Stability Analysis of Probiotic-Containing Infant Formula: Post-Preparation Changes in Nutritional, Chemical, and Microbiological Properties

Abstract

This study evaluated the changes in nutritional content, chemical properties, and microbiological quality of a probiotic-containing commercial powdered infant formula, prepared according to usage instructions and stored for 0-24 hours. Microbiological analyses showed a slight but statistically insignificant increase (P > 0.05) in total mesophilic aerobic bacteria (TMAB) and lactic streptococci counts over time. Coliforms, Enterobacteriaceae, and yeast-mould levels remained consistently below 1 log cfu/mL during the entire storage period. Chemical analysis revealed no significant differences in pH, acidity, thiobarbituric acid (TBA) levels, antioxidant capacity (ABTS, DPPH), or vitamin B12 and folic acid contents (P > 0.05) across different time points. However, fluctuations were noted in the amino acid profile and cholesterol concentrations over time. In terms of color parameters, a statistically significant change was observed along the yellow-blue axis (b*), indicating potential pigment or composition changes during storage. Despite these fluctuations, infant formula largely maintained its microbiological and chemical stability within the first 24 hours post-preparation. These findings are specific to the product in question and, based on them, it is recommended that reconstituted infant formula is consumed within two to six hours of preparation. If longer storage is necessary, the product should be kept under refrigeration at temperatures below 5 °C to minimize any potential quality degradation and ensure infant safety.

Keywords:
Baby foods; infant formula; public health; microbiological quality; chemical quality

HIGHLIGHTS

• Powdered infant formula was monitored for safety and quality up to 24 hours.

• Findings support time-temperature controls for infant formula use.

• Lipid oxidation and antioxidant capacity remained stable during storage.

• Amino acid profile showed compositional shifts without change in total content.

GRAPHICAL ABSTRACTS

INTRODUCTION

Infant nutrition is a critical process with lifelong implications for health. During the neonatal period, breast milk constitutes the primary nutritional source for infants. However, in certain instances, inadequate milk production in mothers, or issues such as sucking difficulties and breast rejection in infants, can lead to an increased requirement for alternative nutritional sources [¹, ²]. In such cases, the use of formulated infant formulas is widespread in order to meet the physiological and developmental needs of infants. The first industrial infant formula was developed by Henri Nestlé in Switzerland in 1860 under the name 'Farine Lactée'. This formula experienced a rapid development process, especially after World War II, due to the increasing infant mortality rates of the time [³].

Formulas for infant milk are designed to contain essential macroand micronutrients such as proteins, lipids, carbohydrates, vitamins and minerals, and to be as close as possible to breast milk in terms of nutrient content. They are produced in powder and liquid form [²]. It is evident that powder formulations are the preferred choice due to their ease of use and extended shelf life. Formula components are highly susceptible to microbial and chemical spoilage due to their high pH values, the presence of certain levels of fermentable carbohydrates, richness in omega-3 fatty acids, protein and other growth factors [⁴]. Notably, inadequate storage conditions and the lack of sterility in these products represent a substantial drawback, given the potential for transmission of pathogenic microorganisms such as Cronobacter sakazakii, Salmonella spp. and Bacillus cereus [⁵-⁷]. These contaminants have the potential to compromise the health of newborns, whose immune and digestive systems are not yet fully developed. Furthermore, factors such as errors during formula preparation, elevated temperatures, and inadequate storage conditions have been demonstrated to result in the degradation of sensitive nutrients, including vitamins and lipids, and chemical deterioration, such as oxidation of lipids [⁸]. In this context, it is imperative to evaluate changes in nutritional value, chemical stability deterioration, and microbiological risks during the process from preparation to consumption of formula foods, in order to safeguard infant health.

In the context of infant feeding, it is imperative to prioritise the safety of infant formula preparation and strict adherence to hygiene regulations to minimise the risk of infection. In 2007, the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) published an international guideline titled 'Safe Preparation, Storage and Use of Powdered Infant Formula' [⁹]. This was due to the occurrence of chemical and microbiological deterioration in infant formula and the increase in infant formula-related deaths. As indicated in this guideline, it is imperative to prioritise hand hygiene prior to formula preparation. All equipment utilised in this process must undergo thorough cleaning and sterilisation. Furthermore, it is crucial to ensure that the formula is prepared with purified water, with a temperature exceeding 70°C, and that the prepared formula is cooled to the appropriate level. These practices are of critical importance in preventing infections, which may occur especially in infants, whose immune systems are not yet developed.

Manufacturers include preparation instructions (e.g. water temperature, formula/water ratio) and ingredient information on formula packaging. However, critical issues such as the duration of infant formula consumption and the optimal storage conditions for unused formula are frequently not addressed in sufficient detail. Preliminary studies and interviews with parents indicate a significant deficit in information regarding this issue. Common practices, such as the one-time consumption of infant formula followed by its reuse in subsequent meals, have the potential to result in nutritional losses and pose a significant risk with regard to chemical and microbiological safety. It is evident that there is a paucity of comprehensive scientific research examining the changes in nutritional values, microbial load and chemical composition during the period following the preparation of infant formulas. Consequently, systematic evaluation of the potential alterations that may ensue in the post-preparation period of infant formula, alongside the delineation of the optimal consumption window, assumes paramount importance from both scientific and public health perspectives.

The objective of the present study was to ascertain the alterations in nutrient content, chemical properties and microbiological quality of infant formula prepared in accordance with the instructions for use under disparate storage conditions for varying periods of time. Furthermore, the study aimed to determine the most suitable time interval for consumption on the basis of scientific evidence.

MATERIAL AND METHODS

Supply of infant formula

This study used a commercially available powdered infant formula containing probiotics (SMA® OPTIPRO®, France). The composition of infant formula is provided in Table 1. Infant formula was purchased commercially. According to the manufacturer's label, the product contains Bifidobacterium animalis subsp. lactis as the declared microbial component. This strain, added by the manufacturer, is a live microorganism that has been clinically proven to provide the host with beneficial effects, in accordance with the probiotic definitions accepted by the FAO/WHO [⁹, ¹⁰]. However, strain-level identification, live cell count at the point of consumption and functional probiotic claims in line with the FAO/WHO definitions are not stated on the product label. Therefore, in the context of the present study, the microbial component was considered a declared microbial component rather than a fully characterised probiotic strain.

Table 1
Detailed ingredient profile of commercial infant formula

Category	Ingredients
Dairy Products	Skimmed milk, lactose, whey, whey permeate
Vegetable Oils	High oleic sunflower oil, low erucic rapeseed oil
Antioxidants	Alpha-tocopherol, L-ascorbyl palmitate
Emulsifier	Soy lecithin
Minerals	Calcium salts of citric acid, sodium salts of orthophosphoric acid, potassium citrate, potassium chloride, magnesium chloride, zinc sulfate, ferrous sulfate, copper sulfate, manganese sulfate, potassium iodide, sodium selenate, calcium carbonate
Acidity Regulator	Citric acid
Fish Oil	Fish oil
Amino Acids	L-threonine, L-lysine, L-isoleucine, L-tryptophan, L-histidine
Vitamins	Vitamin C (sodium L-ascorbate), Vitamin E (DL-alpha-tocopheryl acetate), Niacin (nicotinamide), Vitamin B1 (thiamine mononitrate), Vitamin A (retinyl acetate), Vitamin B2 (riboflavin), Vitamin B6 (pyridoxine hydrochloride), Folate (folic acid), Vitamin K (phytomenadione), Vitamin D (cholecalciferol), Vitamin B12 (cyanocobalamin), Biotin (D-biotin), Pantothenic acid (D-calcium pantothenate)
Probiotic	Bifidobacterium animalis subsp. lactis
Allergen Warning	Contains milk, soy, fish oil

Preparation of infant formula and experimental design

The infant formula was prepared (4.5 g powdered infant formula dissolved in 30 mL of water that had been boiled and then cooled to 70°C) in sterile glass bottles (250 mL) and capped in accordance with the manufacturer's instructions for use. Eight distinct sample groups were established, each corresponding to an individual holding time. For each group, 100 mL of infant formula was prepared. Samples were collected and analysed at 0 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours and 24 hours, respectively. A range of analytical procedures were conducted on the samples obtained at each designated time point: A comprehensive set of microbiological analysis was conducted to assess the microbial profile of the sample. The analysis encompassed various metrics, including the enumeration of total mesophilic aerobic bacteria (TMAB), yeast and mould, as well as specific taxa such as Lactobacillus, Leuconostoc-Pediococcus, lactic Streptococci, Enterobacteriaceae, and Coliform bacteria. A comprehensive suite of analytical procedures was conducted, encompassing the thiobarbituric acid (TBA) determination, antioxidant activity assessment, pH value measurement and % acidity determination, along with colour analysis and nutritional value assessment (total amino acid, amino acid content, B12, folic acid, cholesterol).

Microbiological analysis

Preparation of samples for microbiological analysis. Under aseptic conditions, 10 ml of the prepared infant formula samples were placed in a sterile sample bag (Stomacher 400, Italy), 90 ml of 0.1% peptone water (Merck, Darmstadt, Germany) was added, and the contents were homogenised in a Stomacher (ISOLAB, Germany) for 2-3 min. Thereafter, a 10-1 dilution was prepared. From this dilution, other dilutions of the sample up to 10-7 were prepared using the same diluent. Inoculations were then made in duplicate using the pour plate method for the enumeration of microorganisms, with the exception of yeast mould count. The smear plate method was utilised for the purpose of enumerating yeast moulds. Following the incubation period, plates containing 30-300 colonies were subjected to evaluation [¹¹].

In the context of microbiological analysis, the total mesophilic aerobic bacteria (TMAB) count was determined by inoculating Plate Count Agar (PCA, Merck, Darmstadt, Germany) medium at 35 °C for a 48-hour incubation period, followed by colony count analysis as outlined in the USDA/FSIS methodology [¹⁰]. The Man-Rogosa-Sharpe (MRS) agar was utilised for the identification of Lactobacillus, Leuconostoc and Pediococcus species; petri dishes were subjected to incubation at 30 ± 1 °C for a period of 3 days [¹²]. M17 Agar was the preferred medium for the enumeration of lactic Streptococci, with samples being incubated at 30 ± 1 °C for a period of 3 days. Coliform bacteria were enumerated on Violet Red Bile Agar (VRB) after incubation at 37 ± 1 °C for 24 hours, and dark red-coloured typical colonies were counted in accordance with ISO 4832:2006 [¹³]. The Violet Red Bile Glucose Agar (VRBG) was utilised for the enumeration of Enterobacteriaceae group bacteria. The incubation was maintained at 37 ± 1 °C for a duration of 24 hours, after which the red colonies were enumerated (ISO 21528-2:2004) [¹⁴]. For the enumeration of yeast and mould, Dichloran Rose Bengal Chloramphenicol (DRBC) Agar medium was utilised, with incubation conducted at a temperature of 25 ± 1 °C for a duration of 5 days. The colonies were then enumerated in accordance with the ISO (2008) guidelines [¹⁵].

Chemical Analyses

Determination of thiobarbituric acid (TBA)

The prepared infant formula samples were subjected to a vortex, after which approximately 3 ml of each sample was transferred to a 50 ml Falcon tube. Six millilitres of 7.5% trichloroacetic acid (TCA) (w/v), 0.1% (w/v) ethylene-diamine-tetra-acetic acid (EDTA) and 0.1% (w/v) propyl 3,4,5-trihydroxy-benzoate (propyl gallate) were added to the mixture, which was then subjected to a centrifugation process at 6000 rpm for a period of 10 minutes. For spectrophotometric determination, 1 ml of the sample and 1 ml of TBA reagent (46 mM in 99% glacial acetic acid) were mixed in a test tube and heated in a boiling water bath for 35 minutes. Following the cooling of the reaction mixture, the spectrophotometer (Thermo Scientific, GENESYS 10S UV-Vis, USA) was utilised to measure the extinction coefficient at a wavelength of 532 nm [¹⁶].

Determination of antioxidant activity

The antioxidant activity was determined by DPPH and ABTS radical scavenging techniques according to the methods recommended by Takım [¹⁷]. The DPPH method was employed to prepare appropriate concentrations of the samples, which were then mixed with a 1 mM DPPH solution. The samples were incubated in the dark for 30 minutes, after which the absorbances were measured at 515 nm. In the ABTS method, the ABTS radical was generated using potassium peroxodisulphate, and a diluted solution was prepared by adjusting the absorbance at 734 nm. The samples were subjected to the previously described solution, following which they were placed into an incubator for a period of 30 minutes. The resulting values of light absorption at a specific wavelength of 765 nanometres were then measured. Antioxidant capacity was calculated using the Trolox equivalent method in both experimental approaches.

Measurement of pH value

The pH values of the samples were determined using a digital pH meter (HANNA HI2002-02). A volume of 10 mL of the sample was collected and meticulously measured at a temperature of 25 °C. The resultant data were then recorded in accordance with the AOAC (1990) standard methodology (¹⁸). On each designated analysis day, the pH meter was firstly calibrated (pH 4, pH 7 and pH 10) prior to the measurement of the samples.

The determination of acidity percentage.

In order to ascertain the titration acidity value of the samples, 25 mL of infant formula was placed into a flask and then 3 drops of phenolphthalein were added. The titration was then conducted using a 0.1 N NaOH solution until a pink colouration was observed. The calculation of titration acidity was conducted in accordance with the following infant formula, with the result expressed as a percentage [¹⁹].

% Acidity=V/m x100

V: Amount of NaOH used in titration (mL)

m: Sample amount (mL)

Nutritional value analyses

Total amino acid amounts on infant formulas

Firstly, hydrolysis was performed on the prepared baby foods. For the hydrolysis process, 2 ml of sample was taken and 18 ml of 0.6 M NaOH was added. Subsequently, the temperature was maintained at 120 °C for a duration of 16 hours. After this process, the samples were subjected to a centrifugation procedure, after which the resulting upper layer was extracted for further analysis. The total amino acid content of the hydrolysed samples was determined by TNBS colorimetry, a method originally proposed by Adler-Nissen [²⁰].

Determination of amino acid profile in infant formula

The free amino acid profile of infant formula samples was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Prior to analysis, the hydrolysed infant formula samples were filtered with an Amicon ultra-filtration device with a cut-off value of 3 kDa to remove protein and other macromolecules. The filtrates obtained were then prepared for analysis according to a commercial kit protocol. In this particular instance, 50 μL of infant formula sample was transferred into the analysis tube, followed by the addition of 700 μL of reagent-1 and 50 μL of stable isotope-labelled internal standard mixture. The mixture was then vortexed for a duration of 5 seconds, after which it was prepared for analysis. The prepared samples were then adjusted in accordance with the manufacturer's (Shimadzu, Kyoto, Japan) instructions and injected into the LC-MS/MS system. The instrument parameters were set as follows: gas temperature 150 °C, gas flow rate 10 L/min, nebuliser pressure 40 psi and capillary voltage +2,000 V. Following the analysis, the concentrations of amino acids in the samples were expressed in μmol/L [²¹].

Determination of B12, folic acid and cholesterol levels in infant formula.

The analysis of vitamin B12, folic acid and cholesterol levels in infant formula samples was conducted using an auto analyser (Roche Cobas 8000, Switzerland) with the aid commercial diagnostic kits (Elecsys, Switzerland). The analyses were performed in accordance with the protocols established by the manufacturer, and the sample preparation and measurement procedures were carried out in accordance with the instructions outlined in the device's user manual. The results obtained were then subjected to quantitative evaluation, with the levels of the relevant parameters in infant formula content being calculated.

Colour analysis

The colour of infant formula samples was measured with a CS-10° 8 mm portable digital digital colourimeter (Tuodapu, Inc., China). On each measurement day, the colourimeter was calibrated with a standard white tile. The L* (brightness), b* (yellow-blue) and a* (red-green) colour coordinates were determined according to the CIELab colour scale [²²]. Measurements were done from 3 different points on the surface of each sample and the results were recorded.

Statistical analyses

The statistical analyses were conducted using IBM SPSS Version 16.00 (SPSS Inc., Chicago, IL, USA) software. The study was performed with three replicates for each group. The data obtained from the various sampling times was analysed using a one-way analysis of variance (One-way ANOVA). Following the analysis of variance, the Duncan multiple comparison test was employed to ascertain the differences between the means of the experimental groups. The results of the microbiological analysis were subjected to logarithm transformation for further analysis. The results were presented as mean ± standard deviation.

RESULTS

Results of microbiological analysis

The study revealed that, upon conducting microbiological analyses of infant formula samples over various storage periods, only negligible alterations were detected in TMAB and Lactobacillus streptococci counts over time. However, these alterations were not statistically significant (Table 2). The TMAB count was measured as 4.32 ± 0.22 log cfu/mL immediately after the preparation of the food (at the 0th minute) and increased to 4.95 ± 0.06 log cfu/mL at the end of the 24th hour. It was observed that the number of TMABs increased slightly, especially in the 6th and 24th hour samples, but remained stable in general. This finding suggests that the microbial load in the infant formula remained at a low level. The count of Lactobacillus-Leuconostoc-Pediococcus remained below 1 log cfu/mL. However, the lactic streptococcus count increased from 4.48 ± 0.22 log cfu/mL at the beginning to 4.82 ± 0.32 log cfu/mL at the end of the 24th hour. Coliform bacteria, Enterobacteriaceae and yeast-mould group microorganisms remained <1 log cfu/mL during all sampling periods (Table 2).

Table 2
Results of microbiological analysis (log CFU/mL)

Time	TMAB count (Mean±SD)	Lactic streptococcus count (Mean±SD)	Lactobacillus Leuconostoc count (Mean±SD))	Koliform count (Mean±SD)	Enterobacteriaceae count (Mean±SD)	Yeast and mold count (Mean±SD)
0	4.32±0.22	4.48±0.22	<1	<1	<1	<1
30 min	4.34±0.49	4.56±0.47	<1	<1	<1	<1
1 h	4.57±0.38	4.55±0.57	<1	<1	<1	<1
2 h	4.71±0.33	4.37±0.32	<1	<1	<1	<1
4 h	4.15±0.21	4.43±0.36	<1	<1	<1	<1
6 h	4.98±0.25	4.36±0.47	<1	<1	<1	<1
12 h	4.86±0.12	4.58±0.15	<1	<1	<1	<1
24 h	4.95±0.06	4.82±0.32	<1	<1	<1	<1

Results of chemical analysis

The pH and % acidity values of infant formula during storage are given in Figure 1. Statistically significant differences were not observed in the pH and acidity values of infant formula during storage (P > 0.05).

Figure 1
The pH and % acidity value of infant formula during storage

Statistical analysis revealed no statistically significant difference (P > 0.05) in terms of ABTS and DPPH radical scavenging activities, which are important parameters in the determination of antioxidant capacity (Table 3). Furthermore, no statistically significant difference (P > 0.05) was observed in terms of TBARS values, which indicate the oxidation level in infant formula during storage (Table 3).

Table 3
ABTS, DPPH, TBARS values of infant formula during storage

Time	ABTS values (% Trolox equivalent)(Mean±SD)		DPPH values (% Trolox equivalent)(Mean±SD)	TBARS value (ug MDA g-1 infant formula) (Mean±SD)
0	27.10±4.91	0.82±0.21		0.69±0.17
30 min	28.49±2.16	0.80±0.16		0.60±0.19
1 h	32.89±4.98	0.73±0.26		0.70±0.15
2 h	31.27±4.65	0.87±0.10		0.52±0.20
4 h	32.19±4.51	0.96±0.26		0.83±0.10
6 h	33.82±8.22	0.71±0.21		0.53±0.20
12 h	26.17±6.50	0.79±0.19		0.64±0.22
24 h	25.71±7.52	0.83±0.10		0.68±0.26

Nutritional values

Total amino acid values and amino acid contents of infant formula during storage are given in Table 4. There was no statistically significant difference (P > 0.05) in total amino acid content of infant formula during storage.

Table 4
Total amino acid values of infant formula during storage

Time	Total amino acids (mg leucine eq/g infant formula) (Mean±SD)
0	210.04±12.72
30 min	243.86±18.18
1 h	232.77±26.81
2 h	231.13±28.18
4 h	270.52±18.18
6 h	269.31±25.27
12 h	290.51±18.86
24 h	264.22±32.27

It was observed that the infant formula exhibited a high amino acid content. Despite the total amount of amino acids remaining constant, it has been observed that certain amino acids decrease and others increase 24 hours after the preparation of infant formula (Table 5). A substantial increase or decrease is evident, particularly in certain amino acids. While citrulline, histidine and ethanolamine amino acids demonstrated a significant decrease, alanine, aspartic acid, alloisoleucine, glutamic acid, glycine, leucine, proline and hydroxyproline, valine amino acids exhibited a significant increase. As demonstrated in Table 4, an increase in the 24th hour amino acid results compared to the 0th hour was observed. However, this increase was not deemed to be statistically significant.

Table 5
Amino acid contents of infant formula by LC-MS/MS at different times

Amino Acid	0. Time (ng/mL)	24. Time (ng/mL)
Alanine	439.379	594.635
Arginine	0.809	0.658
Aspartic Acid	353.783	502.161
Citrulline	1.039	0.332
Glutamic Acid	0.576	870.033
Glycine	554.087	767.524
Histidine	0.643	0.416
Leucine	588.621	790.365
Isoleucine	338.938	423.093
Alloisoleucine	1.222	1.614
Lysine	214.251	264.064
Methionine	87.165	118.337
Ornithine	134.05	187.243
Phenylalanine	338.225	400.959
Proline	426.512	595.378
Serine	52.996	92.756
Threonine	4.723	6.367
Tryptophan	41.126	45.045
Tyrosine	243.16	237.184
Valine	332.634	430.747
Anserine	47.622	48.606
Alphaaminobutyric Acid	14.63	17.732
Betaaminoisobutyric Acid	22.687	19.377
Gammaminobutyric Acid	104.433	113.063
Beta-Alanine	12.974	11.179
Sarcosine	48.38	47.937
Thiaproline	26.114	26.998
Hydroxyproline	597.839	616.78
Cystine	0.514	0.174
Ethanolamine	129.005	26.587

As demonstrated in Table 6, the levels of vitamin B12, folic acid and cholesterol in infant formula during the storage period are presented. No statistically significant differences (P > 0.05) were observed for vitamin B12 and folic acid levels during storage. However, cholesterol levels showed statistically significant differences between certain time points (P < 0.05), as indicated in Table 6, although these variations were relatively small.

Table 6
Levels of B12, folic acid, and cholesterol in infant formula during storage

Time	B12 (Mean±SD)) (μg/100mL)	Folik asit (Mean±SD) (μg/100mL)	Cholesterol (Mean±SD) (μg/100mL)
0	0.194±0.001	20.00±0.05	7.1±0.15b
30 min	0.195±0.003	20.00±0.03	6.6±0.17c
1 h	0.197±0.001	20.00 ±0.01	7.6±0.14b
2 h	0.200±0.006	20.00±0.01	8.00±0.21b
4 h	0.197±0.004	20.00±0.04	9.4±0.28ab
6 h	0.198±0.005	20.00±0.02	10.4±0.28a
12 h	0.200±0.007	20.00±0.01	8.9±0.14ab
24 h	0.200±0.005	20.00±0.016	10.6±0.21a

Results of the colour analysis

The colour changes of infant formula during different storage periods are presented in Figure 2. Subsequent to statistical evaluations, no statistically significant changes were observed in L* (brightness) and a* (red-green axis) colour parameters during the storage period (P > 0.05). However, statistically significant fluctuations were detected in b* (yellow-blue axis) value (P < 0.05).

Figure 2
Colour values of infant formula during storage

DISCUSSION

Given that these bacteria may be probiotic bacteria naturally present in the infant formula, a slight increase is expected and does not pose a risk in terms of food safety. It is recommended that the total microbial count (TMAC) in powdered formulae for infants and young children should be less than 5 × 10² CFU/g, according to the Code of Hygienic Practice for Powdered Formulae for Infants and Young Children [²³] standard published by Codex Alimentarius. However, it is stated that these limits are not applicable to products containing live microorganisms (probiotics) and it is expected to be higher [²³]. No coliform bacteria, Enterobacteriaceae, or yeast-mold were detected. Indeed, the regulations pertaining to infant formula explicitly stipulate that the presence of Enterobacteriaceae and pathogenic bacteria is prohibited [²³-²⁵]. Consequently, it was determined that infant formula remained within the prescribed limits of microbiological quality criteria for a maximum period of 24 hours following its preparation and storage. Furthermore, it was found to be in accordance with the stipulated regulations and did not exhibit any indications of spoilage

In the microbiological stability assessment, the presence of general microbial groups related to post-preparation safety and stability was monitored. These groups included TMAB and lactic streptococci. The fact that TMAB counts remained within the range of 4.32-4.95 log CFU/mL during the 24-hour storage period indicates that the overall microbial load in the formulation remained stable. However, the lack of strain-specific viability or selective isolation of the declared Bifidobacterium animalis subsp. lactis component limits the ability to draw direct inferences about the behaviour of the probiotic component over time. Evaluating probiotic functions and potential health effects requires strain-level identification and viability analyses. Therefore, the microbiological findings should be considered in the context of the product's microbiological safety and overall microbial stability after preparation rather than its probiotic efficacy. The relatively high initial TMAB level (4.32 log CFU/mL) observed immediately after preparation may be attributed to the microbiological characteristics of powdered infant formula itself. Powdered infant formulas are not sterile products and may contain background microbiota originating from raw materials, processing environments, or added functional components such as probiotic cultures [²⁶]. In the present study, the formula contained Bifidobacterium animalis subsp. lactis, which may have contributed to the detected microbial load.

A substantial increase in pH and % acidity values is indicative of a significant increase in the number of bacteria. However, as demonstrated in Table 1, there was no significant increase in the number of TMAB and lactic streptococci bacteria. Consequently, it is unsurprising that pH and % acidity remained stable. Conversely, the absence of fluctuations in pH values may have ensured the stability of bacterial numbers. Indeed, bacteria are affected by various factors while growing in the food environment, and pH value, as one of these factors, is known to affect bacterial growth rate, type and proliferation process [²⁷].

There has been no significant change in the antioxidant capacity of the infant formula during storage. This result suggests that the antioxidant substances (alpha-tocopherol and L-ascorbylpalmitate) present in the infant formula are capable of preserving their stability during storage. This is indicative of the infant formula maintaining its nutritional value and quality. Indeed, Lugonja and coauthors [²⁸] reported that antioxidants in infant formula contribute to its nutritional value and provide protection against oxidative damage, thus providing a special insight into the quality of infant formula.

Fluctuations have been observed in the amino acid content of infant formula. This state of affairs was not unexpected. It is important to note that the presence of bacteria in infant formula has the potential to impact the amino acid content. Bacteria have been observed to utilise amino acids present in infant formula for the purpose of growth and multiplication. During this process, some amino acids may be utilised or metabolised by the bacteria, leading to fluctuations in the amount of amino acids within infant formula [²⁹, ³⁰]. Consequently, fluctuations in the quantity of amino acids present in infant formula may be attributable to a multitude of biochemical processes. These processes encompass protein digestion, bacterial metabolism, the capacity of microorganisms to utilise amino acids, and chemical transformations. These alterations are closely related to the composition of infant formula and the microflora conditions. Indeed, microbiological cultivation has been shown to indicate that the amount of bacteria in infant formula is approximately 4 log. These bacteria can be regarded as the primary causative agents of this transformation. In particular a remarkable increase was observed in glutamic acid levels between the initial and 24-hour measurements. This change may be associated with the release of free glutamic acid from protein structures during storage and hydrolysis processes, as well as possible analytical variability inherent to LC-MS/MS quantification of very low initial concentrations. Similar fluctuations in individual amino acids have been reported in protein-rich food matrices during storage and enzymatic or microbial transformations. Similar fluctuations in individual amino acids have been reported in protein-rich food matrices during storage and enzymatic or microbial transformations [³¹,³²].

Fluctuations in cholesterol levels during storage are presumed to be due to microorganisms present in infant formula. While it is evident that cholesterol plays a significant role in bacterial metabolism, these organisms are not typically regarded as cholesterol metabolising or utilising entities. Instead, they are predominantly associated with other lipids and sterols [³³]. However, the literature contains intriguing findings regarding the presence of bacterial enzymes involved in cholesterol metabolism [³⁴,³⁵].

The L* and a* values remained constant during the storage period, indicating that the overall brightness and red-green colour balance of the product was maintained. Conversely, an increase in b* value is indicative of a shift in the yellow hues of the foodstuffs. This was particularly evident at 24 hours, resulting in a substantial shift in the yellow-blue balance of the product. The potential causes of the increase in b* include microbial activity, the Maillard reaction, oxidative processes and temperature changes. The predominance of yellowish tones in the colour of infant formula can be attributed to the combined effect of these factors. The Maillard reaction, in particular, in conjunction with reactions between carbohydrates and proteins, has been observed to result in colour changes from yellow to brown, which is sensitive to storage time and temperature. An increase in temperature has been shown to cause faster degradation of proteins and sugars [³⁶-³⁸]. This, in turn, can have an effect on colour parameters. Furthermore, oxidation of riboflavin (vitamin B2) in infant formula content has been shown to trigger yellowish pigment formation. Indeed, as Liu and coauthors [³⁹] have reported, vitamin B2 has been shown to produce a yellow colour.

This study evaluated a specific commercial infant formula containing Bifidobacterium animalis subsp. lactis. It is important to note that the stability of nutritional components and microbial viability can vary significantly depending on the product in question, the processing techniques used, and the types of probiotic strain present. Therefore, the findings of this study should be interpreted in the context of this specific formulation. Generalisations should not be made directly to other infant formulas with different compositions, production conditions or probiotic strains. Our results support the need for critical time-temperature control, particularly with regard to the post-preparation stability and safety of this specific product. Future studies could expand the knowledge base in this area by comparing different formula types and probiotic strains.

CONCLUSION

This study comprehensively evaluated the changes in microbiological, chemical and nutritional parameters of infant formula over a 24-hour period following preparation. The results demonstrated that the levels of TMAB, coliform and yeast-mould were within the limits of food legislation and microbiologically safe. No significant changes were observed in the chemical parameters (pH, TBA, antioxidant capacity) and vitamin levels (B12, folic acid). However, fluctuations were observed in the amino acid profile, cholesterol content and colour components.

In accordance with the data obtained, it is recommended that infant formulas should be consumed within 2-6 hours after preparation, as this is considered to be the safest interval. In order to ensure the integrity of the products, it is imperative that they are stored under cold chain conditions, with a temperature of below 5 °C, for periods exceeding six hours. This precautionary measure is crucial to prevent potential chemical spoilage and quality degradation. The findings of this study provide scientific substantiation for the World Health Organisation's (WHO, 2007) recommendation that infant formulas can be stored for up to 24 hours under suitable cold chain conditions (≤5 °C). However, given the biochemical changes observed in the formula content over time, it is recommended that the products are consumed as soon as possible after preparation, especially in order to maintain nutritional and sensory integrity.

In conclusion, this study provides evidence specific to infant formula containing Bifidobacterium lactis regarding its microbiological and chemical stability after preparation. While the results provide valuable insights into consumer usage and storage practices, they should not be generalised to other infant formulas with different formulations, probiotic components or production processes. It is therefore important for parents to reconsider their infant formula usage habits in light of the scientific data, and for manufacturers to provide more detailed information on post-preparation storage and consumption periods in their usage instructions. Future studies adopting a comparative approach based on detailed, product-specific stability analyses covering different probiotic strains and infant formula compositions will contribute to a more comprehensive assessment of shelf life and usage safety in infant formulas.

• Funding:
  This study was supported by the Scientific Research Unit of Harran University under project number 23017
• Institutional Review Board Statement: Not applicable.
• Informed Consent Statement: Not applicable.
• Use of Generative Artificial Intelligence: The authors declare that generative artificial intelligence (AI) or AI-assisted tools were used under full human supervision. The tool(s) and version(s) used, and their purpose, are described here: DeepL AI was used to assist with language refinement and editing of the manuscript, and Napkin AI was used to generate part of the graphical abstract. No confidential or sensitive data were uploaded to such tool(s), and all AI-assisted content was checked, corrected and approved by the authors, who take full responsibility for the integrity and originality of the manuscript.

Acknowledgments:

We express our gratitude to the Scientific Research Unit of Harran University.

Data Availability Statement:

Research data are only available upon request for corresponding author.

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• Editor-in-Chief:
  Paulo Vitor Farago

• Associate Editor:
  Marcos Pileggi

Publication Dates

• Publication in this collection
  17 Aug 2026
• Date of issue
  2026

History

• Received
  06 Jan 2026
• Accepted
  21 May 2026