For research Bacillus pumilus was grown in laboratory conditions, the isolation medium served the potato. Appropriate solutions were prepared from pure culture.

Freshly ripened berries (aronia, raspberry and strawberry) were gently washed under water and cleaned with an aseptic knife. The fruits were processed by scalding at 95˚C for 5 min, packed in glass jars and stored at 4.0˚C ± 1.0˚C.

Sea buckthorn, rosehip and hawthorn fruits were dried at 65˚C ± 1˚C to a moisture content of 8.3% ± 0.2% and ground to a powder. The fat-soluble extracts were obtained from powders of sea buckthorn, rose-hip, hawthorn and refined deodorized sunflower oil in a ratio of 1:12, 1:14, 1:20, respectively, at temperature of 45˚C in time of 90 min, under mechanical agitation 60 rpm, in limited conditions of light penetration. The fat-soluble extracts were filtered and stored at 4˚C ± 1˚C.

For the yogurt manufacture the Lyofast YAB 205 starter culture was used for inoculation, which contain Streptococcus thermophilus, Lactobacillus delbrueckii subsp. Bulgaricus, Lactococcus lactis subsp Lactis biovar diacetilactis, sucrose and maltodextrin. The yogurt assortment obtained in laboratory conditins is presented in Table 3.

Samples of wheat bread were made using 1% and 2% fat-soluble extracts of sea buckthorn, rose-hip, and hawthorn fruits. The control sample was prepared

“+” positive; “−” negative; “+/−” variable within species.

without adding any fat-soluble extracts of vegetal material. The wheat bread samples were baked from dough prepared by direct method with the addition of sourdough (control sample) in an amount of 10%.

MRS-broth (medium of Man, Rogosa, Sharpe) (Scharlau); MRS-agar. Composition (g/dm³): MRS-broth (Scharlau), agar, 20; MRSc = MRS-broth + 10% aronia; MRSr = MRS-broth + 10% raspberry; MRSs = MRS-broth + 10% strawberry.

The studied strains of microorganisms were cultured in a liquid medium (MRS-broth) and on an agar medium (MRS-agar) at 37˚C to examine the effect of berries and lactic acid bacteria on growth. The agar diffusion method was used to determine the antimicrobial activity of three prepared fruit supplements.

Nutrient broth Mueller Hinton broth (MHB), use in diffusion method for determining the antimicrobiological properties of berries.

In this study we used inhibition zone test, also called Kirby-Bauer Test. It is a qualitative method used to measure and compare the inhibitory activity levels of the tested substances (berries puree) [42].

The methodology for this experiment was based on the work of Lambert et al. The most common way to assess microbial growth in solution is the measurement of the optical density at 600 nm (OD₆₀₀). The method was based on absorbance detection mode and basically determines which portion of light passes through a sample, more specifically through a suspension of microorganisms [43].

The method of Thompson et al. [44] has been used to research the development of ropiness disease in wheat bread samples.

The variance analysis of the results was carried out by least square method with application of Student test and Microsoft Office Excel program version 2010. The differences were considered statistically significant if probability was greater than 95% (p-value < 0.05). All assays were performed in triplicate. The experimental results were expressed as average ± SD.

Bacillus cereus is an endospore-forming, Gram-positive bacterium able to cause foodborne diseases. Bacillus cereus is also a food poisoning bacterium that can occasionally be an opportunistic human pathogen [45]. The data presented by Branda et al. [46] support the older school of thought, namely that Bacillus cereus is a saprophytic bacterium that is able to grow in the soil and, in addition, is adapted to translocate through the soil. Members of the aerobic spore-forming genus Bacillus are commonly isolated from many types of soil at a range of depths and altitudes, and under various climatic conditions [33] [34] [47] [48]. Bacillus was reported to occur in soil as spores, germinating and becoming active only when readily decomposable organic matter was available [49]. Vegetative cells and especially spores can easily enter the food chain via crop plants. According to this, the range of foods in which Bacillus cereus is detected is broadly diversified [50].

Bacteria of Bacillus cereus and Bacillus pumilus were found in whole wheat flour. Spores of the genus Bacillus, which enter the human body, can cause significant dysfunction of the immune system, gastrointestinal tract, liver, respiratory system, and nervous system. Therefore, even if spore bacteria do not cause ropiness disease in bread, it is better not to have them in the finished product. They may be present in large fractions of whole grain breads. The source of infection may be the air in the bakery [7]. Ropiness disease is mainly due to natural microflora and in particular to Bacillus subtilis or Bacillus pumilus. In the process of baking wheat bread, the bacteria are destroyed, but part of its spores can remain viable. The specific feature of ropiness disease is that its manifestation is most often visible to the user during the summer months when temperatures are noticeably higher, which is a prerequisite for activating it.

It has been found that biological methods of microbial decontamination, namely the use of natural biological substances from leavens have wide technological potential not only to prevent the most dangerous types of bread spoilage, such as ropiness disease, but also as natural additives, improvers of bread quality with intensive methods of its production [8]. Neysens and De Vuyst [51] used a modeling approach to describe the kinetics and production of L471 amylovorin of Lactobacillus amylovorus DCE 471, given its application during the production of type II rye dough. Influence of temperature, pH and undissociated lactic acid concentration on inhibition of Lactobacillus sakei CTC 494 growth in MRS broth.

There are many benefits of the sourdough application in bread making: improvements in the volume of the bread and the structure of the crumb [52] [53], the flavour [54], the nutritional value [55] [56] and shelf life [57] [58] [59], due to the delay of the staling process and the prevention of mould and bacterial spoilage, against saprophytic microorganisms: Bacillus subtilis, Bacillus mesentericus, Aspergillusniger, Penicilliumsp. and Rhizopussp., but none of them inhibited the growth of bakery yeasts Saccharomyces cerevisiae [52] [60]. The addition of sourdough is the best technique to keep the bread from spoilage meeting consumer’s demand for natural food without additives [61]. Sourdough is a mixture of flour (from wheat, rye, rice, etc.) and water, which is fermented by the action of lactic acid bacteria and yeasts [61]. These microorganisms usually come from flour, dough ingredients or the environment.

The objectives to be achieved by the use of sourdough are a significant increase in the shelf life and the nutritional value of bread and improvement of the organoleptic properties of bread. The increase in the retention time of the sourdough bread is due to the higher levels of acidity and the higher concentration of produced organic acids in comparison to the commercial bread produced by using only yeasts [2].

With the inclusion of starter cultures, the pH falls very quickly, so the whole manufacturing process is accelerated, which leads to economic benefits for the producer. The secondary effects of the acidification and the fermentation time acceleration include changes in the enzymes activity of the cereal substrates or the bacterial strains [6]. These positive effects are associated with the metabolic activity of the selected pure cultures, yeast and homo- and heterofermentative lactic acid bacteria in the composition of the sourdough, e.g. lactic acid fermentation, proteolysis, exopolysaccharide production and synthesis of volatile and antimicrobial compounds [52]. Bacillus cereus is an endospore-forming, Gram-positive bacterium able to cause foodborne diseases. Lactic acid bacteria (LAB) are known for their ability to synthesize organic acids and bacteriocins, but the potential of these compounds against Bacillus cereus has been scarcely documented in food models [45].

Various methods to prevent microbiological infection of bread have been developed: technological, chemical, physical, biological and combined [7] [8] [51] [62] [63]. The study by Black et al. [64], includes factors analysis influencing the germination rate of Bacillus subtilis spores by very high pressure. Ropiness disease is mainly due to natural microflora and in particular to Bacillus subtilis or Bacillus mesentericus. When grinding the grain, it is transferred to the resulting flour. In the process of baking wheat bread, the bacteria are destroyed, but part of its spores can remain viable. The specific feature of ropiness disease is that its manifestation is most often visible to the user during the summer months when temperatures are noticeably higher, which is a prerequisite for activating it [37].

Despite developments in the dairy industry, premature milk spoilage due to the contamination by Bacillus cereus continues to be a problem and causes considerable economic losses. Bacillus cereus is ubiquitously present in nature and can contaminate milk through a variety of means from the farm to the processing plant, during transport or distribution [28]. Bacillus cereus was isolated from 9%, 35%, 14% and 48% of raw milk, pasteurized milk, Cheddar cheese and ice cream samples, respectively. Bacillus cereus was not recovered in yogurt. The level of contamination with Bacillus cereus did not exceed 100/mL in raw milk, 1000/mL in pasteurized milk, 200/g in Cheddar cheese and 3800/g in ice cream [65]. In spite of the frequency with which dairy farms are contaminated with Bacillus cereus, no outbreaks of food poisoning have occurred from consumption of milk and milk products, except for a few cases caused by cream and certain desserts. Bacillus cereus can multiply during cheese manufacturing [66], a point that warrants further investigation. Bacillus cereus may contaminate other foods, if milk or a milk product is one of their ingredients, and, if conditions are right, multiply rapidly and produce sufficient toxin to induce symptoms of food poisoning. The possible presence of toxigenic Bacillus cereus is another reason why dairy foods and other foods should be handled appropriately both during processing and in the home. Eleven strains of Bacillus cereus isolated from milk and meat products have been used to study growth and sporulation profiles in detail [50].

A recent example of a microorganism causing undesired growth in consumer milk is Bacillus sporothermodurans producing highly heat-resistant spores (HRS) which may survive ultra-high temperature (UHT) treatment or industrial sterilization [67]. Cortezzo et al. [68] in their studies showed that treatment with oxidizing agents affects the inner membrane of Bacillus subtilis spores and sensitizes the spores to subsequent stress. Treatment of spores with oxidizing agents has been suggested to cause damage to the spore’s inner membrane, a membrane whose integrity is essential for spore viability.

Microorganisms of genus Bacillus, which are incidental in food, but affecting their quality, are increasingly becoming the object of research. This study aimed to characterize the berries potential on reducing the Bacillus pumilus risk in fermented foods. In this study, a three-phase experimental descriptive method was applied on a laboratory scale. The first phase was the observation of the Bacillus pumilus bacterial growth curve and the influence of temperature on the microorganisms’ growth rate (Figure 1 & Figure 2). The second phase was the study of the berry additives influence on reducing the Bacillus pumilus risk infection (Table 4, Figure 3). The third phase was the berry additions influence on the ropiness disease development during the wheat bread storage and in the process of lactic acid fermentation (Table 5, Figure 4). The number of growing bacterial colonies was calculated using the total plaque counting method. The results showed that Bacillus subtilis had an optimal growth point at the eighth hour, achieving a total population of 6.1 × 10¹³ CFU/mL. The temperature of 37˚C gave a growth rate higher than 4.6 compared to the temperature of 20˚C, which is 3.2.

Results of studying the bacteriostatic properties of aronia, strawberry and raspberry in experiments with Bacillus pumilus are presented in Table 4.

A knowledge of the combined technologies with the appropriate concentrations of the “hurdles” in combination can contribute to the process design and control in order to assure the safety and quality of the products demanded by the consumer market. Activity of Lactobacillus cultures against bacteria and fungi is widely observed and frequently reported [69] [70]. Isolation of antimicrobial compounds from cell-free supernatant identified lactic acid, phenyllactic acid and the two cyclic dipeptides cyclo (l-Leu-l-Pro) and cyclo (l-Phe-l-Pro) are the major components responsible for this activity [71] [72]. These data are in accordance with results reported by Arena et al. [73], in which the production of antimicrobial compounds varied between Lp. plantarum strains. In general, Lp.

plantarum S6.3 and S10.12 exhibited the highest inhibitory capability (diameter 23 mm) against Bacillus cereus DSM31.

The ability of selected lactic acid bacteria to inhibit the growth of rope-forming Bacillus strains in laboratory experiments and in wheat bread was investigated. Growth of Bacillus subtilis and Bacillus licheniformis was inhibited by Lactobacillus plantarum VTT E-78076 and Pediococcuspentosaceus VTT E-90390 in an automated turbidometry assay and in test bakings [74].

The introduction of berries into the Bacillus pumilus growth medium reduced the germination rate of bacteria during 8 hours as follows: for aronias up to 37.50%, for raspberries up to 37.78%, and for strawberries up to 36.0%. In the samples without the berries addition, the bacteria germination and growth occurred more during the whole tested period, Figure 3.

The objective of our previous research was to improve the quality of wheat bread and ensure its microbiological stability by using berry additives. The influence of berries (in the form of fat-soluble extracts) on stopping the wheat bread spread ropiness disease was investigated. The results are presented in Table 5.

The analysis of the results showed that 1% of fat-soluble extract from the vegetable matter reduced the ropiness disease risk in wheat bread. For bread samples with 1% of rose-hip, hawthorn and sea buckthorn fruits fat-soluble extracts the spoilage appeared after 96 hours and 120 hours respectively. The manifestation of ropiness disease signs is observed in wheat bread with low acidity. In order to prevent the bread disease with ropiness, lactic bacteria are introduced during the fermentation of wheat dough. Lactic acid bacteria Lactobacillus in the process of development from nutrient mixture carbon-containing components

‘‘−’’ no signs of microbial spoilage; ‘‘+’’ initial signs of ropiness development; ‘‘++’’ average intensity of ropiness development (stickiness, odor); ‘‘+++’’ intense of ropiness development (consolidation of unpleasant odor, stickiness).

form organic acids. The latter reduces active acidity and prevents the reproduction of unwanted microflora. Figure 4 shows the results of the berries influence on the lactic acid bacteria growth.

Growth rate of lactic acid bacteria in modified de Man, Rogosa, and Sharpe (MRSc, MRSr, MRSs) was significantly higher by the 12 h time point compared with traditional de Man, Rogosa, and Sharpe (MRS) broth. Lactic bacteria in the berry additives presence showed a growth rate, measured by OD at 600 from 0.073 to 0.651 (for aronia), from 0.071 to 0.609 (for raspberries), from 0.073 to 0.597 compared to the increase in environments without added fruit, which amounted to −0.050 to 0.410 (Figure 5).

The pH results obtained for the yogurt samples during fermentation varied in each sample, P2 (5.31 - 4.28), P3 (5.27 - 4.25), and P4 (5.19 - 4.27) relative to P1 (5.63 - 4.38). The optical density during fermentation was increased for all yogurt samples tested. The largest share of the increase in optical density was found in the berries yogurt samples. So in the fruit yogurt samples a synergism was created between the lactic bacteria and the chemical composition of the additions of raspberry, strawberry and aronia, which keeps under control the risk of Bacillus products infestation.

The pathogen Bacillus pumilus, which is associated with many foods including dairy products, was studied for its response to acid stress during the exponential phase. Lactic acid bacteria (LAB) are known for their ability to synthesize organic acids and bacteriocins, but the potential of these compounds against Bacillus cereus has been scarcely documented in food models [44].

The results of this research showed that the combined use of lactic acid bacteria with the berries addition (aronia, raspberry and strawberry) had a synergistic effect, as expected, on the risk posed by Bacillus bacteria in food. Aronia was the most effective natural preservative, from the tested fruits, to prevent the damage of yogurt from cow’s and goat’s milk by Bacillus.

Knowledge of the combined technologies with the appropriate concentrations of “obstacles” in combination can contribute to the design and process control, to ensure the products safety and quality required by the consumer market [75] [76].

In studies conducted by [77] it was revealed that Bacillus subtilis were intolerant to acidic condition. During co-culture of Lactobacillus (five strains) or Lactococcus (two strains) with Bacillus cereus, organic acids and other potentially antimicrobial metabolites are produced. The strains that produced lactic acid fastest inhibited Bacillus cereus best [78].

The Pearson correlation [79] between the pH and the lactic acid bacteria growth in the classic yogurt samples fermentation process and with the berries addition was calculated. A high correlation was found a close relationship between variables, inversely proportional, because the values were obtained negative. The results are presented in Table 6.

The interdependence between pH and OD₆₀₀ and the interdependence between

pH and the lactic acid bacteria number in fruit yogurt are shown in Figure 6.

To control the Bacillus bacteria risk in food first is the in-depth knowledge about the survival and growth of Bacillus cereus,Bacillus pumilus and Bacillus subtilis in different food matrices, which depend mainly on pH and a_w (water activity), processing and storage temperatures, oxygen availability and the microflora presence, but also in their production of bacteriocins, diacetyl, carbon dioxide, hydrogen peroxide, ethanol or other food additives [31] [80] [81] [82] [83].

Bacillus cereus was found to adapt to acid stress (pH 4.6) when pre-exposed to a non-lethal, inducing pH of 6.3 or to inducing concentrations of heat, ethanol, salt or hydrogen peroxide. Cells were found to maintain their internal pH at a higher level than the external acid pH and adapted cells had a higher internal pH than unadapted cells [84]. Optimum medium condition of Bacillus subtilis was reached at 37˚C and pH 5.5 [85]. Its ability to form spores makes the bacterium highly resistant towards environmental impacts such as drought, heat or radiation, as well as low pH values or chemical conservation [86].