Application of biosensors in early detection of contamination with lactic acid bacteria during apple juice and concentrate production

The aim of Collective Research Project QUALI-JUICE (COLL-CT-2005, Co Nr 012461) was to introduce the biosensors in control of microbiologically pure apple juice production. Three commercial lactate biosensors were used and compared with enzyme kits assays. Parallel the microbiological analysis of the production process at one juice producing enterprise was done. The results of lactic acid assay with biosensors were in good correlation with those obtained by enzyme kits. The main benefit of biosensor use was shortening of measurement time as compared with assay by enzyme kit and possibility to measure at line. The concentration of l-lactic acid in apple pulp could be correlated with the number of lactic acid bacteria. Pasteurization process was eliminating lactic acid bacteria and the concentration of lactic acid was at this stage not exceeding 0.1 g L⁻¹. The final product (apple concentrate) contained in some cases very high amounts of lactic acid indicating secondary microbiological contamination after pasteurization step. Parallel microbiological analysis of production process and lactate assay indicated that the critical point during production was prolonged vacuum filtration after pasteurization.     

The effects of processing technology on the patulin content of juice during commercial apple juice concentrate production

 The effects of different treatments on the patulin content of apple juice during the production of industrial apple juice concentrate were investigated. Conventional clarification using a rotary vacuum precoat filter was found to be more effective than using ultrafiltration for the removal of patulin from apple juice. The average losses of patulin were 39% and 25% for conventional clarification and filtration, and ultrafiltration, respectively. Washing and handling appeared to be the most critical steps in removing patulin from apples since up to 54% could be removed using high-pressure water spraying. Received: 22 January 1998 / Revised version: 21 April 1998     

The effects of processing technology on the patulin content of juice during commercial apple juice concentrate production

 The effects of different treatments on the patulin content of apple juice during the production of industrial apple juice concentrate were investigated. Conventional clarification using a rotary vacuum precoat filter was found to be more effective than using ultrafiltration for the removal of patulin from apple juice. The average losses of patulin were 39% and 25% for conventional clarification and filtration, and ultrafiltration, respectively. Washing and handling appeared to be the most critical steps in removing patulin from apples since up to 54% could be removed using high-pressure water spraying. Received: 22 January 1998 / Revised version: 21 April 1998     

影响浓缩苹果汁中富马酸因素的研究

采用高效液相色谱法测定不同质量的苹果原料中富马酸含量，获得了不同加工工艺条件下富马酸变化趋势，提出了控制富马酸超标的方法。     

Standardisation of the detection of invert sugar addition to apple juice by capillary gas chromatography

 Capillary gas chromatography (CGC) can be used to check the authenticity of various fruit juices by analysing their oligosaccharide fingerprint profile. Thus, it enables the analyst to detect, for instance, the dilution of apple juice concentrates with cheaper sweeteners such as invert sugar syrup. In order to detect this, two marker peaks (IS1 and IS2) that are present in most invert sugar syrups are monitored in the CGC chromatogram of apple juice. In freshly pressed apple juice, these peaks are normally not found. As a result of apple juice processing and, in particular, the thermal treatment required to concentrate and pasteurise the juice, these peaks may however be present in authentic apple juice concentrate and juice made from it. It is nevertheless possible to distinguish between juice showing this "heat effect" and that to which invert sugar has been added since the IS2/IS1 peak ratio is very different in heated apple juice and in invert sugar syrups. Measuring this ratio and setting a maximum IS2/IS1 cut-off limit enables the analyst to assess whether the sample meets the set norms or has been adulterated. In the past, many laboratories experienced difficulties when comparing the results of this analysis and, in particular, IS2/IS1 peak ratios. We present evidence that this unsatisfactory situation is mainly due to the chromatographic behaviour of the IS1 peak. It appears indeed to be difficult to set up chromatographic conditions in all laboratories in order to produce reproducible results of the IS2/IS1 ratio. We have therefore examined the possibility of standardising results of this analysis and demonstrate that the comparison of any unknown apple juice sample with an external apple juice reference sample enables different laboratories to reach similar conclusions. The IS2/IS1 peak ratio in this reference sample was adjusted to the significant cut-off limit. The assessment (fail or pass) is thus based on a standardised IS2/IS1 ratio that is independent of several important analytical parameters which tend to strongly influence it. Received: 26 December 1997 / Revised version: 30 March 1998     

Standardisation of the detection of invert sugar addition to apple juice by capillary gas chromatography

 Capillary gas chromatography (CGC) can be used to check the authenticity of various fruit juices by analysing their oligosaccharide fingerprint profile. Thus, it enables the analyst to detect, for instance, the dilution of apple juice concentrates with cheaper sweeteners such as invert sugar syrup. In order to detect this, two marker peaks (IS1 and IS2) that are present in most invert sugar syrups are monitored in the CGC chromatogram of apple juice. In freshly pressed apple juice, these peaks are normally not found. As a result of apple juice processing and, in particular, the thermal treatment required to concentrate and pasteurise the juice, these peaks may however be present in authentic apple juice concentrate and juice made from it. It is nevertheless possible to distinguish between juice showing this "heat effect" and that to which invert sugar has been added since the IS2/IS1 peak ratio is very different in heated apple juice and in invert sugar syrups. Measuring this ratio and setting a maximum IS2/IS1 cut-off limit enables the analyst to assess whether the sample meets the set norms or has been adulterated. In the past, many laboratories experienced difficulties when comparing the results of this analysis and, in particular, IS2/IS1 peak ratios. We present evidence that this unsatisfactory situation is mainly due to the chromatographic behaviour of the IS1 peak. It appears indeed to be difficult to set up chromatographic conditions in all laboratories in order to produce reproducible results of the IS2/IS1 ratio. We have therefore examined the possibility of standardising results of this analysis and demonstrate that the comparison of any unknown apple juice sample with an external apple juice reference sample enables different laboratories to reach similar conclusions. The IS2/IS1 peak ratio in this reference sample was adjusted to the significant cut-off limit. The assessment (fail or pass) is thus based on a standardised IS2/IS1 ratio that is independent of several important analytical parameters which tend to strongly influence it. Received: 26 December 1997 / Revised version: 30 March 1998     

Effect of typical sugars on the viscosity and colloidal stability of apple juice

Cloudy apple juice may be modeled as a dilute colloidal dispersion of solid particles in a solution of pectins, sugars, organic acids, and salts. In order to evaluate the effect of sugars on the viscosity and stability of the juice, it was diafiltered by ultrafiltration to remove the native soluble solids, and controlled amounts of sugars (glucose, sucrose, and maltose) were added afterwards. The addition of sugars produced a linear increase of the specific viscosity at decreasing water activities. The rates of increase (slopes) were proportional to the hydration capacity of each sugar. The specific viscosity of a colloidal dispersion of solid particles in sugar solution depends on three types of interactions: particle–particle (p–p), particle–water (p–w), and particle–sugar (p–s). P–p and p–w interactions were estimated from the extended DLVO theory in terms of the energy barrier between pairs of particles, and found to decrease at increasing sugar concentrations. Then, the increase of the specific viscosity was attributed to an increase of p–s interactions. The total energy barrier of the system (a measure of its stability) was modeled to be the sum of a p–p (including p–w) contribution plus a p–s contribution, and estimated from specific viscosity vs. particle volume fraction data at different sugar concentrations. The p–s contribution was estimated by difference. It was found to be positive (which was attributed to hydration repulsion) and higher than the p–p contribution at sugar concentrations ?0.04 mol/mol.     

Compositional characterisation of some apple varieties

 Ten apple varieties, harvested at commercial maturity, were analysed, paying special attention to the level of phenolic compounds and polyphenol oxidase (PPO) activity. The results pointed to great quantitative differences in the composition of the apple varieties, and particularly in their phenolic contents. Their total polyphenol content ranged from 2.3 to 3.6 g/kg. Chlorogenic acid was the main phenolic compound found in the varieties studied, and its concentrations varied from 30 to 430 mg/kg, and were 4–20 times higher than those of catechins. Only in Szampion and Elstar were the concentrations of catechins higher. Amongst the flavanol monomers, the main compound was (–)epicatechin, whose concentration varied from 18 to 89 mg/kg. The (+)catechin concentration ranged from 4 to 21 mg/kg. The dominant proanthocyanidins were oligomers up to trimers. In Warta apples, the concentrations of these oligomers were about 3 times higher than those of monomers. The level of PPO activity ranged from 270 units (U)/g in Szampion to 3120 U/g in Warta. No correlation was found between the concentrations of certain phenolic compounds, nor between PPO activity and phenolic or ascorbic acid concentrations. The results gave important information regarding the best use of the apple varieties investigated, i.e. for direct consumption due to their high nutritional value, or for processing. Received: 7 May 1999     

The reduction of patulin in apple juice by three different types of activated carbon

Three types of activated carbon (NORIT SA 4, NORIT SX 4 and NORIT CA 1) were investigated for their ability to reduce patulin levels in apple juice at various Brix levels and temperatures. The steamactivated carbons (NORIT SA 4 and NORIT SX 4) exhibited similar adsorption isotherms at a dosage level of 1g/l. They achieved patulin reductions of 80% and 70% respectively in 12° Brix juice at 55°C. The similarity in performance between the steam-activated carbons implies that the purity and the surface acidity does not influence the adsorption of patulin. Chemically-activated carbon (NORIT CA 1) was less effective in removing patulin and achieved only a 45% reduction at a dose of 1g/l. Patulin removal was influenced by juice Brix in that higher carbon doses were required at higher Brix levels for equivalent removal efficiency. At a dose of 1g/l, NORIT SA 4 removed only 45% patulin from a 20° Brix juice. The removal of patulin from either 12 or 20° Brix juice by NORIT SA 4 at 1g/l was not influenced by temperature changes in the range 30 to 65°C.     

Application of multivariate statistical methods to extend the authenticity control of flavour constituents of apple juice

 The distribution of 15 aroma constituents, including the (R)- and (S)-enantiomers of ethyl 2-methylbutanoate, in 30 commercially available and laboratory prepared apple juices was evaluated by multivariate statistical methods. Principal component analysis and cluster analysis revealed ethyl 3-methylbutanoate, 3-methylbutyl acetate and (R)-ethyl 2-methylbutanoate as key components for the differentiation of apple juices. In freshly pressed and some commercial juices ethyl 3-methylbutanoate and 3-methylbutyl acetate were not present, and the (S):(R) ratio of ethyl 2-methylbutanoate was found to be >99 : 1%. Another group of commercial products was separated in which the amounts of ethyl 3-methylbutanoate and 3-methylbutyl acetate exceeded 1 μg/l and 180 μg/l, respectively, and in which the (S):(R) ratio of ethyl 2-methylbutanoate was <97 : 3%. The statistical data confirmed the high selectivity of apple fruit tissue in biosynthesizing 2-methylbutyl and 2-methylbutanoate esters, and could also be used to extend the results of enantio-multidimensional gas chromatography and other authenticity analyses. Received: 20 April 1998 / Revised version: 23 June 1998     

The use of malolactic Oenococcus oeni (ATCC 39401) for deacidification of media containing glucose, malic acid and citric acid

 Malolactic fermentation is widely used to reduce the acidity of grape juices in wine production. However, application of the same technology to the production of wine from berries of the northern regions is not straightforward. Unlike grapes, these berries are rich in citric and malic acid while the sugar content is low. An ideal deacidification process for the northern berries would be the microbial degradation of these acids with minimal loss of sugars. Therefore, the co-metabolism of citric acid and glucose was studied under different conditions of malic acid degradation by Oenococcus oeni. At low pH values (pH <4.5) degradation of malic acid always proceeded first to completion with practically no consumption of glucose or citric acid. After the exhaustion of malic acid the degradation of both citric acid and glucose were initiated simultaneously. Following the exhaustion of malic acid and citric acid the remaining glucose remained nonfermentable. Thus, it is concluded that, by maintaining the culture in a resting state by the control of pH, selective degradation of acids can be achieved without subsequent loss of glucose. Received: 20 January 2000     

The choice of strains of Lactobacillus species for the lactic acid fermentation of vegetable juices

 The reasons for using lactic acid bacteria are to make food durable, to improve its taste and to maintain the nutritive, physiological and hygienic value of the fermentation products. Sixteen strains of the genus Lactobacillus were tested on samples of white fresh cabbage and of a sterilized cabbage and carrot juice mixture. After 7 days of lactic acid fermentation at 27  °C or 30  °C, reducing sugars, total acidity, pH value, lactic, citric and acetic acids, ammonia, nitrates and nitrites were measured in the samples. On the basis of the criteria mentioned above three strains were acceptable. These strains reduced the content of nitrates in the original samples. Received: 14 December 1998 / Revised version: 18 March 1999     

Biogenic amines in cold-smoked fish fermented with lactic acid bacteria

 It has been proved in previous studies that fish can be fermented by lactic acid bacteria (LAB). Added glucose must be at concentrations of almost 1% to reduce the pH to <5.3, a level which corresponds to the safe pH values for meat products. Because some LAB and some contaminating microbes form biogenic amines, this study was conducted to examine the possible formation of biogenic amines during the fish fermentation process. Other parameters under study were sensory quality, pH value, titrated acid content, weight loss, a _w value, redox potential and microbiological counts. Three groups of fish (rainbow trout) with three different LAB inocula and a group without any inoculum were made. The fermentation of the products made with LAB succeeded. The products were sensorially accepted, the inoculated LAB grew to >8 log cfu/g, the pH reduced to 5.0–5.3 and a _w to 0.927 and the pseudomonads, the predominant flora of fish raw material, disappeared. The fish raw material and the products contained low amounts of biogenic amines with one exception: cadaverine, histamine and tyramine increased in all product groups in one experimental series (II) out of three. The highest concentrations of these amines were in the control products without any LAB inoculation. The LAB used and the contaminants isolated from the products were unable to produce cadaverine, histamine or tyramine. The appearance of these amines could therefore be caused by the non-isolated contaminants of fish raw material. Received: 18 June 1999     

Screening methods for the proteolytic breakdown of gluten by lactic acid bacteria and enzyme preparations

 Gluten is the major protein in wheat and it is largely responsible for the rheological characteristics of bread dough. The sourdough process is a traditional dough fermentation process with lactic acid bacteria. The proteolytic activity of lactic acid bacteria derived from starter cultures used in sourdough or meat fermentation was tested on gluten as a substrate. The activities of commercial enzyme preparations, which were derived from bacterial or fungal sources, were also evaluated. Proteolytic breakdown of gluten protein in an agar medium by either bacterial culture or enzymes was evident by a clear zone around wells after staining with Coomassie blue. The increase in TCA-soluble material due to gluten breakdown was measured spectrophotometrically. A third test evaluated the release of free amino acids due to exoproteolytic activity. The agar based test showed positive results for the commercial enzyme preparations and one Micrococcus strain. All sourdough strains were positive in the enzyme test whereas some starters from the meat fermentation process were not able to break down gluten under test conditions. Variations in the release of free amino acid indicated differences in the enzyme systems of the lactic acid bacteria tested. Received: 25 February 1999     

Maintaining the quality of sugarcane juice with blanching and ascorbic acid

The physicochemical changes in fresh sugarcane juice stored at 10 °C were studied by determining juice yield, color, reducing sugar, titratable acidity, viscosity, pH, polyphenol oxidase (PPO), sucrose neutral invertase (SNI) and total microbial count. Results showed that blanching of stems before squeezing effectively prevented degreening and/or browning, and reduced activities of PPO and SNI in fresh sugarcane juice. Added ascorbic acid delayed the increase of reducing sugar, titratable acidity, viscosity and total microbial count, and also prevented degreening and/or browning with reduced PPO and SNI activities in fresh sugarcane juice during storage. Addition of 0.1% ascorbic acid seemed to be more effective than blanching of sugarcane stems, and was able to maintain the quality of fresh sugarcane juice for up to 5 days at 10 °C. Deterioration of fresh sugarcane juice was demonstrated as a rapid increase of titratable acidity and viscosity with a obvious browning.     

Applicability of lactic acid and nisin to improve the microbiological quality of cold-smoked rainbow trout

 The effect of lactic acid and nisin whey permeate on the microbiological and sensory quality of cold-smoked rainbow trout was studied. Lactic acid and whey permeate were mixed with salt solution and injected into fish fillets in concentrations of up to 2.5 g/kg lactic acid, 30 g/kg whey permeate and 20 g/kg sodium chloride. After smoking at 28  °C for 6 h the fillets were sliced, vacuum packed and stored at 3  °C. The aerobic and anaerobic/facultative anaerobic counts were measured after 1, 8, 15, 22 and 29 days of storage. The influence of the treatment on the sensory characteristics was analysed after 8 and 22 days by sensory profiling. A triangle test was used in order to ascertain at which stage the sensory quality of the samples changed during storage. Bacterial counts of smoked fish stored for 29 days were lowest in those samples containing a combination of lactic acid, sodium chloride and nisin whey permeate. Lactic acid alone did not affect the odour or flavour characteristics of the fish but did affect their texture as determined by sensory evaluation making it less elastic when stored at +3  °C for 8 days. A combination of lactic acid and nisin whey permeate enhanced the characteristic fishy flavour of the product. Differences between treatments in elasticity and fishy flavour were not detected after 22 days of storage. According to the triangle test, the samples from all treatments remained unchanged over the first 15 days. Received: 23 March 1998 / Revised version: 19 June 1998     

Preparation and characteristics of lactic acid fermented cowpea milk

 Four Lactobacilli were isolated from both commercial cowmilk yoghurt ("Fan" yoghurt) and "wara", a West African soft cheese. They were identified as Lactobacillus acidophilus ("Fan" yoghurt), L. plantarum, L. brevis and unidentified Lactobacillus spp. ("wara"). L. acidophilus and L. plantarum were chosen as starter organisms for the production of a yoghurt-like product from cowpea milk. There was a decrease in the final pH and an increase in the titratable acidity of the cowpea milk samples at the end of the fermentation period. The crude protein, total ash, calcium, potassium and phosphorus values increased in the fermented cowpea milk, while a decrease was obtained for the lipid, moisture and crude fibre contents. A decreasing trend was noted in the phytic acid and trypsin inhibitor of the cowpea flour when processed into milk, followed by a further reduction at the end of fermentation. Consumer acceptability of the cowpea yoghurt varied, with preference being given to the banana and strawberry flavoured samples. Received: 10 June 1996 / Revised version: 17 September 1997     

Bioavailability of antioxidative compounds from Brettacher apple juice in humans

The human bioavailability of ascorbic acid and polyphenolics such as chlorogenic acid and caffeic acid resp. from apple juice produced from the polyphenolic rich variety Brettacher was tested. After intake of 700 ml juice the antioxidant capacity in the plasma of human volunteers assessed by the TRAP-test increased significantly by 52% during the following 2 h. The cumulative urinary excretion of ascorbic acid within 7 h after intake was measured to 104% the ingested dose of the juice. Caffeic acid as the intestinal cleavage product of chlorogenic acid was excreted to only 0.56% of the dose in the juice. The results demonstrate in view of the favourable absorptive attributes and the influence on the plasmatic antioxidative capacity of these juice components that Brettacher apple juice may be suitable as a functional food.     

Antibacterial activity and mechanism of cinnamic acid and chlorogenic acid against Alicyclobacillus acidoterrestris vegetative cells in apple juice

The aim of this study was to investigate the antibacterial activity and mechanism of cinnamic acid and chlorogenic acid against Alicyclobacillus acidoterrestris. Minimum inhibitory concentrations of cinnamic acid and chlorogenic acid were 0.375 and 2.0 mg mL⁻¹, and the minimum bactericidal concentrations were 0.50 and 4.0 mg mL⁻¹, respectively. The apple juice ingredients had little influence on the inactivation of A. acidoterrestris. After treatment with cinnamic acid and chlorogenic acid, the morphology of A. acidoterrestris cells were severely destroyed; the leakage of nucleic acids and proteins increased significantly. SDS-PAGE investigation of bacterial proteins proved that the loss of soluble proteins was obvious as well. These results demonstrated that cinnamic acid and chlorogenic acid exerted their antibacterial activity mainly by an action mode of membrane disruption. This study provides an alternative method for the control of A. acidoterrestris-related spoilage in the fruit juice/beverage industry.     

The influence of enzymatic treatment of mash on the analytical composition of apple juice

Five apple juices were produced on a pilot scale by crushing the fruit, treating the mash with pectolytic enzymes, pressing, extracting water from the pomace, pasteurization, treatment of the juice with enzymes, combined gelatin/silica sol fining and final ultrafiltration. The temperature of the mash treatment was either ambient or one of 30, 40, 50 or 60 °C, in order to test whether this led to different compositional changes in the corresponding juices. The main emphasis was put on detection of D -galacturonic acid as the indicator substance for the extent of the enzymatic treatment. In the pasteurized raw juices after pressing we found values from 226 to 398 mg L⁻¹. A distinct increase of the galacturonic acid levels could be observed during the clarification steps. Enzymatic juice treatment raised the concentrations to 580–720 mg L⁻¹. After ultrafiltration, final values of 790–1100 mg L⁻¹ were measured. Generally, the highest values were found in the 50 °C sample, which is the optimum temperature for pectinase activity. This was also true for the colloid concentrations and polyphenols. The influence of the temperature of the enzymatic mash treatment on other characteristic juice parameters like total titratable acidity, density, sugar, and minerals concentrations was low.