Impact of Gram-negative bacteria in interaction with a complex microbial consortium on biogenic amine content and sensory characteristics of an uncooked pressed cheese

The impact of Gram-negative bacteria on sensory characteristics and production of volatile compounds as well as biogenic amines (BA) in the core of an uncooked pressed type model cheese was investigated in the presence of a defined complex microbial consortium. Eleven strains of Gram-negative bacteria, selected on the basis of their biodiversity and in vitro BA-production ability, were individually tested in a model cheese. Four out of 6 strains of Enterobacteriaceae (Citrobacter freundii UCMA 4217, Klebsiella oxytoca 927, Hafnia alvei B16 and Proteus vulgaris UCMA 3780) reached counts close to 6 log CFU g⁻¹ in the model cheese. In core of cheeses inoculated with Gram-negative bacteria, only slight differences were observed for microbial counts (Enterococcus faecalis or Lactobacillus plantarum count differences below 1 log CFU g⁻¹), acetate concentration (differences below 200 mg kg⁻¹) and texture (greater firmness) in comparison to control cheeses. Cheese core colour, odour and volatile compound composition were not modified. Although ornithine, the precursor of putrescine, was present in all cheeses, putrescine was only detected in cheeses inoculated with H. alvei B16 and never exceeded 2.18 mmol kg⁻¹ cheese dry matter. Cadaverine was only detected in cheeses inoculated with H. alvei B16, K. oxytoca 927, Halomonas venusta 4C1A or Morganella morganii 3A2A but at lower concentrations (<1.05 mmol kg⁻¹ cheese dry matter), although lysine was available. Only insignificant amounts of the detrimental BA histamine and tyramine, as well as isopentylamine, tryptamine or phenylethylamine, were produced in the cheese model by any of the Gram-negative strains, including those which produced these BA at high levels in vitro.     

The microbial flora of Awshari cheese with special reference to lactobacilli

Thirty seven samples of Awshari cheese and 13 of Biza were examined microbiologically. Penicillia occurred in 2 samples of cheese and yeasts in 21, 3 of which contained Saccharomyces lactis. In 8 samples studied for the constitutive microflora, lactobacilli and streptococci were the most prevalent, with L. casei being the only Lactobacillus species isolated. Penicillia and yeasts occurred in all the samples of Biza, with Saccharomyces lactis being encountered in 5 out of 6 samples examined for the type of yeasts. The microbial flora consisted primarily of lactobacilli. Out of 98 lactobacilli isolated from 10 samples 55 were identified as betabacteria: 41 were attached to L. brevis, 13 L. buchneri and 1 L. pasteurianus. The remaining 43 isolates were streptobacteria: 21 belonged to L. coryniformis, 5 L. casei ssp pseudoplantarum, 3 ssp casei and 1 ssp alactosus, 2 L. plantarum ssp plantarum and 1 ssp arabinosus, 1 L. xylosus, 5 motile streptobacteria and 4 unidentifiable.     

Use of Fourier transform infrared spectroscopy to evaluate the proteolytic activity of Yarrowia lipolytica and its contribution to cheese ripening

The interaction between tyrosine-decarboxylase and proteolytic activities of a Lactobacillus curvatus and Staphylococcus xylosus, respectively, on biogenic amine production during the ripening and the storage of dry fermented sausages was investigated. Water content, pH, proteolysis parameters, microbial counts, and biogenic amine contents were monitored in spontaneously and starter fermented sausages. The use of proteolytic staphylococci as starter resulted in a higher content of non-protein nitrogen and total free amino acids. Tyramine was the main amine produced in all batches. However, tyrosine-decarboxylase activity of the L. curvatus starter strain was weak and yielded lower amounts of tyramine than those produced by the wild mioroflora in the control batch. Association between tyramine production and proteolysis could only be established in a defectively dried batch. Putrescine and cadaverine accumulation was efficiently reduced in the starter-mediated fermentation, in agreement with the lower development of enterobacteria. Phenylethylamine and tryptamine were only detected in the spontaneously fermented sausages, while histamine, spermine and spermidine did not vary during the ripening. Biogenic amine levels and related parameters showed significant changes during the storage of dry sausages depending on the temperature and the batch. As a general rule, changes in the pH, proteolysis, microbial counts, and biogenic amine contents were stronger at 19 degrees C than at 4 degrees C. The results suggest that refrigeration would be advisable for preventing further accumulation of biogenic amines during the storage of dry fermented sausages.     

Current methods to evaluate contribution and interactions of components to flavour of solid foods using hard cheese as an example

The flavours of hard cheeses are derived from the balance of odourants and tastants released during their consumption, but the identity, and balance, of these flavour-inducing compounds is still unknown for most cheeses. This can be attributed to the complexity of cheese composition and its analysis, of the processes occurring during consumption and the variations of human perception and expression of flavours. This review addresses some of the issues and methods being used to understand cheese flavour. It discusses factors which determine flavour release in the mouth, methods of preparation of extracts for analysis, sensory assessment of cheese flavour components and fractions, and of whole cheese, identification of odour active compounds and determination of their relative importance, temporal methods of analysis for flavour release and perception and methods of data analysis.     

The behavior of cathepsin D during milk processing and its contribution to bitterness in a model fresh cheese

The bovine endopeptidase cathepsin D was investigated regarding its temperature-dependent inactivation and ability to form bitter peptides within a spiked model fresh cheese. Cathepsin D was found to be more susceptible than other milk endogenous peptidases to temperature treatments in skim milk. Inactivation kinetics revealed decimal reduction times of 5.6 min to 10 s in a temperature range from 60 to 80°C. High temperature and ultra-high temperature (UHT) treatments from 90 to 140°C completely inactivated cathepsin D within 5 s. A residual cathepsin D activity of around 20% was detected under pasteurization conditions (72°C for 20 s). Therefore, investigations were done to estimate the effect of residual cathepsin D activity on taste in a model fresh cheese. The UHT-treated skim milk was spiked with cathepsin D and acidified with glucono-δ-lactone to produce a model fresh cheese. A trained bitter-sensitive panel was not able to distinguish cathepsin D–spiked model fresh cheeses from the control model fresh cheeses in a triangle test. Model fresh cheese samples were also analyzed for known bitter peptides derived from casein fractions using a HPLC–tandem mass spectrometry (MS) approach. In accordance with the sensory evaluation, the MS analyses revealed that the bitter peptides investigated within the cathepsin D–spiked model fresh cheese were not found or were below the limit of detection. Even though cathepsin D may be present during the fermentation of pasteurized milk, it does not seem to be responsible for bitter peptide formation from milk proteins on its own.     

Extruded linseeds,vitamin E and plant extracts in corn silage-based diets of dairy cows: Effects on sensory properties of raw milk and uncooked pressed cheese

Linseed supplementation of dairy cows' diet increases unsaturated fatty acid (FA) concentrations, which could lead to oxidised off-flavours in dairy products if antioxidant content is low. The FA profiles and sensory properties of milk and uncooked pressed cheese (Saint-Nectaire type) from four groups of six cows receiving a corn silage-based diet with no additional lipid (control), or supplemented with extruded linseeds (5% of additional fat-in-dry matter intake; EL), EL and vitamin E (7500 IU per cow per day of dl-α-tocopherol; ELE), or ELE and plant extracts rich in carotenoids and polyphenols (1% of dry matter intake) were compared. Feeding EL decreased milk and cheese 4- to 16-carbon saturated FA, and increased 18:1 cis-9, 18:3n-3 and total trans FA concentrations. Extruded linseeds did not induce oxidised off-flavours but led to a less firm and more meltable texture in cheese. Vitamin E supplementation increased α-tocopherol concentration, but did not affect sensory properties.     

Immunoglobulin A and its significance for mucosa immunity--a contribution to the understanding of microbial interactions

The secretory immunoglobulin A is still dominating with regard to knowledges and further investigations of the causes of mucosal immunity. Origin, formation, structure and mode of action of s-IgA are extensively explored. In the clinical range results of researches on symptoms and consequences of selective IgA deficiency are gaining importance, increasingly. Patients with IgA defect suffer up to 40 times more frequently from allergies and autoimmunopathies. In the induction of the immune response cellular components of mucosa immunity attaining the Lamina propria and the epithelium with the effector cells of Peyer's patches play a particular role.     

Distribution of microbial flora,intracellular enzymes and compositional indices throughout a 12 kg Cheddar cheese block during ripening

Two studies were carried out to investigate the distribution of microbial populations, intracellular enzymes and compositional indices within 12 kg Cheddar cheese blocks. In Study A, at day 21, starter and non-starter lactic acid bacteria viability in the top sub-sections were lower than middle sub-sections, but at day 98 this trend was reversed. Lactate dehydrogenase (LDH) activity in cheese juice was significantly higher in the middle sub-sections of the cheese block, but for Pep X activity there was no apparent trend. In Study B, at day 1 and day 14 of ripening, pH values were greater in the top sub-sections compared with the middle sub-sections. Over ripening, percentage salt-in-moisture was significantly greater in middle sub-sections of the cheese block. A paired comparison of pooled data indicated significantly higher LDH activity in the middle sub-sections, with Pep X activity significantly higher in the top sub-sections of the cheese block. No differences were found in the numbers of either live or dead cells in cheese juice expressed from top or middle sub-sections of cheese blocks.     

Use of principal component analysis to evaluate the physical properties of Mahon cheese

 The texture, colour and water activity of the seven existing types of Mahon cheese, manufactured under the Mahon cheese Appellation of Origin, were studied using eight physical variables. Four of the types are industrially manufactured [fresh (less than 10 days of ripening), half-ripened (2–5 months of ripening), ripened (5–10 months of ripening) and old-ripened (more than 10 months of ripening)] and three are traditionally manufactured (half-ripened, ripened and old-ripened). ANOVA, Tukey's multiple range test and principal component analysis (PCA) were used. Mahon cheeses from different ripening periods exhibited significant differences in water activity and yellowness values (P<0.001). PCA reduced the eight physical variables to two independent components, which accounted for 84.4% of the total variance. The first principal component (PC1) separated cheeses characterised by high water activity, high springiness values and slight yellowness from those with high hardness and puncture force; PC1 therefore distinguished among samples from different ripening periods. Cohesiveness, chewiness and gumminess were highly correlated in the second principal component, which could be interpreted as an index of cohesiveness. Received: 8 February 1999     

Selected lactic acid bacteria as a hurdle to the microbial spoilage of cheese: Application on a traditional raw ewes' milk cheese

To evaluate the efficacy of lactic acid bacteria (LAB) to improve the hygienic safety of a traditional raw milk cheese, the raw ewes' milk protected denomination of origin (PDO) Pecorino Siciliano cheese was used as a model system. Different Pecorino Siciliano curds and cheeses were used as sources of autochthonous LAB subsequently used as starter and non-starter LAB. These were screened for their acidification capacity and autolysis. Starter LAB showing the best performance were genotypically differentiated and identified: two strains of Lactococcus lactis subsp. lactis were selected. From the non-starter LAB, Enterococcus faecalis, Lactococcus garvieae and Streptococcus macedonicus strains were selected. The five cultures were used in individual or dual inocula to produce experimental cheeses in a dairy factory for which production was characterised by high numbers of undesirable bacteria. At 5-month of ripening, the experimental cheeses produced with LAB were characterised by undetectable levels of enterobacteria and pseudomonads and the typical sensory attributes.     

Time-course estimation of evaporated water fluxes together with mean surface water activity in uncooked pressed cheeses during ripening using a purpose-built micro-bioreactor

During cheese ripening, surface water activity controls microbial flora activity and aroma development, essentially shaping the organoleptic properties of the cheese. An experimental micro-bioreactor was set up using air-product water balance to non-destructively estimate the time-course of mean water activity at the cheese surface under well-controlled airflow ripening conditions. Six trials were performed using this experimental device on uncooked pressed cheeses ripened until 30 days. These trials highlighted (1) the effects of a low air velocity and high relative humidity on the water exchanges occurring at the cheese surface, demonstrating strong surface sensitivity to external ripening air conditions, and (2) a close interaction between surface microbial flora development and the water transfers occurring both from cheese core to surface and at the surface itself, although further micro-bioreactor ripening trials are needed to fully clarify this interaction.     

Application of Central Composite Design to evaluate the antilisterial activity of hydro-alcohol berry extract of Myrtus communis L.

The antimicrobial activity of the hydro-alcohol extract of Myrtus communis L. (ME) berries was investigated against six Listeria monocytogenes strains (2 type strains and 4 isolates). Sub-lethal ME concentrations reduced L. monocytogenes counts by at least 2 log cycles. A Central Composite Design was used to investigate the combined effects of sub-lethal concentrations of ME (0.039–0.195 mL/100 mL), NaCl (0–2.0 g/100 mL) and pH (5.0–7.0) on strains growth. ME affected growth parameters, generally extending lag phase length and reducing maximum growth, sometimes with interactive effects with pH. The highest ME concentrations (0.117–0.195 mL/100 mL) combined with the lowest pH values (5.0–6.0) strongly reduced or even inhibited strains growth.     

Characterization of the microbial flora in disinfecting footbaths with hypochlorite

Change or disinfection of footwear are measures to prevent cross contamination between areas with low and high hygienic levels in the food industry. The efficacy of disinfecting footwear is not well documented. Samples of used disinfectant and from swabbing of corners after draining were taken from disinfecting footbaths containing chlorine in four Norwegian cheese factories. Bacteria were present in 9 of 12 footbaths and more positive samples were found from swab samples than from used disinfectant. The microbial flora in footbaths varied between the dairies. In two dairies, the flora was dominated by Pseudomonas spp. and Acinetobacter spp., respectively. In the third dairy, both Bacillus spp. and Staphylococcus spp. were present and in the fourth dairy, the flora was diverse (Acinetobacter sp., Enterococcus faecalis, Klebsiella pneumoniae, and Bacillus sp.). The strains were not resistant to the recommended user concentration of chlorine in bactericidal suspension or surface tests. The degree of attachment to plastic varied between strains and species and bacteria attached to surfaces were in general more resistant than suspended bacteria. The results of the survey indicated that disinfecting footbaths containing chlorine may act as contamination sources in food factories and should not be used without regular hygienic monitoring.     

Effects of treatment with nisin on the microbial flora and sensory properties of a Greek soft acid-curd cheese stored aerobically at 4 °C

The present study evaluated the use of nisin as an antimicrobial treatment for shelf-life extension of Galotyri, a Greek soft acid-curd cheese, stored aerobically under refrigeration for a period of 42 days. Three different treatments were tested: N0, control sample with no nisin added; N1, 50 IU g⁻¹ nisin; and N2, 150 IU g⁻¹ nisin, the latter two treatments added post-production to the Galotyri cheese. Of all microorganisms enumerated, lactobacilli, lactococci and yeasts were the groups that prevailed in cheese samples, irrespective of antimicrobial treatment. Based primarily on sensory evaluation (appearance and taste) and a microbiological acceptability limit for yeasts (5 log cfu g⁻¹), the use of nisin treatments extended the shelf-life of fresh Galotyri cheese stored at 4 °C by ca. 7 days (N1) and 21 days (N2) with cheese maintaining good sensory characteristics.     

Molecular approaches to analysing the microbial composition of raw milk and raw milk cheese

The availability and application of culture-independent tools that enable a detailed investigation of the microbiota and microbial biodiversity of food systems has had a major impact on food microbiology. This review focuses on the application of DNA-based technologies, such as denaturing gradient gel electrophoresis (DGGE), temporal temperature gradient gel electrophoresis (TTGE), single stranded conformation polymorphisms (SSCP), the polymerase chain reaction (PCR) and others, to investigate the diversity, dynamics and identity of microbes in dairy products from raw milk. Here, we will highlight the benefits associated with culture-independent methods which include enhanced sensitivity, rapidity and the detection of microorganisms not previously associated with such products.     

Experimental designs for studying the influence of the raw milk flora on cheese characteristics: a review

To study the influence of native milk flora on the flavour and texture of raw milk cheese it is necessary to produce milk without its native flora. Three procedures to eliminate native flora from milk can be discernedfrom the literature: aseptically drawn milk, heat treatment of whole milk and physical treatment of skim milk with subsequent addition of the heat treated cream. The advantages and disadvantages of these three approaches are discussed.     

Development of a risk assessment model to predict the occurrence of late blowing defect in Gouda cheese and evaluate potential intervention strategies

Late blowing defect (LBD) is an important spoilage issue in semi-hard cheese, with the outgrowth of Clostridium tyrobutyricum spores during cheese aging considered to be the primary cause. Although previous studies have explored the microbial and physicochemical factors influencing the defect, a risk assessment tool that allows for improved and rational management of LBD is lacking. The purpose of this study was to develop a predictive model to estimate the probability of LBD in Gouda cheese and evaluate different intervention strategies. The spore concentration distribution of butyric acid bacteria (BAB) in bulk tank milk was obtained from 8 dairy farms over 12 mo. The concentration of C. tyrobutyricum from raw milk to the end of aging was simulated based on Gouda brined for 2 d in saturated brine at 8°C and aged at 13°C. Predicted C. tyrobutyricum concentrations during aging and estimated concentration thresholds in cheese at onset of LBD were used to predict product loss due to LBD during a simulated 1-yr production. With the estimated concentration thresholds in cheese ranging from 4.36 to 4.46 log most probable number (MPN)/kg of cheese, the model predicted that 9.2% (±1.7%) of Gouda cheese showed LBD by d 60; cheeses predicted to show LBD at d 60 showed a mean pH of 5.39 and were produced with raw milk with a mean BAB spore count of 143 MPN/L. By d 90, 36.1% (±3.4%) of cheeses were predicted to show LBD, indicating that LBD typically manifests between d 60 and 90, which is consistent with observations from the literature and the cheese industry. Sensitivity analysis indicated that C. tyrobutyricum maximum growth rate as well as concentration threshold in cheese at onset of LBD are the most important variables, identifying key data needs for development of more accurate models. The implementation of microfiltration or bactofugation of raw milk (assumed to show 98% efficiency of spore removal) in our model prevented occurrence of LBD during the first 60 d of aging. Overall, our findings provide a framework for predicting the occurrence of LBD in Gouda as well as other cheeses and illustrate the value of developing digital tools for managing dairy product quality.     

晴隆酸菜发酵过程中微生物菌群多样性动态分析

利用高通量测序的方法,对晴隆酸菜四个发酵时期（T1、T2、T3、T4）微生物多样性、群落结构进行动态分析。结果表明,参与发酵的细菌有24门262属,真菌有7门131属;微生物群落多样性随着发酵的进行呈下降趋势,发酵末期的微生物群落多样性最低、微生物种属数量最少;发酵前中期微生物群落结构不稳定相似性较低,而发酵末期微生物群落结构稳定相似性较高;四个发酵时期的酸菜中存在着优势菌属的消长变化,总发酵过程中微生物优势菌属为5个细菌属：魏斯氏菌（Weissella）、乳酸杆菌（Lactobacillus）、明串珠菌（Leuconostoc）、鞘脂单胞菌（Sphingomonas）、根瘤菌（Rhizobium）,9个真菌属：Sampaiozyma、枝孢菌（Cladosporium）、掷孢酵母（Sporobolomyces）、Vishniacozyma、附球菌（Epicoccum）、Plectosphaerella、Filobasidium、红酵母（Rhodotorula）、枝顶孢（Acremonium）。     

河西走廊啤酒大麦附生微生物区系研究

以甘肃河西走廊具有代表性的三个产地的啤酒大麦为研究对象,通过对大麦表面细菌、酵母菌和霉菌的筛查、分离纯化与鉴定,研究大麦附生微生物区系组成及其优势种群。结果显示,河西走廊啤酒大麦附生细菌数量最多,菌落总数在106～107cfu/g之间,酵母总数在104～105cfu/g之间,霉菌菌落总数因产地变化较大,数量在103～105cfu/g。通过对大麦附生微生物区系的鉴定,分离到9株优势细菌,9株优势酵母和11株霉菌,表明大麦附生菌群具有一定的多样性,并因产地不同,微生物组成呈现出一定差异。部分样品大麦中检出了产气肠杆菌(Enterobacter aerogenes)和产色葡萄球菌(Staphylococcus chromogenes)等细菌,以及一些产真菌毒素霉菌菌株,存在一定生物安全隐患。建议在制麦过程中,尤其是浸麦工段应加强洗麦,以减少原料大麦携带的有害病菌,并降低产生真菌毒素的风险。