Roya Rouhi1, Mohammad Reza Koushki2 and Seyed-Ahmad Shahidi3

1Department of Food Science and Technology, Ayatollah Amoli Branch,
Islamic Azad University, Amol, Iran.
2Department of Food Technology Research, National Nutrition and Food Technology
Research Institute, Faculty of Nutrition Sciences and Food Technology,
Shahid Beheshti University of Medical Sciences, Tehran, Iran.
3Department of Food Science and Technology, College of Agriculture and Food Science,
Ayatollah Amoli Branch, Islamic Azad University, Amol, Iran.


The aim of this study was to determine the microbiological and physicochemical properties of raw milk in Mazandaran province, Iran, during 6 months from winter 2013 to spring 2014. A total of 253 raw milk samples were collected, from dairy farms, milk cans, milk collection centers, and delivery milk tankers. The samples were analyzed for microbial quality, total plate count (TPC), and physicochemical properties including titrable acidity, pH, content of fat and protein, solids nonfat (SNF) level, specific gravity, percentage of water adulteration, and alcohol testing. The mean of TPC was 48×107 CFU/mL. The higher TPC (23×108 ) was found in Chalous city during winter season. TPC less than 106 was used as a basic standard limit by Institute of Standards and Industrial Research of Iran (ISIRI). Significant effect of region was observed on all physicochemical properties except pH (p<0.05). The mean counts of titrable acidity, pH, fat, protein, SNF, specific gravity, and percentage of water adulteration were 15.38, 6.66, 3.42%, 3.04%, 8.33%, 1.029 and 2.11%, respectively, and alcohol stability result of 22.1% of the samples was positive. It can be assumed that raw milk in the study area had poor bacteriological quality according to the Iranian National Standard ( ISIRI ; therefore, it may be hazardous for human consumption. This finding shows the necessity to implement good hygiene practices.

Keywords: Raw milk, Physicochemical properties of milk, Microbiological quality of milk, Iran.


The most important matter for the dairy industry is factors affectingthe quality ofmilk products,especially fermenteddairyproducts. High quality raw milk is the  initial  prerequisite to producehigh quality fermented milk products. The quality of raw milk defines by microbial quality  and physicochemical properties that varyin the milk  samples  of different dairy farms;these differences are technologicallyimportant to makefermented milk products (Stulovaet al., 2010).There is a relationship between the properties of used raw milk andproducedpasteurized milk (AbdElrahman et al., 2009).Raw milk attributes and seasonal variation affect the composition and properties of rawmilk  during processing(Chen et al., 2014).  Many  microorganismscan  grow in  milk.Special  composition  of milk, its high water content,andthe neutralpH valueallows the growth of many  microbes (Quigleyet al., 2011). Thesemicroorganisms enter milk from a variety of sources, and can play different roles such ascausing spoilage (Quigley et al., 2013).Milk is  highly  perishable andpoor management causes health threat and economic losses;  therefore,hygienicpractices is needed throughoutthe production to consumer chain (Swai  and Schoonman, 2011).Seasonalvariationaffects raw milk quality.In warmweather,  microbial load of raw milk increases (Yarahmadi et al., 2008).Recently, some studies have been done to assess the quality of raw milk, but no  scientificand comprehensive researchhas been carried outso far onraw milk qualityin Mazandaran province,Iran.The aim of this study was todetermine the microbiological and physicochemicalqualityof rawcow milk inthis provinceduring winter 2013 and spring  2014



Sampling:Totally, 253 raw cow milk samples were collected accordingto random  sampling design, monthly,from winter 2013 to the end of spring 2014.Sampling  was  carried out fromthe  dairy farms, milk cans,milkcollection centers, andmilk  delivery  tankers inMazandaranprovince, Iran. Each milk  sampleconsisted  of 10  mL of raw milk poured into a  sterile  syringe  for  microbial  evaluation and 700 mLin to a sterilecontainer for chemical analysis. The samples were delivered to the laboratoryof  Haraz  Milk Plant, Mazandaranprovincein a cool box,and tested immediately on arrival.


Physicochemical analysis

The pH of milk wasrecorded using pH meter(Metrohm,  Switzerland). pH and titrable acidity weremeasured according toIranian National  Standard (NSI)(ISIRI,2006). Fat content was determined by Gerber method (ISIRI,1991).Protein content was determined  using Kjeldahlmethod, INS No. 639(ISIRI,2013),and SNF was measured according to the  INS No. 637 (ISIRI, 2013).  Specific gravity of the milk samples was measuredusingLactodensimeter (SlW, Germany)  according  tothe INS (ISIRI, 1993);  alsoalcohol test was  conducted according to the NSI(ISIRI, 2005).Water adulteration was measured by Milkoscan(Ekomilk Total EON- Bulgaria).


Microbiological analysis

For microbiological analysis,TPC of  the  samples was  evaluated byplate count agar(Merck, Germany);then they were incubatedat 37Cfor 72  hours. Colony countingwas carried out using colony counter(ISIRI,2008).The  total  plate  count  according  to  ISIRI,  2008  is  maximum  106  CFU/ml acceptable.

Statistical analysis

Statisticalanalysiswas performedusing Minitabsoftware and analysis of variance(ANOVA).TukeyTest wasused for comparison of averages, as well as theeffects  and interactions of factors.Excel and Minitab software were used for drawing charts.




Themean of total plate count ofraw cowmilk in Mazandaran  province duringtwoseasons was 48×107CFU/ml (Table1).However, the microbial count of three regions (Chalous in winter,  and spring  and Tonekabon in winter )werehigherthan theotherregions,but therewas no significant difference in  the totalmicrobial count of differentregions and intwo seasons(p>0.05).


Physicochemical properties

Fat content:Fat contentof the samplesvariedbetween2.95and3.89%withanaverageof 3.42±0.03%(Table 1).Therewasa significantdifference infat content of the raw milk samplesin different regions and two seasons(p<0.05).The samples collected in winter had higher fat content thanthosecollected inspring.Fatcontentof the samplescollectedfromSalmanshahr wassignificantlylower thanthose collected from othercities, andthe samples collectedin Nour, Tonekabon and Chaloushad higher fat content than other regions.

Protein content: Theresults indicated that proteincontentof the samples varied  between 2.47 and3.41%withanaverageof 3.04±0.02% (Table 1).Theresults ofANOVA showeda significant  differencebetween the mean ofprotein contentin different regions (p<0.05). Protein  content  of thesamples collectedfromNowshahr was significantly lower than other cities,the samplescollected  fromNour and Sarihad higher proteincontent than other regions. Nosignificant difference was observed between protein  content of  the samples  collectedin two seasons (p>0.05).

AcidityandpH:Meanof acidityindifferentregions was15.38º D (Table 1).Data analysis showedasignificantdifferencein differentregions, and between the mean of acidityof two seasons(p<0.05).MeanofpHvalue forthe raw milk samples obtained from various regions was 6.66.Therewasnosignificant difference(p>0.05)inthe meanofpH invarious regions (Table1).

SNFcontent: MeanofSNFcontent in different regions was 8.33%(Table 1).However,variousSNF amounts wereobserved indifferent regions(p<0.05) butnot for the samples prepared in two mentioned seasons (p>0.05).

Specific gravity: Meanofthe samples specificgravity for was1.029(Table1).A significant  differencewas observedin the specificgravityof milk samplesin different regions(p<0.05) but  seasondid not have significanteffect  on it (p>0.05).

Water adulterationpercentage:Meanofwater adulteration was2.11% (Table 1).According to the results,therewasa significant differenceinthewater adulteration mean of  various regions (p<0.05), indicating theaddition of water by producers;however, there was  no  significant differencebetween two seasons in this regard(p>0.05).

Alcohol test: Alcohol stability ofthe rawmilk sample wasevaluated, and the result of22.1%of 102sampleswaspositive.

Table 1:  Means  of  microbiological  and  physicochemical  parameters  of  the  raw  milk  samples  according  to  season  and  sampling  region

Sampling  region
TPC  (CFU/ml)
Fat (%)
Protein (%)


SNF (%)


Specific gravity


Water adulteration (%)
1. Sari
23×106 a *
3.64 a
3.15 a
14.08 e
6.70 a
8.60 a
0.00 b
9×106 a
3.27 a
3.07 a
13.66 f
6.75 a
8.64 b
0.25 b
2. Babolsar
18×107 a
3.40 a
3.08 a
15.53 f
6.69 a
8.42 a
0.00 b
28×107 a
3.17 a
3.01 a
13.33 g
6.69 a
8.22 a
1.16 a
3. Amol
28×107 a
3.36 a
3.00 a
14.27 d
6.70 a
8.20 a
3.55 a
13×107 a
3.30 a
3.04 a
14.35 d
6.71 b
8.32 a
1.75 a
4. Noor
78×106 a
3.89 a
3.10 a
14.84 c
6.68 a
8.48 a
25.2 a
19×107 a
3.50 a
3.12 a
15.25 b
6.71 a
8.52 a
0.00 b
5. Nowshahr
29×107 a
3.43 a
2.91 b
15.26 b
6.68 a
7.96 a
5.23 a
26×107 a
3.18 a
3.00 a
16.56 a
6.60 a
8.19 b
4.25 a
6. Chalous
11×108 a
3.72 a
3.07 a
16.03 a
6.64 b
8.39 a
1.38 a
23×108 a
3.39 a
3.05 a
16.42 a
6.61 c
8.35 a
0.53 b
7. Salmanshahr
44×107 a
2.95 b
3.05 a
15.11 b
6.68 a
8.32 a
6.66 a
35×107 a
3.36 a
3.13 a
16.25 a
6.66 a
8.51 a
3.25 a
8. Nashtarood
38×107 a
3.54 a
3.05 a
16.28 a
6.65 b
8.34 a
1.74 a
52×107 a
3.27 a
3.04 a
17.45 a
6.62 b
8.31 a
3.40 a
13×108 a
3.73 a
2.99 a
15.63 a
6.65 b
8.18 a
1.63 a
52×107 a
3.47 a
3.03 a
16.87 a
6.60 e
8.27 a
1.00 a
Total mean

*Different  letters  within  columns  are  significantly  different  at  p<0.05.



Microbiological quality

Total platecountin mostof thepresentsamplesexceededthestandard limits ≥106 CFU/mL(ISIRI,2008).Themilkingprocess (especially themilking-machine, preservation,collection,transportation, cooling, and  equipment associated with it)introduces the greatestproportion of microorganisms in  raw  milk. In order to reduce contamination ofmilk, utensils used for milking should be rinsed, cleaned (using detergent),  and disinfected immediately after use. However,keeping  the milk in refrigerated temperatures immediately after milking process may delay the increaseofthefirst microbial load(Swai and Schoonman,2011). In  the study of raw milk from collection centers  of the three regions in Morocco, 75% of the samples had unsatisfactory quality with respect to TPC and the mean counts of TPC were1.4×10 6  CFU/mL(Belbachir et al 2015). The average TPC of  raw milk 1.03×10 6 CFU/mLand  5.5 (±0.2) log CFU/mLwere reported from Shahrekord, Iran(Fadaei 2014) and Kamana et al (2014) respectively. In another study the mean counts of TPC were between 9.2×10 4 and  3.6×10 7CFU/mL (Pyz-Lukasik et al 2015). Chye et al. (2004)determined the microbiological safety of raw milk from four regions of Malaysia. The meanoftotal plate count of the samples was 12×10 6 CFU/mLthat exceeded the limits; however,  thepresent study showedhigher bacterial contamination of raw milk.The assessing results of 297 samplesfromNew Zealand’s raw milksamples collectedfrom five major dairying regions over a one year period,showedthat raw milk supply inevitably contains pathogens;so, control by thermal treatment of raw milk seems essential  (Hill  et  al.,  2012)even sothecontamination of raw milk in NewZealandwas lower than other countries and the regions of this study.Pyz-Lukasik et  al.  (2015)  examined  the  microbiological  quality  of  raw  cow  milk  samples  in  Poland.  The  mean count of  total  aerobic  bacteria  was  between  9.2×104  and  3.6×107  CFU/ml that  is  lower  than  that  of the  present  study.Kalmus  et  al.  (2015)  determined  the  microbiological  quality  of raw  milk  in Estonia.  The  total  bacterial  count  exceeded  100,000  CFU/mL in  21.4%  ofthe  bulk  milk samples,  and  in  71.4%  of the  collected  samples,it was at the  retail  level.  O,Connell et al.  (2015)  measuredthe total bacterial count(TBC)ofraw milk samples in Ireland. The  average  of  TBC  was  17000  CFU/mL,  and  showed  seasonal  trends.  The  TBC in the above  study was  lower  than  in the  results  of  present  study.It  could  be  concluded  that  more hygienic  practices  are  carried  out  in  the herds  of  Ireland. However, season variation  did  not  affect  the microbiological  qualityof the samples  in  the present  study,  which  can  be  due  to  low  temperature  difference  between the winter  and spring  seasons. In  anotherstudy, 50 samples of rawmilk were collected from Dehradun city in India. Only 8% of thesamples were foundin the categoryofgood quality,and 25% of thesamples contained 41×107CFU/mLbacterial count,and were in poor category (Pant et al., 2013).A total of 100 raw milk samples in Turkey were analyzed for microbiological and chemical quality. The average of total plate count was 3.95 ×106CFU/mL  (Tasci, 2011);which is much lower than our results and very closed to the NSI limit. Themicrobial quality of raw milk produced inEstonia during 4 years was determined;more than 91% ofthesamples involved lessthan 5×104 CFU/mL (Stoluva et al., 2010); this result is betterthan that of the present study in this regard.Different microbial loadsof raw milk samples of different regions are related to milk utensils, water supply, condition and temperature ofraw milk after milking process,especially duringkeeping and transport(Chye et al., 2004). Thesereasons could also,causevaried microbial loadsofthesamples of different regions in this study.


Physicochemical properties

Physicochemical properties of raw milk samplesof the present studyincludingthetitrable acidity,pH, content of fat andprotein, solids nonfat level, specific gravity,percentage of  water adulteration, alcohol testing were determined. The findings showed that all of the mentioned factors have amounts within normal range.Inthepresent study,themean of fat content of thecollected sampleswas  within therange,   andtheminimum limitof 3.2%forfat  content  is  acceptable (ISIRI,  2005);  therefore,  85%  of  theregions  were  within  the  normal  range.  Shojaei and Yadollahi (2008)found an overall average of2.6%fat contentof theraw milksamples ofthree regionsinShahrekord,Iran, which is lower in comparison withthefindings of  the present study  and  the ISIRI (2005).There was a  significant difference in the fat content of samplesof different regions  in  the  present  study(ρ<0.05).Milk fat content variesbecause ofspecies of animal, breed, stage of lactation,  age,  seasonal variations, feeding, management,preservation and transportation  of  milk.Itcan also  be  affected by water adulteration(Javaid et al., 2009).Fat content ofthesamples in the present work  was affected by seasonal variations (ρ<0.05); similar results werefound  by other researchers (Yang et al., 2013).The  protein  contentof3-3.3% wasset asacceptablerange by the ISN (ISIRI,  2005).Shojaei and Yadollahi(2008) reportedthesame parameter  measurement  in Shahrekord, Iran.The results of our study showeda  significant difference between themean of protein content indifferent regions (p<0.05) andinsignificant  difference  in different seasons (p>0.05).In the  present  study,seasonal variation didnot affect onprotein,probabelydue to low temperature difference between winter and spring seasons.Acidity  value  of 14 –16ºD  according  to  ISIRI  (2005)  is  within  the  normal  range . Average ofpH valuefor thepresentsamples was 6.6,which iswithin the normal range, 6.6–6.8  (ISIRI,  2005).  Similar results were obtained by evaluation of pH value forthemilk samplesfrom  Tandojam, Pakistan(Javaid et al., 2009).Evaluation ofthephysical properties of raw milk  samples in Turkeyshowed a meanpH  of6.74(Tasci, 2011)thatis higher thanthe average of pH in the present  investigation.pH variation ofmilk could be due to addition of water, ice or chemical preservatives toimprove its shelf life (Javaid et al., 2009).Mean of SNF content of thesamples was  acceptable (minimum  8%)according to  theISIRI  (2005). The results showed significantdifference in different regions (p<0.05).Water adulteration,as reportedin the present study, could affect on theSNF content of different region samples.There was no difference in theSNF content of samples collected intwodifferent seasons (p>0.05),which is not in agreementwith the reportedresultsby Matutinovic et al. (2011) and Yang et al. (2013).It could be because of low temperature difference betweenthewinter andspring seasonsin the area(Mazandaran province) this studywascarried  out.Mean of specific gravity of  thepresent sampleswas 1.029, which is in the normal  range  (1.029-1.032)according  to  ISIRI (2005). Significant difference was observed in thespecific gravity of milk samples in differentregions (p<0.05). The result showedwater adulteration in milk, since water is lighter than milk, then  its addition reducesthe specific gravity of milk (Javaid et al., 2009).  Mean of specific gravity for 100 raw milk samples in Turkey was1.027 (Tasci, 2011).  The presentstudy showed higher specific gravity, and it can  be  concluded that adulterated water by sellers was lower.Addition  of  wateris carried outcommonly, which affects the physicochemical compositionof milk bychangingthe proportion ofits  different constituents (Javaid et al.,2009).Finally,  alcohol test oftheraw milk samples wasdetermined,and  showed  alcohol  stability  of  22.1 % of the  raw milk sample was nonresistant,  and coagulated therefore,  can not tolerate heat  treatmentduring the  processing.


The results of thepresentstudyshowedthat the raw  milk  samples  had  a  poor  microbiological  quality.  High  contamination  affects  the  keeping  quality  and  safety  of  raw  milk  and milk-based products.  According  to  our results,  the  physicochemical compositionofthe raw milk samples  collectedfrom different regions of Mazandaran provincewas acceptable  according  tothe  ISIRI.Change of milk composition in different seasons could provide scientific pattern for dairyproducers and  manufacturers  to use appropriate raw milk based onthe  type ofproduction.It is recommended that hygienicpractices areimplementedduring the  milking, preservationandtransport.Moreover,avoidingthe consumption ofuntreated rawmilkcould beinstructed.

Acknowledgement: The authors would like to thank Doosheh Dairy Company (Haraz)forproviding facilitiesandlaboratorial supportsduringthisstudy.


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