Rennet is a mixture of proteolytic enzymes (tissue and gastric enzymes) – rennin (chymosin) and pepsin, obtained from gastric mucous membrane of young ruminants . These are the coagulating enzymes used in cheese production, but rennet is widely used. It is the oldest method of producing cheese. Rennin is present in its prorennin form with 42 amino acids at its N-terminal. Rennin (323 amino acids) is made of a single polypeptide chain with a molecular weight of 35,000 Daltons. Rennin breaks the Phenylalanine-methionine bond in kappa casein . The enzyme with high proteolytic activity is not beneficial due to the following reasons-
It breaks down the milk protein casein imparting an unsatisfactory consistency to the cheese.
It is undesirable as it leads to loss of digested protein with the whey.
It results in bitter taste .
With the increase in demand for cheese with the rate of 4% a year and decrease in supply of calf rennet, its substitute plant and microbial rennet has captured the focus. Mucor miehei, Cryptococcus albidus, Mucor pussilus, Bacillus cereus and Endothia parasitica are some microbes which aid in the production of this conventional enzyme. Rennet from fungus Mucor miehei is involved in 33% of cheese production. It is preferred because of its high coagulation rate . In the production process, the organism is grown in nutrient medium, separated by filtration, followed by pH adjustment and then performing ultra filtration and vacuum evaporation .
Easily utilizable sugars such as glucose, lactose or sucrose can be used. Inorganic salts like Calcium, Phosphate and Magnesium are added to support the growth. Assimilable nitrogen source- Ammonium salts, Nitrate salts or amino acids are utilized. The nutrient concentration in the medium varies according to the species and strain . The whey produced from the process is used in milk-based confectionery and for other processed foods .
The plant sources are papain from papaya tree and bromeline from pineapple .Most plant rennet enzyme imparts bitter taste to cheese whereas the microbial enzymes are cheaper and easy to modify. The enzyme activity gets affected with the sugar source. More activity was shown with glucose and less with lactose, when Mucor miehei NRRL 3420 was used. Mucor pusillus QM436 clots milk with higher potential, indicating as a good substitute .
The animal and plant rennet have influence on the amino acid content of cheese. On the 60th day of ripening, the Spanish and Portuguese cheese showed increase in total free amino acid (TFAA) content in cheese, representing >50% of TFAA. The TFAA content was higher in cheese made with plant enzyme (854 mg/100 gm total solids) than with the use of animal enzyme (735 mg/100 gm of total solids). Cheese made with plant enzyme contained high levels of Met, Ser, Ile, Ala, His and Gln .
SUMMARY OF PATENTS-
The action of rennet is to cleave at a specified site in K casein and perform proteolytic function non-specifically. The rennet should have a low Proteolytic Activity (PA) and a high Milk-Coagulating Activity (MCA) i.e., a low PA/MCA ratio. If it is high then it makes the taste bitter, imparts a poor texture and reduces the yield of curd. But practically the microbial rennet has a high PA/MCA ratio than animal rennet. This patent focuses on increasing the MCA and decreasing the proteolytic activity by performing acylation on Mucor pusillus rennet. In this invention, the microbial rennet is succinylated with succinic anhydride which provides better safety and higher stability to the enzyme than maleic acid. The temperature should be between 0ËšC to 40ËšC and pH 4 to 10, preferably 7 to 9. After acylation the mixture is neutralized to 5 to 6.5 and enzyme is obtained by gel filtration, ultra filtration or dialysis.
The preserved stomach of the calves is used to make a crude mixture of enzyme using water. The proenzyme is then activated to form the active version of enzyme. Filtration step is followed to clarify the enzyme and to obtain a concentrated form. Reclarification is done followed by filtration to remove the precipitated impurities. The enzyme is then added with salt and preservatives. The enzymes extracted from old and adult calves have low chymosin content.
Disadvantages of conventional rennet manufacturing process-
High chymosin content requires young calves
Tedious clarifying process
Large residual contaminated waste water
Liquid rennet mixtures have low potential and results in transportation problem.
The enzymes are extracted using a weak DEAE-cellulose ion-exchanger, PEG exchanger and with the use of salts. The drawbacks of using them are-
Difficulty in removing the PEG
Dissolution of salt is difficult, so the solution needs to be warmed.
Cultivation of yeast Cryptococcus albidus, C. diffluens and C. aerius provides the milk coagulating enzyme. The temperature should range from 15ËšC to 40ËšC with aeration and a pH of 3.0–8.0. The process works for 2-7 days. The enzyme is extracted by dialysis, salting out and freeze drying. Precipitation of mixture deprived of solids can be done with organic solvents, purification with ion-exchangers, salting out or low pressure. For salting out NaCl, MgSO4 or NH4NO3 can be used. Further purification is done by performing dialysis against water. The non-dialyzable products are freeze dried and used. The enzymes obtained displayed high clotting activity and low proteolytic action.
The invention focuses on making low cost, high coagulating enzyme with low protease activity by making the use of yeast and their mutants. Yeast species of genus- Torulopsis, Cryptococcus, Rhodotorula, Candida, Kluyveromyces, their natural and artificially induced mutants are used. Strain of Cryptococcus albidus and its variant aerius were inoculated in media containing 2.0% glucose, 0.1% yeast extract, 0.1% Ammonium Sulphate, 0.07% Sodium Phosphate dibasic, 0.2% Potassium Phosphate monobasic and 0.05% Magnesium Sulphate which was sterilized at 115ËšC for 20 minutes. It was incubated at 28ËšC for 72 hrs on shaker condition. Centrifugation was done and enzyme activity of the supernatant was determined which was Ca. 80 Soxhlet units/ml. Precipitation was done with Ammonium Sulphate followed by dialysis and liophilisation. The activity of powder was determined to be Ca. 140 rennet units (RU)/mg. This rennet was used to make parmesan cheese.
This patent aims on preservation and extraction of rennet from stomach of suckling calves. The rennet is recovered using a salt solution. The bovine calf stomachs are stored in a mixture of Ammonium Sulphate, Ammonium Chloride, Sodium Sulphate and Potassium Sulphate followed by refrigeration. The entire stomach is soaked in brine solution and refrigerated (4-5ËšC). Then they are kept in 3:1 ratio of water and meat for weeks at 37ËšC.
The separation of rennet from Endothia parasitica and M. miehei was reported by Kobayashi et al. and its purification was carried using N-acetylpepstatin affinity gel columns. In this patent, Cibacron blue F3GGA, a soluble blue affinity ligand was used. It selectively binds to the enzyme at pH below 4.0. It has no effect no microbial pigment. The dye on binding with the enzyme neutralizes three positive charges and therefore the isoelectric point reduces. In this case, the rennet precipitates in presence of excess dye which can be then centrifuged. It is again mixed with salt and chitosan, a polycation, used to eliminate the dye. Polyethyleneimine can also be used when the R group in the dye is polyethylene glycol. Two-phase process can be used which involves Cibacron blue derived polyethylene glycol (CB-PEG) and dextran in water. This form two phases. The enzyme gets separated in the upper phase of CB-PEG. This portion is separated and added with NaCl to make two more phases.
In this patent, M. miehei NRRL 3420 was grown in nutrient medium. The filtrate was obtained and the pH was adjusted to 3 using conc. HCl. The filtrate was run on a precolumn containing polystyrene sulfonate to remove the pigments. An agarose bead support matrix was linked to Cibacron ligand was used. Sodium citrate of pH 5 eluted the rennet enzyme, giving 85% yield.
Along with the required enzyme i.e., rennet, other protein produces interference. For removing the non-specific enzymes, the filtrate is run over silicate column with a pH of 3 and 9. More than one absorbent can also be made in use. Rennet is stable at a pH range of 3 to 9. As the pH reduces, the stability decreases. The enzyme concentrate is obtained by precipitation or evaporation.
The clotting activity is determined by the following formula- 25X40/n X t u./mg
where, ‘n ’= quantity of rennet in mg/ml
‘t ’= clotting time
After fermentation, white powder is produced which is feasible to transport when compared to liquid form. But, the powder does not render optimum solubility. Addition of 2-3% of fatty acid monoesters of polyoxyethylene sorbitan makes the rennet, obtained from M. miehei, stable. This ester contains 12-22 carbon atoms and 20 oxyethylene units/molecule. This mixture makes it suitable for storage, soluble in liquid, dust free and easy to handle.
The thermal destabilization property of rennet is beneficial as it can be used in pasteurized whey. Rennet shows low thermal destabilization when it is carbamylated with Potassium cyanate. If the whey has to be used for other purposes then the rennet activity should be restricted as it can lead to production of clumps and clots. The half-life of the enzyme is determined by-
T= (t2 –t1) ln2/ lnA1 – lnA2
where, A1 and A2 are enzyme activity on heating at particular temperature at time t1 and t2. More the temperature, shorter is the half-life.
For obtaining microbial rennet, the sequence coding for rennet is expressed in expression vector through recombinant technique. It is then inserted into suitable bacterial host. Most favourable is E. coli. The host cells are grown under proper conditions. The cells are disrupted mechanically, enzymatically or through sonication. The suspension is allowed to centrifuge at 500-5000 g for 10 minutes-2 hours. The obtained pellet contains the desired enzyme. Denaturing agent is added to the pellet needing a pH of 7-8. Quanidine hydrochloride can be used. The mixture is allowed to undergo sulfitolysis. In this reaction, the disulfide bond is broken and sulfide is replaced with sullfonate. It’s a nucleophilic reaction which results in protein-S-SO3 (protein-S-sulfonate) formation. It is then subjected to weakly denaturing medium like urea, which plays role in proper refolding. The extract is purified using molecular sieve or ion-exchange chromatography.
mRNA coding for rennet is extracted from the whole RNA content using oligodT column. Reverse transcriptase is used to make cDNA. Agarose Gel Electrophoresis is run to separate fragments of suitable size. With specific Restriction endonuclease cuts are made in fragments and vector. PolyG tail is added to the vector while polyC to cleaved fragment at termini. The cells are made competent with the help of Calcium Chloride and transformed cells will grow on tetracycline and ampicillin containing plate, if the cloning vector is pBR322. The plasmid from the transformed colonies is obtained by nucleases activity. Suitable host can be- E. coli, B. subtilis or yeast.
Van Kampen V,Lessmann H,Brüning T,Merget R.2013. Occupational allergies against pepsin, chymosin and microbialrennet. 67(5):260-4. Doi: 10.1055/s-0032-1326407. Pubmed.
Chen and Michael Cheng-Yien. 1983. Microbially produced rennet, methods for its production and plasmids used for its production. EP 0 116 778.
Huibert Cornelis, Theiis Moelker and Rutger Matthijsen.1971. Purification of microbial rennets. US 3,591,388.
C.J.B. De Lima, Mariana Cortezi, Roberta B. Lovaglio,.J. Ribeiro, J. Contiero and E.H. De Araújo.2008. Production of Rennet in Submerged Fermentation with the Filamentous Fungus Mucor miehei NRRL 3420. World Applied Sciences Journal 4 (4): 578-585.
Sethuraman Subramanian. 1988. Purification of microbial rennet from Mucor miehei. US 4,743,551.
Alessandro Martini, Federico Federici, Benito Argenti. 1979. Milk Coagulating Microbial Enzyme. US 4,141,791.
Higashi, Toshihiko, Kobayashi, Yoshinori, Iwasaki, Shinjiro. 1988. Process for producing microbial rennet having increased milk coagulating activity. EP 0 091 664 B1.
El-Tanboly el-S,El-Hofi M,Youssef YB,El-Desoki W,Ismail A. 2013. Utilization of salt whey from Egyptian Ras (cephalotyre) cheese in microbial milk clotting enzymesproduction. PMID: 2458486112(1):9-20.
PATENTS REFFERED –
INVENTOR/ COMPANY NAME
YEAR OF PUBLICATION
TITLE OF PATENT
Process for producing microbial rennet having increased milk coagulating activity
EP0 091 664 B1
Robert, Marianne Kirsten, Pal Martin
A process for separating milk clotting enzymes
EP 0 758 380 B1
Alessandro Martini, Federico Federici, Benito Argenti
Milk Coagulating Microbial Enzyme
Richard B. Dardas, Gales Ferry
Preservation of bovine stomachs for Rennet Extraction
Purification of microbial rennet from Mucor miehei
Huibert Cornelis, Theiis Moelker and Rutger
Purification of microbial rennets
Stabilized microbial rennet
Sven Branner-Jiirgensen, Palle Schneider and Peter Eigtved
Thermal destabilization of
Chen and Michael Cheng-Yien
Microbially produced rennet, methods for its production and plasmids used for its production
EP 0 116 778
Kirk J. Hayenga, Virgil B. Lawlis and Bradley R. Snedecor
Preparation of cheese with rennin
From recombinant microbial cells
Problem of Protein Energy Malnutrition in Weaning Infants
This paper will examine the protein energy malnutrition problem amongst weaning children in Niger. By using secondary sources and by looking into precedent practices by different organizations to improve the situation, it will finally conclude with health promotion nutrition intervention plan which will include a collaboration and partnership with stakeholders who will as well have a great impact on the population’s health determinants. For this project we will take the role of three nutritionists hired by Médecins sans frontiers (MSF) to establish a best practice and protocol standardized health system in line with the solution of treatment.
Firstly this paper will provide a background on the country and the subject of protein-energy malnutrition within different regions. Different existing intervention programs will be presented together with a personal health promotion intervention plan. This will be followed by the determinants that will mainly influence the program and its objectives. Secondly the strategies and practices of the intervention plan will be explained in depth. Thirdly, this project will present to collaboration and partnerships with different stakeholders in order to finally indicate how this programs is creating community capacity.
Background context: Niger:
Niger, or officially named the Republic of Niger, is located in Western Africa covering a surface of 1.270.000 km2 of which 80% consists of Sahara. Neighbouring countries are Nigeria, Benin, Burkina Faso, Mali, Algeria, Libya and Chad. Being landlocked it is one of the hottest countries of the world. Fifteen million people live in Niger of which only 5% in the capital Niamsey. The population density is only of 12.1/km2. The population is characterized by its fast growth rate (3rd rank worldwide) and has the number one highest birth rate and fertility rate of 7.2 births per woman which means that 49% of the Nigerien population is under the age of 15. Known also to be one the poorest countries in the world; Niger’s economy has mainly been undercut by the drought cycles, desertification and the strong population growth (Niger, 2010).
Protein-energy under nutrition:
Protein -energy undernutrition (PEU), previously called protein-energy malnutrition is an energy deficit due to chronic deficiency of all macronutrients (which are proteins, fats and carbohydrates). In developed countries, PEU is common among the institutionalized elderly or among patients with decreased appetite. In underdeveloped countries protein malnutrition occurs because of the local diet with protein poor cereal products (Morley, 2007).
The classification is determined by calculating weight as a percentage of expected weight per height using international standards. (Normal: 90-110%; mild PEU: 85-90%; moderate: 75-85%; severe: <75%). In children, chronic primary PEU has two common forms depending on the balance of no protein and protein sources of energy. The first form, Marasmus (also called the dry form of PEU); causes weight loss and exhaustion of fat and muscle. The second form, Kwashiorkor (also called the wet, swollen form of PEU), is associated with premature abandonment of breastfeeding, which typically occurs when a younger sibling is born, displacing the older child from the breast. This phenomenon is also called the "second child spell". Thus, children with Kwashiorkor tend to be older than those with marasmus (Morley, 2007).
Pathophysiologically, the initial response to PEU is decreases metabolic rate. To supply energy, the body first breaks down adipose tissue or body fat. When these tissues are used up, the body may use protein for energy; visceral organs and muscle are broken down and decrease in weight. Loss in organ weight is the greatest in liver and intestine, intermediate in the heart and kidneys and least in the nervous system (Morley, 2007). Total starvation however can be fatal in eight to twelve weeks thus certain symptoms of PEU do not even have time to develop. Patients with protein-energy undernutrition often also have deficiencies of vitamins, essential fatty acids and micro nutrients which contribute to their dermatosis (skin disease) (Scheinfeld, 2010).
Worldwide, the most common cause the malnutrition is inadequate food intake. Another very significant factor however is the ineffective weaning secondary to ignorance, poor hygiene, economic factors and cultural factors. The prognosis is even worse when PEU occurs with HIV infection (Niger, 2005).
Protein-energy malnutrition in Niger:
In Niger, the diet of most children is extremely monotonous, usually consisting of millet based porridge although the diet of older household members might be more diverse. This monotonous diet leads to nutrient deficiencies and consequently diseases such as Kwashiorkor and Marasmus develop.
In 2005, a survey was conducted by MSF which stated that one child on five suffers from malnutrition. That year, the mortality rate of children under five exceeded the emergency threshold; 2 deaths per 10.000 children per day. Through the therapeutic feeding centres of MSF, the presence of doctors enabled to reduce the mortality rate to 6% that year. Care is also provided through 40 mobile nutritional care centres which allow children to be treated closer to home. Many are treated at home with ready-to-use therapeutic food (RUTF) and come to the once a week for a check-up (focus on Niger, 2006).The concept of RUTF will be explained further later.
Due to weather conditions, an annual ‘hunger’ gap exists between April and September when family food stocks run out and hundreds of thousands of children have little access to the nutrients they need for a healthy development (IAR 2007, 2008).
The World Health Organization recorded in the 43rd week of 2009 recorded 2253 cases of moderate malnutrition and 2938 cases of severe malnutrition and 5 deaths caused by malnutrition. On yearly bases for the year 2009, 157.125 cases and 384 deaths were recorded between January 1st 2009 and October 25th 2009. 41% of those patients were diagnosed with severe malnutrition and 23% with moderate malnutrition (Bulletin hebdomadaire, 2009.)
The table in appendix 1 shows the distribution of the different malnutrition diagnoses on patients in the different regions in 2009, the graph on the other hand shows a comparison to the previous years 2006 to 2009. A general decrease is noticeable but sudden peaks and lows are present as well which can be explained by the weather conditions. As in 2005, due to poor rains and severe locust outbreak, Niger registered a record grain deficit of more than 223.000 tons (Niger, 2005).
Nutrition survey data and information in Niger are not compiled and analyzed well according the United States Agency of international development. Most nutrition surveys are conducted on ad hoc basis to meet the needs of varying agency objectives. Currently a joint survey by the Government, UNICEF and the centres for disease control has been conducted regionally. One of the goals of the program will therefore also be to encourage the constant recordkeeping of patients and updating the information.
Determinants: Most important determinants program intends to influence:
In general, protein-energy malnutrition amongst weaning children depends on many aspects of which only a few are biological. The main determinant is that this occurrence is brought upon children in difficult socio-economic conditions, such as those in Niger. Most of these factors are related to poverty which may in turn reason dietary imbalances mainly through the incapability to provide a nutritionally balanced diet.
The following determinants are the main factors that play a role in this health issue:
The work status of the mother and her literacy rate are key in the cause of child malnutrition. If a mother had a good work status and a better education, this would reduce the probability of the child to having a poor nutritional status. The low incomes, the lack of cultivation knowledge are what may cause an unbalanced diet. Therefore, improving a mother and future mother’s education will have a significant impact on their children’s nutrition.
Climate/Topology: Access to food: source to drinking water.
Niger’s hot, desert-dominated topology gives birth to few fruits, vegetables and legumes, and serves as grazing ground for a limited amount of livestock. Consequentially, the few grains and cereals yielded by Niger’s turf epitomize the rural diet. However, such produce provides only a miniscule percentage of the nutritional intake necessary, leading to varying levels of starvation and malnutrition.
Family Size/Second Child Syndrome.
In Niger, statistics show that 75% of girls married before the age of 18 and that 34% of them before 15. According to a source, it can be said that”some as young as ten”. Each woman has on average 7.6 children and statistics further show that there is a 1-in-7 risk of dying during pregnancy or birth (Niger, 2010).
Measurable indicators that can verify whether a child is malnourished.
Before creating a program which proposes a health promotion plan to reduce protein-energy malnutrition amongst weaning children in Niger, it is important to look at the measurable points that can determine whether this malnutrition is the case or not.
According to the pharmaceutical company Merck (Morley, 2007); to determine the severity of protein-energy under nutrition it is important to look at the following points:
Body mass Index.
Total lymphocyte count.
In the table below, many of these points are mentioned and it can be determined whether the child has a normal, mild under nutrition, moderate under nutrition or severe under nutrition (Morley, 2007).
A diagnosis of whether a child has a under nutrition of protein-energy, may be based on the past eating habits of the child. Physical examinations, such as the ones in the table below aid in confirming this diagnosis:
The table above clearly shows which values one has to take into consideration when assessing the severity of protein-energy malnutrition.
Further research has shown that there are other ways to identify malnutrition in a child. This method, used by the UNICEF looks at ways to identify if a child of more than six months is acutely malnourished (Chamois, 2009). First, oedema (swelling) needs to be checked. This is checked by putting your thumb on each foot of the child for three seconds. If the print of your finger creates a shallow hole, then it can be said that the child has oedema. Secondly, the left arm circumference should be measured with a specific kind of measuring device a bit like measuring tape. This left arm circumference can identify according to a colour code, whether the child is very malnourished, moderately malnourished or not malnourished. From both of these identifications, there are different solutions that should take place depending on the result.
Put oedema/left arm circumference picture.
Other tests, as written in the article Protein-Energy Malnutrition: Differential Diagnoses