Occurrence of biogenic amine-forming lactic acid bacteria in wine and cider

A collection of 810 lactic acid bacteria (LAB) strains isolated from wine and cider was screened for potential biogenic amine (BA) producers by combining molecular and phenotypic approaches. A newly developed multiplex PCR method allowed for the simultaneous detection of four genes involved in the production of histamine (histidine decarboxylase, hdc), tyramine (tyrosine decarboxylase, tyrdc) and putrescine (via either ornithine decarboxylase, odc, or agmatine deiminase, agdi) while TLC and HPLC analysis allowed for BA-production determination. One hundred and fifty-eight LAB strains were monitored by the molecular/phenotypic double approach and revealed a good correlation between genotypic and phenotypic data. Eighteen per cent of the tested strains were positive for at least one BA target gene with up to three detected simultaneously, in particular amongst Lactobacillus brevis and Lactobacillus hilgardii isolates for the tyrdc and agdi genes. The most frequent gene corresponded to the agdi gene detected in 112 strains (14% of all LAB strains) of 10 different LAB species. The tyrdc gene was detected in 67 strains represented by 7 different LAB species (8% overall), especially those isolated from wine. Lower levels of hdc⁺ (2% of strains) and especially odc⁺ (0.5% of strains) strains were observed. Interestingly, species that have never been described to carry BA-producing pathway genes were identified in this study. Furthermore, only one cadaverine-producer was detected and corresponded to Lactobacillus 30a, a collection strain not found in fermented beverages, although cadaverine is commonly detected in wines.     

Prevalent lactic acid bacteria in cider cellars and efficiency of Oenococcus oeni strains

Malolactic fermentation (MLF) is an important step in cider production in order to allowing for improvement of microbiological stability and organoleptic characteristics of cider. Induction of this fermentation by using starter cultures enables a better control over this bioprocess, but although it is a common practice in winemaking, starters specifically focussed for cider MLF are not yet commercially available. Proper starter cultures need to present the ability to degrade l-malic acid conferring pleasing sensory characteristics while avoiding toxicological risks. In this work, lactic acid bacteria (LAB) were first isolated from MLF industrial cider samples, obtained in a cellar in the main cider-producing region of Spain, Asturias. Isolates, identified by molecular tools, belonged to the Lactobacillus brevis and Oenococcus oeni species. After a phylogenetic analysis, representative strains of both identified species were evaluated in order to determine their fermentation capacity, showing O. oeni the best behaviour in this cider fermentation, as previously demonstrated for wine in the literature. Consequently, and with the aim to test the influence at strain level, selection of O. oeni isolates as starters for cider fermentation has been undergone. In order to check the influence of geography over biodiversity, O. oeni strains from six different industrial cellars representing the distinct producing areas in the region (located in a ratio of 30 km) were analyzed by using a specific RAPD method. In this way, isolates were typed in five distinct groups, mainly corresponding to each producing area. All strains isolated from the same cellar showed the same RAPD profile revealing the significance of geographical origin in the indigenous cider LAB. Molecular tools were applied to reject those isolates exhibiting presence of genes related to organoleptic spoilage (exopolysaccharides and acrolein production) or food safety (biogenic amine production), as key selection criteria. Representative strains of each of the five O. oeni RAPD groups were tested as pure cultures to evaluate their technological utility for cider production. Experimental data of malic acid degradation and cell concentration obtained were fitted to previously selected kinetic models aimed to optimization and prediction of bioprocess performance. Four strains revealed as suitable potential starter cultures for conducting MLF in cider production.     

NITROSAMINES IN ALCOHOLIC BEVERAGES

A limited survey was carried out to determine the nitrosamine content of several varieties of alcoholic beverages (beer and ale, whiskey, wine, cider, etc.) sold in Canada. Of 22 samples of different beers and ales analyzed all but one contained traces of dimethylnitrosamine; the overall mean level found was 1.5 ppb. Only one Canadian rye and one Scotch whiskey out of a total of 13 samples contained traces of either dimethylnitrosamine or diethylnitrosamine. All the 8 wines and 7 cider samples were negative.     

Characterization of Apples and Apple Cider Produced by a Guelph Area Orchard

Thermal stability of food-borne pathogens in apple cider is influenced by the composition of the product. As a preliminary step to determining the effect of pasteurization of apple cider on survival of E. coli O157:H7, a study was carried out to characterize apples and unpasteurized apple cider produced by a guelph area orchard. Samples of commercial unpasteurized cider and the constituent apples were collected over 13 wk from August to November 1998, and unpasteurized laboratory cider was made from the individual apple varieties. pH, titratable acidity (TA), turbidity, total microbial counts, total solids and °brix for filtered and unfiltered samples were measured. The maximum, minimum, and average values for all unpasteurized commercial cider samples were found to be: pH, 3.71, 3.17, and 3.43; TA, 93.47, 49.46, and 69.95 mL of 0.1 N NaOH/100 mL; total solids, 13.21, 10.93, and 11.90%; °brix, 13.01, 11.17, and 12.02; turbidity, 238.1, 145.1, and 204.9 NTU; and total plate count, 4.91, 2.61, 3.75 log cfu·mL⁻¹. There were no significnat differences (P>0.05) between filtered and unfiltered samples. In addition, in commercial unpasteurized cider, there were no significant differences (P>0.05) with respect to any of the factors with time of processing. The composition of the unpasteurized laboratory cider made form individual apple varieties was dependent on the variety, but was generally within the ranges from published literature values. Mclntosh apples showed a significant (P≤0.05) decrease in TA with time of harvest. The results suggest that it is necessary to take the composition of commercial apple cider into account when developing thermal inactivation models for food-borne pathogens.     

Prevalence of Escherichia coli in apple cider manufactured in Connecticut.

Cider samples obtained from 11 cider mills operating in Connecticut during the 1997 to 1998 production season were tested for the presence of Escherichia coli. Cider production began in mid August and continued through March, with peak production in September and October. Of 314 cider samples tested, 11 (4%) were found to contain E. coli. Of the 11 mills, 6 (55%) tested positive for E. coli in the cider at least once during the production year. E. coli was first observed in cider samples produced in mid to late October and was not detected in samples made after January. A trend was observed for cider to decrease in acidity and increase in Brix (soluble sugars) throughout the production season. No correlation between pH and soluble sugars of cider and the presence of E. coli was detected. Eight mills used both dropped apples and tree-picked apples, whereas three mills used tree-picked apples only. The use of dropped apples in cider production began 5 weeks before the first detection of E. coli in cider. E. coli was isolated from cider samples produced using dropped apples and from samples produced using only tree-picked apples. No direct correlation between the use of dropped apples or tree-picked apples and the presence of E. coli in the cider was observed. An association between the time of apple harvest and the appearance of E. coli in cider was noted. For mills providing adequate records, all contaminated cider was produced from apples harvested between mid October and mid November.     

Characterization of Apples and Apple Cider Produced by a Guelph Area Orchard

Thermal stability of food-borne pathogens in apple cider is influenced by the composition of the product. As a preliminary step to determine the effect of pasteurization of apple cider on the survival of Escherichia coli O157:H7, a study was carried out to characterize apples and unpasteurized apple cider produced by a Guelph area orchard. Samples of commercial unpasteurized cider and the constituent apples were collected over 13 wk from August to November 1998, and unpasteurized laboratory cider was made from the individual apple varieties. pH, titratable acidity, turbidity, total microbial counts, total solids and °Brix for filtered and unfiltered samples were measured. The maximum, minimum, and average values for all unpasteurized commercial cider samples were found as follows: pH, 3.71, 3.17, and 3.43; titratable acidity, 93.47, 49.46, and 69.95 mL of 0.1 N NaOH/100 mL; total solids, 13.21, 10.93, and 11.90%; °Brix, 13.01, 11.17, and 12.02; turbidity, 238.1, 145.1, and 204.9 nephelometric turbidity units; and total plate count, 4.91, 2.61, 3.75 log cfu/mL. There were no significant differences (P>0.05) between filtered and unfiltered samples. In addition, in commercial unpasteurized cider, there were no significant differences (P>0.05) with respect to any of the factors with the time of processing. The composition of the unpasteurized laboratory cider made from individual apple varieties was dependent on the variety, but was generally within the ranges from the published literature values. McIntosh apples showed a significant (P\le0.05) decrease in titratable acidity with time of harvest. The results suggest that it is necessary to take the composition of commercial apple cider into account when developing thermal inactivation models for food-borne pathogens.     

Development of a screening method for biogenic amine producing Bacillus spp

Most of the currently available screening methods for biogenic amine (BA)-producing microorganisms are based on differential media containing a pH indicator; BA production is detected as an increase in pH. However, such acid-alkaline detection methods cannot be adapted directly for use with alkaline bacteria such as Bacillus species. Here we describe an easy, rapid and reliable method for detecting BA-producing Bacillus strains using plate and broth methods. Positive selection using the plate method was achieved within 24 h. The broth method proved more precise and enabled quantitation of the BA production, and correlated well with the plate method. False-negatives were diminished using the broth method, and false-positives could be screened out by an additional test for glycerol metabolism. Of the 12 strains examined using new method, five (B. licheniformis cy2, B. polyfermenticus CJ9, B. licheniformis KCTC1918, B. subtilis KCTC1028, and B. subtilis KCTC1022) were BA-positive with the broth method, among which KCTC1022 was determined to be a false-positive using the glycerol metabolism examination. The suitability and accuracy of the broth method was confirmed by high-performance liquid chromatography detection of BA formation.     

Malolactic Fermentation as a Technique for the Deacidification of Hard Apple Cider

ABSTRACT:  Malolactic fermentation (MLF), the conversion of malate to lactate, is an important process leading to the deacidification of hard apple cider. MLF is dependent on the levels of inhibitory factors such as sulfur dioxide and ethanol. To assess the effect of these 2 factors on MLF, hard apple cider was produced from pasteurized, unfiltered apple cider ( Malus domestica  cvs Red Delicious, Golden Delicious, Braeburn, and Fuji). Apple cider was treated with 2 levels of sulfur dioxide (50 and 80 ppm) and then fermented using  Saccharomyces cerevisiae  montrachet. After the primary fermentation, 1 set of the samples remained unadjusted and 100% ethyl alcohol was used to adjust other sets of samples to 7%, 9%, or 11% (v/v) ethanol. Following the ethanol adjustment,  Oenococcus oeni  MCW was used to initiate the MLF in half of the samples. Cider parameters monitored throughout the fermentations included organic acid content, titratable acidity, pH, ethanol production, and sugar content. Since samples containing either sulfur dioxide level had similar sugar utilization rates and ethanol production it was concluded that sulfur dioxide had no effect on the primary fermentation. Sulfur dioxide content was shown to have an impact on MLF. There was no difference in the rate of malic acid consumption, but lactic acid production was faster in the 50-ppm sulfur dioxide samples. MLF was not inhibited by ethanol content.     

Biogenic amines in natural ciders

Biogenic amines play an important physiological role in mammals, and high amounts of some exogenous amines in human diet may contribute to a wide variety of toxic effects. These amines are commonly found in many foodstuffs, particularly in fermented products such as cheese, meat products, beer, wine, and ciders. Here, the level of biogenic amines in some natural ciders was examined. Twenty-four samples of cider purchased from commercial sources were analyzed by reverse-phase high-performance liquid chromatography and fluorescence detection after precolumn derivatization with o-phthaldialdehyde. Amine levels were variable, ranging from not detected to 23 mg/liter. The average level of total biogenic amines in ciders was 5.94 +/- 8.42 mg/liter. Putrescine, histamine, and tyramine were the prevailing amines being present in 50.0, 37.5, and 33.3% of the ciders studied; very small amounts of ethylamine and phenylethylamine were observed in only one sample. Other cider parameters were analyzed to determine whether they affect the biogenic amine content in ciders, and the results were evaluated by applying cluster analysis and principal component analysis. Ciders that showed lower glycerol contents and higher amounts of 1,3-propanediol had much higher levels of histamine, tyramine, and putrescine, suggesting a high activity of lactic acid bacteria during cider making and thus the need for effective control of lactic acid bacteria.     

模糊综合评判结合响应面法优化苹果酒发酵工艺

采用模糊综合评判结合响应面法优化苹果酒发酵工艺参数,并用顶空固相微萃取气质联用（HS-SPME/GC-MS）技术分析其香气成分。以红富士苹果为原料,结合单因素试验结果,选取了酵母接种量、初始糖度、初始pH值三个主要工艺参数,采用中心组合试验设计,以苹果酒品质的模糊评判结果为目标,构建主因素突出型综合评判模型,进行响应面法分析。得到苹果酒最佳发酵工艺条件为：初始糖度19 g/100 mL,初始pH值3.5,接种量10%,发酵温度22 ℃。此条件下的苹果酒颜色金黄,酒体澄清有光泽,口感丰满协调、风格良好。苹果酒中共检测到33种香气成分,结合气味活性值（OAV）法鉴定出10种关键香气组分,这些香气物质构成了苹果酒的独特的风味。     

Development of an accurate and sensitive gas chromatographic method for the determination of acrolein content in Calvados and cider

Acrolein can be at the origin of an important organoleptic defect in beverages made from apples. Its content was investigated in freshly distilled Calvados and cider. The specificity of 3-methylbenzothiazolone hydrazine (MBTH) towards carbonyl compounds was used in order to derive acrolein in azines. Azines were separated and detected by gas chromatography coupled with a nitrogen-phosphorus Detector. Results showed that acrolein concentrations could be quickly determined in either Calvados or cider. Accuracy of quantification was better than 6% for concentrations about 1 mg/l in freshly distilled Calvados and 10% for concentrations about 10 μg/l in ciders. Acrolein content was found between 0.7 and 5.2 mg/l in samples of freshly distilled Calvados whereas it was between 7 and 15 μg/l in samples of cider. This method of quantification was applied to study disappearance kinetics of acrolein in cider. Acrolein content was found to decrease rapidly during the first hours and to change very slowly after a few days. Its behaviour during a distillation was also investigated showing that in spite of its high volatility it could also be found in the last fractions of distillation.     

Microbial levels in Michigan apple cider and their association with manufacturing practices

In recent decades, apple cider has been implicated in a series of outbreaks of foodborne illness. The objective of this study was to determine the presence and concentrations of pathogenic and indicator microorganisms in apple cider processed in Michigan and to evaluate the impact of thermal pasteurization, UV light radiation, and implementation of hazard analysis critical control point (HACCP) plans on these microbes. Cider samples were obtained from Michigan mills between 1997 and 2004 and analyzed for Escherichia coli O157:H7, Salmonella, generic E. coli, total coliforms, and aerobic bacteria. Neither E. coli O157:H7 nor Salmonella were detected in any tested cider samples, suggesting a very low frequency of pathogens in Michigan apple cider. The persistent and relatively high frequency of generic E. coli observed in samples obtained in all years indicates a continued risk of pathogen contamination in Michigan apple cider, especially when it is untreated. The use of thermal pasteurization or UV light radiation and reported implementation of HACCP plans were associated with lower frequency and counts of generic E. coli, total coliforms, and aerobic microorganisms. However, the relatively high counts of indicator organisms in some cider samples that were claimed to be treated according to these pathogen reduction measures indicates that some processors had inadequate practices, facilities, or equipment for pathogen reduction or did not consistently or adequately apply practices or pathogen-reduction equipment in an effective manner.     

Isolation and characterization of Escherichia coli recovered from Maryland apple cider and the cider production environment

Contaminated apple cider has been implicated in several Escherichia coli O157:H7 outbreaks. In an attempt to investigate sources and modes of entry of E. coli into apple cider, samples of fresh apple, pomace, and cider and equipment and mill floor swabs were analyzed for standard plate counts (SPC), total coliforms (TC), fecal coliforms (FC), and E. coli. E. coli was isolated from 14 (33%) of 42 samples of bottled fresh cider, from food equipment in 6 (67%) of 9 mills, and from apples, pomace, or cider in 7 (78%) of 9 mills. Seventy-five E. coli isolates were further characterized for Shiga toxin-producing E. coli (STEC)-associated virulence factors, antimicrobial susceptibility, and pulsed-field gel electrophoresis (PFGE) type. No E. coli O157:H7 or other STEC was identified. Serotyping and PFGE revealed 64 distinct profiles, suggesting that recovered E. coli arose from multiple independent sources. However, on one occasion, E. coli isolated from the source apple sample was closely related to the E. coli identified in the finished cider sample. E. coli isolates were further tested for antimicrobial susceptibility to 17 antimicrobial agents of human and veterinary importance. Fourteen (19%) of the 75 isolates were resistant to at least one of the antimicrobial agents tested, and 9 (12%) were resistant to at least two of these agents. Of the resistant isolates recovered, 64% were resistant to tetracycline and 57% were resistant to streptomycin. Overall, the level of E. coli contamination in source apple samples did not differ significantly from those in samples of pomace, cider at the press, and cider entering the bottling tank; therefore, source apples cannot be dismissed as a potential contributor of E. coli to the cider-making process.     

Polyphenol profiles of apple juices

Focusing on 17 constituents, the polyphenol profiles of juices freshly made from various dessert (n = 4) and cider apple cultivars (n = 7) as well as commercially available apple juices (n = 24) were investigated using high-performance liquid chromatography-photodiode array detection (HPLC-DAD) and (HPLC)-electrospray ionization-tandem mass spectrometry (ESI(neg)-MS/MS) analyses. Significant differences in the total polyphenol content as well as the profiles of the apple cultivars under study were observed. For dessert apples the total polyphenol content ranged from 154 to 178 mg/L, whereas for 'old' German cider apple cultivars 261-970 mg/L were determined. Boskoop showed the highest (970 mg/L) and Granny Smith the lowest (154 mg/L) polyphenol content of the freshly prepared samples under study. Hydroxycinnamic acids, with chlorogenic acid as dominating constituent, ranged from 57 to 68 mg/L as well as from 134-593 mg/L in juices made from dessert apples and that from cider apples, respectively. Dessert apple juices showed lower contents of dihydrochalcones (10-35 mg/L) and flavan-3-ols (50-95 mg/L) compared to that of cider apples (34-171 mg/L and 70-393 mg/L, respectively). Quercetin and its derivatives were found from 0.4-4 mg/L and 0.4-27 mg/L in juices made from dessert apples and that of cider apples, respectively. Compared with freshly made juices, lower contents of polyphenols were determined in the commercial samples under study. Amounts ranging from 110-459 mg/L, dominated by chlorogenic acid with concentrations from 53-217 mg/L, were determined. Information about cultivar-typical apple polyphenol content and profile is important for bioactivity studies and, consequently, essential for the development of consumer-relevant products with particular nutritional functionalities.     

Changes in headspace volatile attributes of apple cider resulting from thermal processing and storage

One attribute that frequently reflects a product's organoleptic impact is its aroma. In research presented here, a Fox-3000 Electronic Nose was used to determine and compare the aromatic profiles of samples of apple cider subjected to various thermal treatments. Initial results have indicated that exposure to temperatures of up to 90 °C for approximately 28 s acts to stabilize the aromatic properties of apple cider over a 7-day period. In contrast, there are significant changes in the aromatic profiles of fresh apple cider having no such thermal treatment over the same time period. A comparison of samples thermally treated at 60, 70, and 80 °C to non-processed cider showed minimal statistical difference on the basis of similarity indices obtained after 24-h of storage of the samples at 4 °C. Treatment at 90 °C showed significant differences from the unprocessed samples in the same test sequence. After storage for 7-days, samples that were thermally treated exhibited minimal differences from each other, however, were quite different from the non-processed sample on the basis of similarity index analysis. GC analysis was done to confirm potential differences between the samples measured using the electronic nose. Sensory testing is required to determine whether these differences affect the overall quality perception by consumers.     

Biogenic amine formation and bacterial contribution in fish,squid and shellfish

Forty-one species of fish, squid and shellfish were analyzed for biogenic amine (BA) contents. Most of the fish samples showed lower BA contents, whereas some samples showed higher contents than the allowable levels. Shellfish and squid samples had negligible BA levels. Four fish species containing high BA levels were analyzed for changes in histamine contents during storage. In the most samples, the histamine contents remarkably increased up to 36.6–2123.9 mg/kg after 24 h of storage at 25 °C, while the contents began to gradually increase after 2–3 days of storage at 4–10 °C. The dominant microbial group was enterobacteria throughout the storage period. Meanwhile, out of total 119 strains isolated from different fish species showing high BA levels, 23 strains identified as Enterobacter aerogenes produced large amounts of histamine, putrescine and cadaverine, and 33 strains identified as two different Enterobacter spp. produced less histamine but large amounts of putrescine and cadaverine.     

Apple Aminoacid Profile and Yeast Strains in the Formation of Fusel Alcohols and Esters in Cider Production

The amino acid profile in dessert apple must and its effect on the synthesis of fusel alcohols and esters in cider were established by instrumental analysis. The amino acid profile was performed in nine apple musts. Two apple musts with high (>150 mg/L) and low (<75 mg/L) nitrogen content, and four enological yeast strains, were used in cider fermentation. The aspartic acid, asparagine and glutamic acid amino acids were the majority in all the apple juices, representing 57.10% to 81.95%. These three amino acids provided a high consumption (>90%) during fermentation in all the ciders. Principal component analysis (PCA) explained 81.42% of data variability and the separation of three groups for the analyzed samples was verified. The ciders manufactured with low nitrogen content showed sluggish fermentation and around 50% less content of volatile compounds (independent of the yeast strain used), which were mainly 3‐methyl‐1‐butanol (isoamyl alcohol) and esters. However, in the presence of amino acids (asparagine, aspartic acid, glutamic acid and alanine) there was a greater differentiation between the yeasts in the production of fusel alcohols and ethyl esters. High contents of these aminoacids in dessert apple musts are essential for the production of fusel alcohols and most of esters by aromatic yeasts during cider fermentation.     

Inactivation of different strains of Escherichia coli O157:H7 in various apple ciders treated with dimethyl dicarbonate (DMDC) and sulfur dioxide (SO2) as an alternative method

Escherichia coli has been identified as the causative agent in numerous foodborne illness outbreaks associated with the consumption of fresh apple cider. Apple cider has a pH which is normally below 4.0 and would not be considered a medium capable of supporting the growth of foodborne pathogens. The association of unpasteurized apple cider with foodborne illness due to E. coli O157:H7 has however, led to increased interest in potential alternative methods to produce pathogen free cider. Apple cider was prepared from eight different apple cultivars, inoculated with approximately 10⁶–10⁷ CFU of three strains of E. coli O157:H7 per ml (933, ATCC 43889, and ATCC 43895) and tested to determine the effectiveness of sulfur dioxide (SO₂) and dimethyl dicarbonate (DMDC). Bacterial populations for treated and untreated samples were then enumerated by using non-selective media. Eight different ciders were treated with DMDC (125 and 250 ppm) and SO₂ (25, 50, 75, 100 ppm). Greater than a 5-log reduction was achieved at room temperature with 250 ppm of DMDC and 50 ppm of SO₂ after the incubation time of 6 h and 24 h, respectively. Addition of DMDC and/or SO₂ may offer an inexpensive alternative to thermal pasteurization for the production of safe apple cider for small apple cider producers.     

Potential sources of microbial contamination in unpasteurized apple cider

A study was conducted to identify possible sources of microbial contamination and to assess the effect of good cleaning and sanitation practices on the microbial quality and safety of unpasteurized apple cider. Raw unwashed apples, washed apples, cleaning water, fresh cider, and finished cider samples were collected from five Ontario producers over 4 months and microbiologically tested. Total coliforms were found in 31, 71 and 38% of the unwashed apple, water, and washed apple samples, respectively. Escherichia coli was found in 40% of the water samples from one producer alone. The washing step was identified as a potential source of contamination, possibly due to water in the dump tanks seldom being refreshed, and because scrubbers, spray nozzles, and conveyors were not properly cleaned and sanitized. Higher total coliform counts (P < 0.0001) and prevalence (P < 0.0001) in fresh cider compared with those in unwashed apples and washed apples indicated considerable microbial buildup along the process, possibly explained by the lack of appropriate equipment sanitation procedures. Results showed that producers who had better sanitary practices in place had lower (P < 0.001) total coliform prevalence than the rest of the producers. Overall results show that good sanitation procedures are associated with improved microbial quality of fresh cider in terms of total coliforms and that operators who pasteurize and/or UV treat their product should still be required to have a sound good manufacturing practices program in place to prevent recontamination. Cryptosporidium parvum, an important pathogen for this industry, was found in different sample types, including washed apples, water, and fresh and finished cider.