Using lactic acid bacteria for developing novel fish food products

Lactic acid bacteria‐induced fermentation of fish was tested with the purpose of developing a novel fish food product with desired meat properties. Three selected lactic acid bacteria strains, isolated from lightly preserved fish products, were inoculated into minced frozen fillet of yellowfin tuna, and changes in fish properties were observed during 52 days of storage at 8 °C. Organoleptic, bacteriological and biochemical studies (pH, histamine, TVB‐N, malonaldehyde) and shelf‐life measurements suggest that Leuconostoc mesenteroides could be considered as a possible starter culture for fish fermentation, but additional experiments should be performed. © 2000 Society of Chemical Industry     

Characterization of anti-listerial lactic acid bacteria isolated from Thai fermented fish products

Thai fermented fish products were screened for lactic acid bacteria capable of inhibitingListeriasp. (Listeria innocua). Of 4150 assumed lactic acid bacteria colonies from MRS agar plates that were screened by an agar-overlay method 58 (1.4%) were positive. Forty four of these strains were further characterized and 43 strains were inhibitory againstListeria monocytogenes. The strains were inhibitory to other Gram-positive (lactic acid) bacteria probably because of production of bacteriocins. All 44 strains inhibited bothVibrio choleraeandVibrio parahaemolyticusand 37 were inhibitory to a mesophilic fish spoilage bacterium (anAeromonassp.). Inhibition of Gram-negative bacteria was attributed to production of lactic acid. Most strains were identified asLactobacillusspp., and all grew well at ambient temperatures (25–37°C) and tolerated up to 6.5% NaCl. Glucose was fermented rapidly in laboratory media whereas pH decreased only very slowly in fish juice supplemented with 4% glucose and 3.5% NaCl or in a rice–fish mixture. Only four of 44 isolates could degrade and ferment complex carbohydrates such as rice, potatoes and maize starch. This indicates that other types of bacteria may be responsible for the rapid spontaneous fermentation of the products or that other yet-unknown factors ensure rapid fermentation. Overall anti-listerial lactic acid bacteria do occur in fermented fish products and the antibacterial activity against pathogenic bacteria indicates that they may be important in product safety.     

功能性乳酸菌发酵剂在食品发酵工业中的应用

功能性乳酸茼发酵剂是具有一种或几种固有功能特性的发酵剂，其在感官、技术、营养或健康方面的各种优势使之在食品发酵工业中的应用潜力巨大：它可用于食品防腐、提高食品安全性，改善食品质构和风味，加速奶酪成熟，可产生功能因子、去除毒副因子从而使食品具有保健功效；某些抗噬菌体发酵剂还可解决乳品工业中的噬菌体污染问题。通过基因工程或菌株筛选可以获得所需的功能性发酵剂．它使人们可以更好地控制发酵过程，为消费者提供新型健康食品，此外还有利于食品企业的多样化生产。     

The effects of cultivating lactic starter cultures with bacteriocin-producing lactic acid bacteria

The effects of bacteriocins produced by six strains of lactic acid bacteria on 9 mesophilic and 11 thermophilic commercial starter cultures were investigated in mixed cultures of commercial starters with bacteriocin-producing strains in milk. The bacteriocins produced by the test organisms were nisin A, nisin Z, lacticin 481, enterocin AS-48, a novel enterocin, and a novel plantaricin. Mesophilic commercial starters were in most cases tolerant of bacteriocins, with only two of the starters being partially inhibited, one by four and the other by two bacteriocins. The aminopeptidase activities of mesophilic starters were generally low, and only one of the combinations of mesophilic starter-bacteriocin producer gave double the aminopeptidase activity of the starter culture without the bacteriocin producer. Thermophilic commercial starters were more sensitive to bacteriocins than mesophilic starters, with six thermophilic starters being partially inhibited by at least one of the bacteriocins. Their aminopeptidase activities were generally higher than those of the mesophilic starters. The aminopeptidase activities of seven thermophilic starters were increased in the presence of bacteriocins, by factors of up to 9.0 as compared with the corresponding starter cultures alone. Bacteriocin-producing strains may be used as adjunct cultures to mesophilic starters for the inhibition of pathogens in soft and semihard cheeses, because mesophilic starters are rather tolerant of bacteriocins. Bacteriocin producers may also be used as adjunct cultures to thermophilic starters of high aminopeptidase activity, more sensitive to lysis by bacteriocins than mesophilic starters, for the acceleration of ripening in semihard and hard cheeses.     

Characterisation and biochemical properties of predominant lactic acid bacteria from fermenting cassava for selection as starter cultures

A total of 375 lactic acid bacteria were isolated from fermenting cassava in South Africa, Benin, Kenya and Germany, and were characterised by phenotypic and genotypic tests. These could be divided into five main groups comprising strains of facultatively heterofermentative rods, obligately heterofermentative rods, heterofermentative cocci, homofermentative cocci and obligately homofermentative rods, in decreasing order of predominance. Most of the facultatively heterofermentative rods were identified by phenotypic tests as presumptive Lactobacillus plantarum-group strains, which also comprised the most predominant bacteria (54.4% of strains) isolated in the study. The next predominant group of lactic acid bacteria (14.1% of total isolates) consisted of obligately heterofermentative rods belonging either to the genus Lactobacillus or Weissella, followed by the heterofermentative cocci (13.9% of isolates) belonging to the genera Weissella or Leuconostoc. Homofermentative cocci were also isolated (13.3% of isolates). Biochemical properties such as production of alpha-amylase, beta-glucosidase, tannase, antimicrobials (presumptive bacteriocin and H(2)O(2)-production), acidification and fermentation of the indigestible sugars raffinose and stachyose, were evaluated in vitro for selection of potential starter strains. A total of 32 strains with one or more desirable biochemical properties were pre-selected and identified using rep-PCR fingerprinting in combination with 16S rRNA sequencing of representative rep-PCR cluster isolates. Of these strains, 18 were identified as L. plantarum, four as Lactobacillus pentosus, two each as Leuconostoc fallax, Weissella paramesenteroides and Lactobacillus fermentum, one each as Leuconostoc mesenteroides subsp. mesenteroides and Weissella cibaria, while two remained unidentified but could be assigned to the L. plantarum-group. These strains were further investigated for clonal relationships, using RAPD-PCR with three primers, and of the 32 a total of 16 strains were finally selected for the development as starter cultures for Gari production.     

Evaluation of isolated starter lactic acid bacteria in Ras cheese ripening and flavour development

Eighteen cultures of starter lactic acid bacteria with or without added adjunct cultures, isolated from Egyptian dairy products, were evaluated in experimental Ras cheese for flavour development. Chemical composition of experimental cheeses was within the legal limit for Ras cheese in Egypt. All cultures used in this study had no effect on chemical composition of Ras cheese. Very significant variations in free amino acids, free fatty acids and sensory evaluation have been found among the cultures used in Ras cheesemaking. The levels of free amino acids and free fatty acids were correlated well with flavour development in Ras cheese. Seven of the tested cultures produced acceptable flavour and texture of Ras cheese. The highest overall score of flavour intensity, flavour and texture acceptability were in cheese made using YY47 lactic culture in addition to adjunct culture of Lactobacillus helveticus, Lactobacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. lactis and Enterococcus faecium. This culture can be recommended for Ras cheese manufacture using pasteurized milk.     

Evaluation of meat born lactic acid bacteria as protective cultures for the biopreservation of cooked meat products

In this study, 91 strains, originating from meat products, were subjected to a step-by-step screening and characterisation to search for potential protective cultures to be used in the cooked cured meat industry. Strains were first tested on their homofermentative and psychrotrophic character and salt tolerance. Secondly, the antibacterial capacities towards Listeria monocytogenes, Leuconostoc mesenteroides, Leuconostoc carnosum and Brochotrix thermosphacta were determined in an agar spot test. In total, 38% of the tested strains were inhibitory towards all indicator strains. However, 91%, 88% and 74% of the strains could inhibit, respectively, L. monocytogenes, B. thermosphacta and Leuc. mesenteroides. Finally, 12 strains, with the highest antibacterial capacities, were evaluated on their competitive nature by comparing their growth rate, acidifying character and lactic acid production at 7 degrees C under anaerobic conditions in a liquid broth. All 12 strains, except for a bacteriocin producing Lactobacillus plantarum strain and the lactocin S producing Lactobacillus sakei 148, combined a fast growth rate with a deep and rapid acidification caused by the production of high levels of lactic acid. The 12 selected strains were then further investigated for their growth capacity on a model cooked ham product to establish whether the presence of these cultures on the ham did not negatively influence the sensory properties of the ham. All strains grew in 6 days at 7 degrees C from a level of 10(5)-10(6) to 10(7)-10(8) cfu/g and again the bacteriocin producing L. plantarum strain was the slowest growing strain. As the glucose level of the model cooked ham product was low (0.09+/-0.03%), growth of the putative protective cultures resulted in glucose depletion and a limited lactic acid production and accompanying pH decrease. Cooked ham inoculated with isolates 13E, 10A, 14A (all three identified as L. sakei subsp. carnosus by SDS-PAGE) and with strains L. sakei 148 (LS5) and L. sakei subsp. carnosus SAGA 777 (LS8) were not rejected by the sensory panel at the 34th day of the vacuum packaged storage at 7 degrees C. Therefore, these strains could have potential for the use as protective culture in cooked meat products.     

Phenotypic and genetic evaluations of biogenic amine production by lactic acid bacteria isolated from fish and fish products

In this work, biogenic amine production (histamine, tyramine and putrescine) by a collection of 74 lactic acid bacteria of aquatic origin has been investigated by means of amino acid decarboxylation by growth on decarboxylase differential medium, biogenic amine detection by thin-layer chromatography (TLC) and decarboxylase gene detection by PCR. None of the evaluated strains showed neither production of histamine and putrescine, nor presence of the genetic determinants encoding the corresponding decarboxylase activities. However, the tyrosine decarboxylase gene (tdc) was present in all the enterococcal strains, and tyramine production was detected by TLC in all of them but Enterococcus faecium BCS59 and MV5. Analysis of the tyrosine decarboxylase operon of these strains revealed the presence of an insertion sequence upstream tdc that could be responsible for their lack of tyrosine decarboxylase activity.     

Amino acid profiles of lactic acid bacteria, isolated from kefir grains and kefir starter made from them

The characteristics of cell growth, lactic acid production, amino acid release and consumption by single-strain cultures of lactic acid bacteria (isolated from kefir grains), and by a multiple-strain kefir starter prepared from them, were studied. The change in the levels of free amino acids was followed throughout the kefir process: single-strain kefir bacteria and the kefir starter (Lactococcus lactis C15-1%+Lactobacillus helveticus MP12-3%+(Streptococcus thermophilus T15+Lactobacillus bulgaricus HP1 = 1:1)-3%) were cultivated in pasteurized (92 degrees C for 20 min) cow's milk (3% fat content) at 28 degrees C for 5 h (the kefir starter reached pH 4.7) and subsequently grown at 20 degrees C for 16 h; storage was at 4 degrees C for 168 h. The strain L. helveticus MP12 was unrivaled with respect to free amino acid production (53.38 mg (100 g)(-1)) and cell growth (17.8 x 10(8) CFU ml(-1)); however, it manifested the lowest acidification activity. L. bulgaricus HP1 released approximately 3.7 times less amino acids, nearly 5 times lower cell growth, and produced about 1.2 times more lactic acid. S. thermophilus T15 demonstrated dramatically complex amino acid necessities for growth and metabolism. With L. lactis C15, the highest levels of growth and lactic acid synthesis were recorded (18.3 x 10(8) CFU ml(-1) and 7.8 g l(-1) lactic acid at the 21st hour), and as for free amino acid production, it approximated L. bulgaricus HP1 (17.03 mg (100 g)(-1) maximum concentration). In the L. lactis C15 culture, the amino acids were used more actively throughout the first exponential growth phase (by the 10th hour) than during the second growth phase. The unique properties of the L. helveticus MP12 strain to produce amino acids were employed to create a symbiotic bioconsortium kefir culture, which, under conditions of kefir formation, enhanced lactic acid production and shortened the time required to reach pH 4.7; intensified cell growth activity, resulting in a respective 90- and 60-fold increase in the concentration of lactobacilli and cocci in the mixed culture compared to individual cultures; and accumulated free amino acids in the final kefir with higher total concentrations (56.88 mg (100 g)(-1)) and an individual concentration of essential amino acids (1.5 times) greater than that of yogurt.     

冷水鱼肠道乳酸菌的遗传差异分析

为分析新疆额尔齐斯河流域及黑龙江流域冷水鱼肠道中乳酸菌遗传差异,为乳酸菌资源的开发奠定基础。本实验利用MRS、Elliker、M17培养基对冷水鱼肠道中低温乳酸菌进行分离鉴定,并测定其最适生长温度。根据16S rRNA基因序列初步确定低温乳酸菌的系统发育关系,并利用rep-PCR指纹图谱技术进一步区分高度同源性菌株。从冷水鱼的肠道中分离得到134株低温乳酸菌,其最适生长温度在15²4℃之间。16S rRNA测序结果表明,这些菌株分别隶属于Lactobacillus、Lactococcus、Enterococcus、Streptococcus、Leuconostoc、Weissella、Carnobacterium 7个属,19个种,其中Lactobacillus为优势菌。Rep-PCR指纹图谱分析表明同一属的乳酸菌在种水平及同一种的不同菌株之间存在不同程度的遗传差异。      

Factors affecting capsule size and production by lactic acid bacteria used as dairy starter cultures

The effects of sugar substrates on capsule size and production by some capsule-forming nonropy and ropy dairy starter cultures were studied. Test sugars (glucose, lactose, galactose, or sucrose) were used as a sole carbohydrate source and the presence of a capsule and its size were determined by using confocal scanning laser microscopy. Nonropy strains produced maximum capsule size when grown in milk. Strains that did not produce capsules in milk did not produce them in any other growth medium. Specific sugars required for capsule production were strain-dependent. Increasing lactose content of Elliker broth from 0.5 to 5% or adding whey protein or casein digest produced larger capsules. Whey protein concentrate stimulated production of larger capsules than did casamino acids or casitone. Some Streptococcus thermophilus strains produced capsules when grown on galactose only. Nonropy strains of Lactobacillus delbrueckii subsp. bulgaricus produced capsules on lactose, but not on glucose. A ropy strain of Lactobacillus delbrueckii subsp. bulgaricus produced a constant capsule size regardless of the growth medium. The ability of some strains of Streptococcus thermophilus to use galactose in capsule production could reduce browning of mozzarella cheese during baking by removing a source of reducing sugar. Media that do not support capsule production may improve cell harvesting.     

Interactions among lactic acid starter and probiotic bacteria used for fermented dairy products

Interactions among lactic acid starter and probiotic bacteria were investigated to establish adequate combinations of strains to manufacture probiotic dairy products. For this aim, a total of 48 strains of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium spp. (eight of each) were used. The detection of bacterial interactions was carried out using the well-diffusion agar assay, and the interactions found were further characterized by growth kinetics. A variety of interactions was demonstrated. Lb. delbrueckii subsp. bulgaricus was found to be able to inhibit S. thermophilus strains. Among probiotic cultures, Lb. acidophilus was the sole species that was inhibited by the others (Lb. casei and Bifidobacterium). In general, probiotic bacteria proved to be more inhibitory towards lactic acid bacteria than vice versa since the latter did not exert any effect on the growth of the former, with some exceptions. The study of interactions by growth kinetics allowed the setting of four different kinds of behaviors between species of lactic acid starter and probiotic bacteria (stimulation, delay, complete inhibition of growth, and no effects among them). The possible interactions among the strains selected to manufacture a probiotic fermented dairy product should be taken into account when choosing the best combination/s to optimize their performance in the process and their survival in the products during cold storage.     

Characterization of lactic acid bacteria isolated from Italian Bella di Cerignola table olives: selection of potential multifunctional starter cultures

Lactic acid bacteria (19 isolates) from Bella di Cerignola Italian table olives were investigated for their technological and probiotic properties for the selection of multifunctional starter cultures for table olives. The bacteria were first identified by phenotyping and genotyping, then characterized for the production of biogenic amines, growth at different pH, NaCl concentrations, and temperatures. The potentiality of the bacteria to have some probiotic properties (antimicrobial activity against foodborne pathogens, survival in low pH and in the presence of bile salts, ability to adhere to the mammalian cells model IPEC-J2) was also investigated. Eighteen of the studied isolates were identified as Lactobacillus plantarum and one as Enterococcus faecalis. All bacteria were able to grow at a range of pH between 4.0 and 10.0 as well as in media supplemented with 2.5 to 7.5% of NaCl and 0.3% bile salts and survived in MRS broth acidified at pH 2.5; moreover, they inhibited significantly Escherichia coli O157:H7. The adhesion to IPEC-J2 cells was in general low to moderate (5.3 to 8.3%); however, 2 isolates of L. plantarum (c16 and c19) showed interesting higher adhesion values (up to 16%). Our results suggest that at least 3 isolates could be possible multifunctional starters for Bella di Cerignola olives: L. plantarum 16 and 19 for mainly their probiotic properties and L. plantarum 10 for mainly its technological characteristics. Practical Application: A functional starter is a microorganism exerting benefits on human health (probiotic) and able to guide a fermentation (starter). The main goal of this article was to select a functional starter for table olives.     

Use of starter cultures of lactic acid bacteria and yeasts in the preparation of togwa,a Tanzanian fermented food

Starter cultures of lactic acid bacteria (Lactobacillus brevis, Lactobacillus cellobiosus, Lactobacillus fermentum, Lactobacillus plantarum and Pediococcus pentosaceus) and yeasts (Candida pelliculosa, Candida tropicalis, Issatchenkia orientalis and Saccharomyes cerevisiae) isolated from native togwa were tested singly or in combination for their ability to ferment maize-sorghum gruel to produce togwa. All species of bacteria showed an ability to ferment the gruel as judged by lowering the pH from 5.87 to 3.24-3.49 and increasing the titratable acidity from 0.08% to 0.30-0.44% (w/w, lactic acid) in 24 h. Yeasts used singly showed little activity within 12 h, but lowered the pH to 3.57-4.81 and increased the acidity to 0.11-0.21% in 24 h. Yeasts in co-culture with lactic acid bacteria (LAB) had a modest effect on the final acidity (P<0.05). The number of lactic acid bacteria and yeasts increased while the Enterobacteriaceae decreased with fermentation time. The pH was lowered and lactic acid produced significantly (P<0.05) fastest in natural togwa fermentation and in samples fermented by L. plantarum or L. plantarum in co-culture with I. orientalis. The content of fermentable sugars was reduced during fermentation. Most volatile flavour compounds were produced in samples from fermentation by P. pentosaceus and I. orientalis in co-culture with either L. plantarum or L. brevis.     

Suitability ofLactococcus lactissubsp.lactisATCC 11454 as a protective culture for lightly preserved fish products

This study is part of strategy to control the human pathogenListeria monocytogenesin lightly preserved fish products by using food-grade lactic acid bacteria. When the nisin- producingLactococcus lactissubsp.lactisATCC 11454 was cultured in the same vessel asL. monocytogenesScott A in brain–heart infusion broth (BHI) at 30°C, the pathogen declined from 5×10⁵to fewer than 5 cfu ml⁻¹within 31 h. The effect was not due to lactic acid inhibition. Growth and nisin production byL. lactisATCC 11454 were investigated under the conditions of temperature and salt used for light preservation. At 5°C in M17 broth, the organism grew well and produced nisin. In an infusion of cold-smoked salmon the organism did not grow at 5°C, although it did at 10°C. NaCl up to 4% allowed for efficient growth and nisin production, while 5% NaCl resulted in very slow growth and no detectable nisin. On slices of commercial cold-smoked salmon at 10°C, no net propagation ofL. lactisATCC 11454 could be detected within 21 days. However, when salmon slices were inoculated withL. monocytogenesat 10⁴cfu g⁻¹and a 300-fold excess of washed lactococcus cells, the pathogen's population declined a half log the first 1.5 days, then increased at a rate slightly lower than that of the control not inoculated with the lactococcus.     

Biodiversity of lactic acid bacteria isolated from fermented milk products in Xinjiang,China

Fermented dairy products have been produced and consumed for thousands of years in Xinjiang, China. In this study, traditional culture-dependent methods and 16S rRNA gene analyses were performed to analyze the composition of lactic acid bacteria (LAB) from 86 samples of traditional fermented dairy products collected from four regions of Xinjiang. Quantitative polymerase chain reaction (qPCR) was used to quantify Bifidobacterium and Lactobacillus species. The LAB isolates (N = 705) were identified as belonging to seven genera and 26 species or subspecies. The predominant species of all isolates were Lactobacillus (Lb.) delbrueckii subsp. bulgaricus, Lb. fermentum, and Lb. helveticus. The LAB counts of these samples ranged from 5.35 to 10.06 Log CFU/mL. The bacterial counts of seven lactobacilli species and Bifidobacterium ranged from 0.98 ± 0.14 Log CFU/mL (for Lb. sakei from fermented mare milk) to 9.91 ± 0.17 Log CFU/mL (for Lb. helveticus from fermented mare milk). While the microbiota was significantly different between yak and cow milk, there was no significant difference between the Tex and Zhaosu counties. In conclusion, LAB communities in traditional fermented dairy products are complex and differ among local regions within Xinjiang.     

Silage fermentation of fish or fish waste products with lactic acid bacteria

Silage fermentation of minced fish and fish offal after inoculation with cereals prefermented with Pediococcus acidilactici and Lactobacillus plantarum initiates a rapid fall in pH to below 4.5 within 30 h, and the content of competing gram-negative fermenters and fish pathogens, such as Vibrio anguillarum and Aeromonas salmonicida, is eliminated. The addition of 0.1% sorbic acid inhibits the growth of yeasts during the initial fermentation period and during storage but does not affect the lactic acid fermentation. Degradation of nitrogen components proceeds during storage and is manifested as an increase in volatile basic nitrogen compounds, amino acids and peptides. These substances increase the pH and/or force the bacteria to produce more acid. The rate of production of these basic substances is mainly temperature-dependent and cannot be attributed to microbial activity. The proteolytic activity is mainly caused by tissue proteases (e.g., cathepsins) and, to a lower degree, by gut proteases. It is doubtful whether fish fermentation can be utilised on an industrial scale without improving the technique. Such improvements consist of the selection and use of psychotrophic lactic acid fermenters and commercially acceptable inhibitors of proteolytic activity and yeast growth.     

Application of lactic acid bacteria starter cultures for decreasing the biogenic amine levels in sauerkraut

Sauerkrauts from shredded white cabbage of six varieties were prepared in six laboratory experiments by initial fermentation at 22 °C for 14 days, then stored at 5-6 °C and analysed after 6 months. Seven biogenic amines were extracted with perchloric acid and determined as N-benzamides by micellar electrokinetic capillary chromatography. Eight common sauerkraut quality parameters were also determined. In three experiments, a commercial strain of Lactobacillus plantarum and a mixed preparation of Microsil containing L. plantarum, Lactobacillus casei, Enterococcus faecium and Pediococcus pentosaceus, were applied at doses of 5᎒⁴, 1᎒⁵ and 5᎒⁵ CFU/g of cabbage. In three further experiments, L. plantarum, Microsil and a commercial strain of Lactobacillus buchneri were applied at doses of 5᎒⁵ and 5᎒⁶ CFU/g. Spontaneously fermented sauerkrauts were prepared as the control variants in all experiments. L. plantarum at doses of at least 5᎒⁵ CFU/g significantly (P<0.05) suppressed formation of putrescine, tyramine and cadaverine, amines occurring at the highest levels. Spermidine contents varied between 10 and 30 mg/kg and were not affected by the starter cultures. Levels of tryptamine, spermine and histamine were very low, often below the detection limits. For practical application, a dose of at least 5᎒⁶ CFU/g of the tested L. plantarum strain seems to be likely. The tested strains of L. buchneri and E. faecium showed tyrosine decarboxylase activity in an in vitro test.