Mauriello G, Casaburi A, Blaiotta G, Villani F. 2004. Isolation and technological properties of coagulase negative staphylococci from fermented sausages of Southern Italy. Meat Sci 67:149–58. Mounier J, Gelsomino R, Goerges S, Vancanneyt M, Vandemeulebroecke K, Hoste B, Scherer S, Swings J, Fitzgerald GF and Cogan TM. 2005. Surface microflora of four smear-ripened cheeses. Appl Environ Microbiol 71:6489–500. Paludan-M¨uller C, Madsen M, Sophanodora P, Gram L, Møller 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. Rabie M, Simon-Sarkadi L, Siliha H, El-seedy S, El Badawy AA. 2009. Changes in free amino acids and biogenic amines of Egyptian salted-fermented fish (Feseekh) during ripening and storage. Food Chem 115:635–8. Ravyts F, De Vuyst L, Leroy F. 2012. Bacterial diversity and functionalities in food fermentations. Eng Life Sci 12:356–67. Samelis J, Metaxopoulos J, Vlassi M, Pappa A. 1998. Stability and safety of traditional Greek salami-a microbiological ecology study. Int J Food Microbiol 44:69–82. Talon R, Leroy S. 2006. Latest developments in meat bacterial starters. In: Nollet LML, Toldra F, editors. Advanced Technologies for Meat Processing: New York:CRC Press. p 401–18. Ten Brink B, Damink C, Joosten HM, Huis In’t Veld JH. 1990. Occurrence and formation of biologically active amines in foods. Int J Food Microbiol 1:73–84. U.S. FDA. 2001. Scombrotoxin (histamine) formation. In: Fish and fishery products hazards and controls guide. 3rd ed. Washington, DC: Food and Drug Administration. p 73–93. Xu YS. 2012. The researches of the processing technology, quality and safety evaluation of La yu [Research dissertations for Doctor’s degree]. Zhejiang Univ. (in Chinese). Yin YJ, Pan CL, Jiang ST. 2002. Effect of lactic acid bacterial fermentation on the characteristic of minced mackerel. J Food Sci 67:786–92. Zeng X, Xia W, Jiang Q, Yang F. 2013. Chemical and microbial properties of Chinese traditional low-salt fermented whole fish product Suan yu. Food Control 30:590–5.
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Microbiological changes and biodiversity. . . .
Sanbao large yellow croaker is a kind of Chinese salted and fermented seafood processed by adding 50% of salt and fermenting at high temperature [around 30 °C] over 20 d. To get a comprehensive understanding of cultivable microorganism’s diversity present in its fermentation, the chemical and microbial properties of this product, were detected initially, followed by identification of bacteria recovered from different fermentation periods using PCR-RFLP and 16S rRNA gene sequencing analysis. In total, 105 indigenous isolates were recovered with 3 different medium and majority of the isolates picked up from both MRS and MSA medium were finally clustered into the genus of Staphylococcus (S.). Among the 90 Staphylococci, S. xylosus, S. saprophyticus, and S. nepalensis were the most prevailing cultivable species recovered throughout the whole production process (70 isolates, 77.8%), while 5 other species, namely, S. aureus, S. vitulinus, S. sciuri, S. equorum, and S. succinus formed a minor fraction (20 isolates, 22.2%) of the Staphylococcus communities. Lactic acid bacteria, Pseudomonas, Proteus, and Bacillus constituted trivial populations in the initial period of the fermentation and then gave the way to the Staphylococcus immediately. High salt concentration used during the processing is like to have a pronounced influence on the microbial populations involved. Data obtained in this work could be referred in control and optimization of the fermentation process and selecting suitable strains for aquatic product fermentation. Abstract:
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Keywords: diversity, fermentation, lactic acid bacteria, Sanbao large yellow croaker, Staphylococcus
Introduction Traditional spontaneous fermented fish products have long been consumed in Southeast Asian countries and Northeastern regions of Asia, such as Thailand, Korea, Japan, and China (Kopermsub and Yunchalard 2010; Zeng and others 2013). In China, there are more than 100 kinds of fermented fish products manufactured with different processing techniques mainly produced in Zhejiang, Jiangsu, Hubei, Fujian, and Shandong provinces (Xu 2012). In Zhejiang Province, the traditional fermented fish products are always processed with high-salt content according to local preferences and the “Sanbao” (in Chinese) technique is one of the popular traditional fermentation processing methods in this district. During the Sanbao processing, salts are added 3 times on the 0 (fresh fish), 1st , and 5th fermenting days with the salt to fish ratio of 10%, 20%, and 20%, respectively. After 20 d fermentation, the off-odor could become a part of the desirable odor profile in the fermented fish products while the products are free of fishy odor and taste. Previous studies have indicated the microorganisms play a critical role in fish fermentation and the species involved in the fish fermentation is of great variety (Guan and others 2011). Normally, in the low-salt processed fish products, the Gram-negative bacteria are inhibited while the Gram-positive bacteria, particularly
MS 20141515 Submitted 9/9/2014, Accepted 12/4/2014. Authors are with College of Food Science and Biotechnology, Zhejiang Gongshang Univ. , Hangzhou, Zhejiang, 310035, P. R. China and with Inst. of Aquatic Products Processing Zhejiang Gongshang Univ., Hangzhou, Zhejiang Province, 310035, P. R. , China. Direct inquiries to author Jiajia Wu (E-mail: [email protected]). Authors He Zhang and Yan Li contributed equally to this work.
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lactic acid bacteria (LAB), become predominant microorganisms (Paludan-M¨uller and others 2002; Hwanhlem and others 2011; Dai and others 2013). The beneficial effect of LABs involved in the fermentation of those products have been associated with their ability to initiate a fast pH drop by producing lactic acid in the first fermenting days, which contributes to a safe product by preventing the growth of spoilage and pathogenic organisms (Leroy and others 2006). Meanwhile, in the high-salt processed fish product, Staphylococcus constitute the highest number (Guan and others 2011). Generally, gram positive and catalase positive cocci, especially the coagulase-negative Staphylococci, participate in the development of flavor of many high-salt fermented fish and meat products mainly through amino and fatty acid degradation (Hammes and Hertel 1998; Talon and Leroy 2006). All of these desirable proteolytic and lipolytic reactions induced by both microorganisms and endogenous from fish body influence both texture and flavor development through the formation of several low-molecular weight compounds, mainly peptides, amino acids, aldehydes, organic acids, amines, and fatty acids, which are known as important flavor compounds or precursors of flavor compounds (Leroy and others 2006). To date, the fermented fish products are mainly manufactured with the traditional spontaneous fermentation process which relies exclusively on indigenous microorganisms that occur naturally on or in fresh fish or producing environment and thus, it is hard to ensure the consistency of the final products. Moreover, growth of the spoilage and (opportunistic) pathogenic bacteria widely existed in the environment, such as Enterobacteriaceae, Enterovocci, and Staphyloccus aureus (Marty and others 2012), also threaten the safety of the product. Sometimes, activities of the bacterial decarboxylation reactions from precursor amino acids within certain R C 2015 Institute of Food Technologists
doi: 10.1111/1750-3841.12818 Further reproduction without permission is prohibited
Microbiological changes and biodiversity. . . .
Materials and Methods Preparation of sanbao LYC LYCs (weight around 230 to 270 g) were purchased from Yingzhou Aquatic Products Company in Zhoushan city, Zhejiang province and transported to the laboratory on ice within 4 h. Initially, noneviscerated LYCs were cleaned with tap water and smeared with salt (10% of the fish weight) outside of the body. After incubating at 30 °C for 1 d, a partial of salt (8% of the fish weight) was stuffed into the fish belly through the anus with a chopstick and another portion of salt (8% of the fish weight) was coated on the fish body evenly. Later, the fish were placed in a barrel with a layer of salt (4% of the fish weight) at the bottom and all the fish belly was kept upward. The prepared fish layer were subjected to a pressure (40% of the fish weight) and incubated at 30 °C for 4 d. Finally, the treated fish were coated with salt (20% of the fish weight) again and kept with pressure (15% of the fish weight) at 30 °C for another 15 d.
Enumeration and isolation of microorganisms Ten grams of the collected sample were cut into small species and then added into a 90 mL sterile saline solution (0.85% NaCl, w/v) and mixed gently. Aliquots of 100 µL serial decimal dilutions were spread on selective media in duplicate. Aerobic mesophilic bacteria, LAB, Enterobacteriacea, and Staphylococcus were enumerated using Plate Count Agar (PCA), de Man Rogosa and Sharp (MRS) agar, Violet Red Bile Glucose Agar (VRBGA), and Mannitol Salt Agar (MSA, Beijing Land Bridge, Beijing, China), respectively, and all of the plates were incubated at 30 °C for 48 h. Extra 5% of NaCl was added to each of the commercial medium applied in this study. Plates obtained 30 to 300 colonies forming units (CFU) per gram were selected for enumeration. Ten colonies were picked up from the suitable diluted plate based on different morphologies and then purified 2 times. Gram-staining and catalase production test were performed with the purified cultures. For long time preservation, the purified cultures were stored at –80 °C with 30% glycerol. Genomic DNA extraction and gene amplification Genomic DNA was extracted with bacteria genomic DNA kit (Beijing Zoman Biotechnology Co. LTD., Beijing, China) according to the instruction manual. Amplification of the 16S rDNA was accomplished with primers 27F (5 -AGA GTT TGA TCC TGG CTC AG-3 ) and 1492r (5 -GGC TAC CTT GTT ACG ACT T-3 ) according to the protocol described before (Dai and others 2013) using a T100TM Thermal Cycler (Bio-rad laboratories, U.S.A.). Amplicon of partial ropB gene fragments of Staphylococcus were generated using primer pairs of 2643F (5 -CAA TTC ATG GAC CAA GC-3 ) and 3241R (5 -GCD ACD TGD TCC ATA CCT GT-3 ) described previously (Drancourt and Raoult 2002) and the PCR reaction was carried out following the protocols reported by Marty and others (2012). All the PCR products were resolved by electrophoresis in 1% (w/v) agarose gels and the R ChemiDocTM XRS+ gels were visualized with Molecular Image (Bio-rad laboratories, U.S.A.).
Restriction analyses of PCR amplicons Restriction of the amplified fragment Staphylococci isolates were carried out by digestion of the amplicons with Mbo II first. Then the enzyme combination of Hae III and Hha I, or TspR I and Taq I were selected for the further digestion according to the initial results. Enzymes Hae III, Msp I, and Afa I were chosen for the restriction analysis of presumptive Enterobacteriaceae isolates. All the tetrameric restriction endonuclease enzymes used in this study were purchased from Thermo Fisher Scientific Co. (U.S.A.). The restriction patterns were examined using 2% (w/v) agarose gel with the100 bp DNA ladder (Takara biotechnology (Dalian) Co. Sampling and physicochemical characterization Ltd., China). Image analysis of restriction patterns were performed Samples were collected at predetermined days (0, 1st, 5th, 10th, with Image LabTM Software (Bio-Rad Laboratories, U.S.A.). 15th, 20th of the fermenting day) for the measurement of following physical properties: pH value was detected using pH-meter 16S rRNA gene sequencing and molecular identification The amplicons of the 16S rDNA were initially amplified as (Ultra Basic Benchtop, Denver Instrument, U.S.A.) with 10 g samples homogenized in 90 mL sterilized water; salt content was described above and purified with the Gel Mini Purification determined according to the AOAC (1997) procedures and mois- Kit (Zomanbio, China) and then submitted for sequencing ture content was measured by moisture meter (MB45, Ohaus by Nanjing Jinsirui Biotechnology Co. Ltd. The sequencInstrument, N.Y., U.S.A.); histamine content of the fresh fish ing data were analyzed and determined through basic local and the final product were analyzed by high-performance liquid alignment search tool searching in NCBI database (http:// chromatography system following the method proposed by Ben- blast.ncbi.nlm.nih.gov/Blast.cgi) and the assigned GenBank Gigirey and others (1999) with the 5 µm, 150 × 4.6 mm Sunfire Accession number is KJ958195-KJ958217. Determined 16S C18 column (Waters, Milford, U.S.A.). Histamine standard pur- rRNA gene sequences were aligned with the sequences of the type strains obtained from the database and then the phyletic chased from Sigma Chemical Co. was used as positive control. Vol. 80, Nr. 4, 2015 r Journal of Food Science M777
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LABs (Lactobacillus, Pediococcus, and Streptococcus) and Enterobacteriaceae (Klebsiella, Escherichia, Proteus, Pseudomonas, Shigella) could lead to hazardous amounts of biogenic amine formation in fermented meat and fish products ( Ten Brink and others 1990; Anastasio and others 2010). In this case, the use of the starter cultures would be an optimum approach for the control and optimization of the fermentation process (G¨otz 1990). It has been known that the microbial diversity of the spontaneous fermented food products is fundamental for searching starter cultures providing novel properties and the species identification of the fermenting microorganisms would enable a more predictable fermentation process, giving the product greater consistency in quality and decrease in hygienic risks (Kopermsub and Yunchalard 2010; Marty and others 2012). Consequently, investigation of the microbial diversity, technological traits, and hygienic quality of the fermenting microorganisms in spontaneous fermented fish product may provide precious data for the starter selection of the corresponding products. This study was designed to evaluate the diversity of the cultivable indigenous microorganisms involved in the traditional spontaneous fermented Sanbao large yellow croakers (LYC, Pseudosciaena crocea) production, which could provide the basic microbial background knowledge and data for starter culture collection. The Polymerase Chain Reaction-restriction fragment length polymorphism (PCR-RFLP) methods combined with sequencing approach were mainly used as a fast and powerful approach for species identification of the isolates picked up from different fermenting periods during the Sanbao LYC production.
Microbiological changes and biodiversity. . . .
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tree was constructed by the neighbor-joining method using the 2.74 ± 0.05 and 2.06 ± 0.08 log CFU/g. This similar variation trend predicted that the Staphylococci were likely to be the MEGA 4.1 software. dominant bacterial population at the end of the fermenting phase. Among all the tested microorganisms, presumptive EnterobacteResults and Discussion riaceae were most sensitive ones to the salt concentration as their Physicochemical characters along the fermentation counts decreased immediately and rapidly after the salt adding process (Figure 1). The Enterobacteriaceae were progressively eliminated The fermentation was carried out following traditional Sanbao with initial population of 3.8 ± 0.05 log CFU/g and could not be LYC processing technique. Six samples were collected from dif- detected until the 10th fermenting day. As the salt added for the 1st ferent fermenting periods for the physicochemical character tests. time, growths of the natural microbial flora in the fresh fish were The pH value of the fish increased steeply from 6.82 to 7.82 after inhibited obviously with the relatively high salt concentration. Afthe first salt adding step and then decreased gradually to 6.80 at the ter the second salt adding step, the less halophilic and salt-tolerant end of the fermentation. The initial rise in pH was possibly due to microorganisms are eliminated, giving way to the more halophilic the formation of nitrogenous basic compounds as a result of en- and salt-tolerant ones already present in the fish (Anihouvi and zyme and microbial proteolytic activity, while the gradual increase others 2007). in fermenting microbial population may lead to an accumulation of the organic acid and correspond to the following decrease of Identification of Staphylococcus isolates the pH value. In total, 42 presumptive LABs and 52 presumptive StaphyloAs the salt content of the Sanbao LYC increased steadily from cocci were picked up from MRS and MSA agars, respectively, and 0.4% to 35.65% (w/v), the moisture content descended rapidly from 77.58% to 50.93% until the 10th fermenting day as the all of these isolates were gram positive and catalase positive. In this salt was added 3 times. Then, the variation of the salt content case, we tried to amplify the partial rpoB gene of all the isolates and moisture content tended toward stable and stopped finally at gotten from both MRS and MSA medium and 90 of amplicons 36.20% and 46.51%, respectively. This high level of NaCl is likely were obtained. Later, 2-step RFLP analysis was carried out for to have a pronounced influence on the microbial growth and the clustering those isolates. The initial RFLP analysis with the rerate of fermentation, and thereby on the sensory quality and safety striction enzyme Mbo II resulted in 2 clusters, Type H and Type T (Table 1). Isolates grouped into Type H were digested with Hae of the product (Paludan-M¨uller and others 2002). Although there is no histamine detected in the fresh LYC sam- III and Hha I further, while isolates in Type T were distinguished ple, it is noteworthy that the histamine content of the final product by combination of TspR I and Taq I instead. Finally, all of the 90 even reached to 91.67 mg/kg which has exceeded the guidance isolates were classified into 13 different RFLP clusters with fraglevel of 50 mg/kg established by U.S. Food and Drug Administra- ment size ranged from 104 to 591 bp (Table 1). At least 1 isolate tion ( U.S. FDA, 2001) and was also near the limit for human con- from each RFLP profile type was selected for 16S rRNA gene sumption of 100 mg/kg in some fishery products prescribed by the sequencing, which confirmed identity of the isolates on species European Union [Commission Regulation (EC) No 1441/2007]. level (Table 1), and most of the sequences were perfectly identical As far as we knew, the consumption of foods with high levels of to the data in the Gene Bank (99% to 100%). Staphylococcus was histamine can cause allergy-like symptoms in sensitive humans and confirmed as the predominant genus recovered from both MRS the histamine contamination in fish and fish products may cause and MSA agar, which is similar to that occurring in isolates picked food poisoning (Rabie and others 2009; Joint FAO/WHO Expert up from the Plasom (Paludan-M¨uller and others 2002). FurtherMeeing 2012). Therefore, the result obtained in our study sug- more, the Staphylococcus isolates from the Sanbao LYC displayed a gests careful monitoring of histamine contents in these products is pressing. The high value of histamine in Sanbao LYC might be attributed to the fact that the viscera and gills remained in the fish body during the whole fermentation process, which provide more opportunity for the growth of histamine-forming bacteria that usually inhabit in the intestinal tract, viscera, and gills.
Growth of microbial population during Sanbao LYC fermentation The microbial population variation during the Sanbao LYC fermentation was revealed in Figure 1. The data of the initial microbial account in the Sanbao LYC sample indicated that the fresh fish was a good habitat for the microorganisms. With the salt added for the first time, all of the bacterial populations investigated in this study experienced a rapid decrease. Thereafter, the salt added for the 2nd time at the end of the 1st fermenting day, and except for the Enterobacteriaceae, the numbers of the aerobic mesophilic count, LAB, and Staphylococcus counts all rebound to relatively high levels of 5.26 ± 0.06, 4.93 ± 0.10, and 5.10 ± 0.01 log CFU/g, respectively, till the 5th fermenting day. After the 3rd salt adding process, both of the aerobic mesophilic count and Staphylococcus counts decreased gradually in a similar manner and kept at a moderate level at the end of the fermentation, about M778 Journal of Food Science r Vol. 80, Nr. 4, 2015
Figure 1–Growth of the bacteria species during fermentation of Sanbao Large Yellow Croaker. ∗ Total aerobic bacteria counts (open squares), lactic acid bacteria (open circles), Staphylococcus (open triangles), Enterobacteriaceae (open inverted triangles) were counted on PCA, MRS agar, MSA agar, and VRBGA, respectively.
Microbiological changes and biodiversity. . . . Table 1–Species assessment of the Staphylococcus isolates based on the ropB gene RFLP and 16S rDNA sequencing. Restriction profile Cluster B Isolate code
Ampa
Cluster A
SX4, SX8, S5–4, S5–10, L10–8, L10–10 SX2, SX3, SX6, S1–2, S1–4, S1–10, L5–10 SX5, SX7, S5–1, S15–5, LX5,L5–2 S5–5, S10–1, S10–3, S10–4, S10–8 to S10–10, S15–10, S20–1, L1–3, L1–9, L1–10, L5–1, L5–5, L10–1, L10–3 to L10–5, L15–1 S20–9, S20–10, S20–4, S20–8, L10–6, LX6 S5–7, S5–8, S10–6 S5–2, S5–3, S10–7, S15–2, S15–9, S20–7, S20–2, S20–3, L5–4, L10–7, L15–2, L15–4 LX9, L1–2, L1–6, L1–7, L5–3, L5–6, L5–8, L10–2, L20–1 S1–8, S1–9, S5–6, S5–9, S10–5, S15–7, S15–8, S20–5, S20–6, L1–4, L1–8 LX10, L1–1, L5–7, L15–7 S1–3, S1–7, L1–5 S15–1, L15–6 S15–4, S15–6
600 600 600 600
Enzyme a
Enzyme b
Species
T T T T
551 547 300–245 551
315–236 321–289 310–220 279–217–116
S. vitulinus S. aureus S. xylosus S. xylosus
600 600 600
T T T
551 551 366–164
551 292–138 268–116–104
S. xylosus S. saprophyticus S. saprophyticus
600 600
T T
589 319–262
326–140–135 585
S. saprophyticus S. nepalensis
600 600 600 600
T T T H
410–190 273–226 428–198 410–221
315–226 510 570 591
S. nepalensis S. sciuri S. succinus S. equorum
a
great diversity with 8 species identified including S. vitulinus, S. aureus, S. xylosus, S. saoriohyticus, S. nepalensis, S. sciuri, S. succinus, and S. equorum. Moreover, genetic diversity within the species of S. xylosus, S. saprophyticus, and S. nepalensis were also observed as they all possessed varied RFLP profile patterns. The relationship of different Staphylococus species was reflected by the phyletic tree constructed on 16S rRNA gene (Figure 2). Many studies have concluded that coagulase-negative Staphylococci are frequently recovered from food-related sample and especially in fermented products (Cheese or dry sausage; Blaiotta and others 2004; Mounier and others 2005; Mart´ın and others 2006; Coton and others 2010; Irlinger 2008). The species of S. xylosus, S. succinus, S. equorum, and S. saprophyticus are 4 main
Staphylococcus frequently picked up from the fermented sausages and cheese products (Coton and others 2010). In this work, S. xylosus, S. saprophyticus, and S. nepalensis were the most prevailing cultivable species recovered throughout the whole production process (Table 2) with 70 isolate taken part of 77.8% of the Staphylococcal populations. This was partially consistent with studies on traditionally spontaneously fermented sausage showing S. xylosus is frequently dominant in them, followed by S. saprophyticus (Ravyts and others 2012; Janssens and others 2014). The species S. xylosus are eligible as starter cultures for fermented meat products and has been used widely as commercial starter, and characterized by rounded aroma and less acidic taste (Mauriello and others 2004). S. sarprophyticus is often isolated in large numbers from southern European fermented salamis and especially dominant in some of the traditional Greek salami (Samelis and others 1998; Mauriello and others 2004). Although, S. sarprophyticus exhibited the unexcellent ability to reduce nitrate, the first criterion in the selection of strains to be used as starter cultures for sausage manufacture, it processed high values of SOD and catalase activity which could help to prevent off-flavors produced by lipid oxidation during sausage ripening. Therefore, it was suggested to be selected and validated as starter culture in some of the fermented food productions (Samelis and others 1998). However, special careful consideration should be taken before it applied in food industry since this species is deemed to be potentially pathogenic and frequently associated with clinical cases (Hammes and Hertel 1998; Marty and others 2012). S. nepalensis (15 strains, 16%) was the 3rd largest bacterial population recovered from the Sanbao LYC. Although this species was not often reported in the food-associated product, S. nepalensis strains picked up from Japanese fish sauce has been proved to obtain the ability to improve fish sauce odor and is likely to improve the unpleasant odor of fermented seafoods (Fukami and others 2004a,b). Five other species, namely, S. aureus, S. vitulinus, S. sciuri, S. Figure 2–A phylogenetic tree based on 16S rDNA sequences constructed by equorum, and S. succinus formed a minor fraction of the staphylothe neighborhood-joining method. ∗ The type strains M. caseolyticus ATCC coccal communities of the Sanbao LYC biota (Table 2). S. aureus is 13548T was used as outgroup. Numerals at the nodes indicate bootstrap the best known staphylococcal pathogen which produces a great values (%) derived from 1000 replications. Vol. 80, Nr. 4, 2015 r Journal of Food Science M779
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ropB gene amplified product size in bp; restriction fragment under 100 bp are not considered. The isolates with bold mark were subjected to sequencing. PCR-RFLP was based on ropB gene sequence with Mbo II as restriction enzyme first. Cluster A with H type profile was digested by Hae III(a) and Hha I (b) further. Isolates with T type profile were then digested with TspR I (a) and Taq I (b). (Cluster T: 320–280 bp; Cluster H: 330–220 bp).
Microbiological changes and biodiversity. . . . Table 2–Distribution of different isolated Staphylococcus species during the fermentation process. Samples Species S. xylosus S. saprophyticus S.nepalensis S. aureus S. vitulinus S. sciuri S. equorum S. succinus Total isolates
Isolates Total No.
Percentage (%)
0d
1d
5d
10 d
15 d
20 d
31 24 15 7 6 3 2 2 90
34.4% 26.7% 16.7% 7.8% 6.7% 3.3% 2.2% 2.2%
4 1 1 3 2 0 0 0 11
3 3 5 3 0 3 0 1 17
5 8 3 1 2 0 0 0 19
11 4 1 0 2 0 0 0 19
3 4 3 0 0 0 2 1 13
5 4 2 0 0 0 0 0 11
Table 3–Species assessment of the VRBDA isolates based on RFLP analysis and sequencing of the 16S rRNA gene. Restriction profile Isolate code EX7,EX8,EX9, E1–2, E1–4, E1–8 E1–1 E1–6,E1–10 EX4 E1–3
Ampa
Hae III
Msp I
Rsa I
Species
1500 1500 1500 1500 1500
690–240–173–114 286–262–149–108 286–262–149–108 1124–296 678–276–208–157–120
585–520–152–142 611–384–195–141 526–463–218–113 549–484–116–105 540–443–320–140
641–334–200–118–106 494–438–122 487–434–402 486–397–121 475–387–336–128
Ps. putida Pr. vulgaris Pr. penneri Ps. fulva B. subtills
a
16S rRNA gene amplified product size in bp; restriction fragment under 100 bp are not considered. The isolates with bold mark were subjected to sequencing.
M: Food Microbiology & Safety
number of toxins causing foodborne intoxications (Akineden and others 2008). Fortunately, less S. aureus isolates was just detected in the fresh fish and initial fermenting samples and no S. aureus was found after the 5th fermenting day. S. vitulinus, which were deemed as host specific in the fresh LYC, have been also recovered from several kinds of fermented sausages (Coton and others 2010). Generally, S. succinus and S. equorum play important roles in the ripening process of certain fermented dry sausages production (Ravyts and others 2012). Corbi`ere Morot-Bizo and others (2006) have indicated that those 2 species represented 82% of Staphylococcal populations in French naturally fermented sausages, and considerable proportion among the other fermented cheese, meat sausage, as well as fermented fish products (Mauriello and others 2004; Guan and others 2011). It has been predicted that gram positive and catalase positive cocci, especially the coagulase-negative Staphylococci, participate in the development of flavor of many fermented meat products mainly through amino and fatty acid degradation (Talon and Leroy 2006). Berdagu´e and others (1993) suggested that Staphylococcus spp., rather than LAB, could have a predominant effect on sausage flavor development. In this case, considerable high percentage in microbial population and good diverse among the species in the Staphylocuccus investigated in this work could be of great importance to the taste and flavor of the final product.
Identification of LAB and presumptive Enterobacteriacea isolates LABs have been considered as the predominant species in various fermented food products including many fermented fish products (Franc¸oise 2010; Hwanhlem and others 2011), however, in this study a marginal presence of LAB was detected. Four LAB isolates picked up from fresh fish were identified as Macrococcus (M.) caseolyticus and the other one was Carnobacterium divergens through direct 16S rDNA sequencing. The remarkably high NaCl concentration applied for the Sanbao LYC processing may cause a broad inhibition to the nonhalophilic bacteria including LAB, which was consistent with previous studies showing that salt M780 Journal of Food Science r Vol. 80, Nr. 4, 2015
concentration up to 7% results in the inhibition of LAB (Horner 1997). Accordingly, low amount of LAB population could be the reason why no stronger variation in pH was observed during the Sanbao LYC fermentation. Species in Enterobacteriacea family are frequently detected on fresh or processed seafood and responsible for the decomposition of fish (Yin and others 2002). In the work present herein, Enterobacteriacea constituted a small population in the initial period of the Sanbao LYC fermentation. Eleven isolates recovered from the VRBDA medium were finally classified into 3 genus and 5 species, namely, Pseudomonas (Ps.) putida, Ps. fulva, Proteus (Pr.) penneri, Pr. Vulgaris, and Bacillus (B.) subtilis (Table 3). Only 2 Pr. Penneri and 1 Pr. vulgaris belong to Enterobacteriacea family. Note that 55% of the VRBDA isolates belong to the genus Pseudomonas, which has been considered as the most prolific microorganisms during the storage of fish regardless of its origin (Gennari and others 1999). The predominant species Ps. putida was also frequently isolated from fresh and fermented samples and usually involved in spoilage of fish, meat, and milk for producing H2 S and/or biogenic amine.
Conclusion The Sanbao LYC was processed following the traditional Sanbao technique with high concentration of NaCl, which may have a pronounced influence on the microbial populations involved as well as the flavor of the final products. Although, seldom microorganisms could survive under the high osmotic pressure environment, a good species diversity among the predominant bacteria Staphylococus recovered from the Sanbao LYC was still observed. A thorough knowledge of microbiological diversity and variations during the fermentation of the fish product should be helpful for the development of indigenous starters, which is very promising because it enables products to be produced with both high sanitary and sensory qualities. In addition, the major problem related to this product is the considerable high histamine content. The causing of the histamine accumulation in this product should be investigated carefully in future studies. Data obtained in this work could be referred in control and optimization of the fermentation
process and selecting suitable strains for aquatic product fermentation. Further study is required to obtain a better understanding of the metabolic (technological features) characters of the dominant species involved in the fermentation.
Acknowledgment This research work was financially supported by the Zhejiang Provincial Public Program (2014C32048) and National Key Technology R&D Program (2012BAD29B06).
Author Contributions H. Zhang and Y. Li designed the study and interpreted the results. J.J. Wu and Z.Y. Dai collected test data and drafted the manuscript. K.H. Xu attended the bench work.
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