Production of biogenic amines and divercin V41 in cold smoked salmon inoculated with Carnobacterium divergens V41, and specific detection of this strain by multiplex-PCR

AIMS: The objective of this study was to determine the technological behaviour (implantation and biogenic amines production) of Carnobacterium divergens V41, an anti-Listeria bacteriocin producer (divercin V41), after inoculation in cold smoked salmon (CSS). METHODS AND RESULTS: Implantation of the strain was followed by multiplex-PCR during 27 days of storage at 4 degrees C, and biogenic amines were quantified by HPLC. It was found that the strain was able to develop quite well in CSS among lactic wild flora. Divercin V41 (400 AU ml-1) was produced in CSS, and the biogenic amine content was not modified by inoculation of the bacteria. CONCLUSIONS: Carnobacterium divergens V41 is a safe, interesting, bioprotective agent. SIGNIFICANCE AND IMPACT OF THE STUDY: This strain could potentially be used for efficient prevention of L. monocytogenes growth in CSS.     

The effect of biogenic amine production by single bacterial cultures and metabiosis on cold-smoked salmon

AIM: Biogenic amines are important indicators of spoilage in vacuum-packed cold-smoked salmon. It is the aim of this study to identify bacteria responsible for biogenic amine production in cold-smoked salmon. METHODS AND RESULTS: The present study identified spoilage microflora from cold-smoked salmon and determined biogenic amine production of single and co-cultures growing in cold-smoked salmon. Photobacterium phosphoreum was the only species that produced histamine when inoculated on sterile cold-smoked salmon. Production of putrescine was enhanced 10-15 times when cultures of Serratia liquefaciens or Hafnia alvei were grown with Carnobacterium divergens or Lactobacillus sakei subsp. carnosus. This phenomenon was explained by interspecies microbial metabolism of arginine, i.e., metabiosis. CONCLUSIONS: The amounts of biogenic amines produced by single and co-cultures corresponded to those observed during spoilage of naturally-contaminated cold-smoked salmon. Photobacterium phosphoreum and Lact. curvatus were identified as the specific spoilage organisms in cold-smoked salmon. SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of the specific spoilage organism is needed before a model can be developed for shelf-life predictions of cold-smoked salmon.     

Biogenic amine production by lactic acid bacteria isolated from cider

AIMS: To study the occurrence of histidine, tyrosine and ornithine decarboxylase activity in lactic acid bacteria (LAB) isolated from natural ciders and to examine their potential to produce detrimental levels of biogenic amines. METHODS AND RESULTS: The presence of biogenic amines in a decarboxylase synthetic broth and in cider was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). Among the 54 LAB strains tested, six (five lactobacilli and one oenococci) were biogenic amine producers in both media. Histamine and tyramine were the amines formed by the LAB strains investigated. Lactobacillus diolivorans were the most intensive histamine producers. This species together with Lactobacillus collinoides and Oenococcus oeni also seemed to produce tyramine. No ability to form histamine, tyramine or putrescine by Pediococus parvulus was observed, although it is a known biogenic amine producer in wines and beers. CONCLUSIONS: This study demonstrated that LAB microbiota growing in ciders had the ability to produce biogenic amines, particularly histamine and tyramine, and suggests that this capability might be strain-dependent rather than being related to a particular bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of biogenic amines by food micro-organisms has continued to be the focus of intensive study because of their potential toxicity. The main goal was to identify the microbial species capable of producing these compounds in order to control their presence and metabolic activity in foods.     

Free amino acids and biogenic amines in red and white muscle of tuna stored in controlled atmospheres

Summary. This paper analyses the presence of and changes in free amino acids and biogenic amines in red and white muscle of bigeye tuna during storage in controlled atmospheres with 2 gas mixes containing different concentrations of CO₂ and O₂. Levels of amines were generally higher in white than in red muscle, with the exception of putrescine and spermidine. Levels of biogenic amines increased (p<0.05) throughout storage, commencing later in red than in white muscle. A correlation between the amino acid histidine and the biogenic amine histamine was observed, but only in white muscle. Only in the case of tryptophan did white and red muscle differ (p<0.05) in terms of essential free amino acid content. They also differed in anserine content. Concentrations of the non-essential FAAs glutamic acid, glycine and alanine were higher in red than in white muscle. The effectiveness of the atmospheres was reflected in the evolution of both biogenic amines and FAAs. Gas mix 1, containing a higher concentration of CO₂, was the more effective.     

Tyrosine-containing peptides are precursors of tyramine produced by lactobacillus plantarum strain IR BL0076 isolated from wine

ABSTRACT: BACKGROUND: Biogenic amines are molecules with allergenic properties. They are found in fermented products and are synthesized by lactic acid bacteria through the decarboxylation of amino acids present in the food matrix. The concentration of biogenic amines in fermented foodstuffs is influenced by many environmental factors, and in particular, biogenic amine accumulation depends on the quantity of available precursors. Enological practices which lead to an enrichment in nitrogen compounds therefore favor biogenic amine production in wine. Free amino acids are the only known precursors for the synthesis of biogenic amines, and no direct link has previously been demonstrated between the use of peptides by lactic acid bacteria and biogenic amine synthesis. RESULTS: Here we demonstrate for the first time that a Lactobacillus plantarum strain isolated from a red wine can produce the biogenic amine tyramine from peptides containing tyrosine. In our conditions, most of the tyramine was produced during the late exponential growth phase, coinciding with the expression of the tyrDC and tyrP genes. The DNA sequences of tyrDC and tyrP in this strain share 98% identity with those in Lactobacillus brevis consistent with horizontal gene transfer from L. brevis to L. plantarum. CONCLUSION: Peptides amino acids are precursors of biogenic amines for Lactobacillus plantarum strain IR BL0076.     

Evaluation of amino acid-decarboxylative microbiota throughout the ripening of an Italian PDO cheese produced using different manufacturing practices

Aim:  To investigate the presence of biogenic amines (BAs) in Montasio cheese produced by using different cheese manufacturing practices.
Methods and Results:  Three batches of Montasio cheese were made in the following way: batch A using raw milk and natural milk culture, batch B with thermized milk and natural milk culture and batch C with thermized milk and natural milk culture added of a commercial starter culture. During 120 days of ripening analyses were performed for microbial counts and BA content; indeed, the potential to produce BAs was screened in lactic acid bacteria and Enterobacteriaceae isolates. At the end of ripening, the total BA contents of cheeses from batches A, B and C were 166·3, 207·3 and 29·8 mg kg⁻¹, respectively. Amino acid decarboxylase activity was widespread among isolates.
Conclusions:  The BA content of Montasio cheese from the three batches was below the threshold proposed as potentially toxic. The highest BA content was found in cheese produced using thermized milk and natural milk culture; therefore, the thermal treatment of milk was not enough by itself to reduce the counts of decarboxylase-positive bacteria in cheese. The use of selected starters guaranteed a low BA content in Montasio cheese.
Significance and Impact of the Study:  The study of the effects of some technological processes on the incidence of decarboxylative microbiota in 'protected denomination of origin' cheeses could provide useful information on the hygienic risk related to their production.     

Degradation of biogenic amines by vineyard ecosystem fungi. Potential use in winemaking

Aims: To evaluate the ability of grapevine ecosystem fungi to degrade histamine, tyramine and putrescine in synthetic medium and in wines. Methods and Results: Grapevine and vineyard soil fungi were isolated from four locations of Spain and were subsequently identified by PCR. A total of 44 fungi were evaluated for in vitro amine degradation in a microfermentation system. Amine degradation by fungi was assayed by reversed‐phase (RP)‐HPLC. All fungi were able to degrade at least two different primary amines. Species of Pencillium citrinum, Alternaria sp., Phoma sp., Ulocladium chartarum and Epicoccum nigrum were found to exhibit the highest capacity for amine degradation. In a second experiment, cell‐free supernatants of P. citrinum CIAL‐274,760 (CECT 20782) grown in yeast carbon base with histamine, tyramine or putrescine, were tested for their ability to degrade amines in three different wines (red, white and synthetic). The highest levels of biogenic amine degradation were obtained with histamine‐induced enzymatic extract. Conclusion: The study highlighted the ability of grapevine ecosystem fungi to degrade biogenic amines and their potential application for biogenic amines removal in wine. Significance and Impact of Study: The fungi extracts described in this study may be useful in winemaking to reduce the biogenic amines content of wines, thereby preventing the possible adverse effects on health in sensitive individuals and the trade and export of wine.     

Reducing Biogenic-Amine-Producing Bacteria,Decarboxylase Activity,and Biogenic Amines in Raw Milk Cheese by High-Pressure Treatments

Biogenic amines may reach concentrations of public health concern in some cheeses. To minimize biogenic amine buildup in raw milk cheese, high-pressure treatments of 400 or 600 MPa for 5 min were applied on days 21 and 35 of ripening. On day 60, counts of lactic acid bacteria, enterococci, and lactobacilli were 1 to 2 log units lower in cheeses treated at 400 MPa and 4 to 6 log units lower in cheeses treated at 600 MPa than in control cheese. At that time, aminopeptidase activity was 16 to 75% lower in cheeses treated at 400 MPa and 56 to 81% lower in cheeses treated at 600 MPa than in control cheese, while the total free amino acid concentration was 35 to 53% higher in cheeses treated at 400 MPa and 3 to 15% higher in cheeses treated at 600 MPa, and decarboxylase activity was 86 to 96% lower in cheeses treated at 400 MPa and 93 to 100% lower in cheeses treated at 600 MPa. Tyramine, putrescine, and cadaverine were the most abundant amines in control cheese. The total biogenic amine concentration on day 60, which reached a maximum of 1.089 mg/g dry matter in control cheese, was 27 to 33% lower in cheeses treated at 400 MPa and 40 to 65% lower in cheeses treated at 600 MPa. On day 240, total biogenic amines attained a concentration of 3.690 mg/g dry matter in control cheese and contents 11 to 45% lower in cheeses treated at 400 MPa and 73 to 76% lower in cheeses treated at 600 MPa. Over 80% of the histidine and 95% of the tyrosine had been converted into histamine and tyramine in control cheese by day 60. Substrate depletion played an important role in the rate of biogenic amine buildup, becoming a limiting factor in the case of some amino acids.     

Microbial spoilage and formation of biogenic amines in fresh and thawed modified atmosphere-packed salmon (Salmo salar) at 2 degrees C

AIMS: To evaluate the microbial spoilage, formation of biogenic amines and shelf life of chilled fresh and frozen/thawed salmon packed in a modified atmosphere and stored at 2 degrees C. METHODS AND RESULTS: The dominating microflora, formation of biogenic amines and shelf life were studied in two series of storage trials with naturally contaminated fresh and thawed modified atmosphere-packed (MAP) salmon at 2 degrees C. Photobacterium phosphoreum dominated the spoilage microflora of fresh MAP salmon at more than 10(6) cfu g(-1) and the activity of this specific spoilage organism (SSO) limited the shelf life of the product to ca 14 and 21 d in the two experiments. Despite the high levels of P. phosphoreum, less than 20 mg kg(-1) histamine was observed in fresh MAP salmon prior to sensory spoilage. Freezing eliminated P. phosphoreum and extended the shelf life of MAP salmon at 2 degrees C by 1-2 weeks. Carnobacterium piscicola dominated the spoilage microflora of thawed MAP salmon and probably produced the ca 40 mg kg(-1) tyramine detected in this product at the end of its shelf life. CONCLUSIONS: Photobacterium phosphoreum dominated the spoilage microflora of fresh MAP salmon but produced only small amounts of biogenic amines in this product. The elimination of P. phosphoreum by freezing allowed this bacteria to be identified as the SSO in fresh MAP salmon. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of P. phosphoreum as the SSO in fresh MAP salmon facilitates the development of methods to determine and predict the shelf life of this product, as previously shown with fresh MAP cod.     

Monitoring of Staphylococcus xylosus DSM 20266 added as starter during fermentation and ripening of soppressata molisana, a typical Italian sausage

AIMS: "Soppressata molisana", a fermented sausage produced in southern Italy, is commonly obtained without starter addition. However, the use of starter cultures is more and more recommended in meat fermentation processes in order to guarantee stable production performance. In this study, the survival of the Staphylococcus xylosus DSM 20266 was evaluated during the ripening of "soppressata molisana" fermented sausage. METHODS AND RESULTS: The fastest method of RAPD-PCR was employed for discrimination of the added strain from those naturally present during the ripening of the "soppressata molisana". The results obtained were confirmed by analysis of the DNA macrorestriction profile by PFGE. The electrophoretic pattern of bacterial total proteins was also studied, but clear differences between the different strains could not be detected. CONCLUSIONS: The RAPD technique was a valid tool for monitoring Staph. xylosus DSM 20266 in "sopressata molisana". SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the possibility of monitoring the presence of Staph. xylosus strains during the ripening of fermented sausages by a reliable and repeatable technique such as RAPD.     

Biogenic amine-forming microbial communities in cheese

The aim of this study was to screen two cheese starter cultures and cheese-borne microbial communities with the potential to produce biogenic amines in cheese during ripening. Bacteria of the genera Enterococcus and Lactobacillus and coliform bacteria were isolated from Dutch-type semi-hard cheese at the beginning of the ripening period. Statistically significant counts of bacterial isolates were screened for the presence of specific DNA sequences coding for tyrosine decarboxylase (tyrDC) and histidine decarboxylase (hDC) enzymes. The PCR analysis of DNA from 14 Enterococcus and 3 Lactobacillus isolates confirmed the presence of the targetted DNA sequences. Simultaneously, 13 tyrDC- and 3 hDC-positive isolates were grown in decarboxylase screening medium and this was followed by HPLC analysis of the produced tyramine and histamine. Conventional and molecular taxonomic analyses of the above-mentioned isolates identified the following species: Enterococcus durans (7 strains), Enterococcus faecalis (3 strains), Enterococcus faecium (1 strain), Enterococcus casseliflavus (3 strains), Lactobacillus curvatus (1 strain), Lactobacillus lactis (1 strain) and Lactobacillus helveticus (1 strain). All of the above Enterococcus and two of the Lactobacillus strains originated from contaminating microbial communities. The L. helveticus strain, which was tyrosine decarboxylase-positive and exhibited tyramine production, originated from starter culture 1 used for cheese production. Comparison of partial tyrDC sequences of positive Enterococcus isolates revealed 89% sequence similarity, and that of hDC-positive Lactobacillus isolates revealed 99% sequence similarity.     

Biogenic amine production by Lactobacillus

AIMS: The aim of this work was to demonstrate that strains of Lactobacillus may be able to produce putrescine and agmatine from one of the major amino acids present in fruit juices and wine, arginine, and from amino acid-derived ornithine. METHODS AND RESULTS: Biogenic amines were determined by HPLC. Their production in the culture medium was similar under both microaerophilic and anaerobic conditions. The presence of Mn2+ had a minimal influence on the results, whereas the addition of pyridoxal phosphate increased amine production 10-fold. Lactobacillus hilgardii X1B, isolated from wine, was able to degrade arginine by two pathways: arginine deiminase and arginine decarboxylase. The isolate was able to produce putrescine from ornithine and from agmatine. Lactobacillus plantarum strains N4 and N8, isolated from orange, utilized arginine via the arginine deiminase system. Only the N4 strain was able to produce putrescine from ornithine. CONCLUSION: It has been demonstrated that Lact. hilgardii X1B is able to produce the most important biogenic amine found in wine, putrescine, and also agmatine from arginine and ornithine, and that Lactobacillus plantarum, considered to be an innocuous spoilage micro-organism in fruit juices, is able to produce amines. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have significance in relation to food poisoning caused by beverages that have been contaminated with biogenic amines.     

The inverse relationship between the activity of pyridoxal kinase and the level of biogenic amines in rabbit brain

In a series of experiments, an inverse relationship was found to exist between the activity of pyridoxal kinase and the concentrations of brain norepinephrine, dopamine, and serotonin. When the biogenic amine content of the brain is lowered, the activity of pyridoxal kinase rises. Conversely, when the concentration of brain biogenic amines is elevated, a depression occurs in the activity of pyridoxal phosphokinase. This relationship, which resembles an in vivo allosteric inhibition phenomenon, not only is highly suggestive of a definite relation between the brain vitamin B₆ content and the metabolism of the biogenic amines, but also suggests that the concentration of brain biogenic amines might control the production of coenzyme pyridoxal phosphate.     

Amine oxidases in apoptosis and cancer

Amine oxidases, the major enzymes of biogenic amines metabolism, are considered to be biological regulators, especially for cell growth and differentiation. A primary involvement of amine oxidases in cancer growth inhibition and progression, especially by means of aldehydes, H(2)O(2) and other reactive oxygen species, the amine oxidase-mediated products of biogenic amines oxidation, has been demonstrated. Amine oxidases are involved in cancer growth inhibition because of the higher content in tumour cells of biogenic amines in comparison to normal cells. The cytotoxic effect can be explained by a damage to cell membranes and/or nuclei or, indirectly, through modulation of membrane permeability transition and therefore apoptosis. The oxidation products of biogenic amines appears to be also carcinogenic, while acrolein, produced from the oxidation of spermine and spermidine, should be a key compound both carcinogenic and cytotoxic. The cancer inhibition/promotion effect of amine oxidases could be explained by taking into consideration the full pattern of the enzyme content of the cell. The balance of amine oxidases and antioxidant enzymes appear to be a crucial point for cancer inhibition or progression. A long lasting imbalance of these enzymes appears to be carcinogenic, while, for a short time, amine oxidases are cytotoxic for cancer cells.     

The effect of a commercial starter culture addition on the ripening of an artisanal goat's cheese (Cameros cheese)

The evolution of physicochemical parameters, and the most important microbial groups, were determined for the following three batches of 'Cameros' goat's milk cheese during ripening: Batch R elaborated with raw milk, Batch RS elaborated with raw milk and with the addition of a starter culture, and Batch PS elaborated with pasteurized milk and with the addition of the same culture. No differences in total solids (TS) or in the content of NaCl, fat and total nitrogen (expressed as percentages of TS) were found during the ripening. The pH, fat acidity and non-protein nitrogen (NPN, expressed as a percentage of TN) showed significant differences between the batches. The inoculated batches showed the fastest drop in pH at the beginning of the ripening period, but the cheeses of Batch R showed a higher degree of lipolysis and proteolysis. The addition of a starter influenced the microbiological quality of the cheeses. Differences in the counts of Enterobacteriaceae and faecal coliforms were found between Batches R and RS after 15 days. Staphylococcus aureus increased in number during the early period of ripening and attained a population above 6 log cfu g-1 in Batch R in the period from 5 to 10 days. However, enterotoxins were not detected in this Batch. Batch R showed lower values of lactic acid bacteria at the beginning of the ripening period, but no significant differences were found between batches in the period from 5 to 15 days of ripening. At the beginning of the ripening, Lactococcus was the main lactic acid bacteria, with L. lactis lactis being predominant. After 15 days, the lactic acid bacteria counts decreased in the three batches, especially in the cheeses of Batch PS (only 2.2 log cfu g-1 was found at 60 days), as lactococci (the only lactic acid bacteria present in Batch PS) are incapable of growing under the conditions found in cheeses at the end of their ripening period. At this time, Lactobacillus was the predominant genus in Batches R and RS, with L. plantarum predominant. No lactococci were found from day 30 in Batch R and from day 40 in Batch RS. The cheeses of Batch RS received the most favourable scores from the tasting panel for all attributes judged: cut appearance, colour, aroma, taste, texture and general acceptance.     

产生物胺乳酸菌的筛查与检测

目的对上海市场上食品和药品中分离出的20株乳杆菌,13株链球菌,3株乳球菌和3株肠球菌的产生物胺能力进行检测,以揭示其潜在的安全性问题。方法检测的生物胺共包括6种：分别为酪胺、精胺、尸胺、组胺、腐胺和色胺。利用添加了前体氨基酸的氨基酸脱羧酶筛选培养基对各菌株的产胺能力进行初筛,通过培养基中指示剂的颜色变化判定产胺能力。结果在检测的39株菌中,8株菌具有产酪胺的能力,7株菌具有产精胺的能力,1株菌具有产组胺的能力,1株菌具有产腐胺的能力。尤其是精胺和酪胺的产量较为引人关注。结论生物胺的危害水平取决于个体解毒的能力,但在筛选食品药品用菌株时应运用规范的方法来检测其产生物胺的能力,以保障相应食品药品的安全问题。     

Biogenic amine formation and microbial spoilage in chilled garfish (Belone belone belone)--effect of modified atmosphere packaging and previous frozen storage

AIMS: To evaluate biogenic amine formation and microbial spoilage in fresh and thawed chilled garfish. METHODS AND RESULTS: Storage trials were carried out with fresh and thawed garfish fillets at 0 or 5 degrees C in air or in modified atmosphere packaging (MAP: 40% CO2 and 60% N2). During storage, sensory, chemical and microbial changes were recorded and histamine formation by isolates from the spoilage microflora was evaluated at 5 degrees C. Photobacterium phosphoreum was responsible for histamine formation (>1000 ppm) in chilled fresh garfish. The use of MAP did not reduce the histamine formation. Strongly histamine-producing P. phosphoreum isolates formed 2080-4490 ppm at 5 degrees C, whereas below 60 ppm was formed by other P. phosphoreum isolates. Frozen storage inactivated P. phosphoreum and consequently reduced histamine formation in thawed garfish at 5 degrees C markedly. CONCLUSIONS: Photobacterium phosphoreum can produce above 1000 ppm of histamine in chilled fresh garfish stored both in air and in MAP. Freezing inactivates P. phosphoreum, extends shelf life and markedly reduces histamine formation in thawed MAP garfish during chilled storage. SIGNIFICANCE AND IMPACT OF THE STUDY: At 5 degrees C, more than 1000 ppm of histamine was formed in garfish; thus even when it is chilled this product represents a histamine fish-poisoning risk.     

A proteomic approach to studying biogenic amine producing lactic acid bacteria

All fermented foods are subject to the risk of biogenic amine contamination. Histamine and tyramine are among the most toxic amines for consumers' health, exerting undesirable effects on the central nervous and vascular systems, but putrescine and cadaverine can also compromise the organoleptic properties of contaminated foods. These compounds are produced by fermenting microbial flora that decarboxylate amino acids to amines. Little is known of the factors which induce biosynthesis of decarboxylating enzymes and/or which modulate their catalytic activity: the accumulation of amines is generally considered to be a mechanism that contrasts an acidic environment and/or that produces metabolic energy through coupling amino acid decarboxylation with electrogenic amino acid/amine antiporters. Two Lactobacillus strains, Lactobacillus sp. 30a (ATCC 33222), and a Lactobacillus sp. strain (w53) isolated from amine-contaminated wine, carrying genetic determinants for histidine decarboxylase (HDC) and ornithine decarboxylase (ODC), were studied and the influence of some environmental and nutritional parameters on amine production and protein biosynthesis was analyzed through a proteomic approach; this is the first report of a proteomic analysis of amine-producing bacteria. HDC and ODC biosynthesis were shown to be closely dependent on the presence of high concentrations of free amino acids in the growth medium and to be modulated by the growth phase. The stationary phase and high amounts of free amino acids also strongly induced the biosynthesis of an oligopeptide transport protein belonging to the proteolytic system of Lactic Acid Bacteria. At least two isoforms of glyceraldehyde-3-phosphate dehydrogenase, with different M(r), pI and expression profiles, were identified from Lactobacillus sp. w53: the biosynthesis of one isoform, in particular, is apparently repressed by high concentrations of free amino acids. Other proteins were identified from the Lactobacillus proteome, affording a global knowledge of protein biosynthesis modulation during biogenic amine production.     

Microbial content of commercial South African high-moisture dried fruits

AIM: The aim was to evaluate commercially available South African high-moisture dried fruits (HMDF) for the microbial, moisture and SO2 contents, as well as aw and pH. METHODS AND RESULTS: The microbial content of commercially available HMDF was evaluated using nine different growth media. The moisture content, aw) SO2 and pH of each product were determined using standard analytical methods. It was found that the highest total aerobic counts were generated from high-moisture dried (HMD) prunes and raisins. The most frequent spoilers were members of the genus Bacillus. Fungal counts were also very high in the apricot products, exceeding the limit of 1000 CFU g(-1) as set by HMDF producers. Members of the genus Staphylococcus were found in the HMD raisins and Salmonella and thermoduric organisms were isolated from the HMD prunes. CONCLUSIONS: The microbial levels of South African HMDF were within the limits set, with the exception of apricots. SIGNIFICANCE AND IMPACT OF STUDY: The study shows the presence of Salmonella, Staphylococcus and Clostridium in South African HMDF. The presence of thermoduric organisms indicated that the current pasteurization process is not adequate and that the addition of preservatives would be an additional method to ensure safety and quality.