1. Introduction
Nowadays, taking into consideration the current dynamics of development of drug resistance many researchers are working to create new antimicrobial compound combinations for food and beverage industry, which can overcome the problem.
Lactic acid bacteria (LAB) strains are used as technological starters for food preparation because of their important characteristics, especially their antimicrobial properties. Also, LAB can improve sensory properties, as well as nutritional value of food products [
1]. Most of the LAB strains’ antimicrobial properties can be explained by their excretion of different compounds, such as organic acids, bacteriocin-like inhibitory substance (BLIS), and enzymes [
2]. The incorporation of fermented foods and beverages to the main diet is a very important part of balanced nutrition, because fermented foods and beverages have many benefits for consumer health [
3]. Functional beverages could be a healthy alternative in human nutrition. Also, for their preparation, food industry byproducts could be used. Our previous studies showed that fermented milk permeate beverage prototypes with selected LAB strains contained galactooligosaccharides (GOS) (from 8.7 to 26.8 mg
GOS/100mL
sample) [
4]. The above-mentioned study proved a real possibility for milk permeate sustainable valorization to the fermented beverages of higher functional value.
Also, the nutritional value of fermented milk permeate beverages can be further improved by adding natural functional additives. Our previous studies revealed that berries and fruits possess a wide spectrum of antimicrobial properties against pathogenic and opportunistic bacterial strains [
1].
In this study, gooseberries, chokeberries, cranberries, sea buckthorn, and rhubarb were selected as popular berries/vegetables (B/V) in the Nordic European diet.
Gooseberry, widely cultivated in Europe, belongs to the
Ribes L. genus and the
Saxifrgaaceae family [
5]. The composition of this berry is very attractive, as it contains many nutrients, various sugars, organic acids, anthocyanins, inorganic micro- and macro-elements, and vitamins, as well as various amino acids [
6]. For this reason, gooseberry is an important stock for the food industry. Gooseberry is rich in vitamin C, containing 200 mg/100 g [
7]. Furthermore, these berries are rich in flavonoids, which have many desirable properties (antioxidant, diseases prevention, etc.) [
7]. In addition, gooseberry is rich in iron and iodine, which are associated with a lowered risk of atherosclerosis. Gooseberry is used for the prevention of dysentery, foot pain, arthritis, bone dysplasia, and kidney diseases [
7]. For above-mentioned reasons, gooseberry is a considered a medicinal plant [
8].
Black chokeberry (
Aronia melanocarpa) is a fruit with specific taste and dark color, known as a very good source of phenolics, which are associated with many health benefits [
9]. The chemical composition of black chokeberries, as well as their desirable effects on human health, have been very popular subjects of investigation [
9,
10,
11,
12]. The main biological active compounds in black chokeberry are anthocyanins, proanthocyanidins, and hydroxycinnamic acids, whereas quercetin, quercetin glycosides, and epicatechin are minor components [
9,
10]. A previous study found that black chokeberry and its extracts possess cardioprotective, hepatoprotective, anticarcinogenic, antidiabetic, antimutagenic, and many other effects [
9].
Cranberries have a unique flavor and very intense reddish-purple color [
13]. Cranberries sensory properties are related to their composition, which is rich in anthocyanin pigments [
14,
15]. The procyanidins of cranberries possess protective effects against urinary tract infections and cardiovascular diseases [
14,
16].
Sea buckthorn plant (
Hippophae rhamnoides) belongs to the Hippophae genus and to the Elaeagnaceae family, and extracts of the different botanical parts of sea buckthorn are very popular in pharmaceutical preparations. Sea buckthorn is a good source of lipids, vitamins, phenolics, carotenoids, phytosterols, and tocopherols [
17]. Sea buckthorn seeds and oils are used for the prevention of gastric ulcers [
18], cardiovascular diseases [
19], atopic dermatitis [
20], dry mouth in Sjogren Syndrome patients [
21], and depression [
22]. Additionally, this plant and its products possess a wide range of desirable effects, including the effective regeneration of skin and mucous membranes, improved immune functions, reduced oxidation, and a lowered risk of cardiovascular diseases [
23].
Rhubarb (
Rheum rhabarbarum L.) is characterized by strong antioxidant properties. However, this plant is cultivated only for its petiole [
24,
25], because its leaves have a toxic oxalic acid [
26]. In Europe, it is cultivated mainly in Germany, France, and England [
27]. Rhubarb stalks are used for food and drink preparation, as well as in traditional medicine for the treatment of gastrointestinal hemorrhage and constipation jaundice [
28]. A previous study found that this plant has anticancer properties [
29]. However, it should be mentioned that the consumption of rhubarb in large quantities can lead to adverse effects, which are associated with accumulation of calcium in the body [
26].
Finally, we hypothesized that the combination of the selected B/V with fermented milk permeate beverages can increase antimicrobial properties of the end-product.
The aim of this study was to evaluate antimicrobial properties of milk permeate fermented with Lactobacillus plantarum LUHS135, Lactobacillus plantarum LUHS122, and Lactobacillus faraginis LUHS206 strains milk permeate in combination with B/V pomace (gooseberries, chokeberries, cranberries, sea buckthorn, rhubarb) against a variety of pathogenic strains (methicillin-resistant Staphylococcus aureus, Citrobacter freundii, Klebsiella pneumoniae, Salmonella enterica, Bacillus cereus, Pseudomonas aeruginosa, Acinetobacter baumanni, Proteus mirabilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus epidermis, Staphylococcus haemolyticus, Pasteurella multocida, and Enterobacter cloacae) as a potential antimicrobial combination for beverage preparation.
3. Results and Discussion
The DIZ of gooseberry pomace and DIZ of gooseberry pomace with lactic acid bacteria (
Lactobacillus plantarum LUHS135,
Lactobacillus plantarum LUHS122, and
Lactobacillus faraginis LUHS206) strains fermented milk permeate combinations against pathogenic opportunistic microorganisms are shown in
Table 1. Comparing the DIZ of pure gooseberry pomace, it was found that gooseberries inhibited 9 out of 15 tested pathogenic and opportunistic strains, and the highest DIZ against
Pasteurella multocida and
Streptococcus mutans was found (25.6 mm and 23.0 mm, respectively). Comparing the DIZ of pure gooseberry pomace and DIZ of gooseberry pomace combination with LUHS135 permeate combination, it was found that addition of LUHS135 led to a broader spectrum of pathogen inhibition (inhibited 12 out of 15 tested pathogens), and the combination showed antimicrobial activity against
Enterococcus faecalis, Enterobacter cloacae, and
Citrobacter freundii (DIZ 11.6 mm, 12.6 mm, and 12.3 mm, respectively). However, compared to DIZ against pathogens which were inhibited by both samples (pure pomace and the gooseberry pomace combination with LUHS135 beverage), in most of the cases, the DIZ induced by the combination against pathogenic and opportunistic strains was similar or smaller. Comparing pure gooseberry pomace DIZ with gooseberry pomace combination with LUHS122 beverage, it was found that the addition of LUHS135 led to additional inhibition of
Enterococcus faecium, Bacillus cereus,
Enterobacter cloacae, and
Citrobacter freundii (DIZ 12.6 mm, 11.3 mm, 12.4 mm, and 11.1 mm, respectively). However, in most of the cases, the gooseberry pomace combination with LUHS122 showed a lower DIZ, compared to the pure pomace (except against
Salmonella enterica and
Streptococcus mutans). Moreover, the gooseberry pomace combination with LUHS206 showed a broader spectrum of pathogen inhibition (inhibited 12 out of 15 tested pathogens).
Many desirable properties of gooseberries fruit have been described. In addition, this fruit was reported to possess hypolipidemic, hypoglycemic and antimicrobial activities [
31]. However, studies about the physical, chemical, and antimicrobial characteristics of different cultivars are very scarce [
32]. Usually, this fruit is associated with the high concentration of vitamin C [
33]. Gooseberries are also a good source of phytochemicals (polyphenols, tannins, emblicol, linoleic acid, corilagin, phyllemblin, and rutin) [
34], which are related to antimicrobial activity of the fruit.
Pure chokeberries showed antimicrobial activity against 3 out of 15 tested pathogenic and opportunistic strains (
Bacillus cereus,
Streptococcus mutans, and
Pasteurella multocida;
Table 2). The combination of chokeberries with LUHS135, as well as with LUHS206 permeate, additionally showed antimicrobial properties against
Staphylococcus epidermis and
Staphylococcus haemolyticus. However, the combination of chokeberries with LUHS122, compared with the pure berry pomace, additionally inhibited only
Staphylococcus haemolyticus. The highest DIZ of the chokeberry combination with LUHS206 against
Streptococcus mutans was found to be 20.9 mm. For the pure berry pomace combination with LUHS135 and LUHS206 beverages against
Pasteurella multocida, a DIZ higher than 20 mm was determined.
A previous study showed that chokeberry polyphenols differ in their biological activity, and only epicatechin and quercetin show antimicrobial activities against
Candida albicans, but they do not inhibit
Staphylococcus aureus and
Proteus vulgaris. However, whole berries have many health benefits [
35]. Tannin antimicrobial activity can be explained by the inhibition of extracellular microbial enzymes, direct action on microbial metabolism through the inhibition of oxidative phosphorylation, or the deprivation of the substrates required for microbial growth [
36]. Taguri et al. [
37] reported that different types of phenolics, as well as their oxidation products—proanthocyanidins and hydrolyzable tannins—possess antibacterial activities against food pathogens, and pathogen sensitivity to phenolics depends on bacteria species and bioactive compound structure.
Pure cranberry pomace and its combination with fermented milk permeate beverages inhibited the same number of the tested pathogenic and opportunistic strains (10 out of 15 tested pathogens) (
Table 3). Also, in most of the cases, combinations with fermented milk permeate showed lower DIZ, compared to pure cranberry pomace, against
Pseudomonas aeruginosa (except the combination with LUHS135), against
Bacillus cereus (except the combination with LUHS135), against
Streptococcus mutans (except the combination with LUHS122), and against
Streptococcus epidermis.
Berries belonging to the Vaccinium species provide a very good source of compounds, which are associated with antimicrobial properties [
38,
39], e.g., the antimicrobial properties of cranberry concentrates against Staphylococcus aureus and E. coli O157:H7 are well known [
40,
41,
42]. Česonienė et al. [
43] showd that cranberry extracts inhibit a wide range of Gram-negative (
Escherichia coli and
Salmonella typhimurium) and Gram-positive (
Enterococcus faecalis,
Listeria monocytogenes,
Staphylococcus aureus, and
Bacillus subtilis) pathogens. A previous study found that
V. oxycoccus juice showed binding activity with
Streptococcus agalactiae and
Streptococcus pneumoniae due to
S. pneumoniae binding activity to low molecular size fractions of cranberry juices [
38].
Sea buckthorn pomace and its combination with LUHS135 and LUHS206 beverages inhibited 12 out of 15 tested pathogenic and opportunistic strains, and the sea buckthorn combination with LUHS122 additionally showed antimicrobial activity against
Salmonella enterica (
Table 4). Also, the sea buckthorn combination with LUHS206 increased its antimicrobial activity against
Acinetobacter baumanni. The sea buckthorn combination with LUHS135 and LUHS122 beverages showed increased antimicrobial activity against
Proteus mirabilis. The sea buckthorn combinations with all the tested LAB strains showed increased antimicrobial activity against
Enterococcus faecalis. The sea buckthorn combinations with LUHS135 and LUHS122 beverages showed increased antimicrobial activity against
Pasteurella multocida. Opposite tendencies (lower DIZ, compared with pure pomace) of the sea buckthorn combinations with LUHS135 against
Bacillus cereus,
Enterobacter cloacae, Citrobacter freundii, Streptococcus epidermis; combinations with LUHS122 against
Enterobacter cloacae and
Streptococcus epidermis; and combinations with LUHS206 against
Bacillus cereus, Enterobacter cloacae, and
Streptococcus epidermis were established.
The antimicrobial properties of sea buckthorn have the capacity to inhibit
Pseudomonas aeruginosa,
Escherichia coli,
Klebsiella pneumonia,
Staphylococcus aureus,
Bacillus subtilis and
Streptococcus pneumoniae [
44]. The antibacterial properties of sea buckthorn are associated with lipophilic bioactive compounds such as fatty acids (FA) [
23]. FAs have the capacity to kill bacteria or inhibit their growth, and many organisms defend themselves against parasitic or pathogenic bacteria using this mechanism [
17]. A previous study found that long-chain FAs have stronger antimicrobial activities against Gram-positive than Gram-negative bacteria [
45]. Traditionally, buckthorn is used in medicine because of its wide spectrum of biologically active FAs and other compounds, e.g., antibacterial and antioxidant [
46].
The diameters of inhibition zones of rhubarb and fermented milk permeate beverages against pathogenic opportunistic microorganisms are shown in
Table 5. Pure rhubarb pomace, as well as its combination with LUHS135 and LUHS206 beverages, inhibited 12 out of 15 tested pathogenic and opportunistic strains. The rhubarb pomace combination with LUHS122 additionally showed antimicrobial activity against
Salmonella enterica (DIZ 13.1 mm). Moreover, the rhubarb combination with LUHS135 and LUHS122 increased its DIZ against
Proteus mirabilis,
Enterococcus faecalis, and
Pasteurella multocida. The rhubarb combination with LUHS206 showed higher DIZ against
Acinetobacter baumanni,
Enterococcus faecalis, and
Pasteurella multocida. Opposite tendencies of all LAB and rhubarb tested combinations against
Enterobacter cloacae,
Streptococcus epidermis, and
Bacillus cereus (except combination with LUHS122) were also found. The above-mentioned combinations decreased DIZ against the mentioned pathogenic strains.
Usually, the combination of many compounds with different structures is responsible for inhibiting pathogens [
47], antioxidative effects [
48,
49], health benefits [
50,
51] and plant protective properties [
52] in plant materials. A previous study found that rhubarb root composition is rich in phenolic compounds [
53]. Kosikowska et al. [
54] and Raudsepp et al. [
55] found that rhubarb roots possess very strong antimicrobial activity. Hasper et al. [
56] showed that rhubarb root toxicity is very low, and they can be used for food preparation. The antimicrobial activity of the rhubarb is correlated with total polyphenolic concentration, but not with the total content of anthocyanins [
57].
Finally, the highest number of the tested pathogenic and opportunistic strains was inhibited by gooseberries, sea buckthorn, and rhubarb combinations with LUHS122 (13 pathogens out of 15 tested). Twelve out of 15 tested pathogens were inhibited by gooseberries, sea buckthorn, and rhubarb combinations with LUHS135 and LUHS206, as well as with LUHS135 and LUHS206 fermented milk permeate (
Figure 2). Other tested berries/vegetables and their combinations with LAB strains inhibited 10 strains and a lower number of the tested pathogenic and opportunistic strains.