Improvement of the Functionality of Minced Mackerel by Hydrolysis and Subsequent Lactic Acid Bacterial Fermentation

ABSTRACT: To improve functionality and add nutritional value to mackerel, its muscle protein was hydrolyzed by the proteases from Aspergillus oryzae , and further fermented by lactic acid bacteria (LAB). Compared with minced mackerel, the antioxidative ability against linoleic acid (from 10% to 42%), α,α-diphenyl-β-picrylhydrazyl scavenging capability (from 38% to 85%), reducing power (from 0.58 to 0.83), Fe²⁺ chelating capability (from 42% to 72%), and trolox equivalent antioxidant capacity (from 2.5 to 3.5 m M ) of the hydrolysate significantly increased ( P < 0.05) after 1 h of hydrolysis and 12 h of LAB fermentation. Both hydrolysate and LAB fermented samples could stimulate the proliferation of both human hybridoma HB4C5 and mouse macrophage J774.1 cells. These data suggest that the enhancement of cell viability and antioxidative ability could be substantially improved after hydrolysis and 12 h of LAB fermentation.     

Effect of Lactic Acid Bacterial Fermentation on the Characteristics of Minced Mackerel

ABSTRACT: To improve the functionality and quality of seafood, mackerel minces were fermented with lactic acid bacteria (LAB) ∼ Lactobacillus plantarum CCRC10069, Lactococcus lactis subsp. lactis CCRC 12315, Lactobacillus helveticus CCRC 14092, or their combination at 37 °C. Rapid growth of LAB, decline in pH, suppress of main microflora, increases in whiteness, Hunter L, nonproteinous nitrogen, sensory quality, and free amino acids related to taste were observed. However, VBN of samples fermented with LAB were still d ≤ 25 mg/100g after 36 h fermentation. SDS-PAGE indicated the obvious degradation of water- and salt-soluble muscle proteins after 12 h fermentation. Animal test demonstrated the LAB-fermented mince has the functionality on reduction of blood pressure, glucose, and total cholesterol of SHR.     

New Technology for Producing Paste-like Fish Products using Lactic Acid Bacteria Fermentation

ABSTRACT: To develop a new processing technique, mackerel mince was homogenized to obtain different media with protein concentration of 90, 45, 22.5 mg/mL, respectively. Lactic acid bacteria (LAB) were inoculated to the media. During 48 h fermentation at 37 °C, rapid growth of LAB, decline in pH, suppression of main microflora, and increases in whiteness, Hunter L and sensory quality were observed and the VBN of fermented samples was still below 25 mg/100g. After 24 and 48 h fermentation, the sensory evaluation and photographic records indicated the high acceptability of the fermented products. From this study, various LAB fermented fish products can be produced and this technique has very high commercial potential.     

PURIFICATION AND CHARACTERIZATION OF PROTEASES FROM THE VISCERA OF MILKFISH (CHANOS CHANOS)

Proteases in acetone powder prepared from milkfish ( Chanos chanos ) viscera were extracted with deionized water and purified by ammonium sulfate fractionation, Sephadex G-75 gel filtration, repeated DEAE-Sephadex A-50 and CM-Sepharose CL-6B chromatography. Four fractions with caseinolytic activity, named A, B, C and D, were obtained from CM-Sepharose CL-6B and DEAE-Sephadex A-50 chromatography. The four proteases were purified to electrophoretic homogeneity. Substrate specificity studies indicated that proteases A and B were carboxypeptidase A-like and chymotrypsin-like enzymes, respectively; C and D were trypsin-like enzymes .

ABSTRACT

The optimal temperatures of proteases A, B, C and D for hydrolysis of casein were found to be 60, 60, 55 and 60°C, respectively. The optimal pH of protease A for hydrolysis of hippuryl-L-phenylalanine was 9.0, B for hydrolysis of acetyl-L-tyrosine ethyl ester was 8.0, C and D for hydrolysis of tosyl-L-arginine methyl ester was 8.0. The temperatures which inactivated 50% of enzymes in 5 min were 20°C for protease B; 51°C for protease C; 56°C for protease A; and 61°C for protease D. The molecular weights of proteases A, B, C, and D were 14,800, 16,800, 24,800 and 22,000 daltons, respectively.     

Autolysis and Protease Inhibition Effects on Dynamic Viscoelastic Properties during Thermal Gelation of Squid Muscle

ABSTRACT: Viscoelastic changes during thermal gelation of squid ( Loligo vulgaris ) muscle with protease inhibitors were studied, in order to evaluate the contribution of different proteinases to gel degradation. Calcium chloride was also tested as enzyme activator. A minimum in elastic modulus without inhibitors was achieved at 38 to 40°C. Although denaturation temperature was around 55°C, pronounced melting of connective tissue started at 40°C. Proteolysis occurred between 25 and 75°C, with maximum at 40 to 45°C. Addition of PMSF led to highest values of G'. The strong inhibition of autolytic activity by PMSF confirmed the predominance of serine proteases. Addition of CaCl₂ favored thermal protein aggregation from 40°C upwards.     

Pediococcus pentosaceus L and S Utilization in Fermentation and Storage of Mackerel Sausage

ABSTRACT During 72 h fermentation at 37 °C, rapid increase in lactic acid bacteria count (LAB), dramatic decline in pH and suppression in the growth of contaminating Pseudomonas, Staphylococcus, and Enterobacteriaceae of mackerel minces with Pediococcus pentosaceus strains L and S were observed (p < 0.05). When the mackerel mince was processed into Chinese style sausage with 4% sucrose, no significant changes in pH, growth of LAB, Aerobic Plate Count, and Pseudomonas of the fermented mackerel sausage with Pediococcus pentosaceus strains L and S were obtained (p > 0.05) during 3 wk storage at 4 °C, however, the growth of Enterobacteriaceae and Staphylococcus were effectively suppressed. The residual nitrite in sample decreased during processing and fermentation, and was minimally detected after 72 h fermentation. Suppression of VBN and psychrophilic microflora, and lowering residual nitrite suggest the potential of using these 2 strains in the fermented fish products.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF α-GALACTOSIDASE FROM ASPERGILLUS ORYZAE

A crude extract of α-galactosidase obtained by fermenting Aspergillus oryzae on wheat bran was purified 35 fold by ethanol precipitation, gel filtration and ion-exchange chromatography. The final preparation was free of protease activity but contained invertase activity. The molecular weight of the enzyme was estimated as 64,000 daltons. The pH and temperature optima were 4.0 and 60°C, respectively. The enzyme was stable over the pH range 3–7.5 and at temperatures up to 55°C (pH 4.0). The K_m values for p-nitrophenyl α-Dgalactopyranoside (PNPG) and raffinose were 4.0 × 10⁻⁴M and 1 × 10⁻²M, respectively. Divalent cations were not required for activity. More than 80% of the oligosaccharides in soy milk were hydrolyzed after 3 h at 50°C using 0.113 PNPG units/mL milk.     

Effect of Monascus Fermentation on the Characteristics of Mackerel Mince

ABSTRACT: To improve the characteristics of fish muscle, Monascus purpureus or Monascus species was inoculated in 2.1% rice powder broth containing 30% minced mackerel tissue to produce a fermented fish product. Enterbacteriaceae, Staphylococcus , and Pseudomonas were not present during the 7 d of fermentation at 25 °C. The activities of both amylase and acidic proteases were detected after 1 and 2 d of fermentation, respectively, and further increased during fermentation. The volatile base nitrogen of all Monascus fermented samples were lower than the limit of food regulation in Taiwan (< 25 mg/100 g) even after 7 d of fermentation. Hydrolysis of the muscle protein and accumulation of free amino acid were observed. The fermented fish products had good flavor and color that were highly likeable to the sensory panel.     

Effect of Heat-Stable Proteases on the Kinetic Parameters of Milk Clotting by Chymosin

The milk clotting per unit casein hydrolytic activities of proteases from 14 psychrotrophic pseudomonad isolates of raw milk ranged from 0.77 to 9.97 at 30°C. The milk clotting activities of chymodn and T16 protease were not completely additive, especially at high chymosin concentration when clotting time was relatively fast. The T16 protease was not effective in catalyzing the enzymic step of milk clbtting at O°C in the time expected on the basis of its milk clotting at 30°C. Milk incubated with the T25 motease for 8 days at 4°C and then clotted with chymosin at 30°C exhibited weak curd consistency.     

Optimization of the Freezing Conditions on Mackerel and Amberfish for Manufacturing Minced Fish

Optimal freezing, frozen storage and thawing conditions in preserving mackerel and amberfish for producing minced fish products were investigated. Based on assessments of extractability of 0.6M KCl-soluble proteins and actomyosin, Ca-ATPase activity of actomyosin, and gel-forming ability of kamaboko (kind of minced fish meat product), semi-dressed, dressed, and filleted samples showed stable and good quality of gel-forming ability during 3 months storage at −20°C. Optimal ultimate freezing temperatures of mackerel and amberfish were −20°C and between −30°C and −40°C, respectively. The optimal storage temperatures for mackerel and amberfish were −20°C and −40°C, respectively. Appropriate thawing methods for frozen mackerel were microwave and 20°C running water defrosting, while those for frozen amberfish were microwave, 20°C running water, and room temperature defrosting.     

Effect of temperature and salt on the maturation of white-salted cheese

White-salted cheeses were prepared from ultrafiltered (UF) cows' milk and salted to give final salt-in-moisture (SM) levels of 2.5, 3.2 and 4.0%. The cheeses were stored at 5°C and 10°C for up to 15 weeks. The microflora was dominated by lactic acid bacteria (LAB) but some mould growth was evident within 15 weeks at all SM levels and both temperatures. Levels of water-soluble nitrogen (WSN), attributed to chymosin activity, increased significantly with time, the rate being inversely proportional to the SM level and increasing with storage temperature. Similar effects were noted for trichloroacetic acid-soluble nitrogen (TCA-SN) and free amino acid (FAA) levels, both of which would also be affected by bacterial protease activity. The proteolytic activity was reflected by changes in the hardness and fracturability of the cheeses.     

米曲霉和乳酸菌混合制曲对小麦大曲制曲效果的影响

本文研究了米曲霉和乳酸菌混合制曲(KR)及米曲霉单独制曲(KP)条件下,小麦大曲中菌落总数、中性蛋白酶活力、酸性蛋白酶活力、总酸以及发酵液品质,探讨了多菌种制曲的可行性,以期为高品质小麦基调味料的生产提供理论指导。研究结果表明:KR工艺中小麦大曲的乳酸菌和霉菌均繁殖良好,12 h时乳酸菌达到了4.15×108 cfu/g,乳酸菌的繁殖对霉菌的生长有一定的抑制效果;米曲霉和乳酸菌混合制曲比米曲霉单独接种制曲效果好,制曲48 h时,KR工艺所得到的大曲中性和酸性蛋白酶与米曲霉纯种制曲相比,分别提高了22.79%和22.26%;KR大曲发酵液的全氮含量、氨基酸态氮含量均高于KP工艺,发酵60 d时KR发酵液的游离氨基酸含量高于KP,其中谷氨酸含量明显提高,对比KP工艺提高了24.21%,具有更明显的鲜味口感。     

Changes in selected physical property and enzyme activity of rice and barley koji during fermentation and storage

Koji are solid-state fermentation products made by inoculating steamed grains with the spores of fungi, particularly Aspergillus spp. This research was undertaken to identify the fermentation and storage conditions optimal for the production and maintenance of selected hydrolytic enzymes, such as α-amlyase and protease, in koji. Steamed rice and barley were inoculated with 2 × 10 (11) Aspergillus oryzae spores per kilogram of grains and fermented for 118 h in a growth chamber at 28 to 32 °C with controlled relative humidities. Samples were drawn periodically during fermentation and storage at -20, 4, or 32 °C, and α-amylase and protease activity, mold counts, a(w), moisture contents, and pH of collected samples were determined. It was observed that the a(w), moisture contents, and pH of the koji were influenced by the duration of fermentation and temperature of storage. The α-amylase activity of both koji increased as the populations of A. oryzae increased during the exponential growth phase. The enzyme activity of barley koji was significantly higher than that of rice koji, reaching a peak activity of 211.87 or 116.57 U at 46 and 58 h, respectively, into the fermentation process. The enzyme activity in both products started to decrease once the mold culture entered the stationary growth phase. The protease activities of both koji were low and remained relatively stable during fermentation and storage. These results suggest that rice and barley koji can be used as sources of α-amylase and desired enzyme activity can be achieved by controlling the fermentation and storage conditions. PRACTICAL APPLICATION: Amylases and proteases are 2 important hydrolytic enzymes. In the food industry, these enzymes are used to break down starches and proteins while reducing the viscosity of foods. Although amylases and proteases are found in plants and animals, commercial enzymes are often produced using bacteria or molds through solid state fermentation, which is designed to use natural microbial process to produce enzymes in a controlled environment. A properly produced and maintained koji with a high hydrolytic enzyme activity can serve as an important source of the enzymes for the food industry.     

Isolation and Selection of Lactic Acid Bacteria as Biocontrol Agents for Nonacidified, Refrigerated Pickles

ABSTRACT: A nonacidified, deli-type pickle product was used as a model system to study the potential use of biocontrol as a means to prevent the growth of pathogens in minimally processed fruits and vegetables (MPFV). Fresh pickling cucumbers were blanched and brined with sterile spices and garlic oil. The product was stored at 5 °C for 3 wk and then transferred to various abuse temperatures (16 °C, 25 °C, 30 °C). Lactic acid bacteria (LAB) were isolated and characterized as potential biocontrol agents, and the isolates were tested for bacteriocin-like activity. A total of 118 LAB isolates were obtained. Among the LAB identified were species of Lactococcus, Leuconostoc, Lactobacillus, Weissella , and Enterococcus . Three isolates showed transient bacteriocin activity against— Listeria monocytogenes , and 7 isolates ( Lactococcus ) had bacteriocin-like activity against other LAB. Although it did not produce a bacteriocin, a Lactobacillus curvatus isolate (LR55) was found to have desirable characteristics for use as a biocontrol (competitive exclusion) culture to enhance the safety of nonacidified deli-type pickles.     

BACTERIAL FORMATION OF HISTAMINE IN JACK MACKEREL (TRACHURUS SYMMETRICUS)

Peak histamine concentrations of 0.023, 0.031 and 0.027 g histamine/100 g muscle and maximal bacteria concentrations of 1.75, 1.59 and 0.423 g dry cells/100 g muscle were observed in muscles of jack mackerel stored at 25, 15 and 5C, respectively. Incubated fish homogenates suggest rate and transport limitations in histamine formation in muscle. The Mulchandani model predicted bacterial growth in muscle. The Luedeking and Piret expression fitted histamine formation in muscle; α values were 3.0 × 10⁻³, 1.23 × 10⁻² and 4.17 × 10⁻² g histamine/g dry cells, while β-values were 4.5 × 104, 8.0 × 10⁻⁵ and 0 g histamine/g dry cells × h at 25, 15, and 5C, respectively. The model predicts that jack mackerel could be stored from 4.5 to 5.5 days in ice, from 1 to 2 days at 15C and from 17 h to 2 days at 25C before fishmeal quality might be affected.     

Actinomucor elegans、Aspegillus oryzae和Rhizopus oligosporus产蛋白酶条件及蛋白酶性质的比较

比较了腐乳生产菌株Actinomucor elegans、豆酱和酱油生产菌株Aspegillus oryzae以及天培生产菌株Rhizopus oligosporus产生蛋白酶的条件和所产蛋白酶的性质。结果表明,不同的菌株产酶条件及蛋白酶的性质有较大的差异:少孢根霉主要产生酸性蛋白酶,在pH2.5-4.0的酸性介质中、32℃条件下培养时产酶能力较强,所分泌的蛋白酶系在pH5.0时酶活力最高,在pH5.0附近最稳定;米曲霉可以产生酸性、中性及碱性蛋白酶,所产生的蛋白酶活力显著高于少孢根霉和毛霉,米曲霉在酸性条件下产酸性蛋白酶能力强,在中性条件下产中性蛋白酶能力强,在碱性条件下产碱性蛋白酶能力强,在28-32℃时产酶能力强,所分泌的蛋白酶系在pH5.0-9.0的广泛范围内有很强的活力,在pH6.0-8.0的范围内稳定性强;毛霉可以产生酸性、中性及碱性蛋白酶,但酶活力明显低于米曲霉,毛霉在中性偏酸性(pH5.5)的介质中产酸性蛋白酶的能力较强,但介质的酸碱度对毛霉产中性及碱性蛋白酶没有影响,在28℃时产酸性、中性和碱性蛋白酶的能力都比较强,毛霉所分泌的蛋白酶系在pH5.0-9.0的广泛pH范围内有活力,在pH5.0-6.0时酶活力最高,在pH5.0-7.0时稳定强。     

Partial Characterization of Protease Activity in Squid (Todaropsis eblanae) Mantle: Modification by High-pressure Treatment

ABSTRACT: Optimal conditions for proteolytic activity in both nonpressurized and pressurized (300 MPa, 7°C, 20 min) squid ( Todaropsis eblanae ) muscle occurred at acid pH levels (pH 3) over a broad range of temperatures. Pressure treatment did not modify optimal pH and temperatures but did increase proteolytic activity. The acid cysteine proteases, and to a lesser extent the acid serine proteases, were the enzymes mainly affected by the high-pressure treatment. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was indicative of increased protein hydrolysis by pressurization. Myosin heavy chain in both nonpressurized and pressurized squid was degraded at all the temperatures tested, but actin was susceptible only to proteolysis in the pressure-treated muscle at 7°C and 40°C. This behavior was not observed at 55°C.     

粟米酱米曲霉菌的分离、筛选与鉴定

反复纯化培养自然发酵的粟米酱中分离的野生丝状真菌，经形态学鉴定，确认其为米曲霉菌群的米曲霉菌。用福林-酚法测定其蛋白酶活力，并确定活力最高的菌株。实验表明选育优良的米曲霉菌株不仅要考察菌株的蛋白酶活力，也要考虑菌株孢子的比生长速率。     

高温变性豆粕发酵菌株的选育

以米曲霉CICC40186为出发菌株,经紫外诱变处理后,获得对高温变性豆粕分解能力强的突变菌株8421和8422。与出发菌株相比,两突变株所产酸性、中性和碱性三种蛋白酶分别提高了73.2%、91.2%,81.1%、83.7%,37.6%、32.9%;三氯乙酸(TCA)可溶性蛋白含量分别提高了约13.0%和13.2%。经6次传代,遗传性状稳定。     

海洋湿地环境中拮抗水产品腐败菌的乳酸菌筛选与鉴定

从渤海锦州海岸湿地植物根系中筛选对腐败希瓦氏菌、铜绿假单胞菌和副溶血弧菌具有较强抑制作用的乳酸菌,为研发一种海产品防腐保鲜生物制剂奠定基础。应用牛津杯打孔法从碱蓬草、芦苇根部筛选拮抗性乳酸菌菌株,应用生长曲线分析菌株拮抗作用变化,通过pH、蛋白酶、温度、NaCl浓度等因素,分析乳酸菌无细胞上清液(CFS)的拮抗性能。从碱蓬草根部获得1株拮抗能力较强的乳酸菌株JP-12,初步判定其抑菌活性物质存在于CFS中,生长曲线结果显示其抑菌活性在培养28 h时达到峰值。CFS经不同蛋白酶处理后,抑菌活性均有所下降。对胃蛋白酶最为敏感,活性丧失率为24.21%;CFS经40~121 ℃等不同温度处理30 min,其抑菌活性基本不变;在pH3.0~4.5范围内抑菌活性保持稳定;3.0%~3.5% NaCl条件下其抑菌活性较强,抑菌直径达到18.46、18.15 mm。经生理生化实验和16S rRNA菌株JP-12被鉴定为植物乳杆菌(Lactobacillus plantarum)。本文为挖掘拮抗性乳酸菌的资源及在水产品防腐保鲜中的应用提供一条新的途径。