An immunoassay to assess lamb and kid rennets authenticity

The potential adulteration of kid or lamb rennet with calf rennet is of interest for some Protected Designation of Origin cheeses producers and those looking for a specific cheese typicality. The approach proposed here for the authentication of kid or lamb rennet is based on the immuno-detection of bovine pepsin possibly present in calf rennet in varying quantities. The developed immunoassay (indirect ELISA) used a monoclonal antibody (mAb) raised against bovine pepsin. This mAb was found to be specific as it didn't cross-react with the pepsin of animal species other than bovine (kid, lamb, pig) and with other milk-clotting enzymes (chymosin and microbial enzymes). Adulteration tests were conducted with kid and lamb rennets spiked with a wide range of calf rennet (from 0 to 100% v/v). The presence of bovine pepsin was detected at low levels down to 6 mg/L in kid and lamb rennets. Good linear relationships were obtained between added bovine pepsin concentration and the absorbance values over the range 1.25–120 mg/L. Results showed that indirect ELISA proved to be an interesting tool for testing rennets authenticity targeting bovine pepsin as an indicator of the bovine adulteration of kid and lamb rennets.     

产凝乳酶微生物的研究概况

凝乳酶用途广泛,其来源主要有动物凝乳酶、植物凝乳酶和微生物凝乳酶,微生物凝乳酶一直是凝乳酶研究的热点。对近年来国内外产凝乳酶真菌、细菌和放线菌种类以及产凝乳酶微生物的选育的研究情况进行了综述,以期为微生物凝乳酶的研究和开发提供参考。     

Purification and characterization of a pregastric esterase from a hygienized kid rennet paste

Rennet pastes obtained by maceration of gastric tissues from suckling kids are used traditionally to produce some artisanal cheeses in Spain. Besides milk-clotting function, rennet pastes provide proteolytic activity and lipolytic system, essentially pregastric, necessary in the development of piquant flavor typical of these cheeses. A simple and reproducible procedure allows us to obtain a standardized rennet paste that posses the desired activity and is of good microbiological quality. Concomitantly, a kid pregastric esterase (KPGE) was purified to homogeneity. The purification procedure was based on an aqueous extract of hygienized rennet paste (HRP), which was chromatographed on DEAE-Sepharose Fast Flow then adsorbed on phenyl superose followed by a re-chromatography on the same column. The final enzymatic preparation, where the overall activity recovery was 3%, showed a molecular mass of 53 kDa. The highest activity was determined on p-nitrophenyl butyrate, but marked hydrolysis was also detected on beta-naphthyl caprylate. In contrast, low activity on tributyrin (substrate under emulsion form) was detected, thus confirming the esterase character of purified enzyme.     

Standardization of milk using cold ultrafiltration retentates for the manufacture of parmesan cheese

The effects of using cold ultrafiltered (UF) retentates (both whole and skim milk) on the coagulation, yield, composition, and ripening of Parmesan cheese were investigated. Milks for cheese making were made by blending cold UF retentates with partially skimmed milk to obtain blends with 14.2% solids and a casein:fat ratio of 1.1. Cutting times, as selected by the cheese-maker, were approximately 15 and approximately 20 min for experimental and control milks, respectively. Storage modulus values at cutting were similar, but yield stress values were significantly higher in UF retentate standardized milks. Cheese yields were significantly higher in UF retentate standardized milks (approximately 12%) compared with control milk (cream removed) (approximately 7 to 8%). Significantly higher protein recoveries were obtained in cheeses manufactured using cold UF retentates. There were no differences in the pH and moisture contents of the cheeses prior to brining, and there was no residual lactose or galactose left in the cheeses. Using UF retentates resulted in a significant reduction in whey volume as well as a higher proportion of protein in the solids of the whey. Proteolysis, free fatty acids, and sensory properties of the cheeses were similar. The use of milk concentrated by cold UF is a promising way of improving the yield of Parmesan cheese without compromising cheese quality. The question remaining to be answered by the cheesemaker is whether it is economical to do so.     

Mechanisms of Syneresis in Rennet Curd without Mechanical Treatment

An original method for syneresis evaluation was developed, based on image analysis which enabled direct monitoring of curd shrinkage. Image analysis was employed in combination with the customary tracer method to follow the effects of cutting on curd syneresis. The two methods were then utilized to study curd syneresis in the presence of ethanol, a solvent less polar than water. Curd syneresis was reduced in the presence of diluted ethanol which led to the hypothesis that the mechanism which causes rennet curd to synerese probably relates to changes in protein conformation, essentially due to hydrophobic bonding.     

Rennet Coagulability of High-Heat Treated Milk Influenced By Time of pH Adjustment

The coagulability of high-heat treated (91°C for 16 or 60 set) milk was measured with a Formagraph. Coagulation (hysteresis) and curd formation by such milk was increased by direct acidification to pH<6.4 before rennet addition. Coagulation properties were increased greatly when such milk was pH-cycled (i.e., acidified to pH 5.5, held overnight at 4°C, then neutralized to pH 6.2–6.4) before rennet addition.     

Optimization of processing parameters for the ultrasonic extraction of goat kid rennet

Using abomasums of 2- to 3-week-old goat kids as material, the effects of some factors such as ultrasound intensity, extraction time, NaCl concentration in extraction solution and ratio of abomasums mucosa to extraction solution on the extraction yield of goat kid rennet were investigated. On the basis of single factor experiments, the processing parameters for ultrasound extraction were optimized using a four-factor orthogonal and rotatory combinatorial design. The results showed that the optimum processing parameters for the extraction of goat kid rennet were: ultrasound intensity, 44 W/cm²; extraction time, 25 min; NaCl concentration in extraction solution, 16%; and the ratio of abomasumal mucosa to extraction solution, 1 : 30. In addition, the ultrasonic extraction, as compared with traditional extraction, proved to be more effective.     

Partial identification of water-soluble peptides released at early stages of proteolysis in sterilized ovine cheese-like systems: influence of type of coagulant and starter

Cheese-like systems were manufactured from sterilized ovine milk, using crude aqueous extracts of Cynara cardunculus or cardosin A isolated therefrom as clotting agent. The effect of adding a commercial starter culture was also assessed. The impact of the type of coagulant used during the initial 24 h of proteolysis was evaluated via separation of peptides in the water-soluble extracts by reverse-phase HPLC, followed by partial sequencing via Edman degradation. Cardosin A accounted for most events of primary proteolysis. The major cleavage sites were Phe105-Met106 in κ-casein, and Leu127-Thr128, Ser142-Trp143, Leu165-Ser166, and Leu190-Tyr191 in β-casein. The starter culture did not play an active role during the initial stages of ripening.     

The effect of coagulant type on yield and sensory properties of semihard cheese from laboratory‐, pilot‐ and commercial‐scale productions

Using calf rennet or a commercial microbial rennet substitute derived from Rhizomucor miehei cheesemaking experiments were performed at laboratory and pilot scale, and at commercial scale in two industrial dairy plants during regular production. At all levels of scale, the solids transfer from milk to curd was significantly higher (0.50–1.19%) when using calf rennet. There were significant differences in levels of proteolysis during maturation and in levels of bitterness at 12 weeks of ripening between Gouda cheeses produced with calf rennet or with commercial rennet substitute at pilot and at commercial scale.     

Characterization of wine rennet and its kinetics by gel electrophoresis

The rennet of glutinous rice wine (wine rennet) is an exclusive clotting agent for Chinese Royal cheese production. Some characterizations are reported herein in an attempt to provide evidence about the use of the protease as either a rennet substitute or an accelerator in cheese making and ripening. The results showed that wine rennet was a monomeric and unglycosylated protease. The N-sequencing indicated a high degree of similarity to other fungal rennets. The cleavage sites of wine rennet on oxidized insulin B chain identified by HPLC-mass spectrometry included Gln⁴-His⁵, Ala¹⁴-Leu¹⁵, Leu¹⁵-Tyr¹⁶, Tyr¹⁶-Leu¹⁷, and Phe²⁴-Phe²⁵ at pH 6.5, which were similar to those observed for Mucor rennet, but different from calf chymosin except for Leu¹⁵-Tyr¹⁶. A comparison study of the kinetic properties of wine rennet on bovine caseins with that of rennets from calf and Mucor miehei by gel electrophoresis showed that these rennets had similar coagulation efficiency but different reaction rates. Wine rennet exhibited a higher degree of degradation than the calf and Mucor enzymes at pH 6.5 and 40°C. Therefore, wine rennet would be an adjunct for calf rennet or an accelerator in cheese making.