Genotyping of starter cultures of Bacillus subtilis and Bacillus pumilus for fermentation of African locust bean (Parkia biglobosa) to produce Soumbala

Bacillus spp. are the predominant microorganisms in fermented African locust bean called Soumbala in Burkina Faso. Ten strains selected as potential starter cultures were characterised by PCR amplification of the16S-23S rDNA intergenic transcribed spacer (ITS-PCR), restriction fragment length polymorphism of the ITS-PCR (ITS-PCR RFLP), pulsed field gel electrophoresis (PFGE) and sequencing of the 968-1401 region of the 16S rDNA. In previous studies, the isolates were identified by phenotyping as Bacillus subtilis and Bacillus pumilus. The phenotyping was repeated as a reference in the present study.The ITS-PCR and ITS-PCR RLFP allowed a typing at species level. The PFGE was more discriminative and allowed a typing at strain level. Full agreement with the phenotyping was observed in all cases. The sequencing of the 16S rDNA allowed the identification at species level with an identity from 97% to 100% comparing the sequences to those from the GenBank databases. The desired cultures of B. subtilis and B. pumilus from African locust bean fermentation were distinguished by ITS-PCR and ITS-PCR RLFP from Bacillus cereus and Bacillus sphaericus which sometimes occur in the beginning of the fermentation.     

Formation of cereulide and enterotoxins by Bacillus cereus in fermented African locust beans

Afitin, iru and sonru are three spontaneously fermented African locust bean Benin condiments. The fermentation processes are exothermic, with temperatures mostly being above 40 °C. A total of 19 predominant Bacillus cereus isolates from afitin, iru and sonru, were investigated. The enterotoxin genes nhe (A, B, C) were present in all 19 isolates, the hbl (A, C, D) in one (afitin), and the cytK gene in three isolates (afitin). Levels of cytotoxicity to Vero cells and NheA production in BHI-broth was within the range of known diarrheal outbreak strains. Autoclaved cooked African locust beans inoculated with emetic (cereulide producing) B. cereus Ba18H2/RIF supported growth at 25, 30 and 40 °C with highly different maximum cereulide productions of 6 ± 5, 97 ± 3 and 0.04 ± 0.02 μg/g beans, respectively (48 h). For non-autoclaved cooked beans inoculated with 2, 4 and 6 log₁₀B. cereus Ba18H2/RIF spores/g beans, cereulide production was 5 ± 4, 64 ± 8 and 69 ± 34 μg/g beans, respectively at 24 h, while it was 70 ± 43, 92 ± 53 and 99 ± 31 μg/g at 48 h of fermentation at 30 °C. Even though high toxin levels were observed, to date there are no known reports on diarrhea or vomiting due to the consumption or afitin, iru and sonru in Benin, which also according to the present study is likely to be expected from the low levels of cereulide produced at 40 °C.     

Diversity and functionality of Bacillus and related genera isolated from spontaneously fermented soybeans (Indian Kinema) and locust beans (African Soumbala)

A total of 126 isolates of Bacillus and related genera from indigenous, spontaneously fermented soybeans (Kinema) and locust beans (Soumbala) were characterized with the purpose of defining interspecific, as well as intraspecific relationships among the components of their microflora. B. subtilis was the dominant species, and species diversity was more pronounced in Soumbala than in Kinema. While from Kinema, six species were isolated (B. subtilis, B. licheniformis, B. cereus, B. circulans, B. thuringiensis and B. sphaericus), in Soumbala, the species found were B. subtilis, B. thuringiensis, B. licheniformis, B. cereu, B. badius, Paenibacillus alvei, B. firmus, P. larvae, Brevibacillus laterosporus, B. megaterium, B. mycoides and B. sphaericus. Genomic diversity in the isolates of B. subtilis was investigated by random amplified polymorphic DNA (RAPD) analysis using the polymerase chain reaction (PCR). The RAPD-PCR fingerprint analysis showed a high level of diversity. With more than 90% similarity, all 52 RAPD subdivisions were source and continent-wise homogeneous. Profiles of carbon source fermentation also showed a wide but corresponding phenotypic diversity, largely corresponding with RAPD subdivisions. The various strains were tested for several criteria for functionality in soybean fermentation, viz. protein degradation, pH increase, and development of desirable stickiness caused by viscous exopolymers. Profiles of functionality, based upon estimations of pH, free amino nitrogen and stickiness were associated with genotypic and phenotypic profiles. Notwithstanding the heterogenous fermentation results for some genotypic profiles, a ranking of RAPD groups is possible and can be useful in the further selection and study of B. subtilis strains.     

Enterotoxins and emetic toxins production by Bacillus cereus and other species of Bacillus isolated from Soumbala and Bikalga, African alkaline fermented food condiments

The ability of various species of Bacillus from fermented seeds of Parkia biglobosa known as African locust bean (Soumbala) and fermented seeds of Hibiscus sabdariffa (Bikalga) was investigated. The study included screening of the isolates by haemolysis on blood agar, detection of toxins in broth and during the fermentation of African locust bean using the Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA) and the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (BDEVIA). Detection of genes encoding cytotoxin K (CytK), haemolysin BL (Hbl A, Hbl C, Hbl D), non-hemolytic enterotoxin (NheA, NheB, NheC) and EM1 specific of emetic toxin producers was also investigated using PCR with single pair and multiplex primers. Of 41 isolates, 29 Bacillus belonging to the species of B. cereus, Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus showed haemolysis on blood agar. Using RPLA, enterotoxin production was detected for three isolates of B. cereus in broth and all B. cereus (9) in fermented seeds. Using BDEVIA, enterotoxin production was detected in broth as well as in fermented seeds for all B. cereus isolates. None of the isolates belonging to the other Bacillus species was able to produce enterotoxins either by RPLA or BDEVIA. Nhe genes were detected in all B. cereus while Hbl and CytK genes were detected respectively in five and six B. cereus strains. A weak presence of Hbl (A, D) and CytK genes was detected in two isolates of B. subtilis and one of B. licheniformis but results were inconsistent, especially for Hbl genes. The emetic specific gene fragment EM1 was not detected in any of the isolates studied.     

Diversity of lactic acid bacteria from Hussuwa, a traditional African fermented sorghum food

The diversity of lactic acid bacteria associated with Hussuwa fermentation, a Sudanese fermented sorghum food, was studied using a polyphasic taxonomical approach. Predominant strains could be well characterised based on a combination of phenotypic tests and genotypic methods such as ARDRA, rep-PCR and RAPD-PCR, as well as 16S rRNA gene sequencing of representative strains. Thus, the majority (128 of 220, 58.3%) of strains exhibited phenotypic properties typical of heterofermentative lactobacilli and of these, 100 strains were characterised more closely using the genotyping methods. The majority (97/100) strains could be characterised as Lactobacillus fermentum strains. Seventy-two of 220 strains (32.7%) showed phenotypic properties that are characteristic of pediococci. Of 41 selected strains investigated by genotyping techniques, 38 (92.7%) could be characterised as Pediococcus acidilactici strains, while three (7.3%) could be characterised as Pediococcus pentosaceus strains. The Hussuwa fermentation thus appears to be dominated by L. fermentum strains and P. acidilactici strains. For this reason, we selected representative and predominant strains as potential starter cultures for Hussuwa fermentation. These strains, L. fermentum strains BFE 2442 and BFE 2282 and P. acidilactici strain BFE 2300, were shown on the basis of RAPD-PCR fingerprinting to predominate in a model fermentation when used as starter cultures inoculated at 1 × 10⁶ CFU/g and to lower the pH of the fermentation to below pH 4.0 within 48 h. These cultures should be studied for further development as starter preparations in pilot scale studies in actual field fermentations.     

Fate of proteic and lipidic compounds during production of a traditional legume condiment (Soumbala) made from African Locust Bean (Parkia biglobosa) seeds

Soumbala is made from African locust bean seeds (Parkia biglobosa), a legume widely spread in west Africa. It is a lipid and protein-rich condiment obtained by three main steps: cooking, fermentation and drying. Ten different analysis were made on AFLBS samples at each step with a particular focus on protein and lipid fates. Results showed that cooking decreased markedly the carbohydrate content leaving mainly proteins and lipids as substrates for fermentation. During this step, 20% of the proteins were converted into free amino acids, mainly glutamic acid and tyrosine. Significant biogenic amines release occurred and reached 50 mg per 100 g of dried ALBS. Di-glycerides and fatty acids increased markedly and represented 11% and 7% of the total lipids of dried ALBS. These results give a complete view of the nutritional characteristics of dried ALBS, its benefit but also its defects that arise during fermentation and not suppressed by the subsequent processing steps.     

Production of nitric oxide (NO) by lactic acid bacteria isolated from fermented products

In this study, the bacteria which were isolated from various milk and fermented food products were tested for their ability to convert metmyoglobin to nitrosomyoglobin. Lactic acid bacteria were isolated from samples of raw milk, unsalted butter, Beyaz cheese, yoghurt, pickles and silage. The nitric oxide (NO) forming abilities of 1534 isolates were tested using plates of de Man, Rogosa, Sharpe agar supplemented with metmyoglobin (MRS-Mb). Ten isolates formed bright red colonies, brown or clear zones due to the conversion of metmyoglobin to nitrosomyoglobin were identified. Five of the 10 bacteria were identified as Lactobacillus plantarum, three as Pediococcus acidilactici, and two as Leuconostoc mesenteroides subsp. dextranicum. NO formation ability was measured in MRS-Mb broth. There were differences not only among the species, but also among the strains of a species. The highest NO concentrations of 51.5, 51.3, 50.2 μM were produced by P. acidilactici S2, L. plantarum T119, and P. acidilactici S3, respectively.     

Production of -aminobutyric acid (GABA) by Lactobacillus paracasei isolated from traditional fermented foods

Lactobacillus strains that accumulated γ-aminobutyric acid (GABA) in culture medium were screened to determine strains with high GABA-producing ability. One strain, NFRI 7415, which was isolated from a Japanese traditional fermented fish (funa-sushi), showed the highest GABA-producing ability among the screened strains. Identification tests (i.e., 16S rDNA sequencing and sugar assimilation ability) indicated that NFRI 7415 belongs to Lb. paracasei. The GABA production was further improved by the addition of pyridoxal phosphate to the culture medium and pH regulation of culture medium at pH 5.0. Under optimal cultivation conditions, strain NFRI 7415 produced GABA at a concentration of 302 mm when the glutamate concentration in the culture medium was 500 mm.     

Properties and safety aspects of Enterococcus faecium strains isolated from Chungkukjang, a fermented soy product

Enterococcus faecium isolated from Chungkukjang, a Korean traditional fermented soybean food was studied for their functional characteristics as potential new starter culture and safety. Microbiological analysis of ripened Chungkukjang revealed the presence of an enterococcal population in numbers of up to 6 log CFU per g. Seven isolates with higher activity were selected for further study and the strains were identified as E. faecium. The E. faecium strains showed resistance against simulated gastrointestinal conditions such as acidic environment and the presence of bile salts. These strains also showed bile salt hydrolase activity but neither hemolytic activity nor virulence determinant such as gelE and efaAfm. All strains were susceptible to glycopeptides and lacked potential as vancomycin-resistant enterococci (VRE). Two strains, S2C10 and S2C11, showed inhibited the viability of Listeria monocytogenes in vitro. The ability was probably due to the production of bacteriocin. The lipase activity influenced the stability, while either acidic condition or high temperature did not play a significant role in the activity of the antimicrobial substances. The strains also produced thermostable listericidal antimicrobial substance. For this reason, the strains could be used as selected starters or protective cultures in soybean fermented food production.     

Bacteriocin-producing Lactobacillus strains isolated from poto poto, a Congolese fermented maize product, and genetic fingerprinting of their plantaricin operons

Thirty one bacteriocin-producing Lactobacillus isolates were identified among 135 lactobacilli isolated from the Congolese fermented maize product poto poto, during the preparation and from the finished product. Using species-specific PCR and 16S rRNA gene sequencing, 28 and 3 isolates were identified as L. plantarum and L. fermentum, respectively. Cluster analysis of RAPD-PCR fingerprints revealed two main groups (G1 and G2) plus the L. fermentum isolate C4-13. Group G1 contained 23 isolates with a similarity coefficient >74.5%, and could be divided in two subgroups (G1-1, G1-2) each with several branches, plus the L. plantarum isolate C11. Group G2 contained 8 isolates with a similarity coefficient >86%, with two main branches. Using PCR amplification with specific primers, several genes of the plantaricin cluster found in L. plantarum C11 were identified in the isolates. The number of genes that were detected varied between the strains. The L. fermentum isolate EC11 also contained the plnDEFG genes. PCR amplification of DNA from isolates with primers directed to the upstream and downstream region of the plantaricin cluster generated an amplicon identical to that obtained with DNA from the control strain L. plantarum WCFS1. Amplification products from the positive strains were used for restriction analysis with HindIII, EcoRI and KpnI in separate reactions. Cluster analysis of restriction profiles revealed high similarities for EcoRI and HindII digest profiles, and an identical profile for all KpnI digests. The L. fermentum EC11 isolate clustered with L. plantarum strains in a group with a high correlation coefficient. The results suggest a low degree of diversity in the plantarincin gene cluster. However, other strains that tested positive for individual plantaricin genes may present great heterogeneity in the plantaricin operons. Because of their broad spectra of inhibition (including Escherichia coli, Salmonella enterica, Enterobacter aerogenes, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis), isolates from the present study could be used to improve the safety and storage stability of poto poto.     

Extractability of African yam bean (Sphenostylis stenocarpa) protein in acid, salt and alkaline aqueous media

Protein extractability from defatted Africa yam been (Sphenostylis stenocarpa) was studied under various conditions: solid/solvent ratio, time, pH, and salt. Extractable protein from S. stenocarpa was strongly dependent on all these factors. Maximum extractable protein was obtained after 2 h extraction time; the solid to solvent ratio in the range of 1:20–1:50 gave maximum protein extractability. The pH corresponding to maximum and minimum extractable proteins were 10 and 5, respectively, but addition of NaCl changed this slightly. Extractable protein of 92%, 88% and 84% were obtained in aqueous concentration of 0.01 MNa₂SO₄, 1.5 MNaCl and 0.01 MNaOH, respectively while other concentrations gave lower extractability. These salt concentrations, i.e. 0.01 MNa₂SO₄ and 1.5 MNaCl gave slightly lower extractable protein under alkaline condition. S. stenocarpa flour has a higher buffer capacity in acid medium than in alkaline medium.     

Aggregation profile, preparation and nutritional characterization of African yam bean (Sphenostylis stenocarpa) acid and salt protein isolates

Aggregation tendencies of African yam bean (Sphenostylis stenocarpa) protein in the various aqueous extraction media with Ca²⁺, Mg²⁺ or at pI (isoelectric point) were determined. Water extractable S. stenocarpa protein aggregates more with addition of either Ca²⁺ or Mg²⁺ than at pI. In alkaline extractants considered (except at pH 10), aggregation tendency of the protein is in the order: pI > Ca²⁺ > Mg²⁺. The protein aggregation trend in salt media is a function of the salt type, aggregation in NaCl solution is of order: Ca²⁺ = pI > Mg²⁺, while in Na₂SO₄, we had pI > Mg²⁺ > Ca²⁺. S. stenocarpa protein aggregation was significantly (P < 0.05) more in Na₂SO₄ than NaCl. The amount of Ca²⁺ required for maximum precipitation of S. stenocarpa from alkaline (water-pH10) extractant was higher than that of Na₂SO₄ and more Ca-proteinate was obtained from the alkaline aliquot. The crude protein of the Ca-proteinates and isoelectric protein isolates obtained from salt and alkaline extract were in the range 71.7–91.8% (dry basis). Protein isolate from alkaline extract had significantly (P < 0.05) higher fat content than the one from salt extract. Isoelectrically precipitated isolates had lower ash content than Ca-proteinates. The percentage ratio of essential to non-essential amino acid was in the range 45–47%. With reference to FAO/WHO standard, the chemical score showed that most of the essential amino acid were in excess, thus, the amino acid distribution of S. stenocarpa protein isolates showed that it can fulfill the essential amino acid requirement of human especially the acid proteins and can be a good protein supplement in food and a wide range of new food products.     

Broad distribution of enterotoxin genes (hblCDA, nheABC, cytK, and entFM) among Bacillus thuringiensis and Bacillus cereus as shown by novel primers

Eight new pairs of PCR primers were designed and efficiently detect eight toxin genes (hblC, hblD, hblA, nheA, nheB, nheC, cytK, and entFM) in 411 B. cereus strains (121 food- and 290 soil isolates) and 205 B. thuringiensis strains (43 serovars, 10 food- and 152 soil isolates). According to the presence of these eight toxin genes, they were divided into four groups among the total 616 isolates. In Group I, all eight genes occurred simultaneously in 403 (65.42%) isolates, while Group II (134 isolates or 21.75%) and Group III (46 isolates or 7.47%) were devoid of hblCDA and cytK, respectively. In Group IV, there were thirty-three isolates which lacked both hblCDA and cytK. The presence of hblCDA in B. thuringiensis strains (86.80%) was significantly higher (P<0.05) than in B. cereus strains (66.18%) whereas no significant difference in nheABC, cytK and entFM occurrence was detected between both bacterial groups. Both nheABC and entFM genes were found in all B. cereus and B. thuringiensis strains (616 strains in total), while the cytK gene could be detected in 365 (88.80%) of the B. cereus and 172 (83.90%) of the B. thuringiensis strains. None of the 616 tested strains showed the presence of only a single or two genes in either the hbl or nhe operons. The eight primer pairs designed for this multiplex PCR allowed rapid detection of eight toxin genes from boiled cells with high sensitivity, gave 100% reproducibility, and did not cross-react to 32 other bacterial strains.     

Isolation and characterization of a nisin-like bacteriocin produced by a Lactococcus lactis strain isolated from charqui,a Brazilian fermented,salted and dried meat product

A Lactococcus lactis subsp. lactis strain (L. lactis 69) capable to produce a heat-stable bacteriocin was isolated from charqui, a Brazilian fermented, salted and sun-dried meat product. The bacteriocin inhibited, in vitro, Listeria monocytogenes, Staphylococcus aureus, several lactic acid bacteria isolated from foods and spoilage halotolerant bacteria isolated from charqui. The activity of the bacteriocin was not affected by pH (2.0–10.0), heating (100 °C), and chemical agents (1% w/v). Treatment of growing cells of L. monocytogenes ScottA with the cell-free supernatant of L. lactis 69 resulted in complete cell inactivation. L. lactis 69 harbored the gene for the production of a nisin-like bacteriocin, and the amino acid sequence of the active peptide was identical to sequences previously described for nisin Z. However, differences were observed regarding the leader peptide. Besides, the isolate was able to survive and produce bacteriocins in culture medium with NaCl content up to 20%, evidencing a potential application as an additional hurdle in the preservation of charqui.     

用ITS2鉴定芥辣类调味品中山葵、辣根和芥末组分

日式饮食中的芥辣类调味品主要以同属十字花科的山葵(Eutrema wasabi)、辣根(Armoracia rusticana)、芥末(mustard)做原料加工而成。利用核糖体DNA内部转录间隔区Ⅱ(Internal transcribed spacer 2,ITS2)技术快速、准确地鉴定出山葵、芥末和辣根,为该技术应用于芥辣类产品质量检测提供参考。以3种植物材料(山葵、辣根、白芥末)为实验材料,提取基因组DNA,通过引物ITS2_S2进行PCR扩增得到ITS2片段,测序结果通过生物信息学分析进行物种鉴定。MEGA7.0(Molecular evolutionary genetics analysis)分析ITS2序列结果表明,山葵、白芥末和辣根K2P(Kimura 2-parameter)遗传距离在0.088～0.171,均大于0.01,种间变异位点有36个,初步推断利用ITS2序列能将山葵、白芥末和辣根3物种区分开来。另外,GenBank中获得山葵以及近亲西北山嵛菜、辣根、芥末的ITS2序列,MEGA7.0进行种间序列分析,计算K2P,并用邻接法(Neighbor-joining,NJ)构建进化树,山葵与近亲西北山嵛菜聚为一支,棕芥末与白芥末、黑芥末聚为一大支,而辣根单独为一支。通过分析ITS2序列,发现山葵与近亲西北山嵛菜、辣根、芥末的种间K2P遗传距离在0.030～0.105,均大于0.01。研究表明,利用ITS2技术可以鉴别山葵、辣根和芥末等近缘物种,此技术可以用于芥辣类调味品特定原料成分鉴定及定量,为食品质量控制和食品安全提供科学依据。     

Isolation and characterization of a cyanidin-catechin pigment from adzuki bean (Vigna angularis)

Adzuki bean is used to prepare many kinds of foods in east Asia, and the seed coat contains water-soluble anthocyanins, catechins, and flavonols. In the present study, ethyl acetate-soluble purplish pigments were isolated from adzuki bean. Pigments of soaked adzuki bean were extracted with 1% HCl in methanol. Ethyl acetate-soluble purple pigments were obtained from the methanol soluble components. Purple pigments 1 and 2 were purified from the ethyl acetate-soluble pigments by Sephadex LH-20 column chromatography and preparative reversed-phase HPLC. NMR and mass spectra suggested that pigment 1 was a condensation product of cyanidin and (+)-catechin, in which 5-hydroxy and C-4 positions of the cyanidin moiety were substituted by the addition of 5-hydroxy and C-6 positions of the (+)-catechin moiety, respectively. Pigment 2 was an isomer of pigment 1. It is suggested that pigments 1 and 2 contribute to the purplish-red colour of foods prepared using adzuki bean.     

Effects of food additives on biogenic amine formation in Myeolchi-jeot, a salted and fermented anchovy (Engraulis japonicus)

This study was carried out to reduce biogenic amine contents in Myeolchi-jeot, a salted and fermented anchovy (Engraulis japonicus). The effects of various food additives on biogenic amine formation were determined by HPLC. The greatest inhibitory effect on biogenic amine production was observed in the culture treated with glycine. In the culture, the contents of putrescine, cadaverine, histamine, tyramine and spermidine were reduced by 32.6%, 78.4%, 93.2%, 100.0% and 100.0%, respectively, compared to control. The other additives tested had less effect in inhibiting biogenic amine production. Out of food additives tested, glycine was finally applied to the ripening of Myeolchi-jeotin situ, and then overall production of biogenic amines was reduced by up to 63.0% and 73.4%, compared to controls prepared with no and 20% NaCl, respectively. Therefore, it is expected that the findings of this study enhance the safety of not only Myeolchi-jeot but other salted and/or fermented seafood.     

Effect of pork lard content on the chemical, microbiological and sensory properties of a typical fermented meat product (Pitina) obtained from Alpagota sheep

The aim was to investigate the physicochemical, microbiological and sensory properties of Pitina, a typical fermented meat product and evaluate the effect of two levels of pork lard content (Low Fat, LF, 10% vs. High Fat, HF, 30%) on its attributes. HF attained lower pH than LF Pitina, which reached lower water activity. LAB comprised the major flora with substantial counts of micrococci, enterococci and mould and yeast. Gram negative Enterobacteria were recovered as coliforms and faecal coliforms. Listeria monocytogenes was also isolated. The lard level influenced the count of micrococci and some sensory attributes. LF attained higher scores for both hardness and cohesiveness and differed from HF in having a more marked odour of ewe and smoke and sweeter taste. HF had a more pronounced odour and taste of garlic and mould than LF.     

Sweet Basil (Ocimum basilicum) Extracts Obtained by Supercritical Fluid Extraction (SFE): Global Yields, Chemical Composition, Antioxidant Activity, and Estimation of the Cost of Manufacturing

In this work, supercritical technology was used to obtain extracts from Ocimum basilicum (sweet basil) with CO₂ and the cosolvent H₂O at 1, 10, and 20% (w/w). The raw material was obtained from hydroponic cultivation. The extract’s global yield isotherms, chemical compositions, antioxidant activity, and cost of manufacturing were determined. The extraction assays were done for pressures of 10 to 30 MPa at 303 to 323 K. The identification of the compounds present in the extracts was made by GC-MS and ESI-MS. The antioxidant activity of extracts was determined using the coupled reaction of beta-carotene and linolenic acid. At 1% of cosolvent, the largest global yield was obtained at 10 MPa and 303 K (2%, dry basis—d.b.); at 10% of cosolvent the largest global yield was obtained at 10 and 15 MPa (11%, d.b.), and at 20% of cosolvent the largest global yield was detected at 30 MPa and 303 K (24%, d.b.). The main components identified in the extracts were eugenol, germacrene-d, epi–alpha–cadinol, malic acid, tartaric acid, ramnose, caffeic acid, quinic acid, kaempferol, caffeoylquinic acid, and kaempferol 3-O-glucoside. Sweet basil extracts exhibited high antioxidant activity compared to beta-carotene. Three types of SFE extracts from sweet basil were produced, for which the estimated cost of manufacturing (class 5 type) varied from US$ 47.96 to US$ 1,049.58 per kilogram of dry extract.     

Formation of biogenic amines by Gram-negative rods isolated from fresh,spoiled, VP-packed and MAP-packed herring (<Emphasis Type="Italic">Clupea harengus</Emphasis>)

Bacterial strains (120) were isolated from fresh, spoiled, VP-packed and MAP-packed herring. Identified bacterial strains were investigated for their abilities to produce biogenic amines in histidine, lysine and ornithine decarboxylase broth by a rapid high-performance liquid chromatography (HPLC) method. The microflora of fresh herring was dominantly Pseudomonas (30%), Enterobacteriaceae (23.2%), Vibrio (13.3%) and Moraxella (13.3%) but, the microflora of herring stored in VP and MAP was dominated by species belonging to Vibrio (23.3%) and Moraxella (20%), which indicates that these packaging systems prevented the growth of Pseudomonas and Enterobacteriaceae. In a laboratory medium containing amino acid (histidine, ornithine and lysine), most of bacterial strains produced histamine, putrescine and cadaverine. The highest histidine decarboxylase activities were observed in Klebsiella oxytoca, Hafnia alvei and Proteus vulgaris which produced 396, 232 and 54 mg histamine/L, respectively in histidine-enriched broth. The accumulation of cadaverine by Klebsiella oxytoca and Hafnia alvei was 325 and 208 mg/l, respectively. All strains isolated produced putrescine in an ornithine-enriched broth, ranging from 3 to 249 mg/l. The production of putrescine by Klebsiella oxytoca and Hafnia alvei was 249 and 195 mg/l, respectively. Moraxella spp and Acinetobacter spp did not produce histamine which indicates they did not have histidine decarboxylase activity.