Culture-independent study of the diversity of microbial populations in brines during fermentation of naturally-fermented Aloreña green table olives

Aloreña table olives are naturally fermented traditional green olives with a denomination of protection (DOP). The present study focused on Aloreña table olives manufactured by small and medium enterprises (SMEs) from Valle del Guadalhorce (Southern Spain) under three different conditions (cold storage, and ambient temperature fermentations in small vats and in large fermentation tanks). The microbial load of brines during fermentation was studied by plate counting, and the microbial diversity was determined by a culture-independent approach based on PCR-DGGE analysis. The viable microbial populations (total mesophilic counts, yeasts and molds, and lactic acid bacteria — LAB) changed in cell numbers during the course of fermentation. Great differences were also observed between cold, vat and tank fermentations and also from one SME to another. Yeasts seemed to be the predominant populations in cold-fermented olives, while LAB counts increased towards the end of vat and tank fermentations at ambient temperature. According to PCR-DGGE analysis, microbial populations in cold-fermented olives were composed mostly by Gordonia sp./Pseudomonas sp. and Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. together with halophilic archaea (mainly by haloarchaeon/Halosarcina pallida and uncultured archaeon/uncultured haloarchaeon/Halorubrum orientalis) and yeasts (Saccharomyces cerevisiae and Candida cf. apicola). Vat-fermented olives stored at ambient temperature included a more diverse bacterial population: Gordonia sp./Pseudomonas sp., Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. and Thalassomonas agarivorans together with halophilic archaea and yeasts (mainly S. cerevisiae and C. cf. apicola, but also Pichia sp., and Pichia manshurica/Pichia galeiformis). Some LAB were detected towards the end of vat fermentations, including Lactobacillus pentosus/Lactobacillus plantarum and Lactobacillus vaccinostercus/Lactobacillus suebicus. Only the tank fermentation showed a clear predominance of LAB populations (Lactobacillus sp., Lactobacillus paracollinoides, and Pediococcus sp.) together with some halophilic archaea and a more selected yeast population (P. manshurica/P. galeiformis). The present study illustrates the complexity of the microbial populations in naturally-fermented Aloreña table olives.     

Exploring the yeast biodiversity of green table olive industrial fermentations for technological applications

In recent years, there has been an increasing interest in identifying and characterizing the yeast populations associated with diverse types of table olive elaborations because of the many desirable technological properties of these microorganisms. In this work, a total of 199 yeast isolates were directly obtained from industrial green table olive fermentations and genetically identified by means of a RFLP analysis of the 5.8S-ITS region and sequencing of the D1/D2 domains of the 26S rDNA gene. Candida diddensiae, Saccharomyces cerevisiae and Pichia membranifaciens were the most abundant yeast species isolated from directly brined Aloreña olives, while for Gordal and Manzanilla cultivars they were Candida tropicalis, Pichia galeiformis and Wickerhamomyces anomalus. In the case of Gordal and Manzanilla green olives processed according to the Spanish style, the predominant yeasts were Debaryomyces etchellsii, C. tropicalis, P. galeiformis and Kluyveromyces lactis. Biochemical activities of technological interest were then qualitatively determined for isolates belonging to all yeast species. This preliminary screening identified two isolates of W. anomalus with interesting properties, such as a strong β-glucosidase and esterase activity, and a moderate catalase and lipolytic activity, which were also confirmed by quantitative assays. The results obtained in this survey show the potential use that some yeast species could have as starters, alone or in combination with lactic acid bacteria, during olive processing.     

Microbiological stabilization of Aloe vera (Aloe barbadensis Miller) gel by high hydrostatic pressure treatment

The effect of high hydrostatic pressure (HHP) treatment (300, 400 and 500MPa for 1 and 3min at 20°C) on the microbiological shelf-life and microbiota composition of Aloe vera gel during 90days of storage at 4°C was investigated. Aerobic mesophilic and psychrotrophic bacteria, as well as moulds and yeasts, were enumerated after HHP treatment and through cold storage. Randomly selected isolates from the count plates were identified by standard methods and the API identification system. Results showed that HHP treatment at or over 400MPa for 3min were effective to keep the microbial counts to undetectable levels during the whole storage period, and consequently the microbiological shelf-life of A. vera gel was extended for more than 90days at 4°C. The microbiota in the untreated A. vera gel was dominated by Gram-negative bacteria (mostly Rahnella aquatilis) and yeasts (mostly Rhodotorula mucilaginosa). In contrast, Gram-positive bacteria tentatively identified as Arthrobacter spp. and Micrococcus/Kocuria spp. were the predominant microorganisms in samples pressurized at 300MPa for 1 and 3min, while Bacillus megaterium predominating in samples treated at 400MPa for 1min. At 400MPa for 3min and above, the microbial growth was completely suppressed during at least 90days; however, viable spore-formers were detected by enrichment.     

Synergistic impact of sonication and high hydrostatic pressure on microbial and enzymatic inactivation of apple juice

The combination of innovative, non-thermal technologies for the production of safe and quality fruit juices is a recent trend in food processing. The purpose of this study was to evaluate the effects of combined treatment of ultrasound (US) and high hydrostatic pressure (HHP) on enzymes (polyphenolase, peroxidase and pectinmethylesterase), microorganisms (total plate counts, yeasts and molds) and phenolic compounds (total phenols, flavonoids and flavonols) of apple juice. Moreover, its effects on ascorbic acid, antioxidant capacity and DPPH free radical scavenging activity, color values, pH, soluble solids and titratable acidity were investigated. Fresh apple juice was treated with US (25 kHz and 70% amplitude) at 20 °C for 60 min with subsequent HHP treatment at 250, 350 and 450 MPa for 10 min at room temperature. The results revealed that the combined US-HHP450 treatment caused highest inactivation of enzymes with complete inactivation of total plate counts, yeasts and molds. It also significantly improved the phenolic compounds, ascorbic acid, antioxidant capacity, DPPH free radical scavenging activity and color values. The present results suggest that the combination of US and HHP can act as a potential hurdle to produce safe and high quality apple juice with reduced enzymes and microbial activity and improved nutrition.     

Main variables affecting the lactic acid fermentation of table olives

Among the olive varieties, Manzanilla is the most employed as table olives worldwide. Inoculation of the brines of Manzanilla and Gordal olives, which were not treated with alkali, with a strain of Lactobacillus pentosus was carried out and results indicated that variety is one of the main factors to be considered when fermenting olives, even more important than the salt level. This conclusion was based on the presence of a high concentration of antimicrobial compounds, namely the dialdehydic form of decarboxymethyl elenolic acid, either free or linked to hydroxytyrosol in the brines of the Manzanilla variety, as compared to the low level of these substances in the Gordal brines. Likewise, it was also observed that the inhibitory activity of Manzanilla olives against lactic acid bacteria could be counteracted by the addition of nitrogenous supplements such as MRS and yeast extract to their brines. Results obtained from this work are of great interest for the table olive industry in order to develop lactic acid fermentation in olives non‐treated with alkali.     

The effects of high hydrostatic pressure at subzero temperature on the quality of ready-to-eat cured beef carpaccio

We compared the application of high hydrostatic pressure (HHP) on unfrozen carpaccio (HHP at 20 °C) and on previously-frozen carpaccio (HHP at − 30 °C). HHP at 20 °C changed the color. The pressure increase from 400 to 650 MPa and the time increment from 1 to 5 min at 400 MPa increased L* and b*. a* decreased only with 650 MPa for 5 min at 20 °C. The prior freezing of the carpaccio and the HHP at − 30 °C minimized the effect of the HHP on the color and did not change the shear force, but increased expressible moisture as compared to the untreated carpaccio. HHP at 20 °C was more effective in reducing the counts of microorganisms (aerobic total count at 30 °C, Enterobacteriaceae, psychrotrophs viable at 6.5 °C and lactic acid bacteria) than HHP at − 30 º C. With HHP at 20 °C, we observed a significant effect of pressure and time on the reduction of the counts.     

High hydrostatic pressure effect on chemical composition,color, phenolic acids and antioxidant capacity of Cape gooseberry pulp (Physalis peruviana L.)

The aim of this study was to evaluate the effects of high hydrostatic pressure (HHP) at 300, 400 and 500 MPa/1, 3 and 5 min on nutritional and antioxidant properties of Cape gooseberry pulp after immediate application and after 60 days of storage. Proximal analysis, color, phenolic acids content and antioxidant capacity were determined. When analyzing the immediate effect of different treatments, a clear influence of HHP was observed in all the components of the proximal analysis. Regarding color, none of the three chromatic parameters showed significant differences with control leading to a minimum ΔE at 300 MPa/3 min. Changes in bound and free phenolic acids were evidenced after treatments. The maximum levels of TPC as well as antioxidant capacity were observed at 500 MPa/5 min. By the end of storage, all treated samples discolored leading to ΔE = 14.9 at 500 MPa/5 min. The profile of free and bound phenolic acids presented differences compared to Day 0. The antioxidant capacity by means of ORAC increased for treatments above 300 MPa/5 min indicating the effectiveness of these treatments for the production of functional products based on gooseberry pulp. For treatments above 400 MPa/3 min, molds and yeasts were not detected.     

The effects of high hydrostatic pressure at subzero temperature on the quality of ready-to-eat cured beef carpaccio

We compared the application of high hydrostatic pressure (HHP) on unfrozen carpaccio (HHP at 20°C) and on previously-frozen carpaccio (HHP at -30°C). HHP at 20°C changed the color. The pressure increase from 400 to 650MPa and the time increment from 1 to 5min at 400MPa increased L* and b*. a* decreased only with 650MPa for 5min at 20°C. The prior freezing of the carpaccio and the HHP at -30°C minimized the effect of the HHP on the color and did not change the shear force, but increased expressible moisture as compared to the untreated carpaccio. HHP at 20°C was more effective in reducing the counts of microorganisms (aerobic total count at 30°C, Enterobacteriaceae, psychrotrophs viable at 6.5°C and lactic acid bacteria) than HHP at -30o C. With HHP at 20°C, we observed a significant effect of pressure and time on the reduction of the counts.     

Effect of high hydrostatic pressure to sweet potato flour on dough properties and characteristics of sweet potato‐wheat bread

The effect of high hydrostatic pressure (HHP, 100–400 MPa) for 20 min at 25 °C to sweet potato flour (SPF) on dough properties and characteristics of sweet potato‐wheat bread was investigated. The particle size of SPF after HHP was decreased significantly. The obvious rupture was observed in granules of SPF after HHP at 300 and 400 MPa by scanning electron microscopy (SEM). After HHP, significant differences on endothermic peak temperatures (T_P) of SPF were observed by differential scanning calorimetric (DSC), of which the enthalpy change (ΔH) had a slight increase, expect that at 200 MPa. Gas retention of dough with SPF after HHP increased markedly from 1199 (0.1 MPa) to 1246 ml (100 MPa). Specific loaf volume of bread with SPF at 400 MPa was increased significantly, while the hardness and chewiness were reduced. Thus, SPF treated with HHP at 400 MPa could be potentially used in wheat bread production.     

Instability profile of fresh packed “seasoned” Manzanilla-Aloreña table olives

The shelf life of cracked “seasoned” Manzanilla-Aloreña table olives is short; containers may swell and the fruits become progressively brownish. Respiration of cracked fruits continued for a period longer than 48 h after picking and the carbon dioxide produced may be an initial cause of spoilage. As processing progressed, the microbial loads in the olives increased due to successive washings and, together with the microbial load from the added ingredients, led to relatively high viable counts of yeasts and LAB at packing. During shelf life, this microflora showed an increase in viable counts which could also eventually lead to swelling spoilage. This behaviour was favoured by the presence of residual fermentable substrates and a decrease in potassium sorbate over time in both brine and flesh. At the same time, colour darkened, titratable acidity increased and pH decreased. The application of two additional washings to the cracked olives before packing was favourable because fermentable material and polyphenols were removed; however, fruits with as low as possible respiration rates, stronger washing treatments, ingredients with low or no microbial viable counts and higher preservative concentrations would be necessary to achieve complete stabilization of the product.     

高静压对桃汁杀菌、钝化酶活性的效果

研究在不同处理压力和时间条件下,高静压加工技术对桃汁中微生物(细菌总数、霉菌、酵母菌、大肠菌群)以及酶(多酚氧化酶、果胶甲基酯酶、脂肪氧化酶)的影响。结果表明:经400MPa、5min高静压处理即可完全杀灭桃汁中的微生物;在400MPa和500MPa条件下,桃汁中的多酚氧化酶和脂肪氧化酶的活性出现了不同程度的激活现象,但在600MPa时,随着处理时间的延长,其活性逐渐降低,经30min处理后,分别被钝化了0.7662和0.641。而果胶甲基酯酶在400、500、600MPa条件下,出现了不规律的激活或钝化现象。另外,研究表明在高静压加工前增加漂烫工艺,可以有效杀灭桃汁中的微生物及钝化酶活性。     

High hydrostatic pressure treatment for manufacturing of garlic powder with improved microbial safety and antioxidant activity

The traditional method of manufacturing garlic powder (GP) that includes simple grinding of air-dried garlic slices has problems of microbial safety and a pungent flavour for this product. Microbiologically safe GP with a less pungent flavour and better antioxidant activities was manufactured using high hydrostatic pressure (HHP), wet grinding and freeze-drying process. The numbers of total aerobic bacteria and yeasts and molds in untreated (without HHP) GP were 3.64 and 2.47 log CFU/g respectively. Garlic powder treated with 600 MPa HHP for 5 min exhibited a total aerobes count of 1.62 CFU/g and a yeasts and molds count of 1.43 log CFU/g. The diallyl disulfide content, which is responsible for the pungent odour of garlic, was also significantly reduced by HHP due to a decrease in the alliinase activity. Hence, a novel process using HHP can help to produce GP with improved microbial safety, flavour and nutrition.     

Comparison of Microbial Inactivation and Rheological Characteristics of Mango Pulp after High Hydrostatic Pressure Treatment and High Temperature Short Time Treatment

The effects of high hydrostatic pressure (HHP) treatments at pressures of 300–600 MPa for 1–20 min and of high-temperature, short-time (HTST) treatment on the inactivation of natural microorganisms in blanched mango pulp (BMP) and unblanched mango pulp (UBMP) were investigated. No yeasts, molds, or aerobic bacteria were detected in BMP or UBMP after HHP treatments at 300 MPa/15 min, 400 MPa/5 min, 500 MPa/2.5 min, and 600 MPa/1 min and HTST treatment at 110 °C/8.6 s. Therefore, these conditions were selected to study the effects of HHP and HTST treatments on pectin methylesterase (PME) activity, water-soluble pectin (WSP) levels, and the rheological characteristics of UBMP and BMP. HHP treatment at a pressure of 600 MPa for 1 min significantly reduced PME activity in UBMP and significantly activated PME in BMP, whereas pressures of 300–500 MPa activated PME regardless of blanching. However, PME activity was reduced by 97 % in UBMP and was completely inactivated in BMP by HTST treatment. WSP levels were significantly decreased by HHP treatment but were increased by HTST treatment in UBMP and BMP. Both HHP and HTST treatments increased the viscosity, storage modulus, and loss modulus of UBMP and BMP. No significant changes in total sugar, total soluble solids, titratable acid, or pH were found after any treatment.     

高静压处理对黄桃罐头微生物和质构的影响

以黄桃罐头为试材,研究了高静压处理的杀菌效果,并用质地多面分析(TPA)方法,对高静压造成黄桃质构的变化进行分析。结果表明,400MPa下处理25min即可杀灭所有细菌,300MPa下处理15min就能杀灭所有霉菌和酵母菌。高静压处理(600MPa,5min)后黄桃罐头的硬度、凝聚性和咀嚼性与未处理无显著差异,但显著好于热处理。高静压技术能使黄桃罐头达到商业无菌,并保持良好质构。     

Reuterin and High Hydrostatic Pressure Treatments on the Inactivation of Listeria monocytogenes and Effect on the Characteristics of Cold-Smoked Salmon

The effect of reuterin and high hydrostatic pressure (HHP) processing at 450 MPa for 5 min on the inactivation of Listeria monocytogenes and the characteristics of cold-smoked salmon during 35 days at 4 and 10 °C were investigated. The growth rate of the pathogen was reduced by reuterin addition and a synergistic antimicrobial effect against L. monocytogenes was recorded when the biopreservative was applied in combination with HHP at 450 MPa for 5 min. This combined treatment prevented the pathogen recovery observed with individual treatments and delayed the spoilage of smoked salmon maintaining total viable counts under 3.5 log units during 35 days of storage at 4 °C. All treatments assayed induced changes in lightness (L*) and redness (a*), resulting in a brighter appearance of smoked salmon, whereas no modifications were recorded in shear strength values immediately after treatments. Moreover, reuterin and HHP treatments, individually or in combination, avoided the formation of biogenic amines during the 35 days of storage at 4 and 10 °C. The addition of reuterin in combination with HHP at 450 MPa for 5 min might be applied as a hurdle technology to improve the safety and extend the shelf life of lightly preserved seafood products, such as cold-smoked salmon.     

A preliminary study about the influence of high hydrostatic pressure processing on the physicochemical and sensorial properties of a cloudy wheat beer

High hydrostatic pressure (HHP) (400 MPa/15 min, 500 MPa/10 min, 600 MPa/5 min at 20 °C) and heat (60 °C/15 min) processing of wheat beers were evaluated by examining their impacts on microorganisms, colloidal haze, flavour, foam stability and shelf‐life prediction during 84 days of storage at 20 °C. The results obtained showed that the microbiological stability of HHP beers was comparable with heat‐treated samples, and the development of both aerobic bacteria and lactic acid bacteria was inhibited for 84 days of storage. The main parameters of the wheat beer, such as ethanol content, original extract, pH, bitterness and viscosity, were scarcely affected by either treatment compared with the control samples; however, heat pasteurization increased the colour value. Heat‐pasteurized beer resulted in an increase in the phenethyl alcohol concentration and a decrease in isoamyl acetate and ethyl acetate levels compared with the HHP samples. These treatments did not affect the amount of 4‐vinylguaiacol and 4‐vinylphenol in the beer. The HHP‐treated beers had higher colloidal haze and foam stability values than the heat‐pasteurized beers. Dynamic light scattering analysis showed that HHP treatments at 500 MPa/10 min resulted in smaller and more uniform particle sizes, which had a positive effect on beer haze stability during storage. Copyright © 2016 The Institute of Brewing & Distilling     

Effect of Single‐ and Two‐Cycle High Hydrostatic Pressure Treatments on Water Properties,Physicochemical and Microbial Qualities of Minimally Processed Squids (Todarodes pacificus)

This study investigated the effect of single‐ and two‐cycle high hydrostatic pressure (HHP) treatments on water properties, physicochemical, and microbial qualities of squids (Todarodes pacificus) during 4 °C storage for up to 10 d. Single‐cycle treatments were applied at 200, 400, or 600 MPa for 20 min (S‐200, S‐400, and S‐600), and two‐cycle treatments consisted of two 10 min cycles at 200, 400, or 600 MPa, respectively (T‐200, T‐400, and T‐600). HHP‐treated samples had higher (P < 0.05) content of P_2b (immobilized water) and P₂₁ (myofibril water), but lower P₂₂ (free water) than those of control. The single‐ and two‐cycle HHP treatments at the same pressure level caused no significant difference in water state of squids. The two‐cycle HHP treatment was more effective in controlling total volatile basic nitrogen, pH, and total plate counts (TPC) of squids during storage, in which TPC of S‐600 and T‐600 was 2.9 and 1.8 log CFU/g at 10 d, respectively, compared with 7.5 log CFU/g in control. HHP treatments delayed browning discoloration of the squids during storage, and the higher pressure level and two‐cycle HHP were more effective. Water properties highly corresponded with color and texture indices of squids. This study demonstrated that the two‐cycle HHP treatment was more effective in controlling microbial growth and quality deterioration while having similar impact on the physicochemical and water properties of squids in comparison with the single‐cycle treatment, thus more desirable for extending shelf‐life of fresh squids.     

High hydrostatic pressure effect in extraction of 5-methyltetrahydrofolate (5-MTHF) from egg yolk and granule fractions

High hydrostatic pressure (HHP) and centrifugation were combined to produce a 5-methyltetrahydrofolate (5-MTHF)-enriched extract from egg yolk. This study focused on the effect of HHP treatment (200–600 MPa; 5, 10 min) in extraction of 5-MTHF from egg yolk and granule. Specifically, HHP-treatment at 400 MPa for 5 min significantly (p < 0.05) impacted protein extraction and was the optimum condition for 5-MTHF extraction, with recoveries of 230 ± 2.7 μg/100 g and 1264.6 ± 61.4 μg/100 g from yolk and granule, respectively. The applications of HHP combined with centrifugal separation resulted in recovery of free 5-MTHF from granule which was 5.5 times higher compare to yolk. The optimal parameters of HHP at 400 MPa for 5 min improve 93% the recovery of 5-MTHF from non-soluble granule and convert it to a soluble state. Electrophoresis of fractions revealed that high-pressure treatment of yolk and granule at 200–600 MPa resulted in considerable solubilization of low-density lipoproteins (LDL) and high-density lipoproteins (HDL)-phosvitin, which may be due to disruption of hydrophobic interactions. High pressure can be used to further fractionate yolk and granule in the development of new biotechnological applications.Industrial relevance: HHP combined with centrifuge separation was used an efficient and eco-friendly process for improving the recovery of water soluble vitamin 5-MTHF extracted from egg yolk granule. HHP treatment at 400 MPa for 5 min was the optimum condition where a higher concentration of 5-MTHF separated from granule. This study shows promising results in relation to applications of HHP in improving extraction and recovery of bioactive components from egg yolk and the future applications of granules as an ingredient in the food industry.     

Influence of Yeasts on the Oil Quality Indexes of Table Olives

After moisture, fat is the major constituent of table olives. However, scarce studies have been carried out to determine the influence of microorganisms and type of processing on the modification of their quality indexes. The present survey studies the influence of lipolytic (Candida boidinii TOMC Y5 and Wickerhamomyces anomalus TOMC Y10) and nonlipolytic (Debaryomyces etchellsii TOMC Y9 and Pichia galeiformis TOMC Y27) yeasts on the oil quality indexes of Manzanilla and Hojiblanca green fruits processed as directly brined and lye‐treated table olives. Overall, the inocula scarcely used available sugars, except the lipolytic C. boidinii strain in lye‐treated olives. Acetic acid production was limited in all conditions, except for the D. etchellsii strain in directly brined Manzanilla fruits. Ethanol formation was also reduced, although the W. anomalus (in both types of elaboration) and the C. boidinii (in lye‐treated olives) strains produced significantly higher proportions. Apparently, changes in the oil quality indexes of processed olives were not related to the presence of yeasts, and hence, could have been caused by the endogenous activity of the fruits. A principal component analysis using the microbiological, physicochemical, and oil quality data supported this hypothesis, grouping treatments according to olive variety and type of elaboration, while segregation due to yeast inocula was not observed.     

Application of emerging non-thermal technologies to sodium reduction in ready-to-eat fish products

This study investigated the isolated and combined effect of UV-C (0.310 J/cm²) and high hydrostatic pressure (HHP; 300 MPa for 5 min at 25 °C) on the quality parameters of cooked fish batter with reduced salt content by 25% and 50%. The treatments did not affect sodium chloride concentration, redness, yellowness, cohesiveness, springiness, and resilience (p > 0.05). CON (100% sodium chloride - NaCl), UV25%, and HHP25% had similar cooking loss (CL), lightness, hardness, chewiness, and salty taste (p > 0.05). UV + HHP25% showed higher CL and lower salty taste compared to CON, UV25%, and HHP25% (p < 0.05). UV50% and HHP50% had higher CL, lightness, hardness and chewiness, and lower salty taste than CON, UV25%, and HHP25% (p < 0.05). UV + HHP50% showed the highest CL, hardness and chewiness, while the lowest salty taste and higher lightness than CON (p < 0.05). Therefore, UV-C at 0.310 J/cm² or HHP at 300 MPa for 5 min is a potential alternative to developing ready-to-eat fish products reduced by 25% NaCl.