Effects of starter cultures and nitrite levels on formation of biogenic amines in sucuk

The effects of starter cultures, starter A (Lactobacillus sakei+Staphylococcus carnosus) and starter B (Pediococcus acidilactici+Staphylococcus xylosus+Lactobacillus curvatus), nitrite levels (0, 75, and 150ppm) and ripening period on biogenic amine production were investigated in sucuk (Turkish dry-fermented sausage) production. Changes in biogenic amines, pH, water activity, moisture, non-protein nitrogen, nitrite and counts of lactic acid bacteria, Micrococcus/Staphylococcus, Enterobacteriaceae, and moulds-yeasts were determined. Ripening period had a very significant effect (P<0.01) on the all biogenic amines. Sausages produced by fermentation with starter cultures, as compared to natural fermentation (control), had lower amounts of putrescine, cadaverine and tyramine (P<0.05). Starter cultures A and B were found to have almost the same effect on the formation of biogenic amines. Use of nitrite in sucuk production affected the formation of biogenic amines except for spermidine and spermine (P<0.01). Lactic acid bacteria counts in the control group were lower than that of starter culture groups. Lactic acid bacteria, Micrococcus/Staphylococcus, and moulds-yeasts counts increased during ripening period while levels of species of Enterobacteriaceae decreased, which was also found to be under the detectable level (<100cfug(-1)), by day 3 of ripening.     

处理发酵剂对促熟干酪中生物胺和游离氨基酸的影响

以0、30、60、90 d促熟干酪中生物胺（组胺、色胺、苯乙胺、尸胺、酪胺）和游离氨基酸含量为指标,研究了添加复合处理发酵剂对干酪产生游离氨基酸和生物胺量的影响。结果表明：不同处理发酵剂添加量对各种生物胺产生的影响不同;添加处理发酵剂干酪中生物胺和游离氨基酸含量增加,其含量随着复合处理发酵剂添加量增加而增加。     

传统中式香肠成熟过程中生物胺的生物控制

以传统中式香肠成熟过程中生物胺含量的变化为研究对象,重点介绍了色胺、腐胺、尸胺、组胺、酪胺的变化。在香肠加工过程中添加发酵剂和复合植物提取物来控制香肠中生物胺的积累,利用高效液相色谱仪检测成熟过程中生物胺的变化。结果表明,添加发酵剂可有效地抑制色胺、腐胺、尸胺、酪胺的生成;复合植物提取物只是在抑制酪胺含量的增加上有显著效果;同时加入发酵剂和复合植物提取物对色胺、尸胺、组胺和酪胺的抑制效果更显著,特别是组胺,在生产结束时含量为0。发酵剂和复合植物提取物对精胺和亚精胺基本没有影响。     

Formation of biogenic amines in raw milk Hispánico cheese manufactured with proteinases and different levels of starter culture

Two proteinases, a neutral proteinase from Bacillus subtilis and a cysteine proteinase from Micrococcus sp., were used to accelerate the ripening process of raw cow's milk Hispánico cheese, a semihard variety. Two levels (0.1% and 1%) of a commercial starter culture containing Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris were added for cheese manufacture. The influence of both factors, proteinase addition and level of starter culture, on the growth of amino acid-decarboxylating microorganisms and on the formation of biogenic amines during cheese ripening was investigated in duplicate experiments. The population of tyrosine decarboxylase-positive bacteria, which represented less than 1% of the total bacterial population in most cheese samples, and tyrosine decarboxylase-positive lactobacilli was not influenced by proteinase addition or level of starter culture. Tyramine was detected in all batches of cheese from day 30. Its concentration was significantly (P < 0.05) influenced by proteinase addition but not by the level of starter culture and increased with cheese age. After 90 days of ripening, 103 to 191 mg/kg of tyramine was found in the different cheese batches. Histamine was not detected until day 60 in cheese with neutral proteinase and 1% starter culture and until day 90 in the rest of the cheeses. The concentration of this amine did not exceed 20 mg/kg in any of the batches investigated. Phenylethylamine and tryptamine were not found in any of the samples.     

Effects of Lactobacillus plantarum and Staphylococcus xylosus on the Quality Characteristics of Dry Fermented Sausage "Sucuk"

ABSTRACT:  The effect of starter culture, containing the strains Lactobacillus plantarum GM77 and Staphylococcus xylosus GM92, isolated from traditional sucuk, on the quality characteristics of dry fermented sausage (sucuk) during ripening period was studied. The microbiological (lactic acid bacteria, Micrococcus/Staphylococcus , Enterobacteriaceae) and physicochemical (pH, a_w, NPN, TBARS) properties and volatile compounds, extracted by solid phase microextraction and analyzed by gas chromatography-mass spectrometry, were determined in sucuk samples with starter culture and in the control group (without starter culture). Both starter culture and ripening period had significant effects on lactic acid bacteria, Micrococcus/Staphylococcus counts, and pH and a_w values ( P  < 0.01). The highest value for nonprotein nitrogen (NPN) was observed to occur in samples with starter culture ( P  < 0.05). TBARS value increased during ripening, the highest value was determined at 14th day in samples with starter culture. Among volatile compounds, terpenes were the major group in sucuk. Other volatile compounds (aldehydes, ketones, sulfur compounds, acids, esters, aliphatic hydrocarbons, aromatic hydrocarbons, and alcohols) can be found in fewer amounts. While the use of starter culture was affecting a few compounds, ripening period had affected most of the compounds.     

Starter cultures and high-pressure processing to improve the hygiene and safety of slightly fermented sausages

The effectiveness of selected starter cultures and high hydrostatic pressure after ripening was evaluated to improve the safety and quality of slightly fermented sausages. Inhibition of common foodborne pathogens, spoilage bacteria, and biogenic amine content was studied. Random amplification of polymorphic DNA and plasmid profiles were used to monitor the competitiveness of the starter cultures during fermentation and ripening. Lactobacillus sakei CTC6626 and Staphylococcus xylosus CTC6013 dominated L. sakei CTC6469 and S. xylosus CTC6169 independently of the product assayed. Starter cultures were able to control the growth of Listeria monocytogenes, Enterobacteriaceae, Enterococcus, and the biogenic amine content. A pH decrease below 5.3 at the seventh day of fermentation was crucial. Salmonella spp. counts decreased significantly during ripening independently of the use of starter culture and product. High hydrostatic pressure treatment was necessary to ensure absence of Salmonella spp. in final products.     

Formation of Biogenic Amines during Ripening of Dry Sausages as Affected by Starter Culture and Thawing Time of Raw Materials

Raw materials affect formation of biogenic amines in dry sausages. Effects of thawing time of raw materials and amine-negative starter culture on amine formation were studied on a pilot scale. The levels of biogenic amines, precursor amino acids, pH, water activity, and microbial counts were measured. Use of starter culture significantly decreased levels of histamine, tyramine and cadaverine formed. The effect of thawing time on formation of biogenic amines was dependent on the use of starter culture.     

Effects of starter cultures and additives on the quality of Turkish style sausage (sucuk)

The effects of starter cultures and additives on the quality of Turkish style sausage (sucuk) were investigated during ripening and storage. Total aerobic plate counts (aerobic bacteria), mould and yeast counts, pH, thiobarbituric acid (TBA) value and biogenic amine formation were followed. Aerobic bacteria increased (P<0.05) during the first 10 days of ripening to 9.26 log CFU/g and they decreased (P<0.05) to 5.70 at the end of the storage. Larger reductions (P<0.05) were observed in sausages made with high levels of potassium sorbate, nitrite and nitrate than in those containing low levels of additives and those without additives at the end of storage. During the first 3 days of ripening, the pH values of all sausages decreased (P<0.05) from 5.98 to about 4.53. Later, the pH values increased slowly, due to decomposition of acids to an approximately constant value of about 5.20. TBA values were higher (P<0.05) in the sausages made without additives than in the others. Sausages prepared using high concentration of additives had lower (P<0.05) concentrations of biogenic amines than the others. Addition of starter culture with high concentration of additives (nitrite, nitrate, α-tocopherol, ascorbic acid, potassium sorbate, potassium pyrophosphate and di-potassium hydrogenphosphate) decreased the formation of biogenic amine.     

Effect of proteolytic starter cultures of Staphylococcus spp. on biogenic amine formation during the ripening of dry fermented sausages

The effect of proteolytic starter cultures of Staphylococcus carnosus and Staphylococcus xylosus on biogenic amine production was examined during the fermentation process of dry sausages. Microbial counts (lactic acid bacteria, Micrococcaceae and Enterobactenaceae), pH, moisture and proteolysis-related parameters were also studied. The polyamines spermine and spermidine were the main amines found in the raw material and they only showed slight fluctuations during the fermentation. The four elaborated batches presented a significant (P < 0.001) formation of tyramine and putrescine. The main rate of amine production was during the first three days, when a sharp pH decrease and the development of lactic acid bacteria occurred. Sausages fermented with starters had lower amounts of tyramine than naturally fermented sausages (control), but differences in the Micrococcaceae counts were only significant during the first week of the ripening process. A slight formation of diaminopropane, cadaverine, agmatine, tryptamine and phenylethylamine was observed. The amounts of histamine were constant and remained below 0.5 mg/kg of dry matter, while serotonin, octopamine and dopamine were not detected. The sausages with Staphylococcus as starter culture showed strong proteolysis that was correlated with higher pH values than those of the control sausages. However, no positive correlation was found between the proteolysis index and biogenic amine production. Since proteolysis was stronger during the second half of the ripening process, the release of free amino acids as amine precursors occurred later than the early amine production.     

Mixed starter cultures to control biogenic amine production in dry fermented sausages

Several combinations of an amine-negative Lactobacillus sakei strain, along with proteolytic Staphylococcus carnosus or Staphylococcus xylosus strains, were used to study the influence of mixed starter cultures on biogenic amine production during the manufacture of dry fermented sausages. Changes in pH, water content, proteolysis, microbial counts, and biogenic amine contents were simultaneously examined in a spontaneously fermented batch and in three mixed starter-mediated batches. A double-controlled microbial charge initially inoculated as mixed starter culture of L. sakei and Staphylococcus spp. (all amine-negative strains) drastically reduced tyramine, cadaverine, and putrescine accumulation. No production of other aromatic amines such as histamine, phenylethylamine, or tryptamine was observed in any batch. The polyamines, spermine and spermidine, were found in raw materials and their levels decreased slightly in the spontaneously fermented batch. No correlation between proteolysis and biogenic amine production was observed. The use of proper technological conditions favoring starter development and the use of the raw materials with good hygienic quality make it possible to produce fermented sausages nearly free of biogenic amines.     

The influence of sonication time on the biogenic amines formation as a critical point in uncured dry-fermented beef manufacturing

In this study, the influence of sonication time on the biogenic amines formation as a critical point in uncured dry-fermented beef manufacturing was studied. Samples of musculus semimembranosus were sonicated at different times (5 and 10 min) using ultrasound cold bath (4 °C) in acid whey (US 40 kHz and acoustic power 480 W). The effect of sonication on biogenic amine (BA) formation was investigated during 93 days of ripening period. Other parameters (pH value, water activity, microbial counts) that might provide further information on the product under study were also determined. The use of ultrasound during beef marinating in acid whey has a positive effect on retarding histamine (HIS), cadaverine (CAD), tyramine (TYR) and putrescine (PUT) formation. Moreover, the sonication treatment did not inhibit the growth of lactic acid bacteria (LAB) in dry-fermented beef during the whole ripening period. The pathogen bacteria (Staphylococcus aureus, Clostridium sp., Listeria monocytogenes) were not detected in all samples neither after 31 nor after 93 days of ripening period.     

发酵剂对牦牛乳硬质干酪成熟过程中生物胺的影响

乳酸菌产生物胺的能力具有菌株特异性,因此,为了探究不同种类发酵剂对牦牛乳硬质干酪中生物胺形成的影响,该试验利用高效液相色谱对3种不同发酵剂制作的硬质干酪成熟过程中生物胺进行了测定和分析。结果表明,嗜热和嗜温发酵剂牦牛乳硬质干酪中检测出2-苯乙胺、腐胺、尸胺、组胺和酪胺,混合发酵剂干酪中检测出腐胺、2-苯乙胺、尸胺和酪胺。各生物胺之间呈现正相关性。3种不同发酵剂干酪在1～6个月成熟过程中,其各生物胺整体呈现增加趋势,嗜热、嗜温和混合发酵剂干酪中总生物胺最高含量分别为(448.3±9.6)、(456.8±58.4)、(293±24.5)mg/kg。组胺和酪胺是2种毒性相对高的生物胺,嗜热发酵剂干酪中组胺和嗜温发酵剂干酪中酪胺最高,其最高含量分别为(20.8±7.9)、(92.9±6.7)mg/kg,混合发酵干酪中未检测出组胺,酪胺含量次之,3种不同发酵剂干酪中组胺、酪胺含量均低于推荐安全剂量50 mg/kg和100 mg/kg。这为合理选择发酵剂和控制干酪中生物胺形成提供了依据。     

Reduction of biogenic amine formation using a negative amino acid-decarboxylase starter culture for fermentation of Fuet sausages

The ability of Lactobacillus sakei CTC494, a negative amino acid-decarboxylase starter culture, to reduce biogenic amine accumulation during sausage fermentation and storage at 4 and 19 degrees C was studied. The effect on the amine formation of the tyramine producer Lactobacillus curvatus CTC371, as a positive strain, was also examined in comparison to a spontaneous fermentation process without starter culture (control batch). The polyamines spermine, spermidine, and diaminopropane were not influenced by the ripening, and their levels slightly decreased in all the batches throughout the storage. Tyramine, cadaverine, and putrescine were the main amines formed during the ripening. The addition of starter culture resulted in a decrease on the biogenic amine formation, depending on the strain inoculated. A great reduction in tyramine content was achieved when L. sakei CTC494 was inoculated, whereas L. curvatus CTC371 only attenuated tyramine accumulation compared with the control batch. Both starters were able to significantly limit the production of putrescine and cadaverine, and they inhibited tryptamine and phenylethylamine formation by the wild microbial flora. Tyramine levels of the control sausages rose during the storage at both temperatures, whereas those of cadaverine only increased at 19 degrees C. On the contrary, sausages manufactured through the starter controlled fermentation did not show changes of amine contents during the storage. The addition of a proper selected starter culture is advisable to produce safer sausages with low contents of biogenic amines.     

FORMATIONS OF BIOGENIC AMINES IN TURKISH STYLE SAUSAGE DURING RIPENING AND STORAGE PERIODS

Changes of biogenic amine (histamine, β‐phenylethylamine, serotonin, spermine, spermidine, cadaverine, putresine, 1,7‐diamino heptane, tryptamine and tyramine) concentrations and pH values of Turkish style sausages were investigated during ripening and storage. Cadaverine and β‐phenylethylamine were not detected during the ripening and storage periods for all sausage types. Storage time had a significant (P<0.05) effect on the formation of all biogenic amines. Starter culture usage decreased (P<0.05) biogenic amine formation. Histamine levels were higher in sausages prepared without additives and lower in the sausages made with starter culture than the sausages made without starter culture. There was no effect of starter culture and additives on formation of tryptamine, 1,7‐diamino heptane, serotonin or spermidine. Starter culture and additives affected (P<0.05) the formation histamine, putrescine and spermine. pH values of sausages sharply decreased (P< 0.05) from 5.98 to about 4.53 during 3 days of ripening. After that, pH values increased slowly and were nearly constant during the storage period.     

Novel starter cultures to inhibit biogenic amines accumulation during fish sauce fermentation

Bacteria with amine oxidase activity have become a particular interest to reduce biogenic amines concentration in food products such as meat and fish sausages. However, little information is available regarding the application of these bacteria in fish sauce. Hence, our study was aimed to investigate the effect of such starter cultures in reducing biogenic amines accumulation during fish sauce fermentation. Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05 isolated from fish sauce which possess amine oxidase activity were used as starter cultures in this study. Fermentation was held for 120 days at 35 °C. The pH value increased in all samples, while salt concentration remained constant throughout fermentation. Aerobic bacteria count was significantly lower (p < 0.05) in the control than in inoculated samples as a result of starter cultures addition. However, it decreased during fermentation due to the growth inhibition by high salt concentration. Proteolytic bacterial count decreased during fermentation with no significant difference (p > 0.05) among samples. These bacteria hydrolyzed protein in anchovy to produce free amino acid precursors for amines formation by decarboxylase bacteria. The presence of biogenic amines producing bacteria in this study was considered to be indigenous from raw material or contamination during fermentation, since our cultures were negative histamine producers. Amino acid histidine, arginine, lysine and tyrosine concentration decreased at different rates during fermentation as they were converted into their respective amines. In general, biogenic amines concentration namely histamine, putrescine, cadaverine and tyramine increased throughout fermentation. However, their concentrations were markedly higher (p < 0.05) in the control (without starter cultures) as compared to the samples treated with starter cultures. Histamine concentration was reduced by 27.7% and 15.4% by Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05, respectively. Both cultures could also reduce other amines during fermentation. After 120 days of fermentation, the overall biogenic amines concentration was 15.9% and 12.5% less in samples inoculated with Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05, respectively, as compared to control samples. These findings emphasized that application of starter cultures with amines oxidase activity in fish sauce fermentation was found to be effective in reducing biogenic amines accumulation.     

Comparison of the effects of a commercial and an autochthonous Pediococcus acidilactici and Staphylococcus vitulus starter culture on the sensory and safety properties of a traditional Iberian dry‐fermented sausage “salchichón”

The effect of the addition of an autochthonous starter culture and commercial starter culture on the physico‐chemical and sensory characteristics of the dry‐fermented sausage ‘salchichon’ was investigated. Sausages were prepared with a mixture of Pediococcus acidilactici MC184 and Staphylococcus vitulus RS34 as the autochthonous starter culture (P184S34), ripened for 86 days, and compared with a commercial starter batch (CS1). Strains of acid lactic bacteria and Staphylococcus were identified in the samples along the ripening process which demonstrated that the inoculation of sausages assures their implantation during the ripening process. However, P184S34 starter culture showed a higher inhibitory effect on enterobacteria and coliform flora than the CS1 batches, guaranteeing a better quality hygienic sausages. Dry‐fermented sausages ripened with P184S34 showed higher amounts of NPN and volatile compounds derived from amino acid catabolism than the CS1 batches. Especially important was the result found in biogenic amines, since the P184S34 reduced their accumulation compared to the commercial starter batch. The inoculation of a decarboxylase‐negative autochthonous starter culture P184S34 reduced the biogenic amine accumulation guaranteeing safety and homogeneity of the products without producing a negative effect on the sensory characteristics of the traditional fermented sausages.     

Effect of added proteinases and level of starter culture on the formation of biogenic amines in raw milk Manchego cheese

The influence of two proteinases (Bacillus subtilis neutral proteinase and Micrococcus sp. cysteine proteinase) and two starter culture levels (0.1% and 1%) on biogenic amine formation has been studied in raw ewes' milk Manchego cheese. Amino acid decarboxylating micro-organisms were determined on tyrosine enriched selective media. Biogenic amines were analysed by capillary electrophoresis in citrate buffer at pH 3.6. Addition of proteinases and level of starter culture did not influence the population of micro-organisms with amino acid decarboxylating activity, which represented on average 1% of the bacterial population in 30-day-old cheeses. Tyramine and histamine were detected in all batches of cheese from day 30. Concentrations of tyramine and histamine were higher in cheeses made from milk with neutral proteinase (up to 356 and 284 mg kg(-1), respectively, after 90 days) than in cheeses made from milk with cysteine proteinase (up to 269 and 189 mg kg(-1), respectively) or with no proteinase added (up to 305 and 226 mg kg(-1), respectively). Formation of tyramine and histamine was also favoured in cheeses made with 1% starter culture with respect to cheeses made with only 0.1% starter culture, probably due to the higher pH values of the former cheeses. After 90 days of ripening, concentrations of 10-20 mg kg(-1) phenylethylamine were observed in 9 of the 12 batches, and levels < 10 mg kg(-1) tryptamine were only detected in 3 batches, with no significant relationship between the concentration of these amines and proteinase addition or level of starter culture.     

Use of Staphylococcus xylosus as a starter culture in dried sausages: effect on the biogenic amine content

The main objective of this work was to investigate if the use of Staphylococcus xylosus S81 as a starter culture in sausage production can influence the amount of histamine during ripening, and the concentration of the other most important biogenic amines, by influencing the activity of the microbial amino oxidases as well as the aminoacid decarboxylases. The results confirm that the biogenic amines presence in foods is the consequence of a complex equilibrium between the composition of the medium and the enzymatic activities of the microbial population. In addition, the results suggest that the presence and relative activity of amino oxidases should be considered as an important characteristic in the selection of starter cultures used in the production of fermented foods.     

CHANGES IN FREE AMINO ACIDS AND BIOGENIC AMINES DURING RIPENING OF FRESH AND FROZEN SARDINE

A study was carried out on the evolution of free amino acids and biogenic amines throughout the ripening process of fresh and frozen sardine. The effect of the level of fish gutting was also followed. During ripening a general increase of free amino acids content was observed in all samples. Histidine was the exception to this trend and decreased throughout the processing period. On the basis of the ratio of basic/acidic amino acids, as an indicative of the ripened state, the fresh nobbed sardine ripened faster than the gutted fish. During the course of ripening a considerable increase in biogenic amines and particularly histamine occurred until the 20th day in products from fresh fish. The maximum histamine content allowed in ripened fish products in the European Union (400 mg/Kg) was never attained in this period. A general decrease in most amines was observed by the 240th day of ripening. Tyramine behaves differently and increased in the same period. The samples prepared with frozen fish consistently showed the lowest amine values throughout the ripening period. It seems that freezing has a good effect on the final quality of ripened products as it could decrease the bacterial activity responsible for biogenic amine formation.     

The influence of manufacturing and drying conditions on the survival and toxinogenesis of Staphylococcus aureus in two Spanish dry sausages (chorizo and salchichón)

The effects of formulation, starter culture and fermentation temperature on growth and synthesis of toxin A (SEA) and TNase by Staphylococcus aureus during fermentation and drying of Spanish chorizo were investigated. Inhibitory factors able to inhibit SEA synthesis in culture media were unable to prevent SEA production in chorizo fermented at 20 and 30°C, though a lower temperature and starter culture SP318 (Lactobacillus sake, Pediococcus pentosaceous and Staphylococcus xylosus) decreased staphylococcal growth and SEA formation. Reduction and even disappearance of the SEA during ripening was observed. In most batches, TNase was a reliable indicator of staphylococcal growth and SEA production. Dextrose added to the salchichón formulation repressed S. aureus growth during drying. Lactobacillus curvatus in combination with dextrose was an effective anti-staphylococcal agent during fermentation.