Commercial Rennet Research Articles

Improvement of camel milk-clotting: Usefulness of crude extract from green pods of carob (Ceratonia siliqua. L) as a substitute for commercial rennet.

Camel milk transformation into cheese remains an objective to be improved today. This study aimed to improve camel milk clotting using a crude extract from green pods of carob as a substitute for commercial rennet. The composition of the crude carob extract was determined for dry matter and protein content. Milk clotting conditions were studied at different temperatures, pH and CaCl2 concentrations. Milk clotting properties were assessed by milk clotting activity, specific activity and proteolytic activity. Enzymatic hydrolysis of camel milk caseins by crude carob extract and its inhibition were demonstrated by SDS-polyacrylamide gel electrophoresis. Crude carob extract analysis showed a protein and dry matter content of 23.26±0.5 mg/ml and 30.66±0.5 g/l, respectively. Optimal milk clotting activity was observed at 53.6 °C, pH 4.5, and 0.09 M CaCl2. The crude carob extract showed a high milk clotting activity (4.97 U/ml) and a low proteolytic activity (0.04U/ml) with camel milk. The cheese yield of curd produced from camel milk using crude carob extract was the highest (23.95%) compared with that of Camel chymosin (20.5%). The high ratio of milk-clotting to proteolytic activity shows the potential of this extract as a substitute for commercial rennet in the dairy industry.

Chemical, microbiological, textural, and sensory characteristics of pilot-scale Caciofiore cheese curdled with commercial Cynara cardunculus rennet and crude extracts from spontaneous and cultivated Onopordum tauricum

The aim of this study was the chemical, microbiological, textural, and sensory characterization of pilot-scale prototypes of an Italian ewe’s raw milk cheese (Caciofiore) curdled with commercial Cynara cardunculus rennet, used as a control, and crude extracts obtained from flowers of either spontaneous or cultivated Onopordum tauricum. Hence, the control and experimental cheese prototypes produced in two rounds of cheesemaking trials were assayed, at the end of their 60-day maturation, for the following features: pH, titratable acidity, dry matter, fat, total and soluble nitrogen (TN and SN, respectively), ash, salt, protein, lactose, viable plate counts and composition of the bacterial and fungal populations, color, texture, volatile organic compounds (VOCs), and olfactory attributes by sensory analysis (the latter for the sole prototypes curdled with the commercial rennet and the extract obtained from cultivated O. tauricum). The data overall collected showed a very low impact of the type of thistle rennet on the analyzed cheese traits, with significant differences being exclusively found for SN/TN%, titratable acidity, color, and adhesiveness. By contrast, a higher impact of the cheesemaking round was seen, with significant differences being observed for salt content, load of presumptive lactobacilli, thermophilic cocci, and Escherichia coli, and levels of the following VOCs: 2,3-butanedione, 2-pentanone, 1-butanol, 2-heptanone, 3-methyl-1-butanol, 2-heptanol, 2-nonanone, dimethyl trisulfide, 2-methyl propanoic acid, butanoic acid, and 3-methyl butanoic acid. Sensory analysis revealed a strong ewe’s cheese odor, accompanied by other olfactory notes, such as pungent, sour curd, sweet, and Parmesan cheese-like notes, in all the analysed cheese prototypes. Moreover, key odor active compounds, including butanoic acid, ethyl butanoate, 2,3-butanedione, 1-octen-3-one, and dimethyl trisulfide, were identified by GC-olfactometry analysis. Regarding the odor attributes as determined by sensory analysis, again the type of rennet had an almost negligible impact, with significant differences being only perceived for 1 or 2 out of 20 odor attributes, depending on the analytical conditions applied. Although some aspects deserve further investigation, the results herein collected confirm that O. tauricum can be regarded as an alternative source of thistle rennet for the manufacture of Caciofiore cheese, and more in general, Mediterranean ewe’s milk cheeses.

Production, purification, and characterization of a novel milk-clotting metalloproteinase produced by Bacillus velezensis YH-1 isolated from traditional Jiuqu

Bacillus velezensis YH-1 isolated and identified from conventional rice wine starter (Jiuqu) was shown to produce a milk-clotting enzyme (MCE) with remarkable milk-clotting activity (MCA) (642.96 SU/mL). The MCE was produced and purified with a specific 5762.7 SU/mg activity. LC–MS/MS combined with 3D structural modeling was used for the identification of MCE and this analysis indicated that it was a Zn2+ metalloproteinase. The MCE was shown to be relatively stable between pH 6–7 at temperatures below 50 °C. Certain parameters (Km and Vmax are 8.58 mg/mL and 15.65 U/min) were calculated for MCE that could cleave casein at the Lys21-Ile22 and Tyr30-Val31 links. The purpose is to destabilize casein structure and allow coagulation. Whole-genome sequencing and Liquid Chromatograph Mass Spectrometer/Mass Spectrometer (LC-MS/MS) analysis determined the MCE zymogen-producing gene from B.velezensis YH-1 to be 1566 bp long, yielding a protein that is 56.8 kDa in mass and containing 521 amino acid residues. According to an additional structural simulation study, the MCE was hydrophilic and included more-helices, contributing to its structural stability. The signal peptide was in the first 27 amino acids of the MCE. When compared to commercial rennet, MCE was superior in its ability to increase the milk curd's flexibility and viscosity and speed up the coagulation process. This novel study document the presence of a metalloproteinase milk-clotting factor from B. velezensis, the MCE has the potential to replace chymosin in cheese production.

Analysis of the effect of Tenebrio Molitor rennet on the flavor formation of Cheddar cheese during ripening based on gas chromatography-ion mobility spectrometry (GC-IMS)

This study aimed to evaluate the potential application of Tenebrio Molitor rennet (TMR) in Cheddar cheese production, and to use gas chromatography-ion mobility spectrometry (GC-IMS) to monitor flavor compounds and fingerprints of cheese during ripening. The results indicated that Cheddar cheese prepared from TMR (TF) has fat content significantly lower than that of commercial rennet (CF) (p < 0.05). However, the results of the sensory evaluation showed that there were no statistically significant differences between the two kinds of cheese (p > 0.05). Both cheeses were rich in free amino acids and free fatty acids. Compared to the CF cheese, gamma-aminobutyric acid and Ornithine contents of the TF cheese reached 187 and 749 mg/kg, respectively, during 120 days of ripening. Moreover, GC-IMS provided information on the characteristics of 40 flavor substances (monomers and dimers) in the TF cheese during ripening. Only 30 flavor substances were identified in the CF cheese. The fingerprint of the two kinds of cheese during ripening can be established by GC-IMS and principal component analysis based on the identified flavor compounds. Therefore, TMR has potential application in Cheddar cheese production. GC-IMS might be applied for the quick, accurate and comprehensive monitoring of cheese flavor during ripening.

Implementation of Novel Autochthonous Microencapsulated Strains of Lactiplantibacillus plantarum, Lactococcus lactis, and Lamb’s Rennet in the Production of Traditional “Paški Sir” Cheese

This study aimed to implement a microencapsulated form of selected autochthonous lactic-acid bacteria (LAB) isolated from the cheese-production chain and natural rennet obtained from suckling lambs in the traditional production of hard sheep cheese, “Paški sir”, from the island of Pag, Croatia. Two different formulations of microparticles were prepared: (i) microparticles containing the strain of both Lactiplantibacillus plantarum and Lactococcus lactis (S2) and (ii) microparticles containing both strains and natural rennet (S3). These formulations were used in the production of Paški sir cheese simultaneously with standard production using non-encapsulated commercial starter cultures and commercial rennet (S1). The number of Lc. lactis isolates decreased at day 30 and were not isolated during the remaining ripening process, whereas the number of L. plantarum remained stable throughout the ripening process. The level of LAB and the release of the rennet from microsphere formulations at the end allowed for the production of cheese with the same characteristics as the commercial product, indicating no negative interactions of natural rennet, bacterial culture, and chemical components of microparticles. To our knowledge, this is the first report of a microencapsulated L. plantarum (isolated from the abomasum of lambs) coupled with natural lamb’s rennet used in the production of hard sheep cheese. This pilot study showed the great potential for maintaining authenticity in cheese production by combining traditional and sustainable innovative technologies.

Chemical Composition, Fatty Acid Profile, and Lipid Quality Indices in Commercial Ripening of Cow Cheeses from Different Seasons.

Simple SummaryCheese and other dairy products are important components of the diet that have a positive effect on human health. These products include substantial amounts of important nutrients, including proteins, bioactive peptides, amino acids, fat, fatty acids, and vitamins. The quantitative composition of fatty acids in milk fat changes under the influence of various factors, such as animal feeding, breed, lactation stage, individual characteristics, climatic conditions, health, age, and others. Of the above-mentioned factors, the most important influence is the diet. This work presents chemical composition and fatty acid profile, with particular emphasis on trans isomers (cis9trans11 C18:2 (CLA), C18:1 and C18:2 isomers) and lipid quality indices in ripening of cow cheeses from different seasons. The first batch contained cheeses produced in winter and purchased from the market between May and June 2020. The second batch contained cheeses produced in summer and purchased from the market between November and December 2020. Results obtained show differences between summer and winter cheeses in their chemical composition, the content of lipid quality indices, and fatty acids.The aim of the study was to compare and demonstrate whether commercial rennet ripening cheeses available on the market in summer and winter differ in their chemical composition, fatty acid profile, content of cis9trans11 C18:2 (CLA) acid and other trans isomers of C18:1 and C18:2 acid and whether they are characterized by different values of lipid quality assessment indices. The experimental material consisted of rennet ripening of cheeses produced from cow’s milk available in the Polish market. The first batch contained cheeses produced in winter and purchased from the market between May and June. The second batch contained cheeses produced in summer and purchased between November and December. Chemical composition was analyzed by FoodScan apparatus. The gas chromatography (GC) method was used to determine the content of fatty acids. Results obtained in the presented study indicate that the chemical composition, content of fatty acids trans isomers, and lipid quality indices varied between summer and winter cheeses. The summer cheeses were richer sources of MUFA and PUFA compared to winter cheeses. Summer cheeses were also characterized by lower content of SFA, higher content n − 3, lower n − 6/n − 3 ratio, and higher content of DFA. Higher contents of CLA and trans C18:1 and C18:2 were found in summer cheeses.

Effect of pH on the coagulation properties of Tenebrio molitor coagulant

SummaryBecause of the continuous increase in global cheese consumption, there is a continual need to find new coagulants for producing cheeses with different characteristics to suit various consumers. A new coagulant was extracted from Tenebrio molitor larvae. We then investigated the influence of pH on the coagulation properties of Tenebrio molitor coagulant. The results indicate that the rennet flocculation time and whey OD value of Tenebrio molitor coagulant increased with the increase in coagulation pH (P &lt; 0.05). The water holding capacity, turbidity, particle size and storage modulus increased and then decreased (P &lt; 0.05) and reached the maximum at pH 5.6. There was a decrease and then increase for casein glycopeptide in the hydrolysis solution (P &lt; 0.05), and its content reached 0.64 mg/mL at pH 6.2; the minimum hydrolysis of Tenebrio molitor coagulant was found at pH 5.6. SDS‐PAGE, secondary structure and confocal laser scanning microscope indicated that the curd structure was stable at pH 5.6, and the coagulation performance was better. Moreover, there were the smallest differences for the texture structure of Tenebrio molitor coagulant curd with commercial rennet curd at pH 5.6. Therefore, pH 5.6 is the optimum pH of Tenebrio molitor coagulant. The study provides a theoretical basis and reference for the development of a new edible insect coagulant and its application.

Organoleptic Test on Fresh Cheese and Papain Enzyme Rennet Coagulant from Goat's Milk Based on Storage Time

Innovations in milk processing include making it into fresh cheese using local coagulant. This study aims to determine the effect of papain enzyme levels on the coagulation process of milk formaking fresh cheese. This research method used a 4 x 4 factorial completely randomized design with 2 replications of factor A giving papain enzyme levels, namely A0 = 0.2 g (using commercial rennet), A1 = 0.1 g papain enzyme rennet, A2 = 0.2 g papain enzyme rennet, A3 = 0.3 g papain enzyme rennet, and factor B storage time B0 = 0 days B1 = 5 days B2 = 10 days and B3 = 15 days. The variables measured were organoleptic tests including color, aroma, and preference. Based on the results of the study, it was found that from the addition of four levels of papain enzyme used and four variations of storage time, the administration of papain enzyme 0.3 g (A3) and storage time of 15 days (B3) was the optimal treatment to increase color, aroma, and preference for cheese and can be used as an alternative to the use of rennet

Physico-chemical, textural and microstructural properties during the cheese curds manufacturing with different commercial rennets

Amaç: Bu çalışmada, peynir pıhtısı üretimi sırasında farklı ticari rennetlerin fiziko-kimyasal, tekstürel ve mikroyapısal niteliklerde oluşturduğu değişimleri belirlemek amaçlanmıştır.Yöntem ve Bulgular: Süt termize (60 °C 15s) edildikten sonra yaklaşık 6.02 pH’ya kadar ön asitlendirme sonrası 3 eşit kısma ayrılmıştır. Mikrobiyal fermente rennet (M), %100 buzağı renneti (C1) ve rekombinant fermente rennet (C2), maya kuvvetine göre hesaplanan oranlarda her bir kısım süte 33 °C’de ilave edilerek pıhtılaşması sağlanmıştır. Süt jelinden kesim öncesi analizler için örnek alındıktan sonra; peyniraltı suyunu uzaklaştırmak için uygulanan baskılama işleminden sonra peynir pıhtısı 6x6x6 cm3 boyutlarında kalıplara kesilmiştir. Kalıplar, sıcak (60 °C) peyniraltı suyuna daldırılmış ve 30 dk süreyle ısıl işlem uygulanmış ve kalıpların merkez sıcaklığı 55 °C’ye ulaşmıştır. Isıl işlem sonrası 22 °C’ye soğutulan peynir kalıplarından analizler için örneklemeler yapılmıştır. Sonuç olarak, C1 pıhtısında en yüksek kuru madde tespit edilirken; toplam organik asit ve toplam karboksilik asitler en düşük miktarda tespit edilmiştir. M ve C2 pıhtılarında ise propiyonik asit ve heksanoik asit ile heksanal en yüksek tespit edilmiş; mikroyapıda daha az boşluklu yapı gözlemlenmiştir.Genel Yorum: Farklı pıhtılaştırıcılar [mikrobiyal rennet (M), buzağı renneti (C1), ve rekombinant fermente rennet (C2)] kullanılarak üretilen jeller benzer tekstürel nitelikler göstermesine karşın uçucu bileşenlerin oranları bakımından farklılık ortaya koymuşlardır. Peynir pıhtıları ise benzer renk değerlerine sahip olmasına rağmen; tüketiciler tarafından ürünlerin kabul edilebilirliğinde önemli rol oynayan tekstürel parametreler, organik asitler, uçucu bileşenler, asitlik ve pH değerleri açısından önemli farklılık göstermişlerdir. Dolayısıyla pıhtılaştırıcı çeşidinin pıhtı kalitesine üzerine önemli bir rol oynadığı ifade edilebilir.Çalışmanın Önemi ve Etkisi: Tekstür ve mikroyapı birbiri ile oldukça ilişkili niteliklerdir. Toplam kurumadde içeriklerinden bağımsız olarak pıhtıdaki bileşenlerin yapısal organizasyonu, tekstürü etkileyebilmektedir. Hem koruyucu etkisinden hem de lezzete katkısından dolayı yüksek laktik ve propiyonik asit içermesi, en yüksek randıman, en yüksek uçucu karboksilik asit oranı, en düşük pH değeri ve en yüksek sertlik değeri sergileyen pıhtıya neden olduğundan rekombinant fermente peynir mayası diğer mayalara kıyasla tercih edilebilir.

Kluyveromyces lactis and Torulaspora delbrueckii: Probiotic characterization, anti-Salmonella effect, and impact on cheese quality

Cheese presents remarkable diversity and organoleptic richness favored by microbial communities. Different yeasts have already been reported as contributors to cheese characteristics. In this study we aimed to characterize K. lactis B10 and T. delbrueckii B14 probiotic potential and to evaluate their impact on cheese. Yeasts were subjected to hemolysis, mucin degradation, co-aggregation with pathogens, in vivo survival, and antagonism to salmonellosis. Cheeses were produced with pasteurized milk coagulated using commercial rennet. Cheeses were evaluated for texture profile and sensory acceptance up to 40 days of maturation. Both yeasts presented higher co-aggregation to Salmonella than Saccharomyces boulardii. Although both yeasts satisfactorily survived the passage through the gastrointestinal tract, only K. lactis B10 resulted in high survival rate (90%) of mice against salmonellosis. Cheese produced with yeasts on the surface presented no eyes formation, while yeasts inoculated in the mass increased eyes formation. At 21 days of maturation yeasts inoculated in the mass resulted in lower hardness (1787.91), gumminess (1528.70) and chewiness (1339.63). Taste and purchase intention were higher for the cheese produced with yeasts inoculated in the mass. For all maturation times evaluated, most of the tasters expressed a preference for the cheese produced with yeasts inoculation in the mass.

Camel Milk-Clotting Using Plant Extracts as a Substitute to Commercial Rennet

The transformation of camel milk into cheese is an operation considered very delicate because of several difficulties encountered in achieving coagulation. The present study aims to improve coagulation abilities of camel milk using enzyme extracts from pineapple, kiwi, and ginger. Our results concerning the characterization of the enzymatic extract showed an extraction yield that varies according to the type of extract (pineapple: 75.28% ± 4.59, kiwi: 63.97% ± 5.22, and ginger: 28.64% ± 1.47). The optimum coagulation conditions of the 3 types of extract were as follows: for pineapple: pH = 5 and temperature = 45°C; for kiwifruit: pH = 6.6 and temperature = 40°C; and for ginger: pH = 6.6 and temperature = 45°C. A fresh cheese was made from camel milk with a particular nutritional quality and consistency. The kiwi proteases displayed chymosin-like properties and thus hold the best potential for use as a milk coagulant in cheese production.

A novel electrochemical assay for chymosin determination using a label-free peptide as a substrate

Chymosin is a predominant enzyme in rennet and is used in cheese production because of its excellent milk-clotting activity. Herein, we proposed a facile and label-free electrochemical method for determining chymosin activity based on a peptide-based enzyme substrate. The synthesized substrate peptide for chymosin was assembled onto the surface of the Au-deposited grassy carbon electrode.The current was proportional to chymosin activity, and thus chymosin activity could be determined. The detection ranges of chymosin activity were 2.5 to 25 U mL-1. The detection limit of chymosin activity was 0.8 U mL-1. The sensing platform was used to quantify chymosin activity in commercial rennet with high selectivity, excellent stability, and satisfactory reproducibility.We developed a facile, fast, and effective electrochemical assay for detecting chymosin activity, which has potential applications in cheesemaking.

Characterization of Ficus benjamina and Artocarpus heterophyllus Proteases as Potential Rennet Alternatives

Rennet, a milk coagulant exhibiting proteolytic activity, is a crucial component in cheese industries. Its price and availability have discouraged the growth of some small scale cheese industries. Therefore, an alternative for rennet will be beneficial for the industries. Among other sources, plant proteases offer some advantages as rennet alternatives. This study aimed to investigate the potential of plant proteases obtained from the latex as potential rennet alternatives. A total of six plants from the genus Ficus and Artocarpus were screened for their proteolytic activity and milk coagulating ability. The screening indicated that all six tested plants displayed proteolytic activity at various levels, but only Ficus benjamina and Artocarpus heterophyllus produced a firm milk curd. Hence, both F. benjamina and A. heterophyllus were determined to be the most potential. Further characterizations suggested that F. benjamina and A. heterophyllus protease were optimum at pH 7.0 also at 50°C and 40°C, respectively. At their optimum conditions, both proteases exhibited a lower MCA/PA ratio than that of the rennet. This study contributed to scientific knowledge development by becoming the first to characterize the optimum conditions of F. benjamina and A. heterophyllus’ proteases, investigate their MCA/PA ratio, and compare their activity against commercial rennet. The examination of their potentials as rennet alternatives could benefit small cheese industries and the communities.

Vegetable Rennet Tablets for Fresh Cheese Making

Indonesia has a Muslim population of around 200 million and many make religious trips to Arabia and taste fresh cheese. Therefore in Indonesia there is a demand for fresh cheese. In this regard, there is also a need to use a halal cheese starter. The purpose of this study is to prove that the use of plant-based rennet is able to produce fresh cheese which is also a halal rennet because it comes from plants. A1 = 0,1 A2 = 0,2 A3 = 0,3 and the factor B storage time (days) is B0 = 0 days, B1 = 5 B2 = 10 and B3 = 15. Variables measured include water content, dry matter, protein, fat from fresh cheese and analysis of the cost of making fresh cheese with a commercial rennet starter compared to vegetable rennet. The results showed that the dosage of using vegetable rennet papain enzyme was 0.2 g and optimum storage time for 10 days in increasing protein content and dry matter content and being able to reduce fat and water content in fresh cheese from Murrah buffalo milk.&#x0D; &#x0D;

Application of Milk-Clotting Protease from Aspergillus oryzae DRDFS13 MN726447 and Bacillus subtilis SMDFS 2B MN715837 for Danbo Cheese Production

This study aimed to investigate the efficiency, biochemical composition, and sensory quality of Danbo cheese produced using proteases derived from the fungus and bacterium compared to the commercial product. A fungal enzyme from Aspergillus oryzae DRDFS13MN726447 and a bacterial enzyme from Bacillus subtilis SMDFS 2B MN715837 were produced by solid-state and submerged fermentation, respectively. The crude enzyme from A. oryzae DRDFS13 and B. subtilis SMDFS 2B was partially purified by dialysis and used for Danbo cheese production using commercial rennet (CHY-MAX® Powder Extract NB, Christian Hansen, 2235 IMCU/g) as a control. The Danbo cheese produced using dialyzed fungal enzyme (E1) (267 U/mL), dialyzed bacterial enzyme (E2) (522 U/mL), and commercial rennet (C) were analyzed for body property, organoleptic characteristics, and proximate and mineral composition when fresh and after 2 months of ripening. There was no significant difference in the cheese yield (C = 9 kg, E1 = 8.6 kg, and E2 = 8.9 kg) among the three treatments. The body properties of Danbo cheese produced with the fungal enzyme (E1) were firm and acceptable as the control (C), whereas the Danbo cheese produced by bacterial enzymes has shown a watery body. The overall organoleptic characteristics of Danbo cheese produced by the fungal enzyme (5.3) were similar to control cheese produced by commercial rennet (5.5). Both cheese types were significantly different in organoleptic properties from Danbo cheese produced by the bacterial enzyme (4.9). There was no significant difference (p&gt;0.05) in the proximate composition between the ripened Danbo cheese produced by fungal enzyme and the control cheese except for crude protein content. However, the ripened cheese products showed a significant difference in their mineral composition except for sodium. In conclusion, this study demonstrated that the fungal enzyme from Aspergillus oryzae DRDFS 13 is more appropriate for Danbo cheese production than the bacterial enzyme from Bacillus subtilis SMDFS 2B. However, it requires further application of the enzymes for the production of other cheese varieties.

Statistical optimization of B. subtilis MK775302 milk clotting enzyme production using agro-industrial residues, enzyme characterization and application in cheese manufacture

Abstract The agricultural and industrial wastes are environmental pollutants. So, there is an increasing demand for more efficient exploitation of these wastes and their conversion into useful products. Recently, the limited availability of calf rennet led to a search for rennet substitutes for cheese manufacture. In this study, Bacillus subtilis subsp. subtilis strain 168 (B. subtilis MK775302) was isolated from the marine sponge Pseudoceratina Arabica and it was used for milk clotting enzyme (MCE) production using some agro-industrial wastes. The production medium containing agro-industrial residues was optimized by using Plackett-Burman (PB) and central composite designs (CCD). After optimization, the MCE productivity was increased by 2.3-fold compared to the non-optimized medium. The enzyme showed maximum activity at 70 °C, pH 5.0. Ultrafilterated (UF) white soft cheese production was studied by using the crude extract of B. subtilis MK775302 in a comparison with commercial rennet. The cheese produced by the crude extract of B. subtilis MK775302 had a higher ripening index, higher acidity, higher flavor intensity, and acceptable organoleptic score. Therefore, this study offered the production of a promising rennet substitute that is eco-friendly with low production costs and competes with the commercial coagulants for the production of acceptable UF white soft cheese.

Protein composition and textural properties of inulin-enriched tofu produced by hydrothermal process

Tofu protein composition changes induced by a pilot plant processing method involving hydrothermal cooking, commercial rennet as coagulant and inulin as functional ingredient were assessed. Glycoproteins (γ-, β-conglycinin) were firmly bound to the matrix of the obtained gels. Extractable proteins of inulin-enriched tofu were with mainly basic 7S-globulin, and polypeptides of glycinin. Polypeptides A1,2,4 and B1,2,4 were found in higher concentrations in protein extracts of tofu than other 11S polypeptides. Tofu was characterized by high total (417.6–556.5 g/kg) and extractable soluble (155.3–367.5 g/kg) protein content, good protein extractability (37.2–68.6%), both total protein (52–69%) and soluble protein (50–76%) recovery. Increasing concentrations of inulin proportionally improved tofu hardness (6.23–8.98 N/cm2) contributing to the uniformity of gel microstructures. Inulin played an important role in incorporating β- and γ-conglycinins into the gel matrix and by increasing the concentration of inulin in soymilk before gelation, the content of extractable soluble proteins, protein extractability and soluble protein recovery of produced tofu were also proportionally increased. For all tested sensory attributes the grades were increased with increasing inulin content in the samples. Under the applied production method, protein composition and proportion of inulin had important roles in defining tofu quality.

Production of Extracellular Rennin-Like Enzyme by a Newly Isolate Mucor circinelloides (von Tieghem) and its Application in Camembert Cheese Making

Introduction: Aspartic proteases produced by different non-pathogenic fungi belonging to mucorales, are commonly used as milk-coagulants. The present study aims to optimize conditions for milk-clotting protease production by a new strain of Mucor genus and to assess its ability in soft cheese making. Materials and Methods: About 20 fungal strains isolated from soil were investigated for their potential to produce milk-clotting proteases for further applications in cheese making. The hyper producer strain Mucor circinelloides 2095-2047 was selected for optimization of rennin-like enzyme production under solid-state fermentation (SSF) using the stepwise modifications of the selected parameters. The enzyme produced under the optimal conditions was partially purified and then applied in Camembert cheese making trials compared to the crude extract and commercial rennet. Results: The maximum milk-curdling activity achieved after optimization (571.43 SU/mL) was obtained using wheat bran (10 g) as the mainly source of carbon containing 1% galactose; moistened with the M-9 solution (pH 6.0) and incubated at 30°C for 96 hours. The enzyme of M. circinelloides was partially purified with a high recovery of 105% and 6.23-fold purity after (NH4)2SO4 fractionation and dialysis. The physicochemical properties of the three produced cheeses were very close. A low sensory quality of cheese was obtained with the crude extract which was getting better using the pre-purified enzyme. This extract was able to develop a very close or even a similar sensory quality to that obtained by the commercial rennet. Conclusions: According to findings, it is possible to propose the purified enzyme of M. circinelloides as a new alternative for rennet, but further optimization, purification and cheese production tests are required.

CHARACTERIZATION OF MILK CLOTTING PROPERTIES OF ADULT CATTLE RENNET MODIFIED WITH Moringa oleifera SEEDS AND ITS SUITABILITY AS CALF RENNET ALTERNATIVE IN CHEESE MAKING

The present study focused on the mixing of adult cattle rennet with Moringa seedsduring the extraction process to improve its strength and use it as an alternative for the calf rennet incheese production. The Moringa oleifera seeds powder were mixed with adult cattle rennet at a ratioof 2%. Crude and partially purified extracts (using ammonium sulphate 60% concentration) were used.The study include six different milk coagulants; adult cattle rennet extract (ARE), mix of adult cattlerennet with Moringa seeds (AME), partially purified adult cattle rennet extract (ARP), partiallypurified adult rennet mixed with Moringa seeds (AMP), commercial rennet (RRE) and calf rennetextract (CRE). The flocculation time, clotting time, clotting activity, proteolytic activity, the optimumpH and temperature for clotting activity, curdling quality (RCQ) and protein profile of curd formedwere determined (SDS-PAGE). The physical properties include water release (WR) and water holdingcapacity (WHC), yield and sensory attributes of Domiati cheese made by using different coagulantextracts were estimated. The results showed that CRE has shorter flocculation and clotting time andhigher milk clotting activity but ARE had the opposite, in the midst were treatments AME, ARP andAMP. The pH range of 5.5 to 6.5 and temperature between 50 - 60°C were suitable for milk clottingactivity of milk coagulants. The best (RCQ) was found to be for the curd formatted by CRE, AMP,ARP and RRE. Physical properties gave the lowest (WR) and highest (WHC) in CRE curd cheese,followed by AMP, RRE, ARP and AME cheese, whilst it was observed the contrary in ARE.Electrophoretic patterns show that the levels of degradation αs-casein, κ-casein and β-casein differedwith milk coagulant types. Using Moringa seeds and partially purified by ammonium sulphateimproved yield and cheese sensory with characteristic; coherent texture and creamy taste. Inconclusion mixing of adult cattle rennet with Moringa seeds powder during the extraction process and/or partially purified gave an opportunity to be used as an alternative to calf rennet in cheese making.

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

To test the potential of a novel microbial rennet isolated from traditional fermented rice wine for cheese making, Cheddar‐style cheese made with this enzyme was studied for changes in composition, proteolysis, and sensory profile during 90 days of ripening in comparison with a control cheese made with a commercial rennet. The initial proteolysis assay of the microbial rennet on milk proteins indicated a notable increase in the hydrolysis of casein components (α‐, β‐, and κ‐caseins) but no effect on whey proteins upon increasing the concentration of the enzyme. Correspondingly, compared to cheese made with commercial rennet, the use of the microbial rennet in Cheddar‐style cheese resulted in significantly higher primary and secondary proteolysis in the later stages of ripening (60–90 days ripening) and thus a softer texture and the formation of more volatile compounds and free amino acids (FAAs) despite its lower moisture content (41.7%, w/w). Though the cheese made with the microbial rennet was found to contain bitter‐taste FAAs (1,000 mg/100 g), the combined effect of other‐taste FAAs, including sweet (231 mg/100 g), umami (225 mg/100 g), and tasteless (361 mg/100 g) FAAs, in the cheese attenuated the bitter taste of the cheese. This analysis was in accordance with the sensory evaluation, which showed no significantly different sensory scoring between the cheeses made with the microbial and commercial rennets. The present study demonstrated a novel approach to evaluate the bitter taste of ripened cheese. The results of this study suggest the potential of the microbial rennet from rice wine to serve as a new source of milk‐clotting agents in cheese making.