Quality of Fresh Bovine Milk after Addition of Hypothiocyanite-rich-solution from Lactoperoxidase System
Lactoperoxidase system (LPOS) has received high attention for milk preservation in the certain countries, because the system exerts hypothiocyanite ion to inhibit the growth of broad spectrum of bacteria. Since the system might be remained the substrates of thiocyanate (SCN¯) and hydrogen peroxide (H2O2) and the activity of LPO might be inhibited by compounds in milk, this research has been done for generating hypothiocyanite-rich-solution to be added in fresh milk. The solution was obtained from the reaction solution of LPO, SCN¯ and H2O2. The remaining substrates in the reaction solution were detected spectrometrically. LPO was obtained from bovine whey using SP-Sepharose Fast Flow. Untreated bovine fresh milk (1 h after milking) was added with 1% (v/v) of hypothiocyanite-rich-solution and store at 30°C for 6 h. The addition of sterile water has been used instead of the solution for control. It is concluded that hypothiocyanite-rich-solution contained very less amount of substrates. Hypothiocyanite-rich-solution remarkably decreased the total bacteria count in the fresh milk at 6-h incubation. The solution was also kept the pH value in fresh milk during 6-h incubation. This result might open the new way of practical use of LPOS to preserve fresh milk using the hypothiocyanite-rich-solution.
July 25, 2013; Accepted: January 10, 2014;
Published: March 29, 2014
Milk spoilage is a major problem to the dairy sector of tropical countries
since the ambient temperature is preferable for the growth of bacteria (Seifu
et al., 2005; Oghaiki et al., 2007).
Bacterial contamination becomes critical for raw milk especially from the time
between milking until it reaches to the consumers (Saad,
2008). The commonly used of milk preservation to maintain the quality is
cooling method. Since this equipment is costly, the availability of cooling
facilities in developing countries are still limited in number, therefore the
use of another method to preserve the milk quality is required.
Cheese production from bovine milk in Indonesia has increased over the last
one decade and projects aimed at promoting diversification of milk product (DGAH,
2011). As a consequent, whey as a by-product of cheese making has a huge
number of production but very less of handling. Whey is source of biological
and functional protein including ß-lactoglobulin, immunoglobulin, bovine
serum albumin and lactoferrin (Fee and Chand, 2006).
It is well studied that whey generates antimicrobial activity from lactoperoxidase
(LPO) (Madureira et al., 2007; Al-Baarri
et al., 2011). Lactoperoxidase activates the antimicrobial system
named lactoperoxidase system (LPOS). This system will be active only in the
presence of three components: LPO, thiocyanate (SCN¯) and hydrogen peroxide
(H2O2) (Elliot et al., 2004;
Boots and Floris, 2006). Lactoperoxidase catalyses the
oxidation of thiocyanate by hydrogen peroxide and generates antibacterial agent
of hypothiocyanate or OSCN¯ (Seifu et al., 2005;
Madureira et al., 2007). These products have
a broad spectrum of antimicrobial effects against bacteria, fungi and viruses
(Boots and Floris, 2006; Saad, 2008).
LPOS has been used to preserve raw milk quality in areas where it is not possible
to use mechanical cooling unit for technical or economic reasons (FAO/WHO,
2005). Furthermore, FAO legally provides the method to prolong the quality
of milk by LPOS. Many researcher studied the LPOS to preserve the quality of
raw milk (Haddadin et al., 1996; Marks
et al., 2001; Seifu et al., 2004;
Oghaiki et al., 2007; Trujillo
et al., 2007; Dajanta et al., 2008;
Saad, 2008; Zhou and Lim, 2009).
Common method for generating LPOS in raw milk is adding SCN¯ and/or H2O2
separately into milk. However, since H2O2 maybe remained
in the milk and the utilization of H2O2 is limited for
certain country, the objective of this study is to use of hypothiocyanite solution
(OSCN¯) obtained from LPOS of H2O2 and SCN¯
with less/no residue in the LPOS solution for keeping fresh milk quality.
The previous research was succeeded to control the growth of Salmonella
enteritidis using hypothiocyanate-rich-solution from LPOS using immobilized
LPO onto resin, H2O2 and SCN¯ indicating the potent
mass production of antibacterial agent (Al-Baarri et
al., 2010, 2012; Hayashi
et al., 2012). In this study, the investigation is focused on the
production of hypothiocyanate from the reaction solution containing LPO, H2O2
and SCN¯, the remaining concentration of H2O2 and
SCN¯ and the bacterial growth in fresh milk after addition of hypothiocyanite-rich-solution.
MATERIALS AND METHODS
Materials: Fresh bovines milk was provided by campus farm at Faculty
of Animal and Agriculture Sciences, Diponegoro University. H2O2,
KSCN, 2,2-azino-bis(3-ethylbenzthia-zoline-6-sulfonic acid) (ABTS) and were
purchased from Sigma. Rennet was purchased from Singapore. SP Sepharose Fast
Flow was purchased from Amersham Pharmacia Biotech, Sweden. Unless otherwise
specified, all other chemicals were reagent grade.
Purification of LPO: The LPO was purified using the method of Al-Baarri
et al. (2011) with slight modification. Fresh bovines milk
was centrifugated at 8000 rpm and 10°C for 20 min. The whey was separated
from skimmed milk after the addition of 0.02% (w/v) rennet and 2 mL lactic acid/l
milk at 30°C for 30 min using a sterilized filter cloth. The obtained whey
was dialyzed against a large volume (10 L) of 10 mM sodium phosphate buffer
(PB, pH 6.8) overnight at 4°C and loaded into glass column containing 100
g SP-Sepharose FF (Amersham Pharmacia Bio-tech). For removal unwanted compound,
the column was then washed with 500 mL of PB (pH 6.8) containing 0.1 M NaCl.
LPO was eluted with 500 mL of PB containing 0.2 M NaCl. The purification was
conducted in a refrigeration room. The eluate was collected (15 mL tube-1)
and the extinction coefficient at 280 nm of 1.5 cm2 mg-1
for LPO was used to estimate the protein concentration. Each tube was spectrometrically
checked for LPO activity using ABTS as substrate (Al-Baarri
et al., 2011). The highest LPO activity was collected and filtered
through a 0.22 μm filter unit (Millipore, Bedford, USA). The purified LPO
was stored at -20°C.
Production of hypothiocyanite-rich-solution: Hypothiocyanite-rich-solution
was generated from the enzymatic reaction of LPOS that was prepared by mixing
50 μL of LPO (2 U mL-1), 25 μL of of 0.5 mM H2O2
and 25 μL of 0.5 mM KSCN. After one minute storage in the room temperature,
the reaction solution was analyzed for the remaining SCN¯ and H2O2
concentration. This solution was prepared daily without preservation (Al-Baarri
et al., 2010).
Analysis of residual SCN¯ concentration: Analysis for the remaining
SCN¯ in the hypothiocyanate-rich-solution was conducted spectrometrically
according to the method that was performed by Al-Baarri
et al. (2011) with slight modification. Ten gram of Fe (NO3)3•9H2O
was dissolved in 20 mL of concentrated nitric acid. Water was added to the solution
to give the final volume of 200 mL. An aliquot of sample was added to nine volumes
of the ferric nitrate solution. The absorbance of mixture was measured at 460
nm. The SCN¯concentration of sample was calculated from an established
standard curve of KSCN solutions of known concentrations with the scale of 0.05
to 5 mM of KSCN.
Analysis of residual H2O2 concentration: Residual
determination of H2O2 in hypothiocyanite-rich-solution
was measured using spectrophotometer according to the method that was performed
by Al-Baarri et al. (2011). Two hundred micro
liter solution was made from 1.23 mM ABTS and LPO in 0.1 M Phosphate Buffer
(pH 6.8). Enzymatic reaction was determined by adding 800 μL of hypothiocyanite-rich-solution.
Immediately after the enzyme addition, the absorbance of the mixture containing
the enzyme was monitored at 412 nm at 25°C for 20 sec. The absorbance change
at 412 nm was used then to estimate H2O2 concentration,
based on previously established standard curve of ABTS with the scale of 0.5
to 5 mM.
Microbial count: The 3M Petrifilm Aerobic Count Plates (3M Microbiology, St. Paul, Minn., USA) was used to count the total number of bacteria in milk. The number of total bacteria in fresh milk in the presence of LPOS was determined as follows: 1000 μL of the assay mixture containing 900 μL of fresh bovines milk and 100 μL hypothiocyanite-rich-solution were incubated for 6 h in a water bath at 30°C. Subsequently, serial dilutions of the assay mixture were prepared with a sterile 0.88% NaCl solution to enumerate the bacteria. The diluted mixture (1000 μL) was spread onto plates. The plate were incubated at 37°C for 48 h. The CFU of microbes in the sample solution were counted on the plates.
RESULTS AND DISCUSSION
Remaining substrates in hypothiocyanite-rich-solution: It has been known
that in the LPOS reaction, LPO catalyzes the oxidation of SCN¯ by the presence
of H2O2, which leads to the production of hypothiocyanate
as shown in Fig. 1. Therefore, to generate the hypothiocyanate-rich-solution,
LPO, KSCN and H2O2 were mixed for one minute at room temperature.
Figure 2 shows the utilization of 0.1-0.5 mM of substrates
(SCN¯ and H2O2) in the presence of LPO.
Formation of hypothiocyanite solution
(hypothiocyanite rich-solution) with LPOS, consists of LPO, SCN¯
and H2O2. Reaction was carried out at 30°C for
1 h (Three concordant redings were taken. Error bars represent Mean±SD)
After one minute reaction, a hundred microliter of sample from this solution
was added to 900 μL ferric nitrate solution to analyse the remaining concentration
of SCN¯ in the hypothiocyanate-rich-solution. As shown in this figure,
the residue of H2O2 in hypothiocyanite-rich-solution was
0.014 mM when the maximum concentration of H2O2 was employed
indicating almost all of employed H2O2 (97.06%) in LPOS
was reducted to H2O. The presence of H2O2 in
the hypothiocyanite-rich-solution might interfere the antibacterial activity
of the solution since H2O2 has known as a preservative
agent for inhibit the growth of bacteria. However, the detected residue of H2O2
in the solution was in very small amount if compare to the concentration H2O2
for preservative use (50 mM) (Silveira et al., 2008).
On the other hand, detected residue of SCN¯ in hypothiocyanite-rich-solution
was 0.09 mM when 0.5 mM KSCN was employed. This indicate that 92% of SCN¯
was oxidized into OSCN¯. The remaining SCN¯ was higher than the remaining
H2O2 in the solution due to the ion binding of SCN¯
to the heme of LPO resulting in the weakening of LPO activity. The study of
crystal structure of LPO binding clearly concluded that the binding of the SCN
ion at surface of helix protein H3 of LPO presumably disturb the electrical
charge of LPO resulting in the inactivation of LPO which was inhibit reaction
of forming OSCN¯ (Singh et al., 2008, 2009).
Concentration of OSCN¯ is the key for the antimicrobial activity of LPOS.
This reserach used total concentration of 0.5 mM for SCN¯ and H2O2,
respectively. This amount of substrates should produce approximately 0.4 mM
OSCN¯ in the reaction solution based on the our previous experiment on
the production of OSCN¯ using immobilized LPO (Al-Baarri
et al., 2010). The reaction solution containing 0.4 mM OSCN¯
was able to exert antimicrobial activity against S. enteritidis of approximately
5 log CFU mL-1.
Total bacteria in fresh milk: This experiment used direct addition of
sterile hypothiocyanite-rich-solution into fresh bovines milk. Fresh milk
was incubated for 6 h at 30°C. Prior to incubation, fresh milk was added
with with the sterile hypothiocyanite-rich-solution at every hour of incubation
until 4 h.
Effect of hypothiocyanite-rich-solution
to total bacterial counts in fresh milk during 6 h incubation at 30°C.
Hypothiocyanite-rich-solution was added at different intervals. Sterile
pure water was added to the fresh milk instead of hypothiocyanite-rich-solution
as control (Three concordant readings were taken. Error bars represent
The samples were collected hourly for enumeration of total bacteria. Fresh
milk used in this experiment was also counted for total bacteria resulting number
of 4.32±0.67 log CFU mL-1 (data were not presented). The result
of total bacteria in fresh milk after addition of hypothiocyanite-rich-solution
is showed at Fig. 3.
Figure 3 shows the inhibition of hypothiocyanite-rich-solution
against total bacteria in fresh milk. The total bacteria of 8.00±0.80
CFU mL-1 has been detected in the sample with no addition of hypothiocyanite-rich-solution
while the total bacteria of 6.80±0.80 or less has been detected in the
sample with the treatments indicating the suppresive effect of hypothiocyanite-rich-solution
to the bacterial growth. This result is also in line with the findings of Nigussie
and Seifu (2007) who reported that activation of the LPOS in the fresh milk
resulted in supression of the growth of total bacteria from 7.5 log CFU mL-1
from the initial count of 7.73 log CFU mL-1. Based on these results,
the hypothiocyanite-rich-solution showed the higher suppresion effect than those
of activation of LPOS in fresh milk. This can be explained that LPO might be
inhibited by the existing lactose in milk (Al-Baarri et
al., 2011). This result are in agreement with the role of National Standardization
Agency of Indonesia that was announced the maximum total bacteria number for
fresh milk (6 log CFU mL-1), representing the hypothiocyanate-rich-solution
was the potent preservatives for preserving fresh milk.
The inhibition effect of hypothiocyanite-rich-solution to total bacterial count
in fresh milk was detected on the sample after one-hour addition. For instance,
when the solution was added to fresh milk at first-hour incubation, the total
bacteria reduced from 4.23±0.50 to 4.21±0.10 log CFU mL-1.
However the reduction of total bacterial count has only been shown at that sample
while other samples showed the inhibition effect to the growth of bacteria.
This phenomenon could be explained that the population of bacteria is in line
with the number of sulfhydryl group that should be oxidized by OSCN¯ (Al-Baarri
et al., 2010; Hayashi et al., 2012).
Therefore the high population of bacteria, the higher concentration of OSCN¯
might be required.
The direct addition of LPOs substrate ie. KSCN and H2O2
to fresh milk has been guided by FAO for milk preservation in the area with
less refrigeration facility (FAO/WHO, 2005). The addition
of substrates has been proved to extend the shelf life of fresh milk stored
at ambient temperature (Barrett et al., 1999;
FSANZ, 2002; FAO/WHO, 2005; Oghaiki
et al., 2007; Dajanta et al., 2008).
pH value of fresh milk with the addition
of hypothiocyanite-rich-solution after 6 h incubation at 30°C. The
addition was conducted hourly. Sterile pure water was added to the fresh
milk instead of hypothiocyanite-rich-solution as control (Three concordant
readings were taken. Error bars represent Mean±SD)
However the substrates might be remained in the milk since the activity of
LPO was depended on the storage and substrates concentration (Boots
and Floris, 2006; Trujillo et al., 2007;
Singh et al., 2009).
pH value: Figure 4 shows the value of pH in fresh
milk at sixth-hour incubation at 30°C with and without addition of hypothiocyanite-rich-solution.
The pH was analyzed at sixth hour incubation since this point is the critical
value of fresh milk to reach the total bacteria of 6 log CFU mL-1
(Touch et al., 2004). As can be seen on this figure,
hypothiocyanite-rich-solution was able to maintain the pH of fresh milk into
range from 6.66±0.12 to 6.71±0.02 at 6 h incubation while no addition
of the solution decreased pH into 5.90±0.11. Prior to treatment, all
fresh milk were detected on the pH value and resulted in the value of 6.76±0.080.
The suppression of the decrease of pH value was in agreement with the previous
result in the total bacteria. The addition of hypothiocyanite-rich-solution
was able to maintain the total bacteria into maximum of 6.80±0.8 CFU
mL-1 while no addition of the solution increased total bacteria into
from the initial count 6.76±0.08 into 8.00±0.80 CFU mL-1
The hypothiocyanite-rich-solution could be obtained from the reaction solution of LPO, SCN¯ and H2O2. This solution contained very less amount of residual substrates. Addition of this solution into fresh milk remarkably inhibited the growth of total bacteria during 6 h incubation at 30°C but did not change the pH value indicating the hypothiocyanite-rich-solution had the potent preservatives. This result might be opened the new method of keeping the quality of fresh milk using LPOS.
This material is fully based upon work supported by grants from the Indonesian Directorate General of Higher Education (DIKTI). Authors also would like to acknowledge the material support from Professor Shigeru Hayakawa, Professor Masahiro Ogawa and Professor Hirotoshi Tamura (Faculty of Agriculture, Kagawa University, Japan).
Al-Baarri, A.N., M. Hayashi, M. Ogawa and S. Hayakawa, 2011. Effects of mono- and disaccharides on the antimicrobial activity of bovine lactoperoxidase system. J. Food Prot., 74: 134-139.
CrossRef | PubMed | Direct Link |
Al-Baarri, A.N., M. Ogawa and S. Hayakawa, 2010. Scale-up studies on immobilization of lactoperoxidase using milk whey for producing antimicrobial agent. J. Indonesian Trop. Anim. Agric., 35: 185-191.
Direct Link |
Al-Baarri, A.N., M. Ogawa, T. Visalsok and S. Hayakawa, 2012. Lactoperoxidase immobilized onto various beads for producing natural preservatives solution. J. Applied Food Technol., 1: 4-6.
Direct Link |
Barrett, N.E., A.S. Grandison and M.J. Lewis, 1999. Contribution of the lactoperoxidase system to the keeping quality of pasteurized milk. J. Dairy Res., 66: 73-80.
Direct Link |
Boots, J.W. and R. Floris, 2006. Lactoperoxidase: From catalytic mechanism to practical applications. Int. Dairy J., 16: 1272-1276.
DGAH, 2011. Indonesian statistic of animal husbandry. Directorate General of Animal Husbandry, Ministry of Agriculture, Republic of Indonesia.
Dajanta, K., E. Chukeatirote and A. Apichartsrangkoon, 2008. Effect of lactoperoxidase system on keeping quality of raw cow's milk in Thailand. Int. J. Dairy Sci., 3: 112-126.
CrossRef | Direct Link |
Elliot, R.M., J.C. McLay, M.J. Kennedy and R.S. Simmonds, 2004. Inhibition of foodborne bacteria by lactoperoxidase system in a beef cube system. Int. J. Food Microbiol., 91: 73-81.
FAO/WHO, 2005. Benefits and potential risks of the lactoperoxidase system of raw milk preservation. Report of an FAO/WHO Technical Meeting, November 28-December 2, 2005, FAO Headquarters, Rome, Italy, pp: 1-73.
FSANZ, 2002. Application A404 lactoperoxidase system. Food Standards Australia New Zealand Final Assesment Report, December 18, 2002.
Fee, C.J. and A. Chand, 2006. Capture of lactoferrin and lactoperoxidase from raw whole milk by cation exchange chromatography. Separation Purification Technol., 48: 143-149.
Haddadin, M.S., S.A. Ibrahim and R.K. Robinson, 1996. Preservation of raw milk by activation of the natural lactoperoxidase systems. Food Control, 7: 149-152.
CrossRef | Direct Link |
Hayashi, M., S. Naknukool, S. Hayakawa, M. Ogawa and A.B.A. Ni'matulah, 2012. Enhancement of antimicrobial activity of a lactoperoxidase system by carrot extract and β-carotene. Food Chem., 130: 541-546.
CrossRef | Direct Link |
Madureira, A.R., C.I. Pereira, A.M.P. Gomes, M.E. Pintado and F.X. Malcata, 2007. Bovine whey proteins-overview on their main biological properties. Food Res. Int., 40: 1197-1211.
Marks, N.E., A.S. Grandison and M.J. Lewis, 2001. Challenge testing of the lactoperoxidase system in pasteurized milk. J. Applied Microbiol., 91: 735-741.
CrossRef | Direct Link |
Nigussie, H. and E. Seifu, 2007. Effect of the lactoperoxidase system and container smoking on the microbial quality of cows milk produced in Kombolcha woreda, Eastern Ethiopia. Livestock Res. Rural Dev., Vol. 19.
Oghaiki, N.A., F. Fonteh, P. Kamga, S. Mendi and H. Imele, 2007. Activation of the lsctoperoxidise system as a method of preserving raw milk in areas without cooling facilities. Afr. J. Food Agric. Nutr. Dev., 7: 1-14.
Saad, A.H., 2008. Activation of milk lactoperoxidase system for controlling pseudomonas in cow's milk. Int. J. Dairy. Sci., 3: 131-136.
CrossRef | Direct Link |
Seifu, E., E.M. Buys and E.F. Donkin, 2005. Significance of the lactoperoxidase system in the dairy industry and its potential applications: A review. Trends Food Sci. Technol., 16: 137-154.
CrossRef | Direct Link |
Seifu, E., E.M. Buys, E.F. Donkin and I.M. Petzer, 2004. Antibacterial activity of the lactoperoxidase system against food-borne pathogens in Saanen and South African indigenous goat milk. Food Control, 15: 447-452.
CrossRef | Direct Link |
Silveira, A.C., A. Conesa, E. Aguayo and F. Artes, 2008. Alternative sanitizers to chlorine for use on fresh-cut "Galia" (Cucumis melo var. catalupensis) melon. J. Food Sci., 73: M405-M411.
Singh, A.K., N. Singh, S. Sharma, K. Shin and M. Takase et al., 2009. Inhibition of lactoperoxidase by its own catalytic product: Crystal structure of the hypothiocyanate-inhibited bovine lactoperoxidase at 2.3-A resolution. Biophys. J., 96: 646-654.
Singh, A.K., N. Singh, S. Sharma, S.B. Singh and P. Kaur et al., 2008. Crystal Structure of lactoperoxidase at 2.4 A resolution. J. Mol. Biol., 376: 1060-1075.
Touch, V., S. Hayakawa, S. Yamada and S. Kaneko, 2004. Effects of a lactoperoxidase-thiocyanate-hydrogen peroxide system on Salmonella enteritidis in animal or vegetable foods. Int. J. Food Microbiol., 93: 175-183.
CrossRef | PubMed | Direct Link |
Trujillo, A.J., P.I. Pozo and B. Guamis, 2007. Effect of heat treatment on lactoperoxidase activity in caprine milk. Small Rumin. Res., 67: 243-246.
Zhou, Y. and L.T. Lim, 2009. Activation of lactoperoxidase system in milk by glucose oxidase immobilized in electrospun polylactide microfibers. J. Food Sci., 74: C170-C176.