Production of a Milk-Clotting Enzyme by Glutinous Rice Fermentation and Partial Characterization of the Enzyme

Abstract

Fermentation parameters affecting a milk‐clotting enzyme (MCE) production by glutinous rice fermentation were evaluated using selected rice starters called Jiuqu. The MCE was produced under the following conditions: rice inoculation starter at 3%, fermentation time of 5 days, temperature at 30C, initial rice medium pH of 5.0, rice concentration of 70% and rotation speed of 120 rpm. The enzyme preparation obtained by 70% ethanol precipitation of the culture supernatant had milk‐clotting activity (MCA) of 6,160 Su/g and a C/P ratio (the ratio between MCA and proteolytic activity) of 10.81 at a recovery rate of 44.82%. The molecular mass of the MCE was 39.7 kDa. MCE production was optimal at 35C and a pH of 5.4. The presence of Na+, K+, Mg2+, Ca2+ and Al3+ stimulated enzyme activity. The Km and Vmax values were 22.90 g/L and 21.11 mL/s, respectively, with skim milk as the substrate. Practical Applications In the last two decades, different sources of milk‐clotting enzymes (MCEs) have been explored to meet the increasing demand for cheese production worldwide. This study describes a MCE isolated by ethanol fractionation from glutinous rice wine fermented by Jiuqu. The MCE, with a relatively high C/P ratio, is potentially applicable as a coagulant in cheese making. The property of thermal instability of the enzyme is advantageous as it would be inactivated during curd scalding (at about 50C), thus formation of bitter‐taste peptides caused by residual proteolytic activity of the enzyme during cheese ripening could be prevented. In addition, the optimal temperature (35C) of the MCE is within the range of temperature (30–35C) generally used during milk coagulation processes. Therefore, the results suggest that a MCE that could have potential application in cheese production may be obtained by glutinous rice fermentation under the conditions of this study.

Production, purification and characterization of a milk clotting enzyme from Bacillus methanolicus LB-1

“…The values of K m and V max for the MCE from B. methanolicus LB-1 were 0.93 g/L and 20.49 mL/U, respectively. By comparison, the MCEs reported by Hang et al (2016a;2016b) and Zhao et al (2015) had the K m values of 1.36 g/L and 22.90 g/L, respectively. A high K m value generally corresponds to a low affinity, and vice versa.…”

“…The ideal MCE was characterized with a high milk clotting activity (MCA), equivalent to the commercial enzyme with a high ratio of MCA to proteolytic activity (PA) (Ding et al, 2012). Unfortunately, despite many reports on the production, purification and characterization of various bacterial MCEs, few of them met the requirement of an ideal MCE (Sato et al, 2004;Zhao et al, 2015). Production of bacterial MCEs was influenced by cultivation temperature, filled volume of growth medium, cultivation time and rotation speed in shaking-flask cultivation (Hang et al, 2016a;2016b).…”

Utility of Milk Coagulant Enzyme of Moringa oleifera Seed in Cheese Production from Soy and Skim Milks

“…These methods measure the enzymatic and non-enzymatic reactions in milk coagulation. The work of Zhao et al [ 62 ], based on the Arima et al [ 35 ] study, determined MCA, and the results were expressed in Soxhlet units. However, it is not clear if these methods are also regularly used to measure the coagulant activity of plant extracts with other types of milk such as soymilk.…”

Change of proteolysis and sensory profile during ripening of Cheddar‐style cheese as influenced by a microbial rennet from rice wine

“…Hansen, Inc. (Khusham, Denmark). The microbial rennet (1.09 × 10 5 Su/g) was isolated and partially purified from glutinous rice‐fermented wine that had been traditionally produced in southern China for centuries by using a rice starter called Jiuqu that mainly contains yeasts such as Saccharomyces cerevisiae (Zhao et al, ). The skim milk powder was purchased from Fonterra (New Zealand); α‐lactalbumin (≥85%) and β‐lactoglobulin (≥90%) powders were purchased from Sigma‐Aldrich (USA), which were reconstituted to the required concentration using distilled water.…”

“…The selection of suitable MCE could support the desired modification of yield and flavor with increased ripening and better sensory attributes of cheese. Recently, in the authors’ laboratory, a microbial rennet was isolated from glutinous rice‐fermented wine that was traditionally used for making Chinese Royal Cheese (Zhao, Wang, Zheng, Zhao, & Yang, ). The microbial rennet was characterized as having strong milk‐clotting activity and moderate proteolytic activity.…”