The raw materials including beef meat, fallow deer meat and a beef tallow came from a breeding farm. The farm produce was certified as organic by a Polish certifying body according to the article 29(1) of Council Regulation (EC) No. 834/2007 on organic production and labeling of organic products. Additional ingredients used to produce sausages in this study included sea salt (CurodiMare, Apulia, Italy) and acid whey. According to the certificate, the sea salt used in the experiment contained no sodium nitrate and nitrate. Acid whey was obtained from the traditional cottage cheese production (Organic Farm Robert Janowski, Ludwinów, Poland) and contained 93.23%±0.22% of water. Acid whey was used in liquid form and lyophilized in laboratory freeze drier (Labconco Free-Zone, Kansas City, MO, USA). Table 1 presents characteristics of acid whey in liquid and freeze-drying form.

Dry cured sausages were manufactured in two variants from different types of the meat (fallow deer and beef). In both cases beef tallow was used as the fat raw material. The raw materials were chopping and ground through a plate with a mesh diameter 8 mm in a universal machine (MESKO-AGD, Skarżysko-Kamienna, Poland). After that five batches with different additives were prepared from beef and another five from fallow deer meat (the amount of additives is shown in Table 2): C, (a control sample) with the curing mixture (99.5% sea salt, 0.5% sodium nitrite); S, (a reference sample) with the sea salt; AW, with the liquid acid whey; LAW, with the reconstituted, freeze-dried acid whey; 2LAW, with the reconstituted double portion of freeze-dried acid whey. Every time the acid whey was reconstituted from the lyophylizate using saline solution. The meat, fat (in a ratio of 80:20 [w/w]) and the appropriate additives were mixed and stuffed into fibrous casings (95 mm). The sausages were put into a fermentation chamber and kept at 16°C until the weight loss was about 30%. The stuffing and sausages after ripening were used to analyze changes in LAB and Enterobacteriaceae content and BA content.

Samples for microbiological analyses were taken after grinding and mixing the entire sample. All samples were taken in triplicate and carried out at Agrolab Polska Sp. z o. o. (Dęblin, Poland). The measurements were performed in accordance with ISO/IEC Standard 17025:2005 [19]. The results were calculated as a difference between content of bacteria at the beginning and end of process and were expressed as colony forming unit per 1 gram of product (cfu/g).

The BA extraction process was carried out by homogenizing 10 g of each sausage sample with 100 mL of 10% trichloroacetic acid in a homogenizer (15,000 rpm, 1 min, IKA T25D, Staufen, Germany). The homogenate was extracted for 1 hour in fridge. Next the samples were centrifuged (5,000 rpm, 20 min, 4°C, MPW 350R, Warszawa, Poland). The supernatants were filtered through a Whatman filter No. 1, passed back through a 0.22 μm nylon filter (Alfachem, Lublin, Poland) and were stored at 4°C until analysis.

The analysis of BA was performed using an AAA 500 amino acid analyzer (Ingos, Prague, Czech Republic), equipped with an Ostion LG AAA8 ion-exchange column (3.6×100 μm, 8 μm). Separation was by a stepwise gradient elution using Na⁺/K⁺ citric buffers. Colorimetric detection was at 570 nm, after post-column derivatization of ninhydrine. Content of the BA (histamine, tyramine, putrescine, cadaverine, spermidine, agmatine, spermine) was determined with a reference to the amine standards, which were supplied by Ingos, Czech Republic. The BA concentrations were reported as mg/g of product.

The experiment was carried out on two samples from each of the sausages in three replications. The results were expressed as mean±standard deviation. The Dell Statistica v.13 (data analysis software system) was used for the statistical treatment of the data. The main effects and interactions between means were calculated using the two-way analysis of variance method. The significant differences between subgroups have been specified based on post hoc Tukey’s procedure. Pearson’s correlation coefficient was calculated to measure the strength and direction of the relationship between studied variables. All statistical results were considered significant at p<0.05.

The changes in LAB and Enterobacteriaceae content between the beginning and the end of processing of dry cured sausages are presented in Table 3. According to data presented in the Table 4, type of meat, additives used and the interaction between them had significant effect on microbial changes in dry fermented sausages (p<0.01, except that the effect of additives on changes in LAB content had a significance level of p<0.05). During the processing the content of LAB increased in all samples. Nevertheless, in the samples made from fallow deer the increase was clearly higher. In the sausages made from beef, changes in the LAB content are highly correlated with the subsequent applied additives (correlation coefficient = 0.87). Addition of acid whey (especially addition of double portion of acid whey) caused a significantly higher increase of LAB (p<0.05). In case of the sausages made from fallow deer that significant dependence was not observed (p>0.05). Only in the sample with the double portion of acid whey was the increase of LAB content on a similar level as in the control and reference samples. Such results could mean that addition of the higher amount of acid whey inserts more of the desired bacteria into the sausage stuffing. This is in accordance with results obtained by other authors, who observed a higher amount of LAB in samples with acid whey addition [20,21]. During the processing Enterobacteriaceae content decreased in the all samples. The largest decreases were observed in the curing sausage (C) from the beef meat and in the fallow deer sausages with the different addition of acid whey (AW, LAW, 2LAW). Only the Enterobacteriaceae content changes in the fallow deer sausages were indeed correlated with the subsequent additives (correlation coefficient = 0.64). However, changes between the LAB content and Enterobacteriaceae content were significantly negatively correlated (correlation coefficient = −0.58), which may suggest that the increase in LAB content had an influence on inhibiting of growth of Enterobacteriaceae. Similar observations (reduction of the number of Enterobacteriaceae with growth of LAB) have been described by Greppi et al [22] who monitoring of the microbiota of fermented sausages. This dependence results from the activity of LAB which can produce lactic acid (which increases the acidity of the environment) and bacteriocins (which inhibit the growth i.e. Enterobacteriaceae) [23].

The content of BA after processing of dry-cured sausages is presented in the Table 5. Polyamines as spermine, spermidine and agmatine were not detected in any sample. Small amounts of histamine were detected in each variants of sausages. In every type of batch all detected BA content are significantly lower in the sausages made from the fallow deer meat (p<0.05). This could result from different type of meat. The first significant difference between the beef and fallow deer meat is free amino acids content. According to the literature game meat is characterized by lower content of most amino acids precursors for BA, except lysine [24–27]. Paleari et al [28] evaluated a composition of meat products from different animal species. Their results show that after the curing, fermentation and drying, the content of free amino acids (which are precursors for BA) in the deer products are significantly higher than in the beef products (p<0.05) [28]. Changes between the raw and processing products can indicate that, other factors during BA formation could be more important than the amino acids precursors [29]. Another difference that can affect BA formation, depending on the type of meat, is type and quantity of endogenous amino acid decarboxylase and its activity which can contribute to generation of BA [1]. Likewise, in different kinds of meat contaminating bacteria may belong to different species and strains, which can have various amino acid decarboxylase activity [11,30] or have ability to produce detoxifying BA—amine oxidase enzymes [11,12]. The lower level of BA in the sausages made from the fallow deer correlated well with the changes in Enterobacteriaceae and LAB contents. The LAB content increased more in the fallow deer sausages than in the beef sausages, also the Enterobacteriaceae content decreased in the higher level of whey addition in the fallow deer sausages. It confirms, the content of BA in the products is highly related with occurrence of spoilage microorganisms to which belong Enterobacteriaceae [11,31,32]. Pircher et al [32] also researched the formation of most important BA by bacteria isolated from the meat fermented sausages. Their results show that the LAB strains isolated from sausages are not able produce putrescine and cadaverine, but some of them can produce histamine and tyramine, which is similar to the results obtained by Pircher et al [32]. This explains the appearance of histamine in the fallow deer sausages (with the higher increase of LAB content), also the difference in the tyramine content between samples from the fallow deer and beef meat are not as large as in the case of putrescine or cadaverine content.

The most abundant BA in both cases were putrescine and cadaverine, which corresponded with the results obtained by Laranjo et al [33]. However very often as the most abundant BA in dry fermented/dry cured meat products is tyramine with cadaverine and putrescine being next most abundant, respectively [34,35].

The total BA content in samples with the addition of acid whey are significantly lower than in the control or reference sample. The drop in the BA content compared with the reference and the control samples were respectively 19%; 0%; 20% for AW, LAW, and 2 LAW in beef sausage samples and 51%; 31%; and 32% respectively for fallow deer sausages. The changes in the content for each BA are not as clearly visible and not all the differences are significant (p>0.05). However, the biggest impact of acid whey addition on the BA content was observed in the case of cadaverine. Each sample with the liquid acid whey addition (AW) and with the addition of double portion of freeze-dried acid whey (2LAW) had the significantly lower content of cadaverine (p<0.05), in the case of the sample LAW (freeze-dried acid whey) the significant difference was only in the fallow deer sausages (p<0.05). There were no observed significant changes in the putrescine and tyramine content dependent on the applied additive (p>0.05). The addition of organic acid whey causes a drop in a pH value and the increase content of microorganisms (mainly the LAB, which can induce further acidification), thus the growth of decarboxylating microorganisms is inhibited (by unfavourable conditions) resulting in a decreasing formation of BA’s [36]. The LAB as mentioned earlier are mainly related to the formation of tyramine and histamine [32]. Detailed research of Rzepkowska et al [16] concerned organic whey as a source of Lactobacillus strains with selected properties. They investigated microorganisms isolated from the organic acid whey. Results of their study shows that most of Lactobacillus stains present in organic acid whey can metabolise an arginine, which is the amino acid precursor for BA formation [16].

Significant lower level of total BA content in samples with the acid whey addition (p<0.05) may also result from activities of some bacterial strains occurring in acid whey. Rzepkowska et al [16] in their research isolated and identified the strains from similar organic whey as it was used at this study. Their results showed that among isolated species was Lactobacillus plantarum, which is one of many, with proven ability to degrade BA due to production of amine oxidase enzymes [16, 37,38]. To reduce the occurrence of BA in food the EFSA, recommends paying special attention to the hygienic quality of the raw materials and fermentation process as the most important factors for the biogenic accumulation in the fermented products [8]. Our research showed that use of acid whey can be a technological factor preventing the BA formation.