Natural rennet sterilisation by non-thermic methods for fresh cheese manufacture

Abstract Natural rennet is widely used in artisanal cheese shops to produce fresh cheese with particular characteristics such flavour, in comparison to artificial rennet. This rennet is produced by the cheesemakers under non-hygienic conditions. The use of non-thermal technologies such as ultrasound and UV light can be an alternative of rennet sanitisation. The objective of the present work was to evaluate the effect of applying of different times of ultrasound or ultraviolet on the microbial growth in natural rennet for fresh cheese manufacture, its curd yield and curd hardness texture. Natural rennet was prepared and divided into three samples, one of which was exposed to ultrasound for 5, 10 and 15 min, the second one received UV radiation for 30, 60 and 90 min, and the last one was considered as the control treatment. A microbial test was conducted to analyse total coliforms, Escherichia coli, Staphylococcus aereus and Salmonella spp. Fresh cheeses were manufactured using the different rennets and the coagulant effect was tested by way of with a texture test (hardness). The results showed the highest microbial growth in the control treatment; none of the US treatments allowed microbial growth and the UV radiation was effective for all microorganisms at a 90 min exposure. All rennet treatments maintained their coagulant effect. In conclusion, the use of non-thermal methods was effective for rennet sterilisation, although it affected curd yield (2% less). Highlights Ultrasound inhibits pathogenic microorganisms growing in natural rennet. Sterilisation by non-thermal methods does not affect the coagulation process. A 42 kHz ultrasound at, 70 W power caused the best reduction of pathogenic bacteria.


Introduction
The region of the Western Highlands (Altiplano) of the state of San Luis Potos ı, Mexico is characteristically a dairy-producing region, in which most of the milk produced in small production units (PUs) is destined to the production of fresh cheese.In these PUs, cheese is made from raw milk and non-sterile natural calf rennet (calf stomach and milk whey), with the use of nonstandardized traditional methods, scarce technification and in inappropriate facilities (e.g. they use the kitchens in the producers' homes).In this regard, the lack of hygiene in the utensils and equipment in which the cheeses are made, as well as the use of natural rennet, can be a risk to the health of the consumer due to high total coliform (TCs) counts, Staphylococcus aureus, Salmonella spp, yeasts and Enterococcus (Voidarou et al. 2011;Rodr ıguez-Gallegos et al. 2022).Therefore, in Mexico dairy products must comply with the sanitary provisions and specifications of NOM 243.This indicates that Salmonella spp.must be absent in 25 g of cheese or millilitre of milk, a maximum of 10 colony-forming units per millilitre of milk (CFU/mL) is allowed for total coliforms, Escherichia coli must be less than 3 MPN/mL of milk and a maximum of 10 CFU/mL is allowed for Staphylococcus aureus.
Animal (calf and lamb) rennet was the first discovered source of chymosin and pepsin enzymes used to convert liquid milk into a soft gel called curd or cheese (Camin et al. 2019).Chymosin is an enzyme aspartic protease type that breaks j-casein into hydrophobic para-j-casein, which remains on the surface of casein micelle; and hydrophilic glycomacropeptide, which is cleaved from casein micelle, extensive cleavage of j-casein will result in the destabilisation and aggregation of a casein micelles into a coagulum (Li and Zhao 2019).If this enzyme reaches temperatures of up to 60 C to sterilise it can be denaturalised or lost biological functions (Kumar et al. 2006).
Heat is usually used in food processing operations, for instance, to pasteurise liquids.However, if it is not controlled may result in deterioration of the nutritional and sensory qualities of food (Jadhav et al. 2021).The use of non-thermic methods for sterilisation in the food industry is recent (Sonawane et al. 2020).Nonthermal technologies for food applications comprises technologies such as ultrasound, cold plasma, highpressure, pulsed electric field, ozone, UV, electrolysed water, and pulsed UV light.The development of these technologies has been growing in the last decade due to its successful application in sanitisation.However, pasteurisation, sterilisation, and other thermal technologies used to sanitise food products usually affects negatively the nutritional and sensory properties of many products (Fernandes et al. 2021).
Ultrasound (US) is an emerging technology which is considered green technology, since it consumes less energy.In liquid systems, US causes acoustic cavitation, which is the phenomenon of generating, growing and eventual collapsing bubbles (Majid et al. 2015).It has been used to control bacteria and algae in industrial water systems (Broekman et al. 2010).Nowadays, US is being applied as an effective preservation tool in many food-processing fields, namely vegetables and fruits, cereal products, honey, gels, proteins, enzymes, microbial inactivation, cereal technology, water treatment, diary technology, etc. (Majid et al. 2015).Ultraviolet light (UV) has been proposed as a non-thermal alternative for the reduction of micro-organisms in fluids.UV technology has numerous advantages over traditional preservation, such as the use of heat treatment, for example: including the low cost of installation and maintenance, reduction of carbon emissions when compared to traditional thermal pasteurisation systems, and also offers an alternative processing technology in developing countries where the production of milk and cheese are performed on a small scale (Cilliers et al. 2014).
Therefore, the objective of the present work was to evaluate the effect of applying US or UV for different times on the microbial growth in natural rennet for fresh cheese manufacture, its curd yield and curd hardness texture.

Materials and methods
The experiment was conducted in the Microbiological laboratory of the Coordinacion Acad emica Region Altiplano Oeste of the Universidad Aut onoma de San Luis Potosi.Natural rennet was obtained from a local artisanal cheesemaker.The preparation process consisted of washing, then salting and drying one kilogram of calf stomach (abomasum), obtained from a local butcher, followed by fermenting dried tissue in a container with 5.0 L of milk whey obtained from a local farm.The fermentation process took three days at room temperature (20 C); two same rennet batches were prepared with a 15 days period between runs.
After that, 60 samples of 1.5 mL natural rennet were placed in Eppendorf microtubes for ultrasound (US) treatment (Branson 1510, 42 kHz frequency) at 5, 10 or 15 min (n ¼ 20) in distilled water at room temperature (20 C) (Li et al. 2016).Alongside this, five 10 mL of natural rennet samples were verted in sterilised Petri dishes and exposed to ultraviolet (UV) radiation (253.7 nm long wave, distance between UVC lamp and agar medium, 53 cm) in a Biological Safety Cabinet (1300 Class II, SERIES A2, Thermo Scientific) for 30, 60 or 90 min (initial time was taken from the owner's manual for cabinet cleaning).A small fraction of natural rennet (untreated) was considered as the control treatment (À).Also, a commercial rennet (Cuamex V R CHR Hansen de M exico) was used as a positive control (þ) (Table 1).

Microbiological analysis
Microbiological analyses (total coliforms, Salmonella spp., Staphylococcus aereus and Escherichia coli) were performed following to the directions described in the National regulation for milk products (NOM-243-SSA1-20102010).
Agar VRB (Violet red bile lactose, BD-BIOXONTM), Salmonella-Shigella (BD-BIOXONTM), Baird Parker (BD-BIOXONTM) and Eosin-Methylene Blue (EMB) were prepared following the directions of the supplier for the detection, growth and count of total viable microorganisms in a plate (Petri dishes) of total coliforms, Salmonella spp., S. aureus and E. coli, respectively.

Samples preparation and dilutions
Different rennet samples were diluted in 0.1% peptonated water, following the method described in the NOM-110-SSA1-1994(1994).In order to do this, ten glass tubes (20 mL) were previously sterilised for each sample of rennet (n ¼ 3).Dilution was performed with the following process: for total coliforms, E. coli and the Staphylococcus aureus test, three samples (10 mL) from each rennet treatment was placed in tube number one (10 ) and the rest of the tubes contained 9.0 mL of peptonated water 0.1%.Next, 1.0 mL solution from tube one was taken and mixed in tube number two, and the process was repeated until tube ten (10 9 ).For the Salmonella test, three samples of 25 mL were mixed with 225 mL of peptonated water 0.1% and incubated for 18 h at 36 C. Once dilutions were prepared, the Miles and Misra technique modified by Slack and Wheldon (1978) was used; three 20 lL samples of each dilution were placed in a Petri dish for each of the four different agars.The whole experiment was replicated three times with independent rennet batches.
Total coliforms were identified following directions by the NOM-113-SSA1-1994(1995) and Petri dishes were incubated at 37 C for 24 h.Next, plates with 15-150 colonies were reported as Log10 UFC/mL.Salmonella spp.growth was analysed following directions by NOM-114-SSA1-1994(1994) Salmonella-Shigella agar, Petri dishes were incubated at 37 C for 24 h and translucent or shadowed colonies with black dots in the centre were considered as positive (presence).To determine the presence of S. aureus directions of the NOM-115-SSA1-1994(1995) were followed and growth was analysed in Baird Parker (Potassium tellurite þ Egg Yolk Emulsion) agar, incubated at 35 C for 48 h, plates with black coloured colonies between 15 and 150 were selected.To determine the presence of E. coli following directions of the NOM-210-SSA1-2014 (2015) Petri dishes were incubated at 35 C ± 1 C for 24 h; colonies with black points in the centre, flat with or without metallic lustre were considered as positive.

Curd yield
Liquid pasteurised and homogenised whole cow's milk containing 30 g/L fat, 29 g/L protein, pH 6.6 and a density of 1.032 g/mL (LALA V R ) was used to produce small curds.Eighty samples (200 mL) of pasteurised commercial cow's milk were placed in glass beakers (250 mL), heated to 35 C in a water bath (Julabo V R TW20) and 2.0 mL (0.01%) of treated or untreated rennet was added and incubated for 30 min, Later, they were cooled down to 20 C for 20 min.Subsequently, ten miniature curds (2.0 Â 3.0 cm) for each treatment were separated from the whey and were then weighed in fresh and dried at 60 C for 24 h in a forced convection oven (BINDER FD Series) (AOAC 2005).

Texture test
Once fresh curds with the different rennets were obtained, hardness was determined by using a texture analyser (Brookfield V R CT3 Middleboro, MA).Square curd samples 2.0 Â 3.0 cm were cut.Samples were placed into Petri dishes and kept at 20 C for 1 h; afterwards, their height was adjusted to 20 mm with a blade.Curds were compressed using a probe TA53 cutting wire 0.33 mm D, 40 mm L and a crosshead speed of 1 mm/s; five samples of every cheese were analysed.The values of hardness were calculated from the resulting curves using the equipment software (Texture Pro CT Software, Brookfield).

Experimental design and data analysis
Microbiological analyses of the different rennets to identify growth of Escherichia coli, total coliforms and Staphylococcus aureus, curd yield (humid and dry weight) and texture (hardness) were carried out with a completely randomised design with a factorial arrangement 2 Â 4, where factor A was the sterilisation method (US and UV radiation) and factor B was the exposure time (0, 5, 10, 15 or 0, 30, 60, 90 min) both considered as fixed factors.Each Petri dish (n ¼ 15) was considered as a random factor and, the experiment was replicated three times (15 day period between runs).A Tukey HSD test (p < 0.05) was carried out to identify differences between treatments.The statistical software R Core Team (2020) was used to analyse the data.

Results
The pH-value was measured in all rennet treatments (Table 1).All of them were acids below pH 5.0, according with Moschopoulou (2011) chymosin and pepsin enzymes are activated and have proteolytic activity which, at this pH-value, convert milk into curd in a few minutes.

Microbiological analysis
Results of the microbiological analysis are shown in Table 2.For the identification E. coli, statistical differences were found due to sterilisation methods (p < 0.01), exposure time and the interaction (p < 0.0001).The higher value of 4.53 Log10 CFU/mL was found in the untreated rennet (Control À), The UV treatment showed an effect after 60 min of exposure; all UV treatments were effective to inhibit E. coli growing, and as expected of a commercial rennet (Control þ), it did not show E. coli colonies.For the identification of Staphylococcus aureus, statistical differences were found due to sterilisation methods (p < 0.05) and exposure time (p < 0.001).Untreated rennet presented the higher value (4.13 Log10 CFU/mL) and decreased when rennets were irradiated with UV for 30 or 60 min (3.03 or 2.49 Log10 CFU/mL).S. aureus growing was not detected with all ultrasound treatments and commercial rennet (Control þ) did not display S. aureus colonies.The identification of total coliforms (TC) displayed statistical differences due to sterilisation methods (p < 0.0001), exposure time and the interaction (p < 0.0001).Untreated rennet presented the highest value (3.68 Log10 CFU/mL); UV treatment was effective against TC after 60 min and, all US treatments were effective to inhibit TC growing.The identification of Salmonella also displayed statistical differences due to sterilisation methods (p < 0.0001) and exposure time (p < 0.0001).The presence of Salmonella was observed only in the untreated rennet and, commercial rennet, whereas UV and US treatments did not display the presence of Salmonella.

Curd yield
Figure 1 shows the results for curd yield.Statistical differences were found due to factor time of exposure (p < 0.0001) and the factor of sterilisation method showed neither effect (p ¼ 0.757), nor the interaction (p ¼ 0.285).Both control treatments (þ and À) presented the higher weight yields in fresh (24.exposure (p < 0.01), and the factor of sterilisation method did not show effect (p ¼ 0.1287), neither the interaction (p ¼ 0.2784).Control treatment (þ) presented the highest dry weight (13.7 g) that represents 56.7% of solids (43% moisture).UV treatments for 90 min and US for 10 min presented the lowest dry weight (10.2 g).

Texture test
Figure 3 shows the hardness of curds obtained with ultraviolet, ultrasound and untreated rennets as part of the texture test.There were no statistical differences between treatments due to the sterilisation method (p ¼ 0.0833), time of exposure (p ¼ 0.3497) or the interaction (p ¼ 0.3395).

Microbiological analysis
A previous study carried out by Rodr ıguez- Gallegos et al. (2022) showed the high microorganism counts in cheeses and the use of non-sterilised natural rennet in small artisanal cheese shops in the region of the Western Altiplano of the state of San Luis Potos ı due to flavour and aromas that natural rennet confers to the cheeses.As mentioned previously in the introduction section, dairy products in Mexico must comply with the sanitary provisions and specifications of NOM 243.This indicates that Salmonella spp.must be absent in 25 g of cheese or millilitre of milk, a maximum of 10 colony-forming units per millilitre of milk (CFU/mL) is allowed for total coliforms, Escherichia coli must be less than 3 MPN/mL of milk and a maximum of 10 CFU/mL is allowed for Staphylococcus aureus.
Studies about the use and microbiology of natural rennet are scarce.For instance, Fl orez et al. ( 2006) pointed out, that colonies of enterococci, staphylococci and leuconostoc could be occasionally found in natural rennet.Gal an et al. ( 2012) found enterobacteria, coliforms, staphylococci, micrococci in cheeses made with calf rennet.Voidarou et al. (2011) mention that a wide biodiversity of microorganisms can be found in dried artisan inoculants made of the dehydrated rumen of small ruminants (Lactobacilli, Lactococci, Leuconostoc, Pediococci, Streptococci, Bifidobacteria, Enterococci, Clostridia and coliforms).Murgia et al. (2019) studied a peculiar cheese from Sardinia (Italy) made with edible goat rennet namely Caggiu de crabittu, where the kid abomasum is devoid of perivisceral fat and full of suckled milk and is then subjected to a ripening time (30-60 days on average).This process decreases pathogenic bacteria and makes it microbiologically safe, with a high number of live lactic bacteria.In contrast with our results, their lower pathogens counts can be explained due to the ripening effect of time that is important to the growth of lactic bacteria (Lactobacillus spp) and decreases pH.
Ultrasound has attracted great interest in the field of food preservation.Low frequencies (20 kHz) induce damage to E. coli and S. aureus causing cell death (98.14% and 91.68%, respectively) by compromising membrane integrity, inactivating intracellular esterases, and inhibiting metabolic performance (Li et al. 2016).Bartkiene et al. (2018) pointed out that US at low frequency (37 kHz, for 20 min) combined with dehydration methods like lyophilisation or vacuum drying reduces contamination by Enterobacteria, E. coli and yeasts in bovine colostrum that is used as a functional food ingredient.These results coincide with those found in our study, where US treatments (5, 10-or 15min exposure) were effective to inhibit all pathogenic microorganisms possibly due to the force generated during the implosion of water bubbles that cause cell death.
In another study, the effect of US (24 kHz frequency) was proved in raw, thermized (55 C) and pasteurised milk for 16 min.Results suggest that this sterilisation method reduces the total viable count by ffi 37% in the three treatments (Chouliara et al. 2010).The high count of these results in contrast with ours, could be explained to the differences in frequencies used.
Ultraviolet (UV) irradiation is electromagnetic irradiation with a wavelength of 100-400 nm.It is divided into four distinct spectral areas including vacuum UV (100-200 nm), UVC (200-280 nm), UVB (280-315 nm) and UVA (315-400 nm) (V azquez and Hanslmeier 2006).The mechanism of the UVC inactivation of microorganisms is to damage the genetic material in the nucleus of the cell and is the most lethal range of wavelengths for microorganisms, especially the range of 250-270 nm (Chang et al. 1985).UVC at 254 nm is bactericidal (100% inactivation) for antibiotic-resistant strains of Staphylococcus aureus and Enterococcus faecalis at times as short as 5 s, considering a distance between UVC lamp and agar medium of 25.4 mm (Conner-Kerr et al. 1998).Their results were very effective in a short time, in contrast with ours, which took longer, this can be explained considering the distance between the UV lamp and the sample, which was higher in our experiment.

Curd yield
Values of curd yield obtained in this study are higher to those reported by Dussault-Chouinard et al. ( 2019), who used skim milk (6.6 pH) and rennet at pH 6.2, resulting in a cheese yield of 7.47%.In contrast, a higher yield was observed in our study, and these differences could be due to a lower rennet pH value in our trial.Katz et al. (2016) obtained cheese yields ranging between 121-154 g/L of milk (12.1-15.4%)after 60 min incubation.These values are higher than those obtained in our study, and differences could be done to time incubation.Our results of curd yield when rennet was treated with US or UV were similar to those when raw, centrifuged or filtered paste rennet are used to produce artisanal cheeses (9.3-10.0%)(Calvo and Fontecha 2004).

Texture test
The texture of cheese is among the parameters of physical qualities of cheese that influence consumers' acceptance.Fresh curd hardness expressed in gramforce (gf) which, in this trial, was lower compared to other studies, e.g.Setyawardani et al. (2018) found hardness values close to 227 gf in goat cheese, Teter et al. (2021) obtained a curd hardness value of 3.62 N (369 g) and Juan et al. (2013) found a hardness value of 3.97 N (404 gf) in cheeses made with cow's milk (3.5% fat).Wolanciuk et al. (2016) obtained a curd harness value of 4.7 N (479 gf).Authors pointed out that the main reason behind high curd hardness can be associated to a high protein content in milk (3.3%).Ripening and reductions in moisture are another factor to increase hardness in other cheese types, e.g.Manchego-type cheese can display 8.67 N (884 gf) hardness (Lluis-Arroyo et al. 2011) or 3309.8-9507.5 (g) in 90 days for goat surface mould cheese (V azquez-Garc ıa et al. 2020).
Finally, these non-thermic methods could be used to inhibit other pathogens of interest such as Brucella spp in milk of ill animals.Brucellosis is considered as a critical zoonotic disease with global distribution via the consumption of raw milk and unpasteurised dairy products like cheese (Majzobi et al. 2022) or with a deficient pasteurisation process (Rodr ıguez- Gallegos et al. 2022) that causes economic losses and affects human health.It is also important, since it has been reported that Brucella abortus has presented resistance to commonly used antimicrobials (Alwan et al. 2010).

Conclusions
Ultrasound (US) and ultraviolet (UV) are effective sterilisation methods to inhibit pathogenic microorganisms, however both US and UV affected curd yield (2% less) and hardness.These methods can be an alternative for the manufacturing of fresh artisan cheeses where natural rennet is used, without affecting the consumer's health and to fulfil the Mexican regulations for food safety.
3 and 23.0 g/200 mL of milk, respectively) in contrast with the rest of the treatments.It represents around 12% of solids.The rest of treatments presented a weight yield of approximately 20.0 g/200 mL of milk (ffi10% of solids).Curd dry weight results are shown in Figure 2. Differences were found due to the factor of time of

Figure 3 .
Figure 3. Texture analysis by hardness test to curds manufactured with treated (ultraviolet and ultrasound) at different times and untreated rennets.

Table 2 .
Microbiologic analysis of natural rennet treated with different non-thermic methods ultraviolet radiation (UV) or ultrasound (US).Control À UV 30 min UV 60 min UV 90 min US 5 min US 10 min US 15 min Factor effect SEM