Milk-clotting Enzyme Research Articles

Screening of a Bacillus subtilis strain producing both nattokinase and milk-clotting enzyme and its application in fermented milk with thrombolytic activity

Bacillus subtilis is a generally recognized as safe probiotic, which is used as a starter for natto fermentation. Natto is a functional food with antithrombus function due to nattokinase. Compared with natto, fermented milk is a more popular fermented food, which is commonly fermented by Lactobacillus bulgaricus and Streptococcus. However, there is no report on B. subtilis-fermented milk. In this study, to produce a functional fermented milk with antithrombus function, a B. subtilis strain (B. subtilis JNFE0126) that produced both nattokinase and milk-clotting enzyme was isolated from traditionally fermented natto and used as the starter for the functional fermented milk. In liquid fermentation culture, the peak values of thrombolytic activity and milk-clotting activity were 3,511 U/mL at 96 h and 874.5 Soxhlet unit/mL at 60 h, respectively. The optimal pH and temperature were pH 7.0 at 40°C for nattokinase and pH 6.5 and 55°C for milk-clotting enzyme, respectively. The thrombolytic activity in the fermented milk reached 215.1 U/mL after 8 h of fermentation. Sensory evaluation showed that the acceptance of the milk fermented by B. subtilis JNFE0126 was similar to the traditional milk fermented by L. bulgaricus and S. thermophilus. More importantly, oral intake of the fermented milk by the thrombosis-model mice prevented the development of thrombosis. Our results suggest that B. subtilis JNFE0126-fermented milk has potential as a novel, functional food in the prevention of thrombosis-related cardiovascular diseases.

Исследование факторов, влияющих на технологию мягкого сыра из козьего молока

The subject of research. The subject of the research is the production of soft cheese from goat's milk, the features of the technology used for the said, including milk-clotting enzymes and bacterial starter cultures. The purpose of research is to analyze the quality factors and the suitability of the starter culture for the production of soft cheese from goat’s milk to ensure a high yield of finished products, to study the effect of pasteurization modes on the curdling process. Materials and methods. When conducting research on the technological parameters of the production of soft cheese, various amounts of yeast, rennet, and calcium salt were added. The goat’s milk, starter culture, enzyme and calcium chloride available on the market were used. When determining the quality parameters of milk and finished products, we used standard methods for studying sensorial characteristics, determining the content of fat, moisture and dry matter, the mass fraction of protein, density and titratable acidity. Results of research. When conducting research, the duration of cheese mass formation was 8-14 hours. As the amount of added additional raw materials increased in each cheese sample, a decrease in the duration of the formation of the curd mass and the formation of a clot of different consistency were observed. The article also presents the results of studies of sensorial, physical and chemical parameters of samples of the finished soft cheese, describes the technological processes of the production of soft cheese from goat’s milk and the results of studies of factors affecting the quality of products. In particular, the technological parameters of obtaining soft cheese from goat’s milk, the procedure for pasteurization of the milk were studied, the optimal amounts of bacterial yeast, rennet and calcium chloride necessary for the production of cheese mass were determined, the effective concentration values of the enzyme and yeast that improve the physical colloidal properties of the protein clot were determined, these reducing the yield of dry matter during processing, increasing the output of finished cheese, the data on their impact on product quality is also provided. The benefits of the soft cheese made from goat's milk are due to the efficient use of raw materials, proper nutritional value and high yield of finished products. Sensorial, physical and chemical characteristics of ready soft cheeses have been studied. According to the results of the study, a sample of soft cheese from goat's milk was selected that fully meets the regulatory documents, without cheese defects in terms of physical and chemical parameters, fat content, moisture content, salt content, as well as sensorial properties. Scope of research results. The results of the research will be used to improve the technology for the production of milk products, namely, soft cheese from goat’s milk, to improve the food safety and consumer properties of the specified product.

Proteomics analysis of the bio-functions of Dregea sinensis stems provides insights regarding milk-clotting enzyme

Dregea sinensis (D. sinensis) stems have traditionally been used as milk coagulant in Dali of Yunnan Province, China. In this study, proteomics was used to investigate the bio-functions of D. sinensis stem proteins, leading to the purification and identification of the milk-clotting enzyme. A total of 205 proteins mainly involved in the catalytic and metabolic processes were identified, of which 28 proteins exhibited hydrolase activity. Among the 28 proteins, we focused on two enzymes (M9QMC9 and B7VF65). Based on proteomics, a cysteine protease (M9QMC9) with a molecular weight of 25.8 kDa and milk-clotting activity was purified from D. sinensis stems using double ammonium sulfate precipitation and was confirmed using liquid chromatography-mass spectrometry (LC-MS/MS). The milk-clotting temperature using the purified enzyme was around 80 °C (specific activity at 314.38 U/mg), and it was found to be stable in the pH range of 6–9 in NaCl concentration of <0.8 mol/L. These findings indicated that the enzyme isolated from D. sinensis stems has potential in the dairy and food sectors, especially in the cheese-making industry.

Recombinant expression and characterization of Oryctolagus cuniculus chymosin in Komagataella phaffii (Pichia pastoris)

This study was conducted for investigating expression and enzymatic characteristics of recombinant Oryctolagus cuniculus chymosin (ROCC) expressed in Pichia pastoris. SDS-PAGE of partially purified supernatant displayed two distinct molecular bands approximately at the sizes of 40 kDa and 45 kDa corresponding to chymosin and partially glycosylated chymosin, respectively. Proteolysis assay demonstrated that rabbit chymosin was more specific compared to bovine and camel chymosins when it comes to hydrolyzing α, β, and κ-casein. Rabbit chymosin kept its stability in a wide pH range (3.0–6.0) at 37 °C for 8 h. Active chymosin exhibited maximum enzymatic activity at 40 °C and pH 4.0 with the addition of 75 mM CaCl2. The ROCC clotting activity on donkey, cow, goat, lamb, camel milk was determined as 40, 10, 5.7, 3.07, and 2.66 IMCU/mL, respectively. These results revealed that ROCC might possess a potential for incorporation into cheese manufacture technology as a milk-clotting enzyme.

Novel Bacillus Milk-Clotting Enzyme Produces Diverse Functional Peptides in Semihard Cheese.

Although rennet is one of the best choices for cheese manufacturing, its production cannot meet the growing demands of the cheese industry. Thus, new milk-clotting enzymes (MCEs) with similar or better properties as/than those of calf chymosin are needed urgently. Here, three MCEs, BY-2, BY-3, and BY-4, were mined by bioinformatic analysis and then expressed in and isolated from Escherichia coli. BY-4 had the highest milk-clotting activity/proteolytic activity (238.76) with enzyme properties similar to those of calf chymosin. BY-4 cheese had a composition, appearance, consistency/texture, and overall acceptability proximate to calf chymosin cheese. The EC50 values of peptides extracted from BY-4 cheese for 2,2-diphenyl-1-picrylhydrazyl inhibition (antioxidant property), angiotensin-converting enzyme inhibition (antihypertensivity), and growth inhibition of liver cancer cells (antitumor property) were found to be 81, 49, and 238 μg/mL, respectively, which were 2.35, 2.59, and 2.12 folds higher than those of calf chymosin cheese. These results indicated the potential of BY-4 as a supplement to calf chymosin in cheese manufacturing, especially for functional and health care purposes.

Влияние аминокислотных замен в молекуле рекомбинантного химозина коровы на зависимость его коагуляционной активности от концентрации хлорида кальция в молоке

The influence of substitutions of amino acids involved in the formation of salt bridges on the cow chymosin technological properties has been studied. It was shown that point amino acid mutations Asp198→Lys, Asp156→Val and their combination decrease the sensitivity of coagulation activity of engineering recombinant chymosins to change in the concentration of calcium chloride in milk in the range of 1-5 mM. The found effect is a positive technological evidence in terms of cheese making, since it allows varying the content of calcium chloride added to the milk mixture without a threat of significant changes in the undesired proteolytic activity of used milk-clotting enzyme. recombinant chymosin, technological properties, amino acid substitutions, milk-clotting activity, concentration of calcium chloride. The work was performed within the framework of the state task of the Ministry of science and higher education of the Russian Federation (topic number fzmw-2020-0002, "Development of recombinant enzyme producers for cheese making").

The influence of cows stresses resistance on cheese yield and quality

The influence of cows stresses resistance on cheese yield and quality

THE INFLUENCE OF MILK-CLOTTING ENZYMES ON THE FUNCTIONAL PROPERTIES OF PIZZA-CHEESES

The effect of the type and dose of milk-clotting enzymes (Chy-max® M based on recombinant camel chymosin, Fromase® TL based on Rhizomucor miehei protease) on the physicochemical, functional properties and shelf life of pizza-cheeses was studied. When using a low dose of milk-clotting enzymes (MCE) for milk coagulation (250–1100 IMCU per 100 kg of milk), cheeses were obtained with an increased moisture content (55–57%), excessive acidity (pH 4.8–4.9) and texture defects (incoherent, crumbly, with separation of free moisture). This is due to the formation of a weak curd, which releases moisture poorly during processing. The use of an increased dose of MCE makes it possible to obtain a denser curd, better releasing moisture. Cheese produced with a high dose of milk-clotting enzymes (2000–2800 IMCU per 100 kg of milk) had a lower moisture content (52–53%) and lower acidity (pH 5.0–5.1). The protein matrix is more hydrated in these cheeses, which ensures its better water holding capacity and a more homogeneous and cohesive texture. The use of an increased dose of MCE with a high total proteolytic activity (Fromase) gives undesirable consequences in the form of accelerated proteolysis of cheese mass proteins, rapid loss of functional properties of the cheese, and a decrease in the shelf life of cheese (less than 60 days). Cheese production using an increased dose of MCE with a low level of total proteolytic activity (Chy-max M) allows achieving a low level of proteolysis during cheese ripening and increasing its shelf life.

The effects of thickeners upon the viscous properties of sour cream with a low fat content.

Fermented cream is a common and widely used dairy product throughout the world. The structure of sour cream with a low (10 to 15%) fat content is determined by the acid gel of milk proteins, while that of sour cream with a high (≥30%) fat content is determined by the gel formed by fat globules coated with protein. Sour cream with a fat content of about 20% has an intermediate gel structure, which is not characterized with high viscosity. To increase the viscosity of the product, as well as to prevent the separation of whey during storage, thickeners and stabilizers (gelatin, various types of starch, etc.) are added. As these decrease the sensorial characteristics of products, new thickeners (elamin, citrus pectin, flax seed flour, banana puree, and fromase milk-clotting enzyme preparation) are used instead. Sour cream was made by separating whole cow's milk, normalization, homogenization, pasteurization and fermenting by mesophilic lactic acid bacteria. Then thickeners were introduced to the clot: elamin, dry banana, flax seed flour in mass fractions of 0.01%, 0.03% and 0.05%, as well as fromase preparation in the same amount. A control batch of sour cream was made without the use of additives. The ripening process lasted from 8 to 9 hours to obtain a dense consistency. The viscosity of the samples was determined using a rotational viscometer, consisting of two coaxial brass cylinders, between which the test liquid was located. The effective viscosity and its dependence on the shear rate γ in research samples as non-Newtonian fluids were determined. Sour cream is characterized by the presence of a structure quickly destroyed by external forces at shear rates greater than 10 s-1, so exceeding the given value in the production of sour cream is not rational. The dependences of the rheological parameters of sour cream on the content of the additives elamin, flour from flax seeds and dry banana puree, used as thickeners, showed that the greatest strength of intermolecular bonds and the stability of the studied mixtures can be expected with a share of additives ranging from 0.01 to 0.03% (by mass). At the same time, when the said share exceeds 0.05%, the structure of sour cream is weakened - this is especially characteristic in the case of adding dry banana. The research showed that natural thickeners can be chosen to improve the rheological parameters of low fat sour cream without deteriorating its sensorial characteristics. This leads to the conclusion that the studied thickeners can improve the viscosity of sour cream with a low fat content - and dry banana is the best of those studied. Correspondingly, viscous properties are among the principal ones which define customers' perceptions of the product, its low fat content being a possible spoiler of sensorial traits.

Artichoke cv. Francés flower extract as a rennet substitute: effect on textural, microstructural, microbiological, antioxidant properties, and sensory acceptance of miniature cheeses.

The most common milk-clotting enzymes in the cheese industry are recombinant chymosins. Food naturalness is a factor underpinning consumers' food choice. For consumers who avoid food with ingredients from genetically modified organisms (GMOs), the use of vegetable-based rennet substitute in the cheese formulation may be a suitable solution. Artichokes that deviate from optimal products, when allowed to bloom due to flower protease composition, are excellent as raw material for vegetable rennet preparation. As enzymatic milk clotting exerts a significant impact on the characteristics of the final product, this product should be studied carefully. Mature flowers from unharvested artichokes (Cynara scolymus cv. Francés) that did not meet aesthetic standards for commercialization were collected and used to prepare a flower extract. This extract, as a coagulant preparation, enabled the manufacture of cheeses with distinctive characteristics compared with cheeses prepared with chymosin. Rennet substitution did not affect the actual yield but led to significant changes in dry matter yield, humidity, water activity, protein content, and color, and conferred antioxidant activity to the cheeses. The rennet substitution promoted significant modifications in springiness, and in the microstructure of the cheese, with a more porous protein matrix and an increment in the size of the fat globules. Both formulations showed a similar microbiota evolution pattern with excellent microbiological quality and good sensory acceptance. The rennet substitute studied here produced a cheese adapted to specific market segments that demand more natural and healthier products made with a commitment to the environment but well accepted by a general cheese consumer. © 2020 Society of Chemical Industry.

Characteristics of Curds With Milk Clotting Enzyme from Indonesian Local Isolate of Lactic Acid Bacteria

To get the potential of lalcat acid bacteria isolate to produce Milk Clotting Enzyme (MCE), it is necessary to screen milk clotting activity both quantitatively and qualitatively. Through qualitative observation, the characteristics of the curd resulting from enzyme activity can be obtained. MCE is a protease that has the characteristics of milking. Based on the results of this observational research, the curd characteristic produced can be used as a benchmark to determine the length of time of fermentation and optimization of the determination of ammonium sulfate precipitation concentration. Isolate BAL shows the results of a compact curd at a fermentation time of 25 hours at 37 ℃ and the optimization results of the deposition of ammonium sulfate which shows the characteristics of a compact curd by 45% ammonium sulfate.

Optimasi Waktu Inkubasi Lactobacillus rhamnosus SKG 34 Dalam Produksi Enzim Penggumpal Susu

&#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D; This study aims to determine the effect of incubation time on milk clotting enzyme (MCE) activity produced by L. rhamnosus SKG 34 and determine the optimum incubation time to produce the highest its activity. This study used a randomized block design (RBD) with incubation time as a treatment consisting of 8 levels, that were 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, and 48 hours. Data were analyzed with Variance Analysis (ANOVA) than followed by Duncan Multiple Range Test (DMRT). The analyzed were repeated 3 times resulting in 24 experimental units. The results showed that the incubation time significantly affected protease activity, MCE activity, specific protease activity and ratio of MCE to protease but did not affect the total LAB. The optimum incubation time of L. rhamnosus SKG 34 is 12 hours with total LAB 1.83 x 109 CFU/ml and protease activity 180.67 U/ml, MCE activity 595.06 SU, protease spesific activity 73.149 U/mg and ratio of MCE to protease 3.29 SU/U.&#x0D; Keywords : incubation time, Enzyme protease, Lactobacillus rhamnosus SKG 34, Milk clotting enzyme.&#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D;

Selection of starter cultures of direct application for hard cheeses

The article describes and presents the results of research in the selection of direct-application ferments containing lactic acid bacteria and lactic streptococci to optimize the production process of hard cheeses "Melkorisunchatyj 1" and "Melkorisunchatyj 2". The research objects were selected starter cultures of direct application of DSS-275 by " Chr. Hansen" (Denmark) and mos 066 C of Sacco s.r.l (Italy). Based on the results of trial workings, recommendations are given for the use of starter cultures to produce hard cheeses with a small pattern. The possibilities of using direct starter cultures in the production of hard cheeses were studied. As objects of research, conditionally named cheeses "Melkorisunchatyj 1" and "Melkorisunchatyj 2", starter compositions and a milk-clotting preparation were selected. For hard cheeses with fine patterns, the most suitable starter cultures are those consisting of a complex of lactic acid bacteria and milk streptococci. To obtain cheeses with improved organoleptic characteristics, direct starter cultures DSS - 275 from Chr. Hansen "(Denmark) and MOS 066 C of the company" Sacco s.r.l "(Italy). Frozen starter cultures were reconstituted in warm milk and then added to the mixture after filling the bath. A solution of the milk-clotting enzyme was introduced after an increase in titratable acidity by 1.3–1.5 ° T. Good quality cheese was obtained at pH 5.5 at the end of self-pressing. For hard cheeses, it is advisable to use starter cultures combining lactic acid and aromatizing microorganisms.

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.

Integrated method for production of hydrophobin-type protein and milk-clotting enzyme using Coprinus lagopides mushroom

Submerged culture of the mushroomCoprinus lagopideswas studied as a source of surfactant proteins and milk-clotting enzyme. Low molecular fungal proteins called hydrophobins exhibit unusual surfactant properties. They are able to self-assemble at the interface and to change the surface properties. Emulsions formed by these low-molecular proteins are very stable and can be used as stabilizers for edible foams and emulsions. By their taste and mouth feel air emulsions of hydrophobins resemble fats and can be used to substitute up to 50 % of them in large variety of foods. Despite the fact that hydrophobins have great application potential, however, the possibility of their use is currently very limited due to their insufficient output. Screening for new efficient producers of hydrophobins and development of new integrated methods of their production along with other valuable materials can help to overcome this problem. n this paper, we propose a novel producer of highly active surfactant proteins and a novel integrated method of obtaining as a result of a single fermentation surfactant proteins and milk-clotting enzyme.

Fungal Growth Inhibition by Cheese Prepared Using Milk-clotting Crude Enzymes from the Edible Mushroom &lt;i&gt;Hericium erinaceum&lt;/i&gt;

Fungal Growth Inhibition by Cheese Prepared Using Milk-clotting Crude Enzymes from the Edible Mushroom &lt;i&gt;Hericium erinaceum&lt;/i&gt;

Применение микропартикулята сывороточных белков в технологии полутвердых сыров

В данной работе описываются исследования, подтверждающие возможность использования микропартикулята сывороточных белков в технологии полутвердых сыров типа «Российского». Внесение микропартикулята в нормализованную смесь изменяло ее состав, что оказывало негативное влияние на процесс сычужного свертывания, увеличивало продолжительность коагуляции и снижало прочность сгустка. Относительное значение прочности сгустка обосновывает целесообразность использования не более 10% микропартикулята в составе нормализованной молочной смеси. Для получения стандартной продолжительности сычужного свертывания, а также уплотнения сгустка предложены следующие технологические параметры: температура свертывания 35°С, рН 6,4, доля молокосвертывающего ферментного препарата 0,04%, хлорида кальция – 40 г безводной соли на 100 кг нормализованной молочной смеси. При формировании сычужного сгустка частицы микропартикулята проникают внутрь ее ячеек. Для того чтобы частицы микропартикулята не перешли в сыворотку, значение их среднего диаметра должно быть в интервале 0,5-10 мкм. Формирование необходимого диапазона размеров частиц микропартикулята возможно путем направленного изменения технологических параметров микропартикуляции (температуры и интенсивности механического воздействия). На основании данных о содержании растворимого азота показано интенсифицирующее действие микропартикулята на созревание сыра. Готовый продукт характеризовался более высокой массовой долей влаги в сравнении с контролем. Этот факт в совокупности с более высоким содержанием сывороточных белков способствует увеличению выхода сыра. Разработанная технология сычужного полутвердого сыра «Российский» с микропартикулятом сывороточных белков позволяет заменить часть дорогостоящих сырьевых компонентов продуктом модификации сыворотки, а следовательно, вернуть побочное сырье в основное производство.

Plants: A natural solution to enhance raw milk cheese preservation?

Plants have been traditionnally used for centuries in cheese manufacturing, either for their aromatic properties or as technological auxiliaries (e.g. milk-clotting enzyme preparations, cheese wrappers). Some of these plants are known to have antimicrobial and/or antioxidant properties and could also act as natural preservatives for raw milk and derived dairy products. This review examined the traditional uses of plants in dairy processing, and then focuses on known antimicrobial and antioxidant properties of their extracts (e.g. maceration, decoction, essential oil). Known effects of theses plants on technological flora (starter cultures and microorganisms implicated in cheese ripening) were also summarized, and the potential for plant extracts used in combination with hurdle technologies was explored. Then, legal restriction and bioactivity variations from a culture media to a food matrix was reviewed: non-toxic bioactive molecules found in plants, extract preparation modes suitable with foodgrade processing restrictions, the role of the food matrix as a hindrance to the efficiency of bioactive compounds, and a review of food legislation. Finally, some commercial plant extracts for milk preservation were discussed.

PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF ASPARTIC PEPTIDASE PRODUCED BY NOVEL ISOLATE MUCOR CIRCINELLOIDES (VON TIEGHEM) USING SSF PROCESS

Microbial peptidases are among the most important hydrolytic enzymes which have a potential application in a wide number of industrial processes. In this study, the milk-clotting enzyme of a newly isolated strain Mucor circinelloides (von Tieghem) MG603064 was produced by solid-state fermentation using wheat bran as the substrate. The crude extract exhibited a maximum milk-clotting activity of 1500 ± 50.943 SU/mL after 72 h of incubation at 25 °C. Purification of the enzyme using fractionation at 20‒70% (NH4)2SO4 followed by size exclusion chromatography on Sephadex G-100 allowed us to obtain a 20-fold purified peptidase with a recovery of 18.41%. The highest activity of the purified enzyme (30 kDa) was obtained in 25 mM CaCl2, at pH 5.0 and temperature of 60 °C. The enzyme was stable between pH 3.0‒4.0 for 24 h at 4 °C in 0.1 M citrate buffer and retained more than 80% of its maximum activity at 45 °C for 1 h with complete inactivation at 75 °C. The enzyme inhibition of 94.5 and 98.6% by 0.02 and 0.1 mM Pepstatin A, respectively, confirmed that the enzyme is an aspartyl peptidase. A partial inhibition of 78.17% was noted for EDTA at 14 mM. The enzyme activity was improved significantly by Mg2+, Fe2+, Mn2+ and Zn2+ by 40.5, 59.5, 75.6 and 85%, respectively, and strongly inhibited by Al3+ (93.1%) and Hg2+(94.4%), at a concentration of 10 mM. Activity stimulation of 120% was maximum in the presence of Ba2+ using the same concentration. The crude and pre-purified extracts were applied in a trial of semi-hard cheese making (type Edam) as possible rennet substitutes.

An Evaluation of the Clotting Properties of Three Plant Rennets in the Milks of Different Animal Species.

Cynara cardunculus, Carica papaya and Ficus carica extracts are proposed as milk coagulants herein. Their coagulation efficiency was measured in bovine, buffalo, goat and sheep milk incubated at different temperatures. The milk-clotting and proteolytic activities as well as the lactodynamographic parameters were determined considering animal rennet as a reference coagulant. The vegetable coagulant, extracted from C. cardunculus pistils, proved to be the most suitable milk-clotting enzyme for cheesemaking, since it possesses similar milk clotting properties to conventional calf rennet. F. carica latex, but seemed to be a promising alternative coagulant at higher temperatures. The strong proteolytic activity of papain caused poor milk coagulation in all milk samples. To conclude, this result also supports the original hypothesis of this study that the excessive proteolytic nature of plant coagulants can negatively affect the cheesemaking process. The optimization of using a plant rennet in a dairy application can be done by selecting the appropriate plant rennet with a consistent clotting efficiency. These innovative manufacturing processes may also lead to the optimization and production of new cheese varieties.