Background: Breast milk, produced after childbirth during the flow of milk, is food whose composition evolves to adapt to the newborn’s needs. The purpose of this study was to evaluate the amount of non-compliant mother’s milk donations during the study period.

Materials and Methods: We performed a prospective study of milk samples donated by home donors or mothers of hospitalized preterm newborns.

Results: The disposal milk was due to bacterial contamination in 91.6% of cases. The dominant bacteria detected in raw milk were E. coli in 4.2% of samples, S. epidermidis (4.2%), S. aureus (4.2%) and S. hominis (4.2%), and in pasteurized milk were Bacillus spp in 16.2% of samples followed by B. cereus (6.2%), Paenibacillus (6.2%), S. hominis (5%) and P. aeruginosa (5%). 

Conclusion: The role of the milk bank is to provide adequate nutrition for preterm infants. Pasteurized breast milk intended for preterm infants must be bacteriologically consistent with consumption.

APUH: Amiens-Picardie University Hospital; 

BM: breast milk; 

CFU: colony-forming unit; 

DM: donor milk; 

FHPSA: French Health Product Safety Agency; 

HMB: human milk bank; 

HM: human milk; 

MB: milk bank; 

NDHPSA: National Drug and Health Product Safety Agency; 

nICU: neonatal intensive care unit; 

Yrs: years

Healthy breastfeeding mothers and mothers of preterm babies should send their milk to human milk banks (HMB) [1]. The main missions of the HMB are to collect, screen, store, process, pasteurize, and distribute the woman’s milk and promote breastfeeding [2]. Another role of HMB is to obtain enough milk to meet the needs of preterm infants or sick newborns admitted to the neonatal intensive care unit (nICU) [3]. Donor women are screened for Human immunodeficiency virus (HIV-1 and HIV-2); Human T-cell leukemia virus (HTLV-1 and HTLV-2); Hepatitis B virus (HBV), Hepatitis C virus (HCV), and syphilis [2]. 

The need for milk bank (MB) use is globally increasing with more preterm newborns [4]. Personalized milk donation gives the hospitalized newborn the possibility to benefit from their mother’s milk, either directly in the form of raw milk (unpasteurized) that the mother expresses and keeps at home or in MB, or after pasteurization by an MB. This practice depends on the gestational age and the stage of lactation, ensuring an adequate composition of donated milk to preterm infants [5]. 

Human milk (HM), apart from its nutritional qualities, has immunocompetent functions particularly suitable preterm children. HM is known as the most complete nutrition for infants worldwide [6]. If a mother cannot breastfeed her child, either because she is not available or because of the milk produced is insufficient, the best alternative is breast milk (BM) from a healthy breastfeeding mother or milk of HMB [4, 7]. Therefore, in 2005, in the MB, the milk was processed according to the Ministry of Health guidelines [(French Health Product Safety Agency (FHPSA) and National Drug and Health Product Safety Agency (NDHPSA)] [8]. Moreover, the bacteriologic quality of raw milk given to a very preterm infant, even when provided by his/her mother, has to be checked carefully because the newborn’s immature immune system may not be able to combat bacterial contamination. The milk’s fat, protein, and carbohydrate contents are checked to ensure that the nutritional profile is appropriate for a very preterm baby. The purpose of this study was to evaluate the amount of the donated milk non-compliant for consumption during the study period, as well as the reasons for the disposal of these donated milk samples.

Ethical considerations

The study was approved by Ile de France3 Ethics Committee of Amiens-Picardie University Hospital (APUH) (Ref DSLILG/2018-039). Study participants were all breastfeeding mothers, who voluntarily decided to participate in the study. These mothers provided their written informed consent to participate before the study’s initiation. 

Study design and donor population

A prospective study was undertaken on milk samples of healthy breastfeeding mothers. These mothers gave their extra BM to other babies out of their free will. The donors also include mothers attending well-baby clinics, mothers whose babies are in nICUs, and those who have lost their babies. Lactating donors were home donors or mothers of preterm newborns hospitalized in the nICU at APUH from January 1st, 2019 to December 30th, 2020.

During this period, a total of 154 lactating mothers who have their newborns hospitalized at the nICU and produced an excess of milk for their infants were carefully selected and included in this study. Most of them delivered prematurely (84.4%, 130/154), but some had term babies (15.6%, 24/154). Out of 154 milk donors, 112 (72.7%) were personalized, and 42 (27.3%) were anonymous donors. Each mother had a questionnaire to check for absence of formal contraindications to donation (tabacco use, alcohol consumption, blood transfusion) or a positive serologic test for HIV1, 2; HTLV1, 2; HBV, HCV, and Treponema pallidum [1], and, if all requirements are met, the donor is eligible as a milk donor. These women donated a total of 2,000 ML of milk to HMB, including 1,800 ML of personalized milk (90%) and 200 ML of anonymous milk donation (10%). The total quantity of distributed milk was 1,400 ML (70%), and 600 ML (30%) were discarded (Table 1). 

Human milk bank generally follows standardized procedures for the collection and handling of donated milk [1, 9]. Donors are instructed by the MB in breast cleaning and breast pumping procedures. The donor obtains milk by mechanical pump or manual expression and stores it in the freezer compartment of their home refrigerator before delivery to the MB. The milk is transported to the MB either by the mother herself or by a transport service provided by the MB. The milk from 4 donors is pooled in individual 100 ML bottles and stored at -20 °C until performed. Pooling serves the purpose of estimating macronutrients, such as fat, protein, carbohydrate, and energy, using infra-red spectroscopy technology [9]. 

Microbiological testing

Raw and pasteurized milk samples were estimated. They were plated onto 5% Columbia blood agar for facultative anaerobes and onto mannitol salt agar. Following the Holder pasteurization method [10], a final bacteriological check is carried out after pasteurization, by inoculation onto 5% Columbia blood agar incubated aerobically at 35±2 °C 48 times. The raw milk was unsatisfactory when the total viable facultative anaerobe count was ≥106 CFU/ML, the S. aureus count ≥104 CFU/ML, and the total viable facultative anaerobe count in the pasteurized milk ≥101 CFU/ML [11].

A total of 220 milk samples from 154 breastfeeding mothers were studied, including 140 raw and 80 pasteurized milk samples. Of those, 15.7% (22/140) of raw milk samples, and 46.3% (37/80) of pasteurized milk samples were culture-positive. 

During the study period, a total of 750 newborns received BM, and the common indication of recipients was prematurity (100%). Among them, five had malabsorption syndrome (0.6%), three had an intolerance to BM (0.4%), and two had congenital defects (0.2%). The mean birth weight (BW) of the preterm infants was 1150.02±442.7 g, and the median 992.2 g (range: 590—2,600 g). An infant can be classified in extremely low birth weight (ELBW) (<1>

In our hospital, the proportion of discarded donated milk increased yearly, 18% in 2016, 25% in 2017, and 29.4% in 2018. The donated milk discarded for bacterial reasons during the same periods was 86.8%, 87.5%, and 86.4%, respectively (unpublished data). The current results have shown that the quantity of mother’s milk between 2019 and 2020 was 2,000 ML, 1,400 ML (70%) of whom were distributed and 600 ML (30%) thrown, 550 ML (91.6%) for bacterial reasons and 50 ML (8.4%) for other causes (Table 1).

During this period, there were 154 milk donors (112 personalized milk donors and 42 anonymous milk donors), this represented 2000 ML of milk of which (70.0%) were distributed and 600 ML (30.0%) were discarded of whom 550 ML (91.6%) due to bacterial causes and 50 ML (8.4%) due to other reasons. Table 1: Volume of milk (in ML) collected, distributed, and discarded between 2019 and 2020

The most common reason for discarding raw milk was bacterial contamination. All raw milk samples had bacterial load ranging from 1.5X106 to 1.2X107 CFU/ML. The types of bacteria isolated in raw milk cultures were the following: Escherichia coli (E. coli) (4.2%), Staphylococcus epidermidis (S. epidermidis) (4.2%), Staphylococcus. aureus (S. aureus) (4.2%), Staphylococcus hominis (S. hominis) (4.2%), and Enterobacter cloacae (E. cloacae) (3.5%). These bacteria were detected at low levels in pasteurized milk, suggesting possible post-pasteurization contamination (Table 2). 

Bacterial count in pasteurized milk samples ranged from 1.0X102 to 1.2X103 CFU/ML. The most frequent bacteria strains were the following: Bacillus spp. (16.2%), Bacillus cereus (B. cereus) (6.2%), Paenibacillus spp. (6.2%), Staphylococcus hominis (S. hominis) (5.0%), and Pseudomonas aeruginosa (P. aeruginosa) (5.0%) (Table 2).

Most microbial types found both before and after pasteurization of milk samples have seen that the number of Gram-negative bacilli and Gram-

positive cocci recovered decreased after pasteurization: E. coli, E. cloacae, S. epidermidis, S. aureus, and Staphylococcus lugdinensis (S. lugdinensis).  Gram-positive bacilli include: Bacillus spp., B. cereus, Mycobacterium lacticum (M. lacticum), while Gram-negative bacilli include: Stenotrophomonas maltophilia (S. maltophilia), and P. aeruginosa. Gram-positive cocci include: S. hominis, and Streptococcus salivarius (Str. salivarius), whose amount has increased after this process (Table 2). Such results have been observed by Cherif-Antar et al. [14].

This study also showed that the non-spore-forming Gram-negative bacteria heat-sensitive in both raw and pasteurized milk were predominantly represented by P. aeruginosa (5.0%), S. maltophilia (3.7%), E. coli (4.2%), E. cloacae (3.5), and Klebsiella pneumoniae (K. pneumoniae) (1.4%). The non-spore-forming Gam-positive bacteria included M. lacticum (2.5%) and Lactobacillus plantarum (L. plantarum) (1.2%). The psychrotrophic spore-forming bacteria heat-resistant isolated included Bacillus spp. (16.2%), B. cereus (6.2), and Paenibacillus spp. (6.2%) (Table 2).

Six pasteurized milk samples were contaminated by more than one species: Bacillus spp+ Acinetobacter pittii (n=1), Bacillus cereus + Paenibacillus spp (n=1), Klebsiella pneumoniae + Acinetobacter pittii (n=1), S.maltophilia +Acinetobacter pittii (n=1), Streptococcus salivarious + Moraxella osloensis (n=1), and Bacillus cereus, Pantoea agglomerans+ Acinetobacter ursinguii (n=1).Three raw milk samples were contaminated by more than one species: Staphylococcus hominis + Escherichia coli (n=1); Staphylococcus hominis + Stenotrophomonas maltophilia (n=1); Staphylococcus aureus + Enterobacter cloacae (n=1)Table 2: Results of bacterial strains isolated from raw and pasteurized milk samples by culture

The bacterial load detected in raw milk samples ranged from 1.5X106 to 1.2X107 CFU/ML and was slightly lower than that found by Banik et al. (5.2x102 to 1.3X107 CFU/ML) [15]. The results of bacterial count observed in pasteurized milk agree with those by Banik et al. (1.2X102 to 1.8X101 CFU/ML) [15].

The most frequent reason for discarding both raw and pasteurized milk was the non-compliance of quality (collection procedures not complying with guidelines, the lack of information on the label concerning the donor’s identity, the date of collection, and any medications that the donor may have been taking) [16].

The presence of the aforementioned bacteria suggested that the milk samples had been contaminated by people and environmental sources. High bacterial counts indicated that the bacteria had not only contaminated the milk but had grown and multiplied in it [17].

The big problem in ensuring a suitable shelf life of pasteurized milk is to destroy, by pasteurization, a sufficient proportion of the heat-resistant flora, such as Streptococcus, Lactobacillus spp., Paenibacillus spp., and Bacillus spp. and to minimize the contaminating flora, such as coagulase-negative Staphylococcus, S. aureus, and Enterobacteriaceae [15, 18]. Different bacterial species can enter the udder through the teat canal and are excreted with the milk. By developing in the udder, some germs, particularly Staphylococci, Streptococci, and Enterobacteriaceae, cause milk contamination. 

There are several reasons for the occurrence of bacterial contamination in the pasteurized milk samples, such as a defect in pasteurization machinery, or contamination in the post-pasteurization due to a lack of respect for the hygienic conditions defined in the pasteurization protocols by the personnel. 

 Some bacteria, such as E. coli, S. aureus, and Bacillus spp. produce enterotoxins, extracellular enzymes, heat-resistant spores, and can form biofilm [19]. They remain active after pasteurization, even though bacteria have been eliminated. Bacteria within biofilms may attach to tools and equipment, thus persisting in the dairy environment. Biofilms are considered a source of microbial contamination leading to food spoilage and shelf-life reduction of foods, which can be a way of pathogen transmission. 

According to literature data, a variety of pathogenic bacteria have been isolated from raw milk, including Mycobacterium spp., Salmonella spp., Listeria monocytogenes, B. cereus, Campylobacter jejunii, Yersinia enterocolitica, E. coli, S. aureus, Brucella abortus, Klebsiella spp., Proteus spp., and P. aeruginosa [20-22]. These results are different from those found in the present work. Depending on the country of origin, species, climate, and sanitary conditions, raw milk can contain one or more pathogens listed above.

Gram-negative bacteria detected during the processing of donated BM are generally heat-sensitive protease and lipase producers and can cause product deterioration. P. aeruginosa is a psychrotrophic bacterium that lives in cold water, refrigerated milk, and the soil. It is also found as a commensal in the digestive tract. The effective adaptation to cold is probably due to the presence of many unsaturated lipids in the cell membranes [23-25].  The presence of indicator Gram-negative bacteria and some other bacteria in lesser numbers determines the safety and quality of milk and its products [26-28].

In this study, we paid more attention to the quality of BM, including infection control and nutrition density. Some HMB provides fresh-frozen 

raw milk to infants, and only samples containing S. aureus, Klebsiella spp., Enterobacter spp., Serratia spp., E. coli, or a bacteria count of >104 CFU/ML) were pasteurized and supplied when germ-free [29]. At present, however, we still use the protocols for milk pasteurization that meet the FHPSA and NDHPSA criteria [8]. All DM was provided after pasteurization and was germ-free. The bacteriological screen of DM pre- and post-pasteurization resulted in approximately 30% of DM discarded in our MB between 2019 and 2020. We then applied rigorous screening standards because safety should always be paramount. Following the FHPSA and NDHPSA criteria for bacterial screening, only samples ≥106 CFU/ML of microorganisms or ≥104 CFU/ML of S. aureus or Enterobacteriaceae were discarded before pasteurization. That would allow taking corrective measures to increase the volume of collected milk available for consumption and reduce the unnecessary waste of valuable milk.

Most post-pasteurization psychrotrophic spore-formers bacteria found in this study have been reported in the literature [24, 25]. In this category, Bacillus spp. is predominant and can be introduced into milk from water used for rinsed milking machines and equipment. It is an environmental germ, ubiquitous, spore-former, and heat-resistant. These spores remain viable and give rise to vegetative forms (germination) and can develop toxins. Bacillus spp. produces degradation enzymes such as protease, lipases, and phospholipases, which can deteriorate dairy products. 

The presence of pathogens in pasteurized milk may be due to the dysfunction of the pasteurization equipment or insufficient pasteurization temperature and time, untimely handling of the milk after pasteurization [17]. Raw milk may be a prime source of psychrotrophic spore-formers during milk transport, storage, and handling. Additionally, poorly cleaned milk-processing equipment, both raw and post-pasteurized types of milk are a potential source of these organisms, as the latter is more likely to withstand the rigor programs of cleaning [30].

This study shows that the microbiological quality of most of the raw and post-pasteurized milk samples studied were not satisfactory for consumption as indicated by their high loads. As corrective measures, following the results of this work, we decided to strictly and rigorously apply the criteria defined in the FHPSA or NDHPSA guidelines. Further work will allow us to assess the effectiveness of the measures recommended regarding the reduction in the amount of milk bank discarded.

The authors declare that they have no conflict of interest