The Role of Probiotics in Dairy Foods and Strategies to Evaluate Their Functional Modifications

ABSTRACT

Probiotics are widely used in functional foods. However, little has been discussed about its potential application in food fortification. Therefore, researchers have studied the possible products that can come out through the presence and activity of probiotics under certain conditions and examined the increase in their nutritional value. In this review, we discuss the most recent data about the role of probiotics in dairy foods in incorporating minerals into amino acids, their ability to synthesize B-complex vitamins and peptides, and the methodological strategies and emerging technologies for quantifying each of these modifications.

KEYWORDS:

• Probiotics
• fortification
• biosynthesis
• minerals
• vitamins
• peptides

Introduction

Food plays an essential role in health since it is the source of nutrition, provides energy, and maintains the proper functioning of physiological activities. Food is also a carrier that provides probiotics, which are mainly identified as benefiting intestinal health.^[1] Foods that potentially carry these probiotics are dairy products like milk, cheese, yogurt, and even ice cream. Dairy foods are a suitable medium for probiotic bacteria since it allows them to be in an optimal physical environment (temperature, pH, time) with an adequate substrate (chemical composition).^[2] In addition, these foods buffer probiotics when they pass through the intestinal tract; therefore, they can regulate colonization and provide other bioactive compounds that enhance the functionality of probiotics.^[3]

After passing the biological barriers (pH, digestive enzymes) of the gastrointestinal tract, probiotics take action and impact human health.^[1] Some biological activities include antimicrobial, antimutagenic, antigenotoxic, improvement of lactose tolerance, cancer prevention, and immunomodulation.^[1,4,5]

Probiotics commonly used in dairy products belong to the genus Lactobacillus and Bifidobacterium. The microbial interaction with the matrix depends on when they were added to the product and whether they were added during fermentation or afterward. If they are added at the end, the interactions would be less because their activity can occur before or after cooling. If the active participation of bacteria during fermentation is required, aspects such as the chemical composition of media and the starters should be considered. Since there is an antagonistic activity between probiotics and starter cultures, they can result in a retarded growth or even inhibit one of the bacterial components; the production of lactic acid and the reduction of the pH in the fermentation must be monitored could avoid this issue.^[2,6]

Bacteria can also obtain metals, vitamins, and peptides through their biosynthesis, which has made bacteria a potential tool to fortify food. Unfortunately, several countries have not reached the minimum daily dose of essential micronutrients. For example, in South-East Asia countries such as Vietnam and Cambodia, 50% of children have zinc intake deficiency, while Indonesia and Thailand have 30 and 50%, respectively.^[7] These data are alarming because inadequate amounts of micronutrients weaken the immune system and make the population vulnerable to infections and diseases.^[8] In these cases, producing lactic probiotic-fortified products could help solve this problem.

Previously, parameters such as the type of bacteria culture, temperature, matrix or substrate, time, and pH have been studied to optimize fermentation in lactic products.^[2] For all of the above, recent studies have focused on identifying probiotic bacteria from different sources to explore their potential ability to biotransform minerals, production of vitamins and peptides, and the optimization of processes during the production of dairy products to achieve the recommended doses of micronutrients for consumption. This review will show the most relevant studies using lactic acid probiotics to fortify dairy products with minerals, vitamins, and peptides. The data from these reports provide a broad context on the subject and show the most recent strategies that can help identify the opportunity areas for the development of products with a more significant benefit to the health of consumers in countries with macro and micronutrient deficiencies.^[7,8]

The role of probiotics in the biotransformation of minerals

Minerals are part of the nutrients acquired through foods; these can be found in inorganic and organic compounds; however, they are better metabolized organically.^[9] Soil is the primary source of minerals, which provides those to plants, they provide them to animals, and humans obtain them through the consumption of plants and animals.

Minerals are in body tissues, fluids, and much of the skeletal structure.^[10] These have an essential role in human health, and although it is necessary to consume them daily, it requires a small amount to provide all the benefits. The average daily dose for secondary and micro minerals consumption is between 400 to 1500 mg and 45 μg to 11 mg, respectively.^[11] Table 1 shows the amount of minerals that should be consumed daily; their consumption is important since vitamins cannot be absorbed or perform their function without having a certain amount of minerals. For normal nutrition, twelve mineral elements are essential (among them: magnesium, zinc, calcium, manganese, and selenium), which are more important than vitamins, since by not having these, the body can make use of minerals, but if minerals are not available, vitamins are not helpful, and they do not fulfill their function.^[11,18] In addition, minerals transmit nerve impulses and are part of enzymatic structures.^[19]

Table 1. Recommended daily dose of each mineral.

Mineral	Daily dose	Ref.
Selenium	200–400 µg	Haug et al.^[Citation12] and Yang et al.^[Citation13]
Zinc	10–14 mg	Prasad^[Citation14]
Magnesium	320–420 mg	Al Alawi et al.^[Citation15]
Iron	10–20 mg	Alleyne et al.^[Citation16] and Paganini & Zimmermann^[Citation17]

Minerals are present in dairy products in salts or ions and as part of organic molecules. Some minerals can be naturally incorporated as calcium, or they can also be added to a product.^[20] The process of adding nutrients and non-nutritive bioactive components to a product is called fortification.^[20] Habitually, minerals and trace elements in milk are present as inorganic ions, and these arrange a complex or compound with peptides, carbohydrates, lipids, and small molecules.^[10] The mechanism of mineral incorporation has not been well understood, but there are three proposals to explain their integration, including biotransformation, biosorption, and bioaccumulation.

To incorporate minerals in dairy food products, the biotransformation of a mineral salt into a peptide is usual, and it is one of the mechanisms proposed. Lactic acid bacteria (LAB) are used to incorporate minerals into amino acids like cysteine and methionine, forming the basic blocks or enzymes like glutathione peroxidase (Figure 1).^[21,22]

Figure 1. General incorporation of selenium into a protein by Busto et al.^[21] .

This process is carried out in yogurts and other fermented dairy products, specifically for the addition of an essential mineral in human nutrition.^[23] This process consists of adding bacteria cultures to the milk and adding a mineral salt, leaving it to ferment at 37°C. One of the essential minerals in human nutrition is selenite. However, the mechanism of reduction of selenite which is shown in Figure 2, has not yet been understood since, after being reduced by thioredoxin reductases (TrxR), undergoes post-translation, in which the metal-amino acid is synthesized in tRNA from the reduced selenium residue and then make a selenium replacement.^[22,25]

Figure 2. Mechanism of biosynthesis of selenoproteins by bacteria by Metanis & Hilvert^[27].

The other two mechanisms mentioned by Mrvčić et al.^[26] for incorporating the mineral through probiotic bacteria can be seen in Figure 3: biosorption and the bioaccumulation of metal ions. The first one consists of a passive process in which the ion is not metabolized and binds to the cell wall. Moreover, the second metabolizes the ion by passing it through the cell membrane. As a result, it accumulates inside the cell, sometimes with the help of transporter proteins. The mineral can then undergo a reduction, allowing the possibility of exiting the membrane again.^[26]

Figure 3. Mechanism of metal ions binding process by microorganisms by Mrvčić et al.^[26] .

Some studies report the biotransformation of minerals into amino acids, which until now have been carried out with selenium. For example, in Table 2, most studies on selenium in dairy products use Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus bacteria that perform lactic fermentation in yogurt, having mineral accumulation greater than 40%, reaching even 95% of minerals incorporated into amino acids. In addition, other bacteria have been explored to bioaccumulate selenium in dairy derivatives, like Lactobacillus reuteri, Lactobacillus brevis, Enterococcus faecium, Lactobacillus casei, Bifidobacterium, Lactobacillus acidophilus, Lactobacillus helveticus and Fructobacillus tropaeola.^{[28–33,23–24]} Probiotics that also have the potential to biotransform the mineral into an amino acid, since organic species of selenium have been reported from these cultures. However, the study of the selenium integration into amino acids has only been reported for bacteria L. reuteri, L. bulgaricus, S. thermophilus, and Enterococcus faecion, with MeSeCys, SeCys₂, and SeMet as the potential synthesized products. In addition, time could be a significant parameter; a study with bacteria L. reuteri showed less accumulation after fermentation for 6 to 12 hours compared to the other studies that reported their accumulation percentage; the bacteria were allowed to act for more than 12 hours.^[28–32] Therefore, with the results reported with the bacteria mentioned above, it can be said that time proportionally influences their accumulation percentage, regardless of the type of bacteria used. Regarding other bacteria, their ability to biotransform minerals into amino acids and their accumulation remains to be explored to determine which bacterial culture is best for this activity.

Table 2. Incorporation of inorganic salts in probiotic bacteria for mineral supplementation in dairy products.

Mineral	Lactic acid bacteria	Inoculum amount (%)	Matrix	Mineral added	Added mineral concentration	Organic specie	pH	Time (h)	% mineral accumulated	Mineral concentration in the final product	Ref.
Selenium	Lactobacillus reuteri CRL 1101	1	-	Na2SeO3	5 mg/L	SeCys SeMet	6 – 6.5	6 24	58.1% 78.6%	NR	Pescuma et al.^[Citation28]
	Lactobacillus delbrueckii subsp. bulgaricus	1	-	Chitosan-SeNPs, Ethoxylated-SeNPs Hydroxyethyl cellulose-SeNP	1- 10 µg /mL	SeCys2 SeMet SeMetox	NR	24, 48, 72	95% 70% 90%	NR	Palomo-Siguero & Madrid ^[Citation29]
	Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus	NR	Yogurt	Na2SeO3	14 µg/mL	SeCys2 MeSeCys	NR	12, 24, 36	90%	NR	Alzate et al.^[Citation30]
	Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus	NR	Yogurt	Na2SeO3	150 µg/mL	SeCys2	NR	24	40%	NR	Palomo et al.^[Citation24]
	Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus brevis	NR	Yogurt Kefir	Na2SeO3	1- 5 mM	NR	6.8	36	NR	1.28 μg/g 0.02 μg/g 0.02 μg/g	Deng et al.^[Citation31]
	Streptococcus thermophilus Enterococcus faecium	NR	Yogurt	Na2SeO3	50 mg/L	SeMet  MeSeCys	7.32 to 9.9	24-48	93–96%	7348 μg/g 6491 μg/g	Krausova et al.^[Citation32]
	Lactobacillus casei, Streptococcus thermophilus, Bifidobacterium BB-12, Lactobacillus acidophilus (LA-5), Lactobacillus helveticus (LH-B02)	NR	Yogurt	NaHSeO3	200 mg/L	NR	NR	36-48	NR	0.2- 2.25 mg/L	Eszenyi et al.^[Citation33]
	Lactobacillus brevis Fructobacillus tropaeoli	2	Fruit juice-milk	Na2SeO3	5 mg/L	NR	10	24	NR	100 ± 14.14  μg/L Average: 79 μg/L	Martínez et al.^[Citation23]
	Lactobacillus brevis	1	Sheep’s yogurt	Na2SeO3	1.26 mM	NR	NR	NR	NR	160 mg/L of SeO3^2− ions	Shakibaie et al.^[Citation34]
	S. thermophilus	6.0	-	Na2SeO3	80 μg/mL	NR	6.37	24	97.05	NR	Yang et al ^[Citation35]
	S. bulgaricus	6.73	-	Na2SeO3	80 μg/mL	NR	5.96	24	94.34	NR	Yang et al ^[Citation35]
	P. acidilactici	1	-	Na2SeO3	4 μg/mL	SeMet SeCys MeSeCys	6.5	24	—	1.89 mg/g	Kousha et al ^[Citation36]
	E. durans LAB18s	—	-	Na2SeO3	15 μg/mL	NR	7	24	—	2 600 μg/g	Pieniz et al ^[Citation37]
	L. rhamnosus ATCC 53103	1	-	Na2SeO3	10 μg/mL	NR	6.5	24	65.35	—	Xu et al ^[Citation38]
	L. plantarum P2	1	-	Na2SeO3	4 μg/mL	NR	—	24	52.73	—	Chen et al ^[Citation39]
	S. thermophilus CCDM 144	1	-	Na2SeO3	50 μg/mL	SeMet SeCys MeSeCys	—	24	—	7.3 mg/g	Krausova et al ^[Citation32]
	E. faecium CCDM 922A	1	-	Na2SeO3	50 μg/mL	SeMet SeCys MeSeCys	—	24	—	6.5 mg/g	Krausova et al ^[Citation32]
	Lactic acid bacteria	2	-	Na2SeO3	5 μg/mL	SeMet SeCys MeSeCys	—	24	80	—	Martinez et al ^[Citation40]
	E. faecalis QHAE1	1	-	Na2SeO3	600 μg/mL	NR	—	20	—	112.64 mg/g DW	Morales Estrada et al ^[Citation41]
	B. animalis ssp. lactis BB12	1	-	Na2SeO3	1 000 μg/mL	NR	6.8	24	—	60 mg/g DW	Pusztahelyi et al ^[Citation42]
Zinc	Lactobacillus rhamnosus B 442	NR	Ice cream	ZnSO4 x 7H2O	500 μg Zn^2+/mL	NR	5.9 - 6.4	16	NR	NR	Kozłowicz et al.^[Citation43]
	Lactobacillus rhamnosus B 442	NR	Ice cream	ZnSO4 x 7H2O	500 μg Zn^2+/mL	NR	NR	NR	NR	0.962 mg/L 1.81 mg/L	Pankiewicz et al.^[Citation44]
	Bifidobacterium longum CCFM1195	2	-	ZnSO4	200 mg/L	NR	6.5	18	NR	1.7 mg/g	Han et al.^[Citation45]
	L. acidophilus WC 0203	10	-	ZnSO4	1614.7 µg/mL	NR	5.0	48	NR	8.5 mg/g	Leonardi et al. ^[Citation46]
Magnesium	Lactobacillus rhamnosus B 442, Lactobacillus rhamnosus 1937, Lactococcus lactis JBB 500	NR	Ice cream	MgCl2 × 6 H2O	400 μg Mg^2+/mL	NR	NR	48	NR	4.28 mg Mg^2+/g d.m. for L. rhamnosus B 442 1.97 mg Mg^2+/g d.m. for L. rhamnosus 1937  1.86 mg Mg^2+/g d.m. for L. lactis JBB 500	Góral et al.^[Citation47]
	Bifidobacterium animalis ssp. lactis Bb-12	10	Fermented goat’s milk	C6H10MgO6 × H2O  C6H6MgO7	30 mg/100 g	NR	6.5	10	NR	30 mg of magnesium (in 100 g of milk)	Znamirowska et al.^[Citation48]
Iron	Lactobacillus rhamnosus CECT 278, Lactobacillus plantarum 3O9, Bifidobacterium bifidum CECT 870, Bifidobacterium longum CECT 4551, Streptococcus thermophilus CECT 986, Lactobacillus delbrueckii subs. bulgaricus CECT	NR	Almond milk fermented	FeCl3	50 µmol/L	NR	4.4 – 4.6	NR	NR	NR	Bernat et al.^[Citation49]

Only the first four studies in Table 2 depict the incorporation of this mineral into amino acids. The first two examples are L. reuteri and L. d. bulgaricus bacteria were studied without being incorporated into a food, obtaining selenocysteine and selenomethionine as organic species of selenium.^[28,29] It was also observed that L. bulgaricus obtained a higher percentage of accumulated selenium as organic derivatives (seleno-amino acids: selenocysteine and selenomethionine). The following two yogurt studies using the common L. bulgaricus and S. thermophilus were different in the species of selenium since one at slightly higher temperatures (37< T °C < 42) showed selenocysteine and methyl selenium cysteine with a higher accumulation percentage.^[30,24] This can be justified because these bacteria are thermophilic, indicating that at optimal temperatures of 40 and 45°C for Streptococcus thermophilus and Lactobacillus bulgaricus, respectively, they show higher growth and therefore a better fermentation. Therefore, higher biotransformation of selenium can be performed.^[2]

The administered concentrations of sodium selenite in the cultures range from 1–150 µg/mL. However, according to Yang et al.^[13] a dose greater than 80 µg/mL affected cell growth. On the other hand, in another study, it was concluded that the growth of bacteria is not affected by the addition of sodium selenite at concentrations of 50 mg/L. Likewise, it was shown that parameters such as pH and lactic acid production were not affected by the added concentrations.^[32] Therefore, the effect that large amounts of the mineral can have is still under study. One point to check in these studies is to find out what concentration of mineral salt to add since a high concentration of selenium is required to meet the recommended daily dose. Namely, the final product must contain an acceptable daily dose for human ingestion of fermented milk. In the case of selenium, the daily intake recommendation is 200–400 µg (Table 1).

pH effect

There are reports on the effect of pH on Se enrichment. During fermentation, the pH decreases because of the production of lactic acid by LAB strains, it is known that the accumulation of hydrogen ions can affect the uptake of inorganic Se on the cell surface, reducing the capacity for Se enrichment; studies have found that acidic and alkaline conditions can affect Se enrichment.^[35,38,50]

Other studies have explored minerals such as zinc in dairy foods such as ice cream. For this treatment, the authors used Lactobacillus rhamnosus B 442, adding zinc ions as ZnSO₄ · 7 H₂O with a pulsed electric field (PEF) at pH of 5.97 to 6.41.^[43,44] PEF consists of introducing short electrical pulses to the cell to disrupt the membrane cell, increasing porosity to facilitate effective ion permeation.^[51] According to Pankiewicz et al.^[44] the pH is not affected by the addition of zinc ions and the application of PEF, but it can be affected by fermentation; a fermented product is around pH 4.5, and an unfermented product has a pH of 6.3, emphasizing that at high pH, probiotic bacteria survive longer after going through freezing and aeration processes.^[52] This work with PEF technology reported an accumulation in the range of 0.962 mg/L to 1.81 mg/L, which is still considered a low dose since ingestion between 10–14 mg/day is recommended (Table 1).^[16,44]

Adding a suitable concentration of minerals

Undoubtedly one of the factors that greatly affects mineral enrichment is the concentration salt used. Na₂SeO₃ is the most widely used inorganic species in Se enrichment, and it has been shown that the initial concentration can affect biomass formation.

Some LAB and Bifidobacteria strains may have a higher tolerance to these species, such as B. animalis ssp lactis BB12 with a growth capacity at extremely high concentrations of NaHSeO₃ (1000 mg/L).^[42] However, in other bacterial species the tolerance to these inorganic species is very low. Consequently, the effect on biomass formation is one of the keys that impacts mineral enrichment.

In a study for supplementing minerals to ice cream, the magnesium salt was studied using the bacteria Lactobacillus rhamnosus B442, Lactobacillus rhamnosus 1937, Lactococcus lactis JBB 500, obtaining an accumulation of 1.86–4.28 mg Mg/g. Although the mineral concentrations are also low for a daily dose (Table 1), it is possible not to be strict with the requirement for a product that is not for daily consumption.^[47] However, PEF helped increase the concentration of magnesium in ice cream by 30–50% compared to the control samples. The concentration was from 23.02 mg/100 g of ice cream to 31.29 mg/100 g, parallel to the control samples that contained a magnesium concentration of 18.08 mg/100 g of ice cream to 19.26 mg/100 g. It was also seen in another study of the implementation of magnesium with fermented goat milk using Bifidobacterium animalis ssp. lactis Bb-12 and administering C₆H₁₀MgO₆ × H₂O and C₆H₆MgO₇, a concentration of 30 mg of magnesium (per 100 g of milk) were reached with a pH of 6.3–6.5, emphasizing that it was not drastically affected by the addition of magnesium citrate and magnesium pidolate, as well as magnesium citrate, promoted better bacterial growth. However, the same study indicates that Mg-fortification tends to give a bad taste and a slightly bad smell, so it is suggested to study other compounds that supplement magnesium in a way that does not alter the taste of consumers.^[48]

Inoculum amount

One of the factors that influence the ability to enrich Se in LAB is the amount of initial inoculum during bioaccumulation. When there is an excess of inoculum in a microbial culture, the necessary nutrients during fermentation are rapidly depleted. In the absence of nutrients, microorganisms compete, resulting in a significant reduction in biomass formation, which negatively impacts the bioaccumulation and Se enrichment capacity. Some researchers have managed to optimize the amount of inoculum, achieving an increase in Se enrichment.^[35] The amount of inoculum used in these studies varies from 1% to 6.73%, as shown in Table 2.

In the case of Zn enrichment, some reports suggest that using a 10% inoculum it is possible to reach 8.5 mg/g; the recommended amount for daily intake of this mineral.^[46]

Inorganic vs. organic substrate

In studies using both inorganic and organic magnesium compounds pH was not affected, and magnesium concentration increased.^[47,48] However, comparing the works of Góral et al.^[47] and Znamirowska et al.^[48] it can be seen that a concentration of 30 mg/100 g of milk sample was reached using organic compounds. In contrast, using inorganic salts, its concentration ranged from 23–31 mg/100 g, which means that insoluble salts are less absorbable. Nevertheless, in the study by Znamirowska et al.^[48] there were problems with the odor and taste of the final product. Knowing that this sample was fermented milk, the use of organic magnesium compounds for ice creams should be explored since, in this process, freezing and aeration could improve the taste and obtain a less varied magnesium concentration.

The role of probiotics in the enrichment with vitamins

Some studies have shown that probiotic bacteria have the potential to synthesize vitamins, specifically from the B complex, through fermentation. For example, riboflavin is one of the vitamins that chemical strategies have synthesized. However, LAB can ferment and biosynthesize this vitamin in situ, which gives them the character to act as a vitamin supplier to human hosts.^[53]

The biosynthesis of riboflavin in bacteria (Figure 4) is like forming alternative RNA structures, including the mononucleotide RFN, which encodes for other biosynthesis and transport proteins. Then the synthesis proceeds with various catalytic enzymatic activities of guanosine triphosphate (GTP) and ribulose-5-phosphate, which are encoded by four genes (ribG, ribB, ribA, and ribH). At the end, synthase controls the last step, which encodes by the gene operon ribE.^[53,54]

Figure 4. Mechanism of biosynthesis of riboflavin (vitamin B2) in bacteria by Thakur et al.^[53].

It has been explored those probiotics can also synthesize vitamin B9, and a mechanism has been already proposed.^[53] For the synthesis, the precursors DHPPP and pABA are required, and a C-N bond joins these two with a condensation reaction catalyzed by dihydropteroate synthase. Then, the DHP is glutamylated by the enzyme dihydrofolate synthase to form dihydrofolate (DHF), which then is reduced to the active cofactor tetrahydrofolate (THF) and has an addition of polyglutamate to obtain THF-polyglutamate.^[55]

The bacteria commonly used to produce this vitamin B9 are Lactobacillus and Streptococcus, which produced concentrations of 173–700 ng/mL of the synthesized vitamin. Compared to the three studies presented in Table 3, soy milk showed a higher concentration since Lactobacillus plantarum CRL 725 bacteria managed to increase the initial concentration of riboflavin in milk from 309 ± 9 ng/mL to 700 ± 20 ng/mL after 12 h, that is, in less time in contrast to the others.^[56] In the same study, S. thermophilus bacteria was studied, and a significant increase was not shown since it remained at a concentration of 300 ng/mL. However, experimenting with goat milk, using the same bacteria, a concentration greater than 532 ng/mL was obtained, which only increased by 27 ± 3 µg/mL from the initial concentration of riboflavin in the milk.^[19]

Table 3. The use of probiotic bacteria for the synthesis of B-complex vitamins.

Vitamin	Lactic acid bacteria	Matrix/Source	Vitamin Concentration	Time (h)	Ref.
Riboflavin (B2)	Lactobacillus plantarum CRL 725	Soymilk	700 ± 20 ng/mL	12	Juarez del Valle et al.^[Citation56]
	Streptococcus thermophilus	Goat milk	532 ng/mL	48	Pacheco Da Silva et al.^[Citation19]
	Bacillus sp. Lactobacillus helveticus Streptococcus lutetiensis Streptococcus infantarius	Cheese	429 ng/mL 173 ng/mL 508 ng/mL 437 ng/mL	48	Pacheco Da Silva et al.^[Citation19]
	Lactobacillus (heterofermentative) Lactobacillus (homofermentative) Pediococcus	Yogurt	0.45–0.68 mg/L after 24 h 0.55–0.76 mg/L 0.64–0.89 mg/L	24	Jayashree et al.^[Citation57]
Folate (B9)	Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus plantarum	Whey permeate	84 ng/ml 125 ng/ml 397 ng/ml	24	Laiño et al.^[Citation58]
	Lactobacillus delbrueckii subsp. bulgaricus CRL871 + CRL803 + CRL415 Streptococcus thermophilus	Yogurt	180 ± 10 μg/L	16	Laiño et al.^[Citation58]
	Streptococcus thermophilus Lactococcus lactis subsp. Lactis Weissella paramesenteroides	Goat milk	114–311 ng/ml 172 ng/ml 93–111 ng/ml	48	Pacheco Da Silva et al.^[Citation19]
	Bacillus sp. Lactobacillus helveticus Streptococcus themophilus Weissella paramesenteroides Lactococcus lactis subsp. Lactis Streptococcus lutetiensis Streptococcus infantarius	Cheese	169 ng/ml 23 ng/ml 167–294 ng/ml 389 ng/ml 183–253 ng/ml 76 ng/ml 71 ng/ml	48	Pacheco Da Silva et al.^[Citation19]
	Lactobacillus casei Lactobacillus plantarum Lactobacillus paracasei subsp. paracasei Lactobacillus rhamnosus Lactobacillus delbrueckii subsp. bulgaricus	Cheese	4.50–40.74 ng/ml 5.71–89.50 ng/ml 19.97–21.03 ng/ml 2.00–16.00 ng/ml 2.20–18.50 ng/ml	48
	Lactobacillus delbrueckii subsp. bulgaricus CRL 863 Streptococcus thermophilus CRL 415 and 803 Lactococcus lactis subsp. lactis CM28	Artisanal Argentinean yogurts	94.8 µg/L 92.5 and 34.3 µg/L	24	Laiño et al.^[Citation58]
		Cow’s milk	12.5 ng⁄mL 14.2 ng⁄mL	7	Gangadharan et al.^[Citation59]
Cobalamine (B12)	Lactobacillus plantarum	Kanjika	13 ng/g of dry biomass	48	Madhu et al.^[Citation60]
	Lactobacillus reuteri	Soy-yogurt	0.18 μg/mL.	72	Gu et al.^[Citation61]
	Lactobacillus plantarum LZ95 Lactobacillus plantarum CY2	NR	98 ± 15 μg/L 60 ± 9 μg/L (extracellular)	20	Li, Gu, Yang, et al.^[Citation62]
	Enterococcus faecium LZ86	NR	499.8 ± 83.7 μg/L	20	Li, Gu, Wang, et al.^[Citation63]
Thiamine (B1)	L. brevis CRL 2013 Streptococcus thermophilus CRL 986	NR	3.42 ng/mL 2.64 ng/mL (Extracellular)	48	Teran et al.^[Citation64]

For the synthesis of folate, the use of Streptococcus thermophilus is not possible since they cannot synthesize vitamin B6.^[58] However, Streptococcus lutetiensis and Streptococcus infantarius synthesized the vitamin lower than 71 ng/mL.^[19] Furthermore, in Table 3 on studies related to cheese, the concentrations were lower than the others since they require a more aqueous medium and greater availability of lactic acid for their synthesis.^[58,65]

Finally, for the synthesis of vitamin B12, two studies have been carried out with the Lactobacillus plantarum, of which in the first study carried out in a Kanjika matrix (Indian functional food), a concentration of 13 ng/g was achieved. However, being without a matrix, the concentrations were higher, obtaining 98 ± 15 μg/L and 60 ± 9 μg/L (extracellular) for LZ95 and CY2 isolates, respectively.^[62] The same for the study presented with Enterococcus faecium LZ86 without being in a lactose matrix, and a concentration of 499.8 ± 83.7 μg/L was achieved.^[63] Therefore, being in a dry environment reduces its potential for synthesis.

The role of probiotics in the peptides synthesis

As the last point to discuss, probiotic bacteria can synthesize peptides that have different beneficial activities for human health, the best known being an antihypertensive function (Table 4).

Table 4. Bioactive peptides produced by probiotic bacteria.

Function	Mechanism	Lactic acid bacteria	Matrix	(IC50) / concentration	Fraction	Fragments / Sequences	Ref.
Antihypertensive	Angiotensin-I-converting-enzyme (ACE)-inhibitory	Lactobacillus delbrueckii subsp. bulgaricus SS1  Lactobacillus lactis subsp. cremoris FT4	Fermented milk	8.0 - 11.2 mg/L	β-casein (β-CN) β-CN κ-CN	fragment 6-14 (f6-14), f7-14, f73-82, f74-82, and f75-82 f7-14, f47-52, and f169-175 f155-160 and f152-160	Gobbetti et al.^[Citation66]
		Lactobacillus helveticus PR4	Bovine milk	16 to 100 μg/mL	Bovine αS1-casein (αS1-CN) β-CN	24-47 fragment (f24-47), f169-193, f58-76	Minervini et al.^[Citation67]
			Sheep milk		Ovine αS1-CN αS2-CN	f1-6 f182-185 and f186-188
			Goat milk		Caprine β-CN αS2-CN	f58-65 f182-187
			Buffalo milk		Buffalo β-CN	f58-66
			Human milk		three tripeptides originating from human β-CN	f184-210 (antibacterial peptide)
		Kombucha culture	Kombucha fermented milk	0.03 µM 0.03 µM 0.75 µM	NR	VAPFPEVFGK, LVYPFPGPLH, FVAPEPFVFGKEK	Elkhtab et al.^[Citation68]
		Lactobacillus casei	Lactobacillus casei fermented milk	0.11 µM 0.23 µM 0.10 µM	NR	LVESPPELNTVQ, VLESPPELN, WGYLAYGLD
		Lactobacillus delbrueckii QS306	Lactobacillus delbrueckii  fermented milk	12.87 μg/mL	NR	LPYPY	Wu et al.^[Citation69]
		Lactococcus lactis, Lactococcus cremoris, Lactococcus biovar. diacetylactis, Leuconostoc mesenteroides ssp. cremoris, Lactobacillus plantarum, Lactobacillus casei	Caprine Kefir	2.4 ± 0.2 µM 27.9 ± 2.3 µM	αs2-CN β-CN	f203-208 PYVRYL, f58-68 LVYPFTGPIPN	Quirós et al.^[Citation70]
		Lactobacillus helveticus CPN4	Yogurt-Like Product	720 μM.	Tyr-Pro in αs1-casein (CN), β-CN, and κ-CN		Yamamoto et al.^[Citation71]
		Enterococcus faecalis CECT 5727	Milk fermented	5 μm	β-casein	f133-138, LHLPLP f58–76 LVYPFPGPIPNSLPQNIPP	Quirós et al.^[Citation72]
Antioxidant, antihypertensive, and immunomodulatory activities of peptide fractions		Lactobacillus delbrueckii ssp. bulgaricus LB340	Fermented skim milk	67·71±7·62 mg/ml	NR	NR	Qian et al.^[Citation73]
Neurotransmitter	GABA	Lactobacillus paracasei PF6, Lactobacillus delbrueckii subsp. bulgaricus PR1, Lactobacillus lactis PU1, Lactobacillus plantarum C48, Lactobacillus brevis PM17	Italian cheeses	99.9 mg/ kg 63.0 mg/ kg 36.0 mg/ kg 16.0 mg/ kg 15.0 mg/ kg *GABA concentration found in each bacterium	NR	NR	Siragusa et al.^[Citation74]

ACE (antihypertensive activity)

Probiotics have been related by their antihypertensive activity, which is given by the Angiotensin-I-converting-enzyme (ACE) -inhibitory mechanism. Cultures of Lactobacillus and Lactococcus bacteria have been used to inhibit ACE activity but the most used matrix for this synthesis has been milk from different animal sources, with only one study of its synthesis in yogurt and Capri kefir.^[70,71] Peptide fractions were extracted from the milk samples, and the IC₅₀ value was measured to determine the protein concentration necessary to inhibit 50% of the ACE activity.^[75] The casein (CNs) are abundant milk proteins, and their primary sequences are αS₂-CN, αS₁-CN, and β-CN. Furthermore, they show variation between species since they have genes for rapid evolution, and it has also been proposed that they have a common precursor with post-translational modification.^[67]

Regarding the bacteria used, the kombucha and Lactobacillus casei bacteria were the ones that reported a lower IC₅₀ concentration in all the samples, obtaining a result of 0.30 to 0.23 µM.^[68] However, kombucha did not grow in a milk batch but had the highest activity compared to L. casei, obtaining 0.75 µM of IC₅₀. On the other hand, Lactobacillus helveticus bacteria seem to have more potential for antihypertensive activity since, independently of the milk matrix of different animals, a comparison of the other studies showed IC₅₀ of 16 to 100 μg/mL.^[67]

GABA

Another functionality reported about the use of probiotic bacteria in dairy foods is the ability to synthesize some neurotransmitter peptides such as GABA; the synthesis has been reported in an Italian cheese matrix where the bacterium Lactobacillus paracasei PF6 is the one that most synthesized this peptide with 99.9 mg/kg. On the other hand, Lactobacillus plantarum C48 and Lactobacillus brevis PM17 batteries were the ones that had the least capacity to produce this peptide, giving results of 16 mg/kg and 15 mg/kg, respectively. Still, the activity of each peptide extracted from the biosynthesis of each bacterium must be explored to see their effectiveness.

Relevant activities of peptides for human health

Antimicrobial

LAB is known to be nonpathogenic, so they are safe bacteria that can be consumed usually through fermented foods. They can ingest glucose and convert it into lactic acid, acetic acid, ethanol, and CO₂, which provide the product with characteristics such as texture or aroma, among others traits.^[26] One of the qualities for which it is safe to consume is that it prevents the growth of dangerous microorganisms including Staphylococcus aureus KCTC-1621, E. coli O157:H7, Listeria monocytogenes and Salmonella typhi.^[13,76–78] Its antimicrobial property is related to metabolites, hydrogen peroxide, ethanol and antimicrobial proteins or bacteriocins, synthesized by these same.^[79]

Some reports suggest that mineral enrichment such as Se potentiates the antimicrobial effect against common foodborne pathogens compared to bacteria without Se enrichment. The mechanism mentioned but not proven is the increase in the production of selenized proteins (SeCys or SeMet) during Se enrichment.^[35]

Improving lactose digestion

Probiotics have also attracted attention because a relationship with lactose tolerance has been seen with those who are intolerant. There is better digestion of lactose since probiotics promote the hydrolytic capacity of the small intestine by increasing colonial fermentation.^[80] In the study by He et al.^[81] probiotics contribute to this tolerance because they decrease the concentration of lactose in fermented foods and increase the active lactase enzyme when found in the small intestine.

Oak and Jha^[82] analyzed the studies of eight bacteria, which were Bifidobacterium longum, Bifidobacterium animalis, Lactobacillus bulgaricus, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus rhamnosus, Saccharomyces boulardii, and Streptococcus thermophilus. They noted that B. animalis was one of the most effective in developing this activity; this is one of the most common found in gut microbiota after B. longum, which is one of those that the mother passes to the child when it is in gestation. In addition, the B. animalis strain has beneficial properties of mucus adhesion and inhibition of pathogens, among other common to other probiotics, and its mechanism to obtain this effect has not been understood. However, it has been proposed that the influencing factors are intestinal pH, its ability to express beta-galactosidase, and how to intervene to improve intestinal functions and the colonic microbiota.^[82–84]

Elimination of toxic metals

As it is known, the human body is exposed to many chemicals, some of which can damage health; among those chemicals are heavy metals which can be acquired by the air of the environment, water, or even food.^[26] Recently, it has been explored that probiotics can remove heavy metals that are not beneficial. Some heavy metals are essential as iron, copper, and zinc, but others are toxic even in small amounts, such as aluminum, lead, cadmium, mercury, and arsenic.^[85]

Some metals can be present in two forms: sulfide and oxide. Heavy metals like copper, iron, and cobalt that once are present in an acid medium (stomach or water) become acidic. When they have radicals, they can form bonds with biomolecules such as proteins and enzymes, forming strong and stable chemical bonds. Metals are often joined by functional groups such as thiol, which is found in the amino acids cysteine and methionine.^[86]

The accumulation or binding of these toxic metals depends on the microbial strain and the pH since when there is a low pH (2–3), the removal is not good, but when there is a maximum removal, the pH is between 4–6. This occurs due to the competition for negatively charged binding sites with cationic metals and protons.^[85,87] Likewise, the intestinal microbiota alters the absorption of heavy metals since it behaves like a physical barrier, which prevents oxidative stress and controls the pH regulating detoxification enzymes or protein expression.^[88]

Detoxification

LAB have an important capacity to detoxify some foods, their use has been reported in milk to inhibit aflatoxin M1, cereals for Fusarium mycotoxins and in sorghum beverages for aflatoxin^[89,90]; studies report an inhibition of aflatoxin B1 up to 99.8% using Bifidobacterium and Lb. fermentum.^[91] In addition, Peng et al. reported a protective role of sodium selenite against aflatoxin B1-induced apoptosis of jejunum in broilers.^[92]

Analysis of minerals, peptides, and vitamins in dairy foods

The absorbed or biotransformed minerals can be found as part of a protein/peptide or absorbed by the bacteria cell. The following methods can be helpful to determine the amount of mineral, find where it is, and the kind of species that is transformed to analyze the fractions. Some of these standard methods are electrophoresis, spectrophotometry, ICP-MS, HPLC- ICP-MS, and a new one, electrothermal atomic absorption spectrophotometer (ET-AAS) where each technique has a differential scope.

One of the simplest and oldest methods that can be found in some reports is the one developed by Brown and Watkinson with modifications that allow the quantification of the selenite ion by spectrophotometry.^[93,94] Followed, to demonstrate the accumulation of minerals, of the most used techniques is ICP-MS, is highly sensitive and capable of quantitatively determining almost all elements that have an ionization potential less than the ionization potential of argon at very low concentrations (nanogram/liter or part per trillion). It is based on the coupling of a method to generate ions (inductively coupled plasma) and a method to separate and detect ions (mass spectrometer). Another technique that allows us to increase the scope for the quantification of mineral enrichment in LAB, is HPLC coupled to ICP-MS. HPLC-ICP-MS is capable to separate the isotopes of different chemical elements that have the same mass, obtaining retention times and masses of the different minerals that can be compared with analytical standards.^[95–97] Studies of selenium and yogurt have been published on the species that the mineral was transformed into. Once the sample has been fractionated, the protein fractions are characterized by HPLC-ICP-MS using an ammonium citrate solution at pH 5 with 2% methanol as the mobile phase to examine the presence of the different organic selenium species, such as selenocysteine, methylselenocysteine and selenomethionine.^[24,30,98]

HPLC-ICP-MS has been widely used in the field of speciation analysis for its advantage of high separation capacity, low detection limit, wide linear dynamic range, and good analytical precision.^[99] In the case of the study of enrichment with Se, to confirm SeCys species, the use of HPLC-Tandem Mass Spectrometry (HPLC-ESI-MS/MS) has been reported.^[39] LC-ESI-MS/MS combining the separation power of HPLC with the great selectivity, sensitivity and precision in the determination of the molecular mass of mass spectrometry, providing qualitative and quantitative information. The sample components separated on the HPLC pass to the mass spectrometer through an interface where they are ionized. LC-MS equipment can have two different types of interfaces that are included within the so-called atmospheric pressure ionization (API), called electrospray (ESI) and atmospheric pressure chemical ionization (APCI). The ions generated at the interface are accelerated towards an analyzer and separated according to their mass/charge ratio (m/z) by applying electric or magnetic fields or simply determining the time of arrival at a detector. The ions that reach the detector produce an electrical signal that is processed, amplified, and sent to a computer. The record obtained is called the mass spectrum and represents the ionic abundances obtained as a function of the mass/charge ratio of the detected ions.^[100,101] Luo et al., reported an ICP-MS/MS method for the detection of Se species that allows the detection of Se isotopes that are affected by 40Ar40Ar+, 40Ar38Ar+, and 40Ar42Ca+. They reported detection limits of selenite (Se(IV)), selenate (Se(VI)), selenomethionine (SeMet), selenocystine (SeCys2), methylselenocysteine (MeSeCys), and selenoethionine (SeEt) of 0.04, 0.02, 0.05, 0.02, 0.03, and 0.15 ng/mL, respectively.^[102]

The polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS PAGE) is used to determine on which fraction of protein is located the mineral. The samples treated and untreated are commonly separated into fractions, and then, to know the efficiency of the fractionation, an SDS-PAGE is realized. It is expected to see just a line (band) for each fraction; for example, casein fringes are seen from 23.5 kilodaltons (kDa), beta-lactoglobulin at 18 kDa, and alpha-lactalbumin at around 14 kDa.^[24,103] This also reflects a difference in band shift of the standard proteins versus those containing organic species of a specific mineral. Another technique used is ICP-MS which is useful to determine the total mineral in the sample or well and the content of mineral in bacteria cell pellets, using argon as carrier gas. It exhibits a low detection limit (0.9 ng/mL), making it sensitive for quantification.^[30] The sample is put into ICP-MS and what is seen in the spectrum is a characteristic peak of the mineral of interest.

For other minerals, studies about magnesium, ET-AAS was used to measure the concentration of the mineral.^[47] The sample is deposited in small graphite or pyrolytic carbon-coated graphite tube; then, it is treated with heat to vaporize it and make the sample into atoms as an analyte. These atoms absorb the UV-VIS light and make transitions in high energy levels. It is expected to see a peak of absorbance at a specific time, located and characterized by a particular element.

On the other hand, to analyze vitamins like B2, B6, B9, and B12, liquid chromatography (HPLC) is used due to its facility to separate the compounds of the sample.^[19,61] Vitamin B12 is found with B6; that is why it is used with the reversed-phase, which consists of a non-polar stationary phase and a mobile phase of moderate polarity. The retention time is higher for the non-polar molecules, while polar move faster. Therefore, vitamin B12 is detected at peak at the retention time, representing the active cobalamin forms Ado-Cbl and Me-Cbl in 8.5 and 17.0 min, respectively.^[62,63,65] This method is used for vitamins and fractionate peptides by reversed-phase, showing its potential use for multiple analytes.^[66,67]

Challenges

With the data integrated in this review, we were able to show that the development of foods with probiotics for mineral fortification is a growing line of research, where very promising results have been obtained but with great variability. Undoubtedly, the factors associated with mineral fortification require more studies to achieve the desired fortification doses in foods.

For the design of fortified dairy foods using probiotics, it becomes challenging the selection of bacteria, ensuring that the combination to be made of probiotics is adequate so that cultures are not affected in their development and activity. Likewise, ensuring that the conditions of their environment also benefit their potential.

Regarding fortification with minerals, the addition of minerals by bacteria used for different dairy products remains to be explored since they were general studies related to yogurt. In addition, it is necessary to find out if, through the addition of inorganic salts to the bacteria, they have better interaction, which means if they tend to interact better with the presence of salt, nanoparticles, or an organic compound for the incorporation of a mineral. It also remains to be explored whether other minerals can be incorporated into amino acids such as seleno-amino acids and describe the proper amount to be used as part of the substrate to reach the recommended amount for intake.

Regarding vitamins, the biosynthesis mechanism of the other compounds of the B complex still needs to be understood since, as this is understood, it could be analyzed whether it is possible to synthesize vitamins of another type. The same for the biosynthesis of peptides remains to discover the bacteria that could synthesize these products and their activity to exert that function. Finally, it is necessary to design processes that significantly improve the incorporation of nutrients through beneficial bacteria in dairy products since satisfying a daily dose of any bioactive would positively impact consumer health.

Conclusions

Undoubtedly, the fortification of foods is increasing more and more since it helps prevent diseases and helps to have better body function. Probiotics are a good option for adding nutritional value since they not only act for digestive health but are also a source of bioactive compounds since they can synthesize nutrients such as metal-amino acids, vitamins, and peptides. It is only necessary to ensure that these products are not harmful and threatening once consumed. Finally, it is emphasized that it is necessary to have a regulation to guarantee that the fortification is safe and justified according to the nutritional contribution. With the information analyzed in this review, it is possible to optimize the preparation process for the fortification of dairy food, and the proposals of new products and processes, like essential mineral augmentation in yogurt or other dairy foods.

Ethical approval

This study does not involve any human or animal testing.

Acknowledgments

The authors acknowledge the support of the Sciences Department of the School of Engineering and Sciences in Tecnológico de Monterrey. Thanks to Ana Maritza Reyes-González for the support in creating the figures for this document.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

Being this work a review, data sharing is not applicable as no new data were created for this study.