High hydrostatic pressure pasteurization of a draft sake brewed using a Niigata-sake yeast

ABSTRACT

Draft (non-pasteurized) sake typically has a fresh flavor, but usually has to be consumed as soon as possible because of its short shelf life due to the potential for over-fermentation by residual yeast. In contrast, thermally pasteurized sake has a long shelf life, but the fresh flavor is lost during heat sterilization. High hydrostatic pressure (HHP) treatment can be used as a non-thermal pasteurization process while maintaining the characteristics of a draft sake. We evaluated the HHP inactivation behavior of sake yeast for the application of HHP pasteurization as an alternative to thermal pasteurization. The results showed complete pasteurization of sake yeast in the draft sake by HHP treatment at 400?MPa for 10?min. The viable cell count in HHP-pasteurized sake was below the detection limit during storage at 10°C for 3 months. Thus, we have established a HHP pasteurization technology to prevent over-fermentation and succeeded in producing a prototype of HHP-sparkling-cloudy sake, which we have designated AWANAMA.     

Effect of high hydrostatic pressure on enzyme activity in unpasteurized draft sake

ABSTRACT

Unpasteurized draft sake has a potentially high market value, due to its fresh flavor and fruity taste, compared with conventional thermal-pasteurized sake. However, the shelf life of draft sake is limited. To increase the shelf life of draft sake, it is necessary to suppress flavor and taste deterioration resulting from inactivation of enzymes produced by koji-mold. Draft sake was treated with high hydrostatic pressure (HHP) of 200 to 500?MPa at ?7 to 50°C to analyze the inactivation of α-amylases, glucose-forming enzymes, and acid carboxypeptidases. We found significant inactivation of enzymes produced by koji-mold in draft sake subjected to HHP treatment at both high and low temperature. However, HHP treatment at low temperature effectively inactivated enzymes while retaining the fresh flavor and fruity taste of draft sake.     

High-throughput screening of food additives with synergistic effects on high hydrostatic pressure inactivation of budding yeast

ABSTRACT

We focused on food additives that enhance the effect of high hydrostatic pressure (HHP) treatment for microbial inactivation. We previously isolated Saccharomyces cerevisiae ultraviolet (UV) mutant strain a1210H12 that does not cause a growth delay under specific high pressure treatment conditions. We conceived that it is possible to screen effective food additives in a high-throughput manner by combining strain a1210H12 with a viable cell count method based on liquid culture, named high-throughput microbial pressure inactivation kinetics analysis system (HT-PIKAS). Here, we analyzed the synergistic effect between food additives and HHP treatment on inactivation of strain a1210H12 using HT-PIKAS. We calculated the inactivation rate in the condition of additive only, HHP treatment only and HHP treatment with additive. As a result, four compounds, benzoic acid, sorbic acid, adipic acid and caproic acid, showed high synergistic effects with HHP treatment and could be selected from more than 30 compounds as safe food additives.     

High hydrostatic pressure inactivation of Lactobacillus plantarum cells in (O/W)-emulsions is independent from cell surface hydrophobicity and lipid phase parameters

Inactivation efficiency of high hydrostatic pressure (HHP) processing of food is strongly affected by food matrix composition. We investigated effects of fat on HHP inactivation of spoilage-associated Lactobacillus (L.) plantarum strains using defined oil-in-water (O/W)-emulsion model systems. Since fat-mediated effects on HHP inactivation could be dependent on interactions between lipid phase and microbial cells, three major factors possibly influencing such interactions were considered, that is, cell surface hydrophobicity, presence and type of surfactants, and oil droplet size. Pressure tolerance varied noticeably among L. plantarum strains and was independent of cell surface hydrophobicity. We showed that HHP inactivation of all strains tended to be more effective in presence of fat. The observation in both, surfactant-stabilized and surfactant-free (O/W)-emulsion, indicates that cell surface hydrophobicity is no intrinsic pressure resistance factor. In contrast to the presence of fat per se, surfactant type and oil droplet size did not affect inactivation efficiency.     

细菌纤维素菌株超高压诱变选育及其发酵培养基的优化

 为了获得高产细菌纤维素菌株,对初选的细菌纤维素菌株J₂进行超高压诱变,运用Plackett-Burman设计对影响高压诱变菌株生产细菌纤维素的因素效应进行评价,采用Box-Behnken试验优化发酵培养基组成。试验结果表明,超高压诱变压力、时间对细菌纤维素菌株有显著或极显著影响。细菌纤维素菌株高压诱变条件为压力250 MPa、时间15 min、温度25 ℃。经超高压诱变,获得产纤维素能力高、遗传稳定性好的诱变菌株M₄₃₈。影响诱变菌株M₄₃₈发酵生产细菌纤维素的关键因子是酵母浸出汁、MgSO₄和无水乙醇。优化的发酵培养基为碳源5%（葡萄糖∶蔗糖为4∶1）、酵母浸出汁1.25%、CaCl₂0 15%、ZnSO₄ 0.20%、K₂HPO₄ 0.20%、MgSO₄ 0.93%、富马酸0.30%、无水乙醇0.50%。利用此培养基培养诱变细菌纤维素菌株M₄₃₈,其纤维素产量是优化前的1.84倍,是超高压诱变之前的2.69倍。超高压技术用于细菌纤维素菌株的诱变育种是可行的。发酵培养基的优化可显著提高菌株M₄₃₈发酵生产细菌纤维素的能力。     

Inactivation of Staphylococcus aureus using high hydrostatic pressure

The aim of this work was to develop a high-pressure decontamination and sterilization process for pharmaceutical treatments as was developed in food processing in the late eighties. The lack of normalized biological indicators able to validate sterilizing treatments under high pressure led us to select representative pathogenic strains from flora and the European Pharmacopoeia. We selected the following four bacterial strains: Candida albicans (ATCC 10231), Psuedomonas aeruginosa (ATCC 9027), spores of Aspergillus niger (ATCC 16404) and Staphylococcus aureus (ATCC 6538).

This present study is focussed on S. aureus. Successive pressurization and depressurization cycles appeared to be more efficient than a continuous high-pressure treatment. Importantly, these pressure conditions, temperature and process duration are perfectly compatible with current industrial plants. These results show that HHP technology is a new alternative to inactivate pathogenic strains in accordance with pharmaceutical requirements.     

激光诱变选育面包酵母菌的研究

采用He-Ne激光对面包酵母菌进行诱变处理,筛选到3株低糖面团起发活力大幅度提高的菌株XD198、XD193和XD174,其低糖面团起发活力比出发菌株分别提高31.3%、32.7%和35.3%.海藻糖含量分别较出发菌株提高-1.37%、10.83%、5.10%.而且筛选到的高活力菌株遗传性能稳定.证明激光诱变效果明显,是进行面包酵母菌种选育的理想方法之一.     

The usage of high hydrostatic pressure (HHP) to control food-borne pathogens in hummus

ABSTRACT

With the increasing demand for fresher, higher quality, minimally processed and safer food, there is a strong necessity to develop non-thermal processing techniques. Also for hummus, which is popular all around the world. In this work, the effect of refrigerated storage on the survival of pathogens in hummus treated by high hydrostatic pressure (HHP) (500?MPa/10?min/room temperature) was evaluated. The cocktail of two Salmonella, four Listeria monocytogenes and two Escherichia coli strains was used in this study. All pathogen types were able to survive in hummus during 60 days of refrigerated storage. HHP-treated samples plated on day 0 successfully achieved a?>?5 log cfu/g reduction for all pathogen types. No residual survivors were present after 30 and 60 days in any of the HHP-treated samples. These results demonstrate that HHP may be a useful technique for the inactivation of pathogens and therefore helpful in designing non-thermal HHP conditions for pressurization of hummus.     

Thermosensitivity of a barosensitive Saccharomyces cerevisiae mutant obtained by UV mutagenesis

Using UV mutagenesis, a high pressure (HP)-sensitive (barosensitive) mutant of Saccharomyces cerevisiae was obtained. The mutant strain a924E1 showed a significant loss of viability at HP levels of 175 to 250 MPa at 20 °C compared with the parent strain. This strain also showed a significant loss of viability following heat treatment at 50–58 °C at 0.1 MPa. These results showed that the mutation caused a significant thermosensitivity as well as barosensitivity. The activation volume and activation energy values for the inactivation of strain a924E1 were equivalent to those of the parent strain. This suggested that the mechanism for the HP and thermal inactivation reaction of strain a924E1 was basically the same as that of the parent strain. Strain a924E1 showed no deficiency in growth and fermentation ability as well as auxotrophic property. Although the identification of the genetic sites of mutation introduced is underway, these phenotypes are favorable for the application of HP treatment and heat-assisted HP treatment on fermentation control.     

The effect of high hydrostatic pressure on taste substance distribution in fresh green tea leaves

ABSTRACT

High hydrostatic pressure (HHP) can be an alternative method to steaming to inhibit enzymatic fermentation in green tea making process. However, the effect of HHP treatment on green tea taste is not clear. Thus, this study aimed to determine the effect of HHP on substances associated with green tea taste. Fresh green tea leaves were immediately treated with HHP at 300, 500, or 700?MPa for 10, 30, or 60 min at 25°C. The concentration of free amino acids, catechins, and caffeine in HHP-treated samples was quantified by LC-MS. The taste intensity of the samples was detected by taste sensors. HHP resulted in a high accumulation of free amino acids in green tea leaves, which was likely due to proteolysis. In particular, theanine synthesis may have been promoted by an increase in the concentration of substrates during HHP. Compared to steaming, HHP enhanced umami richness, and inhibited bitterness and astringency.     

The variability of times to detect growth from individual Clostridium botulinum type E endospores is differentially affected by high pressure treatments

High pressure thermal (HPT) processing is a candidate technology for the production of safe and stable food. However, little is known about the effect of HPT or high hydrostatic pressure (HHP) treatments at ambient temperature on the variability of times to detect growth from individual spores. We investigated this effect by treating Clostridium botulinum type E spores with HHP (200–600?MPa, 20°C) and HPT (600?MPa, 80°C and 800?MPa, 60°C). Our results indicate that the mean detection times increase and the frequency distribution shifts toward longer times when HHP treatment intensity is increased. HPT treatments result in a highly scattered distribution. In contrast, pressure levels ≤300?MPa decrease detection times and heterogeneity of their distribution, which could lead to an increase in the potential risk originating from C. botulinum type E spores. Data provided here could help to refine risk assessment regarding this important food intoxicator.     

Depletion of arginine in yeast cells decreases the resistance to hydrostatic pressure

High hydrostatic pressure (HP) inhibits growth and inactivates microorganisms by destabilizing non-covalent molecular interactions. Arginine contributes to stress resistance because it has a guanidine side chain, which assists in the refolding of aggregated proteins. We attempted to analyze the contribution of arginine to high HP stress using a pressure-sensitive mutant strain of Saccharomyces cerevisiae and a metabolomics approach. Our results showed that the content of 136 out of 250 detected metabolites differed in the mutant and parent strains. Decreased metabolites were involved in the tricarboxylic acid cycle and arginine biosynthesis. The expression of genes contributing to arginine biosynthesis was significantly lower in the mutant strain than in the parent strain. When arginine was supplemented to the medium, the mutant strain showed more tolerance to pressure. These results suggest that yeast cells survived due to the contribution of arginine to high pressure resistance. This indicates that depletion of arginine caused by decreased activity of the biosynthesis pathway confers sensitivity to HP.     

Non‐thermal plasma‐induced apoptosis in yeast Saccharomyces cerevisiae

The effect of non‐thermal plasma generated by the direct current (DC) corona discharge in the mode of transition spark is studied on a yeast Saccharomyces cerevisiae. The exposure to plasma increases production of reactive oxygen species (ROS) in cells, possibly causing the induction of apoptosis. To clarify the mechanism of apoptosis, its induction is tested not only on a wild strain of S. cerevisiae, but also on mutant strains: A deletion mutant Δyca1 without yeast metacaspase proves that in S. cerevisiae the apoptosis occurs partly by the caspase‐independent pathway. A petite strains with mutation in the mitochondria do not show pronounced ROS formation, but in spite of this, apoptosis is detected. Hence, mitochondrial ROS probably do not play an important role in induction of apoptosis.     

Overexpression of EAR1 and SSH4 that encode PPxY proteins in the multivesicular body provides stability to tryptophan permease Tat2, allowing yeast cells to grow under high hydrostatic pressure

Tryptophan uptake in yeast Saccharomyces cerevisiae is susceptible to high hydrostatic pressure and it limits the growth of tryptophan auxotrophic (Trp^?) strains under pressures of 15–25 MPa. The susceptibility of tryptophan uptake is accounted for by the pressure-induced degradation of tryptophan permease Tat2 occurring in a Rsp5 ubiquitin ligase-dependent manner. Ear1 and Ssh4 are multivesicular body proteins that physically interact with Rsp5. We found that overexpression of either of the EAR1 or SSH4 genes enabled the Trp^? cells to grow at 15–25 MPa. EAR1 and SSH4 appeared to provide stability to the Tat2 protein when overexpressed. The result suggests that Ear1 and Ssh4 negatively regulate Rsp5 on ubiquitination of Tat2. Currently, high hydrostatic pressure is widely used in bioscience and biotechnology for structurally perturbing macromolecules such as proteins and lipids or in food processing and sterilizing microbes. We suggest that hydrostatic pressure is an operative experimental parameter to screen yeast genes specifically for regulation of Tat2 through the function of Rsp5 ubiquitin ligase.     

Combined of ultrasound irradiation with high hydrostatic pressure (US/HHP) as a new method to improve immobilization of dextranase onto alginate gel

In this research work, dextranase was immobilized onto calcium alginate beads by the combination of ultrasonic irradiation and high hydrostatic pressure (US/HHP) treatments. Effects of US/HHP treatments on loading efficiency and immobilization yield of dextranase enzyme onto calcium alginate beads were investigated. Furthermore, the activities of immobilized enzymes prepared with and without US/HHP treatments and that prepared with ultrasonic irradiation (US) and high hydrostatic pressure (HHP), as a function of pH, temperature, recyclability and enzyme kinetic parameters, were compared with that for free enzyme. The maximum loading efficiency and the immobilization yield were observed when the immobilized dextranase was prepared with US (40 W at 25 kHz for 15 min) combined with HHP (400 MPa for 15 min), under which the loading efficiency and the immobilization yield increased by 88.92% and 80.86%, respectively, compared to immobilized enzymes prepared without US/HHP treatment. On the other hand, immobilized enzyme prepared with US/HHP treatment showed V_max, K_M, catalytic and specificity constants values higher than that for the immobilized enzyme prepared with HHP treatment, indicated that, this new US/HHP method improved the catalytic kinetics activity of immobilized dextranase at all the reaction conditions studied. Compared to immobilized enzyme prepared either with US or HHP, the immobilized enzymes prepared with US/HHP method exhibited a higher: pH optimum, optimal reaction temperature, thermal stability and recyclability, and lower activation energy, which, illustrating the effectiveness of the US/HHP method. These results indicated that, the combination of US and HHP treatments could be an effective method for improving the immobilization of enzymes in polymers.     

食品微生物对超高压处理的逆境响应

 超高压（High Hydrostatic Pressure,HHP）作为一种非热杀菌技术,在食品工业中有着很广泛的应用,与传统的热加工处理相比,在保持食品品质、杀菌、钝酶等方面都有其明显的优势。通过对国内外相关文献的分析,对HHP处理后处于亚致死状态食品微生物的细胞膜、遗传与结构物质、机体代谢等方面,以及大部分食品微生物在应对外界的多种刺激时而进行的普遍性调控机制的研究进行了总结,探讨了经过HHP处理后处于亚致死状态的食品微生物在逆境下存活的应激反应。     

蛋白饲料酵母高产菌株的选育及发酵工艺优化研究

饲料中的蛋白含量,是衡量饲料优劣的主要指标,而决定饲料蛋白含量高低的主要因素包括：菌体蛋白含量(酵母菌、乳酸菌等)及饲草中自身蛋白含量;其中饲料酵母菌株蛋白含量直接决定了饲料蛋白的含量,因此获得优良的饲料酵母菌株成为关键。本研究利用辐射能量为80 MeV/u的¹²C⁶⁺重离子束对出发饲料酵母菌种NJ3236 (蛋白质量分数为40.64%)进行辐照诱变,并对辐照后的菌种进行初筛、复筛得到高蛋白含量菌株100G-2,该菌株较NJ3236蛋白含量提高了10.08%。利用响应面分析法对发酵培养基进行优化,通过优化得到的培养基的最优配比为：甜高粱汁20.95 g/L、玉米浆干粉18.17 g/L、硫酸镁1.60 g/L,在此条件下可溶性蛋白浓度达到1.381 mg/mL,较未优化前可溶性蛋白提高了8.7%。     

Identification of a regulatory element for yeast tryptophan permease Tat2 ubiquitination using high hydrostatic pressure

ABSTRACT

Tryptophan uptake in the yeast Saccharomyces cerevisiae is extremely sensitive to high pressure; therefore, the growth of tryptophan auxotrophic strains is impaired. Degradation of tryptophan permease Tat2 is enhanced at 25?MPa, depending on Rsp5 ubiquitin ligase. Any defect in Tat2 ubiquitination confers high pressure growth capacity, which is a luminous phenotype of the yeast used to explore the mechanism by which Rsp5 mediates Tat2 ubiquitination. Here we show that the N-terminal four (K17, K20, K29, and K31) among five lysines are required for efficient Tat2 degradation under high pressure. We found that a domain spanning D70 to S76 is also critical for Tat2 degradation at 25?MPa probably because of the recognition by Bul1, an adaptor protein of Rsp5. Defects in Tat2 ubiquitination do not produce any remarkable mutant phenotype at 0.1?MPa. Therefore, we suggest that high pressure is a unique and advanced tool to explore ubiquitination-dependent Tat2 regulation.     

Sterilization of Liquid and Semi-Solid Pharmaceutical Forms Using High Pressure Technology

The aim of this study was to investigate the use of HHP (High Hydrostatic Pressure) technology as a possible alternative method for decontamination and sterilization of sensitive drugs. We demonstrated the safety of HHP treatment on several sensitive biomolecules. A 10 min. HHP treatment inactivates totally pure suspensions of P. aeruginosa , C. albicans and spores of A. niger , but S. aureus needs a longer HHP duration treatment to be totally inactivated. Endospores appear to be more baroresistant.