**2.2. Yeast and mould counting**

Counting of viable fungi is applicable to honey as it is an acidic food, with a pH of less than 4.5 and relatively low moisture. Fungi are affected little by variations in the pH range 3.0–8.0. The moulds grow below pH 2.0 and several yeasts below 1.5. When the pH deviates from the optimal, which is generally close to 5.0, the growth rate of colonies decreased and, if there are other inhibition factors, such as water or nutrient temperature activity, its restrictive effect on the growth rate becomes stronger [23].

Its presence at high levels in honey can provide various types of information; for example, the poor hygienic conditions of equipment, multiplying in the product due to failures in processing and/or storage. MERCOSUL GMC resolution n° 15 of 1994 approved the Technical Regulations for the Identity and Quality of honey, in view of resolutions n° 18 of 1992 and n °91 of 1993 of the Common Market Group [17], in which, in terms of hygiene, honey must be free of foreign inorganic or organic substances in its composition, such as insects, larvae and grains of sand, and should not exceed the maximum levels tolerable for microbiological contamination or toxic waste. Its preparation should be carried out according to the General Principles of Food Hygiene recommended by the Codex Alimentarius Commission—FAO/ WHO [15]. In terms of fungi, up to 100 colony forming units per gram are tolerated in honey (CFU/g) [17].

Moulds are filamentous, multicellular fungi, and may be present in the soil, air, water and raw organic decomposition. They are generally aerobic and less demanding than other yeasts in terms of humidity, pH, temperature and nutrients. They can absorb any carbon source derived from food. As a nitrogen source, they can use nitrate, ammonia and organic nitrogen. They only grow on the surface of honey when in contact with air, as it is a food rich in carbohydrates and acids [23, 31].

Yeasts are classified as non-filamentous fungi whose form is unicellular and can be spherical, ovoid, cylindrical or triangular. They are usually spread by insect vectors and by wind and air currents [32]. For growth yeasts require moisture more than that required by moulds and less than that required by bacteria, with an ideal temperature range for growth at around 25 and 30°C. The growth of the osmophilic yeasts which are part of the micro-biota of importance of honey is favoured as the liquid substrate provides a greater opportunity for the development of anaerobic conditions, due to possessing the ideal acid pH for use in the fermentation by which the yeast is transformed into sugar, which is used as an energy source in alcohol, when the water activity value is at least 0.65 [31, 33]. According to Pitt and Hocking [34], most osmophilic yeasts are of the genus *Zygosaccharomyces*, including *Z. rouxi, Z. bailii* and *Z. bisporus*. To control these microorganisms in honey the application of good hygiene practices is required, and it must be ensured that water activity or moisture content is within acceptable limits [15]. When the honey extracted from the beehive has a lower water activity than 0.60 the multiplication of osmophilic yeast does not occur.

**Method:** based on the verification of the ability of these microorganisms to develop in a culture media with a pH around 3.5 and incubation temperature 25 ± 1°C. The use of acidified mediums selectively favours the growth of fungi, inhibiting most of the bacteria present in food [2].


**Figure 3.** Procedure for the preparation of dilutions 10−1; 10−2 and 10−3.

**2.2. Yeast and mould counting**

268 Honey Analysis

the growth rate becomes stronger [23].

(CFU/g) [17].

hydrates and acids [23, 31].

Counting of viable fungi is applicable to honey as it is an acidic food, with a pH of less than 4.5 and relatively low moisture. Fungi are affected little by variations in the pH range 3.0–8.0. The moulds grow below pH 2.0 and several yeasts below 1.5. When the pH deviates from the optimal, which is generally close to 5.0, the growth rate of colonies decreased and, if there are other inhibition factors, such as water or nutrient temperature activity, its restrictive effect on

Its presence at high levels in honey can provide various types of information; for example, the poor hygienic conditions of equipment, multiplying in the product due to failures in processing and/or storage. MERCOSUL GMC resolution n° 15 of 1994 approved the Technical Regulations for the Identity and Quality of honey, in view of resolutions n° 18 of 1992 and n °91 of 1993 of the Common Market Group [17], in which, in terms of hygiene, honey must be free of foreign inorganic or organic substances in its composition, such as insects, larvae and grains of sand, and should not exceed the maximum levels tolerable for microbiological contamination or toxic waste. Its preparation should be carried out according to the General Principles of Food Hygiene recommended by the Codex Alimentarius Commission—FAO/ WHO [15]. In terms of fungi, up to 100 colony forming units per gram are tolerated in honey

Moulds are filamentous, multicellular fungi, and may be present in the soil, air, water and raw organic decomposition. They are generally aerobic and less demanding than other yeasts in terms of humidity, pH, temperature and nutrients. They can absorb any carbon source derived from food. As a nitrogen source, they can use nitrate, ammonia and organic nitrogen. They only grow on the surface of honey when in contact with air, as it is a food rich in carbo-

Yeasts are classified as non-filamentous fungi whose form is unicellular and can be spherical, ovoid, cylindrical or triangular. They are usually spread by insect vectors and by wind and air currents [32]. For growth yeasts require moisture more than that required by moulds and less than that required by bacteria, with an ideal temperature range for growth at around 25 and 30°C. The growth of the osmophilic yeasts which are part of the micro-biota of importance of honey is favoured as the liquid substrate provides a greater opportunity for the development of anaerobic conditions, due to possessing the ideal acid pH for use in the fermentation by which the yeast is transformed into sugar, which is used as an energy source in alcohol, when the water activity value is at least 0.65 [31, 33]. According to Pitt and Hocking [34], most osmophilic yeasts are of the genus *Zygosaccharomyces*, including *Z. rouxi, Z. bailii* and *Z. bisporus*. To control these microorganisms in honey the application of good hygiene practices is required, and it must be ensured that water activity or moisture content is within acceptable limits [15]. When the honey extracted from the beehive has a

lower water activity than 0.60 the multiplication of osmophilic yeast does not occur.

**Method:** based on the verification of the ability of these microorganisms to develop in a culture media with a pH around 3.5 and incubation temperature 25 ± 1°C. The use of acidified mediums selectively favours the growth of fungi, inhibiting most of the bacteria present in food [2].


Determine the number of yeast colonies on the plate based on the confirmed percentage. For example, of 30 colonies counted, five were submitted to confirmation and three were confirmed as yeast (60%), so the number of yeast colonies on the plate is 30 × 0.6 = 18. To calculate the number of colony forming units per gram (CFU/g) of yeasts and moulds, multiply the number of colonies by ten and by the inverse of the dilution. The total calculation of both is

**Figure 4.** Procedure for the preparation and inoculation of dilutions 10−1; 10−2 and 10−3 on the plates of potato dextrose agar.

carried out by adding the number of mould colonies and the number of colonies confirmed as yeast and multiplying by the inverse of the dilution according to Eq. (1).

$$\text{CFU/g} \newline \text{\newline = number of colonies} \times \text{Dilution reverse} \times 10 \tag{1}$$

For example:

Dilution 10−2 (inoculated 0.1 mL) Total typical colonies of mould on plate = 30 Presumptive colonies of yeast on plate = 40, five to submit for confirmation, confirmed four (80%) Total yeast colonies on plate = 40 × 0.8 = 32 CFU/g moulds = 30 × 10<sup>2</sup> × 10 = 3.0 × 10<sup>4</sup> CFU/g yeast = 32 × 10<sup>2</sup> × 10 = 3.2 × 10<sup>4</sup> CFU/g of yeasts and moulds = (30+32) × 10<sup>2</sup> × 10 = 6.2 × 10<sup>4</sup>

#### **2.3. Salmonella sp**

Species of the *Salmonella* genus are agents of human and animal intestinal infections. Among the agents of foodborne illnesses, *Salmonella* is one of the most responsible for fatalities and clinical complications. Moreover, the high morbidity and mortality rate and incidence in humans and animals result in significant spending on medications and hospitalizations. The inspection and monitoring of food is aimed at the control and prevention of members of this group and the effects of their presence in food. Compliance with good manufacturing practices and control programs should include a certificate of compliance with the measures adopted, especially for this bacterial genus [23].

**Method**: the method for detecting *Salmonella* in food is based on its presence or absence, developed to guarantee detection even in unfavourable situations. The procedures recommended by various regulatory bodies basically follow five steps that can be applied to any type of food [2, 3, 5, 10, 11, 13].


**Figure 5.** Procedure for the pre-enrichment non-selective broth.

carried out by adding the number of mould colonies and the number of colonies confirmed as

**Figure 4.** Procedure for the preparation and inoculation of dilutions 10−1; 10−2 and 10−3 on the plates of potato dextrose agar.

CFU/g = number of colonies x Dilution reverse x 10 (1)

Presumptive colonies of yeast on plate = 40, five to submit for confirmation, confirmed four (80%)

Species of the *Salmonella* genus are agents of human and animal intestinal infections. Among the agents of foodborne illnesses, *Salmonella* is one of the most responsible for fatalities and clinical complications. Moreover, the high morbidity and mortality rate and incidence in humans and animals result in significant spending on medications and hospitalizations. The inspection and monitoring of food is aimed at the control and prevention of members of this group and the effects of their presence in food. Compliance with good manufacturing practices and control programs should include a certificate of compliance with the measures

**Method**: the method for detecting *Salmonella* in food is based on its presence or absence, developed to guarantee detection even in unfavourable situations. The procedures recommended

× 10 = 6.2 × 10<sup>4</sup>

yeast and multiplying by the inverse of the dilution according to Eq. (1).

× 10 = 3.0 × 10<sup>4</sup>

× 10 = 3.2 × 10<sup>4</sup>

For example:

270 Honey Analysis

Dilution 10−2 (inoculated 0.1 mL)

CFU/g moulds = 30 × 10<sup>2</sup>

CFU/g yeast = 32 × 10<sup>2</sup>

**2.3. Salmonella sp**

Total typical colonies of mould on plate = 30

Total yeast colonies on plate = 40 × 0.8 = 32

CFU/g of yeasts and moulds = (30+32) × 10<sup>2</sup>

adopted, especially for this bacterial genus [23].


**Figure 6.** Procedure for the pre-enrichment selective broth.


**Figure 7.** Procedure for differential plating and biochemical identification.





xylose lysine deoxycholate agar (XLD) and xylose lysine tergitol-4 agar (XLT-4). As there

the second or third plating medium is not based on these characteristics. One option is the brilliant green phenol red lactose sucrose agar (BPLS) or brilliant green agar (BG) based on

used in this step. Add 0.1 mL of novobiocin solution 4% to 100 mL of brilliant green phenol

S production and not lactose fermentation. Rambach agar can also be

are *Salmonella* strains which ferment lactose or do not produce H<sup>2</sup>

red lactose sucrose agar. Incubate all plates at 35°C for 24 hours (**Figure 7**).

the fermentation of lactose but not the production of H<sup>2</sup>

**Figure 7.** Procedure for differential plating and biochemical identification.

S production, such as hektoen enteric agar (HE),

S, it is important that

S, and bismuth sulphite agar (BS),

bath for 24–30 hours.

272 Honey Analysis

which is based on H<sup>2</sup>

the non-fermentation of lactose and by H<sup>2</sup>



**Table 3.** Colour of culture medium and *Salmonella* spp. positive and negative percentage in biochemistry test after 24 h incubated at 35°C.

(1) *Triple sugar iron agar (TSI Agar):* this medium is used to differentiate Gram-negative rods based on fermentation and the gas production from the carbohydrates: glucose, lactose and sucrose and the production of hydrogen sulphide. For the test, inoculate the triple sugar iron agar by deep, grooved stabbing motions and in inclined surface of the bevel. Incubate at 36°C for 18–24 hours. In the presence of *Salmonella*, the glucose is rapidly depleted, and is verified by the appearance of a yellow colour in the base. After the fermentation of glucose, the aerobic degradation of the protein substrate of the medium occurs, producing ammonia, which gives the medium an alkaline pH, changing the bezel colouring to intense pink. Gas production is indicated by the formation of blisters or cracks in the medium. Most *Salmonellas* do not ferment sucrose and lactose. When these two sugars are not fermented, the apex keeps its original colour—amber. The production of H<sup>2</sup> S is indicated by the black colour at the base of the central portion of the tube. Microorganisms such as *Proteus mirabilis, Edwardsiella tarda, C. freundii* and *Salmonella spp* may exhibit a similar behaviour.


(6) *Utilization of citrate (Simmons citrate agar):* characterize microorganisms capable of utilizing citrate as the sole carbon source, which cause the pH of the culture medium to increase due to the metabolism of citrate ions. Transfer by streaking the bacteria to be tested on the inclined surface of the Simmons citrate agar with a needle. Incubate the tubes at 37°C for 24–48 hours. The *Salmonella* strains (95%) are positive, except for the serotypes Typhi, Paratyphi A, Pullorum and Galinarum (100% of negative strains) and Choleraesuis (75% of negative strains), and can utilize the citrate and extract nitrogen ammonium salt, leading to alkalization of the medium from the conversion of the NH<sup>3</sup> in ammonia hydroxide (NH<sup>4</sup> OH). After incubation, examine the cultures contained in the tubes with an inclined medium and assess the presence or absence of bacteria growth, checking for any change in colour: if positive, the medium becomes intense blue, especially at the apex; if negative, the natural colour of the medium does not change, but remains green.

for 18–24 hours. In the presence of *Salmonella*, the glucose is rapidly depleted, and is verified by the appearance of a yellow colour in the base. After the fermentation of glucose, the aerobic degradation of the protein substrate of the medium occurs, producing ammonia, which gives the medium an alkaline pH, changing the bezel colouring to intense pink. Gas production is indicated by the formation of blisters or cracks in the medium. Most *Salmonellas* do not ferment sucrose and lactose. When these two sugars are not fermented, the apex keeps

of the central portion of the tube. Microorganisms such as *Proteus mirabilis, Edwardsiella tarda,* 

S by the appearance of black colouring from the base to the central portion of the tube.

production capacity. Inoculate the culture medium. Incubate at 36°C for 24–30 hours. The motility reading is characterized by the diffusion of growth throughout the medium. If restricted to the line of streaking, it indicates that the microorganism is immobile. After

medium. Bacteria that possess the tryptophanase enzyme are capable of hydrolyzing and deaminating the tryptophan with the production of indole, pyruvic acid and ammonia. To verify its production, add a few drops of Kovac's reactive to the tubes; if there is indole production a red ring will form. In most cases (99%) the strains of *Salmonella* do not pro-

(5) *Voges-Proskauer (VP) test*: determine the ability of some bacteria to oxidize glucose producing organic acid as a final product. Transfer the microorganism to be tested to test tubes with red broth methyl-Voges-Proskauer (Clark and Lubs medium). Incubate the tubes at 37°C for 24–48 hours. To read, add 5 drops methyl red. Positive: red colour (pH < 4.0); Negative: original medium colour (yellow) (pH ≥ 6.0). The *Salmonellas* are VP negative. From the VM-VP medium, remove 2 mL of culture to a new tube and add 15 drops of α-naphthol 5% reagent (reagent A) and 5 drops of KOH 40% solution (Reagent B) to each tube for each ml of culture medium. Agitate the tubes so that there is oxygenation of the medium. Wait for 10–30 minutes. Positive: development of pink-

S production by the development of the black colour in the

(2) *Lysine-iron agar (LIA Agar)*: this medium is used to verify the decarboxylation of lysine which is evidenced by the violet colouration—alkaline—of the base. When this does not occur, the yellow colour indicates only the fermentation of glucose. The positive reaction for the deamination of lysine is visible at the apex (coppered violet) and the production of

(3) *Hydrolysis of urea (Stuart urea broth and Christensen urea agar:* determines the ability of a microorganism to degrade, enzymatically, the urea by urease, with the formation of two molecules of ammonia and carbon dioxide, with the alkalization of the medium and increased pH. Streak only on the surface of the broth or the urea agar. Incubate at 36°C for 18–24 hours. The colour is caused by addition of the phenol red to the medium. The positive reaction turns the yellow (the original colour of the medium) to intense pink. *Proteus* features a more intense reaction; the negative reaction maintains the yellow colour of the medium. A total of 99% of the *Salmonella* strains do not produce urease.

(4) *Indole test (SIM medium):* check the motility of the microorganisms and the H<sup>2</sup>

S and are mobile.

ish to red colouring; Negative: absence of pink or red.

S is indicated by the black colour at the base

S and indole

its original colour—amber. The production of H<sup>2</sup>

the motility reading, verify H<sup>2</sup>

duce indole, do produce H<sup>2</sup>

H2

274 Honey Analysis

*C. freundii* and *Salmonella spp* may exhibit a similar behaviour.


#### Observations:


In general, the various regulatory bodies also recommend the use of miniaturized commercial kits which allow a great number of biochemical tests.


Serologic confirmation verifies the presence of 'O', 'V' and 'H' antigens by agglutination tests with polyvalent antisera:


Classify the reaction as follows:


The cultures with positive results in the agglutination test with the anti*-Salmonella* polyvalent 'O' serum should be sent to certified laboratories for final classification.

#### **2.4. Determination of the antibacterial activity of honey**

With the exaggerated use of certain compounds such as ampicillin, cephalexin and others, bacteria have developed resistance to antibiotics, leading to studies for new compounds with antimicrobial activity from different natural products such as honey [35]. Since the beginning of civilization, honey has had a cultural importance that is not restricted merely to food but also includes use as folk medicine and as a cosmetic [36]. It has different therapeutic properties and is antimicrobial, antifungal, antioxidant, antiviral, anti-parasitic and anti-inflammatory [35, 37].

Observations:

276 Honey Analysis

(2) *S. Typhi* is anaerogenic;

(6) *S. arizonae* absorbs the malonate;


with polyvalent antisera:

and homogenize;

Classify the reaction as follows:


(7) *S. arizonae* does not ferment the dulcitol;

(8) 25% of the *Salmonella* strains are citrate-negative.

kits which allow a great number of biochemical tests.

(1) These percentages indicate the incidence of strains with reactions marked as + or −.

In general, the various regulatory bodies also recommend the use of miniaturized commercial

Serologic confirmation verifies the presence of 'O', 'V' and 'H' antigens by agglutination tests



The cultures with positive results in the agglutination test with the anti*-Salmonella* polyvalent

With the exaggerated use of certain compounds such as ampicillin, cephalexin and others, bacteria have developed resistance to antibiotics, leading to studies for new compounds with





'O' serum should be sent to certified laboratories for final classification.

**2.4. Determination of the antibacterial activity of honey**

sion on the slide and mix, and add one drop of saline to the other;

(3) *Salmonella enterica arizonae*: + or – reaction to lactose, positive β-galactosidase;

(4) *Salmonella enterica salamae*: - reaction to lactose and β-galactosidase;

(5) *Salmonella pullorum* and *Salmonella gallinarum* are immobile;

Honey is a substance prepared from the nectar of flowers (floral honey), plant exudates, or the excretion of sucking insects of plants (honeydew) [18]. The enzyme content present in honey is differentiated, as it depends on the species of bee, soil characteristics, seasonal factors such as temperature, rainfall and bee flora, with the product distinguished by the amount of organic acids, enzymes, vitamins, flavonoids, minerals and an extensive range of organic compounds, contributing to its colour, odour and specific flavour [38].

The antimicrobial action of honey is related to soil characteristics, atmospheric conditions, plant diversification, low water activity (Aw), high osmotic pressure, low pH, the glucose/oxidase system of hydrogen peroxide formation, the presence of phytochemical constituents and volatile substances [39]. These different qualities together create differences in the expression of antimicrobial activity of honey [40]. Molan [41] reported that in super-saturated sugar solutions, honey has a low water activity, which, as well as the natural acidification of the medium, creates unfavourable conditions for bacterial growth. In the presence of water and oxygen, the enzyme glucose oxidase converts glucose into gluconic acid and hydrogen peroxide, which are considered relevant substances for antioxidant action, which affect the microorganisms and preserve the sterility of honey during maturation [42].

**Method:** *Preparation of bacterial inoculum and standardization:* from the pure culture of bacteria preserved under refrigeration at 6°C, proceed to the preparation and standardization of the inoculum, in accordance with to the Clinical and Laboratory Standards Institute CLSI M07-A9 document [43]. Transfer three to five colonies of the selected strain to a test tube with a screw top containing 4–5 mL Miller Hinton broth (MHB), incubate the culture in the broth at 35°C for 18–24 hours and standardize the bacterial suspension in 0.85% saline solution, obtaining an optical turbidity comparable to standard McFarland solution 0.5 to the naked eye under illumination against a white background card with contrasting black lines. Dilute the inoculum at a ratio of 1:10 in saline solution 0.85% resulting in a concentration of 10<sup>7</sup> CFU/ml.


Pipette out 100 μL of Mueller Hinton broth in each well and then perform a dilution series of different samples of honeys, with each honey sample in a different line. For serial dilution, pipette out 100 μL of honey in the first well, homogenize, remove 100 μl from the first well and transfer to the second well, remove 100 μL from the second and transfer to the third; and so on, until the ninth well of each row. This provides the following honey concentrations in percentages (%) (**Table 4**).

**Figure 8.** 96 well U bottom micro-plate with markings indicating the position of the lines (A–H) and columns (1–12).


**Table 4.** Honey concentrations in percentage (%) for wells from 1 to 9.

As a bacterial control (without the addition honey), use well 10, and as a broth control (without the addition of honey and inoculum), use well 11. After adding the honey, inoculate 5 μL of a standardized suspension of the bacteria in question in each well, except the broth control well, so that the end of test bacteria concentration is 5 × 10<sup>4</sup> CFU/well. Identify the microplates, incubate in a bacteriological incubator at 35°C for 24 hour. After 24 hours of incubation the micro-plates are analysed to determine the MIC, which is defined as the lowest concentration of honey in which there is no visible growth after incubation. Finally, analyse the well contents indicated with the minimum inhibitory concentration by microscope to confirm if there is growth or not. Perform the tests in triplicate for each of the bacteria analysed.
