Application of Native Lactic Acid Bacteria (LAB) for Fermentative Recovery of Lipids and Proteins from Fish Processing Wastes: Bioactivities of Fermentation Products

Fermentation using native lactic acid bacteria (LAB) was evaluated for its effectiveness in recovering lipids and proteins simultaneously from freshwater fish visceral waste (FVW). Five different LAB isolated from fish processing waste were employed in a fermentation process that involved 10% (w/w) glucose, 2% (w/w) NaCl, and 10% (v/w) LAB. Cultures evaluated included four native isolates (Pediococcus acidilactici NCIM5368, Enterococcus faecalis NCIM5367, Pediococcus acidilactici FM37, and Pediococcus acidilactici MW2) from FVW with E. faecium NCIM5335 as the reference culture. Fermentation with native LAB resulted in recovery of > 90% oil present in the material as against no recovery in case of raw (unfermented) viscera and resulted in > 50% of degree of hydrolysis of proteins. The fatty acid profile of lipids was not affected by the fermentation process. The fermentation liquor, rich in hydrolyzed protein, exhibited antioxidant as well as antagonistic properties against several bacterial pathogens. The results clearly demonstrate the usefulness of fermentation using native isolates for simultaneous recovery of lipids and proteins from fish processing waste. It also asserts the value of fermentation as an eco-friendly method and aids in minimizing disposal/pollution problems associated with these solid wastes.     

The Effects of Fermentation Process with Acid and Lactic Acid Bacteria Strains on the Biogenic Amine Formation of Wet and Spray-Dried Fish Silages of Discards

The impacts of fermentation process with acid and lactic acid bacteria strains (Lactobacillus plantarum, Lactobacillus brevis, Pediococcus acidilactici, Enterococcus gallinarum, and Streptococcus spp.) on the biogenic amine formation of wet and spray-dried fish silage obtained from whole gibel carp (Carassius gibelio, freshwater discard), whole ponyfish (Equulites klunzingeri, seawater discard), and processing by-products of seabass (Dicentrarchus labrax) were investigated. The results showed that among biogenic amines, cadaverine, putrescine, spermidine, spermine, serotonin, dopamine, and agmatine were predominant in all groups. Significant differences (p < 0.05) in biogenic amine concentrations of wet and spray-dried fish silage were observed. Raw fish and wet silages contained histamine level lower than the allowable limit of 50mg/kg, indicating the use of raw fish material with low microbial counts. In addition, no histamine was detected in spray-dried fish silage, except for seabass by-products with a trace quantity of histamine (<0.04mg/100g). The starter culture used for silage preparation did not effectively retard formation of biogenic amines compared to acid silage. It can be concluded that there is potential use of fermented fish silage as a protein source and possibly as a probiotic ingredient for animal feed in both wet and dry form.     

Isolation,Screening, and Identification of Proteolytic Lactic Acid Bacteria from Indigenous Chao Product

The aim of the study was to isolate, select, and identify proteolytic lactic acid bacteria (LAB) from chao, a traditional fermented fish from Pangkajene and Kepulauan Regency, South Sulawesi, Indonesia. LAB was isolated by poured plate method. Proteolytic LAB were selected using agar skim milk media. Protease activity of LAB was determined based on the amount of tyrosine released in unit/mL. Proteolytic LAB were identified using API 50 CHL kit and 16S rRNA gene sequence analysis. The result showed that a total of 60 isolates were obtained from chao, and 57% of them were cocci-shape. Fifteen isolates were halotolerant proteolytic LAB. Their R values and protease activity were 2.11–3.39 and 0.267–0.304 U/mL, respectively. Identification by API 50 CHL kit showed that four rod-shape isolates were Lactobacillus plantarum, and two others were Lactobacillus curvatus. Cocci-shape isolates could not be identified as cocci bacterium. Rep-PCR results showed that there were two kinds of bands, namely thick and thin. Two isolates were selected from two types of bands that had the highest R for the analysis of 16S rRNA gene sequences, namely Ags1-3 and Ags7-3. The results showed that Ags1-3 isolate was identified as Lactobacillus plantarum and Ags7-3 as Pediococcus acidilactici.     

Effect of Protease Treatment on Oil Extractability of Carotenoids from Shrimp Waste

Crustacean waste is an important source of natural carotenoids. Carotenoids in shrimp waste occur as a complex with proteins. Investigations were carried out to assess the effect of hydrolyzing shrimp waste with different proteases on the subsequent recovery of carotenoids in vegetable oil. Hydrolyzing the shrimp waste with the bacterial protease alcalase gave higher extraction yields in oil (28.6 μg/g waste) compared to hydrolyzing with the plant protease papain (24.8 μg/g) or the animal protease trypsin (25.3 μg/g). Optimized conditions for alcalase treatment and extraction of carotenoids was determined to be hydrolysis of shrimp waste with 0.75% of alcalase at 37°C for 150 min, adding sunflower oil to hydrolyzed waste in a ratio of 2:1, and heating at 70°C for 90 min. Prior hydrolysis of waste considerably reduced the heating time required for maximum extraction of pigments, which may help in preventing the carotenoid degradation during prolonged heating in oil.     

Effects of Lactic Acid Bacteria and Citrus Essential Oil on the Quality of Vacuum-Packed Sea Bass (Dicentrarchus labrax) Fillets During Refrigerated Storage

The preservative effect of refrigerated and vacuum-packed sea bass (Dicentrarchus labrax) fillets inoculated with four mixtures of lactic acid bacteria (LAB) (Lactococcus lactis, Lactobacillus plantarum, and Carnobacterium piscicola) and incorporated with citrus essential oil (CEO) was evaluated on the basis of microbiological and biochemical analysis.

Initially, sea bass fillets showed high nutritional quality. During refrigerated storage, lipid contents did not show a significant decrease in any fish fillets; meanwhile, important proteolysis was observed in untreated control. In addition, results indicated that both CEO and LAB strains exhibited antimicrobial activity against spoilage, pathogenic, and fungi flora. Moreover, the total volatile bases (TVB-N) values were higher in control fillets, and the lowest TVB-N values were observed in Control CEO and C3 + CEO (30.47 ± 0.00 and 32.29 ± 1.12 mg TVB-N/100 g, respectively). Also, the levels of biogenic amines increased in all fillets without exceeding the upper limit of acceptability except for untreated control (sum of about 1396.63 ppm). Furthermore, this combined treatment ameliorated the muscle liquid-holding capacity, which improves technological properties.

Overall, this treatment may open new promising opportunities for the biopreservation of fish products by enhancing the period of storage of refrigerated and vacuum-packed sea bass fillets.     

Isolation,Identification and Characterization of Lactic Acid Bacteria From Shao-jiou-luo (Fermented Zoned Cerith), a Traditional Fermented Food in Taiwan

This study investigated the distribution of the native lactic acid bacteria (LAB) population in shao-jiou-luo (fermented zoned cerith), a popular traditional fermented food in Taiwan. A total of 129 cultures of LAB were isolated from shao-jiou-luo samples, and 14 cultures were isolated from its main raw material, zoned cerith. These isolates were divided into classes by phenotype and then into groups by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. The identification results revealed 11 bacterial species belonging to five genera in shao-jiou-luo and three species belonging to two genera in zoned cerith. The most common bacterial genera in shao-jiou-luo were heterofermentative Weissella and Leuconostoc. On the other hand, Enterococcus aquimarinus was the most common species found in the raw zoned cerith sample. In conclusion, the results suggest that heterofermentative LAB species greatly contributed to the fermentation of shao-jiou-luo. This is the first report describing the distribution and varieties of LAB that exist in the shao-jiou-luo fermentation process and raw zoned cerith.     

混合乳酸菌发酵改善罗非鱼蛋白酶解液风味的研究

通过嗜热乳链球菌（Streptococus thermophilus）和保加利亚乳杆菌（Lactococcus bulgarcus）混合发酵,改善罗非鱼（Oreochromis niloticus）下脚料蛋白酶解液风味,采用响应面设计试验考察乳酸菌发酵时间（X1）、温度（五）及接种量（恐）对酶解液风味感官评分值（Y）的影响,并利用同时蒸馏萃取与气相色谱-质谱（GC—MS）联用技术测定挥发性成分。结果表明,响应面分析法（RSM）优化的乳酸菌发酵条件为时间9h,温度45℃,接种量16％,预测风味感官评分值为4．1;酶解液发酵后的挥发性成分由原来的27种增加至33种,增加的主要是酯类物质,另外产生了辛酸及十六醛等风味物质。乳酸菌发酵能减弱罗非鱼酶解液的腥味,但脱苦效果不明显。     

Development of Dry Artificial Fish Bait for Trap Fishing Using Tuna Red Meat and Shrimp Head Wastes

ABSTRACT

This study developed and evaluated dry artificial fish bait for trap fishing utilizing tuna red meat (TRM) and shrimp head powder (SHP), aiming to replace the usage of edible fishes, such as sardines and squids, as bait. A total of 23 dry baits were prepared and tested for stability in seawater and acceptance by tilapia. Based on the stability and acceptability test results, two baits were selected as ideal and were further tested for protein leaching and dry matter loss in seawater. The tests revealed that the stick-shaped bait containing 61% TRM and 15% SHP was the ideal bait, with a protein leaching rate of 24.82 mg/g/h and dry matter loss of 36.6 mg/g/h. The catch rate of the traps baited with the artificial fish bait was significantly higher (P < 0.05) than those baited with squid meat (17 Nos/trap) and sardines (23 Nos/trap). Further, the catch rate of traps baited with artificial bait (38 Nos/trap) was found to be comparable with those baited with raw shrimp head wastes (SHW) (40 Nos/trap). When the bait was concentrated with 0.1% sodium benzoate, shelf life was extended by up to 6 months.     

分离自腌干鱼的抗氧化发酵菌株的筛选及鉴定

以符合耐盐、耐高低温、产酸等要求和过氧化氢耐受能力为指标,对分离自传统腌干鱼制品的29株乳酸菌进行初筛,再以抗脂质过氧化率、羟自由基清除率和还原力为复筛指标评价了菌株不同组分的体外抗氧化能力,以期获得具有优良抗氧化活性的发酵菌株。结果表明,15株乳酸菌符合腌干鱼发酵要求,菌株间的抗氧化活性差异明显,其中以L4、L11和L21综合抗氧化活性最好。经VITEK-2微生物鉴定系统和16S r DNA分子鉴定,确定L4、L11和L21分别为戊糖片球菌（Pediococcus pentosaceus）、植物乳杆菌（Lactobacillus plantarum）和干酪乳杆菌（L.casei）。 

     

利用虾壳清洁化生产不同脱乙酰度壳寡糖及其抗TMV效果研究

壳寡糖的传统生产工艺是采用HCl和NaOH处理甲壳类制备壳聚糖,再酶解获得壳寡糖,生产过程中含高浓度Cl–和Na+的废水对环境造成严重污染。本研究采用H3PO4和KOH为反应溶液建立壳聚糖的绿色生产工艺,并制得不同脱乙酰度的壳寡糖,探究了不同脱乙酰度壳寡糖抗烟草花叶病毒(TMV)的效果。结果显示,通过该生产工艺制得壳寡糖的脱乙酰度分别为63.79%、72.12%、79.34%和88.15%,分子量均为1500 Da左右。脱乙酰度为79.34%和88.15%的壳寡糖诱导植株对TMV产生抗病性,表现出对TMV进行体外钝化、抑制TMV在寄主内的复制和提高植物体内过氧化氢酶、过氧化物酶和多酚氧化酶的活性。     

3种微藻对海水集约化对虾养殖尾水氮磷的去除效果

基于对虾生物絮团集约化养殖尾水含有高浓度硝态氮和磷酸盐的特征,比较分析钝顶螺旋藻（Spirulina platensis）、牟氏角毛藻（Chaetoceros muelleri）、盐藻（Dunaliella sp.）3种微藻在配制尾水中的存活生长状况及其对无机氮磷的去除效果,以期筛选出适宜的微藻用于后续尾水净化技术。采用显微镜计数法测定藻细胞密度,国标法测定总无机氮、氨氮、硝态氮、亚硝态氮和磷酸盐的含量。结果显示,钝顶螺旋藻在实验前后的藻细胞密度变化不大（P>0.05）,约为3.32×106 个/mL和5.88×106 个/mL;牟氏角毛藻和盐藻细胞密度有明显增加（P<0.05）,分别从初始的4.00×104 个/mL和2.50×105 个/mL升高至实验结束时的1.66×106 个/mL和1.06×107 个/mL。经过16 d实验,钝顶螺旋藻组对硝态氮和总无机氮去除率分别为79.60%和46.06%,显著高于其他各组（P<0.05）,第8天时对磷酸盐的去除率可高达98.55%;牟氏角毛藻组16 d的磷酸盐去除率为98.25%,显著高于其他各组（P<0.05）。研究表明,3种微藻均可在对虾养殖尾水环境中存活,且对尾水氮磷具有较好的净化效果。     

Comparison of four antibiotics with indigenous marine Bacillus spp. in controlling pathogenic bacteria from shrimp and Artemia

Use of antibiotics for the control of bacterial diseases in shrimp culture has caused several adverse impacts to the industry. This has resulted in the search for alternative environment friendly approaches to overcome bacterial infections. This study was conducted to investigate the use of beneficial bacteria as an alternative to antibiotics. Ten pathogenic bacterial species isolated from shrimp, Penaeus monodon, and Artemia cysts were tested for susceptibility to indigenous marine Bacillus subtilis AB65, Bacillus pumilus AB58, Bacillus licheniformis AB69 and compared with oxytetracycline, chloramphenicol, gentamicin and bacitracin, which are common antibiotics used in Asian aquaculture. The Bacillus spp. were isolated from the local marine environment for bioremediation use in shrimp hatcheries and were proven to reduce total ammonium nitrogen. The pathogenic bacterial isolates were 90% susceptible to B. subtilis AB65, 70% susceptible to B. pumilus AB58 and B. licheniformis AB69 and 100% susceptible to oxytetracycline, chloramphenicol and gentamicin but only 40% to bacitracin. Two representative isolates of the vibrio group, Vibrio alginolyticus VaM11 and Vibrio parahaemolyticus VpM1, when tested for competitive exclusion by a common broth method using the marine Bacillus spp., showed decreased viable counts from 10(8) to 10(2) cfu mL(-1). The results suggest that the action of the marine bacteria appears to be significant in protecting the host shrimp against pathogenic bacteria. In addition to the alternative use of antibiotics, the selected marine bacteria had additional bioremediation properties of reducing ammonia.     

Lactic acid bacteria effective for regulating the growth of contaminant bacteria during the fermentation of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> (Phaeophyta)

ABSTRACT:   Lactic acid fermentation of seaweed is a recent topic and quite limited information is available on culture conditions. To know the suitable strains for use as a starter culture for seaweed fermentation, 14 lactic acid bacteria (LAB) strains, including 11 species, were tested in culture conditions prepared with or without salt. A commercial product of Undaria pinnatifida powder was used as a substrate for fermentation without sterilizing. Starter-suitability of the LAB strains was assessed from their predominance after culture. Among the tested strains, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus rhamnosus showed high (>90%) predominance in their cultures, while control cultures prepared without inoculation of LAB did not show any detectable growth of acid producing bacteria and spoiled. A total of 102 strains not showing acid producing activity were isolated and characterized from spoiled cultures, and all the isolates were observed as Bacillus strains, including 64 strains (62.7%) and 16 strains (15.7%) of a Bacillus cereu s-related and B. fusiformis -related species, respectively. The Undaria substrate before fermentation contained culturable microorganisms at 1.4–3.1 × 10² CFU/g, but the Bacillus cereu s-related strain was not a major composition, suggesting a concern of selective growth of the Bacillus cereu s-related strain during the spoiled fermentation.     

Identification and characterization of lactic acid bacteria isolated from rainbow trout (Oncorhynchus mykiss,Walbaum 1792), with inhibitory activity against Vagococcus salmoninarum and Lactococcus garvieae

In this study, a total of 98 lactic acid bacteria isolated from rainbow trout intestines were screened for their probiotic properties. The isolates were tested for their ability to inhibit growth of Vagococcus salmoninarum and Lactococcus garvieae. Based on in vitro antagonism, 10 isolates were selected and evaluated pathogenicity in rainbow trout. Isolates were further investigated for hydrophobicity, bile salts and acid tolerance. These isolates were able to survive low pH and high bile concentrations and showed good adherence characteristics. Isolates were characterized phenotypically, and then, 16S rRNA gene sequence analysis was used for confirmation. Selected strains were administered orally at 10⁸ cfu/g feed, and fish were challenged with V. salmoninarum and L. garvieae. The fish fed with lactic acid bacteria supplemented diets did not improve protection against V. salmoninarum. However, administration of Lactococcus lactis subsp. lactis M17 2‐2 and Lactobacillus sakei 2‐3 resulted in a significant reduction in mortality due to L. garvieae when compared to the control fish. RPS values were calculated as 80 and 53% in fish fed with L. sakei 2‐3 and L. lactis subsp. lactis M17 2‐2, respectively. Our results suggest that these strains could provide an alternative for lactococcosis control in aquaculture.     

Removal of cadmium from scallop processing waste by washing with weak acid solution and utilization of useful constituents for organic fertilizer manufacturing

ABSTRACT:   Hepatopancreases were washed four times with 2% acetic acid solution. In both the washed hepatopancreases and the supernatant of the washing solution, the residual cadmium (Cd) concentration was in accordance with the required standard for organic fertilizers. Hepatopancreases do not naturally contain high levels of nitrogen, but they do contain several amino acids which are useful for cultivation, and these were retained during the washing process. Although the condensed precipitate of the washing solution contained a considerable amount of Cd, Cd elution levels at the time of final disposal met judgment standard for special management industrial waste as a result of kneading and immobilizing with chelating adsorbent for treatment of contaminated soil. The volume of the precipitate of the washing solution was one-fifth of the original hepatopancreas, and the solid waste was suitable for burial at a final disposal site without further treatment.