Milk-clotting properties of plant rennets and their enzymatic, rheological, and sensory role in cheese making: A review

ABSTRACT

Plant rennets hold an important position among various coagulants used in cheese technology. The selection of a suitable plant coagulant is important due to the increasing global demands of cheese alongside reduced supply of calf rennet. Thus, a literature synthesis is presented to investigate recent achievements on their functional properties and enzymatic role in cheese making. Efforts have also been done to compare certain rheological and sensory properties of final products, arising from some plant- and animal-based rennets. In fact, some coagulants such as actinidin or dubiumin produce cheeses with sensory qualities similar to those produced by animal rennet. Others, like ginger, cucumisin, or hieronymain proteases contribute to develop very different textures and flavours, due to excessive proteolytic activity and production of bitter peptides. For milk-clotting enzymes with high non-specific action, several improved strategies have been developed to produce cheeses with sensory properties close to those of animal rennet. For example, the mixture of coagulants (cardosins/chymosin), the selection of appropriate milk or its ultrafiltration, as well as the increase of salting time of cheese during ripening could be efficient ways to improve texture and reduce bitterness. Concerning cheeses with high yield loss, the whey could be used for a traditional production of whey cheese. To conclude, the selection of appropriate plant rennet with high milk-clotting activity/proteolytic activity ratio and the optimisation of all coagulation parameters play a central role in manufacturing cheese with superior rheological and sensory properties.

KEYWORDS:

• Cheese
• Milk-clotting activity
• Plant rennet
• Rheological properties
• Sensory properties

Introduction

Enzymatic coagulation of milk is a crucial step in cheese-making process. After rennet addition, this step begins with an enzymatic cleavage of the phenylalanine₁₀₅–methionine₁₀₆ peptide bond of κ casein.^[1] It is followed by destabilisation of the casein micelles, along with a cooperative aggregation and thus resulting in formation of a three-dimensional protein matrix.^[1,2] In general, milk coagulation using calf rennet is the most used procedure. However, the worldwide increase in cheese production, coupled with reduced supply and increasing prices of calf rennet, has led to search for alternative milk-clotting enzymes, as appropriate rennet substitutes.^[3,4] Apart from this, some religious factors (Islam and Judaism) and others related to vegetarism of some consumers have greatly limited their use.^[4]

Several milk-clotting enzymes of microbial origin have been commercialised and used in cheese making, such as aspartic proteases (APs) obtained from Rhizomucor miehei, Rhizomucor pusillus, and Cryphonectria parasitica.^[5] However, the first formulations of fungal rennet from Rhizomucor sp. were partially suitable for cheese making due to excessive proteolytic activity (PA) and high thermo-stability. Several studies have reported the reduction of the thermo-stability of microbial enzymes by applying chemical modifications to preparations of rennet or by using genetic engineering tools on the microbial organism.^[6] Nowadays, some of the best rennets available are of microbial origin. Nonetheless, this does not exclude the fact that there is no place for plant-derived rennet, most certainly in scientific research.

Plant rennets have become a subject of growing interest in cheese industry, due to their easy availability and simple purification processes.^[7] Furthermore, the use of plant proteases in cheese manufacturing promotes the greater acceptability by the vegetarians and may improve their nutritional intake.^[8] For several years, plant extracts have been widely used in the preparation of various types of artisanal cheeses which are mainly produced in Mediterranean countries, Southern Europe, and West Africa.^[9]

The enzymatic activity of plant rennet is mainly associated with the action of APs or those with serine and cysteine residues.^[10] In fact, the use of different types of plant proteases in cheese technology influences the degradation level of the protein matrix of milk, leading to differences in sensory properties of cheese.^[11] Most of plant rennets produced cheeses with bitter flavours due to excessive PA, which limits their industrial use.^[12] For this reason, the selection of an appropriate plant coagulant and the control of different gelation parameters are of great importance to obtain a better quality of final product.^[13]

Evaluation of enzymatic activities (milk-clotting activity (MCA) and PA) is a crucial step in the selection of an appropriate substitute of calf rennet. Rheological and sensorial properties of the produced cheeses are related to the activities’ ratio. A high value of this ratio reflects an excellent product with desirable firmness and no release of bitter flavours, typical of plant proteases.^[7,14] Thus, the presentation of enzymatic and technological properties of plant rennet, previously studied in literature, could provide a clear vision on key elements for the selection of appropriate plant rennet.

Previous reviews published in this context, particularly that of Shah et al.^[4] have focused mainly on the extraction of coagulant enzymes from plants and the presentation of general enzyme characteristics in cheese making (activities, optimal pH and optimal temperature, thermo-stability). However, a better understanding of the effect of plant coagulants on rheological properties, sensory characteristics (texture, flavour, taste, and colour) as well as yield of the cheese is also of great importance when choosing the best substitute of calf rennet.

In this context, this review is presented to identify the different types of plant proteases involved in milk coagulation and to examine the scientific advances in terms of their enzymatic and functional properties. This review presents, for the first time, both rheological and sensory properties of gels and cheeses produced by plant coagulants. In addition, some factors that can affect these properties, with improvement strategies, were also presented for a better application in cheese technology.

Milk-clotting enzymes from plants

Types and sources of plant proteases involved in milk coagulation

Plant proteases are classified into various groups based on the catalytic mechanism used during the hydrolytic process. The main classes of milk-clotting proteases are aspartic, serine, and cysteine proteases.^[4] The number and the type of enzyme vary from one species to another, and depend on the part within the same plant itself. Examples are presented in Table 1.

Table 1. Examples of milk-clotting proteases from plants: source, classification, and milk-clotting activity.

		Enzyme
Plant	Organ	Type	Name	Number and class	MCA/total MCA/specific MCA	References
Cynara cardunculus	Flowers	Aspartic protease	Cardosin	8 Cardosin A Q9XFX3/AJ132884 Cardosin B Q9XFX4/AJ237674 Cardosin C Cardosin D Cardosin E P85136 Cardosin F P85137 Cardosin G P85138 Cardosin H P85139	Extract: 0.131 ± 0.025 UAC/mL (1 h of maceration) 0.164 ± 0.024 UAC/mL (24 h of maceration) Cardosin A: 1160 UACI/g Cardosin B: 7556 UACI/g	[15–17]
Cynara scolymus	Flowers	Aspartic protease	Cynarase	3 Cynarase A Cynarase B Cynarase C	Extract: Between 60 and 70 CAU/mg 30 CAU/mg 100 CAU/mg Between 30 and 40 CAU/mg	[18,19]
Cynara humilis	Flowers	Aspartic protease	Cardosin A like	–	–	[20]
Silybum marianum	Flowers	Aspartic protease	Enzymatic extract	–	0.083 CAU/mL	[21]
Orytza sativa	Seeds	Aspartic protease	Oryzasin	1	–	[22]
Moringa oleifera	Flowers	Aspartic protease	Enzymatic extract	–	1.9 CAU	[10]
Onopordum acanthium	Flowers	Aspartic protease	Onopordosin	–	0.546 CAU/mL	[23]
Cirisum vulgare	Flowers	Aspartic protease	Cirsin JN703462	–	–	[24]
Centaurea calcitrapa	Cell suspension	Aspartic protease	Enzymatic extract	–	2.023 U/mg	[25]
Albizia lebbeck	Seeds	Cysteine protease	Enzymatic extract	–	Crude extract: 156 × 10^–3 U/mg Concentrated extract: 591 × 10^–3 U/mg	[12]
Helianthus annuus	Seeds	Cysteine protease	Enzymatic extract	–	Crude extract: 5.8 × 10^–3 U/mg Concentrated extract: 39 × 10^–3 U/mg	[12]
Ficus carica sylvestris	Branches latex	Cysteine protease	Ficin (EC3.4.22.3)	2	1.4 U/mg	[26,27]
Sideroxylonobtusifolium	Stems latex	Cysteine protease	-	1	917 U/mg	[28]
Actinidia chinensis	Fruits	Cysteine protease	Actinidin	1	Extract: 244 U/mg	[29,30]
Calotropis gigantea	Latex	Cysteine protease	Calotropain	4 Calotropain FI Calotropain FII Calotropain DI Calotropain DII	Extract: 450 CAU/mL	[3,31]
Zingiber officinale	Rhizome	Cysteine protease	Ginger or Zingibain	3 Ginger proteases GP A GP B GP C	442.2 (CAU/mg) 288.3 (CAU/mg) –	[32]
Bromelia hieronymi Mez	Fruits	Aspartic and cysteine proteases	Hieronymain	3	Extract: 40 ICAU/mL	[33]
Solanum dubium Fresen	Seeds	Serine protease	Dubiumin	1	Extract: 880 CAU/mL	[34]
Cucumis melo	Fruits	Serine protease	Cucumisin	1	25 CAU (enzyme concentration of 1.5 μM)	[35]
Lactuca sativa	Leaves	Serine protease	Lettucin	1	20 UP (enzyme concentration of 1.9 μM) 40 RU (enzyme concentration of 1.9–3.8 μM)	[36]
Ficus religiosa		Serine protease	Religiosin	3 Religiosin Religiosin B Religiosin C	387 CAU/mL 803 CAU/mL –	[37–39]
Streblus asper	Stems latex	Serine protease	Streblin	1	2306 CAU	[40]
Balanites aegyptiaca	Pulp of fruits	Aspartic and serine proteases	–	2 Aspartic protease Serine protease	Crude extract: 2.43 CAU/mL Concentrated extract: 6.80 CAU/mL Discolored extract: 6.88 CAU/mL	[41]
Citrus aurantium	Flowers	–	Enzymatic extract	–	0.18 CAU/mL at 40°C	[42]

Aspartic proteases (APs) are found in many varieties of plant species. They are involved in protein degradation during plant development process, protein storage mechanisms, responses to stress and pathogens, and plant senescence.^[43] These enzymes have two aspartic residues responsible for the catalytic activity, whose action specificity is preferred for cleavage of peptide bonds between hydrophobic amino acid residues.^[44] Most of these enzymes are heterodimeric proteins with large subunit of 28–35 kDa and small subunit of 11–16 kDa, and only very small number of monomeric proteins with molecular mass of 36–65 kDa.^[45]

Serine proteases (SPs) have in their active sites serine residues.^[46] Their main role in plants is almost the same as the APs, with some additional features. SPs are widespread in plants and belong to several taxonomic groups. They were extracted, purified, and characterised from several parts of plants, especially fruits.^[4] Cysteine proteases or thiol-proteases have a catalytic mechanism that involves a cysteine group in their active site.^[4,43] Plant extracts, presented in Table 1, or their purified proteases, have been extensively characterised in many studies, for their potential use as milk coagulants.^[47]

Enzymatic role in milk coagulation and cheese making

After addition of plant rennet, the two main steps of the enzymatic coagulation of milk are carried out in the same manner as coagulation by calf rennet (or calf chymosin). The first phase is entirely proteolytic, during which the coagulant hydrolyses the C-terminal part of the κ-casein, at the Phe₁₀₅–Met₁₀₆ bond. The para-κ-casein, related to α and β caseins, remains integrated with the hydrophobic micelle. The hydrophilic peptide released from the micelle to the whey, is named caseino macro-peptide (CMP). The disposal of 85–90% of κ-casein (COOH κ-casein) causes micelles’ destabilisation which results in the initiation of the secondary coagulation phase.^[48] This corresponds to the aggregation of para-casein micelles in a three-dimensional gel network. After the removal of steric barrier and the reduction of electrostatic repulsion (50% decrease of zeta potential), the destabilised micelles can be grouped progressively through the establishment of hydrophobic and electrostatic bonds.^[1,49]

Several efforts have been made to understand the specificity of APs towards the Phe–Met bond. Studies of Fox^[50] have shown that di-, tri-, or tetra-peptides containing Phe–Met bond were not hydrolysed. Whereas the penta-peptide (Ser-Leu-Phe-Met-Ala-OMe) undergoes easily the enzymatic attack.^[50] Therefore, the peptide length and the residues located around the Phe–Met bond are important for enzyme–substrate interactions. These residues around the cleavage site allowed holding the substrate in its correct orientation within the active site of the enzyme.^[50]

As chymosin, some plant proteases are able to cleave few sites at α_s1 and β caseins, which may occur in maintaining the micellar stability.^[51] These regions of α_s1- and β-caseins are sometimes near to the micellar surface and contribute to electrostatic repulsion between casein micelles. The effect of the removal of these parts could greatly assist the gelling process.^[51] This is translated first by improving the initial destabilisation of micelles and promoting the coagulant access to κ-casein. Second, the removal of these parts increases by increasing the flexibility and/or susceptibility of caseins to readily undergo rearrangements in gel.^[51] An example of action specificity of APs from Cynara cardunculus (cardosins A and B) on peptides bonds of α_s1-, β-, and κ-caseins is detailed in Table 2.

Table 2. Action specificity of cardosin A and cardosin B from Cynara cardunculus upon isolated bovine, caprine, and ovine caseins (α_s1-, β-, and κ).^[52]

	Bovine caseins
Endo-peptidase	αs1-Casein	β-Casein	κ-Casein
Cardosin A	Phe23-Phe24 Phe153-Tyr154 Trp164-Tyr165 Tyr165-Tyr166	Leu127-Thr128 Leu165-Ser166 Leu192-Tyr193	Phe105-Met106
Cardosin B	Phe23-Phe24 Phe150-Arg151 Phe153-Tyr154 Trp164-Tyr165	Leu165-Ser166 Leu192-Tyr193	Phe105-Met106
	Caprine caseins
	αs1-Casein	β-Casein	κ-Casein
Cardosin A and cardosin B	Phe153-Tyr154	Leu127-Thr128 Leu190-Tyr191	Lys116-Thr117
	Ovine caseins
	αs1-Casein	β-Casein	κ-Casein
Cardosin B	Leu156-Asp157 Trp164-Tyr165	Leu127-Thr128 Leu165-Ser166 Leu90-Tyr191	Phe105-Met106

In addition to the main function in milk coagulation, plant proteases play an important role in the beginning of cheese ripening.^[53,54] The hydrolysis of caseins in cheese by residual coagulant produces essential substrates for some bacterial microflora, whose degradation allows flavour development during ripening.^[6] However, the intensity of these effects on cheese quality depends on the type of plant coagulant, its dose, and its enzymatic activities.

In general, the major drawback of most plant rennets is the development of an increased bitterness and the appearance of cheese texture defects during storage and/or ripening.^[12] These defects are mainly due to excessive proteolytic activities and low MCA/PA ratios. For this reason, the evaluation of enzyme activities and their comparison with those of commercial rennet (chymosin) is an important first step in selecting a suitable plant coagulant.

Evaluation of enzymatic activities of plant rennets

MCA is the most important property of proteases used in cheese production. It is the ability of the enzyme to hydrolyse specifically the κ-casein from milk.^[55] MCA can be measured by different methods, namely Berridge 1945, Soxhlet, and IDF Standard 157, using different units such as rennet unit (RU or MCU), Soxhlet unit, and the international milk-clotting unit (IMCU), respectively.^[13] Concerning cheese production using plant rennet, the initial goal was always the production of coagulants with a maximum specific coagulant activity (AC). This activity depends on several factors, such as the plant source, the part in the plant, as well as the type and the concentration of protease.^[56] Milk-clotting activities of some plant extracts and/or their purified proteases, reported in literature, are summarised in Table 1.

In order to detect the optimum of activity, different comparison studies were carried out between extracts of different parts in the same plant. Results of Anusha et al.^[3] revealed that the highest clotting activity was observed in the extract of latex followed by extracts of stems, leaves, and flowers of Calotropis gigantea, in descending order. This is in contradiction with extracts of C. cardunculus, whose clotting activity is concentrated mainly on flowers, because of the predominance of coagulating enzymes (cardosins) in this part of the plant.^[57] In fact, aqueous extracts from flowers have been widely used as substitutes of animal rennet (AR) in some artisanal Italian Spanish and Portuguese cheeses.^[58] Milk-clotting and proteolytic activities, as well as kinetic parameters of C. cardunculus enzymes, reported in literature, are summarised in Table 3.

Table 3. Milk-clotting activities (a), proteolytic activities (b), and kinetics parameters (c) of aspartic proteases from cardoon flowers (C. cardunculus).

(a) Milk-clotting activities
Enzyme		Extraction conditions	Substrate	MCA and/or specific MCA	References
Crude extract of C. cardunculus		0.5 g flowers in 75 mL ultrapure water	Reconstituted skim milk (pH 6.5, T 30°C)	0.131 RU/mL (1 h maceration)	[17]
		7 g of dried flowers in 70 mL of 100 mM sodium citrate buffer (pH 3), then lyophilisation	Reconstituted skim milk (pH 6.5, T 30°C)	0.164 RU/mL (24 h maceration) 103.6 ± 4.1 U/g	[59]
Cardosin A		1 g flowers in 10 mL citric acid (pH 3), then purification	Reconstituted skim milk (pH 6.5, T 30°C)	1160 IMCU/g	[15]
Cardosin B		1 g flowers in 10 mL citric acid (pH 3), then purification	Reconstituted skim milk (pH 6.5, T 30°C)	7556 IMCU/g	[15]
(b)Proteolytic activities
Enzyme		Extraction conditions	Substrate	PA or specific PA	References
Crude extract of C. cardunculus		7 g dried flowers in 70 ml of 100 mM sodium citrate buffer (pH 3), then lyophilisation	Bovine casein in 0.1 M phosphate buffer pH 6.5 at 30°C	16.4 ± 1.3 ΔA280 g^−1.min^−1	[59]
Cardosin A		Purification of cardosins (A, B, E, F, G, and H) after extraction in 100 mM sodium citrate buffer (pH 3.5)	Synthetic peptidesLys-Pro-Ala-Glu-Phe- Phe(NO2)-Ala-LeuKPAEFF(NO2)ALLSF(NO2)NleAL	7.81 ± 0.89 U/mg	[60]
				100–150 mM/min.	16]
				1 mM/min.mg
Cardosin B			LSF(NO2)NleAL	20–25 mM/min.mg	[16]
Cardosin E			KPAEFF(NO2)AL	200–240 mM/min.mg	[16]
			LSF(NO2)NleAL	0.5–1 mM/min.mg
Cardosin F			KPAEFF(NO2)AL	50–100 mM/min.mg	[16]
			LSF(NO2)NleAL	0–0.5 mM/min.mg
Cardosin G			KPAEFF(NO2)AL	250–300 mM/min.mg	[16]
			LSF(NO2)NleAL	1.5 mM/min.mg
Cardosin H			KPAEFF(NO2)AL	100 mM/min.mg	[16]
			LSF(NO2)NleAL	0–0.5 mM/min.mg
(c) Kinetic parameters
Enzyme	Substrate	kcat (s^−1)	Km (mM)	kcat/Km (mM^−1.s^−1)	References
Crude extract of C. cardunculus	FTC–κ-casein	–	0.225 (Vm = 3.912)	–	[61]
Cardosin A	KPAEFF(NO2)AL	18	0.14	129	[16]
	[Leu-Ser-Phe(NO2)-Nle-Ala-Leu-oMe]	23.6	1.08	21.87	[62]
Cardosin B	[Leu-Ser-Phe(NO2)-Nle-Ala-Leu-oMe]	85.5	0.080	1057	[62]
Cardosins (A + B)	κ-casein	1.04	0.00016	6500	[63]
Cardosin E	KPAEFF(NO2)AL	25	0.64	39	[16]
Cardosin G	KPAEFF(NO2)AL	35	0.37	95	[16]

Within the frame of research for a suitable substitute for calf chymosin, comparative studies of coagulant activities and sometimes MCA/PA ratios of plant extracts (or purified proteases) were established. For example, specific activities of crude and concentrated extracts obtained from seeds of Helianthus annus and Albizia lebbeck were evaluated, showing very low values of 156 × 10^–3 and 591 × 10^–3 U/mg for A. lebbeck extracts (crude and concentrated) and 5.8 × 10^–3 and 39 × 10^–3 U/mg for H. annus extracts.^[12]

Mazorra-Manzano et al.^[42] compared the MCA of three crude extracts obtained from kiwi (Actinidia chinensis), ginger (Zingiber officinale), and melon (Cucumis melo). Highly significant differences were observed, which were attributed to different types of proteases (actinidin, ginger, and cucumisin) found in extracts of kiwi, melon, and ginger. The assessment of MCA/PA ratio showed that the chymosin had a value 67, 95, and 500 times higher than those obtained, respectively, for the three extracts, when using casein as substrate.

To face up the problem of reduced MCA/PA ratios found in plant extracts, Ben Amira et al.^[64] have varied the pH of C. cardunculus rennet from 3 to 6 and evaluated enzymatic activities of the crude extracts. They demonstrated that MCA/PA ratio increased with pH drop and reached a maximum value of 28.71 for extract at pH 3, which exceeded that of chymosin (23.59). The lowest ratio attributed to the extract at pH 6 was mainly related to its high PA as well as to its low MCA.^[64] The effect of increasing pH buffer on rennet activity could be explained by the fact that, at high pH, the extraction level of several other compounds, including non-proteolytic enzymes, is more important. These compounds may promote the development of extraneous reactions and interfere with enzyme tests, thus causing underestimation of MCA. In addition, the high content of phenolic compounds involves their swift oxidation to form pigments, which may attach to native enzymes, thus leading to their inactivation.^[59] Moreover, at acidic pH, between 75% and 90% of total extracted enzyme activity correspond to cardosin A.^[52] This enzyme was shown to be similar to chymosin, in terms of kinetic parameters and specificity, by cleaving the same peptide bond (Phe105-Met106) of κ-casein.^[52] So, the specific action of C. cardunculus rennet was promoted at acidic pH. The latter was in close agreement with Withania coagulans extract, for which the highest MCA is recorded at low extraction pH.^[52,65]

Besides, the enzyme composition and the collecting region of C. cardunculus flowers could play a considerable role in promoting the specific activity of coagulant extract.^[66] In fact, only four cardosins were identified in Tunisian cardoon flowers, among them the cardosin A can replace successfully calf chymosin. As concluded by the authors, the absence of the other cardosins (B, C, D, and F) could be an advantage, as it reduces the excessive non-specific PA during cheese making.^[66]

Some studies have also revealed high MCAs of plant rennets as compared with chymosin. Ahmed et al.^[34] concluded that the extract of Solanum dubium is an appropriate calf rennet substitute, as its MCA exceeds significantly that of calf rennet (880 and 249.6 CAU/mL at 37°C, respectively). Kumari et al.^[37] compared also the MCAs of Religiosin and Religiosin B from Ficus religiosa with calf rennet and they found, respectively, values of 387, 803, and 498.9 CAU at 37°C. It was also reported that the ratio of MCA/PA in quixaba latex (5731) was high for commercial cheese production and exceeded that of chymosin (3363).^[28] Moreover, MCA of the partially purified extract (30–50%) from W. coagulans fruits was clearly higher than its PA. This index was sufficiently high to justify the use of this enzyme extract as an appropriate substitute of calf rennet.^[65] According to a recent study on plant rennets,^[67] Calotropis procera and Cryptostegia grandiflora latex fractions were found to be good sources of milk-clotting proteases. Both coagulant fractions were able to produce similar SDS-PAGE and RP-HPLC profiles of the κ-casein peptides, to those obtained for commercial chymosin. These biochemical findings were confirmed later by technological analysis of the cheeses obtained, which showed closer yields and soluble proteins content to those of chymosin cheese.

To conclude on the ability of a plant coagulant to successfully replace AR, comparison studies of enzymatic activities must be completed with rheological studies on milk gels and sensory properties (texture, flavour, colour) of cheeses. This is very important to understand better the effect of plant coagulants on these properties, as compared with AR, then to control well their subsequent use for an industrial application.

Effect of plant coagulants on rheological properties of gels, sensory characteristics, and yields of cheeses

Effect of plant coagulants on rheological properties of gels

Monitoring the evolution of rheological properties is one of the means for measuring gel formation during coagulation. This study evaluates the impact of enzymatic hydrolysis of casein and changes in enzyme specificity on functional properties of milk proteins.^[68] In this context, several parameters can be evaluated using the study of the deformation (γ₀) and measurements of the response to the development of gel. These parameters include the elastic or storage modulus (G′), which is a measure of the energy stored per oscillatory cycle, and reflects the behaviour of the sample as an elastic solid; the viscous or loss modulus (Gʺ), which is a measure of the energy dissipated per cycle and indicates the behaviour of the sample as a viscous liquid; the phase angle (δ) which is a measure of the link type to which the network is based; and the loss factor (tangent δ), which corresponds to the tangent of the phase angle of the response to deformation. This one is connected to the relaxation of the links within the gel during deformation.^[69]

Esteves et al.^[70] studied the rheological properties of milk gels’ coagulated by plant extracts (C. cardunculus L. and C. humilis L.) and chymosin. G′ values recorded at the beginning of gelation by plant coagulants were higher than those of chymosin. According to these authors, this was explained by differences between proteolytic activities of coagulants on casein, which may alter the degree of proteolysis on κ-casein. It is also possible that during gel formation, the hydrolysis of some additional bonds in casein particles may be accompanied by significant rearrangements of gel structure. The latter lead to the increase of strength and number of bonds between adjacent aggregates, resulting in a rapid increase of initial G′ values.^[68] At higher intervals of time (6 h), plant coagulants of C. cardunculus and C. humilis produced G′ values of 74.6 and 72.7 Pa, respectively, which were lower than the value obtained using chymosin as coagulant (96 Pa). This result was related to non-specific proteolytic activities generated by plant enzymes, which consequently would result in some additional hydrolysis in gel network.^[70]

With respect to the loss factor (tan δ), an abrupt decrease in this parameter is observed when the secondary coagulation phase begins, particularly at the time of transition from milk to gel formation. Then, during the increase of the gel strength, this factor is kept constant. It is independent of some physicochemical parameters such as milk pH.^[20,71] According to a study developed by Esteves et al.,^[68] measurements of loss factor, at frequencies of 0.002 Hz, showed high values for gels produced by all coagulants (plant and animal). These high values could be translated by an increased susceptibility of links (protein–protein) to loosen over time.^[68] This explains well the better ability of gels produced by plant coagulants to undergo syneresis and rearrangements.^[72] In addition, to evaluate the firmness of gels obtained from plant coagulants compared with chymosin gel, some authors compared the yield stress within these gels.^[68] They found that chymosin produced firmer and stiffer gels than those of plant proteases, as the value of this parameter was significantly higher.

In the literature, the results of rheological properties of gels produced by plant rennets were sometimes different from one study to another. These variations are due to the use of coagulants from different origins, the application of different values of analysis parameters for rheological measurements (oscillation frequency and stress), and other factors that could intervene in the gelation process and influence the rheological properties of the final product.

Factors influencing rheological properties of gels

Several factors, namely the coagulant dose, pH and temperature of milk, coagulant pH, and heat treatment or ultra-filtration of milk, influence the rheological properties of the obtained gels. Their control could significantly improve the gelation process by plant rennet as well as the final product properties.

The use of a low amount of plant coagulants in milk gelation at low pH, may avoid the negative impact of excessive proteolysis of caseins on the texture and flavour of cheeses.^[20] In fact, by decreasing milk pH, a reduction in the electrostatic repulsion between casein micelles occurs, because of the decrease in charge density on κ-casein. This promotes particles’ aggregation and a faster increase of the initial values of G′.^[20] Silva et al.^[15] showed also that following the addition of CaCl₂ (to low pHs of 6.2 and 6.4), the sigmoïdal increase of the complex modulus (G*) is faster in the case of gels produced by cardosins A and B of C. cardunculus. This was also explained by an increase in Ca²⁺ activity that could lead to faster structural rearrangements and a more favourable subsequent aggregation of micelles.

On the other hand, variation in enzymatic behaviour towards the curd firming rate (dG*/dt) depends on pH. In a comparison study developed by Silva et al.,^[15] the firming rate of a chymosin gel exceeded that of a gel produced by C. cardunculus enzymes (cardosins A and B), after milk coagulation at pHs of 6.2 and 6.4. However, at pH 6.6, cardosins produced higher values (85, 81, and 42 Pa/min). Therefore, a variation of milk pH after CaCl₂ addition could significantly improve the final firmness of the dairy product prepared with plant enzymes. In this way, firmness could exceed in some cases the values obtained for a chymosin curd.

Milk pH could be also affected by rennet pH as reported by Ben Amira et al.,^[64] thus leading to significant variation of all rheological parameters (G′, Gʺ, and curd firmness (CF)) of gels produced by C. cardunculus crude extracts (CEs) at different levels of pH (Fig. 1). By lowering rennet pH, milk pH decreased causing a significant rise of the visco-elastic parameters of skim milk gels and faster initial values of G′ (Fig. 1). The final CF and G′ recorded at the end of gelation (after 2 h) ranged, respectively, from 5.78 and 12.10 Pa for CE at pH 6 to maximums of 55.73 and 64.16 Pa obtained for CE at pH 3.^[64] To explain this, the authors reported that the lowest firmness values of gels made with CE at pH 6 were probably the result of high non-specific proteolytic action, which leads to some hydrolysis in the existing network. According to McMahon et al.,^[73] rennets that are much more proteolytic than chymosin tend to produce slightly softer curds with lower rates of gel firmness.

Figure 1. Evolution of storage modulus (G′) and loss modulus (Gʺ) of skimmed milk gels produced by C. cardunculus extracts prepared, respectively, at pH 3 (A), and pH 6 (B), and by chymosin (C) as a function of time.^[64]

, G′;

, G″.

Results of Ben Amira et al.^[64] were different from those of Esteves et al.,^[20] which showed that plant coagulant had similar values of G′ at the end of gelation, regardless of milk pH. This could be explained by the fact that the pH range (pH 3–6) was larger and more acidic than that used by Esteves et al.^[20] (6–6.7), resulting in a considerable variation of milk pH and therefore a significant effect upon rheological properties.

Regarding milk temperature, the rheological properties of gels produced by plant coagulants are less influenced by temperature changes than chymosin gels.^[74] This is an advantage for cheese production by plant enzymes using a wider range of temperatures. However, the application of a low temperature to the milk leads to a better efficiency of the gelation process by plant proteases and a reduction of non-specific proteolysis. Indeed, at 25°C, milk coagulation is faster with plant coagulants than with chymosin. This results in a cutting time considerably shorter than that of chymosin curds.^[74] Further, the use of low temperature of coagulation could effectively induce conformational changes of casein micelles, leading to the increase of enzymes accessibility to caseins.^[75]

Milk ultra-filtration is also a very important factor that can influence the rheological properties of the gels. This membrane process is generally used to standardise or to increase the protein content in milk. Following the increase of casein concentration, milk properties change during coagulation. The aggregation phase is extended, because of the high viscosity of ultra-filtrated milk and the average free distance between small micelles. This distance is relatively low, which allows a delay of the diffusion rate and the collision frequency between casein micelles.^[76] Gel elasticity is however higher^[77] with a minimum drainage of water and serum proteins. In addition, the presence of serum proteins in cheese may reduce the proteolysis of casein during cheese ripening, which underwent a prior ultrafiltration of milk.^[76]

In addition to membrane separation process, heat treatment of milk also affects the rheological properties of gels. As milk ultrafiltration occurs, the effect of this factor on gel rheology was envisaged only in the case of milk gelation by calf rennet. Blecker et al.^[78] showed that the application of high temperature on milk contributes to the decrease of gel-firming rate during coagulation followed by a reduction in gel viscosity. The values of the maximum gel-firming rate produced by calf chymosin were about 0.0202, 0.0175, and 0.0003 Pa⁻¹, for fresh raw milk and heat-treated milk at 60°C and 80°C for 20 min, respectively. This was explained by the distortion of serum proteins (mainly the α- and β-lactalbumin) resulting in a complex formation between the β-lactalbumin and casein, via intramolecular disulfide bonds. Thus, when using plant rennet in milk coagulation, the application of low temperature on milk could avoid weak gel firmness. The best understanding of the different parameters and all factors, which may influence gelation process using plant rennets, contributes to better rheological properties, connected to the texture and acceptance of the final product.

Effect of plant coagulants on sensory properties of cheeses and some improvement strategies

Determination of the sensory properties of cheese is an essential step to assess the ability of a plant coagulant to successfully replace calf rennet. These properties depend on the type of plant rennet. Some coagulants produce cheeses with organoleptic and sensory qualities similar to those produced by AR. Others contribute to make very different textures and flavours, due to excessive PA and production of bitter peptides. In this context, several research works were carried out to compare texture, taste, and colour of different cheeses produced by plant and ARs. Improvement strategies were also provided for a better application of plant rennet in cheese technology.

Texture, flavour, and taste

Evaluation of sensory properties of semi-hard cheeses made with plant coagulants showed different texture properties. The semi-hard cheese, prepared with Onopordosine extracted from flowers of Onopordum acanthium, showed similar characteristics to other commercial cheeses of the same type. It has a slight bitterness with a sweet aftertaste together with a strong mature taste, which is slightly salty. However, its overall texture is of a good cohesion. Indeed, cheese texture is mainly obtained due to the primary proteolysis of milk caseins by plant coagulants, rather than by the predominant microflora.^[79] The hardness of all cheeses decreases because of the enzymatic hydrolysis of the α_s1 casein within the maturation step.^[80]

The “Peshwari” is a semi-hard fresh cheese, made from wholly or partially skimmed cow’s milk. The coagulation takes place at 32°C for 1 h using a coagulant from ginger rhizomes (Zingiber officinale) called ginger protease (GP). Unlike the Onopordosine cheese, ginger cheese is endowed with a creamy texture, but no defect flavour or bitterness occurred during storage.^[81] Texture results are similar to those of the “Murcia al Vino” cheese prepared with a plant coagulant. This is softer, less grainy, and creamier than the same cheese made by calf rennet.^[82] Similar results were also reported for miniature cheese prepared with coagulant of Bromelia hieronymi coagulant. This one showed a texture which was not very satisfactory compared with chymosin cheese. Hopefully, this type of cheese did not develop an undesirable flavour or bitterness.^[33] Furthermore, cheeses made from sheep milk, using C. cardunculus coagulant presented also similar results.^[83] These texture defects were due to residual coagulant in cheese which is responsible for a high primary proteolysis. Despite the excessive activities and differences in texture, some plant extracts such as those of C. cardunculus were used in the preparation of soft cheeses such as “Roquefort” and “Serra da Estrela.”^[9]

In contrast to this information, several plant extracts could successfully replace calf chymosin in terms of texture properties. For example, curds produced by Kiwi extract and chymosin were characterised by similar values of the parameters describing elasticity (7.10 and 6.38 mm), cohesion (0.328 and 0.349), and chewiness (135 and 122 mJ), respectively. They also had the highest hardness values (6.19 and 5.52 N). However, melon extract produced a curd with lowest values of all textural parameters; particularly the hardness which was about 1.97 N. This was attributed to high proteolytic activities, resulting in low MCA/PA ratio.^[14] Then, when analysing the taste of “Mozzarella” cheese made with Kiwi extract, results showed the absence of bitter taste.^[30] The evaluators classified flavour and acceptability in the range of the hedonic scale from 4 (good) to 5 (very good) and they did not notice any difference between the prepared sample and the commercial “Mozzarella” cheese. The good taste of this type of cheese was mainly due to the ability of Actinidin to degrade undesired cream milk proteins.^[30,84]

Besides Kiwi coagulant, S. dubium extract was used to produce a cheese named “Gibna bayda” which did not differ from cheese made with calf rennet in terms of sensory characteristics, particularly flavour.^[85] It gave better nutritional value to consumers. Nevertheless, it comprised a slight bitter taste. Yousif et al.^[86] reported that it is possible to reduce this slight bitterness by using purified enzyme and optimum concentration of coagulant extract. In this case of traditional cheese, the selection of the cheese type (white cheese) is of great importance as the manufacturing process does not present a ripening step characterised by a primary and secondary proteolysis. Therefore, when avoiding this step and using a coagulant extract with high MCA (880 MCU/ml),^[34] the characteristics of the final product could strongly resemble to those of the same cheese made with calf rennet.

With respect to ripened cheeses, some manufacturing successes using plant extracts were achieved, thanks to the mastery of certain parameters during preparation or ripening. For example, the extract of Cynara scolymus flowers was considered as a good substitute for AR in the production of Gouda-type cheeses, as the physicochemical parameters analysed during ripening as well as organoleptic properties were similar.^[87] Furthermore, cheese salting for a longer period (40 h) during ripening prevents the development of bitterness. To evaluate this, the determination of the ratio (water-soluble nitrogen (WSN)/total nitrogen (TN)) during ripening was a better indicator for measuring the intensity of proteolysis. This ratio was lower in C. scolymus cheeses salted during a period of 40 h than that of those salted for 30 h.^[87]

Galán et al.^[88] compared the sensory characteristics of ripened cheeses made from sheep milk, using as coagulants: AR, coagulant from C. cardunculus (CCP), and a mixture of both (AR/CCP (50:50)). Cheeses prepared with plant coagulants (CCP and AR/CCP) reached faster the biochemical and sensory characteristics of mature cheeses manufactured with the AR. Consequently, the combination of coagulants (AR/CPC) can be exploited to speed up the ripening step. The rennet mixture had no effect on texture, particularly the firmness which remains significantly higher for AR cheeses. However, the bitter taste can be reduced by using a mixture of coagulants (AR/CCP); thus, allowing consumers who detest bitterness to enjoy a better taste.

Generally, the taste of cheeses made with plant rennet is also related to the type of milk and its composition. A better choice of milk could avoid some undesirable taste defects. For example, coagulant extracts of C. cardunculus were successfully used in the production of cheeses prepared from goat and sheep milk.^[89] These latter do not exhibit high bitter taste. While, cheeses produced by cow milk and C. cardunculus extracts tend to develop always undesirable bitterness.^[90] This is probably due to ovine caseins which are less sensitive than bovine caseins to form hydrophobic bitter peptides following proteolytic action.^[91]

Correlation studies were also carried out to better understand the origin of the typical taste of cheese produced by plant rennet. A strong correlation was observed between the different nitrogen fractions (SN, NPN, NH₃N, and AAN) and the intense taste of “Los Pedroches” cheese made from sheep milk and C. cardunculus extract. However, a weak correlation between taste and free fatty acids was detected.^[92] According to Sanjuán et al.,^[54] the SN level in “Los Pedroches” cheese made by vegetable rennet was 30% higher than that made with calf rennet. As reported by these authors, this high content was due to an increased PA of milk-clotting enzymes rather than the involvement of microbial endo- and exo-proteases.

On the other hand, some authors have characterised bitter peptides released during cheese manufacture or ripening to get a clear idea about flavour.^[26] These peptides extracted from cheeses are those with high percentage of aromatic amino acids residues and with elevated hydrophobicity average.^[93] Most of them arise from the hydrolysis of α_s1- and β-casein by residual rennet.^[94] The high concentration of retained peptides in cheese causes undesirable bitterness.^[95] It has been proved that the ratio of hydrophobic to hydrophilic peptides is well correlated with bitter taste. In fact, fractions from Cheddar cheese, separated by RP-HPLC, revealed that hydrophobic peptides were attributed to bitter fractions; while, the savoury fractions were amino acids and hydrophilic peptides.^[96,97]

To face up the problems of bitterness, Agboola^[98] proceeded an ultrafiltration of cow’s milk to keep a solid rate similar to sheep’s milk. Thus, the application of cardoon rennet to ultrafiltered milk resulted in obtaining a semi-hard cheese with similar sensory properties to cheeses made from non-concentrated milk and microbial or AR.

Colour

The improvement of visual quality, mainly the colour of cheeses made from plant rennets is of great importance to meet consumer requirements. For this reason, the reduction of plant extract concentration (solid/liquid) and the volume of coagulant added to milk contributes to better colour results. Chikpah et al.^[99] showed that green colour of soft cheese made in West Africa by Calotropis procera coagulant was mainly influenced by the colour of the plant extract added to milk. It was also affected by the treatment of raw material (leaves and stems: fresh or dried powder) and the concentration of coagulant. In fact, the green colour of the leaf extract of C. procera was responsible for cheese colour. The latter increased with raising the extract concentration and the freshness of the starting raw material (fresh leaves and stems). The use of purified plant extracts in milk coagulation may also reduce the amount of polyphenols and coloured pigments added to milk leading to colour improvement.

Comparison studies of cheese colour were also accomplished in order to verify the ability of plant rennet to produce cheese comparable to that of calf rennet. Results of Agboola et al.^[100] have shown that cheeses prepared with C. cardunculus extract have a darker and more yellow colour than that of cheese manufactured with AR. This was explained by the authors who argued that the volume of cardoon extract (400 ml/100 L of milk) added to milk was higher than that of AR (20 ml/milk 100 L). The use of ultrafiltered milk by the same coagulant (C. cardunculus extract) leads to an improvement in colour which becomes lighter and less yellow.^[100]

Effect of plant coagulants on cheese yield

One of the most important parameters to be considered in choosing an appropriate substitute for calf rennet is determining the yield by weight of cheese. Optimal cheese yield is very important for cheese production process.^[101] Several plant enzymes were studied due to their milk-clotting activities. Most of them are not very suitable for cheese production in an industrial scale. Within this process, cheeses undergo protein loss as a result of excessive proteolytic activities.^[101] Table 4 summarises some cheese yields determined for cheeses produced with plant rennet as compared with those made with commercial chymosin. According to literature, the loss of yield in Portuguese cheeses produced by C. cardunculus coagulant cannot be considered a problem, as whey is used commonly for a traditional production of whey cheese (Requeijão). Consequently, protein and fat loss during cheese making is recovered during the preparation of this by-product.^[102]

Table 4. Examples of cheese yields of some cheeses prepared with plant rennets, as compared with those made with commercial rennet (Chymosin).

Plant	Cheese yield (or curd yield)			References
	Cheese	Plant rennet	Chymosin
Actinidia chinensis Cucumis melo Zingiber officinale	Curd	17.8 ± 1.6 g/100 g Fw 15.1 ± 0.9 g/100 g Fw 15.4 ± 1.9 g/100 g Fw	20.2 ± 2.7 g/100 g Fw	[14]
Bromelia hieronymi	Miniature cheese	14.52 ± 0.31 g/100 g (Fw) 8.63 ± 0.43 g/100 g (Dw)	16.05 ± 0.06 (Fw) 9.26 ± 0.46 g/100 g (Dw)	[33]
Withania coagulans	White cheese	268 g/L	234 g/L	[101]
Calotropis procera	Soft cheese	237.4 g/L	–	[99]

Conclusion

Different types of proteases from plants are used in milk coagulation and cheese-making process. Their industrial use is practically non-existent because of high bitterness and lower cheese yields. However, some developments in the understanding of their action on milk proteins during gelation and the control of the various parameters that influence cheese production process suggest a change. In literature, rheological properties of milk gels and sensory characteristics of cheeses produced by plant rennets varied according to the type of coagulant, its enzymatic activities (MCA, PA), and its concentration. Other physicochemical parameters may also contribute to the improvement of gels qualities and cheeses, such as milk pH, gelation temperature, heat treatment of milk or its ultrafiltration, and the addition of salt during ripening. In fact, the selection of a suitable plant coagulant with the best MCA/PA ratio, the use of a low coagulant dose, the optimisation of various coagulation parameters, and the control of ripening step could promote excellent results in terms of rheological and sensory properties (texture and flavour) of cheeses produced by plant rennets. For milk-clotting enzymes with high proteolytic action, several improvement strategies have been developed to produce cheeses similar to those made with commercial rennet. The good selection of the type of milk for specific cheese variety (non-ripened cheese) plays an important role in the success of cheeses made with these coagulants. Despite some success observed for certain types of cheeses, their production on an industrial scale remains marginal. Subsequent research studies on the production of plant extracts with no associated protease activity or which have undergone specific treatments to reduce excessive proteolysis will be of great importance. The establishment of efficient formulations of plant rennet (high activities ratio) supposed to be commercialised will also be of considerable importance for future uses on an industrial-scale cheese production.

Abbreviations

ΔA	=	delta absorbance
AAN	=	amino acid nitrogen
AP	=	aspartic protease
AR	=	animal rennet
CE	=	crude extract
Dw	=	dried weight
Fw	=	fresh weight
GP	=	ginger protease
IMCAU	=	international milk-clotting unit
kcat	=	catalytic constant
Km	=	Michaëlis constant
MCA	=	milk-clotting activity
NH3	=	N ammoniacal nitrogen
NPN	=	nonprotein nitrogen
PA	=	proteolytic activity
PCC	=	powder of C. cardunculus coagulant
RU	=	rennet unit
SN	=	soluble nitrogen
SP	=	serine protease
Vm	=	maximal velocity

Acknowledgements

We thank Dr. Aman Paul and Mrs. Sawsan Derbel for proofreading this paper.

Funding

We thank University of Liège and University of Sfax for providing financial support to this study.

Additional information

Funding

We thank University of Liège and University of Sfax for providing financial support to this study.